hall. from the cancer research department , university of otago , dunedin , n.z. received for publication july 6 , 1948 . it has been realized for a long time that two processes might be involved in the pathogenesis of neoplasms , one which is concerned with the transformation of normal into neoplastic cells , and the other which promotes the growth of friedewald and rous ( 1944a , 1944b ) used neoplastic cells into a visible tumour . initiating " for the former , and " promoting " for the latter process . the term rous and kidd ( 1941 ) , and mackenzie and rous ( 1941 ) analyzing the effects of tar and carcinogenic hydrocarbons on the skin of rabbits , found that these agents " cause many more cells to become tumour cells than give rise to visible growths . " berenblum ( 1941a ) discovered that the yield of tumours of the skin induced in mice by sub - optimal doses of carcinogenic hydrocarbons could be increased by subsequent application of croton oil , and used the term " epi - carcinogenic action " to describe this effect . 
mottram ( 1944a ) refined berenblum 's technique ; he found that tumours could be obtained by a single application of benzpyrene if the painting with the carcinogen was followed by repeated application of croton oil.. 
they were seen first in a rat killed 10 weeks after the withdrawal of the carcinogen , whereas an animal of the same group sacrificed 3 weeks earlier , showed only the well - known picture of hyperplasia and loss of colloid . 
the number and size of the adenomata increased during the course of the experiment , but their histological pattern remained essentially unaltered there was up to the twenty - first week , when the experiment was terminated . no indication of beginning malignancy , the nodules remaining sharply defined apart from the multiple adenomata of the thyroid , and showing orderly growth . no other lesions were found which could be attributed to the action of a.a.f. even when the duration of the experiment is prolonged for 18 months , the incidence of tumours induced by 10 - 15 mg . 
the only neoplastic changes seen in this group were single adenomata , the first of which was found in a rat which had this was of minute size involving received methyl thiouracil for 21 weeks . in three of the four animals killed after 42 weeks of treatonly a few follicles . ment with methyl thiouracil , single adenomata were also found . 
bielschowsky has allowed me to include in this paper an experiment performed by him in 1945 which he has already mentioned ( bielschowsky , 1947 )  . twenty female rats received daily 6 mg . 
the neoplastic changes found were single adenomata of varying size . these benign neoplasms were indistinguishable from the ones which appear after prolonged stimulation with goitrogenic agents , and which persist after their withdrawal . only in one animal ( rat 3 ) two minute nodules were present in the same lobe . it is of interest that a.a.f. 
did not transform these benign structures into cancers . to test for the existence of latent neoplastic cells in the thyroid the following experiment was set up . fourteen rats received first 4 doses of 2 - 5 mg . 
the existence of such latent cells in mice was demonstrated by applying an unspecific irritant to the skin which had received a single dose of a carcinogenic hydrocarbon ( mottram , 1944a ; berenblum and shubik , 1947a )  . there is excellent agreement between the results of mottram , and of berenblum and shubik except in one point . 
berenblum ( 1941b ) , and berenblum and shubik ( 1947a ) could not find any evidence for a sensitizing action of croton oil applied previous to the carcinogen to.the skthe results obtained in this laboratory agree with those of berenblum and shubik . there was no evidence that pre - treatment of the thyroid with a goitrogenic agent sensitized the gland to the subsequent action of a.a.f. it could be shown that a small dose of a.a.f. , which alone is incapable of inducing visible tumours in any part of the body , must nevertheless transform many normal into neoplastic cells . 
in the doses used , acts on the thyroid in the same way as a single moderate does of a carcinogenic hydrocarbon . there exists , however , one difference between the action of croton oil and the effects obtained with prolonged hormonal croton oil , however long applied to the skin of mice , produces only stimulation . diffuse hyperplasia , but several examples are known where long continued stimulation by hormones leads to formation of benign and malignant tumours . for instance , treatment with oestrogens leads to cancer of the breast in rats ( geschickter , 1942 ; nelson , 1944 ) , and administration of goitrogenic compounds induces cancer of the thyroid after 20 or more months ( purves and griesbach , 1946 ) , benign tumours appearing earlier ( purves and griesbach , 1947 )  . 
similarly in spayed mice , tumours of the ovary can be obtained by transplantation of this organ into the spleen . here the gonadotropic hormones are the stimulating agents ( biskind and biskind , 1944 )  . 
the single adenomata which , as bielschowsky ( 1945 ) has already shown , develop after stimulation with thyrotropic hormone in absence of a chemical carcinogen , very rarely appear as quickly and never with the regularity of the multiple tumours initiated by a.a.f. 
they seem to appear nearly as quickly as the papillomata of the skin in the experiments of mottram ( 1944a ) and berenblum and shubik ( 1947a , 1947b )  . unfortunately it is necessary to kill the animals in order to demonstrate the presence of the adenomata of the thyroid , so that it is difficult to discover the very early lesions . it seems , therefore , justified to assume that , in the experiments reported in this paper , the thyrotropic hormone plays the same role in the pathogenesis of the multiple adenomata of the thyroid as the croton oil in the pathogenesis of the papillomata of the skin . the fact that only benign tumours are induced in both sets of experiments strengthen the analogy , and it seems legitimate to disregard the late effects of hormonal stimulation in the interpretation of the.results. is worth while mentioning that the choice of method is of utmost importance in experiments designed to demonstrate the role of initiating and promoting factors . for instance , when methylcholanthrene is used in high concentrations a single application is sufficient to induce benign or malignant tumours ( cramer and stowell , 1943 )  . also large doses of goitrogenic agents , as used by kuzell , tripi , gardner and laqueur ( 1948 ) , seem to hasten the appearance of multiple adenomata of the thyroid . the induction of multiple adenomata of the thyroid by 10 - 15 mg . 
the conceptions derived from the study of experimental cancer of the skin by rous and collaborators , and by berenblum and shubik can be successfully applied to an analysis of the pathogenesis of experimental neoplasms of the thyroid . i wish to thank t . 
according to these measurements very little difference was observed between the size of the tumours of the control and dibenzanthracene treated animals in the high protein group after 7 days , and the tumours of the treated animals appeared to be only slightly smaller than those of the controls after 10 days . in the low protein group , however , the measurements on the 7th and on the 10th days showed that there was considerable inhibition of tumour growth in the animals treated with dibenzanthracene , while the tumours of the control animals had grown to about the same size as those of the controls of the high after 11 days the animals were killed and the tumours dissected protein group . very little tumour inhibiout and weighed . 
as - there was some doubt as to the absolute validity of this control method , we computed the number of cancer cases which would be expected , based on the morbidity figures from the cancer registry and on the official mortality statistics . 
rather unusual for twin studies of this kind . griginally all hospital cancer cases from the danish population of 4 million through six years , or an annual - number between 5000 and 6000 cases , but this figure must be reduced by about 20 per cent in which the questio ' n about twin birth had been left unanswered , giving a total very near to 30 , 000 cases ( 29 , 458 )  . the system of notification involves a rather heavy toll of omissions , vet it has functioned satisfactorily . 
the special questions asked twins with cancerbesides such items as name , address , ' occupation and the like - were confinea to previous diseases of a more serious character , including diagnosis and the date , in order to identify whether the twins were molloand place of treatment . zygotic or dizygotic , questions were asked about the similarity and about mistakes made by parents or others , and similarly about type and colour of hair , colour of eyes , stature , height and weight on the whole it may be said that we demanded almost full congruity before accepting a pair as monozygotic . 
the final distribution of dizygotic twins on groups of same and opposite sex , and the proportion between monozygotic and dizygotic pairs seem to show that our measures have been justified . it should be stressed that we have not been in - a position to contact the patients or their twins personally . 
the official percentage of twin births in denmark for the years 1926 - 30 is 1 - 64 , and considering the increased mortality among twin babies we ' think this a satisfactory correspondence . another check on the result was obtained through computation of the number of deaths expected from all causes among twins for whom notifications were satisfactory . 
among dizygotics we expected 36 - 3 and found 26 . these differences are not statistically 'significant , and they may be partially due to the less efficient information about those pairs of which one partner has died . in denmark , as elsewhere , the girl - boy ratio at birth is 0 , 97 with a male excess , which in the course of a fev years changes to a slight female preponderance . however , we find among both monozygotic pairs and dizygotic pairs of opposite sex a female preponderance of 1 , 48 to 1 , and 1 , 64 among the same - sexed dizygotic pairs , making 1 ' 53 for the total . 
the explanation of this considerable female excess m our material no doubt must be ascribed to the higher cancer incidence among females than among males in denmark , combined with the earlier age - in which female cancers arise . 
and , if we calculate the percentage of opposite - sexed twin pairs in our material , a figure which should be uninfluenced by the difference in mortality among the two sexes , we find exactly the same value as in the corresponding official birth statistics - 36 , 2 per cent . in spite of these satisfactory results , it should be remembered that in dealing with twin - pairs , of which at least one is suffering from old - age disease like cancer , we must expect insufficient information from twins in cases where the partner died in childhood . therefore we have excluded the twin - pairs where one partner bas died before the age of 5 years . similarly we have lost sight of a number of cc second " twins through emigration and similar events , so that the number has shrunk ' from 336 to 185 pairs . 
3. given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . since 1944 we have taken a detailed family history from all patients reporting with carcinoma of the breast . 
we have 459 family records up to the end of 1947 which are reasonably complete . so far we have not succeeded in supplementing these histories , taken at the time of first visit to hospital , by later questions to the surviving patients or by arranging visits to their homes or relations . 
some attempt has been made to confirm the causes of death of relatives by letters to hospitals , doctors and the registrar - general 's department , but such confirmation has , as yet , been obtained in only a few cases . our figures , therefore , contain all those errors of inadequate information and faulty recollection that one would expect from data collected . 
many people die of cancer without their relations knowing that this was the cause of death . it is in fact surprising how successful a woman can be in concealing a cancer of the breast from her nearest relatives living in the same house with her . it is not , therefore , so surprising that more distant relatives , or even close relatives living away from home , may be unaware of the nature of the illness involved relations , even some of an older generation , who are still living at the time that the patient is first questioned may later develop malignant disease . 
the mixtures which had a ph of about 2 - 5 were polarographed as f - 611ows , : the test solution , into which dipped a dropping mercury cathode , was connected to a saturated calomel reference anode ( s.c.e. ) by salt - agar bridges ( saturated kci in 3 per cent agar - agar ) and the solution deoxygena - ted by passing a stream of water - washed oxygen - free nitrogen through it for 10 minutes . 
the percentage ttc reduced by serum was estimated with reference to the wave height of ttc in the blank reaction mixture . triphenylformazan 1 - 2 volts under the present conditions . gave no reduction wave in the range 0 to 200 to fig . 
2. - scatter diagram showing negative linear correlation between percentage ttc reduction regression lines have been constructed from by serum and percentage tumour weight . the regression equations : in the scatter diagram , fig . 
2 , from which it will be seen that there is a negative linear correlation between percentage ttc reduced and percentage tumour weight . the correlation coefficient ( r ) was calculated in the usual way ( moroney 1953 ) and found to be - 0 - 75 , t test = 5 - 63 ( n = number of animals ) highly significant at the 01 per cent level . reducing capacities of tumour and normal rat sera are show - n in fig . 
serum hastened colour development in iruminated mixtures , the colours being marked after i day . control solutions containing no serurn required 2 to 3 days to develop a pink colour whfle serum mixtures and controls kept in the dark exhibited no colour even after i week . after exposure to dayeght for 2 days tumour serum mixtures were coloured pink whereas normal serum mixtures , orange - pink after i day , were orange366 w . 
neish of 10 tumour sera , 9 ( tumour range , 3 - 4 to 22 - 8 per cent ) yellow in colour . showed pink colours and 1 serum ( 8.1 per cent ) gave the orange - yellow reaction . six normal sera each gave orange - yellow colours . in time , all colours faded to pale yellow and the solutions fluoresced blue in ultra - violet light . 
no difference was found with respect to ttc wave height between tumour and normal serum - ttc mixtures and the illuminated control but a new wave was observed in each solution which had been exposed to light . this wave was never observed in the ttc - alkali reaction mixtures described in sections ( 1 ) and ( 2 ) , or in control mixtures kept in the dark . 
and it was found to be due to another photochemical reaction product of ttc , namely the 2 , 3 , diphenylene - 5 - phenyl tetrazolium compound ( blue fluorescent in ultra - violet light ) described by hausser , jerchel and kuhn ( 1949a ) and by kuhn and jerchel ( 1952 )  . 
the tumour extended outwards for 1 - 5 cm . into the surrounding lung tissue , and in collar - fashion for 4 to 5 calong the bronchus , its branches and the contiguous blood vessels . ' the cut surface of the tumour was finely patterned by white striae , which separated softer , yellow or grey areas . 
the lymph nodes in the hilus of the left lung were replaced by a large mass of partly necrotic tumour tissue which , extended into the posterior mediastinum , compressing and invading the adventitia of the great vessels and of the oesophagus . 
numerous deposits of tumour , up to 3 cin diameter , were present in the left pleura , especially over the intercostal spaces , and the pleural sac contained approxiniately 300 ml . 
the mucosa was fixed to the tumour and slightly elevated ; superficial ulceration involved an area of 2 - 5 x 2 cthe lumen of the bowel was not obstructed , and the mesocolic lymph nodes appeared to be normal . the peritoneal cavity contained a small amount of straw - coloured fluid ; the serosa was sparsely studded with tumour deposits which measured up to i cin diameter . 
the surviving liver tissue was deeply bile - stained and congested . the lymph nodes around the coehac axis and head of the pancreas , in the porta hepatis and in the lesser omentum were enlarged , and their substance was replaced by tumour tissue . 
the para - aortic and iliac lymph nodes were involved to a less degree . there were no relevant abnormahties of the other abdominal and pelvic viscera . the body of the ilth thoracic vertebra was almost completely destroyed by tumour , and had collapsed , with consequent pressure upon the spinal cord . 
the appearances were characteristic of an anaplastic bronchial carcinoma " of the " oat - cell " type , and confirmed the macroscopic diagnosis of primary bronchial carcinoma . the greater part of the colonic tumour was . 
the mucosa overlying the mass , however , showed an abrupt transition at its periphery from the normal structure and cytology to a polypoid , hyperchfomatic columnar - cefl adenocarcinoma , irregular gland - hke extensions of which penetrated deeply into the tunica muscularis ; the structure and cytology were those ' of a primary colonic adenocarcinoma . 
the patient remainecl in seemingly good health for more than 18 months , and then sustained a fracture of the pelvis in an accident . nine weeks later an abscess developed deep in the inter - ischial region , necessitating incision ; its contents were not examined . during the next fortnight a papiroma , about 0 - 8 cin diameter and situated at the medial end of the left inguinal skin fold , began to grow rapidly ; it doubled its size within this period , after which it was excised . 
the patient then remained apparently well until 1949 , when she began to be dyspnoeic ; - examination showed a right - sided pleural effusion . clear , amber - coloured fluid was aspirated 5 times , at intervals of a few weeks , the quantities withdrawn ' ranging from 550 to 1200 ml . the fluid contained between 4 and 5 per cent of protein ; a few cehs were present , mostly lymphocytes , with a very small pro_portion of neutrophile polymorphonuclear leucocytes and normal serosal cells . aerobic and anaerobic cultivation on ordinary media yielded no growth , and , cultures for mycobacterium tuberculosis were also sterile . radiography showed no evidence of neoplasm in the chest , but the effusion persistently obscured the - greater part of the right lung field ; there was never any demonstrable effusion in the left pleural sac . as there was no evidence of recurrence or metastasis of the carcinoma , and as there was no demonstrable infective cause of the recurrent pleural effusion , the discovery of an adnexal tumour on pelvic examination suggested the possibifity that the con ' dition was an at pical variant of the meigs - culhngworth syndrome . 
at laparotomy , 6 months after the onset of dyspnoea , a cystic tumour of the left ovary was fou ' nd and removed ; there was no other abnormality in the pelvis or abdomen . 
the tumour of the left breast was a simple intracanaficular fibro - adenoma . macroscopically , the ovarian tumour was a unilocular cystic teratoma , i 1 cm . in diameter , filled with sebum - hke material and hair . 
the cyst had a smooth lining ; its war was i mor less in thickness , except near the pedicle , where there was an irregular , bone - hard eminence , approximately 2 cin diameter , projecting into the cavity . adjacent to the pediele the remnant of the ovary was identifiable on the external surface of the tumour as a flattened , ovoid area of firm , greyish - white tissue , which measured 3 - 5 x 2 - 5 csuperficially , and up j . 
symmers microscopically the cyst was lined by keratinizing epi - to 0 - 4 cin thickness . dermis , external to which was a narrow zone of fibro - fatty tissue . hair - follicles and large sebaceous glands were numerous in the skin covering the hard projection , which consisted of bone , bron ' chial structures and neuroglia . 
the third hypothesis seems to us to be the only one tenable . if the coexistence of two primary neoplasms is accepted , the significance of the presence of a metastatic deposit from the primary bronchial carcinoma . at the site of the primary colonic carcinoma requires to be considered . this association may have been fortuitous , or the presence of the colonic neoplasrn may have predisposed to the establishment there of the metastasis , or the development of the latter may have inducea cancerous change in the adjacent mucosa ; it seems to be impossible to say which suggestion is correct . 
the induction , by some effect of the existing neoplasm , of sarcomatous change in the stroma of carcinomata has at times been postulated in explanation of the genesis of so - called careinosarcomata : in view of the considerable doubt as to the origin of the sarcoma - like component of these tumours , such a hypothesis has httle practical value . 
the induction of one carcinoma by another was postulated by duboisferri ' ere ( 1939 ) , who suggested that hepatic metastases in his case of islet - cell pancreatic carcinoma had induced local development of bile - duct carcinomata . he was not able to exclude the possibihty that the adenocarcinomatous areas in the liver were metastases with an unusually well - developed tubular structure , such as is sometimes seen in islet - cell neoplasms . seecof ( 1924 ) reported a case which was in some respects similar to ours . 
7. received for publication june 9 , 1947 . the presence and activity of the milk - borne agent of mammary tumours of mice discovered by bittner ( 1936 ) are detectable only by the growth of a mammary tumour . 
found two nodules in one mouse out of nine of the n strain in which mammary tumours had never occurred . also when efforts have been made to exclude the milk - borne agent from high cancer strains by cross - suckling , nodules have nevertheless been found . 
thus bittner , huesby , visscher , ball and smith ( 1944 ) examined the mammae of fostered breeding females ( fostered by cancer - free maternal stock ) from high cancer strains a and c3h . 
all the females had given birth to at least three litters and ranged from 11 to 16 months old , thereby providing suitable conditions for pathogenic activity of the milk - borne agent . nodules were found occasionally , though never more than one to a gland , thus nodules were still present though in smaller numbers when efforts were made to exclude them than in mice which had been suckled on their own cancer - prone mothers . this evidence indicated that nodule incidence is not eliminated by cross suckling with the same regularity that is attainable in regard to tumour incidence . 
6. - regression of hyperplasia induced by 800 y oestradiol in spayed r3 female ( carrying the milk - borne agent ) , and persistence of areas of nodular hyperplasia ( adenomas ) at 6 months old . 
the former discrepant results ( bittner et al . , 1944 ; huesby and bittner , 1946 ) have to be remembered , although subsequent observations ( kirschbaum et al . , 1946 ) provide convincing evidence that hyperplastic mammary nodules do not occur in the absence of the milk - borne tumour agent . the second source of objection to accepting nodular hyperplasia as evidence of activity of the milk - borne agent acting alone is that certain observations , which will be referred to again later , suggest that similar cellular proliferations with acinus formation are stimulated by oestrogenic hormones alone . the problem of the exact origin of the hyperplastic nodules is of great these focal acinar proliferations precede maligimportance for several reasons . nant tumours in time of appearance and are often more numerous . they might thus provide an alternative and earlier means of detecting activity of the tumour they may also represent the earliest sign of tumour growth before it agent . can be described as malignant . if so they would provide material enabling one to determine which influences are causative and which are encouraging , in the sense in which the term is used by rous and kidd ( 1941 ) and mackenzie and rous ( 1941 ) in the development of malignant tumours . 
the discrepant results ( previously referred to ) concerning their incidence often suggest that they may be more sensitive criteria of the presence of the agent . if the nodular hyperplasias are to be used for any of the purposes suggested , it must be proved beyond question that they are in fact dependent for their origin and continued existence on the presence of the milk - borne tumour agent , and that the nodules , or some of them , are but an early stage of eventual malignant tumours . other possible causes of focal cell proliferation such as hormones must be excluded . the possibility has also to be considered that mice of high cancer strains carry more than one formative milk - borne stimulus for the mammary gland . one agent might be the cause of frankly malignant tumours and another the cause of nodular hyperplasias . appears more probable , however , that one and the same agent is responsible for both types of tumour , benign and malignant , but that its virulence varies . greater virulence would be understood as the capacity to stimulate the mammary gland cells to multiply and invade or metastasize within the life of the mouse ; less virulent variants would cause merely focal proliferations . 
whether the cell proliferations which it produces are benign or malignant depends upon the combination of its " virulence " with encouraging or deterrent forces . the experiments here recorded are concerned only with the origin , provocation and continued existence of the adenomatous hyperplastic nodules . was found possible to stimulate the appearance of nodules artificially with measured limited amounts of hormone at an earlier date than that at which they become detectable spontaneously . it was also possible to distinguish responses in the mammary gland due to hormone alone . responses to other formative stimuli were considered . mamnmae of female and rudimentary mammae of male mice respond to 180 b . 
the responses to single and repeated doses ' of ovarian hormones were examined at various intervals after application as described under experimental methods . the existence of a strain difference in response to oestrogens which was independent of the presence of milk - borne tumour agent was confirmed . 
a similar hormonal response was obtained in c3h mice giving rise to focal hyperplasias , but as this strain was not freed from the milk - borne tumour agent , it is uncertain , though probable , that the response to hormone alone would have given this atypical reaction . 
some were derived from pure lines , others from random breeding within the strathe incidence of spontaneous nodules of hyperplasia in the mammae in 26 virgin females segregated from males had previously been found to be 100 per cent after the age of 8 to 9 months . malignant tumour incidence in the same series was 69 per cent nodules in this strain are extremely numerous in both virgin females and breeders . r3x sublines free from the milk - borne tumour agent were bred by foster nursing an original family of 2 females and 1 male on a c57 ( black ) mother from the moment of birth . 
pullinger record was kept of the relationships of every mouse of these sublines so that , if evidence of the presence of milk - borne tumour agent subsequently reappeared , the progeny liable to be affected could be traced . 
from every litter containing more than one female , one or two were used for testing the hormone response this breeding provided further while the remainder were allowed to breed . mice for testing , and also controls to prove the absence of the milk - borne agent . the same breeding females also provided controls for all the male mice that altogether , 44 breeding females reached the average tumour were treated . age ( 81 months for this strain ) ; of these 24 were force bred owing to the circumstance that they failed to rear or destroyed their young , a habit to which many r3 females are prone . in three years no tumours were seen . 
the majority of strong a mice when two months old or over failed to survive the combination of this anaesthetic and ovariectomy . their survival could only be assured after they had reached the age of 2 months , if ether ' ere used . 
the ovaries of all mice were removed by the abdominal route ; they were excised of their oviducts ( uterine horns )  . together with about successful removal of all local oestrogen - producing tissue was judged at autopsy by complete atrophy of oviducts and uterus . vaginal smears were made from some of the females for 2 - 3 weeks before they were killed . it was necessary to examine the former site of uterus and oviducts in every mouse , because if the tissues were not excised as described , cysts containing clear fluid sometimes developed at the cut ends of oviducts after subsequent oestrogen treatment . 
when this was the case atrophy of uterus and oviducts was never complete , and the impression was gained as the result of much experience of mammary gland changes that these cysts were themselves secreting oestrogen . this difficulty was soon overcome at a time when oestrus records were not being made in spayed mice , so the matter was never settled . 
the oviducts became very slender tubes ( brownish in r3 and r3x mice ) and the uterus was greatly shrunken . oestrus cycles ceased in all those mice which were tested and did not reappear . 
any mice that had developed intraabdominal cysts or ligature abscesses , thereby possibly concealing cysts , at the cut ends of the oviducts , or whose oviducts had not undergone atrophy at the 16th to 20th weeks of experiment , were discarded . 
the purpose in using it was to give a limited measured amount of hormone within at most a few hours in order to obtain a limited response rather than a cumulative one due to prolonged irregular absorption as from oily solutions or pellets . 
the results indicate that responses in females vary quantitatively with dosage and qualitatively with strain and , within the strains , with ages younger than 49 to 56 days old . for the purpose of the present experiment most of the females received two doses of 400 microgrammes at 20 days ' interval . 
the response is visible owing to its atypical nature in respect of a milky secretion which renders ducts and clusters of acini visible . mice examined in vivo in early stages and all others were finally killed . all 10 mammary glands ( or fat pads in males ) were stained in bulk , cleared , inspected , and whole mounts made of representative mammae or those showing special features . the method of staining in bulk differed little from that described by gardner ( 1934 )  . after 4 days ' fixation in bouin 's fluid the mammae were dissected off from the skin , muscle and covering fascia with the aid of a dissecting microscope magnifying 7 and 14 times . nerve fibres were removed and some of the large blood vessels . these stripped mammae were then immersed overnight in 50 per cent alcohol , and next stained for about 4 hours in 1 part of ehrlich 's acid haematoxylin freshly diluted with 3 parts of a 2 per cent aqueous solution of after differentiating for about 10 minutes in acid alcohol ( 3 per acetic acid . cent hc1 in 50 per cent alcohol ) the stained glands were rendered blue or blueblack in several changes of 50 , then 70 per cent alcohol for as many hours or days as were required to develop a deep colour . they were next dehydrated and cleared in the usual manner . while in the first change of clearing agent ( xylol ) notes were made by the author after examining all 10 mammae or fat pads with the binocular dissecting microscope . 
the ducts were distended and filled with milky secretion , which rendered the main branches and many smaller ones visible to the naked eye in all 10 mammary glands . there was also a pseudolobular alveolar differentiation with great multiplication of acini arranged in clusters in all 10 mammary glands . 
a similar response was given by the maximum dose of 400 y of oc - oestradiol repeated at 20 days ' interval ( total 800 y ) in 15 out of 15 mice which were examined in ' ivo . 
pullinger limits of outgrowth attained during normal pregnancy . it is pathological also in that the degree of differentiation is suggestive of a 9th - 12th day pregnancy while the milky secretion is voluminous . male glands reacted to larger doses in a similar manner . 
some light on this problem was gained from experiments similar to those here described using the double dose of 400 microgrammes of oestradiol on spayed f.1 hybrid females of the c57 x r3x cross . the curious observation was made that a similar lobular - alveolar response was provokedin all the mammae , but it was localized in approximately half of each gland . 
no inflammatory or metaplastic nodules were encountered . in the course of these experiments the possibility had to be considered that in the females some other sources of oestrogens might be stimulated by ovariectomy and after cessation of the action of artificially applied hormone to produce oestrogens , as smith and bittner ( 1945 ) found in the c3h strain . that no such stimulation had occurred was proved by making vaginal smears during the last two to three weeks of the experimental period in 11 out of 20 mice , and by inspection of uterus and oviducts at autopsy . in no case was there a return of oestrus or failure of uterus and oviducts to atrophy . r3 virgin females deprived of their ovaries but containing the milk - borne tumouqr agent . early changes in the mammae in response to ovariectomy and oestrogen treatment ( ketohydroxyoestrin and oestradiol ) were similar to those described in the same strain deprived of the tumour agent ( 32 out of 32 mice )  . 
by the vaginal smear technique , oestrus in 30 cells were seen on one occasion in each of 2 mice out of 32 examined . mice oestrus cycles or cells did not recur . the contrast between the mammae in the two sets of mice at 16 to 20 weeks there was complete absence of adenomas in those mice which was striking . had been deprived of the milk - borne tumour agent , whereas in mice of the original r3 strains carrying milk agent adenomas were numerous in nearly all fig . 
a few imperfections due to incomplete regression did not cause confusion , and will be referred to in this connection again . the conclusions were drawn that the foci of hyperplasia ( adenomas ) in the r3 females carrying tumour agent , were due in respect of their origin and persistence to the presence of a milk - borne tumour agent , presumably identical with or a variant of bittner 's agent , and that this agent had been forced into activity by the large dose of - hormone . these nodules are considered to be experimentally produced counterparts of those that occur spontaneously in all r3 females after the age of 8 to 9 months . they are similar to those described in great detail , and finely illustrated by gardner ( 1942 ) in other strains . 
an average of 6 mammary glands per mouse developed from rudiments and all examined revealed the atypical response characteristic of females of this strain . irregular lobules were seen after cutaneous application of 8001200 microgrammes of oestradiol with or without 1500 microgrammes of proregression required 6 to 7 months to become complete , leaving gesterone . bare atrophic ducts ( table ii )  . r3 males containing milk - borne tumnour agent . when additional time had been allowed for regression of hormone response , results were similar , and almost as regular as in spayed females containing milkborne tumour agent . either oestrogen alone ( 1200 microgrammes ) or combined with one dose of progesterone ( 1 , 500 microgrammes ) was sufficient to stimulate the growth of adenomas ( table ii )  . 
gagliano. from the cancer research institute and the department of medicine , college of physicians and surgeons , columbia university , new york . received for publication january 30 , 1950 . tetra - sodium mitchell ( 1948 ) recently reported a clinical study on the use of parenterally administered 2 - methyl - i : 4 - naphthohydroquinone diphosphate ( synkayvite ) as an adjuvant to radiotherapy in the management of inoperable and far advanced malignant disease . in a small series he noted prolongation of the mean survival time of patients with inoperable carcinoma of the bronchus treated by the combination of chemical agent and irradiation when compared with that of patients who received x - ray therapy alone , which he found to be statistically significant . the rationale for mitchell 's ( 1948 ) selection of this drug for clinical trial in combination with radiotherapy depended upon his observation that the chemical compound produced mitotic inhibition of chick fibroblasts in tissue culture . although there have been many laboratory studies on synkayvite as a vitamin k substitute , no investigations have been published on the effect of this compound on experimental neoplasms . 
the growth of the tumours in control and treated animals was followed by serial mensuration of the outside diameters , but the conclusions reached in this study rest on the comparison of the wet weight of the tumours in the control and treated series determined at the 104 a . 
from the information included it is possible to estimate with accuracy the actual protocols for the individual experiments , even though the pertinent results are here summarized as averages ; the standard deviation has been calculated in each instance in order to describe the range and the distribution of the individual tumour weights about the mean tumour weight . 
the standard deviation of the mean which is necessary for the determination of statistical significance between two means is derived by dividing the standard deviation by the square root of the number of observations . in several of the experiments 5 - amino - 7 - hydroxy - 1 - h - v - triazolo [ d ] pyrimidine ( 8 - azaguanine ) was also administered to tumour - bearing animals for purposes of comparison with synkayvite . 
gagliano neoplastic disease and malignant tumours in experimental animals is an unsettled issue , it is important to note that , thus far , all of the chemotherapeutic agents which have a demonstrable palliative effect against neoplasms in man have also been shown to inhibit comparable tumours in laboratory animals . it is also appreciated that the observations which we have recorded do not duplicate in the laboratory the clinical experiments of mitchell ( 1948 ) , inasmuch as he studied the combination of radiotherapy and the drug whereas - we investigated the drug alone . evaluation of combination therapy , however , utilizing radiant energy plus a chemical compound , is not feasible in studies on experimental neoplasms due to the ease with which x rays alone can destroy subcutaneous tumours . therefore , in spite of negative experimental evidence of the type presented in this report , the possibility remains that synkayvitein combination with radiotherapy may produce a greater carcinolytic effect than x - ray therapy alone . 
pullinger. from the imperial cancer research fund laboratories and the research department , the glasgow royal cancer hospital , glasgow , c.3. received for publication february 7 , 1952 . the hyperplastic nodules here referred to comprise all varieties found in surveys of spontaneous nodules in previous publications ( pullinger , 1949 , 1952 )  . at the time these surveys were being made specimens of affected nipple regions were almost invariably mounted and kept . it gradually became obvious that one nipple region , the 2nd on either side , was more often the site of nodule formation than any other . 
when the surveys were complete , the 5 pairs of nipple regions were arranged together with the number of times they contained one or more hyperplastic nodules of the various sorts . only those nodules are now included of which whole mounts were kept . 
the period of time covered was continuous from 1945 to the end of 1950 . the number of mice with mounted nodule - containing nipple regions was 116 ; the number of the nipple regions 146 . 
no record had been kept of 13 from which serial sections were made in the earlier stages of the survey ; more were incomplete or have been mislaid . six examples of bilateral nodules in the same gland were counted as only one of the pair . 
belcher. from the wards and the bland - sutton institute of pathology , the middlesex hospital , london , w.1. received for publicatiod , may 16 , 1952 . adenocarcinoma in the oesophagus may arise in three ways : ( 1 ) as an upward extension of a carcinoma of the stomach , ( 2 ) as malignant change in the mucous glands that are normally found in the oesophageal submucosa , and ( 3 ) from ectopic gastric mucosa . the first is not uncommon ; the second is rare . the third , also rare , is the type that concerns us here . in 1950 carrie reported a case of adenocarcinoma of the oesophagus that had arisen in an area of ectopic gastric mucosa . 
he reviewed the literature and likened the lesion to the unicorn in that many authors had described it , but only one had ever seen it . although he makes no reference to the possibility of a hernia being present , the description of the specimen , coupled with the fact that the tumour was in the cricothyroid region ( where ectopic gastric mucous membrane is most frequently encountered - schridde ( 1904 ) ) , makes it certain that this was indeed a genuine example of this rare lesion . bosher and taylor ( 1951 ) describe a case in which ectopic gastric mucosa was present in the oesophagus . there was reflux oesophagitis and stricture formation at the level of the aortic arch . at first sight this would appear to be a case similar to the one about to be described , but it seems more likely that it is another example of hiatus hernia with extreme secondary shortening of the oesophagus , as the barium meal strongly suggests that only a small portion of the stomach is in the abdomen . although they state that at operation the hernia was small , it is notoriously difficult to distinguish stomach from gullet on external appearances alone . 
he had no difficulty with fluids . one week prior to admission food stuck in the same place and was regurgitated . since then solid food would not pass at all . and no glands were palpable . on examination the patient looked well . there was no wasting or dehydration on 14 . 
the oesophagus was removed from a point 2 inches above the tumour to the cardia and the free end implanted into the upper part of the stomach . post - operatively the patient had some fever and pylorospasm but was eventually discharged from hospital well on 4 . 
the mucous membrane above the tumour was entirely squamous in type and was normal for the oesophagus ; that below the growth was entirely glandular apart from a few islands of squamous epithelium about 2 inches above the lower limit of excision . 
the lower half of the oesophagus contains only smooth muscle in its muscular coat . in the case described , striated muscle was present down to and including the site of the adenocarcinoma . this represents a point about 5 inches above the junction of gullet and stomach . 
the normal gullet is about 10 inches long as meastired from the termination of the pharynx at the level of the cricoid cartilage to the cardiac orifice of the stomach . it would thus appear that the striated muscle in this case had a normal distribution . however , the circular muscle - fibres at the squamo - glandular junction appear to have thickened to form a sphincter . 
this constitutes a departure from normal , and it may have prevented regurgitation of acid secreted by the gastric mucosa lining the lower half of the gullet . this may well have prevented the development of oesophagitis earlier in the patient 's life . there was definite histological evidence in this case that the adenocarcinoma had arisen in the glandular mucous membrane immediately adjacent to the junction of the squamous and glandular epithelium . the emphasis is placed on " glandular " , for this showed varied degrees of chronic inflammatory and atrophic changes which obscured the normal picture of a gastric type of mucous membrane . these changes conform to those that may be seen in stomachs that are affected by chronic gastritis , including the atrophic type . 
thus we conclude that the lower half of the gullet in this case was lined by gastric mucous membrane that was the seat of chronic inflammatory and atrophic changes , which may well have been a factor in the development of carcinoma . the rare occurrence of adenocarcinoma in the oesophagus , other than those cases that have obviously spread from a gastric origin , has been put down in the 130 13 . 
mitchell. from the department of radiotherapeuties , university of cambridge . received for publication july 8 , 1953 . attempts to evaluate tetra - sodium 2 - methyl - i : 4 - naphthohydroquinone diphosphate ( compound i , synkavit ) as a radiosensitiser in the radiotherapy of malignant tumours have been in progress since november , 1946 . 
at first the compound was used alone and in conjunction with palliative x - ray therapy in some very it soon became evident that in general the compound alone had advanced cases . no therapeutic effect in malignant tumours . 
at the same time , all cases of inoperable carcinoma of the bronchus were treated , if possible , by the combination of x - ray therapy and compound i ; the results were assessed mainly by survival times from the first x - ray treatment and the first sympton and were compared with those which had been obtained previously in the same department with x - ray therapy only , and which were very similar to the depressing results obtained by most of the workers . slowly the difficulties of this type of clinical investigation were appreciated . moreover in 1950 erratic results were encountered both in the clinical trials and in animal experiments . it seems likely that these findings were attributable to the thermal instability of compound i in aqueous solution in the absence of oxygen ( mitchell and simonreuss , 1952 )  . 
much more consistent results have been obtained since the ampoules have been stored in a refrigerator at about 30 c . the evidence obtained by these preliminary studies was sufficiently suggestive to justify further work , but the methods used were clearly inadequate . it became essential to employ properly designed methods for the clinical evaluation of radiosensitisers . 
the importance of these methods was emphasised at the time by their use in clinical evaluation of chemotherapeutic agents in tuberculosis ( medical research council , 1948 , 1950 )  . investigations of this type in the study of radiosensitisers have been in progress since april 1951 ; as yet the only results available are for the treatment of inoperable carcinoma of the bronchus . the results of the earlier studies have been reported by mitchell ( 1948 , 1949a , 1949b , 1950 , 1951 )  . this work has been criticised by gellhorn and gagliano ( 1950 )  . otte ( 1949 ) reported briefly on the combination of x - ray therapy and some synthetic vitamin k substitutes in the treatment of 300 patients with advanced malignant tumours and claimed improved palliation . 
mitchell selection of types of malignant tumour for investigation . it is suggested that a practical minimum requirement for usefulness of a radiosensitising chemical agent is that its employment in combination with radiotherapy should produce a mean survival time after treatment double that after radiotherapy only , for the type of malignant tumour treated . 
hence taking into account the frequency distribution of the survival times , it is likely to be necessary to observe the cases treated by combined therapy for an interval of at least 6 times the mean survival after radiotherapy only . 
to this time must be added the time necessary to accumulate an adequate number of cases . this approach is limited to the study of types of malignant tumour in which the results of present methods of treatment are bad and in which the time of survival is short . inoperable carcinoma of the bronchus is an obvious choice for investigation it is the only common type of malignant disease with a very short natural history . despite advances , only a small proportion of all cases of carcinoma of the bronuntreated patients are often very miserable . 
the chus are curable by surgery . results of treatment of inoperable cases by present methods , including radical nevertheless x - ray therapy in general has sufficient x - ray therapy , are poor . palliative value apart from prolongation of life per se , to justify attempts to improve this . with x - ray therapy only , inoperable carcinoma ofthe bronchus appears to have a a rather uniform natural history . 
of these , histological evidence was obtained in 245 patients and their average survival from the first symptom was 14 - 3 months and from the beginning of x - ray treatment 4 - 5 months . 
7 of this paper , it can be deduced that of 729 patients , only 45 survived 8 months , 20 survived 12 months , 8 survived 18 months and none survived 3 years following in this series , the best survival rate - 4 - 7 months - was in a x - ray therapy . group of 80 patients who received a total dose as measured in air of 5000 - 6000 r ; higher and lower doses were associated with poorer results . 
the most important factor in determining survival is the extent of spread of the disease when treated . this evidence suggests that in representative series of cases of inoperable carcinoma of the bronchus receiving x - ray therapy , the mean survival from the beginning of treatment is about 4 - 5 months . 
to try to minimise its shortcomings , all the cases to which it is applied have been followed until death , or for at least 3 - 1 years and in assessing the results the proportion of cases showing unexpectedly good response has been compared in the case of patients treated by means of radiotherapy combined with intravenous compound with those treated by radiotherapy combined with intramuscular compound . the results of various types of treatment of patients with advanced malignant tumours classified as inoperable , stages iii and iv , and recurrent of all types except carcinoma of the bronchus using compound i in treatments from november , 1946 to july , 1909 inclusive , and assessed up to 31 december , 1952 , are summarised in table i for the cases verified histoan essentialy similar table has been prepared showing the rather logically . poorer results for 56 cases not confirmed histologically , but with reasonably the cases allocated to this preliminary survey were the great certain diagnosis . majority of the patients with advanced malignant tumours in which it was considered that present methods of treatment were not likely to give satisfactorily results . 
when the survey was started it was not realised that the use of the compound by intravenous injection was likely to be more effective than its use by intramuscular injection . the allocation of the patients to treatments involving the two mnethods of administration of the compound were certainly not a.t random , and the possibility of bias by the use of intramuscular injection in the more seriously ill patients cannot be excluded . however , there is no reason to suppose that such bias would influence the proportion of cases showing unexpectedly good clinical response . the resultsof the preliminary general survey summarised in table i suggests that the proportion of cases showing unexpectedly good clinical response is greater 316 j . 
one case of advanced carcinoma of the body of the uterus was treated by means of palliative x - ray therapy combined with intravenous compound , and one case of carcinoma of the tongue recurrent after previous x - ray therapy was treated successfully by radical x - ray therapy combined with intramuscular compound . of the 56 cases not confirmed histologically three survived more than 4 years after treatment . 
two of these showed an unexpectedly good response ; one was a woman aged 46 , who had a stage iv carcinoma of the breast with supraclavicular glands , and was treated by means of radical x - ray therapy combined with intramuscular compound , and the other was a case of a mixed parotid tumour which had failed to respond to further x - ray therapy , but appeared to be checked and rendered quiescent by intravenous compound onlv . 
the results are treated by statistical methods . it is suggested that estimation of the survival times , especially that from the beginning of x - ray therapy , is the most reliable criterion for objective assessment of the results of treatment . special attention has been paid to the influence of the extent of the disease , age and sex , the histology , the minimum tumour dose and overall time of the x - ray therapy and the details of administration of the compound . the general principles involved in the consideration of these factors are essentially the same in these preliminary studies as in the later clinical trials and except where relevant , will be discussed in this connection . 
the results of treatment were reviewed as from this group of cases of inoperable carcinoma of the bronchus december 31 , 1952 . comprises all those cases referred in which the diagnosis is accepted as proven according to the criteria given below , with the exclusion of patients not likely to benefit by palliative x - ray therapy . 
no technically operable cases and no postoperative cases are included . the control group consists of those cases of inoperable carcinoma of the bronchus selected according to the same criteria , which were treated by x - ray therapy only , using similar methods , in the same department between 1943 and may , 1947 . the difficulties of this method of providing a control group are recognised and include the possibility of introduction of serious errors of sampling . 
the use of alternate cases as controls was attempted but was abandoned as impractical . however , it has been shown that the relevant properties of the control and treated groups apart from the length of survival after treatment are substantially the same . it is found that the results of x - ray treatment in the present control group are substantially identical with those obtained in two large published series . 
mitchell ( b ) cases with positive histology on biopsy specimens of supraclavicular nodes , cutaneous or other metastases but in which there was no bronchoscopy or the bronchoscopic appearances were not definite ; this sub - group consists of 8 control cases and 8 treated cases ; ( c ) one case ( treated ) with typical bronchoscopic appearances together with metastases in the skin , confirmed histologically . these additional 15 control and 18 treated cases are usually more advanced and are in general less comparable than the " histologically verified " cases but must be considered with them to give a complete picture . 
of the combined series of " confirmed cases , " there are in all 34 in the control group , which was treated by x - ray therapy only , and 47 in the treated group , which received x - ray therapy together with the compound . 
as anticipated the results of treatment of the " confirmed cases " are poorer than those of the " histologically verified " cases . other groups of cases which cannot be included in the main part of this investigation may be summarised as follows : ( i ) cases with typical radiological appearances together with metastases at some stage but not verified histologically and in which there was no bronchoscopy or the bronchoscopic appearances were not definite . in these cases the diagnosis can only be regarded as probable . in the control group , there are 12 cases ; the mean time of survival is 8 - 50 months from the first symptom and 3 - 67 months from the first x - ray treatment . in the treated group , there are 13 cases ; the mean times of survival are 12 - 15 months from the first symptom and 6 * 31 months from the first x - ray treatment . ( ii ) cases rejected as unsuitable for palliative x - ray therapy . during the control period , there were 18 of these cases , of which 10 were histologically verified and during the period corresponding to the treated group , there were 7 cases of which only one was verified histologically . the enumeration of rejected cases is unsatisfactory because of selection by the physicians referring these advanced cases . hence the total number of cases with a reasonably certain or probable diagnosis of inoperable carcinoma of the bronchus is 131 . 
of the 7 cases of squamous carcinoma , two showed keratinisation . of the 29 verified treated cases , there are 17 cases of stratified carcinoma , of which 10 are squamous and 7 non - squamous , 3 cases of adenocarcinoma , 8 of anaplastic carcinoma and one an unusual poorly differentiated lesion . of the 10 cases of squamous carcinoma three cases show definite keratinisait is interesting tion ; in two further cases there is a suggestion of keratinisation . to note that all these cases responded well to treatment . the control group was treated by x - ray therapy only , from 1943 to may , 1947 . 
even if the cases which survived only one month from the first x - ray treatment are excluded , to meet the criticism of a possible difference in selection of the cases , the difference in survival after the first treatment between the treated group ( mean survival 9 - 68 months ) and the control group ( mean survival 5 - 23 months ) is still significant ( n = 39 , t 2 * 457 , p is slightly less than 0 - 02 )  . considering the histological classification , the only type providing adequate numbers is the stratified carcinoma . 
the basic principles involved are well understood as a result of the classical statistical studies of fisher ( 1925 , 1935 ) on the design of experiments , and on the work of hill ( 1952 ) on the design of clinical trials . 
in general , in considering therapeutic trials in the cancer field , the design must be capable of giving a completely convincing statistically significant quantitative result as efficiently as possible with a minimum number of patients and must be acceptable and above reproach from the moral and ethical points of view . 
the most important single feature of the design is random allocation of the patients to two or more alternative forms of treatment the value of which is being compared . for assessment of the result of treatment , it is necessary to select relevant criteria capable of objective measurement such as the survival time suitably specified . 
at the same time , it is absolutely essential to provide for each individual patient treatment which is the best possible according to present knowledge . accordingly , it may be , and probably often will be , necessary to plan to depart from what may be termed the theoretically ideal form of experiment with deliberate sacrifice of some information . in the present example of the attempted evaluation of tetra - sodium 2 - methyl1 : 4 - naphthohydroquinone diphosphate ( compound i , synkavit ) as a radiosensitiser in the radio therapy of inoperable carcinoma of the bronchus , cases were allocated at random to one of two alternative forms of treatment , x - ray therapy combined with compound i administered by intravenous injection ( x + i - vs ) and x - ray therapy combined with compound i administered by intramusculrzr injection ( x + i - ms )  . 
the latter is regarded as the control group . the preliminary clinical studies and animal experiments suggested that the results of x - ray therapy combined with intramuscular compound ( x + i - ms ) were slightly better than those of x - ray therapy only , and that with comparable doses of compound it was likely that x + i - vs would produce better results than x + i - ms . 
mitchell tests of heterogeneity must be made . clinical therapeutic trial must be justified a posteriori . it is necessary to - examine the possible importance of all the various factors which may be relevant including age , sex , extent of the disease at treatment , histology and / or cytology , the minimum tumour dose and overall time of radiotherapy , the total dose and overall time of administration of the compound and in particular the certainty of the diagnosis . it does not seem possible to estimate in advance the number of patients required . this will depend on the differences observed between the groups and subgroups . 
the results are likely to be of sufficient importance to necessitate the use of stringent criteria of significance . the randomisation was made on the basis of a provisional diagnosis of inoperable carcinoma of the bronchus , made when the patient was first examined by the staff of the department . 
some of the cases were referred with histological evidence from bronchial biopsy . in other cases , there had been no previous investigation and the provisional diagnosis was based on clinical evidence only . all cases were investigated critically , as necessary , and every effort was made to obtain histological and / or cytological verification of the diagnosis . almost all the sections have been reviewed by a . 
cruickshank , of the sims woodhead memorial laboratory , papworth , for most of the reports on specimens of bronchial aspirate . particular attention has been paid to the establishment of the diagnosis of carcinoma of the bronchus and the identification and exclusion of cases of bronchial adenoma and related tumours of low grade malignancy apparently arising in bronchial glands . 
barrett and may be summarised as follows : ( 1 ) stratified carcinoma , either squamous or non - squamous , the degree of keratinisation or differentiation being noted , ( 2 ) adenocarcinoma , ( 3 ) anaplastic carcinoma , including " oat - celled " carcinoma , and ( 4 ) others , including " alveolar " cell carcinoma and carcinoma not classifiable . 
the histological classification of carcinoma of the bronchus is often difficult because of the existence of intermediate types and is somewhat arbitrary in some cases . on the clinical side , a careful and full medical examination is made , including usually indirect laryngoscopy . it has been considered important to determine the extent of spread of the disease with special reference to the early detection of metastases in bones . the occasional cases originally considered inoperable and in which it has been found possible subsequently to carry out some type of surgery have been considered separately in the assessment of the results . 
and increased daily in steps of any undesirable reaction such as excessive coughing , nausea , faintness the chest is observed after intravenous injection , the dose which there is no such reaction and then cautiously increased again on the following days , sometimes by steps of for most patients , for intravenous injections the best maximum daily dose appears to be 100 mg . ; dose is increased to as high a level as is tolerated . dosage is started with or discomfort or pain for the purpose of + i - ms . administration i - v , is reduced many will mg . 
some cases are still surviving so that additional evidence concerning the diagnosis may be forthcoming in a few of these and the estimates of survival time are necessarily incomplete . it is reasonably certain that any such new evidence will increase the differences observed between the two series . the total numbers of cases are 46 in the x + i - vs series , of which one received compound only with no x - ray therapy and 45 in the x + i - ms series . the fate of every case is accounted for in the groups ( a ) to ( j ) of table ii . 
12 * 4 days ; for the 19 patients in the x + i - ms series , 4 received radical treatment and survived respectively 4 , 6 , 7 and 6 months , the mean m.t.d. 
mitchell by intravenous injection than with radiotherapy combined with the compound administered by intramuscular injection . preliminary clinical studies of the influence of the ancillary use of the compound on the survival times of inoperable cases of carcinoma of the bronchus treated by x - ray therapy are discussed in some detail and the end results summarised . the design of a clinical therapeutic trial of a radiosensitiser is discussed , with special reference to the evaluation of tetra - sodium 2 - methyl - i : 4 - naphthohydroquinone diphosphate m a radiosensitiser in the radiotherapy of inoperable cases of carcinoma of the bronchus . 
the results of this clinical trial are summarised to date . it is concluded that intravenous administration of this compound ( synkavit ) has a small but useful effect as a clinical radiosensitiser . in this work i have been helped by many colleagues at addenbrooke 's hospital . in particular i wish to thank a . 
7. received for publication june 19 , 1948 . the testing of substances for anticarcinogenic properties must proceed on purely empirical lines , or be based upon a hypothetical conception of the nature of some phase of the biochemical mechanism of chemical carcinogenesis . suggestive , but not conclusive , evidence has been obtained that s - metabolism is intimately connected with a primary stage in the interaction of chemical carcinogens with cellular constituents . 
crabtree in the groups receiving either the carcinogen alone or in conjunction with a substance which proved to be innocuous , the number of mice surviving the full all mice were epilated course of the experiment was always 90 per cent or over . over the scapular region once only , two days before the experiments began . the approximate amounts of carcinogen and tested substance used in any one experiment were as follows : 0 - 1l% 3 : 4 - benzpyrene 0 2% / methylcholanthrene 0 20 / 2.0% s - compound 1 : 2 : 5 : 6 - dibenzanthracene solutnused . 
a 0 1 per cent solution has been found most convenient , since with higher concentrations less obvious effects are produced by an inhibitor , and with lower concentrations the experiments are unduly prolonged , thus favouring any possible toxic action of the substance tested . 
with the strain of mice used , the average time for tumour induction with 0.1 per cent 3 : 4 - benzpyrene applied twice weekly is 16 - 0 i 0 1 weeks , a figure obtained repeatedly in many experiments . the substances tested were chosen for no other reason than that of convenience . 
when this occurred in the early part of an experiment and clearly reflected the toxicity of the substance tested , the difficulty was easily met by using smaller doses of the toxic agent . 
spicer. influence of bal on induction time . the results for bal have been analysed in two parts . those for bal and benzpyrene , where several percentages of bal were used , can be analysed using the technique of regression ( fisher , 1947 )  . that is to say a straight line is fitted to the mean induction times by the method of least squares ; if the slope of this line is significantly different from zero , then bal can be said to exert an inhibitory effect on the benzpyrene . the slope of the line is a measure of the efficacy of the bal . 
 " a lesser mass increment " of transplanted tumours was observed when the hosts were injected with diphenylsulphoxide or diphennylsulphone ( hammett , 1930 ) , and cystine disulphoxide proved inhibitory to the growth of spontaneous mouse tumours ( staff of the lankenau hospital , 1936 )  . conversely , compounds containing s in the reduced form , r.sh ( the nature of r appears to be of secondary importance ) , were shown to stimulate cell - division in a wide variety of plant and animal tissues ( hammett , 1931 )  . arising from these results it was anticipated that the increased proliferation of the cells in mouse skin induced by treatment with an sh - compound would render the tissue more susceptible to the action of a p - thiocresol was therefore applied in conjunction with a carcinogen , carcinogen . but the opposite effect was observed , i.e. 
the sh - compound caused a pronounced retardation in the rate of tumour induction ( reimann and hall , 1936 )  . reimann and hall performed a variety such inhibition stands in sharp contrast to the entirely innocuous behaviour towards carcinogenesis of the mono - thiol compounds used in the present work . the discrepancy can hardly be attributed to variations in the activities of the sh - compounds themselves , and must be referred to differences in experimental techniques . of experiments differing only in the amounts of carcinogen and p - thiocresol administered , and the timedistribution of their application . the actual doses of carcinogen varied from experiment to experiment , e.g.. 
the control group of mice received 2 - 4 times as much 1 : 2 : 5 : 6 - dibenzanthracene over the latent period as the groups treated with p - thiocresol , and this feature alone would suffice to account for the wide variations in average induction times , and the apparent inhibitory effect . moreover , the choice of lanoline as a solvent for p - thiocresol was unfortunate , since the work of twort and twort ( 1929 ) , rosicky andhatschek ( 1943 ) , simpson , carruthers and cramer ( 1945 ) , and berenblum and schoental ( 1947 ) , have shown that lanoline greatly retards the carcinogenic action of tar , 3 : 4 - benzpyrene , and methylcholanthrene . complicating factor augments the difficulty of accepting the experiments of reimann and hall as a demonstration of the anti - carcinogenic activity of sh - compounds . of the mono - sh - compounds used in the present work , together with the fact that bal , a di - sh - compound , can cause a moderate retardation of carcinogenic action , clearly signifies that the mere presentation of any sh - containing molecules ( as implied by the philadelphian workers ) is not in itself sufficient to produce a biological effect ascribable to the sh - group . experiences of peters , stocken and thompson ( 1945 ) in the search for a suitable sh - compound , which culminated in the discovery of bal , also emphasizes this . apart from the question of variable toxicity , which rules out a substance like the lack of activity this added 288 h . 
crabtree toluene - 3 : 4 - dithiol , it is evident that the total molecule , with sh - groups appropriately disposed , is a factor of importance . the analogy with the anti - vesicant activity of bal cannot be pushed too far , since experiments in vitro clarified the nature of the reaction between bal and lewisite , whereas any possible reaction between bal and a carcinogen , modified in an unknown way , cannot be attempted at present . lacassagne , buuii - hoi and rudali ( 1945 ) have ostensibly shown that competition between a potent carcinogen and a structurally related substance with their little or no carcinogenic activity can result in delayed tumour induction . work was an outcome of the conception that " the mutation which transforms a normal cell into a malignant cell is the result of the alteration of an organic substrate by the fixation to it of some toxic molecules ( in this case polycyclic chrysene , l : 2 : 5 : 6 - dibenzfluorene , five isomeric dimethyl - benzhydrocarbons )  . " acridines and a trimethyl - benzacridine were chosen as near relations of methylresemblance to cholanthrene , and 1 : 2 : 5 : 6 - dibenzacridine for its structural though they state in general terms that inhibitions 1 : 2 : 5 : 6 - dibenzanthracene . of carcinogenic action were demonstrated , the experimental details hardly justify so wide a conclusion , since only chrvsene and 1 : 2 : 5 : 6 - dibenzfiuorene caused some slight delay in tumour emergence . 
nordling. received for publication december 29 , 1952 . recent research in genetics and pathology has shown an amazing consistency between the agents causing mutations and those causing - or contributing to the one of the more prominent theories , which since the development of - cancer . 1920 's has been advocated by bauer ( 1949 ) , strong ( 1949 ) and others , claims that the original cancerous cell is nothing but an ordinary cell affected by genetic mutation of some kind . 
one of the main objections to this theory has been that it does not explain the age variation of the cancer frequency . in reply , bauer ( 1949 ) contends that the mutated cells apparently remain latent during a long period until the new qualities become evident and the phenomenon can be diagbauer has found this period of latency to average 9 years for nosed as cancer . x - ray cancer , 12 years for paraffin cancer , 18 years for aniline cancer and 40 years for seaman 's cancer ( caused by solar radiation )  . 
the early occurrence of sarcoma and of leukaemia , however , does not conform to the idea of a latent period . moreover , it appears somewhat unreasonable to suppose that the length of the latency period would depend upon whether the mutation is caused by sun - rays writh most forms of carcinoma , furthermore , the frequency at or by x - rays . different ages is such that wre are compelled to consider average latency periods of 70 years or more , in order to explain the actual age frequency curve of cancer these facts still make it difficult for many of the most commortality in man . petent authorities in the field to accept the mutation theory without reservations . dahlberg ( 1943 ) has advanced a completely different explanation concerning the relationship between age and cancer . 
he expressed the opinion that malignant tumours develop increasingly easily with rising age . this implies that , for the development of tumourous cells , it is necessary for a certain number of cellular divisions to have taken place , between each of which there has been a certain period of time . all experience concerning the cancerous influence of chronic irritations promoting cellular divisions seems strongly to support this theory . 
the number of mutations required for experimental cancer in mice may be less , as suggested by the investiobviously , we need not assume that any gations of iversen and arley ( 1950 )  . only mutations which increase the ratio seven mutations will cause cancer . between cellular divisions and cellular loss in a positive direction in the environaccording to fischer ment in question may be expected to have this effect . ( 1930 ) , the short average life - span of cancerous cells is their dominant feature , and that by which all their other qualities can be explained . their rapid propagation , which exceeds their high death - rate , their ability to ferment sugar on a large scale and to disintegrate heterogeneous proteins , constitute normal cellular responses to a high mortality among adjacent cells . it may be added that the frequent variability in the number of chromosomes among cells from the same tumour is also a " normal " feature in the sense that the same phenomenon also occurs in certain normal tissues , as shown by therman and timonen ( 1951 )  . thus , some or most of the mutations required to start an incessant self - stimulating propagation among a group of cells might consist of any of the forms of mutation that weaken the cell , and make it more liable to die from even minor disturbances . if cancer were caused by one mutation only , it might be expected to be equally common among persons of different ages after an age corresponding to the length of the latency period , if this period were interpreted as the time required by the first cancerous cell to multiply in such degree as to be diagnosed as cancer . 
nordling prevails in countries with a high percentage of deaths due to " senility " , " other and unknown causes " and the like , whereas the latter is the case in countries e.g. , the united states , new zealand , with detailed and reliable statistics , australia , great britain and other western european countries . it can be shown , as by nordling ( 1952 ) , that the cancer frequency curve of a population with undependable vital statistics will be restored to the same shape as that of the populations with accurate statistics if , as a rule , about one - fourth of the deaths due to such causes as " senility " are added to the registered cancer deaths . united states white population 1940 united kingdom 1939 france 1947 norway 1941 - 1945 000o = + = : + 14 at = ___x___ 200l asla42w 2 i - = 3 ___ _ - - _ 40 60 80 50 70 60 80 50 70 60 80 50 70 40 60 8u 50 70 fig . 
1. - diagram drawn to double logarithmic ( log / log ) scale showing the cancer death - rate ( in the case of the united kingdom , the carcinoma death - rate ) in males at different ages . deaths per 100 , 000 males are shown on the vertical scale , age figures on the horizontal scale . however , the very highest age - groups , which are always numerically small this is and certainly highly selected , can hardly be taken into consideration . because these individuals are usually already very frail and likely to die from any trivial cause , irrespective of whether or not they have cancer in a more or less developed stage . thus , it seems probable that , in reality , the age - frequency relationship for cancer follows the same general rule everywhere in the civilized world , although in these reliable examples can be given in only a limited number of cases . cases it is obvious , as is shown in fig . 
with regard to cancer among females , it is necessary to distinguish between cancer in the specificially female organs , of which the increase is fairly small above the age of 45 , and cancer at other sites , of which the frequency seems to increase according to the sixth power of age both before and after the forties , but not during the decade of the menopause , when the increase is smaller . it seems possible that altered hormonal conditions in connection with the menopause might play a part in determining the actual cancer frequency curve obviously , more slowly operating hormonal variations might among women . be present in both men and women . 
it is therefore by no means certain that the entire increase in the incidence of cancer with age is to be explained by means of the multiple mutation mechanism . it is possible that a small proportion of the increase is due to a lesser degree of hormonal inhibition of growth of potentially cancerous cells in an older organism than in a younger one . 
the writer is not , however , aware of any evidence definitely substantiating such a variability of natural cancer inhibition with age . during childhood and adolescence cancer is rare , but it occurs considerably in these cases it is more often than the sixth - power - of - age rule would allow . mainly a question of sarcoma , leukaemia and other non - epithelial forms of cancer . such a condition does not , however , imply any contradiction of the hypothesis since it is unlikely that mutaof successive mutations as the cause of cancer . genic noxae reach the bones and other internal tissues , it therefore seems more reasonable in the case of sarcoma to presume spontaneous mutations ( i.e. , mutations due to normal molecular collisions ) and radiation mutations as the such mutations may , of course , occur already in the foetal causative factor . in view of the extremely rapid growth during this period , it is not period . surprising that multiple mutations occasionally have time to occur , giving rise to infantile or even foetal sarcoma . 
the infrequency of malignant tumours in the striated muscles , despite their large volume , may have some connection with the fact that this kind of tissue is composed of multinuclear cells . according to the theory put forward in the present paper , cancer is caused by mutations multiplied and accumulated usually through large - scale proliferation it is easy to explain on this basis such facts as the ( sometimes inherited ) of cells . high susceptibility to cancer of certain organs , especially under experimental this is because every stimulant causing - or congenital capacity conditions . characterized by - a high proliferation rate also increases the number of mutated also the high susceptibility to cancer cells and thus the probability of cancer . of benign tumours is easy to understand in this way . the facts and theories advanced here demonstrate , among other matters , the importance of having reliable vital statistics in order to elucidate casual relationships in the pathogenesis of cancer . 
gutnz of untreated leukaemic cells observed in vitro . only the quantitative aspects of the subject will be dealt with here ; the cytological findings will be described in a separate paper . normal and leukaemic blood and bone marrow have been studied in tissue cultures by many workers since the publication of the first report on tissue culture of leukaemic blood by awrorow and timofejewskij ( 1914 )  . 
among other investigations carried out in recent years the following should be mentioned , as they deal wholly or in part with culture of leukaemic blood or marrow : papers by barnes and furth ( 1937 ) and bichel ( 1940 ) on mouse leukaemia ; doljanski and pikovski ( 1941 ) on fowl leukosis ; and fieschi and astaldi ( 1946 ) , israels ( 1940a , 1940b ) , osgood and brownlee ( 1936 , 1937 , as well as many later papers from the same laboratory ) , pierce ( 1932 , 1942 ) , schrek ( 1946a , 1946b ) , and wallbach ( 1936 ) , on human leukaemia . 
the vaccine caps are washed , and as rubber is toxic to living cells , they are then coated with a very thin layer of paraffin wax , by dipping them into a saturated solution of paraffin in petrol ether and wiping off excess after withdrawal . 
gunz been put into a conical flask , and the cells are now added to it , the aim being to give a final concentration of about 2000 leucocytes per c.msome practice is required in judging the correct volume of cells to be added . three consecutive counts are done on the cell suspension , and when the mean reveals a suitable cell concentration , a quantity of 2 ml . 
3. - mean daily counts of a sample culture of blood ( chronic myeloid leukaemia ) , expressed as per cent of initial count drawn on logarithmic scale . 6 days in the light of these two experiments , the following routine procedure has been thought justifiable : the mean of three counts of the original cell suspension is taken as the 100 per cent initial value for all tubes . 
on each subsequent occasion three separate counts are done on each of three fresh tubes , and the mean and standard error between tubes are calculated for all nine counts . 
means are calculated from the three individual counts and used for constructing the growth curve . in these differential counts a siniplified nomenclature of the myeloid series has been used , cells being divided into the three broad classes of myeloblasts , myelocytes and polymorphonuclears ( eosinophils and basophils being counted separately )  . 
the term " immature cell " comprises myeloblasts and myelocytes , the latter being arbitrarily defined as complying with two or all of the three following characteristics : total cell size exceeding that of a normal polymorphonuclear by 1 / 3 or more ; nucleus oval and only slightly indented ; cytoplasm basophilic . 
some of the patients with chronic myeloid leukaemia were untreated , while others had had various forms of therapy . cultures were either continued until all cells had died , or they were deliberately abandoned at an earlier period . 
when they were continued , there was always a gradual decrease in the total counts until death of the last cells occurred , usually between the sixth and the ninth day of culture . 
no significant increase in total counts has ever been observed . of especial interest is a group of nine cultures , selected because the cells lived for at least 7 days . 
4. - mean daily counts of a sample culture of blood ( chronic myeloid leukaemia ) , expressed as per cent of initial count drawn on linear scale . days log y = 2.0792 - * 0747x 7 days fig . 
6. - effect of 5 per cent rat embryo extract on culture of blood ( chronic myeloid leukaemia )  . total and differential counts expressed as per cent of initial count . 0 1 / 3 3 days of the total count in the two series are probably not significant , they certainly do not follow a straight line . 
of greater interest is the behaviour of the immature cells , in which a striking difference between the two series is apparent . while there is a gradual decrease of the number of immature cells in the control cultures , there is a significant rise in the e.e. 
cultures showing a peak of more than four times the height of that in the control cultures . results similar to these have been found in all cultures in which differential counts were done . 
6 ; the excess polymorphonuclears can have only been formed from immature cells , and the decrease in the number of the latter is explained by their proliferation failing to keep pace with maturation . after the second day , the process is less important and soon stops , mature and immature cells decaying approximately exponentially at the same rate , as shown in fig . 
may be a ribo - nucleoprotein , and experiments are now in progress designed to co - ordinate the growth curve with the nucleic acid metabolism of the cells . the action of ionizing radiations , as well as of many chemotherapeutic agents , is primarily on mitotic cells . in investigating these actions , every effort should therefore be made to provide the maximum number of mitoses in the test object . thus in fluid cultures the addition of e.e. 
de. from the chittaranjan cancer ho8pital , calcutta , india . received for publication january 4 , 1954 . the study of the effect of radiation on cancer cells presents problems of fundamental importance . it is still an open question in the field of radiobiology as to whether the ionising effect of radiation directly induces differentiation in cancer cers . 
he has also shown that radiosensitive and radio - resistant cancer cells react to radiation in a different way after a preliminary radiation . waren and dixon ( 1949 ) are of opinion that radiation does not induce differentiation of cancer cers . johes and koher ( 1950 ) remark that the apparent increase of differentiating cers after radiation on cancer cers , is only relative and is caused by destruction of other types of cells . glucksmann 's method of estimating the radio - sensitivity of cancer cells with prehminary radiation ' has been criticised by gricouroff ( 1952 ) who is of the opinion that radio - sensitivity and differentiation of cancer cells are related to stromal nutrition . thus it is evident from the literature that our knowledge of the mode of action on radiation is not decisive . 
our study was strictly confined to those cancerous areas which were characterised by anisocytosis , anisonucleosis , abundance of mitotic cells , loss of polarity of epithehal cells and just breakage of basement membrane . 
the induction of differentiation in cancer cells is one of such delayed effects . it is for this reason , that we have collected tissues , as mentioned previously , on the 7th day after first radiation with 7000 r , on the 21st day after the second 7000 r radiation and finally 3 months after a total radiation of 21 , 000 r . before coming to our problem proper , we made a comparative study of the cellular population of the differentiating phases of the stratified squamous epithefia of the normal cervix and of epidermoid carcinoma of the cervix . table i shows that the cell population of the differentiating phase of the normal cervix is 48 - 17 0 - 41 per cent compared with 3 - 57 + 0 - 14 per cent of the epidermoid carcinoma of the cervix . we find the incidence of the variation of standard error in post - radiation conditions is high . might be due to variation on the biology of cancer cells , and also to altered conditions of different patients . 
de the trend of distribution shows also significant changes after second and third 7000 r , but the ' t ' test could not be applied because the number of cancer positive cases decreased materially after the 2nd and 3rd radiation . our second problem is concemed with the possibihty of predicting radiosensitivity of epidermoid cancer of cervix after preliminary radiation with 7000 r . it has been shown previously ( table ii ) that after the first radiation with 7000 r , there was a uniform reduction of cancer cell population of both resting and mitotic phases , and similarly a uniform increase of cell population of the differentiating and degenerating phases . 
we have not found any differential variation in the four biological phases of cancer cells after preliminary radiation to distinguish a radio - sensitive case from a radio - resistant one , as has been found by glucksmann ( 1948 )  . in our present series of 91 cases , no cancer cers were found in the final biopsy after 21 , 000 r in 76 cases , which may be considered as radio - sensitive cases . cancer cers were persistent in the remaining 15 cases under similar conditions and they may be termed radio - resistant cases . on further analysis of our data , arranging them into final cancer - positive and cancer - negative groups after full radiation with 21 , 000 r , we do not find any significant variation of cell population in different phases after the preliminary radiation with 7000 r ( table iv )  . 
when the influence of irradiation on the number of cells in the different classes was examined separately after the first irradiation for radio - resistant and radio - sensitive groups of tumours , no significant difference was found between the two groups . we express our gratitude to the atomic energy commission under the government of india for a grant which has given us an opportunity to do this work , and also to the chittaranjan cancer hospital , calcutta , for facilities to work in the laboratories . we also express our sincere thanks to the staff of the chittaranjan cancer hospital , particularly to n . 
the animals were killed by cervical dislocation or by exsanguination under ether anaesthesia and the livers immediately perfused with 0 9 per cent sodium chloride solution before being removed , chilled and homogenized . 
nuclei were also prepared from the grch 15 tumour originally described by peacock ( 1933 )  . for the preparation of fowl erythrocyte nuclei the method of dounce and lan ( 1943 ) was used . in all cases the suspensions of clean nuclei were dried from the frozen state after small measured portions had been taken for dilution and counting in a haemocytometer . 
a weighed portion of dried nuclei acid - soluble 1 ( discarded ) nuclei 10% tca extraction residue lipid extraction phospholipid p residue n - naoh ab 370 insoluble 1 alkali - soluble 1 acidification acid soluble 2 precipitate dnap organic p inorganic p fig . 
2. scheme of separation of phosphorus compounds in nuclei ( modified from schmidt and thaunhauser , 1945 )  . was extracted three times with 10 per cent trichloracetic acid ( tca )  . 
the extracts were combined ( acid soluble 1 ) , but little attention was paid to this fraction since clearly much of the soluble p must already have been removed during the isolation of the nuclei . 
the extracted residue was treated with successive portions of acetone , ethanol , ethanol - chloroform ( 3 : 1 ) , warm ethanol - ether ( 3 : 1 ) and ether . these extracts were taken to dryness and the residue taken up in chloroform to form the phospholipid fraction . the dry nucleoprotein residue was incubated overnight at 37 with n naoh to hydrolyse rna to acid soluble nucleotides . 
the phosphorus which remained in solution was treated as organic phosphate ( fraction " organic p " )  . fractions " acid soluble 2 " and " organic p " should contain phosphorus derived from rna in the form of acid soluble nucleotides ( rntap )  . pentose was therefore estimated in " acid soluble 2 " by the orcinol method of mejbaum nuclei 10% tca extraction acid soluble 1 ( discarded ) residue lipid extraction phospholipid p residue 5% tca at 900 schneider extract schneider residue n nagh at 370 insoluble 2 alkali soluble 2 acidification acid soluble 3 precipitate ester phosphate inorganic p ester phosphate residue fig . 
3. - combined schneider and schmnidt - thannhauser procedure . ( 1939 ) and by the phloroglucinol method of euler and hahn ( 1946 ) , using calibration curves made from a purified sample of yeast rna , so that pentose could be read off directly in terms of ribonucleic acid phosphorus ( rnap )  . fraction " dnap " should contain phosphorus derived from dna only . deoxypentose was therefore estimated by the diphenylamine method ( dische , 1930 ) as employed by davidson and waymouth ( 1944b ) , using a calibration curve prepared from a purified specimen of thymus dna so that readings could be made directly in terms of deoxyribonucleic acid phosphorus ( dnap )  . phosphorus estimations were made on all samples by the method of allen ( 1940 )  . the optical densities of fractions " acid soluble 2 " and " dnap " were read in a beckman model du spectrophotometer at 260 m , u . 
and were read against a reagent blank . the radioactivity of each fraction was determined on a type m6 liquid counter ( 20th century electronics ) attached to a conventional scaling unit . ( ii ) the schneider ( 1945 ) technique . - a weighed portion of dried nuclei was extracted with tca and lipid solvents , as described above . 
the " schneider " residue clearly contained a mixture of phosphorus compounds , and it was therefore submitted to a schmidt - thannhauser separation by incubating in alkaline solution at 370 for 18 hours . 
to demonstrate them a separate run on a strip 57 cx 7 cwas carried out at a potential gradient of 13 - 6 volts / cfor 6 hours . after a run the paper was dried and the bands located by the ultra - violet light method of holiday and johnson ( 1949 ) and marked lightly in pencil . photographic records were made by the method of markham and smith ( 1949 ) , and occasional autoradiographs were made by leaving the paper in contact with kodak industrex type d film for about 18 days . the portions of the paper containing the bands were cut out and eluted according to consden , gordon and martin ( 1947 )  . 
davidson was determined by its position on the paper and its ultraviolet absorption spectrum . total phosphorus and radioactivity were also determined . the results of a series of analyses are shown in table i , in which the composition of bulk nuclei is given in terms of mg . 
although it might be expected that the schneider extract would contain all the phosphorus derived from rna and dna , the figure obtained for this fraction is invariably table ii . - mean values for the composition qf the single cell nucleus pg . 
at the same time the schneider residue , which might be expected to contain only phosphoprotein p , invariably shows a much higher phosphorus content than could be accounted for this discrepancy was observed by schneider ( 1945 , 1946 ) for by fraction p2 . whole tissue and has been commented on by davidson , frazer and hutchison that the schneider residue contains several different types of phosphorus ( 1951 )  . compound is made abundantly clear when it is submitted to a schmidt - thannhauser fractionation ( table i )  . the protein - bound inorganic fraction " p3 " obtained in this way corresponds roughly in amount to fraction " p2 "  . it therefore represents the true " phosphoprotein " phosphorus , and the other phosphorus compounds in the schneider residue are almost certainly derived from the nonnucleotide esters mentioned above , and from nucleic acid phosphorus which has failed to be removed in the schneider extraction although the portions of the nucleic acid molecules containing the reactive sugars have been split off from the this is clear from the fact that , in general , the figures for rnap protein . obtained by pentose estimation on the schneider extract agree with those obtained 206 w . 
davldson by pentose estimation on " acid soluble 2 " , while dnap obtained by the diphenylamine method on the schneider extract gives results which are comparable with those obtained from the schmidt - thannhauser dnap fraction . it is clear from table i that dry preparations of calf thymus and fowl erythrocyte nuclei have a much higher dna concentration and a lower rna concentration than have liver nuclei , but a quite different light is shed on the results when they are expressed as amounts of each component per nucleus ( table ii )  . 
4 , which is a photograph in ultraviolet light taken by the method of markham and smith ( 1949 )  . in the material from one sample of rat liver nuclei each nucleotide was submitted to hydrolysis with perchloric acid ( marshak and vogel , 1950 ) , and the bases so liberated examined by paper chromatography by the method of wyatt ( 1951 )  . in each nucleotide only the expected base and no other was found . it is clear therefore that nuclear rna is similar qualitatively to cytoplasmic rna . 
component b lies immediately behind component a , from which it cannot readily be separated . it is probable that component b consists essentially of the inorganic moiety associated with component a . 
component b should in fact correspond to fraction p2 derived from phosphoprotein . component d lies immediately in front of uridylic acid and component c in the space between b and d ; both c and d are organic in nature . it is clear therefore that fraction " acid soluble 2 " contains in addition to the expected ribonucleotides at least four other components of which three are organic phosphates . these components are clearly visible in the autoradiographs shown in fig . 
the incorporation of 32p into all fractions of fowl erythrocytes was so low as to make the results of the specific activity of the dnap in rabbit and rat liver is little significance . very low indeed as compared with the cytoplasmic fractions , but by contrast the specific activity of the organic p fraction which contains ribonucleotides is very in experiments in which we have compared nuclear rnap and high indeed . cytoplasmic rnap the former has always shown a much higher rate of incorporation of 32p than the latter ( davidson , mclndoe and smellie , 1951 )  . 
the high specific activity of nuclear rna is confirmed by isolation of the individual nucleotides . although there is no great difference between the activity of the nucleotides , adenylic acid tends to show the highest components c and d have activities of the same order as those of the activity . nucleotides , while the activities of components a and b together are considerably higher . 
the general pattern obtained from grch 15 tumour is similar to that for other tissues . the presence of components a , b , c and d is shown clearly in the autoradiograph in fig . 
5 , in which the areas of radioactivity are seen to correspond closely with the ultraviolet absorbing areas in the adjacent ultraviolet print . since the " dnap " fraction has a low specific activity and is precipitated from a solution of much greater specific activity , it is possible that this fraction may be contaminated with small amounts of phosphorus of high activity although it was therefore thought necessary to investigate it is washed several times . the effect of dissolving the " dnap " fraction in alkali and reprecipitating with one such reprecipitation reduced the activity by about 40 per cent , but acid . further reprecipitations proved to be unnecessary and indeed undesirable since in all experiments in which they tended to promote degradation of the material . ionophoresis was employed one reprecipitation of the dnap fraction was used . from table i it is clear that the specific activity of the dnap in rabbit and rat liver is very low indeed , and this is again evident in table iv . 
the dnap of the liver of the normal cock on the other hand has a much higher specific activity , while that of the dnap in the grch 15 tunmour , as might be expected in a rapidly growing tissue , is still higher and approaches the value for the riboit is of interest that the specific activity of the dnap in nucleotides ( table iv )  . the liver of the tumour - bearing bird is higher than that in the normal bird . 
on the other hand fairly convincing evidence for the loss of protein from the nucleus during the process of isolation from aqueous media has been reported by pollister and leuchtenberger ( 1949b ) and by dounce , tishkoff , barnett and frier ( 1950 ) , although this is contested by stedman and stedman ( 1951 )  . 
to what extent loss of rna and other phosphorus - containing constituents may occur during isolation in citric acid is quite unknown . it is clear from the results shown in table i that , as might be expected , the bulk of the phosphorus in the nucleus is present as dna , but the results of the schmidt and thannhauser procedure reveal considerable amounts of phosphorus in the " acid soluble 2 " fraction . that a large proportion of this fraction consists of the ribonucleotides produced by alkaline hydrolysis of rna is evident from the results of pentose estimations by the orcinol and phloroglucinol methods , and the results of the ionophoresis experiments have conclusively established the presence in nuclear rna of the four nucleotides which are found in rna 210 w . 
2. components a , c and .d are organic phosphates , the presence of which is responsible , in part at least , for the difference between the " organic p " fraction in table i and the rnap by the orcinol reaction . the presence of such esters has already been suggested by davidson and mclndoe ( 1949 )  . it is clear that in nuclear tissue , as in whole tissue ( davidson , frazer and hutchison , 1951 ; davidson and smellie , 1952 ) , the method of schmidt and thannhauser ( 1945 ) tends to give rather misleading results , since the rna fraction contains , in addition to ribonucleotides , considerable amounts of non - ribonucleotide phosphate . incorporation studies with 32p have revealed the rapid incorporation of the isotope into nuclear rna and its slow incorporation into dna in nongrowing tissues . although nuclear rna is much less abundant than cytoplasmic rna it is more active , as has been shown by marshak and calvet ( 1949 ) , barnum and huseby ( 1950 ) , jeener and szafarz ( 1950 ) and davidson , mclndoe and smellie ( 1951 ) using 32p and by potter , recknagel and hurlbert ( 1951 ) using labelled orotic acid . 
the present studies show that this more rapid rate of incorporation of 32p into nuclear rna holds for all four nucleotides , which become labeled to practically the same extent . the specific activities of components c and d are of the same order as those of the nucleotides 2 hours after administration of 32p , but that of the inorganic phosphate fraction is much higher ( table iv )  . this component corresponds to fraction p2 in table i and is derived mainly from " phosphoprotein , " although it may contain traces of inorganic phosphate originally present in the tissue . its presence is responsible for the fact that the activity of fraction " acid soluble 2 " is higher than that of " organic p . " the high activity of fraction p2 in whole tissue has already been commented on by davidson , frazer and hutchison ( 1951 )  . the results of the experiments on fowls confirm the observation of kelly and jones ( 1951 ) and kelly , payne , white and jones ( 1951 ) in rats that the presence of a rapidly growing tumour in the animal body leads to an increased incorporation of 32p into the liver dnap . 
this effect is still unexplained and obviously requires much further examination , since it is associated in our experiments with the rather high specific activity of normal fowl liver dna as compared with rat or rabbit liver dna . 
the high activity for the dna of the grch 15 tumour is in agreement with the results of other workers for rapidly growing tissues ( brues , tracey and cohn , 1944 ; davidson and raymond , 1948 )  . the results presented in table ii are of some interest in relation to the claims of vendrely and vendrely ( 1948 , 1949 ) and of mirsky and ris ( 1949 ) that the dna content of the cell nucleus is apparently constant for the cells of the different somatic tissues of any one species although there may be wide variations between one species and another . the implications of these observations have been discussed by davidson and leslie ( 1950a , b )  . 
as might be expected , no loss of dna from the rat liver nucleus too much stress however occurs on fasting , although the rna content falls . should not be laid on this latter observation , since the presence of minute amounts of cytoplasmic contamination could significantly alter the rna content of it has already been shown ( davidson , 1947 ) that fasting causes a the nucleus . marked fall in the total rna content of the rat liver , while the total dna content this would of course be expected if the dna content of the is unchanged . individual nuclei and the number of nuclei per liver were unaltered . in the grch 15 fowl tumour the dna content of the nucleus is approximately double that in the fowl erythrocyte or liver cell . 
the significance of this observation is as yet obscure , but a similar high value for tumour cells has been recorded by klein ( 1951 ) for several mouse tumours . 
the samples were frozen in cyhndrical tubes immersed in a quartz dewar vessel and were irradiated with filtered light from a high pressure mercury arc , either the 3100 a or the 3650 a group of hnes being used . under these conditions afl the molecules examined were strongly luminescent , showing both a short - lived fluorescence ( persisting for less than 10 - 8 seconds ) and longhved phosphorescence ( persisting for several seconds ) in different spectral regions . fluorescence spectra were traced photoelectrically using a hilger e2 spectrograph equipped with a scanning unit . phosphorescence spectra were photographed using a mechanical phosphorescope as described elsewhere ( lewis and kasha , 1944 ; moodie and reid , 1952 )  . 
the phosphorescence spectra are the result of excitation to the very lowest excited state of the carcinogen molecule , a " triplet " state resulting from the fact that there are two unpaired electrons . excitation of this state requires only about 45 k.cal. , much less energy than is necessary to break bonds in the molecule , and its formation may well be an intermediate step in the reaction with protein . in this connection it should be pointed out that the triplet levels , since they cannot be detected in absorption , the common method of observing spectra , have been much neglected by biologists in their considerations of low barrier reaction paths that may be involved in biochemical reactioins . ( 2 ) the second significant correlation comes from heterogeneous system experiments . 
we have found that ff a carcinogen is ' used as the microcrystalfne component and the dissolved component is naphthacene , the ( fluorescent ) emission of the naphthacene is shifted shghtly in wave length because of its interaction with the carcinogen . 
some of the results are it will be observed that the smallest shffts in the naphthacene show - n in table ii . bands occur when the strongest carcinogens are used as the host crystal . there is a moderately good correlation between these shifts and the self - polarizabihties at the position of the substituent calculated by greenwood ( 1951 ) , which values themselves correlate better with carcinogenic power than do an ' other physical constants . 
the technique for painting with the 03 per cent benzpyrene solution in - acetone was that described by calcutt and powell ( 1947 ) , and further treatment was as in earlier work ( weigert , calcutt and powell , 1946 )  . extraction and estimations of the metabolites were by the methods of weigert ( 1948 )  . the nutrient media used were as below : 1 . 
for a period of 8 hours . metabolism of the benzpyrene only occurred in the series on the four buffered in the case of the tap water and the normal saline the media were found media . to be turbid and the skins sodden and waterlogged . 
on the other hand , the variation in explanations of this based on the yields obtained is in itself of some interest . the detailed composition of the media used appear to be of doubtful significance . the outstanding point seems to be the ph of the medium . under the conditions of experiment it is reasonable to assume that the tissues of the skins concemed became adjusted in some degree to the environmental conditions , that is , that the internal state of the cells approximated more nearly to the external ph than would normally be the case . 
the question arises , therefore , as to whether such a state of affairs could influence the metabolic turnover . taking advantage of some recent findings , it is believed that an explanation of the results can be offered along the above lines . it has long been suspected that the process of carcinogenesis is intimately involved with sulphydryl groups . this view has recently been reinforced by crabtree ( 1948 ) , and the weight of evidence is now strongly in its favour . 
at the same time it has been suggested that the metabolism of benzpyrene is also mediated by - sh groups . this has received confirmation by the findings of 432 a . 
extremely active material capable of s " rdng fivsh t clours in less than two weeks has been obtained from both chemically induced and spontaneous sarcom firozen at - 790 c . 
since it involves the quesbon , first raised by ehrlich in 1907 , in connection with a possible bacterial source of cancer , of whether extreme cold can be used to destroy mammalian cells , and multaneously to preserve any posdble intraor extra - cellular agent capable of starting a t aour de novo in if the continued activity of such refiigerated tisme is cells of another host . aour produced in the due to new host is merely a transplant . if , however , it can be shown that the cers in the refrigerated tissue are dead , then the n6w tumour must be produced by an agent ( which would then be the " continuing cause " of cancer ) which is a separate .val of cells of the original anhml , then the t 260 w . 
craigie entity from the cells in which it lives , and which is capable of surviving under such transm , ission would be " cell - free , " certain conditions when they are dead . and would place any tumour so produced in the class of virus tumours , among the known virus chicken tumours , the shope papilloma of rabbits , bittner 's mammary carcinoma and lucke " s adenocarcinoma of the frog . the proof that prolongecl refrigeration kills mammalian tumour cells is difficult , and we are aware that in the past it has always been held , except by cramer ( 1930 ) , that the survival of only a few cells in the tumour mass accounts for the tumour in the new host . we have investigated the problem from a variety ofangles and have concluded , as will appear , that the cells are dead . jn the first place it must be realized that the sarcomas of mice which we have used are three well known to laboratory woikers , one a methylcholanthrene sarcoma ( c.48 in the inbred strain c57 black ) , and the other two arising as stromal transformations in transplants of sporadic mammary cancers investigated by r . 
none of these procedures affect the activity of the refrigerated tissue , which remains as great as or greater thirdly , we have made histological examinations than that of the fresh tissue . of frozen tissue implants at intervals and have examined the material itself , finding , - however , neither any evidence for survival of cells , nor conclusive proof of their total destruction , since one cannot be certain that every cell has been examined . finally , being well aware that to all these methods of approach might be opposed the objection of the remote possibility of the exceptional survival of a few cells , we have proceeded to dxy the refrigerated tissue completely to dust this is universally allowed to be lethal in vacuo at a temperature of - - 25 ' c . to both normal and tumour cells , yet we have obtained highly active dry material it therefore appears impossible to avoid from all the three tumours studied . the conclusion that from both the chemically induced and the sporadic sarcomas a cell - free agent can be recovered , which on injection into appropriate mice is capable of starting the tumour afresh . 
during this period attempts had been made again and again to propagate the tumour with cell - fi - ee filtrates and with dry tissue , and all had previously failed . ( 2 ) a sarcoma of the strain r3 . 
the sarcoma which formed from the stromal connective tissue was separated from the mamm ry cancer by ludford by early transplantations by which the slower growmg carcinoma was lost . ( 3 ) a similar sarcoma derived from the stroma of a carcinoma of the strain all three tumours are soft , rapidly growing sarcomas . 
the ' ampouilie - - s were then transferred to a storage cabinet kept at - 79 ' c . after an appropriate interv - al of hours , days or months , an ampoule was taken out , thawed to room temperature , opened , and 0 - 05 c.c. 
or less of the contents inoculated subcutaneously in the right flank of mice of the appropriate strawhen these ampoules were opened it was always noted that a physical change ha - d taken place in their contents , which had become more homogeneous , softer and slightly sticky after freezing . 
twe - four experiments were made with inoculations at varying intervals with uniform ] ' good results ( table 1 )  . it wiu be noted that in the last experiment on the time frozen makes no difference to the results . the list the material had been frozen 201 days , and was inoculated not only into its mouse of origin , but also into four other atains . 
refrigeration in van ; ow media . from experiments with mouse embryo tissue ( mann , 1949 ) we know that treatment with glucose ( without refrigeration ) reduces the activity of normal mamm lian cells , that storage in glycerol at 4 ' c . them , and that a combination of dextrose and cystein severely damages them , while fiwzing alone for one mour cells using glycerol , hour ik - ill them all . dextrose , eystein , and distifed water with and without fiwzing were therefore performed . table ii shows that these procedures , known to kill normal cells , have no effect on the activity of tumour tissue , dextrose indeed appearing definitely beneficial ( cmigie , 1949 )  . it wiff be noted that the results in table i and table 11 are very similar , and are condstent with the presence of a t nour agent other than a living mamm lian cell . 
as an example of the amount of man pulation which such t aour mince will stand we can cite the protocol of an experimentwith the c48 t ilour , carried various experiments with t 262 w . 
the mortar was then taken out of the c02 snow and solid frozen tissue and buffer pounded vigorously with the pestle , to break up as far as possible any remaining whole t thirty c.c. 
of cold distilled water was added , and when the mixture was finally hquid it was spun for 15 minutes at 10 , 000 revolutions a minute . ' the supernatant fluid was reddi - sh in colour and was of a ph 6 - 0 ; the deposit , firmly . 
of sahne ( - 85 per cent nacl in distffled water ) and the emulsion injected into six c57 mice . the mice developed ical c48 tumours , which were large on the 14th day . fibns of the emulsion were examined under the microscope and ' m none could a whole cell be found ; all that could be seen consisted of naked nuclei and separated masses of cytoplasm . experiments such as this do not appear to lend any aours started bv fi - ozen tissues are transplants , support t - o the belief that the t i.e. 
even there are even fewer apparentl - v intact bv the mincer . cells , while after treatment with glucose or with distified water profound changes appear affecting both nucleus and cytoplasa detailed studv of these changes is being made by one of us and wir be pubhshed later . a series of sections of implants of tumour tissue ( both sarcoma and carcinoma ) frozen to 79 ' c . 
by the fourth day much of this tissue is beiing absorbed and removed by the host , and on the fifth and sixth days and subsequently , the begi of a new sarcoma can be seen in the connective tissues surrounding the ne ; brotic mass . 
the appearances do not suggest survival of any of the implanted tissue , but rather a fresh sarcomatous reaction of the encapsulating in the case of frozen carcinoma also no living subcutaneous tissues of the host . tumour ceus were seen , and by the seventh day the decrotic m was encapsulated and shrunken . 
they were successful , as was also a similar experiment with the or tumour which wasaxied fresh by craigie . it has not so far been possible to obtain active dxied material from the c48 without preliminary storage at - 79 ' c . a brief account of the metbod of drying must be given , though for details of the apparatus used craigie 's paper ( 1949 ) should be consulted . 
the tissue to oule or , if freshly miinced , diluted be dried is either taken from the storage a v ' n ' th an eqltal volume of 5 - 3 per cent dextrose and is run into the drying flask . it sbould be distributed as evenly as possible over the bottom of the flask in a thin layer . 
ofm / 150 eystein ph added after cirying to zero , pumping is then continued for a further half or one hour to be absolutely certain of the removal of the last traces of wate ' r . 
of which is injected subcutaneously in each mouse . before this method of drying was evolved , many unsuccessful experiments with these tumours , using the knox method of drying , had been cam ' ed out . even now a successful res - ult - is not obtained in every case , and - much further experimentation will be required to determine the optimum conditions . 
raven. from the royal cancer hospital ( free ) and the gordon hospital for diseases of the rectum and colon . received for publication march 20 , 1948 . a study of the literature concerning malignant disease of the heart teaches that whilst primary tumours are rare , secondary deposits are not infrequently found . 
the following figures are illustrative . scott and garvin ( 1939 ) , in a series of 11 , 100 autopsies , including 1 , 082 performed in cases of malignant disease , reported secondary malignant disease as follows - heart , 79 cases ( 7 * 3 per cent ) ; pericardium , 61 cases ( 5 7 per cent ) ; heart and pericardium , 140 cases ( 12 * 9 per cent )  . willis reported an incidence of 6 - 2 per cent ; burke , 4 2 per cent ; helwig , 0 9 per cent ; pollia and gogol , 2 per cent ; lymburner , 0 6 per cent ; and symmers , 1 6 per cent . the subject is of more than academic interest , for with a better understanding it may be possible to establish a diagnosis during life in a greater number of patients , and the institution of certain forms of treatment , even though these are of a palliative nature . 
the symptomatology will therefore be reviewed , and attention drawn to the electrocardiographic findings in these patients . this paper is based on a series of 51 cases of secondary malignant disease of the heart which were collected at the royal cancer hospital from the autopsy records for the years 1930 to 1945 inclusive . 
the pericardium is usually involved and various types of disease are described . focal collections of carcinoma cells may occur in the epicardial fat , the cells being transported along the dilated lymphatics . in other cases the pericardium is studded with nodules of growth , whilst a third variety consists of diffuse pericardial infiltration , and this process may extend into the pericardial fat . there may be commencing infiltration near the commencement of the great vessels . another variety comprises flat plaques of growth embedded in the pericardium . in a number of cases there is combined involvement of the pericardium and heart muscle . 
tumour cells were seen in the lumen of a small arteriole ; obviously the disease in the heart was conveyed by the blood stream . in another case nodules of growth were found in the muscle of the right auricle , left ventricle and left auricle ; some of these were situated beneath the endocardium and growing into the auricle . 
the microscopic picture was similar to that of the primary skin tumour . in the third case an amelanotic nodule was present in the upper and posterior part of the left ventricle , from which more extensive spread had occurred into the heart muscle . moragues ( 1939 ) reviewed the subject of cardiac metastases from malignant melanoma and found reports of 23 cases in the literature , to which he added 4 in one of these cases there were signs of cardiac involvement ; personal cases . a loud systolic murmur was present in the pulmonary area , caused by a large tumour mass which almost occluded the pulmonary orifice . the spread of malignancy to the heart . this occurs by the usual four methods , namely , haematogenous , lymphatic , in this combined haematogenous and lymphatic , and by direct extension . series of cases there was definite evidence of all these modes . 
any subsequent abnormality which is noted can be compared with this initial investigation and inferences drawn . in cases with tumours of the heart , carnot and lambling ( 1928 ) have described a clinical syndrome which resembles that of subacute bacterial endoapart from this group of cases , attention is called to certain symptoms carditis . and signs which suggest cardiac involvement in patients known to be suffering from malignant diseases , especially of those organs which are recognized to give rise to secondary cardiac deposits . dyspnoea , tachycardia and cardiac irregularity are frequently outstanding symptoms , the irregularity being of the nature of either auricular fibrillation or auricular flutter . 
the symptoms may be correlated with the location of the tumour in the heart , causing heart block , or due to a pericardial or pleural effusion . it must not be assumed , however , that evidence of pericardial disease in a patient with malignancy is pathognomonic of involvement of the pericardium ; acute pericarditis may occur in the advanced phase from other causes . 
amongst important signs are those of cardiac enlargement and dysfunction , impaired resonance of the chest , the presence of abnormal breath sounds and added sounds . cyanosis may be present and the signs of cardiac failure . in the presence of this symptomatology certain investigations are carried out . 
when a pericardial effusion manifests itself , paracentesis is performed and cytological examination of the fluid is made . in a number of cases malignant tumour cells will be demonstrated . radiological examination of the heart , including tomography , is regarding the electrocardiographic out . findings , the case reports in the literature do not show uniformity ; the results vary according to the position of the tumour in the heart . siegel and young ( 1933 ) , dealing with this aspect of the subject , call attention to the work of lloyd ( 1929 ) , who published the first electrocardiogram in a proved case of tumour of carried r . 
the t waves in leads 1 and 2 were slightly diphasic . had been given before the patient was admitted to hospital , which may account siegel and young ( 1933 ) reported the for the abnormality of the t waves . findings in a case of metastatic sarcoma of the myocardiuthe t waves were inverted constantly in all leads , and there was no significant change daily . 
when the primary tumour is known to be radiosensitive , high - voltage x - irradiation may prove of value in the diagnosis in these cases clinical of metastases in the heart when enlargement is present . and radiological evidence may be forthcoming of a marked reduction in the size of the heart and the disappearance of the pericardial effusion . shelburne and aronson ( 1940 ) report the effects of high - voltage x - irradiation in a patient with metastases in the heart and pericardium from a primary myeloblastoma in the frontal bone . 
1. received for publication january 21 , 1948 . surgery in the form of the so - called radical operation is in general at the present time the principal weapon in the treatment of carcinoma of the breast . what it can achieve and its limitations are now fairly well established . 
thus it is known that if a patient with carcinoma of the breast has microscopical invasion of the axillary glands , the strong probabilities are that the disease has passed beyond the local area and that the patient will sooner or later succumb it is to the disease , although there will be a minority of fortunate exceptions . also known that if the axillary glands are not invaded the chances that the patient will be cured of the disease are appreciably increased . 
with a stroke of 6 cat a rate of 90 per minute , and melanin formation estimated at appropriate intervals by observing the extinction coefficients of the solution with an e.e.l. 
on 15 occasions there was acceleration averaging approximately 0 - 3 per cent as compared with the control . on the assumption that the errors inevitably present will tend to operate equally in either direction , the probability of obtaining 15 results out of 17 in one direction is ( approximately ) only i in 500 , so that the results are statisticany significant . the possibility of the acceleration being due to change in ph was reconsidered . the ph of the various solutions employed was estimated , electrometrically , using a glass electrode . 
1. - lung cancer in denmark , 1931 - 1945 . 1931 33 35 crude mortality rate per 100 , 000 in males 43 45 39 41 alterations in the age distribution of the population might be suggested as the cause of the rise in crude mortality from lung cancer , but if we compute the mortality rate according to age in the capital for periods of five years , and on the base of quinquennial age groups of dead and living persons , as in fig . 
2 , we find no confirmation of the suggestion mentioned . thus , we have found a very steep increase of mortality from lung cancer within a limited area , well furnished with modern medical facilities , available to all social classes . * the increase is found among both sexes , but is far more pronounced for males than for females , and it will be worth while further to analyse the population in question with regard to the incidence of lung cancer , as copenhagen seems to provide a good example of the changes in the mortality from lung cancer found elsewhere in europe . the central tuberculosis station of the city of copenhagen provides excellent opportunities for checking up the reliability of statistics given in the first part in its work to trace tuberculous infections in patients of the present paper . suffering from all sorts of respiratory diseases , this institution performs numerous thorough examinations on such patients referred from general practitioners . annual reports contain tables giving the number of patients , subdivided in decennial age groups for each sex separately . 
the group examinations under * for members of the public health insurance , which comprise 85 - 90 per cent of the danish population , no fees are charged in the municipal hospitals of copenhagen . 
3 , shows no steady rise , and hardly any irregularity is found in the curve for females , which , however , comprises very few individuals , as seen from the absolute figures . the very steep rise of the male curve about 1941 may have been caused by the fact that the efficiency of a central tuberculosis station may also have increased if the duration of the disease is one or two years in a disease like lung cancer . from its earliest possible detection to death , and this is , in fact , the opinion of the tuberculosis station , the steep rise in 1941 given in fig . 
even if we allow for the small absolute values of the figures , it can be said , as with regard to the corresponding mortality figures , that there is no increase of morbidity in the young , as would be expected if the inerease was due to an increase in the inhalation of tobacco smoke , if we are not to assume a latent period for lung cancer of about two decades . in our opinion the figures given illustrate that even a pronounced increase of the crude mortality rate for lung cancer among males , and almost only among males , does not necessarily mean an increase in incidence of that disease . presumably the more frequent detection of the disease is due to improvement of 2s~~~~~~~~~~~~~ __ _ 120 ___ __ 80 __ f - ~i 40 ' j years fig . 
if the results from copenhagen can be generalized , it seems that the apparent increase in lung cancer mortality is , to a very large extent , conditioned by improvements in diagnostic means , and it can be expected to continue until the sex ratio amounts to about 8 males to 1 female . our thanks are due to knud winge , m.d. , the chief physician of the central tuberculosis station , copenhagen , for permitting us to use the material of the station , and for his kind interest in our work , and to kontorchef miss m . 
1. received for publication december 1 , 1952 . it has for long been an accepted principle that the earlier the diagnosis of malignant disease is made the more likely it is that the growth , if in a region of the body accessible to the surgeon , will be in a stage which admits of radical resection and the higher will be the survival rate . 
however , balfour ( 1937 ) recorded that the 5 - year survival rate for 2112 patients after resection of the stomach for carcinoma was 25 per cent for those patients with histories of symptoms for less than 6 months and 35 per cent for those with histories of symptoms for more than one year . 
walters , gray and priestley ( 1942 ) in a later study recorded that 24 * 6 per cent of patients whose symptoms were of less than one year 's duration survived 5 years after resection of the stomach for carcinoma , whereas 32 - 6 per cent of patients the duration of whose symptoms was more than one year survived 5 years . 
the authors investigated the association between the length of history and the grade of malignancy as judged by broders ' classification , and give a table which shows that 39 of the 114 patients had growths which were classified as broders ' groups i and ii , while 75 had growths classified as broders ' groups iii and iv ; in each case the proportion of patients who gave histories of symptoms for more than 2 years was 23 per cent . eggers , de cholnoky and jessop ( 1941 ) reported that while the 5 - year survivals of 235 patients who had undergone radical .mastectomy for cancer of the breast fell steadily from 76 per cent to 20 per cent with delay in treatment up to 2 years , the survival rates rose to 25 per cent of 20 patients in whom the delay was over 2 years , and to 40 * 9 per cent of 22 patients in whose cases - there had been delay of 3 years or over . 
harnett tomies for cancer of the breast , found that 24 - 8 per cent of the 938 5 - year survivors were treated more than one year after recognition of the tumour ; he considered that the majority of these cases were the indolent , slowly - growing types of breast carcinoma , which metastasized late or occurred in hosts whose desmoplastic reaction to the tumour was intense . 
bloom ( 1950a ) in an analysis of 470 cases of cancer of the breast treated by surgery alone or with ancillary radiotherapy found , after classifying them into 3 groups according to histological criteria of degree of malignancy , that 79 per cent of 141 cases graded as of low malignancy survived 5 years , 42 per cent of 191 graded as of moderate malignancy and 25 per cent of 138 graded as of high malignancy ; when the histological grading was subdivided according to the presence or absence of invasion of the axillary lymph nodes , he found that with lymph nodes not invaded the 5 - year survival rates for gradesi , ii and iii were 94 , 61 and 53 per cent respectively , while for patients in whom the lymph nodes were demonstrated histologically to be invaded the survival rates for the three grades were 65 , 30 and 16 per cent respectively . 
as all types of treatment , surgery , radiotherapy and combined treatment , whether radical or palliative , were included , it is not surprising that few significant differences in survival rate were detected , though in most regions it appeared that the patients treated early showed little better survival rates than those who came late . in the present communication tabulations are presented from the same series of cases of the results of treatment of patients in stages i , ii or iii , who were treated by radical surgery alone or combined with radiotherapy , arranged according to duration of symptoms at the time of commencing treatment . 
by the elimination of all patients who were in stage iv , and of those in whose cases only operations of a palliative nature were performed , the series is restricted to those in whom there was a reasonable expectation of 5 - year cure . patients treated by radiotherapy alone have been excluded , because it is difficult to separate those in whose cases the treatment was intended to be curative from those in which it was palliative only , though some of the latter survived 5 years . the cases are grouped by site of the disease and by duration of symptoms at the time of commencing treatment into ( a ) , those in which the interval from first symptom to commencement of treatment was less than 6 months , ( b ) , those in which it was between 6 and 12 months , ( c ) , those in which it was more than 12 months , ( d ) , those in which it was unknown . 
the 5 - year survival rates for each of these groups is expressed as a percentage of the total in the group , and the standard errors of the differences have been calculated to test for statistical significance . in the more important sites the survivals are shown by stages : i . 
survd. over 12 , , not known the differences in survival rate are not statistically significant . for cases of known duration x2 = 1x77 , n = 2 , p < 50 > 30 . . 
among 383 patients treated by radical mastectomy alone , 35 - 5 per cent were in stage i and the best survival rate for all stages combined was 50 9 3 - 3 per cent for those treated during the first 6 months ; among 175 who were treated by radical mastectomy combined with radiotherapy only 21 * 1 per cent were in stage i and 43 * 4 per cent were in stage iii , and the best survival rate for all stages combined was 58 - 8 8 - 4 per cent for patients treated one year or more subsequent to the first symptom , who numbered 34 . 
the difference of 7 - 9 9 0 between these survival rates is not statistically significant . when the sites were subdivided into those in which the highest survival rates were among the patients who came under treatment in less than 3 months and those in which the highest rates were among those treated in the second 3 months , it was found that the former included squamous carcinoma of the skin , corpus uteri , penis and female breast with a survival rate for all groups of 54 - 9 per cent , while the latter included cervix uteri , kidney , .papillary carcinoma of the bladder and carcinoma of the pharynx ( treated by radical surgery or by combined methods ) with a 5 - year survival rate for all groups of 51x0 per cent . the age distribution of 702 patients with cancer of the breast who were . 
the group of 63 in whom the symptoms were of over 12 months ' standing at the time of operation had a survival rate of 14 3 per cent ; the difference of 9 9 7 0 between this figure and that for the group treated in the second 6 months is not statistically significant . if the patients are grouped into those of less than 6 months ' duration , with a survival rate of 11 - 2 per cent , and those of over 6 months ' duration , with a survival rate of 19 - 2 per cent , the difference of 8 - 0 4 - 7 is not significant . the age distribution of the patients with cancer of the stomach and colon were investigated by the x2 test , but in neither case were there any significant differences between the observed and expected survivals in the different age groups ; for the stomach cases p < * 20 > * 10 and for the colon cases p < - 70 > .50 , showing that the differences in survival rates were not due to differences in age distribution . 
the survival rate for cancer of the stomach was 20 - 4 per cent for 108 patients in the age groups 25 - 54 and 11.1 per cent for 133 patients of 55 and upwards , but the difference of 9.3 4 - 7 is not significant , though the prognosis was not so good in the older patients . 
as has been stated above this difference cannot be accounted for by differences in the age distribution of patients in either of the groups , so it must be presumed to be due to variations in the rate of growth of individual tumours all cases of patients with and in their infiltrating and metastatising powers . distant metastases have been eliminated from the present series , which was confined to those in which the tumour was local or had given rise to clinically recognisable metastases in lymph nodes ; they were selected as operable cases , no patient in an advanced stage or suitable only for palliative treatment being small differences were found in the proportion of patients in stage i included . among those with the highest survival rate in each of the three groups ; for group w . 
the mean duration of life in 153 untreated patients in group a was 23 - 1 months , of 176 in group b was 11 - 7 months and of 296 in group c was 17 - 6 months . 
the sites showing the longest survivals among the untreated patients are those in which treatment by radical surgery alone or with radiotherapy gives the best results , although some sites show a short duration of life , either because , as in the case of the lip , very few cases are left untreated , or because , as in the case of the colon and the intrinsic larynx , untreated patients often die of complications , such as intestinal w3w . 
group a included the skin ( squamous carcinoma ) , corpus uteri , penis , cervix uteri , female breast , kidney , bladder ( papillary carcinoma ) and pharynx . for this group of sites , comprising 1097 cases of known duration , the best survival rate was 51 1 per cent of 616 patients treated in the first 6 months against 42 - 0 per cent for the remainder . 
group b included salivary glands , tonsil , testis , colon , skin ( malignant melanoma ) , nasal sinuses , bladder ( infiltrating carcinoma ) and stomach . this group of sites , comprising 718 cases of known duration , the best survival rate was 30 - 6 per cent of 134 patients treated in the second 6 months against 27 1 per cent for the remainder . 
a blank estimation was carried out on the reagents and in the latter half of the investigation a weighed amount of the filter - paper surrounding the discoloured areas was included in the blank , but this addition makes no considerable difference . occasional very high blanks were discarded as due to some unusual contamination . 
3. - mean seasonal variations in arsenic content of air at eight stations . w = winter ( november to march ) ; s = summer ( may to september )  . establish any exact comparison between the seasonal changes in the individual richard doll has pointed out to us that , the average values for the towns . different stations would be more properlv comparable , in view of the seasonal variations , if ( 1 ) the means of two figures for the same month and town were taken to represent that month , and ( 2 ) the mean of the figures for two months which are separated by a gap of one month without data were taken to represent that month . 
one might suggest some such classification as the following of the not very abundant data available upon the types of sunmmation ( table ii ) ; no claim is made that this is in any way a complete summary of the literature . 
the summation of action of two chemical carcinogens is not easy to demonstrate , and we appear to have few indubitable instances of it . * in connection with the subject of cancer of the lung , the work of lynch ( 1935 ) and andervont ( 1937 ) upon summation in the lung of some strains of mice is of especial interest , although the neoplasms of the lung in this species differ from those in man . 
a few small arterioles present in the floor and edges of the ulcer showed no evidence of endarteritis . at one point the tumour had penetrated the periphery of the pancreas for a there was no involvement of the common distance of approximately 2 nun . 
5 , showing invasion of the submucosal connective - tissue strata by adenocarcinoma which has , on left - hand side , lost all resemblance to normal brunner 's gland acini . 
hence there can be no reasonable doubt that it is primarily duodenal . histological appearances clearly indicate that the initial neoplastic change has involved the epithelium throughout the whole thickness of the mucous membrane , including brunner 's glands . 
phillips. from the national cancer in8titute of canada , 800 bay street , toronto 5 , ontario . received for publication april 3 , 1954 . within recent years evidence has been brought forth on variations in the distribution of ' cancer in different parts of the world . cancer of the base of the tongue , for example , is relatively frequent in certain parts of india and primary cancer of the hver is relatively frequent in africa and indonesia . cancer of the stom - ach on the other hand , is relatively infrequent in african negroes , javanese and the indigenous people of french north africa . such observations have focused attention upon racial origin , environment and custom as i ' mportant factors in the study of cancer . in 1950 at a conference on the aeo - araphical pathology and demography of cancer ( clemmesen , 1950a ) it was agreed that a high degree of comparability is desirable in any study of geographical variation . 
phillips the origin of the north american indian remains uncertain , but anthropologists beheve that they came to america in successive migrations in prehistoric times from northern asia , probably by wa of the bering sea . 
the primary function of this branch is to administer the affairs of the indians of canada in a manner that will enable them to become inereasinglv self - supporting and independent members of the community . health services to indians date back to the early part of the seventeenth century when french armv doctors gave medical care to this group . 
the indian health service operates 18 hospitals and 29 nursing stations giving a capacity of approximately 2287 beds . across the country there are 65 full - time doctors caring for indians . there axe also 90 pubhc health nurses assisted by provincial public health nurses , red cross outpost nurses and the ceintres range in size from the 456 - bed charles victorian order of nurses . camser indian hospital at edmpnton , alberta , to small units of 16 to 20 beds . in addition the services of some 380 community ' and private hospitals are utihzed for the treatment of indians . the indian health service has been faced - with a number of problems of which tuberculosis , inadequate nutrition and high infant mortality are the most serious . 
phlllips when an indian receives medical care a record of his case is forwarded to the this record gives , among other data , national office of the indian health service . the patient 's name , band number , age , sex , diagnosis , and days of hospitahzation . with such information available the patient - records in forty - eight hospitals were reviewed for the years 1948 - 1952 inclusive and all cases of cancer were selected for analysis . 
the indian health service has estimated that over 90 per cent of indians with cancer would be referred to ' the hospitals included in this study . in the five years which were reviewed , 327 cases of cancer were re - ported , 125 in males and 202 in females . 
the finding that cancer is less frequent inindians over 30 years of age than in whites over 30 years of age is to be accepted with caution . " ir - defined causes " are reported in death certification more frequently ( table 11 ) for indians and it is possible that illness and deaths from cancer may he within this group and which , if properly reported , would raise the incidence rates for older indians . in other words , the observed difference in total cancer incidence rates between indians and whites may be more apparent than real . it is possible that skin pigmentation of indians acts as a protection against skhi cancer in the same way as for the black race . however , it is also possible that skin cancer cases did not come under medical care but we doubt this because the incidence of other sites , excepting cervix , was the same as in the white population . the most interesting finding is that relating to the high incidence of cancer of the cervix uteri . 
phillips over this age the incidence of cancer among indians is apparently less than among whites . cancer of the skin occurs less frequently among indians . the incidence of cancer of the cervix uteri is much higher among indians than that reported for the white population in canada and the disease tends to occur earlier in life . the incidence rates for all other sites of the disease show no difference between indians and whites . the authors wish to acknowledge the assistance of p . 
the rats were 4 weeks old when these operations were performed and 7 days later the litter - mates were joined in parabiosis . the surgical technique used was the one recommended by jacobsohn ( 1948 )  . in most pairs both partners were females except in those forming groups iii and vii , where a castrated male took the place of the spayed female . 
a total of 10 to 25 doses was given , when possible in a schedule of 3 doses per week . occasionally , this dosage had to be reduced to one or two administrations per week when the pair failed to show the expected gain in weight . twenty - six pairs of albinos ( groups i , ii and iii ) and 3 pairs of piebald rats 332 f . 
by stomach - tube during the first 8 weeks of the experiment . these rats , hke the majority of the parabiotic pairs , were 2 to 21 months old when the administration of the carcinogen was commenced , and served as controls for the assessment of the carcinogenic activity of the amounts of a.a.f. 
the parabiotic rats were sacrificed when declining health , in most instances due to the preserice of large pyometra , made it advisable or when the presence of a tumour was suspected . 
two of the " single " rats were killed when a palpable tumour was present , the remainder when the experiment was terminated at the 52nd week . the material taken for histological study was fixed in zenker , and the sections were stained with haematoxylin - eosin or according to van gieson . 
the tumours originated in gonads which were grossly hyperplastic . the histological changes occurring in the ovaries of rats joined in parabiosis to gonadectomized litter - mates have been well described by zeckwer ( 1944 ) , therefore our findings will be mentioned only briefly . 
the wall of these cysts was smooth , and when an epithelial lining could be recognized the cells were low cuboid or flat without any indication of cellular activity . such cysts occurred equafly in intact partners treated with a.a.f. 
the earliest neoplasms seen were generally round in sfiape and had a diameter of 3 to 4 mthey were found in wistar rats of groups i to iii after 16 to 20 weeks of parabiosis ; in piebalds they occurred even earlier . 
the largest gr ' anulosa cell tumour which had destroyed completely the ovary in which it originated measured 27 x 16 x 25 mand it was the only neoplasm of this kind to produce a metastasis , weighed 3 - 88 g . the secondary being situated in the adhesions linking the primary to neighbouring organs . the material at our disposal allows the study of the development of granulosa fig . 
2 shows a large haemorrhagic cyst cell tumours from their early stages . found in an ovary of rat 1 , group iv , sacrificed at the 13th week of the experiment . this cyst had a thin wall formed b fibrous connective tissue which in most places was poor in cells . in some areas the capsule was thickened , and here signs of previous haemorrhage such as pigment - loaded macrophages were present . into the cavity papillary projections protruded . 
they were of two kinds , some into the former , delicate strands with a narrow and others with a broad base . of spindle - shaped cers entered , forming their core . into the latter , capinaries penetrated surrounded by cers having ample eosinophihc cytoplasm , and a nucleous less rich in chromatin than the cells which covered and formed the bulk of the projections . these had a nucleus rich in chromatin and a scanty cytoplasthey were typical granulosa cells , while the ones having ample cytoplasm this was the earliest neopalstic lesion found in our resembled lutein cells . material , and shows that atypical growths of this kind were not necessarily permanent ones . 
and killed after more prolonged parabiosis hae ' morrhagic cysts , where only small nests of granulosa cells remained embedded in the dense connective tissue of the capsule while the cavity contained only inspissated blood and tissue debris . such chocolate cystlike lesions were absent from the ovaries of the control pairs not treated with a.a.f. another animal of the same group sacrificed 2 weeks , later was found to have in the ovary a macroscopically visible nodule formed almost entirely by granulosa cells . it was of about the same size as the early granulosa cell tumuor depicted in fig . 
3 shows a sharply limited spherical nodule situated in an ovary containing cystic follicles . it was surrounded by a thin fibrous capsule . between the capsule and the neighbouring cystic folhcles were large blood - fifed sinuses . 
5 shows the appearance of the granulosa cells forming the bulk of the nodule , and the tendency towards foci of liquefaction leading to the formation of cystic spaces within the tumour . 
the tumorous right ovary of rat 5 , group 111 , showed histologically an unusual picture . this ovary was also considerably enlarged ( 802 mg . ) and fairly solid with only a few blood - filled cysts only a small rim of ovarian tissue remained ; most of the near its surface . histologically this tumour was composed specimen was obviously neoplastic . mainly of spindle - shaped cells with scanty cytoplasm , and a nucleus which varied considerably from elongated dense to oval or spherical vesicular . 
among them small groups of cells were present having ample cytoplasm which stained well these elements had a larger vesicular nucleus with the chromatin with eosin . displaced towards the nuclear membrane . 
we have classified this neoplasm as a theca cell tumour because the granulosa contributed only little to its make - up . in the 36th week of the experiment the health of the intact partner suddenly declined and therefore in the region of the left ovary there was a large blackish the pair was sacrificed . cyst , on the posterior wall of which some ovarian tissue was recognizable . 
two types of neoplastic cells could be distinguished : bizarre cells of unusually large size having one or , rarely , their cytoplasm was several nuclei with deeply stained prominent nucleoli . intermi ' ngled with these elenients ample and stained only weakly with eosin . these were polyhedral in were nests of much smaller cells of epithelial character . shape and had well - marked cell borders , in cohtrast to the large elements which tended to form svnvctia . 
 ( groups v , vi and vii )  . the only lesion suspicious of atypical growth was found in one of the ovaries of an intact female which survived parabiosis for 42 weeks ( group v , rat 8 )  . 
we are unable to state with certainty that this lesion was of neoplastic nature . as already mentioned , no ovarian tumours were found in the 10 " single albino rats which received 24 doses of 4 mg . 
among many hundred female rats treated with this carcinogen we have found only one instance of a neoplastic lesion in the female gonads . this was discovered in a rat 171 months of age when sacrificed . 
by stomach - tube when 2 months old ; then the animal was kept without further treatment for 1 year , and for the last 16 weeks of her life she received methyl - thiouracil in the drinking water . 
at the post - mortem the left ovary was found to be converted into a large cyst measuring 20 x 15 x 10 mm . on histological examination septa subdividing the cyst in several compartments and covered with granulosa cells were seen . although macroscopicany this cyst resembled the clear cysts which we have frequently found in the ovaries of parabiotic females and also in rats treated with stilboestrol , we have never observed in such structures prohferation of granulosa cells exce ' t in this one instance . the morphological 8ign8of the endocrine imbalance exi8ting in the intact partner . all signs of hyperoestrinism were present in the intact partner ( zeckwer , 1944 )  . 
the vagina was lined by hyperplastic squamous keratinizing epithelium . in the few instances where the uterus was not heavily infected the epithelium lining the cavity was tar cyhndrical and areas of squamous metaplasia were also present , the wall of the organ being thickened . in the infected uteri most of the epithehum was destroyed . 
the breast glands were grossly hyperplastic , and in most animals macroscopicary visible cysts fired with a milky fluid were seen . histologically such glands showed a pronounced cystic hyperplasia of the ducts and periductal fibrosis . 
the thymus was atrophic , and the suprarenals enlarged in size due to a hyperplasia of the cortex . the pituitaries of the intact partners were invariably larger than those of the gonadectomized litter - mates , and already after 15 to 17 weeks of parabiosis had increased to an average of 8 mg . 
the largest observed ( rat 6 , group iii , and rat 8 , group v ) reached the extraordinary weight of 74 - 8 and 139 - 3 mg . 
the pituitaries of the intact partners were mostly solid and of whitish colour and haemorrhagic areas while the majority of these glands showed a symmetrical were not conspicuous . enlargement of the anterior lobe , in females surviving for more than 25 weeks of irregular shape . 
at the experiment the pituitaries assumed an ever - increasin first small whitish or sfightly yerowish nodules appeared which were single or multiple . later on the normal shape of the gland became more and more obscured by irregular growths , which compressed but never invaded the brain . the larger the tumour the greater was the tendency for brownish discoloration . these lesions occurred in intact partners independently of whether a.a.f. 
common to many of these elements and independent of the degree of acidophilic granulation was the pecuhar golgi apparatus which in papanicolaou preparations appeared as a ring - like structure , the negative golgi fig . 
17 , shows that the majority of the cells composing this structure were degranulated with only a few acidophils remaining . in the more advanced adenomata of the pituitary more atypical cens appeared but fundamentary they did not differ from the earlier lesions . in our material we have rarely seen adenomata completely free of acidophils . 
to conclude , we consider the nodular structures to be adenomata derived from cells of the acidophil series . tumour8of other organ8ob8erved in intact partner8and in " 8ingle " rat8of group viii treated with a.a.f. in the rats of group viii three adenocarcinomata of the breast and one squamous ketatinizing epithelioma of the extemal auditory meatus were found ( table iii )  . 
two of the cancers appeared after an interval of 51 to 52 weeks , only one of the i.e. , 9 weeks after the pair surviving longest had been sacrificed . intact partners treated with a.a.f. 
developed tumours of the breast ( rat 6 , group ii )  . in the 35th week of the experiment a mass was felt in the region of it was surgically removed , and was found to be the fifth left mammary gland . a well - encapsulated tumour measuring 20 x 12 x 9 mon cutting , milky histologically it was a fibroadenoma , in which fluid escaped under pressure . the epithelial elements were much more numerous than is common in such tumours except when occurring in pregnant or lactating females . 
the other was a small adenocareinoma of the breast which showed far less secretory activity than the fibroadenoma which had developed simultaneously . benign cystic cholangiomata were present in practically all rats treated with a.a.f. , but in the intact partners they were generally larger and more widespread than in the " single " animals , in which they were limited to a few cysts in lobus caudatus and left lobe . 
we are unable to state why such changes occurred only in isolated instances . another feature in which the spayed parabiont differed from a " single ovariectomized female was the cytology of the suprarenals . 
24 is included for comin animals killed after 15 to 25 weeks of parabiosis . it shows the distribution of lipoids in the cortex of the suprarenal of a parison rat spayed 6 months previously . 
a second experimental method ofobtaining such neoplasms was discovered by biskind and biskind ( 1944 ) , who obtained such tumours by transplanting ovaries into the spleen of gonadectomized rats . 
the gonads of the intact partners were composed of cystic follicles - proof of increased secretion of fouiclestimulating hormone ( f.s.h. ) by the pituitary of the irradiated parabiont . the liver of rodents inactivates ovarian steroids so efficiently that the secretions of a gonad grafted into the spleen do not enter the general circulation . in consequence the pituitaries of animals bearing such grafts are of the castrate type , and have an elevated gonadotropin content , as shown by greep and jones ( 1950 )  . fels ( 1949 ) discovered that a temporary hgature of the blood vessels supplying the ovaries led to an endocrine imbalance characterized by hyperoestrinism in the presence of castration changes in the pituitary . it seems therefore that one factor is common to at least 3 of the 4 methods quoted above ; they induce an endocrine imbalance characterized by pituitaries secreting elevated amounts of gonadotropins . it is therefore not astonishing that the neoplastic changes induced under these experimental conditions are all of the same kind . nearly all growths so obtained belong to the group of granulosa - theca - lutein cell tumours . 
moon , simpson , li and evans ( 1950 ) also regard the neoplastic changes observed in the ovaries of rats treated with growth hormone to be due to a pituitary imbalance . these authors mention a reduction of the acidophils and an increase of the chromophobes in the pituitaries of such rats , and noted that in 2 animals the histological picture of the anterior pituitary was of the castration type . it seems therefore that the basophils and especiary the f.s.h. secreting cells can increase in animals treated with growth hormone . however , more information is required before one can assume that ovarian .eoplasms obtained by treatment with growth hormone are due to the same mechanism as the other experimental tumours of the ovary , i.e. , to an excess of gonadotropins . in our case there can be no doubt that tumour development in the female gonad was the result of increased stimulation by gonadotropins and of the carcinogenic action of a.a.f. it is especially the foricle - stimulating hormone secreted in elevated amounts by the gonadectomized partner which determines the endoadmittedly during the first weeks of parabiosis there is good crine situation . evidence for the action of luteinizin hormone , as indicated b the presence of many healthy corpora lutea in the ovary of the intact litter - mate . later , when the increased secretion of oestrogens from the stimulated ovary has suppressed all the gonadotrophic activity of the intact partner 's pituitary and continuous oestrus has become established , the pituitary hormone dominating the picture is f.s.h. it is therefore not astonishing that granulosa cell tumours are so frequent in our material and that luteinization occurred to only a hmited extent . 
4 , 6 and 7 of their paper resemble closely the early lesions observed by us , and which we have seen to progress to tumours of considerable size . it seems unfortunate that drips and ford ( 1932 ) compared the atypical structures observed - by them in the irradiated ovaries of rats with carcinomatous ovarian cystadenomata of the 342 f . 
and luteinizing hormone ( l.h. ) in the ovarian cycle is greatly disturbed in the parabiotic female joined to a gonadectomized litter - mate . after 3 to 4 months of parabiosis the ripe follicle is not any more transformed into a corpus luteum nor does it involute , but is exposed to continued stimulation , which does not cease after the death of the ovuthis is in our opinion the decisive factor which leads to the development of the granulosa cell tumours . contrary to what is seen in spleen graftecl or irradiated ovaries of mice , in the rat there is not the shghtest evidence that these tumours " arise from proliferation and ingrowth of germinal epithehum " , as assumed by li and gardner ( 1947 )  . we have observed in some glands smar tufts of cells growing out from the surface ' germina of the ovary , but we have never seen tubular ingrowths from origin from embryonic nests seems a most unlikely hypothesis , epithehum "  . since many workers obtained ovarian tumours in 50 to 70 per cent of their experimental animals ( kaplan , 1950 ; furth and sobel , 1948 )  . . 
undoubtedly the stroma cell of the ovary can differentiate into theca or granulosa cells , and therefore one cannot still , we prefer origin from differentiated granulosa exclude this possibility . cers because of the morphology of the early lesions observed by us . 
12 and 13 one has not to fall back on the conception of disturbed embryogenesis . in preovulatory follicles invaginations of the theca accompanying tufts of capifaries are a common feature . the chorion epithelioma - like tumour presented some diagnostic difficulties . we consider it to be an anaplastic carcinoma . 
as pointed out by willis ( 1947 ) , anaplastic carcinoma can simulate chorion epithehoma and therefore more than morphological similarity is required to establish the diagnosis , in the absence of pregnancy . 
the lack of teratomatous elements and the lack of signs of functional activity such as corpus luteum cysts in the unaffected right ovary are in favour of the diagnosis of anaplastic carcinoma . 
was given simultaneously with or previously to the treatment with goitrogen . spontaneous ovarian tumours and especially granulosa cell tumours are exceedingly rare in rats ( iglesias , stemberg and segaloff , 1950 ) , and therefore do not complicate the interpretation of the experiments reported in this paper . finary an explanation has to be given why the incidence of granulosa cell tumours in group i is lower than in groups ii and iii . 
and most probably also our surgicaltechnique . group i represents our first parabiotic pairs , in which we did not succeed as well as later on to obtain wide junctions , measuring 6 to 10 cat the time the animals were sacrificed . the study of a number of organs of the intact partner showed clearly the effects of oestrogens secreted in excessive amounts by the stimulated ovaries . one of the sequels of this hyperoestrinism were the pituitary adenomata which occurred whether or not a.a.f. 
we beheve , however , that originary these benign neoplasms were acidophilic , just as in zondek 's case ( 1940 )  . this author observed an adenomatous lesion composed of acidophils in the pituitary of a woman treated with very high doses of oestradiol . whereas the increased secretion of oestrogens by the stimulated ovaries led to neoplastic changes in the pituitary , no mammary tumours were observed in the control pairs , contrary to the findings of zeckwer ( 1944 ) , and only one of the intact parabionts treated with a.a.f. 
developed tumours of the breast gland . the material at our disposal does not arow the conclusion that the high level of oestrogen in the circulation of the intact partner inhibited the carcinogenic action of a.a.f. 
in intact female rats joined in parabiosis to gonadectomized litter - mates . ovarian tumours developed in about 50 per cent of these animals . ( 2 ) a description of the histology of the ovarian neoplasms has been given and their histogenesis discussed . it has been found that most of them were of the granulosa cell type . 
secreted in excess by the pituitary of the gonadectomized litter - mate is regarded to be the factor essential for their development . ( 3 ) the pituitary tumouis observed in intact parabiotic females are considered to be due to the excess of oestrogens secreted by their stimulated ovaries . 
the gonadectomized partners were always found to be free of neoplastic lesions , whether or not their litter - mates received a.a.f. ( 5 ) the state of hyperoestrinism present in the intact parabiont did not enhance the carcinogenic action of a.a.f. 
were administered to " single " female rats two mammary cancers appeared during the first 40 weeks of the experiment , a period which corresponds to the maximum survival of our parabiotic pairs . ( 6 ) benign cystic cholangiomata were found in nearly all rats treated with in the intact parabionts they were more numerous and larger than in a.a.f. the " single " rats . we wish to thank professor r . 
hamer. from the cancer research laboratories , department of pathology , the medical school , birmingham 15 . received for publication january 13 , 1953 . results for the analysis of thymus histone presented from this laboratory ( hamer , 1950 , 1951 ) have been followed by similar results from other sources ( daly , mirsky and ris , 1951 ; eadie and leaf , 1952 )  . these results all show the same general characteristics in composition but the actual values obtained vary appreciably from worker to worker , suggesting that either the methods of isolation or the source of the material may be affecting the final product . analysis of other nucleo - proteins have been reported by brunish , fairley and luck ( 1951 ) , liver histone , and by khouvine and baron ( 1951 ) , rat epithelioma histone . the last - mentioned paper evidence was presented that different treatments during isolation could yield products of varying composition . 
stedman and stedman ( 1951 ) have also reported qualitative and partial quantitative analyses of a number of basic proteins from isolated nuclei . in the work reported here further analyses of thymus histone are presented along with other results for the non - histone or acid - insoluble protein of the nucleus . all the fractions examined were derived from the same batch of thymus nuclei , and consequently it was possible to study whether the methods of fractionation used caused changes in the amino - acid compositions . 
of defatted calf thymus by the following method : the tissue was homogenised in a blendor , suspended in a total of 2 litres of saline citric acid solution ( 0.14 m nacl , 0 * 025 m citric acid ) , and then centrifuged , discarding the supernatant . 
the sedimented material was washed by alternate suspension and centrifugation in another saline citric acid solution ( 0.14 m nacl , 0 * 01 m citric acid ) , six times in all , the preparation of isolated nuclei was then free of whole cells and had no appreciable cytoplasmic the nitrogen to phosphorus ratio of such a preparation was contamination . 4 - 5 : 1 . 
next a tenth of the volume of a 5 per cent solution of sodium dodecyl sulphate ( sulphonated lauryl alcohol - glover ) in 45 per cent alcohol was added and the ph brought to 5 - 5 . 
the residue from the acid extraction was washed two further times with 0 - 2 m hydrochloric acid , and the remaining insoluble fibrous material was suspended in water , dialysed for 48 hours and freeze - dried ( fraction nr )  . the remaining half of the solution of nuclei in 1 - 2 m saline was shaken with an equal volume of chloroform - butyl alcohol ( 4 : 1 ) to dissociate the nucleic acid and the protethe nucleic acid remains in the aqueous phase ( from which it may be precipitated with alcohol ) , while the protein forms a gel at the interface . this treatment was repeated twice , combining the gel layers obtained . 
the gel was washed three times with the buffered 1 - 2 m saline , agitating and centrifuging each time , and then the protein was thrown down by adding chilled alcohol . 
the precipitate was extracted overnight with 01 m hydrochloric acid , and the acid extract containing histone was worked up as above to give fraction sh , the second histone specimen . 
the residue was washed further with acid and then suspended in water , dialysed and freeze - dried ( fraction sr )  . these four main fractions and the " whole protein " ( dp ) were then analysed . there was insufficient material in fraction r to permit a full analysis , but it is hoped to study this further at a later stage . 
extract with hci whole protein complex ( dp ) nucleic acid histone ( nh ) residue ( nr ) insoluble residue ( r ) shake with chc13 - buoh protein nucleic acid fig . 
the specimens had slightly lower nitrogen contents than those previously examined and contained about 02 per cent phosphorus . the specimen sr of non - histone or acid - insoluble protein was slightly yellowish , and contained a little less nitrogen and more phosphorus than the histones . was insoluble in water , but would dissolve at ph 9 - 10 to give a pale yellow solution 154 d . 
hamer and then precipitated out when this solution was acidified to about ph 5 - 5 . the course of this treatment some unit in the protein is apparently destroyed as evidenced by the production of a strong unpleasant odour on acidification . 
the related specimen nr contains , of course , much nucleic acid in addition to proestimated from the phosphorus content and the absorption spectrum tein . there is approximately 56 per cent nucleic acid in this fraction . in dilute alkalis an opalescent viscous solution is obtained . finally , specimen dp precipitated from the solution of isolated nuclei by sodium dodecyl sulphate has a nitrogen content of about 9 - 8 per cent indicating that the ratio of protein to detergent is around 3 : 2 by weight . 
the act of separating the non - histone protein from the nucleic acid apparently damages the protein , and it does not seem possible to obtain it in other than an insoluble and denatured forthis would suggest that the non - histone protein may be chemioally bound to the nucleic acid while the histone is held by a predominantly ionic union . 
some support for such a structure has recently been presented by fleming and jordan ( 1952 ) , who have found that the results of eleotrophoretic studies on thymus nucleoprotein in solutions of varying ionic strength could be explained by the existence of an equilibrium np np + p ' ( where np is the original nucleoprotein , np a nucleoprotein fraction with a higher nucleic acid content , and p ' a dissociated protein )  . 
however another possibility is that in the cell the histone is loosely combined with the non - histone protein and that acid treatment breaks the compound open . this would be in accord with the close similarity of amino - acid composition , but would require that all the cystine and tryptophane remain with one fraction on fission . 
the first possibility considered seems on the whole to be more probable , as it is more in keeping with the variation in the amounts of histone and non - histone protein in the nucleus of different cell types ( hamer , 1951 ; stedman and stedman , 1951 ; mirsky and ris , 1951 )  . in the case of the thymus nuclei studied here the histone and non - histone proteins occurred in roughly 156 d . 
kreyberg. from the institutt for generell og eksperimentell patologi , universitetet i oslo . received for publication february 19 , 1952 . in many countries the statistical reports show a steady and remarkable increase in the number of deaths from " lung cancer . " the problem puzzles the cancer specialists and troubles the public and the medical profession . does this increase signify a real augmentation of cases of lung cancer , or does it simply mean an increasingly better diagnostic service ? this question will not be discussed in this paper . if we wish , however , to study , by statistical methods , the possible role of certain irritants in the genesis of lung cancer we have to make clear what we mean by the term " lung cancer . " according to kennaway and kennaway ( 1947 ) their statistics from england and wales comprise : " cancer , carcinoma , or sarcoma of lung , bronchus , pleura , root of lung , hilum of lung , lung and mediastinum , or jung and pleura . 
the norwegian " statistisk sentralbyra " has , since the first of january , 1951 , acted accordingly . it is a great step forward . in order to study a possible relationship between lung carcinoma and smoking habits , or industrial irritants , it may be of importance , not only to exclude all other lung tumours than primary lung carcinomas , but even to subdivide the latter into special sub - groups . from our general experience in the field of cancer research we are ever more impressed by the importance of the study of the geographical distribution of the different manifestations of malignant disease . this different geographical distribution examined on the background of the corresponding difference as to races , primary epithelial . 
this report deals with 100 consecutive cases ofprimary epithelial lung tumourb , received for diagnosis at the institute of pathology , at the university all , but also only those , tumours are included , where the piece of tissue of oslo . o 60 1929 1939 fig . 
1. - mortality statistics from norway , 1929 to 1949 ( " statistisk sentralbyroa " )  . was large enough to permit a definite histological classification . the main part of the material ( 97 cases ) was received from the surgical department a , at the university hospital , 2 cases came from the ear , nose and throat department , and 1 case from the roentgen - radium department . hereto are added 5 cases of secondary lung tumours , received within the same period , and clinically regarded as primary tumours , the histological examination , however , revealing their true character ( 2 cases of metastasizing thyroid adenoma , and 1 case each of hypernephroma , adenocarcinoma of uterus and malignant naevus tumour )  . the 100 tumours of the main series were classified according to traditional terminology as follows : 114 l . 
10 the number and the different types of tumours of our main series may be seen , as well as the occurrence in age - groups and the distribution between the two sexes . 
the individual tumours of groups g and f are tabulated in table ii . the groups e , f and g do not show any marked preponderance in the one further , these tumours are rather evenly distributed in the sex or the other . different age - groups , and they occur even in very young people , our youngest patient being a girl of 19 with a malignant adenoma . 
as to the microscopical classfication , he has : adenocarcinomas 4 - 6 per cent , squamous cell carcinoma 62 - 9 per cent , undifferentiated carcinoma 25 1 per cent and uncertain type 7 - 4 per cent . here again the similarity is greater when we examine the material on the basis of the more objective characteristic the distribution in age - groups than on the basis of the more subjectively influenced classification in histological types . it is evident that the surgical material cannot , without a closer examination , be taken as representative for the occurrence and the distribution of lung carcinomas and lung tumours in the population . firstly , cases occurring in old age 118 l . 
kreyberg will not be represented , as these patients are poor operative risks and seldom secondly , in old age the diagnosis may be more difficult and operated upon . possibly not pursued with the same enthusiasm as when the patient is a younger . it is not unlikely that the local thirdly , the sex distribution may be different . doctor , the surgeon and the patient himself will take a greater risk and show a somewhat more heroic attitude towards the question of operation when the patient is a male . 
the women in these age - groups may possibly show more resigfourthly , surgical material may show a greater nation to the blows of fate . number of benign and moderately malignant tumour types than the sum total . this is significant when the material examined includes all primary epithelial lung tumours , but less important when only true bronchogenic carcinomas are this underlines the importance of a careful classification when statisconcerned . tics are to be compared . 30 ~2021 - 30 31 - 40 41 - 50 51 - 60 61 - 70 71 - 80 81 - 90 age groups fig . 
the occurrence of a certain number of cases in the early age - groups indicates that a few adenomas are included also in this post - mortem series . i wish to emphasize that the present paper represents a preliminary orientation only , as to the occurrence of lung carcinomas in norway . 
mottram ( 1945 ) published the results of experiments designed to demonstrate a relationship between the tumour yield induced by benzpyrene and the number of epidermai mitoses present at the time of application of the carcinogen . painting the skin of rnice at midnight resulted in a higher yield ofpapillomata than did painting at midday . 
but not water , and 1 - 4 hours later they were given a single appheation of carcmogen , benzpyrene ( hoffinan - la roche ) in experiment i and methyleholanthrene ( eastman kodak & co in experiment 2 . in the 1 - - ) enzpyrene experiment each animal received 0 - 1 ml . 
of a 0 - 3 per cent solution in acetone apphed to the interseapular region of the skin , and ' m the methyleholanthrene experiments the dose wasalso 0i ml . 
the mice were starved for a total period of 64 hours , after which they three days after the application of the carcinogen painting were fed ad libitum . with croton oil ( 0 - aper cent in acetone ) was commenced , and this was repeated twice weekl for 20 weeks . 
these mice were treated in the same way as those in the group b , except that the period of starvation was omitted altogether . in experiment 3 , group a contained 25 male mice which were deprived of all food 36 hours before each was painted with 0 - 05 ml . 
most of the papillomata being discovered between the 1 : 21th this is in good agreement with the results obtained bv berenand 15th weeks . blum and shubik ( 1947 )  . 
ludford. from the laboratories of the imperial cancer research fund , london . received for publication january 30 , 1948 . in a review devoted to the discussion of colchicine as a possible chemotherapeutic agent for cancer ( ludford , 1945 ) , attention was directed to the dual action of this drug on tumours . it is the most potent of mitotic poisons , and as . 
at the time the experiment to be described was performed , sections exhibited large compact groups of carcinoma cells , surrounded by an abundant and highly cellular stroma , composed of enlarged and hyperchromatinic spindle - shaped and polymorphic cells , presenting the typical cytological appearance of sarcoma cells . 
an aqueous solution was employed in a concentration of 1 part in 10 , 000 of distilled water . this had been prepared six days previously , when it was maintained at the temperature of boiling water for ten minutes to reduce the possibility of infection , and was stored in the dark for use throughout the exp6risince the activity of colchicine is reduced by high temperatures and by ment . keeping , especially when exposed to light , the solution employed for this experiment was rather less potent than a freshly prepared solution . 
1 by comparing the size of the tumours of the controls and the treated mice on the 21st day . control 71141 211301 37 treated 7 14 3 43 10 01 234 fig . 
2. - regression of a sarcoma ( c ) in strong a strain mice after treatment with colchicine . such after the shortest possible interval consistent with survival of the mice . treatment is invariably accompanied by a high mortality . an experiment with a spindle - celled sarcoma in strong a mice demonstrates the possibility of completely inhibiting malignant growth . this sarcoma was one which originated by the sarcomatous transformation of the stroma of a mice into which it is transplanted do not usually survive mammary carcinoma . long , as it rapidly penetrates the skin and leads to considerable superficial ulceraof six young adults transplanted with this sarcoma three were treated tion . 11 and 20 days after transplantation . 
age. - since actively growing tissues are most sensitive to mitotic poisoning , it is to be expected that young animals would be more susceptible than older ones ries ( 1939 ) reported that a 42 - days - old mouse to the toxic action of the drug . will survive more than 17 times the dose of colchicine which is lethal to one 10 days old . the maximum inhibition of malignant growth occurs with the highest tolerated therefore , to obtain the greatest arrest of tumour growth it is doses of the drug . necessary to employ animals at the age when they are most resistant to the the best results general toxic action and the largest doses can be administered . old mice are less able to tolerate have been obtained with young adult mice . thus , 11 strong a mice varying in age from 18 months to 2 years the drug . and 12 young adults were each injected with 0 , 056 mg . 
3. - action of colchicine upon sarcoma 37 grown in strong a and c 57 strain mice . tumours in c 57 mice indicated by outline , tumours in a mice stippled . 
the size of the tumours in the c 57 mice is indicated it will be observed that in outline , and in the a mice by the stippled areas . actually there is little difference in the size of the tumours in the control animals . growth is slightly more rapid in the a mice , but there is a definite difference in the two strains of treated mice . 
a similar acquirement of tolerance by tumours to the haemorrhage - inducing action of bacterial polysaccharides has been reported by shear ( 1944 )  . application of the preceding results for obtaining the maximum " colchicinic effect . " discrepancies in the reports on the action of colchicine on neoplasms which have been published are explicable on the basis of the preceding findings . 
of colchicine over a period of 4hours . two other tumours regressed completely , but the - , remaining two continued to grow progressively . both received the same treatment on the 37th day . one died with a large tumour on the 45th day . 
the five mice which had been completely " cured " were kept under observation for a year and were then killed . post - mortem examination revealed no trace of carcinomas . the charting of all the tumours in these experiments was carried out independently by a . 
chapman , the senior technician in charge of this work . discusston9 with all the tumours which underwent regression in the preceding experiments there was extensive haemorrhage following treatment . this ' was most readily induced in the a strain mice . 
a few hours after the injection of the maximum sub - lethal doses of the drug the tumours appeared blue through the skin . subsequently superficial ulceration was of common occurrence . since boyland and boyland ( 1937 ) first drew attention to the similarity of action of colchicine and bacterial filtrates they have been shown to behave alike in most other respects . 
with both , complete regression of some transplantable and spontaneous tumours has been effected . ' the results acquire significance in that they indicate the limitation of haemorrhage - inducing agents in the treatment of malignant growths . that some form of damage to the capillary system of tumours might be ' the best way to inhibit malignant growth was suggested by woglom ( 1922 )  . 
a critical review of the evidence concerning tumour regression led him to raise the question whether " the receding tumor may not differ from the growing one only in the extent to which its blood vessels have been obliterated by thrombosis . " he pointed out that the blood vessels of many tumours are more subject to thrombosis than those of normal tissues , and he conceived the possibility of both chemical and mechanical factors being responsible . 
the former resulted from the abnormal metabolism of malignant cells , and the latter was described as " the mechanical pressure which the rapidly increasing parenchyma must often exert upon its vessels , with all the possibilities of injury to their walls which this would entail . " more recently algire ( 1945 ) has devised an adaptation of the transparent chamber technique which enables microscopic observations to be made of the development of the blood vessels of tumour transplants . 
he reports that although many vessels in growing tumours become very large , " differentiation in mammary cancers he observed into arterioles and venules was not evident . " the development of " blood - filled cul - de - sacs . " " these appeared to result from r . 
the usefulness of ultraviolet absorption in studying solutions of methylcholanthrene in different solvents and the physico - qhemical significance of spectroscopic data suggested an approach to the standardization problem . it has become necessary for us to discontinue work on the suspensions , and there is no likelihood of our returning to the matter . 
at the end of the exposure time the paste , which had been kept moist through the 42 - day period , was extracted for 24 hours with 35 ml . 
3. - ultraviolet absorption curves from a " protected " suspension of mnethylcholanthrene after 80 days ' exposure to diffuse davlight . curves a , b , c as in fig . 
4. - ultraviolet absorption curves from an " unprotected " suspension of methylcholanthrene after 80 days ' exposure to diffuse daylight . curves a , b , c as in fig . 
4 , a , the contribution of scatter predominates . it is evident that these solutions differ markedly in the type of suspended material , and this might well result in substantial differences in biological behaviour . in order to arrive at a representative selective absorption curve the scatter component must be subtracted from the observed data . 
5. - ultraviolet absorption curves of a saline extract of ground , moist methylcholanthrene crystals which had been exposed to diffuse daylight 42 days before the extract was prepared . curves a , b , c as in fig . 
the procedure for making these approximations is as follows : from an inspection of the curve plotted from the spectrophotometer data the contribution of scatter and turbidity is estimated and a smooth curve characteristic of scatter and turbidity is drawn to approximate this contribution . 
the ratio of the difference obtained as above and the density value at the same wave - length of a known concentration of the solute dissolved in a solvent in which scatter is absent is determined . 
the ratio so found is used as a multiplying factor which is applied to the density values of the reference curve ( known solute in true solution ) at several wave - lengths . this product is subtracted from the density values at corresponding wave - lengths of the mixed scatter - absorption curve to give a series of density values which are plotted - , and ' a smooth curve is fitted to the points so determined . 
the procedure as outlined gives a family of curves , with each member progressing towards a more accurate estimation and being uniquely related to the known absorption curve of the solute , and a mixed curve which is assumed to be composed of true absorption of the solute and a scatter - turbidity component . such a series of approximations can be made when one knows from other sources the true absorption spectrum of the solute , and can use this information as a standard of reference . the above procedure has been used to determine the scatter curve presented in the figures of this paper . 
5 shows the absorption of th3 extract from the ground methylcholanthrene there is a large scatter contribution , otherwise the curve bears little crystals . resemblance to the curves from the suspensions . 
bielschowsky. from the , hugh adam cancer research department of the medical school and the new zealand branch of the british empire cancer campaign , university of otago , dunedin . received for publication april 7 , 1953 . judging from the literature , adenomata occur more frequently in the pituitaries than in the thyroids of aged rats . 
at the routine autopsies of 154 albinos of the wistar strain , 41 of which were males , 86 intact and 27 spayed females , 24 cases of adenoma of the pituitary were discovered . seven of these occurred in the males , 16 in the intact females and only one in an ovariectomised animal . the average age of these rats was 2 years . 
adenomata of the thyroid were found in 5 animals , 4 times in combination with neoplastic lesions of the pituitary . post - mortem examinations performed on old rats of a more recently acquired hooded strain have furnished three additional cases of tumours of thyroid and pituitary . 
the diet consisted of a dry mixture of bran 30 per cent , pollard 35 per cent , meat meal 15 per cent , maize meal 15 per cent and bone flour 5 per cent , supplemented by wheat and kibbled maize , and once weekly by green vegetables and cod liver oil . drinking water was supplied in liberal amounts . 
the weight of the animals was recorded at fortnightly intervals and they wer6 sacrificed when loss of weight and general appearance indicated ill - health . at autopsy the pituitaries were immediately transferred into a pre - weighed bottle containing about 2 ml . 
when treated with gomori 's fuchsin aldehyde reagent , the cytoplasm of the angular cells and especially their granules stained purplish - violet . in other words , these cells strongly resembled and gave the staining reactions of those basophils which purves and griesbach ( 1951a ) named " thyrotrophs . " therefore the tumour is considered to be a basophil adenoma formed by thyrotrophs . in the part of the anterior lobe outside the basophil adenoma the basophils were considerably increased in numbers . angular types again predominated , but here vacuolated forms , extremely rare within the tumour , were numerous . these cells were identical with those seen after partial thyroidectomy . they gave a positive pas reaction of varying intensity , and with the gomori stain some were found to contain coarse violet - purple granules . the large round gomori 206 f . 
5 , the larger of the two protrudes above the anterior surface and is closely connected with the pars internmedia : the other , just appearing in the section , bridges the pituitary cleft and is situated laterally . 
the pituitary was of irregular shape with a small this was a well defined basophil adenoma , the haemorrhagic brownish area . tumour cells giving the same staining reactions as described above . 
the neoplastic changes found in the hypophysis and thyroid of the younger animal were of microscopic size . in the slightly abnormally shaped anterior lobe numerous thyroidectomy cells and also multiple nodules formed by atypical basophils were present , the pars intermedia being normal . 
normal anterior lobe tissue was recognisable in the periphery as a narrow rim surrounding approximately half of the circumference of the tumour . radiating from this rim compressed strands of predominantly acidophilic and chromophobic elements were seen between areas of tumour , an indication that the neoplasm had arisen from different foci . intermediate and posterior lobes appeared normal . 
the cells composing the tumour were large , showed great variation in size and shape , had sharply defined cell borders and vesicular nuclei with one or more large fuchsinophilic nucleoll . multi - nucleated and tumour giant cells were rather numerous and so were mitoses . 
when stained by the pas technique or with gomori 's aldehyde fuchsin after long search an occasional only in this respect , cell was found having pas or gomori positive granules . did the adenoma differ from those described above . in papanicolaou preparations the cytoplasm stained faintly with light green . the histology of the thyroid was that of an inactive gland . 
many mitoses were found in this nodule , considered to be a benign pheochromocytoma . rat 8 , a 26 months old male , was killed because of the presence of an abscess otherwise the animal was in an excellent in the subcutaneous tissue of the neck . state of health and weighed 460 g . 
the first question to be answered is : which cells produce the thyrotrophic hormone and the second , how many types of basophils occur in the anterior lobe of the hypophysis . 
as to the second , romeis ( 1940 ) described two types of basophils both stainable with aniline blue but distinguishable by their affinity to creso - fuschin or resorcinol - fuschthe type stained by these fuchsin dyes is the beta cell , the other is the delta cell of romeis . 
as shown by halmi ( 1950 ) another elastica stain , gomori 's fuchsin aldehyde reagent , also differentiates between two types of basophils , whereas most other techniques like pearse ( 1952a ) does not papanicolaou 's or gram 's method stain all basophils . seem to attach great significance to these different tinctorial qualities and adheres to a unitarian view . 
now the pathogenesis of the basophil tumours can be discussed . chronic iodine deficiency lowers the thyroxine level in time , to which the pituitary responds with an increase in thyrotrophic cells . this compensatory hyperplasia progresses towards the formation of tumours , in this connection it might be which in some instances reach considerable size . mentioned that proliferating normal thyrotrophs , as observed by guyer and claus ( 1937 ) , tend to aggregate in clusters , a mode of growth which might be favourable to nodular hyperplasia , the first stage in adenoma formation . 
the adenoma of this animal was functionally inactive as judged by the appearance of the thyroid which was that of a resting gland . also cells giving a positive reaction for glycoprotein were virtually absent in this tumour . the most convincing evidence for funttional activity is provided by rat 4 . the large pituitary tumour of this animal contained numerous pas and gomori positive elements and its thyroid showed the most pronounced signs of stimulation . in addition , in the pituitary of this animal , so little normal anterior lobe tissue was left that it seems most unlikely that this small rest could have secreted amounts of thyrotrophin large enough to induce a four - five fold enlargement of the thyroid . finally the pituitary lesion of rat 8 has to be considered . in this case the morphological evidence was more in favour of hyperplasia than of neoplasia . here an agglomeration of thyrotrophs had persisted in one area despite the fact that ki had been supplied for 7 months . in the rest of the anterior lobe these cells were found in normal numbers , as was to be expected , and the thyroid had undergone involution . rich glycoprotein content of the cells forming this aggregation was probably due to storage and not a sign of secretory activity . the anomalous behaviour of these cells suggests that they were no longer normal thyrotrophs . the thyroid tumours observed are , in the writer 's opinion , due to stimulation by elevated amounts of thyrotrophic hormone . the stimulus for the increased secretion by the pituitary was a deficiency of thyroxine due to impaired synthesis by an inadequate content of iodine in the diet . in six instances the thyroid tumours were found in activated and twice in resting glands . the latter occurred in animals which had received a supplement of ki for several months and are considered to be evidence of past stimulation . these neoplasms differed morphologically from those observed in stimulated thyroids in the same way as the thyroid adenomata found during administration of thiouracil differ from those seen after the treatment with the goitrogen has ceased . 
their epithelium was rather low , and cystic follicles filled with colloid were a prominent feature . to summarise the pathogenesis and structure of the thyroid adenomata observed is similar to that of the tumours induced by goitrogens which act by blocking the synthesis of thyroxine . the conception that thyroxine deficiency is the ultimate cause of the syndrome described is strengthened by the following observations . fischer ( 1926 ) , writing from bern , switzerland recorded two cases of pituitary tumours found in aged female rats . 
the thyroids of both animals contained multiple tubular adenomata , and in one an adenocarcinoma was also present in this gland . the rats belonged to wegelin 's colony in which goitre was endemic fischer 's paper was published long before the gonadotrophic and thyrotrophic hormones had been discovered . she stressed , however the similarity of the pituitary tumour cells with the ele212 f . 
bielschowsky ments which appear in the rat 's hypophyis after castration or thyroidectomy ; and considered the neoplasms to be carcinomata formed by " castration " cells fischer believed that although the ovaries of one of her animals were normal . the neoplasms originated from the pars intermedia or tuberalis . " spontaneous " tumour formation occurring simultaneously in thyroid and pituitary of rodents seems to have been observed only by fischer and by the writer , but one sees this syndrome under experimental conditions most frequently in animals t ' reated with so purves and griesbach ( 1951a ) mention goitrogens for prolonged periods . a basophil adenoma of a rat treated with methythiouracil for two years . basophil adenomata occur also in thyroidectomised rats treated with acetylaminofluorene into the same class belong also the pituitary tumours ( unpublished results )  . discovered by gorbman ( 1950 )  . 
they appear in mice exposed to high doses of radioactive iodine , but can be prevented by the administration of thyroxine ( goldberg and chaikoff , 1951 ; gorbman , 1952 )  . furth , gadsden and upton ( 1952 ) reported that transplants of such tumours contain large amounts of thyrotrophic hormone . 
of tissue was homogenised in a total volume of 50 ml . , more sucrose being added later to dilute the homogenate to this , as was shown by direct comparison , a final concentration of 10 per cent . had no effect on subsequent fractionation . it was found in preliminary experiments that maximal liberation of cytoplasmic proteins into the supernatant fluid was achieved by homogenisation lasting for 1 to 2 minutes . the separation of the cell components was carried out by means of differential centrifugation at a temperature of 20 to 40 c . 
in a " spinco " ( specialised instruments corporation , belmont , california ) model e ultracentrifuge , except for the preliminary removal of nuclei , unbroken cells , debris , etc . , which was carried out in an ordinary low - speed centrifuge . 
the centrifugal procedure with some modifications was based on those , originally described by claude ( 1943 , 1946 ) and developed by hogeboom , claude , and hotchkiss ( 1946 ) , hogeboom , schneider , and pallade ( 1948 ) , schneider ( 1946b )  . in some of the later experiments it was based on that of schneider and hogeboom ( 1950a )  . 
stickland oxidase activity of each fraction was determined by the method described by umbreit , burris and stauffer ( 1945 ) and the results expressed as q02 ( n )  . 
a concentration of the enzyme in this fraction was also found in normal lactating breast tissue of highand low - cancer - strain mice . while the microsomal fractions of rat and mouse liver showed only a small amount of the enzyme , the same fractions of breast tissue revealed an appreciable quantity of it . in order to establish that this activity was a true property of these fractions , on some occasions the activity was determined after washing and was found to be unaltered . these results expressed as specific activity of the enzyme in the respective fractions are shown in table iii . 
hogeboom and schneider ( 1950 ) observed partial inactivation of succinoxidase after sonic disintegration of mitochondria . the possibility of contamination with mitochondria could not be excluded by histological examination alone . mitochondria were invariably observed in smears of microsome fractions , but in considerably smaller numbers than in the mitochondrial fractions : it is , however , impossible to make such comparisons quantitative . 
the chief arguments against contamination with whole mitochondria are first , that in rat liver no succinoxidase was found in the microsomes ( or negligible quantity ) , and second , that in a number of experiments with mammary tumour tissues the same mitochondria were sedimented in two parallel ways , at 5000 times gravity and at 8000 times gravity , and in the latter case the activity of the microsomes subsequently obtained was only slightly lower than in the former . the possibility of contamination of microsome fractions by fragments of broken mitochondria cannot easily be disposed of . clearly such contamination was not observed in liver microsome fractions , obtained in similar manner as those from normal and malignant mammary tissues . 
the results showed clearly that mitochondria from normal and malignant mammary cells of mice are not more fragile than those from liver , and if anything rather less . it can therefore be deduced that mammary cell mitochondria are not appreciably fragmented during homogenisation , and that consequently the enzymic activity present in the microsome fractions from these cells is likely to be a true property of microsomes present in these fractions . 
the ready fragmentability of liver - cell mitochondria in the potter - elvehjen homogeniser after they have been separated from other cell components is at a first glance not easily reconciled with the fact that no damage appears to occur to these cellular components during the homogenisation of the tissue . this difference is presumably due to the protection of the mitochondria against friction and damage by the presence in the latter case of larger cell fragments . the enzyme activity of microsomal fractions of normal and malignant breast tissues remained practically unaltered in spite of washings in isotonic sucrose 258 l . 
stickland solution , which again points against the possibility of the enzyme being adsorbed on microsomes . from the experiments of price , miller and miller ( 1948 ) , price , miller , miller and weber ( 1949a , 1949b ) on rat hepatoma and of schneider and hogeboom ( 1950b ) on mouse hepatoma , it is now known that in both rat and mouse hepatoma , the protein content of the mitochondrial and microsomal fractions is much lower than that in similar fractions of normal rat and mouse liver . further , it has been shown in these studies that in rat and mouse hepatoma the succinoxidase content of the mitochondrial fraction is lower than that in normal rat and mouse liver , the total activity being 5 times lower and the specific activity 2 times lower , in spite of the same distribution of the enzyme in normal and malignant livers . in the present experiments no significant difference was observed between normal and malignant mammary cells , either in the total amount of succinoxidase or in its distribution between the various fractions of these cells . 
a curious point , so far unexplained , is that although in liver homogenates 100 per cent recovery of succinoxidase could be obtained in the fractions , in mammary tissues the recovery was much lower , amounting on the average to only 70 per cent . no difference was found between highand low - cancer - strain mice either in the protein and succinoxidase content or in the distribution of these in the various fractions , but the mammary tumour tissue in each case showed a reduced content of mitochondria , compared with the normal mammary tissue , which agrees with that found by schneider and hogeboom ( 1950b ) in mouse hepatomas . the reliability of the simplified centrifugal procedure used in the majority of the experiments has been tested by the use on three occasions of the more complicated procedure of schneider and hogeboom ( 1950a ) , which gave substanit may perhaps be of interest to mention that stern tially the same results . ( 1939 ) demonstrated the association of succinoxidase with particles of microsome size in beef heart muscle . a small point worthy of mention is that for greater convenience of making the enzyme assays , the samples of washed particles from rat and mouse liver and mammary tissues were kept overnight at - 79 c . 
korteweg. victorieplein 45 , awkrdam , netherlawi8 received for publication december 8 , 1953 . round about the end of the last war the total number of deaths from cancer recorded in the mortality statistics of the central bureau of statistics for the netherlands showed a conspicuous decrease . in females the decrease amounted to 6 per cent , in males to 9 per cent approximately . in the different age - groups it was about the same . this statistical decrease was very pronounced in lung cancer in males ( table i )  . in this paper the forowing tumours are expressed in terms of headings of the detailed intemational list of causes of death : " lung cancer " : no . 
231 ( revision of ( the figures for deaths from lung cancer in females are too small for a 1948 )  . statistical analysis and will be left out of consideration in this paper )  . 
with cancer of most other organs the decrease was less than with lung cancer , and in breast cancer and in some other cancers there was no decrease at all . in norway , according to kreyberg ( quoted by doll , 1953 ) , there was a similar dip in the mortahty curve for lung cancer in the same years , whereas in england and wales this curve continued without any interruption ( don , 1953 )  . in my report for the symposium on the endemiology of lung cancer in louvain , in 1952 ( korteweg , 1953 ) , i left the question undecided , whether the statistical decrease of mortahty from lung cancer resulted from mis - diagnoses due to war circumstances - non - functioning of the x - ray apparatuses for instance - or whether indeed in these years fewer people died from this disease . according to the central bureau of statistics , during the hunger winter of 1945 1784 males and 609 females died from starvation in amsterdam , a town of about in the first weeks after the liberation the number of persons 800 , 000 inhabitants . suffering from hunger oedema amounted to many tens of thousands . 
he pointed out heading 57 of the detailed intemational list of causes of death , revision of 1938 , under which ar tumours of undetermined nature - those tumours of which it was not reported whether they were mahgnant in one of its subgroups the figure for the tumours of undeor not - are recorded . termined nature of the respiratory organs is given . it appeared that in the same years that the decrease in lung cancer was greatest , those undetermined tumours of the respiratory organs , a very small group under normal circumstances , had strikingly increased . evidently in this group many cases of primary lung cancer were hidden . 
the length of the ordinate for a given age - group , therefore , gives the percentage pertaining to that age - group of the sum of the lengths of the ordinates of all age - groups together . unbroken line : lung cancer . broken line : tumours of undeterxnined nature of the respiratory organs . dotted line : cancer all sites . the vertical dashes at the top of the graph give the averages for the periods in question , i.e. 
the increase of lung cancer mortality in the last 40 years points to an increase of the sum total of cancer promoting influences . the older people of to - day hved a great part of their lives in a period when the menace from these influences was less than it is at present . 
as contrasted with the situation with younger people , therefore , mortality from lung cancer in the higher age - groups does not correspond to the sum total of cancer - promoting influences of to - day , but to this sum in an earlier period . it can be calculated that an inclusion in the group of undetermined tumours of the respiratory organs of even a small number of metastatic cancers possessing the normal age curve would have substantially altered the age curve proper to lung cancer . 
2 gives the crude death rates in males per million living in each year for this combination of recorded and probable ( though not as such recorded ) lung cancer deaths in the netherlands . ' 14t l\ i - i - - i 1938 - i . 
korteweg from the corrected curve . ( d ) the concavity in the curve for the years 1938 to 1941 has disappeared during discussions with officials of the central bureau of statistics on the reliability of the data on which the statistics of the causes of death are based , facts appeared which threw a new hght on our problem . in the netherlands the physician has to fill in two forms for each death . 
one in small vfllages where everyof these goes to the local civil registrar , a layman . body knows everybody the secrecy of this form is not absolutely guaranteed , and this leads , in cases of death from , for instance , venereal disease or cancer , to many physicians reporting meaningless diagnoses , such as heart failure . itnder normal circumstances the official netherlands statistics of causes of death are based on the second forms , which find their way , in a sealed envelope , to the medical official of the central bureau of statistics in the hague . the diagnosis mentioned on this form is incomplete , the medical official makes inquiries of the physician responsible for it as to the complete diagnosis and eventually corrects it . 
as the secrecy of these forms is ensured , there is no reason whatsoever for reserve . in 1944 and 1945 - when there was a general railway strike and other means of transport were extremely scarce and undependable and in 1946 , there was a lack of opportunities for making inquiries about incompletely fired in - forms . this reduces the rehability of the statistics for cancer for these years . 
the statistical decrease of cancer of the cervix uteri mentioned above , as well as the greater part of the statistical decrease of deaths from lung cancer find their explanation in this fact . the situation was by far the worst in 1945 , when the war front cut the netherlands in two . 
the mortality statistics for that year are based exclusively on the diagnoses given on the forms which were sent to the local civil registrars . it is clear that this was detrimental to the netherlands statistics for 1945 , and that to search for other explanations for the big statistical decrease of deaths from lung cancer in 1945 would be a mere waste of time . the only part of the dip for which no explanation can be given is the decrease this is the only indication that of i 0 per cent of lung cancer deaths in 1947 . the low level of tobacco consumption or the severe malnutrition in the foregoing years might have somewhat influenced mortahty from lung cancer . this decrease is rather small , however , and in 1947 eving conditions in the netherlands had not yet quite retumed to normal , so that it would not be wise to attach much importance to it . the cause of the great differences in the numbers of deaths recorded from undetermined tumours of the respiratory organs between the years before and after 1941 could not be discovered . 
maybe changes in the staff or the working methods of the central bureau of statistics or changing over from the revision of 1929 to the revision of 1938 could account for it . 
the straightening of the curve for the crude deathrrates for the years 1938 to 1941 , after correctio ' n for the undetermined tumours has been made , seems an argument in favour of the supposition that most of these tumours were in fact true lung cancers . 
1. received for publication january 21 , 1948 . surgery in the form of the so - called radical operation is in general at the present time the principal weapon in the treatment of carcinoma of the breast . what it can achieve and its limitations are now fairly well established . 
thus it is known that if a patient with carcinoma of the breast has microscopical invasion of the axillary glands , the strong probabilities are that the disease has passed beyond the local area and that the patient will sooner or later succumb it is to the disease , although there will be a minority of fortunate exceptions . also known that if the axillary glands are not invaded the chances that the patient will be cured of the disease are appreciably increased . 
the present article attempts to assess the comparative results of a personal series of cases in which various techniques of treatment were used . the technical surgical problem in carcinoma of the breast has been rendered clearer by modem work on lymphatic anatomy , and in particular by that of gray ( 1939 )  . this has shown , firstly , that the dermis is a plane rich in lvmphatics and hence a rich potential plane of spread of carcinoma , particularly if the growth has spread into or near the skin ; and secondly , that the deep fascia is a plane devoid of or very poor in lymphatics , and hence not an important potential plane of spread . the former fact suggests greater radicalism in removal of skin , and the latter removes the main pathological reason for the routine sacrifice of the pectoralis major muscle . accordingly , since the appearance of gray 's work we have modified our technique to remove more skin , in our more recent cases skin grafting in a high proportion , and we have gradually abandoned routine sacrifice of the pectoralis major , only removing it if actually invaded , which is rare and late . the argument is sometimes used that the removal of the pectoralis major is necessary for the adequate dissection of the axilla , and that in any case its nerve supply is bound to be cut . these technical arguments are with elevation of the arm , retraction of the pectoralis major and not valid . removal of the pectoralis minor it is easy to do a complete clearance of the axillary glands and fatty tissue right up to the apex of the axilla , and at the same time to preserve the lateral pectoral nerve , the main nerve supply to the pectoralis apart from this modified radical operation there are smaller series of major . cases which have been treated in other ways , e.g. 
the comparison between the figures of standard radical and the modified radical operation in this series is rendered more difficult in that the modified radical was evolved out of the standard radical , and hence has not been done for so long . 
survivals it would therefore seem fair to conclude that these figures bring no evidence to support the view that the addition of the removal of the pectoralis major muscle brings any increase in the survival figures , and to raise the question whether the routine removal of this muscle should remain an essential technical point . a further way in which the two operations can be compared is by the comparative incidence of recurrence in the operative area , i.e. 
skin , chest wall and out of 42 traced standard radical operations there were 10 cases with axilla . of these , 7 were recurrence in the operative area , chiefly skin recurrences . cases with original lymphatic glandular involvement and 3 without such involveout of 40 traced modified radical operations there were 7 local recurment . of these , 5 were cases with original lymphatic glandular involvement rences . while the incidence in both is too high , and and 2 without such involvement . approximates to the 22 per cent noted by truscott in the middlesex hospital series , the standard radical shows no lower incidence of local recurrence than the modified . the 3 other types of procedure which have been carried out were : ( 1 ) in the first place there were 10 cases in which partial mastectomy was combined with the usual full axillary dissection after the removal of the pectoralis minor . ( 2 ) secondly , there were 9 cases in which a simple removal of the breast was combined with irradiation to the axilla . ( 3 ) thirdly , there were 7 cases in which irradiation only of the breast was combined with axillary dissection . the last group comprised cases which were all advanced , the glands being invaded in all . 
one case died as a result of the operation ; the other 6 died of the disease at intervals varying from 6 months to 4 years after treatment . moreover , in a high proportion of cases - 4 out of 6 cases which survived operationthere was either failure of the irradiation to get rid of the original primary growth in the breast , or recurrence of the growth locally after apparent disappearance . in view of the advanced character of these cases , the ultimate fatal result was likely whatever form of treatment was adopted . 
of the 2 cases with invaded glands , 1 was alive with the disease at 5 years and 1 died of other causes at 2 of the 5 cases with glands not invaded , 3 were alive and well at 10 years , years . 9 years , and 5 years , 1 was alive with the disease at 8 years , and 1 had died of other causes 1 year after operation . there were also 3 cases in which no histological note was made of the condition of the axillary glands ; of these , 1 was alive and well at 6 years and 2 died of the disease . 
from the point of view of survival after operation , this group compares favourably with other groups . but the procedure has been abandoned because of the occasional further development of carcinoma in the portion of the breast remaining . this happened in 2 of the cases of this series , and it also happened in other similar cases in a private series , in spite of the fact that , as already noted , only small and apparently early cases were considered suitable for this procedure , that a wide area of breast tissue around the growth was removed , and that post - operative irradiation was it was concluded , therefore , that while in an occasional case given as a routine . partial mastectomy combined with a dissection of the axilla might be justifiable a conclusion supporting that of fitzwilliams ( 1940 ) and of mitchener , bailey and price ( 1937 ) - the danger of further development of carcinoma in the remaining breast tissue rendered the procedure an unwise routine . the last group was of 9 cases in which simple removal of the breast was the only surgery performed , the axilla being treated by irradiation . these cases wvere usually elderly patients with apparently early growths . 
the main routine removal is not necessary ? advantages of preserving the pectoralis major are three . firstly , there is the cosmetic consideration , mutilation being much less with the pectoralis major preserved as compared with the skeleton - like prominence of ribs and costal cartilages after - its removal . 
and if there is no curative advantage in removal of the muscle , cosmetic considerations , although secondary , are entitled to a place . secondly , there is less loss of blood if the muscle is not removed , and consequently less operative shock . while in most cases this does not amount to a point of much practical importance , in old or feeble patients it may . with preservation of the pectoralis major combined surgical treatment of the breast and axillary contents is , in our experience , much less upsetting than simple mastectomy combined with irradiation to the axilla , and we have as already stated for this reason largely abandoned the latter procedure . 
1. received for publication january 13 , 1948 . one of the implications underlying the work of huggins and hodges ( 1941 ) on the factors governing prostatic hypertrophy is that malignant prostatic cells are not fully , if at all , autonomous . that is , they are still controlled in some measure by the same factors that control normal prostatic cells . specifically , this is certainly true of their susceptibility to the influence of circulating androgens and oestrogens . carcinoma of the prostate is essentially a disease of old age . the possibility exists that the chief factor in deciding the individual 's susceptibility to this disease may be his androgenic state , that is , the level of circulating androgens in his blood , particularly during the later decades of life . the only practical method for ascertaining androgenic state is a determination of 17 - ketosteroids excreted in the urine of the subject . in the hands of many different workers ( barnett , henly , morris and warren , 1946 ) this method has revealed a very wide range of excretion of 17 - ketosteroids by normal men , at least in the lower age - groups . 
the hvbrid females of each litter , when four to six weeks of age . were mark - ed indi - vidually and di ' %ided into experimental and control n - lice . 
shown bv irregular movements of the indicator between 3 ' and these bursts last for a few seconds , and are separated bv shghtlv longer after the indicator has re - ached zero . 
grow and form large embrvomas which persist for months or indefinitely . the following series of experiments was , undertaken to gain knowledge of the beha - viour of such embrvo grafts under various conditions . 
the teratomas consisted of a me ' lange of normal tissues arranged - in an irregular manner skin , cartilage , bone , around cysts containing hair , skin and various secretions . marrow , muscle , fat , cubical and columnar epithelium , lymphoid tissue , blood vessels , glandular structures and pigment were all found . 
no necrotic patches were present , nor was there evidence of any reaction on the part of the host . in order to discover the best time to transplant such a primary teratoma for passaging mice were killed once a week , beginning a week after inoculation , the ' teratoma removed , lightly minced with scissors and a small portion ( approximately 0 - 03 - 0 - 05 c.c. ) iiioculated with a grafting needle into the right flank of in this generation increase in six cm mice . size of the graft occurred in 66 per cent of transplants done before the 4th week , in 33 per cent or less in those between the 4th and the 8th week , while after the 10th week practically no increase in size took place , the small masses either persisting in practically the same state as when implanted , or actually disappearing . several of those passaged at the first and second weeks were again divided and a portion transplanted . these only increased to between 12 x 6 mand 4 x 4 this is the 2ageneration . 
the e of 8toring findy min - ed embryo tiww in glycerol at 40 c . - the embryo tissue was passed through the craigie pressure mi ioer and then stored in ampoules at 4 ' c . 
extremely active material capable of s " rdng fivsh t clours in less than two weeks has been obtained from both chemically induced and spontaneous sarcom firozen at - 790 c . 
1. received for publication february 6 , 1947 . attempts to find some reliable basis for prognosis in carcinoma of the breast several factors have been thought valuable in have proved disappointing . formulating a prognosis , chief among which have been the presence or absence of secondary deposits in the axillary lymph nodes and the histological character but it has been found that , even in cases with no axillary involveof the growth . ment and carcinomata of low histological malignancy , there has been a mortality from recurrence of about 25 per cent in the first five years after operation . the axillary lymphatics were the only path by which carcinoma cells could escape from the breast , this mortality would be inexplicable . 
a biopsy specimen from one case in the first case the internal mammary gland was removed was also used . post - mortem in 1938 from a patient who died eleven days after a radical mastectomy . 
the internal mammary vessels also lie in this fat in a plane posterior to the glands and usually about half an inch lateral to the edge of the sternuthe glands may be either medial or lateral to the vessels . 
a thin but strong sheet of fascia , the costosternal fascia , intervenes between the glands and vessels and the pleura in the upper two spaces , and the transversus thoracis muscle in the lower spaces . 
the obliquely running the external intercostal membrane and the internal intercostal muscle are reflected as a somewhat ragged flap , either from lateral to medial and hinged on the inner end of the intercostal space : or from medial to lateral and hinged an inch lateral to the edge of the sternuwe prefer the ' former method , since bleeding from the perforating branch of the internal mammary artery seems easier fibres of the intercostal musculature are so loosely bound together that this flap cannot be neatly sutured back into once the intercostal flap has been reflected , position at the end of the operation . dissection should proceed with non - toothed dissecting forceps only . 
they might give rise to troublesome haemorrhage , but this should easily be controllable by packing while the main trunk was being ligated in the spaces above and below . closure of the intercostal space is done as neatly as possible but is seldom complete . 
cushing , my house - surgeon , has in three instances cleared out the anterior mediastinum on one side for recurrent cancer . it is very likely , i think , that we shall , in the near future , remove the mediastinal contents at some of our primary operations . " this idea does not seem to have been followed up . 
sampson handley ( 1922 ) explored the anterior mediastinum in six cases of carcinoma of the breast , finding glands in two , both of which were the seat of metastasis . 
he described in detail a case in which recurrences ' appeared in the inner ends of the intercostal spaces , one after another from above downwards , in a patient who finally succumbed to the disease twelve years after a radical mastectomy ; and he stated his belief that , by the time the axillary glands were enlarged , the internal mammary glands had frequently , and perhaps usually , been invaded . scarff and liandley ( 1938 ) investigated the ten - year results of radical mastectomy in 172 cases of carcinoma of the breast . 
they found that patients without evidence of axillary deposits ( the so - called stage 1 cases ) had a mortality of 25 per cent from recurrence in the first five years after operation , and that this mortality continued at about the same rate between the 5th and 10th years . 
the treatment of these glands must be by some form of radiotherapy . there seems much to commend sampson handley 's method of burying radium tubes in the intercostal spaces at the time of operation , thus securing a localized but intense irradiation of those points where intensity is most needed . it remains to discover whether , in a large series of cases , invasion of the internal mammary glands is as frequent as it would appear to be , what influence the site of a carcinoma in the breast has on internal mammary deposits , and what is the best method of treatment . 
microscopic examination of this gland might greatly increase the accuracy of prognosis , and prove of considerable assistance in post - operative treatment by irradiation . our thanks are due to the director , the bland sutton institute , the middlesex hospital , for permission to use the pathological reports ; to professor r . 
the frankly keratinizing members of our series we have taken as a standard with which to compare the behaviour of our remaining histological types . the morphological features of the more differentiated epitheliomata have been so completely evaluated that any further discussion would be superfluous . 
the mincer is shaken immediately to disperse the mince if the coarse plunger has been used the cap and operating in the suspending fluid . screw are detached and the fine plunger is inserted . tlle cap and screw are replaced and mincing is completmed . the suspension may contain coarse fragments of fibrous tissue or portions of iiiince which have clotted before dispersal in the suspending fluid . these may be removed either by l ' ow speed centrifugation , or by filtering the suspension througli a column of glass ballotini of suitable diameter . the mincer , which is illustrated in fig . 
1 , is the sixth of a series desigiied to all models have proved most satisfactory for operate with grooved plungers . the purpose for which they are intended , and it might be suggested that this tvpe of mincer will , for special purposes , be suitable for preparing suspensions of manimalian tissue or fowl embryos infected with certain viruses or bacteria . i wish toacknowledge my indebtedness to w . 
it is to ' be expected on theoretical grounds that temperatures below the fi - eezing points of the eutectic mixtures of the inorganic salts would favour survival of activitv . 
an equallv pending tumour mince in drying experiments . important reason for the use of dexlrose suspending fluid . tissue mince suspended in saline or ringer solution and frozen m a thin laver progressivelv consequentlv the dr , % - mg process becomes separates from glass during drying . greatlv impeded because of the low rate at which thermai energy is conducted across the intervening vacuuwhile dried fragments mav break off and mav be 22 ' c . 
the thermal conductivity of the dried material is adequate for the rapid drying of thin layers . water is removed from the tumour suspension by the low temperature con79 ' c . 
presents no difficulty , but there densation method . are technical problems in maintaining the drying tissue at a constant temperaif the lead from drying flask to condenser is of sufficient diameter ture level . and the vacuum is adequate , the problem is not one of cooling , but of supplying the maximum of thermal energy for rapid evaporation without heating the frozen it was considered undesirable mass above the chosen maximum t - i.imperature. to attempt to control the temperature of the drying mass on the basis of thermocouple readings . there is no guarantee that a thermocouple junction can be placed at that point in the frozen mass which will be the last to dry . 
the arm of the u - tube distal to the pump is extended horizontally and then downwards , to form the exit tube from the condenser flask . this exit tube is furnished with a ground glass taper which fits the neck of the flask . 
the stirrer rod is attached bv means of a slotted clamp which faeihtates assemblv with the thermos bath and drying flask . the rubber ring which drives the disc if the variable speed gear is mounted on a wheel which can be moved along the horizontal bar . this bar is mounted on two spring - loaded arms . 
a pliotographically reduced protractor scale is cemented to a piece of perspex 2 - 5 x 13 x 65 mand the pivot is centred at the origin of this scale . the vane is a piece of photographic dry mounting tissue 3 x 8 mm . , which is attached by shellac to a piece of fine copper wire 0 - 01 min diameter . 
the combined weight of vane and wire is 3 - 7 mg . , so that the vertical component of its weight at a displacement of v from its zero position is 0 - 065 mg . ( 4 ) vibration dampers . - owing to the design of the glass - drying system and the use of a sensitive vapour flow indicator , it is necessary to check the slight vibration arising from the electric motor driving the pump . 
the distribution of the frozen rnince is readily controlled if the level of ethyl cellosolve in the freezing bath is adjusted so that the flask will float with its neck resting on the rim of the thermos jar and can therefore be spun quickly by hand . the temperature of the freezing bath may it has not yet been determined whether a preliminary 30 ' c . 
shown bv irregular movements of the indicator between 3 ' and these bursts last for a few seconds , and are separated bv shghtlv longer after the indicator has re - ached zero . 
grow and form large embrvomas which persist for months or indefinitely . the following series of experiments was , undertaken to gain knowledge of the beha - viour of such embrvo grafts under various conditions . 
philps. from the department of clinical pathology , university college hospital , london , w.c.1. received for publication july 2 , 1954 . the examination of sputum for carcinoma cells can only be considered a diagnostic method of clinical value if false positive diagnoses be avoided and cases wrongly thought suggestive of carcinoma reduced to a minimuthe method cannot be used to exclude carcinoma as it is evident that malignant cells may not be found if they are present only in small numbers , or in the case of an early it must be accepted , therefore , that growth the sputum may contain none . 20 to 25 per cent of bronchial carcinomata will be missed by this method and the problem is to be sure that a positive report is as reliable as possible . 
1 , 2 and 3 contained a considerable amount of necrotic epithelial tissue , and in one small group of cells , two mitotic figures , one of which is shown in fig . 
4 , 5 , 6 and 7 . both cases the appearance suggested a clump of carcinoma cells but the ciliated border could in places be clearly seen , making the true nature of the cells evident . bronchial epithelial cells sometimes show vacuolation of the cytoplasm and may then give a disorderly appearance with may lead to confusion with carcinoma cells , , such an appearance is illustrated in fig . 
 - on close inspection of this clump , it was clear that the individual cells did not form part a fragment of tissue as there welre spaces between them , and they had more cytoplasm than do oat cells . 
the cells bear a strong resemblance to oat cells but can be distinguished from them because the cytoplasm is relatively abundant and the nuclei of adjacent cells show no tendency to moulld together . 
13. - part of the fragment under higher magnification . the pattern of the cells resembles that seen in oat cell carcinoma , adjacent nuclei showing a tendency to mould together . there is more cytoplasm separating the nuclei than is commonly seen in oat cells , and the chromatin pattern is nowhere visible whereas in oat cells it is usually clearly seen . this appearance was reported as being suggestive of carcinoma . 
17. - a corpus amylaceum enclosed within a cell which appears to be of epithelial origin . the nucleus of the enclosing cell was at the periphery of the enclosed body but is not well shown in the photograph . the corpus amylaceum was semitransparent and was devoid of any nuclear characteristics . 
the nuclei of the individual cells mould together rather as do those of oat cells , giving the whole clump an appearance resembling oat cell carcinoma . all the nuclei however , are dense and the cells possess more cytoplasm than is usually seen in oat cells . 
the specimen was reported as follows : " films show clumps of cells , the arrangement of which is suggestive of oat cell carcinoma though the individual cells are not sufficiently characteristic to indicate malignancy . " the diagnosis of bronchial adenoma was made at bronchoscopy and confirmed at post mortem nine months later following the patient 's death from coronary thrombosis . ( e ) appearances due to macrophages of unusual type . macrophages , when they are seen to contain dust particles , can be easily identified , but when devoid of such particles , they may sometimes be confused with carcinoma cells . 
the nuclei are central , multinucleate cells frequently occur in carcinoma but they and number about 12 . are in no way diagnostic of it , the commonest multinucleate cell seen in sputum films being the macrophage . this example is the largest that i have seen . 
 two recent investigations , both from the united states , upon the arsenic content of tobacco , and the volatilization of this arsenic when tobacco is smoked , have been reported . 
the work of these authors differs from our own in that they used artificial methods of combusting the tobacco , which may or may not repro duce the conditions present when smoking is carried out in the ordinary way , and they found no brands of tobacco containing only minimal quantities of arsenic . 
 ( 1 ) gross and nelson ( 1934 ) employed a method which consisted of digestion in nitric and sulphuric acids , precipitation of magnesium ammonium arsenate , solution in hydrochloric acid , and estimation by the gutzeit method . 
five such analytical groups were analysed for each brand studied . " they found the content of pooled groups of 4 cigarettes of 5 brands to range from 97 to 363 p.p. 
 ( 2 ) thomas and collier ( 1945 ) , using a modified cassil - wichmann method , found that " individual cigarettes and cigars of a given brand varied widely in arsenic content , " which ranged in pools of 2 to 4 cigarettes from 2 packs of 20 ( presumably of the same brand ) from 354 to 114 p.p.m. , while cigars ( 132 to 295 ) and pipe tobaccos ( 227 to 428 ) showed a lower range ( table i )  . 
gross and nelson ( 1934 ) attribute the rather narrower range found in cigarettes and in pipe tobacco , compared with cigars , to the mixing which the former receive , but this difference is not seen in the results for cigarettes and cigars of thom.as and collier ( 1945 )  . 
in american pipe and chewing tobaccos ; using suction by a water - pump , he concluded that " we can say roughly that half of the arsenic is evolved in the smoke gross and nelson ( 1934 ) used an apparatus drawing about 50 ml . 
 each : 207to256 211 , , 267 ' 253 , , 363 97 , , 130 229 , , 261 range in 5 single cigars from each of 5 brands : 229 241 332 116 247 83 to 484 range in 5samples from each of 4 brands : 260 to 500 116 to 266 311 to 451 1 - 5 1 - 4 thomas and collier : cigarettes " a single pack of 20 " " ali.other pack " 1 - 4 " groups of 2 to 4 . " 354 ; 400 ; 405 ; 472 ; 600 ; 61 - 4 * 614 ; 670 ; 714 ; 845 ; 1140 132 ; 225 ; 262 ; 295 cigars pipe tobacco cigars ( about 10%cutoffat ends ) pipe tobacco 1 - 5 396 ; 265 ; 227 ; 345 ; 428 * these figures represent the actual analyses in p.p.m. , on pooled groups of 2 to 4 cigarettes , and hence are not calculated means , though each figure represents the mean composition of the eigarettes in one pool . 
cigars and cigarettes were " smoked down to about 1 / 2 to 3 / 4 " ; the arsenic the " puffed " and " unpuffed " smoke was absorbed and estimated separately . 
 " the pipe smoker who smokes all the tobacco does not therefore have the protection afforded by the butts of cigars and cigarettes . " from the present point of view this question is not of great interest ; the important datum is the amount of arsenic inhaled , and in this country only about one smoker in ten smokes a pipe ( hansard , 1945 )  . 
in their first series ( table ii ) cigarettes of 7 c length were smoked down to 1 c ; in a later serjes only 2 / 3 was burned . 
 the arsenic found was compared with analyses of whole cigarettes from the same pack , " but in view of the large differences they later cut off about os c for analysis ; apparently they failed to discover that this method also can be falla cious . 
 our earlier results ( table iv ) were obtained by the gutzeit method , which depends upon the brown and yellow colours produced in paper impregnated with hgc12 ; it is fitted for the measurement of amounts of as20 3 between 10 and 1 g . , and shows differences most clearly in the range of 8 to 3 g . 
there may be con~ siderable differences in the matches made by different persons ; in these experi ments the mean was taken of the readings made independently by two persons unaware of the nature of the unknowns . 
the iodometric method of thomas and collier ( 1945 ) was adopted later ( tables iii , v and vi ) in view of the difficulty of determining the small amounts of arsenic lost in smoking . 
the method has the advantage that , whatever may be its errors in other respects , there is less disagreement over the very sharp end - point ( titration of iodine and starch ) , about which two observers usually do not differ by more than 005 c.c. 
 the estimations upon brands p and c were usually carried out upon amounts of 20 to 30 g . , as a method which is sufficiently accurate within this range 176 m . 
gross ( 1933 ) has drawn attention to the occurrence of such errors in the estimation of arsenic in tobacco , which are attributed to uncombusted residues of pyridine and nicotine ; we failed to obtain satisfactory results by the method which he devised to overcome this difficulty . 
 in the previous investigations summarized above artificial methods of smoking were employed , air being drawn through a pipe , cigar or cigarette by means of a water - pump . 
we have confined our experiments wholly to cigarettes smoked by one or other of three persons accustomed to this form of smoking , and they were asked to do this in their ordinary way ; the two brands used ( p and c ) are in common use in this country . 
the ash , and stump , were dropped into separate weighing bottles in our earlier experiments , but latterly the two have been analysed together ; the amount of arsenic found , if less than that in the cigarette , should indicate the amount volatilized in the smoke , of which a part must have been inspired . 
 before this width of range was realized we obtained many contradictory results , showing amounts in the ash and stump which were sometimes less , and sometimes more , than that thought to be present originally . 
 8 whole cigarettes 9 cigarettes ( 7 wholes and 2 halves ) 5 pieces from 3 cigarettes 1 piece from each of 4 cigarettes 6 successive pieces cut from 1 cigarette 13 estimations on mixed tobacco of 10 cigarettes e 10 range in 5 packages of brand p brand o . 
 678 823 307 496 461 275 10 pieces from 5 cigarettes 5 successive pieces cut from 1 cigarette a 20 a 20 11_ estimations on mixed tobacco of io cigarettes . 
but as the weight of most cigarettes does not differ very much from one gram the figures give a rough measure of the arsenic in one cigarette , which is of more obvious practical interest . 
the results were again contradictory , and we found that such suc cessive portions may show a range of 388 to 587 , or 100 : 151 in p , and of 306 to 542 , or 100 : 177 in c ( table iv )  . 
 in the hope of obtaining more uniform material , the papers were removed from the cigarettes of a package of 10 or 20 , and the tobacco mixed by hand and 178 m . 
but even this treatment does not give satisfactory results ; it is very difficult to take up samples of such material which will contain constant propor tions of the coarser and finer particles , as the latter tend to fall away from the . 
moreovar , the minced tobacco , even when moistened , is not easy to smoke in the same way as ordinary tobacco : adjacent cigarettes of brand p from a much larger package , containing 100 , were taken ; but these at first showed a wide range ( 495 to 766 , table iv )  . 
 that an average of 158 per cent of the arsenic is lost in the process of smoking and had presumably been volatilized ; this loss is statistically significant ( p = 0001 )  . 
the same method was applied to brand c , of which the largest boxes available contained 50 ; the first two rows showed in 18 cigarettes a loss of only 76 per cent , which was not statistically significant ( p = 02 )  . 
 580 527 623 558 603 418 456 458 568 556 617 } 681 mean 596 631 427 412 471 533 629 662 453 367 527 634 455 367 539 509 lower right row . 
kennaway the 4th row showed a loss of 182 per cent , which is just significant ( p = 005 ) , in spite of the small numbers ( 12 cigarettes ) involved . 
 the most likely source of the arsenic present in some tobaccos , which has been suggested by several authors , is the spraying of the adult plants with insecticides , and this particulate method of application would explain the difficulty in obtaining concordant analyses . 
 of the arsenic volatilized in smoking , a part must escape while the cigarette is not in the smoker 's mouth , and a part of what he inspires is expired again , hence the amount retained is no doubt very small . 
the concentration of arsenic in cigarettes containing the larger amounts is very \rariable ; this irregularity is perhaps due to a method by which the arsenic may have been introduced , namely , by the spraying of the plants with insecticides . 
an estimate was made of the amount of arsenic volatilized in smoking , by comparing the content of cigarettes , and of the ash and stumps derived from them , but the wide range of arsenic content makes it difficult to infer the amount present originally in a cigarette which is to be smoked . 
the results indicated that from 76 to 18 2 per cent of the arsenic present was lost in smoking ; this figure is of the same order as but somewhat lower than that obtained by some previous investigators who have used artificial methods of smoking . 
salaman. from the cancer research department , london hospital medical college , london , e.l. received for publication march 15 , 1952 . if a chemical carcinogen is applied to the skin of mice at a concentration just too low to produce tumours by itself and , after an interval , a co - carcinogen is repeatedly applied to the same site , tumours . 
weekly applications of 2 * 5 per cent croton oil in paraffin to both groups were begun 6 weeks after an initial treatment with 0 - 15 per cent dmba . 
croton oil treatment of group 5 was stopped after 8 applications , when the average tumour incidence in both groups was about 0 - 5 per mouse . incidence in the two groups increased for a further 2 to 3 weeks , after which incidence in group 4 continued to rise , following the curve usual in this type of experiment , while that in group 5 remained almost constant at about 2 tumours per mouse . after 9 weeks without treatment there had been no appreciable change in tumour number in this group , though the tumours already visible had continued to grow . 
tumour incidence in group 5 had , by then , reached about 11 tumours per mouse ; it continued to rise for a short time , as before , then fell a little , and again remained approximately . 
1. - comparison of the effects of continuous and intermittent weekly applications of croton oil to mouse skin , 6 weeks after a single application of a carcinogen . consequently individual points in fig . 
1 , which represent by others , is high . average numbers of tumours per mouse , have high standard errors , and the differences between corresponding points on the curves of groups , 4 and 5 are barely significant . it is easy to show , however , that the curves as a whole differ their slopes between the 15th and 21st week were compared as significantly . follows : the average increments .of tumour number of individual . 
one must suppose that there is a critical size , rather less than 1 mdiameter ; i.e. , that tumours smaller than this do not continue to grow without further stimulation . did these invisible tumours remain unchanged during the remission of treatment ? apparently not , for when the treatment was resumed there was no immediate outburst of newly - visible tumours . after 58 days the interrupted rise in numbers this latent period is not significantly different from that of the continued . response to the first course of croton oil treatment . 
the skin behaved as it would have done if it had had no previous co - carcinogenic stimulation . it is possible to conclude that the change produced by croton oil in mouse skin previously treated with a carcinogen is not sudden , or independently proit is a gradual change , requring repeated applications for its maingressive . tenance and progress , which is reversible during the greater part of its course , but becomes irreversible not long before tumours become visible . it has been previously noted ( shubik , 1950a , 1950b ; salaman and gwynn , 1951 ) that of the tumours produced in mouse skin by croton oil following a single treatment with a carcinogen most are benign , though some reveal or develop a malignant character later . 
the difference between these two latent periods is not significant . ( 3 ) these results are discussed . it is concluded that the change produced by croton oil in mouse skin previously treated with a carcinogen is a gradual one , requiring repeated treatments for progression towards tumour formation , that it is reversible during the greater part of its course , but becomes irreversible not long before tumours become visible . i am indebted to professor s . 
on the 4th day the males were removed from the boxes and 39 females which had shown plugs were placed in separate boxes , these constiforty - three unmated female mice of similar tuting the experimental group . average age , and wherever possible litter mates of the experimental mice , made the control group . on the day of separation , each of the mice received a subcutaneous graft , roughly 2 mx 2 mm . , in the right flank , from a 25 - day - old transplantable methylcholanthrene - induced spindle - cell sarcoma growing in cba mice . this tumour retained the original histological appearance and characters of steady and uniform growth through successive generations , and was chosen because its rapid growth was considered an advantage in view of the shortness of the pregnancy and lactation periods in mice . from the 10th day following grafting the tumours were measured every 4th or 5th day in 2 diameters ( length and breadth ) until the end of lactation , i.e. until 19 days following parturition . 
1 by curves obtained by plotting the average of the sums of 2 diameters ( i + b ) against time , as described by haddow ( 1938 )  . because of the difficulty in obtaining a large number of pregnant mice at one time , the experiment had to be performed on 6 batches of mice , 25 - day - old tumours of successive generations being used for grafting . 
leveaux. * from the westminster hospital , london , s.w.1. received for publication may 12 , 1954 . the earliest recorded description of carcinoma of the pancreas is attributed to mondiere in 1836 . 
da costa ( 1858 ) of philadelphia reviewed 37 cases and gave a comprehensive description of the clinical presentation of this disease with * nmphasis on pain as a prominent sympton in 32 of these patients . 
in not a few cases it is increased by the erect position and hence we find patients seeking relief by stooping and curving their body forward so as to relax the abdominal parietes . " physicians of the french school sustained their interest in this condition , and bard and pic in 1888 observed the common association of a palpable gall bladder with jaundice , and the frequent incidence of cachexia . 
from the descriptions given by these and other observers the concept of painless jaundice as the classical form of presentation of this disease spread widely . mirallie ( 1893 ) found glycosuria in 3 cases , and chauffard ( 1908 ) emphasized distinctive features of growths involving the body of the gland . speed ( 1920 ) , eusterman ( 1922 ) , eusterman and wilbur ( 1933 ) , kiefer ( 1927 ) and other observers have subsequently reported on series of cases , and berk ( 1941 ) has included these in his review of a total of 1449 cases . 
levy and lichtman ( 1940 ) , and duff ( 1939 ) have further clarified the picture of carcinoma of the body and tail of the pancreas , emphasizing early and severe pain , ascites , venous thrombosis , and widespread metastases to liver and peritoneum as important features . towards the end of the last century surgeons became interested in the pancreas , and finney ( 1910 ) quotes a case of total pancreatectomy for carcinoma by billroth in 1884 with recovery of the patient from the operation . gordon - taylor ( 1934 ) successfully removed a carcinoma of the body of the pancreas in 1927 , his patient surviving when the case was reported 7 years later . in spite of such isolated successes , however , attempts at surgical removal gave little promise until , in 1935 , whipple and his colleagues demonstrated the technical feasibility of resection of the head of the gland , and opened a more hopeful era ( whipple , parsons and mullins , 1935 ; whipple , 1938 )  . these and subsequent advances in surgery have greatly intensified the need for early diagnosis in a disease which has been estimated to comprise 1 to 2 per cent of all cancers , and which is by no means a clinical rarity . 
the head was spared in 14 cases ( 30.4 per cent ) , of which 4 cases involved the body alone , 4 cases the tail alone , and 6 cases both regions . 
one case had active thrombo - phlebitis , and 1 presented with paraplegia due to extradural metastases . in many cases the disease was well - advanced by the time that abnormal signs were clear cut . 
per cent in 20 of 40 cases ( 50 per of 8 cases examined by the glucose tolerance test , a diabetic type curve cent )  . was obtained in 6 cases ( 75 per cent )  . these latter cases included 2 of the known cases of diabetes of recent onset , but none of long standing . 
n / 20 sodium hydroxide as the upper limit of normality . johnson and bockus ( 1940 ) reported a raised value in 5 of 8 cases . secretin stimulation of the pancreas was performed in 3 cases , accentuating an already raised level in 2 cases , and producing an abnormal value in 1 . 
per cent in 27 of 33 cases investigated , of which 24 had clinical jaundice . the serum alkaline phosphatase was examined in 26 cases and was raised above 5 bodansky units in 13 , of which all had associated hyperbilirubinaemia . of 13 cases shown to have liver metastases , the alkaline phosphatase was raised in 7 and normal in 6 . the serum cholesterol was raised above 220 mg . 
a normal chloesterol level occurred in 5 cases with associated hyperbilirubinaemia . the cholesterolesters exceeded 50 per cent of the total cholesterol in 20 of 21 cases . it appears , therefore , that these latter three investigations reflect only the presence of biliary obstruction , and beyond this are not helpful in the present diagnostic problem . the plasma proteins were normal in 23 of 25 cases , there being some degree of hypoalbuminaemia in the remaining 2 cases . the cephalin , thymol and colloidal gold flocculation and turbidity tests were normal in all of 22 cases examined . in 3 cases the cephalin flocculations test became positive after cholecysto - enterostomy . the value of these tests in the present connection is that they facilitate exclusion of hepatitis in cases presenting with jaundice , as pointed out by maclagan ( 1947 ) of 46 cases examined , 31 ( 67 - 4 per cent ) had haemoglobin levels below 13 - 0 g . per cent , with an average of 12.13 ( 76 per cent hb . ) for the whole series . 
a relationship between the anatomical position of the tumour and the site of the pain complained of by the patient is suggested . a scheme of investigation is proposed which includes careful radiological studies , a glucose tolerance test , serum lipase and amylase determinations and a bromsulphalein retention test . surgical exploration is advocated where strong clinical grounds alone exist . i wish to express my warm thanks to h . 
bockus , professor of gastroenterology in the graduate hospital of the university of pennsylvania , for all his help and encouragement in this investigation . i am also grateful to a . 
3 received for publication january 11 , 1951 . it has long been known that the rous sarcoma may be transmitted by dried tumour tissue and it has been concluded , therefore , that the tumour agent can survive under such conditions ( rous and murphy , 1914 ; rous , 1911 ; and hoffstadt and tripi , 1946 )  . 
the ampoules were then connected to a manifold and the ice sublimed in vacuo . knox 's ( 1939 ) apparatus and conditions were later used by dmochowski ( 1948 ) for the freeze - drying of rous v - irus suspensions produced by fractional centrifugation and other methods . 
dmochowski ( 1948 ) claimed that similar virus suspensions treated with a drop of fowl or rabbit serum suffered no loss of activity under the same conditions , but that addition of salts , such as nacl , kc1 or caci2 , had no such preservative action . 
by gentle shaking for 35 minutes . if the shaking was carried out in the absence of gas space , albeit with glass beads in the tube , inactivation was negligible . 
gave complete protection for t5 phage for 14 minutes . the duration of the protective effect was found to be a function of the gelatin concentration , since the gelatin itself protection could also be obtained with gum arabic or with is " denatured . " serum albumin although 100 - fold and 10 - fold the gelatin concentration were required . this work may well provide the theory behind the observation that viruses are more stable when diluted in serum or broth than when the dilutions are made equally one might expect that small viruses would be with salt or water . inactivated more rapidly than larger , and that the rate of inactivation would be proportional to temperature , since the forces responsible for bringing the particles to an interface will be thermal forces . such conditions as these have frequently been realized empirically . rivers and vvard ( 1935 ) sought to add a material to vaccinia virus suspension which would ( a ) act as a protective agent , ( b ) add bulk to the final product and ( c ) go back into solution with ease , carrying the virus with it . 
this suspension was clarified on the sharples centrifuge and the virus deposited from the supernatant on to a cellophane bowl lining sheet by deposition at high speed ( carr and harris , 1951 )  . this virus - containing deposit was then resuspended in a ' volume of medium ( concentration medium ) equal v / w to the original weight of tumour tissue . ( 2 ) comentration media . ( a ) buffer - trypsin , b - t . - mcilvaine 's ( 1921 ) phosphate - citric acid buffer ( plff 7 - 5 to 8 - 0 ) prepared from 0 - 2 m na2hp104and 0 - 1 m citric acid and diluted 1 : 19 with water ( buffer ) and a few mg . 
the mccartney bottles were accommodated in drihed compartments in the centrifuge head of the drying chamber of the machine . each bottle was inclined towards the axis of rotation , so that when the head was spinning the contents of each bottle formed a wedge . the virus suspensions were snap - frozen by evaporative cooling , and the purpose for spinning the bottle was 2 - fold . ( a ) frothing was prevented and ( b ) the wedge of frozen material presented a greatly increased surface area with a consequent increase in the rate of drying . 
the rate of drying was high throughout the drying cycle and no evidence was found to suggest that , as the layer of dried virus material increased in thickness , the rate of evaporation of vapour fen off and the underlying frozen mass melted ( bauer and pickels , 1940 )  . the drying cycle occupied 40 to 44 hours . 
the bottles , which were either uncapped , or lightly capped with two or three layers of sterile surgical gauze during the drying , were then screw - capped and sealed with tape and stored at 2 ' c . 
the media used , and here abbreviated , included : ( a ) buffered - 8aline , b - s . - ten per cent sahne adjusted to ph 7 - 2 with 0 - 2 m sodium phosphate . ( b ) buffer , ph 5 - o . - mcilvaine 's phosphate - citric acid , diluted 1 : 19 with water . ( e ) water - trypsin , w - t . - - solution containing a few mg . 
a similar result appears from table vii if the result of 29jr.49 is compared with that of 5.vii.49 , and from table viii , 15ar.49 being compared with 29.iii.49 ; although activation was not found in the experiments with egg albumin or egg white . freeze - drying in salt solutions ( table ii )  . the sharples deposits were resuspended throughout in , 0 - 005 m phosphate buffer , ph 7 - 2 , containing a few mg . 
50 , where lemco is compared with io per cent lemco as a medium for freeze - drying , there is a reduction in the titre with the weaker broth . freeze - drying in carbohydrate media ( table vii )  . the sharples deposits were resuspended in buffer - trypsin and " purified " in the usual way . 
or 0 - 125 per cent neutrahzed cysteine hydrochloride gave protection , and the protection afforded by 2 - 5 per cent dextrose containing 0 - 05 per cent b.a.l. 
the method now adopted , that of freeze - drying virus concentrates in lemeo broth , fulfils the condition , but little hght has been thrown on the mode of action of the stabihzing material . virus concentrates dried in this way have retained their fur activity for minimum periods of 12 months . the experiments with water alone showed that dmochowski 's ( 1948 ) results are confirmed in so far as virus may be recovered after drying - and our preparations had all been treated with 1 : 10 , 000 hcn during the isolation procedurebut it was not possible under these conditions to recover as much as 60 to 80 per cent of the initial virus activity as he claimed . salt solutions had no preservative action , and broth - containing media showed immediate advantages , even where the suspensions had been " purified " by centrifugal fractionation before desiccation . 
the action of the broth appears to be ponfined to the freezing - and - drying cycle or subsequently , since nothing was gained by carrying out the concentration stages in broth or broth - containing media , for which buffer - trypsin is quite satisfactory . it is unhkely , therefore , that the effect is solely an example of the phenomenon described by adams ( 1948 )  . 
the conditions of freeze - drying , moreover , do not involve vigorous shaking of the material but , equally , there is as yet ' no knowledge of the surface conditions which exist at an ice surface which is gradually receding through a drying mass . 
the observation that weaker ( 10 , per cent ) lemco broth is a less effective protective agent is of interest in this respect . it is possible to suppose that the action of broth and similar material is complex , that one or a combination of such factors as the forowing are important : ( a ) the broth pro - vides optimum conditions during freezing - such as an optimum eutectic point ; ( b ) that protection is conferred against the deleterious effect of ' heavy metals or of oxidation systems ; ( c ) that , in some way , the tendenoy of the virus to aggregate is reduced ; or ( d ) that an optimum amount of water only is lost . 
bartholomew 's hospital , london . received for publication may 15 , 1951 the data given below have been collected in continuation of two earlier studies ( kennaway and kennaway , 1936 , 1947 )  . stocks ( 1936 , 1947 ) has compared the death rates from cancer of the lung in males during the years 1921 to 1930 in london , and in the county boroughs , other urban districts , and rural districts of england and wales , and has shown that the rate increased with increasing urban conditions . * the same method has now been applied to the data , for both sexes ( table ii ) , of a later period ( 1946 to 1949 ) , when a great increase in deaths attributed to cancer of the lung has taken place . 
as recently as 1929 the numbers of cancers of the larynx , and of the lung , in males , were about equal ( table i ) ; since then , deaths attributed to the latter have increased enormously . 
the crude death rate for males has been falling in recent years ( 1946 to 1949 ) and in view of the ageing population this indicates an actual decrease in the incidence ; the death rate for females has been remarkably constant since these considerations show that the pooling of data for the larynx and 1935 . lung under any such heading as " respiratory system " is very undesirable . the standardized death rates show no perceptible difference in either sex between the figures for the 14 years 1926 to 1939 immediately preceding the war , and the since 1942 standardized death rates have been means for the period 1921 to 1930 . superseded by comparative mortality indices , which compare the death rate in each year with that of 1938 . . 
22 - 5 = 1 - 0 100 ' 0 82 - 2 17 - 8 100.0 the data at present available on the social incidence of cancer of the larynx show a gradient which is steep in men and absent in women ( table vi )  . 
hence the increased consumption of tobacco in recent years has not affected this form of cancer . ( 5 ) thus cancer of the larynx in men , and in women , differs in geographical , anatomical and social distribution , and in changing prevalence at the present time , and should be regarded as constituting two separate diseases . ( 6 ) the sexual distribution of intrinsic and extrinsic cancer of the larynx appears to be changing . we are greatly indebted to w . 
phillips , of the general register office , for a large part of the material contained in this paper , for help in the preparation of it , and for advice upon many matters . 
campbelt. poultry research centre at the university of edinburgh . * received for publication december 30 , 1948 . the original purpose of this investigation was to study the effects of implanting various tumnours affecting the fowl into the fertile egg . a survey of the literature revealed the rather surprising fact that apart from the rous i sarcoma ( rous and murphy , 1911 ) , and a few leucosis cases ( pierce , 1942 ) , no attempt has been made to grow spontaneous neoplasms of the chicken in fertile eggs . there is a high incidence , relative to disease in general , of spontaneous neoplasia in the fowl . 
campbell ( 1945 ) , in a survey extending over five years , found the relative average incidence to be 18 ' 7 per cent in the breeds examined , the minimum and maximum figures for specific breeds being 10 - 3 per cent and nearly 39 per cent . during this survey a number of chicken tumours were studied which bore a close morphological resemblance to known virus - associated growths . in view ofclaims by various authors ( taylor , 1943 ; heilman and bittner , 1944 ; hungate , snider , taylor and thompson , 1945 ) that mammalian tumours can be easily cultivated in the yolk - sac of the chick embryo , and that a virus - like factor associated with mammary carcinoma of mice has been directly demonstrated by this method , it was thought desirable to apply similar methods to the study of spontaneous chicken tumours . the advantages of using fertile eggs for such cultivation experiments are many . 
the chick embryo , on the other hand , has not yet been reported to be a natural carrier of virus . it has been shown by grasset ( 1929 ) , who worked with diphtheria and tetanus toxoids as antigens , that the chick embryo does not produce antibody . polk , buddingh and goodpasture ( 1938 ) showed that no complement is present in embryonic serum , and murphy ( 1914 ) first demonstrated the ability of normal heteroplastic tissues and tumours to graft on to the chorioallantois without causing an inflammatory reaction until the 18th day of incubation . subsequently some reaction of the host tissues does occur , to the detriment of the graft . the culture of tumour tissues in fertile eggs therefore resembles much more closely tissue culture in vitro than it does in orthodox animal inoculation methods . at an early stage in this investigation the frequent occurrence of typical ectodermal lesions in the vicinity of chicken tumours implanted on the chorioallantois led to the conception that such tumours might contain a factor capable of stimulating cell division . such a factor , if it existed , might be a self - propagating agent within living malignant cells , or something in the nature of a mitotic activator similar to those growth - stimulating substances present in embryonic tissues . with this hypothesis in mind it was decided to study the effects of implanting embryonic tissues , certain viruses , and as wide a range of tumours as possible , derived from a variety of animals . about this time a series of cases of spontaneous liver carcinoma in ducks came under observation , and similar experiments were undertaken with the resultant material , the details of which are recorded in a separate paper . the first record of tumour cultivation in the fertile egg was by rous and murphy ( 1911 ) , who applied it to the newly discovered rous chicken sarcoma , which they succeeded in cultivating in various sites , including the yolk - sac , and on the chorioallantoic membrane . 
they make no mention of any reaction on the part of the chick embryo or its associated membranes , and their plates only illustrate the histology of the cultivated tumours ; the membranes themselves were apparently not examined . stevenson ( 1918 ) then tried various rat , mouse and guinea - pig tumours and had about 30 per cent takes . 
campbell keogh ( 1938 ) found that filtrates prepared from the rous i sarcoma gave rise to ectodermal proliferative lesions when inoculated on the chorioallantois . these focal lesions were fully developed about the 7th day after inoculation , and took the form of flattened pearly opacities varying in diameter from 0 - 5 mm . to 2 mdilute inocula produced these lesions ; when concentrated suspensions were inoculated a proportion of sarcomatous lesions developed . pierce ( 1942 ) transferred leucosis to chick embryos by seeding fragments of leucotic tissue , e.g. 
no lesions developed on the membrane , and inclusion bodhes were absent when filtrates were used , but leucotic tissue caused gross thickening and cloudiness of the membrane . taylor , thacker and penington ( 1942 ) described the growth of cancer tissue ( mammaryv carcinoma of mice ) in the yolk - sac of the chick embryo , and claimed it to be a new method . 
the presence of necrotic tissue in the tumours resulted in the death of the embryo . in a subsequent communication taylor ( 1943 ) described the cultivation of a spontaneous mammary tumour of dba strain mice in the yolk - sac of the fertile egg . 
the yolk from such eggs produced similar tumours in mice upon injection , and taylor found that berkefeld filtrates prepared from this yolk also gave rise to tumours upon injection into mice , thus showing the presence of a virus - like principle in this particular mammary carcinoma . at about the same time heilman and bittner ( 1944 ) injected a 40 per cent suspension of mouse carcinoma into the yolk - sac of the embryonic chick , using an inoculum of 0 - 2 ml . 
they extracted the tumours from the yolk - sac , and showed that fitrates prepared from these and from the yolk itself were capable of causing mammary cancer in mice , thus confirming the work of hungate , taylor and thompson ( 1944 ) , that a virus - like body was involved . following up this line of investigation bittner , evans and green ( 1945 ) showed that the " milk factor " was able to survive in the yolk - sac for 12 days in the absence of mouse mammary carcinoma cells . 
they did not know whether the factor had multiplied in the yolk - sac . twombly and meisel ( 1946 ) attempted to grow several mammalian tumours in the yolk - sacs of fertile eggs . 
the greatest number of takes occurred with the bagg 755 mouse mammary carcinoma , but they report a very high mortality rate ( 73 per cent ) by the 17th day of incubation in their embryos . 
numerous fitration experiments were done in an attempt to demonstrate a cancer virus , but with uniformly negative results . ( a ) the intra - vitelline techniqe . the method adopted for the cultivation of tumours in the yolk - sac was a modification of that described by taylor et al . 
they were then taken direct to 95 per cent alcohol , through absolute alcohol , equal parts absolute alcohol and cedarwood oil , cedarwood oil alone , and finally embedded in paraffin wax . other tissues were fixed in 10 per cent formol saline for 24 hours . 
the eggs were sealed with their own shell flap , using paraffin wax , and this was removed on the fifth day and a cover - glass substituted . tumours were seen growing in each , dependent from the chorioallantois . 
the eggs were opened 9 days later , and both contained large ( 9 mm . ) spherical , richly vascular tumours growing from the chorioallantois and hanging down into the allantoic cavity . 
of this filtrate was injected into each pectoral muscle of a brown leghorn cockerel aged 14 weeks . typical tumours subsequently grew in these sites . hi / to / og . the only histological findings to be given in any detail will be those associated with changes in the membranes in the vicinity of implanted tumours . 
a delicate argyrophil reticulum ramifies throughout the growth , and appears to be derived from the mesenchyme of the membrane . normally the epithelial surface of the chorioallantois consists of low cuboidal cells , and is only one cell thick . 
the tube was shaken in an automatic shaking machine for 1 hour , at the end of which the suspension was filtered through a " technico " filter , using a ford 's " sterimat " grade g.s. the fitrate was used as the inoculum , the amount inoculated on to each membrane varying between 0 - 02 to 0 - 06 ml . four 10 - day embryonated eggs were inoculated , using a graduated capillary pipette as a dropper . in order to secure a wide distribution of the virus over the membrane , it was placed direct on to the shell membrane , and a tear made through this with a glass needle . 
the virus was then drawn into the egg and on to the chorioallantois by applying negative pressure to the air - cell with the aid of a small rubber teat . 
the egg was then sealed in the normal way and incubated for 3 days.when opened and examined , small roughly circular opacities were found on the chorioallantois of 3 embryos . 
an occasional attempt at the formation of a " cell nest " may be found , but usually the cells occur in groups without any sign of a whorled appearance . 
carcinoma simplex , with large round and spindle - shaped cells . experiments 10 and 11 were chorioallantoic implants using methylcholanthrene induced transplantable mammary carcinomas of mice . five 10 - day embryonated eggs were implanted with fragments of tumour and examined 7 days later . all but one contained small tumours 3 - 5 mdiameter . 
of a suspension of mouse sarcoma s.37 in twenty - seven embryos subsequently died at periods varying normal saline . most of these showed congestion and petechial haemorbetween 1 and 8 days . rhages in the skno bacteria were isolated from these eggs . 
the dead embryos showed intense congestion and petechial haemorrhages . in 2 cases tumours were found tumour - bearing membranes were attached to the interior of the yolk - sac . ground up with sand and saline , and 0 - 25 ml . 
a haemorrhagic membrane was treated in the nine days later the mice were found dead , same way and injected into 5 mice . due to a heating failure during a cold spell . six of the mice injected with tumourbearing membranes were found to have small lymphoid turnours growing in the subcutis at the site of injection . 
the membranes appeared normal upon histological examination . ( e ) spontaneouvs chicken tunours . in experiments 19 , 20 and 21 the intra - vitelline method was again tried . these experiments may be briefly dismissed , as in each case contamination with bacteria killed all the eggs . 
coli was isolated from the eggs , although broths inoculated with tumour tissue from the original case remained sterile . experiments 20 and 21 consisted of the injection of a saline suspension prepared from lymphocytomata , involving the thigh and ovary respectively . the embryos died subsequent to injection , and bact . 
hen suffering from aleukaemic lymphoid leucosis . eight days later two of the embryos were found to be dead , namely those with liver and liver plus spleen implants . only the latter had a tumour in the membrane . 
the remaining spleen only implanted embryo was living , and the membrane bore a 4 mdiameter pink tumour . in the case of the dead embryos both were extremely congested , and the livers appeared swollen and leukaemic . histological examination of the membrane bearing the spleen implant shows a vascular growth of endothelial - like structure . all resemblance to normal or even leukaemic spleen has disappeared . 
the growth is composed of an irregular syncytium of calls with round , angular or elongated nuclei , the cytoplasm of which is drawn out into processes which appear to merge with those of adjacent cells . 
the vessels are formed of a single layer of flattened endothelium and are congested . at the periphery of this tumour large numbers of primitive blood cells appear to be wandering into the mesenchymal stroma of the chorioallantois . " cell nests " occur in this region . 
an examination of the heart blood from these two embryos shows in the case of the liver only implant a large number of primitive cells which appear to be differentiating into erythroblasts , i.e. 
the bird was destroyed and immediately opened , and the viscera were exposed to the air in the post - mortem room for about 2 hours before the case was seen , but the liver surface was seared with a hot spatula at the time of implantation , and fragments taken from within the organ with sterile knife and forceps . 
the actual implants were made about 21 hours after the fowl was destroyed . broths inoculated with tumour material at the same time were later seen to be sterile . nine days later two embryos were found to be dead . 
the second method of tissue culture is involved and requires an elaborate technique , and has the great drawback that frequently only one component of the original neoplasm grows , while the rest , e.g. 
more experiments involving sub - inoculation with tissue fitrates and extra - embryonic fluids from dead and haemorrhagic embryos should help to settle this point . the chorioallantoic method has been found to be most successful in the case of the cultivation of avian tumours associated with viruses . thus , the rous i sarcoma and the duran - reynals sarcoma give the biggest growths in the membrane . these are both rapidly growing tumours , and therefore would naturally tend to give better results in the short period ( never more than 14 days ) of cultivation . slow growing tumours usually give disappointingly small growths , whilst normal adult tissues may fail to grow and be absorbed . in the case of the rous and duran - reynals sarcomata , the virus stimulates the mesenchymal tissue of the chorioallantois to participate in the general malignancy , thus augmenting the size of the implant . 
of more interest , however , is the observation , originally by keogh ( 1938 ) , that the rous virus also appears to stimulate the ectodermal cells of the chorioallantois to proliferate in an apparently uncontrolled manner . 
he speculates on the possibility that the virus may be carcinogenic , not only for mesenchymal tissue , but also for epithelium ectodermal proliferation due to rous virus has been under certain conditions . confirmed in the present investigation , and the pathological changes in the membrane ectoderms produced by the rous and duran - reynals tumours have been compared with those produced by dermatrophic viruses such as those of fowl - pox and contagious papillomata of cattle , also with similar changes noted in the membrane adjacent to certain implanted spontaneous tumours of the chicken . as a check on these observations a number of experiments were performed with chemically induced tumours of the mouse , rat and chicken ; also with normal adult and embryonic chicken tissues . the significance of the " cell nests " or " epithelial pearls " is interesting , resembling as they do transverse sections of infiltration cores of epithelial tissue similar lesions were noted by which characterize squamous - cell carcinoma . huxley and murray ( 1924 ) adjacent to the fragments of chick embryos implanted on the chorioallantois . it is well known that embryonic tissue extracts contain growth - promoting substances , and for this reason are frequently added to tissue culture media . claude ( 1938 ) showed that a fraction could be isolated from chick embryos which j . 
campbell possessed the same physical and chemical properties as the purified active fraction despite these similarities , however , the embryo extract of the rous i sarcoma . failed to produce tumours in susceptible chickens . in this connection it is interesting to note that earle ( 1943 ) noticed an apparent malignant transformation of mouse fibroblasts grown on a fibrin clot and bathed in horse serum and chick embryo juice . these observations , together with the production of ectodermal proliferation and cell nest formation found to be associated with so many chorioallantoic implants of tumnours in the present investigation , gave rise to the conception that tumours and embryonic tissue might contain growth - promoting substances capable of causing the overgrowth of the ectodermal layer of cells in the embryonic membrane . if this proved to be the case it might be possible to demonstrate some relationship between the intensity of the membrane reaction and the type of tumour implanted , especially from the aspect of virus content . 
no mention of " epithelial pearls " is made by keogh ( 1938 ) , or by the numerous other workers who have studied ectodermal lesions of the chorioallantoic membrane induced by viruses . a number of experiments were devised in order to test this hypothesis , and especially to study the membrane reaction to a spontaneous liver carcinoma of the duck , which is described in a separate paper . table i shows that the ectodermal lesions do not appear to be as specific as in all cases where tumours known to contain virus , or fitrates of was hoped . such turnours , or dermatrophic viruses , were placed on the membrane , proliferation of the ectoderm took place . in some instances eosinophilic " inclusions " were observed , but the nature of these is not clear , except of course in the case in the main , virus alone caused simple proliferation without the of fowl pox . formation of " cell nests . " in general those tumours which failed to grow subsequent to implantation and were absorbed did not cause any detectable lesions in the membrane ; similar results were obtained for normal adult tissues . also in several cases , although not invariably , an accidental infection of the membrane with bacteria did not result in ectodermal proliferation . the spontaneous chicken tumours which grew successfully all caused proliferation and the formation of " cell nests . " it was thought possible that chemically induced tumours , being presumably free of virus , might give a different membrane reaction . 
the majority of spontaneous tunours give disappointingly small growths . it has been shown that the capacity for indutcing ectodermal proliferation in the chorioallantois is not confined to tumour viruses , or to fowl pox and other non - tumour associated viruses , but occurs in the membranes adjacent to the majority of implanted tumours , whether spontaneous , virus associated , or chemically induced ; or even occasionally in response to bacterial growth . attention has been drawn to the tendency of the proliferating ectoderm to form " cell nests " or " epithelial pearls , " resembling those occurring in squamous - cell carcinoma , and the significance of this has been discussed . the induction has been described of two distinct leukaemic conditions in chick embryos as the result of implanting tissues from a third type of spontaneous leukaemia in a fowl , and has been briefly discussed . i wish to express my gratitude to professor a . 
murray drennan , of the pathology department , university of edinburgh , for his interest in this work , and for the numerous suggestions as to the manner of development of the investigation . all the fertile eggs were obtained from a . 
7. received for publication february 3 , 1949 . one of the surprising elements in bittner 's discovery of the " milk factor " is the apparent entry of the virus into the bodies of the young mice via the it is , however , obviousthat during suckling a small amount alimentary canal . of milk may also enter the nostrils , and that the nasal mucosa may offer an alternative port of entry . it also seems possible that the milk factor might be destroyed by digestion in the stomach . if this were so it would be difficult to recover milk factor from the stomach contents of suckling mice . 
the unfortunately the experiresult was entirely negative in the 40 mice so treated ment was not adequately controlled , in that none of the mice were tested for susceptibility by means of extracts known to contain the virus . 
cohen free , will , in this paper , be designated as ffc , and the reciprocal factor - harbouring ( c3h x cba ) f1 as fhc . all parent strain and hybrid mice were housed in the same environment and maintained on a standard laboratory chow diet with tap water ad libituit was found expedient , in view of ' the temperamental breeding habits of both cba and cm females , to isolate routinely all pregnant mice until after the weaning of consequently , no f , bybrid was ever in contact with the father , which , the litters . as has recently been shown by peacock ( 1953 ) , reduces the probability of " infection " in the ffc with milk - factor from the cm male parent . the incidence of spontaneous mammary tumours in old factor - harbouring ( c3h x stock albino ) f , females , which were observed for over a year and inadvertently force - bred , was at least 70 per cent . 
no mammary tumours were noted in the corresponding , similarly treated , factor - free females , and it was considered that the presence of the milk - factor in the c3h niale parent exerted little or no influence on this group . 
the tumour , retracted from the body onto a wax backing , is fitted into the mouth of a 2 cdiameter open - end applicator , fsd = 25 cm . , and treated at 240 w . 
the mean - log of the volume in cubic millimeters of the fhc group is 2 - 12 ( 0 - 08 ) , while the mean - log - volume for the ffc group is 1 - 85 ( 0 - 08 ) , which is significantly smaller . while unequivocal comparisons between the c3h and hybrid groups could be made with confidence , it was considered possible that , as a result of the variations in growth rate within the hybrid hosts , smaller tumours might be more readily cured , and the ffc group consequently biased in favour of a higher curerate . 
2 , togeth ' er with comparative data previously determined for the parent this design is analogous to a six - point assay , in which both hnearity strain . and parallehsm of the regression hnes can be tested . 
the magnitude of the parameters and the significance of the inter - group differences were computed by finney 's ( i 952 ) methods . a third experiment was designed to determine , whether the increased radiosensitivity of the tuniour in f , hybrids was an inherent change in the tumour cel ' is per se , or a reflection of host - resistance . 
2. - probit diagram showing response of the cm carcinoma growing in various situations . ( a ) control series treated in homozygous cm mice in 8itu . ( d ) irradiated in 8itu in heterogeneous hybrid mice . ( f ) irradiated in factor - free ( ffc ) mice . 
the increased radiosensitivity of the tumour associated with genetic diversification in the host is evident . ( e ) irradiated in factor - harbouring ( fhc ) mice . 6000 7000 5000 cells growing in a relatively alien environment was suggested by barrett and deringer 's ( 1952 ) observation that a permanent adaptive change occurred in the oh mammary carcinoma following one - sub - passage through susceptible f , hybrids . since the object of this experiment was to demonstrate any inherent acquired radiosensitivity in the tumour , a smar homoplast ( first passa - ge from a oh mouse ) , growing slowly in an ffc host , which had attained a volume of only 30mm3 . 
the donor was also re - impladted with this tumour . takes " were 100 per cent in all groups . when each tumour had reached a standard size , about i cin its greatest diameter , it was irradiated in situ with a dose of 4200 r . 
the re - implanted tumour therefore , if the in the ffc donor was also irradiated at this dose , and cured . acquired radiosensitivity was a permanent adaptive change in the tumour cells capable of being carried over into the next transplant generation , it could be expected that the cure - rate of grafts originating from this tumgur at the dose givedwould be about 75 per cent in each genetiegyroup . 
2 , and the probit regression lines derived for the radiosensitivity of the tumour growing each reciprocal group are shown . in the genetically heterozygous environment of the ffc host , excluding the complicating influence of maternal factors , is shown by line f in fig . 
2. this line is virtually parallel to that previously obtained in the parent oh strain ( line a on the diagram ) , the coefficient of variation being 10 per cent . the median effective dose ( ld50 ) is found to be 3950 ( i 10 * ) r , compared to 5700 r previously reported in the oh . 
2. not only is the cure - rate , in the presence of the milk factor , considerably less than that of the factor - free mice , thougli still greater than that of the c3h parent , but the usual sharp response to increasing dosage has become much less sensitive , as evidenced by the considerably flatter slope of the regression line . this change test of the data reveals a is not due to fortuitous fluctuations in cure - rates , as a highly significant departure from parallelism with the other groups . 
the median , effective dose for the tumour growing in fhc mice appears to be 5100 ( + 400 * ) r with the unusually large coefficient of variation of 25 per cent . it must be assumed , therefore , that the maternal influence is not uniform and stochastically independent of other factors . 
was noted in the treatment of radiation - attenuated homoplasts growing in the parent strahi ( cohen and cohen , 1954 )  . at the ld50 level , the relative radiosensitivities , evidenced by the dosage ratio between the c3h and fhc groups is 1 - 13 ( + - 07 * ) , and that between the fhc and ffc groups is 1 - 29 ( - 08 * )  . 
the former ratio , compared with its standard eiror , indicates that the genetic effect per se , in spite of the complicating matemal influence , is probably significant ( p = 0 - 05 ) ; while the latter shows that the effect attributable to the milk factor alone withift the hybrid mice is highly significant ( p < 0 - 001 )  . 
the relative importance of these ratios , however , necessarily varies with the curative level t - ested , the maternal influence having the greater effect at doses above the median effective range . in order to eliminate any possible bias introduced by the greater average volume of the tumours in the fhc mice compared with those of the ffc group , a small number of animals bearing tumours of extreme size were discarded in the manner previously described , so as to equate the mean log - volumes of the two groups , and the probit analyses repeated on the remaining matched tumours . this correction , however , does not alter the magnitude of the parameters or affect materially the significance of the differences between them . further , comparison of the proportion of tumours cured within each class - interval of fig . 
i that within each genetic subgroup , the cured tumours tend to a smaller volume than the non - cures . these differences , however , are not signific ' ant , since the experiment was not designed to test the effect of tumour volume per se on radiosensitivity and contains insufficient data for this purpose . 
the question of the relative importance of genetic factors and of tumour size per se in determining radiosensitivity , is probably meaningless , since the smaller , more slowly - growing homoplast may itself be a manifestation of genetically - conditioned host - resistance ( eichwald , 1953 )  . 
cohen mal factors , therefore , materially affect the response of a tumour homoplast irradiated in situ . subsequent challenge with a second inoculum in those hybrids of both reciprocal groups , previously cured of the tumour , resulted in active growth in all it was also interesting to note , in the course of the experiment , that the cases . spontaneous mammary tumours in factor - harbouring f , females occurred regardless of whether the animal had previously been cured or not . third experiment table 11 shows the results of the third experiment , in which a " radiosensitive homoplast , growing in an ffc and later cured with 4200 r , was sub - passaged through mice of the three genetic categories and treated with the same dose . the cure - rates of these tumours in the ffc , fhc and parent c3h mice are respectesting the null - hypothesis that the radiosensitivity tively 67 , 35 and 0 per cent . of the sub - passaged homoplasts is the same as in the ffc hosts ( 76 per cent at 4200 r , table 1 ) , the probability of obtaining the results observed is shown in the last column of table ii . 
the susceptibility to tumour transplantation is a simple genetic - dominant , shown by the uniformly successful transfer of tlle inbred parent strain tumour to all f , hybrids . however , once such a graft is established , its rate of growth is no longer contingent on host genetics per se , but is dependent on the aternal extrachromosomal influence which , when present , exerts a significantl accelerating effect . likewise , a matemal influence , presumably the milk agent , is sufficient to induce spontaneous mammary tumours in f , hybrid females , the incidence approaching that in the inbred parent strain , in spite of genetic dilution . it has here been shown that the radiosensitivity of a tumour transplant , in this case the c3h mammary adencarcinoma , is dependent on both genetic and maternal ( extrachromosomal ) factors in the hybrid host . it appears , however , that the two factors are not necessarily independent . in the case of the factor - free f1 hybrid , a uniform and uncomphcated response is elicited which seems to be a - clear expression of immunogenetic differences between host and tumour . 
the differences between these groups are significant , although all three groups of mice were uniformly susceptible to the tumour and showed no overt manifestations of resistance . it appears , therefore , that the radiosensitivity of a tumour is a quantitative measure of subhminal host - resistance resulting from immunogenetic differences in the host - tumour relationship , including extrachromosomal factors . the mechanisni involved was further elucidated when a tumour growing in a factor - free f , hybrid , and known to be curable with 4200 r , was sub - passaged to mice of the three genetic groups . 
the homoplasts responded to treatment in the same manne ' r as the directly passaged parent strain tumour in the corresponding hosts , indicating that no inherent radiosensitisation of the tumour cells per se had occurred . all the facilities required for the maintenance of the animals used in this investigation were generously provided at the south african institute for medical 312 a . 
3. received for publication february 6 , 1947 . it has frequently been noted by the early investigators of tumour pathology that besides a high mitotic rate , tumour cells may also show great variation in the details of mitosis ( pianese , 1896 ; hansemann , 1904 )  . 
the abnormalities may concern the behaviour of the chromosomes or the cytoplasm or both , and most of them lead to degeneration and death of the cells . this is largely due to the deficient nuclei which usually result from abnormal mitosis . 
by repeated chromosome multiplication within the nuclear membrane ( biesele , poyner and painter , 1942 ; koller , 1943c )  . polyploid cells are frequent in tumour regions where .cell breakdown is in progress on a large scale . 
x - rays induce stickiness of chromosomes in plants and animals ( markquand , 1938 ; koller , 1943a ; darlington and la cour , 1945 )  . temperature changes and chemical treatment can lead to similar abnormalities ( barber and callan , 1943 ; ostergren , 1944b ) , and complete suppression of the spindle is brought about by low temperature and by specific compounds , such as colchicine . polyploid , binucleate and multinucleate cells , owing to their chromosome balance , survive and may undergo mitosis . their rate of mitosis , however , is always less than that of cells with a normal chromosome number . 
koller until recently it was believed that a nucleus with the diploid chromosome number was necessary for normal cell behaviour , and that cells with a deficient complement could function only under very specific conditions , the life of such cells always being short ( darlington , 1942 )  . 
the adenocarcinoma here described presents an example in which cells with less than the normal number of chromoit is not out of place to somes are still able to divide and to continue dividing . draw attention to recent literature on cytoplasmic control in micro - organisms ( sonneborn , 1943 ; spiegelman and kamen , 1946 ) , the relevance of which , for the origin of cancer , has been dealt with by many authors ( graffi , 1939 , 1940a , b ; darlington , 1944 ; haddow , 1944 ; potter , 1945 ; woods and du buy , 1945 , 1946 ) , who suggest that the permanent change which renders a cell malignant takes place in the cytoplasm . cell behaviour in this adenocarcinoma is a further indication that the nucleus can be so subordinated , and the cell remain active in spite of its deficient nucleus . u0 ' c unromosome numoer fig . 
i presents the absolute number of deatlis from lung cancer in norway in order to avoid the disturbance in comparability introfrom 1929 to 1952 . duced by the 1948 classification , the figures for 1951 and 1952 have been adjusted , according to the analysis of julie e . 
backer , head of the medical statistics division of " statistisk sentralby - rh , " so that they very approximately represent the figures that would have been found in group 47b , if the coding had taken place according to the 1938 classification . 
1 , showing the gradual and pararel rise ' lung cancer in both sexes up to the middle of the 1940 's , however , with a shght lead of the males . 
2 lead , h ' owever , to a more accurate pictureof the registered increase . in males this ' mcfease is from 0 - 11 per 10 , 000 living in 1930 to 0 - 77 in 1950 . 
figures for 1930 a - re given as the mean value of the and 0 - 34 in 1950 . instead of a tenfold increase in males we find a sevenfold , and years 1929 - 1931 . instead of a sixfold increase in females , w - e find a four - and - a - half - fold . in a recent paper kreyberg ( 1954 ) has described 466 primary epithehal lung tumours and typed the tumours in six main types , according to criteria previously stated ( kreyberg , 1952 )  . 
kreyberg these tumours are designated group i tumours . series ( christiansen ( 1953 ) , 134 cases , and jakobsen ( 1953 ) , 100 cases ) , as well as the authors own clinical material , embracing 232 cases . the examination of the histological types , as well as the sex and age occurrence , in the light of our present clinical and epidemiological kiiowledge lead to the following conclusions : ( i ) squamous cell , large - cefl and small - cell carcinomas represent a biological entity . these tumotirs occur with a very marked preponderance in males and they show an age - curve indicating that in the so - called western world potent carcinogenic factors of recent origin are of aetiologic signi ( ii ) adenocarcinomas ficance . occur in norway with the same frequedcyin males and females , and this tumour shows a steadily increasing occurrence with advancing years , indicating a stable and moderate carcinogenic influence . ( iii ) bronchiolar cell carcinomas are likewise evenly distributed among males and females , but there is no increase in the higher age - groups . 
udknown factors hitting at random are supposed responsible ( iv ) lung adenomas and salivary gland tumours of the for their development . lungs sbow an equal sex distribution and occur in all ages . these facts , together with the histological picture point in the direction of developmental factors being responsible for their occurrence . these last four tumour types have been , designated group 11 tuniours . if the three norwegian materials are examined and christiansen 's ( 1953 ) series is divided into two sub - series , according to two different time periods , we get the figures as shown in table ii . 
the consequence of this assumption should again be that an increase registered for group ii tumours would represent a measure of an increase in diagnostic efficacy . as regards the group i tumours , table ii again clearly reveals interesting in the four time periods , tfie two middle partly overlapping , one may facts . observe that females , in accord with clinical experience , constantly present a small number of group i tumours . 
no conclusion in the opposite direction is drawn from the rather declining frequency because of the smafl figures . it may therefore be correct to regard the lung cancer in females to - dav as an expression of what clemmesen , nielsen and jensen ( 1953 ) call " unavoidable lung cancer . this unavoidable lung cancer should include ar the group ii tumours , as well as a smar number of group i tumours . if these assumptions are correct , the increase in lung cancer in females should at least in norway today , be a fair index of the increased diagnostic efficacy . the twenty years ' period 1930 - 1950 this increase is approximately four - and - a - halffold . 
we have , however , probably not yet reached the limitation of our diagnostic although better diagnosis may in some cases lead to the discovery expansion . of a primary site outside the lung , and thereby exercise a depressive effect on the lung cancer mortahty registered , it seems more hkely that further improvements at the present time will mainly result in an increase in the mortahty figures . 
we ought , therefore , to accept even a furtlier increase in the lung cancer mortahty registered in females , without necessarily drawing the conclusion that more lung cancer is actually developing . 
from that period a marked increase in grou ' p i tumours can be observed in males , at the same time as a pronounced deviation in the mortality rates in the two sexes takes place . it seems that the marked additional rise in lung tumours in males is caused solely by the group i tumours . 
the group 11 tumours , on the other hand , closely follow the pattem it may be that in the latest series ( kreyberg 's ) in table ii , the of the females . difference between the group i and ii tumours is too marked to be whory representative , because that material is a clinical one with an underrepresentation of older people liable to develop adenocarcinomas . 
the tendency is clear , however , and the development continues to - day . at present it seems permissible to conclude that in norway , during the twenty years ' period 1930 - 1950 , the four - and - a - half - fold increase registered in lung cancer in females is entirely caused by greater diagnostic efficacy . 
as the development in males initiary closely followed that of the females , it seems reasonable to accept the recent deviation in the mortality rates , as well as in the representation of the histological types , as indicating a new situation , and to regard the added increase in lung cancer in males as an expression of a real increase . this should mean the difference between a sevenfold and a four - and - a - half - fold increase . the importance of this conclusion is not expressed by the magnitude of the real rise to - day , but it is indicated by the tendency and the sliort time it has been manifest . 
1. - lung cancer in denmark , 1931 - 1945 . 1931 33 35 crude mortality rate per 100 , 000 in males 43 45 39 41 alterations in the age distribution of the population might be suggested as the cause of the rise in crude mortality from lung cancer , but if we compute the mortality rate according to age in the capital for periods of five years , and on the base of quinquennial age groups of dead and living persons , as in fig . 
2 , we find no confirmation of the suggestion mentioned . thus , we have found a very steep increase of mortality from lung cancer within a limited area , well furnished with modern medical facilities , available to all social classes . * the increase is found among both sexes , but is far more pronounced for males than for females , and it will be worth while further to analyse the population in question with regard to the incidence of lung cancer , as copenhagen seems to provide a good example of the changes in the mortality from lung cancer found elsewhere in europe . the central tuberculosis station of the city of copenhagen provides excellent opportunities for checking up the reliability of statistics given in the first part in its work to trace tuberculous infections in patients of the present paper . suffering from all sorts of respiratory diseases , this institution performs numerous thorough examinations on such patients referred from general practitioners . annual reports contain tables giving the number of patients , subdivided in decennial age groups for each sex separately . 
the group examinations under * for members of the public health insurance , which comprise 85 - 90 per cent of the danish population , no fees are charged in the municipal hospitals of copenhagen . 
3 , shows no steady rise , and hardly any irregularity is found in the curve for females , which , however , comprises very few individuals , as seen from the absolute figures . the very steep rise of the male curve about 1941 may have been caused by the fact that the efficiency of a central tuberculosis station may also have increased if the duration of the disease is one or two years in a disease like lung cancer . from its earliest possible detection to death , and this is , in fact , the opinion of the tuberculosis station , the steep rise in 1941 given in fig . 
even if we allow for the small absolute values of the figures , it can be said , as with regard to the corresponding mortality figures , that there is no increase of morbidity in the young , as would be expected if the inerease was due to an increase in the inhalation of tobacco smoke , if we are not to assume a latent period for lung cancer of about two decades . in our opinion the figures given illustrate that even a pronounced increase of the crude mortality rate for lung cancer among males , and almost only among males , does not necessarily mean an increase in incidence of that disease . presumably the more frequent detection of the disease is due to improvement of 2s~~~~~~~~~~~~~ __ _ 120 ___ __ 80 __ f - ~i 40 ' j years fig . 
if the results from copenhagen can be generalized , it seems that the apparent increase in lung cancer mortality is , to a very large extent , conditioned by improvements in diagnostic means , and it can be expected to continue until the sex ratio amounts to about 8 males to 1 female . our thanks are due to knud winge , m.d. , the chief physician of the central tuberculosis station , copenhagen , for permitting us to use the material of the station , and for his kind interest in our work , and to kontorchef miss m . 
 the compilation of this paper was prompted by the idea that some investi gators of cancer as it occurs in man do not know the valuable material which is contained in the publications of the general register office , while even those who are acquainted with this literature may find some use for a list of the collec tions of data about cancer which it contains . 
 the material dealt with in these publications consists of the death certificates from england and wales , considered in association with the data on population , civil state , occupation and locality obtained at the census of 1911 , 1921 and 1931 , together with population estimates for intervening and subsequent years . 
the certificate does not state , and is not intended to state , how long the deceased had followed the occupation named , and had lived at the home address given ; this information could be obtained only by inquiry into individual cases . 
the sources of such errors have been discussed in two earlier papers ( henry , kenna way and kenna way , 1931 ; ~eimaway and kennaway , 1936 )  . 
 the _decennial supplements for 1921 and 1931 contain data collected at the census of those years , and were published in 1927 and 1938 respectively ( registrar general , 1927 , 1938 )  . 
_they contain a large amount of tabulated material for periods adjacent to the census , namely 1921 - 23 and 1930 - 32 ; the scope of those tables , in so far as they are concerned with cancer , is summarized in tables i and ii . 
the 240 , 726 deaths of all married women during 1930 - 32 were classified according to the husband 's occupation as stated on the death certificate , while 19 , 422 single women who died during the same period were classified according to their own occupations . 
the reasons for the association of the married woman with the occupation of her husband are that ( 1 ) " only about 10 per cent of married women were recorded as gainfully occupied at the census of 1931 , " and ( 2 ) some indication is obtained of the social , or occupational , nature of the mortality in the husbands ' occupation , "  . 
table ix shows a selection from the supplements for 1921 and 1931 of some very interesting - data for the incidence of cancer of various organs upon men and married women of the five social classes , and in the case of the men two periods ( 1921 - 23 and 1930 - 32 ) can be compared . 
a recent paper by swan ston ( 1950 ) on " the iron and steel industry " shows the use which can be made of the material in the decennial supplements . 
 the registrar - general publishes annually a " statistical review of england and wales for the year  . , " normally in two parts , namely , ( 1 ) text , ( 2 ) tables . 
the " text " now contains a section upon cancer consisting of a commentary upon the data which have been obtained during the year in question , and certain tables . 
this table provides material in answer to the question so often asked - " is cancer increasing ? " the comparative mortality index , which takes account of changes in the sex and age structure of the population , shows that the total mortality from cancer in england and wales has barely increased during the past twenty years . 
the distinction between the entry of " carcinoma " and " cancer " upon death certificates is probably of no value , but the separation of the two at any rate absolves the general register office from any responsibility for the distinction ( table lxxvi )  . 
 ( e ) by regions ( england and wales , greater london , rest of south - east , north , midland , east , south - west , wales ) , and by class of area ( county boroughs , other urban districts , rural districts ) ( table lxxvii )  . 
 such data for the total incidence of cancer must , of couj1se , be recorded , calculated and examined , but they cannot show the peculiar incidence of cancer upon the various organs , and in the two sexes , and in persons living in different areas , and various interesting changes may cancel one another and hence be lost in the totals . 
the great interest of these data is best made plain here by reproduction in table iii of a part of an example ( table lxxviiia , 1940 - 42 )  . 
 all sites lips tongue mouth tonsil pharynx jaw others total oesophagus stomach duodenum other small intestine caecum hepatic flexure , splenic flexure sigmoid flexure large intestine ( colon ) other and undefined intestine rectum ( not anus ) liver gall bladder and ducts pancreas peritoneum , mesentery , others other and unspecified digestive organs 89 , 902 606 2 , 254 703 534 1 , 008 748 529 6 , 382 4 , 498 20 , 778 129 155 860 418 2 , 157 8 , 801 228 10 , 365 2 , 581 703 3 , 267 4 , 053 147 232 199 250 females . 
 other female genital organs total total total total total breast scrotum prostate testis penis others kidney bladder , urethra , ureter : 163 155 6 , 055 339 457 7 , 009 409 3 , 360 3 , 769 147 174 606 623 593 667 219 312 755 2 , 877 202 3 , 834 5 , 482 721 6 , 416 12 , 619 4 , 952 1 , 281 6 , 248 20 , 049 107 184 173 183 107 265 386 162 510 705 499 550 1 , 114 312 1 , 557 1 , 869 452 458 185 the material contained in this table is very significant . 
the cancer problem in its simplest form is reduced to the question , why does a cell divide ? but no single universal " cause of cancer , " such as is announced from time to time , could , even if it were actually discovered , explain all the features of this table . 
the nature of cancer proceeds by means of the most refined cytological methods , there is scope for investigation of factors occurring in everyday life which may explain the 1rery peculiar sexand organ distribution of cancer . 
 breast total cancers of female reproduc tive organs other organs total cancers in females males exce811 in females 18 , 867 20 , 049 38 , 916 58 , 090 97 , 006 89 , 902 7 , 104 164 e . 
kennaway for instance ( table iv ) , the total deaths from carcinoma in males ( 89 , 902 ) and females ( 97 , 006 ) show a difference of only about 7 per cent , yet the latter total includes 38 , 916 deaths , or 40 .per cent of the whole , from carcinoma of organs peculiar to the female , among which organs the female breast , being capable o , f lactation , must for the present purpose be reckoned . 
this calculation illustrates the phenomenon of the " amount " of cancer in man , to which attention was drawn in an earlier paper ( kennaway and kennaway , 1937 )  . 
no better example has been given by later workers than that recorded by the dutch investigators snijders and straub ( 1924 ) in sumatra , who first described this numerical relation of total cancers in populations differing in habitat or in sex ( table v )  . 
 28 27 these figures show that in sumatran and european populations , in which the total incidence of cancer is about the same , the proportion of one form of cancer , in this case that of the liver , may be very different . 
 " there appears to be a general law that when in a given population the incidence of cancer in one particular organ is markedly increased as compared with another population , there is then a compensating decrease in the incidence of cancer in a number of the other organs " ( cramer , 1936 )  . 
 ( b ) the data shown in table lxxviiia and bare subdivided under 15 quin quennial or decennial age - groups from 0 - 5 to 85years ( tables lxxixa , b , e , d , e , f )  . 
thus in men aged 55 - 64 the mortality from cancer of the lip has fallen to 26 per cent of the initial figure , while that attributed to cancer of the lung has increased by more than 17 times . 
 1911 - 20 1921 - 30 1931 - 35 1936 - - 39 1940 - 44 1945 1911 - 20 1921 - 30 1931 - 35 1936 - - 39 1940 - 44 1945 1911 - 20 1921 - 30 1931 - 35 1936 - - 39 1940 - 44 1945 the exact comparison of the prevalence of cancer , or of any form of cancer , at different periods , requires standardized death rates owing to the continual increase in the numbers of older persons , who are of " cancer age . " such data are given at intervals in the statistical reviews ( text ) , and most recently in table lix in the review for 1938 and 1939 ( registrar - general , statistical review ) , which records the standardized rates per million population for cancer of 27 sites in men and women separately in the periods 1901 - 10 , 1911 - 20 , 19.21 - 30 , 1931 - 35 , 1936 , 1937 , 1938 and 1939 . 
these figures for each one of the four most recent years are of especial interest because they show the present state of increase or decrease of the various forms of cancer . 
 in the table in question the great majority of the 52 sex - site combinations show a decrease , or are stationary ; the only quite definite increase is in cahcer of the lung and bronchus in both sexes . 
since 1942 standardized death rates have been superseded by comparative mortality indices , which compare the death rate in each year with that of 1938 , after making allowance for changes in the proportion of the population at different ages . 
for a number of sites of cancer since 1933 have been published in the medical tables volume of the statistical review ( table viii ) for each year since 1942 . 
 ( d ) the deaths from cancer of certain sites ( mouth and oesophagus , stomach and duodenum , respiratory system , breast , uterus , other sites ) per 1000 for all sites in men and women at certain ages , in certain regions and classes of areas named above ( table lxxvii ) during 1940 - - : - 42 and 1943 - 45 ( table lxxxi ) are set forth . 
 the cancers of the respiratory system show the greater liability of urban populations ( stocks , 1936 ) ; this portion of the tabl~ is of such interest that it may be reproduced in extenso here ( table vii , section a )  . 
if one recalculates the figures for the various districts on the basis that " rural districts " = 100 , whereby the data for the two periods 1940 - 42 and 1943 - 45 become more comparable , one obtains the figures shown in section b of the above table , which show a very close similarity between the two series for women , with the single exception of the figures for the south - west region . 
of course this result has no bearing on the accuracy of death certificates , but it does show that two sets of such certificates , gathered in a number of districts during two consecutive periods , give results which have a considerable uniformity . 
 when the mortality from any form of cancer , such as that of the lung , is higher in urban than in rural districts , one must consider whether the difference is due , not to the conditions of life in towns , but to better facilities for diagnosis , and a further extract from table lxxxi is of interest in this respect ( table viii )  . 
 males , 45 - 65 mouth and oesophagus , 1940 - 42 1943 - 45 stomach and duodenum 1940 - 42 1943 - 45 males , all agesrespiratory system females , 45 - 65 breast uterus 1940 - 42 1943 - 45 1940 - 42 1943 - 45 1940 - 42 1943 - 45 238 225 156 183 236 231 165 157 205 187 216 249 261 254 150 137 100 254 235 103 126 236 244 148 144 the diagnosis of cancer of the oesophagus , stomach and duodenum , breast and uterus may be by no means easy , yet these forms of cancer do not show the difference seen in cancer of the respiratory syste this last must be made up largely of cancer of the lung , and hence these data provide further evidence of a carcinogenic factor in urban environment . 
 the annual statistical reviews do not deal with the social and occupational incidence of cancer , as the occupational distribution of the population is ascer tained only at census periods . 
 in this summary of the annual and decennial publications of the general register office in relation to cancer one cannot of course refer to the numerous passages which treat of the material tabulated . 
 the decennial supplement for 1921 ( registrar - general , 1927 ) contains the first detailed study of the social incidence of cancer upon the various organs in men , which study developed from an earlier investigation of the incidence of cancer as a whole ( stevenson , 1923 )  . 
so far as is possible from the material available , class i purports to represent the pro fessional and generally well - to - do section of the population , class iii , skilled artisans and analogous workers , and class v , labourers and other unskilled callings , while classes ii and iv are intermediate , comprising occupations of mixed types , or types not easily assignable to the classes on either side . " the parts of the body affected by cancer were divided into two classes , namely : exposed sites ( buccal cavity , pharynx , oesophagus , stomach , larynx , skin ) , and other sites . 
 the mortality from cancer of the exposed sites increases from social class i to class v , while cancer of the other sites shows no such relationship ( table ix )  . 
 the author of the decennial supplement , 1921 , comments upon these data : " it thus appears that a large proportion , at least , of cancer mortality is of a highly preventable nature , for we must suppose that if the conditions of life of all sections of society could be assimilated to those of its upper ranks , mortality from cancer of the exposed sites would fall for all classes to the class i level . 
 the paragraph made up by these two sentences should be regarded as one of the classics of cancer research , for it brings carcinogenesis in man into relation with factors in everyday life which can be investigated , while the most refined methods of physics and chemistry are applied to the problem of cancer in general . 
 the most extreme instance of a social factor in the incidence of cancer appears in the case of cancer of the scrotum ( kennaway and kennaway , 1946 ) , which could in all probability be eliminated altogether by cleanllness , as is cancer of the penis by circumcision in infancy . 
 the figures ( table ix ) for cancer of the upper alimentary canal in males show a significant reduction in the steepness of the social gradient from 58 , 80 , 99 , 102 , 140 , to 63 , 80 , 97 , 109 , 129 ten years later . 
this may be due in part to " earlier or more effective treatment than before of cancer of the more accessible sites " in men of the poorer classes , as is suggested in the text , but probably also to the improvement of social conditions in other ways . 
the change has been chiefly in the cancers of the buccal cavity ( tongue , tonsil , pharynx ) , for cancer of the oesophagus shows no , and that of the stomach very little , loss of gradient . 
 the steeply graded figures for the larynx , in contrast to those of the buccal cavity , show no change , while those for cancer of the skin show a flattening which one would associate with improved personal cleanliness . 
 figures for the socia ] distribution of cancer in women in 1921 - 23 are not available , but the comparisons of those for men , and women , in 1930 - 32 are 170 e . 
 " of gastric cancer mortality can have little to do with the effects of occupation , and must arise from factors of selection , economics or environment which affect the wives of the men in occupational groups as much as they affect the men themselves . " cancer of the skin also affects men , and married women , of the the employment of a different social classes in much the same way ; "  . 
 proportion , largest no doubt in classes iv and v of married women in textile occupations , may be partly responsible for the mortality gradient for skin cancer . " no attempt is made here to deal with all the indications for inquiry which may be drawn from this table ; the subject is dealt with , and illustrated by graphs , in pp . 
cancers of the buccal cavity , and of the larynx , show a high incidence upon occupations associated with alcohol ( kennaway and kennaway , 1936 ; wassink , 1930 ; registrar - general , 1927 , appendix d ) ; the change in gradient in the former only might suggest that this is due to another factor , namely , better oral hygiene . 
 the lack of influence of social class upon the liability to cancer of the lung , together with the very considerable effect of urban conditions ( stocks , 1936 ; registrar - general , 1947 , table vii ) , suggasts some carcinogenic factor to which all classes are exposed ; owing to the mixing action of the wind there is less difference in the outdoor air breathed by different classes than in other social conditions such 1 , 1 , s food and cleanliness ( kennaway and kennawa.y , 1947 ) , and all classes seem to share in the increased use of tobacco , largely in the form of cigarettes . 
 a good example of the unique value of such studies of social distribution is given by cancer of the uterus , which was examined in this way for the first time in 1930 - 32 . 
thus , at ages 35 - 65 the rate in married women of class v is twice that of class i , and single women in classes iv - v show a mortality 44 per cent greater than that of classes i and ii . 
 thus in 1921 the ratio of registered to 100 calculated births for married males in the five social classes was 85 , 85 , 97 , 109 , 128 , and the social distribution of uterine cancer in married women might be thought to be due to the difference in fertility . 
 the data on the mortality from cancer in the last twenty years should yield very valuable information when correlated with the results of the census in 1951 , the first made since 1931 , in view of the considerable social changes which have taken place . 
 ( 1 ) the british empire cancer campaign has published in its 13th annual report a very elaborate study by stocks ( 1936 ) , il1ustrated by 17 maps , of the distribution of cancer of eight sites ( oesophagus ; stomach ; intestines ; rectum ; liver , gall - bladder and pancreas ; skin ; lung ; breast ) in both sexes at various ages in the counties and county boroughs of england and wales . 
the 14th report of the campaign records an extension of this study ( stocks , 1937 ) , with 7 maps , to cancer of other sites ( tongue , mouth , jaw , larynx , bladder , prostate ) , and a final section ( stocks , 1939 ) , with 9 maps , deals with cancer of the uterus , vagina , ovaries and fallopian tubes , and of the skin , lung , rectum and bones in women . 
 no such study of cancer in relation to this , or any other , geographical area has ever been made , and no summary is attempted here of the results and suggestions for research which it contains . 
 ( 2 ) the changes in mortality from cancer of various organs in england and wales between 1911 and 1944 are described in a paper by stocks and mackay ( 1946 )  . 
 ( 3 ) the general register office has published a study by stocks ( registrar general , 1947 ) of cancer of the stomach , oesophagus , lung , larynx , breast , uterus , ovary and some other sites in relation to a number of localities and environmental factors ( urban and rural districts , counties , the 13 , largest cities in england , 30 large towns , metropolitan boroughs of london , social indices , number of persons per room , amount of sunshine , source of water supply ) during one or other or both of two periods , 1921 - 30 and 1940 - 44 . 
one may mention here two results which provide valuable indications for further research , namely ( a ) an inverse relationship between number of sunshine hours and deaths from cancer of the lung , and ( b ) a high incidence of cancer of the stomach in the northern and central counties of wales , especially in the former . 
the author says : " the purpose of this report is to present the statistical facts , and point out any peculiarities in distribution and correspondences with other measurable factors which appear , so that all possible clues may be followed up by further study . 
 a list has been compiled of the types of information about cancer in man in england and wales which are to be found in the publications of the general register office . 
peacock. froni the research department , glavotr royal cancer hospital . received for publication april 6 , 1949 . it is a remark - able fact that while carcinogenic hydrocarbons readily , induce sarcoma in the connective tissues of fowls , the same agents have not vet been shown to induce epithehal tumours in this species . indeed until recentlv all attempts to induce epithelial tumours in fowls by any means seem to have f.iled. recently gottschalk ( 1948 ) mentioned iin uccessful attempts to induce hepatoma in 89 fowls fed with " massive " doses of p - dimethylaminoazobenzene ( da - ab ) , and maintained for over a year on a diet deficient in protein and riboflavine or enriched with biothe also quoted similar iin uccessu attempts to induce hepatoma with butter yerow by kinosita ( i940 ) , and other failures to induce epithelial tumours in general with various carcinogens . 
one case of hepatoma was observed by ober , guerin and boic ( 1933 ) in a bird inoculated 9 months previously with a mtrate of 3fill hill 2 endothelioma . this hepatoma was not transmissible , but birds grafted with fragments of hepatoma developed leucosis which was transmissible . 
the aetiolo&y of this hepatoma therefore is uncertain.induced epithehal tumours were amongst the teratomas obtained by michalowsky ( i 929 ) following the injection of 5 per cent zinc chloride into the testicles of cocks . duran - reynals ( 1946 ) , while trying to transmit primitive renal tumours in fowls , obtained sarcomas but no epithelial tumours in the inoculated birds . it may be that other workers have made extensive negative tests without them , but it seenis advisable to describe our ow - n mainlv negative recor attempts , which have now extended over a period of about 17 years , and have 3ielded only two carcinomas , apparently caused by 2 - acetylaminofluorene ( 2 - a - a - f )  . in contrast with these negative results , bielchowsky and green ( i945 ) obtained an anaplastic renal carcinoma in i of 5 rhode island red cocks maintained on a mixed diet to which was added 2aaf , at the rate of 3 - 30 mg . 
149 , previously recorded as bearing a tumour , was found to have a necrobiotic lesion in the region of the eneysted tar and a large haematoma in the breast muscle , but no sarcoma . ( 2 ) twenty - seven bpr fowls ' were painted on the right side of the comb witli tar and injected in the right breast muscle with the same sample of tar . while these experiments were running the first recognition of a pure hydrocarbon , 1 : 2 : 5 : 6 - dibenzanthracene ( db ) , as a carcinogen was reported by kennaway and hieger ( i 930 ) , and thereafter when the substance became available these birds were painted with a saturated solution of db in chloroform instead of tar . in view of the lack of obvious reaction to the previous tar painting , light cautery of the comb with a hot platinum wire was applied to survivors immediately before painting with db . 
pellet of 3 : 4 : 5 : 6dibenzearbazole ( dbc ) implanted into the right preening gland , followed by twice - weekly painting of the preening gland area with 0 - 1 per cent solution of croton oil in acetone . 
no tumours occurred in other birds in this experiment . ( 8 ) in preliminary experiments , 7 bl fowls were injected into the preeiiing gland with solutions of dmb in arachis oil . 
the fluorescent solution was expelled through the duct of the preening gland and , as there was no evidence of retention in the gland cavity after a few days , this type of experiment was abandoned . implants into the preening glands . tllree groups of 6 bl hens had the following pellets iniplanted into the left preening gland : 15 - 30 mg . 
the other birds had ar been implanted with similar perets of methylcholanthrene , n : n - dimethylami ioswbene , or 1 : 2 : 5 : 6 - dibenzanthracene into the right preening gland , without obvious effect . of the remaining 6 survivors after 17 months , 2 had palpable nodules in the wah of the crop . 
one of these , a bl hen , was kffled because it was rapidly losing weight and was found to have several ulcers in the crop varying in diameter from about i to 8 mm . , and partly covered by tenaceous slough . there were also numerous oily cysts in the connective tissues around the crop and in the muscular and submucous tissues of the crop itself representing renmant - 8 of previous injections of 2aaf in arachis oil . 
the re ndaer of the alimentarv tract was thoroughly examined but no other lesions were found . the hver and spleen were smafl and paler than normal but showed no locahzed lesions . 
the ovary was in a resting stage and the oviduct was involuted . sections of the ulcers in the crop show that it was the seat of at least 3 apparently separate squamous carcinomass . 
the low incidence of carcinoma in fowls n - iight be explained on these grounds . on the other hand , mammals differ in their susceptibility to the actioli of carcinogens . 
a case of keratinizing squamous carcinoma of the buccal mucosa in a fowl was described and well ifustrated bv puente - duany ( 1932 )  . the orilv case of spontaneous squamous carcinoma se ' en by us in over 3000 post - mortem examinations of fowls , over a period of 20 years , occurred in a bpr fowl that had been unsuccessfully graftedwith fragments firom a chemicauyinduced sarcoma . 
the absence of sebaceous follicles in fowls and the concentration of the sebaceous glands in the paired preening gland prompted us to implant potent carcinogens into the cavity of these glands . 
campbell. from the poultry research centre at the university of edinburgh and from the royal ( dick ) veterinary college , edinburgh . received for publication february 17 , 1949 . in july , 1944 , during the course of a long - term investigation into avian neoplastic disease , a post - mortem examination of a duck showed it to have multiple liver tumours . unfortunately , this case was not seen personally and no record was kept of any details apart from the sex of the subject - a female . however , material was taken for histological examination and a diagnosis was later made of liver - cell carcinoma . primary cancer of the liver is known to occur in chickens , but is relatively uncommon . 
two of these sources provided 3 cases each , a third provided 4 cases ( one of which was not seen personally ) , and a fourth flock provided 8 cases from a flock of 12 ducks . 
one of the latter cases ( case 17 ) had 2 different simultaneous neoplastic conditions , namely a large thoracic histiocytic - sarcoma of undetermined origin , and a much smaller hepatoma . prior to the first observed case in 1944 a total of 21 ducks had been examined in 4 years , and no case of neoplastic disease was encountered . 
no information was obtained regarding the 2 rema several birds suffering from this condition have been examined alive , and in the later stages of the disease the symptoms are very characteristic . there is usuary a history of loss of appetite and of lethargy . 
as the disease progresses , general weakness brings about the loss of locomotory power , and death superv - enes either suddenly from an internal haemorrhage , or slowly from cachexia and exhaustion . if examined during this late stage , a dropsical condition of the abdomen is apparent , and careful palpation shows the prewnee of an enlarged nodular liver projecting wer beyond the caudal extremity of the stemum on the floor of the abdomen . 
usuary one lobe is mainly involved , but there is no definite predilection for right or left ical caw , the fiver tumours are nodular and bright green in colour lobe . 
ina and covered with a tense glistening membrane containing ramifying vessels . other tumours may take the form of firm , cream - coloured nodules , probably reprewnting more recent growths not yet stained with bile . haemorrhagic or necrotic nodules are not uncommon . 
these trabeculae may merge towards the centre of the tumourous lobe to form a broad , well - defined " tnmk " from which the trabeculae radiate outwards like branches wig . 
the liver cells forming these structur ' es - are cuboidal , columnar , or polyhydral with a faintly granular eosinophilic cytoplasm ; they contain a rather small , well defined nucleus , 3 - 4 [ l . 
the nuclei of those cells forming tubules are often situated ceiitrally or adjacent to the lumen . there may be attempts at the formation of a central vein and the cells immediately apposed to the endothelium are connective tissue divides the tumour into irregular usually bolumnar in form . lobules and contains numerous aberrant bile ducts . at the other extreme is the anaplastic type , which is characterized by an almost complete loss of differentiation towards a glandular structure . 
the cells are large and polymorphic with vesicular nuclei varying greatly in size and shape , some exhibiting hyper - chromatisand containing i to as many as 10 typical measurements for such cells are , prominent eosinophilic nucleoli . nucleus 13 - 6 [ l . 
the cells are fi - equently columnar and in such cases the nuclei are usuary situated mitotic figures are common and the proximary , or remote from the lumen . rumour appears to grow mainly by infiltration . 
24 hours old filtrate prepared by grinding up liver tumour tissue in a glass homogenizer , centrifuging , and filtering , using a seitz type filter and ford 's grade g.s. 
a liver fragment and some of the sahne in which it was minced , were placed into broth which - on the fohowing day was found t - o be sterile . on the 20th of the month 2 embrvos were found to be dead . 
of the 5 , 4 had small growths in the membrane - disappointingly smafl considering the cultivation period ( 12 days )  . three of these were removed , dissected awav from the membranes and reimplanted into two ' - d - day - old embrvonated eggs . 
fragments of tissue were immediately implanted into eggs , 4 containing 101 - dav - old embrvos , and i being 9 davs . the eggs were opened 9 davs later ; 4 contained tumours . 
the eggs containing the second passage implants were opened 10 davs later , and 3 contained small tumours between 2 - 3 mdiameter . later a further biopsy on case 20 provided material to implant into three 8 - dav embrvonated eggs . 
the bird was then 8 months old . fourteen we ' eks later it was observed to be weak , losing weight , and to have an enlarged abdomen . it was destroyed and examination showed a large liver tumour weig ' hing 280 g . 
the original tumour was from duck case 18 . on the 3rd day i chicken was found in a dying condition and was kired . the pectoral muscle contained a smar greenish - brown growth which was taken for this was approximately 66 hours subsequent to injection . 
10 shows the same tumour - bearing membrane photographed aaainst a white background , and the lesions are then barely perceptible . upon histological examination , an interesting picture is revealed . 
yolk - sac cultivation did not give satisfact - ory results , this also being the experience of twombly and meisel ( 1946 ) ; although taylor , thacker and p nini ( 1942 ) , and several other workers claimed a high perentage of intraviteuine growths with the particular tumours these were mainly mouse and rat mam iary carcinomata , and the poor used . results obtained by others , using different tumours , seems to indicate that the environ.ment provided by the chick embryo yolk - sac is pecuharly suitable for the metabolic requirements of mam lary cancer cells . on the other hand , most of the tumours implanted on the chorioallantois 208 j . 
the sudden occurrence of a number of cases of hitherto unrecorded primary carcinoma of the duck 's liver provided material for cultivation by various methods , with the main object of searching for a transmissible factor or virus . 
the chorioallantoic method of cultivation , therefore , seemed particularly suitable , and was extensively used . there appear to be two possible explanations of the etiology of this condition . firstly , the disease might be associated with a transmissible factor . 
 ' it is known that the mouse mammary cancer factor of bittner is transmitted from mother to offspring by the milk , and this is one of the very few known instances of a tumour of epithelial nature associated with a virus - like principle . it was felt that the duck liver carcinoma might also contain a factor transmissible probably via the egg , which might be capable of propagation in the same manner as the milk factor of mice . experiments designecl to test this theory , however , have given negative results as far as transmission by cell - free material is concerned . 
but the sui - vival of these heterologous grafts for at le - ast 6 davs in the muscle and peritoneum of chick - ens is rather remarkable . the second possible explanation of this apparent outbreak of liver cancer , is a genetic one . 
a complete circuit of the vascular svstem throu h the pulmonarv circulation and so to the spleen t - ia the splenic arterv . in the one case encountered in the duck , pulmonarv metastases occurred , and once the lungs are involved so this second hypothesis mav be the correct one . tumour cells or emboli could then the tumours there mav also metastasize reach the spleen without much diffieultv . 
a direct spread t - ia the lungs seems iinlikelv in view of the formidable barrier offered bv the pulmonarv capillaries . fox and bartels ( 1928 ) favour the niew that in the human subject the spleen and mesenteric veins mav be invacled bv retrogtade extension along the portal in case 6 , besides seros - al implantation on the wall of the int - estine , tumour vein . emboli were found within the veins of the gut wall . 
greenwood 's flock of brown - legliorns , and i have pleasure in recording niy appreciation of iiis co - operation iii this respect , and for much help in otlier directions . the histological preparations are the work of messrs . 
christiansen. from the institutt for generell og elcperimentell patologi , universitetet i oslo . received for publication february 2 , 1953 . it is a strange fact that in the voluminous literature on breast cancer the prognostic value of the size of the tumour is only cursorily dealt with , a fact mentioned by geschickter ( 1945 ) and underlined by bloom ( 1950 )  . 
the explanation probably is , that the view expressed by kaae ( 1948 ) , namely , " it is generally recognized that the size of the tumour is a very important factor in the prognosis , and that the latter is much more favourable for the small tumours than for the large , " seems so generally sound , even self evident , that the problem has not been considered worthy of a closer study . if we examine the pertinent literature , we find , however , that the findings are not unequivocal . dahl iversen ( 1930 ) states that breast cancers up to the size of plums are without recurrences after 3 years ' observation in 83 per cent of the cases , whereas tumours larger , up to the size of a hen 's egg , show only 13 per cent freedom from recurrences after 3 years . eggers , de cholnoky and jessup ( 1941 ) report upon a 5 - year survival of 73 per cent , if the mass is 2 cor less , and only 24 per cent haagensen and stout ( 1943 ) found a if the mass has reached a size of 3 to 6 cm . 5 - year clinical cure of 62 * 2 per cent if the tumour was under 3 cand only 19 - 8 per cent if the tumour had reached 6 cor more . 
they conclude that " the data indicate , as might be expected , that the prognosis becomes worse as the size of the tumour increases "  . bloom ( 1950 ) found 5 - year survivals in the following percentages : tumour less than 1 inch 59 per cent , 1 to 2 inches 45 per cent , and more than 2 inches 32 per cent , and he finds after grading of the tumours that : " of the tumours with a diameter of 1 inch or less , 37 per cent are classified as grade i and 23 per cent as grade iii . 
on the other hand , in the case of growths of more than 2 inches diameter only 8 per cent belong to grade i whilst 54 per cent are grade iii . when the diameter lies between 1 and 2 inches the incidence of these tumours is practically the same " , and he concludes that : " whether the tumours are small or large , the outlook is uniformly good in the former ( grade i ) and bad in the latter group ( grade iii )  . 
on the other hand , an intermediate result is obtained for the intermediate cases ( grade ii ) , the survival rate being practically halved in in other words , the metastasizing power the presence of the larger neoplasms . for growths for grade i and also grade iii cancers is independent of size . classified as grade ii this power bears a direct relationship to the diameter , the larger the tumour the greater the likelihood of spread having taken place "  . 
christiansen the time factors are so long or so short that their significance is obscured under the usual clinical conditions . when kunath ( 1940 ) says : " size and ma88 . - an analysis of this point failed to reveal that a larger tumour carries any more serious a prognosis than " no does a small one " , and hoopes and mcgraw ( 1942 ) likewise conclude : correlation was found between the post - operative duration of life and the size of the tumour removed " , these findings are not necessarily in contradiction to the opposite conclusions , mentioned above . 
the explanation may be a different composition of the groups of tumours , as regard number of representatives of the again we have to guard ourselves in transferring different grades of malignancy . conclusions from groups to individual cases . considering the importance of assessing the significance of a minimum size of tumour for the prognosis of breast cancer , we have examined breast cancer material consisting of a total of 974 cases . 
we planned to examine the ultimate development of breast cancers as small as practically diagnosable . in order to decide the upper limit of the size to include , theoretical and practical considerafirstly , we considered the smallest lump , tions have been taken into account . that size evidently depends upon the site distinguishable as a definite tumour . and upon the amount of adipose tissue . 
a tumour near the surface and in a shrunken atrophic breast will be more easily discovered than one deeply situated it seems that tumours smaller than those the size in a full and developed breast . of a pea cannot reasonably be distinguished from the many irregular nodosities in a cimacteric , or pre - cimacteric breast , and in most cases it is difficult to feel secondly , we wanted to examine tumours a lump smaller than a small hazel nut . of a size so small that the number of cases would represent a very small fraction of the total , whereby we would obtain a statistical claim to a designation " a very small breast tumour "  . 
the tumours are usually characterized by some object for comparison , usually : pea , bean , nut kernel , hazel nut , date - stone , date , wall nut , plum , etc . 
a tumour the size of a hazel nut will usually measure some 10 to 12 by 15 to 17 mone has to have in mind the small size of a norwegian hazel nut to appreciate the size . when we examined our total material we found that only 56 tumours out of 974 , i.e. , 5 - 7 per cent . 
the fate of the patients has been followed for from 10 to 20 years . the material was examined during the later half of 1952 . the clinical information is not always precise , or detailed , as some of the case histories are very short . 
nor is the pathological material always complete , as some of the surgeons do not submit adipose tissue from the axilla if no suspicious glands are found . in other cases a selection of glands only are sent to the laboratory , and finally , some of our paraffin blocks were destroyed during the war , with the result that for some cases a few slides only could be examined . this means that all the positive findings are minimum recordings . the clinical follow - up has been complete , all patients having been accounted for . reliable death certificates have been received for all the dead , and the patients alive have been personally examined by one of us , or by competent doctors as regards the patients living in remote parts of the country . as only the patients with a definite statement as to the size of the tumour have been included , the size is known in all cases . 
8 died from broncho - pneumonia one year and a half after operation , symptom - free . all the others succumbed to their tumours . this 5 - year oure rate corresponds very closely to the rate quoted by engelstad ( 1948 ) for his group of 768 cases , regarded as stage i and stage ii cases . this indicates that our " small " breast cancers do not present a particularly favourable clinical picture . in one aspect our table ii shows a remarkable deviation from the usual , namely , the small difference between the 5 - year and the 10 - year figures , only 1 out of 37 patients dying in the second 5 - year interval . as stated , the whole group of 56 patients has been followed for 10 years . a smaller group has , however , been followed for a longer period , and in table iii some of the results are summarized . 
even if we select the very smallest tumours present in our series , those the size of a pea or a bean , out of a total of 10 , we find only 5 patients alive after 10 years , or more , and 4 patients dead from metastases . in the present series the combined surgical and radiological treatment seems , in a number of cases , to have effected a considerable delay in a finally fatal this delay is of very great value from a therapeutic standpoint , even outcome . if the treatment fails to effect a complete cure . staging actually is an ambiguous process . the prolonged observation also helps to assess more accurately the real stage the simplest and least of the tumours . reliable staging is the immediate clinical , unreliable to a degree to make it practically useless . 
we have , however , to bear in mind that the value even of negative findings of tumour cells in lymph this grouping is rather limited . nodes removed may be the result of the pathologist not finding the cells in spite or , tumour cells may by - pass the usual lymphatic filters in of their presence . the axillary glands and proceed directly to the supraclavicular or the intrathoracic nodes , or the tumour cells may spread via the blood streathe discrepancy between the number of tumours anatomically staged as stage i and the number of cures effected after a proper local operation shows the degree of failure in the process of staging . 
on the other hand , if radiological treatment has been added to the surgical , even the delayed retrospective clinical staging may be incomplete , because a tumour , anatomically staged as stage i and actually being in stage ii , may be cured by radiological treatment and thereby escape its proper staging . in spite of the incomplete clinical and pathological information , it will be of some interest to examine the state of affairs , as regards stages recorded in this material . 
10 , 27 , 31 died symptom - free after more than 10 years ' observation . the large number of fatal outcomes with negative findings of tumour cells in the axillary lymph nodes confirm our assumption that our figures represent the considerable number of patients alive and symptom - free minimum findings . in spite of positive findings of tumour cells in the lymph nodes , actually 5 patients , further confirm our statement as to a considerable effect of the treatment , but l . 
christiansen also underlines the great number of patients already in stage ii ( or further ) at the moment of presenting a very small breast cancer . if we add these 5 patients with microscopical lymph - node metastases , but still symptom - free , to the group of the dead and those living with metastases , we find that two - thirds of all our patients with small tumours were in stage ii ( or further ) when the operation was performed . the value of bloom 's ( 1950 ) grading and analysis of his material made it natural to try his principles on our material . 
we deliberately use the word principles , because any grading is arbitrary , and the designation in each case is the result of a subjective estimate of a series of characteristics , which themselves are unprecise . 
the explanation may be that the number of cases is too small , that the treatment given may have influenced the final result because a number of very malignant tumours are comparatively radiosensitive , or it may be caused by our incompetency . 
the medium long histories , from a few months up to a year , show an increasingly gloomy picture . if we again deduct the grade i cases we conserve the pattem , and still find indications of a benefit for very quick reaction with immediate treatment after diagnosis , and with a still more pronounced bad prognosis in cases with delays from a few months up to a year . for the very long histories ( more than a year ) the comparatively better prognosis is preserved , and may partly be accounted for by a default in the process of grading , partly by combination of high malignancy and high grade radiosensitivity in certain cases , and partly by inclusion of tumours with a more moderate malignancy ( grade ii )  . besides , a perfect correspondence between morphological grading and biological development is evidently not obtainable . too many unknown factors are involved here . 
we have adopted the following staging , which is similar to that employed at the christie hospital and holt radium institute , manchester : stage i . - primary growth limited in extent to site of origno loss of mobility or function . 
lymph nodes fixed , matted or bilateral , or distant metastases present . table iv shows , as already mentioned , how many cases had already reached stages iii or 1v when first seeking treatment . 
warren and gates ( 1932 ) investigated the problem in considerable detail , and concluded that a patient with one cancer was statistically more likely to develop another cancer than a normal individual . 
watson ( 1939 ) calls attention to the fact that when a patient with carcinoma of the oesophagus has - a second growth this is nearly always situated in the mouth . 
it is therefore of interest to know how often this treatnment succeeds or fails in its objective , and in the latter case , when there is a recurrence , what are the chances of further arresting the disease . these facts cannot be determined with any real accuracy , since each case clearly has to be assessed on its clinical development and course . 
harmer in stage i and ii cancers of the lip , re - treatment is rarely successful . first planned treatment fails , the patient does not often have a second chance . if the response of primary and secondary to irradiation . glucksmann ( 1948 ) has stated that the response to irradiation of the primary and secondary manifestations of cancer of the mouth is about the same , and that those growths " with a tendency to lymph - node involvement differ in their biology and radio - curability from those without such a tendency . " the more generally held view is that while the primary may be " cured , " the secondary is often more resistant to treatment . only stage iii and iv cases can be analysed if the theory that an irradiated primary will recur as frequently as an irradiated secondary is to be examined . there were 248 patients with stage iii and iv growths arising from buccal sites in whom radiotherapy produced regression of both primary and secondary forty - five remained well , and 203 recurred : 61 in the primary only , growths . 69 in the secondary only , 73 in both . 
hartley has kindly done a significance test on these figures . the number of cases has been adjusted so that the proportion of stage iii and iv royal cancer hospital patients is the same as that of the holt radium institute series . 
the accessible growths of the for cervical mouth and lip , and teleradium for the oro - pharyngeal primaries . metastases block dissection hasbeen the method of choice , or radium therapy for the inoaperable cases . x - rays have&been used comparatively rarely . memorial hospital , new york ( martin , 1948 )  . - hayes martin states that surgical excision is the method of choice for cancer of the lip , gum , anterior for oro - pharyngeal portion of the tongue , cheek , floor of mouth or palate . growths a combination of roentgen therapy and interstitial radiation is indicated . cervical metastases are treated by dissection when operable and by x - rays when not . radiumhemmet , stockholm ( berven , 1937 )  . - for more than 20 years - the usual treatment for growths of the tongue and most other buccal sites has been teleradium , with diathermy coagulation of any residual mass . 
sixty - five per cent had symptoms for less than 6 months before treatment commenced , yet ( with the exception of carcinoma of the lip ) in 60 per cent of cases the disease had already metastasized to the cervical lymph nodes when first seen . 
the treatment of these 800 cases has been largely by radiotherapy . comparison of the results of treatment published by different authorities is table xv shows wide difficult because of lack of uniformity in criteria adopted . divergencies for instance in the survivals of patients with growths of the buccal this is largely due to mucosa , and to a lesser extent of the alveoli and palate . the small numbers of the cases . the last section of this paper shows that although very different methods of it is treatment may be employed , the results on the whole are much the same . therefore inadvisable to attempt definite conclusions from the analysis of the cases presented . 
the cases have been under the care of all members of the staff of the hospital , past and present , to whom grateful acknowledgment is made for permission to publish them . in the preparation of the paper much help was derived from copeland - chatterton punch cards , many of which had been completed by j . 
the carcinogen was then placed in contact with the surface of the epithelium , and ingerted into a bashford transplanting needle , care being taken to shield the carcinogen from the connective tissues of the host animal . in some instances as manv as tbxee subcutaneous primarv grafts were made on each side of the belly of a s ' mgle host mouse to give a series of prostatic tumours growing under identical hormonal conditions . 
the technique is similar in some essentials to that - previously employed bv greene ( 1945 ) , and rous and smith ( 1945 ) , except for the important difference that the present experiments involved tiimour production from adult , not embrvonic tissues . the influence of bilateral orchidectomv upon the growth rates of glandular and squamous cefl prostatic carcinomas was mvestigated in 6 - months - old mice , which had pre ' %iously been castrated before attaining pubertv . 
10 were of the squamous variety , and 2 were spindle - cell sarcomas . sixteen homologous grafts failed to gi - ow , and were foiind at autopsy not to have becoi - ne vasettlarized . in order to detern - iine the early phases of carcinogeiiesis , witliin the primary grafts a further 30 mice received prostati c iniplai - its , and , " , - ere sacrificed at intervals ranging from two weeks to five inonths after iniplantation . 
which are practicallv fused together with an anterior portion and an isthmus or middle lobe which , as in the rodent , surrounds the urethra . unlik - e the hu - man crland , the mouse prostate has no uterus masculinus or its equdvalent , nor is it encapsulated . 
thus in the human gladd there are as many as 32 ducts ( 31axiniow and bloom 1948 ) , whilst in the mouse them are onlv six . a fibromuscular stroma consisting of dense connective tissue is common to both ty - pes of gland . 
the cytoplasm of the glandular epithelium in both species contains numerous secretorv granules , the majority cytologically treated mouse maerial shows the secreticn of which are , of lipids . in both forms to consist ot protein w%ith fine lipoidal droplets in suspension . 
iiornixg to a failure in the release of the secretion , siiice the ducts ai - e iio lojitretpatent , and proliferation which might be due to the direct action of the carciilogeii . neoplastic change - s in graft8 implanted with the carcinogen . the carcinogen , which is placed in direct contact with the living tissuewithout solvent such as oil . 
or lard , induces verv little foreign body reaction , necrosis or residue within the graft which iiiight mask or conceal the actual areas where neoplastic changes fii - st arise . 
h perplastic alveolus froni which it has arisen . through a whole graft of dorsal lobe epitheliuni fixed 41 weeks after implantation . there is very little foreign bodv reaction , and it will be iioted how clearly the foci in which iiialignant changes arst arise may be detected . adjacent to alveoli lined with a , low colunuiar epithelium and distended with secretion colitaining epithelial debris and polyiiiorphs are collapsed alveoli in wliieb the epitheliuni has entered an exhaustion phase . 
the tongue - lik - e colony , of earlv malignant cells , of t3 - pe a was t3 - pical of those tumours which finally developed into secreting glandular carcinomas . l [ n the example ihustratc ; 1 the proliferation arose from a single alveolus and was the only fo - cus of later stages from other grafts showed malignant change in this part - icudar gr - aft . the onset of secretorv activitv . 
14 shows three ii - ialignant alveoh in the peripheral engaged in secretion . area of an invading new grom - th of this type from an eight weeks ' old graft.. several epithelial cefls are in process of abnormal cefl division . 
transplanted into n - lice of the s - ame age and sex as the host mouse , retained their histological charact - c - rs for 16 generations of serial transplants before finallv transforming into a squamous - cehed tumour . the type b lesion arose in the various grafts exaniined from a single alveolus . aild the example illustrated in fig . 
the area of the alveolar wafl malignant cells - , contain small nuclei with nunierous atypical mitotic figuxes . apart from the different morphology and staining reactions of the prohferating cells . 
it will be noted that they tak - e origin from an alveoluss in which secretory adjacent alveoli not shor% - ing these proliferative activitv has been inhibited . changes are distended with secretion . in older grafts the type b prohferation leads to the formation of pseudo - alveoli at the margins of the tumour and . although the glandular architecture of the epithelium is thus partly restored , there is no evidence of secretion . 
the secretory epithelium like that in the mouse gland is thrown into folds which project into the glandular lumina . note alveolar concretions , and compare with that of mouse in fig . 
4. - implant of portion of dorsal epithelium from a normal strong a mouse without carcinogen after surviving subcutaneously in a host male mouse of same age and strain for 12 weeks . note distension of alveoli with prostatic fluid which is far in excess of what is typical of the normal gland in 8itu , and frequently leads to cystic dilatation . 
9. - early hyperplastic changes in prostatic epithelium of a strong a mouse , after eight weeks ' subcutaneous implantation with carcinogen in a host mouse of same sex , age and stra ' the glandular epithelium contains patches which are 10 - 20 cells in depth . 
on palpation the tumours undergoing regression were hard and the two which failed to respond to orchidect.omv were soand sin - iilar nodular t.o those growing in the control mice . 
variable influence of orchidectomv on the behaviour and gro - wth of this alandular carcinoma was further demonsirated in another series of experiments which were repeated at a later date . in this instance two groups of mice , 10 in each , lik - ewise castrated before pubertv . 
received transplants of the same tumour . the gro , %th rate of three tuniours in the first group of io mice was entirelv four tumours in the second group also grew at approxitunaffected bv castration . iiiatelv the same rate as the transplants in the intact control mice . 
four tumours - , responded to this treatment bv showing an immediate increase in growth rate while the , remainder were una#ectedsquanwus - cd , l cwrcinonia.. a glandular cell carcinoma which had undergone spontaneous inetaplasia after its 10th generation of serial transplants in non - castrated normal male mice , was chosen to determine the possible influence of orchidectomv on a glandular tumour which was undergoing squamous transformation . microscopic exan - iination of this particular tuniour had sho - w - n that the metaplastic alveolar epithelium was columnar and much of it still retained secretorv activitv . 
a variable degree of keratinization associated with prickle cell transformation of the epithelium occurred in concentric nests of cells in the peripheral alveoli of the tumour . comparison of the grouth rates of this particular tumour transplanted inw intact control mice . 
the occasional slight inhibition of tumour growth in soiiie orchidectomized mice was within the limits of possible normal variation . the histological changes accompanying vaiiations in grawth and behaviours , in castrated and non - castrated mice . 
17 , were ha ' rder and tnore nodular on palpation than those growing in secretory activity as judged from sectioiis had beeil sitpcontrol intact mice . pressed and squamous metaplasia , of variable intensity in different areas of aiiy tumour , was always found . 
 ' portions of this tumour failed to grow when transplanted into normal ho ; t rnice . tumours inhibited bv castration but subsequentlv treated with testosterone . after which wth was renewed . 
the alveolar character of the growth had persisted , but.the epithelial cells were shorter than in the original untreated tumour . these results were confirmed in a second series of experiments involving the same number of castratm mice using the same dosages of testosterone . 
tumours found to be retarded in growtli during the fifth week of treatment were characterized by a decrease in the size of the majoritv of the alveoli , a reduction in the height of the epitheliuni , a coinplete disappearance of cell - divisioii , and an increase in the fibro - mtiscular stroiiia . 
the alveoli diminished in size when this rupture occurred , whereas adjacent alveoli with intact epithelial cells appeared to be so slightly affected by the oestrogen that little more than a reduction in the lieight of the cells could be obsei - ved . 
a detailed study of the hyperplastic ' changes in numerous grafts has shown concltisively that in no single instance has an actively secreting epithelium been the focus of malignant change . 
the technique used in the present without at the same time da experiments is pomibly supenor to that involving injection of carcinogens , dissolved in such solvents as lard , merely because it entails less damage and concentrates the carcinogen at the proper site . since the present technique also involves homologous grafting , however , the reaction between host and graft tissues may play some part in the successful induction of a tumour . in mice the survival of a homologous graft seems to depend on the use of a closely inbred stock mice of incleterminate ancestry will not often tolerate grafts for strain . periods sufficient for the establishment of tumours . recent experiments have indicated fwrthermore that the age of the mice providing the graft tissue , and the age of the host mice into which the grafts are implanted , have an important bearing on the problethus the survival rate of grafts of lung or prostatic epithelium without carcinogen was greater if host mice under eight weeks old were used , and was considerably reduced if the mice were over eight months this was invariably the case if the mice were of mixed or pure line origin , old . but only 12 per cent of the grafts in young mi ed stock mice survived as against in older mixed stock mice of 8 months 85 per cent in the young pure line mice . to one year no graft survivecl after three months ' implantation , whilst in inbred mice of a similar age 25 per cent of grafts survived after the same period . 
combining the carcinogen 20 - methylcholanthrene with grafts of lu or p - rostatic epithelium in 25 eight - weeks - old mice of mixed stock gave only one tumour , a spindle - miled sarcoma , twelve weeks after grafting ; the remaining 24 host mice failed to produce tumours , and at autopsy the grafts were found not to have price ( 1941 ) , using rats of different ages from an albino become vascularizedl strain which was not strictly pure hne , found that ventral lobe prostatic tissue grafted subcutaneousl in the abdominal wall beha - ved in much the same way , prostatic tissues from old rats being very limited in their capacity to survive as homologous grafts . 
many apparently viable grafts from old rats were found to be degenerate on histological examination , but when regrafted into younger hosu the implants underwent a rapid recovery and the pmstatic epithehum even regained its secretory activity . 
one of the most intere8ting features in successful grafting of prostatic epithehum is its ability to continue secreting while growing under the influence of foreign host reactiom and changed vascularity . 
ob - viously androgenic stimulation , as price ( 1941 ) maintains , must be important in this phenomenon . the dependence of prostatic epitheli - um upoii androgen smretion has long been realized and the experimental evidence need not be referred to here , except to mention that in recent years the elegant technique of transplantation of fmgments of prostate into the anterior chamber of the eye ba - s provided additional confirmation . and rosenblum ( 1937 ) , moore , rownblum , tolin and melchionrka ( 1937 ) , and moore and smith ( 1937 ) , grafted rabbit prostatic epithelium in this way and photographed the implants dailv . 
they were able to record fluctuations in size of the grafts which were abohshed by castration , and considerably increased by administration of testosterone propionate . heckel and kretwhmer ( 1935 ) made similar observations of the responses of prostatic grafts to anterior pituitary extracts . 
horning mice or rats will maintain the size and normal histological appearances of the hill and strong ( 1938 ) attribute the androgpnic prostate and seniinal vesicles . effect of the ovary to the lowered temperature of the ear , and deanesly ( 1938 ) has correlated this activit ' with extensive luteinization of the theca intema within the ovarian grafts . another potential source of androgens to be considered in relation to prostatic grafts and tumours is , of course , the adrenal cortex . 
the rare adrenal insufficiency in young children associated with adxenal hypertrophy leads to enlargement of the prostate and differentiation of its glandular tissues to a degree comparable with the adult gland ( dijkhuizen and behr , 1939 - 40 )  . prostatic enlargement also occurs in adults with adxenal cortical tumours . miller ( 1947 ) has found a relative increase in dehydroisoandxosterone , and a decrease in androsterone in the urine of patients with benign hypertrophy of the prostate . according to callow and callow ( 1940 ) the source of dehydroisoandxosterone is probably in the adxenal cortex . it remains in the urine after castration , but disappears in cases where there is destruction of the adrenal cortex . while it niight be assumed that grafts of prostate or transplantable prostatic tumours would have poor chances of survival in female hosts , under certain conditions they do in fact survive and this must be attributed to the influence of androgens produced either in the ovary or in the adxenal cortex . price ( i 941 ) grafted normal prostate into female host rats , and found that in some cases the grafts were similar in histological structure to the prostates of castrate males , whereas other - grafts flourished and underwent secretory activity . this result she maintained could be due to the development of androgenic foci in the ovaries of some , but not all , host animals . 
the grafts failed to differentiate in spayed rats , whereas they survived in a functional condition for prolonged periods if the hosts were over 80 days old . either virgin females or rats which had littered frequently would sustain functional grafts , provided they were not youngei than this apparently critical age . the opinion is often expressed that the prostate gland in rodents is not strictly homologous with the prostate in the human , and thus there can be rttle justification for comparing growth behaviour and endocrine responses in experimentally induced prostatic tumours or hyperplasias with the spontaneously occurr , ingconditionsinthehumangland . greenstein ( i - 947 ) maintainsforinstance , that because certain rodeint prostate tumours grow when transplanted into female mice , whereas human tumours are so sensitive to inhibition by oestrogens , the two types of neoplasm are not fundamentally the same . 
apart from the single case reported by deniing , jenkins and van wagenen ( i935 ) of a spontaneous nodular hyperplasia in the suburethral tissue of an albino rat , there is no record of spontaneous prostatic hyperplasia or cancer in rodents . if it can be shown , however , that the prostate tumou - ts induced by methylcholanthrene behave in essentially the same way to hormone administration or deprivation , the differences between rodent and human neoplasms will be less significant . the glandular carcinomas described in these experiments , when transplanted into rnice ca 'strated before puberty , show complete inhibition of secretion , later followed by squamous differentiation . 
subsequent traimplantation into castrated hosu is not followed by any appreciable inbibition . some glandular celled carcinomas am thus dependent for their sustained growth on an adequate androgen level ; the squamous growths would appear to flourish without testicular androgens and , if they are at all androgen - dependent , it must be concluded that adrenal cortical androgens are in these cases suflicient . and hodges ( 1941 ) buman prostatic cancer falls into two according to it is possible that groups , androgen dependent - and androgen independent . squamous - celled carcinomas in mice belong to the second category , although aours rarely occur in man ( willis , 1948 )  . such t a smah percentage of glandular mouse tumours failed to regress when transplanted into castrated hosts ; they even grew at approxim tely the saine rate m those transplanted into uncaamted mice . 
howard ( 1937 , 1938 ) has demonstrated that removal of the gonads in rats , provided these are sufficiently yoijn , increases the capacity of the adrenals to secrete androgens . 
as ar the mice in the present experiments were castrated before puberty , and an interval of several months elapsed before they received t aour transplants , it seems probable that androgenic foci had developed in the adrenals of those castrated mice in which unfortunately.biochemical assay the tumours showed no inbibition of growth . of androgens secreted from non - testicular sources cannot be undertaken in the mouse to confirm or refute these conjectures . on the chnical side the serum acid phosphatase and urinary 17 - ketosteroid levels can be used respectively as indicators of the activity of mahgnant prostate cells and of androgen smretion . 
thus mn4vs stevens and hodges ( 1941 ) studied cases in which a recurrence of prodatic cancer had fobowed an initial improvement in the condition obtained - by bilateral orchidectomy . there was a rapid reduction in the serum acid phosphatase , and a simil ri marked fall in the urinary 17 - ketosteroids after orchiclectomy . 
the development of androgenic foci in the adrenals was regarded by and swtt ( i945 ) as responsible for the recurrence of the disease . recently , following failure in four - selected cases to control growths by orchidectomy , these worken tried bilateral adrenalectomy ancl , although three of the patients died within a comparatively short fume after operation , the fourth survived for 116 clays . 
cox ( 1947 ) tried unilateral adrenalectomy on three patients with prostatic cancer , all of whom had temporarily responded to treatment with there was an immedi te drop in the 17 - ketosteroid level and a oestrogens . r tumours transplanted into normal male mice showed a varying retardation of growth after oeamgen treatment , but in no single instance did any undergo complete regression , as was the case following orchidectomy . 
horning inhibition of tumour growth with such relatively high doses was the result of non - specific toxic action . it was also found that the amount of stilboestrol tolerated depended upon the genetic constitution of the rnice . 
although in the present experiments the dose of stilboestrol was approximately half of that used by ludford and dmochowski ( 1947 ) , the average loss of weight of the tumourbearing rnice was approximately i ' to 2 g . 
during the total period of treatment . histological examination of regressing glandular tumours after stilboestrol gested that diminution in size of the tumours was primarily due to thera inhibition of secretion , later followed b ' degeneration and collapse of alveoli . schenken , burns and kahle ( 1942 ) and fergusson ( 1946 ) have exandned serial biopsies of human prostatic carcinoma under oestrogen treatment . 
the former authors described in detail such cytological changes as vacuolation of the alveolar epithelium , disintegration of the cell membranes , sometimes accompanied by stratification of the epithelium , and an increase in the stroma . these changes are very similar to those which occur in the glandular mouse tumours during oestrogen treatment . 
the squamous metaplassia , which some of the glandular mouse tumours undergo in response to oestrogen administration , is rarely found in the human gland after sirnilar treatment . recently , however , inglis ( 1948 ) described a pronounced squamous differentiation in a man treated with stilboestrol for prostatic cancer . it is tempting to regard the atrophy of prostatic tumours in the mouse as due to failure of optimum androgen secretion in the castrated hosts , or to a similar drop in gonadotrophin production when intact tumour - bearing mice are treated those tumours whieh fail to regress are possibly receiving with oestrogens . sufficient androgenic stimulation from the adrenal cortex . inhibition of secretion and degeneration of the epithelium in these tumours are so similar to the changes induced in the normal prostate following castration or oestrogen administration that any more complicated mechanism operating in the case of the tumourbearing mice seems unnecessary . 
it bas been possible in grafts , impregnated with the carcinogen , as soon as four to eight weeks following implantation , , to distinguish between three distinct types of early malignaint lesions . detailed swdy of the hyperplastic changes in prostatic alveoh in numerous grafts showed that in no single idstance has the actively secreting epithelium been the focus of mahgnant change . it tberefore appears that the non - secreting exhausted alveolar cells m the prostatic gland are more susceptible to the action of the carcinogen than those at the height of secretory activity . this supports the contention that chemical carcinogens act - more readily on cells following depression of cellular activity . the influence of sex hormones and orchidectomy was determined on the behaviour and growth of glandular and squamous - cell carcinomas of the prosute . a greater number of glandular carcinomas regress when transplanted into male mice cm - trated before puberty , and a small percentage of - the - e tumours resume growth when treated with testosterone propionate . 
tumours which at the time of their induction were diagnosed as squamous growths , or glandular carcinomas which had undergone spontaneous squamous differentiation during serial transplantation , exhibited no appreciable response to these fornvs of therapy . these results indicate that only glandular and not squamous carcinomas are dependent for their sustained growth on an adequate androgen level . glandular carcinomas when treated with dieth - vlstflboeaml respond more re - adily than squamous t aours . 
and bave given additional information on the factors responsible for tumour regremon . particular attention hm been paid to those glandular tumours which failed to respond to either orchidectomy or hormonal theraphy , and the possible endocrine factors inducing resmtance to treatment are discussed in the hght of recent experimental and clinical evidence . this investigation has been supported by grants to the royal cancer hospital , fi - om the british empire cancer campaign , the jane coffin childs memorial fund for medical research , the anna fuller fund , and the u.s. 
at the time it was known that adenomas of this gland produced abnormalities in children and young adults ; it was therefore postulated that in later life , when gigantism and acromegaly were no longer possible , increased activity of the pituitary gland might be responsible for the production of tumours ( funk , 1917 )  . for a long time and for obvious reasons it was impossible to put this hypothesis to a test . 
thanks to the classical work of evans , li and simpson ( 1948 ) we now have the necessary tools for preliminary and systematic appraisal of the importance of various hormones of the anterior pituitary in tumour growth . the purpose of the work reported in this paper was to test the effect of various anterior pituitary and related hormones upon the growth of implanted tumours in hypophysectomized rats . 
as a supplement to this work we also dealt with the removal of certain organs ( mainly glands of internal secretion ) on the rate of growth of a transplanted rat tumour in otherwise intact animals . while there is no doubt that studies on this subject have already been made , it appears doubtful whether a systematic study was ever made of the effect of the removal of these organs upon the growth of one particular tumour . 
to check on the completeness of the surgical procedures used we have excised and weighed , at the time of sacrifice , those organs that are in a physiological relationship to the excised gland . for instance , in orchidectomy the seminal vesicles and the prostate were weighed . 
the diet was the same as in the hypoall animals received tap water , except the adrenaphysectomy experiments . lectomized animals , which received 1 per cent saline . in some experiments rats 282 c . 
no improvement in survival was noted . 1 of the controls 40 per cent had open vaginas , none of the castrates , at time of implant . a mortality was about 30 per cent of the animals operated upon . 8 mortality of 33 per cent of the operated animals , probably due to bartonella muria infection . 4 all animals received 60 mg . 
of alloxan from the time of tumour shaefer - hartman blood - sugar determinations were as implantation to the end of the experiment . follows : pre - implant 461 - 9 mg . 
using the enriched diet in our present work , we have attempted to restore tumour size by the administration of some anterior pituitary hormones and serum gonadotropin . growth hormone increased tumour weight of hypophysectomized animals so treated . however , this increase is directly proportional to the increase in body weight due to the hormone . thus , tumour weight difference per g . 
body weight difference is the same for both growth hormone treated and intact animals using untreated hypophysectomized animals as the base . it would appear that the influence of growth hormone on tumour growth is related to its effect upon the general metabolic condition of the animal . this is not true for acth and thyrotropic hormone . these two hormones cause a greater increase in tumour growth than could be expected from their effect upon body growth , using the same criterion . the ratio of tumour weight to body weight gain in adrenalectomized animals seems to confirm the fact that without adrenal hormones the tumour does not grow as well as in intact rats . this is the opposite of the effect ofacth treatment . 
the restorative effect of the latter hormone and the decreasing effect of the operation , greater than their respective actions upon body growth , show a clear effect on the tumour itself . 
our data are not as clear in the thyroidectomy experiments , however ; in one experiment the tumour - body weight ratio is indicative of the depressant effect of this operation upon the tumour . in the other experiment the administration of thyroxine to a thyroidectomized animal had the same effect as thyro286 c . 
ehrlich tropic hormone given to a hypophysectomized animal . increase in tumour size greater than could be expected from the body growth . this effect was an the effect of fsh and serum gonadotropin on hypophysectomized animals was not very marked either on absolute tumour weight or on tumour weight per g . 
body - weight gathe treatment of castrated males with testosterone may have stimulated tumour growth . splenectomy may have had some effect upon increasing tumour size by both criteria . while it is clear that diabetes per se reduces tumour growth , it is not so clear whether or not this reduction is related to body weight . it has been shown by mceuen and thomson ( 1933 ) that the reduced rate of tumour growth in hypophysectomized animals could not be solely attributable to the concomitant metabolic deficiency . in their studies on intact animals , evans , moon , simpson and li ( 1950 ) have found that only by long - continued treatment with growth hormone were they able to produce neoplasms , while shulman and greenberg ( 1949 ) in a shorter experiment , using intact mice , found no effect upon tumour growth with this hormone . 
daily with their diet ( bielschowsky , in experiment ii the ordinary stock diet ( hall , 1948 ) was 1944 ) for 25 weeks . given and the carcinogens , dissolved in peanut oil , were administered by stomachtube . 
the survivors of experiment i were killed in the 63rd and those of experiment ii in the 52nd week of the experinment . table i summarizes the results of experiment i . for comparison the tumours obtained by feeding 4 mg . 
the first tumour , a hepatoma , was examination . foumd in the 54th week , and the second , a cancer of the meatus acousticus externus , 3 weeks later . 
by the 24th week neoplasms of the breast gland were present also in cancers of the meatus acousticus externus made their the two other groups . appearance in rats treated with the methyl compounds from the 34th week palonwards , whereas in the a.a.f. 
group the first was seen in the 25th week . pable liver tumours , lowever , occurred only in the latter group after the 33rd week of the experiment . these consisted of a few minute cysts . the total yield of cancers induced by d.a. 
induced two hepatomas , definite signs of liver damage and fairly widespread cystic cholangiomata in 2 other males of this group , while 2 of the females showed a few minute cysts in the liver . 
four hepatomas were found in the males and one in the females , cystic cholangiomata being present in all . to summarize , the genetic background determined to a large degree the site of tumour development . 
thus breast tumours appeared in a much higher incidence in the wistar rats than in the piebalds , in which cancers of the small nevertheless in both strains a striking difference intestine were readily induced . in the response of the liver to the three compounds was apparent . in the first experiment 700 mg . 
were given , while in the second the total dose administered amounted to about half of the methyl compounds and to approximately a quarter in the case of a.a.f. yet in both experiments a.a.f. 
is readily split off in vivo and therefore can be only of minor importance for the carcinogenic activity has been substantiated by weisburger , weisburger and morris ( 1950 ) , who showed that after feeding w_ - c14 - 2 - acetylaminofluorene radioactivity appeared in the breath within 6 hours . ray and argus ( 1951 ) found the toluene - sulphonic ester of 2 - aminofluorene to be non - carcinogenic for the rat , only minute amounts of this ester being morris , dubnik , dunn and johnson ( 1947 ) , studying hydrolysed in the body . the action of 2 - nitro , 2 - amino , mono - acetyl and diacetyl - 2 - aminofluorene , reported that the acetyl compounds induced the majority of liver tumours observed , when the substances were given per os . why the acetylated aminofluorenes have such an affinity for the liver , whereas m.a. 
bartholomew 's hospital , london , e.c.1. received for publication january 12 , 1949 . thie carcinogenic activity of ethereal extracts of domestic soot was demonstrated by passey ( 1922 )  . domestic soot may contain as much as 40 per cent of tarry matter ( cohen and ruston , 1925 )  . 
the use of the fluorescence spectrum by mayneord and hieger led to the identification of 3 : 4 - benzpyrene in gas - works pitch ( cook , hewett and hieger , 1933 )  . this discovery suggests that this hydrocarbon might be one of the carcinogenic agents in domestic soot , although the conditions of pyrolysis in the domestic hearth , and in the gas - works retort , are different . berenblum and schoental ( 1947 ) found that carcinogens other than 3 : 4 - benzpyrene were present in coal tar . 
the first attempt to identify 3 : 4 benzpyrene as a contaminant of the atmosphere was made by hieger ( 1946 ) , who exposed non - fluorescing benzene to the air in london , and found that the fluid became fluorescent and showed spectral bands resembling those of benzpyrene . 
for the present purpose , samples showing a ph value of 10 - 0 - 10 - 3 were found to be most suitable . 3 : 4 - benzpyrene . - the commercial product contains a pale - blue - fluorescing impurity which is very strongly adsorbed on alumina . 
benzene. the first attempts to separate benzpyrene from soot were based on the method described by berenblum ( 1945 ) for the extraction of benzpyrene from coal tar . in the original method vacuum distillation of the suitable fraction of tar was followed by solution of the distillate in concentrated sulphuric acid . attempts at vacuum distillation of benzene extracts of soot showed , however , that the composition of the soot extract was very different from that of coal tar . 
evaporation of the benzene extract yielded a residue consisting mainly of non - volatile material , which appeared to decompose on distillation in vacuo with evolution of gas . direct sulphuric acid extraction of the crude tar extracted from soot was also unsuccessful , probably owing to considerable sulphonation . moreover , recovery experiments , with microgram quantities , of pure benzpyrene from icecold sulphuric acid showed that considerable loss of the hydrocarbon occurred , and that the method was useless as a quantitative one for such small amounts . chromatographic adsorption was next tried . 
when an extract of soot was submitted to chromatographic separation , the characteristic fluorescence spectrum experiments with various of benzpyrene was given by the appropriate eluate . samples of alumina showed that spence 's type h alumina , containing adsorbed benzpyrene , could be washed freely with a mixture of benzene - petroleum ether ( 1 : 10 ) without elution , and that the benzpyrene was eluted only when the proportion of benzene was increased ( 3 : 10 )  . 
the extract was given a preliminary purification by passing it through a short column of alumina ( 14 x 25 mm . ) and eluting with the same mixture of solvents . 
the lower band was easily eluted with benzene - petroleum ether ( 1 : 10 ) , while the upper could only be washed down when the benzene content of the eluting fluid was increased ( 3 : 10 )  . this latter fraction is shown in fig . 
cotton treated in a colloid mill . this was prepared from cotton velvet shearings suspended in water and passed through a colloid mill 6 times in succession . after each passage only the finer fraction was slected for further milling . 
5 shows the compact growth of mouse mammary carcinoma obtained in the presence of fibres of colloid milled cotton ; as a contrast the uneven growth of cells in the absence of fibres is shown in fig . 
for 5 minutes , washed with distilled water until free from ammonium ion ( tested by nessler 's reagent ) and sulphide ion ( tested by sodium nitro - prusside )  . they were then bleached 15 minutes at room temperature in sodium hypochlorite solution containing 0 - 1 per cent available chlorine , and finally washed with distilled water until free from chloride ion ( tested by silver nitrate )  . 
they were stored under distilled water . the films were autoclaved in distilled water at 18 lb . / square inch pressure , and before use were rinsed in sterile tyrode solution , excess moisture being removed with sterile filter - paper . cultures were placed directly on the coverslip , a drop of mixed serum and embryonic extract spread over them and the viscose film then applied . 
the culture usually adhered to the glass surface , and on washing or fixation the film floated off . the film is easily cut with a cataract knife when the tissue is subcultured . with a thickness of 30 , u the total thickness of the preparation was considerably less than can be achieved with the double coverslip method of maximow . 
they have found that perforated cellophane is an advantageous addition to flask cultures and hanging - drop preparations ( evans and earle , 1947 ; earle and evans , 1949 ; schilling , earle and evans , 1950 ; earle , evans and schilling , 1950 )  . in our hands perforated cellophane , kindly supplied by w . 
earle , was very siinilar in effect to viscose , but the outgrowth obtained with viscose seemed slightly greater . glass wool has been used for the same purpose by warner , hanawalt and bischoff ( 1949 ) , who used it with the roller tube method . 
viscose ifim , colloid milled cotton and durafil were technicauy suitable for use , and have been found advantageous in improvilg growth of malignant cells and of enabling growth of normal and malignant cells to take place in fluid media . we wish to thank professor astbury of leeds for his advice , and t . 
of the hydrocarbon suspended in arachis oil , by intraperitoneal injection , in no case , however , even with 100 mg . and the rats weighed daily as a group . average 1 : 2 : 5 : 6 - dibenzanthracene , after 9 days a second injection of growth rate of any group of rats observed . 1 : 2 : 5 : 6 - dibenzanthracene was given and again no the same amounts of constant effect was observed , but after several days considerable fluctuations in the growth rates occurred , and eventually 21 days after the first injection all the groups showed a lower average weight than the control group , which had although the average weight of the received injections of arachis oil alone . group which had received the two injections of 100 mg . 
1 : 2 : 5 : 6 - dibenzanthracene was slightly lower than that of the other group , there was no direct relation to the amount of hydrocarbon given , and the rats receiving 50 mg . had a higher average weight than the group which had been given 25 mg . 
of the other five left as controls . pituitary extract daily had practically no effect on the growth of the animals , but when the amount was increased to 1 c.c. 
1 : 2 : 5 : 6 - dibenzanthracene to rats maintained on a 20 per cent protein diet causes no immediate inhibition of growth , a delayed toxic effect occurs which ultimately results in death of all the animals . 
when the length of time survival of the dibenzanthracene - treated rats on the 20 per cent protein diet is compared with that of the similarly treated animals maintained on the 10 per cent protein diet , as shown in fig . 
6 , it is seen that although in the latter group there is an immediate growth inhibitory action , the animals survive for a longer period after treatment . ( mean survival time of 17 animals on the 20 per cent protein diet was 44 days after dibenzanthracene injection , whilst that of the same number of animals on the 10 per cent protein diet was 67 days . ) effect of 1 : 2 : 5 : 6 - dibenzanthracene on tail growth in rats maintained on a 20 per cent protein diet . an attempt was made to see if any growth - inhibiting action of 1 : 2 : 5 : 6 - dibenzanthracene could be demonstrated by measuring the increase in tail length of rats treated with this hydrocarbon . young male rats of about 50 g . 
the animals were lightly anaesthetized with ether , placed on x - ray film in the prone position , and skiagrams taken of the bones from the pelvic region to the tip of the tail . 
of stilboestrol was injected in all but two of the animals growth of the tail was markedly into each rat . inhibited , and in 3 rats tail growth practically ceased . 
the difference in the diets used is mainly of the protein content and , since the fat content of the diets is the same , their total it would thus appear that the procalorific values do not differ appreciably . tective action of the 20 per cent protein diet is directly attributable to its high this is confirmed by the protective action of the 25 per cent protein content . protein diet , which differs from the unprotective 10 per cent protein diet only in protein content . the growth - inhibitory action of the hydrocarbon does not appear to be the result of a direct action on the anterior lobe of the pituitary gland causing interadministration of growth hormone ference with growth hormone secretion . to rats maintained on the high protein diet and treated with dibenzanthracene does , in most cases , bring about some increase in growth , but appears at the same time to increase the toxic action of the hydrocarbon since this growth dibenzanthracene is increase is rapidly followed by the death of the animal . only very slightly soluble in body fluids , and when administered by intraperitoneal injection remains in the peritoneum for a considerable period of time . metabolism of the compound probably takes place by combination with since this combination is sulphydryl - containing substances as its first stage . not the direct cause of its growth - inhibiting action ( elson , goulden and warren , 1947 ) , it is probable that a later stage metabolic product is responsible for this effect . 
one of the effects of the growth hormone is to increase the total metabolism of the animal , and it seems likely that this results in increased absorption and metabolism of the dibenzanthracene from the peritoneum of the treated animal . 
of 1 : 2 : 5 : 6dibenzanthracene per rat usually results in very little growth inhibition for about 14 days , after which the animals , at varying intervals , lose weight rapidly and die . 
1. received for publication july 1 , 1948 . in the course of experiments on a transplantable mouse sarcoma it was observed that fresh saline extracts of tumour tissue agglutinated mouse and rabbit red cells . 
when extract and red cell suspension were mixed on a slide agglutination became visible to the naked eye within 30 seconds and appeared to be complete within 2 minutes . a number of different mouse and rat tumours , and a wide range of normal tissues , have been examined for haemagglutinating activity . 
when quantitative results were required a tube test based on that used by burnet and his co - workers ( burnet , beveridge , mcewin and boake , 1945 ) was adopted . serial two - fold dilutions of tissue extract were made in calcium - saline in round - bottomed tubes of approximately 0 - 8 cinternal diameter . 
of a 2 per cent suspension of washed red cells in calcium - saline ; the tubes were then shaken and allowed to stand at room temperature for 20 - 30 minutes . 
a preliminary reading of the end - point could then be made , if a quick answer was needed , which did not differ by more than one place from the final reading . calcium - saline , 0 - 3 c.c. , was then added , the tubes were shaken and allowed to stand a further 1 hours at room temperature , when the final reading was made . the type of agglutination , and also of the deposition of red cells in saline controls , varied with species . 
mouse cells seldom , and rabbit cells hardly ever , gave the compact button of deposit which is typical of unagglutinated fowl or human cells , and which workers with influenza virus haemagglutination take as negative . rabbit cells , in the absence of an agglutinating agent , settle in a round - bottomed tube as an ill - defined deposit surrounded by a slightly granular film , which would be regarded as evidence of agglutination of fowl cells . resembles closely the " shield pattern " which has been described ( burnet and stone , 1946 ) as typical of agglutination of fowl cells by lipoids . 
proved a it was effective in low concentration ( 0.005 m ) , very satisfactory preservative . and though it increased slightly the granularity of deposits of red cells in control tubes , it had no haemagglutinating action which could be confused with that of tumour extracts , even in concentrations much higher than this . it was slowly lytic for mouse red cells ( though this did not interfere with tests read after the usual period of two hours ) but not for those of other species . 
a final concentration of 0 01 m ( 0 12 per cent ) was generally used ; this was conveniently obtained by adding to a tumour extract 1 / 50th of its volume of a saturated aqueous solution of b.a.l. 
brought about regeneration of the activity of inactive extracts to their original titre . this regeneration was progressive : some activity was apparent in a previously inactive extract a few seconds after the addition of b.a.l. , but the full titre was not reached for about an hour , at room temperature . table i shows the preservation by b.a.l. 
fowl cells of each type , distinguished by a previous test against kahn antigen , were included in this test . it will be seen that rabbit cells were agglutinated by the tumour extract to lipoid agglutinable and far higher titre than those of any other species tried . lipoid non - agglutinable fowl cells were agglutinated to the same low titre . tumour extracts varied somewhat with regard to the ratio between titres for the titre for rabbit different red cells , but these variations were not constant . cells was always between 4 and 16 times the titre for mouse cells . extracts treated in various ways , to be described later , were tested in most cases against rabbit and mouse cells . 
red cells which were agglutinated to low titre by c57 were inagglutinable by s37 extracts . extracts of all the tumours tested lost their activity on standing , though at varying rates , e.g. 
extracts of c57 sarcoma took about 24 hours to become inactive , but extracts of c57 / lh1 , sarcoma , though initially often of similar titre , became inactive in one hour , or less . the loss could be prevented , or reversed , by the addition of b.a.l. occurrence and distribution of haemagglutinating agent in normal tissue . extracts of mouse liver , spleen , kidney , heart , lung , brain , voluntary muscle , lymph gland and thyroid , derived from both normal and tumour - bearing mice , were found not to agglutinate mouse red cells ; and for a time it was thought that the haemagglutinating agent was confined to tumours . examination of a wider range of normal tissues , using rabbit as well as mouse red cells , showed that this was not so . extracts of several tissues , notably adult mouse testicle , ovary , uterus and voluntary muscle , as well as whole mouse embryo and placenta , agglutinated the haemagglutinating rabbit red cells and sometimes mouse red cells also . titres of these extracts were , in general , low compared with those of extracts of mouse sarcomata , but comparable with those of extracts of some of the other table iv gives the range of haemagglutinating titres for tumours examined . rabbit , mouse and human red cells , of a number of normal tissue extracts . haemagglutinating power of these extracts declined on standing , and was preserved , or regenerated , by the addition of b.a.l. 
0 - 01 m added after standing 22 hours at room temperature figures represent reciprocals of haemagglutinating titres for rabbit red cells . some other properties of the haemagglutinating agents of tumrnours and normal tissues . action of normal serum on haemagglutination . - haemagglutination by lipoids is inhibited by normal serum in high dilution , while haemagglutination by viruses is unaffected by normal sera , but inhibited by specific antiviral sera ( burnet and stone , 1946 )  . the effect of normal horse serum on haemagglutination by tumour and normal tissue extracts was tested . extracts were mixed with equal volumes of a 1 in 100 dilution of horse serum , and the mixtures tested for haemagglutination in the usual way . 
an experiment similar to the last showed that addition of either of these sera had no effect on haema gglutination by either c57 or s37 sarcoma extracts . effect of temperature at which the test is performed on haemagglutination . 
than at room temperature ( stone , 1946 ; burnet and stone , 1946 ) , while haemagglutination by influenza virus is more active in the cold than at room temperature or 37 c . 
 ( salk , 1944 )  . haemagglutination by extracts of c57 and s37 sarcomata , and of normal mouse embryo and adult uterus , was tested at approximately 4 c . , at room temperature , and at 37c . 
overnight. neither rabbit nor mouse presence of complement had no detectable effect : cells were lysed and their agglutination by the tumour extract was unaffected . absorption of haemagglutinating agent by red cells . - - c57 sarcoma extracts were absorbed with rabbit , mouse , and fowl red cells , under various conditionts . the following is a representative experiment . samples of 20 per cent extracts of c57 sarcoma , ( i ) fresh , i.e. 
for three hours , and centrifuged ; the supernatant fluids were removed and the cells finally resuspended in the original volumes of saline , and examined for persistent agglutination and for agglutinability . 
fowl cells , which were only very slightly agglutinated , did not absorb appreciably . in all except one case red cells agglutinated during absorption were unsuitable for subsequent tests for agglutinability because they remained agglutinated . the exception was mouse cells used to absorb fresh extract . these , though agglutinated in the presence of the extract ( 30 minutes at room temperature ) redispersed during the subsequent incubation with saline . 
the difference is probably quantitative . gross ( 1947 ) has reported the presence of haemolytic agents in several tumours . haemolysis by such an agent , if of low titre , would be active only in the first one or two tubes of a haemagglutination test and , since these contain only 1 / 450th of their volume of packed red cells , haemolysis might be masked by the small amount of haemoglobin present in all crude tissue extracts . 
had been added before , and those to which it had been added after , heat treatment . a heavy precipitate formed in the heated extract , which was spun out before testing for haemagglutinating power , and it was thought possible that the haemagglutinating agent might be adsorbed on the precipitate . however , no agent could be recovered by extracting the precipitate with m / 15 na2hpo4 . the effect of digestion of tumour extracts by trypsin or papain depends on whether they contain b.a.l. in the absence of b.a.l. , extracts of c57 , or of c57 / lh1 , sarcoma incubated at 37 c . with trypsin or papain ( activated with kcn ) for an hour , were found to be irreversibly inactivated , i.e. 
0005 m 0 - 01 m + , , trypsin 0.5 per cent of commercial powder ( hopkins & williams )  . all mixtures buffered at ph 8 with m / 15 phosphate , 2 drops of chloroform added as baoteriostatic ; neutralized , and b.a.l. 
0.005 0 ' 01 m casein was " light , soluble " of british drug houses , dissolved in m / 15 na , hpo , by heating relevant conditions as in a . 1 hour on a water - bath . it will be noted that there was a considerable irreversible drop in titre of a control sample of sarcoma extract incubated without b.a.l. 
to such extracts gradually falls . it seems probable that this effect is due to the action of tissue proteases . the effect of tryptic digestion on extracts of normal mouse tissues was also tried . it is difficult to obtain such extracts with haemagglutinating titres high enough to make this test satisfactory . in an experiment in which a mouse embryo extract with a haemagglutinating titre for rabbit cells of 1 : 4 was digested with trypsin , with and without b.a.l. , activity appeared to be destroyed in 1 . 
on proteolysis was a 20 per cent extract of c57 sarcoma in m / 15 phosphate buffer ph8 was incubated ( a ) alone , ( b ) with trypsin , ( c ) with trypsin , and b.a.l. 
they show that , with casein as substrate , after 1 hour 's incubation there was no detectable inhibition of proteolysis by b.a.l. ; but with both casein and tumour extract as substrates , after 21 hours ' incubation there was definite inhibition : about 50 per cent by b.a.l. 
elliott , on the haemagglutinating power of a tumour extract , with and without b.a.l. , was essentially similar to that of commercial trypsin . the haemagglutinating agent does not become dialysable through cellophane after digestion of a b.a.l. - containing tumour extract with trypsin . lecithinase : it has been shown by stone ( 1946a ) that the haemagglutinating action of lipoids , and of vaccinia and ectromelia viruses , which probably owe their haemagglutinating action to lipoid components , is destroyed by cl . 
to 0 01 m was then added , and haemagglutinating in a similar experiment mouse embryo extract was treated with the results showed that incubation with lecithinase had no detectable effect on the ' ' haemagglutinating agents of c57 sarcoma or mouse embryo extracts . haemolysis by lecithinase present in the mixtures was not rapid enough , in the presence of b.a.l. , to interfere with haemagglutination . action of heavy metals and oxidizing agents , and its reversal by b.a.l. 
to samples of fresh , actively haemagglutinating , sarcoma extract , not containing b.a.l. , cus0o4 was added , to give final concentrations of m / 300 and m / 3000 . in both mixtures haemagglutinating activity disappeared within a few seconds ; there was a partial return of activity in the later , but none in the former , after addition of b.a.l. 
the haemagglutinating activity of these mixtures declined on standing at the same it thus appeared that the presence of heavy metals rate as that of a control . was not essential for natural inactivation . the addition of various oxidizing agents to a fresh sarcoma extract destroyed or reduced its haemagglutinating activity . 
the list includes serum antibodies ( naturally occurring , acquired , or experimentally induced ) , many viruses ( hirst , 1941 ; nagler , 1942 ; burnet , 1945 ; lush , 1943 ; burnet and stone , 1946 ; mills and dochez , 1944 ) , purified lipoids ( stone , 1946b ) , bacterial extracts ( keogh , north , and warburton , 1947 ) , the globulin fraction of egg - white ( commission on acute respiratory diseases , 1946 ) , some plant proteins such as ricin and abrin , basic proteins such as protamines and histones , inorganic colloidal acids and bases such as colloidal silicic acid , and many metallic salts ( landsteiner , 1945 )  . 
they are labile , while the latter are both fairly stable substances ; their red - cell species specificity is different from that of both lipoids and viruses ; they are unaffected by cl . 
welchii toxin , which destroys the haemaggluthinating power of lipoids and of vaccinia and ectromelia viruses ; their action is unaffected by normal serum , which inhibits that of lipoids but not that of viruses . in the manner in which temperature affects haemagglutinating titre they differ from lipoids . 
a sarcoma extract is absorbed by red cells which it agglutinates , but the cells , when they redisperse , as they do under certain conditions , are fully agglutinable by the same agent ; red cells agglutinated by a virus may also redisperse , but are then inagglutinable by that virus , and often by some others also ( burnet , beveridge , mcewin and boake , 1945 )  . egg - white is more like the tissue extracts in its haemagglutinating properties than are the other haemagglutinating substances referred to , particularly in respect of red cell species range , and effect of temperature on haemagglutinating titre , but it is not - reported to be labile . the general properties of the haemagglutinating agent of tumours suggest that it is a readily oxidizable substance which is active only in the reduced state . it may be a sulphydryl compound , but there is at present no positive evidence of this . neither inactivation by heavy metals , nor by oxidizing agents , is conclusive on this point . 
ascorbic acid , are ineffective , suggests that preservation is a specific effect of sulphydryl . natural inactivation does not appear to depend on the presence of heavy metals in an active state , and therefore it is unlikely that its prevention by b.a.l. 
has been stated not to inhibit the action of trypsin or papain ( webb and van heyningen , 1947 ) , but this work has been repeated here , with a it has been shown that b.a.l. 
protects it from the former . certain physical and chemical properties of the haemagglutinating agent of reasons are given for regarding it as an easily oxidizable it is destroyed by proteolytic b.a.l. 
was found to have definite inhibitory action on proteolysis by trypsin , the haemagglutinating agents of tumours and of normal tissues differ from and on digestion of egg - yolk by lecithinase . haemagglutinating viruses and lipoids in important respects . no qualitative differences have been found between the haemagglutinating agents of different tumours , or between those of tumours and normal tissues . the author gratefully acknowledges his debt to professor j . 
sterile vials , each containing 0 - 7 g . of a mixture of the sodium salts of the four nucleotides of yeast ribonucleic acid preserved with 0 - 3 per cent cresol . the mice used were the f1 cross of the a and c 57 lines . these were chosen because a mice have a relatively high spontaneous lung tumour incidence ( bittner , 1939 ; heston , 1940 ) , and it has been observed that such strains respond more readily to the carcinogenic effect of urethane ( cowen , 1947 )  . 
the hybrids would also tend to have a high spontaneous incidence , since the inheritance of spontaneous lung tumours in this cross seems to be due to a single dominant gene ( bittner and little , 1937 ; bittner , 1938 )  . seventeen a x c 57 males approximately two months old were injected with the pentose nucleotides solution subcutaneously at the flanks , daily at midmorning . 
of pentose nucleotides for a week the water . mice started to show ill effects . both treatments were discontinued ( after 2 weeks of pentose nucleotides and 1 week of urethane treatment ) for two days , on resuming treatments the dosage of pentose nucleotides to rest the mice . was decreased to 0 - 3 c.c. 
the figure clearly shows that a marked inhibition of the carcinogenicity of urethane was produced by the pentose nucleotides . the average number of lung tumours per mouse caused by urethane in controls 1 and 2 separately was 46 in each case , which was reduced to 26 in the p . 
strong. from the school of medicine , yale university , u.s.a. received for publication february 2 , 1949 . the idea of somatic mutation as an explanation for the origin of cancer has had a long and interesting history . its original use as an interpretation of cancer is usually associated with the names of murray and of boveri . these men arrived at the somatic mutation conclusion from different fields , murray from experimental cancer research , and boveri from his classical observations in experimental embryology . 
at that time he stated : " the existence of such tumours , the biological characters of which are retained through long periods of propagation , shows that the cellular transformation which initiates carcinomatous growth may take place in varying degrees . 
1 , the method of analysis employed compensates fully for difference in " size " of the pattern analysed , and the p values for grade and intergrade patterns can therefore be regarded as strictly comparable . if we regard p < .05 as evidence of abnormal patterning , then a glance at the table shows that abnormal patterns occur with considerable frequency . this is best seen by comparing the frequency distribution of the 224 grade and 192 intergrade p values of the table with the strictly equivalent frequency distribution of the 4000 random series ( cruickshank , 1940 )  . 
1. statistical maps often present data by the use of " grades . " the several counties in any one grade may be scattered " at random " over the map , or may be juxtaposed to form small or large these varying types of distribution are described briefly as " patterns . " the exact " groups . " type of pattern to be expected from random distributions has been determined experimentally ( 4000 patterns ) , and from these experiments the " expected frequency " of any specific type of rare types of patterns , i.e. 
patterns with low expected frequency , are observed pattern is known . described as " highly developed . " on cancer maps highly developed patterns occur with excessive frequency . indeed , " statistically significant " patterns , i.e. 
2 and 4 will show that p values across the gradings ( 1 - 12 - 1 - 23 7 ) give rise to curves with clearly defined trends , which include the develop114 d . 
2. the " intensity " of a regional influence can be expressed in terms of the " degree of development " of patterns found within its zone of action ; the more non - random the pattern the more powerful the influences at work . the analysis is best shown graphically ; the expected random frequency of the 7 grades and 6 inter - grades patterns of each map are plotted out on an i * erted logarithmic scale so that " peaks " show high degrees of pattern development - that is , powerful regional influences . the striking feature of the curves so formed is their essential simplicity . in fact they are almost e.g. 
that a significant " zone " is not always associated with a significant peak . in such cases the individual counties of the zone each show a deviation from the mean > 2 s.e. 
that curves of type 2 which develop a peak before the extreme grades and then fall off are usually associated with maps in which the highest ( or lowest ) grades do not form a single zone , but break up into two or more sub - zones , e.g. grade 7 may appear as three separate zones , unrelated to each other , but all related to grade 6 ,  . . 
the frequency of effects " increasing " and " decreasing " the rate are shown separately ; the frequency of effects " modifying " the rate ( significant ] y ) is the sum of these two . in the absence of regional influences we anticipate that a county will participate in " random " significant pattern formations in about 1 / 20 cases , i.e. 
 ( stocks ' data )  . now , if the distributions and trends in each of the 28 maps are unrelated , the result of combining these inter - random distributions should be to flatten out the whole pattern ( irregular multiple hillock picture )  . 
on the other hand , if they are related , their combination may be expected to result in some definite composite pattern or configuration ( hill and valley picture )  . from the entries of table vii three contour maps were prepared ( appendix 7 ) ; these are shown in fig . 
we may therefore conclude that county boundaries have no essential significance in relation to regional influences . in fact these influences behave in every way as continuous variables , and their variations in intensity are most appropriately depicted by means of contour - lines on the surface of the map . to depict these regional influences by contours may serve to drive home another of their characteristics as revealed by this study , viz . 
cornwall , somerset and dorset . while the majority of counties meet at a common county boundary , occasionally they meet only over a very narrow range , the so - called " point contacts " ( solid circles on map )  . 
where the county boundary is a river or an estuary the counties may fail to make a de facto cantact , and yet , it may be necessary to consider these as " touching " for the purpose of studying regional influences ( solid rectangles on map )  . note the distinctly higher values of the contact numbers of the inland counties as contrasted with those of the coastal counties . the mean contact number ( c ) works out at 5 - 12 . , , ~n this is an important constant in theoretical formulae , e.g. 
mark on the glass with grease pencil all " grade 1 " counties . remove glass template and place on white sheet . analyse arrangement of marked counties ; record ; clean off wax pencil mark with chloroform . replace on map and proceed similarly to work and analyse " grade 2 " counties , and so on . 
8 , in conjunction with appendix 4 , indicates the probable , simple explanation , viz . that estimates of the degree of pattern development of an organized pattern must generally fall below the true value when they are based on the examination of only part of the pattern - for the simple reason that the relation of one " piece " of the pattern to a neighbouring " piece " is thereby overlooked . 
the process of " intergrading " was evolved to deal with this contingency . thus , to explain the observed fact that the p grade greater than p intergrade we must assume that the mean " pattern " associated with regional influences covers an area greater than that of a mean grade , i.e. 
regional influences decreasing cancer exist alongside the " positive , " in fact these two types of influence , both of which are represented by peaks and zones , occur with equal negative effects must not be regarded as merely due frequency ( table vii )  . to the absence of positive effects , for the observed type of trend and degree of patterning associated with negative zones is of a much higher order than is to be expected from the mere absence of positive effect . it seems that there must be some general factor other than regional which operates to produce a definite incidence of cancer . regional influences are not themselves the cause of cancer , but merely impress their effect , positively or negatively , upon this general factor . i wish to express my thanks to n . 
the substance , usually dissolved in tyrode saline , is added to a culture after a dividing cell in mitosis therein has been selected fgr study , and the reaction of that cell to the agent is recorded for a period usually less than one hour . 
the cells in prophase then to be found in the outgrowth of the stained cultures were subsequently counted . the effects on mitosis of three phosphorylated naphthohydroquinones have been studied in this work . these are as follows : 1 . 
the di - methyl compound was found to be slightly less active than synkavit as a mitotic inhibitor in vitro . it again potentiates the effects of irradiation . further work on this substance is in progress , including clinical trials . normal mitosis in the chick osteoblast . osteoblasts were chosen for this work on the grounds that clear phase contrast pictures of them can be obtained during mitosis , and that they appear to be much less sensitive to light than are other fibroblast types of chick cells . in previous papers there has been described the normal course of cell division which can be observed under the phase - contrast microscope in living osteoblast cultures ( hughes and fell , 1949 ; hughes , 1949b )  . 
the nucleoli and nuclear membrane disappear remarkably rapidly at the end of prophase , but while the spindle is being formed several minutes pass before the chromosomes assume their equatorial position as a metaphase plate . it is convenient to speak of all this period after the loss of nuclear membrane and nucleoli as metaphase , and to separate an early from a late division of this phase of mitosis , respectively before and after establishment of the metaphase plate . the general cytological effects of compounds , i , ii , and xx viii . in the present work over fifty film records of the behaviour of dividing cells under the influence of these substances have been made . 
20. - cell from the outgrowth of a culture treated with 5 x 10 - 7 m synkavit ( i ) for 40 cell has undergone mitosis with the suppression of cleavage . 
nucleoli still present 5 mlater ditto , ditto , 10 mlater 3.5 x 10 - 6 m metaphase metaphase plate metaphase late prophase early metaphase 4 x 10 - 6 m prophase . 
the methylfree substance exerted this effect in late telophase over a range of dilutions of about one thousand - fold ( 27 , 3 x 10 - 9 m ; 36 , 1 x 10 - 6 m )  . 
at higher concentrations the substance rapidly kills both dividing and resting cells ( 38 , 1 x 1o - 5 m )  . it is noteworthy that although the effect of the methyl - free compound differs considerably from those of the methylated derivatives , both in the types of inhibition and the dilutions at which they are exerted , yet in the present data there are no significant differences in the maximum concentrations of each of the three substances at which a cell has been observed to undergo a normal mitosis ( synkavit , 17 , 18 , 19 ; di - methyl compound 25 , 26 ; non - methyl 36 , 42 )  . one point in the mitotic cycle on which these three substances do not appear to exert any immediate effect is the entry of cells into prophase . early prophases are still found in cultures treated with these substances at the highest concentrations which have been used in the course of this work . since synkavit and the di - methyl compound can interfere with the division of a cell in several ways , a culture which has been treated with these substances at appropriate dilutions for periods of an hour will show a variety of effects . these depend both on the individual sensitivity of the cells and the phase of mitosis which they had reached at the time of adding the substance . 
the fate of a particular cell selected for photography is , therefore , to some extent a matter of chance . it may be that the 55 instances which are recorded in table i do not exhaust these possibilities , though study of the whole cultures after fixation has not so far revealed any other types of inhibition . the variability of the behaviour of cells towards these substances , however , extends further than to inhibition at different stages of mitosis . treatment of a culture with a concentration sufficient to arrest and damage some dividing cells will leave unaffected others which continue a normal mitosis . this fact can best be illustrated by comparison of the effects of synkavit in a number of instances where it was applied to cells early in.prophase. 
at 1 x 10 - 6 m a normal mitosis ( 59 ) , an arrest in metaphase ( 60 ) and a clumping of chromosomes and cell oedema at this stage were recorded ( 52 )  . 
when the di - methyl compound at 4 x 10 - 6 m was added to cells in metaphase , there were seen both a normal mitosis ( 26 ) and an instance of clumping of the chromosomes ( 24 )  . this circumstance must be borne in mind in the search for any further generalisations from however , one other conclusion clearly emerges , namely , that the these data . effect of these substances on dividing cells is greater when they are applied at early stages of mitosis . 
with synkavit at 3 * 5 x 10 - 6 m addition to a cell early in metaphase resulted in clumping of chromosomes and oedema ( 15 ) , while later in metaphase in two instances this concentration was not found to be inhibitory ( 18 , 19 )  . 
it is probably true to say that phase specificity is most marked with substances to which the cell in division is much more sensitive than during the restthus , for instance , the action of colchicine on the dividing cell is ing stage . restricted to its effect on the mitotic spindle , and this is exerted at dilutions much greater than those necessary to affect intermitotic cells . 
the proportion of cells entering prophase thus , this substance appears to penetrate the cell memhas then decreased . brane so slowly that a concentration within the cell sufficient to provoke these effects is not reached for several hours . 
the secondary fusion of the daughter cells in late telophase which has been observed in the course of the present experiments may well be due to an alteration in the properties of the cell surface . 
a large number of binucleate cells were seen in stained cultures after treatment for 6 hours with this substance at concentrations of 1 x 10 - 9 m . this effect may be due either to secondary fusion of the daughter cells , or to an inhibition of cell cleavage in the first instance . finally , the value of the two general methods of investigations of the action of chemical substances on living cells in culture can be compared . in the one mainly used in the present work the effects of the agent on a single living cell in the culture is followed ; in the other a quantitative analysis of the whole culture is made after fixation and staining . 
where the effects of a given treatment often differ from cell to cell , it would be unwise to draw here the quantitative conclusions from the study of only a few examples . analysis of large numbers of cells is indispensable . 
cells may be inhibited at any point in mitosis , though their entry into prophase is apparently unaffected under the conditions of these experiments . arrest in metaphase and clumping of the chromosomes is the most common effect . individual cells vary greatly in their sensitivity to these substances . 
at sub - lethal concentrations the latter does not affect the course of mitosis until late in telophase , when to a varying extent the daughter cell re - unites . we wish to thank a . 
stern. from the general hospital , northampton . received for publication june 12 , 1954 . although a study of the morbid anatomy of carcinoma of the bronchus can have little bearing on the important problem of etiology , nevertheless any ascertainable facts about this increasingly common disease may contribute something to our knowledge of it . recent comprehensive surveys of the pathology of carcinoma of the bronchus have been made in this country by harrison ( 1950 ) , on 353 confirmed cases seen at st . 
the average age at which death occurred was 56.4 years , slightly higher in males ( 56 - 7 years ) than in females ( 54 - 7 years )  . 
on the other hand both willis and bryson and spencer found metastases in the adrenals much more often than they were discovered ( willis 40 per cent , bryson and spencer 23 per cent , present series in this series . 13 - 7 per cent )  . 
the frequency with which cerebral deposits have been recorded in carcinoma of the bronchus has varied widely with different observers . those who have investigated metastatic tumours in the brain have found that a very high proportion of these had their primary source in the lung . elkington ( 1935 ) , who analysed the records of the national hospital for the period 1918 - 1933 , found that of 72 metastatic cerebral tumours , 24 ( 33 per cent ) originated in the bronchus . 
the highest recorded figure is that of fried and buckley ( 1930 ) , who recognized secondary cerebral deposits in other observers have given much lower figures : reingold , ottoman 41 per cent . and konwaler ( 1950 ) 30 - 5 per cent , willis ( 1952 ) 21 per cent , bonser ( 1934 ) 16 per cent , harrison ( 1950 ) 20 per cent , bryson and spencer ( 1951 ) 17 per cent , simpson ( 1929 ) 13.5 per cent ( in a series of 139 cases ) , kilkuth ( 1925 ) 12 - 6 per cent ( in a series of 246 cases )  . few authors have differentiated between multiple cerebral deposits from carcinoma of the bronchus and large single masses except to say that the former are common and the latter rare , though kilkuth ( 1925 ) reported single masses in 13 of 31 cases with cerebral metastases . although the present series is small it is noteworthy that in 14 cases with cerebral metastases only 6 were multiple and 8 single . 
the multiple deposits were widely distributed and showed no predilection for any particular area of the in all except one of the cases with multiple cerebral metastases there were brain . also secondary deposits in the viscera . 
on examination there was a complete hemiplegia and a dubious hemianosevere neck rigidity was present , but examination of the cerebro - spinal fluid revealed pia . a normal fluid except for pressure , which was 190 mthe only abnormality disclosed by examination of the chest was diminished air entry in the upper zone of the left lung with deviation of the trachea to the lethe differential diagnosis made was betweenr tuberculous encephalitis and cerebral thrombosis . 
death took place 2 days after admission . at post mortem dense fibrous adhesions were found almost completely to obliterate the left pleural cavity . there were old fibrous adhesions at the right apex . 
the trachea and arising from , and producing main bronchi contained a considerable amount of muco - pus . almost complete obstruction of the left upper lobe bronchus was a hard , white tumour , distal to this tumour the lobe was collapsed and contained approximately 5 cacross . a number of irregular cavities filled with thick yellow pus . 
the left lower lobe was studded with a number of nodules of tumour up to 0 ' 5 cacross . in the upper half of the right upper lobe there was an irregular tuberculous focus 2 cacross consisting of a central caseous mass enclosed in dense fibrous tissue . 
her main complaint was loss of weight and a cough of 3 months ' duration , which had been accompanied by a blood - stained sputuon examintion the fundi could not be seen . 
an x - ray showed a mass extending from the left hilum and involving the mid and lower portions of the left upper lobe . the patient died 12 days after admission . at post mortem a large , white , firm tumour mass was found involving the whole of the medial portion of the upper lobe of the left lung . 
no mediastinal glands were infiltrated and no other deposits were found in the viscera . the skull was normal , but attached to the flax cerebri at the vertex of the brain , and pressing on the pre - rolandic portion of the left superior frontal gyrus there was a firm hard tumour the size of a cherry ( proved histologically to be a meningioma )  . the cerebral convolutions were somewhat flattened . 
onimmediate examination a weakness of the left face was noted and there was slow nystagmus on lateral and upward deviation of the eyes . the reflexes were all exaggerated but the plantar responses were flexor . a diagnosis of cerebral tumour was made tentatively , but before further investigations could be carried out the patient died suddenly the day after admission . at post mortem a fungating , cauliflower - like growth was found in the upper lobe of the left lung . 
no enlarged mediastinal glands were present . the pelvis showed no evidence of any local or secondary lymphatic involvement from the cervix . the brain was moderately congested and oedematous . the left half of the cerebellum was replaced by a degenerating , cystic tumour . histologically the growth in the lung and the secondary deposit in the cerebellum were found to be an unspecified type of carcinoma of the bronchus . ( sections were not available for personal study . ) chronic bronchitis " and convulsive attacks with loss of consciousness which began a fortnight before admission . six months previously , when he had attended hospital as an out - patient , an x - rayhad been suggestive of bronchial neoplasm , with displacement of the mediastinum to the right side . 
the cerebro - spinal fluid was normal apart from an increased pressure of 250 man x - ray of the chest gave evidence of neoplastic formation in the left hilar region with associated collapse at that base . death occurred 3 days after admission . at post mortem the left lung was found to be collapsed and the left pleural cavity contained about 500 c.c. 
the left main bronchus was compressed by a soft , buff - coloured tumour , about 8 cin diameter , which was covered by a layer of lung 2 cm . this layer of lung was relatively airless and thick yellow pus was expressed from the thick . the oesophagus and aorta were displaced posteriorly by the tumour . 
46 on account of frontal headaches , throbbing in character and especially severe in the mornings , which had been present for 3 weeks . just before admission much nausea and vomiting had developed . there had been no cough at any time . 
the arm and leg sides . reflexes were normal , but some past pointing was noted in the right arlumbar puncture gave a yellow fluid under 300 mof pressure . the protein was increased to 120 mg . 
the patient deteriorated rapidly and died 3 weeks after admission . at post mortem a hard , white tumour , 1 in diameter , was present at the apex of the right lung . 
no abnormalities were found in the abdomen . in the brain there was a secondary deposit , just over 1 in diameter , deep in the left temporo - parietal lobe . the mass impinged on the left lateral ventricle , which was slightly dilated . 
a mixed type of types of cells encountered in these tumours were : alveolar cells and oat cells , 4 cases ; alveolar cells and polygonal cells , 2 cases ; and one case each of spheroidal cells and squamous cells ; polygonal cells and oat cells ;  . 
the alveolar epithelium in this particular graft actually shows the various stages in the secretory cycle of the normal prostatic gland . it is interesting to compare the histology of these successfully vascularised grafts seen in fig . 
horning the prostatic it is relatively easy to detect by palpation non - vascularised grafts , as they are smaller and invariably softer when compared with successfully vascularised prostatic grafts , which are firmer on palpation , larger and invariably cystic in the earlier phases of growth , owing to active secretion of the glandular epithelium . non - vascularised graft fixed5fig . 
in this particular implant this was the only malignant focus within the entire graft . alveoli distended with secretion and with normal epithelium are seen adjacent to this exhausted alveolus from which this early malignant lesion arose . 
the proliferating epithelial components in all the early malignant foci break through the alveolar basement membrane , invariably forming tongue - like lesions which rapidly invade the fibromuscular stroma of the host as depicted in all prostatic grafts which have grown after treatment with 20in fig . 
10. methylcholanthrene , it was found possible to distinguish between three distinct types of early epithelial growth , all of which have been previously described in detail ( horning , 1949 )  . by studying the cytology and mode of growth in serial sections of these early invasive foci through later phases of development in other similar older grafts , it has been possible to predict the types of tumours which would have subsethere is one type which gives rise to a glandular carcinoma , quently arisen . both these types of and a second which develops into a squamous growth . lesions are easily recognised by their histological character , and both arise from the alveolar epithelium . the third type is the most uncommon variety , characterised by a stratified squamous metaplasia of the epithelium in sita , followed by a diffuse marginal growth into the stroma , which clearly arises from the duct occasionally some grafts show two or sometimes three types of epithelium . proliferating foci within the same implant . 
11. this shows a section of a graft treated with crystals of stilboestrol alone and fixed 4weeks after implantation . there is a complete inhibition of secretion after this period of grafting . every individual alveolus within the graft was entirely devoid of prostatic secretion , and the epithelial cells had undergone marked morphological changes . 
the prostatic epithelium at this period of treatment contrasts greatly with similar grafts treated with the female sex hormone after implantation in host mice for the same period of time . 
the alveolar epithelium not only maintains its glandular character , but in the majority of grafts testosterone propionate induces a pronounced stimulation of the prostatic secretions , invariably resulting in a cvstic dilatation of the alveoli . 
grafts combined with the carcinogen and sex hormones . 20 - methylcholanthrene and testosterone propionate . - three squamous - celled carcinomas developed from prostatic grafts impregnated with the carcinogen and the male sex hormone in strain a mice at intervals ranging from 7 to 9 weeks following implantation . one sarcoma arose in a c3h mouse after 8 weeks ( table i , group iii )  . 
the differences in histological structure between prostatic tumours derived from grafts treated with the carcinogen together with either the male or female sex hormone can best be appreciated by comparing fig . 
examination of pituitary and adrenal glands and gonads from host mice bearing grafts . examination of pituitaries from mice bearing grafts from all three groups of rodents treated either with the carcinogen alone or in combination with either of the sex hormones revealed no abnormal histological changes , with the exception of excessive amounts of colloid seen in the clefts of pituitaries from mice treated with stilboestrol and the carcinogen . there was no diminution of acidophiles or increase in chromophobe cells following implantation of the stilboestroltreated grafts . neither did the adrenal cortex reveal any histological abnormalities resulting from this treatment . 
horning of both lung and prostatic tissues have been previously described by the author in mice the survival of an homologous graft seems to be dependent ( 1949 , 1952 )  . upon the use of a closely inbred strain , since stock mice of indeterminate ancestry do not easily tolerate grafts of this kind . 
pan and gardner ( 1948 ) found an increased susceptibility to tumour formation from grafted uterine epithelium in pure line mice as compared with hybrids . recent experiments ( horning , 1952 ) have further indicated that the age of the donor providing the graft , and the age of the host mouse into which the grafts are implanted , also have an important bearing on the problem of graft other factors such as the reaction beween the graft and the host survival . mouse play an important role . it has been previously reported that more difficulty has been experienced in obtaining malignant tumours from lung grafts impregnated with 20 - methylcholanthrene than with similar grafts of other it was interesting to note that tissues which are normally under hormonal tissues . influence are much better tolerated as grafts in host mice of the same sex and strain than tissues which are to a greater extent ( such as the lung ) independent of hormone action ( horning , 1952 )  . nevertheless , a certain number of prostatic grafts , when implanted subcutaneously into host mice of the same strain as those of the donor , fail to survive irrespective as to whether the grafts were impregnated either with the carcinogen alone , or in combination with either the male or female sex hormone . 
the present experiments on the reaction between the implanted prostatic graft and the host bearing mouse have shown conclusively that the survival of the donor graft is dependent upon a rapid vascularisation within the subcutaneous tissues of the host mouse . 
the reason , however , why some grafts fail to become vascularised still remains obscure . the influence of steroid hormones on the behaviour and growth of prostatic cancer has been the subject of intensive investigation . 
1. - the skin of a c3h male mouse is cut and pinned back so as to expose an untreated homologous prostatic graft which had been growing subcutaneously for 6 weeks . 
6. - transverse section through an homologous prostatic graft which failed to become vascularised , fixed 6j weeks after subcutaneous implantation . the relationship of the grafted prostate to that of the skin of the host rodent is clearly shown . 
horning alveoli have completely broken down ; there is also evidence of lymphatic infiltration . the whole graft has been encapsulated and is slowly being absorbed by the tissues of the host mouse . 
meredith. from the christie hospital and holt radiumt institute , manhester . received for publication december 12 , 1948 . ix a series of papers ( dale , 1940 ; dale , meredith and tweedie , 1943 ) it has been shown that the action of x - rays on aqueous solutions of biologically active compounds was indirect , i.e. 
further , it has been shown that in consequence of this indirect action , if two solutes are present , the second solute ( the protector ) reduces the radiation effect on the first one ( the indicator ) ( dale , 1942 ; 1943 ) , by sharing the intermediate product formed from water by the radiation . 
meredith enon revealed certain unexpected quantitative relationships between the protective power of a substance and its concentration , which will be described and discussed in this paper , together with the closely linked ionic yield of the indicators used , the knowledge of which is the necessary basis for the quantitative treatment of the experimental results . 
the fact that these new results were derived from experiments with two indicators of very different molecular weight , for an extended range of protective substances added to both indicators , helps to build up a more complete picture than has been obtained hitherto . 
 ( average a 130xu ) from a 250 kv tube . whenever possible , the solutions , contained in pyrex glass tubes , were spaced at such distances from the target that each received the desired dose during the same overall treatment time , care being taken to avoid shielding of one tube by another . this ensures that the relative doses received by different tubes are not affected by unavoidable fluctuations in the tube output . 
the stock solution of enzyme and the solutions of protective substances were made up with water , glass re - distilled from alkaline potassium permanganate , and the ph adjusted so that a sample reacted pink to phenolphthalein . all necessary dilutions were carried out with glass re - distilled water adjusted to the same ph by the addition of 1 ml . 
were performed during this series of experiments , these arise because very high and some special difficulties were encountered . doses of radiation ( one to two million roentgens ) are required to achieve a reasonable degree of inactivation in solutions with concentrations of the order there is a strong tendenvcy of re - crystallization during of 10 per cent or more . the long exposure to radiation , which makes it almost impossible to keep the enzyme in solution at a ph compatible with its stability . however , it was found that a 6 per cent solution could be maintained for most of the time by stepwise addition of naoh . 
the concentration reached by this method was derived from the known concentration of the suspension by measuring the height of the coluimn of crystals after , and the height of the suspension before centrifugation . 
compound ( warburg and christian , 1938 )  . since various preparations ofthe specific protein of the amino acid oxidase differ in their activity , and even any one preparation gradually loses activity when kept for some weeks , the oxygen uptake with a known amount of d.n. 
1. these results may also be presented , as shown in a previous paper ( dale , gray and meredith , 1949 ) , by the formula : d5 - 2 x 107  . 
calculated by lea ( 1946 ) from earlier experiments by one of us is much lower than the value obtained in the horizontal part of the curve , and this is due to the fact that the concentration then used was of the order of ~~~i ~~~~~~~ii conc . 
for the sake of accuracy the degree of inactivation should be kept between the limits of 25 - 75 per cent . in a previous report ( dale , 1947 ) the value of q was found from the protection afforded by generally lo100 g . 
q was roughly constant , and it was concluded that the protective power per molecule is proportional to the molecular weight - perhaps not a surprising result , since all these substances have roughly the same average composition . it is only when low molecular weight substances of special chemical configuration are examined that remarkable differences are found . oxalic acid , which was earlier ( dale , 1942 ) found to be a poor protector of d.n. , also proved to be a poor protector in the case of c.p. , but if the c - c bond in oxalic acid is broken and the free valency of the carboxylic group linked to an h atom , thus forming formic acid , the protective power is raised about 200 times , and similarly , if w . 
our arguments , however , are not dependent upon its correctness , nor would they be invalidated by its disproof . lea , smith , holmes and markham ( 1944 ) pointed out that , in addition to reacting with solute molecules , radicals may also recombine with one another , having a mean life , as free radicals , of r sees . 
 ( 1944 ) also suggested a modification of equation ( 3 ) to cover the protection effect on the assumption that the indicator and protector molecules " share " the available radicals . 
thus although the protector molecule has removed a radical from the solution , it can " hand on " the effect of the radical to the indicator molecule , and thus fails to fulfil its since we have no clue to the nature of this change function as a protector . we will call it " activation , " and it might be thought of as a sort of meta - stable state . 
and the collision frequency between " activated " protector molecules and indicator molecules , 1 , m and pi have already been defined , whilst pa is the probability that a collision between a radical and a protector molecule destroys the former and activates the latter . 
ps is the probability of an indicator molecule being inactivated in a collision with an " activated " protector molecule . from equations ( 9 ) and ( 4 ) we findao + m rt [ ( ao + t ) pi ( ao + m ) pap . 
i ~~ ( ao + t ) p , + lp , apz~l ( 10 ) some of the constants appearing in equation ( 10 ) can be evaluated from results obtained with solutions of the indicator alone , whilst the others can be evaluated from any two experimental values of q for known protector concentrations . 
the values of ( p ) are indications of the protective power per molecule of each substance for the particular indicator , and the values given show the same tendency as was shown previously for protective power per unit weight by molecules with approximately the same atomic constitution have dale ( 1947 )  . approximately equal protective powers , whilst marked differences occur when special groupings are present . from the results it is also possible to calculate the relative values of pr for d.n. 
not only has the type of therapy most likely to achieve success been the subject of dispute , but also the same measures in the hands of well - known authorities have produced a wide variation in results . 
have we a classification which accomplishes this ? the outlook for a patient with cancer of the breast depends first and foremost upon the degree of malignancy of the tumour and upon its extent . 
it is a wide belief that the prognosis is also influenced by certain other features , but the value of these is open to que ! rtion and will form the subject of a separate inquiry . 
 suffice here to state that such factors as age of patient , site of tumour in the breast , size of primary growth and duration of symptoms were not found , per se , to influence the outlook materiallv . 
 _ - \ttempts have been made in the past to group patients with breast cancer according to histological criteria which , it has been claimed , give some indication as to the degree of malignancy of the tumour . 
 the other is based upon a system of grading , wbilst the results of the former have largely proved disappointing , conflicting views exist as to the value of the latter . 
 it is the prime object of this paper to investigate the significance of the grade of malignancy in mammary cancer in an attempt to establish a more accurate system of classifying patients with this disease . 
 there is great difficulty at the present time in deciding on the line of action to adopt in view of the widely different procedure . ; , advocated by different authorities . 
 , vhilst gordon - taylor ( 1948 ) and cade ( 1948 ) believe radical mastec tomy alone to be the most suitable treatment for these patients , riddell ( 1948 ) and also richards ( 1948 ) advocate ancillary radiotherapy . 
it is now over fifty , ears since : moore of london conceived and introduced the radica.l mastectom , ( rodman , 1904 ) , and still there is controversy in some quarters as to its valu ;  . 
 if we cannot solve the problems of mammary carcinoma , what hope is there for the surgery of malignant disease in less accessible sites ? pretw ' u , , 8 attempts at grading . 
 little attention was directed to the grading of tumours until broders ( 1920 ) applied , on hansemann 's theory to squamous cell carcinoma of the lip , and a year later to similar tumours of the skin ( broders , 1921 )  . 
 l\iaccarty and sistrunk ( 1922 ) and l\iaccarty ( 1922 , 1924 ) also studied the histology of breast tumours , and found the most important prognostic charac teristics to be the degree of cellular differentiation and the presence or absence of certain stromal features . 
 ' white ( 1927 ) was unable to agree with the views of : : \iaccarty , but in a series of 100 cases confirmed the results of greenough ( 1925 )  . 
 smith and bartlett ( 1929 ) , and lat.er simmons , wright , hartwell and greenough ( 1933 ) , also confirmed the value of the syst.em introduced by greenough ( 1925 )  . 
 the five - year survival rat.es for the three grades of malignancy obtained by these authors are comparable , and agree in the main with those of pat.ey and scarff ( 1928 )  . 
they therefore elaborated a " clinical index of malignancy " based upon age , presence of lactation , rat.e of growth of tumour , and extent ( stage ) of the disease . 
perry ( 1925 ) , lane - claypon ( 1928 ) , truscott ( 1947 ) and hamett ( 1948 ) were not able ti < > prove that the position of the growth had any bearing on outcome in breast cancer . 
 flothow ( 1928 ) , in a little over 200 cases of carcinoma of the breast , studied the facrors stated by llaccarty ( 1922 ) to represent a defensive mechanism by the body to a malignant tumour . 
it was shown that the malignancy increased with a rise in the " malignancy index . " for comparison a clinical classification was also elaborated by schmitz , but no att.empt was made to study the effect of correlating these histological and clinical syst.ems ( hueper and schmitz , 1929 )  . 
 opinions regarding the accuracy of hisrological grading have varied from huepe , r , who finds as many as twenty different features of prognostic value ( hueper and schmitz , 1929 ) , ti < > reimann ( 1929 ) , who is unable ti < > find one . 
he considers , however , the broad grouping methods based on general histological stucture - - presumably such as that of greenough ( 1925 ) - ro be of value . 
greenough ( 1925 ) , on the other hand , applying the principal of anaplasia , concentrated on tubule formation , variation in size of cells and nuclei , secretory activity and mitotic and hyperchromatic figures . 
 the principles laid down by greenough ( 1925 ) were closely followed by others , such as smith and bartlett ( 1929 ) , and simmons , wright , hartwell and greenough ( 1933 )  . 
patey and scarff ( 1928 ) and scarff and handley ( 1938 ) , however , attached chief importance to tubule formation , variation in size of nuclei , and mitotic and hyperchromatic figures . 
 delbet and mendaro ( 1927 ) in france , on the other hand , considered the presence or absence of mucous secretion to be the most important factor in ascer taining the outcome of patients with mammary carcinoma . 
leroux and perrot ( 1928 ) andmoureau and lambert ( 1932 ) support the views ofdelbet and mendaro ( 1927 ) , but betrand and de nagey ( 1931 ) consider muco - secretion to be of no value in determining clinical progress . 
 evans ( 1933 ) studied a number of parenchymal and stromal features , and con cluded that , although anaplasia indicates a poor prognosis , it is of no practical value in assessing the outcome for the individual case . 
he concluded that the grade of malignancy is the most important indication ot survival period , and this is more significant when considered together with the state of the axillary glands . 
 complicated , exact numerical systems , such as the " histological malignogram " introduced by hueper ( hueper and schmitz , 1929 ) , are time - consuming and of doubtful value . 
 the series taken for the present investigation consists of 565 cases of carcinoma of the breast treated at the middlesex hospital between the years 1936 and 1942 inclusive , and at the war sector units associated with it during the latter half of this period . 
in spite of the movement of population produced by the recent conflict , all but 11 of the c3888 were traced by the follow - up department of the hospital . 
 for the present study the five - year survival rate will be the chief prognostic yard - stick , though some attention will also be given to ten - year results . 
 " sur vival rate , " however , does not necessarily imply freedom from cancer ; it is in point of fact , merely an indication of the number of patients actually alive . 
this was based on the principles laid down by greenough ( 1925 ) , but chief importance was attached to tubule formation , regularity in size , shape and staining of nuclei and hyperchromatism and mitoses . 
this view is supported by the work of greenough ( 1925 ) , white ( 1927 ) , patey and scarff ( 1928 ) , smith and bartlett ( 1929 ) , sophian ( 1935 ) , and in fact , this is the only histological feature on which scarff and handley ( 1938 )  . 
certain authors , such as evans ( 1933 ) , greenough ( 1925 ) and haagensen ( 1933 ) , paid attention to variation in size of the cells , but the outlines of these are usually indistinct ; it is more reliable to consider the nuclei . 
evans ( 1933 ) was unable to support this view , and plaut ( 1927 ) considers the feature unreliable on the grounds that many cancer cells divide by amitosis . 
nevertheless , as haagensen ( 1933 ) points out , a considerab1 proportion of the cells in cancer of the breast divide by mitosis , and he found a close relationship between the number present and the outcome . 
this is also supported by the work of greenough ( 1925 ) , white ( 1927 ) , patey and scarff ( 1928 ) , smith and bartlett ( 1929 ) , sophian ( 1935 ) , and simmons , wright , hart well and greenough ( 1933 )  . 
 the cases were fairly evenly distributed , 30 per cent of the total being pla - oed in grade i , 41 per cent in grade ii , and 29 per cent in grade iii . 
 227 it is evident that a parallel exists between grading and prognosis , there being more than three times the number of patients alive with grade i than with grade iii tumours . 
it will be noted that they a . , , uree especially with the findings of pat.ey and scarff ( 1928 ) , and table ii . - - grade and prognosis by various authors . 
no grade iii cases remained alive in the series of greenough ( 1925 ) , and also of white ( 1927 ) , but this does not appear to be the general experience . 
 total 218 433 the five - year survival period is a convenient yard - stick with which to gauge prognosis in breast cancer , but as truscott ( 1947 ) and others have pointed out , a large number of fatalities from this disease occur from five to ten years after operation . 
 comparison of the fiveand ten - year results emphasizes the importance of the latter as a more accurate indicator of the control of mammary carcinoma , there being a striking fall in the numbers oi survivals for the corresponding grades of tumour , even with allowance for expected deaths from other causes . 
this is in keeping with the 26 per cent obtained by richards ( 1948 ) , and the 32 per cent by harring t - 0n ( 1946 )  . 
 ( i ) well - marked ( ii ) moderate ( iii ) slight or ml total ( i ) slight ( ii ) moderate ( iii ) marked y ariation in nuclei h : yperchromatic and mitotic  . 
the extent of the growth should also be taken into account , and the most important single factor indicating this is whether the axillary lymph nodes are histologically involved or not . 
it is particularly interesting to note that the prognosis in the lower grade of malignancy with glandular spread is better than for either the higher or intermediate grades without such metastases ( 65 per cent alfre at fi . , e years compared with 53 per cent and 61 per cent respectively )  . 
 these results agree in principle with those of most workers who have employed the system introduced by greenough ( 1925 ) , and are almost identical with the findings of simmons , wright , hartwell and greenough ( 1933 ) ( table viti )  . 
 patey and scarff ( 1928 ) and scarff and handley ( 1938 ) , on the other hand , found a uniformly good result in all three grades when the glands were not involved , and so concluded thatgrading is only of prognostic value in those cases with axillary metastases . 
the figures in table ix refer to the state of the cases at the time of operation , and the time factor involved here is the delay in seeking treatment . 
on the other hand , if for any reason the grade ill .cases tend to present for treatment after a longer interval of time than those belonging to grade i , then this may account for the greater incidence of axillary involvement in the former . 
in addition a higher percentage of the former present within six months of the onset of the disease and fewer after twelve months as compared with the grade i cases . 
thus , in spite of the shorter delay in seeking treatment , there is a greater incidence of axillary involvement among the high and intermediate grad~ of malignancy , consequently emphasizing the greater metastasizing power of these types of growth . 
ewing ( 1940 ) , however , prefers to employ the term " potential malignancy " for a neoplasm as determined by microscopic examination , because the clinical course may be modified by such features as site of tumour , surgical trauma and perhaps changes in rate of growth . 
 several authors , such as hueper and schmitz ( 1929 ) , and also lee and stiiben bord ( 1928 ) , have compared the results of grading and staging in breast cancer . 
 involvement of the skin directly over and in continuitv with the tumour does not affect staging provided that the area ~volved is small in relation to the size of the breast . 
as previously , when the cases were classified according to grade ( tables i , iv ) , a large number of deaths are seen to take place in the interval between the shorter and longer follow - up periods , thus once more emphasizing the importance of the latter in assessing the achievements of breast cancer therapy . 
 a study of grading and also staging indicates that both methods give com parable results ; the latter , however , has the slight advantage of revealing the outcome for the most advanced cases ( stage 4 )  . 
 we ha , e already drawn attention to the relationship in breast cancer between the histological architecture of the tumour and its tendency to involve the axillary glands ( table ix )  . 
exactly half of the grade i tumours have not yet extended beyond the first stage , whilst about a quarter are advanced , belonging to stages 3 or 4 taken together . 
 100 100 100 these results merely gi , e additional support to the view already expressed with regard to the relation between histology and axillary involvement , that the higher the grade of mammary carcinoma , the greater the tendency for early spread beyond the breast . 
the vast majority of workers in this field , however , have argued as to the relati , e merits of either syste but why should the grade and stage be considered only separately , and in the sense of competing with each other to form the basis of a reliable prognostic guide ? a combination of these features would result in a classification which gives information on not only the type of growth , but also its extent when first seen . 
would this not bring about a more accurate grouping of cases than could be produced by either system alone ? indeed , se , eral authors have taken the histological picture into consideration with the state of the axillary lymph nodes , and the value of this has been con firmed here ( table vii )  . 
the percentage of five - year survivors is seen to range from as high as 87 per cent for stage i , grade i cases down to as low as 6 per cent for those belonging to stage 4 , grade ill . 
it is also evident that whilst the prognosis in each stage deteriorates with the rise in histological malignancy , the cases with low grade tumours do not conform to the general trend . 
it reveals that all is by no means lost if a patient presents with a stage 2 or even a state 3 growth provided that it belongs to the lowest grade of malignancy ( grade 1 )  . 
 having revealed the value of combining histological grading with clinical staging for prognosis in mammary carcinoma , the suggestion is now put forward that the wide range in results produced by different authors for identical methods of treatment depends upon the comparison of incomparable groups of cases . 
 w ' hat evidence is there to support these views ? with reference to this question , two groups of cases will be taken for study , one treated by surgery alone , and the other by surgery with ancillary radio therapy . 
although it is not the purpose of this paper to discuss the merits of treatment in breast cancer , the results achieved by these two methods will be compared in order to illustrate the discussion . 
it must be pointed out that the treatment in each group was by no means uniform , there being considerable variation both from the surgical as well as the radiological aspect . 
 irradiation was predominantly in the form of deep x - rays , either pre - operati.e , post - operative , or both , a few cases being treated with radium alone or radium and deep x - rays . 
 according t - 0 grade . - let us first consider the results achieved for each group according to the grade of malignancy ( tables x " \ ' ~i , xviii )  . 
on the other hand , in the radio - surgical group , although this operation was performed in the majority , there were many instances of more conservative surgery , which included 37 simple mastectomies and 17 local excisions . 
 " ~ith this in view , the survivals of a more uniform type of therapy were taken for study , the comparison being made between patients treated by radical surgery alone and radical surgery with irradiation ( tables xxii , xxiit )  . 
 the greater achievement of surgery alone over the combined attack is again seen in harnett 's ( 1948 ) figures which are also for the radical operation and are classified by stages ( table xxi )  . 
 from these facts it is clear that the variation in treatment by surgery in the present material does not appear to interfere with the fair comparison of the 278 h . 
 in this instance a false impres sion may be given when the results of two series of cases are compared owing to the possible unequal distribution of clinical stages in corresponding histological grades . 
here there are small number of seen to be more low and fewer high grade tumours in each stage ot the surgical as compared with the radio - surgical cases . 
furthermore , of all the neoplasms of low grade malignancy ( grade i ) 57 per cent were in this surgical group in contrast to only 30 per cent of the highly malignant type ( grade ill ) ( table xxvi )  . 
what , then , may acco~t for this fact ? is it merely fortuitous chance , it is true , may play a part , but this is not the only possibility  . 
 .although two groups of patients are placed in the same clinical stage , there may be some difference in the extent of the growth for which no allowance is made in our present staging systems . 
in view of the parallel revealed between hist.glogical picture and extent of tumour ( tables ix , xiv ) , it is these unfavourable cases which also tend to be of high grade malig nancy , hence the distribution in fig . 
it i < ; , therefore , useless to compare results obtained by different authors if the cases are taken according to stage and no allowance is in this instance chance is probably the chief factor made for type of growth . 
however , cannot be ruled out , individual surgeons ha , ing , to some extent , personal criteria for deciding on the measures to be adopted for a given case  . 
.y egkcte , d , routes of extension . - reference will be made to avenues of extension of breast cancer which are largely neglected in both prognosis and also treatment . 
of these unfavourable grade iii cases nearly 70 per cent ( table xxvi ) were referred for irradiation , thus emphasizing the disadvantage placed on the radiotherapy group when it comes to the comparison of treatment results . 
 to sum up : it is postulated that in a given stage certain cases haw unfa rnur able clinical features , which influence the surgeon to refer them for radiotherapy . 
 the tumours of these patients tend to be of high grade histological malignancy , and h~ve probably also extended , ia routes , as yet , not given adequate considera tion in prognosis and treatment . 
this may account , at least to some extent , for the unfavourable results achieved here and elsewhere ( adair , 194& ; truscott , 1947 ; harnett , 1948 ) by ancillary radiotherapy . 
in these instances the achievement of radiotherapy is probably even greater than appears on first examination owing to the unfavourable features which tend to be present in cases referred for this treatment . 
 it has been pointed out that the problem of grouping also applies to series treated by identical methods , though here chance rather than selection is prob ably a more important factor accounting for the distribution of the patients . 
with this method it is hoped that further light may be thrown on some of the problems of breast cancer , and possibly the true merits of the various treatments of the disease assessed . 
 when this clinico - pathological system is applied here , will the results achieved by ancillary radiotherapy appear to be any better ? this question , unfortunately , 282 h . 
harries ( personal commanication ) , at the middlesex hospital , quotes 19 per cent for the five - year follow - up and 4 per cent for a period of ten years among some 220 cases . 
these figures are inferior to those obtained by treatment , which is universally accepted as being of definite , aloe in prolonging life , except perhaps in the very advanced cases . 
 once the cases are classified more accurately , such as on the lines indicated earlier in this paper , and only then , shall we be in a position to study the merits of , arious treatments . 
we may find , for example , that less radical surgery may be adequate for the control of certain cases such as those with grade i , stage 1 tumours , whilst radiotherapy may be an important ancillary measure for some cases , but on the other hand of little use for others . 
grace ( 1937 ) in america obtained a 56 per cent fi , e - year surrival rate among 40 cases treated by simple mastectomy and radio tht > rapy . 
this author considers that " the cellular structure of the tumour is the dominant factor in prognosis , and surgical technique , irrespecti.e of its radicalism , plays a definite secondary role . " some support tor this view is perhaps to be found in the paragraphs which follow . 
a group of 45 cases in his series were treated by local excision with implantation of radium , and 63 per cent were alive after a similar period of time . 
 what do these results mean ? can it be possible that more conservative surgical measures than the radical operation are sufficient for the control of mammary cancer ? such , indeed , is the view in this country of keynes ( 1937 ) , and more recently mcwhirter ( 1948a , 1948b )  . 
 we have already suggested that when cases are properly grouped accord.mg to both stage and grade it may be found that less radical surgery may be adequate for certain types of case . 
it is not even necessary to have numerous sections to appreciate this picture ; one or two portions of tissue of reasonable size taken from the periphery of the growth are adequate . 
 thus , patey and scarff ( 1929 ) , in a series of llo cases , found that the metatases in the axillary nodes were of the same grade as the primary in 82 per cent , of a lower grade in 16 per cent , and of a higher grade in only 2 per cent . 
comparable figures to these are also given by other writers , such as harrington ( 1935 ) , lach man ( 1944 ) , riddell ( 1948 ) and ledlie ( 1948 )  . 
little attention appears to have been given to this problem until scarff and handley ( 1938 ) suggest.ed that disappointing results in stage 1 cases may be explained by this mode of spread . 
this applies particularly to growths situated in the inner half of the breast ; of 17 such cases 12 had deposits in the internal mammary chain , in 2 of which the axilla was not also involved . 
 in spite of this , truscott ( 1947 ) and harnett ( 1948 ) were unable to show that the site of the growth plays an important part in prognosis . 
 in those cases where the axilla appears to be free from involvement at the time of operation and the prognosis subsequently turns out to be poor , invasion has probably occurred by routes such as the int.ernal mammary chain , and perhaps the supraclavicular glands . 
 after considering these facts it would appear , as handley and thackray ( 1949 ) point out , that there are but few growths which are really confined to the breast . 
chief success , from the point of accuracy and simplicity , appears to have been from histological grading , on the lines mdicated by pat.ey and scarff ( 1928 ) , combined with the state of the axillary glands . 
stage i ? this question has defeated explanation in the past , and will probably continue to do so until such time as the pathologist plays a part in establishing a prognostic system of the type indicated in this paper . 
 the controversy which exists as to the relative merits of the different methods of treatment for carcinoma of the breast has led to the present investigation , the prime object of which has been to establish a more accurate classification for this disease . 
 the gra < le of the tumour taken together with the state of the axillary lymph nodes gives a more accurate indication of outcome than either grading or staging alone . 
for example , 94 per cent of grade i cases without glandular involvement were ali , e at five years compared with 16 per cent of grade iii with this complication . 
it offers an explanation for disasters in early cases , and also for remarkably good results obtained in advanced in indicates that all is not necessarily lost if a patient presents with a stages . 
 lt has been shown that in a given clinical stage or histological grade the cases treated by surgery alone are not strictly comparable to those subjected to surgery and radiotherapy . 
in addition this has probably also played a major part in bringing about the present - day confusion as to the relative merits of the various measures advanced for the control of the disease . 
sambrook for assessing the clinical st.age of the cases ; to miss chambers of the follow - up department for tracing the patients , and to the photographic department for assistance with the microphotographs . 
a laparotomy was performed on each animal using ether anesthesia for rats and guinea - pigs and nembutal for mioe , and a loop of the intestine adjacent to the pylorus ligated . 
the abdominal cavity was closed with nylon sutures and each animal returned to its original cage . the animals were not treated further until 3 to 3hours following the laparotomy by which time all had emerged from the anesthesia . 
of body - weight of a 0.5 per cent aqueous solution of toluidine blue 0 . animals were anesthetized i an hour following the time of injection and a blood sample withdrawn either by heart puncture or from the aorta . 
the ph of the contents as well as of the gastric mucosa was obtained with hydrion the stomach contents were diluted 1 : 1 with acetone , stirred , and centripaper . fuged for 10 minutes at 2000 r.p.the supernatant was withdrawn and a 1 ml . aliquot diluted with acetone to 10 ml . this was stored at 50 to 100 c . 
overnight following which it was centrifuged 20 minutes at 2000 r.p.m. , and the supernatant removed for spectrophotometric analysis . in some rats the intestinal tract was removed and the contents collected . these as well as the blood samples were analyzed in the same manner as the stomach oontents . for the standard curve , 1 ml . 
an acetone blank was used for all determinations . in the case of gastric samples , the ph of the blank was adjusted to that of the sample using hc1 . the same acetone was used throughout the experiments and was redistilled prior to use . a beckman model du spectrophotometer was employed in making the measurements . 
inasmuch as the the guinea - pig , like the dog , is considered to be susceptible to histamine stimulation , an enhancement of gastric secretion following injection in this species was anticipated . the proportion of formed elements to plasma in the blood was determined for rats and guinea - pigs but not for mice in several of the groups . these results are listed in table i under hematocrit values . it will be observed that among the rats which received histamine ( groups i , ii , v , vi ) , an average value of 69 was obtained . 
whenthe ratswere not injected with histamine ( group iii ) , thehematoneither the ph of the stomach contents nor that of the crit value averaged 65 . gastrio glandular mucosa was altered significantly by the administration of histamine . in the rat a normal hematocrit value of 45 - 8 has been reported for males of the sprague - dawley strain ( gardner , 1947a )  . this value was confirmed in our laboratory . 
thus the histamine appeared to have no additional effect on the hemoconcentration observed in all operated animals . in untreated male guinea - pigs a hematocrit value of 42 has been reported ( gardner , 1947b )  . examination of the blood of treated guinea - pigs in our experiments disclosed a significantly higher hematocrit value in every animal . 
the control and 4 treated animals show no selective absorption in the region in question . similar analyses , using 5 guinea - pigs of group i and 1 guinea - pig corresponding to rat group ii , showed no definite peaks in the range between 375 and 400m , tt . although animals were fasted 48 hours prior to the start of operations no precautions were taken to prevent coprophagy , since it was our intention to duplicate the oonditions which prevailed in the experiments of ray and peters ( 1951 )  . when gastric contents were collected , it was observed that in addition to fluids although some of this may there also was present a varying amount of solids . have been due to residual food , it is more likely that ingestion of feces was responsible . 
18 - not included in any group - rat injected subcutaneously with histamine solution alone . solid lines represent animals given compound iii . analyses of blood from rats injected with compound iii revealed no compound by a method which was shown to detect 001 mg . 
on the other hand , the presence of toluidine blue o may be established by spectrophotometric analysis since intestinal contents give no peaks in the region of maximum absorption of this dye . it is of interest that such analysis from rats injected intraperitoneally with an aqueous solution of toluidine blue 0 failed to reveal this blue dye . ray and peters ( 1951 ) reported that gastric extracts from 3 out of 3 rats treated with compound iii showed substantial absorption at 400 mru . in the present experiments only 1 rat out of 15 showed similar results , while 2 fell in an intermediate , doubtful category . 
lipschutz. from departamento de medicina experimental , servicio nacional de salud , avenida irarrazaval 849 , santiago de chile . received for publication april 2 , 1953 . the tumours which appear in the intrasplenic ovarian graft in castrated rats , mice , guinea - pigs or rabbits apparently are due to the uncontrolled gonadotrophic activity of the prehypophysis , the ovarian steroid hormones which normally control hypophysial activity being inactivated during their passage through the liver before reaching the general circulation ( iglesias , mardones and lipschutz , 1953 )  . 
we shall be able to show ( 1 ) that this is not the case , and ( 2 ) that the hormonal imbalance from which ovarian tumourigenesis under the given experimental conditions derives can , and must , be expressed in quantitative terms . for the purpose of this study the secretory activity of 15 intrasplenic grafts , as listed in table i of our previous paper ( iglesias , mardones and lipschutz , 1953 ) has been checked by the histological examination of the vaginal mucosa . the uterus and the mammary glands . 
the presence of ovarian hormones in the general circulation at a level allowing for the transformation of the vaginal mucosa and of the uterine epithelium , even with the production of cystic glandular hyperplasia and metaplasia . 
rus. * pregnancy = several layers of clear vacuolized epithelia . * * transition = several layers of proliferated cells of the basal epithelium beneath the vacuolized layers as in fig . 
as evidenced by various new observations , we must drop the idea held originally both by biskind and by ourselves as to the gonadotrophic activity of the hypophysis in experiments with intrasplenic ovarian grafts in castrated females : contrary to what we thought before , the hypophysis is , under these experimental conditions , not identical to a castrate hypophysis . thus , the content of gonadotrophic hormones in the hypophysis of animals with grafts is considerably less than in the castrate hypophysis ( jungck , heller and nelson , 1947 )  . indeed this non - identity may be due to a difference , not of production , but only of release of gonadotrophins in castrated and grafted animals ( miller and pfeiffer , 1950 ) ; but non - identity seems to be a fact . castration cells are absent in the hypophysis of the grafted rat , in 224 2 . 
lipschutz any case in advanced stages of ovarian tumourigenesis ( lacour and guerin , 1951 )  . however , as compared to a normal hypophysis , that of a castrated animal with an intrasplenic ovarian graft is functionally impaired . differential tumourigenic thresholk concentrations of oestrogen . the problem of differential levels of oestrogen by which control of the vaginal or uterine mucosa and of the hypophysial gonadotrophic activity is achieved is intimately related to the problem of the differential tumourigenic threshold concentrations of oestrogen . 
how will the hypophysis behave when exposed to the action of these minute quantities of oestrogen in experiments lasting more than 2 years ? a group of 9 guinea - pigs was castrated and one ovary was grafted into the spleen ; sixteen days later a pellet of 23 to 30 mg . 
but the quantities absorbed were not sufficient for a complete control of this hypophysial activity : they were not able to reduce it to normality , as shown by the presence of haemorrhagic follicles in most of the animals of the group . there was in this group control of the vaginal mucosa and of uterine growth . in one animal the uterine weight was only 0o6 g . ; but in the remaining animals a weight of 0 - 8 to 1 - 2 g . 
with these 1 per cent pellets the incidence is not in another great ; new experiment lasting 757 to 875 days , without intrasplenic ovarian grafts , it occurred likewise but once in a group of 10 animals . 
one may raise the question whether some irreversible hypophysial change takes place during those 2 to 3 weeks which are needed for the vascularisation of the graft and for recuperation of hormone production . we have referred above to the three gonadotrophic hormones of the hypobut the possibility must be considered that also the somatotrophic or physis . some other hypophysial organotrophic hormone is partaking in establishing tumoural growth in the ovary ; this question has been discussed especially with reference to tumoural growth of wolffian structures ( iglesias , mardones , bruzzone and lipschutz , 1953 )  . the statement that ovarian tumourigenesis does not presuppose a complete suppression of the steroid control of the hypophysis but is the outcome of an irregular interplay of ovarian and hypophysial hormones may at the first sight however . 
the hormonal imbalance may last for as long as 3 years , and sometimes even more , without norwhereas it was evident , almost from the beginning mality being again attained . of this phase of our work , that the sequels of ovarian fragmentation , i.e. , reducing the mass of two ovaries to a small ovarian fragment , or remnant , in situ , were due to an overthrow of the normal ovarian - hypophysial relationship , we were unable to understand why this hormonal imbalance is in some cases irreversibly maintained . 
gutnz of untreated leukaemic cells observed in vitro . only the quantitative aspects of the subject will be dealt with here ; the cytological findings will be described in a separate paper . normal and leukaemic blood and bone marrow have been studied in tissue cultures by many workers since the publication of the first report on tissue culture of leukaemic blood by awrorow and timofejewskij ( 1914 )  . 
among other investigations carried out in recent years the following should be mentioned , as they deal wholly or in part with culture of leukaemic blood or marrow : papers by barnes and furth ( 1937 ) and bichel ( 1940 ) on mouse leukaemia ; doljanski and pikovski ( 1941 ) on fowl leukosis ; and fieschi and astaldi ( 1946 ) , israels ( 1940a , 1940b ) , osgood and brownlee ( 1936 , 1937 , as well as many later papers from the same laboratory ) , pierce ( 1932 , 1942 ) , schrek ( 1946a , 1946b ) , and wallbach ( 1936 ) , on human leukaemia . 
the vaccine caps are washed , and as rubber is toxic to living cells , they are then coated with a very thin layer of paraffin wax , by dipping them into a saturated solution of paraffin in petrol ether and wiping off excess after withdrawal . 
gunz been put into a conical flask , and the cells are now added to it , the aim being to give a final concentration of about 2000 leucocytes per c.msome practice is required in judging the correct volume of cells to be added . three consecutive counts are done on the cell suspension , and when the mean reveals a suitable cell concentration , a quantity of 2 ml . 
3. - mean daily counts of a sample culture of blood ( chronic myeloid leukaemia ) , expressed as per cent of initial count drawn on logarithmic scale . 6 days in the light of these two experiments , the following routine procedure has been thought justifiable : the mean of three counts of the original cell suspension is taken as the 100 per cent initial value for all tubes . 
on each subsequent occasion three separate counts are done on each of three fresh tubes , and the mean and standard error between tubes are calculated for all nine counts . 
means are calculated from the three individual counts and used for constructing the growth curve . in these differential counts a siniplified nomenclature of the myeloid series has been used , cells being divided into the three broad classes of myeloblasts , myelocytes and polymorphonuclears ( eosinophils and basophils being counted separately )  . 
the term " immature cell " comprises myeloblasts and myelocytes , the latter being arbitrarily defined as complying with two or all of the three following characteristics : total cell size exceeding that of a normal polymorphonuclear by 1 / 3 or more ; nucleus oval and only slightly indented ; cytoplasm basophilic . 
some of the patients with chronic myeloid leukaemia were untreated , while others had had various forms of therapy . cultures were either continued until all cells had died , or they were deliberately abandoned at an earlier period . 
when they were continued , there was always a gradual decrease in the total counts until death of the last cells occurred , usually between the sixth and the ninth day of culture . 
no significant increase in total counts has ever been observed . of especial interest is a group of nine cultures , selected because the cells lived for at least 7 days . 
4. - mean daily counts of a sample culture of blood ( chronic myeloid leukaemia ) , expressed as per cent of initial count drawn on linear scale . days log y = 2.0792 - * 0747x 7 days fig . 
6. - effect of 5 per cent rat embryo extract on culture of blood ( chronic myeloid leukaemia )  . total and differential counts expressed as per cent of initial count . 0 1 / 3 3 days of the total count in the two series are probably not significant , they certainly do not follow a straight line . 
of greater interest is the behaviour of the immature cells , in which a striking difference between the two series is apparent . while there is a gradual decrease of the number of immature cells in the control cultures , there is a significant rise in the e.e. 
cultures showing a peak of more than four times the height of that in the control cultures . results similar to these have been found in all cultures in which differential counts were done . 
6 ; the excess polymorphonuclears can have only been formed from immature cells , and the decrease in the number of the latter is explained by their proliferation failing to keep pace with maturation . after the second day , the process is less important and soon stops , mature and immature cells decaying approximately exponentially at the same rate , as shown in fig . 
may be a ribo - nucleoprotein , and experiments are now in progress designed to co - ordinate the growth curve with the nucleic acid metabolism of the cells . the action of ionizing radiations , as well as of many chemotherapeutic agents , is primarily on mitotic cells . in investigating these actions , every effort should therefore be made to provide the maximum number of mitoses in the test object . thus in fluid cultures the addition of e.e. 
jolles. from the radiotherapy department , general hospital , northampton . received for publication december 16 , 1948 . the focusing of attention on the malignant cell in the study of tumour tissue reactions to radiations and growth inhibitory substances has not brought us nearer the state of affairs where both practical and theoretical issues of oncology and treatment of tumours can be studied with a better understanding and fairer prospects of success . the importance of the stroma reaction to tumour and stromal reaction to radiation has been accepted for a long time , but only with generalizations on changes in " blood supply " and " tumour bed , " leaving the whole problem of connective tissue reaction in a hazy state . as regards tumour tissue reaction to radiation , much stress has been laid in the past on the changes following irradiation in the cells alone , but very little although recent attention has been paid to the intercellular tissue components . advances in cytology and genetics have enhanced the importance of the cellular nucleus , it must not be forgotten that evidence , both experimental and clinical , is available to show that the effect of ionizing radiations on cells alone " is not the whole story of the events leading to the destruction of proliferating tissues and that other factors come into play " ( muller , 1940 )  . the great importance of connective - tissue reactions to x - rays in radiotherapeutics has been raised by windeyer ( 1942 ) , ellis ( 1942 ) , windholz ( 1947 )  . 
the lesion is reproduced by means of a stent impression in a plaster cast , over which a tightly fitting stent or black - tray compound applicator is built in such a way as to be easily mounted over the part of the body , and fitting in such a way as not to allow even minimal shifts of the applicator , and so avoid the x - ray beam striking the alternate " opaque " squares of the lead chess which is recessed in the mould . the radiation has to be delivered at right angles with the x - ray tube head in order to avoid oblique penetration into areas applicator directed vertically . beneath the opaque squares the lead chess must be in direct touch with the tumour it is found in the course of treatment that owing to shrinkage of the surface . tumour the recess sleeve in the stent mould into which the " chess " is slipped has to be filed down from time to time during the course of treatment . the thickness of the lead , which is limited for practical considerations , permits 3 per cent of the radiation to pass through on to the protected areas . 
to that amount of radiation received by the not directly irradiated areas has to be added about 3 - 5 per cent of scatter radiation from the neighbouring exposed areas . this varies according to the size of the apertures , and decreases towards the centre of the " opaque ' , squares . 
the size of the squares has been chosen of not less than 1 cm . , so as to leave an adequate amount of tissue either almost completely non - irradiated or exposed for biopsy taking . sections are taken before treatment is commenced and then at varying intercuts are made into the exposed and protected sectors , and also one section vals . is made cutting across both the exposed and protected areas of tissue . accurate labelling of biopsy material must be insured , as no other way of recognizing the protected and exposed sectors on the histological slide is available . a working chart is found indispensable . prior to taking the biopsy the lead mask is applied and the exposed areas are mapped out by means of gentian violet paint . the biopsy material was fixed in sublimate - formol solution or sousa 's solution . following paraffin embedding , sections were cut at 7l and stained with ( 1 ) harris ' haematoxylin , eosin , ( 2 ) van gieson stain , ( 3 ) by gallego 's method , and ( 4 ) silver impregnation . the irradiation was taking place every day , 5 - 6 days per week . 
hard mass filling right breast , fixed to pectoralis and ulcerating skin in anterior axillary line over an area 8 x 5 cwith a sloughy crater and rolled - up edges . 
at this point " chess " technique abandoned and routine glancing technique reverted to . ( 4 ) section taken when tumour received apart from the 4000r with the chess technique 2500r on each glancing field in 14 days ; 10 treatments . ( 5 ) section taken at end of treatment ( 4000r to chess and 3500r on each glancing field in 5 weeks )  . she is remarkably when last seen , 15 . 
meredith. from the christie hospital and holt radiumt institute , manhester . received for publication december 12 , 1948 . ix a series of papers ( dale , 1940 ; dale , meredith and tweedie , 1943 ) it has been shown that the action of x - rays on aqueous solutions of biologically active compounds was indirect , i.e. 
further , it has been shown that in consequence of this indirect action , if two solutes are present , the second solute ( the protector ) reduces the radiation effect on the first one ( the indicator ) ( dale , 1942 ; 1943 ) , by sharing the intermediate product formed from water by the radiation . 
kennedy. from the endocrinology research department of the new zealand medical research council , medical school , dunedin , new zealand . received for publication july 30 , 1951 . previous communications from this laboratory ( griesbach , kennedy and purves , 1945 ; purves and griesbach , 1946 , 1947 ) described the induction of thyroid adenomata in rats by the long - term administration of goitrogens . bielschowsky , griesbach , hall , kennedy and purves ( 1949 ) showed that these thyroid tumours as well as those appearing under the combined influence of goitrogen and carcinogen could be transplante ' d into rats of the same strain . transplantation was only successful when the recipient animal was kept in a state of thyroxine deficiency produced either by treatment with goitrogen or thyroidectomy . 
from this failure of the transplanted tissue to grow in normal animals it was concluded that an excess of thyrotrophin was needed for the continued progressive growth of such tissue in a similar way as it had been necessary for the induction of the original tumour in the rat 's thyroid . therefore , these tumours were not considered to be " autonomous , " although they showed such signs of mahgnancy as a typical and progressive growth , invasion of surrounding tissues and formation of metastases . 
being injected subcutaneously in the flank of each the majority of rats were continuously supplied with a - 01 per cent rat . special mention is made in the solution of methylthiouracil as drinking water . text when no goitrogen was given . 
the thyroid was very large with a nodule protruding from the isthmus . part of this nodule was removed and two pieces were inoculated into two young rats which had been given 0 - 01 per cent methylthiouracil beginning 5 days previously . 
the acini varied in size , many of them being larger than the acini of the normal rat thyroid . in one line of transplantation this structure has been maintained up to the present day , and ' fig . 
6 shows the histology of a graft from the fourth generation of this fine . it will be noted that surrounding the larger acini there are numbers of small acini which suggest that new acini are being formed by budding from the larger ones . small cellular areas exist without well - defined acini , but these are considered to be areas of prohferation in which differentiation has not yet taken place . 
the acini are well filled with colloid , this colloid accumulation being always present even under conditions of strong thyrotrophic stimulation . this presence of colloid does not , however , indicate that the tumou ' rs are not influenced by thyrotrophic hormone , since when the thyrotrophic hormone production in the host is inhibited , these tumours undergo rapid regression . iodine , metaboli8ln of t , tumour . the iodine metabolism of the transplanted t , tumour was tested in rats bearing second generation grafts . table i shows the results of in vivo tests of the uptake of radioactive iodine in three rats after radioactive iodine had been injected . 
l. - chart of the first 6 generations ' grafting of the tbyroid tumour t , the treatment of the recipient animals is indicated on the cbart . solid black indicates animal bearing tumour . 
kennedy the evidence of secretory function in the t , tumour has been obtained from the results of transplants growing in totally thyroidectomized animals . these animals the ti tumour transplants do not grow continually , but appear to reach a maximum weight of about i g . 
of rats witb tumour growth . growth was obtained in the normal animals . this indicates the dependence on high thyrotrophic hormone levels , a dependence which is also displayed when animals bearing growing tumours during the administration of methylthiouracil have their medication stopped . 
the large irregularly - shaped acini which were prominent in the ti colloid was present in some of the acini but was less tumour were not seen . prominent than in the t , tumour . 
the nuclei were hyperchromatic and irregular in sha e . ( 2 ) embedded in this adenomatous tissue was a nodule of tiss ' ue distinctly different in staining properties . in this tissue , which had a well - developed acinar structure corresponding to a very hype ' rplastic normal rat thyroid , the cells were large with abundant cytoplasthe nuclei were round and did not contain excess chromatthe lesser numbers of nuclei and their weaker staining properties ' ' were responsible for the pafer appearance of these nodules in the 306 h . 
the fact that such tissue always forms microscopic nodules and has not ever formed a large part of the grafts in any of the inoculated animals suggests that this type of tissue is a slow - growing variant which arises spontaneously within the adenomatous tissue . 
the nodules of this type seen in the fourth generation appear to have arisen from the modified adenomatous tissue of these animals mentioned above which differs from that of the original tumour . ( 3 ) the third type of tissue is an anaplastic carcinomatous tissue without any in animal 253 , no . 
in weight being observed after 10 days . in the fourth week after transplantation animals with the rapidly growing tumours began to succumb . in a proportion of the animals , however , the growth was slower and the animals survived 3 or 4 months . 
the histology of ah these tumours was that of the anaplastic carcinoma described above as the third type of tissue seen in the fourth was successfury maintained by serial generation . transplantation , and - in later transplantations successful grafts were obtained in up to 80 per cent of those inoculated . 
the t22 tumour has now been transplanted through 7 consecutive generations and has not undergone any progressive change in either malignancy or histology . histology of tumour t22on the cut surface two regions cciuld be regularly seen , the outer with a shiny grey appearance , 5 to 8 mbroad , and the central one coloured a pale orange frequently a brown albuminuous fluid had replaced with a crumbled surface . mioroscopic examination revealed that , ' generally , the inner the central tissue . zone ofthe tumours consisted ofdegenerating cers or amorphous masses , infiltrated by round ceus , and isolated cers with strongly eosinophihe cytoplasm and pycnotic nucleus , lying , between the cell debris . the outer zone , microscopically , appeared simflar to that of the anaplastic nodule of the original tumour , and this description apphes to both tissues . 
unusuary large tumour cefls were present which had slightly basophilic cytoplasm and nuclei with ir - regulariy dispersed chromatin . giant cells were present with nuclei varying in number from 2 to ' 8 . 
the numbers of giant cers found varied somewhat in different animals and were relatively few they have , however , been a constant feature of in the first animal examined . the tumour ' and we consider that the tumour should , therefore , be classed as a giant cell carci ' noma of the thyroid . mitotic ftgures are frequent and many of them atypical . effe , ct of t22 tumour on the host . the striking feature of the t22 tumour is that it kirs its host in as short a period as 3 weeks . there is , however , a considerable variation in the rate of growth in different animals , some of them surviving for 2 months or more . those animals which died within 3 to 4 weeks after inoculation , the animals showed at autopsy an extreme state of emaciation with atrophic muscles and thin soft bones . 
was lower than ' its weight at the time of transplantation . it appears , therefore , that the rapid growth of these tumours withdraws nutrients from the host and leads to the state of emaciation described . effect of thyrotrophic hormone level on tumour t22 ' the t22 tumour has been found to grow equally wer when inoculated into normal animals or animals receiving methylthiouracil . moreover , in normal animals in which this tumour is growing , no inhibition of the growth rate has been observed when the an ' imals were treated with thyroxine injections . in some selected animals bearing this tumour in which the growth was relatively slow this treatment did not produce any marked some were treated with thiouracil . acceleration in the growth of these tumours . 
the t22tumour , therefore , does not show the dependence upon thyrotrophic stimulation which was shown by its parent tu ' mour . iodine , metabolimof the t22 tumour . rats bearing the t22 tumour were injected with radio - active iodine and 24 hours later were killed , and the radio - active iodine concentrations in the tumour tissue and in the blood plasma were measured . 
kennedy considered that both the action of the carcinogen plus the hyperplasia resulting from the goitrogen were necessary for the induction ofthyroid tumours . however , griesbach , kennedy and purves ( 1945 ) reported that the prolonged action of goitrogens alone led to the formation of tumours , which in a later publication ( bielschowsky , griesbach , hall , kennedy and purves , 1949 ) were shown to be similar in all respectsto those appearing after the carcinogen treatment . 
with goitrogen alone , however , tumours are later in appearing and are fewer in number . these tumours aredependent upon high thyrotrophic hormone level for their in normal animals such tumours , either in the thyroid or as grafted growth . - tumours , undergo rapid regression and eventually disappear , so that they cannot be induced to reappear by the subsequent administration of goitrogen . 
the simplest explanation , therefore , of the role of the goitrogen in the production of these tumours is that it induces the high thyrotrophic hormone production without in this view , the goitrogen would not have which such tumours cannot grow . any influence on the formation of neoplastic cells . it is , therefore , considered that neoplastic cells arl 'se spontaneously in the normal rat thyroid , and that when conditions are suitable for the growth of such cells , visible neoplasms result . 
in weight are commonly produced.. the continuous growth of such tumours 4nd the propagation of them by transplantation leads naturary to the selection of fast - growing types of tissue so that an increase in mahgnancy with transplantation is to be expected . however , modifications of the adenoma are not invariably in the direction of increased mahgnancy . 
we consider that the type of nodule described as the second type of tissue observed in the fourth generation transplants is a relatively slow - growing benig - n structure which reappears in successive generations , and which by virtue of its slow growth and lack ofmetastasizing power cannot be selectively propagated by transplantation . 
on the other hand , the truly malignant , invasive and metastasizing carcinoma will , when it once appears , invariably supplant entirely the benign structure in two , or three at the most , generations of grafting . evolution of malignancy in benign tumour8 . while it has been recognized that transplantable mammalian tumours show increases in mahgnancy on transplantation , it has been often assumed that such increase in mahgnancy results from a gradual adaptation to the host or a gradual modification of the original tumour . 
the thyroid adenomata appearing in - the rat thyroid are all dependent on thyrotrophic hormone for their ' existence , and in fact require higher levels of thyrotrophic hormone for their continued growth than does the normal thyroid tissue . investigation of human thyroid neoplasms with the aid of radio - active iodine have shown that many of these are in fact susceptible to the stimulating influence of thyrotrophic hormone , although neoplasms in human material have not been described which are so entirely dependent upon thyrotrophic hormones as are these primary rat thyroid tumours . 
the existence of undoubted stimulating effects of thyrotrophic hormone in human thyroid tumours supports the view that these rat thyroid tumours are not exceptional , although their high degree of dependence on thyrotrophic hormone sets them somewhat apart from the thyroid tumours encountered clinically . there seems no reason to hold the view , as some people have done , that these rat tumours should not be classed as neoplasms , since in their case the stimulating hormone or factor on which their 310 h . 
kennedy growth depends ' is well characterized and can be artificially manipulated so as to presumably as further knowledge is gained the control the tumour growth . stimulating influences which condition the growth of other tumours win be discovered , but this should in no way affect their classification as neoplasms . it is important to note here that from a tumour at first dependent upon a hormonal imbalance for its growth , there has been derived a mahgnant neoplasm which is no longer dependent upon the stimulating influences which condition the growth of the more benign original tumour . 
thus where mahgnant tumours are found which are not susceptible to any known hormonal influence , the effect of hormonal imbalance in the production of such tumours is not excluded . 
the work was undertaken as a collater ' al contribution to a wider scheme planned in 1952 by the cheshire and north wales branch of the british empire cancer campaign . for research into the pathogenesis of cancer the branch council required among many other things a laboratory analysis of soil taken from the vicinitv there was no precedent for the sort of analysls of the home of each patient . most suited to the project , previous workers in this field having been content with simple and often crude analyses or with the more detailed examination of too few specimens . it was clearly impossible to make a full physical and chemical examination of all the solid , fiquid and gaseous phases of every soil sample as several thousands were to be examined , so with some slight modification the analytical procedure of the soil survey of england and wales * was adopted - at first . it was felt , however , that an investigation of fatal cases of cancer in relation to soils as mapped by the late professor g . 
every case where cancer was mentioned on the death certificate , even when not the immediate cause of death , was included in the analysis , except that those occurring in the county of persons ordinarily resident in other areas were excluded . cancer was defined as any condition named in no . 
the differences between observed and expected values were subjected to the chi - squared test to determine the probabihty of such differences arising as a result of random these are the probabilities quoted in the tables that follow . 
that cancer of site a would be no more associated in an compariwith attribute b than would be the generality of cancer deaths . sons , unless the contrary is indicated , allowance was made for variations attributable to age and sex by calculating separately an expected number for six agesex groups and then adding . 
where it was desired to allow for a possible interaction between associations , expected values were adjusted . for example , if cancer of site a had shown an association with both attribute b and attribute c considered singly , and attributes b and c were known or suspected to be associated one with the other , adjusted distribution of expected values with respect to b were determined as follows . 
thus series having a common parent material geologically , are all indicated by the same capital letter , e.g. , all derived from non - calcareous shales of ordovician age are indicated p . 
the inclusion of many of this large group in social these considerations and our special class ii was therefore open to question . interest being in the association between cancer of the stomach and soil , suggested that it might be advisable to treat separately those occupations which bring the person into intimate connection with the land . 
griffith the public mains are supphed for the most part from surface water such as there is an exception where two small small lakes fed by streams and springs . ceintres get their water from deep bore holes but the populations are too smafl to be separated in the analysis . 
the in group l there social groups , however , show certain interesting differeinces . does not appear to be any association with fl soil . there is a relative excess of deaths in the other three groups although only in group iii is this undoubtedly siginificant in the statistical sense . soil and cancer of the breast . when corrections are applied for social group and water supply it is found that the association betweein cancer of the breast and a soil as compared with all other forms of caincer is stif present ( table x )  . 
0 - 20 > p > 0i i - ii * stomach all sites iii * stomach all sites iv - v * stomach ar sites stomach all sites all social groups : stomach males all sites females stomach ar sites 13 - 8  . 
w.7. received for publication march 14 , 1949 . previous observations ( foulds , 1947 , 1949b ) showed that the growth of some transplanted mammary tumours in hybrid mice depended on a hormonal stimulus which operated in normal female mice but not in normal males ; artificially - administered oestrogen supplied the necessary stimulus for growth in males . 
some of the tumours were independent of the hormonal stimulus at the first transplantation ; others lost their dependence after several transfers . dependence on hormones seemed to characterize one stage in the life - history of mammary tumours in hybrid mice . 
the majority of the mice were br hybrids , mostly of the f3 and f4 generations , and made up of 71 mice of strain br4 ( pink label ) , 90 of strain br4 ( blue label ) , and 69 of strain br6 . 
a preceding paper ( foulds , 1949a ) describes the source of these hybrids and the incidence of tumours in them . the mice were subjected to forced breeding and most of them had pregnancies in rapid succession throughout the period of observation , even when bearing multiple large tumours . 
tumours were recognized usually when about 0 5 cin diameter , and weekly thereafter two main diameters were measured with calipers . growth charts were made by plotting the sum of the two diameters against post - mortem measurements usually agreed time . satisfactorily with those made during life ; measurements on regressing tumours were the most difficult and subject to the largest errors . frequent , often daily , measurements were made on selected mice . litters were recorded each morning except on sundays and public holidays . the time of parturition as shown on the charts was usually later than the actual time by unknown periods of up to 24 hours on weekdays and 48 hours at weekends . 
the mice were examined post - mortem except for a few that were eaten ; the tumours were measured and pieces of them fixed for histological examination . in the charts the first tumour , or largest of contemporaneous tumours , is represented by a continuous line joining crosses which indicate the actual measurements ; the second tumour is represented by a broken line and circles , and the third by a dotted line and triangles . 
178. 8 9 10 weeks ( i ) left axilla ; ( ii ) right axilla . in most of the mice from 1 to 6 new tumours appeared during the course of the first . 
1 shows three different types of behaviour at the same time in one mouse . at least three major factors contributed to the wide diversity of behaviour amongst spontaneous tumours , and even amongst multiple tumours in the same mouse : first , the regulation of tumour growth by the environmental , and presumably hormonal , changes occurring during pregnancy and the puerperium ; second , the specific reactivities of particular tumours to the environmental 348 l . 
foulds changes ; and third the qualitative changes occurring in tumours during their manifest clinical course as evidenced in particular by altered responsiveness to in the remainder of this paper the terms " responsive " and reproduction . " unresponsive " are used in a special and restricted sense to describe tumours which , respectively , do and do not vary their growth in response to the physiological changes occurring in their ho * ts during pregnancy and the puerperium . the term " progression " is used , also in a special sense , to denote qualitative change in tumours as distinguished from mere advancement in size , and as evidenced in particular , but not exclusively , by altered responsiveness to pregnancy and parturition . subsequent paragraphs detail the analysis of the phenomena here outlined . for convenience of description the tumours are classified according to a few main types of behaviour , but transitional and indeterminate types are encountered and preclude a precise statement of the relative frequency of types . 
the peak size , however , was often considerable , as shown in some of the other charts representing type i tumours ( fig . some tumours maintained their 20 )  . course unchanged through several months of observation , whereas others after two or three cycles changed their behaviour , as described in a later section . the growth of responsive type i tumours was strictly conditional and dependent on pregnancy . if breeding stopped the tumours disappeared and , as a rule , did not recur until breeding was resumed . 
51. ( i ) right vulva ( ? recurrence of tumour excised 7 weeks previously ; earlier tumour not shown ) ; ( ii ) right axilla . with a small range of growth and regression at each pregnancy , and a slow but steady increase in average size . another tumour shown in the same diagram ( dotted line ) appeared 8 weeks after the first and then followed an almost parallel course . 
the peaks of growth , or the averages of the maximum and minimum sizes during successive pregnancies , fell close to a straight line . thus , the course of a tumour was represented by a curve which could be resolved into a straight line with superimposed waves ; the straight line indicated the rate of net growth or , for want of a better term , the intrinsic growth rate , and the waves indicated the response to pregnancy and parturition . 
the intrinsic growth rate and the degree of response , however , varied within wide limits and independently similar intrinsic growth rates were of each other from one mouse to another . responsive and linked with different degrees of responsiveness and vice versa . unresponsive tumours in the same mouse sometimes , as an exception to the general rule , had similar intrinsic growth rates as illustrated in fig . 
their differentiation from type i tumours was questionable , but they seemed to correspond with relatively frequent tumours in , nonbreeding mice whose course was represented by a horizontal straight line , and possibly differed essentially from type i tumours in the ability to persist without the stimulus of pregnancy . 
the great diversity of behaviour was attributable to two circumstances : first , the intrinsic growth rates and the responsiveness varied within wide limits and independently of each other as shown in the present section and , second , progression from one type to another occurred variously during the clinically manifest course of the tumour . 
the tumour itself had changed . the recognition of a similar change in responsive tumours was necessarily more difficult because the pregnancy waves obscured the intrinsic growth rate . probably an acceleration of growth often accompanied the loss of responsiveness 4m.3~ 0k 11 i / i i..#4 i 11 9 10 11 12 - 13 14 15 i\it - l 18 19 20 21 22 16 17 weeks fig . 
19 , however , the one that regressed completely between pregnancies ( broken line ) increased its peak size somewhat more steeply than the tumour that regressed incompletely ( continuous line )  . increasing peak size , therefore , was not attributable , in general , to summation . the regression of some tumours was slight and of short duration , being followed almost at once by progressive unresponsive growth . 
the regression was sometimes trivial and probably within the limits of error in measurements , and the tumours were then scarcely distinguishable from those already described which continued as unresponsive tumours from the peak of a pregnancy wave . possibly the two tumours illustrated in fig . 
29 proved unresponsive at a subsequent pregnancy . another tumour ( broken line ) shown in the same figure behaved similarly , except that it apparently began to grow earlier and was less certainly unresponsive at the subsequent pregnancy . some tumours regressed slightly or moderately after parturition and then maintained a constant size . 
the terminal course was represented by a straight line , but the intermediate course through the first and second pregnancies after the resumption of breeding was more fairly indicated by a curved line , suggesting a gradual accession of growth energy in an unresponsive ( or minimally responsive ) tumour . 5 0o d - t . 
more commonly the first tumour was solitary and further tumours developed consecutively . so far as could be seen the spacing of successive tumours and the sequence of types were irregular . 
tumours that remained solitary throughout the period of observation were sometimes unresponsive , but more frequently responsive . in the majority of the mice where two tumours appeared at about the same time both tumours were responsive ; in the minority both were unresponsive , or one was responsive and the other unresponsive . 
as a rule new tumours were registered first during pregnancy . different types of tumours developed independently of each other in the same mouse , and progression from one type to another occurred independently in the several tumours . 
13 , regressed completely within 7 days , but when another pregnancy followed without delay regression was only partial . possibly hormonal changes during the first 2 or 3 days of pregnancy sufficed to check regression , although growth was not apparent until a few days later . perhaps a few of the " recurrent " tumours were , in reality , new primary growths , but so nearly as could be determined the recurrent tumours occupied the same positions as those which had disappeared and were of similar shape . after partial regression the reality of true recurrent growth was not in doubt . some tumours were reduced to thin plaques not greatly different in superficial dimensions from the tumours at their peaks of growth ; recurrence was manifested by a thickening of the whole plaque . other tumours shrank to vague subcutaneous thickenings of doubtful , but probably considerable , extent ; recurrent growth was evidenced by sharpening of the outlines . it seemed that tumours recurred by the simultaneous grow ' th of all parts of a substantial area of previously altered mammary tissue rather than by centrifugal growth from a in consequence , recurrent tumours were often relatively small residual focus . large when first registered and the gradients of growth and regression remarkably steep . a few tumour - bearing mice nursed and weaned their litters . their tumours behaved during lactation like those in recently pregnant mice that did not at once become pregnant again although deprived of their young . 
some dubious tumours , registered once or twice on suspicion , were not seen again , but only 5 tumours , recorded on at least one occasion without query , disappeared permanently from mice that continued to breed . after interruption of breeding the tumours that had regressed recurred , with few fig . 
the recurrences corresponded as a rule with expected pregnancies in continuously breeding mice , and the recurrent tumours , unlike those in segregated females , were responsive ; almost certainly the recurrences were stimulated by unrecorded pregnancies . 
76. male removed at a ; injections three experiments were carried out with oestrogens . in the first , each of 8 mice received 4 daily applications to the skin of 0 - 2 c.c. 
36 shows regression checked and succeeded by growth at the time of the injections , but as similar events occurred without injections during intermissions of breeding the interpretation was doubtful . in other mice an action on tumours was trivial or lacking . in the third experiment cholesterol pellets containing 10 per cent of diethylstilboestrol and weighing on the average 8 - 2 mg . 
were implanted subcutaneously in 7 mice . nine tumours previously observed in these mice had disappeared before the pellets were implanted and none recurred promptly , but eventually each mouse developed a solitary tumour . in 3 mice the late tumours recorded 366 l . 
foulds 54 days , 145 days and 145 days respectively after implantation of the pellets were in the same region as earlier tumours which had regressed , and were possibly , but , in view of the long delays , not probably , recurrent tumours . in the other 4 mice the late tumours were remote from the sites of the earlier tumours and were undoubtedly new primary growths . 
the present section records the transplantation of tumours whose previous course was known and describes experiments with tumours removed surgically with three main objectives , namely to study autotransplantation , to compare successive primary tumours in the same host , and to compare primary tumours with the recurrent tumours which followed the operation . the experiments were few in number , the general plan of this investigation being to study the natural course of tumours with a minimum of experimental interference . tumours of similar size from the same mouse were used to compare the transplantabilities of responsive and unresponsive tumours . 
tumour in the first passage tumour b grew a grew in female hosts but not in males . equally in male and female hosts , although in the second passage the implants grew tardily in males . 
from these experiments it appeared that , in general , unresponsive tumours were transplantable into male and female hosts and responsive tumours only into female hosts , but that the correlation between type of growth in the primary tumour and behaviour after transplantation was not perfect . five tumours were excised and used for autotransplantation , implants being inoculated subcutaneously near the mid - line of the abdomen in order to minimize confusion with new primary tumours . three of the transplantations were unsuccessful . 
foulds each , so far as could be judged from the short period of observation , was " responsive . " two autotransplantations were successful . growth of the implant from a primary tumour measuring about 0 - 2 cin diameter and removed 6 days after detection was evident after 26 days , and continued steadily until the mouse was killed 22 days later . 
haddow , using in the main dealers ' mice of unknown ancestry , recorded frequent measurements during and after pregnancy , and concluded that " no evidence was found to suggest that gestation itself has any influence on the rate of growth of mammary cancer in the mouse , but in approximately half of the available cases it was observed that parturition was followed by a temporary decline in tumour growth rate . " haddow 's two figures illustrate , respectively , a substantial though incomplete regression after parturition and a halting of growth without regression ; they match some curves of mine . 
gardner ( 1941 ) described 5 hybrid ( 057 black x cba ) f1 mice which , repeatedly , had tumours attaining 1 - 2 cin 2 to 5 pregnancies ; the tumours regressed after parturition , but 3 of the mice eventually died with mammary adenocarcinomas . the responses of the tumours in the hybrid mice used in the present investigation are , possibly , exceptional in frequency and degree , but are not essentially different from those described by haddow and by gardner . notable response to pregnancy and parturition is here recorded in hybrids of varied genetic constitution derived from the inbred strains c57 black , riii and a ; it is not correlated with anomalous transmission of the milk - borne mammary tumour agent . the pregnancy effect usually becomes apparent during the second half of gestation , but probably begins during the first week and continues until the ' day before parturition . 
the abrupt regressions about the time of parturition suggest the sudden withdrawal of a stimulus which , though not yet identified , is presumably hormonal , affecting widely - distributed multiple tumours . 
the preliminary experiments with hormones are indecisive ; a simple oestrogenic action seems unlikely , and the possible effect of gonadotropic hormones , placental hormones , and the co - operative action of oestrogenic and luteal hormones require further investigation . lactation has no specific effect , and modifies the course of the tumours only in so far as it delays the next pregnancy . the pregnancy effect on mammary tumours is comparable with the " promoting " action of various procedures on chemically - induced tumours of the skin of rabbits and mice . 
rous and his colleagues ( rous and kidd , 1941 ; mackenzie and rous , 1941 ; friedewald and rous , 1944a , b ) studied promoting factors in rabbits , and berenblum , who has recently adopted rous 's nomenclature , in mice ( berenblum , 1944 ; berenblum and shubik , 1947 )  . 
foulds his colleagues describe the " promoting " action of trauma and irritants in eliciting and maintaining tumour - growth from latent tumour cells present in the skin as a result of the " initiating " action of a carcinogen . 
some " conditional " tumours grow only whilst the promoting factor operates ; they disappear when it is withdrawn , but recur from the irreversibly altered epithelum when the promoting factor is restored . 
rous and kidd discuss whether or not their argument the conditional growths of rabbits ' skin are " true tumours . " in essential applies equally to the conditional mammary tumours of mice . agreement with rous and kidd , i consider that their exclusion from the group of tumours by an arbitrary definition is unwarranted , and i describe as " tumours " all the mammary growths except a few palpable nodules which regress and do not recur , and those nodules , probably similar , that do not survive autotransplantation . they are accountable to a reversible change in mammary tissue , and perhaps to the exceptional persistence in gross nodules of the properties of the hyperplastic nodules or " adenomas " which are widely accepted as precursors of mammary tumours in mice although many of them are abortive ( gardner , 1941 )  . browning ( 1948 ) reported that nodules less than 02 cin diameter in c3h mice were not transplantable , and that about a third of them regressed , whereas nodules larger than 02 cin diameter never regressed . my observations differ from browning 's in revealing no correlation between behaviour and size ; autotransplantation of tumours more than 05 cin diameter was unsuccessful . the remaining mammary tumours develop from an irreversibly altered mammary tissue . 
the great diversity of behaviour is attributable to the interplay of factors which vary within wide limits and independently of each other . classification into types , though convenient for description , is arbitrary , and hampered by gradations from one type to another . 
the primary distinction , although not absolute , is between the " responsive " tumours whose course is notably influenced by pregnancy and parturition and the " unresponsive " tumours whose growth is unaffected by reproduction . the tumours assigned to responsive type i correspond with the " conditional " neoplasms of rabbits ' skthey grow during pregnancy , regress promptly after parturition , and recur , with rare exceptions , in the same place at each successive .pregnancy ; after several cycles of growth and regression they achieve no net increase in size or aggressiveness . 
the responsive type ii tumours are less strictly " conditional " ; they wax and wane at each gestation , but with a net increase of size at each cycle . 
the rate of net increase is steady and characteristic of the individual tumour , depending on a specific property described as the " intrinsic growth rate . " the term is provisional , to be discarded when elucidation of the property suggests a better name , and is used to denote the steady growth , represented by a straight line , upon which the waves of response to pregnancy are superimposed . 
the intrinsic growth rate , measured by the slope of the straight line and the responsiveness as indicated by the amplitude of the pregnancy waves , are constant , often for long periods , in a particular tumour , but vary within wide limits and independently of each other from one tumour to another . 
foulds of tumours , namely the capacity for progression , as above defined , and that general principles or rules of progression , deduced from the study of mammary tumours of mice , are widely applicable . the material for this analysis of progression consists of spontaneous tumours following their natural courses without experimental interference beyond temit is especially favourable on account of the porary interruption of breeding . abundance of multiple tumours , for the comparison of multiple tumours establishes beyond doubt that progression is a change in the individual tumour ; alteration in only one of several contemporaneous growths is not attributable to change in a hypothetical " general resistance . " the varied tumours encountered in this investigation may be arranged in a series having at one extreme the wholly conditional responsive tumours , at the other extreme the rapid - growing unresponsive tumours , and intermediately all indidegrees and combinations of responsiveness and intrinsic growth rate . vidual tumours , however , do not in general traverse this series ; they advance by abrupt steps , vaulting many possible transitional stages . 
at its first clinical manifestation a tumour may occupy any position in the series of types , irrespective of the time of its occurence or its position in a sequence of multiple tumours . 
tumours which develop simultaneously or consecutively in the same mouse conform sometimes to the same type and sometimes to as many types progression occurs independently and unpredictably as there are tumours . in multiple tumours in the same animal irrespective of their size or clinical progression may occur earliest in the first , second , or any subsequent duration . member of a sequence of multiple tumours . 
when two or three tumours , similar in size , duration , and type of behaviour are present , progression occurs in only one of them at a time . moreover , the progression of one tumour has no apparent effect on the course of the others . 
the present observations decisively controvert browning 's ( 1948 ) suggestion that a second tumour may " abrogate the autonomy " of the first . the several recognizable characters of individual tumours undergo progression often , no doubt , responsiveness and intrinsic growth rate independently . change simultaneously , but in some tumours progression in responsiveness occurs transplantation without progression in intrinsic growth rate and vice versa . experiments provide further evidence of independent progression of characters , namely the responsiveness of transplanted tumours to pregnancy and the gross milky secretion in response to oestrogenic stimulation ( foulds , 1949b )  . the development of a tumour does not always reach its end - point within the life - time of the host ; progression advances further during serial transplantation ( foulds , 1949b )  . 
the course of the growth elicited by pregnancy depends on specific properties which change , by progression , in latent tumour cells and in stationary tumours . the manifestation of progression in the mammary tumours is usually abrupt , as it is in the induced skin tumours of rabbits and mice , but progression itself is not necessarily abrupt ; it is possible that cumulative gradual changes reach a threshold level at which they are first manifest . 
some tumours evidence a gradual progression through a graded sequence of types . gradual , continuous progression seems uncommon , but it is less conspicuous than the abrupt type , and more accurate and more frequent measurements might reveal it more frealthough the observations are few and indecisive , it seems that slow quently . gradual progression is apt to result in sluggish , irregular , or dubiously unresponsive growth . 
the unequivocal rapid - growing unresponsive tumours , as a rule , are either primarily unresponsive or the result of abrupt progression from a fully responsive and , often , strictly conditional tumour . apparently two paths of progression are available . 
one path leads directly to unresponsive tumour ; the tumour acquires its definitive properties early without traversing the numerous intermediate stages which are possible , and which are observed on the other path or " responsive detour . " the detour leads ultimately to an unresponsive tumour , but progression along it may be slow and gradual and the end - point indeterminate , or reached only after transplantation . 
abrupt progression switches many tumours from the detour to the direct path ; jumping the intermediate stages , the tumours change from the fully responsive to the definitive unresponsive type . 
a few applications arehere briefly indicated . the behaviour of tumours is the resultant of multiple characters which vary , within wide limits , independently of each other and undergo independent progression . 
the - characters include growth energy , invasiveness , capacity for dissemination , and responsiveness to environmental stimuli , of which the hormones are the most easily recognized , but not necessarily the only , examples . " malignancy " is not a single character . 
independent progression of characters ( rule ii ) , however , results in disproportionate or " out - of - step " development , as for example in the " benign " tumours that metastasize and the " locally malignant " tumours that do not . 
the out - of - step development is especially important in carcinoma of the prostate , which , despite conspicuous growth , invasion and dissemination , willis ( 1948 ) criticizes the sharp division of tumours is responsive to hormones . into innocent and malignant groups , and deprecates the question , " is this tumour innocent or malignant ? " ; the appropriate question in his opinion is , " how it is even more important , i suggest , innocent or malignant is this tumour ? to ask , " in which characters , and to what degree in each of them , is the tumour innocent or malignant ? " errors in the prognosis of " early " tumours are explained by the observations that progression is independent of the size or duration of a tumour , and that progression occurs without manifest growth , as in latent or stationary tumours ( rule iii and corollaries )  . 
progression without manifest growth and independently of the size or duration of tumours probably accounts , too , for the otherwise mysterious but clinically important phenomena of long - delayed recurrence and secondary growth after apparent similarly the ultimate failure of several chemo " cure " of primary tumours . therapeutic methods after favourable early response is reasonably attributed to progression in the inhibited tumours . 
some tumours maintained the same type of behaviour throughout their clinical course ; others changed their course , often abruptly , as the result of an irreversible qualitative change in the tumours termed " progression . " the diversity of behaviour of the spontaneous tumours was attributable , in the main , to varied combinations of responsiveness and intrinsic growth rate , and to progression in one or both of these characters . following generalizations or rules of progression summarize the results of the analysis of the behaviour of tumours , especially multiple tumours , in breeding and non - breeding mice : ( 1 ) progression occurs independently in different ( independent progression of multiple tumours . ) tumours in the same animal . ( 2 ) progression occurs independently in different characters in the same tumour . ( independent progression of characters . ) ( 3 ) progression is independent of growth , occurring in latent tumour cells and in stationary tumours . ( 4 ) pro ( 5 ) progression follows one of altergression is continuous or discontinuous . ( 6 ) progression of a tumour does not always reach its end - point native paths . within the lifetime of the animal . these rules are probably widely applicable to the behaviour of varied tumours in animals and in man , and some applications are mentioned briefly . 
 " malignancy " is not a single character . disproportionate or " out - of - step " development of the various characters which determine malignant behaviour ( independent progression of characters ) accounts for many anomalieg in the behaviour of cancer in man , and progression in tumours whose growth is inhibited probably explains the ultimate failure of therapeutic measures which have a favourable immediate effect . i am grateful to l . 
8. - hormonally - induced hyperplasia on anterior edge of pectoral fin showing an early translucent appearance of tumour produced by my ' xomatous change in stroma . of the mucus - secreting epithelium and a myxomatous change in stroma . 
the present situation has been briefly restated by stocks ( 1953 ) at cardiff . there have been many endeavours to establish a racial factor to account for the known variations in cancer incidence . although they have been invariably inconclusive , as might be expected when consideration is given to the pronounced differences within individual countries , these surveys have shown that where racial types have migrated to other countries , e.g. , negroes to america , they appear to take over the incidence of malignant disease of the natural inhabitants among whom they have come to live . 
1. goitre distribution in holland ( amsterdam university )  . year , three further districts have been added to the list in the northern province of holland ( polak , 1954 ; personal communication )  . the lower incidence of goitre towards the belgian frontier is apparent and almost certainly significant . ( 2 ) united kingdom . variations of goitre and cancer within the united kingdom are striking . 
a recent survey ( stocks , 1950 ) of the incidence of carcinoma of the stomach in 83 county boroughs over a period of 18 years showed notable differences " which cannot be explained by chance variation or by differing accuracy of certification of cause of death "  . these variations were shown to correspond to variations in the hardness of the water supply : those with a moderate hardness tended to have 396 j . 
survey of goitre ( murray et this would be in keeping with the al . , 1948 ) to have a high goitre incidence . known clinical experience with regard to malignant disease in that county . comparing these two maps a striking correlation is at once apparent . outstanding are the heavy incidences of goitre and cancer in mid - wales , westmoreland fig . 
spencer and the fen district and neighbouring counties , and by contrast the light incidence in yorkshire , lincolnshire , herefordshire and pembrokeshire . gradations of correspondence on both maps are evident in comparing norfolk , suffolk and essex ; kent and sussex ; cornwall and devon ; glamorgan and carmarthen . a higher death rate from cancer was shown to exist in two swedish counties , kopparberg and gefieborg , than was found in the total rural areas of sweden ( stocks , 1925 )  . those two counties , as shown by examination of schoolchildren , recruits for the army and candidates for confirmation , were found to have a higher incidence of goitre as compared with the rest of the country . similarly , known goitrous counties in norway were compared by stocks ( 1924 ) with counties showing little goitre , and it was found that the goitrous counties showed a cancer rate for all organs of 113 - 7 as compared with 94.4 for the niiongoitrous counties . in iceland , the thyroid has been shown to contain one of the highest conlcentrations of iodine , and endemic goitre is non - existent ( rundle , 1951 )  . thyroids there weigh on an average 14 g . 
and carcinoma of the stomnach and oesophagus , when considering separate states . ( 10 ) australia and new zealand . australia is considerably more industrialised than new zealand and has many furthermore , rodent ulcer is large cities as compared with the latter country . infinitely more common in australia , where goitre does not obtrude as a major health problem , though there are certain areas where there are definite , if small , districts where goitre is found ( kelly , 1946 )  . 
the more rural and agricultural population of new zealand has here goitre is a major health problem and its a death rate from cancer of 14 - 56 . incidence in europeans , maoris and animals alike has been closely studied for years . 
as metabolic rate is in general a rough indication of thyroid activity , there would be appear to be some relationship between malignant disease and thyroid thus , the lowered metabolic rate often seen in diabetes mellitus dysfunction . ( joslin , 1946 ) may be considered with the increased liability of diabetics to develop carcinoma in any site or organ as compared with non - diabetics ( joslin , 1946 )  . in the same way , the lowered metabolic rate commonly found in patients with all types of peptic ulcers ( kolmer , 1949 ) may be considered with the incidence of carcinoma of the stomach - still one of the commonest neoplasms of man . ( 2 ) it has long been observed that puberty and pregnancy are periods when goitres may appear ( marine and lenhart , 1909 ; marine , 1935 ) , and may disappear when the period of excessive demand ceases or when iodine is given . these are also the periods when melanomata tend to increase in size and may , not infrequently , become malignant . 
at the beginning of this year rwas admitted to this hospital on account of malignant melanoma with spread to glands in the course of her third pregnancy . in her two previous pregnancies she also developed malignant melanoma on each occasion and was in this hospital in consequence . she has a moderate enlargement of the thyroid of the smooth " colloid " type seen commonly in pregnancy . 
the end of pregnancy is also the period when chorionic carcinoma and lactation carcinoma of the breast occur - perhaps the most rapidly growing class of cancer known . ( 3 ) administration of thyroid has been found to improve the action of oestrogens in the treatment of carcinoma of the prostate , permnitting the effective use of smaller doses of oestrogen and delaying the onset of insensibility to oestrogen ( winsbury white , 1948 )  . in support of this clinical experience a paper in endocrinological research is quoted ( chu and you , 1945 ) , but the final conclusions of these workers was that the simultaneous administration of oestrogen and thyroid was the same as that obtained by thyroid feeding alone . ( 4 ) reduction of keloid formation can often be brought about by administration of thyroid . 
the age for the start of the decline in iodine content of the thyroid coincides in general with the age at which malignant disease in mnan becomes a prominent feature . ( 7 ) in the second week of january of this year there were at least three patients in this hospital at the same time with gross goitre and cancer . m - - , carcinoma of breast and a large colloid goitre visible seven or eight beds away down the ward . e - , lymphosarcoma and a thyroid that weighed 80 g . 
the nodular goitre caused some difficulty ing involvement with growth . during resection of glands of neck . since january other patients have been seen in the wards of the hospital with coexistent carcinoma and goitre . it is relevant to note that the area from which this hospital draws its material , gloucester , somerset and cornwall , is not one in which goitre is frequent . ( 8 ) the association of goitre and malignant disease in the post - mortem room was strikingly illustrated by analysis of 1000 post mortems at the middlesex hospital ( stocks , 1924 ) , when anomalies of the thyroid were found in 13.3 per cent of males and 21.2 per cent of females of 500 persons dying of cancer ; whereas only 2 - 0 per cent of males and 6.5 per cent of females dying of non - cancerous conditions showed anomalies of the thyroid . professor barlow excluded from these anomalies secondary invasion of the thyroid by cancer , and the anomalies most frequently found were simple parenchymatous enlargement and adenomatosis , while calcified nodules , cystic changes , atrophy and fibrosis were frequent . 
of dba ( bather , 1952 )  . ( 3 ) rats bearing walker rat carcinoma 256 were treated with natural thyroof these 27 per cent showed complete remission and 12 per cent " favourxine . able " histological response , 68 per cent . 
showing no response at all . another group treated with synthetic hormone gave only 2 per cent of remissions and no response in 98 per cent of animals . 158 animals were involved ( herbut , kraemer , and jacksen , 1950 )  . regression of rat tumours may be induced by thyroxine the natural comment . hormone being superior to the synthetic . ( 4 ) feeding butter yellow ( p - dimethylaminobenzene ) to rats produced malignant neoplasms of the liver . 
the recent work of moon , simpson , li and evans ( 1950a , 1950b , 1952a , 1952b ) has convincingly confirmed the conclusions of earlier workers twenty years ago ( ball and samuels , 1932 ; bischoff , maxwell and ullmann , 1934 ) , that whereas extracts of the anterior pituitary cause tumours to arise in a variety of sites in laboratory 408 j . 
spencer iodine availability , traced by goitre incidence , appears to be one of such factors . closer scrutiny of some of these countries corroborates these conclusions . clinical findings and a review of some of the experimental work available lend further emphasis to these observations . the apparent relationship of thyroid insufficiency and the liability to develop cancer is discussed in connection with such other hormonal influences over cancer as are already known , particularly in respect of the pituitary . lines of investigation and clinical trials have been started , but results of any value cannot be expected for several years . a non - specific organ immunity or susceptibility seems to be the simplest explanation of the facts presented , and the possibilities , preventive and perhaps therapeutic , that are opened up by this line of research are briefly discussed . i am indebted to polak of amsterdam for fig . 
calcutt. from the department of cancer research , mount vernon hospital and the radium institute , northwood , middlesex . received for publication may 3 , 1952 . the staining of living tissues , either in vitro or in the form of freshly excised material , is a well recognised technique . normally it is restricted to the identification of specific morphologic entities such as mitochondria . attempts to determine intracellular biochemical features have been rather unsuccessful as far as living material is concerned , although successes have been achieved with fixed specimens . 
the availability of tetrazolium compounds now has rendered possible further attempts at the identification of intracellular sites of reduction . three compounds are generally available . in their normal state they form colourless solutions , but in the presence of suitable reducing groups they are converted to coloured formazans . 
 it has recently been demonstrated that the most critical factor determining the epidermal mitosis rate in the adult mouse is the carbohydrate supply to the cells , and the evidence at present available all points to the conclusion that the function of this carbohydrate is to supply the energy needed during cell division in extending this work the discovery has been ( bullough , 1949a , 1950a , b )  . 
 made that a restricted diet has a powerful effect in depressing mitotic actirity ( bullough , 1949b ) , and it has been suggested that this obserration may help towards an explanation of tannenbaum 's remarkable results on the effects of restricted diets on tumour genesis ( tannenbaum , 1940a , 1940b , 1942 , 1944 , 1945 , 1947 )  . 
this involved a study of the results obtained with a graded series of diets as regards body weight , blood sugar concentration , liver glycogen content , and epidermal mitotic activity . 
 the two experiments performed involved a total of 55 mice , which were all in the first experiment the 25 adult males of between 3 and 5 months of age . 
 these mice had been reared since weaning on a mixed diet of commercial rat cake with cod - liver oil , flaked maize , and dog biscuit , but during the experiments they were fed on commercial rat cake alone . 
this deter mined the form of their diurnal cycle of mitotic activity , and ensured that a high mitosis rate developed shortly after midday ( bullough , 1948 )  . 
it was unfortunate that the animals in the first experiment were considerably lighter than those in the second experiment , but it was remarkable how little this fact affected the results obtained . 
 on the day when each experiment began a piece of ear was removed from each animal at 14.00 hours , the normal time of maximum epidermal mitotic activity associated with the afternoon sleep period . 
the animals were killed at 1500 hours , it having been previously determined that for a period of about 5 hours after the injection of this weight of colchicine there is no great disturbance of the mitosis rate or of the blood - sugar level ( bullough , 1949c )  . 
the liver was removed , cut into two roughly equal parts , and the glycogen content was converted into glucose by the method of good , kramer and somogyi ( 1933 )  . 
 in estimating the epidermal mitotic activity the pieoos of ear , fixed in bouin 's alcoholic fluid and embedded in ester wax ( steedman , 194 7 ) , were cut into sections 7 thick . 
 255097 217096 200076 199 : : : ! : : 075 197078 100 , , - : 250097 261085 267075 25 - 1 + 092 279 ; 0 - 97 in the second experiment only strong 's cba males were used , and these , being 4 months old when the food restrictions began , were heavier than the mice of the first experiment . 
 252068 244 : : : ! : : 052 218043 21 4052 21206 . ' ; 90 0 ' .o 247150 257118 252130 253075 246070 table ii . - the average body weight ( in grammes ) in group& each of 5 adult male mice fed on restmtetl diets of rat cake . 
 306116 288 : : : : : 115 279075 283070 299094 the results obtained were much as could have been expected , the figures for the groups on the lower diets falling steadily as the weeks passed . 
by the end of the fourth week it is clear that the relationship between body weight and diet is a direct one which can be expressed by a straight line graph . 
 since it is evident that mitotic activity is closely associated with carbohydrate concentration , the carbohydrate reserves were estimated at the close of the experiments in terms of the liver glycogen content and the blood sugar concen tration . 
 233 12 l 214448 200045 183459 172323 240619 218 133 199422 187413 18001 - .~ 175108 superficial examination of table iv might suggest that the poorer the diet the higher the blood - sugar level . 
at that time the blood - sugar level must have been low ( bullough , 1949b ) , and with a coincidentally low secretion rate of insulin the effect of the meal must have been to raise the blood - sugar level to abnormal heights . 
it is thus logical that the poorer the diet the higher the blood - sugar level should be , and once again the relation is a direct one expressible as a straight line graph . 
 75029 36042 25017 23018 22024 one surprising conclusion that can be drawn from these figures is that the imposition of a restricted diet results in an extremely rapid fall in the mitosis in most groups there was a steady reduction throughout the whole experi rate . 
i these results are expressed graphically , the figures chosen for com parison being ~ose of the numbers of mitoses observed after 3 weeks ( without colchicine ) and after 4 weeks ( with colchicine )  . 
it appears that in general the mitosis rates of the lighter mice of the first experiment were lower than those of the second experiment , but the differences are remarkably slight and are of doumful significance . 
 the most significant point emerging from these graphs is the indication given both with and without colchicine that the relationship between resmcted diet and mitotic activity is not a direct one . 
 the present results confirm earlier observations that restricted diets cause a pronounced fall in mitotic activity , and indicate that when less than 70 per cent of the normal food intake is given the epidermal mitosis rate is reduced to about 35 per cent of the normal . 
on the basis of earlier work it can be safely suggested that this reduction is caused by a shortage of carbohydrate , in the form of glycogen or glucose , in the epidermal cells themselves ( bullough , 1949a , 1949b , 1949c , 1950a , 1950b ; bullough and eisa , 1950 )  . 
at this time the fall in the body weight is only just beginning , and it is therefore evident that a mouse is unable to maintain a high rat , e of cell division by means of the utilization of fat deposits . 
 in a fully - fed four - month - old mouse of about 35 g . , as in a fully - fed three - month - old mouse of about 25 g . , the epidermal mitosis rate reaches a sleep maximum of about 75 mitoses per c similarly in either case the food intake remains steady at about 36 g . 
 ' when fed on restricted diets the mice lost weight in a regular manner week by week , the speed of loss being in direct proportion to the degree of underfeeding . 
 in a similar manner the carbohydrate reserves , as indicated by the liver glycogen content , fell after 4 weeks to a level which was directly proportional to the amount of food given . 
2 , in which for comparison a graph of the present results is in both cases the form of the graph is sigmoid , and in both cases the included . 
this adds further support to the thesis put forward by bullough ( 1949b ) , and developed in the following review ( bullough , 1950c ) , that the rate of tumour genesis in a particular tissue or in the body as a whole may be directly related to the mitosis rate . 
the mitosis rate in turn is related to a tariety of factors , of which one of the most important is the carbohydrate , or calorie , content of the food . 
 groups of adult male mice were maintained for 4 weeks on diets varying from 55 per cent to 105 per cent of what they would eat if fed ad libitu weekly records were made of the body weights and epidermal mitosis rates , and at the end of the experiments the carbohydrate reserves were estimated in terms of liver glycogen content and blood - sugar concentration . 
die greatest mitosis depression accompanied a reduction from 80 per cent to 70 per cent of the normal diet , a result which compares closely with that of tannenbaum ( 194 7 ) regarding the effects of restricted diets on carcinogenesis . 
 ( karnofsky , burchenal , armistead , southam , benstein , craver and rhoads , 1951 ) , therefore , aroused much interest . human trials on the hazards of water contamination carried out by the american army medical centre indicated that aliphatic nitrogen mustards might be absorbed after oral administration : hn3 in a total dose of 15 to 18 mg . 
the walker tumours were passages of tumour cell suspension in dextrose , prepared by craigie 's mincer technique ( craigie , 1949 ) and preserved at - 70 ' c . 
the mouse sarcoma was treated daily for 7 days . in the tests against the walker tumour the total dose was given over 7 , 8 or 10 days , and ajso as a . 
single dose ( boyland , cleg , koller , rhoden and warwick , 1948 ; haddow , kon and ross , 1948 )  . eleven to fifteen animals were used for test . the water - soluble drugs were dissolved in distilled water immediately before administration and given orally or intraperitoneally . 
of distilled water was passed through the tube to ensu - re that the whole dose was washed into thl - , stomach . r 48 was given in a single oral dose in arachis oil . control animals received the solvent by stomach - tube or remained untreated . 
the walker tumours and sarcomata 37 were dissected 14 and 9 days respectively after transplantation , fixed in bouin 's fluid and 70 per cent alcohol , dried between filter - paper , and weighed . the difference in weight between control and treated tumours was analysed statisticafly . 
the maximum tolerated oral administered orall dose was about twice the maximum tolerated intraperitoneal dose . using these doses , both oral and parenteral treatment produced similar effects against the walker tumour ( table 11 )  . 
the oral activity seeme ' d to be influenced by the distribution of the total dose ; the weight ratio between control and hn2 - treated tumours became more significant when the total dose was divided over 7 instead of boyland , cleg , koller , rhoden and ' " tarwick ( 1948 ) found over io da - ily doses . that the parenteral activitv of nitrogen mustards increased when the total dose was given as a single injection . 
when given in a single oral do the effect of hn3 was negligible , whereas the activity of hn2 and nor hn2 seemed to be increased to a certain extent . 
the oral activity of nor hn2 seemed at least equal to that of hn2 , and more pronounced than that of hn3 , but even the highest weight ratios between control - tumours and those treated with the ahphatic compounds were only about one - quarter of the ratio recorded after treatment with r 48 ( table ii )  . 
the other aromatic nitrogen mustard tested , nn - bis ( 2 chloroethyl ) p - phenylene diamine hydrochloride , when given as a single oral dose of 4 mg . 
peczenik the adult group ; the ratio was about 3 times that recorded in the young animals - seven tests only were performed in duplicate , six of them against the walker in the three tests with the young rats the duplicat - e results agreed , tumour . whilst in the three tests carried out with the adult rats the duplicates agreed in two but disagreed in the other ( intraperitoneal treatment with hn3 )  . 
the maximum tolerated oral dose of nor hn2 was about 10 times larger than that of hn2 and 14 times larger than that of hn3 ( table 11 )  . 
the therapeutic ratio of hn2 and hn3 , whether the drugs were given intraperitoneally or orally , seemed as low as 2 , or even lower . as estimated from the death - rate , hn2 and hn3 were more toxic to tumourbearing than to normal rats . 
was toxic to adult but tolerated by young tumour - bearers . effect of nitrogen mustard8and triethylene melamine on body - weight . the tumour - inhibitory effect of the agents investigated was frequently assoin some instances , more frequent in ciated with inhibition of somatic growth . the adult than in the young rats . 
the experiment is described here because it has been suggested that urethane potentiates the effect of nitrogen mustards clinically . three groups of adult bearers of walker tumour received hn3 orally in a daily dose of 0 - 4 mg . 
of urethane daily , a dose of urethane whichper8e is known to be inactive against the walker tumour . another group received unstandardized adrenocortical extract ( eucortone ) intraperitoneally , i ml . 
peczenik from that of the controls during the first few days inhibition of the walker tumour became manffest during the second week , suggesting that the agents clid not influence the " take " of the transplants . the therapeutic ratios of hn2 and hn3 are probably not lar er after oral than after intraperitoneal administration . in non - tumour - bearing rats fed with hn3 , roberts ( 1952 , personal communication ) found changes in the intestine very similar to those described by several authors after injections of nitrogen mustards ( selye , 1950 )  . in view of this finding , it is worth noting that most of the tumour - inhibition shown in this paper was associated with significant suppression of somatic growth , whether the drugs that held true even when the net weight were given intraperitoneally or orally . ( body - weight minus tumour - weight ) was considered instead of the gross weight . thus the question arises to what extent the tumour - inhibition recorded after oral administration of the agents was due to a specific effect or to starvation . 
walpole ( 1951 ) has fo - und 40 per cent growth - inhibition of untreated walker tumours when ifthis 40 per cent ofthe observed somatic growth was prevented by underfeeding . tumour - inhibition is deducted from the figures in tables i to iv , the maximum .tolerated oral dose of hn3 would appear to be inactive in adult tumour - bearers ( table iv ) whilst the significance of the other results remains unchanged . it may be assumed , therefore , that the retardation of tumour - growth described above was this view is supported by the finding mainly due to other causes than starvation . that the suppression of tumour - growth after a single oral dose of r 48 or nor hn2 was not associated with suppression of somatic growth . moreover , - whilst effects , on tumour - growth were similar , effects ' on body - growth were more pronounced after intraperitoneal than after oral treatment . 
most of the animals on both diets lost weight during the treatment , and the ratio between the weight losses in rats with the 20 per cent and the 5 per cent protein diet was almost the same as the ratio of the degrees of tumour - inhibition . according to houck , crawford , bannon and smith ( 1947 ) , animals treated with nitrogen mustards lose body weight by loss of protein and water . 
the rats under these experiments , therefore , though fed with loss of protein may also intensify a high protein diet ( 19 - 2 per cent ) , lost protein . according to elson ( 1951 ) , the effect of inhibitors on tumourand body - growth . as an initially large dose of an inhibitor is gradually reduced , the animal will recover from the inhibitory action quicker than the tumour will , and the latter may completely regress under high protein diet . my experiments in which the drug was given in a single dose the loss of protein ceased in time to arow the animal to recover its growth - rate whilst tumour - growth remained retarded . it may well be that in those summary . administered intraperitoneally in about maximum tolerated doses , nor hn2 , hn2 and hn3 had approximately equal activity against the walker carcinosarcoma , although aaainst the sarcoma 37 , hn3 was more active than hn2 . the walker tumour was significantly more affected by triethylene melamine than by the aliphatic nitrogen mustards . 
r 48 was at least 4 times as active as the aliphatic bis ( 2 chloroethyl ) p - phenylenediamine was active after intraperitoneal amines . but inactive after oral administration . bromine substitution in hn2 and hn3 seemed to cause loss of activitv . triethylene melamine given orally was practically inactive in the non - fasting rat , whereas the same dose given to the fasting animal had marked activity but was toxic . tumour - bearing rats tolerated r 48 and nor hn2 in doses respectively 36 and 10 times the maximum tolerated dose of hn2 . 
the smith type is still , however , a relative rarity and less than a score of cases have been reported since the original description in 1934 . the forowing case , the second to be seen in south africa , closely resembles that originally described by smith ( i934 ) ; but it is unique in that the . 
he had suffered since 1942 from recurrent spontaneously heahng tumours of the left face , ear , neck , chest , shoulder and upper arno other member of his family was known to have suffered from any similar disease ; and he was not related to the other south african who suffers from the smith type of morusca pseudocar ' c ' momatosa . skin generally was normal for his age and was not hypersensitive to sunhght . his occupation as a contractor kept him mainly in the open . 
he had never been exposed to tars , mineral oils or arsenic . he stated that the tumours , of wbich he usually had several active , began like festering blackheads " , grew for 4 - 6 weeks , and then began to regress and disappeaxed , leaving scars , in 4 - - 6 months . 
on his first visit he had two active tumours on the back of the left ear and one over the left scapula and thirty depressed wbite scars on the left side of the nose , upper hp , cheek , ear , neck , chest and upper arthe scalp and buccal mucosa were unaffected . 
a month later the ear lesions were distinctly smaher and flatter and that over the scapula had flattened dow - n to a coin - like plaque with rolled edges and a central area of soft keratinous material . all the lesions were chnically indistinguishable from squamous epitheliomata . 
at this visit a fresh lesion , i mm . , was discovered iin the angle of the left ear lobe and cheek it closely resembled , as the patient had described , a festering blackhead . the patient , who b . - ilieved himself to be suffering from skin cancers , has on several occasions been subjected to biopsy or excision of tumours ; and a diagnosis of squamous epithlioma had generally been made . x - ray therapy had also been given niore than once with little impression on the rate of healing of the tumours ; but the scars left in such areas were more mutilating than those seen after spontaneous healing . a portion of one of the lesions on the ear was excised for histological examination and we examined several sections of lesions excised in the past by d . 
the stretched and atrophic epidermis covering the lesion is between the central mass of keratin and the covering epithelium are seen on the left . irregular and well - differentiated masses of epithelium and cell nests . 
the recently developed methods of detection and determination of polycyclic hydrocarbons in micro - gram quantities have provided a more sensitive way of analysis than has yet been employed ( cooper , 1951 , 1953 ; wedgwood and cooper , 1951 , 1953 )  . in view of the suggested connection between carcinoma of the lung and smoking ( menary , 1932 ; schrek , baker , ballard and dolgoff , 1950 ; mirs and porter , 1950 ; dor and hill , 1950 ; rigden and kirschoff , 1952 ; sadowsky , gilliam and cornfield , 1953 ; wynder , graham and croninger , 1953 ) and the estabhshed presence of carcinogenic hydrocarbons in combustion products , it was decided to use these methods to examine cigarette smoke . 
on the other hand several investigators have devised apparatus to collect smoke produced in conditions resembling closely those of normal smoking ( bradford , harlan and hanmer , 1936 ; wenusch and sch6ller , 1938 ; w , ynder , graham and croninger , 1953 )  . the apparatus we have employed . 
the tap c with a glass rod sealed on to it was turned on for the period necessary to simulate a " draw " made by a snioker by means of a cam attached to the periphery of a 3 inch pulley wheel , d . the glass rod was held in the groove of the puuey by means of a stretched rubber this wheel was driven by a gear train from a smar electric motor , the speed band . of which was variable by adjustment of a " variac " auto - transformer connected to the altemating current mains . other cams could be attached to the wheel and thus the length and frequency of the " draws " were adjusted . in the u - tubes suitable solvents were placed so that the smoke was made to bubble tbrough them and the condensable material separated from the gas . a packing of 10 cof pyrex glass wool in the second u - tube served to remove the both u - tubes could be surrounded by beakers last traces of condensable matter . full of solid carbon dioxide to assist in the condensation , but it was found that strong cooling was not always necessary to effect complete removal of the disperse phase . 
each , the average time of draw is about 2 seconds and the average time of smoking such a cigarette to a short butt ( about 1 - 5 clong ) is 12 minutes . these conditions are attained by oiir apparatus when adjusted to give a 2 second draw every 45 seconds with a negative pressure of 25 cwater . 
air. smoke obtained in this way is an aerosol with a viscous fiquid as the disperse phase and a gas , consisting of a mixture of unburnt air , carbon dioxide , carbon monoxide , water vapour and traces of other gases as the dispersing medium . the average amount of condensable material from 100 c ' igarettes weighs about 4 g . we have concemed ourselves only with the condensable disperse phase whicli in bulk , after the evaporation of the solvent ( acetone , chloroform or cyclobexane ) used to trap it , is a dark brown viscous fluid . preparation of apparatus and materials . the solvents ( cyclohexane , acetone , benzene and chloroform of reaaeint grade ) were distilled in an all - glass apparatus , rejecting the first and last tenths . this product was then distilled through a dufton column and corected over a range of - 9jo c . 
the residues were not fluorescent in ultra - violet hgbt . all the glass apparatus employed was cleaned bv immersing overnight in chromic - sulphuric acid mixture , washing and drying . 
500 ultra - violet spectrophotometer . graphy the eluates were examined at a few definite wavelengths only and hydrocarbons were identified by specific absorption peaks and the order in which they appeared in the eluates . 
the solvent was then removed by distillation , the residue boiled twice with cyclohexane ( 5 to 10 ml . ) and the solutions decanted off after cooling . the - united cyclohexane solutions were then shaken with three quantities of 2nsulphuric acid and then with three quantities of 2nsodium hydroxide . solution thus freed from basic and acidic substances was reduced to about 5 ml . 
the eluates from the colunm were examined by the method of wedgwood and cooper ( 1953 ) namely by searching for absorption peaks at wavelengths known to be specific for the various polycyclic hydrocarbons . 
usuahy the initial chromatography only revealed the hydrocarbon peakb as inflexions againbt considerable background absorption and the combined filtrates suspected of containing the compounds had to be passed through fresh columns in order to reveal their presence more satisfactorily . anthracene was recognised by its peak at 376 ma . 
and it was always followed closely by the appearance of a peak at 355 m / % , reveahng pyrene . the compounds were determined bv making the cyclohexane solutions up to known volumes and then measuring the height of the peaks by the base line technique . for this purpose two convenient points on either side of a given peak calibration of the peak heights above the base were used to construct a base line . line was effected by using standard curves of the authentic hydrocarbons prepared from solutions of known strength . the background absorption interfered considerably in these experiments and various attempts were made to reduce its effect . 
when hydrolysis was effected on the cyclohexane extract of the whole smoke , the background absorption was even greate ' r than before , indicating that some hydrolysis products of the smoke constituents were entering the hydrocarbon sections of the column . the presence of pyrene and anthracene indicated in these experients was confirmed by their chromatographic behaviour relative to added methyl ethers . relatively few compounds , absorbing hght in the region studied , are associated with the polycychc hydrocarbons on aluniina columns . 
the peaks used for recognition of the various compounds are labened with their wavelengths . it will be noted that a sequence of peaks of added calibration materials interspersed with the recognisable peaks of substances originary present , gives an infalfble method of identification and determination . 
the results of a number of analyses utilising in all 250 cigarettes of one popular brand showed quantities of anthracene and pyrene equal to 10 - 2 and 9 - 0 itg . 
and to paraffms which he states are present in t e original tobacco and are vaporised unchanged during smoking . acetylene has been determined in tobacco smoke by weighing it as cuprous acetyhde ( fishel and haskins , 1949 )  . its pyrplysis may give a ready explanation 300 b . 
was attained at the hottest part of the periphery . more satisfactory measurements were made by using a calibrated coppernichrome thermocouple which was threaded through the cigarette from one side to the other . after sealing the holes by means of small pieces of paper , the cigarette was smoked in the normal way and the temperature recorded from time to time . 
the temperature rose rapidly when the burning end advanced towards the couple and , when the glowing material was in contact with the couple , a temperature of about 650 c . 
each time air it thus appears that the highest temperatures during was being drawn in . suction are confined to the surface and that the main body of the hot end is always at a temperature between 650 and 700 c . similar temperatures were recorded when a cigarette with a thermocouple inserted was smoked in the mouth . these temperatures are sufficiently high for pyrolysis of simple compounds to polycyclic hydrocarbons . 
the temperatures recorded by us agree remarkably closely with those obtained independently by wynder , graham and croninger ( 1953 )  . our investigations upon the composition of tobacco smoke are being continued . the authors wish to acknowledge the helpful and kindly criticism given by professor sir e . 
legon. from the department of geography , the london school of economics and political science . received for publication april 20 , 1951 . in considering the heavy mortalities from gastric cancer that exist in the counties of northern and western wales ( stocks , 1936 ) , the writer wondered whether mortality was uniform over the area , or whether there were marked local variations within it . 
the low - mortality rural districts of eastern montgomery and of radnor ( 70 deaths ) are well drained , in contrast to the rural districts to the west , which lie on an ill - drained plateau with extensive tracts of bog . 
the impermeability of the rocks of anglesey ( 203 deaths ) , and the extreme flatness of much of the island , make for peat accumulation , despite the fact that rainfall is light compared with that of the highland parts of north wales . case involving the pennant grit . it may be stated , therefore , that the rural districts of low mortality are all on the sheltered east and south of the welsh massif and are well drained . 
legon huntingdon , ely , peterborough and the holland division of lincoln ( stocks , 1947 ) , since these four counties alone in england have proportionally large areas of peaty fen soils . apart from the typically high organic content of their soils , north wales and the fens have little in common . 
as further information ( marston , 1951 ) is made available , it may prove worth while to consider whether a carcinogenic substance exists in food - plants grown on these soils . the possibility of an association between soil organic matter and cancer mortality was expressed verbally to the author by g . 
orr. from the department of pathology , univer8ity of birmingham . received for publication may 7 , 1953 . when a carcinogenic agent is applied to the skin of a mouse epithelial hyperplasia follows in the course of a week or two . if the applications are then stopped , the epidermis reverts to a histologically normal appearance , but if hyperplasia is again induced by the co - carcinogen croton oil , tumours of the skin will in due course be elicited . it is clear that the apparently normal skin has been altered in some way by quite a short treatment with carcinogen ( a single application of a powerful carcinogen will suffice ) , so that further growth - stimulation by an agent not itself carcinogenic completes the process of tumour induction . attention was first drawn to this phenomenon by berenblum ( 1941 )  . 
mottram ( 1944 ) showed that a single application of 3 : 4 - benzpyrene would suffice to produce tumours if followed by a course of croton oil paintings . in a full analysis of the factors involved , berenblum and shubik ( 1947 ) adopted the terminology of friedewald and rous ( 1944 ) , the effect of the preliminary carcinogen being described as the " initiating " factor , and that of the croton oil ( co - carcinogen ) as the " promoting " factor . both these factors were conceived as causing changes in some of the epithelial cells themselves . there is evidence , however , that the effects of carcinogens are not attributable wholly to the direct changes brought about in the epidermis . various authors have drawn attention to the progressive changes that occur in the dermis during experimental carcinogenesis of the skwhen the technique of grafting different components of the skin became available ( billingham and medawar , 1951 ) , an attempt to evaluate the relative importance of epidermal and dermal changes in the evolution of cancer was undertaken by billingham , orr and woodhouse ( 1951 )  . they transferred the fully treated epidermis alone to an untreated site prepared on the opposite side of the mouse 's thorax . in these conditions this epithelium no longer gave rise to tumours , though the reciprocal transfer of untreated epidermis to a bed cut in the carcinogen - treated site gave a yield of tumours approximately equal to what might have been expected had no operative treatment been undertaken . in view of these findings there appears to be another possible explanation of the phenomenon of co - carcinogenesis , namely , that after short treatment with a carcinogen the dermis is sufficiently altered to permit tumour induction when further epidermal hyperplasia is evoked by croton oil . 
the cutting of these grafts was greatly facilitated by the hyperthe rectangular slices plasia of the epidermis due to the carcinogen treatment . were floated raw side down on 0 5 per cent commercial trypsin solution and incubated at 38c . 
i. - experiment j : each horizontal strip represents the survival of a mouse after the grafting operation ( life line )  . oblique shading represents a papilloma , solid black shading a malignant tumour . 
an oblique line cutting the life line indicates operative removal of a tumour . the rate of appearance of tumours on the original methylcholanthrene - treated area after removal of thin thiersch grafts is indicated by the shading in the life line . 
orr treated site after removal of thin thiersch grafts in 16 out of 37 animals . 10 cases these tumours were removed to prolong the life of the animal . in 7 of these further tumours occurred , but 3 of them later regressed . the time range from operation to the appearance of a first persisting tumour was from 0 to 420 days with a mean time of 48 27 - 6 days . 
the survival of these mice from first croton oil painting ranged from 173 to 330 days ; some are still alive and under observation . these times are not , of course , comparable with those for the experimental animals , where the datum line was placed at the time of grafting . the total incidence of tumours on the grafted site is thus not very different from that induced by croton oil alone . there is a qualitative difference in that one of the tumours on the grafted site became conventionally malignant . 
by comparison , the original treated site yielded tumours in 16 of the same animals , 8 of them clinically and histologically malignant , without co - carcinogenic treatment on that site . the results in general of this experiment do not support the view that the transplanted epidermis contained latent tumour cells . 
the result is not , of course , unequivocal , but the occurrence of a single carcinoma does not suggest that many irreversibly altered cells can have been present . an attempt is in progress to find the effect of preliminary grafting of normal skin , followed by treatment with croton oil , in order to see whether the operative treatment of the dermis modifies the effect of croton oil . it is technically difficult to prepare pure epidermal grafts of normal thoracic epidermis , but experiments are in progress with re - implanted thiersch grafts . experiment k : reimplantation of a pinch graft cut from carcinogen - treated skin . billingham , orr and woodhouse ( 1951 ) made a few observations on the effects of cutting pinch grafts from the carcinogen - treated area , and reimplanting them at the same site . these experiments were undertaken to control the results of transplantation and in particular to make certain that the detachment of the superficial skin was not in itself the reason for the paucity of tumours when it was transplanted . 
the grafts were then replaced and the surrounding gap , caused by retraction of the skin around the wound , was dusted with sterile animal charcoal to mark the graft position . 
2. of 17 grafted mice surviving beyond the time of first tumour appearance , 11 produced tumours , but these regressed on 5 animals so that only 6 mice had tumours present at death . 
the tumours appeared after 8 to 200 days ( mean 42 17 - 3 days ) from the time of grafting the experimental group of animals . eight tumours became malignant after 20 to 100 days . 
the mice survived after grafting for 38 to 230 days ( mean 98 11 days )  . it will be seen that 70 days after the reimplantation more tumours were present on experimental animals that controls , and it seemed that the tumour production was indeed being augmented by the grafting operation as in the previously reported experiment . however , after about 100 days several tumours on the experimental animals regressed - a much less frequent occurrence in control animals . the outcome was that the proportion of animals bearing tumours at their deaths was no greater in the grafted animals than in the controls . 
2. - experiment k : the rate of appearance of tumours in the bridge epithelium over the scar around the reimplanted pinch graft of carcinogen - treated skin is indicated by shading in the life line and persistent tumours by the letter s . 
tumours arising on grafts themselves are shown by shaded blocks above the life line and persistent ones by the letter g . tumours arising outside the grafts are shown by shaded blocks below the life line and persistent ones by the letter o . 
1. experiment l : reimplantation of thin thiersch grafts cut from carcinogen - treated skin . in this experiment thin thiersch grafts were removed from the carcinogentreated area as described in experiment j . 
as great an area of the carcinogentreated skin as possible was taken off and the pieces were then planted back again , in some cases after smearing the graft beds with animal charcoal to mark them . the results are shown in fig . 
the mice survived from 69 to 230 days ( mean 124 + 12 - 5 days )  . tumours appeared on 5 out of 18 control ungrafted mice after 16 , 20 , 23 , 24 all became malignant . 
the mice survived from 38 to 230 days and 200 days . ( mean 99 14 - 2 days )  . experimental animals control animals 4222 4223 4225 4246 4247 4248 4249 i 4250 4488 z 4493 4494 4498 4499 4500 4502 4206 4207 4208 4209 4210 4227 = r_ 4229 4230 4474 4475 4476 4477 4478 4479 4480 4481 4483 4495 z 77 days after thiersch graft operation fig . 
3. - experiment l : the rate of appearance on reimplanted thiersch grafts of carcinogenblocks of shading below the line indicate treated skin is indicated by shading in the lifeline . tumours outside the grafted area . 
some of them , such as age , sex and diet are fairly easy to control . others , such as range of temperature and amount of carcinogen administered , may be less easily controlled . 
orr of animals such as were used in the present experiments . may strike at any time . parasites and infections experiment m : the insertion of cotton threads into the dermis of carcinogen - treated skin and their removal after 2 weeks . orr ( 1934 and 1935 ) showed that preliminary alteratioii of the dermis , by the introduction of linen threads and their subsequent removal before treatment with a carcinogen led to a significantly earlier appearance of tumours . 
we thought it of interest to see what happened if the threads were introduced after the standard preliminary carcinogenic treatment of the present experiments . fine white cotton button - thread was threaded into the finest ordinary sewing needle which would take it and then sterilised . the needle was inserted into the skin of the anaesthetised mouse just outside the carcinogen - treated site . was passed through the dermis of the treated area as near to the surface as possible , endeavouring not to injure the treated epidermis itself , and out through the cotton was carefully pulled through and the surface beyond the treated area . the procedure repeated again under another part of the treated area . the thread was passed under the treated skin in both directions about half a dozen times in all and left in situ for 2 weeks . at the end of this time the projecting loops of cotton were cut and the threads carefully pulled out of the skin under anaesthesia . unfortunately the threaded area had not been covered over and many of the mice had scratched and tugged at the loops of thread , resulting in some damage to the carcinogen - treated epidermis itself '  . the results are shown in fig . 
5. persistent tumours appeared on 10 of 19 experimental animals in 9 to 92 days ( mean 35 7 * 9 days )  . eight tumours later showed malignant changes . 
orr croton oil , the tumour yield is only very slightly greater than that obtained by croton oil on otherwise untreated skin . of the few tumours so produced , all except one were papillomata , which appeared somewhat earlier than might have been expected with croton oil on previously untreated intact skthe single exceptional tumour was a carcinoma , an unusual occurrence with simple croton oil treatment , but it has to be remembered that grafting was delayed up to the time that tumours were already beginning to appear on the original methylcholanthreneexperimental animals control animals 77 , , / , , , , z , , 4358 4361 4369 4370 4372 4373 4374 ' .. 
the results of berenblum and shubik ( 1947 ) were explained on this basis by postulating a two - stage process , the first stage being the alteration by a sudden and irreversible process of a few normal epithelial cells into latent tumour cells , the second the conversion of these latent cells into visible tumours . the first stage ( " initiating process " ) requires a carcinogen ; the second ( " promoting process " ) can be brought about by various non - carcinogenic hyperplasia - inducing factors , of which croton oil is the most fully analysed . it appears to us that the facts elicited by berenblum and shubik are explicable without reference to the conception of latent tumour cells , and that an irreversible change inflicted on the carcinogen - treated site as a whole , and perhaps particularly on the supporting tissues , would explain the olbserved phenomena of co - carcinogenesis . in one respect the view we put forward offers a more satisfying basis for one of the berenblum and shubik findings . 
they showed that even after as long an interval as 20 weeks between the initial painting with carcinogen and the subsequent croton oil treatment , the total tumour incidence remained undiminished as compared with shorter intervals . if , therefore , latent tumour cells were there all the time , it would be necessary to assume that they did not participate in the normal processes of desquamation . it is also relevant to point out that blum ( 1944 ) has analysed the results of carcinogenesis by mathematical methods , and reaches the conclusion that the available data are not consistent with the hypothesis of a discontinuous two - stage process . if the dermal and subcuticular changes are of importance to carcinogenesis , the most obvious way in which they could act would be by interference with the nutrition and metabolism of the overlying epitheliuthe latter does not carry blood vessels , and is dependent on the functional effectiveness of the stromal tissues . it has previously been pointed out that the rate of evolution of the dermal changes ( in simple treatment with a carcinogen ) is of the same order as the potency of the carcinogen in terms of time taken to produce tumours . it may therefore be asked why a single application of a carcinogen followed by a co - carcinogen is effective at all in the induction of tumours . 
a possible explanation is that the hyperplastic epithelium following the co - carcinogen is susceptible to degrees of dermal change and vascular impairment which would not effect normal epithelium or epithelium in a less vigorous state of multiplication . 
orr the reimplantation studies of experiments k and l necessitate some modification of the conclusions drawn from the previous reimplantation of billingham , orr and woodhouse ( 1951 )  . it is now found that this treatment does not appreciably alter the ultimate tumour yield , because the apparent increase in the period immediately following the operation is offset by the regression of several of it was suggested that the previous result might be these " explosive " tumours . accounted for if the operative treatment accelerated the establishment of optimal it remains not impossible that this conditions for the evocation of tumours is so , and that subsequent regression of some of these tumours was dependent on the ischa3mic conditions in the dermis and subcutis developing further to a point at which the vascular conditions were inadequate to maintain the tumour . in experiments of the type recorded here , it is necessary to consider the effects experiments such as those of of mechanical trauma on the yield of tumours . lipschuiitz ( 1924 ) , mandl and stohr ( 1924 ) , doderlein ( 1926 ) and deelman ( 1927 ) indicated that trauma of a previously tarred area tended to provoke tumours closely related to the sites of the healed wounds . 
more recently pullinger ( 1943 ) obtained results similar to those of deelman using a pure chemical carcinogen . mackenzie and rous ( 1941 ) and friedewald and rous ( 1944 ) obtained a high incidence of tumours around healing wounds produced by punching holes in the it was on the basis of this work that ears of rabbits after carcinogen treatment . the conception of initiating and promoting factors , later adopted by berenblum and shubik ( 1947 ) was based . in almost all such work , attention has been almost exclusively directed to the epithelial cells , but linell ( 1947 ) showed that there was a difference between superficial and deep trauma . 
numerous tumours , including carcinomata , appeared on the original treated donor site . re - implantation of pinch grafts and thin thiersch grafts into the site from which they were cut in the carcinogen - treated skin did not result in an increased incidence of persistent tumours . there was an increase in regressing tumours . trauma to the treated dermis ( by temporary insertion of threads or by electrically heated wires ) did not increase the incidence of tumours . the present results , like those previously reported , suggest strongly that both the epidermis and dermis ( and possibly deeper structures ) are involved in the carcinogenic action of methylcholanthrene . in particular , they are difficult to reconcile with the two - stage epithelial hypothesis of berenblum and shubik ( 1947 )  . we are indebted to r . 
the column was developed with several litres of light petroleum , followed by mixtures of this with progressively increasing amounts of benzene , and the eluates were collected in separate lots of about 2 l each . these were examined spectrographically , and those possessing similar fluorescent spectra were combined , evaporated to a small bulk , and dissolved in small amounts of benzene , providing the following fractions for testing : pes - a : fractions before the appearance of anthracene bands ; pes - b : those with anthracene bands predominating ; pes - c : those after anthracene , but before benzypyrene ; pes - d : all fractions containing benzypyrene bands ; pes - e : subsequent fractions , with recognizable fluorescence bands at 412 and 430m , u . ; pes - f : later fractions with main fluorescence band at 391 m ,  . ; pes - g : still later fractions with fluorescence band at 385 m ,  . in rabbits , skin tumours were obtained with fractions pes - c , pes - d , pes - e , pes - f , and somewhat more slowly with pes - g , while in mice skin tumours appeared only with fractions pes - d and pes - e . two interesting points arise from these results : the first is the confirmation of the previous experiments , mentioned above , that fractions appearing before and after the benzpyrene - containing fraction , are highly carcinogenic . 
the second is that the one appearing before benzpyrene , is far more potent for the rabbit 's than for the mouse 's skin . in a second fractionation , starting with larger quantities of tar ( 4 l ) , the same procedure was adopted , except that the original petroleum extract was shaken 160 i . 
was somewhat more active , while the highest activity was obtained with the next fraction , coming over at 170 - 180 c . the combined fraction of 160 - 180 c . 
was then chromatographed from light petroleum , and collected into four fractions : ( a ) a very early fraction , containing naphthalene and most of the anthracene . ( b ) an intermediate fraction , containing in addition to the remainder of the anthracene , fluoranthene , a large amount of chrysene , and probably chrysene homologues ( see below )  . ( c ) a large third fraction , containing all the benzyprene , also some chrysene , ( d ) a late fraction , in which 1 : 2 - benzcarbazole was recognized . these four fractions were tested on rabbit skin ; fractions ( b ) and ( c ) ( referred to as pf and ph in table i ) was found to be highly carcinogenic , while ( d ) ( fraction pi ) was less so . these fractions , on concentration , deposited crystalline material , which was collected and recrystallized several times from different solvents . one of the components proved to be chrysene , crystallizing in the first crop . 
they are neither acids nor bases , since preliminary treatment with alkali and acid , respectively , does not remove theby fractional distillation , these carcinogens appear slightly earlier than 3 : 4 - benzpyrene . 
one of these , which appeared just before benzpyrene on the chromatography column , possessed the unusual property of high carcinogenic potency for rabbit skin and none for mouse skin . the final active material obtained by the above methods of fractionation , represented more than a two hundred - fold concentration of the original tar . still consisted , however , of a mixture of substances , and the active constituent has not yet been identified . we are indebted to the city of leeds gas department for a generous supply of horizontal retort tar . 
almost all cases of leukaemia in 2955 days . the aka line are lymphogenous or of a very ' immature type - the so - called stem - cell leukaemia . the low leukaemia line which we have used for the cross - breediilg is called b . this line is also inbred . 
among hundreds of b mice we have previously observed , only one case of lymphogenous leukaemia was found . in the fl there was found 43 per cent of deaths due to leukaemia ; the age distribution of these mice dying from leukaemia is shown in fig . 
the age distribution is somewhat wider than in aka mice and the peak is displaced about 300 days later , so that in th ' is case the average lifetime for mice dying from leukaemia is 59119 - 6 days . in the f2 we encountered 37 per cent cases of leukaemia , and the distribution of age appears to be very wide , as shown by fig . 
the curve may be regarded as a combination of two distributions , one corresponding to the aka mice and one corresponding to the fl . the backcross derived from the fl crossed with aka shows a rise in the frequency of leukaemia to 49 per cent , and the curve concerning the distribut ' ion of age shows , as is seen from fig . 
l. - the age distribution of mice dying from leukaemia within the aka line - - - - - and the a ' ckli ii i t 60days ] days age lunit = 60daysj fig . 
the curve for the f2 shows a tendency to a bimodal form . you will see that , in spite of the genotypical uniformity of the mice of the aka line and the fl , leukaemia only develops.in respectively 63 per cent and 43 per cent of the animals . 
the percentages mentioned as is the case with the figures from previous experiments - calculated by comparing the number of mice dying from leukaemia with the total number of day , ; fig . 
4. - age distribution of leukaemia in backeross of fl mice to b line . dead mice , the animals dying before the first case of leukaemia occurs not being included . it is quite evident that this mode of calculation is entirely inadequate , because deaths caused by other reasons than leukaemia ( such as intercurrent diseases ) , will influence the result to a very high degree . 
the frequency in the fl is not so great as in the aka line , but this can be in part due to the fact that the cases appear at an average age of 300 days later , so that the deaths from other causes will decrease the frequency of leukaemia . the figures found in ' this material , as seen from the following considerations , support the view that the inheritance may depend on one single dominant gene . if we suppose that this is the case , we can calculate the expected numbers of leukaemic animals in the f2 and backcrosses to the parent lines on the basis of the observations in the aka and the fl mice . these results are calculated in table 1 , taking into account the deaths from other causes . these figures agree very well with the observations , but in the calculated mortality curves the agreement was not so evident . 
the difference is not significant . if we assume the presence of two genes the calculated number will only be 8 - 2 '  . these data thus support the hypothesis of a single domi ' nant gene . the variation in the average lifetime of the leukaemic animals has previously been described by macdowell , potter and tayl ' or ( 1945 ) , macdowell and richter ( 1935 ) , furth and barnes ( 1941 ) , kirschbaum ( 1944 ) , and cole and furth ( 1941 ) in similar crossbreeding experiments . 
the phenomenon seems therefore to be independent of the character of the lines used , which fact seems to refute the previous supposition that casual changes in the average lifetime of all the mice may influence the result . i would sooner say that the discovery confirms the supposition that the immature leukaemic cells arise by a somatic mutation in cells which possess a labile 112 g . 
where and how this mutation arises could be a if the animals are homozygous with regard to the labile gene , matter of chance . the mutation will naturally arise earlierand more frequently than ' is the case if the animals are only heterozygous . 
we spent more than eight months working on one such incidental problem , which had to be solved before we could proceed with the main work . this effort was well rewarded , however . 
we not only improved our experimental approach , but also acquired information that will further our understanding of the intricate role played by biological factors in regulating the influence of external agencies on living cells . in view of all this , i should like to point out that our investigations of the genetic potencies of carcinogens are still in an early stage . 
koller these few instances are selected from many clinical and pathological findings to illustrate that the environment of tumours is of great importance , and must be taken into consideration when devising improvements in radiotherapeutic procedures . it may prove to be important not only in surgical and radiotherapeutic practice , but also as regards theoretical problems of oncology . 
the classical study of willis ( 1948 ) emphasizes the fact that potentially neoplastic tissue zone is much greater than the size of the initially appearing tumour would indicate , and brings forward evidence which lends support to the field - theory of the origin of cancer . 
thus the possibility that the " recurrence " of a tumour after surgical treatment may represent an entirely new cancerous change in a predisposed tissue , and is not due to the incomplete removal of the malignant growth , has to be considered . the aim of post - operative x - ray therapy is not only to destroy viable tumour cells which might have been left in situ , but also to reduce the chance of the occurrence of an entirely new malignant growth by producing gross alterations in the histological architecture of particular regions , showing a proneness to neoplasia , and to affect the substrate in which any malignant cell left in situ might progress and defeat the treatment . 
the importance of connective tissue and tumour - environment has also been stressed by vernoni ( 1948 ) , whose views on the connective - tissue role in carcinogenesis find support in recent research work . a better understanding of the behaviour of the connective tissue of the tumour - bed and stroma during and after radiation treatment leads to the revision of fundamental principles on which the rationale of treatment is based . 
the practical application of the latter in the radiotherapy of accessible tumours finds expression in a new technique , described as the " sieve or alternating chess - board method " ( jolles , 1949a )  . for the past two years this method has been tested on a small number of patients , yet the radiation response in these cases has shown characteristics which hitherto have not been seen in tumours irradiated by the usual conventional methods . 
the purpose of the present paper is to describe these phenomena , and to discuss their bearings on the destruction of malignant growth . the biological basis of radiation effects in tumours . the radiation - induced changes in the cellular and stromal part of the tumour may be summarized briefly as follows : the cellular injuries consist of diverse nuclear or chromosome injuries , as well as disturbances in cytoplasmic enzyme activity . 
the chromosome injuries manifest themselves during mitosis which follows treatment , and lead to cell death by the breakdown of the mitotic mechanisthe amount of chromosome injury and the number of injured radiation - induced enzyme disturcells show a direct dependence on the dose . bances , the extent of which also depends on the dose , either lead to reversible injuries , e.g. 
the recent investigations by bloom and jacobson ( 1948 ) , anderson ( 1949 ) , and warren and dixon ( 1949 ) , brought forward further evidence to show that radiation does not produce cell differentiation . in the stroma and tumour - bed two chief events can be distinguished : hypertrophy of the connective - tissue elements , and a reaction closely resembling inflammation . 
the first phenomenon mainly consists in an excessive deposition of intercellular collagen fibres ( fibrosis ) ; the second is represented by an invasion of lymphocytes , polymorphonuclear leucocytes and macrophages into the fine these changes network of connective tissue forming the stroma and tumour - bed . in the normal tissues naturally react on tumour cells and tissue ; the effects in the latter thus produced can be referred to , therefore , as " indirect radiation effects . " they must , however , be clearly distinguished from those " indirect " effects which are assumed to be brought about by injury to the blood supply ( desjardins , 1932 ; harvey , 1942 )  . failla , 1940 ; ellis , 1942 ; windeyer , 1942 ; the importance of the tumour - bed during treatment has been recognized by a great number of investigators ( russ , chambers and scott , 1921 ; kok and vorlaender , 1922 ; caspari , 1922 ; murphy , maisin and sturm , 1923 ; czepa , 1924 ; roussy , 1926 ; ewing , 1926 ; souttar , 1929 ; sugiura and cohen , 1939 ; jolles , 1946 , friedman , 1939 ; evidence was also obtained in various 1948 ; spear , 1946 ; windholz , 1947 )  . experiments which shows the influence of the tumour bed in the radiation response of tumours ( lasnitzki , 1947 ; elson and lamerton , 1949 ) , and it was found that by taking into consideration the " stroma - reaction " , treatment methods could be devised for individual tumours in which the total dose given did not exceed 2700 r ( koller and smithers , 1946 )  . 
in the interdependent tissue structures of tumour and tumour - bed , the different reactions ( cellular and intercellular , direct and indirect ) are knitted together and closely integrated . it was observed that the onset , degree and rate of these reactions vary greatly in different tumours . because the outcome of treatment depends on them , the total dose necessary for the destruction of tumours also varies within very wide limits ( 2700 r and this observation leads to the conclusion that on biological grounds the 9000 r )  . concept of a " standard tumour - lethal dose " is untenable . it is unnecessary , however , to stress that a minimal effective dose which is indispensable for the destruction of a tumour remains of paramount importance , and the enhancement of the effect of this minimal dose , by utilization of the indirect radiation effects , is here discussed . the various reactions induced by the radiation all contribute to the resolution and eventual disappearance of the tumour , therefore the reaction - system must be it is only by the analysis of all these factors and the study considered as a whole . of the behaviour of all tissue structures , that an explanation may be found either for the inadequate response , or for the recurrence of a particular tumour . 
the technique has been fully described in a previous communication ( jolles , 1949b )  . apart from the direct radiation transmitted through the " opaque " lead squares ( and which does not exceed 3 per cent with the thickness of lead used ) , the protected areas also receive a very small amount of scatter irradiation . latter is irregularly distributed , tailing off towards the centre of the protected this contingency must be taken into consideration when studying and areas . conmparing the radiation effects of the directly exposed and protected parts . biopsy specimens are taken simultaneously from the exposed and protected areas , preferably fronm the centre of these regions , and occasionally a third biopsy specimen is obtained by cutting across both the exposed and protected areas in order to follow the transitional stages of the histological changes due to radiation . the great variability of tumours in respect of the histological organization including the structure of stronia and tumour - bed , has to be kept in mind , and our observations were based only on biopsy material showing similarity in these respects . eighteen patients have been treated with the sieve technique , and the radiation reactions in the exposed and protected areas have been compared by the analysis of biopsy specimens . 
two sets of experiments were devised ; in the first instance only one sieve was used throughout the treatment . in the second set , two sieves a and b , were employed ; chess - board b differs from chess - board a in that the order of transparent and opaque squares is reversed . 
xi.48 : 4 days after start of treatment 28.xi.48 : 13 days after start of treatment 8.xii.48 : 23 days after start of treatment 16.xii.48 : 31 days after start of treatment 15 . 
the gland increased in size very slightly during treatment to the lesion on the temple . one month later the gland had grown to the size of a hazel - nut . 
lederman , the behaviour of over 450 accessible tumours has been analysed during and after treatment , and it was established that besides the injuries in dividing cells and the great increase in cytoplasmic volume of differentiating or maturing tumour cells , the most striking change occurs in the connective tissue of the stroma and tumour - bed , which may be described as tissue " fibrosis . " the connective tissue becomes more rigid in architecture , less flexible and adaptable for the development of the inflammatory reaction ( " coarse " tissue fibrosis )  . 
koller to state what is the primary cause of tissue changes in the protected areas ; i may be that the small amount of scatter radiation received in ten fractions o for that reasoi 40 r daily can produce such alterations in the connective tissue . further experiments are in progress to study the stromal effect of such a smal the experimental evidence so far available indicates that tissue reactioi dose . after a dose of this order is negligible ; if there is any , e.g. 
we are of the opinioi that the tissue changes in the protected regions are brought about by a complet mechanism in which , among others , a diffusible substance produced in the directli irradiated tissues might play a role . 
the absence of chromosome fragmentation is another proof that the alteration seen in the protected areas , such as the increase in the size of tumour cells , and the slight degree of fibrosis and inflammatory reaction , must be considered as an indirect reaction to radiation . our investigation has shown that the rate and degree of the indirect radiation reaction depends on the histological organization of stroma , and on the dose . on account of the differences in the histological architecture of tumours , the minimum effective dose varies from tumour to tumour . 
we found that in tumours in which the connective tissue is abundant the minimum effective dose is smaller than in tumours with scanty stroma . this is illustrated in case ii , in which the dosage used ( 2700 r actual dose in 35 days ) did not reach the critical minimum level necessary to produce a reaction of sufficient strength and efficiency in the scanty connective tissue of the " protected " tumour areas . 
on the other hand , the use of the sieve - technique has permitted the subsequent treatment of the residual tumour with a relatively high but adequate dose ( 4500 r actual dose in 18 days ) , because the histological texture of the skin , owing to the small size of the areas directly exposed to irradiation during the initial treatment has been left unimpaired , and could safely be subjected to such a high dose in the second treatment . the complexity of the various cellular and tissue interactions increases when further radiation is carried out through another " chess - board or sieve " in which the position of transparent and opaque areas is reversed . 
we found that while the connective tissue needs protection from excessive radiation , it requires a certain quantity of radiation and a specific distribution in time and space of this quantity in order to elaborate the radiationinduced reaction . 
the stroma and tumour - bed reactions , however , being too obvious to disregard completely , are referred to , but only as concomitant , " independent " events , which have no , or too little , bearing on the radiation response of the malignant growth itself . 
the most cursory histological study of tumours under radiation reveals the fact that the destruction of all malignant cells of the tumour parenchyma rarely , if ever , can it seems , therefore , be achieved by the " direct " action of a therapeutic dose . necessary and logical not only to admit the existence of an " indirect " effect , but to accept all the implications of the generic statement that the purpose of radiation is to kill a variable but significant proportion of active tumour cells , and at the same time to assist the local body defence system by inducing the various responses in the tumour - bed and stroma . it also follows that any view based on changes induced by radiation in some and not all tumour - tissue components is not comprehensive , and that the conclusions drawn from the study of cell behaviour alone are doubtful . 
on " cell count method " of glucksmann and co - workers that account the ( gluicksmann , 1941 , 1948 ; glucksmann and spear , 1945 ; gkicksmann and way , 1948 ) , which per se represents a contribution to radiobiological knowledge , is liable to be misleading when claims are being attached to it , such as that it provides an explanation for the variation of radiation response of tumours , predicts the outcome of treatment , and indicates the most appropriate treatment method , whether surgical or radiological , for a given case . the cell count method is biased by subjective selection and interpretation ; it disregards the great regional heterogeneity shown by tumours both in growth rate and histological organization , and the fact that a malignant growth is composed of tumour and stroma , which form one closely integrated unit . it is more than obvious that for that reason the application of the cell - count method to biopsy specimens taken after a " test dose " of radiation in order to decide whether a tumour be submitted for surgery or radiotherapy is of doubtful value ( glucksmann and way , 1948 )  . it has been stated by spear ( 1946 ) that " radiation affects any given cell of a complex tissue in at least two ways , first by direct action on the cell , and secondly by injuring neighbouring tissues , upon the healthy functioning of which the cell depends . " our investigation leads us to a similar conclusion that the direct cellular effects , e.g. 
fragmentation of chromosomes , represent only the initial phase , and that the most important factors responsible for the disorganization , regression and re - absorption of tumours during and after irradiation are the various , closely integrated and related reactions which are produced by the radiation in the connective tissue of tumour - bed and tumour stroma . the present investigation , in which the alternating chess - board method was employed , has shown the role of the connective - tissue reaction in the destruction of tumours and the way by which this reaction can be favourably influenced and modified . 
we have found that the fractionation of dose in time as well as in space , as carried out by the sieve , induces specific changes in cells and tissues . the intensity and rate of these changes affects the whole architecture of stroma and tumour - bed as well as that of the tumour . our investigation has also shown that when the sieve is used sufficient response can be produced in the local defence system of connective tissue , and can be maintained by a much smaller dose than usually employed . 
roller us in cases where the tumour is radio - resistant , either by its nature or by its environment , to deliver a higher total tumour - dose without the danger of destroyit is our intention to investigate the ing vital structures of the tumour - bed . potentialities of this new procedure by applying the sieve method for the treatment of radio - resistant and deep - seated tumours , especially metastatic deposits in lymph nodes . 
the tumour is divided into a number of " exposed " and " protected " areas , the former receiving direct , the latter receiving only a small amount of scatter and transmitted radiation . 
two chess - boards or sieves , which differ in the order of the transparent ( exposed ) and opaque ( protected ) squares , can be used during treatment . irradiation induces injuries in the tumour cells and alterations in the connective tissue of the tumour - bed and stroma . 
the radiation reaction in the connective tissue plays a very important role in the destruction and reabsorption of tumours . in tumours irradiated through the sieve it was observed that tissue - reaction is reduced in the directly " exposed " regions , but at the same time , fibrosis and inflammatory reactions are induced in the " protected " regions . 
no cellular injuries , due to direct radiation such as chromosome fragmentation , were seen in cells of the " protected " areas . by using two sieves which differ in the order of the " transparent " and " opaque " areas , the reaction of the tumour - bed and stroma in the presence of abundant connective tissue becomes uniform throughout the tumour , irrespective of the different doses which regions may have received during treatment . analysis of the radiation reaction in tumours treated by the sieve has shown that fractionation of dose in time as well as in space induces injuries and specific alterations in the irradiated area , the rate and type of which greatly enhances tumour destruction . the authors are indebted to the british empire cancer campaign for a grant for technical assistance , and thanks are also due to f . 
kirby. from the research department , glasgow royal cancer hospital . received for publication february 8 , 1947 . the exceptional interest of 2 - acetylaminofluorene ( aaf ) as a carcinogen was clearly revealed by wilson , deeds and cox ( 1941 ) , who fed it to inbred albino rats in which only benign mammary tumours had been observed , and found carcinomas of the liver , breast , bladder , ureter , renal pelvis , colon , pancreas , lung and skin , a myogenic sarcoma , and in two cases liver lesions resembling leukaemic infiltration . 
the results of bielschowsky ( 1944 a and b ) and other workers are summarized later in this paper . in cba mice , neoplasms of liver and uterus ( one a sarcoma ) and of the urinary bladder were found by armstrong and bonser ( 1944 )  . bielschowsky and green ( 1945 ) reported a carcinoma of the kidney in a rhode island red cock which had been fed aaf for 45 weeks . the widespread variety of organs affected by aaf led bielschowsky ( 1944b ) to try to induce tumours of the goitrous thyroid gland with this substance . solid , parenchymatous goitre was induced by allyl thiourea ; in conjunction with aaf , also orally administered , allyl - thiourea induced adenoma in 9 out of 10 rats , while malignancy was observed in 3 of these rats . griesbach , kennedy and purves ( 1945 ) claim that the goitrogenic agent in brassica seeds can cause adenoma of the thyroid if given orally for long enough , but only 1 of 8 wistar rats developed such a lesion . subsequently purves and griesbach ( 1946 ) obtained malignant changes in 2 out of 30 wistar rats given thiourea in their drinking water for nearly two years . purves and his co - workers consider that the effect of the goitrogens is indirect , operating by inducing hypersecretion of the thyrotropic pituitary hormone . 
aaf has no specific action even on adenomatous thyroid tissue . the success achieved by bielschowsky in at least accelerating malignant changes .in the thyroid of rats by feeding aaf suggested that other organs apparently untouched by aaf alone might undergo neoplastic changes when stimulated to proliferate by suitable means . 
the sex - organs of the rat can be caused to hypertrophy by the administration of the appropriate sex - hormone , and it was thought that these organs , thus caused to proliferate , might proceed to malignant changes when influenced by orally administered aaf . 
the basal diet for all groups was rat - cake made to the formula of thomson ( 1936 )  . 2 - aminofluorene was synthesized from fluorene by the method of diels ( 1901 ) , * and acetylated as follows . 
up to 1 per cent of concentrated sulphuric acid was added to 2 - aminofiuorene stirring in an excess of acetic anhydride which became very hot and dissolved the amine . after chilling thoroughly the acetylated amine was filtered on a sintered - glass sufficient water was added to cause funnel , and dissolved in hot ethanol . permanent turbidity and the solution well cooled . 
emmens pointed out that absorption during the second month would be much reduced , and recommended the use of 20 mg . therefore thin disc - shaped pellets weighing 20 mg . 
a spindle - cell sarcoma of the left seminal vesicle was found in rat 354 ( group iib ) , and a whorled , spindle - cell sarcoma was found lying above the liver of rat 339 ( group iia )  . 
the reason for this may well be that suggested by these workers , namely that " the hyperplasia of target organs of sex - hormones is a functioning hyperplasia , whereas that induced in the thyroid by goitrogenic agents is non - functionthus the type of hyperplasia induced by bielschowsky in the rat thyroid ing . " in which growth is accompanied by loss of differentiation would appear to have been a suitable basis for carcinogenesis , whereas the hyperplasia associated with increased differentiation induced in sex organs by over - stimulation with sexhormones is apparently unsuitable . 
illustrate a " carcinoma arising adjacent to the external auditory canal , " and discuss the histogenesis of this and other tumours in this locality included by them under " subcutaneous tumours . " bielschowsky first drew attention to tumours of the ductus acusticus externus as a separate entity . 
as wistar rats were also used by bielschowsky , who found these tumours in 18 / 93 rats given aaf orally up to 210 days , the influence of diet seems to have some importance . 
when allyl - thiourea was also given , the incidence of tumours rose to 6 / 10 . lopez ( 1945 ) also found external meatus tumours in 2 / 4 rats . 
hence the action of aaf on proliferating mammary tissue was only slight in our experiments . in the matter of liver tumours , our series has yielded results in full accordance in our series , a . 
the types of liver tumours seem to be essentially the same as in wistar rats . this is also true of the liver tumours obtained by bielschowsky in piebald rats , in which the sex - ratio is also maintained and the in the sherman strain , cantarow et al . 
found the incidence much the same . incidence of liver tumours in female rats raised when oestradiol dipropionate was injected intramuscularly ; but the complete absence of liver neoplasms in rats given thiouracil was much more striking . 
the latter group ate less of the aaf diet than did controls receiving no thiouracil , but more than the oestrogenized rats . the incidence of mammary tumours in piebald rats was very low compared with that in wistar rats on the same diet ( bielschowsky , 1946 )  . but the incidence of tumours of the ductus acousticus externus was much higher in the piebald rats , in which tumours of the small intestine were also much more frequent . moreover , lung tumours were found in this strain , but have not been reported in wistar rats given aaf and were not found in our series , although one control female ( group ia ) was found to have undifferentiated bronchial carcinoma . the albino rats of wilson et al . 
the latter have not been found in any other strain of rat , and tumours of the kidney and bladder only once each . it would seem that , if sufficient strains of rat were employed , possibly no site in the body would be found immune from the carcinogenic action of aaf . 
in the strains so far tested , only mammary tissue among the sex - organs seems to be susceptible , and this tissue is liable to develop carcinoma due to the action of sarcomas are rare , and are not induced by subcutaneous oestrogens alone . injection of aaf , at least in wistar rats . in our series , a whorled , spindle - cell sarcoma was found in the omentum covering the left lateral lobe of the liver in one rat , and a spindle - cell sarcoma was present in the left seminal vesicle of another . it remains to discuss a few other points of interest . toxic damage to kidneys was seen in some animals of all groups ; rats receiving oestradiol benzoate pellets , with or without aaf , tended to show degeneration of the epithelium of the first and second convoluted tubules with the presence of a dark yellow a . 
kirby pigment in these cells . there was no consistent evidence of toxic damage to the kidneys by aaf or its metabolites , nor were any tumours of this organ seen . no tumours of the uterus were found , but all females receiving oestradiol benzoate had pyometra , which often made it necessary to kill the animal ; the 2 females receiving the male sex - hormone had no pyometra . the spleens of the control groups , ia and ib , showed no abnormality . 1 female in group iia and 1 male and 2 females in group iib had enlarged spleens , due to lymphoid hyperplasia . there was no question , however , of the regular , early , gross enlargement of the spleen seen in rats ingesting azo - compounds it may be ( smith , lillie and stohlman , 1943 ; kirby and peacock , in press )  . concluded that no splenotoxic substance passes from the liver into the blood of rats fed aaf ; rats fed p - aminoazobenzene ( aab ) or its n - monomethylor n , n - dimethyl - derivatives ( mab and dab ) have aab circulating in the blood and affecting the spleen ( miller , miller and baumann , 1945 )  . the rats given aaf orally in the experiments described in this paper developed tumours of the liver predominantly in the right lobes ; tumours in the left side of the liver were usually smaller or absent . 
a similar predominance of tumours in the right side of the rat liver has been reported by opie ( 1944 ) in rats fed dab in diets not inducing cirrhosis . 
dmochowski. from the department of experimental pathology and cancer research , medical school , university of leeds . received for publication december 15 , 1952 . the test mice most commonly used for ascertaining the presence of the mammary tumour agent are susceptible hybrid female mice obtained by mating low - breast - cancer - strain females to high - breast - cancer - strain males . in these test mice a low incidence of spontaneous mammary tumours was frequently recorded ( bittner , 1939b ; murray and little , 1939 ; andervont , 1940 ; gardner , 1941 ; dmochowski , 1944a ) , and was frequently found to be higher than that of their maternal parents ( andervont , 1945a )  . 
of even greater interest was the high tumour incidence reported in the progeny of susceptible low - cancer - strain females mated to high - cancer - strain males which had been subjected to forced breeding - that is , bearing a number of litters in quick succession ( andervont , 1945b )  . 
a similar high incidence of breast cancer was also observed in forciblybred hybrid progeny of low - cancer - strain females of low susceptibility to breast tumours mated to high - cancer - strain males ( bagg and jacksen , 1937 ; strong , 1943 )  . attempts to discover the mammary tumour agent in mammary tumours of some of these hybrids proved unsuccessful ( andervont , 1945b )  . 
a subline of c57 black strain is now established in which this subline a number of mammary tumours has been recorded by the author . comes from one of the original threelitters brought from the laboratories of the the progeny of these litters has been kept imperial cancer research fund . separately , and no breast tumours have so far been recorded in the descendants the description of the subline in which tumours have of the other two litters . the riii strain mice appeared will be the subject of a separate communication . were also obtained from the same source and maintained since 1946 . the tumour incidence of 83 per cent at an average age of 8x5 months in breeding females ( dmochowski and gye , 1943 ) has with small variations remained approximately it was known that mice of high - cancer strains derived from the fourth the same . or later litters develop a higher incidence of breast cancer than those from the first two litters ( bittner , 1942 ) , and that the appearance of an active agent in mice deprived of it by foster nursing had been recorded in the progeny obtained from hybrid progeny of the c57 black strain the third or later litters ( bittner , 1943 )  . females was therefore taken only from the fourth or laterlitters for the experiment , mice from earlierlitters being used for various other purposes . 
the ( c57 x riii ) fl hybrid females which were litter mates were divided into two groups . only two hybrid females were taken from each of the c57 females mated to riii strain males , if possible from the same or foliowing litters . whenever two females were not available from the same litter , the mice from later litters were included in this way two groups , each comprising thirty ( c57 x in the control group . riii ) f1 hybrid females , were obtained . mice of the control group were allowed to breed in a normal way and mice of the experimental group were forcibly bred by removing their litters as soon as they were born . mice of one of the later litters from fifteen hybrid females in this group and of one litter of a female from the these mice in turn , control group were saved for brother to sister matings . after saving one litter , were forcibly bred and this procedure was followed for several generations . 
the progeny of six ( c57 x riii ) f1 hybrid females , which had been subjected to forced breeding , was maintained for six generations and the progeny of nine additional ( c57 x riii ) f1 hybrids , treated in a similar manner , for only three generations . 
dmochowski tumour which on histological examination was found to be a lymphosarcoma . the average number of litters bom to these hybrid females was three , and varied from two to seven . 
the number of breast tumours which developed in their litter mate ( c57 x riii ) f1 hybrid females , which had been forcibly bred , is shown in table ii . 
among these hybrids , the tumorous females had an average of 6 * 5 litters and the tumour - free females 6 - 4 litters . there was no difference therefore in the number of litters born to these two types of females which had been forcibly bred . none of the c57 black strain mothers , mated to riii strain males and bred in a normal way , developed mammary cancer , although they lived to an average twelve c57 black strain females , which were litter - mates age of 19 mouths . of some of the c57 females mated to riii strain males , were forcibly bred with their own litter - mate males , and none developed breast cancer , although they had an average of 5 * 2 litters and lived to an average age of 18 months . 
the observation that none of the c57 strain females developed mammary tumours after mating to c57 strain males and forced breeding points against the possibility of c57 black strain females harbouring a weak or attenuated mammary tumour it is now well known that various sublines of c57 black strain differ agent . considerably in their susceptibility to the agent . c57 strain mice of the subline of the present experiments , when foster nursed by riii strain females , developed an incidence of mammary cancer of only 11 per cent ( dmochowski , 1948 )  . this low susceptibility was probably responsible for the failure to induce mammary cancer in mature c57 mice of this subline even by a combined action of large doses of material containing the agent and forced breeding ( dmochowski , 1948 )  . again this low susceptibility may also have been responsible for c57 mice of this subline remaining tumour - free after mating to riii high - cancer - strain males as well as for their hybrid progeny , bred in a normal way , not developing mammary cancer . 
a further indication against the possibility of c57 black mice of this subline harbouring a weak agent or in small quantities was provided by the absence of the agent in mammary tumours induced in both virgin and breeding c57 females by treatment with methyloholanthrene ( dmochowski and orr , 1949 )  . 
the forcibly - bred progeny of four ( c57 x rhij ) f1 hybrid females which had died free from tumours , also continued in these figures , the number of for six generations , is presented in fig . 
3 to 6 that descendants of tumorous hybrid females . females can remain tumour - free for two , three or four successive generations before developing mammary cancer , and that descendants of a non - tumorous female , in spite of reaching tumour age , may show no mammary tumours for as many as five generations before developing breast cancer , in spite of inbreeding it may be of interest to point out that and intensive hormonal stimulation . andervont ( 1949a , 1949b ) found the agent to be transmitted through three or four successive generations of susceptible mice without inducing mammary further , the study of the distribution of mammary tumours in charts cancer . of the progeny of tumorous and non - tumorous hybrids , shown in fig . 
1 to 6 , indicates the possibility of the agent being involved in the development of these tumours . a comparison of the age of the earliest tumour appearance , tumour incidence , average tumour age and average age at death of the progeny of individual tumorous and four tumour - free ( c57 x rii1 ) f1 hybrid females is shown in table iii . as can be seen , there was little , if any , difference between the forcibly - bred progeny of the two ( c57 x riii ) f1 females which developed breast cancer and the forcibly - bred progeny of the four ( c57 x riii ) f1 hybrid females which died without tumours . table iv demonstrates the tumour incidence and tumour age in the forcibly - bred progeny of additional nine non - tumorous ( c57 x rii1 ) f1 females which was continued for three generations . 
the tumour incidence , although varied in the progeny of the individual hybrids , was lower and the age of breast tumour appearance higher than those in the progeny of the two tumorous and also of the four tumour - free ( c57 x r111 ) f1 hybrid females . 
number preceding letters t , n or l = number of female ; number following letters t , n or l = tumour age or life span in days . of the earliest tumour appearance , the appearance of mammary tumours according to months , the tumour incidence , and the average tumour age in six generations of combined forcibly - bred progeny of the thirteen non - tumorous ( c57 x riii ) f1 hybrid females . 
dmochowski generation of the progeny of this non - cancerous hybrid female may be seen in this figure as well as the consecutive number of litter to which the mice belonged which developed tumours . it would have been of interest to obtain the first litter of this female and its progeny and compare any mammary tumour incidence in this progeny with that of the progeny obtained from the sixth litter , but this could not be done owing to limitation of space . table vii shows the age of earliest tumour appearance , the tumour incidence , and the average tumour age in the normally - bred progeny of this non - tumorous ( c57 x riii ) f1 hybrid female . 
the breast tumour incidence in the forcibly - bred progeny of the non - cancerous ( c57 x riii ) f1 hybrid females was lower and the average tumour age considerably higher than those in the forcibly - bred progeny of cancerous ( c57 x riii ) f1 hybrid females , but this difference in tumour incidence and average tumour age gradually became smaller in successive generations of the progeny . 
the age of the earliest tumour appearance and the average tumour age in the progeny bred in a normal way of one cancer - free ( c57 x riii ) f1 hybrid female was distinctly higher than those in the forcibly - bred progeny of both tumorous and tumour - free ( c57 x riii ) f1 hybrids . 
the tumour incidence in the normally - bred progeny was , with one exception , lower than that in the forcibly - bred progeny of tumorous hybrid females and , again with one exception , approximated to that in the forcibly - bred progeny of non - tumorous hybrid females . it should be pointed out that the results of the comparison of the behaviour of normally - bred progeny of one non - tumorous female with that of the forcibly - bred progeny of thirteen non - tumorous hybrid females may not be the same as those which would have been obtained should this comparison have been extended to similarly maintained progeny of a number of other cancer - free hybrids which had been bred in a normal way . this point may be seen from the observations on the forcibly - bred progeny of four nontumorous ( c57 x rii1 ) f1 hybrids compared with those on similarly treated progeny of two tumorous ( c57 x riii ) f1 hybrid females . 
the difference in mammary tumour incidence and average tumour age between these two groups of hybrid females became pronounced only after including the observations on the progeny of nine additional non - tumorous ( c57 x riii ) f1 hybrid females . is therefore possible that the results of the comparison of normally with forciblybred progeny of non - cancerous hybrids would have been different as in the case of the comparison of forcibly - bred progeny of non - tumorous with that of tumorous it is not known whether the results of foulds ' ( 1949 ) comparison of hybrids . forcibly - bred progeny from tumorous and non - tumorous hybrid females would l . 
dmochowski not have been different had the progeny of a greater number of non - tumorous hybrid females been included in his study . from the study of the distribution of cancerous females in the forcibly - bred progeny of tumorous and tumour - free ( c57 x riii ) f1 hybrid females and in the normally - bred progeny of a cancer - free hybrid , shown in fig . 
among the forcibly - bred progeny of the four tumour - free ( c57 x riii ) f1 hybrids , tumorous mice had an average of 7 3 ( 1 to 13 ) and the mice without tumours an average of 8 - 3 ( 1 to 16 ) litters . 
the behaviour of mice in the progeny obtained after the tumour appeared in their mother may greatly vary even in the case of hybrid females of the same origin , probably as a result of variations in the genetic make - up of individual females infiuencing the presence or transmission of the agent . in conolusion it may be stated that both the genetic constitution and hormonal stimulation play an important part in the development of mammary cancer in the hybrid mice which were the subject of the present study , and that the mammary tumour agent also appears to take a part in the origin of these tumours . biological tests for the presence of the mammary tumour agent . as soon as the first mammary tumours appeared in the hybrid mice , biological altogether twenty - three tests for the presence of the agent were carried out . of the mammary tumours were tested and the results are shown in table xiii . four mammary tumours which developed in ( c57 x riii ) f1 hybrid females , two showed the presence of the agent and two others failed to reveal the agent under the conditions of the test . 
at first it appears surprising that the tumour which developed at the age of 10 months failed to show the agent , as the time of storage in desiccated state was equal to that of another breast tumour which appeared in the same generation and which , although injected in smaller quantity , induced tumours in the test mice but in distinctly smaller incidence compared with that induced by other dried agent - harbouring tissues in previous it must be stressed experiments ( dmochowski , 1945a , 1945b , 1946 , 1948 , 1949c )  . that the method of desiccation of these two as well as of all the other tumours was the same , and the age of the test mice used was in all cases approximately the same . the only varying factors were the amount of tissue injected and the time of storage . 
the longest period of storage in the ice - chest was that of a negative tumour in a ( c57 x riii ) f , hybrid female and amounted to 8 months . this should not have made an appreciable difference in the test , as it was previously shown that agent - harbouring tissue induced breast canoer in test mice after two years of storage ( dmochowski , 1946 )  . 
from the results of the tests of the four tumours in the first generation of hybrid females it may be concluded that two of them possessed the agent , and the two other negative tumours either did not contain the agent or harboured only small quantities or a weak agent which could not be shown under the conditions of the test . 
2 , from the study of parent - offspring correlation there was a definite tendency towards breast cancer development in daughters of tumorous as well as non - tumorous mothers . 
9 female had ten litters and her sister had seven litters . it may well be that the considerable hormonal stimulation was responsible for the appearance of breast cancer in these females , and more likely that the agent took part in the development of these tumours but in quantities too small to be detected by the ( c57 x riii ) f3 no . 
it may well be that had the test a : nimals , in which the latter tumour was tested , lived longer , the agent would probably have been detected . in the fourth generation of the progeny of no . 
20 ( c57 x riii ) f3 hybrid , were found to harbour the agent . the first tumour developed at the age of 8 months after the female had six litters , and the second at approximately 17 months after the female had thirteen litters . it is of interest that both tumours showed the agent , although the second developed comparatively late in life . in the fifth generation of the same line three tumours were tested , those of no . 
69 ( c57 x riii ) f1 tumorous hybrid female in whose tumour the agent was not detected , it may be seen how necessaiy it is to adopt a cautious attitude towards the results of biological tests . it appears probable that the agent may have increased gradually in quantity in successive generations of the descendants of this hybrid female , so that it eventually could be detected by the method applied in the present biological tests . it is possible that the treatment , such as desiccation , may have in part been responsible for the negative results when tumours with small quantities of the agent were tested . had the mammary tumour of no . 
69 ( c57 x riii ) f1 hybrid only been tested and the progeny of this female not raised , the conclusion would have been that increased hormonal stimulation of a susceptible genetic substrate was responsible for the appearance of this breast tumour . it may well be stated that hormonal and genetic factors combined with the mammary tumour agent were responsible for the development of the majority of the tested breast tumours in this line , and probably also a weak or attenuated agent in the tumours negative in the biological test . in the progeny of non - tumorous ( c57 x riii ) f1 no . 
33 female at the age of 17 months after she had eleven litters and one which arose in ( c57 x riii ) f4 no . 39 hybrid at the age of 10 - 5 months after she had given birth to six litters . 
21 , 47 , and 49 ( c57 x riii ) f3 hybrids , all of which developed after the eighth litter at the age of 9 , 12 , and 10 months respectively . all four tumours harboured the agent , yet , although they all arose comparatively early in life , the incidence of tumours induced in the test mice by the tumour of no . 
tumours developed in hybrid mice of all three coat colours without particular connection with any coat colour . foulds ( 1949 ) recorded a similar observation in his c57 x riii hybrids . neither the presence or absence of the agent , as shown in biological tests , was connected with any particular coat colour . foulds ( 1949 ) detected the agent in the tumour of a ( 057 x riii ) f1 hybrid muhlbock ( 1952 ) failed to discover the agent in tumours which had been tested . of five ( c57 x d ) hybrids which developed at ages varying from 20 to 30 months . these hybrids came from early litters . 
mammary tumours in hybrids from later litters of c57 females were not tested , but the presence of the agent in these tumours was assumed by muhlbock ( 1952 ) on the basis of a high tumour incidence in the litters , although the average tumour age was 18 months . 
a search for the agent in mammary tumours of hybrid progeny from another ( c ) low - cancer - strain but susceptible females and high - cancer - strain ( 03h ) males led to the detection of the agent in tumours which developed at an early age up to 12 months , although on two occasions even such tumours were negative ( andervont , 1945b ; andervont and dunn , 1950b ) , while mammary tumours arising at a late age , in spite of their high incidence , failed to reveal the agent ( andervont and dunn , 1949 , 1950b ; andervont , 1950a )  . yet , on one occasion a c strain female was found to harbour the agent , although she developed breast cancer at the age of 21 months ( andervont and dunn , 1950b )  . thus there may be exceptions in both early and late developing tumours , as shown in the present study with dried tumour tissue and in the experiments of andervont and dunn with fresh tumour tissues . biological tests of some tumours appearing at a late age , as shown in the present experiments , revealed the agent , although they failed in other . 
tumours of similar it is not known how far the recent observation of andervont ( 1950b ) late age . that small quantities of the agent may only be ascertained by observing at least one or even two generations of the descendants of the inoculated test mice would be helpful , but it is certainly worth trying in any future tests of mammary tumours appearing at a late age . in some mammary cancers which developed up to the age of 12 months , the agent could not be detected on several occasions in the present study . 
69 f1 hybrid were found to possess andervont ( 1945b , 1950a ) also failed to detect the agent in mice with the agent . tumours at 8 , 12 , 14 , 15 and 16 months . 
on the basis of the study of parentoffspring correlation shown in pedigree charts , it appears probable that the negative tumours also harboured the agent possibly in quantities too small to be detected by the tests applied . in conclusion it may be stated that the results of the biological tests for the presence of the agent in desiccated mammary tumours which developed in the hybrid mice indicate a gradual accumulation of factors responsible for the appearance of mammary cancer . it appears that there may be a certain threshold below which it is difficult or not possible to show the agent in some mammary tumours , at least by the methods so far employed , and only after combined influence of inbreeding and intensive hormonal stimulation the presence of the agent may become detectable . there is no reason to consider the biological tests as inadequate , in view of the previously reported constant positive results with dried agent - containing tissue , and also in view of the negative results occasionally recorded by other workers with fresh material even from tumours which developed early inlife . further , negative tests for the agent in dried tissue of an 86th transplant of a mammary tumour which originally harboured the agent ( dmochowski , 1952 ) have been confirmed with fresh tissue of a 101st serial passage of the same tumour . the negative findings may have been due either to small quantities of the agent present in some tumours or to the kind of relationship of the agent to other cell constituents in these tumours , such as close integration with these constituents , different from that in other tumours which were found positive in the biological tests . microscopical appearance of mammary tumours . in view of the availability of a large number of tumours of a comparatively uniform origin a microscopical study of these tumours was made , and also an attempt to correlate the morphological appearance of these tumours with the presence or absence of the agent . 
as can be seen the majority of tumours in this agentharbouring strain could be divided into roughly equal numbers of type a and type b tumours . the results of the study of 377 mammary tumours in 327 c57 x riii hybrid mice are shown in table xiv . 
among the 327 tumorous mice , 32 had two tumours and 9 had three tumours . as shown in table xiv , the majority of most of the tumours ( 70.6 per cent ) developed during tumours were of type b . the first 12 months , and among them 53 were of type a ( 19.7 per cent ) and 215 ( 79.9 per cent ) were type b tumours ; one unclassified mammary carcinoma also after the first 12 months , 21 tumours belonged to this group of early tumours . were of type a ( 19.4 per cent ) , 77 tumours belonged to type b ( 71.4 per cent ) , 9 tumours were of type d ( 8 - 3 per cent ) , and 1 tumour was unclassified . 
an analysis of the distribution of the different types of tumours in the progeny of tumorous and non - tumorous ( c57 x rii ) f1 hybrids revealed that the progeny of both types of females had a number of each type of tumour similar to that found in the total number of all tumours . 
among the 120 mammary tumours of the descendants of tumorous hybrid females , there were 19 ( 15.8 per cent ) of type a , 99 ( 82.5 per cent ) of type b , and 2 ( 1.7 per cent ) of type d . 
among the 225 tumours in the forcibly - bred progeny of non - tumorous hybrid females , 50 ( 22.2 per cent ) belonged to type a , 166 ( 73.8 per cent ) to type b , 7 ( 3.1 per cent ) to type d , and 2 ( 0.9 per cent ) were unclassified . there were 32 mammary tumours examined in the progeny of a non - tumorous hybrid female which had been bred in a normal way ; five ( 15 - 6 per cent ) were of type a and 27 ( 84.4 per cent ) belonged to type b . 
there was no difference in the distribution of types of tumours in the progeny of hybrids which had been subjected to hormonal stimulation of varying intensity . no correlation was observed between the type of tumour and its location or size or rate of growth or litter sequence of the tumour - bearing mice or age of the animal at which the tumour developed , except in the case of type d tumours , further , there was no all of which developed in mice older than 12 months . correlation between the types of tumours in litter mates or between tumours of mothers and offspring . similar observations were reported by andervont and dunn ( 1950b ) on ( c x c3h ) susceptible hybrids , in which they found 38 per cent of type a tumours , 44 per cent of type b , 9 per cent of type c , and 8 per cent of type d . 
they observed a correlation between the age at which mammary tumours appeared and the type encountered , as in mice of up to 19 months of age 63 per cent were type a tumours and 33 per cent type b tumours , and in older mice 31 per cent belonged to type a and 48 per cent to type b . in another study ( andervont , 1950a ) 38 per cent of tumours were of type a and 46 per cent type b , and in 106 l . 
they did not detect the agent in a c strain female with a squamous type mammary tumour at a late age , yet in her progeny there appeared a type a tumour with the agent and one squamous type cancer without the agent . another two females of the same strain , two tumours of type a and b respectively both harboured the agent ; in their progeny all tumours with the agent were of type a . 
the majority of tumours in old mice were similar to those of mice with the agent ( andervont , 1945b , andervont and dunn , 1949 )  . in hybrid mice obtained from crossings of agent - free low - cancer - strains the majority of mammary tumours were of types c and d , and in those obtained from one of the parents belonging to agent - free but high - cancer strain the majority of breast tumours were of types a and b ( andervont and dunn , 1948a )  . it appears that the genetic constitution of strains used for breeding hybrids influences at least the distribution of the various types of breast cancer . there was no correlation in c57 x riii hybrid females between the presence of the agent , as shown in biological tests , and the type of tumour encountered . of the seventeen tumours in which the agent was found , ten were of type b , six of type a , and one of type d , and among the mammary tumours in which the test failed to reveal the agent , four were type b and two type a , cancer ( table xiii )  . 
the finding of the agent in type d mammary tumour which developed at 17 months is of interest , as it appears to indicate that not all squamous type tumours which appear comparatively late need be agent - free tumours , or possibly tumours in which for some as yet unknown reason it is difficult to detect the agent . 
two agent - harbouring tumours of ( c57 x riii ) f1 hybrids were of type a ( 11 months ) and type b ( 16 months ) , and the two other tumours in which the agent was not detected were also of type a ( 10 months ) and type b ( 11 months )  . 
among the fifteen agent - harbouring tumours of c57 x riii hybrid progeny , five were type a and arose at the age of 9 to 13 months , nine were type b and developed at 7 to 17 months , and one type d at the age of 17 months . 
among the tumours in the hybrid progeny in which the agent was not detected , one was of type a ( 18 months ) and three of type b ( 8 to 10 months )  . in andervont and dunn 's experience ( 1950b ) the majority of tumours ( 84 per cent ) in hybrids developed at the age of 18 to 29 months and the rest during the first 17 months . 
they also found no correlation between the microscopical appearance of a tumour and the presence of the agent . some of the early appearing tumours in which the agent could not be detected were of type a or b ( andervont , 1945b , 1950a )  . foulds ( 1949 ) described the microscopical appearance of breast tumours in his c57 x riii hybrids as " unmiihlbock ( 1952 ) found the morphology of tumours in c57 x d remarkable "  . hybrids of little assistance in the study of the part played by the agent in the development of these tumours . 
he also noted more unusual features in tumours presumably free of the agent compared with those harbouring the agent , yet the tumours accumulated in later litters of c57 mice following mating to high - cancerstrain males , in which the agent was presumed to be present , had an appearance like that of agent - harbouring breast tumours , in spite of their late average age 108 l . 
dmochowski of development ( 20 months )  . there appears to be a greater variety in appearance of mammary tumours in mice of strains in which the agent could not be detected ( andervont and dunn , 1950a ; 1950b ; heston , deringer , dunn and levillain , in the present study all tumours of type d developed 1950 ; muhlbock , 1952 )  . after 12 months of age . similarly , andervont and dunn ( 1950b ) found this type of tumour to be very rare in younger hybrid mice in which the agent could be detected , although , as shown in this study , this type of tumour even at a late therefore the greater frequency of squamous age may harbour the agent . metaplasia in tumours of old hybrid mice , if the age of 17 months could be considered as a comparatively late age , need not indicate at least in every case their development without participation of the agent , contrary to the previous sugthere is no doubt , however , about the greater gestion of kirschbaum ( 1949 )  . frequency of squamous type tumours in agent - free high - cancer - strain or susceptible mice ( gardner , 1947 ; heston , 1948 ; heston , deringer , dunn and levillain , 1950 ) than in similar mice with the agent , but they do appear occasionally in agent - carrying strains of mice as observed by dunn ( 1945 ) , andervont and dunn ( 1950b ) , and in the present study . from these observations it may be concluded that no particular microscopical appearance of mammary cancer in hybrid mice can be correlated with the presence or absence of the agent , in spite of the difference in distribution of the particular types in various strains of mice . 
thus a variable but low tumour incidence was reported by andervont and dunn ( 1948a ) in hybrids from reciprocal crosses of low - cancer - strain ( c , i , c57 ) mice and a higher but still comparatively low tumour incidence in the hybrid progeny of low - cancer - strain ( c ) females and agent - free high - cancer - strain ( dba - ) males , in spite of increased hormonal stimulation . 
dmochowski showed a high incidence after mating to one ( c3h ) high - cancer - strain male developed a much lower incidence ( 30 per cent ) when mated to other ( a ) high - cancerstrain males . females of strains with low susceptibility may show similar differences . 
even after the 10th pregnancy of low - cancer - strain females mated to males with and without the agent , these two types of females when used as foster mothers induced a similar tumour incidence in the test mice . 
dmochowski tumour age ( 19 to 25 months ) in both types of hybrids is characteristic and hormonal stimulation was assumed to be the cause of these tumours ( muhlbock , 1952 )  . 
the development of mammary tumours in the descendants of both tumorous and the majority of non - tumorous hybrid progeny ofc57 females which remained tumour - free after mating to agent - carrying males in the present experiments as well as the detection of the agent in the majority of the tumours tested indicate the transfer of the agent by riii high - cancer - strain males . though not all ( 057 x riii ) f1 hybrids developed breast cancer , the transfer of similarly , the agent must have taken place in the majority of the females . bittner ( 1952a ) noted a high incidence of tumours and the presence of the agent in the descendants of cancerous and cancer - free hybrid progeny of certain strain females mated to agent - carrying males . 
the presence of the agent was assumed in mammary tumours which appeared in hybrid progeny of later litters of some c57 females mated to high - cancer - strain ( d ) males ( muhlbock , 1952 ) because of the high tumour incidence , in spite of the late age at which the tumours appeared . 
as a result of this transfer , only few tumours in the hybrid progeny develop early and harbour the agent , while the majority of transfer of the agent tumours develop late without participation of the agent . to embryos , however , appears to be doubtful , because of the absence of the agent in high - cancer - strain embryos ( dmochowski , 1949c ; hummel and little , 1949 )  . the passage of the agent from embryos , should they become infected in utero , is also questionable because of the reported neutralisation of the agent by placenta transmission of the agent in utero would ( hummel , little and eddy , 1949 )  . also contradict the basis of the discovery of the agent itself . 
the increase in tumour incidence in hybrid progeny of later litters ( andervont and dunn , 1949 ; bittner , 1952a ; muhlbock , 1952 ) and the high incidence of tumours observed in the descendants of later litters from hybrid females observed in the present experiments may indicate either a transfer of the agent to females repeatedly mated to agent - carrying males and / or gradual increase in the agent , transferred by mating , during its long latent period under the influence of hormonal stimulation ( repeated pregnancies )  . 
the low incidence of tumours observed in the hybrid progeny of some derivations need not necessarily be interpreted in the same way as the small number of tumours induced in mature mice which had been given the agent , as suggested by muhlbock ( 1952 )  . 
a high incidence of tumours obtained in mature mice of some strains after repeated injections of material containing the agent ( dmochowski , 1945a ; muhlbock , 1952 ) , and a small incidence in mature mice of other strains ( bittner , 1952b ) , indicate that the genetic constitution is the more likely explanation for the different tumour incidences in transmission of the agent by the sperm to lowhybrids of various derivations . cancer - strain females , followed by its transmission to their hybrid progeny in the mnilk of these females ( bittner , 1952a ) , appears to be the most likely way in which the agent gains access to hybrid females , although in females of some strains it may only occasionally take place ( miihlbock , 1952 )  . 
the observation of several generations of descendants of hybrid females can give a clear picture whether the transfer of the agent has taken place or not , as shown in the present study . 
under the same experimental conditions some hybrid females may develop mammary tumours while their litter - mates may fail to show tumours ; their descendants may show a similar variation in their behaviour . thus , the study of the behaviour of the hybrid progeny of low - cancer - strain females , especially of the descendants of later litters of the hybrid progeny , can only decide whether or not the agent has been transferred by the male parent . the observations on tumour development and the presence of the agent in 114 l . 
dmochowsk1 the descendants of the hybrid progeny obtained by mating c57 black straini females to agent - carrying riii strain males , made in the present experiments , appear to indicate an interesting possibility in the agent - host relationship which may be the outoome of gradual mcrease in the amount of the agent or its activation in some hybrid females , while in other hybrid females it appears in quantities large enough to be detected immediately with the development of breast cancer in the first generation of hybrid progeny . this may be on one hand the result of individual variations in the genetic constitution of hybrid females , and on the other hand the outcome of varying quantities of the agent which the hybrid females obtain from their low - cancer - strain mothers mated to high - cancer - strain within the framework of individual differences in the genetic make - up males . and / or quantities of the agent obtained , inbreeding combined with hormonal stimulation also exerts its influence . 
thus we have the situation , encountered in the present experiments , that under the same experimental conditions some hybrid females develop mammary cancer and others ( even their litter mates ) do not show breast cancer ; some of the tumorous females do and others do not reveal the agent ; some of the progeny of the cancerous hybrid females may develop mammary tumours which again may or may not reveal the agent , and other progeny of the same cancerous females do not develop tumours ; the progeny of non - cancerous hybrid females may develop tumours which contain the agent . 
the development of breast cancer in some of the ( c57 x riii ) f1 hybrids only after foroed breeding may be explained by the transmission of small quantities of the agent by at least some of the c57 black strain mothers after mating to riii agent - carrying males . 
the high , although varied , tumour incidence in the progefny of these hybrids may be explained by the origin of these females as well as of their progeny from late litters and therefore an increase or activation of the agent in these litters , enhanced by inbreeding and increased hormonal andervont and dunn ( 1950b ) also observed that under the same stimulation . conditions some low - cancer - strain females developed and others failed to develop breast cancer , and some females with tumours possessed and others lacked the there was also no correlation between the presenco of the agent in these agent . females and its presence in their hybrid progeny , cancerous females with or without the agent giving rise to hybrid progeny with only late tumours apparently without the agent , or to progeny with both early tumours with the agent and late tumours without the agent , although they were litter mates . therefore , again , the presence or absence of the tumour and / or the agent in the mother did not necessarily involve the presence or absence of the agent in her progeny . andervont and dunn ( 1950b ) noted on several occasions among the hybrid progeny with late tumours that their litter mates had early tumours with the agent , and on one occasion a hybrid female with an early tumour harbouring the agent had progeny with either no tumours or only late tumours without the agent , which suggested the disappearance of the agent ( andervont and dunn , 1949 )  . bittner ( 1952a ) also reported on the hybrid progeny of some derivations with low mammary tumour incidence that some of them gave rise to descendants with a high incidence of tumours harbouring the agent . 
thus the variable results appear to be due to variations in the genetic make - up of both the hybrid females and their progeny , which in turn leads to variations in the amount of the agent transmitted . a more favourable genetic make - up may account for an increased amount of the agent and its detection . 
the appearance of mammary tumours in some low - cancer - strain females ( c3hb x ax ) mated to agent - free ( c3hb ) males and the high incidence of tumours in their descendants , on the basis of which the presence of the agent was assumed although the tumours were not tested biologically ( bittner , 1952a ) , is one at the moment rather perplexing sudden " de novo " appearance of the agent was therefore also observation . considered as a possible explanation of some of the findings ( andervont and dunn , 1949 ; bittner , 1952a )  . this consideration was based on previous observations of bittner ( 1941 ) on the sudden appearance of the agent in some susceptible ( ax ) mice , deprived of the agent by foster nursing by low - cancer - strain mothers , which remained free of the agent for seven generations , and on similar observations made recently by bittner ( 1952a ) on susceptible mice of another strain ( c3hb ) which remained free of the agent for sixteen generations following foster nursing before the appearance of breast cancer in one of the females , which then gave rise to progeny with a high incidence of mammary tumours . according to the writer 's opinion , these observations of a sudden appearance of the agent may only show the length of time during which the agent may lie dormant or the time required before the agent , originally present in small amounts , increases in quantity sufficient to induce breast cancer in combination with other known and unknown factors . of the known factors , either a change in the genetic constitution of the animal concerned alone and / or increased hormonal stimulus may , at least in part , be responsible for the activation of the agent . bittner ( 1939a ) reported the appearance of some mammary tumours in mice without the agent , and strong ( 1943 ) stressed the influence of hormonal factors in such tumours . 
the difficulties encountered in the detection of the agent in breast tumours appearing late in the life of mice and the comparative ease with which it could be shown in tumours developing early ( andervont , 1950a ) , further the isolated tumours in hybrid progeny of early litters and the accumulation of tumours in hybrid mice of late litters ( miihlbock , 1952 ) , led to the conclusion that there may be two types of mammary tumours in mice . 
one type of tumour would be the result of all three factors taking part in its development , the other type would be the result of hormonal and genetic factors without participation 116 l . 
dmochowski of the agent ( andervont , 1950a ; muhlbock , 1952 )  . however , the possibility of the agent being involved in both types of tumours was not discounted by andervont ( 1950a )  . 
the genetic constitution and intensive hormonal stimulation were considered adequate by heston , deringer , dunn and levillain ( 1950 ) to give rise to mammary tumours in susceptible ( c3hb ) mice , originally derived from mice deprived of the agent by foster nursing , but they also stressed that absolute proof of the absence of the agent from such tumours was lacking . 
the appearance of only a few tumours in the hybrid progeny from reciprocal matings of these ( c3hb ) mice with low - cancer - strain ( c57 ) mice with no evidence of an increase in tumour incidence in the hybrid progeny of later litters , as originally reported by bittner ( 1944 ) in mice of agent - carrying strains or in hybrid progeny from low - cancer - strain females and agent - carrying males , again led heston and deringer ( 1952 ) to suggest that some mammary tumours develop in the absence yet , in hybrid progeny of similar mice of other strains , bittner of the agent . ( 1952a ) observed the appearance of tumours harbouring the agent . further , the agent has been detected in some mammary tumours appearing in comparatively old hybrids , as shown by bittner ( 1952a ) and in the present study . in other late tumours as well as occasionally also in early - developing breast cancers the agent could not be detected by any , so far , employed testing procedures as noted by andervont ( 1950a ) , andervont and dunn ( 1950b ) , and by the writer . thus , intensive hormonal stimulation combined with a suitable genetic background influence the origin of mammary tumours in hybrid mice , increase their there is no doubt that the mammary incidence , and accelerate their appearance . tumour agent takes a part in the development of breast tumours appearing up to a certain age , although not all tumours of early appearance in hybrid mice reveal its presence . 
 ( c57 x ri11 ) f1 hybrid females , obtained by mating agent - free c57 black strain females to agent - harbouring riii high - cancer - strain males , developed a 14 per cent incidence of mammary tumours at an average age of 13 months after bearing in quick succession an average number of 6 - 4 litters . their litter - mates , bred in a normal way , died free of tumours after rearing an average number of 3 litters . 
analysis of parent - offspring correlation in the distribution of tumours in the descendants of the hybrids revealed that the progeny of tumours and of the majority of non - tumorous hybrids had a greater chance to develop cancer if the parent developed cancer than if the parent died without a tumour . 
thus there was a strong indication of the presence of the agent in these hybrids and of its transmission to their progeny , which showed an increasing number of tumours following the combined influence of inbreeding and intensive hormonal stimulation . 
biological tests for the presence of the agent in twenty - three mammary tumours were positive in seventeen and negative in the remainder of tumours . the age of tumours in which the agent was detected varied from 7 to 17 months , and that of tumours in which the agent could not be detected from 8 to 18 months . these tests , combined with the observed distribution of tumours in the descendants of hybrid mice , showed that under the same experimental conditions some hybrids developed tumours which either harboured the agent or failed to reveal it , while other hybrids , even their litter mates , died without tumours . 
some of the progeny of tumorous hybrids failed to develop tumours and others of the same females developed cancer in which again the agent was either demonstrated or could not be shown . 
dmochowski which as a rule developed in older mice above the age of 12 months . there was also no correlation between the appearance of mammary tumours in parents and their offspring . 
with the advent breast cancer . of x - rays the same effect was brought about by ovarian irradiation ( foveau de courmelles , 1926 ; ahlbom , 1930 )  . on the experimental side , the work of lathrop and loeb ( 1916 ) , loeb ( 1919 ) and murray ( 1928 ) demonstrated the part played by the ovary in the production of mammary tumours , while lacassagne ( 1932 ) showed that , in certain circumstances , mammary carcinomata could be induced by the injection of oestrogenic substances . interest in the problem was further stimulated by the work of huggins and hodges ( 1941 ) on the treatment of carcinoma of the prostate by castration and stilboestrol . it was logical that the next step in the management of advanced breast testosterone propionate was cancer in women should be the use of androgens . tried with success by ulirich ( 1939 ) and by many later workers . the treatment of women suffering from breast cancer with one of the oestrogen preparations was apparently illogical , although zondek ( 1936 ) had put forward the suggestion in 1936 . 
they are rare , however , and in over 4000 cases of breast cancer observed in this department no spontaneous regression has so far been noted . the survival rate will be mentioned in the groups of cases to be considered and , although it seems that life can be prolonged with hormone treatment , no attempt is made to claim success on this account alone . the responses were graded as follows : a . 
good responses were seen in all types of metastases , skin nodules , glands , pulmonary and skeletal in 3 patients with osteolytic bone metastases there was radiological metastases . evidence of new bone formation . in 4 a pleural effusion disappeared and in 4 it diminished in amount . 
5 illustrates the findings on 9.x.46. patients treated by surgical castration and testosterone . in 1941 a small group of patients was treated by surgical castration , accompanied by the administration of testosterone . this was done in an attempt to find out why there were some failures with ovarian irradiation . it was thought that ovarian function might recover to some extent after irradiation and that the effect of o6phorectomy would be more lasting . nine patients with advanced breast carcinoma were treated in this manner by o6phorectomy , followed by daily intramuscular injections of 25 mg . 
testosterone propionate for periods up to 3 months . only one patient showed a slight response . she was a premenopausal woman of 40 with an inoperable breast carcinoma and bone metastases . 
the primary tumour became smaller and the glands disappeared temporarily , but no effect was noted on the skeletal metastases . patients treated by oestrogen therapy . in the latter part of 1942 it was decided to investigate the value of oestrogens in advanced breast carcinoma , and since that time the following oestrogens have been tried : stilboestrol dipropionate , dienoestrol , triphenylchlorethylene and ethinyl oestradiol . 
twice a day sometimes caused sufficient water retention to increase the pleural effusion and add to the patient 's dyspnoea . it was remarkable to find in a few such patients that doses as small as 0 ' 5 mg . 
the number of patients having no symptoms attributable to the hormone while taking stilboestrol was 122 , while many of the remainder had symptoms which did not inconvenience them greatly , the most common being nausea and vomiting at the commencement 36 maryidouglas of treatment . 
even pleural effusions dimished in amount , and in 3 cases disappeared altogether with no treatment other than stilboestrol dipropionate . it is also very interesting to note that in this series 13 patients with bone metastases experienced relief from pain , while in 6 , new bone formation was demonstrated radiologically in osteolytic lesions . 
no post - mortem was carried out , but this gastric tumour was probably metastatic in nature from the carcinoma breast . the following case illustrates the improvement which may take place in a patient with a pleural effusion : ca8e no . 
of testosterone propionate daily , given intramuscularly . treatment was continued for 3 to 4 weeks , and if the patient 's condition showed some improvement , further treatment was given either by means of an implant of 400 to 600 mg . 
one patient who was comatose with cerebral metastases improved sufficiently under treatment with testosterone to subsequent post - mortem examination confirmed attempt crossword puzzles . the presence of large intracerebral metastatic deposits , which presumably had regressed only temporarily under the influence of testosterone . in another patient a large pleural effusion did not cause any further respiratory embarrassment , and required aspiration only at increasingly longer intervals . this patient is still alive 25 months after the commencement of treatment . only one patient experienced relief of pain from skeletal metastases while another patient with metastases in bone stated she receiving testosterone . felt the pain easier when the drug was withdrawn . there was no radiological evidence of repair occurring in bone metastases . 
the series , although small , is in this respect at variance with the results so frequently reported in the literature ( adair et al . , 1949 ; kaae , 1949 ; report of the council on pharmacy and chemistry , 1949 )  . only one patient could not tolerate the drug . she was a woman of 43 with cerebral metastases , who developed very severe vomiting , and treatment had to be discontinued after she had received 150 mg . apart from this one patient no one was upset by treatment . 
treatment was also given to post - menopausal women to determine the effect . many of these patients had already failed to respond to other methods of hormone therapy , and they were beyond the aid of all other forms of treatment . the pituitary was irradiated by two opposed fields , each 6 x 8 cplaced in the temporal region , and a dose of 3000r was delivered in 3 weeks to the region of the sella turcica . a series of 37 patients was treated between october , 1949 , and october , 1950 , and the results are shown in table vi . as will be seen from table vi , only 2 patients showed a good response . 
the recurrence had become flatter for 3 months , but after that had spread again . following pituitary irradiation complete healing took place slowly , but new nodules appeared 10 months later . of the 2 women showing a fair response , the first was aged 46 and premenoshe had a skin recurrence on the chest wall within the irradiated area pausal . and was treated by pituitary irradiation in december , 1949 . slight regression of the recurrence was noted for 3 months , but then the disease became widespread and she died on 6 . 
none had shown any response to the other types of hormone treatment . it would seem from this that pituitary irradiation may be of value in the tylie of case suitable for ovarian irradiation , or in cases where ovarian irradiation has already been given with some good temporary effect . there is no doubt that pituitary irradiation was the least well tolerated of all fourteen patients were very upset by the treatment it is possible that a modification of technique forms of hormone therapy . before the course was completed . and dosage would overcome some of these difficulties . of the 37 patients , 21 have died within one year , the average survival being 2 to 9 months after the commencement of treatment . 
no good results were seen in bone lesions apart from relief of pain in 2 cases , although , of course , the series was small , and treatment may not have been continued over a long enough period . the reports in the literature on the efficacy of testosterone are very variable . farrow ( 1944 ) noted a deleterious effect in female patients , nathanson ( 1944 ) observed no effect at all , and various later workers ( adair , 1947 ; adair et al . , 1949 ; kaae , 1949 ) reported a proportion of good responses in skeletal and extra - skeletal all are agreed that testosterone may produce a beneficial effect metastases . on the general health but this is a non - specific effect , and due partly to the stimulating effect of the hormone on protein metabolism . it is , however , important not to assess improvement in general health as an anti - carcinogenic effect . with both oestrogen and androgen therapy some responses were noted here after the drug was stopped - a withdrawal response . farrow ( 1944 ) also noted this occasionally in the metastases after withdrawal of either oestrogens or androgens . consideration of the mode of action of hormone therapy . wvhile hormone therapy is widely employed , the basis of treatment remains largely empirical . the extension of our knowledge of the exact mode of action would be of great interest , and might result in further advances in this form of therapy.. 
the clinical results obtained , however , appear to be due to some modification of the endocrine status of the patient . for example , in a young patient who has not reached the menopause , oophorectomy or ovarian irradiation causes a sudden withdrawal of the natural ovarian hormones which influence the rate of growth of the cells of breast tissue whether these cells are normal or malignant . 
at this stage of our knowledge , however , it is doubtful if it would be justifiable to use a higher dosage . the administration of an artificial oestrogen again modifies the endocrine status . 
the oestrogens may produce a direct effect on the breast cancer cells , or the effect may be obtained indirectly through the anterior pituitary . if the action is a direct one , the best results would be expected in older women . tn an older patient , for example a woman who is 5 or more years past the nmenopause , there is little or no circulating oestradiol , and the addition of oestrogens produces a change . after the passage of time , however , the body , and in particular the breast cells , may become readjusted , and this might explain why the good effect passes off . the work of zondek suggests that the oestrogens may have an indirect effect through the anterior pituitary . 
zondek ( 1936 ) found that the administration of large doses of oestrin produced a chromophobe adenoma of the pituitary in male rats , that is , in rats not normally subject to the action of large amounts of oestrogen . zondek ( 1947 ) also reports a case in which he gave large doses of oestradiol benzoate to a woman of 216 with metastases from a carcinoma breast . 
the increase in size of the pituitary was due to an eosinophil adenoma . unless the production of an adenoma of the pituitaryr interferes with the output of other hormones the explanation is not very satisfactory in older patients . in a premenopausal patient , a drug such as stilboestrol may cause inhibition of the gonadotrophic hormones , so suppressing natural ovarian secretion , but castration could not be said to be complete not bringing about a true castration . consewhile an artificial oestrogenic substance is circulating in the blood . quently , there is not the same profound change in the endocrine status and no useful therapeutic effect is produced . the question of the administration of testosterone might now be considered . in a premenopausal patient this brings about an artificial menopause and , from this point of view , testosterone might be expected to produce similar results to ovarian irradiation or o6phorectomy . in addition to this , the administration of testosterone must disturb the normal oestrogen - androgen ratio . finally , testosterone , like the oestrogens , causes inhibition of the gonadotrophic hormones of the anterior pituitary ( moore and price , 1932 )  . it may , therefore , like the oestrogens , produce an indirect effect on the breast cells by a disturbance of pituitary function . an explanation must now be sought for the failures . 
how this would affect the response is not known exactly , but it is perhaps significant that few patients with clinical evidence of liver involvement showed any response to any of the methods of treatment mentioned . there may , however , be other factors modifying the response . 
haddow , watkinson and paterson ( 1944 ) suggested that the tumour called " carcinoma of the breast " might in reality comprise walpole and paterson ( 1949 ) also made the same suggestion , several categories . even going so far as to say that a tumour might produce an " anti - hormone " or inhibitory factor antagonising the action of the oestrogen . 
of advanced breast carcinoma treated by hormone therapy has been surveyed and the results obtained have been presented . the findings suggest that , in premenopausal wonmen , the treatment of choice is ovarian irradiation , which was found to produce beneficial results in 30 per cent of patients in this category . in women who are 5 or more years past the menopause , oestrogen therapy would appear to be the most satisfactory form of treatment . the drug found to be most generally useful was stilboestrol dipropionate 5 mg . 
jackson memorial laboratory , bar harbor , maine . received for publication april 20 , 1954 in a comparative study of alkaline phosphatase in tumors , greenstein ( 1942 ) noted that the mouse tumours studied possessed little or no alkahne phosphatase activity with the exception of spontaneous mammary tumors and lymphomas . comparative q values were calculated as the ratio of the percentage of disodiumphenyl phosphate hydrolyzed to milligrams of total n per cubic centimeters of several concentrations of n were taken and the q values remained tissue extract . remarkably constant in relation to nitrogen . 
the highest value for any tumor was the transplanted hepatoma 31 in rats with a q value of ' 542 . kabat and furth ( i 941 ) could not demonstrate alkahne phosphatase by means of gomori procedure in mammary adenocarcinoma spontaneously arising in c3h however , hard , pratt - thomas and belkin ( 1948 ) examined twenty - nine stock . spontaneous mammary carcinomas in c3h , a and dba mice and noted high alkaline phosphatase activity . 
the predominent picture in the mouse tumours examined was the high alkahne phosphatase activity shown by the cells of the acinar units while the stroma and vascular elements were free . 
descenden ' t of a mouse that was bom from a fertilized ovum transplanted into the uterug of a dba mouse this has gone through 320 transplant and was nursed by her dba mother . results from the jackson laboratory indicates 100 per cent transgenerations . plantability in c57bi , and their f , hybrids . 
the histological type is an adenocarcinoma ( sneh , cloudman and woodworth , 1948 )  . the tumor h2712 originated spontaneously in the mammarv gland of ' a c3h mouse in 1948 at the roscoe b . 
the histological type - is an adenocarcinoma . the tumor dbrb originated in the department of genetics , carnegie institute of wasliington in 1918 in the mammary gland of a dba / i mouse and has gone throuo ' h 845 transplant generations . refjults from the roscoe b . 
the histological type is an adenocarcinoma ( strong and little , 1920 ; ' little and strong , 1924 )  . the tumor 15091a originated in the laboratory of the university of a - fichigan in the mammary gland of an a albino mouse in 1928 ( cloudman , 1928 )  . this has gone through 341 transplant generations and shows 100 per cent transplantability in a albino and their f , hybrids . 
the histological type now is that of an anaplastic carcinoma . for the purposes of the experiment the animals were divided into four serieg . series 1 consisted of tumor e0771 transplanted into males and females of c57bl and their f , hybrids between c57bl and a / l . series ii consisted of tumor h2712 transplanted into c3h males and females . series iii consisted of tumor dbrb transplanted into dba male and female . series iv consisted of tumor 15091a transplanted into f , hybrids of c57bl and a / he male and female . the total number of animals of the four series were i 00 and 8 1 . all mice were transplanted with their respective tumor at the roscoe b . 
jackson memorial laboratory and sacrificed at intervals between 7 and 22 days or an average of 16 davs . the am - mals were kired by ether anesthesia and thin slices of the ever , spleen , adrenals , kidney , testes , ovary and the tumor were placed immediately into formoltissues placed in fo ' rmol - saline were stained saline and into ice - cold acetone . by the regular hemotoxyliin and eosin methods . tissues placed in ice - cold acetone were left for 24 hours with one change of acetone . 
they were then placed in cold absolute alcohol over night followed by two changes of benzol of 45 minutes e " ach . they were then imbedded and infiltrated in paraffin at 56 ' c . 
they were then dehydrated and mounted in permount . incubation times in the substrate were as follows : series 1 , ily ill : i minute , 5 miinutes , 15 minutes and 1 hour , and series iv : i.minute , 5 minutes , 15 minutes , i hour , ij hours , 2 hours and 21 hours . 
a total of 1124 shdes was prepared . the shdes were read and visually graded according to their intensities from i to 6 . in this way a relative assay could be arrived at which showed a distribution within each incubation time , and the earliest reaction or end point could be ascercontrols were run . 
at i minute incubation time 29 of the 39 reactions had a reaction intensity of one and hence this was considered as the " end point . " at 5 minutes there were no negati ' ve reactions , the majority of reactions being two or three . 
the distribution was , however , strikingly different from the other tumors and will be referred to in the subsequent section . series iii consisted of 48 transplanted dbrb mammary carcinomas . 
l. - tumor e0771 transplanted into c57bl and f , hydribs ( series i )  . alkaline phosphatase reaction at 15 minutes ' incubation tixne slides show irregular areas of activity . 
3. - tumor dbrb transplanted into dba ( series iii )  . alkaline phosphatase reaction at 15 minutes ' incubation tixne shows a tendency of the peripheral cells of islands to show more activity than central portion . 
7. - tumor h2712 transplanted into c3h ( series ii ) under high power magnification . note the peculiar and intense distribution at the borders of cells lining the lumens . in the center is also seen gomori positive acerular material in a very small lumen . 
4 , 5 , 6 at 60 minutes incubation period . it is quite evident that 1.5091 a exhibits no alkaline phosphatase activity and further extension of the incubation time to 2 - 1 hours showed no reaction except in the vessels . in series i tumor e0771 is composed of irregular islands of tumor tissue . these varv in size and shape . 
the islands are fairly solid with the exception of small areas where itimens can be seen . larger iiimens are lined by several rows and layers of cells . most of ' the lumen8 are very small . 
8 shows a photoreticular material with many large thin - walled vessels . micrograph of this tumor stained by hemotoxyl - in and eosin showing the islands of the interstitiat tumor tissue with an attempt at luminal and tubular forma - tion . tissue seen to the left is loose and reticular in nature . there seem to be attempts to form tubular structures . the phosphatase reaction began during the first minute of incubation time with a blackening or graying of the cytoplasm . this gave a gray appearance to the slide except in such areas where the blackening is much more pronounced . 
at this time enzyme activity large irregular can be demonstrated in many areas in the centres of the island . clefts can be distinguished and stand out prominently bv the activity of the enzyme of the cells lining the lumens . intraluminal material which is not apparent in the hemotoxylin and eosin preparation shows intense activity . small globules are present in the cytoplasm of many cells adjacent to lumens or attempted lumen formation . 
the connective tissue and vessels do not react . tumor dbrb iin series iii under hemotoxylin and eosin stain shows a more lobular distribution than either e0771 or h2712 and is shown in fig . 
the interstitial tissue is composed of loose connective tissue , connective tissue and large thin - walled vessels . with graded gomori stains the reaction has an " end point " at 15 minutes . here the areas of activity are irregular thoughout the tumor . 
some activity is there is a tendency for present in the small lumens but this is not marked . increased activity at the periphery of the lobule as shown in fig . 
the loose supporting connective tissue and vessels show no activity . in series iv tumor 15091a differs somewhat from the other 3 in that it is a fairly solid tumor not composed of islands or lobules . there are numerous mitosis and the cells vary more in size and shape . 
at i hour this showed rather marked activity . the cells of the peripheral portion of the nodules showed most intense activity . there is no essential difference between the various series . the liver shows , for the most part , two types of reaction . 
the ovary begins in most series with the 5 minute incubation time in the theca externa which shows progressive activity to i hour . ksmall stromal vessels also show activity . all the series were approximately the same . 
one difference that occurs in the e0771 is the presence of doubl - e layers of tumor cells in the attempt at acinar formation . in the 15091 a the tumor is solid with very few smar acini . the overall phosphatase reaction was very intense in e07 7 1 , very much less in this was evident in spite of the fact that h2712 and bdrb and none in 15091a . the rate of growth of the tumor was about the same in all the series . 
numerous hard , pratt - thoma 's and belkin ( 1948 ) mitoses were present in all the tumours . felt that there was a loss of alkahne phosphatase activity in the more anaplastic although it would seem in our series that the anaplastic tumor tumor cells . 15091a having no reaction would point toward such contention , the e0771 is a very rapidly growing tumor with numerous mitoses . 
we would prefer to delay our judgment on this issue until a larger variety and strains of tumors have been studied . an interesting and significant paper which has come to our attention as this work was completed was the observation of shelton ( 1952 )  . this investigator found a tumor in a 6 - month - old strain a female mouse that had been exposed to 400 r whole body x - radiation on the date of birth . 
lymphoma 1 was propagated in a mice from a large tumor mass located in the anterior mediastinum . lymphoma 2 was propagated by subcutaneous injection into strain a mice of fragments of lymph nodes from a mouse bearing the original tumor . strain i grew as a localized tumor with local invasion ' but no metastasis , but strain 2 reacted as an acute leukemia with rapid growth infiltration of the organs and high peripheral count . in determination of the alkahne phosphatase activity it was found the lymphoma 1 showed no reaction while lymphoma 2 showed a reaction as expressed by 5 mg . 
no detectable reaction could be seen by the gomori method in lymphoma i whereas lymphoma 2 was here we have a rapidly growing persumably anaplastic tumor strongly positive . showing an increase in alkahne phosphatase reaction . in our laboratory we have also determined the alkahne phosphatase activity as expressed in gamma of phosphorus per mflhgram of nitrogen at 30 minutes incubation time in a large series of these 4 mammary tumours . 
carr. from the poultry re8earch centre , edinburgh , 9 . recieved for publication january 19 , 1953 . most cancer investigations involve the use of small rodents , and it is now clear that many of the " generalisations " obtained from research on this material break down when other animals are considered . while the relevance of any result obtained with grafted tumours to the problem of spontanous cancer may be doubted , it seems desirable to report the results obtained by parallel work on non - rodent material in order to offer proof that similar results are , or are not obtained . the grch / 15 tumour is a non - filterable fibro - sarcoma originated by peacock in 1939 ( peacock , 1946 ) , and by his courtesy made available to other workers it is through the animal breeding unit of the british empire cancer campaign . readily distinguished from the virus - induced tumours by the absence of the mucoid material that they all produce , but unless the section is specially stained for this , microscopic differentiation is less easy . transplantation was always made into brown leghorns of greenwood 's flock ( in one of which the original was induced ) , either by implanting small pieces with a fine trocar and cannula , or injection by syringe of the suspended material left after shaking minced tumour or tumour ground with sand and saline . transplants were usually made into each pectoral muscle , and by the trocar and cannula method " one - sided " negatives occurred about 1 in 20 times , while the second gave less than 1 per 100 " one - sided " failures ; the incorrect negatives due to experimental errors would therefore be 1 in 400 and 1 in 10 , 000 respectively , which can be ignored . 
the material for this account is based upon 60 transplant generations involving some 600 birds over a period of 8 years . in most experiments the birds were 6 to 10 weeks old when the tumour was implanted . in the bird the tumour appears as a hard white lump , though after the alteration in the growth rate the texture was softer , and microscopically the cells appeared rather more compact . 
983 , from which all material for the 32nd and subsequent generations derived . it is interesting that the change coincides with a distinct alteration in the conditions of husbandry , for the facilities for keeping animals at the experiment station were at first not optimuno cages were available for birds , and the food comprised various ration - free products , which produced a series of deficiency diseases ranging from protein to multiple vitamin shortages . 
when the conditions were built up to those nearer first - class management the change in appearance and behaviour of the tumour remained . there is no direct evidence that the change in conditions caused the alteration of the tumour , and it remains possible that it was a coincidence . the most notable change in the tumour was in the rate of growth . 
by contrast , in only one bird out of many thousands has this organ been the site of a rous 1 metastasis . of 352 birds killed and examined with large growths in the breast muscle , 51 had metastic growths in one or more sites . 
nor is it due the conditions for tissue transto a failure of the bird to produce iso - antigens . plantation , however , seem to differ markedly from those of the lower rodents . for example , organ transplantation between fowl varieties is a normal experimental technique , used for example by greenwood ( 1928 ) to investigate the relation of the testis to henny feathering of males of some breeds ; yet transplantation of an adrenal is regarded as a novel event in the mammalian field similarly , the elaborate integumental decorations of birds early ( darcy , 1952 )  . attracted the attention of zoologists , and the results of skin transplant between different genera were being embodied into the general assembly of zoological theory before tissue iso - antigens were recognised . it is possible to suspect that a similar mechanism is indeed operative in the mammalian field , but the methods used are not refined enough to detect it . 
the domesticated rat and mouse have an oestrous cycle of only a few days in length , as compared with the sharply - defined annual cycle in the hen , and lesser cyclic variation of the cock . 
even the pregnancy of 3 weeks is rather short for observations in the rat and mouse , but it is significant that foulds ( 1949 ) found some evidence of a variation in tumour growth and structure in the mouse , and there are many reports of pregnancy influencing the course of tumours in the human . the appearance of many cancers at an age when the sexual functions are failing may be another aspect of this . this would seem to be a point of considerable importance , for it indicates that control of a malignant tumour would be possible within the limits of normal physiological variations ; similarly studies on the cancers induced by chemicals , and more especially by x - rays , indicate that the production of the malignant change and the appearance of a progressive tumour are distinct phenonema , a separation that is even more obvious with the mouse milk factor , where infection takes place soon after birth but the tumour appears months later , and only if the endocrine history of the host has been suitable . 
carr aries in the proventriculus in older birds carrying the " slow " tumour refers to a difference in the biochemical suitability of the organ in the older hosts is it is to be expected that the differences of tissue permeability with unknown . age ( duran - reynals , 1942 ) would also have some influence upon the metastatic growths . the absence of any neutralising action in the sera of fowls immunised against the grch / 15 sarcoma is in contrast to the findings of gottschalk ( 1943 ) for sarcoma 16 . but it is well known for commercial birds suffering from the related " leukosis complex " viruses to have death - rates of up to 40 per cent and carrier rates of nearly 100 per cent , so that such a finding is only of value if it can be stated that such carriers are not a complicating factor . 
a fast - growing mutant strain retained this property , and the mutation was not associated with an increased frequency of takes , though the distribution of secondaries was altered . 
clemmesen. from the danish cancer registry under the national anti - cancer league , copenhagen . received for publication december 10 , 1949 . developments in recent decades have made the term " cancer statistics " inadequate for the description of the ways in which statistics have contributed it is time that this term was reserved for statistical treatment to cancer research . of results , while statistical research into the occurrence of human cancer , be it from racial , genetical , geographical , occupational or other sociological points of view , should be included in the term " cancer demography . " this will be the case in the following . unlike many other branches of science joining in the research of cancer at some stage of its development , cancer sociology can point to a long , though most often neglected , history . 
when percivall pott , in 1775 , described the chimney sweep 's cancer , not only giving its clinical features , its treatment , and its social aetiology , but also taking active steps toward its prevention , in which we have finally succeeded , it was the first step in cancer sociology . pott 's observation of the latent period of scrotal cancer in chimney sweeps contained a very obvious clue to the experimental production of cancer , but experimental workers seem to have been too fascinated by experimental and pathological problems to take a lesson , not to speak of inspiration , from clinical or sociological observations , and 140 years passed before cancer was provoked experimentally by tar and soot . however , it seeins still more surprising that after this experience we have spent decades since young and russell 's demonstration in 1927 of a causal relationship between alcoholic consumption and cancer of the upper digestive tract in examining the carcinogenicity of numerous other compounds , while alcohol has been examnined only in a single experiment on a small scale ( krebs , 1928 )  . similarly it is well known that the first birth increases the possibility of a later cervical cancer , but where are experiments based on this and similar facts ? it would not seem unlikely that the slow progress in obtaining results of practical value in the fight against cancer is to a large extent due to our theoretical way of tackling problems . for instance , it is not unlikely that the future will reveal very interesting facts with regard to the influence of cosmic radiation on the incidence of cancer in heavily inbred animals of some kind , but there is considerable evidence that unless the energy released by such radiation shows a j . 
clemmesen different affinity to individuals of the various social classes , there must be social factors of a far more decisive character influencing the incidence of cancer in man . in order to bring about a closer contact between cancer research and problems of clinical importance , a more thorough investigation into the aetiology of cancer should be attempted by cancer demography through mass observations . 
to this we may add a frequent tendency among the former to take a more national and less universal point of view than is common so far in the medical world . 
an example of this is the variation in the proportion of gastric cancer in different countries , which has been discussed more often than the possible sources of error involved . for instance a first - class textbook of pathology states : " the incidence of gastric cancer in sweden and czechoslovakia apparently is about three times that of england . 
no satisfactory explanation has been offered for these interesting findings " ( anderson , 1948 ) as far as the present author has been able to establish , the reason for some of the differences may be that death certificates in the thinly populated areas of northern sweden for practical reasons are issued by parsons . furthermore , it will appear from the demands already mentioned that a direct comparison of percentages without a further analysis with regard to age distribution is unsatisfactory , especially in the case of a site such as the stomach , in which the establishment of a definite diagnosis is difficult . admittedly , death certificates have in the past rendered valuable information , especially in the hands of the english registrar - general , but this is no guarantee that death certificates from any country will produce similarly reliable details . it is vital to the use of death certificates that they are referred to the domicile of the patient , and not to the locality where death has occurred . 
and it must also be remembered that occupational statistics based on death certificates theoretically demand that the occupation of the deceased person should be stated from the same source as in the census , that is , some registration office , and not from the case - record or the statement of relatives , although tlis might not cause serious errors . comparisons of death certificates of different date are often made without the necessary regard to the dangers of this method . pedigrees of greater length may for instance be misleading because the frequency of cancers of various sites , whether apparent or real , will differ from one generation to the next . for sites of cancer where therapy is improving death certificates are decreasing in value as indicators of frequency , while other sites may show an apparent increase in frequency due to progress in diagnostic technique , and post - mortem figures will also be subject to this influence . 
heady and kennaway ( 1949 ) suggest that the proportional occurrence of lung cancer among the post - mortem total will serve as an indicator of changes in the incidence among the whole population . such figures are , however , influenced not only by the experience and information available to pathologists , but also through progress in the therapy of early complicating pneumonias and of lung cancer itself . * it is generally assumed that progress in diagnostic technique will cause an increase in the number of cancer cases registered , but according to the author 's experience gastric cancer tends to be overestimated , and will often decrease in frequency with improvements in medical facilities . consequently , comparisons between different countries or periods based on death certificates as well as on other sources of medical information demand additional knowledge of a more direct character . * readers of their review are recommended to compare the order of magnitude of the yearly totals criticized with those accepted as a basis for considerations , j . 
clemmesen for years hence , however , death certificates are bound to play an important part in demography of cancer , because they will be the best information available for the many cases not treated in hospital . 
even in the case of compulsory registration there will remain a number of notifications from general practitioners amounting in quality to little more than death certificates . it is also important to.the statistician to realize that the difference in quality of diagnosis between hospitals and general practice will cause a difference in the material , unless practically all cases are examined in hospitals , and this will be the case for only another difference between these groups will appear very few sites of cancer . in the age distribution curves , which must be based on the age at onset of the cancer , theoretically at the onset of symptomns . accurate statements on this item are unfortunately very difficult to obtain on death certificates , and for hospitals it is most practical to state the date of the first admission , which has the advantage of being an accurate reality . for all these reasons it will be evident that complete statistics on the incidence of cancer in a given area should contain figures for the number of cases treated in hospital for each site of cancer , and separate age distribution figures for this it is not uncommon for authors part of the material as well as for the total . dealing with a single site of cancer to leave out the total cancer incidence in the population concerned , but this is not permissible when we do not know the interrelations between cancers of different sites . 
the percentage of cases examined histologically or by autopsy should also be given for each site as an indicator of the basis of the diagnosis , and is particularly valuable in statistics giving figures for diagnoses of a histological character . 
at the moment such figures can only comprise a part of the cases really occurring , and it is most important to know the actual number of microscopical examinations from which they are collected , even in the case of a diagnosis such as " sarcoma , " which sometimes may be founded only on a macroscopical examination . unfortunately , many publications on cancer demography are inadequate with regard to statistics , and amount to little more than a summary of the author 's own conclusions , although they should contain all the basal figures for both the normal and the cancer populations , as well as the formulas applied , not only with regard to an independent judgment by contemporary workers , but also with a probably it is mostly view to retrospective work in the same field in future . editors and not authors who are to blame for such omissions , and the same applies to diagrams , which are as essential to such articles as are photomicrographs to histological or roentgenograms to radiological publications . cost will probably also be blamed for the fact that most of the valuable material on the occurrence of cancer is hidden away in statements from public health authorities . 
a unification of publications in this field , so that they could be collected in journals accessible to workers in experimental cancer research , would contribute far beyond its cost to the vital connection between experimental research and cancer demography . but it is most important in such publications that the author ascribes his findings to factors of some established certainty , and is reticent in advancing readers may find , even in new theories of his own on the aetiology of cancer . modern publications of some standing , that the well - established connection of alcoholic consumption with the incidence of certain cancers is paralleled by the influence of tinned or fried food . 
leveaux. * from the westminster hospital , london , s.w.1. received for publication may 12 , 1954 . the earliest recorded description of carcinoma of the pancreas is attributed to mondiere in 1836 . 
da costa ( 1858 ) of philadelphia reviewed 37 cases and gave a comprehensive description of the clinical presentation of this disease with * nmphasis on pain as a prominent sympton in 32 of these patients . 
in not a few cases it is increased by the erect position and hence we find patients seeking relief by stooping and curving their body forward so as to relax the abdominal parietes . " physicians of the french school sustained their interest in this condition , and bard and pic in 1888 observed the common association of a palpable gall bladder with jaundice , and the frequent incidence of cachexia . 
from the descriptions given by these and other observers the concept of painless jaundice as the classical form of presentation of this disease spread widely . mirallie ( 1893 ) found glycosuria in 3 cases , and chauffard ( 1908 ) emphasized distinctive features of growths involving the body of the gland . speed ( 1920 ) , eusterman ( 1922 ) , eusterman and wilbur ( 1933 ) , kiefer ( 1927 ) and other observers have subsequently reported on series of cases , and berk ( 1941 ) has included these in his review of a total of 1449 cases . 
levy and lichtman ( 1940 ) , and duff ( 1939 ) have further clarified the picture of carcinoma of the body and tail of the pancreas , emphasizing early and severe pain , ascites , venous thrombosis , and widespread metastases to liver and peritoneum as important features . towards the end of the last century surgeons became interested in the pancreas , and finney ( 1910 ) quotes a case of total pancreatectomy for carcinoma by billroth in 1884 with recovery of the patient from the operation . gordon - taylor ( 1934 ) successfully removed a carcinoma of the body of the pancreas in 1927 , his patient surviving when the case was reported 7 years later . in spite of such isolated successes , however , attempts at surgical removal gave little promise until , in 1935 , whipple and his colleagues demonstrated the technical feasibility of resection of the head of the gland , and opened a more hopeful era ( whipple , parsons and mullins , 1935 ; whipple , 1938 )  . these and subsequent advances in surgery have greatly intensified the need for early diagnosis in a disease which has been estimated to comprise 1 to 2 per cent of all cancers , and which is by no means a clinical rarity . 
the head was spared in 14 cases ( 30.4 per cent ) , of which 4 cases involved the body alone , 4 cases the tail alone , and 6 cases both regions . 
one case had active thrombo - phlebitis , and 1 presented with paraplegia due to extradural metastases . in many cases the disease was well - advanced by the time that abnormal signs were clear cut . 
per cent in 20 of 40 cases ( 50 per of 8 cases examined by the glucose tolerance test , a diabetic type curve cent )  . was obtained in 6 cases ( 75 per cent )  . these latter cases included 2 of the known cases of diabetes of recent onset , but none of long standing . 
n / 20 sodium hydroxide as the upper limit of normality . johnson and bockus ( 1940 ) reported a raised value in 5 of 8 cases . secretin stimulation of the pancreas was performed in 3 cases , accentuating an already raised level in 2 cases , and producing an abnormal value in 1 . 
per cent in 27 of 33 cases investigated , of which 24 had clinical jaundice . the serum alkaline phosphatase was examined in 26 cases and was raised above 5 bodansky units in 13 , of which all had associated hyperbilirubinaemia . of 13 cases shown to have liver metastases , the alkaline phosphatase was raised in 7 and normal in 6 . the serum cholesterol was raised above 220 mg . 
a normal chloesterol level occurred in 5 cases with associated hyperbilirubinaemia . the cholesterolesters exceeded 50 per cent of the total cholesterol in 20 of 21 cases . it appears , therefore , that these latter three investigations reflect only the presence of biliary obstruction , and beyond this are not helpful in the present diagnostic problem . the plasma proteins were normal in 23 of 25 cases , there being some degree of hypoalbuminaemia in the remaining 2 cases . the cephalin , thymol and colloidal gold flocculation and turbidity tests were normal in all of 22 cases examined . in 3 cases the cephalin flocculations test became positive after cholecysto - enterostomy . the value of these tests in the present connection is that they facilitate exclusion of hepatitis in cases presenting with jaundice , as pointed out by maclagan ( 1947 ) of 46 cases examined , 31 ( 67 - 4 per cent ) had haemoglobin levels below 13 - 0 g . per cent , with an average of 12.13 ( 76 per cent hb . ) for the whole series . 
a relationship between the anatomical position of the tumour and the site of the pain complained of by the patient is suggested . a scheme of investigation is proposed which includes careful radiological studies , a glucose tolerance test , serum lipase and amylase determinations and a bromsulphalein retention test . surgical exploration is advocated where strong clinical grounds alone exist . i wish to express my warm thanks to h . 
bockus , professor of gastroenterology in the graduate hospital of the university of pennsylvania , for all his help and encouragement in this investigation . i am also grateful to a . 
harnett. published for the clinical cancer research committee of the british empire cancer campaign . received for publication july 16 , 1948 . in 1938 - 39 the clinical cancer research committee of the british empire cancer campaign carried out a clinical survey of all cases of cancer seen in the hospitals , both voluntary and l.c.c. 
in the administrative county of london . this was done by means of questionnaires , one of which was filled in by the registrars in the various hospitals for each patient and returned to the clinical cancer research committee for record . 
the work was interrupted by the outbreak of war after it had been in progress for 17 months . by this time 15 , 200 cases of cancer had been registered , of which 2529 ( 16 * 6 per cent ) were cancer of the breast . these patients have now been followed up for five years or more and the records analysed . 
4 - 556 1 8 + 0 66 . the mean age is 5 - 6 years higher than those given by lane - claypon ( 1926 ) and wainwright ( 1931 ) , due to the fact that the two latter series include only cases coming to hospital for operation , whereas the b.e.c.c. 
the youngest patient in the series was aged 15 , and survived 5 years after enucleation of a tumour of 3 years ' standing , which proved on histological examination to be an early adenocarcinoma . 
expected actual per cent of expected possible x - rays alone or with interstitial radium . number of cases of known duration mean number of months lived j in 5 years from onset maximum  . 
expected lactual possible per cent of expected x - rays with local surgery . number of cases of known duration mean number of months lived in 5 years from onset maximum  . 
it was found that patients in stage iv who were not treated either by surgery or radiotherapy had a 5 - year expectation of life of 46 - 7 per cent of normal , which was not improved by palliative x - ray treatment . all these figures must be considered in the light of the fact that the mean duration of the disease is 38.3 months ( major greenwood , 1926 ) , so that these patients whose mean age was 57 years would have a 5 - year expectation of life of about 56 months ; a 5 - year follow - up is therefore insufficient for assessment of the results of treatment . 
the variations in mean age of the patients submitted to various types of treatment is shown below , and indicates that the more radical methods were used in the younger patients . radiotherapy mean age of 703 patients treated by radical mastectomy alone mean age of 133 patients treated by local mastectomy alone radiotherapy mean age of 174 patients treated by radium alone or with local surgery h.v. 
fodden. from the department of pathology , university of liverpool , and the liverpool cancer control organization . received for publication august 20 , 1948 . in his ' textbook of pathological anatomy ' rokitansky ( 1861 ) made his classical statement that pyloric cancer was exactly bounded by the pyloric ring , and that the growth never reached beyond into the duodenufrom this time it appears that the majority of observers , with the early exception of brinton ( 1864 ) , commented upon the integrity of the duodenum in cases of carcinoma of the stomach . 
many well - known surgical teachers spoke of the habitual immunity of the duodenum from invasion . kocher ( 1893 ) , mikulicz ( 1898 ) and most ( 1899 ) believed it to be always constant . kocher ventured that it was a problem of extreme interest to consider why gastric carcinoma grows in almost all cases towards the cardia , yet stops , on the contrary , at the duodenopyloric junction . it was brinton who first took especial exception to this proposition of rokitansky . 
meredith. from the christie hospital and holt radiumt institute , manhester . received for publication december 12 , 1948 . ix a series of papers ( dale , 1940 ; dale , meredith and tweedie , 1943 ) it has been shown that the action of x - rays on aqueous solutions of biologically active compounds was indirect , i.e. 
further , it has been shown that in consequence of this indirect action , if two solutes are present , the second solute ( the protector ) reduces the radiation effect on the first one ( the indicator ) ( dale , 1942 ; 1943 ) , by sharing the intermediate product formed from water by the radiation . 
meredith enon revealed certain unexpected quantitative relationships between the protective power of a substance and its concentration , which will be described and discussed in this paper , together with the closely linked ionic yield of the indicators used , the knowledge of which is the necessary basis for the quantitative treatment of the experimental results . 
the fact that these new results were derived from experiments with two indicators of very different molecular weight , for an extended range of protective substances added to both indicators , helps to build up a more complete picture than has been obtained hitherto . 
 ( average a 130xu ) from a 250 kv tube . whenever possible , the solutions , contained in pyrex glass tubes , were spaced at such distances from the target that each received the desired dose during the same overall treatment time , care being taken to avoid shielding of one tube by another . this ensures that the relative doses received by different tubes are not affected by unavoidable fluctuations in the tube output . 
the stock solution of enzyme and the solutions of protective substances were made up with water , glass re - distilled from alkaline potassium permanganate , and the ph adjusted so that a sample reacted pink to phenolphthalein . all necessary dilutions were carried out with glass re - distilled water adjusted to the same ph by the addition of 1 ml . 
were performed during this series of experiments , these arise because very high and some special difficulties were encountered . doses of radiation ( one to two million roentgens ) are required to achieve a reasonable degree of inactivation in solutions with concentrations of the order there is a strong tendenvcy of re - crystallization during of 10 per cent or more . the long exposure to radiation , which makes it almost impossible to keep the enzyme in solution at a ph compatible with its stability . however , it was found that a 6 per cent solution could be maintained for most of the time by stepwise addition of naoh . 
the concentration reached by this method was derived from the known concentration of the suspension by measuring the height of the coluimn of crystals after , and the height of the suspension before centrifugation . 
compound ( warburg and christian , 1938 )  . since various preparations ofthe specific protein of the amino acid oxidase differ in their activity , and even any one preparation gradually loses activity when kept for some weeks , the oxygen uptake with a known amount of d.n. 
1. these results may also be presented , as shown in a previous paper ( dale , gray and meredith , 1949 ) , by the formula : d5 - 2 x 107  . 
calculated by lea ( 1946 ) from earlier experiments by one of us is much lower than the value obtained in the horizontal part of the curve , and this is due to the fact that the concentration then used was of the order of ~~~i ~~~~~~~ii conc . 
for the sake of accuracy the degree of inactivation should be kept between the limits of 25 - 75 per cent . in a previous report ( dale , 1947 ) the value of q was found from the protection afforded by generally lo100 g . 
q was roughly constant , and it was concluded that the protective power per molecule is proportional to the molecular weight - perhaps not a surprising result , since all these substances have roughly the same average composition . it is only when low molecular weight substances of special chemical configuration are examined that remarkable differences are found . oxalic acid , which was earlier ( dale , 1942 ) found to be a poor protector of d.n. , also proved to be a poor protector in the case of c.p. , but if the c - c bond in oxalic acid is broken and the free valency of the carboxylic group linked to an h atom , thus forming formic acid , the protective power is raised about 200 times , and similarly , if w . 
our arguments , however , are not dependent upon its correctness , nor would they be invalidated by its disproof . lea , smith , holmes and markham ( 1944 ) pointed out that , in addition to reacting with solute molecules , radicals may also recombine with one another , having a mean life , as free radicals , of r sees . 
 ( 1944 ) also suggested a modification of equation ( 3 ) to cover the protection effect on the assumption that the indicator and protector molecules " share " the available radicals . 
thus although the protector molecule has removed a radical from the solution , it can " hand on " the effect of the radical to the indicator molecule , and thus fails to fulfil its since we have no clue to the nature of this change function as a protector . we will call it " activation , " and it might be thought of as a sort of meta - stable state . 
and the collision frequency between " activated " protector molecules and indicator molecules , 1 , m and pi have already been defined , whilst pa is the probability that a collision between a radical and a protector molecule destroys the former and activates the latter . 
ps is the probability of an indicator molecule being inactivated in a collision with an " activated " protector molecule . from equations ( 9 ) and ( 4 ) we findao + m rt [ ( ao + t ) pi ( ao + m ) pap . 
i ~~ ( ao + t ) p , + lp , apz~l ( 10 ) some of the constants appearing in equation ( 10 ) can be evaluated from results obtained with solutions of the indicator alone , whilst the others can be evaluated from any two experimental values of q for known protector concentrations . 
the values of ( p ) are indications of the protective power per molecule of each substance for the particular indicator , and the values given show the same tendency as was shown previously for protective power per unit weight by molecules with approximately the same atomic constitution have dale ( 1947 )  . approximately equal protective powers , whilst marked differences occur when special groupings are present . from the results it is also possible to calculate the relative values of pr for d.n. 
kerrich from the radiation therapy department , johannesburg general hospital , and the department of mathematics , u niversity of the witwatersrand . received for publication april 20 , 1951 . in a previous paper the writer ( cohen , 1949 ) proposed a biological dosage unit , the " roentgen equivalent clinical " ( rec ) based on an empirical formula d = e.a.tn.l - q , relating the physical dose in roentgens ( d ) to the biological dose in rec ( e ) , taking into account the relative biological efficiency of radiation of various qualities ( a ) , the over - all time in days ( t ) , and the field size in decimetres ( l )  . 
the physical and biological doses could be equated when the specific ion - density is minimal ( a = 1 ) , the treatment is completed in 1 day ( t = 1 ) , and the field diameter is 1 d ( l = 1 )  . 
cohen in diametercan the hypothesis of a " diffusible substance " ( grynkraut and sitkowski , 1936 ; jolles , 1950 ) , for diminishing the treated field by a given factor , or increasing the time interval by the same factor , would allow the same proportion of the " substance " to diffuse across the surface bounding the treated volume . 
the difference between the 220 kv and 180 kv ranges is statistically significant ( p 0 - 01 ) , but of minor importance clinically , so that for practical purposes the two high - voltage qualities may be grouped together taking a as 0 - 7 . 
2 illustrates the variation in biological efficacy with halfvalue layer , and shows the agreement between our estimates and similar data derived in a different manner by maccomb and quimby ( 1937 ) and by mackee and cipollaro ( 1940 )  . each point represents the median erythema dose ( a x 1000 ) estimated from table ii , the vertical lines delimit the 95 per cent confidence range , and the lettering refers to data by the authors quoted . in comparing these results with other published data , it is necessary to note inevitable differences in the definition of the erythema dose . 
from the method we have used the standard erythema dose is that which , delivered to a 10 cm . field in one sitting , produces a " moderate " erythema in 50 per cent of the treated cases . 
with the same dose , however , some slight reddening would be visible in considerably more than half ( probably 90 per cent ) of the treated cases . since this type of " barely perceptible reaction " is a convenient and objective end - point much favoured in erythema studies , there is obviously need for care in comparing such results . it is also necessary to distinguish the transient reddening ( fruiherythems ) appearing within 24 hours of the delivery of quite small doses ( helmke , 1949 ) from the main reaction ( haupterythems ) , which reaches its maximum about three weeks after treatment . for example , the threshold erythema dose ( ted ) is defined ( maccomb and quimby , 1937 ) as that quantity of radiation giving a faint reddening of the skin , visible in two to four weeks , in 80 per cent of treated cases . it would , therefore , differ little from our standard erythema ; hence the fair agreement between our results and the threshold erythema data in fig . 
the customary five - year - cure policy could not be applied since estimates of depth - doses from treatment records prior to 1945 are not sufficiently accurate it was decided instead to use , as a standard criterion , freedom for our purpose . from perceptible recrudescence for not less than three years . the records of 100 suitable cases treated between 1946 and 1948 , and followed with periodic examinations through 1950 , were abstracted and the data collected in table iii . the cases are arranged in order of increasing biological dosage ( rec )  . the dosage data shown are the true mimimum tumour doses , and differed , especially with superficial therapy , from the nominally given doses . in the case of very superficial tumours treated with lightly filtered radiation the significant point was taken to be 5 mbelow the edge of the treated field . with 2 ma1 filter this point receives about 85 per cent of the given dose ; and with the chaoul apparatus , only 67 per cent . with deeper growths treated at higher voltages , standard depth - dose tables were used to assess the tumour dose ; while with radium applicators and implants dosage was calculated on the paterson - parker system . since various qualities of radiation were used , it was necessary to correct the dosage by a factor ( rbe ) which , taking that for y - rays as unity , becomes 0.5 for superficial therapy , and0.7 for the 200 kv range ( tableii )  . in the case of deep - seated tumours treated with 200 kv radiation , the half - value layer ( and hence the rbe ) in the tissues differs from that of the incident beam on account of the increase in the average wave - length brought about by the compton recoil process . this effect is the more marked the harder the incident radiation . 
3 each 188 case site . face orbit neck node larynx back tongue pharynx neck nosoe alveolus neck face scalp larynx tongue , base pharynx orbit face temple post - cricoid  . 
0.5 , dose * ( r )  . 3200 2300 2250 3300 3150 2000 3800 3500 3000 1400 2000 3000 3150 3000 3300 4400 3600 5000 3750 2250 3300  . 
4 illustrates a method for determining the recovery exponent various values of n ranging from zero to 0.5 were ( n ) by a graphical solution . selected , and the resultant estimates of biological dose tabulated in a manner field diameter ( cm . ) therapeutic ratio skin tolerance dose 5000 median cancerocidal dose 4500 time days 1204 a% t% % - iuuut 9000 8000 - 7000 - 6000 - 5000 4000 - 3000 - 2000 1j4mi iolvv 40003500300025002000fig . 
a single line and volume factors . across the diagram gives all the relevant factors simultaneously . similar to table iii but regrouped accordingly . the cure rates so obtained were similarly charted in lognormal probability graphs , and the values of the standard deviation factor ( f ) corresponding to each value of n was determined . it is seen that f is a minimum as n approximates 0 - 25 . 
by means of the method of least squares , estimates of a and n for six different qualities of radiation were obtained , and shown to be in good agreement with previously established data . 
the uncertainty factor in dosage estimated on this basis ranged from 1.21 to 1 - 35 . a further 100 cases of epidermoid cancer were similarly analysed in order to determine the dependence of curability on the biological dose . 
the median lethal dose for this tumour ( 3000 rec ) and its uncertainty factor ( 1.3 ) were determined , and it was shown that , under optimal conditions of time and area , 95 per cent of tumours could be cured with a dose exceeding the median lethal dose by it has also been demonstrated that the prognosis is a mathematical 30 per cent . function of the therapeutic ratio . the author wishes to acknowledge the valuable advice given by j . 
kerrich. from the data in table iii cohen suggests that biological dose = dose / rbe x tn , or x = z / tn say , where xbiological dose , zdose / rbe , t = time in days . 
he goes on to show that if x is the exact dose necessary to effect a cure , then a reasonable assumption is that log x = log z n log t is normally distributed . writing x = log x , z = log z , t = log t this hypothesis becomes x = z nt is normally distributed with , say , mean oc and variance a2 . by a neat graphical method he proceeds to estimate ac , n and a . 
cohen the sizes of the estimated standard deviations show that none of the constants can claim to be very " well determined . " it must , however , be remembered that we are not dealing with a planned experiment , but that the author had to use the data that were to hand . 
as a suggestion to future workers in this field , more observations for small values of t ( say t 0 ) and large values of t ( say t - 1.5 ) would help greatly in fixing c and n more accurately . in more detail , the estimated covariance matrix is var . 
sylvin. fromn the department of radio - pathology , radiumnhemmet , stockholmi , swveden . received for publicationi february 11 , 1947 . our defective knowledge of the biological factors responsible for infiltrative growth is partly due to lack of sufficient information on the biology of the stroma connective tissue in malignant tumours . thus , growing carcinoma vegetations induce different complicated reactions on the part of the surrounding connective tissue , e.g. 
fibroblast proliferation and various inflammatory reactions . in the course of the stroma investigations by the writer , special attention has been paid to the transformation ( " dedifferentiation " ) of the connective tissue preceding the actual infiltration by cancer cells and to the simultaneous appearance of high molecular ester sulphates of unknown composition ( sylvan , 1938 , 1941 , 1945 and unpublished data . ) interpretation of the events taking place in the stroma is rendered almost impossible by the multiplicity of intermingling reactions . 
on this account it was decided to treat each problem separately . consequently , i thought it advisable to study these changes in cartilage , a tissue characterized by very slow reparative and reactive capacities . thus , the actual question was to investigate the morphology and the histochemical changes of normal cartilage when infiltrated by carcinoma cells . 
 - in this way comnparisons could be made with the stroma reactions of the different tissues , and with the alterations in the chondroitin - sulphate of cartilage and the unknown stroma ' ester sulphate mentioned above . 
by selecting suitable cases it was felt that the source of error caused by the connective tissue stroma reaction accompanying cancer vegetations growing from outside the perichondrium , could be avoided . for the sake of brevity , the readers are referred to current literature regarding the normal morphology of cartilage ( maximow and bloom , 1938 )  . 
the resulting newly formed , growing , connective tissue would then " penetrate " into the ground substance of cartilage , which was " dissolved " and replaced by the tissue mentioned above . after this , the cancer cells were believed to grow into this connective tissue , thereby seemingly infiltrating the cartilaginous tissue proper . ribbert emphatically denied that cancer cells ever could infiltrate cartilage directly , the process instead being mediated by connective tissue . 
as to the operating forces ribbert stated that "  . epithelien ( cancerous ) haben nicht die fdhigkeit anders als durch den wachstumsdruck auf ihn ( cartilage ) einzuwirken . " such a restricted mechanical interpretation involving pressure for the explanation of infiltrative growth , is nowadays of little value ( sylv6n , 1945 )  . oestreich ( 1910 ) suggested that the chemical composition of cartilage , mainly the chondroitin - sulphate , would be injurious to the cancer cells and consequently infiltrative growth would become impeded by this substance . since ribbert 's review in 1911 no special articles have been published regarding these questions . 
the statements of ribbert demand a survey of the cooperating factors active in the destruction of cartilage by carcinomatous tumours . the following possible factors have to be considered : 1 . 
the cancer cells might by direct action be destructive . in order to obtain a satisfactory understanding of the morphological factors unfortunately , they should be interpreted in terms of biochemical principles . for lack of complete data the chemical principles can only be touched upon . cartilage has a strongly acid intercellular matrix , due to the presence of large amounts of chondroitin - sulphuric acid . this substance is very sensitive to even slight changes in ph towards the alkaline side ( blix and snellman , 1945 )  . such ph variations cause a rapid depolymerization and disintegration of the long chain molecule . 
a shift in ph resulting in a disappearance of chondroitin - sulphate has recently been suggested to occur in inflammatory lesions of cartilage ( sylven , in press )  . 
when considering the complexity of the problem , i have to admit that no final conclusions regarding the biochemical nature of the operating factors can be drawn from the material presented here . 
some results , however , seem to be conclusive . some information concerning the mutual relationship between cancer cells and the perichondral connective tissue in the absence of any intervening inflammatory stroma reaction was obtained from case 2 , ii . 
the carcinoma strands in question were growing in the perichondrium , which was transformed into a loose metachromatic stroma connective tissue similar to the one met with in most carcinomas ( sylven , 1938 , 1945 and unpublished data . ) in the superficial part of the underlying cartilage a moderate decrease in the content of chondroitin - sulphate was demonstrated , the cartilage in other respects apparently being normal . 
sylvien actual cartilage destruction was seen to be effected either by the sole inflammatory stroma reaction ( case 1 ) or by the combined stroma and cancer strands ( case 2 )  . in the cases under consideration , that part of cartilage just being infiltrated was always devoid of the typical ester sulphate . remains of this substance might be found in the shape of thin pericellular halos . 
1. - the early changes in ear cartilage , previous to the actual infiltration , are demonstrated . at top of picture the perichondral connective tissue is swollen and slightly invaded by lympho - plasmocytes . the superficial part of cartilage appears unstained , indicating that metachromatic material is lacking . 
4. - the disappearance of chondroitin - sulphate precedes the growing tumour . in this picture the stroma reaction has reached the left top corner ; the lightly stained ametachromatic cartilage area contains very few inflammatory cells . 
when investigating the process more carefully , however , it was established that cancer cells really have the capacity of digesting cartilage by their own activities without any co - operating stroma reaction . further , the results indicate , that the first demonstrable alteration of cartilage just being infiltrated is an early disappearance of most of the preexisting ester sulphate . 
the cultures were grown by the hanging - drop technique in a medium consisting of equal parts of fowl plasma and 15 per cent chick embryo extract in ty - rode solution . 
the explants were subcultured every 48 hours for 4 passages to obtain as uniform growth as possible . fresh embryo extract was groups of cultures , each from the 4th passage , were selected and matched used . and used for experimental material and controls . 
agent were observed on the unfixed living cells in culture by cine - photomicrography . for most of the work we used apparatus modified from that described by willmer ( 1933 )  . recently , in collaboration with a . 
hughes of the department of anatomy , we have recorded the effects on the living cells by phase - contrast cine ' - photomicrography , using the techniques described by hugbes ( 1949 , 1952 )  . phase contrast microscopy has been used in a few cases to observe directly the effects of the chemicals on the living cells in culture . ( ii ) radiological details . the primary x - radiation used was of equivalent wave - length 0 - 221k , as deduced from the h.v.l. 
the nominal dose rate , which was always measured within + 2 per cent was in the reaion of 2oor per minute at distance 40 cfrom the focus , and was varied by altering the distance . 
the quality of the radiation was unchainged throughout these experiments , so that the probleof the exact value of the ionisation in the cells of the culture near the surface of the glass coverslip is not of importance here . in the cultures used most of the mitotic cells coudted lie between the glass surface and a distance 201i from it , so that the ionisation in the cells is increased by photo - electrons arising in the glass , probably by a factor in the region of 2 ( spiers , 1949 )  . 
3 , 4 , 5 . - effect of tetra - sodiuni 2 - mothyl - 1 : 4 - naphthohydroquinone cliphosphate ( compound i ) on a living resting cell of a chick fibroblast culture of the fourth passage , 24 hours old . phase - contrast c ' me ' - photomicrographs , taken by a . 
the doses were given in a fixed time , 2 - 00 minutes , except in the case of tlle three points marked , for which the radiation was delivered at 200 r per minute . the mitotic inhibition in probits is plotted against log dos - , ( finney , 1947 ; fisher and yates , 1948 ) there is no indication of departure from a linear relatiodbetween mitotic inhibition in probits and log dose . it is concluded that the relation between iiiitotic inhibition and the radiation dose plotted on a logarithmic scale is a sigmoid curve . 
the dose corresponding to 50 per cent mitot - ic inhibition after 6 hours is 315 the calculated regressiod line gives a sa - tisfactory fit . the calculation of the regression line is somewhat laborious . the mitotic inhibition produced by compound i in the chick fibroblast cultures has been studied in some detail after 24 hours ' application . 
the calculated regression line gives a satisfactory fit . there is no indication of departure from a linear relation between mitotic inhibition in probits and the logarithm of the concentration . thus there is a sigmoid relation between mitotic inhibition and the applied confor 50 per cent mitotic inhibition after 24 hours the concentration centration . of i is 3 - 81 0.15 x 10 - 6molar . for most of the compounds studied such accuracy is not necessary . tt is justifiable to deduce the approximate concentration required to give 50 per cent mitotic inhibition from the curve fitted by eye to relate the observed percentage mitotic inhibition to the molar concentration plotted on a log scale . in the case of compound i this method gave a value of 3 - 84 x 10 - 6m . the first experiments on the antimitotic effects of x - rays and i in chick fibroblast cultures ( mitcher and simon - reuss ' 1947 ) showed much greater mitotic inhibition with the combination of 3 x 10 - 6mcompound and 244 r of x - radiation delivered 18 hours later with fixation and counting after a further period of 6 hours , than with the same amounts of the two agents separately . to test for potentiation rigorously , it is essential to compare the effect of a combination of half amounts ( or other suitable mixtures ) of the two agents with the mean effect of the agents acting independently . this method , which may be termed the summation , method , is well recognised in pharmacology , and has already been applied in radiobiology by liechti and muller ( 1936 ) , gray and read ( 1944 ) , and mitchell ( 1947 )  . our original experiments showed a combined effect of 86 - 7 per cent mitotic inhibition , while the corresponding mean ' calculated for the two agents separately from the regression lines in fig . 
i and 2 , is 78 - 6 per cent . this result suggests that there is at least additivity of the effects , but it does not provide evidence for potentiation . in other experiments , the combined effect of 2 x 10 - 6mcompound followed 310 j . 
the combination of 2 x 10 - 6mcompound followed after 18 hours by 200 r of x - radiation produced 93 - 25 per cent mitotic inhibition after 24 hours . froni the number of mitoses in the individual cultures it is found that for 8 degrees of freedom student 's t is 15 - 71 , so that p is much less thlan - 001 . in this experiment , as in table 1 , the highest values of the mitotic inhibition were associated with no significant reduction of the area of the outgrowth . this finding confirms the unimportance of cell migration under the experimental conditions of the combined action of x - rays and the compound 1 . the possibility of differences in the behaviour of the outgrowth and of the central part of the culture has been , studied by means of the counts made in serial sections of 24 - hour cu - itures . 
 - the method is a laborious one ; by its use , fischer and parker ( 1929 . ) showed that in chick periosteal fibroblasts , the proportion of cells in mitosis was 0 - 79 per cent in the outgrowth , but only about 0 - 09 in our cultures - as in those of jacoby ( 19371 ) - the per ceiit in the central part . mitoses in the central part are limited to the " perichondxium " on its surfaces . studies of the topographical distribution of mitoses in our cultures by a . 
f. phillips showed no appreciable decrease in the number of cells in mitosis per unit the area of the central part is usually area as the central part is approached . not more than 20 per cent of the total area of the culture . in the serial sections of the cultures the central part is easily defined . 
the resting and mitotic cells were counted in the surface layers . in the sections of a control culture , out of 6205 cells counted there were 53 cells in mitosis , of which 13 were in prophase , 31 in metaphase , 3 in anaphase and 6 in telophase . 
at 24 hours after the application of 5 x 10 - 6mcompound 1 , out of 6220 ceus counted , there were 26 cells in mitosis ; of these i was in prophase , 19 were in metaphase , none were in anaphase and 6 were in telophase . 
simon - re ] utss cytological effects . tlio cytological changes produced by compound i in the chick fibi - oblast perhaps the most cultures differ in some ways from those produced by x - rays . fundamental difference is that anaphase bridges are only rarely produced by ' the compound , unlike x - rays . after the application of compound i to the hving cers , the first change observed is temporary cell enlargement , presumably due at least in part to hydration ( " cell oedema " )  . this does not occur with 5 x 10 - 7mconcentration , but has been observed in its early stages at 2 minutes after application of the compound in concentration 5 x 10 - 6m . 
the change is obvious after 5 minutes and probably affects the cytoplasm first . after 10 minutes the effects are still increasing ; ' there is bubbling of the enlarged cytoplasm and enlargement of the mitochondria , nucleus and nucloolus . 
the slight transient shrinkage of the cell immediately after addit - ion of the compound is observed consistently . at the maximum of the cell enlargemei - it the increase in transverse dimensions of the cells is often in the region of 50 per ' cent . mitotic inhibition is obvious after 80 minutes and persists after 24 and 36 counts show that there is no appreciable change in the distribution of hours . the phases of mitosis even in high concentrations such as 5 x 10 5m where mitotic inhibition is almost complete . example at 4 x 10 - 6mconcentration the abnormal mitoses increased slowly from about 5 per cent of the total mitotic count after 80 minutes to about 15 per cent after 24 hours . 
a typical count of the mitotic abnormalities is included in table il and photomicrographs of examples of the abnormal cells after fixation are enlargement of some of the dividing cells is shown in fig . 
2 x 10 - 6m  . compound i followed after 18 hr . by 150 r x - radiation * also 1 tripolar met - aphase . i and x - radiation shows potentiation of chromosome fragmentation . this finding is of particular interest in connection with possible radiotherapeutic applications . instability of compound i in solution . studies of the thermal instability of compound i in aqueous solution were made because of erratic results in animal experiments and clinical trials . 
for 7 days almost completely destroys the antimitotic activity and produces a rather toxic - solution . there is no significant mitotic inhibition , but the outgrowth is poor , many of the resting cells are vacuolated , disconnected and rounded and - a large number of the cells in metaphase show gross chromosome clumpiaig . there is progressive reduction of the mitotic ' hibition + 4 - 1 314 s . 
the outgrowth ig poor , many of the resting cells are rounded , there is some accumulation in metaphase and a number of exploded cells ; there is wide it seems likely that there are variation in the behaviour of different cultures . several unstable intermediate products involved in the process of inactivation of compound i by incubation at 39 ' c . 
experiments. tetra - sodium 2 - methyl - 1 : 4 - naphthohydroquinone diphosphate ( compound 1 ) it is found that there is no has been studied in detail as a reference substance . indication of departure from a linear relation be - tween mitotic inhibition in probits accordingly it is sugand the logarithm of the concentration of the compound . gested that the best estimate of the activity of the compound is the concentration which produces 50 per cent mitotic inhibition under the experimental conditions . this concentration may be termed mi 50 as a convenient abbreviation . 
for compound 1 , there is no indication of departure from a linear relation between the rnitotic inhibition in probits and the logarithm of the concentration . for 50 per cent mitotic inhibition after 24 hours , the concentration is 3 - 81 0 . 
legon. from the department of geography , the london school of economics and political science . received for publication april 20 , 1951 . in considering the heavy mortalities from gastric cancer that exist in the counties of northern and western wales ( stocks , 1936 ) , the writer wondered whether mortality was uniform over the area , or whether there were marked local variations within it . 
the low - mortality rural districts of eastern montgomery and of radnor ( 70 deaths ) are well drained , in contrast to the rural districts to the west , which lie on an ill - drained plateau with extensive tracts of bog . 
the impermeability of the rocks of anglesey ( 203 deaths ) , and the extreme flatness of much of the island , make for peat accumulation , despite the fact that rainfall is light compared with that of the highland parts of north wales . case involving the pennant grit . it may be stated , therefore , that the rural districts of low mortality are all on the sheltered east and south of the welsh massif and are well drained . 
legon huntingdon , ely , peterborough and the holland division of lincoln ( stocks , 1947 ) , since these four counties alone in england have proportionally large areas of peaty fen soils . apart from the typically high organic content of their soils , north wales and the fens have little in common . 
as further information ( marston , 1951 ) is made available , it may prove worth while to consider whether a carcinogenic substance exists in food - plants grown on these soils . the possibility of an association between soil organic matter and cancer mortality was expressed verbally to the author by g . 
daily with their diet ( bielschowsky , in experiment ii the ordinary stock diet ( hall , 1948 ) was 1944 ) for 25 weeks . given and the carcinogens , dissolved in peanut oil , were administered by stomachtube . 
the survivors of experiment i were killed in the 63rd and those of experiment ii in the 52nd week of the experinment . table i summarizes the results of experiment i . for comparison the tumours obtained by feeding 4 mg . 
the first tumour , a hepatoma , was examination . foumd in the 54th week , and the second , a cancer of the meatus acousticus externus , 3 weeks later . 
by the 24th week neoplasms of the breast gland were present also in cancers of the meatus acousticus externus made their the two other groups . appearance in rats treated with the methyl compounds from the 34th week palonwards , whereas in the a.a.f. 
group the first was seen in the 25th week . pable liver tumours , lowever , occurred only in the latter group after the 33rd week of the experiment . these consisted of a few minute cysts . the total yield of cancers induced by d.a. 
induced two hepatomas , definite signs of liver damage and fairly widespread cystic cholangiomata in 2 other males of this group , while 2 of the females showed a few minute cysts in the liver . 
four hepatomas were found in the males and one in the females , cystic cholangiomata being present in all . to summarize , the genetic background determined to a large degree the site of tumour development . 
thus breast tumours appeared in a much higher incidence in the wistar rats than in the piebalds , in which cancers of the small nevertheless in both strains a striking difference intestine were readily induced . in the response of the liver to the three compounds was apparent . in the first experiment 700 mg . 
were given , while in the second the total dose administered amounted to about half of the methyl compounds and to approximately a quarter in the case of a.a.f. yet in both experiments a.a.f. 
is readily split off in vivo and therefore can be only of minor importance for the carcinogenic activity has been substantiated by weisburger , weisburger and morris ( 1950 ) , who showed that after feeding w_ - c14 - 2 - acetylaminofluorene radioactivity appeared in the breath within 6 hours . ray and argus ( 1951 ) found the toluene - sulphonic ester of 2 - aminofluorene to be non - carcinogenic for the rat , only minute amounts of this ester being morris , dubnik , dunn and johnson ( 1947 ) , studying hydrolysed in the body . the action of 2 - nitro , 2 - amino , mono - acetyl and diacetyl - 2 - aminofluorene , reported that the acetyl compounds induced the majority of liver tumours observed , when the substances were given per os . why the acetylated aminofluorenes have such an affinity for the liver , whereas m.a. 
ungar. from the department of pathology , hadassah university hospital , jerusalem , israel . received for publication june 7 , 1949 . carcinoma of the duodenum has been observed in this laboratory at autopsy in three young siblings whose case reports form the subject of this paper . 
the examination revealed several histological aspects which may be regarded as representative of successive stages in the development of polyps . in the area of the prominence , the submucosa was widened and the muscularis mucosae showed a funnel - shaped protrusion in the direction of the intestinal lumen . 
the earlier history contained no fourteen months prior to admission he first began these occurred at irregular contributory information . to complain of bouts of cramp in the abdomen . intervals and were without clear localization . the patient was admitted to the hospital in june , 1939 , three months before his death . 
at admission his chief complaints were shortness of breath , attacks of tachycardia , lack of appetite and abdominal colic localized under the costal arch on either side . during the preceding year the patient had lost a great deal of weight . there were recurrent periods of diarrhoea . 
the duodenal mucosa choledochus as far as 1 cbehind the papilla of vater was without lesions . and pancreatic duct without abnormalities . beyond the papilla of vater , the lumen was considerably distended . there was a nearly circular , crateriform defect , which extended from the posterior wall , the lesion , which including almost the entire circumference of the duodenum . measured 7 to 8 cin diameter , showed a rugged floor ; it was entirely composed of tumour tissue and blended underneath with enlarged lymphatic glands of the peripancreatic group . 
the latter were glued together and had been sections through the floor of the ulceration completely replaced by tumour . revealed a firm and , in some places , a friable tissue which was of a pale greyishyellowish colour . 
the tumour was defined against the head of the pancreas by a thin layer of greyish - red connective tissue . the remaining portion of the duodenum was considerably distended and filled with greenish fluid . 
sections from the large ulcerated tumour revealed a carcinoma which grew in some areas in solid strands , while in others gland - like formations predominated . epithelia showed a considerable degree of pleomorphism and there were numerous typical and atypical mitotic figures . 
the main mass of the node was made up of glandular epithelium tissue with regular lumina which in places were slightly tortuous . was columnar with dark nuclei and oxyphilic cytoplasm . there was no pleomorphism or mitotic activity . 
an x - ray examination of the stomach and duodenum 2 months following the operation likewise failed to reveal pathologic changes . the three younger boys were kept under observation from birth by the infant welfare centres and the school hygiene departments of the hadassah medical organisation . 
nagel ( 1925 ) benign polyposis . duodenal polyp , polyposis recti et colon duodenum , jejunum , ileum duodenum , stomach , colon , duodenum , stomach , caerectum cum , colon adenomas in duodenum , multiple polyps in cae . cum , colon , rectum duodenum , jejunum , colon polyps in duodenum and jejunum duodenum , jejunum 9 . 
the disease is hereditary and affects patients since generalized polyposis is known to of all age groups , including the lowest . involve predominantly the large intestine , it is not infrequently mentioned in textbooks and articles under the name " polyposis or adenomatosis of the colon . " however , in rare cases , the disease was found to be prevalent in the small intestine . besides the instances mentioned in table iii , others of this variant have been described by lubarsch in 1888 ( dohring 1907 ) , schwytzer ( 1924 ) , roan ( 1932 ) , haggard and floyd ( 1935 ) , ravitch ( 1948 )  . some pathologists have taken the view that generalized polyposis is an entity which since it causes characteristic severe dysentery - like symptoms and malignant change in a high percentage of cases , should be separated from that represented by cases in which only few polyps are formed ( oberndoerfer , 1929 ; lockhart - mummery , 1934 ; hullsiek , 1928 )  . 
the designation of all three of our cases as generalized polyposis therefore seems to be justified . the observations reported in this article are relevant to the understanding of factors which govern the manifestation of malignancy in intestinal polyposis . several authors have assumed a casual relationship between the appearance of polyps and carcinoma in any given case . 
gunz. * from the department of radiotherapeutics , university of cambridqe . received for publication june 11 , 1949 . in previous publications ( gunz , 1948a ; b ) a technique of cultivating human leukaemic leucocytes in vitro was described , and the quantitative and cytological findings in untreated cultures made by it were analysed . 
jung. from the cancer research laboratory , university of florida , gainesville , florida . received for publication march 21 , 1951 . the experimental investigation of gastric cancer is seriously impeded by our present inability readily to produce this condition in animals . 
the difficulty and uncertainty of this method have progress in this field would be greatly facilitated if a precluded its general use . chemical compound capable of producing gastric cancer were discovered . that a carcinogen with such specificity is possible may be implied from the frequency with which butter yerow , for example , produces hepatomas ( kinosita , 1940 ) and beta - napthylamine , bladder tumors ( wiley , 1938 )  . 
when this is done it very seldom results in malignant growth in the stomach because of the protective if the carcinogen could be 8ecreted by the stomach it would action of the mucus . be in a much more favourable position to initiate cancer . the first step towards a solution of the problem is to ascertain just what properties of a substance influence its secretion by the glands of the stomach . the optimum properties might then be conferred on - a carcinogen by judicious substitution in the molecule . 
the resulting compound could be expected to initiate neoplastic growth in the stomach . several groups of workers during the past twenty - five years have studied , by means of gastric pouches in dogs , the secretion of intravenously injected dyes . dawson and ivy ( 1925 ) , kobayashi ( 1926 ) , ingraham and visscher ( 1935 ) , and varco and visseher ( 1941 ) , although working under varied conditions , are geneof some sixty dyes rally in agreement as to the dyes secreted by the stomach . studied ( ingraham and visscher , 1935 ) , afl those secreted are electropositive ( basic ) under some conditions . 
on the other hand , dyes not secreted by the gastric glands are acid or amphoteric with the possible exception of four basic triphenylmethane dyes . the basicity of these dyes seems to be the property most likely to determine their degree of gastric secretion . 
at a pkbof 11 - 3 , for instance , this equation yields a concentration ratio of 51 , at a pkb of 7 - 5 a ratio of 2 - 8 x 101 . ratios actuary obtained were 1 - 3 and 26 - 4 . although experimental results indicate a definite tendency for secretion to increase with increasing basicity . 
it is obvious that there is some opposing factor . this opposing factor increases with increasing basicity , so that at pkbof about 6 - 0 the tendency for the basic dve to concentrate in the stomach , due to distribution differences , might be completely overcome . this opposition to gastric secretion of dyes of increasing basicity is nicely explained in terms of the pore theory , or the concept of the ositively charged membrane ( ingraham and visscher , 1935 )  . acid dyes would be retained by chemical combination . basic dyes are assumed to pass through this membrane in the undissociated foras the ionization constants of these dyes increase it would become increasingly more difficult for them to pass through a positively charged membrane . it appears , therefore , that we are dealing with two main effects in the gastric secretion of dyes . 
bartholomew 's hospital , london , e.c.1. received for publication february 11 , 1952 . radium and thorium are widely distributed throughout the earth 's crust , although , as yet , no large deposits have been found ( it has been estimated that uranium and thorium exist in as much as 4 and 12 parts per million respectively )  . both these materials decay through a radioactive series , and in each case one ' these two radioactive gases escape into the member of the series is gaseous . atmosphere where their respective decay products will be formed and these prothere are other sources ducts will probably attach themselves to dust particles . of naturally occurring radioactive material ( actinium , which decays to gaseous actinon ; potassium ; rubidium and carbon 14 ) , but these are insignificant compared to members of the radium and thorium series . the presence of radioactive material in the atmosphere was first observed by their observations were confirmed by other workers elster and geitel ( 1901 )  . most of these early determinations were made by in various parts of the world . the charged wire method , which gave quite good qualitative results . 
the rate of air flow depended on the length of tubing connecting the pump and filter - holder , but was always between 30 and 35 litres a minute . it was measured by a u - tube manometer , and controlled by a screw clip . 
the collecting time was 20 minutes , which does not necessarily give the maximum measurable activity , but allows the experiment to be completed in an hour . the efficiency of the filter was examined in a series of experiments in which one , two or three filter - papers of different types were used in series as a backing for the whatman no . 
as it was not possible to obtain standard samples of each member of the radium and thorium series , we determined the efficiency of the counter for measuring the ft particles from three radioactive these efficiencies were plotted against energy , and the efficiency for the isotopes . , f emitting members of the radium and thorium series were read from this graph . the efficiency of the counter for measuring a particles cannot be determined in the same way as for f8 particles ; since an unknown number of a particles will be absorbed in the filter - paper . in order to calculate the ac efficiency , a dust sample was taken and counted in the usual way ; a piece of aluminium foil ( thick enough to absorb all the ac particles ) was then inserted between the filter - paper and the geiger counter , and the count was continued . this experiment was the proportion of ac to ft particles emitted by repeated for a series of samples . the dust was deduced theoretically , and the proportion of a particles which were counted was determined from the drop in counting rate caused by interposing the aluminium foil . 
dawson the data for kew were taken unless there was a significant difference between these two sites , in which case that day was ignored or mean values were taken . 
dawson than those taken in the morning . rothamsted is a third - order station of the meteorological office and more accurate correlation of weather phenomena and activity were possible ; the conclusions drawn from the hospital results were confirmed . the activity was greater if the ground was dry or drying . 
the floor of the cellar was only 4 below street level ; there was some ventilation from a window and the door leading to the main part of the house . 
the cellar had not been used for several in order to avoid disturbing the dust , the pump and filter - holder were weeks . kept outside the cellar ( table iii )  . 
the proportion of radon atoms that will decay whilst passing through the filter - paper is negligible , but when one considers the very large absorptive capacity of carbon for radon , it is rather surprising that no radon was found . we have not investigated the proportion of the other members of the radioactive series that will adhere to dust particles . carmichael and tunniciffe ( 1948 ) removed the dust from a closed room by fanning the air across a greasy plate and noted the diminution of activity in an air sample . 
on introducing smoke there was an immediate increase in activity , showing that in a small room there is never an appreciable quantity of free radioactive atoms - they either become attached to dust or migrate to the walls . 
shelter tolerance ( maximum concentration ) 11 , 800 100 , 000 the figure for droitwich spa was taken from a brochure issued by the local council . our inquiries as to who made the original measurements were not very successful , but it is believed that they were made by professor h . 
schrodinger , at seeham ; 8 - 9 x 10 - 12 curie / metre3 of air . simpsoin ( 1905 ) made a very exacting survey of the air - borne radioactive material in lapland in 1904 , using the charged wire method ( table vi )  . 
read and mottram ( 1939 ) have published the results of some determinations of the radon concentration in the radium laboratories at mount vernon hospital , at the radium institute , and in the radium workshops of messrs . 
 ( all in or near london )  . in the last case a concentration of 22 x 10 - 7 curie / metre3 of air was observed . evans ( 1950 ) has calculated the dose received by joachimstal ( jachymov ) miners in a working day through inhaling radon . 
bartholomew 's hospital , in the city of london ; rothamsted , a semi - rural area , thirty miles from london ; chiswick , geographically in middlesex but actually a semi - industrial part of london ; and in the centre of manchester . 
the results were similar to those of other observers in different parts ofthe world ( table v )  . large day - to - day variations in activity were observed , but these can be explained by variations in the weather - the more stationary the air , the greater the concentration of active material . 
the slight differences between the four sites mentioned above may be seasonal . the experimental procedure was not sufficiently accurate to show whether thoron or its decay products were present . radon , absorbed on dust particles , was not detected . 
no variation with height up to 100 above ground level was observed . the results obtained in rooms with varying degrees of ventilation are of in the laboratory there was usually twice the activity found at the interest . open - air site on the roof . 
at chiswick , ventilating the room lowered the activity by as much as one - half , whilst in the coal - cellar the activity was 14 times greater than at the site in the garden . in the post office air - raid shelter the ventilation was as restricted as any found in habitable buildings ; the activity was 120 times as high as at the open - air control site , i.e. , nearly one - eighth the tolerance concentration of radon . in every case the large day - to - day variations were similar to those at the control site . these large amounts are due to the greater emanating area in a room ( compared with the open air ) from which the parent radon can escape , and to the restricted ventilation which will allow the radon concentration to build up . 
the amounts show no considerable difference between urban and rural districts , but there are large day - to - day variations which in situations out of doors are affected chiefly by the wind ; the more stationary the air , the greater in closed places , e.g. , an air - raid shelter underground , the amount the activity . may be 100 or more times greater than those in the street outside , but even these are small in comparison with the lowest amounts considered harmful to man . i wish to thank professor j . 
fisk , of the general post office , london , for their kindness in providing facilities for this investigation , which has been supported by grants from the medical research council , the british empire cancer cami wish to express my indebtedness to t . 
usually the ventral prostates were explanted ; they were grown by the watch - glass technique , which is eminently suitable for organized gro - wth ( fell and robinson , 1930 )  . 
the medium was placed in a small watch - glass and allowed to clot ; the watch - glass rested on a layer of sterile cotton - wool soaked with sterile distilled water inside a small petri dish . 
but later this method was improved by explanting one lobe of each gland into medium containing methylcholanthrene while the other was kept as control . the explants were placed well flattened out on th ' e surface of the plasma clot , where they became firmly anchored . after a day or two the growing explants liquefied part of the plasma clot , and while still firmly attached to the surfa - ce of the clot were surrounded by a pool of liquefied mediuthe cultures were after the ninth day no transferred to fresh medium every second or third day . more methy1cholanthrene was added , and the cultures were maintained in normal medium for the rest of the culture period . the used glass - ware was decontaminated by keepino , it in concentrated sulphuric acid at room temperature for at least one week . 
the glands were fixed in 2 per cent acetic zenker after the following periods of growth : 5 days , io days , 14 days and 21 days . five to nine experimental and an equal number of control cultures were used for each point of observation . 
they were embedded in paraffin and seriary sectioned ; the sections were stained with ehrlich 's haematoxylin and eosin . to assess mitosis the number of dividing cells was counted in every second section , i.e. , in at least 20 sections of all single - lobe cultures . 
cultures treated with 20 - methylcholanthrene . cultures treated with methylcbolanthrene show a similar tvpe of growth at the beginning of the culture period : formation of new alveoh at the periphery and some central degeneration . 
the epithehum retains its glandular character and remains actively secreting in the treated explants for a longer time , but the stroma is usually verv poor and reduced in botb . 
one out of fi - ve treated cultures shows squamous metaplasia after the higher mitosis is stfll ' above that in the controls and has , in fact , risen after the dose . higher concentration while it has dropped after the lower . table ii gives th - e number of mitotic cells counted in 20 sections of controls and cultures treated for 9 days with 2 , y / cic . 
the number of dividing cells has fallen and nearly reached the control level . at the same time changes of a more anaplastic type can be observed in one batch of cultures treated with 2y of methylcholanthrene . 
squamous metaplasia develops more fully , particularly after the smaller dose , after the end of treatment . it is noteworthy that the d - ownward bend of the first wave coincides with the end of treatment . this may mean that the first peak is caused by a direct stimulating action of the carcinogen , whfle the second one is due to the increased growth - potential of the changed epitheliua rise in cell division has been reported by cooper and reller ( 1942 ) , who found a ten - fold increase hi the mitotic rate in the skin of mouse ears painted with methylcholanthrene , and by glucksmann ( 1945 ) , who observed a rise in mitosis a few hours after a single painting with benzpyrene . in contrast to the increase in mitosis and proliferation of the epithelial cells is the reduction of the stroma of the treated explants . 
of methylcholanthrene , which indicates that the dose for stimulation of fibroblasts is roughly twenty times lower than that for epithelium if the concentrations of 2 - 4y / / c.c. 
the cultures were grown in the presence of the agent for 9 days , then transferred to a normal medium and fixed at intervals after removal of the carcinogen . in both control and experimental cultures new alveoli were usually formed at the periphery of the explanted glands , while in the centre some of the al - veoli underwent degeneration . in the control cultures the epithelium quickly lost its glandular character , and the alveoli developed wide lumina lined by one layer of low epithelium in which mitosis was present though infrequent . 
of methylcholanthrene , the alveolar epithelium retained its glandular character for a longer time and showed a marked increase in mitosis , leading to hyperplasia and squamous metaplasia , while the stroma was considerably reduced in contrast to that of the controls . the first changes were recognizable 5 days after the beginning of treatment . they became more pronounced as the treatment continued and persisted after removal of the carcinogen . in the later stages partial or complete occlusion of the alveolar lumen with squamous metaplasia were observed . the rise in mitosis showed two distinct peaks , the downward bend of the first wave coinciding with the removal of the c ' arcinogen . 
fell for suggesting this problem and for her criticism in the preparation of the manuscript . i also wish to thank a . howard , radiotherapeutic research unit , hammersmith hospital , ' london , for the generous supply of cx males , c . 
we are probably all agreed that tumours , although usually , or perhaps always , preceded by abnormal local . this change consists in - an increase in conditions , arise by a sudden change . the rate of growth of a cell or a group of cells . in this way there arises a new cell - lineage distinct from its antecedents , and one which can propagate its new it has the character of a genetic property irreversibly and even indefinitelv . secondary changes may follow establishing subchange , a somatic mutation . sidiary cell - lineages , - some involving further increases of growth - rate and dedifferentiation , and all surviving and multiplying subject to natural selection . to these secondary changes i shall return later . the chemical conditions arising - in the changed tissue h4ve been examined bv santesson and caspersson ( 1942 ) , while the processes of its cell division have been described by koller ( 1947a ) for epithelial tumours and by la cour ( 1944 ) for pernicious anaemia , which is an analogous condition depending on the enhanced multiplication of the red blood precursor cells of the bone marrow . 
a single polymitotic gene in maize and extra heterochromatic chromosomes in millet both cause rapid and unwanted nuclear divisi ' ons in the pollen grain . in maize up to fou - r extra mitoses take place before the chromosomes have had time to divide immediately after meiosis . 
the nucleus is forced into division , and the unsplit chromosomes are scattered on a spindle which merely disperses them into a number of deficient nuclei , each of which is aga ' m compelled to divide before it is ready , and in this way the whole pollen grain is brought to ruin . in millet the mitoses ( also up to the nu ' mber of four ) do not occur before the chromosomes are split , but the whole pollen grain is none the i - ess consumed and killed in the end by the production of redundant nuclei . 
the pollen grain is thus , in , a sense , an encapsulated tumour ( darlington , 1947 )  . comparison of these cases of polymitosis in plants with animal tumours polyinitosis is not due to a somatic enables us to clear our minds on one point . it 4ffects every cell of a mutation . particular type in the body . 
the spontaneous tumour on the other hand arises at random both in time and space . it does not affect all cells of one type , but only one cell , nor is it the first cell of a particular normal type to arise which is affected . 
we are therefore confirmed in regarding the spontaneous tumour as due to a genetic change . this conclusion removes the contradiction between development on one side and heredity and infection on the other by withdrawing to a lower level ofanalysis . the organism is now seen from the cell point of view as a vegetatively propagating colony . 
how far does it enable us to distinguish between nucleus and cytoplasm as the seat of the mutation general the most efficient carcmogens ( such as the hydrocarbons ) and the most efficient agents of nuclear mutation ( such as mustard gas ) do not coincide . ' carcinogens do not damage the nucleus in proportion to their effect as carcinogens ( darlington and koller , 1947 )  . 
thus the nucleus again seems to beexcluded , just as it is by the direct evidence . but the evidence of xray effects is a more serious objection to a nuclear origin . x - ray damage leads to the development of cancer only when the dose has been so heavy as to damage the cytoplasm , and only then after prolonged delay . light doses which have a proportionate effect on the breakage of the chromosomes and the mutation of the 120 c . 
different conditions , in differeni tissues and in different stages of development . now plasmagenes , like all other such determinants , are susceptible to mutation . it is not possible certain of them control the formation of plastids in plant cells . to relate here ( as i have elsewhere ) the long detectiv - e story of investigations which have led in the course of 40 years to the revelation of the character and continuity of the plastid plasmagene . suffice it to say that , like the centromere and nucleolar organizer of the chromosomes , it its stock - in - trade on its back , which being a sac of green pigment marks or tags its owner and enables us to count its numbers in the cell and note its changes : it can mutate from green to white and vice versa , so that cells can occur with mixed green and white plasmagenes ; and , moreover , the same plastid may be starved white , as it were , by a stepmother nucleus , and recover its normal colour when it is put back with its own proper parent . prolonged coitus causes a paramecium to be infected by the kappa plasmagene of its mate . transplantation of an organ or of serum from a c02 - sensitive drosophila infects a nonsensitive fly , including its egg , with the sensitive plasmagene ( sonneborn , 1948 ; l ' he ' ritier ; 1948 )  . these infections , it will be seen , are artific ' ial , or at least unnatural . 
a number of aberrant conditions can be trans ' mitted from stock to scioii , and some even have arisen in a scion after it has been grafted on a healthy stock . these are artificial diseases ; they are not transmitted in nature , but only by grafting . 
we make a great mistake therefore in calling them viruses : they are proviruses ( darlington and mather , 1949 )  . conversely it has been know - n for 40 years that the . 
virus ricketmia is carried by the eggs of its vector dermatocentor . the terror of its infection for man has closed our eyes to the fact of its inheritance for the tick . 
what appears as a virus in one host is indistinguishable from a plasmagene in the other - and it may weh be that the plasmagene is the older ( l ' he ' ritier , 1948 )  . at the same time billingham and medawar ( 1948 ) have sh ' own that a selfpropagating particle.differentiated in development may by its diffusibility enjoy certain of the properties of infection - a situation which can come to our notice only when the particle is as innocuous , and the differentiation as trivial , as the black pigmentproducer of a ' piebald mammal . 
they have never been guilty of infection , and are therefore totally unadapted for or against it . the virus type may well have arisen likewise by mutation of a plasmagene , but it must have been a mutation followed by others adapting it to infection . moreover , this type falls into two groups by a most instructive distinction . 
the milk - agent , on the other hand , cannot be imagined as having arisen anywhere save in the moiise , itself , in whieb it is inherited by the milk instead of by the eggs . how accidental is the distinction between the infectious and the non - iilfectious particle is revealed most strikingly by one true virus . 
but in its new host it not only it even ceases to be inoculable . it loses the property ceases to be infectious ; of infection , and becomes transferable , if at all , only in whole cells . this is just the opposite transformation to the one by which proviruses arise or demonstrate their existence when two plants are grafted together . 
ob ection8and di ffil cultie8 . three grounds for suspicion of the plasmagene origin of cancer are now worth first , the cancer agents have one characteristic which will at once discussing . be noticed as marking ' them off from all previous plasmagenes . 
now the inheritance of lethal mutations out of the running . in the nucleus , when they are recessive in their lethality , is mendelian and can therefore be followed in experiment . 
a whole tissue can be destroyed by rendering its there is , however , no contradiction between nuclei unworkable in this way . the nucleus is the assumptions of a cytoplasmic cause and a nuclear cure . that is especially easy the only wheel in the cell that we can put a spoke in . to do in rapidly dividing cells ( darlington and la cour , 1945 )  . 
we are therefore merely attacking the malignant cell where it is weakest . incidentally the effect of irradiation in breaking the chromosomes and stopping the growth of the cells leaves no doubt that the remarkable deficient nuclei found in tumours by koller ( 1947b ) will have a limited life . 
the particle analysis : competitive propagation . the cancer miita - tion , as we saw , is often followed by other changes beyond these may be described under the the primafly one of change in growth rate . headings of dedifferentiation or the loss of tissue character , metastasis or migration , secondary mutation and various breakdowns of mitosis and of chromosome structure . 
what we now have to ask ourselves is how far these secondary changes require secondarv assumptions , and how far they are implied by the primary on.e. recent experiments equally with flies , infusoria and tomatoes liave revealed to us the conditions of normal development . 
thev have shown that it is iiot merely ebaracterized by adjusted between nucleus and cytoplasm , but between different self - propagating constithese adjustments can be broken down under experituents of the cytoplasm . in polymental conditions , with results which have ' been ace - lirately described . mitotic maize , as we saw , the cytoplasm runs away from the nucleus : in polyin paramecium , in mitotic millet the nucleus runs away from the cytoplasm . c02 - sensitive drosophila , and in rogue tomatoes , the whole system at a high temperature can be made to run away from a particular plasmagene . 
bv rapid growth the 250 kappa particles of one cell can be run down to one ( from which they can recover ) and then finally to zero ( from which thev cannot recover ) , so that the cell lineage has mutated . 
and in most virus diseases of course the virus runs away from the rest of the system or vice versa ; only occasionally is equievidently therefore the cell contains self - propagating elements librium reached . these limits are exposed in with different limits to their speeds of reproduction . a protozoan or a plant when the temperature is raised . 
these three classes of particle are conditional and interchangeable . cancerproducing particles fall into all three . the origin of cancer can therefore be ascribed to mutations in cytoplasmic determinants , indifferently infectious or non - infectious , which make themselves visible by causing the resumption of growth . further , the study of plasmagene and virus inheritance in relation to differentiation reveals a competitive propagation of cytoplasmic particles . this explains both the genetic control and the secondary development of cancer with its potential dedifferentiation and metastasis . the discovery of , not the cause , but the system of causation of cancer tells us nothing immediately about the cure that we do not already know . 
althougih biologists have for long been uneasily aware of the existence of a problem of cell heredity , its systematic analysis is the work of very recent cell heredity deals with the origin and maintenance of inherited character years . differences between cells ; more particularly , between cells that set up division its problems present themselves in their most acute , lineages by mitotic fission . if not most easily workable , form in metazoan development . the outcome of cellular differentiation in ( say ) mammalian development is the formation of a limited number of histologically definable cell genera , each one further subdivided into a variety of cellular genetic species . 
the genus " fibroblast , " as yet far from completely analysed , may be subdivided into cells which manufacture bone , cartilage , white connective - tissue fibres , and so on . genus " epidermis , " of which we have made a particular study ; includes the epidermal epithelium of the superficial skin , of the sole of the foot , the tongue , cells of each of these epidermal the claws or nails , the vagina and the cornea . species display a distinctive combination of structural or physiological properties . the epidermis of the sole of the foot , for example , has a characteristically high rate of cell division . 
but this has proved not to be the case . although plausible " phenocopies " of sole epithelium ( in the form of corns and callosities ) can be made by irritating the thin and relatively quiescent skin of the body surface , the difference between the division rates of sole and body epidermis is in fact " intrinsic " and inheritable . 
we have , for example , transplanted sole epidermis to positions in the body where it is protected by neighbouring hair and secure from mechanical irritation ; but even so , its characteristic growth rate has been maintained for at least two years , and thick pads of now functionless cuticle continue to form over it and may periodically be removed . claw epithelium tells the same story - more clearly , since the difference between claw and body epidermis is anatomically crude and obvious . 
hopkin and williams , who supplied the following information : " it is a distillers ' yeast , composed of one race of yeast only . it is not debittered , because it is not bitter in the first instance . the desiccated yeast is active and can be killed by temperabefore use the yeast was heated to6 60 c . 
bielschowsky table ii gives the results of an experiment ( f ) which has been chosen because it is typical of the response of wistar rats receiving 2 - acetyl - amino - fluorene and the original diet . 
two rats dying at an early date ( rats 3 and 6 , experiment b ) had to be omitted from the table . of the 28 rats left , 22 were still alive at the end of the 42nd week ; they seemed to be in excellent health and no tumours could be palpated ; 14 of these were females and 8 males . however , neoplasms were found in 11 at the post - mortem examination . the commonest tumours were small hepatomas , having a diameter of 3 - 6 mm . ; in one wistar rat a small flat intraductal papilloma of the breast was discovered . histological examination of these early neoplasms frequently failed to give proof of their malignancy . 
bielschowsky this difference was best seen when the average weights of fed the yeast diet . only such animals were the females of the piebald strain were compared . selected as were found to be free of hepatomas . 
any effects observed in the yeast series have , therefore , primarily to be attributed to the substitution of milk powder by dried yeast . experiment e is in good agreement with harris 's findings that an increase in milk proteins there exists now failed to protect against the action of 2 - acetyl - amino - fluorene . good evidence that a high casein - high riboflavin diet protects only against dimethyl - amino - azo - benzene . 
even related compounds ( giese , clayton , miller and baumann , 1946 ) are not inhibited to the same extent by such a diet . further , strong and figge ( 1946 ) , who reviewed the literature on this subject , did not succeed in inhibiting by a diet of liver supplemented by a combination of raw milk , riboflavin and xanthine the development of tumours induced by subharris 's failure to protect against cutaneous injection of methylcholanthrene . acetyl - amino - fluorene by a diet which inhibited the action of " butter yellow " has already been mentioned . the results obtained in the yeast experiment are not impressive , but they seem significant since the same trend of delayed tumour growth was found in three experiments involving various litters of two different strains of rats . it would probably have been an advantage to use a smaller dose of the carcinogen in the experiments reported in this paper . 
of acetyl - aminofluorene will still produce a high percentage of cancers . it seems possible that the relatively low incidence of cancers observed by cantarow , paschkis , stasney and rothenberg ( 1946 ) after prolonged treatment of rats with acetyl - amino - fluorene was partly due to a supplement of brewers ' yeast in the diet given . the problem whether certain dietary factors are true anti - carcinogens , or protect only the liver against a damage the reaction to which leads to the establishment of cancers , could not be answered by workers who studied the effect of it was hoped that working diet in rats receiving dimethyl - amino - azo - benzene . with a more versatile carcinogen would give a clue to this question . at first sight it seems that in experiments a , b and c the development of all types of there was , for instance , a significant reduction in the tumours was delayed . incidence of mammary cancers in females of the wistar strain . this finding , however , could be explained also by the assumption that a less damaged liver was better able to cope with an excess of endogenous oestrogen and so prevented the ' development of tumours of the breast . 
the frankly keratinizing members of our series we have taken as a standard with which to compare the behaviour of our remaining histological types . the morphological features of the more differentiated epitheliomata have been so completely evaluated that any further discussion would be superfluous . 
the primary lesion grows slowly and tends to be clinically latent for a considerable period of time , it appears as a especially if it is situated in a region such as the nasopharynx . smooth or finely granular , firm , globular tumour , at first a mere nodule , but later attaining considerable dimensions . infiltrative spread and superficial ulceration occur fairly late in the evolution of the growth . cervical lymph glandular metastases appear at an early date , while the final phase is frequently ushered in by distant visceral extension , osseous deposits being rather characteristic ( kienbock and selka , 1935 ; ch ' in and szutu , 1940 )  . 
the histological picture may be syncytia of large , palely staining cells appear as sheets , irregular quite typical . clumps , or columns , in a lymphocytic stroma of varying density . 
ewing was doubtful of the specificity of schmincke 's description , and found its distinction from other types of undifferentiated pharyngeal growth difficult or impossible . the term " transitional cell carcinoma " was first employed by quick and cutler ( 1927 ) to describe a group of highly radiosensitive buccopharyngeal tumours of uncertain histogenesis which lacked the classical features of epithelioma , but presented transitional epithelial characters . ewing 's writings ( 1929 , 1940 ) have done much to clarify the morphological features of this growth . the most frequently encountered sites are the tonsil , base of tongue , nasopharynx , laryngopharynx , nasal cavity , and accessory nasal sinuses . quick and cutler described the primary lesion as possessing " a finely granular , velvety surface , e . 
fairburn this description is , however , far from being specific . which looks like an erosion of the mucous membrane rather than a frank ulceragives the impression of having originated in the tion . 
the lesion deeper structures and adhered to and eroded the mucous membrane from the clinical beneath . " course resembles that of lympho - epithelioma - a small and often occult primary histologically , transitional focus , and early cervical lymph glandular metastasis . cell cancer is typified by the presence of broad , clearly demarcated , epithelial the cells are cylindrical , polygonal or spindlecolumns and alveolar formations . shaped , with a rather hazy cytoplasmic membrane . 
the oval or fusiform nucleus is more chromatic than that of lympho - epithelioma , while its nucleolus is less frequently the marginal cells appear regimented as though forming distinct . degeneration and necrosis of the central zones of some of the a basal layer . lymphocytic infiltration of the stroma epithelial formations may be observed . may be present , but is never marked . there is considerable divergence of opinion regarding the justification of the classification of lympho - epithelioma and transitional cell carcinoma as specific entities . 
each has been traced to death or for a minimum period of five years following the first examination . purposes of classification we have created the following groups according to the site of the primary lesion : 3 . 
the cells are distributed the cell body is spheroidal or polygonal in solid masses or diffuse infiltrations . in form with palely staining cytoplasm , and a relatively large , oval , hyperchromatic nucleus which frequently shows bizarre , atypical features and mitotic figures . these classifications have presented certain difficulties : 1 . 
compared with the figure for the frankly keratinizing group ( 61.5 years ) , the former two differences are statistically significant , while the latter is not . there is no significant difference between the mean ages of - our cases when distributed according to the site of the primary growth . 
fairburn we have adopted ebenius ' clinical criteria of metastasis . the frankly keratinizing cancers possess a statistically significant lower incidence of metastasis ( 36 per cent ) than the remainder of their fellows , with the exception of the undifferentiated group which only metastasized in 22 per cent of cases . this table also illustrates the influence of glandular metastasis upon the likelihood of death with active growth . 
frank , lev and blahd ( 1941 ) , in their study of transitional cell growths , report a 44 per cent incidence of metastasis . 52 per cent of our cases possessed evidence of glandular extension on first examination . inspection of our data discloses the relative insignificance of tumour histology as a prognostic factor in cases of cancer of the lympho - epithelial sites . 
maccarty ( 1922 ) and plaut ( 1927 ) have emphasized its limitations , stressing the far greater importance , in most instances , of clinical features , such as the age and general condition of the patient , the duration , location , and gross anatomy of the growth , and the presence , or absence , of metastasis . 
the enormous preponderance of the site factor over that of histology is borne out by this study . thus , frankly keratinizing neoplasms of the lip carry a relatively good prognosis , but when arising in a lympho - epithelial region their significance is as sinister as that of their ill - differentiated fellows . 
the presence of lymph - node metastasis on first examination is , in our experience , a factor of far greater prognostic value than tumour histology , although very appreciably influenced by the site of the parent growth and its accessibility to treatment . 
to take a specific example , metastasis in a case of lip cancer markedly decreases the likelihood of cure , whereas when a lympho - epithelial region is involved , death with active malignancy is likely to occur whether the cervical nodes are involved or not . the chance of invasion of the cervical lymph glands appears to be largely independent of the histological grouping , the site of origin of the tumour being of infinitely greater significance . it has been widely assumed that the main factor in dissemination is the inherent growth capacity of the neoplasbut this is not the only influence which has to be considered . 
 two recent investigations , both from the united states , upon the arsenic content of tobacco , and the volatilization of this arsenic when tobacco is smoked , have been reported . 
the work of these authors differs from our own in that they used artificial methods of combusting the tobacco , which may or may not repro duce the conditions present when smoking is carried out in the ordinary way , and they found no brands of tobacco containing only minimal quantities of arsenic . 
 ( 1 ) gross and nelson ( 1934 ) employed a method which consisted of digestion in nitric and sulphuric acids , precipitation of magnesium ammonium arsenate , solution in hydrochloric acid , and estimation by the gutzeit method . 
five such analytical groups were analysed for each brand studied . " they found the content of pooled groups of 4 cigarettes of 5 brands to range from 97 to 363 p.p. 
 ( 2 ) thomas and collier ( 1945 ) , using a modified cassil - wichmann method , found that " individual cigarettes and cigars of a given brand varied widely in arsenic content , " which ranged in pools of 2 to 4 cigarettes from 2 packs of 20 ( presumably of the same brand ) from 354 to 114 p.p.m. , while cigars ( 132 to 295 ) and pipe tobaccos ( 227 to 428 ) showed a lower range ( table i )  . 
gross and nelson ( 1934 ) attribute the rather narrower range found in cigarettes and in pipe tobacco , compared with cigars , to the mixing which the former receive , but this difference is not seen in the results for cigarettes and cigars of thom.as and collier ( 1945 )  . 
in american pipe and chewing tobaccos ; using suction by a water - pump , he concluded that " we can say roughly that half of the arsenic is evolved in the smoke gross and nelson ( 1934 ) used an apparatus drawing about 50 ml . 
 each : 207to256 211 , , 267 ' 253 , , 363 97 , , 130 229 , , 261 range in 5 single cigars from each of 5 brands : 229 241 332 116 247 83 to 484 range in 5samples from each of 4 brands : 260 to 500 116 to 266 311 to 451 1 - 5 1 - 4 thomas and collier : cigarettes " a single pack of 20 " " ali.other pack " 1 - 4 " groups of 2 to 4 . " 354 ; 400 ; 405 ; 472 ; 600 ; 61 - 4 * 614 ; 670 ; 714 ; 845 ; 1140 132 ; 225 ; 262 ; 295 cigars pipe tobacco cigars ( about 10%cutoffat ends ) pipe tobacco 1 - 5 396 ; 265 ; 227 ; 345 ; 428 * these figures represent the actual analyses in p.p.m. , on pooled groups of 2 to 4 cigarettes , and hence are not calculated means , though each figure represents the mean composition of the eigarettes in one pool . 
cigars and cigarettes were " smoked down to about 1 / 2 to 3 / 4 " ; the arsenic the " puffed " and " unpuffed " smoke was absorbed and estimated separately . 
 " the pipe smoker who smokes all the tobacco does not therefore have the protection afforded by the butts of cigars and cigarettes . " from the present point of view this question is not of great interest ; the important datum is the amount of arsenic inhaled , and in this country only about one smoker in ten smokes a pipe ( hansard , 1945 )  . 
in their first series ( table ii ) cigarettes of 7 c length were smoked down to 1 c ; in a later serjes only 2 / 3 was burned . 
 the arsenic found was compared with analyses of whole cigarettes from the same pack , " but in view of the large differences they later cut off about os c for analysis ; apparently they failed to discover that this method also can be falla cious . 
 our earlier results ( table iv ) were obtained by the gutzeit method , which depends upon the brown and yellow colours produced in paper impregnated with hgc12 ; it is fitted for the measurement of amounts of as20 3 between 10 and 1 g . , and shows differences most clearly in the range of 8 to 3 g . 
there may be con~ siderable differences in the matches made by different persons ; in these experi ments the mean was taken of the readings made independently by two persons unaware of the nature of the unknowns . 
the iodometric method of thomas and collier ( 1945 ) was adopted later ( tables iii , v and vi ) in view of the difficulty of determining the small amounts of arsenic lost in smoking . 
the method has the advantage that , whatever may be its errors in other respects , there is less disagreement over the very sharp end - point ( titration of iodine and starch ) , about which two observers usually do not differ by more than 005 c.c. 
 the estimations upon brands p and c were usually carried out upon amounts of 20 to 30 g . , as a method which is sufficiently accurate within this range 176 m . 
gross ( 1933 ) has drawn attention to the occurrence of such errors in the estimation of arsenic in tobacco , which are attributed to uncombusted residues of pyridine and nicotine ; we failed to obtain satisfactory results by the method which he devised to overcome this difficulty . 
 in the previous investigations summarized above artificial methods of smoking were employed , air being drawn through a pipe , cigar or cigarette by means of a water - pump . 
we have confined our experiments wholly to cigarettes smoked by one or other of three persons accustomed to this form of smoking , and they were asked to do this in their ordinary way ; the two brands used ( p and c ) are in common use in this country . 
the ash , and stump , were dropped into separate weighing bottles in our earlier experiments , but latterly the two have been analysed together ; the amount of arsenic found , if less than that in the cigarette , should indicate the amount volatilized in the smoke , of which a part must have been inspired . 
 before this width of range was realized we obtained many contradictory results , showing amounts in the ash and stump which were sometimes less , and sometimes more , than that thought to be present originally . 
 8 whole cigarettes 9 cigarettes ( 7 wholes and 2 halves ) 5 pieces from 3 cigarettes 1 piece from each of 4 cigarettes 6 successive pieces cut from 1 cigarette 13 estimations on mixed tobacco of 10 cigarettes e 10 range in 5 packages of brand p brand o . 
 678 823 307 496 461 275 10 pieces from 5 cigarettes 5 successive pieces cut from 1 cigarette a 20 a 20 11_ estimations on mixed tobacco of io cigarettes . 
but as the weight of most cigarettes does not differ very much from one gram the figures give a rough measure of the arsenic in one cigarette , which is of more obvious practical interest . 
the results were again contradictory , and we found that such suc cessive portions may show a range of 388 to 587 , or 100 : 151 in p , and of 306 to 542 , or 100 : 177 in c ( table iv )  . 
 in the hope of obtaining more uniform material , the papers were removed from the cigarettes of a package of 10 or 20 , and the tobacco mixed by hand and 178 m . 
but even this treatment does not give satisfactory results ; it is very difficult to take up samples of such material which will contain constant propor tions of the coarser and finer particles , as the latter tend to fall away from the . 
moreovar , the minced tobacco , even when moistened , is not easy to smoke in the same way as ordinary tobacco : adjacent cigarettes of brand p from a much larger package , containing 100 , were taken ; but these at first showed a wide range ( 495 to 766 , table iv )  . 
 that an average of 158 per cent of the arsenic is lost in the process of smoking and had presumably been volatilized ; this loss is statistically significant ( p = 0001 )  . 
the same method was applied to brand c , of which the largest boxes available contained 50 ; the first two rows showed in 18 cigarettes a loss of only 76 per cent , which was not statistically significant ( p = 02 )  . 
 580 527 623 558 603 418 456 458 568 556 617 } 681 mean 596 631 427 412 471 533 629 662 453 367 527 634 455 367 539 509 lower right row . 
kennaway the 4th row showed a loss of 182 per cent , which is just significant ( p = 005 ) , in spite of the small numbers ( 12 cigarettes ) involved . 
 the most likely source of the arsenic present in some tobaccos , which has been suggested by several authors , is the spraying of the adult plants with insecticides , and this particulate method of application would explain the difficulty in obtaining concordant analyses . 
 of the arsenic volatilized in smoking , a part must escape while the cigarette is not in the smoker 's mouth , and a part of what he inspires is expired again , hence the amount retained is no doubt very small . 
the concentration of arsenic in cigarettes containing the larger amounts is very \rariable ; this irregularity is perhaps due to a method by which the arsenic may have been introduced , namely , by the spraying of the plants with insecticides . 
an estimate was made of the amount of arsenic volatilized in smoking , by comparing the content of cigarettes , and of the ash and stumps derived from them , but the wide range of arsenic content makes it difficult to infer the amount present originally in a cigarette which is to be smoked . 
the results indicated that from 76 to 18 2 per cent of the arsenic present was lost in smoking ; this figure is of the same order as but somewhat lower than that obtained by some previous investigators who have used artificial methods of smoking . 
korteweg. victorieplein 45 , amskrdam , neturlands . received for publication february 12 , 1951 . most curves indicating the cancer death rates per million living in the different age - groups rise continuously with advancing age . 
the numbers of lung cancer deaths in the netherlands and in england and wales , 1125 and 5941 respectively , were large enough to exclude the possibility that these exceptions to the rule might be caused by chance only . is this decrease of the cancer death rate at a relatively early age a characteristic of lung cancer or is there some other explanation ? cia l%22 r . 
2 , which is derived from table l the age curves for males in all yeargroups given show the same early decline of the lung cancer death rate , with the .2 ^ ^ ^ 35 - 44 45 - 54 55 - 64 65 - 74 75 + years fig . 
3 , constructed from the numbers following one another vertically , show us , for each age - group separately , the increasing lung cancer mortahty in the course of years . 
3 we can derive , by interpolation , for each year and each age - group separately the approximate values of the cancer death rates . this has been done for the years 1915 , 1925 and 1935 . 
4 it is necessary to take the following points into consideration : cancer only develops after a long period of preparation , which in most cases can be counted in decades . those who at the age of 80 contract cancer , do so partly because of a tendency to cancer years before - let us say at the age of 70 . 
they are recruited from the group of the younger ones , whose tendency to cancer is also known , as it is given by the length of the ordinate belonging to that age in fig . 
4. it would be wrong , therefore , to compare in this respect persons who in ' 1945 were 80 years of age with persons surely this comparison should be who , in the same year , were 70 years of age . made with those who , ten years earlier , in 1935 , belonged to that age - group . 
we must presume , therefore , that it is not only the younger people who are endangered by these irritative factors . third : let us suppose that all males were exposed to these irritative factors to about the same degree , and that in each of the last 40 years the quantity of these factors had increased considerably . 
were this so , then really we would expect to find an " age curve " for lung cancer approximately such as we did if one fact proves the reality of the great increase of lung cancer in the find . last 40 years , it is the sbape of its " age curve , " now demoilstrated as being an illusion only . artificiary , with older age , the lung cancer age curve is pushed down . at this moment , the increase of lung cancer in the younger age - groups came to a halt , even then it would - progress in the older age - gr6ups up tir the moment when theillusionary age curve changed into the real one . 
we can ' ut it in another way : at this moment the lung cancer mortalitv is lower than corresponds mr - ith the tota - i amount of the causative irritative factors . the lung cancer " ceifing is lying at a far higher level than the mortality figures would suggest . up to the age - group 55 to 64 inclusive the shape of - the illusionarv age curve is about normal ; it is only after that , that the sharp dechne begins . in 1945 , in england and wales , in males less than 65 years of age , the number of deaths from lung cancer was 33 - 8 per cent of those from " all cancers with the exclusion of lung - cancer " ( registrar - general , 1947 )  . 
6 this also means that in this case the total number of cancer deaths in males would have ' been 30 , 835 + 10422 = 41 , 257 , and not 36 , 776 , which was the number given in the statistical review for 1945 ( registrar - general , 1947 )  . following the same reasoning it appears that in 1949 , in the netherlands , for males this " ceiling " was lying at a level of 1.710 , whereas the lung cancer deaths registered numbered 1125 only . it is clear that the " ceiling " will never be reached except after a long period of rest , with stationary lung cancer death rates in the younger age - groups . 
6. - balance of lung cancer mortality in males , england dnd wales , 1945 : - _ rears ..1 - - lung cancer deaths recorded deficit lung cancer risk lung cancer age curve after correction . ..... 
pseudo lung cancer age curve . 5941 4481 10 , 422 after having studied this table we no longer wonder that in england and wales . cancer in males is again on the increase . 
and still , if in 1948 the " ceiling " of lung cancer had been reached , the total number of cancer deaths would not have been 40 , 026 , but more than 46 , 000 ! in females lung cancer is also increasing . 
an analysis of its age curve , such as i have made in this paper for lung cancer in males , shows us that , just as formales , the real age curve for females closely resembles that for the " non - hormonal cancers " ( cancers other than those of breast and uterus ) in females . the problem which lung cancer presents to us is a very serious one , not only for england and wales , but also for the netherlands and many other countries . * in the review for 1948 ( registrar - general , 1950 ) the numbers for lung cancer are only given in combination with those for cancer of the mediastinum . during the last 40 years cancer of the mediastinum has not changed much in frequency . 
hopkin and williams , who supplied the following information : " it is a distillers ' yeast , composed of one race of yeast only . it is not debittered , because it is not bitter in the first instance . the desiccated yeast is active and can be killed by temperabefore use the yeast was heated to6 60 c . 
bielschowsky table ii gives the results of an experiment ( f ) which has been chosen because it is typical of the response of wistar rats receiving 2 - acetyl - amino - fluorene and the original diet . 
two rats dying at an early date ( rats 3 and 6 , experiment b ) had to be omitted from the table . of the 28 rats left , 22 were still alive at the end of the 42nd week ; they seemed to be in excellent health and no tumours could be palpated ; 14 of these were females and 8 males . however , neoplasms were found in 11 at the post - mortem examination . the commonest tumours were small hepatomas , having a diameter of 3 - 6 mm . ; in one wistar rat a small flat intraductal papilloma of the breast was discovered . histological examination of these early neoplasms frequently failed to give proof of their malignancy . 
bielschowsky this difference was best seen when the average weights of fed the yeast diet . only such animals were the females of the piebald strain were compared . selected as were found to be free of hepatomas . 
any effects observed in the yeast series have , therefore , primarily to be attributed to the substitution of milk powder by dried yeast . experiment e is in good agreement with harris 's findings that an increase in milk proteins there exists now failed to protect against the action of 2 - acetyl - amino - fluorene . good evidence that a high casein - high riboflavin diet protects only against dimethyl - amino - azo - benzene . 
even related compounds ( giese , clayton , miller and baumann , 1946 ) are not inhibited to the same extent by such a diet . further , strong and figge ( 1946 ) , who reviewed the literature on this subject , did not succeed in inhibiting by a diet of liver supplemented by a combination of raw milk , riboflavin and xanthine the development of tumours induced by subharris 's failure to protect against cutaneous injection of methylcholanthrene . acetyl - amino - fluorene by a diet which inhibited the action of " butter yellow " has already been mentioned . the results obtained in the yeast experiment are not impressive , but they seem significant since the same trend of delayed tumour growth was found in three experiments involving various litters of two different strains of rats . it would probably have been an advantage to use a smaller dose of the carcinogen in the experiments reported in this paper . 
of acetyl - aminofluorene will still produce a high percentage of cancers . it seems possible that the relatively low incidence of cancers observed by cantarow , paschkis , stasney and rothenberg ( 1946 ) after prolonged treatment of rats with acetyl - amino - fluorene was partly due to a supplement of brewers ' yeast in the diet given . the problem whether certain dietary factors are true anti - carcinogens , or protect only the liver against a damage the reaction to which leads to the establishment of cancers , could not be answered by workers who studied the effect of it was hoped that working diet in rats receiving dimethyl - amino - azo - benzene . with a more versatile carcinogen would give a clue to this question . at first sight it seems that in experiments a , b and c the development of all types of there was , for instance , a significant reduction in the tumours was delayed . incidence of mammary cancers in females of the wistar strain . this finding , however , could be explained also by the assumption that a less damaged liver was better able to cope with an excess of endogenous oestrogen and so prevented the ' development of tumours of the breast . 
doll. from the statistical research unit of the medical research council , london school of hygiene , keppel stred , kondon , w.c.i. received for publication november 28 , 1953 . attempts to derive theoretical laws from changes in the death rate with age have a long history . 
they have not , in general , been very fruitful and there has - been some hesitation in applying the technique to the study of cancer . recently , however , two hypotheses about the mechanism of carcinogenesis have been put forward , which have been derived from analysis of cancer mortahty fisher and horomon ( 1951 ) used statistics from the united states statistics . for cancer of the stomach in women , and nordling ( i953 ) , classing all sites together , used statistics for cancer in men from britain , france , norway and the u.s.a. both found that , within the age group 25 - 74 years , the logarithm of the death rate increased in direct proportion to the logarithm of the age , but about six times as rapidly ; in other words , the death rate increased proportionany with the sixth power of the age . 
death rates in some age groups under 24 years were . higher than would be expected had this basis been a general law throughout iffe . rates for the age groups above 75 years were considered unreliable and were excluded . in interpreting these observations , both assumed that mortahty gave a good indication of incidence and treated the data as if they referred to age specific incidence rates . 
they considered that cancer in youth might be affected by special factors which were unhkely to operate at later ages and they , therefore , felt justified in basing their hypotheses on the data recorded for adults . fisher and horomon ( 1951 ) pointed out that the observed relationship between age and mortahty could result if a colony of six or seven cancer cells was a critical size below which independent growth was not sustained . thig hypothesis , however , also leads to the conclusion that cancer incidence should be proportional to the fifth or sixth power of the concentration of the effective carcinogen whereas experimental data suggest that , in general , tumour incidence and concentration of the carcinogen vary ' in arithmetical proportion . 
the hypothesis in its simple form is , therefore , untenable . nordling ( 1 - 953 ) , on the other hand , suggested that the observed relationship would be explained if a cancer cell was the end - result of sev ' en successive mutations . this hypothesis does not lead to the observed result in all circumstances . does so only if the probability of occurrence of each mutation remaihs constant throughout hfe . in this case and so long as the occurrence of each mutation p . 
the logarithm of the incidence rate will then be directly proportional to the logarithm of the age and will increase six times as rapidly , ix , log rate 6 log t + a constant . in contrast to fisher and hollomon 's ( 1951 ) hypothesis this leads to the conclusion that the final rate of tumour formation wir be directly proportional to the concentration of an applied carcinogen , so long as the probability of occurrence of a mutation is proportional to the concentration of the carcinogenic factor and different factors are required to effect different mutations . in his analysis , nordling ( 1953 ) grouped all types of cancer in men together and considered them as a whole . 
he gave no detailed figures for cancer in women , but stated that for cancer of the sex organs the increase in mortahty was fairly small above the age of 4.5 and that for other sites the mortality seemed " to increase according to the sixth power of the age both before and after the forties but not during the decade of the menopause when the increase is smaller . " he considered that the entire increase with age in cancer incidence among adults was not wholly explicable by a mechanism of multiple mutations but that hormonal control of growth might play an independent part . 
1 - 4 show the logarithms of the male and female death rates from cancer of the commonest sites in england and wales in 1950 and 1.951 ( registrar general , 19521 1953 ) , plotted against the logarithms of the mid - points of the age groups . for each sex , all those sites are shown for which more than 1000 deaths were recorded in each of the two years . 
are less reliable than those for the subsequent ones since they are based on much smaller numbers , and both standard errors and ( to a lesser extent ) arithmetical errors are much larger . for example , the standard error of the death rate from gastric cancer in women is 18 per cent of the rate for the first age group ( 25 - 29 years ) and .2 per cent of the rate for the last ( 70 - 74 years )  . that is to say , the logarithms of the true rates for these age groups are likely to lie within a range of - .0 - 13 and - .0 - 02 respectively of those showm in fig . 
i. - change in mortality with age for cancer of the oesophagus , stomach and pancreas in men and for cancer of the stomach and pancreas in women shown on a double logarithmic scale , that is , the logarithm of the death rate per million persons plotted against the logarithm of the mid - point of the age group . 
the straight line through the points has been drawn arbitrarily to give the best fit , subject to the gradient being 6 to 1 . cancer of the stomach , colon , rectum and pancreas in women - the observations fall fairly close to the lines . in some , however ( cancer of the colon in men , cancer of the stomach , colon and rectum in women ) , the observations fall closer to lines with less steep gradients . 
the relatively high rates at the younger ages could , however , result if the population contained a group of subjects speciahy susceptible to cancer of these sites in early life - and so far as cancer of the p . 
doll colon and rectum is concerned , such a group is in fact found in subjects of polyposis coli . the actual regression coefficients for all the first group of cancers are shown in all the mortahty rates for this group , table i ; they vary from 4 - 97 to 6 - 48 . therefore , show the same sort of association with age as was reported by fisher andhoromon ( 1951 ) andbynordling ( 1953 )  . 
3. - change in mortality with age for cancer of the lung , bladder and prostate in men and for cancer of the lung in women , shown as in fig . 
1. i 0 - 1 - 6 consists of cancers for which there is already reason to believe that the strengths of some of the factors responsible for their development are variable . cancers of the prostate , breast , ovary , and cervix and corpus uteri are all believed to be influenced by endocrine secretions , which vary 1 ' n each individual in the course of his life ; a proportion of the cases of cancer of tho lung is believed to be related to cigarette smoking which has become more prevalent in the last 50 years and a proportion of the cases of cancer of the bladder is due to occupational hazards , to which men have been exposed for various periods at various ages . 
on the hypothesis that carcinogenesis is a multi - stage process , therefore , cancer at these sites would not be expected to show a uniform relationship between death rates and any power of the age . 
doll the sort of irregularities to be expected will depend on the periods when the carcinogenic factors are most active and on which of the stages in the process of carcinogenesis are affected . 
an increased rate of production for a short time during early life will provide a larger number of altered cells to be acted upon by other factors in the future , and wfll therefore appreciably affect the incidence at , say , age 60 . 
the weight depends both on the time of operation of the carcinogenic factor concemed and on the order of the cellular change which it affects . if t is the age at which the cancer incidence is observed , to the age at which the subjects are exposed to the factor , and s takes a value between i and 6 according as the change effected is the first , or sixth , then the weight is proportional to second , tos - i ( t - to ) 6 - 8 . hence it can be show - n that the weight by which a varying probability of the first mutation would be multiplied is highest at age 0 and decreases rapidly throughout life . 
the weight for the probabihty of the second change reaches its maximum one - fifth of the way through the exposure period ; that for the third change , for factors effecting the seventh change ' , two - fifths of the way ; and so on . the cancer incidence will be directly proportional to the rate of production of this change at the time of observation , and the previous behaviour of this rate will be irrelevant ( i.e. , will have zero weight )  . since the weighted mean of the varying probabihty will in general depend on the age , t , the overall incidence at age t is no longer proportional to the sixth power of t . 
doll old ages , can be attributed to the fact that the populations concerned belong to if mortality generations which smoked fewer cigarettes than subsequent ones . at different ages is examined for cohorts born in different five - year periods , the effect disappears . the concept of carcinogenesis as a multi - stage process also makes it easy to understand the mechanism of the latent period which occurs after exposure to a for example , kennaway carcinogenic agent before the appearance of a tumour . ( 1947 ) has demonstrated that circumcision deferred until the fourteenth year of life fails to give the complete protection against cancer of the penis that it provides when carried out on the eighth day ; in other words some change must take place within the first 14 years of life which eventually leads to the development of the disease after a latent period which may be as long as 70 years . 
on the hypothesis that penile cancer is the end - result of a series of seven successive cellular changes - the first of which occurs only in the presence of the prepuce , it can be shown that at age 53 ( the average age * of all the patients referred to in table ii ) the relative risks of developing the disease when circumcision is carried out at the mid - points of shrek and lenowitz 's ( 1947 ) age periods are 0 - 30 , 0 - 77 , 0 - 98 and 1 - 00 ( see mathematical appendix )  . children circumcised during the first six years of life are , however , most likely to have been operated on within the first few weeks , in which case the theoretical risk for this group would be of the order of 0 - 01 . 
on the other hand , u the present data were held to be equally accurate at all ages ( including the oldest ) it would be possible to obtain a quite different mathematical relationship . thirdly , other mechanisms than that of a multi - stage process can also be postulated to account for the observed relationship - and one has , in fact , been suggested by fisher and hollomon ( 1951 )  . moreover , some other diseases show similar types of increase with age ( e.g. , cerebral haemorrhage , coronary thrombosis and gastric ulcer ) and it is difficult to believe that they should all be dependent on the same type of mechanism . bereilblum and shubik ( 1949 ) , in summarizing the results of their experiments , have , however , stated that " whatever interpretation is adopted as a base line for research , the recognition that careinogenesis is at least a two - stage process , should invariably be bome in mind "  . it is , therefore , natural to see whether a twostage process - or even a more complex multi - stage one can account for the human data . 
from the analysis that has been made here , it would seem that a complex process of perhaps six or seven stages could account for : sites , and ( 1 ) the rapid increase in mortahty with age observed in cancer of some ( 2 ) the irregularity in the increase in cancer of some other sites ( 3 ) the long latent period observed after exposure to a carc ' mogen ( 4 ) clinical observation . 
doll the mortality rates for cancer of the lung , bladder and prostate in men and for cancer of the lung , breast , ovary and cervix and corpus uteri in women also accord with the theory , if it is postulated that the carcinogenic factors responsible have varied in strength . a formula has been obtained which can be used to weight the strengths of the carcinogenic factors at different periods and it is shown that the time when the strength of the factors responsible for the individual changes is of greatest importance varies according to which change in the series is affected . 
the conclusion provides a possible explanation for the observation that circumcision exerts an important protective effect against the development of cancer of the penis only if it be carried out early in life . we are most grateful to r . 
more precisely , we assume that the probability that the sth change occurs in the small interval i changes have already occurred , is p , ( to ) dto where ( tw to + dto ) , given that s all the other p 's are assumed to be constant . 
then p , ( to ) is a function of to . the probability that the sth change occurs in the interval ( to , to + dto ) , and the rth in the interval ( t , t + dt ) , is the product of ( i ) the probability that exactly s i changes occur in the interval  . 
we shall ignore the variation in age amongst the patients , and consider the expected incidence of penile cancer at t = 53 years ( the average age of all the patients referred to in table ii )  . 
w.3. received for publication march 1 , 1952 . some of the effects of ionising radiations are associated with formation of such effects are often reduced free hydroxyl radicals and possibly of peroxides . for these under anaerobic conditions and increased in the presence of oxygen . reasons it appeared possible that conditions which would increase the concentration of hydrogen peroxide in tissues might augment the biological effects of of the possible ways of increasing radiosensitivity by such a radiation . mechanism , two have been investigated . the first depends upon increasing the concentration of substrates , the metabolism of which is known to produce hydrogen peroxide . such substrates are those oxidised by flavine enzymes , including aldehydes , xanthine , hypoxanthine this method is being investigated by administration of and d - amino acids . suitable substrates and riboflavin , the latter in attempts to increase the concentration of flavin enzymes of the tumours . a second method of increasing hydrogen peroxide concentration of tissues depends upon the inhibition of the enzymes involved in hydrogen peroxide thus by inhibition of catalase and peroxidase of tissues this destrucdestruction . tion and utilisation of hydrogen peroxide might be prevented and its concentration in cells should increase . this was attempted without success in the present paper . many of the known catalase inhibitors including sodium azide and cyanide are also respiratory poisons . 
x 77 , 500 so , where ko = the reaction constant at zero time expressed in logarithms to base 10 and so = the h202 concentration at zero time . 
kat.f. qo21 - 29 x 10 - ' mxh20 activity of a tissue to the other metabolic processes expressed in q values . the tables of this catalase activity is expressed as qo21 29 x lothis expression makes it easier to relate the catalase the catalase activity is determined at 0 c . 
the testis probably oxidised primary carbohydrate . recent work has shown that ionising radiations ( taylor , greenstein and radiomimetic hollaender , smith , 1950 ) induce depolymerization of deoxyribonucleic acid . 
some of the biological effects of radiation may be due to such an action occurring in in the case of ionising radiations the effect is probably due to fr~e the cell . hydroxyl radicals which are produced in irradiated water . if increase in hydrogen peroxide concentration should increase sensitivity to x - rays , then addition of hydrogen peroxide to a solution of deoxyribonucleic acid should augment the depolymerising action of x - rays . such an effect has indeed been found by conway and butler ( 1952 )  . a large number of substances have been reported as catalase poisons , and some of these are listed in table i in order of potency . in the present work the activity of catalase poisons is compared with their toxicity to mice and an attempt made to measure the inhibition in vivo . 
the toxicities are also listed for some of the compounds , and the ratio of the concentrations causing inhibition of the enzyme to the toxicity expressed as the ld 50 ( calculated on a molar basis ) is given in table i . 
gallico the poisoning of catalase in vivo is difficult to estimate owing to the reversibility of the poisoning . blaschko ( 1935a ) showed that of a number of catalase poisons only potassium chlorate produced irreversible inhibition . 
the pretreatment with the catalase poisons tried did not modify the effect of radiation on the tumour . reduction of sensitivity of mice treated with sodium azide and hydroxylamine to x - radiation . groups of each stock of 10 mice were exposed to 700 r irradiation from an x - ray tube operated at 220 kvp . 
the result with azide is similar to that obtained by bacq ( 1951 ) , but the result with hydroxylamine appears to be a new finding in general agreement with bacq 's work . a4 [ 2 ' ' ' " 45 1 : contro . 2 : - inece wit 1.5per kg ; nan3 15m.bfoeradato. 
gallico the search for a poison more specific for catalase than is azide was also unsuccessful . sodium azide and hydroxylamine are much the most specific catalase poisons of the compounds examined . p - hydroxyphenylazide had only 1 / 50 of the activity of sodium azide ( calculated on a molar basis )  . this difference is exactiy the same as that found between hydroxylamine and phenylhydroxylamine . on the other hand , phenylhydrazine appears to be a more potent inhibitor than hydrazine ( blaschko , 1935a )  . 
the introduction of a methyl group into the hydroxylamine molecule ( o - methyl - hydroxylamine ) reduced the activity 400 fold . the inhibition produced by diethyl - p - phenylenediamine was of the same order as that described for dimethyl - p - phenylenediamine by homer and betzel ( 1950 )  . the figures on the ratio of catalase inhibiting concentration to lethal dose show that sodium azide is the most favourable catalase poison as well as the most active . 
the median lethal dose of sodium azide should give , a concentration of azide which is one thousand times that causing 50 per cent inhibition of catalase . thus , sodium azide is probably the safest catalase poison for use in vivo in spite of its high toxicity . the increase in poisoning activity of azide with decrease in the ph is similar to that found for the effect of formate and acetate by agner and theorell ( 1946 ) , who showed that anions in general combine with catalase and inactivate it to an extent increasing with decrease in ph . these authors explained this as due to the anion displacing the hydroxyl group of the catalase and it is possible that azide acts in the same way . 
the data of agner and theorell ( 1946 ) on poisoning of catalase with formate at different ph values are analogous to those found for azide ( table iv ) as shown in table xi . 
the replacement of a hydrogen of formic acid by ch3 as in acetic acid reduces the activity 800 fold ( agner and theorell , 1946 ) , which is the same order as that produced by the introduction of an o - methyl group in hydroxylamine . thus the poisons may combine readily with catalase , possibly replacing a hydroxyl group or hydrogen peroxide because of their shape and size . in testing this hypothesis , methylamine was found to be a poison ( of the same order as hydrazine ) , but ethyl formate and formamide , which it was hoped would be as effective as undissociated formic acid , were both found to be inactive . while the poisoning with hydrazoic acid and formic acid may depend upon the entire undissociated molecule , the poisoning with hydroxylamine and hlydrazine appeared to be independent of the ph . 
now in the cases of hydrazoic acid and formic acid , ionisation would result in loss of a hydrogen atom ( and gain of a charge ) , so that the ion would have less structural resemblance to hydrogen peroxide . 
gallico ( 3 ) poisoning of catalase of liver and tumour of rats was demonstrated after injection of azide . ( 4 ) injection of small doses of azide or hydroxylamine into rats immediately before x - irradiation did not increase the radiosensitivity of tumours . injection of large doses of azide or hydroxylamine into mice before irradiation reduced the mortality from x - irradiation of the whole body . ( 5 ) catalase activity could be expressed as a qo21 - 29 x 10h202 value in p11 . 
the investigation was supported by grants to the royal cancer hospital and chester beatty research institute from the british empire cancer campaign , the jane coffin childs memorial fund for medical research , the anna fuller fund , and the national cancer institute of the national institutes of health , u.s. 
i. received for publication january 4 , 1951 . recent work has provided evidence in favour of the supposition that tumours contain , and therefore probably release into circulation , a substance capable of depressing liver catalase activity . 
nakahara and fukuoka ( 1949 ) claimed to have produced a liver catalase depr ' ession in mice after the injection of alcoholprecipitated fractions obtained from human tumours . similar fractions from normal tissue were stated to be inactive . 
adams ( 1950a ) commenced a study of the effect of the injection of homogenized mouse tumours into mice , and showed that after an early ( 24 and 48 hours ) depression , the hver catalase level returned to normal approximately 4 days after injection , and fer once more as the new tumour grew . 
the results were interpreted as indicating that the initial depression is due to some substance or substances present in the , injected material , the second depression only being due to the new tumour . injection of homogenized normal tissues had no effect on catalase level . appleman , skavinski and stein ( i950 ) , in the course of an investigation of the variations in erythrocyte , kidney , and liver catalase activity in rats bearing the jensen sarcoma or the u.c.l.a. 
fibrosarcoma , found that normal rats kept on a protein - free diet showed a drop in liver catalase activity to approximately 50 per cent of normal within 7 to 10 days from the time thev were placed on the diet and this low level wa - s maintained for 53 davs or longer . 
the variatioils in liver catalase level following the subcutaneous injection of coarse suspensions of these tumours into albino mice results in both males and females were similar to those preappear in table 1 . viously obtained with s37 and carcinoma 63 . 
adams two general methods have been employed : the first involved the injection of s37 sarcoma , which had been treated so as to render it incapable of giving rise to tumour growth , and the second , the results of which are presented in the next section , the injection of pure line mouse tumours which do not grow in the strains of mice used . sarcoma 37 tissue was disintegrated by shaking for several minutes with glass beads in a " mickle " tissue disintegrator , made by messrs . 
is a plot of the subsequent variation in hver catalase level over a cers . period of 10 days , the crosses representing the arithmetic mean values of the groups . in the experiment represented iin the left - hand graph the tissue was disintegrated in normal saline ( ph 6 - 8 ) , and it wir be seen that a 24 - and 48 - hour depression was obtained , the level rising to normal at 4 days and remaining so at 7 and 10 days . however , this treatment was not completely succe 'ssful in pre ' venting tumour growth , in spite of failure to discover intact cells in the homogenate microscopicary . 
the experiment was repeated , using m / 100 citric acid as the suspending fluid ( ph 5 - 5 ) , and the results appear in the righthand graph . 
the catalase depressing effect is very similar , but in this case no tumours appeared in 18 mice kept for 3 months , when the experiment was terminated . a second method involved the partial centrifugation of a fine homogenate ofs37 tissue in normal saline , prepared originall with a tenbroeck grinder . 
n. abscissa : time in days . effect of in ' jection of tumours which do not grow in the 8train of mice u8ed . a number of pure line mouse tumours which do not grow , at any rate after the in ection of coarse homogenates , in the stock albinos used , were injected subcutaneously or intraperitoneally in 100 mg . 
the results appear in table il in every case a significant depression in catalase activity was observed 48 hours subsequently , with a return to normal by the 4th to 7th day . in one experiment of this series , not recorded in table 11 , a different result was obtained . 
the catalase level at 48 hours was very low , and remained low at 4 days ; at 10 days , of th - e 6 remaining mice , 3 in which the abscesses were healed had high levels and 3 120 d . 
the results are given in table il no significant variations in catalase level were observed , and with the rat tumours in particular the variations appear to be completely random . ..cb - r = 1 l -  . 
the points lie on a slightly curved line , showing a progressive deorease of catalase activity with dose . the depression is seen to be approximatelv proportional to dosage over the range 10 to 30 per cent . 
4. - liver catalase levels 48 hours after the intraperitoneal injection of varying quantities of coarse homogenate of sarcoma 37 into stock albino male mice . ordinate : catalase level in arbitrary units / mg . 
adams it is not claimed , however , that nutritive effects play no part in producing the depression of catalase activity , particiilarlv in animals bearing large tumours . miller ( 1950 ) has recently shown that although catalase level in animals which are maintained on low or non - protein diets parallels approximately the level of total liver protein , the enzyme soon begins to disappear more rapidly than the consequently , even if the only relevant effect of a large remaining protein . tumour on the nutritional status of an animal were a simple depletion of protein , however , the depression which occurs in a depression of catalase would result . the early stages of tumour growth may be expected to be due mainly to toxic material produced by the tumour , since the importance of purely nutritive factors , e.g. , protein depletion , must be small at this stage , though becoming progressively greater as the tumour size increases . 
the effectof large tumours on catalase is probably the resultant of separate nutritive and toxic actions , which could not be readilv distin uished . the effect of injecting infected c57 sarcoma is quoted as a possible source of error in investigations of this nature . it is not yet known whether catalase depression is a common result of bacterial infections , but this possibility must be reckoned with when using , for example , human intestinal tumours , which are greenstein ( 1947 ) states that infection of mice with certain often infected . strains of poliomyehtis virus , or with streptococci and staphylococei , has no . however , it has been observed in this laboratory effect on catalase activity . during epidemics of a sabnonella infection , and of ectromelia , that catalase level was low when the liver showed signs of involvement . 
dounce and shanewise ( 1950 ) i - lave recently shown that catalase activity is significantly depressed in rats suffering from advanced murine leprosy . apart from these isolated observaticjns , no detailed investigation has beeii made of the effect on liver catalase of infections of the liver , or of injections of infected normal or malignant tissue . although a depression of liver catalase may occur in conditions other than cancer , this does not detract from the importance of the effect as a property of maligiriant but not of nornial tissue , and work on the mechanisbv which the toxic substance exerts its effect may thrcw light on some fundamental difference recently reports of prehminary experibetweennormal and malignant tissue . ments have appeared , indicating the importance of hormones in maintaining a normal level of liver catalase ( begg and reynolds , 1950 ; adams , 1950b )  . present investigation is being follo - vved up by an endeavour to relate the tumourdepressing effect with the hormonal control mechanism . depression of liver catalase activity b - as been shown to follow the injection into mice of mouse tumours , but not of rat and human tumours in single doses of up to 100 mg . 
nakahara and fukuoka ( 1949 ) , on the other hand , observed a depression after the injection of alcohol - precipitated fractions of human tumours however , their dosage , referred to weight of original tissue , was into mice . probably very much bigher than that used here . all that is now claimed is that injection , in the dose used , of mouse tumours into mice produces a considerable depression of liver catalase , while injection of similar doses of tumours of other species produces no apparent change . 
the early variation in injection . females was not significant in one case . ( 2 ) injection into stock albino mice of sarcoma 37 tissue ( a ) disintegrated in m / 100 citric acid , or ( b ) partly centrifuged after coarse homogenization in 70 mg . 
doses , resulted in a depression of liver catalase without any obseirvable tumour growth . ( 3 ) six pure line mouse tumours , which do not grow in the stock mice used , also gave catalase depressions 48 hours after injection in 100 - mg . 
the work was undertaken as a collater ' al contribution to a wider scheme planned in 1952 by the cheshire and north wales branch of the british empire cancer campaign . for research into the pathogenesis of cancer the branch council required among many other things a laboratory analysis of soil taken from the vicinitv there was no precedent for the sort of analysls of the home of each patient . most suited to the project , previous workers in this field having been content with simple and often crude analyses or with the more detailed examination of too few specimens . it was clearly impossible to make a full physical and chemical examination of all the solid , fiquid and gaseous phases of every soil sample as several thousands were to be examined , so with some slight modification the analytical procedure of the soil survey of england and wales * was adopted - at first . it was felt , however , that an investigation of fatal cases of cancer in relation to soils as mapped by the late professor g . 
every case where cancer was mentioned on the death certificate , even when not the immediate cause of death , was included in the analysis , except that those occurring in the county of persons ordinarily resident in other areas were excluded . cancer was defined as any condition named in no . 
the differences between observed and expected values were subjected to the chi - squared test to determine the probabihty of such differences arising as a result of random these are the probabilities quoted in the tables that follow . 
that cancer of site a would be no more associated in an compariwith attribute b than would be the generality of cancer deaths . sons , unless the contrary is indicated , allowance was made for variations attributable to age and sex by calculating separately an expected number for six agesex groups and then adding . 
where it was desired to allow for a possible interaction between associations , expected values were adjusted . for example , if cancer of site a had shown an association with both attribute b and attribute c considered singly , and attributes b and c were known or suspected to be associated one with the other , adjusted distribution of expected values with respect to b were determined as follows . 
thus series having a common parent material geologically , are all indicated by the same capital letter , e.g. , all derived from non - calcareous shales of ordovician age are indicated p . 
the inclusion of many of this large group in social these considerations and our special class ii was therefore open to question . interest being in the association between cancer of the stomach and soil , suggested that it might be advisable to treat separately those occupations which bring the person into intimate connection with the land . 
griffith the public mains are supphed for the most part from surface water such as there is an exception where two small small lakes fed by streams and springs . ceintres get their water from deep bore holes but the populations are too smafl to be separated in the analysis . 
the in group l there social groups , however , show certain interesting differeinces . does not appear to be any association with fl soil . there is a relative excess of deaths in the other three groups although only in group iii is this undoubtedly siginificant in the statistical sense . soil and cancer of the breast . when corrections are applied for social group and water supply it is found that the association betweein cancer of the breast and a soil as compared with all other forms of caincer is stif present ( table x )  . 
0 - 20 > p > 0i i - ii * stomach all sites iii * stomach all sites iv - v * stomach ar sites stomach all sites all social groups : stomach males all sites females stomach ar sites 13 - 8  . 
bruzzone. from the department of experimental medicine , national health service of chile , santiago . received for publication june 9 , 1949 . abdominal fibroids induced in the guinea - pig by a - oestradiol ( nelson , 1937 ; lipschutz and iglesias , 1939 ) are prevented when progesterone or other 3 - ketosteroids are administered simultaneously with the oestrogen ( lipschutz and vargas , 1941 ; lipschutz , 1944 ; lipschutz , iglesias , bruzzone , fuenzalida and riesco , 1948 )  . 
 = fibrous tumoural effect ; units according to lipschutz and vargas ( 1939 ) t tablets of pure progesterone or part of a tablet . in the remaining groups - tablets from experiments of e . 
but these figures of active quantities of progesterone are not very distant from those obtained in our former work with the antifibromatogenic action of progesterone against o - oestradiol . there is also the fact that statements about absorption per day are stultified by its not being uniform throughout the experiment on account of the diminished surface , and by substances entering into the tablet from outside ; errors are 400 s . 
bruzzone this is why it would be daring to consequently considerable ( folley , 1944 )  . interpret the present results as indicative of a greater resistance of artificial oestrogens to the antifibromatogenic action of progesterone . 0o i1111j1 hexestrol hexestrol and progesterone fig . 
 lung tissue is unsuitable for direct injection of carcinogens , consequently pulmonary tumours in laboratory animals have hitherto been induced by applying the carcinogen at some remote site , by injecting it subcutaneously , intratracheally , intravascularly , intraperitoneally , or by including it in the diet . 
 recently the author ( horning , 1947 ) reported the induction of pulmonary tumours after relatively short periods of treatment by the direct application of carcinogens to adult lung tissue growing as subcutaneous homologous grafts in host animals . 
 the method of tumour induction consists in isolating small strips of lung from 6 - 12 weeks strain a mice , impregnating the grafts with crystals of a carcinogen ( 2 - acetylaminofluorene , 2 - aminofluorene , or 20 - methylcholanthrene ) prior to im planting them subcutaneously into host mice of similar age and strain in a manner which has been described elswhere ( horning , 1947 )  . 
small palpable tumours were obtained at intervals ranging from 6 - 12 weeks following grafting , but in order to examine the differences between the responses to individual carcinogens , grafts were fixed before tumours developed at intervals ranging from 1 - 5 weeks after implantation . 
 lung fragments from 6 weeks old strain a mice were grafted , in some instances without the carcinogen , into host mice of similar age , and also into mice of 15 months age which were selected from stock mice of undetermined ancestry . 
 this was done to find out if the survival of homologous grafts was dependent on employing a closely inbred strain , or whether it might be influenced by the age of the host . 
 in another series of experiments a number of lung grafts were treated with 20 - methylcholanthrene , half being implanted into the superficial fascia , and the remainder placed as usual between the skin and the abdominal wall . 
the host mice were again of the same age and strain , and it was thought that some infor mation might be obtained as to whether rapid vascularization played any role in successful survival of the grafts . 
 lung tissue was obtained from 30 strong a male mice aged 6 weeks , and implanted subcutaneously into host mice of the same strain and age , each indi vidual receiving 2 grafts on each side of its belly ( table i )  . 
when the same number of grafts from females were implanted into strong a male mice over 8 months of age , only 14 grafts had survived after 8 weeks and 4 of these contained areas of necrosis ( table i )  . 
grafting the same number of lung implants from strain a mice 6 weeks old into stock mice of indeterminate ancestry , all of which were over io months old , resulted in 58 becoming totally absorbed after the same interval of 8 weeks ' implantation ( table i )  . 
either the nuclei become pycnotic , or as in most grafts they lose their differential stain ing and the cytoplasm becomes homogenous , to be followed in later stages by fragmentation and dispersal . 
the nuclei of the collapsed lung alveoli undergo chromatolysis far more rapidly than those of the adjacent bronchiole epitheliu histological examination of these grafts suggests that their failure to survive in host tissues may be due to a partial failure of vascularization which induces necrosis . 
these results obtained with adult lung grafts indicate that successful survival and growth depends on age of both the donor and host animals , together with employment of a closely inbred strain in which homologous grafting is much better tolerated than in mixed strains . 
 in order to obtain a foreign body reaction fragments of lung from 6 - 7 weeks old donor strong a mice were treated with crystals of commercial cholesterol and subcutaneously implanted in 8 - weeks - old host mice ( table i )  . 
 in every instance in this series of experiments lung tissue was obtained from strong a mice of either sex , 7 - 8 weeks old , and impregnated with the carcinogen and implanted subcutaneously into host mice of the same strain and age ( table i )  . 
 the remaining 13 were no longer translucent , and it was found that eight of these opaque grafts had been accidentally transplanted into the superficial fascia instead of between the skin and the abdominal wall . 
 the histological characteristics of pulmonary tumours induced from sub cutaneous lung grafts treated with carcinogens differ in some respects from those of tumours arising in sitit in the intact lung of rodents under the influence of in pieces of isolated lung growing as . 
 a typical carcinoma of the lung in a graft impregnated with 20 - methylcholan threne fixed 11 weeks following subcutaneous implantation in a strong a host mouse is illustrated in fig . 
the lumen of the bronchiole is occluded by cellular debris , and the epithelium in some regions has become stratified , but in the malignant area the bronchiolar epithelium is ciliated._ the cells comprising this neoplastic focus possess all the histological characteristics of malignant cells . 
 the histological appearance of these carcinomas varies , however , according to the age of the primary gra those recovered from mice 9 - 12 weeks old consist of acini of cells with large oval nuclei . 
 all primary tumours induced by the several carcinogens were obviously bronchogenic carcinomas , with the exception of a squamous - celled growth in duced by 2aaf after 16 weeks ' implantation . 
one was a polymorphic and the other a spindle - celled sarcoma~ as both these tumours were too far advanced in their growth it was impossible to ascertain whether they were derived from the tissues of the implant or from the host , so they were discarded . 
in some which carried six or more graft1 it was frequently noticed that after 8 - 10 weeks the palpable grafts varied considerably in size , and that this variation often occurred irrespective of the particular carcinogen employed . 
those implants which failed to develop into tumours , and were hardly palpable in the living host , were found to contain large inflam matory foci or areas of foreign body reaction . 
in every instance the inflamed grafts were obtained from the same donor mouse grown in the same host and treated with the same carcinogen for a similar period of time as those which developed into tumours . 
 in the majority of grafts ( table i ) necrosis and fibrosis are absent , although these changes are so often associated with inflammation of the intact lung in situ . 
 bronchioles when present in these grafts tend to be obscured , since the lumen is filled with cellular exudate consisting mainly of lymphocytes , plasma cells , and a few macrophages or polymorphs . 
multinucleated giant cells frequently seen in tissues reacting to foreign bodies were absent in grafts treated with car cinogen , but were found in several implants impregnated with commercial choles terol . 
every lung graft was fixed in alcoholic bouin and stained by ehrlich 's haematoxylin and eos f1 . - a typical bronchogenic carcinoma , induced from a graft of adult normal lung impreg nated with crystals of 20 - methylcholanthrene , fixed 11 weeks following subcutaneous implan tation in a host ( strain a ) mouse . 
this is of special significance , as it indicates that the carcinogen has a selective action only for the bronchiolar epitheliu the adjoining alveolar cells remain quiescent to the local presence of the carcinogen . 
the lumen of the bronchiole is occluded with a cellular exudate , and in some regions the epithelium has became stratified , but in area of malignant focus the bronchiolar epithelium is ciliated . 
 since the induction of carcinoma of the prostate in mice , arising from fragments of adult glandular epithelium combined with the carcinogen growing as homologous subcutaneous grafts in host mice , was first reported ( horning , 1946 ) , this method of tumour induction has been successfully extended by other investigators to other tissues and organs . 
more recently hughes ( 1949 a , b ) , using the same technique , has succeeded in inducing transplant able carcinomas from fragments of adult bladder and uterine epithelium combined with methylcholanthrene , as well as tumours of adrenal cortex . 
 one of the several advantages arising from homologous subcutaneous grafting in the presence of a carcinogen is that the tumours growing under the skin are palpable , and can readily be obtained at the required stage of growth . 
the time taken for tumours to appear is shorter most likely because the carcinogen is more concentrated , and lies in close proximity to the bronchiolar epithelium of the gra with indirect methods of induction such as painting the carcinogen of the skin , by injecting it or including it in the diet , there may be changes in organs and tissues throughout the body , and not necessarily only in the region where a tumour arises . 
 thus dunlap and warren ( 1942 ) have reported that if swiss mice are injected subcutaneously with carcinogenic hydrocarbons , the incidence of primary pulmo nary tumours is higher in mice which have also developed tumours at the site of inoculation . 
 it must be admitted , however , that these two methods of induction differ fundamentally , because the changes which occur in the intact lung in situ following treatment at some remote site with a carcinogen are not necessarily the same as those which take place in homologous subcutaneous grafts of isolated , collapsed lung . 
these differences were discussed in a recent paper by orr ( 1947 ) , in which he rightly pointed out that it is difficult to compare results obtained by the two methods of induction . 
 recent eijeriments have shown conclusively that the survival of lung grafts either with or without the carcinogen is dependent upon the age and strain of the donor mouse , together with the age and strain of the host . 
examination of table i shows , however , that donor lung implants impregnated with a carcinogen survive more readily and in greater numbers in older hosts than would be the case if the grafts did not contain any carcinogen . 
it is of course possible that since the lung carcinomas arise from bronchiolar epithelium , some grafts may at the outset contain insufficient epithelium of this region to give a response . 
a separate series of experiments in progress show that pulmonary tumours are obtained more readily and in greater numbers from fragments of lung in which particular care has been taken to isolate portions of a bronchus , discarding the more peripheral parts of the organ . 
otherwise it is difficult to understand why prostatic epithelium should flourish as a graft despite the antagonism between graft and host tissues , and yet lung fragments implanted under the same conditions do not always survive so readily . 
pre liminary experiments reported elswhere ( horning , 1949 ) suggest that tissues which are normally under hormonal influence are much better tolerated as grafts in host mice of the same sex andstrain than tissues which are to a greater extent independent of hormone action . 
a higher incidence was not obtained in males , as seems to be the case in human lung cancer according to clemmensen and busk ( 1947 ) , and in strain a and strain c mice treated with urethane in the experiments of larson and heston ( 1945 )  . 
there is a slight difference between the potency of the three carcinogens used in the present experiments in that a greater number of tumours appeared in grafts treated with 20 - methylcholanthrene . 
 with regard to the histogenesis of the present lung tumours the method of grafting allows examination of the neoplastic changes in graded stages , leaving no doubt that the tumours arise from bronchiolar epitheliu it is curious that no response to the carcinogens is obtained from the walls of the lung alveoli . 
 an alveolar epithelium exists why should it remain entirely quiescent , whilst the closely adjacent epithelium of the bronchioles proliferates in so striking and so specific a manner ? controversy still exists concerning the nature of the lining of the lung alveoli . 
ross ( 1939 ) maintains that although an epithelial lining to the alveoli is not visible in ordinary histological preparations , it becomes appa rent in response to certain abnormal stimuli . 
grady and stewart ( 1940 ) not only affirmed the existence of an alveolar epithelium , but regarded it .as the source of lung tumours in mice treated with methylcholanthrene . 
 thus while they agreed that human tumours are bronchogenic in origin , they were convinced that mouse lung tumours arose from the alveolar epitheliu more recently herbut ( 1946 ) has reviewed the ev ' idence concerning the origin of human lung tumours , and concludes that all " alveolar cell tumours " come from the basal cells of the bronchi and bronchioles ; and that there is no justification for assuming that they arise from septal cells . 
it is unlikely that any fundamental difference exists between the rodent and human lung , and since there is such clear indication of the origin of malignant foci in early grafts combined with a carcinogen , it would appear that the case for bronchogenic origin of all lung carcinomas is strengthened . 
in the presence of chronic inflammation a proliferation of the epithelium in the respiratory bronchioles may extend to form a lining within the alveolar ducts and alveoli in much the same manner as bronchial epithelium will grow into an old abscess cavity . 
 the relationship of pulmonary cancer to inflammatory foci has been studied recently by grady and stewart ( 1940 ) , de eds and cox ( 1941 ) , nettleship , henshaw and meyer ( 1943 ) , orr ( 194 7 ) , orr and bielschowsky ( 194 7 ) , and selbie and thackray ( 1948 )  . 
the effects of urethane on the mouse lung in situ indicate according to nettleship , henshaw and meyer ( 1943 ) and orr ( 1947 ) that inflam mation and adenomas arise together . 
grady and stewart ( 1940 ) and selbie and thackray ( 1948 ) disagree , and the last authors believe that in orr 's series of 433 animals which were examined after they had died from natural causes , there was a susceptibility to terminal inflammatory involvement . 
here also the evidence obtained from grafting clearly supports the contention that there is no relationship between inflammation and lung neoplasia , since the grafts which failed to produce tumours contained large areas of inflammation and those in which tumours developed rapidly were comparatively free . 
 summary , bronchogenic carcinomas have been induced from homologous subcutaneous grafts of lung tissues growing in host mice , after impregnation with either 20methylcholanthrene , 2 - acetylaminofluorene , or 2 - aminofluorene . 
 the period of tumour induction in grafts of homologous lung is considerably reduced when compared with lung tumours which develop in situ , in rodents following treatment with the carcinogen at some remote site on the body . 
 the susceptibility to tumour formation in subcutaneous lung grafts treated with the carcinogen is found to be dependent upon the age and strain of the donor as well as that of the host mouse . 
additional experimental evidence supports the view that the pulmonary alveoli are not lined by a continuous epitheliu factors influencing the survival of lung grafts without using a carcinogen were also investigated ; these showed that the successful survival of subcutaneous grafts is dependent upon the age of both the donor and host mice , together 244 e . 
 this investigation has been supported by grants to the royal cancer hospital from the british empire cancer campaign , the jane coffin childs fund for medical research , the anna fuller fund , and the division of research grants of the u.s. 
hewitt. from the john burford carlill laboratories , westminster hospital , london . received for publication may 26 , 1953 . in a previous paper ( hewitt , 1953 ) a method for the preparation and titration of single - cell suspensions of sarcoma 37 was described . 
each mouse received 4 subcutaneous inoculations of the appropriate cell dose , one in each axilla and one in each grothe new - born mice were allowed to remain with their mothers throughout the experiments . 
1 the points plotted represent the percentage incidence of palpable tumours from inocula containing various numbers of viable tumour cells ( expressed logarithmically ) calculated from the cell count of a dense suspension and the dilution factor . the points shown were obtained from the data of various experiments . although the points are insufficient in number and accuracy to disclose the true character of the curve relating log . 
cell dose and tumour incidence , they serve to show the general trend of the relationship . litters born to mothers which had been rendered completely resistant to s37 by their having regressed a tumour before pregnancy ensued gave results which did not depart appreciably from those indicated in the graph . the td50 , read from the graph , is approximately 9 viable tumour cells . this figure is less than that obtained for the titration of c3h sarcoma in c3h mice over one year old , that is , 18 viable cells . certain technical difficulties have the effect of reducing the observed percentage of tumours obtained from specified cell doses below the maximum value . 
owing to the fact that the tumours which form in the suckling mice quickly reach a size which jeopardises health and movements , some animals die with tumours in 2 or 3 of the inoculated sites before all sites have been observed for a sufficiently long period to exclude tumour formation in thethe incidence of tumours recorded for any group thus tends to be submaximal . 
a further difficulty is that a proportion of each inoculum volume often exudes from the needle track after withdrawl of the needle . cell counts and volume measurements of the exuded fluid have shown that a quarter or more of the inoculated cells are lost in this way . if these known errors were to be eliminated , the points in the graph would be shifted slightly upward and to the left of their present positions , and the td50 would be rather lower than is obtained from the results as they stand . the effect of the diluent on the activity of sarcoma cells in dilute suspensions . craigie ( 1949 ) , discussing various technical requirements for the quantitative transplantation of tumour cells , drew attention to the inimical effects of various 386 h . 
hewitt in a later paper ( craigie , lind , diluents upon the viability of tumour cells . hayward , and begg , 1951 ) an account was given of certain atypical cells which composed about 5 per cent . 
the number of various mean doses of viable 837 cells . beside each point is the number of sites inoculated at that dose level . diluents in which inoculated cells were suspended : o tyrode solution . a , 0 5 per cent gelatine in tyrode solution . * 33 per cent mouse aacitic fluid in tyrode solution . a 10 per cent ascitic fluid in tyrode solution . the suspensions used to obtain the points shown in fig . 
the order of inoculation was so arranged that the interval elapsing between the time of preparation of the dilute suspension and the average time at which the mice of any litter were inoculated was equal for all the litters . 
hewitt quantitative transplantation of s37 in mature mice of different age - groups . a single - cell suspension of s37 was titrated in three series of mice , all from the same albino stock that provided the new - born mice and belonging to the following age - groups : 18 - 25 days ; 143 - 174 days ; 268 - 296 days . 
the incidence of palpable tumours in the inoculated sites was recorded for a period of 27 days following inoculation . in table i the td50 value given by each series is given together with its limits of error . 
the difference between the youngest and the oldest mice is not significant , but the td50 given by the oldest mice is considerably lower than that given by the mice of interthis last different , however , does not attain a significant level by mediate age . the irwin and cheeseman ( 1939 ) test . 
the systemic mechanisms by which resistance is manifested are cellular and humoral . in the case of resistance to tissue grafts , it appears that cells primarily are concerned in the resistance mnechanisms ( murphy , 1926 )  . 
young mice would therefore not be expected to possess resistance against a homologous tumour by virtue of maternal serum factors reaching them by the transplacental route before birth or by the colostrum after birth . in the course of the present studies it has been observed that a litter from a mother which was completely resistant to s37 , a tumour having regressed in her before pregnancy ensued , showed the gross developthe same high susceptibility as the litters of normal mothers . ment of the lymphatic system appears to be quite advanced in new - born animals as indicated by the presence of the thymus and by the existence of lymphocytes if it is assumed that the lymphocyte is responsible for tumour in the circulation . immunity , then the low resistance of new - born mice must be ascribed to functional immaturity of the lymphatic system . the td50 given by the titration of s37 in new - born mice , as will be seen fronm the trend of the points shown in the fig . 
1 , is about 9 viable sarcoma cells . graph suggests that the percentage of tumours to be expected from an average it is of interest to note that furth and inoculum of 1 viable cell is about 7 . kahn ( 1937 ) succeeded in transplanting leukaemia to about 5 per cent . 
kahn and furth ( 1938 ) obtained only about 20 per cent of tumours from subcutaneous sites inoculated with 50 - 100 viable sarcoma cells in circumstances where there was no genetic difference between the animal in which the tumour arose and those to which it was transplanted . 
a c3h sarcoma gave a td50 of 18 viable it is evident from tumour cells when titrated in old c3h mice ( hewitt , 1953 )  . these comparisons that the results of quantitative transplantation of s37 in newborn mice of a heterogeneous strain of which the adults exhibit some resistance to small inocula , are compared with those obtained for similar tumours within a genetically homogeneous tumour - host systethe use of new - born mice therefore provides a more uniform and more sensitive system for the detection or assay of viable tumour cells in circumstances where , owing to limited facilities or to the use of genetically unknown tumours , a genetically homogeneous system cannot be achieved . it will be seen from fig . 
2 , however , that quantitative resistance increases this being so , it is likely that very differperceptibly within the first week of life . ent quantitative results would be obtained from the titration in new - born mice of tumours with a slower growth rate than s37 . in these circumstances resistance factors could produce their effects before the tumours became palpable . 
a lymphoma which occurred spontaneously in a mouse of the albino strain used in these experiments gave a td50 of over 10 , 000 viable cells when titrated in newborn mice ; krebs ' carcinoma ascitic cells gave a td50 of over 2000 viable cells . it must be concluded from the results of titrating s37 in new - born mice that the high td50 levels obtained in adult mice of the same stock ( i.e. , about 1600 ) were due to immunity factors and not to intrinsic limitations of the proliferative power of the cells . an estimate can be made of the proportion of inoculated s37 cells which are capable of continued proliferation if it is assumed that such cells are not subject to any hazard affecting their proliferation once they have been successfully in these circumstances the dose - response curve deposited in the host tissues . would represent the chances of getting one or more " taking units " in random samples ( inocula ) taken from suspensions containing different mean densities of 390 h . 
1 , a suspension which gives 37 per cent of negative sites ( 63 per cent of takes ) contained a mean density of about 17 tumour cells per inoculum , and this evidently constitutes a single " taking unit . " thus , when it is assumed that there are no influences at the inoculated site which inhibit continued proliferation of a cell intrinsically capable of giving rise to a palpable tumour , then the results reported indicate that such cells are present in a proportion of about 1 : 17 ( 6 per cent ) of the population of cells designated as living s37 cells on the grounds of their resistance to staining with trypan blue the frequency with which abnormal mitoses and and their diameters ( > 9 , )  . cellular pleomorphy are found in rapidly growing tumours would seem to indicate that there may be a continuous production of cells which , though alive , are various observations have been intrinsically incapable of indefinite proliferation . fischer ( 1935 ) was unable to cultivate made which suggest that this is the case . solid s37 in vitro , and concluded that dying cells exceeded living cells in this lasnitzki ( 1952 ) found that the majority of s37 ascites cells failed to tumour . she found also that one - third to two - thirds of the mitoses seen divide in vitro . since it is unlikely that all mitotic in the ascitic cells in vivo were abnormal . abnormalities are visible , it is possible that the proportion of mitoses which are koller and smithers ( 1946 ) give experiabnormal in vivo may reach 50 per cent . mental proof that loss of chromosomes or of chromosome segments leads to the death of the cell . 
on the other hand , there is evidence that cells with a defective chromosome complement may survive and divide under conditions where contiguity between the cells is preserved , while being unable to do so when isolated these considerations favour the assumption from one another ( koller , 1947 )  . that a significant proportion of the s37 ascitic cells are unlikely to be capable of furthermore , if the cells should indefinite proliferation and tumour formation . be dependent upon one another for a stimulus to divide , dilution may reduce the proliferative power of an inoculum , not by damaging individual cells , but by abolishing essential mutual influences between the cells . 
quantitative transplantation of s37 in new - born mice of a heterogeneous strain gave results similar to those obtained for the transplantation of a c3h sarcoma in adult c3h mice . 
nielsen. from the danish cancer registry under the national anti - cancer league , copenhagen , denmark . received for publication april 30 , 1951 . in comparing mortality figures for cancer from various countries for the years around 1938 , clemmesen and busk ( 1947a , 1947b , 1948a , 1949a , 1949b ) found that while denmark ranked third for total mortality from cancer , her figures for females were highest among the countries investigated , surpassing even switzerland and england , for which countries the total mortality was higher . appeared that the high mortality figures for danish women were caused primarily by a high mortality from cancers of " accessible site . " from figures given in the series of articles quoted , it is seen that the incidence of cancer as expressed by the figures from the danish cancer registry for 1942 to 1944 was higher for copenhagen than for the provincial towns , which in their turn exceeded the incidence for rural areas . consequently , in the following paper the incidence of cancer in copenhagen will be studied separately , with particular reference to social conditions , social class being expressed in terms of annual house rent for various subdistricts of greater copenhagen . it is an established fact that expenditure on housing is a more reliable indicator of the social status of a family than the annual income of the breadwinner . 
the level of house rent in copenhagen was fixed during the period studied , and the wartime shortage of housing which prevailed during the entire period must have restricted the number of changes of residence . 
hence several factors working together tended to stabilize some of the variables which would be inclined to disturb a study such as this . the city of copenhagen , the capital of denmark , has one million inhabitants , another million live that is , about a quarter of the total for the whole country . in the provincial towns , and two millions in rural areas . medical facilities in copenhagen left very little to be desired during the period examined , in spite of current political and military events . 
nielsen the reference of cases of cancer to distinct geographical location has , however , inforin the present study been made without regard to the place of treatment . mation on cases was collected by the cancer registry , and each case was referred to the subdistrict corresponding to the address of the patient when first seen in hospital for cancer . 
the cancer registry collects notifications from hospitals all over denmark of all cases of cancer seen , and separately of all post - mortem examinations performed on cases of cancer . for patients not entering hospitals it is assumed that death certificates will give sufficient information , but it will appear that such cases amount to rather insignificant numbers in copenhagen . further details of the activities of the danish cancer registry have been given elsewhere by clemmesen and busk ( 1948b , 1948c ) clemmesen , busk and nielsen ( 1949 ) , and clemmesen ( 1950 , 1951 )  . the subdivision of the greater copenhagen area into 22 subdistricts used for the present study had originally been worked out for various administrative purposes , and proved useful because detailed figures for the distribution of the population by age were available for each subdistrict . 
the wealthy boroughs of frederiksberg and gentofte had , however , to be treated as separate entities because ot the absence of such information for their subdistricts . after a careful analysis of the figures for each site of cancer in each of the 22 subdistricts , it became possible to collect subdistricts within the same range of house rent into however , the average annual house rent of five major classes or " districts . " each subdistrict is given together with the relative values for cervical , mammary and pulmonary cancer in table v . in order to describe the material from a medical point of view table i has been worked out . 
we denote the observed number in subdistrict v comprising all age groups . by l we denote the total number of person - years of exposure , and by c the addition of the subscripts v computed number of persons developing cancer . and s to l and c has the same meaning as described for 0 . if the morbidity from cancer were identical for all subdistricts of the town , we would expect the number of cancer cases at a given age to be distributed on the subdistricts in the same manner as the number of person - years of exposure for a given age . consequently we obtain the computed number c , 8 from the equationlvs . and this number should be compared with the observed number ov.. now the number ov , , is usually small ; we therefore take this number for all age groups and make the comparison for all of them at the same time . 
nielsen pondingly the incidence of cervical , mammary and pulmonary cancer is given as a percentage of the values computed for each subdistrict , with full allowance for differences in age distribution of the population o.. 
nielsen between the social status of the various subdistricts and the incidence of cervical cancer demonstrated in table v is striking to anybody familiar with the city . the irregularity presented by district ii , which mainly consists of the borough of frederiksberg , showing an unexpectedly high frequency of cervical cancer , can by no means be ascribed to a particularly high birth rate in the borough , since this rate is lower than for gentofte , and also lower than the average for the - ~~~~~.... . 
from these areas patients with cervical cancer are centralized for treatment in the radiumstations of the anti - cancer league , situated in the towns of copenhagen , aarhus and odense . 
nielsen connection with the development of cancer , it should be pointed out that the cases have been puboccurrence of cervical cancer in virgins is questionable . lished of cervical cancer in children ( heckel , 1950 ) , but these have been adenocarcinomata , so that it may be desirable in statistical work to separate this group from squamous cell cancers of the cervix . 
the mincer is shaken immediately to disperse the mince if the coarse plunger has been used the cap and operating in the suspending fluid . screw are detached and the fine plunger is inserted . tlle cap and screw are replaced and mincing is completmed . the suspension may contain coarse fragments of fibrous tissue or portions of iiiince which have clotted before dispersal in the suspending fluid . these may be removed either by l ' ow speed centrifugation , or by filtering the suspension througli a column of glass ballotini of suitable diameter . the mincer , which is illustrated in fig . 
1 , is the sixth of a series desigiied to all models have proved most satisfactory for operate with grooved plungers . the purpose for which they are intended , and it might be suggested that this tvpe of mincer will , for special purposes , be suitable for preparing suspensions of manimalian tissue or fowl embryos infected with certain viruses or bacteria . i wish toacknowledge my indebtedness to w . 
it is to ' be expected on theoretical grounds that temperatures below the fi - eezing points of the eutectic mixtures of the inorganic salts would favour survival of activitv . 
an equallv pending tumour mince in drying experiments . important reason for the use of dexlrose suspending fluid . tissue mince suspended in saline or ringer solution and frozen m a thin laver progressivelv consequentlv the dr , % - mg process becomes separates from glass during drying . greatlv impeded because of the low rate at which thermai energy is conducted across the intervening vacuuwhile dried fragments mav break off and mav be 22 ' c . 
the thermal conductivity of the dried material is adequate for the rapid drying of thin layers . water is removed from the tumour suspension by the low temperature con79 ' c . 
presents no difficulty , but there densation method . are technical problems in maintaining the drying tissue at a constant temperaif the lead from drying flask to condenser is of sufficient diameter ture level . and the vacuum is adequate , the problem is not one of cooling , but of supplying the maximum of thermal energy for rapid evaporation without heating the frozen it was considered undesirable mass above the chosen maximum t - i.imperature. to attempt to control the temperature of the drying mass on the basis of thermocouple readings . there is no guarantee that a thermocouple junction can be placed at that point in the frozen mass which will be the last to dry . 
the arm of the u - tube distal to the pump is extended horizontally and then downwards , to form the exit tube from the condenser flask . this exit tube is furnished with a ground glass taper which fits the neck of the flask . 
the stirrer rod is attached bv means of a slotted clamp which faeihtates assemblv with the thermos bath and drying flask . the rubber ring which drives the disc if the variable speed gear is mounted on a wheel which can be moved along the horizontal bar . this bar is mounted on two spring - loaded arms . 
a pliotographically reduced protractor scale is cemented to a piece of perspex 2 - 5 x 13 x 65 mand the pivot is centred at the origin of this scale . the vane is a piece of photographic dry mounting tissue 3 x 8 mm . , which is attached by shellac to a piece of fine copper wire 0 - 01 min diameter . 
the combined weight of vane and wire is 3 - 7 mg . , so that the vertical component of its weight at a displacement of v from its zero position is 0 - 065 mg . ( 4 ) vibration dampers . - owing to the design of the glass - drying system and the use of a sensitive vapour flow indicator , it is necessary to check the slight vibration arising from the electric motor driving the pump . 
the distribution of the frozen rnince is readily controlled if the level of ethyl cellosolve in the freezing bath is adjusted so that the flask will float with its neck resting on the rim of the thermos jar and can therefore be spun quickly by hand . the temperature of the freezing bath may it has not yet been determined whether a preliminary 30 ' c . 
shown bv irregular movements of the indicator between 3 ' and these bursts last for a few seconds , and are separated bv shghtlv longer after the indicator has re - ached zero . 
grow and form large embrvomas which persist for months or indefinitely . the following series of experiments was , undertaken to gain knowledge of the beha - viour of such embrvo grafts under various conditions . 
cook. from the bland sutton institute of pathology and the barnato joel laboratories , middlesex hospital , london , w.1. received for publication april 5 , 1951 . the effects of high - frequency electric fields on living tissues , micro - organisms and viruses have been extensively investigated in the last fifty years . short - wave fields were first applied to animal tumours by schereschewsky ( 1928 ) using a transplantable mouse sarcoma , and by schereschewsky and andervont ( 1928 ) working with the rous fowl sarcoma ; these workers claimed to have produced complete regression of a number of the tumours following their results , together with those of subsesubjection to electric fields in vivo . quent workers who have reported investigations into the effects of short waves on animal tumour tissue in vivo and in vitro , were reviewed by dickens , evans and weil - malherbe ( 1936 )  . 
from a consideration of previous studies and as a result of their own extensive investigations performed with tumour tissue to which short - wave fields were applied both in vitro and in vivo , dickens , evans and weil - malherbe ( 1936 , 1937 ) concluded that , when heat effects were eliminated , ultra - short waves had no action on tumours or isolated tissues ; all the findings reported were , in their view , due to the action of heat . the many studies concerned with the effects of high - frequency electric fields on bacteria , bacterial toxins , viruses and unicellular protozoa which followed the original experiments of d ' arsonval and charrin ( 1896a , 1896b ) have been reviewed by burton ( 1949 )  . in commenting on the extreme variation of the results reported , burton ( 1949 ) emphasized that it still remained to be determined whether or not any action of the short waves existed apart from heating effects in the suspending medium . a recent claim was made by nyrop ( 1946 ) to have discovered a so - called specific effect distinct from heat effects , when pulsed fields at a frequency of 20 mc / s were applied to bacterium coli cultures in a fluid medium , the virus of foot and mouth disease , and unspecified tissue cultures . this claim was been criticized on the grounds that energy absorption by ionic and dipolar processes in the medium immediately surrounding the bacterium or virus might have resulted in a local temperature rise sufficiently high to destroy it , even though the general temperature of the test specimen were kept below the lethal level ( jackson , 1946 )  . 
they were between 8 and 14 weeks old when inoculated and belonged to two of the institute 's lines , which have been described by greenwood , blyth and carr ( 1948 ) , or crosses derived from them ( intensity , breeding , breeding hen x intensity cock , intensity hen x breeding cock )  . 
both the age at which the birds were inoculated and their distribution within the various line groups were determined by the exigencies of the supply . preparation of material for irradiation . for each experiment a bird with a large actively growing sarcoma of anything from 8 to 35 days ' growth from the date of inoculation was selected as a source of tumour material . 
the speed and the duration of the run of this machine was varied with each individual tumour , but was so arranged as to give homogenization of the resulting the tissue ; disintegrated material was placed in tubes and centrifuged very lightly ( in a horizontal centrifuge accelerated to 2000 - r.p.and at once decelerated ) , so as to bring all the contained air to the surface as a sharply demarcated layer of foam , which was then removed . 
the material obtained in this way , referred to in this paper as " disintegrated tumour , " was either mounted at once for irradiation , as described below , or was used in the preparation of other material for irradiation as follows : this was usually achieved in less than 1 minute . where a preparation of disintegrated tumour largely free of cells was to be irradiated , it was made by placing 4 c.c. 
the filings and tissue debris were separated off after the period of shaking by centrifuging in a horizontal centrifuge for 5 minutes at 2000 r.p.the supernatant fluid so obtained was drawn off and centrifuged again for 15 minutes at 6000 r.p.in order to throw down at least the greater part of any remaining cells . 
the supernatant fluid resulting from this final centrifugation , designated " virus suspension " , was drawn off ready for irradiation . water flow calorimeter c02 ice specimen standin wave icatoa from magnetron jacket for freezing mixture specimen at centre of waveguide ( maximum field strength ) 1 1 + 1 specimen at side of waveguide ( field of low intensity ) fig . 
with the experimental conditions used this peak field was given by1016v / a volts cm . - 1 , where a - average power in watts . where a section of the guide was filled with carbon dioxide ice the field at the central point of the section depended as well on the length of the section filled and on the dielectric constant of the carbon dioxide ice . this latter had been found ( in a separate experiment ) to be 1.6 at 3000 mc / s , and the length of the guide filled with the carbon dioxide ice was adjusted so as to be half the wave - length of the radiation in the ice . 
under suic ' h conditions the block of carbon dioxide ice was non - reflecting and the field strength at the central point was given bythe field in a frozen specimen at the central point of the carbon diqxide ice blbck differed from that in the ice in the absence of the specimen , since the dielectric constant of the frozen material differed from that of the ice . although the dielectric constant at 3000 mc / s of disintegrated tumour or virus suspension at room temperature is high ( between 40 and 80 ) , in the frozen state it drops to the region of 2 , not far removed from the dielectric constant of the carbon dioxide ice . 
the overall exposure time in each experiment was thirty minutes , corresponding to 9 x 105 pulses of 0.6 x 106 seconds ' duration each . the total time for which the field was operative was therefore 0.54 seconds in each thirty - minute exposure . 
cook surrounding the virus could take place ( experiment 1 , irradiation without cooling ) , and under conditions where precautions had been taken to prevent such energy absorption ( experiments 2 , 3 , 4 , 5 , irradiation with cooling )  . it is considered that the method of cooling employed was such as to keep the temperature of the material exposed to irradiation below 70 c . exposure to a temperature of below 70 c . 
on the other hand , absorption at a resonant frequency increases as the such absorption does not occur in water at microwave temperature falls . frequencies , but may possibly take place in large molecules or parts of them . thus , in the irradiation of frozen material , absorption by ionic conduction and dipolar orientation was avoided . 
at the frequency of 3000 mc / s . the results in table i can be attributed to energy absorption by ionic and dipolar processes in the medium surrounding the virus causing a general temperature rise well above the lethal level . thus the results of the present experiments show that any effects of the microwave field upon the rous no . 
where such heat production was eliminated , microwaves of a frequency of 3000 mc / s were found to have no demonstrable effect on the virus of the rous fowl sarcoma as judged by changes in its tumour - producing activity . 
where heat production was allowed to take place the tum6ur - producing activity was abolished . the expenses of this investigation were borne by the british empire cancer campaign . the authors wish to express their thanks to professor j . 
howatson. * from the cancer research department , royal beatson memorial hospital , glasgow . received for publication july 6 , 1953 . several attempts have been made to purify and isolate the particulate agent or virus associated with mammary carcinoma in certain strains of mice , with a view to its identification and the study of its morphology by electron microscopy . the conclusions reached by different observers , particularly about the average size of the particles identified with the active agent , have been considerably at variance . passey , dmochowski , reed and astbury ( 1950 ) and dmochowski , and passey ( 1952 ) have described an extensive investigation in which biological tests for the presence of the agent in extracts of tissues and milk from high and low cancer strain mice were correlated with the appearance of the same extracts in the electron microscope . 
they conclude that the tumour - inducing activity is associated with typical particles of diameter 200 - 300 a , preparations of high activity containing large numbers of these particles and inactive extracts containing none or very few . graff et al ( 1949 ) , using as starting material milk from riii and c57 mice , and c57 mice foster - nursed by riii mothers , claim to have isolated from infected milk , after treatment with chymotryspin and differential centrifugation , particles of diameter 750 - 1000 a which they regard as the mouse mammary carcinoma virus . 
 ( 1950 ) but this could hardly account for the large difference in size . porter and thompson ( 1948 ) carried out an electron microscope investigation of epithelial cells grown in tissue culture from spontaneous and transplanted adenocarcinoma in c3h mice . 
they found that in three out of six preparations spherical particles were present in the cytoplasm of the cells . these had a double structure - a dense centre portion of diameter about 750 a and a less dense outer zone of diameter approximately 1300 a . 
they conclude that their findings are consonant with the view that the particles represent the milk agent . huseby , barnum and bittner ( 1950 ) , on the other hand , observed particles of diameter 500 - 2500 a in extracts of milk and mammary gland tissue but were unable to distinguish in the active extracts any distinctive particles which could be identified with the virus . in view of the long period required to obtain results from biological tests for the agent , any method of assaying rapidly the activity would be of great value in experimental work . 
howatson it was thought that a new attack on the problem involving a direct comparison of extracts of tumours from agent - carrying and agent - free mice of the same strain might give a conclusive answer . for our experiments we were fortunate in having available , through the courtesy of b . 
a commercially produced atomiser or spray gun operated from the high - pressure air line was found to be satisfactory the specimen was deposited by directing a fine spray of droplets of the extract to be examined on to filmed specimen mounts . 
each droplet is a representative sample of the extract and most are small enough to be included in a single micrograph . thus to obtain information about a given extract it is necessary to photograph only a few droplet patterns instead of searching the whole field . 
as far as possible the treatment of agent - containing and agent - free tumours was identical . a weighed amount of tissue was homogenised for a few minutes in a glass homogeniser , the suspending fluid ( usually distilled water ) being added slowly to give a final tissue concentration of 2 per cent . 
both extracts contain much particulate material together with fragments of mitochondria , collagen fibres and other cellular debris . these larger fragments can be removed if desired by further centrifugation , leaving an array of macromolecular particles on a background composed of low molecular weight material , which is not generally resolved by the electron microscope . 
the particles are approximately spherical and , although covering a range of size , are predominantly of diameter 250 - 300 a , the size attributed to the milk agent by dmochowski and passey ( 1952 )  . the particle - size distribution based in each case on the measurement of 200 particles , is shown in histogram form in fig . 
the number of particles present in a given extract depends somewhat on the type of tumour , the efficiency of homogenisation and the centrifugal forces employed but provided the simple standard procedures outlined were adhered to , the variations were small . despite careful search , it was not found possible to detect any significant difference in the number , size or appearance of the particles present in the droplet patterns obtained from extracts of agent - containing and agent - free tumours . the failure to observe any difference in the tumour extracts is readily understood when similarly prepared extracts of normal organs are investigated . nearly every case examined , including spleen , liver , marrow , brain and lymph 396 a . 
14 ( c ) shows that diameter . in size - distribution , these presumably normal particles do not differ significantly from those derived from tumour tissue . it is apparent that simple fractionation procedures are not sufficient to effect a separation enabling the pathogenic particles to be distinguished from the numerous cell particulates of similar size and shape which are also present . 
14. - size distribution of particles in extracts of ( a ) agent - containing mouse m ary tumour , ( b ) agent - free mouse mammary tumour , and ( c ) normal mouse spleen . the field of the smaller animal viruses , where it has been satisfactorily overcome in only a few cases . 
an attempt was made to differentiate in this way between normal and infective particles by studying the action of the enzymes trypsin , chymotrypsin , ribonuclease and deoxyribonuclease on extracts of agent containing and agent - free tumours . 
the buffer salts , which crystallise on drying , can fairly readily be distinguished from organic material , but the latter shows a tendency to aggregate in the presence of electrolytes . this effect had previously 398 a . 
a comparison was made between extracts of the mill hill 2 endothelioma which has all the attributes of a " virus " tumour and the chemically - induced grch 16 tumour which in addition rous no . 
1 sarcoma and has no such " filterable " properties . biological tests for the infectivity normal chicken tissue extracts were examined . of the various preparations were carried out in many cases . the methods adopted were similar to those previously described , and need not fig . 
extracts of the first type regularly excite tamours on injection into susceptible birds , whereas the second type of extract , though apparently of similar particulate content , is these micrographs may be compared with fig . 
9 , which completely innocuous . shows an extract from a normal chicken spleen . it is again evident that the infective particles , if present , are masked by the large quantity of other particulate material which occurs in normal as well as tumour tissue extracts . 
the lower limit of size ( 600 a ) given by claude ( 1946 ) appears to be arbitrary . certainly most of the particles observed were smaller than this , though some shrinkage during desiccation may occur . 
the presence of still smaller cytoplasmic particles in cells fixed by osmium tetroxide has recently been demonstrated by high resolution electron micrographs of ultra - thin sections ( eaves , 1953 , private communication )  . there is no doubt that the particles observed in the present experiments are products of the disintegrated cells , the spraying method giving consistent and conclusive evidence of their presence in the extracts . 
the dissolving action of trypsin and chymotrypsin is a strong indication that they are protein in nature . the crystalline trypsin used was tested for the presence of particulate material by spraying on to coated specimen mounts a solution 100 times as strong as that used in the experiments . 
12 is shown a partially disintegrated mitochondrion with what appears to be an extrusion of particulate material . a group of mitochondrial membranes largely devoid of contents is illustrated in fig . 
it seems very probable that some of the particles observed in tissue extracts are derived from such disruptions . it is clear that the range of size of the normal cell particulates covers that of the smaller animal viruses . this is a major difficulty in the isolation of any presumed virus by purely physical methods such as differential centrifugation or in chemical composition also , the microsomes are found to be strikfiltration . ingly similar to certain known viruses , so that attempts at chemical or enzymatic separation may also fail . there remains the possibility of using the highly specific methods of serology , though even these may not distinguish normal from pathogenic particles unless the latter are of extraneous origin . the difficulty of identifying tumour - inciting particles , particularly the milk agent , by electron microscopy is enhanced by lack of independent data on size . estmates have been made by finding the centrifugal force required to clear the supernatant of infectious material . there have also been some attempts to deduce the approximate size of the milk agent by measurement of sedimentation rates in an ultracentrifuge ( kahler and bryan ( 1943 ) )  . 
the presence of normal particles and aggregates makes the interpretation of such experiments somewhat doubtful , but the evidence is in favour of the association of the infectivity with rather small particles of molecular weight 3 - 5 million , corresponding to a particle diameter of at most a few hundred angstroms . it is remarkable that no attempts appear to have been made to determine the approximate size of the milk factor particles by means of gradacol membranes , as has been done for example in the case of several fowl tumour viruses . 
by this method it should be possible to determine conclusively whether the infectivity is associated with particles in the large ( > 1000 a ) or small ( < 300 a ) size range . it is clear that further work is necessary to establish the elementary physical properties of the tumour viruses . 
the examination of tissue culture cells and of thin sections of fixed tissue may give valuable information , but is not likely to be conclusive because of the difficulty of relating the observations to biological tests . on the other hand , the method of extracting homogenised tissue , while convenient for biological testing , presents problems arising from the presence of much contaminating material which can only be satisfactorily solved by the discovery of some action highly specific to the virus , which can be used for its isolation . in conclusion it is emphasised that the presence in tumour extracts of particulate material in the size range of the smaller animal viruses is no indication that the particles are in any way related to virus activity , unless it can be conclusively 400 a . 
bartholomew 's hospital , london , e.c.l. received for publication february 11 , 1952 . the use of the fluorescence spectrum by mayneord and hieger led to the detection of 3 - 4 - benzpyrene in coal tar and to the isolation of this compound recently goulden and tipler from pitch ( cook , hewett and hieger , 1933 )  . ( 1949 ) identified it by the same means in domestic soot , which is carcinogenic ( passey , 1922 ) , and their method has been extended to samples of smoke drawn from the atmosphere . 
the apparatus is described fully in " atmospheric pollution in leicester " ( leicester , ' atmospheric pollution , ' 1945 ) , and generally consists of a small electric motor drawing air from outside a building through a filter - paper , upon which most of the suspended smoke is deposited , followed by a bottle containing h202 for the determination of so2 , and a gas meter to measure the volume of air . filter - papers are changed daily , and the " shade " of the resulting stain , as compared with a calibrated " scale of these data are collated shades " , is taken as a measure of the smoke collected . on monthly sheets showing the daily volume of air sampled , the shade number and smoke conceiltration deduced therefrom , with monthly summaries . 
vvaaller in the main experiment acetone extracted up to 50 per cent more benzpyrene . was used , and each extraction was continued until ( after about 4 hours ) no further fluorescent material could be extracted . 
the resultant golden - yellow solution was passed through a short column of activated alumina ( 3 x 1p4 cm . , spence type h ) , which removed any finely divided particles , traces of water , and certain after running more acetone through the column of the unwanted hydrocarbons . the resultant solution was evaporated , leaving a yellow oily droplet with a brilliant blue - violet fluorescence in ultra - violet light , which was dissolved as far as possible in several successive lots of boiling petroleum ether ( 60 - 80 ) ; the insoluble part was redissolved in acetone and retained separately to be checked for the absence of benzpyrene . 
the solution at this stage was pale straw - coloured , with a strong bluish fluorescence in ultra - violet light , and on passing through a column of alumina ( 8 x 1 - 4 cm . ) previously washed with petroleum ether , nmost of the fluorescent material was adsorbed as a narrow , blue - fluorescing band near the top of the column . development with petroleum ether , followed by a mixture of 30 per cent benzene in petroleum ether , produced a series of rather diffuse bands , distinguishable in ultra - violet light by their slightly differing fluorescence . 
 " osira " lamp with separate wood 's glass filter , and spectra were recorded on a hilger e.51.7 spectrograph , using a 0 1 mslit and 2 - mninute exposures with ilford " zenith " plates . all final solutions were prepared in petroleum ether , and no precautions were taken against oxygen quenching ( bowen and williams , 1939 ; miler and baumann , 1943 ; weilmalherbe , 1944 )  . schoental and scott ( 1949 ) give the fluorescence bands of benzpyrene in petroleum ether as a main system at a 4032 , 4272 and 4535 , with subsidiary systems at a 4082 , 4310 and 4149 , 4367 . 
with the weak solutions used here , only those at a 4032 , 4082 , and 4272 appeared in the negatives , and not all of these in reprothe group of hg lines around a 3650 appears in all photographs , and ductions . serves as a useful reference line . 
the position and intensity of the bands varies with the solvent ( chalmers , 1938 ; sambursky and wolfsohn , 1940 ) , but at this stage the difference between petroleum ether and its mixtures with benzene is in most cases , the spectrum of benzpyrene was visible in eluate unimportant . 30% b and not in the others . these former solutions still contained much material other than benzpyrene , some of which fluoresces in the same wave - length region as benzpyrene . 
3. - yearly cycle of benzpyrene at london ( county hall )  . the variations from month to month at any one station are greater than the difference between stations , so that a mean value over at least one year is required for any comparisons . table ii shows appreciable variations from one year to the next ; an average over something like 5 years would be required to give reliable figures . 
a general discussion on recent changes in pollution is included in the 26th report in this series ( ' atmospheric pollution , ' 1949 )  . at sheffield there is less variation during the year than at county hall , suggesting the influence of industrial smoke containing a lower proportion of benzpyrene than domestic smoke , coupled perhaps with a greater use of domestic the smloke at cannock is primarily from coal mines fires during the sumrmer . and domestic fires and shows the highest proportion of benzpyrene , with a range intermediate between that of county hall and sheffield . the seasonal values at the whole range of stations in the ntain confirm and winter concentrations exceed summer extend the conclusions drawn above . ones by a factor of three or four times on the average , and with one exception the proportion of benzpyrene in the smoke is also greater everywhere in the winter by a factor of just under two . the highest values throughout are shown by beckton , which should be regaided apart as a special case . here there is much local pollution from the adjoining gas works , which would appear to contain at least as niuch benzpyrene as domestic smoke since the proportion of benzpyrene in the smoke persists at a level above the average throughout the year . however , the effects of such abnormal conditions fall off rapidly as the distance from the source increases , for at crossness , only two miles a.way , the nmean annual concerntration is one of the lowest observed . these results serve also as a warning r . 
waller that large variations in betnzpyrene concentration can occur even within very short distances , so that to assess the mnean concentration within a town would require readings from a large nunmber of stations . hence the position of a town in the following list , arranged in descending order of concentration , indicates only the conditions close to each station , and not necessarily those in the town as a whole : london ( county hall )  . sheffield leicester bumley bilston cannock hiull bristol  . 
one nmight expect the concentration near the centre of a town to bear some relationi to the population , although many other local conditions play their part . fact , the first six towns are very nearly in descending order of population , and the low position of the large cities of hull and bristol is perhaps noteworthy . results from stations in many other towns not investigated might well be higher than most of those quoted . 
the limitation of an ordinary filter - paper is that it soon becomes blocked with smoke , and must be changed at intervals . the soxhlet thimble mentioned above , when used in series with a normal filterpaper , forms quite an efficient smoke collector , and will retain very much more smoke than a filter - paper alone . however , a more compact filter which serves the same purpose can be made from a pyrene filter disc ( type 2130 ) backed with an ordinary filter - paper . such filters required prolonged washing with acetone in a soxhlet apparatus before use for benzpyrene work . 
they can be used for collecting smoke at a low rate over a long period without being changed , but are not suitable where it is required to weigh the smoke collected , as fibres frequently drop out of the filter during use . 
the use of filter - papers of various kinds has been discussed by bourne and street ( 1950 ) , and silverman and viles ( 1948 ) have described an apparatus using pleated filter - papers . filters consisting of salicylic acid crystals lightly packed in a brass tube have also been used , but again difficulties arise in weighing the smoke , as the usual method of volatilising the acid at about 1050 c . 
a description of various air - sampling devices used in industry has been given by silverman ( 1948 )  . for rapid determinations over a short period some form of impactor is useful ( may , 1945 ; sonkin , 1946 ) , as the smoke can be collected free from all other material on glass slides . samples of smoke weighing a few milligrams were collected in a crude single - jet impactor , but as the smoke began to pile up , so the efficiency of the system dropped , thus limiting its capacity . 
waller air - conditioning plants in hospitals and public buildings , and filters on ment . air compressors in some industrial concerns , collect much " suspended impurity " from the air , which may be used for benzpyrene determinations , although it is often difficult to free the smoke from the filtering medium in order to weigh it , and in general the volume of air from which it has been drawn is unknown . 
a large sample was obtained from the compressed air plant at beckton , and has been used in pilot experiments to determine a suitable method of dealing with to extract the tarry matter without the very finely divided smoke bulk material . particles entering the solution , the sample was contained in a soxhlet thimble completely surrounded by filter - paper powder inside another thimble . since the smoke at beckton is not representative of normal conditions , no final results were obtained from it , as a more normal sample is now available from the royal festival hall , london . some suspended matter is removed from the air by a natural thermal precipitator action , in which warm air rising up the side of a building deposits some of its smoke on projections on the wall . 
a small deposit of soot often builds up near the end of exhaust pipes , and this is probably similar to the material dispersed into the atmosphere . samples taken from a motor cycle , two cars and two diesel compressors have all shown the presence of benzpyrene . uncertain , for the question of whether or not it is present in the fuel or lubricating oil used depends on their source and the treatments to which they have been subjected . 
no benzpyrene was found in particular samples of " pool " petrol , but , after the separation of large quantities of interfering substances , it was detected in a sample of lubricating oil . however , conditions in the combustion chamber may be suitable for the production of benzpyrene , so that it might be emitted in any case . 
as an attempt to identify it in domestic soot proved unsuccessful , this may indicate that a small proportion of smoke collected in towns comes from internal combustion it may also be of importance to note that whilst the particles in coal engines . smoke are distributed over a large range of sizes , those in the exhaust of internal combustion engines are concentrated in a limited range of small sizes , and that this may be quite near to the range which undergoes maximum retention in the lung ( wilson and lamer , 1948 ; landahl and herrmann , 1948 ; hatch and hemeon , 1948 ; davies , 1949 ; owens , 1923 ; boyland , gaddum and macdonald , 1947 )  . hence any further inquiry into this subject might benefit from the segregation of smoke into different size - ranges prior to the estimation of benzpyrene . for this purpose a large capacity cascade impactor would provide a suitable means of collecting the smoke . amongst miscellaneous substances tested for benzpyrene was a sample of the " coke " obtained as a by - product in the refining of petroleum . this showed a trace of benzpyrene which , if not present in the crude oil , may have arisen in a cracking process . the benzpyrene found in certain rubbers ( falk , steiner , goldfein , breslow and hykes , 1951 ) is attributed to the carbon black used as a filler . the literature on the carcinogenic action of tobacco tars and smoke contains somewhat conflicting reports , and has been summarised by flory ( 1941 )  . 
some of the apparent differences may be due to differing conditions of combustion and distillation ; only those simulating natural smoking are of any practical interest . in view of the influence of cigarette smoking upon the incidence of lung cancer ( wynder and graham , 1950 ; doll and hill , 1950 ) a few experiments were carried out on cigarette smoke , with negative results . however , one must emphasise that it was very difficult to free cigarette smoke extracts from relatively large amounts of a yellow substance soluble in petroleum ether and strongly absorbent to ultra - violet light , so that benzpyrene may possibly have been missed in these experiments , which were carried out partly on cigarette ends collected from several smokers who had avoided any contamination of them , and partly on smoke collected by a filter of salicylic acid crystals loosely packed between cotton - wool in a short glass cigarette holder . 
the former method represented normal smoking , and assumed that some of the smoke which had passed through the end would have been retained there ( mulinos and cockrill , 1938 )  . 
none has been found in cigarette smoke . this investigation has been rendered possible only by the enthusiastic cooperation of a number of individuals , including the following and their respective staffs : a . 
as this finding was of great importance not only to the general problem , but more particularly as a possible test of the validity of conclusions which were being formulated , it was decided to confirm their results . initial casual observation by nettleship and henshaw was made on female mice of the c3h strain , which normally shows a low incidence of pulmonary tumours , in an experiment in which urethane had been used as the anaesthetic . 
the urethane group in the first experiment consisted of 100 mice , the other three groups of 60 mice each , and 60 untreated mice were observed over the same period . 
the mice were examined post mortem as they died during the course of the experiment , or when the survivors were killed 185 days urethane 12 per cent ; 312 j . 
the second experiment was made on 100 mice similarly treated with urethane ; no other substances were used , in view of the negative results of the first experiment . in this experiment at the end of treatment the mice were allowed to survive until they died , with a view to assessing the persistence and growth of the adenomata . unfortunately enteritis and cannibalism reduced the amount of histological material below what was hoped for in both experiments , but not to such an extent as to make it inadequate . 
when the nodule projects above the pleural surface , is smooth and uniformly rounded , and greyish - white in colour or semi - translucent , it can be regarded with some confidence as a formed adenoma , but only a proportion of the fiat nodules will prove to be adenomata . 
at a later stage the lungs showed induration from chronic inflammation , more often than not accompanied by acute consolidation of one or more lobes . it was at this stage that subpleural nodules began to appear in numbers , and the pale miliary spots on a deep red background often presented in the latest stages , after treatment had been discontinued , a striking picture . the lungs were free from consolidation , but showed distortion of shape as a result of emphysema . 
not only is it possible to trace the stages of development of a tumour in the chronic foci of inflammation and collapse , but the striking difference in the incidence of pneumonia with urethane as compared with the other anaesthetic substances used is highly suggestive . since this work was done , larsen ( 1946 ) has also shown that urethane is so far unique amongst narcotic compounds tested ; he used a larger selection of such substances than was employed here , but without finding any other which produced a significant increase in pulmonary adenomata . in their original communication nettleship and henshaw ( 1943 ) did not appear to be impressed with the evidence of inflammation . 
they found that conditions such as bronchiectasis , purulent bronchitis and acute pneumonia were distributed alike among the experimental and control animals . they are emphatic that lung tumours and pneumonia were never seen together , it is possible that this difference though they occurred apart from each other . of interpretation may depend on what is meant by the term pneumonia . 
orr. from the department of experimental pathology and cancer research , university of leeds . received for publication july 19 , 1947 . attention was drawn to the frequency of spontaneous tumours in the lungs of mice by tyzzer ( 1907 - 8 , 1909 ) , whose description of their histology has required but little amendment . 
he has shown that bacterial growth can be understood in terms of certain simple mathematical relationships , in spite of the fact that the molecular mechanisms involved are extraordinarily complicated . in this paper an attempt is made to see if such an approacli may help to throw a little light on the problem of tumour formation and growth . 
a brief review of carcinogene8i , 3 . the various ways of producing cancer have been reviewed in a recent paper by boyland ( 1952 )  . but it will help for the purposes of the present communicatioii to provide a summary of the results . ( a ) chemical carcinogens . - the first chemical compound to be identified as a carcinogenic agent was 1 : 2 : 5 : 6 - dibenzanthracene ( cook , hieger , kennaway and mayneord , 1932 )  . since that time , the polycyclic hydrocarbons have been investigated systematically from this point of view . 
a certain degree of specificity has been observed and it has been correlated to some extent with the presence of a region of high electron density ( k region , schmidt , 1939 ) , within the molecule . some recent work has suggested , however , that the carcinogenic activity of the polycyclic hydrocarbons may be related in part to their ability to undergo autooxidation and liberate free radicals ( alexander , 1952 )  . the second class of carcinogenic compounds includes mustard gas ( heston , 1950 ) , nitrogen mustards ( boyland and horning , 1949 ) , epoxides , mesyl glycols ( haddow and timmis , 19051 ) , etc . 
these compounds are all known to act as alkylating or esterifying agents and probably prodtice their effects on the body by this type of reaction . certain inorganic salts of beryllium and zinc can produce tumours ( bagg , 1936 )  . there are also a number of other chemical compoiinds , showing little relationship 260 f . 
urethane it might , from the ( nettleship and henshaw , 1943 ) is an example of this kind . nature of its amide group be expected to attack the secondary forces of cohesion known as hydrogen bonds , which play a most important part in maintaining the structure of biological organeres such as chromosomes ( ambrose and gopalayengar , 1952 )  . ( b ) physical methods of producing cancer . - it appears that the simple process of implanting an inert object such as a disc of bakelite ( tumer , 1941 ) or sheets of cellophane ( oppenheimer , oppenheimer and stout , 1948 ) into a tissue can produce a tumour , while an extreme change of temperature produced by the application of carbon dioxide snow ( berenblum , 1929 ) can have a similar effect . although the ionising radiations can induce cancer , it is probable that their mode of action is indirect and is due to the formation of free radicals ( butler and conway , 1952 )  . ( c ) biological carcinogens . - the well - known rous sarcoma can be propagated by virus particles ( rous , 1936 )  . certain parasitic organisms are able to cause a malignant change to occur in the tissue ( e.g. , phytomonas tumefaciens in plants , smith , 1916 )  . 
the spontaneous tumours , whose origins are unknown should perhaps be considered to be due to the action of a biological carcinogen . it was pointed out by haddow in 1938 that there was a striking multiplicity of tumour - producing agents and , as can be seen from the summary given above , the number has greatly increased since that time ( hartwell , 1951 )  . 
the agents appear to have one feature in common ; they may be expected to lead to a more random configuration of the molecular structure within the cell , than is present in the normal tissue . 
we will try , at a later stage , to see ff the models may have any bearing on the behaviour of real biological systems . ( a ) classical treatment . ( 1 ) simple growth . - we will consider the very simple system shown in fig . 
is considered to contain a dilute solution of metabolite molecules ; the molecules could , for example , be molecules of amino acids . it will be assumed that the solution is ideal ; i.e. , that the molecules are quite independent of each other and do not interact with the solvent molecules . 
the work in thermodynamic which has to be done is generally known as osmotic work . nomenclature the work which must be done on the system is in this case expressed as a change ofentropy . 
1. in real systems the energy required for the decrease of entropy is in some cases balanced by a change in the potential energy , e.g. , the formation of a chemical precipitate is accompanied by a decrease of potential energy , because forces of attraction occur betw ' een the molecules forming the precipitate and a closer approach of the molecules leads to a decrease of potential energy . ( 2 ) growth with differentiation . - we will at this stage consider the problem of growth with differentiation since it has been the subject of a beautiful study by tyler ( 1939 ) , using sea urchin eggs , although the treatment is not in this case strictly thermodynamic . the cells at the two cell stage of embryonic development are isolated . 
2d. it is possible to compare the energy requirements for growth in the case of the normal and dwarf embryos by making use of the principle of dimensional analysis ( galmeo , rayleigh , tolman , buckingham )  . these principles tell us that there is a relationsfii . 
the war thickness is d / a / 2 units and fie , 2 . ( in d the longer line between arrows indicates r . ) suppose that an invaginot d - umits as in the real dwarf embryo shown in fig . 
3b the molecules are shown to be arranged in a hnear sequence , inert gas . in the form of a polymer chain . in this case the chain is coiled in an irregular in fig . 
3a there are very few regtrictions upon they are called , in the three figures . it is only necessary that one molethe arrangement of the metabohte molecules . cule should not coincide with another and that they should an be confined within in fig . 
3b we see that the number of possible ways of the sphere of radius r . arranging the units is greatly reduced , because each unit must be maintained at a 264 e . 
a statistical thermodynamic treatment makes it possible to express the entropy ( s ) of the systems in terms of the number of conformations with the most probable energy . if n is the number of conformations with this energy : k log n , where k is a constant . the logarithmic form occurs because the result is obtained by integration over a large number of terms . it wffl be seen that the simple process of polymerization must lead to a decrease in the entropy of the systems and that any process leading to a preferred orientation of the polymer molecules must lead to a further decrease . let us now consider a case in which we have a number of the spherical units of the kind shown in fig . 
4b is shown a similar corection of units which are now arranged another . if we now consider the configurational entropy in a definite sequence or pattem . of the polymer chlains in an individual spherical unit , we see that in the case of fig . 
4b the restriction in the configurations of the spheres , produced by arranging them in a definite pattern , wfll also restrict the number of configurations of the polymer chain 's inside the spheres . quantitatively , the actual energy difference . 
the building up of biological structures takes place by a complicated process of cefl division in which metabohte molecules are constantly being absorbed from the surroundings , while energy sources are broken down to carbon dioxide and water and subsequently discarded ; i.e. , we are dealing with an open system in which there is a constant flow of material . 
4 and let us suppose for a moment that they represent living cells containin ' g protein , nucleic acid , polysaccharide and lipid molecules . in fig 4a they are considered : to be unicellular organisms which do not interact in any way ; in fig . 
now we may be able to produce a series of reversible processes whereby we could bring metabolite niolecules in dilute solution first within the semi - permeable membrane of the cell by a process of isothermal compression , then into an orientated configuration by a series of compressions and extensions in the sohd state . because no process can be more efficient than a reversible process the energy changes involved would be the least that could possibly occur in the formation of these structures . in the actual biological process of synthesis , where events take place at a finite rate , the energy requirements for synthesis must be greater than they would be in our hypothetical models , we have also neglected for a reversib - le process . changes of internal energy in the systethe formation of polymers involves the synthesis of chemical bonds between the nionomer units . 
the synthesis of the peptide bond - co - nh - from the free amino acids h2n - c - - cooh is an endothermic reaction which requires a supply of energy , and the same is true for other biological polymers . 
the two factors of irreversibility and internal energy changes will require that all growth processes will involve a continuous supply of energy in excess of that which would be required for the entropy component in our hypothetical models . 
but these factors do not prevent us from calculating the entropy term in a real biological system . fowler and guggenheim ( 1939 ) point out that material which is not in an equihbirum state still has a definite entropy . 
ambrose the cell , the number of units of which they are composed is very large and although the number of configurations is restricted it is still very large . in the succeeding sections in the present paper , we shafl attempt to make some purely quahtative observations on what we might expect to be the differences in energy requirements for the growth of normal cells and tumour cells , in so far as the entropy component is concerned . 
m corresponds , as before , to b corresponds to the entropy state of the materials incormetabohte molecules . porated within the embryonic cell . this is represented as a lower entropy than the cell a . 
because the cell contains within itself a molecular structure which is capable of interacting with other cers . b ' + d represents the cell when differentiated and fulfilling its proper function . 
an attack upon the cer which leads to a more randoni condition of its parts can lead to an increase of - entropy . we might expect to find therefore that such an attack would lead to a reversal of the sequence shown in fig . 
a cell ' vh11 remain in an equirbrium state , or wir grow , depending upon whether the rate of synthesis is equal to or greater than the rate of degradation of the striictural components . 
5c the cell is considered to be in an equihbrium state , i.e. , in the condition of dynamic equilibrium in a polypbase system suggested by gowland hopkins ( quoted by baldwin , 1947 )  . 
5c represents the energy requirement for the carrying out of the proper function of the cefl in its differentiated foras for the autosynthetic system , the synthesis of extra - cellular material is also associated with a condition of dynamic equilibriumany of the extracellular proteins , for example , represent unstable svstenis . insuhn exhibts a spontaneous denaturation jaqueous solution which appears to be due to an autocatalytic mechanism ( waugb , 1946 ; ambrose and elliott , 1951 )  . in order that a steady concentration of materials can be present in the organism , continual synthesis must occur to make up for the loss . 
the regionrrepresents the reservoir of energy producing material in the cell , together with its associated system of enzymes . in the normal differentiated cell all the levelsof a , dandrare steady . 
we note that the present simple theory accounts for the existence of the steady state in in any system in which a large number of chemical differentiated organisms . reactions occur in a stepwise sequence , the products of one reaction being utihzed by the next , as occurs in a series of enzymatic reactions in a unicerular orgamsm , the growth curve should obey an exponential law ( hinshelwood , 1952 ) , i.e. , the rate of growth is proportioned to the number of cers present . various possible explanations for this difference have been suggested as for example by rashevsky ( 1945 ) who proposes that each cer prodiices some substance which inhibits the growth of other cells . 
chromosome breakage may therefore be a gross manifestation of the kind of change in the synthetic mechanism which can produce cancer cells . gopal - ayengar ( 1953 ) has made an extensive studv of the chromosome abnormalities which are found in normal cells and in tumour cells , particularly ascites tumour cells . 
the ducts can be seen in cross section ; it will be seen that they are arranged in the form of honow cylinders with the cefls forming a single layer round the wall . in a case of carc ' moma it is the duct cers which become mahgnant . 
6. as in the analysis given in section iib , it would be possible to calculate the energy differences for the three forms of growth , if a reversible process for changing from one form to the other could be visuahzed . 
the increase in surface area associated with the formation of the new surface requires that work must be done to provide the surface energy . in addition , a posit ' lve pressure must be apphed witbin the bubble . 
6 will require still more . were an elastic solid much more work would have to be done to form the hollow it would be similar to blowing up a rubber tube . 
the successive stages in the loss of differentiation , in this case of duct carcinoma , do therefore appear to represe ' nt states of increasing entropy , at least in so far as the observed changes of form are concemed . phenomena of this kind are again clearly irustrated in the very complete account of the genetic origin and morphology of the melanomata by dawson it is generally agreed that the naevi are derived from cefls which would , ( 1925 )  . in normal tissue , be engaged in building up a layer or sheet type of tissue . 
the transition from a layer structure to a warty form and finafly to the smooth malignant tumour represent successive stages in the reduction of surface area / unit mass for the hquid case . 
as in the previous example , the elastic case would require successively increasing amounts of energy for the formation of the structure corresponding to a departure from the uniformly spherical form . we will now examine some changes associated with tumour growth , which are taking place on a molecular scale , and can be considered in terms of the analysis in fig . 
this is because the refractive index of the fibres measured parallel to their length is different from the refractive hidex measured perpendicular to their length ; the brightness of the fibres , for a given thickness , is in these pictures a measure of the degree of molecular orientation which has been achieved during the synthesis , e.g. , the fibres of fig . 
the fibroblast cells are still laying dow - n fibrous material , but it is much more wavy in appearance , as was the case for the tumour derived from the uterine wall . 
the tissue was stained for coragen , using the technique of van gieson . the tumour showed a considerable reduction in the amount of collagen present as compared with the nornial tissue . 
20 still gives a considerable colour due to - collagen . it will be seen that there is practically no detectable birefringence m the tumour region which is on the right of the field . this result suggests that there is a relative decrease in the amount of orientated fibrous material deposited in the tumour region . in fig 9 is shown a section of normal breast tissue , ' hotographed under the polarizing microscope . 
a fibrous structure can be seen but there is a complete absence of any birefringence . the experimental evidence given above therefore appears to be in agreemedt with the general conclusion of section iv , that tumour growth corresponds to disordered states of high entropy and that in so far as the forms of growth and molecular orientation of fibrous protein are concerned the successive stages of malignancy do in fact appear to be associated with successive increases of entropy . it may be pointed out that a number of other instances could be quoted in favour of the general picture . 
the principle may help a httle in understanding the relationship between carcinogenesis and growth inhibition , as first described by haddow ( 1947 )  . if the carcinogens do in fact have a disorganising effect upon the synthetic system , it is more likely that in a given instance they wir disorganise the structure to such an extent that it can no longer function at all . 
 urethane ( ethylcarbamate ) has recently received much attention because of its use in the treatment of chronic myeloid leukaemia and other members of the group of diseases of doubtful nature at present called " reticuloses , " and also because of its action as a carcinogen . 
it was therefore thought to be of interest to study the action of urethane on the oxidation of tyrosine , by tyrosinase , to me1an tyrosine solution of concentration 0 - 02 per cent in buffer ph 7 4 was prepared , and ethyl carbamate solution of 10 per cent concentration in the same buffer . 
the upper limit of urethane concentration inveetigated was 5 per cent ; the lower limit at which inhibition could be detected by the method employed was 0 - 2 per cent . 
 from what is known of the chain of events in the oxidation of tyrosine to melanin ( raper , 1926 ) , it will be evident that this finding does not provide specific evidence for an action of the urethane on the enzyme . 
the further oxidation of dopa to melanin can occur without the aid of the enzyme , though the enzyme produces marked acceleration of the process , and the evidence so far given throws no light on which step in the oxidation is inhibited . 
 the method used was identical with that for tyrosine , with the exceptions that the final concentration of dopa was 0005 per cent , and the absence of the enzyme . 
 120 180 : ? 40 11 : ? 0 3692 - 6105 - 8377 095 : ? 3298 5510 7652 0901 3138 5152 7133 0893 3012 4873 6591 0860 - 2866 4639 6282 0809 2686 4257 5810 from this it appears probable that urethane affects the tyrosine - tyrosinase reaction in two ways : it inhibits the enzymatic conversion of tyrosine to dopa , and it accelerates the conversion of dopa to melan it seemed that further information might be obtained by in , estigating the effect of urethane on the enzymatic oxidation of dopa . 
on the assumption that the errors inevitably present are to be expected to operate equally in either direction , the probability of obtaining 8 results the same is only i in 128 . 
 120 180 240 lncremenu of ' ' e . ' ' 1496 2735 3714 4663 1427 2757 3768 4750 1367 2716 3768 4815 1405 2818 3957 4995 1427 2798 3925 5017 the inhibition of tyrosinase by urethane is , in view of the previously known action of this substance , not surprising . 
the autoxidative processes do not seem to have received the same attention as have the enzymatic ones , and the author has been unable to find any references in the literature to substances affecting their speeds , other than those producing marked changes in ph . 
h the urethane combines with the enzyme , and the ure thane and enzyme are present in such concentrations that they neutralize each other , there should be no difference between the reaction speeds of this solution and the control . 
the only suggestion which at present appears to cover these observations is that urethane has a greater attraction for the enzyme than has dopa , and that one or more of the first reaction products of their interaction acts as an inhibitor of the autoxidation of dopa later , when the enzyme has disposed of the urethane , and the intermediate metabolites have undergone further change to substances which presumably do not affect the progress of the oxidation of dopa , dopa is acted on by the enzyme , hence the acceleration of the reaction in its terminal stages . 
lea it must be emphasized that this explanation is purely hypothetical ; no direct evidence for the combination of tyrosinase with urethane , or of the presence of reaction product.s , either intermediate or final ( other than melanin ) , has been obtained . 
it is not suggested that the oxidation of tyrosine to melanin has any place in either the normal or abnormal metabolism of leucocytes , though there is the interesting fact that these cells do contain a polyphenolase capable of oxidizing dopa to melan but it did seem possible that other similar oxidations might be involved . 
methyl , propyl and butyl carbamates were prepared , the choice being limited to these as they are freely water - soluble , and their actions could be investigated by the method used for ethyl carbamate , and compared directly with that substance . 
it has been found advantageous to reduce these until the present level of 0001 per cent has been reached , as these lower concentrations prolong the period during which melanin formation can be esti mated by the colorimeter . 
the difference , however , is not great , an " d certainly does not seem to be of sufficient magnitude to allow of any explanation of the known action on leukaemia . 
it was thought possible that the effect of another water - soluble carcinogen and chemotherapeutic agent might throw further light on the proble the effect of di ( 2 - chloroethyl ) methylamine hydrochloride on the oxidation of tyrosine was investigated . 
it is of particular interest to compare the differences between these two solutions , when it will be noticed that the changes are , within the limits of accuracy of the method , yery regular . 
 - 0023 0071 0051 0029 - 0069 0172 - 0260 1155 1879 the suggested explanation in this case is that an intermediate metabolite of the interaction between nitrogen mustard and dopa gradually accumulates until it reaches such a concentration that it inhibits the reaction , thus masking the accelerating effect of the nitrogen mustard . 
 it therefore appears that there is a similarity of action between di ( 2 - chloro ethyl ) methylamine hydrochloride and the urethanes , and that this is in their action on autoxidation and not on enzymatic oxidation . 
it is obvious that it would be quite unjustifiable to attempt to explain the action of these substances in the leukaemias and other reticuloses on the grounds of their effects on melanin formation . 
in view of our present lack of knowledge of the reticuloses , and espe cially because of their almost inevitably fatal nature , it might be of ~nsiderable interest and possibly of value to investigate the effect on these diseases of other groups of substances which accelerate the progress of autoxidations . 
 methyl , propyl and butyl carbamat.es have the same effects , but to a lesser the water - soluble nitrogen mustard , di ( 2 - chloroethyl ) methylamine hydro chloride does not inhibit the enzymatic oxidation of tyrosine in a concentration of 0 - 2 per cent . 
pullinger. from the imperial cancer research fund laboratories and the research department , the glasgow royal cancer hospital , glasgow , c.3. received foi publication february 7 , 1952 . the suggestion was previously made that nodular adenomatous hyperplasia in mammary glands of mice of the riii ( paris ) strain , and possibly others , might provide a more sensitive and an earlier indication of the action of bittner 's milk agent than do actual tumours . evidence obtained under experimental conditions of forced activation of the glandular response to combined influence of hormone and agent was givren in support of this suggestion . it seemed probable also that spontaneously occurring nodules as well as those forcibly stimulated would afford reliable evidence of the action of the agent . 
the validity of such evidence would depend upon complete absence of spontaneous adenomatous nodules in the same strain deprived of agent . in sublines of the riiix strain ( renamed riib in accordance with international usage ) , which had been freed from agent by cross - suckling , no nodules had been found in the mammae of young unmated females forcibly stimulated , but two hyperplastic nodules were seen in 2 out of 44 females of the old breeding stock ( pullinger , 1947 )  . 
a survey of the mammae of all old females was in progress and was continued and combined with experiment in an endeavour to find an answer . the same problem , the stimulus for hyperplastic nodules in the zb strain ( c3h cross - suckled to deprive it of milk influence ) , was investigated by huseby and bittner ( 1946 ) , who came to the conclusion that all three factors , namely the milk tumour influence , ovarian hormone and susceptibiltiy that are responsible for the development of heritable mammary tumours in mice are also necessary for the development of these alveolar hyperplasias . 
the same authors gave an account of the morphology of nodules found in the mammae of mice in former high cancer strains and their hybrids after deliberate exclusion of the agent by cross - suckling . the strains examined were c3h and a , designated zb and ax . they compared the nodules with those in high cancer strains described by previous authors before knowledge of the milk agent existed . in the course of the present survey of old rilib ( formerly riiix ) breeders similar types of nodule were seen with the exception focal acinar proliferations were often but of those described as inflammatory . the squamous - celled nodules found in old not invariably discretely lobular . riiib females have been described and their origins discussed ( pullinger , 1949 )  . in relation to the problem of the presence and action of the milk agent these squamous - celled proliferations are not relevant , for it is generally agreed that tumours due to bittner 's agent are characteristically acinar formations and that b . 
the actual average number of litters was 4 to 5 . breeders were then segregated to allow the mammae to involute , thereby permitting detection of nodular hyperplasia of various sorts . under these conditions involution is completed in this straleaving main and a few branch ducts with tapered ends and no acinar structures . 
hybrids of less than i month old . extracts were made by grinding the tumours with sterile sand and distilled water , spinning at 2000 r.p.in a hearson centrifuge for 10 minutes , removing the supernatant fluid and spinning this in an ecco centrifuge at approximately 8000 g . 
the mammae of as many as possible of the rijib mice injected with test tumours were examined in bulk - stained preparations . will be seen from tables ii and iii that the f.i. 
pullinger dilute brown strain and their hybrids which bore more than 3 litters had a higher incidence of mammary tumours than those with less . the parous riiib females attained this average with 4 to 5 litters . of mice injected with riii tumour extracts 13 out of 23 developed mammary tumours . the mammary glands of all 7 which were examined , out of the remaining 10 , contained multiple adenomatous nodules , one as many as 30 . 
thus nodule incidence was increased in the riiib test mice by injection with riii tumour extracts . nodule incidence was not increased in the rilib mice which had been inoculated with extracts of the 2 tumours undergoing test for the agent . 
of 20 mice in the two experiments which were available for examination , the mammae of 3 contained acinar nodules ( 15 per cent ) , 8 contained nodules of other types , and 11 ( 55 per cent ) were free of all nodules . these figures are near the normal incidence . nodules in hvbrids are not recorded because there was no basis for comparison . thus neither from tumour incidence nor from nodule incidence was there any evidence of the presence of bittner 's milk agent in the 2 test tumours . the validity of results of tests in which grafts are used as suspected source of agent rather than primary tumours may be questioned . 
the majority of authors have been able to detect the milk agent in extracts of grafts derived from tumours which were known to have contained it in the first instance . jmochowski ( 1949 ) , who reviewed the various reports , has himself obtained evidence of the presence of agent after 42 serial transplantations . 
on that occasion he used multiple injections , but both he and others have succeeded with single injections of graft extracts . in a total of 69 test mice in the two experiments here recorded , not one tumour arose . these results are in accord with those of other workers who have tested extracts of primary spontaneous mammary adenocarcinomas in othier agent - free strains ( andervont and dunn , 1950 ; heston , deringer , dunn and levillian , 1950 ; dmochowski , 1951 )  . 
 ( personal communication ) were typical adenocarcinomas . assessment of the value of adenomatous nodules as indicators of the action of the milk agent . a comparison of the spontaneous incidence of adenomatous nodules in various groups of riii and ritib mice is given in table iv . it will be seen that among virgin females of the riii strain multiple nodules were present in 100 per cent at 9 months old . 
the relative reduction in number of nodules at 13 months after ovariectomy at 9 months is probably due in part to the difficulty of making accurate counts when the effect of ovarian hormone is still to be seen . among young virgin females of the riiib strain deprived of milk agent no adenomatous or other mammary nodules were found . 
pullinger when these nodules are present in mammae of young females of this strain of less than 12 months old , whether virgin or parous , they appear to provide reliable evidence of the action of the agent . 
this conclusion is more certain if oestrogen has been excluded by ovariectomy several months prior to examination of the mammae . breeders of the riiib strain deprived of agent , of 12 months of age or more , developed 1 to 3 adenomatous nodules in the 10 nipple regions . since these nodules were not associated with agent in the tests recorded , it is clear that they do not invariably provide evidence of the presence of agent in old breeders . they may be due to some other unknown cause . multiple adenomatous nodules in old breeders of this substrain would nevertheless provide presumptive evidence , requiring confirmation , of the presence of the agent . thus for purposes of detection of agent by inoculating suspected substances into susceptible test mice and using the adenomatous nodule as evidence of the action of agent , only young rijib virgins or breeders under 12 months old would be suitable as test animals . that is to say , they would have to be killed and examined at about 9 to 11 months old when positive results could be expected and before the nodules of unknown origin begin to appear . only if counts of nodules were required would ovariectomy have to be done because the naturally occurring hormone under normal conditions does not cause a confusing degree of focal acinar proliferation . 
nodules of purely adenomatous hyperplasia were found in about 19 per cent of parous females of 1 year old and over of the rijib strain deprived of bittner 's milk agent . 
no evidence was found that the nodules or tumours in the mammae of old parous females of the cross - suckled riiib strain owed their persistence to ovarian hormone ; neither could their origin or persistence be attributed to the milk agent . 
ludford , director of the research department , and to the board of management of the mount vernon hospital , northwood , continuity of observation was maintained to the end of these experiments . to the generosity of p . 
allsopp. from guy 's hospital medical school , london , s.e.1. received for publication march 5 , 1951 . in earlier papers ( allsopp and szigeti , 1946 ) an account was given of some of the properties of water - soluble substances which are produced from 3 : 4 - benzpyrene under the influence of monochromatic radiation of wavelength 2537a . aqueous ( bicarbonate ) extracts from the irradiated benzpyrene were found to be carcinogenic when painted on the skins of mice ( allsopp , 1946 )  . these biological tests , however , were very prolonged , four or five paintings weekly for some in an attempt twenty - six weeks being required before growths were obtained . to reduce this " latent period " experiments have been made in which croton oil was used as a co - carcinogen . observations on the effects of croton oil on the mice used in these experiments , when compared with those of p . 
of 0 - 5 per cent croton oil in acetone ( experiment la ) at first caused little superficial change . slight ulceration , as evidenced by patchy encrustation , was visible on the third day , after which epilation began . this was complete by about the tenth day . 
so soon as the encrustation sloughed hair began to grow again , and by the fourteenth day the animals appeared normal . day to day observations are set out in table ii . painting with 0 - 5 per cent croton oil at weekly intervals ( experiment lb ) caused a similar sequence of macroscopic changes , but restoration of hair growth was delayed until the third week , after which the epilation cycle repeated itself 276 c . 
no growths resulted , and histological examination of the painted skins revealed nothing more than a slight hyperplasia on any animal . co - carcinogenic effect of croton oil with 3 : 4 - benzpyrene . ( table i , experiment 3 . ) equal volumes ( 0 ' 1 ml . ) of 0 - 05 per cent benzpyrene in acetone and of 0.5 per cent croton oil in acetone were applied alternately at 3to 4 - day intervals , each animal thus receiving each agent once weekly . 
control animals were painted at the same times with benzpyrene , but with acetone instead of croton oil solution . papillomata were first seen on the experimental animals after 5 weeks ; all the survivors had malignant or " pre - cancerous " lesions by the fourteenth week , a precancerous lesion in this context being one exhibiting irregular epilation , hyperkeratosis , abnormal regeneration of hair follicles , ill - defined epidermal projections , a large increase in the number of mitotic cells , numerous abnormal mitotic figures , and leucocytic infiltration of the dermis ( allsopp , 1946 )  . 
when the experiment was discontinued in the twentieth week , 4 out of 8 surviving control animals had cancerous or pre - cancerous lesions ; the remainder showed marked hyperplasia only . 
the experiments described above , however , suggest that its action is more complicated than this simple description migth indicate . despite a marked difference in the response to croton oil of the mice now used when compared with that of strains used by other workers , berenblum 's findings ( berenblum , 1944 ) have been largely confirmed when 3 : 4 - benzpyrene was the carcinogen ; but when the hydrocarbon was replaced by the water - soluble substances which are obtained from it by the action of ultraviolet light croton oil appeared to exert a different effect since , while papillomata appeared on the mice at a very much earlier stage than they did with the carcinogenic solutions alone , a proportion of these papillomata did not develop into epitheliomata , whereas 278 c . 
the distinction in behaviour , however , was not entirely clear - cut , since the papillomata on 2 out of 9 animals did persist , growing very slowly , until they became malignant . the earlier experiments with the water - soluble substances ( allsopp , 1946 ) showed that with them the process of carcinogenesis was slow , but that it passed through the usual 3 phases of hyperplasia , papillomata , and finally carcinomata . the last stage required 6 to 10 weeks . 
the present experiments suggest that croton oil accelerates the transition from hyperplasia to papillomata - the first papilloma appeared after 13 instead of 26 weeks - but not the transition from papillomata to epitheliomata , which again took about 9 weeks . indeed it may even delay this change , or - in those cases where the papillomata were sloughedin contrast to this , benzpyrene itself appears to act rather more prevent it . quickly ; epitheliomata had developed in the animals recorded in experiment 3b of fig . 
gorer , of guy 's hospital medical school , and to m . salaman , of the london hospital , for comparing with mine the results of their experiments on the effects of croton oil on the skin of mice . 
the tumour incidence in the experimental group ( 9 / 22 , or 41 per cent ) is thus not significantly in fact , a slight falling off in the tumour lower than in the control groups . incidence might have been expected in mice that were 43 weeks older than their controls at the time of tumour development . 
campbell. from ae royal ( dick ) veterinary college , edinburgh , and the poultry re8earch centre , king ' 8buildinp , we8t main8road , edinburgh . received for publication february 5 , 1951 neoplasms involving the ovary are common in the fowl ( olson and bullis , 1942 ; campbell , 1945 )  . 
the great majority far into one or other of two categories , namely adenocareinomatous growths in various stages ofdifferentiation from the frankly anaplastic type to the sclerosing tumour , or , secondly , lymphomatous growths associated with the leucotic complex , e.g. , lymphocytoma , fowl paralysis tumour , aleukaemic lymphoid leucosis , etc . 
most of these lymphoid growths should not be classified as ovarian tumours , since in view of the generalized nature of the leucotic process it is usuary impossible to be certain whether they are primary in that organ or not . 
they are mentioned simply because of the frequent teference in avian pathological literature to " lymphoid ovarian tumours . " in human pathology , besid6s - the common adenocareinomatous types of , ovarian tumour , several other rarer forms are recognized as follows : brenner tumour ( o6phoroma folliculare ) : granulosa - cell , thecal , and luteal tumours arrhenoblastomas ; dysgerminomas . a survey of the hterature deahng with neoplastic disease of the fowl shows the apparent extreme rarity of such tumours . seifried ( 1923 ) recorded a brenner tumour of the fowl and compared it with its human counterpart , a ' nd friedgood and uotila ( i 941 ) detailed 5 cases of - ovarian " tumours " associated with virilism in two of these latter cases , tuberculosis of the ovary compheated in the fowl . the picture , and the remainder were cystic or contained smau growths which appeared to be tumours in various stages of degeneration . tentative diagnoses of arrhenoblastoma were made , but it was also suggested that they might be luteal - cell tumours or " hypernephromata . the only record of an ovarian teratoma in the fowl is by jackson ( 1936 )  . with regard to the male bird , there appear to be no reports of spontaneous seminoma or interstitial - cell tumour , but several cases of spontaneous teratoma testis have been recorded ( sheather , 1911 ; jackson , 1936 ; olson and bulhs , 1942 )  . it seemed reasonable to beheve that the absence of reports of the rarer forms of ovarian and testicular tumours was simply an indication of a lack of intensive study in this brartch of comparative pathology . it was therefore determined to examine as many tumours arising in these sites as possible . 
to date approximately 2000 cases have been submitted to a thorough post - mortem and histological scrutiny , and , as was anticipated , several hitherto unrecorded types of tumour of the fowl gonads have been encountered . it ' is desirable that an account of these should be put on record , not only because of their interest from the viewpoint of comparative oncology , but also j . 
at post - mortem a thorough search was made for metastases , and the possibihty that the gonadal tumour was not the primary growth was ruled out as far as possible . blocks of tissue were taken from many organs for histological examination , whether they showed visible abnormality or not . 
the growth offered some resistance to ' the knife , and its cut surface showed pale yellow fibrous areas containing numerous small cysts and islands of pinkish tissue . there were no implantations in the abdominal cavity and no metastases were found . histologically a large part of the tumour consists of a stroma of interlacing bands and whorls of fibrous tissue , mostly dense , but having a looser texture in there is a certain amount of plain muscle ( characteristic of the some places . avian ovary especially at the hilus ) in one part of a section . 
a comparable structure occurs in the human subject , and is well ifustrated by nicholson ( 1950 )  . ( 2 ) the next ovarian tumour to be considered is the granulosa - cell type , of which 4 cases have been studied in this series . 
on section , it was cream to yellowish in colour and displayed bands of tissue which had a tehdency to radiate from the centre of the growth . the vascularity appeared to be very poor . 
the rest of the ovary seemed normal , with a few atretic follicles , and the oviduct was that of a bird in full lay . histologl ' cally , this tumour is mainly composed of round oval or fusiform cefls , with a j . 
the periphery of the growth is largely necrotic . radiating bands of smooth muscle occur in the interior of the section . wilhs ( 1948 ) states that ovarian fibromata are probably the end result of a in that case , the rarity of the latter in fowls may account theca - cell tumour . for the equary rare occurrence of fibrous tissue tumours associated with this organ . 
as an instance of this the only case of ovarian fibroma encountered in this series is worth mentioning . it was found in a rhode island red hen in her third year , and weighed 570 g . 
maximow and bloom ( 1948 ) state that what are usually considered chromaffin cefls ( sympathicotrophic cells ) are to be found at the hilus of the human ovary . these apparently have much in common with the medullary cells of the adrenal , although they cautiously conclude that until these cells have been shown to elaborate epinephrin , their relationship to adrenal medulla must remain unproven . the chromaffin reaction of such ovarian cells is unrehable but this also applies to the adrenal medullary cells , where the staining by chrome salts may vary from practicary none to a very deep brown . as far as can be ascertained there is no record of chromaffin cells or heterotopic adrenal cortical tissue in the avian ovary . that such cells may in fact be occasionally present seems to be imphed by the following cases , and a thorough cytological examination of the region of the hilus should clear up this point . the first of these tumours to be described occurred in a brown leghorn hen which was brought for examination after developing signs of mascuhnization . the bird was said to have ceased laying , commenced crowing and to have endeavoured to copulate with other hens in the run . the comb was decidedly male in character , and plumage changes were evident especially in the neck and taff . upon post - mortem , a rounded soft yellow - brown tumour measuring 3 - 8 x 3 - 5 x 28 cwas found in the ovary , which was inactive . 
numerous irregular trabeculae containing vessels arise from this and pass into the interior of the growth . between these are sohd islands of large polyhedral epithehal cells which are in turn divided into small irregular groups by fine connective tissue staining as for reticuluthe cytoplasm is eosinophilic and faintly granular . in places it is vacuolated . 
the first view is favoured as being the more probable . ( 5 ) the next case , though not a mascuhnizing growth , is worth recording for its unusual nature , and because its interpretation could mean that besides adrenal cortical ceus , chromaffin cells resembhng those of the adrenal medura may occur in the fowl 's ovary and may undergo neoplasia in common with other heterotopic - tissues . during the examination of a crossbred fowl wbich was found dead , a large intra - abdominal blood clot was found proceeding from a rupture in the substance .of a smoothly lobulated brownish - yerow tumour of soft consistency , measuring 6 - 5 x 5 x 4 - 3 cand occupyi ' ng the site of the left ( functional ) ovary . it was - freelymovable , being only attached at the bilus , and was therefore easily dissected out . 
the healthy remainder is composed of connective tissue cells with spherical to spindle - shaped nuclei , and eosinophilic granular cytoplasm , surrounding aggregations of large polyhedral cells with abundant finely granular eosinophihe cytoplasm , large nuclei and very prominent nucleoli ( nuclear - nucleolar ratio . 1 : 3 to 1 : 5 )  . an abundant infiltration with eosinophils is present in many parts of the smau epithelial - cer moiety . a well - marked but thin layer of connective tissue separates this outer zone . from the inner , which is composed of small lobules of cens separated from each other by fine trabeculae of connective tissu ' e . 
the cefls composing these groups are large , with giant nucleoh similar to those mentioned above , and have a brownish - yerow cytoplasm in bichromate fixed haematoxyhn and eosin preparations . 
at the edge of one series of sections , the only indication of ovarian origin is the presence of a few folhcles . bands of smooth muscle are scattered throughout the section . the presence of such la ' e numbers of cers showing a distinct chromaffin reaction justifies ' a diagnosis of chromaffmoma or paraganghoma . 
the growth appears to have arisen within the adrenal capsule . another small ( 0 - 5 x 0 - 2 cm . ) tumour was found in the site of the rudimentary right goriad and closely adherent to the dorsal wall . this shows not only testiculoid elements , but also a considerable area composed of whorls and strands of rapidly dividing mesenchymatous cers , presumably a metastasis from the experimentary transplanted sarcoma . the histology of both gonadal rowths resembles ewing 's ( 1942 ) fig . 
the nests of cells below the germinal epithehum resemble the primitive sex cords of the indifferent embryonic gonad . this tumour iuustrates well the bisexuality of the embryonic fowl 's ovary . 
the rete ovarii formed from the primitive sex cords is a homologue of the testis and as is well known can develop into one following left ovariectomy of the hen , where the right gonad hypertrophies and differentiates even to the exteht of producing spermatozoa ( benoit , 1923 )  . another tumour of the ovary associated with masculinization in a light sussex hen is shown in fig . 
17. folfculoid formation and luteinization is apparent , and the structure is comparable to a granulosa - cell tumour . in fact , this diagnosis would be justifiable on histological grounds alone , but has to be modified in view of the androgenic effects on the host . it would appear that the rudimentary right ovary is not the only place where in theory at any rate , testiculoid structures could arise in the intact bird . 
champy , lavedan and marquez ( 1 939 ) state that the adrenals of either male or female castrates in the fowl frequently contain " regenerating gonadal cens . " birds ' adrenals constantly contain wolffian duct remnants in the capsule on the ventral these slowly hypertrophy following castration to form an internal aspect . epididymis - hke structure , whilst some form epithelial tubules similar to those found in the right ovarian rudiment subsequent to castration . 
spermatogenesis has not been seen in the adrenal capsule however . there does not seem to be any record of spontaneous arrhenoma arising in the adrenal capsule of the fowl , but it seems possible that the foregoing is a case in point . ( 7 ) the last of the tumours of the female gonad to be described was originally considered to be a teratoma . it is unfortunate that no comparison can be made of this case with the only comparable ovarian tumour of the fowl on record ( jackson , 1936 ) , since apart from stating its didermic character , no other details were given , and the single photograph is of the gross specimen . the present case occurred in a brown leghom hen aged about 3 years , at the poultry research centre . 
of which was withafter death 4 days later , an ovanan tumour was drawn from the abdomen . it was mottled pink , brownish and cream , of found measuring 5 x 4 x 4 cm . other growths , pinkish and soft , firm consistency and mucinous on section . were attached to the wall of the oviduct , and to the serous investment of the duodenum , pancreas and liver . histologically the ovarian tumour is the only one to show complexity of immediately below a thin capsule there are nests oflarge pale staining structure . epithehal cefls , spme of which have differentiated into cysts of varying size , lined with one or several layers of columnar epithehum . in many cases these cells are actively secreting mucus . 
at first sight two embryonic layers appear to be involved , namely mesoderm and ectoderm . the latter may be represented by the cyst epithelium forming mucinous glands , and possibly as the complex epithelial tubular structures . 
the remainder of the tumour is mesodermal in origin . ifthis is the case the presence of these derivatives of two germinal layers presumably puts it in the same category as jackson 's ( 1936 ) " didermic teratoma . " wiuis ( 1948 ) defines a teratoma as " a true tumour or neoplasm composed of multiple tissues of kinds foreign tothe part in which it arises . " the presence of cartilage can be explained by the conversion of connective tissue normary present in the ovary , to cartflage by mucoid degeneration and hyalinization . 
one of their cases exhibited metastases , but no details are given of the histology of this secondary growth or its site . several seminomas have been studied during the past 11 years and two of these are selected for description here . 
the right testis was normal both in size and microscopic structure , and no metastases were found . microscopically the left testis consists of poorly dehneated lobules of pale staining epithelial cells arranged in diffuse sheets and penetrated here and there by blood vessels . the cells are mainly spherical to polygonal through mutual pressure , but the faintness of cytoplasmic outfine often gives a syncytial appearance . 
a second testicular tumour - also involving the left organ - wa 's seen at autopsy in a buff rock cockerel . it measured 4 - 5 x 3 - 8 x 3 - 5 cm . , and on section showed an irregular yeflowish area near the caudal pole , compared with the rest of the tumour which was of a cream colour . 
the cream - coloured area , forming about two - thirds of the tumour . , is composed of typical seminoma cefls , showing in many places a marked acinar structure . luteinization of these cens is proceeding in small isolated areas . 
the question arises whether the interstitial - cell moiety represents a concommitant neoplasia of the stroma , as the acceptance of firket 's ( 1920 ) view on the connective tissue origin of interstitial cells would imply . in other words , is this an example of a " mixed tumour " - or alternatively is it an indication of the teratomatous nature of seminoma , as ewing ( 1942 ) beheves , but which willis ( 1948 ) , nicholson ( 1950 ) , and others dispute ? still another possibility exists , namely that the two cell types have arisen from a common ancestral totipotent germinal cell capable ofgiving rise to a variety of tissues , in accordance with the view put forward by innes , harvey , and dawson ( 1938 )  . a re - examination of fig . 
the last testicular tumour of special interest occurred in a cross - bred fowl , which showed during life a marked atrophy of the comb , and cessation of crowing and of other activities associated with the adult male bird . at post - mortem , a greatly enlarged ( 9 x 10 x 6 cm . ) left gonad was found , a flattened oval in shape and firm in consistency . 
at subsequent autopsy this bird had a small nodule at the site of the right gonad , whose histological structure , in the opinion of the present - writer , resembles that of a thecal - cell tumour . teilum 's ( 1946 ) case of " androma " of the testis in man may be ' another example of a feminizing tumour arising from a persistent remnant of the " ovarian " cortex , and the presence of arrhenomatoid tissue in a testicular teratoma of a .fbwl which lost certain of its male characters has already been described in the present paper . tuming to granulosa - cell and related tumours , the development of the ovary throws some light on the inter - relationship of these oestrogen - secreting growths . in the embryonic ovary it is commonly stated that the granulosa cells arise from coelomic epithehum derived from the genital ridge , whereas the thecal cens , being stromal in nature arise from the mesenchymal elements of the mesoderm . examination of the early ovary , however , shows primary folhcles composed of a central ovocyte surrounded by a ring of indifferent epithehal cens indistinguishable .from the stromal cells composing the rest of the organ . these indifferent cens appear to be the precursors of the granulosa cells , whereas the remainder of the it seems probable that this common ancestry is reflected - stroma forms the theca . in the behaviour of thecal and granulosal cees in the neoplastic state , where both secrete oestrogens as judged by the muscular and glandular hypertrophy of the , oviduct of the tumour - bearing bird , and both undergo the phenomenon of luteinization . granulosa - cell tumours in the fowl are not morphologicary identical to these in man in that they do not exhibit the characteristic " rosettes " which are often j . 
campbell a feature of human tumours , and which are considered to be identical to the bodies of call - exner occurring normally in the stratum granulosum of the human such bodies are not found in the granulosa layer of the ovarian follicle ovary . because this is usuary only one , or at the most two or three cers thick . callexner bodies are usuary considered to be due to cystic degeneration of granulosa , this does not occur in avian tumours where there is ample space for such cers . a change to take place , and it therefore seems possible that these structures may serve some specific purpose as for example a glandular function , in the ovaries of those animals in which they occur . with regard to thecal - luteal cells and the interstitial cells of the testis , it is interesting to note that they appear to have a common ancestory . fell ( 1924 ) states that the ovarian thecal - luteal cells of the fowl are derived from remnants of aborted medulla in the development of the ovary . thus they have the same histogenesis as seminiferous tissue . 
9. - structure of the above , showing vacuolated cells , due to removal of lipoids , to the left ( some nuclei are pyknotic ) and cells resembling fibrous tissue to the right of the figure . 
18. - - - - " mixed ? " ovarian tumour , consisting of areas of tortuous glands , mucinous cysts , cartilage , solid nests of epithelial cells and a myxomatous stroma . 
the medium is left unchanged throughout each experiment . films of concentrated cell suspensions made after gentle centrifugation were in the present series stained by a combined leishman - giemsa stain , and by feulgen 's method . 
about 500 - 600 leishman - giemsastained films were studied in the course of these experiments . the appearances described are those found in the basic culture medium . the addition of 5 per cent rat e.e. 
causes few differencem , apart from raising the number of mitoses . the only divergent feature is the appearance of increased numbers of degenerate polymorphonuclears , probably owing to the metabolic stimulant action of the e.e. from the cytological point of view , the life of each culture of leukaemic blood may be divided into two phases . 
the first , during which there is evidence of multiplication , and to a less extent of maturation , of the immature cells , reaches a climax during the first day , and comes to an end some time during the second 24 hours . 
from this , it does not follow that there is an increase in the total cell count of the cultures , for there is also a concomitant dying - off of mature cells . if , however , the mitotic index is high enough , as in cultures containing e.e. , there may be an increase in the absolute number of immature cells . this means that proliferation outstrips maturation . mitotic figures , very rare in the peripheral blood , are regularly found in cultures from the second hour onwards . 
they occur mainly in myelocytes , but are also seen in myeloblasts . differential counts have been done as a routine , but the total numbers of mitoses are too small to permit precise conclusions on the phase distribution ; further , owing to the technique necessary in making smears , the recognition of telophases is often difficult , and their number probably greater than is apparent in counts . no gross deviation from the ordinary phase distribution has , however , been found . in particular , there is no evidence of a metaphase arrest in untreated cultures . 
the mitotic index reaches a maximum of four to six mitoses per 1000 immature cells at about the eighth hour of culture in basic medium , or of up to 19 per 1000 immature cells at about 24 hours , if e.e. 
an extensive literature deals with tissue culture of normal and leukaemic blood and bone marrow , and has been reviewed by bloom ( 1938 ) , and more recently by fieschi and astaldi ( 1946 )  . it is not proposed to discuss this volume of work in detail , especially in view of the fact that much of it is not strictly comparable , as the methods of culturing and of examining the cells were often different from those employed in this series of experiments . thus most workers have used the cultivation of solid fragments rather than that of cell suspensions , and have described the appearances in sections instead of smears . 
comment will therefore be restricted to a few papers in which results have been obtained by related methods . there is general agreement that survival of human leukaemic cells is possible in cultures , and though the maximum times given vary considerably , no claims of indefinitely prolonged life in vitro have been advanced . 
many authors report the development of non - specific connective - tissue cell strains such as macrophages and fibroblasts from the highly specialized blood cells , but this appears to depend on the presence of a solid supporting framework , and does not occur in cultures made in a fluid medium . mitotic division of immature cells has been frequently observed and generally found normal . it is of interest to note that whereas mitoses are extremely rare in the circulating blood , they begin to appear almost as soon as blood is withdrawn from the body . the rapid change can scarcely be due to the effect of the culture medium alone , but may well be caused by the removal of an inhibitory factor operative in the body . the occurrence of maturation is a more controversial question , and the various answers which have been given appear to be mostly based on impressions . strict proof can only be obtained if total as well as differential counts are done with adequate methods , and this is only possible in fluid cultures . israels ( 1940a , 1940b ) , working along these lines , maintains that maturation occurs normally . fieschi and astaldi , on the strength of smears made from solid cultures , admit a certain degree of maturation , but state that this is always less pronounced than the proliferative processes . 
the findings cited here and in the previous report tend to support the latter view . while the normal maturative changes have not been very striking in this series of experiments , the development of new and abnormal cell types has been it is remarkable that this has not been commented upon by other of interest . workers , except by fieschi and astaldi , who , in their fig . 
7 and 14 it can be seen that the coarsening of abnormal erythropoiesis . the chromatin pattern is accompanied by its breaking up into discrete fragit is possible that ments , the nuclear membrane remaining meanwhile intact . these appearances signify an abnormal prophase , the first stage of endomitosis . the abnormality is conceived to consist in a premature separation of the daughter chromatids , which does not lead to metaphase or to a breakdown of the nuclear membrane . 
the consequence is an increased nuclear size , which may express itself by lobe formation and can , but need not , lead eventually to a more or less regular nuclear division , unaccompanied by division of the cytoplasm . in this connection the disappearance of the nucleolus is noteworthy : it is apparently a prelude to the accumulation in the nucleus of desoxyribose - nucleic acid , which is made evident by the deeper staining in leishman - giemsa and especially in feulgen - stained films . it may be objected that small numbers of polyploid cells occur normally in tissue cultures ( macklin , 1916 ) , and that their formation can be encouraged by manipulations such as the reduction of oxygen tension ( barta , 1926 ) or variations in the temperature of incubation ( stillwell , 1944 )  . 
8 with its double layer of cytoplasm has often been described and pictured as typical of the cells of " monocytic " leukaemia ( d ' antona , 1931 ) , while the resemblance of some of the polymorphs to those seen in pelger 's anomaly has already been remarked upon . it may be concluded that immature blood cells can , in vivo , undergo changes similar to those which are here reported for in vitro work . 
the special conditions prevailing in cultures made in the fluid medium , and - by the technique described , are evidently particularly suitable for encouraging this sequence of abnormal it is probable that at least events , but do not permit full normal maturation . some of the abnormalities in cultures are explained by lack of metabolic factors ordinarily present in the body , and possible that study of the metabolic processes in cultures and of variations in the composition of the medium may elucidate if successful , such a study might lead to the nature of some of these factors . a clearer understanding of the reasons responsible for the varied behaviour in culture of different bloods . 
1. received for publication february 23 , 1948 . in a previous communication we reported that a strain of the shope rabbit papilloma had been transmitted for 10 serial passages in the domestic rabbit ( selbie and robinson , 1947 )  . 
the adaptation of this tumour virus as a transmissible infection in domestic rabbits occurred during experiments in which the simultaneous inoculation of shope papilloma virus and sheep dermatitis virus in domestic rabbits produced papillomas that proved infective to other rabbits ( selbie , 1946 )  . 
the further passage of this virus in domestic rabbits was materially helped by continuing the procedure of mixing a second virus suspension with the infective inoculum of papilloma virus , and also by treating the skin with croton oil before inoculation , but there was no evidence of an increase in the virulence of the virus suspensions during passage . in the present communication we shall show that carcinomatous transformation has occurred regularly in this transmissible papillomatosis , and in a mamner similar to that found in the non - infective papillomatosis that is produced in domestic rabbits by the inoculation of extracts of papillomas from the cotton - tail rabbit , the original host of the shope papilloma virus . 
3. received for publication january 18 , 1949 . the investigation of carcinogenic substances which have been obtained from human tissues ( kleinenberg , neufach and shabad , 1940 ) and from other biological sources has been the subject of earlier publications from this institute ( hieger , 1940 , 1941 , 1946 , 1947 ) , reporting that : 1 . 
technique used in these experiments for testing for carcinogenic activity . unless otherwise stated , the substance is injected subeute ( 15 per cent solution in lard ) into mice of c57 strain or of mixed commercial stock . 
control experiment on reagents used in the saponification of tissue by potassium hydroxide and alcohol . since the great majority of the carcinogenic fractions had undergone saponification at least once , control experiments on the process itself acting on noncarcinogenic tissue components were made . 
distilled alcohol for 2 - 3 hours and then carcinogens might be formed during extracted several times with ether . saponification from precursors in the tissues or from the reagents themselves . in the earlier experiments the alcohol was industrial spirit ( the manufacturers state that it consists of ethyl and methyl alcohols in the ratio of 19 : 1 with 5 per cent water ) , which in separate experiments was found to darken rapidly on refluxing with koh owing to resinous condensation of aldehyde . acetaldehyde was therefore refluxed with koh and spirit under the same conditions as those of tissue saponification . 
 " n on - cancer liver " indicates the liver of a human subject who has died of any cause other than cancer . ( b ) fraction a from mixed cancer livers , partly the same as those used for the preparation in ( a ) , was crystallized from 80 per cent aqueous alcohol after adsorption from petrol ether on to a large column of a1203 eluted with petrol ether - benzene - meoh ( 80 - 20 - 20 )  . 
the possible reasons for the absence of tumours are discussed below . ( d ) a similar fractionation was made on another batch of cancer livers , and mice were injected with material from g , c , f , d , e and h . 
the test on f was repeated on a further 10 mice in which one tumour arose after 2 years . ( f ) the 10 fraction from human non - cancer lung - kidney - muscle ( fraction a ) had given 3 tumours in 25 mice , and was considered a potent it had been in the cold room for over carcinogen - containing fraction . 3 years and was then fractionated according to a simplified scheme ( flow sheet 2 )  . of 52 mice at the beginning of the tests , 43 survived 12 months and 26 , 18 months . 
four sarcomas appeared , 3 in fraction g and 1 in c . ( g ) the crude unsaponifiable fraction ( m ) of mixed livers which had been used in experiment ( d ) was fractionated and tested at j , k , l , g , a , c , f , m , i , using in all 110 mice , which lived well ; 92 survived 12 months and 40 , 18 months . 
a large batch of the crude unsaponifiable matter was worked up to stage a ; a part was kept for injection , and another part was acetylated and crystallized by careful seeding . 
the crystalline acetate was hydrolysed and the regenerated cholesterol tested . in the 12th month one sarcoma appeared in the a series , and in the 19th month one sarcoma developed in the cholesterol group . 20 mice were employed in each test . ( i ) cholesterol - rich fraction from cream ( cow 's milk )  . - kennaway ( unpublished communication ) had found that the unsaponifiable part of cow 's milk produced a sarcoma by injection into c3h mice . 
the experimental mice lived well ; after 29 months a mouse of the f series ( flow sheet i ) developed a sarcoma at the site of injection . ( k ) liver from a lymphosarcomatous human subject gave a highly carcinogenic unsaponifiable fraction in some of the earlier experiments ( hieger , 1940 ) , and the tissues of humans with this type of cancer may possibly be rich sources of carcinogen three livers from lymphosarcoma cases were saponified , fractionated into stages a , j , m , l , and injected into 6 , 5 , 11 and 6 mice of crossed c3h x c57 strain , which survived well , but no tumours developed . the 85 per cent cholesterol fraction ( a ) of cancer livers which had given 2 sarcomas in 10 stock mice was stored in the cold room for 2years , then tested on 10 c57 mice . 
one sarcoma appeared in the first series in the 14th month in a c57 female , and one sarcoma of fibrous type in the second in the 22nd month in a stock female ( experiment 7 , table i )  . when over 300 control tests had been carried out on lard alone without the production of a single tumour , a sarcoma arose at the site of injection in a c57 mouse first injected with lard when already 16 months old . 
fatty extract of walker rat tunmour . aptekman , king and lewis ( 1943 ) induced sarcoma in rats by injection of their results were confirmed here by the fatty fraction of walker rat tumours . tests where the tumours were dried by storing in distilled spirit and extracting with benzene . 
a after parallel group of 10 rats were injected subcutaneously with lard alone . two years one rat in each series developed a fibro - sarcoma , which grew with difficulty on transplantation . 
both tumours arose in the subcutaneous tissue at the same level as the injection site , but on the opposite side this effect has been observed several times in experiments where of the body . the injected substance is very fluid , and is probably pressed round the subcutaneous spaces by the movements of the animal 9 . 
10 stock in the 14th month in group b 2 mice developed sarcomas , and pre - neoplastic changes were seen at the site of injection in 4 other mice . thus in group b , 6 of 10 mice had either developed sarcoma or might have done so if they had lived longer . 
the two sarcomatous mice and two of the incipiently sarcomatous mice were from the same box of 5 mice . one cannot say why only group b responded so readily , but ( 1 ) in a fair number of experiments the saponification of tissue has been done in spirit but the products have produced no tumours , showing that spirit is not essential for carcinogenesis by the unsaponifiable product ; ( 2 ) the absorption spectra of the two final products ( cholesterol saponified in spirit and in alcohol ) are closely similar ; and ( 3 ) while the substance was strongly carcinogenic in group b ( stock mice ) , the same preparation was inactive in group a ( c57 )  . consequently it must be supposed that the b group of mice were either ( 1 ) peculiar genetically ; ( 2 ) peculiar congenitally ; ( 3 ) had become infected or ( 4 ) had become contaminated with carcinogen . ( 4 ) is , on the whole , not very probable , because the 1946 paper reported that in tests on 18 subfractions of the unsaponifiable fraction of human tissue , partly from cancerous , partly from non - cancerous 134 i . 
ieger subjects , of the total yield of 8 sarcomas , 7 were produced by fraction a made from 3 different sources and 1 from an m fraction ( flow sheet 1 )  . 
the concentration of 7 / 8ths of the sarcomas in one particular fraction strongly suggests that the fraction itself was responsible and not a chance contamination , for it would be highly improbable that contamination would occur only in the boxes , holding 5 mice each , containing those treated with the a fraction . the data in table mi are the results when a fraction was divided into 3 or more sub - fractions . 
pre - neoplastic changes at the site of injection of lipid . since the detection of only a few tumours could be important in deciding the technique of further fractionation , it was essential not to miss any tumours not visible macroscopically . 
30 non - cancer livers tested separately ( 6 in 30 were active ) tested separately ( 8 in 37 were active ) ratio : sarcoma mice total mice used . 12 / 56 = 21o 5 / 63 = 8o 44 / 120 = 370to0mean21 17 / 101 = 17o% 12 / 456 = 10 ' 440 = 2 30o thus the effect of testing a number of livers ( 67 ) separately was to reduce the sarcoma production by a factor of 9 , i.e. 
hieger ( 2 ) commercial cholesterol in lard gave sarcomas in about 5 per cent of 213 mice ( table ii )  . ( 3 ) the mixture formed when cholesterol is refluxed with koh and distilled spirit gave no tumnours when injected into 10 c57 mice , but in 10 stock mice there arose 2 sarcomas , and in 4 other mice of the same series changes were detected , post - mortem , suggestive of pre - neoplasia , at the site of injection . 
a total of 69 sarcomas has been obtained at the site of injection of lipoid substances in approximately 2000 mice of c57 and mrc strains and commercial mixed stock mice . 
7. received for publication june 9 , 1947 . the presence and activity of the milk - borne agent of mammary tumours of mice discovered by bittner ( 1936 ) are detectable only by the growth of a mammary tumour . 
found two nodules in one mouse out of nine of the n strain in which mammary tumours had never occurred . also when efforts have been made to exclude the milk - borne agent from high cancer strains by cross - suckling , nodules have nevertheless been found . 
thus bittner , huesby , visscher , ball and smith ( 1944 ) examined the mammae of fostered breeding females ( fostered by cancer - free maternal stock ) from high cancer strains a and c3h . 
all the females had given birth to at least three litters and ranged from 11 to 16 months old , thereby providing suitable conditions for pathogenic activity of the milk - borne agent . nodules were found occasionally , though never more than one to a gland , thus nodules were still present though in smaller numbers when efforts were made to exclude them than in mice which had been suckled on their own cancer - prone mothers . this evidence indicated that nodule incidence is not eliminated by cross suckling with the same regularity that is attainable in regard to tumour incidence . 
6. - regression of hyperplasia induced by 800 y oestradiol in spayed r3 female ( carrying the milk - borne agent ) , and persistence of areas of nodular hyperplasia ( adenomas ) at 6 months old . 
the former discrepant results ( bittner et al . , 1944 ; huesby and bittner , 1946 ) have to be remembered , although subsequent observations ( kirschbaum et al . , 1946 ) provide convincing evidence that hyperplastic mammary nodules do not occur in the absence of the milk - borne tumour agent . the second source of objection to accepting nodular hyperplasia as evidence of activity of the milk - borne agent acting alone is that certain observations , which will be referred to again later , suggest that similar cellular proliferations with acinus formation are stimulated by oestrogenic hormones alone . the problem of the exact origin of the hyperplastic nodules is of great these focal acinar proliferations precede maligimportance for several reasons . nant tumours in time of appearance and are often more numerous . they might thus provide an alternative and earlier means of detecting activity of the tumour they may also represent the earliest sign of tumour growth before it agent . can be described as malignant . if so they would provide material enabling one to determine which influences are causative and which are encouraging , in the sense in which the term is used by rous and kidd ( 1941 ) and mackenzie and rous ( 1941 ) in the development of malignant tumours . 
the discrepant results ( previously referred to ) concerning their incidence often suggest that they may be more sensitive criteria of the presence of the agent . if the nodular hyperplasias are to be used for any of the purposes suggested , it must be proved beyond question that they are in fact dependent for their origin and continued existence on the presence of the milk - borne tumour agent , and that the nodules , or some of them , are but an early stage of eventual malignant tumours . other possible causes of focal cell proliferation such as hormones must be excluded . the possibility has also to be considered that mice of high cancer strains carry more than one formative milk - borne stimulus for the mammary gland . one agent might be the cause of frankly malignant tumours and another the cause of nodular hyperplasias . appears more probable , however , that one and the same agent is responsible for both types of tumour , benign and malignant , but that its virulence varies . greater virulence would be understood as the capacity to stimulate the mammary gland cells to multiply and invade or metastasize within the life of the mouse ; less virulent variants would cause merely focal proliferations . 
whether the cell proliferations which it produces are benign or malignant depends upon the combination of its " virulence " with encouraging or deterrent forces . the experiments here recorded are concerned only with the origin , provocation and continued existence of the adenomatous hyperplastic nodules . was found possible to stimulate the appearance of nodules artificially with measured limited amounts of hormone at an earlier date than that at which they become detectable spontaneously . it was also possible to distinguish responses in the mammary gland due to hormone alone . responses to other formative stimuli were considered . mamnmae of female and rudimentary mammae of male mice respond to 180 b . 
the responses to single and repeated doses ' of ovarian hormones were examined at various intervals after application as described under experimental methods . the existence of a strain difference in response to oestrogens which was independent of the presence of milk - borne tumour agent was confirmed . 
a similar hormonal response was obtained in c3h mice giving rise to focal hyperplasias , but as this strain was not freed from the milk - borne tumour agent , it is uncertain , though probable , that the response to hormone alone would have given this atypical reaction . 
some were derived from pure lines , others from random breeding within the strathe incidence of spontaneous nodules of hyperplasia in the mammae in 26 virgin females segregated from males had previously been found to be 100 per cent after the age of 8 to 9 months . malignant tumour incidence in the same series was 69 per cent nodules in this strain are extremely numerous in both virgin females and breeders . r3x sublines free from the milk - borne tumour agent were bred by foster nursing an original family of 2 females and 1 male on a c57 ( black ) mother from the moment of birth . 
pullinger record was kept of the relationships of every mouse of these sublines so that , if evidence of the presence of milk - borne tumour agent subsequently reappeared , the progeny liable to be affected could be traced . 
from every litter containing more than one female , one or two were used for testing the hormone response this breeding provided further while the remainder were allowed to breed . mice for testing , and also controls to prove the absence of the milk - borne agent . the same breeding females also provided controls for all the male mice that altogether , 44 breeding females reached the average tumour were treated . age ( 81 months for this strain ) ; of these 24 were force bred owing to the circumstance that they failed to rear or destroyed their young , a habit to which many r3 females are prone . in three years no tumours were seen . 
the majority of strong a mice when two months old or over failed to survive the combination of this anaesthetic and ovariectomy . their survival could only be assured after they had reached the age of 2 months , if ether ' ere used . 
the ovaries of all mice were removed by the abdominal route ; they were excised of their oviducts ( uterine horns )  . together with about successful removal of all local oestrogen - producing tissue was judged at autopsy by complete atrophy of oviducts and uterus . vaginal smears were made from some of the females for 2 - 3 weeks before they were killed . it was necessary to examine the former site of uterus and oviducts in every mouse , because if the tissues were not excised as described , cysts containing clear fluid sometimes developed at the cut ends of oviducts after subsequent oestrogen treatment . 
when this was the case atrophy of uterus and oviducts was never complete , and the impression was gained as the result of much experience of mammary gland changes that these cysts were themselves secreting oestrogen . this difficulty was soon overcome at a time when oestrus records were not being made in spayed mice , so the matter was never settled . 
the oviducts became very slender tubes ( brownish in r3 and r3x mice ) and the uterus was greatly shrunken . oestrus cycles ceased in all those mice which were tested and did not reappear . 
any mice that had developed intraabdominal cysts or ligature abscesses , thereby possibly concealing cysts , at the cut ends of the oviducts , or whose oviducts had not undergone atrophy at the 16th to 20th weeks of experiment , were discarded . 
the purpose in using it was to give a limited measured amount of hormone within at most a few hours in order to obtain a limited response rather than a cumulative one due to prolonged irregular absorption as from oily solutions or pellets . 
the results indicate that responses in females vary quantitatively with dosage and qualitatively with strain and , within the strains , with ages younger than 49 to 56 days old . for the purpose of the present experiment most of the females received two doses of 400 microgrammes at 20 days ' interval . 
the response is visible owing to its atypical nature in respect of a milky secretion which renders ducts and clusters of acini visible . mice examined in vivo in early stages and all others were finally killed . all 10 mammary glands ( or fat pads in males ) were stained in bulk , cleared , inspected , and whole mounts made of representative mammae or those showing special features . the method of staining in bulk differed little from that described by gardner ( 1934 )  . after 4 days ' fixation in bouin 's fluid the mammae were dissected off from the skin , muscle and covering fascia with the aid of a dissecting microscope magnifying 7 and 14 times . nerve fibres were removed and some of the large blood vessels . these stripped mammae were then immersed overnight in 50 per cent alcohol , and next stained for about 4 hours in 1 part of ehrlich 's acid haematoxylin freshly diluted with 3 parts of a 2 per cent aqueous solution of after differentiating for about 10 minutes in acid alcohol ( 3 per acetic acid . cent hc1 in 50 per cent alcohol ) the stained glands were rendered blue or blueblack in several changes of 50 , then 70 per cent alcohol for as many hours or days as were required to develop a deep colour . they were next dehydrated and cleared in the usual manner . while in the first change of clearing agent ( xylol ) notes were made by the author after examining all 10 mammae or fat pads with the binocular dissecting microscope . 
the ducts were distended and filled with milky secretion , which rendered the main branches and many smaller ones visible to the naked eye in all 10 mammary glands . there was also a pseudolobular alveolar differentiation with great multiplication of acini arranged in clusters in all 10 mammary glands . 
a similar response was given by the maximum dose of 400 y of oc - oestradiol repeated at 20 days ' interval ( total 800 y ) in 15 out of 15 mice which were examined in ' ivo . 
pullinger limits of outgrowth attained during normal pregnancy . it is pathological also in that the degree of differentiation is suggestive of a 9th - 12th day pregnancy while the milky secretion is voluminous . male glands reacted to larger doses in a similar manner . 
some light on this problem was gained from experiments similar to those here described using the double dose of 400 microgrammes of oestradiol on spayed f.1 hybrid females of the c57 x r3x cross . the curious observation was made that a similar lobular - alveolar response was provokedin all the mammae , but it was localized in approximately half of each gland . 
no inflammatory or metaplastic nodules were encountered . in the course of these experiments the possibility had to be considered that in the females some other sources of oestrogens might be stimulated by ovariectomy and after cessation of the action of artificially applied hormone to produce oestrogens , as smith and bittner ( 1945 ) found in the c3h strain . that no such stimulation had occurred was proved by making vaginal smears during the last two to three weeks of the experimental period in 11 out of 20 mice , and by inspection of uterus and oviducts at autopsy . in no case was there a return of oestrus or failure of uterus and oviducts to atrophy . r3 virgin females deprived of their ovaries but containing the milk - borne tumouqr agent . early changes in the mammae in response to ovariectomy and oestrogen treatment ( ketohydroxyoestrin and oestradiol ) were similar to those described in the same strain deprived of the tumour agent ( 32 out of 32 mice )  . 
by the vaginal smear technique , oestrus in 30 cells were seen on one occasion in each of 2 mice out of 32 examined . mice oestrus cycles or cells did not recur . the contrast between the mammae in the two sets of mice at 16 to 20 weeks there was complete absence of adenomas in those mice which was striking . had been deprived of the milk - borne tumour agent , whereas in mice of the original r3 strains carrying milk agent adenomas were numerous in nearly all fig . 
a few imperfections due to incomplete regression did not cause confusion , and will be referred to in this connection again . the conclusions were drawn that the foci of hyperplasia ( adenomas ) in the r3 females carrying tumour agent , were due in respect of their origin and persistence to the presence of a milk - borne tumour agent , presumably identical with or a variant of bittner 's agent , and that this agent had been forced into activity by the large dose of - hormone . these nodules are considered to be experimentally produced counterparts of those that occur spontaneously in all r3 females after the age of 8 to 9 months . they are similar to those described in great detail , and finely illustrated by gardner ( 1942 ) in other strains . 
an average of 6 mammary glands per mouse developed from rudiments and all examined revealed the atypical response characteristic of females of this strain . irregular lobules were seen after cutaneous application of 8001200 microgrammes of oestradiol with or without 1500 microgrammes of proregression required 6 to 7 months to become complete , leaving gesterone . bare atrophic ducts ( table ii )  . r3 males containing milk - borne tumnour agent . when additional time had been allowed for regression of hormone response , results were similar , and almost as regular as in spayed females containing milkborne tumour agent . either oestrogen alone ( 1200 microgrammes ) or combined with one dose of progesterone ( 1 , 500 microgrammes ) was sufficient to stimulate the growth of adenomas ( table ii )  . 
tumours in the other line of descent behaved similarly as far as the fourth passage . three tumours of the implants of two of the tumours grew in fourth generation were transplanted . female mice but not in males . 
the two lines thus converged , without abrupt changes m beha - viour . one other br tumour ( br4 ) grew onlv in females in the first three pa& - - , ages and in the fourth grew equallv in males and females . 
thev oozed milk - v fluid when cut some discharged similar fluid through ulcerated evsts with milk - v or creamv fluid patches of skin . aahen slices of tumours were fixed in formol sahne the secretorv tumours were evident to cursorv inspection bv reason of the milkiness of th.e histological examination showed aceumulations of secretion , patchv fixative . tumour acini ' distended with secretion were contiguous with in dlistribution small . 
tumours from 12 out of 14 mice were milk - v in greater or less degree : in one of the two mice with non - milkv tumours tl ; e stilboestrol pellet was ' not found post mortem and e ' vidence of oestrogenic action on the sex organs was lacking . secretion was thus dependent on hormonal stimulation . 
the new observations support that conclusion by demonstrating two responses of establisbed transplants to hormonal variations in the host . pregnanev in females and artificiarv administered oestrogen in males elicits gross milky secretion in some transplanted eisen 's observations ( 1941 ) show similarly a pronounced though tumours . patchy secretion , in transplantable mammary carcinoma in rats . 
in response to oestrogenic stimulation above physiological levels , although transplantation was equahy successful in male and female hosts . ' " ithdrawal of oestmgen from male mice stops the secretion but growth continues after only a temporary secretion in response to hormones is a properkv independent of . 
the acceleration of growth in two transplantable stra ' during pregnancv is another specific response ; the growth of transplantable tumours in general is either unchanged or inhibited during pregnancv . 
the two responses , by secretion and by accelerated growth , are independent of each other , and each is manifest in only a minoritv of those transplantable tumours whose growth is inhibited in male hosts . the inbibition of implants in male hosts is sometimes partial or doubtful at the first transplantation or after several transfers . 
wider search would probably " close stih greater variety in the type and degree of response to hormones , and many different combinations of the relatively independent responses exempfified in the establishment of implants and in the growth and secretory activity of established tumours . 
tumour bri , whose growth at first was consistently inbibited in male hosts , lost this characteristic and grew equally in male and female hosts ; concurrently its growth accelerated . 
two other tumouir strains changed similarly under obser there is an evident tendency for hormone - responsive tumours to lose their distinctive quahties ; if suflicient opportunity is provided by repeated transplantations they attain the unresponsive state which characterizes the generahty of transplantable mamm ry carcinomata in mice . 
the hormonedependence was not correlated with lack of milk agent . pm - gnancy in females and an artificial oestrogen in males ehcited gross milky secretion in mammary tumours of some transplantable strains . 
rinaldini. from the strangeways research laboratories , cambridge . received for publication april 25 , 1952 . the nitrogen mustards have been shown to have a marked inhibitory action upon cell division , a property that warrants their inclusion in the miscellaneous group of the " mitotic poisons " ( loveless and revel , 1949 ) and their use as chemotherapeutic agents in certain forms of malignant growth ( haddow , 1947 )  . some of the 2 - chloroethylamines are mutagenic in drosophila ( auerbach and robson , 1947 ) and in neurospora ( tatum , 1947 ) , and more recently both the alkyl ( boyland and horning , 1949 ) and the aryl ( haddow , kon and ross , 1948 ) derivatives have been shown to exhibit tumour - inducing properties . the present work deals primarily with the effects of p - amino n - n - di - ( 2 - chloroethyl ) aniline hydrochloride on the number , duration and distribution of mitoses in tissue cultures during the first 1 to 3 days after exposure . 
though not very adequate for detailed cytological work because of the smallness and large number of the chick chromosomes , this material was found particularly suitable for quantitative treatment as regards variations in mitotic number and duration of the mitotic cycle . 
mitotic counts in fixed and stained cultures . this method furnishes data on the total number of mitoses and on the number of cells undergoing each phase of division at a given moment , and therefore an indirect estimation of the delay or arrest at any stage of the cycle . 
the number of abnormalities can also be ascertained . the cultures that showed the most abundant growth after two transplantations were selected , and the explants were cut in halves as closely similar in size as possible , under the dissecting microscope . 
all the solutions used here were made up in tyrode fluid . the cultures were stained with ehrlich 's haematoxylin after hydrolysis in n hydrochloric acid for 8 to 10 minutes at 600 c . 
 ( hughes and fell , 1949 )  . total mitotic counts were made in every culture . in order to facilitate counting the whole of the mitotic cycle was considered to be comprised within the period between the disappearance and the reappearance of the nucleoli . exception was made , however , for those cells in which the nucleolus persisted in advanced prophase and , conversely , for telophases in which the nucleus was fully reconstructed before completion of cytokinesis . 
low power phase - contrast cinemicrography . this method permits a direct , though only approximate , measurement of the rate of division and duration of each phase in a group of cells . for film recording the cultures were mounted in an air - tight chamber made with a metal frame between two coverslips and sealed with paraffin wax ( hughes and swann , 1948 )  . 
rinaldini the apparatus , devised by canti and lately improved by hughes , consists of a phase - contrast microscope encased in a thermostatically controlled chamber and attached to a 35 mcine camera . 
the chromosome movements can be seen in detail , and it is possible to measure accurately the duration of each mitotic phase . the technical arrangements were similar to those for the low - power film recording , except that critical focusing and phase adjustment required constant observation , and the time interval between frames was much shorter . 
rinaldini less concentrated solutions of the order of 1 : 50 , 000 to 1 : 100 , 000 - 74 x 10 - 6 and 37 x 10 - 6 molar - allowed indefinite survival and successful suboultivation , although the outgrowth was smaller than in normal cultures . 
at this stage the treated cultures showed no sign of recovery , but on the contrary there was a visible difference in the area of the outgrowth as compared with the controls . this was measured with the aid of a camera lucida as shown in fig . 
lindley using test " t " for p = 005 , the x2 analysis of contingency tables and the analysis of the variance on the square root of the counts . 
the percentage of abnormal proand metaphases taken together was approximately 35 per cent after 24 hours and 20 per cent after 48 hours , while the number of abnormal anaand telophases was only 4 per cent after 24 hours and reached 12 per cent after 48 hours . in spite of the normal appearance of the resting cells , all the evidence obtained from the counts points to the fact that almost 100 per cent of the inhibitory action was exerted during interphase . 
rinaldini no difference was found between the duration of 10 normal mitoses photographed during the first 5 hours and the last 5 hours of an experiment , nor between peripheral and central cells . 
a similar case was recorded under high magnification ( see below )  . six cells could be followed from one division to another in the control cultures . the intermitotic period varied from 5 to 20 hours , with an average of 10 - 6 hours . cells in the middle had a longer resting stage than cells in the periphery of the outgrowth . in the treated cultures no cell could be found to divide twice in one film ( 20 to 24 hours ) , but due to the scarcity of cell divisions it was not possible to decide whether the resting stage was prolonged or whether cells were prevented from dividing again . high power phase - contrast cinemicrography . the results have been reported elsewhere by hughes ( 1950 ) , and i shall only briefly summarize them here . 
with dilute solutions a large proportion of cells are prevented from entering mitosis , but no pycnosis or vacuolation appears . proand metaphase abnormalities this effect is exerted mainly during interphase . are conspicuous and anaand telophase abnormalities rare during the first 24 hours , anaphase abnormalities increasing after this period . 
the duration of the first two phases is significantly prolonged in some cells . cultures recover from these effects when transplanted into a fresh medium after 48 hours and hardly any residual effect can be detected . 
on the 4th day the males were removed from the boxes and 39 females which had shown plugs were placed in separate boxes , these constiforty - three unmated female mice of similar tuting the experimental group . average age , and wherever possible litter mates of the experimental mice , made the control group . on the day of separation , each of the mice received a subcutaneous graft , roughly 2 mx 2 mm . , in the right flank , from a 25 - day - old transplantable methylcholanthrene - induced spindle - cell sarcoma growing in cba mice . this tumour retained the original histological appearance and characters of steady and uniform growth through successive generations , and was chosen because its rapid growth was considered an advantage in view of the shortness of the pregnancy and lactation periods in mice . from the 10th day following grafting the tumours were measured every 4th or 5th day in 2 diameters ( length and breadth ) until the end of lactation , i.e. until 19 days following parturition . 
1 by curves obtained by plotting the average of the sums of 2 diameters ( i + b ) against time , as described by haddow ( 1938 )  . because of the difficulty in obtaining a large number of pregnant mice at one time , the experiment had to be performed on 6 batches of mice , 25 - day - old tumours of successive generations being used for grafting . 
and the mice were kept on a balanced diet of commercial food pellets and water ad libidum . eventually the hybrids formed the following groups . group i consists of the offspring of males no . 
1. of the original 2 sisters , the segregated one died after her first litter , and was replaced by a third sister which was also segregated for birth and rearing of her first four litters . she was left with the male for the birth of her fifth litter ( table i . ) group iii consists of the offspring of 2 females , each permanently in the cage the paired segregated sisters of each died with males no . 
two sets of unrelated sisters were mated to male no 11 . ( table ii ) ideally all the segregated females should have been sisters of the female left with the male , but this was not always possible . the result of the experiment is summarised below : c3h . 
 ( mothers kept with male ) cba.c3h 11 mammary tumours out of 82 hybrids . ( mothers segregated from male ) , , 84 the incidence is expressed as the number of mice bearing mammary tumours as numerators over the number of survivors aged 450 days ( age of youngest tumour - bearing mouse in this series ) as denominators . all these mammary tumours appearing in the hybrids were confirmed histologically by p . 
2b , segregated from the male , developcd a mammary tumour at 538 days and 387 days respectively . these tumours could be attributed to transmission of virus in the mothers ' milk , or to infection from the c3h male in each case , or they may be unrelated to the in the case of female no . 
no definite conclusion can be drawn from the occurrence of such sporadic tumours . ( b ) andervont and dunn ( 1948 ) have shown that c3h males can carry the mammary tumour virus in their vesicular glands and secretion , and have suggested that the mother may be infected during copulation . foulds ( 1949 ) has shown a similar transfer of virus by riii males to their fl hybrids with c57 black females . bittner ( 1952 ) has studied similar crosses between high and low mammary cancer strains , with and without the agent , and has reached essentially the same conclusion about the role of the male in transmitting the agent . 
iversen. * from the institute of pathological anatomy , university of copenhagen , denmark . received for publication june 11 , 1948 . earlier investigations by weigert and mottram ( 1946 ) have shown that 3 : 4benzpyrene after administration to the animal organism is metabolized to at least four different derivatives , labelled by weigert and mottram as bpx1 , bpx2 , bpf bpf . 
the diameter of the particles of the emulsion was between 6 to 15k . immediately after the emulsion was prepared it was injected intravenously in patients suffering from myeloid or lymphatic leukaemia . 
emulsion. blood samples ( one part 3 - 8 per cent sodium - citrate solution plus 9 parts blood ) were taken from a cubital vein immediately before , immediately after and at various times after the injection . 
2. - absorption curves of plasma extracted with ethanol before and after intravenous injection of 3 : 4 - benzpyrene . in order to make sure that the emulsifier ( postonal ) itself had no effect on the amount of nitrogen in plasma , 8 g . 
2 shows that the maxima of absorption characteristic of 3 : 4 - benzpyrene are , little by little , altered to maxima of absorption characteristic of the metabolites labelled as bpx1 . 
iversen the results of kjeldahl analysis on the precipitates showed that there was no noticeable difference between the percentage of nitrogen in the different samples of precipitate , which may mean that the composition of protein in plasma does not alter considerably in relation to an intravenous injection of 3 : 4 - benzpyrene , or that it varies so little that it has been impossible , with the technique used , to determine the difference . it would be possible to obtain a quantitative estimation of the proportion between the amount of unchanged benzpyrene and changed benzpyrene , and thus get an expression for the speed with which the metabolism ( or detoxication ? ) of 3 : 4 - benzpyrene takes place by determining the proportions between the extinction at 380 m , u . 
the same quotient for the x1 derivative is unknown and could not be determined , as it was not possible to obtain the x1 derivative in a pure form , but the quotient must be higher than 1 00 , as the absorption of the xl derivative is more intense at 380 m ,  . 
the curves further show that the time at which the rate of conversion is highest nearly coincides with the time at which the decrease in nitrogen - ( protein - ) conadditionally the curves indicate that tent of the plasma is most conspicuous . after the maximum concentration of changed hydrocarbon has been reached , elimination takes place at a constant speed . that the conversion may take place in the liver after an intravenous injection is an obvious consideration , and the following experiment would indicate that this is the case in rabbits : blood is transfused through a living rabbit liver ( nielsen , 1933 ) , and to the circulating blood is added 10 mg . 
3 samples of blood are taken before and at various times after the injection . shows , as in the case of human beings , that the absorption maxima of 3 : 4benzpyrene are altered gradually to the maxima of absorption characteristic of that these curves appear higher and higher on the ordinate the x1 derivatives . axis may be accounted for by the fact that it is not possible for the system to the variations in the quotient 380 / 385 m , u . 
iversen perhaps to be mentioned in this connection that the patients during , or immediately after , but never later than half an hour after the injection , sometimes got slight attacks of pulmonary infarcts . whether one considers the changes in the amount of nitrogen ( protein ) in plasma as attached to the metabolism of 3 : 4 - benzpyrene or as bearing no relation fig . 
 ( left ordinate axis )  . c and d give the corresponding relative amounts of x2 derivative calculated in the way described ( right ordinate axis )  . 1n 135150 us5 so o9 ? 0 150nt240 552t70 from fig . 
no sarcomata were found , however , and the local trauma to the squamous epithelium of the crop apparently favoured the local action of the oily solution of carcinogen . further experiments designed to assess the possible action of 2 - aaf in arachis oil eneysted in the submucous tissues of the crop have been in progress for about 4 years , so far with negative results . 
of 2 - aaf was injected repeatedly into the crop of each bird . sixteen white leghom cockerels of our inbred flock , aged 5 months at the start of the experiment were injected twice weekly for 4 months , then once weekly for a further year , and then at less frequent intervals , determined by their gene ' ral state of health . they were kept on open grass range with access to unheated fowl houses and fed twice daily with commercial poultry mash and pehets . 
no foreign body was present in the ulcer or underlying tissues . halfway along the oesophagus between the buccal ulcer and the crop , a small pedunculated translucent polyp 3 min diameter projected from the posterior wall . 
both kidneys were very mottled with white spots and small retention cysts ; the left kidney also contained a firm white tumour pressing on the sacral plexus ( cause of paralysis )  . 
3412. this bird had repeated i ' ections of a 0 - 5 per cent solution of methylcholanthrene ( mc ) in arachis oil into the wan of the crop with a total dose of 30 mg . 
for 3 months past it had had a palpable tumour in the neck and this had grow - n to a mass about 10 cin diameter when the bird was kined . 
a biopsy taken when the tumour was first noted showed a solid cehular tu ' mour with little differentiation and with a dehcate stroma with little or no intercellular substance . 
the tumour was considered to be an anaplastic carcinoma . cell suspensions from biopsy material were injected into 12 birds with negative results . material taken post - mortem showed a fleshy , spindle - cened tumour very similar histologicary to the chemically - induced sarcomata that are the usual response in fowls to injections of carcinogenic hydrocarbons . grafts of fresh tumour tissue were made into 13 birds with negative results . these 2 tumours contrast with those induced with 2 - aaf in which the diagnosis of carcinoma was in no doubt . n : n - dimethylamino8tilbene ( dmas )  . - as this siibstance has been shown by haddow and his co - workers to be a versatile carcinogen for rats it was thought that it might also prove an epitheliotropic carcinogen for the fowl . thirteen white leghorn cocks aged about 5 months were injected periodically into the pectoral muscles with 0 - 4 per cent solution of dmas in arachis oil with a total dose of from 0 - 5 g . 
to 0 - 8 g . these birds died or were killed between 78 and 208 weeks from the start of the experiment but no tumours were observed in any organ . 
the present experiment shows that 2 - aaf is similarly active against a variety of epithelial tissues ih the fowl and confirms the earlier report by bielschowsky and green ( 1945 ) that it is carcinogenic for the kidney of this species . in contrast with 2 - aaf , n : n - dimethylaminostilbene , which is also a versatile carcinogen for rats ( haddow and kon , 1947 ) , caused no tumours in fowls in our experiments . thus it seems that the epithehal tissues of the fowl are much more resistant to the action of many carcinogenic hydrocarbons than are those of rodents . however , monkeys also seem to be relatively resistant to the action of carcinogenic hydrocarbons ( pfeiffer and allen , 1948 ) , and it may be that we should rather regard the small rodents as being unusually susceptible to these agents . it is unfortunate that all attempts to transmit the 2 - aaf induced carcinomata have failed so far . 
 evidence offered by calcutt ( 1949 ) showed that 3 : 4 - benzpyrene is effective in inducing the oxidation of the - sh groups of a variety of compounds , the benzpyrene itself also undergoing oxidation during this process . 
on the basis of physical properties and fluorescence spectra it was suggested that the benzpyrene derivatives obtained in this fashion were comparable to those obtained as a result of the metabolism of the hydrocarbon by mice . 
this suggestion is important in view of crabtree 's ( 1947 ) conclusions as to the involvement of - sh groups in the carcinogenic process , and also in relation to powell and calcutt 's ( 1949 ) conclusion that the availability of sulphydryl influences the metabolism of benzpyrene . 
 studies by weigert and mottram ( 1946a , 1946b ) showed that the initial step in the metabolism of benzpyrene is conversion to either 8 : 9 - dihydro 8 : 0r1 - 9.0h benzpyrene ( bpx1 ) or 8 : 9 - dihydro 8 : 0r1 - 90r2 benzpyrene ( bpx2 ) , r 1 and r 2 being unidentified radicals . 
at the same time it was shown that these compounds were convertible by simple chemical methods , which have their counterpart in the animal body , to the final excretion products - 8.0h benzpyrene and the 5 : 8 quinone . 
 it was suggested by quick ( 1937 ) that the primary step in the metabolic oxidation of hydrocarbons is a union of cysteine with the unsubstituted aromatic ring , replacement of the mercapturic acid by a hydroxyl group occurring later . 
 there is no direct evidence for this view , but the recognized involvement between carcinogenesis and sulphur metabolism ( crabtree , 1947 ; calcutt , 1949 ) is in its favour . 
it is , however , severely criticized by boyland and weigert ( 1947 ) , who point out that phenylcysteine is excreted as phenylmercapturic acid - - - evidence which suggests that the linkage between cysteine and an aromatic compound is not easily broken in the body . 
it is apparent that such a scheme could apply equally well in the case of the chemically related benzpyrene , bpx1 or bpx2 arising from the substitution of the hydrogen of one or other of the hydroxyl groups of the primarily formed dialcohol . 
an alternative possibility in line with the perhydroxylation view is feasible since autoxidizing thiols are known to give rise to hydrogen peroxide ( schober ! , 1932 ; holtz and triem , 1937 ; schales , 1938 )  . 
 if a perhydroxylation process is the initial step in both the in vivo and in vitro oxidation of benzpyrene it appears that it should be possible - under suitable conditions - to oxidize the hydrocarbon directly with hydrogen peroxide . 
 the addition of 6 per cent hydrogen peroxide to a colloidal suspension of benzpyrene in distilled water had no effect at room temperature even when the two compounds were maintained together for long periods . 
since hydrogen peroxide contains acid as a stabi lizing agent , any derivative of the bpx1 type formed in the reaction would be expected to break down to the phenol and thence to the quinone - a sequence of events which superficially would resemble those found . 
 as an attempt to counteract any influence due to the acid the experiment was repeated using the benzpyrene colloid suspended in a clark and lubbs buffer solution at a ph of 75 . 
this time the hydrogen peroxide was added tilowly ; whilst at intervals the ph was checked , and when necessary was restored to 75 by the further addition of 02m . 
examination of the fluorescent spectra of the solution derived from the three portions gave results as below : surface : - two diffuse maxima at 420 and 440 able from fluorescence spectrum of bpx1 derived from mice . 
the likelihood of it actually being 8.oh benzpyrene is enhanced by the fact that after transfer to petroleum ether and standing for a few days it lost its blue fluorescence and achieved a barely perceptible yellowish tinge , behaviour which would be in keeping with the known autoxidation of 8.oh benzpyrene to benzpyrene 5 : 8 quinone . 
the passage of a weak solution of an authentic sample of 3 : 4 - benzpyrene 5 : 8 - quinone through the same column resulted in concentration over the previous zone , and , although the zone slowly passed down the column , no further elution or variation in solvent allowed any separation of the two zones . 
the similarity of fluorescence spectra , physical pro perties and behaviour suggests then that the material separated from the reaction mixture is similar to , even if not identical with , the bpx1 from mice and the bpx1 - like material formed in the presence of autoxidizing thiols . 
 further examination of the substances extracted from the other two portions of the original chromatograph column produced little of interest other than that the material was water - soluble in both cases . 
attempts to utilize the benzpyrene in solution in organic solvents have so far failed , the only results being the formation of small amounts of apparent 8.oh benzpyrene and quantities of various unidenti fiable products . 
furthermore , the evidence suggests that the primary reaction product is a diol closely resembling that found as the initial product of benzpyrene metabolis at the same time this primary product shows a remarkable resemblance to that found as the result of oxidation of benzpyrene in the presence of autoxidizing thiols . 
 in view of the known forma tion of hydrogen peroxide during the autoxidation of - sh groups , it seems prob able that the primary mechanism of oxidation in both cases consists of the addition of hydrogen peroxide to the hydrocarbon . 
equally , since the formation of peroxide is a phenomenon known to occur during biological oxidations ( oppen heimer and stern , 1939 ) , it seems that such a mechanism could account for the initial steps of the in vivo oxidation of benzpyrene . 
 the oxidation of 3 : 4 - benzpyrene with hydrogen peroxide and the isolation of a product similar to the benzpyrene oxidation products obtained during metabolism and during oxidation in the presence of autoxidizing thiols is de scribed . 
kennaway also of those following each one of the recognized occupations , at the time of the census . the number of deaths attributed to cancer of the lung and larynx occurring in the whole male population in each of these age groups during the years in question was obtained from material at the general register office . the comparison of the various occupations with the general population is then a sum in proportion , which gives the ratio of the number of deaths in any given occupation to those , reckoned as 100 , which occurred in the same number of males in ' the same age groups in the whole population . 
an example of this calculation is given in the earlier paper . sources of error in statistical work on death certificates . these were examined in the earlier paper ( kennaway and kennaway ; 1936 )  . the use of death certificates for statistical purposes is liable to errors which are inherent in the nature of the material and cannot be wholly eliminated . 
one must either recognize and admit these errors and correct them in any way possible , or abandon any attempt to obtain information from these data . ( 1 ) correction for period between census . - in order to compare the incidence of cancer upon different occupations one must know the number of persons following each occupation . this is learned from the census returns . 
the figures for populations used for the earlier period ( 1921 - 32 ) were those of the 1921 and the 1931 census , which were combined by means of a formula described in the earlier paper ( kennaway and kennaway , 1936 ) ; those in the present paper are from the 1931 census , which is the last taken . 
kennaway when large numbers are concerned . the immense amount of labour involved in such enquiries can be seen in the monograph on cancer of the scrotum by henry ( 1946 )  . but as none of the occupations included in the present study , with one exception , shows a very high incidence of cancer of the lung , comparable to that seen in the occupational cancers of ' the skin and bladder , this source of error is not a very serious one . ( 4 ) incorrect diagnos8is is of course a source of error in these , as in all other , in the case of death certificates received in bulk , perhaps some medical data . years after the dates of the deaths recorded , one can do nothing but exclude any certificate of which the wording suggests ( 1 ) that the cause of death did not come within the category under examination , or ( 2 ) that the diagnosis was uncertain or ill - founded ( see next section ; such certificates are made the subject of individual inquiry by the general register office )  . when such exclusions have been made , one must treat the remaining certificates as correct if any use is to be made of the material . death certificates taken for examination . the material utilized in this paper has been restricted as far as possible to cases of primary malignant growth of the lung , and of the larynx . 
when the individual occupations are examined considerable variations are found . in the earlier paper attention was drawn to the high ratio for cancer of the lung in judges , stipendiary magistrates and barristers ( 173 ) and in stockbrokers and stockjobbers ( 187 ) ; in the later period ( 1933 - 38 ) the former fell to 112 and the latter rose to 253 . the stockbrokers and jobbers also have the highest ratio among the professional workers for cancer of the larynx . the figures for roman catholic priests and monks are too small to be significant . the ratio for ministers of other religious bodies has risen ( table xx )  . 
no attempt is made here to deal with all the data on the carcinogenic action of arsenic , as this subject is under investigation by a committee of the industrial health board , but the following quotations from recent annual reports of the senior medical inspector of factories show the possible importance of arsenic in relation to cancer of the lung . 
kennaway there was a special surgical clinic dealing with lung cases , and where full x - ray and bronchoscopic investigation was practised , 42 per cent of the clinical diagnoses were wrong ( table xxiv )  . 
hence the diagnosis of cancer of the lung appears to be still very incomplete , even under favourable conditions , in spite of the great improvements which are generally stated to have been made . 
some possible causes of a real increase are considered in the next section . in the earlier paper ( kennaway and kennaway , 1936 ) we drew attention to the evidence afforded by cases of cancer of the lung in which an erroneous diagnosis was made based on nervous or mental symptoms due to metastases . 
18. ( 2 ) one must look , therefore , for some other factor to explain the increase , if there is a real increase , in cancer of the lung in recent years . 
a similar argumnent might be applied in tile case of cancer of the lung , which is not known to be prevalent in any of the lower animals . the adenoma of the lung of the mouse , and certain affections of the lung of sheep in south africa and iceland , are neoplasms of which the exact nature is uncertain . we know one instance at any rate of the susceptibility of the lung of an animal to a carcinogenic agent , namely the lung of the cat in relation to 2 - acetylaminofluorene given by the mouth ( harding , 1946 ) ; hence there is no reason to think that animals are immune to any such one obvious factor , possibly carcinogenic , to which the lung of mnan agents . alone is exposed is tobacco smoke . during the present century very considerable changes have taken place in this country in the social distribution of the various methods of smoking . there were , of course , exceptions to any rule , but roughly one might say that in the earlier part of this period men of the richer classes smoked pipes , cigarettes and cigars , and men of the poorer classes smoked pipes ; cigarette smoking was increasing among the richer women , while women of the poorer classes did not smoke . one never saw a woman scrubbing her front door - step , or hanging out the washing , with a cigarette in her mouth . the great change which has taken * a . 
of course no claim is made here that the simultaneous increases in the consumption of tobacco , and in cancer of the lung , proves any etiological connection between the two . other changes which have taken place in the same period , which no one proposes to associate with cancer of the lung , e.g. 
kennaway to summarize the evidence brought together in this section on the incidence of cancer of the lung : the higher mortality in towns , the low mortality in agricultural occupations , and the absence of social gradient , are compatible with a factor in the air , such as coal smoke . in any such comparison of urban and rural areas , the question of facilities for diagnosis must be considered . coal smoke , which is probably a decreasing contaminant of the air , does not account well for the increase of cancer of the lung . 
among various possible factors is tobacco smoke ; the consumption of tobacco has risen , and so has the percentage of it smoked in the form of cigarettes , of which the smoke is often inhaled . 
among various possible factors which have been suggested to account for the increase is tobacco smoke ; the consumption of tobacco has risen , and so has the percentage of it smoked in the form of cigarettes , of which the smoke is often inhaled ; such an effect of tobacco would accord well with the absence of social gradient . we are indebted to the registrar - general for the data considered in this paper , and also for kindly permitting us to use some figures which have not yet been made public officially . 
we have received much assistance in the classification of occupations , and on various other questions , from some members of the staff of the general register office , to whom we wish to express our gratitude . we wish to thank e . 
w.3. received for publication july 16 , 1949 . in an investigation of the body growthand tumour growth - inhibiting action of carcinogenic substances , it was found that the nature of the inhibition is profoundly influenced by the protein content of the diet ( elson and warren , 1947 ; elson and haddow , 1947 ; elson , 1949 )  . in rats maintained on a 20 per cent protein diet injection of the carcinogen usually has little immediate effect on body growth although a delayed action , resulting in rapid loss of weight followed by death , may occur later . 
carcinogenic hydrocarbon 1 : 2 : 5 : 6 - dibenzanthracene 24 hours after implantation of walker carcinoma 256 in rats maintained on a 20 per cent protein diet results in very little difference in size and weight of the tumours from those of controls . if , however , the animals are fed a 5 per cent protein diet the tumours of those similarly treated with dibenzanthracene were found to be only one - quarter the weight of those of controls when the rats were killed 13 days after implantation ( elson and haddow , 1947 )  . 
lamerton for the studies involving the localized irradiation of the tumour , it was necessary first to develop .a method whereby the rat could be held firmly in anaesthesia reasonable comfort and the tumour alone irradiated with precision . was contra - indicated , since it may affect the response of the animal to radiation . the device eventually developed is shown in fig . 
a length of transparent plastic " cloth " is held by means of two clamping bars on an aluminium base . the rat is placed under the plastic material , which is then stretched under the clamping bars . 
the edge of the plastic cloth near the rat 's head must be reinforced with a strip of cellophane one to two inches wide , which serves to hold fig . 
1. - photograph of rat in holder designed for x - ray treatment of tumour . the rat 's head down and prevents him biting his way out . the tumour itself projects through a hole in the plastic material , and can be treated by crossfire techniques without irradiation of the rest of the body . a victor kx1o plant was used , run at 140 kvp . 
lamerton direct irradiation of e8tablished tumours . for these experiments the tumours were allowed to grow untreated for 6 days after implantation . daily estimates of the size of the tumour were then made by - measurement with calipers along two axes at right angles . 
2. from the results obtained with more than 20 control animals there appeared to be no very marked difference in rate of growth of the tumour in animals maintained either on 5 per cent or on 20 per cent protein diets . 
the group maintained on the 5 per cent protein diet , however , showed considerable inhibition of tumour growth , and the tumours after the first 7 days of treatment ceased to grow and remained fairly constant in size for a considerable time . 
3. - growth of walker rat carcinoma 256 treated with x - radiation ; 250 r per day , total dose 4000 r . * rats fed on 20 per cent protein diet . rats fed on 5 per cent protein diet . gradually developed areas of necrosis , which usually become infected , and in some cases the tumour had resumed growth shortly before the death of the animal . 
lamerton for the first four days after treatment the tumours of the 5 animals comprising the 20 per cent protein diet group grew more rapidly than those of the 5 per cent protein group , and reached an average size of about one and a half times that of the low protein group . after this period the tumours of the 20 per cent protein diet animals began to decrease in size , and eventually two of them regressed completely by a " shelling - out " process and a third remained very small over a long period . 
the other two , after a considerable period of regression , eventually grew again rapidly and the animals died of the tumour about 45 days after implantation . in the 5 per cent protein group one tumour regressed completely but the others grew fairly steadily at a much reduced rate for a considerable period , eventually resuming a more rapid growth rate before the death of the animal , which occurred on the average 36 days after implantation . died 2400 s 2000 3 1600 z 1200 en 800 perioi fig . 
no cures were obtained in animals of either group . it appeared therefore that regular control of the established walker tumour could not be effected by a daily dose of 400 r , and in the next experiment the effect of a daily dose of 600 r was studied , together with the effect of changing the diet of the low protein group of animals to the high protein content at the completion of the course of radiation . x - radiation , 600 r per treatment ( total 4000 r )  . in these experiments a dose of 600 r was given daily on six days , the seventh dose being reduced to 400 r to maintain the total dose at 4000 r . in the first experiment the animals of the 20 per cent protein diet group responded well at first , and nearly all the tumours began to decrease in size after the fourth dose of radiation . although the tumours then remained small for a considerable time no complete regressions occurred , and all eventually grew again fairly rapidly , leading to the death of the animals . the tumours of the animals maintained on the 5 per cent protein diet again at first responded to the treatment by growing rather more - slowly than those of the 20 per cent protein group . 
the day following the last radiation treatment the 5 per cent protein group animals were changed over to the 20 per cent protein diet and maintained thereafter on this high protein diet . 
5. - growth of walker rat carcinoma 256 treated with x - radiation ; extended treatment with initial high dose ( 1000 r ) ; total dose 4000 r . . 
under these conditions therefore the large initial dose of radiation appears to have such a strong inhibiting effect on the tumours that a marked difference is no longer observed between the initial response of low and high protein diet animals . 
the greatest degree of tumour inhibition was obtained when the animals were maintained on a low protein diet . it has now been shown that the response of rats implanted with walker carcinoma 256 to whole body x - radiation under conditions approaching those of the " walker tumour test " is also greater in those animals maintained on the low protein diet . 
with x - ray treatment , however , it is possible to apply direct to the tumour a sufficiently large dose of radiation to bring about an adequate initial growth - inhibitory response , even in animals maintained on a high protein diet , so that the subsequent outcome of the treatment then depends almost entirely on process ( b ) , and a favourable result is thus to a very large extent dependent on a high protein content of the diet . though the results obtained may be related to some extent to the particular characteristics of the tumour used , it is very interesting to note how closely the two processes distinguished in the radiation response of the tumour follow the concepts of " intra - cellular response " and " inter - cellular response " put forward by koller and smithers ( 1946 ) from studies in clinical radiotherapy . . 
tumour regression as the result of koller and smithers suggest that "  . irradiation is initiated by an intra - cellular response , but is carried on by an inter - cellular response which follows and plays an important part in the success of any treatment . 
the inter - cellular reaction is manifested through the repair processes , and depends on the construction of the tumour tissue as a whole . this reaction is conditioned and influenced by the intra - cellular response ; in this sense it is an indirect effect of radiation it seems likely that this " inter - cellular " response may constitute an important part of what we have described as process ( b ) , and have shown to be favourably if the " inter - cellular response " is related to influenced by a high protein diet . the repair processes , then it is very reasonable to suppose that nutritional factors can affect the elimination of the tumour in radiation treatment , and indeed proteins would be of special importance in view of their known value in the healing of burns and wounds . clearly the present work can only be regarded as a preliminary study . 
lamerton implantation , inhibition of tumour growth was greatest in those animals maintained on the low protein diet , but no complete regressions of the tumour were obtained . with localized irradiation of the tumour , when the total dose of 4000 r was applied in larger daily doses , complete regression of some tumours was observed , and the most favourable results were obtained by application of an initial dose of 1000 r with extended time intervals between successive smaller doses . 
complete regression of the tumours was then obtained in about 90 per cent of the animals maintained on the high protein diet , but in only about 15 per cent of these maintained on the low protein diet . it is suggested that two effects are concerned in the response of tumours to radiation , ( a ) the initial inhibition of tumour growth , ( b ) the elimination of process ( a ) is favoured by a low the inhibited tumour and cure of the animal . protein diet , and process ( b ) by a high protein diet . we would like to express our appreciation of the interest taken in this work and the encouragement given by professor a . 
we also wish to express our thanks for grants supporting this investigation from the british empire cancer - campaign , the anna fuller fund , the jane coffin childs memorial fund for medical research and the u.s. 
powell. from the department of experimental biology , mount vernon hospital , and the radium institute , northwood , middlesex . received for publication july 29 , 1947 . preparatory to commencing some detailed work on the metabolism of 3 : 4 - benzpyrene in the skin of the mouse consideration was given to a number of experimental factors which appeared likely to affect the reproducibility of the results . 
for quantitative work some standardization of the area to be treated was the method finally adopted was to apply the solution of benzpyrene required . through a stencil cut from sheet aluminium ( 16 gauge ) , with a hole of the desired size and shape . 
as it was desired to use a volatile solvent , acetone or benzene , for the application , the question of how much solvent to use in order to apply any given standard amount of the benzpyrene was examined . 
the use of a small volume of solvent , 005 c.c. , resuited in rapid drying and the deposition of the benzpyrene as a crust of crystals over 324 g . 
bearing in mind the points raised above , a series of determinations of the amounts of benzpyrene left in the skin at various time intervals after painting the results are shown diagrammatically in fig . 
on the other hand examination of the dissected animals showed the presence of benzpyrene distributed throughout the length of the digestive tract . this suggested that the benzpvrene was being licked from the skin by the animals . confirmation of this conclusion came from watching the behaviour of the almost immediately after painting and return to the boxes furthermore , when there is more than one painted animals . they start to clean themselves . mouse in a box they lick , not only themselves , but each other . further confirmation was obtained by painting mice and fixing them to a board with adhesive tape . 
 3 : 4 : 5 : 6 - dibenzcarbazole has been tested on numerous occasions and has been shown to be a potent carcinogen in mice . ( boyland and brues , 1937 ; strong , smith and gardner , 1937 ; boyland and mawson , 1938 ; andervont , 1939 ; andervont and edwards , 1941 )  . 
 in the latter organ the changes were very severe and comprised cirrhosis and bile - duct proliferation , which proceeded to the formation of cholangiomas , as well as hepatomas . 
 as boyland and brues ( 1937 ) had suggested that naphthylamine workers might come in contact with dibenzcarbazoles during the course of their work , it was decided to re - investigate 3 : 4 : 5 : 6 - dibenzcarbazole as a potential bladder carcinogen . 
mice of the oba strain were chosen , as this strain had previously given a higher yield of bladder tumours when 2 - acetyl aminofluoi : ene was fed by stomach tube than four other strains tested ( armstrong and bonser , 1947 )  . 
as the mortality in oba mice in the first few weeks was very high , successive groups were tested at different dosage until a reasonable survival was obtained ( table ii )  . 
the oba mice were treated until death ; in strong a mice , when it was evident from the swollen state of the abdomen that much liver damage had taken place , treatment was continued for as long as the mice seemed able to support it and was then withheld until death , the interval in some cases being over 20 weeks ( table iii )  . 
the dose was not found which would permit the females to live for more than 20 weeks , although 27 out of 60 oba males survived from 20 to 49 weeks . 
 in experiment v , strong a female mice showed only a slight tendency to die early from the effects of the chemical with the dose given , 5 out of 18 dying in the first few weeks , but when once tolerance had been established the females survived rather longer than the males ( table ii )  . 
several showed such a degree of cellular irregularity in the basal layers as to be consistent with a diagnosis of early malignancy , but this diagnosis was not made unless there was invasion and destruction of the muscular coat of the stomach of such degree as to leave no doubt as to the invasiveness of the process . 
the livers were on the whole larger in strong a than in cba mice , but it was thought that some reduction in liver size occurred in the mice of the former .strain when the treatment was with drawn . 
no attempt was made to count or measure these tumours , but the impression was gained that they were , on the whole , of larger size in those mice which were treated until death than in those in which a long interval elapsed between the cessation of treatment and death . 
there was focal lymphocytic infiltration of the interstitial - tissue , proceeding to fibrosis , and after 20 weeks of treatment there was usually calcifi cation of some of the cortical tubules and of the adjacent glomerular vessels . 
it is to be noted , however , that no cba mouse survived longer than 49 weeks , while all but one strong a mouse survived into the period 40 to 59 weeks . 
at 49 weeks only one of 18 cba mice treated with the potent bladder carcinogen 2 - acetylaminofluorene was known to have developed a tumour ( armstrong and bonser , 1947 ) , whereas 5 out of 10 strong a mice were known to have hyperplasia or a benign papilloma when dying between 40 and 59 weeks . 
thus , while it cannot be stated with certainty that 3 : 4 : 5 : 6 - dibenzcarbazole administered orally is not a bladder car cinogen , no indication has been obtained which would suggest that it has any action on the bladder epithelium , although from previous experience with these strains of mice evidence of hyperplasia or papillomatosis might be expected to be forthcoming within the experimental period available . 
more strong a than cba mice had tumours at death , but the difference is not statistically significant , and may be due to the greater dose of chemical and the longer survival period in strong a than in cba mice ( table iv )  . 
when it is taken into account that the incidence of carcinomas was identical in the two strains , despite the shorter survival period and smaller dose in cba mice , it would appear probable that cba is the more susceptible stra from table iii it is seen that these tumours of the forestomach are not dependent upon the continued administration of the carcinogen for the main tenance of their growth , as the chemical was discontinued for an average of 10 l weeks before the death of strong a mice , the range being o to 22 weeks . 
 it is rather surprising that papillomas of the forestomach were so rarely observed when tar was applied to the skin of mice , twort and twort ( 1932 ) having observed only occasional papillomas in 60 , 000 mice so treated . 
it seems clear that 20 - methylcholanthrene , 3 : 4 - benzpyrene and 1 : 2 : 5 : 6dibenzanthracene are carcinogenic in this sense , the latter having produced one benign tumour ( beck , 1946 ) and several malignant ones ( lorenz and stewart , 1948 )  . 
from the observations so far reported it is necessary to administer the chemical by the oral route , as no similar tumours were found after skin painting or subcutaneous injection . 
the reasons given by beck ( 1946 ) may account for this : the presence of the mucus barrier , the mucus flow tending to remove the carcinogen from contact with the cells , the possibility that contact between a carcinogen and a resting cell may not be sufficient to induce neoplasia , and finally the possibility that glandular epithelium is essen tially less susceptible than squamous epithelium to the type of carcinogen under discussion . 
there seems to be little support for the latter suggestion when the recent work ( horning , 1946 ; pan and gardner , 1948 ) on the induction of tumours by carcinogens in glandular epithelia in implants is considered . 
carcinomas of the intestine were not observed , in contrast with the results of lorenz and stewart ( 1946 ) , who noted such tumours in strain a and other mice which received aqueous emulsions of 20 - methylcholanthrene and 1 : 2 : 5 : 6 - dibenzanthracene . 
 amines are not without a similar action on the epithelium of the forestomach , papillomas having been observed in one riii , one oba and 2 strong a mice at 77 , 74 , 69 and 65 weeks respectively of treatment with 2 - acetylamhi.ofluorene in addition , otsuka ( 1935 ) described papilloma ( armstrong and bonser , 1947 )  . 
bonser seems possible that perforation of the stomach might have occurred during intu bation , resulting in a false passage of the tube into the muscles of the posterior abdominal wall and consequent injection of . 
in the course of observation of the cba strain for 17 years ( unpublished observation ) , one spontaneous spindle - cell sarcoma has been found and , therefore , the possi bility of a spontaneous origin of the tumour in mouse 10 cannot be completely excluded . 
contrary to the experience of andervont and edwards ( 1941 ) , this sex difference was observed only in the early weeks of the present experiment in strain a mice ( table iii )  . 
 in comparing the degree of liver damage in the two strains , it is evident that both suffered severely , but that the incidence of hepatomas was greater in strain a mice than in cba , the proportion of histologically malignant tumours being also greater . 
not only were hepatomas more abundant and more malignant in strong a mice than in cba with the latter chemical , but cirrhosis and bile - duct proliferation were a marked feature in both strains . 
although a high incidence of hepatomas was observed in cba mice ( but not in strong a mice ) when treated with 2 - acetylaminofluorene ( armstrong and bonser , 1947 ) , cirrhosis was absent and bile duct changes were minimal . 
it would appear possible that the chief action of 2 - acetylaminofluorene is to enhance the tendency to hepatoma already hereditarily present in cba mice , whereas 3 : 4 : 5 : 6 - dibenzcarbazole damages the liver parenchyma in such a way that changes are set in train which eventually lead to tumour formation ( orr , 1940 )  . 
 an experiment is described in which mice of the strong a and cba strains received by stomach tube , twice weekly , an oily solution of 3 : 4 : 5 : 6 - dibenzcar bazole , with a view to testing this substance as a bladder carcinogen . 
 no bladder epithelial changes were observed during an experimental period which would have sufficed to show , in these strains , hyperplasia and early tumour formation had 2 - acetylaminofluorene been the carcinogen . 
3. received for publication april 21 , 1951 . studies of the electrophoretic behaviour of sera from patients bearing malignant and non - malignant tumours have been carried out by various workers with the view to their utilization as diagnostic or prognostic tests for cancer . 
polarography has also been investigated as a possible clinical tool for diagnosis or prognosis following brdicka 's ( 1933 ) discovery that proteins give a characteristic " catalytic double wave " in ammoniacal cobalt salt solutions . applications of this method to the study of sera have shown differences between specimens obtained from normal and pathological cases , and the results of an examination of over 100 sera from cancer subjects has been published by one of us ( butler , although good correlation between the wave height and the pathological 1951 )  . condition was obtained , a number of false positive and false negative results were included , the latter particularly in cases of cancer of the buccal cavity and skin . the electrophoretic behaviour of sera has been discussed by a number of workers ( seibert , seibert , atno and campbell , 1947 ; petermann and hogness , 1948a , 1948b ) , and the most consistent changes found in pathological conditions were increases in the c1and a2 - globulin levels . however , no specific changes have been found for any particular clinical conditions ( mides , alling and morton , 1950 )  . the work of winzler and his colleagues ( winzler , devor and mehl , 1947 ; winzler , devor , mehl and smyth , 1948 ; winzler and smyth , 1948 ; mehl , humphrey and winzler , 1949 ; mehl , golden and winzler , 1949 ; weimer , mehl and winzler , 1950 ) , has shown that a mucoprotein , isolated from normal plasma , appears to be responsible for the polarographic filtrate wave . this mucoprotein migrates in the al - globulin fraction , when examined electrophoretically at ph 8 ' 4 in barbiturate buffer . it has an isoelectric point at ph 1 ' 8 and still has a negative charge at ph 4 ' 5 . 
the acidic protein fraction demonstrated in cases of gastric cancers by petermann and hogness ( 1948b ) has been shown to be a similar mucoprotemayer ( 1942 ) had previously isolated a " mucoid - like substance " from horse serum which he claimed was the polarographically active seibert et al . 
 ( 1947 ) showed that increases in a2 - globulin were paralsubstance . leled by increases in the polysaccharide content of the serum . these results suggest that the polarographic and electrophoretic patterns of cancer sera may be due to mucoproteins which migrate in the a - globulin group . 
the results were expressed either as the height of the filtrate wave , or as the protein index of muller and davis ( 1947 ) or as the blood index ( butler , 1951 )  . the results of the electrophoretic measurements were expressed in terms of the areas ( measured by means of a planimeter ) under each of the peaks of the descending differential patterns ( longsworth and macinnes , 1940 ; svensson , 1943 )  . 
the relative composition was expressed by computing the percentage contribution of the area under any one peak with respect to the area under the whole pattern , excluding the boundary anomaly . 
a statistical evaluation of the results was therefore necessary in order for this an estimation of the change in per cent to draw any valid conclusions . and absolute composition from the normal to the pathological condition was made . 
no such increase in this fraction was observed in the two lymphosarcoma cases investigated here , nor in any of the three cases investigated by petermann , karnofsky and hogness ( 1948 )  . calculation of correlation coefficients between the component peak areas and the polarographic fitrate wave heights ( f ) and blood indices ( bi ) indicate significant relationships between the ocl - globulin area and the filtrate wave heights and blood indices factors in the pathological group . it is of interest and importance that no correlation between the ocl - globulin content and the polarographic factors was found in the normal group , and similarly none of significance if the normal and pathological groups are considered as one population . this is demonstrated in the correlation diagram shown in fig . 
3. correlations between the oq - globulin content and the polarographic indices were not found when percentage composition was considered , indicating that complications by other variable factors may occur . calculations of the correlation coefficients and inspection of the correlation diagrams shown in fig . 
correlations between the serum composition and polarographic factors are discussed , and their relation to the suggested anomalous mucoprotein content in the ocl - globulin fraction is indicated . we wish to thank d . 
the investigation has been supported by grants from the british empire cancer campaign , the jane coffin childs memorial fund for medical research , the anna fuller fund and the u.s. 
kreyberg. from the institutt for generell og eksperimentell patologi , universitetet i oslo . received for publication february 19 , 1952 . in many countries the statistical reports show a steady and remarkable increase in the number of deaths from " lung cancer . " the problem puzzles the cancer specialists and troubles the public and the medical profession . does this increase signify a real augmentation of cases of lung cancer , or does it simply mean an increasingly better diagnostic service ? this question will not be discussed in this paper . if we wish , however , to study , by statistical methods , the possible role of certain irritants in the genesis of lung cancer we have to make clear what we mean by the term " lung cancer . " according to kennaway and kennaway ( 1947 ) their statistics from england and wales comprise : " cancer , carcinoma , or sarcoma of lung , bronchus , pleura , root of lung , hilum of lung , lung and mediastinum , or jung and pleura . 
the norwegian " statistisk sentralbyra " has , since the first of january , 1951 , acted accordingly . it is a great step forward . in order to study a possible relationship between lung carcinoma and smoking habits , or industrial irritants , it may be of importance , not only to exclude all other lung tumours than primary lung carcinomas , but even to subdivide the latter into special sub - groups . from our general experience in the field of cancer research we are ever more impressed by the importance of the study of the geographical distribution of the different manifestations of malignant disease . this different geographical distribution examined on the background of the corresponding difference as to races , primary epithelial . 
this report deals with 100 consecutive cases ofprimary epithelial lung tumourb , received for diagnosis at the institute of pathology , at the university all , but also only those , tumours are included , where the piece of tissue of oslo . o 60 1929 1939 fig . 
1. - mortality statistics from norway , 1929 to 1949 ( " statistisk sentralbyroa " )  . was large enough to permit a definite histological classification . the main part of the material ( 97 cases ) was received from the surgical department a , at the university hospital , 2 cases came from the ear , nose and throat department , and 1 case from the roentgen - radium department . hereto are added 5 cases of secondary lung tumours , received within the same period , and clinically regarded as primary tumours , the histological examination , however , revealing their true character ( 2 cases of metastasizing thyroid adenoma , and 1 case each of hypernephroma , adenocarcinoma of uterus and malignant naevus tumour )  . the 100 tumours of the main series were classified according to traditional terminology as follows : 114 l . 
10 the number and the different types of tumours of our main series may be seen , as well as the occurrence in age - groups and the distribution between the two sexes . 
the individual tumours of groups g and f are tabulated in table ii . the groups e , f and g do not show any marked preponderance in the one further , these tumours are rather evenly distributed in the sex or the other . different age - groups , and they occur even in very young people , our youngest patient being a girl of 19 with a malignant adenoma . 
as to the microscopical classfication , he has : adenocarcinomas 4 - 6 per cent , squamous cell carcinoma 62 - 9 per cent , undifferentiated carcinoma 25 1 per cent and uncertain type 7 - 4 per cent . here again the similarity is greater when we examine the material on the basis of the more objective characteristic the distribution in age - groups than on the basis of the more subjectively influenced classification in histological types . it is evident that the surgical material cannot , without a closer examination , be taken as representative for the occurrence and the distribution of lung carcinomas and lung tumours in the population . firstly , cases occurring in old age 118 l . 
kreyberg will not be represented , as these patients are poor operative risks and seldom secondly , in old age the diagnosis may be more difficult and operated upon . possibly not pursued with the same enthusiasm as when the patient is a younger . it is not unlikely that the local thirdly , the sex distribution may be different . doctor , the surgeon and the patient himself will take a greater risk and show a somewhat more heroic attitude towards the question of operation when the patient is a male . 
the women in these age - groups may possibly show more resigfourthly , surgical material may show a greater nation to the blows of fate . number of benign and moderately malignant tumour types than the sum total . this is significant when the material examined includes all primary epithelial lung tumours , but less important when only true bronchogenic carcinomas are this underlines the importance of a careful classification when statisconcerned . tics are to be compared . 30 ~2021 - 30 31 - 40 41 - 50 51 - 60 61 - 70 71 - 80 81 - 90 age groups fig . 
the occurrence of a certain number of cases in the early age - groups indicates that a few adenomas are included also in this post - mortem series . i wish to emphasize that the present paper represents a preliminary orientation only , as to the occurrence of lung carcinomas in norway . 
parsons. from the department of chemistry , the university , manchester , 13 , and the royal eye hospital , southwark , london . received for publication august 15 , 1949 . the effects of injection of the four nucleotides obtained from ribonucleic acid on the tissues of normal and tumour - bearing mice have been described ( barakan , barker , gulland and parsons , 1948 ; parsons , gulland and barker , further experiments have now been carried out to note the action 1947 )  . of purine and pyrimidine bases on sarcoma growth , and to correlate if possible the biological activities of the compounds with their chemical structure . these experiments fall into two groups : ( a ) those relating to the amino and hydroxy purines , ( b ) those relating to uracil and its derivatives . both pure - line and stock mice were grafted with homologous or heterologous sarcomas , including c57 and s37 and the crocker sarcoma , the latter known to be very refractory to inhibitory action ( badger , elson , haddow , hewett and robinsoir , 1942 )  . 
the technique for treatment of the grafted mice was similar to that previously described , but the standard dose ( 0025 g . ) was reduced in proportion to the toxicity of any particular compound , or where the condition of the mouse necessitated this . insoluble or relatively insoluble compounds such as xanthine , guanine , adenine or hypoxanthine were administered as fine suspensions in the same media as used for the soluble compounds . examination of the tissues at varying intervals showed that all compounds injected as suspensions were rapidly removed within 24 hours with the exception of guanine , the relative inactivity of which on tumour growth seems to be due to its marked insolubility . deposits of the injected guanine were found at the site of injection long after the last dose . table i summarizes the results obtained . 
the total number of control mice was in all cases the same as the total number of treated mice . the amino and hydroxy purines . it had been noted that adenylic acid or adenosine produced shock when injected into mice , and it seems probable that this effect was due to the adenine radical , of which the pyrimidine ring is in a lower state of oxidation than those of the other compounds used which do not induce shock . injection of adenine in doses comparable with those used for adenosine or adenylic acid has been found to cause immediate and profound shock with heavy mortality , the minimum sublethal dose being 0005 g . 
parsons radical , but may be connected with the presence of the amino group in position 6 in the adenine radical . xanthine injected in tumour - bearing mice was removed from the tissues injected into in 24 hours , but no inhibition of sarcoma growth was observed . normal mice in doses of from 0 - 02 - 0 * 025 g . 
kirby. from the research department , glasgow royal cancer hospital . received for publication february 8 , 1947 . the exceptional interest of 2 - acetylaminofluorene ( aaf ) as a carcinogen was clearly revealed by wilson , deeds and cox ( 1941 ) , who fed it to inbred albino rats in which only benign mammary tumours had been observed , and found carcinomas of the liver , breast , bladder , ureter , renal pelvis , colon , pancreas , lung and skin , a myogenic sarcoma , and in two cases liver lesions resembling leukaemic infiltration . 
the results of bielschowsky ( 1944 a and b ) and other workers are summarized later in this paper . in cba mice , neoplasms of liver and uterus ( one a sarcoma ) and of the urinary bladder were found by armstrong and bonser ( 1944 )  . bielschowsky and green ( 1945 ) reported a carcinoma of the kidney in a rhode island red cock which had been fed aaf for 45 weeks . the widespread variety of organs affected by aaf led bielschowsky ( 1944b ) to try to induce tumours of the goitrous thyroid gland with this substance . solid , parenchymatous goitre was induced by allyl thiourea ; in conjunction with aaf , also orally administered , allyl - thiourea induced adenoma in 9 out of 10 rats , while malignancy was observed in 3 of these rats . griesbach , kennedy and purves ( 1945 ) claim that the goitrogenic agent in brassica seeds can cause adenoma of the thyroid if given orally for long enough , but only 1 of 8 wistar rats developed such a lesion . subsequently purves and griesbach ( 1946 ) obtained malignant changes in 2 out of 30 wistar rats given thiourea in their drinking water for nearly two years . purves and his co - workers consider that the effect of the goitrogens is indirect , operating by inducing hypersecretion of the thyrotropic pituitary hormone . 
aaf has no specific action even on adenomatous thyroid tissue . the success achieved by bielschowsky in at least accelerating malignant changes .in the thyroid of rats by feeding aaf suggested that other organs apparently untouched by aaf alone might undergo neoplastic changes when stimulated to proliferate by suitable means . 
the sex - organs of the rat can be caused to hypertrophy by the administration of the appropriate sex - hormone , and it was thought that these organs , thus caused to proliferate , might proceed to malignant changes when influenced by orally administered aaf . 
the basal diet for all groups was rat - cake made to the formula of thomson ( 1936 )  . 2 - aminofluorene was synthesized from fluorene by the method of diels ( 1901 ) , * and acetylated as follows . 
up to 1 per cent of concentrated sulphuric acid was added to 2 - aminofiuorene stirring in an excess of acetic anhydride which became very hot and dissolved the amine . after chilling thoroughly the acetylated amine was filtered on a sintered - glass sufficient water was added to cause funnel , and dissolved in hot ethanol . permanent turbidity and the solution well cooled . 
emmens pointed out that absorption during the second month would be much reduced , and recommended the use of 20 mg . therefore thin disc - shaped pellets weighing 20 mg . 
a spindle - cell sarcoma of the left seminal vesicle was found in rat 354 ( group iib ) , and a whorled , spindle - cell sarcoma was found lying above the liver of rat 339 ( group iia )  . 
the reason for this may well be that suggested by these workers , namely that " the hyperplasia of target organs of sex - hormones is a functioning hyperplasia , whereas that induced in the thyroid by goitrogenic agents is non - functionthus the type of hyperplasia induced by bielschowsky in the rat thyroid ing . " in which growth is accompanied by loss of differentiation would appear to have been a suitable basis for carcinogenesis , whereas the hyperplasia associated with increased differentiation induced in sex organs by over - stimulation with sexhormones is apparently unsuitable . 
illustrate a " carcinoma arising adjacent to the external auditory canal , " and discuss the histogenesis of this and other tumours in this locality included by them under " subcutaneous tumours . " bielschowsky first drew attention to tumours of the ductus acusticus externus as a separate entity . 
as wistar rats were also used by bielschowsky , who found these tumours in 18 / 93 rats given aaf orally up to 210 days , the influence of diet seems to have some importance . 
when allyl - thiourea was also given , the incidence of tumours rose to 6 / 10 . lopez ( 1945 ) also found external meatus tumours in 2 / 4 rats . 
hence the action of aaf on proliferating mammary tissue was only slight in our experiments . in the matter of liver tumours , our series has yielded results in full accordance in our series , a . 
the types of liver tumours seem to be essentially the same as in wistar rats . this is also true of the liver tumours obtained by bielschowsky in piebald rats , in which the sex - ratio is also maintained and the in the sherman strain , cantarow et al . 
found the incidence much the same . incidence of liver tumours in female rats raised when oestradiol dipropionate was injected intramuscularly ; but the complete absence of liver neoplasms in rats given thiouracil was much more striking . 
the latter group ate less of the aaf diet than did controls receiving no thiouracil , but more than the oestrogenized rats . the incidence of mammary tumours in piebald rats was very low compared with that in wistar rats on the same diet ( bielschowsky , 1946 )  . but the incidence of tumours of the ductus acousticus externus was much higher in the piebald rats , in which tumours of the small intestine were also much more frequent . moreover , lung tumours were found in this strain , but have not been reported in wistar rats given aaf and were not found in our series , although one control female ( group ia ) was found to have undifferentiated bronchial carcinoma . the albino rats of wilson et al . 
the latter have not been found in any other strain of rat , and tumours of the kidney and bladder only once each . it would seem that , if sufficient strains of rat were employed , possibly no site in the body would be found immune from the carcinogenic action of aaf . 
in the strains so far tested , only mammary tissue among the sex - organs seems to be susceptible , and this tissue is liable to develop carcinoma due to the action of sarcomas are rare , and are not induced by subcutaneous oestrogens alone . injection of aaf , at least in wistar rats . in our series , a whorled , spindle - cell sarcoma was found in the omentum covering the left lateral lobe of the liver in one rat , and a spindle - cell sarcoma was present in the left seminal vesicle of another . it remains to discuss a few other points of interest . toxic damage to kidneys was seen in some animals of all groups ; rats receiving oestradiol benzoate pellets , with or without aaf , tended to show degeneration of the epithelium of the first and second convoluted tubules with the presence of a dark yellow a . 
kirby pigment in these cells . there was no consistent evidence of toxic damage to the kidneys by aaf or its metabolites , nor were any tumours of this organ seen . no tumours of the uterus were found , but all females receiving oestradiol benzoate had pyometra , which often made it necessary to kill the animal ; the 2 females receiving the male sex - hormone had no pyometra . the spleens of the control groups , ia and ib , showed no abnormality . 1 female in group iia and 1 male and 2 females in group iib had enlarged spleens , due to lymphoid hyperplasia . there was no question , however , of the regular , early , gross enlargement of the spleen seen in rats ingesting azo - compounds it may be ( smith , lillie and stohlman , 1943 ; kirby and peacock , in press )  . concluded that no splenotoxic substance passes from the liver into the blood of rats fed aaf ; rats fed p - aminoazobenzene ( aab ) or its n - monomethylor n , n - dimethyl - derivatives ( mab and dab ) have aab circulating in the blood and affecting the spleen ( miller , miller and baumann , 1945 )  . the rats given aaf orally in the experiments described in this paper developed tumours of the liver predominantly in the right lobes ; tumours in the left side of the liver were usually smaller or absent . 
a similar predominance of tumours in the right side of the rat liver has been reported by opie ( 1944 ) in rats fed dab in diets not inducing cirrhosis . 
 3 : 4 : 5 : 6 - dibenzcarbazole has been tested on numerous occasions and has been shown to be a potent carcinogen in mice . ( boyland and brues , 1937 ; strong , smith and gardner , 1937 ; boyland and mawson , 1938 ; andervont , 1939 ; andervont and edwards , 1941 )  . 
 in the latter organ the changes were very severe and comprised cirrhosis and bile - duct proliferation , which proceeded to the formation of cholangiomas , as well as hepatomas . 
 as boyland and brues ( 1937 ) had suggested that naphthylamine workers might come in contact with dibenzcarbazoles during the course of their work , it was decided to re - investigate 3 : 4 : 5 : 6 - dibenzcarbazole as a potential bladder carcinogen . 
mice of the oba strain were chosen , as this strain had previously given a higher yield of bladder tumours when 2 - acetyl aminofluoi : ene was fed by stomach tube than four other strains tested ( armstrong and bonser , 1947 )  . 
as the mortality in oba mice in the first few weeks was very high , successive groups were tested at different dosage until a reasonable survival was obtained ( table ii )  . 
the oba mice were treated until death ; in strong a mice , when it was evident from the swollen state of the abdomen that much liver damage had taken place , treatment was continued for as long as the mice seemed able to support it and was then withheld until death , the interval in some cases being over 20 weeks ( table iii )  . 
the dose was not found which would permit the females to live for more than 20 weeks , although 27 out of 60 oba males survived from 20 to 49 weeks . 
 in experiment v , strong a female mice showed only a slight tendency to die early from the effects of the chemical with the dose given , 5 out of 18 dying in the first few weeks , but when once tolerance had been established the females survived rather longer than the males ( table ii )  . 
several showed such a degree of cellular irregularity in the basal layers as to be consistent with a diagnosis of early malignancy , but this diagnosis was not made unless there was invasion and destruction of the muscular coat of the stomach of such degree as to leave no doubt as to the invasiveness of the process . 
the livers were on the whole larger in strong a than in cba mice , but it was thought that some reduction in liver size occurred in the mice of the former .strain when the treatment was with drawn . 
no attempt was made to count or measure these tumours , but the impression was gained that they were , on the whole , of larger size in those mice which were treated until death than in those in which a long interval elapsed between the cessation of treatment and death . 
there was focal lymphocytic infiltration of the interstitial - tissue , proceeding to fibrosis , and after 20 weeks of treatment there was usually calcifi cation of some of the cortical tubules and of the adjacent glomerular vessels . 
it is to be noted , however , that no cba mouse survived longer than 49 weeks , while all but one strong a mouse survived into the period 40 to 59 weeks . 
at 49 weeks only one of 18 cba mice treated with the potent bladder carcinogen 2 - acetylaminofluorene was known to have developed a tumour ( armstrong and bonser , 1947 ) , whereas 5 out of 10 strong a mice were known to have hyperplasia or a benign papilloma when dying between 40 and 59 weeks . 
thus , while it cannot be stated with certainty that 3 : 4 : 5 : 6 - dibenzcarbazole administered orally is not a bladder car cinogen , no indication has been obtained which would suggest that it has any action on the bladder epithelium , although from previous experience with these strains of mice evidence of hyperplasia or papillomatosis might be expected to be forthcoming within the experimental period available . 
more strong a than cba mice had tumours at death , but the difference is not statistically significant , and may be due to the greater dose of chemical and the longer survival period in strong a than in cba mice ( table iv )  . 
when it is taken into account that the incidence of carcinomas was identical in the two strains , despite the shorter survival period and smaller dose in cba mice , it would appear probable that cba is the more susceptible stra from table iii it is seen that these tumours of the forestomach are not dependent upon the continued administration of the carcinogen for the main tenance of their growth , as the chemical was discontinued for an average of 10 l weeks before the death of strong a mice , the range being o to 22 weeks . 
 it is rather surprising that papillomas of the forestomach were so rarely observed when tar was applied to the skin of mice , twort and twort ( 1932 ) having observed only occasional papillomas in 60 , 000 mice so treated . 
it seems clear that 20 - methylcholanthrene , 3 : 4 - benzpyrene and 1 : 2 : 5 : 6dibenzanthracene are carcinogenic in this sense , the latter having produced one benign tumour ( beck , 1946 ) and several malignant ones ( lorenz and stewart , 1948 )  . 
from the observations so far reported it is necessary to administer the chemical by the oral route , as no similar tumours were found after skin painting or subcutaneous injection . 
the reasons given by beck ( 1946 ) may account for this : the presence of the mucus barrier , the mucus flow tending to remove the carcinogen from contact with the cells , the possibility that contact between a carcinogen and a resting cell may not be sufficient to induce neoplasia , and finally the possibility that glandular epithelium is essen tially less susceptible than squamous epithelium to the type of carcinogen under discussion . 
there seems to be little support for the latter suggestion when the recent work ( horning , 1946 ; pan and gardner , 1948 ) on the induction of tumours by carcinogens in glandular epithelia in implants is considered . 
carcinomas of the intestine were not observed , in contrast with the results of lorenz and stewart ( 1946 ) , who noted such tumours in strain a and other mice which received aqueous emulsions of 20 - methylcholanthrene and 1 : 2 : 5 : 6 - dibenzanthracene . 
 amines are not without a similar action on the epithelium of the forestomach , papillomas having been observed in one riii , one oba and 2 strong a mice at 77 , 74 , 69 and 65 weeks respectively of treatment with 2 - acetylamhi.ofluorene in addition , otsuka ( 1935 ) described papilloma ( armstrong and bonser , 1947 )  . 
bonser seems possible that perforation of the stomach might have occurred during intu bation , resulting in a false passage of the tube into the muscles of the posterior abdominal wall and consequent injection of . 
in the course of observation of the cba strain for 17 years ( unpublished observation ) , one spontaneous spindle - cell sarcoma has been found and , therefore , the possi bility of a spontaneous origin of the tumour in mouse 10 cannot be completely excluded . 
contrary to the experience of andervont and edwards ( 1941 ) , this sex difference was observed only in the early weeks of the present experiment in strain a mice ( table iii )  . 
 in comparing the degree of liver damage in the two strains , it is evident that both suffered severely , but that the incidence of hepatomas was greater in strain a mice than in cba , the proportion of histologically malignant tumours being also greater . 
not only were hepatomas more abundant and more malignant in strong a mice than in cba with the latter chemical , but cirrhosis and bile - duct proliferation were a marked feature in both strains . 
although a high incidence of hepatomas was observed in cba mice ( but not in strong a mice ) when treated with 2 - acetylaminofluorene ( armstrong and bonser , 1947 ) , cirrhosis was absent and bile duct changes were minimal . 
it would appear possible that the chief action of 2 - acetylaminofluorene is to enhance the tendency to hepatoma already hereditarily present in cba mice , whereas 3 : 4 : 5 : 6 - dibenzcarbazole damages the liver parenchyma in such a way that changes are set in train which eventually lead to tumour formation ( orr , 1940 )  . 
 an experiment is described in which mice of the strong a and cba strains received by stomach tube , twice weekly , an oily solution of 3 : 4 : 5 : 6 - dibenzcar bazole , with a view to testing this substance as a bladder carcinogen . 
 no bladder epithelial changes were observed during an experimental period which would have sufficed to show , in these strains , hyperplasia and early tumour formation had 2 - acetylaminofluorene been the carcinogen . 
as - there was some doubt as to the absolute validity of this control method , we computed the number of cancer cases which would be expected , based on the morbidity figures from the cancer registry and on the official mortality statistics . 
rather unusual for twin studies of this kind . griginally all hospital cancer cases from the danish population of 4 million through six years , or an annual - number between 5000 and 6000 cases , but this figure must be reduced by about 20 per cent in which the questio ' n about twin birth had been left unanswered , giving a total very near to 30 , 000 cases ( 29 , 458 )  . the system of notification involves a rather heavy toll of omissions , vet it has functioned satisfactorily . 
the special questions asked twins with cancerbesides such items as name , address , ' occupation and the like - were confinea to previous diseases of a more serious character , including diagnosis and the date , in order to identify whether the twins were molloand place of treatment . zygotic or dizygotic , questions were asked about the similarity and about mistakes made by parents or others , and similarly about type and colour of hair , colour of eyes , stature , height and weight on the whole it may be said that we demanded almost full congruity before accepting a pair as monozygotic . 
the final distribution of dizygotic twins on groups of same and opposite sex , and the proportion between monozygotic and dizygotic pairs seem to show that our measures have been justified . it should be stressed that we have not been in - a position to contact the patients or their twins personally . 
the official percentage of twin births in denmark for the years 1926 - 30 is 1 - 64 , and considering the increased mortality among twin babies we ' think this a satisfactory correspondence . another check on the result was obtained through computation of the number of deaths expected from all causes among twins for whom notifications were satisfactory . 
among dizygotics we expected 36 - 3 and found 26 . these differences are not statistically 'significant , and they may be partially due to the less efficient information about those pairs of which one partner has died . in denmark , as elsewhere , the girl - boy ratio at birth is 0 , 97 with a male excess , which in the course of a fev years changes to a slight female preponderance . however , we find among both monozygotic pairs and dizygotic pairs of opposite sex a female preponderance of 1 , 48 to 1 , and 1 , 64 among the same - sexed dizygotic pairs , making 1 ' 53 for the total . 
the explanation of this considerable female excess m our material no doubt must be ascribed to the higher cancer incidence among females than among males in denmark , combined with the earlier age - in which female cancers arise . 
and , if we calculate the percentage of opposite - sexed twin pairs in our material , a figure which should be uninfluenced by the difference in mortality among the two sexes , we find exactly the same value as in the corresponding official birth statistics - 36 , 2 per cent . in spite of these satisfactory results , it should be remembered that in dealing with twin - pairs , of which at least one is suffering from old - age disease like cancer , we must expect insufficient information from twins in cases where the partner died in childhood . therefore we have excluded the twin - pairs where one partner bas died before the age of 5 years . similarly we have lost sight of a number of cc second " twins through emigration and similar events , so that the number has shrunk ' from 336 to 185 pairs . 
3. given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . since 1944 we have taken a detailed family history from all patients reporting with carcinoma of the breast . 
we have 459 family records up to the end of 1947 which are reasonably complete . so far we have not succeeded in supplementing these histories , taken at the time of first visit to hospital , by later questions to the surviving patients or by arranging visits to their homes or relations . 
some attempt has been made to confirm the causes of death of relatives by letters to hospitals , doctors and the registrar - general 's department , but such confirmation has , as yet , been obtained in only a few cases . our figures , therefore , contain all those errors of inadequate information and faulty recollection that one would expect from data collected . 
many people die of cancer without their relations knowing that this was the cause of death . it is in fact surprising how successful a woman can be in concealing a cancer of the breast from her nearest relatives living in the same house with her . it is not , therefore , so surprising that more distant relatives , or even close relatives living away from home , may be unaware of the nature of the illness involved relations , even some of an older generation , who are still living at the time that the patient is first questioned may later develop malignant disease . 
orr. from the department of pathology , univer8ity of birmingham . received for publication may 7 , 1953 . when a carcinogenic agent is applied to the skin of a mouse epithelial hyperplasia follows in the course of a week or two . if the applications are then stopped , the epidermis reverts to a histologically normal appearance , but if hyperplasia is again induced by the co - carcinogen croton oil , tumours of the skin will in due course be elicited . it is clear that the apparently normal skin has been altered in some way by quite a short treatment with carcinogen ( a single application of a powerful carcinogen will suffice ) , so that further growth - stimulation by an agent not itself carcinogenic completes the process of tumour induction . attention was first drawn to this phenomenon by berenblum ( 1941 )  . 
mottram ( 1944 ) showed that a single application of 3 : 4 - benzpyrene would suffice to produce tumours if followed by a course of croton oil paintings . in a full analysis of the factors involved , berenblum and shubik ( 1947 ) adopted the terminology of friedewald and rous ( 1944 ) , the effect of the preliminary carcinogen being described as the " initiating " factor , and that of the croton oil ( co - carcinogen ) as the " promoting " factor . both these factors were conceived as causing changes in some of the epithelial cells themselves . there is evidence , however , that the effects of carcinogens are not attributable wholly to the direct changes brought about in the epidermis . various authors have drawn attention to the progressive changes that occur in the dermis during experimental carcinogenesis of the skwhen the technique of grafting different components of the skin became available ( billingham and medawar , 1951 ) , an attempt to evaluate the relative importance of epidermal and dermal changes in the evolution of cancer was undertaken by billingham , orr and woodhouse ( 1951 )  . they transferred the fully treated epidermis alone to an untreated site prepared on the opposite side of the mouse 's thorax . in these conditions this epithelium no longer gave rise to tumours , though the reciprocal transfer of untreated epidermis to a bed cut in the carcinogen - treated site gave a yield of tumours approximately equal to what might have been expected had no operative treatment been undertaken . in view of these findings there appears to be another possible explanation of the phenomenon of co - carcinogenesis , namely , that after short treatment with a carcinogen the dermis is sufficiently altered to permit tumour induction when further epidermal hyperplasia is evoked by croton oil . 
the cutting of these grafts was greatly facilitated by the hyperthe rectangular slices plasia of the epidermis due to the carcinogen treatment . were floated raw side down on 0 5 per cent commercial trypsin solution and incubated at 38c . 
i. - experiment j : each horizontal strip represents the survival of a mouse after the grafting operation ( life line )  . oblique shading represents a papilloma , solid black shading a malignant tumour . 
an oblique line cutting the life line indicates operative removal of a tumour . the rate of appearance of tumours on the original methylcholanthrene - treated area after removal of thin thiersch grafts is indicated by the shading in the life line . 
orr treated site after removal of thin thiersch grafts in 16 out of 37 animals . 10 cases these tumours were removed to prolong the life of the animal . in 7 of these further tumours occurred , but 3 of them later regressed . the time range from operation to the appearance of a first persisting tumour was from 0 to 420 days with a mean time of 48 27 - 6 days . 
the survival of these mice from first croton oil painting ranged from 173 to 330 days ; some are still alive and under observation . these times are not , of course , comparable with those for the experimental animals , where the datum line was placed at the time of grafting . the total incidence of tumours on the grafted site is thus not very different from that induced by croton oil alone . there is a qualitative difference in that one of the tumours on the grafted site became conventionally malignant . 
by comparison , the original treated site yielded tumours in 16 of the same animals , 8 of them clinically and histologically malignant , without co - carcinogenic treatment on that site . the results in general of this experiment do not support the view that the transplanted epidermis contained latent tumour cells . 
the result is not , of course , unequivocal , but the occurrence of a single carcinoma does not suggest that many irreversibly altered cells can have been present . an attempt is in progress to find the effect of preliminary grafting of normal skin , followed by treatment with croton oil , in order to see whether the operative treatment of the dermis modifies the effect of croton oil . it is technically difficult to prepare pure epidermal grafts of normal thoracic epidermis , but experiments are in progress with re - implanted thiersch grafts . experiment k : reimplantation of a pinch graft cut from carcinogen - treated skin . billingham , orr and woodhouse ( 1951 ) made a few observations on the effects of cutting pinch grafts from the carcinogen - treated area , and reimplanting them at the same site . these experiments were undertaken to control the results of transplantation and in particular to make certain that the detachment of the superficial skin was not in itself the reason for the paucity of tumours when it was transplanted . 
the grafts were then replaced and the surrounding gap , caused by retraction of the skin around the wound , was dusted with sterile animal charcoal to mark the graft position . 
2. of 17 grafted mice surviving beyond the time of first tumour appearance , 11 produced tumours , but these regressed on 5 animals so that only 6 mice had tumours present at death . 
the tumours appeared after 8 to 200 days ( mean 42 17 - 3 days ) from the time of grafting the experimental group of animals . eight tumours became malignant after 20 to 100 days . 
the mice survived after grafting for 38 to 230 days ( mean 98 11 days )  . it will be seen that 70 days after the reimplantation more tumours were present on experimental animals that controls , and it seemed that the tumour production was indeed being augmented by the grafting operation as in the previously reported experiment . however , after about 100 days several tumours on the experimental animals regressed - a much less frequent occurrence in control animals . the outcome was that the proportion of animals bearing tumours at their deaths was no greater in the grafted animals than in the controls . 
2. - experiment k : the rate of appearance of tumours in the bridge epithelium over the scar around the reimplanted pinch graft of carcinogen - treated skin is indicated by shading in the life line and persistent tumours by the letter s . 
tumours arising on grafts themselves are shown by shaded blocks above the life line and persistent ones by the letter g . tumours arising outside the grafts are shown by shaded blocks below the life line and persistent ones by the letter o . 
1. experiment l : reimplantation of thin thiersch grafts cut from carcinogen - treated skin . in this experiment thin thiersch grafts were removed from the carcinogentreated area as described in experiment j . 
as great an area of the carcinogentreated skin as possible was taken off and the pieces were then planted back again , in some cases after smearing the graft beds with animal charcoal to mark them . the results are shown in fig . 
the mice survived from 69 to 230 days ( mean 124 + 12 - 5 days )  . tumours appeared on 5 out of 18 control ungrafted mice after 16 , 20 , 23 , 24 all became malignant . 
the mice survived from 38 to 230 days and 200 days . ( mean 99 14 - 2 days )  . experimental animals control animals 4222 4223 4225 4246 4247 4248 4249 i 4250 4488 z 4493 4494 4498 4499 4500 4502 4206 4207 4208 4209 4210 4227 = r_ 4229 4230 4474 4475 4476 4477 4478 4479 4480 4481 4483 4495 z 77 days after thiersch graft operation fig . 
3. - experiment l : the rate of appearance on reimplanted thiersch grafts of carcinogenblocks of shading below the line indicate treated skin is indicated by shading in the lifeline . tumours outside the grafted area . 
some of them , such as age , sex and diet are fairly easy to control . others , such as range of temperature and amount of carcinogen administered , may be less easily controlled . 
orr of animals such as were used in the present experiments . may strike at any time . parasites and infections experiment m : the insertion of cotton threads into the dermis of carcinogen - treated skin and their removal after 2 weeks . orr ( 1934 and 1935 ) showed that preliminary alteratioii of the dermis , by the introduction of linen threads and their subsequent removal before treatment with a carcinogen led to a significantly earlier appearance of tumours . 
we thought it of interest to see what happened if the threads were introduced after the standard preliminary carcinogenic treatment of the present experiments . fine white cotton button - thread was threaded into the finest ordinary sewing needle which would take it and then sterilised . the needle was inserted into the skin of the anaesthetised mouse just outside the carcinogen - treated site . was passed through the dermis of the treated area as near to the surface as possible , endeavouring not to injure the treated epidermis itself , and out through the cotton was carefully pulled through and the surface beyond the treated area . the procedure repeated again under another part of the treated area . the thread was passed under the treated skin in both directions about half a dozen times in all and left in situ for 2 weeks . at the end of this time the projecting loops of cotton were cut and the threads carefully pulled out of the skin under anaesthesia . unfortunately the threaded area had not been covered over and many of the mice had scratched and tugged at the loops of thread , resulting in some damage to the carcinogen - treated epidermis itself '  . the results are shown in fig . 
5. persistent tumours appeared on 10 of 19 experimental animals in 9 to 92 days ( mean 35 7 * 9 days )  . eight tumours later showed malignant changes . 
orr croton oil , the tumour yield is only very slightly greater than that obtained by croton oil on otherwise untreated skin . of the few tumours so produced , all except one were papillomata , which appeared somewhat earlier than might have been expected with croton oil on previously untreated intact skthe single exceptional tumour was a carcinoma , an unusual occurrence with simple croton oil treatment , but it has to be remembered that grafting was delayed up to the time that tumours were already beginning to appear on the original methylcholanthreneexperimental animals control animals 77 , , / , , , , z , , 4358 4361 4369 4370 4372 4373 4374 ' .. 
the results of berenblum and shubik ( 1947 ) were explained on this basis by postulating a two - stage process , the first stage being the alteration by a sudden and irreversible process of a few normal epithelial cells into latent tumour cells , the second the conversion of these latent cells into visible tumours . the first stage ( " initiating process " ) requires a carcinogen ; the second ( " promoting process " ) can be brought about by various non - carcinogenic hyperplasia - inducing factors , of which croton oil is the most fully analysed . it appears to us that the facts elicited by berenblum and shubik are explicable without reference to the conception of latent tumour cells , and that an irreversible change inflicted on the carcinogen - treated site as a whole , and perhaps particularly on the supporting tissues , would explain the olbserved phenomena of co - carcinogenesis . in one respect the view we put forward offers a more satisfying basis for one of the berenblum and shubik findings . 
they showed that even after as long an interval as 20 weeks between the initial painting with carcinogen and the subsequent croton oil treatment , the total tumour incidence remained undiminished as compared with shorter intervals . if , therefore , latent tumour cells were there all the time , it would be necessary to assume that they did not participate in the normal processes of desquamation . it is also relevant to point out that blum ( 1944 ) has analysed the results of carcinogenesis by mathematical methods , and reaches the conclusion that the available data are not consistent with the hypothesis of a discontinuous two - stage process . if the dermal and subcuticular changes are of importance to carcinogenesis , the most obvious way in which they could act would be by interference with the nutrition and metabolism of the overlying epitheliuthe latter does not carry blood vessels , and is dependent on the functional effectiveness of the stromal tissues . it has previously been pointed out that the rate of evolution of the dermal changes ( in simple treatment with a carcinogen ) is of the same order as the potency of the carcinogen in terms of time taken to produce tumours . it may therefore be asked why a single application of a carcinogen followed by a co - carcinogen is effective at all in the induction of tumours . 
a possible explanation is that the hyperplastic epithelium following the co - carcinogen is susceptible to degrees of dermal change and vascular impairment which would not effect normal epithelium or epithelium in a less vigorous state of multiplication . 
orr the reimplantation studies of experiments k and l necessitate some modification of the conclusions drawn from the previous reimplantation of billingham , orr and woodhouse ( 1951 )  . it is now found that this treatment does not appreciably alter the ultimate tumour yield , because the apparent increase in the period immediately following the operation is offset by the regression of several of it was suggested that the previous result might be these " explosive " tumours . accounted for if the operative treatment accelerated the establishment of optimal it remains not impossible that this conditions for the evocation of tumours is so , and that subsequent regression of some of these tumours was dependent on the ischa3mic conditions in the dermis and subcutis developing further to a point at which the vascular conditions were inadequate to maintain the tumour . in experiments of the type recorded here , it is necessary to consider the effects experiments such as those of of mechanical trauma on the yield of tumours . lipschuiitz ( 1924 ) , mandl and stohr ( 1924 ) , doderlein ( 1926 ) and deelman ( 1927 ) indicated that trauma of a previously tarred area tended to provoke tumours closely related to the sites of the healed wounds . 
more recently pullinger ( 1943 ) obtained results similar to those of deelman using a pure chemical carcinogen . mackenzie and rous ( 1941 ) and friedewald and rous ( 1944 ) obtained a high incidence of tumours around healing wounds produced by punching holes in the it was on the basis of this work that ears of rabbits after carcinogen treatment . the conception of initiating and promoting factors , later adopted by berenblum and shubik ( 1947 ) was based . in almost all such work , attention has been almost exclusively directed to the epithelial cells , but linell ( 1947 ) showed that there was a difference between superficial and deep trauma . 
numerous tumours , including carcinomata , appeared on the original treated donor site . re - implantation of pinch grafts and thin thiersch grafts into the site from which they were cut in the carcinogen - treated skin did not result in an increased incidence of persistent tumours . there was an increase in regressing tumours . trauma to the treated dermis ( by temporary insertion of threads or by electrically heated wires ) did not increase the incidence of tumours . the present results , like those previously reported , suggest strongly that both the epidermis and dermis ( and possibly deeper structures ) are involved in the carcinogenic action of methylcholanthrene . in particular , they are difficult to reconcile with the two - stage epithelial hypothesis of berenblum and shubik ( 1947 )  . we are indebted to r . 
salaman. from the cancer research department , london hospital medical college , london , e.l. received for publication march 15 , 1952 . if a chemical carcinogen is applied to the skin of mice at a concentration just too low to produce tumours by itself and , after an interval , a co - carcinogen is repeatedly applied to the same site , tumours . 
weekly applications of 2 * 5 per cent croton oil in paraffin to both groups were begun 6 weeks after an initial treatment with 0 - 15 per cent dmba . 
croton oil treatment of group 5 was stopped after 8 applications , when the average tumour incidence in both groups was about 0 - 5 per mouse . incidence in the two groups increased for a further 2 to 3 weeks , after which incidence in group 4 continued to rise , following the curve usual in this type of experiment , while that in group 5 remained almost constant at about 2 tumours per mouse . after 9 weeks without treatment there had been no appreciable change in tumour number in this group , though the tumours already visible had continued to grow . 
tumour incidence in group 5 had , by then , reached about 11 tumours per mouse ; it continued to rise for a short time , as before , then fell a little , and again remained approximately . 
1. - comparison of the effects of continuous and intermittent weekly applications of croton oil to mouse skin , 6 weeks after a single application of a carcinogen . consequently individual points in fig . 
1 , which represent by others , is high . average numbers of tumours per mouse , have high standard errors , and the differences between corresponding points on the curves of groups , 4 and 5 are barely significant . it is easy to show , however , that the curves as a whole differ their slopes between the 15th and 21st week were compared as significantly . follows : the average increments .of tumour number of individual . 
one must suppose that there is a critical size , rather less than 1 mdiameter ; i.e. , that tumours smaller than this do not continue to grow without further stimulation . did these invisible tumours remain unchanged during the remission of treatment ? apparently not , for when the treatment was resumed there was no immediate outburst of newly - visible tumours . after 58 days the interrupted rise in numbers this latent period is not significantly different from that of the continued . response to the first course of croton oil treatment . 
the skin behaved as it would have done if it had had no previous co - carcinogenic stimulation . it is possible to conclude that the change produced by croton oil in mouse skin previously treated with a carcinogen is not sudden , or independently proit is a gradual change , requring repeated applications for its maingressive . tenance and progress , which is reversible during the greater part of its course , but becomes irreversible not long before tumours become visible . it has been previously noted ( shubik , 1950a , 1950b ; salaman and gwynn , 1951 ) that of the tumours produced in mouse skin by croton oil following a single treatment with a carcinogen most are benign , though some reveal or develop a malignant character later . 
the difference between these two latent periods is not significant . ( 3 ) these results are discussed . it is concluded that the change produced by croton oil in mouse skin previously treated with a carcinogen is a gradual one , requiring repeated treatments for progression towards tumour formation , that it is reversible during the greater part of its course , but becomes irreversible not long before tumours become visible . i am indebted to professor s . 
the carcinogen was then placed in contact with the surface of the epithelium , and ingerted into a bashford transplanting needle , care being taken to shield the carcinogen from the connective tissues of the host animal . in some instances as manv as tbxee subcutaneous primarv grafts were made on each side of the belly of a s ' mgle host mouse to give a series of prostatic tumours growing under identical hormonal conditions . 
the technique is similar in some essentials to that - previously employed bv greene ( 1945 ) , and rous and smith ( 1945 ) , except for the important difference that the present experiments involved tiimour production from adult , not embrvonic tissues . the influence of bilateral orchidectomv upon the growth rates of glandular and squamous cefl prostatic carcinomas was mvestigated in 6 - months - old mice , which had pre ' %iously been castrated before attaining pubertv . 
10 were of the squamous variety , and 2 were spindle - cell sarcomas . sixteen homologous grafts failed to gi - ow , and were foiind at autopsy not to have becoi - ne vasettlarized . in order to detern - iine the early phases of carcinogeiiesis , witliin the primary grafts a further 30 mice received prostati c iniplai - its , and , " , - ere sacrificed at intervals ranging from two weeks to five inonths after iniplantation . 
which are practicallv fused together with an anterior portion and an isthmus or middle lobe which , as in the rodent , surrounds the urethra . unlik - e the hu - man crland , the mouse prostate has no uterus masculinus or its equdvalent , nor is it encapsulated . 
thus in the human gladd there are as many as 32 ducts ( 31axiniow and bloom 1948 ) , whilst in the mouse them are onlv six . a fibromuscular stroma consisting of dense connective tissue is common to both ty - pes of gland . 
the cytoplasm of the glandular epithelium in both species contains numerous secretorv granules , the majority cytologically treated mouse maerial shows the secreticn of which are , of lipids . in both forms to consist ot protein w%ith fine lipoidal droplets in suspension . 
iiornixg to a failure in the release of the secretion , siiice the ducts ai - e iio lojitretpatent , and proliferation which might be due to the direct action of the carciilogeii . neoplastic change - s in graft8 implanted with the carcinogen . the carcinogen , which is placed in direct contact with the living tissuewithout solvent such as oil . 
or lard , induces verv little foreign body reaction , necrosis or residue within the graft which iiiight mask or conceal the actual areas where neoplastic changes fii - st arise . 
h perplastic alveolus froni which it has arisen . through a whole graft of dorsal lobe epitheliuni fixed 41 weeks after implantation . there is very little foreign bodv reaction , and it will be iioted how clearly the foci in which iiialignant changes arst arise may be detected . adjacent to alveoli lined with a , low colunuiar epithelium and distended with secretion colitaining epithelial debris and polyiiiorphs are collapsed alveoli in wliieb the epitheliuni has entered an exhaustion phase . 
the tongue - lik - e colony , of earlv malignant cells , of t3 - pe a was t3 - pical of those tumours which finally developed into secreting glandular carcinomas . l [ n the example ihustratc ; 1 the proliferation arose from a single alveolus and was the only fo - cus of later stages from other grafts showed malignant change in this part - icudar gr - aft . the onset of secretorv activitv . 
14 shows three ii - ialignant alveoh in the peripheral engaged in secretion . area of an invading new grom - th of this type from an eight weeks ' old graft.. several epithelial cefls are in process of abnormal cefl division . 
transplanted into n - lice of the s - ame age and sex as the host mouse , retained their histological charact - c - rs for 16 generations of serial transplants before finallv transforming into a squamous - cehed tumour . the type b lesion arose in the various grafts exaniined from a single alveolus . aild the example illustrated in fig . 
the area of the alveolar wafl malignant cells - , contain small nuclei with nunierous atypical mitotic figuxes . apart from the different morphology and staining reactions of the prohferating cells . 
it will be noted that they tak - e origin from an alveoluss in which secretory adjacent alveoli not shor% - ing these proliferative activitv has been inhibited . changes are distended with secretion . in older grafts the type b prohferation leads to the formation of pseudo - alveoli at the margins of the tumour and . although the glandular architecture of the epithelium is thus partly restored , there is no evidence of secretion . 
the secretory epithelium like that in the mouse gland is thrown into folds which project into the glandular lumina . note alveolar concretions , and compare with that of mouse in fig . 
4. - implant of portion of dorsal epithelium from a normal strong a mouse without carcinogen after surviving subcutaneously in a host male mouse of same age and strain for 12 weeks . note distension of alveoli with prostatic fluid which is far in excess of what is typical of the normal gland in 8itu , and frequently leads to cystic dilatation . 
9. - early hyperplastic changes in prostatic epithelium of a strong a mouse , after eight weeks ' subcutaneous implantation with carcinogen in a host mouse of same sex , age and stra ' the glandular epithelium contains patches which are 10 - 20 cells in depth . 
on palpation the tumours undergoing regression were hard and the two which failed to respond to orchidect.omv were soand sin - iilar nodular t.o those growing in the control mice . 
variable influence of orchidectomv on the behaviour and gro - wth of this alandular carcinoma was further demonsirated in another series of experiments which were repeated at a later date . in this instance two groups of mice , 10 in each , lik - ewise castrated before pubertv . 
received transplants of the same tumour . the gro , %th rate of three tuniours in the first group of io mice was entirelv four tumours in the second group also grew at approxitunaffected bv castration . iiiatelv the same rate as the transplants in the intact control mice . 
four tumours - , responded to this treatment bv showing an immediate increase in growth rate while the , remainder were una#ectedsquanwus - cd , l cwrcinonia.. a glandular cell carcinoma which had undergone spontaneous inetaplasia after its 10th generation of serial transplants in non - castrated normal male mice , was chosen to determine the possible influence of orchidectomv on a glandular tumour which was undergoing squamous transformation . microscopic exan - iination of this particular tuniour had sho - w - n that the metaplastic alveolar epithelium was columnar and much of it still retained secretorv activitv . 
a variable degree of keratinization associated with prickle cell transformation of the epithelium occurred in concentric nests of cells in the peripheral alveoli of the tumour . comparison of the grouth rates of this particular tumour transplanted inw intact control mice . 
the occasional slight inhibition of tumour growth in soiiie orchidectomized mice was within the limits of possible normal variation . the histological changes accompanying vaiiations in grawth and behaviours , in castrated and non - castrated mice . 
17 , were ha ' rder and tnore nodular on palpation than those growing in secretory activity as judged from sectioiis had beeil sitpcontrol intact mice . pressed and squamous metaplasia , of variable intensity in different areas of aiiy tumour , was always found . 
 ' portions of this tumour failed to grow when transplanted into normal ho ; t rnice . tumours inhibited bv castration but subsequentlv treated with testosterone . after which wth was renewed . 
the alveolar character of the growth had persisted , but.the epithelial cells were shorter than in the original untreated tumour . these results were confirmed in a second series of experiments involving the same number of castratm mice using the same dosages of testosterone . 
tumours found to be retarded in growtli during the fifth week of treatment were characterized by a decrease in the size of the majoritv of the alveoli , a reduction in the height of the epitheliuni , a coinplete disappearance of cell - divisioii , and an increase in the fibro - mtiscular stroiiia . 
the alveoli diminished in size when this rupture occurred , whereas adjacent alveoli with intact epithelial cells appeared to be so slightly affected by the oestrogen that little more than a reduction in the lieight of the cells could be obsei - ved . 
a detailed study of the hyperplastic ' changes in numerous grafts has shown concltisively that in no single instance has an actively secreting epithelium been the focus of malignant change . 
the technique used in the present without at the same time da experiments is pomibly supenor to that involving injection of carcinogens , dissolved in such solvents as lard , merely because it entails less damage and concentrates the carcinogen at the proper site . since the present technique also involves homologous grafting , however , the reaction between host and graft tissues may play some part in the successful induction of a tumour . in mice the survival of a homologous graft seems to depend on the use of a closely inbred stock mice of incleterminate ancestry will not often tolerate grafts for strain . periods sufficient for the establishment of tumours . recent experiments have indicated fwrthermore that the age of the mice providing the graft tissue , and the age of the host mice into which the grafts are implanted , have an important bearing on the problethus the survival rate of grafts of lung or prostatic epithelium without carcinogen was greater if host mice under eight weeks old were used , and was considerably reduced if the mice were over eight months this was invariably the case if the mice were of mixed or pure line origin , old . but only 12 per cent of the grafts in young mi ed stock mice survived as against in older mixed stock mice of 8 months 85 per cent in the young pure line mice . to one year no graft survivecl after three months ' implantation , whilst in inbred mice of a similar age 25 per cent of grafts survived after the same period . 
combining the carcinogen 20 - methylcholanthrene with grafts of lu or p - rostatic epithelium in 25 eight - weeks - old mice of mixed stock gave only one tumour , a spindle - miled sarcoma , twelve weeks after grafting ; the remaining 24 host mice failed to produce tumours , and at autopsy the grafts were found not to have price ( 1941 ) , using rats of different ages from an albino become vascularizedl strain which was not strictly pure hne , found that ventral lobe prostatic tissue grafted subcutaneousl in the abdominal wall beha - ved in much the same way , prostatic tissues from old rats being very limited in their capacity to survive as homologous grafts . 
many apparently viable grafts from old rats were found to be degenerate on histological examination , but when regrafted into younger hosu the implants underwent a rapid recovery and the pmstatic epithehum even regained its secretory activity . 
one of the most intere8ting features in successful grafting of prostatic epithehum is its ability to continue secreting while growing under the influence of foreign host reactiom and changed vascularity . 
ob - viously androgenic stimulation , as price ( 1941 ) maintains , must be important in this phenomenon . the dependence of prostatic epitheli - um upoii androgen smretion has long been realized and the experimental evidence need not be referred to here , except to mention that in recent years the elegant technique of transplantation of fmgments of prostate into the anterior chamber of the eye ba - s provided additional confirmation . and rosenblum ( 1937 ) , moore , rownblum , tolin and melchionrka ( 1937 ) , and moore and smith ( 1937 ) , grafted rabbit prostatic epithelium in this way and photographed the implants dailv . 
they were able to record fluctuations in size of the grafts which were abohshed by castration , and considerably increased by administration of testosterone propionate . heckel and kretwhmer ( 1935 ) made similar observations of the responses of prostatic grafts to anterior pituitary extracts . 
horning mice or rats will maintain the size and normal histological appearances of the hill and strong ( 1938 ) attribute the androgpnic prostate and seniinal vesicles . effect of the ovary to the lowered temperature of the ear , and deanesly ( 1938 ) has correlated this activit ' with extensive luteinization of the theca intema within the ovarian grafts . another potential source of androgens to be considered in relation to prostatic grafts and tumours is , of course , the adrenal cortex . 
the rare adrenal insufficiency in young children associated with adxenal hypertrophy leads to enlargement of the prostate and differentiation of its glandular tissues to a degree comparable with the adult gland ( dijkhuizen and behr , 1939 - 40 )  . prostatic enlargement also occurs in adults with adxenal cortical tumours . miller ( 1947 ) has found a relative increase in dehydroisoandxosterone , and a decrease in androsterone in the urine of patients with benign hypertrophy of the prostate . according to callow and callow ( 1940 ) the source of dehydroisoandxosterone is probably in the adxenal cortex . it remains in the urine after castration , but disappears in cases where there is destruction of the adrenal cortex . while it niight be assumed that grafts of prostate or transplantable prostatic tumours would have poor chances of survival in female hosts , under certain conditions they do in fact survive and this must be attributed to the influence of androgens produced either in the ovary or in the adxenal cortex . price ( i 941 ) grafted normal prostate into female host rats , and found that in some cases the grafts were similar in histological structure to the prostates of castrate males , whereas other - grafts flourished and underwent secretory activity . this result she maintained could be due to the development of androgenic foci in the ovaries of some , but not all , host animals . 
the grafts failed to differentiate in spayed rats , whereas they survived in a functional condition for prolonged periods if the hosts were over 80 days old . either virgin females or rats which had littered frequently would sustain functional grafts , provided they were not youngei than this apparently critical age . the opinion is often expressed that the prostate gland in rodents is not strictly homologous with the prostate in the human , and thus there can be rttle justification for comparing growth behaviour and endocrine responses in experimentally induced prostatic tumours or hyperplasias with the spontaneously occurr , ingconditionsinthehumangland . greenstein ( i - 947 ) maintainsforinstance , that because certain rodeint prostate tumours grow when transplanted into female mice , whereas human tumours are so sensitive to inhibition by oestrogens , the two types of neoplasm are not fundamentally the same . 
apart from the single case reported by deniing , jenkins and van wagenen ( i935 ) of a spontaneous nodular hyperplasia in the suburethral tissue of an albino rat , there is no record of spontaneous prostatic hyperplasia or cancer in rodents . if it can be shown , however , that the prostate tumou - ts induced by methylcholanthrene behave in essentially the same way to hormone administration or deprivation , the differences between rodent and human neoplasms will be less significant . the glandular carcinomas described in these experiments , when transplanted into rnice ca 'strated before puberty , show complete inhibition of secretion , later followed by squamous differentiation . 
subsequent traimplantation into castrated hosu is not followed by any appreciable inbibition . some glandular celled carcinomas am thus dependent for their sustained growth on an adequate androgen level ; the squamous growths would appear to flourish without testicular androgens and , if they are at all androgen - dependent , it must be concluded that adrenal cortical androgens are in these cases suflicient . and hodges ( 1941 ) buman prostatic cancer falls into two according to it is possible that groups , androgen dependent - and androgen independent . squamous - celled carcinomas in mice belong to the second category , although aours rarely occur in man ( willis , 1948 )  . such t a smah percentage of glandular mouse tumours failed to regress when transplanted into castrated hosts ; they even grew at approxim tely the saine rate m those transplanted into uncaamted mice . 
howard ( 1937 , 1938 ) has demonstrated that removal of the gonads in rats , provided these are sufficiently yoijn , increases the capacity of the adrenals to secrete androgens . 
as ar the mice in the present experiments were castrated before puberty , and an interval of several months elapsed before they received t aour transplants , it seems probable that androgenic foci had developed in the adrenals of those castrated mice in which unfortunately.biochemical assay the tumours showed no inbibition of growth . of androgens secreted from non - testicular sources cannot be undertaken in the mouse to confirm or refute these conjectures . on the chnical side the serum acid phosphatase and urinary 17 - ketosteroid levels can be used respectively as indicators of the activity of mahgnant prostate cells and of androgen smretion . 
thus mn4vs stevens and hodges ( 1941 ) studied cases in which a recurrence of prodatic cancer had fobowed an initial improvement in the condition obtained - by bilateral orchidectomy . there was a rapid reduction in the serum acid phosphatase , and a simil ri marked fall in the urinary 17 - ketosteroids after orchiclectomy . 
the development of androgenic foci in the adrenals was regarded by and swtt ( i945 ) as responsible for the recurrence of the disease . recently , following failure in four - selected cases to control growths by orchidectomy , these worken tried bilateral adrenalectomy ancl , although three of the patients died within a comparatively short fume after operation , the fourth survived for 116 clays . 
cox ( 1947 ) tried unilateral adrenalectomy on three patients with prostatic cancer , all of whom had temporarily responded to treatment with there was an immedi te drop in the 17 - ketosteroid level and a oestrogens . r tumours transplanted into normal male mice showed a varying retardation of growth after oeamgen treatment , but in no single instance did any undergo complete regression , as was the case following orchidectomy . 
horning inhibition of tumour growth with such relatively high doses was the result of non - specific toxic action . it was also found that the amount of stilboestrol tolerated depended upon the genetic constitution of the rnice . 
although in the present experiments the dose of stilboestrol was approximately half of that used by ludford and dmochowski ( 1947 ) , the average loss of weight of the tumourbearing rnice was approximately i ' to 2 g . 
during the total period of treatment . histological examination of regressing glandular tumours after stilboestrol gested that diminution in size of the tumours was primarily due to thera inhibition of secretion , later followed b ' degeneration and collapse of alveoli . schenken , burns and kahle ( 1942 ) and fergusson ( 1946 ) have exandned serial biopsies of human prostatic carcinoma under oestrogen treatment . 
the former authors described in detail such cytological changes as vacuolation of the alveolar epithelium , disintegration of the cell membranes , sometimes accompanied by stratification of the epithelium , and an increase in the stroma . these changes are very similar to those which occur in the glandular mouse tumours during oestrogen treatment . 
the squamous metaplassia , which some of the glandular mouse tumours undergo in response to oestrogen administration , is rarely found in the human gland after sirnilar treatment . recently , however , inglis ( 1948 ) described a pronounced squamous differentiation in a man treated with stilboestrol for prostatic cancer . it is tempting to regard the atrophy of prostatic tumours in the mouse as due to failure of optimum androgen secretion in the castrated hosts , or to a similar drop in gonadotrophin production when intact tumour - bearing mice are treated those tumours whieh fail to regress are possibly receiving with oestrogens . sufficient androgenic stimulation from the adrenal cortex . inhibition of secretion and degeneration of the epithelium in these tumours are so similar to the changes induced in the normal prostate following castration or oestrogen administration that any more complicated mechanism operating in the case of the tumourbearing mice seems unnecessary . 
it bas been possible in grafts , impregnated with the carcinogen , as soon as four to eight weeks following implantation , , to distinguish between three distinct types of early malignaint lesions . detailed swdy of the hyperplastic changes in prostatic alveoh in numerous grafts showed that in no single idstance has the actively secreting epithelium been the focus of mahgnant change . it tberefore appears that the non - secreting exhausted alveolar cells m the prostatic gland are more susceptible to the action of the carcinogen than those at the height of secretory activity . this supports the contention that chemical carcinogens act - more readily on cells following depression of cellular activity . the influence of sex hormones and orchidectomy was determined on the behaviour and growth of glandular and squamous - cell carcinomas of the prosute . a greater number of glandular carcinomas regress when transplanted into male mice cm - trated before puberty , and a small percentage of - the - e tumours resume growth when treated with testosterone propionate . 
tumours which at the time of their induction were diagnosed as squamous growths , or glandular carcinomas which had undergone spontaneous squamous differentiation during serial transplantation , exhibited no appreciable response to these fornvs of therapy . these results indicate that only glandular and not squamous carcinomas are dependent for their sustained growth on an adequate androgen level . glandular carcinomas when treated with dieth - vlstflboeaml respond more re - adily than squamous t aours . 
and bave given additional information on the factors responsible for tumour regremon . particular attention hm been paid to those glandular tumours which failed to respond to either orchidectomy or hormonal theraphy , and the possible endocrine factors inducing resmtance to treatment are discussed in the hght of recent experimental and clinical evidence . this investigation has been supported by grants to the royal cancer hospital , fi - om the british empire cancer campaign , the jane coffin childs memorial fund for medical research , the anna fuller fund , and the u.s. 
on the other hand the quantity necessary to induce abdominal fibroids is not much above the mentioned hysterotrophic dose ; abdominal fibroids can be induced by three injections per week of 5 lg . 
of oc - oestradiol , or even less , when administered as the 17 - caprylic ester of a - oestradiol , which is absorbed so slowly that a continuous oestrogenic action is made possible ( lipschutz , bellolio , chaume and vargas , 1941 )  . similarly a few micrograms of free oc - oestradiol or oestrone absorbed from a subcutaneously implanted tablet also are sufficient to induce fibroids ( lipschutz , thibaut and vargas , 1942 )  . 
on the contrary , quantities about a hundred times greater are necessary to elicit the same tumorigenic abdominal reaction if administered by subcutaneous injections of the free hormone ( lipschutz et al . , 1939 , 1942 )  . in the present work we shall deal with abdominal fibroids induced by extremely small quantities of estrogens and with the neoplastic action of these quantities on the uterine glands in experiments of long duration . fibromatogenic action of very small quantities of x - oestradiol and of oestradioldipropionate . tablets weighed 33 to 35 mg . 
each. tablets consisting of one part of the oestrogen and 19 parts of cholesterol merck were prepared in the usual way by mixing , dissolving in ether , drying and after drying in vacuo they mixing again . were implanted beneath the skin into castrated female guinea - pigs weighing 380 to 480 g . uterine and other abdominal fibroids were elicited under these experimental conditions ( riesco , 1942 ) , though the incidence was less than with the injection of great quantities of oestradiol administered as the benzoic ester , as in the experiments of iglesias ( 1938 )  . necropsy was made 112 to 118 days later . during necropsy , cleaned and dried subsequently . absorption was calculated from the loss of weight of the tablets recovered calculation was made under * this work has been aided by a grant from the jane coffin childs memorial fund for medical research , grant administered by a . 
the result of these experiments is given in table i . as seen from table i , tablets containing originally 5 per cent of the free cx - oestradiol and remaining for about 4 months in the body of a guinea - pig produce anew oestrogenic and fibromatogenic actions when reimplanted into another guinea - pig . the same result was observed when 4 months afterwards the tablet was implanted for a third time and a fourth time . there was in all these experiments opening of the vagina , growth of the nipples and uterine bleeding . 
should there have been selective absorption , oestrogenic hysterotrophic and fibromatogenic actions or uterine bleeding would have been impossible , in any case , in the third and fourth implantation . 
were obtained for the first 4 months . it is remarkable that in successive implantation smaller figures were obtained . allowance must be made for entering of substances into the tablet as has been shown by different authors ( bishop and folley , 1944 ; deanesly and parkes , 1943 )  . absorption was most probably greater than indicated by figures in our table i . 
but it can easily be demonstrated that daily absorption of oestradiol was certainly less than about 3 to 4 , g . in these experiments the total quantity of oestradiol in the tablet was never more than 1750 , g . , and the tablet remained beneath the skin during 470 days without exhaustion of the oestrogen . 
but in the next paragraph we shall mention experiments in which similar tablets were active for a total of 675 days without the oestrogen being exhausted . histological examination of the uterus in the mentioned animals revealed pronounced cystic glandular hyperplasia of the endometrium , polypous proliferation of the latter , thickening of the vaginal wall and proliferation of mammary these statements coincide with what has been formerly observed with tissue . the prolonged action of estrogens in this department . neoplastic action of small quantities of oestrogens on the uterine glands in experiments of long duration . in the above - mentioned preliminary experiments evidence was given that by continuous absorption of very small quantities of oestrogen , certainly less than 3 to 4 ug . 
fibromatogenic action was rather less pronounced than with the free hormones . but with the fibromatogenic action of oestrogens due allowance has to be made for variation . in the second series in which tablets already used in the first one were again implanted and left in the body for no less than one and a half years , uterine growth was considerable . uterine fibroids were also induced . microscopical examination of the uterus revealed in the series of long duration a condition fundamentally different from that seen at four months ; there was besides cystic glandular hyperplasia of the endometrium also proliferation of glands infiltrating the myometrium , reaching the serosa and even perforating it . the lumen of the proliferating and infiltrating glands was sometimes cystic , sometimes small or absent . 
the neoplastic and infiltrative growth of the glands was , in lipschutz ' experiments with ovarian fragmentation , coincident with those we have described in the last paragraph , with the exception that with ovarian fragmentation the invading glands did not reach the serosa and did not perforate it , as was the case in one of our animals . 
a guinea - pig having been injected for 8 months with oestradiol benzoate was found , 4 months later , to have a " precancerous " transformation of the endometrium in the upper third of the uterine horn . this latter observation of lipschutz and others is so far the only one which may be quoted in favour of irreversible atypical proliferation of the uteririe mucosa elicited in the guinea - pig by oestrogens and persisting after the withdrawal of the hormone . neoplastic malignant growth seems to be more easily elicited in the cervix of the uterus in the mouse , as shown by the work of gardner and others ( gardner , allen , smith and strong , 1938 ) and allen and gardner ( 1941 )  . in the work of these authors the oestrogens - - the benzoic ester of oestrone and oestradiol were given by injection . 
the quantities injected were , when calculated per gram of body weight in the work of the american authors with mice , probably 20 times greater than in our work with guinea - pigs . another interesting aspect of our work may be mentioned here . 
minutes. this degree ' of hydrolysis was found to remove ar non - dna phosphorus as wer as all non - dna adenine from the cells , while leaving dna phosphorus and dna adenine in situ , thus obviating the necessity for the use of the einzyme ribonuclease ( lajtha , 1954b )  . adenine 14c administered to cells appears both as labelled adenine and labelled guanine in the dna ( hamilton , 1953 )  . no attempts to separate labelled adenine from labehed guanine have been made in the experiments ; the activity of the dna has been measured as a whole . the autoradiographs were exposed for 10 - 14 days . 
ar autoradiographs belonging to the same experiment were coated with the same batch of stripping film , exposed the same length of time at the same temperature ( 3 - 4 ' c . ) , and developed in one batch of developer simultaneously , and , finally , were stained simul368 l . 
on each slide 100 - 200 nuclei were counted , and grains were counted over - 5 - 10 showing the strongest autoradiographs . this was to obtain the values for the maximum degree of dna synthesis and was done after surveying repeated counts at different times were performed to tes - t the over 500 nuclei . reprodiicibihty of the counts , and it was found that with the above technique the same observer showed ain error not greater than 10 - 15 per cent . for the calculations on the quantity of isotope i ' ricorporated into the cell , it was assumed ( 1 ) that the geometry of the autoradiographs was such that only 50 per cent of the electrons emitted frothe disintegrations reached the photograpbic emulsion , and ( 2 ) that the 32p electrons produced 0 - 7 grains per incident electron , and the 14c electrons 2 gfai - ns per incident electron ( lamerton , 1950 )  . if the exposure tiine and the half life of the isotope in question as well as the grain count per nueleus and the grain yield per electron of the isotope are known , then the number of the atoms of the isotope withiii the nucleus can be calculated . the technique is sensitive enough to detect 20 - 30 32p atoms per cen . because of the very long half - hfe of 14c ( and because exposure times were limited to the relatively short period of 14 - 30 days ) the minimum necessary number of 14c atoms per nucleus to show an autoradiograph is of the order of 106 . in preliminary experiments the maximum dna 32p uptake per nucleus was found to be the order of 600 atoms ( > i 00 grains in 14 days ' exposure 07 grains / electron )  . 
the labelling obtained in vitro of the dna is therefore of the order of i : 3 x 107 . if the cehs cannot differentiate between 31p and 32p then the total phosphorus pool available for the cens must also have been labered in the ratio of the order of i : 3 x 107 . thus for 5 , ac . 
in seruthe addition of adeniine 14c into the culture medium does not represent the labelling of a pool , but the provision of adenine to the cers which would otherwise have to synthesise it . 
the marked ability of the cellsto take up adenine from the cultur - e medium is shown by the fact that the average maximum uptake per nucleus was of the order of 1 - 2 x 107 atoms dna 14c ( 50 grains in 10 , days ' expostire , 2 grains / electron )  . 
i ' he cell cycle . if the cells svnthesised dna throughout the entire intermitotic period , then , after a few hours of culture in the presence of the isotope all the cers should contain labelled dna . 
ellis fore the cells within a hours of the end of the period of dna synthesis at the start of the culture in isotope wif not be labered , and , similarly , cers enterina the period of dna synthesis during the culture time ( t ) and spending less than a hours 11111hours fig . 
the length of the s period is also indicat - ed by the gradual increase of the grain count / nuclei up to about 15 hours , then reaching the maximum average value for a given concentration of the isotope in the culture mediuthe length of the total cen cycle can also be calculated in a different way . 
3 illustrates the timing this timing , in of the cell cycle obtained with the methods outhned above . principle agrees well with that found in the bean root by howard and pelc ( 1951 )  . it can be seen from tables i and ii and fig . 
the reason for this difference is , firstly the differences betweein eilergy of the beta particles emitted by 14c and the higher energy32p electrons result in a greater scatter and , consequently , in a higher background , thus interfering with the accuracy of the c ' secondly , while the half hfe of 14c is a neghgible problem when the isotope dilutions are added to the culture medium , variations in volume to allow for the decay are a also , diie to the decay of 32p and to the fact that32p is so - lirce of error with32p  . supplied in3lpphosphate buffer , the ratioof 31 to 32pwill not be constant : the degree of labelling of the phosphorus pool will be variable . these factors all contribute to the lesser accuracy of the counts in cultures with 32plabelling as compared with the 14c labelling . 25 - 30hours 12 - 15 hours 3 - 4hours g2 m fig . 
rate , of dna synthe , 8i8 in different type8 of bone marrow cei18 . in these experiments only those types of cells were found to synthesise dna which are known to be capable of mitosis . 
3 represents the average values for the majority of the dividing cells of the bone marrow , i.e. , promyelocytes , myelocytes , pronormoblasts , basophihc normoblasts and early polychromatic normoblasts . in a few experiments , however , where it was found to be possible to count early normoblasts and early myeloid cells separately , a shorter cycle than in fig . 
the affect of x - ray irradiation on dna synthesis large doses of x - rays ( 5000 r in 15 minutes , 140 kv , i . / mal filter ) exerted a marked inhibition on dna synthesis . 
14. - post - irradiation dna synthesis . was not a recovery in the sense of ability to synthesise normal amounts of dna , but the entry of some 0 , cers into the s period for a hmited time . it appears that ar 01 cehs are damaged by a dose of 5000 r . the effect of irradiation on theg2 period is indicated by the fa ' ct that no mitoses were observed in any of the irradiated cultures . a dose of 5000 r produced a marked degeneration of the cers in the cultures , but over 6 hour culture times were needed to show the first morphological signs 378 l . 
in a sense this could be better defined , as the assembly of the dna experiments with the synthesis , of the purines and pyrimidines from molecule . labeued one carbon compound precursors ( e.g. , formate 14c ) are in progress . the timing of the cer cycle is , in principle , in good agreement with that it is expected that described in the bean root cell by howard and pelc ( 1951 )  . the timing of the cer cycle in different . 
the total cycle time was found to be of the order of 40 - 48 hours for the average dividing bone marrow cers ( promyelocytes , myelocytes , pronormoblasts , basophihc and polychromatic normoblasts )  . 
a phenomenon , involving the abnormal growth of two kinds of pigmented cers , was recently found in spontaneous tumours experimentary produced by genetic methods in platyfish - swordtail hybrids . the tumorous hybrids were produced by mating a male spot - sided and rub throated platyfish ( platypoecilw maculatus ) to an albino swordtail ( xiphophoru& hellerii )  . 
the ruby - throated pattern is composed of xantho - erythrophores , which are red pig ' ment cers containing erythropterin in granular form and lutein gordon ( 1950 ) and gordon and nigrelli ( 1950 ) and zeaxanthin in solution . indicated that these colour patterns are inherited and referable to two platyfish sex - linked genes : sp for the black and bt for the red pigment cers . 
the genetic analysis revealed that the pigmented tumours were produced in platyfish - swordtail hybrids containing the hnked genes , sp and bt from the platyfish and a number of sp and bt gene modifiers from the swordtail . some of these pigmented tum ' ours were diagnosed as erythromelanomas in an earher report by gordon ( 1950 ) and nigrelh , jakowska and gordon ( 1950 )  . more extensive study of additional specimens revealed that there were two kinds of pigmented tumours involved , first , a red tumour , a xantho - erythrophoroma of the head and , second , a black tumour , a melanoma of the body . in some of the hybrids , cers of the melanoma reached , penetrated and replaced the tissues of the red pigmented tumour . 
only a few macrophages , however , are evident in these tumours . the relatively few dendritic and rounded cells are similar to those described in the simple xantho - erythrophoroma . 
the various stages in the development of the melanoma within the xantho - erythrophoroma can be demonstrated and traced in one of these fishes ( 243 - 11 )  . 
33. - melanotic area of the tumour ( 243 - 1 1 )  . 1 - n this region one or two cells from the original xantho - erythrophoroma can still be distinguished . in other areas , however , a , region just below the epidermis - note the melanotic they are completely absent . cell ( melanoblast ) in metaphase ; b , typical melanotic cell of this region associated with a cell of the xantho - erythrophoroma ; c . 
the melanotic cells ( melanoblast 's ) are smaller and less dendritic than the typical macromelanophores ; they are usuany rounded , sometimes fusiform , varying from 10 to 40 tl at their widest dimensions . 
the present studies , however , show that two distinct tumours are involved : a xantho - erythrophoroma in the head region and a melanoma of the trunk and caudal peduncle . in some of the hybrids the more actively proliferating cers of the melanoma invade and replace the red pigmented tumours of the head , producing an erythromelanoma . the red and yerow pigment cell characteristic of the xantho - erythrophoroma was first described by kosswig ( 1928 ) from normal fish as an erythro - xanthophore . according to goodrich , hill and arrick ( 1941 ) , the cell ; which they caued a xantho - erythrophore , is a xanthophore in which erythropterin is laid down in addition to lutein and zeaxanthin . in fixed and stained tumorous material the xantho - erythrophores are either non - pigmented dendritic , rounded or fusiform cers . in both normal and tumorous tissues the nuclei of these cers ' are weak in chromatin ; nevertheless , mitotic figures are frequently found . 
the degeneration of the xantho - erythrophoroma probably results from the growth - pressure of the apparently more actively reproducing melanotic cers which infiltrate and penetrate the interstices between the xantho - erythrophores . 
whether or not the metastasizing tissue would have comalthough complete pletely replaced the invaded tumour was not determined . replacement of a xantho - erythrophoroma by melanoma has never been observed in our fishes , the evidence seems to indicate that this would have occurred had the specimens lived longer . an interpretation of the nature of the genic action that modifies pigment cell growth and the physiological effects of the interaction of such genes in platyfish - swordtail hybrids was outhned by gordon ( 1948 , 1950 )  . 
the effect of the various genes for pigment cells upon the development of the red , the black and the red - black pigment cell tumours may now be suggested . in the normal platyfish gordon ( 1928 ) showed that the dominant , sex - finked , spot - sided gene , sp , manifests itself in the development of large black pigment cells or macromelanophores ; the large melanophores first appear in postembryonic platyfish in the posterior region of the body , specifically , in the caudal peduncle . 
the number of macro melanophores produced by the sp gene in the normal , spotted platyfish is influenced strongly by the action of the stippled gene , st , a dominant , autosomal factor for the - presence of many micromelanophores . 
when sp is associated with the dominant gene st ( that is , sp st ) , more macromelanophores develop than when the sp gene is associated with the recessive gene st ( that is sp st )  . furthermore , in both the sp 8t post - embryonic and adult platyfish the macromelanophores are restricted for the most part to the caudal peduncle . 
315. - - below , a platyfish - swordtail hybrid of the genetic constitiition rts , 206 - 15 , showing the early stage of the development of the xantho - erythrophoroma . this shows up as a red patch on the left operculum surrounding the lower half of the eye . 
241 , produced by mating a platyfish - swordtail rtsp hybrid , 206 - 12 , exhibiting an erythromelanoma , back to an albino swordtail , 1822 - 6 ; upper left , an albino with an amelanotic melanoma at the tip of a caudal fin ; lower left , a sibling showing the " ghost " pattern of macromelanophores ; right , typical melanotic hybrid showing the ear ' lv sta - ae of melanosis in the posterior region of the body . 
the minimu ' m requirement for the production of the erythromelanoma in platyfish - swordtail hybrids is the presence of the linked genes rtsp . gordon ( 1950 ) showed that a platyfish parent of the genetic constitution of rtsbidrsp would not , while a rtspidrsb would produce some hybrids with erythromelanomas when mated with an albino swordtail . 
in the tumorous animals the xantho - erythrophoroma develops rapidly in the head region , and at the same time a condition of melanosis develops in the posterior region of the body . in some individuals the melanotic cens show great reproductive activity and produce a melanoma . 
in those animals that develop a melanoma the cells of the melanoma may invade the xantho - erythrophoroma and destroy and replace the cells of the red pigmented tumour . in other cases the melanin - bearing cers are restricted to the posterior part of the body , aflowing the red tumours to develop on the head without the involvement of the black pigment cers . 
the sequence of events in the invasion of one kind of neoplastic tissue by another type is discussed from the points of view of histology and developmental genetics . this work has been aided by a grant to the new york zoological society from the national cancer institute , national institutes of health , united states public health service in support of the project : " genetic and correlated studies of normal and atypical pigment cer growth . " some details in this paper were presented by myron gordon before a meeting , of the royal academy of medicine , london , july 4 , 1950 . we are deeply indebted to ' the american museum of natural history for .laboratory facilities , to j . 
this process is one of the most fundamental of cell mechanisms , so fundamental indeed that , except in minor details , it has never been influenced by evolutionary change . 
obviously the physiology of such a process is particularly fascinating , and in addition it is becoming clear that its study may shed important light on a variety of practical problems . 
 however , the greater part of this work has been from the viewpoint of pathology , and has resulted on the one hand in the mass of observations on the effects of carcinogenic substances , and on the other with the discovery of the so - called mitotic poisons ( loveless and revell , 1949 )  . 
by comparison , the direct study of the physiology of normal mitosis has been surprisingly neglected , presumably because of the difficulty of the techniques involved , and becaube the practical value of such knowledge has not been fully appreciated . 
 the present discussion starts with a consideration of the physiology of normal mitosis in mammalian tissues , and continues with an examination of its relation to modern work on carcinogenesis . 
 in recent years a considerable amount of attention has been paid to the problem of mitotic activity in the epidermis of the mouse ( bullough , 1948a , 1948b , 1949a , 1949b , 1949c , 1949d , 1950a , 1950b , 1950c ) and of the general biology of the skin ( medawar , 1947 , 1948 , 1949 )  . 
the work on the mouse epidermis began with an analysis of the diurnal cycle of mitotic activity , which was found to be a simple rhythm directly determined by the waking and sleeping habits of the animals . 
factors determining the form of the diurnal cycle are the accustomed times of feeding , the quantity and quality of the food given , and the age and sex of the animals . 
it was found that by means of subcutaneous injections of glucose or starch the mitosis rate can be raised in both active and sleeping mice to a level considerably higher than normal . 
 further studies have indicated that the epidermal mitosis rate is related , not to the blood - sugar concentration , which is high during hours of activity and low during hours of sleep , but to the concentration of intracellular glycogen . 
 bullough and eisa ( 1950 ) have shown that the diurnal cycle of glycogen concen tration in the skin is exactly similar to the diurnal cycle of epidermal mitotic activity . 
it is well known that glucose is deposited from the blood during sleep , and , while most of it is stored as glycogen in the liver , it is now evident that significant quantities are also deposited elsewhere . 
the most obvious alternatives were either that carbohydrate is incorporated , for instance as ribose , in the new nucleo plasm or cytoplasm as it is formed , or that it is destroyed to provide energy . 
the second was strengthened when it was found that the stimulus obtained from extra starch can be augmented by coincident injections of phosphate , and that , conversely , mitosis can be almost eliminated by injections of phloridzin , a substance known to inhibit phosphorylation ( bullough , 1949b )  . 
the conclusion that mitotic activity may involve the expenditure of a significant amount of energy also receives support from the results of experiments made with dividing eggs by such men as brachet ( 1932 ) , runnstr5m ( 1933 ) , and zeuthen ( 1946 , 1947 , 1948 )  . 
 in mammalian epidermis the importance of oxygen has been stressed by medawar ( 1947 ) in the rabbit , and by bullough and johnson ( bullough , 1950c ) in the mouse . 
when this tissue is kept in vitro anaerobic conditions inhibit cell division , and it is now clear that glycogen , phosphate and oxygen are all involved at the onset of an epidermal mitosis . 
however , since in a normal body it seems unlikely that either phosphate or oxygen are ever in such short supply as to become limiting factors in cell division , further elaboration of this point is not necessary here . 
 summarizing what has been said above it is evident that , in a normal mouse , carbohydrate in the form of glycogen or glucose is a most important substance determining mitotic activity , and that its apparent function is to supply the energy requirements of cell division . 
as one outcome of these conclusions it was to be expected that diet would be found to have an important effect on the mitosis rate , and this has now been confirmed ( bullough , 1949c )  . 
 it was later confirmed that a restricted diet results in a lowered body weight , a reduced glycogen reserve , and hence a reduced mitosis rate ( bullough and eisa , 1950 )  . 
however , while the body weights and carbohydrate reserves were found to vary in direct proportion to the degree of underfeeding , the rate of epidermal mitosis varied in a complex manner expressible in terms c , f a sigmoid graph . 
 it must be added that while the results primarily concerned the epidermis , other observations showed that similar conditions develop in other tissues , and it can b6 safely concluded that the reduction of mitotic activity by diet is a general effect visible throughout the body . 
 the striking thing about these observations is that they parallel so closely the results of recent work by tannenbaum ( 1940a , 1940b , 1942a , 1942b , 1944a , 19441 > , 1945 , 1947 ) and tannenbaum and silverstone ( 1949a , 1949b ) , who have in introducing this studied the effects of restricted diets on carcinogenesis . 
 subject it is vitally important to emphasize that it is indeed the genesis of tumours that is being considered , and not the growth of tumours once they have been formed . 
the growth of a visible tumour is only influenced in slight degree by variations in diet , and it is only in the process of tumour genesis that diet has any pronounced effect . 
the greater part of the earlier evidence was reviewed by hoffman ( 1937 ) , who came to the conclusion that " ovemutrition is common in the case of cancer patients to a remarkable and exceptional degree . " this statement can be justified by figures such as those of dublin ( 1929 ) , who showed statistically that persons who are overweight during middle age have a higher expectation of death from cancer than those who remain underweight . 
his first experiments involved a simple restriction of diet , and led to the surprising discovery that animals maintained on a 66 per cent diet are more active , develop fewer tumours and fewer diseases , and so live longer on the average than do the fully fed controls . 
 in later experiments all groups were provided with a basic diet of protein , fat , vitamins , and minerals , and the only differences lay in the amount of carbohydrate which they received . 
of male dba mice painted 19 times with 3 : 4 - benzpyrene , one group of 50 fed ad libitum developed 32 tumours , while another group of 50 fed on a carbohydrate restricted diet developed only 11 . 
as a general principle it can be said that the most striking results are obtained with spontaneous tumours , which are often prevented altogether , and it is evident that with induced tumours the modifying effect of the diet can be at least partly masked if sufficiently heavy doses of carcinogens are given . 
 in both cases this relation is expressible by means of a sigmoid curve , and the great.est fall in both the mitosis rate and the tumour yield accompanies a reduction of from 80 per cent to io per cent of the full diet . 
thus with an average daily intake of about 14 calories the latent period in one experiment had an average length of about 18 weeks , while with an intake of 8 c9.lories the latent period was 39 weeks . 
are that carbohydrate , or calorie , shortage causes reduced body weight , a strongly depressed mitosis rate , the restriction or prevention of a wide variety of spon taneous and induced tumours , a considerable delay in the time of appearance of those few tumours which do develop , fewer dise.ases of all kinds , and consequently a healthier and a longer life . 
tannenbaum ( 1947 ) himself has no explanation to offer , but he has rightly emphasized one crucial point that , like the depression of mitotic activity , the prevention of tumour genesis by calorie restriction is the result of a general effect operating throughout the whole body . 
 at this point it is evident that a prima jooie case can be bmlt up to indicate a connexion between the three factors carbohydrate lack , mitosis depression , and reduced tumour incidence . 
further , it appears possible that these factors may be related to each other in this sequence , carbohydrate lack limiting mitotic activity , and low mitotic activity limiting carcinogenesis . 
however , before attempting this analysis it is important to consider at what possible point in the sequence of events leading to the formation of a tumour the mitosis rate may be able to exert an effect . 
 in the first , whether through the action of a carcinogen , a virus , or some unknown factor , a normal cell is transformed into a cancerous cell , which then lies dormant . 
 of these two steps , the first need not be considered further because , although it was once suggested that the mitosis rate at the time of application of a carcinogen has an effect on the number of resulting tumours ( mottram , 1945 ) , this theory has now been abandoned ( bilsechowsky and bullough , 1949 )  . 
they must compete with the healthy cells for the nutrients in the fluids of the tissue spaces . " some of them may even succumb , and it seems highly probable that those which do survive do not begin to multiply actively until they are stimulated to do so . 
the theory developed below is that the stimulus to multiplication may he any one of the several factors that are known to promote cell division in normal cells , and there fore that a close and direct connexion may well exist between normal mitotic activity and carcinogenesis . 
it is well known , for instance , that carcinomas , derived from mitotically active epithelia , are of much commoner occurrence than sarcomas , derived from mitotically inert connective tissues . 
on the one hand , the only extensive study of normal mitosis rates in a wide varietv of tissues concerns the female mome ( bullough , 1946 ) , while on the other the only extensive analysis of the natural tumour yield tissue by tissue concerns man ( annual statistical reviews of the registrar - general )  . 
first , it is evident that epithelia such as those of the vagina , uterus , and : rectum , which are naturally most highly active mito tically , are also most liable to develop tumours , while such mitotically inert tissues as striped muscle and brain do so ra : re ] y if at all . 
one such exception is the duo denal mucosa , which , though it has a normal mitosis rate as high or higher than that of the : rectal mucosa , shows a relatively low tumour yield . 
the mammary gland , which , though it shows far less mitotic activity than the lining epithelium of the vagina , has a tumour yield which is : relatively extremely high . 
willis ( 1948 ) emphasu , ed this strongly when he noted that " in the breast , fibro - adenomas are almost always situated in a bed of hyperplastic tissue , and persistent cystic hyperplasia is an important pre - cancerous state . 
 persist , eventually evoke progressive neoplasia as well . ' ' incidentally this seems to he the explanation of the action of croton oil , which , after the application of a carcinogen , greatly increases the tumour yield in mice . 
 it is a substance which has been shown to cause a local increase of as much as six - fold in the epidermal mitosis rate ( bullough , unpublished )  . 
 h now it is admitt.ed that some connexion may be traceable between local mitotic activity and local tumour genesis , it is reasonable to consider that some connexion may also be found between the general mitosis rat.e of the body as a whole , and the chance that somewhere within that body a tumour may develop . 
 as for the suppression of mitosis and of carcinogenesis , it is evident that both can be achieved by means of a restricted diet , and in view of the apparent role of carbohydrat.e during cell division , it may be expected that anything which limits the production of energy in the tissues will have a similar effect . 
one such substance is phloridzin , which inhibits phosphorylation , and another is dinitro phenol , which is said to uncouple the processes of phosphorylation and oxidation ( loomis and lipmann , 1948 )  . 
as regards phloridzin , bullough ( 1949b ) has described its effect in depressing the mitosis rat.e , and he has also obtained results showing a lowered yield of spontaneous mamma " " y tumours . 
as regards dinitro phenol , clowes and krahl ( 1936 ) first noticed its power to inhibit.mitosis , and tannenbaum and stlverstone ( 1949b ) have recently shown that it , too , reduces the yield of spontaneous mammary tumours  . 
environmental cold and muscular exercise are also known to depress mitotic activity , apparently by di , erting t - 0 other uses the energy produced from carbo hydrat.e ( bullough , 1949a )  . 
tannenbaum and silverstone ( 1949b ) have now shown that cold has a similar depressing effect on carcinogenesis , but the effect of prolonged muscular exercise has apparently not yet been determined . 
thus the explanation of the connection between hyperplasia and cancer may rest quite simply with the fact that with a higher mitosis rat.e there is a correspondingly higher chance that some lat.ant tumour cell will be stimulated to multiply , an effect which will become evident both in the earlier development of tumours , and in the appearance of many which would otherwise never have formed . 
conversely , hypoplasia may be expected to reduce the chances of multiplication , and so , as tannenbaum 's ( 1947 ) results show , to delay the development of those few tumours which do form and to prevent the appearance of many which otherwise would have formed . 
it appears possible that the average length of the period of dormancy , which every newly - formed tum.our cell appears to experience , is determined by the mitosis rate of the tissue in which the cell lies . 
 the mitosis rate itself is determined by a variety of factors , local and general , normal and abnormal , of which one is the carbohydrate , or calorie , supply . 
 this conclusion is in agreement with a suggestion that the practical problem of cancer can be divided into two distinct parts : the formation of the latent tumour cell , and the breaking of its period of dormancy . 
already it is known that the period of dormancy can be lengthened simply by means of a restricted diet , and with a more detailed understanding of those factors which control normal mitotic activity other practical methods may be devised . 
 already it is known that the incidence of human cancer varies in direct proportion to body weight , and therefore presumably to food intake , and it does not seem impossible that the idea of strict dieting to maintain the body weight of the middle - aged at an optimum level may some day be an accepted and normal practice . 
 as a postscript , attention may be drawn once more to tannenbaum 's ( 194 : 7 ) observation that with restricted diets disease incidence as well as tum.our incidence is markedly lowered . 
in considering this further question it seems possible that parasites penetrating into a tissue may , like the latent tumour cells , have to compete with the normal cells for the available nutrients . 
already it is known that in conditions of hypoglycaemia canaries are less liable to contract malaria ( hegner , 1937 ) and rats are less liable to contract tuberculosis ( steinbach and duca , 194 : 2 )  . 
 it now appears that the whole subject of competition between cells , and perhaps also between tissues , is worthy of the most serious consideration for the practical results which it may yield . 
this process is one of the most fundamental of cell mechanisms , so fundamental indeed that , except in minor details , it has never been influenced by evolutionary change . 
obviously the physiology of such a process is particularly fascinating , and in addition it is becoming clear that its study may shed important light on a variety of practical problems . 
 however , the greater part of this work has been from the viewpoint of pathology , and has resulted on the one hand in the mass of observations on the effects of carcinogenic substances , and on the other with the discovery of the so - called mitotic poisons ( loveless and revell , 1949 )  . 
by comparison , the direct study of the physiology of normal mitosis has been surprisingly neglected , presumably because of the difficulty of the techniques involved , and becaube the practical value of such knowledge has not been fully appreciated . 
 the present discussion starts with a consideration of the physiology of normal mitosis in mammalian tissues , and continues with an examination of its relation to modern work on carcinogenesis . 
 in recent years a considerable amount of attention has been paid to the problem of mitotic activity in the epidermis of the mouse ( bullough , 1948a , 1948b , 1949a , 1949b , 1949c , 1949d , 1950a , 1950b , 1950c ) and of the general biology of the skin ( medawar , 1947 , 1948 , 1949 )  . 
the work on the mouse epidermis began with an analysis of the diurnal cycle of mitotic activity , which was found to be a simple rhythm directly determined by the waking and sleeping habits of the animals . 
factors determining the form of the diurnal cycle are the accustomed times of feeding , the quantity and quality of the food given , and the age and sex of the animals . 
it was found that by means of subcutaneous injections of glucose or starch the mitosis rate can be raised in both active and sleeping mice to a level considerably higher than normal . 
 further studies have indicated that the epidermal mitosis rate is related , not to the blood - sugar concentration , which is high during hours of activity and low during hours of sleep , but to the concentration of intracellular glycogen . 
 bullough and eisa ( 1950 ) have shown that the diurnal cycle of glycogen concen tration in the skin is exactly similar to the diurnal cycle of epidermal mitotic activity . 
it is well known that glucose is deposited from the blood during sleep , and , while most of it is stored as glycogen in the liver , it is now evident that significant quantities are also deposited elsewhere . 
the most obvious alternatives were either that carbohydrate is incorporated , for instance as ribose , in the new nucleo plasm or cytoplasm as it is formed , or that it is destroyed to provide energy . 
the second was strengthened when it was found that the stimulus obtained from extra starch can be augmented by coincident injections of phosphate , and that , conversely , mitosis can be almost eliminated by injections of phloridzin , a substance known to inhibit phosphorylation ( bullough , 1949b )  . 
the conclusion that mitotic activity may involve the expenditure of a significant amount of energy also receives support from the results of experiments made with dividing eggs by such men as brachet ( 1932 ) , runnstr5m ( 1933 ) , and zeuthen ( 1946 , 1947 , 1948 )  . 
 in mammalian epidermis the importance of oxygen has been stressed by medawar ( 1947 ) in the rabbit , and by bullough and johnson ( bullough , 1950c ) in the mouse . 
when this tissue is kept in vitro anaerobic conditions inhibit cell division , and it is now clear that glycogen , phosphate and oxygen are all involved at the onset of an epidermal mitosis . 
however , since in a normal body it seems unlikely that either phosphate or oxygen are ever in such short supply as to become limiting factors in cell division , further elaboration of this point is not necessary here . 
 summarizing what has been said above it is evident that , in a normal mouse , carbohydrate in the form of glycogen or glucose is a most important substance determining mitotic activity , and that its apparent function is to supply the energy requirements of cell division . 
as one outcome of these conclusions it was to be expected that diet would be found to have an important effect on the mitosis rate , and this has now been confirmed ( bullough , 1949c )  . 
 it was later confirmed that a restricted diet results in a lowered body weight , a reduced glycogen reserve , and hence a reduced mitosis rate ( bullough and eisa , 1950 )  . 
however , while the body weights and carbohydrate reserves were found to vary in direct proportion to the degree of underfeeding , the rate of epidermal mitosis varied in a complex manner expressible in terms c , f a sigmoid graph . 
 it must be added that while the results primarily concerned the epidermis , other observations showed that similar conditions develop in other tissues , and it can b6 safely concluded that the reduction of mitotic activity by diet is a general effect visible throughout the body . 
 the striking thing about these observations is that they parallel so closely the results of recent work by tannenbaum ( 1940a , 1940b , 1942a , 1942b , 1944a , 19441 > , 1945 , 1947 ) and tannenbaum and silverstone ( 1949a , 1949b ) , who have in introducing this studied the effects of restricted diets on carcinogenesis . 
 subject it is vitally important to emphasize that it is indeed the genesis of tumours that is being considered , and not the growth of tumours once they have been formed . 
the growth of a visible tumour is only influenced in slight degree by variations in diet , and it is only in the process of tumour genesis that diet has any pronounced effect . 
the greater part of the earlier evidence was reviewed by hoffman ( 1937 ) , who came to the conclusion that " ovemutrition is common in the case of cancer patients to a remarkable and exceptional degree . " this statement can be justified by figures such as those of dublin ( 1929 ) , who showed statistically that persons who are overweight during middle age have a higher expectation of death from cancer than those who remain underweight . 
his first experiments involved a simple restriction of diet , and led to the surprising discovery that animals maintained on a 66 per cent diet are more active , develop fewer tumours and fewer diseases , and so live longer on the average than do the fully fed controls . 
 in later experiments all groups were provided with a basic diet of protein , fat , vitamins , and minerals , and the only differences lay in the amount of carbohydrate which they received . 
of male dba mice painted 19 times with 3 : 4 - benzpyrene , one group of 50 fed ad libitum developed 32 tumours , while another group of 50 fed on a carbohydrate restricted diet developed only 11 . 
as a general principle it can be said that the most striking results are obtained with spontaneous tumours , which are often prevented altogether , and it is evident that with induced tumours the modifying effect of the diet can be at least partly masked if sufficiently heavy doses of carcinogens are given . 
 in both cases this relation is expressible by means of a sigmoid curve , and the great.est fall in both the mitosis rate and the tumour yield accompanies a reduction of from 80 per cent to io per cent of the full diet . 
thus with an average daily intake of about 14 calories the latent period in one experiment had an average length of about 18 weeks , while with an intake of 8 c9.lories the latent period was 39 weeks . 
are that carbohydrate , or calorie , shortage causes reduced body weight , a strongly depressed mitosis rate , the restriction or prevention of a wide variety of spon taneous and induced tumours , a considerable delay in the time of appearance of those few tumours which do develop , fewer dise.ases of all kinds , and consequently a healthier and a longer life . 
tannenbaum ( 1947 ) himself has no explanation to offer , but he has rightly emphasized one crucial point that , like the depression of mitotic activity , the prevention of tumour genesis by calorie restriction is the result of a general effect operating throughout the whole body . 
 at this point it is evident that a prima jooie case can be bmlt up to indicate a connexion between the three factors carbohydrate lack , mitosis depression , and reduced tumour incidence . 
further , it appears possible that these factors may be related to each other in this sequence , carbohydrate lack limiting mitotic activity , and low mitotic activity limiting carcinogenesis . 
however , before attempting this analysis it is important to consider at what possible point in the sequence of events leading to the formation of a tumour the mitosis rate may be able to exert an effect . 
 in the first , whether through the action of a carcinogen , a virus , or some unknown factor , a normal cell is transformed into a cancerous cell , which then lies dormant . 
 of these two steps , the first need not be considered further because , although it was once suggested that the mitosis rate at the time of application of a carcinogen has an effect on the number of resulting tumours ( mottram , 1945 ) , this theory has now been abandoned ( bilsechowsky and bullough , 1949 )  . 
they must compete with the healthy cells for the nutrients in the fluids of the tissue spaces . " some of them may even succumb , and it seems highly probable that those which do survive do not begin to multiply actively until they are stimulated to do so . 
the theory developed below is that the stimulus to multiplication may he any one of the several factors that are known to promote cell division in normal cells , and there fore that a close and direct connexion may well exist between normal mitotic activity and carcinogenesis . 
it is well known , for instance , that carcinomas , derived from mitotically active epithelia , are of much commoner occurrence than sarcomas , derived from mitotically inert connective tissues . 
on the one hand , the only extensive study of normal mitosis rates in a wide varietv of tissues concerns the female mome ( bullough , 1946 ) , while on the other the only extensive analysis of the natural tumour yield tissue by tissue concerns man ( annual statistical reviews of the registrar - general )  . 
first , it is evident that epithelia such as those of the vagina , uterus , and : rectum , which are naturally most highly active mito tically , are also most liable to develop tumours , while such mitotically inert tissues as striped muscle and brain do so ra : re ] y if at all . 
one such exception is the duo denal mucosa , which , though it has a normal mitosis rate as high or higher than that of the : rectal mucosa , shows a relatively low tumour yield . 
the mammary gland , which , though it shows far less mitotic activity than the lining epithelium of the vagina , has a tumour yield which is : relatively extremely high . 
willis ( 1948 ) emphasu , ed this strongly when he noted that " in the breast , fibro - adenomas are almost always situated in a bed of hyperplastic tissue , and persistent cystic hyperplasia is an important pre - cancerous state . 
 persist , eventually evoke progressive neoplasia as well . ' ' incidentally this seems to he the explanation of the action of croton oil , which , after the application of a carcinogen , greatly increases the tumour yield in mice . 
 it is a substance which has been shown to cause a local increase of as much as six - fold in the epidermal mitosis rate ( bullough , unpublished )  . 
 h now it is admitt.ed that some connexion may be traceable between local mitotic activity and local tumour genesis , it is reasonable to consider that some connexion may also be found between the general mitosis rat.e of the body as a whole , and the chance that somewhere within that body a tumour may develop . 
 as for the suppression of mitosis and of carcinogenesis , it is evident that both can be achieved by means of a restricted diet , and in view of the apparent role of carbohydrat.e during cell division , it may be expected that anything which limits the production of energy in the tissues will have a similar effect . 
one such substance is phloridzin , which inhibits phosphorylation , and another is dinitro phenol , which is said to uncouple the processes of phosphorylation and oxidation ( loomis and lipmann , 1948 )  . 
as regards phloridzin , bullough ( 1949b ) has described its effect in depressing the mitosis rat.e , and he has also obtained results showing a lowered yield of spontaneous mamma " " y tumours . 
as regards dinitro phenol , clowes and krahl ( 1936 ) first noticed its power to inhibit.mitosis , and tannenbaum and stlverstone ( 1949b ) have recently shown that it , too , reduces the yield of spontaneous mammary tumours  . 
environmental cold and muscular exercise are also known to depress mitotic activity , apparently by di , erting t - 0 other uses the energy produced from carbo hydrat.e ( bullough , 1949a )  . 
tannenbaum and silverstone ( 1949b ) have now shown that cold has a similar depressing effect on carcinogenesis , but the effect of prolonged muscular exercise has apparently not yet been determined . 
thus the explanation of the connection between hyperplasia and cancer may rest quite simply with the fact that with a higher mitosis rat.e there is a correspondingly higher chance that some lat.ant tumour cell will be stimulated to multiply , an effect which will become evident both in the earlier development of tumours , and in the appearance of many which would otherwise never have formed . 
conversely , hypoplasia may be expected to reduce the chances of multiplication , and so , as tannenbaum 's ( 1947 ) results show , to delay the development of those few tumours which do form and to prevent the appearance of many which otherwise would have formed . 
it appears possible that the average length of the period of dormancy , which every newly - formed tum.our cell appears to experience , is determined by the mitosis rate of the tissue in which the cell lies . 
 the mitosis rate itself is determined by a variety of factors , local and general , normal and abnormal , of which one is the carbohydrate , or calorie , supply . 
 this conclusion is in agreement with a suggestion that the practical problem of cancer can be divided into two distinct parts : the formation of the latent tumour cell , and the breaking of its period of dormancy . 
already it is known that the period of dormancy can be lengthened simply by means of a restricted diet , and with a more detailed understanding of those factors which control normal mitotic activity other practical methods may be devised . 
 already it is known that the incidence of human cancer varies in direct proportion to body weight , and therefore presumably to food intake , and it does not seem impossible that the idea of strict dieting to maintain the body weight of the middle - aged at an optimum level may some day be an accepted and normal practice . 
 as a postscript , attention may be drawn once more to tannenbaum 's ( 194 : 7 ) observation that with restricted diets disease incidence as well as tum.our incidence is markedly lowered . 
in considering this further question it seems possible that parasites penetrating into a tissue may , like the latent tumour cells , have to compete with the normal cells for the available nutrients . 
already it is known that in conditions of hypoglycaemia canaries are less liable to contract malaria ( hegner , 1937 ) and rats are less liable to contract tuberculosis ( steinbach and duca , 194 : 2 )  . 
 it now appears that the whole subject of competition between cells , and perhaps also between tissues , is worthy of the most serious consideration for the practical results which it may yield . 
woodyatt. * from the meyerstein institute of radiotherapy and the bland sutton institute of pathology , middlesex hospital , london , w.1. received for publication february 15 , 1950 . failure in the treatment of malignant disease is most often due to the occurthere rence of metastasis before treatment of the primary lesion is instituted . are , however , a number of cases in which the tumour remains localized for a considerable time , but cannot be removed surgically on account of its size and anatomical situation , and cannot be destroyed by radiotherapy without irreit is the aim of the radioparable damage to neighbouring normal tissues . therapist to exploit the differences in sensitivity between normal and neoplastic tissues , and it is the object of this research to see whether the effects of radiotherapy on certain tumours can be enhanced by modification of the techniques of therapy in common use at present . a course of x - ray treatment involves a number of variable factors . 
the segments , usually 2 or 4 in number , and as nearly as possible of equal size , were then treated by one or more experimental techniques , while one segment was treated by a control technique . segments not being treated were protected by lead shields while neighbouring segments were irradiated , and care was taken to avoid overlap of adjacent segments or an untreated gap * b.m.a. 
woodyatt between them by careful skin markings and " setting up . " the question of side - scattering of x - rays from one segment into adjacent ones was investigated by physical measurements with micro - ionization chambers in a wax " phantom . " the amount of scattered radiation was found to be less than 5 per cent at 1 cm . from the edge of a treated segment , and this was considered small enough to be ignored . in this way erroneous conclusions resulting from unpredictable individual variations in response are largely eliminated , and each case constitutes a complete separate experiment . 
the results of individual experiments can later be compared to see whether the experimental technique in question regularly produces a particular effect . the tumours and surrounding skin were examined at frequent intervals to follow progress , and to detect and note differences in response between the areas . in addition , biopsies were taken from all areas before , during and after treatment . at first , weekly biopsies were taken , but it soon became obvious that in the earlier stages more frequent examinations were necessary , while after completion of the course of treatment intervals of a month or more might elapse . 
the sections were examined by a pathologist who not only had no preconceived views about the various techniques of irradiation employed , but was kept in ignorance of the area from which each specimen had been taken . biopsies from each area before treatment were studied carefully to provide a base line , and also to give an idea of the degree of variation between different this variability was often found to be marked , and areas of the same tumour . differences in biopsies subsequently taken from the several segments had often to be discounted as representing no more than possible chance variations . 
fractionation is , however , usually by equal daily doses given on 5 or 6 days each week , and this fractionation technique is applied with little discrimination to widely different sorts of tumour . certain theoretical considerations , mentioned below , suggested that a different method of fractionation , leaving longer intervals between the fractions , might be more effective . 
an important direct effect of a dose of irradiation is temporary suppression of mitotic activity . koller ( 1948 ) found that in squamousand basal - cell carcinomas of the skin the number of dividing cells was considerably reduced 6 hours after 200r , and that after doses of 400r and 500r mitotic suppression might last for several days . 
the susceptibility of cells to irradiation damage varies at different periods of their life cycle , being greatest in early prophase ( lea , 1947 ) , and irradiation given spear and during the period of mitotic suppression is relatively ineffective . glucksmann ( 1938 , 1939 ) have demonstrated by experiments on irradiation of tissue cultures and animal tissues in vivo that a greatly enhanced lethal effect is obtained if fractions are spaced so as to coincide with the recovery of mitotic activity following the suppressive effect of the previous dose . information concerning the duration of mitotic suppression in human tumours it probably varies in different tumours , but will after different doses is scanty . depend largely on the size of the dose . 
a clinical investigation indicating advantages from a wider spacing of fractions has been reported by koller and smithers ( 1946 )  . koller ( 1948 ) considers that the nature of the tumour bed and the indirect effects of irradiation on the stromal reaction in the surrounding normal tissue exercises an important influence on the response of the tumour itself . 
a further limitation was , of course , imposed by consideration for the patient 's welfare , and no case was undertaken where it was felt that there would be less chance of effecting a cure by the method . tumours of three types were treated - large rodent ulcers ( 2 cases ) , carcinomas of the skin ( 5 cases ) , and carcinomas of the breast involving the skin ( 4 cases )  . most tumours of the first two types are radio - sensitive , and a successful result could normally be expected from the use of an orthodox technique . these tumours were treated in order to assess the relative efficiency of the experimental techniques , and to determine any differences in the reaction of normal tissue at the edge of the tumour . the cases of carcinoma of the breast were of special interest , for , as is well known , a considerable proportion are markedly radio - resistant . 
the usual erythematous reaction developed , and proceeded to moist desquamation over the tumour and surrounding skthe area of skin at the periphery which was only covered by the original 71 x 15 cfields showed pigmentation , but no desquamation . 
the dose to this skin area had only been about 2000r when the smaller fields were substituted . no difference in reaction or tumour response in the two areas was apparent at this stage . on october 6 , as viable tumour was still feared to be present , further treatment was given as described . three weeks after the conclusion of treatment a marked difference was apparent between the two areas . 
the area treated by the control technique ( area 1 ) was completely healed over by healthy new epithelium except for a small ulcer at the site of the first surgical attack . 
the remaining tumour consisted of some firm pink papillary processes , almost flush with the surface . in area 2 much of the tumour and surrounding skin covered by the treatment applicator was still raw and denuded of epitheliuthe remaining tumour was a little flatter than in the control area . eight weeks after treatment further flattening was apparent . area 1 was healed except for the remains of the ulcer . area 2 showed very little progress of healing , although a few islands of epithelium had appeared in the raw skin area ; there was some discharge from a fissure between two sessile processes . four months after treatment a striking difference between the two areas was apparent . 
the control area was healed and showed marked pigmentation , all that remained of the tumour being some fibrotic nodularity beneath the surface . area 2 showed up as a sharply outlined , completely unpigmented square , corresponding to the treatment applicator . there was still an indurated fissure at the site of the tumour , and an area of skin below the tumour remained unhealed and had clearly undergone necrosis . five and a half months after treatment the patient complained of burning pain in the lower part ( area 2 ) , and an increase in discharge from the fissure which was still present . 
a month later ( april , 1948 ) radionecrosis was obviously developing in area 2 ; the fissure had deepened , its edges were grey and necrotic , and the surrounding tissues were pale and avascular , and as hard as cartilage . by may a large necrotic ulcer corresponding to area 2 had developed . local treatment failed to bring about healing , so in november , 1948 , the whole treated area was excised , after a preliminary colostomy . this was done partly because residual foci of active growth were suspected . 
woodyatt there was a moderate degree of associated inflammatory mitotic activity . biopsies taken from both areas during and after treatment showed reaction . progressive differentiation of the tumour cells with reduction in the percentage of viable cells and their final disappearance . 
no sign of tumour could be seen in the biopsies taken at 6months , and no significant differences between the series of sections from areas 1 and 2 were detected . 
a small nodule of recurrent growth ( 1.25 cdiameter ) had appeared , adjacent to the mastectomy scar , in february , 1945 . superficial x - ray treatment was given ( 4 daily doses of 800r ) and the nodule regressed , being quite healed by september , 1945 . 
3. - carcinoma of breast ( case 3 ) divided into four areas . 11111 11111 11111 total 3000r . in 27days area area area area total 4000 r in 26 days total 3000r in 27 days total 3680r in 24 days 11111 11111 11111 days i = biopsies taken from all areas , and on day 50 fig . 
the control technique was modified in this case , 150r being given daily instead of the usual 200r . this was done to bring it more into line with the average daily tumour dose administered in this clinic for carcinoma of the breast . 
by the end of treatment , february 18th , the greater part of the surface was covered by smooth granulation tissue . there was still a considerable amount of tumour present , with a subcutaneous shelf of induration palpable all round the edge . there was severe erythema of the surrounding skin , with much pain and discharge , and for these reasons treatment was curtailed . it is probable that the unusual severity of the reaction was due to infection of the raw surface , as the patient had fever up to 1010 f . 
the tumour had shrunk to 10 - 5 x 6 - 5 cm . the ulcer floor was covered by necrotic slough and its edges were still firm , but were softer and less raised in areas 1 and 3 . seven weeks after treatment the skin had begun to heal in area 3 . 
the skin in area 1 showed well - marked pigmentation , but was perfectly healed . well - marked differences were now apparent in the different areas of the tumour , which had shrunk further to 9 x 6 cboth areas treated by the control technique showed clinical evidence of active residual tumour , the ulcer in these areas having a hard raised edge with much adjacent subcutaneous induration . area 1 showed some suspicious induration , but less than in the areas treated by the control technique . area 3 was soft and nearly flat , with no definite sign of residual growth . 
6 at the end of treatment . the tumour cell mass in the upper part of the field shows extensive hyalinization , and there is heavy lymphocytic infiltration of the stroma . note that the magnification is the same as in fig . 
8 , 9 and 10 . - squamous - cell carcinoma of the ear ( case 8 )  . biopsies taken before , at about the middle of , and at the end of the course of treatment . 
the upper half was treated by technique i ( 500r twice weekly ) to a total of 3500r in 24 days , and the lower half by the control technique ( 200r daily ) to a total of 3600r in 24 days ; 95 k.v. 
x - rays clinically there was rapid regression of the lesion in both areas , were used . and the tumour had disintegrated completely before the course was finished . the serial biopsies showed a very satisfactory response in both areas , no viable cells remaining within two weeks of starting treatment . 
any conclusions we may draw from this small series of cases must be purely tentative ; the final evaluation of these techniques may require the elapse of a considerable interval of time , and in view of the possibility of variation in different parts of the same growth many cases must be studied before definite conclusions can be reached . complete equality of total dose and protraction were seldom obtained , and this introduces another variation from strict experimental conditions . 
with these facts in mind the following conclusions are cautiously presented . technique i ( 500r twice a week ) was used in 9 cases ; 3 squamous - cell carcinomas , 4 carcinomas of the breast , and 2 rodent ulcers . 
the clinical result in the 3 cases of epithelioma was uniformly good , and in one case this technique gave slightly quicker results than the control . in 2 cases of carcinoma of the breast this technique gave a better response , judged clinically and histologically , than the control ; in the other 2 cases the results were equally good . 
the cases of basal cell carcinoma responded better both clinically and histologically to the control technique ( daily fractions of 200r )  . technique ii ( looor once a week ) was tried in 4 cases ; 2 squamous - cell carcinomas of the skin , one carcinoma of the breast , and one rodent ulcer . 
he was considered too old for successful operative treatment so he was kept under observation and in february , 1954 , was readmitted to hospital with evidence of bone metastasis . 
apart from this , there is little irregularity of nuclear shape , size or staining density . these cells clearly form part of a fragment of tissue in the sputum , and because of the vacuolation of the cytoplasm , were considered to be diagnostic of adenocarcinoma . 
they are of adenocarcinomatous type and do not resemble cells usually seen in primary bronchial carcinomata . " an enlarged cervical gland was later removed for histological examination and was reported as showing a mucus - secreting adenocarcinoma , probably of colonic origin . the patient died , and post mortem examination was refused . it will be noted that the cells shown in the clump in fig . 
4 appear to surround a number of vacuoles , but gross vacuolation is not evident in the cells themselves . the larger mass is hollow , the wall being formed of a single layer of cells . i make it a practice only to report adenocarcinoma from sputum examination it cannot be too strongly emphawhen clumps of the types described are seen . sised that single cells containing vacuoles as shown in fig . 
the tracheo - bronchial glands may occasionally arow small amounts of the carbon to pass through into the thoracic duct or into lymphatic efferents from the glands which communicate directly with the great veins within the thorax , and thus the carbon reaches the such carbon in the blood stream is taken up by the cells of the blood stream . reticulo - endothehal system in organs hke the spleen and hver , which very occasionally at autopsy may show shght stippling with carbon , though in six spleens examined chemically we have found no weighable carbon in our residues . the amount of carbon deposited in a lifetime in the lungs and tracheo - bronchial glands depends on many variable factors such as age and occupation . 
where the degree of atmospheric pollution is great the excessive amount of carbon inhaled clogs the ciha of the respiratory mucous membrane and a large amount is not expelled even in the normal subject . in the subject who suffers from chronic catarrhal inflammation of the respiratory mucosa , there occurs ' a progressive destruction of the normal ciliated epithelium which is partially replaced by a non - cifiated type of a lower order , such as cuboidal or even squamous cens , which are unable to expel carbon particles . 
the scarring so caused is usuary the seat of dense areas of diffuse fibrosis resulting from bronchiectasis accumulation of carbon . or from the imperfect resolutions of acute pneumonias are often relatively free thus the only from carbon . 
why this should be so is not altogether clear . accurate means of assessing the total amount of carbon in the lungs and tracheobronchial glands as a whole is by estimating chemically all the carbon in these structures , and it is from this angle that we have approached the problethe amount found in these organs at autopsy thus represents what has been retained during a hfetime . 
the carbon itself may be innocuous , but from our studies of atmospheric pollution ( goulden and tipler , 1949 ; warer , 1952 ; goulden , kennaway and urquhart , 1952 ) , one can form some idea of the amounts of the carcinogens , 3 : 4 benzpyrene and arsenic , which would accompany it , and may possibly have some effect , either in association with , or apart from , that of tobacco . the black coloration of the lungs and bronchial glands must have become increasingly famihar to british pathologists since the industrial revolution which in the earlier part of the nineteenth century converted britain , by the utilisation of coal and iron , from an agricultural to a largely industrial country . although abundant material has been available in britain during the last 150 years , no one has , so far as we know , attempted to estimate the amount of carbon in human lungs , except in the special case of coal - miners ( king and gilchrist , 1945 )  . 
a questionnaire on the following points was circulated : sex ; age ; diagnosis and post - mortem findings ; places of residence for more than three years and the period lived in each ; occupations followed for more than three years ; the smoking history as regards the use of a pipe or cigarettes ; the age at which smoking was begun and stopped ; the maximum and average amounts smoked per day , and whether inhalation was practised always , occasionally or never . considerable difficulties were encountered in obtaining lungs from persons without bronchial carcinoma for controls ; those were needed from whom an accurate history could be obtained , but who were not likely to live long . 
joules of the central middlesex hospital , london , for the supply of material ; also to professor neils dungal of reykjavik for the lungs of eight persons who had always lived in iceland . 
the pulmonary glands in the lung substance were separated as far as possible from lung tissue ; no claim is made that this division , was carried out very accurately , but the much lower concentration of carbon in the lung makes some contamination with it unimportant . 
the separation oflymph glands is of course still more difficult when a large invasive mass of bronchial carcinoma is present ; the practice has been to regard as lymph glands any deep black stippled areas of simflar size and shape , and to class the remainder as lung . 
histology. - the lungs submitted to us had been fixed whole in formalin and thus the fixation from a histological point of view was not perfect . blocks of lung tissue were nearly always taken from the pleural region and from deep in the lung substance , and occasionally from the tracheo - bronchial glands . these blocks were subsequently refixed in formol - corrosive and the paraffin sections were stained by haemotoxylin , by carmalum or neutral red , by van gieson or mallory , and , in a certain number of cases referred to later , by potassium ferrocyanide to demonstrate any iron . 
the cases from hospitals in london ( tables ii and iii )  . - the places of residence and occupations given are those of longest duration only . in the case of persons hving in london , the part of london in which they hved is stated in brackets after a capital l ; for those living in other towns , the county is given i " cigarettes " expresses tobacco consumption in this form even where brackets . cigars or a pipe were smoked . 
tobacco is equivalent to 28 cigarettes . the small number of cases prevents any firm conclusions about differences in the amounts of carbon in the lungs ofpersons with and without bronchial carcinoma . these differences are not statistically significant , but are nevertheless of interest . the average amount of carb ' on in the lungs of females with bronchial carcinoma was greater than that in the controls ( table iii ) , although the carbon content of the glands did not differ appreciably between the two groups of women ; one might expect this result if some carcinogen such as 3 : 4 benzpyrene adsorbed on to the carbon particles in soot is concemed in the aetiology of lung cancer . 
the again , the carbon in two groups of men , however , showed the opposite result . the glands was approximately the same for the two groups , but the control group had the higher carbon content in the lungs . there was no apparent reason why men and women should differ in this way . the total carbon content of a lung must vary according to three chief factors , ( a ) age , ( b ) the size of the lung ( since a larger person will ' mspire more air and hence more carbon ) , and ( c ) the environment in which a person lived . allowance for the first of these variables is easily made . 
when the weight of carbon in the lungs and glands was divided by the age ( table iv ) the differences between the four groups remained and were still of the same order . 
the cases from iceland ( table v . ) - since these , specimens did not include the whole trachea , some of the bronchial glands may not have been present ; the figures in table v therefore represent the carbon and ash content of the lungs and glands together . the content of both carbon and ash in the women - 's lungs was considerably greater than that in the men 's , apart from the exceptional value for the man no . 
carbon in lungs / age in years . 25 - 1 ( 12 - 6 ) 13 - 8 15 - 4 11.9 bracketed averages include the abnormal figures for no . 
50. tthis very high value for a - sh was omitted from the average . microscopicary , no silica was found in sections of the lung . those obtained in london , shows that the lungs of icelandic men had a lower average carbon content , while those of the icelandic women had a much larger amount . 
the same differences obtained on correction of these figures for variation in age distributio ' n . the presence of such amounts of carbon in these lungs from iceland seemed at first sight surprising , for in iceland general atmospheric pollution as understood in this country is very small . there is no native coal or oil , and these are costly to import . 
was a non - smoker . in the group of women with bronchial carcinoma , i out of 5 did not smoke , and in the control group , 9 out these figures , especiary for the women , show a higher proportion of nonof 1 1 . smokers among persons without bronchial carcinoma . 
the total carbon content of the lungs varied over a wide range all the points indicating a total consumption of more than for non - smokers ; 200 , 000 cigarettes refer to men and women with bronchial carcinoma . 
the average daily consumptions were : males with bronchial carcinoma , 24 / day ( 16 cases ) ; males without bron ' chial carcinoma , 12 / day ( 5 cases ) ; females with j . 
urquhart bronchial carcinoma , 19 / day ( 5 cases ) ; and females without bronchial carcinoma , 2 / day ( 10 cases )  . ash content of the lungs and bronchial glands . the nature of the ash deposited from the hydrolysates of these lungs has not although about 10 per cent of the total carbon was contained been determined . in the glands , the fraction of the total ash present in them was about 20 per cent . this suggested that the bronchial glands were even more efficient in concentrating particles of mineral matter than they were in accumulating carbon particles . 
for comparison with the histological findings the chemical results were divided into three groups , namely , those with a carbon content of ( a ) 0 - 49 g . 
the carbon in the sections showed the normal distribution , being heavier under the pleura , around the bronchi and blood vessels and in the fibrous septa , than in the vesicular portions of the lung . the rocks of iceland are of volcanic origin and the roads are metaned with such material which , due to weathering , quickly breaks down so that with the passing probably road dust has become considerable of traffic much dust is created . in iceland , where there are now 11 , 000 motor vehicles , only since the acquisition in view of this the icelandic lungs were of american cars after the last war . submitted to a more intense microscopic study for the presence of sihca and of iron . 
no evidence of silicosis was noted in any and doubly refractile granules of in only one case was iron silica were very scanty or absent in all the sections . found , apart from occasional heart - failure cells in the others . in this case the iron was present chiefly in the fibrous tissue of the septa and around the bronchi and blood - vessels in close association with carbon particles which the iron pigment appeared to surround . 
is retained ; this gives some measure of the efficiency of the nasal filter and of the mechanism by which particles which have reached the deeper respiratory passages are swept upwards again by ciliary action and expectorated or swallowed . 
when the floor of a room is swept towards the door , the part adjacent to the door is exposed to dirt from a much larger area . similarly , when particles of dust are arrested on the walls of the smaller bronchi and bronchioles , they are swept upwards by ciliary action through the main bronchi ( the door ) into the trachea , and carcinogens might diffuse from the particles into the layer of mucus covering the bronchial mucous membrane during transit . 
some such mechanism would be in accord with the statement about the site quoted above ; but some recent investigators ( westermark , 1938 ; raebum , 1951 ; raebum and spencer , 1953 ) think that tumours arise - with about equal frequency in any part of the lung . the estimations of arsenic and of 3 : 4 benzpyrene in the air of large towns given in table viii are those of goulden , kennaway and urquhart ( 1952 ) and table viii . - concentrations of arsenic and of 3 : 4 benzpyrene in town air . london ( becton ) bilston manchester liverpool . sheffield london ( county hall ) hull bristol mean concentrations , pg . 
a8203per cigarette ( daff and kennaway , 1950 )  . ini ' mum amount of a strong carcinogen such as i : 2 : 5 : 6 ' dibenzanthracene which will produce * our earlier results , which were confirmed subsequently , were demonstrated at a meeting of the pathology section of the royal society of medicine held at this hospital on january 15 , 1952 . shear ( 1936 ) found that the j . 
of 3 : 4 benzpyrene which may be inspired in ' a lifetime , but in view of the vastly greater area over which this may be spread and the different time relations , one may doubt whether this comparison has any value . the size and arrest of smoke particlm . the available data about the arrest of smoke particles in the air passages are very defective because we have not , to begin with , any adequate information about the size of these particles . 
the passages quoted verbatim below contain all the information that we have found on this subject . the leicester report says : " the size distribution of smoke is a subject which still requires investigation . green ( 1936 ) gives estimates for five industrial dusts from which it can be inferred that 50 per cent by weight of such dusts is less than 3 to 10 microns , and 80 per cent is less than 5 to 15 microns in mean diameter . atmospheric smoke may be similar in size distribution but a direct investigation is  . 
some of the properties of smoke depend on the area it can cover ; half the surface or sectional area of the smoke was in particles larger than about 0 - 35 micron . there is a distinct tendency for smoke particles to stick together in chains , perhaps one micron long . " the comparison of such results obtained by different methods of arrest is not e . 
are in the range of size where there is minimum retention in the lung , 80 per cent of them being exhaled and 20 per cent arrested in the alveoli . ( b ) the mean diameter of smoke particles and the range of particle size of carbon black which shows the greatest adsorption of 3 : 4 benzpyrene from benzene ( falk and steiner , 1952 )  . 
though the material was not statisticary adequate , the smoking histories of the cases examined showed a higher consumption of tobacco among both men and women with bronchial carcinoma compared with the controls . 
if the carbon in the lungs and bronchial glands does not leave the body by any channel the amount found post - mortem represents what is retained out of the intake of a lifetime . 
the data now available for atmospheric poflution in some english towns arows an estimate to be made of the amount inspired during a hfetime in them ; this might be of the order of i 00 g . , while the quantity found in the lungs and bronchial glands was approximately 0 - 5 to i - 0 g . 
estimations of 3 : 4 benzpyrene and of arsenic in the suspended matter of the air of various english towns enable one to calculate the amounts of these compounds which would accompany the carbon inspired , and retained , in a lifetime . we wish to express our gratitude for the materials and case histories which have been the subject of this investi ation to professor niels dungal of revkiavik ; to horace joules of the central middlesex hospital , london , and to two members of his staff , a . 
a fuller account will be found in appendix i . it is sufficient to note here that it is a slightly modified form of the ordinary death rate which would be calculated as 2 l ( x ) m ( x ) l ( z ) ' the reasons for introducing such a modification are twofold : firstly , it makes allowance for the fact that the specific death rate considered is underestimated owing to deaths from other causes , and secondly , it measures the rate at which animals are dying out from the cause under investigation at the moment , x , not the bulk death rate over a month . the latter property allows a continuous curve to be drawn through the calculated values whose ordinates measure the rate of mortality at any moment of life . 
the monthly death rates on the other hand are strictly discontinuous , being , as is shown in appendix i , functions of the area under the , ( x ) curve over the month in question . using the method of haldane given in appendix i , the values of , ( x ) for cancer were calculated for a number of strains . those for three strains whose life tables were given by murray and hoffman ( 1941 ) are graphed in fig . 
1. the three curves bear out the conclusion of maurray and hoffman that the dba mice were the most susceptible , then the bittner albinos , and finally the marsh time in months fig . 
spicer albinos - a result which is the reverse of that indicated by the percentages dying the method of approach used here indicates that the cause of the of tumour . discrepancy is the difference that exists between the non - tumour death rates of the three strains . for if , using the methods outlined below , we calculate the percentage of cancer deaths that would be expected if eachl strain had the same non - cancer death rate as the marsh albinos , we find the following values : bittnera marsh a  . 
~. corrected to marsh - a . 90 5 per cent 87 - 7 76 - 6 uncorrected . 67 7 per cent 73.7 76 - 6 the process of calculating the p ( x ) 's has involved some smoothing of the data , and in the graphs no attempt has been made to draw a smooth curve ; the points have merely been joined by straight lines . 
the irregularities in the curves are mostly due to random errors , though the flattening of the , u ( x ) curve for dba mice from the 12th to the 15th month is repeated in the curve for another group of the same strain , and is therefore probably due to some real interruption of the steady increase in the tumour mortality . a further series of , l ( x ) curves are given in fig . 
3. allowing for this by subjecting the a - ro mice to the a - fc non - cancer , ( x ) , it is found that they would have almost the same percentage of cancer deaths . the percentage of cancer expected in the a - ro mice at the a - fc death rate is in fact 86 , while the percentage found in the a - fc is 88 . it appears then that almost the whole 302 c . 
the most obvious method of comparison is to compare the numbers or proportion of cancer deaths in any strain with those of a strain having a standard non - tumour death rate . alternatively the number of deaths over a similar period of time may be calculated when all non - tumour mortality has been eliminated . both of these indices can be calculated using the , ( x ) values . the difficulty of this approach is to find a standard which is equally useful for all strains , since there are wide variations in life span and in o 15 iii 0 ' 30 0.25 0 20 - o * i 0.10 time in months fig . 
murray and hoffman ( 1941 ) have attempted to overcome the first difficulty by using a life span as a unit and expressing time in percentiles of this value . this method seems to be unsatisfactory in that the determination of the length of span is to some extent arbitrary . 
murray and hoffman use the time at which 1 per cent of animals are surviving , adding that it would be possible to use the 0.1 per cent point if larger numbers of animals were available . 
each of these two cut - off points would give a different system furthermore these authors make no direct allowance for differences of time units . in non - tumour mortality , though they discuss the merits of a number of arbitrary weighting factors for calculating averages of the monthly cancer death rates . to avoid the use of arbitrary systems of weights it seemed best to calculate indices of susceptibility after the non - tumour death rate has been eliminated . this was done using a method due to dublin and lotka ( 1920 , 1921 ) , which has also been discussed by karn ( 1933 ) and irwin ( 1935 )  . 
spicer quently pointed out that the estimates might be improved by using the method of maximum likelihood , which gives results having a smaller standard error . the full calculations are rather more laborious than in the case of dublin and lotka 's method . 
6. it will be seen that the process of summation has resulted in a considerable smoothing of the data , and though the original , u ( x ) curves are not all similar in form , the integrated curves approach more closely to this ideal which is essential if strict comparisons of tumour susceptibility are to be made . after some initial confusion the curves spread out from left to right in order of decreasing susceptibility . 
any horizontal line drawn across the figures will give , by its intersections with the curves , the times of equal proportional mortality . vertical lines will give the proportional mortality corresponding to equal times of life . 
they are : 25 6 for the cross with a dba mother , and 37 8 for the reciprocal . from a study of the , ( x ) graphs it is obvious that the differences in the form of the curves from strain to strain make strict comparisons of tumour susceptibility , of the same kind as are made in biological assay , impossible . future work will probably reveal some of the factors which determine the shape of the curves , but in the meantime it should be emphasized that susceptibility to mammary tumour is determined in a different way in each strain , though the similarity of the , u ( x ) curves of different groups of mice of the same pure line does enable more exact comparisons to be made in this case . 
haldane , who first suggested that the , t ( x ) curves could be used for the investigation of tumour susceptibility , and who provided me with the method of calculation given in appendix i . 
 ( - ) = log.e 2 = 0 693 gives the half life . ( 4 ) modified expectation of life . the number obtained by taking the reciprocal of the sum x log . 
 ( 1 / px ) for the whole table is of interest since it gives an estimate of the expectation of life . suppose the table extends over ages from o t so that the sum of probabilities is then we can twrite the reciprocal of this sum t1 log . 
 ( 1 / p ) = t , j ( x ) dx ' ( x ) 1 ( x ) dx if there is no cause of death but cancer . so that the reciprocal is a weighted mean of the [ ( x ) 's . multiplying this by t , the interval over which the table extends , gives an estimate of the expectation of life . this estimate is rather lower than that given by the correct method of calculation since the values of 1 ( x ) are weighted in favour of the low values at the higher ages where the , u ( x ) 's are large . it might be preferable to use this modified life expectation instead of the half - life as it takes into account all the data and can be given a standard error . 
 ( p ) and the variance of the sum is equal to the sum of the variances since the p 's are independent . if we call this sum 2 then the variance of the half life will be given by and since and the standard error of x is var . 
levvis. from the chemi8try department , university of adelaide . received for publication may 23 , 1952 . soon after the discovery of the carcinogenic activity of 1 : 2 : 5 : 6 - dibenzanthracene by cook , kennaway and colleagues , yoshida ( 1932 , 1933 , 1934 ) and sasaki and yoshida ( 1935 ) reported that the addition of o - aminoazotoluene to the food leads to the production of malignant liver tumours in rats . during the past two decades scores of other azocompounds have been tested for carcinogenic activity of this type and many have been shown to be active . for reviews see shear ( 1937 ) , kinosita ( 1937 ) , cook ( 1939 , 1943 , 1948 ) , cook and kennaway ( 1938 )  . these carcinogenic azocompounds form a very interesting group . 
unhke the polycyclic aromatic hydrocarbons , they do not , as a rule , produce tumours at the site of injection , but mostly affect the liver . furthermore , the induction of liver tumours with azocompounds is markedl influenced bv the dieb , and if a cc protective " diet is supplied , the production of tumours can be strongly inhibited or even entirely prevented . all the azocompounds are , of course , artificial synthetic substances ; and they are highly coloured ( mainly orange to red )  . 
some are used as textile dyestuffs , and certain members of the group are used for colouring foodstuffs . the most part such compounds are probably harmless ( cook , 1948 )  . 
most of the food - colouring matters which have been tested have failed to induce liver tumours in rats ; and in any case it is most unlikely that anyone could ever consume enough of an azocompound in food to have any deleterious effect . 
to some extent the compound is also carcinogenic towards the bladder ( yoshida , 1935 ) , and it is interesting that the deaminated compound , namely 2 : 3 ' - azotoluene ( iii ) , was found to be non - carcinogenic to the liver of the rat , but produced numerous papillomas in the bladder ( otsuka and nagao , 1936 )  . many derivatives of azobenzene , containing substituent aminoand methylgroups , were tested for carcinogenic activity by kinosita ( 1937 )  . 
none of these produced cancer of the liver except o - aminoazotoluene , its mono - acetyland diacetylderivatives , and an isomer of o - aminoazotoluene , namely 4 - dimethylaminoazobenzene ( iv )  . 
the latter compound , which is also known as p - dimethylaminoazobenzene , and as n , n - dimethyl - p - aminoazobenzene , was formerly used as a food - colouring matter under the name of " butter yellow "  . 
the effects of such structural alterations are described in a following section . most of the known carcinogenic azocompounds are derivatives of 4 - dimethylnevertheless , a 4 - aminoaminoazobenzene , or at least of 4 - aminoazobenzene . this was shown for example , by group is not essential for activity of this type . cook , hewett , kennaway and kennaway ( 1940 ) , who examined the action of azonaphthalenes on mice . 
manv liver tumours , mostly of the type of cholangioma , were obtained with 2 : 2 ' - azonaphthalene ( vi ) , and a few tumours were obtained with 1 : 1 ' - azonaphthalene ( vii )  . 
lewis the same relative potencies do not always apply in other laborat - ory animals . it is well known , for example , that 4 - dimethylaminoazobenzene is much less active in producing ' hver tumours in mice than it is in rats . 
agents with therapeutic possibilities such as radio - phosphorus and the radio - iodines , and probably much better , suitable organic compounds containing radio - active isotopes , whose action depends on selective concentration of the radio - activity in particular cells . the separation on the industrial scale of non - radio - active isotopes and the practical development of the mass spectrometer open new possibilities of isotopic tracer research where radio - active effects are undesirable , as in many clinical investigations , or where , as in the case of nitrogen and oxygen , no suitable radioactive isotopes exist . the cyclotron still remains an essential instrument both for the production of small amounts of certain isotopes and as a source of fast neutrons for therait is by no means certain that the peutic trials in cancer and related diseases . frequency modulated cyclotron will be useful as a therapeutic source of fast j . 
mitchell neutrons , on account of the possibility of reduced neutron output and of the undesirability of a pulsed output . a promising field for radiotherapeutic investigation appears to be the study of high energy , 20 - 50 mev . 
prove more suitable than the synchrotron . the electron beam can now be extracted from these machines , the therapeutic possibilities of the high energy beta particles are rather uncertain and must be investigated with caution . the development of high energy generators opens the interesting possibility of the radiotherapeutic application of fast protons of energy in the region of 140 mev . 
 ( wilson , 1946 )  . the results of selected investigations are best summarized in the discussion of these newer agents . properties and production of isotopes , radio - active and stable . the properties of selected isotopes of especial interest are summarized in recent information on isotopes , which may be useful as radio - active table i . tracers , and which are produced in the pile , is summarized in table ii . 
a list of recent references on isotopes is also given . the pile is without a rival for producing isotopes in the very large number of cases where , during irradiation in the pile , a simple slow neutron capture process occurs , e.g. 
and for s35 10 millicuries per g . there is one slow neutron ( np ) reaction of especial importance : n14 + it is of interest that this process is probably responsible for n - > c14 + p . 50 - 80 per cent of the biological effects of thermal neutrons , the remainder of the effects being due almost entirely to the n - y reaction in hydrogen ( mitchell , however , the n , p reaction in nitrogen is of extreme importance for 1947 )  . isotopic tracer research as the basis for the preparation of c14 , the long - lived it has been shown by yankwich , rollefson and radio - active isotope of carbon . norris ( 1946 ) that the c14 is produced by slow neutron irradiation of compounds such as ammonium nitrate and urea in the form of very simple compounds such as c02 , co , ch3oh , h.cooih , and of especial importance as a starting - point for organic syntheses , hcn . in addition to the preparation of materials by direct irradiation , a very considerable number of radio - active isotopes are found as fission products in the uranium rods in a pile . 
a most valuable detailed list of these has been given by the plutonium project ( 1946 )  . in general , for the preparation of isotopes the yields with the cyclotron are very much smaller than those with the pile . 
the oxygen isotopes have been concentrated by distillation of water and by exchange reactions . sulphur has been separated by chemical exchange between so2 and the bisulphite ion . potassium has been also separated by chemical exchange methods . the development of the mass - spectrometer as a practical instrument and its routine use for hydrocarbon analyses by industrial laboratories in u.s.a. show clearly the possibility in medicine and biology of non - radio - active isotopic tracer investigations . 
an interesting possible diagnostic application of measurements of p32 in breast tumours in situ has been reported by low - beer , bell , mccorkle , stone , steinbach and hill ( 1946 )  . * the application of p32 as a tracer in metabolic investigations exemplifies the in order to avoid short - term problems raised by this method of investigation . effects , it is essential to calculate the dose of radiation received by the tissues for example , with p32 , one microcurie per g . from the radio - active isotope . * one line of metabolic tracer investigation insufficiently well known is the use of radio - active brominated compounds , e.g. 
of animal with times of observation of the order of 6 hours . of especial interest is the work of abels , kenney , craver , marinelli and rhoads ( 1941 ) , who accidentally found significant metabolic changes in the leucocytes in chronic leukaemias after total body x - radiation with doses as small as 3 r . , as a result of the radiation given by sub - therapeutic doses of p32 . of much greater importance in the clinical applications of isotopic tracer research is the avoidance of all possible risk of carcinogenesis as a long - term in a recent report on the result of the radio - active material introduced . " health protection activities of the plutonium project , " robert s . 
stone ( 1946 ) states that " it is now well established that ( radio ) strontium given to mice and rats in amounts insufficient to kill them for many months can cause bone and lymphatic tissue tumours to develop in significant numbers of animals . over long periods of time , plutonium has been shown to cause atrophy of the bone and bone sarcomas . " it is evident that great caution is necessary however , before introducing any radio - active materials into human subjects . it is likely that for many isotopes safe conditions for tracer work will be established after dosimetric studies and prolonged animal experiments . 
mayneord ( march 1 , 1946 - unpublished ) that the dose rate at 1 cfrom a point source of 1 millicurie of co60 enclosed in a platinum envelope of thickness 0 5 mis 11 - 1 r . 
however , it may prove more convenient to measure co60 sources in terms of equivalent radium . the production of large amounts of these isotopes by means of the pile raises the practical possibility of the therapeutic application of large gamma ray sources - " mass radiation units " equivalent to 50 - 100 g . 
mitchell in the form of na2hpo4 solution soaked in blotting - paper and dried . in addition to therapeutic applications , these beta - ray sources may be useful in experimental cancer research , e.g. 
stone ( 1946 ) states that " it has been established that single large doses of beta rays and multiple small doses can cause cancers in the skin without the animal being killed by the effect of the beta rays . " therapeutic possibilities of selectively concentrated radio - active agents . radio - active isotope therapy has as yet been limited to inorganic compounds . radio - phosphorus ( p32 ) , and to a much smaller extent the radio - iodines ( 1130 andi1131 ) , are the only isotopes with which limited clinical trials appear justified without further preliminary animal experiments . in general it seems unlikely that inorganic radio - active isotope therapy will stand the test of time . it seems much more hopeful to study the selective concentration by malignant cells of suitable organic compounds carrying radio - active isotopes of many elements ( rhoads , 1946 )  . the usual therapeutic applications of p32 in the form of na2hpo4 appear to depend upon its synthesis into nucleic acids by the multiplying cells . one other method of therapeutic application ofp32 reported ( jones , wrobel and lyons , see low - beer , lawrence and stone , 1942 ) depends on the selective concentration of colloidal anhydrous chromic phosphate by the cells of the reticulo - endothelial system . it is easy to understand the concentration of the radio - iodines by the thyroid in hyperthyroidism and in the rare cases of carcinoma of the thyroid , where the primary and sometimes also the metastases retain the function of secretion . 
one must be sceptical of the therapeutic possibilities in bone sarcoma of sr89 or ca45 ; it is interesting to note that stone ( 1946 ) reports that radio " strontium , barium , zirconium , yttrium and others locate quite selectively in the bones , and many of them stay for long periods of time . " radio - phosphorus , p32 , has been used since 1936 mainly in u.s.a. , in the treatment of patients with chronic myeloid and lymphatic leukaemia , polycythemia vera , lymphosarcoma and various related ( low - beer , lawrence and stone , 1942 ; reinhard , moore , bierbaum , kenney , 1942 ; lindgren ( 1944 ) showed thatp32 , which is selectively moore and kamen , 1946 )  . concentrated , gave better results than na24 , which is not selectively concentrated . the therapeutic possibilities and dosage of radio - phosphorus has been reviewed recently ( mitchell , 1947b )  . it is considered that the present position with regard to its therapeutic applications may be summarized as follows : diseases 1 . 
in the treatment of chronic myeloid and chronic lymphatic leukaemia , p32 is probably as satisfactory as , but no better than x - radiait is important to investigate the possibility of development of tion . methods of routine treatment of these diseases by means ofp32 . the average , each case is likely to require 10 to 15 millicuries ofp32 given , preferably intravenously , in the form of a solution of na2hpo4 in fractions in an over - all time of 10 - 12 weeks . it is desirable to correlate the dose with roentgen units ( marinelli , 1942 ) , and to measure the differential absorption ratio in different tissues ( kenney , marinelli and woodard , 1941 )  . 
phillips. * from the department of radiotherapeutics , university of cambridge . received for publication june 3 , 1952 . for the development of quantitative methods of study of mitotic inhibition by x - rays and chemical a ents , it has been necessary to examine the distribution of mitoseg in tissue cultures ' of chick fibroblasts . 
a statistical analysis of the distribution of , the mitoses has been made . previous quantitative work on tissue culture colonies in vitro has been reviewed by mayer ( 1939 )  . afitoses in fibroblast colonies were mapped by fischer and parker ( 1929 ) , by ' gairard ( 1935 ) , by jacoby ( 1937 ) , and in epithelial colonies by ephrussi and litvae ( 1934 )  . 
the colony had been grow - n for 24 hours in the usual ' mixture of one drop of fowl plasma and one drop of 15 per cent chick embryo extract ' then fixed in susa , stained with heidenhain 's haematoxyhn , and mounted whole . it was selected from a number of " control colonies of another experiment , asbeing of average growth and mitot - ic activity , and free from any obvious abnormahty . the colony was mapped using the 1 / 6 inch ( dry ) objective and an eye - piece micrometer which divided the field of view into 16 squares each of 901l side . 
the number of ceus in each square was counted , and at the same time a record was made of cers in mitosis , and of the phase of mitosis . 
4 by which the colony is divided into five arbitrary zones according to the cer density . the area of the uncounted central part is 22 per cent of the total area of the colony . 
the proportions of the phases of mitosis over the whole area are : prophase 17 per cent , metaphase 34 per cent , anaphase 7 per cent , telophase 42 per cent , and within the hmits of statistical variation they are the same in each of the five zones . in order to detect any departure from random distr ' lbution of mitoses within any one zone , the numbers of squares containing 0 , 1 , 2 , 3 , etc . 
mitoses were c ' ompared with the numbers expected on the poisson distribution , and were there was a suggestion found to agree within the limits of statistical significance . that the number of squares containing more than two mitoses wag shghtly greater than expected , but the total numbers were too small to establish that this was more than random variation . 
the total value of " chi squared " is 16 - 5 , which , for 7 degrees of freedom , gives a probabihty p this strongly suggests a factor other than chance causing non - uniform distribution . 
the number of mitoses per unit area can be seen in the map of his colony 27646 to be lower in the zone nearest the central part than the average value by at least a factor of 2 , which again is in marked contrast with the present work . the map published by jacoby ( 1937 ) can be compared as regards the relative number of mitoses per unit area in the different parts of the colony , and shows a similar trend to that found in the present work . it is interesting to note that the fairly constant level reached outside the central zone is maintained in jacoby 's colony across the central zone itsell if this was also the case in the present colony , there would have been about 120 mitoses in the central part which was not counted . willmer ( 1933 ) found that the frequency of mitosis was great - est in regions of moderate cell density , as is the case in the present colony . the distribution of cells and of mitoses in the outgrowth of a typical fibroblast colony is presented in the form of a map . 
the maximum mitotic index is in the region of moderate cell density , and the number of mitoses per unit area rises from the periphery to a steady value near the central part . 
the distribution of mitoses within the regions of constant average mitotic frequency agrees in general with a random distribution , which suggests that chance is the main factor controlling it . 
some of the authors misrepresent their own data , misquote those of others , are defective in arithmetic and make the task of anyone who wishes to examine their conclusions difficult . some authors , when giving data for the incidence of cancer other than uterine , do not say whether the figures refer to one or both sexes ; and in statistics based on admissions to a hospital they do not say whether this is a women 's hospital or a general hospital , or where the hospital is situated , data which are not always easy to obtain in another country . 
the age distribution of these 7 cases was as follows : 30 - 40 , 1 ; 40 - 50 , 2 ; 50 - 60 , 2 ; 60 - 70 , 2 . the seven cases of uterine cancer in jewesses which appear in table ii appear to be identical with those in table iii . * this is a very high percentage , for which theilhaber shows no detailed evidence . deaths of women from all forms of malignant disease deaths from malignant disease of the uterus the registrargeneral 's statistical review ( 1935 - 1939 ) gives the following figures for england and wales for the five years 1935 - 1939 :  . 
sanders ( 1916 ) gives data from rotterdam ( table x ) which show a low incidence of cancer of the uterus in jews , and a still lower incidence upon members of two of the protestant churches ( christian reformed and reformed )  . 
about one per thousand of the population . the radiumhemmet serves a well - defined area of the country which covers about half of the population . since 1914 practically all cases of cancer of the cervix occurring within that area are referred to the radiumhemmet for treatment . from 1914 to 1947 inclusive i have seen about 7000 cases of cervical cancer . among those were three jewish women . 
he considered that this was almost certainly an hereditary factor and not likely to be due to special observances of the jewish law , the same facts being noted in unorthodox jews . * unfortunately karplus gave no figures in support of this statement . 
data showing an equally low incidence of cancer of the uterus , which is more difficult to demonstrate than a high incidence , in populations known to consist of orthodox , and of unorthodox jews must have been difficult to obtain and would be of great interest . * i am indebted to d . 
100 table xi shows that cancer of the uterus , reckoned as a percentage of all cancers ( fishberg does not make clear whether the figures refer to females only ) is less than one - half as common among jewish women as among other races , while cancer of the breast shows a smaller difference in the same direction . smith ( 1941 ) studied the racial incidence of 3106 cases of cancer of the cervix observed at the memorial hospital , new york , from 1916 to 1937 . 
he says : " in an attempt to compare the nationality incidence in cervical cancer in this series with that of patients suffering from other gynecological lesions , consecutive records as filed have been studied , but the numbers used in each classification are not the entire number of patients filed under that diagnosis ; i.e. , this is a cross section of the clinic in the years studied rather than the total number of cases in the clinic . " i have been unable to learn from the memorial hospital the system upon which the cases making up this " cross section " were selected . apparently a number of cases of other gynaecological conditions roughly equal in number ( 3062 ) to those of cancer of the cervix ( 3106 ) were taken . 
the results are summarized by smith in tables xii and xiii ( his tables iv and iva )  . the significance of these figures is affected by the very high proportion of benign tumours , hence in table xiv and xiva smith 's figures have been recalculated for the malignant tumours only . in table xiv the result of the whole investigation , which for some reason smith does not give , namely , the ratio of cervix cancers to other conditions , is calculated from his figures . 
3106 6168 3062 vineberg ( 1919 ) gives data for the incidence of cancer of the cervix based upon material from 80 , 000 female patients at the mount sinai dispensary and hospital , table xv represents an attempt to reduce his rather confused statenew york . ment to a clearer forthe higher proportion of cancer in the hospital cases as against those of the dispensary is attributed to better conditions for examination . vineberg ( 1919 ) points out that jewesses of the poorer classes , to which most of those dealt with in his paper belong , are most likely to be orthodox in religious matters . 
the authors suggest the possibility that this difference certainly the age at which some forms of cancer depends upon racial factors . occur is affected by genetic differences ; examples have been given in detail in another paper ( kennaway and kennaway , 1944 )  . but there is nothing in these data for age distribution to indicate whether the liability to uterine cancer , which is the chief subject considered here , is affected by racial or by extrinsic factors . 
cancer of the uterus in hindu , moslem , parsee and indian christian women . statistics based upon attendance at hospitals are notoriously liable to error , more especially when they refer to people differing in race and religion . however , the following figures ( table xxi1 ) should at any rate encourage further investigation . 
they are taken from the valuable collection of data on cancer in india obtained by nath and grewal ( 1935 ) from the records of in - patients of 13 hospitals in punjab , delhi , united provinces , and bihar and orissa , comprising 6395 cases of cancer in all . 
cancer of the uterus in china . maxwell ( 1929 ) in his book " the diseases of china " says : " it is probable that uterine cancer ranks highest of all in the malignant growths in this country . 
the women are classified according to the husbands ' occupation into four main classes , with 14 numbered and 35 named sub - divisions . in table xxvii , only the four main classes are quoted . theilhaber does not state the populations of these various groups , and makes figures for the age distribution no comment upon this very serious deficiency . of the populations of these classes were not available . 
the object of the preliminary bath was not cleanliness per se , but the removal of anything which would intervene between the skin and the water during the total immersion , and thus deprive this ritual of its efficacy.t some medical writers have emphasized , in rather vague terms , the importance of the cleanliness enforced by the mosaic law , hence it is important to note exactly what the law requires . 
the washing , in a normal woman , at the beginning and end of the seven days , adds two baths a month to whatever other ablutions she may carry out . * this account of jewish ritual immersion is taken from various articles in the jewish encyclopedia ( 1891 ) , from ploss , bartels and bartels ( 1927 ) and from preuss ( 1923 )  . 
one might form some estimate in london from the number of public ritual baths available , which are said to be not more than half a dozen ; there are also some private ones . 
but it seems probable that many women who do not go to the ritual bath may conform to the law in other respects , namely , the first and second bath , and abstention from intercourse , which are matters of purely private conduct . the mosaic law ( leviticus xii , 2 , 5 ) relating to child - birth is important in view of the association of cancer of the cervix with parity . 
 " if a woman conceive seed , and bear a man child , then she shall be unclean seven days ; as in the days of the impurity of her sickness shall she be unclean . . 
but if she bear a maid child , then shall she be unclean two weeks as in her impurity : and she shall continue in the blood of her impurity three score and six days . " thus the whole period of impurity was either 7 + 33 = 40 , or 14 + 66 = 80 days . 
the practice in this country at the present time is said to be , to observe rather longer periods , of two and three months respectively . ( b ) moslem law . the koran ( sura 2 ) gives directions for the purification of women which are much less precise than those of the jewish law . 
a flow of blood lasting more than nine nights is the work of evil spirits . modi ( 1922 ) , a parsee , quotes these laws and says : " at present also , most of the parsee women generally observe the above practices . there are no separate dastanistans or houses for menses in parsee towns or streets , but generally a sequestered part of one 's own house is chosen for the purpose . the downfloor of the house was thought to be the proper place . 
but nowadays , in a crowded city like bombay , the down - floor , instead of being a quiet and healthy place such as that contemplated by the early injunctions of the vendidad , is so , most women in menses pass the period of generally quite the contrary . menstruation on their upper floors , but in an isolated way . 
in the matter of taking food , very few use spoons now , though up to about 25 years ago , that was generally the case . in the matter of purification , they observe the bath enjoined by the early books , but the vendidad injunction of bathing over the three " magas " is not observed at all . 
a separate place of bathing and for purposes of nature for women in this condition is generally provided in parsee houses . " ( d ) hindu ritual . khanolkar ( personal communication ) says that the parsee laws regarding menstruation are almost identical with those practised by orthodox hindus and probably both of them are derived from early aryan ritual . 
the abbe dubois ( beauchamp , 1906 ) , writing about the beginning of the nineteenth century , quotes from the book padma - purama , reputed to be the work of the hermit vasishta , a rule for a 3 - day p3riod of isolation for the menstruating woman , followed by a day of ceremonies and ablutions , including 36 total immersions in a river . 
during the three days "  . the mere wish to cohabit with her husband would be a serious sin . " elsewhere he says , " the mother of the newly - born child lives entirely apart for a whole month or more , during which time she may touch neither the vessels nor the furniture of the house , nor any clothes , and still less any person whatsoever . the time of her seclusion being over , she is immersed in a bath , or else a great quantity of water is poured over her head and body . women are similarly isolated during the time of their periodical uncleanness . in all decent houses there is a sort of small gynaeceum set apart for them ; but amongst the poor , in whose huts there is no such accommodation , the women are turned into the street , under a sort of shed or outhouse , or else they are allowed a corner of the cowshed . . 
when the time of uncleanness is passed , all the garments that the woman has worn are given to the washerwoman . her clothes are not allowed inside the house ; in fact , no one would even dare to look on them . " jhaveri ( 1910 ) says that in all hindu religious books minute directions for during the first four days of the first baths on very many occasions are given . menstrual period everything which the girl touches must be washed and " 202 e . 
nor do married women abstain procreation is almost a matter of religious observance with from child - bearing . uterine cancer ought to be , if anything , more the mass of jewesses . common among jewish women than among non - jewish . " various talmudic writers ( epstein , 1936 , 29b - 30a ) laid down that a father should cause his sons to marry at 16 to 24 , or 18 to 24 , and that a daughter should " be dowered , clothed and adorned , that men should eagerly desire her " ; no age is stated at which a woman should marry . 
a modern compiler ( abramowitz , 1900 ) states the law of israel on this matter thus : " after a man has arrived at the age of 18 it is his duty to take unto himself a wife in order that he may be fruitful and multiply , at any rate he should not pass his 20th year without having taken a wife . having begotten a son and a daughter who are also generative , one has fulfilled  . 
the command " to the commandment " to be fruitful and multiply . " be fruitful and multiply " is not obligatory upon women ; nevertheless a it is woman should not remain single lest she be liable to suspicion . one of the mandates of the sages that a man shall give his sons and daughters in marriage immediately they approach maturity . these jewish practices thus laid down in the law , and described by sorsby as prevailing at the present day , should encourage early marriage . the abb6 dubois ( beauchamp , 1906 ) says that "  . . 
thus 45 per cent of women with cancer of the cervix , and only 16 per cent of those with cancer of the breast , were married before that age . " variables which might be considered are : earlier childbirth , multiple children , poor obstetrical service , longer duration of married life , syphilis , immaturity of tissues at time of marriage , and excessive hormonal stimulation . " of these factors , longer married life could be eliminated by comparison with other groups . 
circumcision. some authors ( handley , 1936 , 1947 ) consider that no explanation of the lower incidence of cervical cancer upon jewesses need be sought beyond the ( assumed ) difference in the bacteriali flora of the female genital tract brought about by this factor , a matter which it would be possible to investigate . more data from moslem populations who practise circumcision , and from parsees , who do not , are obviously very desirable . handley ( 1936 ) has emphasized the value of statistics from fiji , where the natives practise circumcision , in contrast to the hindu immigrants , in whose native country phimosis and cancer of the penis are very common ( kennaway , 1947 ) ; certainly if these two races could receive equal medical attention interesting results might be obtained.t circumcision might act in another way , not dependent upon bacterial action . phimosis , cancer of the penis and cancer of the cervix appear to be common in * see " the racial origins of jewish types , " by radcliffe n . 
we have no data which give directly the respective social incidence of cancer of the cervix , and of the corpus , but information should be available from hospitals which have separate accommodation for paying and other patients . 
2. - age at death from oanoer of uterus in bamberg , augsburg , wuirzburg , erlangen , niirnberg and the provinoe of unterfranken , 1908 , and in munioh , 1906 - 7 - 8 ( theilhaber , 1910 )  . 
the jewish ritual has some unique features . more or less complete isolation of menstruating women is practised by many other peoples and is laid downi in the religious literature of some of these ( koran , zend - avesta , dharmasindhu )  . some quite primitive peoples ( aleuts ; natives of the congo and of australia ; for references see ploss , bartels and bartels ( 1927 ) ) are stated to enforce isolation at the menstrual period for as long as 6 to 7 days . 
one might make the following suggestions for further investigations : ( a ) an inquiry into the incidence of cancer of the cervix in jewish women who observe the 12 - day period , the ritual immersion being disregarded . 
all of the twenty collections of data which have been found in the literature show an incidence of uterine cancer which is greater on non - jewish than on jewish women . 
these data are calculated upon several different bases and hence are not all comparable . seven authors express the figures for cancer of the uterus as a percentage of all cancers in women , which percentage ranges from 28 to 14 in non - jews , and from 10 to 4 in jews , the mean values being 20 and 7 . 
the very scanty data available ( 25 cases only ) show a much lower incidence of cancer of the cervix on parsees . this might signify that so short a post - menstrual period does not affect the matter one way or the other , anid this difference in liability to cancer must then be due to other factors . 
cancer of the uterus appears to be prevalent in some peoples ( hindus , chinese ) among whom phimosis and cancer of the penis are also common , while the moslem women of india show a lower incidence . 
but any attribution of this difference to the practice of circumcision by moslems is very doubtful in view of the similar low incidence on peoples ( parsees , indian christians , possibly some dutch ) in whom this factor is absent . 
the only numerical data on the social incidence of cancer of the uterus appear to be those from bavaria 40 years ago , and from england and wales , after the last census , in 1930 - 32 . 
the unavoidable mixture , in large - scale statistics , of cancers of the cervix and of the corpus uteri , is unfortunate , as these two forms probably differ in etiology . 
harnett. published for the clinical cancer research committee of the british empire cancer campaign . received for publication july 16 , 1948 . in 1938 - 39 the clinical cancer research committee of the british empire cancer campaign carried out a clinical survey of all cases of cancer seen in the hospitals , both voluntary and l.c.c. 
in the administrative county of london . this was done by means of questionnaires , one of which was filled in by the registrars in the various hospitals for each patient and returned to the clinical cancer research committee for record . 
the work was interrupted by the outbreak of war after it had been in progress for 17 months . by this time 15 , 200 cases of cancer had been registered , of which 2529 ( 16 * 6 per cent ) were cancer of the breast . these patients have now been followed up for five years or more and the records analysed . 
a laparotomy was performed on each animal using ether anesthesia for rats and guinea - pigs and nembutal for mioe , and a loop of the intestine adjacent to the pylorus ligated . 
the abdominal cavity was closed with nylon sutures and each animal returned to its original cage . the animals were not treated further until 3 to 3hours following the laparotomy by which time all had emerged from the anesthesia . 
of body - weight of a 0.5 per cent aqueous solution of toluidine blue 0 . animals were anesthetized i an hour following the time of injection and a blood sample withdrawn either by heart puncture or from the aorta . 
the ph of the contents as well as of the gastric mucosa was obtained with hydrion the stomach contents were diluted 1 : 1 with acetone , stirred , and centripaper . fuged for 10 minutes at 2000 r.p.the supernatant was withdrawn and a 1 ml . aliquot diluted with acetone to 10 ml . this was stored at 50 to 100 c . 
overnight following which it was centrifuged 20 minutes at 2000 r.p.m. , and the supernatant removed for spectrophotometric analysis . in some rats the intestinal tract was removed and the contents collected . these as well as the blood samples were analyzed in the same manner as the stomach oontents . for the standard curve , 1 ml . 
an acetone blank was used for all determinations . in the case of gastric samples , the ph of the blank was adjusted to that of the sample using hc1 . the same acetone was used throughout the experiments and was redistilled prior to use . a beckman model du spectrophotometer was employed in making the measurements . 
inasmuch as the the guinea - pig , like the dog , is considered to be susceptible to histamine stimulation , an enhancement of gastric secretion following injection in this species was anticipated . the proportion of formed elements to plasma in the blood was determined for rats and guinea - pigs but not for mice in several of the groups . these results are listed in table i under hematocrit values . it will be observed that among the rats which received histamine ( groups i , ii , v , vi ) , an average value of 69 was obtained . 
whenthe ratswere not injected with histamine ( group iii ) , thehematoneither the ph of the stomach contents nor that of the crit value averaged 65 . gastrio glandular mucosa was altered significantly by the administration of histamine . in the rat a normal hematocrit value of 45 - 8 has been reported for males of the sprague - dawley strain ( gardner , 1947a )  . this value was confirmed in our laboratory . 
thus the histamine appeared to have no additional effect on the hemoconcentration observed in all operated animals . in untreated male guinea - pigs a hematocrit value of 42 has been reported ( gardner , 1947b )  . examination of the blood of treated guinea - pigs in our experiments disclosed a significantly higher hematocrit value in every animal . 
the control and 4 treated animals show no selective absorption in the region in question . similar analyses , using 5 guinea - pigs of group i and 1 guinea - pig corresponding to rat group ii , showed no definite peaks in the range between 375 and 400m , tt . although animals were fasted 48 hours prior to the start of operations no precautions were taken to prevent coprophagy , since it was our intention to duplicate the oonditions which prevailed in the experiments of ray and peters ( 1951 )  . when gastric contents were collected , it was observed that in addition to fluids although some of this may there also was present a varying amount of solids . have been due to residual food , it is more likely that ingestion of feces was responsible . 
18 - not included in any group - rat injected subcutaneously with histamine solution alone . solid lines represent animals given compound iii . analyses of blood from rats injected with compound iii revealed no compound by a method which was shown to detect 001 mg . 
on the other hand , the presence of toluidine blue o may be established by spectrophotometric analysis since intestinal contents give no peaks in the region of maximum absorption of this dye . it is of interest that such analysis from rats injected intraperitoneally with an aqueous solution of toluidine blue 0 failed to reveal this blue dye . ray and peters ( 1951 ) reported that gastric extracts from 3 out of 3 rats treated with compound iii showed substantial absorption at 400 mru . in the present experiments only 1 rat out of 15 showed similar results , while 2 fell in an intermediate , doubtful category . 
de. from the chittaranjan cancer ho8pital , calcutta , india . received for publication january 4 , 1954 . the study of the effect of radiation on cancer cells presents problems of fundamental importance . it is still an open question in the field of radiobiology as to whether the ionising effect of radiation directly induces differentiation in cancer cers . 
he has also shown that radiosensitive and radio - resistant cancer cells react to radiation in a different way after a preliminary radiation . waren and dixon ( 1949 ) are of opinion that radiation does not induce differentiation of cancer cers . johes and koher ( 1950 ) remark that the apparent increase of differentiating cers after radiation on cancer cers , is only relative and is caused by destruction of other types of cells . glucksmann 's method of estimating the radio - sensitivity of cancer cells with prehminary radiation ' has been criticised by gricouroff ( 1952 ) who is of the opinion that radio - sensitivity and differentiation of cancer cells are related to stromal nutrition . thus it is evident from the literature that our knowledge of the mode of action on radiation is not decisive . 
our study was strictly confined to those cancerous areas which were characterised by anisocytosis , anisonucleosis , abundance of mitotic cells , loss of polarity of epithehal cells and just breakage of basement membrane . 
the induction of differentiation in cancer cells is one of such delayed effects . it is for this reason , that we have collected tissues , as mentioned previously , on the 7th day after first radiation with 7000 r , on the 21st day after the second 7000 r radiation and finally 3 months after a total radiation of 21 , 000 r . before coming to our problem proper , we made a comparative study of the cellular population of the differentiating phases of the stratified squamous epithefia of the normal cervix and of epidermoid carcinoma of the cervix . table i shows that the cell population of the differentiating phase of the normal cervix is 48 - 17 0 - 41 per cent compared with 3 - 57 + 0 - 14 per cent of the epidermoid carcinoma of the cervix . we find the incidence of the variation of standard error in post - radiation conditions is high . might be due to variation on the biology of cancer cells , and also to altered conditions of different patients . 
de the trend of distribution shows also significant changes after second and third 7000 r , but the ' t ' test could not be applied because the number of cancer positive cases decreased materially after the 2nd and 3rd radiation . our second problem is concemed with the possibihty of predicting radiosensitivity of epidermoid cancer of cervix after preliminary radiation with 7000 r . it has been shown previously ( table ii ) that after the first radiation with 7000 r , there was a uniform reduction of cancer cell population of both resting and mitotic phases , and similarly a uniform increase of cell population of the differentiating and degenerating phases . 
we have not found any differential variation in the four biological phases of cancer cells after preliminary radiation to distinguish a radio - sensitive case from a radio - resistant one , as has been found by glucksmann ( 1948 )  . in our present series of 91 cases , no cancer cers were found in the final biopsy after 21 , 000 r in 76 cases , which may be considered as radio - sensitive cases . cancer cers were persistent in the remaining 15 cases under similar conditions and they may be termed radio - resistant cases . on further analysis of our data , arranging them into final cancer - positive and cancer - negative groups after full radiation with 21 , 000 r , we do not find any significant variation of cell population in different phases after the preliminary radiation with 7000 r ( table iv )  . 
when the influence of irradiation on the number of cells in the different classes was examined separately after the first irradiation for radio - resistant and radio - sensitive groups of tumours , no significant difference was found between the two groups . we express our gratitude to the atomic energy commission under the government of india for a grant which has given us an opportunity to do this work , and also to the chittaranjan cancer hospital , calcutta , for facilities to work in the laboratories . we also express our sincere thanks to the staff of the chittaranjan cancer hospital , particularly to n . 
 although urethane has been mentioned as carcinogenic for mouse ( nettleship and henshaw , 1943 ) and rat lung ( jaffe , 1947 ) this action has not yet been reported in other animals . 
since the inherited susceptibility to most known cancers is due to dominant genes this was anticipated here , and con sequently a large number of the c57 backcross mice were bred in order to note segregation , which should be manifest in the group on this assumption . 
preliminary experiments indicated that the various strains and crosses of mice used did not differ in their fluid intake , and consequently strain differences due to this treatment were not due to a different intake of the carcinogen . 
they were from another experiment and were only given 7 weeks ' urethane treatment , though they were killed 7 months from the beginning of treatment , the same as the rest . 
 when the mice were sacrificed their lungs were removed and fixed in 10 per cent formol - saline for 24 hours , which preserved the material and made handling easy . 
since nearly all lung tumours in mice tend to occur on the periphery of the lobes ( grady and stewart , 1940 ) the lobes were separated and the number of nodules on their surface visible to the naked eye were counted . 
in a previous paper ( cowen , 194 7 ) it was noted that strains of mice which gave a low number oflung tumours had tumours of a smaller size . 
 since the tumours in a lobe varied in size from microscopic dimensions to nodules about 4 m in diameter , it was not feasible to use the average size of all these tumours . 
the best way of gauging the response on a basis of tumour size was considered to be that of determining the size attained by the largest tumour in the total lungs of a mouse . 
this was tested statistically using the formula v ' ; ; ; 2 on the other hand , the fl hybrids were slightly but significantly lower than both of these classes . 
the polygon for the 057 backcross mice is obviously bimodal , and roughly consists of a combination of the figures for the 057 and fl hybrid mice in approximately equal proportions . 
 it may be mentioned that the a backcross mice which segregated for colour giving three phenotypes , albino , non - agouti and non - agouti brown , showed no apparent linkage of susceptibility with colour . 
 - ~ number of tumours f 3 . - - scatter diagram showing the relationship between the 2 groups of c57 backcross mice with respect to number and size of tumours . 
 thus , of all the chickens and guinea - pigs treated with urethane , only one chicken developed a neoplasm which was lymphoid in orig this type of tumour is not uncommon in chickens normally , and is not considered to be due to treatment with urethane . 
the first ( number of tumours ) gave more obvious results than the second ( size of the largest tumour per mouse ) , though both results closely paralleled each other . 
 these results point most significantly to the existence of a single dominant gene in a mice which is responsible for urethane - induced lung tumour suscepti bility , the c57 mice carrying the recessive . 
this does not entirely explain the results , for if a single dominant gene only were responsible , then a , fl , a back cross and the high group of the c57 backcross mice would all have exactly the same tumour incidence , which is not the case . 
firstly , the tumour susceptibility was genetically ina.ependent of mortality , and secondly , as the mortality was considerable only in the a and a backcross mice , a higher tumour susceptibility in the dead mice would merely have emphasized the line differences . 
 heston ( 1940 , 1942a , 1942b ) reported somewhat similar results using spon taneous and hydrocarbon - induced lung tumours in a and l mice ( high and low strains respectively )  . 
a possible reason for this is that the incidence of lung tumours in these mice was much lower than in the urethane - treated mice of the c57 backcross as reported here . 
 from the results of these workers , it appears that certain carcinogenic chemicals cause different lung tumour incidences in different strains of mice depending on the incidence of spontaneous lung tumours in these strains . 
that is , a strain of mice which develops few spontaneous lung tumours does not respond so well to a carcinogen as a strain which has a high spontaneous incidence . 
from this , it seems that environmental factors ( certain carcinogens ) do not simply cause tumours , but increase the incidence of them in mice depending on the inherited tendency of mice to develop the this argument can most probably be applied to other animals as well as mice . 
 it seems rather significant that no reports have been found in the literature of spontaneous ( or induced ) lung tumours in guinea - pigs , and out of the many neoplasms that arise in chickens , only one report exists of a spontaneous lung tumour in these animals ( apperly , 1935 )  . 
for example , guinea - pigs , which have a relatively frequent incidence of spontaneous liposarcomas ( warren and gates , 1941 ) , develop these tumours on treatment with hydrocarbons ( shimkin and mider , 1941 )  . 
it was considered that the resistance of mice to infections in general was lowered , and at the same time a specific carcinogenic virus was responsible for the lung tumours . 
if a single recessive gene is assumed to be the cause of the different mortalities , then the a backcross would be the segregating group , and should have a survival rate equal to the average of those for the a and c57 mice . 
from table i it is seen to be 45 , which agrees reasonably with the theoretical figure considering the small number of mice involved ( x2 = 317 , p > 005 )  . 
 it can thus be safely concluded that the mechanism whereby strain a mice are genetically susceptible to lung tumours is not the same as the one responsible for their susceptibility to general infections . 
correlation between leucopaenicity causing susceptibility to infection on one hand and a carcinogenic virus causing lung tumours on the other is therefore considered most unlikely in mice given urethane treatment . 
 two criteria which parallel each other closely are used as an index of response of mice to the carcinogenic action of urethane - the number of tumours per mouse and the size attained by the largest tumour in total lungs . 
of 9 : 10dimethyl - 1 : 2 - benzanthracene into the lungs of mice of strain street formed a part of a comprehensive study ( rask - nielsen , 1948 ) carried out in an endeavour to confirin or repudiate the hypothesis that tumours can be induced only in tissues that produce tumours spontaneously , whereas tissues that do not produce tumours spontaneously are unable to develop tumours even following very powerful carcinogenic action . the experiments showed that direct application of 0 - 5 mg . 
to this end , 3 : 4 - benzpyrene , 1 : 2 : 5 : 6 dibenzanthracene , or 20 - methylcholanthrene , all in doses of 0 5 mg . , were introduced directly into the lungs of street mice . since application of 9 : 10 - dimethyl - 1 : 2 - benzanthracene to the lung had previously induced thymic tumours ( rask - nielsen , 1948 ) , the present experiments were presumed to afford information also about the tumours which the three hydrocarbons would be capable of inducing in the thymus . these experiments will be reported in the present paper , which also includes the previous experiments with 9 : 10 - dimethyl - 1 : 2 - benzanthracene for comparison . benzpyrene , dibenzanthracene , or methylcholanthrene , 0 5 mg . , was injected , suspended in 0 01 c.c. 
the technique was as follows : in order to make the path of the needle in the lung tissue as long as possible , the needlewas inserted through the abdominal wall , immediately below the right costal border , and plunged through the diaphragm and longitudinally upwards through about two - thirds of the chest , before the suspension was injected . the mice used were litter mates of strain street , aged 5 to 7 weeks , divided into four lots . one lot was left untreated as controls , whereas the other three were injected with the respective hydrocarbons . 
as far as possible , the litters were divided equally in all four lots , an equal number of males and females in each in the corresponding experiments carried out with 9 : 10 - dimethyl - 1 : 2lot . benzanthracene ( rask - nielsen , 1948 ) litters of street mice were divided into lots of equal number , one of which was set aside as a control group . 
the effective total is the number of mice living to be as old as the youngest tumour - bearing mouse , namely , 12 months . in the sections prepared for the study of the spindle - cell sarcomas and thymic tumours , small adenomas were observed in a few mice . 
as shown in table ii , this phenomenon ' occurred following injection of dibenzanthracene in 4 out of 21 mice aged 5 - 7 months , following injection of methylcholanthrene in 2 out of 29 mice aged 6 and 7 months , and following injection of 9 : 10 - dimethyl - 1 : 2 - benzanthracene in 1 out of 9 mice aged 5 months . these figures apply only to the adenomas , one , or at most two or three , in each mouse , observed in one section through the lungs . nothing is known about microscopically visible adenomas in the remaining part of the lungs '  . neither is it known whether a corresponding production of adenomas had taken place in the mice showing no grossly visible it is worth mentioning that untreated tumours , but it is considered probable . street mice do not usually exhibit microscopic adenomas . serial sections from 48 mice , ranging in age from four to twelve months ( rask - nielsen , 1948 ) , showed one adenoma , only microscopically visible , in a mouse aged 12 months . 
the present experiments , therefore , appear to indicate that 9 : 10 - dimethyl - 1 : 2 - benzanthracene , methylcholanthrene , and dibenzanthracene , and presumably also benzpyrene , have induced pulmonary adenomas . 
the explanation why these growths were in most cases visible only upon microscopic examination is probably afforded by the short survival time of the mice ( table i )  . although the experiments allow of but an estimate of the development of pulmonary adenoma , they appear to indicate that injection of dibenzanthracene has induced a more marked increase in the development of adenoma than did injection of benzpyrene , methylcholanthracene and 9 : 10 - dimethyl - 1 : 2 - benzanthracene . the macroscopic adenomas as well as those visible only upon microscopic examination were of the ordinary , typical sub - pleural variety . in addition to the adenomas of the right lung , adenomas were also present in the left lung of a 7 - month - old mouse injected with dibenzanthracene and in the two 12 - month - old mice injected with 9 : 10 - dimethyl - 1 : 2 - benzanthracene . 
1rask - nielsen sarcomas of varying differentiation . associated with lymphosarcomatous thymic tumours . in two mice the spindle - cell sarcomas were table ii shows that spindle - cell sarcomas were observed following injection of benzpyrene in 2 out of 81 ( 2 - 5 per cent ) , following injection of dibenzanthracene in 14 out of 73 ( 19 per cent ) , and following injection of methylcholanthrene in 19 out of 59 mice ( 32 per cent ) , the effective total of experimental mice being the number of mice living to be as old as the youngest tumour - bearing mouse of all 4 groups , namely four months . 
the effective total of experimental mice was calculated on the basis of the number of mice living to be as old as the youngest mouse exhibiting a thymic tumour within all four groups , namely three months . with the exception of one spindle - cell sarcoma of the thymus , observed in a mouse injected with methylcholanthrene , all the growths were typical lymphosarcomas , made up of stem cells and accompanied by a frequently highly pronounced perivascular infiltration , particularly in the central areas of the lungs . 
one mouse injected with 9 : 10 - dimethyl - 1 : 2benzanthracene exhibited such violent infiltration without actual thymic tumour . the latent period of thymic tumours is given in table iii , which shows that the average latent period following injection of benzpyrene , dibenzanthracene , methylcholanthrene , and 9 : 10 - dimethyl - 1 : 2 - benzanthracene was 20 , 16 , 10 and 16 weeks respectively . the experiments revealed that the carcinogenicity of the hydrocarbons used , in doses of 0 - 5 mg . 
none of the six cases of generalized leukaemia was affected with thymic tumour . on the whole , the experiments showed that the order of carcinogenicity of the four hydrocarbons , as estimated from the incidence of tumours induced , varied , when 0 5 mg . 
of the same four hydrocarbons ( rask - nielsen , 1950a ) which showed that the lung tissue of street mice was susceptible to this dose of dibenzanthracene , less so to this dose of 9 : 10 - dimethyl - 1 : 2 - benzanthracene , but failed to respond to benzpyrene and iiethylcholanthrene . these recent experiments also showed that the thymus was particularly susceptible to direct application of 0 - 02 mg . 
of 9 : 10 - dimethyl - 1 : 2 - benzanthracene and methylcholanthrene , less to the same dose of dibenzanthracene and least to the same dose of benzpyrene . the incidence of thymic tumours induced by the four hydrocarbons was therefore decreasing in the same order following injection of a small dose into the thymus ( rask - nielsen , 1950a ) and a large dose into the lung . the results cannot be compared with earlier experiments , since thymic tumours following a direct carcinogenic action on the lung or thymus have not been reported before , with the exception of a few instances of thymic lymuphosarcoma observed after injection of methylcholanthrene into the lung ( esmarch , 1940b )  . but such remote effect was not observed . it is evident that the carcinogenicity of 9 : 10 - dimethyl - 1 : 2 - benzanthracene for the interstitial connective tissue of the lungs differed from that of the other three hydrocarbons . 9 : 1 0 - dimethyl1 : 2 - benzanthracene proved to be non - carcinogenic for this tissue , whereas the carcinogenicity of the other three hydrocarbons increased in the same order as their carcinogenicity for the subcutaneous connective tissue following application of the same dose into this tissue ( rask - nielsen , 19501 ) , i.e. 
they take advantage of the spontaneous adhesion on plastic membranes of the free cells present in physiological fluids such as blood , cerebro - spinal fluid , peritoneal and pleural effusions . in brief : glass slides coated with formvar are placed in contact with the fluid ( with addition of heparin if necessary ) in an incubator at 37 ' c . 
for 15 to 60 minutes . the slides are then rinsed with tyrode solution at 37 ' , fixed 10 minutes to several hours in acid osmic vapour and washed thoroughly in distined water . the formvar is stripped from the slide , mounted on grids and anowed to dry at room temperature for at least 24 hours . photographs were taken with a triib - taiiber electron microscope at 60 kv . we adopted stringent criteria for purposes of comparison and considered only undamaged cells , that is to say cers having clear and well defined chondriosomes and with a satisfactory cytoplasmic transparency . 
as the appearance of a cell may vary from one area to another ( because of differences in spreading , lipid constituents , etc . ) we only considered the best portions . counts were made in each cell of osmiophihe bodies less than 150 mp . 
at least a few such bodies are to be found in every cell , but to consider them as true ultrachondrioma we adopted as a minimum 10 such particles 354 j . 
oberling showing a certain degree of polymorphism ( to distinguish them from merely lipidic granules ) per i oo #2  . for each preparation at least 1000 cells were counted . 
the same submicroscopic corpuscles as in leukocytes are encountered , but frequently they appear as bent or twisted filaments which may have swelhngs or a beaded structure . sometimes these filaments are extremely long . their thickness may vary from 30 mlt . 
they may aggregate in groups of two , three or more , or may appear to arise from an ordinary chondriocont . the statistical analysis shows that those submicroscopic bodies are not a ebaracteristic only of nialignant effusions but are seen in various inflammatory or circulatory disturbances . 
but when those figures are an almost constant finding , when it becomes impossible to estabhsh a distinction between chondrioconts and those structures on any other basis than the rather arbitrary one of size and thickness , then it seems difficult to deny the mitochondrial nature of the described structure . they have the same characteristics and the same polymorphism as certain types of mitochondria ( especially in plant cells )  . 
even the specifity of janus green stain depends on certain criteria ( showacre , 1953 )  . furthermore there is a question of principle involved in the discussion about the significance of these structures . if we require for the identification of ultramicroscopic structures the same characteristics as those used in classical cytology , ff certain mitochondria can no ion er be considered as such because their size prevents the use of janus green and the apphcation of more or less debatable functional tests , then we shau have two cytologies : orie optical and one electronit is highly improbable that cytoplasmic conical , which is not satisfactory . stituents are of a different nature simply because they are too smar to be seen with the ordinary microscope . on the contrary it should be our aim to show the links between microscopic and - ultramicroscopic structures . this has been done with great benefit with basophihc structures , ergastoplasm and intracytoplasmic network . it would be curious if the same did not hold true for the mitochondrial structures . furthermore the concept of mitochondria which are invisible witli the optical microscope is not new and it can account for certain results . many cytologists consider the chondrioma as fixed both in amount and morphology . 
but this stabihty is dependant upon the material studied , and it is in fact true of speciahzed tissues observed under standard conditions . botanists have long noted the morphological variations of the chondrioma in active cens ( guilhermond , 1934 ; gautheret , 1950 )  . no6l ( 1924 ) and many others bave made similar observations with animal cells . the researches of levi ( 1934 ) , lewis and 1 - jewis ( 1915 ) , etc . , seemed in favour of the de novo appearance of mitochondrioma in cells of tissue cultures . recently chevremont and frederic ( 1952 ) observing tissue culture cells during mitosis with the phase contrast - microscope , noted the transformation of chondrioconts into very minute rods and granules which resemble the structures described here . ' these formations sometimes disappeared and appeared again in daughter - cehs and then growing thicker and longer , reconstituted ordinary chondriosomes . the existence of the ultrachondrioma may well explain the apparent appear - ance de novo of mitochondrioma as a result of their regeneration from the ultra.chondrioma. it is not impossible that the absence of the ultrachondrioma in many cers may be more apparent than real -  . 
the degree to which ordinary mitochondriaare susceptible to variations of physicochemical conditions such as ph , osmotic afinor variations of conditions that are uncontrollable pressure , is well known . by present methods might cause a cavitation of the ultrachondrioma that would make it impossible to distinguish from the surrounding endoplasm . actually we never observed ultrachondrioma in cells where swelling of the ordinary chondrioma occurred . furthermore we sometimes found ultrachondrioma in the perinuclear region wben very favourable conditions permit observation in this area . but usually the thickness and high rpid content of this area in spread cens pre - ntent its study with the electron microscope . 
oberling there are two fields in which the existence of the ultrachondrioma raises special problems . in cytology , the presence of corpuscles resembling viruses in normal ( or presumably normal ) cells should be a warning against mistaken interpretations and will complicate the task of those workers who are engaged on the problem of the interaction of cell and virus . in biochemistry it introduces a new factor into the study of cytoplasmic it seems obvious that a large part of fractions isolated by ultracentrifugation . the ultrachondrioma will be deposited in the so - called microsomal fraction and would therefore lead the erroneous conclusions concerning the homogeneity of this fraction . the concept of the ultrachondrioma limited though it appears should entail a critical revision of some of the present data of cytology . we wish to thank madame dontcheff for the tissue cultures , and madame arnoult and martin for technical assistance . 
ungar. from the department of pathology , hadassah university hospital , jerusalem , israel . received for publication june 7 , 1949 . carcinoma of the duodenum has been observed in this laboratory at autopsy in three young siblings whose case reports form the subject of this paper . 
the examination revealed several histological aspects which may be regarded as representative of successive stages in the development of polyps . in the area of the prominence , the submucosa was widened and the muscularis mucosae showed a funnel - shaped protrusion in the direction of the intestinal lumen . 
the earlier history contained no fourteen months prior to admission he first began these occurred at irregular contributory information . to complain of bouts of cramp in the abdomen . intervals and were without clear localization . the patient was admitted to the hospital in june , 1939 , three months before his death . 
at admission his chief complaints were shortness of breath , attacks of tachycardia , lack of appetite and abdominal colic localized under the costal arch on either side . during the preceding year the patient had lost a great deal of weight . there were recurrent periods of diarrhoea . 
the duodenal mucosa choledochus as far as 1 cbehind the papilla of vater was without lesions . and pancreatic duct without abnormalities . beyond the papilla of vater , the lumen was considerably distended . there was a nearly circular , crateriform defect , which extended from the posterior wall , the lesion , which including almost the entire circumference of the duodenum . measured 7 to 8 cin diameter , showed a rugged floor ; it was entirely composed of tumour tissue and blended underneath with enlarged lymphatic glands of the peripancreatic group . 
the latter were glued together and had been sections through the floor of the ulceration completely replaced by tumour . revealed a firm and , in some places , a friable tissue which was of a pale greyishyellowish colour . 
the tumour was defined against the head of the pancreas by a thin layer of greyish - red connective tissue . the remaining portion of the duodenum was considerably distended and filled with greenish fluid . 
sections from the large ulcerated tumour revealed a carcinoma which grew in some areas in solid strands , while in others gland - like formations predominated . epithelia showed a considerable degree of pleomorphism and there were numerous typical and atypical mitotic figures . 
the main mass of the node was made up of glandular epithelium tissue with regular lumina which in places were slightly tortuous . was columnar with dark nuclei and oxyphilic cytoplasm . there was no pleomorphism or mitotic activity . 
an x - ray examination of the stomach and duodenum 2 months following the operation likewise failed to reveal pathologic changes . the three younger boys were kept under observation from birth by the infant welfare centres and the school hygiene departments of the hadassah medical organisation . 
nagel ( 1925 ) benign polyposis . duodenal polyp , polyposis recti et colon duodenum , jejunum , ileum duodenum , stomach , colon , duodenum , stomach , caerectum cum , colon adenomas in duodenum , multiple polyps in cae . cum , colon , rectum duodenum , jejunum , colon polyps in duodenum and jejunum duodenum , jejunum 9 . 
the disease is hereditary and affects patients since generalized polyposis is known to of all age groups , including the lowest . involve predominantly the large intestine , it is not infrequently mentioned in textbooks and articles under the name " polyposis or adenomatosis of the colon . " however , in rare cases , the disease was found to be prevalent in the small intestine . besides the instances mentioned in table iii , others of this variant have been described by lubarsch in 1888 ( dohring 1907 ) , schwytzer ( 1924 ) , roan ( 1932 ) , haggard and floyd ( 1935 ) , ravitch ( 1948 )  . some pathologists have taken the view that generalized polyposis is an entity which since it causes characteristic severe dysentery - like symptoms and malignant change in a high percentage of cases , should be separated from that represented by cases in which only few polyps are formed ( oberndoerfer , 1929 ; lockhart - mummery , 1934 ; hullsiek , 1928 )  . 
the designation of all three of our cases as generalized polyposis therefore seems to be justified . the observations reported in this article are relevant to the understanding of factors which govern the manifestation of malignancy in intestinal polyposis . several authors have assumed a casual relationship between the appearance of polyps and carcinoma in any given case . 
gunz. * from the department of radiotherapeutics , university of cambridqe . received for publication june 11 , 1949 . in previous publications ( gunz , 1948a ; b ) a technique of cultivating human leukaemic leucocytes in vitro was described , and the quantitative and cytological findings in untreated cultures made by it were analysed . 
the mice which survived to tumour age , though not themselves used for experiment , were not certainly representative of the whole colony and , owing to the concentration of tumours in famihes , the inclusion or omission of a single litter could greatly alter the tumour incidence calculated on the small nurabers available . 
the " effective total " ' is the number of mice which surv - ived to the age at which the earfiest tumour was detected in any of the groups , namely i 10 days in an f4 mouse of strain br4 ( blue label )  . 
many of the mice recorded as dead without tumour succumbed to epidemic infection about a year after the beginning of the experiment . tables 1 - 111 show that the incidence of tumours in the strains br4 ( pink label ) , br4 ( blue label ) , and br6 was unequivocally " high . " the three strains were substantially alike . 
tumours developed , indiscriminately as it seemed , in 11 wild , " 11 white , " and " black " mice in the f . - f4generations and continue to develop in " white " and " black " sublines up to the f6 generation . 
the later age - incidence of tumours in the f2 females was probably attributable to the breeding history ; the f2 females , in contrast with later generations , were not subjected to forced breeding . the incidence of tumours in the br16 strain ( table iv ) so far is extremely low . 
the great majority of the mice survive and more tumours may yet develop , but the average age is already higher than th ' e average age of tumour development in the other three strains . 
tumours were ground in - a mortar with sand and saline to make suspensions which ranged in strength , in different experiments , from 13 to 20 per cent w / v . 
the debris was removed by spinning in an ordinary centrifuge , and the supematant fluid was injected , at weekly intervals , into the dorsal subcutaneous tissues , each dose being 1 - 0 c.c. sixteen mice reeeived each 4 injections of extracts of bri tumours of the first or third transplanted generations , five received 3 injections and two received 2 injections . twelve mice received each i injection of an extract of a primary spontaneous c3h tumour and 3 injections of primary spontaneous r3 tumours , and seven mice received 3 injections of the r3 extracts only . all the extracts were similarly prepared . the females were caged with males and subjected to forced breeding , litters being removed within 48 hours ( usually within 24 hours ) of birth . table v summarizes the results at 89 weeks from the beginning of the experiment . 
the " effective totals " comprise the mice which survived to the age when the first tumour was detected ( 180 days in a mouse injected with bri extract )  . 
the average number of tumours per mouse was 2 - 5 , 24 mice having from 2 to 6 t aours each . aour was 25 weeks ; the earhest the average age at the first detection of a t tumour wa - s recorded at the age of 17 weeks , and the latest at 54 weeks . the 3 mice without tumours are alive at ages of 81 , 81 and 85 weeks respecit is noteworthy that although continuously with males they have had tively . no litters for about a year ; previoxwly litters were not recorded . 
no observations were made on virgin mice . incidence of tumours in ( c57 black ? x agew - free r3 ct ) f ] l hybri& . remale mice taken at random from the laboratorystock of c57 black were mated with males : firom a colony of r3 mice free from milk agent . i am indebted for the male mice to my coreague b . 
the f , , hybrids were subjected to forced breeding throughout their reproductive lives . one hundred female hybrids were reared ; 21 died without tumours at an average age of 21 weeks and 79 are alive at an average age of 57 weeks . 
the diversity of genetic constitution in the f2 and subsequent generations did not noticeably affect the incidence of tumours which developed indiscriminately in " black , " " white " and " wild " mice 4 ' white " and " w - fld " subhnes estabhshed by and in each of the " black , ly 31 selective breeding . 
the patemal contribution was not probably genetic for , as shown in this paper and in previous reports from this laboratory ( dmochowski , 1944 , 1945 ) , the c57 black x a hybrids developed tumours abundantly when tumour agent was supphed to them artificiafly . 
the uncompleted observ - ations on hybrids having fathers belonging to a subline of r3 mice deprived of tumour agent accorded with similar observations bv anderv - ont and dinnn ( 1948a , b , c ) in showing a substantial reduction in the incidence of tumours comparison with similar hybrids whose fathers derived from a line carrying the tumour agent . 
the experiment was open to the criticism , which anderv - ont and dunn apphed to their ow - n experiment , that the agent - fiee and agentbearing malet ; were separated by several generations of inbreeding and possibly differed geneticary . 
tumour agent was not demonstrable in the hybrids or in their tumours , and the offspring of tumour - bearing mice , whether obtained by back - crossing or by brother - sister matings , rarely developed tumours . 
two litters of - ( c x c3h ) f , were exceptional in that tumours developed in sisters at unusually early ages ( 4 - 10 months )  . the backcross offspring of two tumour - bearing females of one litter all developed tumours at average ages of 7 and 9 months respectively . 
the progeny of two tumour - bearing ( c57 black y x r3 ct ) - p . , females had a high incidence of mammary tuinours ( about 70 per cent ) through at least 5 generations . 
5 ( peacock , 1946 )  . since our last publication on this subject we have successfully propagated three new induced sarcomata , grch 16 , 17 and 18 . these three tumours occurred in the course of attempts to induce epithelial tumours by injecting into adult white leghorn fowls a mixture of tissues from 10 to 14 - day chick embryos of the same flock , with methylcholanthrene and sudan iv dissolved in olive oil , following the technique used by rous and smith ( 1945 ) for inducing epithelial tumours in mammalian embryo tissues . 
we used a variety of embryonic epithelia , but the only tumours that resulted from these procedures were spindle cell sarcomata of precisely the same familiar pattern as when the carcinogens were inoculated directly into the adult breast muscles . 
they are made up of fleshy spindle cells arranged in whorls and interlacing bundles , the individual cells being elongated , sometimes with branched ends , with finely granular acidophil cytoplasm , and with an oval nucleus containing generally one small sometimes the cells are multinucleate , in which case the nuclei are nucleolus . in line one behind another , never in the form of giant cells of the foreign body mitoses occur very frequently , and are often somewhat irregular with type . bridging and incomplete separation of the chromosomes ; but many apparently occasionally longitudinal fibrillation of the normal mitoses are also to be seen . cytoplasm can be made out , but cross - striation has never been observed in metastases in viscera free from striated muscle such as the liver . 
48 : ( a ) liver and ( b ) squamous epithelium from the crop of a 10 - dayold chick embryo were mixed with 1 per cent methylcholanthrene dissolved in a saturated solution of sudan iv in olive oil . 
3086 was killed in moribund condition . there was an oval tumour 5 cin greatest diameter in the left pectoral muscles . there was no tumour in the right pectoral muscles , and no residual fluorescence was detected at either site of injection . there was a chronic plastic peritonitis , probably from a healed perforation of the gut , which was bound down with adhesions . the gall bladder was greatly distended . 
of fresh tumour was ground smooth in a sterile mortar , and 10 ml . of 5 per cent dextrose saline were added and well stirred to give a turbid cell suspension . ten 6 - week - old white leghorn chicks were inoculated with doses of 0 - 2 ml . to 1 * 0 ml . 
a 1 in 10 fresh cell suspension in 5 per cent dextrose saline was injected into the breast muscles of 6 white leghorns , 9 to 12 months old , in 2 of which tumours were palpable in about 4 weeks . subsequently the tumour was maintained through 3 serial passages , by cell suspension inoculation . 
the lowest 1 cwas cut off and discarded , and the remainder of the paper was laid on a sterile agar plate and flooded with melted agar at 460 c . 
prodigiosus was present only in the lower 3 cm . all levels of wet paper , however , were infective in the case of of the wet strip . the mh2 tumour , but in the case of the grch 16 tumour no level above the meniscus of the tumour extract was infective . as in the case of ea ' rlier chemically induced sarcomata in which the test was made by similar technique , using paper at levels above the first centimetre from the free fluid , grch 16 and 17 homogenates yielded only negative results . this observation is completely in keeping with the results of the more conventional filtrations through gradacol membranes or seitz clarifying pads . 
on the other hand , the tumour itself might be supposed to contain such an agent , which for some reason or other was adsorbed strongly by the filter . in the case of paper chromatography the whole paper can be injected either intact or homogenised in saline , and this might be thought to favour the demonstration of viruses where filtrates yield negative results . 
as there are many variable factors in such exposures , it is convenient to express dosage as time related to sterilising effect on bacteria . under our conditions suspensions of b . 
for 5 to 10 minutes were exposed in plastic tubes to a focused beam of ultrasonic waves with a frequency of one megacycle per second transmitted through a cooling water bath . 
the plastic tube was transparent to the waves , and was so placed that maximum disturbance occurred in the centre of the tube . in order to concentrate the effect of the waves as much as possible 2 to 5 ml . of 1 / 10 homogenate of fresh tumour in normal saline were used . 
1 in the resulting proliferative reaction tissue . we have previously reported the localisation of virus - induced fowl sarcomata at remote sites of x - ray or radium burns ( peacock , 1946 )  . cauterisation with a red hot platinum wire or with fuming nitric acid applied to the skin with a glass rod ( 2 to 3 mdiameter ) has more recently been found to localise the virus in birds bearing rous sarcoma no . 
a sterile cotton thread was inserted in the left pectoral muscles of each chick and also ( a ) in 5 the left wing was injected with 0 - 1 ml . 
50 : ( a ) four out of 5 chicks had tumours in the right breast but no ( b ) all 5 chicks had tumours in the right breast . 
50 : the 9 chicks with large tumours at the site of rous sarcoma three chicks out of 6 had localisation at site of cautery , inoculation were killed . and 1 of them had localisation at site of control thread . none of the remaining 3 tumour - bearing chicks had localisation either at the site of sudan iv injection or at the site of the control thread . 
on the other hand , occasionally local inflammatory reaction occurs around the metal wing tabs used for numbering the birds , and we have seen 2 cases of localisation of rous sarcoma no . 
1 , and had 1 feather plucked from the leading edge of the wing on the day of injection and on the 4th , 6th , 13th and 15th days thereafter . 
no localisation occurred at the sites of trauma . it was thought that histamine - like action following cautery might cause local blood stasis and so favour the metastatic growth of blood - borne emboli from the tumour histamine was therefore injected into the left wing web at the damaged site . of 6 white leghorn chicks 4 weeks old bearing rous sarcoma no . 
50 : a large tumour was present in the right breast and the bird was there was direct spread from the grch 17 tumour injection site to the killed . liver , but no tumour at the site of rous no . 
3312 , 3316 and 3317 , which had not grown tumours at the site of injection of the seitz filtrate ( see above )  . typical grch 17 sarcoma grew in no . 
on the other hand , no such localisation has been observed in the case of the grch series of tumours . it seemed that the electron microscope , which can undoubtedly reveal particles of the size of viruses , might afford conclusive evidence of their presence or absence in our limited experience of this technique , however , there are in tumour cells . even less differences to be seen between infective and non - infective tumours and even normal tissues and in homogenates and filtrates prepared from them than can be seen by ordinary visual microscopy or even by direct visual observafurther work may overcome this initial difficulty of recognising viruses tion . from normal cell constituents ; but in the meantime circumstantial evidence points to essential differences between certain induced and spontaneous tumours . the occasional occurrence of a chemically - induced tumour which on propagation yields a virus does not in our opinion justify the assumption that all indeed , the existence of spontaneous filterable tumours contain such viruses . tumours would suggest that ultimately one is likely to encounter such agents in duran - reynals ( 1952 ) has recently suggested , birds injected with carcinogens . however , that viruses not usually associated with tumours may become adapted to cells conditioned by chemical carcinogens , and so play a part in the aetiology of cancer . 
our failure to induce any epidermoid cancers in the skin of fowls after repeated painting with chemical carcinogens may be due , as he suggests , to the absence of fowl pox in our flock ; but we have had no difficulty in inducing upwards of 60 sarcomata at the sites of injection of chemical carcinogens , and have seen a few carcinomata of the crop following exposure to 2 - acetylaminofluorene ( peacock and peacock , 1949 )  . 
though we failed in attempts to transmit the carcinomata and therefore cannot assess their possible viral content , we saw no inclusions of the kind described and illustrated by duran - reynals ( 1952 ) in his fowl pox methylcholanthrene carcinomata . the study of a wider range of chemically - induced fowl tumours , preferably including carcinomata of recognisable patterns , seems to offer the best prospects for obtaining more information about the aetiological factors concerned . 
1 , mill hill 2 endothelioma and the grch 16 , 17 , 18 sarcomata show that the two former tumours are readily transmissible by filtrates and by other extracts in which cells are probably destroyed or excluded , whereas the grch tumours are only transmissible by extracts containing cells . at the time of writing the authors were unaware that loewenthal ( 1931 ) had used capillary ascent on strips of filter paper of extracts of rous sarcoma no . 
w.3. received for publication july 16 , 1949 . in an investigation of the body growthand tumour growth - inhibiting action of carcinogenic substances , it was found that the nature of the inhibition is profoundly influenced by the protein content of the diet ( elson and warren , 1947 ; elson and haddow , 1947 ; elson , 1949 )  . in rats maintained on a 20 per cent protein diet injection of the carcinogen usually has little immediate effect on body growth although a delayed action , resulting in rapid loss of weight followed by death , may occur later . 
carcinogenic hydrocarbon 1 : 2 : 5 : 6 - dibenzanthracene 24 hours after implantation of walker carcinoma 256 in rats maintained on a 20 per cent protein diet results in very little difference in size and weight of the tumours from those of controls . if , however , the animals are fed a 5 per cent protein diet the tumours of those similarly treated with dibenzanthracene were found to be only one - quarter the weight of those of controls when the rats were killed 13 days after implantation ( elson and haddow , 1947 )  . 
lamerton for the studies involving the localized irradiation of the tumour , it was necessary first to develop .a method whereby the rat could be held firmly in anaesthesia reasonable comfort and the tumour alone irradiated with precision . was contra - indicated , since it may affect the response of the animal to radiation . the device eventually developed is shown in fig . 
a length of transparent plastic " cloth " is held by means of two clamping bars on an aluminium base . the rat is placed under the plastic material , which is then stretched under the clamping bars . 
the edge of the plastic cloth near the rat 's head must be reinforced with a strip of cellophane one to two inches wide , which serves to hold fig . 
1. - photograph of rat in holder designed for x - ray treatment of tumour . the rat 's head down and prevents him biting his way out . the tumour itself projects through a hole in the plastic material , and can be treated by crossfire techniques without irradiation of the rest of the body . a victor kx1o plant was used , run at 140 kvp . 
lamerton direct irradiation of e8tablished tumours . for these experiments the tumours were allowed to grow untreated for 6 days after implantation . daily estimates of the size of the tumour were then made by - measurement with calipers along two axes at right angles . 
2. from the results obtained with more than 20 control animals there appeared to be no very marked difference in rate of growth of the tumour in animals maintained either on 5 per cent or on 20 per cent protein diets . 
the group maintained on the 5 per cent protein diet , however , showed considerable inhibition of tumour growth , and the tumours after the first 7 days of treatment ceased to grow and remained fairly constant in size for a considerable time . 
3. - growth of walker rat carcinoma 256 treated with x - radiation ; 250 r per day , total dose 4000 r . * rats fed on 20 per cent protein diet . rats fed on 5 per cent protein diet . gradually developed areas of necrosis , which usually become infected , and in some cases the tumour had resumed growth shortly before the death of the animal . 
lamerton for the first four days after treatment the tumours of the 5 animals comprising the 20 per cent protein diet group grew more rapidly than those of the 5 per cent protein group , and reached an average size of about one and a half times that of the low protein group . after this period the tumours of the 20 per cent protein diet animals began to decrease in size , and eventually two of them regressed completely by a " shelling - out " process and a third remained very small over a long period . 
the other two , after a considerable period of regression , eventually grew again rapidly and the animals died of the tumour about 45 days after implantation . in the 5 per cent protein group one tumour regressed completely but the others grew fairly steadily at a much reduced rate for a considerable period , eventually resuming a more rapid growth rate before the death of the animal , which occurred on the average 36 days after implantation . died 2400 s 2000 3 1600 z 1200 en 800 perioi fig . 
no cures were obtained in animals of either group . it appeared therefore that regular control of the established walker tumour could not be effected by a daily dose of 400 r , and in the next experiment the effect of a daily dose of 600 r was studied , together with the effect of changing the diet of the low protein group of animals to the high protein content at the completion of the course of radiation . x - radiation , 600 r per treatment ( total 4000 r )  . in these experiments a dose of 600 r was given daily on six days , the seventh dose being reduced to 400 r to maintain the total dose at 4000 r . in the first experiment the animals of the 20 per cent protein diet group responded well at first , and nearly all the tumours began to decrease in size after the fourth dose of radiation . although the tumours then remained small for a considerable time no complete regressions occurred , and all eventually grew again fairly rapidly , leading to the death of the animals . the tumours of the animals maintained on the 5 per cent protein diet again at first responded to the treatment by growing rather more - slowly than those of the 20 per cent protein group . 
the day following the last radiation treatment the 5 per cent protein group animals were changed over to the 20 per cent protein diet and maintained thereafter on this high protein diet . 
5. - growth of walker rat carcinoma 256 treated with x - radiation ; extended treatment with initial high dose ( 1000 r ) ; total dose 4000 r . . 
under these conditions therefore the large initial dose of radiation appears to have such a strong inhibiting effect on the tumours that a marked difference is no longer observed between the initial response of low and high protein diet animals . 
the greatest degree of tumour inhibition was obtained when the animals were maintained on a low protein diet . it has now been shown that the response of rats implanted with walker carcinoma 256 to whole body x - radiation under conditions approaching those of the " walker tumour test " is also greater in those animals maintained on the low protein diet . 
with x - ray treatment , however , it is possible to apply direct to the tumour a sufficiently large dose of radiation to bring about an adequate initial growth - inhibitory response , even in animals maintained on a high protein diet , so that the subsequent outcome of the treatment then depends almost entirely on process ( b ) , and a favourable result is thus to a very large extent dependent on a high protein content of the diet . though the results obtained may be related to some extent to the particular characteristics of the tumour used , it is very interesting to note how closely the two processes distinguished in the radiation response of the tumour follow the concepts of " intra - cellular response " and " inter - cellular response " put forward by koller and smithers ( 1946 ) from studies in clinical radiotherapy . . 
tumour regression as the result of koller and smithers suggest that "  . irradiation is initiated by an intra - cellular response , but is carried on by an inter - cellular response which follows and plays an important part in the success of any treatment . 
the inter - cellular reaction is manifested through the repair processes , and depends on the construction of the tumour tissue as a whole . this reaction is conditioned and influenced by the intra - cellular response ; in this sense it is an indirect effect of radiation it seems likely that this " inter - cellular " response may constitute an important part of what we have described as process ( b ) , and have shown to be favourably if the " inter - cellular response " is related to influenced by a high protein diet . the repair processes , then it is very reasonable to suppose that nutritional factors can affect the elimination of the tumour in radiation treatment , and indeed proteins would be of special importance in view of their known value in the healing of burns and wounds . clearly the present work can only be regarded as a preliminary study . 
lamerton implantation , inhibition of tumour growth was greatest in those animals maintained on the low protein diet , but no complete regressions of the tumour were obtained . with localized irradiation of the tumour , when the total dose of 4000 r was applied in larger daily doses , complete regression of some tumours was observed , and the most favourable results were obtained by application of an initial dose of 1000 r with extended time intervals between successive smaller doses . 
complete regression of the tumours was then obtained in about 90 per cent of the animals maintained on the high protein diet , but in only about 15 per cent of these maintained on the low protein diet . it is suggested that two effects are concerned in the response of tumours to radiation , ( a ) the initial inhibition of tumour growth , ( b ) the elimination of process ( a ) is favoured by a low the inhibited tumour and cure of the animal . protein diet , and process ( b ) by a high protein diet . we would like to express our appreciation of the interest taken in this work and the encouragement given by professor a . 
we also wish to express our thanks for grants supporting this investigation from the british empire cancer - campaign , the anna fuller fund , the jane coffin childs memorial fund for medical research and the u.s. 
the hvbrid females of each litter , when four to six weeks of age . were mark - ed indi - vidually and di ' %ided into experimental and control n - lice . 
the largest part has been obtained from the rikshospital , which receives patients from approximately one - sixth of the material has been placed at the whole country . ' cal depaxtment iii and by proniy disposal by professor c . 
a few cases onl come from - other hospitals . in addition to the two clinical , two post - mortem series have been used in this analysis , namely , t6 material of jacobsen ( 1953 ) from wo city hos itals , covering the years 1937 to 1946 , and the material of christiansen ( 1953 ) from the rikshospital , covering the years 1925 to 1952 / 1 . all the tumours of these post - mortem materials have been typed by kreyberg with no knowledge ofthe clinical data . 
figures of each series are comparatively small , but a few feature - s seem to be significant , and possibly important . the main differences between the chnical and the post - mortem series are ( 1 ) the relatively much higher number of adenocareinomas , 30 per cent of an cases in the latter against less than 10 per cent in the former , and ( 2 ) the lower number of squamous cell carcinomas in the post mortem series . 
a study of the two papers dealing with the post - mortem series reveals a greater number of old persona i these materials , as compared to the clinical group . 
the significance of these differences will be discussed later . in kreyberg 's ( 1952 ) first paper a comparison was made between the occurrence of the different histological types in the norwegian material and a sipailar recent material from the mayo clinic . 
kreyberg mainly as a result of differences in criteria for typing . in the present table 1 , three series from foot ( 1952 ) have been included in order to stress this point . 
the distribution of histological types in this american material is remarkably similar to the norwegian clinical series , with one exception , the group adenomas and salivary gland tumours . these tumours occur more than ten times as frequeiltly in all the norwegian series . 
the cause of the relatively greater number of these suffice it to mention at this tumours may be manifold and will be discussed later . point that even in america certain investigators have found these tumours ratheifrequently . in table ii is presented the distribution of the different histological types as regards age and sex . 
t. uncertain group ii : 17 in table iii the findings regarding the sex distribution have been summarized . it will be seen that the different histological types in this chnical material form two main groups with markedly different sex ratio . 
the fact that the tumour material has been collected during the years 1948 to 1953 , while the population figures refer females 3 4567891 4 5 67 891 i1 1 3 456789 figa . 
1. - age distribution at the time of diagnosis of all lung tumours ( l ) and the mortality figures for stomach carcinomas ( v ) in males and females separately . 
1 curves have been drawn on double logarithmic paper , representing the whole material of the clinical series for males and females separately . for comparison one other curve for each sex has been drawn , namely , the corresponding curves for stomach carcinomas , based upon the mortality figures for norway , 1950 . 
2 two curves have been plotted in the traditional manner , namely , the curves for the relative age frequency of squamous cell carcinomas and for large cell lo~ ~ ~ fig . 
the figures are given in table iv the two curves are nearly identical and they indicate that the group i tumours biologically actually represent a uniform group , in spite of unquestionable histoit will further be seen that these group i tumours mainly are logical differences . responsible for the characteristic features of the lung " cancer " curve in general . the other lung tumour types have been examined in a similar manner . 
the adenocarcinomas and the sub - group lung adenomas and salivary gland tumours are , however , comparatively few in number in each of all the series , and accordingly subject to considerable chance fluctuations . in table v the figures for these tumours in all the three norwegian materials the basic figures are given for each material separately . 
the unusual fall in the higher age groups is taken to indicate a new carcinogenic situation in development , and the future stages are forecast by the direction of the curve for the age groups 30 - 39 and 50 - 59 . the adenocarcinomas , according to our diagnostic criteria : malignant tumours coniposed of more or less atypical , secreting or non - secreting , columnar cefls , with their nearly equal sex distribution and their increasing frequency with the advancing age , indicate that they are caused bv comparatively weak carcinogenic agents , well established in the society , and acting with equal strength in both sexes , at least in large parts of the country . the small group of bronchiolar cell carcinomas occur in all age groups and with no definite sex preference . 
they may be caused by unknown agents , hitting at random . the histological pictures , the equal sex incidence , as well as the appearance with no preference for any age group , together indicate that lung adenomas , benign and malignant , and sahvary gland tumours are caused by accidental factors , presumably of developmental origin . at this stage a few more words about the latter tumours may be justified . 
some surprise has been voiced as to the considerable number of adenomas and sahvary gland tumours in norwegian materials . firstly , a survey of the lung tumour problem i : ft general in norway shows that the total number of lung tumours is very moderate , as compared to the figures for england and wales , the netherlands , the united states , and even denmark . if , as assumed and actually found , the recent increase in norway is caused mainly by the ever larger number of group i tumours , it follows that the other histological this development has been types must show a proportionate decrease in number . observed in the ordinary columnar cer carcinomas . 
a decrease in the number of lung adenomas and sahvary gland tumours in relation to the sum total is predicted , but it may for a while be less marked in chnical and especiary surgical materials , because these tumours represent comparatively favourable clinical cases and are therefore selected . secondly , the tumours in question are , actually , observed in considerable numbers also in other materials . fried ( 1948 ) in the massachusetts general hospital found 17 adenomas out of 175 cases of bronchogenic neoplasms , and carlens ( 1952 ) reported 8 - 10 per cent in a swedish material . tbirdly , these tumours are most probably present in many other materials without beino , recognized as such . true columnar cen careindmas and group i tumours very rarely occur before the age of 35 years . in most 1 - ung cancer statistics some tumours are registered in younger and verv young persons . 
some of them have been diagnosed as adenocareinomas , others as sman cer carcinomas , especially if unfavourable preparations have been the base for diagnosis , or if the tumours are infiltrating . 
any academic discussion as to the propriety of the designation cc mahgnant adenoma " ought to be silenced by the very important fact , that if the proper criteria are observed and the material typed without clinical information , a grou ' of tumours will emerge , wit - h a sex ratio 1 : 1 , with no accumulation in anv a - ae aro - ud . 
each histological type has been analysed , especiany as to distribution according to age and sex , and the findings correlated to our additional present knowledge regarding the development of these tumours . 
they do very seldom occur before the age of 35 years and they present a very characteristic and , for human carcinomas , singular age distribution . this age curve as wer as the vital statistical evidence indicate that the main part of these tumours are caused by a recently estabhshed carcinogenic situation in certain ' parts of the world , notably the united states of america and westem europe . columnar cell adenocarcinomas appear with httle sex difference and they show a steadily increasing ffequ - ency with advancing age , a development in conformity these tumours are probably caused by with a great many human carcinomas . comparatively weak carcinogenic influences , evenly distributed over large areas , well established in the society and striking both sexes with equal force . bronchiolar cell carcinomas occur with equal frequency in both sexes and strike individuals in ar ages . 
hewitt. from the john burford carlill laboratories , westminster hospital , london . received for publication may 26 , 1953 . from the fact that certain mouse tumours could be transplanted using freezedried tumour tissue , gye , begg , mann and craigie ( 1949 ) concluded that they had evidence of virus transmission of their tumours . subsequently , it was shown ( passey , dmochowski , lasnitzki and milard , 1950 ) that a small proportion of the cells in these tumours were able to withstand freeze - drying as indicated by the proliferation in vitro of tumour tissue so treated . these findings focused attention on the quantitative aspects of tumour transplantation , and various observations were made which suggested that the malignant cell population in sarcomacraigie , lind , hayward and begg , ( 1951 ) tous ascitic fluids is heterogeneous . reported that a small proportion of the sarcoma cells in certain ascites tumours could be distinguished from the modal cells by their peculiar appearance under phase - contrast microscopy , and by their relative resistance to freezing and to the inimical effects of exposure to isotonic glucose . lasnitzki ( 1952 ) showed that a high proportion of the cells of sarcoma 37 ( hereafter referred to as " s37 " ) ascitic fluid undergo morphological transformation in tissue - culture and do not subsequently divide ; she found that this change was accompanied by a reduction in the power of the cells to give rise to a tumour on transplantation , and considered her observations to be evidence of bimorphism of the s37 cells . histological studies of the fate of implants of tumour tissue have not given conclusive evidence that the cells of the new tumour are direct descendants of the cells in the implant . 
 ( 1949 ) stated that the appearance of implants which had been frozen previous to inoculation , although they subsequently would give rise to tumours , " did not suggest survival of any of the implanted tissue . " zahl and drasher ( 1947 ) drew similar conclusions from their observations of the fate of implants of sarcoma 180 in mice . these authors state that there is no increase in the size of the implant and that it undergoes immediate cytological degeneration . several previous workers have studied the variation of tumour incidence with the number of cells inoculated . 
de gaetani and blothner ( 1936 ) found that over 100 , 000 cells were generally required for the successful transplantation of mouse carcinoma and sarcoma . furth and kahn ( 1937 ) , working with an inbred strain of mouse and a spontaneous leukaemia derived from that strain succeeded in transplanting leukaemia to about 5 per cent of their mice by the intravenous inoculation of a single leukamic cellw kahn and furth ( 1938 ) , using the trypan 368 h . 
hewitt a reaction usually regarded as being incompatible with cell viability ( schrek , 1936b ; pappenheimer , 1917 )  . counts of suspensions prepared from solid tumours were tberefore made after mixing equal volumes of the suspension and 0 5 per cent trypan blue ( b.d.h. , biologically tested ) in m / 15 sorensen buffer at ph 7 - 2 . no ascitic cell suspension has been found to contain more than 3 per cent of stained cells , and counts of these suspensions were made in the absence of trypan blue . trypan blue was preferred to eosin for the differentiation of non - viable cells because counts were made in a fuchsit gave more definite staining at non - toxic levels . rosenthal haemocytometer using phase - contrast microscopy . the absence of any reliable morphological criterion by which to identity a viable tumour cell makes it impossible to signify with certainty whether a given cell is a malignant cell or a normal cell contributed by the cellular reaction of the host . it has been found convenient to make an arbitrary distinction between normal tissue cells and sarcoma cells in fresh single - cell preparations of the tumours used in this study , on the basis of cell diameter . it was noted that 90 per cent of the nucleated single cells in sedimented preparations of minces of normal mouse lymph gland , thymus and spleen , and 70 per cent of the cells in saline washings of normal mouse peritoneum had diameters of 9 , t or less . 
on the other hand , a relatively small proportion ( 10 - 20 per cent ) of the cells in many single cell preparations of the solid sarcomas or in some s37 ascitic fluids , had diameters of less than 9 , u . 
the density of viable cells in a suspension has therefore been taken as equivalent to the density of cells having a diameter of over 9 , t and failing to stain with after considerable experience had been gained of the measurement trypan blue . of the cells in fresh preparations , actual measurement of the cells was abandoned and the larger cells were selectively counted by estimation of their size . frequency distribution curves of the diameters of the single cells of sarcoma preparations showed a modal diameter in the region 14 - 16 , t . 
a smaller , second peak was sometimes seen at about 7 , u , and this was considered to represent the mode of the inflammnatory cell population in the suspension . the effect of elimination from the cell count of all cells of diameter less than 9 , u was to exclude from the count the great majority of the non - tumour cells . 
on the other hand , a count of tumour cells made using this criterion might represent only 80 per cent , but no less , of the actual number of tumour cells present . titration of sarcoma cell suspensions . if necessary , the counted s37 cell suspensions were diluted to contain about 20 , 000 tumour cells in 0q1 ln . 
 ( the inoculum volume )  . of this suspension , five 3 - fold dilutions were made in the gelatine - tyrode medium , giving a series of six suspensions with cell densities varying from about 20 , 000 / 0 1 ml . 
to facilitate accurate placing of the inocula , all inoculations were given under ether anaesthesia . throughout all manipulations from the time of setting up the second phase of the sedimentation procedure until inoculation , the suspensions were kept at 2 - 5 c . full aseptic technique was employed throughout and special precautions were taken to keep the suspended celis in homogeneous distribution . recording of tumour incidence and calculation of end - points . inoculated mice were examined for palpable tumours at 2 - 3 day intervals from the 6th till the 30th day after inoculation in the case of s37 and from the 6th to the 40th day after inoculation in the case of the c3h sarcoma . it was found that , with very few exceptions , the smallest detectable mass in an inoculated site would become a definite tumour within a few days of its presentation . regression occurred in a fairly high proportion of the s37 tumours derived from the smaller inocula , but was unusual except in tumours which had attained moderate size . 
the proportion of sites in which a palpable tumour formed was recorded for each of the 6 different tumour cell in an ideal titration , all sites inoculated with the highest dose doses inoculated . of cells gave tumours and all those inoculated with the lowest dose failed to give tumours . 
from these data , the number of tumour cells which would have given tumours in 50 per cent of the inoculated sites ( hereafter referred to as " td50 " ) was calculated by the method of reed and muench ( 1938 )  . 
variation in td50 values obtained in ( i ) separate titrations of different s37 cell suspensions and ( ii ) parallel titrations qf the same suspension . in 16 titrations of different s37 cell suspensions performed in the manner described and using 4 - 6 mice per group , the td50 values ranged from 603 to 3162 cells , with a mean of 1641 and a coefficient of variation of 51 per cent . 
the ratio of the highest to the lowest value obtained in this series was 5 - 2 . it has not been possible to correlate this variability with any one factor known to vary from one experiment to another . suspensions prepared from tumours during their early growth phase and those prepared from older tumours gave results which were not consistently different . it is clear , however , that the variability in results from one experiment to another is contributed to both by unspecified differences between the suspensions and by random differences in the batches of mice used for the titrations . in order to assess the order of variability in results which may be expected in parallel titrations of the same suspension , a number of paired titrations were performed using 2 series each of 6 groups of 2 - 3 mice . in these experiments the mice were of comparable age but the members of corresponding groups were not litter mates . 
the values 2 sd and 3sd corresponded to ratios respectively of 2 - 0 and 2 - 82 , so that a significant difference between the td50s obtained for parallel titrations in control and treated animals should be considered when this figure was used as a guide ; the following further their ratio exceeds 2 - 0 . test of significance being applied when the ratio of the results exceeded 2 - 0 : the td50 and its standard error were calculated from the results of each titration by the approximate method of irwin and cheeseman ( 1939 ) ; the limits of error of each td50 were defined from a value 3 x s.e. , and a significant difference was inferred when the limits of error of the td50 with the greatest s.e. 
did not include this criterion of significance was the td50 value given by the other titration . used by the authors of the method to compare ld50s in certain toxicity tests . the adequacy of the test in the present context appears to be justified by the the multiple difference of td50 values obtained in following considerations : 8 pairs of titrations using only 2 - 3 mice per group , and with only random pairing of mice between the two series , did not exceed 2 * 0 ; in the parallel titrations to the results of which the test was applied , however , there were 4 - 6 mice per group , and each mouse of one series was carefully paired with a mouse in the corresponding group of the other series , the members of a pair being usually litter mates but occasionally mice from two litters born on the same day . 
each mouse received four equal inocula in the following sites : subcutaneously in the right axilla and right groin ; intramuscularly in the medial muscles of the proximal segment of the left forelimb and in the adductor muscles of the left leg . 
the inter - actions of multiple inocula in a single mouse . no normal mouse of the age and stock used has failed to develop a palpable tumour from the inoculation of undiluted s37 mince or of single - cell suspensions nevertheless , the behaviour of s37 transplants in of over 100 , 000 viable cells . the mice used displays the immunological phenomena characteristic of most strain - heterologus transplantations : regression occurs in a high percentage of tumours , and solid immunity to a second transplant usually follows upon the regression . 
from the fact that growth of s37 in the mice used results in the induction of resistance it must be assumed that each of several simultaneous inoculations into the same animal , whether of equal or of graduated doses , may be subject to influences of a constitutional nature induced by the others . to investigate this possibility a suspension of s37 cells was titrated in two series of mice . 
each series consisted of 6 groups of 5 mice each , and corresponding groups of the two series received the same dilution of the suspension and the same cell dose per inoculueach mouse of a group in one series ( s ) received a single inoculum of the appropriate dilution subcutaneously in the right axilla . 
each mouse of a group in the other series ( m ) received 6 subcutaneous inocula , all of the same size , one of these being in the right axilla . 
when the td50s were calculated from the right axilla results alone for each series their ratio was 1 - 6 , the difference it was concluded that the practice of giving four equal being not significant inoculations per mouse , in order to increase the number of sites used in the calcula374 h . 
hewitt tions , was probably not associated with results peculiar to the multiple inoculation procedure itself provided that the several inocula in each mouse were of equal sizes . a further experiment was designed to show whether the resistance induced by a large inoculum was capable of preventing a smaller inoculum of cells , placed simultaneously elsewhere in the same animal , from becoming a palpable tumour . a number of 3 - 5 - week - old albino mice were randomised to give one series ( c ) of 6 groups of 5 mice each and a second series ( m ) of 20 mice . 
the dilutions were inoculated into the mice of the successive groups of the c series in the usual way , 4 equal inocula being given to each mouse subcutaneously in the groin and axillary sites . 
the mean latent periods of all tumours arising from inocula of 247 , 741 and 2222 cells ( i.e. , in the region of the td50 ) in the s series and in the m series were respectively 13 - 7 days and 11 - 4 days . 
the 30 per cent incidence of tumours from 82 cells in the s series is fortuitously high , as may be seen from the trend of inoidence in this series . it is clear that the relatively high total inocula per animal with which the 247 , 741 and 2222 cell inocula were associated in the m series ( see last horizontal column of table ii ) has not significantly diminished the incidence or extended the latent periods of the tumours produced by these inocula . 
the effect of " hyalase " of the tumour incidence from small inocula of s37 cells . a single - cell suspension of s37 was titrated in two parallel series of mice . 
the influence of dead s37 cells upon the fate of small inocula of viable cells . in the following two experiments the effect of dead tumour tissue upon the fate of minimal inocula s37 cells has been studied under two conditions : ( i ) where the dead tissue was inoculated intraperitoneally previous to the inoculation of viable tumour cells subcutaneously , and ( ii ) where the dead tissue was incorporated in the inocula of viable cells . a s37 single - cell suspension was titrated in a control series of mice and in a parallel series each mouse of which had been inoculated intraperitoneally with 0 5 ml . 
of 20 per cent s37 mince freshly killed by repeated freezing and thawing . the inoculation with dead tumour tissue was made 48 hours before titration of the viable cell suspension . 
no significant difference between the td50s was obtained in the two titrations , their ratio being 1 - 07 . the second experiment was designed to show whether tumour production from minimal inocula is depressed or enhanced by the presence in the inoculum of a large preponderance of dead cells . 
hewitt the td50 values obtained in the two series were as follows : dilutions in gelatine - tyrode diluent dilutions in dead cell suspension 2818 although the difference ( ratio 4.3 ) was considerable , it did not quite attain significance by the irwin and cheeseman ( 1939 ) test . 
an experiment was therefore designed to compare under more carefully controlled conditions the activities on transplantation of two suspensions prepared respectively from these two sources . malignant ascitic fluid and four moderate sized subcutaneous tumours of s37 - were harvested from a single mouse inoculated intraperitoneally and subcutaneously with s37 mince 15 days previously . 
215 it will be seen from table iv that correlation between the td50 values in the two series is highest when the cell - determining tumour incidence is taken to be the larger unstained cell . 
1 shows the relationship between viable tumour cell dose and the proportion of inoculated sites giving tumours , for the c3h sarcoma and for s37 . the case of each tumour the points shown were obtained from two separate titraaverage tumour cells per inoculum ( loglo ) fig . 
the accuracy of the points is not sufficient to reveal the true character of the curves relating cell dose and tumour incidence but it will be seen that the rate of change of tumours incidence with log . 
white and loeb ( 1910 ) have described the successful transplantation of regressing tumours , and schrek ( 1936a ) has found no effect of immunity on the growth capicity of the tumour cells . these findings do not suggest the presence of humoral factors in regressing tumours which are active against the tumour cells themselves and encourage the view that regression is the result of primary breakdown of the stroma . the significantly lower td50 values given by intramuscular sites of inoculation compared with subcutaneous sites , for s37 , might ascribed to the greater vascularity of muscle . 
on the other hand , this site difference was not demonstrable for the c3h sarcoma , and a more likely explanation of the site difference found for s37 would appear to be that local resistance mechanisms are less readily manifested in the intramuscular sites . from the results of the experiments recorded in section 3 , it is clear that a small inoculum of cells is not suppressed , in the period before it forms a palpable tumour , by multiple and larger inocula of the same tumour placed simultaneously elsewhere in the same animal . this being the case , it seems probable that the failure to give rise to palpable tumours which is displayed by half the inocula of about 2000 cells of s37 , is not due to induction of systemic immunity by these 380 h . 
methods are described of preparing single - cell suspensions of mouse sarcomata from solid and ascitic tumours , of finding the viable tumour cell density in the suspensions , and of " titrating " them for tumour producing activity on inoculation . 
the implication of this finding for the problem of cell - free transfer is discussed . i wish to express my gratitude to miss catherine fysh , b.sc. , for skilled technical assistance , and to professor r . 
1. received for publication january 13 , 1948 . one of the implications underlying the work of huggins and hodges ( 1941 ) on the factors governing prostatic hypertrophy is that malignant prostatic cells are not fully , if at all , autonomous . that is , they are still controlled in some measure by the same factors that control normal prostatic cells . specifically , this is certainly true of their susceptibility to the influence of circulating androgens and oestrogens . carcinoma of the prostate is essentially a disease of old age . the possibility exists that the chief factor in deciding the individual 's susceptibility to this disease may be his androgenic state , that is , the level of circulating androgens in his blood , particularly during the later decades of life . the only practical method for ascertaining androgenic state is a determination of 17 - ketosteroids excreted in the urine of the subject . in the hands of many different workers ( barnett , henly , morris and warren , 1946 ) this method has revealed a very wide range of excretion of 17 - ketosteroids by normal men , at least in the lower age - groups . 
robinson the prostates of individuals who excrete at the higher or lower end of this scale will be subjected to grossly different amounts of androgenic stimulation . most of the available data on the excretion of 17 - ketosteroids is confined to results obtained in studies of men under the age of 30 - years . although there is a general impression that the excretion of these substances decreases with advancing age of the individual , there is a paucity of data on the range of excretion in the higher age - groups . the ideal method of study would be to make repeated determinations of the 17 - ketosteroid output in a large number of individuals over a period of m%ny years , until , in fact , the subjects did or did not develop prostatic cancer . 
the colorimetric measurements - were made on a hilger spekker photoelectric absorptiometer using a green ( ilford spectrum 604 ) and a violet ( ilford spectrum 601 ) light filter . 
1 clearly shows the distribution of excretion of 17 - ketosteroids with age . the output in childhood is low , rising rapidly at or about the age of puberty and reaching a maximum in middle age . 
2 , in which are plotted the as men between 30 and 40 . mean excretions for the third to the eighth decade , together with the upper and the ranges are computed lower limits of the ranges encountered at those ages . as the mean values + 2a . the fact that men continue to show comparatively wide individual variations in their outputs of 1 7 - ketosteroids and hence , probably , in the degree of androgenic stimulation to which their prostates are subjected , until very advanced age , must obviously be taken into account in any consideration of the aetiology of prostatic it will clearly be of importance to know whether known sufferers from cancer . this disease are characterized by excretions at the upper or lower ranges of their appropriate age - groups , or whether they are not significantly distinguishable a . 
clemmesen. from the danish cancer registry under the national anti - cancer league , copenhagen . received for publication december 10 , 1949 . developments in recent decades have made the term " cancer statistics " inadequate for the description of the ways in which statistics have contributed it is time that this term was reserved for statistical treatment to cancer research . of results , while statistical research into the occurrence of human cancer , be it from racial , genetical , geographical , occupational or other sociological points of view , should be included in the term " cancer demography . " this will be the case in the following . unlike many other branches of science joining in the research of cancer at some stage of its development , cancer sociology can point to a long , though most often neglected , history . 
when percivall pott , in 1775 , described the chimney sweep 's cancer , not only giving its clinical features , its treatment , and its social aetiology , but also taking active steps toward its prevention , in which we have finally succeeded , it was the first step in cancer sociology . pott 's observation of the latent period of scrotal cancer in chimney sweeps contained a very obvious clue to the experimental production of cancer , but experimental workers seem to have been too fascinated by experimental and pathological problems to take a lesson , not to speak of inspiration , from clinical or sociological observations , and 140 years passed before cancer was provoked experimentally by tar and soot . however , it seeins still more surprising that after this experience we have spent decades since young and russell 's demonstration in 1927 of a causal relationship between alcoholic consumption and cancer of the upper digestive tract in examining the carcinogenicity of numerous other compounds , while alcohol has been examnined only in a single experiment on a small scale ( krebs , 1928 )  . similarly it is well known that the first birth increases the possibility of a later cervical cancer , but where are experiments based on this and similar facts ? it would not seem unlikely that the slow progress in obtaining results of practical value in the fight against cancer is to a large extent due to our theoretical way of tackling problems . for instance , it is not unlikely that the future will reveal very interesting facts with regard to the influence of cosmic radiation on the incidence of cancer in heavily inbred animals of some kind , but there is considerable evidence that unless the energy released by such radiation shows a j . 
clemmesen different affinity to individuals of the various social classes , there must be social factors of a far more decisive character influencing the incidence of cancer in man . in order to bring about a closer contact between cancer research and problems of clinical importance , a more thorough investigation into the aetiology of cancer should be attempted by cancer demography through mass observations . 
to this we may add a frequent tendency among the former to take a more national and less universal point of view than is common so far in the medical world . 
an example of this is the variation in the proportion of gastric cancer in different countries , which has been discussed more often than the possible sources of error involved . for instance a first - class textbook of pathology states : " the incidence of gastric cancer in sweden and czechoslovakia apparently is about three times that of england . 
no satisfactory explanation has been offered for these interesting findings " ( anderson , 1948 ) as far as the present author has been able to establish , the reason for some of the differences may be that death certificates in the thinly populated areas of northern sweden for practical reasons are issued by parsons . furthermore , it will appear from the demands already mentioned that a direct comparison of percentages without a further analysis with regard to age distribution is unsatisfactory , especially in the case of a site such as the stomach , in which the establishment of a definite diagnosis is difficult . admittedly , death certificates have in the past rendered valuable information , especially in the hands of the english registrar - general , but this is no guarantee that death certificates from any country will produce similarly reliable details . it is vital to the use of death certificates that they are referred to the domicile of the patient , and not to the locality where death has occurred . 
and it must also be remembered that occupational statistics based on death certificates theoretically demand that the occupation of the deceased person should be stated from the same source as in the census , that is , some registration office , and not from the case - record or the statement of relatives , although tlis might not cause serious errors . comparisons of death certificates of different date are often made without the necessary regard to the dangers of this method . pedigrees of greater length may for instance be misleading because the frequency of cancers of various sites , whether apparent or real , will differ from one generation to the next . for sites of cancer where therapy is improving death certificates are decreasing in value as indicators of frequency , while other sites may show an apparent increase in frequency due to progress in diagnostic technique , and post - mortem figures will also be subject to this influence . 
heady and kennaway ( 1949 ) suggest that the proportional occurrence of lung cancer among the post - mortem total will serve as an indicator of changes in the incidence among the whole population . such figures are , however , influenced not only by the experience and information available to pathologists , but also through progress in the therapy of early complicating pneumonias and of lung cancer itself . * it is generally assumed that progress in diagnostic technique will cause an increase in the number of cancer cases registered , but according to the author 's experience gastric cancer tends to be overestimated , and will often decrease in frequency with improvements in medical facilities . consequently , comparisons between different countries or periods based on death certificates as well as on other sources of medical information demand additional knowledge of a more direct character . * readers of their review are recommended to compare the order of magnitude of the yearly totals criticized with those accepted as a basis for considerations , j . 
clemmesen for years hence , however , death certificates are bound to play an important part in demography of cancer , because they will be the best information available for the many cases not treated in hospital . 
even in the case of compulsory registration there will remain a number of notifications from general practitioners amounting in quality to little more than death certificates . it is also important to.the statistician to realize that the difference in quality of diagnosis between hospitals and general practice will cause a difference in the material , unless practically all cases are examined in hospitals , and this will be the case for only another difference between these groups will appear very few sites of cancer . in the age distribution curves , which must be based on the age at onset of the cancer , theoretically at the onset of symptomns . accurate statements on this item are unfortunately very difficult to obtain on death certificates , and for hospitals it is most practical to state the date of the first admission , which has the advantage of being an accurate reality . for all these reasons it will be evident that complete statistics on the incidence of cancer in a given area should contain figures for the number of cases treated in hospital for each site of cancer , and separate age distribution figures for this it is not uncommon for authors part of the material as well as for the total . dealing with a single site of cancer to leave out the total cancer incidence in the population concerned , but this is not permissible when we do not know the interrelations between cancers of different sites . 
the percentage of cases examined histologically or by autopsy should also be given for each site as an indicator of the basis of the diagnosis , and is particularly valuable in statistics giving figures for diagnoses of a histological character . 
at the moment such figures can only comprise a part of the cases really occurring , and it is most important to know the actual number of microscopical examinations from which they are collected , even in the case of a diagnosis such as " sarcoma , " which sometimes may be founded only on a macroscopical examination . unfortunately , many publications on cancer demography are inadequate with regard to statistics , and amount to little more than a summary of the author 's own conclusions , although they should contain all the basal figures for both the normal and the cancer populations , as well as the formulas applied , not only with regard to an independent judgment by contemporary workers , but also with a probably it is mostly view to retrospective work in the same field in future . editors and not authors who are to blame for such omissions , and the same applies to diagrams , which are as essential to such articles as are photomicrographs to histological or roentgenograms to radiological publications . cost will probably also be blamed for the fact that most of the valuable material on the occurrence of cancer is hidden away in statements from public health authorities . 
a unification of publications in this field , so that they could be collected in journals accessible to workers in experimental cancer research , would contribute far beyond its cost to the vital connection between experimental research and cancer demography . but it is most important in such publications that the author ascribes his findings to factors of some established certainty , and is reticent in advancing readers may find , even in new theories of his own on the aetiology of cancer . modern publications of some standing , that the well - established connection of alcoholic consumption with the incidence of certain cancers is paralleled by the influence of tinned or fried food . 
and storing at that temperature and after drying following freezing to from the frozen state ( gye , begg , mann and craigie , 1949 )  . in addition , mouse breast carcinoma which had been frozen to - 79 ' c . 
from these results it was concluded that since the physical treatments kired the normal embryo cells , they must also have killed the tumour cers , and that the tumours which arose from tho inoculations of treated m ' aterial must have been caused by a viius liberated from the dead cells . the claims of gye and his co - workers have been much criticized ; hirschberg and rusch ( 1950 ) , passey and dmochowski ( 1950 ) , passey , dmochowski , lasnitzki and millard ( 1950 ) and wamer and gostfing ( 1950 ) have all pointed out the great weakness of the assumption that since embryonic cells failed to grow after exposure these recent authors to low temperature , such treatment is lethal to ar cells . have each reviewed some of the ' extensive work reported in the last forty years , which shows that of both normal and neoplastic tissues in many species , some will and some will not survive exposure to extreme cold . in view of this great variation in response to cold shown by different tissues , it was felt that an investigation into the effects of freezing and drying upon a tumour affecting a non - mammalian host would be of value ; the rous no . 
i since it is a tumour from which a virus is easily fowl sarcoma was selected . separable , it was considered that the effects of cold and desiccation should be investigated upon the virus and the cers separately . that the virus was able to survive freezing and drying has been estabhshed for some time ( knox , 1939a ; hoffstadt and tripi , 1946 ; dmochowski , 1948 ; carr and harris , 1951 ) , and it has also been know - n to retain its tumour - producing activity after the ap ' plication of cold by repeated freezing and thawing ( cramer and foulds , 1930 ; selbie and mcintosh , 1939 )  . however , the response of the cells of the rous sarcoma to freezing and desiccation has not hitherto been ascertained . 
nakahara and yaoi ( 1930 ) found that rous sarcoma material which had been subjected to repeated freezing and thawing was less active when injected than similar untreated material , whidst selbie and mcintosh ( 1939 ) were able to prepare from rous tumour treated in this way filtrates with a greater tumour - producing activity than those made from untreated samples . selbie and mcintosh ( 1939 ) considered that repeated freezing and thawing broke up the cers and allowed a greater liberation of virus than would otherwise occur , and the method has been used for obtaining good yields of rous virus in various investigations ( knox , 1939b ; porard , 1939 )  . the work reported here consists ofexperiments upon the effects oftemperatures ' 70 ' c . 
they were between eight and nineteen weeks old when inoculated , apart from four exceptions which were slightly older , and belonged to three susceptible inbred lines developed at edinburgh , or crosses derived from thethe fines and crosses used were intensity , breeding , non - moult , breeding hen x intensity ' cock , and intensity hen x breeding cock , and have been described by greenwood , blyth and carr ( i948 ) ; both the age at which the birds were inoculated and the line or cross to which they belonged were determined by the exigencies of supply . preparation and treatment of tumour material . for each experiment a bird with a large actively growing sarcoma of anything from 15 to 27 days ' growth from the date of inoculation was selected as a source of tumour material and kired by wringing of the neck . 
when prepared , the disintegrated tumour was loaded into a record syringe for easy manipulation . where disintegrated tumour was to be subjected to cold or desiccation , the treatment was apphed as follows : freezing and storing . - amounts of i c.c. 
by starting the motor and setting the ampoule rapidly spinning on its longitudinal axis , a thin even film of disintegrated tumour was obtained on the inside of the bulb of the ampoule . 
the forceps w ' ere then released , causing a rapid rise of pressure within the apparatus ; this fell within five minutes to the original level , which was then maintained until the end of the period of drying , which lasted for 3 to 5 hours . 
epstein were separated off by centrifuging in an angle centrifuge at 3500 r.p.for 15 minutes , decanting the supernatant fluid obtained , and recentrifuging it aga ' in in a horizontal centrifuge at 6000 r.p.for a further 15 minutes . 
of saline and centrifuging in a horizontal centrifuge at 6000 r.p.for 15 minutes , resuspending the deposit in fresh saline , and performing the procedure either three ( experiment 1 ) or six - ( experiment 4 ) times . 
one sample of each preparation was mixed and incubated with an equal volume of saline and the other with an equal volume of serum ( diluted i in i 0 with sahne )  . 
the serum used ( e ) was from a fowl whose rous tumour had regressed and was one which had been shown to have strong anti - rous virus neutralizing powers in experiments 2 and 3 . that part of the experiment in which the virus and cer preparations were made from disintegrated tumour subjected to freezing and storing ( experiment 4c ) , samples of the preparations were mixed with an equal volume of pooled normal serum ( diluted 1 in io with saline ) , as well as with safine and neutrahzing serum e . all the mixtures were incubated in a water - bath at 37 ' c . 
in volume , and four inoculations of each preparation or dilution of a preparation were made . when inoculating preparations whose tumour - producing potencies were to be compared with one another , the injections of material from each preparation were made into separate groups of birds ; the scheme for the distribution of the injections of the various dilutions of each preparation amongst the birds in a group was the same in every case . 
the method of ' calculation used was that of reed and muench ( 1938 ) , and the figure obtained has been called by warner and gostling ( i950 ) the tpd 50 ; it is show - n in the tables comparison of this by its negative logarithm , designated the " tpd 50 index . " tpd 50 index of various preparations offers a comparison of their tumourproducing activities . strict aseptic technique was maintained throughout all the experimental procedures . all the experimental procedures were timed . 
where the virus suspension was made from disintegrated tumour which had been frozen and stored , the tumour - producing activity was in one case greater ( experiment id ) and in the other case less than that made from untreated material ( experiment 4d )  . it will be seen from table ii that washed cells of the rous sarcoma which had been exposed to any of the three physical treatments employed had less tumour322 m . 
on the other hand , in the case of washed cells which had been subjected to repeated freezing and thawing or freezing and storing ( experiments 4b and 4d ) , incubation with neutralizing serum , in contrast with incubation with saline or normal serum , totally deprived them of their tumour - producing activity . 
at the very least the centrifugation employed must be allowed to have separated off the overwhelming majority of the cefls , and the great tumour - producing activity remaining with the virus suspensions cannot be attributed to occasional cells left in them . accepting therefore that the virus suspensions were almost entirely if not wholly cell - free , the ability of the rous virus to retain some of its tumour - producing activity after exposure to cold has been demonstrated , thus confirming the work of cramer and foulds ( 1930 ) and selbie and mcintosh ( 1939 ) , who were able to prepare active filtrates from rous tumour material subjected to repeated confirmation has also been obtained of the ability of the freezing and thawing . virus to survive freezing and drp ' ng , which has been demonstrated by a number of previous investigators ( knox , 1939a ; hoffstadt and tripi , 1946 ; dmochowski , 1948 ; carr and harris , 1951 )  . 
as in the work of knox ( 1939a ) , so here the activity of virus preparations obtained from frozen and dried tumour material was considerably less than those made from untreated material ( table i , experiments ic and 4c )  . 
the contention , however , that preparations of rous virus made from repeatedly frozen and thawed sarcoma had a greater tumour - producing activity than those made from untreated material on account of the treatment breaking up.the tissue fragments and hberating the virus ( selbie and mcintosh , 1939 ) has not been borne out . 
the failure of the neutralizing ' serum to abolish the tumour - pro - ducing activity of washed cells which had been left untreated ( table iv , experiment 4a ) is considered to be evidence of the presence of live cells in the preparation . the slight reduction in the tumour - producing activity of the untreated washed cells which was brought about by incubation with neutralizing serum is considered to be due to the action of the serum on free virus liberated from or attached to cells damaged during the disintegration of the tumour material . the washed tumour cells subjected to freezing and drying were deprived of most of their tumour - producing activity after incubation with neutralizing serum ( table iv , experiment 4c ) ; out of the inoculations made with all the various dilutions of the frozen and dried washed cells , only one gave rise to a tumour . this was from one of the inocula of the 10 - 1 dilution of the mixture of cells and antiserunow , it has long been known that viruses can be recovered from neutral mixtures with antiserum simply by dilution of the mixture with sahne ; such reactivation has been demonstrated with vaccinia ( andrewes , 1928 ) and fowl plague ( todd , 1928 ) , and although this " dilution phenomenon " is harder to obtain in the case of the rous virus , andrewes ( 1932 ) was able to show it conclusively . 
japha. received for publication february 2 , 1949 . martn and reese ( 1936 , 1942 , 1945 ) were the first to describe an extensive series of cases of retinal glioma treated by deep x - ray therapy . in their latest paper they report 75 per cent 5 - year cures in 8 cases of bilateral disease , one eye having previously been removed by surgery . 
at the middlesex hospital 19 cases of glioma retinae were seen between 1929 and january , 1946 . all cases were treated , 4 by surgery alone ; in the others radiotherapy of one type or another played a major part in the treatment . 
the numbers are too small to allow comparisons to be made between different types of treatment , but as regards deep x - ray therapy certain conclusions have emerged , and a technique has been evolved which differs in some respects from that advocated by the workers of the new york memorial hospital . the results of surgery have greatly improved in the last 30 years according to ray and mcclean ( 1943 )  . collecting their figures from the literature they estimate that between 1868 and 1941 , 18 per cent cures were obtained by enucleation in 677 cases , whereas 50 per cent cures are claimed for the 105 cases so treated between 1911 and 1941 . this progress is ascribed to the more radical and improved technique , often with partial excision of the optic nerve , used in later years . 
the number of bilateral cases included , if any , is , however , not stated . the authors themselves advocate a combined intracranial and orbital operation in view of reese 's ( 1931 ) statement that 52 per cent of 119 cases showed invasion of the optic nerve beyond the lamina cribrosa . duke - elder ( 1940 ) also states that 50 - 57 per cent of unilateral cases may expect to be cured . reese ( 1940 ) cites statistics of 95 cases collected by the division of ophthalmic pathology of the american registry at the army medical museum , 26 of which had bilateral disease . 
at the time of review 21 cases were untraced , 39 had died , 13 had lived 5 years , 2 had lived 3 - 4 years , 10 between 1 and 3 years , and another 10 just 1 year . besides x - ray therapy , which is historically reviewed by martin and reese ( 1936 , 1942 , 1945 ) , attempts have been made at treatment of the second eye m bilateral cases by radon - seeds . these are usually introduced either into the growth itself or sown on to the sclera over the neoplasm . stalhard , in his gifford edmond prize essay ( 1933 ) , reports 5 cases treated primarily by these methods . one of his cases - first reported on by moore ( 1930 , 1935 ) , was treated in 1929 ( twice by the insertion of a seed into the growth and also by episcleral seeds ) , is still alive , but has a cataract . three other cases , including an advanced case treated only experimentally and intended for subsequent histological investigation , came to excision . in a further report stallard ( 1948 ) gives the result of 15 cases so treated . 
deep x - ray therapy . - as long as radium moulds were being used it was inevitable that the whole of the globe was being irradiated to a high dose , but with the change - over to and use of small beams that were developed in deep x - ray therapy it became possible to limit the volume ofhigh dose to a certain extent . 
our technique has been directed at eradicating the focus of disease present rather than treating the whole of the posterior half of the retina ( where the majority of the growths are situated )  . 
a recent case , however ( not included in this survey ) , showed us that a certain risk isrun thereby in that shortly after the treatment and subsequent disappearance of a small lesion below the equator by fields of 2 and 3 cdiameter a further nodule appeared in the upper half of the retina . in an endeavour to treat as small a part of the eye as possible we have used circular fields of 2 and 3 cdiameter by means of special tubular applicators primarily intended for intrabuccal lesions as described by roberts and quick these are used in connection with our deep x - ray tubes , treatment ( 1943 )  . being carried out at 190 - 220 kv . 
lateral ( temporal ) , medial ( tansnasal ) , superior and inferior orbitals . abandoned as soon as it became apparent that the cornea was the most vulnerable structure of the eye , and disintegration of the eye had occurred in 2 or 3 cases in which this approach had been used . the lens , too , will receive a relatively large dose of irradiation when the anterior field is used and the lens dose should be kept at aminimuwe endeavour to ensure this by our technique of " by - passing " the lens as much as possible , which can be achieved in all the other fields mentioned . the anterior field was fields used so that accuracy ofalignment positioning . - special problems ofset - up arise because of the age of the patients and the small is of much greater importance . most of the children under the age of 4 need steadying during each treatment , and the older ones atleast during the first 2 or 3 sessions . we have in a few cases tried to make individual plaster casts into which the child is put during treatment , the cast including a headpiece with a frontal process to obviate any possibility of head movement . in practice , however , even the smallestchildren have been found to wriggle around in these casts human hands have had to help . the casts are usefuil , nevertheless , for steadying at least the trunk , and one escapes the otherwise necessary manoeuvre of binding the child on to the couch . children this can be done best by first wrapping the child tightly into a blanket , arms included . all cases - - and at each treatment - are set up by a medical officer , and during treatment , if necessary , the head steadied either by himself , by a radiographer , or by a nurse - or , if available , one of the parents . 
and by treatments lasting 16 or 57 days , but the number of cases observed is too small to confirm the impression that neither dose rate nor the total time are of critical importance within a wide range . 
irrespective of whether radium - moulds or deep x - rays were used , including one case in whom radium - moulds of increasing strengths were applied at short intervals within 5 months , the highest single dose being 3800 r . two main treatment schemes have been used : either a single " radical " course or repeated courses of deep x - rays , in each one of which a dose was given just sufficient to make the tumour disappear . this was usually achieved by doses of 2000 - 3000 r . , to be repeated as soon as there was renewed activity . the latter scheme is somewhat similar to that recommended by martin and reese ( 1936 , 1942 , 1945 ) , but in our hands it has never been successful , whereas the single radical course of treatment has given us outright cures . we have only once had the opportunity of treating a unilateral case ab initio . this was a girl , aged 3 years and 9 months , who came with a history of over 2 years but still only a comparatively small growth . 
ten months later , however , there was a large recurrence and the eye was excised . histology confirmed the presence of active growth . sometimes it has been necessary to continue the first course up to a much higher tumour dose . there was one case in our series where even the second eye carried a growth covering almost the whole of the retina - he had come to us with simultaneous affection of both eyes . after the excision of the first eye , which was completely filled by growth , a tumour dose of 5730 r . 
japha in this part of the body as anywhere else - but perhaps even with greater force - is one struck by the confirmation of the hypothesis most prevalent for the explanation of failures through over - dosage . 
we have not as yet given postoperative treatment to the orbit of the removed eye in bilateral cases . this may be advisable as a general policy in view of the ultimate fate of some of them , i.e. 
local recurrence even where no apparent extra - ocular extension was originally present , unless one regards these deposits as due to retrograde extension from the second eye , which by that time has got completely out of control . 
one should also remember , as duke - elder ( 1940 ) points out , citing meighan and michaelson ( 1938 ) , that there may be discontinuous spread in the nerve , so that the excised specimen ( even if part of the nerve is included ) need not necessarily show the extra - ocular extension which has in fact already taken place . carrying out this suggestion the contribution that the lateral fields will make to the retained retina or the enucleated orbit respectively and which will amount to a depth dose of about 20 per cent must be taken into account . this technique has been used in a recent case which had a recurrence in the excised orbit as well as growth in the second eye , and is also applicable in the " prophylactic " irradiation of a case in which bilateral enucleation has been performed . when we first collected these cases it appeared as if the unilateral cases treated post - operatively by irradiation would not be of very great interest because surgery alone might have been responsible for the good results . 
we have , however , in our series one case of recurrence within 6 / 12 of the operation ( this was treated by a combination of deep x - rays and implantation of radon - seeds , but recurred again and proved rapidly fatal ) , and the above - mentioned case , which may be regarded as having been inadequately treated in the post - operative period . this case had a remarkably long latent period of 5 years , and a very large dose of irradiation was necessary to arrest the growth . 
no growth was found on histological examination . one case of massive recurrence in the orbit with bone involvement after enucleation of the only affected eye failed to respond to combined irradiation by deep x - rays and implantation of radon needles ; another case , successfually treated , is of considerable interest . it is the case already mentioned which received a post - operative prophylactic dose of 660 r . 
by deep x - rays in 16 days for a recurrence after treatment by radon - seeds elsewhere ( tumnour dose estimated at 2000 r . ) , was complicated by chronic iritis and needed extraction of this the remaining eye 4 months later ( now 52 years ago ) - no growth was found on histological examination , as already mentioned . 
 ( in 57 days ) ; this was extracted 4 years ago and vision is only partial another cataract that was removed was obvious already 9 months after now . a dose of 3000 r . 
hfirst came under observation at the middlesex hospital in july , 1945 , at the age of 9 / 12 , having had his left eye excised elsewhere 3 months before ( histology showed retinoblastoma without involvement of the optic nerve ) , and at the same time a focus the size of a disc in the periphery of the right retina had been isolated by diathermy barrage . 
about a fortnight after this examination the boy began to vomit ( apparently in a rather projectile manner ) and became drowsy . lvhen examined again on 20.xi.46 there were signs of brain - stem involvement , and a course of deep x - rays directed there was some temporary improvement , but the at this region was started . child died within 3 months of the onset of the final stage . 
lumbar puncture during this time showed the presence of peculiar mononuclear cells in the cerebrospinal fluid , which in the light of the later post - mortemn findigs must be regarded as retinoblasts . 
m - 1 , a boy , 5 years of age , was sent to the middlesex hospital in 1929 from jersey , where 5 months previously his right eye had been removed for glioma retinae . 
a radium mould was applied as a " delayed post - operative measure , " but the surface dose when recalculated recently was found to have been only 660 r . 
 ( in 36 hours )  . nearly 5 years later there was a large swelling in the orbit whiceh was treated by x - rays in jersey ( details unknown ) and the swelling disappeared . 
a few months later a further course of x - rays was given for a " small hard bean - shaped swelling " in the lower half of the orbit . seven months after this the mass was again thought to be larger and the boy referred back to the middlesex hospital in september , 1936 . 
the boy was lost sight of during the war , but reported again as a young man in october , 1946 , that is , 10 years later , when he presented with marked telangiectases on the right cheek around the orbit and a small sinus over the zygomatic arch . 
the results of radiotherapy are summarized in table vii . ( a ) post - operative irradiation has been successful in the two cases where adequate treatment was given irrespective of whether a radium mould or deep x - rays were applied . in the one case described at length above , the first treatment , where 660 r . 
only were given , must be regarded as quite inadequate , but the recurrence which developed 5 years later responded well to a radical course . ( b ) primary irradiation . - the two cases treated by radium moulds and two of the three cases treated by deep x - rays more than 5 years ago are alive and e . 
cotton treated in a colloid mill . this was prepared from cotton velvet shearings suspended in water and passed through a colloid mill 6 times in succession . after each passage only the finer fraction was slected for further milling . 
5 shows the compact growth of mouse mammary carcinoma obtained in the presence of fibres of colloid milled cotton ; as a contrast the uneven growth of cells in the absence of fibres is shown in fig . 
for 5 minutes , washed with distilled water until free from ammonium ion ( tested by nessler 's reagent ) and sulphide ion ( tested by sodium nitro - prusside )  . they were then bleached 15 minutes at room temperature in sodium hypochlorite solution containing 0 - 1 per cent available chlorine , and finally washed with distilled water until free from chloride ion ( tested by silver nitrate )  . 
they were stored under distilled water . the films were autoclaved in distilled water at 18 lb . / square inch pressure , and before use were rinsed in sterile tyrode solution , excess moisture being removed with sterile filter - paper . cultures were placed directly on the coverslip , a drop of mixed serum and embryonic extract spread over them and the viscose film then applied . 
the culture usually adhered to the glass surface , and on washing or fixation the film floated off . the film is easily cut with a cataract knife when the tissue is subcultured . with a thickness of 30 , u the total thickness of the preparation was considerably less than can be achieved with the double coverslip method of maximow . 
they have found that perforated cellophane is an advantageous addition to flask cultures and hanging - drop preparations ( evans and earle , 1947 ; earle and evans , 1949 ; schilling , earle and evans , 1950 ; earle , evans and schilling , 1950 )  . in our hands perforated cellophane , kindly supplied by w . 
earle , was very siinilar in effect to viscose , but the outgrowth obtained with viscose seemed slightly greater . glass wool has been used for the same purpose by warner , hanawalt and bischoff ( 1949 ) , who used it with the roller tube method . 
viscose ifim , colloid milled cotton and durafil were technicauy suitable for use , and have been found advantageous in improvilg growth of malignant cells and of enabling growth of normal and malignant cells to take place in fluid media . we wish to thank professor astbury of leeds for his advice , and t . 
dmochowski. from the department of experimental pathology and cancer research , medical school , university of leeds . received for publication december 15 , 1952 . the test mice most commonly used for ascertaining the presence of the mammary tumour agent are susceptible hybrid female mice obtained by mating low - breast - cancer - strain females to high - breast - cancer - strain males . in these test mice a low incidence of spontaneous mammary tumours was frequently recorded ( bittner , 1939b ; murray and little , 1939 ; andervont , 1940 ; gardner , 1941 ; dmochowski , 1944a ) , and was frequently found to be higher than that of their maternal parents ( andervont , 1945a )  . 
of even greater interest was the high tumour incidence reported in the progeny of susceptible low - cancer - strain females mated to high - cancer - strain males which had been subjected to forced breeding - that is , bearing a number of litters in quick succession ( andervont , 1945b )  . 
a similar high incidence of breast cancer was also observed in forciblybred hybrid progeny of low - cancer - strain females of low susceptibility to breast tumours mated to high - cancer - strain males ( bagg and jacksen , 1937 ; strong , 1943 )  . attempts to discover the mammary tumour agent in mammary tumours of some of these hybrids proved unsuccessful ( andervont , 1945b )  . 
a subline of c57 black strain is now established in which this subline a number of mammary tumours has been recorded by the author . comes from one of the original threelitters brought from the laboratories of the the progeny of these litters has been kept imperial cancer research fund . separately , and no breast tumours have so far been recorded in the descendants the description of the subline in which tumours have of the other two litters . the riii strain mice appeared will be the subject of a separate communication . were also obtained from the same source and maintained since 1946 . the tumour incidence of 83 per cent at an average age of 8x5 months in breeding females ( dmochowski and gye , 1943 ) has with small variations remained approximately it was known that mice of high - cancer strains derived from the fourth the same . or later litters develop a higher incidence of breast cancer than those from the first two litters ( bittner , 1942 ) , and that the appearance of an active agent in mice deprived of it by foster nursing had been recorded in the progeny obtained from hybrid progeny of the c57 black strain the third or later litters ( bittner , 1943 )  . females was therefore taken only from the fourth or laterlitters for the experiment , mice from earlierlitters being used for various other purposes . 
the ( c57 x riii ) fl hybrid females which were litter mates were divided into two groups . only two hybrid females were taken from each of the c57 females mated to riii strain males , if possible from the same or foliowing litters . whenever two females were not available from the same litter , the mice from later litters were included in this way two groups , each comprising thirty ( c57 x in the control group . riii ) f1 hybrid females , were obtained . mice of the control group were allowed to breed in a normal way and mice of the experimental group were forcibly bred by removing their litters as soon as they were born . mice of one of the later litters from fifteen hybrid females in this group and of one litter of a female from the these mice in turn , control group were saved for brother to sister matings . after saving one litter , were forcibly bred and this procedure was followed for several generations . 
the progeny of six ( c57 x riii ) f1 hybrid females , which had been subjected to forced breeding , was maintained for six generations and the progeny of nine additional ( c57 x riii ) f1 hybrids , treated in a similar manner , for only three generations . 
dmochowski tumour which on histological examination was found to be a lymphosarcoma . the average number of litters bom to these hybrid females was three , and varied from two to seven . 
the number of breast tumours which developed in their litter mate ( c57 x riii ) f1 hybrid females , which had been forcibly bred , is shown in table ii . 
among these hybrids , the tumorous females had an average of 6 * 5 litters and the tumour - free females 6 - 4 litters . there was no difference therefore in the number of litters born to these two types of females which had been forcibly bred . none of the c57 black strain mothers , mated to riii strain males and bred in a normal way , developed mammary cancer , although they lived to an average twelve c57 black strain females , which were litter - mates age of 19 mouths . of some of the c57 females mated to riii strain males , were forcibly bred with their own litter - mate males , and none developed breast cancer , although they had an average of 5 * 2 litters and lived to an average age of 18 months . 
the observation that none of the c57 strain females developed mammary tumours after mating to c57 strain males and forced breeding points against the possibility of c57 black strain females harbouring a weak or attenuated mammary tumour it is now well known that various sublines of c57 black strain differ agent . considerably in their susceptibility to the agent . c57 strain mice of the subline of the present experiments , when foster nursed by riii strain females , developed an incidence of mammary cancer of only 11 per cent ( dmochowski , 1948 )  . this low susceptibility was probably responsible for the failure to induce mammary cancer in mature c57 mice of this subline even by a combined action of large doses of material containing the agent and forced breeding ( dmochowski , 1948 )  . again this low susceptibility may also have been responsible for c57 mice of this subline remaining tumour - free after mating to riii high - cancer - strain males as well as for their hybrid progeny , bred in a normal way , not developing mammary cancer . 
a further indication against the possibility of c57 black mice of this subline harbouring a weak agent or in small quantities was provided by the absence of the agent in mammary tumours induced in both virgin and breeding c57 females by treatment with methyloholanthrene ( dmochowski and orr , 1949 )  . 
the forcibly - bred progeny of four ( c57 x rhij ) f1 hybrid females which had died free from tumours , also continued in these figures , the number of for six generations , is presented in fig . 
3 to 6 that descendants of tumorous hybrid females . females can remain tumour - free for two , three or four successive generations before developing mammary cancer , and that descendants of a non - tumorous female , in spite of reaching tumour age , may show no mammary tumours for as many as five generations before developing breast cancer , in spite of inbreeding it may be of interest to point out that and intensive hormonal stimulation . andervont ( 1949a , 1949b ) found the agent to be transmitted through three or four successive generations of susceptible mice without inducing mammary further , the study of the distribution of mammary tumours in charts cancer . of the progeny of tumorous and non - tumorous hybrids , shown in fig . 
1 to 6 , indicates the possibility of the agent being involved in the development of these tumours . a comparison of the age of the earliest tumour appearance , tumour incidence , average tumour age and average age at death of the progeny of individual tumorous and four tumour - free ( c57 x rii1 ) f1 hybrid females is shown in table iii . as can be seen , there was little , if any , difference between the forcibly - bred progeny of the two ( c57 x riii ) f1 females which developed breast cancer and the forcibly - bred progeny of the four ( c57 x riii ) f1 hybrid females which died without tumours . table iv demonstrates the tumour incidence and tumour age in the forcibly - bred progeny of additional nine non - tumorous ( c57 x rii1 ) f1 females which was continued for three generations . 
the tumour incidence , although varied in the progeny of the individual hybrids , was lower and the age of breast tumour appearance higher than those in the progeny of the two tumorous and also of the four tumour - free ( c57 x r111 ) f1 hybrid females . 
number preceding letters t , n or l = number of female ; number following letters t , n or l = tumour age or life span in days . of the earliest tumour appearance , the appearance of mammary tumours according to months , the tumour incidence , and the average tumour age in six generations of combined forcibly - bred progeny of the thirteen non - tumorous ( c57 x riii ) f1 hybrid females . 
dmochowski generation of the progeny of this non - cancerous hybrid female may be seen in this figure as well as the consecutive number of litter to which the mice belonged which developed tumours . it would have been of interest to obtain the first litter of this female and its progeny and compare any mammary tumour incidence in this progeny with that of the progeny obtained from the sixth litter , but this could not be done owing to limitation of space . table vii shows the age of earliest tumour appearance , the tumour incidence , and the average tumour age in the normally - bred progeny of this non - tumorous ( c57 x riii ) f1 hybrid female . 
the breast tumour incidence in the forcibly - bred progeny of the non - cancerous ( c57 x riii ) f1 hybrid females was lower and the average tumour age considerably higher than those in the forcibly - bred progeny of cancerous ( c57 x riii ) f1 hybrid females , but this difference in tumour incidence and average tumour age gradually became smaller in successive generations of the progeny . 
the age of the earliest tumour appearance and the average tumour age in the progeny bred in a normal way of one cancer - free ( c57 x riii ) f1 hybrid female was distinctly higher than those in the forcibly - bred progeny of both tumorous and tumour - free ( c57 x riii ) f1 hybrids . 
the tumour incidence in the normally - bred progeny was , with one exception , lower than that in the forcibly - bred progeny of tumorous hybrid females and , again with one exception , approximated to that in the forcibly - bred progeny of non - tumorous hybrid females . it should be pointed out that the results of the comparison of the behaviour of normally - bred progeny of one non - tumorous female with that of the forcibly - bred progeny of thirteen non - tumorous hybrid females may not be the same as those which would have been obtained should this comparison have been extended to similarly maintained progeny of a number of other cancer - free hybrids which had been bred in a normal way . this point may be seen from the observations on the forcibly - bred progeny of four nontumorous ( c57 x rii1 ) f1 hybrids compared with those on similarly treated progeny of two tumorous ( c57 x riii ) f1 hybrid females . 
the difference in mammary tumour incidence and average tumour age between these two groups of hybrid females became pronounced only after including the observations on the progeny of nine additional non - tumorous ( c57 x riii ) f1 hybrid females . is therefore possible that the results of the comparison of normally with forciblybred progeny of non - cancerous hybrids would have been different as in the case of the comparison of forcibly - bred progeny of non - tumorous with that of tumorous it is not known whether the results of foulds ' ( 1949 ) comparison of hybrids . forcibly - bred progeny from tumorous and non - tumorous hybrid females would l . 
dmochowski not have been different had the progeny of a greater number of non - tumorous hybrid females been included in his study . from the study of the distribution of cancerous females in the forcibly - bred progeny of tumorous and tumour - free ( c57 x riii ) f1 hybrid females and in the normally - bred progeny of a cancer - free hybrid , shown in fig . 
among the forcibly - bred progeny of the four tumour - free ( c57 x riii ) f1 hybrids , tumorous mice had an average of 7 3 ( 1 to 13 ) and the mice without tumours an average of 8 - 3 ( 1 to 16 ) litters . 
the behaviour of mice in the progeny obtained after the tumour appeared in their mother may greatly vary even in the case of hybrid females of the same origin , probably as a result of variations in the genetic make - up of individual females infiuencing the presence or transmission of the agent . in conolusion it may be stated that both the genetic constitution and hormonal stimulation play an important part in the development of mammary cancer in the hybrid mice which were the subject of the present study , and that the mammary tumour agent also appears to take a part in the origin of these tumours . biological tests for the presence of the mammary tumour agent . as soon as the first mammary tumours appeared in the hybrid mice , biological altogether twenty - three tests for the presence of the agent were carried out . of the mammary tumours were tested and the results are shown in table xiii . four mammary tumours which developed in ( c57 x riii ) f1 hybrid females , two showed the presence of the agent and two others failed to reveal the agent under the conditions of the test . 
at first it appears surprising that the tumour which developed at the age of 10 months failed to show the agent , as the time of storage in desiccated state was equal to that of another breast tumour which appeared in the same generation and which , although injected in smaller quantity , induced tumours in the test mice but in distinctly smaller incidence compared with that induced by other dried agent - harbouring tissues in previous it must be stressed experiments ( dmochowski , 1945a , 1945b , 1946 , 1948 , 1949c )  . that the method of desiccation of these two as well as of all the other tumours was the same , and the age of the test mice used was in all cases approximately the same . the only varying factors were the amount of tissue injected and the time of storage . 
the longest period of storage in the ice - chest was that of a negative tumour in a ( c57 x riii ) f , hybrid female and amounted to 8 months . this should not have made an appreciable difference in the test , as it was previously shown that agent - harbouring tissue induced breast canoer in test mice after two years of storage ( dmochowski , 1946 )  . 
from the results of the tests of the four tumours in the first generation of hybrid females it may be concluded that two of them possessed the agent , and the two other negative tumours either did not contain the agent or harboured only small quantities or a weak agent which could not be shown under the conditions of the test . 
2 , from the study of parent - offspring correlation there was a definite tendency towards breast cancer development in daughters of tumorous as well as non - tumorous mothers . 
9 female had ten litters and her sister had seven litters . it may well be that the considerable hormonal stimulation was responsible for the appearance of breast cancer in these females , and more likely that the agent took part in the development of these tumours but in quantities too small to be detected by the ( c57 x riii ) f3 no . 
it may well be that had the test a : nimals , in which the latter tumour was tested , lived longer , the agent would probably have been detected . in the fourth generation of the progeny of no . 
20 ( c57 x riii ) f3 hybrid , were found to harbour the agent . the first tumour developed at the age of 8 months after the female had six litters , and the second at approximately 17 months after the female had thirteen litters . it is of interest that both tumours showed the agent , although the second developed comparatively late in life . in the fifth generation of the same line three tumours were tested , those of no . 
69 ( c57 x riii ) f1 tumorous hybrid female in whose tumour the agent was not detected , it may be seen how necessaiy it is to adopt a cautious attitude towards the results of biological tests . it appears probable that the agent may have increased gradually in quantity in successive generations of the descendants of this hybrid female , so that it eventually could be detected by the method applied in the present biological tests . it is possible that the treatment , such as desiccation , may have in part been responsible for the negative results when tumours with small quantities of the agent were tested . had the mammary tumour of no . 
69 ( c57 x riii ) f1 hybrid only been tested and the progeny of this female not raised , the conclusion would have been that increased hormonal stimulation of a susceptible genetic substrate was responsible for the appearance of this breast tumour . it may well be stated that hormonal and genetic factors combined with the mammary tumour agent were responsible for the development of the majority of the tested breast tumours in this line , and probably also a weak or attenuated agent in the tumours negative in the biological test . in the progeny of non - tumorous ( c57 x riii ) f1 no . 
33 female at the age of 17 months after she had eleven litters and one which arose in ( c57 x riii ) f4 no . 39 hybrid at the age of 10 - 5 months after she had given birth to six litters . 
21 , 47 , and 49 ( c57 x riii ) f3 hybrids , all of which developed after the eighth litter at the age of 9 , 12 , and 10 months respectively . all four tumours harboured the agent , yet , although they all arose comparatively early in life , the incidence of tumours induced in the test mice by the tumour of no . 
tumours developed in hybrid mice of all three coat colours without particular connection with any coat colour . foulds ( 1949 ) recorded a similar observation in his c57 x riii hybrids . neither the presence or absence of the agent , as shown in biological tests , was connected with any particular coat colour . foulds ( 1949 ) detected the agent in the tumour of a ( 057 x riii ) f1 hybrid muhlbock ( 1952 ) failed to discover the agent in tumours which had been tested . of five ( c57 x d ) hybrids which developed at ages varying from 20 to 30 months . these hybrids came from early litters . 
mammary tumours in hybrids from later litters of c57 females were not tested , but the presence of the agent in these tumours was assumed by muhlbock ( 1952 ) on the basis of a high tumour incidence in the litters , although the average tumour age was 18 months . 
a search for the agent in mammary tumours of hybrid progeny from another ( c ) low - cancer - strain but susceptible females and high - cancer - strain ( 03h ) males led to the detection of the agent in tumours which developed at an early age up to 12 months , although on two occasions even such tumours were negative ( andervont , 1945b ; andervont and dunn , 1950b ) , while mammary tumours arising at a late age , in spite of their high incidence , failed to reveal the agent ( andervont and dunn , 1949 , 1950b ; andervont , 1950a )  . yet , on one occasion a c strain female was found to harbour the agent , although she developed breast cancer at the age of 21 months ( andervont and dunn , 1950b )  . thus there may be exceptions in both early and late developing tumours , as shown in the present study with dried tumour tissue and in the experiments of andervont and dunn with fresh tumour tissues . biological tests of some tumours appearing at a late age , as shown in the present experiments , revealed the agent , although they failed in other . 
tumours of similar it is not known how far the recent observation of andervont ( 1950b ) late age . that small quantities of the agent may only be ascertained by observing at least one or even two generations of the descendants of the inoculated test mice would be helpful , but it is certainly worth trying in any future tests of mammary tumours appearing at a late age . in some mammary cancers which developed up to the age of 12 months , the agent could not be detected on several occasions in the present study . 
69 f1 hybrid were found to possess andervont ( 1945b , 1950a ) also failed to detect the agent in mice with the agent . tumours at 8 , 12 , 14 , 15 and 16 months . 
on the basis of the study of parentoffspring correlation shown in pedigree charts , it appears probable that the negative tumours also harboured the agent possibly in quantities too small to be detected by the tests applied . in conclusion it may be stated that the results of the biological tests for the presence of the agent in desiccated mammary tumours which developed in the hybrid mice indicate a gradual accumulation of factors responsible for the appearance of mammary cancer . it appears that there may be a certain threshold below which it is difficult or not possible to show the agent in some mammary tumours , at least by the methods so far employed , and only after combined influence of inbreeding and intensive hormonal stimulation the presence of the agent may become detectable . there is no reason to consider the biological tests as inadequate , in view of the previously reported constant positive results with dried agent - containing tissue , and also in view of the negative results occasionally recorded by other workers with fresh material even from tumours which developed early inlife . further , negative tests for the agent in dried tissue of an 86th transplant of a mammary tumour which originally harboured the agent ( dmochowski , 1952 ) have been confirmed with fresh tissue of a 101st serial passage of the same tumour . the negative findings may have been due either to small quantities of the agent present in some tumours or to the kind of relationship of the agent to other cell constituents in these tumours , such as close integration with these constituents , different from that in other tumours which were found positive in the biological tests . microscopical appearance of mammary tumours . in view of the availability of a large number of tumours of a comparatively uniform origin a microscopical study of these tumours was made , and also an attempt to correlate the morphological appearance of these tumours with the presence or absence of the agent . 
as can be seen the majority of tumours in this agentharbouring strain could be divided into roughly equal numbers of type a and type b tumours . the results of the study of 377 mammary tumours in 327 c57 x riii hybrid mice are shown in table xiv . 
among the 327 tumorous mice , 32 had two tumours and 9 had three tumours . as shown in table xiv , the majority of most of the tumours ( 70.6 per cent ) developed during tumours were of type b . the first 12 months , and among them 53 were of type a ( 19.7 per cent ) and 215 ( 79.9 per cent ) were type b tumours ; one unclassified mammary carcinoma also after the first 12 months , 21 tumours belonged to this group of early tumours . were of type a ( 19.4 per cent ) , 77 tumours belonged to type b ( 71.4 per cent ) , 9 tumours were of type d ( 8 - 3 per cent ) , and 1 tumour was unclassified . 
an analysis of the distribution of the different types of tumours in the progeny of tumorous and non - tumorous ( c57 x rii ) f1 hybrids revealed that the progeny of both types of females had a number of each type of tumour similar to that found in the total number of all tumours . 
among the 120 mammary tumours of the descendants of tumorous hybrid females , there were 19 ( 15.8 per cent ) of type a , 99 ( 82.5 per cent ) of type b , and 2 ( 1.7 per cent ) of type d . 
among the 225 tumours in the forcibly - bred progeny of non - tumorous hybrid females , 50 ( 22.2 per cent ) belonged to type a , 166 ( 73.8 per cent ) to type b , 7 ( 3.1 per cent ) to type d , and 2 ( 0.9 per cent ) were unclassified . there were 32 mammary tumours examined in the progeny of a non - tumorous hybrid female which had been bred in a normal way ; five ( 15 - 6 per cent ) were of type a and 27 ( 84.4 per cent ) belonged to type b . 
there was no difference in the distribution of types of tumours in the progeny of hybrids which had been subjected to hormonal stimulation of varying intensity . no correlation was observed between the type of tumour and its location or size or rate of growth or litter sequence of the tumour - bearing mice or age of the animal at which the tumour developed , except in the case of type d tumours , further , there was no all of which developed in mice older than 12 months . correlation between the types of tumours in litter mates or between tumours of mothers and offspring . similar observations were reported by andervont and dunn ( 1950b ) on ( c x c3h ) susceptible hybrids , in which they found 38 per cent of type a tumours , 44 per cent of type b , 9 per cent of type c , and 8 per cent of type d . 
they observed a correlation between the age at which mammary tumours appeared and the type encountered , as in mice of up to 19 months of age 63 per cent were type a tumours and 33 per cent type b tumours , and in older mice 31 per cent belonged to type a and 48 per cent to type b . in another study ( andervont , 1950a ) 38 per cent of tumours were of type a and 46 per cent type b , and in 106 l . 
they did not detect the agent in a c strain female with a squamous type mammary tumour at a late age , yet in her progeny there appeared a type a tumour with the agent and one squamous type cancer without the agent . another two females of the same strain , two tumours of type a and b respectively both harboured the agent ; in their progeny all tumours with the agent were of type a . 
the majority of tumours in old mice were similar to those of mice with the agent ( andervont , 1945b , andervont and dunn , 1949 )  . in hybrid mice obtained from crossings of agent - free low - cancer - strains the majority of mammary tumours were of types c and d , and in those obtained from one of the parents belonging to agent - free but high - cancer strain the majority of breast tumours were of types a and b ( andervont and dunn , 1948a )  . it appears that the genetic constitution of strains used for breeding hybrids influences at least the distribution of the various types of breast cancer . there was no correlation in c57 x riii hybrid females between the presence of the agent , as shown in biological tests , and the type of tumour encountered . of the seventeen tumours in which the agent was found , ten were of type b , six of type a , and one of type d , and among the mammary tumours in which the test failed to reveal the agent , four were type b and two type a , cancer ( table xiii )  . 
the finding of the agent in type d mammary tumour which developed at 17 months is of interest , as it appears to indicate that not all squamous type tumours which appear comparatively late need be agent - free tumours , or possibly tumours in which for some as yet unknown reason it is difficult to detect the agent . 
two agent - harbouring tumours of ( c57 x riii ) f1 hybrids were of type a ( 11 months ) and type b ( 16 months ) , and the two other tumours in which the agent was not detected were also of type a ( 10 months ) and type b ( 11 months )  . 
among the fifteen agent - harbouring tumours of c57 x riii hybrid progeny , five were type a and arose at the age of 9 to 13 months , nine were type b and developed at 7 to 17 months , and one type d at the age of 17 months . 
among the tumours in the hybrid progeny in which the agent was not detected , one was of type a ( 18 months ) and three of type b ( 8 to 10 months )  . in andervont and dunn 's experience ( 1950b ) the majority of tumours ( 84 per cent ) in hybrids developed at the age of 18 to 29 months and the rest during the first 17 months . 
they also found no correlation between the microscopical appearance of a tumour and the presence of the agent . some of the early appearing tumours in which the agent could not be detected were of type a or b ( andervont , 1945b , 1950a )  . foulds ( 1949 ) described the microscopical appearance of breast tumours in his c57 x riii hybrids as " unmiihlbock ( 1952 ) found the morphology of tumours in c57 x d remarkable "  . hybrids of little assistance in the study of the part played by the agent in the development of these tumours . 
he also noted more unusual features in tumours presumably free of the agent compared with those harbouring the agent , yet the tumours accumulated in later litters of c57 mice following mating to high - cancerstrain males , in which the agent was presumed to be present , had an appearance like that of agent - harbouring breast tumours , in spite of their late average age 108 l . 
dmochowski of development ( 20 months )  . there appears to be a greater variety in appearance of mammary tumours in mice of strains in which the agent could not be detected ( andervont and dunn , 1950a ; 1950b ; heston , deringer , dunn and levillain , in the present study all tumours of type d developed 1950 ; muhlbock , 1952 )  . after 12 months of age . similarly , andervont and dunn ( 1950b ) found this type of tumour to be very rare in younger hybrid mice in which the agent could be detected , although , as shown in this study , this type of tumour even at a late therefore the greater frequency of squamous age may harbour the agent . metaplasia in tumours of old hybrid mice , if the age of 17 months could be considered as a comparatively late age , need not indicate at least in every case their development without participation of the agent , contrary to the previous sugthere is no doubt , however , about the greater gestion of kirschbaum ( 1949 )  . frequency of squamous type tumours in agent - free high - cancer - strain or susceptible mice ( gardner , 1947 ; heston , 1948 ; heston , deringer , dunn and levillain , 1950 ) than in similar mice with the agent , but they do appear occasionally in agent - carrying strains of mice as observed by dunn ( 1945 ) , andervont and dunn ( 1950b ) , and in the present study . from these observations it may be concluded that no particular microscopical appearance of mammary cancer in hybrid mice can be correlated with the presence or absence of the agent , in spite of the difference in distribution of the particular types in various strains of mice . 
thus a variable but low tumour incidence was reported by andervont and dunn ( 1948a ) in hybrids from reciprocal crosses of low - cancer - strain ( c , i , c57 ) mice and a higher but still comparatively low tumour incidence in the hybrid progeny of low - cancer - strain ( c ) females and agent - free high - cancer - strain ( dba - ) males , in spite of increased hormonal stimulation . 
dmochowski showed a high incidence after mating to one ( c3h ) high - cancer - strain male developed a much lower incidence ( 30 per cent ) when mated to other ( a ) high - cancerstrain males . females of strains with low susceptibility may show similar differences . 
even after the 10th pregnancy of low - cancer - strain females mated to males with and without the agent , these two types of females when used as foster mothers induced a similar tumour incidence in the test mice . 
dmochowski tumour age ( 19 to 25 months ) in both types of hybrids is characteristic and hormonal stimulation was assumed to be the cause of these tumours ( muhlbock , 1952 )  . 
the development of mammary tumours in the descendants of both tumorous and the majority of non - tumorous hybrid progeny ofc57 females which remained tumour - free after mating to agent - carrying males in the present experiments as well as the detection of the agent in the majority of the tumours tested indicate the transfer of the agent by riii high - cancer - strain males . though not all ( 057 x riii ) f1 hybrids developed breast cancer , the transfer of similarly , the agent must have taken place in the majority of the females . bittner ( 1952a ) noted a high incidence of tumours and the presence of the agent in the descendants of cancerous and cancer - free hybrid progeny of certain strain females mated to agent - carrying males . 
the presence of the agent was assumed in mammary tumours which appeared in hybrid progeny of later litters of some c57 females mated to high - cancer - strain ( d ) males ( muhlbock , 1952 ) because of the high tumour incidence , in spite of the late age at which the tumours appeared . 
as a result of this transfer , only few tumours in the hybrid progeny develop early and harbour the agent , while the majority of transfer of the agent tumours develop late without participation of the agent . to embryos , however , appears to be doubtful , because of the absence of the agent in high - cancer - strain embryos ( dmochowski , 1949c ; hummel and little , 1949 )  . the passage of the agent from embryos , should they become infected in utero , is also questionable because of the reported neutralisation of the agent by placenta transmission of the agent in utero would ( hummel , little and eddy , 1949 )  . also contradict the basis of the discovery of the agent itself . 
the increase in tumour incidence in hybrid progeny of later litters ( andervont and dunn , 1949 ; bittner , 1952a ; muhlbock , 1952 ) and the high incidence of tumours observed in the descendants of later litters from hybrid females observed in the present experiments may indicate either a transfer of the agent to females repeatedly mated to agent - carrying males and / or gradual increase in the agent , transferred by mating , during its long latent period under the influence of hormonal stimulation ( repeated pregnancies )  . 
the low incidence of tumours observed in the hybrid progeny of some derivations need not necessarily be interpreted in the same way as the small number of tumours induced in mature mice which had been given the agent , as suggested by muhlbock ( 1952 )  . 
a high incidence of tumours obtained in mature mice of some strains after repeated injections of material containing the agent ( dmochowski , 1945a ; muhlbock , 1952 ) , and a small incidence in mature mice of other strains ( bittner , 1952b ) , indicate that the genetic constitution is the more likely explanation for the different tumour incidences in transmission of the agent by the sperm to lowhybrids of various derivations . cancer - strain females , followed by its transmission to their hybrid progeny in the mnilk of these females ( bittner , 1952a ) , appears to be the most likely way in which the agent gains access to hybrid females , although in females of some strains it may only occasionally take place ( miihlbock , 1952 )  . 
the observation of several generations of descendants of hybrid females can give a clear picture whether the transfer of the agent has taken place or not , as shown in the present study . 
under the same experimental conditions some hybrid females may develop mammary tumours while their litter - mates may fail to show tumours ; their descendants may show a similar variation in their behaviour . thus , the study of the behaviour of the hybrid progeny of low - cancer - strain females , especially of the descendants of later litters of the hybrid progeny , can only decide whether or not the agent has been transferred by the male parent . the observations on tumour development and the presence of the agent in 114 l . 
dmochowsk1 the descendants of the hybrid progeny obtained by mating c57 black straini females to agent - carrying riii strain males , made in the present experiments , appear to indicate an interesting possibility in the agent - host relationship which may be the outoome of gradual mcrease in the amount of the agent or its activation in some hybrid females , while in other hybrid females it appears in quantities large enough to be detected immediately with the development of breast cancer in the first generation of hybrid progeny . this may be on one hand the result of individual variations in the genetic constitution of hybrid females , and on the other hand the outcome of varying quantities of the agent which the hybrid females obtain from their low - cancer - strain mothers mated to high - cancer - strain within the framework of individual differences in the genetic make - up males . and / or quantities of the agent obtained , inbreeding combined with hormonal stimulation also exerts its influence . 
thus we have the situation , encountered in the present experiments , that under the same experimental conditions some hybrid females develop mammary cancer and others ( even their litter mates ) do not show breast cancer ; some of the tumorous females do and others do not reveal the agent ; some of the progeny of the cancerous hybrid females may develop mammary tumours which again may or may not reveal the agent , and other progeny of the same cancerous females do not develop tumours ; the progeny of non - cancerous hybrid females may develop tumours which contain the agent . 
the development of breast cancer in some of the ( c57 x riii ) f1 hybrids only after foroed breeding may be explained by the transmission of small quantities of the agent by at least some of the c57 black strain mothers after mating to riii agent - carrying males . 
the high , although varied , tumour incidence in the progefny of these hybrids may be explained by the origin of these females as well as of their progeny from late litters and therefore an increase or activation of the agent in these litters , enhanced by inbreeding and increased hormonal andervont and dunn ( 1950b ) also observed that under the same stimulation . conditions some low - cancer - strain females developed and others failed to develop breast cancer , and some females with tumours possessed and others lacked the there was also no correlation between the presenco of the agent in these agent . females and its presence in their hybrid progeny , cancerous females with or without the agent giving rise to hybrid progeny with only late tumours apparently without the agent , or to progeny with both early tumours with the agent and late tumours without the agent , although they were litter mates . therefore , again , the presence or absence of the tumour and / or the agent in the mother did not necessarily involve the presence or absence of the agent in her progeny . andervont and dunn ( 1950b ) noted on several occasions among the hybrid progeny with late tumours that their litter mates had early tumours with the agent , and on one occasion a hybrid female with an early tumour harbouring the agent had progeny with either no tumours or only late tumours without the agent , which suggested the disappearance of the agent ( andervont and dunn , 1949 )  . bittner ( 1952a ) also reported on the hybrid progeny of some derivations with low mammary tumour incidence that some of them gave rise to descendants with a high incidence of tumours harbouring the agent . 
thus the variable results appear to be due to variations in the genetic make - up of both the hybrid females and their progeny , which in turn leads to variations in the amount of the agent transmitted . a more favourable genetic make - up may account for an increased amount of the agent and its detection . 
the appearance of mammary tumours in some low - cancer - strain females ( c3hb x ax ) mated to agent - free ( c3hb ) males and the high incidence of tumours in their descendants , on the basis of which the presence of the agent was assumed although the tumours were not tested biologically ( bittner , 1952a ) , is one at the moment rather perplexing sudden " de novo " appearance of the agent was therefore also observation . considered as a possible explanation of some of the findings ( andervont and dunn , 1949 ; bittner , 1952a )  . this consideration was based on previous observations of bittner ( 1941 ) on the sudden appearance of the agent in some susceptible ( ax ) mice , deprived of the agent by foster nursing by low - cancer - strain mothers , which remained free of the agent for seven generations , and on similar observations made recently by bittner ( 1952a ) on susceptible mice of another strain ( c3hb ) which remained free of the agent for sixteen generations following foster nursing before the appearance of breast cancer in one of the females , which then gave rise to progeny with a high incidence of mammary tumours . according to the writer 's opinion , these observations of a sudden appearance of the agent may only show the length of time during which the agent may lie dormant or the time required before the agent , originally present in small amounts , increases in quantity sufficient to induce breast cancer in combination with other known and unknown factors . of the known factors , either a change in the genetic constitution of the animal concerned alone and / or increased hormonal stimulus may , at least in part , be responsible for the activation of the agent . bittner ( 1939a ) reported the appearance of some mammary tumours in mice without the agent , and strong ( 1943 ) stressed the influence of hormonal factors in such tumours . 
the difficulties encountered in the detection of the agent in breast tumours appearing late in the life of mice and the comparative ease with which it could be shown in tumours developing early ( andervont , 1950a ) , further the isolated tumours in hybrid progeny of early litters and the accumulation of tumours in hybrid mice of late litters ( miihlbock , 1952 ) , led to the conclusion that there may be two types of mammary tumours in mice . 
one type of tumour would be the result of all three factors taking part in its development , the other type would be the result of hormonal and genetic factors without participation 116 l . 
dmochowski of the agent ( andervont , 1950a ; muhlbock , 1952 )  . however , the possibility of the agent being involved in both types of tumours was not discounted by andervont ( 1950a )  . 
the genetic constitution and intensive hormonal stimulation were considered adequate by heston , deringer , dunn and levillain ( 1950 ) to give rise to mammary tumours in susceptible ( c3hb ) mice , originally derived from mice deprived of the agent by foster nursing , but they also stressed that absolute proof of the absence of the agent from such tumours was lacking . 
the appearance of only a few tumours in the hybrid progeny from reciprocal matings of these ( c3hb ) mice with low - cancer - strain ( c57 ) mice with no evidence of an increase in tumour incidence in the hybrid progeny of later litters , as originally reported by bittner ( 1944 ) in mice of agent - carrying strains or in hybrid progeny from low - cancer - strain females and agent - carrying males , again led heston and deringer ( 1952 ) to suggest that some mammary tumours develop in the absence yet , in hybrid progeny of similar mice of other strains , bittner of the agent . ( 1952a ) observed the appearance of tumours harbouring the agent . further , the agent has been detected in some mammary tumours appearing in comparatively old hybrids , as shown by bittner ( 1952a ) and in the present study . in other late tumours as well as occasionally also in early - developing breast cancers the agent could not be detected by any , so far , employed testing procedures as noted by andervont ( 1950a ) , andervont and dunn ( 1950b ) , and by the writer . thus , intensive hormonal stimulation combined with a suitable genetic background influence the origin of mammary tumours in hybrid mice , increase their there is no doubt that the mammary incidence , and accelerate their appearance . tumour agent takes a part in the development of breast tumours appearing up to a certain age , although not all tumours of early appearance in hybrid mice reveal its presence . 
 ( c57 x ri11 ) f1 hybrid females , obtained by mating agent - free c57 black strain females to agent - harbouring riii high - cancer - strain males , developed a 14 per cent incidence of mammary tumours at an average age of 13 months after bearing in quick succession an average number of 6 - 4 litters . their litter - mates , bred in a normal way , died free of tumours after rearing an average number of 3 litters . 
analysis of parent - offspring correlation in the distribution of tumours in the descendants of the hybrids revealed that the progeny of tumours and of the majority of non - tumorous hybrids had a greater chance to develop cancer if the parent developed cancer than if the parent died without a tumour . 
thus there was a strong indication of the presence of the agent in these hybrids and of its transmission to their progeny , which showed an increasing number of tumours following the combined influence of inbreeding and intensive hormonal stimulation . 
biological tests for the presence of the agent in twenty - three mammary tumours were positive in seventeen and negative in the remainder of tumours . the age of tumours in which the agent was detected varied from 7 to 17 months , and that of tumours in which the agent could not be detected from 8 to 18 months . these tests , combined with the observed distribution of tumours in the descendants of hybrid mice , showed that under the same experimental conditions some hybrids developed tumours which either harboured the agent or failed to reveal it , while other hybrids , even their litter mates , died without tumours . 
some of the progeny of tumorous hybrids failed to develop tumours and others of the same females developed cancer in which again the agent was either demonstrated or could not be shown . 
dmochowski which as a rule developed in older mice above the age of 12 months . there was also no correlation between the appearance of mammary tumours in parents and their offspring . 
calcutt. from the department of cancer research , mount vernon hospital , and the radium institute , northwood , middlesex . received for publication april 16 , 194 ' the question of the involvement of the polycyclic hydrocarbons with sulphydryl groups during the process of carcinogenesis has been under discussion for a ceitain indirect evidence exists and has been reviewed by crabtree long time . ( 1947 ) , who concludes that the available evidence does not warrant any decision as to whether the involvement is direct or not . in other directions mueller and rusch ( 194t3 ) could obtain no effect upon the activity of urease - - this being dependent upon the integrity of the - sh groups - with benzpyrene solution . 
on the other hand rondoni and bassi ( 1948 ) have found benzpyrene to inhibit the papainaise digestion of gelatin and also the cathepsins of rat sarcoma and horse liver . 
the non - carcinogenic hydrocarbons , phenanthrene , anthracene and pyrene , had no action . calcutt and newhouse ( 1948 ) also found that the photodynamic action of benzpyrene was inhibited by cysteine hydrochloride . further work ( calcutt , 1948 ) showed that certain other - sh - containing compounds were also effective as inhibitors of photodynamic action . in the attempt to find the mechanism of this action it was found that after incubation of a colloidal suspension of benzpyrene in a solution of cysteine hydrochloride the characteristic yellow - green fluorescence colour of the benzpyrene changed to preliminary attempts to isolate this blue fluorescing compound were pale blue . unsuccessful , but it was found that the mixture no longer gave a positive test this implied the inhibition of the - sh group of the with sodium nitroprusside . cysteine and formed the starting - point for further work . the inhibition of - sh groups . using colloidal benzpyrene in distilled water , tests were carried out with a in each case the technique was the same . equal aniounts series of compounds . of a solution of the sulphydryl - containing compound were placed in each of two tubes . 
with slightly alkahne nlixtures , however , a small amount of blue fluorescing compound was obtained in solution . for all the cases mentioned above incubation has to be continued for upwards of 11 hours at a temperature of 65 ' c . 
before any inhibition became apparent . at lower temperatures the action was very slow , and at room temperature - 25 ' c . - there appeared to be no effect whatever . prelim ' mary trials having indicated that caffeine solutions had fittle effect upon - sh as detected by nitroprusside , some of the previous experiments were repeated , using a solution of benzpyrene in a 3 per cent caffeine solution . 
inhibition of - sh was achieved as before , but this time inuch more rapidly - 30 nlinutes at 650 c . isolation of the reaction products . under the conditions so far investigated the yield of the pale blue fluorescing product of the reactions has only been very small . nevertheless it has been found possible to isolate products from both the alkahne and acid reaction rmxtures . 
from the alkahne reaction raixture a water soluble compound was derived . this is soluble in organic reagents , strongly adsorb6d to silica , strongly adsorbed to alurnina , fluoresces a whitish - blue colour in ultra - violet light . 
on addition of dilute acid it changes to a blue fluorescing compound which is soluble in organic reagents insoluble in water but soluble in dilute alkali . from the acid reaction mixture a com ound has been separated wliich is soluble in organic reagents , soluble in dilute alka ' li , insoluble in water , adsorbed strongly to alumina and is weakly adsorbed to silica , moving down slowly as elution is continued . thus in their physical properties these compounds show a remarkable similarity to those separated from aninials and designated by weigert and mottrani ( 1946 ) as bpx . , , bpfj aind bpf21 this latter apparently being identical with the compound characterized by berenblum , crowfoot , holiday and schoental ( 1943 ) as 8 . 
furthermore the evidence suggests a considerable parallelism , even if not identity , with the course of events after the introduction of benzpyrene into the animal body . whilst the apparent identity of the fluorescence spectra does not - imply strict cheniical identity , it does mean the very close relationship of the substances concerned . from this it is reasonable to suggest that the interaction of benzpyrene with a - sh containing compound gives rise to a substance of the bpxj type . this , bowever , is in the presence of acid successively broken to compounds of bpfj and bpf2 type . it has already been suggested by crabtree ( 1947 ) that the detoxication of benzpyrene is by way of - sh compounds . in view of the evidence presented above this must now be regarded as being most probable . crabtree ( 1940 , 1944 , 1945 ) has as a result of his work with - sh inhibitors suggested the involvement of sulphur in carcinogenesis caused by the polycyclic hydrocarbons . this is further reinforced by the recent work of lusky , braun and woodard ( 1947 ) , who inhibited the carcinogenic action - of the benzpyrene by application of b.a.l. 
 ( 2 : 3 - dimercaptopropanol )  . additionally , evidence has been put forward by weigert , calcutt and powell ( 1947 ) to the effect that detoxication occurs at the site of careinogenesis . 
modem technique - 3 of microscopy , particularly phase - contrast , by greatly facilitating the study of fiving cells have resulted in greater use being made of tissue cultures for morphological studies . 
when cells migrate from an explant in vitro , they tend to spread out so that around the periphery of an outgrowth they are considerably flattened . this is most obvious in fluid culture media where the cells furthest away from the explant may be reduced to little such cells are ideal for the study of cytoplasmic structure , more than a fine film . as many investigators have realised . 
the necessity of rapid fixation , in order to obtain perfect preservation of chromosome structure has also resulted in the common practice of using squash preparations and smears for the study of nuclear strucsuch procedures possess the further merit of eliminating the possibihty tures . of slight morphological alterations being brought about by the prolonged dehydrating , clearing and imbedding necessary for the preparation of sections . nevertheless ah these methods have the common disadvantage of rendering difficult the correct appreciation of the spatial arrangement of intranuclear structures . in this respect the older method of studying stained sections of fixed tissues presents definite advantages . 
by cutting sections of approximately the same thickness as the average diameter of the cell nuclei , one obtains preparations with whole nuclei and shces of nuclei of varying thickness . 
yet with all fixed preparations of cers there arise doubts as to the validity of the finest structural details which are detectable , especially when they approach the limits of mic ' roscopic resolution . so the results of the study of fixed and stained cells have been correlated with observations made by other techniques , which have included the microscopical examination of living cers immediately after their removal from the animal body . structural organization of the nucleus seen in sections . the ideal type of cell with which to commence an investigation of this kind would be one with a large nucleus , single nucleolus and well - defined chromatin granules . 
most nearly fulfilling these requirements were the cells of a mouse adenocareinoma ( t27 )  . its selection was further influenced by the fact that it had been employed in several previous researches ( ludford , 1924 , 1930 , 1932 a , 1932 b , 1933 , 1934 ) so that the structure of its cells , and their behaviour under a variety of experimental conditions , as well as their growth characteristics , both in vivo and in vitro , were already well known . the same methods of fixation and staining were adopted as were formerly used for the study of chromosomes ( ludford , 1930 ) , except that after iron - alumhaematoxylin staining the differentiation was not carried so far as is usual in making chromos ' ome preparations . frozen sections stained by the feulgen technique were also found most useful . for demonstrating the smallest structural details it is doubtful whether any method can excel the results obtained by flemming fixation followed by intense staining with iron - alum - haematox.ylin when the surface of a nucleus from such a preparation is examined there are to be seen numerous minute granules varying in size . 
4 depicts a slice through the membranes in the process of section cutting . here are seen the same peripheral granules , and also the centre of a nucleus . strands connecting the two nucleoli with the membrane . 
3 , which is another slice from in this one , some well - defined strings of granules are the surface of a nucleus . discernible , while in the adjacent fig . 
the latter obviously represent an early stage in the formation of the prophase ch - romosomes . actually in a section of a rapidly growing tumour it is impo - ssible to determine ex ' actly when the prophase does . 
8 they appear as single threads , and granules are perceptible on some of them . it thus seems that the nucleoli are connected with the periphery of the nucleus by chromosomes , and also by threads of a different nature . frequently the latter appear to be attached distally to a chromosome . examination of prophases at the stage when the nuclear membrane is undergoing dissolution will sometime reveal chromosomes connected with remnants of nucleoli . 
13 represents part of a cell of the same carcinoma on the edge of a 2 - days - old culture , which had been fixed in flemming 's fluid , and intensely stained with iron - alum - haematoxylthe fine peripheral granules beneath the nuclear membrane which are shown in fig . 
16 and 17 , these are projection drawings ( indicated by " p " in the legends )  . there are included in the plane of each drawing bodies distributed throughout the whole thus each drawing constitutes a diagrammatic representation of the nucleus . structures as if they were projected on to the equatorial plane . 
this does not mean none is present , because extremely thin threads might only be visible when coated with dyestuff , and this may have beeil removed in the early stage of differentiating with the iron - aluthe nucleus seen in fig . 
each of the nucleoli is surrounded by particles which appear to have been emitted froin its surface . if observation is now extended to feulgen - stained cultures it becomes evident that the nucleolus is of a dual nature . 
the latter constitutes what caspersson ( 1947 ) has termed " the nucleolar associated chromat - in " and to which he attributes a special role in it can be distinguished from the material of which the bulk protein synthesis . of the nucleolus is composed in the living condition , and in fixed preparations after a variety of staining methods . 
on the basis of the reaction of the two nucleolar components to staining by the feulgen and the pyronine - methyl - green techniques it is concluded that the chromatin is composed of appreciable amounts of deoxyribose nucleotides , and the remainder of the nucleolus of ribose nucleotides . there is considerable variation in the amount of nucleolar chromatin in different cers , as is demonstrated by fig . 
20. in one form of cellular degeneration as has previously been mentioned , particles are discharged from the surface of the nucleolus . comparison of cells stained by the iron - alum - haematoxylin technique , such as that show - n in fig . 
the latter are stained by haematoxylin , but are invisible in feulgen preparations . there are also distinguishable in nucleoli varying numbers of argentophile bodies . page , regan and maccarty ( i 938 ) found that both types of inclusions were more numerous in malignant than in non - malignant cells , and that " the more ma - lignant the neoplasm , the greater the number of intra - nucleolar bodies . " examination of nucleoh with the electron microscope has suggested the existence of a more comphcated structure . 
the hmit of resolution attained with the ultra - violet this is the best resolution represented in microscope is only about 0 - 1 micron . the figures ' illustrating this paper . occasionaby when a nucleolus is highly vacuolated its appearance is rather deceptive , and might erroneously be interpreted as being reticulate . 
one wonders whether the coniphcated internal structure which has been described may be the result of a slight distortion induced by the procedures involved in preparing sections for examination by the electron microscope . 
the vacuolated appearance is not an artefact of fixation as it is visible in hving cells . distribution of nucleic acids in the nucleus . as a matter of convenience , for the purpose of description , it is proposed to refer to the material of which the nucleolus is mainly composed as " plasmosomin "  . 
the nucleolar chromatin contains sufficient deoxyribose nucleotides to stain intensely by the feulgen techiiique , while plasmosomin this does not ehminate the possibility that in the latter deoxyribose does not . nucleotides may be present in such small quantities as to be below the limit of sensitivity of the feulgen techn ' ique , upon the vahdity of which as a tes t for deoxyribonucleic acid this only definitely known chemical difference between nucleolar chromatin and plasmosomin depends . cytogeneticists find it convenient to use the terms " euchromatin " and their usage is based upon the conclusion that at the heterochromatin "  . end of mitosis as the chromosomes uncoil deoxyribonucleic acid remains attached at certain loci ( heterochromatin ) , and is discharged from the rest of since it has been impossible to see whether the chromosome ( euchromatin )  . this actually happens in the cells employed in this investigation no attempt has been made to distinguish between two types of chromatthe term " chromatin " is used in this paper to describe nuclear material which gives a positive reaction with the feulgen test , and which is not visibly connected with the nucleolus . whether one studies fixed and stained feulgen preparations , or ultra - violet micrographs of living cells , it becomes obvious that there must be an increase of deoxyribonucleic acid at prophase , and a corresponding decrease during the evidence has already been adduced that the reconstruction phase of mitosis . chromosomes are represented in the " resting " nucleus by the fine granules at its surface , and on strands connecting the nucleolus with the nuclear membrane . these granules appear to be faintly stained by the feulgen technique . 
but it is doubtful whether any definite conclusion can be justifiably drawn from pale staining , as it has been pointed out , most of these minute particles are just ifthey fail to stain that does above , or below , the liniits ofmicroscopic resolution . not necessarily mean that they contain no deoxyribonucleic acid . it might well be that the amounts present in such small particles would be undetectable by the techni ' que . also very faint staining could result from absorption of traces of diffuse dyestuff . 
the latter usually appears to be very faintly coloured in feulgen preparations , and the nuclear periphery is always most clearly defined there is by comparison an even in the absence of cytoplasmic counterstaining . intense staining of larger peripheral particles , and of others on the filaments connecting the nucleoli with one another and with the nuclear membrane . 
30 is an ultra - violet micrograph of some flattened coagulated nuclei from another mouse carcinoma ( af )  . it will be noticed that the superficial structure is very similar to that draw - n in fig . 
they have been regarded as the chromosomes , or fragments of chromosomes , of the interphase nucleus , but this interpretation of their nature has been disputed by lamb ( 1950 )  . it is highly probable that they are identical with the strands of material exhibiting strong absorption of ultra - violet radiations of 2570 a wave - length , which are seen in fig . 
the absorbing material presumably comprises the fine granules , which for reasons already stated are regarded as representing the chromosomes of the interphase probably their volume is augmented at the time of nuclear coagulation nucleus . as the result of the accretion of additionaldeoxyribonucleic acid from the nuclear sap , by a piocess analogous to that which occurs at the prophase of mitosis . relationship between nucleolar chromatin and plasmosomin . on the basis of the preceding considerations it is concluded that the highest concentration of deoxyribonucleic acid is in the nucleolar chromatin and ' bodies deoxyribonucleic acid is also present in the minute granules derived from it . which represent the interphase chromosomes and in the nuclear sap . considerable evidence has been adduced by other investigators to prove that the plasmosome is particularly rich in oxyribonucleic acid . the functional relationship between nucleolar chromatin and plasmosomin requires furtber investigation . 
the observations that are reported in this paper are consistent with two possibifities . either nucleolar chromatin and plasmosomin are built up from the same simple precursors , and there are altemate pathways of metabolism the 122 r . 
ludford course of which can be deflected so as to lead to the formation of either oiie or or , there are contained in plasmosomin enzynie systems the other substance . responsible for the synthesis of the substances comprising nucleolar chromatin from certain of its chemicary simpler constituents ; or perhaps for breaking dow - n plasmosomin and converting it into nucleolar chromatthe latter notion finds support from the behaviour of the nucleolus during the formation of the chromofig . 
36 and 37 are ultra - violet micrographs of hving prophase somes at prophase . nuclei of a sarcoma ( rb )  . in the former the surface of the nucleus is in focus and the peripheral chromosomes are seen in process of formation . in the latter the nucleoli in the interior of the nucleus are in focus . 
the darker nucleolar chromatin is distinguishable from the lighter plasmosomthe nucleoli are in process of disintegration , and there is a considerable production of nucleolar after the chromosomes are fully formed , small spherical masses of chromatin . plasmosomin are often distinguishable devoid of any chromatat this stage all the feulgen positive material has been deposited on the chromosomes . 
the fonner was photograpbed from a 3 - days - old primary culture of mammary carcinoma ( ad ) in a strain mice ; the latter from a 5 - days - old primary culture of mammary gland from a low - cancerstrain mouse ( c57 )  . 
38 exhibited the greatest degree of polymorphisnote also in this figure the derangement of mitosis which has resulted in two daughter nuclei with numerous karyomeres instead of nuclei . despite the similaritv of the nuclear structure of the cers of fig . 
38 and 39 explants of carcinomata give rise to sheet their behaviour in vitro differs . growths of cells with greater reg - ularity and rapidity than do those of mammary glands . 
the cehs of other mammary cancers induced by carcinogenic compounds , independently of the milk factor , exhibit greater abnormalities , which distinguish them from normal mammary gland cells . there is corresponding variation in the extent to which sarcoma cells differ from fibroblasts with respect to nuclear structure . 
the former is a photomicrograph of a 7 - days - old primary culture of mouse - embryo - heart fibroblasts , and the latter of a 3 - days - old culture of a sarcoma ( aa ) which originated fig . 
34 and 35 mosomin and nucleolar chromatin are included in the drawing . are ultra - violet micrographs of a cell of the same sarcoma , which was the most rapidly growing of the tumours which have been studied in the course of this since all the fig . 
ludford of these the " simplest and rarest type " involved the formation reconstruction . of chromosomal vesicles , or karyomeres . this was " long ago described by biitschli and fol in the blastomeres of segmenting eggs and since observed in embryonic cells of many species.. in this process each chromosome is converted into a small vesicle exactly hke a minature nucleus , the whole group then fusing together progressively so as to form first an irregular , chambered structure and finally a smgyle nucleus . 
from the outer wall of this , apparently , arises the nuclear menibrane , while the inner walls of the vesicles break down irregularly to form the nuclear network "  . 
he considers that the interphase nucleus " probably consists of closely packed , firmly adherent , swollen chromosomes ( chromosomal vesicles or karyomeres )  . " this condition is brought about by the telophase chromosomes swelling to form vesicles whicli adhere to one another . 
each vesicle retains its identity throughout the interphase . " during prophase each chromosome sinks into a dense gel which is separated from its neighbours by the fluid which collects between them . " lewis ( 1947 ) points out that amitosis and nuclear fragmentation would be readily explicable as resulting from the loss of adhesion between chromosomal vesicles . since nuclear formation by the fusion of chromosomal vesicles is said to be speciary characteristic of embryonic cells , it would not be surprising to find the same process occurring in malignant cells which they resemble in many respects . karyomeres are often clearly distinguishable in dividing cancer cells . attention has already been directed to the example in the middle of fig . 
28 and 29 could originate from the cohesion of karyomeres , formed , not as lewis ( 1947 ) suggested , by the swelhng of chromosomes , but by their spreading out over the surface of after coalescence of the latter , the chromosomes would adhere to the vesicles . internal surface of the external walls of the vesicles , which form the nuclear membrane . 
instead of the inner walls of the vesicles breaking down irregularly to form a nuclear network as wilson ( 1925 ) suggested , they would constitute the strands which extend between the nucleoli and the cell membrane . 
to bring ' about such an arrangement it would be necessary to postulate the existence of some mutually repellant force between the chromosomes and the nucleolus at this stage , so that the latter was forced into the centre of the coherent vesicles . from a cursory examination which has been made of different tissues of a number of mammals , including man , it appears that their nuclear structure is essentially the same as that which has been described , but no comparable study has been made of the nuclei of lower animals , or plants . apparently there is a fundamental difference between the nuclear structure of the higher animals and the higher plants . according to the recent work of chayen , davies and miles ( 1953 ) the interphase nucleus of plants ( vicia , allium , zea , tradescantia ) consists of a peripheral zone containing the chromosomes , a middle zone with a protein content , the width of which varies with the state of mitotic activity , and an inner zone occupied by the nucleoli . 
the most striking morphological feature is the manner in which most of the chromosomes are spread out over a spherical area , therebv brinain - a about maximum exposure to the cytoplasm on the outside , and to subsiances emitted from the nucleolus on the inside . 
some of the mitochondria and other granules are applied to the surface of the nucleus , and only clearly , the thickness of its membrane separates them from the chromosomes . substances which penetrate the plasma membrane must pass through the cordon of mitochondria before reaching the nucleus , and between the chromosomes if they are to get to the nucleolar system . in pioneer studies on living cells in tissue cultures , lewis and lewis ( 1915 ) followed the movements of mitochondria backwards and forwards between the nucleus and the cell periphery , and recently pomerat ( 1953 ) has observed rotary movements of the nucleus in epithelial cells growing in vitro . reasons have already been adduced for believing that the mitochondria are involved in protein synthesis ( ludford , smiles and welch , 1948a , 1948b . ) , and attention has been directed to the correlation between . 
hypertrophy of the nucleolar system , proliferation of mitochondria and increased nucleotide content of the cytoplasm ( ludford , 1951 )  . such observations as these tend to emphasise that the cell as a whole is a unified functional system . it is reasonable to suppose that the performance of its fundamental vital processes necessitates intimate correlation of the functional activities of all its organerae . recently brenner ( 1953 ) has deduced from the appearance presented by the nucleus in phase - contrast photomicrographs and ultra - violet micrographs ( ludford and smiles , 1950a , 1950b . ) , and from the results of ultra - centrifugation experiments carried out by himself and others ( beams , 1948 ; claude , 1943 ) that segments of chromosomes are attached to the inner surface of the nuclear mem " these segments maintain their continuity with the remaining parts of brane . the chromosomes which , as uncoiled threads , occupy the inside of the interphase nucleus . " on the basis of caspersson 's ( 1950 ) hypothesis that heterochromatic regions of the chromosomes control protein synthesis brenner ( 1953 ) has proposed that " the nuclear membrane heterochromatin with its specific genes controls the synthesis of equally specific microsomes , each desoxyribonucleic acid directing the synthesis of a characteristic type of ribonucleoprotein "  . " protein synthesis is a two phase reaction " , according to haurowitz and crampton ( 1952 )  . in its first phase , an expanded peptide film acts as a template on which a layer of amino acids is absorbed . 
the peptide film is maintained in the expanded state , its only efficient form , by combining with nucleic acids to form a nucleoin the second phase of protein synthesis , the peptide chain is folded protein . to form ' a three - dimensional globular molecule . haurowitz and crampton ( 1952 ) suggest that the highly specific peptide chains are probably first formed in the nucleus , and that they pass out into the cytoplasm where they are specifically since antigens are deposited mainly in cytoplasmic granules in liver folded . cells it is concluded that the folding of the peptide chains takes place in these granules . 
as haurowitz and crampton ( 1952 ) admit , their evidence that the first phase occurs inside the nucleus is equivocal , while certain of their experimental results indicate that it takes place in the cytoplasmic granules . 
the peptide ffim which haurowitz and crampton ( i952 ) suggest acts as a template in protein synthesis might be locahsed on the surface of the occasionally fibroblasts migrating from explants in tissue cultures mitochondria . leave behind them small pieces of cytoplasm which contain mitochondria and other granules . 
one possible reason for this is that the function of mitochondria is controlled by substances produced by the chromosomes . certain observations also suggest that the latter are dependent upon substances supphed by the nucleolar system . in conclusion , it should be pointed out that cells may undergo malignant transformation without any alteration occurring in their nuclear structure that can be detected by the present methods of microscopy . this is understandable if as has been suggested ( lufford , 1952 ) the cytological basis of malignancy ie ; an imbalance in the genes brought about by processes which result in ( 1 ) the loss of some chromosomes , or parts of chromosomes , and ( 2 ) the intranuclear duphhcation of others . 
this implies that with improvements in microscopical technique it should be possible to distinguish differences between the chromosomes which are spread out on the inner surface of the nuclear membranes of malignant cells and their non - mahgnant prototypes . 
accorcling to caspersson and santesson ( 1942 ) the heterochromatin system of malignant cers is stimulated to abnormal activity . this leads to disturbances in the formation of cytoplasmic proteins and in the reproduction of gene protein , which result in abnormal growth . 
it would be more compatible with the results of the present study if hypertrophy of the nucleolar system and a hyperchromatinic condition of the nucleus were the morphological expression of an increased growth rate rather than being indicative of malignant growth . it has not been possible to identify the nucleolar organizer on the chromosomes of the cells examined during the course of this work . so it remains to be deter ' mined whether increase in size of the nucleolar system is the result of duplication of chromosomes bearing nucleolar orgadizers . there is , however , indirect evidence that this may be the case ( biesele , poyner and painter , 1942 )  . the studies with ultra - violet microscopy described in this paper were carried out in the national institute for medical research and constitute part of a wider 130 r . 
korteweg. from ae antoni vanleeuwenhoek - hui8 , am8terdam . given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . it was shown by little et al . 
korteweg ( 1936a , b ) predicted that chromosomal factors must also be active . this prediction was based on the fact that differences in susceptibility to cancer exist between dba females and the fl hybrids of the dba x c57 black cross . 
the present paper concems these chromosomal factors . the fact that chromosomal factors exist is important , but the way they act , their mechanism , is of still greater inter ' est . 
a number of investigators have considered the role of the follicular hormone , and checked the possibility of differences between the oestrus cycles of the high - cancer and low - cancer strains of mice . 
as no differences were found this form of experiment was discontinued . yet i was not convinced . i spayed females of different strains , and afterwards determined the sensitivity of the vaginal epithelium to oestrone by means of the vaginal smear method . i found - that , to cause oestrus , it was necessary to inject three times as much oestrone into females of the high - cancer strain dba as in those of the low - cancer c57 black . 
the ovaries of our high - cancer strain greatly sur ass those of our low - cancer ammals this is partly caused by the somewhat greater num ' ber of graafian in volume . follicles , partly by the much greater number of corpora lutea , and partly by the r . 
hooker ( 1945 ) recorded that the effect of progesterone can be demonstrated in ' the structure of .the endometrial stroma just as well as the effect of i have found that the epithelium of the uterus of the virginal mouse oestrone . also shows a response to progesterone . 
as in the endometrium of the human female , in which on the 17th day of the cycle , shortly after the bursti ' ng of the follicle when the luteinizing process is beginning , a vacuole becomes visible in the basal pait of the epithelial cells , so also this phenomenon occurs in the virginal it is the ' refore necessary to be aware always of the possibilitythat mouse . progesterone too may influence susceptibility to cancer ( by its synergistic ' action with oestrone )  . if the mammary glands ' also of high - cancer strain mice should be relatively insensitive to oestrone , as is the vaginal epithelium of high - cancer strain animals , then the excess of oestrone produced would probably be harmless to the mammary glands . if , on the contrary , the sensitivity of the mammary glands of highand low - cancer strain animals should be the same , the excess of oestrone produced in high - cancer strain animals might be injurious to the mammary glands . it therefore became necessary to determine the 'sensitivity to oestrone of the mammary glands . 
of oestrone into 21 - months - old spayed females of different str ' ains , it seemed evident to van gulik and myself that our high - cancer strain females dba reacted to a lesser degree to oestrone than our low - cancer strain animals when judged by the extent of development of the glands . in 21 - m ' onths - old castrated males injected with 13 i.u. 
of oestrone , we found more growth also in the low - cancer than in the high - cancer miihlbock then drew our attention to the fact that in our experiments strains . we had injected large doses . if one wishes to determine the sensitivit ' y of an organ it is preferable to determine the threshold dose '  . 
as the first visible sign of an effect of oestrone , miihlbock ( 1948a ) took the beginning of budding of the end of the milk ducts ( swelling of the end bulbs )  . 
firstly in the males of the three strains c57 black , dba and 020 , the sensitivity of the mammary gland is about the same according to the threshold test . secondly , the minimal dose causing budding ( swelling of end bulbs ) in castrated males is approximately five times less than in non - castrated males . obviously in non - castrated males the testosterone largely counteracts the oestrone . this supposition proved to be right , as in castrated males which were injected both with oestrone and testosterone , the presence of the latter suppressed the action of the former . in noncastrated males three times as much oestrone is needed to cause budding ( swelling of end bulbs ) in the glands of high - cancer strain dba mice than in those of lowcancer strain mice ; in castrated males the sensitivity is the same . 
anderson. from the research department , glasgow royal cancer hospital . received for publication february 22 , 1949 . the need for controlling the influence of solvents used as vehicles for carcinogenic hydrocarbons was recognized by burrows , hieger and kennaway ( 1932 ) , who reported experiments designed to test the fat solvents then in use for this pui - pose , for possible carcinogenic action on the connective tissues of rats and subsequent work showed that in fact lard , which had been previously mice . heated to 140 ' c . , was a potential carcinogen for the connective tissues of fowls ( peacock , 1933 ) , and that lard alone could give rise to spindle - cell tumours in rats ( barry and cook , 1934 ) ; and later that lard , olive oil ' and other fatty materials could induce connective - tissue tumours , some of which appeared to be malignant in rats , but not in mice , in the experience of burrows , hieger and kennaway ( 1936 )  . the first instance of undoubted sarconia induction at the site of the injection of lard in fowls cited above was complicated by the fact that the same birds had been injected with a solution of 1 : 2 : 5 : 6 - dibenzanthracene in lard in the right breast and with lard alone in the left breast . 
of incidentally these tumours in a susceptible bird cannot be excluded . experiments suggest that the quantity of dibenzanthracene actually required to initiate sarcomatous growth must be remarkably small , since the bulk of the the injection appears to remain encapsulated throughout the expei - inient " ( peacock , 1933 )  . 
the latter assumption subsequently proved to be wrong . to test this point , chalmers ( 1934 ) analysed the contents of encapsulated niatter from the site of dibenzanthracene lard injection in one of peacock 's sarcoiiia - bearing fowls , which died 7 months after the last injectioii . 
dibenzanmoreover , in this group no tissue reaction thracene in 0 - 4 per cent solution . could be found at the site of injection after a few months , indicating that the homologous fat had caused httle disturbance . however , with egg yolk fat as vehicle for dibenzanthmmne m 0 - 4 per cent solution , 2 metastasising sarcomas occurred among 4 birds that s ' urvived 9 months or more after injection . birds inject - ed with yolkfat showed fibrous cysts at the site of injection , sinular t - 0 those observed in the lard experiment , and in contrast with the absence of such local reaction in birds injected with chicken - fat . there seemed , therefore , to be some aetiological connection bet - w - een the " foreignness " of the solvent used as vehicle and the local tissue reaction and the ultimate carcinogenic response t - o dibenzantli - racene . owing to the costlv and time - consuming nature of long - term experinients on fowls work was begun on similar lines on mice . while these experiments were in progress berenblum ( 1938 ) reported the co - carcinogenic action of croton oil . he had previouslv shown ( 1929 ) that mustard gas in dilute solution and ( 1935 ) cantharidin had anticarcinogenic properties for the skin of mice , and in the interim had found that no simple relationship between irritant and coor anticarcinogenic action was demonstrable . 
olive oil , tended to retain the dissolved benzp - % - rene as judged by the persistence of violet fluorescence , where - as in the case of those that were rapidlv absorbed , e.g. 
mouse fat or ether , fluorescence could not , be detected after a few weeks ( peacock and beck , 1938 )  . moreover , in the fornier group the incidence of sarcomas was high , and in the latter group it was low . this suggested that there mav be an optimal period for the retention of a carcinogen at the site of injection for the induction of a tumour , and that in mice this period was about a months . dickens and weil - malherbe ( 1942 ) at first confirmed our observation with mouse fat ' , but later ( 1946a ) , as a result of intensive experiments , found that the effect was not constant and was related to the phospho - lipid content of the 298 p . 
the mice were killed after different periods ( table 1 ) , the subcutaneous tissues exposed and examined for ultra - violet sixteen of 20 mice killed at various times over a period of 7 months fluorescence . had fluorescent material located at the original site of injection ; ulceration and sepsis at the site of injection killed 4 mice early in the experiment . in 3 of these mice no sign of the injected material was found , probably due to sloughing and leakage from the site of injection . 
the fourth had a small spot of fluorescence . these mice were not examined further ; in the remainder the fluorescent material was extracted with benzene , with the exception of 2 mice , which died after 412 - and 51 months , both showing persistent fluorescence and tumours at the site of injection . after the third month of experiinent tumours began to be clinically recognized at the sites of injection . as most of these tumours were similar to many such induced sarcomas in mice , not a 11 were histologically examined . 
one tumour in the abdominal cavity associated with intraperitoneal deposits of fluorescent material was probably induced by benzpyrene injected accidentally through the abdominal w , - , 11 . another tumour occurred in an ulcerated area at the site of injection . this proved to be a squamous carcinoma . 
benzene , and the benzene extract dried over anhydrous sodium sulphate . since the main object of these experiments was to demonstrate the pre - sence vne after various periods , the experiments were not conor absence of benz ducted quantitatively . however , the limit of sensitivity of the method of detection provides some indication of the quantities involved ( vi& infra )  . 
to eliminate the possibihty of a false positive reswt due to traces of benzpvrene adhering to the apparatus used in the experiments , benzene washings of each piece of chemical apparatus or other instrument used , were examined under the ultraviolet lamp imniediately before its use . attention was drawn to the necessity for such precautions ri_ - , cently ( anderson , 1947a )  . 
owing t - o the difficulty of eliminating minute traces of material from apparatus , such traces may be recorded by the fluorescence method of detection , and lead to a false positi ' ve result . 
the absenee of benzpyrene from extracts of two tumour - bearing mice killed - 4 months after a siiigle injection of benzpyrene does not preclude the possibilitv that the uneliaiiored hvdrocarbon was still present at the inception of the tuiiiour process as it was in tfie other tumour - bearing animals killed after shorter periodss . thus the results of this experiment accord well  . , a - ith otir previous observations ( peacock and beck , 1938 ) that when fluorescence remained at the site of injectioii in iiiiee for 5 months or more there was a hiah incidence of sarcoma . 
on this basis dickens ( 1947 ) states that " the surprising result was obtained that the more rapid elimination of benzpyrene was associated witli the higher carcinogenic activity , and slower elimination mith lower acti - 6ty . " fact the graphs published bv dickens and weil - ' - nlalherbe ( 1946b ) show . 
in the case of tricaprylin alone , and of tricaprylin plus phospholipids as solvents for benzpyrene , a consistent rate of elimination of the hvdrocarbon from the sit ' e of injection , but not to the point of extinction within s5 davs and 10 - 10 davs extrapolation of the tricaprvlin graph would in&ate total etim " lrespectively . nation in the region of 26 weeks . as w out of 26 mice had tumours bv the 20th week , it seems reasonable to , suppose , that some benzpyrene was present in these mice throughout the latent period of careinogenesis . this would correspond closely with our own experience . in the graph illustrating the course of events following injection of benzpyrene dissolved in tricaprylin plus 3 per cent choleste , rol , dickens and weil - malherbe ( 1946b ) show a considerable variation in the persistence of benzpyrene in different mice . 
as no t - umours were recorded before about the 19th week of their experiment , there is no positive evidence about the persistence or absence of benzpyrene at the site of injeetion in their tumourbearing mice . ou - r own repetition of their experiment . 
anderson mice , and must therefore have been present throughout the latent period of carcinogenesis . while it seems certain that partition of benzpyrene between the solvent used - is vehicle and the body fluids must be largely responsible for the rate of elimination of benzpyrene , the local tissue reaction to different solvents may also help to determine the local response . 
they established that for benzpyrene the partition coefficient between human sera and various lipoid solvents was directly related to the carcinogenic response of mice injected with benzpyrene dissolved in those solvents that readily parted with benzpyrene in vitro gave fewer solvents . ttimours when used as vehicles for injection of benzpyrene in mice . their conclusions support the conception that there is a significant relationship between the retention at the site of injection of benzpyrene and the ultimate carcinogenic response . whatever may be the essential mechanism of conversion of a normal cell to a malignant one , it seems certain that some local consumption of energy by some part of the cell must be involved in the transformation . it may well be that the liberation of energy at the optimum time in the life - cycle of a stisceptible cell is the essential carcinogenic stimulus , and this may be provided by the metabolism of a carcinogen . in this event neither the parent hydrocarbon nor its metabolites , but the transition from one to the other , may provide the essential carcinogenic stimulus . 
kreyberg spondence is established between the patient group and the control material with regard to such factors as age and sex composition , distribution of broad occupational groups and place of residence , which may influence smoking habits and / or lung cancer frequency , and a large number of factors clearly may be considered irrelevant , a field of uncertainty of considerable extent still remains . two points of practical importance emerge from the above discussion . 
the first is that control material consistin - g of non lung cancer cases among patients in a chest department , or even among hospital patients generary , may introduce a bias if , for instance , the factor " visiting a chest department for reasons other than lung cancer " - present in a control material but not among the lung cancer patients - is associated with the level of tobacco smoking . there may thus be a tendency towards excessive smoking in such a control material , or patients in chest departments may on the contrary tend to be persons whose reaction even to mild forms of chest trouble is a visit to the hospital , and who are careful if both these tendencies not to expose themselves to irritants sucli as tobacco . are present among the patients , they may neutralize each other , but the net result will obviously depend on their relative occurrence in the control material . similar objections may be brought against the use of hospital patients generally this point has been stressed by mirs and porter ( 1953 ) as control material . who believe that the bias probably wfll obscure an excess of smoking among lung cancer cases . 
at least , it is not established that hospital patients do not differ systematically from the general population with regard to smoking habits . the second point to be made is that a careful survey of all possible cancer producing factors must precede the instigation of a control study , and that prehminary surveys are needed to estabhsh the relationships between these factors and the level of tobacco smoking . there is thus a priori reason to consider the need for relevant definitions and differentiation of occupational groups among the cancer patients as well as in the control material , in view of the frequently indicated possibilities of specific occupational hazards with respect to lung cancer . assume , for instance , that a particular type of industrial occupation has a definite but unrecognized excess incidence of lung cancer , and that the carcinogenic agent is connected with the nature of the work . 
assume further that this particular occupation also is associated with an excess of tabocco smoking , because of higher pay , better opportunities of smoking during working hours , psychological stress or other causes . if the occupational selection criteria are such that this occupation does not exphcitly constitute a stratum in the control material it may be under - represented in a control material with random selection within each stratum , and the controls may , for the separate occupational groups and in toto , show a lower smoking level than the cancer such a difference will clearly be misleading for an evaluation of patient group . the carcinogenic effect of tobacco smoking . if , on the other hand , the particular occupation in our example is associated with an exceptionally low smoking level , a possibly significant excess smoking in the cancer patient group may be obscured . 
kreyberg composition of tobacco consumption are available only after 1927 . since then , tobacco smoked , i.e. , cigars , cigarettes and pipe tobacco , has accounted for a steadily increasing proportion of the total tobacco consumption at the expense of chewing tobacco and snuff , as shown in fig . 
the amount smoked is here calculated as production plus imports minus exports of cigars , cigarettes and pipe tobacco . these changes will year to year inventory changes have not been corrected for . be small , however , and without influence on the trend even if the vearlv figures may contain erratic deviations from the true consumption level . it should be permissible to assume that chewing tobacco and snuff played a greater r ' ole before 1927 . 
even if some of the chewing tobacco in fact has been smoked in pipe , it is reasonable to assu ' me that the amount of tobacco smoked , per person 15 years and older , has increased to some extent during the whole period from 1901 to 1940 . 1800 1600 1400 1200 1000 11 1% in600 1928 year fig . 
2. - consumption of smoking tobacco in granis per person 15 years and older . 38 39 after the last world war the yearly amount of tobacco smoked , i.e. , cigars , cigarettes and pipe tobacco , has reached a level of approximately 1650 g . 
3 for 1928 - 29 , 193 7 - 39 and the post - war years . the black parts of the columns represent the proportion of ready made cigarettes , which was 34 - 7 per cent in 1928 , rose to 42 - 9 per cent in 1947 and has since declined to 35 - 8 per cent in 1951 . 
patients , ear - nose - throat department , the rikshospital ( whole country ) kongsberg is a smar industrial town in central southem nor - way , s smar industrial and mining commuiiity ( iron - ore ) in the extreme north . varanger ( kirkenes ) a in the groups 1 , 3 , 4 and 5 among the males and 1 , 2 and 3 among the females the number of returned questionnaires falls substantially short of the total number of individuals in the groups selected . 
the age distribution of the separate groups is given appendix tables iii and iv . incidence of each of the forowing were computed . each of the above - mentioned groups were divided into age - classes with a for each group and age - class the percentage for males : 1 . 
4 is shown the range of variation found for the frequency of smokers in each age - class among the groups investigated . it will be noted that this range is fairly narrow , especially in the age - class 45 - 54 years in which the lowest percentage smokers ( found among the visitors at the oslo first aid station ) is 83 - 5 and the highest ( among industrial workers , oslo ) is 91 - 9 . 
the latter group , on the other hand , reaches its highest frequency at a i ' ater age than most of the other groups . generally the percentage smokers is lower among industrial workers in kongsberg , than in the other groups . per cent pure cigarette smokers . 
5 we find a very wide range of variation for the occurrence of pure cigarette smokers in the material , especially in the lower age - classes . among the industrial workers in kongsberg in the age - class 25 - 34 years only 28 - 4 per cent smoke cigarettes exclusively , whereas 75 - 9 per cent of their colleagues in s6r - varanger do so . 
6 is similar to that found for the percentage pure cigarette smokers , but the level has been shifted upwards and the range is a little less wide , corresponding to the tendency for the cigarette - pipe smoker combination to be more frequent in groups with relatively few pure cigarette smokers than in those with a high pure cigarette smoker percentage . 
8 the range of variation in the percentage medium and heavy smokers is large in all age classes , but the frequency does in no case exceed 50 per cent . 
12 is very rare among women , occuriing only among physicians and the patients and followers at the oslo first aid station , in the age - classes 35 - 54 years , with frequencies ranging from 1 - 4 per cent to 2 - 3 . per cent heavy and medium smokers . 
how much of this variation is real , i.e. , caused by actual differences in smoking habits in the groups under consideration , and how much may be caused by a systematic bias in those groups whose degree of co - operation in retur ' n ' mg questionnaires was low , cannot be decided on the basis of available inforrnation . 
the question is of considerable interest however , since for most of the smoking habit criteria , the range of variation among males would narrow considerably if the figures for the industrial workers in kongsberg are left out . this group comprises questionnaires from only a part of the total number of workers in the arms factory selected for analysis , and ff it could be shown that the degree of co - operation in this group is systematically related to smoking habits , the deviations shown would not reflect a true difference in smoking habits between these factory workers and the other groups . there are , however , certain indications that part of the difference at least between the kongsberg workers and the other male groups is real . first of all the percentage smokers in this group ( appendix table v ) are not substantially below those of the other groups . 
even if there is a tendency for smokers to be under - represented among those workers who answered the questionnaire , such a bias can not account for the very low percentages pure and pure and mixed cigarette rsmokers found in the kongsberg group , and is not likely to expla ' entirely the low percentages found for the quantitative criteria ( per cent heavy and per cent heavy and medium smokers )  . secondly , groups other than the kongsberg workers have failed to co - operate fully , without any resulting systematic deviation from the complete groups . 
an explanation of the result for the kongsberg group in terms of a systematic bias , would thus imply that this bias was a characteristic of the kongsberg workers but not of the others . thirdly , the material from the male physicians seems to indicate that there is no strong tendency that individuals who fail to fir a questionaire deviate appreciably i their smoking habits from those who co - operate readily . 
a new questionnaire was sent out approximately a year later with an appeal to those who had not answered previously this appeal resulted in 601 new questionnaires which were to do so now . analysed separately , however , the two groups showed added to the old material . the following figures for the separate smoking habit criteria . there is thus cause to believe that the range of variation found in the material largely reflects real differences in smoking habits . special menti ' on may be made of the male group of patients at the rikshospital ear - nose - throat department which displays exceptionally low percentages heavy and heavy and medium smokers ( both generally and cigarette ) in the lower age - classes and rather high percentages in the higher age - classes . the material is , however , too small to permit any conclusion as to whether or not this reflects a characteristic pattem in this group . conclusion as was pointed out initiahy , the present study has been undertaken in order to provide background information on factors which must be taken into conh . 
the present study may serve as a basis of comparison with the smoking habits of lung cancer patients , in the sense that findings generally outside the range of variation exhibited by the present material would be indicative of real deviations in the smoking habits of lung cancer patients , but quantitative statements about differences found cannot be inferred . the low frequency of smokers in the higher age - classes among most groups of women may indicate that the increasing trend in total tobacco consumption h . 
fibroid with advanced sclerosis ; patch of oedematous tissue ' probably smooth muscle fibres ; proliferation of vascular endothelium ; invasion diahypertrophic endophragm ; thelium on the surface of the tumour . . 
the peripheral belt of fibroblasts originating most probably from subendothelial cells of the serosa and so characteristic of the oestrogeninduced abdominal fibroid ( lipschutz , 1942 ) was present in a diaphragmatic tumour belonging to the animal of fig . 
as with the abdominal fibroids we have described formerly , there were in thoracic tumours also some cells resembling there was a proliferation of the cells of the wall of vascular smooth muscle fibres . spaces ; there was an hypertrophy of the endothelium at some places on the there was invasion of the muscular tissue of the diasurface of the tumour . phragm , or the thoracic wall , by the tumours . 
but there were in this tumour also accumulations of cells resembling fibroblasts which seemed to originate from the proliferation of the subendothelial cells of the wall of vascular spaces , or the endothelium proper . 
the tumour was covered by various layers of flattened cells . there were besides the animals described , 4 females and 2 males in which small nodules just visible to the naked eye were found on the surface of the only 2 animals of this group have been examined lung or on the parietal pleura . microscopically ( table i ) and in one of these a tiny fibrous nodule was found on the surface of the lung ; the structure of this nodule was identical with that of similar oestrogen - induced nodules of the abdominal serosa . 
note thickened border of opening in the diaphragm , prolapsed liver and intestine . fibrous reaction also on the right though the opening is on the left . small nodules on the pericardium in lxiii . 
the tablet occupied in some cases a retrosternal or mediastinal position ; it was surrounded by a thin capsule and in other cases the capsule adhered to the costal serosa , thin fibrous strands . where the fibrous reaction around the oestradiol tablet was more conspicuous . but there was not a single animal with thoracic tumours in this group . 
lipschutz oestrogen - induced thoracic fibroids increased very considerably ; thoracic fibroids in 7 out of 18 animals with such a communication . there were in most of these animals part of the liver and of the intestine prolapsed to but the mechanical stimulus due to this accident was the thoracic cavity . seemingly not responsible for the increase of the incidence of oestrogen - induced this was shown by the fact that in 24 animals without an thoracic fibroids . abdominal - thoracic communication the capsule enveloping a foreign body introduced in the thoracic cavity ( metallic tablet or a tablet of oc - oestradiol ) did not , or only exceptionally , become surrounded , under the influence of the oestrogen , by a fibroid as was the case with a similar foreign body ( metallic tablet ) introduced in the abdominal cavity of these animals . the above experimental results suggest that the oestrogen - induced fibrous tumoral reaction of the peritoneum in the guinea - pig is mediated by an internal ambiental factor normally present in the abdominal serosal fluid , or a factor originating under the influence of the oestrogen and given up to the abdominal it is seemingly the absence of this mediating factor which protects the cavity . animals against production of fibroids in the thoracic cavity . aided by grants from the the jane coffin childs memorial fund for medical research . 
lipschutz. from departmento de medicina experimental , servicio nacional de salud , avenida irarrdzaval 849 , santiago de chile . received for publication february 2 , 1953 . the behaviour of the intrasplenic ovarian graft in the castrated guinea - pig has been studied in this department since 1942 . the graft shows two particularities : blood follicles which may appear as early as 3 weeks after transplantation and at 2 months are frequently cystically enlarged ; and small nodules and cords of luteal cells , i.e. , of eells whieh resemble those of corpora lutea , scattered in the ovarian stroma . 
later gn , at say 10 months , the graft consists , predominantly , of very large corpora lutea embedded in irregularly shaped masses of cells similar to those of corpora lutea ( ponce de leon , 1944 ; woywood , 1944 ; lipschutz , 1946 ; lipschutz , ponce de leon , woywood and gay , 1946 ; iglesias , lipschutz and mardones , 1950 )  . 
the condition has been termed by us as luteomatous . the luteal cells originate partly from small follicles whose theca - in some cases , but in a minor degree , possibly also the granulosa - undergoes precocious luteinization , and partly , according to our evidence , from cells of the stroma . blood follicles and graafian follicles were still present at 10 and even 20 months . on the contrary , blood follioles fail to appear in the intrasplenic graft when the seoond ovary is left in situ ( gay , 1944 ) or grafted into the kidney ( ramirez , 1950 )  . 
they appear only exceptionally when a minute ovarian fragment is left in situ ( gay , 1944 ; niedmann , 1947 ; bruzzone , lipschutz and niedman.n , 1952 ) , whereas they invariably appear when both ovaries are grafted into the spleen ( ramirez , 1950 ; ramirez , iglesias , mardones and lipschutz , 1952 )  . there is thus full evidence that the whole gamut of atypical changes , including tumourigenesis , is due to the hypophysial gonadotrophic activity not duly controlled by ovarian hormones , the latter having been inactivated to a considerable degree in the liver . the luteomatous or tumoural structure described in the guinea - pig is different from that in other laboratory animals . granulosa - cell tumours and luteomata appear in the intrasplenic graft in castrated rats ( biskind and biskind , 1944 , 1949 ; van lancker and maisin , 1950 ; lacour , oberling and guerin , 1951 )  . mice granulosa - cell tumours are more prevalent than luteoma ( li and gardner , 1947a , 1947b ; li , 1948 ; furth and sobel , 1947 )  . metastases may occur in mice and the tumour is transplantable ( li , 1948 )  . 
in there but haeinorrhagic follicles were present in all the growth of nipples and uterine growth . one of these animals there was a luteoma . t in the animals of this group tiny pellets containinig 40 per cent . 
15 - the graft divided into two halves - could be due to proliferation of capsular endothelium ? in several cases we found a zone of necrotio appearance between the fibrous capsule of the graft and the splenic tissue , possibly due to luteal tissue invading the spleen and undergoing necrosis after a period of more or less abundant proliferation . rupture of the capsule is possibly not the only route by which invasion is in fig . 
23 what seems to be splenic tissue was found in the loose part of the ovarian stroma which corresponds to the hilus of the ovary . we have not seen metastases in our work in the guinea - pig . 
bartholomew 's hospital , london , e.c.1. received for publication january 12 , 1949 . thie carcinogenic activity of ethereal extracts of domestic soot was demonstrated by passey ( 1922 )  . domestic soot may contain as much as 40 per cent of tarry matter ( cohen and ruston , 1925 )  . 
the use of the fluorescence spectrum by mayneord and hieger led to the identification of 3 : 4 - benzpyrene in gas - works pitch ( cook , hewett and hieger , 1933 )  . this discovery suggests that this hydrocarbon might be one of the carcinogenic agents in domestic soot , although the conditions of pyrolysis in the domestic hearth , and in the gas - works retort , are different . berenblum and schoental ( 1947 ) found that carcinogens other than 3 : 4 - benzpyrene were present in coal tar . 
the first attempt to identify 3 : 4 benzpyrene as a contaminant of the atmosphere was made by hieger ( 1946 ) , who exposed non - fluorescing benzene to the air in london , and found that the fluid became fluorescent and showed spectral bands resembling those of benzpyrene . 
for the present purpose , samples showing a ph value of 10 - 0 - 10 - 3 were found to be most suitable . 3 : 4 - benzpyrene . - the commercial product contains a pale - blue - fluorescing impurity which is very strongly adsorbed on alumina . 
benzene. the first attempts to separate benzpyrene from soot were based on the method described by berenblum ( 1945 ) for the extraction of benzpyrene from coal tar . in the original method vacuum distillation of the suitable fraction of tar was followed by solution of the distillate in concentrated sulphuric acid . attempts at vacuum distillation of benzene extracts of soot showed , however , that the composition of the soot extract was very different from that of coal tar . 
evaporation of the benzene extract yielded a residue consisting mainly of non - volatile material , which appeared to decompose on distillation in vacuo with evolution of gas . direct sulphuric acid extraction of the crude tar extracted from soot was also unsuccessful , probably owing to considerable sulphonation . moreover , recovery experiments , with microgram quantities , of pure benzpyrene from icecold sulphuric acid showed that considerable loss of the hydrocarbon occurred , and that the method was useless as a quantitative one for such small amounts . chromatographic adsorption was next tried . 
when an extract of soot was submitted to chromatographic separation , the characteristic fluorescence spectrum experiments with various of benzpyrene was given by the appropriate eluate . samples of alumina showed that spence 's type h alumina , containing adsorbed benzpyrene , could be washed freely with a mixture of benzene - petroleum ether ( 1 : 10 ) without elution , and that the benzpyrene was eluted only when the proportion of benzene was increased ( 3 : 10 )  . 
the extract was given a preliminary purification by passing it through a short column of alumina ( 14 x 25 mm . ) and eluting with the same mixture of solvents . 
the lower band was easily eluted with benzene - petroleum ether ( 1 : 10 ) , while the upper could only be washed down when the benzene content of the eluting fluid was increased ( 3 : 10 )  . this latter fraction is shown in fig . 
lane. from the department of pathology , royal cancer ho8pital , london . received for publication june 7 , 1952 . scepticism is a healthy response to diagnosis of any tumour as angiosarcoin ' - 11 . to dispel all doubts , full necropsy and thorough histological exaniination of all lesions with indisputable evidence of the vasoformative natuire of the tumour and proof of metastasis are essential . 
many lymph nodes in the cervical regions , supraclavicular fossae and posterior triangles were enlarged ( up to 2 - 0 cmain diameter ) , soft , dark red and cystic . 
on the surface and in the substance of both lungs were numerous discrete , firm , red , spherical nodules ( up to 3 - 0 cdiameter ) whose cut surfaces resembled blood clot . other parts the paratracheal and mediastinal lymph nodes of the lungs were oedematous . were greyish - black and slightly enlarged . 
the common bile - duct was dilated and contained several small pigment calculi . the spleen was small , and on its surface was a cyst ( 1 - 2 cdiameter ) containing clear fluid.. 
the nuclei are pale - staining , vesicular and variable in size : in shape 's.pherical , ovoid or indented . mitotic figures are few , but within such aggregations capillary channels are forming by separation of the cells . 
he interpreted his case as a benign angioma producing metastases , and separated it sharply from a case described by theile ( 1904 ) in which all the lesions showed sarcomatous areas . his interpretation is not acceptable in pathology , and the histology of the lesions in shennan 's ( 1914 ) case was no less benign , yet penetration of a pulmonary vein shennan suggested that our criteria of malignancy were at fault in was found . regarding such lesions as histologically benign . 
livingstone and klemperer ( i 926 ) concluded that the lesions in these cases were not as benign histolozicallv as the authors believed , and they thought that all showed atypical areas suggestive of malignancy . stout ( i 943 ) confirmed this , and considered the same applied to the original lesion in robinson and castleman 's ( 1936 ) case . however , in this connection it is well know - n that other malignant tumours and their metastases sometimes show deceptively benign histological appearances ( e.g. , chondrosarcoma and carcinoma of prostate , thyroid or kidney )  . the present case seems to be of the same nature as these and , in addition , has fe ' atiires similar to some others in which the histological structure was more malignant . kettle ( 1918 ) described an angioblastic tumour of the leg with metastases in the inguinal lymph nodes , ulrich ( 1921 ) a similar tumour of the lumbar muscles with metastases in the lungs , and downing and manory ( 1930 ) multiple angiomatoid lesions appearing in successive crops over an area which had been bumt some months previously . in the last two cases intravascular extensions were present , which strongly suggests that the lesions other than the primary mallory ( 1 914 ) , however , states that some caverwere blood - bome metastases . nous haema - ngiomas - not only arise in veins but permeate their wa ' ils and , at various placeg , rupture them , infiltrate the tissues and produce lesions which , while simulating metastases , are , in reality , in contiguity . this must be unusual , and would clearly be an incorrect interpretation of some of the cases cited where the lesions are far apart and particularly where peripheral lesions are associated with pulmonary ones . 
on the other hand , if benign angiomatous lesions extend into the lumina of veins , it cannot be concluded from observing intravascular extension alone that multiple lesions are metastatic rather than multifocal . in the present case , however , tumour tissue , not in cont ' lguity with large masses , is demonstrable within pulmonary vessels and , of special interest , witbin perivascular lymphatics and afferents to cervical lymph nodes . 
the only acceptable explanation of such findings is that the lesions in the cervical lymph nodes and lungs are embolic metastases . this case does not appear to shed any new light on those angiomatous conditions having a " system " distrib - ution described by theile ( 1904 ) , jores ( 1908 ) , wright ( 1928 ) and de nava , squez ( 1936 )  . in all these the authors described primary angioblastic tumours of the spleen with metastases in the liver . in theile 's case there were additional lesions in the lungs and stomach . willis ( 1948 ) , while c ' onceding these authors ' interpretations may have been correct , points out the possibility of the lesions in their cases being multifocal in origin . evidence suggestive of such cases being malignant metastasizing neoplasms ( local infiltration , poor differentiation , mitoses and intravascular extensions ) is not sufficient to classify them , witb certainty , as such . 
tumour tissue was found within perineural lymphatics of the scalp , in the afferent lymphatics of cervical lymph nodes ' and within pulmonary blood - vessels . i am indebted to j . 
a. - ( 1934 ) ' the spread of tumours in the human body , ' london ( chiirchill ) , p 148 . - ( 1948 ) ' the pathology of tumours , ' london ( biitterworth ) , p . 
at that time research into their pharmacological and toxicological properties showed that certain of their effects were strikingly similar to those of ionizing radiation ( gilman and philips , 1946 ; kamofsky , graef and smith , 1948 ; philips , 1950 )  . this led to further investigations of their action as mutagenic agents and as growth inhibitors and their importance in these connections quickly became apparent . 
tumours were selected for treatment on the 6th or 7th day after implantation . the standard dose level adapted as the basis for comparison of chemical and x - ray effects was 1 mg . 
casarini analyses showed that 200 r might be considered to be an " equivalent " dose , i.e. , it produced in tumour and bone marrow the same amount and the same type of cellular injuries up to 24 hours after administration ( koller , unpublished )  . 
body weight throughout the experiments . for cytological analysis small pieces of tumours were fixed in acetic alcohol mixture ( 1 : 3 ) , and squash preparations were made and stained with feulgen 's for histological study fragments were fixed in bouin 's solution , basic fuchsin . embedded in paraffin wax , and sections cut at 5 . 
were stained with haematoxylin and orange - g . cytological effects : method of analysis . the method used for estimating cellular injuries is the same as was employed by devik , elson , koller and lamerton ( 1950 )  . cells in post - metaphase stages were selected . 
the importance of some biological variables ( age , sex , diet , etc . ) has already been discussed ; devil and coworkers ( 1950 ) demonstrated by analysis that the data , obtained by the same method as used in the present study , do in fact provide a valid basis for comparison . but as will be seen , the effects observed 72 hours after treatment by x ray and hn2 were in any case qualitatively so different that they could not be compared on a quantitative basis . x - ray effects : localised treatment ( 200 r )  . the tumours only were irradiated and cytological effects were analysed at 6 , 12 , 24 , 48 , 72 , 96 , 120 , 144 and 168 hours after treatment . 
date when the animal was killed . this is also the sample in which the number of chromosome fragments per cell is at a maximuthe damage is less 24 hours after irradiation , and it diminishes further in the 48 and 72 - hour samples . in tumours 4 to 6 days after treatment the number of dividing tumour cells with abnormalities is very small . our observations indicate that 200 r is insufficient to inhibit the growth of the walker carcinoma ; the growth rate of treated tumours 6 days after irradiation was found this finding is in close agreement with that to be the same as that of the controls . of devik and co - workers ( 1950 )  . hn2 - induced cytological effects . it has already been reported by the senior author that a dose of 1 mg . 
by including such cells in the analysis , the true number of injured cells is reduced ( compare the 144 and 168 hour samples , table iib )  . ' 0060 - / time after treatment in hours fig . 
they are particularly evident in young tumour implants and are found in large numbers at the periphery of growalthough these cells are normally present in the walker carcinoma , ing tumours . they play a small part in the histological organisation of the tumour . 
the great and rapid increase in histiocytes after hn2 administration , which takes place in the cellular tumour parenchyma , has important consequences . table iib shows the drastic change which occurs in the cell composition of the tumour . 
the proliferation of those tumours cells which may recover from nuclear or cytoplasmic disturbances was found to be seriously hindered by the altered environmental conditions . establishment of a solid tumour parenchyma does occur occasionally in the region of the connective - tissue capsule . 
the histological structure of this particular region is known to differ greatly from that of the tumour , and the different type of reaction to hn2 is presumably correlated with this . in conclusion we wish to emphasize the important fact that 72 hours after hn2 administration a series of changes takes place which result in the complete transformation of the histological structure of the walker carcinoma . 
the effect is , however , temporary ; the animals were already gaining weight again 7 days after treatment . in this respect the effect of hn2 is much more drastic than was observed after  . 
body weight ( intraperitoneal injection )  . while the cellular injury in the femoral bone marrow induced by 500 r was much more drastic than that produced by the hn2 " ld50 - equivalent " this x - ray dose was found to be still insufficient to bring about by way of the systemic effects the same amount of cellular and the same kind of histological changes which have been observed after hn2 administration . 
the delay in the developmental cycle may last 12 hours . this is indicated by the fact that in the 12 - hour sample 6 cells with chromosome injuries were observed out of 27 in which the chromosome abnormality suggested that they were already the frequency distribution of the injured cells in mitosis during irradiation . observed in the various samples is a further indication that cell development has been retarded . while the highest proportion of injured cells is seen 12 hours after exposure to 300 r , the maximum damage was in the 48 - hour tumour sample 182 p . 
the true significance of this observation lies in the relationship which it has to the primary reactions induced by the two ' agents . the view is now widely accepted that during irradiation ionisation takes place in the cell , followed immediately by the formation of free radicals of the water molecules ( lea , 1946 )  . this event represents the first step in the complex similarly reaction - chain which in time leads to a definite biological phenomenon . there is no substantial delay between administration of hn2 and its primary reaction in the organisthe rate of hydrolysis of nitrogen mustard is extremely high in a biological system , and it has been shown recently by batemann , klopp and cromer ( 1951 ) that it remains " active " only for a few minutes after intravenous injection . this is the main reason which allows us to compare the cellular effects of x ray and hn2 in tumour samples taken at the same time after treatment and to determine the relative sensitivity of the various stages of the mitotic cycle . the measure of sensitivity of a cell is the frequency of chromosome injuries observed at a given time . 
the interval between treatment and fixing the cell for analysis indicates the cell stage during treatment . using this criterion the radiation sensitivity of the mitotic cycle has been determined in pollen grains of tradescantia ( koller , 1946 )  . if our present data are interpreted on the same criterion , then we must draw the conclusion that tumour cells of the walker carcinoma are most sensitive to x rays at the end ofthe resting stage or beginning of prophase , while they are least sensitive to hn2 at this stage . 
revell ( 1952 ) employed a new and critical method in the study of chromosome effects of several substances ( mustards , epoxides , etc . ) and came to the same conclusion . 
the fact that x ray ' and hn2 initiate their effects at different stages strongly suggests that their mode of action is different . thus , for instance , we may assume that x rays act directly on definite linkages of the already synthesized polypeptide structure of the prophase chromosome thread , while a chemical agent affects the ' resting stage chromosome - filament either before or during the process of synthesis . this aspect of the chemical effects is discussed in more detail by revell ( 1952 )  . the higher sensitivity of the resting stage to the chemical agents has another implication . 
the non - dividing ( intermitotic or " resting " ) cells are ' believed to be functionally the most active cells , and often referred to as cells in the " metait is not unlikely that the degeneration of many bolic phase " ( hughes , 1952 )  . non - dividing tumour cells which we observed after hn2 administration is due to a gross disturbance in this metabolic phase . if it is found that chemical agents act preferentially on cells in the resting stage , a new approach may possibly be opened to the chemotherapy of cancer . because the functional or morphological characteristics of cells are determined 184 p . 
our experiment demonstrated such difference between tumour cells and histiocytes . the study of the cytological behaviour of the walker carcinoma under x ray and hn2 treatment has accordingly led to the conclusion that the mode of action of these agents differs , and there is other experimental evidence of different kinds to support this conclusion . 
no such reduction was observed when nitrogen mustard in barley , mutations of particular genes was used ( auerbach and moser , 1951 )  . were induced with a higher frequency by hn2 than was expected , indicating some specificity of action ( gustafsson and mackey , 1948 )  . 
cytological analysis of the femoral bone marrow and implanted walker carcinoma of the rat has shown that up to 24 hours after treatment , 200 roentgens produce the same amount of chromosome injuries as 1 mg . 
the difference in behaviour shown by the walker carcinoma after irradiation and after nitrogen mustard treatment indicates that the primary basic reactions initiated by these agents are fundamentally different . this investigation has been supported by grants to the royal cancer hospital and chester beatty research institute from the british empire cancer campaign , the jane coffin childs memorial fund for medical research , the anna fuller fund , and the national cancer institute of the national institutes of health , u.s. 
harris , coal tar research association , who had purified it by chromatography and checked it by ultra - violet spectroscopy for impurities . these were below the level of detection . fluorewene mea8urement& the fluorescence values for solutions of the various hydrocarbons were determined by constructing curves using a hilger " spekker " photoelectric fluorimeter with cers of capacity approximately 11 ml . , and with filter chance 0 x 7 348 d . 
mouse protein after 6 days ' benzpyrene treatment appear to be of the same order as that recovered by mller ( 1951 ) although it is not possible to assess gravimetricallv the data given by this worker since her figures are relative and apply to the instrument and filters employed . berenblum and schoental ( 1942 ) found that 24 hours after one intraperitoneal injection of 10 mg . 
woodhouse benzpyrene was added to denatured , fat - free , normal skin - protein and refluxed with alkah , toluene and zinc , it could be completely and quantitatively removed from the alkahne solution by benzene extraction . 
the extra - cellular bound ilydrocarbon might consist of metabohtes " excreted " from the cells - or it might be derived from dead or damaged cells . although the fluorescence technique was not sufficiently sensitive for accurate estimations with the small amounts of protein available , the occurence of bound hydrocarbon in cellular constituents has been confirmed by extracting the dna and rna skin proteins from a batch of benzpyrene - treated mice . 
of " fixed " benzpyrene was extracted and a trace was found in the dna protein sample which , however , weighed only 2 mg . a corroborative experiment was also carried out based on the observations of calcutt aind payne ( 1953 ) who showed that nuclei and mitochondria isolated from the livers of mice which had been given a single intraperitoneal injection of benzpyrene in finely dispersed eiuspension , contained fluorescent hydrocarbon 2 hours to 21 weeks after the injection . 
the believed , therefore , that benzpvrene injected in this way is rapidly transported to the mitochondria and nuclei and is held there in an unchanged state for prolonged periods . 
they did not examine the effect of alkahne hydrolysis on alcohol - extracted nuclei . the experiment was repeated in this laboratory but the prelinu ' nary extractions were made as described previously for the skin tissues and continued with alkaline hydrolysis . 
a small amount of fluorescent material was extracted from the hydrolysate but nuclei froni control mice did not yield comparable fluorescent extracts at any stage . this experiment showed that at least a portion of the benzpyrene can be it was repeated , therefore , employing fixed in vivo by nuclear components . perylene , and , after alkaline hvdrolysis an extract with fluorescence characteristic of perylene was obtained from the separated , alcohol - treated nuclei . 
the free hydrocarbon was thoroughly removed and the " bound hydrocarbon " was extracted after hydrolysing the protein with koh and measured fluorimetrically . although these species of animals vary considerably in their response to this hydrocarbon as a carcinogenic agent the amounts of bound hydrocarbon obtained from 25 mg . 
samples of extracted and dried tissue were very similar in all instances . other polycyclic hydrocarbons which are less carcinogenic to mice , e.g. , 2 ' 6 - dimethyl - benzanthracene , and perylene which is non - carcinogenic , have also been tested , and in all instances comparable amounts of bound hydrocarbon could be extracted from the alkahne bydrolysate . using similar extraction techniques a smar amount of fluorescent , bound hydrocarbon could be removed from the nuclei isolated from hvers of mice 24 hours after a single intraperitoneal injection of either 3 4 - benzpyrene or perylene . it is concluded that further evidence is needed to substantiate the view that 352 d . 
harris. * from the chester beatty research in.3titute , royal cancer hospital , london , s.w.3. received for publication january 11 , 1951 . themajor requirement for the investigation of the physical and chemical properties of the rous no . 
i sarcoma agent is a source of " purified " agent of a uniformly high infectivity , and which may be stored without loss of this activity , since it is not always convenient to compress the investigation to within the relatively short period in which the purified agent is stable in vitro . this paper describes a method for the production of concentrates of the vir ' us in a form suitable for storage , and the storage problem itself is considered in the following paper . a variety of chemical and physical methods have hitherto been used for separating and concentrating the agent from biologically - inactive tumour protein and nucleoproteins . claude ( 1935a ) found that such contaminants could be removed by adsorption , on to freshlv prepared aluminium bvdroxide gel , and the carbohydrate contaminant by precipitation with gelatthe agent was not directly affected , and a - combined chemical fractionation and dialysis gave a 25 - fold concentration in - terms of dry weight ( claude , 1935b )  . this method was also investigated by dmochowski ( 1948b ) , who recorded a 50 per cent increase in the activity of the agent compared with the startino , c5 filtrate . shemin , sproul and jobling ( 1940 ) , and shemin and sproul ( 1942 ) precipitated the agent from filtrates by addition of papain or of histone . 
nitrogen ( the range was 1 - 3 x 10 - 3 mg . to 7 x 10 - 7 mg . ) were found to be infective . concentration of the agent by centrifugation was first described by ledingham and gye in 1935 and fractional centrifugation methods have since been widely used ( mcintosh , 1935 ; amies , 1937 ; claude , 1937 , 1938 , 1939 , 1940 ; claude and rothen , 1940 ; pollard , 1938 , 1939 ; stern and duran - reynals , 1939 ; dmochowski , 1948a )  . claude ( 1938 , 1939 ) isolated a fraction corresponding to less than 3 - 5 per cent of the dry weight of the original extract which , in amounts corresponding to 4 - 0 x 10 - 10 mg . 
harris pollard ( 1939 ) and amies and carr ( 1939 ) were able to overcome the first difficulty by precipitat - ing the agent from a clarified extract by adjusting the ph to 5.0 to 5 - 5 . 
the isoelectric point of the purified agent is 3 - 5 , but tumour extracts deposit a mucoprotein at ph 5 - 0 , which takes down the agent with it . the aggregated agent , etc . , was then further concentrated by a low - speed centrithe aggregates were.broken down by tryptic digestion at fugation procedure . ph 8 - 8 to 9 - 0 and the released agent further " purified " by fractional centrifugation . alternatively , the ' mucoprotein may be hydrolysed by incubation of the extract - with hyaluronidase , which has no deleterious effect upon the agent ( siturm , and duran - reynals , 1932 ; shemin and sproul , 1942 )  . 
the agent may then be deposited directly from the non - viscous extract . bryan , riley , deihl and voorhees ( 1947 ) have avoided this difficulty by the simple , but uneconomical , procedure of rejecting the first , viscous extract . a further method which has been claimed to apply to the purification of small arrlounts of agent is that of chromatography on celite ( riley , 1948 )  . 
the agent is strongly adsorbed to celite from physiological salt solution , but may be eluted intermsoftheratio , biologicalactivity : withverydilute ( o - 001m ) saltsolution . nitrogen content , a 3to 7 - fold enrichment was obtained from a partially ' - purified three of the above methods , papain precipitation , fractional tumour extract . centrifugation after precipitation at ph 5 - 0 and direct deposition after treatment of the initial extract with hyaluronidase , have been reinvestigated from the point of view of the preparation of large quantities of agent in a form suitable for storage . 
a further method , methanol precipitation , hitherto applied to a number of other animal viruses , has also been investigated . cox , van der scheer , aiston and bohnel ( 1947 ) found that influenza virus ( from arantoic fluid ) could be precipitated in active form from suspensions in phosphate buffer at ph 7 - 0 to 8 - 0 by addition of methanol to a final concentration of 15 to 30 per cent . 
sharples air - driven supercentrifuge , stainles - 3 the rous agent becomes progressively more thermolabile as purification centrifuge b was used in a cold - room at o ' c . , and c was adjusted to the sharples centrifuge ( a ) was cooled with the centrifugal procedures used are listed below and steel bowls . - ( i ) clarifica ( ii ) deposition at ( iii ) deposition at ph 7 - 0 . 
the bulk of the tumour was extracted in the waring " blendor " with 10 volumes of water containing 1 : 10 , 000 hcn , and the small sample " minced with scissors " and extracted into a similar medium . suitable dilutions of these extracts were injected into fowls . 
0.005 m buffer at ph 7 - 2 containing hyaluronidase and 1 : 10 , 000 ticn . the tissue suspension was clarified ( b , i ) and the agent deposited from the supernatant ( b , ii )  . 
the control suspension ( a ) was ' assayed comparatively against ( b ) - resuspended papain - agent complex , etc . , ( b ) - supematant and ( c )  . 
and methanolic buffer solutions prepared , and cooled to these buffers contained ( a ) 40 per cent methancil , ( b ) 50 per cent methanol and ( c ) 60 per cent . methanol respectively in 0 - 005 m buffer . equal volumes of ( a ) , ( b ) , ( c ) or buffer ( d ) - control , were added to volumes of the supernatant . 
an equal volume of 50 per cent methanolic buffer was added under the same conditions as method 1 and the resuspended deposit assayed against the extract . the assay showed that the alcohol - deposited agent had only i per cent of the activity of the control suspension . ( 3 ) a similar clarified extract was prepared , cooled to o ' c . 
the deposit was resuspended in 0 - 005 m buffer at ph 7 - 2 , clarified ( b , i ) and the agent in the supernatant deposited ( b , ii )  . the pellet was finary resuspended in 0 - 005 m buffer , ph 7 - 2 , adjusted in concentration to the equivalent of i g . 
de osition after precipitation at ph 5 - 0 . a 10 per cent fresh tumour tissue suspension was prepared in the usual way , but with the omission of hyaluronidase . 
the air - driven sharples supereentrifuge with a maximum speed of rotation of 50 , 000 r.p. ( 62 , 000 g . ) was used by stanley in 1945 for the concentration of influenza virus for vaccine production . 
for deposition of the virus the stainlesssteel centrifuge bowl was invariably lined with cell ' ophane ( 7 / 1000 in . ) which may ( a ) be readily withdrawn and which ( b ) minimizes contact of the virus with metals . when unrolled this " liner " invariably showed a boundary , the distance of which from the bottom of the bowl , varied with the rate of flow of the virus in terms of total nitrogen distribution , the suspension through the centrifuge . deposit was uniformly distributed above and below the boundary , although the virus content of the top and bottom fractions differed considerably . 
the " liner was therefore divided at the boundary and the deposits removed and separately resuspended in 0 - 01 m buffer at ph 7 - 2 ( dband dt )  . 
per cent crystalline trypsin was added to each , the suspensions incubated at room temperature for 40 minutes and the virus re - deposited ( a , iii ) ( sb , st - supernatants and dbj , dt , - deposits )  . 
fresh tumour tissue was disintegrated with 10 volumes of 0 - 005 m phosphate buffer at ph 7 - 2 containing 1 : 10 , 000 hcn - but without hyaluronidase - and the suspension clarified ( a , i )  . 
88. the " liner " was removed and the total deposit ( d . , ) resuspended in 375 ml . 0 - 005 m phosphate buffer at ph 7 - 5 to 8 - 0 containing a few mg . 
harris constant standard could be obtained or that the subsequent comparison would be unaffected by such factors as the season of the year or the nature of the medium in which the virus was suspended . 
the first group ( " limiting dilution " methods ) had , also , the advantage of giving some degree of absolute measure corresponding to the actual number of tumour - producing particles . two injection routines have been favoured by prev - ious workers ; either the intradermal or the intramuscular . 
the former allows several different dilutions to be tested in the same bird , but accurate localization of the injection is rendered difficult by the paper - thin structure of the avian skin . moreover , the frequent occurrence of resistant fowls necessitates replication of the tests . intramuscular injections involve a more readily standardized reaction site , but 6 - week - old birds frequently show a variable and unpredictable resistance to the virus . such resistant birds may not react to 1000 times the virus dose which will produce tumours in susceptible birds , and if the end - point , ( m.i.d. ) is dependent , as may be shown , upon a poisson distribution of virus particles , then a large number of 6 - week - old fowls would be required for an accurate assay . it is inconvenient to use large numbers of birds of this age for a single assay and for such reasons the use of embryos or of day - old chicks , which may readily be obtained in large numbers , was investigated . 
the survivors are killed and examined at 28 - days . sudden deaths in the young birds as a result of the " haemorrhagic disease " described by milford and duranreynals ( 1943 ) did not occur when the inoculum was given into the leg muscle . irregular results were obtained in the assay when the . 
harris trypsin ( which removes more than 60 per cent of the non - virus proteins ) gives a product containing only 2 per cent of the nitrogen of the clarified tumour extracts , but possessing almost ar the virus activity . 
a biological assay method using very young chicks has beerr developed . these chicks are found to be more sensitive to the virus than older birds and are obviously more convenient to handle and maintain . the authors wish to express their thanks to professor a . 
the tumour extended outwards for 1 - 5 cm . into the surrounding lung tissue , and in collar - fashion for 4 to 5 calong the bronchus , its branches and the contiguous blood vessels . ' the cut surface of the tumour was finely patterned by white striae , which separated softer , yellow or grey areas . 
the lymph nodes in the hilus of the left lung were replaced by a large mass of partly necrotic tumour tissue which , extended into the posterior mediastinum , compressing and invading the adventitia of the great vessels and of the oesophagus . 
numerous deposits of tumour , up to 3 cin diameter , were present in the left pleura , especially over the intercostal spaces , and the pleural sac contained approxiniately 300 ml . 
the mucosa was fixed to the tumour and slightly elevated ; superficial ulceration involved an area of 2 - 5 x 2 cthe lumen of the bowel was not obstructed , and the mesocolic lymph nodes appeared to be normal . the peritoneal cavity contained a small amount of straw - coloured fluid ; the serosa was sparsely studded with tumour deposits which measured up to i cin diameter . 
the surviving liver tissue was deeply bile - stained and congested . the lymph nodes around the coehac axis and head of the pancreas , in the porta hepatis and in the lesser omentum were enlarged , and their substance was replaced by tumour tissue . 
the para - aortic and iliac lymph nodes were involved to a less degree . there were no relevant abnormahties of the other abdominal and pelvic viscera . the body of the ilth thoracic vertebra was almost completely destroyed by tumour , and had collapsed , with consequent pressure upon the spinal cord . 
the appearances were characteristic of an anaplastic bronchial carcinoma " of the " oat - cell " type , and confirmed the macroscopic diagnosis of primary bronchial carcinoma . the greater part of the colonic tumour was . 
the mucosa overlying the mass , however , showed an abrupt transition at its periphery from the normal structure and cytology to a polypoid , hyperchfomatic columnar - cefl adenocarcinoma , irregular gland - hke extensions of which penetrated deeply into the tunica muscularis ; the structure and cytology were those ' of a primary colonic adenocarcinoma . 
the patient remainecl in seemingly good health for more than 18 months , and then sustained a fracture of the pelvis in an accident . nine weeks later an abscess developed deep in the inter - ischial region , necessitating incision ; its contents were not examined . during the next fortnight a papiroma , about 0 - 8 cin diameter and situated at the medial end of the left inguinal skin fold , began to grow rapidly ; it doubled its size within this period , after which it was excised . 
the patient then remained apparently well until 1949 , when she began to be dyspnoeic ; - examination showed a right - sided pleural effusion . clear , amber - coloured fluid was aspirated 5 times , at intervals of a few weeks , the quantities withdrawn ' ranging from 550 to 1200 ml . the fluid contained between 4 and 5 per cent of protein ; a few cehs were present , mostly lymphocytes , with a very small pro_portion of neutrophile polymorphonuclear leucocytes and normal serosal cells . aerobic and anaerobic cultivation on ordinary media yielded no growth , and , cultures for mycobacterium tuberculosis were also sterile . radiography showed no evidence of neoplasm in the chest , but the effusion persistently obscured the - greater part of the right lung field ; there was never any demonstrable effusion in the left pleural sac . as there was no evidence of recurrence or metastasis of the carcinoma , and as there was no demonstrable infective cause of the recurrent pleural effusion , the discovery of an adnexal tumour on pelvic examination suggested the possibifity that the con ' dition was an at pical variant of the meigs - culhngworth syndrome . 
at laparotomy , 6 months after the onset of dyspnoea , a cystic tumour of the left ovary was fou ' nd and removed ; there was no other abnormality in the pelvis or abdomen . 
the tumour of the left breast was a simple intracanaficular fibro - adenoma . macroscopically , the ovarian tumour was a unilocular cystic teratoma , i 1 cm . in diameter , filled with sebum - hke material and hair . 
the cyst had a smooth lining ; its war was i mor less in thickness , except near the pedicle , where there was an irregular , bone - hard eminence , approximately 2 cin diameter , projecting into the cavity . adjacent to the pediele the remnant of the ovary was identifiable on the external surface of the tumour as a flattened , ovoid area of firm , greyish - white tissue , which measured 3 - 5 x 2 - 5 csuperficially , and up j . 
symmers microscopically the cyst was lined by keratinizing epi - to 0 - 4 cin thickness . dermis , external to which was a narrow zone of fibro - fatty tissue . hair - follicles and large sebaceous glands were numerous in the skin covering the hard projection , which consisted of bone , bron ' chial structures and neuroglia . 
the third hypothesis seems to us to be the only one tenable . if the coexistence of two primary neoplasms is accepted , the significance of the presence of a metastatic deposit from the primary bronchial carcinoma . at the site of the primary colonic carcinoma requires to be considered . this association may have been fortuitous , or the presence of the colonic neoplasrn may have predisposed to the establishment there of the metastasis , or the development of the latter may have inducea cancerous change in the adjacent mucosa ; it seems to be impossible to say which suggestion is correct . 
the induction , by some effect of the existing neoplasm , of sarcomatous change in the stroma of carcinomata has at times been postulated in explanation of the genesis of so - called careinosarcomata : in view of the considerable doubt as to the origin of the sarcoma - like component of these tumours , such a hypothesis has httle practical value . 
the induction of one carcinoma by another was postulated by duboisferri ' ere ( 1939 ) , who suggested that hepatic metastases in his case of islet - cell pancreatic carcinoma had induced local development of bile - duct carcinomata . he was not able to exclude the possibihty that the adenocarcinomatous areas in the liver were metastases with an unusually well - developed tubular structure , such as is sometimes seen in islet - cell neoplasms . seecof ( 1924 ) reported a case which was in some respects similar to ours . 
the tumour incidence in the experimental group ( 9 / 22 , or 41 per cent ) is thus not significantly in fact , a slight falling off in the tumour lower than in the control groups . incidence might have been expected in mice that were 43 weeks older than their controls at the time of tumour development . 
some important records of pedigrees , showing apparent . transmission from mother to daughter , were published nearly a century ago . the most remarkable was recorded by broca ( 1866 ) , in which four generations of females had the disease . in spite of evidence brought forward by a great many observers that mammary cancer can frequently be found affecting several of the female members of a family group , doubt , still remains as to whether heredity plays any significant ' aetiological part . this doubt arises because of the great prevalence of the disease in the general population , where it is responsible for nearly 3 per cent of all female deaths . 
with so comm ' on a condition , it is difficult to demonstrate convincingly that the occas ' lonal familial conoentration is not merely the result of a random distribution of cases . to obviate this uncertainty , most investigators in recent years have collected a series of cases , noted the incidence of similar disease among their relatives , and compared it with the corresponding incidence among a control group of cases free from the disease , selected so far as possible at random . in spite of elaborate precautions , - the difficulties , involved in obtaining a satigfactory control group of family histories to match those of the propositae , have repeatedly proved almost insuperable . 
the result is that many workers ( hanhart , 1943 ; passey , 1942 ) still ' consider that there is , insufficient evidence to prove the reality of familial transmission of mammary cancer or of aay type of human ma " lignant disease . stocks and karn ( 1933 ) found no evidence that a history of cancer in relatives increased the risk of cancer to anymeasurable extent . several extensive surveys have been published , all of which give some evidence for believing that mammary cancer is unusually prevalent amon the mothers and sisters of affected cases . 
the patients were attending either university college ilospital or the london hospital , and they were personally interviewed . all possible hospital records and reports were searched or checked by correspondence . this procedure applied b6th to the propositae and to any of their relatives who were reported to have suffered from malignant disease . unfortunately , in a number ' of instances , records had been destroyed by enemy action during the war . ideally , the death certifica ' te of every relative believed to have died of malignant disease should have been obtained , as corroborative evidence , but th ' is we have not been able to do . 
the distribution of these 116 " familial " cases and the mean age of onset of their illness ( 5 1 0 years ) was extremely similar to that of the this finding does not support jacobsen 's remaining " non - familial " cases . assertion that familial cases have an earlier onset than others . 
the infertility ratio , 91 out of 408 , or 22 - 3 per cent , significantly exceeds the value of 17 - 3 per cent for married women of the same mean age given by the registrargeneral ( 1938 ) , and supports the view that nulliparity is an etiological factor . the mean age of the patients when first interviewed for purposes of this this indicates that they had , on the average , lived survey was 55 - 2 years . undoubtedly three - and - a - half years since first gigns of the disease had appeared . the survival of the patient was a property , which determined her inclusion in the investigation , and the longer the survival , the more probably would she be selected as a proposita . 
the date of death and the age at the time were recorded in a few instances the dates were approximate , but could hardly for each case . be more than a few years in error . 
the expected proportion of deaths due to mammary cancer and other types of malignancy in each year and at each age can be obtained on the basis of the general population statistics . 
the distribution of deaths by year and age is shown in table iii . from ' this table the number of deaths due to mammary cancer if the women had been subject to the mortality in the general population was estimated at 11 - 17 , a figure significantly less than the observed 25 . 
51 observed as compared with 48 - 76 expected . there were 102 mothers still living at the time of investigation a ' nd , of these , six were know - n to be under treatment for mammary cancer while four had some other type of malignant disease . 
hence any likeness of onset age of mammary cancer in sisters is not peculiar to this type of malignancy . another interesting correlation concerns the sites affected in pairs of sisters and other relatives . 
the tracheo - bronchial glands may occasionally arow small amounts of the carbon to pass through into the thoracic duct or into lymphatic efferents from the glands which communicate directly with the great veins within the thorax , and thus the carbon reaches the such carbon in the blood stream is taken up by the cells of the blood stream . reticulo - endothehal system in organs hke the spleen and hver , which very occasionally at autopsy may show shght stippling with carbon , though in six spleens examined chemically we have found no weighable carbon in our residues . the amount of carbon deposited in a lifetime in the lungs and tracheo - bronchial glands depends on many variable factors such as age and occupation . 
where the degree of atmospheric pollution is great the excessive amount of carbon inhaled clogs the ciha of the respiratory mucous membrane and a large amount is not expelled even in the normal subject . in the subject who suffers from chronic catarrhal inflammation of the respiratory mucosa , there occurs ' a progressive destruction of the normal ciliated epithelium which is partially replaced by a non - cifiated type of a lower order , such as cuboidal or even squamous cens , which are unable to expel carbon particles . 
the scarring so caused is usuary the seat of dense areas of diffuse fibrosis resulting from bronchiectasis accumulation of carbon . or from the imperfect resolutions of acute pneumonias are often relatively free thus the only from carbon . 
why this should be so is not altogether clear . accurate means of assessing the total amount of carbon in the lungs and tracheobronchial glands as a whole is by estimating chemically all the carbon in these structures , and it is from this angle that we have approached the problethe amount found in these organs at autopsy thus represents what has been retained during a hfetime . 
the carbon itself may be innocuous , but from our studies of atmospheric pollution ( goulden and tipler , 1949 ; warer , 1952 ; goulden , kennaway and urquhart , 1952 ) , one can form some idea of the amounts of the carcinogens , 3 : 4 benzpyrene and arsenic , which would accompany it , and may possibly have some effect , either in association with , or apart from , that of tobacco . the black coloration of the lungs and bronchial glands must have become increasingly famihar to british pathologists since the industrial revolution which in the earlier part of the nineteenth century converted britain , by the utilisation of coal and iron , from an agricultural to a largely industrial country . although abundant material has been available in britain during the last 150 years , no one has , so far as we know , attempted to estimate the amount of carbon in human lungs , except in the special case of coal - miners ( king and gilchrist , 1945 )  . 
a questionnaire on the following points was circulated : sex ; age ; diagnosis and post - mortem findings ; places of residence for more than three years and the period lived in each ; occupations followed for more than three years ; the smoking history as regards the use of a pipe or cigarettes ; the age at which smoking was begun and stopped ; the maximum and average amounts smoked per day , and whether inhalation was practised always , occasionally or never . considerable difficulties were encountered in obtaining lungs from persons without bronchial carcinoma for controls ; those were needed from whom an accurate history could be obtained , but who were not likely to live long . 
joules of the central middlesex hospital , london , for the supply of material ; also to professor neils dungal of reykjavik for the lungs of eight persons who had always lived in iceland . 
the pulmonary glands in the lung substance were separated as far as possible from lung tissue ; no claim is made that this division , was carried out very accurately , but the much lower concentration of carbon in the lung makes some contamination with it unimportant . 
the separation oflymph glands is of course still more difficult when a large invasive mass of bronchial carcinoma is present ; the practice has been to regard as lymph glands any deep black stippled areas of simflar size and shape , and to class the remainder as lung . 
histology. - the lungs submitted to us had been fixed whole in formalin and thus the fixation from a histological point of view was not perfect . blocks of lung tissue were nearly always taken from the pleural region and from deep in the lung substance , and occasionally from the tracheo - bronchial glands . these blocks were subsequently refixed in formol - corrosive and the paraffin sections were stained by haemotoxylin , by carmalum or neutral red , by van gieson or mallory , and , in a certain number of cases referred to later , by potassium ferrocyanide to demonstrate any iron . 
the cases from hospitals in london ( tables ii and iii )  . - the places of residence and occupations given are those of longest duration only . in the case of persons hving in london , the part of london in which they hved is stated in brackets after a capital l ; for those living in other towns , the county is given i " cigarettes " expresses tobacco consumption in this form even where brackets . cigars or a pipe were smoked . 
tobacco is equivalent to 28 cigarettes . the small number of cases prevents any firm conclusions about differences in the amounts of carbon in the lungs ofpersons with and without bronchial carcinoma . these differences are not statistically significant , but are nevertheless of interest . the average amount of carb ' on in the lungs of females with bronchial carcinoma was greater than that in the controls ( table iii ) , although the carbon content of the glands did not differ appreciably between the two groups of women ; one might expect this result if some carcinogen such as 3 : 4 benzpyrene adsorbed on to the carbon particles in soot is concemed in the aetiology of lung cancer . 
the again , the carbon in two groups of men , however , showed the opposite result . the glands was approximately the same for the two groups , but the control group had the higher carbon content in the lungs . there was no apparent reason why men and women should differ in this way . the total carbon content of a lung must vary according to three chief factors , ( a ) age , ( b ) the size of the lung ( since a larger person will ' mspire more air and hence more carbon ) , and ( c ) the environment in which a person lived . allowance for the first of these variables is easily made . 
when the weight of carbon in the lungs and glands was divided by the age ( table iv ) the differences between the four groups remained and were still of the same order . 
the cases from iceland ( table v . ) - since these , specimens did not include the whole trachea , some of the bronchial glands may not have been present ; the figures in table v therefore represent the carbon and ash content of the lungs and glands together . the content of both carbon and ash in the women - 's lungs was considerably greater than that in the men 's , apart from the exceptional value for the man no . 
carbon in lungs / age in years . 25 - 1 ( 12 - 6 ) 13 - 8 15 - 4 11.9 bracketed averages include the abnormal figures for no . 
50. tthis very high value for a - sh was omitted from the average . microscopicary , no silica was found in sections of the lung . those obtained in london , shows that the lungs of icelandic men had a lower average carbon content , while those of the icelandic women had a much larger amount . 
the same differences obtained on correction of these figures for variation in age distributio ' n . the presence of such amounts of carbon in these lungs from iceland seemed at first sight surprising , for in iceland general atmospheric pollution as understood in this country is very small . there is no native coal or oil , and these are costly to import . 
was a non - smoker . in the group of women with bronchial carcinoma , i out of 5 did not smoke , and in the control group , 9 out these figures , especiary for the women , show a higher proportion of nonof 1 1 . smokers among persons without bronchial carcinoma . 
the total carbon content of the lungs varied over a wide range all the points indicating a total consumption of more than for non - smokers ; 200 , 000 cigarettes refer to men and women with bronchial carcinoma . 
the average daily consumptions were : males with bronchial carcinoma , 24 / day ( 16 cases ) ; males without bron ' chial carcinoma , 12 / day ( 5 cases ) ; females with j . 
urquhart bronchial carcinoma , 19 / day ( 5 cases ) ; and females without bronchial carcinoma , 2 / day ( 10 cases )  . ash content of the lungs and bronchial glands . the nature of the ash deposited from the hydrolysates of these lungs has not although about 10 per cent of the total carbon was contained been determined . in the glands , the fraction of the total ash present in them was about 20 per cent . this suggested that the bronchial glands were even more efficient in concentrating particles of mineral matter than they were in accumulating carbon particles . 
for comparison with the histological findings the chemical results were divided into three groups , namely , those with a carbon content of ( a ) 0 - 49 g . 
the carbon in the sections showed the normal distribution , being heavier under the pleura , around the bronchi and blood vessels and in the fibrous septa , than in the vesicular portions of the lung . the rocks of iceland are of volcanic origin and the roads are metaned with such material which , due to weathering , quickly breaks down so that with the passing probably road dust has become considerable of traffic much dust is created . in iceland , where there are now 11 , 000 motor vehicles , only since the acquisition in view of this the icelandic lungs were of american cars after the last war . submitted to a more intense microscopic study for the presence of sihca and of iron . 
no evidence of silicosis was noted in any and doubly refractile granules of in only one case was iron silica were very scanty or absent in all the sections . found , apart from occasional heart - failure cells in the others . in this case the iron was present chiefly in the fibrous tissue of the septa and around the bronchi and blood - vessels in close association with carbon particles which the iron pigment appeared to surround . 
is retained ; this gives some measure of the efficiency of the nasal filter and of the mechanism by which particles which have reached the deeper respiratory passages are swept upwards again by ciliary action and expectorated or swallowed . 
when the floor of a room is swept towards the door , the part adjacent to the door is exposed to dirt from a much larger area . similarly , when particles of dust are arrested on the walls of the smaller bronchi and bronchioles , they are swept upwards by ciliary action through the main bronchi ( the door ) into the trachea , and carcinogens might diffuse from the particles into the layer of mucus covering the bronchial mucous membrane during transit . 
some such mechanism would be in accord with the statement about the site quoted above ; but some recent investigators ( westermark , 1938 ; raebum , 1951 ; raebum and spencer , 1953 ) think that tumours arise - with about equal frequency in any part of the lung . the estimations of arsenic and of 3 : 4 benzpyrene in the air of large towns given in table viii are those of goulden , kennaway and urquhart ( 1952 ) and table viii . - concentrations of arsenic and of 3 : 4 benzpyrene in town air . london ( becton ) bilston manchester liverpool . sheffield london ( county hall ) hull bristol mean concentrations , pg . 
a8203per cigarette ( daff and kennaway , 1950 )  . ini ' mum amount of a strong carcinogen such as i : 2 : 5 : 6 ' dibenzanthracene which will produce * our earlier results , which were confirmed subsequently , were demonstrated at a meeting of the pathology section of the royal society of medicine held at this hospital on january 15 , 1952 . shear ( 1936 ) found that the j . 
of 3 : 4 benzpyrene which may be inspired in ' a lifetime , but in view of the vastly greater area over which this may be spread and the different time relations , one may doubt whether this comparison has any value . the size and arrest of smoke particlm . the available data about the arrest of smoke particles in the air passages are very defective because we have not , to begin with , any adequate information about the size of these particles . 
the passages quoted verbatim below contain all the information that we have found on this subject . the leicester report says : " the size distribution of smoke is a subject which still requires investigation . green ( 1936 ) gives estimates for five industrial dusts from which it can be inferred that 50 per cent by weight of such dusts is less than 3 to 10 microns , and 80 per cent is less than 5 to 15 microns in mean diameter . atmospheric smoke may be similar in size distribution but a direct investigation is  . 
some of the properties of smoke depend on the area it can cover ; half the surface or sectional area of the smoke was in particles larger than about 0 - 35 micron . there is a distinct tendency for smoke particles to stick together in chains , perhaps one micron long . " the comparison of such results obtained by different methods of arrest is not e . 
are in the range of size where there is minimum retention in the lung , 80 per cent of them being exhaled and 20 per cent arrested in the alveoli . ( b ) the mean diameter of smoke particles and the range of particle size of carbon black which shows the greatest adsorption of 3 : 4 benzpyrene from benzene ( falk and steiner , 1952 )  . 
though the material was not statisticary adequate , the smoking histories of the cases examined showed a higher consumption of tobacco among both men and women with bronchial carcinoma compared with the controls . 
if the carbon in the lungs and bronchial glands does not leave the body by any channel the amount found post - mortem represents what is retained out of the intake of a lifetime . 
the data now available for atmospheric poflution in some english towns arows an estimate to be made of the amount inspired during a hfetime in them ; this might be of the order of i 00 g . , while the quantity found in the lungs and bronchial glands was approximately 0 - 5 to i - 0 g . 
estimations of 3 : 4 benzpyrene and of arsenic in the suspended matter of the air of various english towns enable one to calculate the amounts of these compounds which would accompany the carbon inspired , and retained , in a lifetime . we wish to express our gratitude for the materials and case histories which have been the subject of this investi ation to professor niels dungal of revkiavik ; to horace joules of the central middlesex hospital , london , and to two members of his staff , a . 
althougih biologists have for long been uneasily aware of the existence of a problem of cell heredity , its systematic analysis is the work of very recent cell heredity deals with the origin and maintenance of inherited character years . differences between cells ; more particularly , between cells that set up division its problems present themselves in their most acute , lineages by mitotic fission . if not most easily workable , form in metazoan development . the outcome of cellular differentiation in ( say ) mammalian development is the formation of a limited number of histologically definable cell genera , each one further subdivided into a variety of cellular genetic species . 
the genus " fibroblast , " as yet far from completely analysed , may be subdivided into cells which manufacture bone , cartilage , white connective - tissue fibres , and so on . genus " epidermis , " of which we have made a particular study ; includes the epidermal epithelium of the superficial skin , of the sole of the foot , the tongue , cells of each of these epidermal the claws or nails , the vagina and the cornea . species display a distinctive combination of structural or physiological properties . the epidermis of the sole of the foot , for example , has a characteristically high rate of cell division . 
but this has proved not to be the case . although plausible " phenocopies " of sole epithelium ( in the form of corns and callosities ) can be made by irritating the thin and relatively quiescent skin of the body surface , the difference between the division rates of sole and body epidermis is in fact " intrinsic " and inheritable . 
we have , for example , transplanted sole epidermis to positions in the body where it is protected by neighbouring hair and secure from mechanical irritation ; but even so , its characteristic growth rate has been maintained for at least two years , and thick pads of now functionless cuticle continue to form over it and may periodically be removed . claw epithelium tells the same story - more clearly , since the difference between claw and body epidermis is anatomically crude and obvious . 
comeal epithelium is very transparent , and its mode of cell - packing and cuticle formation is characteristic . these properties might well be the outcome of the peculiar situation in which it lives - lying taut over the unvascularized comeal dermis , relatively low in temperature , and nourished under conditions of ' ow 02 tension by the aqueous humour , and perhaps to some extent through its outer ( cuticular ) surface as well . if the difference between comeal and body epidermis were merely a difference of " nurture , " then the heter6topic transplantation of the comeal epidermis to ( say ) the chest wall should bring about its reversion to the cell type characthis does not happen . 
expand its substance and area fifty - fold by cell division and outward spread ; but it still retains its characteristic cell packing , mode of cuticle formation and exquisite transparency , so that fine regenerating dermal blood - vessels corneal grafts have are to be seen through it as clearly as through a window . proved to form a rather unstable epithelium , which never seems to achieve the firm union with the underlying corium characteristic of body skin . for this reason we have not yet been able to " ' cultivate ' " corneal epithelium as a heterotopic graft long enough to be quite sure that ' it represents a distinct epidermal species . 
we can only say that it has given no hint of reversion after fiv ' e weeks of cultivation . in general , the technique of tissue culture confirms the results of heterotopic transplantation ; but it is far less critical in its discrimination between cells of different species , because cultivation in vitro has a tendency to make different ciiltivation in vitro is not known to bring about any cells look rather alike . inherited change in differentiated cells , unless we take into account the fact that prolonged and rapid growth may perhaps cause individual cells to lose specific properties in the " growth rate dilution process " recently reported on by preer ( 1946 )  . 
the " antigenic simplification " ( gorer , 1938 , 1942 ) and anaplastic tendencies of long established and ra idly growing transplanted tumours may conceivably represent a phenomenon of similar origiia . it will be clear from the foregoing examples that the techniques we have so far used to discriminate between the several true - breeding species of epidermal cells are , in their use of heterotopic transplantation , formally identical with those used in classical experimental embryology to distinguish between cell systems undergo ' ing " self " and " dependent " differentiation . 
the study of cell heredity is likewise , in effect , the study of cellular transformation - that is , of the origin of inherited differences between the phenotypes of cells . from our point of view , the most important of the several different types of cell transformation is that which , in a rough preliminary classification , has been called infective ( medawar , 1947 )  . 
the second , a corollary of the first , is that infective transformations can be serially propagated - a cell once transformed can in its ttirn transform another . the special importance of infective transformations is that they provide clear direct evidence of the particulate nature of cell heredity , and so , indirectly , of the fact that the differences between cell species are combinatorial in nature and not smoothly blended . 
 ' the authors ' work on the inheritance of colour differences in spotted guinea - pigs ( as , in the cells of guinea - pigs ' skin is relevant here . apparently , in spotted pigs and friesian cattle ) the - pigmentation of the black skin areas can be seen during life to encroach upon the white . 
the spread is even in density and snioothly progressive , but it notmally leaves untouched the liairs of the white area that are being encroached upon , so - that - the transitional zone of black skin that was formerly white is distinguished by bearing white liairs on a black skin background . 
our interpretation of the phenomenon ( billingham and medawar , 1948 ) is , in outline , as follows : the origin and seat of pigmentary function in black or other coloured skins is the pigmentary dendritic cell , a branching cell which lives in the lower reaches of the epidermis and which in some unexplained manner " feeds " - or , as masson ( 1948 ) puts it , " injects " - rnelanin granules into the malpighian layer cells on which its branches end . pigmentary dendritic cells form a facultatively syncytial system , since the branches of neighbouring cells are sometimes confluent , and a branch from one dendritic cell , instead of ending on a malpighian cell , may sometimes apply itself to the cell body of another . in the white areas a dendritic cell system is present which is similar in every respect save one : the cells lack both melanin granules and the enzyme apparatus required for making it , and no form of merelv physical stimulus will cause them to acquire pigmentary ftinction . we have shown , not yet with final precision , that the phenomenon of pigment encroachment or spread at the pigmentation boundaries of spotted guinea - pigs is due to the transformation of white ( non - pigmentary ) dendritic cells into melaninforming cells by contact with their pigmentary neighbours . 
the transformation , once achieved , is permanent in the transformed cell and its division lineage and a white cell transformed to pigmentary function can in its tum tramform its neighbours . 
we - have now to ask whether the pigment - spread phenomenon we have analysed is a mere curiosity of nature , or whether it exemplifies a principle of general importance . the experimental system we bave been using is a very unusual one indeed , for the following reasons : most cells liberate their secretory products externally or into the general circulation . dendritic cells pass their secretory product , melanin , into the cells around them - " cytocrine " activity , as masson calls it . their neighbours are norinally the malpighian cells of the epidermis ; but , as inlasson lias pointed out , dendritic cells may also inject melanin granules into cells of quite different origin - into the cells of gut epithelium for example , if a tumour metastasis happens to bring them within reach of its branches . 
the casual leakage of enzymes from one cell into another would make nonsense of embryo ] ogical de - velopment ; no organ could exist under these conditions , with its great variety of cells of different types in intimate physical contact . 
an infective transformation can only be secured by taking advantage of peculiar cell properties , like the cytocrine properties of dendritic cells and their transiently syncytial lay - o - ut ; of peculiar animals , like spotted guinea - pigs ; or of technical tricks , like grafting claw epithelium to the skin of the chest , or - to choose an example from the illuminating pioneer work of sonneborn ( 1943a , b ) - by delaying the conjugation process of paramecium so that the conjugant pairs may 130 r . 
the relevance of these ideas of cellular heredity to cancer etiology has been dealt with by other speakers , so that only special aspects of the problem need be considered here . the centre of gravity of modern speculat - ion about tumours has altogether shifted from - the idea of the malignant cell as one which proliferates faster than its neighbours , towards the simpler and more general fact that it has acquired their rapid rate an inherited character difference from its normal homologues . of growth and their invasiveness may be the most important clinical facts about tumours , but they are not necessarily the most important biological facts . suppose , now , that we accept one variant or another of the view , that malignant cells differ from their normal homologues by the multiplication within them of an exogenous or proprietary virus or mutant plasmagene . if that view is correct , the infective propagation of tumours should be the rule rather than the ( for if we disregard the intervention of virus - like particles in rare exception . the initiation of mammary tumours in mice and in the propagation of the brownpearce carcinoma ( kidd , 1946 ) , the list of tumours or tumour - like growths initiated by viruses seems to be exhausted by the shope papilloma and a variety of chicken sarcomas . ) but what has been said above about the inherent difficulty of demonstrating infective transformationsin normal cells applies without any qualification to those which happen to be mahgnant . 
the limiting factor must be supposed to be , not the actual ' rarity of tumours rendered malignant by the propagation within them of specific virus - like particles , but our technical facilities for extracting them from malignant cells and introducing them into normal cells of a type in which they are competent to flourish ' ; in other words , not the material existence of such particles , but thoir reluctance to display infective behaviour . luckily , infection techniques are not the only ones . 
at our " disposal ; kidd 's ( 1946 , 1948 ) subtle serological analysis of the brow - n - pearce carcinoma has the great theoretical merit of overcoming their almost crippling defects , though it has certain limitations of its own . the interpretation we have put upon the phenomenon of pigment spread in guinea - pigs ' skin was said earlier to have been incompletely demonstrated for this vory reason ; altho - ligh we have the strongest circumstantial and anatomically factual evidence for supposing that the transformation we have been studying is infective in character , we have not yet been able to produce a cell - fiee extract ofpigmentary dendritic cells which will convert white dendritic cells to pigmentary function . 
of extraction are formidable enough ; we may be dealing with a small battery of oxidases , and the existence of at least two , a tyrosinase and a dopa - oxidase , is likely . 
to reproduce its action artificially cannot be technically easy ; we may be obliged in the end to resort to the crude solution offered by microinjection methods . in spite of the rarity of overt cases of infective propagation of tumours , it may nevertheless occur under circumstances in which , being clinically of trivial if the so - called melanotic carcinoma of significance , it has been overlooked . skin is in reality a tumour of dendritic cells , then it is at least conceivable that some melanomas spread infectively by the transformation of their non - malignant neighbours . such spread would be so slow as to be wholly outweighed in clinical importance by infiltrative growth and metastasis , but clinicians may know of certain peculiarities of melanotic growths which such a concept would help to it would be a mistake here , of course , to suppose that the melanotic interpret . character of such growths was itself of anything but secondary importance : some melanomas are recognizable histologically which are barely melanotic . epidermal dendritic cells of human beings , barring albinos , can be provoked into manufacturing melanin ; and an irreversible malignant or pre - malignant change might be initiated in normal dendritic cells by the cytocrine activities of their malignant neighbours before their rapid growth , and rapid elaboration of melanin , gave outward evidence of the fact . since this is a privileged occasion , speculation may be allowed to become wilder still . 
the ordinary literature of tumour transplantation is unhelpful on this point , since stock epidermal carcinomas are normally propagated by subcutaneous ( or some other form of heterotopic ) transplantation from mouse to mouse . 
my reasonsfor considering that the risk is a real one , and of importance , are perhaps best discussed in relation to my own experiments , as i am naturally most familiar with this material . i was iniectin - a dibenzanthracene into some dominant ( lethal ) yellow mice of mainly inbred type , and intercrossing for up to four generations , in order to study the type . 
the tests were on 400 - 800 f . , these results , though consistent , are not significant , males in each group . statistically tinless all three treatments are combined into one group . 
of some interest was the incidental finding of two visible mutants found in the casual inspection of cib c - liltures , which would be an extraordinary occurrence bv rhe drosophila work , too , indicated that the mutants were mere chance . appearing in sperm after removal from the mutagen . 
carr which tends to mutate only those loci that require a relatively small energy this would also imply that these comchange to give a new stable mutation . pounds will tend especially to mutate genes that have already mutated sponthus , although not really specific for any given gene , their effects taneously . may be normally limited to only a small fraction of the genes on the chromosome . but i think that there is a risk with chemicals not encountered with radiation , and this is that a given chemical can react with all , or at any rate a major part of the genes of one type in the animal exposed , as this is merely a repetition of a possible chemical reaction . 
then they might also react with the same genes in another exposed individual equally well , and thus the offspring of two people exposed to carcinogenic mutagens could immediately show an induced recessive . this gives a new problem in human genetics - inbreeding with an occupation . and i have suggested that mutant sperm may be produced long after exposure i don ' t know whether any data on this could be accuto the mutagen ceases . mulated - all i can recall in the literature is the so - called " industrial melanism " of insects described by harrison ( 1920 ) , but i think we should be aware of the risk . 
jackson memorial laboratory , bar harbor , me . received for publication december 16 , 1948 . it is known from the work of andervont and bryan ( 1944 ) and green , moosey and bittner ( 1946 ) that the milk agent is antigenic for the rabbit and the rat . if , as is probable , the agent , is a virus one would expect it to be antigenic for the susceptible species . 
for 30 minutes and then cooled prior to incubation . in experiments 4 and 5 serum from hyper - immunized blacks was used . c57 black mice were injected at weekly intervals as follows : 0 - 1 ml . , 0 - 2 ml . , 0 - 4 ml . , 0 - 8 ml . , 0 - 8 ml . , 0 - 8 ml . 
a few papillomata also occurred . there is a suggestion of neutralization in experiment 2 . however , the probability of obtaining such a result by chance alone is of the order of 10 per it is specially regrettable that this experiment was terminated , as a higher cent . degree of significance might well have been obtained . on the other hand , there is no suggestion of any neutralization in experiments 4 and 5 , where hyper - immune serum was used together with a relatively weak tumour fitrate . 
of special pertinence to experiments with serum from hyper - immunized blacks may be the observations of webster ( 1938 ) and hodes and webster ( 1938 ) with the virus of st . 
louis here a high degree of immunity developed about 6 weeks after encephalitis . vaccination , and had waned before antibodies appeared some 20 weeks after injection . it may be that we bled our mice too soon . possibly our technique was entirely inadequate to show any neutralization . however , identical techniques were used by andervont and bryan ( 1944 ) and green , moosey and bittner ( 1946 )  . 
a strain it may be that this very mice are being constantly immunized since birth . prolonged stimulation is needed for the production of protective antibodies . the fact that the agent can be demonstrated in serum may be due to dilution if this is . 
hamer. from the cancer research laboratories , department of pathology , the medical school , birmingham 15 . received for publication january 13 , 1953 . results for the analysis of thymus histone presented from this laboratory ( hamer , 1950 , 1951 ) have been followed by similar results from other sources ( daly , mirsky and ris , 1951 ; eadie and leaf , 1952 )  . these results all show the same general characteristics in composition but the actual values obtained vary appreciably from worker to worker , suggesting that either the methods of isolation or the source of the material may be affecting the final product . analysis of other nucleo - proteins have been reported by brunish , fairley and luck ( 1951 ) , liver histone , and by khouvine and baron ( 1951 ) , rat epithelioma histone . the last - mentioned paper evidence was presented that different treatments during isolation could yield products of varying composition . 
stedman and stedman ( 1951 ) have also reported qualitative and partial quantitative analyses of a number of basic proteins from isolated nuclei . in the work reported here further analyses of thymus histone are presented along with other results for the non - histone or acid - insoluble protein of the nucleus . all the fractions examined were derived from the same batch of thymus nuclei , and consequently it was possible to study whether the methods of fractionation used caused changes in the amino - acid compositions . 
of defatted calf thymus by the following method : the tissue was homogenised in a blendor , suspended in a total of 2 litres of saline citric acid solution ( 0.14 m nacl , 0 * 025 m citric acid ) , and then centrifuged , discarding the supernatant . 
the sedimented material was washed by alternate suspension and centrifugation in another saline citric acid solution ( 0.14 m nacl , 0 * 01 m citric acid ) , six times in all , the preparation of isolated nuclei was then free of whole cells and had no appreciable cytoplasmic the nitrogen to phosphorus ratio of such a preparation was contamination . 4 - 5 : 1 . 
next a tenth of the volume of a 5 per cent solution of sodium dodecyl sulphate ( sulphonated lauryl alcohol - glover ) in 45 per cent alcohol was added and the ph brought to 5 - 5 . 
the residue from the acid extraction was washed two further times with 0 - 2 m hydrochloric acid , and the remaining insoluble fibrous material was suspended in water , dialysed for 48 hours and freeze - dried ( fraction nr )  . the remaining half of the solution of nuclei in 1 - 2 m saline was shaken with an equal volume of chloroform - butyl alcohol ( 4 : 1 ) to dissociate the nucleic acid and the protethe nucleic acid remains in the aqueous phase ( from which it may be precipitated with alcohol ) , while the protein forms a gel at the interface . this treatment was repeated twice , combining the gel layers obtained . 
the gel was washed three times with the buffered 1 - 2 m saline , agitating and centrifuging each time , and then the protein was thrown down by adding chilled alcohol . 
the precipitate was extracted overnight with 01 m hydrochloric acid , and the acid extract containing histone was worked up as above to give fraction sh , the second histone specimen . 
the residue was washed further with acid and then suspended in water , dialysed and freeze - dried ( fraction sr )  . these four main fractions and the " whole protein " ( dp ) were then analysed . there was insufficient material in fraction r to permit a full analysis , but it is hoped to study this further at a later stage . 
extract with hci whole protein complex ( dp ) nucleic acid histone ( nh ) residue ( nr ) insoluble residue ( r ) shake with chc13 - buoh protein nucleic acid fig . 
the specimens had slightly lower nitrogen contents than those previously examined and contained about 02 per cent phosphorus . the specimen sr of non - histone or acid - insoluble protein was slightly yellowish , and contained a little less nitrogen and more phosphorus than the histones . was insoluble in water , but would dissolve at ph 9 - 10 to give a pale yellow solution 154 d . 
hamer and then precipitated out when this solution was acidified to about ph 5 - 5 . the course of this treatment some unit in the protein is apparently destroyed as evidenced by the production of a strong unpleasant odour on acidification . 
the related specimen nr contains , of course , much nucleic acid in addition to proestimated from the phosphorus content and the absorption spectrum tein . there is approximately 56 per cent nucleic acid in this fraction . in dilute alkalis an opalescent viscous solution is obtained . finally , specimen dp precipitated from the solution of isolated nuclei by sodium dodecyl sulphate has a nitrogen content of about 9 - 8 per cent indicating that the ratio of protein to detergent is around 3 : 2 by weight . 
the act of separating the non - histone protein from the nucleic acid apparently damages the protein , and it does not seem possible to obtain it in other than an insoluble and denatured forthis would suggest that the non - histone protein may be chemioally bound to the nucleic acid while the histone is held by a predominantly ionic union . 
some support for such a structure has recently been presented by fleming and jordan ( 1952 ) , who have found that the results of eleotrophoretic studies on thymus nucleoprotein in solutions of varying ionic strength could be explained by the existence of an equilibrium np np + p ' ( where np is the original nucleoprotein , np a nucleoprotein fraction with a higher nucleic acid content , and p ' a dissociated protein )  . 
however another possibility is that in the cell the histone is loosely combined with the non - histone protein and that acid treatment breaks the compound open . this would be in accord with the close similarity of amino - acid composition , but would require that all the cystine and tryptophane remain with one fraction on fission . 
the first possibility considered seems on the whole to be more probable , as it is more in keeping with the variation in the amounts of histone and non - histone protein in the nucleus of different cell types ( hamer , 1951 ; stedman and stedman , 1951 ; mirsky and ris , 1951 )  . in the case of the thymus nuclei studied here the histone and non - histone proteins occurred in roughly 156 d . 
it seemed likely that 1131 would prove so since it is specifically concentrated in the thyroid , whose cells are thereby submitted to a course of ~ - ray irradiation . 
the problem is important , sirice we do not yet know the carcinogenic hazard of 1131 in clinical medicine , where it is being increasingly used both tracer and therapeutic doses . 
hyperplasia and adenomas of the thyroid have been produced by goitro gens ( griesbach , kennedy and purves , 1945 ) , and by goitrogens together with the carcinogen acetylaminofluorene ( a.a.f. ) ( bielschowsky , 1944 )  . 
when the following work was started in february , 1948 , there were discrepancies in the literature as to the incidence of thyroid tumours in untreated rats and the carcinogenic potencies of goitrogens , which were due to the different rat strains , ages and living conditions , times of application of the in view of the discrepancies and in order drugs and criteria of malignancy used . 
the rats were killed by coal gas_ at an average age of 15 months , having had treatment for an average of 13 months ( including the rest period )  . 
the trachea and thyroid attached was fixed in helly 's fluid ; the thyroid was then dissected off the trachea and weighed to the nearest milligra the thyroids were embedded in wax , serially cut at 5 and mounted on to slides as ribbons of 8 to 14 sections , including both lobes . 
the number of rats used was 98 to obtain data for the main experiment , at the end of which the remaining 15 from various groups were put aside for study of the thyroid ! 131 uptake by direct measurement and autoradiography . 
 the indication of adenomas by plus signs in tables i , ii , iii , iv and v is not devised to be any more than a crude indication of tumour incidence ; the object was to seek for major rather than minor differences resulting from the various treatments of these small numbers of rats . 
 i have not given a detailed description of the histology and histogenesis of the adenomas , because in addition to the references cited in the introduction , there have appeared further detailed illustrated descriptions by purves and griesbach ( 1947 ) , money and rawson ( 1947 ) , laqueur ( 1949 ) , and hall and bielschowsky ( 1949 )  . 
 nil + + + + + + + + + nil female * number of months before the rats were killed when they received the first of their two injections + represents i adenoma per gland ; + + , 2 or 3 adenomas ; + + + , multiple adenomas . 
in many areas it was difficult to differentiate the adenomas , and the number of plus signs awarded in the thyroid adenoma column of table ii is probably an underestimate . 
however , the cells of these extra and intracapsular and juxta - venous follicles were quite innocent in appear ance , and in no way different from those constituting the main mass of the glands . 
the effect of the 1131 was such that every one of the glands in this admittedly small group showed a most striking increase in adenoma formation as compared with the rats treated with methylthiouracil alone . 
 female male 10 14 14 14 14 17 11 11 14 14 14 15 14 14 14 11 12 12 11 11 died killed died killed died kiiied di~d killed di~ died killed died killed 229 thyroid adenomas . 
 100 168 100 129 136 116 120 160 125 130 110 125 130 155 110 120 130 125 140 130 175 117 119 125 120 108 124 107 acetylaminofluorene + methylthi , ouracil + radioactive iodine . 
 the adenomas were larger in size as well as increased in number , and showed evidence of malignancy by a gross increase in size and by dissemination outside the thyroid in two instances . 
secondly , as shown by autoradiography ( leblond and gross , 1948 ; doniach and pelc , 1949 ) , there is a wide variation in concentration from follic ] e to follicle ; the peripheral follicles in the rat take up considerably less iodine than the central ones . 
the radiation effect~ are due mostly to radiation , since most of the y rays , which are much more penetrating , will not be absorbed in the small thyroid of the rat . 
they found that this radiation did not interfere at all with the ability of the thyroids to take up further iodine , produced no histological changes , and showed no alteration of thyroid function as gauged by various physiological tests . 
their rats averaged 40 per cent maximal uptake of psi , ours was nearer 20 per cent , and our total psi injected was 32 , theirs was 30 c . , therefore our dosage was roughly 15 , 000 roentgen equivalents physical . 
from the long - term view in our experiments this dosage proved damaging , as shown in table vi , where it can be seen that the thyroid weights of all psi treated rats in all groups was one - half to one - third of their respective controls . 
clearly though , enough thyroid had survived to maintain normal growth of the rats , since the body - weights did not differ from their respective controls in spite of the fact that the first dose of psi was administered at an average age of 2 months . 
methylthiouracil rat killed 12 days after cessation of 13 months ' treatment , showing intense blackening over t , he colloid of the normal follicles and less intense but definite blackening over the colloid secreted by the adenoma lying in the centre of the photomicrograph . 
this is the accepted mechanism for the goitrogenic action of anti thyroid " drugs , such as thiouracil ( astwood , sullivan , bissell and tyslowitz , 1943 ; mackenzie and mackenzie , 1943 )  . 
nevertheless , this nodular hyperplasia must be regarded as a definite deviation from the normal , akin to tumour production both on morphology , and on the grounds that it may lead to malignant change as shown by previous and the present work . 
this may well be due to the fact that the dosage of pat used was destructive enough to the thyroid temporarily to diminish thyroxine formation , and thus step up thyrotrophic hormone production . 
it is interesting to note in this connection that the same number of rats showed adenomas in the pat group ( table ii ) as in the methyl thiouracil group ( table iii ) , and that the number of adenomas was greater in the former group . 
 the problem remains as to why the controls show an occasional deviation from the normal in the formation of adenomata , and how does prolonged excessive thyroid stimulation by the pituitary intensify this deviation , and by what means additional carcinogen leads to a further increase in adenomas . 
experimentally , gorb man ( 1947 ) found hyperplastic thyroid tissue within the lumens of pulmonary vessels of chronically thiouracil treated mice as well as parenchymatous pul monary deposits considered to be probably thyroid in orig yet a return to normal diet after prolonged goitrogen treatment was followed by involution of the supposedly cancerous thyroids . 
one may assume , therefore , that two types of metastatic thyroid tissue exist , one , possibly resulting from a mechanical breakaway , still dependent upon thyrotrophic hormone for its survival , the other independent . 
neverthe less , from the host 's point of view the first type of metastasis may prove just as embarrassing as the second , so long as thyrotrophic hormone stimulation is main tained . 
prolonged thyroxine treatment , used in clinical medicine in the therapy of nodular goitre , may , by inhibiting thyrotrophic hormone production , not only shrink the goitre , but lessen the likelihood of mechanical production of thyroid metastases and of their survival . 
the radioactive iodine was found to significantly increase the formation of thyroid adenomas as compared with non ! 1 31 treated controls in the following groups : those treated with ! 131 alone , those treated with methylthiouracil and those treated with methylthiouracil plus acetylaminofl.uorene. 
some of the authors misrepresent their own data , misquote those of others , are defective in arithmetic and make the task of anyone who wishes to examine their conclusions difficult . some authors , when giving data for the incidence of cancer other than uterine , do not say whether the figures refer to one or both sexes ; and in statistics based on admissions to a hospital they do not say whether this is a women 's hospital or a general hospital , or where the hospital is situated , data which are not always easy to obtain in another country . 
the age distribution of these 7 cases was as follows : 30 - 40 , 1 ; 40 - 50 , 2 ; 50 - 60 , 2 ; 60 - 70 , 2 . the seven cases of uterine cancer in jewesses which appear in table ii appear to be identical with those in table iii . * this is a very high percentage , for which theilhaber shows no detailed evidence . deaths of women from all forms of malignant disease deaths from malignant disease of the uterus the registrargeneral 's statistical review ( 1935 - 1939 ) gives the following figures for england and wales for the five years 1935 - 1939 :  . 
sanders ( 1916 ) gives data from rotterdam ( table x ) which show a low incidence of cancer of the uterus in jews , and a still lower incidence upon members of two of the protestant churches ( christian reformed and reformed )  . 
about one per thousand of the population . the radiumhemmet serves a well - defined area of the country which covers about half of the population . since 1914 practically all cases of cancer of the cervix occurring within that area are referred to the radiumhemmet for treatment . from 1914 to 1947 inclusive i have seen about 7000 cases of cervical cancer . among those were three jewish women . 
he considered that this was almost certainly an hereditary factor and not likely to be due to special observances of the jewish law , the same facts being noted in unorthodox jews . * unfortunately karplus gave no figures in support of this statement . 
data showing an equally low incidence of cancer of the uterus , which is more difficult to demonstrate than a high incidence , in populations known to consist of orthodox , and of unorthodox jews must have been difficult to obtain and would be of great interest . * i am indebted to d . 
100 table xi shows that cancer of the uterus , reckoned as a percentage of all cancers ( fishberg does not make clear whether the figures refer to females only ) is less than one - half as common among jewish women as among other races , while cancer of the breast shows a smaller difference in the same direction . smith ( 1941 ) studied the racial incidence of 3106 cases of cancer of the cervix observed at the memorial hospital , new york , from 1916 to 1937 . 
he says : " in an attempt to compare the nationality incidence in cervical cancer in this series with that of patients suffering from other gynecological lesions , consecutive records as filed have been studied , but the numbers used in each classification are not the entire number of patients filed under that diagnosis ; i.e. , this is a cross section of the clinic in the years studied rather than the total number of cases in the clinic . " i have been unable to learn from the memorial hospital the system upon which the cases making up this " cross section " were selected . apparently a number of cases of other gynaecological conditions roughly equal in number ( 3062 ) to those of cancer of the cervix ( 3106 ) were taken . 
the results are summarized by smith in tables xii and xiii ( his tables iv and iva )  . the significance of these figures is affected by the very high proportion of benign tumours , hence in table xiv and xiva smith 's figures have been recalculated for the malignant tumours only . in table xiv the result of the whole investigation , which for some reason smith does not give , namely , the ratio of cervix cancers to other conditions , is calculated from his figures . 
3106 6168 3062 vineberg ( 1919 ) gives data for the incidence of cancer of the cervix based upon material from 80 , 000 female patients at the mount sinai dispensary and hospital , table xv represents an attempt to reduce his rather confused statenew york . ment to a clearer forthe higher proportion of cancer in the hospital cases as against those of the dispensary is attributed to better conditions for examination . vineberg ( 1919 ) points out that jewesses of the poorer classes , to which most of those dealt with in his paper belong , are most likely to be orthodox in religious matters . 
the authors suggest the possibility that this difference certainly the age at which some forms of cancer depends upon racial factors . occur is affected by genetic differences ; examples have been given in detail in another paper ( kennaway and kennaway , 1944 )  . but there is nothing in these data for age distribution to indicate whether the liability to uterine cancer , which is the chief subject considered here , is affected by racial or by extrinsic factors . 
cancer of the uterus in hindu , moslem , parsee and indian christian women . statistics based upon attendance at hospitals are notoriously liable to error , more especially when they refer to people differing in race and religion . however , the following figures ( table xxi1 ) should at any rate encourage further investigation . 
they are taken from the valuable collection of data on cancer in india obtained by nath and grewal ( 1935 ) from the records of in - patients of 13 hospitals in punjab , delhi , united provinces , and bihar and orissa , comprising 6395 cases of cancer in all . 
cancer of the uterus in china . maxwell ( 1929 ) in his book " the diseases of china " says : " it is probable that uterine cancer ranks highest of all in the malignant growths in this country . 
the women are classified according to the husbands ' occupation into four main classes , with 14 numbered and 35 named sub - divisions . in table xxvii , only the four main classes are quoted . theilhaber does not state the populations of these various groups , and makes figures for the age distribution no comment upon this very serious deficiency . of the populations of these classes were not available . 
the object of the preliminary bath was not cleanliness per se , but the removal of anything which would intervene between the skin and the water during the total immersion , and thus deprive this ritual of its efficacy.t some medical writers have emphasized , in rather vague terms , the importance of the cleanliness enforced by the mosaic law , hence it is important to note exactly what the law requires . 
the washing , in a normal woman , at the beginning and end of the seven days , adds two baths a month to whatever other ablutions she may carry out . * this account of jewish ritual immersion is taken from various articles in the jewish encyclopedia ( 1891 ) , from ploss , bartels and bartels ( 1927 ) and from preuss ( 1923 )  . 
one might form some estimate in london from the number of public ritual baths available , which are said to be not more than half a dozen ; there are also some private ones . 
but it seems probable that many women who do not go to the ritual bath may conform to the law in other respects , namely , the first and second bath , and abstention from intercourse , which are matters of purely private conduct . the mosaic law ( leviticus xii , 2 , 5 ) relating to child - birth is important in view of the association of cancer of the cervix with parity . 
 " if a woman conceive seed , and bear a man child , then she shall be unclean seven days ; as in the days of the impurity of her sickness shall she be unclean . . 
but if she bear a maid child , then shall she be unclean two weeks as in her impurity : and she shall continue in the blood of her impurity three score and six days . " thus the whole period of impurity was either 7 + 33 = 40 , or 14 + 66 = 80 days . 
the practice in this country at the present time is said to be , to observe rather longer periods , of two and three months respectively . ( b ) moslem law . the koran ( sura 2 ) gives directions for the purification of women which are much less precise than those of the jewish law . 
a flow of blood lasting more than nine nights is the work of evil spirits . modi ( 1922 ) , a parsee , quotes these laws and says : " at present also , most of the parsee women generally observe the above practices . there are no separate dastanistans or houses for menses in parsee towns or streets , but generally a sequestered part of one 's own house is chosen for the purpose . the downfloor of the house was thought to be the proper place . 
but nowadays , in a crowded city like bombay , the down - floor , instead of being a quiet and healthy place such as that contemplated by the early injunctions of the vendidad , is so , most women in menses pass the period of generally quite the contrary . menstruation on their upper floors , but in an isolated way . 
in the matter of taking food , very few use spoons now , though up to about 25 years ago , that was generally the case . in the matter of purification , they observe the bath enjoined by the early books , but the vendidad injunction of bathing over the three " magas " is not observed at all . 
a separate place of bathing and for purposes of nature for women in this condition is generally provided in parsee houses . " ( d ) hindu ritual . khanolkar ( personal communication ) says that the parsee laws regarding menstruation are almost identical with those practised by orthodox hindus and probably both of them are derived from early aryan ritual . 
the abbe dubois ( beauchamp , 1906 ) , writing about the beginning of the nineteenth century , quotes from the book padma - purama , reputed to be the work of the hermit vasishta , a rule for a 3 - day p3riod of isolation for the menstruating woman , followed by a day of ceremonies and ablutions , including 36 total immersions in a river . 
during the three days "  . the mere wish to cohabit with her husband would be a serious sin . " elsewhere he says , " the mother of the newly - born child lives entirely apart for a whole month or more , during which time she may touch neither the vessels nor the furniture of the house , nor any clothes , and still less any person whatsoever . the time of her seclusion being over , she is immersed in a bath , or else a great quantity of water is poured over her head and body . women are similarly isolated during the time of their periodical uncleanness . in all decent houses there is a sort of small gynaeceum set apart for them ; but amongst the poor , in whose huts there is no such accommodation , the women are turned into the street , under a sort of shed or outhouse , or else they are allowed a corner of the cowshed . . 
when the time of uncleanness is passed , all the garments that the woman has worn are given to the washerwoman . her clothes are not allowed inside the house ; in fact , no one would even dare to look on them . " jhaveri ( 1910 ) says that in all hindu religious books minute directions for during the first four days of the first baths on very many occasions are given . menstrual period everything which the girl touches must be washed and " 202 e . 
nor do married women abstain procreation is almost a matter of religious observance with from child - bearing . uterine cancer ought to be , if anything , more the mass of jewesses . common among jewish women than among non - jewish . " various talmudic writers ( epstein , 1936 , 29b - 30a ) laid down that a father should cause his sons to marry at 16 to 24 , or 18 to 24 , and that a daughter should " be dowered , clothed and adorned , that men should eagerly desire her " ; no age is stated at which a woman should marry . 
a modern compiler ( abramowitz , 1900 ) states the law of israel on this matter thus : " after a man has arrived at the age of 18 it is his duty to take unto himself a wife in order that he may be fruitful and multiply , at any rate he should not pass his 20th year without having taken a wife . having begotten a son and a daughter who are also generative , one has fulfilled  . 
the command " to the commandment " to be fruitful and multiply . " be fruitful and multiply " is not obligatory upon women ; nevertheless a it is woman should not remain single lest she be liable to suspicion . one of the mandates of the sages that a man shall give his sons and daughters in marriage immediately they approach maturity . these jewish practices thus laid down in the law , and described by sorsby as prevailing at the present day , should encourage early marriage . the abb6 dubois ( beauchamp , 1906 ) says that "  . . 
thus 45 per cent of women with cancer of the cervix , and only 16 per cent of those with cancer of the breast , were married before that age . " variables which might be considered are : earlier childbirth , multiple children , poor obstetrical service , longer duration of married life , syphilis , immaturity of tissues at time of marriage , and excessive hormonal stimulation . " of these factors , longer married life could be eliminated by comparison with other groups . 
circumcision. some authors ( handley , 1936 , 1947 ) consider that no explanation of the lower incidence of cervical cancer upon jewesses need be sought beyond the ( assumed ) difference in the bacteriali flora of the female genital tract brought about by this factor , a matter which it would be possible to investigate . more data from moslem populations who practise circumcision , and from parsees , who do not , are obviously very desirable . handley ( 1936 ) has emphasized the value of statistics from fiji , where the natives practise circumcision , in contrast to the hindu immigrants , in whose native country phimosis and cancer of the penis are very common ( kennaway , 1947 ) ; certainly if these two races could receive equal medical attention interesting results might be obtained.t circumcision might act in another way , not dependent upon bacterial action . phimosis , cancer of the penis and cancer of the cervix appear to be common in * see " the racial origins of jewish types , " by radcliffe n . 
we have no data which give directly the respective social incidence of cancer of the cervix , and of the corpus , but information should be available from hospitals which have separate accommodation for paying and other patients . 
2. - age at death from oanoer of uterus in bamberg , augsburg , wuirzburg , erlangen , niirnberg and the provinoe of unterfranken , 1908 , and in munioh , 1906 - 7 - 8 ( theilhaber , 1910 )  . 
the jewish ritual has some unique features . more or less complete isolation of menstruating women is practised by many other peoples and is laid downi in the religious literature of some of these ( koran , zend - avesta , dharmasindhu )  . some quite primitive peoples ( aleuts ; natives of the congo and of australia ; for references see ploss , bartels and bartels ( 1927 ) ) are stated to enforce isolation at the menstrual period for as long as 6 to 7 days . 
one might make the following suggestions for further investigations : ( a ) an inquiry into the incidence of cancer of the cervix in jewish women who observe the 12 - day period , the ritual immersion being disregarded . 
all of the twenty collections of data which have been found in the literature show an incidence of uterine cancer which is greater on non - jewish than on jewish women . 
these data are calculated upon several different bases and hence are not all comparable . seven authors express the figures for cancer of the uterus as a percentage of all cancers in women , which percentage ranges from 28 to 14 in non - jews , and from 10 to 4 in jews , the mean values being 20 and 7 . 
the very scanty data available ( 25 cases only ) show a much lower incidence of cancer of the cervix on parsees . this might signify that so short a post - menstrual period does not affect the matter one way or the other , anid this difference in liability to cancer must then be due to other factors . 
cancer of the uterus appears to be prevalent in some peoples ( hindus , chinese ) among whom phimosis and cancer of the penis are also common , while the moslem women of india show a lower incidence . 
but any attribution of this difference to the practice of circumcision by moslems is very doubtful in view of the similar low incidence on peoples ( parsees , indian christians , possibly some dutch ) in whom this factor is absent . 
the only numerical data on the social incidence of cancer of the uterus appear to be those from bavaria 40 years ago , and from england and wales , after the last census , in 1930 - 32 . 
the unavoidable mixture , in large - scale statistics , of cancers of the cervix and of the corpus uteri , is unfortunate , as these two forms probably differ in etiology . 
harnett. published for the clinical cancer research committee of the british empire cancer campaign . received for publication july 16 , 1948 . in 1938 - 39 the clinical cancer research committee of the british empire cancer campaign carried out a clinical survey of all cases of cancer seen in the hospitals , both voluntary and l.c.c. 
in the administrative county of london . this was done by means of questionnaires , one of which was filled in by the registrars in the various hospitals for each patient and returned to the clinical cancer research committee for record . 
the work was interrupted by the outbreak of war after it had been in progress for 17 months . by this time 15 , 200 cases of cancer had been registered , of which 2529 ( 16 * 6 per cent ) were cancer of the breast . these patients have now been followed up for five years or more and the records analysed . 
 * from the department of experimental pathology and cancer research , school of medicine , leeds , 2 . received for publication august 11 , 1951 . ' thf , need for devising a technique for testing the carcinogenic properties of chemical compounds by direct application to the bladder epithelium has been felt for some time . 
yamazaki and sato ( 1937 ) described a method of introduction of oily solutions of chemicals into the rabbit bladder by catheterization . interpretation of the true nature of the one acceptable tumour described by these authors after the introduction of o - aminoazotoluol is complicated by the presence in an extensive series of of lipoid in phagocytes in the subepithehal tissues . experiments , hughes ( 1949 ) , by means of epithelial grafts wrapped around 20methylcholanthrene crystals , obtained 14 transplantable carcinomas from 120 attempts . 
by this method an even distribution of the carcinogen was difficult to obtain , and so considerable variation in concentration may have occurred . under ether anaesthesia the bladder was exposed by a small incision through the skin and abdominal wall about i cabove the urethral opening . 
the bladder was drawn out of the abdomen and supported on a small pad of gauze ; an incision was then made through the dome of the bladder and the edges held apart by small metal retractors . 
the suture passed through all the layers of the bladder wall , and the edges were drawn together so that the two epithelial surfaces were bound closely to one another ; thus a continuous epithelial layer only and no muscle nor connective tissue was exposed to the direct action of the carcinogen in the pellet . 
the abdominal wall and skin were closed separately by sutures of cotton or thick silk . the operative mortality was negligible , but a number ' of animals died within a few weeks of the operation due to the pellets blocking the urethra . 
the latter concentration had been found previously to be effective in inducing subcutaneou 's tumours , both sarcomas and breast adenocarcinomas ( bonser , personal communication )  . in the present experiment pellets containing 2 per cent of the carcinogen induced bladder epithelial h erplasia but not tumours , whereas a pellet containing 30 per cent induced an infiltrating cancer . 
no doubt lower concentrations would be equally effective . this technique has the advantage that an ' substance so apphed to th ' e bladder is subject only to the action of the urine be ' fore coming in contact with the bladder epithelium ; thus metabohc changes in chemical structure subsequent to feeding or injection are obviated . 
a reasonably high i ' ncidence of tumours should be obtained with an active substance without incidental induction of sarcomas . it should now be possible to determine whether metabolites of bladder carcinogens such as 2 - naphthylamine are ' tho active agents in inducing bladder cancer . economy in time , animals and material can be effected , and this is important when investigating the carcinogenicity of small quantities of metabolites isolated from urine . 
bartholomew 's hospital , and the statistical research unit of the me - dical research council , london school of hygiene and tropical medicine . received for publication february 20 , 1951 further data on the arsenic content of cigarette - 3 . in an earher paper ( daff and kennaway , 1950 ) data were given for the arsenic content of 5 brands of cigarettes of british and american types , of 8 brands of turkish type , and of two - others ( french , rhodesian ) , and also for the amount of arsenic volatilized in smoking . 
and germany are those of saglam ( 1944 ) from turkey and of dungal ( 1950 ) from iceland . ( 4 ) population by aores . ( 2 ) number of autopsies showing cancer . the data which one would like to obtain from any given country are : a . 
from univer8ity clinic8 ( for men and women separatelv - preferably by ages )  . - ( i ) number of autopsies . number of autopsies showing cancer of lung . in the countries where a ( 5 ) is not available one must rely on b . 
le hastaliklari klinigi in 23 months ( among 2084 patients , 53 in a thousand )  . " cc all these statistics show that in our country , within the last 30 years , pulmonary carcinoma has increased in a great proportion . " saglam rejects cigarette - smoking as a factor in the incidence of bronchial " some authors believe that there exists a relationship between carcinoma . smoking tobacco and inhahng the smoke and pulmonary carcinoma . 
we do not hold for a long time , at least since 50 years , almost only cigarettes the same view . in spite of this the increase in pulmonary ca . 
kennaway data for 1934 and have removed 3 cases , all in men , described as benign tumours , from the total of primary lung tumours ( a chondroma in 1935 , another in 1939 , and a polyp in 1937 )  . 
the official figures for tobacco consumption ( hutson , 1937 ) and for population ( statesman 's year book , 1931 ) indicate an annual consumption of about 17 lb . 
he writes that the kinds of tobacco smoked in yugoslavia ( b ) type hercegovina ( cigarettes ) ; and are : " ( a ) type makedonija ( cigarettes ) ; ( c ) type vojcodina ( pipe and cigars )  . " these are of the oriental type , i.e. , they have a natural ' bouquet ' which gives the tobacco type makedonija . " only in the north - western part of yugoslavia there are smoked cigars and pipe . 
use of tobacco for snuffing is quite insignificant . before the last war the only import was some dutch tobacco for cigars - ; during the war some bulgarian and after it some american tobacco ( through u.n.r.r.a. ) was imported , but all these amounts were very small . we are also indebted to kosir and to f . 
among 20 , 681 autopsies , there were 3584 cases of cancer and 276 cases of cancer of the lung . these figures are compared with those from other countries in table ix . ( 3 ) it is , however , not necessary to be dependent on hospital statistics for an estimate of the incidence of cancer of the lung in switzerland , as vital statistics are available for - the whole country . 
the calculated death rates from cancer of the lung are shown in table x . the national figures are reasonably reliable ' as , according to jakobsen , " the doctor who has treated the deceased during his last illness is required to which must be fill in an official certificate stating the cause of death stated in medical terms as precisel.v as possible , and it is not permitted to use general diagnoses such as " disease of the heart " or " disease of the lungs . " the 16fl ravii i 12 . 
kennaway clearl no exact proportionaht however , reasonably close to the difference in cigarette consumption . in switzerland tobacco consumption was much the same as in england and wales , but death rates were only half as high . differences in cigarette consumption again agree with the differences in death rates better than do differences in total tobacco conthis is because a much smaher proportion of the tobacco is consumed sumption . in the form of cigarettes in switzerland than in england . has been found in the data so far available between the amount of tobacco smoked and the prevalence of cancer of the lung cancer of the lung appears to in different countries and at different periods . increase more rapidly than does the use of tobacco ; such a change in effect may occur at a certain level in the dosage of a drug . 
the figures from a single hospital in yugoslavia suggest an incidence of cancer of the lung comparable to the british figures of 30 years ago . we have no figures for the incidence of cancer of the lung upon the whole population of any east european country . 
the e - vidence available at the moment is against the direct carcinogenic importance of tobacco smoke , because it tends to exclude the two most obvious carcinogens , arsenic and benzpyrene , which one might expect to be present . 
known carcinogens is now so wide that one must consider other possibilities . ( 3 ) all the older data about cancer of the lung must be reviewed in the light of this connection with smoking ; one must reconsider the inverse relationship with sunhght ( stocks , 1947 ) and the very low incidence upon a rather curious - assortment of occupations , namely , agriculture , coal - niining , and mule - spinning ( kennaway and kennaway , 1947 )  . 
one cannot answer this question at present , but it is especiary important in countries where smokers buy the cheaper pipe tobacco to make their own cigarettes . norway one obtains in this way nearly twice as many cigarettes for a given sum ( jakobsen , personal communication )  . 
the incidence of cancer of the lung upon the two sexes is not very different ( death rate , male to female , i : 07 ) ; it is greater in urban than in rural districts , and this difference is greater in men than in women . 
the increase in mortality in the last 20 years has been greater in men than in women , and greater in the towns than in the country . ( 5 ) in sweden , snuff makes up a much larger fraction ( one - third in 1949 ) of the total tobacco products consumed than is recorded in other countries . ( 6 ) a comparison is made of the data available for the increases since 1931 in ( a ) deaths attributed to cancer of the lung , and ( b ) in the consumption of tobacco , in england and wales , norway and switzerland . 
the consumption of tobacco per head has been for the last 10 years rather higher in switzerland than in the united kingdom , and in norway has been about one - half that in the other two countries , while the crude death rates at the beginning and end of the period were m . 
kennaway rou - ahlv in the proportion of i 0 ( england and wales ) to 5 ( switzerland ) , and 2 cigarette consumption was approximately in the proportion of 4 ( norway )  . ( england and wales ) to 2 ( switzerland ) and i ( norway ) and was more in accord with the relative death rates . 
the increase in the number of deaths has been about the same ( twofold ) in all three countries , but the increase in consumption oftobacco and cigarettes has , been ' less . 
the differences in the incidence of cancer of the lung are therefore quite different in extent from those in the quantity of tobacco consumed ; they are m ' ore hke ( though stir different from ) those in the quantity of cigarettes consumed . 
the study of the relation between the national consumption of tobacco and the national incidence of cancer of , the lung has scarcely begun . we wish to thank saglam and prol p . 
westlake avenue , l08angele8 , california . received for publication january 19 , 1951 . huggins , miller and jensen ( 1949 ) studied the thermocoagulability of the serum protein in health and disease in the presence of an extrinsic inhibitortheir results , however , while showing an abnormal behaviour iodoacetic acid . fo ' r mahgnant and certain non - mahgnant diseases , tend to establish a seeming relationship between cancer and tuberculosis . 
serum m micromoles of sodium iodoacetate in the nearest where 05 tube containing a firm clot preceding a tube in which the coagulum is a " shapeless viscous mass , .. 
or in the ori inal state before boiling . the serum protein determinations were carried out by the semi - micro kjeldahl procedure recommended by huggins , miller and jensen ( 1949 )  . every fifth determination was duplicated with an average agreement of 0 - 5 per cent . procedure was frequently checked for accuracy for the recovery of 5 - 0 mg . quantities of nitrogen by carrying out a complete determination with 23 - 6 mg . quantities of anhydrous ammonium sulfate with recovery in duplicate being 0 - 02 to 0 , 03 mg . 
the two myelomas were r - eceiving nitrogen mustards ; the leucaemias , x - rays and / or transfusions ; some of the advanced cases , polysaccharides or chymotrypsin ; eight of the breast cancer cases , testosterone . 
none of the patients in groups i and ii received any special diet , except that the most advanced , uncontrolled cases were getting either plasma transfusions or protein hydrolysates of one kind or another . all of them received an iron - vitamin preparation . 
for the series of cases in group iv merely reflects the ratio of concentration of iodoacetate as an inhibitor of coagulation for decreasing quantities of serum protein . there is no evidence that age distribution of the group tested played any significant role in the ratios obtained for the i.i. discussion analysis of the factors involved in the procedure showed that the coagulability of 52 per cent of the group i sera is abnormal for entirely different reasons from the sera ofgroups 11 and iii . 
evidence for this lack ofhomogeneity between the protein values and the degree of positive h.m.j. , obtained for tubercular sera and the sera from new untreated cancer soon became apparent , i.e. 
to the group i patients . in some the " positive " was altered to " negative , " andin there was good agreeothers the positive results became more or less positive . ment , however , for the i.i. 
values established by both kjeldahl and falhng - drop methods of protein estimations , for the remah - iing groups of cases . this led us to consider an " aberrant " or " combined - protein " as being responsible for the striking disagreement with the h.m.j. 
for the two types of disease . the possibihty that we were deahng with an increase in one of the normal fractions of the serum , the alpha globulins , reported to be increased in mahgnancy , led us to carry out the test on a sample of mucoprotein from cancerous serum , generously supphed by richard winzler , of the department of biochemistry , of the university of southem califomia , and a " bence jones " protein , isolated from the urine of one of the two myeloma cases in this series . 
neither ofthese substances yielded a positive i.i. if , as others have observed , the degree of increase of alpha globuhn is proportional to the severity of active tissue destruction , then apparently the alpha globulin plays no part in the huggins test , either for advanced tuberculosis , which certainly involves active tissue destruction , or in the early and new untreated cancer cases in our study . the question of the existence of modified serum protein molecules in various disease states , including mahgnancy , has been discussed recently by one of those whose published data estabhshed a close correlation between plasma viscosity , and the severity of the organic changes ( harkness , 1949 )  . 
added emphasis that we may be dealing with an altered protein in malignant tumours is given by the experimental observations ofhellwig ( 1949 ) , who found that as a rule the size ofspherical bodies in the form ofclusters , short - chains and aggregates from tumour extracts when examined by electron microscope was exceeffingly larger than that in benign or normal tissue . 
he concludes that these globules are not virus - like causative agents of cancer , but rather globular proteins , probable aggregates of the cytoplasmic globules due to an alteration in the cohoidal state of the cancer it is quite possible that this cytoplasmic material may be introduced into cell . the blood serum and may play a role in accounting for the positive i.i. 
duboff subjective element in the estiniation of the coaguluthe task for the future is the elimination of this factor , perhaps , by measuring the degree of coagulation by an appropriate electronic procedure characterized by the electrical resistance or potential of the coagulum . can one explain the paradox of the 22 negative h.m.j. 
tests have a pattern of deformation in one direction , it is not inconceivable that the former 22 cases have - a pattern of deformation in another direction that is not manifested in the test through the present combination ofreagents . 
furthermore , if the mechanism of action of this method is due to the increase of net negative charges on the protein molecule by the addition of sodium iodoacetate , then our results strongly suggest variations in the polar character of the proteiii molecule in the serum of certain phases of tumour development . the magnitude of relationship between absolute protein per cent and the ll demonstrated by group iii serum does not reflect the magnitude of the effect , when apphed to group i . 
the term " false positive " cannot be apphed ' there - t fore , to such tuberculosis sera , in the same sense as the term " positive " is applied to that of early cancerous sera , since the term " positive " itself is not tenableon the same basis . 
the serum of individuals undergoing some type of degenerative process , appears to possess an admixture of some elements , the source of which we do not yet comprehend , but the presence of which has been demonstrated by measurable function of the thermocoagulation of protein in the presence of an extrinsic inhibitor , iodoacetate . tentatively , we are inclined toward the conclusiozi that abnormality in the blood serum of some cancer patients , unhke that of some tuberculosis blood with a corresponding i.i. 
it is suggested that the use of the expression " false positive " or " pseudo positive " be elimiiaated from the terminology of cancer tests . the ' author wishes to tender his sincere thanks to clyde k . 
lipschutz. from departamento de medicina experimental , servicio nacional de salud , avenida irarrazaval 849 , santiago de chile . received for publication april 2 , 1953 . the tumours which appear in the intrasplenic ovarian graft in castrated rats , mice , guinea - pigs or rabbits apparently are due to the uncontrolled gonadotrophic activity of the prehypophysis , the ovarian steroid hormones which normally control hypophysial activity being inactivated during their passage through the liver before reaching the general circulation ( iglesias , mardones and lipschutz , 1953 )  . 
we shall be able to show ( 1 ) that this is not the case , and ( 2 ) that the hormonal imbalance from which ovarian tumourigenesis under the given experimental conditions derives can , and must , be expressed in quantitative terms . for the purpose of this study the secretory activity of 15 intrasplenic grafts , as listed in table i of our previous paper ( iglesias , mardones and lipschutz , 1953 ) has been checked by the histological examination of the vaginal mucosa . the uterus and the mammary glands . 
the presence of ovarian hormones in the general circulation at a level allowing for the transformation of the vaginal mucosa and of the uterine epithelium , even with the production of cystic glandular hyperplasia and metaplasia . 
rus. * pregnancy = several layers of clear vacuolized epithelia . * * transition = several layers of proliferated cells of the basal epithelium beneath the vacuolized layers as in fig . 
as evidenced by various new observations , we must drop the idea held originally both by biskind and by ourselves as to the gonadotrophic activity of the hypophysis in experiments with intrasplenic ovarian grafts in castrated females : contrary to what we thought before , the hypophysis is , under these experimental conditions , not identical to a castrate hypophysis . thus , the content of gonadotrophic hormones in the hypophysis of animals with grafts is considerably less than in the castrate hypophysis ( jungck , heller and nelson , 1947 )  . indeed this non - identity may be due to a difference , not of production , but only of release of gonadotrophins in castrated and grafted animals ( miller and pfeiffer , 1950 ) ; but non - identity seems to be a fact . castration cells are absent in the hypophysis of the grafted rat , in 224 2 . 
lipschutz any case in advanced stages of ovarian tumourigenesis ( lacour and guerin , 1951 )  . however , as compared to a normal hypophysis , that of a castrated animal with an intrasplenic ovarian graft is functionally impaired . differential tumourigenic thresholk concentrations of oestrogen . the problem of differential levels of oestrogen by which control of the vaginal or uterine mucosa and of the hypophysial gonadotrophic activity is achieved is intimately related to the problem of the differential tumourigenic threshold concentrations of oestrogen . 
how will the hypophysis behave when exposed to the action of these minute quantities of oestrogen in experiments lasting more than 2 years ? a group of 9 guinea - pigs was castrated and one ovary was grafted into the spleen ; sixteen days later a pellet of 23 to 30 mg . 
but the quantities absorbed were not sufficient for a complete control of this hypophysial activity : they were not able to reduce it to normality , as shown by the presence of haemorrhagic follicles in most of the animals of the group . there was in this group control of the vaginal mucosa and of uterine growth . in one animal the uterine weight was only 0o6 g . ; but in the remaining animals a weight of 0 - 8 to 1 - 2 g . 
with these 1 per cent pellets the incidence is not in another great ; new experiment lasting 757 to 875 days , without intrasplenic ovarian grafts , it occurred likewise but once in a group of 10 animals . 
one may raise the question whether some irreversible hypophysial change takes place during those 2 to 3 weeks which are needed for the vascularisation of the graft and for recuperation of hormone production . we have referred above to the three gonadotrophic hormones of the hypobut the possibility must be considered that also the somatotrophic or physis . some other hypophysial organotrophic hormone is partaking in establishing tumoural growth in the ovary ; this question has been discussed especially with reference to tumoural growth of wolffian structures ( iglesias , mardones , bruzzone and lipschutz , 1953 )  . the statement that ovarian tumourigenesis does not presuppose a complete suppression of the steroid control of the hypophysis but is the outcome of an irregular interplay of ovarian and hypophysial hormones may at the first sight however . 
the hormonal imbalance may last for as long as 3 years , and sometimes even more , without norwhereas it was evident , almost from the beginning mality being again attained . of this phase of our work , that the sequels of ovarian fragmentation , i.e. , reducing the mass of two ovaries to a small ovarian fragment , or remnant , in situ , were due to an overthrow of the normal ovarian - hypophysial relationship , we were unable to understand why this hormonal imbalance is in some cases irreversibly maintained . 
laws. from the department of pathology , guy 's hospital medical school . received for publication august 8 , 1952 . in a previous paper laws and wright ( 1952 ) have shown that - the growth of an implanted sarcoma in the bagg albino strain of mouse is associated with a reduction in the mitotic activity of the normal epidermis of the pinna . this fall was apparent when the tumour reached about 10 per cent of the body weight of the host . 
the tumour was the sarcoma 37 of the imperial epidermal samples were obtained from the pinna , and cancer research fund . after their separation from the underlying connective tissues they were staiiied by feulgen 's method . 
aetotic activity was estimated by counting the division figures seen in 100 microscopic fields uiider a magnification of 540 diameters . blood - sugar determinations were made on tail vein blood bv the method of haslewood and strookman ( 1939 )  . 
blood - sugar values in control and tumour - bearing mice . in experiments previouslv recorded ( laws and wright , 1952 ) , a significant difference was observed between the mitotic activities in the epidermis of the pinna in contiol and tumour - bearing mice . in order to compare the bloodsugar values in these two groups of animals , samples of blood were obtained at the same time of day ( 10 a.m. ) as the skin biopsies . 
correlatiom between tumour size , mitotic activity and blood - 8ugar value . since the growth of the tumour seemed to affect detrimentally the n - iitotic activity of the epidermis , coefficients of correlation were calculated between proportionate tumour size , niitotic activity and - blood - sugar value for a group 244 j . 
black. from the postgraduate medical school of london . received for publication march 25 , 1952 . from perusal of the literature available it appears that chronic ulceration associated with varicose veins is the most rare cause of malignant disease of the skin of the lower limb . 
we therefore wish to present 6 cases which undoubtedly occurred in conjunction with a varicose ulcer or eczema which had been present for many years . cutaneous neoplasia of the leg is rare , and study of the literature demonstrates that such skin cancers as do occur very rarely arise de novo , but usually in connection with chronic inflammation following injury , in old scars and sinuses , or after some chronic skin infections such as lupus vulgaris as well as in the varicose conditions mentioned . since the latter are without question the most commonly found benign lesions of the leg the excessive rarity of malignant degeneration is surprising and interesting . in recent years papers on this subject have been few . 
on examination she was a wasted , frail old woman not well orientated . there were no large nodes in the groin , the general examination showed senile but several small soft nodes could be palpated . changes but no obvious disease . biopsy of the tumour revealed a well - differentiated squamous carcinoma with cell - nests . 
b - , aged 71 years , was referred to us for treatment of a very large varicose ulcer of 20 years ' duration . four months before his admission to hospital the lower edge had begun to hypertrophy and the ulcer to spread on to the dorsum of the foot . 
h - , a widow of 67 years , was referred to us for treatment to secondary neoplastic nodes in the right inguinal region . she had been treated by mid - thigh amputation , at another hospital , for a 30 - year - old decubitus ulcer which , at the time of amputation , extended from the dorsum of the right foot to one - third of the distance up the tibial shaft and involved the bone . there had been no recent change in the ulcer prior to amputation but it had steadily increased in size during about 7 years before admission to hospital . amputation had been advised for pain , and because of the large size and failure to heal with support and dressings . on examination her right leg had been removed through the thigh . 
hard mobile nodes were present in both groins and in the right axilla . the left leg showed marked varicose veins and the scar of a healed ulcer above the left internal malleolus . her heart was fibrillating ( she had been receiving treatment for this for about 6 years ) , but there were no signs of cardiac failure . a report on sections taken from the ulcer after amputation showed a well - differentiated keratinising squamous - celled carcinoma . a biopsy excision of nodes palpable in the axilla was performed . 
no other radiological evidence of malignancy was found . biopsy of the tumour showed a general structure like granulation tissue , with wellformed capillaries and abundant reticulin . cytologically it was definitely sarcomatous , with large , pleomorphic , usually spindle - shaped cells with large irregular often multiple nuclei and numerous mitoses . although the size and histology of the growth and the necrosis of bone underlying made primary mid - thigh amputation the treatment of choice , his general condition was so bad that it was decided to attempt more by radiotherapy . 
lateral and medial fields were used and skin doses of 3300 and 3000 r respectively given using 220 kv . however , only slight response to this therapy couldbe found on serial biopsy , the tumour cells and stroma remaining as in the first biopsy , but mitoses became less frequent and some collagen was deposited . his blood urea , reduced by diet and conservative measures , again rose above 200 mg . 
he remained well for one year , when he was readmitted for treatment of a mass in the posterior flap of the stump . this mass was excised locally , a wide wedge of tissue being removed down to the bone . this local operation was again performed at the patient 's insistence . section of the tumour contained in this block of tissue revealed the identical histological picture presented by the original tumour and the nodule found in the inguinal lymph node . healing was by first intention . 
black this will prevent loss of time , decrease the period of pain for the treatment . patient , and allow the patient to acclimatize himself to the loss of his leg without radiotherapy should be the disappointment of failed conservative treatment . held in reserve for those patients whose general condition does not permit of operation , and these must be very few now . the treatment ofthe inguinal nodes depends upon the age and general condition extensive block dissection is a formidable undertaking in the of the patient . very old , and in these cases irradiation should be advised . 
where the patient will bear the operation this represents the safest method of eradicating growth . sarcoma , largely owing to its extreme rarity is difficult to recognise both clinically and histologically . it appears to occur more towards the centre of an ulcer than at the edge , to produce a florid fungating cauliflower growth and to these bleed readily . 
as with the squamous growths , sarcomata in varicose ulcers appear less ready to metathis may be due stasise than similar tumours elsewhere or in younger patients . to the fibrosis and scarring present after years of ulceration . 
 from the department of experimental , pathology and ganeer research , university of leeds , and department of pharmacology , guy 's hospital , medical school , university of london . 
greenblatt and kupper man ( 1947 ) , also using the rat , observed squamous metaplasia of the horn and a doubtful precancerous lesion in the cervix uteri , but no tumours , when a similar dose of methylcholanthrene was introduced . 
pan and gardner ( 1948 ) reported the development of numerous carcinomas and sarcomas in mice of several strains , when subcutaneous implants of cervical and uterine epithelium - were made together with small crystals of methylcholanthrene . 
 in the experiments described here uterine tumours were induced by the intro duction of methylcholanthrene directly into the uterine horn in two types of mice and the effects of various hormonal factors were studied . 
 the experiments were performed on two types of mice : inbred cba mice , maintained by brother - sister mating in the department of cancer research at leeds , and white mice obtained from dealers . 
the gun was intro duced through the incision , a fine silk ligature placed around it a short distance below the incision , and the carcinogen pushed out into the lumen of the horn . 
the amount of material introduced in solution in lard varied considerably , depending on the size of the uterus , but there is no doubt that the weight of methylcholanthrene introduced with method ( 2 ) was less , and sometimes considerably less , than that introduced into the uterus with method ( 1 )  . 
 ( 3 ) this method was similar to ( 1 ) and ( 2 ) , except that the upper part of the horn through which the material was introduced was removed . 
the upper part of the vagina was then exposed and dissected free from surrounding tissue and partly separated from the rectu a stout ligature was loosely placed around the vagina just above the symphysis pubis . 
the needle was introduced into the vagina and pushed up above the level of the ligature , which was then tied ( with one knot ) around the needle and held tied by an assistant . 
the ligature around the lower end of the left horn was tied , the ligature around the vagina rele : ased and removed , and the needle with syringe withdrawn . 
 the abdominal organs were carefully palpated at regular intervals to deter mine the first appearance of uterine tumours , but the mice were not killed until they appeared to be ill or until there was no doubt about the presence of an abdominal tumour . 
 of 15 immature oba mice , 12 developed tumours of the uterus , as follows : 4 had carcinomas , 6 sarcomas , and 2 both types of tumour ( table i )  . 
 the tumours presented a homogeneous translucent appearance , sometimes with haemorrhagic and necrotic areas , except in the case of the squamous cancers , table 1 . - 0oourrenoe of tumours in immature oba mice . 
 the object of the experiment was to find out whether the direct introduction of a carc~ogen into the uterine horn would be effective in inducing carcinoma of the endometrium in mice , and if so , whether the incidence of tumours could in 109 mice be influenced by hormonal factors , or by differences in technique . 
 effect of stra a higher incidence of both carcinoma and sarcoma occurred in oba than in laboratory white mice ( tables i , ii ) , in spite of the fact that all the oba mice were immature at the time of implantation . 
the difference in carcinoma incidence ( oba 40 per cent , white 149 per cent 134 ) is nearly but not quite statistically significant ( table iii ) ; that in sarcoma incidence ( oba 533 per cent , white 74 per cent 134 ) exceeds the conventional level of statistical significance . 
but when immature oba mice are compared with immature white mice ( table iii ) , then the difference in carcinoma incidence becomes significant ( oba 40 per cent , white 83 per cent 134 )  . 
above and to the right are the rectal glands and submucosa invaded by loose sarcoma tissue ; passing obliquely across the figure is the muscle of the rectal wall ; below is the main mass of sarcoma . 
 it was rather surprising that carcinoma could be induced by this means in immature mice , spayed at the time of implantation , when they weighed ll - 15 g . 
it is evident , however , that maturity at the start tended to improve the tumour yield , though it did not lower the period of induction in white mice ( table iv )  . 
 spaying had no apparent effect on the incidence of all tumours either in oba mice ( table i ) , where the numbers were very small , or in white mice ( table iv )  . 
 16 85 27 85 14 8 l 26 8 l 86 113 114 25 81 13 81 the yield of sarcomas was , however , greater in intact than in spayed white mice , though the difference was not statistically significant . 
mice which received the larger dose of carcinogen , are considered ( table v ) , it is noteworthy that the lowest incidence of tumours occurred in spayed immature mice . 
even though the numbers are small , the agreement between the three other groups in table v suggests that the lower incidence of tumours in spayed immature mice is of significance . 
it is probably comparable with the dose used by pan and gardner ( 1948 ) for the induction of tumours in subcutaneous implants ( " small crystals " )  . 
price. from the medical research laboratory , department of pathology , university of bristol . received for publication december 8 , 1951 . during the past thirty years much useful progress has been made in the this work , of histological grading of malignant tumours of epithelial origin . which one practical application has been the attempted correlation of prognosis with histological structure of a tumour , has had many valuable results ; and although there may be not infrequent discrepancies between the histological appreciation and the outcome of treatment in any given type of neoplasm , the results have at any rate led to some attempt being made by the histo - pathologist to guide the clinician at least in qualitative terms of high or low malignancy ; and to more precise premises for the comnparison of differing methods of treatment in series of comparable cases . the starting point of this present analysis was the observation that irrespective of the mode of treatment applied , the overall five year survival rates in most published dissections of groups of cases of osteogenic sarcomata were approxinmately of a similar order . 
some of these features can , it is true , be visualised by x - ray plates . however , the interpretation of these is mainly the proper province of the radiologist , and they are seldom to be seen in theroutine pathological laboratory . for the fortunate histo - pathologist who may be granted access to such aids , the position is onlyinmproved to such an extent as the experience which he may have in the interpretation of skiagrams , which is a field with c . 
the ideal state no doubt is the close liaison between surgeon , radiodiagnostician , radiotherapist and histologist with personal conference in each and every case . such states of collaboration seldom exist under conditions of normal routine work . the histological specimens from the present series of cases have been considered under the following headings : 1 . 
to anyone working in this field the fallacies of diagnosis are only too well known , and the cases included have all been subjected to free discussion and unanimous acceptance as osteogenic sarcoma by the group of radiologists , radiotherapists , surgeons and pathologists which forms the working panel of the register . in each case the diagnosis has been based upon consideration of the full history , clinical examination , adequate skiagrams and histological sections . in some instances the histological material is derived from biopsy , in others from surgical or autopsy specimens . this has been indicated in table i , also the time relationship of the histological specimen to any pre - operative x - radiation . for histological purposes the specimens were fixed , cut and stained by the usual routine methods . 
owing to the presence in sonle sections of many multinucleated tumour giant cells , and the indistinct outlines of a large proportion of the polyhedral cells , the nucleus was taken as the unit rather than the cell . in each case a minimum of 2500 neoplastic cell nuclei were examined in each section , using a ruled internal screen in one ocular of a binocular microscope . 
as there are also 8 cases included in the series that are still alive and when last reported were well , but for periods less than 5 years , the 5 - year survival rate is best expressed as 6 in 28 cases - i.e. , 21 - 4 per cent . two of these cases died at respectively 77 and 96 months with multiple pulmonary metastases , and the writer fully supports the view that 5 years is too short a period on which to base estimates of rate of cure in patients with osteogenic sarcoma . these brief data may be compared with those published on larger series by the following : christensen ( 1925 ) , coley and pool ( 1940 ) , the results of the new york memorial hospital series mentioned by coley ( 1949 ) , geschickter and copeland ( 1949 ) , the series reported by the british empire cancer campaign ( 1949 ) , and platt ( 1951 )  . as may be expected with a relatively small group of cases , there are some differences from the larger published series , e.g. , an unduly high incidence of femoral tumtours , but the broad agreement would suggest that this group may be regarded as a fair sample from which certain conclusions may tentatively be reached . clinical grading of the tumours . the data on which the clinical assessment of degree of malignancy has been based has been considered from the following points of view : 1 . 
from observations on these tumours and other bony conditions , the writer has acquired the view that one of the essential functions of the osteoblast is the formation of osteoid tissue - the precursor of bone , from which under suitable conditions of substrate the formation of bone may occur , possibly with no further part being taken in the process by the osteoblast . 
price callus and in paget 's disease . both normal and neoplastic , may produce phosphatase . it is also generally accepted that the osteoblast , from the grounds that this cell therefore is actively concerned in the laying down of osteoid arises the essential stromal feature of this histological sub - type of osteogenic sarcoma , i.e. , the characteristic osteoid stroma , which mav also be accompanied by considerable amounts of collagen and ill - formed cartilage . however , the osteoid is the essential feature . this stromal material may , however , sometimes occur in considerable amount in a tumour which is predominantly spindle - celled , but is then usually less evident than the collagenous fibrous matrix produced by the spindle cells . 
under suitable conditions the osteoid matrix may either calcify or ossify , more often the latter , and then produce the appearance of the osteoblastic or sclerotic type of tumour . it is thus possible to subdivide this group further into i . 
from the viewpoint of the histologist the essential histological structure of any neoplasmn occurring in a bone is more characteristically shown in an area of active growth , the radiological manifestations of which are more frequently lytic than blastic . hence this feature of the amount of bony matrix is not always readily appreciable from even a large biopsy . for this reason this subdivision will not be pursued further in the present analysis ; but it should be added , however , that a markedly bony stroma is often indicative of a tumour of relatively low malignancy , and this point should receive due consideration by the person who may attempt to correlate the histological , radiological and clinical findings in any particular case . from inspection of table iv the following points are evident : 1 . 
in an approximately quantitative assessment of the amount of osteoid stroma present in these sarcomata it is seen that there is usually a rich matrix in tumours of lower grade malignancv , and conversely a relatively smaller amount in the high - grade group . there are noteworthy exceptions , and the specimens showing perhaps this stromal feature in the greatest degree were those from btr / 66 , r . 
66 , 213 , 221 and 226 , but generally this feature was less evident in the more actively growing tumours . it is emphasized that a change in ph of the substrate is only one of a number of factors concerned it is also wise to bear in mind a fallacy that may in this biochemical change . arise in sampling a bony tumour , as the more easily sectioned and hence less bony areas are frequently selected for routine diagnostic examination . quite a nuimber of these sarcomata also show chondroid stromal areas which may either calcify or ossify , both changes often being seen in adjacent areas and largely governed by two factors : 1 . 
the iimean mitotic ratio of these sarcomata decreases as one passes from this is mluch more evident in the separation of clinical grade i to grade iii , clinical grade i from the tumours of grades ii and iii . it may be noted here that based upon clinical data case btr / 136 , y . 
price by the american registry of bone sarcoma in their 1939 ( revised ) classification , and they continued to regard intrinsic fibro - sarcoma as a variant of the osteogenic group ( ewing , 1939 )  . jaffe ( 1947 ) , on the other hand , commenting upon this adheres to the stricter view and classifies fibro - sarcoma as a separate entity . from consideration of the small group of cases here reported it would seem that this differentiation is indistinct and serves no useful purpose . table v gives details of these 7 cases . this grouip is far too small to be the basis for any definite conclusions , but the following tentative points emerge : 1 . 
in the spindle - celled group there were no tumours which appeared to merit a clinical or histological grading of i on comparison with those of the larger polyhedral - celled series . this appears to be confirmed on considering the figures obtained for the mitotic ratio in these growths . .5. 
one may perhaps from this draw the conclusion that there is some evidence to suggest that in a series of these two histological types of osteogenic sarconma if composed of tumours of average or more than average malignancy that the tinme of survival is rather longer in favour of the spindle - celled type . in this small series of cases , one obvious difficulty that has arisen has been the determination of mean survival time of the sub - groups , and it is fully appreciated that the figures shown are only minimal inasmuch as cases have been included which are still living . 
on reference to table iii again it will be seen that 7 of the 9 tuiyours showed a mitotic ratio considerably greater than the average for their appropriate clinical grade . 
g - , and it is strongly suggested that the aoverning factor in the survival of any case of osteogenic sarcoma is the combination of site of origin and histological grading of the tumour . table vi shows the 36 cases of osteogenic sarcoma arranged in ascending order according to the mitotic ratio , i.e. , in order of diminishing mitotic activity . this has been subdivided into three purely histological grades of malignancy , c . 
price into those cases with a mitotic ratio less than 100 : 1 , from 100 : 1 to 400 : 1 , and greater than 400 : 1 . this may be taken as approximnating to those of greater , average and lesser degrees of nuclear activity . reference back to tables iv and v will show that all the cases there clinically graded as iii appear again as grade iii ( histological ) plus one case , btr / 157 , d . 
a - : probably favourable result of treatment has led ta unduly low clinical grading ; tumour in lower end of femur . group based on range of mitotic ratio of less than 100 . mean survival period of grade iii ( 11 ) 114 + months . 
g - , aged 14 , mitotic ratio 83 : 1 , sarcoma of lower end of femur , who survived for a total period of 18 months . another case supporting this view was btr / 157 , d . 
mean survival 11 4 months . the marked difference in the prognosis in these separated groups of cases can it would be of the greatest value to subject many again be well seen from fig . 
2. further cases to this type of analysis , to confirm if possible the significance of the mitotic ratio and to find its mean value based upon the examination of a much larger series of tumours . 
the mean mitotic ratio for this series is 238 resting nuclei to one nucleus in mitosis . analysis of the group gives the following ranges : grade i : grade ii : grade iii : m.r. 
the mean mitotic ratio for this group of sarcomata is markedly different from that which has been found in the examination of presumably related cells in non - neoplastic conditions . the author wishes to record his great indebtedness to the late s . 
much thanks are also justly due to the members of the committee of the register for their co - operation in the collection and discussion of all the cases included in this series . material has been derived from the sourceindicated below , and to the surgeons , radiotherapists , radiologists and pathologists who have referred cases to the register , due recognition and thanks for their assistance are here rendered : 1 . 
we have adopted the following staging , which is similar to that employed at the christie hospital and holt radium institute , manchester : stage i . - primary growth limited in extent to site of origno loss of mobility or function . 
lymph nodes fixed , matted or bilateral , or distant metastases present . table iv shows , as already mentioned , how many cases had already reached stages iii or 1v when first seeking treatment . 
warren and gates ( 1932 ) investigated the problem in considerable detail , and concluded that a patient with one cancer was statistically more likely to develop another cancer than a normal individual . 
watson ( 1939 ) calls attention to the fact that when a patient with carcinoma of the oesophagus has - a second growth this is nearly always situated in the mouth . 
it is therefore of interest to know how often this treatnment succeeds or fails in its objective , and in the latter case , when there is a recurrence , what are the chances of further arresting the disease . these facts cannot be determined with any real accuracy , since each case clearly has to be assessed on its clinical development and course . 
harmer in stage i and ii cancers of the lip , re - treatment is rarely successful . first planned treatment fails , the patient does not often have a second chance . if the response of primary and secondary to irradiation . glucksmann ( 1948 ) has stated that the response to irradiation of the primary and secondary manifestations of cancer of the mouth is about the same , and that those growths " with a tendency to lymph - node involvement differ in their biology and radio - curability from those without such a tendency . " the more generally held view is that while the primary may be " cured , " the secondary is often more resistant to treatment . only stage iii and iv cases can be analysed if the theory that an irradiated primary will recur as frequently as an irradiated secondary is to be examined . there were 248 patients with stage iii and iv growths arising from buccal sites in whom radiotherapy produced regression of both primary and secondary forty - five remained well , and 203 recurred : 61 in the primary only , growths . 69 in the secondary only , 73 in both . 
hartley has kindly done a significance test on these figures . the number of cases has been adjusted so that the proportion of stage iii and iv royal cancer hospital patients is the same as that of the holt radium institute series . 
the accessible growths of the for cervical mouth and lip , and teleradium for the oro - pharyngeal primaries . metastases block dissection hasbeen the method of choice , or radium therapy for the inoaperable cases . x - rays have&been used comparatively rarely . memorial hospital , new york ( martin , 1948 )  . - hayes martin states that surgical excision is the method of choice for cancer of the lip , gum , anterior for oro - pharyngeal portion of the tongue , cheek , floor of mouth or palate . growths a combination of roentgen therapy and interstitial radiation is indicated . cervical metastases are treated by dissection when operable and by x - rays when not . radiumhemmet , stockholm ( berven , 1937 )  . - for more than 20 years - the usual treatment for growths of the tongue and most other buccal sites has been teleradium , with diathermy coagulation of any residual mass . 
sixty - five per cent had symptoms for less than 6 months before treatment commenced , yet ( with the exception of carcinoma of the lip ) in 60 per cent of cases the disease had already metastasized to the cervical lymph nodes when first seen . 
the treatment of these 800 cases has been largely by radiotherapy . comparison of the results of treatment published by different authorities is table xv shows wide difficult because of lack of uniformity in criteria adopted . divergencies for instance in the survivals of patients with growths of the buccal this is largely due to mucosa , and to a lesser extent of the alveoli and palate . the small numbers of the cases . the last section of this paper shows that although very different methods of it is treatment may be employed , the results on the whole are much the same . therefore inadvisable to attempt definite conclusions from the analysis of the cases presented . 
the cases have been under the care of all members of the staff of the hospital , past and present , to whom grateful acknowledgment is made for permission to publish them . in the preparation of the paper much help was derived from copeland - chatterton punch cards , many of which had been completed by j . 
iversen. * from the institute of pathological anatomy , university of copenhagen , denmark . received for publication june 11 , 1948 . earlier investigations by weigert and mottram ( 1946 ) have shown that 3 : 4benzpyrene after administration to the animal organism is metabolized to at least four different derivatives , labelled by weigert and mottram as bpx1 , bpx2 , bpf bpf . 
the diameter of the particles of the emulsion was between 6 to 15k . immediately after the emulsion was prepared it was injected intravenously in patients suffering from myeloid or lymphatic leukaemia . 
emulsion. blood samples ( one part 3 - 8 per cent sodium - citrate solution plus 9 parts blood ) were taken from a cubital vein immediately before , immediately after and at various times after the injection . 
2. - absorption curves of plasma extracted with ethanol before and after intravenous injection of 3 : 4 - benzpyrene . in order to make sure that the emulsifier ( postonal ) itself had no effect on the amount of nitrogen in plasma , 8 g . 
2 shows that the maxima of absorption characteristic of 3 : 4 - benzpyrene are , little by little , altered to maxima of absorption characteristic of the metabolites labelled as bpx1 . 
iversen the results of kjeldahl analysis on the precipitates showed that there was no noticeable difference between the percentage of nitrogen in the different samples of precipitate , which may mean that the composition of protein in plasma does not alter considerably in relation to an intravenous injection of 3 : 4 - benzpyrene , or that it varies so little that it has been impossible , with the technique used , to determine the difference . it would be possible to obtain a quantitative estimation of the proportion between the amount of unchanged benzpyrene and changed benzpyrene , and thus get an expression for the speed with which the metabolism ( or detoxication ? ) of 3 : 4 - benzpyrene takes place by determining the proportions between the extinction at 380 m , u . 
the same quotient for the x1 derivative is unknown and could not be determined , as it was not possible to obtain the x1 derivative in a pure form , but the quotient must be higher than 1 00 , as the absorption of the xl derivative is more intense at 380 m ,  . 
the curves further show that the time at which the rate of conversion is highest nearly coincides with the time at which the decrease in nitrogen - ( protein - ) conadditionally the curves indicate that tent of the plasma is most conspicuous . after the maximum concentration of changed hydrocarbon has been reached , elimination takes place at a constant speed . that the conversion may take place in the liver after an intravenous injection is an obvious consideration , and the following experiment would indicate that this is the case in rabbits : blood is transfused through a living rabbit liver ( nielsen , 1933 ) , and to the circulating blood is added 10 mg . 
3 samples of blood are taken before and at various times after the injection . shows , as in the case of human beings , that the absorption maxima of 3 : 4benzpyrene are altered gradually to the maxima of absorption characteristic of that these curves appear higher and higher on the ordinate the x1 derivatives . axis may be accounted for by the fact that it is not possible for the system to the variations in the quotient 380 / 385 m , u . 
iversen perhaps to be mentioned in this connection that the patients during , or immediately after , but never later than half an hour after the injection , sometimes got slight attacks of pulmonary infarcts . whether one considers the changes in the amount of nitrogen ( protein ) in plasma as attached to the metabolism of 3 : 4 - benzpyrene or as bearing no relation fig . 
 ( left ordinate axis )  . c and d give the corresponding relative amounts of x2 derivative calculated in the way described ( right ordinate axis )  . 1n 135150 us5 so o9 ? 0 150nt240 552t70 from fig . 
and the mice were kept on a balanced diet of commercial food pellets and water ad libidum . eventually the hybrids formed the following groups . group i consists of the offspring of males no . 
1. of the original 2 sisters , the segregated one died after her first litter , and was replaced by a third sister which was also segregated for birth and rearing of her first four litters . she was left with the male for the birth of her fifth litter ( table i . ) group iii consists of the offspring of 2 females , each permanently in the cage the paired segregated sisters of each died with males no . 
two sets of unrelated sisters were mated to male no 11 . ( table ii ) ideally all the segregated females should have been sisters of the female left with the male , but this was not always possible . the result of the experiment is summarised below : c3h . 
 ( mothers kept with male ) cba.c3h 11 mammary tumours out of 82 hybrids . ( mothers segregated from male ) , , 84 the incidence is expressed as the number of mice bearing mammary tumours as numerators over the number of survivors aged 450 days ( age of youngest tumour - bearing mouse in this series ) as denominators . all these mammary tumours appearing in the hybrids were confirmed histologically by p . 
2b , segregated from the male , developcd a mammary tumour at 538 days and 387 days respectively . these tumours could be attributed to transmission of virus in the mothers ' milk , or to infection from the c3h male in each case , or they may be unrelated to the in the case of female no . 
no definite conclusion can be drawn from the occurrence of such sporadic tumours . ( b ) andervont and dunn ( 1948 ) have shown that c3h males can carry the mammary tumour virus in their vesicular glands and secretion , and have suggested that the mother may be infected during copulation . foulds ( 1949 ) has shown a similar transfer of virus by riii males to their fl hybrids with c57 black females . bittner ( 1952 ) has studied similar crosses between high and low mammary cancer strains , with and without the agent , and has reached essentially the same conclusion about the role of the male in transmitting the agent . 
laws. from the department of pathology , guy 's hospital medical school . received for publication august 8 , 1952 . in a previous paper laws and wright ( 1952 ) have shown that - the growth of an implanted sarcoma in the bagg albino strain of mouse is associated with a reduction in the mitotic activity of the normal epidermis of the pinna . this fall was apparent when the tumour reached about 10 per cent of the body weight of the host . 
the tumour was the sarcoma 37 of the imperial epidermal samples were obtained from the pinna , and cancer research fund . after their separation from the underlying connective tissues they were staiiied by feulgen 's method . 
aetotic activity was estimated by counting the division figures seen in 100 microscopic fields uiider a magnification of 540 diameters . blood - sugar determinations were made on tail vein blood bv the method of haslewood and strookman ( 1939 )  . 
blood - sugar values in control and tumour - bearing mice . in experiments previouslv recorded ( laws and wright , 1952 ) , a significant difference was observed between the mitotic activities in the epidermis of the pinna in contiol and tumour - bearing mice . in order to compare the bloodsugar values in these two groups of animals , samples of blood were obtained at the same time of day ( 10 a.m. ) as the skin biopsies . 
correlatiom between tumour size , mitotic activity and blood - 8ugar value . since the growth of the tumour seemed to affect detrimentally the n - iitotic activity of the epidermis , coefficients of correlation were calculated between proportionate tumour size , niitotic activity and - blood - sugar value for a group 244 j . 
the original premise on which the present study was built was that if these three groups have any real existence it should be possible to demonstrate some differences in their natural history . a diagnosis that does not imply a prognosis is traditionally worthless ; not only is it of no practical value , but the difficulty of testing it leaves every opportunity it was proposed therefore to collect a series of rodent ulcers , classify for error . them according to foot 's ( 1947 ) criteria , and then to apply to them all available touchstones of clinical behaviour in the hope of being able to separate them . some such differences as those demonstrated by schrek and gates ( 1941 ) and schrek ( 1941a , 1941b ) between rodent ulcer and squamous carcinoma were sought . the attempt to do this , however , broke down unexpectedly early , for after our series had been collected it proved impossible to classify it on the lines proposed by foot ( 1947 )  . 
wells lesions with which foot ( 1947 ) dealt , it became clear that the great majority of tumours at the stage at which they are usually received by the pathologist are typical examples too various in appearance to admit of such simple treatment . of each class could be found easily enough , but there were so many mixed and intermediate and aberrant forms that no progress was possible . 
no modification of the classification could be devised that would avoid them . during a long interval in which the matter was left in this discouraging state , interest was deflected into a study of the pigmentation of rodent ulcers and of epithelial skin tumours generally which has already been reported ( lennox , 1949 )  . there emerged from this one possible way of classifying rodent ulcers , namely , into the pigmented and the non - pigmented . such a classification is almost wholly objective ; there either is or is not melanin in the tumour . contrast with the process of matching a tumour against a series of idealized types in the hope of being able to recognize a greater or less resemblance to one of them is very marked . it is true that the matching technique is reliable enough in the ordinary process of histological diagnosis , but where the types themselves are of doubtful existence it becomes open to great error . 
they are a large proportion of those received in this department in the years 1936 to 1948 . most have come from the radio - therapeutic research unit which was established in 1942 ; without its very full record system this study would have been impossible . cases have been rejected if the biopsy specimen was inadequate or if the history was deficient in any important detail , but an effort was made to include as many of the cases found as possible , partly because the available material was small , partly because any form of selection may profoundly and unpredictably influence the results of any study of this type . it was not possible to insist on more than a year 's follow - up , though most cases have had considerably longer ; the recurrence rate is therefore falsely low , though sufficient to provide some useful data for comparative purposes . surgical excision supplied 26 specimens ; diagnostic biopsies , multiple in 30 cases , were provided by 124 cases . most of the material was fixed in plain formalin - saline ; recent biopsies have been fixed in halfsaturated mercuric chloride in 15 per cent formalin - saline ( fss )  . 
the typical examples of this group seem distinct enough , yet it soon became obvious that transitional forms are numerous . wte therefore provisionally included the sub - group , and found later no reason to regret it ; we believe that these are merely the most benign and well - differentiated end of the rodent ulcer series ( the multiple and often familial tumours of brooke ( 1892 ) and fordyce ( 1892 ) we believe also to be of the same nature , though here we are extrapolating beyond our personal experience )  . in the second place , at the other end of the scale we find 11 tumours of the type illustrated in fig . 
we believe the evidence against the squamous nature of this sub - group is strong . within the typically squamous tumours a close parallel between histological dedifferentiation and clinical malignancy can be the most malignant epithelial skin tumour seen in this hospital demonstrated . in the last 4 years is that illustrated in fig . 
3 ; it grew very rapidly under observation and killed its host within 18 months of first appearance . less well differentiated squamous carcinomata should be even more malignant ; but all the tumours we have seen of the type of fig . 
2 have been relatively benign , and can be shown only by careful statistical analysis to differ in behaviour from the general run of rodent ulcers . inclusion of these two sub - groups harmonizes with the view of the rodent ulcer group developed later in this paper , as a series in which a gradation of differentiation can be recognized , with a definite though not very striking parallel gradation in behaviour . 
removal of these two sub - groups would not affect the general conclusions drawn , though it would reduce the level of significance of some of our figures . forms of differentiation chosen for analysis . only four common histological forms of differentiation in rodent ulcers could be defined well enough for analysis . 
some obvious features , especially those based on cell size and shape and on tumour cell group size and shape , had to be rejected because of the difficulty of establishing exact descriptive criteria and , more important , their great variability within any one tumour . 
some very interesting features were too infrequent in our series to justify detailed separate analysis : these were ( a ) keratin formation ( 11 cases , 7 ' 3 per cent ) , ( b ) hyaline stroma ( 11 cases , 7 ' 3 per cent ) , ( c ) squamous differentiation ( 4 cases , 2 ' 7 per cent ) , ( d ) " sebaceous " differentiation ( 4 cases , 2 - 7 per cent ; lipoid was not identified , and the recognition of these occasional foamy cell groups as sebaceous remains doubtful ) , ( e ) duct formation ( 1 case , 0 ' 67 per cent )  . the four forms of differentiation finally chosen were , in order of frequency , palisading , fluid formation , whorls and pigmentation . 
these presented the following advantages : ( a ) they were all common , so that sufficient cases were available for analysis , ( b ) their presence or absence could be readily determined , ( c ) between them they were responsible for a large part of the variation in appearance assumed by rodent ulcers , and ( d ) they were constant throughout the same tumour , varying 198 b . 
what form of degeneration of the stroma it can be we fail to see ; it is not myxomatous , it is only occasionally hyaline , and it is curiously localized if it is oedema . if one believes it to be stromal in origin one is driven to the proposition that it is an active secretion of the stroma - an unprecedented occurrence in an epithelial tumour , and one that in any case does not explain cysts within the epithelial masses . massive fluid production such as that of fig . 
by whorls we mean small spherical groups of concentrically arranged flattened cells , more eosinophilic than the bulk of the tumour and centred on an even more eosinophilic core ; this core often appears to be converted into a laminated plug of keratwe have counted nothing that did not have at least a trace of central eosinophilia , but think it possible to recognize even earlier stages of which the giant - cell - like structure seen above left centre in fig . 
9 may be an example . at least three interpretations of these structures have been offered : ( a ) they are cell nests ( parakeratotic pearls ) of basically the same nature as those of squamous carcinoma ( darier and ferrand , 1922 ; montgomery , 1928 )  . 
they differ from these in their smaller size , sharper demarcation , more concentric structure and lack of prickle cells , and usually also in the smaller amount of central keratin . ( b ) they are abortive hair follicles ( haythorn , 1931 ; warren , gates and butterfield , 1936 )  . 
the term " abortive " here is a convenient one ; it conceals the fact that the structures resemble no part of the follicle in any respect which will bear analysis . 
1. - a nodular tumour of the epithelioma adenoides cysticum type , only slightly cystic . note the position immediately deep to the epidermis , unrelated to hair matrices , and also the focus of abnormal epithelium , similar to that seen in early rodent ulcers , to the right of case 219 : from the scalp of a man of 33 , duration 22 years . 
14. - colonization of hair follicles by homologous epidermis in the rabbit . the epidermis and the epithelium of the hair follicles of the graft have died , but the stroma survives . 
and post - auricular area from pinna postauricular , 2 . is justified by the obvious difference in exposure , and by a difference in behaviour well marked even in the small number of cases involved . 
a preliminary test by standard error of the differences of the percentages in the 0 and 9 - 15 columns for each other and from the mean figures for the whole series gave in the event results differing little from those of more elaborate methods . 
wells it casts some doubt on the opinion earlier histological diagnosis is most difficult . it would , however , be statisexpressed that these tumours are all rodent ulcers . tically equally suspicious to find no such tumours . perhaps the group does include some spurious cases , but we have found no satisfactory way of determining which they are , and at least two alternative explanations , apart from chance numerical variation , offer themselves . 
boag , to whom we submitted the figures , pointed out that from nition . the statistical point of view they could equally be explained by a slight tendency to aggregation of the separate differentiations ; he was able to show that three of the differentiations ( melanin being the exception ) were rather more frequent in the presence of one than in the presence of no other differentiation , and a little more frequent still in the presence of two others . 
our four main differentiations are seen to associate in what appears to be an entirely random manner . put the case that there exists any histologically separable sub - group of rodent it is exceedingly improbable that ulcers , and that it is of any substantial size . any significant definition of such a group could be devised that would not involve at least one of our four main features . 
then infallibly the random grouping only if it is extremely small , or if it depends on criteria would be disturbed . which have so far escaped definition , can any such sub - group exist . of such possible sub - groups the most important , because the most widely accepted , is the " type intermediare mixte " of darier and ferrand ( 1922 ) which is the " baso - squamous " form of montgomery ( 1928 )  . 
we can find no real criterion of the diagnosis of this type of rodent ulcer except the presence of the " pearls , " and these do not appear to be anything except our whorls in their higher grades . montgomery ( 1928 ) states that his cases are more malignant and radio - resistant than other rodent ulcers . 
he gives no statistical evidence for this , basing it as far as one can tell from his original article on impressions of a small number of it will be clear that our series gives no support for the idea either of the cases . separate existence of such a group , or of the abnormal behaviour of tumours showing whorls . 
of our 54 cases showing whorls , 4 recurred ( 7 ' 4 per cent , compared to a general recurrence rate of 11 ' 3 per cent ) and 3 responded poorly to irradiation ( 5 - 6 per cent compared to a general rate of 9 ' 7 per cent )  . 
an analogy which seems justifiable is that of the parathyroids and the thymus ( the liver and the pancreas , or the enamel organ and the sublingual glands , or several other pairs might be used ) , which are equally closely related in their manner of origin , but yet , being histologically totally distinct structures , produce totally distinct tumours . foot 's ( 1947 ) manner of using the term primordium for the conical downgrowth of epidermal cells in which both begin obscures the fact that such primordia are not usually multipotential ; except in the few areas where sweat glands open into the mouths of hair follicles the two structures arise quite independently , and in most places at different times . we are impressed with the lack of truly organized structure in these tumours . we have already expressed our doubts about the so - called abortive hair follicles . one solitary tumour in this series ( unique so far in an experience of roughly twice the number of cases here reported ) showed reasonably convincing duct - like structures . 
wells hair matrix origin of rodent ulcers . mallory ( 1910 ) suggested that the rodent ulcer might arise from the hair matrix , and haythorn ( 1931 ) asserted that all rodent ulcers are simply hair matrix subsequent authors ( except wallace and halpert , 1950 ) have not tumours . accepted this completely , but no detailed refutation of his arguments has been attempted . 
they may , however , be shown to include several major flaws . ( a ) haythorn ( 1931 ) regards the connection of the tumour with the epidermis as purely accidental . 
we are not very impressed with the fibrils which we have been able to demonstrate ; in any case it seems probable that they are homologues of the tonofibrils of the rete malpighii , and that such fibrils form readily in any epidermal derivative , forming intercellular bridges which will be demonstrable as prickles wherever the cells are sufficiently separated . ( c ) haythorn 's ( 1931 ) identification of hair shafts we have already discussed . ( d ) we have been unable to confirm haythorn 's ( 1931 ) observations on the resemblance of the silver - stained basement membrane of rodent ulcers to that indeed it would be surprising if a hair matrix tumour of the hair follicle . developed a basement membrane like the " membrana vitrea " of the root sheath . we have not examined the neurofibril content of our tumours , but willis ( 1948 ) has indicated the unreliability of the evidence they provide . ( e ) haythorn ( 1931 ) describes atypical proliferations of hair follicles producing appearances which he interprets as the early stages of rodent ulcer formation . but nearly all these changes occurred in hairs surrounded by squamous carcinotheir occurrence is therefore rather to be taken as evidence that hair mata . follicles react in this sort of way ( it may be merely a disturbance of ordinary cyclical regeneration ) to any injury , and to be a warning that apparent transitional changes in hair follicles even within a rodent ulcer may be quite meaningless . ( f ) an argument which haythorn ( 1931 ) does not consider is that based on the site of origin of the tumours . 
where we find it necessary to disagree with them is in the assumption that this necessarily involves a specifically basal - cell origin . as von hansemann ( 1907 ) was first to point out , the surface epithelium and squamous carcinoma arise equally from the basal layer of the epidermis . seems , however , to be generally believed that if squamous carcinoma and rodent ulcer are different tumours they must have separate cells of origin . this is not necessarily so . 
at the time of first formation of hair follicles the epidermis possesses only a rudimentary " peritrichous " outer layer , and all the capacity for growth in either of its two main directions resides in one layer of cells . 
the stimulus to active keratin - ward proliferation ( whatever its proximate mechanism ) must be found in some contact with the external world - though there must be a basal level of activity of the degree seen in implantation cysts . 
the stimulus to the adnexa - ward proliferation almost certainly comes from some form of direct evidence for this in mammals is organizer - like action of the mesoderm . lacking , studies of hair genesis so far available ( danneel , 1931 ; trotter , 1932 ) being purely anatomical . 
but in birds , where the large size of the feather papilla makes transplantation experiments possible , the work of lillie and his school has produced a great deal of evidence on the process of morphogenesis ( lillie , 1942 ) , and it is known that the original stimulus to feather growth arises in the dermis ( wang , 1943 ) , though the type of epithelium responding to the stimulus partly determines the type of feather to be formed . in the absence of evidence to the contrary it seems reasonable to assume that in mammals hairs and sweat glands are evoked from the epidermis by similar stimuli derived from the dermis . thus we have two stimuli , two responses , and two very different forms of correspondingly we have two forms tissue formed from the same layer of cells . it is simplest ( though perhaps not entirely necessary ) to state the of tumour . resultant hypothesis in terms of pullinger 's ( 1949 ) theory of tumour origin in general . according to this , squamous carcinoma would be the result of the acquirement by epidermal cells of the ability to produce within themselves some stimulating substance which is normally only derived by external irritation , and rodent ulcer is the result of the acquirement by the same cells of the ability to produce an altogether different substance - a substance which is the normal stimulus to growth of the adnexae and which is normally derived only from the dermis . why then , if it results from the application of the same stimulus to the same tissue , is the rodent ulcer so poor a copy of hair follicle or sweat gland ? a possible answer may lie in the importance of the connective - tissue framework in the differentiation of adnexae , a matter concerning which there is some experimental evidence . 
a hypothesis is proposed ascribing the rodent ulcer to the autogenous production by the epidermis of a stimulus to proliferation normally supplied by the foetal dermis . our thanks are due to professor j . 
mark 's hospital , london . received for publication december 29 , 1949 . it is well known that different histological types of cancer vary in their rate of growth and liability to metastasise , but this relationship between histology cancer and spread is easier to study in some organs of the body than in others . of the intestine provides exceptionally good material for investigating this question becauise ; surgical operations are planned to remove a good stretch of bowel above and below the tumour as well as a wide field of lymphatic drainage in the mesentery . 
it happens , therefore , that the pathologist is presented not only with the tumour , but ' also with its surroundings , so he can first classify the tumour according to ' its histology , then measure the extent ' qf local spread by direct continuity , and finally record the number and position - of lymphatic metastases . when the histology and spread are compared in ' this way it soon becomes obvious that different histological ' varieties often behave differently , but a fairly big series of cases must be examined in order to express these differences quantiit takes a long time tatively and give them anything like precise significance . to collect many cases of cancer of one organ of the body such as the bowel at a general hospital admitting patients of all sorts , ' but this is done much more quickly in a special hospital like st . 
mark 's , which is intended primarily for in the last 21 years ( 1928 - 1948 inclusive ) diseases of the rectum and colon . 3 , 220 operation specimens of intestinal cancer were examined and dissected in the laboratory of st . 
mark 's hospital , the general distribution being as follows : 2 , 642 cases of cancer of the rectum , 429 of the colon , 51 of the anal canal , and 98 cases of multiple malignancy . in each case the records describe the extent of local spread and the n ' umber and position of all lymphatic metastases . preparation for this paper i ' have re - examined the sections of all these tumours and compared their histology ' and their local and lymphatic spread . since rectal cancer is much the largest of these groups , forming 82 per cent ' of all cases , it will be convenient to start with this . histological varieties of rectal cancer . the three main histological varieties of rectal cancer are adenocarcinoma , colloid ( or mucinous ) carcinoma and an undifferentiated variety which i shall call " anaplastic carcinoma simplex . " aden ? ocarcinoma is much the commonest variety , constituting 85 per cent its histological characteristics are well known , and it is sufficient of all cases . to point out that though the growth usually shows a tubular or acinar pattern there is as a rule very little evidence of mucous secretion , either in the cells or 60cuthbert e . 
the last two varieties were too few in number for analysis , but comparisons between adenocarcimoma and colloid showed once more that mucus thus , secreting or colloid tumours spread more rapidly than adenocarcinoma . at the time of operation , only 5 per cent of the colloid carcinomas were still stage 1 , i.e. 
dukes such relationship between histology and spread can only be established if the histological varieties are clearly distinct : as definitely different , let us say , as adenocarcinoma , colloid carcinoma and carcinoma simplex of the intestine . this is seldom the case . another handicap arises from the fact that in some organs of the body the injury caused by a malignant growth is much more attributable to its anatomical position than to any niceties of histology . this is particularly true of tumours of the kidney , ureter and bladder . 
jackson memorial laboratory , bar harbor , me . received for publication december 16 , 1948 . it is known from the work of andervont and bryan ( 1944 ) and green , moosey and bittner ( 1946 ) that the milk agent is antigenic for the rabbit and the rat . if , as is probable , the agent , is a virus one would expect it to be antigenic for the susceptible species . 
for 30 minutes and then cooled prior to incubation . in experiments 4 and 5 serum from hyper - immunized blacks was used . c57 black mice were injected at weekly intervals as follows : 0 - 1 ml . , 0 - 2 ml . , 0 - 4 ml . , 0 - 8 ml . , 0 - 8 ml . , 0 - 8 ml . 
a few papillomata also occurred . there is a suggestion of neutralization in experiment 2 . however , the probability of obtaining such a result by chance alone is of the order of 10 per it is specially regrettable that this experiment was terminated , as a higher cent . degree of significance might well have been obtained . on the other hand , there is no suggestion of any neutralization in experiments 4 and 5 , where hyper - immune serum was used together with a relatively weak tumour fitrate . 
of special pertinence to experiments with serum from hyper - immunized blacks may be the observations of webster ( 1938 ) and hodes and webster ( 1938 ) with the virus of st . 
louis here a high degree of immunity developed about 6 weeks after encephalitis . vaccination , and had waned before antibodies appeared some 20 weeks after injection . it may be that we bled our mice too soon . possibly our technique was entirely inadequate to show any neutralization . however , identical techniques were used by andervont and bryan ( 1944 ) and green , moosey and bittner ( 1946 )  . 
a strain it may be that this very mice are being constantly immunized since birth . prolonged stimulation is needed for the production of protective antibodies . the fact that the agent can be demonstrated in serum may be due to dilution if this is . 
sionally. 197 3 67 0 364 - 0 102 - 6 . 41 - 0 used purgatives ; 1 - 7 x 34 or 63 to have used purgatives occasionary in a hke manner the " expected 34 or 6 - 1 to have taken purgatives often . f7 - 7 x purgative consumption was calculated separately for each age - group in each sex . 
the differences in the senna rates - while showing tlle same trendwere not so marked or consistent , and though the age - specific rates of beecham 's pffls ' usage were without exception higher amongst gastro - intestinal cancer patient 's , the individual differences were , on the whole , small . direct comparison between the gastro - intestinal cancer and non - gastrointestinal groups was carried out for each of the three purgatives , making appropriate adjustment as before for the differing age and sex structures of the two populations ( table vi )  . 
the comparisons relating to the use of senna and beecham 's pills show similar , but less marked , trends towards heavier usage by the gastro - intestinal cancer patients . 
the excess of senna in the gastrointestinal cancer group just reached the level of technical significance and while a similar picture was presented by the separate consideration of cancer of the large bowel ( x2 - 7.21 , n - 2 , p = 0 03 ) , - there was no significant difference between the experience of patients with cancer of the stomach and those suffering from a non - gastro - intestinal complaint . 
my reasonsfor considering that the risk is a real one , and of importance , are perhaps best discussed in relation to my own experiments , as i am naturally most familiar with this material . i was iniectin - a dibenzanthracene into some dominant ( lethal ) yellow mice of mainly inbred type , and intercrossing for up to four generations , in order to study the type . 
the tests were on 400 - 800 f . , these results , though consistent , are not significant , males in each group . statistically tinless all three treatments are combined into one group . 
of some interest was the incidental finding of two visible mutants found in the casual inspection of cib c - liltures , which would be an extraordinary occurrence bv rhe drosophila work , too , indicated that the mutants were mere chance . appearing in sperm after removal from the mutagen . 
carr which tends to mutate only those loci that require a relatively small energy this would also imply that these comchange to give a new stable mutation . pounds will tend especially to mutate genes that have already mutated sponthus , although not really specific for any given gene , their effects taneously . may be normally limited to only a small fraction of the genes on the chromosome . but i think that there is a risk with chemicals not encountered with radiation , and this is that a given chemical can react with all , or at any rate a major part of the genes of one type in the animal exposed , as this is merely a repetition of a possible chemical reaction . 
then they might also react with the same genes in another exposed individual equally well , and thus the offspring of two people exposed to carcinogenic mutagens could immediately show an induced recessive . this gives a new problem in human genetics - inbreeding with an occupation . and i have suggested that mutant sperm may be produced long after exposure i don ' t know whether any data on this could be accuto the mutagen ceases . mulated - all i can recall in the literature is the so - called " industrial melanism " of insects described by harrison ( 1920 ) , but i think we should be aware of the risk . 
h there had been equal nijmbers of men and women , and equaltiijmbers in the age - groups , on an average the male mortahty would have amounted to 24 , 855 . 
environment and occupation may , therefore , be expected to exert a greater influence in men . bt say that , ta average age at death from carcinoma of each site . for each organ we can assess not only the fi - equency with which it is attacked , but also the average ao , , e at which this occurs . 
among jewesses we find a low incidence for carcinomas of the stomach , bile pwmges and cerv and a high incidence for carcinomas of the breast , ovary , intestines and kidneys . it would be more conrect to compare the jews , not with the average population , but with that of the big towns , for 80 - 4 per cent and 80 - 7 per cent of jews and jewesses respectively live in the big towns . if we do so , we find an increased incidence for carcinomas of the liver , gall - bladder , intestines , rectum , lung , ovary and breast , and a decrease for the mouth , oesophagus , stomach , uterus and skin . from the age - group distribution of jews and jewesses we should expect 228 and 296 cases of carcinoma respectively . 
versluys carcinoma and trade or profe , 88ion . we now come to the real object of this research - to trace the influence of trade or profession on the origin and the localization of carcinoma . 
our research covers a wider field than that of the so - called occupational cancers , and all that has gone before is necessary to enable us to interpret what follows . before we can proceed , we shall have to examine some problems a little more this will be facihtated by taking a specific occupation as an example , closely . and i have chosen coal - mining . the mortality cards show usa . 
how many people who had formerly been miners died of carcinoma from 1931 to 1935 , their ages , and the sites of their carcinomas . the census of 1930 ( there was no census in 1940 on account of the german occupation , consequently - an interpolation is - impossible for the years between 1930 and 1940 ) shows us ilow many miners there were in the netherlands and their ages , but ' thin ' about ex - miners . now if we take groups a and b together we include all miners who got carcinoma while still working or aftei . 
leaving the trade , with their ages , but t , leir distribution over the various age - classes cannot be compared with the census of 1930 , which gives no information about ex - miners . 
then the average error is when y i - s small this is practically equal to - % / .. this average error has always been taken into account in this research , and a difference is cared real only when the percentages found and those to be expected differ by at least 3 times the average error . fi - equency of carcinoma of the stomach and a low prostate fi - equency . 9 and 50 - 59 we find a total mortality of 50 cases , whereas we should expect 32 , so that the total carcinoma mortality of coal miners is very high . 
the fi - equency of carcinoma of the stomach is greatly increased , for we find 33 cases instead of the 12 we should expect . the number of cases of carcinoma of the prostate in these age - classes is too small to reach a conclusion . the question now arises , is this high stomach fi - equency amongst miners bound up with their trade or with their environment ; is it an " occupational or a " smial cancer " ? ( cramer )  . 
to answer this question we must caiieer study wives of miners " ( from the mortality cards of those married women where the trade of the head of the - fanifly is given as " coal miner . the manded woman practically always lodges with her husband )  . the percentage distribution of carcinoma over the various organs in " wives of miners " compared with the distribution in married women amongst the general population is shown in table xi . taking the " average error " into account , we see from table xi that only 172 j . 
the smaller the unit in our research the more chance we have of getting results of value . if we gather the trades into groups their typical characteristics will become less and less obvious until at last the ratios obtaining among the average population are reached . i have tried taking some trades together , but the typical characteristics of a certain trade always get lost , and the whole group misses what individual component parts show as typical . it is , however , worth while compari ' ng any one trade with another of the same kind , and i have therefore tried to arrange the order of the trad - es - discussed so that trades 6f a kind come near tog ' ether . table xii compares the actual deaths of men of different occupations with the numbers expected if the total carcinoma deaths had in each instance been distributed over the various sites in the same way as amongst all m - ales - that is to say , according to the percentages shown in table 1 . table xllla gives a similar comparison , for wives of men in different occupations , with the deaths expected if the total carcl ' no ' ma deaths had been distributed in the same way as amongst all married women . 
cervix uteri lung prostate lung prostate oil vender8 are only a small group but have an excessi - % , , ely high stomach frequency ' ( 26 carcinomas )  . . 
faxmers. baxmen butch ' ers soldiers - cigax makers soldiers university men tobacconists dock labourers shoemakers office - staff .iall porters officestaffs car men barinen teachers soldiers fisherinen plumbers greengrocers barinen plumbers commer . 
froiii the figures in this paper , but that would take up too much space . besides , in mv opinion conclusions will only be justifiable when we have sufficient international material at our disposal for comparison . this material will have to consist of the results of research on manv , relativelv small , areas during a period shortlv before the second world war , with due observance of the nece , - , sarv precautions . 
with larger areas racial , geographical and economic differences in the same trade plkv a prominent part . , and a trade during and after the war may be quite different in one countrv from another . there was too much changing of occupation . i wish to express mv gratitude to w . 
in 1940 , however , an important change was made in the method of selecting the main cause when more than one was mentioned , a disease certified as merely contributory and not in the direct line of causation being ignored if another well - defined condition was stated as the direct cause of death . 
for breast cancer in women , for example , the factor to be applied to a rate at all ages prior to 1935 was * 941 , signifying that the new procedure resulted in about 6 per cent of deaths which would previously have been credited to breast cancer being classified from 1940 onwards to some other cause . when it is desired to study the trend of mortality at particular ages it is not safe to assume that the correction to be applied to rates before 1940 is the same at every age , until that has been tested , as may be done by examining the ratios at different age groups between the deaths in 1939 based upon the old and new methods , tabulated for that purpose in table 21 and appendix bl of the statistical review for that year . 
for certain diseases where it was important , such as diabetes , more exact correction factors based on the dual classification carried out during the four years 1936 - 1939 were used in the registrar - general 's analyses of past death rates at separate age - groups , and on page 135 of the text volume of the review for 1938 - 1939 it was shown that for cancer of all sites combined the conversion ratios for each sex were slightly above unity at ages before 45 and below unity at ages after , diminishing regularly from 1 - 17 at 5 - 14 to * 95 at 75 and over for males . 
for cancer of the breast in females it can be deduced from the tables in the review for 1939 referred to above that the correcting factor diminishes progressively from * 997 at ages 25 - 29 to * 970 at 55 - 59 and * 810 at ages 85 and over . this is easy to understand because of the increasing frequency of the presence of breast cancer , in its primary or secondary manifestations , as age advances , coupled with increasing liability to die of some intercurrent disease which is certified as the direct cause of death . throughout these studies of the trend of death rates according to age , correction of the rates in years prior to 1940 has been carried out by means of factors derived jointly from the age - by - age comparison of the deaths in 1939 and the conversion ratio forall ages combined obtained from the dual tabulations of 19361939 . 
in 1950 - 51 it was possible for the first time to distinguish mortality from cancer of the cervix and corpus uteri as a result of the inquiries by the general register office concerning the large number of deaths certified as cancer of " uterus " without further definition , and the rates for cervix uteri show still lower proportions of single women and greater contrasts with cancer of the breast and other causes of death . amongst women who died of cancer of the cervix in 1950 - 51 at ages under 55 the proportions who had never married were about one - third of the corresponding proportions amongst women who died of all causes . 
at ages 55 - 74 the proportion was about two - fifths , and after 75 it was about three - fifths . in the registrar - general 's statistical review for 1940 - 45 ( registrar - general , 1951 ) deaths of married women from various causes were analysed by age groups according to whether the deceased women had borne any children or not , and the resulting proportions stated at death registration not to have had a child are shown in table iv for cancer of the uterus , breast and ovary , non - genital cancer and all causes . 
2 , in sharp contrast with those for uterine cancer , show on the whole very little change through 25 10 o > 9a t > 7 - id 5 - s = ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~i 1921 - 26 1926 - 30 1931 - 35 1936 - 40 1941 - 45 1946 - 50 40 period when death occurred age when death occurred fig . 
at ages over 65 the rates increased up to 1940 and then fell back slightly , and between ages 30 and 45 the rates have increased since 1940 . this may be seen by expressing the rates in 1936 - 40 as percentages of those in 1926 - 30 , and the rates in 1946 - 50 as percentages of those in 1936 - 40 ( table x )  . in the right - hand part of fig . 
the increase in breast cancer mortality of married women to be expected at ages over 45 in the last 20 years would be , therefore , about 2 per cent of the rate . information as to the numbers of pregnancies experienced by women who later develop breast cancer at various ages is scanty , and the only data found suitable for the purpose of this study were in a tabulation by smithers et al . 
at ages under 45 the effect seems to be even greater , but the numbers on which the ratios are based are small , and there might be other reasons contributing to the low frequency of women with four or more children amongst patients seen during the period of childbearing . it is desirable that this inverse correlation with number of children should be confirmed by other and larger data , and such information is in process of collection in the liverpool hospital region and north wales by the british empire cancer campaign . assuming it to be correct , what effect would the decline in family size since 1905 be expected to have upon the incidence of breast cancer amongst all married women ? this can be calculated by applying the ratios for women over 45 to the successive distributions in table via and aggregating the products , as was done for uterine cancer . 
lung cancer death rates and also those from other forms of cancer tend to increase steadily with advancing age , so movements of the ratio will give no indication of that part of the upward progression which lung cancer shares with other cancer . 
the years of birth of the cohorts attaining these ages in 1901 would be 1882 - 1886 , centred at 1884 as the table indicates ; in 1931 the men of this cohort would be aged 45 - 49 , in 1933 their ages would be in calculating the ratio for the 1884 cohort in 1933 , three times 47 - 51 , and so on . the deaths at 45 - 49 were added to twice the deaths at 50 - 54 in 1933 , both for lung cancer and all other cancer , and the first total divided by the second . 
the " nil " increases in table xvi from 1937 to 1940 mean that in the cohorts bom before 1875 the lung cancer death rate was then rising with advancing age after 65 at about the same gradient as the death rate for all other cancer , and the actual gradient at that age and time period has been shown to be about 33 per cent increase in 5 years or 20 per cent in a trienniuthe preceding explanation for what occurred between 1925 and 1937 is far more probable , but the hypothesis of influenza as a contributory factor cannot be excluded as a possibility . from 1937 onwards sulphonamides began to be used in increasing amount , and pneumonia deaths soon began to fall . could this have affected lung cancer rates appreciably by keeping alive longer many persons who without sulphonamides would have died of a complicating pneumonia before the cancer could be recognised ? pneumonia death rates of males fell between 1937 and 1942 from 477 to 255 per nillion at ages 35 - 44 , from 950 to 457 at 45 - 54 and from 1535 to 1024 at 55 - 64 , and the fall continued till 1946 . during that period the lung cancer ratio continued to rise steadily in men born about 1880 , the gradient being steeper amongst men at ages around 40 and 50 than around 60 , and only slight after 65 . 
the distribution according to number of children born for hospital patients with breast cancer , in such data as are available , differs from what would be expected , and it is estimated tentatively that the bearing of 2 or 3 children reduces the likelihood of this form of cancer developing after 45 by about onefifth and that 4 or more children reduces it by about two - fifths . 
from 1937 to 1946 the lung cancer ratio continued to rise by about 7 per cent of the ratio annually in men born since 1890 , more slowly in men born between 1875 and 1890 , and inappreciably in cohorts born before that ; but after 1946 the increase again spread to all ages . sulphonamides , and later penicillin , must have contributed something to this by preventing some men from dying from penumonia without cancer having been diagnosed and allowing them to die a year or so later of recognised lung cancer . 
one of these paths is through the physiology of hormone production , a second is through the physiology of the propagation and transmission of the milk agent , and a third possible path is through the physiology within the mammary tissue cell governing its reaction to the hormonal stimulation or the milk agent . other paths may exist , and it is probable that these three are interwoven . proof of the effect of genic action in relation to the hormonal stimulation resulted from crosses between strain a in which the incidence in the virgin females is low althougb that of the breeders is high , and strain ch , in which the incidence in the virgin females as well as the breeders is high . results of such crosses were reported by bittner , h ' useby , visscber , ball and smith ( 1944 ) , heston and andervont ( i 944 ) , and bittner and huseby ( i 946 )  . 
heston incidence occurred among the virgin f , females resulting from mating strain a females to ' ch males as well as in the reciprocal f , virgins , together with evidence of segregation in the f2 generation , indicated that the difference between the strains was primarily a genetic difference . such a difference was to be regarded as either a difference in horinone metabolism , or a difference in the reaction of the mammary gland cell to horinone such evidence as the fact that added oestrogen stimulation yielded stimulation . tumours ' m ' strain a virgins , ' and the relationship of number of litters to tumour incidence in strain a reported by jones ( 1940 ) , strongly indicated a difference studies of the oestrus cycles of virgin females of strains in hormone stimulation . a and ch and their reciprocal f , hybrids reported by deringer , heston and andervont ( 1945 ) , however , failed to show any difference in the cycles , except that in the low - tumout group of virgin strain a females the onset of oestrus was these observations , therefore , suggested later than in the high - tubiour groups . that at least a part of the genetic difference could be manifested through the physiology within the mammary gland cell . in an attempt to determine the physiological mechanisms through which the genetic differences are manifest , huseby and bittner ( 1947 ) also have carried out a number of studies , including ovarian transplantation to f , hybrid mice , ovariectomy , vaginal smearing , and histologic examination of the genital tissues . these studies have indicated that more than ' one endocrine organ is affected . we have become more interested in the second path of gene action - that it has concemed with the propagation and transmission of the milk agent . been observed by a number of investigators that certain strains will propagate all the . 
high - mammary tumour strains the agent , whereas others will not . normally transmit the agent from generation to generation , and even low - tumour strains may have the same innate capacity as andervont ( 1945 ) has reported for strain c . 
on the other hand , bittner ( 1948 ) has found tumours in his descendants of strain c57 black females into which the agent was introduced , and fekete and little ( 1942 ) bave reported mammary tumours in descendants of c , , 7 black females that had developed in strain dba uteri from transferred ova . 
thus the situat ' lon m regard to the capacities of the various strains to propagate the agent is not clear . by crossing high - tumour strain c , h females with low tumour strain c57 black males , and comparing the two . 
types of backeross females produced by backcrossmg the f , females to both the ch and c.7black males , definite evidence of genetic differences in the ability to propagate and transmit the agent was revealed ( heston , deringer and andervont , 1945 )  . 
the possibility of an intracellular origin of the agent also enters into consideration . in some respects the picture is comparable to the gene - kappa relationship stuclied by sonneborn ( 1945 ) and co - workers in parameciuthe picture is not as , simple , however , in that there is . 
our attention has been directed toward tumours that arise in females in which the agent cannot be demonstrated ( heston , deringer and levillain , unpublished data )  . in order to obtain a line of strain ch friae of the milk agent a litter of 3 females and 3 males were removed from their c3h mother by cesarean section and fostered upon c57 black female . 
these females were later mated to their male littermates , and subsequently the line has been maintained by brother x sister matings , the young being nursed upon their own mothers . in this line ; designated as c3hb , mammary tumours are arising . in 194 of the females of this line 14 months of age and older and extending through the first 5 generations to date , 47 , or 24 per cent , have developed mammary tumours at an average age of 18 - 9 months ; seventy - eight have died without mammary tumours at an average age of 16 months , and 69 are still living at an average age of 18 months . study of the histologic section of 37 of these tumours has not revealed mucli dissimilarity between these and the usual ch mammary tumours . nine , however , ' have show - n areas of squamous metaplasia with pearl formation similar to that described by kirschbaum , williams and bittner ( 1946 ) in mammary tumours induced by methylcholanthrene in the absence of the milk agent such areas also occasionally occur in normal strain ch females of older age , suggesting that the occurrence of squamous areas may be characteristic of older age rather than specifically owing to the possible absence of the milk agent . three of the tumours arising in these c3hb females have been transplanted into other c3hb females , and in each case the transplant has grown . we have no evidence , however , of a milk agent in the etiology of these tumours . distribution of the tumour - bearing females in the pedigree chart does not show segregation of tumour families , as ' would be expected if the agent were present . furthermore , we have found no evidence of the agent in cell - free extracts of these these extracts varied from 2 to 5 per cent , and were prepared in 0 - 005 m tumours . .phosphate buffer ph 7 - 0 with a waring blendor ; cleared in a multispeed centrifuge at 18 , 000 g.. 
heston extracts premonths of age and older , and none has yet developed a tumour . pared with the same technique from tumours from c3h females produce tumours in c3hb test females in approximately 8 to 10 months . this is not presented as absolute proof that the agent is not present . if it is , however , it is not comparable to that in c3h females , nor is there any evidence of its being built up comparable to that of c3h . if the agent is not present we have proof that the accumulation of genetic factors and of non - genetic factors other than the milk agent can cause the tumour threshold to be surpassed . this light the milk agent would be comparable to the carcinogens in the induction of lung tumours , where the presence of the carcinogen appears to merely increase the probability that the tumour will appear and at an earlier age ( heston , 1942a , b )  . while all of these factors have been shown to alter the probability that the normal mammary tissue cell may be transformed into a malignant cell , these investigations have not answered the question as to what constitutes this change to malignancy . 
the largest part has been obtained from the rikshospital , which receives patients from approximately one - sixth of the material has been placed at the whole country . ' cal depaxtment iii and by proniy disposal by professor c . 
a few cases onl come from - other hospitals . in addition to the two clinical , two post - mortem series have been used in this analysis , namely , t6 material of jacobsen ( 1953 ) from wo city hos itals , covering the years 1937 to 1946 , and the material of christiansen ( 1953 ) from the rikshospital , covering the years 1925 to 1952 / 1 . all the tumours of these post - mortem materials have been typed by kreyberg with no knowledge ofthe clinical data . 
figures of each series are comparatively small , but a few feature - s seem to be significant , and possibly important . the main differences between the chnical and the post - mortem series are ( 1 ) the relatively much higher number of adenocareinomas , 30 per cent of an cases in the latter against less than 10 per cent in the former , and ( 2 ) the lower number of squamous cell carcinomas in the post mortem series . 
a study of the two papers dealing with the post - mortem series reveals a greater number of old persona i these materials , as compared to the clinical group . 
the significance of these differences will be discussed later . in kreyberg 's ( 1952 ) first paper a comparison was made between the occurrence of the different histological types in the norwegian material and a sipailar recent material from the mayo clinic . 
kreyberg mainly as a result of differences in criteria for typing . in the present table 1 , three series from foot ( 1952 ) have been included in order to stress this point . 
the distribution of histological types in this american material is remarkably similar to the norwegian clinical series , with one exception , the group adenomas and salivary gland tumours . these tumours occur more than ten times as frequeiltly in all the norwegian series . 
the cause of the relatively greater number of these suffice it to mention at this tumours may be manifold and will be discussed later . point that even in america certain investigators have found these tumours ratheifrequently . in table ii is presented the distribution of the different histological types as regards age and sex . 
t. uncertain group ii : 17 in table iii the findings regarding the sex distribution have been summarized . it will be seen that the different histological types in this chnical material form two main groups with markedly different sex ratio . 
the fact that the tumour material has been collected during the years 1948 to 1953 , while the population figures refer females 3 4567891 4 5 67 891 i1 1 3 456789 figa . 
1. - age distribution at the time of diagnosis of all lung tumours ( l ) and the mortality figures for stomach carcinomas ( v ) in males and females separately . 
1 curves have been drawn on double logarithmic paper , representing the whole material of the clinical series for males and females separately . for comparison one other curve for each sex has been drawn , namely , the corresponding curves for stomach carcinomas , based upon the mortality figures for norway , 1950 . 
2 two curves have been plotted in the traditional manner , namely , the curves for the relative age frequency of squamous cell carcinomas and for large cell lo~ ~ ~ fig . 
the figures are given in table iv the two curves are nearly identical and they indicate that the group i tumours biologically actually represent a uniform group , in spite of unquestionable histoit will further be seen that these group i tumours mainly are logical differences . responsible for the characteristic features of the lung " cancer " curve in general . the other lung tumour types have been examined in a similar manner . 
the adenocarcinomas and the sub - group lung adenomas and salivary gland tumours are , however , comparatively few in number in each of all the series , and accordingly subject to considerable chance fluctuations . in table v the figures for these tumours in all the three norwegian materials the basic figures are given for each material separately . 
the unusual fall in the higher age groups is taken to indicate a new carcinogenic situation in development , and the future stages are forecast by the direction of the curve for the age groups 30 - 39 and 50 - 59 . the adenocarcinomas , according to our diagnostic criteria : malignant tumours coniposed of more or less atypical , secreting or non - secreting , columnar cefls , with their nearly equal sex distribution and their increasing frequency with the advancing age , indicate that they are caused bv comparatively weak carcinogenic agents , well established in the society , and acting with equal strength in both sexes , at least in large parts of the country . the small group of bronchiolar cell carcinomas occur in all age groups and with no definite sex preference . 
they may be caused by unknown agents , hitting at random . the histological pictures , the equal sex incidence , as well as the appearance with no preference for any age group , together indicate that lung adenomas , benign and malignant , and sahvary gland tumours are caused by accidental factors , presumably of developmental origin . at this stage a few more words about the latter tumours may be justified . 
some surprise has been voiced as to the considerable number of adenomas and sahvary gland tumours in norwegian materials . firstly , a survey of the lung tumour problem i : ft general in norway shows that the total number of lung tumours is very moderate , as compared to the figures for england and wales , the netherlands , the united states , and even denmark . if , as assumed and actually found , the recent increase in norway is caused mainly by the ever larger number of group i tumours , it follows that the other histological this development has been types must show a proportionate decrease in number . observed in the ordinary columnar cer carcinomas . 
a decrease in the number of lung adenomas and sahvary gland tumours in relation to the sum total is predicted , but it may for a while be less marked in chnical and especiary surgical materials , because these tumours represent comparatively favourable clinical cases and are therefore selected . secondly , the tumours in question are , actually , observed in considerable numbers also in other materials . fried ( 1948 ) in the massachusetts general hospital found 17 adenomas out of 175 cases of bronchogenic neoplasms , and carlens ( 1952 ) reported 8 - 10 per cent in a swedish material . tbirdly , these tumours are most probably present in many other materials without beino , recognized as such . true columnar cen careindmas and group i tumours very rarely occur before the age of 35 years . in most 1 - ung cancer statistics some tumours are registered in younger and verv young persons . 
some of them have been diagnosed as adenocareinomas , others as sman cer carcinomas , especially if unfavourable preparations have been the base for diagnosis , or if the tumours are infiltrating . 
any academic discussion as to the propriety of the designation cc mahgnant adenoma " ought to be silenced by the very important fact , that if the proper criteria are observed and the material typed without clinical information , a grou ' of tumours will emerge , wit - h a sex ratio 1 : 1 , with no accumulation in anv a - ae aro - ud . 
each histological type has been analysed , especiany as to distribution according to age and sex , and the findings correlated to our additional present knowledge regarding the development of these tumours . 
they do very seldom occur before the age of 35 years and they present a very characteristic and , for human carcinomas , singular age distribution . this age curve as wer as the vital statistical evidence indicate that the main part of these tumours are caused by a recently estabhshed carcinogenic situation in certain ' parts of the world , notably the united states of america and westem europe . columnar cell adenocarcinomas appear with httle sex difference and they show a steadily increasing ffequ - ency with advancing age , a development in conformity these tumours are probably caused by with a great many human carcinomas . comparatively weak carcinogenic influences , evenly distributed over large areas , well established in the society and striking both sexes with equal force . bronchiolar cell carcinomas occur with equal frequency in both sexes and strike individuals in ar ages . 
the present work was undertaken to determine whether any such action would affect the process of malignant transformation as brought about by painting a carcinogenic hydrocarbon on mouse sk material .and me ' l ' hods . 
 a drop of the solution was applied interscapularly on the skin of the mice , by paint - brush , once a week , for one year till the mice died or were sacrificed . 
at the end of the experiment one mouse group3 urethane group2 } : 2 = 5 = 6 - dibem : anthracene groupl 1 : 2 : 5 : 6 - dibenxanthracene and urethane 3 - .... 
it was found that all mice in groups 1 and 3 after six or more months ' treatment , had developed multiple pulmonary adenomas which appeared to be typically urethane - induced . 
 the results show that , under the experimental conditions described here , urethane ca~ no obvious change in the frequency or time of appearance of papillomas or epitheliomas induced by 1 : 2 : 5 : 6 - dibenzanthracene . 
consequently a better test to detect a weak inhibiting activity of urethane might have consisted of treating mouse skin with one application of the hydrocarbon , and thereafter more frequent urethane applications . 
no studies were made of the histological effects of urethane on either normal skin or skin under going malignant transformation , since the experiments suggest that any such effects have little action on the production of cancer . 
 it is most interesting to note that those mice which received urethane treat ment ( groups 1 and 3 ) had multiple lung tumours , whereas those receiving 1 : 2 : 5 : 6 - dibenzanthracene alone ( group 2 ) had none . 
 when urethane was administered to mice in their drinking water in previous experiments , a reduced fluid consumption was noted for the first few days , showing their aversion to it . 
an estimate was made of amount of urethane in a paint - brush full of the solution ( as painted on the mice ) by applying it to a cover slip and weighing after the acetone had evaporated . 
for previous studies ( cowen , 1947 ) , lung tumours in comparable numbers to those obtained here were induced in various inbred jines of mice by injecting 025 c.c. 
since total dosage was not the same , and since there are marked strain differences with respect to lung tumour response by different lines of mice , no accurate comparison can be made . 
it would be safe to say , however , that a large part of the urethane applied to the skin was absorbed by the mice in this experiment , judging by their tumour response . 
 it is not unreasonable to suppose that urethane is absorbed at least as readily from human as from mouse sk since urethane causes leucopenia in man ( moeschlin and melli , 1947 ) and lung cancer in mice ( nettleship and henshaw , 1943 ) and rats ( jaffe , 1947 ) , it would be well for those who handle this substance frequently in the laboratory or commercially to consider it a possible risk to health and consequently take effective precautions . 
 three groups of mice were painted interscapularly with an acetone solution of 1 : 2 : 5 : 6 - dibenzanthracene , urethane and a mixture of both each week . 
 this points out that most of the urethane applied to the skin was absorbed .all expenses in connection with this work were home by the british empire by it  . 
according to these measurements very little difference was observed between the size of the tumours of the control and dibenzanthracene treated animals in the high protein group after 7 days , and the tumours of the treated animals appeared to be only slightly smaller than those of the controls after 10 days . in the low protein group , however , the measurements on the 7th and on the 10th days showed that there was considerable inhibition of tumour growth in the animals treated with dibenzanthracene , while the tumours of the control animals had grown to about the same size as those of the controls of the high after 11 days the animals were killed and the tumours dissected protein group . very little tumour inhibiout and weighed . 
strong. from the school of medicine , yale university , u.s.a. received for publication february 2 , 1949 . the idea of somatic mutation as an explanation for the origin of cancer has had a long and interesting history . its original use as an interpretation of cancer is usually associated with the names of murray and of boveri . these men arrived at the somatic mutation conclusion from different fields , murray from experimental cancer research , and boveri from his classical observations in experimental embryology . 
at that time he stated : " the existence of such tumours , the biological characters of which are retained through long periods of propagation , shows that the cellular transformation which initiates carcinomatous growth may take place in varying degrees . 
strong this biological alteration colours permanently their whole biological behaviour . is of such a kind that the cells are able to take up nourishment , increase in size and multiply indefinitely . 
they acquire an individuality and powers of resistance to injurious agencies superior to those of normal tissue elements . " boveri 's idea on the nature of the origin of cancer stemmed from his observations on atypical mitoses . 
he had suggested the possibility that by this means an unequal distribution of the chromosomes would ensue and thus lead to unconhe was loath to expand upon this concept until aichel trolled growth . ( 1911 ) , using boveri 's own observations on the development of the sea urchin egg , arrived at the conclusion that a malignant tumour was caused by a fusion boveri 's concept has not received wide recogof a tissue cell with a leucocyte . the essential feature of the idea is expressed in his own words ( boveri , nition . 1929 ) : " the essence of my theory is not abnormal mitosis , but in general , a however this may arise , the result definite abnormal chromosome - complex . beside the multipolar mitoses , which might would always be a definite tumour . depend either on a simultaneous multidivision of the centrosome or on distorting the parallelism between the division of the centrosome and the cell - division , asymmetrical mitosis should be chiefly considered for the origin of tumours . indeed , according to analogy with certain occurrences in sea urchins , these would depend on a lack in certain chromosomes of the power to divide ; this would result far more surely in tumour than would multipolar mitoses , which depend agencies which would act on chance for the distribution of their chromatin . most directly would be those that have the power of destroying definite chromoboveri , therefore , saw quite somes.of a cell , while leaving the others uninjured . " distinctly that irrespective of the mechanism of origin , the disturbed chromatin content is the essential feature in cellular physiology , including the abnormal condition of a tumorous growth . 
the pleomorphism of cancer cells and the irregular atypical mitoses so characteristic of their nuclei had been observed and carefully studied , however , since the middle of the nineteenth century . whitman ( 1919 ) wrote a critical review of von hansemann 's theory of aiiatyzzer ( 1916 ) expressed plasia in the origin of cancer in terms of modern genetics . the opinion that a somatic mutation may be involved in the origin of cancer . tyzzer stated , " there are marked differences in the behaviour of various tumours on transplantation in given classes of mice . 
even tumours arising in homogeneous races show such differences , and this may be attributed to the acquisition of new characteristics by the soma which are manifested in the development of the tumour . 
as a matter of fact some investigators express the opinion that tb.e two genetic phenomena are entirely antagonistic to each other . for example , a very recent article by bauer ( 1948 ) of heidelberg discusses at length that susceptibility to cancer in the human population is not inherited , and at the same time elaborates on the development of the concept that cancer arises as a somatic mutation . 
that a tendency to undergo somatic mutation may have a definite genetic basis in normal tissues has been discussed by east ( 1917 )  . this phenomenon has been encountered especially in plants , but also has been reported in the higher animals . an early example of a genetic influence on somatic mutations is discussed by whitman ( 1919 )  . 
a yellow chrysanthemum carrying white flowers on one branch . it is obvious that , since the colour of the flower is known to depend on definite factors , the development of a branch carrying flowers of a colour different from that of the flowers on the other branches could only arise through the failure of the factors to pass over , during mitosis , into the cell from which that branch developed . the branch , in other words , represents a somatic mutation due to asymmetrical mitosis . " many tumours have been described in a variety of genetic material . 
many of these tumnours are pigmented and occur especially in the early development of the individual , and some are associated with or are identical to lethal mutathese have been recorded by stark and bridges ( 1926 ) , wilson ( 1924 ) tions . and others in drosophila , by federly ( 1936 ) in lepidopterous larvae , and by many other investigators . most of these tumours are inherited by a multiple factor complex and , in the opinion of t . 
morgan , expressed to the author many years ago , at least one of the drosophila tumours arose as a mutation . time alone prevents the further discussion of this interesting genetic material and its bearing on the somatic mutation hypothesis . the next advance in the somatic mutation idea in relation to neoplastic lesions was undertaken by the present author , in 1918 . 
from that time onward until 1930 , a considerable amount of evidence was accumulated from the study of the transplantation of adenocarcinomata of the mammary gland in mice that eventually provided the first experimental evdence that a somatic mutation may be involved in the origin of cancer ( strong , 1926a , b )  . 
some of these data have been published , but a considerable amount has not . the development of this experimental approach to the nature of the origin of cancer was as follows : tyzzer and little investigated the transplantation of a sarcoma that originally arose in a japanese waltzing mouse . 
they found that the progressive growth of this transplantable tumour was dependent upon thesimultaneous presence of multiple mendelizing units strong and little , in 1920 , and later in 1924 , determined that two transplantable adenocarcinomata of the mammary gland in mice were dependent upon different genetic complexes for their continued the dbrb tumour was dependent upon the simultaneous presence of growth . two genes , whereas the dbra tumour was dependent upon the simultaneous presence of these same two genes , but required the presence , in the host , of a third gene . 
the presence of the third gene had no detectable influence on the growth of the dbrb tumnour . this geneticsimilarity and difference was obtained in the same series of mice in spite of the fact that the two tumours were histologically indistinguishable ( strong andlittle , 1920 ; little and strong , 1924 )  . the opinion of identical diagnosis was given by the late james ewing , as well as by francis carter wood . 
a third spontaneous tumour that arose in a dilute brown mouse ( given symbol dbrc ) provided evidence that six genes were involved originally in the growth of this tumour when transplanted into a series of suitable f2 individuals derived from a cross between mice of the dilute brown stock and mice of a totally unrelated stock , the well - known a stock . 
growth rates on the various transplantable tumours derived from the dbrc tumrour were determined . after several transplant generations a tumour was obtained that grew so rapidly that it was considered out of the normal range of growth for the original transplantable tumour . 
when the derived transplant was tested it was found that now its growth was dependent upon the simultaneous presence of only two genes . this sudden acquirement of growth capacity also resulted consequently in a new transplantability characteristic . 
a second sudden change subsequently took place , and a new derived ( mutant ) type of tumour would now grow in 75 per cent of all inoculated f2 mice ( observation 75 - 00 + : 23 - 002 - 79 )  . 
theprobable error difference between this newratio and the actual observation for the original dbrc tumour was 5813 per cent 3 ' 67 or 15 - 84 x p.e. this new derived transplantable tumnour continued to give a 3 : 1 ratio in a series of f2 mice for several transplant generations . eventually a new sudden change took place at which time the - new mutant type grew in practically all f2 mice ( observation 243 ' 00 + : 2 - 004 0 ' 94 )  . these new data in the f2 deviated from the ratio obtained with the original dbrc tumour as follows : 80 - 78 per cent 2 - 48 or 32 - 57 x p.e. there can be no doubt that sudden genetic changes are taking place somewhere in the relationship between the host and the transplanted tumour . since the mutant types and the original transplantable tumour were being compared in the same series of f2 mice , these sudden changes could not be taking place in the host but must be taking place within the tumour cell ( strong , 1926a , 1926b )  . another point that should also be emphasized is the fact that when the derived mutant transplantable tumour would grow in all f2 mice derived from a cross between the dilute brown and the a stocks , then , and not till then , would it grow in all mice irrespective of genetic relationship . in other words , it had lost all characteristics of tissue specificity . the transplanted tumour would grow invasively at a tremendous rate and would metastasize extensively . 
thus evidence was obtained that a highly specific , relatively benign , slow - growing transplantable tumour had progressively and suddenly acquired periodically new biological characteristics until finally it had been converted over into a carcinoma . 
toward the end of the series , however , especially at the terminal non - specific state , the tumour had become more cellular and had lost most of its original adenocarcinomatous arrangement . another series of investigations was performed on the transplantation of the 13 spontaneous tumours that mouse f1 79 gave rise to . this mouse was not only the common ancestor of the well known c , c3h , cba , c121 and chi strains of inbred mice , but also gave rise to a wealth of neoplastic tissue as follows : all 13 of her spontaneous tumours of mammary origin were tested out by trans102 l . 
and if one tumour tissue has deviated from the genetic constitution of the somatic tissue from which it arose , then possibly all tumours undergo the same process in their origin . the above phenomena especially of sudden changes taking place during the transplantability of spontaneous adenocarcinomas of the mammary gland in mice has been amply verified , not only by the investigation of the 11 other tumours derived from female f179 referred to previously , but also by many other all the phenomena of tumours derived from other mice in my laboratory . transplantation consistent with the genetic theory of transplantation were also verified by the independent work of bittner ( 1931 ) and by cloudman ( 1932a , b )  . eight years later little ( 1934 ) wrote a review on the genetic work on the transplanted tumour in mice in which he included a discussion of the somatic mutation in this paper little points out that he had outlined the genetic basis for idea . susceptibility and nonsusceptibility to transplanted tumours in 1914 ( little , 1914 )  . 
he states again in 1934 that " the bit of tumnour tissue to be transplanted has a genetic composition , which is determined by that of the animal in which it if this idea was exclusively true , then it is obvious that a somatic arises . " mutation could not have been involved in its origin since any mutational process it is indeed true that in the should alter or change the original genetic state . early transfer generations , the transplanted tumour derived originally from a spontaneous source does retain the characteristic of tissue specificity determined by the genetic constitution of the mouse , inthat it will grow only in individuals which are genetically related to the mouse that gave rise to the tumour . 
but it has now been definitely proven that this phenomenon of tissue specificity is periodically and progressively lost by sudden changes resulting in simpler and simpler mendelian ratios , until eventually the tumour retains no or very little tissue specificity which it originally had . these sudden or mutational changes have always produced more and more malignancy , as determined by percentage of takes , growth rate and eventual invasive and metastatic characteristics . has also been indicated that no two tumours , even though they be derived from the same mouse and present the same histological appearance , ever showed the same mendelian ratio when transplanted into a series of appropriate f2 individuals . 
when two transplantable tumours were derived from - the same mouse , some of the genetic complex involved in their successful transplantation is common to both tumours , whereas some are unique to either the one or the similarity of genetic transplantability complex indicates genetic other tumour . in this particular little 's concept of 1914 is relationship of origin for tumours . it is also evident that the more recently chemicallytherefore partially correct . induced tumours show , from their very origin , on an average , more malignancy , and thus less tissue specificity than do the spontaneous tumours that arise in it is probably true , although at present the same series of inbred or hybrid mice . impossible of proof , that there is some genetic mechanism within cells that determines to some extent normal cell relationship , but that under certain conditions this controlling genetic mechanism alters or changes , and by so doing permits an uncontrolled or cancerouscondition to arise somewhere in the biological this phenomenon of break of co - ordinating influence of system ( the organism )  . a definitive part is , i believe , the essential feature in the origin of cancer . 
strong following the experimental evidence reported by strong ( 1926a , b ) , and verified later by bittner ( 1931 ) and by cloudman ( 1932a , b )  . little ( 1941 ) stated the problem in different terms from those he used in 1914 . he now states : " in accepting as a working hypothesis the statement that cancer ilfils the requirements of a somatic mutation , namely , a sudden change which is perpetuated in succeeding cell generations , it must be noted that there are already on record two distinct general types of somatic mutation , either of which might be involved in the appearance of cancer . 
the other has a sporadic etiology dependent on various influences which affect individual cells or groups in the first category belong such cases as mutations in colour of cells directly . genes in rodents described by castle , pincus , bittner and others . 
one of castle 's cases is particularly interesting in that it shows several cases of mutations occurring in several generations of a single family of rabbits , thus indicating a definite cases of the second type have been recorded as the result of genetic basis . lockhart - mummery ( 1934 ) has published a exposure to radio - active agents . " book on mutations and cancer , and there have been several other articles dealing with this problem . these cannot be discussed at this time . the disovery that carcinogenic compounds can induce mutations when injected into suitable experimental animals has not originated the idea that cancer may arise by a process of somatic mutation , but rather has reopened interest in an old concept . 
some experimental evidence , although of an indirect nature , has been obtained , and it is clear that there is no evidence contrary to such a somatic mutation concept . 
on the other hand , the somatic - mutation theory hardly explains the embryonic features of malignant tumours ; and it does not explain why growths are more frequent in older than in younger individuals , because it has not been observed that mutations in cells of the gonads are more frequent in older than in younger persons . 
by this means he has reported many mutations ( notably sex - linked lethals ) with the carcinogenic hydrocarbons , and has stated that he has obtained a few chromosome breaks in his experimental material . it is an established fact that the term mutagenic is not an absolute characteristic for any physical or chemical agent . for example , it may be said that x - rays are a powerful mutagen . they are very effective in inducing mutations in drosophila and in neurospora , and to acertain extent in maize , but do so very poorly in mice . 
on the other hand , ultra - violet light appears to be more effective in inducing mutations in maize than in drosophila . again it must be taken into consideration that the different physical and chemical agentsbring about mutations in various ways . x - rays break chromosomes , thus leading to rearrangements of parts , transpositions and inversions , to deletions , etc . ultra - violet light , however , seems more effective in inducing point mutations in both corn and drosophila , possibly by a specific wave - length , 2600 , that is selectively absorbed by the nucleoproviding proteins of the chromosomes . 
strong sex ratios and in distorted mendelian ratios which may be later proven to be chromosomal , but so far all mutations induced by methylancholthrene in mice finally , the which have been tested have been proven to be point mutations . mustard gas derivatives , the fourth group of powerful mutagens seem very effective in breaking chromosomes , producing embryonic disturbances very similar to the effects of x - rays , but apparently may have another action on mutagenesis , since they appear to affect cells even before there be any evidence of cell division ( action therefore similar , but possibly different from that obtained with x - rays )  . carcinogenesis again is a relative rather than an absolute property . 
on the other hand , other species are extremely liable to great resistance to experimentally for example , the rhesus monkeys in new haven have been induced tumours . injected with methylcholanthrene for 15 years without producing a single neoone must conclude , therefore , that for this species methylplastic tumour . cholanthrene is not a carcinogen , or at least , if it is , the evidence for such a conclusion is still not available . 
the problem is even more complex than this , as will be indicated in the following discussion . not all of the biological variants obtained in mice with methylcholanthrene are genetic . 
the somatic mosaic may be due to a chromosomal aberration rather than a point mutation in somatic differentiation , although the actual mechanism involved in their origin cannot be determined . we have obtained , therefore , a multiplicity of biological effects when methylcholanthrene has been injected into a series of mice over many generations . whether all these phenomena are due to the original methylcholanthrene or to one or many of the derived metabolites is still not known . 
some of these effects are definite point mutations , some are non - genetic , some are non - genetic produced possibly by inhibitions of embryonic morphogenesis at critical periods , and other biological effects may have other mechanisms in their origin , such as abnormal distribution of chromosomes during morphogenesis . but these mice whose ancestry has been injected with methylcholanthrene for many generations are not only giving rise to a wealth of biological effects , but also to a great variety of cancers . 
the first genetic result was the production of germinal mutations which changed the rate at which fibrosarcomas appeared at the site of injection of methylcholanthrene ( a decreased latent period )  . 
the second genetic effect was the production of spontaneous lesions involving the mucus - secreting cells of the gastric mucosa following the induction of this same gastric lesion from the subcutaneous injection of methylcholanthrene . 
the appearance of this specific type of pathological lesion was brought about by a mutation on the " brown tagged " chromosome . several new sudden changes have appeared in the gastric lesion subline changing the latent period for the appearance of the lesion - a phenomenon that was obtained in the transplantation of adenocarcinomata of the mammary gland in mice and discussed in this same paper . 
1. received for publication march 25 , 1947 . tins investigation was undertaken to bring up to date the records of the middlesex hospital , and to attempt to discover whether there was any statistical evidence to support commonly held views on factors affecting prognosis and methods of treatment . 836 cases of carcinoma of the female breast treated during the years 1926 - 35 were analysed and a smaller group of 265 cases , treated during the years 1936 - 40 , were the subject of a further investigation to discover the effect of more modern methods of treatment . the hopes held at the start of this work have not been completely fulfilled . for such an investigation to give a clear cut statistical answer to all of the controversial questions , careful planning and equal distribution of cases , according to the extent of the disease and type of treatment used , would be necessary . this was not so in this series of cases . 
as a result the evidence on some points is too inconclusive to give an answer . careful analysis of results would seem to be essential if methods of treatment are to be guided by fact rather than by impressions . if , in the future , reliable figures on a large scale are to be obtained , co - ordination of policy in treatment and planning ahead in large medical formations will be necessary . whether this is acceptable or desirable may be open to argument , for it must inevitably mean some acceptance by each individual surgeon of the combined opinion of his colleagues . only thus , however , can the successful analysis of large series be substituted for the analysis of smaller series for individual surgeons . despite the difficulties that have arisen owing to the many variations in technique during the years covering this investigation , certain facts that may be of interest and value have emerged . 
some previously held views have been supported ; others appear to be incorrect . the histological findings in this series have been omitted owing to the impracticability of examining over 1000 sections in the time available . this in itself would make the basis of a separate inquiry . from the statistical point of view therefore it has been assumed that any variations in the histological grouping of the growths have been spread evenly throughout the series . the presentation of results of an investigation such as this is difficult . while the actual detailed figures and tables by which conclusions have been reached are of importance , their inclusion in the text would make reading wearisome . therefore simplified tables only have been included . * late surgical registrar ( demobilized )  . 
a case dying of other causes or of unknown causes cannot really be called a success or a failure . two ways of expressing the result seem to be possible - the survival rate or the mortality rate . 
the former has the advantage that it allows easier comparison with previous series that are almost universally expressed in terms of survival it would appear , however , that results expressed in terms of ( or cure ) rates . those actually dying of carcinoma of the breast will give a more accurate picture of effects of various factors influencing the prognosis . for these reasons the " mortality rate " has been used as a routine in this it has been expressed as the percentage of those cases dead from carcinoma paper . of the breast of all traced cases . 
when both ulceration and axillary gland involvement are present as in stage iiib cases the outlook is gloomy in the extreme . the survival rate for stage i cases is 51 - 4 per cent . this appears poor when compared with other series , but it should be remembered that this figure includes only those cases alive . 
any case dying of unknown cause or other cause in this method of calculating the survival rate would be counted as a failure . if such cases are excluded from the total the figure would be very close to 60 per cent . a point that is striking is the number of cases that die from a carcinoma of the breast during the 5 - 10 - year period . there is a consistent increase in mortality rates . 
a further subdivision was made of cases with slight attachment into those with attachment to the skin and those with attachment to deeper tissues . the mortality rate for these sub - groups was : deep attachment skin attachment percentage ~~~~~stage . 
when a growth lay between two quadrants it was described as lying in a " mid " position . thus there were the " mid - medial , " the " mid - inferior , " the " mid - lateral " and thbe " mid - superior " positions . 
the average age for stage i growths was 55 years , for stage ii growths 54 years , and for stage iii growths 59 years . the incidence of growths in decades was : decade . 0 - 39 40 - 49 50 - 59 60 - 69 70 - 79 80 - 89  . 
when associated with surgery the radiotherapy was either the implantation of radium into the wound at the time of operation according to the method described by sampson handley ( 1922 ) or prophylactic deep x - ray therapy . the latter was given in multiple intermittent sub - erythemal doses with medium voltage . 
as would be expected , this interval was increased with the stages of the disease . thus stage i cases had an average delay of 7 months , stage ii cases of 10 months and stage iii cases of 18 months . one can therefore reason that an average case has between 7 and 10 months ' unforgrace before the catastrophe of axillary gland involvement takes place . tunately there is too much variance in individual growths for this figure to be of anly real value . 
they should probably be called " insufficiently treated , " but have those under the heading " breast , been included in this analysis for convenience . contralateral " are those in which the evidence strongly suggests a true recurrence rather than a second primary . the low incidence of mediastinal recurrences is probably due to the fact that practitioners were likely to call any intrathoracic recurrences " pulmonary metathese figures , however , do not lend support to rowlands and turner stasis . " ( 1936 ) , who state that the mediastinum is the commonest site for a recurrence . this investigation has cleared up one point that has been debated in the past . there is a direct path of spread from the breast to the supraclavicular glands . of 91 cases that developed recurrences in those glands 17 were stage i cases , treated by radical mastectomy , and with axillary glands proved to be free from in view of the fact that the axillary glands were removed growth histologically . at operation and that path of spread permanently closed , the subsequent invasion of the supraclavicular glands must have been through a more direct path . relation of site of primary growth to site of recurrence . some quadrants of the breast have been considered to be more likely to metathus , inner quadrant growths have been held to give stasize in certain areas . secondaries in the chest more frequently . 
the ten to fifteen year period . no surgeon who has followed cases of carcinoma of the breast can have failed to see cases of recurrence occurring at long intervals after operation . these are often regarded as interesting rarities . 
the actual number that do develop these late recurrences is small owing to the cases that die early , and this has given the false impression that survival to five years means that the chance of recurrence is negligible . 
a few small arterioles present in the floor and edges of the ulcer showed no evidence of endarteritis . at one point the tumour had penetrated the periphery of the pancreas for a there was no involvement of the common distance of approximately 2 nun . 
5 , showing invasion of the submucosal connective - tissue strata by adenocarcinoma which has , on left - hand side , lost all resemblance to normal brunner 's gland acini . 
hence there can be no reasonable doubt that it is primarily duodenal . histological appearances clearly indicate that the initial neoplastic change has involved the epithelium throughout the whole thickness of the mucous membrane , including brunner 's glands . 
burn re8earch laboratory , royal victoria infirmary , newcastle - upon - tyne . received for publication january 30 , 1954 . this is the first of a series of reports on an experiment suggested by the work strong hybridised three strains of mice , and after 3 of , strong ( 1940 , 1945 )  . inbred generations he injected subcutaneously i mg . 
to f12 injections were continued but there was selection towards resistance to tumours at the site of injection ; five sublines were then spht off and the mice injected for a further 4 generations , after which one group was set aside and bred from without further treatment . the experiment to be reported here differs in several respects from that of strong ( 1940 , 1945 )  . 
towards the ' end of 1945 two inbred strains of mice , the nbt ( newcastle bone tumour ) and the cba ( one of the ancestral strains used by strong ) were crossed in both directions . half the mice in each f , htter were injected subcutaneously in the right flank at the age of 2 months with i mg . methylcholanthrene in 0 - 1 c.c. 
sesame oil ( one inject ' lon only ) ; the other half were not treated and were bred from ( brother - sister matings only ) for 12 generations , ar untreated . 
the untreated reciprocal - hybrid strains were known as the cba / nbt and nbt / cba strains , or cn and nc for short , the maternal strain being given first . the injected f , mice were also bred from , their inbred descendants likewise being injected and subsequently bred from for a total of 10 generations . 
from the beginning an attempt was made to breed selectively for resistance to local tumours , but entirely without success ; offspring of apparently resistant inice appeared to be just as susceptible as those of susceptible mice . 
onwards etters from mice which early developed local tumours were neither injected nor bred from . finauy , as the failure to produce resistant lines seemed likely to spoil the purpose of the experiment , 2 final generations ( f , , and f.2 ) were raised , in which no mice were injected . the injected reciprocal hybrids together with their untreated descendants formed the m / cba / nbt and m / nbt , / cba strains , or mcn and amc for short . mice were fed on " rat cake " compounded to the formula of the rowett institute by an aberdeen firm , supplemented with cabbage and carrot , with drinking water ad lib . the purpose of the experiment was , like strong 's ( i 940 , 1945 ) , the development of lines of mice resistant to tumours at the place of injection , so that the mice would live long enough to produce tunio ' urs at ' remote sites . 
the origin of the strain has been described ( pybus and mffler , 1938 ) and the various types of bone tumours have been described and illustrated ( pybus and mfller , 1940a , 1940b )  . afice of inbred generations 28 and 29 , coming from i pair of mice of generation 22 , were used as parents of the hybrid stra ' ms ; 131 mice ( 59 females and 72 males ) of generations 28 to 30 were treated with methylcholanthrene . 
one generation of 81 females and 94 males , an untreated , was raised from the treated mice ; 28 p , , .irs , belonging to 9 famihes , were successfuey bred from , a further 17 pairs proving sterfle . the last bone sarcomata to be seen in this strain were 4 which occurred in f27 prior to these there were 3 in f22and i in f24 . 
the purpose of the experiment was not to investigate the occurrence of local t ' umours , but ( as already stated ) to try to develop strains of mice that would be resistant to the local action of the carcinogen and would therefore live long enough to produce neoplasms at places remote from the site of injection . 
ar mice were therefore allowed to live as long as possible , and those with local tumours were killed only when the tumour grew large or showed signs of ulceration . there was a certain amount of variation in size of tumours at death , those close to the skin or in a position to rub against the box having a tendency to ulcerate 166 e . 
at first the mice were charted regularly , outhne drawings of tumours being made when they appeared and their growth followed week by week , but as the numbers of injected animals increased this became impossible and tumour sizes at death only were charted . it was observed from the initial weekly chartings that there was great variation in the , rate of growth of different tumours , but it cannot be stated whether this variation depended to any extent on the sex of the h ' ost ; it did not appear to do so . 
the males might be more susceptible than the females to the local action of the carcinogen ; i.e. , tumours might appear after a shorter latent period in males , and , even if tumour growth - rate was the same in both sexes , the males would be killed at an earlier age . 
with a very few exceptions in certain generations , many non - tumour males died prematurely and usuahy earlier than non - tumour females ( i.e. , mean age at death was less for males in ar cases except m / cba , mcn fl , and mnc f9 and flo ) , due mainly to wounds received in fighting . in some generations some of the non - tumour males which died prematurely might have developed tumours had they lived longer . this would have raised the tumour incidence and would proba - bly also have raised the mean tumour age at death in these instances . although causes i and 2 cannot be completely dismissed , there is evidence for the fourth possibihty in a comparison between the two pure strains . 
the local response o methylcholanthrene . two inbred strains to one subcutaneous injection of nbt strain . - as shown in table 1 , 59 females and 72 males were treated at local tumours appeared in 27 females ( 45 - 8 per cent ) and the age of 2 months . in 25 males ( 34 - 7 per cent )  . 
the non - tumour males in this strain died young from various causes , the principal bein - a filrhting ( 17 mice ) , while diarrhoea diarrhoea was responsible for the death of a number ( 7 ) of accounted for 8 . other causes of death in both sexes were kidney diseases , young females . pneumonia and pseudo - tubercle , and there were a few cases of neoplasms ( leukaemia , lung adenoma , mammary carcinoma and skin papillomata )  . there was no significant difference between the ages at death of tumour and non - tumour males ; from this it may be concluded that more of the males might have developed tumours had they lived longer . 
f6was a small generation of only 25 mi ' ce and 8 of th ' e io non - tumour males were dead by 6 months ; this could account partly for the lo ' w incidence . 
but f2was a very large generation with 134 females and 120 males , there being 96 tumour females ( 71 - 6 per cent ) and 94 tumour males ( 78 - 3 per cent ) , the combined incidence being 74 - 8 per cent . 
why this generation should be so susceptible is not clear at the moment . age at death : the result of inbreeding on the survival age of injected mice was evident in this experiment . in generations i to 5 , 74 mice died at 21 months and over , the oldest at 28 months ; in generations 6 to 10 only 3 mice ( non - tumour ) except for fl , to be discussed later , the ages at lived over the age of 17 months . death in each generation were not analysed separately . 
 - minbticba8train ( table iii )  . - each generation was analysed to find out whether the sexes were equally susceptible to the development oflocal tumours . with the exception of the first and third generations , the differences in each generation between the tumour incidences i ' n males and females were not significant . in fl , more than half the non - tumour males died at ages up to 7 months ( mainly due to fighting ) and the tumour incidence in the males was probably too low ; f , , was a smar generation and the early death of a number of non - tumour males had a disproportionate influence on the result . 
was a smah generation of 36 males 66 - 4 per cent ( 511 tumours in 770 males )  . and many of the non - tumour males died young , while in f4 half the non - tumour males were dead by 8 months . 
the incidences for these two generations of males were very probably too low , as some of the non - tumour mice would certainly have developed tumours had they eved longer . although it is therefore probable that the sex - difference in incidence was due to a lack of fur opportunity for the development of tumours in the males owing to a disproportionate number of the latter dying young , this cannot be presumed to be certain . a comparison of the reciprocal crosses regarding their susceptibility to local tumours was thus a comparison between an incidence of 62 - 8 per cent in the 538 mcn females and 73 - 4 per cent in the 669 mnc females ; and between 59 - 6 per cent in the 542 mcn males and 66 - 4 per cent in the 770 mnc males . 
the tumours seen were of the various types described by bonser and orr ( 1939 ) ; the greatest number were subcutaneous fibrosarcomata , of varying degrees of fibrosis ; there were a few pure fibromata , and many spindle and polymorphous - celled sarcomata . giant cells were frequently present . 
persons. 0 - 020 0 090 0 219 the difference between any pair of rates for subjects of the same age group men . 0 031 0 070 0 - 509 0 - 018 0091 0 203 is , in no instance , as great as twice the standard error of the difference . * between 0 000 and 0 - 017 , depending on the proportion of the 14 female patients dying of lung cancer who were non - smokers . all areas , england and wales . a____ - __ an opportunity to test the first assumption is provided through the courtesy of the government social survey by the use of figures for the proportions of nonsmokers in the population , obtained in the course of an independent inquiry . the subjects were interviewed in september 1951 and were a random sample of the whole population ; the definition of a " non - smoker " was the same as that used by doll and hill ( 1952 )  . 
the total number of subjects interviewed between the ages of 21 and 74 was 2 , 268 ; the numbers in each age and sex group were proportional to the numbers in the whole population and the numbers of men interviewed - particularly in the older age groupswere considerably smaller than the numbers used for the previous calculations . the results obtained by the use of the government social survey figures for the estimation of the numbers of non - smokers living at june 30 , 1950 , are shown in table v . 
1 it is seen that the estimated rates for non - smokers are ( with one exception ) slightly less than the rates for women in rural districts - that is , the lowest recorded by the registrar - general . 
the exception , the rate for non - smokers aged 25 to 44 , is derived from the experience of the smallest number of bronchial carcinoma patients and is likely to be the least reliable ; this rate lies between the lowest and the lowest but one of the registrar - general 's rates . the estimated rates are , therefore , of the order which could be expected if they represented the basic minima independent of variable environmental factors . proportion of deaths due to causes other than smoking . if the estimated rates for non - smokers are accepted as appoximately accurate , it is possible to calculate the number of deaths from lung cancer in persons between the ages of 25 and 74 which would have been expected , in the absence of smoking , by multiplying the populations given in table i by the rates for all persons given in table iv and adding the results for each age group . the number expected in 1950 would have been 1 , 875 ; the number which actually occurred was 11 , 189 . it must be presumed that the other causes of lung cancer continued to operate irrespective of the use of tobacco , and it is , therefore , concluded that about one in five of the lung cancer deaths in persons aged 25 to 74 in 1950 , were attributable to causes other than smoking . sex ratio among non - smokers . table iv shows no consistent difference between the estimated rates for men and for women ; and in the age - group 45 to 64 , for which the rates must be presumed to be the most reliable because derived from interviews with the greatest number of patients , the rates are most nearly equal . 
the data are , therefore , not grossly inconsistent with the hypothesis that in the absence of smoking there is no appreciable sex difference in the incidence of lung cancer . the closeness with which the data fit the hypothesis can be seen more readily if the rates for women ( derived from greater numbers of non - smokers than the rates for men ) are used to calculate the number of non - smokers among men with bronchial carcinoma who would have been expected to have been interviewed , if men and women suffered the same mortality . for example , the mortality rate for women aged 45 to 64 , and resident in greater london , is estimated to be 60.6 / 673 , 000 ( table iii ) ; the estimated number of male non - smokers in the same age and area of residence sub - group is 38 , 100 ( table iii ) , so that the expected number of male non - smokers dying of lung cancer in 1950 is ( 60.6 / 673 , 000 ) x 38 , 100 = 3 * 43 . altogether 1 , 473 men in this sub - group died of lung cancer , 308 r . 
the expected numbers are calculated similarly for each sub - group when , by addition , the total number of male non - smokers expected to have been interviewed is found to be 6 - 1 . 
the number actually observed was 7 . the conclusion that in the absence of smoking there is no appreciable sex difference in the incidence of lung cancer appears to be contrary to the observations reported earlier that the mortality among women in greater london was lower than that among men at each level of tobacco consumption ( doll and hill , 1952 , table xii )  . 
the observations recorded among smokers are , therefore , not incompatible with the findings among non - smokers . mortality among non - smokers in different areas . from table iv it appears that there are no consistent differences in mortality among persons resident in areas of different density of population . moreover , the rates for greater london , other urban areas and rural districts are most nearly equal in the age - group 45 to 64 in which they are likely to be subject to the least error . the essential similarity of the rates , in the absence of smoking , can be seen more readily by a method similar to that used for comparing the mortality in men and in women . 
since there is no reason to presume that any one set of rates is more reliable than another , it is preferable to calculate the number of nonsmokers with bronchial carcinoma , resident in each type of area , who would have been expected to have been interviewed , if the estimated rates for men and 310 r . 
the data are not accurate enough for an aetiological relationship to be postulated . " no difference was found in the incidence of past attacks of asthma , chronic nasal catarrh , pleural effusion and pulmonary tuberculosis between the patients with lung carcinoma and those with other forms of cancer . 
the data did not suggest that the effect of smoking was through the intermediary of other respiratory diseases . should , however , pneumonia and chronic bronchitis really predispose to the development of bronchial carcinoma independently of any association they may have with smoking , it might be expected that their effect would be seen more clearly among non - smokers than among the whole population , in whom another cause can frequently be implicated . 
the past history of respiratory illnesses has , therefore , been analysed in the 47 patients who were non - smokers and the incidence of each disease compared with the incidence which would have been expected from the experience of all patients with bronchial carcinoma of the same sex and age groups ( table viii )  . it is clear that the data provide no evidence 312 r . 
pected. 5 * 2 62 summary . a " non - smoker " is defined as a person who has never consistently smoked for as long as one year at the rate of as much as one cigarette or one gramme of tobacco a day . estimates of the mortality rates from lung cancer among non - smokers are obtained from the registrar - general 's figures for the number of deaths attributed to lung cancer and for the total population ; and from the data , obtained in a clinical inquiry into the proportion of non - smokers among patients with bronchial carcinoma and among patients with other diseases ( excluding cancer of the oral cavity , respiratory tract or intrathoracic organs )  . the assump - tions required to enable the estimates to be made are bold and the number of cases of bronchial carcinoma among non - smokers is small . the rates obtained are , therefore , highly speculative , but it is thought that they are likely to be reasonably reliable since they are consistent with other experience . it is concluded : 1 . 
that occupational hazards and the previous occurrence of certain respiratory diseases are unlikely to be of frequent aetiological importance . i am most grateful to the officers of the government social survey for permission to make use of data collected during one of their inquiries and to trade sources for data of tobacco consumption , summarised in table vi . i am deeply indebted to professor a . 
the present work was undertaken to determine whether any such action would affect the process of malignant transformation as brought about by painting a carcinogenic hydrocarbon on mouse sk material .and me ' l ' hods . 
 a drop of the solution was applied interscapularly on the skin of the mice , by paint - brush , once a week , for one year till the mice died or were sacrificed . 
at the end of the experiment one mouse group3 urethane group2 } : 2 = 5 = 6 - dibem : anthracene groupl 1 : 2 : 5 : 6 - dibenxanthracene and urethane 3 - .... 
it was found that all mice in groups 1 and 3 after six or more months ' treatment , had developed multiple pulmonary adenomas which appeared to be typically urethane - induced . 
 the results show that , under the experimental conditions described here , urethane ca~ no obvious change in the frequency or time of appearance of papillomas or epitheliomas induced by 1 : 2 : 5 : 6 - dibenzanthracene . 
consequently a better test to detect a weak inhibiting activity of urethane might have consisted of treating mouse skin with one application of the hydrocarbon , and thereafter more frequent urethane applications . 
no studies were made of the histological effects of urethane on either normal skin or skin under going malignant transformation , since the experiments suggest that any such effects have little action on the production of cancer . 
 it is most interesting to note that those mice which received urethane treat ment ( groups 1 and 3 ) had multiple lung tumours , whereas those receiving 1 : 2 : 5 : 6 - dibenzanthracene alone ( group 2 ) had none . 
 when urethane was administered to mice in their drinking water in previous experiments , a reduced fluid consumption was noted for the first few days , showing their aversion to it . 
an estimate was made of amount of urethane in a paint - brush full of the solution ( as painted on the mice ) by applying it to a cover slip and weighing after the acetone had evaporated . 
for previous studies ( cowen , 1947 ) , lung tumours in comparable numbers to those obtained here were induced in various inbred jines of mice by injecting 025 c.c. 
since total dosage was not the same , and since there are marked strain differences with respect to lung tumour response by different lines of mice , no accurate comparison can be made . 
it would be safe to say , however , that a large part of the urethane applied to the skin was absorbed by the mice in this experiment , judging by their tumour response . 
 it is not unreasonable to suppose that urethane is absorbed at least as readily from human as from mouse sk since urethane causes leucopenia in man ( moeschlin and melli , 1947 ) and lung cancer in mice ( nettleship and henshaw , 1943 ) and rats ( jaffe , 1947 ) , it would be well for those who handle this substance frequently in the laboratory or commercially to consider it a possible risk to health and consequently take effective precautions . 
 three groups of mice were painted interscapularly with an acetone solution of 1 : 2 : 5 : 6 - dibenzanthracene , urethane and a mixture of both each week . 
 this points out that most of the urethane applied to the skin was absorbed .all expenses in connection with this work were home by the british empire by it  . 
cohen. from the experimental oncology laboratory , radiation therapy department , johannesburg general hospital . received for publication february 7 , 1953 . an apparent discrepancy exists between the multiplicity of experimental cancer cures which have appeared in the literature since homologous transmission of tumours was first demonstrated , and the difficulties encountered in the therapy of human cancer . 
among the basic reasons for this are differences in the hosttumour relationship which presents a range of characteristics , from the completely unstable heterologous relationship , where the host and tumour are of different strains , or even species , to the relatively stable situation , as with human between these two extremes are the many , now or other autogenous tumours . widely used , homologous tumours displaying varying degrees of stability . 
the facile experimental cure , or spontaneous regression of a tumour followed by absolute immunity to further implants , is invariably a feature of the relatively unstable host - tumour relationship , and the less stable this relationship the more since this effective the " cure , " be it mechanical , chemical or radiological . propitious situation rarely , if ever , occurs in humans bearing autogenous tumours , experimental investigation of an animal tumour can have at best only remote application to clinical cancer therapy unless it applies in the first instance to a stable host - tumour relationship . 
as a prerequisite to any such investigation , therefore , the following criteria for stability of the host - tumour relationship are considered indispensable : ( 1 ) there shall be unequivocal evidence of malignancy , characterised by progressive growth , invasion or metastasis , and death of the host . ( 2 ) the tumour shall be genetically compatible with the host , either having arisen spontaneously or been induced in the tissues of the animal being investigated , or be transmitted by implantation into a homologous animal closely related by inbreeding to the host in which the tumour first arose . ( 3 ) the tumour shall " take " in practically 100 per cent of recipients on irnplantation , and there shall be neither spontaneous regression nor regression induced by non - specific trauma or intoxication of the host . ( 4 ) on irradiation of the tumour in situ , the curative dose shall be of the same order of magnitude as that of human tumours ( 103 to 104 r )  . ( 5 ) removal or destruction of the tumour in situ shall not evoke absolute resistanoe to further implants of the same tumour . since both human and experimental arlimal tumours can be made to regress following exposure to ionising radiations , one of the more promising avenues of 232 a . 
cohen research would appear to be the investigation of all factors affecting the radioalthough earlier work in this field has formulated certain sensitivity of tumours . fundamental principles ( krebs , 1929 ; cramer , 1932 ; crabtree and cramer , 1934 ; sugiura , 1937 ; sugiura and cohen , 1939 ; goldfeder , 1942 ) , it has been largely limited by inaccurate dosage estimation or lack of suitable biologic it is now possible for the radiobiologist to combine precise quantitamaterials . tive radiotherapy with a judicious choice of experimental hosts and tumours ; and it was with this in mind that a study of the radiobiology of the c3h mouse mammary adenocarcinoma was undertaken . 
with no added filters ( the inherent filtration of the tube - head consisting of approximately 3 moil and 1 mbakelite in addition to the glass envelope ) , giving a half - value layer of 0 34 mcu . 
of 25 cthe dose rate was 500 r / m ( 3 per cent ) measured in air . dose rates were measured in air with a secondary standard ( victoreen ) dosemeter , and corrected for atmospheric density . 
when completely filled with tissue fragments this arrangement provides full back - scatter for the field used , and avoids any heterogeneity due to variations in density or atomic number such as would be encountered with a glass slide . 
the tumour is relatively radioresistant , requiring doses of the order of 6000 r in one treatment to produce a significant number of this is essentially similar to goldfeder 's findings ( 1951 )  . 
the discrepancy isprobably due to ionisation contributed by the glass slide in which her tumour fragments were irradiated , instead of the plastic used in this experiment . it is of interest to note that the effective dosage for some other strains of mouse mammary carcinomas irradiated prior to implantation are of a similar magnitude ( lawrence , horn and strong , 1937 ; reinhard , goltz and warner , 1952 )  . considering that their more heavily filtered radiation ( hvl 1 mcu . ) has a relative biological efficiency of about four - fifths of that used in this experiment , the reported effective dosage of 3600 r is virtually identical with our findings . the results of both types of irradiation are analysed graphically in fig . 
2 using the " probit " method , from which it can be deduced that , in the case of treatment in situ ( line a ) the ld50 is 5700 ( + 140 * ) r , and for attenuation prior to implantation ( line b ) the ld50 is 2850 ( i 50 * ) r . it will be noted that the two dosage - response lines are apparently parallel , their slopes corresponding in both cases to a coefficient of variation of about 9 per cent . attenuation of tumour homoplasts by x rays prior to implantation into animals was described as early as 1909 by haaland . 
on occasion , this procedure was shown to induce a state in which the recipient became resistant to challenge with unirradiated implants of the same tumour , although rarely were adequately inbred strains immunised against homologous carcinoma ( hauschka , 1952 )  . 
 urethane ( ethylcarbamate ) has recently received much attention because of its use in the treatment of chronic myeloid leukaemia and other members of the group of diseases of doubtful nature at present called " reticuloses , " and also because of its action as a carcinogen . 
it was therefore thought to be of interest to study the action of urethane on the oxidation of tyrosine , by tyrosinase , to me1an tyrosine solution of concentration 0 - 02 per cent in buffer ph 7 4 was prepared , and ethyl carbamate solution of 10 per cent concentration in the same buffer . 
the upper limit of urethane concentration inveetigated was 5 per cent ; the lower limit at which inhibition could be detected by the method employed was 0 - 2 per cent . 
 from what is known of the chain of events in the oxidation of tyrosine to melanin ( raper , 1926 ) , it will be evident that this finding does not provide specific evidence for an action of the urethane on the enzyme . 
the further oxidation of dopa to melanin can occur without the aid of the enzyme , though the enzyme produces marked acceleration of the process , and the evidence so far given throws no light on which step in the oxidation is inhibited . 
 the method used was identical with that for tyrosine , with the exceptions that the final concentration of dopa was 0005 per cent , and the absence of the enzyme . 
 120 180 : ? 40 11 : ? 0 3692 - 6105 - 8377 095 : ? 3298 5510 7652 0901 3138 5152 7133 0893 3012 4873 6591 0860 - 2866 4639 6282 0809 2686 4257 5810 from this it appears probable that urethane affects the tyrosine - tyrosinase reaction in two ways : it inhibits the enzymatic conversion of tyrosine to dopa , and it accelerates the conversion of dopa to melan it seemed that further information might be obtained by in , estigating the effect of urethane on the enzymatic oxidation of dopa . 
on the assumption that the errors inevitably present are to be expected to operate equally in either direction , the probability of obtaining 8 results the same is only i in 128 . 
 120 180 240 lncremenu of ' ' e . ' ' 1496 2735 3714 4663 1427 2757 3768 4750 1367 2716 3768 4815 1405 2818 3957 4995 1427 2798 3925 5017 the inhibition of tyrosinase by urethane is , in view of the previously known action of this substance , not surprising . 
the autoxidative processes do not seem to have received the same attention as have the enzymatic ones , and the author has been unable to find any references in the literature to substances affecting their speeds , other than those producing marked changes in ph . 
h the urethane combines with the enzyme , and the ure thane and enzyme are present in such concentrations that they neutralize each other , there should be no difference between the reaction speeds of this solution and the control . 
the only suggestion which at present appears to cover these observations is that urethane has a greater attraction for the enzyme than has dopa , and that one or more of the first reaction products of their interaction acts as an inhibitor of the autoxidation of dopa later , when the enzyme has disposed of the urethane , and the intermediate metabolites have undergone further change to substances which presumably do not affect the progress of the oxidation of dopa , dopa is acted on by the enzyme , hence the acceleration of the reaction in its terminal stages . 
lea it must be emphasized that this explanation is purely hypothetical ; no direct evidence for the combination of tyrosinase with urethane , or of the presence of reaction product.s , either intermediate or final ( other than melanin ) , has been obtained . 
it is not suggested that the oxidation of tyrosine to melanin has any place in either the normal or abnormal metabolism of leucocytes , though there is the interesting fact that these cells do contain a polyphenolase capable of oxidizing dopa to melan but it did seem possible that other similar oxidations might be involved . 
methyl , propyl and butyl carbamates were prepared , the choice being limited to these as they are freely water - soluble , and their actions could be investigated by the method used for ethyl carbamate , and compared directly with that substance . 
it has been found advantageous to reduce these until the present level of 0001 per cent has been reached , as these lower concentrations prolong the period during which melanin formation can be esti mated by the colorimeter . 
the difference , however , is not great , an " d certainly does not seem to be of sufficient magnitude to allow of any explanation of the known action on leukaemia . 
it was thought possible that the effect of another water - soluble carcinogen and chemotherapeutic agent might throw further light on the proble the effect of di ( 2 - chloroethyl ) methylamine hydrochloride on the oxidation of tyrosine was investigated . 
it is of particular interest to compare the differences between these two solutions , when it will be noticed that the changes are , within the limits of accuracy of the method , yery regular . 
 - 0023 0071 0051 0029 - 0069 0172 - 0260 1155 1879 the suggested explanation in this case is that an intermediate metabolite of the interaction between nitrogen mustard and dopa gradually accumulates until it reaches such a concentration that it inhibits the reaction , thus masking the accelerating effect of the nitrogen mustard . 
 it therefore appears that there is a similarity of action between di ( 2 - chloro ethyl ) methylamine hydrochloride and the urethanes , and that this is in their action on autoxidation and not on enzymatic oxidation . 
it is obvious that it would be quite unjustifiable to attempt to explain the action of these substances in the leukaemias and other reticuloses on the grounds of their effects on melanin formation . 
in view of our present lack of knowledge of the reticuloses , and espe cially because of their almost inevitably fatal nature , it might be of ~nsiderable interest and possibly of value to investigate the effect on these diseases of other groups of substances which accelerate the progress of autoxidations . 
 methyl , propyl and butyl carbamat.es have the same effects , but to a lesser the water - soluble nitrogen mustard , di ( 2 - chloroethyl ) methylamine hydro chloride does not inhibit the enzymatic oxidation of tyrosine in a concentration of 0 - 2 per cent . 
they are of twofold character : ( 1 ) mortality statistics and post - mortem examinations ; ( 2 ) systematic bloodpressure examinations among people of various ages . relying on mortality statistics of the united states , fahr ( 1928 ) estimates essential hypertension to be the cause of death of 23 per cent of the population aged over 50 years , a figure which is held by volhard ( 1931 ) as being too small . bell and clawson ( 1928 ) , on the strength of exact anatomical examinations , came to the conclusion that in all cases of mortality about 7 per cent in the 40 to 49 agegroup and 13 per cent of people over 50 years are dying from hypertension . 
zondek the attempt to come to a definite estimation of the incidence of essential hypertension based on the numerous blood - pressure examinations among various classes of a certain population undoubtedly encounters certain difficulties ; one of the main difficulties lies , in my opinion , in the precise definition of essential not every rise in blood pressure , even a permanent one , is , of hypertension . it is rather easy to exclude course , connected with true essential hypertension . cases of secondary hypertension , such as those caused by bright 's disease , aortic valvular disease and hyperthyroidism ; moreover , from a statistical point of view , these cases do not play a major part , at least not in the age - group which is of particular interest to us . 
a person was not classified as suffering from essential hypertension unless , constantly , or at least in most of the exaniinations carried out , a blood pressure of more than 160 / 90 mhg was detected , i.e. , 165 / 90 or 160 / 95 and more . though a permanent diastolic pressure of 95 mhg may be regarded as a very reliable proof of the existence of essential hypertension , if diseases such as bright 's disease can be excluded , we based our examination nevertheless on a somewhat lower pressure , i.e. , 90 mhg ; we did so because people suffering from hypertension in the course of their illness may not infrequently display a lowering of their diastolic pressure , probably as a result of developing arteriosclerosis ( fishberg , 1939 )  . 
the decrease of diastolic pressure from 95 to 90 mhg as a minimum value for essential hypertension will , of course , increase the figure for the morbidity of hypertension ; but , apparently , a compensation has been created - at least in men - by relating the diastolic pressure to a minimal systolic 134 s . 
zondek pressure of 165 mhg . ( blood - pressure has always been taken while there is no statistical method , based on blood - pressure patient was sitting . ) examinations alone , which will include every case of essential hypertension , or will , with certainty , exclude every other type of hypertension ; but this is not of paramount importance , since in view of the high incidence of essential hypertension , small degrees of error will probably not be significant . it may reasonably be assumed that the figures contained in our statistics , and especially in that of master , marks and dack ( 1943 ) , indicate fairly accurately the incidence of essential hypertension in the various age - groups ; confirmation of this assumption may be noted by the figures contained in the mortality statistics ; nor do the anatomical examinations quoted above contradict this . what , in comparison , is the incidence of essential hypertension in cancer patients ? we shall not attach any importance to anything but a great deviation from the normal rate . 
the number of cancer patients referred to in this paper is 1490 ; a certain proportion of the cases has been observed by us ; as to the remainder , we obtained the necessary data from the cards of the cases put at our disposal by various hospitals of the country . 
the number of other types of carcinoma , such as carcinoma of the gall - bladder , pancreas , liver , kidneys and respiratory tract , was not great enough to be used for separate statistical elaboration . for the same reason , in men , only the cases of carcinoma of the digestive tract were statistically elaborated . essential hypertension proved to be a rather infrequent occurrence in all it is more or less the age - groups , but especially between the ages of 40 to 59 . same picture as that reproduced in the general statistics ( table i )  . though the number of cases of carcinoma of the respiratory tract examined by us was relatively small , we tended to find the same relationship to hypertension as in table i . 
we examined 37 cases in the age - group 40 to 59 , of whom only one showed hypertension . two factors may be held responsible for the rare occurrence of a combination of carcinoma and essential hypertension : ( 1 ) carcinoma may have a depressing influence on a previously existing hypertension ; ( 2 ) a person suffering from essential hypertension may have smaller tendency to develop carcinoma . the possibility of a depressing action of carcinoma cannot be denied . 
in this connection we may quote the investigations of rosenfeld ( 1929 ) , feldweg ( 1929 ) and fortunati ( 1936 ) , who were among the few who carried out systematic blood - pressure investigations in cases of cancer ; in the course of the illness a fall in blood pressure could be detected , though the fall , if present , was in most cases only moderate . 
the number of cases examined by fortunati ( 1936 ) was very small ; yet it occurred to him that high blood pressure is more often met with in cases of cancer of breast and female genitals than in cases of cancer of the digestive tract ; he failed , however , to take into consideration the normal incidence of hypertension ; therefore , he could not even try to answer the decisive question as to whether the frequent occurrence of hypertension in carcinoma of the breast and female genitals or its rare occurrence in carcinoma of the digestive tract are to be considered as extraordinary . 
ritchie. from the division of oncology , the chicago medical school , 2755 west 15th street , chicago 8 , illinois . received for publication july 31 , 1953 . by studying the life of various kinds of spontaneous and induced tumors in animals , it has been found that they sometimes undergo irreversible changes in character . for example , a papilloma induced in mouse skin by applications of carcinogen , may become a carcinoma , so changing its character ; or a spontaneous tumor of the mouse breast responding to pregnancy by an increase in growth rate may change its character and no longer respond to this stimulus . foulds ( 1949 , 1950 , 1951 ) made a special study of these changes in character , calling them " progressions " , and defining " progression " as an " irreversible , qualitative change " in a tumor . foulds then went further , claiming that different characters of the same tumor could progress independently of one another . for example , he found that in spontaneous mammary tumours in mice one character , the growth rate , might progress , and the tumor grow faster , while another , the responsiveness to pregnancy remained unchanged . foulds ( 1949 ) studied not only the spontaneous mammary tumors of mice , but also bladder tumors induced by feeding 2 - acetylaminofluorene to mice ( foulds , 1950 ) and transplantable mammary fibroadenomata in rats ( foulds , 1951 )  . in each case he found that the tumors did sometimes progress , and that progression could occur independently in different characters of the same tumor . these findings seemed so interesting as to make desirable further confirmation , and this paper records an investigation of the life of tumors induced in mouse skin by repeated applications of 9 , 10 - dimethyl - 1 , 2 - benzanthracene . 
ritchie the second type of sessile papilloma consisted mainly of epithelial tissue . the architectural pattern of the normal human epidermis was imitated , the epidermis being thickened from four to six times . 
the surface of the tumor was often hyperkeratotic . in a few cases a tumor of this type arose in a pedunculated papilloma . sheets of closely packed cells invaded the dermis and muscularis . 
most of the tumors grew at a steady rate throughout , in so far as the growth rate could be determined by the crude method used . however , definite changes in growth rate did occur . 
8 to 11 , and will be described individually , each mouse having tumors with differing behaviors demonstrating the validity of one or other of fould 's proposals . all charts illustrate the validity of rule 1 : namely , that progression occurs independently in different tumors in the same animal . 
9 also confirms fould 's rule 2 , showing three tumors , a , b and c progressing from one morphological type to another while growing at a constant rate . this type of progression , from one morphological type to another , without progression in growth rate , was much more common than progression in growth rate alone , as seen in this chart in tumor d . 
10 , and confirms the validity of fould 's rule 3 , that progression is independent of growth . in the same chart , in tumors a and b may be seen again a change in growth rate without a corresponding change in morphological type . * n 5 31_ * - - __ + * - - + b ..od / , ' / ___ _ , / " ... 
11. - showing independent progression of multiple tumors . tulmor a was malignant at its first clinical appearance , showing that at this period of its development a tumor can be all other tumors began as sessile papillomata . 
a well - known example of progression affecting only part of a tumor is the carcinomatous change which sometimes affects the tip of an adenomatous polyp of the colon in man . foulds ( 1949 ) remarked that progression could occur by gradual change or by abrupt steps . in general , the changes seen in the tumors of this experiment occurred abruptly . 
a step - wise transformation of different characters in these tumors was observed throughout their existence . this investigation was supported by a cancer control grant from the national cancer institute of the national institutes of health , u.s. 
price. from the medical research laboratory , department of pathology , university of bristol . received for publication december 8 , 1951 . during the past thirty years much useful progress has been made in the this work , of histological grading of malignant tumours of epithelial origin . which one practical application has been the attempted correlation of prognosis with histological structure of a tumour , has had many valuable results ; and although there may be not infrequent discrepancies between the histological appreciation and the outcome of treatment in any given type of neoplasm , the results have at any rate led to some attempt being made by the histo - pathologist to guide the clinician at least in qualitative terms of high or low malignancy ; and to more precise premises for the comnparison of differing methods of treatment in series of comparable cases . the starting point of this present analysis was the observation that irrespective of the mode of treatment applied , the overall five year survival rates in most published dissections of groups of cases of osteogenic sarcomata were approxinmately of a similar order . 
some of these features can , it is true , be visualised by x - ray plates . however , the interpretation of these is mainly the proper province of the radiologist , and they are seldom to be seen in theroutine pathological laboratory . for the fortunate histo - pathologist who may be granted access to such aids , the position is onlyinmproved to such an extent as the experience which he may have in the interpretation of skiagrams , which is a field with c . 
the ideal state no doubt is the close liaison between surgeon , radiodiagnostician , radiotherapist and histologist with personal conference in each and every case . such states of collaboration seldom exist under conditions of normal routine work . the histological specimens from the present series of cases have been considered under the following headings : 1 . 
to anyone working in this field the fallacies of diagnosis are only too well known , and the cases included have all been subjected to free discussion and unanimous acceptance as osteogenic sarcoma by the group of radiologists , radiotherapists , surgeons and pathologists which forms the working panel of the register . in each case the diagnosis has been based upon consideration of the full history , clinical examination , adequate skiagrams and histological sections . in some instances the histological material is derived from biopsy , in others from surgical or autopsy specimens . this has been indicated in table i , also the time relationship of the histological specimen to any pre - operative x - radiation . for histological purposes the specimens were fixed , cut and stained by the usual routine methods . 
owing to the presence in sonle sections of many multinucleated tumour giant cells , and the indistinct outlines of a large proportion of the polyhedral cells , the nucleus was taken as the unit rather than the cell . in each case a minimum of 2500 neoplastic cell nuclei were examined in each section , using a ruled internal screen in one ocular of a binocular microscope . 
as there are also 8 cases included in the series that are still alive and when last reported were well , but for periods less than 5 years , the 5 - year survival rate is best expressed as 6 in 28 cases - i.e. , 21 - 4 per cent . two of these cases died at respectively 77 and 96 months with multiple pulmonary metastases , and the writer fully supports the view that 5 years is too short a period on which to base estimates of rate of cure in patients with osteogenic sarcoma . these brief data may be compared with those published on larger series by the following : christensen ( 1925 ) , coley and pool ( 1940 ) , the results of the new york memorial hospital series mentioned by coley ( 1949 ) , geschickter and copeland ( 1949 ) , the series reported by the british empire cancer campaign ( 1949 ) , and platt ( 1951 )  . as may be expected with a relatively small group of cases , there are some differences from the larger published series , e.g. , an unduly high incidence of femoral tumtours , but the broad agreement would suggest that this group may be regarded as a fair sample from which certain conclusions may tentatively be reached . clinical grading of the tumours . the data on which the clinical assessment of degree of malignancy has been based has been considered from the following points of view : 1 . 
from observations on these tumours and other bony conditions , the writer has acquired the view that one of the essential functions of the osteoblast is the formation of osteoid tissue - the precursor of bone , from which under suitable conditions of substrate the formation of bone may occur , possibly with no further part being taken in the process by the osteoblast . 
price callus and in paget 's disease . both normal and neoplastic , may produce phosphatase . it is also generally accepted that the osteoblast , from the grounds that this cell therefore is actively concerned in the laying down of osteoid arises the essential stromal feature of this histological sub - type of osteogenic sarcoma , i.e. , the characteristic osteoid stroma , which mav also be accompanied by considerable amounts of collagen and ill - formed cartilage . however , the osteoid is the essential feature . this stromal material may , however , sometimes occur in considerable amount in a tumour which is predominantly spindle - celled , but is then usually less evident than the collagenous fibrous matrix produced by the spindle cells . 
under suitable conditions the osteoid matrix may either calcify or ossify , more often the latter , and then produce the appearance of the osteoblastic or sclerotic type of tumour . it is thus possible to subdivide this group further into i . 
from the viewpoint of the histologist the essential histological structure of any neoplasmn occurring in a bone is more characteristically shown in an area of active growth , the radiological manifestations of which are more frequently lytic than blastic . hence this feature of the amount of bony matrix is not always readily appreciable from even a large biopsy . for this reason this subdivision will not be pursued further in the present analysis ; but it should be added , however , that a markedly bony stroma is often indicative of a tumour of relatively low malignancy , and this point should receive due consideration by the person who may attempt to correlate the histological , radiological and clinical findings in any particular case . from inspection of table iv the following points are evident : 1 . 
in an approximately quantitative assessment of the amount of osteoid stroma present in these sarcomata it is seen that there is usually a rich matrix in tumours of lower grade malignancv , and conversely a relatively smaller amount in the high - grade group . there are noteworthy exceptions , and the specimens showing perhaps this stromal feature in the greatest degree were those from btr / 66 , r . 
66 , 213 , 221 and 226 , but generally this feature was less evident in the more actively growing tumours . it is emphasized that a change in ph of the substrate is only one of a number of factors concerned it is also wise to bear in mind a fallacy that may in this biochemical change . arise in sampling a bony tumour , as the more easily sectioned and hence less bony areas are frequently selected for routine diagnostic examination . quite a nuimber of these sarcomata also show chondroid stromal areas which may either calcify or ossify , both changes often being seen in adjacent areas and largely governed by two factors : 1 . 
the iimean mitotic ratio of these sarcomata decreases as one passes from this is mluch more evident in the separation of clinical grade i to grade iii , clinical grade i from the tumours of grades ii and iii . it may be noted here that based upon clinical data case btr / 136 , y . 
price by the american registry of bone sarcoma in their 1939 ( revised ) classification , and they continued to regard intrinsic fibro - sarcoma as a variant of the osteogenic group ( ewing , 1939 )  . jaffe ( 1947 ) , on the other hand , commenting upon this adheres to the stricter view and classifies fibro - sarcoma as a separate entity . from consideration of the small group of cases here reported it would seem that this differentiation is indistinct and serves no useful purpose . table v gives details of these 7 cases . this grouip is far too small to be the basis for any definite conclusions , but the following tentative points emerge : 1 . 
in the spindle - celled group there were no tumours which appeared to merit a clinical or histological grading of i on comparison with those of the larger polyhedral - celled series . this appears to be confirmed on considering the figures obtained for the mitotic ratio in these growths . .5. 
one may perhaps from this draw the conclusion that there is some evidence to suggest that in a series of these two histological types of osteogenic sarconma if composed of tumours of average or more than average malignancy that the tinme of survival is rather longer in favour of the spindle - celled type . in this small series of cases , one obvious difficulty that has arisen has been the determination of mean survival time of the sub - groups , and it is fully appreciated that the figures shown are only minimal inasmuch as cases have been included which are still living . 
on reference to table iii again it will be seen that 7 of the 9 tuiyours showed a mitotic ratio considerably greater than the average for their appropriate clinical grade . 
g - , and it is strongly suggested that the aoverning factor in the survival of any case of osteogenic sarcoma is the combination of site of origin and histological grading of the tumour . table vi shows the 36 cases of osteogenic sarcoma arranged in ascending order according to the mitotic ratio , i.e. , in order of diminishing mitotic activity . this has been subdivided into three purely histological grades of malignancy , c . 
price into those cases with a mitotic ratio less than 100 : 1 , from 100 : 1 to 400 : 1 , and greater than 400 : 1 . this may be taken as approximnating to those of greater , average and lesser degrees of nuclear activity . reference back to tables iv and v will show that all the cases there clinically graded as iii appear again as grade iii ( histological ) plus one case , btr / 157 , d . 
a - : probably favourable result of treatment has led ta unduly low clinical grading ; tumour in lower end of femur . group based on range of mitotic ratio of less than 100 . mean survival period of grade iii ( 11 ) 114 + months . 
g - , aged 14 , mitotic ratio 83 : 1 , sarcoma of lower end of femur , who survived for a total period of 18 months . another case supporting this view was btr / 157 , d . 
mean survival 11 4 months . the marked difference in the prognosis in these separated groups of cases can it would be of the greatest value to subject many again be well seen from fig . 
2. further cases to this type of analysis , to confirm if possible the significance of the mitotic ratio and to find its mean value based upon the examination of a much larger series of tumours . 
the mean mitotic ratio for this series is 238 resting nuclei to one nucleus in mitosis . analysis of the group gives the following ranges : grade i : grade ii : grade iii : m.r. 
the mean mitotic ratio for this group of sarcomata is markedly different from that which has been found in the examination of presumably related cells in non - neoplastic conditions . the author wishes to record his great indebtedness to the late s . 
much thanks are also justly due to the members of the committee of the register for their co - operation in the collection and discussion of all the cases included in this series . material has been derived from the sourceindicated below , and to the surgeons , radiotherapists , radiologists and pathologists who have referred cases to the register , due recognition and thanks for their assistance are here rendered : 1 . 
the animals were killed by cervical dislocation or by exsanguination under ether anaesthesia and the livers immediately perfused with 0 9 per cent sodium chloride solution before being removed , chilled and homogenized . 
nuclei were also prepared from the grch 15 tumour originally described by peacock ( 1933 )  . for the preparation of fowl erythrocyte nuclei the method of dounce and lan ( 1943 ) was used . in all cases the suspensions of clean nuclei were dried from the frozen state after small measured portions had been taken for dilution and counting in a haemocytometer . 
a weighed portion of dried nuclei acid - soluble 1 ( discarded ) nuclei 10% tca extraction residue lipid extraction phospholipid p residue n - naoh ab 370 insoluble 1 alkali - soluble 1 acidification acid soluble 2 precipitate dnap organic p inorganic p fig . 
2. scheme of separation of phosphorus compounds in nuclei ( modified from schmidt and thaunhauser , 1945 )  . was extracted three times with 10 per cent trichloracetic acid ( tca )  . 
the extracts were combined ( acid soluble 1 ) , but little attention was paid to this fraction since clearly much of the soluble p must already have been removed during the isolation of the nuclei . 
the extracted residue was treated with successive portions of acetone , ethanol , ethanol - chloroform ( 3 : 1 ) , warm ethanol - ether ( 3 : 1 ) and ether . these extracts were taken to dryness and the residue taken up in chloroform to form the phospholipid fraction . the dry nucleoprotein residue was incubated overnight at 37 with n naoh to hydrolyse rna to acid soluble nucleotides . 
the phosphorus which remained in solution was treated as organic phosphate ( fraction " organic p " )  . fractions " acid soluble 2 " and " organic p " should contain phosphorus derived from rna in the form of acid soluble nucleotides ( rntap )  . pentose was therefore estimated in " acid soluble 2 " by the orcinol method of mejbaum nuclei 10% tca extraction acid soluble 1 ( discarded ) residue lipid extraction phospholipid p residue 5% tca at 900 schneider extract schneider residue n nagh at 370 insoluble 2 alkali soluble 2 acidification acid soluble 3 precipitate ester phosphate inorganic p ester phosphate residue fig . 
3. - combined schneider and schmnidt - thannhauser procedure . ( 1939 ) and by the phloroglucinol method of euler and hahn ( 1946 ) , using calibration curves made from a purified sample of yeast rna , so that pentose could be read off directly in terms of ribonucleic acid phosphorus ( rnap )  . fraction " dnap " should contain phosphorus derived from dna only . deoxypentose was therefore estimated by the diphenylamine method ( dische , 1930 ) as employed by davidson and waymouth ( 1944b ) , using a calibration curve prepared from a purified specimen of thymus dna so that readings could be made directly in terms of deoxyribonucleic acid phosphorus ( dnap )  . phosphorus estimations were made on all samples by the method of allen ( 1940 )  . the optical densities of fractions " acid soluble 2 " and " dnap " were read in a beckman model du spectrophotometer at 260 m , u . 
and were read against a reagent blank . the radioactivity of each fraction was determined on a type m6 liquid counter ( 20th century electronics ) attached to a conventional scaling unit . ( ii ) the schneider ( 1945 ) technique . - a weighed portion of dried nuclei was extracted with tca and lipid solvents , as described above . 
the " schneider " residue clearly contained a mixture of phosphorus compounds , and it was therefore submitted to a schmidt - thannhauser separation by incubating in alkaline solution at 370 for 18 hours . 
to demonstrate them a separate run on a strip 57 cx 7 cwas carried out at a potential gradient of 13 - 6 volts / cfor 6 hours . after a run the paper was dried and the bands located by the ultra - violet light method of holiday and johnson ( 1949 ) and marked lightly in pencil . photographic records were made by the method of markham and smith ( 1949 ) , and occasional autoradiographs were made by leaving the paper in contact with kodak industrex type d film for about 18 days . the portions of the paper containing the bands were cut out and eluted according to consden , gordon and martin ( 1947 )  . 
davidson was determined by its position on the paper and its ultraviolet absorption spectrum . total phosphorus and radioactivity were also determined . the results of a series of analyses are shown in table i , in which the composition of bulk nuclei is given in terms of mg . 
although it might be expected that the schneider extract would contain all the phosphorus derived from rna and dna , the figure obtained for this fraction is invariably table ii . - mean values for the composition qf the single cell nucleus pg . 
at the same time the schneider residue , which might be expected to contain only phosphoprotein p , invariably shows a much higher phosphorus content than could be accounted for this discrepancy was observed by schneider ( 1945 , 1946 ) for by fraction p2 . whole tissue and has been commented on by davidson , frazer and hutchison that the schneider residue contains several different types of phosphorus ( 1951 )  . compound is made abundantly clear when it is submitted to a schmidt - thannhauser fractionation ( table i )  . the protein - bound inorganic fraction " p3 " obtained in this way corresponds roughly in amount to fraction " p2 "  . it therefore represents the true " phosphoprotein " phosphorus , and the other phosphorus compounds in the schneider residue are almost certainly derived from the nonnucleotide esters mentioned above , and from nucleic acid phosphorus which has failed to be removed in the schneider extraction although the portions of the nucleic acid molecules containing the reactive sugars have been split off from the this is clear from the fact that , in general , the figures for rnap protein . obtained by pentose estimation on the schneider extract agree with those obtained 206 w . 
davldson by pentose estimation on " acid soluble 2 " , while dnap obtained by the diphenylamine method on the schneider extract gives results which are comparable with those obtained from the schmidt - thannhauser dnap fraction . it is clear from table i that dry preparations of calf thymus and fowl erythrocyte nuclei have a much higher dna concentration and a lower rna concentration than have liver nuclei , but a quite different light is shed on the results when they are expressed as amounts of each component per nucleus ( table ii )  . 
4 , which is a photograph in ultraviolet light taken by the method of markham and smith ( 1949 )  . in the material from one sample of rat liver nuclei each nucleotide was submitted to hydrolysis with perchloric acid ( marshak and vogel , 1950 ) , and the bases so liberated examined by paper chromatography by the method of wyatt ( 1951 )  . in each nucleotide only the expected base and no other was found . it is clear therefore that nuclear rna is similar qualitatively to cytoplasmic rna . 
component b lies immediately behind component a , from which it cannot readily be separated . it is probable that component b consists essentially of the inorganic moiety associated with component a . 
component b should in fact correspond to fraction p2 derived from phosphoprotein . component d lies immediately in front of uridylic acid and component c in the space between b and d ; both c and d are organic in nature . it is clear therefore that fraction " acid soluble 2 " contains in addition to the expected ribonucleotides at least four other components of which three are organic phosphates . these components are clearly visible in the autoradiographs shown in fig . 
the incorporation of 32p into all fractions of fowl erythrocytes was so low as to make the results of the specific activity of the dnap in rabbit and rat liver is little significance . very low indeed as compared with the cytoplasmic fractions , but by contrast the specific activity of the organic p fraction which contains ribonucleotides is very in experiments in which we have compared nuclear rnap and high indeed . cytoplasmic rnap the former has always shown a much higher rate of incorporation of 32p than the latter ( davidson , mclndoe and smellie , 1951 )  . 
the high specific activity of nuclear rna is confirmed by isolation of the individual nucleotides . although there is no great difference between the activity of the nucleotides , adenylic acid tends to show the highest components c and d have activities of the same order as those of the activity . nucleotides , while the activities of components a and b together are considerably higher . 
the general pattern obtained from grch 15 tumour is similar to that for other tissues . the presence of components a , b , c and d is shown clearly in the autoradiograph in fig . 
5 , in which the areas of radioactivity are seen to correspond closely with the ultraviolet absorbing areas in the adjacent ultraviolet print . since the " dnap " fraction has a low specific activity and is precipitated from a solution of much greater specific activity , it is possible that this fraction may be contaminated with small amounts of phosphorus of high activity although it was therefore thought necessary to investigate it is washed several times . the effect of dissolving the " dnap " fraction in alkali and reprecipitating with one such reprecipitation reduced the activity by about 40 per cent , but acid . further reprecipitations proved to be unnecessary and indeed undesirable since in all experiments in which they tended to promote degradation of the material . ionophoresis was employed one reprecipitation of the dnap fraction was used . from table i it is clear that the specific activity of the dnap in rabbit and rat liver is very low indeed , and this is again evident in table iv . 
the dnap of the liver of the normal cock on the other hand has a much higher specific activity , while that of the dnap in the grch 15 tunmour , as might be expected in a rapidly growing tissue , is still higher and approaches the value for the riboit is of interest that the specific activity of the dnap in nucleotides ( table iv )  . the liver of the tumour - bearing bird is higher than that in the normal bird . 
on the other hand fairly convincing evidence for the loss of protein from the nucleus during the process of isolation from aqueous media has been reported by pollister and leuchtenberger ( 1949b ) and by dounce , tishkoff , barnett and frier ( 1950 ) , although this is contested by stedman and stedman ( 1951 )  . 
to what extent loss of rna and other phosphorus - containing constituents may occur during isolation in citric acid is quite unknown . it is clear from the results shown in table i that , as might be expected , the bulk of the phosphorus in the nucleus is present as dna , but the results of the schmidt and thannhauser procedure reveal considerable amounts of phosphorus in the " acid soluble 2 " fraction . that a large proportion of this fraction consists of the ribonucleotides produced by alkaline hydrolysis of rna is evident from the results of pentose estimations by the orcinol and phloroglucinol methods , and the results of the ionophoresis experiments have conclusively established the presence in nuclear rna of the four nucleotides which are found in rna 210 w . 
2. components a , c and .d are organic phosphates , the presence of which is responsible , in part at least , for the difference between the " organic p " fraction in table i and the rnap by the orcinol reaction . the presence of such esters has already been suggested by davidson and mclndoe ( 1949 )  . it is clear that in nuclear tissue , as in whole tissue ( davidson , frazer and hutchison , 1951 ; davidson and smellie , 1952 ) , the method of schmidt and thannhauser ( 1945 ) tends to give rather misleading results , since the rna fraction contains , in addition to ribonucleotides , considerable amounts of non - ribonucleotide phosphate . incorporation studies with 32p have revealed the rapid incorporation of the isotope into nuclear rna and its slow incorporation into dna in nongrowing tissues . although nuclear rna is much less abundant than cytoplasmic rna it is more active , as has been shown by marshak and calvet ( 1949 ) , barnum and huseby ( 1950 ) , jeener and szafarz ( 1950 ) and davidson , mclndoe and smellie ( 1951 ) using 32p and by potter , recknagel and hurlbert ( 1951 ) using labelled orotic acid . 
the present studies show that this more rapid rate of incorporation of 32p into nuclear rna holds for all four nucleotides , which become labeled to practically the same extent . the specific activities of components c and d are of the same order as those of the nucleotides 2 hours after administration of 32p , but that of the inorganic phosphate fraction is much higher ( table iv )  . this component corresponds to fraction p2 in table i and is derived mainly from " phosphoprotein , " although it may contain traces of inorganic phosphate originally present in the tissue . its presence is responsible for the fact that the activity of fraction " acid soluble 2 " is higher than that of " organic p . " the high activity of fraction p2 in whole tissue has already been commented on by davidson , frazer and hutchison ( 1951 )  . the results of the experiments on fowls confirm the observation of kelly and jones ( 1951 ) and kelly , payne , white and jones ( 1951 ) in rats that the presence of a rapidly growing tumour in the animal body leads to an increased incorporation of 32p into the liver dnap . 
this effect is still unexplained and obviously requires much further examination , since it is associated in our experiments with the rather high specific activity of normal fowl liver dna as compared with rat or rabbit liver dna . 
the high activity for the dna of the grch 15 tumour is in agreement with the results of other workers for rapidly growing tissues ( brues , tracey and cohn , 1944 ; davidson and raymond , 1948 )  . the results presented in table ii are of some interest in relation to the claims of vendrely and vendrely ( 1948 , 1949 ) and of mirsky and ris ( 1949 ) that the dna content of the cell nucleus is apparently constant for the cells of the different somatic tissues of any one species although there may be wide variations between one species and another . the implications of these observations have been discussed by davidson and leslie ( 1950a , b )  . 
as might be expected , no loss of dna from the rat liver nucleus too much stress however occurs on fasting , although the rna content falls . should not be laid on this latter observation , since the presence of minute amounts of cytoplasmic contamination could significantly alter the rna content of it has already been shown ( davidson , 1947 ) that fasting causes a the nucleus . marked fall in the total rna content of the rat liver , while the total dna content this would of course be expected if the dna content of the is unchanged . individual nuclei and the number of nuclei per liver were unaltered . in the grch 15 fowl tumour the dna content of the nucleus is approximately double that in the fowl erythrocyte or liver cell . 
the significance of this observation is as yet obscure , but a similar high value for tumour cells has been recorded by klein ( 1951 ) for several mouse tumours . 
payne. from the department of cancer research , mount vernon hospital and the radium institute , northwood , middx . received for publication may 2 , 1953 . in the course of their original work on the metabolism of 3 : 4 benzpyrene weigert and mottram ( 1946 ) noted that once the hydrocarbon had been introduced into a tissue it could only be extracted with difficulty . repeated and prolonged washing of the finely minced tissue was found necessary to remove the hydrocarbon and its metabolites . in view of this " binding " of the carcinogen and its derivatives within the cell an attempt has been made to fractionate mouse liver after treatment with 3 : 4 benzpyrene . 
the liver was chosen for detailed work as being easily handled , of adequate bulk , and as a site where metabolism of 3 : 4 benzpyrene is known to occur . 
the hydrocarbon was introduced as a colloidal susthis method was deliberately chosen as pension by intraperitoneal injection . offering the best chance of avoiding the presence of undissolved particles of hydrocarbon in the blood vessels as occurs after intravenous injection . all mice received one intraperitoneal injection of 1 c.c. 
the best known method for isolation of intact mitochondria is that devised by hogeboom , schneider and pallade this involves maceration of the tissue in 0 - 88 m sucrose solution , removal ( 1948 )  . of nuclei by low speed centrifugation and then isolation of the mitochondria by further differential centrifugation . 
payne as they caused agglutination of the mitochondria and their consequent sedimentation with the nuclear fraction . methods , such as the modified behrens technique used by mayer and gulick ( 1942 ) , where fractionation is done in organic solvents , were considered impracticable on the grounds of being liable to remove any bound benzpyrene attention was then turned to the standard methods for isofrom the tissues . these involve maceration in citric acid solutions lation of nuclear fractions . since it is generally accepted that mitochondria followed by centrifugation . however , it was found are destroyed by acids these did not appear hopeful . that using ice - cold 1 per cent citric acid as advocated by mirsky and pollister ( 1946 ) it was possible to remove a clean nuclear fraction , and then by further centrifugation at higher speed to obtain a mitochondrial fraction . 
the mitochondria obtained in this fashion were a bit swollen and distorted , but still stained readily in very dilute janus green b solutions . in bulk the fraction obtained in this fashion compared favourably with similar fractions obtained using sucrose this result was rendered less surprising , however , when it was found solutions . that the literature references to the solubility of mitochondria in acid only refer to acetic acid . on the basis of the above findings a technique using citric acid was developed . at selected intervals after injection the mice were killed by a blow on the head , this was examined under a u.v. 
lamp opened , and the entire liver removed . with wood 's glass filter , and any particles of benzpyrene detected - their fluorescence shows very distinctly against the background dull blue fluorescence of the liver - were washed off with normal saline . 
the 70 per cent acetone was selected , as previous experience had shown this to be the best solvent for benzpyrene in tissues . all fractions were spun down again to clear them , and fluorescence spectrograms were made according to the method devised by doniach , mottram and weigert ( 1943 )  . 
payne an acid mediuat ph values below about 7 - 1 both these compounds lose water to form 8 - 0 h benzpyrene . this latter has a rather indistinct spectrum , and may easily have been missed against the background fluoresence due to the tissue equally it may have been oxidised still further to the residues themselves . 5 - 8 quinone during the violent agitation associated with the maceration process . in any event it was considered that the experimental conditions employed were not suited to the quest for benzpyrene metabolites . the significance of the results obtained is difficult to assess . 
althougih biologists have for long been uneasily aware of the existence of a problem of cell heredity , its systematic analysis is the work of very recent cell heredity deals with the origin and maintenance of inherited character years . differences between cells ; more particularly , between cells that set up division its problems present themselves in their most acute , lineages by mitotic fission . if not most easily workable , form in metazoan development . the outcome of cellular differentiation in ( say ) mammalian development is the formation of a limited number of histologically definable cell genera , each one further subdivided into a variety of cellular genetic species . 
the genus " fibroblast , " as yet far from completely analysed , may be subdivided into cells which manufacture bone , cartilage , white connective - tissue fibres , and so on . genus " epidermis , " of which we have made a particular study ; includes the epidermal epithelium of the superficial skin , of the sole of the foot , the tongue , cells of each of these epidermal the claws or nails , the vagina and the cornea . species display a distinctive combination of structural or physiological properties . the epidermis of the sole of the foot , for example , has a characteristically high rate of cell division . 
but this has proved not to be the case . although plausible " phenocopies " of sole epithelium ( in the form of corns and callosities ) can be made by irritating the thin and relatively quiescent skin of the body surface , the difference between the division rates of sole and body epidermis is in fact " intrinsic " and inheritable . 
we have , for example , transplanted sole epidermis to positions in the body where it is protected by neighbouring hair and secure from mechanical irritation ; but even so , its characteristic growth rate has been maintained for at least two years , and thick pads of now functionless cuticle continue to form over it and may periodically be removed . claw epithelium tells the same story - more clearly , since the difference between claw and body epidermis is anatomically crude and obvious . 
comeal epithelium is very transparent , and its mode of cell - packing and cuticle formation is characteristic . these properties might well be the outcome of the peculiar situation in which it lives - lying taut over the unvascularized comeal dermis , relatively low in temperature , and nourished under conditions of ' ow 02 tension by the aqueous humour , and perhaps to some extent through its outer ( cuticular ) surface as well . if the difference between comeal and body epidermis were merely a difference of " nurture , " then the heter6topic transplantation of the comeal epidermis to ( say ) the chest wall should bring about its reversion to the cell type characthis does not happen . 
expand its substance and area fifty - fold by cell division and outward spread ; but it still retains its characteristic cell packing , mode of cuticle formation and exquisite transparency , so that fine regenerating dermal blood - vessels corneal grafts have are to be seen through it as clearly as through a window . proved to form a rather unstable epithelium , which never seems to achieve the firm union with the underlying corium characteristic of body skin . for this reason we have not yet been able to " ' cultivate ' " corneal epithelium as a heterotopic graft long enough to be quite sure that ' it represents a distinct epidermal species . 
we can only say that it has given no hint of reversion after fiv ' e weeks of cultivation . in general , the technique of tissue culture confirms the results of heterotopic transplantation ; but it is far less critical in its discrimination between cells of different species , because cultivation in vitro has a tendency to make different ciiltivation in vitro is not known to bring about any cells look rather alike . inherited change in differentiated cells , unless we take into account the fact that prolonged and rapid growth may perhaps cause individual cells to lose specific properties in the " growth rate dilution process " recently reported on by preer ( 1946 )  . 
the " antigenic simplification " ( gorer , 1938 , 1942 ) and anaplastic tendencies of long established and ra idly growing transplanted tumours may conceivably represent a phenomenon of similar origiia . it will be clear from the foregoing examples that the techniques we have so far used to discriminate between the several true - breeding species of epidermal cells are , in their use of heterotopic transplantation , formally identical with those used in classical experimental embryology to distinguish between cell systems undergo ' ing " self " and " dependent " differentiation . 
the study of cell heredity is likewise , in effect , the study of cellular transformation - that is , of the origin of inherited differences between the phenotypes of cells . from our point of view , the most important of the several different types of cell transformation is that which , in a rough preliminary classification , has been called infective ( medawar , 1947 )  . 
the second , a corollary of the first , is that infective transformations can be serially propagated - a cell once transformed can in its ttirn transform another . the special importance of infective transformations is that they provide clear direct evidence of the particulate nature of cell heredity , and so , indirectly , of the fact that the differences between cell species are combinatorial in nature and not smoothly blended . 
 ' the authors ' work on the inheritance of colour differences in spotted guinea - pigs ( as , in the cells of guinea - pigs ' skin is relevant here . apparently , in spotted pigs and friesian cattle ) the - pigmentation of the black skin areas can be seen during life to encroach upon the white . 
the spread is even in density and snioothly progressive , but it notmally leaves untouched the liairs of the white area that are being encroached upon , so - that - the transitional zone of black skin that was formerly white is distinguished by bearing white liairs on a black skin background . 
our interpretation of the phenomenon ( billingham and medawar , 1948 ) is , in outline , as follows : the origin and seat of pigmentary function in black or other coloured skins is the pigmentary dendritic cell , a branching cell which lives in the lower reaches of the epidermis and which in some unexplained manner " feeds " - or , as masson ( 1948 ) puts it , " injects " - rnelanin granules into the malpighian layer cells on which its branches end . pigmentary dendritic cells form a facultatively syncytial system , since the branches of neighbouring cells are sometimes confluent , and a branch from one dendritic cell , instead of ending on a malpighian cell , may sometimes apply itself to the cell body of another . in the white areas a dendritic cell system is present which is similar in every respect save one : the cells lack both melanin granules and the enzyme apparatus required for making it , and no form of merelv physical stimulus will cause them to acquire pigmentary ftinction . we have shown , not yet with final precision , that the phenomenon of pigment encroachment or spread at the pigmentation boundaries of spotted guinea - pigs is due to the transformation of white ( non - pigmentary ) dendritic cells into melaninforming cells by contact with their pigmentary neighbours . 
the transformation , once achieved , is permanent in the transformed cell and its division lineage and a white cell transformed to pigmentary function can in its tum tramform its neighbours . 
we - have now to ask whether the pigment - spread phenomenon we have analysed is a mere curiosity of nature , or whether it exemplifies a principle of general importance . the experimental system we bave been using is a very unusual one indeed , for the following reasons : most cells liberate their secretory products externally or into the general circulation . dendritic cells pass their secretory product , melanin , into the cells around them - " cytocrine " activity , as masson calls it . their neighbours are norinally the malpighian cells of the epidermis ; but , as inlasson lias pointed out , dendritic cells may also inject melanin granules into cells of quite different origin - into the cells of gut epithelium for example , if a tumour metastasis happens to bring them within reach of its branches . 
the casual leakage of enzymes from one cell into another would make nonsense of embryo ] ogical de - velopment ; no organ could exist under these conditions , with its great variety of cells of different types in intimate physical contact . 
an infective transformation can only be secured by taking advantage of peculiar cell properties , like the cytocrine properties of dendritic cells and their transiently syncytial lay - o - ut ; of peculiar animals , like spotted guinea - pigs ; or of technical tricks , like grafting claw epithelium to the skin of the chest , or - to choose an example from the illuminating pioneer work of sonneborn ( 1943a , b ) - by delaying the conjugation process of paramecium so that the conjugant pairs may 130 r . 
the relevance of these ideas of cellular heredity to cancer etiology has been dealt with by other speakers , so that only special aspects of the problem need be considered here . the centre of gravity of modern speculat - ion about tumours has altogether shifted from - the idea of the malignant cell as one which proliferates faster than its neighbours , towards the simpler and more general fact that it has acquired their rapid rate an inherited character difference from its normal homologues . of growth and their invasiveness may be the most important clinical facts about tumours , but they are not necessarily the most important biological facts . suppose , now , that we accept one variant or another of the view , that malignant cells differ from their normal homologues by the multiplication within them of an exogenous or proprietary virus or mutant plasmagene . if that view is correct , the infective propagation of tumours should be the rule rather than the ( for if we disregard the intervention of virus - like particles in rare exception . the initiation of mammary tumours in mice and in the propagation of the brownpearce carcinoma ( kidd , 1946 ) , the list of tumours or tumour - like growths initiated by viruses seems to be exhausted by the shope papilloma and a variety of chicken sarcomas . ) but what has been said above about the inherent difficulty of demonstrating infective transformationsin normal cells applies without any qualification to those which happen to be mahgnant . 
the limiting factor must be supposed to be , not the actual ' rarity of tumours rendered malignant by the propagation within them of specific virus - like particles , but our technical facilities for extracting them from malignant cells and introducing them into normal cells of a type in which they are competent to flourish ' ; in other words , not the material existence of such particles , but thoir reluctance to display infective behaviour . luckily , infection techniques are not the only ones . 
at our " disposal ; kidd 's ( 1946 , 1948 ) subtle serological analysis of the brow - n - pearce carcinoma has the great theoretical merit of overcoming their almost crippling defects , though it has certain limitations of its own . the interpretation we have put upon the phenomenon of pigment spread in guinea - pigs ' skin was said earlier to have been incompletely demonstrated for this vory reason ; altho - ligh we have the strongest circumstantial and anatomically factual evidence for supposing that the transformation we have been studying is infective in character , we have not yet been able to produce a cell - fiee extract ofpigmentary dendritic cells which will convert white dendritic cells to pigmentary function . 
of extraction are formidable enough ; we may be dealing with a small battery of oxidases , and the existence of at least two , a tyrosinase and a dopa - oxidase , is likely . 
to reproduce its action artificially cannot be technically easy ; we may be obliged in the end to resort to the crude solution offered by microinjection methods . in spite of the rarity of overt cases of infective propagation of tumours , it may nevertheless occur under circumstances in which , being clinically of trivial if the so - called melanotic carcinoma of significance , it has been overlooked . skin is in reality a tumour of dendritic cells , then it is at least conceivable that some melanomas spread infectively by the transformation of their non - malignant neighbours . such spread would be so slow as to be wholly outweighed in clinical importance by infiltrative growth and metastasis , but clinicians may know of certain peculiarities of melanotic growths which such a concept would help to it would be a mistake here , of course , to suppose that the melanotic interpret . character of such growths was itself of anything but secondary importance : some melanomas are recognizable histologically which are barely melanotic . epidermal dendritic cells of human beings , barring albinos , can be provoked into manufacturing melanin ; and an irreversible malignant or pre - malignant change might be initiated in normal dendritic cells by the cytocrine activities of their malignant neighbours before their rapid growth , and rapid elaboration of melanin , gave outward evidence of the fact . since this is a privileged occasion , speculation may be allowed to become wilder still . 
the ordinary literature of tumour transplantation is unhelpful on this point , since stock epidermal carcinomas are normally propagated by subcutaneous ( or some other form of heterotopic ) transplantation from mouse to mouse . 
the purely acinar foci found in rlllb females were shown to ' persist in the absence of milk factor in this cross - suckled substrain and in the absence of ovarian hormone . 
they were referred to as benign adenomas rather than nodular hyperplasias on account of their eventual independence of ovarian in addition some small invasive tumours were found by microscopic hormone . examination that appeared to represent intermediate stages in progression from benign to malignant growth ( pulhnger , 1952 )  . afitoses were found in both types of growth with the exception of the keratinised foci . all the morphological varieties persisted after ovariectomy in old animals . ' in order to ' test the capacity of the benign growths for progression to malignancy , transplantation of as many as could be found in the fresh state was attempted . grafts were transferred to young unmated females and to others of the same strain breeding rapidly . 
the nipple area on cork was searched under a glass cover using a dissecting microscope magnifying 7 and 14 times and with sterile forceps for the presence of benign tumours . ( these are seldom all adjacent surfaces of dissecting visible to the naked eye unless stained . ) microscope and glass cover were thoroughly wiped with absolute alcohol . the event no trouble arose from sepsis . 
as each benign growth was found it was cut out with fine scissors and placed in a sterile embryo dish with a drop of sterile water on the inside of the cover glass to provide a moist atmosphere . 
the next nipple area was then excised and similarly grafting was done either under anaesthesia through an incision or by treated . injection with needle and trocar into subcutaneous tissue or peritoneal cavity . the inoculated hosts were unmated females about 2 months old or breeding females that had already bome one litter . all were of the same strain bred by brother and sister matings . 
they were left in the latter from 12 - 18 months , with 15 as the average . fourteen subcutaneous grafts of bem ' gn growths minced with scissors were made from 8 donors into 9 rapidly breeding females . 
shujbik. from the oxford university reseach centre of tje british empire cancer campaign , sir william dunn scoo of pathology , unirersity of oxford . received for publication november 20 , 1948 . though non - carcinogenic to normal skin , croton oil elicits tumours in skin previously treated with a carcinogen for an inadequate period ( berenblum , 1941 )  . this procedure , with the modification of mottram ( 1944 ) , using only one single application of carcinogen prior to the croton oil treatment , served as the basis of a quantitative analysis of carcinogenic response ( berenblum and shubik , 1947b )  . 
the results led to the establishment of the essential difference in mechanism between the preliminary " initiating process " and the subsequent " promoting process " of carcinogenesis . for reviews of the literature dealing with earlier work on the stages of carcinogenesis , and with the previous terminologies used , see berenblum , 1944 , 1947 ; rusch , 1944 ; berenblum and shubik , 1947a and b . from this analysis ( berenblum and shubik , 1947b ) , it was concluded that the initiating process represents a sudden and irreversible change in a small minority of the cells of the treated area , giving rise to isolated " latent tumour cells , " apparently mdtinguishable morphologically from the surrounding non - neoplastic cells . 
the presence of these latent tumour cells is only demonstrable by subsequent promoting action , which converts them into morphological tumours . this promoting action is less specific than the initiating action , in that it can as readily be induced by croton oil as by continued applications of a true carcinogen . the irreversible nature of the initiating process was demonstrated by the fact that the number of tumours elicited by croton oil was as great after an interval of 20 weeks ( between the carcinogen application and the commencement of croton oil treatment ) as after an interval of only 3 days . according to this new concept , the latent period of carcinogenesis is dependent on the efficacy ' of the promoting action , while the actual turnour yield is prethis was borne out by comparative tests , determined by the initiating action . using 3 : 4 - benzpyrene , and 9 : 10 - dimethyl - 1 : 2 - benzit was found , as anthracene , as initiators , followed by croton oil treatment . expected , that whereas the percentage of tumour - bearing animals differed in the three series , the latent periods were approximately the same . 1 : 2 : 5 : 6 - dibenzanthracene , unfortunately , owing to considerations of solubilities , it was necessary , in these comparisons , to use a different concentration for each carcinogen . consequently , more than one variable was involved in the experiment . 
while the results obtained provided an adequate basis for the general conclusions referred to above , it was felt , nevertheless , that unequivocal evidence could best be 110 i . 
shubik provided by studying the effects of a series of graded concentrations of one and for this investigation , described below , the same carcinogen as initiator . 9 : 10 - dimethyl - 1 : 2 - benzanthracene was chosen as initiator . an additional purpose of this investigation was that the quantitative data thus obtained could , at the same time , serve as indirect evidence for or against the possibility of a " mutation - like change " for the initiating stage of carcinohowever , for a re - assessment of the validity of the " somatic cell genesis . mutation theory of cancer " in the light of the dual - stage mechanism of carcinogenesis , a more direct experimental approach was required . such an approach seemed possible from the following considerations . mustard gas is known to possess mutagenic properties when tested on drosophila ( auerbach and robson , 1946 , 1947 ) , and on neurospora and e . 
coli ( tatum , 1947 ) , so that , according to the somatic cell mutation theory , this compound admittedly , mustard gas is a potent should act as a carcinogenic initiator . anti - carcinogenic agent ( berenblum , 1929 ) , but this effect was shown to be confined to the later stages of carcinogenesis ( berenblum , 1929 , 1931 ) , and could thus , mustard gas should , thus be attributed to an anti - promoting action ; theoretically , still be capable of initiating action . 
the experimental areas of skin , in the inter - scapular regions , were clipped periodically all test solutions were applied with a with fine scissors for removal of hair . glass rod , and colourless , non - fluorescent , liquid paraffin ( " liquid petrolatum " ) was used as solvent throughout , for standardization of response ( berenblum and schoental , 1947 ; berenblum and shubik , 1947a )  . the main departure from the technique of the previous experiments was that instead of merely noting the numbers of tumour - bearing mice , the individual tumours were also recorded . 
from these results , and from other evidence brought forward , it was concluded that the ultimate tumour yield in carcinogenesis is pre - determined by an irreversible initiating process , while the latent period of carcinogenesis , i.e. 
the speed with which the induced " latent tumnour cells " are converted into morphological tumours , is a function of the subsequent promoting process . the idea of the possible existence of " latent tumour cells " requiring additional stimulation , i.e. 
the results suggest that the acetylaminofluorene acted as initiator , and the goitrogenic agent as promoter , in an analogous fashion to the carcinogen and the croton oil , respectivelv.y , in the case of skin experiments . in the present investigation , one and the same carcinogen was applied to the mouse 's skonce only in different ( graded ) concentrations , to serve as initiator , and then followed by repeated applications of croton oil . expressed in ratios , the concentrations of initiator ( 9 : 10 - dimethvl - l : 2 - benzanthracene in liquid paraffin ) in the four experimental groups were 1 : 3 : 9 : 27 , and the total yields of tumours for the corresponding groups were in the ratio of 1 : 6 - 6 : 12 - 4 : 26 - 5 ( table yet the average latent periods for these 4 groups were closely similar , ii )  . namely , 14 - 4 , 12 - 1 , 10 - 6 , and 11 - 8 weeks , respectively . 
not only was there a progressive increase in the total number of tumours , with increase in concentration of initiator , and also in the number of tumour - bearing animals . 
many different experimental approaches have , however , been devised in recent years to obtain , at least , indirect evidence in support of the theory , and these may conveniently be considered under the following headings : 1 . 
induction , by knonn carcinogenic agents , of mutations in louer organisms . the early studies in this field were concerned with x - ravs and ultraviolet although radiation , the literature of which is well reviewed by muller ( 1941 )  . most of this work was not directly concerned with correlating carcinogenicity 114 i . 
genetic analysis of tumour transplantation phenomena in pure strain mice . the genetic control of tumour transplantation in pure strain mice has been investigated in some detail , and the earlier work in this field is well smmarized it has been shown that a number of genes control the behaviour by little ( 1941 )  . of certain tumours , and this approach has been extended further by gorer ( 1937 , 1947 ) , showing that these genes probably determine their antigenic proalso , according to strong ( 1926 ) , bittner ( 1931 ) and others , the observed perties . sudden change in the transplantation properties of tumnours could be explained on the basis of the occurrence of mutations . 
quantitative interpretation of carcinogenic experiment . several attempts have been made to evaluate quantitatively some of the more reliable carcinogenic data , to determine whether or not such data would be in keeping with a sudden irrev%ersible change of a mutation - like nature . in the investigation of dunning , curtis and wood ( 1940 ) , the production of sarcomas in rats , by subcutaneous injection of 3 : 4 - benzpyrene , was submitted to quantitative analysis . 
the concentration of carcinogen and the volume of fluid injected , both of which were variables , were correlated with the number of tumours induced and with the latent period of tumour induction . it was found that increasing the number of foci injected did increase the number of tumours induced , whereas increasing the volume of fluid injected in one locus did not . in view of the variables involved , and for other reasons to be discussed later , these results do not lend themselves to an exact interpretation of a single response to a single stimulus . charles and luce - clausen ( 1942 ) analysed data of a mouse skin carcinogenesis experiment , using continued painting with 3 : 4 - benzpyrene , to determine whether or not this was in keeping with the concept of the somatic cell mutation theory . in their introduction , they make the primary assumption that if a mutation were the basis of carcinogenesis , it must , of necessity , be of a recessive nature , thus postulating two successive mutations to bring to light an actual tumour . there is , however , no valid reason for this assumption , which is , in fact , at variance with the theory of dominancy , as proposed by fisher ( 1930 )  . in all these previous attempts at analysing carcinogenic experiments , in which the carcinogen is allowed to act continuously , there is the inevitable complication arising from the fact that , of the two stages of carcinogenesis , only one - the initiating stage - could possibly be ascribed to a mutation , since this is the stage which involves a sudden and irreversible change , possibly affecting one cell only . 
shlbik in the present investigation , the correlation of concentration of carcinogen , as initiator , with the total number of turnours induced , has revealed approximately a direct 1 : 1 ratio . this could be taken as being in keeping with the somatic cell mutation theory . however , the other investigation , described above , does not lend support to as already mentioned , mustard gas is a potent mutagenic agent this theory . on drosophila ( auerbach and robson , 1946 , 1947 )  . since its anti - carcinogenic properties ( berenblum , 1929 , 1931 ) can undoubtedly be attributed to antipromoting action , it was to be expected , according to the somatic cell mutation theory , that when allowed to act once only on mouse 's skin , followed by repeated croton oil treatment , tumours should have arisen , the mustard gas acting as an initiator by virtue of its mutagenic properties . 
when tested , this was found not to be the case . in view of this negative finding , and in the light of the highly conflicting evidence of recent experiment in an attempt to adduce more indirect support of the theory , it becomes necessary to re - assess the validity of the theory . basically , neither proof nor disproof of the somatic cell mutation theorv of cancer is possible ( haldane , 1934 ) , as referred to above . the accumulating indirect evidence would seem to run counter to the theorinstead of supporting of the evidence presented in the present communicationi , the first experiment is consistent with the theory , while the second is strongly against it . the force of past reasoning in support of the theory has rested largely on the assumption that , given an irreversible change as the basis of carcinogenesis , the only known biological phenomenon to explain this would be a mutation . however , a closer examination of other common biological phenomena instantly reveals that this is not so . for example , in the course of the development of the embryo , the divergent differentiation that ultimately results in the irreversible cell types , e.g. 
horning. from the chester beally research institute of the royal cancer hospital , london , s.w.3. received for publication december 12 , 1948 . recfnt experience has shown that many of the growth - inhibiting agents used in the palliative treatment of cancer are carcinogenic . 
they also cause specific damage to cell nuclei and chromosomes and are able to induce mutations . the association of these biological effects of ( 1 ) growth inhibition , ( 2 ) chromosome damage , ( 3 ) production of mutations and ( 4 ) induction of cancer suggests if the that they may have a common fimundamental biochemical mechanism . induction of cancer is indeed a somatic mutation , then cancer induction might be included as a special mutation . 
the fact that x - rays can produce cancer in man was published in 1902 ( frieben , 1902 ) , seven years after rontgen 's discovery muiiller ( 1928 ) found that x - rays are mutagenic , and later mather of x - rays . and stone ( 1933 ) and koller ( 1934 ) described the chromosome damage following irradiation . 
the idea that cancer arises as a somatic mutation was supported by the demonstration of an increased incidence of mutations occurring in mice this was shown with methylcholanthrene treated with chemical carcinogens . by strong ( 1945 ) and with 1 : 2 : 5 : 6 - dibenzanthracene by carr ( 1947 )  . carcinogenic hydrocarbons inhibit the growth of animals and the establishment and growth of transplanted tumours ( haddow , scott and scott , 1937 )  . 
niicleoprotein , are believed to play an important part in cell reproduction , but their basic functions have recently developed analytical techniques , particularly not been elucidated . chromatographic analysis and the use of the ultra - violet spectrophotoilieter , have produced considerable fresh data on the composition of the nucleic acids , but much less research has been done on the protein fractions . this paper collects some information on this topic and some analytical results obtained in our laboratory are presented . the proteins found in the nucleus . mirsky and pollister ( 1946 ) were able to dissolve nuclear nucleoprotein in 1 m sodium chloride , after first removing the cytoplasniic material with 014 m saline , and to isolate this nucleoprotein by diluting the solution to a salt concentration of 014 m , when it precipitates in fibrous form . similar material could be obtained from isolated nuclei ( dounce , 1943 ) confirming that these nucleoproteins were of this extracted material was found to consist of deoxyribonuclear origin . nucleic acid and two protein fractions , one of which was classified as a histone , being readily soluble in acid and precipitated out of solution by ammonia . 
from isolated nuclei , apparently failed to do so from the chromosomes , and therefore claims respecting the identity of tr.pr. and the residual chronlosomes must await analytical confirmation . 1 - t is of special interest that cells which could be considered metabolically active ( liver , kid ' ney ) contain as mucb . 
as 50 per cent of the total isolated chromosome material in the form of residual chromosome . stedman and stedman ( 1943 , 1947 ) also isolated an acid - insoliible protein from the nucleus which they termed " chromosomin , " believing that it was the structural protein of the chromosome and that in the nucleus it was embedded in a support of nucleo - bistone . 
the work of both mirsky ( 1947 ) and stedman and stedman ( 1943 , 1947 ) seems to indicate that a protein forms the basic structure of the ebromosomes though the part played by the nucleic acid and the histone this is an important aspect but cannot be discussed here . 
from this material they isolated nucleic acid and histone , and in addition a fraction with isoelectric point between 5 - 8 and 6 - 15 , which coiistituted an appreciable proportion of the nuclear proteins . 
the evidence suggested that the fraction contained a sulphur - containing protein with an acidic isoelectric point and a protein of globulin typeif this material was , indeed , of nuclear origin , then this fraction appears to correspond in properties with the " chromosomin " ' of8tedmanandstedman ( 1943 , 1947 )  . 
the nuclei and chromosomes were fi - rst extracted with i m sodi - um chloride and then the residue was extracted with dilute sodium hydroxide . acidification cf this extract precipitated a protein which contained about 6 per cent arginine and since the nucleoprotein extracted from the nuclei has been no nucleic acid . shown to contain nucleic acid histone and tr.pr. , the alkali soluble protein here must be of different character and this is more likely to be the protein of the finally , two russian workers residual chromosomes than the tr.pr. 
fraction. ( zbarskii and debov , 1948 ) have reported that the protein remaining after the extraction of the nucleoprotein consists of two fractions , one of which is weakly acidic in character , being soluble in dilute alh - ah and having an isoelectric point 132 d . 
da - vidson and lawrie ( 1948 ) , using paper chromatography , carried out qualitative analyses of specimens of histone and non - histone ( acid - insoluble ) proteins from nuclei of calf thymus , rat liver and fowl erythrocytes . the histones contained alanine , arginine , aspartic acid , glutamic acid , isoleucine , leucine , ivsine , phenylalanine , proline , serine , tyrosine , valine and one unidentified constituent . in addition to the above amino - acids the acid - insoluble portein contained i per cent tryptophane , glycine but no lysine , and a further unidentified component . in silnilar studies khouvine and gregoire ( 1949 ) have analysed nuclear nucleoprotein from rat epithelioma and compared it with that foiind in the surrounding nectrotic the acid - extractable proteins nn - ere compared , using paper chromatotissues . graphy and found to be qualitatively the same . very recently stedman and stedman ( 1950 ) have reported arginine contents of histones from various tissues and found they all contained about 28 to 30 per cent arginine ( as per cent of total nitrogen )  . in addition these workers claim to have isolated sub - fractions of the histone of different composition , but no details of the n - iethod of extraction or fractionation are given . importance has often been attached to the tryptopbane content of the nuclear generally speaking , histone specimens have been found to contain proteins . 0 - 0 per cent to 0 - 1 per cent , while the acid - insoluble proteins have about 1 - 0 per cent tryptophane . stedman and stedman ( 1 - 947 ) believe that the presence of tryptopbane in histone is an indication of impurity . mirsky and pollister ( 1946 ) found very small amounts of tryptophane in histone , but about 0 - 8 per cent in the tr.pr. 
of the other histochemical studies reference will only be made to the recent work of thomas and steinitz ( 1950 ) using a method which will stain proteins of high arginine content in paraffin sections . 
the application of this method has indicated that the ratio of arginine - containing protein ( histone type ) to total possibly a combination of this technique protein , is greater in normal epidermis . with spectrophotometric methods will give further data on the localization of the basic proteins in the nucleus . extracted histone has been tested for bactericidal action ( nnleissman and graf , 1947 ) and the pharmacological properties of histone from avian erythrocytes bave also been studied . 
the nitrogen content of histone from both the above materials was 18 - 2 per cent . the acid - insoluble protein was prepared from the whole protein of the nucleoprotein , separated from the nucleic acid by the method of sevag , lackman and smollens ( 1938 )  . 
the solvent n - bu ' tanol - acetic acid - water ( 4 : 1 : 5 ) was used , followed bv either collidine - lutidine - water ( 1 : 1 : 4 ) or phenol - water . the papers were sprayed with 0 - 2 per cent ninhydrin and heated for 6 minutes at 100 ' c . 
to develop the characteristic ninhydrin - amino - acid colours . reference mixtures , " , , ere alwavs run at the same time . in the acid - hydrolysates of three specimens , histone and the acid - insoluble protein from thymus gland and histone from rat sarcoma , exactly the same comindeed , by this qualitative method it was not possible to ponents were found . detect any differences in composition in three fractions . all contained the following aniino - acids : alanine , arginine , aspartic acid , glutamic acid , glycine , bistidine , isoleucine and leucine , lysine , phenylalanine , proline , serine , threonine , tyrosine , were detected . 
for each analysis except in the case of the solvent ( c ) , wben half this amount was used to avoid excess of water on the coluinn . columns of potato stai - ch i cdiameter and 30 clong were used in conjunction with the following solvents : ( a ) n - butanol - n - propanol - 0 . 
moore and stein ( i 949 ) studied the recovery of individual amino - acids from mixtures and found that the chromatographic procedure on starch columns is capable of yielding recoveries of 100 3 per cent . their average recoveries were well within this rang - e and the sum of the aminoacids was almost invariably accurate to i per cent . 
by separate analysis no appreciable amount of trytophane could be found in the histone specimens . less than 0 - 04 per cent was found using the colorimetric method described by spies and chambers ( 1948 )  . 
protein was indicated in the rat sarcoma histone . the thymus histone specimen was quite negative in this test . comparing the two specimens analysed , there are no striking differences in composition . 
stedman and stedman ( 1943 ) believed that their acidinsoluble material ( chromosomin ) , despite a high content of the basic amino - acids must also contain a relatively large amount of glutamic acid . 
the present analyses do not support this claim . since the two proteins , histone and the acid - insoluble ty ] pe are so similar and are extracted together , it seems possible that they may be bonded together in the cell and are subsequently split by the extraction techniques to give basic soluble histone and a denatured " sulphurhistone " product . no considerable differences have as vet been found in the nuclear proteins of normal and neoplastic tissues . there are some small differences in the aminoacid compositions and there is evidence of ' variation in the proportions of the this may be connected with mirsky 's protein fractions in different tissues . ( 1947 ) observation on the variation of the proportion of " residual chromosomes " in the cell in relation to its metabolic acti - vity . 
complete amino - acid analyses of histone from calf thymus and from rat only small quantitative differences v - ere found , but there is sarcoma are given . evidence of a difference in the ] proportion of the protein components in different materials . this work is part of the research programme of the birmingham branch of the british empire cancer campaign . 
they reported an increase of urinary cholesterol in 19 out of 92 cancer patients . later , bruger and ehrlich ( 1943 ) , using a similar method of estimation , found a higher incidence of hypercholeshowever , the teroluria , 11 of their 32 cases giving results above normal limits . actual concentration of cholesterol reported by bruger and ehrlich in normal urine was approximately twice as great as that found by sobotka , bloch and rosenbloom . it appeared , therefore , that the methods used in these two studies must have been significantly different , and we felt that further work on the technique of estimation was needed . while admitting the possible non - specificity of the liebermann - burchard reaction , we felt that this type of method was the only one likely to find immediate clinical application , and it does appear to have given results consistent with the original work of bloch and sobotka . 
an attempt was made to correct for this error by the introduction of a blank reading , which was obtained by adding to half the urinary extract in 5 ml . 
the general procedure was to filter the urine slowly through a 3 - inch wide column of adsorbent from 1 to 3 inches in length , and after washing the column with water , to elute with 40 ml . 
maclagan treated with the liebermann - burchard reagent as described below under aluminthe final chloroform solution was invariably colourless , ium tungstate method . and gave negligible blank values when treated with 09 ml . 
the large tarry residue after alcohol - ether extraction was difficult to extract with petroleum ether and recovery of added cholesterol was poor , ranging from 30 to 70 per cent in various experiments . in this method a gross excess of sulphuric acid is used which is difficult to wash out of the precipitate and results in charring . 
for 20 minutes , and the green colour compared in the photoelectric absorptiometer ( king - gallenkamp ) with the ilford spectrum red filter ( maximum transmission 660mi . ) a preliminary calibration curve showed that beer 's law was well obeyed up to optical densities of 0 - 5 . 
proteose. the results given above under adsorption methods indicate a definite association between urinary cholesterol and protein when present , and suggest a possible association between cholesterol and proteose in normal urine . further evidence of the latter association was sought by estimating the nitrogen content of the aluminium tungstate precipitate obtained ( a ) after washing the precipitate with water as above , and ( b ) after dialysis of the original urine before precipitation . it was assumed that any nitrogen remaining in the precipitate after these procedures was attributable to urinary proteose . ( a ) washing of aluminium tungstate precipitate . - the relationship between cholesterol and proteose was investigated by serial washing of the aluminium in this experiment four identical samples of 100 ml . 
some support for this assumption was obtained by treating the washed precipitate with biuret reagent ( hiller , 1927 ) and also with the phenol reagent of folin and ciocalteu ( 1927 ) as follows : the precipitate from 5 ml . 
of normal urine with added cholesterol ( 0 - 5 mg . ) were duplicate dialysed in a collodion sac against running tap - water for 16 hours . samples were similarly dialysed for 40 and 64 hours . 
of the dialysed urine were precipitated with aluminium tungstate , and the nitrogen content of the precipitate determined by the kjeldahl method as above , without further washing . from the result of this experiment , as shown in table x , it can be seen that the m . 
the precipitated protein was removed by centrifugation , washed once with 0 - 5 per cent acetic acid and extracted three times with boiling acetone , the acetone extract evaporated to dryness and the residue extracted with petroleum ether for cholesterol determination as above . 
the experiments reported give some support to the hypothesis that the urinary cholesterol is normally associated with a urinary proteose fraction , and also with heat - coagulable protein if present . removal of heat coagulable protein does not , however , carry down the whole of the native cholesterol . urine is not a homogeneous substance , and the varying distribution of cholesterol between the deposit and supernatant urine which we have demonstrated is obviously relevant to clinical studies . this factor has been recognized previously , e.g. 
bruger in 1935 reported up to 40 per cent of the cholesterol in the urinary deposit in cases of nephritis . however , most authors have not recognized its importance , and have included the deposit even though pathological in certain cases ( sobotka , bloch and rosenbloom , 1940 ; bruger and ehrlich , 1943 )  . 
much or all of the urinary cholesterol may at times be found in the urinary deposit , or associated with heat - coagulable protein when present . this work was aided by a grant from the british empire cancer campaign to westminster hospital . 
the diet was supplemented with cod liver oil , and greens once weekly , and 2 - 3 g . bread ( 80 - 85 per cent extraction ) daily . thirty - four female and 12 male control rats received the same diet without pda or a mixed diet of rat cubes , bread , and once weekly greens . 
the value for total proteins obtained in the present experiments , in which only mature animals were employed , are higher than those found by lippman for mature animals , but lower than the values of metcoff and favour . 
the deviations of the values obtained by the specific gravity method were within + 6 - 5 and 3 - 5 per cent in 10 cases . the sera were fractionated by electrophoresis in the tiselius apparatus with the cylindrical lens schlieren optical system ( thovert , 1914 ; philpot , 1938 ; the buffer solution svensson , 1939 )  . 
the final patterns and the base lines were traced under magnification , superposed in such a way that those parts which did not show any elevation of the base line coincided . both the ascending and descending patterns were analysed . 
the aand the r - globulins appear frequently as broad regions ( moore , 1945 ) , which by overlapping with the 5 - globulin peak , made the separation of the globulins into individual groups uncertathe uncertainty in the values given for the x - , por - - globulins was + 15 per cent or less . wherever the uncertainty was greater , the combined areas of pand y - globulin were measured . 
of ascites was found together with high serum protein . the sera from 6 treated and 10 control animals were analysed by electrophoresis , and the results are given in table ii and fig . 
no difference in the patterns was found between the normal albino and blackhooded rats , on the semisynthetic diet , or on the mixed laboratory diet . all these patterns showed a double albumin peak , the components of which were separated to varying extent . ~~~~~~~~~~~~~~~i ~~01o0 ~~~~~~~~~~~~~~~~~~~~~i c0 - 3 5 - 0 - o ' - o ! a very ~asmiil fig . 
1. 20 b 30 50 gtc 5o.tu.g in the descending pattern the peak of the slower albumin component was in some cases higher than the corresponding peak in the ascending pattern , and in some cases this region in the descending side indicated gravitational instability , reminisin contrast cent of the , - anomaly , which is often seen with normal human sera . the patterns of sera from rats which developed hepatic tumours due to the ingesin no . 
the gravitational disturbance on the descending side was seen in cases 15 and 16 , and to a smaller extent in cases 13 and 14 . the concentration of the total globnlins was significantly increased in the group fed pda , while the concentration of the albumin remained unchanged . the increase occurred mainly in the [ and y - globulins with the exception of case 12 . 
3. - - e : lectrophoretic patterns of rat sera~ 1 - 10 , cotrol rats , 11 - 16 , rate fed pda._ left : = a~ - ~ling , rit = descenamig patterns . cending , right = descendig patterns . threpeaks in the ascending limb , remaining single in the descending limb - and convectional disturbances were observed near the m - globulin region of the ascending limb , but not in the descending or any other part of the ascending limb . ( similar disturbances near the 4 - globulin region have been observed with some human sera in more concentrated solution in the same buffer . ) these disturbances were not due to heat effects caused by the electric current , but probably to the formation of a gravitationally unstable region . 
the figures in brackets are percentages of the total protein . the remarkable sudden increase in the serum proteins at the time of the development of a tumour and the fact that the increase is independent of the size of the tumour seem to exclude the possibility that the increased serum protein has its source in the disintegration of tumour tissue : this sudden increase in the serum proteins has an analogy with the reaction of the body to foreign protein , where the amount of antibody produced is largely independent of the amount of antigen present . 
the results would suggest that , at least in hepatic tumours , the first pathological change in the serum proteins is an increase in the globulins , and a decrease is only the expression of the general deterioration of the diseased individual . the general impression that the serum protein of patients suffering from malignant diseases is decreased may be erroneously gained from reports ( a ) giving 146 c . 
the increase of the yand p - globulins of these rats might be due to an increase in immune bodies frequently associated with these fractions ( tiselius this explanation would be in accordance with the concept and kabat , 1939 )  . that the increase of the total serum protein in rats developing hepatic tumours is an immunological phenomenon . 
powell. from the department of experimental biology , mount vernon hospital , and the radium institute , northwood , middlesex . received for publication july 29 , 1947 . preparatory to commencing some detailed work on the metabolism of 3 : 4 - benzpyrene in the skin of the mouse consideration was given to a number of experimental factors which appeared likely to affect the reproducibility of the results . 
for quantitative work some standardization of the area to be treated was the method finally adopted was to apply the solution of benzpyrene required . through a stencil cut from sheet aluminium ( 16 gauge ) , with a hole of the desired size and shape . 
as it was desired to use a volatile solvent , acetone or benzene , for the application , the question of how much solvent to use in order to apply any given standard amount of the benzpyrene was examined . 
the use of a small volume of solvent , 005 c.c. , resuited in rapid drying and the deposition of the benzpyrene as a crust of crystals over 324 g . 
bearing in mind the points raised above , a series of determinations of the amounts of benzpyrene left in the skin at various time intervals after painting the results are shown diagrammatically in fig . 
on the other hand examination of the dissected animals showed the presence of benzpyrene distributed throughout the length of the digestive tract . this suggested that the benzpvrene was being licked from the skin by the animals . confirmation of this conclusion came from watching the behaviour of the almost immediately after painting and return to the boxes furthermore , when there is more than one painted animals . they start to clean themselves . mouse in a box they lick , not only themselves , but each other . further confirmation was obtained by painting mice and fixing them to a board with adhesive tape . 
pyrene per animal and the same procedure followed . 1 : 2 : 5 : 6 - dibenzanthracene . for the determination of lipid and nucleic acid phosphorus the animal was weighed and then killed by dislocation of the neck . the liver was removed immediately , weighed , and a sample of about 500 mg . 
from each of two lobes was analysed for lipid , " pentosenucleic acid " ( p.n.a.p. ) and " desoxypentosenucleic acid " ( d.n.a.p. ) phosphorus by a modification of the method of schmidt and thannhauser ( 1945 )  . in this modification the whole of the separation is carried out in a single centrifuge tube and transfer of material is thereby avoided . the tubes have a ground neck to which may be fitted a condenser for refiuxing the solvent in the lipid extraction stage . the dry weight of the liver was determined by drying a weighed sample of the organ in an air oven at 110 c . 
has occurred , and the decrease is due solely to an increase in the concentration of d.n.a.p. there was no significant difference in the liver weight : body weight ratio between the groups of animals on 5 per cent and 20 per cent protein diets . influence of 1 : 2 : 5 : 6 - dibenzanthracene . administration diet on either l : 2 : 5 : 6 - dibenzanthracene produced a marked increase in the p.n.a.p. 
1 , which shows the growth curves of six animals , three of which were killed before any very marked growth inhibitory effect of the carcinogen was observed , and three after considerable loss in weight had the p.n.a.p. 
concentration , and a decrease in p.n.a.p. concentration was also found in the low protein group . administration of the non - carcinogenic hydrocarbon pyrene to rats maintained on the 5 per cent protein diet produced no significant change in the nucleic acid balance . the increase which was found in the liver weight : body weight ratio in the carcinogen - treated rats did not appear to be a result of any increase in the water or neutral fat content of the livers . kennaway , kennaway and warren ( 1944 ) have observed a similarly - increased ratio in mice treated with carcinogenic hydrocarbons . non - carcinogenic substances gave only irregular results . 
no explanation of this increase in liver weight was advanced . the possibility that the increase is related to a differential inhibitory action of the carcinogen on the growth of the animal body as a whole compared with the growth of the liver is being investigated . in considering the possible relationship of the decrease in the d.n.a.p ; concentration in the livers of the carcinogen - treated rats to growth inhibition and carcinogenesis , it is of interest that gopal - ayengar ( 1947 ) has shown that in epidermal carcinogenesis , induced experimentally in mice by application of 20 - methylcholanthrene , the initial effect of the compound was to produce a decrease in the desoxyribosenucleic content of chromosome threads isolated from the epidermal cells . the low desoxyribosenucleic acid content persisted during.the precancerous ( hyperplastic ) stage and was then followed by an increase when malignancy ensued . stowell ( 1945 ) found that x - irradiation of transplanted mouse mammary carcinoma brought about an initial decrease in cellular desoxyribosenucleic acid content , and mitchell ( 1942 ) found a small increase in cytoplasmic pentose nucleotides in biopsied specimens of x - irradiated human tumours . 
she complained of cough , haemoptyses and loss of weight for about four months , but since october , 1944 , she had been feelmg listless and noticing palpitations and dyspnoea on exertion . 
a little lymphatic permeation was traced to the base of this lobe and involved the diaphragthe lower part of the lower lobe showed a fairly marked collapse together with a slight fibrosis . the left upper lobe was well aerated , but showed a sharply defined , firm area of congestion ( suggesting venous infarction )  . 
adjacent broncho - pulmonary lymph nodes are almost completely infiltrated . section of a main pulmonary vein shows neoplastic cells infiltrating the wall of the vessel ; in places these have reached the lumen and lined the wall with a thin layer of squamous carcinoma . 
ante - mortem thrombi have resulted , and some regions show early organization of the thrombus , which itself contains typical neoplastic cells . section of the left lower lobe shows lymphatic permeation by neoplasm , marked collapse and areas of oedema and haemorrhage . 
intra - alveolar phagocytes containing iron and carbon are numerous , and foreign body giant cells are seen in relation to the typical golden - yellow asbestos bodies lying in the alveoli and the finer lung trabeculae . there is marked subpleural fibrosis not related to neoplastic infiltration , but a consequence of the asbestosis . 
one section of this lobe shows that the neoplastic cells , although preserving most of the features of squamous carcinoma , nevertheless show a definite tendency to acinus formation , and here there is much secretion of mucus into the intercellular 252 r . 
the period of exposure also was long ( seven years ) , and although this is usual , cases have been reported of a neop ] asm developing after considerably shorter exposures . 
1. received for publication july 1 , 1948 . in the course of experiments on a transplantable mouse sarcoma it was observed that fresh saline extracts of tumour tissue agglutinated mouse and rabbit red cells . 
when extract and red cell suspension were mixed on a slide agglutination became visible to the naked eye within 30 seconds and appeared to be complete within 2 minutes . a number of different mouse and rat tumours , and a wide range of normal tissues , have been examined for haemagglutinating activity . 
the column was developed with several litres of light petroleum , followed by mixtures of this with progressively increasing amounts of benzene , and the eluates were collected in separate lots of about 2 l each . these were examined spectrographically , and those possessing similar fluorescent spectra were combined , evaporated to a small bulk , and dissolved in small amounts of benzene , providing the following fractions for testing : pes - a : fractions before the appearance of anthracene bands ; pes - b : those with anthracene bands predominating ; pes - c : those after anthracene , but before benzypyrene ; pes - d : all fractions containing benzypyrene bands ; pes - e : subsequent fractions , with recognizable fluorescence bands at 412 and 430m , u . ; pes - f : later fractions with main fluorescence band at 391 m ,  . ; pes - g : still later fractions with fluorescence band at 385 m ,  . in rabbits , skin tumours were obtained with fractions pes - c , pes - d , pes - e , pes - f , and somewhat more slowly with pes - g , while in mice skin tumours appeared only with fractions pes - d and pes - e . two interesting points arise from these results : the first is the confirmation of the previous experiments , mentioned above , that fractions appearing before and after the benzpyrene - containing fraction , are highly carcinogenic . 
the second is that the one appearing before benzpyrene , is far more potent for the rabbit 's than for the mouse 's skin . in a second fractionation , starting with larger quantities of tar ( 4 l ) , the same procedure was adopted , except that the original petroleum extract was shaken 160 i . 
was somewhat more active , while the highest activity was obtained with the next fraction , coming over at 170 - 180 c . the combined fraction of 160 - 180 c . 
was then chromatographed from light petroleum , and collected into four fractions : ( a ) a very early fraction , containing naphthalene and most of the anthracene . ( b ) an intermediate fraction , containing in addition to the remainder of the anthracene , fluoranthene , a large amount of chrysene , and probably chrysene homologues ( see below )  . ( c ) a large third fraction , containing all the benzyprene , also some chrysene , ( d ) a late fraction , in which 1 : 2 - benzcarbazole was recognized . these four fractions were tested on rabbit skin ; fractions ( b ) and ( c ) ( referred to as pf and ph in table i ) was found to be highly carcinogenic , while ( d ) ( fraction pi ) was less so . these fractions , on concentration , deposited crystalline material , which was collected and recrystallized several times from different solvents . one of the components proved to be chrysene , crystallizing in the first crop . 
they are neither acids nor bases , since preliminary treatment with alkali and acid , respectively , does not remove theby fractional distillation , these carcinogens appear slightly earlier than 3 : 4 - benzpyrene . 
one of these , which appeared just before benzpyrene on the chromatography column , possessed the unusual property of high carcinogenic potency for rabbit skin and none for mouse skin . the final active material obtained by the above methods of fractionation , represented more than a two hundred - fold concentration of the original tar . still consisted , however , of a mixture of substances , and the active constituent has not yet been identified . we are indebted to the city of leeds gas department for a generous supply of horizontal retort tar . 
keyser. from the department of pathology and bacteriology , wel - sh national school qf medicine , cardiff . receivecl for publication march 13 , 1954 . many observations have been published on the effect of disease or injury on the concentration of protein - bound carbohydrate ( polysaccharide , protein sugar ) or various carbohydrate - rich protein fractions in the blood serum , both in human beings and in animals . 
a study of the level of serum polysaccharide in various diseases led seibert , seibert , atno and campbell ( 1947 ) to suggest that a raised however , shetlar , bryan , concentration is associated with tissue destruction . foster , shetlar and everett ( 1949 ) put forward an apparently opposing view , namely , that such increases are a reflection of tissue proliferation or repair rather than of destruction . this paper deals with an investigation undertaken to find out what effect nitrogen mustard ( the bis compound , di ( 2 - chloroethyl ) methylamine hydrochloride , was used throughout ) or irradiation treatment might have on the serum polysaccharide level in patients with various neoplastic and allied diseases . ( in such patients raised values are usually found . ) it was hoped that sonie light might be thrown on any relationship between the polysaccharide level and tissue proliferation or destruction , since the nitrogen mustards and x - irradiation are known to cause tissue destruction or at least to inhibit actively growing tissues . another ob ' ect was to find out how much of any increase was contributed by the mucoprotein fraction . 
p - , a man , aged 50 , suffering from hodgkin 's disease , was admitted to bospital on 30.iii.50 for bis second coursfof nitrogen mustard . since the first course of - treatment a few months previously there had been an increase in essential case notes are as fohows 240 j . 
keyser dailv injections resulted in improvement correspoindidg the size of the glands . with a fall in the total white cell count and total polysaccharide , which was down nearly to normal a week after the beginning of treatment ( see table i )  . 
no significant fall in the polycourse . saccharide level occuited during the course of injections , but within 3 weeks after the end of the course it had faren by 40 mg . 
on examination it was found that the whole of the left breast was involved by growth , and biopsy showed there was no radiological evidence of secondary deposits squamous carcinoma . in chest , thorax or pelvis but there was body destruction of the radicles and laminae of vertebra u . 
at the end of 2 months , however , the total polysaccharide had fallen considerably and the mucoprotefn fraction to a lesser extent . healing of the medial half of the right breast area was progressing satisfactorily . 
on examination ( about september , 1950 ) the patient had a large mass of glands involving the whole left side of the neck , and the left tonsil was enlarged and fleshy - looking . biopsy of the neck glands showed the presence of hodgkin 's disease . 
the polysaccharide ( total and mueoprotein ) soon began to increase again , though the patient appeared to be in good health . after several months , however , she began to complain of tiredness and aching associated with a feeling of pyrexia in the evenings , for several clays at a time . these symptoms were apparently unconnected with the menstrual cycle . 
a temperature chart kept by the patient from january to may , 1952 , showed that on these days there was in fact a rise of temperature idthe evenings , usually to 100 - 101 ' f . 
normal figures found ' m this laboratory for the distribution of polysaccharide betweedthese fractions are given in table il the polysaccharide soon began to increase again and the patient 's condition deteriorated . 
 ( 1947 ) found a correlation between the levels of serum a - globulin and polysaccharide in their series of cases . using the tryptophan - perchloric acid test ( cohen , 1944 ; seibert et al . , 1948 ) we obtained evidence of a rise in the level of a serum constituent , that was believed 248 j . 
keyser to be probably polysaccharide in nature , after buming ( keyser , p949 , 1950 )  . the reaction has since been shown to be due to a polyhydroxylic acid of unknown structure ( " sialic acid " ) occurring in the proteins ( werner and odin , 1952 )  . more recently we have employed the orcinol method : both in burns and in fractitre cases pronounced rises in the level of serum polysaccharide ( total and mucoprotein ) were found ( keyser , 1952b )  . this is consistent with the tissue destruction hypothesis , but the increase might equary wer bave been connected with healing processes . that increases in the serum polysaccharide concentration occur in adimals after experimental injury was shown by shetlar , bryan et al . 
the response was a delayed one , with a maximal elevation in 3 to 6 days after the increases in polysaccharide were produced by ( among other methods ) injury . injection of talc suspensions or intrapleural injection of turpentine , causing conditions in which little tissue destructions was thought to occur ; fever was not essential for the elevation ; and the length of time that elapsed before the polysaccharide level returned to normal seemed to be correlated with the extent of repair taking place . 
they give no details , however , except in one case in which a small temporary rise is attributed this patient had previously undergone surgical removal of to inflammation . increases in the level of blood " polypeptides " as a result of the carcinoma . radium and x - ray treatment were reported by cristol and puech in 1930 . 
effect due to inhibition of a breakdown process in and around the tumour and depending on tumour growth . such a break - down process might be expected to be greatest when the tumour is growmg rapidly , and to be retarded when the tumour is subjected to an inhibitory influence such as that of the nitrogen mustards or irradiation . 
working with mice , catchpole ( 1950 ) found that connective tissue bordering on tumours contained increased amounts of water - soluble mueoproteins ; and histochemical studies suggest that in the region of tumours a process of depolymerization is taking place , whereby the components of the ground substance may become water - soluble and enter the general circulation as mucoproteins ( catchpole , 1950 ; gersh and catchpole , 1949 )  . 
the existence of such a mechanism would go far tciwards reconciling the " tissue destruction " and " tissue proliferation " hypotheses , since the two processes - growth of the tumour and consequent depolymerizait remains to be determined what tion - would be associated with one another . bearing the depolymerization hypothesis has on the rises in the level of serum this and other aspects of the problem are mucoprotein observed after in ' now being studied . in four of our cases urinary total nitrogen and nitrogenous constituents were measured in the hope that they might provide an indication of any tissue destruction taking place . that no consistent changes were found was perhaps to be expected , in view of the results of danowski et al . 
wood of the radiotherapy department for calculating the estimnated integral doses referred to in table i ; to dodwell of whitchurch hospital for much help in obtaining specimens of blood ; to our haematology laboratory for blood counts ; to a . 
philps. from the department of clinical pathology , univer8ity college , ho8pital . received for publication december 5 , 1953 . owing to recent improvements in surgical technique the chances of successful intervention in bronchial carcinoma have much improved , and the need for early diagnosis has correspondingly increased . 
the early recognition of mahgnancy , however , may be very difficult and it is hoped that the method described it does not replace histological in this paper may be a help towards diagnosis . examination of material removed at bronchoscopy , but when no tumour can be seen through the bronchoscope , or when histological examination reveals no tumour tissue it can be of great value . when there is some contra - indication to bronchoseopy , the demonstration of carcinoma cells in the sputum may be the sole means of estabhshing a diagnosis . the demonstration of atypical epithehal cers in the sputum of a patient suffering from bronchial carcinoma is not new . 
a positive result was obtained in 13 of the 25 cases . in 1935 dudgeon and wrigley pubfished details of a simple staining method that could be used on sputum films , in which they employed haemalum and eosin . they also described some varieties of neoplastic cell found in the sputum . subsequent papers ( dudgeon , 1936 ; barrett , 1938 ) enlarged upon the original work . at about the same time , gloyne ( 1937 ) described the cytology of sputum with special reference to neoplastic cells . since then , papers on the subject have been pubhshed in a number ofcountries , including britain ( gowar , 1943 ; bamforth , 1946 ; schuster , 1947 ; perrin and littlejohn , 1950 ) , america ( herbut and clerf , 1946 ; mckay , ware , atwood and harken , ' i948 ; liebow , lindskog and bloomer , 1948 ; papanicolaou , 1949 ; foot , 1952 ) , germany ( schmidtmann and sauer , 1952 ) , russia ( altgauzen , 1939 ) and denmark ( wandall 1943 , 1944 )  . 
the lastmentioned paper is quite outstanding and forms a valuable work of reference on this subject . it has a full bibliography , particularly of the continental literature . the proportion of cases of bronchial carcinoma that can be discovered by for example , sputum examination has varied in the hands of different workers . perrin and littlejohn ( 1950 ) obtained a positive result in 60 per cent whereas wandall ( 1944 ) discovered 84 per cent in this way . all of the reports to which i have had access have shown that occasionary a false positive diagnosis is made by sputum examination . while it is clear f . 
philps that such mistakes are more fikely to be made by an inexperienced worker , there remains the risk that even when the pathologist has considerable experience , errors may still occur . 
herbut and clerf ( 1946 ) point out that patients with early bronchial carcinoma frequently have no sputum and advocate the examination of bronchial secretion . in my hands the examination of sputum has been more successful and has sometimes been positive when examination of bronchial secretion has yielded a negative result . 
the routine is simple for patients who are in hospital . a specimen coughed up in the early morning , before any food or drink is taken , and before the teeth are cleaned , is sent to the laboratory . 
very often , although the specimen is produced before breakfast , it is found to contain particles of food material unless the patient has been warned to eat nothing on waking . specimens that contain particles of food visible to the naked eye should usually be rejected , but when a patient has very rttle sputum , any specimen that can be obtained should be examined . out - patients can be given containers and instructed to bring their sputum in them , but it is important that specimens so produced reach the laboratory as soon as possible , and certainly within 24 hours of being coughed up , otherwise infection and autolysis may destroy the cytological picture . specimens sent through the post , particularly in the winter ,  . 
are occasionally satisfactory , but if long delayed they are useless . the whole success of sputum examination for carcinoma cells depends in the first instance upon the selection of a suitable piece of the specimen for microscopical examination . in order that it may be clearly seen , the specimen is transferred to a petri dish and examined against a dark background . 
a mucopurulent piece that is slightly tinged should be taken if it can be found . if the specimen be frankly and uniformly purulent , then there is no alternative but to take a piece of it at randounder these circumstances the chance of finding neoplastic cells is not good , but on occasion they may be found , and are often of squamous type . it has been found that sputum produced during the first few days after the patient has had a bronchoscopy is unsuitable for examination for carcinoma cells . it contains a great deal of cellular material shed from the tracheal lining and the chance of finding neoplastic cells is therefore reduced . i have made it a practice not to examine sputum until a week has elapsed after bronchoscopy . the most suitable instrument with which to handle sputum is a pair of dissecting forceps that have been sharpened to a point on a grindstone . after a little practice , minute pieces of the specimen can be cut out and manipulated with ease . the piece of sputum removed from the gross specimen is placed on a slide , thoroughly mixed with the help of the forceps , and half of it transferred to a second slide . 
one of these is stained with methylene blue ( see below ) , and the other smeared carefully into as even and thin a layer as possible , and placed while still wet into fixative preparatory to staining with haemalum and eosin . it is essential that precautions be taken to avoid disseminating infection from tuberculous sputa . 
philps group has the advantage that films may be stored and re - examined in the light of subsequent findings , or compared with histological preparations made from a tumour . 
the precise technique used differs only in minor detail from that described by schuster ( 1947 )  . it was taught to me at the london chest hospital , and is as follows : a 0 - 5 per cent aqueous solution of methylene blue is used . 
the shde is then warmed by holding it about 8 above a bunsen flame , and at the same time the two are rapidly mixed with the forceps , until the excess moisture contributed by the stain has evaporated . 
the stained sputum is then heaped into a ridge along the middle of the slide , and a cover glass of suitable length immediately put on it and pressed down so that an even thin film is formed . such films tend to fade after an hour or so , the precise time apparently depending upon the amount of infection present in the specimen , but if it is desired that they last longer , this can sometimes be achieved by sealing round the edge of the cover glass with paraffin wax . schuster ( 1947 ) recommends the addition of glycerine to the stain in order that the preparation may keep longer . all sputa sent for examination are stained first by this technique , which is quick . 
a few crystals of mercuric chloride are allowed to remain in the bottom of the vessel containing the mixture to saturate any moisture that may be absorbed by the alcohol . 
the stain is ready for use in 24 hours and can be used repeatedly for 3 or 4 months . it should be filtered if it shows a deposit . it is essential that the film be fixed immediately after it has been spread and while it is still wet , nor at any subsequent stage must it be abowed to dry . it sometimes happens that a specially characteristic cer or clump of cers is seen in the mothylene blue film , and it is desired to make a permanent preparathis can frequently be done in the following way . 
the cover glass is slid off in a direction exactly at right angles to the long axis of the shde , an operation which occasionally destroys the preparation but more frequently leaves the film in place though somewhat smeared in a transverse direction . the slide is immediately dropped into ' the fixative , where it is left for 5 min . the methylene blue becomes purple during fixation . 
undoubtedly this is desirable , but it ' has been found in the present series that between 20 and 30 minutes are required for the preparation and complete examination of a film and it has been impracticable to examine six films from each specimen . 
the writer has found it more profitable to look at it with the naked - eye first so that any parts of it that show a streaky appearance may be especially selected for it has been found that carcinoma cells tend to occur in minute examination . streaks and when these are seen they should be searched throughout their entire length and depth . normally , films are examined with the 1 - inch objective , the i - inch objective being used only when likely cers are seen with the low power . there is little - inch objective in this work , though it is occasionany useful for demand for the examining the chromatin pattem , especiary in oat cells . 
a rather powerful lamp should be used ( a 200 watt bulb is satisfactory ) so that the condenser of the microscope may be lowered shghtly in order to obtain maximum resolution and contrast . 
the nuclear border is smooth and devoid of wrinkhng or irregularity . sometimes , particularly in infected sputum that has stood on the bench for some time , squamous cells are seen which are infiltrated with neutrophil leucocytes . 
39 shows such a carcinoma cells and can ' be a serious source of confusion . cell from a patient in whom there was no evidence of carcinoma . less commonly , a smaller type of squamous cell is seen , which stains more deeply than that just described . it is about 20 microns in diameter , and usually occurs as part of a small sheet of epithelium in the spututhe cytoplasm stains pure b ' lue ( as opposed to green ) with methylene blue , or fairly deep pink with eosthe outline of the cefl is frequently angular - a feature that is evident when such cells are seen lying singly . 
normal epithelial cells tend to be regular in shape , whereas carcinoma cells , particularly those of squamous type , show an irregularity which is best appreciated when many are seen together in the low - power field . 
macrophages. macrophages are constantly found in the sputum in large or small numbers , and are sometimes rather difficult to distinguish from carcinoma cens . many macrophages contain dust particles , which appear as black or brown granules in the haemalum and eosin - stained preparation , and as black , greenish , or purple granules in a film stained with methylene blue . sometimes in a film stained by the latter method , the macrophages are seen to contain a large number of highly refractile fat droplets . 
the presence of dust particles in a cell enables it to be identified as a macrophage , but confusion may arise through the presence of free dust particles overlying a cell and giving the impression of being contained within it . normally they are from 10 to 50 microns in diameter , but , exceptionally , large forms occur . 
as a general rule , they are more or less circular in outline , but this feature is bv no means constant and they assume a variety of shapes . spindle shaped and crescentic forms are not uncommon , and occasionally they may assume a tadpole - fike shape , and may thus be confused with some types of carcinoma cell if shape alone be relied usually the cytoplasm is eosinophilic when stained upon for identification . with haemalum and eosin , but ' it may stain somewhat brownish owing to the presence of ingested particles . 
with methylene blue , it is usuall ' stained blue but when there is a large amount of ingested material the colour may be anything from green to pinkish purple . 
the cytoplasm stains blue with methylene blue , and pink with eosin . the nucleus stains rather deeply and may show some unevenness in the distriit is circular in shape and the nuclei of neighbouring bution of the chromatin . cells in a clump maintain their shape and do not indent each other . cells from oat cell carcinomata , when seen in the sputum , are similar in size to lymphocytes , but differ from them in the following ways : ( a ) the nucleus of an oat cell is larger in relation to the cell area than that of a lymphocyte . 
the cytoplasm of an oat cer appears as only a thin rim round the nucleus , or it may not be visible at all . ( b ) the chromatin of an oat cell sometimes stains more deeply than that of a lymphocyte , and when seen under the - inch objective , shows as a reticulum as opposed to the rather lumpy chromatin of the lymphocyte . ( c ) oat cells nearly always appear in clumps ; lymphocytes seldom do . frequently , oat cers are so closely opposed to each other that they appear t - 0 form a syncytiuthe individual cer borders of lymphocytes are always seen . ( d ) the individual nuclei of oat cers , though usually fairly constant in size , vary considerably in shape . it will be seen that neighbouring nuclei compress and indent each other . 
the only normal cell found in the sputum which occasionally shows this compression effect is the macrophage , which is unhkely to be confused with an oat cell . lymphocytes are shown in fig . 
they are circular bodies , from 10 to 20 microns in diameter . they stain deep blue with methylene blue , and pink , yellowish or - brown with eosalmost always their non - cellular nature is clear : they may show concentric rings , - radial streaking or fissures , or they may be structureless . 
43. - a drawing of a cell seen in a methylene blue filthe cell " body " is a macrophage , and the " nucleus " is a corpus amylaceum which has been taken up . this appearance should not lead to difficulty , a - s the lack of any nuclear details and the concentric rings in the corpus amylaceum make its nature obvious . 
26. - two cell bodies united by a filamentous cytoplasmic bridge . this appearance is not commonly seen , but when present it is considered by the writer to be diagnostic of squamous cell carcinoma . carcinoma whenever considerable pleomorphism is seen in epithelial cells , it should be stressed that a moderate degree may be seen in conditions unassociated fig . 
35 shows an appearance which was mistakenly thought with carcinoma . to be due to carcinoma in a patient who showed no other evidence of a tumour at the time , who has been followed for a year and has developed no sign of the disease . the staining reaction of the cytoplasm is somewhat variable , but may be of some help in identification . 
with methylene blue , it frequently stains greener than that of normal cells , but this property is shared by any degenerate epithelial cell , be it from a carcinoma or from a non - malignant condition , so it is in no way diagnostic . 
the cytoplasm of many squamous carcinoma cells is peculiar in that it appears to be more refractile than that of normal epithelial cells . the methylene blue preparation , this property shows itself by the presence of a dark , rather emerald green outline to the cell and sometimes also to the nucleus within it . 
the cytoplasm itself appears lighter and slightly luminous compared with that of the surrounding normal cells . when stained with haemalum and eosin , the cytoplasm as a general rule stains bright pink , occasionally with a tinge of orange . frequently , as in the methylene blue film , the cell appears brighter than those round it , and if it is put slightly out of focus it may shine up quite brilliantly . cells of this type have been named " bright cells " and are in my opinion diagnostic of squamous cen carcinoma . 
25. - cells from squamous cell carcinomata that give the appearance of wrinkling often , squamous carcinoma cells show a series of irregular concentric lines in the cytothese may be seen most commonly at the periphery , although they are seen to the best advantage in the methylene blue film , they show in of the cytoplasm . plasm , which appear to be minute folds . but they may also occur round the nucleus . the permanent film as well . the nuclei of squamous carcinoma cers have few constant features . another feature frequently seen in the methylene blue film is a mass of minute refractile bodies which are usually disposed in a circular manner round the nucleus . on close inspection , the individual particles sometimes appear to be angular in outhne rather than circular . 
24. - cells showing collections of minute refractile particles in the cytoplasm . these particles are transparent and tend to occur most commonly in a circular arrangement round the nucleus , but they may be seen in any part of the cell . 
normal squamous epithelial cells not uncommonly show sirnilar particles in the neighbourhood of the nucleus , but in the writer 's experience , these are not seen in such large numbers as in carcinoma cells , nor are they concentrated so definitely in one portion of the cell . these particles must be clearly distinguished from the larger , usually black , dust particles and from the large circular fat droplets that are commonly seen in macropltages . dust particles usually present no difficulty owing to their larger size , dark colour and the fact that they are evenly distributed throughout the cell . fat droplets are usually evenly distributed , and appear globular . the six drawings , and those in fig . 
the presence of mitoses in epithehal cells does not imply malignancy , as it may occur in any condition in which there is a considerable amount of epithelial regeneration . in addition to the features mentioned above , a nuihber of other characteristics may be seen which are of considera - ble help in the diagnosis of carcinoma of squamous cell type . 
a particular type of clump has been found to occur in both squamous cer and adenocarcinoma , and though not absolutely diagnostic of mahgnancv appears to be nearly always associated with it . 
the cells all appear to spring from a central point and the clump gives the impression that it may have formed part of a polypoid mass that has broken off into the sputum . if this is correct then any condition associated with papilliferous projections into a bronchus could give rise to such clumps and they might be expected in the sputum in bronchiectasis . i have not seen such clumps in bronchiectatic sputum , but a clump of this type has , nevertheless , led to a mistake . 
philps in the presence of such a change , cells from the metaplastic area would probably be seen in the sputum , but their morphology is not clearlv known . occasionary , round cells of squamous type which have the pecuhar glistening cytoplasm that one associates with keratinisation are seen in the methylene blue preparation . these cells differ from carcinoma cells in that their nuclei are regular in size and shape , though they are often somewhat large in relation to the size of the cell . 
they do not suggest carcinoma in the latter preparation , though they may give rise to suspicion in the methylene blue film . it appears that cells of this type are young squamous cells which may have been derived from the mouth or pharynx or may possibly have come from a metaplastic area in a bronchus . 
the cvtoplasm is geanty , forming at the most a thin rim round the nucleus , which nearly fills the cell . in niany cells , no cytoplasm at all is seen : in others , when the clump is examinedlinder the - j - - inch objective , cell borders cannot be defined , the ceus appearmg to form a svncvtiiithe cytoplasm when seen stains pink with eosin . the nucleus stains deeply with haemalum , and is seldom circular as the nuclei of neighbouring cells indent each other , giving the appearance of having been pressed together . this feature is of great importance in the identification of these cells , and in conjunction with the clumpiing and relatively large nuclear size , appears to be diagnostic and makes the cers easy to recognise . 
the nuclear chromatin is usually clearly defined when thecell is seen under the - ! - - inch objective , and is reminiscent of the appearance of the , reticulum of a stained reticulocyte . less commonly , particularly in degenerate cells , the nucleus may be denser and the chromatin network difficult to define . oat cells are shown in fig . 
the round cers were about twice the size of an pat cell , that is , from 20 to 25 microns in diameter , and they showed greater variation in some , the nucleus was vesicular and nearly fined the cell : than do oat cells . in others , it appeared somewhat contracted and stained more deeply , while in the third and commonest type the nucleus was quite smah and stained nearly spindle cells were seen in clumps amona the streaks of round cens . 
typically , the cells appear in clumps and show vacuolation ' of the cytoplasm , the vacuoles firequently filling the cell and pushing the nucleus to one side , often flatten ' mg ' it against the cell wall . the appearance shown in fig . 
31 , which is from the sputum of a patient diagnosed histoloyicallv as suffering from alveolar cell carcinoma , is very si ' milar to that described by the above - mentioned writers in adenocarcinoma . the presence of cells showing vacuolation is not in itself evidence of carcinoma . the cells shown in fig . 
38 were from the sputum of a patient who showed n ' o evidence of carcinoma , and who , because of th.e presence of these and similar cells has been followed for a - year and has ' developed no tumour . therefore , unless the ceus showing vacuolation occur in clumps and show other characteristics associated with malignancy , they must be considered to be unreliable as evidence of carcinoma . quite frequently , in sputa showing ample evidence of carcinoma of squamous cell type , clumps of cells are seen which surround a single vacuole . 
cers seen which constitute clear evidence of carcinoma . can be g ' iven . categories 1 , 3 and 4 appear from the results to be valuable as an indication of whether the patient has carcinoma , but the value of category 2 , judging from the analysis of results which follows , appears open to question . it will be seen that of the . 
untraced. no cells suggestive of carcinoma seen category 1 . in sputum catego , ry 2 . atypical epithelial cells seen upon which no certain opinion can be given category 3 . cells seen which are highly suggestive of carcinoma category 4 . cells seen which constitute clear evidence of carcinoma of the 9 patients untraced , 8 were out - patients in whose sputum no cells suggestive of carcinoma were found , and who did not return to the hospital . 
he left the hospital soon afterwards and the final diagnosis is not at present known to the writer . of the 8 cases recorded as not yet diagnosed , 3 had no cells suggestive of carcinoma in their sputum , 3 showed epithelial cells which were considered to be f . 
philps atypical but were not thought to constitute evidence of carcinoma , i showed cells that were highly suggestive of carcinoma and 1 , cells that were held to constitute clear evidence of it . 
the 2 patients who were diagnosed as sputum positive are both elderly men in one of whom there is strongly suggestive radiological evidence of carcinoma with persistent collapse of the right lower lobe but no tumour demonstrable on bronchoscopy . 
the other has a shadow persisting in the left mid - zone but has acid - fast bacilli in his sputuit is thought quite probable by those in clinical charge of him that he has carcinoma as well as tuberculosis . 
no bronchoscopy has been performed . * of the 123 patients , there are 106 in whom a final diagnosis has been reached . the results of sputum examination on these 106 will be considered in the analysis which follows . for the purpose of analysis , it is considered that categories 1 and 2 constitute a negative , and 3 and 4 a positive sputum diagnosis , and the terms " sputum negative " and " sputum positive " are used in this sense . it wfll be seen from table i that in two cases , cells that were highly suggestive of carcinoma were seen in the sputum of patients who were finally diagnosed as not having suffered from the condition . one of these patients has now come to post - mortem at which a diagnosis of haemorrhagic bronchitis was made : the other , who was admitted complaining of haemoptysis , is now well , with no evidence of any pulmonary lesion . 
the films ofthe sputa of both these patients have been reviewed and a single clump of cells in the former would still be held to be highly suggestive of carcinoma . in the case of the latter patient , there was a large amoudt ofrather eosinophilic epithelial debris , in the film , the appearance being one which would not now be thought by the writer to be suggestive of carcinoma unless some indication of the mahgnant nature of the cells were also present . 
of the two errors described here , the first must be held to be due to a limitation of the method , whereas the second was caused by lack of experience . of the 106 patients under review , , 39 were finally diagnosed as suffering from bronchial carcinoma , and 67 from other conditions . 
41. total cases in the series cases finally diagnosed as positive cases finally diagnosed as negative total positive diagnoses made by sputum examination correct positive diagnoses false positive diagnoses percentage of negative cases incorrectlydiagnosed as positive 30 false positive diagnoses expressed as a percentage of the total series positive cases in which carcinoma cells were not found in the sputum 9 percentage of positive cases in which carcinoma cells were not found 23percentage of positive cases correctly diagnosed by sputum examination addendum - at the time of going to press , these two patients a - re still udder observation . 
the one whose sputum contained cells that were considered clear evidence of carcinoma has now been readmitted to hospital a year after the cells were first found with clear clinical evidence of carcinoma with multiple bone metatases . 
b is sputum now contains cells similar to , but more pleomorphic than those first found . the other , whose sputum contained cells that were thought highly suggestive of carcinoma has been treated for a year for pulmonary tuberculosis and has improved , ' though the radiological picture is little changed . 
patient8 correctly diagno8ed a8 8putum negative . total patients one speciinen only examined two speciinens examined three four five total number of specimens examlined from these 65 patients mean of number of specimens examined in sputum negative patients j . 
of those who had carcinomata , 30 ( 76 - 9 per cent ) showed carcinoma cells in the sputum . sputum specimens from two patients in the series were wrongly reported as positive . 
one of these patients complained of haemoptysis but recovered rapidly and subsequently showed no evidence of any lung lesion . his sputum contained a great deal of eosinophihc cell debris which was thought to be the remains of necrotic carcinoma cells . 
he died and at post - mortem examination was diagnosed as having suffered from haemorrhagic bronchitis , no evidence of carcinoma being found . those from the first would not now be considered suggestive of carcinoma as the cells showed no other appearance associated with mahgnancy . 
the clump of cells from the second patient would still be held to be suggestive of carcinoma if seen again , which indicates that the method has limitations . further investigation is required into the significance of clumps of cells of this type . it ha - s been my experience that when a confident diagnosis of carcinoma can be made from sputum examination ' , it is usually possible to tell the predominant cell type in the tumour , and so far , when there has been an opportunity for histological confirmation , this has been show ' n to be correct . it has been stressed that in a series of this type , it is possible that misdiagnoses of carcinoma may go undiscovered because the proportion of positive cases is high . it is not certain that there are no such mistakes in the present series but since only about one third of the patients were in fact suffering from bronchial carcinoma , the chances are in favour of such errors being discovered . it is necessary that the limitation of a diagnostic technique of this type be clearly understood . 
a negative finding is of no significance in the exclusion of carcinoma , and may be due simply to the fact that the miinute portion of the specimen chosen for examination contains no cells that can be recognised as malignant . this is likely to be the commonest cause of failure . 
of these , a final diagnosis had been reached at the time of writing in 106 , 39 being shown to suffer from bronchial carcinoma . cells which were either highly suggestive of carcinoma or were considered to be clear evidence of it were seen in the sputum of 30 ( 76 - 9 per cent ) of these . 
an attempt is made to analyse the effect of the number of specimens examined upon the accuracy of diagnosis . where possible , it has been the writer 's practice to state the predominant cell type when a positive report is made . this was done in 23 of the 30 cases so reported . 
an opportun ' ity for histological confirmation has occurred in 10 of these , and there has been agreement . two sputa were erroneously thought to contain cells which were highly suggestive of carcinoma . 
the reasons for these two misdiagnoses are critically examined and it is concluded that one could have been avoided and one could not . further research is necessary to eliminate false positive diagnoses . my thanks are due to professor m . 
3. - normal bronchial epithelial cells obtained by aspiration at bronchoscopy . characteristically these cells are elongated with a ciliated free border and a filiform point of attachment to the ba - sement membrane . 
the nucleus , which ir , regular in size and oval in shape , is nearer to the attached end than to the free border of the cell and tends to make a small expansion in the cell body . 
on close inspection , however , it will be seen that a number of cells in the clump have the general shape of bronchial epithelial cells , showing a wide free border and a filiform point of attachment , and the nuclear distortion is mainly due to the presence of it is possible to discern gradations from those which appear vacuoles in the cytoplasm . nearly normal to those which seem grossly abnormal . 
the strongly eosinophilic cytoplasm is also suggestive of carcinoma , but may occasionally be seen in other degenerate epithelial cells . it should be noted that the appearance suggestive of inclusions seen in this cell differs from that shown in fig . 
the nucleus shows no unusual characteristics and is situated in the wider " body " of the cell . sometimes , chromatin masses are seen in the tail , and on occasion , two cell bodies , each containing a nucleus , are united by a long filamentous bridge . 
the writer has seen them only in cases of squamous cell carcinoma , and nothing resembling them has been seen in any sputum specimen from a non - malignant condition . they appear considerably more refractile than do normal cells , and by virtue of this property , look bright when compared with the cells that surround thewhen put slightly out of focus they shine brilliantly . the high refractility of these cells is possibly associated with keratinisation , and may be related to the appearance shown in fig . 
and the cell itself is surrounded by a greenish coloured band which is probably due to some optical property of the cytoplasm . its brilliance is enhanced compared with the cells in the surrounding field . 
the coloured band which surrounds the cell stands out clearly . in addition to the cell described , there is a considerable amount of eosinophilic debris shown , similar to that seen in fig . 
34 , which is from the sputum of a patient diagnosed clinically as suffering from bronchial carcinoma , but which is considered by the writer to constitute insufficient evidence to enable a diagnosis of carcinoma to be made . 
the present writer now shows considerable caution in the interpretation of the significance of such clumps as two mistakes have been made . before this it was thought that clumps of epithelial cells of this type constituted strong evidence of carcinoma . 
41 and 42 , which were both thought to be strongly suggestive of carcinoma , were from patients who had no other evidence of carcinoma at the tiirne , and in one of whom there wa - s later no post - mortem evidence . 
they have the following chaxacteristics which , taken together , distinguish them clearly from any other cell that is seen in the sputum : the cytopla - sm may be visible as a thin nvhere neighbouring cells are closely applied to each other , riirn , or it may not be seen at all . as is often the case , cell borders are not seen , the appearance being that of a syncytium . ( 2 ) the nuclei of all the cells in a clump are of approxiinately the same size , but they vary considerably in shape , many of them tending to be rectangular . 
the reason for this is that the nuclei of neighbouring cells indent each other . this is the most important single feature in the identification of oat cells . ( 3 ) the nucleus usually stains deeply , but the chromatin pattern can nearly alway 's be distinguished . 
29. - ( 2 ) spheroidal and 8pindle cell carcinonw . a field showing a group of cells similar tov but larger than , oat cells and a small bundle of spindle cells . 
34. - a clump of cells , the individual members of which have no definite characteristics of malignancy though one of the cells seems to possess large nucleoh - an appearance which may be due to the superimposition of pus cells . 
at one time , clumps of this type were thought by the writer to be diagnostic of carcinoma , but a clump of rather similar appearance led to a false positive diagnosis in a patient who was found at postmortem examination not to be suffering from carcinoma . for clumps of cells that are considered to be diagnostic of carcinoma , see fig . 
35. - a group of epithelial cells showing a considerable degree of pleomorphism and nuclear degeneration . this is the most confusing appearance that has been seen in the whole series . 
the cells resemble carcinoma cells closely , but in no case is the nucleus large . this field , and the three that follow , were from the sputum of a single patient , in whom a positive diagnosis was made as a ' result . 
41. - a clump of cells which appear to surround a vacuole from the sputum of a patient who , on the strength of this evidence , was thought by the writer to be suffering from bronchial carcinoma . 
eye hospital , southwark , london . received for publication december 15 , 1950 . in a previous communication ( barker , dhar and parsons , 1949 ) the effects on sarcoma growth in mice produced by treatment with some derivatives of purine and pyrimidine compounds were recorded . further experiments described below have extended the range of pyrimidine compounds studied , and an attempt has been made to correlate molecular structure with biological activity . 
the compounds employed fall into the forowing groups : ( 1 ) cytidyfic acid and alhed compounds . ( 2 ) barbituric acid , its imino derivatives and related compounds . ( 3 ) acyclic analogues of some of the above compounds . pure line or stock mice were grafted with the homologous c57 or the heterologous 837 or crocker sarcomas . 
the technique for treatment was similar to that previously described ( barakan , barker , guband and parsons , 1948 ) , the standard dose ( 0 - 025 g . ) being reclueed in proportion to the toxicity of the particular compound . 
table i summarizes the statistical analysis has shown that the differences in tumour results obtained . weights for treated and untreated grafted mice are significant for groups 2 , 10 , ii and 12 . 
cytosine and uracil , on the other hand , are not utilized in nucleic acid synthesis . this suggested that examination should be made of the action of cytosine when injected into grafted mice . 
the behaviour of 4 - aminouracil appears to be specifically associated with its chemical structure , since the introduction of the sulphur atom at position 2 to give 2 - thio - 4 - amino - 6oxypyrimidine is sufficient to abohsh its inhibitory action . it is considered possible that the marked difference in activity between 4 - aminouracil and 5aminouracil is due to the difference in the electron - availability at the free positions of the two molecules ( positions 4 and 5 respectively ) , which would influence the abihty of the tissues to metabohze the compounds by oxidation . since 4 - aminouracil may also be regarded in its tautomeric form as 4 - iminobarbituric acid , a systematic study was made of barbituric acid and its derivatives , all of which are characterized by high chemical reactivity at position 5 . 
parsons ( 3 ) the 8pecificity of the heterocyclic ring . in the above discussio ' n of the biological act ' ivity of the varl ' ous constituent groups in the pyrimidine derivati - ves the structures were considered only as part of the heterocychc system . in order to determi e whether the ring structure of the compounds is essential to the activities observed three acychc compounds in the first , carbamido - acetamidine , the whole of the structure have been tested . of 4 - aniinouracil is present except for the carbon atom at position 2 . in the second , acetyl urea , the configurations at positions 1 , 2 , 3 and 6 of 4 - aminouracil the third , acetamidine , possesses a similar grouping of atoms to are inaitated . that at position 4 of 4 - aminouracil . 
hall. from the cancer research laboratories of the new zealand branch of the british empire cancer campaign , medical school , university of otago , dunedin . received for publication january 1 , 1951 . kseveralmethods are available for studying the effect of prolonged stimu ( 1 ) repeated implanlation of the sex organs by gonadotropic hormones : tation of pituitaries obtained from castrates , ( 2 ) implantation of testis or ovary into the spleen of a castrated animal ( biskind and biskind , 1944 , 1945 ) and ( 3 ) parabiotic junction of a normal animal to a gonadectomized litter - mate . the first method is not very effective owing to the generally short survival of the grafted pituitaries . 
the ingenious method of the biskinds ( 1944 , 1945 ) , however , does not allow one to study how the increased hormonal output of the stimulated gonads affects the accessory sex organs , whereas in parabiotic animals this effect can be observed . 
we have fouiid in the literature only one reference to an investigation in which a similar experimental procedure has been used . strombeck and ekman ( 1949 ) fed a diet containing a.a.f. 
a breakdown of the junction is a rare event . operative mortality was practically nil , but in the second or third week after the operation a great number of pairs died due to incompatibility of the partners no pair sbowing signs of " parabiotic intoxication " survived longer than 5 weeks . the use of more closely inbred rats ( three generations of brother - sister mating ) did not lower the rate of mortality at this critical period . in most experiments acetylaminofluorene - vvas given by stomach tube to the normal partner , who received the carcinogen in doses of 4 mg . 
the treatment with the carcinogen was started 3 to 4 weeks after parabiosis had been established , when consistent gain in weight and the good condition of the animals showed that the operation had been successful . 
were given on 6 consecutive days by stomach tube and in another the carcinogen was given to the twins with their diet ( 4 mg . dailv to each rat for a period of 4 weeks )  . all the rats which received a.a.f. by stomach tube were fed the ordinary stock diet , consisting of meat meal , pollard , bran , maize meal , bone flour and whole wheat supplemented by cabbage . 
was administered with their food were kept for 4 w - eeks on a diet consisting of skimmed milk and whole meal flour supplemented by codliver oil and cabbage . subsequently they received the same food as the other pairs . the twins were killed when a tumour was suspected or when loss of weight indicated that they were in a precarious state of health . pituitary , testis , accessory sex organs , breast , suprarenal and liver of most pairs were studied histologically . 
the pituitaries were fixed in mercury - saline ( 6 per cent ) , the other organs in zenker or neutralized formol - saline . for staining of the pituitaries a modified papanicolau tecbnique was used ' the other organs were stained with ha , matoxylin - eosin or according to weigert - vail cxieson . the material presented consists of 20 r . , airs of parabiotic rats treated with a.a.f. 
the remaining 2 pairs consisted of litter - mates , none of which had been castrated . of the 6 pairs not treated with a.a.f. , 4 were combinations of a normal male joined to a castrated brother , while in the other 2 the male was joined to a spayed female . in addition , cc single " ' rats were treated with a.a.f. 
 - in a similar manner as the normal partners of parabiotic pairs . five young males received by stomach tube 15 doses of the carcinogen and to a group of 10 , 24 doses ofa.a.f. 
only benign lesions of the liver were seen when the animals were killed in the 52nd week of the experiment . in the group treated with 24 dosos malignant hepatomas were also found from the 37th week onwards . 
was seen in any of the rats joined to a partner to whom the carcinogen had been administered . these livers were macroscopically and histologically normal . in the pair of rats who had received a.a.f. 
the hyperplastic seminal vesicles contained macroscopically visible iiodules which were situated near the cephalad margin of the organ ; in addition a fifth neoplasm of microscopic size was found . in all 5 instances the fundamental histological change was the invasion of the muscular coat of the seminal vesicle by atypical glandular epithelium and therefore the lesion must be considered an adenocarcinoma . 
11 and 12 illustrate the appearance of the only tumour of the seminal vesicle found previously in our material . this large anaplastic carcinoma arose in a seminal vesicle of an albino rat of the " sheffield " strain which had received a diet containing a.a.f. 
and para - amino benzoic acid . in another case both seminal vesicles and prostates formed one large mass containing multiple cysts filled with yellowish creamy material . adhesions connected this mass with loops of the bowel . this - lesion was of inflammatory origin and no neoplastic changes were found in it although many sections were studied . as already mentioned not a single neoplastic lesion was found in the castrates joined to brothers treated with a.a.f. in the pair where both received the carcinogen with their food , the kidnev of the castrate showed a lesion of microscopic size which we have not found so far in rats treated with a.a.f. 
the whole picture resembled that of a benign tubular a4enoma of the kidney . morphological sign8 of increa8ed androgen secretion in the normal parabiotic male partner . the changes to be described were found in most cases and were i ' ndependent of the administration of a.a.f. 
the testes were frequently increased in size . their tubules were normal ; the leydig cers were large , having ample cytoplasm and in some animals their numbers h ' ad increased . 
the hyperplasia of prostate and in tlle latter the glandular part of seminal vesicle has already been mentioned . the organ was more stimulated than the outer mesenchyma ' l layer . this disproportionate growth led to compression of the epithehal folds lined by high cylindrical cells . 
the suprarenals of the intact partner differed histologically from those of the castrated twthe glomerulo 'sa of the former was denser and consisted mainly of cells the cytoplasm of which stained wen with eosin . however , thev - were not as frequent the fascicularis contained signet - ring cells . as in the example ifustrated by selye ( 1947 ) on p . 
133 of his text - book of endoin the castrate the glomerulosa was formed by large pale cells , the crinology . fascicularis was free of signet - ring cells and the reticularis was hyperaemic and better developed than in the normal twthe kidneys of the normal partner their surfa ' e was more granular and were larger than the ones of the castrate . histologically the tubular apparatus showed slight degenerative changes , the , connective tissue being more prominent , especially in the outer layer of the cortex . the pituitaries of the norma - i litter - mates were consistently smaher than in the castrates and were sometimes even ' below the weights normally found in our in these pituitaries the numbers of acidophils were increased rats of similar size . with a corresponding decrease in chromophobes . 
griesbach with faintl the periodate - feulgen technique , the cells gave a slight schiff reaction . this agglomeration of basophils was probably not a - permanent structure . zahler ( 1950 ) has described the appearance of nests of basophils in the pituitaries of rats treated with testosterone . the state of parabiosis did not seem to influence the growth of the rats , nor to increase their suseeptibihty tor a.a.f. in the two pairs where two normal males had been joined together , the animals treated with 24 doses of a.a.f. reached the same size as " single " rats , which had received the same dosage of the carcinogen . 
amounts as used in this investigation are administered . in this respect the carcinogen behaves like the steroid hormones which ass only from one to the other partner when extremely high concentrations are reached in the eirculat ' ion of one of them ( zeckwer , 1946 )  . the liver tumours obtained appeared after approximately the same interval as in " single " rats treated with similar amounts of a.a.f. metastases occurred in two instances , proving the malignant character of these hepatomas . 
although the hepatomatous liver was situated in the same peritoneal cavity as the hver of the untreated partner , the latter remained perfectly normal . there was not the slightest indication for an agent derived from the malignant hepatoma affecting the liver of the litter - mate . as mentioned in the introduction a constant flow of gonadotropins passes from the castrate to the normal partner stimulating its gonads . however , no tumours in this respect our experiments failed where of the testicles have been found . those of biskind and bisldnd ( 1945 ) and of twombly , meisel and stout ( 1949 ) these authors obtained tumours in testes which had been grafted succeeded . into the splee ' n . 
two further points should be considered . spermatogenesis is inhibited in testes situated in the abdomen and consequently the morphology of such gonads is considerably altered ; in additionil transplantation of a testis into the spleen involves traumatic injury . these two factors further studies seem are not present in the experimental procedure used by us . to be required to elucidate the conditions essential for tumour induction in the male gonad , a problem which has been recently reviewed by lipschutz ( 1950 )  . the androgens secreted by a testis transplanted into the spleen do not reach the general circulation and therefore the accessory sex organs of rats bearing such the normal parabiotic male joined to a gonadecgrafts are not stimulated . tomized litter - mate shows the effects of increased androgen secretion . in the pairs not treated with a.a.f. 
this stimulation did not lead to tumour formation in any of the target organs , but in the group treated with the carcinogen five tumours appeared in the seminal vesicles of normal partners . 
we have never found this type of tumour occurring spontaneously in the new zealand strain , nor have we seen it in " single " rats only once a carcinoma of the seminal vesicle was observed treated with a.a.f. in an albino rat of the " sheffield " strain treated with a.a.f. our combined material represents the post - mortem findings of nearly a thousand male rats treated with a.a.f. in the rat spontaneous tumours of the seminal vesicle are extremely rare and we have found in the literature only the case reported by flexner and jobling ( 1907 )  . in men , tumours of this organ are also of very lazarus ( 1946 ) has critically reviewed the cases reported , infrequent occurrence . and since then only a few additional observations have been recorded ( mccree , ] 1948 ; gee , 1948 )  . in the case of the thyroid , the carcinogenic action of a.a.f. 
the liyperplasia induced by increased amounts of androgens secreted by the stimulated testis enhances tlle susceptibflity of the seminal vesicle to the carcinogenic action of ' a.a.f. the ' fact that tumours of macroscopic size were found only in rats treated with 24 or more doses of 4 mg . 
and not in animals receiving less suggests that considerably more of the carcinogen is required to bring about tumour development in the stimulated seminal vesicle than was needed for the induction of adenomata in the hyperplastic thyroid ( hall , 1948 )  . that only one minute tumour of the seminal vesicle was found in an intact male joined to a spayed female is probably due to the lower dosage of a.a.f. given to this group and not to the nature of the gonadotropins of the spayed female . only 17 to 20 doses of the carcinogen were administered because great difficulties were encountered in keeping alive pairs of opposite sex for periods necessary for tumour development . however , our material is not large enough to decide this question . to summarize , we consider the tumours of the seminal vesicle to be due to a hormonal imbalance created by gonadotropins in the normal partner . these hormones , by stimulating the gonads of the ' normal partner , increase the androgen production of its testes and therefore are responsible for the hyperplasia of the although unable to promote tumour growth in the testes they , seminal vesicles . bring about conditions in which an accessory sex organ becomes susceptible to the . 
the possibility cannot be excluded that stin more prolonged androgenic stimulation may ultimately bring about tumour formation in the seminal vesicle without the aid of a.a.f. addend - um ( received for publication february 7 , 1951 )  . after the above paper had been submitted an additional pair of parabiotic rats had to be sacrificed . in this pair a normal male was joined in parabiosis to a castrate , the former having received 20 doses of a.a.f. 
left seminal vesicle with loops of small intestine and a hard small nodule was found embedded in these adhesions at approximately 3 mdistance from the upper margin of the fig . 
in normal male rats joined in parabiosis neoplasms were only to castrated or spayed litter - mates have been described . obtained in animals receiving the carcinogen and never in their untreated littermates . ( 2 ) benign or malignant tumours of the liver were found depending on the amounts of the carcinogen given and on the duration of the experiment . 
the mahgnant hepatomas metastasized to the lungs of the animal bearing the cancer of the liver , but never " infected " the other partner . these neoplasms are considered to be due to two factors ; tlle carcinogenic action of a.a.f. 
agents with therapeutic possibilities such as radio - phosphorus and the radio - iodines , and probably much better , suitable organic compounds containing radio - active isotopes , whose action depends on selective concentration of the radio - activity in particular cells . the separation on the industrial scale of non - radio - active isotopes and the practical development of the mass spectrometer open new possibilities of isotopic tracer research where radio - active effects are undesirable , as in many clinical investigations , or where , as in the case of nitrogen and oxygen , no suitable radioactive isotopes exist . the cyclotron still remains an essential instrument both for the production of small amounts of certain isotopes and as a source of fast neutrons for therait is by no means certain that the peutic trials in cancer and related diseases . frequency modulated cyclotron will be useful as a therapeutic source of fast j . 
mitchell neutrons , on account of the possibility of reduced neutron output and of the undesirability of a pulsed output . a promising field for radiotherapeutic investigation appears to be the study of high energy , 20 - 50 mev . 
prove more suitable than the synchrotron . the electron beam can now be extracted from these machines , the therapeutic possibilities of the high energy beta particles are rather uncertain and must be investigated with caution . the development of high energy generators opens the interesting possibility of the radiotherapeutic application of fast protons of energy in the region of 140 mev . 
 ( wilson , 1946 )  . the results of selected investigations are best summarized in the discussion of these newer agents . properties and production of isotopes , radio - active and stable . the properties of selected isotopes of especial interest are summarized in recent information on isotopes , which may be useful as radio - active table i . tracers , and which are produced in the pile , is summarized in table ii . 
a list of recent references on isotopes is also given . the pile is without a rival for producing isotopes in the very large number of cases where , during irradiation in the pile , a simple slow neutron capture process occurs , e.g. 
and for s35 10 millicuries per g . there is one slow neutron ( np ) reaction of especial importance : n14 + it is of interest that this process is probably responsible for n - > c14 + p . 50 - 80 per cent of the biological effects of thermal neutrons , the remainder of the effects being due almost entirely to the n - y reaction in hydrogen ( mitchell , however , the n , p reaction in nitrogen is of extreme importance for 1947 )  . isotopic tracer research as the basis for the preparation of c14 , the long - lived it has been shown by yankwich , rollefson and radio - active isotope of carbon . norris ( 1946 ) that the c14 is produced by slow neutron irradiation of compounds such as ammonium nitrate and urea in the form of very simple compounds such as c02 , co , ch3oh , h.cooih , and of especial importance as a starting - point for organic syntheses , hcn . in addition to the preparation of materials by direct irradiation , a very considerable number of radio - active isotopes are found as fission products in the uranium rods in a pile . 
a most valuable detailed list of these has been given by the plutonium project ( 1946 )  . in general , for the preparation of isotopes the yields with the cyclotron are very much smaller than those with the pile . 
the oxygen isotopes have been concentrated by distillation of water and by exchange reactions . sulphur has been separated by chemical exchange between so2 and the bisulphite ion . potassium has been also separated by chemical exchange methods . the development of the mass - spectrometer as a practical instrument and its routine use for hydrocarbon analyses by industrial laboratories in u.s.a. show clearly the possibility in medicine and biology of non - radio - active isotopic tracer investigations . 
an interesting possible diagnostic application of measurements of p32 in breast tumours in situ has been reported by low - beer , bell , mccorkle , stone , steinbach and hill ( 1946 )  . * the application of p32 as a tracer in metabolic investigations exemplifies the in order to avoid short - term problems raised by this method of investigation . effects , it is essential to calculate the dose of radiation received by the tissues for example , with p32 , one microcurie per g . from the radio - active isotope . * one line of metabolic tracer investigation insufficiently well known is the use of radio - active brominated compounds , e.g. 
of animal with times of observation of the order of 6 hours . of especial interest is the work of abels , kenney , craver , marinelli and rhoads ( 1941 ) , who accidentally found significant metabolic changes in the leucocytes in chronic leukaemias after total body x - radiation with doses as small as 3 r . , as a result of the radiation given by sub - therapeutic doses of p32 . of much greater importance in the clinical applications of isotopic tracer research is the avoidance of all possible risk of carcinogenesis as a long - term in a recent report on the result of the radio - active material introduced . " health protection activities of the plutonium project , " robert s . 
stone ( 1946 ) states that " it is now well established that ( radio ) strontium given to mice and rats in amounts insufficient to kill them for many months can cause bone and lymphatic tissue tumours to develop in significant numbers of animals . over long periods of time , plutonium has been shown to cause atrophy of the bone and bone sarcomas . " it is evident that great caution is necessary however , before introducing any radio - active materials into human subjects . it is likely that for many isotopes safe conditions for tracer work will be established after dosimetric studies and prolonged animal experiments . 
mayneord ( march 1 , 1946 - unpublished ) that the dose rate at 1 cfrom a point source of 1 millicurie of co60 enclosed in a platinum envelope of thickness 0 5 mis 11 - 1 r . 
however , it may prove more convenient to measure co60 sources in terms of equivalent radium . the production of large amounts of these isotopes by means of the pile raises the practical possibility of the therapeutic application of large gamma ray sources - " mass radiation units " equivalent to 50 - 100 g . 
mitchell in the form of na2hpo4 solution soaked in blotting - paper and dried . in addition to therapeutic applications , these beta - ray sources may be useful in experimental cancer research , e.g. 
stone ( 1946 ) states that " it has been established that single large doses of beta rays and multiple small doses can cause cancers in the skin without the animal being killed by the effect of the beta rays . " therapeutic possibilities of selectively concentrated radio - active agents . radio - active isotope therapy has as yet been limited to inorganic compounds . radio - phosphorus ( p32 ) , and to a much smaller extent the radio - iodines ( 1130 andi1131 ) , are the only isotopes with which limited clinical trials appear justified without further preliminary animal experiments . in general it seems unlikely that inorganic radio - active isotope therapy will stand the test of time . it seems much more hopeful to study the selective concentration by malignant cells of suitable organic compounds carrying radio - active isotopes of many elements ( rhoads , 1946 )  . the usual therapeutic applications of p32 in the form of na2hpo4 appear to depend upon its synthesis into nucleic acids by the multiplying cells . one other method of therapeutic application ofp32 reported ( jones , wrobel and lyons , see low - beer , lawrence and stone , 1942 ) depends on the selective concentration of colloidal anhydrous chromic phosphate by the cells of the reticulo - endothelial system . it is easy to understand the concentration of the radio - iodines by the thyroid in hyperthyroidism and in the rare cases of carcinoma of the thyroid , where the primary and sometimes also the metastases retain the function of secretion . 
one must be sceptical of the therapeutic possibilities in bone sarcoma of sr89 or ca45 ; it is interesting to note that stone ( 1946 ) reports that radio " strontium , barium , zirconium , yttrium and others locate quite selectively in the bones , and many of them stay for long periods of time . " radio - phosphorus , p32 , has been used since 1936 mainly in u.s.a. , in the treatment of patients with chronic myeloid and lymphatic leukaemia , polycythemia vera , lymphosarcoma and various related ( low - beer , lawrence and stone , 1942 ; reinhard , moore , bierbaum , kenney , 1942 ; lindgren ( 1944 ) showed thatp32 , which is selectively moore and kamen , 1946 )  . concentrated , gave better results than na24 , which is not selectively concentrated . the therapeutic possibilities and dosage of radio - phosphorus has been reviewed recently ( mitchell , 1947b )  . it is considered that the present position with regard to its therapeutic applications may be summarized as follows : diseases 1 . 
in the treatment of chronic myeloid and chronic lymphatic leukaemia , p32 is probably as satisfactory as , but no better than x - radiait is important to investigate the possibility of development of tion . methods of routine treatment of these diseases by means ofp32 . the average , each case is likely to require 10 to 15 millicuries ofp32 given , preferably intravenously , in the form of a solution of na2hpo4 in fractions in an over - all time of 10 - 12 weeks . it is desirable to correlate the dose with roentgen units ( marinelli , 1942 ) , and to measure the differential absorption ratio in different tissues ( kenney , marinelli and woodard , 1941 )  . 
3. given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . since 1944 we have taken a detailed family history from all patients reporting with carcinoma of the breast . 
we have 459 family records up to the end of 1947 which are reasonably complete . so far we have not succeeded in supplementing these histories , taken at the time of first visit to hospital , by later questions to the surviving patients or by arranging visits to their homes or relations . 
some attempt has been made to confirm the causes of death of relatives by letters to hospitals , doctors and the registrar - general 's department , but such confirmation has , as yet , been obtained in only a few cases . our figures , therefore , contain all those errors of inadequate information and faulty recollection that one would expect from data collected . 
many people die of cancer without their relations knowing that this was the cause of death . it is in fact surprising how successful a woman can be in concealing a cancer of the breast from her nearest relatives living in the same house with her . it is not , therefore , so surprising that more distant relatives , or even close relatives living away from home , may be unaware of the nature of the illness involved relations , even some of an older generation , who are still living at the time that the patient is first questioned may later develop malignant disease . 
many patients with cancer of the breast survive for long periods following treatment , and may ultimately die from some other cause ; we are , however , unable to include patients living who either have , or have had , cancer in our figures for comparison with expected incidence in the general population because no adequate morbidity statistics are available . in our series , for instance , there are the same number of sisters alive following treatment for cancer of the breast as there are sisters who died of the disease . ' comparisons must be made on a basis of mor164 d . 
3 shows a family where the mother of the patient died of cancer of the uterus aged 67 - she had two sisters , one who died of " internal cancer , " and one 166 d . 
smithers this sister had a daughter of cancer of the breast ' the latter aged about 60 . who had her breast amputated for cancer aged 45 , and who died aged 60 of lymphatic leukaemia . 
we find no evidence that cancer of the breast tends to develop earlier in patients whose relations are known to have suffered from the disease . the greater part of the work presented in this paper was done by p . rigby - jones and h . 
hartley , whose valuable assistance made this contribution to the symposium possible . i am also indebted to my colleagues on the staff of the royal cancer hospital whose patients were interrogated during this investigation , and particutlarly to r . 
the centrifugary - cleared extract was adjusted to i per cent with 20 per cent saline and the crude mucoprotein , etc . , precipitated by addition of two volumes of alcohol . 
the washed deposit was redissolved in 1 per cent saline , adjusted to ph 7 - 5 , and digested for 24 hours at room temperature with commercial trypsthe mixture was then filtered with the aid of hyflo super - cel and reprecipitated with 2 volumes of alcohol . 
the yield of protein - free , crude polysaccharide was about 2 per cent of the acetone - dried tumour tissue . the material contained polysaccharide and pentosenucleic acid , and the analytical data were : nitrogen ( kjeldahl ) varying between 5 - 2 and 6 - 3 per cent ; phosphorus ( fiske and subbarow ) 1 - 7 to 1 - 8 per cent ; and sulphur ( paulson ) 0 - 6 per cent . 
the ninhydrin reaction was negative . metachromatic sta ' m ' mg with azure a ( sylv6n and malmgren , 1952 ) revealed the presence of one orthochromatic component and another presenting alcohol - resistant metachromatic precipitates . 
d was also degraded by testis hyaluronidase at a similar rate to other such hyaluronates . glucose ( glucosamine ) could be demonstrated by following acid hvdrolvsis onl paper electrophoresis in borate buffer ( consden and stanier , 1952 )  . in addition , infra - red spectral measurements kindly performed by s . 
the two spectra were identical in the region 680 - 980 cm . - ' , showing the two materials to have the same molecular skeleton ( orr , 1954 )  . 
the only difference lay in the relative intensities of the bands due to the acid and amide carbonyl groups . however , the acid : amide ratio in sample d cannot be more than 5 per cent greater than that in the purified umbilical hyaluronate . nature of the_8uiphur - containing material . the metachromatic precipitate formed by azure a treatment of sample b was almost insoluble and further information about its composition could not be obtained . 
attempts were therefore made to remove the nucleic acid and the hyaluronate from the starting material by successive ribonuclease and hyaluroafter removal of the hydrolysis products by dialysis it was nidase treatments . thought possible to obtain the s - bearing material in a purified and soluble state . however , this latter material exhibited a powerful inhibitory effect on hyaluroin one experiment , when 30 mg . 
the s content of the remaining non - digested material was 0 - 49 per in the course of this long digestion , however , a small precipitate was cent . formed , which had a n content of 10 - 0 per cent and s content of 4 - 8 per cent . these figures suggested that a new product had been formed between the protein hyaluronidase and a s - containing polysaccharide . if the polysaccharide is assumed to contain 3 per cent nitrogen , the nitrogen value of the precipitate would fit a protein content of about 55 per cent , and hence a polybaccharide content of about 45 per cent . 
the intermediate protein - free material ( sample a ) contained about 70 per cent hyaluronate , 25 per cent nucleic acid , and about 5 per cent of a sulphur - be ' aring polysaccharide presenting some biological characteristics of heparthe largest part of the polysaccharide material present is thus beyond doubt hyaluronic acid with identical molar characteristics with that obtained from mammahan sources . 
the rous hyaluronate has probably a lower particle size than the hyaluronate from normal tissues , and the high viscosity of the intercellular rous material seems partly due to the protein components . the identification of heparin in the sulphur - bearing polysaccharide has not been fury established . 
a single male introduced the ay gene into all three lines , and the yellow mice in the offspring were crossed again to the respective pure line , and this process carried on indefinitely . 
were he responsible , he would have had to be heterozygous for these 7 genes in addition to his own ayat make - up , which would be rather an admirable genetic achievement , even if deliberately attempted . ( 3 ) contamination by stray mice : the whole experiment was carried out in a small room of the mousery , in which only this " yellow experiment " and inbred mice are maintained . 
none of the mutants was of this type , but all of the contamination by strays does not therefore appear to be usual " inbred " size . a satisfactory explanation , and true genetic mutation . 
though considerable discussion has been given to the question of mutations produced by atomic bomb explosions , it is possible that a much more frequent cause of these disturbances may have been unsuspectedly in action for many years on persons exposed to the influences of carcinogenic hydrocarbons . while mutations analogous to some described in this paper , e.g. 
reduction of hair and eye pigments , may be unimportant in humans , other types of mutants , some not detectable by the hydrocephalus , found methods used , would be a serious factor in public health . in this work , certainly would be . dominant lethals , leading to sterility or miscarriages , may also be produced , unimportant in mice where a large litter size is found , but of serious concern in man . it is possible that some mutants , such as the brain hernia found as an embryo , would normally be missed by the scheme used above , as mice usually eat abnormal and inviable young . the same unconcerned and easy solution of such difficulties is not the case in humans . there are possible also dominant mutations , which would appear in the first generation . though none was found in the series above , several were reported by strong it is obviously desirable that this additional ( 1945 ) using methylcholanthrene . hazard of exposure to these carcinogens should be evaluated . that this need is urgent is also suggested by the following line of argument : radiation mutations are almost entirely random , i.e. 
if a certain gene is mutated by an ionization in one sperm , the chance that the same gene will mutate in another sperm in the same or another individual is not increased . 
the efficiency of but this is not mutation with regard to any given gene is thus almost zero . if a chemical distributed via the blood necessarily the case with chemicals . stream reacts with a given locus to produce a mutation in one sperm , it is bbviously liable to do the same with all other similar loci in other sperms ( or ova )  . 
an efficiency of mutation at a given locus at 100 per cent can thus be imagined , and then all offspring of an exposed individual will carry the abnormal gene . a mating of any two exposed individuals would then give all f1 of the mutated type , a state which does not arise until the f4 of close inbreeding with radiation156 j . 
they could therefore have a high efficiency at certain loci resulting in a high frequency of abnormal offspring without causing death of the cell , as happens when concentrated doses of irradiation are given to increase mutation rate . combination with genetic material is thus likely . it is obviously tempting to ascribe the ability of the carcinogenic hydrocarbons to produce mutations to their complex system of conjugated double bonds . this system would readily pick up energy from the surroundings , and liberate it with destructive effect at one point . 
the hydrocarbons are possibly even less energetic , and thus may only be able to produce mutations in genes that are of less stability than most . this would produce some degree of specificity as required above , and it is significant that most of the mutations produced both here and by strong are already known , i.e. 
no sarcomata were found , however , and the local trauma to the squamous epithelium of the crop apparently favoured the local action of the oily solution of carcinogen . further experiments designed to assess the possible action of 2 - aaf in arachis oil eneysted in the submucous tissues of the crop have been in progress for about 4 years , so far with negative results . 
of 2 - aaf was injected repeatedly into the crop of each bird . sixteen white leghom cockerels of our inbred flock , aged 5 months at the start of the experiment were injected twice weekly for 4 months , then once weekly for a further year , and then at less frequent intervals , determined by their gene ' ral state of health . they were kept on open grass range with access to unheated fowl houses and fed twice daily with commercial poultry mash and pehets . 
no foreign body was present in the ulcer or underlying tissues . halfway along the oesophagus between the buccal ulcer and the crop , a small pedunculated translucent polyp 3 min diameter projected from the posterior wall . 
both kidneys were very mottled with white spots and small retention cysts ; the left kidney also contained a firm white tumour pressing on the sacral plexus ( cause of paralysis )  . 
3412. this bird had repeated i ' ections of a 0 - 5 per cent solution of methylcholanthrene ( mc ) in arachis oil into the wan of the crop with a total dose of 30 mg . 
for 3 months past it had had a palpable tumour in the neck and this had grow - n to a mass about 10 cin diameter when the bird was kined . 
a biopsy taken when the tumour was first noted showed a solid cehular tu ' mour with little differentiation and with a dehcate stroma with little or no intercellular substance . 
the tumour was considered to be an anaplastic carcinoma . cell suspensions from biopsy material were injected into 12 birds with negative results . material taken post - mortem showed a fleshy , spindle - cened tumour very similar histologicary to the chemically - induced sarcomata that are the usual response in fowls to injections of carcinogenic hydrocarbons . grafts of fresh tumour tissue were made into 13 birds with negative results . these 2 tumours contrast with those induced with 2 - aaf in which the diagnosis of carcinoma was in no doubt . n : n - dimethylamino8tilbene ( dmas )  . - as this siibstance has been shown by haddow and his co - workers to be a versatile carcinogen for rats it was thought that it might also prove an epitheliotropic carcinogen for the fowl . thirteen white leghorn cocks aged about 5 months were injected periodically into the pectoral muscles with 0 - 4 per cent solution of dmas in arachis oil with a total dose of from 0 - 5 g . 
to 0 - 8 g . these birds died or were killed between 78 and 208 weeks from the start of the experiment but no tumours were observed in any organ . 
the present experiment shows that 2 - aaf is similarly active against a variety of epithelial tissues ih the fowl and confirms the earlier report by bielschowsky and green ( 1945 ) that it is carcinogenic for the kidney of this species . in contrast with 2 - aaf , n : n - dimethylaminostilbene , which is also a versatile carcinogen for rats ( haddow and kon , 1947 ) , caused no tumours in fowls in our experiments . thus it seems that the epithehal tissues of the fowl are much more resistant to the action of many carcinogenic hydrocarbons than are those of rodents . however , monkeys also seem to be relatively resistant to the action of carcinogenic hydrocarbons ( pfeiffer and allen , 1948 ) , and it may be that we should rather regard the small rodents as being unusually susceptible to these agents . it is unfortunate that all attempts to transmit the 2 - aaf induced carcinomata have failed so far . 
the technique for painting with the 03 per cent benzpyrene solution in - acetone was that described by calcutt and powell ( 1947 ) , and further treatment was as in earlier work ( weigert , calcutt and powell , 1946 )  . extraction and estimations of the metabolites were by the methods of weigert ( 1948 )  . the nutrient media used were as below : 1 . 
for a period of 8 hours . metabolism of the benzpyrene only occurred in the series on the four buffered in the case of the tap water and the normal saline the media were found media . to be turbid and the skins sodden and waterlogged . 
on the other hand , the variation in explanations of this based on the yields obtained is in itself of some interest . the detailed composition of the media used appear to be of doubtful significance . the outstanding point seems to be the ph of the medium . under the conditions of experiment it is reasonable to assume that the tissues of the skins concemed became adjusted in some degree to the environmental conditions , that is , that the internal state of the cells approximated more nearly to the external ph than would normally be the case . 
the question arises , therefore , as to whether such a state of affairs could influence the metabolic turnover . taking advantage of some recent findings , it is believed that an explanation of the results can be offered along the above lines . it has long been suspected that the process of carcinogenesis is intimately involved with sulphydryl groups . this view has recently been reinforced by crabtree ( 1948 ) , and the weight of evidence is now strongly in its favour . 
at the same time it has been suggested that the metabolism of benzpyrene is also mediated by - sh groups . this has received confirmation by the findings of 432 a . 
nordling. received for publication december 29 , 1952 . recent research in genetics and pathology has shown an amazing consistency between the agents causing mutations and those causing - or contributing to the one of the more prominent theories , which since the development of - cancer . 1920 's has been advocated by bauer ( 1949 ) , strong ( 1949 ) and others , claims that the original cancerous cell is nothing but an ordinary cell affected by genetic mutation of some kind . 
one of the main objections to this theory has been that it does not explain the age variation of the cancer frequency . in reply , bauer ( 1949 ) contends that the mutated cells apparently remain latent during a long period until the new qualities become evident and the phenomenon can be diagbauer has found this period of latency to average 9 years for nosed as cancer . x - ray cancer , 12 years for paraffin cancer , 18 years for aniline cancer and 40 years for seaman 's cancer ( caused by solar radiation )  . 
the early occurrence of sarcoma and of leukaemia , however , does not conform to the idea of a latent period . moreover , it appears somewhat unreasonable to suppose that the length of the latency period would depend upon whether the mutation is caused by sun - rays writh most forms of carcinoma , furthermore , the frequency at or by x - rays . different ages is such that wre are compelled to consider average latency periods of 70 years or more , in order to explain the actual age frequency curve of cancer these facts still make it difficult for many of the most commortality in man . petent authorities in the field to accept the mutation theory without reservations . dahlberg ( 1943 ) has advanced a completely different explanation concerning the relationship between age and cancer . 
he expressed the opinion that malignant tumours develop increasingly easily with rising age . this implies that , for the development of tumourous cells , it is necessary for a certain number of cellular divisions to have taken place , between each of which there has been a certain period of time . all experience concerning the cancerous influence of chronic irritations promoting cellular divisions seems strongly to support this theory . 
the number of mutations required for experimental cancer in mice may be less , as suggested by the investiobviously , we need not assume that any gations of iversen and arley ( 1950 )  . only mutations which increase the ratio seven mutations will cause cancer . between cellular divisions and cellular loss in a positive direction in the environaccording to fischer ment in question may be expected to have this effect . ( 1930 ) , the short average life - span of cancerous cells is their dominant feature , and that by which all their other qualities can be explained . their rapid propagation , which exceeds their high death - rate , their ability to ferment sugar on a large scale and to disintegrate heterogeneous proteins , constitute normal cellular responses to a high mortality among adjacent cells . it may be added that the frequent variability in the number of chromosomes among cells from the same tumour is also a " normal " feature in the sense that the same phenomenon also occurs in certain normal tissues , as shown by therman and timonen ( 1951 )  . thus , some or most of the mutations required to start an incessant self - stimulating propagation among a group of cells might consist of any of the forms of mutation that weaken the cell , and make it more liable to die from even minor disturbances . if cancer were caused by one mutation only , it might be expected to be equally common among persons of different ages after an age corresponding to the length of the latency period , if this period were interpreted as the time required by the first cancerous cell to multiply in such degree as to be diagnosed as cancer . 
nordling prevails in countries with a high percentage of deaths due to " senility " , " other and unknown causes " and the like , whereas the latter is the case in countries e.g. , the united states , new zealand , with detailed and reliable statistics , australia , great britain and other western european countries . it can be shown , as by nordling ( 1952 ) , that the cancer frequency curve of a population with undependable vital statistics will be restored to the same shape as that of the populations with accurate statistics if , as a rule , about one - fourth of the deaths due to such causes as " senility " are added to the registered cancer deaths . united states white population 1940 united kingdom 1939 france 1947 norway 1941 - 1945 000o = + = : + 14 at = ___x___ 200l asla42w 2 i - = 3 ___ _ - - _ 40 60 80 50 70 60 80 50 70 60 80 50 70 40 60 8u 50 70 fig . 
1. - diagram drawn to double logarithmic ( log / log ) scale showing the cancer death - rate ( in the case of the united kingdom , the carcinoma death - rate ) in males at different ages . deaths per 100 , 000 males are shown on the vertical scale , age figures on the horizontal scale . however , the very highest age - groups , which are always numerically small this is and certainly highly selected , can hardly be taken into consideration . because these individuals are usually already very frail and likely to die from any trivial cause , irrespective of whether or not they have cancer in a more or less developed stage . thus , it seems probable that , in reality , the age - frequency relationship for cancer follows the same general rule everywhere in the civilized world , although in these reliable examples can be given in only a limited number of cases . cases it is obvious , as is shown in fig . 
with regard to cancer among females , it is necessary to distinguish between cancer in the specificially female organs , of which the increase is fairly small above the age of 45 , and cancer at other sites , of which the frequency seems to increase according to the sixth power of age both before and after the forties , but not during the decade of the menopause , when the increase is smaller . it seems possible that altered hormonal conditions in connection with the menopause might play a part in determining the actual cancer frequency curve obviously , more slowly operating hormonal variations might among women . be present in both men and women . 
it is therefore by no means certain that the entire increase in the incidence of cancer with age is to be explained by means of the multiple mutation mechanism . it is possible that a small proportion of the increase is due to a lesser degree of hormonal inhibition of growth of potentially cancerous cells in an older organism than in a younger one . 
the writer is not , however , aware of any evidence definitely substantiating such a variability of natural cancer inhibition with age . during childhood and adolescence cancer is rare , but it occurs considerably in these cases it is more often than the sixth - power - of - age rule would allow . mainly a question of sarcoma , leukaemia and other non - epithelial forms of cancer . such a condition does not , however , imply any contradiction of the hypothesis since it is unlikely that mutaof successive mutations as the cause of cancer . genic noxae reach the bones and other internal tissues , it therefore seems more reasonable in the case of sarcoma to presume spontaneous mutations ( i.e. , mutations due to normal molecular collisions ) and radiation mutations as the such mutations may , of course , occur already in the foetal causative factor . in view of the extremely rapid growth during this period , it is not period . surprising that multiple mutations occasionally have time to occur , giving rise to infantile or even foetal sarcoma . 
the infrequency of malignant tumours in the striated muscles , despite their large volume , may have some connection with the fact that this kind of tissue is composed of multinuclear cells . according to the theory put forward in the present paper , cancer is caused by mutations multiplied and accumulated usually through large - scale proliferation it is easy to explain on this basis such facts as the ( sometimes inherited ) of cells . high susceptibility to cancer of certain organs , especially under experimental this is because every stimulant causing - or congenital capacity conditions . characterized by - a high proliferation rate also increases the number of mutated also the high susceptibility to cancer cells and thus the probability of cancer . of benign tumours is easy to understand in this way . the facts and theories advanced here demonstrate , among other matters , the importance of having reliable vital statistics in order to elucidate casual relationships in the pathogenesis of cancer . 
bielschowsky. from the hugh adam cancer research department of the , medical school and the , new zealand branch of the british empire cancer campaign , university of otago , dunedin . received for publication december 5 , 1953 . chromophmic tumours of the pituitary are more frequent in the rat than in man . 
thus chronic thyroxine deficiency can lead to the formation of basophihc adenomata and the morphological signs of hyperoestrinism are found often in conjunction with acidophilic adenomata . when the normal level of oestrogen in the body of the female rat is raised for prolonged periods , vagina , uterus and mammary glands undergo a series of changes one of which is cystic hyperplasia of the breast . 
growth had taken place mainly in direction of the cerebellum with which the tumour was in close contact without invading uterus ( 125 mg . ) and vagina were atrophic , it . 
the kidneys had a granulated surface and the adrenals were of a slightly darker colour than normally . large fat deposits were present in subcutaneous and retroperitoneal tissues , as well as in the abdomen . 
respectively , when killed after 10 months of parabiosis . smears taken from the spayed female indicated persistent anoestrous , whereas those of the intact littermate showed the picture of continuous oestrous . 
at the post mortem the pituitary of the intact parabiont ( rat 2 ) was found to be considerably enlarged ( 139 - 3 mg )  . nodular structures protruded above the surface which showed haemorrhagic discoloration . 
the ovaries were large and cystic , of a yerowish colour and apparently free of corpora lutea . there was after removal of the capsule fluid between the capsule and the ovarian tissue . the combined weight of the ovaries was 299 mg . 
the breast glands were remarkably hyperplastic and contained large milk cysts . in sections of the pituitary normal anterior lobe tissue was not recognisable and the presence of basophihc cells could not be demonstrated by any method . the cleft separating intermediate and anterior lobes was enlarged ' and filled with 156 f . 
the great majority of the tumour cells appeared to be larger than normal chromophobes or acidophils and most of them had a very prominent golgi apparatus the negative image of which was alreadv recogni 'sable at low power . 
the frequency of mitoses varied in different parts of the tumour , but rarely more than two were seen in one field at low magnification . blood - filled sinuses and areas of haemorrhage were present in all sections and so were strands of dense connective tissue , originating from the capsule or from larger blood vessels . 
the vagina was lined by layers of stratified , squamous , keratinising epithehuthe outstanding feature of the greatly hyperplastic mammary glands were cysts filled with eosinophilic material which apparently exercised pressure on the lining epithehum , flattening it . smaller cysts were surrounded by cuboid epithelium containing secretion droplets . 
the histological examination of the breast confirmed that the changes seen at autopsy were of a similar kind in both partners as far as their secretory activity was coneemed . to recapitulate , a remarkable secretory activity was found in the breast of the ovariectomised parabiont , the uterus and vagina of which showed the typical castration atrophy . in this case the stimulus responsible for " lactation " is oestrogenic stimulation can be assumed to have come from the intact partner . excluded because of the state of pituitary , vagina and uterus of the spayed animal . at the time this observation was published we were unable to explain whv such changes in the breast were seen only occasionally in spayedanimals joined in parabiosis to an intact female . revision of the material has shown that there exists a good parahelism between the presence of a pituitary tumour in the intact partner and stimulation of the breast of the ovariectomised parabiont . 
a fortnight later vaginal smears revealed the presence of cornified unfortunately cells which remained the predominant cell type for 13 months . no smears were taken during the following 3 months , but during the week preceding death the vaginal smears were those of anoestrous . 
ar other organs appeared normal except the lungs which contained small areas of consolidation . histological investigation showed that little non - neoplastic tissue remained in the pituitary . this consisted of chromophobes and acidophils of fairly normal appearance , but the presence of basophils could not be demonstrated in the compressed rim of normal anterior lobe . 
the tumour itself consisted of atypical large acidophils which in some regions were nearly as numerous as non - granulated cells , whereas in others the latter predominated . bizarre cells with giant nuclei were a common occurrence and one or more mitoses could be found in nearly an fields . 
the glands contained follicles in various stages of development groups of pale vacuolated cells , separated by with healthy appearing ova . strands of spindle - shaped elements predominated in the stroma . in frozen section the pale cells were found to contain ample sudanophilic material . 
of the target organs of the adenohypophysis only the breast glands were found to be stimulated and of the known pituitary hormones only prolactin can cause secretion in the breasts of ovariectomised adult rats ( desclin , 1952 )  . the functional activity of the pituitary tumours of the rat is at variance with that of the acidophilic adenomata of human pathology . 
two types of acidophils can be easily distinguished in the pituitaries of several species as for instance the rabbit ( friedgood and dawson , 1938 )  . here cells with a strong affinity for carmine are found besides others which do not retain this dye . 
the former increase in numbers after coitus , towards the end of pregnancy and during the first days of lactation . in man , romeis ( 1940 ) demonstrated the presence of one type of acidophils staining red , and of a second staining yerow in azan preparations , his a and e cefls . in addition he recognised a third variety of acidophils , the q or pregnancy cers of erdheiin the rat the histological methods with which the writer is familiar unfortunately do not allow differential staining of two types of acidophils . 
why in man the growth hormone producing cells undergo neoplastic changes and in rodents the prolactin secreting acidophils , i am unable to explain . the interrelation between pituitary tumours and secretory activity of the mammary glands has been studied by lacour ( 1950 ) in the rat . in a series of 37 oestrogen induced adenomata she found in 23 granulated as well as " chromophobic " cells with a hypertrophic golgi apparatus . 
whenever the granulated elements were present , the breast had the aspect of a " lactating " gland . used romeis ' kresazan technique which stained the granulated tumour cells according to lacour a few cells , having the same tinctorial qualities orange . occur in the pituitary of the normal female rat . 
they become more numerous early in pregnancy and increase considerably in numbers 3 days ante partum . from this date onwards until weaning these ora ' ngeophil acidophils outnumber the other type . 
the close correlation between the presence of orangeophilic cells in the normal and tumourous pituitary and lactation changes in the breast suggested to lacour that these elements were the sourc ' e of prolactin . the hterature on experimental and spontaneous pituitary adenomata in rodents has been recently reviewed by horning ( 1952 ) and by gardner ( 1953 )  . in contrast to the opinion of the writer , gardner considers the oestrogen induced pituitary tumours as chromophobic adenomata , a view still shared by most admittedly , degranulated elements are more numerous in many authorities . of these tumours than granulated forms , but can - one classify all cells without granula as chromophobes ? the difficulty lies in the morphological resemblance of degranulated acidophils to chromophobes . 
the oestrogen induced pituitary growths with functional activity cannot be chromophobic tumours because the acidophils and not the chromophobes are the source of lactogenic hormone . another point of controversy is the nature of these pituitary growths . 
they are considered by some authors to be true neoplasms and conditioned growths by others , because they have been seen to regress when stimulation ceased ( nelson , 1944 )  . 
the " spontaneous " tumour described in this paper did not differ histologically or functionary from the two oestrogen mduced growths . this adenoma certainly was not dependant on oestrogen since it showed no sign of regression 51 months after ovariectomy . 
one occurred in an ovariectomised rat , the second in an intact female joined in parabiosis to a spayed partner and the third in a female treated with stilboestrol , in which , however , the histology of the vagina indicated a very low oestrogen level at the end of the experiment . the pituitary growths were found in animals having secreting mammary 160 f . 
pullinger. from the imperial cancer research fund laboratories and the research department , the glasgow royal cancer hospital , glasgow , c.3. received foi publication february 7 , 1952 . the suggestion was previously made that nodular adenomatous hyperplasia in mammary glands of mice of the riii ( paris ) strain , and possibly others , might provide a more sensitive and an earlier indication of the action of bittner 's milk agent than do actual tumours . evidence obtained under experimental conditions of forced activation of the glandular response to combined influence of hormone and agent was givren in support of this suggestion . it seemed probable also that spontaneously occurring nodules as well as those forcibly stimulated would afford reliable evidence of the action of the agent . 
the validity of such evidence would depend upon complete absence of spontaneous adenomatous nodules in the same strain deprived of agent . in sublines of the riiix strain ( renamed riib in accordance with international usage ) , which had been freed from agent by cross - suckling , no nodules had been found in the mammae of young unmated females forcibly stimulated , but two hyperplastic nodules were seen in 2 out of 44 females of the old breeding stock ( pullinger , 1947 )  . 
a survey of the mammae of all old females was in progress and was continued and combined with experiment in an endeavour to find an answer . the same problem , the stimulus for hyperplastic nodules in the zb strain ( c3h cross - suckled to deprive it of milk influence ) , was investigated by huseby and bittner ( 1946 ) , who came to the conclusion that all three factors , namely the milk tumour influence , ovarian hormone and susceptibiltiy that are responsible for the development of heritable mammary tumours in mice are also necessary for the development of these alveolar hyperplasias . 
the same authors gave an account of the morphology of nodules found in the mammae of mice in former high cancer strains and their hybrids after deliberate exclusion of the agent by cross - suckling . the strains examined were c3h and a , designated zb and ax . they compared the nodules with those in high cancer strains described by previous authors before knowledge of the milk agent existed . in the course of the present survey of old rilib ( formerly riiix ) breeders similar types of nodule were seen with the exception focal acinar proliferations were often but of those described as inflammatory . the squamous - celled nodules found in old not invariably discretely lobular . riiib females have been described and their origins discussed ( pullinger , 1949 )  . in relation to the problem of the presence and action of the milk agent these squamous - celled proliferations are not relevant , for it is generally agreed that tumours due to bittner 's agent are characteristically acinar formations and that b . 
the actual average number of litters was 4 to 5 . breeders were then segregated to allow the mammae to involute , thereby permitting detection of nodular hyperplasia of various sorts . under these conditions involution is completed in this straleaving main and a few branch ducts with tapered ends and no acinar structures . 
hybrids of less than i month old . extracts were made by grinding the tumours with sterile sand and distilled water , spinning at 2000 r.p.in a hearson centrifuge for 10 minutes , removing the supernatant fluid and spinning this in an ecco centrifuge at approximately 8000 g . 
the mammae of as many as possible of the rijib mice injected with test tumours were examined in bulk - stained preparations . will be seen from tables ii and iii that the f.i. 
pullinger dilute brown strain and their hybrids which bore more than 3 litters had a higher incidence of mammary tumours than those with less . the parous riiib females attained this average with 4 to 5 litters . of mice injected with riii tumour extracts 13 out of 23 developed mammary tumours . the mammary glands of all 7 which were examined , out of the remaining 10 , contained multiple adenomatous nodules , one as many as 30 . 
thus nodule incidence was increased in the riiib test mice by injection with riii tumour extracts . nodule incidence was not increased in the rilib mice which had been inoculated with extracts of the 2 tumours undergoing test for the agent . 
of 20 mice in the two experiments which were available for examination , the mammae of 3 contained acinar nodules ( 15 per cent ) , 8 contained nodules of other types , and 11 ( 55 per cent ) were free of all nodules . these figures are near the normal incidence . nodules in hvbrids are not recorded because there was no basis for comparison . thus neither from tumour incidence nor from nodule incidence was there any evidence of the presence of bittner 's milk agent in the 2 test tumours . the validity of results of tests in which grafts are used as suspected source of agent rather than primary tumours may be questioned . 
the majority of authors have been able to detect the milk agent in extracts of grafts derived from tumours which were known to have contained it in the first instance . jmochowski ( 1949 ) , who reviewed the various reports , has himself obtained evidence of the presence of agent after 42 serial transplantations . 
on that occasion he used multiple injections , but both he and others have succeeded with single injections of graft extracts . in a total of 69 test mice in the two experiments here recorded , not one tumour arose . these results are in accord with those of other workers who have tested extracts of primary spontaneous mammary adenocarcinomas in othier agent - free strains ( andervont and dunn , 1950 ; heston , deringer , dunn and levillian , 1950 ; dmochowski , 1951 )  . 
 ( personal communication ) were typical adenocarcinomas . assessment of the value of adenomatous nodules as indicators of the action of the milk agent . a comparison of the spontaneous incidence of adenomatous nodules in various groups of riii and ritib mice is given in table iv . it will be seen that among virgin females of the riii strain multiple nodules were present in 100 per cent at 9 months old . 
the relative reduction in number of nodules at 13 months after ovariectomy at 9 months is probably due in part to the difficulty of making accurate counts when the effect of ovarian hormone is still to be seen . among young virgin females of the riiib strain deprived of milk agent no adenomatous or other mammary nodules were found . 
pullinger when these nodules are present in mammae of young females of this strain of less than 12 months old , whether virgin or parous , they appear to provide reliable evidence of the action of the agent . 
this conclusion is more certain if oestrogen has been excluded by ovariectomy several months prior to examination of the mammae . breeders of the riiib strain deprived of agent , of 12 months of age or more , developed 1 to 3 adenomatous nodules in the 10 nipple regions . since these nodules were not associated with agent in the tests recorded , it is clear that they do not invariably provide evidence of the presence of agent in old breeders . they may be due to some other unknown cause . multiple adenomatous nodules in old breeders of this substrain would nevertheless provide presumptive evidence , requiring confirmation , of the presence of the agent . thus for purposes of detection of agent by inoculating suspected substances into susceptible test mice and using the adenomatous nodule as evidence of the action of agent , only young rijib virgins or breeders under 12 months old would be suitable as test animals . that is to say , they would have to be killed and examined at about 9 to 11 months old when positive results could be expected and before the nodules of unknown origin begin to appear . only if counts of nodules were required would ovariectomy have to be done because the naturally occurring hormone under normal conditions does not cause a confusing degree of focal acinar proliferation . 
nodules of purely adenomatous hyperplasia were found in about 19 per cent of parous females of 1 year old and over of the rijib strain deprived of bittner 's milk agent . 
no evidence was found that the nodules or tumours in the mammae of old parous females of the cross - suckled riiib strain owed their persistence to ovarian hormone ; neither could their origin or persistence be attributed to the milk agent . 
ludford , director of the research department , and to the board of management of the mount vernon hospital , northwood , continuity of observation was maintained to the end of these experiments . to the generosity of p . 
malowan ( 1932 ) - also found blood changes of the same low order , though becher and herrmann ( 1932 ) stated that they were unable to find any significant change that could be attributed to the presence of the tumour . all the above workers used danielson 's modification of folin 's well - known colorimetric method for blood amino acids . 
2. these observations show that the growth of the transplantable carcinoma ' m 2146 is accompanied by an elevation in the blood ac - amino nitrogen . while the mean value in normal mice was 4 76 mg . 
the corresponding regression equation between these variables is : oc - amino nitrogen values = 0 - 1213 tumour percentage size + 5 - 63 . these changes in the level of the blood ac - amino nitrogen probably account for the increase in the blood non - protein nitrogen in tumour - bearing animals found by other authors ( greenstein , 1947 )  . blood ac - amino nitrogen in mice with regressing tumours . in about 10 per cent of the mice used in these experiments the tumour , after having grown and reached a definite size , regressed completely . it seemed of interest to estimate the blood amino acid concentrations in these animals . 
the animals consumed this food freely for the first two days , but seemed to lose appetite during the remainder of the experimental five - day period . the loss of weight , which had become severe by the fifth day , together with the blood ac - amino nitrogen values , are shown in table iv . with the introduction of the deficient diet there was a marked loss of weight in both groups of mice ; the tumours , however , continued to increase in size even at the expense of the host , which was clearly in negative nitrogen balance . 
per cent throughout most of the experiment seems to show that this represents the basal level for this group of substances , below which further in the tumour - bearing group , on the other hand , the concentrafall is resisted . tion of the ac - amino nitrogen fell rapidly to between 4 and 5 mg . 
the similarity of the fall to that found for the control group would seem to show that whatever metabolic change was responsible for the characteristically elevated blood oc - amino nitrogen level was independent of the state of alimentation , 100 m . 
there is little evidence that the free ac - amino acids are increased in tumorous animals , but observations on rats with a transplantable hepatoma have shown that the second fraction may on average be raised seven or eight fold ( winzler and burk , 1944 )  . 
the elevation continues almost unabated when the animals are placed on a diet wholly deficient in protein . it seems , therefore , that the increase in oc - amino nitrogen must be attributable to one or other of the following factors : a local increase in the production of amino acids or of polypeptides by the tumour itself , either through the degeneration of its cells or the hydrolysis of normal body proteins brought to it by the circulation ; or to some hepatic dysfunction that is present in tumour - bearing animals which interferes with the normal disposal of these substances , and thus permits them to accumulate in the blood . this latter possibility will be considered first . fujiwara ( 1929 ) , weil ( 1935 ) , greenstein , jenrette , mider and white ( 1941 ) , and el mehairy ( 1949 ) found little inhibition of liver arginase in tumorous animals . greenstein ( 1947 ) also noted no dimunition in cystine , desulphurase activity in the same organ . arginase and cystine desulphurase are two of the main liver enzymes concerned with the intermediary metabolism of amino acids , and the study of their behaviour provides valuable information upon disturbances in the hepatic enzyme pattern generally . robertson and kahler ( 1942 ) and lan ( 1944 ) have also failed to observe any decrease in the enzymic activity of livers in animals bearing tumours . 
tumours of less than 5 per cent of the total body weight were not accompanied by any significant change in the oc - amino nitrogen concentration in the blood . the tumour tissue itself is believed to be the source of extra a - amino nitrogen in the blood . i wish to record my indebtedness to professor g . 
kirby , research department , glasgow royal cancer hospital . received for publication march 4 , 1948 . althoulgh the claims of roffo ( 1938 , 1939a , 1939b , 1941 ) to have induced adenocarcinoma of the stomach in rats given orally fats or cholesterol which had been heated to 3500 c . 
for half - an - hour have not been confirmed ( beck and peacock , 1941 ; kirby , 1943 , 1944 , 1945 ; morris , larsen and lippincott , 1943 ) , the positive results obtained by other routes in mice by other workers make a good case for regarding the products of such pyrolyses as carcinogenic . thus beck ( 1941 ) found sarcomas in 2 of 12 mice at the site of injection of cottonseed oil , previously heated to 340 - 360 c . 
for 1 hour , and steiner , steele and koch ( 1943 ) reported sarcomas in 3 of 9 mice injected with sesame oil heated to 3500 c . these latter workers found no tumours in similar mice injected with cholesterol that had been heated to 2000 c . 
for half - an - hour , and another sarcoma in 1 of 9 mice surviving more than 340 days after subcutaneous injection with a residue left after removing dicholesteryl ether and a4cholestenone from cholesterol heated in the same way ; these tumours were closely associated with the sites of injection . negative results were obtained by these workers when mice were injected subcutaneously with cholesterol that had been heated to 4300 c . 
of the remaining 14 , 5 died or were killed between 400 and 500 days , 1 was killed at 533 days , 1 died and 2 were killed between 500 and 600 days and 3 were killed at 735 days . bronchiectatic abscesses were found in most animals surviving 400 days ; there was no other frequent lesion . 
the stomachs were all normal , save for that of a rat dying after 616 days which had the haemorrhagic erosions of the glandular zone commonly found in rats in a state of inanition . 
shortly after the paintings had been commenced , 0 5 per cent of croton oil was added to the benzene solution in the hope of speeding up or enhancing the action of any carcinogen present in the c.o.h. after 258 days ' experimentation , a 15 per cent benzene solution of c.o.h , containing 0 5 per cent croton oil , replaced the original 10 per cent solution ; 2 males and 5 females survived long enough to be treated with the stronger three other male mice were painted with the 10 per cent - solution solution . for 168 days and then with the 15 per cent solution . atrophic changes at the site of painting were seen in mice dying at 80 days after the beginning of the experiment , but even in 2 females painted 258 days with the 10 per cent solution and then 374 days with the 15 per cent solution , and surviving totals of 640 and 656 days respectively , no further lesion was found . fourteen mice showed fatty changes in the liver , sometimes with extensive necrosis , but no hyperplastic changes were seen in this organ . 
the first mouse to die survived 80 days after the first injection ; 2 others died before 100 days , 10 survived 100 to 200 days , 2 from 200 to 300 days , 2 from 300 to 400 days , 5 from 400 to 500 days , and 1 survived 518 days . a mild histiocytic reaction was seen around sites of injection , but no sign of fatty changes in the liver were rare , but in mice any tumour was ever found . dying at 126 , 130 and 384 days after the first injection there were leukemic changes involving the liver and spleen in the first , the liver in the second , and the liver , spleen , kidney and lungs in the third mouse . ( c ) cholesteryl linoleate ( heated ) in mice . linoleic acid was prepared from cottonseed oil ( organic syntheses , 1942 ) and converted to the acid chloride by refluxing with oxalyl chloride . purified cholesterol was heated with linoleyl chloride on the water - bath for 3 hours , and purified as for the oleate . 
in the left flank , 31 days after the injection in the other flank . eight mice survived 100 to 200 days after the first injection , 1 for 247 days , 1 for 357 days , 2 for 470 days , 1 for 509 days , 1 for 558 days and 1 for 653 days . at the site of injection nothing beyond a mild histiocytic reaction was found in any mouse . 
and the time was 2 hours . as the esters were prepared by a non - pyrolytic method and were subsequently purified till non - fluorescent , any polycyclic hydrocarbon present in the residue after heating would have arisen as a direct result of the heating . 
no chemical experiments have been made to determine the nature of the components of the but the biological tests reported here gave no evidence of carcinoresidue . genicity in these residues for the skin or subcutaneous tissues of stock mice . such tumours as did arise in the mice used were almost certainly spontaneous . damage to the liver and the spleen was frequent , and the occurrence of leukemic infiltration was significant and probably attributable to the injections . it now seems that cholesterol and its naturally occurring esters may not be an important source of carcinogens in heated food . 
the results of the experiments described in this paper lend no support to the results reported by beck , kirby and peacock ( 1945 ) , who administered cholesterol heated to 3000 c . 
for the same period , as recorded by those authors , also of the negative findings of steiner , steele and koch ( 1943 ) with cholesterol heated to 2000 and to 300 c . 
it is concluded from this work and from other reports in the literature that cholesterol and its esters heated to reasonable dfooking temperatures for a reasonable cooking period have not been proved to yield carcinogenic residues . the author is indebted to p . 
i. - cancer of the lung and of the larynx . males , england and wales , 1921 - 1951 . ascribed to lung cancer , for cancer of the larynx there has been little change over the past twenty years . the preceding papers in this series ( kennaway and kennaway , 1951 ; kennaway and waller , 1953 ) compared the incidence during the period 1946 - 1949 . 
kennaway cancer of the lung and larynx as shown by death certificates in four classes of area ( london , county boroughs , other urban ' districts , rural districts ) in england and wales . 
for a particular sub - division of the country expresses the mortality in that area as a percentage of that for the whole of england and wales , allowance being made for any differences in the age - distribution . 
given as an indication of the relative importance of the two causes of death ; these measures of mortality will not be used in this analysis . the most striking feature of table 11 is the behaviour of the figures for the female larynx as previously described . whereas for cancer of the lung the male and female figures show similar trends the corresponding figures for the larynx show no such similarity . 
the next step is to see whether the geographical or the urbanization factor is the more important ; this can be done by a study of table iv and fig . 
3 and table iv that urbanization is the predominating factor : in each region , the mortality due to lung cancer increases from the rural to the urban areas and although the same is true of the male larynx , when we look at the figures foi the female larynx we find the exact contrary ; in every case the highest mortahty is in the fural districts . 
the comparison is particularly striking when we look at the s.m.r.s for the county of london ( the most densely populat - ed of all our areas ) and then at those for the sparsely populated rural districts inv.i ' ales ancl the north . for the purposes of this comparison the differences between the c.m.r. 
we cannot say whether the extremely high figures for the female larynx in wales ( where the population at risk is in any case small ) are more than an extreme manifestation of the reverse urbanization gradient or whether they m , ight be due , for instance , to some ethnographical factor . further study of eancer of the lung . another feature that emerges from the diagram is , that for cancer of the lung the urbanization gradient is consistently steeper in the men thad . 
of 160 for the county of london . it would be interesting to make a similar analysis with the urban districts ( or better with the county boroughs and urban districts combined ) but the labour would be prohibitive . it may be mentioned in this connection that since 1950 the registrar general has adopted a more realistic classification , which wfll considerably simphfy the study of many problems . 
with its large dormitory areas , the male s.m.r.s for cancer of the lung are all higher than in the sparsely populated s.w. , wales ii and north il in females the absence of such a clear association is partly a reflexion of the lesser gradient already noticed for women and partly due to the larger sampling errors to which these s.m.r.s are subject ; on the other hand the remarkably low female s.m.r. 
a possible explanation is that there are two aetiological factors at work , the one positively correlated with urbanization ( a ) and the other negatively ( b )  . 
the mortahty ratio for the larynx ranged from 60 for males of class i to 143 for males of class v , but it is significant that married wo ' men showed no significant social class gradient , thus suggesting that the factors responsible for the excess of cancee of this site amongst unskflled males are directly associated with occupation . " on the other hand there was in 1931 no marked social gradient for cancer of the lung . what connection is there between the occupational factor and urbanization ? we cannot answef that question in the absence of mortality rates analysed by urbanization simultaneously with occupation . 
if the figures in table viii are representative , they may be said to show ( a ) cancer of the larynx is four times as common in men as in women ; in men , intrinsic cancers are nearly twice as common as extrinsic , whereas in women extrinsic are four times as common as intrinsic ; as a consequence of ( a ) and ( b ) , intrinsic cancers appear nearly thirteen times as often in men as in women * and the extrinsic twice as often in men as in women . the conventional inclusion of cancers of the retro - cricoid region among laryngeal tumours is very unfortunate . " the term ( cancer of the larynx ) should be restricted to the so - called ' intrinsic ' group of cancers . 
kennaway mouth , tongue , and pharynx , angular stomatitis , hypochromic anaemia , achlorhydria , splenomegaly , and spoon nails , in which carcinoma ofthe post - cricoid region this condition was first described independedtly by may develop in later years . paterson from cardiff ( 1919 ) and by brown kery from the westem infirmary , glasgow ( 1919 ) and has become know - n by the names of two latee investigators as the " plummer - vinson " syndrome . neitherpaterson ( 1919 , 1937 ) nor brown kelly ( 1919 , 1931 ) suggest , either in their earher or later papers , that this affectioin . is especiafly common in their own districts , though any such frequency would have this syndrome is of qu ' ite especial interest in cancer research assisted recognition . because the features present during the earlier years may indicate a precancerous metabolic state , which may be treated succes - sfury by improved feeding and administration of iron ( see for instance hare ' n , 1938 ; waldenstrom , 1938 , and elkelesg 1942 )  . 
the forowing section represents an attempt to collect the literature on the paterson - kery syndrome in relation to cancer ; no attempt has been made to introduce any uniformity into the anatomical classificatioin adopted by the various authors . ahlbom ( 1937 ) records , from the radiumhemmet , stockholm ( 1931 - 1936 ) , the incidence of carcinoma of the post - cricoid region . ( a ) cancers of the hypopharynx are more frequent in women , in whom also a mucb higher proportion ( 90 per cent ) of these tumours affect the post - cricoid area , than in men . 
was in der diiit der drmeren schwedischen bevblkerung fehlt , sind vor allem eisenhaltige vegetabilien , obst und fleisch . " bingham and logan ( 1953 ) say " the incidence of hypochromic anaemia in northern ireland is considerable and . 
these trends apply equally when the figures are analysed according to the separate regions , but there are differences between the regions , which may or may not be due to differences in degrees of urbanization undetected by the classiin particular the s.m.r.s for wales are very low for the fication we have used . lung and male larynx but very high for the female larynx . 
the literature upon the relation of the paterson - kelly syndrome to cancer , and upon possible dietetic and social factors in this condition , is summarized . we are greatly indebted to w . 
calcutt. from the department of cancer research , mount vernon hospital and the radium institute , northwood , middlesex . received for publication may 3 , 1952 . the staining of living tissues , either in vitro or in the form of freshly excised material , is a well recognised technique . normally it is restricted to the identification of specific morphologic entities such as mitochondria . attempts to determine intracellular biochemical features have been rather unsuccessful as far as living material is concerned , although successes have been achieved with fixed specimens . 
the availability of tetrazolium compounds now has rendered possible further attempts at the identification of intracellular sites of reduction . three compounds are generally available . in their normal state they form colourless solutions , but in the presence of suitable reducing groups they are converted to coloured formazans . 
orr. from the department of experimental pathology and cancer research , university of leeds . received for publication july 19 , 1947 . attention was drawn to the frequency of spontaneous tumours in the lungs of mice by tyzzer ( 1907 - 8 , 1909 ) , whose description of their histology has required but little amendment . 
a small group of stock mice was treated intranasally with 1 : 2 : 5 : 6 - dibenzanthracene in almond oil , but owing to intercurrent disease these had all died before adenomata were present in any except one . 
the present communication is based on the histological investigation of the lungs of a representative sample of these mice , together with the urethanetreated mice of the previous paper ( orr , 1947 ) , and a number of mice showing spontaneous pulmonary adenomata . 
the outline of the whole tumour is usually more or less circumscribed , though there is no definite capsule ; sometimes it is irregular , but the appearance is not that of true infiltration . 
the two types of tumour have also been noted by grady and stewart ( 1940 )  . occasionally , both types of structure have been seen in the same nodule , and there appear to be adequate grounds for regarding them as morphological variants of an identical process . it is not uncommon to find alveolar macrophages of the " dust cell " type in the adenomata . 
they are often so heavily loaded with carbon as to obscure all nuclear and cytoplasmic detail . less often other evidence of inflammation in the form of lymphocytic or leucocytic infiltration is seen . but in addition a very large number of lesions show appearances which make the impression inescapable that they are stages in the development of adenomata within inflammatory foci . this evidence is more striking where the tubulo - papillary structure is being evolved , in view of its highly characteristic and easily recognized pattern . 
the inflammatory changes resulting from urethane are relatively chronic , and take a considerable time to clear up on withdrawal of treatment . moreover , the proportionate share played by polymorphs in the reaction even in its early stages seemed smaller than average : it may be that urethane exerts some depressant effect on them , and thus necessitates greater activity by the so - called fixed tissues . the source of the epithelium in the tumours has not been determined with certainty . indeed , in many instances even the simple statement that it is epithelium involves a measure of assumption ; the most that can be said with 320 j . 
the actual existence in the fully developed lung of such a tissue is denied by many authorities , and some of those who claim to find it describe morphological appearances very different from those found in the tumour cells . if the alveolar epithelium is implicated , it can only be by reversion to the embryonic type , and if this occurs , such epithelium might owe its genesis , as it does in the embryo , to the bronchial epithelium . it appears to the present author that some workers have been inclined to regard as alveolar epithelium any lepidic tissue in the lung which could not be . 
shown directly to be bronchial in origin . the present results are therefore in agreement with those of magnus ( 1939 ) in respect of the tissue of origin , without accepting his view regarding the intrabronchial papillomata . 
orr does not suggest that it is malignant ; the rate of growth is slow once the characbut there can be little doubt that a malignant teristic size has been attained . " variant " occasionally turns up . the role of antecedent inflammation . slye , holmes and wells ( 1914 ) thought that adenomata arise from areas of lung tissue in which inflammatory hyperplasia has occurred . grady and stewart ( 1940 ) consider that they are not associated with inflammation . 
the present author 's observations have convinced him that these tumours invariably originate in foci of chronic collapse inflammation , which become invaded by bronrchial epithelium , and that it is the extent of the original inflammatory focus which this would explain the paradox of its determines that of the formed adenoma . comparatively rapid appearance and slow subsequent growth . 
ludford. from the laboratories of the imperial cancer research fund , london . received for publication january 30 , 1948 . in a review devoted to the discussion of colchicine as a possible chemotherapeutic agent for cancer ( ludford , 1945 ) , attention was directed to the dual action of this drug on tumours . it is the most potent of mitotic poisons , and as . 
at the time the experiment to be described was performed , sections exhibited large compact groups of carcinoma cells , surrounded by an abundant and highly cellular stroma , composed of enlarged and hyperchromatinic spindle - shaped and polymorphic cells , presenting the typical cytological appearance of sarcoma cells . 
an aqueous solution was employed in a concentration of 1 part in 10 , 000 of distilled water . this had been prepared six days previously , when it was maintained at the temperature of boiling water for ten minutes to reduce the possibility of infection , and was stored in the dark for use throughout the exp6risince the activity of colchicine is reduced by high temperatures and by ment . keeping , especially when exposed to light , the solution employed for this experiment was rather less potent than a freshly prepared solution . 
1 by comparing the size of the tumours of the controls and the treated mice on the 21st day . control 71141 211301 37 treated 7 14 3 43 10 01 234 fig . 
2. - regression of a sarcoma ( c ) in strong a strain mice after treatment with colchicine . such after the shortest possible interval consistent with survival of the mice . treatment is invariably accompanied by a high mortality . an experiment with a spindle - celled sarcoma in strong a mice demonstrates the possibility of completely inhibiting malignant growth . this sarcoma was one which originated by the sarcomatous transformation of the stroma of a mice into which it is transplanted do not usually survive mammary carcinoma . long , as it rapidly penetrates the skin and leads to considerable superficial ulceraof six young adults transplanted with this sarcoma three were treated tion . 11 and 20 days after transplantation . 
age. - since actively growing tissues are most sensitive to mitotic poisoning , it is to be expected that young animals would be more susceptible than older ones ries ( 1939 ) reported that a 42 - days - old mouse to the toxic action of the drug . will survive more than 17 times the dose of colchicine which is lethal to one 10 days old . the maximum inhibition of malignant growth occurs with the highest tolerated therefore , to obtain the greatest arrest of tumour growth it is doses of the drug . necessary to employ animals at the age when they are most resistant to the the best results general toxic action and the largest doses can be administered . old mice are less able to tolerate have been obtained with young adult mice . thus , 11 strong a mice varying in age from 18 months to 2 years the drug . and 12 young adults were each injected with 0 , 056 mg . 
3. - action of colchicine upon sarcoma 37 grown in strong a and c 57 strain mice . tumours in c 57 mice indicated by outline , tumours in a mice stippled . 
the size of the tumours in the c 57 mice is indicated it will be observed that in outline , and in the a mice by the stippled areas . actually there is little difference in the size of the tumours in the control animals . growth is slightly more rapid in the a mice , but there is a definite difference in the two strains of treated mice . 
a similar acquirement of tolerance by tumours to the haemorrhage - inducing action of bacterial polysaccharides has been reported by shear ( 1944 )  . application of the preceding results for obtaining the maximum " colchicinic effect . " discrepancies in the reports on the action of colchicine on neoplasms which have been published are explicable on the basis of the preceding findings . 
of colchicine over a period of 4hours . two other tumours regressed completely , but the - , remaining two continued to grow progressively . both received the same treatment on the 37th day . one died with a large tumour on the 45th day . 
the five mice which had been completely " cured " were kept under observation for a year and were then killed . post - mortem examination revealed no trace of carcinomas . the charting of all the tumours in these experiments was carried out independently by a . 
chapman , the senior technician in charge of this work . discusston9 with all the tumours which underwent regression in the preceding experiments there was extensive haemorrhage following treatment . this ' was most readily induced in the a strain mice . 
a few hours after the injection of the maximum sub - lethal doses of the drug the tumours appeared blue through the skin . subsequently superficial ulceration was of common occurrence . since boyland and boyland ( 1937 ) first drew attention to the similarity of action of colchicine and bacterial filtrates they have been shown to behave alike in most other respects . 
with both , complete regression of some transplantable and spontaneous tumours has been effected . ' the results acquire significance in that they indicate the limitation of haemorrhage - inducing agents in the treatment of malignant growths . that some form of damage to the capillary system of tumours might be ' the best way to inhibit malignant growth was suggested by woglom ( 1922 )  . 
a critical review of the evidence concerning tumour regression led him to raise the question whether " the receding tumor may not differ from the growing one only in the extent to which its blood vessels have been obliterated by thrombosis . " he pointed out that the blood vessels of many tumours are more subject to thrombosis than those of normal tissues , and he conceived the possibility of both chemical and mechanical factors being responsible . 
the former resulted from the abnormal metabolism of malignant cells , and the latter was described as " the mechanical pressure which the rapidly increasing parenchyma must often exert upon its vessels , with all the possibilities of injury to their walls which this would entail . " more recently algire ( 1945 ) has devised an adaptation of the transparent chamber technique which enables microscopic observations to be made of the development of the blood vessels of tumour transplants . 
he reports that although many vessels in growing tumours become very large , " differentiation in mammary cancers he observed into arterioles and venules was not evident . " the development of " blood - filled cul - de - sacs . " " these appeared to result from r . 
cravetz. from the lawall memorial laboratory of pharmacology and biochemi8try , philadelphia college of pharmacy and science , philadelphia , penn8ylvania . received for publication june 15 , 1951 . caspersson and his associates advanced a hypothesis on the ' role of nucleic acids in the biological synthesis of proteins ( caspersson , 1947 )  . several other results suggested the role of nucleotides in the metabolism of tumors as well , as a result of which many structural analogues of pyrimidine and purine derivatives were synthesized and tested for anti - cancer and growth - inhibiting activity ( roblin , lampen , english , cole and vaughan , 1945 ; kidder and dewey , 1949 ; kidder , dewey , parks and woodside , 1949 ; sugiura , hitchings , cavalieri and stock , 1950 ; burchenal , bendich , brown , clian , hitchings , rhoads and stock , 1949 ; skipper , bennet , edwards , bryan , hutchison , chapman and bell , 1950 ; law , 1950 ; lewis and crossley , 1950 . ) however , little is known about the effect of the naturally occurring nucleotides on tumor growth . parsons , gulland and barker ( 1946 , 1947 ) described that in c57 mice the growth of homologous methylcholanthrene sarcoma grafts was inhibited by ( yeast ) ad ' enylic acid or guanylic acid ; uridyhc acid showed a growth - promoting effect , whereas cytidylic acid had no effect . 
tumor cells are free in the ascitic ascitic fluid was harvested by fluid , usuar abdominal puncture under aseptic conditions , and the number of cells present in the ascitic fluid counted in a hemacytometer . 
cravetz the method used in this study offers the forowing advantages : ' ( a ) cancer cells of known number and equal virulence are inoculated into the control , as ( b ) upon intraperitoneal inoculation similar well as the experimental animals ; ascitic tumors are produced , while subcutaneous inoculation results in the development of solid tumors ; ( c ) in case of intraperitoneal inoculation the action . 
the error in thi 's method is that there is always some microscopic infiltration of sohd tissues in the peritoneal cavity ( klein , 1950 ; klein and klein 1951 )  . cells are , of course , not included in our cell counts . these infiltratin ' swiss mice , bred in our colony , were used for these studies . these mice were maintained o ' n rockland complete rat diet and water , ad libitum , and kept in air - conditioned quarte ' rs at 2 p c . 
the strain aspecificity of this tumor was discussed by klein ( 1950 ) and klein and klein ( 1951 )  . subcutaneous inoculations were twenty - one days after inoculation the made into the caudal third of the back . groups animals were sacrificed , and their tumors dissected and weighed . receiving intraperitoneal injections of cancer cehs were observed for mortailty animals dying were autopsied , their ascitic fluid harvested and measured , rate . and the number of cancer cehs per cubic mihimetre estabhshed as previously descr ' lbed . nucleotides were administered by daily subcutaneous injection , alternating adenosine - 3 - phosphoric acid was injected in between the right and left flank . solution in 3 - 2 per cent disodium phosphate , adenosine triphosphate and adenosine - 5 - phosphoric acid in normal saline warmed to about 37 ' c . 
mean tumor weight8 in gram8 . serim i ( 2 , 000 , 000 cells ) meem mean mean mean serim 11 ( 2 , 000 , 000 cells ) : serim ih ( 2 , 000 , 000 cells ) seriew .1 v ( 200 , 000 cells ) : adenosine - 3adenosine - 5adenosine phosphoric acid phosphoric acid triphosphate i 00 mg . 
the number of cancer cers , however , was smaher in the ascitic fluid of the nucleotide - treated mice than in the control animals . we are greatly indebted to margaret r . 
anderson. from the research department , glasgow royal cancer hospital . received for publication february 22 , 1949 . the need for controlling the influence of solvents used as vehicles for carcinogenic hydrocarbons was recognized by burrows , hieger and kennaway ( 1932 ) , who reported experiments designed to test the fat solvents then in use for this pui - pose , for possible carcinogenic action on the connective tissues of rats and subsequent work showed that in fact lard , which had been previously mice . heated to 140 ' c . , was a potential carcinogen for the connective tissues of fowls ( peacock , 1933 ) , and that lard alone could give rise to spindle - cell tumours in rats ( barry and cook , 1934 ) ; and later that lard , olive oil ' and other fatty materials could induce connective - tissue tumours , some of which appeared to be malignant in rats , but not in mice , in the experience of burrows , hieger and kennaway ( 1936 )  . the first instance of undoubted sarconia induction at the site of the injection of lard in fowls cited above was complicated by the fact that the same birds had been injected with a solution of 1 : 2 : 5 : 6 - dibenzanthracene in lard in the right breast and with lard alone in the left breast . 
of incidentally these tumours in a susceptible bird cannot be excluded . experiments suggest that the quantity of dibenzanthracene actually required to initiate sarcomatous growth must be remarkably small , since the bulk of the the injection appears to remain encapsulated throughout the expei - inient " ( peacock , 1933 )  . 
the latter assumption subsequently proved to be wrong . to test this point , chalmers ( 1934 ) analysed the contents of encapsulated niatter from the site of dibenzanthracene lard injection in one of peacock 's sarcoiiia - bearing fowls , which died 7 months after the last injectioii . 
dibenzanmoreover , in this group no tissue reaction thracene in 0 - 4 per cent solution . could be found at the site of injection after a few months , indicating that the homologous fat had caused httle disturbance . however , with egg yolk fat as vehicle for dibenzanthmmne m 0 - 4 per cent solution , 2 metastasising sarcomas occurred among 4 birds that s ' urvived 9 months or more after injection . birds inject - ed with yolkfat showed fibrous cysts at the site of injection , sinular t - 0 those observed in the lard experiment , and in contrast with the absence of such local reaction in birds injected with chicken - fat . there seemed , therefore , to be some aetiological connection bet - w - een the " foreignness " of the solvent used as vehicle and the local tissue reaction and the ultimate carcinogenic response t - o dibenzantli - racene . owing to the costlv and time - consuming nature of long - term experinients on fowls work was begun on similar lines on mice . while these experiments were in progress berenblum ( 1938 ) reported the co - carcinogenic action of croton oil . he had previouslv shown ( 1929 ) that mustard gas in dilute solution and ( 1935 ) cantharidin had anticarcinogenic properties for the skin of mice , and in the interim had found that no simple relationship between irritant and coor anticarcinogenic action was demonstrable . 
olive oil , tended to retain the dissolved benzp - % - rene as judged by the persistence of violet fluorescence , where - as in the case of those that were rapidlv absorbed , e.g. 
mouse fat or ether , fluorescence could not , be detected after a few weeks ( peacock and beck , 1938 )  . moreover , in the fornier group the incidence of sarcomas was high , and in the latter group it was low . this suggested that there mav be an optimal period for the retention of a carcinogen at the site of injection for the induction of a tumour , and that in mice this period was about a months . dickens and weil - malherbe ( 1942 ) at first confirmed our observation with mouse fat ' , but later ( 1946a ) , as a result of intensive experiments , found that the effect was not constant and was related to the phospho - lipid content of the 298 p . 
the mice were killed after different periods ( table 1 ) , the subcutaneous tissues exposed and examined for ultra - violet sixteen of 20 mice killed at various times over a period of 7 months fluorescence . had fluorescent material located at the original site of injection ; ulceration and sepsis at the site of injection killed 4 mice early in the experiment . in 3 of these mice no sign of the injected material was found , probably due to sloughing and leakage from the site of injection . 
the fourth had a small spot of fluorescence . these mice were not examined further ; in the remainder the fluorescent material was extracted with benzene , with the exception of 2 mice , which died after 412 - and 51 months , both showing persistent fluorescence and tumours at the site of injection . after the third month of experiinent tumours began to be clinically recognized at the sites of injection . as most of these tumours were similar to many such induced sarcomas in mice , not a 11 were histologically examined . 
one tumour in the abdominal cavity associated with intraperitoneal deposits of fluorescent material was probably induced by benzpyrene injected accidentally through the abdominal w , - , 11 . another tumour occurred in an ulcerated area at the site of injection . this proved to be a squamous carcinoma . 
benzene , and the benzene extract dried over anhydrous sodium sulphate . since the main object of these experiments was to demonstrate the pre - sence vne after various periods , the experiments were not conor absence of benz ducted quantitatively . however , the limit of sensitivity of the method of detection provides some indication of the quantities involved ( vi& infra )  . 
to eliminate the possibihty of a false positive reswt due to traces of benzpvrene adhering to the apparatus used in the experiments , benzene washings of each piece of chemical apparatus or other instrument used , were examined under the ultraviolet lamp imniediately before its use . attention was drawn to the necessity for such precautions ri_ - , cently ( anderson , 1947a )  . 
owing t - o the difficulty of eliminating minute traces of material from apparatus , such traces may be recorded by the fluorescence method of detection , and lead to a false positi ' ve result . 
the absenee of benzpyrene from extracts of two tumour - bearing mice killed - 4 months after a siiigle injection of benzpyrene does not preclude the possibilitv that the uneliaiiored hvdrocarbon was still present at the inception of the tuiiiour process as it was in tfie other tumour - bearing animals killed after shorter periodss . thus the results of this experiment accord well  . , a - ith otir previous observations ( peacock and beck , 1938 ) that when fluorescence remained at the site of injectioii in iiiiee for 5 months or more there was a hiah incidence of sarcoma . 
on this basis dickens ( 1947 ) states that " the surprising result was obtained that the more rapid elimination of benzpyrene was associated witli the higher carcinogenic activity , and slower elimination mith lower acti - 6ty . " fact the graphs published bv dickens and weil - ' - nlalherbe ( 1946b ) show . 
in the case of tricaprylin alone , and of tricaprylin plus phospholipids as solvents for benzpyrene , a consistent rate of elimination of the hvdrocarbon from the sit ' e of injection , but not to the point of extinction within s5 davs and 10 - 10 davs extrapolation of the tricaprvlin graph would in&ate total etim " lrespectively . nation in the region of 26 weeks . as w out of 26 mice had tumours bv the 20th week , it seems reasonable to , suppose , that some benzpyrene was present in these mice throughout the latent period of careinogenesis . this would correspond closely with our own experience . in the graph illustrating the course of events following injection of benzpyrene dissolved in tricaprylin plus 3 per cent choleste , rol , dickens and weil - malherbe ( 1946b ) show a considerable variation in the persistence of benzpyrene in different mice . 
as no t - umours were recorded before about the 19th week of their experiment , there is no positive evidence about the persistence or absence of benzpyrene at the site of injeetion in their tumourbearing mice . ou - r own repetition of their experiment . 
anderson mice , and must therefore have been present throughout the latent period of carcinogenesis . while it seems certain that partition of benzpyrene between the solvent used - is vehicle and the body fluids must be largely responsible for the rate of elimination of benzpyrene , the local tissue reaction to different solvents may also help to determine the local response . 
they established that for benzpyrene the partition coefficient between human sera and various lipoid solvents was directly related to the carcinogenic response of mice injected with benzpyrene dissolved in those solvents that readily parted with benzpyrene in vitro gave fewer solvents . ttimours when used as vehicles for injection of benzpyrene in mice . their conclusions support the conception that there is a significant relationship between the retention at the site of injection of benzpyrene and the ultimate carcinogenic response . whatever may be the essential mechanism of conversion of a normal cell to a malignant one , it seems certain that some local consumption of energy by some part of the cell must be involved in the transformation . it may well be that the liberation of energy at the optimum time in the life - cycle of a stisceptible cell is the essential carcinogenic stimulus , and this may be provided by the metabolism of a carcinogen . in this event neither the parent hydrocarbon nor its metabolites , but the transition from one to the other , may provide the essential carcinogenic stimulus . 
as this finding was of great importance not only to the general problem , but more particularly as a possible test of the validity of conclusions which were being formulated , it was decided to confirm their results . initial casual observation by nettleship and henshaw was made on female mice of the c3h strain , which normally shows a low incidence of pulmonary tumours , in an experiment in which urethane had been used as the anaesthetic . 
the urethane group in the first experiment consisted of 100 mice , the other three groups of 60 mice each , and 60 untreated mice were observed over the same period . 
the mice were examined post mortem as they died during the course of the experiment , or when the survivors were killed 185 days urethane 12 per cent ; 312 j . 
the second experiment was made on 100 mice similarly treated with urethane ; no other substances were used , in view of the negative results of the first experiment . in this experiment at the end of treatment the mice were allowed to survive until they died , with a view to assessing the persistence and growth of the adenomata . unfortunately enteritis and cannibalism reduced the amount of histological material below what was hoped for in both experiments , but not to such an extent as to make it inadequate . 
when the nodule projects above the pleural surface , is smooth and uniformly rounded , and greyish - white in colour or semi - translucent , it can be regarded with some confidence as a formed adenoma , but only a proportion of the fiat nodules will prove to be adenomata . 
at a later stage the lungs showed induration from chronic inflammation , more often than not accompanied by acute consolidation of one or more lobes . it was at this stage that subpleural nodules began to appear in numbers , and the pale miliary spots on a deep red background often presented in the latest stages , after treatment had been discontinued , a striking picture . the lungs were free from consolidation , but showed distortion of shape as a result of emphysema . 
not only is it possible to trace the stages of development of a tumour in the chronic foci of inflammation and collapse , but the striking difference in the incidence of pneumonia with urethane as compared with the other anaesthetic substances used is highly suggestive . since this work was done , larsen ( 1946 ) has also shown that urethane is so far unique amongst narcotic compounds tested ; he used a larger selection of such substances than was employed here , but without finding any other which produced a significant increase in pulmonary adenomata . in their original communication nettleship and henshaw ( 1943 ) did not appear to be impressed with the evidence of inflammation . 
they found that conditions such as bronchiectasis , purulent bronchitis and acute pneumonia were distributed alike among the experimental and control animals . they are emphatic that lung tumours and pneumonia were never seen together , it is possible that this difference though they occurred apart from each other . of interpretation may depend on what is meant by the term pneumonia . 
orr. from the department of experimental pathology and cancer research , university of leeds . received for publication july 19 , 1947 . attention was drawn to the frequency of spontaneous tumours in the lungs of mice by tyzzer ( 1907 - 8 , 1909 ) , whose description of their histology has required but little amendment . 
bruzzone. from the department of experimental medicine , national health service of chile , santiago . received for publication june 9 , 1949 . abdominal fibroids induced in the guinea - pig by a - oestradiol ( nelson , 1937 ; lipschutz and iglesias , 1939 ) are prevented when progesterone or other 3 - ketosteroids are administered simultaneously with the oestrogen ( lipschutz and vargas , 1941 ; lipschutz , 1944 ; lipschutz , iglesias , bruzzone , fuenzalida and riesco , 1948 )  . 
 = fibrous tumoural effect ; units according to lipschutz and vargas ( 1939 ) t tablets of pure progesterone or part of a tablet . in the remaining groups - tablets from experiments of e . 
but these figures of active quantities of progesterone are not very distant from those obtained in our former work with the antifibromatogenic action of progesterone against o - oestradiol . there is also the fact that statements about absorption per day are stultified by its not being uniform throughout the experiment on account of the diminished surface , and by substances entering into the tablet from outside ; errors are 400 s . 
bruzzone this is why it would be daring to consequently considerable ( folley , 1944 )  . interpret the present results as indicative of a greater resistance of artificial oestrogens to the antifibromatogenic action of progesterone . 0o i1111j1 hexestrol hexestrol and progesterone fig . 
5. continued immersion in the agent gave rise to a more intense staining in this region , with the formation of dense , highly coloured figures . these are shown in fig . 
7 , and are strictly comparable with the golgi bodies of cells of the same tumour ( sarcoma 37s ) illustrated by ludford ( 1932 )  . it was found that the golgi body of cells of the two fast - growing tumours , i.e. , beta and 37s , was 262 g . 
the intense , and rapid staining may also be associated with lipoid constituents in the mitochondrial structure , since fatty substances within the cells very rapidly absorb and reduce the tetrazolium salts . consideration of the staining reactions of nuclei suggests that the nuclear membrane is in two distinct layers . 
the outer is a continuous hyaline sheath , whilst the inner is a structure of irregular thickness . it is this inner structure which so readily stains with the tetrazoliuthe appearance of the nuclear membrane under these conditions corresponds with that described by ludford and smiles ( 1950 )  . these authors ascribed the granular material on the inner side of the nuclear membrane to chromatthe present technique indicates by virtue of the differential staining intensities that it is chemically different from whatever material comprises the chromonemata , and more nearly resembles the nucleolar material in the distribution of reducing groups . 
the method does not , however , offer any indications as to exact chemical nature of the stained substrate . the falling off in staining intensity exhibited by the chromosomes as division proceeds is interesting in that tumour cells are known to be more sensitive to irradiation during the early stages of mitosis . it appears possible that this is associated with the greater number of reducing groups available at this stage , since radiation effects are known to include oxidation of such groups as - sh . since the evidence on this point can only be regarded as equivocal this issue will be the subject of further experiment . the uptake of tetrazolium by the golgi region of these cells is perhaps not surprising , since the classical techniques for demonstration of the golgi apparatus rely on the reduction of metallic compounds to insoluble derivatives . 
the very slow but continuous manner in which the staining takes place suggests that there is no great array of reducing groups available at any one time , but that as a result of metabolic processes these are steadily being rendered accessible . 
lucasanda.c.thackray. from the , department of pathology , the royal dental hospital of london school of dental surgery and the bland - sutton institute of pathology , the , middlesex hospital , london , w . 
the tumour occurs in the lower jaw much more frequently than in the upper ( i 7 cases and 3 cases respectively in this series ) , is usually a central lesion and gradually enlarges , in the majonty of cases without pain . in the course of time the bone becomes absorbed , and if a cystic loculus is opened infection and discharge forow . local recurrence iscommon fohowing inadequate treatment , but metastases are very rare . 
a case of pulmonary metastases has been reported by waterworth and purar ( 1948 ) , who review previous reports . the natural history of the disease is long ; it is not always easy therefore to assess the efficacy of various methods of treatment . most authorities consider radical excision of the tumour to be the treatment of choice . 
thus the least evolved type of tumour consists of strands of more or less undifferentiated epithehal cers growing in a fibrous stroma , while a tumour which has undergone a greater degree of entiation consists of folhclea 290 r . 
the epithelial follicles in such cases consist of an outer layer of columnar cells resembling the similarly situated cells of the enamel organ , while towards the centre the cells become stellate in appearance and cyst formation may occur . on the other hand , the trend of differentiation may be towards a more highly developed type of squamous epithelium , in which case the tumour consists of sheets of squamous cells showing a tendency to cornification , or a basal cell type of growth may be produced . these tumours may thus present a variety of appearances , from case to case , or in different areas of the same growth , and while attempts at classification on a histological basis are helpful in so far as they indicate the varying degrees of differentiation whicli can be observed , they are seldom entirely applicable to any given specimen . 
tboma ( 1950 ) has proposed one such classification , as follows , though he notes that pure types are but infrequently seen : ( 1 ) primitive type , composed of strands of epithelial cells showing little tendency to differentiation , growing in a loose fibrous stroma . 
a very rare type of tumour in which haemangiomatous characters are present . ( 8 ) melano - amelobla , 3toma . also a very rare tumour , in which the epithelial cells contain pigment . in our series 4 cases were of the primitive type , whilst the remainder were fig . 
2 ( case 5 ) shows an largely follicular or showed mixed appearances . example of the primitive type of tumour , cords of epithelial cells growing in a fibrous stroma . 
6 ( case 5 ) , in which the growth is composed of follicles or nests of epithelial cells with outer columnar layer and inner stellate area . microcyst formation , a common occurrence in adamantinoma , was present in typically , these cysts occur in the area of stellate cells towards the 14 cases . centres of the follicles , but they also form in the stroma . 
the occurrence of stromal cysts is a point to which very little previous consideration appears to siegmund ( 1929 ) noted their occurrence in 2 of his cases , but bave been given . apart from this instance , and a passing reference by kronfeld ( 1930 ) to the same fig . 
8 ( case 1 )  . in the latter case , however , it will be seen that though there is some breakdown within the follicles , the majority of the cysts have formed on the side of the columnar cell layer opposite to that on which fig . 
even in the 6 cases showing epithelial cysts only , evidence could be found in 3 cases of incipient degenerative changes in the stroma . the cause of cyst formation is a matter of conjecture . it is believed to be the result of progressive degeneration in originally solid growths . the solid tumour closely resembles the developing tooth up to the point at which differentiation of adjacent connective - tissue cells into odontoblasts , and subsequently amelogenesis , would occur in the case of the tooth , but at this stage the tumour cannot develop further . degeneration therefore takes place ( kronfeld , 1930 ; robinson , the ensuing accumulation of fluid of relatively high osmotic tension is 1937a )  . probably a factor in the progressive enlargement of the microcysts , a mechanism investigated by toller ( 1948 ) in the case of simple dental cysts , and quite possibly a factor also in the case of cyst formation in these tumours . with regard to cyst formation in the stroma , it appears that again a process of degeneration is at work , and all stages of breakdown can be seen from a slight loosening of the stroma to mucinous - like degeneration and the formation of completely clear cystic spaces . 
the inner enamel epithelium is that which lines the concavity of the enamel organ , a single layer of tan columnar cells which will produce enamel , while the outer enamel epithelium consists of cuboidal cells covering the convexity of the organ . 
on the other hand , bland - sutton ( 1922 ) thought that the enamel organ could not be the origin of these growths , since the majority of patients were well into adult life when their tumours first appeared . however , robinso ' n ( 1937b ) in his survey of 379 cases found that the tumour was first noticed between the tenth and thirty - fifth years in 70 per cent of cases . considered together with the fact that these tumours are very slow growmg and have a long natural history , it is evident that the majority of growths originate at quite an early age . robinson ( 1937b ) also found that the tumour occurred most frequently in the premolar and molar regions , where supernumerary tooth germs most often occur , kegel ( 1932 ) and and that many cases were associated with a missing tooth . geschickter ( 1935 ) also support the view that the tumour arises from the enamel byers and sarnat ( 1945 ) believe that the tumour originates from that organ . structure during the first few years of life , though active growth may not become manifest till later . if these tumours , or at least the majority of them , originated directly from the enamel organ , it might be expected that on occasion enamel would be formed . this is never the case , though thoma ( i 950 ) considers that the homogeneous zone sometimes to be seen between the basal membrane of the columnar cells in the follicular type of adamantinoma and the stroma , which appears yellow with van gieson 's stain , must be considered as abortive enamel formation , though it is possible that precystic changes in the stroma could account for such appearances . 
thackray these tumours resemble rodent ulcers in several respects . tumour without involvement of the oral epithelium , is , however , evidence against an origin from the oral mucosa . both are related to derivatives of the covering ectoderm , the one to teeth and the other to hair follicles , though discussion continues as to the stage of development of the appendage at which they arise . 
they are composed of relatively undifferentiated cells which can give rise to any of the appearances found in these tumours , and moreover will produce a central growth . it is not necessary to postulate tumour formation from these rests in early life , with a subsequent interval of dormancy to account for those cases in which the tumour first appears at a comparatively late age . being present from the formative phase of the dental tissues onwards they can proliferate at any time , and this seems a more satisfactory hypothesis than the dormancy theory . the rests of serre , those left by the dental lamina , are situated quite superficially . 
here the cells tend to take on a sterate appearance towarcls the centres of the cords and become elongated and columnar at the periphery . there is some cystic degeneration in the stellate areas and also in the stroma . case 4 : - 11 . 
1893. - 1910 : dentigerous cyst of one year 's duration 1928 : swelling had gradually increased in removed . size till there was now a hard tumour of the right mandible . this was excised and the cavity of a multilocular cyst exposed . 
radium tubes forced up through harcl palate . october , 1925 : no improvement . tubes left in 18 hours . december , 1925 : left side of face swollen over superior maxilla . severe paon left side of hard palate is an irregular swelling extending from mid - line to , and including alveolar margjanuary , 1926 : alveolar part of superior maxilla and the palatal tumour excised . 
1892. - 1926 : noticed a lump in the right upper jaw which she thought to be due to ill - fitting dentures . this was treated with " caustic and disappeared , but recurred within 4 months . it was again similarly treated . 
1928 on admission to hospital an oval area of enlargement of the right maxina was noted , situatecl between the alveolar margin of the maxilla and the inner surface of the cheek , the long axis clirected antero - posteriorly for about an inch . 
1899. - 1928 : operated on for " abscess of j aw . 1937 : painful swelling appeared at site of previous operation . x - ray examination showed a multflocular cyst in the 3 - / - 6 region . 
1887. - 1929 : the patient complained of a lump on the right side of the jaw of 10 years ' duration , which had been operated on 3 times , 10 , 6 and 3 years previously . it had increased in size , painlessly , but rather rapidly since the last operation . 
1881. - 1937 : three years ' history of swelling of the right lower jaw after extraction of 3 teeth . this was considered to be due to a dental cyst , and the jaw was scraped on two occasions . after the first operation the swelling disappeared . 
thackra ' y examination showed a large swelling on the lingual and buccal aspects of the pain . x - ray showed expansion of the jaw by a loculated , premolar and molar regions . 1950 : died from other causes . 
1883. - 1937 : admitted to hospital for a fracture of the this appeared elbow - joint , when a large swelling of the right mandible was noticed . to have started some 5 years previously , and had been gradually increasing in size . x - ray examination showed the typical appearances of adamantinoma , and local excision of the cyst was undertaken . 
3. given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . since 1944 we have taken a detailed family history from all patients reporting with carcinoma of the breast . 
we have 459 family records up to the end of 1947 which are reasonably complete . so far we have not succeeded in supplementing these histories , taken at the time of first visit to hospital , by later questions to the surviving patients or by arranging visits to their homes or relations . 
some attempt has been made to confirm the causes of death of relatives by letters to hospitals , doctors and the registrar - general 's department , but such confirmation has , as yet , been obtained in only a few cases . our figures , therefore , contain all those errors of inadequate information and faulty recollection that one would expect from data collected . 
many people die of cancer without their relations knowing that this was the cause of death . it is in fact surprising how successful a woman can be in concealing a cancer of the breast from her nearest relatives living in the same house with her . it is not , therefore , so surprising that more distant relatives , or even close relatives living away from home , may be unaware of the nature of the illness involved relations , even some of an older generation , who are still living at the time that the patient is first questioned may later develop malignant disease . 
many patients with cancer of the breast survive for long periods following treatment , and may ultimately die from some other cause ; we are , however , unable to include patients living who either have , or have had , cancer in our figures for comparison with expected incidence in the general population because no adequate morbidity statistics are available . in our series , for instance , there are the same number of sisters alive following treatment for cancer of the breast as there are sisters who died of the disease . ' comparisons must be made on a basis of mor164 d . 
3 shows a family where the mother of the patient died of cancer of the uterus aged 67 - she had two sisters , one who died of " internal cancer , " and one 166 d . 
smithers this sister had a daughter of cancer of the breast ' the latter aged about 60 . who had her breast amputated for cancer aged 45 , and who died aged 60 of lymphatic leukaemia . 
we find no evidence that cancer of the breast tends to develop earlier in patients whose relations are known to have suffered from the disease . the greater part of the work presented in this paper was done by p . rigby - jones and h . 
hartley , whose valuable assistance made this contribution to the symposium possible . i am also indebted to my colleagues on the staff of the royal cancer hospital whose patients were interrogated during this investigation , and particutlarly to r . 
5. continued immersion in the agent gave rise to a more intense staining in this region , with the formation of dense , highly coloured figures . these are shown in fig . 
7 , and are strictly comparable with the golgi bodies of cells of the same tumour ( sarcoma 37s ) illustrated by ludford ( 1932 )  . it was found that the golgi body of cells of the two fast - growing tumours , i.e. , beta and 37s , was 262 g . 
the intense , and rapid staining may also be associated with lipoid constituents in the mitochondrial structure , since fatty substances within the cells very rapidly absorb and reduce the tetrazolium salts . consideration of the staining reactions of nuclei suggests that the nuclear membrane is in two distinct layers . 
the outer is a continuous hyaline sheath , whilst the inner is a structure of irregular thickness . it is this inner structure which so readily stains with the tetrazoliuthe appearance of the nuclear membrane under these conditions corresponds with that described by ludford and smiles ( 1950 )  . these authors ascribed the granular material on the inner side of the nuclear membrane to chromatthe present technique indicates by virtue of the differential staining intensities that it is chemically different from whatever material comprises the chromonemata , and more nearly resembles the nucleolar material in the distribution of reducing groups . 
the method does not , however , offer any indications as to exact chemical nature of the stained substrate . the falling off in staining intensity exhibited by the chromosomes as division proceeds is interesting in that tumour cells are known to be more sensitive to irradiation during the early stages of mitosis . it appears possible that this is associated with the greater number of reducing groups available at this stage , since radiation effects are known to include oxidation of such groups as - sh . since the evidence on this point can only be regarded as equivocal this issue will be the subject of further experiment . the uptake of tetrazolium by the golgi region of these cells is perhaps not surprising , since the classical techniques for demonstration of the golgi apparatus rely on the reduction of metallic compounds to insoluble derivatives . 
the very slow but continuous manner in which the staining takes place suggests that there is no great array of reducing groups available at any one time , but that as a result of metabolic processes these are steadily being rendered accessible . 
the coal miners form an interesting group for comparison with the professional classes , for the miner 's daily bath , often obtained under very adverse conditions , secures cleanliness for about two - thirds qf the time , while in other respects his skin receives very rough treatment . 
the census returne ( registrar - general , 1938 ) give the age distribution , in 5and 10 - yearly periods , of the whole population of males , and also of those following each one of the recognized occupations , at the time of the census . the number of deaths attributed to cancer of the skin and lip occurring in the whole male population in each of these age groups during the years in question was obtained from the statistical reviews of the registrar - general . 
 " cancer of the thigh , " " cancer of the neck and face , " " cancer of the face and antrum . " certificates of persons under 14 years of age were excluded . 
1 shows the percentages , arranged in ascending order from left to right , for 41 occupations in which more than 5 deaths from cancer of the skin occurred in the 34 - year period ; this quite arbitrary limit in number of deaths is drawn to lessen sampling error . 
the percentage in 15 out of 16 professional occupations selected thus is below 100 , while that in 9 out of 13 agricultural occupations is above 100 , and in 5 is above 150 . 
the occupations of mining engineers , and mechanical and electrical engineers , show no deaths from cancer of the lip , and low percentages for cancer of the skin , but the numbers of deaths ( 5 and 2 respectively ) are small . the high standard of cleanliness maintained by the coal miner at the end of his day 's work , and his lack of exposure to sunlight , perhaps compensate for the various injurious factors , of which an account has been given by knowles ( 1944 ) , first - hand descriptions of the coal - miner 's life to which his skin is exposed . are given in two recent books ( shaw , 1946 ; agnew , 1947 )  . 
the mortality in the hewers and getters , who carry out much of the most severe work of the mine , is , if anything , lower ( 97 - 7 per cent ) than it is in the general population . the percentage is below 100 in 5 coal - mining occupations , comprising 828 , 246 out of the 939 , 378 men , or 88 per cent of those engaged in the 7 divisions of the industry . 
six out of 17 occupations , with a population of nearly 700 , 000 , have percentages from 131 to 249 ; this last figure ( gardeners ) is the highest of all those recorded in this paper . 
fingers , hand , forearm , and so on , might seem to give more information , but the sampling errors would be increased , and in such material the assignment to the smaller sites has not been carried out on any uniform system ; e.g. 
there would be no certainty that some cancers of the fingers had not been classed as of the hand . the numbers of cancers at these sites in the three groups of occupations , and their percentage distribution , are given in table iv and fig . 
1 provides a comparison of the percentages for cancer of the skin , and of the lip , selected and arranged in the same way , except that all cancers of the lip are included and not only those shown by occupation yielding more than 5 deaths . 
the number of fatal cases of cancer of the lip in the professional classes is so small ( 27 in 34 years in about 400 , 000 men ) that these have been pooled m . 
2. - occupational distrnbution of cancers of the skin according to site . show that no one of the seven groups of coal and shale miners gives a percentage above 100 ; the figures are low ( 38 - 3 to 56 - 0 per cent ) except in one large group which is the most exposed to light ( other workers above ground , population 106 , 549 ) , in which the incidence ( 97 - 5 per cent ) is approximately the same as in the general population . 
1 shows that the incidence of fatal cancer of the skin is high upon agricultural workers , and very low upon the professional classes , while the miners take an intermediate position not far removed from that of the general population . 
the especial liability of the out - door workers is generally supposed to be due to exposure to sunlight ( blum , 1948 )  . the results given in table iv and fig . 
2 show that the cancers of the whole region of neck , head , and face together , selected on the basis described above , make up from 70 to 78 per cent of cancers of the skin in all three occupational groups . 
the face is the only part of the body which is never clothed , and those parts which are shaved receive more thorough cleansing than do any others ; these characters are common to all social classes . 
the most notable result of this tabulation is the lack of any great difference between the three groups . another factor which may be of considerable importance is that the cosmetic effect of cancer of exposed portions of the skin and of the lip may attract attention sooner in richer subjects . ingram ( 1947 ) , in a comment on the paper by ryle and russell ( 1947 ) , says : " prognosis in skin cancer depends largely upon the size of the lesion , and negligence is probably an important factor in relation to death from this cause . i should , in the ordinary course of events , expect neglect of a symptomless lesion to be higher in unskilled workers and labourers ( iv and v of the registrar - general 's social classes ) than in the other groups . " obviously one cannot assess the quantitative importance of this difference . data in equal detail for the distribution of skin cancer in married women whose husbands follow these occupations would be of great interest . 
the great preponderance of tumours of the lower lip is explained as due to direct exposure of its mucocutaneous junction to sunlight , while the corresponding part of the upper lip is relatively shaded . " some factor in addition to exposure to sunlight is required smoking , and especially pipe to explain the differences shown in table vii . smoking , is , of course , regarded as another possible factor in the production of cancer of the lip . during the earlier part of the period ( 1911 to 1944 ) cigarette smoking was practically restricted to the richer classes , which produce fewest cancers of the lip ; data on the change in this matter in recent years have been given elsewhere ( kennaway and kennaway , 1947 )  . 
the data collected by lane - claypon ( 1930 ) from the literature , summarized in table vii , show the great preponderance of cancers of the lower lip , especially in males . 
 ( lanecilaypon , 1930 )  . all occupied and retired males agricultural labourers farmers deaths . death rate per million per annul . 212 798 55 - 0 comparison of cancer of the skin and of the lip . the data given in fig . 
of the 14 agricultural occupations which produce any cancers of the lip , 11 show agreement in regard to cancer of the skin and of the lip in that both percentages are either below , or above , 100 . this suggests that the aetiological factors are to some extent the same in some occupations . 
the incidence upon the coal miner is of especial interest in view of the cleanliness secured by his daily bath , and of his minimal exposure to sunlight . in occupations in which 88 per cent of the coal - mining population are engaged the liability to cancer of the skin is less than it is in the general population . only one occupation of social class i ( civil engineers and surveyors ) shows a mortality from cancer of the skin higher than that in the whole population ; hence these results illustrate the social distribution of cancer of exposed sites discovered by stevenson . 
the comparative distribution of cancer upon the various parts of the skin does not show any great differences in the three groups ; from 70 to 80 per cent of these cancers are situated on the head and neck . 
campbell. from ae royal ( dick ) veterinary college , edinburgh , and the poultry re8earch centre , king ' 8buildinp , we8t main8road , edinburgh . received for publication february 5 , 1951 neoplasms involving the ovary are common in the fowl ( olson and bullis , 1942 ; campbell , 1945 )  . 
the great majority far into one or other of two categories , namely adenocareinomatous growths in various stages ofdifferentiation from the frankly anaplastic type to the sclerosing tumour , or , secondly , lymphomatous growths associated with the leucotic complex , e.g. , lymphocytoma , fowl paralysis tumour , aleukaemic lymphoid leucosis , etc . 
most of these lymphoid growths should not be classified as ovarian tumours , since in view of the generalized nature of the leucotic process it is usuary impossible to be certain whether they are primary in that organ or not . 
they are mentioned simply because of the frequent teference in avian pathological literature to " lymphoid ovarian tumours . " in human pathology , besid6s - the common adenocareinomatous types of , ovarian tumour , several other rarer forms are recognized as follows : brenner tumour ( o6phoroma folliculare ) : granulosa - cell , thecal , and luteal tumours arrhenoblastomas ; dysgerminomas . a survey of the hterature deahng with neoplastic disease of the fowl shows the apparent extreme rarity of such tumours . seifried ( 1923 ) recorded a brenner tumour of the fowl and compared it with its human counterpart , a ' nd friedgood and uotila ( i 941 ) detailed 5 cases of - ovarian " tumours " associated with virilism in two of these latter cases , tuberculosis of the ovary compheated in the fowl . the picture , and the remainder were cystic or contained smau growths which appeared to be tumours in various stages of degeneration . tentative diagnoses of arrhenoblastoma were made , but it was also suggested that they might be luteal - cell tumours or " hypernephromata . the only record of an ovarian teratoma in the fowl is by jackson ( 1936 )  . with regard to the male bird , there appear to be no reports of spontaneous seminoma or interstitial - cell tumour , but several cases of spontaneous teratoma testis have been recorded ( sheather , 1911 ; jackson , 1936 ; olson and bulhs , 1942 )  . it seemed reasonable to beheve that the absence of reports of the rarer forms of ovarian and testicular tumours was simply an indication of a lack of intensive study in this brartch of comparative pathology . it was therefore determined to examine as many tumours arising in these sites as possible . 
to date approximately 2000 cases have been submitted to a thorough post - mortem and histological scrutiny , and , as was anticipated , several hitherto unrecorded types of tumour of the fowl gonads have been encountered . it ' is desirable that an account of these should be put on record , not only because of their interest from the viewpoint of comparative oncology , but also j . 
at post - mortem a thorough search was made for metastases , and the possibihty that the gonadal tumour was not the primary growth was ruled out as far as possible . blocks of tissue were taken from many organs for histological examination , whether they showed visible abnormality or not . 
the growth offered some resistance to ' the knife , and its cut surface showed pale yellow fibrous areas containing numerous small cysts and islands of pinkish tissue . there were no implantations in the abdominal cavity and no metastases were found . histologically a large part of the tumour consists of a stroma of interlacing bands and whorls of fibrous tissue , mostly dense , but having a looser texture in there is a certain amount of plain muscle ( characteristic of the some places . avian ovary especially at the hilus ) in one part of a section . 
a comparable structure occurs in the human subject , and is well ifustrated by nicholson ( 1950 )  . ( 2 ) the next ovarian tumour to be considered is the granulosa - cell type , of which 4 cases have been studied in this series . 
on section , it was cream to yellowish in colour and displayed bands of tissue which had a tehdency to radiate from the centre of the growth . the vascularity appeared to be very poor . 
the rest of the ovary seemed normal , with a few atretic follicles , and the oviduct was that of a bird in full lay . histologl ' cally , this tumour is mainly composed of round oval or fusiform cefls , with a j . 
the periphery of the growth is largely necrotic . radiating bands of smooth muscle occur in the interior of the section . wilhs ( 1948 ) states that ovarian fibromata are probably the end result of a in that case , the rarity of the latter in fowls may account theca - cell tumour . for the equary rare occurrence of fibrous tissue tumours associated with this organ . 
as an instance of this the only case of ovarian fibroma encountered in this series is worth mentioning . it was found in a rhode island red hen in her third year , and weighed 570 g . 
maximow and bloom ( 1948 ) state that what are usually considered chromaffin cefls ( sympathicotrophic cells ) are to be found at the hilus of the human ovary . these apparently have much in common with the medullary cells of the adrenal , although they cautiously conclude that until these cells have been shown to elaborate epinephrin , their relationship to adrenal medulla must remain unproven . the chromaffin reaction of such ovarian cells is unrehable but this also applies to the adrenal medullary cells , where the staining by chrome salts may vary from practicary none to a very deep brown . as far as can be ascertained there is no record of chromaffin cells or heterotopic adrenal cortical tissue in the avian ovary . that such cells may in fact be occasionally present seems to be imphed by the following cases , and a thorough cytological examination of the region of the hilus should clear up this point . the first of these tumours to be described occurred in a brown leghorn hen which was brought for examination after developing signs of mascuhnization . the bird was said to have ceased laying , commenced crowing and to have endeavoured to copulate with other hens in the run . the comb was decidedly male in character , and plumage changes were evident especially in the neck and taff . upon post - mortem , a rounded soft yellow - brown tumour measuring 3 - 8 x 3 - 5 x 28 cwas found in the ovary , which was inactive . 
numerous irregular trabeculae containing vessels arise from this and pass into the interior of the growth . between these are sohd islands of large polyhedral epithehal cells which are in turn divided into small irregular groups by fine connective tissue staining as for reticuluthe cytoplasm is eosinophilic and faintly granular . in places it is vacuolated . 
the first view is favoured as being the more probable . ( 5 ) the next case , though not a mascuhnizing growth , is worth recording for its unusual nature , and because its interpretation could mean that besides adrenal cortical ceus , chromaffin cells resembhng those of the adrenal medura may occur in the fowl 's ovary and may undergo neoplasia in common with other heterotopic - tissues . during the examination of a crossbred fowl wbich was found dead , a large intra - abdominal blood clot was found proceeding from a rupture in the substance .of a smoothly lobulated brownish - yerow tumour of soft consistency , measuring 6 - 5 x 5 x 4 - 3 cand occupyi ' ng the site of the left ( functional ) ovary . it was - freelymovable , being only attached at the bilus , and was therefore easily dissected out . 
the healthy remainder is composed of connective tissue cells with spherical to spindle - shaped nuclei , and eosinophilic granular cytoplasm , surrounding aggregations of large polyhedral cells with abundant finely granular eosinophihe cytoplasm , large nuclei and very prominent nucleoli ( nuclear - nucleolar ratio . 1 : 3 to 1 : 5 )  . an abundant infiltration with eosinophils is present in many parts of the smau epithelial - cer moiety . a well - marked but thin layer of connective tissue separates this outer zone . from the inner , which is composed of small lobules of cens separated from each other by fine trabeculae of connective tissu ' e . 
the cefls composing these groups are large , with giant nucleoh similar to those mentioned above , and have a brownish - yerow cytoplasm in bichromate fixed haematoxyhn and eosin preparations . 
at the edge of one series of sections , the only indication of ovarian origin is the presence of a few folhcles . bands of smooth muscle are scattered throughout the section . the presence of such la ' e numbers of cers showing a distinct chromaffin reaction justifies ' a diagnosis of chromaffmoma or paraganghoma . 
the growth appears to have arisen within the adrenal capsule . another small ( 0 - 5 x 0 - 2 cm . ) tumour was found in the site of the rudimentary right goriad and closely adherent to the dorsal wall . this shows not only testiculoid elements , but also a considerable area composed of whorls and strands of rapidly dividing mesenchymatous cers , presumably a metastasis from the experimentary transplanted sarcoma . the histology of both gonadal rowths resembles ewing 's ( 1942 ) fig . 
the nests of cells below the germinal epithehum resemble the primitive sex cords of the indifferent embryonic gonad . this tumour iuustrates well the bisexuality of the embryonic fowl 's ovary . 
the rete ovarii formed from the primitive sex cords is a homologue of the testis and as is well known can develop into one following left ovariectomy of the hen , where the right gonad hypertrophies and differentiates even to the exteht of producing spermatozoa ( benoit , 1923 )  . another tumour of the ovary associated with masculinization in a light sussex hen is shown in fig . 
17. folfculoid formation and luteinization is apparent , and the structure is comparable to a granulosa - cell tumour . in fact , this diagnosis would be justifiable on histological grounds alone , but has to be modified in view of the androgenic effects on the host . it would appear that the rudimentary right ovary is not the only place where in theory at any rate , testiculoid structures could arise in the intact bird . 
champy , lavedan and marquez ( 1 939 ) state that the adrenals of either male or female castrates in the fowl frequently contain " regenerating gonadal cens . " birds ' adrenals constantly contain wolffian duct remnants in the capsule on the ventral these slowly hypertrophy following castration to form an internal aspect . epididymis - hke structure , whilst some form epithelial tubules similar to those found in the right ovarian rudiment subsequent to castration . 
spermatogenesis has not been seen in the adrenal capsule however . there does not seem to be any record of spontaneous arrhenoma arising in the adrenal capsule of the fowl , but it seems possible that the foregoing is a case in point . ( 7 ) the last of the tumours of the female gonad to be described was originally considered to be a teratoma . it is unfortunate that no comparison can be made of this case with the only comparable ovarian tumour of the fowl on record ( jackson , 1936 ) , since apart from stating its didermic character , no other details were given , and the single photograph is of the gross specimen . the present case occurred in a brown leghom hen aged about 3 years , at the poultry research centre . 
of which was withafter death 4 days later , an ovanan tumour was drawn from the abdomen . it was mottled pink , brownish and cream , of found measuring 5 x 4 x 4 cm . other growths , pinkish and soft , firm consistency and mucinous on section . were attached to the wall of the oviduct , and to the serous investment of the duodenum , pancreas and liver . histologically the ovarian tumour is the only one to show complexity of immediately below a thin capsule there are nests oflarge pale staining structure . epithehal cefls , spme of which have differentiated into cysts of varying size , lined with one or several layers of columnar epithehum . in many cases these cells are actively secreting mucus . 
at first sight two embryonic layers appear to be involved , namely mesoderm and ectoderm . the latter may be represented by the cyst epithelium forming mucinous glands , and possibly as the complex epithelial tubular structures . 
the remainder of the tumour is mesodermal in origin . ifthis is the case the presence of these derivatives of two germinal layers presumably puts it in the same category as jackson 's ( 1936 ) " didermic teratoma . " wiuis ( 1948 ) defines a teratoma as " a true tumour or neoplasm composed of multiple tissues of kinds foreign tothe part in which it arises . " the presence of cartilage can be explained by the conversion of connective tissue normary present in the ovary , to cartflage by mucoid degeneration and hyalinization . 
one of their cases exhibited metastases , but no details are given of the histology of this secondary growth or its site . several seminomas have been studied during the past 11 years and two of these are selected for description here . 
the right testis was normal both in size and microscopic structure , and no metastases were found . microscopically the left testis consists of poorly dehneated lobules of pale staining epithelial cells arranged in diffuse sheets and penetrated here and there by blood vessels . the cells are mainly spherical to polygonal through mutual pressure , but the faintness of cytoplasmic outfine often gives a syncytial appearance . 
a second testicular tumour - also involving the left organ - wa 's seen at autopsy in a buff rock cockerel . it measured 4 - 5 x 3 - 8 x 3 - 5 cm . , and on section showed an irregular yeflowish area near the caudal pole , compared with the rest of the tumour which was of a cream colour . 
the cream - coloured area , forming about two - thirds of the tumour . , is composed of typical seminoma cefls , showing in many places a marked acinar structure . luteinization of these cens is proceeding in small isolated areas . 
the question arises whether the interstitial - cell moiety represents a concommitant neoplasia of the stroma , as the acceptance of firket 's ( 1920 ) view on the connective tissue origin of interstitial cells would imply . in other words , is this an example of a " mixed tumour " - or alternatively is it an indication of the teratomatous nature of seminoma , as ewing ( 1942 ) beheves , but which willis ( 1948 ) , nicholson ( 1950 ) , and others dispute ? still another possibility exists , namely that the two cell types have arisen from a common ancestral totipotent germinal cell capable ofgiving rise to a variety of tissues , in accordance with the view put forward by innes , harvey , and dawson ( 1938 )  . a re - examination of fig . 
the last testicular tumour of special interest occurred in a cross - bred fowl , which showed during life a marked atrophy of the comb , and cessation of crowing and of other activities associated with the adult male bird . at post - mortem , a greatly enlarged ( 9 x 10 x 6 cm . ) left gonad was found , a flattened oval in shape and firm in consistency . 
at subsequent autopsy this bird had a small nodule at the site of the right gonad , whose histological structure , in the opinion of the present - writer , resembles that of a thecal - cell tumour . teilum 's ( 1946 ) case of " androma " of the testis in man may be ' another example of a feminizing tumour arising from a persistent remnant of the " ovarian " cortex , and the presence of arrhenomatoid tissue in a testicular teratoma of a .fbwl which lost certain of its male characters has already been described in the present paper . tuming to granulosa - cell and related tumours , the development of the ovary throws some light on the inter - relationship of these oestrogen - secreting growths . in the embryonic ovary it is commonly stated that the granulosa cells arise from coelomic epithehum derived from the genital ridge , whereas the thecal cens , being stromal in nature arise from the mesenchymal elements of the mesoderm . examination of the early ovary , however , shows primary folhcles composed of a central ovocyte surrounded by a ring of indifferent epithehal cens indistinguishable .from the stromal cells composing the rest of the organ . these indifferent cens appear to be the precursors of the granulosa cells , whereas the remainder of the it seems probable that this common ancestry is reflected - stroma forms the theca . in the behaviour of thecal and granulosal cees in the neoplastic state , where both secrete oestrogens as judged by the muscular and glandular hypertrophy of the , oviduct of the tumour - bearing bird , and both undergo the phenomenon of luteinization . granulosa - cell tumours in the fowl are not morphologicary identical to these in man in that they do not exhibit the characteristic " rosettes " which are often j . 
campbell a feature of human tumours , and which are considered to be identical to the bodies of call - exner occurring normally in the stratum granulosum of the human such bodies are not found in the granulosa layer of the ovarian follicle ovary . because this is usuary only one , or at the most two or three cers thick . callexner bodies are usuary considered to be due to cystic degeneration of granulosa , this does not occur in avian tumours where there is ample space for such cers . a change to take place , and it therefore seems possible that these structures may serve some specific purpose as for example a glandular function , in the ovaries of those animals in which they occur . with regard to thecal - luteal cells and the interstitial cells of the testis , it is interesting to note that they appear to have a common ancestory . fell ( 1924 ) states that the ovarian thecal - luteal cells of the fowl are derived from remnants of aborted medulla in the development of the ovary . thus they have the same histogenesis as seminiferous tissue . 
9. - structure of the above , showing vacuolated cells , due to removal of lipoids , to the left ( some nuclei are pyknotic ) and cells resembling fibrous tissue to the right of the figure . 
18. - - - - " mixed ? " ovarian tumour , consisting of areas of tortuous glands , mucinous cysts , cartilage , solid nests of epithelial cells and a myxomatous stroma . 
moroney. from the leicester general h08pital and the colleges qf technology and commerce , leicester . received for publication juno 3 , 1952 . does chronic g " tric ulcer tend to undergo malignant change ? the importance of this question is beyond doubt , both because of the high incidence of gastric ulcer , and because many patients suffering from it are still treated medically over long periods before reference to a surgeon . 
the answer to it is also of considerable importance to the surgeon , owing to the great difficulty of assessing at laparotomy whether early malignant change may have already if the development of secondary carcinoma supervened on chronic ulceration . is really as common as some pathologists ' figures would appear to indicate , there might be a strong case for an e ' ven more radical gastrectomy than is usually carried out for such lesions . conflicting clinical and pathological evidence . the consensus of opinion in the medical literature is that gastric ulcer is a definite predisposing cause of carcinoma , but there is profound disagreement among pathologists about the percentage of cases ' which undergo such malignant change . 
udaondo ( 1939 ) , in an extensive survey of the world literatu ' re , found estimat - es of the incidence of " ulcer - cancer " which ranged frotn 0 to 100 per obviously , some statistical cent , with most intermediate values represented . uncertainty must be present in the majority of these assessmeints , yet there is usually quite inadequate evidence in the published data to lead to its detection . the only general conclusion that can be drawn from a study of these papers is that the authors ' confidence in the more extraordinary estimates seems to vary inversely with the amount of evidence at their disposal . since , according to the registrar - general 's returns , s ' omething like 14 , 000 people die each year from cancer of the stomach , it is not surprising that the heavy incidence of gastric ulcer in the population should have suggested that there was some connection between the two conditions . 
moroney as regards the stomach , and suggests that the incidence of mahgnant change must be very considerably lower than is suggested by most pathologists ' figures . livingstone and pack ( 1939 ) assess the average survival of patients admitted to hospital with inoperable cancer of the stomach at under 4 months , whilst the total duration of untreated primary gastric carcinoma is often under a year , from the onset of the first chnical symptoms until death supervenes . chronic gastric ulcer , on the other hand , appears to be relatively benign . 
the importance of this is amply justified in view of the gravity of the problem at issue ; particularly so since the ultimate verdict of malignancy must so frequently depend on a but , apart ' from the fact that no two histologists will microscopical opinion . whollv agree on the evidence which justifies such an opinion , the same pathologist not infrequently changes his criteria on reviewing the same series of specimens after a lapse of years . this , of course , is bound to occur as the result of increasing experience ; yet it means that the findings of an individual pathologist over a lengthy series of cases are only likely to be sufficiently homogeneous for statistical purposes if the time i - nvolved in the assessment of his data is not too lengthy , in terms of his tendency to change his pathological criteria . equally , it means that there is grave statistical objection to " averaging " blindly the data of until pathologists in general are able to come to an different pathologists . agreement to assess their findings on common criteria as to what constitutes malignant change in an ulcer and what differentiates this from a cancer arising de novo , each set of figures must be judged independently on internal statistical criteria , 1 4 . 
the only statistical task then remaining would be that of estimating the frequency of its occurrence . but no such agreement on the microscopical findings and their histological interpretation appears probable as ven amongst the most experienced workers . 
on the contrary , the conflicting evidence and extreme variations in the published pathological data make any attempt at mathematical analysis of them futile . as has already been suggested , the chnical evidence of malignant change in pre - existing gastric ulcer is a relative rarity in the collective experience of both thus , as soon as doubts are cast on the infauibility surgeons ' and pathologists . of histological diagnosis , it would seem that the only logical basis for continued belief in " ulcer - cancer " as a comparatively common entity is the analogy of the causative influence of chronic inflammation on subsequent development of carcinoma elsewhere in the body . such arguments by analogy may , however , be most fallacious , and can directly contravene the famous principle first enunciated by william of occam several centuries ago - " entia non sunt multiplicanda praeter nece88itatem . " the importance of this principle in scientific thought , of which it forms one of the main pillars , arises from the fact that it is impossible in practice to prove a universal negative by inductive processes . 
an example will make this clear . our ancestors , influenced by superstition rather than by scientific method , were constantly inventing ad hoc explanations for things in terms of witches , goblins , evil eyes , fairies and leprechauns which the whole weight of philosophy and christianity has not yet succeeded in completely eradicating . 
the survival of such hypotheses is understandable on the grounds that they are easy to conceive , better than no explanation at all and , once accepted , virtually impossible to disprove . 
and then we shall always be countered by the fellow who is positive he saw several the other night such hypotheses are by their very nature invulnerable . in contrast , the opposite scientific hypothesis that the genu8 leprechaun does not exist , except possibly as a freak of natural development in isolated instancesis extremely vulnerable . indisputable evidence of widespread loprechaun activity will entirely refute it , even if no actual leprechaun can be produced for examination . it is essential that this characteristic be present in any scientific hypothesis , so that the collection of sufficient opposition evidence can establish its falsity . adoption o the null hypothesi8 . applying these basic principles to the problem of " ulcer - cancer , " can it be said that this conception has really been adequately demonstrated clinically , 218 a . 
moroney or have we or even that it forms an essential entity in morbid anatomy perhaps accepted its existence with little more justification than our ancestors had in welcoming the leprechaun ? the very phraseology of the relevant literature - " the incidence of ulcer - cancer probably does not exceed x per cent suggests that this ma be the case , and that pathologists are themselves engaged in an attempt to depreciate its frequency of occurrence . it would seem , therefore , that a more profitable approach to the problem will be to attack it from the opposite direction , by the assumption of a " nufl hypothesis "  . in other words , to investigate the situation on the postulate that ulcer and cancer always occur independently in the stomach , in the aetiological sense , and that their occasional intimate association is entirply a matter of pure chance . failing the demonstration of " ulcer - cancer " as a necessary and unmistakable microscopical entity , such a null hypothesis could still be chanenged by statistical evidence that the relative frequency of occurrence of associated ulcer if the frequency and cancer is significantlv areater than the hypothesis predicts . of such associated lesions is so high that it is unlikely to have arisen by chance , the aetiological connection would be estabhshed , but even then this might only indicate that ulcer is a favoured site for the development of a carcinoma destined however , since it is on the relative frequencies of these to arise ' m any case . lesions that the null hypothesis must stand or fall , it is now important to survey the position statistically . 
and this is best carried out ' m general terms in order to avoid errors present in the conflicting pathological figures , and any profitless mathematical resolution of them . in - such an investigation , general figures of cancer incidence are obviously valueless . 
the only data worthy of consideration are thos ' e which have been verified pathologically , and the analysis must therefore be carried from the population at large up to the point where gastric specimens arrive at the patholoin the statistical model of the situation about to be constructed , it is gist . assumed that the specimens eventually arriving at the pathologist are drawn from a population ' whose characteristics with respect to both gastric ulcer and carcinoma may be regarded as stable over a period of time covered by any particular analysis ; and that , by virtue both of its size and its autogenerative properties , the composition of this population remains substantially unaffected by the withdrawal of the specimens . it will also be assumed that u1cer and cancer occur quite independently . development of the statistical model . in the population postulated therewill . 
i with the frequencies stated , the left - hand compartments of the diagrams representing the pyloric zone and the right - hand ones the lesser curvature ; the two lower sections showing the position with respect to the relative frequencies of double lesions occurring in the same region , compared with double lesions which are sited in separate regions of the stomach . 
we shall refer to such double lesions occurring in the same region of the stomacb as " amalgamated lesions " throughout the remainder of this paper , even if they are actually separate . within this limited selection of the whole data evidence might be found of a tendency for co - existing ulcer and cancer lesions to become amalgamated in this sense more frequently thad predicted by the null hypothesis , on wbicb basis the expected value of the ratio of amalgamat - ed lesions to non - amalgamated lesions is seen from fig . 
moroney the developmaint of a cancer , if not an actual aetiolooical factor in its developit will be - seen that this ratio has the statistical advantage that it does ment . not include the imponderable quantities u and c , but depends only on the site it wfll . 
i gives the expected distribution of an cases of if the null hypothesis is true , ulcer and carcinoma into the various categories . we should expect that a randoni collection of pathological specimens from the population at large would agree with the predicted ratios within the linlits of sampling error . but , of course , pathological specimens are not collected at random , and it is therefore essential to consider in some detail the effect of the selection chain along which patients have to pass before their stomachs become pathological specimens . the first group illustrated in the model shown in fig . 
1 , those cases which have neither ulcer nor cancer , will , of course , be entirely missing from the pathologist 's data . is it likely that ulcer of consider next the two categories of simple ulcer . the pyloric canal will have the same probability of pass ' mg along the reference chain to the pathologist as ulcer at the lesser curve ? prepyloric ulcer , in its uncomplicated form , is notoriously difficult to demonstrate radiologicany but , once diagnosed with certainty , is usually operated on without delay , since so many of these lesions prove eventually to have been malignant since their benign ulcers at this site tend to give rise to early symptoms from the onset . secondary pylorospasm which they cause ; yet they are often undetected until pyloric obstruction supervenes later , as the result of gradual stenosis , or unless acute perforation occurs . 
moroney hand , it is more often inoperable at laparotomy , and therefore is less hkely to reach the pathologist as a specimen . there is thus a complex selection mechanism operating along the whole of the reference chain , from the differential symptomatology in the patient through only after all the medical practitioner or consulting physician to the surgeon . this does the stomach become a potential pathological specimen , the likehhood of which is again govemed by the surgeon 's technical skill , and the influence of this on his decision whether to operate and finally to resect . moreover , still other factors may influence this selection of specimens , which will be different for each pathologist . 
the number of patients with gastric ulcer under observation by the medical side of the hospital wif be one of these , for associated " ulcercancer " lesions will have a better chance of being diagnosed early if the chronic ulcer lesions are kept under continual scrutiny by the consultant physician rather than being referred back to their own farnily doctors for routine treatment . further , the alteration in symptomatology resulting from the development of a secondary cancer in these cases is often relatively smah in the early stages . much depends on the vigilance and diagnostic skill of the physician concerned . the almost universal tendency among reporters is to ignore these very important weightings of their figures , and to hope that in the end they win cancel each other out over a large series . comparison of one series with another - or even the first and second part of the same observer 's series - soon demonstrates that this does not happen . 
the fact is that each pathologist 's series must be considered a unique set , not only because of his particular pathological criteria for " ulcer - cancer " but , perhaps even more , because his experience will include a unique set of weights in the data . introduction of the weighting factors . it is now evident that the simple model shown in fig . 
2 appropriate weighting factors have been introduced , which will take unique values for each individual pathologist in accordance with the varying nature of the particular selection chain along which his specimens have passed . 
the group of the population having neither ulcer nor cancer will be completely absent from such specimens and so will have weight zero . this is the only weight that the data of all pathologists will . 
and w6 , are the weightings for referability of associated ulcer and cancer losions in separate compartments of the same stomach - the pathological data for the frequency of wbich is scanty and its clinical recognition difficult . 
moroney if the individual pathologist could be given this ratio of the phcit values . weiahts concerned , and the values for u and c , he could then calculate the expected values for a and d3 , against which the observed values for these ratios in his own series could be tested . the evaluation of u and c from existin - a fi - aures is , however , rendered impossible by the effect of the reference chain and the different criteria adopted for the boundaries of the various stomach zones , nearly all the pubhshed work on stomach pathology conflicting in this respect . minor differences on the latter score between different series - may be partially taken up in the weights w7 and w8 , since all comparisons wif be intemal ones within each set of individual data , but the question is of academic interest only so long as marked reference chain separate independent research , deliberately designed to eliminate effects persist . moreover , the effect of reference chains , would be necessary for their estimation . even if the individual pathologist was supplied with values for u and c , he would still be unable to determine whether his observed values for a and a differed from the null hypothesis expectation for these ratios . 
the ratio w7 is inherently a property of his own series , representing the effect of the reference chain operating in his particular case . it can thorefore only be determined by using his own observed frequencies for u and c and checking them against those established manifestly , he cannot use his own data through such an independent survey . both to establish the expected value of the expressions d2 and d3 , and then to see if his observed values differ from expectation . 
the position , from the point of view of the individual pathologist , is that significant departures in this respect from the null hyporthesis predictions are inextricably confused with the effect of the reference chain ; and the same applies , even mo ' re , to the ratios a2 and a 35 which are under the influence of the reference chain to a greater extent . its implication is that no pathologist , however competent , and no consensus of opinion among pathologists , however widespread , can refute the null hypothesis on the basis of existing figures . it follows inevitably that the solution to this problem of the incidence of cc ulcer - cancer " must await a research project specifically designed to remove the insuperable obstacle facino , the individual pathologist - the effect of the reference chain represented by the weights w in our analysis . 
the results of such an investigation could then be evaluated by means of the ratios al and dl , which only involve the site ' distribution factors u and c . it would be essential for this purpose to establish some sufficiently accurate convention defining the admittedly , these are not boundaries between the various stomach zones . always easy to demarcate in practice , but the contrivance ot ' some effective standard in this respect ought not to present insuperable difficulties . the most hopeful way of establishing reliable values for u and c would appear to be by planned , clinical research at a number of large centres . the assiduous co - operation of all the local general practitioners surrounding these centres would be an essential , so that every case of a suspected stomach lesion reaches them at the earliest possible moment . 
the research would have to be based on several centres , whose respective findings could finally be checked for statistical homogeneity , and these cent - res ought to be carefully select ' ed . 
they should be situated at large - population centres which are growing , to minimise loss of patients during prolonged observation from drift to other areas ; and in provincial towns rather than in very large cities , where it might be difficult to trace their admission to , or treatment at , a large number of alternative hospitals for a gastric emergency or some intercurrent disease . if the establishment of such centres were practicable , it might be possible to eliminate the reference chain weigbts w for all practical purposes , and thus - trrive at reliable values for the site distribution factors u and c . there are , of course , dangers of leakage in the procedure outlined . in addition to the inevitable loss of some patients during the prolonged observation necessary , there is the certainty that some cases would not reach autopsy . moreover , there will be a fraction of the population who will not seek any medical advice until the disease has progressed too far for any statistical value to be elicited regarding the site diagnosis . this is not the place to discuss the details of such an investigation , but the essential problem regarding such losses is not so much their magnitude as whether they are likely to introduce bias in the determination of u and c . .if unbiased estimates of u and c could be ob - tained in this way , the data collected would suffice to test the null hypothesis expectations . cases with single lesions could be used to provide values for these quantities , and thus for the expected values of al and dl , of a high degree of accuracy deriving from the large number of observations on which they would be based . cases with double lesions would then give an adequate check on such results , using the incidence figures of ulcer and cancer in different stomach zones for this purpose in addition to those of amalgamated lesions in the sa - me zone . 
butler. from the chester beatty research institute , the royal cancer hospital , london , s.w.3. received for publication april 21 , 1951 . the observation of brdicka ( 1933 ) that proteins gave catalytic double waves when added to an ammoniacal cobalt buffer and examined polarographically was soon applied to an examination of the serum proteins , and brdicka ( 1937 ) himself was the first to show that differences could be found in this way between normal and cancerous sera . 
there is no need to relate in detail here the earlier studies on the protein reaction as these have already been adequately reported ( brdicka , 1939 , 1947 ; brdicka , novak and klumpar , 1939 ) , but it will suffice to state briefly that pathological sera after hydrolysis with potassium hydroxide and polarographed in cobaltous buffer show a decreased wave height as compared with normal sera , but after deproteination with sulphosalicylic acid and the filtrates polarographed in cobaltic buffer show an increased wave height . 
brdicka ( 1939 ) found that the latter test , known as the " filtrate test , " was more reliable , but the readings from both tests are dependent on the temperature , and on the characteristics of the dropping mercury electrode . muiller and davis ( 1947 ) introduced an index , known as the " protein index , " derived from the ratio of the two tests multiplied by a constant , which was found to be independent of the temperature and of the capillary characteristics , and also of the age and sex of the subjects . the fact that the polarographic test is not specific for malignancy has been well established and it is therefore of no use diagnostically , but more recent results robinson ( 1948 ) , using the indicate that it may be of value as a prognostic tool . filtrate test , has studied its application in the prognosis of prostate cancers , and has obtained results which are superior to those obtained with the acid phosphatase test normally used for this type of cancer . 
the purpose of the work to be reported below was to gain further information concerning the usefulness of the method , and to compare it with other methods which might be more applicable in general routine . over 100 sera have been examined polarographically , and a smaller number examined by means of the " huggins test " and by the tryptophane - perchloric acid reaction . since the publication by huggins , miller and jensen ( 1949 ) of the " huggins test , " depending on the relative ease of the thermal clotting of serum in the presence of various concentrations of iodoacetate , many workers have investigated its application ( bodansky and mcinnes , 1950 ; homburger , pfeiffer , page , rizzone and benotti , 1950 )  . 
the original authors stated that the " iodoacetate index , " obtained by dividing the highest concentration of iodoacetate in which clotting could still occur by the total protein content of the serum , was lower in 226 l . 
the results obtained from an examination of a limited number of sera by means of the modified method ( huggins , miller and jensen , 1949 , private communication ) are reported below . increases in the polysaccharide content of sera have been observed by a number ofworkers in cases ofmalignancy , tuberculosis , nephrosis , hepatic cirrhosis andpneumonia ( shetlar , foster , kelly , shetlar , bryan and everett , 1949 )  . using the carbazole method seibert , pfaff and seibert ( 1948 ) showed that the globulins ( particularly the a2 - globulin ) contained the most of the protein bound polysaccharide . in an attempt to determine the nature of the polysaccharide seibert and her co - workers ( 1948 ) found that the tryptophane - perchloric acid ( tpa ) test of cohen ( 1944 ) was more useful . using this reaction the authors claimed a correlation between increases in the polysaccharide and increases in the o2 - globulin in cases of tuberculosis and malignancy . moreover , changes in the tpa readings were found in minimal tuberculosis when no change was observed with the carbazole reaction , and it was suggested that this was probably due to the former reaction being more specific although the latter reaction is probably more sensitive . the identity of the polysaccharide involved in the tpa reaction is not known , but only negligible colour formation is obtained with glucose , mannose , galactose and glucosamine , while a positive reaction is obtained with desoxyribose - nucleic acid and fructose . 
a new index was then formulated derived from dividing the value of the filtrate test ( test ii ) by the difference between tests iii and iv and multiplying by ten to bring it to a round number , i.e. , blood index ( b.i. ) = test ( ii ) test ( iii ) test ( iv ) huggins reaction . the modified method ( huggins , miller and jensen , 1949 , private communication ) was used . the iodoacetic acid was recrystallized from a mixture of equal volumes of benzene and 80 - 100 c . 
the contents of the tubes were gently mixed , the tubes covered and heated in a boiling water - bath for 10 minutes . alter cooling quickly they were allowed to stand for 40 minutes and then filtered through a no . 
the upper limit for normal values was fixed at an arbitrary level below which the majority of the normals were grouped ( 21 table i is divided into two groups , and considering the negative , 3 positive )  . cases placed in group ( a ) , it can be seen that 93 per cent gave a positive reaction . 
the former were ( i ) a patient with a 5 years ' history who had been successfully treated by surgery and by androgen , and who was clinically normal at the time the blood sample was taken ; ( ii ) a male case who gave a positive reading 2 weeks later ; and ( iii ) a patient with an advanced tumour who gave a negative reading again 2 weeks later . 
the negative prostate case had already been receiving stilboestrol treatment . these cases show a high correlation with the clinical observations , but group ( b ) of table i gives figures for two malignant groups which appear to give an even chance of positive results . classified in " buccal cavity " are carcinomas of the fauces , palate , tonsil , tongue , mouth , larynx and pharynx , and in " skin " are classified carcinomas of nose and scalp . 
great improvement was observed clinically while under androgen treatment ( after radiotherapy followed by radical mastectomy ) , and the polarographic however , the last five readings , taken readings altered from positive to normal . over a period of eight months , gave positive reactions , although it was only at the time of the last reading that a relapse was clinically observed . in this case , therefore , the relapse was forecast by the polarographic readings . 
youden ( 1950 ) has suggested the use of an index for the comparison of so - called diagnostic methods , which has a value of unity if there are no false positives or false negatives , and a value of zero when the test gives equal proportions of positives for the condition under investigation and the controls . 
the polarographic method gives a value for the index of 0 - 219 , the huggins reaction , 0 - 250 , and the tryptophane - perchloric acid method , 0 - 253 . 
the end - point as described by huggins was not easily determinable , as it was not always obvious what constituted a " well - defined mass . " this difficulty was also noted by bodansky and mcinnes ( 1950 )  . also , the end - point often did not occur within the limits of the recommended iodoacetate concentrations - a problem which presumably would have been overcome by using a larger range of concentrations , making the test much more tedious . 
the results obtained support the conclusions of bodansky and mcinnes ( 1950 ) and of homburger , pfeiffer , page , rizzone and benotti ( 1950 ) , which were reached after a much more intensive study of the reaction than has been attempted here , and it can be stated further that the test is too unreliable to be used in prognosis . the results given in table iii showed that the polarographic readings followed to a very large degree the clinical findings , and it can be stated that within certain limitations this method is useful as a prognostic agent . it can be seen from table i that apart from the two groups of cancers , a high correlation was obtained and the proportion of false negatives was low . there was , in fact , only one case in the negatives shown in table i ( a ) , which remains unexplained , since the prostate case was being treated with stilboestrol , and of the breast patients , one was clinically normal and the other case gave a positive reading two weeks later . this last case supports the view that the serial examination of serum gives far greater information than a single test , a view also supported by brdicka both positive and negative cases should be followed . ( 1947 ) and robinson ( 1948 )  . a persistently positive test or a negative test becoming positive would indicate that the patient requires investigation , a positive test becoming negative indicates that the patient has improved , while a persistently negative test may be misleading ; this last fact is the main weakness of the method . 
excluding tumours of the buccal cavity and skin , which gave an equal chance of giving positive or negative readings , polarographic examination gave a 93 per cent correlation with the clinical diagnosis of malignancy . 
a high correlation with the clinical diagnosis of malignancy was obtained with the tryptophane - perchloric acid test , although it is considered too unspecific to be of use in prognosis . i wish to thank professor e . 
the concentration of aaf in the food was 0.03 per cent for 5 weeks and then 0 - 05 per cent for 20 weeks , the total period of administration extending over 25 weeks from october , 1945 , to april , 1946 . thereafter the mice received the standard diet of rat cubes for the rest of their lives and the control mice received that diet throughout . all mice had an the experiment was terminated in unrestricted - supply of food and water . the eighty - fourth week by killing the mice then surviving , namely 2 males which had eaten aaf and 3 control males . several attempts to control the localization of tumours were made on small during the third to sixth weeks of experiment 5 females of groups of mice . the aaf group and 5 control females were mated with vasectomized males and silk threads were tied in the pseudo - pregnant uterine horns to elicit deciduomata . in the twenty - second week of experiment , pellets containing 25 per cent of diethylstilboestrol ( b.d.h. ) in cholesterol and weighing 5 or 6 mg . 
the stilboestrol pellets shortened life , the testosterone tablets were harmless ; especially by causing pyometra . their possible effect on the induction of bladder tumours is mentioned below . granulation tissue , fibrosis and abscess were found in uterine horns where silk threads were tied but tumours were not recognized there . " spontaneous " tumours were found in 3 male control mice as follows : osteogenic sarcoma of the thigh , with secondary deposits in liver and lungs , in the forty - eighth week ; hepatoma in the sixty - third week ; and lymphoma in the sixty - eighth week . 
the average time was 50.7 weeks , but 8 of the tumours only 1 tumour occurred were found from the thirty - ninth to forty - sixth weeks . in an effective total of 8 mice carrying testosterone tablets ; 6 of the mice survived from 58 to 64 weeks without tumours . 
the incidence as a percentage of the effective total was 12.5 for aaf males with testosterone tablets , 73 - 3 for aaf males without testosterone , and 52 - 2 for all aaf males . most of the tumours were intravesical , pedunculated or sessile papilliferous tumours of transitionalor squamous - cell types , and conformed with the descriptions of armstrong and bonser ( 1944 ) , who distinguished benign and malignant forms but observed no extra - vesical extension . 
the distribution of tumours amongst these and less common sites depends on the species and on the strain of the animals used ( bielschowsky , 1946 , 1947 ; armstrong and bonser , 1947 ; dunning , curtis and madsen , 1947 ; harris , 1947 )  . harris concludes that " the site at which tumours may be induced by fluorene compounds is probably determined by the genetic constitution of the rat employed . " this statement , though true , is not a definitive explanation . 
the genetic constitution determines how particular cells react to particular stimuli ; remains to determine what reactions are iminportant in the production of tumours by acetylaminofluorene and how the reactions are variable in kind or degree . the importance of the genotype is accepted ; the urgent task is to elucidate its phenotypic expression . carcinogenic hydrocarbons affect tissues remote from the site of application and increase the incidence of certain tumours , notably tumours of the breast , long adenomas and leukaemia . 
many observations , summarized recently by enge ] breth - holm and rask - nielsen ( 1947 ) , suggest that the action is , essentially , an acceleration of spontaneous tumour development . 
some actions of acetylaminofluorene may be attributable to acceleration of a spontaneous disease as , for examnple , the increase of mammary tumours recorded by armstrong and in my experiment the evidence for an increase in the naturally high bonser . incidence of breast tumours in r3 mice was not decisive , and acetylaminofluorene did not accelerate or increase the incidence of lymphoma or sarcoma which occur , uncommonly , in normal r3 mice . 
the unequivocal effect of aminofluorene was the growth of tumours of the bladder which are not found in normal controls and which , as yet , have not been induced in mice by other carcinogens ( bonser , 1947 )  . in some strains of rats , acetylaminofluorene notably induces carcinoma of the external auditory canal , previously unrecorded . these and other comparable observations show that tumours induced by acetylaminofluorene do not grow preferentially in tissues prone to develop " spontaneous " tumours and are not accelerated " spontaneous " tumours . 
foulds hepatoma is much less frequent and , as my observations show , develops later than tumours of the bladder by about 20 weeks - a substantial fraction of the life of a mouse . consequently , intercurrent diseases , mammary tumours in females or bladder tumours in males kill most r3 mice before the response of the liver to aminofluorene is manifested by the growth of hepatoma . 
some geneticists maintain that the processes directed by allelomorphic genes differ only in speed . speed of reaction is probably one of the most important expressions of the genetic factors which govern the distribution of the tumours induced by acetylaminofluorene . 
the high and relatively early incidence of mammary tumours in female r3 mice presumably reduces the opportunities for other tumours to become evident , but it does not account for the complete absence of bladder tumours from the females in my experiment , or for the lower incidence in females compared with males in the 5 strains studied by armstrong and bonser . 
tumours of the bladder grew in about half the male mice which survived 9 months or longer from the beginning of the experiment . testosterone seemed to inhibit the induction of bladder tumours in males and none grew in females . 
stern. from the general hospital , northampton . received for publication june 12 , 1954 . although a study of the morbid anatomy of carcinoma of the bronchus can have little bearing on the important problem of etiology , nevertheless any ascertainable facts about this increasingly common disease may contribute something to our knowledge of it . recent comprehensive surveys of the pathology of carcinoma of the bronchus have been made in this country by harrison ( 1950 ) , on 353 confirmed cases seen at st . 
the average age at which death occurred was 56.4 years , slightly higher in males ( 56 - 7 years ) than in females ( 54 - 7 years )  . 
on the other hand both willis and bryson and spencer found metastases in the adrenals much more often than they were discovered ( willis 40 per cent , bryson and spencer 23 per cent , present series in this series . 13 - 7 per cent )  . 
the frequency with which cerebral deposits have been recorded in carcinoma of the bronchus has varied widely with different observers . those who have investigated metastatic tumours in the brain have found that a very high proportion of these had their primary source in the lung . elkington ( 1935 ) , who analysed the records of the national hospital for the period 1918 - 1933 , found that of 72 metastatic cerebral tumours , 24 ( 33 per cent ) originated in the bronchus . 
the highest recorded figure is that of fried and buckley ( 1930 ) , who recognized secondary cerebral deposits in other observers have given much lower figures : reingold , ottoman 41 per cent . and konwaler ( 1950 ) 30 - 5 per cent , willis ( 1952 ) 21 per cent , bonser ( 1934 ) 16 per cent , harrison ( 1950 ) 20 per cent , bryson and spencer ( 1951 ) 17 per cent , simpson ( 1929 ) 13.5 per cent ( in a series of 139 cases ) , kilkuth ( 1925 ) 12 - 6 per cent ( in a series of 246 cases )  . few authors have differentiated between multiple cerebral deposits from carcinoma of the bronchus and large single masses except to say that the former are common and the latter rare , though kilkuth ( 1925 ) reported single masses in 13 of 31 cases with cerebral metastases . although the present series is small it is noteworthy that in 14 cases with cerebral metastases only 6 were multiple and 8 single . 
the multiple deposits were widely distributed and showed no predilection for any particular area of the in all except one of the cases with multiple cerebral metastases there were brain . also secondary deposits in the viscera . 
on examination there was a complete hemiplegia and a dubious hemianosevere neck rigidity was present , but examination of the cerebro - spinal fluid revealed pia . a normal fluid except for pressure , which was 190 mthe only abnormality disclosed by examination of the chest was diminished air entry in the upper zone of the left lung with deviation of the trachea to the lethe differential diagnosis made was betweenr tuberculous encephalitis and cerebral thrombosis . 
death took place 2 days after admission . at post mortem dense fibrous adhesions were found almost completely to obliterate the left pleural cavity . there were old fibrous adhesions at the right apex . 
the trachea and arising from , and producing main bronchi contained a considerable amount of muco - pus . almost complete obstruction of the left upper lobe bronchus was a hard , white tumour , distal to this tumour the lobe was collapsed and contained approximately 5 cacross . a number of irregular cavities filled with thick yellow pus . 
the left lower lobe was studded with a number of nodules of tumour up to 0 ' 5 cacross . in the upper half of the right upper lobe there was an irregular tuberculous focus 2 cacross consisting of a central caseous mass enclosed in dense fibrous tissue . 
her main complaint was loss of weight and a cough of 3 months ' duration , which had been accompanied by a blood - stained sputuon examintion the fundi could not be seen . 
an x - ray showed a mass extending from the left hilum and involving the mid and lower portions of the left upper lobe . the patient died 12 days after admission . at post mortem a large , white , firm tumour mass was found involving the whole of the medial portion of the upper lobe of the left lung . 
no mediastinal glands were infiltrated and no other deposits were found in the viscera . the skull was normal , but attached to the flax cerebri at the vertex of the brain , and pressing on the pre - rolandic portion of the left superior frontal gyrus there was a firm hard tumour the size of a cherry ( proved histologically to be a meningioma )  . the cerebral convolutions were somewhat flattened . 
onimmediate examination a weakness of the left face was noted and there was slow nystagmus on lateral and upward deviation of the eyes . the reflexes were all exaggerated but the plantar responses were flexor . a diagnosis of cerebral tumour was made tentatively , but before further investigations could be carried out the patient died suddenly the day after admission . at post mortem a fungating , cauliflower - like growth was found in the upper lobe of the left lung . 
no enlarged mediastinal glands were present . the pelvis showed no evidence of any local or secondary lymphatic involvement from the cervix . the brain was moderately congested and oedematous . the left half of the cerebellum was replaced by a degenerating , cystic tumour . histologically the growth in the lung and the secondary deposit in the cerebellum were found to be an unspecified type of carcinoma of the bronchus . ( sections were not available for personal study . ) chronic bronchitis " and convulsive attacks with loss of consciousness which began a fortnight before admission . six months previously , when he had attended hospital as an out - patient , an x - rayhad been suggestive of bronchial neoplasm , with displacement of the mediastinum to the right side . 
the cerebro - spinal fluid was normal apart from an increased pressure of 250 man x - ray of the chest gave evidence of neoplastic formation in the left hilar region with associated collapse at that base . death occurred 3 days after admission . at post mortem the left lung was found to be collapsed and the left pleural cavity contained about 500 c.c. 
the left main bronchus was compressed by a soft , buff - coloured tumour , about 8 cin diameter , which was covered by a layer of lung 2 cm . this layer of lung was relatively airless and thick yellow pus was expressed from the thick . the oesophagus and aorta were displaced posteriorly by the tumour . 
46 on account of frontal headaches , throbbing in character and especially severe in the mornings , which had been present for 3 weeks . just before admission much nausea and vomiting had developed . there had been no cough at any time . 
the arm and leg sides . reflexes were normal , but some past pointing was noted in the right arlumbar puncture gave a yellow fluid under 300 mof pressure . the protein was increased to 120 mg . 
the patient deteriorated rapidly and died 3 weeks after admission . at post mortem a hard , white tumour , 1 in diameter , was present at the apex of the right lung . 
no abnormalities were found in the abdomen . in the brain there was a secondary deposit , just over 1 in diameter , deep in the left temporo - parietal lobe . the mass impinged on the left lateral ventricle , which was slightly dilated . 
a mixed type of types of cells encountered in these tumours were : alveolar cells and oat cells , 4 cases ; alveolar cells and polygonal cells , 2 cases ; and one case each of spheroidal cells and squamous cells ; polygonal cells and oat cells ;  . 
it seemed likely that 1131 would prove so since it is specifically concentrated in the thyroid , whose cells are thereby submitted to a course of ~ - ray irradiation . 
the problem is important , sirice we do not yet know the carcinogenic hazard of 1131 in clinical medicine , where it is being increasingly used both tracer and therapeutic doses . 
hyperplasia and adenomas of the thyroid have been produced by goitro gens ( griesbach , kennedy and purves , 1945 ) , and by goitrogens together with the carcinogen acetylaminofluorene ( a.a.f. ) ( bielschowsky , 1944 )  . 
when the following work was started in february , 1948 , there were discrepancies in the literature as to the incidence of thyroid tumours in untreated rats and the carcinogenic potencies of goitrogens , which were due to the different rat strains , ages and living conditions , times of application of the in view of the discrepancies and in order drugs and criteria of malignancy used . 
the rats were killed by coal gas_ at an average age of 15 months , having had treatment for an average of 13 months ( including the rest period )  . 
the trachea and thyroid attached was fixed in helly 's fluid ; the thyroid was then dissected off the trachea and weighed to the nearest milligra the thyroids were embedded in wax , serially cut at 5 and mounted on to slides as ribbons of 8 to 14 sections , including both lobes . 
the number of rats used was 98 to obtain data for the main experiment , at the end of which the remaining 15 from various groups were put aside for study of the thyroid ! 131 uptake by direct measurement and autoradiography . 
 the indication of adenomas by plus signs in tables i , ii , iii , iv and v is not devised to be any more than a crude indication of tumour incidence ; the object was to seek for major rather than minor differences resulting from the various treatments of these small numbers of rats . 
 i have not given a detailed description of the histology and histogenesis of the adenomas , because in addition to the references cited in the introduction , there have appeared further detailed illustrated descriptions by purves and griesbach ( 1947 ) , money and rawson ( 1947 ) , laqueur ( 1949 ) , and hall and bielschowsky ( 1949 )  . 
 nil + + + + + + + + + nil female * number of months before the rats were killed when they received the first of their two injections + represents i adenoma per gland ; + + , 2 or 3 adenomas ; + + + , multiple adenomas . 
in many areas it was difficult to differentiate the adenomas , and the number of plus signs awarded in the thyroid adenoma column of table ii is probably an underestimate . 
however , the cells of these extra and intracapsular and juxta - venous follicles were quite innocent in appear ance , and in no way different from those constituting the main mass of the glands . 
the effect of the 1131 was such that every one of the glands in this admittedly small group showed a most striking increase in adenoma formation as compared with the rats treated with methylthiouracil alone . 
 female male 10 14 14 14 14 17 11 11 14 14 14 15 14 14 14 11 12 12 11 11 died killed died killed died kiiied di~d killed di~ died killed died killed 229 thyroid adenomas . 
 100 168 100 129 136 116 120 160 125 130 110 125 130 155 110 120 130 125 140 130 175 117 119 125 120 108 124 107 acetylaminofluorene + methylthi , ouracil + radioactive iodine . 
 the adenomas were larger in size as well as increased in number , and showed evidence of malignancy by a gross increase in size and by dissemination outside the thyroid in two instances . 
secondly , as shown by autoradiography ( leblond and gross , 1948 ; doniach and pelc , 1949 ) , there is a wide variation in concentration from follic ] e to follicle ; the peripheral follicles in the rat take up considerably less iodine than the central ones . 
the radiation effect~ are due mostly to radiation , since most of the y rays , which are much more penetrating , will not be absorbed in the small thyroid of the rat . 
they found that this radiation did not interfere at all with the ability of the thyroids to take up further iodine , produced no histological changes , and showed no alteration of thyroid function as gauged by various physiological tests . 
their rats averaged 40 per cent maximal uptake of psi , ours was nearer 20 per cent , and our total psi injected was 32 , theirs was 30 c . , therefore our dosage was roughly 15 , 000 roentgen equivalents physical . 
from the long - term view in our experiments this dosage proved damaging , as shown in table vi , where it can be seen that the thyroid weights of all psi treated rats in all groups was one - half to one - third of their respective controls . 
clearly though , enough thyroid had survived to maintain normal growth of the rats , since the body - weights did not differ from their respective controls in spite of the fact that the first dose of psi was administered at an average age of 2 months . 
methylthiouracil rat killed 12 days after cessation of 13 months ' treatment , showing intense blackening over t , he colloid of the normal follicles and less intense but definite blackening over the colloid secreted by the adenoma lying in the centre of the photomicrograph . 
this is the accepted mechanism for the goitrogenic action of anti thyroid " drugs , such as thiouracil ( astwood , sullivan , bissell and tyslowitz , 1943 ; mackenzie and mackenzie , 1943 )  . 
nevertheless , this nodular hyperplasia must be regarded as a definite deviation from the normal , akin to tumour production both on morphology , and on the grounds that it may lead to malignant change as shown by previous and the present work . 
this may well be due to the fact that the dosage of pat used was destructive enough to the thyroid temporarily to diminish thyroxine formation , and thus step up thyrotrophic hormone production . 
it is interesting to note in this connection that the same number of rats showed adenomas in the pat group ( table ii ) as in the methyl thiouracil group ( table iii ) , and that the number of adenomas was greater in the former group . 
 the problem remains as to why the controls show an occasional deviation from the normal in the formation of adenomata , and how does prolonged excessive thyroid stimulation by the pituitary intensify this deviation , and by what means additional carcinogen leads to a further increase in adenomas . 
experimentally , gorb man ( 1947 ) found hyperplastic thyroid tissue within the lumens of pulmonary vessels of chronically thiouracil treated mice as well as parenchymatous pul monary deposits considered to be probably thyroid in orig yet a return to normal diet after prolonged goitrogen treatment was followed by involution of the supposedly cancerous thyroids . 
one may assume , therefore , that two types of metastatic thyroid tissue exist , one , possibly resulting from a mechanical breakaway , still dependent upon thyrotrophic hormone for its survival , the other independent . 
neverthe less , from the host 's point of view the first type of metastasis may prove just as embarrassing as the second , so long as thyrotrophic hormone stimulation is main tained . 
prolonged thyroxine treatment , used in clinical medicine in the therapy of nodular goitre , may , by inhibiting thyrotrophic hormone production , not only shrink the goitre , but lessen the likelihood of mechanical production of thyroid metastases and of their survival . 
the radioactive iodine was found to significantly increase the formation of thyroid adenomas as compared with non ! 1 31 treated controls in the following groups : those treated with ! 131 alone , those treated with methylthiouracil and those treated with methylthiouracil plus acetylaminofl.uorene. 
3 , and the cancer re8earch laboratories , department cf pathology , the medical sclwol , birmingham , 15 . received for publication may 28 , 1952 . the experiments recorded in this article were carried out in the course of investigations on behalf of the medical research council , a sub - committee having been set up in 1949 to inquire into the careinogeiuc properties of mineral oils and allied products . 
the arrangements for co - ordinating the research have been briefly described by auld ( 1950 )  . the entire series of tests have been made in duphcate at the cancer research laboratories , department of pathology , university of birmingham , and at , the chester beatty research institute , the royal cancer hospital , london . 
at each centre the activity of 3 selected crude oils 4 fractions derived from each crude by methods of distiration specially designed to avoid high temperature cracking , and the final residues , have been tested on groups of 50 mice for each sample and also on a total of 105 rabbits . 
he had already shown ( berenblum , 1945a ) that tumours were readily elicited on the rabbit by 9 : 10 - dimethyl - i : 2 - benzanthraceiie , which was found to be more potent to rabbits than to rats or guinea - pigs , in which positive results were , however , obtained ( berenblum , 1945b )  . 
he also subsequently observed ( 1947 ) that this hyd - r - ocarbon when injected subcutaneously did not induce rabbit tumours , tbough rats and guinea - pigs responded by this method . 
much earlier , oberling , sannie ' , guerin and guerin ( 1937 ) had shown that a i per cent solution of 3 : 4 - benzpyrene in benzene was active on rabbits ' skins , and berenblum ( 1945a ) observed that 9 : 10 - dimethyl - i : 2 - benzanthracene was much more active than benzpyrene at a similar ( o 5 per cent ) concentration . experiments using a high boiling fraction of mineral oil from an experimental catalytic cracking operatiori have recently been carried out by smith , sunderland and sugiura ( 1951 ) , testing the residue after removing the lower boiling naphtha and li - aht aas oil cuts , upon mice , rats , guinea - pigs , rabbits and rhesus monkeys . the rats and guinea - pigs were found to be refractory to skin applications but papillomata were elicited in all of the 6 monkeys , 2 being proved cancerous by papillomata were produced on biopsy 4 years after the start of the experiment . the inner surface of the ears of the 21 rabbits within 100 days . 
the number and size of such growths tended to increase during the 2 years in which painting was continued , and in 3 of the 6 surviving animals cancerous changes in the growths for tests on the type of material which they intended to study , were observed . namely , samples containing oils which had been subjected to a process of fluid catalysis up to 950 ' f . 
at birminorham they were selected , 25 of each sex froni an outbred laboratory albino strain previously employed in grading a series of oil fractions ( woodhouse and irwin , 1950 )  . in london the mice used were from laboratory stocks randomised so that each group of 50 contained some of each colour and genetic constitution . 
they were housed in fairly small cages of usual type and fed on greens alternated with crushed oats and a little bran . applications of the oils to patches of skin about 3 csquare from which the hair had been removed by electric clippers were made twice weekly . six areas on each of 30 animals were used for testing the 3 crudes , and 75 animals were used for the 15 derived fractions employing 7 areas on each animal . 
the 6 areas comprised the 2 ears , left and right thoracic , and the left and right lateral abdominal . the seventh area was the inter - scapular reorion . these sites are shown in fig . 
irwin ( medical research council 's statistical research unit , university of london ) , so that a statistical evaluation of the effects of site variation and differences in response by the individual animals might be possible . it will be apparent that each crude was applied to 60 sites and each of the other fractions to 35 sites . appheations were made twice weekly using approximately 0 3 ml . 
woodhouse in contrast , there was a total of 61 tumour - bearing rabbit sites in the birmingham series and 39 in the london series out of a possible 705 sites in each case , or , excluding the crudes and residues , which altogether yielded only 4 tumours ( london series ) , the remaining 12 fractions produced 61 and 35 tumours in the birmingham and london sen ' es respectively on a possible 420 sites . 
the addition of methionine to the basal diet affords good protection against fatty cystine is not as livers and the health of the animals was much improved . reliable as methionine in this respect , probably on account of its toxic action ( curtis and newburgh , 1927 ) , and the growth rates of the animals in the cystinesupplemented diet show considerably more variation than those of the animals on the basal and methionine - supplemented diets . in experiments on the growth - inhibitory action of 1 : 2 : 5 : 6 - dibenzanthracene in rats maintained on a 5 per cent protein diet it was noticed that the treated animals had fatty livers . this 5 per cent protein diet ( for composition see table ii ) contained considerably less fat than the white basal diet , and the control animals maintained on it did not develop fatty livers during the course of the experiment ( up to 56 days )  . although combination of the hydrocarbon with essential sulphur - containing amino acids has been shown not to be a direct cause of its growth - inhibitory action , it was considered possible that on the very low ( 5 per cent ) protein diet such a combination might be sufficient to prevent the lipotropic action of these amino - acids , and thus result in the development of fatty livers . 
 - in the present investigation it has been found that the addition of cystine or methionine to the basal diet without incorporation of the growth inhibitory hydrocarbon brings about an increase in growth rate of the animal ; hence if the effect of this addition is also to negative the growth - inhibiting action of added hydrocarbon , the resultant growth rate should be greater than that produced by the basal diet alone , which , on white 's results , is not the case . the white basal diet is a very poor diet for maintaining the health of the animals , and a number of deaths occurred after a few weeks . 
campbell. from the poultry research centre at the university of edinburgh and from the royal ( dick ) veterinary college , edinburgh . received for publication february 17 , 1949 . in july , 1944 , during the course of a long - term investigation into avian neoplastic disease , a post - mortem examination of a duck showed it to have multiple liver tumours . unfortunately , this case was not seen personally and no record was kept of any details apart from the sex of the subject - a female . however , material was taken for histological examination and a diagnosis was later made of liver - cell carcinoma . primary cancer of the liver is known to occur in chickens , but is relatively uncommon . 
two of these sources provided 3 cases each , a third provided 4 cases ( one of which was not seen personally ) , and a fourth flock provided 8 cases from a flock of 12 ducks . 
one of the latter cases ( case 17 ) had 2 different simultaneous neoplastic conditions , namely a large thoracic histiocytic - sarcoma of undetermined origin , and a much smaller hepatoma . prior to the first observed case in 1944 a total of 21 ducks had been examined in 4 years , and no case of neoplastic disease was encountered . 
no information was obtained regarding the 2 rema several birds suffering from this condition have been examined alive , and in the later stages of the disease the symptoms are very characteristic . there is usuary a history of loss of appetite and of lethargy . 
as the disease progresses , general weakness brings about the loss of locomotory power , and death superv - enes either suddenly from an internal haemorrhage , or slowly from cachexia and exhaustion . if examined during this late stage , a dropsical condition of the abdomen is apparent , and careful palpation shows the prewnee of an enlarged nodular liver projecting wer beyond the caudal extremity of the stemum on the floor of the abdomen . 
usuary one lobe is mainly involved , but there is no definite predilection for right or left ical caw , the fiver tumours are nodular and bright green in colour lobe . 
ina and covered with a tense glistening membrane containing ramifying vessels . other tumours may take the form of firm , cream - coloured nodules , probably reprewnting more recent growths not yet stained with bile . haemorrhagic or necrotic nodules are not uncommon . 
these trabeculae may merge towards the centre of the tumourous lobe to form a broad , well - defined " tnmk " from which the trabeculae radiate outwards like branches wig . 
the liver cells forming these structur ' es - are cuboidal , columnar , or polyhydral with a faintly granular eosinophilic cytoplasm ; they contain a rather small , well defined nucleus , 3 - 4 [ l . 
the nuclei of those cells forming tubules are often situated ceiitrally or adjacent to the lumen . there may be attempts at the formation of a central vein and the cells immediately apposed to the endothelium are connective tissue divides the tumour into irregular usually bolumnar in form . lobules and contains numerous aberrant bile ducts . at the other extreme is the anaplastic type , which is characterized by an almost complete loss of differentiation towards a glandular structure . 
the cells are large and polymorphic with vesicular nuclei varying greatly in size and shape , some exhibiting hyper - chromatisand containing i to as many as 10 typical measurements for such cells are , prominent eosinophilic nucleoli . nucleus 13 - 6 [ l . 
the cells are fi - equently columnar and in such cases the nuclei are usuary situated mitotic figures are common and the proximary , or remote from the lumen . rumour appears to grow mainly by infiltration . 
24 hours old filtrate prepared by grinding up liver tumour tissue in a glass homogenizer , centrifuging , and filtering , using a seitz type filter and ford 's grade g.s. 
a liver fragment and some of the sahne in which it was minced , were placed into broth which - on the fohowing day was found t - o be sterile . on the 20th of the month 2 embrvos were found to be dead . 
of the 5 , 4 had small growths in the membrane - disappointingly smafl considering the cultivation period ( 12 days )  . three of these were removed , dissected awav from the membranes and reimplanted into two ' - d - day - old embrvonated eggs . 
fragments of tissue were immediately implanted into eggs , 4 containing 101 - dav - old embrvos , and i being 9 davs . the eggs were opened 9 davs later ; 4 contained tumours . 
the eggs containing the second passage implants were opened 10 davs later , and 3 contained small tumours between 2 - 3 mdiameter . later a further biopsy on case 20 provided material to implant into three 8 - dav embrvonated eggs . 
the bird was then 8 months old . fourteen we ' eks later it was observed to be weak , losing weight , and to have an enlarged abdomen . it was destroyed and examination showed a large liver tumour weig ' hing 280 g . 
the original tumour was from duck case 18 . on the 3rd day i chicken was found in a dying condition and was kired . the pectoral muscle contained a smar greenish - brown growth which was taken for this was approximately 66 hours subsequent to injection . 
10 shows the same tumour - bearing membrane photographed aaainst a white background , and the lesions are then barely perceptible . upon histological examination , an interesting picture is revealed . 
yolk - sac cultivation did not give satisfact - ory results , this also being the experience of twombly and meisel ( 1946 ) ; although taylor , thacker and p nini ( 1942 ) , and several other workers claimed a high perentage of intraviteuine growths with the particular tumours these were mainly mouse and rat mam iary carcinomata , and the poor used . results obtained by others , using different tumours , seems to indicate that the environ.ment provided by the chick embryo yolk - sac is pecuharly suitable for the metabolic requirements of mam lary cancer cells . on the other hand , most of the tumours implanted on the chorioallantois 208 j . 
the sudden occurrence of a number of cases of hitherto unrecorded primary carcinoma of the duck 's liver provided material for cultivation by various methods , with the main object of searching for a transmissible factor or virus . 
the chorioallantoic method of cultivation , therefore , seemed particularly suitable , and was extensively used . there appear to be two possible explanations of the etiology of this condition . firstly , the disease might be associated with a transmissible factor . 
 ' it is known that the mouse mammary cancer factor of bittner is transmitted from mother to offspring by the milk , and this is one of the very few known instances of a tumour of epithelial nature associated with a virus - like principle . it was felt that the duck liver carcinoma might also contain a factor transmissible probably via the egg , which might be capable of propagation in the same manner as the milk factor of mice . experiments designecl to test this theory , however , have given negative results as far as transmission by cell - free material is concerned . 
but the sui - vival of these heterologous grafts for at le - ast 6 davs in the muscle and peritoneum of chick - ens is rather remarkable . the second possible explanation of this apparent outbreak of liver cancer , is a genetic one . 
a complete circuit of the vascular svstem throu h the pulmonarv circulation and so to the spleen t - ia the splenic arterv . in the one case encountered in the duck , pulmonarv metastases occurred , and once the lungs are involved so this second hypothesis mav be the correct one . tumour cells or emboli could then the tumours there mav also metastasize reach the spleen without much diffieultv . 
a direct spread t - ia the lungs seems iinlikelv in view of the formidable barrier offered bv the pulmonarv capillaries . fox and bartels ( 1928 ) favour the niew that in the human subject the spleen and mesenteric veins mav be invacled bv retrogtade extension along the portal in case 6 , besides seros - al implantation on the wall of the int - estine , tumour vein . emboli were found within the veins of the gut wall . 
greenwood 's flock of brown - legliorns , and i have pleasure in recording niy appreciation of iiis co - operation iii this respect , and for much help in otlier directions . the histological preparations are the work of messrs . 
whitfield jarcho ( i936 ) also states : has often biased the clinician in favour of a purely haematological diagnosis . despite the frequent difficulties in chnical diagnosis the primary growth and / or its metastases were always readily visible at autopsy . jarcho ( 1936 ) stated that many patients dying from mahgnant thrombocytopenic purpura also showed lymphangitic carcinomatosis of the lungs althou - ah histological examination was often necessary to demonstrate its presence . 
he adopted the term " diffusely infiltrating carcinoma " to cover both mahgnant purpura and lymphangitic carcinomatosis of the lungs , suggesting that they were manifestations of the same disease process . 
on boxing day , 1952 , she noticed profuse painless haematuria which lasted for 19 days . investigation showed no casts in the urine , a normal blood urea and normal intravenous and retrograde pyelograms . 
the urine contained a few red cells , polymorphs and cohform bacilli . hess 's test was negative , the bleeding and clotting times were normal and the prothrombin was 100 per cent . there were 2 , 180 , 000 red cells , 46 per cent haemo lobin , 5 , 000 white cells and a again there was normal differential count . 
no cause for the purpura had been found during life . po - st - mortem finding8 . there were many skin ecchymoses over the legs , thighs and arms , some of which extended deeply into the underlying tissues . there were petechial haemorrhages on the posterior third of the tongue and soft palate . 
the left breast and axilla were normal . the pleural surfaces of the lung8 were normal . their cut surfaces were unusually bloodless , firm in texture and grey in colour . these gross appearances suggested diffuse pulmonary fibrosis . there was no evidence of carcinomatous infiltration . 
the hilar lymph node8 were enlarged and firm and their cut surfaces in view of the previous history showed several tiny grey homogeneous areas . of breast carcinoma a frozen section was prepared from one of these glands . it showed secondary polyhedral - celled carcinoma consistent with a primary origin in the breast . these nodes were not adherent to the hilar blood vessels which were normal in all respects . 
two similar para - tracheal lymph nodes were found i cinferior to the thyroid gland . the upper 12 cof both femoral shafts contained pink marrow which sank in water and which on close inspection was flecked with several pale homogeneous foci measuriaig 1 - 2 min diameter . there was absorption ofthe bony trabeculae . a smear prepared from this marrow showed irregular clumps of malignant cells . the rest of the marrow was fatty . immediately inferior to the left lesser trochanter was a smooth walled cyst 2 x i cdiameter . 
the femoral marrow would have been accepted as showing hyperplasia and a simple cyst resulting from previous haemorrhage if carcinomatous deposits in the pulmonary lymph nodes had not been confirmed histologically earlier in the course of the autopsy . the 8ternum showed hyperplasia of red marrow and absorption of tlle bony trabeculae . the brain showed symmetrical swelling of both frontal lobes and bilateral sectioning revealed extensive tentorial hemiations . iiitracerebral haemorrhages involving the white matter dorsal ancl anterior to the genu of the bilateral 100 w . 
whitfield corpus callosuthe haemorrhages extended backwards on both sides as far as the post - central gyrus , becoming continuous at this level by involvement of the body of the corpus callosuboth lateral ventricles contained recent bloodclot . 
the basal gangha , brain - stem , cerebellum , venous sinuses and pituitary showed no macroseopical changes apart from occasional petechial haemorrhages . the vessels of the circle of wilhs and their major branches showed no evidence of embolism or thrombosis . the thyroid was small , hard , had an irregularly scarred outer surface and showed several brownish areas - each 1 - 2 min diameter - on its cut surface . these areas resembled old areas of haemorrhage . 
the gall bladder was normal . several para - aortic lymph nodes felt firm , their cut surfaces were a uniform pinkish - grey colour and were devoid of definite metastatic deposits . the pericardium , heart , skmmch , intestines , spleen , pancreas , suprarenals , urinary bladder , fallopian tubes and ovaries all appeared normal macroscopically . the uterus contained several small intra - mural fibroids . although there had been histological confirmation of secondary deposits in the pulmonary lymph nodes and bone - marrow during the autopsy , it should be emphasised that there was no other definite evidence of gross metastases , despite extensive examination of all viscera . 
the axillary lymph nodes were extensively infiltrated with similar carcinoma . ( 2 ) tissues taken at autopsy . - the pulmonary and right axillary lymph nodes and femoral marrow contained microscopical metastases which resembled the primary tumour morphologically . the abdominal lymph nodes , thyroid and posterior lobe of pituitary showed scanty microscopical metastases nevertheless still identifiable with the primary growth . 
the above tissues also showed intra - vascular carcinomatous einboli , which had not effected extra - vasc - ular extension . all other post - mortem material examined showed numerous intra - vascular , micro - emboli composed of compact clumps of polyhedral carcinoma cells devoid of stroma . 
the cells were cytologically identical with those which constituted the primary tumour , but because of the minute size of the emboli the general pattem of the primary was lacking . 
the renal tubules contained red blood cells or occasional clumps of tumour cells - " tumour casts . " intracapillary carcinoma plugs were seen in the dermi8and8ub - cutaneousfat in sections prepared from areas of skin ecchymoses . 
the post - operative history of the case may be explained in two ways . either ( 1 ) viable carcinoma cells survived in unextirpated glands or other sites of pre - operative metastasis during the major part of the long period of apparent surgical cure , and only later - perhaps related in time to the onset of purpura 6 months before deathgained access to the general circulation by venous invasion , or ( 2 ) a degree of intra - vascular dissemination was also present since the original operation , but for 71 years had not resu - ited in manifest embolic phenomena . 
the lung was the main exception to this statewidespread intra - vascular deposits , in places apparently resulting in ment . complete occlusion , had not given r ' ise to obvious symptoms or signs during life , even though cardio - respiratory manifestations arising on this basis are well recognised ( jarcho 1936 , storstein 1951 )  . neither did the lung show histological evidence of embolic phenomena . carcinoma cells had undoubtedly passed through the pulmonary vascular bed and into the systemic circulation without interruption or the formation of metastases . thik ; is a rare phenomenon and is discussed in detail by wilhs ( i 952 , page 45 )  . 
the experimental evidence of zeidman and buss ( i952 ) suggests that the transpulmonary passage of tumour emboli may occur more commonly than was previously supposed . the reason for the absence of the usual type of gross metastases in this patient is unknown . 
the fact that all organs examined showed tumour emboli , often in large numbers , does not support the view of coman ( 1953 ) that the distribution of metastases i 's dependant upon the number of embohc cells reaching the various factors other than the frequency - distribution of tumour cells appear to organs . have played a part in our case . 
the ancestral neoplastic cells constituting the primary growth clearly possessed infiltrative propensities . unless the intra - vascular descendants of these cehs had lost these inherent aggressive characteristics - and the presence of mitoses , lack of degenerative changes and the scanty but nevertheless definite microscopical foci of extravascular infiltration that were found does not support this view - one is left to assume that the unusual behaviour of this neoplasm resulted from a lack of the host stromal response which is such an essential factor in successful metastasisation . 
the possibility that " stroma - inducing " properties of the tumour cells were lacking , cannot of course be excluded by histological criteria . it is repolted ( willis , 1952 , page 272 ) that carcinoma of the breast produces intra - thyroid metastases in about 20 per cent of cases . it has also been show - n ( wilhs , 1931b ) that pre - existing abnormahties in the gland were prominent in these findings favour the cases of carcinoma showing intra - thyroid deposits . view that the associated chronic non - specific thyroiditis in our case , preceded the secondary deposits . in a recent paper describing four cases of sub - acute cerebellar degeneration occurring in association with carcinoma elsewhere in the body , brain , daniel and greenfield ( 1951 ) recorded non - specific thyroid changes in one of their patients , similar to those found in our patient . 
the status of the thyroid gland in carcinomatosis seems to merit further investigation . it will depend upon the results of such studies as to whether relationship can be seen between the thyroid changes and the unusual features of our case . the presenting symptoms of haematuria and skin purpura were related to the presence of tumour emboli in the skin and glomerular capillaries . 
haematological investigations during the life failed to elucidate any other aetiological vascular changes in the skin in malignant disease have recently been factors . described ( forman 1952 ) and may have contributed to the skin lesions shown by our p ' atient . the terminal acute massive cerebral haemorrhage may have resulted from the rupture of a small degenerative blood vessel traversing a pre - haemorrhagic focus of softening , a mechanism postulated by globus ( 1937 ) and further substantiated by globus and epstein ( 1953 )  . 
the presence of embohc phenomena elsewhere in the body , the unusual distribution of the effused blood , and the absence of cerebro - vascular disease excluded the possibility that this wa - s a coincident haemorrhage of the classical spontaneous variety . 
madow and alpers ( 1952 ) have described a case of cerebral softening due to multiple cerebral carcinomatous emboli , the primary tumour being a squamous carcinoma of bronchus . as in our case there were no macroseopical cerebral deposits but other visceral metastases were however abundant . 
the above authors also refer to three previously recorded cases of cerebral softening resulting from blood - bome cerebral lesions due to massive neoplastic embohsm carcinomatous emboli . are reviewed by till and fairbum ( 1947 )  . 
the petechial haemorrhages , microscopical softeiiings and focal demylination in the cerebral white matter of our patient resembled experimental lesions produced by the injection of calibrated paraffin emboli ( swank and hain , 1952 ) and sterile cod - liver oil ( lumsden , 1950 ) into the cerebral circulation . cerebellar degeneration ( brain et al . , 1951 ) and peripheral neuritis ( dennybrown , 1948 ) occurring in association with carcinoma elsewhere in the body have been described . 
the changes in the cerebellum and peripheral nerves of our case bear only a superficial resemblance to the lesions described in the above reports as they were focal , associated with carcinomatous emboli and did not include systematised tract degeneration as furthermore , in our case - apart from the terminal far as we could ascertain . apoplexy - the neurological lesions gave rise to no definite symptoms or signs . the platelet thrombosis syndrome ( " thrombotic microangiopathic haemolytic anaemia " ) has recently been reviewed by symmers ( 1952 ) , who states that this condition was first described by moschowitz ( 1924 )  . 
the neurohistological lesions in this syndrome were described by adams , cammermeyer and fitzgerald ( 1948 ) and are easily differentiated from those noted in our case by the presence of platelet thrombi , endothelial and probably adventitial hyperplasia in the walls of the involved vessels and less severe damage to the neural thrombocytopenia is usually present both in previously recorded cases tissue . of malignant purpura and in the platelet thrombosis syndrome . 
at autopsy gross metastases were not present and yet histology revealed intravascular carcinoma in the liver , lungs , one suprarenal , lymph nodes , bone - marrow and dura - mater . 
the degree of intra - vascular spread noted both in oertel 's and our own cases leads us to suggest that a search for carcinoma cells in marrow , skin or muscle biopsies and perhaps in urine , sputum or even blood specimens may be of diagnostic value in suspected cases of intra - vascular carcinomatosis either associated with an occult primary growth or occurring after radical excision such investigations may also help in differentiating the of a known primary . condition from the platelet thrombosis syndrome . 
we feel that even though features such as thrombocytopenia were present , tumour emboli may have been a factor in the mechanism of the purpura in these cases . the evidence that we have presented shows that our patient finauy died from the mechanical effects of tumour emboli that had not resulted in macroscopical metastases , and also that carcinoma cers may have existed " commensary " i the blood stream for many years . 
the patient died from massive intracerebral detailed histological examination estabhshed that the purpura haemorrhage . it is suggested that resulted from the effects of blood - bome carcinoma emboli . carcinoma cells may have existed in the blood stream since the original operation and that similar embohsation may also play some part in the mechanism of malignant purpura . 
the clinical picture of this case is compared with that of the platelet thrombosis syndrome . neurological lesions were present and are discussed in relation to previously recorded cases that show points of similarity . we wish to express our gratitude to professor w . 
whittick. from the department of pathology , royal cancer hospital , london . received for publication december 13 , 1952 . there is an astonishing neglect in the literature of the benign cutaneous condition described by maccormac and scarff in 1936 , and named by them molluscum sebaceuan alternative name , kerato - acanthoma , has since been applied , and this has the merit of preventing confusion with molluscum contagiosum , an unrelated condition . it has been the subject of only 4 papers , all by british authors , maccormac and scarff ( 1936 ) , musso and gordon ( 1950 ) , rook and whimster ( 1950 ) and beare ( 1953 ) , and does not yet appear to have reached text - books of dermatology or pathology . that this in no way reflects the incidence of molluscum sebaceum is certain . indeed , it is not an uncommon condition , and is still very frequently misdiagnosed as squamous - cell carcinoma by both dermatologist and pathologist . smith , in 1934 , described the occurrence in a young man of multiple primary squamous - cell carcinomas of the skin which healed spontaneously and he was unable to find any comparable case in the literature . since that time there have been eight papers on the subject ( table ii ) with descriptions of similar lesions in 11 patients . comparison of the clinical and pathological descriptions of these cases with the natural history and the structure of molluscum sebaceum at once suggests the probability that all the recorded cases of spontaneously healing epithelioma of the skin are in fact instances of molluscum sebaceum . the following account is based on a series of 7 cases of molluscum sebaceum which after biopsy diagnosis were untreated and allowed to run their natural course to spontaneous regression . 
whittick that the lesion of molluscum sebaceum closely simulates highly differentiated squamous carcinoma will be apparent from the histological description and illustrations . there is no single feature which will allow differentiation . 
about this central plug of keratin is a broad , dull red , convex and smooth - surfaced epidermal riwith regression there is gradual flattening of the lesion , and finally the keratin plug falls off to leave a clean , slightly depressed scar . ordinary squamous carcinoma usually has a much slower rate of growth with progressive increase in size for several months . although rapid growth may occur , it is usually accompanied by early involvement of the regional lymph that rapidity of growth has not been accompanied by metastasis to the glands . local lymph nodes in the spontaneously regressing cases has indeed been stressed by some of the authors . 
whittick local and lymphatic spread . were the alleged cases of spontaneously healing epithelioma true instances of squamous carcinoma there should be good evidence of local destructive invasion and lymphatic extension , but this is lacking . in over half the recorded cases the regional glands are not mentioned and , in view of the long survival period in these cases , it may reasonably be presumed that they were not involved . example in smith 's ( 1948 ) case 1 there is a history of lesions for 21 years and in his case 2 for 29 years . 
lack of lymphatic involvement was mentioned by dunn and smith ( 1934 ) , and grzybowski ( 1950 ) comments on " the lack of involvement of lymph nodes in spite of the 9 years ' duration of in only one case , charteris ' second ( charteris , 1951 ) , is enlargethe disease . " ment of lymphatic glands mentioned , and these were found , histologically , to be tumour - free . local invasion , too , appears to have been very limited in the spontaneously healing cases , being restricted to local penetration into the dermis . thus , dunn and smith ( 1934 ) , " the invasive process at its height never appears to transgress this feature applies equally well to molluscum the limits of the true skin . " description by two authors of local tissue destruction in sponsebaceum . taneously healing epithelioma requires comment . smith ( 1948 ) , when recording the subsequent history of his case 1 , states that he was an irregular attender and " permitted much further destruction of the soft tissues of his face , ' and that he now wore a prosthesis to cover extensive destruction of his nose . this on first reading seems proof of an infiltrating and destructive malignant growth , but it must be pointed out that in the previous paragraph we are told that following treatment of a lesion of the right leg by radium , the same patient had to undergo amputation of his leg because of necrosis of the tibia . lesions of his face had been treated by contact x - ray therapy . if this extensive destruction of the nose , requiring a prosthesis , was indeed due to squamous carcinoma , then the lack of lymphatic extension and the ultimate spontaneous healing ( sic ) make this case even more remarkable . 
chadwin. since completion of this paper two other relevant communications have currie and smith ( 1952 ) , under the title " multiple primary sponappeared . taneous - healing squamous - cell carcinomata of the skin " , describe 2 new cases . they maintain that the lesions they describe are unrelated to molluscum sebaceum . whittle and lyell ( 1952 ) , in commenting on a case of molluscum 'sebaceum which f . 
powell. from the department of experimental pathology , mount vernon hospital , northwood , middlesex . received for publication april 30 , 1951 . in previous communications ( powell , 1944 , 1946a , 1946b , 1947 ) it has been suggested that many of the typical properties of tumour cells can be explained by the occurrence of a derangement of the submicroscopic protein framework it was also suggested that the growth of malignant cells of their protoplasm . might be inhibited by chemical compounds able to link together the ultrastructural protein fibrils of tumour cells , and thus simulate the arrangement of comparable fibrils in normal cells . disulphide bridges constitute one of the more important classes of interconnections between the fibrils of native proteins . 
the quinone was recovered from the fitered bisulphite solution by precipitation with sodium carbonate or sulphuric acid . further purification was carried out by repeated formation of the additive bisulphite compound and subsequent precipitation as above . 
the following transplantable tumours were used : carcinoma 63 , a fibrosarcoma ( ac ) of strong a mice , a spindle cell tumour and a squamous cell carcinoma of cba mice , a rapidly growing undifferentiated sarcoma ( rb ) and a mammary adenocarcinoma of riii mice . the tumours were grafted subcutaneously in the right flanks of the mice , in each experiment the implants being portions taken from one tumour . in each experiment mice with actively growing tumours were divided into groups , control and experimental . 
1. - effect of 1 per cent phenanthraquinone upon the growth of carcinoma 63 in a strain in all the text - figures the horizontally placed numbers indicate days after inoculation , mice . and the concentration of phenanthraquinone in the food is given as a percentage of the combined weights of flour and rat cake powder . the quinone used in this experiment possibly contained a trace of the hot tasting impurity , but treated mice showed no harmful effects from it . 
2 and 3 demonstrate the similarity of the results obtained in different experiments with a particular strain of tumour when the rate of tumour growth is approximately the same , and the concentration of the drug is constant . 
1 , in which the growth of the control tumours is appreciably faster . here , with a higher concentration of the drug , smaller initial sizes and slower intrinsic growth rates of the tumours , the inhibitory effects of the treatment are greater than in c ontrol a . 
the inhibition of several spontaneous mammary tumours of a and riii strains of mice by the quinone was comparable with that of the transplanted tumours . also , the growth of a transplantable mammary adeno . carcinoma of riii mice and a transplantable squamous cell carcinoma of cba mice was strongly inhibited . thus the growth of a variety of different tumours can be inhibited by phenanthraquinone under the conditions described . unpalatable cause loss of weight of the treated animals . samples of phenanthraquinone containing sufficient impurity to make it gastro - enteritis some270 a . 
the latter causes prepared food to be unpalatable , and can give rise , if present in sufficient amount , to gastrobut , by comparing the effects of samples of quinone of varying degrees enteritis . of palatability , it has been established that the purest samples are the least toxic this greater inhibition to mice and inhibit tumour growth to the greatest extent . of tumour growth may be due in part to a greater intake of food , and therefore detailed investigation of the effects of phenanthraquinone upon of quinone . tumours and of its possible value in tumour chemotherapy is dependent upon the preparation of the drug in a pure state . its further purification and alternative methods of synthesis are under investigation . the additive compound of phenanthraquinone with sodium metabisulphite is serial intraperitoneal very readily hydrolyzed in vivo to yield the free quinone . injections in mice of aqueous solutions of the quinone - bisulphite compound in sub - lethal doses at frequent intervals indicate that the liberated quinone is very rapidly metabolized in the body . in view of the rapid destruction of phenanthraquinone in vivo and of the rapid growth rates of many tumours , it is essential that continuous administration of the drug be maintained in order to expose the tumour cells to an effective concentration of the drug for sufficiently long periods . failure to achieve this could permit a proportion of the tumour cells to grow and in the early stages of an experiment cessation of treatment with the divide . quinone may result , in some instances , in stationary tumours recommencing growth . 
on the other hand , similar tumours continually subjected to the action of the quinone fail to grow and often regress completely . in these preliminary experiments the simplest method of ensuring an adequate and continual supply of the quinone , namely , by adding it to the food , was adopted . however , this method is not entirely satisfactory . adequately controlled experiments in which individual mice receive known amounts of quinone at definite times are necessary for detailed studies of the action of the drug . very little of the quinone in a concentration of 1 to 2 per cent in the food ingested by a mouse can be available for action on substrates in tumour cells , since there is a very great disparity between this amount and the lethal dose given as the bisulphite derivative by injection . 
much of the fed quinone may not be absorbed from the food , and the greater part of that reaching the body fluids may be metabolized . sodium salts of the phosphoric and sulphuric acid esters of 9 : 10 - phenanthrahydroquinone have been synthesized and tested for possible inhibitory effects on tumour growth , but they have been found to be unsuitable for the treatment of tumour - bearing mice . 
the former very readily hydrolyzes to give the hydroquinone , which readily oxidizes to the quinone , and the latter is extremely resistant to hydrolysis . preliminary experiments upon the administration of thiol compounds concurrently with phenanthraquinone to tumour - bearing mice appear to indicate that its inhibitory action on tumour the latter reacts with thiol groups in vivo . growth may be due , at least in part , to this effect . 
powell may enable phenanthraquinone to function as an oxidation - reduction catalyst and so disturb the natural oxidation - reduction balance of tumour cells , possibly to a greater extent than that of most normal cells . in view of its considerable chemical reactivity , as well as of its affinity for proteins , phenanthraquinone despite the possibility that would be expected to have multiple effects on cells . phenanthraquinone has in vivo the mode of action postulated earlier , that is , of forming cross - linkages between ultra - structural protein fibrils , it is emphasized that its actual mode of action remains a completely open question . 
the available evidence favours the view that over a certain range of concentration tumour cells are more sensitive than normal cells to the effects of phenanthraquinone . it has been reported by mitchell ( 1948 ) that the combined antimitotic effect of " synkavit " and x - rays is greater than the additive effects of both agents used singly . 
kreyberg. from the institutt for generell og eksperimentell patologi , universitetet i oslo . received for publication april 24 , 1952 . the importance of using pure , inbred strains in cancer research is now generally accepted . strong ( 1942 ) , emphasizing this point , gave a survey of the origin of some of the best known strains , originating from his laboratory . 
the new informal document , mouse news letter , acts as a most valuable source of information as to a great many strains used all over the world . the characteristics of the different strains as to susceptibility to development of spontaneous tumours , reaction to carcingenic agents , as well as susceptibility to transplantation and other biological responses may be very marked and very different . it is therefore of great importance to have a great number of different strains with marked characteristics , in order to supply research workers with the best material possible for their special needs . the purpose of the present paper is to describe the origin , development and some of the features of our strain " white label "  . the first animals were obtained from a commercial dealer in oslo , a young man who used to supply a number of laboratories with albino mice , and who , from time to time , added animals of different origin to his stock . 
the figure shows , however , very clearly how two lines were developing with extreme differences as to the formation of spontaneous mammary carcinomas . " line 27 " produced a high , and an increasingly higher percentage of mammary tumours . 
from f2 and up to and including f14 , when this line became extinct , mammary tumours occurred in every generation , and from fg until f14 16 out of 18 females developed such tumours . from the sister of no . 
2 indicated by an arrow . in the 11 generations ( f5 - f15 ) in which there were 102 females , not a single instance of mammary carcinoma was observed . such was the situation in 1935 . in the following years the brother - to - sister matings were continued and many sidelines died out , or were not continued , but " line 426 " was kept until the war interrupted the experiment at f37 . in table i is given a complete record of the females of all the generations descending from pair ( 2 + 3 ) up to f37 . in the first column is indicated the generation . 
in the second column is given the total number of females in each generation . in the third column is given the number of females belonging to " line 426 " ; from f15 all the females belong to this line , as this is the only line surviving . in the fourth column is given the total number of mammary carcinomas occurring in each generation . finally the 24 columns show the number of animals dying in each month after birth , as well as the occurrence of mammary carcinomas , the figures in brackets . at the bottom of the table the sum total is recorded , as well as the reversed figures , indicating the number of females alive at the end of each month . table i gives a clear picture of the great number of mammary tumours occurring in the first heterozygous generations . 
from f12 it is only " line 27 " which still contributes to the presentation of mammary tumours , and from f14 when this line becomes extinct , only one single mammary tumour occurs ( that is in " line 426 " )  . 
an exception , indicated by a cross , will be mentioned later . this means that " line 426 " , during 34 generations , comprising 1595 females , presents one single case of mammary carcinoma . 
bonser , university of leeds , wrote me in august , 1949 , regarding this question as follows : " i still keep ' white label ' mice in this laboratory . 
by suckling ' white label ' babies on riii mothers i showed that they were susceptible to the riii milk factor , which i regard as a potent factor . never published these experiments , and they were only done on small numbers of mice , but they point strongly to the fact that ' white label ' mice in leeds have the milk factor , and are susceptible to it . " bonser wrote me a further letter in february , 1951 , stating that " mammary cancers still occur in our line and we regard the incidence as being about 30 per cent in mice which live to be 12 months of age or more , but i have not any recent accurate estimate of the mammary cancer incidence , because we have been very short of accommodation lately . addition : " now , in 1952 , we find that 7 of 33 females over 10 months of age have developed mammary cancer , usually rather late in life . but we make no attempt to assess the mammary cancer incidence accurately as we usually dispose of breeding females when they reach the age of one year . " i also had the following information , dated january , 1951 , from w . 
s. bullough : " my experienoe with these mice falls into two periods . first when i was working in the zoology department in leeds and when i obtained the mice from bonser . then mammary carcinoma was common in females of from 11 months of age onwards . i have no figures now , but my recollection is that it developed in not less than 40 per cent of the older females . also there was the odd occurrence in a male , though this was of course rare . " further : " this colony was allowed to die out in 1944 , but in 1946 i obtained two females and a male from bonser and started a new colony in the university of sheffield . since then a colony of some 200 - 300 mice has been maintained . . 
kreyberg further , in 1952 : " the final chapter in the story starts in 1949 when , in the autumn , i took the milk factor out by fostering one family with a strong 's cba female . 
my present colony is entirely derived from this one family , and now mammary cancer is exceedingly rare . so my present stock of your mice indeed i cannot remember when i last had a case . is without the milk factor , and i suppose it is no use for your own present purposes . " i regret very much that i cannot definitely state so , but i have strong reasons to believe that the animals sent to leeds in may , 1934 , were descendants from the pair ( 4 + 5 ) ofmy " white label "  . 
at present one " white label " strain ( leeds ) carries the milk factor and produces about 30 per cent of mammary carcinomas in females reaching the age of 12 months or more . this strain has the characteristics of the branch ( 4 + 5 ) of the original material . 
another " white label " strain ( oslo ) is devoid of the milk factor and produces practically no mammary carcinomas spontaneously . this strain is probably one of the lowest mammary for future references it is therefore of great imcancer strains in existence . therefore portance to distinguish between these two " white label " strains ; the leeds strain will be designated wll and the oslo strain wlo . 
the two strains most probably have only a heterozygous founding mother in common . this experiment has been carried out for more than twenty years . it would not have been possible without the assistance of my collaborator miss hj . 
aasland , who continued the experiment during the first years of the war , until illness and an untimely death ended her devoted and loyal partnership . during the remaining years of the war the strain was maintained at a reduced scale through the initiative and faithful service of e . 
orr. from the department of experimental pathology and cancer research , medical school , university of leeds , and the de7partment of pathology , university of birmingham.. received for publication , april 23 , 1949 . the discovery of the milk factor or mammary tumour agent ( bittner , 1936 ) and the demonstration that three factors , namely , the genetic factor , the hormonal factor , and the milk factor , are essential for the development of spontaneous breast cancer in certain inbred strains of mice ( bittner , 1939 ) , was followed by investigations of the part played by the agent in the induction of breast cancer by oestrogenic hormone . 
lacassagne ( 1939a ) , lacassagne and danysz ( 1939 ) , twombly ( 1939 , 1940 ) , and later bittner ( 1940 , 1941 ) , gardner ( 1941a ) , shimnkin and andervont ( 1941 , 1942 ) , dmochowski and gye ( 19.44 ) , found that fosternursing of low - breast - cancer strain mice by high - breast - cancer strain females increases the incidence of breast cancer in the low - breast - cancer strain mice treated with oestrogenic hormones . 
tlhus male mice do not develop breast cancer , even if they are susceptible and have the mammary tumour agent , unless the hormonal or oestrogenic factor is supplied , and only few or no breast tumours are induced in low - breast - cancer strain mice , i.e. 
it is probable that a quantitative deficiency of any one factor can be overcome by a relative quantitative increase in the other two factors ( shimkin , 1945 )  . the accelerating influence of carcinogenic hydrocarbons on the appearance of breast cancer in mice of unmknow - n genetic constitution was first observed by maisin and coolen ( 1936 ) and perry and ginzton ( 1937 ) , while dobrovolskaiazavadskala and adamova ( 1938 , 1939 ) , were unable to observe a similar effect on inbred mice employed by theexperiments carried out by mider and morton ( 1939 ) , bonser and orr ( 1939 ) , strong and smith ( 1939 ) , bonser ( 1940 ) , strong and williams ( 1941 ) , engelbreth - holm ( 1941 ) , engelbreth - holm and lefevre ( 1941 ) , orr ( 1943 ) , kirschbaum , lawrason , kaplan and bittner ( 1944 ) , kirschbaum , williams and bittner ( 1946 ) , shimkin and bryan ( 1943 ) , strong ( 1945 ) , orr ( 1946 ) , demonstrated that carcinogenic hydrocarbons induce the appearance of breast cancer in females of low - cancer strains or accelerate the development of breast tumours in female mice of high - cancer strains . 
in these experiments it was also shown that oestrogenic influence is important in the induction of breast cancer by carcinogenic hydrocarbons , since mice developing these tumours in the absence of treatment with oestrogens were females , with one exception , a cba male ( orr , 1943 )  . 
the male mice of both strains , when 60 - 70 days old , were divided into three groups : mice of the first group received oestrone and methylcholanthrene , mice of the second group methylcholanthrene alone , and mice of the third group oestrone alone . 
c57 strain females , when 6 weeks old , were divided into two groups : mice of the first group were forcibly bred , mice of the second group were maintained as virgins ; both groups were treated with methylcholanthrene alone . oestrone was applied as a 0.02 per cent solution of keto - hydroxy - oestrone in alcohol , by painting the mice twice weekly for a period of 12 months . 
methylcholanthrene was administered according to the method described by orr ( 1946 )  . sixteen drops of 0 - 5 per cent solution in sweet almond oil were applied , at fortnightly intervals , to the skin of mice , four on each side of the dorsal and ventral surfaces of each animal . the c57 females of both groups received fortnightly applications of methylcholanthrene from the age of 6 weeks till death or the appearance of a tumour . in male mice the mammary gland is in a rudimentary state and they do not have nipples ( lacassagne , 1936 )  . 
administration of oestrogenic hormones leads to the progressive development of the mammary glands described in detail by turner and gomez ( 1934 ) , burrows ( 1935 , 1936 ) , gardner ( 1935 , 1939 , 1941b ) , lacassagne ( 1939b ) , loeb ( 1940 ) , loeb and suntzeff ( 1941 )  . 
because of these observations and the findings that breast cancer can be induced by methylcholanthrene in males treated simultaneously with oestrogens ( orr , 1943 ) , the c57 and c3h strain males were painted for 3 months with oestrone before the treatment with methylcholanthrene . 
after 3 months of painting with oestrone both the 057 and c3h males received their first application of methylcholanthrene which was then continued at fortnightly intervals until death or appearance of a tumour , except in the group of c3h males painted with oestrone which received only 14 applications of methylcholanthrene . 
two 057 males and one c57 female , which were treated with methylcholanthrene , developed general enlargement of lymphoid tissue , and in the c57 male which developed breast cancer a lymphosarcoma was found in the abdominal cavity . 
one c57 virgin female developed a spindle - celled sarcoma in the subcutaneous tissue . skin tumours were found in 10 out of 18 c57 males treated with methylcholanthrene alone and in 10 out of 35 c57 males treated with oestrone and methylcholanthrene . 
squamous metaplasia was observed in two tumours induced in 03h males by oestrone and .methylcholanthrene. the breast tumours induced in c57 mice , both male and female mice , showed a much greater degree of squamous metaplasia than the breast tumours induced in some of the induced tumours , as in the case of spontaneous in c3h male mice . tumours , variations in structure between different parts of the same tumour were found . 
sometimes the tumour cells were packed in solid sheets while elsewhere they occurred singly or - in small groups surrounded by an abundant stroma ; in some parts acinar structure could be clearly seen while in other parts it was lost entirely ; in places elongated cords of spindle - shaped tumour cells were found . 
thus the observations recorded by previous workers in this field were confirmed and the influence of hormonal factors on the development of carcinogen - induced breast cancer in mice shown . mammary tumours were also induced in c57 low - breast - cancer strain females , both breeding and non - breeding . 
orr results of other investigators . in mice , both males and females , which lack the mammary tumour agent . it is , therefore , possible to induce breast cancer in the present experiments breast cancer was induced in mice of a sub - line of c57 black low - breast - cancer strain which has a low susceptibility to spontaneous breast cancer . 
breast tumours developed in c57 black strain females , both breeding and non - breeding , after treatment with methylcholanthrene , and a c57 black strain male after combined treatment with oestrone and methylcholanthrene . it is not known whether the failure of kirschbaum , williams and bittner ( 1946 ) to induce breast cancer in breeding c57 black strain females with methylcholanthrene was due to the different method of application of methylcholanthrene or to differences in susceptibility of the two sub - lines of the c57 strano tumours were observed in either c3h or c57 black strain males treated with methylcholanthrene alone . this supports the findings of other workers that hormonal factors play a part in the development of tumours induced by methylcholanthrene . orr ( 1943 ) , however , reported the induction of breast cancer in one out of 16 cba low - breast - cancer strain males after intranasal administration of methylcholanthrene in the absence of treatment with oestrogen . 
he also obtained breast cancer in virgin mice of two low - breast - cancer strains ( if and cba ) after treatment with methylcholanthrene . in the present experiments tumours developed in two non - breeding c57 females treated with methylcholanthrene . mider and morton ( 1939 ) failed to induce breast cancer in non - breeding dba high - breast - cancer strain mice , while strong and smith ( 1939 ) , strong and williams ( 1941 ) and strong ( 1945 ) in their successful experiments on the induction of breast cancer in jk , nh and nho low - breast - cancer strain mice employed breeding females only . the present experiments confirm the findings of other workers that breast tumours can be induped in male mice of susceptible strains after treatment with oestrogenic hormones ( shimkin and andervont , 1941 , 1942 ; bittner , 1941 ; dmochowski and gye , 1944 ; bonser , 1944a , 1944b )  . the present findings also confirm the observation that mammary tumours develop in mice of resistant strains following treatment with methylcholanthrene . 
the c57 low - breastcancer strain mice of the sub - line used , when foster - nursed soon after their birth by riii high - breast - cancer strain females , develop an incidence of 11 per cent of breast tumours , but fail to develop breast cancer when given the tumour agent at the age of 4 - 6 weeks , even after large quantities of the agent and in spite of increased hormonal stimulation by repeated pregnancies ( dmochowski , thus mature mice of this strain though resistant to the mammary 1948 )  . tumour agent are susceptible to a different agent , i.e. 
methylcholanthrene. the treatment with oestrogenic hormones failed to induce breast cancer in c57 low - cancer strain males . this again confirms the previous findings ( twombly , 1939 , 1940 ; bittner , 1940 , 1941 ; shimkin and andervont , 1941 , 1942 ; dmochowski and gye , 1944 ) that an increased oestrogenic stimulation overcomes the threshold of lowor non - susceptibility and results in the development of breast tumours only in the presence of the mammary tumour agent . treatment with methylcholanthrene induced in c57 black low - cancer strain females , breast tumours which , although similar in their structure to spontaneous breast cancer of highand of low - breast - cancer strain mice , show an increased incidence of squamous metaplasia . 
the samples were frozen in cyhndrical tubes immersed in a quartz dewar vessel and were irradiated with filtered light from a high pressure mercury arc , either the 3100 a or the 3650 a group of hnes being used . under these conditions afl the molecules examined were strongly luminescent , showing both a short - lived fluorescence ( persisting for less than 10 - 8 seconds ) and longhved phosphorescence ( persisting for several seconds ) in different spectral regions . fluorescence spectra were traced photoelectrically using a hilger e2 spectrograph equipped with a scanning unit . phosphorescence spectra were photographed using a mechanical phosphorescope as described elsewhere ( lewis and kasha , 1944 ; moodie and reid , 1952 )  . 
the phosphorescence spectra are the result of excitation to the very lowest excited state of the carcinogen molecule , a " triplet " state resulting from the fact that there are two unpaired electrons . excitation of this state requires only about 45 k.cal. , much less energy than is necessary to break bonds in the molecule , and its formation may well be an intermediate step in the reaction with protein . in this connection it should be pointed out that the triplet levels , since they cannot be detected in absorption , the common method of observing spectra , have been much neglected by biologists in their considerations of low barrier reaction paths that may be involved in biochemical reactioins . ( 2 ) the second significant correlation comes from heterogeneous system experiments . 
we have found that ff a carcinogen is ' used as the microcrystalfne component and the dissolved component is naphthacene , the ( fluorescent ) emission of the naphthacene is shifted shghtly in wave length because of its interaction with the carcinogen . 
some of the results are it will be observed that the smallest shffts in the naphthacene show - n in table ii . bands occur when the strongest carcinogens are used as the host crystal . there is a moderately good correlation between these shifts and the self - polarizabihties at the position of the substituent calculated by greenwood ( 1951 ) , which values themselves correlate better with carcinogenic power than do an ' other physical constants . 
harnett. published for the clinical cancer research committee of the british empire cancer campaign . received for publication july 16 , 1948 . in 1938 - 39 the clinical cancer research committee of the british empire cancer campaign carried out a clinical survey of all cases of cancer seen in the hospitals , both voluntary and l.c.c. 
in the administrative county of london . this was done by means of questionnaires , one of which was filled in by the registrars in the various hospitals for each patient and returned to the clinical cancer research committee for record . 
the work was interrupted by the outbreak of war after it had been in progress for 17 months . by this time 15 , 200 cases of cancer had been registered , of which 2529 ( 16 * 6 per cent ) were cancer of the breast . these patients have now been followed up for five years or more and the records analysed . 
4 - 556 1 8 + 0 66 . the mean age is 5 - 6 years higher than those given by lane - claypon ( 1926 ) and wainwright ( 1931 ) , due to the fact that the two latter series include only cases coming to hospital for operation , whereas the b.e.c.c. 
the youngest patient in the series was aged 15 , and survived 5 years after enucleation of a tumour of 3 years ' standing , which proved on histological examination to be an early adenocarcinoma . 
expected actual per cent of expected possible x - rays alone or with interstitial radium . number of cases of known duration mean number of months lived j in 5 years from onset maximum  . 
expected lactual possible per cent of expected x - rays with local surgery . number of cases of known duration mean number of months lived in 5 years from onset maximum  . 
it was found that patients in stage iv who were not treated either by surgery or radiotherapy had a 5 - year expectation of life of 46 - 7 per cent of normal , which was not improved by palliative x - ray treatment . all these figures must be considered in the light of the fact that the mean duration of the disease is 38.3 months ( major greenwood , 1926 ) , so that these patients whose mean age was 57 years would have a 5 - year expectation of life of about 56 months ; a 5 - year follow - up is therefore insufficient for assessment of the results of treatment . 
the variations in mean age of the patients submitted to various types of treatment is shown below , and indicates that the more radical methods were used in the younger patients . radiotherapy mean age of 703 patients treated by radical mastectomy alone mean age of 133 patients treated by local mastectomy alone radiotherapy mean age of 174 patients treated by radium alone or with local surgery h.v. 
fodden. from the department of pathology , university of liverpool , and the liverpool cancer control organization . received for publication august 20 , 1948 . in his ' textbook of pathological anatomy ' rokitansky ( 1861 ) made his classical statement that pyloric cancer was exactly bounded by the pyloric ring , and that the growth never reached beyond into the duodenufrom this time it appears that the majority of observers , with the early exception of brinton ( 1864 ) , commented upon the integrity of the duodenum in cases of carcinoma of the stomach . 
many well - known surgical teachers spoke of the habitual immunity of the duodenum from invasion . kocher ( 1893 ) , mikulicz ( 1898 ) and most ( 1899 ) believed it to be always constant . kocher ventured that it was a problem of extreme interest to consider why gastric carcinoma grows in almost all cases towards the cardia , yet stops , on the contrary , at the duodenopyloric junction . it was brinton who first took especial exception to this proposition of rokitansky . 
1. received for publication may 10 , 1951 . there is still some disagreement on the question whether the early changes produced in epidermis by chemical carcinogens are specific , i.e. , whether they can be distinguished from the effects of non - carcinogenic irritants . the chemical evidence is inconclusive . 
cowdry and his school have been able to show that the chemical constituents of mouse epidermis made hyperplastic by several applications of 20 methylcholanthrene differ quantitatively from those of normal epidermis ( carruthers , 1950 ) , but it does not appear to have been proved that these changes are produced only by carcinogenic substances . histological evidence is more definite . 
the only significant way in which such skin has hitherto been found to differ from untreated skin is in its ability to produce tumours when treated with a co - carcinogen such as croton oil . 
the " latent tumour cells , " if present , must be so few that they are very unlikely to be detected in sections , or perhaps they may not be recognizable if seen . 
the skin has passed through a state known to be produced only by carcinogenic agents ; this state has apparently passed away , yet the skin will then react to treatment with a non - carcinogenic substance in a manner quite different from normal skin . the early reaction of normal mouse skin to croton oil is similar in general type to its reaction to other non - carcinogenic irritants , and differs in important respects from the reaction to carcinogenic substances . 
the chief criteria on which this distinction is based have been fully described by pullinger ( 1940 ) and by gluiicksmann ( 1945 ) , and only a summary will be given here . 
gwynn the epidermis is stratified . in the normal skin of the body of the mouse there are no strata , and the various types of cell are arranged apparently at random in a layer one cell , or in places two cells , thick . of the nucleated cells in the epidermis of the back of a normal adult mouse , about 14 per cent are resting , 80 per cent differentiating , and the rest are mitotic or degenerating . this classification was used because it gives definite information about the state of the epidermis which is not obtainable by any other method known to this information is not dependent on any hypothesis about the function or potentialities of the different classes of cells . 
for the purpose of this work they are simply types of cells which can be distinguished , the absolute and relative numbers of which vary in different states of the skin . one of the most striking features of the hyperplasia produced by chemical carcinogens in mouse epidermis is the rise in absolute and relative numbers of the resting cells . non - carcinogenic irritants , on the other hand , produce a hyperplasia in which the absolute number of all epidermal cells is increased but their relative numbers remain approximately the same as in normal skin ( gliicksmann , 1945 )  . 
the percentage of resting cells in the epidermis was chosen as a criterion for comparison of the hyperplasia produced by the different treatments used in the following experiments . it provides a measure , which can be handled statistically , of the specificity of the reaction of the epidermis , i.e. , of its resemblance to the characteristic reaction to carcinogenic substances . in the work now reported the effect of croton oil on normal mouse skin has been compared histologically with its effect on skin after the reaction to previous treatment with a chemical carcinogen has apparently disappeared . three strains of mice and two different carcinogens were used . 
 " t " and " s " mice were purchased from dealers . " p " mice were of an institute - bred strain originally obtained from the national institute for medical research ; they had been used before for studies on skin carcinogenesis by gluiicksmann ( 1945 ) and salaman ( 1943 )  . 
the standard errors of means were of the same order in this experiment as in the other two . the mice were examined weekly for the appearance of tumours , for periods of 200 to 250 days . 
gwynn high level and remained well above normal . in group 2 there was a parallel but smaller increase . in group 3 application of croton oil without previous treatment was followed by an initial slight rise in resting cell percentage , which then these results are listed in table i and charted in fig . 
the first tumour appeared on the further trials have 63rd day in group 1 , but not till the 170th day in group 2 . confirmed the superiority of acetone over paraffin as a solvent for the carcinogen comparisons of various concentrations of croton used as the primary treatment . oil in acetone and paraffin have shown that a 0.5 per cent solution in the former is almost as powerful a co - carcinogen as a 2.5 per cent solution in the latter . 
13. - experiment 2 : 0 group 2 : 10 mice at beginning of experiment , 8 survivors at 217th day . e group 4 : 12 mice at beginning of experiment , 11 survivors at 217th day . weekly croton oil applications without previous treatment ( group 3 ) caused only a transient rise to 21 per cent on the 3rd day , and thereafter the level declined gradually to 16 per cent on the 115th day . 
13. since no tumour it will be seen that tumour appeared in the other groups , they are omitted . production in group 4 began earlier than in group 2 , but later proceeded more this suggests a possible parallelism between the early rise of resting rapidly . cell percentage and the tumour production which follows about 50 days later . ( 3 ) in a third experiment , four groups , each of 8 male and 8 female mice of group 1 , in this experiment , the " s " strain , were treated as shown in table iii . has the same function as in the last : a test of the carcinogenicity of the primary treatment with the carcinogen . 
14. as in the last two experiments , the difference between the effects of croton oil treatment with and without a previous carcinogenic stimulus is clear ( groups 2 and 3 )  . in group 4 , resting cell percentages were at first lower than , but later rose above , those of group 2 . 
15 shows the rate of tumour production in these groups . dimethyl - 1 : 2 - benzanthracene in paraffin oil appears to have been less effective than 1 per cent benzpyrene in acetone ( group 4 of experiment 2 , but note that different strains of mice were used )  . tumours appeared earlier in group 2 than in group 4 ; thus , as in the former experiment , there appears to be a parallelism between resting cell percentage and subsequent tumour production . 
gwynn except one in which early malignant change was probably present . during a considerable experience of the application to mouse skin of croton oil alone , a very few small tumours have been seen , but never as many as in this experiment . precautions against contamination of cages , etc . , with carcinogenic substances are taken , but the possibility that such a technical mistake occurred cannot , of course , be entirely excluded . it is perhaps significant that the resting cell percentage in this group , after falling to 16.6 per cent on the 132nd day , rose again to 20 per cent on the 216th day . tumours produced by the application of substances generally regarded as non - carcinogenic have been occasionally reported . 
9 and 10 are high - power views of regions of the epidermis consisting predominantly of resting cells and differentiating cells , respectively , taken from the same specimen as fig . 
many substances are as powerful hyperplastic agents as croton oil , without being co - carcinogenic . croton oil is much the most powerful co - carcinogen for the mouse 's skin yet discovered . its action appears to be highly specific with respect to animal species , and perhaps also with respect to the carcinogen with which it acts . it may be that it combines very weak carcinogenic with powerful co - carcinogenic power . 
a number of tumours subsequently appear . this number varies widely according to strain of mouse and strength of initial carcinogenic stimulus ; there may be an average of only 2 or 3 , or as many as 20 , per mouse . 
but even if the higher figure is taken , and it is assumed that each tumour was represented before croton oil treatment began by a single " latent tumour cell , " or a small group of such cells , these cannot be responsible for the generalized rise in restingcell percentage which is observed throughout the epidermis . in fact the chance of finding such a cell or group in a section is small . 
it appears that mouse epidermis which has been treated with a carcinogen , though it returns to a state closely resembling the normal , has suffered a general change which does not consist merely in the presence of a few latent tumour cells . we are indebted to professor s . 
w.3. received for publication april 3 , 1954 . the development of the search for endogenic carcinogens has been reported in earher publications from this institute , ( see hieger ( 1949 ) for references to previous papers )  . it has been shown that the cholesterol - rich fraction of ' tissues from human subjects who had died of cancer or of other causes , and even commercial cholesterol . 
the first tumour in table i was carried through 93 generations and the first tumour in table iv ( highly purified cholesterol ) has been growing vigorously in its 25th generation . 
the control tests on solvents are described further on in this paper . purification of the cholesterol was next undertaken , as a first step using the simplest technique . table ii gives the results of tests on fractions of commercial cholesterol obtained by crystalhsation from acetone . 
the method involved several stages . these were : ( 1 ) acetylation . this step provided several advantages , nameiy ( a ) the oh group was protected during bromination ; ( b ) the acetate can be differentially eluted from a1203by mild eluents the unacetylated sterol when absorbed on a1203 requires more powerful eluents . ( 2 ) bromination , filtration and debromination . ( 3 ) adsorption on a1203colunin and elution with benzene - pet . 
orr there was , of course , no guarantee that contamination could have been completely avoided in the " carcinogen - free " room ; for example , 'sawdust from a box - containing benzpyrene - treated mice might accidentally be carried in to the noncarcinogen room on the shoes of anyone who had walked through the carcinogen however , the technicians who serviced the mice in the . 
consequently the controls had more injections than the sterol injected mice in order to maintain a stationary nodule . is the carcinogenic activity of commercial cholesterol due ( a ) to the sterol itself , or ( b ) to highly active impurities ? the evidence presented in tables iv and v provides support for ( a ) , i.e. , cholesterol per se is the true carcinogen in the commercial product . if the incidence of sarcomas be expressed as the ratio ( percentage ) of tumour mice to injmted mice which survived to a minimal latent period level , say i year , then the result can be stated thus : ( a ) purified cholesterol gives rise to 4 per cent sarcomas in q ,  . 
the mice did not survive well , and in fact the single sarcoma appeared at the 27th month in the sole survivor of the 34 mice initioy injected with purified cholesterol . twenty - seven months is near the outsidei limit of the range of latent periods found with the carcinogens of the cholesterol categpyy , , in an experiment with commercial cholesterol ( table ii , section a ) 3 of the 4 280 1 . 
orr in 1947 ( unpubhshed ) , would provide an attractive theory of the mode of action of cholesterol as carcinogen . however , it would be necessary to postulate that the carcinogen is held by adsorption on the cholesterol 292 1 . 
op.p. although stif in a - n active stage , and that in sucha condition it is not roadily eluted , for clearly if it were a case of simple solubility there would be no more reason for ' the factor dissolving in the cholesterol than for it to be dissolved out secondly i ' ected cholesterol after a year or so in the body should be again . thirdly , if such an assumption were accepted , there rich in absorbed carcinogen . would be httle necessity to proceed with the question of whether pure or impure fourthly , if the carcinogen ' which is concencholesterol is the true carcinogen . trated in the cholesterol originated in the environment , it should be easy to test the hypothesis by purposely arowing the mice injected with the cholesterol to hve an atmosphere rich in carcinogen . this test has been put into operation , and constitutes a secoind stage to the investigation on atmospheric carcinogens as a factor in sarcoma induction ( see above )  . one hundred stock mice were injected with ohve oil solution of commercial cholesterol and kept in a carcinogen - rich atmosphere as described above . 
the control mice ( 135 ) were kept in the room free from carcinogen . the results are shown in table v . control te8t8 on the 801vent8 . lard was employed as solvent from 1936 to 1949 . during this time a total of over 360 mice was tested with the solvent , lard ' alone . these mice did not survive remarkably well ; they gave no positive result until the series had been almost completed , when among the last few batches a single sarcoma developed . 
when in 1950 , a change was made from lard to olive oil ( hospital dispensary grade ) , a control series of 134 mice was set up for injection with the oil alone . 
the old c " mice were 12 ( and upwards ) months of age at the start of the experiments ; their effective number should therefore be raised by a factor of the order of 2 since the initial number , 19 , would represent the survivors at 12 months . 
the siingle sarcoma occurred in an oldc57moim at the unexpectedly low latent period of 6 months . * however , s ' mce this mouse was 16 months of age at the start of the experiment some ageing factor for tumour induction is obviously operating . 
this preparation gave 2 sarcomas in 20 mice initiary . other work on carcinogenic activity of chole8terol . truhaut ( 1947 ) could find no activity in his sample of purified cholesterol . truhaut injected 20 mice , of unnamed genetic origin , with cholesterol " purifie ' , " 20 mice with cholesterol irradiated with 10 , 000 r , and 20 with solvent alone , namelv ohve oil . 
 " " si la susceptibilite ' individuelle des animaux n ' est pas en cause , cela tendrait ' a d6monter la pre 'sence , dans le choleste ' rol commercial , d ' impurete 's fortement cance ' rig ' enes qm se rapprochent vraisemblablement des substancek ; responsables de i ' activite ' cance ' rig ' ene de la fraction des ste ' rols precipitables par le digitonoside dans l ' insaponifiable du foie de sujets cancereux . steiner ( 1951 ) has also reported that no carcinogenic activity could be detected in a sample of cholesterol . 
from the carbonyl absorption found in cholesterol fractions , therefore , the type of impurity and its approximate amount can be deduced . cholesterol preparation numbers , 3 , 4 , 6 , 7 and 9 ( for key , vide supra ) showed no appreciable carbonyl absorption ( less than 0 - 25 per cent )  . 
number 8 ( glaxo sample 5989 ) had a band at 1744 cm . - ' , assigned to a 17 - ketone , equivalent to about 1 per cent of such an impurity . 
number 11 ( glaxo sample 1201 ) had three bands at 1744 , 1710 and 1674 cm - ' ; these indicate about 9 per cent of a 17 - ketone , 4 per cent of a 20 - ketone ( or possibly in a 6 - membered ring ) and 3 per cent of an ac , unsaturated ketone , probably a a4 : 3 - ketone . number 5 , which was the least soluble fraction obtained by fractional crystallisation of this latter commer290 i . 
the most soluble fraction in this crystallisation and used for the test in section d , table ii ( number 13 ) contained the impurities concentrated about five - fold . 
1. received for publication january 21 , 1949 . kennaway , kennaway and warren ( 1944 ) found that injection of certain carcinogenic substances , including 1 : 2 : 5 : 6 - dibenzanthracene , caused an increase in the concentration of ascorbic acid in the liver of mice , whilst some noncareinogenic , chemically related substances ( naphthalene , anthracene , phenanthrene ) did not show this effect . in an investigation of the growth - inhibitory action of the carcinogenic hydrocarbon 1 : 2 : 5 : 6 - dibenzanthracene ( elson and warren , 1947 ) it was found that the inhibition of rat body growth and of the growth of walker rat carcinoma 256 ( elson and haddow , 1947 ) by this substance is dependent on the protein content of the diet of the animals . 
1 : 2 : 5 : 6 - dibenzanthracene in the rat usually results in little growth inhibition for about the first 14 days , after which the animals at varying intervals lose weight and die . 
on a 10 per cent protein diet after the same dose of the carcinogen , immediate inhibition of growth occurs and persists for a prolonged period , usually until the death of the animal . in considering the cause of the rise in liver ascorbic acid brought about by 1 : 2 : 5 : 6 - dibenzanthracene , it appeared possible that this was a result of the growthinhibitory action of the compound . 
there was some evidence ( elson , unpublished communication ) that the growth inhibition was accompanied by a fall in the succinic dehydrogenase component of the succinoxidase enzyme system of since the cytochrome oxidase component of this enzyme system is the liver . capable of rapid oxidation of ascorbic acid , it seemed possible that the rise in ascorbic acid after 1 : 2 : 5 : 6 - dibenzanthracene might be the result of less destruction by such oxidation occurring owing to the decreased content of the oxidizing enzyme . 
they were weighed every 2 or 3 days . high and low protein tabls . in later experiments it was found necessary to estimate the amount of food consumed by each rat , and in order to facilitate this 25 per cent and 5 per cent protein diets were made up in 5 g . 
tablets in the shape of cylinders of approximately 3 cdiameter and 0 - 5 chigh . this necessitated replacement of some of the starch in the original diets by cane sugar and glucose in order to obtain a firm tablet . 
from the control experiments , in which only arachis oil was injected , .it is seen that the variation of the protein content of the diet itself has practically no influence on the level of ascorbic acid in the liver . 
the animals fed the 5 per cent protein diet grow only very slowly , but the effect of dibenzanthracene on them is not to make them lose weight , as would be expected if the magnitude of the growth inhibition was proportionate to that observed in animals this smaller effect is presumably maintained on the 10 per cent protein diet . related to the lower total metabolism of the 5 per cent protein diet animals , and 152 l . 
5 , although complete inhibition of growth occurred , and at one time actual loss of weight , the food intake , after the initial drop , rose steadily to a value higher than the level before administration of dibenzanthracene at the time the animals were killed for determination of liver ascorbic acid the actual degree of growth inhibition was greater than the maximum previously obtained with the 10 per cent protein diet animals . 
the degree of growth inhibition is dependent on an equilibrium being established between the rate at which the animal is metabo - _wa% growth rate fooie ratn ? j final liver wt ascorbic acid o * + |~~ 1i 135 6 - 14 ( 1 * 1 [ oo llll , , illlllllllllll , lll ( 134 1i111 . , g14 3 ) o3 ) _ t .%1 5 days 20 25 7 - 02 ' 10 - ~ _ ~3124 8 - 17 7 - 63 fig . 
in animals fed 20 per cent protein diet liver ascorbic acid increases before any marked growth - inhibition is observed , and the increase is thus not an indirect consequence of the inhibition of growth . the growth - inhibition is not merely the result of a lowered intake of food . the animals may react to treatment with dibenzanthracene by maintaining or even increasing their daily food consumption , although growth is completely inhibited . this suggests that a profound change in basal metabolism must have taken place , and the rise in liver ascorbic acid may be associated with this change . we wish to express our thanks for generous grants supporting this investigation , from the british empire cancer campaign , the anna fuller fund , the jane coffin childs memorial fund for medical research , and the u.s. 
carr. from the poultry re8earch centre , edinburgh , 9 . recieved for publication january 19 , 1953 . most cancer investigations involve the use of small rodents , and it is now clear that many of the " generalisations " obtained from research on this material break down when other animals are considered . while the relevance of any result obtained with grafted tumours to the problem of spontanous cancer may be doubted , it seems desirable to report the results obtained by parallel work on non - rodent material in order to offer proof that similar results are , or are not obtained . the grch / 15 tumour is a non - filterable fibro - sarcoma originated by peacock in 1939 ( peacock , 1946 ) , and by his courtesy made available to other workers it is through the animal breeding unit of the british empire cancer campaign . readily distinguished from the virus - induced tumours by the absence of the mucoid material that they all produce , but unless the section is specially stained for this , microscopic differentiation is less easy . transplantation was always made into brown leghorns of greenwood 's flock ( in one of which the original was induced ) , either by implanting small pieces with a fine trocar and cannula , or injection by syringe of the suspended material left after shaking minced tumour or tumour ground with sand and saline . transplants were usually made into each pectoral muscle , and by the trocar and cannula method " one - sided " negatives occurred about 1 in 20 times , while the second gave less than 1 per 100 " one - sided " failures ; the incorrect negatives due to experimental errors would therefore be 1 in 400 and 1 in 10 , 000 respectively , which can be ignored . 
the material for this account is based upon 60 transplant generations involving some 600 birds over a period of 8 years . in most experiments the birds were 6 to 10 weeks old when the tumour was implanted . in the bird the tumour appears as a hard white lump , though after the alteration in the growth rate the texture was softer , and microscopically the cells appeared rather more compact . 
983 , from which all material for the 32nd and subsequent generations derived . it is interesting that the change coincides with a distinct alteration in the conditions of husbandry , for the facilities for keeping animals at the experiment station were at first not optimuno cages were available for birds , and the food comprised various ration - free products , which produced a series of deficiency diseases ranging from protein to multiple vitamin shortages . 
when the conditions were built up to those nearer first - class management the change in appearance and behaviour of the tumour remained . there is no direct evidence that the change in conditions caused the alteration of the tumour , and it remains possible that it was a coincidence . the most notable change in the tumour was in the rate of growth . 
by contrast , in only one bird out of many thousands has this organ been the site of a rous 1 metastasis . of 352 birds killed and examined with large growths in the breast muscle , 51 had metastic growths in one or more sites . 
nor is it due the conditions for tissue transto a failure of the bird to produce iso - antigens . plantation , however , seem to differ markedly from those of the lower rodents . for example , organ transplantation between fowl varieties is a normal experimental technique , used for example by greenwood ( 1928 ) to investigate the relation of the testis to henny feathering of males of some breeds ; yet transplantation of an adrenal is regarded as a novel event in the mammalian field similarly , the elaborate integumental decorations of birds early ( darcy , 1952 )  . attracted the attention of zoologists , and the results of skin transplant between different genera were being embodied into the general assembly of zoological theory before tissue iso - antigens were recognised . it is possible to suspect that a similar mechanism is indeed operative in the mammalian field , but the methods used are not refined enough to detect it . 
the domesticated rat and mouse have an oestrous cycle of only a few days in length , as compared with the sharply - defined annual cycle in the hen , and lesser cyclic variation of the cock . 
even the pregnancy of 3 weeks is rather short for observations in the rat and mouse , but it is significant that foulds ( 1949 ) found some evidence of a variation in tumour growth and structure in the mouse , and there are many reports of pregnancy influencing the course of tumours in the human . the appearance of many cancers at an age when the sexual functions are failing may be another aspect of this . this would seem to be a point of considerable importance , for it indicates that control of a malignant tumour would be possible within the limits of normal physiological variations ; similarly studies on the cancers induced by chemicals , and more especially by x - rays , indicate that the production of the malignant change and the appearance of a progressive tumour are distinct phenonema , a separation that is even more obvious with the mouse milk factor , where infection takes place soon after birth but the tumour appears months later , and only if the endocrine history of the host has been suitable . 
carr aries in the proventriculus in older birds carrying the " slow " tumour refers to a difference in the biochemical suitability of the organ in the older hosts is it is to be expected that the differences of tissue permeability with unknown . age ( duran - reynals , 1942 ) would also have some influence upon the metastatic growths . the absence of any neutralising action in the sera of fowls immunised against the grch / 15 sarcoma is in contrast to the findings of gottschalk ( 1943 ) for sarcoma 16 . but it is well known for commercial birds suffering from the related " leukosis complex " viruses to have death - rates of up to 40 per cent and carrier rates of nearly 100 per cent , so that such a finding is only of value if it can be stated that such carriers are not a complicating factor . 
a fast - growing mutant strain retained this property , and the mutation was not associated with an increased frequency of takes , though the distribution of secondaries was altered . 
koller. from the chester beatty research institute , the royal cancer hospital , london . given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . xeroderma pigmentosum ( hence referred to as x.p. ) , is marked by rougheniing , dryness , pigmentation and ulceration of the skthe condition is attributed to hypersensitivity of the skin , particularly that of the basal cell layer , to sunlight . the disease was first described adequately by kaposi in 1874 ( hebra and kaposi , 1874 )  . 
occurs in the first year of life , often within a few , months after birth . first , erythema develops on the skin , on those regions which are exposed to light , followed by freckling and frequently by telangiectasis . from these regions epithelioma or basal - cell carcinoma develops . 
the malignant change may take place not only in the skin , but also in the cornea and conjunctiva the genetical and chromosomal basis of x.p. the hereditary basis of x.p. 
was first investigated by siemens and kohn ( 1925 )  . they analysed 76 families , and cockayne ( 1933 ) brought this more up - to - date with a further 43 family histories . in both groups of data the incidence of consanguineous marriages was very high : 24 out of 76 and 16 out of 43 respectively , were first - cousin marriages . 
is one of those which is borne in that particular region of the sex chromosome , common to x and y . it may not be superfluous to describe the cytological mechanism which underlies incomplete sex - linkage . the genetical basis of incomplete sex - linkage is clear , and is already described 150 p . 
they found thait during the first meiotic d ' ivi ' lsion of the male rat , one or two chiasmata are forined between the x and y chromosomes , and that the shape of the sex bivalent is determined by the position of chiasmata in relation to the centromere and differential segment . 
the structure of the bivalent is determined by the position of the chiasmata ; the frequencies of the various bivalents are shown by the number of percentages beside the figures . similar chromosome behaviour wak ; observed in man . 
the fact that the heteromorphic pair present in the chromosome set of the human male , forms a very easily observable unequal or asymmetrical bivalent during meiosis , is - well known from the - works of painter ( 1923 ) , evans and swezy ( 1929 )  . 
when a ' man inherits such incompletely sex - linked gene from his mother , it will be in his x chromosome , but occasionally it may cross - o ' ver to the y consequently the man will transmit the gene to most of his daughters and to a similarly , if he inherits it from his . 
father , he , will transmit it few of his sons . to most of his sons and to a few of his daughters . human genetics is greatly indebted to j . 
he analysed 82 family histories collected from various sources , and by using ingemoug statistical methods drew the conclusion thatthe incomplete sex - linkage of the g ' ene x.p. 
he made the suggestion tha ' t one of the 82 families on which analysis was based should be excluded on intemal evidence , which would further loosen linkage , and in this case the true position of x.p. 
the data obtained from these cases has given further information on the hereditary basis of this morbid condition , and has shown the 'significance of the pathological symptoms , including the degree of manifestation and the time of onset of the disease . 
of one year the whole skin of the left 'side of her face came up in red blisters after being exposed to the sun . she was treated with calomel lotion . 
the condition it was treated by " caustic pencl ' l , " and six months later , was diagnosed as x.p. in 1943 another lesion developed on the right when it recurred , by radium . cheek , which was similarly treated by radiuthe patient 's skin of the exposed both the pathological skin conparts ( face , hands , legs ) shows some scarring . ditions and the histological analysis of the lesions indicated x.p. 
the parents are unrelated ; father is of a dark complexion , mother has some slight freckling search through family histories on the cheek , which is of the common type . of near relatives and sibs did not reveal any similar conditions . 
at that time some of the freckles developed into small ulcers , which healed slowly . she found out , by experience , that strong sunlight accentuates the lesions , and since the age of 40 she has taken care not to expose herself to direct sunli.ght. 
the matemal and patemal grandfathers were brothers.. the eldest brother , frank , hischildren and grandchildren are all normal . isabel , the patient concemed , was the second child , but before her birth one ' miscarriage took place . 
her sister , dorothy , is one year younger ; she is afflicted with the dorothy shows freckling on the face disease in the same mild manner as isabel . and forearm , which is somewhat less severe than that of - her elder sister '  . a younger brother , thomas , was born after isabel . 
the high number of miscarriages and the death of the sixth child , a girl , on the day following birth , may , not unlikely , find its sidney died at the age of 25 explanation in the consanguineous marr ' lage . up to his death he appeared to be normal . there are two unusual features in the history of the hfamily which require explanation . 
in the hfamily does not conform closely to expectations ; the number of cross - overs is higher than could be expected . in order to explain the exceptional pathological features and the unexpected segregation of x.p. 
in the sexe - s in this particular family , the followina possibilities may be considered : ( a ) this condition - may be due to an independent autosomal gene , or ( b ) the gene x.p. 
the increased distance of the gene from the differential segment would loosen linkage with sex , and its altered position may also lead to a lower degree of malignancy . the data at our disposal from this one family , however , is too meagre to draw definite conclusions , whether we are dealing here with a distinct autosomal gene or with position effect . 
gunz. * from the department of radiotherapeutics , university of cambridqe . received for publication june 11 , 1949 . in previous publications ( gunz , 1948a ; b ) a technique of cultivating human leukaemic leucocytes in vitro was described , and the quantitative and cytological findings in untreated cultures made by it were analysed . 
he did not , however , determine the mitotic rates in his cultures , because of the small number of dividing cells present . osgood 's findings may be compared with those of schrek ( 1946a , b ) , who irradiated suspensions of thymus and spleen lymphocytes , and of bone marrow cells ( rabbit ) , and of normal and leukaemic human blood cells in " phosphate ringer solution " with and without homologous serum , with doses of x - rays of using as a criterion of survival a failure of cells to stain with a 20 - 4000 r . 1 : 1000 solution of eosin in tyrode solution , schrek found exposure to x - rays to cause a decreased survival time vitro in lymphocytes from the thymus , normal blood and four of five bloods from patients with chronic lymphatic leukaemia . such an effect was noted after a dose of 50 r , and increased with an increase in the dose up to 1000 r , remaining approximately constant at higher doses . 
the author , discussing possible reasons for this lack of effect , attributes it to an absence of dividing myeloid cells in his preparations , x - rays leaving resting myeloid cells unaffected . interference with mitosis , as a result of x - irradiation , was definitely demonstrated by rachmilewitz , rosin , goldhaber and doljanski ( 1945 , 1947 ) in experiments with fragments of explanted rabbit bone marrow . following the application of 250 - 1000 r there was a depression of mitosis lasting for two to four hours , with subsequent recovery . 
many mitotic abnormalities were caused , and the marrow became hypocelhular , or almost completely aplastic , according to the size of the dose given . the observations in the literature may be summarized by stating that x - rays have been found to damage blood and haemopoietic cells , especially lymphocytes and all classes of immature cells , when applied directly in vitro , and that disturbances of mitosis appear to be among the earliest signs of injury in cells so exposed . 
two patients had not received any x - ray treatment when first seen ; the others had had various amounts of treatment in the past . the technique of culture was that previously described ( gunz , 1948a )  . brief , it consisted of the distribution of small equal quantities of leukaemic leucocytes , suspended in the culture medium , over a series of culture tubes , and the withdrawal of some of the tubes for counting after varying periods of incubaat these times total and differential counts were made , and the total tion . number and phase distribution of mitoses determined . the culture medium used consisted either of 50 per cent dried , reconstituted human plasma , with the addition of 5 per cent rat embryo extract , or of 50 per cent fresh homologous human serum , both diluted in ringer solution . the duration of experiments was two days , and counts were done at 8 , 24 and 48 hours , unless otherwise stated . the technique of irradiation was as follows : after the cultures had been set up , they were incubated at 370 for about 15 minutes , to give them time to reach body temperature . in some experiments some of the tubes were also incubated for longer periods before irradiation . 
the tubes were exposed at two fixed distances ( approximately 21 and 81 cfrom the target ) , the dose rates , measured in air , at these points being 622 and 62r / nirespectively . 
the actual dose rate to which cells were exposed depended on three factors : the dose rate in air , the absorption and scatter due to the glass , and the absorption and scatter due to the fluid . 
the dose rate in air was measured by means of a victoreen dose meter in the normal way ; it was found that when the chamber of the dose meter was inserted into empty culture tubes , the average decrease in the dose rate was 6 - 5 per cent . this enabled a correction to be made for the glass of the tubes . 
the volume of the liquid being only of the order of 1 or 2 ml . , the correction for the absorption and scatter due to it would be less than 5 per cent , and was ignored in estimating the dose rate . 
the results of irradiation with 100 , 1000 , and 10 , 000 r are set out in tables i - vi . if the contents of tables i - vi be summarized and analysed , we obtain the following findings : immature cell8 . with a dose of 100 r the ratio , at 8 hours , of immature cells in irradiated to in five experiments it those in control cultures varied from 65 - 113 per cent . was greater , and in seven experiments less than 100 per cent . 
a significance in the analyses of immature cell counts test ( student 's t - test ) was employed . percentages were first converted to logarithms , in order to obtain a more normal distribution of the series . 
the actual formula used was t - s - - = where x is the arithmetic mean of the series , 8x its standard error , and m = log10100 = 2 . this gave a value of t = 0 * 97 , which is not significant . at 24 hours the ratio varied from 71 to 111 per cent . in four experiments it was greater , in one equal to , and in seven less than 100 per cent . 
the difference is not significant . at 48 hours the ratio varied from 48 to 112 per cent . in one experiment it was greater , and in ten experiments less than 100 per cent . 
from this t = 5 - 25 , and p is less than 0 * 001 . there was , therefore , a highly significant reduction in the number of immature cells in cultures 48 hours after irradiation with 100 r . with a dose of 1000 r the ratio , at 8 hours , of immature cells in irradiated to those in control cultures varied from 76 to 111 per cent . in three experiments it was greater , and in three experiments less than 100 per cent . 
no analysis was carried out for the 48 - hour values . with a dose of 1000 r the ratio was less than 100 per cent in all experiments , and at all times . 
no mitoses were present in irradiated cultures in 4 out of 7 experiments at 8 hours , in 5 out of 8 experiments at 24 hours , and in 4 out of 6 experiments at 48 hours . 
the results with 10 , 000 r were similar . it must be emphasized that these experiments were performed with varying culture media , and that the cells in some of them came from untreated , and in others from previously treated patients . this explains many of the numerical differences between individual experiments , particularly in the mitotic counts . however , in spite of the non - homogeneity of the material , the results show clearly the following points : irradiation of cultures of leucocytes from chronic myeloid leukaemias with 100 r produced a significant reduction in the number of dividing cells for at mitosis then recovered , and did not differ significantly from least 8 hours . controls at 24 hours or later . 
1. - in vitro irradiation of leucocytes ( chronic myeloid leukaemia )  . irradiation with 1000 and 10 , 000 r produced a more marked depression of mitosis at 8 hours , and this did not recover subsequently . 
at that stage there were practically no dividing cells present , and the vast majority of all immature leucocytes in the cultures were truly " resting . " only after the lapse of several hours could any considerable numbers of them be expected to be approaching or entering it is , however , well known that chromosomes are particularly liable mitosis . to damage from radiation in the late pre - mitotic or early mitotic stages ( lea , 1946 )  . 
the following experiments were made in an attempt to irradiate cells at such a moment of increased sensitivity to x - rays , and to determine whether chromosome abnormalities could thereby be induced . - ~~~~ _ ~~~ ~ ~~~~1 ' ~~~~~~~~~~~~~~~~~~~~~ 338 f . 
gunz effect of varying the time of irradiation . in two experiments different groups of tubes were irradiated with 100 r at 0 , 2 and 4 hours from the time of setting up the cultures . 
3 , from which it is clear that no significant difference in the mitotic rate or the number of immature cells was produced as a result of varying the time at which the cultures were irradiated by up to 4 hours ; neither was the number of mitotic abnormalities in stained films altered . in another group of three experiments cultures were irradiated with 100 or 1000 r 7 hours after they had been set up , and counts and filmns were made at it was known that at 7 hours , appreciable frequent intervals after irradiation . numbers of cells would be entering mitosis . 
4. - effect of x - rays on leukaemic leucoeytes in vitro . irradiation 7 hours after explanta ' tion . that they only appeared when cultures were irradiated at a time when many mitoses were present , suggests that they arose from dividing cells , and it is likely that they originated in early prophase . 
15. it would seem that the findings at 8 and 24 hours are most likely to reflect the character of the cells themselves ; later on , artefacts due to the composition of the medium become superadded . taking only the 8 - hour counts into consideration , there was a good correlation between the patient 's blood count and the number of mitoses in cultures . 
 ( 1945 ) for normal rabbit bone marrow , but these authors worked with a different culture technique . no evidence was found that 100 r produced different results when given at this is in agreement with the finding of lasnitzki ( 1946 ) different dose rates . that in cultures of chick fibroblasts the initial inhibition of mitosis was independent of the dose rate ( 9.3 - 103 r / min . ) for doses up to 100 r . 
the explanation in the present experiments is probably that with both the dose rates used ( 62 and 622 r / min . ) , the time of irradiation - 10 or 97 seconds - was short compared to the overall time required for mitosis ( 35 - 60 minutes )  . no significant increase in the number of mitotic abnormalities was found to follow irradiation of cultures . 
the explanation for this is not clear ; it is , however , possible that technical difficulties prevented the recognition of finer structural alterations , like chromosome breaks . when cultures were irradiated 7 hours after their start , it was found that many cells in mitosis at the moment of irradiation completed their division . this agrees with the observations of many other workers , such as strangeways and oakley ( 1923 ) , canti and donaldson ( 1926 ) , kemp and juul ( 1930 ) , and furthermore , with 1000 r , a new kind of breaking - down cell love ( 1931 )  . appeared 3 hours after irradiation , and rapidly increased in numbers . 
assuming , for instance , that a similar process took place in the irradiated spleen , we should expect a definite diminution in the number of new leukaemic cells produced in that organ ; moreover , any further similar treatments would repeat , and possibly augment the effect . 
one could therefore picture the manner in which x - rays cause the enlarged spleen to involute . it is , however , more difficult to account by this means for the fall in circulating leucocytes which is such a prominent feature of the response to local radiotherapy in leukaemia , or for the involution of distant leukaemic lesions which had not been directly irradiated ( for examples , see piney and riach , 1932 )  . 
some light may be thrown on the latter problem by the findings in section b of the experimental part . here it was seen that leukaemic leucocytes , taken from the peripheral blood , showed a dininished power of proliferation in vitro after therapeutic irradiation of the patient , and that this effect was reversed as the clinical effects of treatment began to wear off . it thus seemed as if the biological make - up of the cells had been changed by the treatment . 
pullinger. from the imperial cancer research fund laboratories and the research department , the glasgow royal cancer hospital , glasgow , c.3. received for publication february 7 , 1952 . the hyperplastic nodules here referred to comprise all varieties found in surveys of spontaneous nodules in previous publications ( pullinger , 1949 , 1952 )  . at the time these surveys were being made specimens of affected nipple regions were almost invariably mounted and kept . it gradually became obvious that one nipple region , the 2nd on either side , was more often the site of nodule formation than any other . 
when the surveys were complete , the 5 pairs of nipple regions were arranged together with the number of times they contained one or more hyperplastic nodules of the various sorts . only those nodules are now included of which whole mounts were kept . 
the period of time covered was continuous from 1945 to the end of 1950 . the number of mice with mounted nodule - containing nipple regions was 116 ; the number of the nipple regions 146 . 
no record had been kept of 13 from which serial sections were made in the earlier stages of the survey ; more were incomplete or have been mislaid . six examples of bilateral nodules in the same gland were counted as only one of the pair . 
but for the extremes prognosis is definitelv affected in group statistics by the age at time of operation . in our e - xperience below the age of fortv vears axillary metastasis is much more apt to be present at the time of operation than bevond that age . in the s - ame , wav postoperative survivals . 
and the average postoperative hfe is shorter ( 16 - 7 months ) than in similar cases in the adult . " duk - es ( 1940 ) found that patients under the age of 40 vears had a higher incidence of lymphatic metastases than those in the age gmup 40 - 59 vears . 
they gave their opinion that the most malignant forms , both chnically and histologically , tended to occur at the younger ages . frissell and knox ( 1937 ) gave details of 46 cases of primarv lung cancer which on examining included - the age , and the duration of the - disease for each patient . these data we have found that there is no relation at all between age and the duration of the disease . 
he divided them into a carcinoma  . " group of 89 patients and an " epithelioma " group of 23 patients . he had no doubt that the clinical mahgnanev of the tumoiirs in the carcinoma group was greater in these patients than in older patients . 
undifferentiated tissue , often with manv mitotic figures in each field . " he found that h - valinization was never present that 1.ymphocytic infilltration and fibrosis were present to a moderate degree and only in the less mahgnant and that cellular differentiation was present mainlv in ovarian and tumours th - vroid carcinomas where . 
carried on through the cards and chose the first carcinoma recorded in the same organ and of the same sex in an individual in age group 46 - - 55 and again in a high age group . 
rectum ; and 61 vears and over for lung ( where the middle age group was 41 - 50 )  . for each tumour type we thus had three randomlv chosen groups of slides . 
the middle - aged and the old . ' the slides were then taken out of their holders and shuffled . this made identification of the sections impossible without reference to the laboratorv register of the numbers etched on the slides . 
ceu polymorphim and degree of fibrost - 3 ( scirrhous reaction ) the principle adopt . - d was to allocate grades i and iv to cases which were strikinglv low and strikinglv high respectivelv in these features . grades 11 and iii formed intermediate groups of " low moderate " and " high moderate respectivelv . for epidern " d differentiation the scale . 
 ' after completing the ' experiment , we were qiiite convinced that there was no such gross systematic change in type of material or standards of diagnosis over the years . 
we have , therefore , not considered that the difference between blocks arranged by date of diagnosis would appreciably reduce the residual error on which the significance of inter - age differences would be estimated . the experiment was therefore designed to allow for an analysis of variance . for example , in the sub - experiment " degree of glanduliform differentiation in rectal carcinoma " there was a total of 42 units or slides divided into 3 groups of 14 each for patients of 30 ygars or less , 46 to 55 years , and 70 years or more respectively , and divisible also into 14 groups of 3 each , i in each age group , diagnosed in an analysis of the variance in this balanced design in the same calendar year . there would then have been a total of 41 " degrees of freedom " comprising 2 for age , 13 for date of diagnosis , and 26 for residual error . 
of the 2 degrees of freedom for age , one could have been used to test the linearity of the difference in fact there was no need for this since a relatively coarse by age if necessary . analysis of the data indicated that the figures might we ] i have occurred by chance - on the hypothesis that there was no correlation between age and any of the factors we estimated . colloid ' formation " and " cell polymorphism , " using the total figures of each factor for for example , in the case of " epidermoid differentiation " there each age group . was a contingency table of the formthe x2 test was carried out for each factor , with the exception of the results for the various factors were epidermoid differentiation : 6 x2 4 - 95 p 0 - 7 - 0 - 5 . glanduliform differentiation ( consolidating grades iii and iv ) : 4 x2 = 4 - 55 p = c . 
at the begin9of the experiment , however , we laid down the hypothesis that we had no reason to expect significant results from one tumour type or characteristic more than another . it foflows that there is , so far as our results per se are coneemed , no rea - son to regard this as indicating a real correlation between age and increas ' epidermoid differentiation in bronchogenic carcinoma . it is notable , however , that in our reading of the literature the tumour in which the correlation between increasing differentiation and increa - sing age ha - s been most strikingly demonstrated ( geschickter and denison , 1934 ) is also bronchogenic carcinoma . consequently our figures , in so far as they are confirmatory , are also suggestive . we did , in fact , although we have not given the table , estimate , using the method of analysis of variance , the probabihty with which the figures for epidermoid differentiation and age could have occurred by chance for epidermoid differentiation of ( a ) bronchogeiiic carcinoma , ( b ) carcinoma of the cervix , ( c ) carcinoma oflip . 
a blank estimation was carried out on the reagents and in the latter half of the investigation a weighed amount of the filter - paper surrounding the discoloured areas was included in the blank , but this addition makes no considerable difference . occasional very high blanks were discarded as due to some unusual contamination . 
3. - mean seasonal variations in arsenic content of air at eight stations . w = winter ( november to march ) ; s = summer ( may to september )  . establish any exact comparison between the seasonal changes in the individual richard doll has pointed out to us that , the average values for the towns . different stations would be more properlv comparable , in view of the seasonal variations , if ( 1 ) the means of two figures for the same month and town were taken to represent that month , and ( 2 ) the mean of the figures for two months which are separated by a gap of one month without data were taken to represent that month . 
one might suggest some such classification as the following of the not very abundant data available upon the types of sunmmation ( table ii ) ; no claim is made that this is in any way a complete summary of the literature . 
the summation of action of two chemical carcinogens is not easy to demonstrate , and we appear to have few indubitable instances of it . * in connection with the subject of cancer of the lung , the work of lynch ( 1935 ) and andervont ( 1937 ) upon summation in the lung of some strains of mice is of especial interest , although the neoplasms of the lung in this species differ from those in man . 
3. received for publication january 21 , 1948 . during the war the pharmacology of the nitrogen mustards or chloroethylamines was studied intensively ( gilman and philips , 1946 )  . in the course of these investigations it was observed that tissues with a high proportion of dividing cells were particularly injured . this discovery led to the application of these substances in the treatment of malignant growth . 
of dimethyl - , - chloroethylamine hydrochloride by subcutaneous injection . such treatment when given repeatedly caused inhibition of some spontaneous mammary tumours ( table i ) , but had very little effect in prolonging the life of mice with transplanted lymphosarcoma . this compound given in single doses of 100 mg . 
the effect is similar to that induced by radioactive substances ; it is identical with the phenomenon of depigmentation in mice caused by subcutaneous implantation of plutonium ( brues , 19 , 47 , personal thus " bleaching of hair - colour " is another example of the communication )  . radiomimetic actions of the bis - chloroethylamines . the metabolism of walker carcinoma tissue from rats dosed with 1 mg . 
a variant of this compound , dimethyl - ] 3 - chloroethylamine hydrochloride , was used in 3 other cases , reported here because of the favourable response in 1 case . 
on two consecutive days in 30 cases , in 10 of which treatment was repeated once or several times at 3 - week or longer intervals depending upon the length of remission from symptoms . 
the majority of patients with advanced bronchogenic carcinoma have white cell counts above 10 , 000 per c.mm. , and such patients do not develop the moderate to severe leucopenia observed in cases of hodgkin 's disease ( apthomas and cullumbine , 1947 )  . in only 2 of our patients has the white cell count fallen below 4000 per c.mm. , in these cases to 1000 and 1800 with restoration to normal counts in 3 weeks ' time . the first of these cases had had previous radiation therapy , and at the commencement of treatment had a white blood cell count of 4000 per c.mhaemorrhagic nianifestations have not been observed in this group , nor has any significant change in haemoglobin dosages of 0 - 2 mg . 
at 10 - day intervals can be tolerated for been noted . periods of at least 10 weeks , as judged by the 4 cases we have treated over such a period . 
the presence of fever , pleural exudation or jaundice are not , in our experience , contraindications to the use of the drug . the adverse effects of the dimethyl compound were observed in 3 patients , 2 of whom are not included in this group as their diagnosis was not histologically proven . 
one patient was carried on for a period of 5 months with marked relief of pain , cough and dyspnoea and decrease of sputum for 18 to 21 days following each course of injections . 
a patient with remission of symptoms for 6 to 8 weeks after each treatment is still being observed one patient receiving dimethyl - , 3 - chloroethylamine is still in after 9 months . remission 7 months after treatment and leading a normal life . 
the relief of dyspnoea in some cases is almost complete - an observation very difficult to explain , for in such cases there may not be corresponding observable changes in the lungs . relief of general malaise and anorexia is also noted in over half the cases following the initial 1 - or 2 - day period of adverse effects . 
warwick sputum usually increases in amount for several days , and is said " to come up more easily . " the amount may then fall off rapidly , in some cases from 90 - 120 ml . 
the sputum may also change in character from purulent to colourless . reabsorption of pleural exudate or decreased rate of formation has been these observations are of interest in that most radioseen in 5 of 13 cases . ' therapists hesitate to give treatment to cases with pleural exudation because of the tendency to increase the amount of exudate . superficial metastases of measurable size decreased in 3 of 10 cases . 
a larger total number for such observations wouild have been obtained had not metastatic nodules been removed for microscopic study at various intervals following injection in 8 cases . twenty - two patients who had weight loss were weighed weekly following in 9 of these cases weight loss continued for approximately 1 week , treatment . following which a gain in weight occurred amounting uisually to 2 to 8 lb . four patients gained 10 to 15 lb . 
in 2 months , and 1 patient who lost 40 lb . in the 18 months prior to treatment with dimethyl - 3 - chloroethylamine has in 7 months regained all her lost weight . radiological evidence of decrease in size of pulmonary metastases and re - aeration of segments or lobes was observed in 8 of 35 cases . 
hb , 55 per cent . 3 , 800 , 000 . heart , mediastinum and trachea displaced to the right ; appearance of atelectasis with probable cavitation near 2nd right carina broadened rib . and invaded on the right by a large nodular friable mass completely occluding the right main bronchus . bronchoscopy report from harefield in april , 1946 . pathological report : adenocarcinoma with numerous mitotic figures . treatment : dimethyl - chloroethylamine 2 mg . 
a corresponding effect in a tumour of the breast might reasonably be expected not to be noticed in support of such a suggestion is the fact that a similar relief by the patient . of respiratory symptoms following chloroethylamine without radiological evidence of changes in the lungs has been observed in a case of carcinoma of the breast with pulmonary metastases . 
by repeating the treatment at suitable intervals the life of animals with tumours can be prolonged , but cessation of treatment leads to recurrence of tumour growth . forty - one cases of histologically proven carcinoma of the bronchus unsuitable for other forms of therapy have been treated with the chloroethylamines . 
the original premise on which the present study was built was that if these three groups have any real existence it should be possible to demonstrate some differences in their natural history . a diagnosis that does not imply a prognosis is traditionally worthless ; not only is it of no practical value , but the difficulty of testing it leaves every opportunity it was proposed therefore to collect a series of rodent ulcers , classify for error . them according to foot 's ( 1947 ) criteria , and then to apply to them all available touchstones of clinical behaviour in the hope of being able to separate them . some such differences as those demonstrated by schrek and gates ( 1941 ) and schrek ( 1941a , 1941b ) between rodent ulcer and squamous carcinoma were sought . the attempt to do this , however , broke down unexpectedly early , for after our series had been collected it proved impossible to classify it on the lines proposed by foot ( 1947 )  . 
wells lesions with which foot ( 1947 ) dealt , it became clear that the great majority of tumours at the stage at which they are usually received by the pathologist are typical examples too various in appearance to admit of such simple treatment . of each class could be found easily enough , but there were so many mixed and intermediate and aberrant forms that no progress was possible . 
no modification of the classification could be devised that would avoid them . during a long interval in which the matter was left in this discouraging state , interest was deflected into a study of the pigmentation of rodent ulcers and of epithelial skin tumours generally which has already been reported ( lennox , 1949 )  . there emerged from this one possible way of classifying rodent ulcers , namely , into the pigmented and the non - pigmented . such a classification is almost wholly objective ; there either is or is not melanin in the tumour . contrast with the process of matching a tumour against a series of idealized types in the hope of being able to recognize a greater or less resemblance to one of them is very marked . it is true that the matching technique is reliable enough in the ordinary process of histological diagnosis , but where the types themselves are of doubtful existence it becomes open to great error . 
they are a large proportion of those received in this department in the years 1936 to 1948 . most have come from the radio - therapeutic research unit which was established in 1942 ; without its very full record system this study would have been impossible . cases have been rejected if the biopsy specimen was inadequate or if the history was deficient in any important detail , but an effort was made to include as many of the cases found as possible , partly because the available material was small , partly because any form of selection may profoundly and unpredictably influence the results of any study of this type . it was not possible to insist on more than a year 's follow - up , though most cases have had considerably longer ; the recurrence rate is therefore falsely low , though sufficient to provide some useful data for comparative purposes . surgical excision supplied 26 specimens ; diagnostic biopsies , multiple in 30 cases , were provided by 124 cases . most of the material was fixed in plain formalin - saline ; recent biopsies have been fixed in halfsaturated mercuric chloride in 15 per cent formalin - saline ( fss )  . 
the typical examples of this group seem distinct enough , yet it soon became obvious that transitional forms are numerous . wte therefore provisionally included the sub - group , and found later no reason to regret it ; we believe that these are merely the most benign and well - differentiated end of the rodent ulcer series ( the multiple and often familial tumours of brooke ( 1892 ) and fordyce ( 1892 ) we believe also to be of the same nature , though here we are extrapolating beyond our personal experience )  . in the second place , at the other end of the scale we find 11 tumours of the type illustrated in fig . 
we believe the evidence against the squamous nature of this sub - group is strong . within the typically squamous tumours a close parallel between histological dedifferentiation and clinical malignancy can be the most malignant epithelial skin tumour seen in this hospital demonstrated . in the last 4 years is that illustrated in fig . 
3 ; it grew very rapidly under observation and killed its host within 18 months of first appearance . less well differentiated squamous carcinomata should be even more malignant ; but all the tumours we have seen of the type of fig . 
2 have been relatively benign , and can be shown only by careful statistical analysis to differ in behaviour from the general run of rodent ulcers . inclusion of these two sub - groups harmonizes with the view of the rodent ulcer group developed later in this paper , as a series in which a gradation of differentiation can be recognized , with a definite though not very striking parallel gradation in behaviour . 
removal of these two sub - groups would not affect the general conclusions drawn , though it would reduce the level of significance of some of our figures . forms of differentiation chosen for analysis . only four common histological forms of differentiation in rodent ulcers could be defined well enough for analysis . 
some obvious features , especially those based on cell size and shape and on tumour cell group size and shape , had to be rejected because of the difficulty of establishing exact descriptive criteria and , more important , their great variability within any one tumour . 
some very interesting features were too infrequent in our series to justify detailed separate analysis : these were ( a ) keratin formation ( 11 cases , 7 ' 3 per cent ) , ( b ) hyaline stroma ( 11 cases , 7 ' 3 per cent ) , ( c ) squamous differentiation ( 4 cases , 2 ' 7 per cent ) , ( d ) " sebaceous " differentiation ( 4 cases , 2 - 7 per cent ; lipoid was not identified , and the recognition of these occasional foamy cell groups as sebaceous remains doubtful ) , ( e ) duct formation ( 1 case , 0 ' 67 per cent )  . the four forms of differentiation finally chosen were , in order of frequency , palisading , fluid formation , whorls and pigmentation . 
these presented the following advantages : ( a ) they were all common , so that sufficient cases were available for analysis , ( b ) their presence or absence could be readily determined , ( c ) between them they were responsible for a large part of the variation in appearance assumed by rodent ulcers , and ( d ) they were constant throughout the same tumour , varying 198 b . 
what form of degeneration of the stroma it can be we fail to see ; it is not myxomatous , it is only occasionally hyaline , and it is curiously localized if it is oedema . if one believes it to be stromal in origin one is driven to the proposition that it is an active secretion of the stroma - an unprecedented occurrence in an epithelial tumour , and one that in any case does not explain cysts within the epithelial masses . massive fluid production such as that of fig . 
by whorls we mean small spherical groups of concentrically arranged flattened cells , more eosinophilic than the bulk of the tumour and centred on an even more eosinophilic core ; this core often appears to be converted into a laminated plug of keratwe have counted nothing that did not have at least a trace of central eosinophilia , but think it possible to recognize even earlier stages of which the giant - cell - like structure seen above left centre in fig . 
9 may be an example . at least three interpretations of these structures have been offered : ( a ) they are cell nests ( parakeratotic pearls ) of basically the same nature as those of squamous carcinoma ( darier and ferrand , 1922 ; montgomery , 1928 )  . 
they differ from these in their smaller size , sharper demarcation , more concentric structure and lack of prickle cells , and usually also in the smaller amount of central keratin . ( b ) they are abortive hair follicles ( haythorn , 1931 ; warren , gates and butterfield , 1936 )  . 
the term " abortive " here is a convenient one ; it conceals the fact that the structures resemble no part of the follicle in any respect which will bear analysis . 
1. - a nodular tumour of the epithelioma adenoides cysticum type , only slightly cystic . note the position immediately deep to the epidermis , unrelated to hair matrices , and also the focus of abnormal epithelium , similar to that seen in early rodent ulcers , to the right of case 219 : from the scalp of a man of 33 , duration 22 years . 
14. - colonization of hair follicles by homologous epidermis in the rabbit . the epidermis and the epithelium of the hair follicles of the graft have died , but the stroma survives . 
and post - auricular area from pinna postauricular , 2 . is justified by the obvious difference in exposure , and by a difference in behaviour well marked even in the small number of cases involved . 
a preliminary test by standard error of the differences of the percentages in the 0 and 9 - 15 columns for each other and from the mean figures for the whole series gave in the event results differing little from those of more elaborate methods . 
wells it casts some doubt on the opinion earlier histological diagnosis is most difficult . it would , however , be statisexpressed that these tumours are all rodent ulcers . tically equally suspicious to find no such tumours . perhaps the group does include some spurious cases , but we have found no satisfactory way of determining which they are , and at least two alternative explanations , apart from chance numerical variation , offer themselves . 
boag , to whom we submitted the figures , pointed out that from nition . the statistical point of view they could equally be explained by a slight tendency to aggregation of the separate differentiations ; he was able to show that three of the differentiations ( melanin being the exception ) were rather more frequent in the presence of one than in the presence of no other differentiation , and a little more frequent still in the presence of two others . 
our four main differentiations are seen to associate in what appears to be an entirely random manner . put the case that there exists any histologically separable sub - group of rodent it is exceedingly improbable that ulcers , and that it is of any substantial size . any significant definition of such a group could be devised that would not involve at least one of our four main features . 
then infallibly the random grouping only if it is extremely small , or if it depends on criteria would be disturbed . which have so far escaped definition , can any such sub - group exist . of such possible sub - groups the most important , because the most widely accepted , is the " type intermediare mixte " of darier and ferrand ( 1922 ) which is the " baso - squamous " form of montgomery ( 1928 )  . 
we can find no real criterion of the diagnosis of this type of rodent ulcer except the presence of the " pearls , " and these do not appear to be anything except our whorls in their higher grades . montgomery ( 1928 ) states that his cases are more malignant and radio - resistant than other rodent ulcers . 
he gives no statistical evidence for this , basing it as far as one can tell from his original article on impressions of a small number of it will be clear that our series gives no support for the idea either of the cases . separate existence of such a group , or of the abnormal behaviour of tumours showing whorls . 
of our 54 cases showing whorls , 4 recurred ( 7 ' 4 per cent , compared to a general recurrence rate of 11 ' 3 per cent ) and 3 responded poorly to irradiation ( 5 - 6 per cent compared to a general rate of 9 ' 7 per cent )  . 
an analogy which seems justifiable is that of the parathyroids and the thymus ( the liver and the pancreas , or the enamel organ and the sublingual glands , or several other pairs might be used ) , which are equally closely related in their manner of origin , but yet , being histologically totally distinct structures , produce totally distinct tumours . foot 's ( 1947 ) manner of using the term primordium for the conical downgrowth of epidermal cells in which both begin obscures the fact that such primordia are not usually multipotential ; except in the few areas where sweat glands open into the mouths of hair follicles the two structures arise quite independently , and in most places at different times . we are impressed with the lack of truly organized structure in these tumours . we have already expressed our doubts about the so - called abortive hair follicles . one solitary tumour in this series ( unique so far in an experience of roughly twice the number of cases here reported ) showed reasonably convincing duct - like structures . 
wells hair matrix origin of rodent ulcers . mallory ( 1910 ) suggested that the rodent ulcer might arise from the hair matrix , and haythorn ( 1931 ) asserted that all rodent ulcers are simply hair matrix subsequent authors ( except wallace and halpert , 1950 ) have not tumours . accepted this completely , but no detailed refutation of his arguments has been attempted . 
they may , however , be shown to include several major flaws . ( a ) haythorn ( 1931 ) regards the connection of the tumour with the epidermis as purely accidental . 
we are not very impressed with the fibrils which we have been able to demonstrate ; in any case it seems probable that they are homologues of the tonofibrils of the rete malpighii , and that such fibrils form readily in any epidermal derivative , forming intercellular bridges which will be demonstrable as prickles wherever the cells are sufficiently separated . ( c ) haythorn 's ( 1931 ) identification of hair shafts we have already discussed . ( d ) we have been unable to confirm haythorn 's ( 1931 ) observations on the resemblance of the silver - stained basement membrane of rodent ulcers to that indeed it would be surprising if a hair matrix tumour of the hair follicle . developed a basement membrane like the " membrana vitrea " of the root sheath . we have not examined the neurofibril content of our tumours , but willis ( 1948 ) has indicated the unreliability of the evidence they provide . ( e ) haythorn ( 1931 ) describes atypical proliferations of hair follicles producing appearances which he interprets as the early stages of rodent ulcer formation . but nearly all these changes occurred in hairs surrounded by squamous carcinotheir occurrence is therefore rather to be taken as evidence that hair mata . follicles react in this sort of way ( it may be merely a disturbance of ordinary cyclical regeneration ) to any injury , and to be a warning that apparent transitional changes in hair follicles even within a rodent ulcer may be quite meaningless . ( f ) an argument which haythorn ( 1931 ) does not consider is that based on the site of origin of the tumours . 
where we find it necessary to disagree with them is in the assumption that this necessarily involves a specifically basal - cell origin . as von hansemann ( 1907 ) was first to point out , the surface epithelium and squamous carcinoma arise equally from the basal layer of the epidermis . seems , however , to be generally believed that if squamous carcinoma and rodent ulcer are different tumours they must have separate cells of origin . this is not necessarily so . 
at the time of first formation of hair follicles the epidermis possesses only a rudimentary " peritrichous " outer layer , and all the capacity for growth in either of its two main directions resides in one layer of cells . 
the stimulus to active keratin - ward proliferation ( whatever its proximate mechanism ) must be found in some contact with the external world - though there must be a basal level of activity of the degree seen in implantation cysts . 
the stimulus to the adnexa - ward proliferation almost certainly comes from some form of direct evidence for this in mammals is organizer - like action of the mesoderm . lacking , studies of hair genesis so far available ( danneel , 1931 ; trotter , 1932 ) being purely anatomical . 
but in birds , where the large size of the feather papilla makes transplantation experiments possible , the work of lillie and his school has produced a great deal of evidence on the process of morphogenesis ( lillie , 1942 ) , and it is known that the original stimulus to feather growth arises in the dermis ( wang , 1943 ) , though the type of epithelium responding to the stimulus partly determines the type of feather to be formed . in the absence of evidence to the contrary it seems reasonable to assume that in mammals hairs and sweat glands are evoked from the epidermis by similar stimuli derived from the dermis . thus we have two stimuli , two responses , and two very different forms of correspondingly we have two forms tissue formed from the same layer of cells . it is simplest ( though perhaps not entirely necessary ) to state the of tumour . resultant hypothesis in terms of pullinger 's ( 1949 ) theory of tumour origin in general . according to this , squamous carcinoma would be the result of the acquirement by epidermal cells of the ability to produce within themselves some stimulating substance which is normally only derived by external irritation , and rodent ulcer is the result of the acquirement by the same cells of the ability to produce an altogether different substance - a substance which is the normal stimulus to growth of the adnexae and which is normally derived only from the dermis . why then , if it results from the application of the same stimulus to the same tissue , is the rodent ulcer so poor a copy of hair follicle or sweat gland ? a possible answer may lie in the importance of the connective - tissue framework in the differentiation of adnexae , a matter concerning which there is some experimental evidence . 
a hypothesis is proposed ascribing the rodent ulcer to the autogenous production by the epidermis of a stimulus to proliferation normally supplied by the foetal dermis . our thanks are due to professor j . 
but for the extremes prognosis is definitelv affected in group statistics by the age at time of operation . in our e - xperience below the age of fortv vears axillary metastasis is much more apt to be present at the time of operation than bevond that age . in the s - ame , wav postoperative survivals . 
and the average postoperative hfe is shorter ( 16 - 7 months ) than in similar cases in the adult . " duk - es ( 1940 ) found that patients under the age of 40 vears had a higher incidence of lymphatic metastases than those in the age gmup 40 - 59 vears . 
they gave their opinion that the most malignant forms , both chnically and histologically , tended to occur at the younger ages . frissell and knox ( 1937 ) gave details of 46 cases of primarv lung cancer which on examining included - the age , and the duration of the - disease for each patient . these data we have found that there is no relation at all between age and the duration of the disease . 
he divided them into a carcinoma  . " group of 89 patients and an " epithelioma " group of 23 patients . he had no doubt that the clinical mahgnanev of the tumoiirs in the carcinoma group was greater in these patients than in older patients . 
undifferentiated tissue , often with manv mitotic figures in each field . " he found that h - valinization was never present that 1.ymphocytic infilltration and fibrosis were present to a moderate degree and only in the less mahgnant and that cellular differentiation was present mainlv in ovarian and tumours th - vroid carcinomas where . 
carried on through the cards and chose the first carcinoma recorded in the same organ and of the same sex in an individual in age group 46 - - 55 and again in a high age group . 
rectum ; and 61 vears and over for lung ( where the middle age group was 41 - 50 )  . for each tumour type we thus had three randomlv chosen groups of slides . 
the middle - aged and the old . ' the slides were then taken out of their holders and shuffled . this made identification of the sections impossible without reference to the laboratorv register of the numbers etched on the slides . 
ceu polymorphim and degree of fibrost - 3 ( scirrhous reaction ) the principle adopt . - d was to allocate grades i and iv to cases which were strikinglv low and strikinglv high respectivelv in these features . grades 11 and iii formed intermediate groups of " low moderate " and " high moderate respectivelv . for epidern " d differentiation the scale . 
 ' after completing the ' experiment , we were qiiite convinced that there was no such gross systematic change in type of material or standards of diagnosis over the years . 
we have , therefore , not considered that the difference between blocks arranged by date of diagnosis would appreciably reduce the residual error on which the significance of inter - age differences would be estimated . the experiment was therefore designed to allow for an analysis of variance . for example , in the sub - experiment " degree of glanduliform differentiation in rectal carcinoma " there was a total of 42 units or slides divided into 3 groups of 14 each for patients of 30 ygars or less , 46 to 55 years , and 70 years or more respectively , and divisible also into 14 groups of 3 each , i in each age group , diagnosed in an analysis of the variance in this balanced design in the same calendar year . there would then have been a total of 41 " degrees of freedom " comprising 2 for age , 13 for date of diagnosis , and 26 for residual error . 
of the 2 degrees of freedom for age , one could have been used to test the linearity of the difference in fact there was no need for this since a relatively coarse by age if necessary . analysis of the data indicated that the figures might we ] i have occurred by chance - on the hypothesis that there was no correlation between age and any of the factors we estimated . colloid ' formation " and " cell polymorphism , " using the total figures of each factor for for example , in the case of " epidermoid differentiation " there each age group . was a contingency table of the formthe x2 test was carried out for each factor , with the exception of the results for the various factors were epidermoid differentiation : 6 x2 4 - 95 p 0 - 7 - 0 - 5 . glanduliform differentiation ( consolidating grades iii and iv ) : 4 x2 = 4 - 55 p = c . 
at the begin9of the experiment , however , we laid down the hypothesis that we had no reason to expect significant results from one tumour type or characteristic more than another . it foflows that there is , so far as our results per se are coneemed , no rea - son to regard this as indicating a real correlation between age and increas ' epidermoid differentiation in bronchogenic carcinoma . it is notable , however , that in our reading of the literature the tumour in which the correlation between increasing differentiation and increa - sing age ha - s been most strikingly demonstrated ( geschickter and denison , 1934 ) is also bronchogenic carcinoma . consequently our figures , in so far as they are confirmatory , are also suggestive . we did , in fact , although we have not given the table , estimate , using the method of analysis of variance , the probabihty with which the figures for epidermoid differentiation and age could have occurred by chance for epidermoid differentiation of ( a ) bronchogeiiic carcinoma , ( b ) carcinoma of the cervix , ( c ) carcinoma oflip . 
causes an almost complete cessation ofepidermal mitotic activity , and the same effect is caused by phloridzin , which interferes with the phosphorylation of sugar . since insulin and phloridzin both exert their mitosis depressing effect bv reducing the availabifity of sugar , it appeared probable that a similar depression would also be induced bv starvation . 
12 hours of starvation the blood sugar level remained fairlv high , but the drop in the mitosis rate was pronounced . however , during the afternoon sleep period there was a marked rise in mitotic activitv at 14.00 hours to a figure of 4 - 1 0 - 35 , which is about half the figum reachc ; l by the well - fed controls . - after 24 hours ' starvation the blood sugar level had faren seriously , and mitotic .1gain , however , there was a slight activitv had reached a very low level . response to the period of rest , so that at 14.00 hours a figure of 2.0 0 - 21 was this is about a quarter of the figure for the control animals obtained . ..a&r 36 hours the blood sugar concentration had dropped to 100 mg . 
observations were next made on the effects of such diets on epidermal mitotic acti - %ity . in prehminary experiments determinati ' ons were made of the dailv food intake of threeor four - monthold strong cba males which were allowe ; 1 to feed ad libitum on a diet of rat cake . 
13ttllo ' ug14 66 per cent diet remaitied active aiid in excelleiit health , btit those which received a 50 per cent diet showed sigiis of ill - health , and it is dotibtftil whether they could have stirvived the treatnieiit for i - iiore tliaii another few ' weeks , at the end of the experiment at 20.00 hours all the niice wei - e killed and blood sugaiestimations were made . 
that middle age is characterized bv a generallv raised mitosis rate which must itself assist in the development of any latent tumour cells which may be present . ll ' hfle the reason for the increased initotic activitv is still obscure . 
this observation mav afford some explanation of the fact that middle age is characteristically the cancer age . since the development of latent tumour cells is assisted bv conditions of hyperplasia , it is reasonable to suppose that conditions of hypoplasia wih have the hyperplasia increases the chances that the latent tumour cells opposite effect . will be stimulated to multiplv . 
and so delavs the development of those tumours which do forand prevents altogether the appearance of manv which otherwise would forthus it is possible to provide a logical explanation of tannenbaum 's re - sults . 
and the fact that carbohvdrate - supply is the critical factor in both cell division and tumour genesis can be taken as corroborative evidence . at last it iis becoming possible to split the cancer problem into two separate parts : that which is coneemed with the formation of latent tumour cells . 
but it now appears possible that a practical control of cancer mav be developed from a thorough understanding of those factors which govern normal cell division . if hypoplasia can be maintained without damage to health . 
and reveal fewer pathological changes in the tissues . " this mav well be related to , the fact that hypoglyeaemia not onlv reduces the rate of cell division of a host . but also that of a parasite . 
a sin - iilar effect is produeed bv restricted diets . - kninials rationed to 66 per cent of what thev would eat if fed .4 , libitunt have an epidermal mitosis rate which is less than 40 per cent of that of well fed controls . if rationed to 50 per cent . 
the mitosis rate dxops to about 15 per cent of that of the controls . it is suggested that these observations provide an explanation of tannenbaum 's results on the prevention of tumour genesis bv diet restriction . 
mottram ( 1945 ) published the results of experiments designed to demonstrate a relationship between the tumour yield induced by benzpyrene and the number of epidermai mitoses present at the time of application of the carcinogen . painting the skin of rnice at midnight resulted in a higher yield ofpapillomata than did painting at midday . 
of 9 : 10 : dimethyl - 1 : 2 - benzanthracene ; specific tumours of the thymus and lung , which are prone to spontaneous tulour developmnent in this strain , were induced by this treatment , whereas the mammary and subcutaneous tissues which are also prone to spontaneous tumour developmnent did not respond . the testis and kidney , in which spontaneous tumours have never been observed , and the spleen , lymph nodes and bone marrow , inl which tumours are not presumed to develop spontaneously , also proved insusceptible to this slight carcinogenic influence , and did not respond to a very powerful direct carcinogeniic influence . 
paraffin of the same batch as that used for analogous experiments performed with 9 : 10 - dimethyl - 1 : 2 - benzanthracene ( engelbreth - holm and rask - nielsen , 1947 ; rask - nielsen , 1948 )  . the technique of application was the same as that used in earlier experiments . 
rask - nielsen zanthracene - induced specific tumours , lymphosarcomatous hyperplasia of the thymic gland in 1i7 per cent , 6.7 per cent , 10 4 per cent and 13 per cent , the effective total being the number of mice whose survival time was at least that of the youngest tumour - bearing animal within all four groups , namely , three months . in addition a thymic spindle - cell sarcoma was found in one animal injected with methylcholanthrene . injections of three of the hydrocarbons into the lung induced thymic tumours in 2 , 2 and 4 per cent following the injection of benzpyrene , dibenzanthracene and 9 : 10 - dimethyl - 1 : 2 - benzanthracene respectively . 
the average latent period , the interval between injection and the death of the animal , following injections into the thymus of benzpyrene , dibenzanthracene , methylcholanthrene , and 9 : 10 - dimethyl - 1 : 2 - benzanthracene was 15 , 14 , 16 and 21 weeks , respectively ; the minimum latent period of the three latter hydrocarbons was 11 , 14 and 10 weeks , respectively . 
analogous negative results were observed by others , using mice from various strains ( esmarch , 1940 ; strong and smith , 1939 )  . experiments including the nho strain of mice , however , exhibit positive results ( strong and smith , 1939 ; strong and williams , 1941 ; strong , 1945 )  . 
from these experiments it is rather difficult to form an estimate of the susceptibility of the mammary tissue to carcinogenic hydrocarbons , since susceptibility is dependent on simultaneous hormonal stimulation as well as on the presence of the milk agent . since the present experinments included only non - breeding mice . 
the hormonal stimulation was at least not maximal . street mice have been used in all the experiments recorded here and the deductions made apply only to mice of that strain . the susceptibility to carcinogenic action of various tissues being genetically determined ( lefevre , 1945 ) it is necessary , in order to elucidate the susceptibility to a direct carcinogenic action of a particular tissue , to examine animals from different strains under identical experinmental conditions . such investigations , for the time being with application of 9 : 10 - dimethyl - 1 : 2 - benzanthracene to the thymus , lung and subcutaneous 116 r . 
fodden. from the department of pathology , university of liverpool , and the liverpool cancer control organization . received for publication august 20 , 1948 . in his ' textbook of pathological anatomy ' rokitansky ( 1861 ) made his classical statement that pyloric cancer was exactly bounded by the pyloric ring , and that the growth never reached beyond into the duodenufrom this time it appears that the majority of observers , with the early exception of brinton ( 1864 ) , commented upon the integrity of the duodenum in cases of carcinoma of the stomach . 
many well - known surgical teachers spoke of the habitual immunity of the duodenum from invasion . kocher ( 1893 ) , mikulicz ( 1898 ) and most ( 1899 ) believed it to be always constant . kocher ventured that it was a problem of extreme interest to consider why gastric carcinoma grows in almost all cases towards the cardia , yet stops , on the contrary , at the duodenopyloric junction . it was brinton who first took especial exception to this proposition of rokitansky . 
he brought against it criticisms founded upon numerous personal caseobservations : " we may justifiably apply to it a criticism of unusual severitya criticism which , even if it weigh every word , will scarcely do more than the author 's ( rokitansky 's ) terse and weighty proposition really deserves . " from 125 cancers of the pylorus studied by brinton , there were no less than ten cases in which the disease was not bounded by the valve , but passed beyond it for a variable distance , often an inch or two inches , into the duodenuhe gave no information concerning the method of this spread . brinton concluded by saying the rules which rokitansky had the merit of laying down were , in this respect , like many others in pathology , of general though not of universal importance ; their value was not much affected by occasional exceptions . this question of duodenal invasion by pyloric cancer seemed to present a surgical problem of out of a large series of gastric carcinoma cun6o ( 1903 ) took 11 j . 
fodden some magnitude , and the frequency and degree of its occurrence has been the object of several subsequent researches . carle and fantino ( 1898 ) , discussing the pathology and treatment of carcinoma of the stomach , expressed disagreement with kocher . they mention three cases out of fourteen gastric resections in which neoplastic infiltration had spread under brunner 's glands for a distance of one to three centimetres . in addition , these authors stated that such a progression of the growth towards the duodenum necessitates an extension of the surgical resection as far as the second part of the duodenum . other later surgical authors , notably borrmann ( 1901 ) and cuneo ( 1903 ) , attached considerable importance to brinton 's " occasional exceptions " with reference to the spread of cancer into the duodenum . cuneo ( 1900 ) , during his classical work on the lymphatic propagation of pyloric cancer , was already investigating the accepted state of immunity of the duodenuhe remarked on the disaccord between the macroscopic aspect - the abrupt termination of a gastric cancer at the level of the pylorus and the results of the histological examination . this examination systematically diminished , he said , the percentage of the duodenal integrity . cancers of the pylorus , and subjected their distal extension to a critical macroscopic and microscopic examination . 
but , he goes on to say , it is impossible to ignore the fact that such a wide resection complicates the operation , and necessitates a dissection of the duodenum from the pancreas . the researches of fenwick and fenwick ( 1902 ) do not lend support to brinton 's findings , as they discovered out of a series of 87 pyloric cancers only 2 cases which demonstrated lymphatic spread into the duodenum . these authors remarked that though malignant growths of the pylorus may project into the lumen of the duodenum , they rarely implicated its walls . however , it is open to question whether their histological investigations were sufficiently careful to give reliable evidence , and the conclusions they advanced could scarcely have been founded upon their own results . moynihan ( 1926 ) was very familiar with the possibility of duodenal permeation in gastric cancer , and stressed that the duodenal integrity was more apparent than real . 
he quoted several of the authors already mentioned , and advocated the removal in all cases of pyloric cancer of the whole of the first portion of the duodenum . sherren ( 1932 ) , describing the methods of spread of gastric cancer , mentioned his own surgical familiarity with duodenal infiltration by carcinoma . 
a study of the microscopical anatomy of this region , in relation to the morbid process in situ , may help to clear up some components of each problem . to cun6o ( 1900 ) the apparent arrest of a cancer at the pylorus was due , for him it depended upon an absence of conabove all , to a mechanical cause . tinuity between the gastric and duodenal submucous spaces - a continuity broken by the annular muscle of the pylorus , and by a condensation of cellular tissue within the submucosa to form a dividing septum . his gelatine injections into the gastric submucosa near the pylorus met with an " invincible obstacle . " at the same time he admitted that though it would be ludicrous to consider such barriers as absolute in opposing gastric cancer , they still played the principal role in the protection of the duodenum against neoplastic invasion . for brinton ( 1864 ) and for fenwick and fenwick ( 1902 ) the reason was again anatomical . 
the muscular coats of the two viscera along the line of fusion were so distinct from one another that a direct continuity could scarcely be said to these authors made the analogy between the duodenal attachment to exist . the stomach and the manner in which the vagina embraces the neck of the uterus . to them a growth of the pylorus was more apt to infiltrate the contiguous walls of . 
in their opinion it was obvious that the duodenum may possibly be invaded from the pylorus by the in addition , extension of cancer along the submucous and subserous channels . both jamieson and dobson and cun6o emphasized the final possibility of retrograde lymphatic spread . 
as these nodes also receive vessels from the that this may be of some duodenum , an indirect communication also exists . importance was stressed by moynihan ( 1926 ) when he says that the implication of a single gland may so sufficiently disturb the direction of the lymph current that cancer cells from a primary growth may pursue an erratic course . horton ( 1928 ) working upon stomachs removed from young subjects one to three hours after death , injected 35 with coloured gelatine or indian ink . 
two of the present surgical specimens were small cancer - ulcers upon the lesser curvature of the pyloric antrum , and cancer had invaded on the gastric side only the submucosa , with extension in one case into the pyloric sphincter muscle . 
yet both these specimens showed cancer islands within duodenal lymphatics at a distance of almost 1 ' 5 cfrom the pyloro - duodenal junction . this was also instanced by a cabot case no . 
25472 ( 1939 ) of the massachusetts general hospital , in which specimen a small malignant ulcer of the pylorus was limited to the mucosa except for a small extension below the muscularis mucosae . 
the only structure apparently offering any kind of barrier action was the gland layer of brunner . it has been the experience of the majority of authors that the layer of brunner glands remains free , in spite of many , and long , extensions of gastric cancer beyond the pylorus . in addition , whatever the method of propagation , it is exceptional that the mucosal epithelium of the 246 j . 
a resection of almost 3 cof the duodenum was made . microscopically the duodenal growth was so proliferative in the muscular and serous layers that one could not avoid an impression that an even greater length of duodenal resection would have been advisable . 
davie for his help with the plan of this research during its initial stages , and those pathologists and surgeons of the liverpool regional hospitals who have so kindly supplied me with specimens and helpful data . 
as none of these other cell types are seen in the typical rodent ulcer , the lesion must be considered as being completely undifferentiated . but such a tumour should be highly malignant , more so at any rate than the ordinary keratinizing squamous cell carcinoma . this difficulty is resolved by the more recent view , apparently a revival of krompecher 's first thoughts on the matter , which relates these tumour , not to the basal layer of the epidermis , but 214 a . 
thackray to those accessory structures of the skin derived from the surface epithelium , the haythorn ( 1931 ) for example , relates the tumours hair follicles and sweat glands . in nearly every case to existing hair follicles and matrix , regarding any connection between epidermis and tumour tissue as secondary and fortuitous . 
some workers find it difficult to reconcile an origin in the hair matrix , which is comparatively deeply situated , with the superficial situation of early tumours . but hairs have a definite life span , at the end of which they atrophy , are shed and replaced . 
the pilar type , which included about threequarters of the tumours in his series , he further subdivided into a pilar type proper , a primordial type , a cylindrical celled or " ribbon " type , and a cystic type . 
from this large number 200 cases were finally selected to form the basis of the present investigation . the selected cases were all those that fulfilled the following conditions : 1 . 
the sections available did not always include the whole lesion , but where this was so a note was made as to whether excision appeared to be complete , incomplete or doubtful . 
of the 12 cases which recurred , 8 were obviously incompletely excised , one appeared to have been completely removed from the section studied , and 3 were doubtful . of the 51 successful excisions , 42 had been removed with an adequate margin of healthy tissue , one tumour reached to the limit of excision , and 3 came so near to the edge that the outcome seemed doubtful . in the remainder the sections available did not cover the whole lesion and no opinion as to completeness of removal could be given . of the 12 failures with surgery , one was cured by further excision , and 4 more by irradiation . three of the remaining 7 died as a result of the disease , unchecked by further surgery or irradiation , 2 patients are still alive with the ulcer active , and the other 2 died of other causes with the ulcer uncured . 
of these 39 ulcers , 19 were soon cured by further treatment , leaving 20 which proved radio - resistant . it is proposed to compare the incidence of various histological features in the biopsies from the 82 lesions cured by a single course of radiotherapy with their these figures incidentally introduced incidence in the 20 radio - resistant ulcers . must not be considered as representative of the results of radiotherapy by modern methods , for it must be remembered that these cases were all followed for 10 years , the most recent treatment being therefore in 1938 . influence of size of lesion on prognosis . before considering the histological analysis of these cases it is interesting to note the influence of the size of the lesion on the prognosis . 
the greatest measurement of the ulcer in inches was noted on the cards , and these sizes were then divided into two groups , large and small , taking one inch or more as the criterion of largeness . this boundary between large and small was placed quite arbitrarily and in fact there proved to be about twice as many small lesions as large . of the surgically excised lesions , the size had been noted in 51 cases , and of these 16 were large and 35 small . 
thackray influence of type of lesion on prognosis . as has been described already , the lesions were classed on their histological appearance as being circumscribed ( group i ) , predominantly circumscribed , but with early infiltration present ( group ii ) , or infiltrative ( group iii ) in their type of growth . it was hoped that this classification might give an indication of the malignancy of the tumours without introducing controversial problems concerning their origin . it may be suggested that the infiltrative lesions are the large and advanced ones , and that any correlation we may note between type of growth and prognosis will merely be a repetition of what we saw in the preceding section . 
the whole series of surgical cases , including also those in which surgical excision was combined with radiotherapy in the first instance , was therefore grouped as to size of lesion and type of growth . 
the great majority of the lesions with no palisade formation apparent on section were of the infiltrative variety , whereas in the group with well - marked palisades 30 were circumscribed ( group i ) and 19 infiltrative ( group iii )  . 
the question of metastasis does not arise , and it is only necessary to be sure that the margin of the excised tissue is free from growth . this can be done by cutting serial sections of the excised tissue , which is well worth while if there is any doubt . if serial sections are not prepared , then the nature of the growth itself gives a fair indication , for tumours of the circumscribed type , as we have called it , are relatively compact and their extent is apparent from the surface , whereas infiltrative growths extend beyond their apparent limits . when considering the results of radiotherapy it is important to distinguish clearly between radio - sensitivity and radio - curability . 
a tumour which melts away when irradiated may often recur , whereas the tumour which can be destroyed curing the patient , may show no dramatic immediate response . in this investigation the ten - year cure is the criterion of successful treatment , so that radiocurability is all we are concerned with . it is well known that the histological grade of malignancy as applied to carcinomas of the skin , say , or breast , has some bearing on the outcome of radiotherapy - those of low - grade malignancy often being curable , those of high malignancy usually being sensitive , but recurring . in carcinomas of the breast such features as the degree of tubule formation , variability in size and staining of the nuclei and the frequency of mitoses are all significant , and are taken into account in determining the grade . 
the results of the present investigation have been largely negative , for most of the histological features of rodent ulcers considered have been shown to be without significance in assessing the probable outcome of treatment . 
thackray the presence of cysts , pigmentation , nor even the number of mitoses seem to have any bearing on the question . the only method of dividing up the tumours which seemed to bear any relation to the prognosis was that based on the habit of growth , into circumscribed or infiltrative , and the fact that an intermediate group was necessary indicates grading as practised in connection the difficulty in making this distinction . with carcinomas of the breast and elsewhere is an indication of the degree of malignancy of malignant tumours ; the term " group " has been used here rather than " grade " in view of the suspicion that many of the group i tumours are probably in actual fact benign . 
of the other histological features studied , only the extent to which growth is infiltrative seemed to have a direct bearing on the prognosis and prospect of cure by radiotherapy . my thanks are due to the surgeons of the middlesex hospital and to professor b . 
korteweg. from ae antoni vanleeuwenhoek - hui8 , am8terdam . given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . it was shown by little et al . 
korteweg ( 1936a , b ) predicted that chromosomal factors must also be active . this prediction was based on the fact that differences in susceptibility to cancer exist between dba females and the fl hybrids of the dba x c57 black cross . 
the present paper concems these chromosomal factors . the fact that chromosomal factors exist is important , but the way they act , their mechanism , is of still greater inter ' est . 
a number of investigators have considered the role of the follicular hormone , and checked the possibility of differences between the oestrus cycles of the high - cancer and low - cancer strains of mice . 
as no differences were found this form of experiment was discontinued . yet i was not convinced . i spayed females of different strains , and afterwards determined the sensitivity of the vaginal epithelium to oestrone by means of the vaginal smear method . i found - that , to cause oestrus , it was necessary to inject three times as much oestrone into females of the high - cancer strain dba as in those of the low - cancer c57 black . 
the ovaries of our high - cancer strain greatly sur ass those of our low - cancer ammals this is partly caused by the somewhat greater num ' ber of graafian in volume . follicles , partly by the much greater number of corpora lutea , and partly by the r . 
hooker ( 1945 ) recorded that the effect of progesterone can be demonstrated in ' the structure of .the endometrial stroma just as well as the effect of i have found that the epithelium of the uterus of the virginal mouse oestrone . also shows a response to progesterone . 
as in the endometrium of the human female , in which on the 17th day of the cycle , shortly after the bursti ' ng of the follicle when the luteinizing process is beginning , a vacuole becomes visible in the basal pait of the epithelial cells , so also this phenomenon occurs in the virginal it is the ' refore necessary to be aware always of the possibilitythat mouse . progesterone too may influence susceptibility to cancer ( by its synergistic ' action with oestrone )  . if the mammary glands ' also of high - cancer strain mice should be relatively insensitive to oestrone , as is the vaginal epithelium of high - cancer strain animals , then the excess of oestrone produced would probably be harmless to the mammary glands . if , on the contrary , the sensitivity of the mammary glands of highand low - cancer strain animals should be the same , the excess of oestrone produced in high - cancer strain animals might be injurious to the mammary glands . it therefore became necessary to determine the 'sensitivity to oestrone of the mammary glands . 
of oestrone into 21 - months - old spayed females of different str ' ains , it seemed evident to van gulik and myself that our high - cancer strain females dba reacted to a lesser degree to oestrone than our low - cancer strain animals when judged by the extent of development of the glands . in 21 - m ' onths - old castrated males injected with 13 i.u. 
of oestrone , we found more growth also in the low - cancer than in the high - cancer miihlbock then drew our attention to the fact that in our experiments strains . we had injected large doses . if one wishes to determine the sensitivit ' y of an organ it is preferable to determine the threshold dose '  . 
as the first visible sign of an effect of oestrone , miihlbock ( 1948a ) took the beginning of budding of the end of the milk ducts ( swelling of the end bulbs )  . 
firstly in the males of the three strains c57 black , dba and 020 , the sensitivity of the mammary gland is about the same according to the threshold test . secondly , the minimal dose causing budding ( swelling of end bulbs ) in castrated males is approximately five times less than in non - castrated males . obviously in non - castrated males the testosterone largely counteracts the oestrone . this supposition proved to be right , as in castrated males which were injected both with oestrone and testosterone , the presence of the latter suppressed the action of the former . in noncastrated males three times as much oestrone is needed to cause budding ( swelling of end bulbs ) in the glands of high - cancer strain dba mice than in those of lowcancer strain mice ; in castrated males the sensitivity is the same . 
the substance , usually dissolved in tyrode saline , is added to a culture after a dividing cell in mitosis therein has been selected fgr study , and the reaction of that cell to the agent is recorded for a period usually less than one hour . 
the cells in prophase then to be found in the outgrowth of the stained cultures were subsequently counted . the effects on mitosis of three phosphorylated naphthohydroquinones have been studied in this work . these are as follows : 1 . 
the di - methyl compound was found to be slightly less active than synkavit as a mitotic inhibitor in vitro . it again potentiates the effects of irradiation . further work on this substance is in progress , including clinical trials . normal mitosis in the chick osteoblast . osteoblasts were chosen for this work on the grounds that clear phase contrast pictures of them can be obtained during mitosis , and that they appear to be much less sensitive to light than are other fibroblast types of chick cells . in previous papers there has been described the normal course of cell division which can be observed under the phase - contrast microscope in living osteoblast cultures ( hughes and fell , 1949 ; hughes , 1949b )  . 
the nucleoli and nuclear membrane disappear remarkably rapidly at the end of prophase , but while the spindle is being formed several minutes pass before the chromosomes assume their equatorial position as a metaphase plate . it is convenient to speak of all this period after the loss of nuclear membrane and nucleoli as metaphase , and to separate an early from a late division of this phase of mitosis , respectively before and after establishment of the metaphase plate . the general cytological effects of compounds , i , ii , and xx viii . in the present work over fifty film records of the behaviour of dividing cells under the influence of these substances have been made . 
20. - cell from the outgrowth of a culture treated with 5 x 10 - 7 m synkavit ( i ) for 40 cell has undergone mitosis with the suppression of cleavage . 
nucleoli still present 5 mlater ditto , ditto , 10 mlater 3.5 x 10 - 6 m metaphase metaphase plate metaphase late prophase early metaphase 4 x 10 - 6 m prophase . 
the methylfree substance exerted this effect in late telophase over a range of dilutions of about one thousand - fold ( 27 , 3 x 10 - 9 m ; 36 , 1 x 10 - 6 m )  . 
at higher concentrations the substance rapidly kills both dividing and resting cells ( 38 , 1 x 1o - 5 m )  . it is noteworthy that although the effect of the methyl - free compound differs considerably from those of the methylated derivatives , both in the types of inhibition and the dilutions at which they are exerted , yet in the present data there are no significant differences in the maximum concentrations of each of the three substances at which a cell has been observed to undergo a normal mitosis ( synkavit , 17 , 18 , 19 ; di - methyl compound 25 , 26 ; non - methyl 36 , 42 )  . one point in the mitotic cycle on which these three substances do not appear to exert any immediate effect is the entry of cells into prophase . early prophases are still found in cultures treated with these substances at the highest concentrations which have been used in the course of this work . since synkavit and the di - methyl compound can interfere with the division of a cell in several ways , a culture which has been treated with these substances at appropriate dilutions for periods of an hour will show a variety of effects . these depend both on the individual sensitivity of the cells and the phase of mitosis which they had reached at the time of adding the substance . 
the fate of a particular cell selected for photography is , therefore , to some extent a matter of chance . it may be that the 55 instances which are recorded in table i do not exhaust these possibilities , though study of the whole cultures after fixation has not so far revealed any other types of inhibition . the variability of the behaviour of cells towards these substances , however , extends further than to inhibition at different stages of mitosis . treatment of a culture with a concentration sufficient to arrest and damage some dividing cells will leave unaffected others which continue a normal mitosis . this fact can best be illustrated by comparison of the effects of synkavit in a number of instances where it was applied to cells early in.prophase. 
at 1 x 10 - 6 m a normal mitosis ( 59 ) , an arrest in metaphase ( 60 ) and a clumping of chromosomes and cell oedema at this stage were recorded ( 52 )  . 
when the di - methyl compound at 4 x 10 - 6 m was added to cells in metaphase , there were seen both a normal mitosis ( 26 ) and an instance of clumping of the chromosomes ( 24 )  . this circumstance must be borne in mind in the search for any further generalisations from however , one other conclusion clearly emerges , namely , that the these data . effect of these substances on dividing cells is greater when they are applied at early stages of mitosis . 
with synkavit at 3 * 5 x 10 - 6 m addition to a cell early in metaphase resulted in clumping of chromosomes and oedema ( 15 ) , while later in metaphase in two instances this concentration was not found to be inhibitory ( 18 , 19 )  . 
it is probably true to say that phase specificity is most marked with substances to which the cell in division is much more sensitive than during the restthus , for instance , the action of colchicine on the dividing cell is ing stage . restricted to its effect on the mitotic spindle , and this is exerted at dilutions much greater than those necessary to affect intermitotic cells . 
the proportion of cells entering prophase thus , this substance appears to penetrate the cell memhas then decreased . brane so slowly that a concentration within the cell sufficient to provoke these effects is not reached for several hours . 
the secondary fusion of the daughter cells in late telophase which has been observed in the course of the present experiments may well be due to an alteration in the properties of the cell surface . 
a large number of binucleate cells were seen in stained cultures after treatment for 6 hours with this substance at concentrations of 1 x 10 - 9 m . this effect may be due either to secondary fusion of the daughter cells , or to an inhibition of cell cleavage in the first instance . finally , the value of the two general methods of investigations of the action of chemical substances on living cells in culture can be compared . in the one mainly used in the present work the effects of the agent on a single living cell in the culture is followed ; in the other a quantitative analysis of the whole culture is made after fixation and staining . 
where the effects of a given treatment often differ from cell to cell , it would be unwise to draw here the quantitative conclusions from the study of only a few examples . analysis of large numbers of cells is indispensable . 
cells may be inhibited at any point in mitosis , though their entry into prophase is apparently unaffected under the conditions of these experiments . arrest in metaphase and clumping of the chromosomes is the most common effect . individual cells vary greatly in their sensitivity to these substances . 
at sub - lethal concentrations the latter does not affect the course of mitosis until late in telophase , when to a varying extent the daughter cell re - unites . we wish to thank a . 
niicleoprotein , are believed to play an important part in cell reproduction , but their basic functions have recently developed analytical techniques , particularly not been elucidated . chromatographic analysis and the use of the ultra - violet spectrophotoilieter , have produced considerable fresh data on the composition of the nucleic acids , but much less research has been done on the protein fractions . this paper collects some information on this topic and some analytical results obtained in our laboratory are presented . the proteins found in the nucleus . mirsky and pollister ( 1946 ) were able to dissolve nuclear nucleoprotein in 1 m sodium chloride , after first removing the cytoplasniic material with 014 m saline , and to isolate this nucleoprotein by diluting the solution to a salt concentration of 014 m , when it precipitates in fibrous form . similar material could be obtained from isolated nuclei ( dounce , 1943 ) confirming that these nucleoproteins were of this extracted material was found to consist of deoxyribonuclear origin . nucleic acid and two protein fractions , one of which was classified as a histone , being readily soluble in acid and precipitated out of solution by ammonia . 
from isolated nuclei , apparently failed to do so from the chromosomes , and therefore claims respecting the identity of tr.pr. and the residual chronlosomes must await analytical confirmation . 1 - t is of special interest that cells which could be considered metabolically active ( liver , kid ' ney ) contain as mucb . 
as 50 per cent of the total isolated chromosome material in the form of residual chromosome . stedman and stedman ( 1943 , 1947 ) also isolated an acid - insoliible protein from the nucleus which they termed " chromosomin , " believing that it was the structural protein of the chromosome and that in the nucleus it was embedded in a support of nucleo - bistone . 
the work of both mirsky ( 1947 ) and stedman and stedman ( 1943 , 1947 ) seems to indicate that a protein forms the basic structure of the ebromosomes though the part played by the nucleic acid and the histone this is an important aspect but cannot be discussed here . 
from this material they isolated nucleic acid and histone , and in addition a fraction with isoelectric point between 5 - 8 and 6 - 15 , which coiistituted an appreciable proportion of the nuclear proteins . 
the evidence suggested that the fraction contained a sulphur - containing protein with an acidic isoelectric point and a protein of globulin typeif this material was , indeed , of nuclear origin , then this fraction appears to correspond in properties with the " chromosomin " ' of8tedmanandstedman ( 1943 , 1947 )  . 
the nuclei and chromosomes were fi - rst extracted with i m sodi - um chloride and then the residue was extracted with dilute sodium hydroxide . acidification cf this extract precipitated a protein which contained about 6 per cent arginine and since the nucleoprotein extracted from the nuclei has been no nucleic acid . shown to contain nucleic acid histone and tr.pr. , the alkali soluble protein here must be of different character and this is more likely to be the protein of the finally , two russian workers residual chromosomes than the tr.pr. 
fraction. ( zbarskii and debov , 1948 ) have reported that the protein remaining after the extraction of the nucleoprotein consists of two fractions , one of which is weakly acidic in character , being soluble in dilute alh - ah and having an isoelectric point 132 d . 
da - vidson and lawrie ( 1948 ) , using paper chromatography , carried out qualitative analyses of specimens of histone and non - histone ( acid - insoluble ) proteins from nuclei of calf thymus , rat liver and fowl erythrocytes . the histones contained alanine , arginine , aspartic acid , glutamic acid , isoleucine , leucine , ivsine , phenylalanine , proline , serine , tyrosine , valine and one unidentified constituent . in addition to the above amino - acids the acid - insoluble portein contained i per cent tryptophane , glycine but no lysine , and a further unidentified component . in silnilar studies khouvine and gregoire ( 1949 ) have analysed nuclear nucleoprotein from rat epithelioma and compared it with that foiind in the surrounding nectrotic the acid - extractable proteins nn - ere compared , using paper chromatotissues . graphy and found to be qualitatively the same . very recently stedman and stedman ( 1950 ) have reported arginine contents of histones from various tissues and found they all contained about 28 to 30 per cent arginine ( as per cent of total nitrogen )  . in addition these workers claim to have isolated sub - fractions of the histone of different composition , but no details of the n - iethod of extraction or fractionation are given . importance has often been attached to the tryptopbane content of the nuclear generally speaking , histone specimens have been found to contain proteins . 0 - 0 per cent to 0 - 1 per cent , while the acid - insoluble proteins have about 1 - 0 per cent tryptophane . stedman and stedman ( 1 - 947 ) believe that the presence of tryptopbane in histone is an indication of impurity . mirsky and pollister ( 1946 ) found very small amounts of tryptophane in histone , but about 0 - 8 per cent in the tr.pr. 
of the other histochemical studies reference will only be made to the recent work of thomas and steinitz ( 1950 ) using a method which will stain proteins of high arginine content in paraffin sections . 
the application of this method has indicated that the ratio of arginine - containing protein ( histone type ) to total possibly a combination of this technique protein , is greater in normal epidermis . with spectrophotometric methods will give further data on the localization of the basic proteins in the nucleus . extracted histone has been tested for bactericidal action ( nnleissman and graf , 1947 ) and the pharmacological properties of histone from avian erythrocytes bave also been studied . 
the nitrogen content of histone from both the above materials was 18 - 2 per cent . the acid - insoluble protein was prepared from the whole protein of the nucleoprotein , separated from the nucleic acid by the method of sevag , lackman and smollens ( 1938 )  . 
the solvent n - bu ' tanol - acetic acid - water ( 4 : 1 : 5 ) was used , followed bv either collidine - lutidine - water ( 1 : 1 : 4 ) or phenol - water . the papers were sprayed with 0 - 2 per cent ninhydrin and heated for 6 minutes at 100 ' c . 
to develop the characteristic ninhydrin - amino - acid colours . reference mixtures , " , , ere alwavs run at the same time . in the acid - hydrolysates of three specimens , histone and the acid - insoluble protein from thymus gland and histone from rat sarcoma , exactly the same comindeed , by this qualitative method it was not possible to ponents were found . detect any differences in composition in three fractions . all contained the following aniino - acids : alanine , arginine , aspartic acid , glutamic acid , glycine , bistidine , isoleucine and leucine , lysine , phenylalanine , proline , serine , threonine , tyrosine , were detected . 
for each analysis except in the case of the solvent ( c ) , wben half this amount was used to avoid excess of water on the coluinn . columns of potato stai - ch i cdiameter and 30 clong were used in conjunction with the following solvents : ( a ) n - butanol - n - propanol - 0 . 
moore and stein ( i 949 ) studied the recovery of individual amino - acids from mixtures and found that the chromatographic procedure on starch columns is capable of yielding recoveries of 100 3 per cent . their average recoveries were well within this rang - e and the sum of the aminoacids was almost invariably accurate to i per cent . 
by separate analysis no appreciable amount of trytophane could be found in the histone specimens . less than 0 - 04 per cent was found using the colorimetric method described by spies and chambers ( 1948 )  . 
protein was indicated in the rat sarcoma histone . the thymus histone specimen was quite negative in this test . comparing the two specimens analysed , there are no striking differences in composition . 
stedman and stedman ( 1943 ) believed that their acidinsoluble material ( chromosomin ) , despite a high content of the basic amino - acids must also contain a relatively large amount of glutamic acid . 
the present analyses do not support this claim . since the two proteins , histone and the acid - insoluble ty ] pe are so similar and are extracted together , it seems possible that they may be bonded together in the cell and are subsequently split by the extraction techniques to give basic soluble histone and a denatured " sulphurhistone " product . no considerable differences have as vet been found in the nuclear proteins of normal and neoplastic tissues . there are some small differences in the aminoacid compositions and there is evidence of ' variation in the proportions of the this may be connected with mirsky 's protein fractions in different tissues . ( 1947 ) observation on the variation of the proportion of " residual chromosomes " in the cell in relation to its metabolic acti - vity . 
complete amino - acid analyses of histone from calf thymus and from rat only small quantitative differences v - ere found , but there is sarcoma are given . evidence of a difference in the ] proportion of the protein components in different materials . this work is part of the research programme of the birmingham branch of the british empire cancer campaign . 
3. received for publication february 6 , 1947 . it has frequently been noted by the early investigators of tumour pathology that besides a high mitotic rate , tumour cells may also show great variation in the details of mitosis ( pianese , 1896 ; hansemann , 1904 )  . 
the abnormalities may concern the behaviour of the chromosomes or the cytoplasm or both , and most of them lead to degeneration and death of the cells . this is largely due to the deficient nuclei which usually result from abnormal mitosis . 
by repeated chromosome multiplication within the nuclear membrane ( biesele , poyner and painter , 1942 ; koller , 1943c )  . polyploid cells are frequent in tumour regions where .cell breakdown is in progress on a large scale . 
x - rays induce stickiness of chromosomes in plants and animals ( markquand , 1938 ; koller , 1943a ; darlington and la cour , 1945 )  . temperature changes and chemical treatment can lead to similar abnormalities ( barber and callan , 1943 ; ostergren , 1944b ) , and complete suppression of the spindle is brought about by low temperature and by specific compounds , such as colchicine . polyploid , binucleate and multinucleate cells , owing to their chromosome balance , survive and may undergo mitosis . their rate of mitosis , however , is always less than that of cells with a normal chromosome number . 
koller until recently it was believed that a nucleus with the diploid chromosome number was necessary for normal cell behaviour , and that cells with a deficient complement could function only under very specific conditions , the life of such cells always being short ( darlington , 1942 )  . 
the adenocarcinoma here described presents an example in which cells with less than the normal number of chromoit is not out of place to somes are still able to divide and to continue dividing . draw attention to recent literature on cytoplasmic control in micro - organisms ( sonneborn , 1943 ; spiegelman and kamen , 1946 ) , the relevance of which , for the origin of cancer , has been dealt with by many authors ( graffi , 1939 , 1940a , b ; darlington , 1944 ; haddow , 1944 ; potter , 1945 ; woods and du buy , 1945 , 1946 ) , who suggest that the permanent change which renders a cell malignant takes place in the cytoplasm . cell behaviour in this adenocarcinoma is a further indication that the nucleus can be so subordinated , and the cell remain active in spite of its deficient nucleus . u0 ' c unromosome numoer fig . 
1. given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . most people would agree that studies with transplanted tumours have given information concerning the autonomy of malignant tumours and concerning their histogenesis . however , there is a tendency to regard such studie8 as somewhat old - fashioned and this field of study more or less closed . in this paper it is hoped to show that there is still valuable information to be gathered of significance for general biology as well as oncology . general biology and tumour transplantation . the genetical laws governing the transplantation of normal and malignant tissues are now generally accepted . in both cases a complex of dominant genes is concerned which snell ( 1948 ) has called histocompatibility genes . there have always been two schools of thought conceming the nature of these genes , gne that the genes determine iso - antigenic differences , the other that they deterinine " individual differentials " - substances with somewhat mysterious and ill - defined properties . it is not easy to study these questions with normal tissues for technical reasons , one of which is that the number of factors is always high . 
with tumours the number varies , but may be low , and in addition tumours stimulate a better antibody response . iso - immune reactions are most easily studied in the erythrocytes , and it so happens that certain important iso - antigens in strong 's a strain of mice are shared by the erythrocytes and fixed tissues . 
four tumours from this strain have been studied genetically and serologically . the first two were studied in england ; in both it appeared that two histocompatibility genes were concerned , one of which was identical with a gene for an antigen known as antigen ii . 
by serological means it was shown that the occasionally iso - antibodies tumours contained another antigen in addition to 11 . were formed against it , but too irregularly 4nd at too low a titre to be of value for genetical work ( gorer , 1937 , 1938 , 1942 )  . those studied in america were both found to give single gene ratios in backcrosses . 
furthermore , in conjunction with snell and lyman ( gorer , snell and lyman , 1948 ) it was possible to show close linkage between antigen 11 and the locus for " fused " ( a tail anomaly )  . full information is to be published elsewhere , but it can be seen from table 11 that the linkage is close . 
the sera were produced by inoculation of c57 blacks . the similarity is not shown to the same degree by all sera . provisionally called h2 and h2d ( table 111 )  . closely related antigens occur in strains cba and - cm , so perhaps , there is a long series of alleles at this locus . animals carrying the we are not certain how many cross - overs there were . gene for fused may have norm ' al tails , and there are other difficulties associated with tumour transplantation that will be discussed later on . in backerosses involving 257 animals the recombinations were between 0 and 5 per cent . previous to these studies strong ( 1929 ) had used tumour transplantation to show sex linkage of a histocompatibility gene , and bittner ( 1933 ) found loose linkage with " dilution . " the location of h2 was first found by snell and lyman using tumour . 
inoculation without serological testing , and i hope i am betraying no confidences by saying that they have other instances of linkage as well . will be seen that tumour inoculation is - a most useful weapon in mapping chromoit is best to use serological tests as well . 
without going into details it may be said that they were not serologically unifortumour transplantation indicates that this polymorphism may be maintained by a high pure strains unless kept on a very small scale automatically mutation rate . it was shown by bittner ( 1935 ) that a dba crystallize into various sub - strains . tumour would not take in all sub - strains , and this has been found to be a common finding , having been seen with various tumours arising in strains c57 black ( law , gorer , unpublished ) and cm ( gross , 1947 )  . 
we have as yet no ' evidence that liability to disease is influenced bv antigemc constitution , or indeed very little knowledge of the antigens ' perhaps the polymorphism is connected with the risk of functions in the cell . iso - i ' mmunization during pregnancy ; this must be great where foetal absorption is common . 
if a species were completely uniform the risk would vanish , but it is unlikely that any species would be completely so over the whole of its rangle . on the other hand , if it were sufficiently polymorphic , the danger may be minimized . 
if a male has a rare antigen he would probably be heterozygous , and only half the litter would perish if iso - immunization took place . further , in matings with different males , a female would be unlikely to receive successive doses of lastly , it is possible that if a mixture of a large - number of the same antigen . antigens is received , dangerous titres of antibodies are not formed against any antigen ii does not appear to be very mutable , but here mice one of them . also seem very ' polymorphic . eight pure strains have been examined ; all are somewhat different . 
both the tumours tested in bar harbor gave such a ratio , but it was shown conclusively that they both contained at least two antigens . this is easily explained immunologically . antigens differ greatly in potency . 
gorer it is now well known that tumours may " mutate . " strain , we would get about 50 per cent susceptible progeny ( macdowell and richter , 1932 )  . errors of the above t - vpe are counterbalanced by precisely the opposite effect . some tumours may grow and kill the host in spite of iso - antigenic differences . there is considerable indirect evidence of this , but recently it has been possible to show that an animal succumbing to a tumour derived from any other strain may form high titres of antibodies ( gorer , 1947 )  . 
when first tested a tumour may appear to have upwards of 7 factors ; later on , only one or two . differences of this order cannot be accounted for by errors of the type just mentioned . there this might be due to mutations of domimust be some real antigenic alteration . this is extremely improbable ; one would have to nants to recessive alleles . have 6 or 7 homozygous mutations . 
what may happen is that one or more antigens crowd the others if for the sake of argument we assume that antigen 11 occupies 10 per cent out . of the surface of a normal cell , in a malignant one it may occupy 90 per cent . these experiments have the disadvantage that we have no really satisfactory for example , we can show that the cells of a myeloblastic normal control.. leukaemia contain increased amounts of antigen 11 , but we cannot get enough there is evidence that analogous antigenic normal myeloblasts for comparison . alterations may occur in human tumours . 
it is believed that all mammals ( and perhaps many other forms ) are highly polymorphic for histocompatibility genes . there is evidence that some of these genes have a high mutation rate in mice . 
sylvin. fromn the department of radio - pathology , radiumnhemmet , stockholmi , swveden . received for publicationi february 11 , 1947 . our defective knowledge of the biological factors responsible for infiltrative growth is partly due to lack of sufficient information on the biology of the stroma connective tissue in malignant tumours . thus , growing carcinoma vegetations induce different complicated reactions on the part of the surrounding connective tissue , e.g. 
fibroblast proliferation and various inflammatory reactions . in the course of the stroma investigations by the writer , special attention has been paid to the transformation ( " dedifferentiation " ) of the connective tissue preceding the actual infiltration by cancer cells and to the simultaneous appearance of high molecular ester sulphates of unknown composition ( sylvan , 1938 , 1941 , 1945 and unpublished data . ) interpretation of the events taking place in the stroma is rendered almost impossible by the multiplicity of intermingling reactions . 
on this account it was decided to treat each problem separately . consequently , i thought it advisable to study these changes in cartilage , a tissue characterized by very slow reparative and reactive capacities . thus , the actual question was to investigate the morphology and the histochemical changes of normal cartilage when infiltrated by carcinoma cells . 
 - in this way comnparisons could be made with the stroma reactions of the different tissues , and with the alterations in the chondroitin - sulphate of cartilage and the unknown stroma ' ester sulphate mentioned above . 
by selecting suitable cases it was felt that the source of error caused by the connective tissue stroma reaction accompanying cancer vegetations growing from outside the perichondrium , could be avoided . for the sake of brevity , the readers are referred to current literature regarding the normal morphology of cartilage ( maximow and bloom , 1938 )  . 
the resulting newly formed , growing , connective tissue would then " penetrate " into the ground substance of cartilage , which was " dissolved " and replaced by the tissue mentioned above . after this , the cancer cells were believed to grow into this connective tissue , thereby seemingly infiltrating the cartilaginous tissue proper . ribbert emphatically denied that cancer cells ever could infiltrate cartilage directly , the process instead being mediated by connective tissue . 
as to the operating forces ribbert stated that "  . epithelien ( cancerous ) haben nicht die fdhigkeit anders als durch den wachstumsdruck auf ihn ( cartilage ) einzuwirken . " such a restricted mechanical interpretation involving pressure for the explanation of infiltrative growth , is nowadays of little value ( sylv6n , 1945 )  . oestreich ( 1910 ) suggested that the chemical composition of cartilage , mainly the chondroitin - sulphate , would be injurious to the cancer cells and consequently infiltrative growth would become impeded by this substance . since ribbert 's review in 1911 no special articles have been published regarding these questions . 
the statements of ribbert demand a survey of the cooperating factors active in the destruction of cartilage by carcinomatous tumours . the following possible factors have to be considered : 1 . 
the cancer cells might by direct action be destructive . in order to obtain a satisfactory understanding of the morphological factors unfortunately , they should be interpreted in terms of biochemical principles . for lack of complete data the chemical principles can only be touched upon . cartilage has a strongly acid intercellular matrix , due to the presence of large amounts of chondroitin - sulphuric acid . this substance is very sensitive to even slight changes in ph towards the alkaline side ( blix and snellman , 1945 )  . such ph variations cause a rapid depolymerization and disintegration of the long chain molecule . 
a shift in ph resulting in a disappearance of chondroitin - sulphate has recently been suggested to occur in inflammatory lesions of cartilage ( sylven , in press )  . 
when considering the complexity of the problem , i have to admit that no final conclusions regarding the biochemical nature of the operating factors can be drawn from the material presented here . 
some results , however , seem to be conclusive . some information concerning the mutual relationship between cancer cells and the perichondral connective tissue in the absence of any intervening inflammatory stroma reaction was obtained from case 2 , ii . 
the carcinoma strands in question were growing in the perichondrium , which was transformed into a loose metachromatic stroma connective tissue similar to the one met with in most carcinomas ( sylven , 1938 , 1945 and unpublished data . ) in the superficial part of the underlying cartilage a moderate decrease in the content of chondroitin - sulphate was demonstrated , the cartilage in other respects apparently being normal . 
sylvien actual cartilage destruction was seen to be effected either by the sole inflammatory stroma reaction ( case 1 ) or by the combined stroma and cancer strands ( case 2 )  . in the cases under consideration , that part of cartilage just being infiltrated was always devoid of the typical ester sulphate . remains of this substance might be found in the shape of thin pericellular halos . 
1. - the early changes in ear cartilage , previous to the actual infiltration , are demonstrated . at top of picture the perichondral connective tissue is swollen and slightly invaded by lympho - plasmocytes . the superficial part of cartilage appears unstained , indicating that metachromatic material is lacking . 
4. - the disappearance of chondroitin - sulphate precedes the growing tumour . in this picture the stroma reaction has reached the left top corner ; the lightly stained ametachromatic cartilage area contains very few inflammatory cells . 
when investigating the process more carefully , however , it was established that cancer cells really have the capacity of digesting cartilage by their own activities without any co - operating stroma reaction . further , the results indicate , that the first demonstrable alteration of cartilage just being infiltrated is an early disappearance of most of the preexisting ester sulphate . 
the smith type is still , however , a relative rarity and less than a score of cases have been reported since the original description in 1934 . the forowing case , the second to be seen in south africa , closely resembles that originally described by smith ( i934 ) ; but it is unique in that the . 
he had suffered since 1942 from recurrent spontaneously heahng tumours of the left face , ear , neck , chest , shoulder and upper arno other member of his family was known to have suffered from any similar disease ; and he was not related to the other south african who suffers from the smith type of morusca pseudocar ' c ' momatosa . skin generally was normal for his age and was not hypersensitive to sunhght . his occupation as a contractor kept him mainly in the open . 
he had never been exposed to tars , mineral oils or arsenic . he stated that the tumours , of wbich he usually had several active , began like festering blackheads " , grew for 4 - 6 weeks , and then began to regress and disappeaxed , leaving scars , in 4 - - 6 months . 
on his first visit he had two active tumours on the back of the left ear and one over the left scapula and thirty depressed wbite scars on the left side of the nose , upper hp , cheek , ear , neck , chest and upper arthe scalp and buccal mucosa were unaffected . 
a month later the ear lesions were distinctly smaher and flatter and that over the scapula had flattened dow - n to a coin - like plaque with rolled edges and a central area of soft keratinous material . all the lesions were chnically indistinguishable from squamous epitheliomata . 
at this visit a fresh lesion , i mm . , was discovered iin the angle of the left ear lobe and cheek it closely resembled , as the patient had described , a festering blackhead . the patient , who b . - ilieved himself to be suffering from skin cancers , has on several occasions been subjected to biopsy or excision of tumours ; and a diagnosis of squamous epithlioma had generally been made . x - ray therapy had also been given niore than once with little impression on the rate of healing of the tumours ; but the scars left in such areas were more mutilating than those seen after spontaneous healing . a portion of one of the lesions on the ear was excised for histological examination and we examined several sections of lesions excised in the past by d . 
the stretched and atrophic epidermis covering the lesion is between the central mass of keratin and the covering epithelium are seen on the left . irregular and well - differentiated masses of epithelium and cell nests . 
1. received for publication may 10 , 1951 . there is still some disagreement on the question whether the early changes produced in epidermis by chemical carcinogens are specific , i.e. , whether they can be distinguished from the effects of non - carcinogenic irritants . the chemical evidence is inconclusive . 
cowdry and his school have been able to show that the chemical constituents of mouse epidermis made hyperplastic by several applications of 20 methylcholanthrene differ quantitatively from those of normal epidermis ( carruthers , 1950 ) , but it does not appear to have been proved that these changes are produced only by carcinogenic substances . histological evidence is more definite . 
the only significant way in which such skin has hitherto been found to differ from untreated skin is in its ability to produce tumours when treated with a co - carcinogen such as croton oil . 
the " latent tumour cells , " if present , must be so few that they are very unlikely to be detected in sections , or perhaps they may not be recognizable if seen . 
the skin has passed through a state known to be produced only by carcinogenic agents ; this state has apparently passed away , yet the skin will then react to treatment with a non - carcinogenic substance in a manner quite different from normal skin . the early reaction of normal mouse skin to croton oil is similar in general type to its reaction to other non - carcinogenic irritants , and differs in important respects from the reaction to carcinogenic substances . 
the chief criteria on which this distinction is based have been fully described by pullinger ( 1940 ) and by gluiicksmann ( 1945 ) , and only a summary will be given here . 
gwynn the epidermis is stratified . in the normal skin of the body of the mouse there are no strata , and the various types of cell are arranged apparently at random in a layer one cell , or in places two cells , thick . of the nucleated cells in the epidermis of the back of a normal adult mouse , about 14 per cent are resting , 80 per cent differentiating , and the rest are mitotic or degenerating . this classification was used because it gives definite information about the state of the epidermis which is not obtainable by any other method known to this information is not dependent on any hypothesis about the function or potentialities of the different classes of cells . 
for the purpose of this work they are simply types of cells which can be distinguished , the absolute and relative numbers of which vary in different states of the skin . one of the most striking features of the hyperplasia produced by chemical carcinogens in mouse epidermis is the rise in absolute and relative numbers of the resting cells . non - carcinogenic irritants , on the other hand , produce a hyperplasia in which the absolute number of all epidermal cells is increased but their relative numbers remain approximately the same as in normal skin ( gliicksmann , 1945 )  . 
the percentage of resting cells in the epidermis was chosen as a criterion for comparison of the hyperplasia produced by the different treatments used in the following experiments . it provides a measure , which can be handled statistically , of the specificity of the reaction of the epidermis , i.e. , of its resemblance to the characteristic reaction to carcinogenic substances . in the work now reported the effect of croton oil on normal mouse skin has been compared histologically with its effect on skin after the reaction to previous treatment with a chemical carcinogen has apparently disappeared . three strains of mice and two different carcinogens were used . 
 " t " and " s " mice were purchased from dealers . " p " mice were of an institute - bred strain originally obtained from the national institute for medical research ; they had been used before for studies on skin carcinogenesis by gluiicksmann ( 1945 ) and salaman ( 1943 )  . 
the standard errors of means were of the same order in this experiment as in the other two . the mice were examined weekly for the appearance of tumours , for periods of 200 to 250 days . 
gwynn high level and remained well above normal . in group 2 there was a parallel but smaller increase . in group 3 application of croton oil without previous treatment was followed by an initial slight rise in resting cell percentage , which then these results are listed in table i and charted in fig . 
the first tumour appeared on the further trials have 63rd day in group 1 , but not till the 170th day in group 2 . confirmed the superiority of acetone over paraffin as a solvent for the carcinogen comparisons of various concentrations of croton used as the primary treatment . oil in acetone and paraffin have shown that a 0.5 per cent solution in the former is almost as powerful a co - carcinogen as a 2.5 per cent solution in the latter . 
13. - experiment 2 : 0 group 2 : 10 mice at beginning of experiment , 8 survivors at 217th day . e group 4 : 12 mice at beginning of experiment , 11 survivors at 217th day . weekly croton oil applications without previous treatment ( group 3 ) caused only a transient rise to 21 per cent on the 3rd day , and thereafter the level declined gradually to 16 per cent on the 115th day . 
13. since no tumour it will be seen that tumour appeared in the other groups , they are omitted . production in group 4 began earlier than in group 2 , but later proceeded more this suggests a possible parallelism between the early rise of resting rapidly . cell percentage and the tumour production which follows about 50 days later . ( 3 ) in a third experiment , four groups , each of 8 male and 8 female mice of group 1 , in this experiment , the " s " strain , were treated as shown in table iii . has the same function as in the last : a test of the carcinogenicity of the primary treatment with the carcinogen . 
14. as in the last two experiments , the difference between the effects of croton oil treatment with and without a previous carcinogenic stimulus is clear ( groups 2 and 3 )  . in group 4 , resting cell percentages were at first lower than , but later rose above , those of group 2 . 
15 shows the rate of tumour production in these groups . dimethyl - 1 : 2 - benzanthracene in paraffin oil appears to have been less effective than 1 per cent benzpyrene in acetone ( group 4 of experiment 2 , but note that different strains of mice were used )  . tumours appeared earlier in group 2 than in group 4 ; thus , as in the former experiment , there appears to be a parallelism between resting cell percentage and subsequent tumour production . 
gwynn except one in which early malignant change was probably present . during a considerable experience of the application to mouse skin of croton oil alone , a very few small tumours have been seen , but never as many as in this experiment . precautions against contamination of cages , etc . , with carcinogenic substances are taken , but the possibility that such a technical mistake occurred cannot , of course , be entirely excluded . it is perhaps significant that the resting cell percentage in this group , after falling to 16.6 per cent on the 132nd day , rose again to 20 per cent on the 216th day . tumours produced by the application of substances generally regarded as non - carcinogenic have been occasionally reported . 
9 and 10 are high - power views of regions of the epidermis consisting predominantly of resting cells and differentiating cells , respectively , taken from the same specimen as fig . 
many substances are as powerful hyperplastic agents as croton oil , without being co - carcinogenic . croton oil is much the most powerful co - carcinogen for the mouse 's skin yet discovered . its action appears to be highly specific with respect to animal species , and perhaps also with respect to the carcinogen with which it acts . it may be that it combines very weak carcinogenic with powerful co - carcinogenic power . 
a number of tumours subsequently appear . this number varies widely according to strain of mouse and strength of initial carcinogenic stimulus ; there may be an average of only 2 or 3 , or as many as 20 , per mouse . 
but even if the higher figure is taken , and it is assumed that each tumour was represented before croton oil treatment began by a single " latent tumour cell , " or a small group of such cells , these cannot be responsible for the generalized rise in restingcell percentage which is observed throughout the epidermis . in fact the chance of finding such a cell or group in a section is small . 
it appears that mouse epidermis which has been treated with a carcinogen , though it returns to a state closely resembling the normal , has suffered a general change which does not consist merely in the presence of a few latent tumour cells . we are indebted to professor s . 
woodyatt. * from the meyerstein institute of radiotherapy and the bland sutton institute of pathology , middlesex hospital , london , w.1. received for publication february 15 , 1950 . failure in the treatment of malignant disease is most often due to the occurthere rence of metastasis before treatment of the primary lesion is instituted . are , however , a number of cases in which the tumour remains localized for a considerable time , but cannot be removed surgically on account of its size and anatomical situation , and cannot be destroyed by radiotherapy without irreit is the aim of the radioparable damage to neighbouring normal tissues . therapist to exploit the differences in sensitivity between normal and neoplastic tissues , and it is the object of this research to see whether the effects of radiotherapy on certain tumours can be enhanced by modification of the techniques of therapy in common use at present . a course of x - ray treatment involves a number of variable factors . 
the segments , usually 2 or 4 in number , and as nearly as possible of equal size , were then treated by one or more experimental techniques , while one segment was treated by a control technique . segments not being treated were protected by lead shields while neighbouring segments were irradiated , and care was taken to avoid overlap of adjacent segments or an untreated gap * b.m.a. 
woodyatt between them by careful skin markings and " setting up . " the question of side - scattering of x - rays from one segment into adjacent ones was investigated by physical measurements with micro - ionization chambers in a wax " phantom . " the amount of scattered radiation was found to be less than 5 per cent at 1 cm . from the edge of a treated segment , and this was considered small enough to be ignored . in this way erroneous conclusions resulting from unpredictable individual variations in response are largely eliminated , and each case constitutes a complete separate experiment . 
the results of individual experiments can later be compared to see whether the experimental technique in question regularly produces a particular effect . the tumours and surrounding skin were examined at frequent intervals to follow progress , and to detect and note differences in response between the areas . in addition , biopsies were taken from all areas before , during and after treatment . at first , weekly biopsies were taken , but it soon became obvious that in the earlier stages more frequent examinations were necessary , while after completion of the course of treatment intervals of a month or more might elapse . 
the sections were examined by a pathologist who not only had no preconceived views about the various techniques of irradiation employed , but was kept in ignorance of the area from which each specimen had been taken . biopsies from each area before treatment were studied carefully to provide a base line , and also to give an idea of the degree of variation between different this variability was often found to be marked , and areas of the same tumour . differences in biopsies subsequently taken from the several segments had often to be discounted as representing no more than possible chance variations . 
fractionation is , however , usually by equal daily doses given on 5 or 6 days each week , and this fractionation technique is applied with little discrimination to widely different sorts of tumour . certain theoretical considerations , mentioned below , suggested that a different method of fractionation , leaving longer intervals between the fractions , might be more effective . 
an important direct effect of a dose of irradiation is temporary suppression of mitotic activity . koller ( 1948 ) found that in squamousand basal - cell carcinomas of the skin the number of dividing cells was considerably reduced 6 hours after 200r , and that after doses of 400r and 500r mitotic suppression might last for several days . 
the susceptibility of cells to irradiation damage varies at different periods of their life cycle , being greatest in early prophase ( lea , 1947 ) , and irradiation given spear and during the period of mitotic suppression is relatively ineffective . glucksmann ( 1938 , 1939 ) have demonstrated by experiments on irradiation of tissue cultures and animal tissues in vivo that a greatly enhanced lethal effect is obtained if fractions are spaced so as to coincide with the recovery of mitotic activity following the suppressive effect of the previous dose . information concerning the duration of mitotic suppression in human tumours it probably varies in different tumours , but will after different doses is scanty . depend largely on the size of the dose . 
a clinical investigation indicating advantages from a wider spacing of fractions has been reported by koller and smithers ( 1946 )  . koller ( 1948 ) considers that the nature of the tumour bed and the indirect effects of irradiation on the stromal reaction in the surrounding normal tissue exercises an important influence on the response of the tumour itself . 
a further limitation was , of course , imposed by consideration for the patient 's welfare , and no case was undertaken where it was felt that there would be less chance of effecting a cure by the method . tumours of three types were treated - large rodent ulcers ( 2 cases ) , carcinomas of the skin ( 5 cases ) , and carcinomas of the breast involving the skin ( 4 cases )  . most tumours of the first two types are radio - sensitive , and a successful result could normally be expected from the use of an orthodox technique . these tumours were treated in order to assess the relative efficiency of the experimental techniques , and to determine any differences in the reaction of normal tissue at the edge of the tumour . the cases of carcinoma of the breast were of special interest , for , as is well known , a considerable proportion are markedly radio - resistant . 
the usual erythematous reaction developed , and proceeded to moist desquamation over the tumour and surrounding skthe area of skin at the periphery which was only covered by the original 71 x 15 cfields showed pigmentation , but no desquamation . 
the dose to this skin area had only been about 2000r when the smaller fields were substituted . no difference in reaction or tumour response in the two areas was apparent at this stage . on october 6 , as viable tumour was still feared to be present , further treatment was given as described . three weeks after the conclusion of treatment a marked difference was apparent between the two areas . 
the area treated by the control technique ( area 1 ) was completely healed over by healthy new epithelium except for a small ulcer at the site of the first surgical attack . 
the remaining tumour consisted of some firm pink papillary processes , almost flush with the surface . in area 2 much of the tumour and surrounding skin covered by the treatment applicator was still raw and denuded of epitheliuthe remaining tumour was a little flatter than in the control area . eight weeks after treatment further flattening was apparent . area 1 was healed except for the remains of the ulcer . area 2 showed very little progress of healing , although a few islands of epithelium had appeared in the raw skin area ; there was some discharge from a fissure between two sessile processes . four months after treatment a striking difference between the two areas was apparent . 
the control area was healed and showed marked pigmentation , all that remained of the tumour being some fibrotic nodularity beneath the surface . area 2 showed up as a sharply outlined , completely unpigmented square , corresponding to the treatment applicator . there was still an indurated fissure at the site of the tumour , and an area of skin below the tumour remained unhealed and had clearly undergone necrosis . five and a half months after treatment the patient complained of burning pain in the lower part ( area 2 ) , and an increase in discharge from the fissure which was still present . 
a month later ( april , 1948 ) radionecrosis was obviously developing in area 2 ; the fissure had deepened , its edges were grey and necrotic , and the surrounding tissues were pale and avascular , and as hard as cartilage . by may a large necrotic ulcer corresponding to area 2 had developed . local treatment failed to bring about healing , so in november , 1948 , the whole treated area was excised , after a preliminary colostomy . this was done partly because residual foci of active growth were suspected . 
woodyatt there was a moderate degree of associated inflammatory mitotic activity . biopsies taken from both areas during and after treatment showed reaction . progressive differentiation of the tumour cells with reduction in the percentage of viable cells and their final disappearance . 
no sign of tumour could be seen in the biopsies taken at 6months , and no significant differences between the series of sections from areas 1 and 2 were detected . 
a small nodule of recurrent growth ( 1.25 cdiameter ) had appeared , adjacent to the mastectomy scar , in february , 1945 . superficial x - ray treatment was given ( 4 daily doses of 800r ) and the nodule regressed , being quite healed by september , 1945 . 
3. - carcinoma of breast ( case 3 ) divided into four areas . 11111 11111 11111 total 3000r . in 27days area area area area total 4000 r in 26 days total 3000r in 27 days total 3680r in 24 days 11111 11111 11111 days i = biopsies taken from all areas , and on day 50 fig . 
the control technique was modified in this case , 150r being given daily instead of the usual 200r . this was done to bring it more into line with the average daily tumour dose administered in this clinic for carcinoma of the breast . 
by the end of treatment , february 18th , the greater part of the surface was covered by smooth granulation tissue . there was still a considerable amount of tumour present , with a subcutaneous shelf of induration palpable all round the edge . there was severe erythema of the surrounding skin , with much pain and discharge , and for these reasons treatment was curtailed . it is probable that the unusual severity of the reaction was due to infection of the raw surface , as the patient had fever up to 1010 f . 
the tumour had shrunk to 10 - 5 x 6 - 5 cm . the ulcer floor was covered by necrotic slough and its edges were still firm , but were softer and less raised in areas 1 and 3 . seven weeks after treatment the skin had begun to heal in area 3 . 
the skin in area 1 showed well - marked pigmentation , but was perfectly healed . well - marked differences were now apparent in the different areas of the tumour , which had shrunk further to 9 x 6 cboth areas treated by the control technique showed clinical evidence of active residual tumour , the ulcer in these areas having a hard raised edge with much adjacent subcutaneous induration . area 1 showed some suspicious induration , but less than in the areas treated by the control technique . area 3 was soft and nearly flat , with no definite sign of residual growth . 
6 at the end of treatment . the tumour cell mass in the upper part of the field shows extensive hyalinization , and there is heavy lymphocytic infiltration of the stroma . note that the magnification is the same as in fig . 
8 , 9 and 10 . - squamous - cell carcinoma of the ear ( case 8 )  . biopsies taken before , at about the middle of , and at the end of the course of treatment . 
the upper half was treated by technique i ( 500r twice weekly ) to a total of 3500r in 24 days , and the lower half by the control technique ( 200r daily ) to a total of 3600r in 24 days ; 95 k.v. 
x - rays clinically there was rapid regression of the lesion in both areas , were used . and the tumour had disintegrated completely before the course was finished . the serial biopsies showed a very satisfactory response in both areas , no viable cells remaining within two weeks of starting treatment . 
any conclusions we may draw from this small series of cases must be purely tentative ; the final evaluation of these techniques may require the elapse of a considerable interval of time , and in view of the possibility of variation in different parts of the same growth many cases must be studied before definite conclusions can be reached . complete equality of total dose and protraction were seldom obtained , and this introduces another variation from strict experimental conditions . 
with these facts in mind the following conclusions are cautiously presented . technique i ( 500r twice a week ) was used in 9 cases ; 3 squamous - cell carcinomas , 4 carcinomas of the breast , and 2 rodent ulcers . 
the clinical result in the 3 cases of epithelioma was uniformly good , and in one case this technique gave slightly quicker results than the control . in 2 cases of carcinoma of the breast this technique gave a better response , judged clinically and histologically , than the control ; in the other 2 cases the results were equally good . 
the cases of basal cell carcinoma responded better both clinically and histologically to the control technique ( daily fractions of 200r )  . technique ii ( looor once a week ) was tried in 4 cases ; 2 squamous - cell carcinomas of the skin , one carcinoma of the breast , and one rodent ulcer . 
w.7. received for publication march 14 , 1949 . previous observations ( foulds , 1947 , 1949b ) showed that the growth of some transplanted mammary tumours in hybrid mice depended on a hormonal stimulus which operated in normal female mice but not in normal males ; artificially - administered oestrogen supplied the necessary stimulus for growth in males . 
some of the tumours were independent of the hormonal stimulus at the first transplantation ; others lost their dependence after several transfers . dependence on hormones seemed to characterize one stage in the life - history of mammary tumours in hybrid mice . 
the majority of the mice were br hybrids , mostly of the f3 and f4 generations , and made up of 71 mice of strain br4 ( pink label ) , 90 of strain br4 ( blue label ) , and 69 of strain br6 . 
a preceding paper ( foulds , 1949a ) describes the source of these hybrids and the incidence of tumours in them . the mice were subjected to forced breeding and most of them had pregnancies in rapid succession throughout the period of observation , even when bearing multiple large tumours . 
tumours were recognized usually when about 0 5 cin diameter , and weekly thereafter two main diameters were measured with calipers . growth charts were made by plotting the sum of the two diameters against post - mortem measurements usually agreed time . satisfactorily with those made during life ; measurements on regressing tumours were the most difficult and subject to the largest errors . frequent , often daily , measurements were made on selected mice . litters were recorded each morning except on sundays and public holidays . the time of parturition as shown on the charts was usually later than the actual time by unknown periods of up to 24 hours on weekdays and 48 hours at weekends . 
the mice were examined post - mortem except for a few that were eaten ; the tumours were measured and pieces of them fixed for histological examination . in the charts the first tumour , or largest of contemporaneous tumours , is represented by a continuous line joining crosses which indicate the actual measurements ; the second tumour is represented by a broken line and circles , and the third by a dotted line and triangles . 
178. 8 9 10 weeks ( i ) left axilla ; ( ii ) right axilla . in most of the mice from 1 to 6 new tumours appeared during the course of the first . 
1 shows three different types of behaviour at the same time in one mouse . at least three major factors contributed to the wide diversity of behaviour amongst spontaneous tumours , and even amongst multiple tumours in the same mouse : first , the regulation of tumour growth by the environmental , and presumably hormonal , changes occurring during pregnancy and the puerperium ; second , the specific reactivities of particular tumours to the environmental 348 l . 
foulds changes ; and third the qualitative changes occurring in tumours during their manifest clinical course as evidenced in particular by altered responsiveness to in the remainder of this paper the terms " responsive " and reproduction . " unresponsive " are used in a special and restricted sense to describe tumours which , respectively , do and do not vary their growth in response to the physiological changes occurring in their ho * ts during pregnancy and the puerperium . the term " progression " is used , also in a special sense , to denote qualitative change in tumours as distinguished from mere advancement in size , and as evidenced in particular , but not exclusively , by altered responsiveness to pregnancy and parturition . subsequent paragraphs detail the analysis of the phenomena here outlined . for convenience of description the tumours are classified according to a few main types of behaviour , but transitional and indeterminate types are encountered and preclude a precise statement of the relative frequency of types . 
the peak size , however , was often considerable , as shown in some of the other charts representing type i tumours ( fig . some tumours maintained their 20 )  . course unchanged through several months of observation , whereas others after two or three cycles changed their behaviour , as described in a later section . the growth of responsive type i tumours was strictly conditional and dependent on pregnancy . if breeding stopped the tumours disappeared and , as a rule , did not recur until breeding was resumed . 
51. ( i ) right vulva ( ? recurrence of tumour excised 7 weeks previously ; earlier tumour not shown ) ; ( ii ) right axilla . with a small range of growth and regression at each pregnancy , and a slow but steady increase in average size . another tumour shown in the same diagram ( dotted line ) appeared 8 weeks after the first and then followed an almost parallel course . 
the peaks of growth , or the averages of the maximum and minimum sizes during successive pregnancies , fell close to a straight line . thus , the course of a tumour was represented by a curve which could be resolved into a straight line with superimposed waves ; the straight line indicated the rate of net growth or , for want of a better term , the intrinsic growth rate , and the waves indicated the response to pregnancy and parturition . 
the intrinsic growth rate and the degree of response , however , varied within wide limits and independently similar intrinsic growth rates were of each other from one mouse to another . responsive and linked with different degrees of responsiveness and vice versa . unresponsive tumours in the same mouse sometimes , as an exception to the general rule , had similar intrinsic growth rates as illustrated in fig . 
their differentiation from type i tumours was questionable , but they seemed to correspond with relatively frequent tumours in , nonbreeding mice whose course was represented by a horizontal straight line , and possibly differed essentially from type i tumours in the ability to persist without the stimulus of pregnancy . 
the great diversity of behaviour was attributable to two circumstances : first , the intrinsic growth rates and the responsiveness varied within wide limits and independently of each other as shown in the present section and , second , progression from one type to another occurred variously during the clinically manifest course of the tumour . 
the tumour itself had changed . the recognition of a similar change in responsive tumours was necessarily more difficult because the pregnancy waves obscured the intrinsic growth rate . probably an acceleration of growth often accompanied the loss of responsiveness 4m.3~ 0k 11 i / i i..#4 i 11 9 10 11 12 - 13 14 15 i\it - l 18 19 20 21 22 16 17 weeks fig . 
19 , however , the one that regressed completely between pregnancies ( broken line ) increased its peak size somewhat more steeply than the tumour that regressed incompletely ( continuous line )  . increasing peak size , therefore , was not attributable , in general , to summation . the regression of some tumours was slight and of short duration , being followed almost at once by progressive unresponsive growth . 
the regression was sometimes trivial and probably within the limits of error in measurements , and the tumours were then scarcely distinguishable from those already described which continued as unresponsive tumours from the peak of a pregnancy wave . possibly the two tumours illustrated in fig . 
29 proved unresponsive at a subsequent pregnancy . another tumour ( broken line ) shown in the same figure behaved similarly , except that it apparently began to grow earlier and was less certainly unresponsive at the subsequent pregnancy . some tumours regressed slightly or moderately after parturition and then maintained a constant size . 
the terminal course was represented by a straight line , but the intermediate course through the first and second pregnancies after the resumption of breeding was more fairly indicated by a curved line , suggesting a gradual accession of growth energy in an unresponsive ( or minimally responsive ) tumour . 5 0o d - t . 
more commonly the first tumour was solitary and further tumours developed consecutively . so far as could be seen the spacing of successive tumours and the sequence of types were irregular . 
tumours that remained solitary throughout the period of observation were sometimes unresponsive , but more frequently responsive . in the majority of the mice where two tumours appeared at about the same time both tumours were responsive ; in the minority both were unresponsive , or one was responsive and the other unresponsive . 
as a rule new tumours were registered first during pregnancy . different types of tumours developed independently of each other in the same mouse , and progression from one type to another occurred independently in the several tumours . 
13 , regressed completely within 7 days , but when another pregnancy followed without delay regression was only partial . possibly hormonal changes during the first 2 or 3 days of pregnancy sufficed to check regression , although growth was not apparent until a few days later . perhaps a few of the " recurrent " tumours were , in reality , new primary growths , but so nearly as could be determined the recurrent tumours occupied the same positions as those which had disappeared and were of similar shape . after partial regression the reality of true recurrent growth was not in doubt . some tumours were reduced to thin plaques not greatly different in superficial dimensions from the tumours at their peaks of growth ; recurrence was manifested by a thickening of the whole plaque . other tumours shrank to vague subcutaneous thickenings of doubtful , but probably considerable , extent ; recurrent growth was evidenced by sharpening of the outlines . it seemed that tumours recurred by the simultaneous grow ' th of all parts of a substantial area of previously altered mammary tissue rather than by centrifugal growth from a in consequence , recurrent tumours were often relatively small residual focus . large when first registered and the gradients of growth and regression remarkably steep . a few tumour - bearing mice nursed and weaned their litters . their tumours behaved during lactation like those in recently pregnant mice that did not at once become pregnant again although deprived of their young . 
some dubious tumours , registered once or twice on suspicion , were not seen again , but only 5 tumours , recorded on at least one occasion without query , disappeared permanently from mice that continued to breed . after interruption of breeding the tumours that had regressed recurred , with few fig . 
the recurrences corresponded as a rule with expected pregnancies in continuously breeding mice , and the recurrent tumours , unlike those in segregated females , were responsive ; almost certainly the recurrences were stimulated by unrecorded pregnancies . 
76. male removed at a ; injections three experiments were carried out with oestrogens . in the first , each of 8 mice received 4 daily applications to the skin of 0 - 2 c.c. 
36 shows regression checked and succeeded by growth at the time of the injections , but as similar events occurred without injections during intermissions of breeding the interpretation was doubtful . in other mice an action on tumours was trivial or lacking . in the third experiment cholesterol pellets containing 10 per cent of diethylstilboestrol and weighing on the average 8 - 2 mg . 
were implanted subcutaneously in 7 mice . nine tumours previously observed in these mice had disappeared before the pellets were implanted and none recurred promptly , but eventually each mouse developed a solitary tumour . in 3 mice the late tumours recorded 366 l . 
foulds 54 days , 145 days and 145 days respectively after implantation of the pellets were in the same region as earlier tumours which had regressed , and were possibly , but , in view of the long delays , not probably , recurrent tumours . in the other 4 mice the late tumours were remote from the sites of the earlier tumours and were undoubtedly new primary growths . 
the present section records the transplantation of tumours whose previous course was known and describes experiments with tumours removed surgically with three main objectives , namely to study autotransplantation , to compare successive primary tumours in the same host , and to compare primary tumours with the recurrent tumours which followed the operation . the experiments were few in number , the general plan of this investigation being to study the natural course of tumours with a minimum of experimental interference . tumours of similar size from the same mouse were used to compare the transplantabilities of responsive and unresponsive tumours . 
tumour in the first passage tumour b grew a grew in female hosts but not in males . equally in male and female hosts , although in the second passage the implants grew tardily in males . 
from these experiments it appeared that , in general , unresponsive tumours were transplantable into male and female hosts and responsive tumours only into female hosts , but that the correlation between type of growth in the primary tumour and behaviour after transplantation was not perfect . five tumours were excised and used for autotransplantation , implants being inoculated subcutaneously near the mid - line of the abdomen in order to minimize confusion with new primary tumours . three of the transplantations were unsuccessful . 
foulds each , so far as could be judged from the short period of observation , was " responsive . " two autotransplantations were successful . growth of the implant from a primary tumour measuring about 0 - 2 cin diameter and removed 6 days after detection was evident after 26 days , and continued steadily until the mouse was killed 22 days later . 
haddow , using in the main dealers ' mice of unknown ancestry , recorded frequent measurements during and after pregnancy , and concluded that " no evidence was found to suggest that gestation itself has any influence on the rate of growth of mammary cancer in the mouse , but in approximately half of the available cases it was observed that parturition was followed by a temporary decline in tumour growth rate . " haddow 's two figures illustrate , respectively , a substantial though incomplete regression after parturition and a halting of growth without regression ; they match some curves of mine . 
gardner ( 1941 ) described 5 hybrid ( 057 black x cba ) f1 mice which , repeatedly , had tumours attaining 1 - 2 cin 2 to 5 pregnancies ; the tumours regressed after parturition , but 3 of the mice eventually died with mammary adenocarcinomas . the responses of the tumours in the hybrid mice used in the present investigation are , possibly , exceptional in frequency and degree , but are not essentially different from those described by haddow and by gardner . notable response to pregnancy and parturition is here recorded in hybrids of varied genetic constitution derived from the inbred strains c57 black , riii and a ; it is not correlated with anomalous transmission of the milk - borne mammary tumour agent . the pregnancy effect usually becomes apparent during the second half of gestation , but probably begins during the first week and continues until the ' day before parturition . 
the abrupt regressions about the time of parturition suggest the sudden withdrawal of a stimulus which , though not yet identified , is presumably hormonal , affecting widely - distributed multiple tumours . 
the preliminary experiments with hormones are indecisive ; a simple oestrogenic action seems unlikely , and the possible effect of gonadotropic hormones , placental hormones , and the co - operative action of oestrogenic and luteal hormones require further investigation . lactation has no specific effect , and modifies the course of the tumours only in so far as it delays the next pregnancy . the pregnancy effect on mammary tumours is comparable with the " promoting " action of various procedures on chemically - induced tumours of the skin of rabbits and mice . 
rous and his colleagues ( rous and kidd , 1941 ; mackenzie and rous , 1941 ; friedewald and rous , 1944a , b ) studied promoting factors in rabbits , and berenblum , who has recently adopted rous 's nomenclature , in mice ( berenblum , 1944 ; berenblum and shubik , 1947 )  . 
foulds his colleagues describe the " promoting " action of trauma and irritants in eliciting and maintaining tumour - growth from latent tumour cells present in the skin as a result of the " initiating " action of a carcinogen . 
some " conditional " tumours grow only whilst the promoting factor operates ; they disappear when it is withdrawn , but recur from the irreversibly altered epithelum when the promoting factor is restored . 
rous and kidd discuss whether or not their argument the conditional growths of rabbits ' skin are " true tumours . " in essential applies equally to the conditional mammary tumours of mice . agreement with rous and kidd , i consider that their exclusion from the group of tumours by an arbitrary definition is unwarranted , and i describe as " tumours " all the mammary growths except a few palpable nodules which regress and do not recur , and those nodules , probably similar , that do not survive autotransplantation . they are accountable to a reversible change in mammary tissue , and perhaps to the exceptional persistence in gross nodules of the properties of the hyperplastic nodules or " adenomas " which are widely accepted as precursors of mammary tumours in mice although many of them are abortive ( gardner , 1941 )  . browning ( 1948 ) reported that nodules less than 02 cin diameter in c3h mice were not transplantable , and that about a third of them regressed , whereas nodules larger than 02 cin diameter never regressed . my observations differ from browning 's in revealing no correlation between behaviour and size ; autotransplantation of tumours more than 05 cin diameter was unsuccessful . the remaining mammary tumours develop from an irreversibly altered mammary tissue . 
the great diversity of behaviour is attributable to the interplay of factors which vary within wide limits and independently of each other . classification into types , though convenient for description , is arbitrary , and hampered by gradations from one type to another . 
the primary distinction , although not absolute , is between the " responsive " tumours whose course is notably influenced by pregnancy and parturition and the " unresponsive " tumours whose growth is unaffected by reproduction . the tumours assigned to responsive type i correspond with the " conditional " neoplasms of rabbits ' skthey grow during pregnancy , regress promptly after parturition , and recur , with rare exceptions , in the same place at each successive .pregnancy ; after several cycles of growth and regression they achieve no net increase in size or aggressiveness . 
the responsive type ii tumours are less strictly " conditional " ; they wax and wane at each gestation , but with a net increase of size at each cycle . 
the rate of net increase is steady and characteristic of the individual tumour , depending on a specific property described as the " intrinsic growth rate . " the term is provisional , to be discarded when elucidation of the property suggests a better name , and is used to denote the steady growth , represented by a straight line , upon which the waves of response to pregnancy are superimposed . 
the intrinsic growth rate , measured by the slope of the straight line and the responsiveness as indicated by the amplitude of the pregnancy waves , are constant , often for long periods , in a particular tumour , but vary within wide limits and independently of each other from one tumour to another . 
foulds of tumours , namely the capacity for progression , as above defined , and that general principles or rules of progression , deduced from the study of mammary tumours of mice , are widely applicable . the material for this analysis of progression consists of spontaneous tumours following their natural courses without experimental interference beyond temit is especially favourable on account of the porary interruption of breeding . abundance of multiple tumours , for the comparison of multiple tumours establishes beyond doubt that progression is a change in the individual tumour ; alteration in only one of several contemporaneous growths is not attributable to change in a hypothetical " general resistance . " the varied tumours encountered in this investigation may be arranged in a series having at one extreme the wholly conditional responsive tumours , at the other extreme the rapid - growing unresponsive tumours , and intermediately all indidegrees and combinations of responsiveness and intrinsic growth rate . vidual tumours , however , do not in general traverse this series ; they advance by abrupt steps , vaulting many possible transitional stages . 
at its first clinical manifestation a tumour may occupy any position in the series of types , irrespective of the time of its occurence or its position in a sequence of multiple tumours . 
tumours which develop simultaneously or consecutively in the same mouse conform sometimes to the same type and sometimes to as many types progression occurs independently and unpredictably as there are tumours . in multiple tumours in the same animal irrespective of their size or clinical progression may occur earliest in the first , second , or any subsequent duration . member of a sequence of multiple tumours . 
when two or three tumours , similar in size , duration , and type of behaviour are present , progression occurs in only one of them at a time . moreover , the progression of one tumour has no apparent effect on the course of the others . 
the present observations decisively controvert browning 's ( 1948 ) suggestion that a second tumour may " abrogate the autonomy " of the first . the several recognizable characters of individual tumours undergo progression often , no doubt , responsiveness and intrinsic growth rate independently . change simultaneously , but in some tumours progression in responsiveness occurs transplantation without progression in intrinsic growth rate and vice versa . experiments provide further evidence of independent progression of characters , namely the responsiveness of transplanted tumours to pregnancy and the gross milky secretion in response to oestrogenic stimulation ( foulds , 1949b )  . the development of a tumour does not always reach its end - point within the life - time of the host ; progression advances further during serial transplantation ( foulds , 1949b )  . 
the course of the growth elicited by pregnancy depends on specific properties which change , by progression , in latent tumour cells and in stationary tumours . the manifestation of progression in the mammary tumours is usually abrupt , as it is in the induced skin tumours of rabbits and mice , but progression itself is not necessarily abrupt ; it is possible that cumulative gradual changes reach a threshold level at which they are first manifest . 
some tumours evidence a gradual progression through a graded sequence of types . gradual , continuous progression seems uncommon , but it is less conspicuous than the abrupt type , and more accurate and more frequent measurements might reveal it more frealthough the observations are few and indecisive , it seems that slow quently . gradual progression is apt to result in sluggish , irregular , or dubiously unresponsive growth . 
the unequivocal rapid - growing unresponsive tumours , as a rule , are either primarily unresponsive or the result of abrupt progression from a fully responsive and , often , strictly conditional tumour . apparently two paths of progression are available . 
one path leads directly to unresponsive tumour ; the tumour acquires its definitive properties early without traversing the numerous intermediate stages which are possible , and which are observed on the other path or " responsive detour . " the detour leads ultimately to an unresponsive tumour , but progression along it may be slow and gradual and the end - point indeterminate , or reached only after transplantation . 
abrupt progression switches many tumours from the detour to the direct path ; jumping the intermediate stages , the tumours change from the fully responsive to the definitive unresponsive type . 
a few applications arehere briefly indicated . the behaviour of tumours is the resultant of multiple characters which vary , within wide limits , independently of each other and undergo independent progression . 
the - characters include growth energy , invasiveness , capacity for dissemination , and responsiveness to environmental stimuli , of which the hormones are the most easily recognized , but not necessarily the only , examples . " malignancy " is not a single character . 
independent progression of characters ( rule ii ) , however , results in disproportionate or " out - of - step " development , as for example in the " benign " tumours that metastasize and the " locally malignant " tumours that do not . 
the out - of - step development is especially important in carcinoma of the prostate , which , despite conspicuous growth , invasion and dissemination , willis ( 1948 ) criticizes the sharp division of tumours is responsive to hormones . into innocent and malignant groups , and deprecates the question , " is this tumour innocent or malignant ? " ; the appropriate question in his opinion is , " how it is even more important , i suggest , innocent or malignant is this tumour ? to ask , " in which characters , and to what degree in each of them , is the tumour innocent or malignant ? " errors in the prognosis of " early " tumours are explained by the observations that progression is independent of the size or duration of a tumour , and that progression occurs without manifest growth , as in latent or stationary tumours ( rule iii and corollaries )  . 
progression without manifest growth and independently of the size or duration of tumours probably accounts , too , for the otherwise mysterious but clinically important phenomena of long - delayed recurrence and secondary growth after apparent similarly the ultimate failure of several chemo " cure " of primary tumours . therapeutic methods after favourable early response is reasonably attributed to progression in the inhibited tumours . 
some tumours maintained the same type of behaviour throughout their clinical course ; others changed their course , often abruptly , as the result of an irreversible qualitative change in the tumours termed " progression . " the diversity of behaviour of the spontaneous tumours was attributable , in the main , to varied combinations of responsiveness and intrinsic growth rate , and to progression in one or both of these characters . following generalizations or rules of progression summarize the results of the analysis of the behaviour of tumours , especially multiple tumours , in breeding and non - breeding mice : ( 1 ) progression occurs independently in different ( independent progression of multiple tumours . ) tumours in the same animal . ( 2 ) progression occurs independently in different characters in the same tumour . ( independent progression of characters . ) ( 3 ) progression is independent of growth , occurring in latent tumour cells and in stationary tumours . ( 4 ) pro ( 5 ) progression follows one of altergression is continuous or discontinuous . ( 6 ) progression of a tumour does not always reach its end - point native paths . within the lifetime of the animal . these rules are probably widely applicable to the behaviour of varied tumours in animals and in man , and some applications are mentioned briefly . 
 " malignancy " is not a single character . disproportionate or " out - of - step " development of the various characters which determine malignant behaviour ( independent progression of characters ) accounts for many anomalieg in the behaviour of cancer in man , and progression in tumours whose growth is inhibited probably explains the ultimate failure of therapeutic measures which have a favourable immediate effect . i am grateful to l . 
we spent more than eight months working on one such incidental problem , which had to be solved before we could proceed with the main work . this effort was well rewarded , however . 
we not only improved our experimental approach , but also acquired information that will further our understanding of the intricate role played by biological factors in regulating the influence of external agencies on living cells . in view of all this , i should like to point out that our investigations of the genetic potencies of carcinogens are still in an early stage . 
when i learned , therefore , that vapours could reach the sperm of drosophila i was confident that it would be possible to produce fine aerosols of chemical solutions it was evident tbat , if we could develop an aerosol that could do the same thing . method of inducing mutations , we could work with a wide selection of chemicals that are soluble in solvents not injurious to the ' flies , and would not be limited to chemicals that can be vaporized at temperatures not lethal to the flies . after much experimentation we finally succeeded in developing an aerosol technique that is effective in inducing mutations in drosophila males . males are kept for several hours ( from 6 to 200 ) in an atmosphere containing an aerosol of the desired solution ; and after this treatment their sperm is tested for induced genetic changes ' by standard genetical methods . 
the flies are treated in a milk bottle , and the aerosol is generated intermittently , for 30 seconds every 30 minutes , by a de vilbiss nebulizer . in our work with chemicals we have concentrated on carcinogens , because there is a feeling.among geneticists that these coffipounds should also be mutagenic . 
what classifies them as carcinogens is their ability to change normal cells into cancerous cells - that is , to produce permanent changes in living cells , which this definition can also be applied are transmitted to the progeny of those cells . to mutagens , which cause permanent and heritable changes , or mutations , in cells . this series of experiments with carcinogens also included chemically related tests were made with nine polycyclic hydrocarbons ( four non - carcinogens . carcinogens and five ' non - carcinogens ) and with seven azo compounds ( three of them carcinogenic )  . 
the observations were made on induction of x - chromosome .onclusions lethals and of chromosomal aberrations coincident with lethals . reached on the basis of the experimental results are summarized in table i . from table t a definite correlation between mutagenicity and carcinogenicity is evident . 
of the seven carcinogens tested , six were found to be mutagenic ; and of the nine non - carcinogens onlv two were found to be mutagens , and one is still classified as doubtful . the evidence now available suggests that some chemicals ( dibenzanthracene , benzpyrene , and hydroxyazobenzene ) induce both gene changes - le . 
lethals and chromosomal aberrations , whereas some others ( benzanthracene , dimethylaminoazobenzene , and 2 - amino - 5 - azobenzene ) are more effective in inducing chromosomal aberrations . during our experiinentation it was observed that different males treated at the sametime quite frequently showed different results ; that is , genetic changes could be induced in some males m ' ore readily than in others . table ti gives the 116 m . 
at present the most likely solution seems to be that a gene is responsible for the observed variability of effect between different males and between different , experiments . i shall conclude this presentation by quoting freely the conclusions given in a paper presented at the meeting of the fourth intemational cancer research congress in st . 
from these results it seems reasonable to infer a common causative mechanism relating mutagenicity this inference is further strengthened by the behaviour and carcinogenicity . of all known non - chemical carcinogens , such as x - rays and related radiations . ultraviolet rays , and heat - all of which are also mutagenic . 
the most obvious and probable relation ' between mutagenicity and carcinogenicity is the one suggested by the hypothesis that cancer may originate through a gene mutation occurring in a somatic cell . 
such a cell , and the cells derived from it by division , would have their properties changed so that they would behave as cancerous . this would mean that higher organisms possess a gene - - or , more likely , a number of genes - whose mutations can initiate a cancer - type cell . it may be assumed that such mutations , like a great majority of mutations in other genes , would occur spontaneously with a very low frequency . 
the human body has a tremendously large num ' ber of cells , however , so that - it is probable that a cancertype mutation would occur a number of times among the cells of an individual . not all of these need give rise to cancer , since a large proportion of the cancertype cells might be prevented by normal cells from dividing , or might be eliminated in some other way . if gene mutation is responsible for the origin of cancer , then all mutagenic agents may be expected to increase the frequency with which such mutation occurs , and consequently to act as carcinogens . in our fight against cancer , therefore , precautions should be taken to avoid exposure to ' all mutagens chemicals as well as radiations . such precautions , however , even if rigorously enforced , would only lower the incidence of cancer ; they could not entirely prevent its occurrence . there would still be a chance left for cancer - type mutations to occur among the billion 's of cells that constitute the human body , and a mutated cell that continued to divide would give rise to cancerous growth . 
we know that mutations do occur with great regularity , caused by some force not yet explained , and that we have no means of stopping or controlling their occurconsequently , if cancer originates through a genetic change , our chances of finding ways to prevent it are very , very slight . 
a phenomenon , involving the abnormal growth of two kinds of pigmented cers , was recently found in spontaneous tumours experimentary produced by genetic methods in platyfish - swordtail hybrids . the tumorous hybrids were produced by mating a male spot - sided and rub throated platyfish ( platypoecilw maculatus ) to an albino swordtail ( xiphophoru& hellerii )  . 
the ruby - throated pattern is composed of xantho - erythrophores , which are red pig ' ment cers containing erythropterin in granular form and lutein gordon ( 1950 ) and gordon and nigrelli ( 1950 ) and zeaxanthin in solution . indicated that these colour patterns are inherited and referable to two platyfish sex - linked genes : sp for the black and bt for the red pigment cers . 
the genetic analysis revealed that the pigmented tumours were produced in platyfish - swordtail hybrids containing the hnked genes , sp and bt from the platyfish and a number of sp and bt gene modifiers from the swordtail . some of these pigmented tum ' ours were diagnosed as erythromelanomas in an earher report by gordon ( 1950 ) and nigrelh , jakowska and gordon ( 1950 )  . more extensive study of additional specimens revealed that there were two kinds of pigmented tumours involved , first , a red tumour , a xantho - erythrophoroma of the head and , second , a black tumour , a melanoma of the body . in some of the hybrids , cers of the melanoma reached , penetrated and replaced the tissues of the red pigmented tumour . 
only a few macrophages , however , are evident in these tumours . the relatively few dendritic and rounded cells are similar to those described in the simple xantho - erythrophoroma . 
the various stages in the development of the melanoma within the xantho - erythrophoroma can be demonstrated and traced in one of these fishes ( 243 - 11 )  . 
33. - melanotic area of the tumour ( 243 - 1 1 )  . 1 - n this region one or two cells from the original xantho - erythrophoroma can still be distinguished . in other areas , however , a , region just below the epidermis - note the melanotic they are completely absent . cell ( melanoblast ) in metaphase ; b , typical melanotic cell of this region associated with a cell of the xantho - erythrophoroma ; c . 
the melanotic cells ( melanoblast 's ) are smaller and less dendritic than the typical macromelanophores ; they are usuany rounded , sometimes fusiform , varying from 10 to 40 tl at their widest dimensions . 
the present studies , however , show that two distinct tumours are involved : a xantho - erythrophoroma in the head region and a melanoma of the trunk and caudal peduncle . in some of the hybrids the more actively proliferating cers of the melanoma invade and replace the red pigmented tumours of the head , producing an erythromelanoma . the red and yerow pigment cell characteristic of the xantho - erythrophoroma was first described by kosswig ( 1928 ) from normal fish as an erythro - xanthophore . according to goodrich , hill and arrick ( 1941 ) , the cell ; which they caued a xantho - erythrophore , is a xanthophore in which erythropterin is laid down in addition to lutein and zeaxanthin . in fixed and stained tumorous material the xantho - erythrophores are either non - pigmented dendritic , rounded or fusiform cers . in both normal and tumorous tissues the nuclei of these cers ' are weak in chromatin ; nevertheless , mitotic figures are frequently found . 
the degeneration of the xantho - erythrophoroma probably results from the growth - pressure of the apparently more actively reproducing melanotic cers which infiltrate and penetrate the interstices between the xantho - erythrophores . 
whether or not the metastasizing tissue would have comalthough complete pletely replaced the invaded tumour was not determined . replacement of a xantho - erythrophoroma by melanoma has never been observed in our fishes , the evidence seems to indicate that this would have occurred had the specimens lived longer . an interpretation of the nature of the genic action that modifies pigment cell growth and the physiological effects of the interaction of such genes in platyfish - swordtail hybrids was outhned by gordon ( 1948 , 1950 )  . 
the effect of the various genes for pigment cells upon the development of the red , the black and the red - black pigment cell tumours may now be suggested . in the normal platyfish gordon ( 1928 ) showed that the dominant , sex - finked , spot - sided gene , sp , manifests itself in the development of large black pigment cells or macromelanophores ; the large melanophores first appear in postembryonic platyfish in the posterior region of the body , specifically , in the caudal peduncle . 
the number of macro melanophores produced by the sp gene in the normal , spotted platyfish is influenced strongly by the action of the stippled gene , st , a dominant , autosomal factor for the - presence of many micromelanophores . 
when sp is associated with the dominant gene st ( that is , sp st ) , more macromelanophores develop than when the sp gene is associated with the recessive gene st ( that is sp st )  . furthermore , in both the sp 8t post - embryonic and adult platyfish the macromelanophores are restricted for the most part to the caudal peduncle . 
315. - - below , a platyfish - swordtail hybrid of the genetic constitiition rts , 206 - 15 , showing the early stage of the development of the xantho - erythrophoroma . this shows up as a red patch on the left operculum surrounding the lower half of the eye . 
241 , produced by mating a platyfish - swordtail rtsp hybrid , 206 - 12 , exhibiting an erythromelanoma , back to an albino swordtail , 1822 - 6 ; upper left , an albino with an amelanotic melanoma at the tip of a caudal fin ; lower left , a sibling showing the " ghost " pattern of macromelanophores ; right , typical melanotic hybrid showing the ear ' lv sta - ae of melanosis in the posterior region of the body . 
the minimu ' m requirement for the production of the erythromelanoma in platyfish - swordtail hybrids is the presence of the linked genes rtsp . gordon ( 1950 ) showed that a platyfish parent of the genetic constitution of rtsbidrsp would not , while a rtspidrsb would produce some hybrids with erythromelanomas when mated with an albino swordtail . 
in the tumorous animals the xantho - erythrophoroma develops rapidly in the head region , and at the same time a condition of melanosis develops in the posterior region of the body . in some individuals the melanotic cens show great reproductive activity and produce a melanoma . 
in those animals that develop a melanoma the cells of the melanoma may invade the xantho - erythrophoroma and destroy and replace the cells of the red pigmented tumour . in other cases the melanin - bearing cers are restricted to the posterior part of the body , aflowing the red tumours to develop on the head without the involvement of the black pigment cers . 
the sequence of events in the invasion of one kind of neoplastic tissue by another type is discussed from the points of view of histology and developmental genetics . this work has been aided by a grant to the new york zoological society from the national cancer institute , national institutes of health , united states public health service in support of the project : " genetic and correlated studies of normal and atypical pigment cer growth . " some details in this paper were presented by myron gordon before a meeting , of the royal academy of medicine , london , july 4 , 1950 . we are deeply indebted to ' the american museum of natural history for .laboratory facilities , to j . 
oliver. from the radiotherapy and cancer research departments , london ho8piw , london , e.i. received for publication january 25 , 1951 . the distribution of radioactive phosphorus can be investigated in vivo using a thin - walled g.m. 
the maximum range for beta paitioles of p32 in soft tissue is 8 mm . , but the half - value thickness is only of the order of 0 - 6 to 0 - 8 mconsequently , measurements on the skin can only indicate the amount of phosphorus in the superficial layers of the body ( effectively some 3 mdeep )  . however , under favourable conditions , such measurements can be used to demonstrate the increased phosphorus up - take by malignant tumours . low - beer , glenn - bell , mccorkle and stone ( 1946 ) have used this method with some success , as a diagnostic procedure , but its value is reduced by the low penetration of the beta rays and by the increased up - take also found in inflammatory conditions . 
phosphate buffer at ph 7 - 35 . it has been found advisable to check , by counter measurements , that the radioactive material has actuary left the site of injection , as , even after taking more than ordinary care , some unsatisfactory injections have been detected . this is particularly important , of course , when giving the isotope therapeutically , as dose calculations assume that the whole of the material enters the blood streano side effects of the injection were observed . 
the tumour may have received 4 to 8 times the dose calculated above , a total which is in excess of the generally agreed " tolerance " level . this was , however , of no consequence as the site was to re ' ceive an x - ray dose of 3000 to 4000 r . a number of sites over the tumour , on the diseased breast , and at corresponding points on the normal breast , were marked with gentian violet , and measurements of the activities were made with a counter tube applied directly f . 
the background rate was in each case the complete set of measurechecked before and after the series . ments was repeated and a mean value for each site was used . it was not feasible to prolong the counting time at any site ab ' ove about 3 minutes . 
as the count rates with this tracer dose were rather low , the measurements were usually limited to 1000 counts ( 3 per cent standard deviation ) or , after several days and over normal tissue , to only 100 counts ( 10 per cent standard deviation )  . consequently , the ratios of count rates over tumour and normal skin were not obtained very accurately , but the results sufficed to demonstrate large changes . series of measurements were made before , during , and 4 to 6 weeks after the treatment , at which time the x - ray erythema had subsided but the skin of the irradiated breast remained highly pigmented . observations were made on altemate days during 2 to 3 weeks . after intravenous injection , the radioactive phosphorus could be demonstrated immediately ( 5 to 8 seconds ) in the tissues of the breast , but the concentrations continued to increase in both healthy and dis6ased breasts for 10 to considerably lower count rates were observed when the next series 15 minutes . of measurements were made after an interval of 2 days , a change probably due to the excretion of p32 ' thereafter the fall was found to follow a roughly exponential form , corresponding to a " biological half - hfe " of between 7 and 9 typical cases are ifustrated in fig . 
i. and over both axillae are also included . it is of interest to note that the readings , over the forehead ( indicating thep . , 2 content of the frontal bone ) increased to the level found over the tumour and then decreased more slowly than the latter . values of the ratio of count rates over the diseased breast to those over the healthy breast are presented in fig . 
a sudden drop in the ratio occurred after the third irradiation . four weeks after the end of the treatment a second tracer dose was given , and the results obtained are shown in fig . 
for the first three readings the skin was still very red . by the time the erythema had subsided the count rates were rather low , but they appeared to be higher than normal over the whole of the diseased breast , without any locahzed . 
at a later date the authors hope to continue the investigation , although working at different institutions . the countrate due to p..2 was fodnd to be very much higher ( between 3 and 8 times ) over malignant mammary tumours attached to the skin , than over the corresponding parts of the healthy breast . 
no such , change occurred during after erythema stilboestrol treatment in a case which did not respond clinically . had subsided the ratios of measurements over the two breasts were found to be similar to those before treatmentbut the primary growth was still present in all these cases . the authors wish to thank sykes , the chief pharmacist at the hos ital , for preparing the material for intravenous injections , and k . 
argus. from the cancer research laboratory , university of florida , gainesville , florida . received for publication april 7 , 1953 . it has been observed that when certain sulfonic acid dyes , such as trypan blue , are injected into animals with tumors , the neoplasms become more highly unfortunately these dyes concentrate in colored than the surrounding tissue . some vital organs to a much greater extent . 
an investigation by moore , tobin and aub ( 1943 ) of br82 derivatives of evans blue and trypan blue in tumorbearing mice showed that the tumor took up considerably more dye than the surrounding tissue . 
the liver , however , took up about three times as much stevens , lee , stewart , quinlin and gilson ( 1949 ) radioactivity as the tumor . studied the distribution of radioactive iodinated trypan blue , one of the compounds first prepared by bloch and ray ( 1946 )  . 
the concentration of radioactivity in tumor tissue was several times greater than that in skeletal muscle or skin , but considerably less than in the liver , spleen and kidneys . the purpose of the present study was to deternine if such localization in vital organs is a necessary concomitant to tumor localization by aromatic sulfonic acids . because radioactivity offers a ready means of detection , the need of a colored derivative no longer exists and sulfonic acids other than dyes may be employed . since certain derivatives of fluorene are known to produce tumors in various sites in the animal body ( bielschowsky , 1944 , 1947 ; morris , dubnik and johnson , 1950 ; wilson , deeds and cox , 1941 ) , it is possible that other derivatives of this molecule might be directed to tumors already present . 
the 2 , 7 - disulfonic acid of fluorene was selected and this compound was prepared labelled with s35 . benzene sulfonic acid injected into dogs can be recovered unchanged in the urine ( williams , 1947 )  . 
sammons , shelswell and williams ( 1941 ) report that p - hydroxybenzenesulfonic acid is excreted unchanged from rabbits . it does not even form an ethereal sulfate despite the fact that it possesses a phenolic hydroxy zehender ( 1943 ) found that the amino group of sulfanilic acid ( p - aminogroup . benzenesulfonic acid ) is acetylated to the extent of 75 per cent in the guinea - pig , but that no cleavage of the sulfonic acid group from the ring occurs . 
a study of various aminophenolsulfonic acids by sammons , shelswell and williams ( 1941 ) also indicates that these compounds are eliminated unchanged . it is , therefore , apparent that aromatic sulfonic acids are stable compounds . since there is no evidence to indicate that the sulfonic acid groups of fluorene2 , 7 - disulfonic acid would be removed from the molecule , the distribution of radioactivity following administration of the compound labelled with s35 should 274 mary f . 
2 , 7 - fds35 having a specific activity of 39 , 660 counts per minute base experiments in which the animals received twice this concentraper mg . tion of the compound failed to reveal any toxicity symptoms . the animals were sacrificed at 2 - , 8and 32 - hour intervals following treatment , and pooled samples from 2 animals were used for each determination . 
samples of blood were plated directly on planchets . radioactivity measurements were made in an internal - type counter ( q - gas chamber and nuclear instrument and chemical corporation scaler , unit model 162 ) with an efficiency of 45 per cent . 
the concentration of 2 , 7 - fds35 in the tumor was over three and one - half times that in the spleen and two and onehalf times that in the liver . 
with this same compound the kidneys and spleen concentrated over three times as much of the dye as did the tumor . a much more favorable situation is found in the distribution of 2 , 7 - fds35 . at 8 and 32 hours only the kidneys had a slightly higher concentration of this compound than did the tumor . 
at no time was the localization of the fluorene derivative greater in the liver than in the tumor , and after 32 hours the concentration was two and one - half times greater in the tumor tissue than in the liver . 
by comparison with similar ratios for 1131 - labelled trypan blue , the fluorene compound has increased the ratio of localization in tumor tissue compared to liver , kidneys , spleen , blood and muscle . this investigation showed that the localization of a sulfonic acid in the liver and spleen of tumor - bearing mice 278 mary f . 
ray for his valuable suggestions and to hilda banks for technical assistance . this work was supported by a research grant from the national cancer institute of the national institutes of health , u.s. 
ritchie. from the division of oncology , the chicago medical school , 2755 west 15th street , chicago 8 , illinois . received for publication july 31 , 1953 . by studying the life of various kinds of spontaneous and induced tumors in animals , it has been found that they sometimes undergo irreversible changes in character . for example , a papilloma induced in mouse skin by applications of carcinogen , may become a carcinoma , so changing its character ; or a spontaneous tumor of the mouse breast responding to pregnancy by an increase in growth rate may change its character and no longer respond to this stimulus . foulds ( 1949 , 1950 , 1951 ) made a special study of these changes in character , calling them " progressions " , and defining " progression " as an " irreversible , qualitative change " in a tumor . foulds then went further , claiming that different characters of the same tumor could progress independently of one another . for example , he found that in spontaneous mammary tumours in mice one character , the growth rate , might progress , and the tumor grow faster , while another , the responsiveness to pregnancy remained unchanged . foulds ( 1949 ) studied not only the spontaneous mammary tumors of mice , but also bladder tumors induced by feeding 2 - acetylaminofluorene to mice ( foulds , 1950 ) and transplantable mammary fibroadenomata in rats ( foulds , 1951 )  . in each case he found that the tumors did sometimes progress , and that progression could occur independently in different characters of the same tumor . these findings seemed so interesting as to make desirable further confirmation , and this paper records an investigation of the life of tumors induced in mouse skin by repeated applications of 9 , 10 - dimethyl - 1 , 2 - benzanthracene . 
ritchie the second type of sessile papilloma consisted mainly of epithelial tissue . the architectural pattern of the normal human epidermis was imitated , the epidermis being thickened from four to six times . 
the surface of the tumor was often hyperkeratotic . in a few cases a tumor of this type arose in a pedunculated papilloma . sheets of closely packed cells invaded the dermis and muscularis . 
most of the tumors grew at a steady rate throughout , in so far as the growth rate could be determined by the crude method used . however , definite changes in growth rate did occur . 
8 to 11 , and will be described individually , each mouse having tumors with differing behaviors demonstrating the validity of one or other of fould 's proposals . all charts illustrate the validity of rule 1 : namely , that progression occurs independently in different tumors in the same animal . 
9 also confirms fould 's rule 2 , showing three tumors , a , b and c progressing from one morphological type to another while growing at a constant rate . this type of progression , from one morphological type to another , without progression in growth rate , was much more common than progression in growth rate alone , as seen in this chart in tumor d . 
10 , and confirms the validity of fould 's rule 3 , that progression is independent of growth . in the same chart , in tumors a and b may be seen again a change in growth rate without a corresponding change in morphological type . * n 5 31_ * - - __ + * - - + b ..od / , ' / ___ _ , / " ... 
11. - showing independent progression of multiple tumors . tulmor a was malignant at its first clinical appearance , showing that at this period of its development a tumor can be all other tumors began as sessile papillomata . 
a well - known example of progression affecting only part of a tumor is the carcinomatous change which sometimes affects the tip of an adenomatous polyp of the colon in man . foulds ( 1949 ) remarked that progression could occur by gradual change or by abrupt steps . in general , the changes seen in the tumors of this experiment occurred abruptly . 
a step - wise transformation of different characters in these tumors was observed throughout their existence . this investigation was supported by a cancer control grant from the national cancer institute of the national institutes of health , u.s. 
murray. from the radiation therapy department , johannesburg general hospital , the non - european hospital , and the south african institute for medical research , johannesburg . received for publication january 10 , 1953 . skin cancer is relatively infrequent in the african . in the three - year period 1949 to 1951 , during which time 590 cases of malignant disease in africans were referred to the radiation therapy department , only 50 cases of skin cancer were encountered . 
the pigmented skin of the african would , of course , be largely protected from this effect , and indeed when skin cancer does make its appearance , it is almost invariably in areas exposed , not to sunlight , but to other types of chronic irritation . we have also observed distinct differences in pathogenesis , and the rate of growth and dissemination of skin tumours in the african , as compared with the m . 
murray classical description of the disease in europeans . these factors have compelled us to adopt a different approach to the management of skin tumours in the two races , and to revise our views on the surgical and radiological curability of skin cancers of various histopathological types . since these racial differences might be of some interest in the study of the demography , endemiology , and management of cancer in africa , it was considered worth while putting our experiences on record at this stage . the population from which this material is drawn consists of approximately 2 , 000 , 000 africans of the bantu race inhabiting the southern half of the transvaal , and originating for the most part from the sotho and zulu tribes . 
about onequarter of this population is urbanised and resident on the witwatersrand , but a much larger fraction of the hospital population gives an urban address . this is due , in part , to many rural patients living with relatives in the urban area prior to entering the hospital and witholding their true addresses , and also to the greater tendency among rural natives to receive treatment from indigenous herbalists in the absence of suitable vital statistics it is impossible rather than in hospitals . to state the age distribution of the bantu population , although it is safe to assume that the average age is somewhat lower than that of the european group . 
the ratio of males to females at the time of the 1946 census was 1 - 33 , the predominance of males presumably reflecting the migratory labouring class . from the genetic point of view it is important to bear in mind that the bantu inhabitants of south africa originate from central and east african tribes , who have , at least within historic memory , had no admixture of european blood . 
by comparison , the negro of the united states originates from west african ancestry and is largely a product of prolonged hybridisation . although environmental conditions in the southern states of the u.s.a. 
are not dissimilar to those encountered by the bantu in the transvaal , the two groups generally show a very different incidence and distribution of malignant disease ( higginson , 1951 ; and robinson , 1951 )  . 
the cells are arranged in the form of distorted , branching tubules or of alveolar masses . in some areas the cells are separated from the surrounding stroma by a well - defined basement membrane while in other areas the cells appear to infiltrate into the stroma . 
as the cells approach the lumen they become larger , the cytoplasm becomes faintly eosinophilio and sometimes vacuolated . the nucleus becomes smaller , more dense , and shows a well - defined nuclear membrane . the nucleus in these cells lies centrally , and their whole appearance is that of lipoid containing cells resembling those of the sebaceous glands . 
he was admitted from an outside hospital with a history of having had a tumour of the right upper arm excised 10 months previously , and that the lesion has subsequently recurred . there was a massive tumour present on the right upper arm and shoulder region , surrounding a vertical scar of the old excision . 
he became very ill , and was transferred home after further palliative x - ray therapy to the lumbar spine . the 2 cases of " sebaceous gland carcinoma " , although showing advanced disease , clearly illustrate the extreme radiosensitivity of these tumours , with conventional large field x - ray therapy . 
one patient is free from disease , while the second has extensive and incurable tumour . albinism and cancer of the skin . it is remarkable that there were 12 albinos among the 50 cases of skin cancer reviewed , although the albino trait is rare in the african . 
the exact incidence of albinism in the bantu is not known , but a cursory review of recent hospital records suggests that it cannot be greater than 1 in 5000 , and is possible less than consequently the 12 patients seen , representing the appearance of 1 in 10 , 000 . 4 new new cases annually ( and probably several more who do not attend hospital ) , implies that a large proportion , if not all of the albinos among the transvaal bantu necessarily develop skin cancer in early adult life . this observation is in agreement with the experimental findings of findlay ( 1928 ) , beard , boggess and von haam , ( 1936 ) and the recent precise quantitative investigations of blum ( 1950 ) , who have demonstrated that albino animals exposed to daily insolation or ultraviolet irradiation for a significant fraction of their life - spans invariably develop malignant skin tumours . all 12 patients showed complete albinism of the whole skin surface including the hair and eyes . 
we did not encounter any cases of partial albinism , such as those described by gelfand ( 1944 ) and shown to have the same propensity for skin cancer as the true albino . however , there was one case of xeroderma pigmentosum ( not included in table i ) occurring in a 4 - year - old bantu child in whom the whole skin surface showed the characteristic regular mottling caused by depigmented patches alternating with areas of increased pigmentation . 
murray pigment - free zones , especially those of the head and neck , possessed the characteristic hypersensitivity to light and were the origin of several histologically proved squamous carcinomas . there were 8 males and 4 females in the series . the age distribution of skin cancer in albinos shows a distinct tendency for the disease to occur in younger age - groups than in normally pigmented persons . there was 1 patient in the second decade , 3 in the third , 5 in the fourth and 3 in the fifth decade . 
we have not had the opportunity of observing the subsequent history of skin areas cleared of all macroscopic keratoses by podophyllin , and , in consequence , are not able to assert that the onset of skin cancer is , in fact , prevented . however , berenblum 's ( 1951 ) observations on the definite anticarcinogenic effect of podophyllin , even in the presence of actively carcinogenic hydrocarbons , suggests that this material may have a true prophylactic action . 
two to 3 weeks before admission black lumps appeared on the skin in the elbow region . on admission the patient had a typical malignant melanoma over the thenar eminence of the right hand . 
the primary growth and axillary glands were given 250 r daily to a total of 5500 r , other areas were treated with 1500 r and 1000 r daily to totals of 4000 r , 4500 r , and 6000 r , and six small areas were treated with three daily doses of 2000 r . all treated areas responded clinically by regression of the tumours and some necrosis of the tumour surface , but histological examination after the radiation showed minimal changes , a few lesions treated with 1000 r daily for 6 days disappeared completely . weeks after admission it was noted that the supraclavicular glands were enlarged and painful , but the intervening skin appeared normal . ten days later small black nodules were visible in this skin area . 
in 1940 , however , an important change was made in the method of selecting the main cause when more than one was mentioned , a disease certified as merely contributory and not in the direct line of causation being ignored if another well - defined condition was stated as the direct cause of death . 
for breast cancer in women , for example , the factor to be applied to a rate at all ages prior to 1935 was * 941 , signifying that the new procedure resulted in about 6 per cent of deaths which would previously have been credited to breast cancer being classified from 1940 onwards to some other cause . when it is desired to study the trend of mortality at particular ages it is not safe to assume that the correction to be applied to rates before 1940 is the same at every age , until that has been tested , as may be done by examining the ratios at different age groups between the deaths in 1939 based upon the old and new methods , tabulated for that purpose in table 21 and appendix bl of the statistical review for that year . 
for certain diseases where it was important , such as diabetes , more exact correction factors based on the dual classification carried out during the four years 1936 - 1939 were used in the registrar - general 's analyses of past death rates at separate age - groups , and on page 135 of the text volume of the review for 1938 - 1939 it was shown that for cancer of all sites combined the conversion ratios for each sex were slightly above unity at ages before 45 and below unity at ages after , diminishing regularly from 1 - 17 at 5 - 14 to * 95 at 75 and over for males . 
for cancer of the breast in females it can be deduced from the tables in the review for 1939 referred to above that the correcting factor diminishes progressively from * 997 at ages 25 - 29 to * 970 at 55 - 59 and * 810 at ages 85 and over . this is easy to understand because of the increasing frequency of the presence of breast cancer , in its primary or secondary manifestations , as age advances , coupled with increasing liability to die of some intercurrent disease which is certified as the direct cause of death . throughout these studies of the trend of death rates according to age , correction of the rates in years prior to 1940 has been carried out by means of factors derived jointly from the age - by - age comparison of the deaths in 1939 and the conversion ratio forall ages combined obtained from the dual tabulations of 19361939 . 
in 1950 - 51 it was possible for the first time to distinguish mortality from cancer of the cervix and corpus uteri as a result of the inquiries by the general register office concerning the large number of deaths certified as cancer of " uterus " without further definition , and the rates for cervix uteri show still lower proportions of single women and greater contrasts with cancer of the breast and other causes of death . amongst women who died of cancer of the cervix in 1950 - 51 at ages under 55 the proportions who had never married were about one - third of the corresponding proportions amongst women who died of all causes . 
at ages 55 - 74 the proportion was about two - fifths , and after 75 it was about three - fifths . in the registrar - general 's statistical review for 1940 - 45 ( registrar - general , 1951 ) deaths of married women from various causes were analysed by age groups according to whether the deceased women had borne any children or not , and the resulting proportions stated at death registration not to have had a child are shown in table iv for cancer of the uterus , breast and ovary , non - genital cancer and all causes . 
2 , in sharp contrast with those for uterine cancer , show on the whole very little change through 25 10 o > 9a t > 7 - id 5 - s = ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~i 1921 - 26 1926 - 30 1931 - 35 1936 - 40 1941 - 45 1946 - 50 40 period when death occurred age when death occurred fig . 
at ages over 65 the rates increased up to 1940 and then fell back slightly , and between ages 30 and 45 the rates have increased since 1940 . this may be seen by expressing the rates in 1936 - 40 as percentages of those in 1926 - 30 , and the rates in 1946 - 50 as percentages of those in 1936 - 40 ( table x )  . in the right - hand part of fig . 
the increase in breast cancer mortality of married women to be expected at ages over 45 in the last 20 years would be , therefore , about 2 per cent of the rate . information as to the numbers of pregnancies experienced by women who later develop breast cancer at various ages is scanty , and the only data found suitable for the purpose of this study were in a tabulation by smithers et al . 
at ages under 45 the effect seems to be even greater , but the numbers on which the ratios are based are small , and there might be other reasons contributing to the low frequency of women with four or more children amongst patients seen during the period of childbearing . it is desirable that this inverse correlation with number of children should be confirmed by other and larger data , and such information is in process of collection in the liverpool hospital region and north wales by the british empire cancer campaign . assuming it to be correct , what effect would the decline in family size since 1905 be expected to have upon the incidence of breast cancer amongst all married women ? this can be calculated by applying the ratios for women over 45 to the successive distributions in table via and aggregating the products , as was done for uterine cancer . 
lung cancer death rates and also those from other forms of cancer tend to increase steadily with advancing age , so movements of the ratio will give no indication of that part of the upward progression which lung cancer shares with other cancer . 
the years of birth of the cohorts attaining these ages in 1901 would be 1882 - 1886 , centred at 1884 as the table indicates ; in 1931 the men of this cohort would be aged 45 - 49 , in 1933 their ages would be in calculating the ratio for the 1884 cohort in 1933 , three times 47 - 51 , and so on . the deaths at 45 - 49 were added to twice the deaths at 50 - 54 in 1933 , both for lung cancer and all other cancer , and the first total divided by the second . 
the " nil " increases in table xvi from 1937 to 1940 mean that in the cohorts bom before 1875 the lung cancer death rate was then rising with advancing age after 65 at about the same gradient as the death rate for all other cancer , and the actual gradient at that age and time period has been shown to be about 33 per cent increase in 5 years or 20 per cent in a trienniuthe preceding explanation for what occurred between 1925 and 1937 is far more probable , but the hypothesis of influenza as a contributory factor cannot be excluded as a possibility . from 1937 onwards sulphonamides began to be used in increasing amount , and pneumonia deaths soon began to fall . could this have affected lung cancer rates appreciably by keeping alive longer many persons who without sulphonamides would have died of a complicating pneumonia before the cancer could be recognised ? pneumonia death rates of males fell between 1937 and 1942 from 477 to 255 per nillion at ages 35 - 44 , from 950 to 457 at 45 - 54 and from 1535 to 1024 at 55 - 64 , and the fall continued till 1946 . during that period the lung cancer ratio continued to rise steadily in men born about 1880 , the gradient being steeper amongst men at ages around 40 and 50 than around 60 , and only slight after 65 . 
the distribution according to number of children born for hospital patients with breast cancer , in such data as are available , differs from what would be expected , and it is estimated tentatively that the bearing of 2 or 3 children reduces the likelihood of this form of cancer developing after 45 by about onefifth and that 4 or more children reduces it by about two - fifths . 
from 1937 to 1946 the lung cancer ratio continued to rise by about 7 per cent of the ratio annually in men born since 1890 , more slowly in men born between 1875 and 1890 , and inappreciably in cohorts born before that ; but after 1946 the increase again spread to all ages . sulphonamides , and later penicillin , must have contributed something to this by preventing some men from dying from penumonia without cancer having been diagnosed and allowing them to die a year or so later of recognised lung cancer . 
ghadially. from the department of pathology , the univemity of sheffleld . received for publication may 18 , 1951 . havingobserved that regeneration of hair in a clipped area on the back of a rabbit did not occur uniformly over the entire area , and occurred after varying intervals of time in different animals , it was decided to investigate the mode of hence , a band of hair , approximately regeneration of hair on the rabbit trunk . 4 inches wide , completely encircling the trunk , was removed from a few rabbits , and it was observed that the depilated skin was not uniformly pink , but that there were roughly linear bluish - black bilaterally symmetrical zones . besides these major dark zones , there were , in some animals , a few small irregular patches of a similar colour . 
the skin in these dark zones felt thicker than the rest of the after two days , when the animals were inspected , it was found that there skin . was prohfic hair growth in the dark zones , which continued until the hair reached its normal length , but during all this time no hair growth was observed in the pink zones . 
for a further period of 12 weeks with 2 per cent w / w , 9 : 10 - dimethyl - 1 : 2 - benzanthracene in lanohne , which was warmed to melting - point to facihtate apphcation . 
the animals were photographed at the beginning ofthe experiment and on numerous subsequent occasions . skin biopsies were taken at intervals from 3 experimental and 2 control animals . these were fixed in alcoholic bouin and sections were stained with haematoxylin 354 h . 
with the appearance of the papillomata changes began to occur in the surrounding skin ; the hair on the painted side now failed to show the vigorous growth it had shown so far . 
as already stated , by this time the whole of the skin on the painted side had attained a uniform appearance , and no distinction between active and quiescent zones was seen . 
the epidermis was much thicker than either the this change appeared to extend down active or quiescent skin before painting . into the folhcles , which were more prominent and deeply seated and showed the usual arrangement in groups . 
mottram ( 1944a , 1944b ) showed that the skin of mice can be sensitized by painting with croton oil , so that a subsequent single application of 3 : 4 - benzpyrene leads to an abundant production of tumours and a single application of carcinogen followed by repeated applications of croton oil also procluces tumours . 
hence mottram ( 1944b ) concluded that carcinogenesis was composed of three phases : ( 1 ) a " sensitizing factor " which could be brought about by preliminary treatment with croton oil , acting presumably in a non - specific manner by causina hyperplasia , on the supposition that proliferating cells were more responsive than resting cells to the specific ( 2 ) a " specific cellular reaction " induced carcinogenic action which followed . by the specific action ( even of a single application ) of a carcinogen , this representing the essential neoplastic change . ( 3 ) a " developing factor " responsible for the actual appearance of a visible wart , and produced by croton oil of by a carberenblum and shubik ( 1947a , 1947b ) confirmed the latter part of his cilnogen . observations , but found no evidence to suggest that a prelimi - nary croton oil hyperplasia had an augmenting influence on tumour production , which would 356 h . 
the depilated skin showed bila ' terally symmetrical dark zones which showed prolific hair growth within a few days , the rest of the skin remaining 9 : 10 - dimethyl - i : 2 - benzanthracene.in lanoline was painted on the left bald . half of 6 animals ; the remaining 4 were painted with lanoline . 
the following changes were observed : ( 1 ) within 30 days the painted area in the experimental animals became dark and showed prolific hair growth , while on the opposite side there was suppression of hair growth , the skin becoming bald . ( 2 ) in all animals initial papiromata appeared at the site of an originary active zone . 
the first papilloma appeared after 53 days . ( 3 ) after the appearance of papillomata hair growth ceased and zones of activity appeared on the opposite side . ( 4 ) the first carcinoma appeared after 81 days at the site of an originary active zone . it was concluded that ( a ) the naturally occurring zones of increased mitotic activity which continued for some time after the first painting must have acted as a promoting agent ; ( b ) it seems more probable that the tumours arose from hair folfcles , and not the epidermis , as the former are the site of increased mitotic activity during hair growth . it gives us great pleasure to express our thanks to professor h . 
keyser. from the department of pathology and bacteriology , wel - sh national school qf medicine , cardiff . receivecl for publication march 13 , 1954 . many observations have been published on the effect of disease or injury on the concentration of protein - bound carbohydrate ( polysaccharide , protein sugar ) or various carbohydrate - rich protein fractions in the blood serum , both in human beings and in animals . 
a study of the level of serum polysaccharide in various diseases led seibert , seibert , atno and campbell ( 1947 ) to suggest that a raised however , shetlar , bryan , concentration is associated with tissue destruction . foster , shetlar and everett ( 1949 ) put forward an apparently opposing view , namely , that such increases are a reflection of tissue proliferation or repair rather than of destruction . this paper deals with an investigation undertaken to find out what effect nitrogen mustard ( the bis compound , di ( 2 - chloroethyl ) methylamine hydrochloride , was used throughout ) or irradiation treatment might have on the serum polysaccharide level in patients with various neoplastic and allied diseases . ( in such patients raised values are usually found . ) it was hoped that sonie light might be thrown on any relationship between the polysaccharide level and tissue proliferation or destruction , since the nitrogen mustards and x - irradiation are known to cause tissue destruction or at least to inhibit actively growing tissues . another ob ' ect was to find out how much of any increase was contributed by the mucoprotein fraction . 
p - , a man , aged 50 , suffering from hodgkin 's disease , was admitted to bospital on 30.iii.50 for bis second coursfof nitrogen mustard . since the first course of - treatment a few months previously there had been an increase in essential case notes are as fohows 240 j . 
keyser dailv injections resulted in improvement correspoindidg the size of the glands . with a fall in the total white cell count and total polysaccharide , which was down nearly to normal a week after the beginning of treatment ( see table i )  . 
no significant fall in the polycourse . saccharide level occuited during the course of injections , but within 3 weeks after the end of the course it had faren by 40 mg . 
on examination it was found that the whole of the left breast was involved by growth , and biopsy showed there was no radiological evidence of secondary deposits squamous carcinoma . in chest , thorax or pelvis but there was body destruction of the radicles and laminae of vertebra u . 
at the end of 2 months , however , the total polysaccharide had fallen considerably and the mucoprotefn fraction to a lesser extent . healing of the medial half of the right breast area was progressing satisfactorily . 
on examination ( about september , 1950 ) the patient had a large mass of glands involving the whole left side of the neck , and the left tonsil was enlarged and fleshy - looking . biopsy of the neck glands showed the presence of hodgkin 's disease . 
the polysaccharide ( total and mueoprotein ) soon began to increase again , though the patient appeared to be in good health . after several months , however , she began to complain of tiredness and aching associated with a feeling of pyrexia in the evenings , for several clays at a time . these symptoms were apparently unconnected with the menstrual cycle . 
a temperature chart kept by the patient from january to may , 1952 , showed that on these days there was in fact a rise of temperature idthe evenings , usually to 100 - 101 ' f . 
normal figures found ' m this laboratory for the distribution of polysaccharide betweedthese fractions are given in table il the polysaccharide soon began to increase again and the patient 's condition deteriorated . 
 ( 1947 ) found a correlation between the levels of serum a - globulin and polysaccharide in their series of cases . using the tryptophan - perchloric acid test ( cohen , 1944 ; seibert et al . , 1948 ) we obtained evidence of a rise in the level of a serum constituent , that was believed 248 j . 
keyser to be probably polysaccharide in nature , after buming ( keyser , p949 , 1950 )  . the reaction has since been shown to be due to a polyhydroxylic acid of unknown structure ( " sialic acid " ) occurring in the proteins ( werner and odin , 1952 )  . more recently we have employed the orcinol method : both in burns and in fractitre cases pronounced rises in the level of serum polysaccharide ( total and mucoprotein ) were found ( keyser , 1952b )  . this is consistent with the tissue destruction hypothesis , but the increase might equary wer bave been connected with healing processes . that increases in the serum polysaccharide concentration occur in adimals after experimental injury was shown by shetlar , bryan et al . 
the response was a delayed one , with a maximal elevation in 3 to 6 days after the increases in polysaccharide were produced by ( among other methods ) injury . injection of talc suspensions or intrapleural injection of turpentine , causing conditions in which little tissue destructions was thought to occur ; fever was not essential for the elevation ; and the length of time that elapsed before the polysaccharide level returned to normal seemed to be correlated with the extent of repair taking place . 
they give no details , however , except in one case in which a small temporary rise is attributed this patient had previously undergone surgical removal of to inflammation . increases in the level of blood " polypeptides " as a result of the carcinoma . radium and x - ray treatment were reported by cristol and puech in 1930 . 
effect due to inhibition of a breakdown process in and around the tumour and depending on tumour growth . such a break - down process might be expected to be greatest when the tumour is growmg rapidly , and to be retarded when the tumour is subjected to an inhibitory influence such as that of the nitrogen mustards or irradiation . 
working with mice , catchpole ( 1950 ) found that connective tissue bordering on tumours contained increased amounts of water - soluble mueoproteins ; and histochemical studies suggest that in the region of tumours a process of depolymerization is taking place , whereby the components of the ground substance may become water - soluble and enter the general circulation as mucoproteins ( catchpole , 1950 ; gersh and catchpole , 1949 )  . 
the existence of such a mechanism would go far tciwards reconciling the " tissue destruction " and " tissue proliferation " hypotheses , since the two processes - growth of the tumour and consequent depolymerizait remains to be determined what tion - would be associated with one another . bearing the depolymerization hypothesis has on the rises in the level of serum this and other aspects of the problem are mucoprotein observed after in ' now being studied . in four of our cases urinary total nitrogen and nitrogenous constituents were measured in the hope that they might provide an indication of any tissue destruction taking place . that no consistent changes were found was perhaps to be expected , in view of the results of danowski et al . 
wood of the radiotherapy department for calculating the estimnated integral doses referred to in table i ; to dodwell of whitchurch hospital for much help in obtaining specimens of blood ; to our haematology laboratory for blood counts ; to a . 
cohen. from the experimental oncology laboratory , radiation therapy department , johannesburg general ho8pital . received for publication february 11 , 1954 . there are many reported examples of animals having been rendered immune or resistant , by various procedures , to subsequent inoculations of a tumour to which they are normally susceptible . 
with an adequately inbred strain eke the c3h mouse , however , and using homologous mammary tumour which has not lost its genetic specificity by repeated serial transplantation , absolute resistance to further implants of the same tumour cannot be evoked . it was previously shown that the radiosensitivity of a homologously - transmitted tumour is a function of the state of resistance of the host . 
when the tumours had reached the standard size of about i cin the largest diameter ( overall average volume 250 481 mm.3 ) , thev were irradiated in situ with our standardised techni.qu ' e as previously reported in detail . all mice were individually numbered and examined at regular intervals for at least 3 months after treatment . with the doses used in this series , the tumoiir has , within this observation period , either disappeared completely or bas resumed active growth , ulcerating the skin and killing the mouse by exsanguination or infection . in order to detect any acquired increase in radiosensitivity , tumours arising from each attenuation - dosage group were treated at three dose levels : 3500 , 4200 and 5000 r , making a total of six categories tested . 
1 - 27 ( 0 - 07 * )  . since this ratio exceeds unit the ld , for the attenuated homoplasts is found to be 4500 ( 200 * ) r , compared with 5700 r previously determined in the controls , and the coefficient of variation is 26 per cent compared to 9 per cent in the controls . 
the relative radiosensitivity of attenuated tumours with reference to the controls , that is the ratio of the ld50 's is four times its standard error , the result is highly significant ( p < 0 - 0001 ) , indicating a definite acquired radiosensitivity of the tumour arising from an attenuated implant . as has been previously shown , a changed radiosensitivity generally reflects an underlying change in host - resistance to the tumour . 
it would appear , therefore . , that the attenuation procedure , while not producing any absolute immunity to further implants of the same tumour , nevertheless induces within the host , albeit the strain of origin of the tumour , a subliminal resistant state . 
of the 22 " recipients " ( 2 to each donor ) , 1 1 were similarly treated in situ with 4200 r in order to detect persistent acquired radiosensitivity in the tumour transplants , and the remaining ii were treated with 7500 r in8ituto exclude the possibihty of an acquired radioresistance . 
the added disadvantage of bilateral treatments , even when the immediate mortahty could be reduced , was the possibility that the increased body dose would lower the expected percentage of cures , as previously demonstrated ( cohen and cohen , 1953b )  . 
the results of ' treatment of the attenuated homoplasts in the four experimental categories are shown in table ii , and the proportion of cures in each group plotted in fig . 
1 it can be seen that the proportion of cures to be expecied at this dosage with unattenuated homoplasts ( line a ) is exceedingly small , while in animals bearing attenuated implants ( line g ) it is 40 per cent , from which this result . 
the attenuation procedure , therefore , seems to induce a degree of resistance , presumably a similar antigenic difference between host and tumotir , at least as great as that engendered by the tumour growing in f , ilybrids harbouring the milk factor . the following facts conceming the nature of this phenomenon have been elucidated : ( 1 ) while not excluding the possibihty that the tumour cells have been inherently changed by this procedure , it is obvious that any such change is not , in itself " sufficient to affect the radiosensitivity of the transplanted attenuated conversely , there is no evidence of " takes " in the environment of a new host . acqiiired radioresistance in the tumour as a result of previous irradiation , in conformity with montgomery and warren 's findings ( 1953 )  . ( 2 ) it is unlikely that any local change in the tumour bed contributes to the changed radiosensitivity since no cures were obtained when recurrences followina the local excision at the original site were treated . it can probably be assumed , too , that damage to the tumour bed by the rsurgical excision was sufficient to abrogate the effect of systemic resistance . ( 3 ) since the increased radiosensitivity persists even where the tumour has 318 a . 
cohen been transplanted to another site in the original donor , it must be concluded that the effect is due to a systemic , generalised resistance mecbanism . the phenomenon of induced resistance to experimentalfy transmitted . 
cancer has been extensively investigated . in the past , results in this field were often contradictory and confusing ( woglom , 1929 ) , due to the use of heterogeneous strains of animals and gentically alien tumours . 
the long transplantation history of these and other commonly used tumours , maintained solely by serial transmission , is surveyed by dunham and it is generally realised that such procedures may perrnit antistewart ( 1953 )  . genic diversification or mutation in the tumour , which , in effect , results in a relatively unstable host - tumour relationship . consequently , many of these findings are largely invalidated in so far as their applicability to human cancer therapy is concemed . it would appear that only when a supply of spontaneous tumours is constantly availdble within a given strain , and is routinely transplanted through no more than a few subpassages to experimental animals of the same strain , can the hosttuniour relationship be considered adequately stabilised . 
where such stringent conditions prevail , absolute immunity to a tumour cannot be evoked ( bittner , 1936 ; barrett , 1940 ; lewis , 1940 ; fardon and prince , 1953 ; foley , 1953 )  . 
a recent review by hauschka ( 1952 ) contains an extensive analysis of the immunogenetic complexities of host - tumour relationships used in experimental cancer reseal - ch . with respect to the cm mammary carcinoma , the authors have shown that , even where some degree of immunogenetic difference existed between the tumour and the host , as in the case of f , hybrids bearing the parent strain tumour , no overt - immunity could be elicited by curing the comparatively radiosensitive tumour in8itu ( coben and coben , 1954 ) , or by previouslv inoculating the animals with radiation - attenuated fragments ( unpubhshed data )  . when one correlates these facts with the findings of the foregoing experiment , it become 's evident that resistance to tumour is by no means an " all - or - none " pbenomenon . it seems likely that partial or subliminal states of resistance can be induced in the stable host - tumour relationship which , while not obviously detectable by conventional methods , nevertheless yield significant results in the form of a perceptible increase in radiosensivity . in this connection , it is significant that gorer ( 1947 ) demonstrated high titres of circulating anti - tumour iso - antibodies in mice dying of foreign strain tumour transplants , which grew progressively in spite of antigenic differences with the hosts . 
when the previously attenuated homoplasts were transplanted from the original hosts to new hosts , however , the curative dose reverted to that of unattenuated tumour . it is considered probable that the inoculation of a viable tumour fragment , attenuated by sublethal doses of radiation prior to implantation , induces a state of subliminal resistance in the host which enhances the radiosensitivity of the resultant tumour . facilities for the maintenance of our experimental animals were generously provided at the south african institute for medical research by j . 
3. received for publication january 18 , 1949 . the investigation of carcinogenic substances which have been obtained from human tissues ( kleinenberg , neufach and shabad , 1940 ) and from other biological sources has been the subject of earlier publications from this institute ( hieger , 1940 , 1941 , 1946 , 1947 ) , reporting that : 1 . 
technique used in these experiments for testing for carcinogenic activity . unless otherwise stated , the substance is injected subeute ( 15 per cent solution in lard ) into mice of c57 strain or of mixed commercial stock . 
control experiment on reagents used in the saponification of tissue by potassium hydroxide and alcohol . since the great majority of the carcinogenic fractions had undergone saponification at least once , control experiments on the process itself acting on noncarcinogenic tissue components were made . 
distilled alcohol for 2 - 3 hours and then carcinogens might be formed during extracted several times with ether . saponification from precursors in the tissues or from the reagents themselves . in the earlier experiments the alcohol was industrial spirit ( the manufacturers state that it consists of ethyl and methyl alcohols in the ratio of 19 : 1 with 5 per cent water ) , which in separate experiments was found to darken rapidly on refluxing with koh owing to resinous condensation of aldehyde . acetaldehyde was therefore refluxed with koh and spirit under the same conditions as those of tissue saponification . 
 " n on - cancer liver " indicates the liver of a human subject who has died of any cause other than cancer . ( b ) fraction a from mixed cancer livers , partly the same as those used for the preparation in ( a ) , was crystallized from 80 per cent aqueous alcohol after adsorption from petrol ether on to a large column of a1203 eluted with petrol ether - benzene - meoh ( 80 - 20 - 20 )  . 
the possible reasons for the absence of tumours are discussed below . ( d ) a similar fractionation was made on another batch of cancer livers , and mice were injected with material from g , c , f , d , e and h . 
the test on f was repeated on a further 10 mice in which one tumour arose after 2 years . ( f ) the 10 fraction from human non - cancer lung - kidney - muscle ( fraction a ) had given 3 tumours in 25 mice , and was considered a potent it had been in the cold room for over carcinogen - containing fraction . 3 years and was then fractionated according to a simplified scheme ( flow sheet 2 )  . of 52 mice at the beginning of the tests , 43 survived 12 months and 26 , 18 months . 
four sarcomas appeared , 3 in fraction g and 1 in c . ( g ) the crude unsaponifiable fraction ( m ) of mixed livers which had been used in experiment ( d ) was fractionated and tested at j , k , l , g , a , c , f , m , i , using in all 110 mice , which lived well ; 92 survived 12 months and 40 , 18 months . 
a large batch of the crude unsaponifiable matter was worked up to stage a ; a part was kept for injection , and another part was acetylated and crystallized by careful seeding . 
the crystalline acetate was hydrolysed and the regenerated cholesterol tested . in the 12th month one sarcoma appeared in the a series , and in the 19th month one sarcoma developed in the cholesterol group . 20 mice were employed in each test . ( i ) cholesterol - rich fraction from cream ( cow 's milk )  . - kennaway ( unpublished communication ) had found that the unsaponifiable part of cow 's milk produced a sarcoma by injection into c3h mice . 
the experimental mice lived well ; after 29 months a mouse of the f series ( flow sheet i ) developed a sarcoma at the site of injection . ( k ) liver from a lymphosarcomatous human subject gave a highly carcinogenic unsaponifiable fraction in some of the earlier experiments ( hieger , 1940 ) , and the tissues of humans with this type of cancer may possibly be rich sources of carcinogen three livers from lymphosarcoma cases were saponified , fractionated into stages a , j , m , l , and injected into 6 , 5 , 11 and 6 mice of crossed c3h x c57 strain , which survived well , but no tumours developed . the 85 per cent cholesterol fraction ( a ) of cancer livers which had given 2 sarcomas in 10 stock mice was stored in the cold room for 2years , then tested on 10 c57 mice . 
one sarcoma appeared in the first series in the 14th month in a c57 female , and one sarcoma of fibrous type in the second in the 22nd month in a stock female ( experiment 7 , table i )  . when over 300 control tests had been carried out on lard alone without the production of a single tumour , a sarcoma arose at the site of injection in a c57 mouse first injected with lard when already 16 months old . 
fatty extract of walker rat tunmour . aptekman , king and lewis ( 1943 ) induced sarcoma in rats by injection of their results were confirmed here by the fatty fraction of walker rat tumours . tests where the tumours were dried by storing in distilled spirit and extracting with benzene . 
a after parallel group of 10 rats were injected subcutaneously with lard alone . two years one rat in each series developed a fibro - sarcoma , which grew with difficulty on transplantation . 
both tumours arose in the subcutaneous tissue at the same level as the injection site , but on the opposite side this effect has been observed several times in experiments where of the body . the injected substance is very fluid , and is probably pressed round the subcutaneous spaces by the movements of the animal 9 . 
10 stock in the 14th month in group b 2 mice developed sarcomas , and pre - neoplastic changes were seen at the site of injection in 4 other mice . thus in group b , 6 of 10 mice had either developed sarcoma or might have done so if they had lived longer . 
the two sarcomatous mice and two of the incipiently sarcomatous mice were from the same box of 5 mice . one cannot say why only group b responded so readily , but ( 1 ) in a fair number of experiments the saponification of tissue has been done in spirit but the products have produced no tumours , showing that spirit is not essential for carcinogenesis by the unsaponifiable product ; ( 2 ) the absorption spectra of the two final products ( cholesterol saponified in spirit and in alcohol ) are closely similar ; and ( 3 ) while the substance was strongly carcinogenic in group b ( stock mice ) , the same preparation was inactive in group a ( c57 )  . consequently it must be supposed that the b group of mice were either ( 1 ) peculiar genetically ; ( 2 ) peculiar congenitally ; ( 3 ) had become infected or ( 4 ) had become contaminated with carcinogen . ( 4 ) is , on the whole , not very probable , because the 1946 paper reported that in tests on 18 subfractions of the unsaponifiable fraction of human tissue , partly from cancerous , partly from non - cancerous 134 i . 
ieger subjects , of the total yield of 8 sarcomas , 7 were produced by fraction a made from 3 different sources and 1 from an m fraction ( flow sheet 1 )  . 
the concentration of 7 / 8ths of the sarcomas in one particular fraction strongly suggests that the fraction itself was responsible and not a chance contamination , for it would be highly improbable that contamination would occur only in the boxes , holding 5 mice each , containing those treated with the a fraction . the data in table mi are the results when a fraction was divided into 3 or more sub - fractions . 
pre - neoplastic changes at the site of injection of lipid . since the detection of only a few tumours could be important in deciding the technique of further fractionation , it was essential not to miss any tumours not visible macroscopically . 
30 non - cancer livers tested separately ( 6 in 30 were active ) tested separately ( 8 in 37 were active ) ratio : sarcoma mice total mice used . 12 / 56 = 21o 5 / 63 = 8o 44 / 120 = 370to0mean21 17 / 101 = 17o% 12 / 456 = 10 ' 440 = 2 30o thus the effect of testing a number of livers ( 67 ) separately was to reduce the sarcoma production by a factor of 9 , i.e. 
hieger ( 2 ) commercial cholesterol in lard gave sarcomas in about 5 per cent of 213 mice ( table ii )  . ( 3 ) the mixture formed when cholesterol is refluxed with koh and distilled spirit gave no tumnours when injected into 10 c57 mice , but in 10 stock mice there arose 2 sarcomas , and in 4 other mice of the same series changes were detected , post - mortem , suggestive of pre - neoplasia , at the site of injection . 
a total of 69 sarcomas has been obtained at the site of injection of lipoid substances in approximately 2000 mice of c57 and mrc strains and commercial mixed stock mice . 
kreyberg. from the institutt for generell og ek8perimentell patologi , universitetet i oslo . received for publication march 9 , 1953 . the clinical problems of breast carcinoma have been under intensive discussion for decades . 
a great deal of detailed information has been accumulated , but still widely different views are entertained as to diagnosis , treatment and theraour present knowledge was amply illustrated during the panel peutic results . discussion on treatment and results in cancer of the breast at the thirtieth annual meeting of the american radium society , chicago ( 1948 ) , and at the twenty - fifth meeting of the northern surgical association , copenhagen ( 1951 ) , to which readers may be referred . a few points seem to be universally acknowledged , as regards treatment . firstly , a careful and extensive surgical intervention will , in a number of cases , be life saving , or life prolonging . 
on the other hand , if the surgeon operates in a field where cancerous cells are present and the operation is incomplete , the surgeon may by his intervention precipitate an outburst of metastases and shorten the it may further be agreed that the development of the special life of the patient . surgical technique since the days of halstead and meyer is comparatively small , and that the advances , as manifested by a higher cure rate , are modest and mostly caused by improvements of surgery in general , for instance the introduction of penicillin and a better postoperative regime , thereby reducing the immediate it was this state of affairs that caused mcwhirter ( 1949 ) operative dangers . to state : " when radical mastectomy is the only method of treatment available and when all cases coming to a large general hospital are taken into account , the five - year survival rate is unlikely to exceed 25 per cent "  . secondly , it seems generally accepted that radiological treatment in a certain , restricted number of cases and under certain conditions , is able to effect a complete destruction of the tumour cells and thereby be life - saving . it further seems generally accepted that radiological treatment in other cases may , for a shorter or longer period , retard the development and the spread of the tumour cells , and that radiological treatment thereby may be life prolonging . kreyberg ( 1938 ) microscopically examined breast carcinomas and axillary metastases which had been submitted to pre - operative irradiation . 
the material came from three different hospitals , using different radiological techniques and different doses . the conclusions were that - : a pre - operative treatment , with the doses used , may 158 l . 
kreyberg in a series of cases damage the tumour cells to a degree , histologically visible , and in rare cases lead to complete disappearance of the tumour cells . this effect is dependent upon the dose , the effect being more pronounced after stronger doses . also the axillary metastases are damaged , but in a considerably lower degree than the primary tumour . but , even after the strongest doses ( with tele - radium ) , a great number of tumours show such small changes that we must conclude that the cells are growing during and in spite of the treatment . these observations have been generally confirmed . 
kaae ( 1952 ) stated that only 15 per cent of all breast carcinomas are really radiosensitive . baclesse ( 1949 ) had greater effects , but he used very heavy doses . 
an observation period of 5 years only is , however , too brief , since one of the radiological effects is fibrosis and scarring , after which cancer cells , retarded in growth , may regain their vitality and proliferative possibilities at a later date . haagensen ( 1949 ) stated " our reliance upon irradiation for the cure of the disease ( breast carcinoma ) has faltered until we have come to the point where we reserve irradiation for cases in which palliation is all that can be hoped for . " an attempt has been made to combine surgical and radiological treatment , in the hope of obtaining better results . 
the absolute or over - all cure rate is the number of patients alive and symptom - free out of the total number of patients received in the hospital and suffering from the disease under investigation . all dead are counted as victims of said disease , regardless of the real cause of death . 
even if this strict attitude is adopted , we meet , however , with factors which influence the statistics . firstly , the general cancer consciousness of the society , which to a certain degree will influence the relative number of early and late cases . secondly , social and racial factors , which may influence the soil of the cancer cells , and thirdly , the relative number of cases with moderate and with high malignancy , as shown by bloom ( 1950 )  . kreyberg ( 1952 ) in a survey of lung cancer in norway attempted to show that the difference in the sex distribution of this form of cancer in norway , as compared to england and wales , may be explained by the difference in the relative number of the different histological types . 
a similar situation may hold it is probable , therefore , that besides a plea good for breast carcinoma as well . for the use of absolute cure rates and tumour stages already in use , also a correction for tumour types and grades should be attempted . nielsen ( 1951 ) gave as reasonable the following figures as the average chances for a breast carcinoma patient in scandinavia : 5 - year survival rate , symptom free ( operable cases ) , 35 - 50 per cent . 5 - year survival rate , symptom free ( absolute ) , 20 - 30 per cent . these figures are valid for a population with a generally fair cancer consciousness and with a rather high standard of the doctors and the hospitals . individual surgeons and special clinics may present better results , but such material is more or less selected . the cause of the depressingly low cure rate in breast carcinoma is two - fold : ( 1 ) the tumour cells have spread beyond surgical control before the operation is performed and even before the diagnosis is made , and / or ( 2 ) the tumour cells are not sufficiently radiosensitive . this state of affairs has naturally led to a quest for other means of improving the therapeutic results , and as one of those , the importance of " early diagnosis " has been universally stressed and accepted . it may be useful therefore to examine the meaning of the designation " early diagnosis "  . 
in the present paper it is intended to examine different definitions of the term " early diagnosis " commonly used in order to ascertain our position today as regards the criteria for the immediate recognition of a breast carcinoma in a stage where a complete cure is possible , if a proper treatment actually at our disposal is used . especially will be examined the foundation of a propoganda promising great hopes of cure if the patient arrives for treatment on the basis of an " early diagnosis "  . when a breast carcinoma develops morbid changes have usually been present in the tissue for a shorter or a longer period . 
even if the essential change is considered to be a somatic mutation in a single cell , this mutation has its local further , the tissue is not clinically cancerous before the proliferation cause . has reached a certain extent . 
from a therapeutic standpoint to - day the breast carcinoma is in its early phase as long as the tumour is localized to a degree that makes a complete cure possible by such a relatively simple local operation as this is the background for the designation " stage i " , and in the mastectomy . absolute sense mastectomy should by definition in such cases result in 100 per 160 l . 
the discrepancy between the number of tumours histologically classified as stage i and the number of cures effected in this group , after a proper local operation , shows the degree of failure in the process of staging . this failure is approximately 20 - 30 per cent . in spite of this serious inaccuracy the histological staging is the method used for grouping of tumours for comparing different therapeutic interventions . the most accurate staging is the retrospective clinical , taking into consideration the final development of the cancerous process in each individual . even this staging is not perfect . it has been mentioned that patients supposed to be stage i eventually are shown to be in stageii or further . 
on the other hand , patients believed to be stagei , though actually in stage ii , may never be recognized as such if radiological treatment cures the patient . this brief survey shows that the decisive determination of the stage of a certain tumour is neither a direct nor an immediate process . 
kreyberg this table shows that even in these groups , where the time lag between diagnosis and commencement of treatment is reduced to the practical minimum , only half of the average patients can count upon a 5 years ' survival symptom free . it seems , therefore , that the definition p2 is of rather limited usefulness because a great number of the tumours are not any longer curable and the definition has not contained the criteria necessary to obtain this goal . 
a limited usefulness of p2 is demonstrated by the fact that some patients , an unknown number , have benefited from the shorter time lag - a question which will be discussed later . if we analyse the usual symptoms , some of them ( a feeling of heaviness , pains , stinging sensation and similar ) are rather vague and strictly subjective and little fitted for attempts at earlier detection . 
but among the symptoms analysed , a lump in the breast is the first in at least three - fourths of all cases , and this symptom is more material , more objective and more fitted for diagnostic research . 
here a regular and systematic palpation may facilitate the discovery of comparatively small nodules , but there evidently is a certain lower size limit . kaae ( 1948 ) voiced the traditional opinion when he stated that : another attempt at a useful definition may accordingly be : a diagnosis of a very small tumour ( p3 ) , in the hope that all such tumours actually are curable . " it is generally recognized that the size of the tumour is a very important factor in the prognosis , and that the latter is much more favourable for the small tumours than for the large . " if we , however , examine the pertinent literature , we shall discover that the students of this problem are not at all unanimous . 
the fate of the patients was followed and the long range survey , ten to twenty year 's observation , showed that nearly two - thirds of the patients had metastases when arriving for treatment . 
even the very smallest carcinomas , those the size of a pea or a bean , had a fatal outcome in four out of ten cases . these facts show that our attempt to define " early diagnosis " as a diagnosis of a very small tumour ( p3 ) , actually a tumour as small as practically diagnosable , also fails miserably . taking into consideration the three commonly used or accepted definitions of " early diagnosis " , pi fails , because the criteria are not immediately available , and p2 and p3 fail , because the criteria do not enable a patient to recognize a tumour still in a curable stage . 
nor is any other definition known , which makes such a recognition possible to - day . what p2 and p3 give are indications for obtaining the earliest possible diagnosis with our present diagnostic means . 
an examination of the actual degree of earliness shows that only half of the tumours occurring in a population can , under the best possible conditions , be diagnosed early enough to be cured with our present therapeutic means . 
the high percentage of cures is the result of a very strict selection of cases . first all cases proved to belong to stage ii , or further , are excluded . this would leave a curability of approximately 75 per cent for the remaining . next , larger tumours , and finally especially malignant - looking tumours , are excluded . 
a further improvement in the statistics may be obtained by a still greater efficiency in the process of selection , not necessarily being an expression of increased chances for the patient at large to survive . it is not correct and not fair to publish such statistics in a manner that makes 164 l . 
kreyberg the public believe that if the women examine themselves regularly and carefully , and pay a visit to a competent doctor at the first suspicion of a symptom from the breast , their chances of a complete cure is in the vicinity of 90 per cent . 
kaae ( 1948 ) arrived at the same standpoint , and wrote : " it is indicated by these investigations that a 5 - year symptom - free survival rate of about 40 per cent is obtainable in denmark to - day , and that these results may be improved from 40 to 50 per cent , if all patients see a doctor as soon as they notice the first symptom , and if the doctor makes the correct diagnosis and institutes adequate treatment without delay "  . the present study seems to show that a diagnosis as early as possible is of great importance to a certain , yet unknown , number of individual breast carcinoma patients . 
as we do not beforehand know who will benefit and who will not , a general plea for " early diagnosis " is fully substantiated , as every individual salvaged is important . 
we must , however , always have in mind that the benefit is statistically moderate , and we ought to be modest in our claims as to the possibility of materially altering the situation for the breast carcinoma patients by this approach to the problem . it has finally to be added that the present conclusion is based upon the use of the word " early " as an expression of curability seen on the background of our present diagnostic and therapeutic means . 
the earliness is , term is not used in the sense of a more or less absolute time unit . actually , here synonymous with curability to - day , and this again , in most cases , means a carcinoma in stage i . if we examine the means to diagnose a carcinoma still in stage i , we will discover that such a diagnosis can with a reasonable certainty be made only after a retrospective clinical survey , lasting some 15 to 20 years . the analysis further shows that we have no criteria enabling an immediate " eary diagnosis " , in the sense required . 
1949. although convenient and entirely suitable for the purposes for which they are employed , the u8ual methods of maintaining and utilizing transplantable tumours are not well adapted to special needs in experimental oncology , particularly when quantitative refinements and the desirability of repeating experiments with aliquot portions of the same tumour suspension are important considerations . the basis of any method which might meet such requirements must necessarily be one in which a number of separate and sin - iilar portions of a tumour can be preserved with some assurance that different portions tested at different - times will exhibit sirnilar activities . this means that the tumour tissue must be reduced to a fine state of division and homogeneous suspension and subjected to a method of preservation whereby its capacity to induce tumours remains constant for a long time . it is well known that growth may be obtained on transplantation of frozen and thawed tu ' mour tissue . although breedis and furth ( 1938 ) demonstrated the feasibility of preserving neoplastic cells in the frozen state for periods up to 400 days , this useful method does not seem to have been adopted to the extent that might have been expected . 
most of the observations have been made using the c3h sarcoma referred to by gye , begg , mann and craigie ( 1949 )  . pr " ration of tumour mmpennow . - the tumours were reduced to the necessary - state of subdivision by means of the pressure mincer described by craigie ( 1949 ) and the mince dispersed immediately in diluting fluid . 
the diluting fluids were freshly prepared from reboiled sterile distilled water and concentrated preparations of the required solutes . in experiments in which different diluting fluids were compared , a low primary dilution was first made in fluid of suitable composition and from this the secondary dilutions in the other fluids were prepared . 
were given subcutaneously in the approexcept in limited experiments , four sites in the anterior priate strain of mice . abdominal wall were used , these being approximately the centres of the upper and lower right and left quadrants . the " rtival of tumour 8uspen8iow in sau 8olulion& when a solution of sodium chloride is frozen at a temperature above 21t . ice crystalhzes out and the salt is concentrated as a strong solution ( 30 per cent ) between the cryistals . if a cer suspension in 34 volumes of 0 - 85 per cent nacl 20 ' c . 
in the strong salt solutions , in others the material was held in the frozen state for several hours at selected temperatures above and below that of the eutectic of nacl ; the results showed that although complete inactivation might require several hours , the initial rate of inactivation referable to electrolyte concentration was undesirably high . survival of tumour 8u8pen8ion8 in dextro8e solutiom . in view of the difficulties that might be expected to arise in attempts to obtain adequate preservation of activity in tumour suspensions frozen in inorganic salt solutions , the possibihty of substituting a suitable non - electrolyte was considered . of water - soluble organic materials , accordingly it was planned to test a variet but dextrose , the first of these selected for test , was found to have such a favourabl . 
effect that no attempt has yet been made to find substances more suitable for the purpose . because of the varied nature of the comparative tests which have been carried out and the introduction of a number of improvements in technique , it is not possible to present a synopsis of the results on a quantitative basis . table i provides a brief summary which , at best , merely shows that there is no contraindication to the use of - dextrose solution as a suspending fluid . 
16 - 25 m / 150cysteinehcim / 15onaohm / l5ona2hpoi , s = 0 - 85pereentn&o ; r four compamtive injections were msule in each mouse and the figure given in the cohnnn headed mouse group " ] indicates the arrangement of injections , eg . 
1 : 300 n&c1 the tumour mince was first thoroughly mixed with an equal volume of 0 - 85 per cent nacl and the mi iture was diluted i in 15 in the stated diluents . 
the first test was made after the suspensions had been kept frozen for 4 days . the lag periods observed in this test are not comparable w ' ith those in the later tests because of changes in technique of thawing , diluting and injecting , and also because of the use of a eysteine - phosphate.saline mixture in the later tests . however , it would appear legitimate to compare the activity of the material frozen for 253 days in the mixture of dextrose and 40 per cent glycerol withthe average activity of fresh c3h sarcoma suspension in ringer . 
further investigation is desirable not onlv with a view to improvements that might facifitate studies in which the tise of preserved tumour material is preferable , but also with the object of dete - riiiining the mecha i ms of surv - ival of activity . 
the varietv of pkvsical and physico - chemical factors which may he involved and the potential coniplexitv of their interplav need not be detailed , but some of the points which require investigation from the practical point of view mav be mentioned . thev are as follows : 1 . 
the development of a suitable fluid for dilution of thawed material . tumour ' suspensions which have been frozen may show a definite drop in activitv in 15 to 30 minutes after thawing . 
on the degree to which the material is diluted . it will have been noted from tables 11 and iii that cysteine phosphate buffer mixtures have been used as diluting fluids . 
the experimental evidence for the use of dilutina fluid of this composition is contradictorv and suggests a different approach to this problem . another matter which merits further investigation is the effect of dextrose on the nuclei of tumour c , - - ils as viewed with the phase contrast microscope . 
after preservation in the frozen state , sarcoma and c#rcinoma suspensions in dextrose solution show a greater and more consistent retention of activity than suspensions prepared with saline and ringer.. 
malowan ( 1932 ) - also found blood changes of the same low order , though becher and herrmann ( 1932 ) stated that they were unable to find any significant change that could be attributed to the presence of the tumour . all the above workers used danielson 's modification of folin 's well - known colorimetric method for blood amino acids . 
2. these observations show that the growth of the transplantable carcinoma ' m 2146 is accompanied by an elevation in the blood ac - amino nitrogen . while the mean value in normal mice was 4 76 mg . 
the corresponding regression equation between these variables is : oc - amino nitrogen values = 0 - 1213 tumour percentage size + 5 - 63 . these changes in the level of the blood ac - amino nitrogen probably account for the increase in the blood non - protein nitrogen in tumour - bearing animals found by other authors ( greenstein , 1947 )  . blood ac - amino nitrogen in mice with regressing tumours . in about 10 per cent of the mice used in these experiments the tumour , after having grown and reached a definite size , regressed completely . it seemed of interest to estimate the blood amino acid concentrations in these animals . 
the animals consumed this food freely for the first two days , but seemed to lose appetite during the remainder of the experimental five - day period . the loss of weight , which had become severe by the fifth day , together with the blood ac - amino nitrogen values , are shown in table iv . with the introduction of the deficient diet there was a marked loss of weight in both groups of mice ; the tumours , however , continued to increase in size even at the expense of the host , which was clearly in negative nitrogen balance . 
per cent throughout most of the experiment seems to show that this represents the basal level for this group of substances , below which further in the tumour - bearing group , on the other hand , the concentrafall is resisted . tion of the ac - amino nitrogen fell rapidly to between 4 and 5 mg . 
the similarity of the fall to that found for the control group would seem to show that whatever metabolic change was responsible for the characteristically elevated blood oc - amino nitrogen level was independent of the state of alimentation , 100 m . 
there is little evidence that the free ac - amino acids are increased in tumorous animals , but observations on rats with a transplantable hepatoma have shown that the second fraction may on average be raised seven or eight fold ( winzler and burk , 1944 )  . 
the elevation continues almost unabated when the animals are placed on a diet wholly deficient in protein . it seems , therefore , that the increase in oc - amino nitrogen must be attributable to one or other of the following factors : a local increase in the production of amino acids or of polypeptides by the tumour itself , either through the degeneration of its cells or the hydrolysis of normal body proteins brought to it by the circulation ; or to some hepatic dysfunction that is present in tumour - bearing animals which interferes with the normal disposal of these substances , and thus permits them to accumulate in the blood . this latter possibility will be considered first . fujiwara ( 1929 ) , weil ( 1935 ) , greenstein , jenrette , mider and white ( 1941 ) , and el mehairy ( 1949 ) found little inhibition of liver arginase in tumorous animals . greenstein ( 1947 ) also noted no dimunition in cystine , desulphurase activity in the same organ . arginase and cystine desulphurase are two of the main liver enzymes concerned with the intermediary metabolism of amino acids , and the study of their behaviour provides valuable information upon disturbances in the hepatic enzyme pattern generally . robertson and kahler ( 1942 ) and lan ( 1944 ) have also failed to observe any decrease in the enzymic activity of livers in animals bearing tumours . 
tumours of less than 5 per cent of the total body weight were not accompanied by any significant change in the oc - amino nitrogen concentration in the blood . the tumour tissue itself is believed to be the source of extra a - amino nitrogen in the blood . i wish to record my indebtedness to professor g . 
 lung tissue is unsuitable for direct injection of carcinogens , consequently pulmonary tumours in laboratory animals have hitherto been induced by applying the carcinogen at some remote site , by injecting it subcutaneously , intratracheally , intravascularly , intraperitoneally , or by including it in the diet . 
 recently the author ( horning , 1947 ) reported the induction of pulmonary tumours after relatively short periods of treatment by the direct application of carcinogens to adult lung tissue growing as subcutaneous homologous grafts in host animals . 
 the method of tumour induction consists in isolating small strips of lung from 6 - 12 weeks strain a mice , impregnating the grafts with crystals of a carcinogen ( 2 - acetylaminofluorene , 2 - aminofluorene , or 20 - methylcholanthrene ) prior to im planting them subcutaneously into host mice of similar age and strain in a manner which has been described elswhere ( horning , 1947 )  . 
small palpable tumours were obtained at intervals ranging from 6 - 12 weeks following grafting , but in order to examine the differences between the responses to individual carcinogens , grafts were fixed before tumours developed at intervals ranging from 1 - 5 weeks after implantation . 
 lung fragments from 6 weeks old strain a mice were grafted , in some instances without the carcinogen , into host mice of similar age , and also into mice of 15 months age which were selected from stock mice of undetermined ancestry . 
 this was done to find out if the survival of homologous grafts was dependent on employing a closely inbred strain , or whether it might be influenced by the age of the host . 
 in another series of experiments a number of lung grafts were treated with 20 - methylcholanthrene , half being implanted into the superficial fascia , and the remainder placed as usual between the skin and the abdominal wall . 
the host mice were again of the same age and strain , and it was thought that some infor mation might be obtained as to whether rapid vascularization played any role in successful survival of the grafts . 
 lung tissue was obtained from 30 strong a male mice aged 6 weeks , and implanted subcutaneously into host mice of the same strain and age , each indi vidual receiving 2 grafts on each side of its belly ( table i )  . 
when the same number of grafts from females were implanted into strong a male mice over 8 months of age , only 14 grafts had survived after 8 weeks and 4 of these contained areas of necrosis ( table i )  . 
grafting the same number of lung implants from strain a mice 6 weeks old into stock mice of indeterminate ancestry , all of which were over io months old , resulted in 58 becoming totally absorbed after the same interval of 8 weeks ' implantation ( table i )  . 
either the nuclei become pycnotic , or as in most grafts they lose their differential stain ing and the cytoplasm becomes homogenous , to be followed in later stages by fragmentation and dispersal . 
the nuclei of the collapsed lung alveoli undergo chromatolysis far more rapidly than those of the adjacent bronchiole epitheliu histological examination of these grafts suggests that their failure to survive in host tissues may be due to a partial failure of vascularization which induces necrosis . 
these results obtained with adult lung grafts indicate that successful survival and growth depends on age of both the donor and host animals , together with employment of a closely inbred strain in which homologous grafting is much better tolerated than in mixed strains . 
 in order to obtain a foreign body reaction fragments of lung from 6 - 7 weeks old donor strong a mice were treated with crystals of commercial cholesterol and subcutaneously implanted in 8 - weeks - old host mice ( table i )  . 
 in every instance in this series of experiments lung tissue was obtained from strong a mice of either sex , 7 - 8 weeks old , and impregnated with the carcinogen and implanted subcutaneously into host mice of the same strain and age ( table i )  . 
 the remaining 13 were no longer translucent , and it was found that eight of these opaque grafts had been accidentally transplanted into the superficial fascia instead of between the skin and the abdominal wall . 
 the histological characteristics of pulmonary tumours induced from sub cutaneous lung grafts treated with carcinogens differ in some respects from those of tumours arising in sitit in the intact lung of rodents under the influence of in pieces of isolated lung growing as . 
 a typical carcinoma of the lung in a graft impregnated with 20 - methylcholan threne fixed 11 weeks following subcutaneous implantation in a strong a host mouse is illustrated in fig . 
the lumen of the bronchiole is occluded by cellular debris , and the epithelium in some regions has become stratified , but in the malignant area the bronchiolar epithelium is ciliated._ the cells comprising this neoplastic focus possess all the histological characteristics of malignant cells . 
 the histological appearance of these carcinomas varies , however , according to the age of the primary gra those recovered from mice 9 - 12 weeks old consist of acini of cells with large oval nuclei . 
 all primary tumours induced by the several carcinogens were obviously bronchogenic carcinomas , with the exception of a squamous - celled growth in duced by 2aaf after 16 weeks ' implantation . 
one was a polymorphic and the other a spindle - celled sarcoma~ as both these tumours were too far advanced in their growth it was impossible to ascertain whether they were derived from the tissues of the implant or from the host , so they were discarded . 
in some which carried six or more graft1 it was frequently noticed that after 8 - 10 weeks the palpable grafts varied considerably in size , and that this variation often occurred irrespective of the particular carcinogen employed . 
those implants which failed to develop into tumours , and were hardly palpable in the living host , were found to contain large inflam matory foci or areas of foreign body reaction . 
in every instance the inflamed grafts were obtained from the same donor mouse grown in the same host and treated with the same carcinogen for a similar period of time as those which developed into tumours . 
 in the majority of grafts ( table i ) necrosis and fibrosis are absent , although these changes are so often associated with inflammation of the intact lung in situ . 
 bronchioles when present in these grafts tend to be obscured , since the lumen is filled with cellular exudate consisting mainly of lymphocytes , plasma cells , and a few macrophages or polymorphs . 
multinucleated giant cells frequently seen in tissues reacting to foreign bodies were absent in grafts treated with car cinogen , but were found in several implants impregnated with commercial choles terol . 
every lung graft was fixed in alcoholic bouin and stained by ehrlich 's haematoxylin and eos f1 . - a typical bronchogenic carcinoma , induced from a graft of adult normal lung impreg nated with crystals of 20 - methylcholanthrene , fixed 11 weeks following subcutaneous implan tation in a host ( strain a ) mouse . 
this is of special significance , as it indicates that the carcinogen has a selective action only for the bronchiolar epitheliu the adjoining alveolar cells remain quiescent to the local presence of the carcinogen . 
the lumen of the bronchiole is occluded with a cellular exudate , and in some regions the epithelium has became stratified , but in area of malignant focus the bronchiolar epithelium is ciliated . 
 since the induction of carcinoma of the prostate in mice , arising from fragments of adult glandular epithelium combined with the carcinogen growing as homologous subcutaneous grafts in host mice , was first reported ( horning , 1946 ) , this method of tumour induction has been successfully extended by other investigators to other tissues and organs . 
more recently hughes ( 1949 a , b ) , using the same technique , has succeeded in inducing transplant able carcinomas from fragments of adult bladder and uterine epithelium combined with methylcholanthrene , as well as tumours of adrenal cortex . 
 one of the several advantages arising from homologous subcutaneous grafting in the presence of a carcinogen is that the tumours growing under the skin are palpable , and can readily be obtained at the required stage of growth . 
the time taken for tumours to appear is shorter most likely because the carcinogen is more concentrated , and lies in close proximity to the bronchiolar epithelium of the gra with indirect methods of induction such as painting the carcinogen of the skin , by injecting it or including it in the diet , there may be changes in organs and tissues throughout the body , and not necessarily only in the region where a tumour arises . 
 thus dunlap and warren ( 1942 ) have reported that if swiss mice are injected subcutaneously with carcinogenic hydrocarbons , the incidence of primary pulmo nary tumours is higher in mice which have also developed tumours at the site of inoculation . 
 it must be admitted , however , that these two methods of induction differ fundamentally , because the changes which occur in the intact lung in situ following treatment at some remote site with a carcinogen are not necessarily the same as those which take place in homologous subcutaneous grafts of isolated , collapsed lung . 
these differences were discussed in a recent paper by orr ( 1947 ) , in which he rightly pointed out that it is difficult to compare results obtained by the two methods of induction . 
 recent eijeriments have shown conclusively that the survival of lung grafts either with or without the carcinogen is dependent upon the age and strain of the donor mouse , together with the age and strain of the host . 
examination of table i shows , however , that donor lung implants impregnated with a carcinogen survive more readily and in greater numbers in older hosts than would be the case if the grafts did not contain any carcinogen . 
it is of course possible that since the lung carcinomas arise from bronchiolar epithelium , some grafts may at the outset contain insufficient epithelium of this region to give a response . 
a separate series of experiments in progress show that pulmonary tumours are obtained more readily and in greater numbers from fragments of lung in which particular care has been taken to isolate portions of a bronchus , discarding the more peripheral parts of the organ . 
otherwise it is difficult to understand why prostatic epithelium should flourish as a graft despite the antagonism between graft and host tissues , and yet lung fragments implanted under the same conditions do not always survive so readily . 
pre liminary experiments reported elswhere ( horning , 1949 ) suggest that tissues which are normally under hormonal influence are much better tolerated as grafts in host mice of the same sex andstrain than tissues which are to a greater extent independent of hormone action . 
a higher incidence was not obtained in males , as seems to be the case in human lung cancer according to clemmensen and busk ( 1947 ) , and in strain a and strain c mice treated with urethane in the experiments of larson and heston ( 1945 )  . 
there is a slight difference between the potency of the three carcinogens used in the present experiments in that a greater number of tumours appeared in grafts treated with 20 - methylcholanthrene . 
 with regard to the histogenesis of the present lung tumours the method of grafting allows examination of the neoplastic changes in graded stages , leaving no doubt that the tumours arise from bronchiolar epitheliu it is curious that no response to the carcinogens is obtained from the walls of the lung alveoli . 
 an alveolar epithelium exists why should it remain entirely quiescent , whilst the closely adjacent epithelium of the bronchioles proliferates in so striking and so specific a manner ? controversy still exists concerning the nature of the lining of the lung alveoli . 
ross ( 1939 ) maintains that although an epithelial lining to the alveoli is not visible in ordinary histological preparations , it becomes appa rent in response to certain abnormal stimuli . 
grady and stewart ( 1940 ) not only affirmed the existence of an alveolar epithelium , but regarded it .as the source of lung tumours in mice treated with methylcholanthrene . 
 thus while they agreed that human tumours are bronchogenic in origin , they were convinced that mouse lung tumours arose from the alveolar epitheliu more recently herbut ( 1946 ) has reviewed the ev ' idence concerning the origin of human lung tumours , and concludes that all " alveolar cell tumours " come from the basal cells of the bronchi and bronchioles ; and that there is no justification for assuming that they arise from septal cells . 
it is unlikely that any fundamental difference exists between the rodent and human lung , and since there is such clear indication of the origin of malignant foci in early grafts combined with a carcinogen , it would appear that the case for bronchogenic origin of all lung carcinomas is strengthened . 
in the presence of chronic inflammation a proliferation of the epithelium in the respiratory bronchioles may extend to form a lining within the alveolar ducts and alveoli in much the same manner as bronchial epithelium will grow into an old abscess cavity . 
 the relationship of pulmonary cancer to inflammatory foci has been studied recently by grady and stewart ( 1940 ) , de eds and cox ( 1941 ) , nettleship , henshaw and meyer ( 1943 ) , orr ( 194 7 ) , orr and bielschowsky ( 194 7 ) , and selbie and thackray ( 1948 )  . 
the effects of urethane on the mouse lung in situ indicate according to nettleship , henshaw and meyer ( 1943 ) and orr ( 1947 ) that inflam mation and adenomas arise together . 
grady and stewart ( 1940 ) and selbie and thackray ( 1948 ) disagree , and the last authors believe that in orr 's series of 433 animals which were examined after they had died from natural causes , there was a susceptibility to terminal inflammatory involvement . 
here also the evidence obtained from grafting clearly supports the contention that there is no relationship between inflammation and lung neoplasia , since the grafts which failed to produce tumours contained large areas of inflammation and those in which tumours developed rapidly were comparatively free . 
 summary , bronchogenic carcinomas have been induced from homologous subcutaneous grafts of lung tissues growing in host mice , after impregnation with either 20methylcholanthrene , 2 - acetylaminofluorene , or 2 - aminofluorene . 
 the period of tumour induction in grafts of homologous lung is considerably reduced when compared with lung tumours which develop in situ , in rodents following treatment with the carcinogen at some remote site on the body . 
 the susceptibility to tumour formation in subcutaneous lung grafts treated with the carcinogen is found to be dependent upon the age and strain of the donor as well as that of the host mouse . 
additional experimental evidence supports the view that the pulmonary alveoli are not lined by a continuous epitheliu factors influencing the survival of lung grafts without using a carcinogen were also investigated ; these showed that the successful survival of subcutaneous grafts is dependent upon the age of both the donor and host mice , together 244 e . 
 this investigation has been supported by grants to the royal cancer hospital from the british empire cancer campaign , the jane coffin childs fund for medical research , the anna fuller fund , and the division of research grants of the u.s. 
cohen. from the experimental oncology laboratory , radiation therapy department , johannesburg general ho8pital . received for publication february 11 , 1954 . there are many reported examples of animals having been rendered immune or resistant , by various procedures , to subsequent inoculations of a tumour to which they are normally susceptible . 
with an adequately inbred strain eke the c3h mouse , however , and using homologous mammary tumour which has not lost its genetic specificity by repeated serial transplantation , absolute resistance to further implants of the same tumour cannot be evoked . it was previously shown that the radiosensitivity of a homologously - transmitted tumour is a function of the state of resistance of the host . 
when the tumours had reached the standard size of about i cin the largest diameter ( overall average volume 250 481 mm.3 ) , thev were irradiated in situ with our standardised techni.qu ' e as previously reported in detail . all mice were individually numbered and examined at regular intervals for at least 3 months after treatment . with the doses used in this series , the tumoiir has , within this observation period , either disappeared completely or bas resumed active growth , ulcerating the skin and killing the mouse by exsanguination or infection . in order to detect any acquired increase in radiosensitivity , tumours arising from each attenuation - dosage group were treated at three dose levels : 3500 , 4200 and 5000 r , making a total of six categories tested . 
1 - 27 ( 0 - 07 * )  . since this ratio exceeds unit the ld , for the attenuated homoplasts is found to be 4500 ( 200 * ) r , compared with 5700 r previously determined in the controls , and the coefficient of variation is 26 per cent compared to 9 per cent in the controls . 
the relative radiosensitivity of attenuated tumours with reference to the controls , that is the ratio of the ld50 's is four times its standard error , the result is highly significant ( p < 0 - 0001 ) , indicating a definite acquired radiosensitivity of the tumour arising from an attenuated implant . as has been previously shown , a changed radiosensitivity generally reflects an underlying change in host - resistance to the tumour . 
it would appear , therefore . , that the attenuation procedure , while not producing any absolute immunity to further implants of the same tumour , nevertheless induces within the host , albeit the strain of origin of the tumour , a subliminal resistant state . 
of the 22 " recipients " ( 2 to each donor ) , 1 1 were similarly treated in situ with 4200 r in order to detect persistent acquired radiosensitivity in the tumour transplants , and the remaining ii were treated with 7500 r in8ituto exclude the possibihty of an acquired radioresistance . 
the added disadvantage of bilateral treatments , even when the immediate mortahty could be reduced , was the possibility that the increased body dose would lower the expected percentage of cures , as previously demonstrated ( cohen and cohen , 1953b )  . 
the results of ' treatment of the attenuated homoplasts in the four experimental categories are shown in table ii , and the proportion of cures in each group plotted in fig . 
1 it can be seen that the proportion of cures to be expecied at this dosage with unattenuated homoplasts ( line a ) is exceedingly small , while in animals bearing attenuated implants ( line g ) it is 40 per cent , from which this result . 
the attenuation procedure , therefore , seems to induce a degree of resistance , presumably a similar antigenic difference between host and tumotir , at least as great as that engendered by the tumour growing in f , ilybrids harbouring the milk factor . the following facts conceming the nature of this phenomenon have been elucidated : ( 1 ) while not excluding the possibihty that the tumour cells have been inherently changed by this procedure , it is obvious that any such change is not , in itself " sufficient to affect the radiosensitivity of the transplanted attenuated conversely , there is no evidence of " takes " in the environment of a new host . acqiiired radioresistance in the tumour as a result of previous irradiation , in conformity with montgomery and warren 's findings ( 1953 )  . ( 2 ) it is unlikely that any local change in the tumour bed contributes to the changed radiosensitivity since no cures were obtained when recurrences followina the local excision at the original site were treated . it can probably be assumed , too , that damage to the tumour bed by the rsurgical excision was sufficient to abrogate the effect of systemic resistance . ( 3 ) since the increased radiosensitivity persists even where the tumour has 318 a . 
cohen been transplanted to another site in the original donor , it must be concluded that the effect is due to a systemic , generalised resistance mecbanism . the phenomenon of induced resistance to experimentalfy transmitted . 
cancer has been extensively investigated . in the past , results in this field were often contradictory and confusing ( woglom , 1929 ) , due to the use of heterogeneous strains of animals and gentically alien tumours . 
the long transplantation history of these and other commonly used tumours , maintained solely by serial transmission , is surveyed by dunham and it is generally realised that such procedures may perrnit antistewart ( 1953 )  . genic diversification or mutation in the tumour , which , in effect , results in a relatively unstable host - tumour relationship . consequently , many of these findings are largely invalidated in so far as their applicability to human cancer therapy is concemed . it would appear that only when a supply of spontaneous tumours is constantly availdble within a given strain , and is routinely transplanted through no more than a few subpassages to experimental animals of the same strain , can the hosttuniour relationship be considered adequately stabilised . 
where such stringent conditions prevail , absolute immunity to a tumour cannot be evoked ( bittner , 1936 ; barrett , 1940 ; lewis , 1940 ; fardon and prince , 1953 ; foley , 1953 )  . 
a recent review by hauschka ( 1952 ) contains an extensive analysis of the immunogenetic complexities of host - tumour relationships used in experimental cancer reseal - ch . with respect to the cm mammary carcinoma , the authors have shown that , even where some degree of immunogenetic difference existed between the tumour and the host , as in the case of f , hybrids bearing the parent strain tumour , no overt - immunity could be elicited by curing the comparatively radiosensitive tumour in8itu ( coben and coben , 1954 ) , or by previouslv inoculating the animals with radiation - attenuated fragments ( unpubhshed data )  . when one correlates these facts with the findings of the foregoing experiment , it become 's evident that resistance to tumour is by no means an " all - or - none " pbenomenon . it seems likely that partial or subliminal states of resistance can be induced in the stable host - tumour relationship which , while not obviously detectable by conventional methods , nevertheless yield significant results in the form of a perceptible increase in radiosensivity . in this connection , it is significant that gorer ( 1947 ) demonstrated high titres of circulating anti - tumour iso - antibodies in mice dying of foreign strain tumour transplants , which grew progressively in spite of antigenic differences with the hosts . 
when the previously attenuated homoplasts were transplanted from the original hosts to new hosts , however , the curative dose reverted to that of unattenuated tumour . it is considered probable that the inoculation of a viable tumour fragment , attenuated by sublethal doses of radiation prior to implantation , induces a state of subliminal resistance in the host which enhances the radiosensitivity of the resultant tumour . facilities for the maintenance of our experimental animals were generously provided at the south african institute for medical research by j . 
hewitt. from the john burford carlill laboratories , westminster hospital , london . received for publication may 26 , 1953 . from the fact that certain mouse tumours could be transplanted using freezedried tumour tissue , gye , begg , mann and craigie ( 1949 ) concluded that they had evidence of virus transmission of their tumours . subsequently , it was shown ( passey , dmochowski , lasnitzki and milard , 1950 ) that a small proportion of the cells in these tumours were able to withstand freeze - drying as indicated by the proliferation in vitro of tumour tissue so treated . these findings focused attention on the quantitative aspects of tumour transplantation , and various observations were made which suggested that the malignant cell population in sarcomacraigie , lind , hayward and begg , ( 1951 ) tous ascitic fluids is heterogeneous . reported that a small proportion of the sarcoma cells in certain ascites tumours could be distinguished from the modal cells by their peculiar appearance under phase - contrast microscopy , and by their relative resistance to freezing and to the inimical effects of exposure to isotonic glucose . lasnitzki ( 1952 ) showed that a high proportion of the cells of sarcoma 37 ( hereafter referred to as " s37 " ) ascitic fluid undergo morphological transformation in tissue - culture and do not subsequently divide ; she found that this change was accompanied by a reduction in the power of the cells to give rise to a tumour on transplantation , and considered her observations to be evidence of bimorphism of the s37 cells . histological studies of the fate of implants of tumour tissue have not given conclusive evidence that the cells of the new tumour are direct descendants of the cells in the implant . 
 ( 1949 ) stated that the appearance of implants which had been frozen previous to inoculation , although they subsequently would give rise to tumours , " did not suggest survival of any of the implanted tissue . " zahl and drasher ( 1947 ) drew similar conclusions from their observations of the fate of implants of sarcoma 180 in mice . these authors state that there is no increase in the size of the implant and that it undergoes immediate cytological degeneration . several previous workers have studied the variation of tumour incidence with the number of cells inoculated . 
de gaetani and blothner ( 1936 ) found that over 100 , 000 cells were generally required for the successful transplantation of mouse carcinoma and sarcoma . furth and kahn ( 1937 ) , working with an inbred strain of mouse and a spontaneous leukaemia derived from that strain succeeded in transplanting leukaemia to about 5 per cent of their mice by the intravenous inoculation of a single leukamic cellw kahn and furth ( 1938 ) , using the trypan 368 h . 
hewitt a reaction usually regarded as being incompatible with cell viability ( schrek , 1936b ; pappenheimer , 1917 )  . counts of suspensions prepared from solid tumours were tberefore made after mixing equal volumes of the suspension and 0 5 per cent trypan blue ( b.d.h. , biologically tested ) in m / 15 sorensen buffer at ph 7 - 2 . no ascitic cell suspension has been found to contain more than 3 per cent of stained cells , and counts of these suspensions were made in the absence of trypan blue . trypan blue was preferred to eosin for the differentiation of non - viable cells because counts were made in a fuchsit gave more definite staining at non - toxic levels . rosenthal haemocytometer using phase - contrast microscopy . the absence of any reliable morphological criterion by which to identity a viable tumour cell makes it impossible to signify with certainty whether a given cell is a malignant cell or a normal cell contributed by the cellular reaction of the host . it has been found convenient to make an arbitrary distinction between normal tissue cells and sarcoma cells in fresh single - cell preparations of the tumours used in this study , on the basis of cell diameter . it was noted that 90 per cent of the nucleated single cells in sedimented preparations of minces of normal mouse lymph gland , thymus and spleen , and 70 per cent of the cells in saline washings of normal mouse peritoneum had diameters of 9 , t or less . 
on the other hand , a relatively small proportion ( 10 - 20 per cent ) of the cells in many single cell preparations of the solid sarcomas or in some s37 ascitic fluids , had diameters of less than 9 , u . 
the density of viable cells in a suspension has therefore been taken as equivalent to the density of cells having a diameter of over 9 , t and failing to stain with after considerable experience had been gained of the measurement trypan blue . of the cells in fresh preparations , actual measurement of the cells was abandoned and the larger cells were selectively counted by estimation of their size . frequency distribution curves of the diameters of the single cells of sarcoma preparations showed a modal diameter in the region 14 - 16 , t . 
a smaller , second peak was sometimes seen at about 7 , u , and this was considered to represent the mode of the inflammnatory cell population in the suspension . the effect of elimination from the cell count of all cells of diameter less than 9 , u was to exclude from the count the great majority of the non - tumour cells . 
on the other hand , a count of tumour cells made using this criterion might represent only 80 per cent , but no less , of the actual number of tumour cells present . titration of sarcoma cell suspensions . if necessary , the counted s37 cell suspensions were diluted to contain about 20 , 000 tumour cells in 0q1 ln . 
 ( the inoculum volume )  . of this suspension , five 3 - fold dilutions were made in the gelatine - tyrode medium , giving a series of six suspensions with cell densities varying from about 20 , 000 / 0 1 ml . 
to facilitate accurate placing of the inocula , all inoculations were given under ether anaesthesia . throughout all manipulations from the time of setting up the second phase of the sedimentation procedure until inoculation , the suspensions were kept at 2 - 5 c . full aseptic technique was employed throughout and special precautions were taken to keep the suspended celis in homogeneous distribution . recording of tumour incidence and calculation of end - points . inoculated mice were examined for palpable tumours at 2 - 3 day intervals from the 6th till the 30th day after inoculation in the case of s37 and from the 6th to the 40th day after inoculation in the case of the c3h sarcoma . it was found that , with very few exceptions , the smallest detectable mass in an inoculated site would become a definite tumour within a few days of its presentation . regression occurred in a fairly high proportion of the s37 tumours derived from the smaller inocula , but was unusual except in tumours which had attained moderate size . 
the proportion of sites in which a palpable tumour formed was recorded for each of the 6 different tumour cell in an ideal titration , all sites inoculated with the highest dose doses inoculated . of cells gave tumours and all those inoculated with the lowest dose failed to give tumours . 
from these data , the number of tumour cells which would have given tumours in 50 per cent of the inoculated sites ( hereafter referred to as " td50 " ) was calculated by the method of reed and muench ( 1938 )  . 
variation in td50 values obtained in ( i ) separate titrations of different s37 cell suspensions and ( ii ) parallel titrations qf the same suspension . in 16 titrations of different s37 cell suspensions performed in the manner described and using 4 - 6 mice per group , the td50 values ranged from 603 to 3162 cells , with a mean of 1641 and a coefficient of variation of 51 per cent . 
the ratio of the highest to the lowest value obtained in this series was 5 - 2 . it has not been possible to correlate this variability with any one factor known to vary from one experiment to another . suspensions prepared from tumours during their early growth phase and those prepared from older tumours gave results which were not consistently different . it is clear , however , that the variability in results from one experiment to another is contributed to both by unspecified differences between the suspensions and by random differences in the batches of mice used for the titrations . in order to assess the order of variability in results which may be expected in parallel titrations of the same suspension , a number of paired titrations were performed using 2 series each of 6 groups of 2 - 3 mice . in these experiments the mice were of comparable age but the members of corresponding groups were not litter mates . 
the values 2 sd and 3sd corresponded to ratios respectively of 2 - 0 and 2 - 82 , so that a significant difference between the td50s obtained for parallel titrations in control and treated animals should be considered when this figure was used as a guide ; the following further their ratio exceeds 2 - 0 . test of significance being applied when the ratio of the results exceeded 2 - 0 : the td50 and its standard error were calculated from the results of each titration by the approximate method of irwin and cheeseman ( 1939 ) ; the limits of error of each td50 were defined from a value 3 x s.e. , and a significant difference was inferred when the limits of error of the td50 with the greatest s.e. 
did not include this criterion of significance was the td50 value given by the other titration . used by the authors of the method to compare ld50s in certain toxicity tests . the adequacy of the test in the present context appears to be justified by the the multiple difference of td50 values obtained in following considerations : 8 pairs of titrations using only 2 - 3 mice per group , and with only random pairing of mice between the two series , did not exceed 2 * 0 ; in the parallel titrations to the results of which the test was applied , however , there were 4 - 6 mice per group , and each mouse of one series was carefully paired with a mouse in the corresponding group of the other series , the members of a pair being usually litter mates but occasionally mice from two litters born on the same day . 
each mouse received four equal inocula in the following sites : subcutaneously in the right axilla and right groin ; intramuscularly in the medial muscles of the proximal segment of the left forelimb and in the adductor muscles of the left leg . 
the inter - actions of multiple inocula in a single mouse . no normal mouse of the age and stock used has failed to develop a palpable tumour from the inoculation of undiluted s37 mince or of single - cell suspensions nevertheless , the behaviour of s37 transplants in of over 100 , 000 viable cells . the mice used displays the immunological phenomena characteristic of most strain - heterologus transplantations : regression occurs in a high percentage of tumours , and solid immunity to a second transplant usually follows upon the regression . 
from the fact that growth of s37 in the mice used results in the induction of resistance it must be assumed that each of several simultaneous inoculations into the same animal , whether of equal or of graduated doses , may be subject to influences of a constitutional nature induced by the others . to investigate this possibility a suspension of s37 cells was titrated in two series of mice . 
each series consisted of 6 groups of 5 mice each , and corresponding groups of the two series received the same dilution of the suspension and the same cell dose per inoculueach mouse of a group in one series ( s ) received a single inoculum of the appropriate dilution subcutaneously in the right axilla . 
each mouse of a group in the other series ( m ) received 6 subcutaneous inocula , all of the same size , one of these being in the right axilla . 
when the td50s were calculated from the right axilla results alone for each series their ratio was 1 - 6 , the difference it was concluded that the practice of giving four equal being not significant inoculations per mouse , in order to increase the number of sites used in the calcula374 h . 
hewitt tions , was probably not associated with results peculiar to the multiple inoculation procedure itself provided that the several inocula in each mouse were of equal sizes . a further experiment was designed to show whether the resistance induced by a large inoculum was capable of preventing a smaller inoculum of cells , placed simultaneously elsewhere in the same animal , from becoming a palpable tumour . a number of 3 - 5 - week - old albino mice were randomised to give one series ( c ) of 6 groups of 5 mice each and a second series ( m ) of 20 mice . 
the dilutions were inoculated into the mice of the successive groups of the c series in the usual way , 4 equal inocula being given to each mouse subcutaneously in the groin and axillary sites . 
the mean latent periods of all tumours arising from inocula of 247 , 741 and 2222 cells ( i.e. , in the region of the td50 ) in the s series and in the m series were respectively 13 - 7 days and 11 - 4 days . 
the 30 per cent incidence of tumours from 82 cells in the s series is fortuitously high , as may be seen from the trend of inoidence in this series . it is clear that the relatively high total inocula per animal with which the 247 , 741 and 2222 cell inocula were associated in the m series ( see last horizontal column of table ii ) has not significantly diminished the incidence or extended the latent periods of the tumours produced by these inocula . 
the effect of " hyalase " of the tumour incidence from small inocula of s37 cells . a single - cell suspension of s37 was titrated in two parallel series of mice . 
the influence of dead s37 cells upon the fate of small inocula of viable cells . in the following two experiments the effect of dead tumour tissue upon the fate of minimal inocula s37 cells has been studied under two conditions : ( i ) where the dead tissue was inoculated intraperitoneally previous to the inoculation of viable tumour cells subcutaneously , and ( ii ) where the dead tissue was incorporated in the inocula of viable cells . a s37 single - cell suspension was titrated in a control series of mice and in a parallel series each mouse of which had been inoculated intraperitoneally with 0 5 ml . 
of 20 per cent s37 mince freshly killed by repeated freezing and thawing . the inoculation with dead tumour tissue was made 48 hours before titration of the viable cell suspension . 
no significant difference between the td50s was obtained in the two titrations , their ratio being 1 - 07 . the second experiment was designed to show whether tumour production from minimal inocula is depressed or enhanced by the presence in the inoculum of a large preponderance of dead cells . 
hewitt the td50 values obtained in the two series were as follows : dilutions in gelatine - tyrode diluent dilutions in dead cell suspension 2818 although the difference ( ratio 4.3 ) was considerable , it did not quite attain significance by the irwin and cheeseman ( 1939 ) test . 
an experiment was therefore designed to compare under more carefully controlled conditions the activities on transplantation of two suspensions prepared respectively from these two sources . malignant ascitic fluid and four moderate sized subcutaneous tumours of s37 - were harvested from a single mouse inoculated intraperitoneally and subcutaneously with s37 mince 15 days previously . 
215 it will be seen from table iv that correlation between the td50 values in the two series is highest when the cell - determining tumour incidence is taken to be the larger unstained cell . 
1 shows the relationship between viable tumour cell dose and the proportion of inoculated sites giving tumours , for the c3h sarcoma and for s37 . the case of each tumour the points shown were obtained from two separate titraaverage tumour cells per inoculum ( loglo ) fig . 
the accuracy of the points is not sufficient to reveal the true character of the curves relating cell dose and tumour incidence but it will be seen that the rate of change of tumours incidence with log . 
white and loeb ( 1910 ) have described the successful transplantation of regressing tumours , and schrek ( 1936a ) has found no effect of immunity on the growth capicity of the tumour cells . these findings do not suggest the presence of humoral factors in regressing tumours which are active against the tumour cells themselves and encourage the view that regression is the result of primary breakdown of the stroma . the significantly lower td50 values given by intramuscular sites of inoculation compared with subcutaneous sites , for s37 , might ascribed to the greater vascularity of muscle . 
on the other hand , this site difference was not demonstrable for the c3h sarcoma , and a more likely explanation of the site difference found for s37 would appear to be that local resistance mechanisms are less readily manifested in the intramuscular sites . from the results of the experiments recorded in section 3 , it is clear that a small inoculum of cells is not suppressed , in the period before it forms a palpable tumour , by multiple and larger inocula of the same tumour placed simultaneously elsewhere in the same animal . this being the case , it seems probable that the failure to give rise to palpable tumours which is displayed by half the inocula of about 2000 cells of s37 , is not due to induction of systemic immunity by these 380 h . 
methods are described of preparing single - cell suspensions of mouse sarcomata from solid and ascitic tumours , of finding the viable tumour cell density in the suspensions , and of " titrating " them for tumour producing activity on inoculation . 
the implication of this finding for the problem of cell - free transfer is discussed . i wish to express my gratitude to miss catherine fysh , b.sc. , for skilled technical assistance , and to professor r . 
korteweg. victorieplein 45 , awkrdam , netherlawi8 received for publication december 8 , 1953 . round about the end of the last war the total number of deaths from cancer recorded in the mortality statistics of the central bureau of statistics for the netherlands showed a conspicuous decrease . in females the decrease amounted to 6 per cent , in males to 9 per cent approximately . in the different age - groups it was about the same . this statistical decrease was very pronounced in lung cancer in males ( table i )  . in this paper the forowing tumours are expressed in terms of headings of the detailed intemational list of causes of death : " lung cancer " : no . 
231 ( revision of ( the figures for deaths from lung cancer in females are too small for a 1948 )  . statistical analysis and will be left out of consideration in this paper )  . 
with cancer of most other organs the decrease was less than with lung cancer , and in breast cancer and in some other cancers there was no decrease at all . in norway , according to kreyberg ( quoted by doll , 1953 ) , there was a similar dip in the mortahty curve for lung cancer in the same years , whereas in england and wales this curve continued without any interruption ( don , 1953 )  . in my report for the symposium on the endemiology of lung cancer in louvain , in 1952 ( korteweg , 1953 ) , i left the question undecided , whether the statistical decrease of mortahty from lung cancer resulted from mis - diagnoses due to war circumstances - non - functioning of the x - ray apparatuses for instance - or whether indeed in these years fewer people died from this disease . according to the central bureau of statistics , during the hunger winter of 1945 1784 males and 609 females died from starvation in amsterdam , a town of about in the first weeks after the liberation the number of persons 800 , 000 inhabitants . suffering from hunger oedema amounted to many tens of thousands . 
he pointed out heading 57 of the detailed intemational list of causes of death , revision of 1938 , under which ar tumours of undetermined nature - those tumours of which it was not reported whether they were mahgnant in one of its subgroups the figure for the tumours of undeor not - are recorded . termined nature of the respiratory organs is given . it appeared that in the same years that the decrease in lung cancer was greatest , those undetermined tumours of the respiratory organs , a very small group under normal circumstances , had strikingly increased . evidently in this group many cases of primary lung cancer were hidden . 
the length of the ordinate for a given age - group , therefore , gives the percentage pertaining to that age - group of the sum of the lengths of the ordinates of all age - groups together . unbroken line : lung cancer . broken line : tumours of undeterxnined nature of the respiratory organs . dotted line : cancer all sites . the vertical dashes at the top of the graph give the averages for the periods in question , i.e. 
the increase of lung cancer mortality in the last 40 years points to an increase of the sum total of cancer promoting influences . the older people of to - day hved a great part of their lives in a period when the menace from these influences was less than it is at present . 
as contrasted with the situation with younger people , therefore , mortality from lung cancer in the higher age - groups does not correspond to the sum total of cancer - promoting influences of to - day , but to this sum in an earlier period . it can be calculated that an inclusion in the group of undetermined tumours of the respiratory organs of even a small number of metastatic cancers possessing the normal age curve would have substantially altered the age curve proper to lung cancer . 
2 gives the crude death rates in males per million living in each year for this combination of recorded and probable ( though not as such recorded ) lung cancer deaths in the netherlands . ' 14t l\ i - i - - i 1938 - i . 
korteweg from the corrected curve . ( d ) the concavity in the curve for the years 1938 to 1941 has disappeared during discussions with officials of the central bureau of statistics on the reliability of the data on which the statistics of the causes of death are based , facts appeared which threw a new hght on our problem . in the netherlands the physician has to fill in two forms for each death . 
one in small vfllages where everyof these goes to the local civil registrar , a layman . body knows everybody the secrecy of this form is not absolutely guaranteed , and this leads , in cases of death from , for instance , venereal disease or cancer , to many physicians reporting meaningless diagnoses , such as heart failure . itnder normal circumstances the official netherlands statistics of causes of death are based on the second forms , which find their way , in a sealed envelope , to the medical official of the central bureau of statistics in the hague . the diagnosis mentioned on this form is incomplete , the medical official makes inquiries of the physician responsible for it as to the complete diagnosis and eventually corrects it . 
as the secrecy of these forms is ensured , there is no reason whatsoever for reserve . in 1944 and 1945 - when there was a general railway strike and other means of transport were extremely scarce and undependable and in 1946 , there was a lack of opportunities for making inquiries about incompletely fired in - forms . this reduces the rehability of the statistics for cancer for these years . 
the statistical decrease of cancer of the cervix uteri mentioned above , as well as the greater part of the statistical decrease of deaths from lung cancer find their explanation in this fact . the situation was by far the worst in 1945 , when the war front cut the netherlands in two . 
the mortality statistics for that year are based exclusively on the diagnoses given on the forms which were sent to the local civil registrars . it is clear that this was detrimental to the netherlands statistics for 1945 , and that to search for other explanations for the big statistical decrease of deaths from lung cancer in 1945 would be a mere waste of time . the only part of the dip for which no explanation can be given is the decrease this is the only indication that of i 0 per cent of lung cancer deaths in 1947 . the low level of tobacco consumption or the severe malnutrition in the foregoing years might have somewhat influenced mortahty from lung cancer . this decrease is rather small , however , and in 1947 eving conditions in the netherlands had not yet quite retumed to normal , so that it would not be wise to attach much importance to it . the cause of the great differences in the numbers of deaths recorded from undetermined tumours of the respiratory organs between the years before and after 1941 could not be discovered . 
maybe changes in the staff or the working methods of the central bureau of statistics or changing over from the revision of 1929 to the revision of 1938 could account for it . 
the straightening of the curve for the crude deathrrates for the years 1938 to 1941 , after correctio ' n for the undetermined tumours has been made , seems an argument in favour of the supposition that most of these tumours were in fact true lung cancers . 
the same study indicated that an excessive output of ethanolamine frequently occurred in the urine of patients with severe parenchymal liver disease , but that in such cases it was always accompanied by an excess of many other amino - acids . 
the presence in the urine of large quantities of ethanolamine , as an isolated abnormality , in the patient with primary carcinoma of the liver was therefore of some interest . its excretion might have resulted from a disorder of metabolism consequent on non - specific liver damage or from some peculiar form of neoplastic cell metabolism ; or it might even have represented a hitherto unrecognised error of metabolism which preceded and led to the development of a primary carcinoma in the liver . it has for some years been recognised , as a result mainly of animal experiment , that ethanolamine plays an important role as an intermediary in the metabolism of phospholipids and amino - acids . 
1. - some metabolic inter - relationships involving ethanolamine and other compounds . experimental dietary deficiency of choline has also been extensively studied . in rats it has been shown to lead to the production of fatty livers , followed after some time by cirrhosis . 
more recent work has shown that this may be followed eventually by the development of hepatoma - like tumours ( copeland and salmon , 1946 ; staub , viollier and werthemann , 1948 )  . furthermore , choline deficiency of a severity not sufficient to produce cirrhosis predisposes the liver to the carcinogenic action of butter yellow ( opie , 1944 ; buckley , buckley and snipes , 1951 )  . in the human hepatomata that occur in tropical countries there is ample evidence that dietary factors are concerned ( berman , 1951 )  . 
il - , a housewife , was aged 45 years at the time of her death in 1950 . apart from an attack of jaundice at the age of 7 she was in good health until april , 1939 , when , at the age of 34 , she had a febrile illness with delirium and a she had a second similar attack in october that year , and productive cough . this , like the first , lasted about 3 weeks . she suffered from similar attacks each in that year her illness was more severe and was associated winter until 1942 . with generalised aches and pains and vomiting . since that time she suffered from morning vomiting and also a burning pain , relieved by alkalis , behind the despite these regular winter illnesses and an increasing lower end of her sternum . sense of tiredness she continued with her work in a munitions factory until 1945 . from then until her death in 1950 she worked as a housewife . in january , 1947 , she first noticed a painless lump in her abdomen . in march of that year , following an illness with fever and malaise , she noticed that her abdomen was becoming tender to touch , her appetite was failing and the morning vomiting was becoming more severe . it was at this time that she was first seen and admitted for investigation at university college hospital . both parents and her 4 siblings were all alive and well . examination revealed a pale , slightly cyanosed , nonicteric woman of 8 st . 
the pulse was regular at 90 and the blood - pressure was 110 / 60 mhg . there were rhonchi over both lung fields and bilateral basal rales . in the nervous system the pupils were normal , the kneeand ankle - jerks were absent and the plantar responses were flexor . 
was normal , the circulation time was 17 seconds from arm to lung and 35 seconds from arm to tongue . the cyanosis was rapidly and completely relieved by breathing oxygen . whilst in hospital she ran a continuous low - grade fever . 
the metabolic studies carried out during this period are described later . opportunity was also taken at this time to examine chromatographically the urines of both parents and the other 4 siblings ; all showed a normal concentration and pattern of amino - acids . at the conclusion of the metabolic studies the patient went home much improved for her fortnight 's rest and largely pain - free for the first time for 2 years . she was instructed to take methionine 1 g . 
ethanolamine labelled with 50 per cent atom excess of 15n . on june 21 , 1950 , she was readmitted to hospital with an attack of bronchopneumonia . apart from her chest infection and gross oedema of the legs her general condition was unchanged . she volunteered the information that since taking choline her abdomen had felt looser and had been less painful . pneumonia was successfully treated with penicillin , and whilst on this her fever was . 
cameron reported on the histological sections of the liver as follows : " the tumour is undoubtedly a hepatoma ; it has not changed in character between 1947 , when the biopsy was taken , and 1950 . the most notable 170 c . 
each 24 - hour urine was analysed chromatographically for amino - acids and chemically for in addition blood specimens were drawn soon after the last dose ethanolamine . of each supplement had been taken and were analysed for amino - acids . further 24 - hour urine specimens were collected from her as an outpatient after she had been for 4 weeks on 10 g . 
7. apart from the very strong spot given by ethanolamine the remaining spots are of similar strength and pattern to those found in many normal urines . there was , however , an increase to a slightly abnormal level of the strength of the cystine spot during the patient 's last few months of life , and the unidentified weak " under proline " is not commonly found in urine whether normal or pathological . 
the only clearly identified gross abnormality was , nevertheless , the increased excretion of ethanolamine . these results clearly suggested that the ethanolamine was not the result of variable extrinsic factors ( such as diet ) , but was more likely to be due to a continuing metabolic abnormality . the plasma chromatograms were quite normal , except for a faint trace of ethanolamine . this indicated a definite , if slight , increase in the ethanolamine concentration of the plasma since the quantity ( 625 ' , d . ) of desalted plasma ultrafiltrate analysed was not sufficient to reveal the ethanolamine present in the plasma of a normal person . 
the urine to be analysed was placed at the right - hand bottom comer of the paper . phenol was run as the first solvent in the direction from right to left , followed by collidine - lutidine in an upward direction as the second solvent . 
8. - section of rat hepatoma kindly provided by copeland . hepatocellular type of tumour produced by prolonged feeding on a choline deficient diet ; ( no carcinogens added )  . note the difference from the histology shown in the illustrations of the tumour of c . 
9. it is seen that the output of " apparent ethanolamine " increased most on the day after the ethanolamine had been given and was probably still increased on the second day . 
the " increase " after serine was almost certainly due entirely to serine and not to additional ethanolamine , since the chromatograms showed a greatly increased serine output on this day and serine also gives rise , in the assay method , to its equivalent of " apparent ethanolamine " ( see above )  . 
walshe it has been involved were very small and the " spots " only just detectable . shown that many of the known amino - acidurias result mainly , if not entirely , from a raised renal clearance ( low threshold ) for amino - acids ( dent , 1951b )  . is not necessary to invoke this mechanism as a cause for the ethanolamine excretion here , since the normal subject who took ethanolamine by mouth produced a comparable urinary output at an induced blood level roughly similar to that of the patient . 
we suggest that it can best be understood in terms of an increased membrane permeability , on the part of the tumour cells , for ethanolamine . if such abnormnal permeability existed , a larger percentage of the loading dose of ethanolamine would pass from the extra - cellular fluid to the cells of the patient than it would in the case of the normal control . 
the urine from 7 cases of extensive secondary carcinoma of the liver ( appendix b ) was also investigated with negative results . it is not clear from the evidence at present available whether there is a specific , though rare , type of hepatic neoplasm characterised by an increased urinary excretion of ethanolamine or whether any tumour , if large enough , would give rise to a similar finding . 
ch3 + ch3 ch20h ( c . ) ch2n ( ch3 ) 2 + ch3 ch20h ( d . ) ch2n ( ch3 ) 3 ch20h ( choline )  . but , as has been mentioned already , we now prefer the theory of an increased membrane permeability for ethanolamine . 
the principal objection , perhaps , to the theory of a metabolic block is that if it were appreciable im extent it should have resulted in some degree of choline deficiency as well as in the accumulation of unmetabolisable ethanolamine . in experimental animals choline deficiency leads to fatty infiltration of the liver followed by fibrosis and , if sufficiently 176 c . 
we are , therefore , unable to accept the possibility that the large urinary excretion of ethanolamine resulted from a disturbance of the known metabolism of choline or one of its important intermediate metabolites . 
we prefer to believe that it was caused by some other property of this particular type of heptoma , such as a disturbance of " cell membrane permeability . " a further problem has been posed by the complete failure to increase the excretion of ethanolamine in the experiments in which large doses of its known biochemical precursors ( glycine and serine ) were given by mouth . this fact appears to underline further the difficulties encountered in attempting to interpret the results of this type of " classical " experiment . presumably the compounds failed to reach the cellular site where they would have been converted into ethanolamine and , therefore , that our patient did not provide a suitable opportunity to study normal ethanolamine metabolism by the rather crude methods at out disposal . 
the prospect of success would have been much greater using isotopically labelled precursors , since isolation from urine of pure samples of ethanolamine for isotype analysis presents no great difficulty . in conclusion we wish to thank sir harold himsworth for permission to publish this case , professor m . 
copeland for his very great kindness in lending us his original blocks of rat hepatoma tissue ; also the following for permission to investigate the cases referrred to in the appendices : a . 
a chemical method of analysis suggested that the output of ethanolamine this was confirmed by a rough quantitative was of the order of 0 - 5 to 1 - 0 g . 
as this substance is probably not readily taken up even in normals from the extracellular fluids by the tissue cells or from the glomerular filtrate by the renal tubule cells , it was therefore readily excreted into the urine . 
the presumed source of the ethanolamine was the neoplastic liver cell , and the enormous mass of these cells ( about a quarter of her body weight ) in the patient was probably sufficient to account for a greatly excessive production of this compound . 
he had a cholecystectomy for gall in 1950 he had an attack of renal colic and a ureteric calculus was stones in 1942 . in 1951 he was admitted to hospital with severe upper abdominal and demonstrated . laparotomy revealed a moderate enlargement of the liver , with left shoulder pain . very numerous white opaque nodules scattered throughout the liver substance . 
h - , aged 53 years . apart from a cholecysfectomy in 1950 he was in good health until shortly before admission to hospital when he was seized with a severe low back pain , greatly aggravated by movement . 
a diagnosis was made of carcinoma of the bronchus with secondaries in the lumbar spine and liver . his course was marked by very rapid enlargement of the liver and progressive cachexia . 
the medium is left unchanged throughout each experiment . films of concentrated cell suspensions made after gentle centrifugation were in the present series stained by a combined leishman - giemsa stain , and by feulgen 's method . 
about 500 - 600 leishman - giemsastained films were studied in the course of these experiments . the appearances described are those found in the basic culture medium . the addition of 5 per cent rat e.e. 
causes few differencem , apart from raising the number of mitoses . the only divergent feature is the appearance of increased numbers of degenerate polymorphonuclears , probably owing to the metabolic stimulant action of the e.e. from the cytological point of view , the life of each culture of leukaemic blood may be divided into two phases . 
the first , during which there is evidence of multiplication , and to a less extent of maturation , of the immature cells , reaches a climax during the first day , and comes to an end some time during the second 24 hours . 
from this , it does not follow that there is an increase in the total cell count of the cultures , for there is also a concomitant dying - off of mature cells . if , however , the mitotic index is high enough , as in cultures containing e.e. , there may be an increase in the absolute number of immature cells . this means that proliferation outstrips maturation . mitotic figures , very rare in the peripheral blood , are regularly found in cultures from the second hour onwards . 
they occur mainly in myelocytes , but are also seen in myeloblasts . differential counts have been done as a routine , but the total numbers of mitoses are too small to permit precise conclusions on the phase distribution ; further , owing to the technique necessary in making smears , the recognition of telophases is often difficult , and their number probably greater than is apparent in counts . no gross deviation from the ordinary phase distribution has , however , been found . in particular , there is no evidence of a metaphase arrest in untreated cultures . 
the mitotic index reaches a maximum of four to six mitoses per 1000 immature cells at about the eighth hour of culture in basic medium , or of up to 19 per 1000 immature cells at about 24 hours , if e.e. 
an extensive literature deals with tissue culture of normal and leukaemic blood and bone marrow , and has been reviewed by bloom ( 1938 ) , and more recently by fieschi and astaldi ( 1946 )  . it is not proposed to discuss this volume of work in detail , especially in view of the fact that much of it is not strictly comparable , as the methods of culturing and of examining the cells were often different from those employed in this series of experiments . thus most workers have used the cultivation of solid fragments rather than that of cell suspensions , and have described the appearances in sections instead of smears . 
comment will therefore be restricted to a few papers in which results have been obtained by related methods . there is general agreement that survival of human leukaemic cells is possible in cultures , and though the maximum times given vary considerably , no claims of indefinitely prolonged life in vitro have been advanced . 
many authors report the development of non - specific connective - tissue cell strains such as macrophages and fibroblasts from the highly specialized blood cells , but this appears to depend on the presence of a solid supporting framework , and does not occur in cultures made in a fluid medium . mitotic division of immature cells has been frequently observed and generally found normal . it is of interest to note that whereas mitoses are extremely rare in the circulating blood , they begin to appear almost as soon as blood is withdrawn from the body . the rapid change can scarcely be due to the effect of the culture medium alone , but may well be caused by the removal of an inhibitory factor operative in the body . the occurrence of maturation is a more controversial question , and the various answers which have been given appear to be mostly based on impressions . strict proof can only be obtained if total as well as differential counts are done with adequate methods , and this is only possible in fluid cultures . israels ( 1940a , 1940b ) , working along these lines , maintains that maturation occurs normally . fieschi and astaldi , on the strength of smears made from solid cultures , admit a certain degree of maturation , but state that this is always less pronounced than the proliferative processes . 
the findings cited here and in the previous report tend to support the latter view . while the normal maturative changes have not been very striking in this series of experiments , the development of new and abnormal cell types has been it is remarkable that this has not been commented upon by other of interest . workers , except by fieschi and astaldi , who , in their fig . 
7 and 14 it can be seen that the coarsening of abnormal erythropoiesis . the chromatin pattern is accompanied by its breaking up into discrete fragit is possible that ments , the nuclear membrane remaining meanwhile intact . these appearances signify an abnormal prophase , the first stage of endomitosis . the abnormality is conceived to consist in a premature separation of the daughter chromatids , which does not lead to metaphase or to a breakdown of the nuclear membrane . 
the consequence is an increased nuclear size , which may express itself by lobe formation and can , but need not , lead eventually to a more or less regular nuclear division , unaccompanied by division of the cytoplasm . in this connection the disappearance of the nucleolus is noteworthy : it is apparently a prelude to the accumulation in the nucleus of desoxyribose - nucleic acid , which is made evident by the deeper staining in leishman - giemsa and especially in feulgen - stained films . it may be objected that small numbers of polyploid cells occur normally in tissue cultures ( macklin , 1916 ) , and that their formation can be encouraged by manipulations such as the reduction of oxygen tension ( barta , 1926 ) or variations in the temperature of incubation ( stillwell , 1944 )  . 
8 with its double layer of cytoplasm has often been described and pictured as typical of the cells of " monocytic " leukaemia ( d ' antona , 1931 ) , while the resemblance of some of the polymorphs to those seen in pelger 's anomaly has already been remarked upon . it may be concluded that immature blood cells can , in vivo , undergo changes similar to those which are here reported for in vitro work . 
the special conditions prevailing in cultures made in the fluid medium , and - by the technique described , are evidently particularly suitable for encouraging this sequence of abnormal it is probable that at least events , but do not permit full normal maturation . some of the abnormalities in cultures are explained by lack of metabolic factors ordinarily present in the body , and possible that study of the metabolic processes in cultures and of variations in the composition of the medium may elucidate if successful , such a study might lead to the nature of some of these factors . a clearer understanding of the reasons responsible for the varied behaviour in culture of different bloods . 
1. received for publication february 23 , 1948 . in a previous communication we reported that a strain of the shope rabbit papilloma had been transmitted for 10 serial passages in the domestic rabbit ( selbie and robinson , 1947 )  . 
the adaptation of this tumour virus as a transmissible infection in domestic rabbits occurred during experiments in which the simultaneous inoculation of shope papilloma virus and sheep dermatitis virus in domestic rabbits produced papillomas that proved infective to other rabbits ( selbie , 1946 )  . 
the further passage of this virus in domestic rabbits was materially helped by continuing the procedure of mixing a second virus suspension with the infective inoculum of papilloma virus , and also by treating the skin with croton oil before inoculation , but there was no evidence of an increase in the virulence of the virus suspensions during passage . in the present communication we shall show that carcinomatous transformation has occurred regularly in this transmissible papillomatosis , and in a mamner similar to that found in the non - infective papillomatosis that is produced in domestic rabbits by the inoculation of extracts of papillomas from the cotton - tail rabbit , the original host of the shope papilloma virus . 
2 , where it wir be seen that the addition to the medium of the drug in a concentration of 60 mg . / litre reduces mitosis to less than one half . 
the drug was given intraperitoneary in doses of 5 y twice daily over a 5 - day period . there was no difference between treated and untreated mice in the time of disposal . ar experimental animals have shown a temporary loss of weight after being given the drug . effed , 8 on human malignant di8ea8e . this report , which is prehminary in nature , describes the testing of the compound in 17 patients suffering from leukaemia , polycvthaemia vera , lymphadenoma , multiple myeloma and three types of carcinoma . cases were chosen because they were unfavourable or too advanced for other forms of treatment . this choice of the least favourable cases - makes it harder to compare the effects ofthe drug with effects obtained with other forms oftherapy . it is , however , of value to compare in any individual patient the effects of the drug against those of any other type of appropriate treatment given either prior or subsequent to 9500 . the drug was given intravenously in doses ranging from 0 - 09 mg . 
probably represents a working range within which toxic effects on the bone marrow are not serious . side effects were slight ; half the patients had anorexia or nausea , generary very shght . 
the dose used in this case resulted in a temporary fan in haemoglobin and a fall in the white blood count to 1000 per c.mm. , at which time she was given a simple blood transfusion . 
at the end of this remission she was suffering from lassitude and following treatment with abdominal discomfort , and a rising white blood , count . 9500 her well - being improved ; her white cell count fell and the haemoglobin , which was 86 per cent , rose slightlv . primitive cells fell from 26 per cent to 14 per cent within 4 davs . 
the siibsequent historyof this patient was complicated , 2 months - after treatment , by , ' hepatogenous jaundice with leukopenia . four months after treatment the ' haemoglobin began to fall and the white cell count commenced to rise , and sore throat appeared , primitive cells rising above the she was re - treated a month later with 9500 , no beneficial pre - treatment level . effects being observed in the week that elapsed between treatment and death . post - mortem examination showed a massive leukaemic infiltration of liver , kidneys and spleen . 
the bone - marrow from femur and sternum appeared active . multiple capillary haemorrhages were present in the myocardium and brain . is difficult to assess the effect of treatment in this case owing to the complicating factors . 
the i - emission , however , was not as satisfactory as that previously obtained with x - rays . myeloblastic leukaemia of naegeli type . - this was a terminal case which had been treated previously only with transfusions . within 7 days after treatment the haemoglobin rose and the white cell count fell from 145 , 000 to within normal limits . primitive cells fell from a pre - treatment level of 76 per cent to 37 per cent and the myeloblasts and premyelocytes disappeared completely . however , this remission , although dramatic , was short . 
an improvement in the leukaemic condition was prompt . five days after the first treatment the total white cell count was within normal limits and immature cells had been reduced from 40 per cent to ' 6 per cent of the total . 
a good remission was noted 20 davs after treatment , which compared well with her first and best remission fohowing splenic irradiation . the patient felt better within a few days . 
the remission as far as the blood count was concerned after 9500 a similar reduction in red cells and haemoglobin lasted one month . this las ' ted 4 months , after which the symptoms recurred . 
none ( transfusion ) ( bleeding from tumour ) ca8e ix . generalized lymphadenoma ( section proved )  . - this patient was nevertheless the enlarged nodes decreased given a rather low dose of 9500 . very temporarily and the skin itching from which he suffered disappeared for - about a week . 
a drill - biopsy of the mass did not yield conclusive proof of lymphadenoma , which was nevertheless following treatment the itching was reduced and probable on clinical grounds . the skin , which was dry and iethyotic , became more normal in texture . 
the relief of symptoms this patient showed an interesting initial rise in the lasted less than 3 months . polymorphonuclear count at 4 days , prior to the onset of leukopenia . 
on both these occasions improvement in well - being occurred and some reduction took place in the size of his enlarged liver , - the improvement after the second course of nitrogen mustard being much less . on admission for treatment with 9500 , the liver was enlarged and oedema of the legs was present . 
none of them responded to the drug . case xv was later given a second course of treatment simultaneously with x - ray therapy , both agents being given as weekly treatments for growth - restraint . 
a leukopenia is generary seen , especiary at higher dose levels . this is due to a fall in both granular and lymphocyte series . the fafl in the granular cells may be preceded by a transitory rise over 3 to 4 days , not seen unless blood counts are repeated soon after treatment . 
effect of 9500 on peripheral blood count in a non - leukaemic case . no initial rise has been seen in the lymphocyte count , which reaches the lowest level 1 to 7 days after the end of treatment . complete recovery is slow and would seem to take about 2 months . 
for these two reasons we have waited for several weeks before instituting a second course of treatment in any patient . an extended clinical trial of 9500 would , however , seem to be justified on several grounds : first because it may be found effective in diseases other than those quoted ; even among the cases described there would seem to be a place for secondly , it could be regarded as a it in the treatment of polycythaemia . pleasanter alternative to nitrogen mustard , since , unlike nitrogen mustard , 9500 does not produce gastric effects and there is no fear of local thrombosis . 
thirdly , the drug is active ifgiven by mouth ; we have so far not tried its clinical effectiveness by this method , since , animal experiments have demonstrated that by this route the haematological response was less predictable . there is one disadvantage to an easily given drug of such potency . it is possible that it might be used without the constant supervision and check of blood counts which is necessary . 
as with all cytotoxic agents , its use should be contemplated only by those with facilities for adequate laboratory examinations . the tissue culture work has been carried out by m . 
the purely acinar foci found in rlllb females were shown to ' persist in the absence of milk factor in this cross - suckled substrain and in the absence of ovarian hormone . 
they were referred to as benign adenomas rather than nodular hyperplasias on account of their eventual independence of ovarian in addition some small invasive tumours were found by microscopic hormone . examination that appeared to represent intermediate stages in progression from benign to malignant growth ( pulhnger , 1952 )  . afitoses were found in both types of growth with the exception of the keratinised foci . all the morphological varieties persisted after ovariectomy in old animals . ' in order to ' test the capacity of the benign growths for progression to malignancy , transplantation of as many as could be found in the fresh state was attempted . grafts were transferred to young unmated females and to others of the same strain breeding rapidly . 
the nipple area on cork was searched under a glass cover using a dissecting microscope magnifying 7 and 14 times and with sterile forceps for the presence of benign tumours . ( these are seldom all adjacent surfaces of dissecting visible to the naked eye unless stained . ) microscope and glass cover were thoroughly wiped with absolute alcohol . the event no trouble arose from sepsis . 
as each benign growth was found it was cut out with fine scissors and placed in a sterile embryo dish with a drop of sterile water on the inside of the cover glass to provide a moist atmosphere . 
the next nipple area was then excised and similarly grafting was done either under anaesthesia through an incision or by treated . injection with needle and trocar into subcutaneous tissue or peritoneal cavity . the inoculated hosts were unmated females about 2 months old or breeding females that had already bome one litter . all were of the same strain bred by brother and sister matings . 
they were left in the latter from 12 - 18 months , with 15 as the average . fourteen subcutaneous grafts of bem ' gn growths minced with scissors were made from 8 donors into 9 rapidly breeding females . 
we spent more than eight months working on one such incidental problem , which had to be solved before we could proceed with the main work . this effort was well rewarded , however . 
we not only improved our experimental approach , but also acquired information that will further our understanding of the intricate role played by biological factors in regulating the influence of external agencies on living cells . in view of all this , i should like to point out that our investigations of the genetic potencies of carcinogens are still in an early stage . 
when i learned , therefore , that vapours could reach the sperm of drosophila i was confident that it would be possible to produce fine aerosols of chemical solutions it was evident tbat , if we could develop an aerosol that could do the same thing . method of inducing mutations , we could work with a wide selection of chemicals that are soluble in solvents not injurious to the ' flies , and would not be limited to chemicals that can be vaporized at temperatures not lethal to the flies . after much experimentation we finally succeeded in developing an aerosol technique that is effective in inducing mutations in drosophila males . males are kept for several hours ( from 6 to 200 ) in an atmosphere containing an aerosol of the desired solution ; and after this treatment their sperm is tested for induced genetic changes ' by standard genetical methods . 
the flies are treated in a milk bottle , and the aerosol is generated intermittently , for 30 seconds every 30 minutes , by a de vilbiss nebulizer . in our work with chemicals we have concentrated on carcinogens , because there is a feeling.among geneticists that these coffipounds should also be mutagenic . 
what classifies them as carcinogens is their ability to change normal cells into cancerous cells - that is , to produce permanent changes in living cells , which this definition can also be applied are transmitted to the progeny of those cells . to mutagens , which cause permanent and heritable changes , or mutations , in cells . this series of experiments with carcinogens also included chemically related tests were made with nine polycyclic hydrocarbons ( four non - carcinogens . carcinogens and five ' non - carcinogens ) and with seven azo compounds ( three of them carcinogenic )  . 
the observations were made on induction of x - chromosome .onclusions lethals and of chromosomal aberrations coincident with lethals . reached on the basis of the experimental results are summarized in table i . from table t a definite correlation between mutagenicity and carcinogenicity is evident . 
of the seven carcinogens tested , six were found to be mutagenic ; and of the nine non - carcinogens onlv two were found to be mutagens , and one is still classified as doubtful . the evidence now available suggests that some chemicals ( dibenzanthracene , benzpyrene , and hydroxyazobenzene ) induce both gene changes - le . 
lethals and chromosomal aberrations , whereas some others ( benzanthracene , dimethylaminoazobenzene , and 2 - amino - 5 - azobenzene ) are more effective in inducing chromosomal aberrations . during our experiinentation it was observed that different males treated at the sametime quite frequently showed different results ; that is , genetic changes could be induced in some males m ' ore readily than in others . table ti gives the 116 m . 
at present the most likely solution seems to be that a gene is responsible for the observed variability of effect between different males and between different , experiments . i shall conclude this presentation by quoting freely the conclusions given in a paper presented at the meeting of the fourth intemational cancer research congress in st . 
from these results it seems reasonable to infer a common causative mechanism relating mutagenicity this inference is further strengthened by the behaviour and carcinogenicity . of all known non - chemical carcinogens , such as x - rays and related radiations . ultraviolet rays , and heat - all of which are also mutagenic . 
the most obvious and probable relation ' between mutagenicity and carcinogenicity is the one suggested by the hypothesis that cancer may originate through a gene mutation occurring in a somatic cell . 
such a cell , and the cells derived from it by division , would have their properties changed so that they would behave as cancerous . this would mean that higher organisms possess a gene - - or , more likely , a number of genes - whose mutations can initiate a cancer - type cell . it may be assumed that such mutations , like a great majority of mutations in other genes , would occur spontaneously with a very low frequency . 
the human body has a tremendously large num ' ber of cells , however , so that - it is probable that a cancertype mutation would occur a number of times among the cells of an individual . not all of these need give rise to cancer , since a large proportion of the cancertype cells might be prevented by normal cells from dividing , or might be eliminated in some other way . if gene mutation is responsible for the origin of cancer , then all mutagenic agents may be expected to increase the frequency with which such mutation occurs , and consequently to act as carcinogens . in our fight against cancer , therefore , precautions should be taken to avoid exposure to ' all mutagens chemicals as well as radiations . such precautions , however , even if rigorously enforced , would only lower the incidence of cancer ; they could not entirely prevent its occurrence . there would still be a chance left for cancer - type mutations to occur among the billion 's of cells that constitute the human body , and a mutated cell that continued to divide would give rise to cancerous growth . 
we know that mutations do occur with great regularity , caused by some force not yet explained , and that we have no means of stopping or controlling their occurconsequently , if cancer originates through a genetic change , our chances of finding ways to prevent it are very , very slight . 
the addition of methionine to the basal diet affords good protection against fatty cystine is not as livers and the health of the animals was much improved . reliable as methionine in this respect , probably on account of its toxic action ( curtis and newburgh , 1927 ) , and the growth rates of the animals in the cystinesupplemented diet show considerably more variation than those of the animals on the basal and methionine - supplemented diets . in experiments on the growth - inhibitory action of 1 : 2 : 5 : 6 - dibenzanthracene in rats maintained on a 5 per cent protein diet it was noticed that the treated animals had fatty livers . this 5 per cent protein diet ( for composition see table ii ) contained considerably less fat than the white basal diet , and the control animals maintained on it did not develop fatty livers during the course of the experiment ( up to 56 days )  . although combination of the hydrocarbon with essential sulphur - containing amino acids has been shown not to be a direct cause of its growth - inhibitory action , it was considered possible that on the very low ( 5 per cent ) protein diet such a combination might be sufficient to prevent the lipotropic action of these amino - acids , and thus result in the development of fatty livers . 
 - in the present investigation it has been found that the addition of cystine or methionine to the basal diet without incorporation of the growth inhibitory hydrocarbon brings about an increase in growth rate of the animal ; hence if the effect of this addition is also to negative the growth - inhibiting action of added hydrocarbon , the resultant growth rate should be greater than that produced by the basal diet alone , which , on white 's results , is not the case . the white basal diet is a very poor diet for maintaining the health of the animals , and a number of deaths occurred after a few weeks . 
they are of twofold character : ( 1 ) mortality statistics and post - mortem examinations ; ( 2 ) systematic bloodpressure examinations among people of various ages . relying on mortality statistics of the united states , fahr ( 1928 ) estimates essential hypertension to be the cause of death of 23 per cent of the population aged over 50 years , a figure which is held by volhard ( 1931 ) as being too small . bell and clawson ( 1928 ) , on the strength of exact anatomical examinations , came to the conclusion that in all cases of mortality about 7 per cent in the 40 to 49 agegroup and 13 per cent of people over 50 years are dying from hypertension . 
zondek the attempt to come to a definite estimation of the incidence of essential hypertension based on the numerous blood - pressure examinations among various classes of a certain population undoubtedly encounters certain difficulties ; one of the main difficulties lies , in my opinion , in the precise definition of essential not every rise in blood pressure , even a permanent one , is , of hypertension . it is rather easy to exclude course , connected with true essential hypertension . cases of secondary hypertension , such as those caused by bright 's disease , aortic valvular disease and hyperthyroidism ; moreover , from a statistical point of view , these cases do not play a major part , at least not in the age - group which is of particular interest to us . 
a person was not classified as suffering from essential hypertension unless , constantly , or at least in most of the exaniinations carried out , a blood pressure of more than 160 / 90 mhg was detected , i.e. , 165 / 90 or 160 / 95 and more . though a permanent diastolic pressure of 95 mhg may be regarded as a very reliable proof of the existence of essential hypertension , if diseases such as bright 's disease can be excluded , we based our examination nevertheless on a somewhat lower pressure , i.e. , 90 mhg ; we did so because people suffering from hypertension in the course of their illness may not infrequently display a lowering of their diastolic pressure , probably as a result of developing arteriosclerosis ( fishberg , 1939 )  . 
the decrease of diastolic pressure from 95 to 90 mhg as a minimum value for essential hypertension will , of course , increase the figure for the morbidity of hypertension ; but , apparently , a compensation has been created - at least in men - by relating the diastolic pressure to a minimal systolic 134 s . 
zondek pressure of 165 mhg . ( blood - pressure has always been taken while there is no statistical method , based on blood - pressure patient was sitting . ) examinations alone , which will include every case of essential hypertension , or will , with certainty , exclude every other type of hypertension ; but this is not of paramount importance , since in view of the high incidence of essential hypertension , small degrees of error will probably not be significant . it may reasonably be assumed that the figures contained in our statistics , and especially in that of master , marks and dack ( 1943 ) , indicate fairly accurately the incidence of essential hypertension in the various age - groups ; confirmation of this assumption may be noted by the figures contained in the mortality statistics ; nor do the anatomical examinations quoted above contradict this . what , in comparison , is the incidence of essential hypertension in cancer patients ? we shall not attach any importance to anything but a great deviation from the normal rate . 
the number of cancer patients referred to in this paper is 1490 ; a certain proportion of the cases has been observed by us ; as to the remainder , we obtained the necessary data from the cards of the cases put at our disposal by various hospitals of the country . 
the number of other types of carcinoma , such as carcinoma of the gall - bladder , pancreas , liver , kidneys and respiratory tract , was not great enough to be used for separate statistical elaboration . for the same reason , in men , only the cases of carcinoma of the digestive tract were statistically elaborated . essential hypertension proved to be a rather infrequent occurrence in all it is more or less the age - groups , but especially between the ages of 40 to 59 . same picture as that reproduced in the general statistics ( table i )  . though the number of cases of carcinoma of the respiratory tract examined by us was relatively small , we tended to find the same relationship to hypertension as in table i . 
we examined 37 cases in the age - group 40 to 59 , of whom only one showed hypertension . two factors may be held responsible for the rare occurrence of a combination of carcinoma and essential hypertension : ( 1 ) carcinoma may have a depressing influence on a previously existing hypertension ; ( 2 ) a person suffering from essential hypertension may have smaller tendency to develop carcinoma . the possibility of a depressing action of carcinoma cannot be denied . 
in this connection we may quote the investigations of rosenfeld ( 1929 ) , feldweg ( 1929 ) and fortunati ( 1936 ) , who were among the few who carried out systematic blood - pressure investigations in cases of cancer ; in the course of the illness a fall in blood pressure could be detected , though the fall , if present , was in most cases only moderate . 
the number of cases examined by fortunati ( 1936 ) was very small ; yet it occurred to him that high blood pressure is more often met with in cases of cancer of breast and female genitals than in cases of cancer of the digestive tract ; he failed , however , to take into consideration the normal incidence of hypertension ; therefore , he could not even try to answer the decisive question as to whether the frequent occurrence of hypertension in carcinoma of the breast and female genitals or its rare occurrence in carcinoma of the digestive tract are to be considered as extraordinary . 
sterile vials , each containing 0 - 7 g . of a mixture of the sodium salts of the four nucleotides of yeast ribonucleic acid preserved with 0 - 3 per cent cresol . the mice used were the f1 cross of the a and c 57 lines . these were chosen because a mice have a relatively high spontaneous lung tumour incidence ( bittner , 1939 ; heston , 1940 ) , and it has been observed that such strains respond more readily to the carcinogenic effect of urethane ( cowen , 1947 )  . 
the hybrids would also tend to have a high spontaneous incidence , since the inheritance of spontaneous lung tumours in this cross seems to be due to a single dominant gene ( bittner and little , 1937 ; bittner , 1938 )  . seventeen a x c 57 males approximately two months old were injected with the pentose nucleotides solution subcutaneously at the flanks , daily at midmorning . 
of pentose nucleotides for a week the water . mice started to show ill effects . both treatments were discontinued ( after 2 weeks of pentose nucleotides and 1 week of urethane treatment ) for two days , on resuming treatments the dosage of pentose nucleotides to rest the mice . was decreased to 0 - 3 c.c. 
the figure clearly shows that a marked inhibition of the carcinogenicity of urethane was produced by the pentose nucleotides . the average number of lung tumours per mouse caused by urethane in controls 1 and 2 separately was 46 in each case , which was reduced to 26 in the p . 
strong. from the school of medicine , yale university , u.s.a. received for publication february 2 , 1949 . the idea of somatic mutation as an explanation for the origin of cancer has had a long and interesting history . its original use as an interpretation of cancer is usually associated with the names of murray and of boveri . these men arrived at the somatic mutation conclusion from different fields , murray from experimental cancer research , and boveri from his classical observations in experimental embryology . 
at that time he stated : " the existence of such tumours , the biological characters of which are retained through long periods of propagation , shows that the cellular transformation which initiates carcinomatous growth may take place in varying degrees . 
the ratio of the concentration of the compounds in the stomach to that in the blood in this range was about 20 : 1 or greater . by selecting carcinogens that have a pk , , falhng in this range it should be possible to circumvent the mucus barrier and expose the glandular stomach to attack . 
the animals had been fasted for 48 hours preceding the operation to simultaneously with the injecavoid contamination of the secretions with food . tion of the compounds to be tested 0 - 2 mg . 
with acetone and this extract allowed to stand overnight it was then centrifuged for 20 minutes at 2000 r.p.and the superat 5 - 10 ' c . natant used for spectrographic analysis . measurements were made on a beckman model du spectrophotometer against acetone - hci standard having the same ph as the gastric extracts . standard curves were obtained which essentially followed beer 's law . 
the 2 , 5 - isomer is soluble , whereas the 2 , 7 - isomer is not ( morgan and thomason , 1926 )  . yields of 55 to 60 per cent were obtained of 2 , 7 - dinitrofluorene decomposing at 293 - 4 '  . the 2 , 7 - dinitrofluorene was reduced with tin and hydrochloric acid by the method of schulman ( 1949 )  . 
the pure compound melts at 165 ' after recrystalthis was then converted to the dihydrochloride and lization from benzene . 2 - acetylamino - 7 - aminomono - acetylated in aqueous solution ( schulman , 1949 )  . fluorene hydrochloride was then slurried in 500 ml . 
the almost clear solution was filtered hot and a cream - white product precipitated from the cooled three recrystallizations from 50 per cent acetic acid resulted in a filtrate . goulden and kon ( 1945 ) reported that 2 - hydroxy - 7compound melting at 232 '  . acetylaminofluorene melts at 232 '  . the dyes of this series were prepared by coupling the components in acid buffered , or basic aqueous solution , neutralization and filtration . 
the yields ranged from 70 to 90 per cent . products could be recrystallized from dilute cellosolve , but highest purity was obtained from aniline , or benzyl alcohol . these compounds have not been previously reported . constants and analyses are given in table il 368 f . 
compound iv seems to be secreted to a slightly lesser extent , but this may not be significant . this was entirely unexpected , because it has a pkb ( 8 - 5 ) which hes in the optimum secretion range ( ray and jung , 1951 )  . this indicates that the pkbof maximum secretion in the fluorene series is much higher than it is in the benz ' ene series . because the serosa was colored , it was thought that this dye might have been reduced in the stomach at the azo link . 
an examination of the stomach contents for aminofluorene by the method of westfall and morris ( 1947 ) by miss mary f . argus failed to show the presence of this substance , either free or coniulyated . while , successful in leading us to potential carcinogens that are secreted in large amounts by the stomach , it is obvious that some factor ( or factors ) are involved other than the pkbit is true that the stomach of the rat may be less acidic than that of the dog , but this should only reduce the total concentration and not the relative order of absorption . the use of a tricyclic molecule hke fluorene in place of benzene leads to compounds with greatly reduced solubilities in dilute acid . 
the introduction of a third ( insoluble ) phase might well cause the stomach barrier to operate to exclude compounds of lower pkb ( greater basicity )  . the demonstration that some of these compounds are secreted in rather large amounts in the stomach has encouraged us to set up long - time experiments for testing their carcinogenicity . 
 data have been published which indicate that the average latent period for all methylcholanthrene - induced fibrosarcomas ( strong , 1948 ) and the survival time ( strong , 1950 ) of mice developing such tumours are both influenced by litter seriation . 
the latent period ( time between the subcutaneous injection of the carcinogen and the initiation or the initial growth of the ensuing fibrosarcoma ) was variouslv affected in the different strains bv the litter to which the mouse in mice of some strains ( prunt and f : of c67 x brs ) the latent period belonged . 
 ( strong , 1948 ) for the appearance of fibrosarcomas decreased in the successirn litters of the same pair of mice , while in other strains susceptibility to fibro sarcomas was relatively constant . 
the sunival time ( strong , 1950 ) of mice growing a chemically - induced fibrosarcoma ( time between the initial growth of the malig nancy and the death of the individual ) is also affected by litter seriation . 
 ' \\nen a mouse developed a fibrosarcoma in less than 100 days following the subcutaneous injection of methylcholanthrene it sunived an average of 52 days with the progressive growth of the tumour ifit belonged to a first litter . 
in the succeeding litters mice of the same age and latent i : eriod survived longer and longer with growing fibrosarcomas until , if they belonged to an eighth litter , they lived on an average of 130 days . 
these genes were derived from mice of the ancestral stocks jk and n which were used in the origin of the : xho stra the first three generations ( cba... " x jk ) were used for breeding purposes only . 
at the time of the separation of prunt from pbr descent , the average latent period for the appearance of methylcholanthrene - induced fibrosarcomas of the direct in the f 20 generation of the experimental maternal ancestry was 3695 days . 
these two untreated descents , prunt and 2prunt , should possess genetic similarity , and any biological differences that they have may be due either to divergent segregation from a residuum of genetic heterozygosity ( probably very small after 17 generations of brother - to - sistier matings ) or to new biological variability which has appeared in one or both untreated deseents following their separation from a common ancestry . 
the mice were fed a standard diet of nurishmix pellets ( pratt food company ) and water ad libitu a supplement of mixed grains ( oats , wheat and sunflower seeds ) and calf meal pellets were given to the mice once a week . 
the mice were examined periodically for tumoun ; , and the latent period taken to be the time at which a firm nodule at the site of the injection of the carcinogen began to increase progressively in si mice which developed tumours at sites other than the one at which the carcinogen had been injected were tabulated separately . 
 table i gives the percentage incidence of tumours in mice which developed in less than 100 days ( column e ) , together with the data obtained on all tumours at the site of injection of the carcinogen irrespective of latent period ( column d )  . 
 data on the total number of mice injected with methylcholanthrene ( column a ) , the number of mice which died without developing any tumour ( column b ) , and the total number of mice showing tumours irrespective of latent period and site are also given ( column c )  . 
since the two separate descents disclose similar genetic origin and show similar trends by which the females are consistently more susceptible to fibrosarcomas than the males , the data for both series may be added together . 
however , the present analysis of trends of susceptibility in s1icoessi.e litters discloses differences , and therefore the two strains , the prunt and the 2prunt , should be kept distinct . 
thus 27 2 pe : r cent of the females developed fi.brosa : rcomas within less than 100 days of latent period , whereas only 92 pe : r cent of the males developed similar tumou : rs during the same period , a difference of 180 pe : r cent in tumour susceptibility  . 
the trend for the percentage incidence of tumours developing in less than 100 days among females of successive litters was found to be y = 85 + 5 lz , with a standard deviation of the slope of 0 - 741 . 
comparison of this value with a no - trend or 0 - slope value by t test reveals that the observed trend is statistically significant ( since p = < 001 )  . 
 similarly the trend for the percentage incidence of tum.ours developing in less than 100 days among males of successive litters was found to be y = 132 - 12x , with a standard deviation of the slope c , f 0968 . 
 the analysis of the data thus discloses that there is an increasing sex differential in relation to chemically induced : fi.brosarcomas in successive litters in mice of the prunt descent . 
the difference between the mice of the two descents must , therefore , be associated with a mechanism which in the 2prunt descent changes in litter seriation at least in some strains of mice . 
 the maximal sexual differential is found in mice of the second litters ( 302 per cent ) , and this sex differential fluctuates in the succeeding litters and thus shows no significant trend . 
however , the data of the 2prunt descent are complicated by a high value for females of the second litter ( 382 per cent ) and a high value for males of the sixth litter ( 143 per cent )  . 
in one strain ( the chi ) males were more susceptible to chemically induced fibrosarcomas than were the females , whereas in the 15th inbred strain ( the cul ) the females were more susceptible to tumours than were the males . 
 the mechanism involved in the development of this new sexual differential in relation to chemically - induced fibrosarcomas in mice of the prunt descent gives a slight sex difference in mice belonging to the early litters of a breeding female , and apparently gradually increases in the succeeding litters . 
h the sex pattern of an individual is the reflection of the genetic constitution of the individua1 , then it is not clear whether mice of the early litters or mice of the later litters are to be considered the pattern for the species . 
but sex physiology is also under the influence of several hormones , and the sex pattern can be influenced significantly when a hormone is administered at an early age , preferably before birth . 
 the present investigation with methylcholanthrene has disclosed that several aspects of malignancy ( the average latent period , the survival time and the per centage susceptibility of tumours in mice of latent periods of less than 100 days ) are influenced by litter seriation . 
do hormones fluctuate in the female body with advancing litter frequency , pass the placental barrier and influence the subsequent physiology of the offspring at least in relation to malignancy and perhaps to other characteristics as well ? or are we to conclude that maturation phenomena in cytoplasm ( perhaps the mito chondria or other constituents ) are responsible for this transmission from mother to offspring which apparently is not through the genes ? perhaps another biological reason may eventually be found for the data at hand . 
these mice belonged to two separate descents derived from a common genetic orig in one descent , the prunt , there was an increasing sexual differential in relation to chemically - induced fibrosarcoma.s in the succeed320 l . 
cravetz. from the lawall memorial laboratory of pharmacology and biochemi8try , philadelphia college of pharmacy and science , philadelphia , penn8ylvania . received for publication june 15 , 1951 . caspersson and his associates advanced a hypothesis on the ' role of nucleic acids in the biological synthesis of proteins ( caspersson , 1947 )  . several other results suggested the role of nucleotides in the metabolism of tumors as well , as a result of which many structural analogues of pyrimidine and purine derivatives were synthesized and tested for anti - cancer and growth - inhibiting activity ( roblin , lampen , english , cole and vaughan , 1945 ; kidder and dewey , 1949 ; kidder , dewey , parks and woodside , 1949 ; sugiura , hitchings , cavalieri and stock , 1950 ; burchenal , bendich , brown , clian , hitchings , rhoads and stock , 1949 ; skipper , bennet , edwards , bryan , hutchison , chapman and bell , 1950 ; law , 1950 ; lewis and crossley , 1950 . ) however , little is known about the effect of the naturally occurring nucleotides on tumor growth . parsons , gulland and barker ( 1946 , 1947 ) described that in c57 mice the growth of homologous methylcholanthrene sarcoma grafts was inhibited by ( yeast ) ad ' enylic acid or guanylic acid ; uridyhc acid showed a growth - promoting effect , whereas cytidylic acid had no effect . 
tumor cells are free in the ascitic ascitic fluid was harvested by fluid , usuar abdominal puncture under aseptic conditions , and the number of cells present in the ascitic fluid counted in a hemacytometer . 
cravetz the method used in this study offers the forowing advantages : ' ( a ) cancer cells of known number and equal virulence are inoculated into the control , as ( b ) upon intraperitoneal inoculation similar well as the experimental animals ; ascitic tumors are produced , while subcutaneous inoculation results in the development of solid tumors ; ( c ) in case of intraperitoneal inoculation the action . 
the error in thi 's method is that there is always some microscopic infiltration of sohd tissues in the peritoneal cavity ( klein , 1950 ; klein and klein 1951 )  . cells are , of course , not included in our cell counts . these infiltratin ' swiss mice , bred in our colony , were used for these studies . these mice were maintained o ' n rockland complete rat diet and water , ad libitum , and kept in air - conditioned quarte ' rs at 2 p c . 
the strain aspecificity of this tumor was discussed by klein ( 1950 ) and klein and klein ( 1951 )  . subcutaneous inoculations were twenty - one days after inoculation the made into the caudal third of the back . groups animals were sacrificed , and their tumors dissected and weighed . receiving intraperitoneal injections of cancer cehs were observed for mortailty animals dying were autopsied , their ascitic fluid harvested and measured , rate . and the number of cancer cehs per cubic mihimetre estabhshed as previously descr ' lbed . nucleotides were administered by daily subcutaneous injection , alternating adenosine - 3 - phosphoric acid was injected in between the right and left flank . solution in 3 - 2 per cent disodium phosphate , adenosine triphosphate and adenosine - 5 - phosphoric acid in normal saline warmed to about 37 ' c . 
mean tumor weight8 in gram8 . serim i ( 2 , 000 , 000 cells ) meem mean mean mean serim 11 ( 2 , 000 , 000 cells ) : serim ih ( 2 , 000 , 000 cells ) seriew .1 v ( 200 , 000 cells ) : adenosine - 3adenosine - 5adenosine phosphoric acid phosphoric acid triphosphate i 00 mg . 
the number of cancer cers , however , was smaher in the ascitic fluid of the nucleotide - treated mice than in the control animals . we are greatly indebted to margaret r . 
7. received for publication june 19 , 1948 . the testing of substances for anticarcinogenic properties must proceed on purely empirical lines , or be based upon a hypothetical conception of the nature of some phase of the biochemical mechanism of chemical carcinogenesis . suggestive , but not conclusive , evidence has been obtained that s - metabolism is intimately connected with a primary stage in the interaction of chemical carcinogens with cellular constituents . 
crabtree in the groups receiving either the carcinogen alone or in conjunction with a substance which proved to be innocuous , the number of mice surviving the full all mice were epilated course of the experiment was always 90 per cent or over . over the scapular region once only , two days before the experiments began . the approximate amounts of carcinogen and tested substance used in any one experiment were as follows : 0 - 1l% 3 : 4 - benzpyrene 0 2% / methylcholanthrene 0 20 / 2.0% s - compound 1 : 2 : 5 : 6 - dibenzanthracene solutnused . 
a 0 1 per cent solution has been found most convenient , since with higher concentrations less obvious effects are produced by an inhibitor , and with lower concentrations the experiments are unduly prolonged , thus favouring any possible toxic action of the substance tested . 
with the strain of mice used , the average time for tumour induction with 0.1 per cent 3 : 4 - benzpyrene applied twice weekly is 16 - 0 i 0 1 weeks , a figure obtained repeatedly in many experiments . the substances tested were chosen for no other reason than that of convenience . 
when this occurred in the early part of an experiment and clearly reflected the toxicity of the substance tested , the difficulty was easily met by using smaller doses of the toxic agent . 
spicer. influence of bal on induction time . the results for bal have been analysed in two parts . those for bal and benzpyrene , where several percentages of bal were used , can be analysed using the technique of regression ( fisher , 1947 )  . that is to say a straight line is fitted to the mean induction times by the method of least squares ; if the slope of this line is significantly different from zero , then bal can be said to exert an inhibitory effect on the benzpyrene . the slope of the line is a measure of the efficacy of the bal . 
 " a lesser mass increment " of transplanted tumours was observed when the hosts were injected with diphenylsulphoxide or diphennylsulphone ( hammett , 1930 ) , and cystine disulphoxide proved inhibitory to the growth of spontaneous mouse tumours ( staff of the lankenau hospital , 1936 )  . conversely , compounds containing s in the reduced form , r.sh ( the nature of r appears to be of secondary importance ) , were shown to stimulate cell - division in a wide variety of plant and animal tissues ( hammett , 1931 )  . arising from these results it was anticipated that the increased proliferation of the cells in mouse skin induced by treatment with an sh - compound would render the tissue more susceptible to the action of a p - thiocresol was therefore applied in conjunction with a carcinogen , carcinogen . but the opposite effect was observed , i.e. 
the sh - compound caused a pronounced retardation in the rate of tumour induction ( reimann and hall , 1936 )  . reimann and hall performed a variety such inhibition stands in sharp contrast to the entirely innocuous behaviour towards carcinogenesis of the mono - thiol compounds used in the present work . the discrepancy can hardly be attributed to variations in the activities of the sh - compounds themselves , and must be referred to differences in experimental techniques . of experiments differing only in the amounts of carcinogen and p - thiocresol administered , and the timedistribution of their application . the actual doses of carcinogen varied from experiment to experiment , e.g.. 
the control group of mice received 2 - 4 times as much 1 : 2 : 5 : 6 - dibenzanthracene over the latent period as the groups treated with p - thiocresol , and this feature alone would suffice to account for the wide variations in average induction times , and the apparent inhibitory effect . moreover , the choice of lanoline as a solvent for p - thiocresol was unfortunate , since the work of twort and twort ( 1929 ) , rosicky andhatschek ( 1943 ) , simpson , carruthers and cramer ( 1945 ) , and berenblum and schoental ( 1947 ) , have shown that lanoline greatly retards the carcinogenic action of tar , 3 : 4 - benzpyrene , and methylcholanthrene . complicating factor augments the difficulty of accepting the experiments of reimann and hall as a demonstration of the anti - carcinogenic activity of sh - compounds . of the mono - sh - compounds used in the present work , together with the fact that bal , a di - sh - compound , can cause a moderate retardation of carcinogenic action , clearly signifies that the mere presentation of any sh - containing molecules ( as implied by the philadelphian workers ) is not in itself sufficient to produce a biological effect ascribable to the sh - group . experiences of peters , stocken and thompson ( 1945 ) in the search for a suitable sh - compound , which culminated in the discovery of bal , also emphasizes this . apart from the question of variable toxicity , which rules out a substance like the lack of activity this added 288 h . 
crabtree toluene - 3 : 4 - dithiol , it is evident that the total molecule , with sh - groups appropriately disposed , is a factor of importance . the analogy with the anti - vesicant activity of bal cannot be pushed too far , since experiments in vitro clarified the nature of the reaction between bal and lewisite , whereas any possible reaction between bal and a carcinogen , modified in an unknown way , cannot be attempted at present . lacassagne , buuii - hoi and rudali ( 1945 ) have ostensibly shown that competition between a potent carcinogen and a structurally related substance with their little or no carcinogenic activity can result in delayed tumour induction . work was an outcome of the conception that " the mutation which transforms a normal cell into a malignant cell is the result of the alteration of an organic substrate by the fixation to it of some toxic molecules ( in this case polycyclic chrysene , l : 2 : 5 : 6 - dibenzfluorene , five isomeric dimethyl - benzhydrocarbons )  . " acridines and a trimethyl - benzacridine were chosen as near relations of methylresemblance to cholanthrene , and 1 : 2 : 5 : 6 - dibenzacridine for its structural though they state in general terms that inhibitions 1 : 2 : 5 : 6 - dibenzanthracene . of carcinogenic action were demonstrated , the experimental details hardly justify so wide a conclusion , since only chrvsene and 1 : 2 : 5 : 6 - dibenzfiuorene caused some slight delay in tumour emergence . 
 from the department of experimental , pathology and ganeer research , university of leeds , and department of pharmacology , guy 's hospital , medical school , university of london . 
greenblatt and kupper man ( 1947 ) , also using the rat , observed squamous metaplasia of the horn and a doubtful precancerous lesion in the cervix uteri , but no tumours , when a similar dose of methylcholanthrene was introduced . 
pan and gardner ( 1948 ) reported the development of numerous carcinomas and sarcomas in mice of several strains , when subcutaneous implants of cervical and uterine epithelium - were made together with small crystals of methylcholanthrene . 
 in the experiments described here uterine tumours were induced by the intro duction of methylcholanthrene directly into the uterine horn in two types of mice and the effects of various hormonal factors were studied . 
 the experiments were performed on two types of mice : inbred cba mice , maintained by brother - sister mating in the department of cancer research at leeds , and white mice obtained from dealers . 
the gun was intro duced through the incision , a fine silk ligature placed around it a short distance below the incision , and the carcinogen pushed out into the lumen of the horn . 
the amount of material introduced in solution in lard varied considerably , depending on the size of the uterus , but there is no doubt that the weight of methylcholanthrene introduced with method ( 2 ) was less , and sometimes considerably less , than that introduced into the uterus with method ( 1 )  . 
 ( 3 ) this method was similar to ( 1 ) and ( 2 ) , except that the upper part of the horn through which the material was introduced was removed . 
the upper part of the vagina was then exposed and dissected free from surrounding tissue and partly separated from the rectu a stout ligature was loosely placed around the vagina just above the symphysis pubis . 
the needle was introduced into the vagina and pushed up above the level of the ligature , which was then tied ( with one knot ) around the needle and held tied by an assistant . 
the ligature around the lower end of the left horn was tied , the ligature around the vagina rele : ased and removed , and the needle with syringe withdrawn . 
 the abdominal organs were carefully palpated at regular intervals to deter mine the first appearance of uterine tumours , but the mice were not killed until they appeared to be ill or until there was no doubt about the presence of an abdominal tumour . 
 of 15 immature oba mice , 12 developed tumours of the uterus , as follows : 4 had carcinomas , 6 sarcomas , and 2 both types of tumour ( table i )  . 
 the tumours presented a homogeneous translucent appearance , sometimes with haemorrhagic and necrotic areas , except in the case of the squamous cancers , table 1 . - 0oourrenoe of tumours in immature oba mice . 
 the object of the experiment was to find out whether the direct introduction of a carc~ogen into the uterine horn would be effective in inducing carcinoma of the endometrium in mice , and if so , whether the incidence of tumours could in 109 mice be influenced by hormonal factors , or by differences in technique . 
 effect of stra a higher incidence of both carcinoma and sarcoma occurred in oba than in laboratory white mice ( tables i , ii ) , in spite of the fact that all the oba mice were immature at the time of implantation . 
the difference in carcinoma incidence ( oba 40 per cent , white 149 per cent 134 ) is nearly but not quite statistically significant ( table iii ) ; that in sarcoma incidence ( oba 533 per cent , white 74 per cent 134 ) exceeds the conventional level of statistical significance . 
but when immature oba mice are compared with immature white mice ( table iii ) , then the difference in carcinoma incidence becomes significant ( oba 40 per cent , white 83 per cent 134 )  . 
above and to the right are the rectal glands and submucosa invaded by loose sarcoma tissue ; passing obliquely across the figure is the muscle of the rectal wall ; below is the main mass of sarcoma . 
 it was rather surprising that carcinoma could be induced by this means in immature mice , spayed at the time of implantation , when they weighed ll - 15 g . 
it is evident , however , that maturity at the start tended to improve the tumour yield , though it did not lower the period of induction in white mice ( table iv )  . 
 spaying had no apparent effect on the incidence of all tumours either in oba mice ( table i ) , where the numbers were very small , or in white mice ( table iv )  . 
 16 85 27 85 14 8 l 26 8 l 86 113 114 25 81 13 81 the yield of sarcomas was , however , greater in intact than in spayed white mice , though the difference was not statistically significant . 
mice which received the larger dose of carcinogen , are considered ( table v ) , it is noteworthy that the lowest incidence of tumours occurred in spayed immature mice . 
even though the numbers are small , the agreement between the three other groups in table v suggests that the lower incidence of tumours in spayed immature mice is of significance . 
it is probably comparable with the dose used by pan and gardner ( 1948 ) for the induction of tumours in subcutaneous implants ( " small crystals " )  . 
as none of these other cell types are seen in the typical rodent ulcer , the lesion must be considered as being completely undifferentiated . but such a tumour should be highly malignant , more so at any rate than the ordinary keratinizing squamous cell carcinoma . this difficulty is resolved by the more recent view , apparently a revival of krompecher 's first thoughts on the matter , which relates these tumour , not to the basal layer of the epidermis , but 214 a . 
thackray to those accessory structures of the skin derived from the surface epithelium , the haythorn ( 1931 ) for example , relates the tumours hair follicles and sweat glands . in nearly every case to existing hair follicles and matrix , regarding any connection between epidermis and tumour tissue as secondary and fortuitous . 
some workers find it difficult to reconcile an origin in the hair matrix , which is comparatively deeply situated , with the superficial situation of early tumours . but hairs have a definite life span , at the end of which they atrophy , are shed and replaced . 
the pilar type , which included about threequarters of the tumours in his series , he further subdivided into a pilar type proper , a primordial type , a cylindrical celled or " ribbon " type , and a cystic type . 
from this large number 200 cases were finally selected to form the basis of the present investigation . the selected cases were all those that fulfilled the following conditions : 1 . 
the sections available did not always include the whole lesion , but where this was so a note was made as to whether excision appeared to be complete , incomplete or doubtful . 
of the 12 cases which recurred , 8 were obviously incompletely excised , one appeared to have been completely removed from the section studied , and 3 were doubtful . of the 51 successful excisions , 42 had been removed with an adequate margin of healthy tissue , one tumour reached to the limit of excision , and 3 came so near to the edge that the outcome seemed doubtful . in the remainder the sections available did not cover the whole lesion and no opinion as to completeness of removal could be given . of the 12 failures with surgery , one was cured by further excision , and 4 more by irradiation . three of the remaining 7 died as a result of the disease , unchecked by further surgery or irradiation , 2 patients are still alive with the ulcer active , and the other 2 died of other causes with the ulcer uncured . 
of these 39 ulcers , 19 were soon cured by further treatment , leaving 20 which proved radio - resistant . it is proposed to compare the incidence of various histological features in the biopsies from the 82 lesions cured by a single course of radiotherapy with their these figures incidentally introduced incidence in the 20 radio - resistant ulcers . must not be considered as representative of the results of radiotherapy by modern methods , for it must be remembered that these cases were all followed for 10 years , the most recent treatment being therefore in 1938 . influence of size of lesion on prognosis . before considering the histological analysis of these cases it is interesting to note the influence of the size of the lesion on the prognosis . 
the greatest measurement of the ulcer in inches was noted on the cards , and these sizes were then divided into two groups , large and small , taking one inch or more as the criterion of largeness . this boundary between large and small was placed quite arbitrarily and in fact there proved to be about twice as many small lesions as large . of the surgically excised lesions , the size had been noted in 51 cases , and of these 16 were large and 35 small . 
thackray influence of type of lesion on prognosis . as has been described already , the lesions were classed on their histological appearance as being circumscribed ( group i ) , predominantly circumscribed , but with early infiltration present ( group ii ) , or infiltrative ( group iii ) in their type of growth . it was hoped that this classification might give an indication of the malignancy of the tumours without introducing controversial problems concerning their origin . it may be suggested that the infiltrative lesions are the large and advanced ones , and that any correlation we may note between type of growth and prognosis will merely be a repetition of what we saw in the preceding section . 
the whole series of surgical cases , including also those in which surgical excision was combined with radiotherapy in the first instance , was therefore grouped as to size of lesion and type of growth . 
the great majority of the lesions with no palisade formation apparent on section were of the infiltrative variety , whereas in the group with well - marked palisades 30 were circumscribed ( group i ) and 19 infiltrative ( group iii )  . 
the question of metastasis does not arise , and it is only necessary to be sure that the margin of the excised tissue is free from growth . this can be done by cutting serial sections of the excised tissue , which is well worth while if there is any doubt . if serial sections are not prepared , then the nature of the growth itself gives a fair indication , for tumours of the circumscribed type , as we have called it , are relatively compact and their extent is apparent from the surface , whereas infiltrative growths extend beyond their apparent limits . when considering the results of radiotherapy it is important to distinguish clearly between radio - sensitivity and radio - curability . 
a tumour which melts away when irradiated may often recur , whereas the tumour which can be destroyed curing the patient , may show no dramatic immediate response . in this investigation the ten - year cure is the criterion of successful treatment , so that radiocurability is all we are concerned with . it is well known that the histological grade of malignancy as applied to carcinomas of the skin , say , or breast , has some bearing on the outcome of radiotherapy - those of low - grade malignancy often being curable , those of high malignancy usually being sensitive , but recurring . in carcinomas of the breast such features as the degree of tubule formation , variability in size and staining of the nuclei and the frequency of mitoses are all significant , and are taken into account in determining the grade . 
the results of the present investigation have been largely negative , for most of the histological features of rodent ulcers considered have been shown to be without significance in assessing the probable outcome of treatment . 
thackray the presence of cysts , pigmentation , nor even the number of mitoses seem to have any bearing on the question . the only method of dividing up the tumours which seemed to bear any relation to the prognosis was that based on the habit of growth , into circumscribed or infiltrative , and the fact that an intermediate group was necessary indicates grading as practised in connection the difficulty in making this distinction . with carcinomas of the breast and elsewhere is an indication of the degree of malignancy of malignant tumours ; the term " group " has been used here rather than " grade " in view of the suspicion that many of the group i tumours are probably in actual fact benign . 
of the other histological features studied , only the extent to which growth is infiltrative seemed to have a direct bearing on the prognosis and prospect of cure by radiotherapy . my thanks are due to the surgeons of the middlesex hospital and to professor b . 
this proportion insured chrom - atograms of satisfactory color intensity . ethanol was used for the extraction because it is believed to be a solvent which causes minimum proteolysis during denaturation of protea 78 per cent , solution has been found to be optimal for solubility of the amino - acids and for the extraction of non - protein nitrogen only . a two - dimensional chromatogram on whatman no . 
new amino - acids appeared at varying intervals in each of the four tissues 'studied , and - the changes were consistent fot each tissue in ar of the rabbits studied . 
they found that muscle temperature and ph decreased with isch - aemia . proteolysis found during ischaemia may be.caused by accelerated enzyjnic degradation in a more acid medium as the optimum h of tissue cathepsins hes between 4 and 5 ( willstiitter and bamann , 1929 )  . , in any event ischaemia m , 4y alter the chemical characteristics of a tissue . 8ummaim ' the number of free amino - acids extractable from clinical cancers of similar histogenesis - varied widely , due apparently to deliberate occlusion of the arterial supply in the , course . 
bielschowsky. from the hugh adam cancer research department of the , medical school and the , new zealand branch of the british empire cancer campaign , university of otago , dunedin . received for publication december 5 , 1953 . chromophmic tumours of the pituitary are more frequent in the rat than in man . 
thus chronic thyroxine deficiency can lead to the formation of basophihc adenomata and the morphological signs of hyperoestrinism are found often in conjunction with acidophilic adenomata . when the normal level of oestrogen in the body of the female rat is raised for prolonged periods , vagina , uterus and mammary glands undergo a series of changes one of which is cystic hyperplasia of the breast . 
growth had taken place mainly in direction of the cerebellum with which the tumour was in close contact without invading uterus ( 125 mg . ) and vagina were atrophic , it . 
the kidneys had a granulated surface and the adrenals were of a slightly darker colour than normally . large fat deposits were present in subcutaneous and retroperitoneal tissues , as well as in the abdomen . 
respectively , when killed after 10 months of parabiosis . smears taken from the spayed female indicated persistent anoestrous , whereas those of the intact littermate showed the picture of continuous oestrous . 
at the post mortem the pituitary of the intact parabiont ( rat 2 ) was found to be considerably enlarged ( 139 - 3 mg )  . nodular structures protruded above the surface which showed haemorrhagic discoloration . 
the ovaries were large and cystic , of a yerowish colour and apparently free of corpora lutea . there was after removal of the capsule fluid between the capsule and the ovarian tissue . the combined weight of the ovaries was 299 mg . 
the breast glands were remarkably hyperplastic and contained large milk cysts . in sections of the pituitary normal anterior lobe tissue was not recognisable and the presence of basophihc cells could not be demonstrated by any method . the cleft separating intermediate and anterior lobes was enlarged ' and filled with 156 f . 
the great majority of the tumour cells appeared to be larger than normal chromophobes or acidophils and most of them had a very prominent golgi apparatus the negative image of which was alreadv recogni 'sable at low power . 
the frequency of mitoses varied in different parts of the tumour , but rarely more than two were seen in one field at low magnification . blood - filled sinuses and areas of haemorrhage were present in all sections and so were strands of dense connective tissue , originating from the capsule or from larger blood vessels . 
the vagina was lined by layers of stratified , squamous , keratinising epithehuthe outstanding feature of the greatly hyperplastic mammary glands were cysts filled with eosinophilic material which apparently exercised pressure on the lining epithehum , flattening it . smaller cysts were surrounded by cuboid epithelium containing secretion droplets . 
the histological examination of the breast confirmed that the changes seen at autopsy were of a similar kind in both partners as far as their secretory activity was coneemed . to recapitulate , a remarkable secretory activity was found in the breast of the ovariectomised parabiont , the uterus and vagina of which showed the typical castration atrophy . in this case the stimulus responsible for " lactation " is oestrogenic stimulation can be assumed to have come from the intact partner . excluded because of the state of pituitary , vagina and uterus of the spayed animal . at the time this observation was published we were unable to explain whv such changes in the breast were seen only occasionally in spayedanimals joined in parabiosis to an intact female . revision of the material has shown that there exists a good parahelism between the presence of a pituitary tumour in the intact partner and stimulation of the breast of the ovariectomised parabiont . 
a fortnight later vaginal smears revealed the presence of cornified unfortunately cells which remained the predominant cell type for 13 months . no smears were taken during the following 3 months , but during the week preceding death the vaginal smears were those of anoestrous . 
ar other organs appeared normal except the lungs which contained small areas of consolidation . histological investigation showed that little non - neoplastic tissue remained in the pituitary . this consisted of chromophobes and acidophils of fairly normal appearance , but the presence of basophils could not be demonstrated in the compressed rim of normal anterior lobe . 
the tumour itself consisted of atypical large acidophils which in some regions were nearly as numerous as non - granulated cells , whereas in others the latter predominated . bizarre cells with giant nuclei were a common occurrence and one or more mitoses could be found in nearly an fields . 
the glands contained follicles in various stages of development groups of pale vacuolated cells , separated by with healthy appearing ova . strands of spindle - shaped elements predominated in the stroma . in frozen section the pale cells were found to contain ample sudanophilic material . 
of the target organs of the adenohypophysis only the breast glands were found to be stimulated and of the known pituitary hormones only prolactin can cause secretion in the breasts of ovariectomised adult rats ( desclin , 1952 )  . the functional activity of the pituitary tumours of the rat is at variance with that of the acidophilic adenomata of human pathology . 
two types of acidophils can be easily distinguished in the pituitaries of several species as for instance the rabbit ( friedgood and dawson , 1938 )  . here cells with a strong affinity for carmine are found besides others which do not retain this dye . 
the former increase in numbers after coitus , towards the end of pregnancy and during the first days of lactation . in man , romeis ( 1940 ) demonstrated the presence of one type of acidophils staining red , and of a second staining yerow in azan preparations , his a and e cefls . in addition he recognised a third variety of acidophils , the q or pregnancy cers of erdheiin the rat the histological methods with which the writer is familiar unfortunately do not allow differential staining of two types of acidophils . 
why in man the growth hormone producing cells undergo neoplastic changes and in rodents the prolactin secreting acidophils , i am unable to explain . the interrelation between pituitary tumours and secretory activity of the mammary glands has been studied by lacour ( 1950 ) in the rat . in a series of 37 oestrogen induced adenomata she found in 23 granulated as well as " chromophobic " cells with a hypertrophic golgi apparatus . 
whenever the granulated elements were present , the breast had the aspect of a " lactating " gland . used romeis ' kresazan technique which stained the granulated tumour cells according to lacour a few cells , having the same tinctorial qualities orange . occur in the pituitary of the normal female rat . 
they become more numerous early in pregnancy and increase considerably in numbers 3 days ante partum . from this date onwards until weaning these ora ' ngeophil acidophils outnumber the other type . 
the close correlation between the presence of orangeophilic cells in the normal and tumourous pituitary and lactation changes in the breast suggested to lacour that these elements were the sourc ' e of prolactin . the hterature on experimental and spontaneous pituitary adenomata in rodents has been recently reviewed by horning ( 1952 ) and by gardner ( 1953 )  . in contrast to the opinion of the writer , gardner considers the oestrogen induced pituitary tumours as chromophobic adenomata , a view still shared by most admittedly , degranulated elements are more numerous in many authorities . of these tumours than granulated forms , but can - one classify all cells without granula as chromophobes ? the difficulty lies in the morphological resemblance of degranulated acidophils to chromophobes . 
the oestrogen induced pituitary growths with functional activity cannot be chromophobic tumours because the acidophils and not the chromophobes are the source of lactogenic hormone . another point of controversy is the nature of these pituitary growths . 
they are considered by some authors to be true neoplasms and conditioned growths by others , because they have been seen to regress when stimulation ceased ( nelson , 1944 )  . 
the " spontaneous " tumour described in this paper did not differ histologically or functionary from the two oestrogen mduced growths . this adenoma certainly was not dependant on oestrogen since it showed no sign of regression 51 months after ovariectomy . 
one occurred in an ovariectomised rat , the second in an intact female joined in parabiosis to a spayed partner and the third in a female treated with stilboestrol , in which , however , the histology of the vagina indicated a very low oestrogen level at the end of the experiment . the pituitary growths were found in animals having secreting mammary 160 f . 
steiner. from the university of chicago , chicago 37 , illinois , u.s.a. received for publication april 15 , 1954 . in an aae of high specialization there is a tendency to achieve understanding in oncology this by the study of progressively smaller and stif smaher units . has taken the form of going fiom caincer as a whole to its various sites and types , and from tissues to cells to intracellular components and , finaffy , to molecules . much progress has been made in this way in an understanding of the disease . this dissective analytical approach has , however , tended to divert attention from some of the larger units within individuals and in societv which are still capable of teaching much about the natural history of the disease , including its etiology . 
the endemiological approach to etiology in cancer , wbich makes use of groups of cases , appears to be neglected . the present purpose is to show by a statistical study of large bodily units , namely , organ systems , an endemiological pattem in the natural frequency of cancers that appears to have etiological impfications . 
each major body system shows a harmonious pattem in its principal tumours , different from other systems , suggesting that they have similar etiological factors . in a recent statistical study , it was found that the curves fof cancer to autopsy ratios by age were similar for all major sites in an organ system and different from those of other organ systems . 
the study was then extended to the mortality statistics of the united states , which corroborated the previous observations . it is believed that this behaviour pattern has etiological significance . necropsy data , the basic data from which the curves in fig . 
1 , and the number of cancers at each site can be found in the principal table of tlle appropriate chapters of another publication ( steiner , 1954 )  . in that material of 35 , 293 necropsies , there were 6 , 072 malignant neoplasms . 
the twenty commonest cancers comprised 91 - 3 per cent of the total . sixteen of the twenty ( omitting carcinomas of the prostate , skin , pancreas , andthe intracranial tumors because they are lone representatives of systems ) far into six organ systems . 
they form the basis for the present study . if the curves of ratios of necropsies for cancer to total necropsies in the twenty tumors are plotted together , as in fig . 
the relationships of the can ' cers appear haphazard and disorderly . however , if now the curves for each organ system are withdrawn from this melange and plotted separately , as in fig . 
an attractive order emerges from chaos . thus , the tumors of the four principal sites in the ahmentary tract which give carcinomas of the mouth , esophagus , rise to carcinomas are plotted in fig . 
2. stomach and large intestine had low mortahties early in life but they increased to a maximum in the seventh decade , after which their importance dechned in relation to other causes of death . 
the mortahty from carcinomas of the mammary glands , uterus and ovaries began in the third decade , increased quickly as cancer to necropsy ratios to a maximum in the fifth decade , and then fell . 
the peaks and general configuration of the three curves are the same , differing only in amplitude . the mortafity began soon after puberty , increased to the time of the menopause , and then fell off relative to other causes of death - more in the uterus and ovary than in breast . 
steiner peak was at least four decades later than in the feinale generative tract . mortality began in the prostate when in the female generative organs it had already reached its peak . 
the curve for the prostate shows an inverse relationship to the period of most active sexual iffe , but a direct relationship is found in the female generative system . the two principal tumours of the respira ' tory tract , free from the confusion of fig . 
except for the sman peak m renal cancers in the first decade , caused by the wilm 's tumors , the configuration of the curves is the same . there was a gradual increase with age in these cancer necropsy ratios . 
7. although mortahtv from carcinoma of the hver began earlier than from the gafl - bladder , in both it mcreased with age , and there is a similarity in the curves which is not obvious in the apparent anarchy in fig . 
the cancers of the stomach intestine ( including rectum ) , esophagus and the buccal cavity ( including the pbarynx ) had totals of 24 , 258 , 33 , 383 , 3 , 866 if cancer death to ar death ratios are made by and 5 , 138 cases respectively . decenniums or bv quinquenniums , the configurations of the curves are the same for all four - tumours and they resemble those in fig . 
cavity show a virtual plateau in the two decades 50 - 59 and 60 - 69 . in the respiratory tract , 18 , 277 cancers were reported in the ' trachea , bronchi and lung ( some unspecified as primary or secondary ) and 1 , 852 in the larynx . the cancer to death ratios show peaks in the deca - de 50 - 59 as in fig . 
4. these peaks , also , lie between those found in the alimentary tract and the female gellefative organs . similarly , the three principal cancers of the female generative organs _ ( mammary glands , uterus and ovary plus fallopian tube and broad hgament ) show curves which are similar in configuration to each other and to - those in fig . 
3 , there were reported in 1950 18 , 973 cases of with peaks in the decade 40 - 49 . cancer of the breast , 16 085 of the uterus , and 5 , 652 of the ovary , faropian tube and broad ligament combined . ln the mesoblastic series of mahgnant tumours , those of bone ( 2 , 180 cases ) showed a curve resembling that in fig . 
5 with a peak in the decade 10 - 19 , a sman depression in the decade 30 - 39 ' and a small secondary rise followed by a gradual dechne . 
the mahgnant lymphatic diseases ( code numbers 200 - 205 ; total of 16 , 756 cases ) showed a peak in the first decade forowed by a gradual decline . the ratios to all deaths were higher during the first three decades than in the los angeles material . 
the sarcomas of ar soft tissues are not combined in the u.s. vital statistics , so this category cannot be stuclied . in the ul - inary tract , kidney tuiriiors ( 3 , 643 cases ) had a maximum in the decade 50 - 59 while bladder tumors ( 6 , 401 cases ) had a peak a decade later . neither curve showed the persistent rise of fig . 
6 in the older age groups . because all tumors of the hver and biliary passages are combined in the u.s. vital statistics , comparisons cannot be made between individual sites or with fig . 
mortality figures for 1950 conform to , with minor exceptions , the same endemiological pattems as the los angeles necropsies , showing that the latter were not exceptional in this regard . it should be noted that cancer to au ' topsy ratios were used in one set of data thus , autopsies for all causes and an and cancer to death ratios in the other . deaths were used , respectively , as the points of reference . cancer to autopsy ratios may be ofhmited . 
value in expressing total quantity but they are excenent for contrasting one site with another within a material because the point of reference however , essentially the same endemiowithin each age group is the same for all . logical facts are revealed by other methods ofanalysis , altbough less clearly . thus , the age distribution figures ( steiner , 1954 ) for the cases in the los angeles necropsies show - similarities within organ systems . 
figures gave sinfflar results in so far as they were compared . thus , if the data of clemmesen and nielsen ( 1952 ) from the danish cancer registry are used , the incidence curves for cancers of the stomach , esophagus and large intestine ( who code 153.0 - 3 , 153.9 plus 154 ) are similar in configuration , as are those of the uterus and ovary but not of the mammary gland . the question arises as to wbat , if anything , these statistical findings indicate regarding etiology . are they explained by chance ? that they probably are not is shown by the absence of exception in sixteen tumor sites representing six systems in the los angeles necropsy series , a ; nd an essential concordance by most such a high degree of thirteen tumors in five systems in the u.s. 
2 to 7 that the organs in some systems develop a greater proportion of their it is not known whether this earlier cancers at an earlier age than in others . occurrence represents an earher exposure to etiological factors or a shorter tumor induction time after the beginning of the exposure . induction time isthe resultant of many factors including type , potency and amount of carcinogen , number of ca - rcinogens , conditions of exposure and susceptibifity of the cens . that differences in induction time exist in man is indicated by the exposures - to ionizing radiation in japan ( hiroshima and nagasaki ) where to date only leukemias have occurred although ionizing radiations are known to induce tumors of other types . it is also apparent from the figures that the tumor yield by sites within systems varies greatly although the induction time appears to be the same . 
tumor yield is govemed , in general , by the same factors as the induction time just mentioned . in addition the number of cells being exposed is important in deter number of tumors obtained . 
steiner organ system can , in theory , be correlated in part with the number of cells at thus , in the alimentary tract each site exposed to hypothetical carcinogens . there would appear to be , on the basis of the gross and microscopical anatomy , approximately equal numbers of epithelial cells available for neoplastic conversion in the mouth and esophagus , many more in the stomach , and still more in the this is roughly parallel to the numbers of cancers that occur large intestine . in these locations ( in 1950 5 , 138 , 3 , 866 , 24 , 258 and 33 , 383 respectively in the united states )  . similar agreements between the relative numbers of epithelial cells and tumors are found in the larynx and lung , in the female generative organs ( if one assumes that most mammary carcinomas are ductal ) , and in the mesoblastic tissues if nuclei rather than total volume are considered . 
the curves of cancer to total mortality ratios by age in thirteen common tumors found in five systems showed a harmonious behavior within each system , which differed from that of other systems . some case distribution and some incidence data show the same statistical facts although less clearly . the etiological significance of these endemiological observations is unknown . however , it is suggested that they could be interpreted as signifying common etiological factors within each system with the differences in quantity in tumor by sites accounted for in part by the unequal numbers of cell populations at risk . comments are made about the interpretation of the quantitative differences in tumors between organ systems and in their age distribution . the author is grateful to the research committee of the los angeles county hospital staff for permission to study their material and to e . 
8. - hormonally - induced hyperplasia on anterior edge of pectoral fin showing an early translucent appearance of tumour produced by my ' xomatous change in stroma . of the mucus - secreting epithelium and a myxomatous change in stroma . 
pda was all rats received greens once ' %eekly . added in a concentration of 0 - 06 per cent . the diet and water were given ad libitum . twenty of 50 rats r ' eceiving this diet served as controls , whilst the remaining 30 rats each received - a daily addition of 10 ml . 
in two of the tumours ( rat ca 801 on the pda control diet and rat cl 917 receiving fresh milk ) pancreatic tissue was recognized and the origin of the tuniours . 
he describes these extraas being similar to reticulo - sarcomas or " composed of spindleordinarv tiimours shaped cells and multi - nucleated giant cells . as another deviiation some tumour cells . 
grouped in bulbous masses , show an evident tendenev to concentiric arrangement of cells which are more or less keratinized towards the centre sin - tilar to ordinarv squamous cell epithelionias . " the hver of rat cl 912 was infiltrated , %ith tumour cells . over the two - vear period in which the semi - synthetic diet was used , some 150 rats receivmg this diet without the addition of pda have been dissected . during this period carcinomas of the no pancreatic tumour was encountered . small intestine were found in three piebald and in one albino rat . in the present series of 50 rats fed pda one gimilar intestinal carcinoma was found . 
tumours not localized in the liver have not been observed previously , partly because of the preference of the compound for the liver and partly because experiments in which the liver has been protected ' were terminated too early for ttimours to develop elsewhere . whether or not the substances in the diet which defer or prevent tlle produetion of hepatic tun ' iours by feeding pda are really anticarcinogenic or only protect the liver has been questioned . it seems that in the case of milk at any rate only the liver is protected from the carcinogenic activity of pda , and that tumour development generally is not prevented . it is remarkable that the extra - hepatic tumours should arise in an intestinal gland , and this seems to siiggest that organs which might be connected with the metabolism of pda are attacked . the conception that milk has a protective action on the liver only is interesting in ' view of the general plan of these experiments , the object of which was to find , if possible , an explanation of the prevalence of hepatic tumours in the natives in it might be assumed that the effect of a careinocertain parts of africa and asia . - genic factor which produces hepatic tumours , given a certain set of dietary habits and environmental conditions can be modified when the conditions differ . 
as a differerit method of approach , studies are in progress on the antagonisation of the antimitotic action of tetra - sodium 2 - methyl - 1 : 4 - naphthohyd - roquinone diphosphate ( compound 1 ) by nucleotides , nucleosides , purines and pyrimidines and some other compounds ( mitchell , 1950 , 1951 )  . this paper is an account of the cytological effects of a series of 29 selectect 318 j . 
marrian. details of preparations have been published as follows : compounds 11 and iv ( friedmann , marrian and simon - reuss , 1948a ) , compounds xx - xxiii ( friedmann , marrian and simon - reuss , 1948b ) , compounds viii , ix and xxiv ( friedmann , marrian and simon - reuss 1952a )  . we are indebted to f . 
the cytological effects are considered in relation to those of compound 1 . of especial interest from the point of view of the selection of compounds as possible therapeutic radiosensitisers is the appearance of mitotic abnormalities in concentrations which produce mitotic inhibition without gross toxic effects on the resting cells . 
the most important abnormalities studied are chromosome fragmentation , anaphase bridges , metaphase chromosomes with regions of impaired stainabihty and with irregular and beaded structure , metaphases with clumping of the chromosomes , arrested metaphases and other spindle abnormalities , of which many of the types described by barber and callan ( 1943 ) have been noted . 
as already discussed ( mitchell and simonreuss ' 1952 ) , it seems likely that the most relevant cytological abnormalities are chromosome fragmentation , anaphase bridges and chromosome aberrations in examples of the various types of cytological effects observed with this general . series of compounds are show - n in lb ' ig . 
on the basis of the concentration required to procluce 50 per cent mitotic inhibition , hydroquinone diphosphate ( xvi ) is about twice as active as hydroquinone ( xv ) , but the increase of mitotic inhibition with concentration is much more rapid in the case of the former than with the latter . 
somewhat sirnilarly , for 50 per cent mitotic inhibition , phenyl phosphate ( xviii ) is more active than phenol ( xvii ) by a factor of about 20 , but the increase of mitotic inhibition with concentration is much less rapid in the case of phenyl phosphate than for phenol . the only exception so far to the finding of increased antimitotic activity on phosphorylation is in the case of resorcinol . friedmann and simonreuss found that 50 per cent mitotic inhibition is produced by resorcinol at 6 x 10 - 8m concentration but by resoreinol diphosphate at about 5 x 10 - 7m . - kt these concentrations both compounds showed few abnormal mitoses . the usual increase in antimitotic activity on phosphorylation is not associated with any change in character of the cytological effects . 
the frequency of cytological abnormalities appears to be of the same order for the phospborylated and non - phosphorylated compound at concentrations which produce the same mitotic inhibition . ( 2 ) methyl group . the methyl group plays an important part in modifying the biological action , especially from the point of view of practical applications . moreover , among methyl derivatives , the limitations of the use of mitotic inhibition as a sorting test are emphasised . in the chick fibroblast cultures , compound 11 is more active than compound i as a mitotic inhibitor by a factor of about 1000 , and it also shows potentiation of mitotic inhibition in combination with x - rays . however , compound ii appears to be ineffective as a radiosensitiser for the jensen rat sarcoma , and did not produce a focal reaction in the tumour after intravenous injection in a few it may be mentioned that its use in man in high doses may be cases in man . undesirable because of the possibility of induction of cataract , analogous to the experimental naphthalene cataract in rabbits ( boume , 1937 )  . it is of interest to compare the methyl free compound 11 , tetra - sodium 1 : 4 - naphthohydroquinone diphosphate , with the 2 - methyl derivative , compound 1 , and with the 2 : 3 - dimethyl derivative , compound xxviii . 
no other compound examined showed this behaviour . compound 11 showed an increase in the number of metaphases by a factor of about 2 - 5 on increasing the concentration for 50 per cent mitotic inhibition by a factor of io . it bas been show - n in the preceding paper ( mitcher and simon - reuss , 1952 ) that compound i is somewhat unstable in aqileous solution in the absence of oxygen at 39 ' c . 
one may speculate and suggest the possibihty of combination of the phosphates with arginine side cha ' ms of histones the chromosome structure . biological action of 2 : 3 - dimethyl - 1 : 4 - naphthohydroquinone diphosphate , tetrasodium salt ( compound xxviii )  . - the biological action of compound xxviii differs considerably from that of ar the other c ' , ompounds . 
even in concentration 2 x 10 - 6m ( though not at 5 x 10 - 7m ) , which produces no detectable mitotic inhibition , there is metaphase accumulation by about 50 per cent above the fraction of metaphases in the controls , together with spindle abnormalities . as an example of the frequency of relevant chromosome aberrations , it will be seen from table iii that after application in concentration 4 x 10 - 6mfor 24 hours , out of 534 cells in mitosis , there were 74 cells in metaphase showing chromosome fragmentation and 17 anaphase bridges . it appears that with compound xxviii there is more rejoining of chromosome breaks than with moreover , compound xxviii only any of the other compounds examined . starts to show effects on resting cells in high con ' entrations associated with almost complete mitot - ic inhibition ; this behaviour suggests low toxicity . detailed studies of the combination of the effects of compound xxviii and x - radiation on the chick fibroblast cultures have been made by the summation method . 
the results of one experiment are given in table iii . it is concluded that under the experimental conditions , the combination of the action of compound xxviii and of x - radiation shows potentiation of mitotic inhibition and that the mechanism of action of the two agents is different . ( 3 ) carboxyl group . the compound most closely resembling compound xxviii in biological action is compound xi , the hexasodium salt of 1 : 4 - dihydroxy - 2 : 3 - naphthalene dicarboxylic acid diphosphate . replacement of the methyl groups in the 2 and 3 positions in compound xxviii by carboxyl groups slightly reduces the activity as measured by mitotic inhibition and considerably reduces the metaphase effects , so that at 50 per cent mitotic inhibition there is metaphase accumulation by about 25 per cent and sphadle abnormalities are much less frequent . 
compound vi produces clumped metaphases with some chromosome fragmentation and many undivided telophases , but practically no anaphase bridges . metaphase accumulation appears at a concentration which produces about 70 per cent mitotic inhibition ; this is associated with negligible effect on resting ceus . the weak metaphase arrest with compound vi is perhaps associated . 
simon - reuss 5 - methyl - 4 : 7 - thionaphthene quinone ( xxvii ) is active when tested in solution in cerosolve and gum ghatti , and produces 50 per cent rnitotic inhibiti ' on m approximately 5 x 10 - 6m concentration . 
mitotic inhibitor than compound ( xiv ) by a factor of 2 , but produces metaphase accumulation and abnormal mitoses , mainly clumped metaphases , chromosome fragmentation and multipolar cells , at all concentrations showing mitotic inhibition . the tetra - sodium salt of hydroquinone diphosphate ( xvi ) is a very active compound , and produces 50 per cent mitotic inhibition at almost exactly the same concentrat - ion , 3 x 10 - 9m , as does compound il in this region of concentratioin it also produces few abnormal mitoses . 
simon - reuss variably increased by phosphorylation , often by a factor of 5 or more . these results and those with dinitrophenol suggest that blockage of the entry of cells into mitosis depends upon inhibition of synthetic processes mvolving phosit is interesting that from the non - protein moiety of a phosphorylation . phorylase has been isolated a yellow pigment tentatively identified as 2 - methylit seems unlikely that at the concentrations 1 : 4 - naphthoquinone ( buell , 1952 )  . used there is interference with glycolysis ( gemmill , 1949 )  . the comparison of the cytological effects of different compounds of closely related chemical constitution and also of ionising radiations makes it possible to separate some of the main types of mechanism of action of these agents on proliferating cells as follows : ( a ) blockage of the entry of ceus into mitosis , without specification of the exact time of the action and presumably including pre - prophase and antephase this is the characteristic action of compound 1 , and in this case is inhibition . other less clearly associated with relatively few chromosomal abnormalities . defined examples are compounds x and xv . ( b ) metapbase arrest and disturbances of the spindle mechanism apparent - ly examples of this type of action in association similar to the effects of coichicine . with ( a ) are compounds 11 , vi , viii , xi , xxvii and xxx at the higher concentrations , compound ix at low concentrations and compound xxviii iso - naphthazarin produces metaphase over a wide range of concentrations . arrest with little or no inhibition of the entry of cells into mitosis . ( c ) chromosome , including chromatid , brea - kage - with varyina degrees of rejoinability of the broken ends . 
these experiments suggest that the tetra - godium salt of 2 : 3 - dimethyl - 1 : 4naphthohydroquinone diphosphate , compound xxviii , is the most suitable compound for further investigation as a possible therapeutic radiosensitiser . in addition to the help acknowledged in the text , e . 
wiltshire. from the biochemical liaboratory , university of cambridge . received for publication january 22 , 1953 . in 1939 k6gl and erxleben ( 1939a ) published the first of a series of papers it was on the optical configuration of amino - acids in proteins from tumours . found that the optical rotations of some amino - acids , which had been isolated by the classical methods and purified by recrystallisation , did not correspond with it was concluded that the preparations contained the values in the literature . the " unnatural " d - isomer in addition to the " natural " or l - isomer . 
the calculated proportion of d - isomer was largest in serine and glutamic acid . authors considered that serine had probably been partly racemised during hydroglutamic acid on the lysis of the proteins or during subsequent purification . glutamic other hand was considered resistant to racemisation in acid solution . acid from normal tissues and benign tumours had the specific rotation [ a ] 200d + attention was directed to this apparent difference in the optical form 31 - 60 . of glutamic acid from malignant and non - malignant tissues , which promised to be a lead to the long - sought chemical difference between normal and neoplastic tissue , and one that could be used for diagnostic purposes . 
a theory of malignity was proposed ( kogl , 1939 ) depending on the inability of most peptidases to hydrolyse peptides of d - amino - acids . the experiments of kogl and collaborators were repeated in many other laboratories with variable results , which on the whole showed smaller amounts of d - isomer than had been found by kogl . 
the considerable controversy which developed between different schools was suspended by the recent war , but dodds and dickens ( 1940 ) had already concluded from a review of the results then available that the appearance of the d - isomer of glutamic acid was not characteristic of since the war further work by kogl , barendregt and klein tumour tissues . ( 1948 ) and kogl ( 1949 , 1950 ) has renewed discussion of this problem . indirect evidence was produced in support of the original hypothesis . it was decided to limit the present study to the problem of whether or not the d - isomer is found in protein hydrolysates , and if so whether it exists as such in the proteins or is an artifact . 
a similar study using different methods ( boulanger and osteux , 1950 ) has appeared since the experimental work was completed . preliminary experiments ( wiltshire , 1953 ) showed that glutainic acid is racemised to the extent of about 5 per cent in the preparation of protein hydrolysates . 
found for calf lung protein total glutamic - n equivalent to 5 59 per cent and the l - isomer to 554 per cent of the protein - n compared with the present 5 69 per cent and 5 49 per cent respectively . for the carcinoma of the bronchus they found 6 * 40 per cent and 6 29 per cent both methods compared with the present 6 * 23 per cent and 6 29 per cent . therefore would appear to account for all the glutamic acid ofthe proteins . this is of some importance , since in much of the earlier work involving purification of the amino - acid for polarimetry a very small proportion of the total glutamic acid was finally isolated and analysed . 
the same criticism may be made ofthe aluminachromatographic methods of wieland ( 1942 ) and boulanger and osteux ( 1950 ) , where only 70 to 80 per cent of the original glutamic was recovered . 
the isolation of a small and unrepresentative sample may account for the discrepancy between the results of experiments by graff , rittenberg and foster ( 1940 ) and wieland and paul ( 1944 ) , who , using n15 - labelled glutamic acid , could find only 2 to 5 per cent d - isomer in the glutamio acid from tumours , and by k6gl , erxleben and van veersen ( 1943 ) , who with deuterium - labelled glutamic found over 20 per cent . 
the latter experiments have been criticised on other grounds by rittenberg and shemin ( 1946 )  . soon after the discovery of d - amino - acids in tumours several workers tested the activity of d - amino - acid oxidase from kidney on hydrolysates of tumour and other tissue proteins . 
among others k6gl , herken and erxleben ( 1940 ) ; lipmann , behrens , kabat and burk ( 1940 ) ; lipmann , hotchkiss and dubos ( 1941 ) ; arnow and opsahl ( 1940a , 1940b ) and boulanger ( 1944 ) could find no d - amino - acid in whole hydrolysates by this means . 
the percentage of d - glutamic acid found was in all cases very small , and not more than would be formed by inversion of the l - isomer during hydrolysis . i wish to thank b . 
holmes for the jensen sarcomata , the bland - sutton institute of pathology , middlesex hospital , london , for the liver metastases from a case of carcinoma of the colon , and professor f . 
with the advent breast cancer . of x - rays the same effect was brought about by ovarian irradiation ( foveau de courmelles , 1926 ; ahlbom , 1930 )  . on the experimental side , the work of lathrop and loeb ( 1916 ) , loeb ( 1919 ) and murray ( 1928 ) demonstrated the part played by the ovary in the production of mammary tumours , while lacassagne ( 1932 ) showed that , in certain circumstances , mammary carcinomata could be induced by the injection of oestrogenic substances . interest in the problem was further stimulated by the work of huggins and hodges ( 1941 ) on the treatment of carcinoma of the prostate by castration and stilboestrol . it was logical that the next step in the management of advanced breast testosterone propionate was cancer in women should be the use of androgens . tried with success by ulirich ( 1939 ) and by many later workers . the treatment of women suffering from breast cancer with one of the oestrogen preparations was apparently illogical , although zondek ( 1936 ) had put forward the suggestion in 1936 . 
they are rare , however , and in over 4000 cases of breast cancer observed in this department no spontaneous regression has so far been noted . the survival rate will be mentioned in the groups of cases to be considered and , although it seems that life can be prolonged with hormone treatment , no attempt is made to claim success on this account alone . the responses were graded as follows : a . 
good responses were seen in all types of metastases , skin nodules , glands , pulmonary and skeletal in 3 patients with osteolytic bone metastases there was radiological metastases . evidence of new bone formation . in 4 a pleural effusion disappeared and in 4 it diminished in amount . 
5 illustrates the findings on 9.x.46. patients treated by surgical castration and testosterone . in 1941 a small group of patients was treated by surgical castration , accompanied by the administration of testosterone . this was done in an attempt to find out why there were some failures with ovarian irradiation . it was thought that ovarian function might recover to some extent after irradiation and that the effect of o6phorectomy would be more lasting . nine patients with advanced breast carcinoma were treated in this manner by o6phorectomy , followed by daily intramuscular injections of 25 mg . 
testosterone propionate for periods up to 3 months . only one patient showed a slight response . she was a premenopausal woman of 40 with an inoperable breast carcinoma and bone metastases . 
the primary tumour became smaller and the glands disappeared temporarily , but no effect was noted on the skeletal metastases . patients treated by oestrogen therapy . in the latter part of 1942 it was decided to investigate the value of oestrogens in advanced breast carcinoma , and since that time the following oestrogens have been tried : stilboestrol dipropionate , dienoestrol , triphenylchlorethylene and ethinyl oestradiol . 
twice a day sometimes caused sufficient water retention to increase the pleural effusion and add to the patient 's dyspnoea . it was remarkable to find in a few such patients that doses as small as 0 ' 5 mg . 
the number of patients having no symptoms attributable to the hormone while taking stilboestrol was 122 , while many of the remainder had symptoms which did not inconvenience them greatly , the most common being nausea and vomiting at the commencement 36 maryidouglas of treatment . 
even pleural effusions dimished in amount , and in 3 cases disappeared altogether with no treatment other than stilboestrol dipropionate . it is also very interesting to note that in this series 13 patients with bone metastases experienced relief from pain , while in 6 , new bone formation was demonstrated radiologically in osteolytic lesions . 
no post - mortem was carried out , but this gastric tumour was probably metastatic in nature from the carcinoma breast . the following case illustrates the improvement which may take place in a patient with a pleural effusion : ca8e no . 
of testosterone propionate daily , given intramuscularly . treatment was continued for 3 to 4 weeks , and if the patient 's condition showed some improvement , further treatment was given either by means of an implant of 400 to 600 mg . 
one patient who was comatose with cerebral metastases improved sufficiently under treatment with testosterone to subsequent post - mortem examination confirmed attempt crossword puzzles . the presence of large intracerebral metastatic deposits , which presumably had regressed only temporarily under the influence of testosterone . in another patient a large pleural effusion did not cause any further respiratory embarrassment , and required aspiration only at increasingly longer intervals . this patient is still alive 25 months after the commencement of treatment . only one patient experienced relief of pain from skeletal metastases while another patient with metastases in bone stated she receiving testosterone . felt the pain easier when the drug was withdrawn . there was no radiological evidence of repair occurring in bone metastases . 
the series , although small , is in this respect at variance with the results so frequently reported in the literature ( adair et al . , 1949 ; kaae , 1949 ; report of the council on pharmacy and chemistry , 1949 )  . only one patient could not tolerate the drug . she was a woman of 43 with cerebral metastases , who developed very severe vomiting , and treatment had to be discontinued after she had received 150 mg . apart from this one patient no one was upset by treatment . 
treatment was also given to post - menopausal women to determine the effect . many of these patients had already failed to respond to other methods of hormone therapy , and they were beyond the aid of all other forms of treatment . the pituitary was irradiated by two opposed fields , each 6 x 8 cplaced in the temporal region , and a dose of 3000r was delivered in 3 weeks to the region of the sella turcica . a series of 37 patients was treated between october , 1949 , and october , 1950 , and the results are shown in table vi . as will be seen from table vi , only 2 patients showed a good response . 
the recurrence had become flatter for 3 months , but after that had spread again . following pituitary irradiation complete healing took place slowly , but new nodules appeared 10 months later . of the 2 women showing a fair response , the first was aged 46 and premenoshe had a skin recurrence on the chest wall within the irradiated area pausal . and was treated by pituitary irradiation in december , 1949 . slight regression of the recurrence was noted for 3 months , but then the disease became widespread and she died on 6 . 
none had shown any response to the other types of hormone treatment . it would seem from this that pituitary irradiation may be of value in the tylie of case suitable for ovarian irradiation , or in cases where ovarian irradiation has already been given with some good temporary effect . there is no doubt that pituitary irradiation was the least well tolerated of all fourteen patients were very upset by the treatment it is possible that a modification of technique forms of hormone therapy . before the course was completed . and dosage would overcome some of these difficulties . of the 37 patients , 21 have died within one year , the average survival being 2 to 9 months after the commencement of treatment . 
no good results were seen in bone lesions apart from relief of pain in 2 cases , although , of course , the series was small , and treatment may not have been continued over a long enough period . the reports in the literature on the efficacy of testosterone are very variable . farrow ( 1944 ) noted a deleterious effect in female patients , nathanson ( 1944 ) observed no effect at all , and various later workers ( adair , 1947 ; adair et al . , 1949 ; kaae , 1949 ) reported a proportion of good responses in skeletal and extra - skeletal all are agreed that testosterone may produce a beneficial effect metastases . on the general health but this is a non - specific effect , and due partly to the stimulating effect of the hormone on protein metabolism . it is , however , important not to assess improvement in general health as an anti - carcinogenic effect . with both oestrogen and androgen therapy some responses were noted here after the drug was stopped - a withdrawal response . farrow ( 1944 ) also noted this occasionally in the metastases after withdrawal of either oestrogens or androgens . consideration of the mode of action of hormone therapy . wvhile hormone therapy is widely employed , the basis of treatment remains largely empirical . the extension of our knowledge of the exact mode of action would be of great interest , and might result in further advances in this form of therapy.. 
the clinical results obtained , however , appear to be due to some modification of the endocrine status of the patient . for example , in a young patient who has not reached the menopause , oophorectomy or ovarian irradiation causes a sudden withdrawal of the natural ovarian hormones which influence the rate of growth of the cells of breast tissue whether these cells are normal or malignant . 
at this stage of our knowledge , however , it is doubtful if it would be justifiable to use a higher dosage . the administration of an artificial oestrogen again modifies the endocrine status . 
the oestrogens may produce a direct effect on the breast cancer cells , or the effect may be obtained indirectly through the anterior pituitary . if the action is a direct one , the best results would be expected in older women . tn an older patient , for example a woman who is 5 or more years past the nmenopause , there is little or no circulating oestradiol , and the addition of oestrogens produces a change . after the passage of time , however , the body , and in particular the breast cells , may become readjusted , and this might explain why the good effect passes off . the work of zondek suggests that the oestrogens may have an indirect effect through the anterior pituitary . 
zondek ( 1936 ) found that the administration of large doses of oestrin produced a chromophobe adenoma of the pituitary in male rats , that is , in rats not normally subject to the action of large amounts of oestrogen . zondek ( 1947 ) also reports a case in which he gave large doses of oestradiol benzoate to a woman of 216 with metastases from a carcinoma breast . 
the increase in size of the pituitary was due to an eosinophil adenoma . unless the production of an adenoma of the pituitaryr interferes with the output of other hormones the explanation is not very satisfactory in older patients . in a premenopausal patient , a drug such as stilboestrol may cause inhibition of the gonadotrophic hormones , so suppressing natural ovarian secretion , but castration could not be said to be complete not bringing about a true castration . consewhile an artificial oestrogenic substance is circulating in the blood . quently , there is not the same profound change in the endocrine status and no useful therapeutic effect is produced . the question of the administration of testosterone might now be considered . in a premenopausal patient this brings about an artificial menopause and , from this point of view , testosterone might be expected to produce similar results to ovarian irradiation or o6phorectomy . in addition to this , the administration of testosterone must disturb the normal oestrogen - androgen ratio . finally , testosterone , like the oestrogens , causes inhibition of the gonadotrophic hormones of the anterior pituitary ( moore and price , 1932 )  . it may , therefore , like the oestrogens , produce an indirect effect on the breast cells by a disturbance of pituitary function . an explanation must now be sought for the failures . 
how this would affect the response is not known exactly , but it is perhaps significant that few patients with clinical evidence of liver involvement showed any response to any of the methods of treatment mentioned . there may , however , be other factors modifying the response . 
haddow , watkinson and paterson ( 1944 ) suggested that the tumour called " carcinoma of the breast " might in reality comprise walpole and paterson ( 1949 ) also made the same suggestion , several categories . even going so far as to say that a tumour might produce an " anti - hormone " or inhibitory factor antagonising the action of the oestrogen . 
of advanced breast carcinoma treated by hormone therapy has been surveyed and the results obtained have been presented . the findings suggest that , in premenopausal wonmen , the treatment of choice is ovarian irradiation , which was found to produce beneficial results in 30 per cent of patients in this category . in women who are 5 or more years past the menopause , oestrogen therapy would appear to be the most satisfactory form of treatment . the drug found to be most generally useful was stilboestrol dipropionate 5 mg . 
of 9 : 10 - dimethyl - 1 : 2 - benzanthracene into the flank and below a nipple in strain street mice ( rask - nielsen , 1948 )  . in both sites the injection induced only spindle - cell sarcomas and , in a few instances , squamous - cell carcinomas , but no mammary carcinomas . 
the ratio of cases of isolated thymic tumours to cases of generalized leukaemia was found to be 1 : 1 following injection into the flank and 1 : 5 following injection into the mammary region . 
of 9 : 10 - dimethyl - 1 : 2 - benzanthracene were performed . the leukaemic manifestations induced have been described in detail in a recent publication ( rask - nielsen , 1949 ) and the local tumours will be dealt with in the present paper . for the purpose of ascertaining whether the failing development of mammary carcinoma might be a phenomenon specific for injection of 9 : 10 - dimethyl - 1 : 2benzanthracene , mice of strain street were injected with 05 mg . 
of a mixture of the same hard and liquid paraffin as used in the analogous experiments with 9 : 10 - dimethyl - 1 : 2 - benzanthracene ( rask - nielsen , 1948 , 1949 )  . 
an equal number of females and males were injected into the flank , but only female mice were injected into the mammary region , except in a few 9 : 10 - dimethyl - 1 : 2benzanthracene experiments , where an equal number of each sex was used . more than half the experiments were carried out with litter mates , one half of each litter being left untreated as a control group . 
rask - nielsen sarcomas of a varying degree of differentiation ; a few were polymorphocellular , and a few rhabdomvosarcomas occurred . in the squamous - cell carcinomas marked cornification was a frequent finding ; no microscopic changes indicated that the growths might have originated in mammary tissue . 
however , the growths were considered to be most probably spontaneous and were , therefore , not included in table iii . following injection in the flank , spindle - cell sarcomas were induced by benzpyrene , dibenzanthracene , methylcholanthrene and 9 : 10 - dimethyl - 1 : 2 - benzanthracene in 27 per cent , 50 per cent , 39 per cent and 12 per cent of the effective total of mice respectively . squamous - cell carcinomas were observed in the same experiment in 6 per cent , 7 per cent and 0 - 8 per cent following the injection of benzpyrene , methylcholanthrene and 9 : 10 - dimethyl - 1 : 2 - benzanthracene respectivelv . 
as regards the application into the mammary region , spindle - cell sarcomas were observed following injection of benzpvrene , dibenzanthracene , methvlcholanthrene , and 9 : 10 - dimethyl - 1 : 2 - benzanthracene in 4 per cent , 22 per cent , in this experiment , squamous - cell 37 per cent and 8 - 3 per cent respectively . carcinoma was observed in 2 per cent , 2 per cent , 22 per cent and 2 - 4 per cent respectively . the effective total of experimental mice is the number of mice living to be as old as the youngest tumour - bearing mouse , namely 4 months . the minimum , maximum and average latent period , the interval from the injection until death , is presented in table iv . 
the higher carcinogenicity of methylcholanthrene than of the other hydrocarbons apparent from the present experiments accords with previous findings ( greenstein , it accords also with the recent observation ( rask - nielsen , 1950a ) that 1947 )  . subcutaneous application of 0 - 02 mg . 
of 9 : 10 - dimethyl - 1 : 2 - benzanthracene was followed by leukaemic manifestations in 20 - 4 per cent with an average latent period of 20 to 25 weeks ( rask - nielsen , 1949 ) , injection of dibenzanthracene and methylocholanthrene had no accelerating effect on the development of leukamia , and the doubtful , extremely faint acceleration observed following injection of benzpyrene affected at any rate only the incidence ; not the latent period . the absence of leukaemic lesions following injection of the three last - mentioned hydrocarbons is all the more remarkable , as the development of leukaemia has been found to be accelerated following painting with benzyprene in dilute brown mice ( morton and mider , 1941 ) and in strain f mice ( kirschbaum and strong , 1942 ) following painting with dibenzanthracene , without , however , decreasing the latent period in strain f mice and with methylcholanthrene in strain f mice ( kirschbaum and strong , 1942 ) , in dilute brown mice ( mider and morton , 1939b ; morton and mider , 1941 ; lefevre , 1945 ) , in strains rf , and rf / ak ( mcendy , boon and furth , 1942 ) , in c3h mice ( kirschbaum , strong and gardner , 1940 ; morton and mider , 1941 ) , and in old and new buffalo mice painting with 9 : 10 - dimethyl - 1 : 2 - benzanthracene ( morton and mider , 1941 )  . also accelerated the development of leukaemia in aka mice ( engelbreth - holm and lefevre , 1941 ; lefevre , 1945 ) , in dilute brown mice ( engelbreth - holm and lefevre , 1941 ; law , 1941 ) , and in swiss mice ( law , 1941 )  . 
into the lung ( rask - nielsen , 1950a ) , and by the induction of only microscopically visible adenomas in mice , 4 - 11 months of age , injected with 05 mg . 
of benzpyrene , dibenzanthracene , methylcholanthrene , and 9 : 10 - dimethyl - 1 : 2 - benzanthracene into the flank of strain street mice induced local spindle - cell sarcoma in 27 per cent , 50 per cent , 39 per cent and 12 per cent ; and squamous - cell carcinoma in 6 per cent , 0 per cent , 7 per cent and 0 - 8 per cent respectively . injection of the same hydrocarbons into the mammary region induced spindle - cell sarcoma in 4 per cent , 22 per cent , 37 per cent and 8 ' 3 per cent and squamous - cell carcinoma in 2 per cent , 2 per cent , 22 per cent and 2 - 4 per cent respectively . local mammary carcinoma was not induced . leukaemia developed following subcutaneous injection into the flank and into 132 r . 
rask - nielsen the mammary region of benzpyrene , dibenzanthracene , methylcholanthrene and 9 : 10 - dimethyl - 1 : 2 - benzanthracene in 6 - 7 per cent , 1 - 2 per cent , 0 per cent and 20a4 per cent respectively . 
on the other hand the quantity necessary to induce abdominal fibroids is not much above the mentioned hysterotrophic dose ; abdominal fibroids can be induced by three injections per week of 5 lg . 
of oc - oestradiol , or even less , when administered as the 17 - caprylic ester of a - oestradiol , which is absorbed so slowly that a continuous oestrogenic action is made possible ( lipschutz , bellolio , chaume and vargas , 1941 )  . similarly a few micrograms of free oc - oestradiol or oestrone absorbed from a subcutaneously implanted tablet also are sufficient to induce fibroids ( lipschutz , thibaut and vargas , 1942 )  . 
on the contrary , quantities about a hundred times greater are necessary to elicit the same tumorigenic abdominal reaction if administered by subcutaneous injections of the free hormone ( lipschutz et al . , 1939 , 1942 )  . in the present work we shall deal with abdominal fibroids induced by extremely small quantities of estrogens and with the neoplastic action of these quantities on the uterine glands in experiments of long duration . fibromatogenic action of very small quantities of x - oestradiol and of oestradioldipropionate . tablets weighed 33 to 35 mg . 
each. tablets consisting of one part of the oestrogen and 19 parts of cholesterol merck were prepared in the usual way by mixing , dissolving in ether , drying and after drying in vacuo they mixing again . were implanted beneath the skin into castrated female guinea - pigs weighing 380 to 480 g . uterine and other abdominal fibroids were elicited under these experimental conditions ( riesco , 1942 ) , though the incidence was less than with the injection of great quantities of oestradiol administered as the benzoic ester , as in the experiments of iglesias ( 1938 )  . necropsy was made 112 to 118 days later . during necropsy , cleaned and dried subsequently . absorption was calculated from the loss of weight of the tablets recovered calculation was made under * this work has been aided by a grant from the jane coffin childs memorial fund for medical research , grant administered by a . 
the result of these experiments is given in table i . as seen from table i , tablets containing originally 5 per cent of the free cx - oestradiol and remaining for about 4 months in the body of a guinea - pig produce anew oestrogenic and fibromatogenic actions when reimplanted into another guinea - pig . the same result was observed when 4 months afterwards the tablet was implanted for a third time and a fourth time . there was in all these experiments opening of the vagina , growth of the nipples and uterine bleeding . 
should there have been selective absorption , oestrogenic hysterotrophic and fibromatogenic actions or uterine bleeding would have been impossible , in any case , in the third and fourth implantation . 
were obtained for the first 4 months . it is remarkable that in successive implantation smaller figures were obtained . allowance must be made for entering of substances into the tablet as has been shown by different authors ( bishop and folley , 1944 ; deanesly and parkes , 1943 )  . absorption was most probably greater than indicated by figures in our table i . 
but it can easily be demonstrated that daily absorption of oestradiol was certainly less than about 3 to 4 , g . in these experiments the total quantity of oestradiol in the tablet was never more than 1750 , g . , and the tablet remained beneath the skin during 470 days without exhaustion of the oestrogen . 
but in the next paragraph we shall mention experiments in which similar tablets were active for a total of 675 days without the oestrogen being exhausted . histological examination of the uterus in the mentioned animals revealed pronounced cystic glandular hyperplasia of the endometrium , polypous proliferation of the latter , thickening of the vaginal wall and proliferation of mammary these statements coincide with what has been formerly observed with tissue . the prolonged action of estrogens in this department . neoplastic action of small quantities of oestrogens on the uterine glands in experiments of long duration . in the above - mentioned preliminary experiments evidence was given that by continuous absorption of very small quantities of oestrogen , certainly less than 3 to 4 ug . 
fibromatogenic action was rather less pronounced than with the free hormones . but with the fibromatogenic action of oestrogens due allowance has to be made for variation . in the second series in which tablets already used in the first one were again implanted and left in the body for no less than one and a half years , uterine growth was considerable . uterine fibroids were also induced . microscopical examination of the uterus revealed in the series of long duration a condition fundamentally different from that seen at four months ; there was besides cystic glandular hyperplasia of the endometrium also proliferation of glands infiltrating the myometrium , reaching the serosa and even perforating it . the lumen of the proliferating and infiltrating glands was sometimes cystic , sometimes small or absent . 
the neoplastic and infiltrative growth of the glands was , in lipschutz ' experiments with ovarian fragmentation , coincident with those we have described in the last paragraph , with the exception that with ovarian fragmentation the invading glands did not reach the serosa and did not perforate it , as was the case in one of our animals . 
a guinea - pig having been injected for 8 months with oestradiol benzoate was found , 4 months later , to have a " precancerous " transformation of the endometrium in the upper third of the uterine horn . this latter observation of lipschutz and others is so far the only one which may be quoted in favour of irreversible atypical proliferation of the uteririe mucosa elicited in the guinea - pig by oestrogens and persisting after the withdrawal of the hormone . neoplastic malignant growth seems to be more easily elicited in the cervix of the uterus in the mouse , as shown by the work of gardner and others ( gardner , allen , smith and strong , 1938 ) and allen and gardner ( 1941 )  . in the work of these authors the oestrogens - - the benzoic ester of oestrone and oestradiol were given by injection . 
the quantities injected were , when calculated per gram of body weight in the work of the american authors with mice , probably 20 times greater than in our work with guinea - pigs . another interesting aspect of our work may be mentioned here . 
daily , add the remainder received twice this dose . table i shows the result of this experiment on the group receiving the lower dose level ; the treated tumours were completely inhibited . 
smar nodules persisted in the majority of these animals without change for about 2 weeks , after whicb tumours began to grow ; they were well developed in 70 per cent of the group 1 month after cessation of treatment . 
i and it seems reasonable to suppose that the same applies to all such tumours developing from inhibited nodules . when drug treatment was commenced 3 - 5 days after transplantation , both the sensitive walker tumour and its refractory derivative were always actively growing . 
the growth of the derived tumour was affected to a reasonably consistent extent by triethylene melamine , comparable to that observed with the lymphosarcoma . the subsequent development of inhibited walker tumour nodules has been surprisingly , growth was found to occur after a similar repeatedly confirmed . latent period in the majority of animals even though drug treatment was continued 340 h . 
ad example of this is shown id table iv , from wbich it is evident that tumours developed in 8 - 10 animals following the usual inbibitory period of nearly 3 weeks . it proceeded rapidly and then slowed quite markedly . in spite of the prolonged treatment in which 31 doses were given , there were no deaths which could be directly attributed to the effect of the drug . this faflure of continued treatment to maintain the state of inhibition id the tumour has also been confirmecl in other experiments . the contrast in susceptibihty between the original and derived walker tumours to the triazine , has also been found . 
as was mentioned earlier , the development of mouse leukaemia resistant to fohc acid analogues is wen known and there has been a recent report that the mouse sarcoma 180 , whicb is moderately inbibited by 6 - mercaptopurine , rapidly develops some resistance to this compound ( clarke , philips , stemberg , stock , elion and hitchings , 1953 )  . on the - whole , solid rat and mouse tumours appear to be relatively resistant to chemical inhibitors , although the response of different tumours to the same drug varies widely . 
not often does the arrest of growth extend to all tumours in an adequate group of experimental animals , in spite of the administration of dose levels of near lethal magnitude ( suguira and , stock , 1952a , 1952b )  . appears that rat tumours are more susceptible than those of the mouse to trie - thylene melamine , for complete regression has been obtained in nearly 100 per cent , of animals with the jensen rat sarcoma , sarcoma r39 and the flexner joblidg it is generally agreed that the longer carcinoma ( suguira and stock , 1952b )  . initiation of treatment is delayed , the less chance thei - e is of total regression . - the action of triethylene melamine on the walker carcinosarcoma is striking - even when treatment is delayed for as long as 5 days after implantation , by which time the tumour is actively growing . in the present series of experiments regression occurrecl in every animal and usuary little or no palpable material this confirms the work of hendry , homer , - remained at the site of the implant . rose and walpole ( 1951 ) and peczenik ( 1952 ) , who however , commenced treatment curiously , 24 hr . 
after transplantation of the walker tumour into wistar rats . suguira and stock ( 1952b ) were less successful with this tumour in wistar rats , .obtaining inhibition of growth in 70 per cent of animals with day old implants and regression in only 20 per cent when treatment was delayed for 7 days . 
the results clearly show that in effectively 4h animals the inhibitory effect is only temporary and that growth subsequently occurred irrespective of the duration of treatment , even though this may be continued for several weeks instead of the usual 7 - 10 in either case the suppression of growth continued for upwards of 2 weeks days . before renewal of activity could be detected . 
the derived tumour on which most of the work has been carried out , was selected at random from a group of tumours growing subsequent to one short course of treatment applied to the original walker tumour . 
the resistance is thus considered to be a stable and permanent change . another member of a group of tumours similarly derived from the original walker carcinosarcoma has also been shown to possess comparable resistance to further treatment . it is reasonable to suppose that the same will apply to all such tumours . 
a noticeable difference between the original walker and the derived tumours is the more rapid growth of the latter in untreated animals . the growth of tumours after a latent period followino , a short course of treatment with triethylene melamine , must be due to the survival of a small number of cells which are naturally resistant to the drug . 
the fact that this sequence of events is not changed by the continued administration of triethylene melamine to animals bearing implants of the original walker tumour , suggests that the resistance of the residual cells is maximal at the outset . additional support for this view emerges from the failure to enhance the resistance by further exposure to the drug . 
how far mutagenic effects of the ethyleneimine may be concerned remains to be investigated , but the fact that such a bigli proportiod of inhibited tumours ultimately grow seems to make this factor less likely . 
the resistant tumour has also been shown to be refractory to two other ethyleneimines ( compounds ii and iii ) at dose levels which completely inhibit the original walker thus resistance to triethylene melamine confers resistance to carcinosarcoma . a diethyleneimine , and this may be true of active ethyleneimines in general . how far the same statement may apply to other kinds of chemical inhibitors active against the ordinary walker tumour is being investigated . it is interesting that the walker tumour should be so sensitive to triethylene melamine , whilst two other tumours which have been tested in the same strain of animal ( a mammary adenocarcinoma and the lymphosarcoma referred to in the crossley , allison , wainio text ) were only little affected by the same treatment . and muenzen ( 1951 ) studied the effect of triethylene melamine on a sarcoma ( 231 ) in the king a rat . 
of the triazine without undue toxic treatment of 7 - day old transplants of the flexner jobhng careinomaeffects . with this dose rate for 7 days produced regressions in 90 per cent of animals in there was no reappearance of tumours during the next 3 months , 1 to 2 weeks . which was taken to indicate complete cure . 
not only different kinds of tumour but various strains of rat vary in their susceptibihty to triethylene melamine ; the growth of an occasional tumour in animals bearino , a neoplasm susceptible to the drug , may be connected with some unsuspected ability of the host animal to dispose of the administered compound . it appears reasonable to suppose that ethyleneimino - compounds as a whole owe their anti - tumour activity to these highly reactive groups , so that it is difficult to conceive a protective mechanism possessed by some cefls but not by the great majority it is equally difficult to imagine a high degree of susceptibility in a tumour . in one variety of neoplasm and indifference in another kind to chemical inhibitors hke triethyleneimino - phospboramide ( iii ) , which is highly soluble in water and hkely to diffuse with ease into cells . 
then too , there is the fact which has been estabhshed on many occasions , that the susceptibility of sensitive tumours to these drugs diminishes markedly with the age ( i.e. , size ) of the tumour . it is hoped that a study of the mechanism by which the resistant walker tumour is derived from the original variety and an investigation of the reason for the insusceptibihty of the formee tumour may throw light on these interesting and important phenomena . inhibited nodules from the walker carcinosarcoma treated with triethylene melamine , 24 hr . 
after the conclusion of a course of treatment show intact tumour cells . no mitotic figures were seen . estimation of the mitotic index of the original and derived forms , 48 hr . 
after the administration of the last of 2 doses of the same drug reveals that the growth rates of the two tumours are similarly depressed . the drug would therefore , appear to be exerting a cytotoxic effect on the resistant form , although the overall growth of this tumour is not much hindered by such treatment . 
steiner. from the university of chicago , chicago 37 , illinois , u.s.a. received for publication april 15 , 1954 . in an aae of high specialization there is a tendency to achieve understanding in oncology this by the study of progressively smaller and stif smaher units . has taken the form of going fiom caincer as a whole to its various sites and types , and from tissues to cells to intracellular components and , finaffy , to molecules . much progress has been made in this way in an understanding of the disease . this dissective analytical approach has , however , tended to divert attention from some of the larger units within individuals and in societv which are still capable of teaching much about the natural history of the disease , including its etiology . 
the endemiological approach to etiology in cancer , wbich makes use of groups of cases , appears to be neglected . the present purpose is to show by a statistical study of large bodily units , namely , organ systems , an endemiological pattem in the natural frequency of cancers that appears to have etiological impfications . 
each major body system shows a harmonious pattem in its principal tumours , different from other systems , suggesting that they have similar etiological factors . in a recent statistical study , it was found that the curves fof cancer to autopsy ratios by age were similar for all major sites in an organ system and different from those of other organ systems . 
the study was then extended to the mortality statistics of the united states , which corroborated the previous observations . it is believed that this behaviour pattern has etiological significance . necropsy data , the basic data from which the curves in fig . 
1 , and the number of cancers at each site can be found in the principal table of tlle appropriate chapters of another publication ( steiner , 1954 )  . in that material of 35 , 293 necropsies , there were 6 , 072 malignant neoplasms . 
the twenty commonest cancers comprised 91 - 3 per cent of the total . sixteen of the twenty ( omitting carcinomas of the prostate , skin , pancreas , andthe intracranial tumors because they are lone representatives of systems ) far into six organ systems . 
they form the basis for the present study . if the curves of ratios of necropsies for cancer to total necropsies in the twenty tumors are plotted together , as in fig . 
the relationships of the can ' cers appear haphazard and disorderly . however , if now the curves for each organ system are withdrawn from this melange and plotted separately , as in fig . 
an attractive order emerges from chaos . thus , the tumors of the four principal sites in the ahmentary tract which give carcinomas of the mouth , esophagus , rise to carcinomas are plotted in fig . 
2. stomach and large intestine had low mortahties early in life but they increased to a maximum in the seventh decade , after which their importance dechned in relation to other causes of death . 
the mortahty from carcinomas of the mammary glands , uterus and ovaries began in the third decade , increased quickly as cancer to necropsy ratios to a maximum in the fifth decade , and then fell . 
the peaks and general configuration of the three curves are the same , differing only in amplitude . the mortafity began soon after puberty , increased to the time of the menopause , and then fell off relative to other causes of death - more in the uterus and ovary than in breast . 
steiner peak was at least four decades later than in the feinale generative tract . mortality began in the prostate when in the female generative organs it had already reached its peak . 
the curve for the prostate shows an inverse relationship to the period of most active sexual iffe , but a direct relationship is found in the female generative system . the two principal tumours of the respira ' tory tract , free from the confusion of fig . 
except for the sman peak m renal cancers in the first decade , caused by the wilm 's tumors , the configuration of the curves is the same . there was a gradual increase with age in these cancer necropsy ratios . 
7. although mortahtv from carcinoma of the hver began earlier than from the gafl - bladder , in both it mcreased with age , and there is a similarity in the curves which is not obvious in the apparent anarchy in fig . 
the cancers of the stomach intestine ( including rectum ) , esophagus and the buccal cavity ( including the pbarynx ) had totals of 24 , 258 , 33 , 383 , 3 , 866 if cancer death to ar death ratios are made by and 5 , 138 cases respectively . decenniums or bv quinquenniums , the configurations of the curves are the same for all four - tumours and they resemble those in fig . 
cavity show a virtual plateau in the two decades 50 - 59 and 60 - 69 . in the respiratory tract , 18 , 277 cancers were reported in the ' trachea , bronchi and lung ( some unspecified as primary or secondary ) and 1 , 852 in the larynx . the cancer to death ratios show peaks in the deca - de 50 - 59 as in fig . 
4. these peaks , also , lie between those found in the alimentary tract and the female gellefative organs . similarly , the three principal cancers of the female generative organs _ ( mammary glands , uterus and ovary plus fallopian tube and broad hgament ) show curves which are similar in configuration to each other and to - those in fig . 
3 , there were reported in 1950 18 , 973 cases of with peaks in the decade 40 - 49 . cancer of the breast , 16 085 of the uterus , and 5 , 652 of the ovary , faropian tube and broad ligament combined . ln the mesoblastic series of mahgnant tumours , those of bone ( 2 , 180 cases ) showed a curve resembling that in fig . 
5 with a peak in the decade 10 - 19 , a sman depression in the decade 30 - 39 ' and a small secondary rise followed by a gradual dechne . 
the mahgnant lymphatic diseases ( code numbers 200 - 205 ; total of 16 , 756 cases ) showed a peak in the first decade forowed by a gradual decline . the ratios to all deaths were higher during the first three decades than in the los angeles material . 
the sarcomas of ar soft tissues are not combined in the u.s. vital statistics , so this category cannot be stuclied . in the ul - inary tract , kidney tuiriiors ( 3 , 643 cases ) had a maximum in the decade 50 - 59 while bladder tumors ( 6 , 401 cases ) had a peak a decade later . neither curve showed the persistent rise of fig . 
6 in the older age groups . because all tumors of the hver and biliary passages are combined in the u.s. vital statistics , comparisons cannot be made between individual sites or with fig . 
mortality figures for 1950 conform to , with minor exceptions , the same endemiological pattems as the los angeles necropsies , showing that the latter were not exceptional in this regard . it should be noted that cancer to au ' topsy ratios were used in one set of data thus , autopsies for all causes and an and cancer to death ratios in the other . deaths were used , respectively , as the points of reference . cancer to autopsy ratios may be ofhmited . 
value in expressing total quantity but they are excenent for contrasting one site with another within a material because the point of reference however , essentially the same endemiowithin each age group is the same for all . logical facts are revealed by other methods ofanalysis , altbough less clearly . thus , the age distribution figures ( steiner , 1954 ) for the cases in the los angeles necropsies show - similarities within organ systems . 
figures gave sinfflar results in so far as they were compared . thus , if the data of clemmesen and nielsen ( 1952 ) from the danish cancer registry are used , the incidence curves for cancers of the stomach , esophagus and large intestine ( who code 153.0 - 3 , 153.9 plus 154 ) are similar in configuration , as are those of the uterus and ovary but not of the mammary gland . the question arises as to wbat , if anything , these statistical findings indicate regarding etiology . are they explained by chance ? that they probably are not is shown by the absence of exception in sixteen tumor sites representing six systems in the los angeles necropsy series , a ; nd an essential concordance by most such a high degree of thirteen tumors in five systems in the u.s. 
2 to 7 that the organs in some systems develop a greater proportion of their it is not known whether this earlier cancers at an earlier age than in others . occurrence represents an earher exposure to etiological factors or a shorter tumor induction time after the beginning of the exposure . induction time isthe resultant of many factors including type , potency and amount of carcinogen , number of ca - rcinogens , conditions of exposure and susceptibifity of the cens . that differences in induction time exist in man is indicated by the exposures - to ionizing radiation in japan ( hiroshima and nagasaki ) where to date only leukemias have occurred although ionizing radiations are known to induce tumors of other types . it is also apparent from the figures that the tumor yield by sites within systems varies greatly although the induction time appears to be the same . 
tumor yield is govemed , in general , by the same factors as the induction time just mentioned . in addition the number of cells being exposed is important in deter number of tumors obtained . 
steiner organ system can , in theory , be correlated in part with the number of cells at thus , in the alimentary tract each site exposed to hypothetical carcinogens . there would appear to be , on the basis of the gross and microscopical anatomy , approximately equal numbers of epithelial cells available for neoplastic conversion in the mouth and esophagus , many more in the stomach , and still more in the this is roughly parallel to the numbers of cancers that occur large intestine . in these locations ( in 1950 5 , 138 , 3 , 866 , 24 , 258 and 33 , 383 respectively in the united states )  . similar agreements between the relative numbers of epithelial cells and tumors are found in the larynx and lung , in the female generative organs ( if one assumes that most mammary carcinomas are ductal ) , and in the mesoblastic tissues if nuclei rather than total volume are considered . 
the curves of cancer to total mortality ratios by age in thirteen common tumors found in five systems showed a harmonious behavior within each system , which differed from that of other systems . some case distribution and some incidence data show the same statistical facts although less clearly . the etiological significance of these endemiological observations is unknown . however , it is suggested that they could be interpreted as signifying common etiological factors within each system with the differences in quantity in tumor by sites accounted for in part by the unequal numbers of cell populations at risk . comments are made about the interpretation of the quantitative differences in tumors between organ systems and in their age distribution . the author is grateful to the research committee of the los angeles county hospital staff for permission to study their material and to e . 
 ( karnofsky , burchenal , armistead , southam , benstein , craver and rhoads , 1951 ) , therefore , aroused much interest . human trials on the hazards of water contamination carried out by the american army medical centre indicated that aliphatic nitrogen mustards might be absorbed after oral administration : hn3 in a total dose of 15 to 18 mg . 
the walker tumours were passages of tumour cell suspension in dextrose , prepared by craigie 's mincer technique ( craigie , 1949 ) and preserved at - 70 ' c . 
the mouse sarcoma was treated daily for 7 days . in the tests against the walker tumour the total dose was given over 7 , 8 or 10 days , and ajso as a . 
single dose ( boyland , cleg , koller , rhoden and warwick , 1948 ; haddow , kon and ross , 1948 )  . eleven to fifteen animals were used for test . the water - soluble drugs were dissolved in distilled water immediately before administration and given orally or intraperitoneally . 
of distilled water was passed through the tube to ensu - re that the whole dose was washed into thl - , stomach . r 48 was given in a single oral dose in arachis oil . control animals received the solvent by stomach - tube or remained untreated . 
the walker tumours and sarcomata 37 were dissected 14 and 9 days respectively after transplantation , fixed in bouin 's fluid and 70 per cent alcohol , dried between filter - paper , and weighed . the difference in weight between control and treated tumours was analysed statisticafly . 
the maximum tolerated oral administered orall dose was about twice the maximum tolerated intraperitoneal dose . using these doses , both oral and parenteral treatment produced similar effects against the walker tumour ( table 11 )  . 
the oral activity seeme ' d to be influenced by the distribution of the total dose ; the weight ratio between control and hn2 - treated tumours became more significant when the total dose was divided over 7 instead of boyland , cleg , koller , rhoden and ' " tarwick ( 1948 ) found over io da - ily doses . that the parenteral activitv of nitrogen mustards increased when the total dose was given as a single injection . 
when given in a single oral do the effect of hn3 was negligible , whereas the activity of hn2 and nor hn2 seemed to be increased to a certain extent . 
the oral activity of nor hn2 seemed at least equal to that of hn2 , and more pronounced than that of hn3 , but even the highest weight ratios between control - tumours and those treated with the ahphatic compounds were only about one - quarter of the ratio recorded after treatment with r 48 ( table ii )  . 
the other aromatic nitrogen mustard tested , nn - bis ( 2 chloroethyl ) p - phenylene diamine hydrochloride , when given as a single oral dose of 4 mg . 
peczenik the adult group ; the ratio was about 3 times that recorded in the young animals - seven tests only were performed in duplicate , six of them against the walker in the three tests with the young rats the duplicat - e results agreed , tumour . whilst in the three tests carried out with the adult rats the duplicates agreed in two but disagreed in the other ( intraperitoneal treatment with hn3 )  . 
the maximum tolerated oral dose of nor hn2 was about 10 times larger than that of hn2 and 14 times larger than that of hn3 ( table 11 )  . 
the therapeutic ratio of hn2 and hn3 , whether the drugs were given intraperitoneally or orally , seemed as low as 2 , or even lower . as estimated from the death - rate , hn2 and hn3 were more toxic to tumourbearing than to normal rats . 
was toxic to adult but tolerated by young tumour - bearers . effect of nitrogen mustard8and triethylene melamine on body - weight . the tumour - inhibitory effect of the agents investigated was frequently assoin some instances , more frequent in ciated with inhibition of somatic growth . the adult than in the young rats . 
the experiment is described here because it has been suggested that urethane potentiates the effect of nitrogen mustards clinically . three groups of adult bearers of walker tumour received hn3 orally in a daily dose of 0 - 4 mg . 
of urethane daily , a dose of urethane whichper8e is known to be inactive against the walker tumour . another group received unstandardized adrenocortical extract ( eucortone ) intraperitoneally , i ml . 
peczenik from that of the controls during the first few days inhibition of the walker tumour became manffest during the second week , suggesting that the agents clid not influence the " take " of the transplants . the therapeutic ratios of hn2 and hn3 are probably not lar er after oral than after intraperitoneal administration . in non - tumour - bearing rats fed with hn3 , roberts ( 1952 , personal communication ) found changes in the intestine very similar to those described by several authors after injections of nitrogen mustards ( selye , 1950 )  . in view of this finding , it is worth noting that most of the tumour - inhibition shown in this paper was associated with significant suppression of somatic growth , whether the drugs that held true even when the net weight were given intraperitoneally or orally . ( body - weight minus tumour - weight ) was considered instead of the gross weight . thus the question arises to what extent the tumour - inhibition recorded after oral administration of the agents was due to a specific effect or to starvation . 
walpole ( 1951 ) has fo - und 40 per cent growth - inhibition of untreated walker tumours when ifthis 40 per cent ofthe observed somatic growth was prevented by underfeeding . tumour - inhibition is deducted from the figures in tables i to iv , the maximum .tolerated oral dose of hn3 would appear to be inactive in adult tumour - bearers ( table iv ) whilst the significance of the other results remains unchanged . it may be assumed , therefore , that the retardation of tumour - growth described above was this view is supported by the finding mainly due to other causes than starvation . that the suppression of tumour - growth after a single oral dose of r 48 or nor hn2 was not associated with suppression of somatic growth . moreover , - whilst effects , on tumour - growth were similar , effects ' on body - growth were more pronounced after intraperitoneal than after oral treatment . 
most of the animals on both diets lost weight during the treatment , and the ratio between the weight losses in rats with the 20 per cent and the 5 per cent protein diet was almost the same as the ratio of the degrees of tumour - inhibition . according to houck , crawford , bannon and smith ( 1947 ) , animals treated with nitrogen mustards lose body weight by loss of protein and water . 
the rats under these experiments , therefore , though fed with loss of protein may also intensify a high protein diet ( 19 - 2 per cent ) , lost protein . according to elson ( 1951 ) , the effect of inhibitors on tumourand body - growth . as an initially large dose of an inhibitor is gradually reduced , the animal will recover from the inhibitory action quicker than the tumour will , and the latter may completely regress under high protein diet . my experiments in which the drug was given in a single dose the loss of protein ceased in time to arow the animal to recover its growth - rate whilst tumour - growth remained retarded . it may well be that in those summary . administered intraperitoneally in about maximum tolerated doses , nor hn2 , hn2 and hn3 had approximately equal activity against the walker carcinosarcoma , although aaainst the sarcoma 37 , hn3 was more active than hn2 . the walker tumour was significantly more affected by triethylene melamine than by the aliphatic nitrogen mustards . 
r 48 was at least 4 times as active as the aliphatic bis ( 2 chloroethyl ) p - phenylenediamine was active after intraperitoneal amines . but inactive after oral administration . bromine substitution in hn2 and hn3 seemed to cause loss of activitv . triethylene melamine given orally was practically inactive in the non - fasting rat , whereas the same dose given to the fasting animal had marked activity but was toxic . tumour - bearing rats tolerated r 48 and nor hn2 in doses respectively 36 and 10 times the maximum tolerated dose of hn2 . 
wiltshire. from the biochemical liaboratory , university of cambridge . received for publication january 22 , 1953 . in 1939 k6gl and erxleben ( 1939a ) published the first of a series of papers it was on the optical configuration of amino - acids in proteins from tumours . found that the optical rotations of some amino - acids , which had been isolated by the classical methods and purified by recrystallisation , did not correspond with it was concluded that the preparations contained the values in the literature . the " unnatural " d - isomer in addition to the " natural " or l - isomer . 
the calculated proportion of d - isomer was largest in serine and glutamic acid . authors considered that serine had probably been partly racemised during hydroglutamic acid on the lysis of the proteins or during subsequent purification . glutamic other hand was considered resistant to racemisation in acid solution . acid from normal tissues and benign tumours had the specific rotation [ a ] 200d + attention was directed to this apparent difference in the optical form 31 - 60 . of glutamic acid from malignant and non - malignant tissues , which promised to be a lead to the long - sought chemical difference between normal and neoplastic tissue , and one that could be used for diagnostic purposes . 
a theory of malignity was proposed ( kogl , 1939 ) depending on the inability of most peptidases to hydrolyse peptides of d - amino - acids . the experiments of kogl and collaborators were repeated in many other laboratories with variable results , which on the whole showed smaller amounts of d - isomer than had been found by kogl . 
the considerable controversy which developed between different schools was suspended by the recent war , but dodds and dickens ( 1940 ) had already concluded from a review of the results then available that the appearance of the d - isomer of glutamic acid was not characteristic of since the war further work by kogl , barendregt and klein tumour tissues . ( 1948 ) and kogl ( 1949 , 1950 ) has renewed discussion of this problem . indirect evidence was produced in support of the original hypothesis . it was decided to limit the present study to the problem of whether or not the d - isomer is found in protein hydrolysates , and if so whether it exists as such in the proteins or is an artifact . 
a similar study using different methods ( boulanger and osteux , 1950 ) has appeared since the experimental work was completed . preliminary experiments ( wiltshire , 1953 ) showed that glutainic acid is racemised to the extent of about 5 per cent in the preparation of protein hydrolysates . 
found for calf lung protein total glutamic - n equivalent to 5 59 per cent and the l - isomer to 554 per cent of the protein - n compared with the present 5 69 per cent and 5 49 per cent respectively . for the carcinoma of the bronchus they found 6 * 40 per cent and 6 29 per cent both methods compared with the present 6 * 23 per cent and 6 29 per cent . therefore would appear to account for all the glutamic acid ofthe proteins . this is of some importance , since in much of the earlier work involving purification of the amino - acid for polarimetry a very small proportion of the total glutamic acid was finally isolated and analysed . 
the same criticism may be made ofthe aluminachromatographic methods of wieland ( 1942 ) and boulanger and osteux ( 1950 ) , where only 70 to 80 per cent of the original glutamic was recovered . 
the isolation of a small and unrepresentative sample may account for the discrepancy between the results of experiments by graff , rittenberg and foster ( 1940 ) and wieland and paul ( 1944 ) , who , using n15 - labelled glutamic acid , could find only 2 to 5 per cent d - isomer in the glutamio acid from tumours , and by k6gl , erxleben and van veersen ( 1943 ) , who with deuterium - labelled glutamic found over 20 per cent . 
the latter experiments have been criticised on other grounds by rittenberg and shemin ( 1946 )  . soon after the discovery of d - amino - acids in tumours several workers tested the activity of d - amino - acid oxidase from kidney on hydrolysates of tumour and other tissue proteins . 
among others k6gl , herken and erxleben ( 1940 ) ; lipmann , behrens , kabat and burk ( 1940 ) ; lipmann , hotchkiss and dubos ( 1941 ) ; arnow and opsahl ( 1940a , 1940b ) and boulanger ( 1944 ) could find no d - amino - acid in whole hydrolysates by this means . 
the percentage of d - glutamic acid found was in all cases very small , and not more than would be formed by inversion of the l - isomer during hydrolysis . i wish to thank b . 
holmes for the jensen sarcomata , the bland - sutton institute of pathology , middlesex hospital , london , for the liver metastases from a case of carcinoma of the colon , and professor f . 
bather. from the poultry research centre , edinburgh , 9 . received for publication november 18 , 1953 . considerable interest has been centred lately on the use of ascites tumours in rats and mice for biological research . 
such tumours include , in mice , the ebrlich and krebs - 2 carcinomata ( loewenthal and jahn , 1932 ; klein and klein , 1951 ) and s - 37 and malignant lymphoma ( goldie and felix , 1951 ) and , in rats , the yoshida sarcoma , the mtk sarcomata i and 11 ( tanaka and kan ' o , 1951 ) and the hirosaki sarcoma ( makino and kano ' , 1953 )  . 
the growth of these tumours is characterized by the formation of large numbers of discrete tumour cers , living in a compatible medium ( peritoneal fluid ) which lend themselves admirably to various biochemical studies . during the course ofinvestigations into the nature of the virus of rous sarcoma , an ascitic variant of the tumour was estabhshed early in 1953 in order to see if infective virus could be obtained in a more pure forthe fact that rous no . 
of the suspension was no evidence of necrosis . inoculated into the abdominal cavity of each of 2 6 - week old chickens from a known rous susceptible strain maintained at this centre . 
most of the negative results can be attributed to failure of the inoculum to enter the abdomen and , usually , subcutaneous tumours grew at the site of entry of the needle . the rapid growth of the rous tissue and its invasion of the intemal organs has been observed in all subsequent passages . 
smar nodules of tumour tissue attach themselves to the surface of the organs and then proceed to infiltrate the membrane and musculature , often penetrating to the interior of the organ itsell of 30 birds from the ist to the 12th passages autopsied 9 - 19 days after the inoculation of the tumour , the following tissues showed invasion microscopically and grossly : gizzard ( 30 ) , pancreas ( 27 ) , duodenum ( 27 ) , spleen ( 25 ) , ovary ( 17 out of 20 ) , testis ( 5 out of 10 ) , liver ( 12 ) , heart ( 10 ) , adrenal ( 10 ) , kidney ( 7 ) , and lungs in the cases of the ovary and pancreas , invasion has frequently pr ' ogressed ( 1 )  . to such an extent that only a few fragments of normal tissue remain , almost the whole organ having been replaced by tumour tissue . 
pale yellow , viscous fluid present in a 6 - week - old bird after 10 - 1 2 days ' incubation , and , on occasion , as much as 60 ml . 
the cells account for approximately 10 per cent of the fluid volume . counts of the cells made from stained smears ( giemsa ) at variou 's times during the passaging of the tumour show that of over 2000 cells counted approximately 50 per cent are round tumour cehs , and the remainder leukocytes , lymphocytes and connective tissue cehs . this proportion has remained unchanged throughout the 18 passages so far maintained . 
on various occasions , virus or whole cells from ascitic fluid have been inoculated into the muscles of birds and , again , there is an immediate return to the normal histological picture of a solid rous no . 
1 shows the typical appearance of the fluid using iron haematoxylin stathe cytoplasmic and nuclear changes in rous sarcoma cells cultivated in vitro have been described ( tenenbaum and doljanski , 1941 ; doljanski and tenenbaum , 1943 ) and many of the descriptions apply to the appearance of the cells grown intraperitoneahy . 
the blue staining granule and thread network in the peripheral cytoplasm ( giemsa stain ) and the dense central zone can be seen , as well as the large cytoplasmic vacuoles and clubshaped pseudopodia . 
bather scattered at random and sometimes arranged round the periphery of the nucleus . the gathering of this granular material into the centre of the nucleus , leaving a clear zone between it and the nuclear membrane as described by tenenbaum and doljanski ( 1941 ) , is only rarely seen in these preparations . large round or rod - shaped , densely staining nucleoh are common and irregularly shaped ones are frequently found . multinucleate cells are present and many contain fragmented nuclei with as many as 15 - 20 fragments . measurement of the frequency of multinucleate cells is given in a later section . ascitic fluid examined microscopically by dark field illumination on the warm stage provides an excellent opportunity to observe the living cells for long periods of time . 
the lace - edge appearance of the .cells is typical of fresh preparations and the cell membrane tends to become more regular after a few hours . in the peripheral area of the cytoplasm , and in the pseudopodia , a very fine " mist " of minute particles can sometimes be seen , although this does not occur in every cell . 
the club - shaped pseudopodia described by doljanski and te ' nenbaum ( 1943 ) are a common feature and sometimes become detached , forming free spherical " bubbles " of protoplasm , usuary shimmering with the rapid brownian movement of very fine particles . similar vesicles are seen , especially in the clumps of aggregated cells in which either particles or an interwoven mass of fine filaments appears . 
they have been show - n to be present not only in pathological blood conditions , b ' ut also in normal blood after it has been kept for several hours under sterile conditions . in the rous ascitic tumour and in erythroleukaemic blood ( campbell , 1952 ) filamentous structures are present i fresh preparations and may be associated with the high rate of c ' ell degeneration . the possibility that they may represent a method of virus release from infected cells must be borne in mind . similar structures have been observed associated with cells infected with influenza virus by hoyle ( 1950 ) and wyckoff ( 1951 )  . a peculiar filamentous structure encountered , only occasionauy , is represented by the spear - shaped process shown in fig . 
the outer part of the thread becomes an external , free floating filament and the inner part withdraws to the cell , forming a small protrusion on the cell membrane . 
this type of structure usually occurs in cells having a poorly defined nucleas under conditions of dark - ground inumination . the fluid surrounding the cers also contains numerous tiny rapidly moving particles which scatter light . multinucleate cells show up very well and , occaascitic rous no . 
the samples were taken between the 10th and only cers containing morphologically 14th days after inoculation of the tumour . such cells arise because normal nuclei are included in the multinucleate forms . of failure of cell cleavage after nuclear division . 
the incidences of karyorrhexis and mitosis after 10 - 1 4 days ' tumour growth have not altered to any significant extent . number of nuclei per cell . karyorrhexis mitosis infective viru8 in ascite8 and 8olid rou8 8arcomata . the amounts of infective virus contained in either the cytoplasm of the washed cells or in the cell - free fluid have been determined by means of the day - old chick titration method first described by carr and harris ( 1951 )  . 
bather table ii shows the results for the first i 0 passages , along with two sets of figures obtained for solid rous sarcoma tissue and cell - free exudate . 
the virus titres of fluid and cell contents follow each other fairly closely . in 4 of the 7 pairs of results , however , the intracellular virus shows a higher titre by one logio dose . in this respect the ascitic tumour behaves in the same way as the sohd sarcoma and its exudate . edimation of total 4cpurified " viru8 material in cell8 and cell - free fluid of the rou8 in view of the differences encountered in the infectivity titres of the fluid and cehs , estimates have been made of the amounts of virus material obtainable from both sources . 
the " purified " virus was isolated by the fractional centrifugation method of carr and harris ( 1951 )  . ascites fluid was first treated with hyaluronidase to reduce the viscosity . 
the cells were then centrifuged off , washed in 0 - 85 per cent saline , lysed in distilled water and the cell debris discarded . both the cell extract and the fluid were then deposited in the servall s.s. 
the resulting pellets were treated with trypsin in m / 5 phosphate solution , clarified and deposited agathe amounts of virus material obtained in this way were estimated by the biuret method described in a previous pubhcation ( bather , 1953 )  . virus content is expressed as ml . 
dry weight per unit of tissue or fluid . during the washing of the cers , a cell count was done in a haemacytometer . table iii summarizes the results and includes an estimate of the virus per cell in solid rous sarcoma tissue . 
the figure is based on the average virus yields , as reported elsewhere ( bather , 1953 ) and an approximate cer count using the method of afizen and petermann ( 1952 )  . this method utifizes controlled homogenization in sucrose and acetic acid solutions containhag calcium chloride , ( 0 - 0023 m cac12 is used here )  . after suitable dilution , a count can be made of the whole cells and nuclei in a haemacytometer . 
an average figure for rous sarcoma , based on five different tumours was found to be 32 - 2 x 107 cells per g . with a standard deviation of + 2 - 8 . 
bather peritoneal fluid containing suspended round cehs . it seems probable that this type of conversion does not depend on the selection of round cells since when the tumour invades the abdominal organs , the secondary growth reverts to the normal spindle - cell form immediately . moreover , virus or whole fluids , from the ascites tumour , when inoculated intramuscularly , produces a typical solid rous doljanski and tenenbaum ( 1941 ) have described the reversible transsarcoma . formation of the one type olf rous cell to the other in tissue culture . 
thus the rous sarcoma , in chickens , closely resembles s37 sarcoma and t2146 tumour in mice , both of which have been shown to form ascites tumours by reversible adaptation ( lasnitzki , 1953 )  . results from the first ten passages of the ascites tumour indicate that the cell - free ascitic fluid contains infective virus to an extent , roughly , of that contained in a 10 per cent extract of the cehs . similar observations have been made on the liquid exudate from sohd rous sarcomata . there are various ways in the first place , of course , in which the virus may be released from the cells . virus might be released upon necrosis and disintegration of the cell . secondly , there is the possibihty that the filaments , which are abundant even in fresh preparations of rous ascites fluid , may represent a method of eiectinlr virus particles . 
of the sohd tumour growing in birds of the same age . in terms of dry weight of virus material , this infective titre was given by 0 - 30 m.g " purified " virus for the ascites tumour and 0 - 46 mg . 
the ascites produced is made up of cells ( 10 per cent ) and a pale yellow viscous fluid ( 90 per cent ) both of which contain infective virus . 
the cells are mostly dispersed singly but some are aggregated into small clumps . approximately 50 per cent of them are round tumour cells , the remainder lymphocytes , leucocytes and connective tissue cehs . 
after 10 - 14 days ' growth , 10 - 5 per cent of the tumour cells are multinucleate ( with 2 - 4 nuclei ) 1 - 8 per cent contain pyknotic , fragmentary nuclei ( karyorrhexis ) and 1 - 1 per cent are undergoing mitosis . 
no improvement in the yields of infective virus , as detectable by the day - old chick titration method , has been obtained from either washed cehs or cell - free fluid of the rous ascites tumour when compared with the sohd rous no . 
86 x 10 9 mg fluid exhibits the same , or more often , all expenses in connection with this work were borne by the british empire cancer campaign . i should fike to thank j . 
5 ( peacock , 1946 )  . since our last publication on this subject we have successfully propagated three new induced sarcomata , grch 16 , 17 and 18 . these three tumours occurred in the course of attempts to induce epithelial tumours by injecting into adult white leghorn fowls a mixture of tissues from 10 to 14 - day chick embryos of the same flock , with methylcholanthrene and sudan iv dissolved in olive oil , following the technique used by rous and smith ( 1945 ) for inducing epithelial tumours in mammalian embryo tissues . 
we used a variety of embryonic epithelia , but the only tumours that resulted from these procedures were spindle cell sarcomata of precisely the same familiar pattern as when the carcinogens were inoculated directly into the adult breast muscles . 
they are made up of fleshy spindle cells arranged in whorls and interlacing bundles , the individual cells being elongated , sometimes with branched ends , with finely granular acidophil cytoplasm , and with an oval nucleus containing generally one small sometimes the cells are multinucleate , in which case the nuclei are nucleolus . in line one behind another , never in the form of giant cells of the foreign body mitoses occur very frequently , and are often somewhat irregular with type . bridging and incomplete separation of the chromosomes ; but many apparently occasionally longitudinal fibrillation of the normal mitoses are also to be seen . cytoplasm can be made out , but cross - striation has never been observed in metastases in viscera free from striated muscle such as the liver . 
48 : ( a ) liver and ( b ) squamous epithelium from the crop of a 10 - dayold chick embryo were mixed with 1 per cent methylcholanthrene dissolved in a saturated solution of sudan iv in olive oil . 
3086 was killed in moribund condition . there was an oval tumour 5 cin greatest diameter in the left pectoral muscles . there was no tumour in the right pectoral muscles , and no residual fluorescence was detected at either site of injection . there was a chronic plastic peritonitis , probably from a healed perforation of the gut , which was bound down with adhesions . the gall bladder was greatly distended . 
of fresh tumour was ground smooth in a sterile mortar , and 10 ml . of 5 per cent dextrose saline were added and well stirred to give a turbid cell suspension . ten 6 - week - old white leghorn chicks were inoculated with doses of 0 - 2 ml . to 1 * 0 ml . 
a 1 in 10 fresh cell suspension in 5 per cent dextrose saline was injected into the breast muscles of 6 white leghorns , 9 to 12 months old , in 2 of which tumours were palpable in about 4 weeks . subsequently the tumour was maintained through 3 serial passages , by cell suspension inoculation . 
the lowest 1 cwas cut off and discarded , and the remainder of the paper was laid on a sterile agar plate and flooded with melted agar at 460 c . 
prodigiosus was present only in the lower 3 cm . all levels of wet paper , however , were infective in the case of of the wet strip . the mh2 tumour , but in the case of the grch 16 tumour no level above the meniscus of the tumour extract was infective . as in the case of ea ' rlier chemically induced sarcomata in which the test was made by similar technique , using paper at levels above the first centimetre from the free fluid , grch 16 and 17 homogenates yielded only negative results . this observation is completely in keeping with the results of the more conventional filtrations through gradacol membranes or seitz clarifying pads . 
on the other hand , the tumour itself might be supposed to contain such an agent , which for some reason or other was adsorbed strongly by the filter . in the case of paper chromatography the whole paper can be injected either intact or homogenised in saline , and this might be thought to favour the demonstration of viruses where filtrates yield negative results . 
as there are many variable factors in such exposures , it is convenient to express dosage as time related to sterilising effect on bacteria . under our conditions suspensions of b . 
for 5 to 10 minutes were exposed in plastic tubes to a focused beam of ultrasonic waves with a frequency of one megacycle per second transmitted through a cooling water bath . 
the plastic tube was transparent to the waves , and was so placed that maximum disturbance occurred in the centre of the tube . in order to concentrate the effect of the waves as much as possible 2 to 5 ml . of 1 / 10 homogenate of fresh tumour in normal saline were used . 
1 in the resulting proliferative reaction tissue . we have previously reported the localisation of virus - induced fowl sarcomata at remote sites of x - ray or radium burns ( peacock , 1946 )  . cauterisation with a red hot platinum wire or with fuming nitric acid applied to the skin with a glass rod ( 2 to 3 mdiameter ) has more recently been found to localise the virus in birds bearing rous sarcoma no . 
a sterile cotton thread was inserted in the left pectoral muscles of each chick and also ( a ) in 5 the left wing was injected with 0 - 1 ml . 
50 : ( a ) four out of 5 chicks had tumours in the right breast but no ( b ) all 5 chicks had tumours in the right breast . 
50 : the 9 chicks with large tumours at the site of rous sarcoma three chicks out of 6 had localisation at site of cautery , inoculation were killed . and 1 of them had localisation at site of control thread . none of the remaining 3 tumour - bearing chicks had localisation either at the site of sudan iv injection or at the site of the control thread . 
on the other hand , occasionally local inflammatory reaction occurs around the metal wing tabs used for numbering the birds , and we have seen 2 cases of localisation of rous sarcoma no . 
1 , and had 1 feather plucked from the leading edge of the wing on the day of injection and on the 4th , 6th , 13th and 15th days thereafter . 
no localisation occurred at the sites of trauma . it was thought that histamine - like action following cautery might cause local blood stasis and so favour the metastatic growth of blood - borne emboli from the tumour histamine was therefore injected into the left wing web at the damaged site . of 6 white leghorn chicks 4 weeks old bearing rous sarcoma no . 
50 : a large tumour was present in the right breast and the bird was there was direct spread from the grch 17 tumour injection site to the killed . liver , but no tumour at the site of rous no . 
3312 , 3316 and 3317 , which had not grown tumours at the site of injection of the seitz filtrate ( see above )  . typical grch 17 sarcoma grew in no . 
on the other hand , no such localisation has been observed in the case of the grch series of tumours . it seemed that the electron microscope , which can undoubtedly reveal particles of the size of viruses , might afford conclusive evidence of their presence or absence in our limited experience of this technique , however , there are in tumour cells . even less differences to be seen between infective and non - infective tumours and even normal tissues and in homogenates and filtrates prepared from them than can be seen by ordinary visual microscopy or even by direct visual observafurther work may overcome this initial difficulty of recognising viruses tion . from normal cell constituents ; but in the meantime circumstantial evidence points to essential differences between certain induced and spontaneous tumours . the occasional occurrence of a chemically - induced tumour which on propagation yields a virus does not in our opinion justify the assumption that all indeed , the existence of spontaneous filterable tumours contain such viruses . tumours would suggest that ultimately one is likely to encounter such agents in duran - reynals ( 1952 ) has recently suggested , birds injected with carcinogens . however , that viruses not usually associated with tumours may become adapted to cells conditioned by chemical carcinogens , and so play a part in the aetiology of cancer . 
our failure to induce any epidermoid cancers in the skin of fowls after repeated painting with chemical carcinogens may be due , as he suggests , to the absence of fowl pox in our flock ; but we have had no difficulty in inducing upwards of 60 sarcomata at the sites of injection of chemical carcinogens , and have seen a few carcinomata of the crop following exposure to 2 - acetylaminofluorene ( peacock and peacock , 1949 )  . 
though we failed in attempts to transmit the carcinomata and therefore cannot assess their possible viral content , we saw no inclusions of the kind described and illustrated by duran - reynals ( 1952 ) in his fowl pox methylcholanthrene carcinomata . the study of a wider range of chemically - induced fowl tumours , preferably including carcinomata of recognisable patterns , seems to offer the best prospects for obtaining more information about the aetiological factors concerned . 
1 , mill hill 2 endothelioma and the grch 16 , 17 , 18 sarcomata show that the two former tumours are readily transmissible by filtrates and by other extracts in which cells are probably destroyed or excluded , whereas the grch tumours are only transmissible by extracts containing cells . at the time of writing the authors were unaware that loewenthal ( 1931 ) had used capillary ascent on strips of filter paper of extracts of rous sarcoma no . 
some authors ( willis , 1948 ) believe that the association is mainly indirect , and that the nutritional features result chiefly from anorexia , ulceration and infection , to which , with cancers of the alimentary tract especially , are added disordered digestion and absorption of food . 
others consider that the connection may be morp , direct , perhaps through metabolic competition for circulating nutrients , and in - recent years animal experiments have lent some support for this hypothesis . a reduction in plasma albumin concentration has been noted both in cancer patients and in tumour - bearing animals ( abols , rekers , binkley , pack and rhoads , 1942 ; siebert , siebert , atno and campbell , i 947 ; huggins , 1949 )  . 
a - n early fall in the blood haemoglobin level ( greenstein , andervont and thompson , 11942 taylor and pollack , 1942 ; el mahairy , 1950 ) and marked reductions in tissue enzyme activity , e.g. , in liver catalase , in organs far d - istant from the growth have been similarly recorded ( greenstein , 1947 )  . while tb - ere is no doubt that a growing cancer often exerts widespread effects on general metabolism , no study has yet been made of any process common to both normal and tumour cells in whicb any such metabolic competition might be expected to disclose itself . 
with this in mind , therefore , it seemed profitable to niake a comparative study in normal and tumour - bearing animals of the occurrence of mitosis - a phase of the growth process which to the best of our present knowledge presents similar features in botb types of cell . 
in some experiments the protein content was doubled at the expense of the carbohydrate ; this " high - protein " diet contained 40 per cent of casewith the aid of a little water all the ingredients were mixed into a stiff paste which the mice ate readily . 
present work , bullough ( 1949b ) stated that changes in temperature within the range normally found indoors in this country had no noticeable effect on mitotic activitv in the skin of the ear . 
this anaplastic tumour grows readily in the strain of mouse used ; it has a high percentage of " takes , " and shows very little tendency to regress when once established . 
immediately after its removal the piece was trimmed all round in order to expose a raw edge which facilitated the penetration of the cold i per cent acetic acid solution in which it was at once immersed . 
after i to 2 hours in this solution ( the time depending on the size of the piece ) , it w ' as possible to slide the epithelium from both sides of the underlying connective tissues . 
after it had been covered with a similar piece of filter - paper the whole was moistened with fixative , compressed lightly between two coverslips held together with an elastic band , and immersed in fixative for at least an hour . the fixative used was a mixture of one part of glacial acetic acid with two parts of absolute alcohol ; exposure to this solul . - jon for several days did not greatly harm the cells , biit a period of not more than 24 hours gave optimal results . 
the " sandwich " was therefore removed intact from the coverslips , washed free from fixative in 33 per cent alcohol followed by water , and then soaked for a short time inn / 1 hci before it was replaced between the coverslipa and their rubber band . 
after about i hour the maximum degree of staining was reached , and a subsequent washing for a similar period in s02 water was needed to remove unchanged stain which would otherwise have become re - oxidized in the later treatment . 
changes in the mitotic activit qf ear epithelium in tumour - bearing m - ice . the results of three similar experiments which show the general effect of the growth of the tumour on the mitotic activity of the epidermal cells of the pinna are given in table i . 
the relation of the reduction in the mitotic activity of epidermal ce118 in tumoursince it is possible , as some authors have suggested , that systemic effects in persons or animals bearing tumours may be due more to depression of appetite and lowered food intake than to the direct competitive effect of the tumour itself , some experiments were performed to determine how far such indirect factors may be - the cause of the reduction in mitotic activity . 
since the tumours had reached about 10 per cent of the animal 's body weight at the time of death , the neoplastic tissue was resected before the final carcass weight was determined . from table ii it can be seen that even wlien the tumours had reached this lai - ge proportion of the body weight , the average carcass weight had not fallen from the time of implantation . 
although there was no sign of wasting in these tumour - bearing mice , and their behaviour continued to be typically active , the frequency of mitosis in the epidermal cells had fallen significantly . a further test of the inanition theory of reduction of mitosis lay in determining whetlier there was any correlation between the change in the weiglit of the mouse in the ' final 24 hours and the frequency of mitosis . 
since the mouse has a very higil food intake - 15 to 20 per cent of its body weight daily - any abst ' inence from eating is quickly revealed by a . 
in table iii the control and tumourbearing mic - e have been subdivided into two aroups on the basis of change of weight in the preceding 24 hours , and the mean number of mitoses per i 00 fields of the ear epidermis recorded . 
these general findings are in conformity with those of sherman , morton and mider ( i 9 , 50 ) , who observed that in tumour - bearing rats the nitrogen required for the nutrition of the tumour was derived from the diet until the growth reached about 10 per cent of the liost 's body weight , and that it was only after this proportion had been exceeded that this requirement was obtained at the expense of wasting of the body tissues . 
it is difficult , consequently , to attribute the decreased rate of multiplication of the epidermal cells in our mi - ce to any change ' comparable with ihe intoxication and cachexia often present in the later stages of neoplastic disease in man . an alternative explanatioii for our observations is that during the growth of such implanted . 
tumours nutrient substances essentia ' l for cell division may be diverted from normal to neoplastic cells to the detriment of the former . during periods of nutritional stress , such as severe malnutrition or pregnancy ( barcroft , 1946 ) , it is nowbelieved that a competition for circulating anabolites develops between cells , which be ' ars unequally on those of different types . 
should a coniparable contest for nutrients develop in neoplasia , the processes associated with the multiplication of ' normal cells might be amongst the first to reflect a deterioration in the metabolic state of the host . 
further , as cortisone fails to produce this effect when it is applied in the " pre - induction " and " induction " phase of carcinogenesis , no support is given to mottram 's hypothesis , that the inducing action of a carcinogen is exerted on a dividing cell . 
the number of cells in mitosis in the cortisone pre - treated animals during the period of " induction " would be few or none and on this hypothesis far less papillomata should have appeared whereas the numbers equalled that of the controls . baserga and shubik ( 1954 ) have obtained essentially siniilar results . 
the findings of green and savigear ( 1951 ) may bear on this point . they showed that after 4 applications of dmb , over 14 days , the epidermal cells of the mouse ear became relatively refractory to the antimitotic action of cortisone given systematically . this was confirmed by an in vitro technique ( green , 1952 )  . it might be expected therefore that chemical carcinogenesis would not be delayed or prevented by cortisone , whereas in fact the present results show that it was . 
a possible explanation of this apparent discrepancy is that the time of appearance of resistance to cortisone depends directly on the rate of mitosis . if this rate is strongly diminished ( by cortisone ) from the outset of carcinogen treatment resistance would then appear much more slowly . 
at the time of its appearing however the treated area of epidermis would be rich in cells in the " induction " phase of carcinogenesis since cortisone does not affect this process . such a mass of cells , after a long exposure to the carcinogen , could be envisaged , when finally they had become completely resistant to cortisone , as emerging as a frank carcinoma . 
a single male introduced the ay gene into all three lines , and the yellow mice in the offspring were crossed again to the respective pure line , and this process carried on indefinitely . 
were he responsible , he would have had to be heterozygous for these 7 genes in addition to his own ayat make - up , which would be rather an admirable genetic achievement , even if deliberately attempted . ( 3 ) contamination by stray mice : the whole experiment was carried out in a small room of the mousery , in which only this " yellow experiment " and inbred mice are maintained . 
none of the mutants was of this type , but all of the contamination by strays does not therefore appear to be usual " inbred " size . a satisfactory explanation , and true genetic mutation . 
though considerable discussion has been given to the question of mutations produced by atomic bomb explosions , it is possible that a much more frequent cause of these disturbances may have been unsuspectedly in action for many years on persons exposed to the influences of carcinogenic hydrocarbons . while mutations analogous to some described in this paper , e.g. 
reduction of hair and eye pigments , may be unimportant in humans , other types of mutants , some not detectable by the hydrocephalus , found methods used , would be a serious factor in public health . in this work , certainly would be . dominant lethals , leading to sterility or miscarriages , may also be produced , unimportant in mice where a large litter size is found , but of serious concern in man . it is possible that some mutants , such as the brain hernia found as an embryo , would normally be missed by the scheme used above , as mice usually eat abnormal and inviable young . the same unconcerned and easy solution of such difficulties is not the case in humans . there are possible also dominant mutations , which would appear in the first generation . though none was found in the series above , several were reported by strong it is obviously desirable that this additional ( 1945 ) using methylcholanthrene . hazard of exposure to these carcinogens should be evaluated . that this need is urgent is also suggested by the following line of argument : radiation mutations are almost entirely random , i.e. 
if a certain gene is mutated by an ionization in one sperm , the chance that the same gene will mutate in another sperm in the same or another individual is not increased . 
the efficiency of but this is not mutation with regard to any given gene is thus almost zero . if a chemical distributed via the blood necessarily the case with chemicals . stream reacts with a given locus to produce a mutation in one sperm , it is bbviously liable to do the same with all other similar loci in other sperms ( or ova )  . 
an efficiency of mutation at a given locus at 100 per cent can thus be imagined , and then all offspring of an exposed individual will carry the abnormal gene . a mating of any two exposed individuals would then give all f1 of the mutated type , a state which does not arise until the f4 of close inbreeding with radiation156 j . 
they could therefore have a high efficiency at certain loci resulting in a high frequency of abnormal offspring without causing death of the cell , as happens when concentrated doses of irradiation are given to increase mutation rate . combination with genetic material is thus likely . it is obviously tempting to ascribe the ability of the carcinogenic hydrocarbons to produce mutations to their complex system of conjugated double bonds . this system would readily pick up energy from the surroundings , and liberate it with destructive effect at one point . 
the hydrocarbons are possibly even less energetic , and thus may only be able to produce mutations in genes that are of less stability than most . this would produce some degree of specificity as required above , and it is significant that most of the mutations produced both here and by strong are already known , i.e. 
the mice used for transplantation undoubtedly came from therefore , additional experiments were different sublines of these two strains . carried , out in an attempt to elucidate the forowing questions : 1 . 
does the regression of a well - developed transplanted tumour forowing irradiation in vivo render the animal resistant to the development of spontaneous tumours ? this study was made possible by the use of the irradiation technique ( goldfeder , 1950a ) which facihtates the exposure of the tumour and protects the rest of the body of the animal from secondary scattered radiation , thus permitting the treated animals to eve their life span . previous work on the irradiation of tumours has been summarized by the writer ( 1945a )  . there is no doubt that results obtained with tumours heterogenous to the animal bosts - that is , tumours which have a different genetic constitution from that of the host , are not comparable with those obtained with tumours autogenous to the animal hosts - that is with tumours having the same genetic constitution as that of the host , or with , those in patients suffering from cancer . 
memorial laboratory on julv 5 , 1933 , and they were designated at that these mice have been inbred since : they behave time as inbred strain ' d '  . immunologicary like dba / 212 , and have an incidence of over 85 pe ' r cent of spontaneous mammary tumours among breeding females . 
a tumour of about 5 min diameter usuauy develops from an implant within a period of 12 to 14 days , and grows progressively . a spontaneous mammary tumour of an i 1 - month - old female mouse of the dba this tumour was : firmer in consistency than that strain was excised asepticary . fragments of about 2 mdissected removed from the c3h mouse described . from the edges of the tumour were implanted subcutaneously into 10 dba mice ( 8 males and 2 females )  . 
1 round coverslip 7 inch in diameter , wbich had been previously attached by a drop of sterile water to a mica sheet and covered with a maximow depression slide , in the concavity of wl - lich moist filter paper had been placed to maintain the water content of the tissue . 
the absorption of radiation by the cover glass , mica sheet and maximow slide was about io per cent , which was taken into consideration in calculating the x - ray dose delivered to the tumour tissue . immediately after irradiation the tumour fragments were implanted into about 2 - 3 - month - old mice , most in two additional experiments the mice were injected of which were litter mates . intradermally with aliquots of irradiated . 
tumour cells , suspended in physiological this was done because , according to observations made by several investisaline . gators , the intradermal route of immunization appears to produce more effective and lasting results ( besredka , magat , laval and besnard , 1936 ; kolmar and tuft , for the latter experiments the tumour fragments following irradiation 1941 )  . were cut with sharp curved scissors until an almost homogeneous mass was obtained . 
doses by mea ' ns of a tuberculiin syringe and .25 gauge needle in two or three sites of the abdominal wall of each mouse . the latent period and the number of " takes " produced by tumciur grafts irradiated in vitro served as criteria of the effect of a given dose of x - radiation . the procedure of irradiation of the estabhshed tumours in v& ' vo was as follows actively growing tumours ranging in size from 1 - 0 to 1 - 5 cin diameter were exposed to filtered x - radiation while the rest of the animal orgamsm was shielded bv a lead chamber ( goldfeder , 1950a )  . during the early experiments the tumours were irradiated in vivo under the same physical conditions as the tumour grafts during the later experiments , the following physical factors were in vitro . employed : the radiation was generated by a westinghouse " quadrocondex " constant potential unit at 200 kv , 15 ma ; the incident beam was filtered by 05 mcu plus i 0 mal ; hvl equalled i 0 mcu ; fsd , 20 . 
the dose rate measured in air bv a thimble ionization chamber during the coiirse of the investigations varied from 395 r / mto 402 r / min . special measurements were made with and without the lead ( 5 per cent )  . shield , the ionizatioil chamber being placed in the same position . 
observations on c3hb transplantable mammary tumour . the results obtained on implantation of the irradiated c3hb tumour - grafts a - re recorded in table i are presented graphically in fig . 
the first ' retardation of tumour growth , as indicated by a slight increase in the latent period of tumour appearance , was prodiiced by a dose of 2000 r . 
ii , an implant irradiated with 5000 r developed a tumour after a latent period of 117 days . in this , as wer as in other similar instances , the question arises whether the tumour cells had been attenuated by x - radiation to such an extent that they remained dormant for this comparatively long ' time , or whether only a few cers had escaped injury and took , therefore , a much longer time to develop a palpable tumour . this question cannot be answered with any certainty . it is certain , however , that the tumour grew on the site of grafting and that the rate of growth and the histologic ' al pattem were similar to those of a tumour developed by a non - irradiated implant . in no instance did a tumour , developed by an irradiated implant , regress spontaneously , contrary to observations frequently made when the tumoiir implant and the host are not of the same genetic origin . search for induced re8i8tance the mice in which the irradiated tumour grafts failed to produce tumours ( experiments 7 - 14 , table i ) were reimplanted with fresh , viable tumour grafts . due to the long latent period , the second implantation into mice of this series was performed 3 to 4 months after the implantation of the irradiated tumour grafts . 
a dose of 12 , 000 r completely destroyed all the treated tumours . sections of the tumours removed within 10 to 12 days after irradiation showed only necrotic tissue . it appears , therefore , that a dose within the range of i 1 , 000 r to 12 , 000 r is lethal for the c3hb tumour of 1 - 0 to 1 - 5 cni . 
the irradiated tumours which resumed their growth after initial decrease in size , grew steadily and in no instance did a tumour regress once it began to grow again . occasionary secondary tumours developed in the treated mice either in the inguinal or axillary reg - ions within 2 or 3 weeks after the regression of the irradiated in some instances , new growth developed on the periphery of the tuniours . irradiated tumour , probably from tumour cells which bad not been included in the exposed area . 
the histological appearance of these secondary tumours resembled that of the primary , non - irradiated c3hb tumour . epilation and regrowth of gray hair appeared in the exposed areas 3 to 6 weeks after regression of the irradiated tumours . 
no ulceration of the skin in the irradiated area was ever noted in the c3h mice once gray hair appeared . a significant number of c3h mice hved about i year after regression of the tumours and spontaneous tumours appeared in some of the c3h females . 
observation8on dbah transplantable mammary titmour . this tumour had been carried for 8 generations of serial transplants in the dba mice from which it originated before it was used for irradiation . although loo per cent " takes " resulted in most instances , occasionany , however , an iniplant failed to produce a tumour in a dba mouse which later proved to be resistant to further viable tumour grafts . according to hauschka , this may be the result of a mutant histocompatibility factor present in the dba strain ; in other words , an outcome of genetic incompatibihty between the bost and the transplanted tumour . 
to secure a better statistical evaluation of the results , a number of mice were grafted with viable , non - irradiated dbah tumour fragments to serve as controls to those grafted with irradiated dbah tumour implants . 
the delay in the latent period is probably a more rehable criterion than the tumour " take " for re - evaluation of the radiation effects in this case , because it could be argued that the 2 mice in which the irradiated tumourcells failed to produce tumours possessed the mutant - histocompatibility factor . in one experinient , a dose of 4500 r resulted in 22 - 9 per cent of " takes " , while a dose of 4800 r resulted in 13 - 0 per cent of " takes " and a dose of 5000 r gave only 5 - 1 per cent of " takes however , in two additional experiments , after doses of 5000 r and 5200 r no " takes ocetirred . 
none of these 7 produced tumours fohowing implantations of viable tumour grafts . in the experimental - mice , the number which failed to develop tumours was 150 out of a total of 341 mice which had been implanted with irradiated tumour grafts . 
none of these eighteen dayis later these - mice were reimplanted 25 niiee developed tumours . subcutaneously with fiesh , viable tumour grafts . palpable tumours , whicb , grew progressively , appeared within 9 to 13 days in 14 out of the 15 reimplanted male and in 9 out of 10 re - implanted female mice . two mice , one male and one female , in which the irradiated tumour cells failed to produce tumours in the first place , remained refractory to twice repeated implantations of viable tumour grafts . this result may also be ascribed to the mutant histocompatibihty factor present in these 2 mice . re8u1t8qf x - radiation o dbah tumour8in vivo . table v records the observations made on a total of 227 mice ( 164 males and 63 females )  . 
a rather sudden increase in the number of tumours which showed regression was noted forowing the application of doses higher than 6000 r . thus , 60 per cent of the tumours regressed after a dose of 7000 r . 
while a dose of 6000 r destroyed only 25 per cent of the treated tumours . the larger percentage of the regressed tumours which had been treated with a dose of 10 , 000 r indicates that this is the destructive dose for the dbah transplantable tiimours , ranging in size.from 1 - 0 to 1 - 5 cin cliameter . 
as in the case of the c3hb tumour , this dose is twice as large as the dose necessary for the attenuation of grafts of this tumour in vitro . the majority of the mice . ' m which tumours had regressed eved in apparent good health for i year or longer , and the young female ' mice produced apparently normal litters . 
the ulceration - s persisted iintil the death of the - ani ' mals . these two instances may parallel radiation necrosis occasionally noted in human patients treated with x - rays . 
the turnours in the remaining 7 mice grew steadily . fourteen tumours decreased considerably in size within 10 days after irradiation increased tumours grew steadily after exposure . five tumours regressed within 3 to 4 weeks following exposure . 
two females developed spontaneous months after regression of the treated tumours . in 27 mice ( 20 males and 7 females ) the tumours regressed completely within 3 weeks after irradiation . 
microscopic sections of the regressing tumours consisted mainly of fibrous tissue . the tumours regressed completely in 30 treated mice ( 21 males and 9 females )  . five fexnales developed spontaneous tumours 6 - 7 months after regression of the treated tumours . the treated m1ce lived for as long as i year and more after treatment . 
the mice lived in apparent good health up to 1 year and more . of ar treated mice only in two - instances did ulceration of the skin in the irradiated area occur 11 months after regression of the tumour . should be noted that in the experiments cited above , geneticary impure ammals and tumours geneticary different from the hosts were used . 
the writer 's interest in the problem of induced resistance arose from investigations on mouse sarcoma 180 grown in white mice of the swiss strain ( golffeder , 1942 )  . 
the writer explored the possibihty of inducing a resistant state in inbred animals by implanting into these animals irradiated grafts of a tumour which originary developpd in an animal of the same inbred line . these expenments were carried out hi an attempt to ascertain whether ionizing radiation induces genetic and antigeriie changes in the tiimour cells which make them foreign to the host . 
the irradiation of tumour cells in the present experiments did not make them foreign or antigenic to the host in which they originahy developed . an analysis of the results recorded in table i and in table iii reveals a difference in the radiosensitivity between the c3hb and dbah tumours . for example , a dose of 3500 r gave 92 per cent " takes " of the c3hb tumour grafts and only 57 1 . 
determined for implants of other mammary adenocarcinomas in inbred strains of mice ( lawrence , horn , strong , 1937 ; goldfeder , 1945a ) and for a mammary adenocarcinoma of an inbred line of rats ( eisen , 1940 )  . 
on the other hand , the c3hb mammary adenocarcinoma used in t - he present studies proved to be more resistant to irradiation than the cm mammary adenocarcinoma used in previous experiments ( goldfeder , 1947 ) , the latter requiring only 2700 r to prevent its grafts from " takes "  . 
the difference in the radiosensitivity between the c3hb and c3h mammary adenocarcinomas may be explained by ( a ) a difference in degree of differentiation : the c3hb mammary adenocarcinoma in the present experiments is a more anaplastic type of tumour consisting of fiheets and bands of closely packed cells , with relatively little fibrous connective tissue , while the cm mammary adenocarcinoma previously reported ( goldfeder 1947 ) consisted of acini , uniformly distributed and separated by connective tissue ; ( b ) by a difference in genetic make - up between the tumours and their hosts : the c3hb niammary tumour was carried by serial transplants in pure inbred cm mice from which it originated , while the mammary tumour , used in previous studies ( 1947.1950 ) , was carried in c3h mice of different sublines and thus the tumoiir and the hosts were not of the same genetic origin . failure of the tumour to " tak - e " in about 10 per cent of the hosts indicated that some of the cm mice used in the experiments of 1947 - 1950 had a different genetic constitution than that of the .mouse in which the tumour developed . 
the skewed curve illustrated in that publication ( goldfeder , 1947 ) in contrast to the typical s - shaped curve obtained in the present experiment may also be the result of the difference in the genetic make - up between the hosts and the tumour . 
the radiosensitivity of tumours depends to a large extent on whether they are growing in hosts of the same strain from which they originated , or in foreign hosts as shown by aughterson , tennant , irradiation of a transplantable mammary carcinoma in and lawrence ( 1940 )  . strain a mice in which the tumour originated induced regression only in i per cent of the treated mice when 2500 r were apphed and in 48 per cent of mice forowing the application of 5000 r . 
the antigenic reaction of ' the foreign host is thus a contributing f ' actor in rendering the tumour more vulnerable to ' regression after irradiation . the more anaplastic c3hb tumour proved to be more resistant to radiatioii than the relatively more differentiated dbah tumour . this corroborates the observations made by the writer on another mouse mammary adenocarcinonia ( goldfeder , 1950a ) and those noted on human tumours . for example , cathie ( 1939 ) , lenz ( 1942 ) and gliicksmann ( 1948 ) found that well differentiated cancers regressed more readily and offered a better prognosis than the less differentiated tumours . the r ' ole of connective tissue in the response of animal tumours to irradiation and its relationship to the host was brought forth by cramer ( 1932 ) aind by aughterson , tennant and lawrence ( 1940 )  . 
the relatively more pronounced proliferation of the connective tissue in the dbah tiiniour thanin the c3hb tumour may also be regarded as a contributing factor rendering the dbah tumour more vulnerable to irradiation . 
the observation of secondary tumours occurring in the treated mice also seems to parallel observations made on human mammary this observatioin indicates the failure of the disintegrated irradiated cancers . tumour tissue to induce resistance in the hosts to new cancerous growths . it has been noted that untreated metastatic tumours often regress after x - ravv treatment of primary tumours ( sugiura , 1937 ; aughterson , tennant and lawthis observation has been attribiited to a supposed indirect effect rence , 1940 )  . of irradiation ( sugiura , 1937 )  . these observations could probably be explained by the fact that the tumours were grown in genetically foreign mice . 
the present experiments on mice of geneticafly pure strains and their autogenous tumours indicate that the indirect effect of irradiation is not sufficient to affect secondary growths . regression of the secondary tumours in x - ray treated heterozygous mice may be attributed to an antigenic effect ' ehcited by the genetically differing tumour tissue . 
the inability of the irradiated tumour tissue to induce " immunity " in hosts from which it originated may indicate that no quahtative change of a genetic or antigenic character occurred in the irradiated tumour cells which would render them foreign to the hosts . inbred hnes of mice and their tumours offer far more suitable material for investigations of this type than heterozygous mice . 
the observations made from such studies seem to pararel those noted on human mammary cancers . in earlier publications ( goldfeder 1950b , 1952 ) , the writer showed that a fast growing , less differentiated mouse mammary adenocarcinoma also possessed a more vigorous metabolic rate and suggested that this propertv may have been one of the factors influencing the radiosensitivity of the mammary adenocarcinomas then used . 
a smaller irradiation dose was found to prevent the irradiated dbah tumour grafts from growing in vivo than was required to produce a similar effect on c3hb tumour similarly , for treatment of established tumours in vivo , the dose of irragrafts . diation which produced regression of the dbah tumour was found to be smaller than that which had a similar effect on the c3hb tumour . 
some factors which are involved in the difference in the radiosensitivity of these two tumours are the state of differentiation of the tumour cells and the amount of stroma . in both instances about half of the radiation dose , producing destruction of the established tumours grown in their respective hosts , was required to obtain the attenuation of the grafts of both c3hb and dbah tumours . the c3h mice in which the irradiated in vitro c3hb tumour grafts failed to " take " or in which a well developed transplanted c3hb tumour regressed after irradiation in vivo remained susceptible to reimplanted viable c3hb tumour grafts . 
a small number of the dba mice in which the viable dbah tumour grafts failed to " take " in vivo remained resistant to viable tumour grafts . similarly , a small number of the dba mice in which the irradiated in vitro tumour grafts failed to " take " was found resistant to re - implantation of fresh viable tumour implants . this is explained by a difference between the genetic make - up of some of the dba mice and the dbah tumour . a significant number of mice of both strains lived in apparent good health for 1 year and longer after regression of the in vivo irradiated tumours ; among these survivors the young females produced apparently normal litters . spontaneous tumours also occurred among these female survivors on reaching cancer age . thus , under the experimental conditions described , the question about the possibility of inducing immunity in homozygous hosts to endogenous tumours by irradiation of these tumours appears to have been answered in the negative.. the part played by the genetic relationship between the tumour and host in the induction of resistance to the appearance of tumours was brought forth . 
butler. from the chester beatty research institute , the royal cancer hospital , london , s.w.3. received for publication april 21 , 1951 . the observation of brdicka ( 1933 ) that proteins gave catalytic double waves when added to an ammoniacal cobalt buffer and examined polarographically was soon applied to an examination of the serum proteins , and brdicka ( 1937 ) himself was the first to show that differences could be found in this way between normal and cancerous sera . 
there is no need to relate in detail here the earlier studies on the protein reaction as these have already been adequately reported ( brdicka , 1939 , 1947 ; brdicka , novak and klumpar , 1939 ) , but it will suffice to state briefly that pathological sera after hydrolysis with potassium hydroxide and polarographed in cobaltous buffer show a decreased wave height as compared with normal sera , but after deproteination with sulphosalicylic acid and the filtrates polarographed in cobaltic buffer show an increased wave height . 
brdicka ( 1939 ) found that the latter test , known as the " filtrate test , " was more reliable , but the readings from both tests are dependent on the temperature , and on the characteristics of the dropping mercury electrode . muiller and davis ( 1947 ) introduced an index , known as the " protein index , " derived from the ratio of the two tests multiplied by a constant , which was found to be independent of the temperature and of the capillary characteristics , and also of the age and sex of the subjects . the fact that the polarographic test is not specific for malignancy has been well established and it is therefore of no use diagnostically , but more recent results robinson ( 1948 ) , using the indicate that it may be of value as a prognostic tool . filtrate test , has studied its application in the prognosis of prostate cancers , and has obtained results which are superior to those obtained with the acid phosphatase test normally used for this type of cancer . 
the purpose of the work to be reported below was to gain further information concerning the usefulness of the method , and to compare it with other methods which might be more applicable in general routine . over 100 sera have been examined polarographically , and a smaller number examined by means of the " huggins test " and by the tryptophane - perchloric acid reaction . since the publication by huggins , miller and jensen ( 1949 ) of the " huggins test , " depending on the relative ease of the thermal clotting of serum in the presence of various concentrations of iodoacetate , many workers have investigated its application ( bodansky and mcinnes , 1950 ; homburger , pfeiffer , page , rizzone and benotti , 1950 )  . 
the original authors stated that the " iodoacetate index , " obtained by dividing the highest concentration of iodoacetate in which clotting could still occur by the total protein content of the serum , was lower in 226 l . 
the results obtained from an examination of a limited number of sera by means of the modified method ( huggins , miller and jensen , 1949 , private communication ) are reported below . increases in the polysaccharide content of sera have been observed by a number ofworkers in cases ofmalignancy , tuberculosis , nephrosis , hepatic cirrhosis andpneumonia ( shetlar , foster , kelly , shetlar , bryan and everett , 1949 )  . using the carbazole method seibert , pfaff and seibert ( 1948 ) showed that the globulins ( particularly the a2 - globulin ) contained the most of the protein bound polysaccharide . in an attempt to determine the nature of the polysaccharide seibert and her co - workers ( 1948 ) found that the tryptophane - perchloric acid ( tpa ) test of cohen ( 1944 ) was more useful . using this reaction the authors claimed a correlation between increases in the polysaccharide and increases in the o2 - globulin in cases of tuberculosis and malignancy . moreover , changes in the tpa readings were found in minimal tuberculosis when no change was observed with the carbazole reaction , and it was suggested that this was probably due to the former reaction being more specific although the latter reaction is probably more sensitive . the identity of the polysaccharide involved in the tpa reaction is not known , but only negligible colour formation is obtained with glucose , mannose , galactose and glucosamine , while a positive reaction is obtained with desoxyribose - nucleic acid and fructose . 
a new index was then formulated derived from dividing the value of the filtrate test ( test ii ) by the difference between tests iii and iv and multiplying by ten to bring it to a round number , i.e. , blood index ( b.i. ) = test ( ii ) test ( iii ) test ( iv ) huggins reaction . the modified method ( huggins , miller and jensen , 1949 , private communication ) was used . the iodoacetic acid was recrystallized from a mixture of equal volumes of benzene and 80 - 100 c . 
the contents of the tubes were gently mixed , the tubes covered and heated in a boiling water - bath for 10 minutes . alter cooling quickly they were allowed to stand for 40 minutes and then filtered through a no . 
the upper limit for normal values was fixed at an arbitrary level below which the majority of the normals were grouped ( 21 table i is divided into two groups , and considering the negative , 3 positive )  . cases placed in group ( a ) , it can be seen that 93 per cent gave a positive reaction . 
the former were ( i ) a patient with a 5 years ' history who had been successfully treated by surgery and by androgen , and who was clinically normal at the time the blood sample was taken ; ( ii ) a male case who gave a positive reading 2 weeks later ; and ( iii ) a patient with an advanced tumour who gave a negative reading again 2 weeks later . 
the negative prostate case had already been receiving stilboestrol treatment . these cases show a high correlation with the clinical observations , but group ( b ) of table i gives figures for two malignant groups which appear to give an even chance of positive results . classified in " buccal cavity " are carcinomas of the fauces , palate , tonsil , tongue , mouth , larynx and pharynx , and in " skin " are classified carcinomas of nose and scalp . 
great improvement was observed clinically while under androgen treatment ( after radiotherapy followed by radical mastectomy ) , and the polarographic however , the last five readings , taken readings altered from positive to normal . over a period of eight months , gave positive reactions , although it was only at the time of the last reading that a relapse was clinically observed . in this case , therefore , the relapse was forecast by the polarographic readings . 
youden ( 1950 ) has suggested the use of an index for the comparison of so - called diagnostic methods , which has a value of unity if there are no false positives or false negatives , and a value of zero when the test gives equal proportions of positives for the condition under investigation and the controls . 
the polarographic method gives a value for the index of 0 - 219 , the huggins reaction , 0 - 250 , and the tryptophane - perchloric acid method , 0 - 253 . 
the end - point as described by huggins was not easily determinable , as it was not always obvious what constituted a " well - defined mass . " this difficulty was also noted by bodansky and mcinnes ( 1950 )  . also , the end - point often did not occur within the limits of the recommended iodoacetate concentrations - a problem which presumably would have been overcome by using a larger range of concentrations , making the test much more tedious . 
the results obtained support the conclusions of bodansky and mcinnes ( 1950 ) and of homburger , pfeiffer , page , rizzone and benotti ( 1950 ) , which were reached after a much more intensive study of the reaction than has been attempted here , and it can be stated further that the test is too unreliable to be used in prognosis . the results given in table iii showed that the polarographic readings followed to a very large degree the clinical findings , and it can be stated that within certain limitations this method is useful as a prognostic agent . it can be seen from table i that apart from the two groups of cancers , a high correlation was obtained and the proportion of false negatives was low . there was , in fact , only one case in the negatives shown in table i ( a ) , which remains unexplained , since the prostate case was being treated with stilboestrol , and of the breast patients , one was clinically normal and the other case gave a positive reading two weeks later . this last case supports the view that the serial examination of serum gives far greater information than a single test , a view also supported by brdicka both positive and negative cases should be followed . ( 1947 ) and robinson ( 1948 )  . a persistently positive test or a negative test becoming positive would indicate that the patient requires investigation , a positive test becoming negative indicates that the patient has improved , while a persistently negative test may be misleading ; this last fact is the main weakness of the method . 
excluding tumours of the buccal cavity and skin , which gave an equal chance of giving positive or negative readings , polarographic examination gave a 93 per cent correlation with the clinical diagnosis of malignancy . 
a high correlation with the clinical diagnosis of malignancy was obtained with the tryptophane - perchloric acid test , although it is considered too unspecific to be of use in prognosis . i wish to thank professor e . 
1. received for publication july 1 , 1948 . in the course of experiments on a transplantable mouse sarcoma it was observed that fresh saline extracts of tumour tissue agglutinated mouse and rabbit red cells . 
when extract and red cell suspension were mixed on a slide agglutination became visible to the naked eye within 30 seconds and appeared to be complete within 2 minutes . a number of different mouse and rat tumours , and a wide range of normal tissues , have been examined for haemagglutinating activity . 
when quantitative results were required a tube test based on that used by burnet and his co - workers ( burnet , beveridge , mcewin and boake , 1945 ) was adopted . serial two - fold dilutions of tissue extract were made in calcium - saline in round - bottomed tubes of approximately 0 - 8 cinternal diameter . 
of a 2 per cent suspension of washed red cells in calcium - saline ; the tubes were then shaken and allowed to stand at room temperature for 20 - 30 minutes . 
a preliminary reading of the end - point could then be made , if a quick answer was needed , which did not differ by more than one place from the final reading . calcium - saline , 0 - 3 c.c. , was then added , the tubes were shaken and allowed to stand a further 1 hours at room temperature , when the final reading was made . the type of agglutination , and also of the deposition of red cells in saline controls , varied with species . 
mouse cells seldom , and rabbit cells hardly ever , gave the compact button of deposit which is typical of unagglutinated fowl or human cells , and which workers with influenza virus haemagglutination take as negative . rabbit cells , in the absence of an agglutinating agent , settle in a round - bottomed tube as an ill - defined deposit surrounded by a slightly granular film , which would be regarded as evidence of agglutination of fowl cells . resembles closely the " shield pattern " which has been described ( burnet and stone , 1946 ) as typical of agglutination of fowl cells by lipoids . 
proved a it was effective in low concentration ( 0.005 m ) , very satisfactory preservative . and though it increased slightly the granularity of deposits of red cells in control tubes , it had no haemagglutinating action which could be confused with that of tumour extracts , even in concentrations much higher than this . it was slowly lytic for mouse red cells ( though this did not interfere with tests read after the usual period of two hours ) but not for those of other species . 
a final concentration of 0 01 m ( 0 12 per cent ) was generally used ; this was conveniently obtained by adding to a tumour extract 1 / 50th of its volume of a saturated aqueous solution of b.a.l. 
brought about regeneration of the activity of inactive extracts to their original titre . this regeneration was progressive : some activity was apparent in a previously inactive extract a few seconds after the addition of b.a.l. , but the full titre was not reached for about an hour , at room temperature . table i shows the preservation by b.a.l. 
fowl cells of each type , distinguished by a previous test against kahn antigen , were included in this test . it will be seen that rabbit cells were agglutinated by the tumour extract to lipoid agglutinable and far higher titre than those of any other species tried . lipoid non - agglutinable fowl cells were agglutinated to the same low titre . tumour extracts varied somewhat with regard to the ratio between titres for the titre for rabbit different red cells , but these variations were not constant . cells was always between 4 and 16 times the titre for mouse cells . extracts treated in various ways , to be described later , were tested in most cases against rabbit and mouse cells . 
red cells which were agglutinated to low titre by c57 were inagglutinable by s37 extracts . extracts of all the tumours tested lost their activity on standing , though at varying rates , e.g. 
extracts of c57 sarcoma took about 24 hours to become inactive , but extracts of c57 / lh1 , sarcoma , though initially often of similar titre , became inactive in one hour , or less . the loss could be prevented , or reversed , by the addition of b.a.l. occurrence and distribution of haemagglutinating agent in normal tissue . extracts of mouse liver , spleen , kidney , heart , lung , brain , voluntary muscle , lymph gland and thyroid , derived from both normal and tumour - bearing mice , were found not to agglutinate mouse red cells ; and for a time it was thought that the haemagglutinating agent was confined to tumours . examination of a wider range of normal tissues , using rabbit as well as mouse red cells , showed that this was not so . extracts of several tissues , notably adult mouse testicle , ovary , uterus and voluntary muscle , as well as whole mouse embryo and placenta , agglutinated the haemagglutinating rabbit red cells and sometimes mouse red cells also . titres of these extracts were , in general , low compared with those of extracts of mouse sarcomata , but comparable with those of extracts of some of the other table iv gives the range of haemagglutinating titres for tumours examined . rabbit , mouse and human red cells , of a number of normal tissue extracts . haemagglutinating power of these extracts declined on standing , and was preserved , or regenerated , by the addition of b.a.l. 
0 - 01 m added after standing 22 hours at room temperature figures represent reciprocals of haemagglutinating titres for rabbit red cells . some other properties of the haemagglutinating agents of tumrnours and normal tissues . action of normal serum on haemagglutination . - haemagglutination by lipoids is inhibited by normal serum in high dilution , while haemagglutination by viruses is unaffected by normal sera , but inhibited by specific antiviral sera ( burnet and stone , 1946 )  . the effect of normal horse serum on haemagglutination by tumour and normal tissue extracts was tested . extracts were mixed with equal volumes of a 1 in 100 dilution of horse serum , and the mixtures tested for haemagglutination in the usual way . 
an experiment similar to the last showed that addition of either of these sera had no effect on haema gglutination by either c57 or s37 sarcoma extracts . effect of temperature at which the test is performed on haemagglutination . 
than at room temperature ( stone , 1946 ; burnet and stone , 1946 ) , while haemagglutination by influenza virus is more active in the cold than at room temperature or 37 c . 
 ( salk , 1944 )  . haemagglutination by extracts of c57 and s37 sarcomata , and of normal mouse embryo and adult uterus , was tested at approximately 4 c . , at room temperature , and at 37c . 
overnight. neither rabbit nor mouse presence of complement had no detectable effect : cells were lysed and their agglutination by the tumour extract was unaffected . absorption of haemagglutinating agent by red cells . - - c57 sarcoma extracts were absorbed with rabbit , mouse , and fowl red cells , under various conditionts . the following is a representative experiment . samples of 20 per cent extracts of c57 sarcoma , ( i ) fresh , i.e. 
for three hours , and centrifuged ; the supernatant fluids were removed and the cells finally resuspended in the original volumes of saline , and examined for persistent agglutination and for agglutinability . 
fowl cells , which were only very slightly agglutinated , did not absorb appreciably . in all except one case red cells agglutinated during absorption were unsuitable for subsequent tests for agglutinability because they remained agglutinated . the exception was mouse cells used to absorb fresh extract . these , though agglutinated in the presence of the extract ( 30 minutes at room temperature ) redispersed during the subsequent incubation with saline . 
the difference is probably quantitative . gross ( 1947 ) has reported the presence of haemolytic agents in several tumours . haemolysis by such an agent , if of low titre , would be active only in the first one or two tubes of a haemagglutination test and , since these contain only 1 / 450th of their volume of packed red cells , haemolysis might be masked by the small amount of haemoglobin present in all crude tissue extracts . 
had been added before , and those to which it had been added after , heat treatment . a heavy precipitate formed in the heated extract , which was spun out before testing for haemagglutinating power , and it was thought possible that the haemagglutinating agent might be adsorbed on the precipitate . however , no agent could be recovered by extracting the precipitate with m / 15 na2hpo4 . the effect of digestion of tumour extracts by trypsin or papain depends on whether they contain b.a.l. in the absence of b.a.l. , extracts of c57 , or of c57 / lh1 , sarcoma incubated at 37 c . with trypsin or papain ( activated with kcn ) for an hour , were found to be irreversibly inactivated , i.e. 
0005 m 0 - 01 m + , , trypsin 0.5 per cent of commercial powder ( hopkins & williams )  . all mixtures buffered at ph 8 with m / 15 phosphate , 2 drops of chloroform added as baoteriostatic ; neutralized , and b.a.l. 
0.005 0 ' 01 m casein was " light , soluble " of british drug houses , dissolved in m / 15 na , hpo , by heating relevant conditions as in a . 1 hour on a water - bath . it will be noted that there was a considerable irreversible drop in titre of a control sample of sarcoma extract incubated without b.a.l. 
to such extracts gradually falls . it seems probable that this effect is due to the action of tissue proteases . the effect of tryptic digestion on extracts of normal mouse tissues was also tried . it is difficult to obtain such extracts with haemagglutinating titres high enough to make this test satisfactory . in an experiment in which a mouse embryo extract with a haemagglutinating titre for rabbit cells of 1 : 4 was digested with trypsin , with and without b.a.l. , activity appeared to be destroyed in 1 . 
on proteolysis was a 20 per cent extract of c57 sarcoma in m / 15 phosphate buffer ph8 was incubated ( a ) alone , ( b ) with trypsin , ( c ) with trypsin , and b.a.l. 
they show that , with casein as substrate , after 1 hour 's incubation there was no detectable inhibition of proteolysis by b.a.l. ; but with both casein and tumour extract as substrates , after 21 hours ' incubation there was definite inhibition : about 50 per cent by b.a.l. 
elliott , on the haemagglutinating power of a tumour extract , with and without b.a.l. , was essentially similar to that of commercial trypsin . the haemagglutinating agent does not become dialysable through cellophane after digestion of a b.a.l. - containing tumour extract with trypsin . lecithinase : it has been shown by stone ( 1946a ) that the haemagglutinating action of lipoids , and of vaccinia and ectromelia viruses , which probably owe their haemagglutinating action to lipoid components , is destroyed by cl . 
to 0 01 m was then added , and haemagglutinating in a similar experiment mouse embryo extract was treated with the results showed that incubation with lecithinase had no detectable effect on the ' ' haemagglutinating agents of c57 sarcoma or mouse embryo extracts . haemolysis by lecithinase present in the mixtures was not rapid enough , in the presence of b.a.l. , to interfere with haemagglutination . action of heavy metals and oxidizing agents , and its reversal by b.a.l. 
to samples of fresh , actively haemagglutinating , sarcoma extract , not containing b.a.l. , cus0o4 was added , to give final concentrations of m / 300 and m / 3000 . in both mixtures haemagglutinating activity disappeared within a few seconds ; there was a partial return of activity in the later , but none in the former , after addition of b.a.l. 
the haemagglutinating activity of these mixtures declined on standing at the same it thus appeared that the presence of heavy metals rate as that of a control . was not essential for natural inactivation . the addition of various oxidizing agents to a fresh sarcoma extract destroyed or reduced its haemagglutinating activity . 
the list includes serum antibodies ( naturally occurring , acquired , or experimentally induced ) , many viruses ( hirst , 1941 ; nagler , 1942 ; burnet , 1945 ; lush , 1943 ; burnet and stone , 1946 ; mills and dochez , 1944 ) , purified lipoids ( stone , 1946b ) , bacterial extracts ( keogh , north , and warburton , 1947 ) , the globulin fraction of egg - white ( commission on acute respiratory diseases , 1946 ) , some plant proteins such as ricin and abrin , basic proteins such as protamines and histones , inorganic colloidal acids and bases such as colloidal silicic acid , and many metallic salts ( landsteiner , 1945 )  . 
they are labile , while the latter are both fairly stable substances ; their red - cell species specificity is different from that of both lipoids and viruses ; they are unaffected by cl . 
welchii toxin , which destroys the haemaggluthinating power of lipoids and of vaccinia and ectromelia viruses ; their action is unaffected by normal serum , which inhibits that of lipoids but not that of viruses . in the manner in which temperature affects haemagglutinating titre they differ from lipoids . 
a sarcoma extract is absorbed by red cells which it agglutinates , but the cells , when they redisperse , as they do under certain conditions , are fully agglutinable by the same agent ; red cells agglutinated by a virus may also redisperse , but are then inagglutinable by that virus , and often by some others also ( burnet , beveridge , mcewin and boake , 1945 )  . egg - white is more like the tissue extracts in its haemagglutinating properties than are the other haemagglutinating substances referred to , particularly in respect of red cell species range , and effect of temperature on haemagglutinating titre , but it is not - reported to be labile . the general properties of the haemagglutinating agent of tumours suggest that it is a readily oxidizable substance which is active only in the reduced state . it may be a sulphydryl compound , but there is at present no positive evidence of this . neither inactivation by heavy metals , nor by oxidizing agents , is conclusive on this point . 
ascorbic acid , are ineffective , suggests that preservation is a specific effect of sulphydryl . natural inactivation does not appear to depend on the presence of heavy metals in an active state , and therefore it is unlikely that its prevention by b.a.l. 
has been stated not to inhibit the action of trypsin or papain ( webb and van heyningen , 1947 ) , but this work has been repeated here , with a it has been shown that b.a.l. 
protects it from the former . certain physical and chemical properties of the haemagglutinating agent of reasons are given for regarding it as an easily oxidizable it is destroyed by proteolytic b.a.l. 
was found to have definite inhibitory action on proteolysis by trypsin , the haemagglutinating agents of tumours and of normal tissues differ from and on digestion of egg - yolk by lecithinase . haemagglutinating viruses and lipoids in important respects . no qualitative differences have been found between the haemagglutinating agents of different tumours , or between those of tumours and normal tissues . the author gratefully acknowledges his debt to professor j . 
the possibility then had to be considered whether the hereditary condition acted through genetically transmitted differences in ovarian functional activity , or through an unequal response of mammary glands or genital tract to ovarian hormone . 
numerous and extensive observations were made with somewhat conflicting results , both on the nature of the oestrous cycle and on variations in organ sensitivity to hormonal stimulation , in strains of mice with differing mammary cancer incidence . when the existence of the third factor , i.e. 
as the number of determinations was so small , no statistical analysis of the figures was made , but there appears to be no real difference between the strains themselves , nor could the changes produced by foster - nursing be correlated with the presence or absence of the milk factor . 
armstrong ( b ) the character of the oestrous cycle . ( i ) virgin mice . - daily vaginal smearing of virgin mice of all groups was continued for periods ranging from 56 - 137 days immediately following canalization or weaning . 
the number of complete cycles during the observation period in addition , the average length of the whole cycle and of the was recorded . comparison actual period of heat for each mouse was calculated ( table ii )  . was then made between the fostered and non - fostered groups ( table iii )  . all cases comparisons show no significant difference . 
the p factor being of the order of o 1 - 0 5 . a second series of estimations was then made , in which two types of atypical cycle were excluded , in view , of the fact that such cycles might have been caused in the one type , a prolonged dioestrous by the repeated smearing technique . interval is observed , a state termed pseudo - pregnaney . this occurs naturally following sterile copulation and artificially following a great variety of stimuli . parkes ( 1926 ) regarded 7 days as the shortest duration of natural pseudo - pregtherefore all cycles with dioestrus greater than 7 days have been nancy . in the other type there is prolonged cornification or regarded as atypical . heat , a condition which has been produced experimentally by repeated vaginal smearing . 
the death of so many mice while still showing oestrous cycles detracts from the value of the results , but there was some indication that the range of age at which the menoe . 
from table i it is seen that vaginal canalization occurred later in group 3 ( free from milk factor ) than in group 1 ( having natural milk factor ) , but that the effect of foster - nursing was to increase the age in both cases , so that this cannot be correlated with the administration or withholding of milk factor . 
no undue importance has been attached to this as it occurs in the riii groups , in which foster - nursing is an unsatisfactory procedure , due to the possibility of the offspring receiving milk factor from their mothers during the interval between birth and transference to foster - parent ; also the difference is not statisticllay significant when all cycles are included and the samples are small . on comparing the two strains ( table iii ) there is some indication that the nature of the cycle shows a strain difference , which is unaffected by foster - nursing . comparing these results with the features of the oestrous cycle as described by other workers , there are , in general , appreciable strain differences in this respect ( table vi )  . it may be noted that the average duration of the cycle in young cba mice as observed by bonser ( 1935 ) is closely comparable with that obtained in the present experiment - 5 - 8 and 5 - 3 days respectively . foster - nursing as a means of administering the milk factor appears to be an entirely satisfactory procedure . thus the fact that no significant change in the character of the oestrous cycle was induced by this means , would seem to indicate that the factor does not influence the cycle . it is possible , however , that some adjustment to the new conditions created by the foster - mother 's milk is made during the period of suckling . the milk factor effect might be counteracted by the time puberty is reached and the hereditary condition alone revealed in the characteristics of the sexual cycle . the distribution and extent of two forms of atypical cycle were stuidied , as it was thought that , if they were caused by smearing technique , their incidence might vary in the different groups of mice . should , however , such cycles occur spontaneously , any observed strain difference in the duration of the cycle or e . 
bonser for her guidance and interest in the planning of this work and for her co - operation in the experimental procedure , which made consecutive daily observations over such a long period possible . i also wish to thank j . 
jv.7. reeeived for publication march 5 , 1949 . the pressure mincer described in this article was designed to facilitate the preparation of tumour suspensions for a quantitative studv of the survival of tumours in the frozen state . 
the choice of principle and design was determined by the foflowing requirements : ( a ) rapid reduction of tumour tissue to a suspension of fine fragments and separated ceus , ( b ) ease of aseptic manipulation , ( c ) mechanical simpficitv and ( d ) ease of cleaning . the essential feature of this instrument is a grooved plunger which operates in a closed eyfinder . 
the tissue is placed below ' the plunger , and on the application of pressure is forced through the grooves to the upper face . the niincer and its component parts are shown in fig . 
1. all parts are machined from brass and are chromium plated . * it is important to have the plungers ( c and d ) fitted to the body tube ( a ) with the minimum clearance consistent with smooth working . 
the nose of each plunger fits accumtely into the recess in the base plate ( b )  . similarlv the nose of the fine pliiin er fits accurately into the base of the coarse plunger . 
the diameter of the bodv tube is 1 - 6 cm . thus i mof displacement of the plung - er corresponds to a volume of 0 - 2 ml . cleaning is facilitated and design of the plungers is simplified bv the prov - ision of a detachable base plate . the operating screw ( f ) is of sufficient length to extrude the plungers from the lower end of the body tube . the various parts of the mincer are sterilized separately in glass tubes . 
the mincer is shaken immediately to disperse the mince if the coarse plunger has been used the cap and operating in the suspending fluid . screw are detached and the fine plunger is inserted . tlle cap and screw are replaced and mincing is completmed . the suspension may contain coarse fragments of fibrous tissue or portions of iiiince which have clotted before dispersal in the suspending fluid . these may be removed either by l ' ow speed centrifugation , or by filtering the suspension througli a column of glass ballotini of suitable diameter . the mincer , which is illustrated in fig . 
1 , is the sixth of a series desigiied to all models have proved most satisfactory for operate with grooved plungers . the purpose for which they are intended , and it might be suggested that this tvpe of mincer will , for special purposes , be suitable for preparing suspensions of manimalian tissue or fowl embryos infected with certain viruses or bacteria . i wish toacknowledge my indebtedness to w . 
at the time it was known that adenomas of this gland produced abnormalities in children and young adults ; it was therefore postulated that in later life , when gigantism and acromegaly were no longer possible , increased activity of the pituitary gland might be responsible for the production of tumours ( funk , 1917 )  . for a long time and for obvious reasons it was impossible to put this hypothesis to a test . 
thanks to the classical work of evans , li and simpson ( 1948 ) we now have the necessary tools for preliminary and systematic appraisal of the importance of various hormones of the anterior pituitary in tumour growth . the purpose of the work reported in this paper was to test the effect of various anterior pituitary and related hormones upon the growth of implanted tumours in hypophysectomized rats . 
as a supplement to this work we also dealt with the removal of certain organs ( mainly glands of internal secretion ) on the rate of growth of a transplanted rat tumour in otherwise intact animals . while there is no doubt that studies on this subject have already been made , it appears doubtful whether a systematic study was ever made of the effect of the removal of these organs upon the growth of one particular tumour . 
to check on the completeness of the surgical procedures used we have excised and weighed , at the time of sacrifice , those organs that are in a physiological relationship to the excised gland . for instance , in orchidectomy the seminal vesicles and the prostate were weighed . 
the diet was the same as in the hypoall animals received tap water , except the adrenaphysectomy experiments . lectomized animals , which received 1 per cent saline . in some experiments rats 282 c . 
no improvement in survival was noted . 1 of the controls 40 per cent had open vaginas , none of the castrates , at time of implant . a mortality was about 30 per cent of the animals operated upon . 8 mortality of 33 per cent of the operated animals , probably due to bartonella muria infection . 4 all animals received 60 mg . 
of alloxan from the time of tumour shaefer - hartman blood - sugar determinations were as implantation to the end of the experiment . follows : pre - implant 461 - 9 mg . 
using the enriched diet in our present work , we have attempted to restore tumour size by the administration of some anterior pituitary hormones and serum gonadotropin . growth hormone increased tumour weight of hypophysectomized animals so treated . however , this increase is directly proportional to the increase in body weight due to the hormone . thus , tumour weight difference per g . 
body weight difference is the same for both growth hormone treated and intact animals using untreated hypophysectomized animals as the base . it would appear that the influence of growth hormone on tumour growth is related to its effect upon the general metabolic condition of the animal . this is not true for acth and thyrotropic hormone . these two hormones cause a greater increase in tumour growth than could be expected from their effect upon body growth , using the same criterion . the ratio of tumour weight to body weight gain in adrenalectomized animals seems to confirm the fact that without adrenal hormones the tumour does not grow as well as in intact rats . this is the opposite of the effect ofacth treatment . 
the restorative effect of the latter hormone and the decreasing effect of the operation , greater than their respective actions upon body growth , show a clear effect on the tumour itself . 
our data are not as clear in the thyroidectomy experiments , however ; in one experiment the tumour - body weight ratio is indicative of the depressant effect of this operation upon the tumour . in the other experiment the administration of thyroxine to a thyroidectomized animal had the same effect as thyro286 c . 
ehrlich tropic hormone given to a hypophysectomized animal . increase in tumour size greater than could be expected from the body growth . this effect was an the effect of fsh and serum gonadotropin on hypophysectomized animals was not very marked either on absolute tumour weight or on tumour weight per g . 
body - weight gathe treatment of castrated males with testosterone may have stimulated tumour growth . splenectomy may have had some effect upon increasing tumour size by both criteria . while it is clear that diabetes per se reduces tumour growth , it is not so clear whether or not this reduction is related to body weight . it has been shown by mceuen and thomson ( 1933 ) that the reduced rate of tumour growth in hypophysectomized animals could not be solely attributable to the concomitant metabolic deficiency . in their studies on intact animals , evans , moon , simpson and li ( 1950 ) have found that only by long - continued treatment with growth hormone were they able to produce neoplasms , while shulman and greenberg ( 1949 ) in a shorter experiment , using intact mice , found no effect upon tumour growth with this hormone . 
persons. 0 - 020 0 090 0 219 the difference between any pair of rates for subjects of the same age group men . 0 031 0 070 0 - 509 0 - 018 0091 0 203 is , in no instance , as great as twice the standard error of the difference . * between 0 000 and 0 - 017 , depending on the proportion of the 14 female patients dying of lung cancer who were non - smokers . all areas , england and wales . a____ - __ an opportunity to test the first assumption is provided through the courtesy of the government social survey by the use of figures for the proportions of nonsmokers in the population , obtained in the course of an independent inquiry . the subjects were interviewed in september 1951 and were a random sample of the whole population ; the definition of a " non - smoker " was the same as that used by doll and hill ( 1952 )  . 
the total number of subjects interviewed between the ages of 21 and 74 was 2 , 268 ; the numbers in each age and sex group were proportional to the numbers in the whole population and the numbers of men interviewed - particularly in the older age groupswere considerably smaller than the numbers used for the previous calculations . the results obtained by the use of the government social survey figures for the estimation of the numbers of non - smokers living at june 30 , 1950 , are shown in table v . 
1 it is seen that the estimated rates for non - smokers are ( with one exception ) slightly less than the rates for women in rural districts - that is , the lowest recorded by the registrar - general . 
the exception , the rate for non - smokers aged 25 to 44 , is derived from the experience of the smallest number of bronchial carcinoma patients and is likely to be the least reliable ; this rate lies between the lowest and the lowest but one of the registrar - general 's rates . the estimated rates are , therefore , of the order which could be expected if they represented the basic minima independent of variable environmental factors . proportion of deaths due to causes other than smoking . if the estimated rates for non - smokers are accepted as appoximately accurate , it is possible to calculate the number of deaths from lung cancer in persons between the ages of 25 and 74 which would have been expected , in the absence of smoking , by multiplying the populations given in table i by the rates for all persons given in table iv and adding the results for each age group . the number expected in 1950 would have been 1 , 875 ; the number which actually occurred was 11 , 189 . it must be presumed that the other causes of lung cancer continued to operate irrespective of the use of tobacco , and it is , therefore , concluded that about one in five of the lung cancer deaths in persons aged 25 to 74 in 1950 , were attributable to causes other than smoking . sex ratio among non - smokers . table iv shows no consistent difference between the estimated rates for men and for women ; and in the age - group 45 to 64 , for which the rates must be presumed to be the most reliable because derived from interviews with the greatest number of patients , the rates are most nearly equal . 
the data are , therefore , not grossly inconsistent with the hypothesis that in the absence of smoking there is no appreciable sex difference in the incidence of lung cancer . the closeness with which the data fit the hypothesis can be seen more readily if the rates for women ( derived from greater numbers of non - smokers than the rates for men ) are used to calculate the number of non - smokers among men with bronchial carcinoma who would have been expected to have been interviewed , if men and women suffered the same mortality . for example , the mortality rate for women aged 45 to 64 , and resident in greater london , is estimated to be 60.6 / 673 , 000 ( table iii ) ; the estimated number of male non - smokers in the same age and area of residence sub - group is 38 , 100 ( table iii ) , so that the expected number of male non - smokers dying of lung cancer in 1950 is ( 60.6 / 673 , 000 ) x 38 , 100 = 3 * 43 . altogether 1 , 473 men in this sub - group died of lung cancer , 308 r . 
the expected numbers are calculated similarly for each sub - group when , by addition , the total number of male non - smokers expected to have been interviewed is found to be 6 - 1 . 
the number actually observed was 7 . the conclusion that in the absence of smoking there is no appreciable sex difference in the incidence of lung cancer appears to be contrary to the observations reported earlier that the mortality among women in greater london was lower than that among men at each level of tobacco consumption ( doll and hill , 1952 , table xii )  . 
the observations recorded among smokers are , therefore , not incompatible with the findings among non - smokers . mortality among non - smokers in different areas . from table iv it appears that there are no consistent differences in mortality among persons resident in areas of different density of population . moreover , the rates for greater london , other urban areas and rural districts are most nearly equal in the age - group 45 to 64 in which they are likely to be subject to the least error . the essential similarity of the rates , in the absence of smoking , can be seen more readily by a method similar to that used for comparing the mortality in men and in women . 
since there is no reason to presume that any one set of rates is more reliable than another , it is preferable to calculate the number of nonsmokers with bronchial carcinoma , resident in each type of area , who would have been expected to have been interviewed , if the estimated rates for men and 310 r . 
the data are not accurate enough for an aetiological relationship to be postulated . " no difference was found in the incidence of past attacks of asthma , chronic nasal catarrh , pleural effusion and pulmonary tuberculosis between the patients with lung carcinoma and those with other forms of cancer . 
the data did not suggest that the effect of smoking was through the intermediary of other respiratory diseases . should , however , pneumonia and chronic bronchitis really predispose to the development of bronchial carcinoma independently of any association they may have with smoking , it might be expected that their effect would be seen more clearly among non - smokers than among the whole population , in whom another cause can frequently be implicated . 
the past history of respiratory illnesses has , therefore , been analysed in the 47 patients who were non - smokers and the incidence of each disease compared with the incidence which would have been expected from the experience of all patients with bronchial carcinoma of the same sex and age groups ( table viii )  . it is clear that the data provide no evidence 312 r . 
pected. 5 * 2 62 summary . a " non - smoker " is defined as a person who has never consistently smoked for as long as one year at the rate of as much as one cigarette or one gramme of tobacco a day . estimates of the mortality rates from lung cancer among non - smokers are obtained from the registrar - general 's figures for the number of deaths attributed to lung cancer and for the total population ; and from the data , obtained in a clinical inquiry into the proportion of non - smokers among patients with bronchial carcinoma and among patients with other diseases ( excluding cancer of the oral cavity , respiratory tract or intrathoracic organs )  . the assump - tions required to enable the estimates to be made are bold and the number of cases of bronchial carcinoma among non - smokers is small . the rates obtained are , therefore , highly speculative , but it is thought that they are likely to be reasonably reliable since they are consistent with other experience . it is concluded : 1 . 
that occupational hazards and the previous occurrence of certain respiratory diseases are unlikely to be of frequent aetiological importance . i am most grateful to the officers of the government social survey for permission to make use of data collected during one of their inquiries and to trade sources for data of tobacco consumption , summarised in table vi . i am deeply indebted to professor a . 
of particles of 3 diameter or less during a 6 - weeks period : seven rabbits survived the injections for 7 months or more , and all developed malignant osteosarcomas , often with multiple primary sites . 
 we have repeated these experiments , using zinc beryllium silicate and beryl lium silicate and confirm the findings of gardner and heslington ( 1946 ) , although the proportion of survivors developing tumours is less in our series . 
x - ray analysis has shown that zinc beryllium silicate is really a solid solution of z11iisio4 and be2sio4 an analysis of the zinc beryllium silicate used in experimental groups b and c ( table i ) gave a composition zno 67 per cent , sio2 31 per cent , beo 2 per cent . 
the complex injected in experimental group a ( table i ) contained manganese , and had a composition zno 67 per cent , sio2 28 per cent , beo 2 per cent , mno 3 per cent . 
 examination of the tissues of animals dying immediately after injection and between 14 and 83 weeks after the injection of the silicates has shown the presence of silicate particles in the lungs , spleen and liver of all injected animals . 
the macro phages of the spleen first form giant cells of the foreign body type and even of the langhans type , but later the particles are contained in large syncytial masses , which show up to 100 nuclei in a single section . 
for some months the particles are present in isolated kupffer cells , but 3 to 4 months after injection the kupffer cells have increased in size and number to form cellular masses that fill and often distend the sinusoids . 
 the nodules are present in all injected animals whose bones have been searched for them , and in many cases are not associated with any other abnormality , either histological or radiological . 
these : findings correspond closely with those for rabbit 4 , where consideration of histological material in conjunction with radiographs leads to the conclusion that the radiographic appearances result from occupation of the marrow cavity by ossifying tumour tissue . 
histological examination of the femoral shaft shows a complete replacement of the normal marrow by tumour tissue , quite comparable to that seen in more advanced lesions in other animals , but not yet expanding the periosteum or causing erosion of the cortical bone of the sha part of the tumour is bony , part cartilaginous , while some areas consist merely of anaplastic spindle - celled tissue . 
numerous fat cells are scattered through the area , and the tissue varies from an almost acellular mass of fibres to a tissue composed largely of rounded and elongated fibroblasts . 
5 shows such an area , where a network of recently - formed bone - trabeculae is present , and where the osteoblastic cells covering the surfaces of the trabeculae are linked to the intervening fibroblasts by numerous intermediate forms . 
on the left side of the bone a few areas show a similar pattern , but there is also some more diffuse aggregation of radio - opaque material , corresponding to the formation of calcified cartilage by the tumourtissue . 
 in the lower part of each femur the marrow is completely replaced by a mass of bone - forming tumour tissue , which has begun to extend into the haversian canals of the dense cortical bone . 
some areas consist entirely of cartilaginous tissue , others show extensive osteoid and bony differentiation , while in some places the two tissues merge into a composite " osteochondroid " pattern . 
in several situations this was beginning to invade the dense cortex of the bone concerned , and to expand the covering periosteu in this animal the body of one lumbar vertebra contained a small rounded area of calcifying spindl~ - celled tumour tissue , 2 m in diameter , sharply demarcated from the surrounding normal marrow . 
this suggests that a continuous process is concerned in the development of frankly malignant tissue from the earlier pre - invasive lesion seen , for example , in the humerus of rabbit 4 . 
of soluble berylliu treatment of the refractory beryijium silicates with n , 100 hydrochloric acid produces a 10 to 20 - fold increase in the proportion of soluble beryllium in the aqueous suspension . 
 when stained by the method developed by denz ( 1949 ) for the histochemical detec tion of beryllium these sections showed that some soluble beryllium had left the necrotic area and stained the surrounding fibrous tissue faintly an_d diffusely . 
while the fully _developed tumours present a very different appearance from the medul lary bone formation that has been described as an early stage of malignancy , all stages of transition between the two can be traced . 
it is suggested that these indicate a continuous process of transformation from the early medullary : fibrosis to the later obviously malignant lesions , but this is only the interpretation of a number of morbid anatomical observations . 
 the relationship between the small nodules which are the first change seen within the bone marrow and the subsequent : fibrosis and malignant change is uncerta the nodules produced by the silicates containing beryllium and those produced by the zinc silicate are indistinguishable by the usual histological methods . 
it seems clear , however , that it is the presence ofthe beryllium within the nodule that is in some way responsible for the progressive changes that lead eventually to malignancy . 
although the beryllium silicates are considered to be insoluble , the observations on material injected intradermally into a rabbit showed that beryllium can be liberated from the particles and can diffuse into the surrounding tissues . 
 _therefore , if beryllium is to be held directly responsible for the tumours , it is much more likely that this beryllium is liberated from deposits in the marrow rather than derived from more distant deposits . 
the possibility that the silicate may play some part in the development of the tumours is excluded by the fact that gardner and heslington ( 1946 ) produced tumours with beryllium oxide , and more recently barnes ( 1950 ) has reported that two rabbits that received intravenous injections of a suspension of beryllium metal particles have developed characteristic sarcomas . 
 it is also apparent that the tissues themselves must contribute a factor , because similar nodules in the liver and lungs arouse very little : fibrous tissue reaction , while in the spleen : fibrosis and atrophy may go to extreme lengths without showing evidence of malignant change . 
 since this paper was submitted one further rabbit has died with a sarcoma of the pelvis 30 months after the last of a series of injections of zinc beryllium silicate . 
 the materials used in these experiments were supplied to us by the research laboratories of the general electric company , wembley , to whom we would like to express our thanks . 
1. given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . most people would agree that studies with transplanted tumours have given information concerning the autonomy of malignant tumours and concerning their histogenesis . however , there is a tendency to regard such studie8 as somewhat old - fashioned and this field of study more or less closed . in this paper it is hoped to show that there is still valuable information to be gathered of significance for general biology as well as oncology . general biology and tumour transplantation . the genetical laws governing the transplantation of normal and malignant tissues are now generally accepted . in both cases a complex of dominant genes is concerned which snell ( 1948 ) has called histocompatibility genes . there have always been two schools of thought conceming the nature of these genes , gne that the genes determine iso - antigenic differences , the other that they deterinine " individual differentials " - substances with somewhat mysterious and ill - defined properties . it is not easy to study these questions with normal tissues for technical reasons , one of which is that the number of factors is always high . 
with tumours the number varies , but may be low , and in addition tumours stimulate a better antibody response . iso - immune reactions are most easily studied in the erythrocytes , and it so happens that certain important iso - antigens in strong 's a strain of mice are shared by the erythrocytes and fixed tissues . 
four tumours from this strain have been studied genetically and serologically . the first two were studied in england ; in both it appeared that two histocompatibility genes were concerned , one of which was identical with a gene for an antigen known as antigen ii . 
by serological means it was shown that the occasionally iso - antibodies tumours contained another antigen in addition to 11 . were formed against it , but too irregularly 4nd at too low a titre to be of value for genetical work ( gorer , 1937 , 1938 , 1942 )  . those studied in america were both found to give single gene ratios in backcrosses . 
furthermore , in conjunction with snell and lyman ( gorer , snell and lyman , 1948 ) it was possible to show close linkage between antigen 11 and the locus for " fused " ( a tail anomaly )  . full information is to be published elsewhere , but it can be seen from table 11 that the linkage is close . 
the sera were produced by inoculation of c57 blacks . the similarity is not shown to the same degree by all sera . provisionally called h2 and h2d ( table 111 )  . closely related antigens occur in strains cba and - cm , so perhaps , there is a long series of alleles at this locus . animals carrying the we are not certain how many cross - overs there were . gene for fused may have norm ' al tails , and there are other difficulties associated with tumour transplantation that will be discussed later on . in backerosses involving 257 animals the recombinations were between 0 and 5 per cent . previous to these studies strong ( 1929 ) had used tumour transplantation to show sex linkage of a histocompatibility gene , and bittner ( 1933 ) found loose linkage with " dilution . " the location of h2 was first found by snell and lyman using tumour . 
inoculation without serological testing , and i hope i am betraying no confidences by saying that they have other instances of linkage as well . will be seen that tumour inoculation is - a most useful weapon in mapping chromoit is best to use serological tests as well . 
without going into details it may be said that they were not serologically unifortumour transplantation indicates that this polymorphism may be maintained by a high pure strains unless kept on a very small scale automatically mutation rate . it was shown by bittner ( 1935 ) that a dba crystallize into various sub - strains . tumour would not take in all sub - strains , and this has been found to be a common finding , having been seen with various tumours arising in strains c57 black ( law , gorer , unpublished ) and cm ( gross , 1947 )  . 
we have as yet no ' evidence that liability to disease is influenced bv antigemc constitution , or indeed very little knowledge of the antigens ' perhaps the polymorphism is connected with the risk of functions in the cell . iso - i ' mmunization during pregnancy ; this must be great where foetal absorption is common . 
if a species were completely uniform the risk would vanish , but it is unlikely that any species would be completely so over the whole of its rangle . on the other hand , if it were sufficiently polymorphic , the danger may be minimized . 
if a male has a rare antigen he would probably be heterozygous , and only half the litter would perish if iso - immunization took place . further , in matings with different males , a female would be unlikely to receive successive doses of lastly , it is possible that if a mixture of a large - number of the same antigen . antigens is received , dangerous titres of antibodies are not formed against any antigen ii does not appear to be very mutable , but here mice one of them . also seem very ' polymorphic . eight pure strains have been examined ; all are somewhat different . 
both the tumours tested in bar harbor gave such a ratio , but it was shown conclusively that they both contained at least two antigens . this is easily explained immunologically . antigens differ greatly in potency . 
gorer it is now well known that tumours may " mutate . " strain , we would get about 50 per cent susceptible progeny ( macdowell and richter , 1932 )  . errors of the above t - vpe are counterbalanced by precisely the opposite effect . some tumours may grow and kill the host in spite of iso - antigenic differences . there is considerable indirect evidence of this , but recently it has been possible to show that an animal succumbing to a tumour derived from any other strain may form high titres of antibodies ( gorer , 1947 )  . 
when first tested a tumour may appear to have upwards of 7 factors ; later on , only one or two . differences of this order cannot be accounted for by errors of the type just mentioned . there this might be due to mutations of domimust be some real antigenic alteration . this is extremely improbable ; one would have to nants to recessive alleles . have 6 or 7 homozygous mutations . 
what may happen is that one or more antigens crowd the others if for the sake of argument we assume that antigen 11 occupies 10 per cent out . of the surface of a normal cell , in a malignant one it may occupy 90 per cent . these experiments have the disadvantage that we have no really satisfactory for example , we can show that the cells of a myeloblastic normal control.. leukaemia contain increased amounts of antigen 11 , but we cannot get enough there is evidence that analogous antigenic normal myeloblasts for comparison . alterations may occur in human tumours . 
it is believed that all mammals ( and perhaps many other forms ) are highly polymorphic for histocompatibility genes . there is evidence that some of these genes have a high mutation rate in mice . 
we are probably all agreed that tumours , although usually , or perhaps always , preceded by abnormal local . this change consists in - an increase in conditions , arise by a sudden change . the rate of growth of a cell or a group of cells . in this way there arises a new cell - lineage distinct from its antecedents , and one which can propagate its new it has the character of a genetic property irreversibly and even indefinitelv . secondary changes may follow establishing subchange , a somatic mutation . sidiary cell - lineages , - some involving further increases of growth - rate and dedifferentiation , and all surviving and multiplying subject to natural selection . to these secondary changes i shall return later . the chemical conditions arising - in the changed tissue h4ve been examined bv santesson and caspersson ( 1942 ) , while the processes of its cell division have been described by koller ( 1947a ) for epithelial tumours and by la cour ( 1944 ) for pernicious anaemia , which is an analogous condition depending on the enhanced multiplication of the red blood precursor cells of the bone marrow . 
a single polymitotic gene in maize and extra heterochromatic chromosomes in millet both cause rapid and unwanted nuclear divisi ' ons in the pollen grain . in maize up to fou - r extra mitoses take place before the chromosomes have had time to divide immediately after meiosis . 
the nucleus is forced into division , and the unsplit chromosomes are scattered on a spindle which merely disperses them into a number of deficient nuclei , each of which is aga ' m compelled to divide before it is ready , and in this way the whole pollen grain is brought to ruin . in millet the mitoses ( also up to the nu ' mber of four ) do not occur before the chromosomes are split , but the whole pollen grain is none the i - ess consumed and killed in the end by the production of redundant nuclei . 
the pollen grain is thus , in , a sense , an encapsulated tumour ( darlington , 1947 )  . comparison of these cases of polymitosis in plants with animal tumours polyinitosis is not due to a somatic enables us to clear our minds on one point . it 4ffects every cell of a mutation . particular type in the body . 
the spontaneous tumour on the other hand arises at random both in time and space . it does not affect all cells of one type , but only one cell , nor is it the first cell of a particular normal type to arise which is affected . 
we are therefore confirmed in regarding the spontaneous tumour as due to a genetic change . this conclusion removes the contradiction between development on one side and heredity and infection on the other by withdrawing to a lower level ofanalysis . the organism is now seen from the cell point of view as a vegetatively propagating colony . 
how far does it enable us to distinguish between nucleus and cytoplasm as the seat of the mutation general the most efficient carcmogens ( such as the hydrocarbons ) and the most efficient agents of nuclear mutation ( such as mustard gas ) do not coincide . ' carcinogens do not damage the nucleus in proportion to their effect as carcinogens ( darlington and koller , 1947 )  . 
thus the nucleus again seems to beexcluded , just as it is by the direct evidence . but the evidence of xray effects is a more serious objection to a nuclear origin . x - ray damage leads to the development of cancer only when the dose has been so heavy as to damage the cytoplasm , and only then after prolonged delay . light doses which have a proportionate effect on the breakage of the chromosomes and the mutation of the 120 c . 
different conditions , in differeni tissues and in different stages of development . now plasmagenes , like all other such determinants , are susceptible to mutation . it is not possible certain of them control the formation of plastids in plant cells . to relate here ( as i have elsewhere ) the long detectiv - e story of investigations which have led in the course of 40 years to the revelation of the character and continuity of the plastid plasmagene . suffice it to say that , like the centromere and nucleolar organizer of the chromosomes , it its stock - in - trade on its back , which being a sac of green pigment marks or tags its owner and enables us to count its numbers in the cell and note its changes : it can mutate from green to white and vice versa , so that cells can occur with mixed green and white plasmagenes ; and , moreover , the same plastid may be starved white , as it were , by a stepmother nucleus , and recover its normal colour when it is put back with its own proper parent . prolonged coitus causes a paramecium to be infected by the kappa plasmagene of its mate . transplantation of an organ or of serum from a c02 - sensitive drosophila infects a nonsensitive fly , including its egg , with the sensitive plasmagene ( sonneborn , 1948 ; l ' he ' ritier ; 1948 )  . these infections , it will be seen , are artific ' ial , or at least unnatural . 
a number of aberrant conditions can be trans ' mitted from stock to scioii , and some even have arisen in a scion after it has been grafted on a healthy stock . these are artificial diseases ; they are not transmitted in nature , but only by grafting . 
we make a great mistake therefore in calling them viruses : they are proviruses ( darlington and mather , 1949 )  . conversely it has been know - n for 40 years that the . 
virus ricketmia is carried by the eggs of its vector dermatocentor . the terror of its infection for man has closed our eyes to the fact of its inheritance for the tick . 
what appears as a virus in one host is indistinguishable from a plasmagene in the other - and it may weh be that the plasmagene is the older ( l ' he ' ritier , 1948 )  . at the same time billingham and medawar ( 1948 ) have sh ' own that a selfpropagating particle.differentiated in development may by its diffusibility enjoy certain of the properties of infection - a situation which can come to our notice only when the particle is as innocuous , and the differentiation as trivial , as the black pigmentproducer of a ' piebald mammal . 
they have never been guilty of infection , and are therefore totally unadapted for or against it . the virus type may well have arisen likewise by mutation of a plasmagene , but it must have been a mutation followed by others adapting it to infection . moreover , this type falls into two groups by a most instructive distinction . 
the milk - agent , on the other hand , cannot be imagined as having arisen anywhere save in the moiise , itself , in whieb it is inherited by the milk instead of by the eggs . how accidental is the distinction between the infectious and the non - iilfectious particle is revealed most strikingly by one true virus . 
but in its new host it not only it even ceases to be inoculable . it loses the property ceases to be infectious ; of infection , and becomes transferable , if at all , only in whole cells . this is just the opposite transformation to the one by which proviruses arise or demonstrate their existence when two plants are grafted together . 
ob ection8and di ffil cultie8 . three grounds for suspicion of the plasmagene origin of cancer are now worth first , the cancer agents have one characteristic which will at once discussing . be noticed as marking ' them off from all previous plasmagenes . 
now the inheritance of lethal mutations out of the running . in the nucleus , when they are recessive in their lethality , is mendelian and can therefore be followed in experiment . 
a whole tissue can be destroyed by rendering its there is , however , no contradiction between nuclei unworkable in this way . the nucleus is the assumptions of a cytoplasmic cause and a nuclear cure . that is especially easy the only wheel in the cell that we can put a spoke in . to do in rapidly dividing cells ( darlington and la cour , 1945 )  . 
we are therefore merely attacking the malignant cell where it is weakest . incidentally the effect of irradiation in breaking the chromosomes and stopping the growth of the cells leaves no doubt that the remarkable deficient nuclei found in tumours by koller ( 1947b ) will have a limited life . 
the particle analysis : competitive propagation . the cancer miita - tion , as we saw , is often followed by other changes beyond these may be described under the the primafly one of change in growth rate . headings of dedifferentiation or the loss of tissue character , metastasis or migration , secondary mutation and various breakdowns of mitosis and of chromosome structure . 
what we now have to ask ourselves is how far these secondary changes require secondarv assumptions , and how far they are implied by the primary on.e. recent experiments equally with flies , infusoria and tomatoes liave revealed to us the conditions of normal development . 
thev have shown that it is iiot merely ebaracterized by adjusted between nucleus and cytoplasm , but between different self - propagating constithese adjustments can be broken down under experituents of the cytoplasm . in polymental conditions , with results which have ' been ace - lirately described . mitotic maize , as we saw , the cytoplasm runs away from the nucleus : in polyin paramecium , in mitotic millet the nucleus runs away from the cytoplasm . c02 - sensitive drosophila , and in rogue tomatoes , the whole system at a high temperature can be made to run away from a particular plasmagene . 
bv rapid growth the 250 kappa particles of one cell can be run down to one ( from which they can recover ) and then finally to zero ( from which thev cannot recover ) , so that the cell lineage has mutated . 
and in most virus diseases of course the virus runs away from the rest of the system or vice versa ; only occasionally is equievidently therefore the cell contains self - propagating elements librium reached . these limits are exposed in with different limits to their speeds of reproduction . a protozoan or a plant when the temperature is raised . 
these three classes of particle are conditional and interchangeable . cancerproducing particles fall into all three . the origin of cancer can therefore be ascribed to mutations in cytoplasmic determinants , indifferently infectious or non - infectious , which make themselves visible by causing the resumption of growth . further , the study of plasmagene and virus inheritance in relation to differentiation reveals a competitive propagation of cytoplasmic particles . this explains both the genetic control and the secondary development of cancer with its potential dedifferentiation and metastasis . the discovery of , not the cause , but the system of causation of cancer tells us nothing immediately about the cure that we do not already know . 
althougih biologists have for long been uneasily aware of the existence of a problem of cell heredity , its systematic analysis is the work of very recent cell heredity deals with the origin and maintenance of inherited character years . differences between cells ; more particularly , between cells that set up division its problems present themselves in their most acute , lineages by mitotic fission . if not most easily workable , form in metazoan development . the outcome of cellular differentiation in ( say ) mammalian development is the formation of a limited number of histologically definable cell genera , each one further subdivided into a variety of cellular genetic species . 
the genus " fibroblast , " as yet far from completely analysed , may be subdivided into cells which manufacture bone , cartilage , white connective - tissue fibres , and so on . genus " epidermis , " of which we have made a particular study ; includes the epidermal epithelium of the superficial skin , of the sole of the foot , the tongue , cells of each of these epidermal the claws or nails , the vagina and the cornea . species display a distinctive combination of structural or physiological properties . the epidermis of the sole of the foot , for example , has a characteristically high rate of cell division . 
but this has proved not to be the case . although plausible " phenocopies " of sole epithelium ( in the form of corns and callosities ) can be made by irritating the thin and relatively quiescent skin of the body surface , the difference between the division rates of sole and body epidermis is in fact " intrinsic " and inheritable . 
we have , for example , transplanted sole epidermis to positions in the body where it is protected by neighbouring hair and secure from mechanical irritation ; but even so , its characteristic growth rate has been maintained for at least two years , and thick pads of now functionless cuticle continue to form over it and may periodically be removed . claw epithelium tells the same story - more clearly , since the difference between claw and body epidermis is anatomically crude and obvious . 
bartholomew 's hospital , london , e.c.1. received for publication december 22 , 1948 . in an earlier publication ( robinson , 1948 ) the results obtained in a study of 17 - ketosteroid excretion in men of different age - groups were reported . special attention was paid to quantitative dlifferences in total ketosteroid excretion during the later decades of life . 
the method involves adsorption of the 17 - ketosteroids on a column of alumina from benzene solution followed by fractional elution with benzene of increasing ethanol content . the amount of 17 - ketosteroid present in each eluate is determined by the zimmerpreliminary experiments with the method described by these mann reaction . authors showed that separation and estimation of the steroid components could be achieved , but that reproducible results depended on careful standardization of the alumina and of the conditions of elution . 
of total 17 - ketosteroid was found to be the most suitable amount , and sufficient urine from a 24 or 48 - hour collection was hydrolysed and extracted to furnish a quantity in excess of 5 mg . 
was evaporated to dryness , and two successive portions of pure benzene ( 5 ml . ) added and distilled off to remove the last traces of alcohol prior to solution in benzene for adsorption on alumina . ( 2 ) stamndardization of alumina and adsorption columns . since merck 's alumina was not available , many samples of alumina were examined in order to obtain a product with the desired activity . spence " type h " alumina was finally selected for this purpose . 
when samples of this alumina are exposed to air ( by spreading the material in thin layers and exposing it to the moist air of the laboratory ) they gradually lose their adsorptive activity . this loss of activity is accompanied by a decrease in ph of aqueous suspensions of the alumina . suspensions prepared by mixing alumina with distilled water ( 1 part a1203 with 2 parts distilled water , by weight ) show an initial ph of 105 before deactivation and a gradual decrease to ph 9 - 3 after 2 to 3 days ' deactivation . the adsorptive power of the alumina was checked at intervals during the deactivating process by employing brockman 's method of standardization , using mixtures of dyes ( brockmann and schodder , 1941 )  . 
gurr , and further purified by solution in benzene and filtration through alumina followed by crystallization from benzene ( sudan red ) or alcohol ( sudan yellow )  . for standardization , 1 ml . of this dye solution is adsorbed on a column ( 50 x 14 mm . ) prepared from a suspension of alumina ( 7 g . ) in benzene - petroleum ether ( 1 : 4 ) and eluted with 30 ml . 
the alumina is considered to have suitable adsorptive power when the yellow band reaches the bottom of the column , while the red band moves down about 10 mm . the alumina columns for adsorption of the urine extracts are contained in glass tubes of 14 minternal diameter fitted with glass taps at the lower end . above the tap a plug of cotton - wool is inserted and the alnmina filling is added above the plug . 
fractions. these fractions are numbered successively 1 - 43 . ( 4 ) assay of eluted fractions . the solvent is removed from each fractional eluate and the residue dissolved in 2 ml . 
the observed colorimetric values are corrected when necessary for interfering chromogens ( robinson , 1948 )  . ( 5 ) recording of chronatograms and identification of con8tituents . the amount of 17 - ketosteroid in each eluate is plotted as the ordinate against an abscissa corresponding to the serial number of the eluate . 
with normal urine as many as nine peaks may occur , but in no case was the number less than seven . dingemanse , huis in ' t veld and de laat ( 1946 ) adopted a system of numbering in which successive peaks on the curve were indicated by roman numerals i - vi , the lowest numeral corresponding to the peak with the lowest eluate serial numbers . this system was used in the present work to facilitate comparison with the results of these authors . in some urines , however , small quantities of a component occurred which appeared to be different from any of the 8 main fractions , and this component was allocated to a sub - group hia . 
the substance originarlly referred to as " 17 - ketosteroid hi " ( dingemanse , huis in ' t veld and de laat , 1946 ; dingemanse , huis in ' t veld and hartogh - katz , 1948a ) was later shown to be i - androsten - 6 - ol - 17 - one ( dingemanse , huis in ' t veld and hartoghkatz , 1948b ; barton and klyne , 1948 )  . the dutch workers isolated this substance from the urine of a two - year - old girl with an adrenal tumour , but found only small quantities in the urine of normal persons . in the presence of hydrochloric acid this substance gives rise to chlorodehydroisoandrrone and dehydroisoandrosterone , and such a conversion may occur durijg the preliminary hydrolysis of the urine . in the present investigation confirmation of these identities for peaks mn , rv and v was obtained ( a ) by the isolation of the constituents of peaks rv and v and the preparation of crystalline acetates and benzoates therefrom , ( b ) by the urinary 17 - ketosteroids application of a colour reaction ( patterson , 1947 ) to the constituents of peak ni , ( c ) by identification of the peaks formed by authentic specimens of dehydroi&oandrosterone , androsterone and aetiocholan - 3 - ( x ) - ol - 17 - one . in order to study the reproducibility of the method and the type of curves to be expected , known amounts of dehydroisoandrosterone , androsterone and aetiocholan - 3 - ( x ) - ol - 17 - one were chromstographed alone and in mixtures containing varying proportions of these three steroids . 
the curves obtained in these experiments revealed immediately a problem that has to be faced in determining the amount of ketosteroid represented by any one peak . this is the complication introduced by the fact that the minimum values in the depressions between peaks are not always zero . 
the question therefore arises as to how the amount of ketosteroid corresponding to such minimum values should be partitioned between two peaks on either side of the minimuthree treatments were considered : ( i ) half of the minimum value might be allotted to each of the adjacent peaks ; ( ii ) the whole of the mimmum value might be added to the preceding peak ; ( iii ) the descending curve of the preceding peak might be projected down to the abscissa axis , and the additional values thus formed added to the preceding peak while corresponding deductions are made from the succeeding peak . these three methods were tested by their application to the model chromatograms obtained with the known mixtures of the three steroids menin general it was found that the projection method ( iii ) gave a tioned above . closer approximation to the actual amounts of the individual steroids than did either method ( i ) or method ( ii )  . this method was therefore adopted as standard for computing the amounts of 17 - ketosteroids represented by different peaks . a striking example of the reproducibility of the method has recently been encountered . 
1. the curve of case 0 was obtained on an extract from a 10 - day collection of urine from a boy aged three . in cases 1 and 2 it was necessary to collect urine for 2 to 3 days in order to obtain sufficient ketosteroid for analysis . 
the percentage distribution of the total ketosteroids among the different peaks is given in table i for all the normal males investigated with the exception of case 0 , in which the peaks normally present in adult curves could not be differentiated . urines from 2 cases of benign hyperplasia of the prostate and from 6 cases of carcinoma of this gland were also examined , and the corresponding curves and percentage compositions of these extracts are shown in fig . 
1. - 17 - ketosteroid content of eluates from chromatograms of urines from normal males case 0 ( not included in table i )  . three - year - old boy . incomplete differentiation into cases 1 , 8 , 17 represent the predominant type in which androsterone exceeds aetiocases 2 and 5 are unusual types in which aetiocholanolone exceeds androsterone . case 13 is a man , aged 50 ; 17 - ketosteroid output 20 mg . 
per day ; dehydroi / androcholanolone . sterone 34 per cent of total ketosteroid . .420 urinary 17 - ktogsrerrds case 20 case 19 ly~~~~~~~~~~~l case21 case22 case 23 case 24 case 25 case 26 eluate number fio . 
peak ii , average value 11 - 7 per cent , ranges from 2 to 34 per cent of the total , peak iv averages 30 per cent , with a range from 8 - 4 to 49 per cent , and peak v has a mean it will be seen that the of 24 - 2 per cent , with a range from 11 to 38 per cent . average output of androsterone exceeds that of actiocholanolone in the ratio only in three of the 18 cases shown in table i does the output of 1 - 25 : 1 - 0 . aetiocholanolone notably exceed that of androsterone ( cases 2 , 3 and 5 ) ; in the other 15 cases the amount of the former ketosteroid is either approximately equal to or less than the amount of androsterone . luriary 17 - ketosteroimds a comparison of the data shown in table ii with those of table i shows , however , what appears to be a significant change in pattern . in the urines from the cases of prostatic disease there is a consistent preponderance of peak v over peak iv . this is illustrated in fig . 
3. - relative excretions of dehydroisoandrosterone , androsterone and aetiocholanolone in normal males and in males with prostatic disease . cases 13 - 18 , normal . cases 19 - 20 , prostatic hyperplasia . cases 21 - 26 , prostatic carcinoma . amounts of dehydroisoandrosterone and aetiocholanolone are shown relative to the same amount ( 100ug . ) of androsterone in all cases . three major peaks are shown for extracts prepared from urines of six normal males over 50 years of age ( cases 13 - 18 ) , and from the urines of two subjects with prostatic hyperplasia ( cases 19 and 20 ) , and six patients with carcinoma of the prostate ( cases 21 - 26 )  . utrines from the carcinoma cases were obtained before oestrogen therapy was begun , and those from the benign hyperplasias before operation . 
for convenience of comparison , the amounts of peaks in and v are shown relative to the same amount ( 100lg . ) of peak rv ( androsterone ) in it will be seen that the most striking feature is the relative increase in all cases . peak v in the cases suffering from prostatic disease . in the normal males over 50 years of age the amount of this peak is equal to or less than the amount of androsterone , but in the 8 cases of prostatic disease it is present in considerable excess . as mentioned earlier , the preliminary experiments with known mixtures of 17 - ketosteroids showed that peak v normally indicates the presence of aetiocholanolone , and the total amount represented by this peak is a measure of the quantity of this steroid in the original mixture . it may be provisionally assumed , therefore , that the increased size of this peak in prostatic disease indicates a a . 
goulden relative increase in the output of aetiocholan - 3 - ( x ) - ol - 17 - one , although the possibility is not excluded that the incrased peak size is caused by the inclusion of a second substance ( of at present unknown nature ) which is eluted with the aetiocholanolone . 
a decision on this question can be made only when sufficient of the material present in this peak has been accumulated to allow of its identification , and experiments are in progress with this object in view . if it is assumed that aetiocholanolone is the substance that is increased in relative amount in prostatic disease , the cause of this increase is at the moment obscure . this ketosteroid has been recognized as a transformation product of more than one other steroid . callow ( 1939 ) isolated aetiocholanolone and androsterone in approximately equ%l amounts from the urine of normal men , and also much larger , but still approximately equal , amounts from the urine of a man treated with testosterone . these two isomers appear to be end - products of testosterone metabolism , and to be formed in about equal amounts from this hormone . 
an explanation of increased testosterone metabolism does.not appear to fit the present observations , however , since they show an increase in aetiocholanolone relative to androsterone . in this connection it may be remarked that callow 's finding of approximately equal amounts of these two ketosteroids in normal male urine was based on experiments with a pooled sample of urine , and an examination of table i shows that in individual urines the ratio between the amounts of androsterone and aetiocholanolone is not constant , although the average values are approximately equal . aetiocholan - 3 - ( c ) - ol - 17 - one has also been shown to be a metabolite of dehydrosoandrosterone . 
mason and kepler ( 1945 , 1947 ) found that administration of dehydroandsterone acetate by intramuscular injection in oil to patients with very low outputs of 17 - ketosteroids ( a man with anterior pituitary deficiency and a man and a woman with addison 's disease ) led to greatly increased excretion of both aetiocholanolone and androsterone . in the two male subjects the amount of androsterone excreted exceeded that of aetiocholanolone by approximately 75 per cent , while in the female patient the aetiocholanolone was about 4 - 5 times as much as the androsterone . the observed increase in the ratio of aetiocholanolone to androsterone in the cases of prostatic disease might be produced by an increase in the absolute amount of aetiocholanolone , or by a decrease in the absolute amount of androsterone excreted , or by both simultaneously . the amounts of 3 - ( , ) - hydroxyketosteroids , androsterone and aetiocholanolone excreted in 24 - hours were calculated for the cases illustrated in fig . 
3. normal males in the later decades have been examined . such a comparison will be possible when further in a study of z - ketosteroid excretion in patients with neoplastic disease dobriner , rhoads , lieberman , hill and fieser ( 1944 ) found abnormal patterns in in the one case of prostatic cancer reported in several cancerous conditions . their paper there was a great excess of aetiocholanolone as compared with androthey found a similar relation in a case of carcinoma of the larynx and sterone . a case of carcinoma of the breast . 
kennedy. from the endocrinology research department of the new zealand medical research council , medical school , dunedin , new zealand . received for publication july 30 , 1951 . previous communications from this laboratory ( griesbach , kennedy and purves , 1945 ; purves and griesbach , 1946 , 1947 ) described the induction of thyroid adenomata in rats by the long - term administration of goitrogens . bielschowsky , griesbach , hall , kennedy and purves ( 1949 ) showed that these thyroid tumours as well as those appearing under the combined influence of goitrogen and carcinogen could be transplante ' d into rats of the same strain . transplantation was only successful when the recipient animal was kept in a state of thyroxine deficiency produced either by treatment with goitrogen or thyroidectomy . 
from this failure of the transplanted tissue to grow in normal animals it was concluded that an excess of thyrotrophin was needed for the continued progressive growth of such tissue in a similar way as it had been necessary for the induction of the original tumour in the rat 's thyroid . therefore , these tumours were not considered to be " autonomous , " although they showed such signs of mahgnancy as a typical and progressive growth , invasion of surrounding tissues and formation of metastases . 
being injected subcutaneously in the flank of each the majority of rats were continuously supplied with a - 01 per cent rat . special mention is made in the solution of methylthiouracil as drinking water . text when no goitrogen was given . 
the thyroid was very large with a nodule protruding from the isthmus . part of this nodule was removed and two pieces were inoculated into two young rats which had been given 0 - 01 per cent methylthiouracil beginning 5 days previously . 
the acini varied in size , many of them being larger than the acini of the normal rat thyroid . in one line of transplantation this structure has been maintained up to the present day , and ' fig . 
6 shows the histology of a graft from the fourth generation of this fine . it will be noted that surrounding the larger acini there are numbers of small acini which suggest that new acini are being formed by budding from the larger ones . small cellular areas exist without well - defined acini , but these are considered to be areas of prohferation in which differentiation has not yet taken place . 
the acini are well filled with colloid , this colloid accumulation being always present even under conditions of strong thyrotrophic stimulation . this presence of colloid does not , however , indicate that the tumou ' rs are not influenced by thyrotrophic hormone , since when the thyrotrophic hormone production in the host is inhibited , these tumours undergo rapid regression . iodine , metaboli8ln of t , tumour . the iodine metabolism of the transplanted t , tumour was tested in rats bearing second generation grafts . table i shows the results of in vivo tests of the uptake of radioactive iodine in three rats after radioactive iodine had been injected . 
l. - chart of the first 6 generations ' grafting of the tbyroid tumour t , the treatment of the recipient animals is indicated on the cbart . solid black indicates animal bearing tumour . 
kennedy the evidence of secretory function in the t , tumour has been obtained from the results of transplants growing in totally thyroidectomized animals . these animals the ti tumour transplants do not grow continually , but appear to reach a maximum weight of about i g . 
of rats witb tumour growth . growth was obtained in the normal animals . this indicates the dependence on high thyrotrophic hormone levels , a dependence which is also displayed when animals bearing growing tumours during the administration of methylthiouracil have their medication stopped . 
the large irregularly - shaped acini which were prominent in the ti colloid was present in some of the acini but was less tumour were not seen . prominent than in the t , tumour . 
the nuclei were hyperchromatic and irregular in sha e . ( 2 ) embedded in this adenomatous tissue was a nodule of tiss ' ue distinctly different in staining properties . in this tissue , which had a well - developed acinar structure corresponding to a very hype ' rplastic normal rat thyroid , the cells were large with abundant cytoplasthe nuclei were round and did not contain excess chromatthe lesser numbers of nuclei and their weaker staining properties ' ' were responsible for the pafer appearance of these nodules in the 306 h . 
the fact that such tissue always forms microscopic nodules and has not ever formed a large part of the grafts in any of the inoculated animals suggests that this type of tissue is a slow - growing variant which arises spontaneously within the adenomatous tissue . 
the nodules of this type seen in the fourth generation appear to have arisen from the modified adenomatous tissue of these animals mentioned above which differs from that of the original tumour . ( 3 ) the third type of tissue is an anaplastic carcinomatous tissue without any in animal 253 , no . 
in weight being observed after 10 days . in the fourth week after transplantation animals with the rapidly growing tumours began to succumb . in a proportion of the animals , however , the growth was slower and the animals survived 3 or 4 months . 
the histology of ah these tumours was that of the anaplastic carcinoma described above as the third type of tissue seen in the fourth was successfury maintained by serial generation . transplantation , and - in later transplantations successful grafts were obtained in up to 80 per cent of those inoculated . 
the t22 tumour has now been transplanted through 7 consecutive generations and has not undergone any progressive change in either malignancy or histology . histology of tumour t22on the cut surface two regions cciuld be regularly seen , the outer with a shiny grey appearance , 5 to 8 mbroad , and the central one coloured a pale orange frequently a brown albuminuous fluid had replaced with a crumbled surface . mioroscopic examination revealed that , ' generally , the inner the central tissue . zone ofthe tumours consisted ofdegenerating cers or amorphous masses , infiltrated by round ceus , and isolated cers with strongly eosinophihe cytoplasm and pycnotic nucleus , lying , between the cell debris . the outer zone , microscopically , appeared simflar to that of the anaplastic nodule of the original tumour , and this description apphes to both tissues . 
unusuary large tumour cefls were present which had slightly basophilic cytoplasm and nuclei with ir - regulariy dispersed chromatin . giant cells were present with nuclei varying in number from 2 to ' 8 . 
the numbers of giant cers found varied somewhat in different animals and were relatively few they have , however , been a constant feature of in the first animal examined . the tumour ' and we consider that the tumour should , therefore , be classed as a giant cell carci ' noma of the thyroid . mitotic ftgures are frequent and many of them atypical . effe , ct of t22 tumour on the host . the striking feature of the t22 tumour is that it kirs its host in as short a period as 3 weeks . there is , however , a considerable variation in the rate of growth in different animals , some of them surviving for 2 months or more . those animals which died within 3 to 4 weeks after inoculation , the animals showed at autopsy an extreme state of emaciation with atrophic muscles and thin soft bones . 
was lower than ' its weight at the time of transplantation . it appears , therefore , that the rapid growth of these tumours withdraws nutrients from the host and leads to the state of emaciation described . effect of thyrotrophic hormone level on tumour t22 ' the t22 tumour has been found to grow equally wer when inoculated into normal animals or animals receiving methylthiouracil . moreover , in normal animals in which this tumour is growing , no inhibition of the growth rate has been observed when the an ' imals were treated with thyroxine injections . in some selected animals bearing this tumour in which the growth was relatively slow this treatment did not produce any marked some were treated with thiouracil . acceleration in the growth of these tumours . 
the t22tumour , therefore , does not show the dependence upon thyrotrophic stimulation which was shown by its parent tu ' mour . iodine , metabolimof the t22 tumour . rats bearing the t22 tumour were injected with radio - active iodine and 24 hours later were killed , and the radio - active iodine concentrations in the tumour tissue and in the blood plasma were measured . 
kennedy considered that both the action of the carcinogen plus the hyperplasia resulting from the goitrogen were necessary for the induction ofthyroid tumours . however , griesbach , kennedy and purves ( 1945 ) reported that the prolonged action of goitrogens alone led to the formation of tumours , which in a later publication ( bielschowsky , griesbach , hall , kennedy and purves , 1949 ) were shown to be similar in all respectsto those appearing after the carcinogen treatment . 
with goitrogen alone , however , tumours are later in appearing and are fewer in number . these tumours aredependent upon high thyrotrophic hormone level for their in normal animals such tumours , either in the thyroid or as grafted growth . - tumours , undergo rapid regression and eventually disappear , so that they cannot be induced to reappear by the subsequent administration of goitrogen . 
the simplest explanation , therefore , of the role of the goitrogen in the production of these tumours is that it induces the high thyrotrophic hormone production without in this view , the goitrogen would not have which such tumours cannot grow . any influence on the formation of neoplastic cells . it is , therefore , considered that neoplastic cells arl 'se spontaneously in the normal rat thyroid , and that when conditions are suitable for the growth of such cells , visible neoplasms result . 
in weight are commonly produced.. the continuous growth of such tumours 4nd the propagation of them by transplantation leads naturary to the selection of fast - growing types of tissue so that an increase in mahgnancy with transplantation is to be expected . however , modifications of the adenoma are not invariably in the direction of increased mahgnancy . 
we consider that the type of nodule described as the second type of tissue observed in the fourth generation transplants is a relatively slow - growing benig - n structure which reappears in successive generations , and which by virtue of its slow growth and lack ofmetastasizing power cannot be selectively propagated by transplantation . 
on the other hand , the truly malignant , invasive and metastasizing carcinoma will , when it once appears , invariably supplant entirely the benign structure in two , or three at the most , generations of grafting . evolution of malignancy in benign tumour8 . while it has been recognized that transplantable mammalian tumours show increases in mahgnancy on transplantation , it has been often assumed that such increase in mahgnancy results from a gradual adaptation to the host or a gradual modification of the original tumour . 
the thyroid adenomata appearing in - the rat thyroid are all dependent on thyrotrophic hormone for their ' existence , and in fact require higher levels of thyrotrophic hormone for their continued growth than does the normal thyroid tissue . investigation of human thyroid neoplasms with the aid of radio - active iodine have shown that many of these are in fact susceptible to the stimulating influence of thyrotrophic hormone , although neoplasms in human material have not been described which are so entirely dependent upon thyrotrophic hormones as are these primary rat thyroid tumours . 
the existence of undoubted stimulating effects of thyrotrophic hormone in human thyroid tumours supports the view that these rat thyroid tumours are not exceptional , although their high degree of dependence on thyrotrophic hormone sets them somewhat apart from the thyroid tumours encountered clinically . there seems no reason to hold the view , as some people have done , that these rat tumours should not be classed as neoplasms , since in their case the stimulating hormone or factor on which their 310 h . 
kennedy growth depends ' is well characterized and can be artificially manipulated so as to presumably as further knowledge is gained the control the tumour growth . stimulating influences which condition the growth of other tumours win be discovered , but this should in no way affect their classification as neoplasms . it is important to note here that from a tumour at first dependent upon a hormonal imbalance for its growth , there has been derived a mahgnant neoplasm which is no longer dependent upon the stimulating influences which condition the growth of the more benign original tumour . 
thus where mahgnant tumours are found which are not susceptible to any known hormonal influence , the effect of hormonal imbalance in the production of such tumours is not excluded . 
1. received for publication january 21 , 1949 . kennaway , kennaway and warren ( 1944 ) found that injection of certain carcinogenic substances , including 1 : 2 : 5 : 6 - dibenzanthracene , caused an increase in the concentration of ascorbic acid in the liver of mice , whilst some noncareinogenic , chemically related substances ( naphthalene , anthracene , phenanthrene ) did not show this effect . in an investigation of the growth - inhibitory action of the carcinogenic hydrocarbon 1 : 2 : 5 : 6 - dibenzanthracene ( elson and warren , 1947 ) it was found that the inhibition of rat body growth and of the growth of walker rat carcinoma 256 ( elson and haddow , 1947 ) by this substance is dependent on the protein content of the diet of the animals . 
1 : 2 : 5 : 6 - dibenzanthracene in the rat usually results in little growth inhibition for about the first 14 days , after which the animals at varying intervals lose weight and die . 
on a 10 per cent protein diet after the same dose of the carcinogen , immediate inhibition of growth occurs and persists for a prolonged period , usually until the death of the animal . in considering the cause of the rise in liver ascorbic acid brought about by 1 : 2 : 5 : 6 - dibenzanthracene , it appeared possible that this was a result of the growthinhibitory action of the compound . 
there was some evidence ( elson , unpublished communication ) that the growth inhibition was accompanied by a fall in the succinic dehydrogenase component of the succinoxidase enzyme system of since the cytochrome oxidase component of this enzyme system is the liver . capable of rapid oxidation of ascorbic acid , it seemed possible that the rise in ascorbic acid after 1 : 2 : 5 : 6 - dibenzanthracene might be the result of less destruction by such oxidation occurring owing to the decreased content of the oxidizing enzyme . 
they were weighed every 2 or 3 days . high and low protein tabls . in later experiments it was found necessary to estimate the amount of food consumed by each rat , and in order to facilitate this 25 per cent and 5 per cent protein diets were made up in 5 g . 
tablets in the shape of cylinders of approximately 3 cdiameter and 0 - 5 chigh . this necessitated replacement of some of the starch in the original diets by cane sugar and glucose in order to obtain a firm tablet . 
from the control experiments , in which only arachis oil was injected , .it is seen that the variation of the protein content of the diet itself has practically no influence on the level of ascorbic acid in the liver . 
the animals fed the 5 per cent protein diet grow only very slowly , but the effect of dibenzanthracene on them is not to make them lose weight , as would be expected if the magnitude of the growth inhibition was proportionate to that observed in animals this smaller effect is presumably maintained on the 10 per cent protein diet . related to the lower total metabolism of the 5 per cent protein diet animals , and 152 l . 
5 , although complete inhibition of growth occurred , and at one time actual loss of weight , the food intake , after the initial drop , rose steadily to a value higher than the level before administration of dibenzanthracene at the time the animals were killed for determination of liver ascorbic acid the actual degree of growth inhibition was greater than the maximum previously obtained with the 10 per cent protein diet animals . 
the degree of growth inhibition is dependent on an equilibrium being established between the rate at which the animal is metabo - _wa% growth rate fooie ratn ? j final liver wt ascorbic acid o * + |~~ 1i 135 6 - 14 ( 1 * 1 [ oo llll , , illlllllllllll , lll ( 134 1i111 . , g14 3 ) o3 ) _ t .%1 5 days 20 25 7 - 02 ' 10 - ~ _ ~3124 8 - 17 7 - 63 fig . 
in animals fed 20 per cent protein diet liver ascorbic acid increases before any marked growth - inhibition is observed , and the increase is thus not an indirect consequence of the inhibition of growth . the growth - inhibition is not merely the result of a lowered intake of food . the animals may react to treatment with dibenzanthracene by maintaining or even increasing their daily food consumption , although growth is completely inhibited . this suggests that a profound change in basal metabolism must have taken place , and the rise in liver ascorbic acid may be associated with this change . we wish to express our thanks for generous grants supporting this investigation , from the british empire cancer campaign , the anna fuller fund , the jane coffin childs memorial fund for medical research , and the u.s. 
this proportion insured chrom - atograms of satisfactory color intensity . ethanol was used for the extraction because it is believed to be a solvent which causes minimum proteolysis during denaturation of protea 78 per cent , solution has been found to be optimal for solubility of the amino - acids and for the extraction of non - protein nitrogen only . a two - dimensional chromatogram on whatman no . 
new amino - acids appeared at varying intervals in each of the four tissues 'studied , and - the changes were consistent fot each tissue in ar of the rabbits studied . 
they found that muscle temperature and ph decreased with isch - aemia . proteolysis found during ischaemia may be.caused by accelerated enzyjnic degradation in a more acid medium as the optimum h of tissue cathepsins hes between 4 and 5 ( willstiitter and bamann , 1929 )  . , in any event ischaemia m , 4y alter the chemical characteristics of a tissue . 8ummaim ' the number of free amino - acids extractable from clinical cancers of similar histogenesis - varied widely , due apparently to deliberate occlusion of the arterial supply in the , course . 
mitchell. from the department of radiotherapeuties , university of cambridge . received for publication july 8 , 1953 . attempts to evaluate tetra - sodium 2 - methyl - i : 4 - naphthohydroquinone diphosphate ( compound i , synkavit ) as a radiosensitiser in the radiotherapy of malignant tumours have been in progress since november , 1946 . 
at first the compound was used alone and in conjunction with palliative x - ray therapy in some very it soon became evident that in general the compound alone had advanced cases . no therapeutic effect in malignant tumours . 
at the same time , all cases of inoperable carcinoma of the bronchus were treated , if possible , by the combination of x - ray therapy and compound i ; the results were assessed mainly by survival times from the first x - ray treatment and the first sympton and were compared with those which had been obtained previously in the same department with x - ray therapy only , and which were very similar to the depressing results obtained by most of the workers . slowly the difficulties of this type of clinical investigation were appreciated . moreover in 1950 erratic results were encountered both in the clinical trials and in animal experiments . it seems likely that these findings were attributable to the thermal instability of compound i in aqueous solution in the absence of oxygen ( mitchell and simonreuss , 1952 )  . 
much more consistent results have been obtained since the ampoules have been stored in a refrigerator at about 30 c . the evidence obtained by these preliminary studies was sufficiently suggestive to justify further work , but the methods used were clearly inadequate . it became essential to employ properly designed methods for the clinical evaluation of radiosensitisers . 
the importance of these methods was emphasised at the time by their use in clinical evaluation of chemotherapeutic agents in tuberculosis ( medical research council , 1948 , 1950 )  . investigations of this type in the study of radiosensitisers have been in progress since april 1951 ; as yet the only results available are for the treatment of inoperable carcinoma of the bronchus . the results of the earlier studies have been reported by mitchell ( 1948 , 1949a , 1949b , 1950 , 1951 )  . this work has been criticised by gellhorn and gagliano ( 1950 )  . otte ( 1949 ) reported briefly on the combination of x - ray therapy and some synthetic vitamin k substitutes in the treatment of 300 patients with advanced malignant tumours and claimed improved palliation . 
mitchell selection of types of malignant tumour for investigation . it is suggested that a practical minimum requirement for usefulness of a radiosensitising chemical agent is that its employment in combination with radiotherapy should produce a mean survival time after treatment double that after radiotherapy only , for the type of malignant tumour treated . 
hence taking into account the frequency distribution of the survival times , it is likely to be necessary to observe the cases treated by combined therapy for an interval of at least 6 times the mean survival after radiotherapy only . 
to this time must be added the time necessary to accumulate an adequate number of cases . this approach is limited to the study of types of malignant tumour in which the results of present methods of treatment are bad and in which the time of survival is short . inoperable carcinoma of the bronchus is an obvious choice for investigation it is the only common type of malignant disease with a very short natural history . despite advances , only a small proportion of all cases of carcinoma of the bronuntreated patients are often very miserable . 
the chus are curable by surgery . results of treatment of inoperable cases by present methods , including radical nevertheless x - ray therapy in general has sufficient x - ray therapy , are poor . palliative value apart from prolongation of life per se , to justify attempts to improve this . with x - ray therapy only , inoperable carcinoma ofthe bronchus appears to have a a rather uniform natural history . 
of these , histological evidence was obtained in 245 patients and their average survival from the first symptom was 14 - 3 months and from the beginning of x - ray treatment 4 - 5 months . 
7 of this paper , it can be deduced that of 729 patients , only 45 survived 8 months , 20 survived 12 months , 8 survived 18 months and none survived 3 years following in this series , the best survival rate - 4 - 7 months - was in a x - ray therapy . group of 80 patients who received a total dose as measured in air of 5000 - 6000 r ; higher and lower doses were associated with poorer results . 
the most important factor in determining survival is the extent of spread of the disease when treated . this evidence suggests that in representative series of cases of inoperable carcinoma of the bronchus receiving x - ray therapy , the mean survival from the beginning of treatment is about 4 - 5 months . 
to try to minimise its shortcomings , all the cases to which it is applied have been followed until death , or for at least 3 - 1 years and in assessing the results the proportion of cases showing unexpectedly good response has been compared in the case of patients treated by means of radiotherapy combined with intravenous compound with those treated by radiotherapy combined with intramuscular compound . the results of various types of treatment of patients with advanced malignant tumours classified as inoperable , stages iii and iv , and recurrent of all types except carcinoma of the bronchus using compound i in treatments from november , 1946 to july , 1909 inclusive , and assessed up to 31 december , 1952 , are summarised in table i for the cases verified histoan essentialy similar table has been prepared showing the rather logically . poorer results for 56 cases not confirmed histologically , but with reasonably the cases allocated to this preliminary survey were the great certain diagnosis . majority of the patients with advanced malignant tumours in which it was considered that present methods of treatment were not likely to give satisfactorily results . 
when the survey was started it was not realised that the use of the compound by intravenous injection was likely to be more effective than its use by intramuscular injection . the allocation of the patients to treatments involving the two mnethods of administration of the compound were certainly not a.t random , and the possibility of bias by the use of intramuscular injection in the more seriously ill patients cannot be excluded . however , there is no reason to suppose that such bias would influence the proportion of cases showing unexpectedly good clinical response . the resultsof the preliminary general survey summarised in table i suggests that the proportion of cases showing unexpectedly good clinical response is greater 316 j . 
one case of advanced carcinoma of the body of the uterus was treated by means of palliative x - ray therapy combined with intravenous compound , and one case of carcinoma of the tongue recurrent after previous x - ray therapy was treated successfully by radical x - ray therapy combined with intramuscular compound . of the 56 cases not confirmed histologically three survived more than 4 years after treatment . 
two of these showed an unexpectedly good response ; one was a woman aged 46 , who had a stage iv carcinoma of the breast with supraclavicular glands , and was treated by means of radical x - ray therapy combined with intramuscular compound , and the other was a case of a mixed parotid tumour which had failed to respond to further x - ray therapy , but appeared to be checked and rendered quiescent by intravenous compound onlv . 
the results are treated by statistical methods . it is suggested that estimation of the survival times , especially that from the beginning of x - ray therapy , is the most reliable criterion for objective assessment of the results of treatment . special attention has been paid to the influence of the extent of the disease , age and sex , the histology , the minimum tumour dose and overall time of the x - ray therapy and the details of administration of the compound . the general principles involved in the consideration of these factors are essentially the same in these preliminary studies as in the later clinical trials and except where relevant , will be discussed in this connection . 
the results of treatment were reviewed as from this group of cases of inoperable carcinoma of the bronchus december 31 , 1952 . comprises all those cases referred in which the diagnosis is accepted as proven according to the criteria given below , with the exclusion of patients not likely to benefit by palliative x - ray therapy . 
no technically operable cases and no postoperative cases are included . the control group consists of those cases of inoperable carcinoma of the bronchus selected according to the same criteria , which were treated by x - ray therapy only , using similar methods , in the same department between 1943 and may , 1947 . the difficulties of this method of providing a control group are recognised and include the possibility of introduction of serious errors of sampling . 
the use of alternate cases as controls was attempted but was abandoned as impractical . however , it has been shown that the relevant properties of the control and treated groups apart from the length of survival after treatment are substantially the same . it is found that the results of x - ray treatment in the present control group are substantially identical with those obtained in two large published series . 
mitchell ( b ) cases with positive histology on biopsy specimens of supraclavicular nodes , cutaneous or other metastases but in which there was no bronchoscopy or the bronchoscopic appearances were not definite ; this sub - group consists of 8 control cases and 8 treated cases ; ( c ) one case ( treated ) with typical bronchoscopic appearances together with metastases in the skin , confirmed histologically . these additional 15 control and 18 treated cases are usually more advanced and are in general less comparable than the " histologically verified " cases but must be considered with them to give a complete picture . 
of the combined series of " confirmed cases , " there are in all 34 in the control group , which was treated by x - ray therapy only , and 47 in the treated group , which received x - ray therapy together with the compound . 
as anticipated the results of treatment of the " confirmed cases " are poorer than those of the " histologically verified " cases . other groups of cases which cannot be included in the main part of this investigation may be summarised as follows : ( i ) cases with typical radiological appearances together with metastases at some stage but not verified histologically and in which there was no bronchoscopy or the bronchoscopic appearances were not definite . in these cases the diagnosis can only be regarded as probable . in the control group , there are 12 cases ; the mean time of survival is 8 - 50 months from the first symptom and 3 - 67 months from the first x - ray treatment . in the treated group , there are 13 cases ; the mean times of survival are 12 - 15 months from the first symptom and 6 * 31 months from the first x - ray treatment . ( ii ) cases rejected as unsuitable for palliative x - ray therapy . during the control period , there were 18 of these cases , of which 10 were histologically verified and during the period corresponding to the treated group , there were 7 cases of which only one was verified histologically . the enumeration of rejected cases is unsatisfactory because of selection by the physicians referring these advanced cases . hence the total number of cases with a reasonably certain or probable diagnosis of inoperable carcinoma of the bronchus is 131 . 
of the 7 cases of squamous carcinoma , two showed keratinisation . of the 29 verified treated cases , there are 17 cases of stratified carcinoma , of which 10 are squamous and 7 non - squamous , 3 cases of adenocarcinoma , 8 of anaplastic carcinoma and one an unusual poorly differentiated lesion . of the 10 cases of squamous carcinoma three cases show definite keratinisait is interesting tion ; in two further cases there is a suggestion of keratinisation . to note that all these cases responded well to treatment . the control group was treated by x - ray therapy only , from 1943 to may , 1947 . 
even if the cases which survived only one month from the first x - ray treatment are excluded , to meet the criticism of a possible difference in selection of the cases , the difference in survival after the first treatment between the treated group ( mean survival 9 - 68 months ) and the control group ( mean survival 5 - 23 months ) is still significant ( n = 39 , t 2 * 457 , p is slightly less than 0 - 02 )  . considering the histological classification , the only type providing adequate numbers is the stratified carcinoma . 
the basic principles involved are well understood as a result of the classical statistical studies of fisher ( 1925 , 1935 ) on the design of experiments , and on the work of hill ( 1952 ) on the design of clinical trials . 
in general , in considering therapeutic trials in the cancer field , the design must be capable of giving a completely convincing statistically significant quantitative result as efficiently as possible with a minimum number of patients and must be acceptable and above reproach from the moral and ethical points of view . 
the most important single feature of the design is random allocation of the patients to two or more alternative forms of treatment the value of which is being compared . for assessment of the result of treatment , it is necessary to select relevant criteria capable of objective measurement such as the survival time suitably specified . 
at the same time , it is absolutely essential to provide for each individual patient treatment which is the best possible according to present knowledge . accordingly , it may be , and probably often will be , necessary to plan to depart from what may be termed the theoretically ideal form of experiment with deliberate sacrifice of some information . in the present example of the attempted evaluation of tetra - sodium 2 - methyl1 : 4 - naphthohydroquinone diphosphate ( compound i , synkavit ) as a radiosensitiser in the radio therapy of inoperable carcinoma of the bronchus , cases were allocated at random to one of two alternative forms of treatment , x - ray therapy combined with compound i administered by intravenous injection ( x + i - vs ) and x - ray therapy combined with compound i administered by intramusculrzr injection ( x + i - ms )  . 
the latter is regarded as the control group . the preliminary clinical studies and animal experiments suggested that the results of x - ray therapy combined with intramuscular compound ( x + i - ms ) were slightly better than those of x - ray therapy only , and that with comparable doses of compound it was likely that x + i - vs would produce better results than x + i - ms . 
mitchell tests of heterogeneity must be made . clinical therapeutic trial must be justified a posteriori . it is necessary to - examine the possible importance of all the various factors which may be relevant including age , sex , extent of the disease at treatment , histology and / or cytology , the minimum tumour dose and overall time of radiotherapy , the total dose and overall time of administration of the compound and in particular the certainty of the diagnosis . it does not seem possible to estimate in advance the number of patients required . this will depend on the differences observed between the groups and subgroups . 
the results are likely to be of sufficient importance to necessitate the use of stringent criteria of significance . the randomisation was made on the basis of a provisional diagnosis of inoperable carcinoma of the bronchus , made when the patient was first examined by the staff of the department . 
some of the cases were referred with histological evidence from bronchial biopsy . in other cases , there had been no previous investigation and the provisional diagnosis was based on clinical evidence only . all cases were investigated critically , as necessary , and every effort was made to obtain histological and / or cytological verification of the diagnosis . almost all the sections have been reviewed by a . 
cruickshank , of the sims woodhead memorial laboratory , papworth , for most of the reports on specimens of bronchial aspirate . particular attention has been paid to the establishment of the diagnosis of carcinoma of the bronchus and the identification and exclusion of cases of bronchial adenoma and related tumours of low grade malignancy apparently arising in bronchial glands . 
barrett and may be summarised as follows : ( 1 ) stratified carcinoma , either squamous or non - squamous , the degree of keratinisation or differentiation being noted , ( 2 ) adenocarcinoma , ( 3 ) anaplastic carcinoma , including " oat - celled " carcinoma , and ( 4 ) others , including " alveolar " cell carcinoma and carcinoma not classifiable . 
the histological classification of carcinoma of the bronchus is often difficult because of the existence of intermediate types and is somewhat arbitrary in some cases . on the clinical side , a careful and full medical examination is made , including usually indirect laryngoscopy . it has been considered important to determine the extent of spread of the disease with special reference to the early detection of metastases in bones . the occasional cases originally considered inoperable and in which it has been found possible subsequently to carry out some type of surgery have been considered separately in the assessment of the results . 
and increased daily in steps of any undesirable reaction such as excessive coughing , nausea , faintness the chest is observed after intravenous injection , the dose which there is no such reaction and then cautiously increased again on the following days , sometimes by steps of for most patients , for intravenous injections the best maximum daily dose appears to be 100 mg . ; dose is increased to as high a level as is tolerated . dosage is started with or discomfort or pain for the purpose of + i - ms . administration i - v , is reduced many will mg . 
some cases are still surviving so that additional evidence concerning the diagnosis may be forthcoming in a few of these and the estimates of survival time are necessarily incomplete . it is reasonably certain that any such new evidence will increase the differences observed between the two series . the total numbers of cases are 46 in the x + i - vs series , of which one received compound only with no x - ray therapy and 45 in the x + i - ms series . the fate of every case is accounted for in the groups ( a ) to ( j ) of table ii . 
12 * 4 days ; for the 19 patients in the x + i - ms series , 4 received radical treatment and survived respectively 4 , 6 , 7 and 6 months , the mean m.t.d. 
mitchell by intravenous injection than with radiotherapy combined with the compound administered by intramuscular injection . preliminary clinical studies of the influence of the ancillary use of the compound on the survival times of inoperable cases of carcinoma of the bronchus treated by x - ray therapy are discussed in some detail and the end results summarised . the design of a clinical therapeutic trial of a radiosensitiser is discussed , with special reference to the evaluation of tetra - sodium 2 - methyl - i : 4 - naphthohydroquinone diphosphate m a radiosensitiser in the radiotherapy of inoperable cases of carcinoma of the bronchus . 
the results of this clinical trial are summarised to date . it is concluded that intravenous administration of this compound ( synkavit ) has a small but useful effect as a clinical radiosensitiser . in this work i have been helped by many colleagues at addenbrooke 's hospital . in particular i wish to thank a . 
campbelt. poultry research centre at the university of edinburgh . * received for publication december 30 , 1948 . the original purpose of this investigation was to study the effects of implanting various tumnours affecting the fowl into the fertile egg . a survey of the literature revealed the rather surprising fact that apart from the rous i sarcoma ( rous and murphy , 1911 ) , and a few leucosis cases ( pierce , 1942 ) , no attempt has been made to grow spontaneous neoplasms of the chicken in fertile eggs . there is a high incidence , relative to disease in general , of spontaneous neoplasia in the fowl . 
campbell ( 1945 ) , in a survey extending over five years , found the relative average incidence to be 18 ' 7 per cent in the breeds examined , the minimum and maximum figures for specific breeds being 10 - 3 per cent and nearly 39 per cent . during this survey a number of chicken tumours were studied which bore a close morphological resemblance to known virus - associated growths . in view ofclaims by various authors ( taylor , 1943 ; heilman and bittner , 1944 ; hungate , snider , taylor and thompson , 1945 ) that mammalian tumours can be easily cultivated in the yolk - sac of the chick embryo , and that a virus - like factor associated with mammary carcinoma of mice has been directly demonstrated by this method , it was thought desirable to apply similar methods to the study of spontaneous chicken tumours . the advantages of using fertile eggs for such cultivation experiments are many . 
the chick embryo , on the other hand , has not yet been reported to be a natural carrier of virus . it has been shown by grasset ( 1929 ) , who worked with diphtheria and tetanus toxoids as antigens , that the chick embryo does not produce antibody . polk , buddingh and goodpasture ( 1938 ) showed that no complement is present in embryonic serum , and murphy ( 1914 ) first demonstrated the ability of normal heteroplastic tissues and tumours to graft on to the chorioallantois without causing an inflammatory reaction until the 18th day of incubation . subsequently some reaction of the host tissues does occur , to the detriment of the graft . the culture of tumour tissues in fertile eggs therefore resembles much more closely tissue culture in vitro than it does in orthodox animal inoculation methods . at an early stage in this investigation the frequent occurrence of typical ectodermal lesions in the vicinity of chicken tumours implanted on the chorioallantois led to the conception that such tumours might contain a factor capable of stimulating cell division . such a factor , if it existed , might be a self - propagating agent within living malignant cells , or something in the nature of a mitotic activator similar to those growth - stimulating substances present in embryonic tissues . with this hypothesis in mind it was decided to study the effects of implanting embryonic tissues , certain viruses , and as wide a range of tumours as possible , derived from a variety of animals . about this time a series of cases of spontaneous liver carcinoma in ducks came under observation , and similar experiments were undertaken with the resultant material , the details of which are recorded in a separate paper . the first record of tumour cultivation in the fertile egg was by rous and murphy ( 1911 ) , who applied it to the newly discovered rous chicken sarcoma , which they succeeded in cultivating in various sites , including the yolk - sac , and on the chorioallantoic membrane . 
they make no mention of any reaction on the part of the chick embryo or its associated membranes , and their plates only illustrate the histology of the cultivated tumours ; the membranes themselves were apparently not examined . stevenson ( 1918 ) then tried various rat , mouse and guinea - pig tumours and had about 30 per cent takes . 
campbell keogh ( 1938 ) found that filtrates prepared from the rous i sarcoma gave rise to ectodermal proliferative lesions when inoculated on the chorioallantois . these focal lesions were fully developed about the 7th day after inoculation , and took the form of flattened pearly opacities varying in diameter from 0 - 5 mm . to 2 mdilute inocula produced these lesions ; when concentrated suspensions were inoculated a proportion of sarcomatous lesions developed . pierce ( 1942 ) transferred leucosis to chick embryos by seeding fragments of leucotic tissue , e.g. 
no lesions developed on the membrane , and inclusion bodhes were absent when filtrates were used , but leucotic tissue caused gross thickening and cloudiness of the membrane . taylor , thacker and penington ( 1942 ) described the growth of cancer tissue ( mammaryv carcinoma of mice ) in the yolk - sac of the chick embryo , and claimed it to be a new method . 
the presence of necrotic tissue in the tumours resulted in the death of the embryo . in a subsequent communication taylor ( 1943 ) described the cultivation of a spontaneous mammary tumour of dba strain mice in the yolk - sac of the fertile egg . 
the yolk from such eggs produced similar tumours in mice upon injection , and taylor found that berkefeld filtrates prepared from this yolk also gave rise to tumours upon injection into mice , thus showing the presence of a virus - like principle in this particular mammary carcinoma . at about the same time heilman and bittner ( 1944 ) injected a 40 per cent suspension of mouse carcinoma into the yolk - sac of the embryonic chick , using an inoculum of 0 - 2 ml . 
they extracted the tumours from the yolk - sac , and showed that fitrates prepared from these and from the yolk itself were capable of causing mammary cancer in mice , thus confirming the work of hungate , taylor and thompson ( 1944 ) , that a virus - like body was involved . following up this line of investigation bittner , evans and green ( 1945 ) showed that the " milk factor " was able to survive in the yolk - sac for 12 days in the absence of mouse mammary carcinoma cells . 
they did not know whether the factor had multiplied in the yolk - sac . twombly and meisel ( 1946 ) attempted to grow several mammalian tumours in the yolk - sacs of fertile eggs . 
the greatest number of takes occurred with the bagg 755 mouse mammary carcinoma , but they report a very high mortality rate ( 73 per cent ) by the 17th day of incubation in their embryos . 
numerous fitration experiments were done in an attempt to demonstrate a cancer virus , but with uniformly negative results . ( a ) the intra - vitelline techniqe . the method adopted for the cultivation of tumours in the yolk - sac was a modification of that described by taylor et al . 
they were then taken direct to 95 per cent alcohol , through absolute alcohol , equal parts absolute alcohol and cedarwood oil , cedarwood oil alone , and finally embedded in paraffin wax . other tissues were fixed in 10 per cent formol saline for 24 hours . 
the eggs were sealed with their own shell flap , using paraffin wax , and this was removed on the fifth day and a cover - glass substituted . tumours were seen growing in each , dependent from the chorioallantois . 
the eggs were opened 9 days later , and both contained large ( 9 mm . ) spherical , richly vascular tumours growing from the chorioallantois and hanging down into the allantoic cavity . 
of this filtrate was injected into each pectoral muscle of a brown leghorn cockerel aged 14 weeks . typical tumours subsequently grew in these sites . hi / to / og . the only histological findings to be given in any detail will be those associated with changes in the membranes in the vicinity of implanted tumours . 
a delicate argyrophil reticulum ramifies throughout the growth , and appears to be derived from the mesenchyme of the membrane . normally the epithelial surface of the chorioallantois consists of low cuboidal cells , and is only one cell thick . 
the tube was shaken in an automatic shaking machine for 1 hour , at the end of which the suspension was filtered through a " technico " filter , using a ford 's " sterimat " grade g.s. the fitrate was used as the inoculum , the amount inoculated on to each membrane varying between 0 - 02 to 0 - 06 ml . four 10 - day embryonated eggs were inoculated , using a graduated capillary pipette as a dropper . in order to secure a wide distribution of the virus over the membrane , it was placed direct on to the shell membrane , and a tear made through this with a glass needle . 
the virus was then drawn into the egg and on to the chorioallantois by applying negative pressure to the air - cell with the aid of a small rubber teat . 
the egg was then sealed in the normal way and incubated for 3 days.when opened and examined , small roughly circular opacities were found on the chorioallantois of 3 embryos . 
an occasional attempt at the formation of a " cell nest " may be found , but usually the cells occur in groups without any sign of a whorled appearance . 
carcinoma simplex , with large round and spindle - shaped cells . experiments 10 and 11 were chorioallantoic implants using methylcholanthrene induced transplantable mammary carcinomas of mice . five 10 - day embryonated eggs were implanted with fragments of tumour and examined 7 days later . all but one contained small tumours 3 - 5 mdiameter . 
of a suspension of mouse sarcoma s.37 in twenty - seven embryos subsequently died at periods varying normal saline . most of these showed congestion and petechial haemorbetween 1 and 8 days . rhages in the skno bacteria were isolated from these eggs . 
the dead embryos showed intense congestion and petechial haemorrhages . in 2 cases tumours were found tumour - bearing membranes were attached to the interior of the yolk - sac . ground up with sand and saline , and 0 - 25 ml . 
a haemorrhagic membrane was treated in the nine days later the mice were found dead , same way and injected into 5 mice . due to a heating failure during a cold spell . six of the mice injected with tumourbearing membranes were found to have small lymphoid turnours growing in the subcutis at the site of injection . 
the membranes appeared normal upon histological examination . ( e ) spontaneouvs chicken tunours . in experiments 19 , 20 and 21 the intra - vitelline method was again tried . these experiments may be briefly dismissed , as in each case contamination with bacteria killed all the eggs . 
coli was isolated from the eggs , although broths inoculated with tumour tissue from the original case remained sterile . experiments 20 and 21 consisted of the injection of a saline suspension prepared from lymphocytomata , involving the thigh and ovary respectively . the embryos died subsequent to injection , and bact . 
hen suffering from aleukaemic lymphoid leucosis . eight days later two of the embryos were found to be dead , namely those with liver and liver plus spleen implants . only the latter had a tumour in the membrane . 
the remaining spleen only implanted embryo was living , and the membrane bore a 4 mdiameter pink tumour . in the case of the dead embryos both were extremely congested , and the livers appeared swollen and leukaemic . histological examination of the membrane bearing the spleen implant shows a vascular growth of endothelial - like structure . all resemblance to normal or even leukaemic spleen has disappeared . 
the growth is composed of an irregular syncytium of calls with round , angular or elongated nuclei , the cytoplasm of which is drawn out into processes which appear to merge with those of adjacent cells . 
the vessels are formed of a single layer of flattened endothelium and are congested . at the periphery of this tumour large numbers of primitive blood cells appear to be wandering into the mesenchymal stroma of the chorioallantois . " cell nests " occur in this region . 
an examination of the heart blood from these two embryos shows in the case of the liver only implant a large number of primitive cells which appear to be differentiating into erythroblasts , i.e. 
the bird was destroyed and immediately opened , and the viscera were exposed to the air in the post - mortem room for about 2 hours before the case was seen , but the liver surface was seared with a hot spatula at the time of implantation , and fragments taken from within the organ with sterile knife and forceps . 
the actual implants were made about 21 hours after the fowl was destroyed . broths inoculated with tumour material at the same time were later seen to be sterile . nine days later two embryos were found to be dead . 
the second method of tissue culture is involved and requires an elaborate technique , and has the great drawback that frequently only one component of the original neoplasm grows , while the rest , e.g. 
more experiments involving sub - inoculation with tissue fitrates and extra - embryonic fluids from dead and haemorrhagic embryos should help to settle this point . the chorioallantoic method has been found to be most successful in the case of the cultivation of avian tumours associated with viruses . thus , the rous i sarcoma and the duran - reynals sarcoma give the biggest growths in the membrane . these are both rapidly growing tumours , and therefore would naturally tend to give better results in the short period ( never more than 14 days ) of cultivation . slow growing tumours usually give disappointingly small growths , whilst normal adult tissues may fail to grow and be absorbed . in the case of the rous and duran - reynals sarcomata , the virus stimulates the mesenchymal tissue of the chorioallantois to participate in the general malignancy , thus augmenting the size of the implant . 
of more interest , however , is the observation , originally by keogh ( 1938 ) , that the rous virus also appears to stimulate the ectodermal cells of the chorioallantois to proliferate in an apparently uncontrolled manner . 
he speculates on the possibility that the virus may be carcinogenic , not only for mesenchymal tissue , but also for epithelium ectodermal proliferation due to rous virus has been under certain conditions . confirmed in the present investigation , and the pathological changes in the membrane ectoderms produced by the rous and duran - reynals tumours have been compared with those produced by dermatrophic viruses such as those of fowl - pox and contagious papillomata of cattle , also with similar changes noted in the membrane adjacent to certain implanted spontaneous tumours of the chicken . as a check on these observations a number of experiments were performed with chemically induced tumours of the mouse , rat and chicken ; also with normal adult and embryonic chicken tissues . the significance of the " cell nests " or " epithelial pearls " is interesting , resembling as they do transverse sections of infiltration cores of epithelial tissue similar lesions were noted by which characterize squamous - cell carcinoma . huxley and murray ( 1924 ) adjacent to the fragments of chick embryos implanted on the chorioallantois . it is well known that embryonic tissue extracts contain growth - promoting substances , and for this reason are frequently added to tissue culture media . claude ( 1938 ) showed that a fraction could be isolated from chick embryos which j . 
campbell possessed the same physical and chemical properties as the purified active fraction despite these similarities , however , the embryo extract of the rous i sarcoma . failed to produce tumours in susceptible chickens . in this connection it is interesting to note that earle ( 1943 ) noticed an apparent malignant transformation of mouse fibroblasts grown on a fibrin clot and bathed in horse serum and chick embryo juice . these observations , together with the production of ectodermal proliferation and cell nest formation found to be associated with so many chorioallantoic implants of tumnours in the present investigation , gave rise to the conception that tumours and embryonic tissue might contain growth - promoting substances capable of causing the overgrowth of the ectodermal layer of cells in the embryonic membrane . if this proved to be the case it might be possible to demonstrate some relationship between the intensity of the membrane reaction and the type of tumour implanted , especially from the aspect of virus content . 
no mention of " epithelial pearls " is made by keogh ( 1938 ) , or by the numerous other workers who have studied ectodermal lesions of the chorioallantoic membrane induced by viruses . a number of experiments were devised in order to test this hypothesis , and especially to study the membrane reaction to a spontaneous liver carcinoma of the duck , which is described in a separate paper . table i shows that the ectodermal lesions do not appear to be as specific as in all cases where tumours known to contain virus , or fitrates of was hoped . such turnours , or dermatrophic viruses , were placed on the membrane , proliferation of the ectoderm took place . in some instances eosinophilic " inclusions " were observed , but the nature of these is not clear , except of course in the case in the main , virus alone caused simple proliferation without the of fowl pox . formation of " cell nests . " in general those tumours which failed to grow subsequent to implantation and were absorbed did not cause any detectable lesions in the membrane ; similar results were obtained for normal adult tissues . also in several cases , although not invariably , an accidental infection of the membrane with bacteria did not result in ectodermal proliferation . the spontaneous chicken tumours which grew successfully all caused proliferation and the formation of " cell nests . " it was thought possible that chemically induced tumours , being presumably free of virus , might give a different membrane reaction . 
the majority of spontaneous tunours give disappointingly small growths . it has been shown that the capacity for indutcing ectodermal proliferation in the chorioallantois is not confined to tumour viruses , or to fowl pox and other non - tumour associated viruses , but occurs in the membranes adjacent to the majority of implanted tumours , whether spontaneous , virus associated , or chemically induced ; or even occasionally in response to bacterial growth . attention has been drawn to the tendency of the proliferating ectoderm to form " cell nests " or " epithelial pearls , " resembling those occurring in squamous - cell carcinoma , and the significance of this has been discussed . the induction has been described of two distinct leukaemic conditions in chick embryos as the result of implanting tissues from a third type of spontaneous leukaemia in a fowl , and has been briefly discussed . i wish to express my gratitude to professor a . 
murray drennan , of the pathology department , university of edinburgh , for his interest in this work , and for the numerous suggestions as to the manner of development of the investigation . all the fertile eggs were obtained from a . 
7. received for publication february 3 , 1949 . one of the surprising elements in bittner 's discovery of the " milk factor " is the apparent entry of the virus into the bodies of the young mice via the it is , however , obviousthat during suckling a small amount alimentary canal . of milk may also enter the nostrils , and that the nasal mucosa may offer an alternative port of entry . it also seems possible that the milk factor might be destroyed by digestion in the stomach . if this were so it would be difficult to recover milk factor from the stomach contents of suckling mice . 
the unfortunately the experiresult was entirely negative in the 40 mice so treated ment was not adequately controlled , in that none of the mice were tested for susceptibility by means of extracts known to contain the virus . 
he was considered too old for successful operative treatment so he was kept under observation and in february , 1954 , was readmitted to hospital with evidence of bone metastasis . 
apart from this , there is little irregularity of nuclear shape , size or staining density . these cells clearly form part of a fragment of tissue in the sputum , and because of the vacuolation of the cytoplasm , were considered to be diagnostic of adenocarcinoma . 
they are of adenocarcinomatous type and do not resemble cells usually seen in primary bronchial carcinomata . " an enlarged cervical gland was later removed for histological examination and was reported as showing a mucus - secreting adenocarcinoma , probably of colonic origin . the patient died , and post mortem examination was refused . it will be noted that the cells shown in the clump in fig . 
4 appear to surround a number of vacuoles , but gross vacuolation is not evident in the cells themselves . the larger mass is hollow , the wall being formed of a single layer of cells . i make it a practice only to report adenocarcinoma from sputum examination it cannot be too strongly emphawhen clumps of the types described are seen . sised that single cells containing vacuoles as shown in fig . 
shujbik. from the oxford university reseach centre of tje british empire cancer campaign , sir william dunn scoo of pathology , unirersity of oxford . received for publication november 20 , 1948 . though non - carcinogenic to normal skin , croton oil elicits tumours in skin previously treated with a carcinogen for an inadequate period ( berenblum , 1941 )  . this procedure , with the modification of mottram ( 1944 ) , using only one single application of carcinogen prior to the croton oil treatment , served as the basis of a quantitative analysis of carcinogenic response ( berenblum and shubik , 1947b )  . 
the results led to the establishment of the essential difference in mechanism between the preliminary " initiating process " and the subsequent " promoting process " of carcinogenesis . for reviews of the literature dealing with earlier work on the stages of carcinogenesis , and with the previous terminologies used , see berenblum , 1944 , 1947 ; rusch , 1944 ; berenblum and shubik , 1947a and b . from this analysis ( berenblum and shubik , 1947b ) , it was concluded that the initiating process represents a sudden and irreversible change in a small minority of the cells of the treated area , giving rise to isolated " latent tumour cells , " apparently mdtinguishable morphologically from the surrounding non - neoplastic cells . 
the presence of these latent tumour cells is only demonstrable by subsequent promoting action , which converts them into morphological tumours . this promoting action is less specific than the initiating action , in that it can as readily be induced by croton oil as by continued applications of a true carcinogen . the irreversible nature of the initiating process was demonstrated by the fact that the number of tumours elicited by croton oil was as great after an interval of 20 weeks ( between the carcinogen application and the commencement of croton oil treatment ) as after an interval of only 3 days . according to this new concept , the latent period of carcinogenesis is dependent on the efficacy ' of the promoting action , while the actual turnour yield is prethis was borne out by comparative tests , determined by the initiating action . using 3 : 4 - benzpyrene , and 9 : 10 - dimethyl - 1 : 2 - benzit was found , as anthracene , as initiators , followed by croton oil treatment . expected , that whereas the percentage of tumour - bearing animals differed in the three series , the latent periods were approximately the same . 1 : 2 : 5 : 6 - dibenzanthracene , unfortunately , owing to considerations of solubilities , it was necessary , in these comparisons , to use a different concentration for each carcinogen . consequently , more than one variable was involved in the experiment . 
while the results obtained provided an adequate basis for the general conclusions referred to above , it was felt , nevertheless , that unequivocal evidence could best be 110 i . 
shubik provided by studying the effects of a series of graded concentrations of one and for this investigation , described below , the same carcinogen as initiator . 9 : 10 - dimethyl - 1 : 2 - benzanthracene was chosen as initiator . an additional purpose of this investigation was that the quantitative data thus obtained could , at the same time , serve as indirect evidence for or against the possibility of a " mutation - like change " for the initiating stage of carcinohowever , for a re - assessment of the validity of the " somatic cell genesis . mutation theory of cancer " in the light of the dual - stage mechanism of carcinogenesis , a more direct experimental approach was required . such an approach seemed possible from the following considerations . mustard gas is known to possess mutagenic properties when tested on drosophila ( auerbach and robson , 1946 , 1947 ) , and on neurospora and e . 
coli ( tatum , 1947 ) , so that , according to the somatic cell mutation theory , this compound admittedly , mustard gas is a potent should act as a carcinogenic initiator . anti - carcinogenic agent ( berenblum , 1929 ) , but this effect was shown to be confined to the later stages of carcinogenesis ( berenblum , 1929 , 1931 ) , and could thus , mustard gas should , thus be attributed to an anti - promoting action ; theoretically , still be capable of initiating action . 
the experimental areas of skin , in the inter - scapular regions , were clipped periodically all test solutions were applied with a with fine scissors for removal of hair . glass rod , and colourless , non - fluorescent , liquid paraffin ( " liquid petrolatum " ) was used as solvent throughout , for standardization of response ( berenblum and schoental , 1947 ; berenblum and shubik , 1947a )  . the main departure from the technique of the previous experiments was that instead of merely noting the numbers of tumour - bearing mice , the individual tumours were also recorded . 
from these results , and from other evidence brought forward , it was concluded that the ultimate tumour yield in carcinogenesis is pre - determined by an irreversible initiating process , while the latent period of carcinogenesis , i.e. 
the speed with which the induced " latent tumnour cells " are converted into morphological tumours , is a function of the subsequent promoting process . the idea of the possible existence of " latent tumour cells " requiring additional stimulation , i.e. 
the results suggest that the acetylaminofluorene acted as initiator , and the goitrogenic agent as promoter , in an analogous fashion to the carcinogen and the croton oil , respectivelv.y , in the case of skin experiments . in the present investigation , one and the same carcinogen was applied to the mouse 's skonce only in different ( graded ) concentrations , to serve as initiator , and then followed by repeated applications of croton oil . expressed in ratios , the concentrations of initiator ( 9 : 10 - dimethvl - l : 2 - benzanthracene in liquid paraffin ) in the four experimental groups were 1 : 3 : 9 : 27 , and the total yields of tumours for the corresponding groups were in the ratio of 1 : 6 - 6 : 12 - 4 : 26 - 5 ( table yet the average latent periods for these 4 groups were closely similar , ii )  . namely , 14 - 4 , 12 - 1 , 10 - 6 , and 11 - 8 weeks , respectively . 
not only was there a progressive increase in the total number of tumours , with increase in concentration of initiator , and also in the number of tumour - bearing animals . 
many different experimental approaches have , however , been devised in recent years to obtain , at least , indirect evidence in support of the theory , and these may conveniently be considered under the following headings : 1 . 
induction , by knonn carcinogenic agents , of mutations in louer organisms . the early studies in this field were concerned with x - ravs and ultraviolet although radiation , the literature of which is well reviewed by muller ( 1941 )  . most of this work was not directly concerned with correlating carcinogenicity 114 i . 
genetic analysis of tumour transplantation phenomena in pure strain mice . the genetic control of tumour transplantation in pure strain mice has been investigated in some detail , and the earlier work in this field is well smmarized it has been shown that a number of genes control the behaviour by little ( 1941 )  . of certain tumours , and this approach has been extended further by gorer ( 1937 , 1947 ) , showing that these genes probably determine their antigenic proalso , according to strong ( 1926 ) , bittner ( 1931 ) and others , the observed perties . sudden change in the transplantation properties of tumnours could be explained on the basis of the occurrence of mutations . 
quantitative interpretation of carcinogenic experiment . several attempts have been made to evaluate quantitatively some of the more reliable carcinogenic data , to determine whether or not such data would be in keeping with a sudden irrev%ersible change of a mutation - like nature . in the investigation of dunning , curtis and wood ( 1940 ) , the production of sarcomas in rats , by subcutaneous injection of 3 : 4 - benzpyrene , was submitted to quantitative analysis . 
the concentration of carcinogen and the volume of fluid injected , both of which were variables , were correlated with the number of tumours induced and with the latent period of tumour induction . it was found that increasing the number of foci injected did increase the number of tumours induced , whereas increasing the volume of fluid injected in one locus did not . in view of the variables involved , and for other reasons to be discussed later , these results do not lend themselves to an exact interpretation of a single response to a single stimulus . charles and luce - clausen ( 1942 ) analysed data of a mouse skin carcinogenesis experiment , using continued painting with 3 : 4 - benzpyrene , to determine whether or not this was in keeping with the concept of the somatic cell mutation theory . in their introduction , they make the primary assumption that if a mutation were the basis of carcinogenesis , it must , of necessity , be of a recessive nature , thus postulating two successive mutations to bring to light an actual tumour . there is , however , no valid reason for this assumption , which is , in fact , at variance with the theory of dominancy , as proposed by fisher ( 1930 )  . in all these previous attempts at analysing carcinogenic experiments , in which the carcinogen is allowed to act continuously , there is the inevitable complication arising from the fact that , of the two stages of carcinogenesis , only one - the initiating stage - could possibly be ascribed to a mutation , since this is the stage which involves a sudden and irreversible change , possibly affecting one cell only . 
shlbik in the present investigation , the correlation of concentration of carcinogen , as initiator , with the total number of turnours induced , has revealed approximately a direct 1 : 1 ratio . this could be taken as being in keeping with the somatic cell mutation theory . however , the other investigation , described above , does not lend support to as already mentioned , mustard gas is a potent mutagenic agent this theory . on drosophila ( auerbach and robson , 1946 , 1947 )  . since its anti - carcinogenic properties ( berenblum , 1929 , 1931 ) can undoubtedly be attributed to antipromoting action , it was to be expected , according to the somatic cell mutation theory , that when allowed to act once only on mouse 's skin , followed by repeated croton oil treatment , tumours should have arisen , the mustard gas acting as an initiator by virtue of its mutagenic properties . 
when tested , this was found not to be the case . in view of this negative finding , and in the light of the highly conflicting evidence of recent experiment in an attempt to adduce more indirect support of the theory , it becomes necessary to re - assess the validity of the theory . basically , neither proof nor disproof of the somatic cell mutation theorv of cancer is possible ( haldane , 1934 ) , as referred to above . the accumulating indirect evidence would seem to run counter to the theorinstead of supporting of the evidence presented in the present communicationi , the first experiment is consistent with the theory , while the second is strongly against it . the force of past reasoning in support of the theory has rested largely on the assumption that , given an irreversible change as the basis of carcinogenesis , the only known biological phenomenon to explain this would be a mutation . however , a closer examination of other common biological phenomena instantly reveals that this is not so . for example , in the course of the development of the embryo , the divergent differentiation that ultimately results in the irreversible cell types , e.g. 
horning. from the chester beally research institute of the royal cancer hospital , london , s.w.3. received for publication december 12 , 1948 . recfnt experience has shown that many of the growth - inhibiting agents used in the palliative treatment of cancer are carcinogenic . 
they also cause specific damage to cell nuclei and chromosomes and are able to induce mutations . the association of these biological effects of ( 1 ) growth inhibition , ( 2 ) chromosome damage , ( 3 ) production of mutations and ( 4 ) induction of cancer suggests if the that they may have a common fimundamental biochemical mechanism . induction of cancer is indeed a somatic mutation , then cancer induction might be included as a special mutation . 
the fact that x - rays can produce cancer in man was published in 1902 ( frieben , 1902 ) , seven years after rontgen 's discovery muiiller ( 1928 ) found that x - rays are mutagenic , and later mather of x - rays . and stone ( 1933 ) and koller ( 1934 ) described the chromosome damage following irradiation . 
the idea that cancer arises as a somatic mutation was supported by the demonstration of an increased incidence of mutations occurring in mice this was shown with methylcholanthrene treated with chemical carcinogens . by strong ( 1945 ) and with 1 : 2 : 5 : 6 - dibenzanthracene by carr ( 1947 )  . carcinogenic hydrocarbons inhibit the growth of animals and the establishment and growth of transplanted tumours ( haddow , scott and scott , 1937 )  . 
it would be wrong to give the impression that undesirable sequelae are common , yet such complications as wound sepsis , necrosis of the skin edges and a poor functional result are still seen with greater frequency than one would expect following an operation which has been abundantly practised by innumerable surgeons all over the world for over half a century . 
these particular sequelae are essentially a reflection on the judgment or the executive skill of the operator and not upon the operation itself , because with care they need not happen . 
furthermore , such indifferent results become known , and because they lose nothing in the telling may have a serious deterrent effect upon patients contemplating advice about a newly acquired lump in the breast . 
it is to be remembered that we are operating under almost ideal conditions - the patients are constitutionally well and often near the prime of life , the anatomy of the area is normal and not distorted as it might be , and there are none of the embarrassments associated with operations within a body cavity . 
a very high degree of technical excellence should therefore be our aim , both in regard to the thoroughness of the dissection which has as its objective the extirpation of the disease , and in regard to the care with which this is carried out , which can so much determine the degree of ultimate functionaf disability . 
 there are three features of the " skin short " case which need some explana i will call them ( i ) anticipating the gap , ( ii ) siting the gap , ( iii ) covering tion . 
riddell surgery - may be influenced ; second , the surgeon is forewarned , and so can avoid being surprised into an awkward situation at the end of a long operation . 
 an assessment - an estimate - should be made in all new cases of cancer of the breast of the amount of skin that will be left for closing the wound after the breast and tumour have been removed . 
it will be found that patients fall into two categories : ( 1 ) a larger group in whom it is judged that it will be possible to approximate the skin edges in the usual way without tension . 
h we remove too little skin , secondary nodules will appear for certain and quickly ; if we remove a more generous amount - - as we must - we shall have to be prepared to deal with a gap . 
 in these patients two alternatives are open to us : either we can proceed to radical mastectomy , skin graft the resulting gap , and hope that the skin covering it will heal in a reasonable time and so allow early post - operative irradiation , or we can anticipate the probable delay in healing by giving the x - ray treatment before the operation . 
 personally in this small group i prefer to give the x - ray treatment first , because in practice unfortunately every skin graft does not take uniformly , so that a proportion of cases are left to heal - at least in part - by granulation , and this takes time ; indeed , sometimes so much time that post - operative x - ray treat ment may be delayed to a point where its usefulness is open to question . 
for this reason it is suggested that if by careful pre - operative clinical assessment we can anticipate and so forecast such a defect arising , we should consider giving the x - ray treatment before the operation . 
the use of pre - operative irradiation does not in any way absolve the operator subsequently from still adhering to the principle of excising a wide area clear of the growth . 
this patient was treated by pre - operatilre irradiation followed by radical mastectomy with an immediate skin - gra the satisfactory functional result can be attributed to ( i ) adoption of the " arm raised " position before approximation of the skin flaps , ( ii ) the early recognition of the inevitability of " a gap " in this type of case and of the need for a skin - gra fro . 
2. - full range of movement : the " arm raised " position is the most delicate test of function at the shoulder following mastectomy ; six months should be allowed after the termination of all treatment for the skin to regain its suppleness beforp 8 - ing function ; inability to raise the arm vertically after such an interval must be regarded as an imperfect functional result . 
3. - llarginal flap necrosis : the illustration shows an area of skin loss as a result of damage to the blood supply of the lateral flap during its elevation . 
at the same time it is not vital to graft ; what is vital is that a gap should be left in a skin short case ; whether a dressing of skin is put on that gap or not is almost a secondary consideration , although skin is very clearly to be preferred to scar tissue if the gap is a large one . 
 the time lo gra the decision as to whether the graft is applied at the time of the mastectomy or not depends solely on the condition of the patient at the end of the operation . 
 h this is unsatisfactory , as it may be in an elderly patient , no harm is done by leaving a raw area and grafting early in convalescence , usually at the time of the first major dressing . 
 if , in addition , we have made our pre - operative reconnaissance , the procedure is robbed of its only disagreeable feature , the element of surprise , since a donor area will have been chosen and prepared and the few special instruments and dressings will be immediately available . 
this accident most commonly occurs as the dissection approaches the anterior border oflatissimus dorsi , which corresponds with the deepest and darkest part of the wound ; it is usually due to hurrying this part of the reflection in a field obscured by haemorrhage ; the blood 811pply may also be cut off from a portion of the flap by the use of tension sutures and with the same result . 
riddell displacement of sk in the second category the skin is not lost in the sense that it is destroyed , but is lost by displacement , because skin is borrowed from the axilla and upper arm to close the axillary part of the wound . 
 ( 2 ) slide the skin of the lateral flap well up into the axilla ; by making use of the longer contour of the lateral flap in this way an adequate supply of skin in in suturing the flaps together exactly opposite points on the axilla is assured . 
 in other words , lend skin from the lateral flap to the axilla ; do not borrow it from the axilla and upper arm by bringing the arm down to a right angle or less . 
 i should perhaps add that in my experience functional disability at the shoulder is not related to loss of the pectoral muscles ; indeed , it can be quite severe following a local mast.ectomy , and is not related t - 0 failure to employ early fig . 
the drawing shows the correct position of the upper limb before closing the wound ; the skin of the lateral flap should be pushed well up into the axilla before suturing ( arrow )  . 
in a series of cases examined in which the subscapular nerve had been divided , no functional disability could be found by comparison with control cases and with the opposite side , although , of course , there was a positive r.d. 
the aim is to display the anterior border of the latissimus dorsi muscle , and in " skin short " cases to mobilize the lateral flap still further by carrying the dissection backwards for two or three inches beyond this point , keeping on a plane buperficial to the muscle . 
the aim is to raise a flap of even thinness , but if by a careless stroke of the knife the flap is cut too thin , the blood supply will be damaged and the whole course of convalescence unnecessarily prolonged . 
h too thick a flap is raised the dissection may in error proceed on the deep face of latissimus dorsi into the space between this muscle and the serratus anterior , a space which is filled with large veins . 
h , on admission , the patient is severely anaemic , blood should be given , but let the transfusion be given before the operation ; it can be supplemented by plasma or other blood substitute during the operation if necessary . 
this is a partial contradiction of views ex pressed earlier in my experience ( riddell , 1948 ) , and is based on the fact that blood transfusion to an unconscious patient carries with it an extra hazard - slight though it may be - for the patient cannot proclaim against the blood if it is incom patible , as would be possible if she were conscious . 
the towelling of the operation area should be so arranged , by placing a towel behind the patient 's back , that there is no unprotected area on the flank from which the main wound might be contaminated ; hot wet packs , which so quickly become cold wet packs and chill the patient , should only be used for haemostatic purposes at the end of the operation ; dry hot packs 296 v . 
 drainage should never be employed through any part of the original incision , because it can so easily lead to cross infection of the main length of the wound ; it is best carried out through a separate stab incision in the lateral flap , pre ferably well clear of the hair and sweat - bearing skin of the axilla . 
lastly , let us banish for all time the use of tension sutures , and above all the expression f 10 . - the composite dra ' lring illustrates ( a ) the position of the leg for cutting an imme it is a sound precaution to towel up all " skin - short " cases with the diate skin - gra donor area prepared and &ec8111111 > le . 
functional disability usually takes the form of restricted movement at the shoulder and is doubly undesirable , since it not only seriously reduces the range of w ! efo1ness of the limb concerned , but aggravates by constant reminder the psychological trauma resulting from the operation . 
3. received for publication january 21 , 1948 . during the war the pharmacology of the nitrogen mustards or chloroethylamines was studied intensively ( gilman and philips , 1946 )  . in the course of these investigations it was observed that tissues with a high proportion of dividing cells were particularly injured . this discovery led to the application of these substances in the treatment of malignant growth . 
of dimethyl - , - chloroethylamine hydrochloride by subcutaneous injection . such treatment when given repeatedly caused inhibition of some spontaneous mammary tumours ( table i ) , but had very little effect in prolonging the life of mice with transplanted lymphosarcoma . this compound given in single doses of 100 mg . 
the effect is similar to that induced by radioactive substances ; it is identical with the phenomenon of depigmentation in mice caused by subcutaneous implantation of plutonium ( brues , 19 , 47 , personal thus " bleaching of hair - colour " is another example of the communication )  . radiomimetic actions of the bis - chloroethylamines . the metabolism of walker carcinoma tissue from rats dosed with 1 mg . 
a variant of this compound , dimethyl - ] 3 - chloroethylamine hydrochloride , was used in 3 other cases , reported here because of the favourable response in 1 case . 
on two consecutive days in 30 cases , in 10 of which treatment was repeated once or several times at 3 - week or longer intervals depending upon the length of remission from symptoms . 
the majority of patients with advanced bronchogenic carcinoma have white cell counts above 10 , 000 per c.mm. , and such patients do not develop the moderate to severe leucopenia observed in cases of hodgkin 's disease ( apthomas and cullumbine , 1947 )  . in only 2 of our patients has the white cell count fallen below 4000 per c.mm. , in these cases to 1000 and 1800 with restoration to normal counts in 3 weeks ' time . the first of these cases had had previous radiation therapy , and at the commencement of treatment had a white blood cell count of 4000 per c.mhaemorrhagic nianifestations have not been observed in this group , nor has any significant change in haemoglobin dosages of 0 - 2 mg . 
at 10 - day intervals can be tolerated for been noted . periods of at least 10 weeks , as judged by the 4 cases we have treated over such a period . 
the presence of fever , pleural exudation or jaundice are not , in our experience , contraindications to the use of the drug . the adverse effects of the dimethyl compound were observed in 3 patients , 2 of whom are not included in this group as their diagnosis was not histologically proven . 
one patient was carried on for a period of 5 months with marked relief of pain , cough and dyspnoea and decrease of sputum for 18 to 21 days following each course of injections . 
a patient with remission of symptoms for 6 to 8 weeks after each treatment is still being observed one patient receiving dimethyl - , 3 - chloroethylamine is still in after 9 months . remission 7 months after treatment and leading a normal life . 
the relief of dyspnoea in some cases is almost complete - an observation very difficult to explain , for in such cases there may not be corresponding observable changes in the lungs . relief of general malaise and anorexia is also noted in over half the cases following the initial 1 - or 2 - day period of adverse effects . 
warwick sputum usually increases in amount for several days , and is said " to come up more easily . " the amount may then fall off rapidly , in some cases from 90 - 120 ml . 
the sputum may also change in character from purulent to colourless . reabsorption of pleural exudate or decreased rate of formation has been these observations are of interest in that most radioseen in 5 of 13 cases . ' therapists hesitate to give treatment to cases with pleural exudation because of the tendency to increase the amount of exudate . superficial metastases of measurable size decreased in 3 of 10 cases . 
a larger total number for such observations wouild have been obtained had not metastatic nodules been removed for microscopic study at various intervals following injection in 8 cases . twenty - two patients who had weight loss were weighed weekly following in 9 of these cases weight loss continued for approximately 1 week , treatment . following which a gain in weight occurred amounting uisually to 2 to 8 lb . four patients gained 10 to 15 lb . 
in 2 months , and 1 patient who lost 40 lb . in the 18 months prior to treatment with dimethyl - 3 - chloroethylamine has in 7 months regained all her lost weight . radiological evidence of decrease in size of pulmonary metastases and re - aeration of segments or lobes was observed in 8 of 35 cases . 
hb , 55 per cent . 3 , 800 , 000 . heart , mediastinum and trachea displaced to the right ; appearance of atelectasis with probable cavitation near 2nd right carina broadened rib . and invaded on the right by a large nodular friable mass completely occluding the right main bronchus . bronchoscopy report from harefield in april , 1946 . pathological report : adenocarcinoma with numerous mitotic figures . treatment : dimethyl - chloroethylamine 2 mg . 
a corresponding effect in a tumour of the breast might reasonably be expected not to be noticed in support of such a suggestion is the fact that a similar relief by the patient . of respiratory symptoms following chloroethylamine without radiological evidence of changes in the lungs has been observed in a case of carcinoma of the breast with pulmonary metastases . 
by repeating the treatment at suitable intervals the life of animals with tumours can be prolonged , but cessation of treatment leads to recurrence of tumour growth . forty - one cases of histologically proven carcinoma of the bronchus unsuitable for other forms of therapy have been treated with the chloroethylamines . 
3. received for publication february 6 , 1947 . following the discovery by haddow and sexton ( 1946 ) of the effects of ethylcarbamate ( urethane ) on the growth and differentiation of the walker rat carcinoma , the action of this substance in human leukaemia was described by paterson , apthomas , haddow and watkinson ( 1946 )  . since the early experiments of warburg in 1910 , ethylphenylcarbamate ( phenylurethane ) had been known to arrest the division of the sea - urchin egg at metaphase . 
a similar effect in plant cells , closely resembling that of colchicine , was discovered in 1939 by lefevre and by simonet and guinochet ( 1939 ) , and was further studied by gavaudan ( 1943 ) and deysson ( 1944 )  . ludford ( 1936 ) treated tissue cultures with a 2 per cent solution of ethylcarbamate and noted abnormal metaphases and a considerable amount of nuclear and cellular degeneration . similar results were obtained by geiersbach ( 1939 ) , and ostergren ( 1944 ) also claimed that ethylcarbamate arrested mitosis at metaphase in alliutempleman and sexton ( 1945 ) , investigating the growth - inhibiting properties of numerous carbamic esters in plants , found , however , that ethylcarbamate was inactive in this respect , although marked activity was shown by ethyl phenylcarbamate and it was this work which led haddow and sexton isopropyl phenylcarbamate . to investigate the action of carbamic esters on tumour growth , and to the discovery that in this case ethylcarbamate itself is effective . these authors suggested that this substance might belong to the group of caryoclastic ( nuclear ) poisons studied by a . 
described by gorer ( 1946 ) was utilized , in which large stem - cells produced a diffuse infiltration of lymph nodes , spleen , liver and kidneys , leading to death of the mice in from 14 to 22 days after subcutaneous grafting of leukaemic tissue . enumeration of reticulocytes was made from dry blood smears , following postvital staining with * associe du fonds national belge de la recherche scientifique . 
the nucleus then disintegrates , the thymonucleic acid material forming shell - like structures covering an acidophil sphere , which also contains some ribonucleic acid ( by the further destruction ribonuclease method ) and the remnant of the nucleolus . leads to irregular fragments , with a positive or negative feulgen reaction . 
52 and within 3 days the number of reticulocytes was close to the initial value . no significant change in the numbers of red blood cells , or in the leucocytes , was observed . these results may be compared with those following the injection of colchicine , when the reticulocyte fall is marked and the minimum is also reached in 48 hours . 
the erythroblastic mitoses are arrested in metaphase by colchicine during the first hours following its injection , and the delayed decrease of the number of reticulocytes is a consequence of the time needed for the maturation of the erythroblasts . 
by comparing the histological pictures found in these last two groups it is possible to appreciate the specific effects of ethylcarbamate . ( ii ) blood - picture . - the principal changes were an increase in the percentage of granulocytes and a decrease in the numbers of reticulocytes . 
no evident atrophy of the mucosa , nor ulceration . leukaemic infiltrations : no evidence of increased cell - destruction nor mitotic abnormalities . in animals losing weight during the experiment , splenic and lymphoid atrophy is more pronounced . 
the bone - marrow remains very cellular , with a there is no evidence of aplasia . high proportion of immature granulocytes . in the thymus the atrophy of the cortical region is complete , the epithelial cells this picture of " inverted thymus " is found after the pycnotic only remaining . destruction of thymus cells by mitotic poisons ( dustin , 1929 ) or non - specific in this case it is probable that the stimuli ( selye , 1937 ) , and in inanition . ethylcarbamate injections and the loss of body weight have additive effects . no marked changes are found in other organs . * rosin and doljanski ( 1944 ) have described inclusions in liver cells of rats treated with urethane , which they believe are red blood cells engulfed in cytoplasm and even in the nucleus . 
for 5 days , there was an increase in leucocytes from 9200 to 22 , 800 , and in granulocytes from 33 to 58 per cent . the bone - marrow showed very active granulopoiesis . bring about any aplasia of the bone - marrow in the rabbit . these preliminary results indicate that large doses of ethylcarbamate do not 2 . 
the possible relations between mitotic poisoning and the therapeutic action of ethylcarbamate in human leukaemia are discussed . this work has been supported by grants to the royal cancer hospital ( free ) from the british empire cancer campaign , the anna fuller fund , and the jane coffin childs memorial fund . 
bartholomew 's hospital , london . received for publication february 3 , 1953 . this investigation was planned at a meeting on august 29 , 1949 , at the baggeridge colliery , staffordshire , ' arranged by a . 
some small series of cases suggest that silicosis may predispose to cancer of the ' lung , but the general indication of the literature is that silicosis is not active in this respect . 
hence the area where there is most pneumokoniosis shows a low incidence of lung cancer . the prevalence of pneumokoniosis in any area is indicated by the number of compensation cases arising under the workmen 's compensation acts and the industrial injuries scheme . 
4. - numbers of deaths from cancer of the lung in coal miners , and of wage - earners on colliery books , from 1921 . 1930 1940 face may be included , but this criterion must be difficult to apply to non - stationary however , in practice , the doubtful cases may not be very numerous , workers . and one must match the entry on the death certificate as nearly as possible with one of the official occupations . the increase in deaths from cancer of the lung . the increase in deaths per annum attributed to cancer of the lung from 1921 to 1950 is shown in tables iii and ix and in fig . 
some possible reasons for these differences are discussed . ( 2 ) in the last 30 years the mortality from cancer of the lung has increased in coal miners in the same way as in the general population ; in view of the peculiarities of the miner 's life , this fact calls for further inquiry . ( 3 ) the incidence of cancer of the lung differs ( a ) in face - workers and in other it is low coal miners , and ( b ) in the various coalfields of england and wales . in the south wales coalfield , where pneumokoniosis is most prevalent . we are indebted to the officers of the national coal board named at the begining of this paper for the suggestions which initiated this inquiry . 
of these large towns other than london 42 are located in the northern region of england and 41 in the remainder of england and wales , these groups being designated as " north " and " south " in this paper . 
the total deaths in all county boroughs , numbering 70 , 110 , were divided by the census population at the centre of the period , 1931 , and " expected ratios " of deaths to population obtained for each of the 16 sex - age groups . 
the census population of each town at each sex - age group was then multiplied by the corresponding " expected ratio , " giving the " expected " deaths in that group in 1921 - 39 . 
the actual deaths in a particular age group , or at all ages , divided by the expected deaths at the corresponding ages , or by the summation of them in all the age groups , and multiplied by 100 , gave for each sex comparative mortality ratios ( c.m.r. ) at each age group and at all ages . 
these are only approximate indices , since no correction has been made for irregular population trends in some of the towns ; but in no instance would the error from that cause exceed 5 per cent , and for only a few towns would it exceed 2 per cent . 
the values range from 130 to 55 , those which are greater than 100 by at least twice the standard deviation being denoted by a + sign , and those less than 100 by twice the standard deviation are denoted by a sign . 
stocks as a percentage of the expected deaths , giving a series of group c.m.r. 's , indi cating how the relative excess or deficiency of mortality characterizing the towns within the group changed according to age . 
 other digestive indices : similar ratios for deaths from cancer of the liver and causes included in the international groups for diseases of the diges tive system except appendicitis and peptic ulcer . 
 in table i 23 of the towns had c.m.r. 's exceeding 100 by twice their standard deviation or more , and 30 had c.m.r. 's less than 100 by that amount . 
no more than 4 out of the 83 would be expected to give values differing from 100 by as much as this on account of random variations due to a small number of deaths , so it is evident that the deviations for 49 of the towns cannot be so accounted for . 
was from 142 to 61 , with 27 towns showing significant excess over 100 and 27 a significant deficiency , so the deviations for 50 of the towns cannot be accollnted for by random variation . 
 and the social class index on the one hand and the age index on the other have been calculated separately for the 42 northern towns and 41 other towns , and also for the 83 together . 
the values given in table iii show that cancer of stomach mortality is positively correlated with the proportion of unskilled workers in the populapion , more so in south than north ; and in england and wales as a whole the coefficients are about + 5 for each sex . 
there is , however , a strong negative correlation between the social class index and age index , mean ing that towns with larger proportions of unskilled labour tend to have smaller proportions of their adult populations with ages over 55 . 
when the age index is kept constant cancer of stomach is still positively correlated with social class index to the extent of + 3 to + 4 , and this agrees with a similar result for the metropolitan boroughs of london in 1921 - 30 ( registrar - general , 1948 )  . 
 it is now seen that there is also a small negative correlation between cancer of stomach and the proportion of people of the same sex alive at ages 55 and over , and that this does not disappear when the social class index is made constant . 
 this is important , because it shows that the great variation in cancer of stomach rates in the towns is not due to mere differences in the correctness of certification of this cause of death by doctors . 
it has been suggested that the notable differences between cancer of stomach rates recorded in britain , denmark and switzerland may be due to excessive statement of this as cause of death in parts of those countries . 
if that were so it would be expected that the over - state ment would be greater amongst old than amongst young people , since the care devoted to diagnosis and correct statement of cause of death inevitably diminishes after about age 55 . 
consequently , if the great differences in standardized death rates in different towns , and in social grades , were due to excessive statement of cancer of stomach in certain of them , those with high rates would tend to have larger proportions of old people . 
for males the contrast is greatest at ages between 35 and 55 , when the death rates in the aggregate of 20 towns forming group ( a ) were just double those in the 18 towns forming group ( d ) ; and after 55 the relative excess diminishes to only 35 per cent at ages 75 and over . 
for females the excess in group ( a ) compared with group ( d ) was around 90 per cent up to age 65 , and then diminished to 45 per cent at 75 and over . 
 a pronounced decrease in the dispersion of death rates as age advances after 45 in males and after 55 in females is to be seen in table v , where rates for cancer of the stomach in the different social classes of the population of england and wales in 1930 - 32 are compared in the same way . 
for males the excess mor tality in class v , unskilled labourers , compared with that in classes i - ii , pro fessional and higher graded occupations , was as great as 86 per cent at 35 - 45 , and diminished progressively to only 8 per cent at 70 and over . 
for the wives of men in class v and single women in unskilled occupations the excess was 80 per cent at 45 - 55 , and fell to 37 per cent at 70 and over . 
 these relations with age are the reverse of what would be expected if the large differences in causes of stomach mortality between various large towns and between social classes arose from differing accuracy of death certification . 
 it will be seen from table 13 of that publication that the dispersion of rates according to a housing density grouping were much smaller at ages over 65 than at 45 - 65 . 
if the differences between cancer rates in groups ( a ) and ( d ) of table iv were due to more complete diagnosis , or excessive diagnosis , of stomach cancer in ( a ) than ( d ) , there must have been corresponding deficiencies in causes of death other than cancer in the towns of group ( a )  . 
the total deaths in all the county boroughs during 1931 - 39 from the various groups of causes concerned , and from cancer of the stomach , at ages 45 - 65 and at 65 and over were as shown in table vii . 
10 , 568 at the age - groups 45 - 65 , 65 and over , the totals of cancer of stomach deaths were not much different from the totals of digestive diseases excluding peptic ulcer and appendicitis , and since the great bulk of any transfer of deaths into or out of the cancer group by wrong certification must pass out of or into the " other digestive " group , this means that a strong negative correlation must exist between death rates attributed to the two groups if the large variation in stomach cancer rates arises from diagnostic differences amongst the towns . 
 indeed , if this were the main cause , doubling the stomach cancer rate ( as in group ( a ) compared with group ( d ) towns ) would involve almost abolishing the rate for other digestive diseases . 
transfer might also occur between stomach cancer and peptic ulcer as a result of mis taken diagnosis , but the numbers of deaths attributed to the latter group were small in comparison with those attributed to the former , except amongst males aged 45 - 65 . 
it follows that the transfer of the whole of them to cancer , if that were possible , in certain towns would fail to account for the excess of certified cancer in those towns ; and even large negative correlations between the two causes could not account for the variation in stomach cancer amongst males over 65 or amongst females of either age group . 
 table vi shows that for males of both age groups cancer of stomach death rates had no appreciable correlation with either peptic ulcer or other digestive diseases ; for females of both age groups there was a small but statistically insig nificant negative correlation with peptic ulcer , and a small just significant positive correlation with other digestive diseases in the country as a whole . 
 since none of the factors tested above can account for the differences in tables i and ii , the remaining explanation is that some extrinsic irritant factor is concerned which differed in intensity in the various towns . 
the curious contrasts in cancer of stomach mortality in london boroughs when grouped according to their source of water supply ( registrar - general , 1947 ) , correlation coefficients were calculated between the c.m.r. 
for cancer of the stomach during 1921 - 39 and the estimated total hardness of thewater supply of the 83 towns during 1911 - 31 , as shown in tables i and ii . 
it has to be remembered that the location of certain industries has been influenced to some extent in the past by the need for a soft water supply , and that in the north of england much of the industrial population is served by soft water . 
of the 42 northern towns 23 had water supplies with less than 7 degrees , or 10 parts per 100 , 000 of total hardness ; their average social class index was about 37 per cent unskilled and partly skilled workers . 
the 9 northern towns with water supplies of 20 or more parts per 100 , 000 total hardness had an average social class ' lildex about 42 per cent ; and the correlation between hardness and social index was in the 41 towns elsewhere there was no correlation between significantly positive . 
 the coefficients of correlation in table iii between cancer of stomach mor tality and hardness were positive in the north for males and negative in the south for each sex ; and when all the towns are grouped according to an ascending scale of water hardness , it appears that the c.m.r. 
no expla nation is offered for this , but the : figures are placed on record because they might at some future time fit in with other facts ( table viii )  . 
 102 100 llo 100 100 whatever irritant may be concerned in the production of gastric cancer , the effect of greater intensity of the irritant in one environment than in another might be ( 1 ) to increase the proportion of people developing cancer at each age without changing the latent period of develop : ment ; ( 2 ) to shorten the average latent period before cancer appears without changing the proportions of people affected , or ( 3 ) to increase the proportion affected and also shorten the latent period . 
if it is a long - continued mechanical or chemical injury to cells the predominant effect to be expected from increased intensity of action would be a shortening of the latent period , without necessarily increasing the proportion of people affected , although that might also occur . 
the ratios between death rates in groups of towns and between social groups would not be expected to fall with advancing age after 50 or 60 as in tables iv and v , and this suggests that the irritant is not an infective organis assuming some form of chemical or mechanical irritant is concerned , the latent period before cancer appears will vary rather widely about a mean value in the manner characteristic of most measurable vital factors ; and if the latent periods of gastric cancers in residents of all towns could be measured they would form a distribution approximating to the " normal " type . 
 it would be possible to reproduce the observed age - incidence of deaths from gastric cancer on the supposition that the irritant begins to affect a certain pro portion , p , of people who are susceptible to it at about 15 years of age , and th.at at each year of age thereafter an additional proportion p / k become susceptible to it and begin to be affected . 
 if , for example , the mean latent period before death were 20 years , with a standard deviation of 5 and normal distribution , k being a suitable constant , the numbers of deaths at successive quinquennial age periods from 25 - 29 to 65 - 69 would give a reasonably good fit to the deaths registered in england and wales in 194 7 . 
a shortening of the latent period in the towns where the irritant was most intense and a lengthening of it in those towns where it was least intense would then result in the ratio between the respective death rates diminishing with advancing age after about 40 , as in table iv . 
the observed facts appear to be consistent with such an explanation , but a careful study of the mathematics of the assumptions outlined above is needed before definite conclusions can be reached . 
 the death rates from cancer of the stomach in the 83 county boroughs of england and wales in the period 1921 - 39 show great differences , which cannot be explained either by chance variations , or by differing accuracy of certification of the cause of death . 
standardized mortality tends to be greater in the northern towns , in towns with a low proportion of people of advanced age and in towns with a high proportion of men in unskilled and partly skilled occupations . 
 cancer of the stomach death rates of men were unrelated with those for peptic ulcer or other digestive diseases ; but amongst women there was a small negative correlation with peptic ulcer and a positive correlation with other digestive diseases . 
the present situation has been briefly restated by stocks ( 1953 ) at cardiff . there have been many endeavours to establish a racial factor to account for the known variations in cancer incidence . although they have been invariably inconclusive , as might be expected when consideration is given to the pronounced differences within individual countries , these surveys have shown that where racial types have migrated to other countries , e.g. , negroes to america , they appear to take over the incidence of malignant disease of the natural inhabitants among whom they have come to live . 
1. goitre distribution in holland ( amsterdam university )  . year , three further districts have been added to the list in the northern province of holland ( polak , 1954 ; personal communication )  . the lower incidence of goitre towards the belgian frontier is apparent and almost certainly significant . ( 2 ) united kingdom . variations of goitre and cancer within the united kingdom are striking . 
a recent survey ( stocks , 1950 ) of the incidence of carcinoma of the stomach in 83 county boroughs over a period of 18 years showed notable differences " which cannot be explained by chance variation or by differing accuracy of certification of cause of death "  . these variations were shown to correspond to variations in the hardness of the water supply : those with a moderate hardness tended to have 396 j . 
survey of goitre ( murray et this would be in keeping with the al . , 1948 ) to have a high goitre incidence . known clinical experience with regard to malignant disease in that county . comparing these two maps a striking correlation is at once apparent . outstanding are the heavy incidences of goitre and cancer in mid - wales , westmoreland fig . 
spencer and the fen district and neighbouring counties , and by contrast the light incidence in yorkshire , lincolnshire , herefordshire and pembrokeshire . gradations of correspondence on both maps are evident in comparing norfolk , suffolk and essex ; kent and sussex ; cornwall and devon ; glamorgan and carmarthen . a higher death rate from cancer was shown to exist in two swedish counties , kopparberg and gefieborg , than was found in the total rural areas of sweden ( stocks , 1925 )  . those two counties , as shown by examination of schoolchildren , recruits for the army and candidates for confirmation , were found to have a higher incidence of goitre as compared with the rest of the country . similarly , known goitrous counties in norway were compared by stocks ( 1924 ) with counties showing little goitre , and it was found that the goitrous counties showed a cancer rate for all organs of 113 - 7 as compared with 94.4 for the niiongoitrous counties . in iceland , the thyroid has been shown to contain one of the highest conlcentrations of iodine , and endemic goitre is non - existent ( rundle , 1951 )  . thyroids there weigh on an average 14 g . 
and carcinoma of the stomnach and oesophagus , when considering separate states . ( 10 ) australia and new zealand . australia is considerably more industrialised than new zealand and has many furthermore , rodent ulcer is large cities as compared with the latter country . infinitely more common in australia , where goitre does not obtrude as a major health problem , though there are certain areas where there are definite , if small , districts where goitre is found ( kelly , 1946 )  . 
the more rural and agricultural population of new zealand has here goitre is a major health problem and its a death rate from cancer of 14 - 56 . incidence in europeans , maoris and animals alike has been closely studied for years . 
as metabolic rate is in general a rough indication of thyroid activity , there would be appear to be some relationship between malignant disease and thyroid thus , the lowered metabolic rate often seen in diabetes mellitus dysfunction . ( joslin , 1946 ) may be considered with the increased liability of diabetics to develop carcinoma in any site or organ as compared with non - diabetics ( joslin , 1946 )  . in the same way , the lowered metabolic rate commonly found in patients with all types of peptic ulcers ( kolmer , 1949 ) may be considered with the incidence of carcinoma of the stomach - still one of the commonest neoplasms of man . ( 2 ) it has long been observed that puberty and pregnancy are periods when goitres may appear ( marine and lenhart , 1909 ; marine , 1935 ) , and may disappear when the period of excessive demand ceases or when iodine is given . these are also the periods when melanomata tend to increase in size and may , not infrequently , become malignant . 
at the beginning of this year rwas admitted to this hospital on account of malignant melanoma with spread to glands in the course of her third pregnancy . in her two previous pregnancies she also developed malignant melanoma on each occasion and was in this hospital in consequence . she has a moderate enlargement of the thyroid of the smooth " colloid " type seen commonly in pregnancy . 
the end of pregnancy is also the period when chorionic carcinoma and lactation carcinoma of the breast occur - perhaps the most rapidly growing class of cancer known . ( 3 ) administration of thyroid has been found to improve the action of oestrogens in the treatment of carcinoma of the prostate , permnitting the effective use of smaller doses of oestrogen and delaying the onset of insensibility to oestrogen ( winsbury white , 1948 )  . in support of this clinical experience a paper in endocrinological research is quoted ( chu and you , 1945 ) , but the final conclusions of these workers was that the simultaneous administration of oestrogen and thyroid was the same as that obtained by thyroid feeding alone . ( 4 ) reduction of keloid formation can often be brought about by administration of thyroid . 
the age for the start of the decline in iodine content of the thyroid coincides in general with the age at which malignant disease in mnan becomes a prominent feature . ( 7 ) in the second week of january of this year there were at least three patients in this hospital at the same time with gross goitre and cancer . m - - , carcinoma of breast and a large colloid goitre visible seven or eight beds away down the ward . e - , lymphosarcoma and a thyroid that weighed 80 g . 
the nodular goitre caused some difficulty ing involvement with growth . during resection of glands of neck . since january other patients have been seen in the wards of the hospital with coexistent carcinoma and goitre . it is relevant to note that the area from which this hospital draws its material , gloucester , somerset and cornwall , is not one in which goitre is frequent . ( 8 ) the association of goitre and malignant disease in the post - mortem room was strikingly illustrated by analysis of 1000 post mortems at the middlesex hospital ( stocks , 1924 ) , when anomalies of the thyroid were found in 13.3 per cent of males and 21.2 per cent of females of 500 persons dying of cancer ; whereas only 2 - 0 per cent of males and 6.5 per cent of females dying of non - cancerous conditions showed anomalies of the thyroid . professor barlow excluded from these anomalies secondary invasion of the thyroid by cancer , and the anomalies most frequently found were simple parenchymatous enlargement and adenomatosis , while calcified nodules , cystic changes , atrophy and fibrosis were frequent . 
of dba ( bather , 1952 )  . ( 3 ) rats bearing walker rat carcinoma 256 were treated with natural thyroof these 27 per cent showed complete remission and 12 per cent " favourxine . able " histological response , 68 per cent . 
showing no response at all . another group treated with synthetic hormone gave only 2 per cent of remissions and no response in 98 per cent of animals . 158 animals were involved ( herbut , kraemer , and jacksen , 1950 )  . regression of rat tumours may be induced by thyroxine the natural comment . hormone being superior to the synthetic . ( 4 ) feeding butter yellow ( p - dimethylaminobenzene ) to rats produced malignant neoplasms of the liver . 
the recent work of moon , simpson , li and evans ( 1950a , 1950b , 1952a , 1952b ) has convincingly confirmed the conclusions of earlier workers twenty years ago ( ball and samuels , 1932 ; bischoff , maxwell and ullmann , 1934 ) , that whereas extracts of the anterior pituitary cause tumours to arise in a variety of sites in laboratory 408 j . 
spencer iodine availability , traced by goitre incidence , appears to be one of such factors . closer scrutiny of some of these countries corroborates these conclusions . clinical findings and a review of some of the experimental work available lend further emphasis to these observations . the apparent relationship of thyroid insufficiency and the liability to develop cancer is discussed in connection with such other hormonal influences over cancer as are already known , particularly in respect of the pituitary . lines of investigation and clinical trials have been started , but results of any value cannot be expected for several years . a non - specific organ immunity or susceptibility seems to be the simplest explanation of the facts presented , and the possibilities , preventive and perhaps therapeutic , that are opened up by this line of research are briefly discussed . i am indebted to polak of amsterdam for fig . 
allsopp. from guy 's hospital medical school , london , s.e.1. received for publication march 5 , 1951 . in earlier papers ( allsopp and szigeti , 1946 ) an account was given of some of the properties of water - soluble substances which are produced from 3 : 4 - benzpyrene under the influence of monochromatic radiation of wavelength 2537a . aqueous ( bicarbonate ) extracts from the irradiated benzpyrene were found to be carcinogenic when painted on the skins of mice ( allsopp , 1946 )  . these biological tests , however , were very prolonged , four or five paintings weekly for some in an attempt twenty - six weeks being required before growths were obtained . to reduce this " latent period " experiments have been made in which croton oil was used as a co - carcinogen . observations on the effects of croton oil on the mice used in these experiments , when compared with those of p . 
of 0 - 5 per cent croton oil in acetone ( experiment la ) at first caused little superficial change . slight ulceration , as evidenced by patchy encrustation , was visible on the third day , after which epilation began . this was complete by about the tenth day . 
so soon as the encrustation sloughed hair began to grow again , and by the fourteenth day the animals appeared normal . day to day observations are set out in table ii . painting with 0 - 5 per cent croton oil at weekly intervals ( experiment lb ) caused a similar sequence of macroscopic changes , but restoration of hair growth was delayed until the third week , after which the epilation cycle repeated itself 276 c . 
no growths resulted , and histological examination of the painted skins revealed nothing more than a slight hyperplasia on any animal . co - carcinogenic effect of croton oil with 3 : 4 - benzpyrene . ( table i , experiment 3 . ) equal volumes ( 0 ' 1 ml . ) of 0 - 05 per cent benzpyrene in acetone and of 0.5 per cent croton oil in acetone were applied alternately at 3to 4 - day intervals , each animal thus receiving each agent once weekly . 
control animals were painted at the same times with benzpyrene , but with acetone instead of croton oil solution . papillomata were first seen on the experimental animals after 5 weeks ; all the survivors had malignant or " pre - cancerous " lesions by the fourteenth week , a precancerous lesion in this context being one exhibiting irregular epilation , hyperkeratosis , abnormal regeneration of hair follicles , ill - defined epidermal projections , a large increase in the number of mitotic cells , numerous abnormal mitotic figures , and leucocytic infiltration of the dermis ( allsopp , 1946 )  . 
when the experiment was discontinued in the twentieth week , 4 out of 8 surviving control animals had cancerous or pre - cancerous lesions ; the remainder showed marked hyperplasia only . 
the experiments described above , however , suggest that its action is more complicated than this simple description migth indicate . despite a marked difference in the response to croton oil of the mice now used when compared with that of strains used by other workers , berenblum 's findings ( berenblum , 1944 ) have been largely confirmed when 3 : 4 - benzpyrene was the carcinogen ; but when the hydrocarbon was replaced by the water - soluble substances which are obtained from it by the action of ultraviolet light croton oil appeared to exert a different effect since , while papillomata appeared on the mice at a very much earlier stage than they did with the carcinogenic solutions alone , a proportion of these papillomata did not develop into epitheliomata , whereas 278 c . 
the distinction in behaviour , however , was not entirely clear - cut , since the papillomata on 2 out of 9 animals did persist , growing very slowly , until they became malignant . the earlier experiments with the water - soluble substances ( allsopp , 1946 ) showed that with them the process of carcinogenesis was slow , but that it passed through the usual 3 phases of hyperplasia , papillomata , and finally carcinomata . the last stage required 6 to 10 weeks . 
the present experiments suggest that croton oil accelerates the transition from hyperplasia to papillomata - the first papilloma appeared after 13 instead of 26 weeks - but not the transition from papillomata to epitheliomata , which again took about 9 weeks . indeed it may even delay this change , or - in those cases where the papillomata were sloughedin contrast to this , benzpyrene itself appears to act rather more prevent it . quickly ; epitheliomata had developed in the animals recorded in experiment 3b of fig . 
gorer , of guy 's hospital medical school , and to m . salaman , of the london hospital , for comparing with mine the results of their experiments on the effects of croton oil on the skin of mice . 
 data have been published which indicate that the average latent period for all methylcholanthrene - induced fibrosarcomas ( strong , 1948 ) and the survival time ( strong , 1950 ) of mice developing such tumours are both influenced by litter seriation . 
the latent period ( time between the subcutaneous injection of the carcinogen and the initiation or the initial growth of the ensuing fibrosarcoma ) was variouslv affected in the different strains bv the litter to which the mouse in mice of some strains ( prunt and f : of c67 x brs ) the latent period belonged . 
 ( strong , 1948 ) for the appearance of fibrosarcomas decreased in the successirn litters of the same pair of mice , while in other strains susceptibility to fibro sarcomas was relatively constant . 
the sunival time ( strong , 1950 ) of mice growing a chemically - induced fibrosarcoma ( time between the initial growth of the malig nancy and the death of the individual ) is also affected by litter seriation . 
 ' \\nen a mouse developed a fibrosarcoma in less than 100 days following the subcutaneous injection of methylcholanthrene it sunived an average of 52 days with the progressive growth of the tumour ifit belonged to a first litter . 
in the succeeding litters mice of the same age and latent i : eriod survived longer and longer with growing fibrosarcomas until , if they belonged to an eighth litter , they lived on an average of 130 days . 
these genes were derived from mice of the ancestral stocks jk and n which were used in the origin of the : xho stra the first three generations ( cba... " x jk ) were used for breeding purposes only . 
at the time of the separation of prunt from pbr descent , the average latent period for the appearance of methylcholanthrene - induced fibrosarcomas of the direct in the f 20 generation of the experimental maternal ancestry was 3695 days . 
these two untreated descents , prunt and 2prunt , should possess genetic similarity , and any biological differences that they have may be due either to divergent segregation from a residuum of genetic heterozygosity ( probably very small after 17 generations of brother - to - sistier matings ) or to new biological variability which has appeared in one or both untreated deseents following their separation from a common ancestry . 
the mice were fed a standard diet of nurishmix pellets ( pratt food company ) and water ad libitu a supplement of mixed grains ( oats , wheat and sunflower seeds ) and calf meal pellets were given to the mice once a week . 
the mice were examined periodically for tumoun ; , and the latent period taken to be the time at which a firm nodule at the site of the injection of the carcinogen began to increase progressively in si mice which developed tumours at sites other than the one at which the carcinogen had been injected were tabulated separately . 
 table i gives the percentage incidence of tumours in mice which developed in less than 100 days ( column e ) , together with the data obtained on all tumours at the site of injection of the carcinogen irrespective of latent period ( column d )  . 
 data on the total number of mice injected with methylcholanthrene ( column a ) , the number of mice which died without developing any tumour ( column b ) , and the total number of mice showing tumours irrespective of latent period and site are also given ( column c )  . 
since the two separate descents disclose similar genetic origin and show similar trends by which the females are consistently more susceptible to fibrosarcomas than the males , the data for both series may be added together . 
however , the present analysis of trends of susceptibility in s1icoessi.e litters discloses differences , and therefore the two strains , the prunt and the 2prunt , should be kept distinct . 
thus 27 2 pe : r cent of the females developed fi.brosa : rcomas within less than 100 days of latent period , whereas only 92 pe : r cent of the males developed similar tumou : rs during the same period , a difference of 180 pe : r cent in tumour susceptibility  . 
the trend for the percentage incidence of tumours developing in less than 100 days among females of successive litters was found to be y = 85 + 5 lz , with a standard deviation of the slope of 0 - 741 . 
comparison of this value with a no - trend or 0 - slope value by t test reveals that the observed trend is statistically significant ( since p = < 001 )  . 
 similarly the trend for the percentage incidence of tum.ours developing in less than 100 days among males of successive litters was found to be y = 132 - 12x , with a standard deviation of the slope c , f 0968 . 
 the analysis of the data thus discloses that there is an increasing sex differential in relation to chemically induced : fi.brosarcomas in successive litters in mice of the prunt descent . 
the difference between the mice of the two descents must , therefore , be associated with a mechanism which in the 2prunt descent changes in litter seriation at least in some strains of mice . 
 the maximal sexual differential is found in mice of the second litters ( 302 per cent ) , and this sex differential fluctuates in the succeeding litters and thus shows no significant trend . 
however , the data of the 2prunt descent are complicated by a high value for females of the second litter ( 382 per cent ) and a high value for males of the sixth litter ( 143 per cent )  . 
in one strain ( the chi ) males were more susceptible to chemically induced fibrosarcomas than were the females , whereas in the 15th inbred strain ( the cul ) the females were more susceptible to tumours than were the males . 
 the mechanism involved in the development of this new sexual differential in relation to chemically - induced fibrosarcomas in mice of the prunt descent gives a slight sex difference in mice belonging to the early litters of a breeding female , and apparently gradually increases in the succeeding litters . 
h the sex pattern of an individual is the reflection of the genetic constitution of the individua1 , then it is not clear whether mice of the early litters or mice of the later litters are to be considered the pattern for the species . 
but sex physiology is also under the influence of several hormones , and the sex pattern can be influenced significantly when a hormone is administered at an early age , preferably before birth . 
 the present investigation with methylcholanthrene has disclosed that several aspects of malignancy ( the average latent period , the survival time and the per centage susceptibility of tumours in mice of latent periods of less than 100 days ) are influenced by litter seriation . 
do hormones fluctuate in the female body with advancing litter frequency , pass the placental barrier and influence the subsequent physiology of the offspring at least in relation to malignancy and perhaps to other characteristics as well ? or are we to conclude that maturation phenomena in cytoplasm ( perhaps the mito chondria or other constituents ) are responsible for this transmission from mother to offspring which apparently is not through the genes ? perhaps another biological reason may eventually be found for the data at hand . 
these mice belonged to two separate descents derived from a common genetic orig in one descent , the prunt , there was an increasing sexual differential in relation to chemically - induced fibrosarcoma.s in the succeed320 l . 
 evidence offered by calcutt ( 1949 ) showed that 3 : 4 - benzpyrene is effective in inducing the oxidation of the - sh groups of a variety of compounds , the benzpyrene itself also undergoing oxidation during this process . 
on the basis of physical properties and fluorescence spectra it was suggested that the benzpyrene derivatives obtained in this fashion were comparable to those obtained as a result of the metabolism of the hydrocarbon by mice . 
this suggestion is important in view of crabtree 's ( 1947 ) conclusions as to the involvement of - sh groups in the carcinogenic process , and also in relation to powell and calcutt 's ( 1949 ) conclusion that the availability of sulphydryl influences the metabolism of benzpyrene . 
 studies by weigert and mottram ( 1946a , 1946b ) showed that the initial step in the metabolism of benzpyrene is conversion to either 8 : 9 - dihydro 8 : 0r1 - 9.0h benzpyrene ( bpx1 ) or 8 : 9 - dihydro 8 : 0r1 - 90r2 benzpyrene ( bpx2 ) , r 1 and r 2 being unidentified radicals . 
at the same time it was shown that these compounds were convertible by simple chemical methods , which have their counterpart in the animal body , to the final excretion products - 8.0h benzpyrene and the 5 : 8 quinone . 
 it was suggested by quick ( 1937 ) that the primary step in the metabolic oxidation of hydrocarbons is a union of cysteine with the unsubstituted aromatic ring , replacement of the mercapturic acid by a hydroxyl group occurring later . 
 there is no direct evidence for this view , but the recognized involvement between carcinogenesis and sulphur metabolism ( crabtree , 1947 ; calcutt , 1949 ) is in its favour . 
it is , however , severely criticized by boyland and weigert ( 1947 ) , who point out that phenylcysteine is excreted as phenylmercapturic acid - - - evidence which suggests that the linkage between cysteine and an aromatic compound is not easily broken in the body . 
it is apparent that such a scheme could apply equally well in the case of the chemically related benzpyrene , bpx1 or bpx2 arising from the substitution of the hydrogen of one or other of the hydroxyl groups of the primarily formed dialcohol . 
an alternative possibility in line with the perhydroxylation view is feasible since autoxidizing thiols are known to give rise to hydrogen peroxide ( schober ! , 1932 ; holtz and triem , 1937 ; schales , 1938 )  . 
 if a perhydroxylation process is the initial step in both the in vivo and in vitro oxidation of benzpyrene it appears that it should be possible - under suitable conditions - to oxidize the hydrocarbon directly with hydrogen peroxide . 
 the addition of 6 per cent hydrogen peroxide to a colloidal suspension of benzpyrene in distilled water had no effect at room temperature even when the two compounds were maintained together for long periods . 
since hydrogen peroxide contains acid as a stabi lizing agent , any derivative of the bpx1 type formed in the reaction would be expected to break down to the phenol and thence to the quinone - a sequence of events which superficially would resemble those found . 
 as an attempt to counteract any influence due to the acid the experiment was repeated using the benzpyrene colloid suspended in a clark and lubbs buffer solution at a ph of 75 . 
this time the hydrogen peroxide was added tilowly ; whilst at intervals the ph was checked , and when necessary was restored to 75 by the further addition of 02m . 
examination of the fluorescent spectra of the solution derived from the three portions gave results as below : surface : - two diffuse maxima at 420 and 440 able from fluorescence spectrum of bpx1 derived from mice . 
the likelihood of it actually being 8.oh benzpyrene is enhanced by the fact that after transfer to petroleum ether and standing for a few days it lost its blue fluorescence and achieved a barely perceptible yellowish tinge , behaviour which would be in keeping with the known autoxidation of 8.oh benzpyrene to benzpyrene 5 : 8 quinone . 
the passage of a weak solution of an authentic sample of 3 : 4 - benzpyrene 5 : 8 - quinone through the same column resulted in concentration over the previous zone , and , although the zone slowly passed down the column , no further elution or variation in solvent allowed any separation of the two zones . 
the similarity of fluorescence spectra , physical pro perties and behaviour suggests then that the material separated from the reaction mixture is similar to , even if not identical with , the bpx1 from mice and the bpx1 - like material formed in the presence of autoxidizing thiols . 
 further examination of the substances extracted from the other two portions of the original chromatograph column produced little of interest other than that the material was water - soluble in both cases . 
attempts to utilize the benzpyrene in solution in organic solvents have so far failed , the only results being the formation of small amounts of apparent 8.oh benzpyrene and quantities of various unidenti fiable products . 
furthermore , the evidence suggests that the primary reaction product is a diol closely resembling that found as the initial product of benzpyrene metabolis at the same time this primary product shows a remarkable resemblance to that found as the result of oxidation of benzpyrene in the presence of autoxidizing thiols . 
 in view of the known forma tion of hydrogen peroxide during the autoxidation of - sh groups , it seems prob able that the primary mechanism of oxidation in both cases consists of the addition of hydrogen peroxide to the hydrocarbon . 
equally , since the formation of peroxide is a phenomenon known to occur during biological oxidations ( oppen heimer and stern , 1939 ) , it seems that such a mechanism could account for the initial steps of the in vivo oxidation of benzpyrene . 
 the oxidation of 3 : 4 - benzpyrene with hydrogen peroxide and the isolation of a product similar to the benzpyrene oxidation products obtained during metabolism and during oxidation in the presence of autoxidizing thiols is de scribed . 
the frankly keratinizing members of our series we have taken as a standard with which to compare the behaviour of our remaining histological types . the morphological features of the more differentiated epitheliomata have been so completely evaluated that any further discussion would be superfluous . 
the primary lesion grows slowly and tends to be clinically latent for a considerable period of time , it appears as a especially if it is situated in a region such as the nasopharynx . smooth or finely granular , firm , globular tumour , at first a mere nodule , but later attaining considerable dimensions . infiltrative spread and superficial ulceration occur fairly late in the evolution of the growth . cervical lymph glandular metastases appear at an early date , while the final phase is frequently ushered in by distant visceral extension , osseous deposits being rather characteristic ( kienbock and selka , 1935 ; ch ' in and szutu , 1940 )  . 
the histological picture may be syncytia of large , palely staining cells appear as sheets , irregular quite typical . clumps , or columns , in a lymphocytic stroma of varying density . 
ewing was doubtful of the specificity of schmincke 's description , and found its distinction from other types of undifferentiated pharyngeal growth difficult or impossible . the term " transitional cell carcinoma " was first employed by quick and cutler ( 1927 ) to describe a group of highly radiosensitive buccopharyngeal tumours of uncertain histogenesis which lacked the classical features of epithelioma , but presented transitional epithelial characters . ewing 's writings ( 1929 , 1940 ) have done much to clarify the morphological features of this growth . the most frequently encountered sites are the tonsil , base of tongue , nasopharynx , laryngopharynx , nasal cavity , and accessory nasal sinuses . quick and cutler described the primary lesion as possessing " a finely granular , velvety surface , e . 
fairburn this description is , however , far from being specific . which looks like an erosion of the mucous membrane rather than a frank ulceragives the impression of having originated in the tion . 
the lesion deeper structures and adhered to and eroded the mucous membrane from the clinical beneath . " course resembles that of lympho - epithelioma - a small and often occult primary histologically , transitional focus , and early cervical lymph glandular metastasis . cell cancer is typified by the presence of broad , clearly demarcated , epithelial the cells are cylindrical , polygonal or spindlecolumns and alveolar formations . shaped , with a rather hazy cytoplasmic membrane . 
the oval or fusiform nucleus is more chromatic than that of lympho - epithelioma , while its nucleolus is less frequently the marginal cells appear regimented as though forming distinct . degeneration and necrosis of the central zones of some of the a basal layer . lymphocytic infiltration of the stroma epithelial formations may be observed . may be present , but is never marked . there is considerable divergence of opinion regarding the justification of the classification of lympho - epithelioma and transitional cell carcinoma as specific entities . 
each has been traced to death or for a minimum period of five years following the first examination . purposes of classification we have created the following groups according to the site of the primary lesion : 3 . 
the cells are distributed the cell body is spheroidal or polygonal in solid masses or diffuse infiltrations . in form with palely staining cytoplasm , and a relatively large , oval , hyperchromatic nucleus which frequently shows bizarre , atypical features and mitotic figures . these classifications have presented certain difficulties : 1 . 
compared with the figure for the frankly keratinizing group ( 61.5 years ) , the former two differences are statistically significant , while the latter is not . there is no significant difference between the mean ages of - our cases when distributed according to the site of the primary growth . 
fairburn we have adopted ebenius ' clinical criteria of metastasis . the frankly keratinizing cancers possess a statistically significant lower incidence of metastasis ( 36 per cent ) than the remainder of their fellows , with the exception of the undifferentiated group which only metastasized in 22 per cent of cases . this table also illustrates the influence of glandular metastasis upon the likelihood of death with active growth . 
frank , lev and blahd ( 1941 ) , in their study of transitional cell growths , report a 44 per cent incidence of metastasis . 52 per cent of our cases possessed evidence of glandular extension on first examination . inspection of our data discloses the relative insignificance of tumour histology as a prognostic factor in cases of cancer of the lympho - epithelial sites . 
maccarty ( 1922 ) and plaut ( 1927 ) have emphasized its limitations , stressing the far greater importance , in most instances , of clinical features , such as the age and general condition of the patient , the duration , location , and gross anatomy of the growth , and the presence , or absence , of metastasis . 
the enormous preponderance of the site factor over that of histology is borne out by this study . thus , frankly keratinizing neoplasms of the lip carry a relatively good prognosis , but when arising in a lympho - epithelial region their significance is as sinister as that of their ill - differentiated fellows . 
the presence of lymph - node metastasis on first examination is , in our experience , a factor of far greater prognostic value than tumour histology , although very appreciably influenced by the site of the parent growth and its accessibility to treatment . 
to take a specific example , metastasis in a case of lip cancer markedly decreases the likelihood of cure , whereas when a lympho - epithelial region is involved , death with active malignancy is likely to occur whether the cervical nodes are involved or not . the chance of invasion of the cervical lymph glands appears to be largely independent of the histological grouping , the site of origin of the tumour being of infinitely greater significance . it has been widely assumed that the main factor in dissemination is the inherent growth capacity of the neoplasbut this is not the only influence which has to be considered . 
 * from the department of experimental pathology and cancer research , school of medicine , leeds , 2 . received for publication august 11 , 1951 . ' thf , need for devising a technique for testing the carcinogenic properties of chemical compounds by direct application to the bladder epithelium has been felt for some time . 
yamazaki and sato ( 1937 ) described a method of introduction of oily solutions of chemicals into the rabbit bladder by catheterization . interpretation of the true nature of the one acceptable tumour described by these authors after the introduction of o - aminoazotoluol is complicated by the presence in an extensive series of of lipoid in phagocytes in the subepithehal tissues . experiments , hughes ( 1949 ) , by means of epithelial grafts wrapped around 20methylcholanthrene crystals , obtained 14 transplantable carcinomas from 120 attempts . 
by this method an even distribution of the carcinogen was difficult to obtain , and so considerable variation in concentration may have occurred . under ether anaesthesia the bladder was exposed by a small incision through the skin and abdominal wall about i cabove the urethral opening . 
the bladder was drawn out of the abdomen and supported on a small pad of gauze ; an incision was then made through the dome of the bladder and the edges held apart by small metal retractors . 
the suture passed through all the layers of the bladder wall , and the edges were drawn together so that the two epithelial surfaces were bound closely to one another ; thus a continuous epithelial layer only and no muscle nor connective tissue was exposed to the direct action of the carcinogen in the pellet . 
the abdominal wall and skin were closed separately by sutures of cotton or thick silk . the operative mortality was negligible , but a number ' of animals died within a few weeks of the operation due to the pellets blocking the urethra . 
the latter concentration had been found previously to be effective in inducing subcutaneou 's tumours , both sarcomas and breast adenocarcinomas ( bonser , personal communication )  . in the present experiment pellets containing 2 per cent of the carcinogen induced bladder epithelial h erplasia but not tumours , whereas a pellet containing 30 per cent induced an infiltrating cancer . 
no doubt lower concentrations would be equally effective . this technique has the advantage that an ' substance so apphed to th ' e bladder is subject only to the action of the urine be ' fore coming in contact with the bladder epithelium ; thus metabohc changes in chemical structure subsequent to feeding or injection are obviated . 
a reasonably high i ' ncidence of tumours should be obtained with an active substance without incidental induction of sarcomas . it should now be possible to determine whether metabolites of bladder carcinogens such as 2 - naphthylamine are ' tho active agents in inducing bladder cancer . economy in time , animals and material can be effected , and this is important when investigating the carcinogenicity of small quantities of metabolites isolated from urine . 
payne. from the department of cancer research , mount vernon hospital and the radium institute , northwood , middx . received for publication may 2 , 1953 . in the course of their original work on the metabolism of 3 : 4 benzpyrene weigert and mottram ( 1946 ) noted that once the hydrocarbon had been introduced into a tissue it could only be extracted with difficulty . repeated and prolonged washing of the finely minced tissue was found necessary to remove the hydrocarbon and its metabolites . in view of this " binding " of the carcinogen and its derivatives within the cell an attempt has been made to fractionate mouse liver after treatment with 3 : 4 benzpyrene . 
the liver was chosen for detailed work as being easily handled , of adequate bulk , and as a site where metabolism of 3 : 4 benzpyrene is known to occur . 
the hydrocarbon was introduced as a colloidal susthis method was deliberately chosen as pension by intraperitoneal injection . offering the best chance of avoiding the presence of undissolved particles of hydrocarbon in the blood vessels as occurs after intravenous injection . all mice received one intraperitoneal injection of 1 c.c. 
the best known method for isolation of intact mitochondria is that devised by hogeboom , schneider and pallade this involves maceration of the tissue in 0 - 88 m sucrose solution , removal ( 1948 )  . of nuclei by low speed centrifugation and then isolation of the mitochondria by further differential centrifugation . 
payne as they caused agglutination of the mitochondria and their consequent sedimentation with the nuclear fraction . methods , such as the modified behrens technique used by mayer and gulick ( 1942 ) , where fractionation is done in organic solvents , were considered impracticable on the grounds of being liable to remove any bound benzpyrene attention was then turned to the standard methods for isofrom the tissues . these involve maceration in citric acid solutions lation of nuclear fractions . since it is generally accepted that mitochondria followed by centrifugation . however , it was found are destroyed by acids these did not appear hopeful . that using ice - cold 1 per cent citric acid as advocated by mirsky and pollister ( 1946 ) it was possible to remove a clean nuclear fraction , and then by further centrifugation at higher speed to obtain a mitochondrial fraction . 
the mitochondria obtained in this fashion were a bit swollen and distorted , but still stained readily in very dilute janus green b solutions . in bulk the fraction obtained in this fashion compared favourably with similar fractions obtained using sucrose this result was rendered less surprising , however , when it was found solutions . that the literature references to the solubility of mitochondria in acid only refer to acetic acid . on the basis of the above findings a technique using citric acid was developed . at selected intervals after injection the mice were killed by a blow on the head , this was examined under a u.v. 
lamp opened , and the entire liver removed . with wood 's glass filter , and any particles of benzpyrene detected - their fluorescence shows very distinctly against the background dull blue fluorescence of the liver - were washed off with normal saline . 
the 70 per cent acetone was selected , as previous experience had shown this to be the best solvent for benzpyrene in tissues . all fractions were spun down again to clear them , and fluorescence spectrograms were made according to the method devised by doniach , mottram and weigert ( 1943 )  . 
payne an acid mediuat ph values below about 7 - 1 both these compounds lose water to form 8 - 0 h benzpyrene . this latter has a rather indistinct spectrum , and may easily have been missed against the background fluoresence due to the tissue equally it may have been oxidised still further to the residues themselves . 5 - 8 quinone during the violent agitation associated with the maceration process . in any event it was considered that the experimental conditions employed were not suited to the quest for benzpyrene metabolites . the significance of the results obtained is difficult to assess . 
horning. from the chester beally research institute of the royal cancer hospital , london , s.w.3. received for publication december 12 , 1948 . recfnt experience has shown that many of the growth - inhibiting agents used in the palliative treatment of cancer are carcinogenic . 
they also cause specific damage to cell nuclei and chromosomes and are able to induce mutations . the association of these biological effects of ( 1 ) growth inhibition , ( 2 ) chromosome damage , ( 3 ) production of mutations and ( 4 ) induction of cancer suggests if the that they may have a common fimundamental biochemical mechanism . induction of cancer is indeed a somatic mutation , then cancer induction might be included as a special mutation . 
the fact that x - rays can produce cancer in man was published in 1902 ( frieben , 1902 ) , seven years after rontgen 's discovery muiiller ( 1928 ) found that x - rays are mutagenic , and later mather of x - rays . and stone ( 1933 ) and koller ( 1934 ) described the chromosome damage following irradiation . 
the idea that cancer arises as a somatic mutation was supported by the demonstration of an increased incidence of mutations occurring in mice this was shown with methylcholanthrene treated with chemical carcinogens . by strong ( 1945 ) and with 1 : 2 : 5 : 6 - dibenzanthracene by carr ( 1947 )  . carcinogenic hydrocarbons inhibit the growth of animals and the establishment and growth of transplanted tumours ( haddow , scott and scott , 1937 )  . 
body - weight of freshly made aqueous solutions of nitrogen the first group was given methyl di ( 2 - chloroethyl ) amine hydromustard . chloride ( hn2 ) in a concentration of 1 mg . 
the second group received the same dose of tri ( 2 - chloroethyl ) amine hydrochloride ( hn3 ) for 10 weeks , after which time only 4 mice remained alive and injections were stopped . post - mortem findings of the mice which died within a week of an injection usually showed congestion of the alimentary tract or lungs . 
with the doses used all the coloured mice showed the effect of no nervous symptoms were seen . greying of hair ( boyland et al . , 1948 ) similar to that produced by x - rays ( hance and murphy , 1926 ) or by subcutaneously implanted plutonium ( prosser , painter , lisco , brues , jacobson and swift , 1947 )  . 
hornin - g occur spontaneously , and the tumours were larger and more malignant than those generally encountered in untreated mice . the carcinogenic action of the nitrogen mustards is very slow , the earliest lung tumour and lymphosarcoma being seen at 284 and 347 days respectively . berenblum and shubik ( 1947 ) have shown that carcinogenesis can be divided into an initial stage in which cells are irreversibly changed into latent tumrnour cells , and a development stage which can be brought about by cocarcinogens such as croton oil . 
the incidence of tumours expressed as quantal response is probably dependent on the initiating action , but the rate of appearance of the tumours depends upon cocarcinogenic action . the present authors consider that the initial irreversible conversion to latent tumour cells is the change which is associated with chromosome damage and mutations . 
the nitrogen mustards are possibly active in initiating the carcinoin order to test this hypothesis genic change but poor in developing action . experiments are in hand in which mice are being treated with nitrogen mustard and croton oil . a possible explanation of the association between inhibition of growth and carcinogenic power is that the inhibition is due to interference with cell division in growing tissues . 
the drugs under discussion reduce the rate of growth by impeding mitosis . this interference with mitosis is likely to increase the chances of a somatic mutation from normal to malignant cells occurring . 
of 14 mice which survived more than 250 days , 10 had tumours including lung tumours ( 8 ) , lymphosarcomas ( 2 ) , a uterine fibromyoma and a spindle - celled sarcoma at site of injection . this investigation has been supported by grants to the royal cancer hospital from the british empire cancer campaign , the jane coffin childs memorial fund for medical research , the anna fuller fund , and the u.s. 
3. received for publication january 18 , 1949 . the investigation of carcinogenic substances which have been obtained from human tissues ( kleinenberg , neufach and shabad , 1940 ) and from other biological sources has been the subject of earlier publications from this institute ( hieger , 1940 , 1941 , 1946 , 1947 ) , reporting that : 1 . 
a fuller account will be found in appendix i . it is sufficient to note here that it is a slightly modified form of the ordinary death rate which would be calculated as 2 l ( x ) m ( x ) l ( z ) ' the reasons for introducing such a modification are twofold : firstly , it makes allowance for the fact that the specific death rate considered is underestimated owing to deaths from other causes , and secondly , it measures the rate at which animals are dying out from the cause under investigation at the moment , x , not the bulk death rate over a month . the latter property allows a continuous curve to be drawn through the calculated values whose ordinates measure the rate of mortality at any moment of life . 
the monthly death rates on the other hand are strictly discontinuous , being , as is shown in appendix i , functions of the area under the , ( x ) curve over the month in question . using the method of haldane given in appendix i , the values of , ( x ) for cancer were calculated for a number of strains . those for three strains whose life tables were given by murray and hoffman ( 1941 ) are graphed in fig . 
1. the three curves bear out the conclusion of maurray and hoffman that the dba mice were the most susceptible , then the bittner albinos , and finally the marsh time in months fig . 
spicer albinos - a result which is the reverse of that indicated by the percentages dying the method of approach used here indicates that the cause of the of tumour . discrepancy is the difference that exists between the non - tumour death rates of the three strains . for if , using the methods outlined below , we calculate the percentage of cancer deaths that would be expected if eachl strain had the same non - cancer death rate as the marsh albinos , we find the following values : bittnera marsh a  . 
~. corrected to marsh - a . 90 5 per cent 87 - 7 76 - 6 uncorrected . 67 7 per cent 73.7 76 - 6 the process of calculating the p ( x ) 's has involved some smoothing of the data , and in the graphs no attempt has been made to draw a smooth curve ; the points have merely been joined by straight lines . 
the irregularities in the curves are mostly due to random errors , though the flattening of the , u ( x ) curve for dba mice from the 12th to the 15th month is repeated in the curve for another group of the same strain , and is therefore probably due to some real interruption of the steady increase in the tumour mortality . a further series of , l ( x ) curves are given in fig . 
3. allowing for this by subjecting the a - ro mice to the a - fc non - cancer , ( x ) , it is found that they would have almost the same percentage of cancer deaths . the percentage of cancer expected in the a - ro mice at the a - fc death rate is in fact 86 , while the percentage found in the a - fc is 88 . it appears then that almost the whole 302 c . 
the most obvious method of comparison is to compare the numbers or proportion of cancer deaths in any strain with those of a strain having a standard non - tumour death rate . alternatively the number of deaths over a similar period of time may be calculated when all non - tumour mortality has been eliminated . both of these indices can be calculated using the , ( x ) values . the difficulty of this approach is to find a standard which is equally useful for all strains , since there are wide variations in life span and in o 15 iii 0 ' 30 0.25 0 20 - o * i 0.10 time in months fig . 
murray and hoffman ( 1941 ) have attempted to overcome the first difficulty by using a life span as a unit and expressing time in percentiles of this value . this method seems to be unsatisfactory in that the determination of the length of span is to some extent arbitrary . 
murray and hoffman use the time at which 1 per cent of animals are surviving , adding that it would be possible to use the 0.1 per cent point if larger numbers of animals were available . 
each of these two cut - off points would give a different system furthermore these authors make no direct allowance for differences of time units . in non - tumour mortality , though they discuss the merits of a number of arbitrary weighting factors for calculating averages of the monthly cancer death rates . to avoid the use of arbitrary systems of weights it seemed best to calculate indices of susceptibility after the non - tumour death rate has been eliminated . this was done using a method due to dublin and lotka ( 1920 , 1921 ) , which has also been discussed by karn ( 1933 ) and irwin ( 1935 )  . 
spicer quently pointed out that the estimates might be improved by using the method of maximum likelihood , which gives results having a smaller standard error . the full calculations are rather more laborious than in the case of dublin and lotka 's method . 
6. it will be seen that the process of summation has resulted in a considerable smoothing of the data , and though the original , u ( x ) curves are not all similar in form , the integrated curves approach more closely to this ideal which is essential if strict comparisons of tumour susceptibility are to be made . after some initial confusion the curves spread out from left to right in order of decreasing susceptibility . 
any horizontal line drawn across the figures will give , by its intersections with the curves , the times of equal proportional mortality . vertical lines will give the proportional mortality corresponding to equal times of life . 
they are : 25 6 for the cross with a dba mother , and 37 8 for the reciprocal . from a study of the , ( x ) graphs it is obvious that the differences in the form of the curves from strain to strain make strict comparisons of tumour susceptibility , of the same kind as are made in biological assay , impossible . future work will probably reveal some of the factors which determine the shape of the curves , but in the meantime it should be emphasized that susceptibility to mammary tumour is determined in a different way in each strain , though the similarity of the , u ( x ) curves of different groups of mice of the same pure line does enable more exact comparisons to be made in this case . 
haldane , who first suggested that the , t ( x ) curves could be used for the investigation of tumour susceptibility , and who provided me with the method of calculation given in appendix i . 
 ( - ) = log.e 2 = 0 693 gives the half life . ( 4 ) modified expectation of life . the number obtained by taking the reciprocal of the sum x log . 
 ( 1 / px ) for the whole table is of interest since it gives an estimate of the expectation of life . suppose the table extends over ages from o t so that the sum of probabilities is then we can twrite the reciprocal of this sum t1 log . 
 ( 1 / p ) = t , j ( x ) dx ' ( x ) 1 ( x ) dx if there is no cause of death but cancer . so that the reciprocal is a weighted mean of the [ ( x ) 's . multiplying this by t , the interval over which the table extends , gives an estimate of the expectation of life . this estimate is rather lower than that given by the correct method of calculation since the values of 1 ( x ) are weighted in favour of the low values at the higher ages where the , u ( x ) 's are large . it might be preferable to use this modified life expectation instead of the half - life as it takes into account all the data and can be given a standard error . 
 ( p ) and the variance of the sum is equal to the sum of the variances since the p 's are independent . if we call this sum 2 then the variance of the half life will be given by and since and the standard error of x is var . 
1. received for publication january 13 , 1948 . one of the implications underlying the work of huggins and hodges ( 1941 ) on the factors governing prostatic hypertrophy is that malignant prostatic cells are not fully , if at all , autonomous . that is , they are still controlled in some measure by the same factors that control normal prostatic cells . specifically , this is certainly true of their susceptibility to the influence of circulating androgens and oestrogens . carcinoma of the prostate is essentially a disease of old age . the possibility exists that the chief factor in deciding the individual 's susceptibility to this disease may be his androgenic state , that is , the level of circulating androgens in his blood , particularly during the later decades of life . the only practical method for ascertaining androgenic state is a determination of 17 - ketosteroids excreted in the urine of the subject . in the hands of many different workers ( barnett , henly , morris and warren , 1946 ) this method has revealed a very wide range of excretion of 17 - ketosteroids by normal men , at least in the lower age - groups . 
robinson the prostates of individuals who excrete at the higher or lower end of this scale will be subjected to grossly different amounts of androgenic stimulation . most of the available data on the excretion of 17 - ketosteroids is confined to results obtained in studies of men under the age of 30 - years . although there is a general impression that the excretion of these substances decreases with advancing age of the individual , there is a paucity of data on the range of excretion in the higher age - groups . the ideal method of study would be to make repeated determinations of the 17 - ketosteroid output in a large number of individuals over a period of m%ny years , until , in fact , the subjects did or did not develop prostatic cancer . 
the colorimetric measurements - were made on a hilger spekker photoelectric absorptiometer using a green ( ilford spectrum 604 ) and a violet ( ilford spectrum 601 ) light filter . 
1 clearly shows the distribution of excretion of 17 - ketosteroids with age . the output in childhood is low , rising rapidly at or about the age of puberty and reaching a maximum in middle age . 
2 , in which are plotted the as men between 30 and 40 . mean excretions for the third to the eighth decade , together with the upper and the ranges are computed lower limits of the ranges encountered at those ages . as the mean values + 2a . the fact that men continue to show comparatively wide individual variations in their outputs of 1 7 - ketosteroids and hence , probably , in the degree of androgenic stimulation to which their prostates are subjected , until very advanced age , must obviously be taken into account in any consideration of the aetiology of prostatic it will clearly be of importance to know whether known sufferers from cancer . this disease are characterized by excretions at the upper or lower ranges of their appropriate age - groups , or whether they are not significantly distinguishable a . 
jolles. from the radiotherapy department , general hospital , northampton . received for publication december 16 , 1948 . the focusing of attention on the malignant cell in the study of tumour tissue reactions to radiations and growth inhibitory substances has not brought us nearer the state of affairs where both practical and theoretical issues of oncology and treatment of tumours can be studied with a better understanding and fairer prospects of success . the importance of the stroma reaction to tumour and stromal reaction to radiation has been accepted for a long time , but only with generalizations on changes in " blood supply " and " tumour bed , " leaving the whole problem of connective tissue reaction in a hazy state . as regards tumour tissue reaction to radiation , much stress has been laid in the past on the changes following irradiation in the cells alone , but very little although recent attention has been paid to the intercellular tissue components . advances in cytology and genetics have enhanced the importance of the cellular nucleus , it must not be forgotten that evidence , both experimental and clinical , is available to show that the effect of ionizing radiations on cells alone " is not the whole story of the events leading to the destruction of proliferating tissues and that other factors come into play " ( muller , 1940 )  . the great importance of connective - tissue reactions to x - rays in radiotherapeutics has been raised by windeyer ( 1942 ) , ellis ( 1942 ) , windholz ( 1947 )  . 
the lesion is reproduced by means of a stent impression in a plaster cast , over which a tightly fitting stent or black - tray compound applicator is built in such a way as to be easily mounted over the part of the body , and fitting in such a way as not to allow even minimal shifts of the applicator , and so avoid the x - ray beam striking the alternate " opaque " squares of the lead chess which is recessed in the mould . the radiation has to be delivered at right angles with the x - ray tube head in order to avoid oblique penetration into areas applicator directed vertically . beneath the opaque squares the lead chess must be in direct touch with the tumour it is found in the course of treatment that owing to shrinkage of the surface . tumour the recess sleeve in the stent mould into which the " chess " is slipped has to be filed down from time to time during the course of treatment . the thickness of the lead , which is limited for practical considerations , permits 3 per cent of the radiation to pass through on to the protected areas . 
to that amount of radiation received by the not directly irradiated areas has to be added about 3 - 5 per cent of scatter radiation from the neighbouring exposed areas . this varies according to the size of the apertures , and decreases towards the centre of the " opaque ' , squares . 
the size of the squares has been chosen of not less than 1 cm . , so as to leave an adequate amount of tissue either almost completely non - irradiated or exposed for biopsy taking . sections are taken before treatment is commenced and then at varying intercuts are made into the exposed and protected sectors , and also one section vals . is made cutting across both the exposed and protected areas of tissue . accurate labelling of biopsy material must be insured , as no other way of recognizing the protected and exposed sectors on the histological slide is available . a working chart is found indispensable . prior to taking the biopsy the lead mask is applied and the exposed areas are mapped out by means of gentian violet paint . the biopsy material was fixed in sublimate - formol solution or sousa 's solution . following paraffin embedding , sections were cut at 7l and stained with ( 1 ) harris ' haematoxylin , eosin , ( 2 ) van gieson stain , ( 3 ) by gallego 's method , and ( 4 ) silver impregnation . the irradiation was taking place every day , 5 - 6 days per week . 
hard mass filling right breast , fixed to pectoralis and ulcerating skin in anterior axillary line over an area 8 x 5 cwith a sloughy crater and rolled - up edges . 
at this point " chess " technique abandoned and routine glancing technique reverted to . ( 4 ) section taken when tumour received apart from the 4000r with the chess technique 2500r on each glancing field in 14 days ; 10 treatments . ( 5 ) section taken at end of treatment ( 4000r to chess and 3500r on each glancing field in 5 weeks )  . she is remarkably when last seen , 15 . 
meredith. from the christie hospital and holt radiumt institute , manhester . received for publication december 12 , 1948 . ix a series of papers ( dale , 1940 ; dale , meredith and tweedie , 1943 ) it has been shown that the action of x - rays on aqueous solutions of biologically active compounds was indirect , i.e. 
further , it has been shown that in consequence of this indirect action , if two solutes are present , the second solute ( the protector ) reduces the radiation effect on the first one ( the indicator ) ( dale , 1942 ; 1943 ) , by sharing the intermediate product formed from water by the radiation . 
sionally. 197 3 67 0 364 - 0 102 - 6 . 41 - 0 used purgatives ; 1 - 7 x 34 or 63 to have used purgatives occasionary in a hke manner the " expected 34 or 6 - 1 to have taken purgatives often . f7 - 7 x purgative consumption was calculated separately for each age - group in each sex . 
the differences in the senna rates - while showing tlle same trendwere not so marked or consistent , and though the age - specific rates of beecham 's pffls ' usage were without exception higher amongst gastro - intestinal cancer patient 's , the individual differences were , on the whole , small . direct comparison between the gastro - intestinal cancer and non - gastrointestinal groups was carried out for each of the three purgatives , making appropriate adjustment as before for the differing age and sex structures of the two populations ( table vi )  . 
the comparisons relating to the use of senna and beecham 's pills show similar , but less marked , trends towards heavier usage by the gastro - intestinal cancer patients . 
the excess of senna in the gastrointestinal cancer group just reached the level of technical significance and while a similar picture was presented by the separate consideration of cancer of the large bowel ( x2 - 7.21 , n - 2 , p = 0 03 ) , - there was no significant difference between the experience of patients with cancer of the stomach and those suffering from a non - gastro - intestinal complaint . 
kerrich from the radiation therapy department , johannesburg general hospital , and the department of mathematics , u niversity of the witwatersrand . received for publication april 20 , 1951 . in a previous paper the writer ( cohen , 1949 ) proposed a biological dosage unit , the " roentgen equivalent clinical " ( rec ) based on an empirical formula d = e.a.tn.l - q , relating the physical dose in roentgens ( d ) to the biological dose in rec ( e ) , taking into account the relative biological efficiency of radiation of various qualities ( a ) , the over - all time in days ( t ) , and the field size in decimetres ( l )  . 
the physical and biological doses could be equated when the specific ion - density is minimal ( a = 1 ) , the treatment is completed in 1 day ( t = 1 ) , and the field diameter is 1 d ( l = 1 )  . 
cohen in diametercan the hypothesis of a " diffusible substance " ( grynkraut and sitkowski , 1936 ; jolles , 1950 ) , for diminishing the treated field by a given factor , or increasing the time interval by the same factor , would allow the same proportion of the " substance " to diffuse across the surface bounding the treated volume . 
the difference between the 220 kv and 180 kv ranges is statistically significant ( p 0 - 01 ) , but of minor importance clinically , so that for practical purposes the two high - voltage qualities may be grouped together taking a as 0 - 7 . 
2 illustrates the variation in biological efficacy with halfvalue layer , and shows the agreement between our estimates and similar data derived in a different manner by maccomb and quimby ( 1937 ) and by mackee and cipollaro ( 1940 )  . each point represents the median erythema dose ( a x 1000 ) estimated from table ii , the vertical lines delimit the 95 per cent confidence range , and the lettering refers to data by the authors quoted . in comparing these results with other published data , it is necessary to note inevitable differences in the definition of the erythema dose . 
from the method we have used the standard erythema dose is that which , delivered to a 10 cm . field in one sitting , produces a " moderate " erythema in 50 per cent of the treated cases . 
with the same dose , however , some slight reddening would be visible in considerably more than half ( probably 90 per cent ) of the treated cases . since this type of " barely perceptible reaction " is a convenient and objective end - point much favoured in erythema studies , there is obviously need for care in comparing such results . it is also necessary to distinguish the transient reddening ( fruiherythems ) appearing within 24 hours of the delivery of quite small doses ( helmke , 1949 ) from the main reaction ( haupterythems ) , which reaches its maximum about three weeks after treatment . for example , the threshold erythema dose ( ted ) is defined ( maccomb and quimby , 1937 ) as that quantity of radiation giving a faint reddening of the skin , visible in two to four weeks , in 80 per cent of treated cases . it would , therefore , differ little from our standard erythema ; hence the fair agreement between our results and the threshold erythema data in fig . 
the customary five - year - cure policy could not be applied since estimates of depth - doses from treatment records prior to 1945 are not sufficiently accurate it was decided instead to use , as a standard criterion , freedom for our purpose . from perceptible recrudescence for not less than three years . the records of 100 suitable cases treated between 1946 and 1948 , and followed with periodic examinations through 1950 , were abstracted and the data collected in table iii . the cases are arranged in order of increasing biological dosage ( rec )  . the dosage data shown are the true mimimum tumour doses , and differed , especially with superficial therapy , from the nominally given doses . in the case of very superficial tumours treated with lightly filtered radiation the significant point was taken to be 5 mbelow the edge of the treated field . with 2 ma1 filter this point receives about 85 per cent of the given dose ; and with the chaoul apparatus , only 67 per cent . with deeper growths treated at higher voltages , standard depth - dose tables were used to assess the tumour dose ; while with radium applicators and implants dosage was calculated on the paterson - parker system . since various qualities of radiation were used , it was necessary to correct the dosage by a factor ( rbe ) which , taking that for y - rays as unity , becomes 0.5 for superficial therapy , and0.7 for the 200 kv range ( tableii )  . in the case of deep - seated tumours treated with 200 kv radiation , the half - value layer ( and hence the rbe ) in the tissues differs from that of the incident beam on account of the increase in the average wave - length brought about by the compton recoil process . this effect is the more marked the harder the incident radiation . 
3 each 188 case site . face orbit neck node larynx back tongue pharynx neck nosoe alveolus neck face scalp larynx tongue , base pharynx orbit face temple post - cricoid  . 
0.5 , dose * ( r )  . 3200 2300 2250 3300 3150 2000 3800 3500 3000 1400 2000 3000 3150 3000 3300 4400 3600 5000 3750 2250 3300  . 
4 illustrates a method for determining the recovery exponent various values of n ranging from zero to 0.5 were ( n ) by a graphical solution . selected , and the resultant estimates of biological dose tabulated in a manner field diameter ( cm . ) therapeutic ratio skin tolerance dose 5000 median cancerocidal dose 4500 time days 1204 a% t% % - iuuut 9000 8000 - 7000 - 6000 - 5000 4000 - 3000 - 2000 1j4mi iolvv 40003500300025002000fig . 
a single line and volume factors . across the diagram gives all the relevant factors simultaneously . similar to table iii but regrouped accordingly . the cure rates so obtained were similarly charted in lognormal probability graphs , and the values of the standard deviation factor ( f ) corresponding to each value of n was determined . it is seen that f is a minimum as n approximates 0 - 25 . 
by means of the method of least squares , estimates of a and n for six different qualities of radiation were obtained , and shown to be in good agreement with previously established data . 
the uncertainty factor in dosage estimated on this basis ranged from 1.21 to 1 - 35 . a further 100 cases of epidermoid cancer were similarly analysed in order to determine the dependence of curability on the biological dose . 
the median lethal dose for this tumour ( 3000 rec ) and its uncertainty factor ( 1.3 ) were determined , and it was shown that , under optimal conditions of time and area , 95 per cent of tumours could be cured with a dose exceeding the median lethal dose by it has also been demonstrated that the prognosis is a mathematical 30 per cent . function of the therapeutic ratio . the author wishes to acknowledge the valuable advice given by j . 
kerrich. from the data in table iii cohen suggests that biological dose = dose / rbe x tn , or x = z / tn say , where xbiological dose , zdose / rbe , t = time in days . 
he goes on to show that if x is the exact dose necessary to effect a cure , then a reasonable assumption is that log x = log z n log t is normally distributed . writing x = log x , z = log z , t = log t this hypothesis becomes x = z nt is normally distributed with , say , mean oc and variance a2 . by a neat graphical method he proceeds to estimate ac , n and a . 
cohen the sizes of the estimated standard deviations show that none of the constants can claim to be very " well determined . " it must , however , be remembered that we are not dealing with a planned experiment , but that the author had to use the data that were to hand . 
as a suggestion to future workers in this field , more observations for small values of t ( say t 0 ) and large values of t ( say t - 1.5 ) would help greatly in fixing c and n more accurately . in more detail , the estimated covariance matrix is var . 
cohen free , will , in this paper , be designated as ffc , and the reciprocal factor - harbouring ( c3h x cba ) f1 as fhc . all parent strain and hybrid mice were housed in the same environment and maintained on a standard laboratory chow diet with tap water ad libituit was found expedient , in view of ' the temperamental breeding habits of both cba and cm females , to isolate routinely all pregnant mice until after the weaning of consequently , no f , bybrid was ever in contact with the father , which , the litters . as has recently been shown by peacock ( 1953 ) , reduces the probability of " infection " in the ffc with milk - factor from the cm male parent . the incidence of spontaneous mammary tumours in old factor - harbouring ( c3h x stock albino ) f , females , which were observed for over a year and inadvertently force - bred , was at least 70 per cent . 
no mammary tumours were noted in the corresponding , similarly treated , factor - free females , and it was considered that the presence of the milk - factor in the c3h niale parent exerted little or no influence on this group . 
the tumour , retracted from the body onto a wax backing , is fitted into the mouth of a 2 cdiameter open - end applicator , fsd = 25 cm . , and treated at 240 w . 
the mean - log of the volume in cubic millimeters of the fhc group is 2 - 12 ( 0 - 08 ) , while the mean - log - volume for the ffc group is 1 - 85 ( 0 - 08 ) , which is significantly smaller . while unequivocal comparisons between the c3h and hybrid groups could be made with confidence , it was considered possible that , as a result of the variations in growth rate within the hybrid hosts , smaller tumours might be more readily cured , and the ffc group consequently biased in favour of a higher curerate . 
2 , togeth ' er with comparative data previously determined for the parent this design is analogous to a six - point assay , in which both hnearity strain . and parallehsm of the regression hnes can be tested . 
the magnitude of the parameters and the significance of the inter - group differences were computed by finney 's ( i 952 ) methods . a third experiment was designed to determine , whether the increased radiosensitivity of the tuniour in f , hybrids was an inherent change in the tumour cel ' is per se , or a reflection of host - resistance . 
2. - probit diagram showing response of the cm carcinoma growing in various situations . ( a ) control series treated in homozygous cm mice in 8itu . ( d ) irradiated in 8itu in heterogeneous hybrid mice . ( f ) irradiated in factor - free ( ffc ) mice . 
the increased radiosensitivity of the tumour associated with genetic diversification in the host is evident . ( e ) irradiated in factor - harbouring ( fhc ) mice . 6000 7000 5000 cells growing in a relatively alien environment was suggested by barrett and deringer 's ( 1952 ) observation that a permanent adaptive change occurred in the oh mammary carcinoma following one - sub - passage through susceptible f , hybrids . since the object of this experiment was to demonstrate any inherent acquired radiosensitivity in the tumour , a smar homoplast ( first passa - ge from a oh mouse ) , growing slowly in an ffc host , which had attained a volume of only 30mm3 . 
the donor was also re - impladted with this tumour . takes " were 100 per cent in all groups . when each tumour had reached a standard size , about i cin its greatest diameter , it was irradiated in situ with a dose of 4200 r . 
the re - implanted tumour therefore , if the in the ffc donor was also irradiated at this dose , and cured . acquired radiosensitivity was a permanent adaptive change in the tumour cells capable of being carried over into the next transplant generation , it could be expected that the cure - rate of grafts originating from this tumgur at the dose givedwould be about 75 per cent in each genetiegyroup . 
2 , and the probit regression lines derived for the radiosensitivity of the tumour growing each reciprocal group are shown . in the genetically heterozygous environment of the ffc host , excluding the complicating influence of maternal factors , is shown by line f in fig . 
2. this line is virtually parallel to that previously obtained in the parent oh strain ( line a on the diagram ) , the coefficient of variation being 10 per cent . the median effective dose ( ld50 ) is found to be 3950 ( i 10 * ) r , compared to 5700 r previously reported in the oh . 
2. not only is the cure - rate , in the presence of the milk factor , considerably less than that of the factor - free mice , thougli still greater than that of the c3h parent , but the usual sharp response to increasing dosage has become much less sensitive , as evidenced by the considerably flatter slope of the regression line . this change test of the data reveals a is not due to fortuitous fluctuations in cure - rates , as a highly significant departure from parallelism with the other groups . 
the median , effective dose for the tumour growing in fhc mice appears to be 5100 ( + 400 * ) r with the unusually large coefficient of variation of 25 per cent . it must be assumed , therefore , that the maternal influence is not uniform and stochastically independent of other factors . 
was noted in the treatment of radiation - attenuated homoplasts growing in the parent strahi ( cohen and cohen , 1954 )  . at the ld50 level , the relative radiosensitivities , evidenced by the dosage ratio between the c3h and fhc groups is 1 - 13 ( + - 07 * ) , and that between the fhc and ffc groups is 1 - 29 ( - 08 * )  . 
the former ratio , compared with its standard eiror , indicates that the genetic effect per se , in spite of the complicating matemal influence , is probably significant ( p = 0 - 05 ) ; while the latter shows that the effect attributable to the milk factor alone withift the hybrid mice is highly significant ( p < 0 - 001 )  . 
the relative importance of these ratios , however , necessarily varies with the curative level t - ested , the maternal influence having the greater effect at doses above the median effective range . in order to eliminate any possible bias introduced by the greater average volume of the tumours in the fhc mice compared with those of the ffc group , a small number of animals bearing tumours of extreme size were discarded in the manner previously described , so as to equate the mean log - volumes of the two groups , and the probit analyses repeated on the remaining matched tumours . this correction , however , does not alter the magnitude of the parameters or affect materially the significance of the differences between them . further , comparison of the proportion of tumours cured within each class - interval of fig . 
i that within each genetic subgroup , the cured tumours tend to a smaller volume than the non - cures . these differences , however , are not signific ' ant , since the experiment was not designed to test the effect of tumour volume per se on radiosensitivity and contains insufficient data for this purpose . 
the question of the relative importance of genetic factors and of tumour size per se in determining radiosensitivity , is probably meaningless , since the smaller , more slowly - growing homoplast may itself be a manifestation of genetically - conditioned host - resistance ( eichwald , 1953 )  . 
cohen mal factors , therefore , materially affect the response of a tumour homoplast irradiated in situ . subsequent challenge with a second inoculum in those hybrids of both reciprocal groups , previously cured of the tumour , resulted in active growth in all it was also interesting to note , in the course of the experiment , that the cases . spontaneous mammary tumours in factor - harbouring f , females occurred regardless of whether the animal had previously been cured or not . third experiment table 11 shows the results of the third experiment , in which a " radiosensitive homoplast , growing in an ffc and later cured with 4200 r , was sub - passaged through mice of the three genetic categories and treated with the same dose . the cure - rates of these tumours in the ffc , fhc and parent c3h mice are respectesting the null - hypothesis that the radiosensitivity tively 67 , 35 and 0 per cent . of the sub - passaged homoplasts is the same as in the ffc hosts ( 76 per cent at 4200 r , table 1 ) , the probability of obtaining the results observed is shown in the last column of table ii . 
the susceptibility to tumour transplantation is a simple genetic - dominant , shown by the uniformly successful transfer of tlle inbred parent strain tumour to all f , hybrids . however , once such a graft is established , its rate of growth is no longer contingent on host genetics per se , but is dependent on the aternal extrachromosomal influence which , when present , exerts a significantl accelerating effect . likewise , a matemal influence , presumably the milk agent , is sufficient to induce spontaneous mammary tumours in f , hybrid females , the incidence approaching that in the inbred parent strain , in spite of genetic dilution . it has here been shown that the radiosensitivity of a tumour transplant , in this case the c3h mammary adencarcinoma , is dependent on both genetic and maternal ( extrachromosomal ) factors in the hybrid host . it appears , however , that the two factors are not necessarily independent . in the case of the factor - free f1 hybrid , a uniform and uncomphcated response is elicited which seems to be a - clear expression of immunogenetic differences between host and tumour . 
the differences between these groups are significant , although all three groups of mice were uniformly susceptible to the tumour and showed no overt manifestations of resistance . it appears , therefore , that the radiosensitivity of a tumour is a quantitative measure of subhminal host - resistance resulting from immunogenetic differences in the host - tumour relationship , including extrachromosomal factors . the mechanisni involved was further elucidated when a tumour growing in a factor - free f , hybrid , and known to be curable with 4200 r , was sub - passaged to mice of the three genetic groups . 
the homoplasts responded to treatment in the same manne ' r as the directly passaged parent strain tumour in the corresponding hosts , indicating that no inherent radiosensitisation of the tumour cells per se had occurred . all the facilities required for the maintenance of the animals used in this investigation were generously provided at the south african institute for medical 312 a . 
3 received for publication january 11 , 1951 . it has long been known that the rous sarcoma may be transmitted by dried tumour tissue and it has been concluded , therefore , that the tumour agent can survive under such conditions ( rous and murphy , 1914 ; rous , 1911 ; and hoffstadt and tripi , 1946 )  . 
the ampoules were then connected to a manifold and the ice sublimed in vacuo . knox 's ( 1939 ) apparatus and conditions were later used by dmochowski ( 1948 ) for the freeze - drying of rous v - irus suspensions produced by fractional centrifugation and other methods . 
dmochowski ( 1948 ) claimed that similar virus suspensions treated with a drop of fowl or rabbit serum suffered no loss of activity under the same conditions , but that addition of salts , such as nacl , kc1 or caci2 , had no such preservative action . 
by gentle shaking for 35 minutes . if the shaking was carried out in the absence of gas space , albeit with glass beads in the tube , inactivation was negligible . 
gave complete protection for t5 phage for 14 minutes . the duration of the protective effect was found to be a function of the gelatin concentration , since the gelatin itself protection could also be obtained with gum arabic or with is " denatured . " serum albumin although 100 - fold and 10 - fold the gelatin concentration were required . this work may well provide the theory behind the observation that viruses are more stable when diluted in serum or broth than when the dilutions are made equally one might expect that small viruses would be with salt or water . inactivated more rapidly than larger , and that the rate of inactivation would be proportional to temperature , since the forces responsible for bringing the particles to an interface will be thermal forces . such conditions as these have frequently been realized empirically . rivers and vvard ( 1935 ) sought to add a material to vaccinia virus suspension which would ( a ) act as a protective agent , ( b ) add bulk to the final product and ( c ) go back into solution with ease , carrying the virus with it . 
this suspension was clarified on the sharples centrifuge and the virus deposited from the supernatant on to a cellophane bowl lining sheet by deposition at high speed ( carr and harris , 1951 )  . this virus - containing deposit was then resuspended in a ' volume of medium ( concentration medium ) equal v / w to the original weight of tumour tissue . ( 2 ) comentration media . ( a ) buffer - trypsin , b - t . - mcilvaine 's ( 1921 ) phosphate - citric acid buffer ( plff 7 - 5 to 8 - 0 ) prepared from 0 - 2 m na2hp104and 0 - 1 m citric acid and diluted 1 : 19 with water ( buffer ) and a few mg . 
the mccartney bottles were accommodated in drihed compartments in the centrifuge head of the drying chamber of the machine . each bottle was inclined towards the axis of rotation , so that when the head was spinning the contents of each bottle formed a wedge . the virus suspensions were snap - frozen by evaporative cooling , and the purpose for spinning the bottle was 2 - fold . ( a ) frothing was prevented and ( b ) the wedge of frozen material presented a greatly increased surface area with a consequent increase in the rate of drying . 
the rate of drying was high throughout the drying cycle and no evidence was found to suggest that , as the layer of dried virus material increased in thickness , the rate of evaporation of vapour fen off and the underlying frozen mass melted ( bauer and pickels , 1940 )  . the drying cycle occupied 40 to 44 hours . 
the bottles , which were either uncapped , or lightly capped with two or three layers of sterile surgical gauze during the drying , were then screw - capped and sealed with tape and stored at 2 ' c . 
the media used , and here abbreviated , included : ( a ) buffered - 8aline , b - s . - ten per cent sahne adjusted to ph 7 - 2 with 0 - 2 m sodium phosphate . ( b ) buffer , ph 5 - o . - mcilvaine 's phosphate - citric acid , diluted 1 : 19 with water . ( e ) water - trypsin , w - t . - - solution containing a few mg . 
a similar result appears from table vii if the result of 29jr.49 is compared with that of 5.vii.49 , and from table viii , 15ar.49 being compared with 29.iii.49 ; although activation was not found in the experiments with egg albumin or egg white . freeze - drying in salt solutions ( table ii )  . the sharples deposits were resuspended throughout in , 0 - 005 m phosphate buffer , ph 7 - 2 , containing a few mg . 
50 , where lemco is compared with io per cent lemco as a medium for freeze - drying , there is a reduction in the titre with the weaker broth . freeze - drying in carbohydrate media ( table vii )  . the sharples deposits were resuspended in buffer - trypsin and " purified " in the usual way . 
or 0 - 125 per cent neutrahzed cysteine hydrochloride gave protection , and the protection afforded by 2 - 5 per cent dextrose containing 0 - 05 per cent b.a.l. 
the method now adopted , that of freeze - drying virus concentrates in lemeo broth , fulfils the condition , but little hght has been thrown on the mode of action of the stabihzing material . virus concentrates dried in this way have retained their fur activity for minimum periods of 12 months . the experiments with water alone showed that dmochowski 's ( 1948 ) results are confirmed in so far as virus may be recovered after drying - and our preparations had all been treated with 1 : 10 , 000 hcn during the isolation procedurebut it was not possible under these conditions to recover as much as 60 to 80 per cent of the initial virus activity as he claimed . salt solutions had no preservative action , and broth - containing media showed immediate advantages , even where the suspensions had been " purified " by centrifugal fractionation before desiccation . 
the action of the broth appears to be ponfined to the freezing - and - drying cycle or subsequently , since nothing was gained by carrying out the concentration stages in broth or broth - containing media , for which buffer - trypsin is quite satisfactory . it is unhkely , therefore , that the effect is solely an example of the phenomenon described by adams ( 1948 )  . 
the conditions of freeze - drying , moreover , do not involve vigorous shaking of the material but , equally , there is as yet ' no knowledge of the surface conditions which exist at an ice surface which is gradually receding through a drying mass . 
the observation that weaker ( 10 , per cent ) lemco broth is a less effective protective agent is of interest in this respect . it is possible to suppose that the action of broth and similar material is complex , that one or a combination of such factors as the forowing are important : ( a ) the broth pro - vides optimum conditions during freezing - such as an optimum eutectic point ; ( b ) that protection is conferred against the deleterious effect of ' heavy metals or of oxidation systems ; ( c ) that , in some way , the tendenoy of the virus to aggregate is reduced ; or ( d ) that an optimum amount of water only is lost . 
the rats were 4 weeks old when these operations were performed and 7 days later the litter - mates were joined in parabiosis . the surgical technique used was the one recommended by jacobsohn ( 1948 )  . in most pairs both partners were females except in those forming groups iii and vii , where a castrated male took the place of the spayed female . 
a total of 10 to 25 doses was given , when possible in a schedule of 3 doses per week . occasionally , this dosage had to be reduced to one or two administrations per week when the pair failed to show the expected gain in weight . twenty - six pairs of albinos ( groups i , ii and iii ) and 3 pairs of piebald rats 332 f . 
by stomach - tube during the first 8 weeks of the experiment . these rats , hke the majority of the parabiotic pairs , were 2 to 21 months old when the administration of the carcinogen was commenced , and served as controls for the assessment of the carcinogenic activity of the amounts of a.a.f. 
the parabiotic rats were sacrificed when declining health , in most instances due to the preserice of large pyometra , made it advisable or when the presence of a tumour was suspected . 
two of the " single " rats were killed when a palpable tumour was present , the remainder when the experiment was terminated at the 52nd week . the material taken for histological study was fixed in zenker , and the sections were stained with haematoxylin - eosin or according to van gieson . 
the tumours originated in gonads which were grossly hyperplastic . the histological changes occurring in the ovaries of rats joined in parabiosis to gonadectomized litter - mates have been well described by zeckwer ( 1944 ) , therefore our findings will be mentioned only briefly . 
the wall of these cysts was smooth , and when an epithelial lining could be recognized the cells were low cuboid or flat without any indication of cellular activity . such cysts occurred equafly in intact partners treated with a.a.f. 
the earliest neoplasms seen were generally round in sfiape and had a diameter of 3 to 4 mthey were found in wistar rats of groups i to iii after 16 to 20 weeks of parabiosis ; in piebalds they occurred even earlier . 
the largest gr ' anulosa cell tumour which had destroyed completely the ovary in which it originated measured 27 x 16 x 25 mand it was the only neoplasm of this kind to produce a metastasis , weighed 3 - 88 g . the secondary being situated in the adhesions linking the primary to neighbouring organs . the material at our disposal allows the study of the development of granulosa fig . 
2 shows a large haemorrhagic cyst cell tumours from their early stages . found in an ovary of rat 1 , group iv , sacrificed at the 13th week of the experiment . this cyst had a thin wall formed b fibrous connective tissue which in most places was poor in cells . in some areas the capsule was thickened , and here signs of previous haemorrhage such as pigment - loaded macrophages were present . into the cavity papillary projections protruded . 
they were of two kinds , some into the former , delicate strands with a narrow and others with a broad base . of spindle - shaped cers entered , forming their core . into the latter , capinaries penetrated surrounded by cers having ample eosinophihc cytoplasm , and a nucleous less rich in chromatin than the cells which covered and formed the bulk of the projections . these had a nucleus rich in chromatin and a scanty cytoplasthey were typical granulosa cells , while the ones having ample cytoplasm this was the earliest neopalstic lesion found in our resembled lutein cells . material , and shows that atypical growths of this kind were not necessarily permanent ones . 
and killed after more prolonged parabiosis hae ' morrhagic cysts , where only small nests of granulosa cells remained embedded in the dense connective tissue of the capsule while the cavity contained only inspissated blood and tissue debris . such chocolate cystlike lesions were absent from the ovaries of the control pairs not treated with a.a.f. another animal of the same group sacrificed 2 weeks , later was found to have in the ovary a macroscopically visible nodule formed almost entirely by granulosa cells . it was of about the same size as the early granulosa cell tumuor depicted in fig . 
3 shows a sharply limited spherical nodule situated in an ovary containing cystic follicles . it was surrounded by a thin fibrous capsule . between the capsule and the neighbouring cystic folhcles were large blood - fifed sinuses . 
5 shows the appearance of the granulosa cells forming the bulk of the nodule , and the tendency towards foci of liquefaction leading to the formation of cystic spaces within the tumour . 
the tumorous right ovary of rat 5 , group 111 , showed histologically an unusual picture . this ovary was also considerably enlarged ( 802 mg . ) and fairly solid with only a few blood - filled cysts only a small rim of ovarian tissue remained ; most of the near its surface . histologically this tumour was composed specimen was obviously neoplastic . mainly of spindle - shaped cells with scanty cytoplasm , and a nucleus which varied considerably from elongated dense to oval or spherical vesicular . 
among them small groups of cells were present having ample cytoplasm which stained well these elements had a larger vesicular nucleus with the chromatin with eosin . displaced towards the nuclear membrane . 
we have classified this neoplasm as a theca cell tumour because the granulosa contributed only little to its make - up . in the 36th week of the experiment the health of the intact partner suddenly declined and therefore in the region of the left ovary there was a large blackish the pair was sacrificed . cyst , on the posterior wall of which some ovarian tissue was recognizable . 
two types of neoplastic cells could be distinguished : bizarre cells of unusually large size having one or , rarely , their cytoplasm was several nuclei with deeply stained prominent nucleoli . intermi ' ngled with these elenients ample and stained only weakly with eosin . these were polyhedral in were nests of much smaller cells of epithelial character . shape and had well - marked cell borders , in cohtrast to the large elements which tended to form svnvctia . 
 ( groups v , vi and vii )  . the only lesion suspicious of atypical growth was found in one of the ovaries of an intact female which survived parabiosis for 42 weeks ( group v , rat 8 )  . 
we are unable to state with certainty that this lesion was of neoplastic nature . as already mentioned , no ovarian tumours were found in the 10 " single albino rats which received 24 doses of 4 mg . 
among many hundred female rats treated with this carcinogen we have found only one instance of a neoplastic lesion in the female gonads . this was discovered in a rat 171 months of age when sacrificed . 
by stomach - tube when 2 months old ; then the animal was kept without further treatment for 1 year , and for the last 16 weeks of her life she received methyl - thiouracil in the drinking water . 
at the post - mortem the left ovary was found to be converted into a large cyst measuring 20 x 15 x 10 mm . on histological examination septa subdividing the cyst in several compartments and covered with granulosa cells were seen . although macroscopicany this cyst resembled the clear cysts which we have frequently found in the ovaries of parabiotic females and also in rats treated with stilboestrol , we have never observed in such structures prohferation of granulosa cells exce ' t in this one instance . the morphological 8ign8of the endocrine imbalance exi8ting in the intact partner . all signs of hyperoestrinism were present in the intact partner ( zeckwer , 1944 )  . 
the vagina was lined by hyperplastic squamous keratinizing epithelium . in the few instances where the uterus was not heavily infected the epithelium lining the cavity was tar cyhndrical and areas of squamous metaplasia were also present , the wall of the organ being thickened . in the infected uteri most of the epithehum was destroyed . 
the breast glands were grossly hyperplastic , and in most animals macroscopicary visible cysts fired with a milky fluid were seen . histologically such glands showed a pronounced cystic hyperplasia of the ducts and periductal fibrosis . 
the thymus was atrophic , and the suprarenals enlarged in size due to a hyperplasia of the cortex . the pituitaries of the intact partners were invariably larger than those of the gonadectomized litter - mates , and already after 15 to 17 weeks of parabiosis had increased to an average of 8 mg . 
the largest observed ( rat 6 , group iii , and rat 8 , group v ) reached the extraordinary weight of 74 - 8 and 139 - 3 mg . 
the pituitaries of the intact partners were mostly solid and of whitish colour and haemorrhagic areas while the majority of these glands showed a symmetrical were not conspicuous . enlargement of the anterior lobe , in females surviving for more than 25 weeks of irregular shape . 
at the experiment the pituitaries assumed an ever - increasin first small whitish or sfightly yerowish nodules appeared which were single or multiple . later on the normal shape of the gland became more and more obscured by irregular growths , which compressed but never invaded the brain . the larger the tumour the greater was the tendency for brownish discoloration . these lesions occurred in intact partners independently of whether a.a.f. 
common to many of these elements and independent of the degree of acidophilic granulation was the pecuhar golgi apparatus which in papanicolaou preparations appeared as a ring - like structure , the negative golgi fig . 
17 , shows that the majority of the cells composing this structure were degranulated with only a few acidophils remaining . in the more advanced adenomata of the pituitary more atypical cens appeared but fundamentary they did not differ from the earlier lesions . in our material we have rarely seen adenomata completely free of acidophils . 
to conclude , we consider the nodular structures to be adenomata derived from cells of the acidophil series . tumour8of other organ8ob8erved in intact partner8and in " 8ingle " rat8of group viii treated with a.a.f. in the rats of group viii three adenocarcinomata of the breast and one squamous ketatinizing epithelioma of the extemal auditory meatus were found ( table iii )  . 
two of the cancers appeared after an interval of 51 to 52 weeks , only one of the i.e. , 9 weeks after the pair surviving longest had been sacrificed . intact partners treated with a.a.f. 
developed tumours of the breast ( rat 6 , group ii )  . in the 35th week of the experiment a mass was felt in the region of it was surgically removed , and was found to be the fifth left mammary gland . a well - encapsulated tumour measuring 20 x 12 x 9 mon cutting , milky histologically it was a fibroadenoma , in which fluid escaped under pressure . the epithelial elements were much more numerous than is common in such tumours except when occurring in pregnant or lactating females . 
the other was a small adenocareinoma of the breast which showed far less secretory activity than the fibroadenoma which had developed simultaneously . benign cystic cholangiomata were present in practically all rats treated with a.a.f. , but in the intact partners they were generally larger and more widespread than in the " single " animals , in which they were limited to a few cysts in lobus caudatus and left lobe . 
we are unable to state why such changes occurred only in isolated instances . another feature in which the spayed parabiont differed from a " single ovariectomized female was the cytology of the suprarenals . 
24 is included for comin animals killed after 15 to 25 weeks of parabiosis . it shows the distribution of lipoids in the cortex of the suprarenal of a parison rat spayed 6 months previously . 
a second experimental method ofobtaining such neoplasms was discovered by biskind and biskind ( 1944 ) , who obtained such tumours by transplanting ovaries into the spleen of gonadectomized rats . 
the gonads of the intact partners were composed of cystic follicles - proof of increased secretion of fouiclestimulating hormone ( f.s.h. ) by the pituitary of the irradiated parabiont . the liver of rodents inactivates ovarian steroids so efficiently that the secretions of a gonad grafted into the spleen do not enter the general circulation . in consequence the pituitaries of animals bearing such grafts are of the castrate type , and have an elevated gonadotropin content , as shown by greep and jones ( 1950 )  . fels ( 1949 ) discovered that a temporary hgature of the blood vessels supplying the ovaries led to an endocrine imbalance characterized by hyperoestrinism in the presence of castration changes in the pituitary . it seems therefore that one factor is common to at least 3 of the 4 methods quoted above ; they induce an endocrine imbalance characterized by pituitaries secreting elevated amounts of gonadotropins . it is therefore not astonishing that the neoplastic changes induced under these experimental conditions are all of the same kind . nearly all growths so obtained belong to the group of granulosa - theca - lutein cell tumours . 
moon , simpson , li and evans ( 1950 ) also regard the neoplastic changes observed in the ovaries of rats treated with growth hormone to be due to a pituitary imbalance . these authors mention a reduction of the acidophils and an increase of the chromophobes in the pituitaries of such rats , and noted that in 2 animals the histological picture of the anterior pituitary was of the castration type . it seems therefore that the basophils and especiary the f.s.h. secreting cells can increase in animals treated with growth hormone . however , more information is required before one can assume that ovarian .eoplasms obtained by treatment with growth hormone are due to the same mechanism as the other experimental tumours of the ovary , i.e. , to an excess of gonadotropins . in our case there can be no doubt that tumour development in the female gonad was the result of increased stimulation by gonadotropins and of the carcinogenic action of a.a.f. it is especially the foricle - stimulating hormone secreted in elevated amounts by the gonadectomized partner which determines the endoadmittedly during the first weeks of parabiosis there is good crine situation . evidence for the action of luteinizin hormone , as indicated b the presence of many healthy corpora lutea in the ovary of the intact litter - mate . later , when the increased secretion of oestrogens from the stimulated ovary has suppressed all the gonadotrophic activity of the intact partner 's pituitary and continuous oestrus has become established , the pituitary hormone dominating the picture is f.s.h. it is therefore not astonishing that granulosa cell tumours are so frequent in our material and that luteinization occurred to only a hmited extent . 
4 , 6 and 7 of their paper resemble closely the early lesions observed by us , and which we have seen to progress to tumours of considerable size . it seems unfortunate that drips and ford ( 1932 ) compared the atypical structures observed - by them in the irradiated ovaries of rats with carcinomatous ovarian cystadenomata of the 342 f . 
and luteinizing hormone ( l.h. ) in the ovarian cycle is greatly disturbed in the parabiotic female joined to a gonadectomized litter - mate . after 3 to 4 months of parabiosis the ripe follicle is not any more transformed into a corpus luteum nor does it involute , but is exposed to continued stimulation , which does not cease after the death of the ovuthis is in our opinion the decisive factor which leads to the development of the granulosa cell tumours . contrary to what is seen in spleen graftecl or irradiated ovaries of mice , in the rat there is not the shghtest evidence that these tumours " arise from proliferation and ingrowth of germinal epithehum " , as assumed by li and gardner ( 1947 )  . we have observed in some glands smar tufts of cells growing out from the surface ' germina of the ovary , but we have never seen tubular ingrowths from origin from embryonic nests seems a most unlikely hypothesis , epithehum "  . since many workers obtained ovarian tumours in 50 to 70 per cent of their experimental animals ( kaplan , 1950 ; furth and sobel , 1948 )  . . 
undoubtedly the stroma cell of the ovary can differentiate into theca or granulosa cells , and therefore one cannot still , we prefer origin from differentiated granulosa exclude this possibility . cers because of the morphology of the early lesions observed by us . 
12 and 13 one has not to fall back on the conception of disturbed embryogenesis . in preovulatory follicles invaginations of the theca accompanying tufts of capifaries are a common feature . the chorion epithelioma - like tumour presented some diagnostic difficulties . we consider it to be an anaplastic carcinoma . 
as pointed out by willis ( 1947 ) , anaplastic carcinoma can simulate chorion epithehoma and therefore more than morphological similarity is required to establish the diagnosis , in the absence of pregnancy . 
the lack of teratomatous elements and the lack of signs of functional activity such as corpus luteum cysts in the unaffected right ovary are in favour of the diagnosis of anaplastic carcinoma . 
was given simultaneously with or previously to the treatment with goitrogen . spontaneous ovarian tumours and especially granulosa cell tumours are exceedingly rare in rats ( iglesias , stemberg and segaloff , 1950 ) , and therefore do not complicate the interpretation of the experiments reported in this paper . finary an explanation has to be given why the incidence of granulosa cell tumours in group i is lower than in groups ii and iii . 
and most probably also our surgicaltechnique . group i represents our first parabiotic pairs , in which we did not succeed as well as later on to obtain wide junctions , measuring 6 to 10 cat the time the animals were sacrificed . the study of a number of organs of the intact partner showed clearly the effects of oestrogens secreted in excessive amounts by the stimulated ovaries . one of the sequels of this hyperoestrinism were the pituitary adenomata which occurred whether or not a.a.f. 
we beheve , however , that originary these benign neoplasms were acidophilic , just as in zondek 's case ( 1940 )  . this author observed an adenomatous lesion composed of acidophils in the pituitary of a woman treated with very high doses of oestradiol . whereas the increased secretion of oestrogens by the stimulated ovaries led to neoplastic changes in the pituitary , no mammary tumours were observed in the control pairs , contrary to the findings of zeckwer ( 1944 ) , and only one of the intact parabionts treated with a.a.f. 
developed tumours of the breast gland . the material at our disposal does not arow the conclusion that the high level of oestrogen in the circulation of the intact partner inhibited the carcinogenic action of a.a.f. 
in intact female rats joined in parabiosis to gonadectomized litter - mates . ovarian tumours developed in about 50 per cent of these animals . ( 2 ) a description of the histology of the ovarian neoplasms has been given and their histogenesis discussed . it has been found that most of them were of the granulosa cell type . 
secreted in excess by the pituitary of the gonadectomized litter - mate is regarded to be the factor essential for their development . ( 3 ) the pituitary tumouis observed in intact parabiotic females are considered to be due to the excess of oestrogens secreted by their stimulated ovaries . 
the gonadectomized partners were always found to be free of neoplastic lesions , whether or not their litter - mates received a.a.f. ( 5 ) the state of hyperoestrinism present in the intact parabiont did not enhance the carcinogenic action of a.a.f. 
were administered to " single " female rats two mammary cancers appeared during the first 40 weeks of the experiment , a period which corresponds to the maximum survival of our parabiotic pairs . ( 6 ) benign cystic cholangiomata were found in nearly all rats treated with in the intact parabionts they were more numerous and larger than in a.a.f. the " single " rats . we wish to thank professor r . 
philps. from the department of clinical pathology , university college hospital , london , w.c.1. received for publication july 2 , 1954 . the examination of sputum for carcinoma cells can only be considered a diagnostic method of clinical value if false positive diagnoses be avoided and cases wrongly thought suggestive of carcinoma reduced to a minimuthe method cannot be used to exclude carcinoma as it is evident that malignant cells may not be found if they are present only in small numbers , or in the case of an early it must be accepted , therefore , that growth the sputum may contain none . 20 to 25 per cent of bronchial carcinomata will be missed by this method and the problem is to be sure that a positive report is as reliable as possible . 
1 , 2 and 3 contained a considerable amount of necrotic epithelial tissue , and in one small group of cells , two mitotic figures , one of which is shown in fig . 
4 , 5 , 6 and 7 . both cases the appearance suggested a clump of carcinoma cells but the ciliated border could in places be clearly seen , making the true nature of the cells evident . bronchial epithelial cells sometimes show vacuolation of the cytoplasm and may then give a disorderly appearance with may lead to confusion with carcinoma cells , , such an appearance is illustrated in fig . 
 - on close inspection of this clump , it was clear that the individual cells did not form part a fragment of tissue as there welre spaces between them , and they had more cytoplasm than do oat cells . 
the cells bear a strong resemblance to oat cells but can be distinguished from them because the cytoplasm is relatively abundant and the nuclei of adjacent cells show no tendency to moulld together . 
13. - part of the fragment under higher magnification . the pattern of the cells resembles that seen in oat cell carcinoma , adjacent nuclei showing a tendency to mould together . there is more cytoplasm separating the nuclei than is commonly seen in oat cells , and the chromatin pattern is nowhere visible whereas in oat cells it is usually clearly seen . this appearance was reported as being suggestive of carcinoma . 
17. - a corpus amylaceum enclosed within a cell which appears to be of epithelial origin . the nucleus of the enclosing cell was at the periphery of the enclosed body but is not well shown in the photograph . the corpus amylaceum was semitransparent and was devoid of any nuclear characteristics . 
the nuclei of the individual cells mould together rather as do those of oat cells , giving the whole clump an appearance resembling oat cell carcinoma . all the nuclei however , are dense and the cells possess more cytoplasm than is usually seen in oat cells . 
the specimen was reported as follows : " films show clumps of cells , the arrangement of which is suggestive of oat cell carcinoma though the individual cells are not sufficiently characteristic to indicate malignancy . " the diagnosis of bronchial adenoma was made at bronchoscopy and confirmed at post mortem nine months later following the patient 's death from coronary thrombosis . ( e ) appearances due to macrophages of unusual type . macrophages , when they are seen to contain dust particles , can be easily identified , but when devoid of such particles , they may sometimes be confused with carcinoma cells . 
the nuclei are central , multinucleate cells frequently occur in carcinoma but they and number about 12 . are in no way diagnostic of it , the commonest multinucleate cell seen in sputum films being the macrophage . this example is the largest that i have seen . 
they received a diet consisting of whole meal flour ( 7 parts ) and skimmed milk powder ( 3 parts ) , supplemented by cod - liver oil and cabbage once weekly . for each animal 12 g . of food were provided daily . 
the dose of methylthiouracil given to the males was doubled during the last 10 days before they were sacrificed . four males and two females received methylthiouracil only ; two males and two females methylthiouracil plus a.a.f. , and four females a.a.f. 
only. thyroxine injections were started when the animals had stopped growing . this occurred in the females after three weeks of methylthiouracil treatment , in the case of the males , after four weeks . dl - thyroxine was dissolved in saline with na2co3 to ' give a solution containing 10 , ug . 
griesbach during the period of thyroxine treatment the rats were weighed were killed . twice weekly and the injected amounts adjusted to the weights . five female rats , 7 to 8 weeks old , were thyroidectomized . three weeks after operation , when the constant weight of the animals suggested completeness of thyroid ablation , these rats were started on the milk - flour diet containing 0 02 per this concentration of a.a.f. 
are badly tolerated the carcinogen to each rat per day . after eight weeks of this regime , thyroxine treatment by thyroidectomized rats . was started , each animal receiving one daily injection of 2 - 5 , ug . 
body weight completely compensated for the thyroxine deficiency produced by methylthiouracil , independent of the presence of a.a.f. twenty - two rats ( 6 males and 16 females ) were treated with 2 - 5 , ug . 
nor the controls reacted as uniformly as the animals injected with the higher dose of thyroxine ( table iii )  . in the males , the thyroid weights did not show any significant differences , the values being slightly above normal in both groups . 
with the exception of two animals , all showed an increase in cell all pituitaries were normal , showing heights far above the normal values . basophil counts of the same order as the controls . comparison of the values obtained in the animals with and without a.a.f. statistical shows that the greatest differences were found for the cell heights . analysis of these values proved that the difference between the means was highly the difference found for the means of the thyroid weights significant ( p < 0 0 1 )  . of the two groups , however , was of doubtful significance ( p > 0 05 )  . the pituitaries of the five thyroidectomized females treated with a.a.f. 
the weight increase of the thyroid induced by goitrogens is a complex phenomenon . it is due to increased numbers and size of the epithelial cells , and may be partially offset by the loss of colloid . 
when the supply of thyrotropin is reduced either by withdrawal of the goitrogen , or by administration of thyroxine , cell size decreases within a short time , whereas , the number of cells remains high over prolonged periods and colloid reappears . 
the latter factor must introduce uncontrollable errors when the thyroid weight is used as a criterion of thyrotropic action . in our experience , as in that of rawson and starr ( 1938 ) , the estimation of the mean acinar cell height has been found to give more reproducible results in assaying the degree of thyroid stimulation than the weight method . since a close correlation exists between the size of thyroid cells and the number of pituitary basophil cells , the percentage count of basophils has been used for assaying states of thyroxine deficiency in combination with the measurement of epithelial cell heights ( griesbach and purves , 1945 )  . in the state of fully developed thyroxine deficiency the percentage of basophils reaches values of 30 to 45 per cent . 
by excluding from the count masses of basophilic colloid free from cellular structures , we believe to have eliminated this possible source of error . all rats injected with 5 - 0 yig . 
griesbach the goitrogen - treated rat enlargement of thyroid cells is linked with an increase in numbers of pituitary basophil cells , indicating elevated thyrotropic activity . this correlation breaks down under the influence of a.a.f. 
one half of the animals still had large goitres and , histologically , in all but one the epithelium was almost as high as in the rats which did not receive thyroxine . no evidence for an increased demand for thyroxine or for higher sensitivity to thyrotropin was found in the animals treated with a.a.f. 
when a thyroxine deficiency was induced in a.a.f. treated animals by surgical or chemical means , their pituitaries responded in the similarly , the growth same way to thyroxine as did the animals without a.a.f. response of the female rats to thyroxine was independent of the presence of a.a.f. in the males , the pronounced liver damage prevented normal growth . these facts show that the action of thyroxine on the pituitary was not inhibited by a.a.f. 
the problem can therefore be reduced to that of an anomalous behaviour of the thyroid epithelium . in the hands of paschkis , cantarow and stasney ( 1948 ) l - thyroxine did not prevent the change from normal to high epithelium . in our experiments , physiological amounts of dlthyroxine did not reduce a high cylindrical epithelium to normal . it must , however , be remembered that the hyperplastic thyroid epithelium of the a.a.f. treated animals was not completely refractory to thyroxine . 
and storing at that temperature and after drying following freezing to from the frozen state ( gye , begg , mann and craigie , 1949 )  . in addition , mouse breast carcinoma which had been frozen to - 79 ' c . 
from these results it was concluded that since the physical treatments kired the normal embryo cells , they must also have killed the tumour cers , and that the tumours which arose from tho inoculations of treated m ' aterial must have been caused by a viius liberated from the dead cells . the claims of gye and his co - workers have been much criticized ; hirschberg and rusch ( 1950 ) , passey and dmochowski ( 1950 ) , passey , dmochowski , lasnitzki and millard ( 1950 ) and wamer and gostfing ( 1950 ) have all pointed out the great weakness of the assumption that since embryonic cells failed to grow after exposure these recent authors to low temperature , such treatment is lethal to ar cells . have each reviewed some of the ' extensive work reported in the last forty years , which shows that of both normal and neoplastic tissues in many species , some will and some will not survive exposure to extreme cold . in view of this great variation in response to cold shown by different tissues , it was felt that an investigation into the effects of freezing and drying upon a tumour affecting a non - mammalian host would be of value ; the rous no . 
i since it is a tumour from which a virus is easily fowl sarcoma was selected . separable , it was considered that the effects of cold and desiccation should be investigated upon the virus and the cers separately . that the virus was able to survive freezing and drying has been estabhshed for some time ( knox , 1939a ; hoffstadt and tripi , 1946 ; dmochowski , 1948 ; carr and harris , 1951 ) , and it has also been know - n to retain its tumour - producing activity after the ap ' plication of cold by repeated freezing and thawing ( cramer and foulds , 1930 ; selbie and mcintosh , 1939 )  . however , the response of the cells of the rous sarcoma to freezing and desiccation has not hitherto been ascertained . 
nakahara and yaoi ( 1930 ) found that rous sarcoma material which had been subjected to repeated freezing and thawing was less active when injected than similar untreated material , whidst selbie and mcintosh ( 1939 ) were able to prepare from rous tumour treated in this way filtrates with a greater tumour - producing activity than those made from untreated samples . selbie and mcintosh ( 1939 ) considered that repeated freezing and thawing broke up the cers and allowed a greater liberation of virus than would otherwise occur , and the method has been used for obtaining good yields of rous virus in various investigations ( knox , 1939b ; porard , 1939 )  . the work reported here consists ofexperiments upon the effects oftemperatures ' 70 ' c . 
they were between eight and nineteen weeks old when inoculated , apart from four exceptions which were slightly older , and belonged to three susceptible inbred lines developed at edinburgh , or crosses derived from thethe fines and crosses used were intensity , breeding , non - moult , breeding hen x intensity ' cock , and intensity hen x breeding cock , and have been described by greenwood , blyth and carr ( i948 ) ; both the age at which the birds were inoculated and the line or cross to which they belonged were determined by the exigencies of supply . preparation and treatment of tumour material . for each experiment a bird with a large actively growing sarcoma of anything from 15 to 27 days ' growth from the date of inoculation was selected as a source of tumour material and kired by wringing of the neck . 
when prepared , the disintegrated tumour was loaded into a record syringe for easy manipulation . where disintegrated tumour was to be subjected to cold or desiccation , the treatment was apphed as follows : freezing and storing . - amounts of i c.c. 
by starting the motor and setting the ampoule rapidly spinning on its longitudinal axis , a thin even film of disintegrated tumour was obtained on the inside of the bulb of the ampoule . 
the forceps w ' ere then released , causing a rapid rise of pressure within the apparatus ; this fell within five minutes to the original level , which was then maintained until the end of the period of drying , which lasted for 3 to 5 hours . 
epstein were separated off by centrifuging in an angle centrifuge at 3500 r.p.for 15 minutes , decanting the supernatant fluid obtained , and recentrifuging it aga ' in in a horizontal centrifuge at 6000 r.p.for a further 15 minutes . 
of saline and centrifuging in a horizontal centrifuge at 6000 r.p.for 15 minutes , resuspending the deposit in fresh saline , and performing the procedure either three ( experiment 1 ) or six - ( experiment 4 ) times . 
one sample of each preparation was mixed and incubated with an equal volume of saline and the other with an equal volume of serum ( diluted i in i 0 with sahne )  . 
the serum used ( e ) was from a fowl whose rous tumour had regressed and was one which had been shown to have strong anti - rous virus neutralizing powers in experiments 2 and 3 . that part of the experiment in which the virus and cer preparations were made from disintegrated tumour subjected to freezing and storing ( experiment 4c ) , samples of the preparations were mixed with an equal volume of pooled normal serum ( diluted 1 in io with saline ) , as well as with safine and neutrahzing serum e . all the mixtures were incubated in a water - bath at 37 ' c . 
in volume , and four inoculations of each preparation or dilution of a preparation were made . when inoculating preparations whose tumour - producing potencies were to be compared with one another , the injections of material from each preparation were made into separate groups of birds ; the scheme for the distribution of the injections of the various dilutions of each preparation amongst the birds in a group was the same in every case . 
the method of ' calculation used was that of reed and muench ( 1938 ) , and the figure obtained has been called by warner and gostling ( i950 ) the tpd 50 ; it is show - n in the tables comparison of this by its negative logarithm , designated the " tpd 50 index . " tpd 50 index of various preparations offers a comparison of their tumourproducing activities . strict aseptic technique was maintained throughout all the experimental procedures . all the experimental procedures were timed . 
where the virus suspension was made from disintegrated tumour which had been frozen and stored , the tumour - producing activity was in one case greater ( experiment id ) and in the other case less than that made from untreated material ( experiment 4d )  . it will be seen from table ii that washed cells of the rous sarcoma which had been exposed to any of the three physical treatments employed had less tumour322 m . 
on the other hand , in the case of washed cells which had been subjected to repeated freezing and thawing or freezing and storing ( experiments 4b and 4d ) , incubation with neutralizing serum , in contrast with incubation with saline or normal serum , totally deprived them of their tumour - producing activity . 
at the very least the centrifugation employed must be allowed to have separated off the overwhelming majority of the cefls , and the great tumour - producing activity remaining with the virus suspensions cannot be attributed to occasional cells left in them . accepting therefore that the virus suspensions were almost entirely if not wholly cell - free , the ability of the rous virus to retain some of its tumour - producing activity after exposure to cold has been demonstrated , thus confirming the work of cramer and foulds ( 1930 ) and selbie and mcintosh ( 1939 ) , who were able to prepare active filtrates from rous tumour material subjected to repeated confirmation has also been obtained of the ability of the freezing and thawing . virus to survive freezing and drp ' ng , which has been demonstrated by a number of previous investigators ( knox , 1939a ; hoffstadt and tripi , 1946 ; dmochowski , 1948 ; carr and harris , 1951 )  . 
as in the work of knox ( 1939a ) , so here the activity of virus preparations obtained from frozen and dried tumour material was considerably less than those made from untreated material ( table i , experiments ic and 4c )  . 
the contention , however , that preparations of rous virus made from repeatedly frozen and thawed sarcoma had a greater tumour - producing activity than those made from untreated material on account of the treatment breaking up.the tissue fragments and hberating the virus ( selbie and mcintosh , 1939 ) has not been borne out . 
the failure of the neutralizing ' serum to abolish the tumour - pro - ducing activity of washed cells which had been left untreated ( table iv , experiment 4a ) is considered to be evidence of the presence of live cells in the preparation . the slight reduction in the tumour - producing activity of the untreated washed cells which was brought about by incubation with neutralizing serum is considered to be due to the action of the serum on free virus liberated from or attached to cells damaged during the disintegration of the tumour material . the washed tumour cells subjected to freezing and drying were deprived of most of their tumour - producing activity after incubation with neutralizing serum ( table iv , experiment 4c ) ; out of the inoculations made with all the various dilutions of the frozen and dried washed cells , only one gave rise to a tumour . this was from one of the inocula of the 10 - 1 dilution of the mixture of cells and antiserunow , it has long been known that viruses can be recovered from neutral mixtures with antiserum simply by dilution of the mixture with sahne ; such reactivation has been demonstrated with vaccinia ( andrewes , 1928 ) and fowl plague ( todd , 1928 ) , and although this " dilution phenomenon " is harder to obtain in the case of the rous virus , andrewes ( 1932 ) was able to show it conclusively . 
christiansen. from the institutt for generell og elcperimentell patologi , universitetet i oslo . received for publication february 2 , 1953 . it is a strange fact that in the voluminous literature on breast cancer the prognostic value of the size of the tumour is only cursorily dealt with , a fact mentioned by geschickter ( 1945 ) and underlined by bloom ( 1950 )  . 
the explanation probably is , that the view expressed by kaae ( 1948 ) , namely , " it is generally recognized that the size of the tumour is a very important factor in the prognosis , and that the latter is much more favourable for the small tumours than for the large , " seems so generally sound , even self evident , that the problem has not been considered worthy of a closer study . if we examine the pertinent literature , we find , however , that the findings are not unequivocal . dahl iversen ( 1930 ) states that breast cancers up to the size of plums are without recurrences after 3 years ' observation in 83 per cent of the cases , whereas tumours larger , up to the size of a hen 's egg , show only 13 per cent freedom from recurrences after 3 years . eggers , de cholnoky and jessup ( 1941 ) report upon a 5 - year survival of 73 per cent , if the mass is 2 cor less , and only 24 per cent haagensen and stout ( 1943 ) found a if the mass has reached a size of 3 to 6 cm . 5 - year clinical cure of 62 * 2 per cent if the tumour was under 3 cand only 19 - 8 per cent if the tumour had reached 6 cor more . 
they conclude that " the data indicate , as might be expected , that the prognosis becomes worse as the size of the tumour increases "  . bloom ( 1950 ) found 5 - year survivals in the following percentages : tumour less than 1 inch 59 per cent , 1 to 2 inches 45 per cent , and more than 2 inches 32 per cent , and he finds after grading of the tumours that : " of the tumours with a diameter of 1 inch or less , 37 per cent are classified as grade i and 23 per cent as grade iii . 
on the other hand , in the case of growths of more than 2 inches diameter only 8 per cent belong to grade i whilst 54 per cent are grade iii . when the diameter lies between 1 and 2 inches the incidence of these tumours is practically the same " , and he concludes that : " whether the tumours are small or large , the outlook is uniformly good in the former ( grade i ) and bad in the latter group ( grade iii )  . 
on the other hand , an intermediate result is obtained for the intermediate cases ( grade ii ) , the survival rate being practically halved in in other words , the metastasizing power the presence of the larger neoplasms . for growths for grade i and also grade iii cancers is independent of size . classified as grade ii this power bears a direct relationship to the diameter , the larger the tumour the greater the likelihood of spread having taken place "  . 
christiansen the time factors are so long or so short that their significance is obscured under the usual clinical conditions . when kunath ( 1940 ) says : " size and ma88 . - an analysis of this point failed to reveal that a larger tumour carries any more serious a prognosis than " no does a small one " , and hoopes and mcgraw ( 1942 ) likewise conclude : correlation was found between the post - operative duration of life and the size of the tumour removed " , these findings are not necessarily in contradiction to the opposite conclusions , mentioned above . 
the explanation may be a different composition of the groups of tumours , as regard number of representatives of the again we have to guard ourselves in transferring different grades of malignancy . conclusions from groups to individual cases . considering the importance of assessing the significance of a minimum size of tumour for the prognosis of breast cancer , we have examined breast cancer material consisting of a total of 974 cases . 
we planned to examine the ultimate development of breast cancers as small as practically diagnosable . in order to decide the upper limit of the size to include , theoretical and practical considerafirstly , we considered the smallest lump , tions have been taken into account . that size evidently depends upon the site distinguishable as a definite tumour . and upon the amount of adipose tissue . 
a tumour near the surface and in a shrunken atrophic breast will be more easily discovered than one deeply situated it seems that tumours smaller than those the size in a full and developed breast . of a pea cannot reasonably be distinguished from the many irregular nodosities in a cimacteric , or pre - cimacteric breast , and in most cases it is difficult to feel secondly , we wanted to examine tumours a lump smaller than a small hazel nut . of a size so small that the number of cases would represent a very small fraction of the total , whereby we would obtain a statistical claim to a designation " a very small breast tumour "  . 
the tumours are usually characterized by some object for comparison , usually : pea , bean , nut kernel , hazel nut , date - stone , date , wall nut , plum , etc . 
a tumour the size of a hazel nut will usually measure some 10 to 12 by 15 to 17 mone has to have in mind the small size of a norwegian hazel nut to appreciate the size . when we examined our total material we found that only 56 tumours out of 974 , i.e. , 5 - 7 per cent . 
the fate of the patients has been followed for from 10 to 20 years . the material was examined during the later half of 1952 . the clinical information is not always precise , or detailed , as some of the case histories are very short . 
nor is the pathological material always complete , as some of the surgeons do not submit adipose tissue from the axilla if no suspicious glands are found . in other cases a selection of glands only are sent to the laboratory , and finally , some of our paraffin blocks were destroyed during the war , with the result that for some cases a few slides only could be examined . this means that all the positive findings are minimum recordings . the clinical follow - up has been complete , all patients having been accounted for . reliable death certificates have been received for all the dead , and the patients alive have been personally examined by one of us , or by competent doctors as regards the patients living in remote parts of the country . as only the patients with a definite statement as to the size of the tumour have been included , the size is known in all cases . 
8 died from broncho - pneumonia one year and a half after operation , symptom - free . all the others succumbed to their tumours . this 5 - year oure rate corresponds very closely to the rate quoted by engelstad ( 1948 ) for his group of 768 cases , regarded as stage i and stage ii cases . this indicates that our " small " breast cancers do not present a particularly favourable clinical picture . in one aspect our table ii shows a remarkable deviation from the usual , namely , the small difference between the 5 - year and the 10 - year figures , only 1 out of 37 patients dying in the second 5 - year interval . as stated , the whole group of 56 patients has been followed for 10 years . a smaller group has , however , been followed for a longer period , and in table iii some of the results are summarized . 
even if we select the very smallest tumours present in our series , those the size of a pea or a bean , out of a total of 10 , we find only 5 patients alive after 10 years , or more , and 4 patients dead from metastases . in the present series the combined surgical and radiological treatment seems , in a number of cases , to have effected a considerable delay in a finally fatal this delay is of very great value from a therapeutic standpoint , even outcome . if the treatment fails to effect a complete cure . staging actually is an ambiguous process . the prolonged observation also helps to assess more accurately the real stage the simplest and least of the tumours . reliable staging is the immediate clinical , unreliable to a degree to make it practically useless . 
we have , however , to bear in mind that the value even of negative findings of tumour cells in lymph this grouping is rather limited . nodes removed may be the result of the pathologist not finding the cells in spite or , tumour cells may by - pass the usual lymphatic filters in of their presence . the axillary glands and proceed directly to the supraclavicular or the intrathoracic nodes , or the tumour cells may spread via the blood streathe discrepancy between the number of tumours anatomically staged as stage i and the number of cures effected after a proper local operation shows the degree of failure in the process of staging . 
on the other hand , if radiological treatment has been added to the surgical , even the delayed retrospective clinical staging may be incomplete , because a tumour , anatomically staged as stage i and actually being in stage ii , may be cured by radiological treatment and thereby escape its proper staging . in spite of the incomplete clinical and pathological information , it will be of some interest to examine the state of affairs , as regards stages recorded in this material . 
10 , 27 , 31 died symptom - free after more than 10 years ' observation . the large number of fatal outcomes with negative findings of tumour cells in the axillary lymph nodes confirm our assumption that our figures represent the considerable number of patients alive and symptom - free minimum findings . in spite of positive findings of tumour cells in the lymph nodes , actually 5 patients , further confirm our statement as to a considerable effect of the treatment , but l . 
christiansen also underlines the great number of patients already in stage ii ( or further ) at the moment of presenting a very small breast cancer . if we add these 5 patients with microscopical lymph - node metastases , but still symptom - free , to the group of the dead and those living with metastases , we find that two - thirds of all our patients with small tumours were in stage ii ( or further ) when the operation was performed . the value of bloom 's ( 1950 ) grading and analysis of his material made it natural to try his principles on our material . 
we deliberately use the word principles , because any grading is arbitrary , and the designation in each case is the result of a subjective estimate of a series of characteristics , which themselves are unprecise . 
the explanation may be that the number of cases is too small , that the treatment given may have influenced the final result because a number of very malignant tumours are comparatively radiosensitive , or it may be caused by our incompetency . 
the medium long histories , from a few months up to a year , show an increasingly gloomy picture . if we again deduct the grade i cases we conserve the pattem , and still find indications of a benefit for very quick reaction with immediate treatment after diagnosis , and with a still more pronounced bad prognosis in cases with delays from a few months up to a year . for the very long histories ( more than a year ) the comparatively better prognosis is preserved , and may partly be accounted for by a default in the process of grading , partly by combination of high malignancy and high grade radiosensitivity in certain cases , and partly by inclusion of tumours with a more moderate malignancy ( grade ii )  . besides , a perfect correspondence between morphological grading and biological development is evidently not obtainable . too many unknown factors are involved here . 
usually the ventral prostates were explanted ; they were grown by the watch - glass technique , which is eminently suitable for organized gro - wth ( fell and robinson , 1930 )  . 
the medium was placed in a small watch - glass and allowed to clot ; the watch - glass rested on a layer of sterile cotton - wool soaked with sterile distilled water inside a small petri dish . 
but later this method was improved by explanting one lobe of each gland into medium containing methylcholanthrene while the other was kept as control . the explants were placed well flattened out on th ' e surface of the plasma clot , where they became firmly anchored . after a day or two the growing explants liquefied part of the plasma clot , and while still firmly attached to the surfa - ce of the clot were surrounded by a pool of liquefied mediuthe cultures were after the ninth day no transferred to fresh medium every second or third day . more methy1cholanthrene was added , and the cultures were maintained in normal medium for the rest of the culture period . the used glass - ware was decontaminated by keepino , it in concentrated sulphuric acid at room temperature for at least one week . 
the glands were fixed in 2 per cent acetic zenker after the following periods of growth : 5 days , io days , 14 days and 21 days . five to nine experimental and an equal number of control cultures were used for each point of observation . 
they were embedded in paraffin and seriary sectioned ; the sections were stained with ehrlich 's haematoxylin and eosin . to assess mitosis the number of dividing cells was counted in every second section , i.e. , in at least 20 sections of all single - lobe cultures . 
cultures treated with 20 - methylcholanthrene . cultures treated with methylcbolanthrene show a similar tvpe of growth at the beginning of the culture period : formation of new alveoh at the periphery and some central degeneration . 
the epithehum retains its glandular character and remains actively secreting in the treated explants for a longer time , but the stroma is usually verv poor and reduced in botb . 
one out of fi - ve treated cultures shows squamous metaplasia after the higher mitosis is stfll ' above that in the controls and has , in fact , risen after the dose . higher concentration while it has dropped after the lower . table ii gives th - e number of mitotic cells counted in 20 sections of controls and cultures treated for 9 days with 2 , y / cic . 
the number of dividing cells has fallen and nearly reached the control level . at the same time changes of a more anaplastic type can be observed in one batch of cultures treated with 2y of methylcholanthrene . 
squamous metaplasia develops more fully , particularly after the smaller dose , after the end of treatment . it is noteworthy that the d - ownward bend of the first wave coincides with the end of treatment . this may mean that the first peak is caused by a direct stimulating action of the carcinogen , whfle the second one is due to the increased growth - potential of the changed epitheliua rise in cell division has been reported by cooper and reller ( 1942 ) , who found a ten - fold increase hi the mitotic rate in the skin of mouse ears painted with methylcholanthrene , and by glucksmann ( 1945 ) , who observed a rise in mitosis a few hours after a single painting with benzpyrene . in contrast to the increase in mitosis and proliferation of the epithelial cells is the reduction of the stroma of the treated explants . 
of methylcholanthrene , which indicates that the dose for stimulation of fibroblasts is roughly twenty times lower than that for epithelium if the concentrations of 2 - 4y / / c.c. 
the cultures were grown in the presence of the agent for 9 days , then transferred to a normal medium and fixed at intervals after removal of the carcinogen . in both control and experimental cultures new alveoli were usually formed at the periphery of the explanted glands , while in the centre some of the al - veoli underwent degeneration . in the control cultures the epithelium quickly lost its glandular character , and the alveoli developed wide lumina lined by one layer of low epithelium in which mitosis was present though infrequent . 
of methylcholanthrene , the alveolar epithelium retained its glandular character for a longer time and showed a marked increase in mitosis , leading to hyperplasia and squamous metaplasia , while the stroma was considerably reduced in contrast to that of the controls . the first changes were recognizable 5 days after the beginning of treatment . they became more pronounced as the treatment continued and persisted after removal of the carcinogen . in the later stages partial or complete occlusion of the alveolar lumen with squamous metaplasia were observed . the rise in mitosis showed two distinct peaks , the downward bend of the first wave coinciding with the removal of the c ' arcinogen . 
fell for suggesting this problem and for her criticism in the preparation of the manuscript . i also wish to thank a . howard , radiotherapeutic research unit , hammersmith hospital , ' london , for the generous supply of cx males , c . 
w.3. received for publication april 3 , 1954 . the development of the search for endogenic carcinogens has been reported in earher publications from this institute , ( see hieger ( 1949 ) for references to previous papers )  . it has been shown that the cholesterol - rich fraction of ' tissues from human subjects who had died of cancer or of other causes , and even commercial cholesterol . 
the first tumour in table i was carried through 93 generations and the first tumour in table iv ( highly purified cholesterol ) has been growing vigorously in its 25th generation . 
the control tests on solvents are described further on in this paper . purification of the cholesterol was next undertaken , as a first step using the simplest technique . table ii gives the results of tests on fractions of commercial cholesterol obtained by crystalhsation from acetone . 
the method involved several stages . these were : ( 1 ) acetylation . this step provided several advantages , nameiy ( a ) the oh group was protected during bromination ; ( b ) the acetate can be differentially eluted from a1203by mild eluents the unacetylated sterol when absorbed on a1203 requires more powerful eluents . ( 2 ) bromination , filtration and debromination . ( 3 ) adsorption on a1203colunin and elution with benzene - pet . 
orr there was , of course , no guarantee that contamination could have been completely avoided in the " carcinogen - free " room ; for example , 'sawdust from a box - containing benzpyrene - treated mice might accidentally be carried in to the noncarcinogen room on the shoes of anyone who had walked through the carcinogen however , the technicians who serviced the mice in the . 
consequently the controls had more injections than the sterol injected mice in order to maintain a stationary nodule . is the carcinogenic activity of commercial cholesterol due ( a ) to the sterol itself , or ( b ) to highly active impurities ? the evidence presented in tables iv and v provides support for ( a ) , i.e. , cholesterol per se is the true carcinogen in the commercial product . if the incidence of sarcomas be expressed as the ratio ( percentage ) of tumour mice to injmted mice which survived to a minimal latent period level , say i year , then the result can be stated thus : ( a ) purified cholesterol gives rise to 4 per cent sarcomas in q ,  . 
the mice did not survive well , and in fact the single sarcoma appeared at the 27th month in the sole survivor of the 34 mice initioy injected with purified cholesterol . twenty - seven months is near the outsidei limit of the range of latent periods found with the carcinogens of the cholesterol categpyy , , in an experiment with commercial cholesterol ( table ii , section a ) 3 of the 4 280 1 . 
orr in 1947 ( unpubhshed ) , would provide an attractive theory of the mode of action of cholesterol as carcinogen . however , it would be necessary to postulate that the carcinogen is held by adsorption on the cholesterol 292 1 . 
op.p. although stif in a - n active stage , and that in sucha condition it is not roadily eluted , for clearly if it were a case of simple solubility there would be no more reason for ' the factor dissolving in the cholesterol than for it to be dissolved out secondly i ' ected cholesterol after a year or so in the body should be again . thirdly , if such an assumption were accepted , there rich in absorbed carcinogen . would be httle necessity to proceed with the question of whether pure or impure fourthly , if the carcinogen ' which is concencholesterol is the true carcinogen . trated in the cholesterol originated in the environment , it should be easy to test the hypothesis by purposely arowing the mice injected with the cholesterol to hve an atmosphere rich in carcinogen . this test has been put into operation , and constitutes a secoind stage to the investigation on atmospheric carcinogens as a factor in sarcoma induction ( see above )  . one hundred stock mice were injected with ohve oil solution of commercial cholesterol and kept in a carcinogen - rich atmosphere as described above . 
the control mice ( 135 ) were kept in the room free from carcinogen . the results are shown in table v . control te8t8 on the 801vent8 . lard was employed as solvent from 1936 to 1949 . during this time a total of over 360 mice was tested with the solvent , lard ' alone . these mice did not survive remarkably well ; they gave no positive result until the series had been almost completed , when among the last few batches a single sarcoma developed . 
when in 1950 , a change was made from lard to olive oil ( hospital dispensary grade ) , a control series of 134 mice was set up for injection with the oil alone . 
the old c " mice were 12 ( and upwards ) months of age at the start of the experiments ; their effective number should therefore be raised by a factor of the order of 2 since the initial number , 19 , would represent the survivors at 12 months . 
the siingle sarcoma occurred in an oldc57moim at the unexpectedly low latent period of 6 months . * however , s ' mce this mouse was 16 months of age at the start of the experiment some ageing factor for tumour induction is obviously operating . 
this preparation gave 2 sarcomas in 20 mice initiary . other work on carcinogenic activity of chole8terol . truhaut ( 1947 ) could find no activity in his sample of purified cholesterol . truhaut injected 20 mice , of unnamed genetic origin , with cholesterol " purifie ' , " 20 mice with cholesterol irradiated with 10 , 000 r , and 20 with solvent alone , namelv ohve oil . 
 " " si la susceptibilite ' individuelle des animaux n ' est pas en cause , cela tendrait ' a d6monter la pre 'sence , dans le choleste ' rol commercial , d ' impurete 's fortement cance ' rig ' enes qm se rapprochent vraisemblablement des substancek ; responsables de i ' activite ' cance ' rig ' ene de la fraction des ste ' rols precipitables par le digitonoside dans l ' insaponifiable du foie de sujets cancereux . steiner ( 1951 ) has also reported that no carcinogenic activity could be detected in a sample of cholesterol . 
from the carbonyl absorption found in cholesterol fractions , therefore , the type of impurity and its approximate amount can be deduced . cholesterol preparation numbers , 3 , 4 , 6 , 7 and 9 ( for key , vide supra ) showed no appreciable carbonyl absorption ( less than 0 - 25 per cent )  . 
number 8 ( glaxo sample 5989 ) had a band at 1744 cm . - ' , assigned to a 17 - ketone , equivalent to about 1 per cent of such an impurity . 
number 11 ( glaxo sample 1201 ) had three bands at 1744 , 1710 and 1674 cm - ' ; these indicate about 9 per cent of a 17 - ketone , 4 per cent of a 20 - ketone ( or possibly in a 6 - membered ring ) and 3 per cent of an ac , unsaturated ketone , probably a a4 : 3 - ketone . number 5 , which was the least soluble fraction obtained by fractional crystallisation of this latter commer290 i . 
the most soluble fraction in this crystallisation and used for the test in section d , table ii ( number 13 ) contained the impurities concentrated about five - fold . 
lucasanda.c.thackray. from the , department of pathology , the royal dental hospital of london school of dental surgery and the bland - sutton institute of pathology , the , middlesex hospital , london , w . 
the tumour occurs in the lower jaw much more frequently than in the upper ( i 7 cases and 3 cases respectively in this series ) , is usually a central lesion and gradually enlarges , in the majonty of cases without pain . in the course of time the bone becomes absorbed , and if a cystic loculus is opened infection and discharge forow . local recurrence iscommon fohowing inadequate treatment , but metastases are very rare . 
a case of pulmonary metastases has been reported by waterworth and purar ( 1948 ) , who review previous reports . the natural history of the disease is long ; it is not always easy therefore to assess the efficacy of various methods of treatment . most authorities consider radical excision of the tumour to be the treatment of choice . 
thus the least evolved type of tumour consists of strands of more or less undifferentiated epithehal cers growing in a fibrous stroma , while a tumour which has undergone a greater degree of entiation consists of folhclea 290 r . 
the epithelial follicles in such cases consist of an outer layer of columnar cells resembling the similarly situated cells of the enamel organ , while towards the centre the cells become stellate in appearance and cyst formation may occur . on the other hand , the trend of differentiation may be towards a more highly developed type of squamous epithelium , in which case the tumour consists of sheets of squamous cells showing a tendency to cornification , or a basal cell type of growth may be produced . these tumours may thus present a variety of appearances , from case to case , or in different areas of the same growth , and while attempts at classification on a histological basis are helpful in so far as they indicate the varying degrees of differentiation whicli can be observed , they are seldom entirely applicable to any given specimen . 
tboma ( 1950 ) has proposed one such classification , as follows , though he notes that pure types are but infrequently seen : ( 1 ) primitive type , composed of strands of epithelial cells showing little tendency to differentiation , growing in a loose fibrous stroma . 
a very rare type of tumour in which haemangiomatous characters are present . ( 8 ) melano - amelobla , 3toma . also a very rare tumour , in which the epithelial cells contain pigment . in our series 4 cases were of the primitive type , whilst the remainder were fig . 
2 ( case 5 ) shows an largely follicular or showed mixed appearances . example of the primitive type of tumour , cords of epithelial cells growing in a fibrous stroma . 
6 ( case 5 ) , in which the growth is composed of follicles or nests of epithelial cells with outer columnar layer and inner stellate area . microcyst formation , a common occurrence in adamantinoma , was present in typically , these cysts occur in the area of stellate cells towards the 14 cases . centres of the follicles , but they also form in the stroma . 
the occurrence of stromal cysts is a point to which very little previous consideration appears to siegmund ( 1929 ) noted their occurrence in 2 of his cases , but bave been given . apart from this instance , and a passing reference by kronfeld ( 1930 ) to the same fig . 
8 ( case 1 )  . in the latter case , however , it will be seen that though there is some breakdown within the follicles , the majority of the cysts have formed on the side of the columnar cell layer opposite to that on which fig . 
even in the 6 cases showing epithelial cysts only , evidence could be found in 3 cases of incipient degenerative changes in the stroma . the cause of cyst formation is a matter of conjecture . it is believed to be the result of progressive degeneration in originally solid growths . the solid tumour closely resembles the developing tooth up to the point at which differentiation of adjacent connective - tissue cells into odontoblasts , and subsequently amelogenesis , would occur in the case of the tooth , but at this stage the tumour cannot develop further . degeneration therefore takes place ( kronfeld , 1930 ; robinson , the ensuing accumulation of fluid of relatively high osmotic tension is 1937a )  . probably a factor in the progressive enlargement of the microcysts , a mechanism investigated by toller ( 1948 ) in the case of simple dental cysts , and quite possibly a factor also in the case of cyst formation in these tumours . with regard to cyst formation in the stroma , it appears that again a process of degeneration is at work , and all stages of breakdown can be seen from a slight loosening of the stroma to mucinous - like degeneration and the formation of completely clear cystic spaces . 
the inner enamel epithelium is that which lines the concavity of the enamel organ , a single layer of tan columnar cells which will produce enamel , while the outer enamel epithelium consists of cuboidal cells covering the convexity of the organ . 
on the other hand , bland - sutton ( 1922 ) thought that the enamel organ could not be the origin of these growths , since the majority of patients were well into adult life when their tumours first appeared . however , robinso ' n ( 1937b ) in his survey of 379 cases found that the tumour was first noticed between the tenth and thirty - fifth years in 70 per cent of cases . considered together with the fact that these tumours are very slow growmg and have a long natural history , it is evident that the majority of growths originate at quite an early age . robinson ( 1937b ) also found that the tumour occurred most frequently in the premolar and molar regions , where supernumerary tooth germs most often occur , kegel ( 1932 ) and and that many cases were associated with a missing tooth . geschickter ( 1935 ) also support the view that the tumour arises from the enamel byers and sarnat ( 1945 ) believe that the tumour originates from that organ . structure during the first few years of life , though active growth may not become manifest till later . if these tumours , or at least the majority of them , originated directly from the enamel organ , it might be expected that on occasion enamel would be formed . this is never the case , though thoma ( i 950 ) considers that the homogeneous zone sometimes to be seen between the basal membrane of the columnar cells in the follicular type of adamantinoma and the stroma , which appears yellow with van gieson 's stain , must be considered as abortive enamel formation , though it is possible that precystic changes in the stroma could account for such appearances . 
thackray these tumours resemble rodent ulcers in several respects . tumour without involvement of the oral epithelium , is , however , evidence against an origin from the oral mucosa . both are related to derivatives of the covering ectoderm , the one to teeth and the other to hair follicles , though discussion continues as to the stage of development of the appendage at which they arise . 
they are composed of relatively undifferentiated cells which can give rise to any of the appearances found in these tumours , and moreover will produce a central growth . it is not necessary to postulate tumour formation from these rests in early life , with a subsequent interval of dormancy to account for those cases in which the tumour first appears at a comparatively late age . being present from the formative phase of the dental tissues onwards they can proliferate at any time , and this seems a more satisfactory hypothesis than the dormancy theory . the rests of serre , those left by the dental lamina , are situated quite superficially . 
here the cells tend to take on a sterate appearance towarcls the centres of the cords and become elongated and columnar at the periphery . there is some cystic degeneration in the stellate areas and also in the stroma . case 4 : - 11 . 
1893. - 1910 : dentigerous cyst of one year 's duration 1928 : swelling had gradually increased in removed . size till there was now a hard tumour of the right mandible . this was excised and the cavity of a multilocular cyst exposed . 
radium tubes forced up through harcl palate . october , 1925 : no improvement . tubes left in 18 hours . december , 1925 : left side of face swollen over superior maxilla . severe paon left side of hard palate is an irregular swelling extending from mid - line to , and including alveolar margjanuary , 1926 : alveolar part of superior maxilla and the palatal tumour excised . 
1892. - 1926 : noticed a lump in the right upper jaw which she thought to be due to ill - fitting dentures . this was treated with " caustic and disappeared , but recurred within 4 months . it was again similarly treated . 
1928 on admission to hospital an oval area of enlargement of the right maxina was noted , situatecl between the alveolar margin of the maxilla and the inner surface of the cheek , the long axis clirected antero - posteriorly for about an inch . 
1899. - 1928 : operated on for " abscess of j aw . 1937 : painful swelling appeared at site of previous operation . x - ray examination showed a multflocular cyst in the 3 - / - 6 region . 
1887. - 1929 : the patient complained of a lump on the right side of the jaw of 10 years ' duration , which had been operated on 3 times , 10 , 6 and 3 years previously . it had increased in size , painlessly , but rather rapidly since the last operation . 
1881. - 1937 : three years ' history of swelling of the right lower jaw after extraction of 3 teeth . this was considered to be due to a dental cyst , and the jaw was scraped on two occasions . after the first operation the swelling disappeared . 
thackra ' y examination showed a large swelling on the lingual and buccal aspects of the pain . x - ray showed expansion of the jaw by a loculated , premolar and molar regions . 1950 : died from other causes . 
1883. - 1937 : admitted to hospital for a fracture of the this appeared elbow - joint , when a large swelling of the right mandible was noticed . to have started some 5 years previously , and had been gradually increasing in size . x - ray examination showed the typical appearances of adamantinoma , and local excision of the cyst was undertaken . 
orr. from the department of experimental pathology and cancer research , university of leeds . received for publication july 19 , 1947 . attention was drawn to the frequency of spontaneous tumours in the lungs of mice by tyzzer ( 1907 - 8 , 1909 ) , whose description of their histology has required but little amendment . 
a small group of stock mice was treated intranasally with 1 : 2 : 5 : 6 - dibenzanthracene in almond oil , but owing to intercurrent disease these had all died before adenomata were present in any except one . 
the present communication is based on the histological investigation of the lungs of a representative sample of these mice , together with the urethanetreated mice of the previous paper ( orr , 1947 ) , and a number of mice showing spontaneous pulmonary adenomata . 
the outline of the whole tumour is usually more or less circumscribed , though there is no definite capsule ; sometimes it is irregular , but the appearance is not that of true infiltration . 
the two types of tumour have also been noted by grady and stewart ( 1940 )  . occasionally , both types of structure have been seen in the same nodule , and there appear to be adequate grounds for regarding them as morphological variants of an identical process . it is not uncommon to find alveolar macrophages of the " dust cell " type in the adenomata . 
they are often so heavily loaded with carbon as to obscure all nuclear and cytoplasmic detail . less often other evidence of inflammation in the form of lymphocytic or leucocytic infiltration is seen . but in addition a very large number of lesions show appearances which make the impression inescapable that they are stages in the development of adenomata within inflammatory foci . this evidence is more striking where the tubulo - papillary structure is being evolved , in view of its highly characteristic and easily recognized pattern . 
the inflammatory changes resulting from urethane are relatively chronic , and take a considerable time to clear up on withdrawal of treatment . moreover , the proportionate share played by polymorphs in the reaction even in its early stages seemed smaller than average : it may be that urethane exerts some depressant effect on them , and thus necessitates greater activity by the so - called fixed tissues . the source of the epithelium in the tumours has not been determined with certainty . indeed , in many instances even the simple statement that it is epithelium involves a measure of assumption ; the most that can be said with 320 j . 
the actual existence in the fully developed lung of such a tissue is denied by many authorities , and some of those who claim to find it describe morphological appearances very different from those found in the tumour cells . if the alveolar epithelium is implicated , it can only be by reversion to the embryonic type , and if this occurs , such epithelium might owe its genesis , as it does in the embryo , to the bronchial epithelium . it appears to the present author that some workers have been inclined to regard as alveolar epithelium any lepidic tissue in the lung which could not be . 
shown directly to be bronchial in origin . the present results are therefore in agreement with those of magnus ( 1939 ) in respect of the tissue of origin , without accepting his view regarding the intrabronchial papillomata . 
orr does not suggest that it is malignant ; the rate of growth is slow once the characbut there can be little doubt that a malignant teristic size has been attained . " variant " occasionally turns up . the role of antecedent inflammation . slye , holmes and wells ( 1914 ) thought that adenomata arise from areas of lung tissue in which inflammatory hyperplasia has occurred . grady and stewart ( 1940 ) consider that they are not associated with inflammation . 
the present author 's observations have convinced him that these tumours invariably originate in foci of chronic collapse inflammation , which become invaded by bronrchial epithelium , and that it is the extent of the original inflammatory focus which this would explain the paradox of its determines that of the formed adenoma . comparatively rapid appearance and slow subsequent growth . 
7. received for publication february 7 , 1947 . it is a widespread doctrine among students of cancer that any tissue or organ capable of cell division may be the site of a malignant neoplasm , but it is well known to clinicians that certain tumours , theoretically possible , do not in fact occur . 
the most striking example of an organ which never gives rise to a neonotwithstanding the fact that mitoses occur in the lens plasm is the lens . throughout life , having been seen even in the lenses of patients 80 years old , no tumour of the lens has ever been discovered . the lens is an entirely epithelial structure embryologically derived from the surface ectoderm , devoid of blood supply and enclosed in a semi - permeable although mammalian lenses are enclosed membrane , the hyaline lens capsule . during embryonic life in a capillary net ( the vascular lens capsule ) this does not constitute a blood supply , since the vessels do not come in contact with the lens in the majority of vertebrates cells , being always outside the hyaline capsule . the lens can therefore be looked even this embryonic vascular capsule is absent . on as a segregated group of specialized epithelial cells growing and differentiating in a closed system , separated alike from the blood - stream and from the surface of the body . 
by carefill dissection of the eyes it is possible to get the lenses in their hyaline capsules completely free from vascular capsule or uveal tissue . these lenses were inserted under the skin of the flank of mice of the same inbred strain together with methylcholanthrene . in some cases the hyaline capsules were ruptured and the lenses mixed with a few crystals of methylcholanin others the unruptured lenses coated with a solution of the threne . in others again teased lenses were mixed carcinogen in oil were used . with methylcholanthrene in solution in equal parts of soft and liquid the exact method employed did not appear to be important , paraffin . two of the tumours being obtained with a solution of 1 mg . 
the tumours appeared between two and three months after implantation . characteristics of tumrnours of lens epithelium . the tumours are anaplastic carcinomas possessing certain characteristics suggestive of their origin from the subcapsular epithelium of the lens . 
a brief consideration of the normal structure and behaviour of this in mice is a necessary preliminary to the study of the tumours . the normal lens and its epithelium is seen in fig . 
the first stage of this differentiation is the lengthening of the cell and the displacement of the once this has occurred the cell cannot divide again , but nucleus to one end . proceeds to complete differentiation into a lens fibre , the nucleus of which evenfig . 
4 and 5 show results at three and at two days . thle ruptured capsule is in culture of the whole lens of a late mouse embryo . the centre of the explant and is surrounded by degenerating lens fibres . 
the cells are large , with voluminous spherical or oval nuclei , showing one or more nucleoli and a prominent reticular chromatin in the young cells the nucleus is central , in structure . the older cells it moves to the edge of the cell just before becoming pyknotic . metastases occurring in the inguinal lymph nodes show the same structure , but with less necrosis . 
6 and 7 is apparent . extending edge showed fibrillar outgrowths somewhat similar to those described this by kirby in tissue cultures of chick lenses . fixed and stained preparation of a 5 - day tissue culture of the same tumour also these are seen in fig . 
1. received for publication february 23 , 1948 . in a previous communication we reported that a strain of the shope rabbit papilloma had been transmitted for 10 serial passages in the domestic rabbit ( selbie and robinson , 1947 )  . 
the adaptation of this tumour virus as a transmissible infection in domestic rabbits occurred during experiments in which the simultaneous inoculation of shope papilloma virus and sheep dermatitis virus in domestic rabbits produced papillomas that proved infective to other rabbits ( selbie , 1946 )  . 
the further passage of this virus in domestic rabbits was materially helped by continuing the procedure of mixing a second virus suspension with the infective inoculum of papilloma virus , and also by treating the skin with croton oil before inoculation , but there was no evidence of an increase in the virulence of the virus suspensions during passage . in the present communication we shall show that carcinomatous transformation has occurred regularly in this transmissible papillomatosis , and in a mamner similar to that found in the non - infective papillomatosis that is produced in domestic rabbits by the inoculation of extracts of papillomas from the cotton - tail rabbit , the original host of the shope papilloma virus . 
thus in all rabbits frqm passage 4 in table i , area 1 represents normal skin inoculated with a 10 per cent extract of papillomas , area 2 , normal skin inoculated with a mixed extract of papillomas and sheep dermatitis lesions , area 3 , skin pre - treated with croton oil and inoculated with a 10 per cent extract of papillomas , and area 4 , skin pre - treated with croton oil and inoculated with the mixed virus suspensions . rabbit 2 , passage 2 was inoculated with papilloma extract on normal skin only , and in rabbit 4 , passage 3 , the procedures for areas 1 and 3 were omitted . the rabbits were examined at weekly intervals from the 7th week onwards . the tumours were charted and their size was measured by callipers at regular intervals . 
as was noted in the earlier communication ( selbie and robinson , 1947 ) , there was a tendency for individual papillomas to unite during development , and in some instances papillomas regressed completely . 
hence in this communication only those papillomas that were recognizable as single entities at 6 months in areas with confluent or semi - confluent papillomatosis will be considered . only a few papillomas could be distinguished as independent entities at 6 months , and no accurate count could be made , so that a fair estimate was given of 30 papillomas for confluent papillomatosis and 20 for semi - confluent papillomatosis . it became apparent early in this investigation that if the development of individual papillomas was to be studied over a prolonged period , cancers which arose in adjoining papillomas should be removed . 
1 are shown the times at which carcinomatous growths were first detected in passages 2 to 7 , passage 9 being omitted because the surviving rabbits have been observed for only 40 weeks after inoculation . it will be seen that the passage rabbit 2 __ to ~~ * : 6s weeks after inoculation fig . 
1. - times of observations of carcinomas in all rabbits from the 2nd to the 7th passage . each rabbit is represented by a line indicating the period of observation from the 40th week blocked circles indicate carcinomas arising from papillomas on skin treated onwards . with croton oil before inoculation , and open circles indicate carcinomas arising from papillomas on untreated skin . x = removal by operation of all remaining papillomas . 
the time at which glandular metastases were observed varied with the time at which the primary carcinomatous growth occurred , the limits being 33 weeks after inoculation in rabbit 2 , passage 9 , and 89 weeks in rabbit 2 , passage 2 , while the average time was 67 weeks for all the rabbits in the series . 
to draw any definite conclusions from this series concerning the incidence of glandular metastases as many of the primary growths were removed , and in consequence the opportunity for metastases to occur was reduced . 
2 , area 2 , the central papilloma with a concurrent thickening of the base . had slowly enlarged from the time of its original appearance to attain a diameter nine weeks later it measured 1 * 8 cin diameter and of 1 - 4 cat 53 weeks . the base had now thickened and was fleshy in appearance and consistency , in three weeks later , at 65 contras , t to the dry horny surface shown in fig . 
19. the secondary deposits in glands and lungs have all proved to be of the in many of squamous cell type showing varying degrees of differentiation . the lung metastases it can be seen that the infiltrating carcinoma cells form nodules within the alveolar spaces , so that the stroma of the deposit retains the architectural structure of the lung tissue . 
the time at which this change first occurs has tended to become shorter in successive passages , and has been reduced to 45 and 28 weeks in single rabbits of the 7th and 9th passages respecthis may indicate an increase in the virulence of the virus , but it is more tively . likely to be due to variations in the sensitivity of the rabbits . it has also been shown that the first occurrence of carcinomatous transformation in a rabbit is usually followed by others up to at least 5 in number and up to 85 weeks after indeed carcinomatous transformation occurs so regularly in this inoculation . transmissible infection that it would appear that all papillomas would show this change , provided that their evolution was not interrupted by the death of the host or by other factors such as spontaneous regression , infiltration from nearbv carcinomatous growth , or operative removal . carcinomatous transformation should then be regarded as a natural stage in the development of infective papillomatosis , and attributable only to the activity of the virus which initiates the infection . it is also clear from the work of kidd and rous ( 1938a , 1938b ) and mcintosh and selbie ( 1940 ) , who observed rapid transformation to malignancy in tar - induced papillomas infected with shope papilloma virus , that this virus is capable of causing immediate carcinomatous change . it is not therefore necessary to postulate an extraneous precipitating factor such as bacterial infection or an alteration in the virus itself to account for the change to malignancy , as suggested by rous , beard and kidd ( 1936 )  . although carcinomatous transformation would appear to be a natural stage in the development of this infection , it presents such a radical change in behaviour that it is difficult to see how it could be part of a continuous process . 
robinson cidence of tar tumours in mouse skin was increased at the site of injury by deep incision , numerous investigators have demonstrated the enhancing effect of unspecific irritation on the tumour - producing activity of carcinogenic substances . that chemical agents can also have this stimulating effect was shown by twort and twort ( 1928 ) , who accelerated the production of tar tumours in mice by pretreatment with oleic acid , and by berenblum ( 1941a , 1941b ) , who obtained a higher proportion of mice with tumours after painting with a dilute solution of benzpyrene by concurrent treatment with croton oil or by applying croton oil after a limited course of benzpyrene treatment . 
the possibility then had to be considered whether the hereditary condition acted through genetically transmitted differences in ovarian functional activity , or through an unequal response of mammary glands or genital tract to ovarian hormone . 
numerous and extensive observations were made with somewhat conflicting results , both on the nature of the oestrous cycle and on variations in organ sensitivity to hormonal stimulation , in strains of mice with differing mammary cancer incidence . when the existence of the third factor , i.e. 
 it has been found ( elson and lamerton , 1949 ) that the protein content of the diet has a profound influence on the response of the , valk.er rat carcinoma 256 to x - radiation . 
two effects were considered to be concerned in this response : ( a ) the initial inhibition of tumour growth and ( b ) the elimination of the inhibited tumour and cure of the animal . 
 although variations occur within a group of animals maintained on a standard diet of fixed composition , the general type of response to a suitably fractionated in animals maintained on the 5 per x - ray treatment is illustrated in fig . 
growth may be nearly stopped for a consider able time , but the tumour usually begins to grow again , at first slowly in animals maintained on a 20 and then more rapidly , finally causing death . 
the rate at which it decreases in size is at first fairly rapid , but gradually becomes slower , and in many cases the animal is then able to rid itself entirely of the tumour by a " shelling out " process or sometimes by the gradual complete regression of the tumour . 
 a detailed histological and c : ytological examination of tumours , particularly under conditions presented by a and b , was therefore undertaken in an attempt to elucidate the part played by diet in influencing the ultimate response of these tumours to radiation . 
they were kept in individual cages , and maintained on the 20 per cent or the 5 per cent protein diet for at lea.st 7 * r - arch fellow of the xorwegian defence research establishment . 
15 c under these conditions the dosage rate at the centre of the tumour is about 140 r / m h~ tuhnique . - - cross sections of the implanted tumours were made by means of a special cutter with parallel kni , es giving a section of about 5 m thickness . 
 the conventional method of propagation of the walker carcinoma consists of the insertion of small pieces of tumour ( approximately 7 x 5 x 5 mm . ) under the skin of rats . 
although factors such as age , sex , weight , etc . , and general condition may account for some of this variation , it is probably mainly an expression of genotypic differences present in the colony from which the rats used in these experiments were drawn . 
 with our present routine for breeding and selection of animals for tumour implantation , when the tumours of a group of ten rats are measured 8 days after implantation the measurements show a distribution of i or 2 large tumours , about 5 medium - sized tumours and the rest considerably smaller . 
that this is largely the result of genetical variation is shown by comparison of the growth rates of tumours in litter mates and by implantation of similar sized pieces of tumour in both flanks of the same animal . 
the animals were divided into 4 groups , each group consisting of 4 litter mates , 2 male and 2 female ; they were killed 14 days after implantation and the weights of their tumours recorded . 
it is seen that the 3 most rapidly growing and largest tumours ( over 35 g . ) all occurred in group 1 , whilst in group 3 none of the tumours grew well . 
the growth rates of both tum , ours were remarkably similar , and if a tumour implant in one flank failed to grow the corresponding tumour in the opposite flank also failed . 
the average daily weight increase of rat.s maintained on our 20 per cent protein diet is about 3 to 4 : g . , whilst that of those maintained on our 5 per cent protein diet is less than 05 g . 
 weight of tumours 14 : days after implantation taken from 85 animals maintained on the 20 per cent protein diet was 24 : g . , whilst that of 86 animals maintained on the 5 per cent protein diet was 20 g . 
thus the ratio between the tumour weight.s on 20 and on 5 per cent protein diet is 12 , and the average growth rate of tumours is only about 20 per cent higher in anima1s maintained on high prot.ein than in those receiving a low protein diet . 
la.merton for comparison of the histology of tumour grafts in animals maintained on high and low protiein diets transverse sections were made through the tumour implants and their surrounding tissue 4 , 7 , 8 , 10 and 15 days after implantation . 
 it was found that 4 days after grafting the int , ensity of the inflammatory reaction as estimated by the number of cells of the reticulo - endothelium accumulated around the graft was great.er in rats maintained on the 5 per cent than in those on the 20 per cent prot , ein diet . 
on the other hand , the capsule of connective tissue was much more distinct in rats on the 20 per cent than on the 5 per cent prot , ein diet . 
the boundary of the tumour is well defined in rats on the 20 per cent prot , ein diei , the migration of tumour cells is negligible and the tumour grows by the very high rat.a of cell proliferation at the periphery of the tumour parenchyma . 
on the 5 per cent prot , ein diet , how ever , there is no definit , e tumour boundary and many isolated tumour cells can be seen scatt , ered amongst the loose , primitive network of connective tissue , which included in this " incomplet , e " capsule are also many surrounds the tumour . 
 during the early stages of the " take " and growth of the implanted tumours the reactions of the surrounding tissues may be very complex and int , erpretation of the exact course of events is difficult , but in older tumours the histological features of the process are much more clearly defined . 
the cell - elements ofthe capsule undergo progressive : fibrous differentiation , which can be clearly seen in high prot , ein diet rats with 10 to 15 - days - old tumours . 
the : fibrous nature of the capsule is much less obvious in animals maintained on a low prot , ein diet , and its clear demarcation is obscured by the migrating tumour cells . 
 it should be emphasized that on both diets there may be a considerable variation in the " take " of the graft and in the spread of the implanted tumour in the " host , and instances have occasionally been encount , ered in which the tumour graft took and grew more rapidly in animals maintained on low than in those fed on high prot , ein diet . 
such cases were , however , rare , and the effect of the diet is so marked that the differences in capsule - formation and organization between animals maintained on high and low prot , eins diets can easily be det , ected in most cases . 
 in the study of the reaction of the host rat to the implantation of tumour grafts the fact that , the tumour graft is itself growing may very often obscure the observations of the organization of the newly formed connective tissue capsule . 
 it was felt that the introduction into the subcutaneous tissues of a " passive " non - growing hetierologous tissue fragment such as muscle inst , ead of the " active " tumour fragment , would make it easier to analyse the influence of environmental factors , etc . , on the host reaction . 
 pieces of muscle of about the sam~ size as the usual tumour implants , taken from the leg of a second rat were implanted under the skin of the host animal . 
 305 in the mbsequent reaction to this muscle implant two phases could be clearly distinguished : ( a ) the inflammatory tissue response , ( b ) the formation of a capsule around the implant . 
 the in : oammatory reaction ( a ) is intense 3 days after implantation in rats receiving either the 20 per cent or 5 per cent protein diet , but in those main tained on the 20 per cent protein diet it rapidly subsides and has almost dis appeared 5 days after grafting . 
 the 5 per cent protein diet rats , however , still show persistence of the inflam matory reaction at a time when the capsule is already well developed in 20 per cent protein diet animals . 
it appears that the prolonged persistence of the inflammatory reaction around the implant is related to the absence of the connective tissue capsu1e , the development of which is impaired or delayed in animals maintained on a diet deficient in prote the evidence derived from the histological investigation of muscle implants thus supports the conclusion reached from the tumour implant experiments that while maintenence of th~ animals on a low protein diet has no deleterious influence on the inflammatory reaction to the implant , it does interfere to a very large extent with the organization and development of the connective tissue capsule . 
 a quantitative analysis of radiation - induced injuries in cells of the walker carcinoma is difficult owing to the large number of chromosomes ( 2n = 40 ) in the relatively small tumour cell . 
 injuries to the chromosomes can be detected only in ana - telc : , phase during which the lagging of ' " acentric " chromosome fragments ( segments without the centromere ) can be seen . 
13. - dividing tumour cell in late anaphase with three acentric fragments , one lying out of focus , 6 hours after 300 r , 5 per cent protein diet . 
27. - the same tumour ( l . ) , showing the active tumour cell island which lies beyond the periphery of the tumour which occupies the bottom right hand corner . 
28. - the same tumour ( l . ) at a higher magnification to show that tumour cells which were scattered in the loose connective tissue capsule form foci of renewed activity after irradiation . 
 a comparison has been made of the frequency of tumour cells with various cytological abnormalities , such as chromosome fragments , bridges and micro nuclei at different times after irradiation of a six - day old walker tumour in rats kept on high and low protein diets . 
the data obtained ( table i ) do not represent the full extent of the radiation injury resulting from any particular treat ment , since an unknown proportion of injuries undergo restitution before the cytological examination is made and are thus lost for analysis . 
moreover , the acentric chromosome fragments can only be seen when they lie freely in the cytoplasm , and consequently those fragments which are mixed up with the normal chromosomes and moved to the pole escape detection . 
a similar difficulty arises when we attempt to estimate the true extent of the radiation injury to cells by the number of micro nuclei , since some of the chromosome fragments may be included in the daughter nuclei and some of the micronuclei can contain more than one fragment . 
on the assumption , however , that the chance for such events is the same in all the samples , it is justifiable to use the quantitative differences shown by the data as a basis for comparing differences in cell behaviour which may be induced by maintaining the animals on different diets . 
 the data of table i shows that the greatest amount of injury was found in tumour samples taken 12 hours after irradiation , suggesting that these samples contained cells which were , at the time of irradiation in the most sensitive period of the mitotic cycje . 
it is inferred on experimental evidence , derived from x - rayed pollen grains and root - tips cells ( darlington and lacour , 1945 ) that the most sensitive period coincides with the duplication of the chromosome filament , which process marks the beginning of prophase . 
since the injury in the cells is at a maximum at 12 hours after both 100 and 300 r , we may conclude that the duration of mitotic suppression does not differ over this range of dosage . 
 the number of cells with chromosome injuries and with micronuclei was found to be already quite high in the 6 hours ' sample , suggesting that the time interval between treatment and the first appearance of chromosome fragments may be shorter than 6 hours . 
therefore some rats were killed 4 hours after treatment , but although both chromosome fragments and bridges were observed in this early sample , their frequency could not be estimated because the so - called " physiological " effects of radiation grossly interfered with the analysis ( mar quardt , 1938 ; koller , 1943 )  . 
l. - uierton the number of resting cells with micronuclei increases up to 24 hours after treatment , but at 72 hours such cells have almost disappeared from the tumour . 
 comparison of the results obtained at different times after irradiation with both 100 rand 300 r ( table i ) , in all samples except those taken after 12 hours , shows no significant difference in the number of cells injured or the number of chromosome fragments per cell , between animals maintained on high or low protein diets . 
 the inconsistent behaviour observed in the 12 - hour samples may be related to the fact that all the mitotic cells in this sample represent those cells which were in their most sensitive stat , e when irradiated . 
it was , therefore , felt that the results obtained with the 12 - hour samples could not be relied upon and should be disregarded in view of the consistent results obtained with all the other samples . 
it is thus possible to compare the length of the inter - mitotic period on the two diets in order to find whether the duration of the mitotic cvcle is altered bv the diet or not . 
 the greatest amount of injury are those which at the time of irradiation were at in the x - rayed \\ ' alker carcinoma the the beginning of prophase of mitosis . 
 greatest amount of injury is found in the - 12 - hour sample and consequently the duration of mitosis from prophase to the end of telophase cannot be longer than 12 hours ; the cycle of the second division ( from prophase to telophase ) would occupy another 12 hours , thus lea , ing 20 hours for the duration of the inter mitotic period , if an allowance of 4 hours is made for the time lost by the radiation induced mitotic suppression . 
thus we may conclude that the active cells in the \\ ' alker carcinoma have a mitotic cycle of the order of 32 hours ( 20 hours spent in resting and 12 hours in mit - 0sis ) : this agrees with data obtained in similar experi ments in which irradiation is replaced by radiomimetic chemicals , such as the nitrogen mustards ( koller , unpublished communication )  . 
 from the observations of the frequency of cells in second mitosis 48 and 72 hours after irradiation with 100 or 300 r there was no indication that diet affects the duration of the mitotic cycle . 
at 6 days after the radiation treatment tissue fibrosis was much more developed , and the connective tissue capsule could be clearly distinguished from the actual tumour , since no tumour cells were present in this capsule zone . 
 it does , however , contain islands of active tumour cells , which , since the growth - inhibiting effect of a single dose of 1000 r is usually only temporary , no doubt represent centres from which the renewed growth of the tumour develops . 
 in the animals receiving the 5 per cent protein diet , the loosely organized con nective tissue capsule , 3 days after 1000 r , was not undergoing fibrosis to the same extent as the capsule in the 20 per cent protein diet animals . 
 six days after irradiation the tumour boundary which was previously very ill defi.ned , becomes more distinct , presumably owing to the fact that some of those tumour cells which were scattered in the connective tissue network and have survived the radiation treatment , have now undergone proliferation and brought about the formation of a new continuous tumour parenchyma . 
 w ' hile a single dose of 1000 r may cause considerable inhibition of growth of the walker rat carcinoma , and the inhibition may last for several days , complete regression of the tumour rarely occurs . 
the experiments with the single dose have shown , however , that at least in the 20 per cent protein diet animals , whilst the extent and rate of histological changes induced in the capsule or tumour bed is very favourable , it is insufficient to enable the animal to effect a cure . 
it is obvious , therefore , that the amount of radiation injury in the tumour cells must be increased without at the same time adversely affecting the favourable tissue response . 
it was therefore decided to increase the dose to 4000 r , which is of the order employed in the radiotherapy of human cancer , and a suitable method of fractionation for applying this dose was devised based on observations of the response of tumour capsule and tumour bed obtained in the single dose experi ments . 
the method employed can be illustrated as follows : 1000 r 500 r , 500 r 500 r , 500 r 500 r 250 r 250 r , in which represents two consecutive days on which no treatment was given . 
lamerton for the histological investigations 15 rats were treated with this fractionated dose , 7 of them being maintained on the 20 per cent and 8 on the 5 per cent protein diet . 
 two rats of each diet group h 1 , h ! ( high protein ) and l 1 , l 2 ( low protein ) were selected for histological examination ; of the remaining rats 4 out of the 5 maindied " 's e .. 
h 1 and h , indicate animals maintained on high protein diet and li and l , animals maintained on low protein diet at the time they were killed for histological examination . 
from similar results obtained in previous experiments ( elson and lamerton , 1949 ) and from a com parison of the growth curves of the tumours selected for histological investigation with those of the remainder of the groups shown in fig . 
22 it is reasonable to suppose that animals h 1 and h 2 on the high protein diet would have been cured , whereas li and l 2 maintained on the low protein diet would not . 
those of the high protein diet animals ( h1 and hz ) , however , had grown to a considerable maximum size and were then regressing , whilst those of the low protein diet animals ( l1 and lz ) had shown earlier retardation of growth , but were beginning to assume a more rapid growth rate . 
the animals h 1 and li had received a total dose of 3000 r ( in 5 fractions ) , whereas hz and lz had received the foll total dose of 4000 r ( in 8 fractions )  . 
 in assessing the response of tumours to radiation two effects must be con sidered : ( i ) the initial inhibition of tumour growth and ( 2 ) the elimination of the inhibited tumour and cure of the animal . 
in work with the walker rat carcinoma 256 it has previously been shown that the diet of the animal has a definite influence on each of these processes ( elson and lamerton , 1949 )  . 
 dealing first with process ( i ) , it has been shown that maintenance of the animal on a low protein diet favours the immediate growth inhibitory response of the implanted walker rat carcinoma 256 to radiation . 
it might be thought that this diet effect is related to an increased sensitivity of dividing tumour ce ] ls to radiation induced in some manner by the poor protein diet . 
furthermore , we do not feel that such differences as have been revealed in the course of the histological investigation of the tumour bed reported here offer any very satisfactory explanation of the greater immediat.e tumour response in the low protein diet animals , and we are inclined to believe that some more fundamental biochemical relation is involved . 
lamerton investigations with carcinogenic chemicals have suggest , ed that these sub stances may inhibit cellular growth by interfering with the normal processes of protein synthesis ( elson and warren , 1947 ; elson , 1949 )  . 
h this is the case it might be expected that animals in which the full capacity for protein synthesis is already restricted by a deficient protein diet would probably show an immediate growth inhibitory response of both animal and tumour to treatment . 
with ample protein to furnish more than suffi cient amino acid " building blocks ' for the full efficiency of protein synthesis would not be expected to show such an immediate reaction . 
it is not unlikely that x - radiation may act in a similar way and in order to throw more light on this problem the protein metabolism of irradiated animals maintained . 
 of the walker rat carcinoma a high protein diet has been shown to be of great assistance to the animal in the process of elimination of the inhibited tumour and the replacement of the tumour area by healthy tissue . 
that in the case of the implanted walker rat carcinoma 256 the main effect of the high protein diet is to ensure the development of a well organized . , extensive capsule of connective tissue around the growing tumour . 
response to radiation treat ment the ill - defined tissue capsule zone does not undergo fibrosis in the same way as does the firm capsule of the high protein diet animals . 
altho~h its main mass usually becomes necrotic , new tumour growth often develops in the incomplete capsule zone , from cells which have escaped the lethal action of the radiation . 
on a diet deficient in prote the effect of addition to the low protein diet of substances such as amino acids , particularly cystine and methionine , amino sugars , etc . , which may conceivably be involved . 
 313 taking into consideration the connective tissue reaction , devised treatment methods for epitheliomata , in which the total dose is of the order of 3000 r , far below the so - called " standard tumour lethal dose . " also it has been shown by jolles and koller ( 1950 ) that the tumour bed reaction can be influenced by a method of fractionation of the dose in space as well as time to give favourable therapeutic resu.jts. 
 the present animal experiments have shown that the character and organization of the tumour bed can be influenced by nutritional factors , and suggest that suitable regulation of nutritional conditions , such as , for instance , supplying a high protein diet , or supplementing the diet by protein hydrolysates , etc . , and by increasing protein anabolism by hormone administration , etc . , may jead to improvements in clinical response . 
 the effect of the protein content of the diet on the establishment and growth of implants of the walker rat carcinoma 256 , and on the response of this tumour to x - radiation , has been investigated . 
 the nature and extent of the inflammatory reaction has been found to be similar in animals maintained on both high ( 20 per cent ) and low ( 5 per cent ) protein diets , but in the low protein diet animals it persists for a longer period after implantation owing to slower and less complete development of the connec tive tissue capsule . 
 in assessing the response of the tumour to radiation two effects have been considered , ( a ) the initial inhibition of tumour growth , which is favoured by a low protein diet , and ( b ) the elimination of the inhibited tumour which is favoured by a high protein diet . 
 histological investigation has suggested that the favourable effect of a high protein diet on the elimination of the tumour is related to the development of the well organized capsule of connective tissue around the growing tumour . 
radia tion treatment , besides inhibiting division of the tumour cells , also increases the fibrosis in this capsu.je , which aids in the eventual elimination of the damaged tumour . 
 we wish to express our thanks for grants supporting this investigation from the british empire cancer campaign , the anna fuller fund , the jane coffin childs memorial fund for medical research and the u.s. 
of 9 : 10 - dimethyl - 1 : 2 - benzanthracene into the flank and below a nipple in strain street mice ( rask - nielsen , 1948 )  . in both sites the injection induced only spindle - cell sarcomas and , in a few instances , squamous - cell carcinomas , but no mammary carcinomas . 
the ratio of cases of isolated thymic tumours to cases of generalized leukaemia was found to be 1 : 1 following injection into the flank and 1 : 5 following injection into the mammary region . 
of 9 : 10 - dimethyl - 1 : 2 - benzanthracene were performed . the leukaemic manifestations induced have been described in detail in a recent publication ( rask - nielsen , 1949 ) and the local tumours will be dealt with in the present paper . for the purpose of ascertaining whether the failing development of mammary carcinoma might be a phenomenon specific for injection of 9 : 10 - dimethyl - 1 : 2benzanthracene , mice of strain street were injected with 05 mg . 
of a mixture of the same hard and liquid paraffin as used in the analogous experiments with 9 : 10 - dimethyl - 1 : 2 - benzanthracene ( rask - nielsen , 1948 , 1949 )  . 
an equal number of females and males were injected into the flank , but only female mice were injected into the mammary region , except in a few 9 : 10 - dimethyl - 1 : 2benzanthracene experiments , where an equal number of each sex was used . more than half the experiments were carried out with litter mates , one half of each litter being left untreated as a control group . 
rask - nielsen sarcomas of a varying degree of differentiation ; a few were polymorphocellular , and a few rhabdomvosarcomas occurred . in the squamous - cell carcinomas marked cornification was a frequent finding ; no microscopic changes indicated that the growths might have originated in mammary tissue . 
however , the growths were considered to be most probably spontaneous and were , therefore , not included in table iii . following injection in the flank , spindle - cell sarcomas were induced by benzpyrene , dibenzanthracene , methylcholanthrene and 9 : 10 - dimethyl - 1 : 2 - benzanthracene in 27 per cent , 50 per cent , 39 per cent and 12 per cent of the effective total of mice respectively . squamous - cell carcinomas were observed in the same experiment in 6 per cent , 7 per cent and 0 - 8 per cent following the injection of benzpyrene , methylcholanthrene and 9 : 10 - dimethyl - 1 : 2 - benzanthracene respectivelv . 
as regards the application into the mammary region , spindle - cell sarcomas were observed following injection of benzpvrene , dibenzanthracene , methvlcholanthrene , and 9 : 10 - dimethyl - 1 : 2 - benzanthracene in 4 per cent , 22 per cent , in this experiment , squamous - cell 37 per cent and 8 - 3 per cent respectively . carcinoma was observed in 2 per cent , 2 per cent , 22 per cent and 2 - 4 per cent respectively . the effective total of experimental mice is the number of mice living to be as old as the youngest tumour - bearing mouse , namely 4 months . the minimum , maximum and average latent period , the interval from the injection until death , is presented in table iv . 
the higher carcinogenicity of methylcholanthrene than of the other hydrocarbons apparent from the present experiments accords with previous findings ( greenstein , it accords also with the recent observation ( rask - nielsen , 1950a ) that 1947 )  . subcutaneous application of 0 - 02 mg . 
of 9 : 10 - dimethyl - 1 : 2 - benzanthracene was followed by leukaemic manifestations in 20 - 4 per cent with an average latent period of 20 to 25 weeks ( rask - nielsen , 1949 ) , injection of dibenzanthracene and methylocholanthrene had no accelerating effect on the development of leukamia , and the doubtful , extremely faint acceleration observed following injection of benzpyrene affected at any rate only the incidence ; not the latent period . the absence of leukaemic lesions following injection of the three last - mentioned hydrocarbons is all the more remarkable , as the development of leukaemia has been found to be accelerated following painting with benzyprene in dilute brown mice ( morton and mider , 1941 ) and in strain f mice ( kirschbaum and strong , 1942 ) following painting with dibenzanthracene , without , however , decreasing the latent period in strain f mice and with methylcholanthrene in strain f mice ( kirschbaum and strong , 1942 ) , in dilute brown mice ( mider and morton , 1939b ; morton and mider , 1941 ; lefevre , 1945 ) , in strains rf , and rf / ak ( mcendy , boon and furth , 1942 ) , in c3h mice ( kirschbaum , strong and gardner , 1940 ; morton and mider , 1941 ) , and in old and new buffalo mice painting with 9 : 10 - dimethyl - 1 : 2 - benzanthracene ( morton and mider , 1941 )  . also accelerated the development of leukaemia in aka mice ( engelbreth - holm and lefevre , 1941 ; lefevre , 1945 ) , in dilute brown mice ( engelbreth - holm and lefevre , 1941 ; law , 1941 ) , and in swiss mice ( law , 1941 )  . 
into the lung ( rask - nielsen , 1950a ) , and by the induction of only microscopically visible adenomas in mice , 4 - 11 months of age , injected with 05 mg . 
of benzpyrene , dibenzanthracene , methylcholanthrene , and 9 : 10 - dimethyl - 1 : 2 - benzanthracene into the flank of strain street mice induced local spindle - cell sarcoma in 27 per cent , 50 per cent , 39 per cent and 12 per cent ; and squamous - cell carcinoma in 6 per cent , 0 per cent , 7 per cent and 0 - 8 per cent respectively . injection of the same hydrocarbons into the mammary region induced spindle - cell sarcoma in 4 per cent , 22 per cent , 37 per cent and 8 ' 3 per cent and squamous - cell carcinoma in 2 per cent , 2 per cent , 22 per cent and 2 - 4 per cent respectively . local mammary carcinoma was not induced . leukaemia developed following subcutaneous injection into the flank and into 132 r . 
rask - nielsen the mammary region of benzpyrene , dibenzanthracene , methylcholanthrene and 9 : 10 - dimethyl - 1 : 2 - benzanthracene in 6 - 7 per cent , 1 - 2 per cent , 0 per cent and 20a4 per cent respectively . 
gunz. * from the department of radiotherapeutics , university of cambridqe . received for publication june 11 , 1949 . in previous publications ( gunz , 1948a ; b ) a technique of cultivating human leukaemic leucocytes in vitro was described , and the quantitative and cytological findings in untreated cultures made by it were analysed . 
he did not , however , determine the mitotic rates in his cultures , because of the small number of dividing cells present . osgood 's findings may be compared with those of schrek ( 1946a , b ) , who irradiated suspensions of thymus and spleen lymphocytes , and of bone marrow cells ( rabbit ) , and of normal and leukaemic human blood cells in " phosphate ringer solution " with and without homologous serum , with doses of x - rays of using as a criterion of survival a failure of cells to stain with a 20 - 4000 r . 1 : 1000 solution of eosin in tyrode solution , schrek found exposure to x - rays to cause a decreased survival time vitro in lymphocytes from the thymus , normal blood and four of five bloods from patients with chronic lymphatic leukaemia . such an effect was noted after a dose of 50 r , and increased with an increase in the dose up to 1000 r , remaining approximately constant at higher doses . 
the author , discussing possible reasons for this lack of effect , attributes it to an absence of dividing myeloid cells in his preparations , x - rays leaving resting myeloid cells unaffected . interference with mitosis , as a result of x - irradiation , was definitely demonstrated by rachmilewitz , rosin , goldhaber and doljanski ( 1945 , 1947 ) in experiments with fragments of explanted rabbit bone marrow . following the application of 250 - 1000 r there was a depression of mitosis lasting for two to four hours , with subsequent recovery . 
many mitotic abnormalities were caused , and the marrow became hypocelhular , or almost completely aplastic , according to the size of the dose given . the observations in the literature may be summarized by stating that x - rays have been found to damage blood and haemopoietic cells , especially lymphocytes and all classes of immature cells , when applied directly in vitro , and that disturbances of mitosis appear to be among the earliest signs of injury in cells so exposed . 
two patients had not received any x - ray treatment when first seen ; the others had had various amounts of treatment in the past . the technique of culture was that previously described ( gunz , 1948a )  . brief , it consisted of the distribution of small equal quantities of leukaemic leucocytes , suspended in the culture medium , over a series of culture tubes , and the withdrawal of some of the tubes for counting after varying periods of incubaat these times total and differential counts were made , and the total tion . number and phase distribution of mitoses determined . the culture medium used consisted either of 50 per cent dried , reconstituted human plasma , with the addition of 5 per cent rat embryo extract , or of 50 per cent fresh homologous human serum , both diluted in ringer solution . the duration of experiments was two days , and counts were done at 8 , 24 and 48 hours , unless otherwise stated . the technique of irradiation was as follows : after the cultures had been set up , they were incubated at 370 for about 15 minutes , to give them time to reach body temperature . in some experiments some of the tubes were also incubated for longer periods before irradiation . 
the tubes were exposed at two fixed distances ( approximately 21 and 81 cfrom the target ) , the dose rates , measured in air , at these points being 622 and 62r / nirespectively . 
the actual dose rate to which cells were exposed depended on three factors : the dose rate in air , the absorption and scatter due to the glass , and the absorption and scatter due to the fluid . 
the dose rate in air was measured by means of a victoreen dose meter in the normal way ; it was found that when the chamber of the dose meter was inserted into empty culture tubes , the average decrease in the dose rate was 6 - 5 per cent . this enabled a correction to be made for the glass of the tubes . 
the volume of the liquid being only of the order of 1 or 2 ml . , the correction for the absorption and scatter due to it would be less than 5 per cent , and was ignored in estimating the dose rate . 
the results of irradiation with 100 , 1000 , and 10 , 000 r are set out in tables i - vi . if the contents of tables i - vi be summarized and analysed , we obtain the following findings : immature cell8 . with a dose of 100 r the ratio , at 8 hours , of immature cells in irradiated to in five experiments it those in control cultures varied from 65 - 113 per cent . was greater , and in seven experiments less than 100 per cent . 
a significance in the analyses of immature cell counts test ( student 's t - test ) was employed . percentages were first converted to logarithms , in order to obtain a more normal distribution of the series . 
the actual formula used was t - s - - = where x is the arithmetic mean of the series , 8x its standard error , and m = log10100 = 2 . this gave a value of t = 0 * 97 , which is not significant . at 24 hours the ratio varied from 71 to 111 per cent . in four experiments it was greater , in one equal to , and in seven less than 100 per cent . 
the difference is not significant . at 48 hours the ratio varied from 48 to 112 per cent . in one experiment it was greater , and in ten experiments less than 100 per cent . 
from this t = 5 - 25 , and p is less than 0 * 001 . there was , therefore , a highly significant reduction in the number of immature cells in cultures 48 hours after irradiation with 100 r . with a dose of 1000 r the ratio , at 8 hours , of immature cells in irradiated to those in control cultures varied from 76 to 111 per cent . in three experiments it was greater , and in three experiments less than 100 per cent . 
no analysis was carried out for the 48 - hour values . with a dose of 1000 r the ratio was less than 100 per cent in all experiments , and at all times . 
no mitoses were present in irradiated cultures in 4 out of 7 experiments at 8 hours , in 5 out of 8 experiments at 24 hours , and in 4 out of 6 experiments at 48 hours . 
the results with 10 , 000 r were similar . it must be emphasized that these experiments were performed with varying culture media , and that the cells in some of them came from untreated , and in others from previously treated patients . this explains many of the numerical differences between individual experiments , particularly in the mitotic counts . however , in spite of the non - homogeneity of the material , the results show clearly the following points : irradiation of cultures of leucocytes from chronic myeloid leukaemias with 100 r produced a significant reduction in the number of dividing cells for at mitosis then recovered , and did not differ significantly from least 8 hours . controls at 24 hours or later . 
1. - in vitro irradiation of leucocytes ( chronic myeloid leukaemia )  . irradiation with 1000 and 10 , 000 r produced a more marked depression of mitosis at 8 hours , and this did not recover subsequently . 
at that stage there were practically no dividing cells present , and the vast majority of all immature leucocytes in the cultures were truly " resting . " only after the lapse of several hours could any considerable numbers of them be expected to be approaching or entering it is , however , well known that chromosomes are particularly liable mitosis . to damage from radiation in the late pre - mitotic or early mitotic stages ( lea , 1946 )  . 
the following experiments were made in an attempt to irradiate cells at such a moment of increased sensitivity to x - rays , and to determine whether chromosome abnormalities could thereby be induced . - ~~~~ _ ~~~ ~ ~~~~1 ' ~~~~~~~~~~~~~~~~~~~~~ 338 f . 
gunz effect of varying the time of irradiation . in two experiments different groups of tubes were irradiated with 100 r at 0 , 2 and 4 hours from the time of setting up the cultures . 
3 , from which it is clear that no significant difference in the mitotic rate or the number of immature cells was produced as a result of varying the time at which the cultures were irradiated by up to 4 hours ; neither was the number of mitotic abnormalities in stained films altered . in another group of three experiments cultures were irradiated with 100 or 1000 r 7 hours after they had been set up , and counts and filmns were made at it was known that at 7 hours , appreciable frequent intervals after irradiation . numbers of cells would be entering mitosis . 
4. - effect of x - rays on leukaemic leucoeytes in vitro . irradiation 7 hours after explanta ' tion . that they only appeared when cultures were irradiated at a time when many mitoses were present , suggests that they arose from dividing cells , and it is likely that they originated in early prophase . 
15. it would seem that the findings at 8 and 24 hours are most likely to reflect the character of the cells themselves ; later on , artefacts due to the composition of the medium become superadded . taking only the 8 - hour counts into consideration , there was a good correlation between the patient 's blood count and the number of mitoses in cultures . 
 ( 1945 ) for normal rabbit bone marrow , but these authors worked with a different culture technique . no evidence was found that 100 r produced different results when given at this is in agreement with the finding of lasnitzki ( 1946 ) different dose rates . that in cultures of chick fibroblasts the initial inhibition of mitosis was independent of the dose rate ( 9.3 - 103 r / min . ) for doses up to 100 r . 
the explanation in the present experiments is probably that with both the dose rates used ( 62 and 622 r / min . ) , the time of irradiation - 10 or 97 seconds - was short compared to the overall time required for mitosis ( 35 - 60 minutes )  . no significant increase in the number of mitotic abnormalities was found to follow irradiation of cultures . 
the explanation for this is not clear ; it is , however , possible that technical difficulties prevented the recognition of finer structural alterations , like chromosome breaks . when cultures were irradiated 7 hours after their start , it was found that many cells in mitosis at the moment of irradiation completed their division . this agrees with the observations of many other workers , such as strangeways and oakley ( 1923 ) , canti and donaldson ( 1926 ) , kemp and juul ( 1930 ) , and furthermore , with 1000 r , a new kind of breaking - down cell love ( 1931 )  . appeared 3 hours after irradiation , and rapidly increased in numbers . 
assuming , for instance , that a similar process took place in the irradiated spleen , we should expect a definite diminution in the number of new leukaemic cells produced in that organ ; moreover , any further similar treatments would repeat , and possibly augment the effect . 
one could therefore picture the manner in which x - rays cause the enlarged spleen to involute . it is , however , more difficult to account by this means for the fall in circulating leucocytes which is such a prominent feature of the response to local radiotherapy in leukaemia , or for the involution of distant leukaemic lesions which had not been directly irradiated ( for examples , see piney and riach , 1932 )  . 
some light may be thrown on the latter problem by the findings in section b of the experimental part . here it was seen that leukaemic leucocytes , taken from the peripheral blood , showed a dininished power of proliferation in vitro after therapeutic irradiation of the patient , and that this effect was reversed as the clinical effects of treatment began to wear off . it thus seemed as if the biological make - up of the cells had been changed by the treatment . 
koller these few instances are selected from many clinical and pathological findings to illustrate that the environment of tumours is of great importance , and must be taken into consideration when devising improvements in radiotherapeutic procedures . it may prove to be important not only in surgical and radiotherapeutic practice , but also as regards theoretical problems of oncology . 
the classical study of willis ( 1948 ) emphasizes the fact that potentially neoplastic tissue zone is much greater than the size of the initially appearing tumour would indicate , and brings forward evidence which lends support to the field - theory of the origin of cancer . 
thus the possibility that the " recurrence " of a tumour after surgical treatment may represent an entirely new cancerous change in a predisposed tissue , and is not due to the incomplete removal of the malignant growth , has to be considered . the aim of post - operative x - ray therapy is not only to destroy viable tumour cells which might have been left in situ , but also to reduce the chance of the occurrence of an entirely new malignant growth by producing gross alterations in the histological architecture of particular regions , showing a proneness to neoplasia , and to affect the substrate in which any malignant cell left in situ might progress and defeat the treatment . 
the importance of connective tissue and tumour - environment has also been stressed by vernoni ( 1948 ) , whose views on the connective - tissue role in carcinogenesis find support in recent research work . a better understanding of the behaviour of the connective tissue of the tumour - bed and stroma during and after radiation treatment leads to the revision of fundamental principles on which the rationale of treatment is based . 
the practical application of the latter in the radiotherapy of accessible tumours finds expression in a new technique , described as the " sieve or alternating chess - board method " ( jolles , 1949a )  . for the past two years this method has been tested on a small number of patients , yet the radiation response in these cases has shown characteristics which hitherto have not been seen in tumours irradiated by the usual conventional methods . 
the purpose of the present paper is to describe these phenomena , and to discuss their bearings on the destruction of malignant growth . the biological basis of radiation effects in tumours . the radiation - induced changes in the cellular and stromal part of the tumour may be summarized briefly as follows : the cellular injuries consist of diverse nuclear or chromosome injuries , as well as disturbances in cytoplasmic enzyme activity . 
the chromosome injuries manifest themselves during mitosis which follows treatment , and lead to cell death by the breakdown of the mitotic mechanisthe amount of chromosome injury and the number of injured radiation - induced enzyme disturcells show a direct dependence on the dose . bances , the extent of which also depends on the dose , either lead to reversible injuries , e.g. 
the recent investigations by bloom and jacobson ( 1948 ) , anderson ( 1949 ) , and warren and dixon ( 1949 ) , brought forward further evidence to show that radiation does not produce cell differentiation . in the stroma and tumour - bed two chief events can be distinguished : hypertrophy of the connective - tissue elements , and a reaction closely resembling inflammation . 
the first phenomenon mainly consists in an excessive deposition of intercellular collagen fibres ( fibrosis ) ; the second is represented by an invasion of lymphocytes , polymorphonuclear leucocytes and macrophages into the fine these changes network of connective tissue forming the stroma and tumour - bed . in the normal tissues naturally react on tumour cells and tissue ; the effects in the latter thus produced can be referred to , therefore , as " indirect radiation effects . " they must , however , be clearly distinguished from those " indirect " effects which are assumed to be brought about by injury to the blood supply ( desjardins , 1932 ; harvey , 1942 )  . failla , 1940 ; ellis , 1942 ; windeyer , 1942 ; the importance of the tumour - bed during treatment has been recognized by a great number of investigators ( russ , chambers and scott , 1921 ; kok and vorlaender , 1922 ; caspari , 1922 ; murphy , maisin and sturm , 1923 ; czepa , 1924 ; roussy , 1926 ; ewing , 1926 ; souttar , 1929 ; sugiura and cohen , 1939 ; jolles , 1946 , friedman , 1939 ; evidence was also obtained in various 1948 ; spear , 1946 ; windholz , 1947 )  . experiments which shows the influence of the tumour bed in the radiation response of tumours ( lasnitzki , 1947 ; elson and lamerton , 1949 ) , and it was found that by taking into consideration the " stroma - reaction " , treatment methods could be devised for individual tumours in which the total dose given did not exceed 2700 r ( koller and smithers , 1946 )  . 
in the interdependent tissue structures of tumour and tumour - bed , the different reactions ( cellular and intercellular , direct and indirect ) are knitted together and closely integrated . it was observed that the onset , degree and rate of these reactions vary greatly in different tumours . because the outcome of treatment depends on them , the total dose necessary for the destruction of tumours also varies within very wide limits ( 2700 r and this observation leads to the conclusion that on biological grounds the 9000 r )  . concept of a " standard tumour - lethal dose " is untenable . it is unnecessary , however , to stress that a minimal effective dose which is indispensable for the destruction of a tumour remains of paramount importance , and the enhancement of the effect of this minimal dose , by utilization of the indirect radiation effects , is here discussed . the various reactions induced by the radiation all contribute to the resolution and eventual disappearance of the tumour , therefore the reaction - system must be it is only by the analysis of all these factors and the study considered as a whole . of the behaviour of all tissue structures , that an explanation may be found either for the inadequate response , or for the recurrence of a particular tumour . 
the technique has been fully described in a previous communication ( jolles , 1949b )  . apart from the direct radiation transmitted through the " opaque " lead squares ( and which does not exceed 3 per cent with the thickness of lead used ) , the protected areas also receive a very small amount of scatter irradiation . latter is irregularly distributed , tailing off towards the centre of the protected this contingency must be taken into consideration when studying and areas . conmparing the radiation effects of the directly exposed and protected parts . biopsy specimens are taken simultaneously from the exposed and protected areas , preferably fronm the centre of these regions , and occasionally a third biopsy specimen is obtained by cutting across both the exposed and protected areas in order to follow the transitional stages of the histological changes due to radiation . the great variability of tumours in respect of the histological organization including the structure of stronia and tumour - bed , has to be kept in mind , and our observations were based only on biopsy material showing similarity in these respects . eighteen patients have been treated with the sieve technique , and the radiation reactions in the exposed and protected areas have been compared by the analysis of biopsy specimens . 
two sets of experiments were devised ; in the first instance only one sieve was used throughout the treatment . in the second set , two sieves a and b , were employed ; chess - board b differs from chess - board a in that the order of transparent and opaque squares is reversed . 
xi.48 : 4 days after start of treatment 28.xi.48 : 13 days after start of treatment 8.xii.48 : 23 days after start of treatment 16.xii.48 : 31 days after start of treatment 15 . 
the gland increased in size very slightly during treatment to the lesion on the temple . one month later the gland had grown to the size of a hazel - nut . 
lederman , the behaviour of over 450 accessible tumours has been analysed during and after treatment , and it was established that besides the injuries in dividing cells and the great increase in cytoplasmic volume of differentiating or maturing tumour cells , the most striking change occurs in the connective tissue of the stroma and tumour - bed , which may be described as tissue " fibrosis . " the connective tissue becomes more rigid in architecture , less flexible and adaptable for the development of the inflammatory reaction ( " coarse " tissue fibrosis )  . 
koller to state what is the primary cause of tissue changes in the protected areas ; i may be that the small amount of scatter radiation received in ten fractions o for that reasoi 40 r daily can produce such alterations in the connective tissue . further experiments are in progress to study the stromal effect of such a smal the experimental evidence so far available indicates that tissue reactioi dose . after a dose of this order is negligible ; if there is any , e.g. 
we are of the opinioi that the tissue changes in the protected regions are brought about by a complet mechanism in which , among others , a diffusible substance produced in the directli irradiated tissues might play a role . 
the absence of chromosome fragmentation is another proof that the alteration seen in the protected areas , such as the increase in the size of tumour cells , and the slight degree of fibrosis and inflammatory reaction , must be considered as an indirect reaction to radiation . our investigation has shown that the rate and degree of the indirect radiation reaction depends on the histological organization of stroma , and on the dose . on account of the differences in the histological architecture of tumours , the minimum effective dose varies from tumour to tumour . 
we found that in tumours in which the connective tissue is abundant the minimum effective dose is smaller than in tumours with scanty stroma . this is illustrated in case ii , in which the dosage used ( 2700 r actual dose in 35 days ) did not reach the critical minimum level necessary to produce a reaction of sufficient strength and efficiency in the scanty connective tissue of the " protected " tumour areas . 
on the other hand , the use of the sieve - technique has permitted the subsequent treatment of the residual tumour with a relatively high but adequate dose ( 4500 r actual dose in 18 days ) , because the histological texture of the skin , owing to the small size of the areas directly exposed to irradiation during the initial treatment has been left unimpaired , and could safely be subjected to such a high dose in the second treatment . the complexity of the various cellular and tissue interactions increases when further radiation is carried out through another " chess - board or sieve " in which the position of transparent and opaque areas is reversed . 
we found that while the connective tissue needs protection from excessive radiation , it requires a certain quantity of radiation and a specific distribution in time and space of this quantity in order to elaborate the radiationinduced reaction . 
the stroma and tumour - bed reactions , however , being too obvious to disregard completely , are referred to , but only as concomitant , " independent " events , which have no , or too little , bearing on the radiation response of the malignant growth itself . 
the most cursory histological study of tumours under radiation reveals the fact that the destruction of all malignant cells of the tumour parenchyma rarely , if ever , can it seems , therefore , be achieved by the " direct " action of a therapeutic dose . necessary and logical not only to admit the existence of an " indirect " effect , but to accept all the implications of the generic statement that the purpose of radiation is to kill a variable but significant proportion of active tumour cells , and at the same time to assist the local body defence system by inducing the various responses in the tumour - bed and stroma . it also follows that any view based on changes induced by radiation in some and not all tumour - tissue components is not comprehensive , and that the conclusions drawn from the study of cell behaviour alone are doubtful . 
on " cell count method " of glucksmann and co - workers that account the ( gluicksmann , 1941 , 1948 ; glucksmann and spear , 1945 ; gkicksmann and way , 1948 ) , which per se represents a contribution to radiobiological knowledge , is liable to be misleading when claims are being attached to it , such as that it provides an explanation for the variation of radiation response of tumours , predicts the outcome of treatment , and indicates the most appropriate treatment method , whether surgical or radiological , for a given case . the cell count method is biased by subjective selection and interpretation ; it disregards the great regional heterogeneity shown by tumours both in growth rate and histological organization , and the fact that a malignant growth is composed of tumour and stroma , which form one closely integrated unit . it is more than obvious that for that reason the application of the cell - count method to biopsy specimens taken after a " test dose " of radiation in order to decide whether a tumour be submitted for surgery or radiotherapy is of doubtful value ( glucksmann and way , 1948 )  . it has been stated by spear ( 1946 ) that " radiation affects any given cell of a complex tissue in at least two ways , first by direct action on the cell , and secondly by injuring neighbouring tissues , upon the healthy functioning of which the cell depends . " our investigation leads us to a similar conclusion that the direct cellular effects , e.g. 
fragmentation of chromosomes , represent only the initial phase , and that the most important factors responsible for the disorganization , regression and re - absorption of tumours during and after irradiation are the various , closely integrated and related reactions which are produced by the radiation in the connective tissue of tumour - bed and tumour stroma . the present investigation , in which the alternating chess - board method was employed , has shown the role of the connective - tissue reaction in the destruction of tumours and the way by which this reaction can be favourably influenced and modified . 
we have found that the fractionation of dose in time as well as in space , as carried out by the sieve , induces specific changes in cells and tissues . the intensity and rate of these changes affects the whole architecture of stroma and tumour - bed as well as that of the tumour . our investigation has also shown that when the sieve is used sufficient response can be produced in the local defence system of connective tissue , and can be maintained by a much smaller dose than usually employed . 
roller us in cases where the tumour is radio - resistant , either by its nature or by its environment , to deliver a higher total tumour - dose without the danger of destroyit is our intention to investigate the ing vital structures of the tumour - bed . potentialities of this new procedure by applying the sieve method for the treatment of radio - resistant and deep - seated tumours , especially metastatic deposits in lymph nodes . 
the tumour is divided into a number of " exposed " and " protected " areas , the former receiving direct , the latter receiving only a small amount of scatter and transmitted radiation . 
two chess - boards or sieves , which differ in the order of the transparent ( exposed ) and opaque ( protected ) squares , can be used during treatment . irradiation induces injuries in the tumour cells and alterations in the connective tissue of the tumour - bed and stroma . 
the radiation reaction in the connective tissue plays a very important role in the destruction and reabsorption of tumours . in tumours irradiated through the sieve it was observed that tissue - reaction is reduced in the directly " exposed " regions , but at the same time , fibrosis and inflammatory reactions are induced in the " protected " regions . 
no cellular injuries , due to direct radiation such as chromosome fragmentation , were seen in cells of the " protected " areas . by using two sieves which differ in the order of the " transparent " and " opaque " areas , the reaction of the tumour - bed and stroma in the presence of abundant connective tissue becomes uniform throughout the tumour , irrespective of the different doses which regions may have received during treatment . analysis of the radiation reaction in tumours treated by the sieve has shown that fractionation of dose in time as well as in space induces injuries and specific alterations in the irradiated area , the rate and type of which greatly enhances tumour destruction . the authors are indebted to the british empire cancer campaign for a grant for technical assistance , and thanks are also due to f . 
levvis. from the chemi8try department , university of adelaide . received for publication may 23 , 1952 . soon after the discovery of the carcinogenic activity of 1 : 2 : 5 : 6 - dibenzanthracene by cook , kennaway and colleagues , yoshida ( 1932 , 1933 , 1934 ) and sasaki and yoshida ( 1935 ) reported that the addition of o - aminoazotoluene to the food leads to the production of malignant liver tumours in rats . during the past two decades scores of other azocompounds have been tested for carcinogenic activity of this type and many have been shown to be active . for reviews see shear ( 1937 ) , kinosita ( 1937 ) , cook ( 1939 , 1943 , 1948 ) , cook and kennaway ( 1938 )  . these carcinogenic azocompounds form a very interesting group . 
unhke the polycyclic aromatic hydrocarbons , they do not , as a rule , produce tumours at the site of injection , but mostly affect the liver . furthermore , the induction of liver tumours with azocompounds is markedl influenced bv the dieb , and if a cc protective " diet is supplied , the production of tumours can be strongly inhibited or even entirely prevented . all the azocompounds are , of course , artificial synthetic substances ; and they are highly coloured ( mainly orange to red )  . 
some are used as textile dyestuffs , and certain members of the group are used for colouring foodstuffs . the most part such compounds are probably harmless ( cook , 1948 )  . 
most of the food - colouring matters which have been tested have failed to induce liver tumours in rats ; and in any case it is most unlikely that anyone could ever consume enough of an azocompound in food to have any deleterious effect . 
to some extent the compound is also carcinogenic towards the bladder ( yoshida , 1935 ) , and it is interesting that the deaminated compound , namely 2 : 3 ' - azotoluene ( iii ) , was found to be non - carcinogenic to the liver of the rat , but produced numerous papillomas in the bladder ( otsuka and nagao , 1936 )  . many derivatives of azobenzene , containing substituent aminoand methylgroups , were tested for carcinogenic activity by kinosita ( 1937 )  . 
none of these produced cancer of the liver except o - aminoazotoluene , its mono - acetyland diacetylderivatives , and an isomer of o - aminoazotoluene , namely 4 - dimethylaminoazobenzene ( iv )  . 
the latter compound , which is also known as p - dimethylaminoazobenzene , and as n , n - dimethyl - p - aminoazobenzene , was formerly used as a food - colouring matter under the name of " butter yellow "  . 
the effects of such structural alterations are described in a following section . most of the known carcinogenic azocompounds are derivatives of 4 - dimethylnevertheless , a 4 - aminoaminoazobenzene , or at least of 4 - aminoazobenzene . this was shown for example , by group is not essential for activity of this type . cook , hewett , kennaway and kennaway ( 1940 ) , who examined the action of azonaphthalenes on mice . 
manv liver tumours , mostly of the type of cholangioma , were obtained with 2 : 2 ' - azonaphthalene ( vi ) , and a few tumours were obtained with 1 : 1 ' - azonaphthalene ( vii )  . 
lewis the same relative potencies do not always apply in other laborat - ory animals . it is well known , for example , that 4 - dimethylaminoazobenzene is much less active in producing ' hver tumours in mice than it is in rats . 
cohen. from the experimental oncology laboratory , radiation therapy department , johannesburg general hospital . received for publication february 7 , 1953 . an apparent discrepancy exists between the multiplicity of experimental cancer cures which have appeared in the literature since homologous transmission of tumours was first demonstrated , and the difficulties encountered in the therapy of human cancer . 
among the basic reasons for this are differences in the hosttumour relationship which presents a range of characteristics , from the completely unstable heterologous relationship , where the host and tumour are of different strains , or even species , to the relatively stable situation , as with human between these two extremes are the many , now or other autogenous tumours . widely used , homologous tumours displaying varying degrees of stability . 
the facile experimental cure , or spontaneous regression of a tumour followed by absolute immunity to further implants , is invariably a feature of the relatively unstable host - tumour relationship , and the less stable this relationship the more since this effective the " cure , " be it mechanical , chemical or radiological . propitious situation rarely , if ever , occurs in humans bearing autogenous tumours , experimental investigation of an animal tumour can have at best only remote application to clinical cancer therapy unless it applies in the first instance to a stable host - tumour relationship . 
as a prerequisite to any such investigation , therefore , the following criteria for stability of the host - tumour relationship are considered indispensable : ( 1 ) there shall be unequivocal evidence of malignancy , characterised by progressive growth , invasion or metastasis , and death of the host . ( 2 ) the tumour shall be genetically compatible with the host , either having arisen spontaneously or been induced in the tissues of the animal being investigated , or be transmitted by implantation into a homologous animal closely related by inbreeding to the host in which the tumour first arose . ( 3 ) the tumour shall " take " in practically 100 per cent of recipients on irnplantation , and there shall be neither spontaneous regression nor regression induced by non - specific trauma or intoxication of the host . ( 4 ) on irradiation of the tumour in situ , the curative dose shall be of the same order of magnitude as that of human tumours ( 103 to 104 r )  . ( 5 ) removal or destruction of the tumour in situ shall not evoke absolute resistanoe to further implants of the same tumour . since both human and experimental arlimal tumours can be made to regress following exposure to ionising radiations , one of the more promising avenues of 232 a . 
cohen research would appear to be the investigation of all factors affecting the radioalthough earlier work in this field has formulated certain sensitivity of tumours . fundamental principles ( krebs , 1929 ; cramer , 1932 ; crabtree and cramer , 1934 ; sugiura , 1937 ; sugiura and cohen , 1939 ; goldfeder , 1942 ) , it has been largely limited by inaccurate dosage estimation or lack of suitable biologic it is now possible for the radiobiologist to combine precise quantitamaterials . tive radiotherapy with a judicious choice of experimental hosts and tumours ; and it was with this in mind that a study of the radiobiology of the c3h mouse mammary adenocarcinoma was undertaken . 
with no added filters ( the inherent filtration of the tube - head consisting of approximately 3 moil and 1 mbakelite in addition to the glass envelope ) , giving a half - value layer of 0 34 mcu . 
of 25 cthe dose rate was 500 r / m ( 3 per cent ) measured in air . dose rates were measured in air with a secondary standard ( victoreen ) dosemeter , and corrected for atmospheric density . 
when completely filled with tissue fragments this arrangement provides full back - scatter for the field used , and avoids any heterogeneity due to variations in density or atomic number such as would be encountered with a glass slide . 
the tumour is relatively radioresistant , requiring doses of the order of 6000 r in one treatment to produce a significant number of this is essentially similar to goldfeder 's findings ( 1951 )  . 
the discrepancy isprobably due to ionisation contributed by the glass slide in which her tumour fragments were irradiated , instead of the plastic used in this experiment . it is of interest to note that the effective dosage for some other strains of mouse mammary carcinomas irradiated prior to implantation are of a similar magnitude ( lawrence , horn and strong , 1937 ; reinhard , goltz and warner , 1952 )  . considering that their more heavily filtered radiation ( hvl 1 mcu . ) has a relative biological efficiency of about four - fifths of that used in this experiment , the reported effective dosage of 3600 r is virtually identical with our findings . the results of both types of irradiation are analysed graphically in fig . 
2 using the " probit " method , from which it can be deduced that , in the case of treatment in situ ( line a ) the ld50 is 5700 ( + 140 * ) r , and for attenuation prior to implantation ( line b ) the ld50 is 2850 ( i 50 * ) r . it will be noted that the two dosage - response lines are apparently parallel , their slopes corresponding in both cases to a coefficient of variation of about 9 per cent . attenuation of tumour homoplasts by x rays prior to implantation into animals was described as early as 1909 by haaland . 
on occasion , this procedure was shown to induce a state in which the recipient became resistant to challenge with unirradiated implants of the same tumour , although rarely were adequately inbred strains immunised against homologous carcinoma ( hauschka , 1952 )  . 
the conversion of the cohesive subcutaneous tumour forms into a homogenous suspension of free cells may be due either to a selective proliferation of a few round cells present in the solid tumour or to a gradual adaptation to their new environment of the original spindle or polymorphous cells typical of sarcomas . 
two mouse ascites tumours were used for the experiments , the s 37 sarcoma and the t 2146 tumour which originated as a benzpyrene induced epithelioma but has both these tumours were developed since undergone sarcomatous transformation . from the subcutaneous form by craigie at the imperial cancer research fund laboratories . the paper is divided into three parts : i . 
no tumour smears of cell suspensions were nodules were present in the abdominal cavity . obtained by withdrawing peritoneal fluid at daily intervals from 24 inoculated mice and staining either by the feulgen method or that of papanicolaou . 
the whole of the ascites fluid was withdrawn and centrifuged at low speed for 3 minutes , which caused the heavier tumour cells to sink down while leucocytes and erythrocytes , if present , formed a top layer which could easily be removed with the supernatant fluid . 
the culture medium consisted of equal parts of fowl plasma , ascites fluid and chick embryo extract to which one drop of either s 37 or t 2146 tumour cell suspension was added . 
the undulating movements of the peripheral cytoplasmic area become more rapid , spikes and blunt processes are pushed out and withdrawn in quick succession . finally the cells elongate and assume pear and then spindle shape . for several hours after incubation the process may be reversed and spindle cells are seen to return to the round shape . examination of the fixed and stained specimens confirms the observations made on the living cells and shows that the process of transformation continues for 24 hours after incubation . 
the latter may represent a differentiated form which can no longer proliferate actively in contrast to the free round elements from which they are derived . to decide this question cultures of ascites tumour cells were inoculated subcutaneously into mice before and after 24 hours ' incubation , i.e. , before and after establishment of the spindle forms . 
the clots were cut into strips 2 x 4 min size containing approximately 2000 cells , and these were inoculated subcutaneously into the flank of c3h mice 2 - 3 months old . 
and that mitosis is only a third of that found in round cell grafts . after 24 hours the elongated cells revert to the round form but mitosis remains low until the fourth day ( fig . 31 , dotted line )  . 
the development of the implants can be followed from an early stage , and is described from 5 hours after inoculation until the fourth grafts derived from spindle cells showed a lower mitotic rate day of growth . after 5 hours in vivo , a significant delay in the appearance of tumours , and were of smaller size as compared with tumours obtained from round cells . 
3 , and the cancer re8earch laboratories , department cf pathology , the medical sclwol , birmingham , 15 . received for publication may 28 , 1952 . the experiments recorded in this article were carried out in the course of investigations on behalf of the medical research council , a sub - committee having been set up in 1949 to inquire into the careinogeiuc properties of mineral oils and allied products . 
the arrangements for co - ordinating the research have been briefly described by auld ( 1950 )  . the entire series of tests have been made in duphcate at the cancer research laboratories , department of pathology , university of birmingham , and at , the chester beatty research institute , the royal cancer hospital , london . 
at each centre the activity of 3 selected crude oils 4 fractions derived from each crude by methods of distiration specially designed to avoid high temperature cracking , and the final residues , have been tested on groups of 50 mice for each sample and also on a total of 105 rabbits . 
he had already shown ( berenblum , 1945a ) that tumours were readily elicited on the rabbit by 9 : 10 - dimethyl - i : 2 - benzanthraceiie , which was found to be more potent to rabbits than to rats or guinea - pigs , in which positive results were , however , obtained ( berenblum , 1945b )  . 
he also subsequently observed ( 1947 ) that this hyd - r - ocarbon when injected subcutaneously did not induce rabbit tumours , tbough rats and guinea - pigs responded by this method . 
much earlier , oberling , sannie ' , guerin and guerin ( 1937 ) had shown that a i per cent solution of 3 : 4 - benzpyrene in benzene was active on rabbits ' skins , and berenblum ( 1945a ) observed that 9 : 10 - dimethyl - i : 2 - benzanthracene was much more active than benzpyrene at a similar ( o 5 per cent ) concentration . experiments using a high boiling fraction of mineral oil from an experimental catalytic cracking operatiori have recently been carried out by smith , sunderland and sugiura ( 1951 ) , testing the residue after removing the lower boiling naphtha and li - aht aas oil cuts , upon mice , rats , guinea - pigs , rabbits and rhesus monkeys . the rats and guinea - pigs were found to be refractory to skin applications but papillomata were elicited in all of the 6 monkeys , 2 being proved cancerous by papillomata were produced on biopsy 4 years after the start of the experiment . the inner surface of the ears of the 21 rabbits within 100 days . 
the number and size of such growths tended to increase during the 2 years in which painting was continued , and in 3 of the 6 surviving animals cancerous changes in the growths for tests on the type of material which they intended to study , were observed . namely , samples containing oils which had been subjected to a process of fluid catalysis up to 950 ' f . 
at birminorham they were selected , 25 of each sex froni an outbred laboratory albino strain previously employed in grading a series of oil fractions ( woodhouse and irwin , 1950 )  . in london the mice used were from laboratory stocks randomised so that each group of 50 contained some of each colour and genetic constitution . 
they were housed in fairly small cages of usual type and fed on greens alternated with crushed oats and a little bran . applications of the oils to patches of skin about 3 csquare from which the hair had been removed by electric clippers were made twice weekly . six areas on each of 30 animals were used for testing the 3 crudes , and 75 animals were used for the 15 derived fractions employing 7 areas on each animal . 
the 6 areas comprised the 2 ears , left and right thoracic , and the left and right lateral abdominal . the seventh area was the inter - scapular reorion . these sites are shown in fig . 
irwin ( medical research council 's statistical research unit , university of london ) , so that a statistical evaluation of the effects of site variation and differences in response by the individual animals might be possible . it will be apparent that each crude was applied to 60 sites and each of the other fractions to 35 sites . appheations were made twice weekly using approximately 0 3 ml . 
woodhouse in contrast , there was a total of 61 tumour - bearing rabbit sites in the birmingham series and 39 in the london series out of a possible 705 sites in each case , or , excluding the crudes and residues , which altogether yielded only 4 tumours ( london series ) , the remaining 12 fractions produced 61 and 35 tumours in the birmingham and london sen ' es respectively on a possible 420 sites . 
the cultures were grown by the hanging - drop technique in a medium consisting of equal parts of fowl plasma and 15 per cent chick embryo extract in ty - rode solution . 
the explants were subcultured every 48 hours for 4 passages to obtain as uniform growth as possible . fresh embryo extract was groups of cultures , each from the 4th passage , were selected and matched used . and used for experimental material and controls . 
agent were observed on the unfixed living cells in culture by cine - photomicrography . for most of the work we used apparatus modified from that described by willmer ( 1933 )  . recently , in collaboration with a . 
hughes of the department of anatomy , we have recorded the effects on the living cells by phase - contrast cine ' - photomicrography , using the techniques described by hugbes ( 1949 , 1952 )  . phase contrast microscopy has been used in a few cases to observe directly the effects of the chemicals on the living cells in culture . ( ii ) radiological details . the primary x - radiation used was of equivalent wave - length 0 - 221k , as deduced from the h.v.l. 
the nominal dose rate , which was always measured within + 2 per cent was in the reaion of 2oor per minute at distance 40 cfrom the focus , and was varied by altering the distance . 
the quality of the radiation was unchainged throughout these experiments , so that the probleof the exact value of the ionisation in the cells of the culture near the surface of the glass coverslip is not of importance here . in the cultures used most of the mitotic cells coudted lie between the glass surface and a distance 201i from it , so that the ionisation in the cells is increased by photo - electrons arising in the glass , probably by a factor in the region of 2 ( spiers , 1949 )  . 
3 , 4 , 5 . - effect of tetra - sodiuni 2 - mothyl - 1 : 4 - naphthohydroquinone cliphosphate ( compound i ) on a living resting cell of a chick fibroblast culture of the fourth passage , 24 hours old . phase - contrast c ' me ' - photomicrographs , taken by a . 
the doses were given in a fixed time , 2 - 00 minutes , except in the case of tlle three points marked , for which the radiation was delivered at 200 r per minute . the mitotic inhibition in probits is plotted against log dos - , ( finney , 1947 ; fisher and yates , 1948 ) there is no indication of departure from a linear relatiodbetween mitotic inhibition in probits and log dose . it is concluded that the relation between iiiitotic inhibition and the radiation dose plotted on a logarithmic scale is a sigmoid curve . 
the dose corresponding to 50 per cent mitot - ic inhibition after 6 hours is 315 the calculated regressiod line gives a sa - tisfactory fit . the calculation of the regression line is somewhat laborious . the mitotic inhibition produced by compound i in the chick fibroblast cultures has been studied in some detail after 24 hours ' application . 
the calculated regression line gives a satisfactory fit . there is no indication of departure from a linear relation between mitotic inhibition in probits and the logarithm of the concentration . thus there is a sigmoid relation between mitotic inhibition and the applied confor 50 per cent mitotic inhibition after 24 hours the concentration centration . of i is 3 - 81 0.15 x 10 - 6molar . for most of the compounds studied such accuracy is not necessary . tt is justifiable to deduce the approximate concentration required to give 50 per cent mitotic inhibition from the curve fitted by eye to relate the observed percentage mitotic inhibition to the molar concentration plotted on a log scale . in the case of compound i this method gave a value of 3 - 84 x 10 - 6m . the first experiments on the antimitotic effects of x - rays and i in chick fibroblast cultures ( mitcher and simon - reuss ' 1947 ) showed much greater mitotic inhibition with the combination of 3 x 10 - 6mcompound and 244 r of x - radiation delivered 18 hours later with fixation and counting after a further period of 6 hours , than with the same amounts of the two agents separately . to test for potentiation rigorously , it is essential to compare the effect of a combination of half amounts ( or other suitable mixtures ) of the two agents with the mean effect of the agents acting independently . this method , which may be termed the summation , method , is well recognised in pharmacology , and has already been applied in radiobiology by liechti and muller ( 1936 ) , gray and read ( 1944 ) , and mitchell ( 1947 )  . our original experiments showed a combined effect of 86 - 7 per cent mitotic inhibition , while the corresponding mean ' calculated for the two agents separately from the regression lines in fig . 
i and 2 , is 78 - 6 per cent . this result suggests that there is at least additivity of the effects , but it does not provide evidence for potentiation . in other experiments , the combined effect of 2 x 10 - 6mcompound followed 310 j . 
the combination of 2 x 10 - 6mcompound followed after 18 hours by 200 r of x - radiation produced 93 - 25 per cent mitotic inhibition after 24 hours . froni the number of mitoses in the individual cultures it is found that for 8 degrees of freedom student 's t is 15 - 71 , so that p is much less thlan - 001 . in this experiment , as in table 1 , the highest values of the mitotic inhibition were associated with no significant reduction of the area of the outgrowth . this finding confirms the unimportance of cell migration under the experimental conditions of the combined action of x - rays and the compound 1 . the possibility of differences in the behaviour of the outgrowth and of the central part of the culture has been , studied by means of the counts made in serial sections of 24 - hour cu - itures . 
 - the method is a laborious one ; by its use , fischer and parker ( 1929 . ) showed that in chick periosteal fibroblasts , the proportion of cells in mitosis was 0 - 79 per cent in the outgrowth , but only about 0 - 09 in our cultures - as in those of jacoby ( 19371 ) - the per ceiit in the central part . mitoses in the central part are limited to the " perichondxium " on its surfaces . studies of the topographical distribution of mitoses in our cultures by a . 
f. phillips showed no appreciable decrease in the number of cells in mitosis per unit the area of the central part is usually area as the central part is approached . not more than 20 per cent of the total area of the culture . in the serial sections of the cultures the central part is easily defined . 
the resting and mitotic cells were counted in the surface layers . in the sections of a control culture , out of 6205 cells counted there were 53 cells in mitosis , of which 13 were in prophase , 31 in metaphase , 3 in anaphase and 6 in telophase . 
at 24 hours after the application of 5 x 10 - 6mcompound 1 , out of 6220 ceus counted , there were 26 cells in mitosis ; of these i was in prophase , 19 were in metaphase , none were in anaphase and 6 were in telophase . 
simon - re ] utss cytological effects . tlio cytological changes produced by compound i in the chick fibi - oblast perhaps the most cultures differ in some ways from those produced by x - rays . fundamental difference is that anaphase bridges are only rarely produced by ' the compound , unlike x - rays . after the application of compound i to the hving cers , the first change observed is temporary cell enlargement , presumably due at least in part to hydration ( " cell oedema " )  . this does not occur with 5 x 10 - 7mconcentration , but has been observed in its early stages at 2 minutes after application of the compound in concentration 5 x 10 - 6m . 
the change is obvious after 5 minutes and probably affects the cytoplasm first . after 10 minutes the effects are still increasing ; ' there is bubbling of the enlarged cytoplasm and enlargement of the mitochondria , nucleus and nucloolus . 
the slight transient shrinkage of the cell immediately after addit - ion of the compound is observed consistently . at the maximum of the cell enlargemei - it the increase in transverse dimensions of the cells is often in the region of 50 per ' cent . mitotic inhibition is obvious after 80 minutes and persists after 24 and 36 counts show that there is no appreciable change in the distribution of hours . the phases of mitosis even in high concentrations such as 5 x 10 5m where mitotic inhibition is almost complete . example at 4 x 10 - 6mconcentration the abnormal mitoses increased slowly from about 5 per cent of the total mitotic count after 80 minutes to about 15 per cent after 24 hours . 
a typical count of the mitotic abnormalities is included in table il and photomicrographs of examples of the abnormal cells after fixation are enlargement of some of the dividing cells is shown in fig . 
2 x 10 - 6m  . compound i followed after 18 hr . by 150 r x - radiation * also 1 tripolar met - aphase . i and x - radiation shows potentiation of chromosome fragmentation . this finding is of particular interest in connection with possible radiotherapeutic applications . instability of compound i in solution . studies of the thermal instability of compound i in aqueous solution were made because of erratic results in animal experiments and clinical trials . 
for 7 days almost completely destroys the antimitotic activity and produces a rather toxic - solution . there is no significant mitotic inhibition , but the outgrowth is poor , many of the resting cells are vacuolated , disconnected and rounded and - a large number of the cells in metaphase show gross chromosome clumpiaig . there is progressive reduction of the mitotic ' hibition + 4 - 1 314 s . 
the outgrowth ig poor , many of the resting cells are rounded , there is some accumulation in metaphase and a number of exploded cells ; there is wide it seems likely that there are variation in the behaviour of different cultures . several unstable intermediate products involved in the process of inactivation of compound i by incubation at 39 ' c . 
experiments. tetra - sodium 2 - methyl - 1 : 4 - naphthohydroquinone diphosphate ( compound 1 ) it is found that there is no has been studied in detail as a reference substance . indication of departure from a linear relation be - tween mitotic inhibition in probits accordingly it is sugand the logarithm of the concentration of the compound . gested that the best estimate of the activity of the compound is the concentration which produces 50 per cent mitotic inhibition under the experimental conditions . this concentration may be termed mi 50 as a convenient abbreviation . 
for compound 1 , there is no indication of departure from a linear relation between the rnitotic inhibition in probits and the logarithm of the concentration . for 50 per cent mitotic inhibition after 24 hours , the concentration is 3 - 81 0 . 
not only has the type of therapy most likely to achieve success been the subject of dispute , but also the same measures in the hands of well - known authorities have produced a wide variation in results . 
have we a classification which accomplishes this ? the outlook for a patient with cancer of the breast depends first and foremost upon the degree of malignancy of the tumour and upon its extent . 
it is a wide belief that the prognosis is also influenced by certain other features , but the value of these is open to que ! rtion and will form the subject of a separate inquiry . 
 suffice here to state that such factors as age of patient , site of tumour in the breast , size of primary growth and duration of symptoms were not found , per se , to influence the outlook materiallv . 
 _ - \ttempts have been made in the past to group patients with breast cancer according to histological criteria which , it has been claimed , give some indication as to the degree of malignancy of the tumour . 
 the other is based upon a system of grading , wbilst the results of the former have largely proved disappointing , conflicting views exist as to the value of the latter . 
 it is the prime object of this paper to investigate the significance of the grade of malignancy in mammary cancer in an attempt to establish a more accurate system of classifying patients with this disease . 
 there is great difficulty at the present time in deciding on the line of action to adopt in view of the widely different procedure . ; , advocated by different authorities . 
 , vhilst gordon - taylor ( 1948 ) and cade ( 1948 ) believe radical mastec tomy alone to be the most suitable treatment for these patients , riddell ( 1948 ) and also richards ( 1948 ) advocate ancillary radiotherapy . 
it is now over fifty , ears since : moore of london conceived and introduced the radica.l mastectom , ( rodman , 1904 ) , and still there is controversy in some quarters as to its valu ;  . 
 if we cannot solve the problems of mammary carcinoma , what hope is there for the surgery of malignant disease in less accessible sites ? pretw ' u , , 8 attempts at grading . 
 little attention was directed to the grading of tumours until broders ( 1920 ) applied , on hansemann 's theory to squamous cell carcinoma of the lip , and a year later to similar tumours of the skin ( broders , 1921 )  . 
 l\iaccarty and sistrunk ( 1922 ) and l\iaccarty ( 1922 , 1924 ) also studied the histology of breast tumours , and found the most important prognostic charac teristics to be the degree of cellular differentiation and the presence or absence of certain stromal features . 
 ' white ( 1927 ) was unable to agree with the views of : : \iaccarty , but in a series of 100 cases confirmed the results of greenough ( 1925 )  . 
 smith and bartlett ( 1929 ) , and lat.er simmons , wright , hartwell and greenough ( 1933 ) , also confirmed the value of the syst.em introduced by greenough ( 1925 )  . 
 the five - year survival rat.es for the three grades of malignancy obtained by these authors are comparable , and agree in the main with those of pat.ey and scarff ( 1928 )  . 
they therefore elaborated a " clinical index of malignancy " based upon age , presence of lactation , rat.e of growth of tumour , and extent ( stage ) of the disease . 
perry ( 1925 ) , lane - claypon ( 1928 ) , truscott ( 1947 ) and hamett ( 1948 ) were not able ti < > prove that the position of the growth had any bearing on outcome in breast cancer . 
 flothow ( 1928 ) , in a little over 200 cases of carcinoma of the breast , studied the facrors stated by llaccarty ( 1922 ) to represent a defensive mechanism by the body to a malignant tumour . 
it was shown that the malignancy increased with a rise in the " malignancy index . " for comparison a clinical classification was also elaborated by schmitz , but no att.empt was made to study the effect of correlating these histological and clinical syst.ems ( hueper and schmitz , 1929 )  . 
 opinions regarding the accuracy of hisrological grading have varied from huepe , r , who finds as many as twenty different features of prognostic value ( hueper and schmitz , 1929 ) , ti < > reimann ( 1929 ) , who is unable ti < > find one . 
he considers , however , the broad grouping methods based on general histological stucture - - presumably such as that of greenough ( 1925 ) - ro be of value . 
greenough ( 1925 ) , on the other hand , applying the principal of anaplasia , concentrated on tubule formation , variation in size of cells and nuclei , secretory activity and mitotic and hyperchromatic figures . 
 the principles laid down by greenough ( 1925 ) were closely followed by others , such as smith and bartlett ( 1929 ) , and simmons , wright , hartwell and greenough ( 1933 )  . 
patey and scarff ( 1928 ) and scarff and handley ( 1938 ) , however , attached chief importance to tubule formation , variation in size of nuclei , and mitotic and hyperchromatic figures . 
 delbet and mendaro ( 1927 ) in france , on the other hand , considered the presence or absence of mucous secretion to be the most important factor in ascer taining the outcome of patients with mammary carcinoma . 
leroux and perrot ( 1928 ) andmoureau and lambert ( 1932 ) support the views ofdelbet and mendaro ( 1927 ) , but betrand and de nagey ( 1931 ) consider muco - secretion to be of no value in determining clinical progress . 
 evans ( 1933 ) studied a number of parenchymal and stromal features , and con cluded that , although anaplasia indicates a poor prognosis , it is of no practical value in assessing the outcome for the individual case . 
he concluded that the grade of malignancy is the most important indication ot survival period , and this is more significant when considered together with the state of the axillary glands . 
 complicated , exact numerical systems , such as the " histological malignogram " introduced by hueper ( hueper and schmitz , 1929 ) , are time - consuming and of doubtful value . 
 the series taken for the present investigation consists of 565 cases of carcinoma of the breast treated at the middlesex hospital between the years 1936 and 1942 inclusive , and at the war sector units associated with it during the latter half of this period . 
in spite of the movement of population produced by the recent conflict , all but 11 of the c3888 were traced by the follow - up department of the hospital . 
 for the present study the five - year survival rate will be the chief prognostic yard - stick , though some attention will also be given to ten - year results . 
 " sur vival rate , " however , does not necessarily imply freedom from cancer ; it is in point of fact , merely an indication of the number of patients actually alive . 
this was based on the principles laid down by greenough ( 1925 ) , but chief importance was attached to tubule formation , regularity in size , shape and staining of nuclei and hyperchromatism and mitoses . 
this view is supported by the work of greenough ( 1925 ) , white ( 1927 ) , patey and scarff ( 1928 ) , smith and bartlett ( 1929 ) , sophian ( 1935 ) , and in fact , this is the only histological feature on which scarff and handley ( 1938 )  . 
certain authors , such as evans ( 1933 ) , greenough ( 1925 ) and haagensen ( 1933 ) , paid attention to variation in size of the cells , but the outlines of these are usually indistinct ; it is more reliable to consider the nuclei . 
evans ( 1933 ) was unable to support this view , and plaut ( 1927 ) considers the feature unreliable on the grounds that many cancer cells divide by amitosis . 
nevertheless , as haagensen ( 1933 ) points out , a considerab1 proportion of the cells in cancer of the breast divide by mitosis , and he found a close relationship between the number present and the outcome . 
this is also supported by the work of greenough ( 1925 ) , white ( 1927 ) , patey and scarff ( 1928 ) , smith and bartlett ( 1929 ) , sophian ( 1935 ) , and simmons , wright , hart well and greenough ( 1933 )  . 
 the cases were fairly evenly distributed , 30 per cent of the total being pla - oed in grade i , 41 per cent in grade ii , and 29 per cent in grade iii . 
 227 it is evident that a parallel exists between grading and prognosis , there being more than three times the number of patients alive with grade i than with grade iii tumours . 
it will be noted that they a . , , uree especially with the findings of pat.ey and scarff ( 1928 ) , and table ii . - - grade and prognosis by various authors . 
no grade iii cases remained alive in the series of greenough ( 1925 ) , and also of white ( 1927 ) , but this does not appear to be the general experience . 
 total 218 433 the five - year survival period is a convenient yard - stick with which to gauge prognosis in breast cancer , but as truscott ( 1947 ) and others have pointed out , a large number of fatalities from this disease occur from five to ten years after operation . 
 comparison of the fiveand ten - year results emphasizes the importance of the latter as a more accurate indicator of the control of mammary carcinoma , there being a striking fall in the numbers oi survivals for the corresponding grades of tumour , even with allowance for expected deaths from other causes . 
this is in keeping with the 26 per cent obtained by richards ( 1948 ) , and the 32 per cent by harring t - 0n ( 1946 )  . 
 ( i ) well - marked ( ii ) moderate ( iii ) slight or ml total ( i ) slight ( ii ) moderate ( iii ) marked y ariation in nuclei h : yperchromatic and mitotic  . 
the extent of the growth should also be taken into account , and the most important single factor indicating this is whether the axillary lymph nodes are histologically involved or not . 
it is particularly interesting to note that the prognosis in the lower grade of malignancy with glandular spread is better than for either the higher or intermediate grades without such metastases ( 65 per cent alfre at fi . , e years compared with 53 per cent and 61 per cent respectively )  . 
 these results agree in principle with those of most workers who have employed the system introduced by greenough ( 1925 ) , and are almost identical with the findings of simmons , wright , hartwell and greenough ( 1933 ) ( table viti )  . 
 patey and scarff ( 1928 ) and scarff and handley ( 1938 ) , on the other hand , found a uniformly good result in all three grades when the glands were not involved , and so concluded thatgrading is only of prognostic value in those cases with axillary metastases . 
the figures in table ix refer to the state of the cases at the time of operation , and the time factor involved here is the delay in seeking treatment . 
on the other hand , if for any reason the grade ill .cases tend to present for treatment after a longer interval of time than those belonging to grade i , then this may account for the greater incidence of axillary involvement in the former . 
in addition a higher percentage of the former present within six months of the onset of the disease and fewer after twelve months as compared with the grade i cases . 
thus , in spite of the shorter delay in seeking treatment , there is a greater incidence of axillary involvement among the high and intermediate grad~ of malignancy , consequently emphasizing the greater metastasizing power of these types of growth . 
ewing ( 1940 ) , however , prefers to employ the term " potential malignancy " for a neoplasm as determined by microscopic examination , because the clinical course may be modified by such features as site of tumour , surgical trauma and perhaps changes in rate of growth . 
 several authors , such as hueper and schmitz ( 1929 ) , and also lee and stiiben bord ( 1928 ) , have compared the results of grading and staging in breast cancer . 
 involvement of the skin directly over and in continuitv with the tumour does not affect staging provided that the area ~volved is small in relation to the size of the breast . 
as previously , when the cases were classified according to grade ( tables i , iv ) , a large number of deaths are seen to take place in the interval between the shorter and longer follow - up periods , thus once more emphasizing the importance of the latter in assessing the achievements of breast cancer therapy . 
 a study of grading and also staging indicates that both methods give com parable results ; the latter , however , has the slight advantage of revealing the outcome for the most advanced cases ( stage 4 )  . 
 we ha , e already drawn attention to the relationship in breast cancer between the histological architecture of the tumour and its tendency to involve the axillary glands ( table ix )  . 
exactly half of the grade i tumours have not yet extended beyond the first stage , whilst about a quarter are advanced , belonging to stages 3 or 4 taken together . 
 100 100 100 these results merely gi , e additional support to the view already expressed with regard to the relation between histology and axillary involvement , that the higher the grade of mammary carcinoma , the greater the tendency for early spread beyond the breast . 
the vast majority of workers in this field , however , have argued as to the relati , e merits of either syste but why should the grade and stage be considered only separately , and in the sense of competing with each other to form the basis of a reliable prognostic guide ? a combination of these features would result in a classification which gives information on not only the type of growth , but also its extent when first seen . 
would this not bring about a more accurate grouping of cases than could be produced by either system alone ? indeed , se , eral authors have taken the histological picture into consideration with the state of the axillary lymph nodes , and the value of this has been con firmed here ( table vii )  . 
the percentage of five - year survivors is seen to range from as high as 87 per cent for stage i , grade i cases down to as low as 6 per cent for those belonging to stage 4 , grade ill . 
it is also evident that whilst the prognosis in each stage deteriorates with the rise in histological malignancy , the cases with low grade tumours do not conform to the general trend . 
it reveals that all is by no means lost if a patient presents with a stage 2 or even a state 3 growth provided that it belongs to the lowest grade of malignancy ( grade 1 )  . 
 having revealed the value of combining histological grading with clinical staging for prognosis in mammary carcinoma , the suggestion is now put forward that the wide range in results produced by different authors for identical methods of treatment depends upon the comparison of incomparable groups of cases . 
 w ' hat evidence is there to support these views ? with reference to this question , two groups of cases will be taken for study , one treated by surgery alone , and the other by surgery with ancillary radio therapy . 
although it is not the purpose of this paper to discuss the merits of treatment in breast cancer , the results achieved by these two methods will be compared in order to illustrate the discussion . 
it must be pointed out that the treatment in each group was by no means uniform , there being considerable variation both from the surgical as well as the radiological aspect . 
 irradiation was predominantly in the form of deep x - rays , either pre - operati.e , post - operative , or both , a few cases being treated with radium alone or radium and deep x - rays . 
 according t - 0 grade . - let us first consider the results achieved for each group according to the grade of malignancy ( tables x " \ ' ~i , xviii )  . 
on the other hand , in the radio - surgical group , although this operation was performed in the majority , there were many instances of more conservative surgery , which included 37 simple mastectomies and 17 local excisions . 
 " ~ith this in view , the survivals of a more uniform type of therapy were taken for study , the comparison being made between patients treated by radical surgery alone and radical surgery with irradiation ( tables xxii , xxiit )  . 
 the greater achievement of surgery alone over the combined attack is again seen in harnett 's ( 1948 ) figures which are also for the radical operation and are classified by stages ( table xxi )  . 
 from these facts it is clear that the variation in treatment by surgery in the present material does not appear to interfere with the fair comparison of the 278 h . 
 in this instance a false impres sion may be given when the results of two series of cases are compared owing to the possible unequal distribution of clinical stages in corresponding histological grades . 
here there are small number of seen to be more low and fewer high grade tumours in each stage ot the surgical as compared with the radio - surgical cases . 
furthermore , of all the neoplasms of low grade malignancy ( grade i ) 57 per cent were in this surgical group in contrast to only 30 per cent of the highly malignant type ( grade ill ) ( table xxvi )  . 
what , then , may acco~t for this fact ? is it merely fortuitous chance , it is true , may play a part , but this is not the only possibility  . 
 .although two groups of patients are placed in the same clinical stage , there may be some difference in the extent of the growth for which no allowance is made in our present staging systems . 
in view of the parallel revealed between hist.glogical picture and extent of tumour ( tables ix , xiv ) , it is these unfavourable cases which also tend to be of high grade malig nancy , hence the distribution in fig . 
it i < ; , therefore , useless to compare results obtained by different authors if the cases are taken according to stage and no allowance is in this instance chance is probably the chief factor made for type of growth . 
however , cannot be ruled out , individual surgeons ha , ing , to some extent , personal criteria for deciding on the measures to be adopted for a given case  . 
.y egkcte , d , routes of extension . - reference will be made to avenues of extension of breast cancer which are largely neglected in both prognosis and also treatment . 
of these unfavourable grade iii cases nearly 70 per cent ( table xxvi ) were referred for irradiation , thus emphasizing the disadvantage placed on the radiotherapy group when it comes to the comparison of treatment results . 
 to sum up : it is postulated that in a given stage certain cases haw unfa rnur able clinical features , which influence the surgeon to refer them for radiotherapy . 
 the tumours of these patients tend to be of high grade histological malignancy , and h~ve probably also extended , ia routes , as yet , not given adequate considera tion in prognosis and treatment . 
this may account , at least to some extent , for the unfavourable results achieved here and elsewhere ( adair , 194& ; truscott , 1947 ; harnett , 1948 ) by ancillary radiotherapy . 
in these instances the achievement of radiotherapy is probably even greater than appears on first examination owing to the unfavourable features which tend to be present in cases referred for this treatment . 
 it has been pointed out that the problem of grouping also applies to series treated by identical methods , though here chance rather than selection is prob ably a more important factor accounting for the distribution of the patients . 
with this method it is hoped that further light may be thrown on some of the problems of breast cancer , and possibly the true merits of the various treatments of the disease assessed . 
 when this clinico - pathological system is applied here , will the results achieved by ancillary radiotherapy appear to be any better ? this question , unfortunately , 282 h . 
harries ( personal commanication ) , at the middlesex hospital , quotes 19 per cent for the five - year follow - up and 4 per cent for a period of ten years among some 220 cases . 
these figures are inferior to those obtained by treatment , which is universally accepted as being of definite , aloe in prolonging life , except perhaps in the very advanced cases . 
 once the cases are classified more accurately , such as on the lines indicated earlier in this paper , and only then , shall we be in a position to study the merits of , arious treatments . 
we may find , for example , that less radical surgery may be adequate for the control of certain cases such as those with grade i , stage 1 tumours , whilst radiotherapy may be an important ancillary measure for some cases , but on the other hand of little use for others . 
grace ( 1937 ) in america obtained a 56 per cent fi , e - year surrival rate among 40 cases treated by simple mastectomy and radio tht > rapy . 
this author considers that " the cellular structure of the tumour is the dominant factor in prognosis , and surgical technique , irrespecti.e of its radicalism , plays a definite secondary role . " some support tor this view is perhaps to be found in the paragraphs which follow . 
a group of 45 cases in his series were treated by local excision with implantation of radium , and 63 per cent were alive after a similar period of time . 
 what do these results mean ? can it be possible that more conservative surgical measures than the radical operation are sufficient for the control of mammary cancer ? such , indeed , is the view in this country of keynes ( 1937 ) , and more recently mcwhirter ( 1948a , 1948b )  . 
 we have already suggested that when cases are properly grouped accord.mg to both stage and grade it may be found that less radical surgery may be adequate for certain types of case . 
it is not even necessary to have numerous sections to appreciate this picture ; one or two portions of tissue of reasonable size taken from the periphery of the growth are adequate . 
 thus , patey and scarff ( 1929 ) , in a series of llo cases , found that the metatases in the axillary nodes were of the same grade as the primary in 82 per cent , of a lower grade in 16 per cent , and of a higher grade in only 2 per cent . 
comparable figures to these are also given by other writers , such as harrington ( 1935 ) , lach man ( 1944 ) , riddell ( 1948 ) and ledlie ( 1948 )  . 
little attention appears to have been given to this problem until scarff and handley ( 1938 ) suggest.ed that disappointing results in stage 1 cases may be explained by this mode of spread . 
this applies particularly to growths situated in the inner half of the breast ; of 17 such cases 12 had deposits in the internal mammary chain , in 2 of which the axilla was not also involved . 
 in spite of this , truscott ( 1947 ) and harnett ( 1948 ) were unable to show that the site of the growth plays an important part in prognosis . 
 in those cases where the axilla appears to be free from involvement at the time of operation and the prognosis subsequently turns out to be poor , invasion has probably occurred by routes such as the int.ernal mammary chain , and perhaps the supraclavicular glands . 
 after considering these facts it would appear , as handley and thackray ( 1949 ) point out , that there are but few growths which are really confined to the breast . 
chief success , from the point of accuracy and simplicity , appears to have been from histological grading , on the lines mdicated by pat.ey and scarff ( 1928 ) , combined with the state of the axillary glands . 
stage i ? this question has defeated explanation in the past , and will probably continue to do so until such time as the pathologist plays a part in establishing a prognostic system of the type indicated in this paper . 
 the controversy which exists as to the relative merits of the different methods of treatment for carcinoma of the breast has led to the present investigation , the prime object of which has been to establish a more accurate classification for this disease . 
 the gra < le of the tumour taken together with the state of the axillary lymph nodes gives a more accurate indication of outcome than either grading or staging alone . 
for example , 94 per cent of grade i cases without glandular involvement were ali , e at five years compared with 16 per cent of grade iii with this complication . 
it offers an explanation for disasters in early cases , and also for remarkably good results obtained in advanced in indicates that all is not necessarily lost if a patient presents with a stages . 
 lt has been shown that in a given clinical stage or histological grade the cases treated by surgery alone are not strictly comparable to those subjected to surgery and radiotherapy . 
in addition this has probably also played a major part in bringing about the present - day confusion as to the relative merits of the various measures advanced for the control of the disease . 
sambrook for assessing the clinical st.age of the cases ; to miss chambers of the follow - up department for tracing the patients , and to the photographic department for assistance with the microphotographs . 
mottram ( 1945 ) published the results of experiments designed to demonstrate a relationship between the tumour yield induced by benzpyrene and the number of epidermai mitoses present at the time of application of the carcinogen . painting the skin of rnice at midnight resulted in a higher yield ofpapillomata than did painting at midday . 
but not water , and 1 - 4 hours later they were given a single appheation of carcmogen , benzpyrene ( hoffinan - la roche ) in experiment i and methyleholanthrene ( eastman kodak & co in experiment 2 . in the 1 - - ) enzpyrene experiment each animal received 0 - 1 ml . 
of a 0 - 3 per cent solution in acetone apphed to the interseapular region of the skin , and ' m the methyleholanthrene experiments the dose wasalso 0i ml . 
the mice were starved for a total period of 64 hours , after which they three days after the application of the carcinogen painting were fed ad libitum . with croton oil ( 0 - aper cent in acetone ) was commenced , and this was repeated twice weekl for 20 weeks . 
these mice were treated in the same way as those in the group b , except that the period of starvation was omitted altogether . in experiment 3 , group a contained 25 male mice which were deprived of all food 36 hours before each was painted with 0 - 05 ml . 
most of the papillomata being discovered between the 1 : 21th this is in good agreement with the results obtained bv berenand 15th weeks . blum and shubik ( 1947 )  . 
the methylcholanthrene was introduced into average age of the mice was 6 months . the prostate gland in the following manner : under general anaesthesia , a median abdominal incision was made , exposing the prostate lobes . 
8. - the same after 3 weeks ' time . 10 or more layers deep , keratin pearl formation still absent . cells acquiring more pronounced squamous metaplasia . development of stratified squamous epithelium , h . 
they varied between i to 3 of an inch in diameter and frequently more than a single prostate lobe was involved . a tumour of average size is shown in fig . 
7. received for publication february 7 , 1947 . it is a widespread doctrine among students of cancer that any tissue or organ capable of cell division may be the site of a malignant neoplasm , but it is well known to clinicians that certain tumours , theoretically possible , do not in fact occur . 
the most striking example of an organ which never gives rise to a neonotwithstanding the fact that mitoses occur in the lens plasm is the lens . throughout life , having been seen even in the lenses of patients 80 years old , no tumour of the lens has ever been discovered . the lens is an entirely epithelial structure embryologically derived from the surface ectoderm , devoid of blood supply and enclosed in a semi - permeable although mammalian lenses are enclosed membrane , the hyaline lens capsule . during embryonic life in a capillary net ( the vascular lens capsule ) this does not constitute a blood supply , since the vessels do not come in contact with the lens in the majority of vertebrates cells , being always outside the hyaline capsule . the lens can therefore be looked even this embryonic vascular capsule is absent . on as a segregated group of specialized epithelial cells growing and differentiating in a closed system , separated alike from the blood - stream and from the surface of the body . 
may be important factors in the pathogenesis of cancer of deeply seated as well as of superficial organs indeed , early in the 19th century a careful local study of is hardly a new one . cancer deaths in verona was made by stern ( 1842 ) , the notion of cancer houses was looked into and fragmentary studies of cancer endemiology were made in the concept of cancer as a unitary disease switzerland , america and elsewhere . affecting organs rather at random has tended to discourage detailed research on site distributions . the general register office in london worked patiently upon the occupational aspects at successive census periods with increasing detail as death certification recently the danish cancer registry , initially of primary cancer sites improved . rather sceptical of some of the conclusions reached , has admitted that danish data tend to confirm them ( clemmesen , 1951 ) , and similar data have been pubin 1950 and 1951 the council for lished from the netherlands ( versluys , 1949 )  . co - ordination of international congresses of medical sciences arranged conferences on what was at first called " geographical pathology " , but has now become " endemiology " of cancer . very little has been written , however , on lung cancer from this aspect except with respect to tobacco , although it came under discussion at the first of the above - mentioned conferences ( c.c.i.c.m.s. , 1951 ; daff , doll and kennaway , 1951 )  . in the present paper the short term " lung cancer " is used to mean carcinoma and other malignant neoplasm of the bronchus , lung and pleura ; and when " mortality " is mentioned it is understood that whatever rate or index is used has been standardized for age . * the substance of this paper was communicated to the metropolitan brench of the society of medical officers of health on may 9 , 1952 , 100 p . 
at that time i still had to work upon the rates for 1921 - 30 , and failed to find any significant correlation either in the large towns or in the metropolitan boroughs between lung cancer mortality and the social class or overcrowding indices . recent research on correlation with tobacco smoking . between 1930 and 1946 the recorded rates in england and wales as a whole had been rising steadily , and to a surprising extent even when increasing use of x rays was considered . consequently the medical research council called a conference to advise whether any useful research could be undertaken to throw light on the causes . 
the result of that study was published in september , 1950 ( doll and hill , 1950 ) , and in my view it proved beyond doubt , in conjunction with american evidence of the same kind ( wynder and graham , 1950 ) that 102 p . 
stocks tobacco smoking is a very important factor in the causation of lung cancer . did not prove , however , that tobacco smoking is the only important factor in it has been suggested that the figures could mean that 90 per cent causation . of the lung cancer occurring in greater london is caused by tobacco ; but they do not necessarily mean that , and there are reasons for thinking that the proportion is - much lower , and that atmospheric pollution may be an additional factor . these reasons derive from some statistical facts not hitherto published . mortality in london in 1946 to 1949 . in the four years 1946 to 1949 the deaths of males attributed to lung cancer in in 1950 england and wales averaged 8183 annually , and of females 1774 . deaths of males exceeded 10 , 250 and those of females were just short of 2000 , and in 1951 the total reached 13 , 000 . 
the national rates at different ages are compared in table iv , which shows that at ages 35 - 45 the increase was 6 - fold , and at 55 - 75 it was more than 11 - fold . how much of this enormous increase has been due to more complete detection of the disease and how much to increased incidence we can only surmise . london county as a whole , allowing for age changes , male mortality increased about 8 - fold between the two periods ; and it is now about 60 per cent greater than in the country as a whole , and 21 times the rate for rural districts . 
1 shows the mortality distribution in 1946 - 49 . for 1921 - 30 there were two areas of specially high certified mortality , one in bermondsey , stepney , bethnal green , hackney and stoke newington , the other in hampstead , st . 
stocks wind direction as a possible factor . returning now to greater london , clark ( 1951 ) has shown that the logarithm of the population density per acre in successive zones diminishes in arithmetic progression on passing outwards from the business centre , and the same is true supposing domestic smoke to be an important factor in of other large cities . producing lung cancer , if there was no wind at all one would expect to find the highest incidence at the centre , diminishing in all directions outwards . if there is wind and it comes from all directions equally , a more diffused but still symmetrical distribution would be expected , with less contrast near the centre and more near the periphery . however , wind does not blow equally from all directions , and greenwich records over 12 - hour periods throughout the year 1950 show that the wind direction , when analysed into 8 sectors , was s.w. 
jensen. frm the danish cancer regi8try under the national anti - cancer league , kobenhavn 0 . received for publication january 31 , 1953 . in 1947 clemmesen and busk reviewed the material of the danish death certificates with a diagnosis of cancer of the lung from the years 1931 to 1945 ( clemmesen and busk , 1947 )  . 
they found an increase in the numbers for males , particularly pronounced in copenhagen , the capital , less striking in provincial towns , and somewhat smaller for rural areas . however , the incidence of cancer of the lung among persons subject to routine examination in the central tuberculosis station for the city of copenhagen did not reflect the heavy increase in mortality found in the city as a whole . 
diagnose a number of cases of cancer of the lung , so that the graph illustrating the incidence rates in its material should be changed , as shown in fig . 
1. during the years up to 1950 the increase in mortality rates for cancer of the lung among men in the various areas of denmark continued , and from fig . 
women. of the disease , and only to a small extent attributable to improvements of diagnosis , which can be expected to cause a proportional increase for both sexes in the number of cases diagnosed . 
the possibility that the deciding factor might be an increase in medical attention for all categories of diagnosis can be excluded because the patients from the tuberculosis station were unselected and referred directly for diagnosis with a view primarily to a disease different from bronchial carcinoma , and finally were examined in a uniform way . further analysis of the material from the danish cancer registry confirm the results from mortality statistics ( clemmesen and busk , 1947 ) that the increase in incidence of cancer of the lung among men is particularly strong for the relatively younger age classes ( clemmesen and nielsen , 1952a , 1952b )  . this seems to speak against the assumption that atmospheric pollution is a causative factor of primary importance to cancer of the lung as far as denmark is concerned . if this were the case we would expect an increase in incidence for all age classes . it might also be expected that the windy climate of copenhagen could cause regional differences in incidence of the disease within the city analogous to the findings of stocks ( 1952 ) for london , but in fact it appears that the distribution of cancer of the lung among men in copenhagen varies with the social level expressed in terms of annual house rent , much like cervical cancer of the uterus being most frequent in the classes of subdistricts with the lowest level of house rent , although the single subdistricts show no such parallelism . however , there is no tendency for leeward areas to exceed the weatherside with regard to incidence rates for carcinoma of the lung ( clemmesen and nielsen , 1951 )  . thus , it would seem that the main reason for assuming atmospheric pollution to be causative to cancer of the lung in denmark would be the excess of cases in towns , particularly in copenhagen . 
5 represents mortality rates at various ages for lung cancer among men in copenhagen arranged in quinquennial cohorts born in the period about the years given in the diagram . it would seem reasonable to assume that the carcinogenic influences determining the incidence of cancer of the lung , whether they be of customary , occupational or hormonal character , will not begin before the age of 15 or perhaps a few years later . it appears from the tables that the cohorts of highest mortality at an conearly age do not show an increase in rates until after the 35th year of age . sequently it will be justifiable to assume a minimal period of about 20 years from the beginning of the influence to the death of the patient from cancer of the lung under the conditions usual in copenhagen . it follows logically that if we assume a later or earlier beginning of the carcinogenic influence we must respectively shorten or prolong the assumed period with a corresponding number of years . it should also be remembered that we must allow for wide variations either way . under these assumptions and from the observations illustrated in fig . 
5 that the graphs for the quinquennial cohorts do not coincide as they would for most other sites of cancer , but are moving to the left , although now at a decreasing pace . this would not be explained by assuming that all cohorts were from a certain age subject to some carcinogenic influence unless we add that the influence was strengthened at some point of time , or during a limited period of years . now it is seen that the result of the carcinogenic influence increased particularly for men born between 1875 and 1885 , while the effect on the following cohorts progressed at a slower rate . 
2 , it will be seen that the differences in the level of the graphs indicating crude mortality rates for cancer of the lung between capital , provincial towns and rural areas can be ascribed to a later beginning of the rise in the latter categories , i.e. , 8 years for provincial towns and 10 for the rural areas , which is in full conformity with the result of the cohort analysis . hence it appears that the assumption of atmospheric pollution as a cause of cancer of the lung in denmark in order to explain the differences between towns and country is unnecessary , if we suppose that the carcinogenic influence began a few years later in the provincial towns and a few years later still in the country . quite apart from the etiological implications of our cohort study it is in the field of practical hygiene we find the widest consequences of the diagram of fig . 
calcutt oestrogens : oestrone and diethystilboestrol . a group of arsenicals chosen in view of neubauer 's ( i947 ) implication of arsenicals as carcinogem ' c agents . three unrelated compounds : nitrogen mustard ( shown to be carcinogenic by boyland and homing , 1949 ) , urethane ( shown to be carcinogenic by jaff6 , 1947 ) and light green f.s. 
numbers represent duration of exposure in minutes to filtered ultraviolet radiatioxi required to cause death of 90 per cent of the cultures . with the exception of the oestrogens none of the agents tested compare in comparisons among the compounds tested effectiveness with 3 : 4 benzpyrene . suggest a certain degree of correlation with carcinogenicity . 
whether the effects achieved are a true photodynamic response as occurs with fluorescein and other dye stuffs , or ate a consequence of intracellular action by the radiation leading to increased susceptibility to a toxic agent , as calcutt ( i 950 ) has show - n to occur in the case of certain - sh inhibitors must remain in doubt . 
on the other hand , it appears possible that the radiation has brought about such an intracenular change that the chemical carcinogen is now able to attack a much greater amount of substrate than is normary the case . 
thus complete disruption of the intracellular functioning and death occurs , rather than ' a partial disruption which manifests itself as the carcinogenic change . certain evidence supporting such a view exists calcutt ( 1950 ) showed that irradiation with wave - lengths in the literature . greater than 3300 a increased the availabihty of intracellular - sh groups . such treatment prior to the addition of a photosensitising agent was show - n by calcutt ( 1951 ) to enhance the photodynamic response . 
jung. from the cancer research laboratory , university of florida , gainesville , florida . received for publication march 21 , 1951 . the experimental investigation of gastric cancer is seriously impeded by our present inability readily to produce this condition in animals . 
the difficulty and uncertainty of this method have progress in this field would be greatly facilitated if a precluded its general use . chemical compound capable of producing gastric cancer were discovered . that a carcinogen with such specificity is possible may be implied from the frequency with which butter yerow , for example , produces hepatomas ( kinosita , 1940 ) and beta - napthylamine , bladder tumors ( wiley , 1938 )  . 
when this is done it very seldom results in malignant growth in the stomach because of the protective if the carcinogen could be 8ecreted by the stomach it would action of the mucus . be in a much more favourable position to initiate cancer . the first step towards a solution of the problem is to ascertain just what properties of a substance influence its secretion by the glands of the stomach . the optimum properties might then be conferred on - a carcinogen by judicious substitution in the molecule . 
the resulting compound could be expected to initiate neoplastic growth in the stomach . several groups of workers during the past twenty - five years have studied , by means of gastric pouches in dogs , the secretion of intravenously injected dyes . dawson and ivy ( 1925 ) , kobayashi ( 1926 ) , ingraham and visscher ( 1935 ) , and varco and visseher ( 1941 ) , although working under varied conditions , are geneof some sixty dyes rally in agreement as to the dyes secreted by the stomach . studied ( ingraham and visscher , 1935 ) , afl those secreted are electropositive ( basic ) under some conditions . 
on the other hand , dyes not secreted by the gastric glands are acid or amphoteric with the possible exception of four basic triphenylmethane dyes . the basicity of these dyes seems to be the property most likely to determine their degree of gastric secretion . 
at a pkbof 11 - 3 , for instance , this equation yields a concentration ratio of 51 , at a pkb of 7 - 5 a ratio of 2 - 8 x 101 . ratios actuary obtained were 1 - 3 and 26 - 4 . although experimental results indicate a definite tendency for secretion to increase with increasing basicity . 
it is obvious that there is some opposing factor . this opposing factor increases with increasing basicity , so that at pkbof about 6 - 0 the tendency for the basic dve to concentrate in the stomach , due to distribution differences , might be completely overcome . this opposition to gastric secretion of dyes of increasing basicity is nicely explained in terms of the pore theory , or the concept of the ositively charged membrane ( ingraham and visscher , 1935 )  . acid dyes would be retained by chemical combination . basic dyes are assumed to pass through this membrane in the undissociated foras the ionization constants of these dyes increase it would become increasingly more difficult for them to pass through a positively charged membrane . it appears , therefore , that we are dealing with two main effects in the gastric secretion of dyes . 
kennaway also of those following each one of the recognized occupations , at the time of the census . the number of deaths attributed to cancer of the lung and larynx occurring in the whole male population in each of these age groups during the years in question was obtained from material at the general register office . the comparison of the various occupations with the general population is then a sum in proportion , which gives the ratio of the number of deaths in any given occupation to those , reckoned as 100 , which occurred in the same number of males in ' the same age groups in the whole population . 
an example of this calculation is given in the earlier paper . sources of error in statistical work on death certificates . these were examined in the earlier paper ( kennaway and kennaway ; 1936 )  . the use of death certificates for statistical purposes is liable to errors which are inherent in the nature of the material and cannot be wholly eliminated . 
one must either recognize and admit these errors and correct them in any way possible , or abandon any attempt to obtain information from these data . ( 1 ) correction for period between census . - in order to compare the incidence of cancer upon different occupations one must know the number of persons following each occupation . this is learned from the census returns . 
the figures for populations used for the earlier period ( 1921 - 32 ) were those of the 1921 and the 1931 census , which were combined by means of a formula described in the earlier paper ( kennaway and kennaway , 1936 ) ; those in the present paper are from the 1931 census , which is the last taken . 
kennaway when large numbers are concerned . the immense amount of labour involved in such enquiries can be seen in the monograph on cancer of the scrotum by henry ( 1946 )  . but as none of the occupations included in the present study , with one exception , shows a very high incidence of cancer of the lung , comparable to that seen in the occupational cancers of ' the skin and bladder , this source of error is not a very serious one . ( 4 ) incorrect diagnos8is is of course a source of error in these , as in all other , in the case of death certificates received in bulk , perhaps some medical data . years after the dates of the deaths recorded , one can do nothing but exclude any certificate of which the wording suggests ( 1 ) that the cause of death did not come within the category under examination , or ( 2 ) that the diagnosis was uncertain or ill - founded ( see next section ; such certificates are made the subject of individual inquiry by the general register office )  . when such exclusions have been made , one must treat the remaining certificates as correct if any use is to be made of the material . death certificates taken for examination . the material utilized in this paper has been restricted as far as possible to cases of primary malignant growth of the lung , and of the larynx . 
when the individual occupations are examined considerable variations are found . in the earlier paper attention was drawn to the high ratio for cancer of the lung in judges , stipendiary magistrates and barristers ( 173 ) and in stockbrokers and stockjobbers ( 187 ) ; in the later period ( 1933 - 38 ) the former fell to 112 and the latter rose to 253 . the stockbrokers and jobbers also have the highest ratio among the professional workers for cancer of the larynx . the figures for roman catholic priests and monks are too small to be significant . the ratio for ministers of other religious bodies has risen ( table xx )  . 
no attempt is made here to deal with all the data on the carcinogenic action of arsenic , as this subject is under investigation by a committee of the industrial health board , but the following quotations from recent annual reports of the senior medical inspector of factories show the possible importance of arsenic in relation to cancer of the lung . 
kennaway there was a special surgical clinic dealing with lung cases , and where full x - ray and bronchoscopic investigation was practised , 42 per cent of the clinical diagnoses were wrong ( table xxiv )  . 
hence the diagnosis of cancer of the lung appears to be still very incomplete , even under favourable conditions , in spite of the great improvements which are generally stated to have been made . 
some possible causes of a real increase are considered in the next section . in the earlier paper ( kennaway and kennaway , 1936 ) we drew attention to the evidence afforded by cases of cancer of the lung in which an erroneous diagnosis was made based on nervous or mental symptoms due to metastases . 
18. ( 2 ) one must look , therefore , for some other factor to explain the increase , if there is a real increase , in cancer of the lung in recent years . 
a similar argumnent might be applied in tile case of cancer of the lung , which is not known to be prevalent in any of the lower animals . the adenoma of the lung of the mouse , and certain affections of the lung of sheep in south africa and iceland , are neoplasms of which the exact nature is uncertain . we know one instance at any rate of the susceptibility of the lung of an animal to a carcinogenic agent , namely the lung of the cat in relation to 2 - acetylaminofluorene given by the mouth ( harding , 1946 ) ; hence there is no reason to think that animals are immune to any such one obvious factor , possibly carcinogenic , to which the lung of mnan agents . alone is exposed is tobacco smoke . during the present century very considerable changes have taken place in this country in the social distribution of the various methods of smoking . there were , of course , exceptions to any rule , but roughly one might say that in the earlier part of this period men of the richer classes smoked pipes , cigarettes and cigars , and men of the poorer classes smoked pipes ; cigarette smoking was increasing among the richer women , while women of the poorer classes did not smoke . one never saw a woman scrubbing her front door - step , or hanging out the washing , with a cigarette in her mouth . the great change which has taken * a . 
of course no claim is made here that the simultaneous increases in the consumption of tobacco , and in cancer of the lung , proves any etiological connection between the two . other changes which have taken place in the same period , which no one proposes to associate with cancer of the lung , e.g. 
kennaway to summarize the evidence brought together in this section on the incidence of cancer of the lung : the higher mortality in towns , the low mortality in agricultural occupations , and the absence of social gradient , are compatible with a factor in the air , such as coal smoke . in any such comparison of urban and rural areas , the question of facilities for diagnosis must be considered . coal smoke , which is probably a decreasing contaminant of the air , does not account well for the increase of cancer of the lung . 
among various possible factors is tobacco smoke ; the consumption of tobacco has risen , and so has the percentage of it smoked in the form of cigarettes , of which the smoke is often inhaled . 
among various possible factors which have been suggested to account for the increase is tobacco smoke ; the consumption of tobacco has risen , and so has the percentage of it smoked in the form of cigarettes , of which the smoke is often inhaled ; such an effect of tobacco would accord well with the absence of social gradient . we are indebted to the registrar - general for the data considered in this paper , and also for kindly permitting us to use some figures which have not yet been made public officially . 
we have received much assistance in the classification of occupations , and on various other questions , from some members of the staff of the general register office , to whom we wish to express our gratitude . we wish to thank e . 
1. received for publication december 1 , 1952 . it has for long been an accepted principle that the earlier the diagnosis of malignant disease is made the more likely it is that the growth , if in a region of the body accessible to the surgeon , will be in a stage which admits of radical resection and the higher will be the survival rate . 
however , balfour ( 1937 ) recorded that the 5 - year survival rate for 2112 patients after resection of the stomach for carcinoma was 25 per cent for those patients with histories of symptoms for less than 6 months and 35 per cent for those with histories of symptoms for more than one year . 
walters , gray and priestley ( 1942 ) in a later study recorded that 24 * 6 per cent of patients whose symptoms were of less than one year 's duration survived 5 years after resection of the stomach for carcinoma , whereas 32 - 6 per cent of patients the duration of whose symptoms was more than one year survived 5 years . 
the authors investigated the association between the length of history and the grade of malignancy as judged by broders ' classification , and give a table which shows that 39 of the 114 patients had growths which were classified as broders ' groups i and ii , while 75 had growths classified as broders ' groups iii and iv ; in each case the proportion of patients who gave histories of symptoms for more than 2 years was 23 per cent . eggers , de cholnoky and jessop ( 1941 ) reported that while the 5 - year survivals of 235 patients who had undergone radical .mastectomy for cancer of the breast fell steadily from 76 per cent to 20 per cent with delay in treatment up to 2 years , the survival rates rose to 25 per cent of 20 patients in whom the delay was over 2 years , and to 40 * 9 per cent of 22 patients in whose cases - there had been delay of 3 years or over . 
harnett tomies for cancer of the breast , found that 24 - 8 per cent of the 938 5 - year survivors were treated more than one year after recognition of the tumour ; he considered that the majority of these cases were the indolent , slowly - growing types of breast carcinoma , which metastasized late or occurred in hosts whose desmoplastic reaction to the tumour was intense . 
bloom ( 1950a ) in an analysis of 470 cases of cancer of the breast treated by surgery alone or with ancillary radiotherapy found , after classifying them into 3 groups according to histological criteria of degree of malignancy , that 79 per cent of 141 cases graded as of low malignancy survived 5 years , 42 per cent of 191 graded as of moderate malignancy and 25 per cent of 138 graded as of high malignancy ; when the histological grading was subdivided according to the presence or absence of invasion of the axillary lymph nodes , he found that with lymph nodes not invaded the 5 - year survival rates for gradesi , ii and iii were 94 , 61 and 53 per cent respectively , while for patients in whom the lymph nodes were demonstrated histologically to be invaded the survival rates for the three grades were 65 , 30 and 16 per cent respectively . 
as all types of treatment , surgery , radiotherapy and combined treatment , whether radical or palliative , were included , it is not surprising that few significant differences in survival rate were detected , though in most regions it appeared that the patients treated early showed little better survival rates than those who came late . in the present communication tabulations are presented from the same series of cases of the results of treatment of patients in stages i , ii or iii , who were treated by radical surgery alone or combined with radiotherapy , arranged according to duration of symptoms at the time of commencing treatment . 
by the elimination of all patients who were in stage iv , and of those in whose cases only operations of a palliative nature were performed , the series is restricted to those in whom there was a reasonable expectation of 5 - year cure . patients treated by radiotherapy alone have been excluded , because it is difficult to separate those in whose cases the treatment was intended to be curative from those in which it was palliative only , though some of the latter survived 5 years . the cases are grouped by site of the disease and by duration of symptoms at the time of commencing treatment into ( a ) , those in which the interval from first symptom to commencement of treatment was less than 6 months , ( b ) , those in which it was between 6 and 12 months , ( c ) , those in which it was more than 12 months , ( d ) , those in which it was unknown . 
the 5 - year survival rates for each of these groups is expressed as a percentage of the total in the group , and the standard errors of the differences have been calculated to test for statistical significance . in the more important sites the survivals are shown by stages : i . 
survd. over 12 , , not known the differences in survival rate are not statistically significant . for cases of known duration x2 = 1x77 , n = 2 , p < 50 > 30 . . 
among 383 patients treated by radical mastectomy alone , 35 - 5 per cent were in stage i and the best survival rate for all stages combined was 50 9 3 - 3 per cent for those treated during the first 6 months ; among 175 who were treated by radical mastectomy combined with radiotherapy only 21 * 1 per cent were in stage i and 43 * 4 per cent were in stage iii , and the best survival rate for all stages combined was 58 - 8 8 - 4 per cent for patients treated one year or more subsequent to the first symptom , who numbered 34 . 
the difference of 7 - 9 9 0 between these survival rates is not statistically significant . when the sites were subdivided into those in which the highest survival rates were among the patients who came under treatment in less than 3 months and those in which the highest rates were among those treated in the second 3 months , it was found that the former included squamous carcinoma of the skin , corpus uteri , penis and female breast with a survival rate for all groups of 54 - 9 per cent , while the latter included cervix uteri , kidney , .papillary carcinoma of the bladder and carcinoma of the pharynx ( treated by radical surgery or by combined methods ) with a 5 - year survival rate for all groups of 51x0 per cent . the age distribution of 702 patients with cancer of the breast who were . 
the group of 63 in whom the symptoms were of over 12 months ' standing at the time of operation had a survival rate of 14 3 per cent ; the difference of 9 9 7 0 between this figure and that for the group treated in the second 6 months is not statistically significant . if the patients are grouped into those of less than 6 months ' duration , with a survival rate of 11 - 2 per cent , and those of over 6 months ' duration , with a survival rate of 19 - 2 per cent , the difference of 8 - 0 4 - 7 is not significant . the age distribution of the patients with cancer of the stomach and colon were investigated by the x2 test , but in neither case were there any significant differences between the observed and expected survivals in the different age groups ; for the stomach cases p < * 20 > * 10 and for the colon cases p < - 70 > .50 , showing that the differences in survival rates were not due to differences in age distribution . 
the survival rate for cancer of the stomach was 20 - 4 per cent for 108 patients in the age groups 25 - 54 and 11.1 per cent for 133 patients of 55 and upwards , but the difference of 9.3 4 - 7 is not significant , though the prognosis was not so good in the older patients . 
as has been stated above this difference cannot be accounted for by differences in the age distribution of patients in either of the groups , so it must be presumed to be due to variations in the rate of growth of individual tumours all cases of patients with and in their infiltrating and metastatising powers . distant metastases have been eliminated from the present series , which was confined to those in which the tumour was local or had given rise to clinically recognisable metastases in lymph nodes ; they were selected as operable cases , no patient in an advanced stage or suitable only for palliative treatment being small differences were found in the proportion of patients in stage i included . among those with the highest survival rate in each of the three groups ; for group w . 
the mean duration of life in 153 untreated patients in group a was 23 - 1 months , of 176 in group b was 11 - 7 months and of 296 in group c was 17 - 6 months . 
the sites showing the longest survivals among the untreated patients are those in which treatment by radical surgery alone or with radiotherapy gives the best results , although some sites show a short duration of life , either because , as in the case of the lip , very few cases are left untreated , or because , as in the case of the colon and the intrinsic larynx , untreated patients often die of complications , such as intestinal w3w . 
group a included the skin ( squamous carcinoma ) , corpus uteri , penis , cervix uteri , female breast , kidney , bladder ( papillary carcinoma ) and pharynx . for this group of sites , comprising 1097 cases of known duration , the best survival rate was 51 1 per cent of 616 patients treated in the first 6 months against 42 - 0 per cent for the remainder . 
group b included salivary glands , tonsil , testis , colon , skin ( malignant melanoma ) , nasal sinuses , bladder ( infiltrating carcinoma ) and stomach . this group of sites , comprising 718 cases of known duration , the best survival rate was 30 - 6 per cent of 134 patients treated in the second 6 months against 27 1 per cent for the remainder . 
japha. received for publication february 2 , 1949 . martn and reese ( 1936 , 1942 , 1945 ) were the first to describe an extensive series of cases of retinal glioma treated by deep x - ray therapy . in their latest paper they report 75 per cent 5 - year cures in 8 cases of bilateral disease , one eye having previously been removed by surgery . 
at the middlesex hospital 19 cases of glioma retinae were seen between 1929 and january , 1946 . all cases were treated , 4 by surgery alone ; in the others radiotherapy of one type or another played a major part in the treatment . 
the numbers are too small to allow comparisons to be made between different types of treatment , but as regards deep x - ray therapy certain conclusions have emerged , and a technique has been evolved which differs in some respects from that advocated by the workers of the new york memorial hospital . the results of surgery have greatly improved in the last 30 years according to ray and mcclean ( 1943 )  . collecting their figures from the literature they estimate that between 1868 and 1941 , 18 per cent cures were obtained by enucleation in 677 cases , whereas 50 per cent cures are claimed for the 105 cases so treated between 1911 and 1941 . this progress is ascribed to the more radical and improved technique , often with partial excision of the optic nerve , used in later years . 
the number of bilateral cases included , if any , is , however , not stated . the authors themselves advocate a combined intracranial and orbital operation in view of reese 's ( 1931 ) statement that 52 per cent of 119 cases showed invasion of the optic nerve beyond the lamina cribrosa . duke - elder ( 1940 ) also states that 50 - 57 per cent of unilateral cases may expect to be cured . reese ( 1940 ) cites statistics of 95 cases collected by the division of ophthalmic pathology of the american registry at the army medical museum , 26 of which had bilateral disease . 
at the time of review 21 cases were untraced , 39 had died , 13 had lived 5 years , 2 had lived 3 - 4 years , 10 between 1 and 3 years , and another 10 just 1 year . besides x - ray therapy , which is historically reviewed by martin and reese ( 1936 , 1942 , 1945 ) , attempts have been made at treatment of the second eye m bilateral cases by radon - seeds . these are usually introduced either into the growth itself or sown on to the sclera over the neoplasm . stalhard , in his gifford edmond prize essay ( 1933 ) , reports 5 cases treated primarily by these methods . one of his cases - first reported on by moore ( 1930 , 1935 ) , was treated in 1929 ( twice by the insertion of a seed into the growth and also by episcleral seeds ) , is still alive , but has a cataract . three other cases , including an advanced case treated only experimentally and intended for subsequent histological investigation , came to excision . in a further report stallard ( 1948 ) gives the result of 15 cases so treated . 
deep x - ray therapy . - as long as radium moulds were being used it was inevitable that the whole of the globe was being irradiated to a high dose , but with the change - over to and use of small beams that were developed in deep x - ray therapy it became possible to limit the volume ofhigh dose to a certain extent . 
our technique has been directed at eradicating the focus of disease present rather than treating the whole of the posterior half of the retina ( where the majority of the growths are situated )  . 
a recent case , however ( not included in this survey ) , showed us that a certain risk isrun thereby in that shortly after the treatment and subsequent disappearance of a small lesion below the equator by fields of 2 and 3 cdiameter a further nodule appeared in the upper half of the retina . in an endeavour to treat as small a part of the eye as possible we have used circular fields of 2 and 3 cdiameter by means of special tubular applicators primarily intended for intrabuccal lesions as described by roberts and quick these are used in connection with our deep x - ray tubes , treatment ( 1943 )  . being carried out at 190 - 220 kv . 
lateral ( temporal ) , medial ( tansnasal ) , superior and inferior orbitals . abandoned as soon as it became apparent that the cornea was the most vulnerable structure of the eye , and disintegration of the eye had occurred in 2 or 3 cases in which this approach had been used . the lens , too , will receive a relatively large dose of irradiation when the anterior field is used and the lens dose should be kept at aminimuwe endeavour to ensure this by our technique of " by - passing " the lens as much as possible , which can be achieved in all the other fields mentioned . the anterior field was fields used so that accuracy ofalignment positioning . - special problems ofset - up arise because of the age of the patients and the small is of much greater importance . most of the children under the age of 4 need steadying during each treatment , and the older ones atleast during the first 2 or 3 sessions . we have in a few cases tried to make individual plaster casts into which the child is put during treatment , the cast including a headpiece with a frontal process to obviate any possibility of head movement . in practice , however , even the smallestchildren have been found to wriggle around in these casts human hands have had to help . the casts are usefuil , nevertheless , for steadying at least the trunk , and one escapes the otherwise necessary manoeuvre of binding the child on to the couch . children this can be done best by first wrapping the child tightly into a blanket , arms included . all cases - - and at each treatment - are set up by a medical officer , and during treatment , if necessary , the head steadied either by himself , by a radiographer , or by a nurse - or , if available , one of the parents . 
and by treatments lasting 16 or 57 days , but the number of cases observed is too small to confirm the impression that neither dose rate nor the total time are of critical importance within a wide range . 
irrespective of whether radium - moulds or deep x - rays were used , including one case in whom radium - moulds of increasing strengths were applied at short intervals within 5 months , the highest single dose being 3800 r . two main treatment schemes have been used : either a single " radical " course or repeated courses of deep x - rays , in each one of which a dose was given just sufficient to make the tumour disappear . this was usually achieved by doses of 2000 - 3000 r . , to be repeated as soon as there was renewed activity . the latter scheme is somewhat similar to that recommended by martin and reese ( 1936 , 1942 , 1945 ) , but in our hands it has never been successful , whereas the single radical course of treatment has given us outright cures . we have only once had the opportunity of treating a unilateral case ab initio . this was a girl , aged 3 years and 9 months , who came with a history of over 2 years but still only a comparatively small growth . 
ten months later , however , there was a large recurrence and the eye was excised . histology confirmed the presence of active growth . sometimes it has been necessary to continue the first course up to a much higher tumour dose . there was one case in our series where even the second eye carried a growth covering almost the whole of the retina - he had come to us with simultaneous affection of both eyes . after the excision of the first eye , which was completely filled by growth , a tumour dose of 5730 r . 
japha in this part of the body as anywhere else - but perhaps even with greater force - is one struck by the confirmation of the hypothesis most prevalent for the explanation of failures through over - dosage . 
we have not as yet given postoperative treatment to the orbit of the removed eye in bilateral cases . this may be advisable as a general policy in view of the ultimate fate of some of them , i.e. 
local recurrence even where no apparent extra - ocular extension was originally present , unless one regards these deposits as due to retrograde extension from the second eye , which by that time has got completely out of control . 
one should also remember , as duke - elder ( 1940 ) points out , citing meighan and michaelson ( 1938 ) , that there may be discontinuous spread in the nerve , so that the excised specimen ( even if part of the nerve is included ) need not necessarily show the extra - ocular extension which has in fact already taken place . carrying out this suggestion the contribution that the lateral fields will make to the retained retina or the enucleated orbit respectively and which will amount to a depth dose of about 20 per cent must be taken into account . this technique has been used in a recent case which had a recurrence in the excised orbit as well as growth in the second eye , and is also applicable in the " prophylactic " irradiation of a case in which bilateral enucleation has been performed . when we first collected these cases it appeared as if the unilateral cases treated post - operatively by irradiation would not be of very great interest because surgery alone might have been responsible for the good results . 
we have , however , in our series one case of recurrence within 6 / 12 of the operation ( this was treated by a combination of deep x - rays and implantation of radon - seeds , but recurred again and proved rapidly fatal ) , and the above - mentioned case , which may be regarded as having been inadequately treated in the post - operative period . this case had a remarkably long latent period of 5 years , and a very large dose of irradiation was necessary to arrest the growth . 
no growth was found on histological examination . one case of massive recurrence in the orbit with bone involvement after enucleation of the only affected eye failed to respond to combined irradiation by deep x - rays and implantation of radon needles ; another case , successfually treated , is of considerable interest . it is the case already mentioned which received a post - operative prophylactic dose of 660 r . 
by deep x - rays in 16 days for a recurrence after treatment by radon - seeds elsewhere ( tumnour dose estimated at 2000 r . ) , was complicated by chronic iritis and needed extraction of this the remaining eye 4 months later ( now 52 years ago ) - no growth was found on histological examination , as already mentioned . 
 ( in 57 days ) ; this was extracted 4 years ago and vision is only partial another cataract that was removed was obvious already 9 months after now . a dose of 3000 r . 
hfirst came under observation at the middlesex hospital in july , 1945 , at the age of 9 / 12 , having had his left eye excised elsewhere 3 months before ( histology showed retinoblastoma without involvement of the optic nerve ) , and at the same time a focus the size of a disc in the periphery of the right retina had been isolated by diathermy barrage . 
about a fortnight after this examination the boy began to vomit ( apparently in a rather projectile manner ) and became drowsy . lvhen examined again on 20.xi.46 there were signs of brain - stem involvement , and a course of deep x - rays directed there was some temporary improvement , but the at this region was started . child died within 3 months of the onset of the final stage . 
lumbar puncture during this time showed the presence of peculiar mononuclear cells in the cerebrospinal fluid , which in the light of the later post - mortemn findigs must be regarded as retinoblasts . 
m - 1 , a boy , 5 years of age , was sent to the middlesex hospital in 1929 from jersey , where 5 months previously his right eye had been removed for glioma retinae . 
a radium mould was applied as a " delayed post - operative measure , " but the surface dose when recalculated recently was found to have been only 660 r . 
 ( in 36 hours )  . nearly 5 years later there was a large swelling in the orbit whiceh was treated by x - rays in jersey ( details unknown ) and the swelling disappeared . 
a few months later a further course of x - rays was given for a " small hard bean - shaped swelling " in the lower half of the orbit . seven months after this the mass was again thought to be larger and the boy referred back to the middlesex hospital in september , 1936 . 
the boy was lost sight of during the war , but reported again as a young man in october , 1946 , that is , 10 years later , when he presented with marked telangiectases on the right cheek around the orbit and a small sinus over the zygomatic arch . 
the results of radiotherapy are summarized in table vii . ( a ) post - operative irradiation has been successful in the two cases where adequate treatment was given irrespective of whether a radium mould or deep x - rays were applied . in the one case described at length above , the first treatment , where 660 r . 
only were given , must be regarded as quite inadequate , but the recurrence which developed 5 years later responded well to a radical course . ( b ) primary irradiation . - the two cases treated by radium moulds and two of the three cases treated by deep x - rays more than 5 years ago are alive and e . 
for example , greenstein and his associates have shown that the activity of certain liver enzyme systems is affected to a considerable extent by the presence of a distant tumour , and of those investigated , liver catalase appeared to be the most sensitive . 
in fact a marked decrease in the activity of this enzyme has been observed in rats and mice bearing a variety of spontaneous , transplanted , and chemically induced tumours ( greenstein and andervont , 1942 )  . 
the evidence connecting the liver catalase depression with the presence of a tumour has recently been reviewed , both by greenstein ( 1947 ) and by weil - malherbe and schade ( 1948 )  . 
either the tumour may release some toxic material into the circulation , or its excessive nutritive demands may abstract from the circulation some substance essential for the maintenance of a normal liver catalase level . 
 consequently , quantities of tissue breakdown products are likely to be released into the circulation , and any toxic material associated with tumour growth may be due to such relatively non - specific processes . 
there is in fact evidence ( winzler and burk , 1944 ) that abnormally large quantities of circulating protein break down products such as proteoses and polypeptides are found in tumour - bearing animals , and these authors did tentatively suggest that liver catalase depression might be due to this cause . 
excessive nutritive demands of the tumour , greenstein and andervont ( 1943 ) showed that the depression was not simply due to the presence of growing tissue within the host , since catalase activity remained normal both in pregnant mice , and in mice bearing progres sively growing , subcutaneously implanted , embryonic tissue mash . 
 greenstein ( 1943 ) looked for a direct inhibitor of catalase in tumours , arul in the livers of tumour - bearing animals , but the results were all negative . 
greenstein interpreted his experiments as indicating that the affected livers simply contained less catalase than normal , rather than the same ' amount of catalase plus an inhibitor , and_put forwal ' _d the interesting l ' ! lj.ggestion that the decrease in activity was due to an interference with the _synthesis of the enzyme . 
however , no depression of liver catalase activity was observed even after long periods of administration of commercial peptone or sheep seru sheep serum in fact resulted in a rise in activity , the significance of which is unknown . 
further , these authors in vestigated the possibility that the depression might be due to the exaggerated protein requirements oi the tumour , by maintaining two series of tumour - bearing rats on high and low protein diets respectively , but no significant differential effect on the course of the process was observed . 
 most previous work appears to have been carried out with relatively massive tumours ( roughly within the range of 5 per cent to 50 per cent of body weight )  . 
 it is true , however , that while miller ( 1947 ) found that starvation up to about 7 days caused a marked depression in liver catalase level , he also showed that total liver protein fell in an approximately parallel manner . 
there appears to be a possibility that the effect is of a relatively specific nature , given only by malignant tissue , but the evidence presented in the literature does not ; enable a decision to be made on this queetion . 
so far as the results go they are in support of the supposition that tumours contain a substance not present , or - present in much lower concentration , in normal tissue , which is capable of depressing catalase activity~ animals . 
during the period of the experiment the diet of the animals consisted of " rat cubes " ( north - eastern agricultural co - operative society , aberdeen ) and water ad libitu as a routine the mice were starved for 12 hours before catalase determination  . 
 most of the work has been carried out with the transplanted tumour sarcoma 37 ( obtained from the chester beatty research institute ) , which grows rapidly in both strains of mice after subcutaneous inoculation . 
since , however , the non - protein nitrogen amounts to less than io per cent of the protein nitrogen and appears relatively constant , total nitrogen has been employed as a standard in this investigation . 
f measurement , but to construct a standard curve of enzyme concentration in arbitrary units ( using different volumes of the same liver homogenate ) against hydrogen peroxide decomposed in 4 minutes . 
since the rate of hydrogen peroxide decomposition falls off rapidly with increasing enzyme concentration , it was found necessary to construct a curve covering a relatively wide range of concentra tions . 
of 15 per cent w / v potassium iodide and 3 drops of 5 per cent w / v ammonium molybdate were then added , and the liberated iodine titrated with n / 20 sodium thiosulphate . 
all enzyme determinations have been made in duplicate , and , for routine estimations , the quantity of enzyme taken has normally decomposed between 1 / 3 and 2 / 3 of the substrate during the course of the reaction . 
adams determinations , catalase activity in arbitrary units has been calculated by sub stituting the quantity of hydrogen peroxide decomposed in the equation to this line , and the result divided by the nitrogen content in mg . 
this was obtained by dissecting out tumours previously inoculated into ff mice , removing any necrotic areas , and homogenizing the actively growing tissue in a loose - fitting ten broeck grinder just sufficiently to give a coarse suspension . 
it may be seen that at 24 and 48 hours after injection the level is considerably depressed , that it rises approximately to normal at 4 days , and then falls progressively until 14 days , when the experiment was terminated . 
 because af this , and also since the amount of the initial ( 24and 48 - hour ) depression is not approached again until the new tumours are of a considerable size , it seems not unreasonable to suggest that the initial drop is due to some material contained in the injected tumour homogenate , that this effect is rapidly exhausted ( 4 days ) and that only the second depression ( > 4 days ) is due to the presence of the new tumour . 
although the treated groups become very small when split up , the initial ( 24and 48 - hour ) percentage depression in catalase activity appears to be considerably greater in the males . 
the experiment was repeated with schofield albinos , using s37 taken from the same strain , and larger treated groups to enable the sex difference in initial effect to be examined further . 
3 , where it will be seen that there is an early depression in enzyme level , a rise approximately to normal in 4 days , and a subsequent progressive fall . 
each point represents 6 tumour - bearing animals , and has been obtained from the arithmetic mean of the catalase level for the treated animals , and the arithmetic mean of the appropriate control group . 
this method of presenting the experimental data is by no means ideal , but the individual results would be very difficult to obtain , since no informa tion is available regarding the original oatalase level of a tumour - bearing animal . 
according to the graph , these authors obtained a rapid fall in 2 days , after which the level remained approximately constant until 4 days , and then fell once more . 
the salient point is that their results cannot be separated into " early " and " late " effects in the absence of the 4 - day rise to normal . 
their tumour must have grown extremely rapidly since they obtained a 90 per cent depression in 7 days , and greenstein and ander vont ( 1942 ) probably employed the usual technique of grafting small pieces of tumour subcutaneously rather than injecting a homogenate . 
it is fairly obvious , from the results already given ; that if the onset of tumour growth is sufficiently rapid to affect the catalase level at 4 days , the rise to normal after the initial drop will be obscured , or perhaps entirely obliterated . 
 a number of normal tissues have been coarsely homogenized ; and injected subcutaneously into male mice , which , as has been already pointed out , are more sensitive than females . 
 since the initial ( 24 and 48 hour ) depression is of primary interest , the effects on catalase level have been followed over a period of 4 days , the results appearing in table ii . 
the results in table ii show that a 24and 48 - hour depres sion was observed , although an appreciably smaller one than with s37 , and that the catalase level rose to normal at 4 days . 
 because of the much delayed onset of tumour growth , and because " takes " only occurred in a.bout half the animals it is not possible to state definitely whether the females showed a greater resistance to tumour growth , although there appeared to be a tendency in this direction . 
140 25 carcinoma 63 105 21 101 16 113 14 123 22 131 34 101 13 75 19 145 28 125 22 108 26 113 17 123 29 127 19 95 13 79 13 92 33 113 33 116 14 114 19 128 17 131 27 125 20 95 11 117 6 an experiment with coarsely homogenized necrotic s37 tissue yielded the following result : no significant depression was observed at 24 hours , but by 48 hours the level was considerably below normal , beginning to rise at 4 days . 
 since attention does not appear to have been drawn to a sex_ difference in normal mouse catalase levels , some results for males and females drawn from the same batches are given in table ill . 
 80 21 123 18 90 14 as pointed out in the introduction , the alternative mechanisms by which tumours may cause a catalase depression can be given thus : discussion . 
it is hardly possible that this early depression can be a result of any nutritive demands on the organism , and can only be due to some substance or substances present in the injected tumour tissue . 
assuming that the early and late depres sions are not entirely unconnected , it is suggested that the tumour is exerting its effect on catalase by continuously releasing this material into the circulation . 
of homogenized tumour tissue ( administered by the relatively inefficient method of subcutaneous injection ) is sufficiently active to depress the catalase level very considerably for approximately 2 days , it is probable that only a comparatively small continuous production would be required to account for the observed results . 
further , the point at which the graph flattens corresponds approximately to the tumour weight at which necrosis commences , and , for example , a tumour weighing 2 to ; 3 g . 
also , after subcutaneous injection , these tumours grow in the subcutaneous space and do not , as a rule , perforate the abdominal wall until they weigh 4 to 5 g . 
consequently little or no normal tissue can be destroyed by the growth of the tumour ; and even if this process were occurring to some small extent , the flattening of the curve at a given point would be unexplained . 
schultz and kuiken ( 1941 ) briefly mention that normal male rats have higher liver and kidney levels than females , and serfaty ( 1946 ) , working with the erythrocytes of fowls , found the level to be higher in cocks than in hens . 
 it seems reasonable to suppose that the maintenance of a normal catalase level is to some extent under hormonal control , and the different initial response of males and females to tumour tissue may indicate that the primary effect is not on the liver . 
these graphs make it clear that not only does homogenization enhance the initial depression ( as might be expected if the effect is due to some substance contained in the tumour cells ) but that such a procedure 194 d . 
their results show only a progressive fall in catalase level after the tumour implantation , and provide no evidence which could suggest that tumour tissue contains a toxic product capable of depressing liver catalsea activity . 
in some experiments the arithmetic mean of the results for the treated animals have been slightly lower than the arithmetic mean values for the controls , in others , slightly higher . 
it is impossible at present , therefore , to say whether tissues are capable of causing a slight depression , or whether normal the effect is wholly specific to tumours . 
after this process the material could be compared with similar fractions from normal tissue , and a sufficiently large dose given to show whether the cata ] ase depressing material is present at all in normal tissue . 
 it is already known ( greenstein and andervont , 1942 ) that , in general , the greatest falls in liver catalase level are produced by rapidly growing tumours , and , in fact , certain s ] ow - growing tumours produced little or no effect . 
 in the present investigation it appears that s37 tissue , which grows readily , gave a greater 24 and 48 - hour depression than carcinoma 63 , which does not . 
 following the subcutaneous injection of homogenized sarcoma 37 tissue into two strains of mice , the liver catalase activity fell significantly at 24 and 48 hours , subsequently rose to normal by the 4th day , and diminished again during the growth of the new tumours . 
there was also a sex difference in the initial response to the injection of tumour material , the males , which have the higher normal level , being more sensitive . 
bielschowsky. from the , hugh adam cancer research department of the medical school and the new zealand branch of the british empire cancer campaign , university of otago , dunedin . received for publication april 7 , 1953 . judging from the literature , adenomata occur more frequently in the pituitaries than in the thyroids of aged rats . 
at the routine autopsies of 154 albinos of the wistar strain , 41 of which were males , 86 intact and 27 spayed females , 24 cases of adenoma of the pituitary were discovered . seven of these occurred in the males , 16 in the intact females and only one in an ovariectomised animal . the average age of these rats was 2 years . 
adenomata of the thyroid were found in 5 animals , 4 times in combination with neoplastic lesions of the pituitary . post - mortem examinations performed on old rats of a more recently acquired hooded strain have furnished three additional cases of tumours of thyroid and pituitary . 
the diet consisted of a dry mixture of bran 30 per cent , pollard 35 per cent , meat meal 15 per cent , maize meal 15 per cent and bone flour 5 per cent , supplemented by wheat and kibbled maize , and once weekly by green vegetables and cod liver oil . drinking water was supplied in liberal amounts . 
the weight of the animals was recorded at fortnightly intervals and they wer6 sacrificed when loss of weight and general appearance indicated ill - health . at autopsy the pituitaries were immediately transferred into a pre - weighed bottle containing about 2 ml . 
when treated with gomori 's fuchsin aldehyde reagent , the cytoplasm of the angular cells and especially their granules stained purplish - violet . in other words , these cells strongly resembled and gave the staining reactions of those basophils which purves and griesbach ( 1951a ) named " thyrotrophs . " therefore the tumour is considered to be a basophil adenoma formed by thyrotrophs . in the part of the anterior lobe outside the basophil adenoma the basophils were considerably increased in numbers . angular types again predominated , but here vacuolated forms , extremely rare within the tumour , were numerous . these cells were identical with those seen after partial thyroidectomy . they gave a positive pas reaction of varying intensity , and with the gomori stain some were found to contain coarse violet - purple granules . the large round gomori 206 f . 
5 , the larger of the two protrudes above the anterior surface and is closely connected with the pars internmedia : the other , just appearing in the section , bridges the pituitary cleft and is situated laterally . 
the pituitary was of irregular shape with a small this was a well defined basophil adenoma , the haemorrhagic brownish area . tumour cells giving the same staining reactions as described above . 
the neoplastic changes found in the hypophysis and thyroid of the younger animal were of microscopic size . in the slightly abnormally shaped anterior lobe numerous thyroidectomy cells and also multiple nodules formed by atypical basophils were present , the pars intermedia being normal . 
normal anterior lobe tissue was recognisable in the periphery as a narrow rim surrounding approximately half of the circumference of the tumour . radiating from this rim compressed strands of predominantly acidophilic and chromophobic elements were seen between areas of tumour , an indication that the neoplasm had arisen from different foci . intermediate and posterior lobes appeared normal . 
the cells composing the tumour were large , showed great variation in size and shape , had sharply defined cell borders and vesicular nuclei with one or more large fuchsinophilic nucleoll . multi - nucleated and tumour giant cells were rather numerous and so were mitoses . 
when stained by the pas technique or with gomori 's aldehyde fuchsin after long search an occasional only in this respect , cell was found having pas or gomori positive granules . did the adenoma differ from those described above . in papanicolaou preparations the cytoplasm stained faintly with light green . the histology of the thyroid was that of an inactive gland . 
many mitoses were found in this nodule , considered to be a benign pheochromocytoma . rat 8 , a 26 months old male , was killed because of the presence of an abscess otherwise the animal was in an excellent in the subcutaneous tissue of the neck . state of health and weighed 460 g . 
the first question to be answered is : which cells produce the thyrotrophic hormone and the second , how many types of basophils occur in the anterior lobe of the hypophysis . 
as to the second , romeis ( 1940 ) described two types of basophils both stainable with aniline blue but distinguishable by their affinity to creso - fuschin or resorcinol - fuschthe type stained by these fuchsin dyes is the beta cell , the other is the delta cell of romeis . 
as shown by halmi ( 1950 ) another elastica stain , gomori 's fuchsin aldehyde reagent , also differentiates between two types of basophils , whereas most other techniques like pearse ( 1952a ) does not papanicolaou 's or gram 's method stain all basophils . seem to attach great significance to these different tinctorial qualities and adheres to a unitarian view . 
now the pathogenesis of the basophil tumours can be discussed . chronic iodine deficiency lowers the thyroxine level in time , to which the pituitary responds with an increase in thyrotrophic cells . this compensatory hyperplasia progresses towards the formation of tumours , in this connection it might be which in some instances reach considerable size . mentioned that proliferating normal thyrotrophs , as observed by guyer and claus ( 1937 ) , tend to aggregate in clusters , a mode of growth which might be favourable to nodular hyperplasia , the first stage in adenoma formation . 
the adenoma of this animal was functionally inactive as judged by the appearance of the thyroid which was that of a resting gland . also cells giving a positive reaction for glycoprotein were virtually absent in this tumour . the most convincing evidence for funttional activity is provided by rat 4 . the large pituitary tumour of this animal contained numerous pas and gomori positive elements and its thyroid showed the most pronounced signs of stimulation . in addition , in the pituitary of this animal , so little normal anterior lobe tissue was left that it seems most unlikely that this small rest could have secreted amounts of thyrotrophin large enough to induce a four - five fold enlargement of the thyroid . finally the pituitary lesion of rat 8 has to be considered . in this case the morphological evidence was more in favour of hyperplasia than of neoplasia . here an agglomeration of thyrotrophs had persisted in one area despite the fact that ki had been supplied for 7 months . in the rest of the anterior lobe these cells were found in normal numbers , as was to be expected , and the thyroid had undergone involution . rich glycoprotein content of the cells forming this aggregation was probably due to storage and not a sign of secretory activity . the anomalous behaviour of these cells suggests that they were no longer normal thyrotrophs . the thyroid tumours observed are , in the writer 's opinion , due to stimulation by elevated amounts of thyrotrophic hormone . the stimulus for the increased secretion by the pituitary was a deficiency of thyroxine due to impaired synthesis by an inadequate content of iodine in the diet . in six instances the thyroid tumours were found in activated and twice in resting glands . the latter occurred in animals which had received a supplement of ki for several months and are considered to be evidence of past stimulation . these neoplasms differed morphologically from those observed in stimulated thyroids in the same way as the thyroid adenomata found during administration of thiouracil differ from those seen after the treatment with the goitrogen has ceased . 
their epithelium was rather low , and cystic follicles filled with colloid were a prominent feature . to summarise the pathogenesis and structure of the thyroid adenomata observed is similar to that of the tumours induced by goitrogens which act by blocking the synthesis of thyroxine . the conception that thyroxine deficiency is the ultimate cause of the syndrome described is strengthened by the following observations . fischer ( 1926 ) , writing from bern , switzerland recorded two cases of pituitary tumours found in aged female rats . 
the thyroids of both animals contained multiple tubular adenomata , and in one an adenocarcinoma was also present in this gland . the rats belonged to wegelin 's colony in which goitre was endemic fischer 's paper was published long before the gonadotrophic and thyrotrophic hormones had been discovered . she stressed , however the similarity of the pituitary tumour cells with the ele212 f . 
bielschowsky ments which appear in the rat 's hypophyis after castration or thyroidectomy ; and considered the neoplasms to be carcinomata formed by " castration " cells fischer believed that although the ovaries of one of her animals were normal . the neoplasms originated from the pars intermedia or tuberalis . " spontaneous " tumour formation occurring simultaneously in thyroid and pituitary of rodents seems to have been observed only by fischer and by the writer , but one sees this syndrome under experimental conditions most frequently in animals t ' reated with so purves and griesbach ( 1951a ) mention goitrogens for prolonged periods . a basophil adenoma of a rat treated with methythiouracil for two years . basophil adenomata occur also in thyroidectomised rats treated with acetylaminofluorene into the same class belong also the pituitary tumours ( unpublished results )  . discovered by gorbman ( 1950 )  . 
they appear in mice exposed to high doses of radioactive iodine , but can be prevented by the administration of thyroxine ( goldberg and chaikoff , 1951 ; gorbman , 1952 )  . furth , gadsden and upton ( 1952 ) reported that transplants of such tumours contain large amounts of thyrotrophic hormone . 
1 , the method of analysis employed compensates fully for difference in " size " of the pattern analysed , and the p values for grade and intergrade patterns can therefore be regarded as strictly comparable . if we regard p < .05 as evidence of abnormal patterning , then a glance at the table shows that abnormal patterns occur with considerable frequency . this is best seen by comparing the frequency distribution of the 224 grade and 192 intergrade p values of the table with the strictly equivalent frequency distribution of the 4000 random series ( cruickshank , 1940 )  . 
1. statistical maps often present data by the use of " grades . " the several counties in any one grade may be scattered " at random " over the map , or may be juxtaposed to form small or large these varying types of distribution are described briefly as " patterns . " the exact " groups . " type of pattern to be expected from random distributions has been determined experimentally ( 4000 patterns ) , and from these experiments the " expected frequency " of any specific type of rare types of patterns , i.e. 
patterns with low expected frequency , are observed pattern is known . described as " highly developed . " on cancer maps highly developed patterns occur with excessive frequency . indeed , " statistically significant " patterns , i.e. 
2 and 4 will show that p values across the gradings ( 1 - 12 - 1 - 23 7 ) give rise to curves with clearly defined trends , which include the develop114 d . 
2. the " intensity " of a regional influence can be expressed in terms of the " degree of development " of patterns found within its zone of action ; the more non - random the pattern the more powerful the influences at work . the analysis is best shown graphically ; the expected random frequency of the 7 grades and 6 inter - grades patterns of each map are plotted out on an i * erted logarithmic scale so that " peaks " show high degrees of pattern development - that is , powerful regional influences . the striking feature of the curves so formed is their essential simplicity . in fact they are almost e.g. 
that a significant " zone " is not always associated with a significant peak . in such cases the individual counties of the zone each show a deviation from the mean > 2 s.e. 
that curves of type 2 which develop a peak before the extreme grades and then fall off are usually associated with maps in which the highest ( or lowest ) grades do not form a single zone , but break up into two or more sub - zones , e.g. grade 7 may appear as three separate zones , unrelated to each other , but all related to grade 6 ,  . . 
the frequency of effects " increasing " and " decreasing " the rate are shown separately ; the frequency of effects " modifying " the rate ( significant ] y ) is the sum of these two . in the absence of regional influences we anticipate that a county will participate in " random " significant pattern formations in about 1 / 20 cases , i.e. 
 ( stocks ' data )  . now , if the distributions and trends in each of the 28 maps are unrelated , the result of combining these inter - random distributions should be to flatten out the whole pattern ( irregular multiple hillock picture )  . 
on the other hand , if they are related , their combination may be expected to result in some definite composite pattern or configuration ( hill and valley picture )  . from the entries of table vii three contour maps were prepared ( appendix 7 ) ; these are shown in fig . 
we may therefore conclude that county boundaries have no essential significance in relation to regional influences . in fact these influences behave in every way as continuous variables , and their variations in intensity are most appropriately depicted by means of contour - lines on the surface of the map . to depict these regional influences by contours may serve to drive home another of their characteristics as revealed by this study , viz . 
cornwall , somerset and dorset . while the majority of counties meet at a common county boundary , occasionally they meet only over a very narrow range , the so - called " point contacts " ( solid circles on map )  . 
where the county boundary is a river or an estuary the counties may fail to make a de facto cantact , and yet , it may be necessary to consider these as " touching " for the purpose of studying regional influences ( solid rectangles on map )  . note the distinctly higher values of the contact numbers of the inland counties as contrasted with those of the coastal counties . the mean contact number ( c ) works out at 5 - 12 . , , ~n this is an important constant in theoretical formulae , e.g. 
mark on the glass with grease pencil all " grade 1 " counties . remove glass template and place on white sheet . analyse arrangement of marked counties ; record ; clean off wax pencil mark with chloroform . replace on map and proceed similarly to work and analyse " grade 2 " counties , and so on . 
8 , in conjunction with appendix 4 , indicates the probable , simple explanation , viz . that estimates of the degree of pattern development of an organized pattern must generally fall below the true value when they are based on the examination of only part of the pattern - for the simple reason that the relation of one " piece " of the pattern to a neighbouring " piece " is thereby overlooked . 
the process of " intergrading " was evolved to deal with this contingency . thus , to explain the observed fact that the p grade greater than p intergrade we must assume that the mean " pattern " associated with regional influences covers an area greater than that of a mean grade , i.e. 
regional influences decreasing cancer exist alongside the " positive , " in fact these two types of influence , both of which are represented by peaks and zones , occur with equal negative effects must not be regarded as merely due frequency ( table vii )  . to the absence of positive effects , for the observed type of trend and degree of patterning associated with negative zones is of a much higher order than is to be expected from the mere absence of positive effect . it seems that there must be some general factor other than regional which operates to produce a definite incidence of cancer . regional influences are not themselves the cause of cancer , but merely impress their effect , positively or negatively , upon this general factor . i wish to express my thanks to n . 
jensen. frm the danish cancer regi8try under the national anti - cancer league , kobenhavn 0 . received for publication january 31 , 1953 . in 1947 clemmesen and busk reviewed the material of the danish death certificates with a diagnosis of cancer of the lung from the years 1931 to 1945 ( clemmesen and busk , 1947 )  . 
they found an increase in the numbers for males , particularly pronounced in copenhagen , the capital , less striking in provincial towns , and somewhat smaller for rural areas . however , the incidence of cancer of the lung among persons subject to routine examination in the central tuberculosis station for the city of copenhagen did not reflect the heavy increase in mortality found in the city as a whole . 
diagnose a number of cases of cancer of the lung , so that the graph illustrating the incidence rates in its material should be changed , as shown in fig . 
1. during the years up to 1950 the increase in mortality rates for cancer of the lung among men in the various areas of denmark continued , and from fig . 
women. of the disease , and only to a small extent attributable to improvements of diagnosis , which can be expected to cause a proportional increase for both sexes in the number of cases diagnosed . 
the possibility that the deciding factor might be an increase in medical attention for all categories of diagnosis can be excluded because the patients from the tuberculosis station were unselected and referred directly for diagnosis with a view primarily to a disease different from bronchial carcinoma , and finally were examined in a uniform way . further analysis of the material from the danish cancer registry confirm the results from mortality statistics ( clemmesen and busk , 1947 ) that the increase in incidence of cancer of the lung among men is particularly strong for the relatively younger age classes ( clemmesen and nielsen , 1952a , 1952b )  . this seems to speak against the assumption that atmospheric pollution is a causative factor of primary importance to cancer of the lung as far as denmark is concerned . if this were the case we would expect an increase in incidence for all age classes . it might also be expected that the windy climate of copenhagen could cause regional differences in incidence of the disease within the city analogous to the findings of stocks ( 1952 ) for london , but in fact it appears that the distribution of cancer of the lung among men in copenhagen varies with the social level expressed in terms of annual house rent , much like cervical cancer of the uterus being most frequent in the classes of subdistricts with the lowest level of house rent , although the single subdistricts show no such parallelism . however , there is no tendency for leeward areas to exceed the weatherside with regard to incidence rates for carcinoma of the lung ( clemmesen and nielsen , 1951 )  . thus , it would seem that the main reason for assuming atmospheric pollution to be causative to cancer of the lung in denmark would be the excess of cases in towns , particularly in copenhagen . 
5 represents mortality rates at various ages for lung cancer among men in copenhagen arranged in quinquennial cohorts born in the period about the years given in the diagram . it would seem reasonable to assume that the carcinogenic influences determining the incidence of cancer of the lung , whether they be of customary , occupational or hormonal character , will not begin before the age of 15 or perhaps a few years later . it appears from the tables that the cohorts of highest mortality at an conearly age do not show an increase in rates until after the 35th year of age . sequently it will be justifiable to assume a minimal period of about 20 years from the beginning of the influence to the death of the patient from cancer of the lung under the conditions usual in copenhagen . it follows logically that if we assume a later or earlier beginning of the carcinogenic influence we must respectively shorten or prolong the assumed period with a corresponding number of years . it should also be remembered that we must allow for wide variations either way . under these assumptions and from the observations illustrated in fig . 
5 that the graphs for the quinquennial cohorts do not coincide as they would for most other sites of cancer , but are moving to the left , although now at a decreasing pace . this would not be explained by assuming that all cohorts were from a certain age subject to some carcinogenic influence unless we add that the influence was strengthened at some point of time , or during a limited period of years . now it is seen that the result of the carcinogenic influence increased particularly for men born between 1875 and 1885 , while the effect on the following cohorts progressed at a slower rate . 
2 , it will be seen that the differences in the level of the graphs indicating crude mortality rates for cancer of the lung between capital , provincial towns and rural areas can be ascribed to a later beginning of the rise in the latter categories , i.e. , 8 years for provincial towns and 10 for the rural areas , which is in full conformity with the result of the cohort analysis . hence it appears that the assumption of atmospheric pollution as a cause of cancer of the lung in denmark in order to explain the differences between towns and country is unnecessary , if we suppose that the carcinogenic influence began a few years later in the provincial towns and a few years later still in the country . quite apart from the etiological implications of our cohort study it is in the field of practical hygiene we find the widest consequences of the diagram of fig . 
some important records of pedigrees , showing apparent . transmission from mother to daughter , were published nearly a century ago . the most remarkable was recorded by broca ( 1866 ) , in which four generations of females had the disease . in spite of evidence brought forward by a great many observers that mammary cancer can frequently be found affecting several of the female members of a family group , doubt , still remains as to whether heredity plays any significant ' aetiological part . this doubt arises because of the great prevalence of the disease in the general population , where it is responsible for nearly 3 per cent of all female deaths . 
with so comm ' on a condition , it is difficult to demonstrate convincingly that the occas ' lonal familial conoentration is not merely the result of a random distribution of cases . to obviate this uncertainty , most investigators in recent years have collected a series of cases , noted the incidence of similar disease among their relatives , and compared it with the corresponding incidence among a control group of cases free from the disease , selected so far as possible at random . in spite of elaborate precautions , - the difficulties , involved in obtaining a satigfactory control group of family histories to match those of the propositae , have repeatedly proved almost insuperable . 
the result is that many workers ( hanhart , 1943 ; passey , 1942 ) still ' consider that there is , insufficient evidence to prove the reality of familial transmission of mammary cancer or of aay type of human ma " lignant disease . stocks and karn ( 1933 ) found no evidence that a history of cancer in relatives increased the risk of cancer to anymeasurable extent . several extensive surveys have been published , all of which give some evidence for believing that mammary cancer is unusually prevalent amon the mothers and sisters of affected cases . 
the patients were attending either university college ilospital or the london hospital , and they were personally interviewed . all possible hospital records and reports were searched or checked by correspondence . this procedure applied b6th to the propositae and to any of their relatives who were reported to have suffered from malignant disease . unfortunately , in a number ' of instances , records had been destroyed by enemy action during the war . ideally , the death certifica ' te of every relative believed to have died of malignant disease should have been obtained , as corroborative evidence , but th ' is we have not been able to do . 
the distribution of these 116 " familial " cases and the mean age of onset of their illness ( 5 1 0 years ) was extremely similar to that of the this finding does not support jacobsen 's remaining " non - familial " cases . assertion that familial cases have an earlier onset than others . 
the infertility ratio , 91 out of 408 , or 22 - 3 per cent , significantly exceeds the value of 17 - 3 per cent for married women of the same mean age given by the registrargeneral ( 1938 ) , and supports the view that nulliparity is an etiological factor . the mean age of the patients when first interviewed for purposes of this this indicates that they had , on the average , lived survey was 55 - 2 years . undoubtedly three - and - a - half years since first gigns of the disease had appeared . the survival of the patient was a property , which determined her inclusion in the investigation , and the longer the survival , the more probably would she be selected as a proposita . 
the date of death and the age at the time were recorded in a few instances the dates were approximate , but could hardly for each case . be more than a few years in error . 
the expected proportion of deaths due to mammary cancer and other types of malignancy in each year and at each age can be obtained on the basis of the general population statistics . 
the distribution of deaths by year and age is shown in table iii . from ' this table the number of deaths due to mammary cancer if the women had been subject to the mortality in the general population was estimated at 11 - 17 , a figure significantly less than the observed 25 . 
51 observed as compared with 48 - 76 expected . there were 102 mothers still living at the time of investigation a ' nd , of these , six were know - n to be under treatment for mammary cancer while four had some other type of malignant disease . 
hence any likeness of onset age of mammary cancer in sisters is not peculiar to this type of malignancy . another interesting correlation concerns the sites affected in pairs of sisters and other relatives . 
the conversion of the cohesive subcutaneous tumour forms into a homogenous suspension of free cells may be due either to a selective proliferation of a few round cells present in the solid tumour or to a gradual adaptation to their new environment of the original spindle or polymorphous cells typical of sarcomas . 
two mouse ascites tumours were used for the experiments , the s 37 sarcoma and the t 2146 tumour which originated as a benzpyrene induced epithelioma but has both these tumours were developed since undergone sarcomatous transformation . from the subcutaneous form by craigie at the imperial cancer research fund laboratories . the paper is divided into three parts : i . 
no tumour smears of cell suspensions were nodules were present in the abdominal cavity . obtained by withdrawing peritoneal fluid at daily intervals from 24 inoculated mice and staining either by the feulgen method or that of papanicolaou . 
the whole of the ascites fluid was withdrawn and centrifuged at low speed for 3 minutes , which caused the heavier tumour cells to sink down while leucocytes and erythrocytes , if present , formed a top layer which could easily be removed with the supernatant fluid . 
the culture medium consisted of equal parts of fowl plasma , ascites fluid and chick embryo extract to which one drop of either s 37 or t 2146 tumour cell suspension was added . 
the undulating movements of the peripheral cytoplasmic area become more rapid , spikes and blunt processes are pushed out and withdrawn in quick succession . finally the cells elongate and assume pear and then spindle shape . for several hours after incubation the process may be reversed and spindle cells are seen to return to the round shape . examination of the fixed and stained specimens confirms the observations made on the living cells and shows that the process of transformation continues for 24 hours after incubation . 
the latter may represent a differentiated form which can no longer proliferate actively in contrast to the free round elements from which they are derived . to decide this question cultures of ascites tumour cells were inoculated subcutaneously into mice before and after 24 hours ' incubation , i.e. , before and after establishment of the spindle forms . 
the clots were cut into strips 2 x 4 min size containing approximately 2000 cells , and these were inoculated subcutaneously into the flank of c3h mice 2 - 3 months old . 
and that mitosis is only a third of that found in round cell grafts . after 24 hours the elongated cells revert to the round form but mitosis remains low until the fourth day ( fig . 31 , dotted line )  . 
the development of the implants can be followed from an early stage , and is described from 5 hours after inoculation until the fourth grafts derived from spindle cells showed a lower mitotic rate day of growth . after 5 hours in vivo , a significant delay in the appearance of tumours , and were of smaller size as compared with tumours obtained from round cells . 
the recently developed methods of detection and determination of polycyclic hydrocarbons in micro - gram quantities have provided a more sensitive way of analysis than has yet been employed ( cooper , 1951 , 1953 ; wedgwood and cooper , 1951 , 1953 )  . in view of the suggested connection between carcinoma of the lung and smoking ( menary , 1932 ; schrek , baker , ballard and dolgoff , 1950 ; mirs and porter , 1950 ; dor and hill , 1950 ; rigden and kirschoff , 1952 ; sadowsky , gilliam and cornfield , 1953 ; wynder , graham and croninger , 1953 ) and the estabhshed presence of carcinogenic hydrocarbons in combustion products , it was decided to use these methods to examine cigarette smoke . 
on the other hand several investigators have devised apparatus to collect smoke produced in conditions resembling closely those of normal smoking ( bradford , harlan and hanmer , 1936 ; wenusch and sch6ller , 1938 ; w , ynder , graham and croninger , 1953 )  . the apparatus we have employed . 
the tap c with a glass rod sealed on to it was turned on for the period necessary to simulate a " draw " made by a snioker by means of a cam attached to the periphery of a 3 inch pulley wheel , d . the glass rod was held in the groove of the puuey by means of a stretched rubber this wheel was driven by a gear train from a smar electric motor , the speed band . of which was variable by adjustment of a " variac " auto - transformer connected to the altemating current mains . other cams could be attached to the wheel and thus the length and frequency of the " draws " were adjusted . in the u - tubes suitable solvents were placed so that the smoke was made to bubble tbrough them and the condensable material separated from the gas . a packing of 10 cof pyrex glass wool in the second u - tube served to remove the both u - tubes could be surrounded by beakers last traces of condensable matter . full of solid carbon dioxide to assist in the condensation , but it was found that strong cooling was not always necessary to effect complete removal of the disperse phase . 
each , the average time of draw is about 2 seconds and the average time of smoking such a cigarette to a short butt ( about 1 - 5 clong ) is 12 minutes . these conditions are attained by oiir apparatus when adjusted to give a 2 second draw every 45 seconds with a negative pressure of 25 cwater . 
air. smoke obtained in this way is an aerosol with a viscous fiquid as the disperse phase and a gas , consisting of a mixture of unburnt air , carbon dioxide , carbon monoxide , water vapour and traces of other gases as the dispersing medium . the average amount of condensable material from 100 c ' igarettes weighs about 4 g . we have concemed ourselves only with the condensable disperse phase whicli in bulk , after the evaporation of the solvent ( acetone , chloroform or cyclobexane ) used to trap it , is a dark brown viscous fluid . preparation of apparatus and materials . the solvents ( cyclohexane , acetone , benzene and chloroform of reaaeint grade ) were distilled in an all - glass apparatus , rejecting the first and last tenths . this product was then distilled through a dufton column and corected over a range of - 9jo c . 
the residues were not fluorescent in ultra - violet hgbt . all the glass apparatus employed was cleaned bv immersing overnight in chromic - sulphuric acid mixture , washing and drying . 
500 ultra - violet spectrophotometer . graphy the eluates were examined at a few definite wavelengths only and hydrocarbons were identified by specific absorption peaks and the order in which they appeared in the eluates . 
the solvent was then removed by distillation , the residue boiled twice with cyclohexane ( 5 to 10 ml . ) and the solutions decanted off after cooling . the - united cyclohexane solutions were then shaken with three quantities of 2nsulphuric acid and then with three quantities of 2nsodium hydroxide . solution thus freed from basic and acidic substances was reduced to about 5 ml . 
the eluates from the colunm were examined by the method of wedgwood and cooper ( 1953 ) namely by searching for absorption peaks at wavelengths known to be specific for the various polycyclic hydrocarbons . 
usuahy the initial chromatography only revealed the hydrocarbon peakb as inflexions againbt considerable background absorption and the combined filtrates suspected of containing the compounds had to be passed through fresh columns in order to reveal their presence more satisfactorily . anthracene was recognised by its peak at 376 ma . 
and it was always followed closely by the appearance of a peak at 355 m / % , reveahng pyrene . the compounds were determined bv making the cyclohexane solutions up to known volumes and then measuring the height of the peaks by the base line technique . for this purpose two convenient points on either side of a given peak calibration of the peak heights above the base were used to construct a base line . line was effected by using standard curves of the authentic hydrocarbons prepared from solutions of known strength . the background absorption interfered considerably in these experiments and various attempts were made to reduce its effect . 
when hydrolysis was effected on the cyclohexane extract of the whole smoke , the background absorption was even greate ' r than before , indicating that some hydrolysis products of the smoke constituents were entering the hydrocarbon sections of the column . the presence of pyrene and anthracene indicated in these experients was confirmed by their chromatographic behaviour relative to added methyl ethers . relatively few compounds , absorbing hght in the region studied , are associated with the polycychc hydrocarbons on aluniina columns . 
the peaks used for recognition of the various compounds are labened with their wavelengths . it will be noted that a sequence of peaks of added calibration materials interspersed with the recognisable peaks of substances originary present , gives an infalfble method of identification and determination . 
the results of a number of analyses utilising in all 250 cigarettes of one popular brand showed quantities of anthracene and pyrene equal to 10 - 2 and 9 - 0 itg . 
and to paraffms which he states are present in t e original tobacco and are vaporised unchanged during smoking . acetylene has been determined in tobacco smoke by weighing it as cuprous acetyhde ( fishel and haskins , 1949 )  . its pyrplysis may give a ready explanation 300 b . 
was attained at the hottest part of the periphery . more satisfactory measurements were made by using a calibrated coppernichrome thermocouple which was threaded through the cigarette from one side to the other . after sealing the holes by means of small pieces of paper , the cigarette was smoked in the normal way and the temperature recorded from time to time . 
the temperature rose rapidly when the burning end advanced towards the couple and , when the glowing material was in contact with the couple , a temperature of about 650 c . 
each time air it thus appears that the highest temperatures during was being drawn in . suction are confined to the surface and that the main body of the hot end is always at a temperature between 650 and 700 c . similar temperatures were recorded when a cigarette with a thermocouple inserted was smoked in the mouth . these temperatures are sufficiently high for pyrolysis of simple compounds to polycyclic hydrocarbons . 
the temperatures recorded by us agree remarkably closely with those obtained independently by wynder , graham and croninger ( 1953 )  . our investigations upon the composition of tobacco smoke are being continued . the authors wish to acknowledge the helpful and kindly criticism given by professor sir e . 
for example , greenstein and his associates have shown that the activity of certain liver enzyme systems is affected to a considerable extent by the presence of a distant tumour , and of those investigated , liver catalase appeared to be the most sensitive . 
in fact a marked decrease in the activity of this enzyme has been observed in rats and mice bearing a variety of spontaneous , transplanted , and chemically induced tumours ( greenstein and andervont , 1942 )  . 
the evidence connecting the liver catalase depression with the presence of a tumour has recently been reviewed , both by greenstein ( 1947 ) and by weil - malherbe and schade ( 1948 )  . 
either the tumour may release some toxic material into the circulation , or its excessive nutritive demands may abstract from the circulation some substance essential for the maintenance of a normal liver catalase level . 
 consequently , quantities of tissue breakdown products are likely to be released into the circulation , and any toxic material associated with tumour growth may be due to such relatively non - specific processes . 
there is in fact evidence ( winzler and burk , 1944 ) that abnormally large quantities of circulating protein break down products such as proteoses and polypeptides are found in tumour - bearing animals , and these authors did tentatively suggest that liver catalase depression might be due to this cause . 
excessive nutritive demands of the tumour , greenstein and andervont ( 1943 ) showed that the depression was not simply due to the presence of growing tissue within the host , since catalase activity remained normal both in pregnant mice , and in mice bearing progres sively growing , subcutaneously implanted , embryonic tissue mash . 
 greenstein ( 1943 ) looked for a direct inhibitor of catalase in tumours , arul in the livers of tumour - bearing animals , but the results were all negative . 
greenstein interpreted his experiments as indicating that the affected livers simply contained less catalase than normal , rather than the same ' amount of catalase plus an inhibitor , and_put forwal ' _d the interesting l ' ! lj.ggestion that the decrease in activity was due to an interference with the _synthesis of the enzyme . 
however , no depression of liver catalase activity was observed even after long periods of administration of commercial peptone or sheep seru sheep serum in fact resulted in a rise in activity , the significance of which is unknown . 
further , these authors in vestigated the possibility that the depression might be due to the exaggerated protein requirements oi the tumour , by maintaining two series of tumour - bearing rats on high and low protein diets respectively , but no significant differential effect on the course of the process was observed . 
 most previous work appears to have been carried out with relatively massive tumours ( roughly within the range of 5 per cent to 50 per cent of body weight )  . 
 it is true , however , that while miller ( 1947 ) found that starvation up to about 7 days caused a marked depression in liver catalase level , he also showed that total liver protein fell in an approximately parallel manner . 
there appears to be a possibility that the effect is of a relatively specific nature , given only by malignant tissue , but the evidence presented in the literature does not ; enable a decision to be made on this queetion . 
so far as the results go they are in support of the supposition that tumours contain a substance not present , or - present in much lower concentration , in normal tissue , which is capable of depressing catalase activity~ animals . 
during the period of the experiment the diet of the animals consisted of " rat cubes " ( north - eastern agricultural co - operative society , aberdeen ) and water ad libitu as a routine the mice were starved for 12 hours before catalase determination  . 
 most of the work has been carried out with the transplanted tumour sarcoma 37 ( obtained from the chester beatty research institute ) , which grows rapidly in both strains of mice after subcutaneous inoculation . 
since , however , the non - protein nitrogen amounts to less than io per cent of the protein nitrogen and appears relatively constant , total nitrogen has been employed as a standard in this investigation . 
f measurement , but to construct a standard curve of enzyme concentration in arbitrary units ( using different volumes of the same liver homogenate ) against hydrogen peroxide decomposed in 4 minutes . 
since the rate of hydrogen peroxide decomposition falls off rapidly with increasing enzyme concentration , it was found necessary to construct a curve covering a relatively wide range of concentra tions . 
of 15 per cent w / v potassium iodide and 3 drops of 5 per cent w / v ammonium molybdate were then added , and the liberated iodine titrated with n / 20 sodium thiosulphate . 
all enzyme determinations have been made in duplicate , and , for routine estimations , the quantity of enzyme taken has normally decomposed between 1 / 3 and 2 / 3 of the substrate during the course of the reaction . 
adams determinations , catalase activity in arbitrary units has been calculated by sub stituting the quantity of hydrogen peroxide decomposed in the equation to this line , and the result divided by the nitrogen content in mg . 
this was obtained by dissecting out tumours previously inoculated into ff mice , removing any necrotic areas , and homogenizing the actively growing tissue in a loose - fitting ten broeck grinder just sufficiently to give a coarse suspension . 
it may be seen that at 24 and 48 hours after injection the level is considerably depressed , that it rises approximately to normal at 4 days , and then falls progressively until 14 days , when the experiment was terminated . 
 because af this , and also since the amount of the initial ( 24and 48 - hour ) depression is not approached again until the new tumours are of a considerable size , it seems not unreasonable to suggest that the initial drop is due to some material contained in the injected tumour homogenate , that this effect is rapidly exhausted ( 4 days ) and that only the second depression ( > 4 days ) is due to the presence of the new tumour . 
although the treated groups become very small when split up , the initial ( 24and 48 - hour ) percentage depression in catalase activity appears to be considerably greater in the males . 
the experiment was repeated with schofield albinos , using s37 taken from the same strain , and larger treated groups to enable the sex difference in initial effect to be examined further . 
3 , where it will be seen that there is an early depression in enzyme level , a rise approximately to normal in 4 days , and a subsequent progressive fall . 
each point represents 6 tumour - bearing animals , and has been obtained from the arithmetic mean of the catalase level for the treated animals , and the arithmetic mean of the appropriate control group . 
this method of presenting the experimental data is by no means ideal , but the individual results would be very difficult to obtain , since no informa tion is available regarding the original oatalase level of a tumour - bearing animal . 
according to the graph , these authors obtained a rapid fall in 2 days , after which the level remained approximately constant until 4 days , and then fell once more . 
the salient point is that their results cannot be separated into " early " and " late " effects in the absence of the 4 - day rise to normal . 
their tumour must have grown extremely rapidly since they obtained a 90 per cent depression in 7 days , and greenstein and ander vont ( 1942 ) probably employed the usual technique of grafting small pieces of tumour subcutaneously rather than injecting a homogenate . 
it is fairly obvious , from the results already given ; that if the onset of tumour growth is sufficiently rapid to affect the catalase level at 4 days , the rise to normal after the initial drop will be obscured , or perhaps entirely obliterated . 
 a number of normal tissues have been coarsely homogenized ; and injected subcutaneously into male mice , which , as has been already pointed out , are more sensitive than females . 
 since the initial ( 24 and 48 hour ) depression is of primary interest , the effects on catalase level have been followed over a period of 4 days , the results appearing in table ii . 
the results in table ii show that a 24and 48 - hour depres sion was observed , although an appreciably smaller one than with s37 , and that the catalase level rose to normal at 4 days . 
 because of the much delayed onset of tumour growth , and because " takes " only occurred in a.bout half the animals it is not possible to state definitely whether the females showed a greater resistance to tumour growth , although there appeared to be a tendency in this direction . 
140 25 carcinoma 63 105 21 101 16 113 14 123 22 131 34 101 13 75 19 145 28 125 22 108 26 113 17 123 29 127 19 95 13 79 13 92 33 113 33 116 14 114 19 128 17 131 27 125 20 95 11 117 6 an experiment with coarsely homogenized necrotic s37 tissue yielded the following result : no significant depression was observed at 24 hours , but by 48 hours the level was considerably below normal , beginning to rise at 4 days . 
 since attention does not appear to have been drawn to a sex_ difference in normal mouse catalase levels , some results for males and females drawn from the same batches are given in table ill . 
 80 21 123 18 90 14 as pointed out in the introduction , the alternative mechanisms by which tumours may cause a catalase depression can be given thus : discussion . 
it is hardly possible that this early depression can be a result of any nutritive demands on the organism , and can only be due to some substance or substances present in the injected tumour tissue . 
assuming that the early and late depres sions are not entirely unconnected , it is suggested that the tumour is exerting its effect on catalase by continuously releasing this material into the circulation . 
of homogenized tumour tissue ( administered by the relatively inefficient method of subcutaneous injection ) is sufficiently active to depress the catalase level very considerably for approximately 2 days , it is probable that only a comparatively small continuous production would be required to account for the observed results . 
further , the point at which the graph flattens corresponds approximately to the tumour weight at which necrosis commences , and , for example , a tumour weighing 2 to ; 3 g . 
also , after subcutaneous injection , these tumours grow in the subcutaneous space and do not , as a rule , perforate the abdominal wall until they weigh 4 to 5 g . 
consequently little or no normal tissue can be destroyed by the growth of the tumour ; and even if this process were occurring to some small extent , the flattening of the curve at a given point would be unexplained . 
schultz and kuiken ( 1941 ) briefly mention that normal male rats have higher liver and kidney levels than females , and serfaty ( 1946 ) , working with the erythrocytes of fowls , found the level to be higher in cocks than in hens . 
 it seems reasonable to suppose that the maintenance of a normal catalase level is to some extent under hormonal control , and the different initial response of males and females to tumour tissue may indicate that the primary effect is not on the liver . 
these graphs make it clear that not only does homogenization enhance the initial depression ( as might be expected if the effect is due to some substance contained in the tumour cells ) but that such a procedure 194 d . 
their results show only a progressive fall in catalase level after the tumour implantation , and provide no evidence which could suggest that tumour tissue contains a toxic product capable of depressing liver catalsea activity . 
in some experiments the arithmetic mean of the results for the treated animals have been slightly lower than the arithmetic mean values for the controls , in others , slightly higher . 
it is impossible at present , therefore , to say whether tissues are capable of causing a slight depression , or whether normal the effect is wholly specific to tumours . 
after this process the material could be compared with similar fractions from normal tissue , and a sufficiently large dose given to show whether the cata ] ase depressing material is present at all in normal tissue . 
 it is already known ( greenstein and andervont , 1942 ) that , in general , the greatest falls in liver catalase level are produced by rapidly growing tumours , and , in fact , certain s ] ow - growing tumours produced little or no effect . 
 in the present investigation it appears that s37 tissue , which grows readily , gave a greater 24 and 48 - hour depression than carcinoma 63 , which does not . 
 following the subcutaneous injection of homogenized sarcoma 37 tissue into two strains of mice , the liver catalase activity fell significantly at 24 and 48 hours , subsequently rose to normal by the 4th day , and diminished again during the growth of the new tumours . 
there was also a sex difference in the initial response to the injection of tumour material , the males , which have the higher normal level , being more sensitive . 
the coal miners form an interesting group for comparison with the professional classes , for the miner 's daily bath , often obtained under very adverse conditions , secures cleanliness for about two - thirds qf the time , while in other respects his skin receives very rough treatment . 
the census returne ( registrar - general , 1938 ) give the age distribution , in 5and 10 - yearly periods , of the whole population of males , and also of those following each one of the recognized occupations , at the time of the census . the number of deaths attributed to cancer of the skin and lip occurring in the whole male population in each of these age groups during the years in question was obtained from the statistical reviews of the registrar - general . 
 " cancer of the thigh , " " cancer of the neck and face , " " cancer of the face and antrum . " certificates of persons under 14 years of age were excluded . 
1 shows the percentages , arranged in ascending order from left to right , for 41 occupations in which more than 5 deaths from cancer of the skin occurred in the 34 - year period ; this quite arbitrary limit in number of deaths is drawn to lessen sampling error . 
the percentage in 15 out of 16 professional occupations selected thus is below 100 , while that in 9 out of 13 agricultural occupations is above 100 , and in 5 is above 150 . 
the occupations of mining engineers , and mechanical and electrical engineers , show no deaths from cancer of the lip , and low percentages for cancer of the skin , but the numbers of deaths ( 5 and 2 respectively ) are small . the high standard of cleanliness maintained by the coal miner at the end of his day 's work , and his lack of exposure to sunlight , perhaps compensate for the various injurious factors , of which an account has been given by knowles ( 1944 ) , first - hand descriptions of the coal - miner 's life to which his skin is exposed . are given in two recent books ( shaw , 1946 ; agnew , 1947 )  . 
the mortality in the hewers and getters , who carry out much of the most severe work of the mine , is , if anything , lower ( 97 - 7 per cent ) than it is in the general population . the percentage is below 100 in 5 coal - mining occupations , comprising 828 , 246 out of the 939 , 378 men , or 88 per cent of those engaged in the 7 divisions of the industry . 
six out of 17 occupations , with a population of nearly 700 , 000 , have percentages from 131 to 249 ; this last figure ( gardeners ) is the highest of all those recorded in this paper . 
fingers , hand , forearm , and so on , might seem to give more information , but the sampling errors would be increased , and in such material the assignment to the smaller sites has not been carried out on any uniform system ; e.g. 
there would be no certainty that some cancers of the fingers had not been classed as of the hand . the numbers of cancers at these sites in the three groups of occupations , and their percentage distribution , are given in table iv and fig . 
1 provides a comparison of the percentages for cancer of the skin , and of the lip , selected and arranged in the same way , except that all cancers of the lip are included and not only those shown by occupation yielding more than 5 deaths . 
the number of fatal cases of cancer of the lip in the professional classes is so small ( 27 in 34 years in about 400 , 000 men ) that these have been pooled m . 
2. - occupational distrnbution of cancers of the skin according to site . show that no one of the seven groups of coal and shale miners gives a percentage above 100 ; the figures are low ( 38 - 3 to 56 - 0 per cent ) except in one large group which is the most exposed to light ( other workers above ground , population 106 , 549 ) , in which the incidence ( 97 - 5 per cent ) is approximately the same as in the general population . 
1 shows that the incidence of fatal cancer of the skin is high upon agricultural workers , and very low upon the professional classes , while the miners take an intermediate position not far removed from that of the general population . 
the especial liability of the out - door workers is generally supposed to be due to exposure to sunlight ( blum , 1948 )  . the results given in table iv and fig . 
2 show that the cancers of the whole region of neck , head , and face together , selected on the basis described above , make up from 70 to 78 per cent of cancers of the skin in all three occupational groups . 
the face is the only part of the body which is never clothed , and those parts which are shaved receive more thorough cleansing than do any others ; these characters are common to all social classes . 
the most notable result of this tabulation is the lack of any great difference between the three groups . another factor which may be of considerable importance is that the cosmetic effect of cancer of exposed portions of the skin and of the lip may attract attention sooner in richer subjects . ingram ( 1947 ) , in a comment on the paper by ryle and russell ( 1947 ) , says : " prognosis in skin cancer depends largely upon the size of the lesion , and negligence is probably an important factor in relation to death from this cause . i should , in the ordinary course of events , expect neglect of a symptomless lesion to be higher in unskilled workers and labourers ( iv and v of the registrar - general 's social classes ) than in the other groups . " obviously one cannot assess the quantitative importance of this difference . data in equal detail for the distribution of skin cancer in married women whose husbands follow these occupations would be of great interest . 
the great preponderance of tumours of the lower lip is explained as due to direct exposure of its mucocutaneous junction to sunlight , while the corresponding part of the upper lip is relatively shaded . " some factor in addition to exposure to sunlight is required smoking , and especially pipe to explain the differences shown in table vii . smoking , is , of course , regarded as another possible factor in the production of cancer of the lip . during the earlier part of the period ( 1911 to 1944 ) cigarette smoking was practically restricted to the richer classes , which produce fewest cancers of the lip ; data on the change in this matter in recent years have been given elsewhere ( kennaway and kennaway , 1947 )  . 
the data collected by lane - claypon ( 1930 ) from the literature , summarized in table vii , show the great preponderance of cancers of the lower lip , especially in males . 
 ( lanecilaypon , 1930 )  . all occupied and retired males agricultural labourers farmers deaths . death rate per million per annul . 212 798 55 - 0 comparison of cancer of the skin and of the lip . the data given in fig . 
of the 14 agricultural occupations which produce any cancers of the lip , 11 show agreement in regard to cancer of the skin and of the lip in that both percentages are either below , or above , 100 . this suggests that the aetiological factors are to some extent the same in some occupations . 
the incidence upon the coal miner is of especial interest in view of the cleanliness secured by his daily bath , and of his minimal exposure to sunlight . in occupations in which 88 per cent of the coal - mining population are engaged the liability to cancer of the skin is less than it is in the general population . only one occupation of social class i ( civil engineers and surveyors ) shows a mortality from cancer of the skin higher than that in the whole population ; hence these results illustrate the social distribution of cancer of exposed sites discovered by stevenson . 
the comparative distribution of cancer upon the various parts of the skin does not show any great differences in the three groups ; from 70 to 80 per cent of these cancers are situated on the head and neck . 
the mice used for transplantation undoubtedly came from therefore , additional experiments were different sublines of these two strains . carried , out in an attempt to elucidate the forowing questions : 1 . 
does the regression of a well - developed transplanted tumour forowing irradiation in vivo render the animal resistant to the development of spontaneous tumours ? this study was made possible by the use of the irradiation technique ( goldfeder , 1950a ) which facihtates the exposure of the tumour and protects the rest of the body of the animal from secondary scattered radiation , thus permitting the treated animals to eve their life span . previous work on the irradiation of tumours has been summarized by the writer ( 1945a )  . there is no doubt that results obtained with tumours heterogenous to the animal bosts - that is , tumours which have a different genetic constitution from that of the host , are not comparable with those obtained with tumours autogenous to the animal hosts - that is with tumours having the same genetic constitution as that of the host , or with , those in patients suffering from cancer . 
memorial laboratory on julv 5 , 1933 , and they were designated at that these mice have been inbred since : they behave time as inbred strain ' d '  . immunologicary like dba / 212 , and have an incidence of over 85 pe ' r cent of spontaneous mammary tumours among breeding females . 
a tumour of about 5 min diameter usuauy develops from an implant within a period of 12 to 14 days , and grows progressively . a spontaneous mammary tumour of an i 1 - month - old female mouse of the dba this tumour was : firmer in consistency than that strain was excised asepticary . fragments of about 2 mdissected removed from the c3h mouse described . from the edges of the tumour were implanted subcutaneously into 10 dba mice ( 8 males and 2 females )  . 
1 round coverslip 7 inch in diameter , wbich had been previously attached by a drop of sterile water to a mica sheet and covered with a maximow depression slide , in the concavity of wl - lich moist filter paper had been placed to maintain the water content of the tissue . 
the absorption of radiation by the cover glass , mica sheet and maximow slide was about io per cent , which was taken into consideration in calculating the x - ray dose delivered to the tumour tissue . immediately after irradiation the tumour fragments were implanted into about 2 - 3 - month - old mice , most in two additional experiments the mice were injected of which were litter mates . intradermally with aliquots of irradiated . 
tumour cells , suspended in physiological this was done because , according to observations made by several investisaline . gators , the intradermal route of immunization appears to produce more effective and lasting results ( besredka , magat , laval and besnard , 1936 ; kolmar and tuft , for the latter experiments the tumour fragments following irradiation 1941 )  . were cut with sharp curved scissors until an almost homogeneous mass was obtained . 
doses by mea ' ns of a tuberculiin syringe and .25 gauge needle in two or three sites of the abdominal wall of each mouse . the latent period and the number of " takes " produced by tumciur grafts irradiated in vitro served as criteria of the effect of a given dose of x - radiation . the procedure of irradiation of the estabhshed tumours in v& ' vo was as follows actively growing tumours ranging in size from 1 - 0 to 1 - 5 cin diameter were exposed to filtered x - radiation while the rest of the animal orgamsm was shielded bv a lead chamber ( goldfeder , 1950a )  . during the early experiments the tumours were irradiated in vivo under the same physical conditions as the tumour grafts during the later experiments , the following physical factors were in vitro . employed : the radiation was generated by a westinghouse " quadrocondex " constant potential unit at 200 kv , 15 ma ; the incident beam was filtered by 05 mcu plus i 0 mal ; hvl equalled i 0 mcu ; fsd , 20 . 
the dose rate measured in air bv a thimble ionization chamber during the coiirse of the investigations varied from 395 r / mto 402 r / min . special measurements were made with and without the lead ( 5 per cent )  . shield , the ionizatioil chamber being placed in the same position . 
observations on c3hb transplantable mammary tumour . the results obtained on implantation of the irradiated c3hb tumour - grafts a - re recorded in table i are presented graphically in fig . 
the first ' retardation of tumour growth , as indicated by a slight increase in the latent period of tumour appearance , was prodiiced by a dose of 2000 r . 
ii , an implant irradiated with 5000 r developed a tumour after a latent period of 117 days . in this , as wer as in other similar instances , the question arises whether the tumour cells had been attenuated by x - radiation to such an extent that they remained dormant for this comparatively long ' time , or whether only a few cers had escaped injury and took , therefore , a much longer time to develop a palpable tumour . this question cannot be answered with any certainty . it is certain , however , that the tumour grew on the site of grafting and that the rate of growth and the histologic ' al pattem were similar to those of a tumour developed by a non - irradiated implant . in no instance did a tumour , developed by an irradiated implant , regress spontaneously , contrary to observations frequently made when the tumoiir implant and the host are not of the same genetic origin . search for induced re8i8tance the mice in which the irradiated tumour grafts failed to produce tumours ( experiments 7 - 14 , table i ) were reimplanted with fresh , viable tumour grafts . due to the long latent period , the second implantation into mice of this series was performed 3 to 4 months after the implantation of the irradiated tumour grafts . 
a dose of 12 , 000 r completely destroyed all the treated tumours . sections of the tumours removed within 10 to 12 days after irradiation showed only necrotic tissue . it appears , therefore , that a dose within the range of i 1 , 000 r to 12 , 000 r is lethal for the c3hb tumour of 1 - 0 to 1 - 5 cni . 
the irradiated tumours which resumed their growth after initial decrease in size , grew steadily and in no instance did a tumour regress once it began to grow again . occasionary secondary tumours developed in the treated mice either in the inguinal or axillary reg - ions within 2 or 3 weeks after the regression of the irradiated in some instances , new growth developed on the periphery of the tuniours . irradiated tumour , probably from tumour cells which bad not been included in the exposed area . 
the histological appearance of these secondary tumours resembled that of the primary , non - irradiated c3hb tumour . epilation and regrowth of gray hair appeared in the exposed areas 3 to 6 weeks after regression of the irradiated tumours . 
no ulceration of the skin in the irradiated area was ever noted in the c3h mice once gray hair appeared . a significant number of c3h mice hved about i year after regression of the tumours and spontaneous tumours appeared in some of the c3h females . 
observation8on dbah transplantable mammary titmour . this tumour had been carried for 8 generations of serial transplants in the dba mice from which it originated before it was used for irradiation . although loo per cent " takes " resulted in most instances , occasionany , however , an iniplant failed to produce a tumour in a dba mouse which later proved to be resistant to further viable tumour grafts . according to hauschka , this may be the result of a mutant histocompatibility factor present in the dba strain ; in other words , an outcome of genetic incompatibihty between the bost and the transplanted tumour . 
to secure a better statistical evaluation of the results , a number of mice were grafted with viable , non - irradiated dbah tumour fragments to serve as controls to those grafted with irradiated dbah tumour implants . 
the delay in the latent period is probably a more rehable criterion than the tumour " take " for re - evaluation of the radiation effects in this case , because it could be argued that the 2 mice in which the irradiated tumourcells failed to produce tumours possessed the mutant - histocompatibility factor . in one experinient , a dose of 4500 r resulted in 22 - 9 per cent of " takes " , while a dose of 4800 r resulted in 13 - 0 per cent of " takes " and a dose of 5000 r gave only 5 - 1 per cent of " takes however , in two additional experiments , after doses of 5000 r and 5200 r no " takes ocetirred . 
none of these 7 produced tumours fohowing implantations of viable tumour grafts . in the experimental - mice , the number which failed to develop tumours was 150 out of a total of 341 mice which had been implanted with irradiated tumour grafts . 
none of these eighteen dayis later these - mice were reimplanted 25 niiee developed tumours . subcutaneously with fiesh , viable tumour grafts . palpable tumours , whicb , grew progressively , appeared within 9 to 13 days in 14 out of the 15 reimplanted male and in 9 out of 10 re - implanted female mice . two mice , one male and one female , in which the irradiated tumour cells failed to produce tumours in the first place , remained refractory to twice repeated implantations of viable tumour grafts . this result may also be ascribed to the mutant histocompatibihty factor present in these 2 mice . re8u1t8qf x - radiation o dbah tumour8in vivo . table v records the observations made on a total of 227 mice ( 164 males and 63 females )  . 
a rather sudden increase in the number of tumours which showed regression was noted forowing the application of doses higher than 6000 r . thus , 60 per cent of the tumours regressed after a dose of 7000 r . 
while a dose of 6000 r destroyed only 25 per cent of the treated tumours . the larger percentage of the regressed tumours which had been treated with a dose of 10 , 000 r indicates that this is the destructive dose for the dbah transplantable tiimours , ranging in size.from 1 - 0 to 1 - 5 cin cliameter . 
as in the case of the c3hb tumour , this dose is twice as large as the dose necessary for the attenuation of grafts of this tumour in vitro . the majority of the mice . ' m which tumours had regressed eved in apparent good health for i year or longer , and the young female ' mice produced apparently normal litters . 
the ulceration - s persisted iintil the death of the - ani ' mals . these two instances may parallel radiation necrosis occasionally noted in human patients treated with x - rays . 
the turnours in the remaining 7 mice grew steadily . fourteen tumours decreased considerably in size within 10 days after irradiation increased tumours grew steadily after exposure . five tumours regressed within 3 to 4 weeks following exposure . 
two females developed spontaneous months after regression of the treated tumours . in 27 mice ( 20 males and 7 females ) the tumours regressed completely within 3 weeks after irradiation . 
microscopic sections of the regressing tumours consisted mainly of fibrous tissue . the tumours regressed completely in 30 treated mice ( 21 males and 9 females )  . five fexnales developed spontaneous tumours 6 - 7 months after regression of the treated tumours . the treated m1ce lived for as long as i year and more after treatment . 
the mice lived in apparent good health up to 1 year and more . of ar treated mice only in two - instances did ulceration of the skin in the irradiated area occur 11 months after regression of the tumour . should be noted that in the experiments cited above , geneticary impure ammals and tumours geneticary different from the hosts were used . 
the writer 's interest in the problem of induced resistance arose from investigations on mouse sarcoma 180 grown in white mice of the swiss strain ( golffeder , 1942 )  . 
the writer explored the possibihty of inducing a resistant state in inbred animals by implanting into these animals irradiated grafts of a tumour which originary developpd in an animal of the same inbred line . these expenments were carried out hi an attempt to ascertain whether ionizing radiation induces genetic and antigeriie changes in the tiimour cells which make them foreign to the host . 
the irradiation of tumour cells in the present experiments did not make them foreign or antigenic to the host in which they originahy developed . an analysis of the results recorded in table i and in table iii reveals a difference in the radiosensitivity between the c3hb and dbah tumours . for example , a dose of 3500 r gave 92 per cent " takes " of the c3hb tumour grafts and only 57 1 . 
determined for implants of other mammary adenocarcinomas in inbred strains of mice ( lawrence , horn , strong , 1937 ; goldfeder , 1945a ) and for a mammary adenocarcinoma of an inbred line of rats ( eisen , 1940 )  . 
on the other hand , the c3hb mammary adenocarcinoma used in t - he present studies proved to be more resistant to irradiation than the cm mammary adenocarcinoma used in previous experiments ( goldfeder , 1947 ) , the latter requiring only 2700 r to prevent its grafts from " takes "  . 
the difference in the radiosensitivity between the c3hb and c3h mammary adenocarcinomas may be explained by ( a ) a difference in degree of differentiation : the c3hb mammary adenocarcinoma in the present experiments is a more anaplastic type of tumour consisting of fiheets and bands of closely packed cells , with relatively little fibrous connective tissue , while the cm mammary adenocarcinoma previously reported ( goldfeder 1947 ) consisted of acini , uniformly distributed and separated by connective tissue ; ( b ) by a difference in genetic make - up between the tumours and their hosts : the c3hb niammary tumour was carried by serial transplants in pure inbred cm mice from which it originated , while the mammary tumour , used in previous studies ( 1947.1950 ) , was carried in c3h mice of different sublines and thus the tumoiir and the hosts were not of the same genetic origin . failure of the tumour to " tak - e " in about 10 per cent of the hosts indicated that some of the cm mice used in the experiments of 1947 - 1950 had a different genetic constitution than that of the .mouse in which the tumour developed . 
the skewed curve illustrated in that publication ( goldfeder , 1947 ) in contrast to the typical s - shaped curve obtained in the present experiment may also be the result of the difference in the genetic make - up between the hosts and the tumour . 
the radiosensitivity of tumours depends to a large extent on whether they are growing in hosts of the same strain from which they originated , or in foreign hosts as shown by aughterson , tennant , irradiation of a transplantable mammary carcinoma in and lawrence ( 1940 )  . strain a mice in which the tumour originated induced regression only in i per cent of the treated mice when 2500 r were apphed and in 48 per cent of mice forowing the application of 5000 r . 
the antigenic reaction of ' the foreign host is thus a contributing f ' actor in rendering the tumour more vulnerable to ' regression after irradiation . the more anaplastic c3hb tumour proved to be more resistant to radiatioii than the relatively more differentiated dbah tumour . this corroborates the observations made by the writer on another mouse mammary adenocarcinonia ( goldfeder , 1950a ) and those noted on human tumours . for example , cathie ( 1939 ) , lenz ( 1942 ) and gliicksmann ( 1948 ) found that well differentiated cancers regressed more readily and offered a better prognosis than the less differentiated tumours . the r ' ole of connective tissue in the response of animal tumours to irradiation and its relationship to the host was brought forth by cramer ( 1932 ) aind by aughterson , tennant and lawrence ( 1940 )  . 
the relatively more pronounced proliferation of the connective tissue in the dbah tiiniour thanin the c3hb tumour may also be regarded as a contributing factor rendering the dbah tumour more vulnerable to irradiation . 
the observation of secondary tumours occurring in the treated mice also seems to parallel observations made on human mammary this observatioin indicates the failure of the disintegrated irradiated cancers . tumour tissue to induce resistance in the hosts to new cancerous growths . it has been noted that untreated metastatic tumours often regress after x - ravv treatment of primary tumours ( sugiura , 1937 ; aughterson , tennant and lawthis observation has been attribiited to a supposed indirect effect rence , 1940 )  . of irradiation ( sugiura , 1937 )  . these observations could probably be explained by the fact that the tumours were grown in genetically foreign mice . 
the present experiments on mice of geneticafly pure strains and their autogenous tumours indicate that the indirect effect of irradiation is not sufficient to affect secondary growths . regression of the secondary tumours in x - ray treated heterozygous mice may be attributed to an antigenic effect ' ehcited by the genetically differing tumour tissue . 
the inability of the irradiated tumour tissue to induce " immunity " in hosts from which it originated may indicate that no quahtative change of a genetic or antigenic character occurred in the irradiated tumour cells which would render them foreign to the hosts . inbred hnes of mice and their tumours offer far more suitable material for investigations of this type than heterozygous mice . 
the observations made from such studies seem to pararel those noted on human mammary cancers . in earlier publications ( goldfeder 1950b , 1952 ) , the writer showed that a fast growing , less differentiated mouse mammary adenocarcinoma also possessed a more vigorous metabolic rate and suggested that this propertv may have been one of the factors influencing the radiosensitivity of the mammary adenocarcinomas then used . 
a smaller irradiation dose was found to prevent the irradiated dbah tumour grafts from growing in vivo than was required to produce a similar effect on c3hb tumour similarly , for treatment of established tumours in vivo , the dose of irragrafts . diation which produced regression of the dbah tumour was found to be smaller than that which had a similar effect on the c3hb tumour . 
some factors which are involved in the difference in the radiosensitivity of these two tumours are the state of differentiation of the tumour cells and the amount of stroma . in both instances about half of the radiation dose , producing destruction of the established tumours grown in their respective hosts , was required to obtain the attenuation of the grafts of both c3hb and dbah tumours . the c3h mice in which the irradiated in vitro c3hb tumour grafts failed to " take " or in which a well developed transplanted c3hb tumour regressed after irradiation in vivo remained susceptible to reimplanted viable c3hb tumour grafts . 
a small number of the dba mice in which the viable dbah tumour grafts failed to " take " in vivo remained resistant to viable tumour grafts . similarly , a small number of the dba mice in which the irradiated in vitro tumour grafts failed to " take " was found resistant to re - implantation of fresh viable tumour implants . this is explained by a difference between the genetic make - up of some of the dba mice and the dbah tumour . a significant number of mice of both strains lived in apparent good health for 1 year and longer after regression of the in vivo irradiated tumours ; among these survivors the young females produced apparently normal litters . spontaneous tumours also occurred among these female survivors on reaching cancer age . thus , under the experimental conditions described , the question about the possibility of inducing immunity in homozygous hosts to endogenous tumours by irradiation of these tumours appears to have been answered in the negative.. the part played by the genetic relationship between the tumour and host in the induction of resistance to the appearance of tumours was brought forth . 
burn re8earch laboratory , royal victoria infirmary , newcastle - upon - tyne . received for publication january 30 , 1954 . this is the first of a series of reports on an experiment suggested by the work strong hybridised three strains of mice , and after 3 of , strong ( 1940 , 1945 )  . inbred generations he injected subcutaneously i mg . 
to f12 injections were continued but there was selection towards resistance to tumours at the site of injection ; five sublines were then spht off and the mice injected for a further 4 generations , after which one group was set aside and bred from without further treatment . the experiment to be reported here differs in several respects from that of strong ( 1940 , 1945 )  . 
towards the ' end of 1945 two inbred strains of mice , the nbt ( newcastle bone tumour ) and the cba ( one of the ancestral strains used by strong ) were crossed in both directions . half the mice in each f , htter were injected subcutaneously in the right flank at the age of 2 months with i mg . methylcholanthrene in 0 - 1 c.c. 
sesame oil ( one inject ' lon only ) ; the other half were not treated and were bred from ( brother - sister matings only ) for 12 generations , ar untreated . 
the untreated reciprocal - hybrid strains were known as the cba / nbt and nbt / cba strains , or cn and nc for short , the maternal strain being given first . the injected f , mice were also bred from , their inbred descendants likewise being injected and subsequently bred from for a total of 10 generations . 
from the beginning an attempt was made to breed selectively for resistance to local tumours , but entirely without success ; offspring of apparently resistant inice appeared to be just as susceptible as those of susceptible mice . 
onwards etters from mice which early developed local tumours were neither injected nor bred from . finauy , as the failure to produce resistant lines seemed likely to spoil the purpose of the experiment , 2 final generations ( f , , and f.2 ) were raised , in which no mice were injected . the injected reciprocal hybrids together with their untreated descendants formed the m / cba / nbt and m / nbt , / cba strains , or mcn and amc for short . mice were fed on " rat cake " compounded to the formula of the rowett institute by an aberdeen firm , supplemented with cabbage and carrot , with drinking water ad lib . the purpose of the experiment was , like strong 's ( i 940 , 1945 ) , the development of lines of mice resistant to tumours at the place of injection , so that the mice would live long enough to produce tunio ' urs at ' remote sites . 
the origin of the strain has been described ( pybus and mffler , 1938 ) and the various types of bone tumours have been described and illustrated ( pybus and mfller , 1940a , 1940b )  . afice of inbred generations 28 and 29 , coming from i pair of mice of generation 22 , were used as parents of the hybrid stra ' ms ; 131 mice ( 59 females and 72 males ) of generations 28 to 30 were treated with methylcholanthrene . 
one generation of 81 females and 94 males , an untreated , was raised from the treated mice ; 28 p , , .irs , belonging to 9 famihes , were successfuey bred from , a further 17 pairs proving sterfle . the last bone sarcomata to be seen in this strain were 4 which occurred in f27 prior to these there were 3 in f22and i in f24 . 
the purpose of the experiment was not to investigate the occurrence of local t ' umours , but ( as already stated ) to try to develop strains of mice that would be resistant to the local action of the carcinogen and would therefore live long enough to produce neoplasms at places remote from the site of injection . 
ar mice were therefore allowed to live as long as possible , and those with local tumours were killed only when the tumour grew large or showed signs of ulceration . there was a certain amount of variation in size of tumours at death , those close to the skin or in a position to rub against the box having a tendency to ulcerate 166 e . 
at first the mice were charted regularly , outhne drawings of tumours being made when they appeared and their growth followed week by week , but as the numbers of injected animals increased this became impossible and tumour sizes at death only were charted . it was observed from the initial weekly chartings that there was great variation in the , rate of growth of different tumours , but it cannot be stated whether this variation depended to any extent on the sex of the h ' ost ; it did not appear to do so . 
the males might be more susceptible than the females to the local action of the carcinogen ; i.e. , tumours might appear after a shorter latent period in males , and , even if tumour growth - rate was the same in both sexes , the males would be killed at an earlier age . 
with a very few exceptions in certain generations , many non - tumour males died prematurely and usuahy earlier than non - tumour females ( i.e. , mean age at death was less for males in ar cases except m / cba , mcn fl , and mnc f9 and flo ) , due mainly to wounds received in fighting . in some generations some of the non - tumour males which died prematurely might have developed tumours had they lived longer . this would have raised the tumour incidence and would proba - bly also have raised the mean tumour age at death in these instances . although causes i and 2 cannot be completely dismissed , there is evidence for the fourth possibihty in a comparison between the two pure strains . 
the local response o methylcholanthrene . two inbred strains to one subcutaneous injection of nbt strain . - as shown in table 1 , 59 females and 72 males were treated at local tumours appeared in 27 females ( 45 - 8 per cent ) and the age of 2 months . in 25 males ( 34 - 7 per cent )  . 
the non - tumour males in this strain died young from various causes , the principal bein - a filrhting ( 17 mice ) , while diarrhoea diarrhoea was responsible for the death of a number ( 7 ) of accounted for 8 . other causes of death in both sexes were kidney diseases , young females . pneumonia and pseudo - tubercle , and there were a few cases of neoplasms ( leukaemia , lung adenoma , mammary carcinoma and skin papillomata )  . there was no significant difference between the ages at death of tumour and non - tumour males ; from this it may be concluded that more of the males might have developed tumours had they lived longer . 
f6was a small generation of only 25 mi ' ce and 8 of th ' e io non - tumour males were dead by 6 months ; this could account partly for the lo ' w incidence . 
but f2was a very large generation with 134 females and 120 males , there being 96 tumour females ( 71 - 6 per cent ) and 94 tumour males ( 78 - 3 per cent ) , the combined incidence being 74 - 8 per cent . 
why this generation should be so susceptible is not clear at the moment . age at death : the result of inbreeding on the survival age of injected mice was evident in this experiment . in generations i to 5 , 74 mice died at 21 months and over , the oldest at 28 months ; in generations 6 to 10 only 3 mice ( non - tumour ) except for fl , to be discussed later , the ages at lived over the age of 17 months . death in each generation were not analysed separately . 
 - minbticba8train ( table iii )  . - each generation was analysed to find out whether the sexes were equally susceptible to the development oflocal tumours . with the exception of the first and third generations , the differences in each generation between the tumour incidences i ' n males and females were not significant . in fl , more than half the non - tumour males died at ages up to 7 months ( mainly due to fighting ) and the tumour incidence in the males was probably too low ; f , , was a smar generation and the early death of a number of non - tumour males had a disproportionate influence on the result . 
was a smah generation of 36 males 66 - 4 per cent ( 511 tumours in 770 males )  . and many of the non - tumour males died young , while in f4 half the non - tumour males were dead by 8 months . 
the incidences for these two generations of males were very probably too low , as some of the non - tumour mice would certainly have developed tumours had they eved longer . although it is therefore probable that the sex - difference in incidence was due to a lack of fur opportunity for the development of tumours in the males owing to a disproportionate number of the latter dying young , this cannot be presumed to be certain . a comparison of the reciprocal crosses regarding their susceptibility to local tumours was thus a comparison between an incidence of 62 - 8 per cent in the 538 mcn females and 73 - 4 per cent in the 669 mnc females ; and between 59 - 6 per cent in the 542 mcn males and 66 - 4 per cent in the 770 mnc males . 
the tumours seen were of the various types described by bonser and orr ( 1939 ) ; the greatest number were subcutaneous fibrosarcomata , of varying degrees of fibrosis ; there were a few pure fibromata , and many spindle and polymorphous - celled sarcomata . giant cells were frequently present . 
the possibility then had to be considered whether the hereditary condition acted through genetically transmitted differences in ovarian functional activity , or through an unequal response of mammary glands or genital tract to ovarian hormone . 
numerous and extensive observations were made with somewhat conflicting results , both on the nature of the oestrous cycle and on variations in organ sensitivity to hormonal stimulation , in strains of mice with differing mammary cancer incidence . when the existence of the third factor , i.e. 
as the number of determinations was so small , no statistical analysis of the figures was made , but there appears to be no real difference between the strains themselves , nor could the changes produced by foster - nursing be correlated with the presence or absence of the milk factor . 
armstrong ( b ) the character of the oestrous cycle . ( i ) virgin mice . - daily vaginal smearing of virgin mice of all groups was continued for periods ranging from 56 - 137 days immediately following canalization or weaning . 
the number of complete cycles during the observation period in addition , the average length of the whole cycle and of the was recorded . comparison actual period of heat for each mouse was calculated ( table ii )  . was then made between the fostered and non - fostered groups ( table iii )  . all cases comparisons show no significant difference . 
the p factor being of the order of o 1 - 0 5 . a second series of estimations was then made , in which two types of atypical cycle were excluded , in view , of the fact that such cycles might have been caused in the one type , a prolonged dioestrous by the repeated smearing technique . interval is observed , a state termed pseudo - pregnaney . this occurs naturally following sterile copulation and artificially following a great variety of stimuli . parkes ( 1926 ) regarded 7 days as the shortest duration of natural pseudo - pregtherefore all cycles with dioestrus greater than 7 days have been nancy . in the other type there is prolonged cornification or regarded as atypical . heat , a condition which has been produced experimentally by repeated vaginal smearing . 
the death of so many mice while still showing oestrous cycles detracts from the value of the results , but there was some indication that the range of age at which the menoe . 
from table i it is seen that vaginal canalization occurred later in group 3 ( free from milk factor ) than in group 1 ( having natural milk factor ) , but that the effect of foster - nursing was to increase the age in both cases , so that this cannot be correlated with the administration or withholding of milk factor . 
no undue importance has been attached to this as it occurs in the riii groups , in which foster - nursing is an unsatisfactory procedure , due to the possibility of the offspring receiving milk factor from their mothers during the interval between birth and transference to foster - parent ; also the difference is not statisticllay significant when all cycles are included and the samples are small . on comparing the two strains ( table iii ) there is some indication that the nature of the cycle shows a strain difference , which is unaffected by foster - nursing . comparing these results with the features of the oestrous cycle as described by other workers , there are , in general , appreciable strain differences in this respect ( table vi )  . it may be noted that the average duration of the cycle in young cba mice as observed by bonser ( 1935 ) is closely comparable with that obtained in the present experiment - 5 - 8 and 5 - 3 days respectively . foster - nursing as a means of administering the milk factor appears to be an entirely satisfactory procedure . thus the fact that no significant change in the character of the oestrous cycle was induced by this means , would seem to indicate that the factor does not influence the cycle . it is possible , however , that some adjustment to the new conditions created by the foster - mother 's milk is made during the period of suckling . the milk factor effect might be counteracted by the time puberty is reached and the hereditary condition alone revealed in the characteristics of the sexual cycle . the distribution and extent of two forms of atypical cycle were stuidied , as it was thought that , if they were caused by smearing technique , their incidence might vary in the different groups of mice . should , however , such cycles occur spontaneously , any observed strain difference in the duration of the cycle or e . 
bonser for her guidance and interest in the planning of this work and for her co - operation in the experimental procedure , which made consecutive daily observations over such a long period possible . i also wish to thank j . 
thus , administration of the milk factor from two different highbreast - cancer strains to the same susceptible hybrid mice , kept under controlled conditions , has shown differences in either quantity and / or quality of the factor present in these two high - breast - cancer strains . l , dmocrowski bittner and huseby ( 1946 ) reported similar findings by a different experimental procedure ,  . 
namely , by reciprocal crossings of two high - breast - cancer strains ( c3h and strong a ) , and by recording the tumour incidence and tumour age in virgin hybrids obtained from these strains . 
they described the recorded d ' ifferences in the tumour incidence and in the tumour age in these hybrids as a result of different concentration and / or activity of the milk factor present in these heston and andervont ( 1944 ) in a similarly two high - breast - cancer strains . conducted experiment on sublines of the same strains found no conclusive andervont and eviden ' ce of a difference in the milk factor of these strains . mceleney ( 1941 ) pointed out , however , that his high - breast - cancer strain - ( c3h ) must be regarded as a separate line , and the results obtained with it cannot be controlled by data procured from other lines of this strawamer , reinhard and goltz ( 1945 ) in experiments on virgin hybrids of two other high - breastcancer strains ( m and a ) obtained similar results ' and concluded that the milk factor was more concentrated in one of these stra ' ms than in the other . bittner and huseby ( 1946 ) found , however , the same incidence of breast cancer in breeding hybrids of the two strains tested . 
but they rightly pointed out that the ultimate result of the changes in the structure of breast tissue acted upon by the milk factor of another strain is determined also by the genetic constitution and responsiveness of the breast tissue to the particular milk factor . heston , deringer and andervont ( 1945 ) , b ' y employing susceptible hybrid mice obtained from high - breast - cancer strain mothers and low - breast - cancer strain fathers , and by backcrossing them to highand low - breast - cancer strain males , showed that differences in the milk factor were produced by differences in the genetic constitution of the mice from which the milk factor originated . 
3. as possible into three groups . mice of the first group , when 4r - 5 months old , received one subcutaneous injection of a distilled water suspension of dried riii breast tumour tissue corresponding to 0125 g . 
of fresh breast tumour tissue ; mice of the second group , when of the same age , received one subcutaneous injection ' of a distilled water suspension of dried 63 ca transplantable breast tumour tissue ( from a 428th transplant of the tumour obtained from the imperial cancer research fund ) corresponding to 0125 g . 
no tumours developed in any of the s low - breast - cancer strain females , whether injected with riii or 63 ca dried breast tumour tissue . in the fl generation of s strain females injected with 63 ca tissue and of the control females , no tumours were observed in any of the females , or in any of the males painted with oestrone . in the fl generation of s strain females injected with riii dried tumour tissue , 7 out of 16 females developed breast cancer , and 5 out of 15 males , which were painted , also developed breast tumours . in s x riii hybrid females obtained from daughters of s strain females injected with dried riii breast tumour tissue , 27 out of 32 females showed breast tumours , while s x riii hybrid males of the same origin , painted with oestrone , gave tumours in 10 out of 16 mice . 
observed by the method ' employed in the present experiment . it is impossible to state , however , whether the milk factor was originally absent in the primary tumour or whether it was lost during the course of so many transplantations . .in conclusion , it can be stated that only an analysis of the action and interaction of all know - n factors with allowance for other possible factors can make 102 l . 
the diet was supplemented with cod liver oil , and greens once weekly , and 2 - 3 g . bread ( 80 - 85 per cent extraction ) daily . thirty - four female and 12 male control rats received the same diet without pda or a mixed diet of rat cubes , bread , and once weekly greens . 
the value for total proteins obtained in the present experiments , in which only mature animals were employed , are higher than those found by lippman for mature animals , but lower than the values of metcoff and favour . 
the deviations of the values obtained by the specific gravity method were within + 6 - 5 and 3 - 5 per cent in 10 cases . the sera were fractionated by electrophoresis in the tiselius apparatus with the cylindrical lens schlieren optical system ( thovert , 1914 ; philpot , 1938 ; the buffer solution svensson , 1939 )  . 
the final patterns and the base lines were traced under magnification , superposed in such a way that those parts which did not show any elevation of the base line coincided . both the ascending and descending patterns were analysed . 
the aand the r - globulins appear frequently as broad regions ( moore , 1945 ) , which by overlapping with the 5 - globulin peak , made the separation of the globulins into individual groups uncertathe uncertainty in the values given for the x - , por - - globulins was + 15 per cent or less . wherever the uncertainty was greater , the combined areas of pand y - globulin were measured . 
of ascites was found together with high serum protein . the sera from 6 treated and 10 control animals were analysed by electrophoresis , and the results are given in table ii and fig . 
no difference in the patterns was found between the normal albino and blackhooded rats , on the semisynthetic diet , or on the mixed laboratory diet . all these patterns showed a double albumin peak , the components of which were separated to varying extent . ~~~~~~~~~~~~~~~i ~~01o0 ~~~~~~~~~~~~~~~~~~~~~i c0 - 3 5 - 0 - o ' - o ! a very ~asmiil fig . 
1. 20 b 30 50 gtc 5o.tu.g in the descending pattern the peak of the slower albumin component was in some cases higher than the corresponding peak in the ascending pattern , and in some cases this region in the descending side indicated gravitational instability , reminisin contrast cent of the , - anomaly , which is often seen with normal human sera . the patterns of sera from rats which developed hepatic tumours due to the ingesin no . 
the gravitational disturbance on the descending side was seen in cases 15 and 16 , and to a smaller extent in cases 13 and 14 . the concentration of the total globnlins was significantly increased in the group fed pda , while the concentration of the albumin remained unchanged . the increase occurred mainly in the [ and y - globulins with the exception of case 12 . 
3. - - e : lectrophoretic patterns of rat sera~ 1 - 10 , cotrol rats , 11 - 16 , rate fed pda._ left : = a~ - ~ling , rit = descenamig patterns . cending , right = descendig patterns . threpeaks in the ascending limb , remaining single in the descending limb - and convectional disturbances were observed near the m - globulin region of the ascending limb , but not in the descending or any other part of the ascending limb . ( similar disturbances near the 4 - globulin region have been observed with some human sera in more concentrated solution in the same buffer . ) these disturbances were not due to heat effects caused by the electric current , but probably to the formation of a gravitationally unstable region . 
the figures in brackets are percentages of the total protein . the remarkable sudden increase in the serum proteins at the time of the development of a tumour and the fact that the increase is independent of the size of the tumour seem to exclude the possibility that the increased serum protein has its source in the disintegration of tumour tissue : this sudden increase in the serum proteins has an analogy with the reaction of the body to foreign protein , where the amount of antibody produced is largely independent of the amount of antigen present . 
the results would suggest that , at least in hepatic tumours , the first pathological change in the serum proteins is an increase in the globulins , and a decrease is only the expression of the general deterioration of the diseased individual . the general impression that the serum protein of patients suffering from malignant diseases is decreased may be erroneously gained from reports ( a ) giving 146 c . 
the increase of the yand p - globulins of these rats might be due to an increase in immune bodies frequently associated with these fractions ( tiselius this explanation would be in accordance with the concept and kabat , 1939 )  . that the increase of the total serum protein in rats developing hepatic tumours is an immunological phenomenon . 
black. from the postgraduate medical school of london . received for publication march 25 , 1952 . from perusal of the literature available it appears that chronic ulceration associated with varicose veins is the most rare cause of malignant disease of the skin of the lower limb . 
we therefore wish to present 6 cases which undoubtedly occurred in conjunction with a varicose ulcer or eczema which had been present for many years . cutaneous neoplasia of the leg is rare , and study of the literature demonstrates that such skin cancers as do occur very rarely arise de novo , but usually in connection with chronic inflammation following injury , in old scars and sinuses , or after some chronic skin infections such as lupus vulgaris as well as in the varicose conditions mentioned . since the latter are without question the most commonly found benign lesions of the leg the excessive rarity of malignant degeneration is surprising and interesting . in recent years papers on this subject have been few . 
on examination she was a wasted , frail old woman not well orientated . there were no large nodes in the groin , the general examination showed senile but several small soft nodes could be palpated . changes but no obvious disease . biopsy of the tumour revealed a well - differentiated squamous carcinoma with cell - nests . 
b - , aged 71 years , was referred to us for treatment of a very large varicose ulcer of 20 years ' duration . four months before his admission to hospital the lower edge had begun to hypertrophy and the ulcer to spread on to the dorsum of the foot . 
h - , a widow of 67 years , was referred to us for treatment to secondary neoplastic nodes in the right inguinal region . she had been treated by mid - thigh amputation , at another hospital , for a 30 - year - old decubitus ulcer which , at the time of amputation , extended from the dorsum of the right foot to one - third of the distance up the tibial shaft and involved the bone . there had been no recent change in the ulcer prior to amputation but it had steadily increased in size during about 7 years before admission to hospital . amputation had been advised for pain , and because of the large size and failure to heal with support and dressings . on examination her right leg had been removed through the thigh . 
hard mobile nodes were present in both groins and in the right axilla . the left leg showed marked varicose veins and the scar of a healed ulcer above the left internal malleolus . her heart was fibrillating ( she had been receiving treatment for this for about 6 years ) , but there were no signs of cardiac failure . a report on sections taken from the ulcer after amputation showed a well - differentiated keratinising squamous - celled carcinoma . a biopsy excision of nodes palpable in the axilla was performed . 
no other radiological evidence of malignancy was found . biopsy of the tumour showed a general structure like granulation tissue , with wellformed capillaries and abundant reticulin . cytologically it was definitely sarcomatous , with large , pleomorphic , usually spindle - shaped cells with large irregular often multiple nuclei and numerous mitoses . although the size and histology of the growth and the necrosis of bone underlying made primary mid - thigh amputation the treatment of choice , his general condition was so bad that it was decided to attempt more by radiotherapy . 
lateral and medial fields were used and skin doses of 3300 and 3000 r respectively given using 220 kv . however , only slight response to this therapy couldbe found on serial biopsy , the tumour cells and stroma remaining as in the first biopsy , but mitoses became less frequent and some collagen was deposited . his blood urea , reduced by diet and conservative measures , again rose above 200 mg . 
he remained well for one year , when he was readmitted for treatment of a mass in the posterior flap of the stump . this mass was excised locally , a wide wedge of tissue being removed down to the bone . this local operation was again performed at the patient 's insistence . section of the tumour contained in this block of tissue revealed the identical histological picture presented by the original tumour and the nodule found in the inguinal lymph node . healing was by first intention . 
black this will prevent loss of time , decrease the period of pain for the treatment . patient , and allow the patient to acclimatize himself to the loss of his leg without radiotherapy should be the disappointment of failed conservative treatment . held in reserve for those patients whose general condition does not permit of operation , and these must be very few now . the treatment ofthe inguinal nodes depends upon the age and general condition extensive block dissection is a formidable undertaking in the of the patient . very old , and in these cases irradiation should be advised . 
where the patient will bear the operation this represents the safest method of eradicating growth . sarcoma , largely owing to its extreme rarity is difficult to recognise both clinically and histologically . it appears to occur more towards the centre of an ulcer than at the edge , to produce a florid fungating cauliflower growth and to these bleed readily . 
as with the squamous growths , sarcomata in varicose ulcers appear less ready to metathis may be due stasise than similar tumours elsewhere or in younger patients . to the fibrosis and scarring present after years of ulceration . 
1. received for publication february 6 , 1947 . attempts to find some reliable basis for prognosis in carcinoma of the breast several factors have been thought valuable in have proved disappointing . formulating a prognosis , chief among which have been the presence or absence of secondary deposits in the axillary lymph nodes and the histological character but it has been found that , even in cases with no axillary involveof the growth . ment and carcinomata of low histological malignancy , there has been a mortality from recurrence of about 25 per cent in the first five years after operation . the axillary lymphatics were the only path by which carcinoma cells could escape from the breast , this mortality would be inexplicable . 
a biopsy specimen from one case in the first case the internal mammary gland was removed was also used . post - mortem in 1938 from a patient who died eleven days after a radical mastectomy . 
the internal mammary vessels also lie in this fat in a plane posterior to the glands and usually about half an inch lateral to the edge of the sternuthe glands may be either medial or lateral to the vessels . 
a thin but strong sheet of fascia , the costosternal fascia , intervenes between the glands and vessels and the pleura in the upper two spaces , and the transversus thoracis muscle in the lower spaces . 
the obliquely running the external intercostal membrane and the internal intercostal muscle are reflected as a somewhat ragged flap , either from lateral to medial and hinged on the inner end of the intercostal space : or from medial to lateral and hinged an inch lateral to the edge of the sternuwe prefer the ' former method , since bleeding from the perforating branch of the internal mammary artery seems easier fibres of the intercostal musculature are so loosely bound together that this flap cannot be neatly sutured back into once the intercostal flap has been reflected , position at the end of the operation . dissection should proceed with non - toothed dissecting forceps only . 
they might give rise to troublesome haemorrhage , but this should easily be controllable by packing while the main trunk was being ligated in the spaces above and below . closure of the intercostal space is done as neatly as possible but is seldom complete . 
cushing , my house - surgeon , has in three instances cleared out the anterior mediastinum on one side for recurrent cancer . it is very likely , i think , that we shall , in the near future , remove the mediastinal contents at some of our primary operations . " this idea does not seem to have been followed up . 
sampson handley ( 1922 ) explored the anterior mediastinum in six cases of carcinoma of the breast , finding glands in two , both of which were the seat of metastasis . 
he described in detail a case in which recurrences ' appeared in the inner ends of the intercostal spaces , one after another from above downwards , in a patient who finally succumbed to the disease twelve years after a radical mastectomy ; and he stated his belief that , by the time the axillary glands were enlarged , the internal mammary glands had frequently , and perhaps usually , been invaded . scarff and liandley ( 1938 ) investigated the ten - year results of radical mastectomy in 172 cases of carcinoma of the breast . 
they found that patients without evidence of axillary deposits ( the so - called stage 1 cases ) had a mortality of 25 per cent from recurrence in the first five years after operation , and that this mortality continued at about the same rate between the 5th and 10th years . 
the treatment of these glands must be by some form of radiotherapy . there seems much to commend sampson handley 's method of burying radium tubes in the intercostal spaces at the time of operation , thus securing a localized but intense irradiation of those points where intensity is most needed . it remains to discover whether , in a large series of cases , invasion of the internal mammary glands is as frequent as it would appear to be , what influence the site of a carcinoma in the breast has on internal mammary deposits , and what is the best method of treatment . 
microscopic examination of this gland might greatly increase the accuracy of prognosis , and prove of considerable assistance in post - operative treatment by irradiation . our thanks are due to the director , the bland sutton institute , the middlesex hospital , for permission to use the pathological reports ; to professor r . 
the frankly keratinizing members of our series we have taken as a standard with which to compare the behaviour of our remaining histological types . the morphological features of the more differentiated epitheliomata have been so completely evaluated that any further discussion would be superfluous . 
philps. from the department of clinical pathology , univer8ity college , ho8pital . received for publication december 5 , 1953 . owing to recent improvements in surgical technique the chances of successful intervention in bronchial carcinoma have much improved , and the need for early diagnosis has correspondingly increased . 
the early recognition of mahgnancy , however , may be very difficult and it is hoped that the method described it does not replace histological in this paper may be a help towards diagnosis . examination of material removed at bronchoscopy , but when no tumour can be seen through the bronchoscope , or when histological examination reveals no tumour tissue it can be of great value . when there is some contra - indication to bronchoseopy , the demonstration of carcinoma cells in the sputum may be the sole means of estabhshing a diagnosis . the demonstration of atypical epithehal cers in the sputum of a patient suffering from bronchial carcinoma is not new . 
a positive result was obtained in 13 of the 25 cases . in 1935 dudgeon and wrigley pubfished details of a simple staining method that could be used on sputum films , in which they employed haemalum and eosin . they also described some varieties of neoplastic cell found in the sputum . subsequent papers ( dudgeon , 1936 ; barrett , 1938 ) enlarged upon the original work . at about the same time , gloyne ( 1937 ) described the cytology of sputum with special reference to neoplastic cells . since then , papers on the subject have been pubhshed in a number ofcountries , including britain ( gowar , 1943 ; bamforth , 1946 ; schuster , 1947 ; perrin and littlejohn , 1950 ) , america ( herbut and clerf , 1946 ; mckay , ware , atwood and harken , ' i948 ; liebow , lindskog and bloomer , 1948 ; papanicolaou , 1949 ; foot , 1952 ) , germany ( schmidtmann and sauer , 1952 ) , russia ( altgauzen , 1939 ) and denmark ( wandall 1943 , 1944 )  . 
the lastmentioned paper is quite outstanding and forms a valuable work of reference on this subject . it has a full bibliography , particularly of the continental literature . the proportion of cases of bronchial carcinoma that can be discovered by for example , sputum examination has varied in the hands of different workers . perrin and littlejohn ( 1950 ) obtained a positive result in 60 per cent whereas wandall ( 1944 ) discovered 84 per cent in this way . all of the reports to which i have had access have shown that occasionary a false positive diagnosis is made by sputum examination . while it is clear f . 
philps that such mistakes are more fikely to be made by an inexperienced worker , there remains the risk that even when the pathologist has considerable experience , errors may still occur . 
herbut and clerf ( 1946 ) point out that patients with early bronchial carcinoma frequently have no sputum and advocate the examination of bronchial secretion . in my hands the examination of sputum has been more successful and has sometimes been positive when examination of bronchial secretion has yielded a negative result . 
the routine is simple for patients who are in hospital . a specimen coughed up in the early morning , before any food or drink is taken , and before the teeth are cleaned , is sent to the laboratory . 
very often , although the specimen is produced before breakfast , it is found to contain particles of food material unless the patient has been warned to eat nothing on waking . specimens that contain particles of food visible to the naked eye should usually be rejected , but when a patient has very rttle sputum , any specimen that can be obtained should be examined . out - patients can be given containers and instructed to bring their sputum in them , but it is important that specimens so produced reach the laboratory as soon as possible , and certainly within 24 hours of being coughed up , otherwise infection and autolysis may destroy the cytological picture . specimens sent through the post , particularly in the winter ,  . 
are occasionally satisfactory , but if long delayed they are useless . the whole success of sputum examination for carcinoma cells depends in the first instance upon the selection of a suitable piece of the specimen for microscopical examination . in order that it may be clearly seen , the specimen is transferred to a petri dish and examined against a dark background . 
a mucopurulent piece that is slightly tinged should be taken if it can be found . if the specimen be frankly and uniformly purulent , then there is no alternative but to take a piece of it at randounder these circumstances the chance of finding neoplastic cells is not good , but on occasion they may be found , and are often of squamous type . it has been found that sputum produced during the first few days after the patient has had a bronchoscopy is unsuitable for examination for carcinoma cells . it contains a great deal of cellular material shed from the tracheal lining and the chance of finding neoplastic cells is therefore reduced . i have made it a practice not to examine sputum until a week has elapsed after bronchoscopy . the most suitable instrument with which to handle sputum is a pair of dissecting forceps that have been sharpened to a point on a grindstone . after a little practice , minute pieces of the specimen can be cut out and manipulated with ease . the piece of sputum removed from the gross specimen is placed on a slide , thoroughly mixed with the help of the forceps , and half of it transferred to a second slide . 
one of these is stained with methylene blue ( see below ) , and the other smeared carefully into as even and thin a layer as possible , and placed while still wet into fixative preparatory to staining with haemalum and eosin . it is essential that precautions be taken to avoid disseminating infection from tuberculous sputa . 
philps group has the advantage that films may be stored and re - examined in the light of subsequent findings , or compared with histological preparations made from a tumour . 
the precise technique used differs only in minor detail from that described by schuster ( 1947 )  . it was taught to me at the london chest hospital , and is as follows : a 0 - 5 per cent aqueous solution of methylene blue is used . 
the shde is then warmed by holding it about 8 above a bunsen flame , and at the same time the two are rapidly mixed with the forceps , until the excess moisture contributed by the stain has evaporated . 
the stained sputum is then heaped into a ridge along the middle of the slide , and a cover glass of suitable length immediately put on it and pressed down so that an even thin film is formed . such films tend to fade after an hour or so , the precise time apparently depending upon the amount of infection present in the specimen , but if it is desired that they last longer , this can sometimes be achieved by sealing round the edge of the cover glass with paraffin wax . schuster ( 1947 ) recommends the addition of glycerine to the stain in order that the preparation may keep longer . all sputa sent for examination are stained first by this technique , which is quick . 
a few crystals of mercuric chloride are allowed to remain in the bottom of the vessel containing the mixture to saturate any moisture that may be absorbed by the alcohol . 
the stain is ready for use in 24 hours and can be used repeatedly for 3 or 4 months . it should be filtered if it shows a deposit . it is essential that the film be fixed immediately after it has been spread and while it is still wet , nor at any subsequent stage must it be abowed to dry . it sometimes happens that a specially characteristic cer or clump of cers is seen in the mothylene blue film , and it is desired to make a permanent preparathis can frequently be done in the following way . 
the cover glass is slid off in a direction exactly at right angles to the long axis of the shde , an operation which occasionally destroys the preparation but more frequently leaves the film in place though somewhat smeared in a transverse direction . the slide is immediately dropped into ' the fixative , where it is left for 5 min . the methylene blue becomes purple during fixation . 
undoubtedly this is desirable , but it ' has been found in the present series that between 20 and 30 minutes are required for the preparation and complete examination of a film and it has been impracticable to examine six films from each specimen . 
the writer has found it more profitable to look at it with the naked - eye first so that any parts of it that show a streaky appearance may be especially selected for it has been found that carcinoma cells tend to occur in minute examination . streaks and when these are seen they should be searched throughout their entire length and depth . normally , films are examined with the 1 - inch objective , the i - inch objective being used only when likely cers are seen with the low power . there is little - inch objective in this work , though it is occasionany useful for demand for the examining the chromatin pattem , especiary in oat cells . 
a rather powerful lamp should be used ( a 200 watt bulb is satisfactory ) so that the condenser of the microscope may be lowered shghtly in order to obtain maximum resolution and contrast . 
the nuclear border is smooth and devoid of wrinkhng or irregularity . sometimes , particularly in infected sputum that has stood on the bench for some time , squamous cells are seen which are infiltrated with neutrophil leucocytes . 
39 shows such a carcinoma cells and can ' be a serious source of confusion . cell from a patient in whom there was no evidence of carcinoma . less commonly , a smaller type of squamous cell is seen , which stains more deeply than that just described . it is about 20 microns in diameter , and usually occurs as part of a small sheet of epithelium in the spututhe cytoplasm stains pure b ' lue ( as opposed to green ) with methylene blue , or fairly deep pink with eosthe outline of the cefl is frequently angular - a feature that is evident when such cells are seen lying singly . 
normal epithelial cells tend to be regular in shape , whereas carcinoma cells , particularly those of squamous type , show an irregularity which is best appreciated when many are seen together in the low - power field . 
macrophages. macrophages are constantly found in the sputum in large or small numbers , and are sometimes rather difficult to distinguish from carcinoma cens . many macrophages contain dust particles , which appear as black or brown granules in the haemalum and eosin - stained preparation , and as black , greenish , or purple granules in a film stained with methylene blue . sometimes in a film stained by the latter method , the macrophages are seen to contain a large number of highly refractile fat droplets . 
the presence of dust particles in a cell enables it to be identified as a macrophage , but confusion may arise through the presence of free dust particles overlying a cell and giving the impression of being contained within it . normally they are from 10 to 50 microns in diameter , but , exceptionally , large forms occur . 
as a general rule , they are more or less circular in outline , but this feature is bv no means constant and they assume a variety of shapes . spindle shaped and crescentic forms are not uncommon , and occasionally they may assume a tadpole - fike shape , and may thus be confused with some types of carcinoma cell if shape alone be relied usually the cytoplasm is eosinophilic when stained upon for identification . with haemalum and eosin , but ' it may stain somewhat brownish owing to the presence of ingested particles . 
with methylene blue , it is usuall ' stained blue but when there is a large amount of ingested material the colour may be anything from green to pinkish purple . 
the cytoplasm stains blue with methylene blue , and pink with eosin . the nucleus stains rather deeply and may show some unevenness in the distriit is circular in shape and the nuclei of neighbouring bution of the chromatin . cells in a clump maintain their shape and do not indent each other . cells from oat cell carcinomata , when seen in the sputum , are similar in size to lymphocytes , but differ from them in the following ways : ( a ) the nucleus of an oat cell is larger in relation to the cell area than that of a lymphocyte . 
the cytoplasm of an oat cer appears as only a thin rim round the nucleus , or it may not be visible at all . ( b ) the chromatin of an oat cell sometimes stains more deeply than that of a lymphocyte , and when seen under the - inch objective , shows as a reticulum as opposed to the rather lumpy chromatin of the lymphocyte . ( c ) oat cells nearly always appear in clumps ; lymphocytes seldom do . frequently , oat cers are so closely opposed to each other that they appear t - 0 form a syncytiuthe individual cer borders of lymphocytes are always seen . ( d ) the individual nuclei of oat cers , though usually fairly constant in size , vary considerably in shape . it will be seen that neighbouring nuclei compress and indent each other . 
the only normal cell found in the sputum which occasionally shows this compression effect is the macrophage , which is unhkely to be confused with an oat cell . lymphocytes are shown in fig . 
they are circular bodies , from 10 to 20 microns in diameter . they stain deep blue with methylene blue , and pink , yellowish or - brown with eosalmost always their non - cellular nature is clear : they may show concentric rings , - radial streaking or fissures , or they may be structureless . 
43. - a drawing of a cell seen in a methylene blue filthe cell " body " is a macrophage , and the " nucleus " is a corpus amylaceum which has been taken up . this appearance should not lead to difficulty , a - s the lack of any nuclear details and the concentric rings in the corpus amylaceum make its nature obvious . 
26. - two cell bodies united by a filamentous cytoplasmic bridge . this appearance is not commonly seen , but when present it is considered by the writer to be diagnostic of squamous cell carcinoma . carcinoma whenever considerable pleomorphism is seen in epithelial cells , it should be stressed that a moderate degree may be seen in conditions unassociated fig . 
35 shows an appearance which was mistakenly thought with carcinoma . to be due to carcinoma in a patient who showed no other evidence of a tumour at the time , who has been followed for a year and has developed no sign of the disease . the staining reaction of the cytoplasm is somewhat variable , but may be of some help in identification . 
with methylene blue , it frequently stains greener than that of normal cells , but this property is shared by any degenerate epithelial cell , be it from a carcinoma or from a non - malignant condition , so it is in no way diagnostic . 
the cytoplasm of many squamous carcinoma cells is peculiar in that it appears to be more refractile than that of normal epithelial cells . the methylene blue preparation , this property shows itself by the presence of a dark , rather emerald green outline to the cell and sometimes also to the nucleus within it . 
the cytoplasm itself appears lighter and slightly luminous compared with that of the surrounding normal cells . when stained with haemalum and eosin , the cytoplasm as a general rule stains bright pink , occasionally with a tinge of orange . frequently , as in the methylene blue film , the cell appears brighter than those round it , and if it is put slightly out of focus it may shine up quite brilliantly . cells of this type have been named " bright cells " and are in my opinion diagnostic of squamous cen carcinoma . 
25. - cells from squamous cell carcinomata that give the appearance of wrinkling often , squamous carcinoma cells show a series of irregular concentric lines in the cytothese may be seen most commonly at the periphery , although they are seen to the best advantage in the methylene blue film , they show in of the cytoplasm . plasm , which appear to be minute folds . but they may also occur round the nucleus . the permanent film as well . the nuclei of squamous carcinoma cers have few constant features . another feature frequently seen in the methylene blue film is a mass of minute refractile bodies which are usually disposed in a circular manner round the nucleus . on close inspection , the individual particles sometimes appear to be angular in outhne rather than circular . 
24. - cells showing collections of minute refractile particles in the cytoplasm . these particles are transparent and tend to occur most commonly in a circular arrangement round the nucleus , but they may be seen in any part of the cell . 
normal squamous epithelial cells not uncommonly show sirnilar particles in the neighbourhood of the nucleus , but in the writer 's experience , these are not seen in such large numbers as in carcinoma cells , nor are they concentrated so definitely in one portion of the cell . these particles must be clearly distinguished from the larger , usually black , dust particles and from the large circular fat droplets that are commonly seen in macropltages . dust particles usually present no difficulty owing to their larger size , dark colour and the fact that they are evenly distributed throughout the cell . fat droplets are usually evenly distributed , and appear globular . the six drawings , and those in fig . 
the presence of mitoses in epithehal cells does not imply malignancy , as it may occur in any condition in which there is a considerable amount of epithelial regeneration . in addition to the features mentioned above , a nuihber of other characteristics may be seen which are of considera - ble help in the diagnosis of carcinoma of squamous cell type . 
a particular type of clump has been found to occur in both squamous cer and adenocarcinoma , and though not absolutely diagnostic of mahgnancv appears to be nearly always associated with it . 
the cells all appear to spring from a central point and the clump gives the impression that it may have formed part of a polypoid mass that has broken off into the sputum . if this is correct then any condition associated with papilliferous projections into a bronchus could give rise to such clumps and they might be expected in the sputum in bronchiectasis . i have not seen such clumps in bronchiectatic sputum , but a clump of this type has , nevertheless , led to a mistake . 
philps in the presence of such a change , cells from the metaplastic area would probably be seen in the sputum , but their morphology is not clearlv known . occasionary , round cells of squamous type which have the pecuhar glistening cytoplasm that one associates with keratinisation are seen in the methylene blue preparation . these cells differ from carcinoma cells in that their nuclei are regular in size and shape , though they are often somewhat large in relation to the size of the cell . 
they do not suggest carcinoma in the latter preparation , though they may give rise to suspicion in the methylene blue film . it appears that cells of this type are young squamous cells which may have been derived from the mouth or pharynx or may possibly have come from a metaplastic area in a bronchus . 
the cvtoplasm is geanty , forming at the most a thin rim round the nucleus , which nearly fills the cell . in niany cells , no cytoplasm at all is seen : in others , when the clump is examinedlinder the - j - - inch objective , cell borders cannot be defined , the ceus appearmg to form a svncvtiiithe cytoplasm when seen stains pink with eosin . the nucleus stains deeply with haemalum , and is seldom circular as the nuclei of neighbouring cells indent each other , giving the appearance of having been pressed together . this feature is of great importance in the identification of these cells , and in conjunction with the clumpiing and relatively large nuclear size , appears to be diagnostic and makes the cers easy to recognise . 
the nuclear chromatin is usually clearly defined when thecell is seen under the - ! - - inch objective , and is reminiscent of the appearance of the , reticulum of a stained reticulocyte . less commonly , particularly in degenerate cells , the nucleus may be denser and the chromatin network difficult to define . oat cells are shown in fig . 
the round cers were about twice the size of an pat cell , that is , from 20 to 25 microns in diameter , and they showed greater variation in some , the nucleus was vesicular and nearly fined the cell : than do oat cells . in others , it appeared somewhat contracted and stained more deeply , while in the third and commonest type the nucleus was quite smah and stained nearly spindle cells were seen in clumps amona the streaks of round cens . 
typically , the cells appear in clumps and show vacuolation ' of the cytoplasm , the vacuoles firequently filling the cell and pushing the nucleus to one side , often flatten ' mg ' it against the cell wall . the appearance shown in fig . 
31 , which is from the sputum of a patient diagnosed histoloyicallv as suffering from alveolar cell carcinoma , is very si ' milar to that described by the above - mentioned writers in adenocarcinoma . the presence of cells showing vacuolation is not in itself evidence of carcinoma . the cells shown in fig . 
38 were from the sputum of a patient who showed n ' o evidence of carcinoma , and who , because of th.e presence of these and similar cells has been followed for a - year and has ' developed no tumour . therefore , unless the ceus showing vacuolation occur in clumps and show other characteristics associated with malignancy , they must be considered to be unreliable as evidence of carcinoma . quite frequently , in sputa showing ample evidence of carcinoma of squamous cell type , clumps of cells are seen which surround a single vacuole . 
cers seen which constitute clear evidence of carcinoma . can be g ' iven . categories 1 , 3 and 4 appear from the results to be valuable as an indication of whether the patient has carcinoma , but the value of category 2 , judging from the analysis of results which follows , appears open to question . it will be seen that of the . 
untraced. no cells suggestive of carcinoma seen category 1 . in sputum catego , ry 2 . atypical epithelial cells seen upon which no certain opinion can be given category 3 . cells seen which are highly suggestive of carcinoma category 4 . cells seen which constitute clear evidence of carcinoma of the 9 patients untraced , 8 were out - patients in whose sputum no cells suggestive of carcinoma were found , and who did not return to the hospital . 
he left the hospital soon afterwards and the final diagnosis is not at present known to the writer . of the 8 cases recorded as not yet diagnosed , 3 had no cells suggestive of carcinoma in their sputum , 3 showed epithelial cells which were considered to be f . 
philps atypical but were not thought to constitute evidence of carcinoma , i showed cells that were highly suggestive of carcinoma and 1 , cells that were held to constitute clear evidence of it . 
the 2 patients who were diagnosed as sputum positive are both elderly men in one of whom there is strongly suggestive radiological evidence of carcinoma with persistent collapse of the right lower lobe but no tumour demonstrable on bronchoscopy . 
the other has a shadow persisting in the left mid - zone but has acid - fast bacilli in his sputuit is thought quite probable by those in clinical charge of him that he has carcinoma as well as tuberculosis . 
no bronchoscopy has been performed . * of the 123 patients , there are 106 in whom a final diagnosis has been reached . the results of sputum examination on these 106 will be considered in the analysis which follows . for the purpose of analysis , it is considered that categories 1 and 2 constitute a negative , and 3 and 4 a positive sputum diagnosis , and the terms " sputum negative " and " sputum positive " are used in this sense . it wfll be seen from table i that in two cases , cells that were highly suggestive of carcinoma were seen in the sputum of patients who were finally diagnosed as not having suffered from the condition . one of these patients has now come to post - mortem at which a diagnosis of haemorrhagic bronchitis was made : the other , who was admitted complaining of haemoptysis , is now well , with no evidence of any pulmonary lesion . 
the films ofthe sputa of both these patients have been reviewed and a single clump of cells in the former would still be held to be highly suggestive of carcinoma . in the case of the latter patient , there was a large amoudt ofrather eosinophilic epithelial debris , in the film , the appearance being one which would not now be thought by the writer to be suggestive of carcinoma unless some indication of the mahgnant nature of the cells were also present . 
of the two errors described here , the first must be held to be due to a limitation of the method , whereas the second was caused by lack of experience . of the 106 patients under review , , 39 were finally diagnosed as suffering from bronchial carcinoma , and 67 from other conditions . 
41. total cases in the series cases finally diagnosed as positive cases finally diagnosed as negative total positive diagnoses made by sputum examination correct positive diagnoses false positive diagnoses percentage of negative cases incorrectlydiagnosed as positive 30 false positive diagnoses expressed as a percentage of the total series positive cases in which carcinoma cells were not found in the sputum 9 percentage of positive cases in which carcinoma cells were not found 23percentage of positive cases correctly diagnosed by sputum examination addendum - at the time of going to press , these two patients a - re still udder observation . 
the one whose sputum contained cells that were considered clear evidence of carcinoma has now been readmitted to hospital a year after the cells were first found with clear clinical evidence of carcinoma with multiple bone metatases . 
b is sputum now contains cells similar to , but more pleomorphic than those first found . the other , whose sputum contained cells that were thought highly suggestive of carcinoma has been treated for a year for pulmonary tuberculosis and has improved , ' though the radiological picture is little changed . 
patient8 correctly diagno8ed a8 8putum negative . total patients one speciinen only examined two speciinens examined three four five total number of specimens examlined from these 65 patients mean of number of specimens examined in sputum negative patients j . 
of those who had carcinomata , 30 ( 76 - 9 per cent ) showed carcinoma cells in the sputum . sputum specimens from two patients in the series were wrongly reported as positive . 
one of these patients complained of haemoptysis but recovered rapidly and subsequently showed no evidence of any lung lesion . his sputum contained a great deal of eosinophihc cell debris which was thought to be the remains of necrotic carcinoma cells . 
he died and at post - mortem examination was diagnosed as having suffered from haemorrhagic bronchitis , no evidence of carcinoma being found . those from the first would not now be considered suggestive of carcinoma as the cells showed no other appearance associated with mahgnancy . 
the clump of cells from the second patient would still be held to be suggestive of carcinoma if seen again , which indicates that the method has limitations . further investigation is required into the significance of clumps of cells of this type . it ha - s been my experience that when a confident diagnosis of carcinoma can be made from sputum examination ' , it is usually possible to tell the predominant cell type in the tumour , and so far , when there has been an opportunity for histological confirmation , this has been show ' n to be correct . it has been stressed that in a series of this type , it is possible that misdiagnoses of carcinoma may go undiscovered because the proportion of positive cases is high . it is not certain that there are no such mistakes in the present series but since only about one third of the patients were in fact suffering from bronchial carcinoma , the chances are in favour of such errors being discovered . it is necessary that the limitation of a diagnostic technique of this type be clearly understood . 
a negative finding is of no significance in the exclusion of carcinoma , and may be due simply to the fact that the miinute portion of the specimen chosen for examination contains no cells that can be recognised as malignant . this is likely to be the commonest cause of failure . 
of these , a final diagnosis had been reached at the time of writing in 106 , 39 being shown to suffer from bronchial carcinoma . cells which were either highly suggestive of carcinoma or were considered to be clear evidence of it were seen in the sputum of 30 ( 76 - 9 per cent ) of these . 
an attempt is made to analyse the effect of the number of specimens examined upon the accuracy of diagnosis . where possible , it has been the writer 's practice to state the predominant cell type when a positive report is made . this was done in 23 of the 30 cases so reported . 
an opportun ' ity for histological confirmation has occurred in 10 of these , and there has been agreement . two sputa were erroneously thought to contain cells which were highly suggestive of carcinoma . 
the reasons for these two misdiagnoses are critically examined and it is concluded that one could have been avoided and one could not . further research is necessary to eliminate false positive diagnoses . my thanks are due to professor m . 
3. - normal bronchial epithelial cells obtained by aspiration at bronchoscopy . characteristically these cells are elongated with a ciliated free border and a filiform point of attachment to the ba - sement membrane . 
the nucleus , which ir , regular in size and oval in shape , is nearer to the attached end than to the free border of the cell and tends to make a small expansion in the cell body . 
on close inspection , however , it will be seen that a number of cells in the clump have the general shape of bronchial epithelial cells , showing a wide free border and a filiform point of attachment , and the nuclear distortion is mainly due to the presence of it is possible to discern gradations from those which appear vacuoles in the cytoplasm . nearly normal to those which seem grossly abnormal . 
the strongly eosinophilic cytoplasm is also suggestive of carcinoma , but may occasionally be seen in other degenerate epithelial cells . it should be noted that the appearance suggestive of inclusions seen in this cell differs from that shown in fig . 
the nucleus shows no unusual characteristics and is situated in the wider " body " of the cell . sometimes , chromatin masses are seen in the tail , and on occasion , two cell bodies , each containing a nucleus , are united by a long filamentous bridge . 
the writer has seen them only in cases of squamous cell carcinoma , and nothing resembling them has been seen in any sputum specimen from a non - malignant condition . they appear considerably more refractile than do normal cells , and by virtue of this property , look bright when compared with the cells that surround thewhen put slightly out of focus they shine brilliantly . the high refractility of these cells is possibly associated with keratinisation , and may be related to the appearance shown in fig . 
and the cell itself is surrounded by a greenish coloured band which is probably due to some optical property of the cytoplasm . its brilliance is enhanced compared with the cells in the surrounding field . 
the coloured band which surrounds the cell stands out clearly . in addition to the cell described , there is a considerable amount of eosinophilic debris shown , similar to that seen in fig . 
34 , which is from the sputum of a patient diagnosed clinically as suffering from bronchial carcinoma , but which is considered by the writer to constitute insufficient evidence to enable a diagnosis of carcinoma to be made . 
the present writer now shows considerable caution in the interpretation of the significance of such clumps as two mistakes have been made . before this it was thought that clumps of epithelial cells of this type constituted strong evidence of carcinoma . 
41 and 42 , which were both thought to be strongly suggestive of carcinoma , were from patients who had no other evidence of carcinoma at the tiirne , and in one of whom there wa - s later no post - mortem evidence . 
they have the following chaxacteristics which , taken together , distinguish them clearly from any other cell that is seen in the sputum : the cytopla - sm may be visible as a thin nvhere neighbouring cells are closely applied to each other , riirn , or it may not be seen at all . as is often the case , cell borders are not seen , the appearance being that of a syncytium . ( 2 ) the nuclei of all the cells in a clump are of approxiinately the same size , but they vary considerably in shape , many of them tending to be rectangular . 
the reason for this is that the nuclei of neighbouring cells indent each other . this is the most important single feature in the identification of oat cells . ( 3 ) the nucleus usually stains deeply , but the chromatin pattern can nearly alway 's be distinguished . 
29. - ( 2 ) spheroidal and 8pindle cell carcinonw . a field showing a group of cells similar tov but larger than , oat cells and a small bundle of spindle cells . 
34. - a clump of cells , the individual members of which have no definite characteristics of malignancy though one of the cells seems to possess large nucleoh - an appearance which may be due to the superimposition of pus cells . 
at one time , clumps of this type were thought by the writer to be diagnostic of carcinoma , but a clump of rather similar appearance led to a false positive diagnosis in a patient who was found at postmortem examination not to be suffering from carcinoma . for clumps of cells that are considered to be diagnostic of carcinoma , see fig . 
35. - a group of epithelial cells showing a considerable degree of pleomorphism and nuclear degeneration . this is the most confusing appearance that has been seen in the whole series . 
the cells resemble carcinoma cells closely , but in no case is the nucleus large . this field , and the three that follow , were from the sputum of a single patient , in whom a positive diagnosis was made as a ' result . 
41. - a clump of cells which appear to surround a vacuole from the sputum of a patient who , on the strength of this evidence , was thought by the writer to be suffering from bronchial carcinoma . 
daily , add the remainder received twice this dose . table i shows the result of this experiment on the group receiving the lower dose level ; the treated tumours were completely inhibited . 
smar nodules persisted in the majority of these animals without change for about 2 weeks , after whicb tumours began to grow ; they were well developed in 70 per cent of the group 1 month after cessation of treatment . 
i and it seems reasonable to suppose that the same applies to all such tumours developing from inhibited nodules . when drug treatment was commenced 3 - 5 days after transplantation , both the sensitive walker tumour and its refractory derivative were always actively growing . 
the growth of the derived tumour was affected to a reasonably consistent extent by triethylene melamine , comparable to that observed with the lymphosarcoma . the subsequent development of inhibited walker tumour nodules has been surprisingly , growth was found to occur after a similar repeatedly confirmed . latent period in the majority of animals even though drug treatment was continued 340 h . 
ad example of this is shown id table iv , from wbich it is evident that tumours developed in 8 - 10 animals following the usual inbibitory period of nearly 3 weeks . it proceeded rapidly and then slowed quite markedly . in spite of the prolonged treatment in which 31 doses were given , there were no deaths which could be directly attributed to the effect of the drug . this faflure of continued treatment to maintain the state of inhibition id the tumour has also been confirmecl in other experiments . the contrast in susceptibihty between the original and derived walker tumours to the triazine , has also been found . 
as was mentioned earlier , the development of mouse leukaemia resistant to fohc acid analogues is wen known and there has been a recent report that the mouse sarcoma 180 , whicb is moderately inbibited by 6 - mercaptopurine , rapidly develops some resistance to this compound ( clarke , philips , stemberg , stock , elion and hitchings , 1953 )  . on the - whole , solid rat and mouse tumours appear to be relatively resistant to chemical inhibitors , although the response of different tumours to the same drug varies widely . 
not often does the arrest of growth extend to all tumours in an adequate group of experimental animals , in spite of the administration of dose levels of near lethal magnitude ( suguira and , stock , 1952a , 1952b )  . appears that rat tumours are more susceptible than those of the mouse to trie - thylene melamine , for complete regression has been obtained in nearly 100 per cent , of animals with the jensen rat sarcoma , sarcoma r39 and the flexner joblidg it is generally agreed that the longer carcinoma ( suguira and stock , 1952b )  . initiation of treatment is delayed , the less chance thei - e is of total regression . - the action of triethylene melamine on the walker carcinosarcoma is striking - even when treatment is delayed for as long as 5 days after implantation , by which time the tumour is actively growing . in the present series of experiments regression occurrecl in every animal and usuary little or no palpable material this confirms the work of hendry , homer , - remained at the site of the implant . rose and walpole ( 1951 ) and peczenik ( 1952 ) , who however , commenced treatment curiously , 24 hr . 
after transplantation of the walker tumour into wistar rats . suguira and stock ( 1952b ) were less successful with this tumour in wistar rats , .obtaining inhibition of growth in 70 per cent of animals with day old implants and regression in only 20 per cent when treatment was delayed for 7 days . 
the results clearly show that in effectively 4h animals the inhibitory effect is only temporary and that growth subsequently occurred irrespective of the duration of treatment , even though this may be continued for several weeks instead of the usual 7 - 10 in either case the suppression of growth continued for upwards of 2 weeks days . before renewal of activity could be detected . 
the derived tumour on which most of the work has been carried out , was selected at random from a group of tumours growing subsequent to one short course of treatment applied to the original walker tumour . 
the resistance is thus considered to be a stable and permanent change . another member of a group of tumours similarly derived from the original walker carcinosarcoma has also been shown to possess comparable resistance to further treatment . it is reasonable to suppose that the same will apply to all such tumours . 
a noticeable difference between the original walker and the derived tumours is the more rapid growth of the latter in untreated animals . the growth of tumours after a latent period followino , a short course of treatment with triethylene melamine , must be due to the survival of a small number of cells which are naturally resistant to the drug . 
the fact that this sequence of events is not changed by the continued administration of triethylene melamine to animals bearing implants of the original walker tumour , suggests that the resistance of the residual cells is maximal at the outset . additional support for this view emerges from the failure to enhance the resistance by further exposure to the drug . 
how far mutagenic effects of the ethyleneimine may be concerned remains to be investigated , but the fact that such a bigli proportiod of inhibited tumours ultimately grow seems to make this factor less likely . 
the resistant tumour has also been shown to be refractory to two other ethyleneimines ( compounds ii and iii ) at dose levels which completely inhibit the original walker thus resistance to triethylene melamine confers resistance to carcinosarcoma . a diethyleneimine , and this may be true of active ethyleneimines in general . how far the same statement may apply to other kinds of chemical inhibitors active against the ordinary walker tumour is being investigated . it is interesting that the walker tumour should be so sensitive to triethylene melamine , whilst two other tumours which have been tested in the same strain of animal ( a mammary adenocarcinoma and the lymphosarcoma referred to in the crossley , allison , wainio text ) were only little affected by the same treatment . and muenzen ( 1951 ) studied the effect of triethylene melamine on a sarcoma ( 231 ) in the king a rat . 
of the triazine without undue toxic treatment of 7 - day old transplants of the flexner jobhng careinomaeffects . with this dose rate for 7 days produced regressions in 90 per cent of animals in there was no reappearance of tumours during the next 3 months , 1 to 2 weeks . which was taken to indicate complete cure . 
not only different kinds of tumour but various strains of rat vary in their susceptibihty to triethylene melamine ; the growth of an occasional tumour in animals bearino , a neoplasm susceptible to the drug , may be connected with some unsuspected ability of the host animal to dispose of the administered compound . it appears reasonable to suppose that ethyleneimino - compounds as a whole owe their anti - tumour activity to these highly reactive groups , so that it is difficult to conceive a protective mechanism possessed by some cefls but not by the great majority it is equally difficult to imagine a high degree of susceptibility in a tumour . in one variety of neoplasm and indifference in another kind to chemical inhibitors hke triethyleneimino - phospboramide ( iii ) , which is highly soluble in water and hkely to diffuse with ease into cells . 
then too , there is the fact which has been estabhshed on many occasions , that the susceptibility of sensitive tumours to these drugs diminishes markedly with the age ( i.e. , size ) of the tumour . it is hoped that a study of the mechanism by which the resistant walker tumour is derived from the original variety and an investigation of the reason for the insusceptibihty of the formee tumour may throw light on these interesting and important phenomena . inhibited nodules from the walker carcinosarcoma treated with triethylene melamine , 24 hr . 
after the conclusion of a course of treatment show intact tumour cells . no mitotic figures were seen . estimation of the mitotic index of the original and derived forms , 48 hr . 
after the administration of the last of 2 doses of the same drug reveals that the growth rates of the two tumours are similarly depressed . the drug would therefore , appear to be exerting a cytotoxic effect on the resistant form , although the overall growth of this tumour is not much hindered by such treatment . 
 although urethane has been mentioned as carcinogenic for mouse ( nettleship and henshaw , 1943 ) and rat lung ( jaffe , 1947 ) this action has not yet been reported in other animals . 
since the inherited susceptibility to most known cancers is due to dominant genes this was anticipated here , and con sequently a large number of the c57 backcross mice were bred in order to note segregation , which should be manifest in the group on this assumption . 
preliminary experiments indicated that the various strains and crosses of mice used did not differ in their fluid intake , and consequently strain differences due to this treatment were not due to a different intake of the carcinogen . 
they were from another experiment and were only given 7 weeks ' urethane treatment , though they were killed 7 months from the beginning of treatment , the same as the rest . 
 when the mice were sacrificed their lungs were removed and fixed in 10 per cent formol - saline for 24 hours , which preserved the material and made handling easy . 
since nearly all lung tumours in mice tend to occur on the periphery of the lobes ( grady and stewart , 1940 ) the lobes were separated and the number of nodules on their surface visible to the naked eye were counted . 
in a previous paper ( cowen , 194 7 ) it was noted that strains of mice which gave a low number oflung tumours had tumours of a smaller size . 
 since the tumours in a lobe varied in size from microscopic dimensions to nodules about 4 m in diameter , it was not feasible to use the average size of all these tumours . 
the best way of gauging the response on a basis of tumour size was considered to be that of determining the size attained by the largest tumour in the total lungs of a mouse . 
this was tested statistically using the formula v ' ; ; ; 2 on the other hand , the fl hybrids were slightly but significantly lower than both of these classes . 
the polygon for the 057 backcross mice is obviously bimodal , and roughly consists of a combination of the figures for the 057 and fl hybrid mice in approximately equal proportions . 
 it may be mentioned that the a backcross mice which segregated for colour giving three phenotypes , albino , non - agouti and non - agouti brown , showed no apparent linkage of susceptibility with colour . 
 - ~ number of tumours f 3 . - - scatter diagram showing the relationship between the 2 groups of c57 backcross mice with respect to number and size of tumours . 
 thus , of all the chickens and guinea - pigs treated with urethane , only one chicken developed a neoplasm which was lymphoid in orig this type of tumour is not uncommon in chickens normally , and is not considered to be due to treatment with urethane . 
the first ( number of tumours ) gave more obvious results than the second ( size of the largest tumour per mouse ) , though both results closely paralleled each other . 
 these results point most significantly to the existence of a single dominant gene in a mice which is responsible for urethane - induced lung tumour suscepti bility , the c57 mice carrying the recessive . 
this does not entirely explain the results , for if a single dominant gene only were responsible , then a , fl , a back cross and the high group of the c57 backcross mice would all have exactly the same tumour incidence , which is not the case . 
firstly , the tumour susceptibility was genetically ina.ependent of mortality , and secondly , as the mortality was considerable only in the a and a backcross mice , a higher tumour susceptibility in the dead mice would merely have emphasized the line differences . 
 heston ( 1940 , 1942a , 1942b ) reported somewhat similar results using spon taneous and hydrocarbon - induced lung tumours in a and l mice ( high and low strains respectively )  . 
a possible reason for this is that the incidence of lung tumours in these mice was much lower than in the urethane - treated mice of the c57 backcross as reported here . 
 from the results of these workers , it appears that certain carcinogenic chemicals cause different lung tumour incidences in different strains of mice depending on the incidence of spontaneous lung tumours in these strains . 
that is , a strain of mice which develops few spontaneous lung tumours does not respond so well to a carcinogen as a strain which has a high spontaneous incidence . 
from this , it seems that environmental factors ( certain carcinogens ) do not simply cause tumours , but increase the incidence of them in mice depending on the inherited tendency of mice to develop the this argument can most probably be applied to other animals as well as mice . 
 it seems rather significant that no reports have been found in the literature of spontaneous ( or induced ) lung tumours in guinea - pigs , and out of the many neoplasms that arise in chickens , only one report exists of a spontaneous lung tumour in these animals ( apperly , 1935 )  . 
for example , guinea - pigs , which have a relatively frequent incidence of spontaneous liposarcomas ( warren and gates , 1941 ) , develop these tumours on treatment with hydrocarbons ( shimkin and mider , 1941 )  . 
it was considered that the resistance of mice to infections in general was lowered , and at the same time a specific carcinogenic virus was responsible for the lung tumours . 
if a single recessive gene is assumed to be the cause of the different mortalities , then the a backcross would be the segregating group , and should have a survival rate equal to the average of those for the a and c57 mice . 
from table i it is seen to be 45 , which agrees reasonably with the theoretical figure considering the small number of mice involved ( x2 = 317 , p > 005 )  . 
 it can thus be safely concluded that the mechanism whereby strain a mice are genetically susceptible to lung tumours is not the same as the one responsible for their susceptibility to general infections . 
correlation between leucopaenicity causing susceptibility to infection on one hand and a carcinogenic virus causing lung tumours on the other is therefore considered most unlikely in mice given urethane treatment . 
 two criteria which parallel each other closely are used as an index of response of mice to the carcinogenic action of urethane - the number of tumours per mouse and the size attained by the largest tumour in total lungs . 
kemp. from ae university institute , for human genetics , copenhagen . given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . from ancient times it has been observed that cancer often occurs familially the inheritance of tumour 's in human beings has been studied and discussed for centuries , and it has especiallv been the object of extensive systematic investigations during the last decade , , about 6 years ago , a group of collaborators at the university institute for human genetics - in copenhagen decided to make an attempt to investigate the the ' investigation has partlybeen carried genetics of various aspects of cancer . out in co - operation with the danish cancer registry , under the direction of j . 
clemmesen , the university institute of pathological anatomy , and the radium centre in copenhagen and numerous danish hospitals and clinics . the work was supported by kong christian x 's fond , landsforenigen til kraeftens bekaempelse and anders hasselbalchs fond . these investigations include genetic experiments ' in mice and studies in man . twin examinations were also initiated in co - operation with the cancer registry . the statistical - genealogical investigations have been performecl as a series of surveys each concerning a considerable number , usually sevetal hundreds , of probands or propositi with cancer of a ce - rtain - type , e.g. 
canc - er mammae , cancer uteri , cancer oesophagi , multiple cancer or leukaemia , picked at random from the population . these investigations were made by a number of physicians , each being a ecialist in the field his study coneems . 
the families have been studied thoroughly , and every case of cancer in the relatives has , so far as possible , been verified ; in that respeot these investigations differ from earlier , more compre hensive statistical studies in the heredity of cancer . 
cancer of the breast , uterus , stomach and rectum , their occasionally familial occurrence has been know - n for in order to elucidate the etiologic significance of hered ' itary many years . factors in these tumours , proband investigation has been carried out . jacobsen 's ( 1946 ) study on the heredity in breast cancer includes ' 200 probands , ( 197 women and 3 men ) with cancer of the breast . 
the investigations of the probands ' families comprise the following categories of relationsi : parents , brothers and sisters , grandparents and brothers and sisters of the parents . videbaek ( 1947 ) has investigated ' edigrees of 20 ' 9 patients with leukaemia and 200 control probands , using the same methods as jacobsen . the propositi - method of investigation is , of course , rather complicated and leaves room for several sources of error . it must be realized that the diagnoses and the information on the whole as regards the older or more distant groups of relations are rather unreliable . 
some of the grandparents , for instance , died more than 50 years ago , and at that time , diaanostic skill was more limited and hospital records and death certificates were more insufficient than to - day . furthermore , it is difficult to procure a comparable control , material , because the probands are normally more interested in the occurrence of cancer in the family than normal control probands are ; probably that is the reason why the information conceming the distant groups of relations in jacobsen 's control material is insufficient . also it must be considered that during recent years many cancer patients , e.g. 
suffering from skin cancer , get cured without knowing they have had cancer , and thus escapenotice by the investigator . it should also be pointed out that the calculation of the morbidity risk is a complicated method , and cannot always be relied on . 
lip and skin cancer , are not represented in the families of the probands , but cancer of most other forms , sites and types occurs in the relatives in about the same proportion as among the r ' elations of the control probands , and is comprised under the collect ' lve term " endogenous cancer as a whole . " according to jacobsen ( 1946 ) , a study of the case histories of the probands gives no basis for supposing that extemal factors play any important role in the development of breast cancer . 
an excess incidence of breast cancer among the female relatives of the probands and likewise a significant excess incidence of " endogenous cancer as a whole " in all the categories of relatives , both male and female , were found . this indicates hereditary predisposition as the chief factor in the development of breast cancer . breast cancer is dependent o ' n hereditary factors , and the tendency to this particular form of the diseas.e is bound up with an inherited predisposition to endogenous cancers endogenous cancer in general . 
the localization of the tumour is determined either by hereditary or extemal factors . in many pedigrees the general hereditary predisposition appears to be inherited as a dominant character . in the leukaemia proband material , the familial incidence of the disease was found to be at least 8 per cent . several types of leukaemia ( acute or chronic lymphoge ' neous or myelogenous , monocytic or stem - cell leukaemia ) may occur in the same family -  . 
the multiple occurrence of leukaemia in an individual family is usually not confined to some particular type , which indicates that leukal - imia is probably genetically a morbid - entity . among members of a family there seems to be an age correlation as regards the onset of leukaemia.. 
according to videbaek ( 1947 ) leukaemia , as such , is not simple domininherited ; it is a question of inherited disposition to the disease . ance or recessivity may possibly be excluded ; it may have a genetical - basis with incomplete dominance or polymeria ( homologous polymeric factors )  . hereditary predisposition to disease is generally regarded as polymeric . 
the expression of the character is furthermore dependent on the interaction between the genotype and environment . the incidence of pemicious anaemia is significantly higher among the relatives of patients with leukaemia than among the relatives of the control probands . the relation is probably due . 
among the relatives of the leukaemia patients there is a significantly excessive incidence cancer , of cancer as a whole , due to a high incidence of all forms of cancer . including leukaemia , is piobably a disease entity genetically dependent on a dominant gene common for all the different forms of " endogenous cancer . " according to videbaek 's oplinlon the - development of leukaemia seems to depend on various conditions , among others , on a non - specific hereditary predisposition to cancer , which is believed to be present in at least 20 per cent of thepopulation in general , and partly on one or several genes , the activity of which plays a role for the localization of the cancer to the leukon ; leukaemia seems to constitute an entity genetically and environmental changes influence the type of the disease . there is evidence that extemal factors also ' play a role in the development of leukaemia . 
of several cases of leukaemia in the same family is rather slight ; but the risk of getting cancer in near relatives of a patient with leukaemia is as high as almost 50 per cent . table iii shows some of the data from the breast cancer and leuka6mia material in relation to cancer risk . 
among the relatives of probands with cancer of the body of the uterus the incidence of " endogenous cancer " was high , and the relatives of probands with cancer of the cervix show the same frequency as the relations in the families of the patients with cancor of the cervix , of control probands . on the other hand , a relatively increased frequency of cancer of the oesophagus in this connection it is worth while to draw attention to the simiwas present . larity of the histological structure of cancer of the cervix and cancer of the in the families of probands suffering from cancer of the oesophagus oesophagus . investigated by mogensen ( unpublished data ) , cancer of the oesophagus was often found in a close male relative , the father or a brother of the proband . in these families , both the proband and the relative with cancer of the oesophagus 148 t . 
the differences could , however , not be significantly proved , owing to the insufficiency of the material , and the differences may be due to the fact that - the old propositi generally have less detailed and exact information about their relatives in earlier generations than the young propositi have . still the accordance of the experimental results and the clinical observations makes the significance of the latter probable . our investigations into the heredity of various aspects of cancer , have show - n that hereditary disposition is an important tumour - causing factor . it was found that it is the chief factor in the development of breast cancer , and many other tumours are more or less dependent on hereditary factors . 
the general cancer - tendency " ( likely not a general blastoma tendency ) occurs with considerable frequency in the population ( it is probablv higher than 20 per cent ) , and is possibly inherited as a character showing incomplete dominance . 
our investigations have shown that the tendency of tumour - localization has also a hereditary basis . the development of leu ' kaemia and cancer is probably due to a common hereditary . 
koller. from the chester beatty research institute , the royal cancer hospital , london . given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . xeroderma pigmentosum ( hence referred to as x.p. ) , is marked by rougheniing , dryness , pigmentation and ulceration of the skthe condition is attributed to hypersensitivity of the skin , particularly that of the basal cell layer , to sunlight . the disease was first described adequately by kaposi in 1874 ( hebra and kaposi , 1874 )  . 
occurs in the first year of life , often within a few , months after birth . first , erythema develops on the skin , on those regions which are exposed to light , followed by freckling and frequently by telangiectasis . from these regions epithelioma or basal - cell carcinoma develops . 
the malignant change may take place not only in the skin , but also in the cornea and conjunctiva the genetical and chromosomal basis of x.p. the hereditary basis of x.p. 
was first investigated by siemens and kohn ( 1925 )  . they analysed 76 families , and cockayne ( 1933 ) brought this more up - to - date with a further 43 family histories . in both groups of data the incidence of consanguineous marriages was very high : 24 out of 76 and 16 out of 43 respectively , were first - cousin marriages . 
i. received for publication january 4 , 1951 . recent work has provided evidence in favour of the supposition that tumours contain , and therefore probably release into circulation , a substance capable of depressing liver catalase activity . 
nakahara and fukuoka ( 1949 ) claimed to have produced a liver catalase depr ' ession in mice after the injection of alcoholprecipitated fractions obtained from human tumours . similar fractions from normal tissue were stated to be inactive . 
adams ( 1950a ) commenced a study of the effect of the injection of homogenized mouse tumours into mice , and showed that after an early ( 24 and 48 hours ) depression , the hver catalase level returned to normal approximately 4 days after injection , and fer once more as the new tumour grew . 
the results were interpreted as indicating that the initial depression is due to some substance or substances present in the , injected material , the second depression only being due to the new tumour . injection of homogenized normal tissues had no effect on catalase level . appleman , skavinski and stein ( i950 ) , in the course of an investigation of the variations in erythrocyte , kidney , and liver catalase activity in rats bearing the jensen sarcoma or the u.c.l.a. 
fibrosarcoma , found that normal rats kept on a protein - free diet showed a drop in liver catalase activity to approximately 50 per cent of normal within 7 to 10 days from the time thev were placed on the diet and this low level wa - s maintained for 53 davs or longer . 
the variatioils in liver catalase level following the subcutaneous injection of coarse suspensions of these tumours into albino mice results in both males and females were similar to those preappear in table 1 . viously obtained with s37 and carcinoma 63 . 
adams two general methods have been employed : the first involved the injection of s37 sarcoma , which had been treated so as to render it incapable of giving rise to tumour growth , and the second , the results of which are presented in the next section , the injection of pure line mouse tumours which do not grow in the strains of mice used . sarcoma 37 tissue was disintegrated by shaking for several minutes with glass beads in a " mickle " tissue disintegrator , made by messrs . 
is a plot of the subsequent variation in hver catalase level over a cers . period of 10 days , the crosses representing the arithmetic mean values of the groups . in the experiment represented iin the left - hand graph the tissue was disintegrated in normal saline ( ph 6 - 8 ) , and it wir be seen that a 24 - and 48 - hour depression was obtained , the level rising to normal at 4 days and remaining so at 7 and 10 days . however , this treatment was not completely succe 'ssful in pre ' venting tumour growth , in spite of failure to discover intact cells in the homogenate microscopicary . 
the experiment was repeated , using m / 100 citric acid as the suspending fluid ( ph 5 - 5 ) , and the results appear in the righthand graph . 
the catalase depressing effect is very similar , but in this case no tumours appeared in 18 mice kept for 3 months , when the experiment was terminated . a second method involved the partial centrifugation of a fine homogenate ofs37 tissue in normal saline , prepared originall with a tenbroeck grinder . 
n. abscissa : time in days . effect of in ' jection of tumours which do not grow in the 8train of mice u8ed . a number of pure line mouse tumours which do not grow , at any rate after the in ection of coarse homogenates , in the stock albinos used , were injected subcutaneously or intraperitoneally in 100 mg . 
the results appear in table il in every case a significant depression in catalase activity was observed 48 hours subsequently , with a return to normal by the 4th to 7th day . in one experiment of this series , not recorded in table 11 , a different result was obtained . 
the catalase level at 48 hours was very low , and remained low at 4 days ; at 10 days , of th - e 6 remaining mice , 3 in which the abscesses were healed had high levels and 3 120 d . 
the results are given in table il no significant variations in catalase level were observed , and with the rat tumours in particular the variations appear to be completely random . ..cb - r = 1 l -  . 
the points lie on a slightly curved line , showing a progressive deorease of catalase activity with dose . the depression is seen to be approximatelv proportional to dosage over the range 10 to 30 per cent . 
4. - liver catalase levels 48 hours after the intraperitoneal injection of varying quantities of coarse homogenate of sarcoma 37 into stock albino male mice . ordinate : catalase level in arbitrary units / mg . 
adams it is not claimed , however , that nutritive effects play no part in producing the depression of catalase activity , particiilarlv in animals bearing large tumours . miller ( 1950 ) has recently shown that although catalase level in animals which are maintained on low or non - protein diets parallels approximately the level of total liver protein , the enzyme soon begins to disappear more rapidly than the consequently , even if the only relevant effect of a large remaining protein . tumour on the nutritional status of an animal were a simple depletion of protein , however , the depression which occurs in a depression of catalase would result . the early stages of tumour growth may be expected to be due mainly to toxic material produced by the tumour , since the importance of purely nutritive factors , e.g. , protein depletion , must be small at this stage , though becoming progressively greater as the tumour size increases . 
the effectof large tumours on catalase is probably the resultant of separate nutritive and toxic actions , which could not be readilv distin uished . the effect of injecting infected c57 sarcoma is quoted as a possible source of error in investigations of this nature . it is not yet known whether catalase depression is a common result of bacterial infections , but this possibility must be reckoned with when using , for example , human intestinal tumours , which are greenstein ( 1947 ) states that infection of mice with certain often infected . strains of poliomyehtis virus , or with streptococci and staphylococei , has no . however , it has been observed in this laboratory effect on catalase activity . during epidemics of a sabnonella infection , and of ectromelia , that catalase level was low when the liver showed signs of involvement . 
dounce and shanewise ( 1950 ) i - lave recently shown that catalase activity is significantly depressed in rats suffering from advanced murine leprosy . apart from these isolated observaticjns , no detailed investigation has beeii made of the effect on liver catalase of infections of the liver , or of injections of infected normal or malignant tissue . although a depression of liver catalase may occur in conditions other than cancer , this does not detract from the importance of the effect as a property of maligiriant but not of nornial tissue , and work on the mechanisbv which the toxic substance exerts its effect may thrcw light on some fundamental difference recently reports of prehminary experibetweennormal and malignant tissue . ments have appeared , indicating the importance of hormones in maintaining a normal level of liver catalase ( begg and reynolds , 1950 ; adams , 1950b )  . present investigation is being follo - vved up by an endeavour to relate the tumourdepressing effect with the hormonal control mechanism . depression of liver catalase activity b - as been shown to follow the injection into mice of mouse tumours , but not of rat and human tumours in single doses of up to 100 mg . 
nakahara and fukuoka ( 1949 ) , on the other hand , observed a depression after the injection of alcohol - precipitated fractions of human tumours however , their dosage , referred to weight of original tissue , was into mice . probably very much bigher than that used here . all that is now claimed is that injection , in the dose used , of mouse tumours into mice produces a considerable depression of liver catalase , while injection of similar doses of tumours of other species produces no apparent change . 
the early variation in injection . females was not significant in one case . ( 2 ) injection into stock albino mice of sarcoma 37 tissue ( a ) disintegrated in m / 100 citric acid , or ( b ) partly centrifuged after coarse homogenization in 70 mg . 
doses , resulted in a depression of liver catalase without any obseirvable tumour growth . ( 3 ) six pure line mouse tumours , which do not grow in the stock mice used , also gave catalase depressions 48 hours after injection in 100 - mg . 
fibroid with advanced sclerosis ; patch of oedematous tissue ' probably smooth muscle fibres ; proliferation of vascular endothelium ; invasion diahypertrophic endophragm ; thelium on the surface of the tumour . . 
the peripheral belt of fibroblasts originating most probably from subendothelial cells of the serosa and so characteristic of the oestrogeninduced abdominal fibroid ( lipschutz , 1942 ) was present in a diaphragmatic tumour belonging to the animal of fig . 
as with the abdominal fibroids we have described formerly , there were in thoracic tumours also some cells resembling there was a proliferation of the cells of the wall of vascular smooth muscle fibres . spaces ; there was an hypertrophy of the endothelium at some places on the there was invasion of the muscular tissue of the diasurface of the tumour . phragm , or the thoracic wall , by the tumours . 
but there were in this tumour also accumulations of cells resembling fibroblasts which seemed to originate from the proliferation of the subendothelial cells of the wall of vascular spaces , or the endothelium proper . 
the tumour was covered by various layers of flattened cells . there were besides the animals described , 4 females and 2 males in which small nodules just visible to the naked eye were found on the surface of the only 2 animals of this group have been examined lung or on the parietal pleura . microscopically ( table i ) and in one of these a tiny fibrous nodule was found on the surface of the lung ; the structure of this nodule was identical with that of similar oestrogen - induced nodules of the abdominal serosa . 
note thickened border of opening in the diaphragm , prolapsed liver and intestine . fibrous reaction also on the right though the opening is on the left . small nodules on the pericardium in lxiii . 
the tablet occupied in some cases a retrosternal or mediastinal position ; it was surrounded by a thin capsule and in other cases the capsule adhered to the costal serosa , thin fibrous strands . where the fibrous reaction around the oestradiol tablet was more conspicuous . but there was not a single animal with thoracic tumours in this group . 
lipschutz oestrogen - induced thoracic fibroids increased very considerably ; thoracic fibroids in 7 out of 18 animals with such a communication . there were in most of these animals part of the liver and of the intestine prolapsed to but the mechanical stimulus due to this accident was the thoracic cavity . seemingly not responsible for the increase of the incidence of oestrogen - induced this was shown by the fact that in 24 animals without an thoracic fibroids . abdominal - thoracic communication the capsule enveloping a foreign body introduced in the thoracic cavity ( metallic tablet or a tablet of oc - oestradiol ) did not , or only exceptionally , become surrounded , under the influence of the oestrogen , by a fibroid as was the case with a similar foreign body ( metallic tablet ) introduced in the abdominal cavity of these animals . the above experimental results suggest that the oestrogen - induced fibrous tumoral reaction of the peritoneum in the guinea - pig is mediated by an internal ambiental factor normally present in the abdominal serosal fluid , or a factor originating under the influence of the oestrogen and given up to the abdominal it is seemingly the absence of this mediating factor which protects the cavity . animals against production of fibroids in the thoracic cavity . aided by grants from the the jane coffin childs memorial fund for medical research . 
harris , coal tar research association , who had purified it by chromatography and checked it by ultra - violet spectroscopy for impurities . these were below the level of detection . fluorewene mea8urement& the fluorescence values for solutions of the various hydrocarbons were determined by constructing curves using a hilger " spekker " photoelectric fluorimeter with cers of capacity approximately 11 ml . , and with filter chance 0 x 7 348 d . 
mouse protein after 6 days ' benzpyrene treatment appear to be of the same order as that recovered by mller ( 1951 ) although it is not possible to assess gravimetricallv the data given by this worker since her figures are relative and apply to the instrument and filters employed . berenblum and schoental ( 1942 ) found that 24 hours after one intraperitoneal injection of 10 mg . 
woodhouse benzpyrene was added to denatured , fat - free , normal skin - protein and refluxed with alkah , toluene and zinc , it could be completely and quantitatively removed from the alkahne solution by benzene extraction . 
the extra - cellular bound ilydrocarbon might consist of metabohtes " excreted " from the cells - or it might be derived from dead or damaged cells . although the fluorescence technique was not sufficiently sensitive for accurate estimations with the small amounts of protein available , the occurence of bound hydrocarbon in cellular constituents has been confirmed by extracting the dna and rna skin proteins from a batch of benzpyrene - treated mice . 
of " fixed " benzpyrene was extracted and a trace was found in the dna protein sample which , however , weighed only 2 mg . a corroborative experiment was also carried out based on the observations of calcutt aind payne ( 1953 ) who showed that nuclei and mitochondria isolated from the livers of mice which had been given a single intraperitoneal injection of benzpyrene in finely dispersed eiuspension , contained fluorescent hydrocarbon 2 hours to 21 weeks after the injection . 
the believed , therefore , that benzpvrene injected in this way is rapidly transported to the mitochondria and nuclei and is held there in an unchanged state for prolonged periods . 
they did not examine the effect of alkahne hydrolysis on alcohol - extracted nuclei . the experiment was repeated in this laboratory but the prelinu ' nary extractions were made as described previously for the skin tissues and continued with alkaline hydrolysis . 
a small amount of fluorescent material was extracted from the hydrolysate but nuclei froni control mice did not yield comparable fluorescent extracts at any stage . this experiment showed that at least a portion of the benzpyrene can be it was repeated , therefore , employing fixed in vivo by nuclear components . perylene , and , after alkaline hvdrolysis an extract with fluorescence characteristic of perylene was obtained from the separated , alcohol - treated nuclei . 
the free hydrocarbon was thoroughly removed and the " bound hydrocarbon " was extracted after hydrolysing the protein with koh and measured fluorimetrically . although these species of animals vary considerably in their response to this hydrocarbon as a carcinogenic agent the amounts of bound hydrocarbon obtained from 25 mg . 
samples of extracted and dried tissue were very similar in all instances . other polycyclic hydrocarbons which are less carcinogenic to mice , e.g. , 2 ' 6 - dimethyl - benzanthracene , and perylene which is non - carcinogenic , have also been tested , and in all instances comparable amounts of bound hydrocarbon could be extracted from the alkahne bydrolysate . using similar extraction techniques a smar amount of fluorescent , bound hydrocarbon could be removed from the nuclei isolated from hvers of mice 24 hours after a single intraperitoneal injection of either 3 4 - benzpyrene or perylene . it is concluded that further evidence is needed to substantiate the view that 352 d . 
whitfield jarcho ( i936 ) also states : has often biased the clinician in favour of a purely haematological diagnosis . despite the frequent difficulties in chnical diagnosis the primary growth and / or its metastases were always readily visible at autopsy . jarcho ( 1936 ) stated that many patients dying from mahgnant thrombocytopenic purpura also showed lymphangitic carcinomatosis of the lungs althou - ah histological examination was often necessary to demonstrate its presence . 
he adopted the term " diffusely infiltrating carcinoma " to cover both mahgnant purpura and lymphangitic carcinomatosis of the lungs , suggesting that they were manifestations of the same disease process . 
on boxing day , 1952 , she noticed profuse painless haematuria which lasted for 19 days . investigation showed no casts in the urine , a normal blood urea and normal intravenous and retrograde pyelograms . 
the urine contained a few red cells , polymorphs and cohform bacilli . hess 's test was negative , the bleeding and clotting times were normal and the prothrombin was 100 per cent . there were 2 , 180 , 000 red cells , 46 per cent haemo lobin , 5 , 000 white cells and a again there was normal differential count . 
no cause for the purpura had been found during life . po - st - mortem finding8 . there were many skin ecchymoses over the legs , thighs and arms , some of which extended deeply into the underlying tissues . there were petechial haemorrhages on the posterior third of the tongue and soft palate . 
the left breast and axilla were normal . the pleural surfaces of the lung8 were normal . their cut surfaces were unusually bloodless , firm in texture and grey in colour . these gross appearances suggested diffuse pulmonary fibrosis . there was no evidence of carcinomatous infiltration . 
the hilar lymph node8 were enlarged and firm and their cut surfaces in view of the previous history showed several tiny grey homogeneous areas . of breast carcinoma a frozen section was prepared from one of these glands . it showed secondary polyhedral - celled carcinoma consistent with a primary origin in the breast . these nodes were not adherent to the hilar blood vessels which were normal in all respects . 
two similar para - tracheal lymph nodes were found i cinferior to the thyroid gland . the upper 12 cof both femoral shafts contained pink marrow which sank in water and which on close inspection was flecked with several pale homogeneous foci measuriaig 1 - 2 min diameter . there was absorption ofthe bony trabeculae . a smear prepared from this marrow showed irregular clumps of malignant cells . the rest of the marrow was fatty . immediately inferior to the left lesser trochanter was a smooth walled cyst 2 x i cdiameter . 
the femoral marrow would have been accepted as showing hyperplasia and a simple cyst resulting from previous haemorrhage if carcinomatous deposits in the pulmonary lymph nodes had not been confirmed histologically earlier in the course of the autopsy . the 8ternum showed hyperplasia of red marrow and absorption of tlle bony trabeculae . the brain showed symmetrical swelling of both frontal lobes and bilateral sectioning revealed extensive tentorial hemiations . iiitracerebral haemorrhages involving the white matter dorsal ancl anterior to the genu of the bilateral 100 w . 
whitfield corpus callosuthe haemorrhages extended backwards on both sides as far as the post - central gyrus , becoming continuous at this level by involvement of the body of the corpus callosuboth lateral ventricles contained recent bloodclot . 
the basal gangha , brain - stem , cerebellum , venous sinuses and pituitary showed no macroseopical changes apart from occasional petechial haemorrhages . the vessels of the circle of wilhs and their major branches showed no evidence of embolism or thrombosis . the thyroid was small , hard , had an irregularly scarred outer surface and showed several brownish areas - each 1 - 2 min diameter - on its cut surface . these areas resembled old areas of haemorrhage . 
the gall bladder was normal . several para - aortic lymph nodes felt firm , their cut surfaces were a uniform pinkish - grey colour and were devoid of definite metastatic deposits . the pericardium , heart , skmmch , intestines , spleen , pancreas , suprarenals , urinary bladder , fallopian tubes and ovaries all appeared normal macroscopically . the uterus contained several small intra - mural fibroids . although there had been histological confirmation of secondary deposits in the pulmonary lymph nodes and bone - marrow during the autopsy , it should be emphasised that there was no other definite evidence of gross metastases , despite extensive examination of all viscera . 
the axillary lymph nodes were extensively infiltrated with similar carcinoma . ( 2 ) tissues taken at autopsy . - the pulmonary and right axillary lymph nodes and femoral marrow contained microscopical metastases which resembled the primary tumour morphologically . the abdominal lymph nodes , thyroid and posterior lobe of pituitary showed scanty microscopical metastases nevertheless still identifiable with the primary growth . 
the above tissues also showed intra - vascular carcinomatous einboli , which had not effected extra - vasc - ular extension . all other post - mortem material examined showed numerous intra - vascular , micro - emboli composed of compact clumps of polyhedral carcinoma cells devoid of stroma . 
the cells were cytologically identical with those which constituted the primary tumour , but because of the minute size of the emboli the general pattem of the primary was lacking . 
the renal tubules contained red blood cells or occasional clumps of tumour cells - " tumour casts . " intracapillary carcinoma plugs were seen in the dermi8and8ub - cutaneousfat in sections prepared from areas of skin ecchymoses . 
the post - operative history of the case may be explained in two ways . either ( 1 ) viable carcinoma cells survived in unextirpated glands or other sites of pre - operative metastasis during the major part of the long period of apparent surgical cure , and only later - perhaps related in time to the onset of purpura 6 months before deathgained access to the general circulation by venous invasion , or ( 2 ) a degree of intra - vascular dissemination was also present since the original operation , but for 71 years had not resu - ited in manifest embolic phenomena . 
the lung was the main exception to this statewidespread intra - vascular deposits , in places apparently resulting in ment . complete occlusion , had not given r ' ise to obvious symptoms or signs during life , even though cardio - respiratory manifestations arising on this basis are well recognised ( jarcho 1936 , storstein 1951 )  . neither did the lung show histological evidence of embolic phenomena . carcinoma cells had undoubtedly passed through the pulmonary vascular bed and into the systemic circulation without interruption or the formation of metastases . thik ; is a rare phenomenon and is discussed in detail by wilhs ( i 952 , page 45 )  . 
the experimental evidence of zeidman and buss ( i952 ) suggests that the transpulmonary passage of tumour emboli may occur more commonly than was previously supposed . the reason for the absence of the usual type of gross metastases in this patient is unknown . 
the fact that all organs examined showed tumour emboli , often in large numbers , does not support the view of coman ( 1953 ) that the distribution of metastases i 's dependant upon the number of embohc cells reaching the various factors other than the frequency - distribution of tumour cells appear to organs . have played a part in our case . 
the ancestral neoplastic cells constituting the primary growth clearly possessed infiltrative propensities . unless the intra - vascular descendants of these cehs had lost these inherent aggressive characteristics - and the presence of mitoses , lack of degenerative changes and the scanty but nevertheless definite microscopical foci of extravascular infiltration that were found does not support this view - one is left to assume that the unusual behaviour of this neoplasm resulted from a lack of the host stromal response which is such an essential factor in successful metastasisation . 
the possibility that " stroma - inducing " properties of the tumour cells were lacking , cannot of course be excluded by histological criteria . it is repolted ( willis , 1952 , page 272 ) that carcinoma of the breast produces intra - thyroid metastases in about 20 per cent of cases . it has also been show - n ( wilhs , 1931b ) that pre - existing abnormahties in the gland were prominent in these findings favour the cases of carcinoma showing intra - thyroid deposits . view that the associated chronic non - specific thyroiditis in our case , preceded the secondary deposits . in a recent paper describing four cases of sub - acute cerebellar degeneration occurring in association with carcinoma elsewhere in the body , brain , daniel and greenfield ( 1951 ) recorded non - specific thyroid changes in one of their patients , similar to those found in our patient . 
the status of the thyroid gland in carcinomatosis seems to merit further investigation . it will depend upon the results of such studies as to whether relationship can be seen between the thyroid changes and the unusual features of our case . the presenting symptoms of haematuria and skin purpura were related to the presence of tumour emboli in the skin and glomerular capillaries . 
haematological investigations during the life failed to elucidate any other aetiological vascular changes in the skin in malignant disease have recently been factors . described ( forman 1952 ) and may have contributed to the skin lesions shown by our p ' atient . the terminal acute massive cerebral haemorrhage may have resulted from the rupture of a small degenerative blood vessel traversing a pre - haemorrhagic focus of softening , a mechanism postulated by globus ( 1937 ) and further substantiated by globus and epstein ( 1953 )  . 
the presence of embohc phenomena elsewhere in the body , the unusual distribution of the effused blood , and the absence of cerebro - vascular disease excluded the possibility that this wa - s a coincident haemorrhage of the classical spontaneous variety . 
madow and alpers ( 1952 ) have described a case of cerebral softening due to multiple cerebral carcinomatous emboli , the primary tumour being a squamous carcinoma of bronchus . as in our case there were no macroseopical cerebral deposits but other visceral metastases were however abundant . 
the above authors also refer to three previously recorded cases of cerebral softening resulting from blood - bome cerebral lesions due to massive neoplastic embohsm carcinomatous emboli . are reviewed by till and fairbum ( 1947 )  . 
the petechial haemorrhages , microscopical softeiiings and focal demylination in the cerebral white matter of our patient resembled experimental lesions produced by the injection of calibrated paraffin emboli ( swank and hain , 1952 ) and sterile cod - liver oil ( lumsden , 1950 ) into the cerebral circulation . cerebellar degeneration ( brain et al . , 1951 ) and peripheral neuritis ( dennybrown , 1948 ) occurring in association with carcinoma elsewhere in the body have been described . 
the changes in the cerebellum and peripheral nerves of our case bear only a superficial resemblance to the lesions described in the above reports as they were focal , associated with carcinomatous emboli and did not include systematised tract degeneration as furthermore , in our case - apart from the terminal far as we could ascertain . apoplexy - the neurological lesions gave rise to no definite symptoms or signs . the platelet thrombosis syndrome ( " thrombotic microangiopathic haemolytic anaemia " ) has recently been reviewed by symmers ( 1952 ) , who states that this condition was first described by moschowitz ( 1924 )  . 
the neurohistological lesions in this syndrome were described by adams , cammermeyer and fitzgerald ( 1948 ) and are easily differentiated from those noted in our case by the presence of platelet thrombi , endothelial and probably adventitial hyperplasia in the walls of the involved vessels and less severe damage to the neural thrombocytopenia is usually present both in previously recorded cases tissue . of malignant purpura and in the platelet thrombosis syndrome . 
at autopsy gross metastases were not present and yet histology revealed intravascular carcinoma in the liver , lungs , one suprarenal , lymph nodes , bone - marrow and dura - mater . 
the degree of intra - vascular spread noted both in oertel 's and our own cases leads us to suggest that a search for carcinoma cells in marrow , skin or muscle biopsies and perhaps in urine , sputum or even blood specimens may be of diagnostic value in suspected cases of intra - vascular carcinomatosis either associated with an occult primary growth or occurring after radical excision such investigations may also help in differentiating the of a known primary . condition from the platelet thrombosis syndrome . 
we feel that even though features such as thrombocytopenia were present , tumour emboli may have been a factor in the mechanism of the purpura in these cases . the evidence that we have presented shows that our patient finauy died from the mechanical effects of tumour emboli that had not resulted in macroscopical metastases , and also that carcinoma cers may have existed " commensary " i the blood stream for many years . 
the patient died from massive intracerebral detailed histological examination estabhshed that the purpura haemorrhage . it is suggested that resulted from the effects of blood - bome carcinoma emboli . carcinoma cells may have existed in the blood stream since the original operation and that similar embohsation may also play some part in the mechanism of malignant purpura . 
the clinical picture of this case is compared with that of the platelet thrombosis syndrome . neurological lesions were present and are discussed in relation to previously recorded cases that show points of similarity . we wish to express our gratitude to professor w . 
gagliano. from the cancer research institute and the department of medicine , college of physicians and surgeons , columbia university , new york . received for publication january 30 , 1950 . tetra - sodium mitchell ( 1948 ) recently reported a clinical study on the use of parenterally administered 2 - methyl - i : 4 - naphthohydroquinone diphosphate ( synkayvite ) as an adjuvant to radiotherapy in the management of inoperable and far advanced malignant disease . in a small series he noted prolongation of the mean survival time of patients with inoperable carcinoma of the bronchus treated by the combination of chemical agent and irradiation when compared with that of patients who received x - ray therapy alone , which he found to be statistically significant . the rationale for mitchell 's ( 1948 ) selection of this drug for clinical trial in combination with radiotherapy depended upon his observation that the chemical compound produced mitotic inhibition of chick fibroblasts in tissue culture . although there have been many laboratory studies on synkayvite as a vitamin k substitute , no investigations have been published on the effect of this compound on experimental neoplasms . 
the growth of the tumours in control and treated animals was followed by serial mensuration of the outside diameters , but the conclusions reached in this study rest on the comparison of the wet weight of the tumours in the control and treated series determined at the 104 a . 
from the information included it is possible to estimate with accuracy the actual protocols for the individual experiments , even though the pertinent results are here summarized as averages ; the standard deviation has been calculated in each instance in order to describe the range and the distribution of the individual tumour weights about the mean tumour weight . 
the standard deviation of the mean which is necessary for the determination of statistical significance between two means is derived by dividing the standard deviation by the square root of the number of observations . in several of the experiments 5 - amino - 7 - hydroxy - 1 - h - v - triazolo [ d ] pyrimidine ( 8 - azaguanine ) was also administered to tumour - bearing animals for purposes of comparison with synkayvite . 
gagliano neoplastic disease and malignant tumours in experimental animals is an unsettled issue , it is important to note that , thus far , all of the chemotherapeutic agents which have a demonstrable palliative effect against neoplasms in man have also been shown to inhibit comparable tumours in laboratory animals . it is also appreciated that the observations which we have recorded do not duplicate in the laboratory the clinical experiments of mitchell ( 1948 ) , inasmuch as he studied the combination of radiotherapy and the drug whereas - we investigated the drug alone . evaluation of combination therapy , however , utilizing radiant energy plus a chemical compound , is not feasible in studies on experimental neoplasms due to the ease with which x rays alone can destroy subcutaneous tumours . therefore , in spite of negative experimental evidence of the type presented in this report , the possibility remains that synkayvitein combination with radiotherapy may produce a greater carcinolytic effect than x - ray therapy alone . 
the presence of the agent in mammary tumour cells after 42 transplantations in c57 black low - cancer - strain mice cbaracterised by low susceptibility to mammary cancer and lack of the agent again raised the question what part is played by the mammary tumour inducing agent im the propagation of the transplanted breast tumour cells , and whether bho cells will continue to multiply in the same way should the agent disappear from the bittner , evans and green ( 1945 ) reported the presence of the agent after cells . 10 serial passages of high - cancer - strain tumour cells in ag - ent - free but susceptible mice , and concluded that the agent is continuously produced in the transplanted tumour cells . in an attempt to ascertain whether the presence of the agent is a permanent feature of the transplanted cells , the c57 x transplantable mammary tumour was again tested for the presence of the agent after 86 serial transplant 's in c57 black low - cancer - strain mice . 
the method of preparation of the tissue for testing and the test itself were the same as those employed for the 42nd transplant of the tumour in whicb the agent was found to be present . 
 ' they succeeded in the recovery of small amounts of papilloma v ' irus from the first and second transplant of carcinoma cells in now - bom rabbits , but failed to recover the virus from tumours of the third and fourth passage . 
the same authors reported positive neutralisation and complement - fixation test - s between papiroma vi ' lrug iand sera of adult rabbits in which a carcinoma originally derived from a v ' irus papilloma was transplanted for 22 the 46th and 50th tumour transsuccessive passages . plants failed to give a positi - ve compl - e ' ment - fixation test . 
they concluded that the " virus was - no longer present in the attempts tumour , although the morphology of - the tumour , remained unaltered . to reinfect the tumours by placing them in a v ' l ' r - us s - aspension or mjecting papiltransplants of the carcinoma loma virus int - ravenously into rabbits be - ar ' m ' yielded dubious results . serological tests m mice similar to those carried out in rabbits by smith , kidd ancl rous ( 1947 ) in order to ascertain the . 
presence of a lat - ent or masked mammary tumour induc ' mg agent in cells of the , 86th transplant could not be of any help in this respect . 
no atte - mpts were made to test the nells of the 86th transplant c57 xmanimar tumou ' r by grafting the tumour tisgue into susceptible mice in view of , the previous observations by various authors . 
thus transplantation of mammary tumour tissue with the agent into susceptible mice has either failed to transfer the agent ( andervont , 1949 ) , or has induced only a low incidence of tumours ( humniel and little , 1949 )  . it has also been found to be a poor source of the a ' gent ( barnum , ball and bittner , 1947 )  . it was therefore considered an unsatisfactory method for testing the transplantreinfection of the tumour able c57 x tumour for the presence of the agent . tissue wit - h the mammary tumour agent was not carried out because of the observations of smith , kidd and rous ( 1947 ) , although it is quite posgible that it would in connection with the negative result obtained have yielded unequivocal results . with the 86th transplant , another test for the presence of the agent in cells of the so far no tumours have been observed 101st tumour transplant was carried out . in an of the test mice , but it is too early to draw a conclusion , as all the mice bittner ( 1952 , personal comare still alive , havi ' n ' g lived now for 11 months . munication ) has found the agent present in the 15th transplant of a c3h mammary tumour in agent - free cm mice , while in t - he cells , of the 7th transplant , of the same tumour it was apparently difficult to detect . it is not known whether a similar situation may arise with the c57 x transplant ' able mammary tumour , although the agent could not be detected in the 86th s ' en ' al passage by the same means which revealed its presence in the 42nd transplant . 
the agent or bittner virus , as it is described by some authors , may have been altered in some unknown way , possibly by mutation . it may have become closely associated with one of the particulate this close integration of the agent with the tumour elements of the tumour cells . cers may have produced a state in which it was unable to survive the death of the tumour cells during extraction procedures preparatory to the test . 
in giemsa preparations feulgen staining was the membrane appeared as a faintly stained pink line . completely ineffective , whilst after iron haematoxylin the membrane lost its stain early in the decolorising process . 
the mercuric bromphenol blue staining technique of mazia , brewer and alfert ( 1953 ) was tried and found to stain the membrane feebly . this method , however , proved useful for determining the relationship of the membrane to the spindle structure . examinat - ion of a large number of preparations has allowed the following description to be pieced together : 1 . 
telopha8e. - as the cytoplasm constricts to form two daughter cells , the membrane also constricts and finally , after fusion at the centre , separates into two complete membranes - - one to each daughter cell . 
7. examination of fresh smears of the tumour by phase contrast microscopy demonstrates the occurrence of this membrane within the living cell and dispels ' any possibility of its origin being that of a fixation artefact . 
the membrane , under these conditions , appears as a thin clear zone form ' mg a sharp line of demarcation between the karyoplasm and the cytoplasm . because of the overlying cytoplasm it is difficult to focus and photograph . in this particular tumour mitosis within a persistent membrane appears to be the standard type of division . 
3. other than that it was taken from a section of a hepatic metastasis of a carcinoma of the tonsil no further comment or information is it appears then that the phenomenon as described is not a completely given . isolated case but does occur occasionally in other material , but not with the regularity shown in this instance . it would not appear to be a fixation ar - tefact since it can be determined by phase contrast microscopy in living cells . further , the fact that such a wide variety of fixatives will preserve it is against such a view . 
on the basis of the evidence available it seems unhkely that such an event has taken place , and if so , takes no cogmsance of the fact that similar appearances do occur irregularly in other tumours . although no evidence can be adduced to show that this membrane arises de novo within each dividing cell it is possible to offer a fairly plausible explanation it has been demonstrated by chambers and of its appearance in this fashion . ludford ( 1932 ) that the cytoplasm of malignant cells has a ph value lying between 6 - 4 and 7 - 0 whilst the intranuclear ph is in excess of 7 - 2 . 
under such conditions a gradient of ph would occur . it is suggested that at some point on this gradi ' ent the isoelectric point of neighbouring cytoplasmic structural protein is reached . this would involve the localised precipitation of protein and the ensuing membrane appearance . if such an event does occur it suggests that intracytoplasmic disturbance during mitosis is minimal , otherwise one would expect distortion and breakage of the membrane . a mechanism of origin as suggested above would account for the isolated examples of similar appearances in other tumours . 
thus , administration of the milk factor from two different highbreast - cancer strains to the same susceptible hybrid mice , kept under controlled conditions , has shown differences in either quantity and / or quality of the factor present in these two high - breast - cancer strains . l , dmocrowski bittner and huseby ( 1946 ) reported similar findings by a different experimental procedure ,  . 
namely , by reciprocal crossings of two high - breast - cancer strains ( c3h and strong a ) , and by recording the tumour incidence and tumour age in virgin hybrids obtained from these strains . 
they described the recorded d ' ifferences in the tumour incidence and in the tumour age in these hybrids as a result of different concentration and / or activity of the milk factor present in these heston and andervont ( 1944 ) in a similarly two high - breast - cancer strains . conducted experiment on sublines of the same strains found no conclusive andervont and eviden ' ce of a difference in the milk factor of these strains . mceleney ( 1941 ) pointed out , however , that his high - breast - cancer strain - ( c3h ) must be regarded as a separate line , and the results obtained with it cannot be controlled by data procured from other lines of this strawamer , reinhard and goltz ( 1945 ) in experiments on virgin hybrids of two other high - breastcancer strains ( m and a ) obtained similar results ' and concluded that the milk factor was more concentrated in one of these stra ' ms than in the other . bittner and huseby ( 1946 ) found , however , the same incidence of breast cancer in breeding hybrids of the two strains tested . 
but they rightly pointed out that the ultimate result of the changes in the structure of breast tissue acted upon by the milk factor of another strain is determined also by the genetic constitution and responsiveness of the breast tissue to the particular milk factor . heston , deringer and andervont ( 1945 ) , b ' y employing susceptible hybrid mice obtained from high - breast - cancer strain mothers and low - breast - cancer strain fathers , and by backcrossing them to highand low - breast - cancer strain males , showed that differences in the milk factor were produced by differences in the genetic constitution of the mice from which the milk factor originated . 
3. as possible into three groups . mice of the first group , when 4r - 5 months old , received one subcutaneous injection of a distilled water suspension of dried riii breast tumour tissue corresponding to 0125 g . 
of fresh breast tumour tissue ; mice of the second group , when of the same age , received one subcutaneous injection ' of a distilled water suspension of dried 63 ca transplantable breast tumour tissue ( from a 428th transplant of the tumour obtained from the imperial cancer research fund ) corresponding to 0125 g . 
no tumours developed in any of the s low - breast - cancer strain females , whether injected with riii or 63 ca dried breast tumour tissue . in the fl generation of s strain females injected with 63 ca tissue and of the control females , no tumours were observed in any of the females , or in any of the males painted with oestrone . in the fl generation of s strain females injected with riii dried tumour tissue , 7 out of 16 females developed breast cancer , and 5 out of 15 males , which were painted , also developed breast tumours . in s x riii hybrid females obtained from daughters of s strain females injected with dried riii breast tumour tissue , 27 out of 32 females showed breast tumours , while s x riii hybrid males of the same origin , painted with oestrone , gave tumours in 10 out of 16 mice . 
observed by the method ' employed in the present experiment . it is impossible to state , however , whether the milk factor was originally absent in the primary tumour or whether it was lost during the course of so many transplantations . .in conclusion , it can be stated that only an analysis of the action and interaction of all know - n factors with allowance for other possible factors can make 102 l . 
murray. from the radiation therapy department , johannesburg general hospital , the non - european hospital , and the south african institute for medical research , johannesburg . received for publication january 10 , 1953 . skin cancer is relatively infrequent in the african . in the three - year period 1949 to 1951 , during which time 590 cases of malignant disease in africans were referred to the radiation therapy department , only 50 cases of skin cancer were encountered . 
the pigmented skin of the african would , of course , be largely protected from this effect , and indeed when skin cancer does make its appearance , it is almost invariably in areas exposed , not to sunlight , but to other types of chronic irritation . we have also observed distinct differences in pathogenesis , and the rate of growth and dissemination of skin tumours in the african , as compared with the m . 
murray classical description of the disease in europeans . these factors have compelled us to adopt a different approach to the management of skin tumours in the two races , and to revise our views on the surgical and radiological curability of skin cancers of various histopathological types . since these racial differences might be of some interest in the study of the demography , endemiology , and management of cancer in africa , it was considered worth while putting our experiences on record at this stage . the population from which this material is drawn consists of approximately 2 , 000 , 000 africans of the bantu race inhabiting the southern half of the transvaal , and originating for the most part from the sotho and zulu tribes . 
about onequarter of this population is urbanised and resident on the witwatersrand , but a much larger fraction of the hospital population gives an urban address . this is due , in part , to many rural patients living with relatives in the urban area prior to entering the hospital and witholding their true addresses , and also to the greater tendency among rural natives to receive treatment from indigenous herbalists in the absence of suitable vital statistics it is impossible rather than in hospitals . to state the age distribution of the bantu population , although it is safe to assume that the average age is somewhat lower than that of the european group . 
the ratio of males to females at the time of the 1946 census was 1 - 33 , the predominance of males presumably reflecting the migratory labouring class . from the genetic point of view it is important to bear in mind that the bantu inhabitants of south africa originate from central and east african tribes , who have , at least within historic memory , had no admixture of european blood . 
by comparison , the negro of the united states originates from west african ancestry and is largely a product of prolonged hybridisation . although environmental conditions in the southern states of the u.s.a. 
are not dissimilar to those encountered by the bantu in the transvaal , the two groups generally show a very different incidence and distribution of malignant disease ( higginson , 1951 ; and robinson , 1951 )  . 
the cells are arranged in the form of distorted , branching tubules or of alveolar masses . in some areas the cells are separated from the surrounding stroma by a well - defined basement membrane while in other areas the cells appear to infiltrate into the stroma . 
as the cells approach the lumen they become larger , the cytoplasm becomes faintly eosinophilio and sometimes vacuolated . the nucleus becomes smaller , more dense , and shows a well - defined nuclear membrane . the nucleus in these cells lies centrally , and their whole appearance is that of lipoid containing cells resembling those of the sebaceous glands . 
he was admitted from an outside hospital with a history of having had a tumour of the right upper arm excised 10 months previously , and that the lesion has subsequently recurred . there was a massive tumour present on the right upper arm and shoulder region , surrounding a vertical scar of the old excision . 
he became very ill , and was transferred home after further palliative x - ray therapy to the lumbar spine . the 2 cases of " sebaceous gland carcinoma " , although showing advanced disease , clearly illustrate the extreme radiosensitivity of these tumours , with conventional large field x - ray therapy . 
one patient is free from disease , while the second has extensive and incurable tumour . albinism and cancer of the skin . it is remarkable that there were 12 albinos among the 50 cases of skin cancer reviewed , although the albino trait is rare in the african . 
the exact incidence of albinism in the bantu is not known , but a cursory review of recent hospital records suggests that it cannot be greater than 1 in 5000 , and is possible less than consequently the 12 patients seen , representing the appearance of 1 in 10 , 000 . 4 new new cases annually ( and probably several more who do not attend hospital ) , implies that a large proportion , if not all of the albinos among the transvaal bantu necessarily develop skin cancer in early adult life . this observation is in agreement with the experimental findings of findlay ( 1928 ) , beard , boggess and von haam , ( 1936 ) and the recent precise quantitative investigations of blum ( 1950 ) , who have demonstrated that albino animals exposed to daily insolation or ultraviolet irradiation for a significant fraction of their life - spans invariably develop malignant skin tumours . all 12 patients showed complete albinism of the whole skin surface including the hair and eyes . 
we did not encounter any cases of partial albinism , such as those described by gelfand ( 1944 ) and shown to have the same propensity for skin cancer as the true albino . however , there was one case of xeroderma pigmentosum ( not included in table i ) occurring in a 4 - year - old bantu child in whom the whole skin surface showed the characteristic regular mottling caused by depigmented patches alternating with areas of increased pigmentation . 
murray pigment - free zones , especially those of the head and neck , possessed the characteristic hypersensitivity to light and were the origin of several histologically proved squamous carcinomas . there were 8 males and 4 females in the series . the age distribution of skin cancer in albinos shows a distinct tendency for the disease to occur in younger age - groups than in normally pigmented persons . there was 1 patient in the second decade , 3 in the third , 5 in the fourth and 3 in the fifth decade . 
we have not had the opportunity of observing the subsequent history of skin areas cleared of all macroscopic keratoses by podophyllin , and , in consequence , are not able to assert that the onset of skin cancer is , in fact , prevented . however , berenblum 's ( 1951 ) observations on the definite anticarcinogenic effect of podophyllin , even in the presence of actively carcinogenic hydrocarbons , suggests that this material may have a true prophylactic action . 
two to 3 weeks before admission black lumps appeared on the skin in the elbow region . on admission the patient had a typical malignant melanoma over the thenar eminence of the right hand . 
the primary growth and axillary glands were given 250 r daily to a total of 5500 r , other areas were treated with 1500 r and 1000 r daily to totals of 4000 r , 4500 r , and 6000 r , and six small areas were treated with three daily doses of 2000 r . all treated areas responded clinically by regression of the tumours and some necrosis of the tumour surface , but histological examination after the radiation showed minimal changes , a few lesions treated with 1000 r daily for 6 days disappeared completely . weeks after admission it was noted that the supraclavicular glands were enlarged and painful , but the intervening skin appeared normal . ten days later small black nodules were visible in this skin area . 
carr it is considered that the same proportion of the reproductive life of a - human female would bring her to 21 years of age , and that of a mouse to 41 moiiths , this emphasizes the very restricted nature of the sample usually studied . most of the data on spontaneous tumours comes from reports from poultry pathology laboratories , and is given as a percentage of all cases of deaths sent in for examination . this causes a serious under - estimate for several reasons for example , many of the deaths are due to s . 
pullorum and coccidial infections which strike the immature chick long before cancer age ; further , pathological investigations are usually only requested when a si - idden rise in mortality suggests an epizootic is beginning , while isolated deaths , which would include those due to cancer , are not always submitted for investigation post mortem . thirdly , there is little agreement among poultry pathologists upon the classification of cancer . 
the data available are thus no guide to " cancer incidence " in fowls , in the sense that this can be defined for mice and man , and are , for the reasons suggested above , usually a gross underestimate . 
although many such figures are available from various parts of the world it will perhaps be sufficient to compare the tumour incidence in the edinburgh flock , where all birds dying are examinedpodmortem , with data given bv goss ( 1940 ) , where a similar analysis has been made with flocks totalling 24 , 900 birds . 
out of 675 female birds dying since 1932 in the edinburgh flock at ages ranging from i to 9 years , only 50 , or 7 - 4per cent of the total adult mortality , gave evidence of neoplastic or allied conditions . 
the edinburgh flock in general is thus a " low cancer " line of considerable ' interest . at present it is sufficient to indicate that complications due to spontaneous cancer can be neglected in considering the resiilts which follow a preliminary 8tudy0f 8u8ceptibility . the first analysis was made upon a series of 429 birds inoculated with cell - free rous no . 
the susceptibility of each bird was graded a ' ceording to the following scheme : negative result ( 0 ) , regression of tumours ( r ) , tumour response to i to 4 , for increasing lasses in all . 
the largest size and malignancy of the induced sarcoma at death or after 28 days was used as an indicator of the susceptibility of the bird . the grading was ' done by inspection . weighing the tumour is unlikely to be more accurate because of the difficulty of clean dissection , and the neglect of such factors as the size and condition of the host , the invasiveness of the tumour , and the presence of large blood - clots and mucoid fluids within its substance . in addition to differences in susceptibility of the host other possible causes of variation were considered , such as sex , season , site of inoculation , number of inoculations , dose of virus , and presence of other factors , such as antibodies injected with the virus . 
no consistent sex difference in susceptibility was noted during the work , and , as was later seen in the analysis , sex does not seem to affect the susceptibility to any detectable extent . 
teratoma testis ( michaelowsky , 1928 ; falin and gromzewa , 1939 ; bagg , 1936 ) ; transplantable sarcoma ( peacock , 1935 ) ; chemical induction ( murphy and sturm , 1941 ) , an analysis of the susceptibility of these young chicks to the rous no . 
i sarcoma showed no seasonal trend ( carr , 1942 )  . that the site of inocul - ation is of little importance in influencing tumour growth was shown by gye and purdy ( 1 931 ) ; it was claimed by them that the resistance depends upon the dose of virus inoculation . no support for this claim could be found by carr ( 1942 , 1943a and b ) , and the results described below indicate that any such variation is inconsiderable . that the other factors mentioned , viz . 
number of inoculations - and presence of other materials , were probably of sl ' l ' ght importance was concluded from several experiments in which only one of these factors varied in an otherwise identically treated g ' roup . 
a large number of the experiinents found in the literature illustrate this point , which is seen to be confirmed in the analyses that follow . the birds comprising the first group of tested animals were from most - of the " all the year round " hatching had been necessary to maintain institute lines . an even supply of experimental chicks , but not many matingswere represented by more than a few tested chicks . in fact , the 429 birds were descended from 33 sires and 147 dams , and many matings had but one tested offspring . 
carr the various institute lines ; these were all selected for physiological characters associated with egg production . dividing each line into two clear classes , negatives and non - negatives , the difference between the breeding line ( with the largest number ) and non - moult line was examined . 
a similar test on the most conspicuous of the other negative tumour groups , intensity , gave a value of chisquare 1 - 23 , which is of no significance , being the variation that would occur in i out of 4 random samples . it was therefore unnecessary to test the rest of the since the regressio ' ns ( r ) are equal to the negatives in the breeding line , groups . but more frequent than the negatives in the non - moult line , the frequency of regressions in the non - moult line is therefore even more significantly disproportionate than the number of negatives , so that this line has an obviously high resistance to the production of fatal cancer by the rous no . 
at about the close of the period when the first analy 'sis was made , the breeding programme at edinburgh resulted in a large proportion of chicks provided for experimental inoculations being derived from a single pen of the breeding line . these chicks were all offspring of a single sire , f651 , and dams fairly closely related to him . .the annual population of the breeding line is larger than that in any of the other groups constituting the flock , and the method of selection used is such that they are also less inbred . in table iv the responses of the groups of progeny from 14 females mated to f651 are illustrated . it is obvious that there is considerably variation among the groups ; in the first three the responses are mainly restricted to the two highest types , and in the fourth the same tendency is only slightly less marked . in the next six progenies ( g196 - g866 ) they are distributed fairly evenly over all the classifications except the completely negative one . 
carr this tendency for the susceptibility of chicks from certain individual matings to fall into a rather narrow range has been encountered in other pens of birds , and knowledge of its existence has been made use of both in providing susceptible chicks for experimentation 4nd avo - iding the progeny of dams whose offspring were known to give a poor response . 
it had been noted that when the non - moult chicks included in the original analysis were grouped according to sires , one of the latter , h573 , stood out as producing particularly resistant progeny ( table v )  . fact , when the data from his chicks were omitted the difference between the responses of chicks from the breeding and non - moult lines were no longer signifiit seemed probable then that the disproportionate number of regressions cant . and negatives produced by h573 was not due to the character non - moult as such , but to some mechanism which can . 
on the other hand , to have carried out an experiment on a large number of birds in the hope that one of the rare resistant class was included would require labour and accommodation far beyond the facilities of any place engaged in fowl tumour work at this time.. the dams of the offspring of h573 analysed in table v were all fairly close relatives of the cock himself , and the way was thus opened for breeding a line of birds which would include a large proportion of resistant animals . it was arranged to carry out a small series of tests on the progeny of h573 , and almost all the birds tested gave either very ismall tumours , or more often , complete regressions . 
the slight shift towards greater resistance in the first 1941 test in both halves ofthe table suggests that some environmental factor leading t ' o a greater inhibitive effect on tumour growth may have been operative then . when the line had been thus established , a number of females , which had been tested and found to produce resistant offspring with a male of their own line , were then outcrossed to a non - moult cock , a descendant of the group from which the non - susceptible ( n - s ) line had been extracted . 
1 sarcoma virus varies from 40 per cent ( fischer , 1927 ) to 4 = 5 per cent ( gye and purdy , these figures may be complicated , however , by different rates of spon1931 )  . taneous sarcoma occurring in the population , either causing " natural " - or this complication is " induced " antibodies to be formed in the tested animal . probably absent at 6 weeks ( the age used here ) as no antibodies are present at this stage ( amies , 1937 ; andrewes , 1939 ; carr , 1943b ) , though they occur in resistance also varies with the age of the bird ( duranyounger and older chicks . reynals , 1940 ; rous and murphy , 1914 ; carr , 1943b ) , so a standard of age for testing is requisite for comparison . in the selection experiment for high and low susceptibility to jungherr sarcoma , carried out by cole ( 1941 ) for example , a resistant line with only 12 und impossible to raise the per cent susceptibility was obtained , but it was high incidence line much above the . 
original 70 per cent ; the cause of this may have been acquired r ' ather than a genetic immunity , since transmission from the immune female to the chick via the egg - yolk ( andrewes , 1939 ; carr , 1944 ) cannot be excluded , for progeny were tested at 7 - 18 days ,  . 
a time when yolk antibody is possibly still present in the chick . acquired immunity was ruled out as a cause of resistance of the n - s line . perhaps the most useful comparison which can be ' made is that between the present analysis of responses of the edinburgh flock to the filtrable tumour rous no . 
1 , and those of birds from the same stock to chemical tumour inducing material as demonstrated in the work of peacock , a report on which has been published ( greenwood and peacock , 1945 )  . 
the data , which cover the behaviour of 4 lines for the 10 - year period from 1935 - 1944 , are interesting both in their points of si ' milarity and dissimilarity to the material already discussed : they were classified only into positive and negative groups , but the non - moult line again showed itself the most resistant to cbemically induced tumours with a susceptibility of about 30 per cent , while breeding and large egg gave a figure close to 40 per cent and intensity had the highest susceptibility with 60 per cent since the early analysis of rous data places breeding ' as highest in posit ' lves . order of susceptibility , this difference in rank order might reflect inconsistency in the relative responses of the two lines to the different types of tumou ' r , but it appears more likely that with continued inbreeding the intensity line has chanced to become more susceptible . 
when most of the inbred lines were tested it was found that one in particular showed an obviously high resistance to the production of fatal cancer by the rous no . 
h there had been equal nijmbers of men and women , and equaltiijmbers in the age - groups , on an average the male mortahty would have amounted to 24 , 855 . 
environment and occupation may , therefore , be expected to exert a greater influence in men . bt say that , ta average age at death from carcinoma of each site . for each organ we can assess not only the fi - equency with which it is attacked , but also the average ao , , e at which this occurs . 
among jewesses we find a low incidence for carcinomas of the stomach , bile pwmges and cerv and a high incidence for carcinomas of the breast , ovary , intestines and kidneys . it would be more conrect to compare the jews , not with the average population , but with that of the big towns , for 80 - 4 per cent and 80 - 7 per cent of jews and jewesses respectively live in the big towns . if we do so , we find an increased incidence for carcinomas of the liver , gall - bladder , intestines , rectum , lung , ovary and breast , and a decrease for the mouth , oesophagus , stomach , uterus and skin . from the age - group distribution of jews and jewesses we should expect 228 and 296 cases of carcinoma respectively . 
versluys carcinoma and trade or profe , 88ion . we now come to the real object of this research - to trace the influence of trade or profession on the origin and the localization of carcinoma . 
our research covers a wider field than that of the so - called occupational cancers , and all that has gone before is necessary to enable us to interpret what follows . before we can proceed , we shall have to examine some problems a little more this will be facihtated by taking a specific occupation as an example , closely . and i have chosen coal - mining . the mortality cards show usa . 
how many people who had formerly been miners died of carcinoma from 1931 to 1935 , their ages , and the sites of their carcinomas . the census of 1930 ( there was no census in 1940 on account of the german occupation , consequently - an interpolation is - impossible for the years between 1930 and 1940 ) shows us ilow many miners there were in the netherlands and their ages , but ' thin ' about ex - miners . now if we take groups a and b together we include all miners who got carcinoma while still working or aftei . 
leaving the trade , with their ages , but t , leir distribution over the various age - classes cannot be compared with the census of 1930 , which gives no information about ex - miners . 
then the average error is when y i - s small this is practically equal to - % / .. this average error has always been taken into account in this research , and a difference is cared real only when the percentages found and those to be expected differ by at least 3 times the average error . fi - equency of carcinoma of the stomach and a low prostate fi - equency . 9 and 50 - 59 we find a total mortality of 50 cases , whereas we should expect 32 , so that the total carcinoma mortality of coal miners is very high . 
the fi - equency of carcinoma of the stomach is greatly increased , for we find 33 cases instead of the 12 we should expect . the number of cases of carcinoma of the prostate in these age - classes is too small to reach a conclusion . the question now arises , is this high stomach fi - equency amongst miners bound up with their trade or with their environment ; is it an " occupational or a " smial cancer " ? ( cramer )  . 
to answer this question we must caiieer study wives of miners " ( from the mortality cards of those married women where the trade of the head of the - fanifly is given as " coal miner . the manded woman practically always lodges with her husband )  . the percentage distribution of carcinoma over the various organs in " wives of miners " compared with the distribution in married women amongst the general population is shown in table xi . taking the " average error " into account , we see from table xi that only 172 j . 
the smaller the unit in our research the more chance we have of getting results of value . if we gather the trades into groups their typical characteristics will become less and less obvious until at last the ratios obtaining among the average population are reached . i have tried taking some trades together , but the typical characteristics of a certain trade always get lost , and the whole group misses what individual component parts show as typical . it is , however , worth while compari ' ng any one trade with another of the same kind , and i have therefore tried to arrange the order of the trad - es - discussed so that trades 6f a kind come near tog ' ether . table xii compares the actual deaths of men of different occupations with the numbers expected if the total carcinoma deaths had in each instance been distributed over the various sites in the same way as amongst all m - ales - that is to say , according to the percentages shown in table 1 . table xllla gives a similar comparison , for wives of men in different occupations , with the deaths expected if the total carcl ' no ' ma deaths had been distributed in the same way as amongst all married women . 
cervix uteri lung prostate lung prostate oil vender8 are only a small group but have an excessi - % , , ely high stomach frequency ' ( 26 carcinomas )  . . 
faxmers. baxmen butch ' ers soldiers - cigax makers soldiers university men tobacconists dock labourers shoemakers office - staff .iall porters officestaffs car men barinen teachers soldiers fisherinen plumbers greengrocers barinen plumbers commer . 
froiii the figures in this paper , but that would take up too much space . besides , in mv opinion conclusions will only be justifiable when we have sufficient international material at our disposal for comparison . this material will have to consist of the results of research on manv , relativelv small , areas during a period shortlv before the second world war , with due observance of the nece , - , sarv precautions . 
with larger areas racial , geographical and economic differences in the same trade plkv a prominent part . , and a trade during and after the war may be quite different in one countrv from another . there was too much changing of occupation . i wish to express mv gratitude to w . 
daab seemed to interfere with vitamin - a storage and hence to cut down food consumption , leading to a deficiency of some other kind , such as that suggested by zucker , berg and zucker ( 1945 ) , which was the immediate cause of the forestomach lesions . the present author ( 1947 ) was , however , unable to elicit such lesions in the stomachs of stock mice given daab orally , even up to 294 days , in either a balanced or one type of unbalanced diet . 
he concluded that otsuka 's diet ( which was grossly deficient in several essentials ) allowed a deficiency not existing in his own diets but necessary for eliciting such forestomach lesions in mice . he also found no tumours in similar mice injected subcutaneously with daab in arachis oil , on three occasions , up to 329 days after the first injection . 
daab thus appears not to be a carcinogen for the subcutaneous tissues of mice . hiowever , in another experiment which apparently broke fresh ground , the present author applied daab in acetone , in increasing strengths , to the nape of the neck of stock mice , and obtained two squamous carcinomas , one with spindlecell metaplasia , in 2 of 8 mice surviving 346 days or more . this seemed to be sufficient grounds for classing daab as a carcinogen for the skin of mice . a further series of experiments has now been carried out to check the previous findings with daab painted in acetone ; at the same time , the effect , if any , of added croton oil was investigated . 
at the commencement , all mice were painted with the solution containing croton oil ; after 15 days , the croton oil was withdrawn . croton oil was re - introduced for one - third of the mice after a further 167 days , and the experiments therefore fall into two groups : ( a ) initial croton oil , and ( b ) initial - plus - subsequent croton oil . applications were made to the nape of the neck , after clipping away hair , thrice weekly for a total period of 545 days . the basal diet throughout was rat - cake ( thomson , 1936 )  . ( a ) initial croton oil series . - the survival rates of mice surviving more than 200 days are shown in fig . 
kirby the commencement of painting ; of these , two showed acute yellow atrophy of hyperthe liver and one showed slight hyperkeratosis at the site of painting . keratosis , with or without necrosis or ulceration , was common after 200 days , but the first papilloma was found in a mouse killed after 320 days ' painting ; this papilloma grew as a coxcomb - shaped wart . 
the other mouse had a similar , though smaller , lesion which also proved to be a squamous carcinomna of low - grade malignancy . although a mouse dying after 435 days showed no gross abnormality at the site of painting , another dying after 449 days had two tumours in the painted area ; one of these was found to be a squamous carcinoma with invasion down to the depth of the muscle . 
a necrotic , warty outgrowth , showing parakeratosis beneath , and a multiple papillary growth , were found at the site of painting in mice painted for 495 and 545 days respectively , the latter dying at mice dying at 524 , 528 and 588 days , i.e. 
33 days after painting was stopped . 549 days respectively showed nothing more than a little hyperkeratosis at the the last survivor and one of the mice from group ( b ) unfortusites of painting . nately died at the same time ( 611 days ) and were examined post - mortem in the absence of the author . 
one of these mice showed early squamous carcinoma , and the other simple infected ulceration ; as there is no way of deciding which histological report belongs to which mouse , they will be excluded from consideration in comparing the results in the two groups . 
a mouse dying at 623 days showed no lesion at the site of injection . thus in 16 mice surviving more than 300 days ' painting with 5 per cent daab in acetone , 3 developed simple papilloma , and 3 others developed squamous hyperkeratosis and / or ulceration was found carcinoma , at the site of painting . in nearly all the rest . abdominal organs were only examined histologically when gross damage was visible , but the male dying at 449 days and one of the mice dying at 611 days showed amyloid changes in spleen , liver and kidney similar to those referred to in the previous paper ( kirby , 1947 )  . 
the mouse dying at 623 days showed pulmonary adenoma . ( b ) initial - plus - subsequent croton oil series . - survival rates , beyond 200 days , only slight are shown in fig . 
1. hyperkeratosis was found at the site of painting in 4 mice dying at 215 days and in 1 dying at 305 days . of 3 mice dying at 435 , 442 and 443 days respectively , all had papillomas , keratinized and partly necrotic , at the site of painting . 
of 17 mice painted for more than 400 days , 5 developed squamous carcinoma , and 5 others squamous papiiloma , at the site of painting , a tumour incidence of 60 per cent . 
the malignancy appears to have been low - grade in all cases , and metastases were never found . no evidence of spindle - cell metaplasia was seen in this series . 
as this substance is an intermediate in the manufacture of p - aminoazobenzene , the possible danger to workers handling it , especially in organic solvents , must not be overlooked . induction by daab of tumours at sites remote from that of application seems to be confined to the lung . 
no tumours were found in the liver of any mouse . co - carcinogen tests : neither the incidence of skin carcinomas - 3 / 11 ( surviving 400 days ) in group ( a ) and 1 / 5 in group ( b ) - nor the time of appearanceabout 400 days in either group - lends any weight to the view that croton oil acted as a co - carcinogen when applied concurrently with daab in group ( b )  . on the contrary , it would seem that croton oil had no effect either way on the induction of skin tumours . this suggests that the mechanism involved in carcinogenesis by daab differs from that of the polycyclic hydrocarbons . 
croton oil painted concurrently caused no increase in the incidence of the significance of the skin tumours , nor did it decrease the latent period ; absence of co - carcinogenic action of croton oil is discussed . the author is greatly indebted to p . 
kirby. from the research department , glasgow royal cancer hospital . received for publication , june 28 , 1984 . activity the carcinogenic of orally - administered 2 - acetylaminofluorene , aaf , for a number of tissues , mainly epithelial , was first demonstrated by wilson , deeds and cox ( 1941 ) for the rat , and subsequently confirmed for this species by bielschowsky ( 1944 , 1946 ) and others , for mice of various strains ( armstrong and bonser , 1944 , 1947 ; foulds , 1947 ) , for the fowl ( bielschowsky and green , 1945 ) and for the cat ( harding , quoted by bielschowsky , 1947 )  . recently wilson , deeds and cox ( 1947a ) have reported their own findings in mice of three pure strains . 
aaf has now been shown to be a carcinogen ( when given per os ) in eight strains of mice : cba , if , riii , white label , strong a , c57 black , c3h , and bagg albino . 
of the first five strains mentioned , cba were most prone to both liver and bladder tumours ; strong a were the least susceptible ( armstrong and bonser , 1947 )  . 
they received a diet consisting of whole meal flour ( 7 parts ) and skimmed milk powder ( 3 parts ) , supplemented by cod - liver oil and cabbage once weekly . for each animal 12 g . of food were provided daily . 
the dose of methylthiouracil given to the males was doubled during the last 10 days before they were sacrificed . four males and two females received methylthiouracil only ; two males and two females methylthiouracil plus a.a.f. , and four females a.a.f. 
only. thyroxine injections were started when the animals had stopped growing . this occurred in the females after three weeks of methylthiouracil treatment , in the case of the males , after four weeks . dl - thyroxine was dissolved in saline with na2co3 to ' give a solution containing 10 , ug . 
griesbach during the period of thyroxine treatment the rats were weighed were killed . twice weekly and the injected amounts adjusted to the weights . five female rats , 7 to 8 weeks old , were thyroidectomized . three weeks after operation , when the constant weight of the animals suggested completeness of thyroid ablation , these rats were started on the milk - flour diet containing 0 02 per this concentration of a.a.f. 
are badly tolerated the carcinogen to each rat per day . after eight weeks of this regime , thyroxine treatment by thyroidectomized rats . was started , each animal receiving one daily injection of 2 - 5 , ug . 
body weight completely compensated for the thyroxine deficiency produced by methylthiouracil , independent of the presence of a.a.f. twenty - two rats ( 6 males and 16 females ) were treated with 2 - 5 , ug . 
nor the controls reacted as uniformly as the animals injected with the higher dose of thyroxine ( table iii )  . in the males , the thyroid weights did not show any significant differences , the values being slightly above normal in both groups . 
with the exception of two animals , all showed an increase in cell all pituitaries were normal , showing heights far above the normal values . basophil counts of the same order as the controls . comparison of the values obtained in the animals with and without a.a.f. statistical shows that the greatest differences were found for the cell heights . analysis of these values proved that the difference between the means was highly the difference found for the means of the thyroid weights significant ( p < 0 0 1 )  . of the two groups , however , was of doubtful significance ( p > 0 05 )  . the pituitaries of the five thyroidectomized females treated with a.a.f. 
the weight increase of the thyroid induced by goitrogens is a complex phenomenon . it is due to increased numbers and size of the epithelial cells , and may be partially offset by the loss of colloid . 
when the supply of thyrotropin is reduced either by withdrawal of the goitrogen , or by administration of thyroxine , cell size decreases within a short time , whereas , the number of cells remains high over prolonged periods and colloid reappears . 
the latter factor must introduce uncontrollable errors when the thyroid weight is used as a criterion of thyrotropic action . in our experience , as in that of rawson and starr ( 1938 ) , the estimation of the mean acinar cell height has been found to give more reproducible results in assaying the degree of thyroid stimulation than the weight method . since a close correlation exists between the size of thyroid cells and the number of pituitary basophil cells , the percentage count of basophils has been used for assaying states of thyroxine deficiency in combination with the measurement of epithelial cell heights ( griesbach and purves , 1945 )  . in the state of fully developed thyroxine deficiency the percentage of basophils reaches values of 30 to 45 per cent . 
by excluding from the count masses of basophilic colloid free from cellular structures , we believe to have eliminated this possible source of error . all rats injected with 5 - 0 yig . 
griesbach the goitrogen - treated rat enlargement of thyroid cells is linked with an increase in numbers of pituitary basophil cells , indicating elevated thyrotropic activity . this correlation breaks down under the influence of a.a.f. 
one half of the animals still had large goitres and , histologically , in all but one the epithelium was almost as high as in the rats which did not receive thyroxine . no evidence for an increased demand for thyroxine or for higher sensitivity to thyrotropin was found in the animals treated with a.a.f. 
when a thyroxine deficiency was induced in a.a.f. treated animals by surgical or chemical means , their pituitaries responded in the similarly , the growth same way to thyroxine as did the animals without a.a.f. response of the female rats to thyroxine was independent of the presence of a.a.f. in the males , the pronounced liver damage prevented normal growth . these facts show that the action of thyroxine on the pituitary was not inhibited by a.a.f. 
the problem can therefore be reduced to that of an anomalous behaviour of the thyroid epithelium . in the hands of paschkis , cantarow and stasney ( 1948 ) l - thyroxine did not prevent the change from normal to high epithelium . in our experiments , physiological amounts of dlthyroxine did not reduce a high cylindrical epithelium to normal . it must , however , be remembered that the hyperplastic thyroid epithelium of the a.a.f. treated animals was not completely refractory to thyroxine . 
 it has recently been demonstrated that the most critical factor determining the epidermal mitosis rate in the adult mouse is the carbohydrate supply to the cells , and the evidence at present available all points to the conclusion that the function of this carbohydrate is to supply the energy needed during cell division in extending this work the discovery has been ( bullough , 1949a , 1950a , b )  . 
 made that a restricted diet has a powerful effect in depressing mitotic actirity ( bullough , 1949b ) , and it has been suggested that this obserration may help towards an explanation of tannenbaum 's remarkable results on the effects of restricted diets on tumour genesis ( tannenbaum , 1940a , 1940b , 1942 , 1944 , 1945 , 1947 )  . 
this involved a study of the results obtained with a graded series of diets as regards body weight , blood sugar concentration , liver glycogen content , and epidermal mitotic activity . 
 the two experiments performed involved a total of 55 mice , which were all in the first experiment the 25 adult males of between 3 and 5 months of age . 
 these mice had been reared since weaning on a mixed diet of commercial rat cake with cod - liver oil , flaked maize , and dog biscuit , but during the experiments they were fed on commercial rat cake alone . 
this deter mined the form of their diurnal cycle of mitotic activity , and ensured that a high mitosis rate developed shortly after midday ( bullough , 1948 )  . 
it was unfortunate that the animals in the first experiment were considerably lighter than those in the second experiment , but it was remarkable how little this fact affected the results obtained . 
 on the day when each experiment began a piece of ear was removed from each animal at 14.00 hours , the normal time of maximum epidermal mitotic activity associated with the afternoon sleep period . 
the animals were killed at 1500 hours , it having been previously determined that for a period of about 5 hours after the injection of this weight of colchicine there is no great disturbance of the mitosis rate or of the blood - sugar level ( bullough , 1949c )  . 
the liver was removed , cut into two roughly equal parts , and the glycogen content was converted into glucose by the method of good , kramer and somogyi ( 1933 )  . 
 in estimating the epidermal mitotic activity the pieoos of ear , fixed in bouin 's alcoholic fluid and embedded in ester wax ( steedman , 194 7 ) , were cut into sections 7 thick . 
 255097 217096 200076 199 : : : ! : : 075 197078 100 , , - : 250097 261085 267075 25 - 1 + 092 279 ; 0 - 97 in the second experiment only strong 's cba males were used , and these , being 4 months old when the food restrictions began , were heavier than the mice of the first experiment . 
 252068 244 : : : ! : : 052 218043 21 4052 21206 . ' ; 90 0 ' .o 247150 257118 252130 253075 246070 table ii . - the average body weight ( in grammes ) in group& each of 5 adult male mice fed on restmtetl diets of rat cake . 
 306116 288 : : : : : 115 279075 283070 299094 the results obtained were much as could have been expected , the figures for the groups on the lower diets falling steadily as the weeks passed . 
by the end of the fourth week it is clear that the relationship between body weight and diet is a direct one which can be expressed by a straight line graph . 
 since it is evident that mitotic activity is closely associated with carbohydrate concentration , the carbohydrate reserves were estimated at the close of the experiments in terms of the liver glycogen content and the blood sugar concen tration . 
 233 12 l 214448 200045 183459 172323 240619 218 133 199422 187413 18001 - .~ 175108 superficial examination of table iv might suggest that the poorer the diet the higher the blood - sugar level . 
at that time the blood - sugar level must have been low ( bullough , 1949b ) , and with a coincidentally low secretion rate of insulin the effect of the meal must have been to raise the blood - sugar level to abnormal heights . 
it is thus logical that the poorer the diet the higher the blood - sugar level should be , and once again the relation is a direct one expressible as a straight line graph . 
 75029 36042 25017 23018 22024 one surprising conclusion that can be drawn from these figures is that the imposition of a restricted diet results in an extremely rapid fall in the mitosis in most groups there was a steady reduction throughout the whole experi rate . 
i these results are expressed graphically , the figures chosen for com parison being ~ose of the numbers of mitoses observed after 3 weeks ( without colchicine ) and after 4 weeks ( with colchicine )  . 
it appears that in general the mitosis rates of the lighter mice of the first experiment were lower than those of the second experiment , but the differences are remarkably slight and are of doumful significance . 
 the most significant point emerging from these graphs is the indication given both with and without colchicine that the relationship between resmcted diet and mitotic activity is not a direct one . 
 the present results confirm earlier observations that restricted diets cause a pronounced fall in mitotic activity , and indicate that when less than 70 per cent of the normal food intake is given the epidermal mitosis rate is reduced to about 35 per cent of the normal . 
on the basis of earlier work it can be safely suggested that this reduction is caused by a shortage of carbohydrate , in the form of glycogen or glucose , in the epidermal cells themselves ( bullough , 1949a , 1949b , 1949c , 1950a , 1950b ; bullough and eisa , 1950 )  . 
at this time the fall in the body weight is only just beginning , and it is therefore evident that a mouse is unable to maintain a high rat , e of cell division by means of the utilization of fat deposits . 
 in a fully - fed four - month - old mouse of about 35 g . , as in a fully - fed three - month - old mouse of about 25 g . , the epidermal mitosis rate reaches a sleep maximum of about 75 mitoses per c similarly in either case the food intake remains steady at about 36 g . 
 ' when fed on restricted diets the mice lost weight in a regular manner week by week , the speed of loss being in direct proportion to the degree of underfeeding . 
 in a similar manner the carbohydrate reserves , as indicated by the liver glycogen content , fell after 4 weeks to a level which was directly proportional to the amount of food given . 
2 , in which for comparison a graph of the present results is in both cases the form of the graph is sigmoid , and in both cases the included . 
this adds further support to the thesis put forward by bullough ( 1949b ) , and developed in the following review ( bullough , 1950c ) , that the rate of tumour genesis in a particular tissue or in the body as a whole may be directly related to the mitosis rate . 
the mitosis rate in turn is related to a tariety of factors , of which one of the most important is the carbohydrate , or calorie , content of the food . 
 groups of adult male mice were maintained for 4 weeks on diets varying from 55 per cent to 105 per cent of what they would eat if fed ad libitu weekly records were made of the body weights and epidermal mitosis rates , and at the end of the experiments the carbohydrate reserves were estimated in terms of liver glycogen content and blood - sugar concentration . 
die greatest mitosis depression accompanied a reduction from 80 per cent to 70 per cent of the normal diet , a result which compares closely with that of tannenbaum ( 194 7 ) regarding the effects of restricted diets on carcinogenesis . 
kreyberg. from the institutt for generell og ek8perimentell patologi , universitetet i oslo . received for publication march 9 , 1953 . the clinical problems of breast carcinoma have been under intensive discussion for decades . 
a great deal of detailed information has been accumulated , but still widely different views are entertained as to diagnosis , treatment and theraour present knowledge was amply illustrated during the panel peutic results . discussion on treatment and results in cancer of the breast at the thirtieth annual meeting of the american radium society , chicago ( 1948 ) , and at the twenty - fifth meeting of the northern surgical association , copenhagen ( 1951 ) , to which readers may be referred . a few points seem to be universally acknowledged , as regards treatment . firstly , a careful and extensive surgical intervention will , in a number of cases , be life saving , or life prolonging . 
on the other hand , if the surgeon operates in a field where cancerous cells are present and the operation is incomplete , the surgeon may by his intervention precipitate an outburst of metastases and shorten the it may further be agreed that the development of the special life of the patient . surgical technique since the days of halstead and meyer is comparatively small , and that the advances , as manifested by a higher cure rate , are modest and mostly caused by improvements of surgery in general , for instance the introduction of penicillin and a better postoperative regime , thereby reducing the immediate it was this state of affairs that caused mcwhirter ( 1949 ) operative dangers . to state : " when radical mastectomy is the only method of treatment available and when all cases coming to a large general hospital are taken into account , the five - year survival rate is unlikely to exceed 25 per cent "  . secondly , it seems generally accepted that radiological treatment in a certain , restricted number of cases and under certain conditions , is able to effect a complete destruction of the tumour cells and thereby be life - saving . it further seems generally accepted that radiological treatment in other cases may , for a shorter or longer period , retard the development and the spread of the tumour cells , and that radiological treatment thereby may be life prolonging . kreyberg ( 1938 ) microscopically examined breast carcinomas and axillary metastases which had been submitted to pre - operative irradiation . 
the material came from three different hospitals , using different radiological techniques and different doses . the conclusions were that - : a pre - operative treatment , with the doses used , may 158 l . 
kreyberg in a series of cases damage the tumour cells to a degree , histologically visible , and in rare cases lead to complete disappearance of the tumour cells . this effect is dependent upon the dose , the effect being more pronounced after stronger doses . also the axillary metastases are damaged , but in a considerably lower degree than the primary tumour . but , even after the strongest doses ( with tele - radium ) , a great number of tumours show such small changes that we must conclude that the cells are growing during and in spite of the treatment . these observations have been generally confirmed . 
kaae ( 1952 ) stated that only 15 per cent of all breast carcinomas are really radiosensitive . baclesse ( 1949 ) had greater effects , but he used very heavy doses . 
an observation period of 5 years only is , however , too brief , since one of the radiological effects is fibrosis and scarring , after which cancer cells , retarded in growth , may regain their vitality and proliferative possibilities at a later date . haagensen ( 1949 ) stated " our reliance upon irradiation for the cure of the disease ( breast carcinoma ) has faltered until we have come to the point where we reserve irradiation for cases in which palliation is all that can be hoped for . " an attempt has been made to combine surgical and radiological treatment , in the hope of obtaining better results . 
the absolute or over - all cure rate is the number of patients alive and symptom - free out of the total number of patients received in the hospital and suffering from the disease under investigation . all dead are counted as victims of said disease , regardless of the real cause of death . 
even if this strict attitude is adopted , we meet , however , with factors which influence the statistics . firstly , the general cancer consciousness of the society , which to a certain degree will influence the relative number of early and late cases . secondly , social and racial factors , which may influence the soil of the cancer cells , and thirdly , the relative number of cases with moderate and with high malignancy , as shown by bloom ( 1950 )  . kreyberg ( 1952 ) in a survey of lung cancer in norway attempted to show that the difference in the sex distribution of this form of cancer in norway , as compared to england and wales , may be explained by the difference in the relative number of the different histological types . 
a similar situation may hold it is probable , therefore , that besides a plea good for breast carcinoma as well . for the use of absolute cure rates and tumour stages already in use , also a correction for tumour types and grades should be attempted . nielsen ( 1951 ) gave as reasonable the following figures as the average chances for a breast carcinoma patient in scandinavia : 5 - year survival rate , symptom free ( operable cases ) , 35 - 50 per cent . 5 - year survival rate , symptom free ( absolute ) , 20 - 30 per cent . these figures are valid for a population with a generally fair cancer consciousness and with a rather high standard of the doctors and the hospitals . individual surgeons and special clinics may present better results , but such material is more or less selected . the cause of the depressingly low cure rate in breast carcinoma is two - fold : ( 1 ) the tumour cells have spread beyond surgical control before the operation is performed and even before the diagnosis is made , and / or ( 2 ) the tumour cells are not sufficiently radiosensitive . this state of affairs has naturally led to a quest for other means of improving the therapeutic results , and as one of those , the importance of " early diagnosis " has been universally stressed and accepted . it may be useful therefore to examine the meaning of the designation " early diagnosis "  . 
in the present paper it is intended to examine different definitions of the term " early diagnosis " commonly used in order to ascertain our position today as regards the criteria for the immediate recognition of a breast carcinoma in a stage where a complete cure is possible , if a proper treatment actually at our disposal is used . especially will be examined the foundation of a propoganda promising great hopes of cure if the patient arrives for treatment on the basis of an " early diagnosis "  . when a breast carcinoma develops morbid changes have usually been present in the tissue for a shorter or a longer period . 
even if the essential change is considered to be a somatic mutation in a single cell , this mutation has its local further , the tissue is not clinically cancerous before the proliferation cause . has reached a certain extent . 
from a therapeutic standpoint to - day the breast carcinoma is in its early phase as long as the tumour is localized to a degree that makes a complete cure possible by such a relatively simple local operation as this is the background for the designation " stage i " , and in the mastectomy . absolute sense mastectomy should by definition in such cases result in 100 per 160 l . 
the discrepancy between the number of tumours histologically classified as stage i and the number of cures effected in this group , after a proper local operation , shows the degree of failure in the process of staging . this failure is approximately 20 - 30 per cent . in spite of this serious inaccuracy the histological staging is the method used for grouping of tumours for comparing different therapeutic interventions . the most accurate staging is the retrospective clinical , taking into consideration the final development of the cancerous process in each individual . even this staging is not perfect . it has been mentioned that patients supposed to be stage i eventually are shown to be in stageii or further . 
on the other hand , patients believed to be stagei , though actually in stage ii , may never be recognized as such if radiological treatment cures the patient . this brief survey shows that the decisive determination of the stage of a certain tumour is neither a direct nor an immediate process . 
kreyberg this table shows that even in these groups , where the time lag between diagnosis and commencement of treatment is reduced to the practical minimum , only half of the average patients can count upon a 5 years ' survival symptom free . it seems , therefore , that the definition p2 is of rather limited usefulness because a great number of the tumours are not any longer curable and the definition has not contained the criteria necessary to obtain this goal . 
a limited usefulness of p2 is demonstrated by the fact that some patients , an unknown number , have benefited from the shorter time lag - a question which will be discussed later . if we analyse the usual symptoms , some of them ( a feeling of heaviness , pains , stinging sensation and similar ) are rather vague and strictly subjective and little fitted for attempts at earlier detection . 
but among the symptoms analysed , a lump in the breast is the first in at least three - fourths of all cases , and this symptom is more material , more objective and more fitted for diagnostic research . 
here a regular and systematic palpation may facilitate the discovery of comparatively small nodules , but there evidently is a certain lower size limit . kaae ( 1948 ) voiced the traditional opinion when he stated that : another attempt at a useful definition may accordingly be : a diagnosis of a very small tumour ( p3 ) , in the hope that all such tumours actually are curable . " it is generally recognized that the size of the tumour is a very important factor in the prognosis , and that the latter is much more favourable for the small tumours than for the large . " if we , however , examine the pertinent literature , we shall discover that the students of this problem are not at all unanimous . 
the fate of the patients was followed and the long range survey , ten to twenty year 's observation , showed that nearly two - thirds of the patients had metastases when arriving for treatment . 
even the very smallest carcinomas , those the size of a pea or a bean , had a fatal outcome in four out of ten cases . these facts show that our attempt to define " early diagnosis " as a diagnosis of a very small tumour ( p3 ) , actually a tumour as small as practically diagnosable , also fails miserably . taking into consideration the three commonly used or accepted definitions of " early diagnosis " , pi fails , because the criteria are not immediately available , and p2 and p3 fail , because the criteria do not enable a patient to recognize a tumour still in a curable stage . 
nor is any other definition known , which makes such a recognition possible to - day . what p2 and p3 give are indications for obtaining the earliest possible diagnosis with our present diagnostic means . 
an examination of the actual degree of earliness shows that only half of the tumours occurring in a population can , under the best possible conditions , be diagnosed early enough to be cured with our present therapeutic means . 
the high percentage of cures is the result of a very strict selection of cases . first all cases proved to belong to stage ii , or further , are excluded . this would leave a curability of approximately 75 per cent for the remaining . next , larger tumours , and finally especially malignant - looking tumours , are excluded . 
a further improvement in the statistics may be obtained by a still greater efficiency in the process of selection , not necessarily being an expression of increased chances for the patient at large to survive . it is not correct and not fair to publish such statistics in a manner that makes 164 l . 
kreyberg the public believe that if the women examine themselves regularly and carefully , and pay a visit to a competent doctor at the first suspicion of a symptom from the breast , their chances of a complete cure is in the vicinity of 90 per cent . 
kaae ( 1948 ) arrived at the same standpoint , and wrote : " it is indicated by these investigations that a 5 - year symptom - free survival rate of about 40 per cent is obtainable in denmark to - day , and that these results may be improved from 40 to 50 per cent , if all patients see a doctor as soon as they notice the first symptom , and if the doctor makes the correct diagnosis and institutes adequate treatment without delay "  . the present study seems to show that a diagnosis as early as possible is of great importance to a certain , yet unknown , number of individual breast carcinoma patients . 
as we do not beforehand know who will benefit and who will not , a general plea for " early diagnosis " is fully substantiated , as every individual salvaged is important . 
we must , however , always have in mind that the benefit is statistically moderate , and we ought to be modest in our claims as to the possibility of materially altering the situation for the breast carcinoma patients by this approach to the problem . it has finally to be added that the present conclusion is based upon the use of the word " early " as an expression of curability seen on the background of our present diagnostic and therapeutic means . 
the earliness is , term is not used in the sense of a more or less absolute time unit . actually , here synonymous with curability to - day , and this again , in most cases , means a carcinoma in stage i . if we examine the means to diagnose a carcinoma still in stage i , we will discover that such a diagnosis can with a reasonable certainty be made only after a retrospective clinical survey , lasting some 15 to 20 years . the analysis further shows that we have no criteria enabling an immediate " eary diagnosis " , in the sense required . 
the mixtures which had a ph of about 2 - 5 were polarographed as f - 611ows , : the test solution , into which dipped a dropping mercury cathode , was connected to a saturated calomel reference anode ( s.c.e. ) by salt - agar bridges ( saturated kci in 3 per cent agar - agar ) and the solution deoxygena - ted by passing a stream of water - washed oxygen - free nitrogen through it for 10 minutes . 
the percentage ttc reduced by serum was estimated with reference to the wave height of ttc in the blank reaction mixture . triphenylformazan 1 - 2 volts under the present conditions . gave no reduction wave in the range 0 to 200 to fig . 
2. - scatter diagram showing negative linear correlation between percentage ttc reduction regression lines have been constructed from by serum and percentage tumour weight . the regression equations : in the scatter diagram , fig . 
2 , from which it will be seen that there is a negative linear correlation between percentage ttc reduced and percentage tumour weight . the correlation coefficient ( r ) was calculated in the usual way ( moroney 1953 ) and found to be - 0 - 75 , t test = 5 - 63 ( n = number of animals ) highly significant at the 01 per cent level . reducing capacities of tumour and normal rat sera are show - n in fig . 
serum hastened colour development in iruminated mixtures , the colours being marked after i day . control solutions containing no serurn required 2 to 3 days to develop a pink colour whfle serum mixtures and controls kept in the dark exhibited no colour even after i week . after exposure to dayeght for 2 days tumour serum mixtures were coloured pink whereas normal serum mixtures , orange - pink after i day , were orange366 w . 
neish of 10 tumour sera , 9 ( tumour range , 3 - 4 to 22 - 8 per cent ) yellow in colour . showed pink colours and 1 serum ( 8.1 per cent ) gave the orange - yellow reaction . six normal sera each gave orange - yellow colours . in time , all colours faded to pale yellow and the solutions fluoresced blue in ultra - violet light . 
no difference was found with respect to ttc wave height between tumour and normal serum - ttc mixtures and the illuminated control but a new wave was observed in each solution which had been exposed to light . this wave was never observed in the ttc - alkali reaction mixtures described in sections ( 1 ) and ( 2 ) , or in control mixtures kept in the dark . 
and it was found to be due to another photochemical reaction product of ttc , namely the 2 , 3 , diphenylene - 5 - phenyl tetrazolium compound ( blue fluorescent in ultra - violet light ) described by hausser , jerchel and kuhn ( 1949a ) and by kuhn and jerchel ( 1952 )  . 
they take advantage of the spontaneous adhesion on plastic membranes of the free cells present in physiological fluids such as blood , cerebro - spinal fluid , peritoneal and pleural effusions . in brief : glass slides coated with formvar are placed in contact with the fluid ( with addition of heparin if necessary ) in an incubator at 37 ' c . 
for 15 to 60 minutes . the slides are then rinsed with tyrode solution at 37 ' , fixed 10 minutes to several hours in acid osmic vapour and washed thoroughly in distined water . the formvar is stripped from the slide , mounted on grids and anowed to dry at room temperature for at least 24 hours . photographs were taken with a triib - taiiber electron microscope at 60 kv . we adopted stringent criteria for purposes of comparison and considered only undamaged cells , that is to say cers having clear and well defined chondriosomes and with a satisfactory cytoplasmic transparency . 
as the appearance of a cell may vary from one area to another ( because of differences in spreading , lipid constituents , etc . ) we only considered the best portions . counts were made in each cell of osmiophihe bodies less than 150 mp . 
at least a few such bodies are to be found in every cell , but to consider them as true ultrachondrioma we adopted as a minimum 10 such particles 354 j . 
oberling showing a certain degree of polymorphism ( to distinguish them from merely lipidic granules ) per i oo #2  . for each preparation at least 1000 cells were counted . 
the same submicroscopic corpuscles as in leukocytes are encountered , but frequently they appear as bent or twisted filaments which may have swelhngs or a beaded structure . sometimes these filaments are extremely long . their thickness may vary from 30 mlt . 
they may aggregate in groups of two , three or more , or may appear to arise from an ordinary chondriocont . the statistical analysis shows that those submicroscopic bodies are not a ebaracteristic only of nialignant effusions but are seen in various inflammatory or circulatory disturbances . 
but when those figures are an almost constant finding , when it becomes impossible to estabhsh a distinction between chondrioconts and those structures on any other basis than the rather arbitrary one of size and thickness , then it seems difficult to deny the mitochondrial nature of the described structure . they have the same characteristics and the same polymorphism as certain types of mitochondria ( especially in plant cells )  . 
even the specifity of janus green stain depends on certain criteria ( showacre , 1953 )  . furthermore there is a question of principle involved in the discussion about the significance of these structures . if we require for the identification of ultramicroscopic structures the same characteristics as those used in classical cytology , ff certain mitochondria can no ion er be considered as such because their size prevents the use of janus green and the apphcation of more or less debatable functional tests , then we shau have two cytologies : orie optical and one electronit is highly improbable that cytoplasmic conical , which is not satisfactory . stituents are of a different nature simply because they are too smar to be seen with the ordinary microscope . on the contrary it should be our aim to show the links between microscopic and - ultramicroscopic structures . this has been done with great benefit with basophihc structures , ergastoplasm and intracytoplasmic network . it would be curious if the same did not hold true for the mitochondrial structures . furthermore the concept of mitochondria which are invisible witli the optical microscope is not new and it can account for certain results . many cytologists consider the chondrioma as fixed both in amount and morphology . 
but this stabihty is dependant upon the material studied , and it is in fact true of speciahzed tissues observed under standard conditions . botanists have long noted the morphological variations of the chondrioma in active cens ( guilhermond , 1934 ; gautheret , 1950 )  . no6l ( 1924 ) and many others bave made similar observations with animal cells . the researches of levi ( 1934 ) , lewis and 1 - jewis ( 1915 ) , etc . , seemed in favour of the de novo appearance of mitochondrioma in cells of tissue cultures . recently chevremont and frederic ( 1952 ) observing tissue culture cells during mitosis with the phase contrast - microscope , noted the transformation of chondrioconts into very minute rods and granules which resemble the structures described here . ' these formations sometimes disappeared and appeared again in daughter - cehs and then growing thicker and longer , reconstituted ordinary chondriosomes . the existence of the ultrachondrioma may well explain the apparent appear - ance de novo of mitochondrioma as a result of their regeneration from the ultra.chondrioma. it is not impossible that the absence of the ultrachondrioma in many cers may be more apparent than real -  . 
the degree to which ordinary mitochondriaare susceptible to variations of physicochemical conditions such as ph , osmotic afinor variations of conditions that are uncontrollable pressure , is well known . by present methods might cause a cavitation of the ultrachondrioma that would make it impossible to distinguish from the surrounding endoplasm . actually we never observed ultrachondrioma in cells where swelling of the ordinary chondrioma occurred . furthermore we sometimes found ultrachondrioma in the perinuclear region wben very favourable conditions permit observation in this area . but usually the thickness and high rpid content of this area in spread cens pre - ntent its study with the electron microscope . 
oberling there are two fields in which the existence of the ultrachondrioma raises special problems . in cytology , the presence of corpuscles resembling viruses in normal ( or presumably normal ) cells should be a warning against mistaken interpretations and will complicate the task of those workers who are engaged on the problem of the interaction of cell and virus . in biochemistry it introduces a new factor into the study of cytoplasmic it seems obvious that a large part of fractions isolated by ultracentrifugation . the ultrachondrioma will be deposited in the so - called microsomal fraction and would therefore lead the erroneous conclusions concerning the homogeneity of this fraction . the concept of the ultrachondrioma limited though it appears should entail a critical revision of some of the present data of cytology . we wish to thank madame dontcheff for the tissue cultures , and madame arnoult and martin for technical assistance . 
the ratio of the concentration of the compounds in the stomach to that in the blood in this range was about 20 : 1 or greater . by selecting carcinogens that have a pk , , falhng in this range it should be possible to circumvent the mucus barrier and expose the glandular stomach to attack . 
the animals had been fasted for 48 hours preceding the operation to simultaneously with the injecavoid contamination of the secretions with food . tion of the compounds to be tested 0 - 2 mg . 
with acetone and this extract allowed to stand overnight it was then centrifuged for 20 minutes at 2000 r.p.and the superat 5 - 10 ' c . natant used for spectrographic analysis . measurements were made on a beckman model du spectrophotometer against acetone - hci standard having the same ph as the gastric extracts . standard curves were obtained which essentially followed beer 's law . 
the 2 , 5 - isomer is soluble , whereas the 2 , 7 - isomer is not ( morgan and thomason , 1926 )  . yields of 55 to 60 per cent were obtained of 2 , 7 - dinitrofluorene decomposing at 293 - 4 '  . the 2 , 7 - dinitrofluorene was reduced with tin and hydrochloric acid by the method of schulman ( 1949 )  . 
the pure compound melts at 165 ' after recrystalthis was then converted to the dihydrochloride and lization from benzene . 2 - acetylamino - 7 - aminomono - acetylated in aqueous solution ( schulman , 1949 )  . fluorene hydrochloride was then slurried in 500 ml . 
the almost clear solution was filtered hot and a cream - white product precipitated from the cooled three recrystallizations from 50 per cent acetic acid resulted in a filtrate . goulden and kon ( 1945 ) reported that 2 - hydroxy - 7compound melting at 232 '  . acetylaminofluorene melts at 232 '  . the dyes of this series were prepared by coupling the components in acid buffered , or basic aqueous solution , neutralization and filtration . 
the yields ranged from 70 to 90 per cent . products could be recrystallized from dilute cellosolve , but highest purity was obtained from aniline , or benzyl alcohol . these compounds have not been previously reported . constants and analyses are given in table il 368 f . 
compound iv seems to be secreted to a slightly lesser extent , but this may not be significant . this was entirely unexpected , because it has a pkb ( 8 - 5 ) which hes in the optimum secretion range ( ray and jung , 1951 )  . this indicates that the pkbof maximum secretion in the fluorene series is much higher than it is in the benz ' ene series . because the serosa was colored , it was thought that this dye might have been reduced in the stomach at the azo link . 
an examination of the stomach contents for aminofluorene by the method of westfall and morris ( 1947 ) by miss mary f . argus failed to show the presence of this substance , either free or coniulyated . while , successful in leading us to potential carcinogens that are secreted in large amounts by the stomach , it is obvious that some factor ( or factors ) are involved other than the pkbit is true that the stomach of the rat may be less acidic than that of the dog , but this should only reduce the total concentration and not the relative order of absorption . the use of a tricyclic molecule hke fluorene in place of benzene leads to compounds with greatly reduced solubilities in dilute acid . 
the introduction of a third ( insoluble ) phase might well cause the stomach barrier to operate to exclude compounds of lower pkb ( greater basicity )  . the demonstration that some of these compounds are secreted in rather large amounts in the stomach has encouraged us to set up long - time experiments for testing their carcinogenicity . 
the histology was typical of lymphosarcoma , the cells being chiefly of the " lymphoblastic " type . local spread had occurred upwards in the submucosa for a short distance , and there was infiltration of adjacent rectal muscle but no noticeable spread in perirectal fat . 
the tumour was softer in consistence than most carcinomas and the base of the ulcer was covered by a greyish slough . both " lymphoblastic " and " lymphocytic " cells present . extensive spread in rectal muscle and commencing invasion of perirectal fat . 
the adenocarcinoma had infiltrated more deeply , but there was no sign of venous spread and the lymphatic glands were all free from metastases . we have given careful consideration to the possibility that this might actually be only one tumour , exhibiting an undifferentiated pattern in one part and a differentiated glandular pattern in another , as was suggested by gr . 
tumour b is a melanoma with four lymphatic metastases containing melantumour a is an adenocarcinoma with two lymphatic metastases free from pigment . section of tumour a showing adenocarcinoma , fig . 
1945 , and found to have a fibrous nodular tumour on posterior quadrant of lower third of rectum . biopsy showed spindle cell sarcoma apparently of low grade malignancy . seven years previously patient had had the coccyx removed in another hospital , and three years later a small tumour had been found in the rectum which was reported as a mass of fibrous tissue . synchronous combined excision of rectum by o . 
more than 4 inches of healthy bowel both tumours had given separated the melanoma from the adenocarcinoma . rise to lymphatic metastases , those from the melanoma being pigmented , whereas those from the adenocarcinoma contained no melana unique pathological specimen . 
twenty years ago weeks ( 1927 ) found reference to 100 cases of rectal sarcoma , but he regarded many as of doubtful validity and remarked - " if the questionable cases were discarded it is certain that the number would be reduced by one half . " we agree with this comment and have formed the impression that many of the cases which until recent years were recorded in the literature as " sarcomas " were probably examples of anaplastic carcinoma . this criticism does not apply to the cases reported in the last 15 to 20 years . most authors who have written about this subject seeinm to take the view that all varieties of sarcoma tend to run a rapidly fatal course , but we think that each of the four varieties of malignancy has this attitude needs qualification . to some extent its own characteristics and peculiarities and the prognosis differs considerably in each . lymphosarcoma is the commonest form of sarcoma of the rectum . it may manifest itself either as a single localized tumour or give rise to a crop of tumours bensaude ( 1929 ) was one of the first to spread over a long stretch of the bowel . draw attention to the diffuse or generalized variety of lymphosarcoma . in one of our cases ( case 3 ) the disease affected the whole rectum and distal end of the pelvic colon , so that in describing the primary focus we could not speak of a particular site of origin , but only of a region of involvement . 
the largest tumour was in the lower third of the rectum , but the other smaller tumours at a higher level were certainly not secondary growths resulting from the transportation of tumour cells , but were obviously new foci of lymphosarcoma developing indein spite of this unfavourable feature of lymphosarcoma the prognosis pendently . after surgical treatment would seem to be fairly good on the whole . 
at present there is insufficient evidence on which to base a comparison because it is only within recent years that these tumours interest in the subject have been separated out from other varieties of sarcoma . of reticulosis , stimulated by the writings of robb - smith ( 1938 ) , has had considerreticulum cell sarcoma is certainly a rarer tumour able influence in this respect . in the rectum than lymphosarcoma , and we think that it will probably be found to have a worse prognosis . spindle cell sarcoma is also a rare form of malignancy in the rectum . it is the one most easy to distinguish clinically from carcinoma because it obviously arises from the tissues of the bowel wall and not from the surface mucous membrane . the firm solid structure and covering of intact mucosa are of special significance . it has already been mentioned that in their histology these tumours give the impression of being of a relatively benign character . all who have written about melanoma speak of it as the most malignant of all the tumours now being considered . 
we agree with the view expressed by linder and wood ( 1936 ) that melanomas arise from the skin of the anal canal and tend to grow up into the rectum and that they metastasize early . 
the lymphatic glands may contain more pigment than the primary tumour , but it is worth mentioning that the haemorrhoidal and paracolic lymphatic glands may contain melanin apart altogether from the presence of a melanotic tumour . 
bussey although the cases we have examined personally are only ten in number they do provide material for comparison with the much commoner tumour , carcinoma of the rectum . in the end , of course , these rarer forms of malignancy can only be identified by detailed study of their histology , but there are some features in their gross characters which would make an experienced surgeon suspect that he was dealing with something " out of the ordinary . " we have summarized these by setting them out in tabular form ( table i )  . finally we have left to the end what we consider to be the most interesting feature of these rare tumours , namely , the frequency of multiple foci of malignancy . in four of our ten cases more than one tumour was found in the rectum , and in two cases the second tumour was of a completely different histological type . 
this raises the question both of multiple malignancy and dissimilar primary tumours . multiple malignancy is commoner in the rectum than is usually supposed . it so happens that we are in a position to give facts about this question because for many years we have been making observations on the frequency of the occurrence of multiple primary carcinoma and have found that in operation specimens of rectal cancer removed by a combined operation more than one primary rectal carcinoma is present in 4 per cent of cases ( over 2 , 000 examined )  . moreover we have records of many additional cases in which a second carcinoma was present in the colon in association with rectal cancer , so we were not surprised to find multiple tumours in our cases of lymphosarcomas ; in fact , we expected this to be the case . the occurrence of dissimilar primary tumours was , however , a complete surprise , and as far as we have been able to find out has not been previously recorded as a characteristic of these rarer forms of malignancy . in any organ of the body the existence of two tumours of a different histological type is a rare phenomenon . it is indeed remarkable that this unusual combination should have occurred twice in a series of only ten cases . 
we have searched through the literature but found no reference to the simultaneous occurrence of carcinoma and sarcoma in the rectum , though kreibig ( 1929 ) mentions a case of carcinoma plus lymphosarcoma in the ileo - caecal region . as far as the general relationship between carcinoma and sarcoma is concerned we note that warren and gates ( 1932 ) in a study of this subject were only able to collect from the literature 25 instances of the co - occurrence of carcinoma and sarcoma as separate tumours in the same organ . three were cases of sarcoma and carcinoma of the breast , twelve were of the uterus , and ten were in miscellaneous organs , including four cases of the combination of carcinoma and 1ynphosarcoma of the stomach . they found no recorded case of carcinoma and sarcoma of the rectum or indeed of carcinoma of the rectum associated with sarcoma of any other part of the intestine . 
melanoma arises always from the ano - rectal region and spreads upwards into the rectum . it metastasizes early and runs a rapid course . in four of the ten cases we are reporting more than one malignant tumour was present in the rectuone of these was an example of multiple lymphosarcoma and another of multiple reticulum cell sarcoma . in these cases tlhe associated tumours were of the same histological type , but in two other cases the associated tumours were of completely different histological type . 
rinaldini. from the strangeways research laboratories , cambridge . received for publication april 25 , 1952 . the nitrogen mustards have been shown to have a marked inhibitory action upon cell division , a property that warrants their inclusion in the miscellaneous group of the " mitotic poisons " ( loveless and revel , 1949 ) and their use as chemotherapeutic agents in certain forms of malignant growth ( haddow , 1947 )  . some of the 2 - chloroethylamines are mutagenic in drosophila ( auerbach and robson , 1947 ) and in neurospora ( tatum , 1947 ) , and more recently both the alkyl ( boyland and horning , 1949 ) and the aryl ( haddow , kon and ross , 1948 ) derivatives have been shown to exhibit tumour - inducing properties . the present work deals primarily with the effects of p - amino n - n - di - ( 2 - chloroethyl ) aniline hydrochloride on the number , duration and distribution of mitoses in tissue cultures during the first 1 to 3 days after exposure . 
though not very adequate for detailed cytological work because of the smallness and large number of the chick chromosomes , this material was found particularly suitable for quantitative treatment as regards variations in mitotic number and duration of the mitotic cycle . 
mitotic counts in fixed and stained cultures . this method furnishes data on the total number of mitoses and on the number of cells undergoing each phase of division at a given moment , and therefore an indirect estimation of the delay or arrest at any stage of the cycle . 
the number of abnormalities can also be ascertained . the cultures that showed the most abundant growth after two transplantations were selected , and the explants were cut in halves as closely similar in size as possible , under the dissecting microscope . 
all the solutions used here were made up in tyrode fluid . the cultures were stained with ehrlich 's haematoxylin after hydrolysis in n hydrochloric acid for 8 to 10 minutes at 600 c . 
 ( hughes and fell , 1949 )  . total mitotic counts were made in every culture . in order to facilitate counting the whole of the mitotic cycle was considered to be comprised within the period between the disappearance and the reappearance of the nucleoli . exception was made , however , for those cells in which the nucleolus persisted in advanced prophase and , conversely , for telophases in which the nucleus was fully reconstructed before completion of cytokinesis . 
low power phase - contrast cinemicrography . this method permits a direct , though only approximate , measurement of the rate of division and duration of each phase in a group of cells . for film recording the cultures were mounted in an air - tight chamber made with a metal frame between two coverslips and sealed with paraffin wax ( hughes and swann , 1948 )  . 
rinaldini the apparatus , devised by canti and lately improved by hughes , consists of a phase - contrast microscope encased in a thermostatically controlled chamber and attached to a 35 mcine camera . 
the chromosome movements can be seen in detail , and it is possible to measure accurately the duration of each mitotic phase . the technical arrangements were similar to those for the low - power film recording , except that critical focusing and phase adjustment required constant observation , and the time interval between frames was much shorter . 
rinaldini less concentrated solutions of the order of 1 : 50 , 000 to 1 : 100 , 000 - 74 x 10 - 6 and 37 x 10 - 6 molar - allowed indefinite survival and successful suboultivation , although the outgrowth was smaller than in normal cultures . 
at this stage the treated cultures showed no sign of recovery , but on the contrary there was a visible difference in the area of the outgrowth as compared with the controls . this was measured with the aid of a camera lucida as shown in fig . 
lindley using test " t " for p = 005 , the x2 analysis of contingency tables and the analysis of the variance on the square root of the counts . 
the percentage of abnormal proand metaphases taken together was approximately 35 per cent after 24 hours and 20 per cent after 48 hours , while the number of abnormal anaand telophases was only 4 per cent after 24 hours and reached 12 per cent after 48 hours . in spite of the normal appearance of the resting cells , all the evidence obtained from the counts points to the fact that almost 100 per cent of the inhibitory action was exerted during interphase . 
rinaldini no difference was found between the duration of 10 normal mitoses photographed during the first 5 hours and the last 5 hours of an experiment , nor between peripheral and central cells . 
a similar case was recorded under high magnification ( see below )  . six cells could be followed from one division to another in the control cultures . the intermitotic period varied from 5 to 20 hours , with an average of 10 - 6 hours . cells in the middle had a longer resting stage than cells in the periphery of the outgrowth . in the treated cultures no cell could be found to divide twice in one film ( 20 to 24 hours ) , but due to the scarcity of cell divisions it was not possible to decide whether the resting stage was prolonged or whether cells were prevented from dividing again . high power phase - contrast cinemicrography . the results have been reported elsewhere by hughes ( 1950 ) , and i shall only briefly summarize them here . 
with dilute solutions a large proportion of cells are prevented from entering mitosis , but no pycnosis or vacuolation appears . proand metaphase abnormalities this effect is exerted mainly during interphase . are conspicuous and anaand telophase abnormalities rare during the first 24 hours , anaphase abnormalities increasing after this period . 
the duration of the first two phases is significantly prolonged in some cells . cultures recover from these effects when transplanted into a fresh medium after 48 hours and hardly any residual effect can be detected . 
the nature of the s35 substance . the presence of s35 in the cytoplasm of myeloid cells after culture in s35 sulphate was all the more interesting since most mammalian cells will not utilise experiments were designed to obtain information on the nature of sulphate . the s25 substance in the myeloid cells . 
the summarised findings are presented in table i . ( 1 ) since 2 - 3 hours ' washing of the slides in running water , or 1 hours ' incubation at 370 c . 
a search was therefore made for a sulphate compound in these cells . although most sulphates form an insoluble precipitate with barium , it has been reported that the barium salt of chondroitin sulphate is soluble in water it was thought that the s35 in the cells might ( bray , gregory and stacey , 1944 )  . be in a form related to the chondroitin sulphates , therefore experiments were carried out to test the barium solubility of the su compound in the cells . both 1 m and 0 - 1 m ba ( oh ) 2 removed all the cells from the slides , but this could be prevented by m.aking up the ba ( oh ) 2 solutions in 10 per cent formalin . the morphology of the cells on the slides thus treated was unaltered , and it was found that 15 minutes ' treatment with 1 m or 0 - 1 m ba ( oh ) 2 formalin at 20 c . removed all isotope from the cells . formalin alone did not appear to remove any s35 . it had to be ascertained whether the effect of ba ( oh ) 2 was a specific ba effect slides subjected to distilled water alkalinised with or due to the alkaline ph . nh3 to ph 9 - 10 for 15 minutes at 200 c . 
for one hour or longer . that the s35 substance is not closely related to hyaluronic acids . ( 6 ) no parallelism could be detected between the behaviour of the s35 substance and those polysaccharides and other substances which give a positive hotchkiss reaction ( periodic acid schiff reagent )  . 
for 1 hour , the s3 substance was not . all hotchkiss - positive substances ( fresh human saliva , 1 hour at 370 c . ) , but it did not affect the isotope content of the cells . 
the uptake of methionine s35 . at the beginning of the experiments it was considered to be unlikely that s35 administered as sulphate to the culture medium would be transformed by the cells to sh groups . 
oliver s35 sulphate administered in vivo to rats has been shown to be taken up into the chondroitin sulphate of the growing cartilage ( dziewiatkowsky , benesch and benesch , 1949 ; dziewiatkowsky , 1949 ; 1951a , 1951b , 1952 )  . irrespective of whether sulphate or sulphite s35 compounds were given to rats in vivo , activity this finding has was found in the bone marrow by singher and marinelli ( 1945 )  . been confirmed by dziewiatkowsky ( 1949 , 1952 ) using whole bone autoradiographs and it was thought not to be chondroitin sulphate , but to be inorganic sulphate this sulphur after the first 30 minutes sulphur which is precipitated by ba ( oh ) 2 . of administration , is incorporated into a more complex compound from which the sulphur is slowly removed . the s35 substance in the myeloid cells of the bone marrow cultured in vitro it is possible that the dose of s35 sulphate given is removed by barium salts . to the rats by dziewiatkowsky was too small to be detectable in whole bone autoradiographs in the acid formalin fixed bones ( 5 / tc . / 7 - day - old rat in one series and 1 - 25 , tc . / 7 - day - old - rat in the other series of experiments )  . at this stage of the work it is probably too speculative to attempt to correlate the specific s35 sulphate uptake of the myeloid cells with the specific action of myleran ( 1 - 4 dimethanesulphonyloxybutane ) on the myeloid cells . 
raven. from the royal cancer hospital ( free ) and the gordon hospital for diseases of the rectum and colon . received for publication march 20 , 1948 . a study of the literature concerning malignant disease of the heart teaches that whilst primary tumours are rare , secondary deposits are not infrequently found . 
the following figures are illustrative . scott and garvin ( 1939 ) , in a series of 11 , 100 autopsies , including 1 , 082 performed in cases of malignant disease , reported secondary malignant disease as follows - heart , 79 cases ( 7 * 3 per cent ) ; pericardium , 61 cases ( 5 7 per cent ) ; heart and pericardium , 140 cases ( 12 * 9 per cent )  . willis reported an incidence of 6 - 2 per cent ; burke , 4 2 per cent ; helwig , 0 9 per cent ; pollia and gogol , 2 per cent ; lymburner , 0 6 per cent ; and symmers , 1 6 per cent . the subject is of more than academic interest , for with a better understanding it may be possible to establish a diagnosis during life in a greater number of patients , and the institution of certain forms of treatment , even though these are of a palliative nature . 
the symptomatology will therefore be reviewed , and attention drawn to the electrocardiographic findings in these patients . this paper is based on a series of 51 cases of secondary malignant disease of the heart which were collected at the royal cancer hospital from the autopsy records for the years 1930 to 1945 inclusive . 
the pericardium is usually involved and various types of disease are described . focal collections of carcinoma cells may occur in the epicardial fat , the cells being transported along the dilated lymphatics . in other cases the pericardium is studded with nodules of growth , whilst a third variety consists of diffuse pericardial infiltration , and this process may extend into the pericardial fat . there may be commencing infiltration near the commencement of the great vessels . another variety comprises flat plaques of growth embedded in the pericardium . in a number of cases there is combined involvement of the pericardium and heart muscle . 
tumour cells were seen in the lumen of a small arteriole ; obviously the disease in the heart was conveyed by the blood stream . in another case nodules of growth were found in the muscle of the right auricle , left ventricle and left auricle ; some of these were situated beneath the endocardium and growing into the auricle . 
the microscopic picture was similar to that of the primary skin tumour . in the third case an amelanotic nodule was present in the upper and posterior part of the left ventricle , from which more extensive spread had occurred into the heart muscle . moragues ( 1939 ) reviewed the subject of cardiac metastases from malignant melanoma and found reports of 23 cases in the literature , to which he added 4 in one of these cases there were signs of cardiac involvement ; personal cases . a loud systolic murmur was present in the pulmonary area , caused by a large tumour mass which almost occluded the pulmonary orifice . the spread of malignancy to the heart . this occurs by the usual four methods , namely , haematogenous , lymphatic , in this combined haematogenous and lymphatic , and by direct extension . series of cases there was definite evidence of all these modes . 
any subsequent abnormality which is noted can be compared with this initial investigation and inferences drawn . in cases with tumours of the heart , carnot and lambling ( 1928 ) have described a clinical syndrome which resembles that of subacute bacterial endoapart from this group of cases , attention is called to certain symptoms carditis . and signs which suggest cardiac involvement in patients known to be suffering from malignant diseases , especially of those organs which are recognized to give rise to secondary cardiac deposits . dyspnoea , tachycardia and cardiac irregularity are frequently outstanding symptoms , the irregularity being of the nature of either auricular fibrillation or auricular flutter . 
the symptoms may be correlated with the location of the tumour in the heart , causing heart block , or due to a pericardial or pleural effusion . it must not be assumed , however , that evidence of pericardial disease in a patient with malignancy is pathognomonic of involvement of the pericardium ; acute pericarditis may occur in the advanced phase from other causes . 
amongst important signs are those of cardiac enlargement and dysfunction , impaired resonance of the chest , the presence of abnormal breath sounds and added sounds . cyanosis may be present and the signs of cardiac failure . in the presence of this symptomatology certain investigations are carried out . 
when a pericardial effusion manifests itself , paracentesis is performed and cytological examination of the fluid is made . in a number of cases malignant tumour cells will be demonstrated . radiological examination of the heart , including tomography , is regarding the electrocardiographic out . findings , the case reports in the literature do not show uniformity ; the results vary according to the position of the tumour in the heart . siegel and young ( 1933 ) , dealing with this aspect of the subject , call attention to the work of lloyd ( 1929 ) , who published the first electrocardiogram in a proved case of tumour of carried r . 
the t waves in leads 1 and 2 were slightly diphasic . had been given before the patient was admitted to hospital , which may account siegel and young ( 1933 ) reported the for the abnormality of the t waves . findings in a case of metastatic sarcoma of the myocardiuthe t waves were inverted constantly in all leads , and there was no significant change daily . 
when the primary tumour is known to be radiosensitive , high - voltage x - irradiation may prove of value in the diagnosis in these cases clinical of metastases in the heart when enlargement is present . and radiological evidence may be forthcoming of a marked reduction in the size of the heart and the disappearance of the pericardial effusion . shelburne and aronson ( 1940 ) report the effects of high - voltage x - irradiation in a patient with metastases in the heart and pericardium from a primary myeloblastoma in the frontal bone . 
1. received for publication january 21 , 1948 . surgery in the form of the so - called radical operation is in general at the present time the principal weapon in the treatment of carcinoma of the breast . what it can achieve and its limitations are now fairly well established . 
thus it is known that if a patient with carcinoma of the breast has microscopical invasion of the axillary glands , the strong probabilities are that the disease has passed beyond the local area and that the patient will sooner or later succumb it is to the disease , although there will be a minority of fortunate exceptions . also known that if the axillary glands are not invaded the chances that the patient will be cured of the disease are appreciably increased . 
of particles of 3 diameter or less during a 6 - weeks period : seven rabbits survived the injections for 7 months or more , and all developed malignant osteosarcomas , often with multiple primary sites . 
 we have repeated these experiments , using zinc beryllium silicate and beryl lium silicate and confirm the findings of gardner and heslington ( 1946 ) , although the proportion of survivors developing tumours is less in our series . 
x - ray analysis has shown that zinc beryllium silicate is really a solid solution of z11iisio4 and be2sio4 an analysis of the zinc beryllium silicate used in experimental groups b and c ( table i ) gave a composition zno 67 per cent , sio2 31 per cent , beo 2 per cent . 
the complex injected in experimental group a ( table i ) contained manganese , and had a composition zno 67 per cent , sio2 28 per cent , beo 2 per cent , mno 3 per cent . 
 examination of the tissues of animals dying immediately after injection and between 14 and 83 weeks after the injection of the silicates has shown the presence of silicate particles in the lungs , spleen and liver of all injected animals . 
the macro phages of the spleen first form giant cells of the foreign body type and even of the langhans type , but later the particles are contained in large syncytial masses , which show up to 100 nuclei in a single section . 
for some months the particles are present in isolated kupffer cells , but 3 to 4 months after injection the kupffer cells have increased in size and number to form cellular masses that fill and often distend the sinusoids . 
 the nodules are present in all injected animals whose bones have been searched for them , and in many cases are not associated with any other abnormality , either histological or radiological . 
these : findings correspond closely with those for rabbit 4 , where consideration of histological material in conjunction with radiographs leads to the conclusion that the radiographic appearances result from occupation of the marrow cavity by ossifying tumour tissue . 
histological examination of the femoral shaft shows a complete replacement of the normal marrow by tumour tissue , quite comparable to that seen in more advanced lesions in other animals , but not yet expanding the periosteum or causing erosion of the cortical bone of the sha part of the tumour is bony , part cartilaginous , while some areas consist merely of anaplastic spindle - celled tissue . 
numerous fat cells are scattered through the area , and the tissue varies from an almost acellular mass of fibres to a tissue composed largely of rounded and elongated fibroblasts . 
5 shows such an area , where a network of recently - formed bone - trabeculae is present , and where the osteoblastic cells covering the surfaces of the trabeculae are linked to the intervening fibroblasts by numerous intermediate forms . 
on the left side of the bone a few areas show a similar pattern , but there is also some more diffuse aggregation of radio - opaque material , corresponding to the formation of calcified cartilage by the tumourtissue . 
 in the lower part of each femur the marrow is completely replaced by a mass of bone - forming tumour tissue , which has begun to extend into the haversian canals of the dense cortical bone . 
some areas consist entirely of cartilaginous tissue , others show extensive osteoid and bony differentiation , while in some places the two tissues merge into a composite " osteochondroid " pattern . 
in several situations this was beginning to invade the dense cortex of the bone concerned , and to expand the covering periosteu in this animal the body of one lumbar vertebra contained a small rounded area of calcifying spindl~ - celled tumour tissue , 2 m in diameter , sharply demarcated from the surrounding normal marrow . 
this suggests that a continuous process is concerned in the development of frankly malignant tissue from the earlier pre - invasive lesion seen , for example , in the humerus of rabbit 4 . 
of soluble berylliu treatment of the refractory beryijium silicates with n , 100 hydrochloric acid produces a 10 to 20 - fold increase in the proportion of soluble beryllium in the aqueous suspension . 
 when stained by the method developed by denz ( 1949 ) for the histochemical detec tion of beryllium these sections showed that some soluble beryllium had left the necrotic area and stained the surrounding fibrous tissue faintly an_d diffusely . 
while the fully _developed tumours present a very different appearance from the medul lary bone formation that has been described as an early stage of malignancy , all stages of transition between the two can be traced . 
it is suggested that these indicate a continuous process of transformation from the early medullary : fibrosis to the later obviously malignant lesions , but this is only the interpretation of a number of morbid anatomical observations . 
 the relationship between the small nodules which are the first change seen within the bone marrow and the subsequent : fibrosis and malignant change is uncerta the nodules produced by the silicates containing beryllium and those produced by the zinc silicate are indistinguishable by the usual histological methods . 
it seems clear , however , that it is the presence ofthe beryllium within the nodule that is in some way responsible for the progressive changes that lead eventually to malignancy . 
although the beryllium silicates are considered to be insoluble , the observations on material injected intradermally into a rabbit showed that beryllium can be liberated from the particles and can diffuse into the surrounding tissues . 
 _therefore , if beryllium is to be held directly responsible for the tumours , it is much more likely that this beryllium is liberated from deposits in the marrow rather than derived from more distant deposits . 
the possibility that the silicate may play some part in the development of the tumours is excluded by the fact that gardner and heslington ( 1946 ) produced tumours with beryllium oxide , and more recently barnes ( 1950 ) has reported that two rabbits that received intravenous injections of a suspension of beryllium metal particles have developed characteristic sarcomas . 
 it is also apparent that the tissues themselves must contribute a factor , because similar nodules in the liver and lungs arouse very little : fibrous tissue reaction , while in the spleen : fibrosis and atrophy may go to extreme lengths without showing evidence of malignant change . 
 since this paper was submitted one further rabbit has died with a sarcoma of the pelvis 30 months after the last of a series of injections of zinc beryllium silicate . 
 the materials used in these experiments were supplied to us by the research laboratories of the general electric company , wembley , to whom we would like to express our thanks . 
kreyberg spondence is established between the patient group and the control material with regard to such factors as age and sex composition , distribution of broad occupational groups and place of residence , which may influence smoking habits and / or lung cancer frequency , and a large number of factors clearly may be considered irrelevant , a field of uncertainty of considerable extent still remains . two points of practical importance emerge from the above discussion . 
the first is that control material consistin - g of non lung cancer cases among patients in a chest department , or even among hospital patients generary , may introduce a bias if , for instance , the factor " visiting a chest department for reasons other than lung cancer " - present in a control material but not among the lung cancer patients - is associated with the level of tobacco smoking . there may thus be a tendency towards excessive smoking in such a control material , or patients in chest departments may on the contrary tend to be persons whose reaction even to mild forms of chest trouble is a visit to the hospital , and who are careful if both these tendencies not to expose themselves to irritants sucli as tobacco . are present among the patients , they may neutralize each other , but the net result will obviously depend on their relative occurrence in the control material . similar objections may be brought against the use of hospital patients generally this point has been stressed by mirs and porter ( 1953 ) as control material . who believe that the bias probably wfll obscure an excess of smoking among lung cancer cases . 
at least , it is not established that hospital patients do not differ systematically from the general population with regard to smoking habits . the second point to be made is that a careful survey of all possible cancer producing factors must precede the instigation of a control study , and that prehminary surveys are needed to estabhsh the relationships between these factors and the level of tobacco smoking . there is thus a priori reason to consider the need for relevant definitions and differentiation of occupational groups among the cancer patients as well as in the control material , in view of the frequently indicated possibilities of specific occupational hazards with respect to lung cancer . assume , for instance , that a particular type of industrial occupation has a definite but unrecognized excess incidence of lung cancer , and that the carcinogenic agent is connected with the nature of the work . 
assume further that this particular occupation also is associated with an excess of tabocco smoking , because of higher pay , better opportunities of smoking during working hours , psychological stress or other causes . if the occupational selection criteria are such that this occupation does not exphcitly constitute a stratum in the control material it may be under - represented in a control material with random selection within each stratum , and the controls may , for the separate occupational groups and in toto , show a lower smoking level than the cancer such a difference will clearly be misleading for an evaluation of patient group . the carcinogenic effect of tobacco smoking . if , on the other hand , the particular occupation in our example is associated with an exceptionally low smoking level , a possibly significant excess smoking in the cancer patient group may be obscured . 
kreyberg composition of tobacco consumption are available only after 1927 . since then , tobacco smoked , i.e. , cigars , cigarettes and pipe tobacco , has accounted for a steadily increasing proportion of the total tobacco consumption at the expense of chewing tobacco and snuff , as shown in fig . 
the amount smoked is here calculated as production plus imports minus exports of cigars , cigarettes and pipe tobacco . these changes will year to year inventory changes have not been corrected for . be small , however , and without influence on the trend even if the vearlv figures may contain erratic deviations from the true consumption level . it should be permissible to assume that chewing tobacco and snuff played a greater r ' ole before 1927 . 
even if some of the chewing tobacco in fact has been smoked in pipe , it is reasonable to assu ' me that the amount of tobacco smoked , per person 15 years and older , has increased to some extent during the whole period from 1901 to 1940 . 1800 1600 1400 1200 1000 11 1% in600 1928 year fig . 
2. - consumption of smoking tobacco in granis per person 15 years and older . 38 39 after the last world war the yearly amount of tobacco smoked , i.e. , cigars , cigarettes and pipe tobacco , has reached a level of approximately 1650 g . 
3 for 1928 - 29 , 193 7 - 39 and the post - war years . the black parts of the columns represent the proportion of ready made cigarettes , which was 34 - 7 per cent in 1928 , rose to 42 - 9 per cent in 1947 and has since declined to 35 - 8 per cent in 1951 . 
patients , ear - nose - throat department , the rikshospital ( whole country ) kongsberg is a smar industrial town in central southem nor - way , s smar industrial and mining commuiiity ( iron - ore ) in the extreme north . varanger ( kirkenes ) a in the groups 1 , 3 , 4 and 5 among the males and 1 , 2 and 3 among the females the number of returned questionnaires falls substantially short of the total number of individuals in the groups selected . 
the age distribution of the separate groups is given appendix tables iii and iv . incidence of each of the forowing were computed . each of the above - mentioned groups were divided into age - classes with a for each group and age - class the percentage for males : 1 . 
4 is shown the range of variation found for the frequency of smokers in each age - class among the groups investigated . it will be noted that this range is fairly narrow , especially in the age - class 45 - 54 years in which the lowest percentage smokers ( found among the visitors at the oslo first aid station ) is 83 - 5 and the highest ( among industrial workers , oslo ) is 91 - 9 . 
the latter group , on the other hand , reaches its highest frequency at a i ' ater age than most of the other groups . generally the percentage smokers is lower among industrial workers in kongsberg , than in the other groups . per cent pure cigarette smokers . 
5 we find a very wide range of variation for the occurrence of pure cigarette smokers in the material , especially in the lower age - classes . among the industrial workers in kongsberg in the age - class 25 - 34 years only 28 - 4 per cent smoke cigarettes exclusively , whereas 75 - 9 per cent of their colleagues in s6r - varanger do so . 
6 is similar to that found for the percentage pure cigarette smokers , but the level has been shifted upwards and the range is a little less wide , corresponding to the tendency for the cigarette - pipe smoker combination to be more frequent in groups with relatively few pure cigarette smokers than in those with a high pure cigarette smoker percentage . 
8 the range of variation in the percentage medium and heavy smokers is large in all age classes , but the frequency does in no case exceed 50 per cent . 
12 is very rare among women , occuriing only among physicians and the patients and followers at the oslo first aid station , in the age - classes 35 - 54 years , with frequencies ranging from 1 - 4 per cent to 2 - 3 . per cent heavy and medium smokers . 
how much of this variation is real , i.e. , caused by actual differences in smoking habits in the groups under consideration , and how much may be caused by a systematic bias in those groups whose degree of co - operation in retur ' n ' mg questionnaires was low , cannot be decided on the basis of available inforrnation . 
the question is of considerable interest however , since for most of the smoking habit criteria , the range of variation among males would narrow considerably if the figures for the industrial workers in kongsberg are left out . this group comprises questionnaires from only a part of the total number of workers in the arms factory selected for analysis , and ff it could be shown that the degree of co - operation in this group is systematically related to smoking habits , the deviations shown would not reflect a true difference in smoking habits between these factory workers and the other groups . there are , however , certain indications that part of the difference at least between the kongsberg workers and the other male groups is real . first of all the percentage smokers in this group ( appendix table v ) are not substantially below those of the other groups . 
even if there is a tendency for smokers to be under - represented among those workers who answered the questionnaire , such a bias can not account for the very low percentages pure and pure and mixed cigarette rsmokers found in the kongsberg group , and is not likely to expla ' entirely the low percentages found for the quantitative criteria ( per cent heavy and per cent heavy and medium smokers )  . secondly , groups other than the kongsberg workers have failed to co - operate fully , without any resulting systematic deviation from the complete groups . 
an explanation of the result for the kongsberg group in terms of a systematic bias , would thus imply that this bias was a characteristic of the kongsberg workers but not of the others . thirdly , the material from the male physicians seems to indicate that there is no strong tendency that individuals who fail to fir a questionaire deviate appreciably i their smoking habits from those who co - operate readily . 
a new questionnaire was sent out approximately a year later with an appeal to those who had not answered previously this appeal resulted in 601 new questionnaires which were to do so now . analysed separately , however , the two groups showed added to the old material . the following figures for the separate smoking habit criteria . there is thus cause to believe that the range of variation found in the material largely reflects real differences in smoking habits . special menti ' on may be made of the male group of patients at the rikshospital ear - nose - throat department which displays exceptionally low percentages heavy and heavy and medium smokers ( both generally and cigarette ) in the lower age - classes and rather high percentages in the higher age - classes . the material is , however , too small to permit any conclusion as to whether or not this reflects a characteristic pattem in this group . conclusion as was pointed out initiahy , the present study has been undertaken in order to provide background information on factors which must be taken into conh . 
the present study may serve as a basis of comparison with the smoking habits of lung cancer patients , in the sense that findings generally outside the range of variation exhibited by the present material would be indicative of real deviations in the smoking habits of lung cancer patients , but quantitative statements about differences found cannot be inferred . the low frequency of smokers in the higher age - classes among most groups of women may indicate that the increasing trend in total tobacco consumption h . 
it would be wrong to give the impression that undesirable sequelae are common , yet such complications as wound sepsis , necrosis of the skin edges and a poor functional result are still seen with greater frequency than one would expect following an operation which has been abundantly practised by innumerable surgeons all over the world for over half a century . 
these particular sequelae are essentially a reflection on the judgment or the executive skill of the operator and not upon the operation itself , because with care they need not happen . 
furthermore , such indifferent results become known , and because they lose nothing in the telling may have a serious deterrent effect upon patients contemplating advice about a newly acquired lump in the breast . 
it is to be remembered that we are operating under almost ideal conditions - the patients are constitutionally well and often near the prime of life , the anatomy of the area is normal and not distorted as it might be , and there are none of the embarrassments associated with operations within a body cavity . 
a very high degree of technical excellence should therefore be our aim , both in regard to the thoroughness of the dissection which has as its objective the extirpation of the disease , and in regard to the care with which this is carried out , which can so much determine the degree of ultimate functionaf disability . 
 there are three features of the " skin short " case which need some explana i will call them ( i ) anticipating the gap , ( ii ) siting the gap , ( iii ) covering tion . 
riddell surgery - may be influenced ; second , the surgeon is forewarned , and so can avoid being surprised into an awkward situation at the end of a long operation . 
 an assessment - an estimate - should be made in all new cases of cancer of the breast of the amount of skin that will be left for closing the wound after the breast and tumour have been removed . 
it will be found that patients fall into two categories : ( 1 ) a larger group in whom it is judged that it will be possible to approximate the skin edges in the usual way without tension . 
h we remove too little skin , secondary nodules will appear for certain and quickly ; if we remove a more generous amount - - as we must - we shall have to be prepared to deal with a gap . 
 in these patients two alternatives are open to us : either we can proceed to radical mastectomy , skin graft the resulting gap , and hope that the skin covering it will heal in a reasonable time and so allow early post - operative irradiation , or we can anticipate the probable delay in healing by giving the x - ray treatment before the operation . 
 personally in this small group i prefer to give the x - ray treatment first , because in practice unfortunately every skin graft does not take uniformly , so that a proportion of cases are left to heal - at least in part - by granulation , and this takes time ; indeed , sometimes so much time that post - operative x - ray treat ment may be delayed to a point where its usefulness is open to question . 
for this reason it is suggested that if by careful pre - operative clinical assessment we can anticipate and so forecast such a defect arising , we should consider giving the x - ray treatment before the operation . 
the use of pre - operative irradiation does not in any way absolve the operator subsequently from still adhering to the principle of excising a wide area clear of the growth . 
this patient was treated by pre - operatilre irradiation followed by radical mastectomy with an immediate skin - gra the satisfactory functional result can be attributed to ( i ) adoption of the " arm raised " position before approximation of the skin flaps , ( ii ) the early recognition of the inevitability of " a gap " in this type of case and of the need for a skin - gra fro . 
2. - full range of movement : the " arm raised " position is the most delicate test of function at the shoulder following mastectomy ; six months should be allowed after the termination of all treatment for the skin to regain its suppleness beforp 8 - ing function ; inability to raise the arm vertically after such an interval must be regarded as an imperfect functional result . 
3. - llarginal flap necrosis : the illustration shows an area of skin loss as a result of damage to the blood supply of the lateral flap during its elevation . 
at the same time it is not vital to graft ; what is vital is that a gap should be left in a skin short case ; whether a dressing of skin is put on that gap or not is almost a secondary consideration , although skin is very clearly to be preferred to scar tissue if the gap is a large one . 
 the time lo gra the decision as to whether the graft is applied at the time of the mastectomy or not depends solely on the condition of the patient at the end of the operation . 
 h this is unsatisfactory , as it may be in an elderly patient , no harm is done by leaving a raw area and grafting early in convalescence , usually at the time of the first major dressing . 
 if , in addition , we have made our pre - operative reconnaissance , the procedure is robbed of its only disagreeable feature , the element of surprise , since a donor area will have been chosen and prepared and the few special instruments and dressings will be immediately available . 
this accident most commonly occurs as the dissection approaches the anterior border oflatissimus dorsi , which corresponds with the deepest and darkest part of the wound ; it is usually due to hurrying this part of the reflection in a field obscured by haemorrhage ; the blood 811pply may also be cut off from a portion of the flap by the use of tension sutures and with the same result . 
riddell displacement of sk in the second category the skin is not lost in the sense that it is destroyed , but is lost by displacement , because skin is borrowed from the axilla and upper arm to close the axillary part of the wound . 
 ( 2 ) slide the skin of the lateral flap well up into the axilla ; by making use of the longer contour of the lateral flap in this way an adequate supply of skin in in suturing the flaps together exactly opposite points on the axilla is assured . 
 in other words , lend skin from the lateral flap to the axilla ; do not borrow it from the axilla and upper arm by bringing the arm down to a right angle or less . 
 i should perhaps add that in my experience functional disability at the shoulder is not related to loss of the pectoral muscles ; indeed , it can be quite severe following a local mast.ectomy , and is not related t - 0 failure to employ early fig . 
the drawing shows the correct position of the upper limb before closing the wound ; the skin of the lateral flap should be pushed well up into the axilla before suturing ( arrow )  . 
in a series of cases examined in which the subscapular nerve had been divided , no functional disability could be found by comparison with control cases and with the opposite side , although , of course , there was a positive r.d. 
the aim is to display the anterior border of the latissimus dorsi muscle , and in " skin short " cases to mobilize the lateral flap still further by carrying the dissection backwards for two or three inches beyond this point , keeping on a plane buperficial to the muscle . 
the aim is to raise a flap of even thinness , but if by a careless stroke of the knife the flap is cut too thin , the blood supply will be damaged and the whole course of convalescence unnecessarily prolonged . 
h too thick a flap is raised the dissection may in error proceed on the deep face of latissimus dorsi into the space between this muscle and the serratus anterior , a space which is filled with large veins . 
h , on admission , the patient is severely anaemic , blood should be given , but let the transfusion be given before the operation ; it can be supplemented by plasma or other blood substitute during the operation if necessary . 
this is a partial contradiction of views ex pressed earlier in my experience ( riddell , 1948 ) , and is based on the fact that blood transfusion to an unconscious patient carries with it an extra hazard - slight though it may be - for the patient cannot proclaim against the blood if it is incom patible , as would be possible if she were conscious . 
the towelling of the operation area should be so arranged , by placing a towel behind the patient 's back , that there is no unprotected area on the flank from which the main wound might be contaminated ; hot wet packs , which so quickly become cold wet packs and chill the patient , should only be used for haemostatic purposes at the end of the operation ; dry hot packs 296 v . 
 drainage should never be employed through any part of the original incision , because it can so easily lead to cross infection of the main length of the wound ; it is best carried out through a separate stab incision in the lateral flap , pre ferably well clear of the hair and sweat - bearing skin of the axilla . 
lastly , let us banish for all time the use of tension sutures , and above all the expression f 10 . - the composite dra ' lring illustrates ( a ) the position of the leg for cutting an imme it is a sound precaution to towel up all " skin - short " cases with the diate skin - gra donor area prepared and &ec8111111 > le . 
functional disability usually takes the form of restricted movement at the shoulder and is doubly undesirable , since it not only seriously reduces the range of w ! efo1ness of the limb concerned , but aggravates by constant reminder the psychological trauma resulting from the operation . 
the methylcholanthrene was introduced into average age of the mice was 6 months . the prostate gland in the following manner : under general anaesthesia , a median abdominal incision was made , exposing the prostate lobes . 
8. - the same after 3 weeks ' time . 10 or more layers deep , keratin pearl formation still absent . cells acquiring more pronounced squamous metaplasia . development of stratified squamous epithelium , h . 
they varied between i to 3 of an inch in diameter and frequently more than a single prostate lobe was involved . a tumour of average size is shown in fig . 
7. received for publication february 7 , 1947 . it is a widespread doctrine among students of cancer that any tissue or organ capable of cell division may be the site of a malignant neoplasm , but it is well known to clinicians that certain tumours , theoretically possible , do not in fact occur . 
the most striking example of an organ which never gives rise to a neonotwithstanding the fact that mitoses occur in the lens plasm is the lens . throughout life , having been seen even in the lenses of patients 80 years old , no tumour of the lens has ever been discovered . the lens is an entirely epithelial structure embryologically derived from the surface ectoderm , devoid of blood supply and enclosed in a semi - permeable although mammalian lenses are enclosed membrane , the hyaline lens capsule . during embryonic life in a capillary net ( the vascular lens capsule ) this does not constitute a blood supply , since the vessels do not come in contact with the lens in the majority of vertebrates cells , being always outside the hyaline capsule . the lens can therefore be looked even this embryonic vascular capsule is absent . on as a segregated group of specialized epithelial cells growing and differentiating in a closed system , separated alike from the blood - stream and from the surface of the body . 
7. received for publication february 7 , 1947 . it is a widespread doctrine among students of cancer that any tissue or organ capable of cell division may be the site of a malignant neoplasm , but it is well known to clinicians that certain tumours , theoretically possible , do not in fact occur . 
the most striking example of an organ which never gives rise to a neonotwithstanding the fact that mitoses occur in the lens plasm is the lens . throughout life , having been seen even in the lenses of patients 80 years old , no tumour of the lens has ever been discovered . the lens is an entirely epithelial structure embryologically derived from the surface ectoderm , devoid of blood supply and enclosed in a semi - permeable although mammalian lenses are enclosed membrane , the hyaline lens capsule . during embryonic life in a capillary net ( the vascular lens capsule ) this does not constitute a blood supply , since the vessels do not come in contact with the lens in the majority of vertebrates cells , being always outside the hyaline capsule . the lens can therefore be looked even this embryonic vascular capsule is absent . on as a segregated group of specialized epithelial cells growing and differentiating in a closed system , separated alike from the blood - stream and from the surface of the body . 
by carefill dissection of the eyes it is possible to get the lenses in their hyaline capsules completely free from vascular capsule or uveal tissue . these lenses were inserted under the skin of the flank of mice of the same inbred strain together with methylcholanthrene . in some cases the hyaline capsules were ruptured and the lenses mixed with a few crystals of methylcholanin others the unruptured lenses coated with a solution of the threne . in others again teased lenses were mixed carcinogen in oil were used . with methylcholanthrene in solution in equal parts of soft and liquid the exact method employed did not appear to be important , paraffin . two of the tumours being obtained with a solution of 1 mg . 
the tumours appeared between two and three months after implantation . characteristics of tumrnours of lens epithelium . the tumours are anaplastic carcinomas possessing certain characteristics suggestive of their origin from the subcapsular epithelium of the lens . 
a brief consideration of the normal structure and behaviour of this in mice is a necessary preliminary to the study of the tumours . the normal lens and its epithelium is seen in fig . 
the first stage of this differentiation is the lengthening of the cell and the displacement of the once this has occurred the cell cannot divide again , but nucleus to one end . proceeds to complete differentiation into a lens fibre , the nucleus of which evenfig . 
4 and 5 show results at three and at two days . thle ruptured capsule is in culture of the whole lens of a late mouse embryo . the centre of the explant and is surrounded by degenerating lens fibres . 
the cells are large , with voluminous spherical or oval nuclei , showing one or more nucleoli and a prominent reticular chromatin in the young cells the nucleus is central , in structure . the older cells it moves to the edge of the cell just before becoming pyknotic . metastases occurring in the inguinal lymph nodes show the same structure , but with less necrosis . 
6 and 7 is apparent . extending edge showed fibrillar outgrowths somewhat similar to those described this by kirby in tissue cultures of chick lenses . fixed and stained preparation of a 5 - day tissue culture of the same tumour also these are seen in fig . 
 the compilation of this paper was prompted by the idea that some investi gators of cancer as it occurs in man do not know the valuable material which is contained in the publications of the general register office , while even those who are acquainted with this literature may find some use for a list of the collec tions of data about cancer which it contains . 
 the material dealt with in these publications consists of the death certificates from england and wales , considered in association with the data on population , civil state , occupation and locality obtained at the census of 1911 , 1921 and 1931 , together with population estimates for intervening and subsequent years . 
the certificate does not state , and is not intended to state , how long the deceased had followed the occupation named , and had lived at the home address given ; this information could be obtained only by inquiry into individual cases . 
the sources of such errors have been discussed in two earlier papers ( henry , kenna way and kenna way , 1931 ; ~eimaway and kennaway , 1936 )  . 
 the _decennial supplements for 1921 and 1931 contain data collected at the census of those years , and were published in 1927 and 1938 respectively ( registrar general , 1927 , 1938 )  . 
_they contain a large amount of tabulated material for periods adjacent to the census , namely 1921 - 23 and 1930 - 32 ; the scope of those tables , in so far as they are concerned with cancer , is summarized in tables i and ii . 
the 240 , 726 deaths of all married women during 1930 - 32 were classified according to the husband 's occupation as stated on the death certificate , while 19 , 422 single women who died during the same period were classified according to their own occupations . 
the reasons for the association of the married woman with the occupation of her husband are that ( 1 ) " only about 10 per cent of married women were recorded as gainfully occupied at the census of 1931 , " and ( 2 ) some indication is obtained of the social , or occupational , nature of the mortality in the husbands ' occupation , "  . 
table ix shows a selection from the supplements for 1921 and 1931 of some very interesting - data for the incidence of cancer of various organs upon men and married women of the five social classes , and in the case of the men two periods ( 1921 - 23 and 1930 - 32 ) can be compared . 
a recent paper by swan ston ( 1950 ) on " the iron and steel industry " shows the use which can be made of the material in the decennial supplements . 
 the registrar - general publishes annually a " statistical review of england and wales for the year  . , " normally in two parts , namely , ( 1 ) text , ( 2 ) tables . 
the " text " now contains a section upon cancer consisting of a commentary upon the data which have been obtained during the year in question , and certain tables . 
this table provides material in answer to the question so often asked - " is cancer increasing ? " the comparative mortality index , which takes account of changes in the sex and age structure of the population , shows that the total mortality from cancer in england and wales has barely increased during the past twenty years . 
the distinction between the entry of " carcinoma " and " cancer " upon death certificates is probably of no value , but the separation of the two at any rate absolves the general register office from any responsibility for the distinction ( table lxxvi )  . 
 ( e ) by regions ( england and wales , greater london , rest of south - east , north , midland , east , south - west , wales ) , and by class of area ( county boroughs , other urban districts , rural districts ) ( table lxxvii )  . 
 such data for the total incidence of cancer must , of couj1se , be recorded , calculated and examined , but they cannot show the peculiar incidence of cancer upon the various organs , and in the two sexes , and in persons living in different areas , and various interesting changes may cancel one another and hence be lost in the totals . 
the great interest of these data is best made plain here by reproduction in table iii of a part of an example ( table lxxviiia , 1940 - 42 )  . 
 all sites lips tongue mouth tonsil pharynx jaw others total oesophagus stomach duodenum other small intestine caecum hepatic flexure , splenic flexure sigmoid flexure large intestine ( colon ) other and undefined intestine rectum ( not anus ) liver gall bladder and ducts pancreas peritoneum , mesentery , others other and unspecified digestive organs 89 , 902 606 2 , 254 703 534 1 , 008 748 529 6 , 382 4 , 498 20 , 778 129 155 860 418 2 , 157 8 , 801 228 10 , 365 2 , 581 703 3 , 267 4 , 053 147 232 199 250 females . 
 other female genital organs total total total total total breast scrotum prostate testis penis others kidney bladder , urethra , ureter : 163 155 6 , 055 339 457 7 , 009 409 3 , 360 3 , 769 147 174 606 623 593 667 219 312 755 2 , 877 202 3 , 834 5 , 482 721 6 , 416 12 , 619 4 , 952 1 , 281 6 , 248 20 , 049 107 184 173 183 107 265 386 162 510 705 499 550 1 , 114 312 1 , 557 1 , 869 452 458 185 the material contained in this table is very significant . 
the cancer problem in its simplest form is reduced to the question , why does a cell divide ? but no single universal " cause of cancer , " such as is announced from time to time , could , even if it were actually discovered , explain all the features of this table . 
the nature of cancer proceeds by means of the most refined cytological methods , there is scope for investigation of factors occurring in everyday life which may explain the 1rery peculiar sexand organ distribution of cancer . 
 breast total cancers of female reproduc tive organs other organs total cancers in females males exce811 in females 18 , 867 20 , 049 38 , 916 58 , 090 97 , 006 89 , 902 7 , 104 164 e . 
kennaway for instance ( table iv ) , the total deaths from carcinoma in males ( 89 , 902 ) and females ( 97 , 006 ) show a difference of only about 7 per cent , yet the latter total includes 38 , 916 deaths , or 40 .per cent of the whole , from carcinoma of organs peculiar to the female , among which organs the female breast , being capable o , f lactation , must for the present purpose be reckoned . 
this calculation illustrates the phenomenon of the " amount " of cancer in man , to which attention was drawn in an earlier paper ( kennaway and kennaway , 1937 )  . 
no better example has been given by later workers than that recorded by the dutch investigators snijders and straub ( 1924 ) in sumatra , who first described this numerical relation of total cancers in populations differing in habitat or in sex ( table v )  . 
 28 27 these figures show that in sumatran and european populations , in which the total incidence of cancer is about the same , the proportion of one form of cancer , in this case that of the liver , may be very different . 
 " there appears to be a general law that when in a given population the incidence of cancer in one particular organ is markedly increased as compared with another population , there is then a compensating decrease in the incidence of cancer in a number of the other organs " ( cramer , 1936 )  . 
 ( b ) the data shown in table lxxviiia and bare subdivided under 15 quin quennial or decennial age - groups from 0 - 5 to 85years ( tables lxxixa , b , e , d , e , f )  . 
thus in men aged 55 - 64 the mortality from cancer of the lip has fallen to 26 per cent of the initial figure , while that attributed to cancer of the lung has increased by more than 17 times . 
 1911 - 20 1921 - 30 1931 - 35 1936 - - 39 1940 - 44 1945 1911 - 20 1921 - 30 1931 - 35 1936 - - 39 1940 - 44 1945 1911 - 20 1921 - 30 1931 - 35 1936 - - 39 1940 - 44 1945 the exact comparison of the prevalence of cancer , or of any form of cancer , at different periods , requires standardized death rates owing to the continual increase in the numbers of older persons , who are of " cancer age . " such data are given at intervals in the statistical reviews ( text ) , and most recently in table lix in the review for 1938 and 1939 ( registrar - general , statistical review ) , which records the standardized rates per million population for cancer of 27 sites in men and women separately in the periods 1901 - 10 , 1911 - 20 , 19.21 - 30 , 1931 - 35 , 1936 , 1937 , 1938 and 1939 . 
these figures for each one of the four most recent years are of especial interest because they show the present state of increase or decrease of the various forms of cancer . 
 in the table in question the great majority of the 52 sex - site combinations show a decrease , or are stationary ; the only quite definite increase is in cahcer of the lung and bronchus in both sexes . 
since 1942 standardized death rates have been superseded by comparative mortality indices , which compare the death rate in each year with that of 1938 , after making allowance for changes in the proportion of the population at different ages . 
for a number of sites of cancer since 1933 have been published in the medical tables volume of the statistical review ( table viii ) for each year since 1942 . 
 ( d ) the deaths from cancer of certain sites ( mouth and oesophagus , stomach and duodenum , respiratory system , breast , uterus , other sites ) per 1000 for all sites in men and women at certain ages , in certain regions and classes of areas named above ( table lxxvii ) during 1940 - - : - 42 and 1943 - 45 ( table lxxxi ) are set forth . 
 the cancers of the respiratory system show the greater liability of urban populations ( stocks , 1936 ) ; this portion of the tabl~ is of such interest that it may be reproduced in extenso here ( table vii , section a )  . 
if one recalculates the figures for the various districts on the basis that " rural districts " = 100 , whereby the data for the two periods 1940 - 42 and 1943 - 45 become more comparable , one obtains the figures shown in section b of the above table , which show a very close similarity between the two series for women , with the single exception of the figures for the south - west region . 
of course this result has no bearing on the accuracy of death certificates , but it does show that two sets of such certificates , gathered in a number of districts during two consecutive periods , give results which have a considerable uniformity . 
 when the mortality from any form of cancer , such as that of the lung , is higher in urban than in rural districts , one must consider whether the difference is due , not to the conditions of life in towns , but to better facilities for diagnosis , and a further extract from table lxxxi is of interest in this respect ( table viii )  . 
 males , 45 - 65 mouth and oesophagus , 1940 - 42 1943 - 45 stomach and duodenum 1940 - 42 1943 - 45 males , all agesrespiratory system females , 45 - 65 breast uterus 1940 - 42 1943 - 45 1940 - 42 1943 - 45 1940 - 42 1943 - 45 238 225 156 183 236 231 165 157 205 187 216 249 261 254 150 137 100 254 235 103 126 236 244 148 144 the diagnosis of cancer of the oesophagus , stomach and duodenum , breast and uterus may be by no means easy , yet these forms of cancer do not show the difference seen in cancer of the respiratory syste this last must be made up largely of cancer of the lung , and hence these data provide further evidence of a carcinogenic factor in urban environment . 
 the annual statistical reviews do not deal with the social and occupational incidence of cancer , as the occupational distribution of the population is ascer tained only at census periods . 
 in this summary of the annual and decennial publications of the general register office in relation to cancer one cannot of course refer to the numerous passages which treat of the material tabulated . 
 the decennial supplement for 1921 ( registrar - general , 1927 ) contains the first detailed study of the social incidence of cancer upon the various organs in men , which study developed from an earlier investigation of the incidence of cancer as a whole ( stevenson , 1923 )  . 
so far as is possible from the material available , class i purports to represent the pro fessional and generally well - to - do section of the population , class iii , skilled artisans and analogous workers , and class v , labourers and other unskilled callings , while classes ii and iv are intermediate , comprising occupations of mixed types , or types not easily assignable to the classes on either side . " the parts of the body affected by cancer were divided into two classes , namely : exposed sites ( buccal cavity , pharynx , oesophagus , stomach , larynx , skin ) , and other sites . 
 the mortality from cancer of the exposed sites increases from social class i to class v , while cancer of the other sites shows no such relationship ( table ix )  . 
 the author of the decennial supplement , 1921 , comments upon these data : " it thus appears that a large proportion , at least , of cancer mortality is of a highly preventable nature , for we must suppose that if the conditions of life of all sections of society could be assimilated to those of its upper ranks , mortality from cancer of the exposed sites would fall for all classes to the class i level . 
 the paragraph made up by these two sentences should be regarded as one of the classics of cancer research , for it brings carcinogenesis in man into relation with factors in everyday life which can be investigated , while the most refined methods of physics and chemistry are applied to the problem of cancer in general . 
 the most extreme instance of a social factor in the incidence of cancer appears in the case of cancer of the scrotum ( kennaway and kennaway , 1946 ) , which could in all probability be eliminated altogether by cleanllness , as is cancer of the penis by circumcision in infancy . 
 the figures ( table ix ) for cancer of the upper alimentary canal in males show a significant reduction in the steepness of the social gradient from 58 , 80 , 99 , 102 , 140 , to 63 , 80 , 97 , 109 , 129 ten years later . 
this may be due in part to " earlier or more effective treatment than before of cancer of the more accessible sites " in men of the poorer classes , as is suggested in the text , but probably also to the improvement of social conditions in other ways . 
the change has been chiefly in the cancers of the buccal cavity ( tongue , tonsil , pharynx ) , for cancer of the oesophagus shows no , and that of the stomach very little , loss of gradient . 
 the steeply graded figures for the larynx , in contrast to those of the buccal cavity , show no change , while those for cancer of the skin show a flattening which one would associate with improved personal cleanliness . 
 figures for the socia ] distribution of cancer in women in 1921 - 23 are not available , but the comparisons of those for men , and women , in 1930 - 32 are 170 e . 
 " of gastric cancer mortality can have little to do with the effects of occupation , and must arise from factors of selection , economics or environment which affect the wives of the men in occupational groups as much as they affect the men themselves . " cancer of the skin also affects men , and married women , of the the employment of a different social classes in much the same way ; "  . 
 proportion , largest no doubt in classes iv and v of married women in textile occupations , may be partly responsible for the mortality gradient for skin cancer . " no attempt is made here to deal with all the indications for inquiry which may be drawn from this table ; the subject is dealt with , and illustrated by graphs , in pp . 
cancers of the buccal cavity , and of the larynx , show a high incidence upon occupations associated with alcohol ( kennaway and kennaway , 1936 ; wassink , 1930 ; registrar - general , 1927 , appendix d ) ; the change in gradient in the former only might suggest that this is due to another factor , namely , better oral hygiene . 
 the lack of influence of social class upon the liability to cancer of the lung , together with the very considerable effect of urban conditions ( stocks , 1936 ; registrar - general , 1947 , table vii ) , suggasts some carcinogenic factor to which all classes are exposed ; owing to the mixing action of the wind there is less difference in the outdoor air breathed by different classes than in other social conditions such 1 , 1 , s food and cleanliness ( kennaway and kennawa.y , 1947 ) , and all classes seem to share in the increased use of tobacco , largely in the form of cigarettes . 
 a good example of the unique value of such studies of social distribution is given by cancer of the uterus , which was examined in this way for the first time in 1930 - 32 . 
thus , at ages 35 - 65 the rate in married women of class v is twice that of class i , and single women in classes iv - v show a mortality 44 per cent greater than that of classes i and ii . 
 thus in 1921 the ratio of registered to 100 calculated births for married males in the five social classes was 85 , 85 , 97 , 109 , 128 , and the social distribution of uterine cancer in married women might be thought to be due to the difference in fertility . 
 the data on the mortality from cancer in the last twenty years should yield very valuable information when correlated with the results of the census in 1951 , the first made since 1931 , in view of the considerable social changes which have taken place . 
 ( 1 ) the british empire cancer campaign has published in its 13th annual report a very elaborate study by stocks ( 1936 ) , il1ustrated by 17 maps , of the distribution of cancer of eight sites ( oesophagus ; stomach ; intestines ; rectum ; liver , gall - bladder and pancreas ; skin ; lung ; breast ) in both sexes at various ages in the counties and county boroughs of england and wales . 
the 14th report of the campaign records an extension of this study ( stocks , 1937 ) , with 7 maps , to cancer of other sites ( tongue , mouth , jaw , larynx , bladder , prostate ) , and a final section ( stocks , 1939 ) , with 9 maps , deals with cancer of the uterus , vagina , ovaries and fallopian tubes , and of the skin , lung , rectum and bones in women . 
 no such study of cancer in relation to this , or any other , geographical area has ever been made , and no summary is attempted here of the results and suggestions for research which it contains . 
 ( 2 ) the changes in mortality from cancer of various organs in england and wales between 1911 and 1944 are described in a paper by stocks and mackay ( 1946 )  . 
 ( 3 ) the general register office has published a study by stocks ( registrar general , 1947 ) of cancer of the stomach , oesophagus , lung , larynx , breast , uterus , ovary and some other sites in relation to a number of localities and environmental factors ( urban and rural districts , counties , the 13 , largest cities in england , 30 large towns , metropolitan boroughs of london , social indices , number of persons per room , amount of sunshine , source of water supply ) during one or other or both of two periods , 1921 - 30 and 1940 - 44 . 
one may mention here two results which provide valuable indications for further research , namely ( a ) an inverse relationship between number of sunshine hours and deaths from cancer of the lung , and ( b ) a high incidence of cancer of the stomach in the northern and central counties of wales , especially in the former . 
the author says : " the purpose of this report is to present the statistical facts , and point out any peculiarities in distribution and correspondences with other measurable factors which appear , so that all possible clues may be followed up by further study . 
 a list has been compiled of the types of information about cancer in man in england and wales which are to be found in the publications of the general register office . 
i. - cancer of the lung and of the larynx . males , england and wales , 1921 - 1951 . ascribed to lung cancer , for cancer of the larynx there has been little change over the past twenty years . the preceding papers in this series ( kennaway and kennaway , 1951 ; kennaway and waller , 1953 ) compared the incidence during the period 1946 - 1949 . 
kennaway cancer of the lung and larynx as shown by death certificates in four classes of area ( london , county boroughs , other urban ' districts , rural districts ) in england and wales . 
for a particular sub - division of the country expresses the mortality in that area as a percentage of that for the whole of england and wales , allowance being made for any differences in the age - distribution . 
given as an indication of the relative importance of the two causes of death ; these measures of mortality will not be used in this analysis . the most striking feature of table 11 is the behaviour of the figures for the female larynx as previously described . whereas for cancer of the lung the male and female figures show similar trends the corresponding figures for the larynx show no such similarity . 
the next step is to see whether the geographical or the urbanization factor is the more important ; this can be done by a study of table iv and fig . 
3 and table iv that urbanization is the predominating factor : in each region , the mortality due to lung cancer increases from the rural to the urban areas and although the same is true of the male larynx , when we look at the figures foi the female larynx we find the exact contrary ; in every case the highest mortahty is in the fural districts . 
the comparison is particularly striking when we look at the s.m.r.s for the county of london ( the most densely populat - ed of all our areas ) and then at those for the sparsely populated rural districts inv.i ' ales ancl the north . for the purposes of this comparison the differences between the c.m.r. 
we cannot say whether the extremely high figures for the female larynx in wales ( where the population at risk is in any case small ) are more than an extreme manifestation of the reverse urbanization gradient or whether they m , ight be due , for instance , to some ethnographical factor . further study of eancer of the lung . another feature that emerges from the diagram is , that for cancer of the lung the urbanization gradient is consistently steeper in the men thad . 
of 160 for the county of london . it would be interesting to make a similar analysis with the urban districts ( or better with the county boroughs and urban districts combined ) but the labour would be prohibitive . it may be mentioned in this connection that since 1950 the registrar general has adopted a more realistic classification , which wfll considerably simphfy the study of many problems . 
with its large dormitory areas , the male s.m.r.s for cancer of the lung are all higher than in the sparsely populated s.w. , wales ii and north il in females the absence of such a clear association is partly a reflexion of the lesser gradient already noticed for women and partly due to the larger sampling errors to which these s.m.r.s are subject ; on the other hand the remarkably low female s.m.r. 
a possible explanation is that there are two aetiological factors at work , the one positively correlated with urbanization ( a ) and the other negatively ( b )  . 
the mortahty ratio for the larynx ranged from 60 for males of class i to 143 for males of class v , but it is significant that married wo ' men showed no significant social class gradient , thus suggesting that the factors responsible for the excess of cancee of this site amongst unskflled males are directly associated with occupation . " on the other hand there was in 1931 no marked social gradient for cancer of the lung . what connection is there between the occupational factor and urbanization ? we cannot answef that question in the absence of mortality rates analysed by urbanization simultaneously with occupation . 
if the figures in table viii are representative , they may be said to show ( a ) cancer of the larynx is four times as common in men as in women ; in men , intrinsic cancers are nearly twice as common as extrinsic , whereas in women extrinsic are four times as common as intrinsic ; as a consequence of ( a ) and ( b ) , intrinsic cancers appear nearly thirteen times as often in men as in women * and the extrinsic twice as often in men as in women . the conventional inclusion of cancers of the retro - cricoid region among laryngeal tumours is very unfortunate . " the term ( cancer of the larynx ) should be restricted to the so - called ' intrinsic ' group of cancers . 
kennaway mouth , tongue , and pharynx , angular stomatitis , hypochromic anaemia , achlorhydria , splenomegaly , and spoon nails , in which carcinoma ofthe post - cricoid region this condition was first described independedtly by may develop in later years . paterson from cardiff ( 1919 ) and by brown kery from the westem infirmary , glasgow ( 1919 ) and has become know - n by the names of two latee investigators as the " plummer - vinson " syndrome . neitherpaterson ( 1919 , 1937 ) nor brown kelly ( 1919 , 1931 ) suggest , either in their earher or later papers , that this affectioin . is especiafly common in their own districts , though any such frequency would have this syndrome is of qu ' ite especial interest in cancer research assisted recognition . because the features present during the earlier years may indicate a precancerous metabolic state , which may be treated succes - sfury by improved feeding and administration of iron ( see for instance hare ' n , 1938 ; waldenstrom , 1938 , and elkelesg 1942 )  . 
the forowing section represents an attempt to collect the literature on the paterson - kery syndrome in relation to cancer ; no attempt has been made to introduce any uniformity into the anatomical classificatioin adopted by the various authors . ahlbom ( 1937 ) records , from the radiumhemmet , stockholm ( 1931 - 1936 ) , the incidence of carcinoma of the post - cricoid region . ( a ) cancers of the hypopharynx are more frequent in women , in whom also a mucb higher proportion ( 90 per cent ) of these tumours affect the post - cricoid area , than in men . 
was in der diiit der drmeren schwedischen bevblkerung fehlt , sind vor allem eisenhaltige vegetabilien , obst und fleisch . " bingham and logan ( 1953 ) say " the incidence of hypochromic anaemia in northern ireland is considerable and . 
these trends apply equally when the figures are analysed according to the separate regions , but there are differences between the regions , which may or may not be due to differences in degrees of urbanization undetected by the classiin particular the s.m.r.s for wales are very low for the fication we have used . lung and male larynx but very high for the female larynx . 
the literature upon the relation of the paterson - kelly syndrome to cancer , and upon possible dietetic and social factors in this condition , is summarized . we are greatly indebted to w . 
croton oil painted concurrently caused no increase in the incidence of the significance of the skin tumours , nor did it decrease the latent period ; absence of co - carcinogenic action of croton oil is discussed . the author is greatly indebted to p . 
kirby. from the research department , glasgow royal cancer hospital . received for publication , june 28 , 1984 . activity the carcinogenic of orally - administered 2 - acetylaminofluorene , aaf , for a number of tissues , mainly epithelial , was first demonstrated by wilson , deeds and cox ( 1941 ) for the rat , and subsequently confirmed for this species by bielschowsky ( 1944 , 1946 ) and others , for mice of various strains ( armstrong and bonser , 1944 , 1947 ; foulds , 1947 ) , for the fowl ( bielschowsky and green , 1945 ) and for the cat ( harding , quoted by bielschowsky , 1947 )  . recently wilson , deeds and cox ( 1947a ) have reported their own findings in mice of three pure strains . 
aaf has now been shown to be a carcinogen ( when given per os ) in eight strains of mice : cba , if , riii , white label , strong a , c57 black , c3h , and bagg albino . 
of the first five strains mentioned , cba were most prone to both liver and bladder tumours ; strong a were the least susceptible ( armstrong and bonser , 1947 )  . 
 ( 1941 ) were at that time unable to elicit tumours in rats by subcutaneous implantation of aaf , and , in view of the probable hydrolysis of aaf in the intestine to 2 - aminofluorene , af , they suggested that aaf might be converted to af and that the latter product was the real carcinogen . might be expected that af would be more carcinogenic for rats than aaf in however , in a recent paper , wilson , deeds and similar feeding experiments . cox ( 1947b ) report tumours to have arisen more slowly and in fewer numbers in rats given af orally than in similar rats receiving aaf in the same basal diet . they suggest that af , being more soluble than aaf , may have been absorbed more rapidly and then excreted rapidly . 
af derived from aaf by hydrolysis in the intestine could only be absorbed as fast as it was liberated . however , against this view it must be said that af , once absorbed , should be excreted as fast in either case and that tissues in remote sites will stand the same chance of receiving af , whether af or aaf be given orally , provided the presentation it seems possible that the hydrolysis takes place after absorption ; is continuous . studies on the liver after ingestion of aaf , using the extraction technique of miller and baumann ( 1945 ) and the estimation method of westfall ( 1945 ) , should yield information to test this point . it is significant that wilson , deeds and cox ( 1947a ) report tumours and other changes after implantation of crystalline aaf for long periods . bielschowsky ( 1944 ) painted af in acetone thrice weekly on the epilated contrary to expectation , no tumours at the site of painting skin of wistar rats . occurred , but all 5 rats developed liver tumour by 280 days and one had a tumour of the ductus acusticus externus . 
at first , arachis oil was used as vehicle , but later , tricaprylin became available and was used in place of arachis oil . injections were made at intervals of approximately 4 weeks , in alternate flanks ; 0 5 ml . 
a female dying at 351 days showed no abnormality , but of 7 mice dying between 530 and 594 days , all showed one or more liver tumours , some solid , some diffuse , with three cases being notably vascular . six of these seven mice were females , the other mouse being the only male to survive more than 125 days . 
one female , killed at 582 days , showed a mammary adenocarcinoma , and another , killed the same day , had a granulosa - cell tumour of the ovary . ( c ) c3h mice . - thirteen mice survived 52 to 126 days ; the rest survived 206 days or more . three females , killed at 206 , 235 and 235 days respectively , had one or two mammary adenocarcinomas . 
a fourth female was killed at 242 days without any gross tumour . three males survived 409 , 426 and 506 days respectively ; the first and the last of these showed hepatoma ; the other had primary renal carcinoma . 
ten male and two female mice survived over 500 days , four of these dying between 602 and 616 days ; extensive leukaemic infiltration was seen in two of these mice , but no liver tumours ( or tumours in other sites ) were found in any . 
kidney and bladder tumours were not found , nor were mammary tumours . ( b ) cba mice . - only cysts with minimal tissue reaction were found at the sites of injection in these mice . 
a variety of degenerative changes were found in the liver in most mice ; a male , injected eight times and dying at 285 days , had hepatoma at the edge of the left lobe . 
hepatomas were also found in three mice injected 11 times ; in two males dying at 565 and 574 days respectively , and in a female dying at 630 days . 
no tumours were found in the bladder or kidney in any of these mice . ( c ) c3h mice . - no tumours were found at the sites of injection , only minimal tissue reaction . 
one injected male of this strain had hepatomas by 242 days ; two males of the painted group had hepatomas by 409 and 506 days respectively . burns and schenken ( 1943 ) record a spontaneous primary hepatoma in a c311 male 14.5 months ( 440 days ) old . thus the mouse dying at 506 days may well have borne spontaneous tumours , and this possibility may also apply to that dying at 409 days . 
the earliest case seems to be undoubtedly induced by af which is therefore probably carcinogenic for the liver when injected subcutaneously in male c3h mice . among 12 cba mice given 2 - acetylaminofluorene ( aaf ) orally by armstrong and bonser ( 1944 ) , which survived more than 12 weeks , four showed liver tumours , but two were females dying at 63 and 65 weeks respectively and therefore liable to bear spontaneous tumnours . armstrong and bonser ( 1947 ) record three malignant hepatomas in seven male mice and five benign hepatomas in eight females given aaf orally . 
the time required for these tumours to develop is not recorded , but the incidence is well above that for spontaneous hepatoma in this species and , moreover , the latter always appear to remain benign . 
hence aaf and af both appear to be carcinogenic for the liver of cba mice ; af is slightly active also in c3h and c57 black mice . armstrong and bonser ( 1944 , 1947 ) record a high incidence of bladder tumours , wilson et al . 
in the present series no bladder tumours were found in mice injected subcutaneously with af and only in one female cba mouse , painted with af and killed at 582 thus af seems to be a bladder carcinogen when given orally to c57 black days . mice , but not when painted or injected subcutaneously . 
c3h mice appear to be unaffected in this organ . the kidney was free from tumour in all three strains after injection of af . painted mice of the c57 black and cba strains likewise had no kidney tumours , but a probable renal carcinoma was found in a painted c3h mouse , a male dying wilson et al . 
 ( 1947b ) record a carcinoma of the kidney in one of at 426 days . their c57 black mice given af orally . armstrong and bonser do not record any kidney tumour in cba mice given aaf orally . neither compound is very active for mouse kidney . armstrong and bonser ( 1947 ) record one mammary cancer in 9 females given aaf orally and surviving more than 20 weeks . 
one cba female in the present series , painted with af , showed mammary adenocarcinoma at 582 days . spontaneous mammary tumours in this strain seem not to occur and the solitary tumour found in each series referred to above is therefore considered to be significant . 
extremely active material capable of s " rdng fivsh t clours in less than two weeks has been obtained from both chemically induced and spontaneous sarcom firozen at - 790 c . 
since it involves the quesbon , first raised by ehrlich in 1907 , in connection with a possible bacterial source of cancer , of whether extreme cold can be used to destroy mammalian cells , and multaneously to preserve any posdble intraor extra - cellular agent capable of starting a t aour de novo in if the continued activity of such refiigerated tisme is cells of another host . aour produced in the due to new host is merely a transplant . if , however , it can be shown that the cers in the refrigerated tissue are dead , then the n6w tumour must be produced by an agent ( which would then be the " continuing cause " of cancer ) which is a separate .val of cells of the original anhml , then the t 260 w . 
craigie entity from the cells in which it lives , and which is capable of surviving under such transm , ission would be " cell - free , " certain conditions when they are dead . and would place any tumour so produced in the class of virus tumours , among the known virus chicken tumours , the shope papilloma of rabbits , bittner 's mammary carcinoma and lucke " s adenocarcinoma of the frog . the proof that prolongecl refrigeration kills mammalian tumour cells is difficult , and we are aware that in the past it has always been held , except by cramer ( 1930 ) , that the survival of only a few cells in the tumour mass accounts for the tumour in the new host . we have investigated the problem from a variety ofangles and have concluded , as will appear , that the cells are dead . jn the first place it must be realized that the sarcomas of mice which we have used are three well known to laboratory woikers , one a methylcholanthrene sarcoma ( c.48 in the inbred strain c57 black ) , and the other two arising as stromal transformations in transplants of sporadic mammary cancers investigated by r . 
none of these procedures affect the activity of the refrigerated tissue , which remains as great as or greater thirdly , we have made histological examinations than that of the fresh tissue . of frozen tissue implants at intervals and have examined the material itself , finding , - however , neither any evidence for survival of cells , nor conclusive proof of their total destruction , since one cannot be certain that every cell has been examined . finally , being well aware that to all these methods of approach might be opposed the objection of the remote possibility of the exceptional survival of a few cells , we have proceeded to dxy the refrigerated tissue completely to dust this is universally allowed to be lethal in vacuo at a temperature of - - 25 ' c . to both normal and tumour cells , yet we have obtained highly active dry material it therefore appears impossible to avoid from all the three tumours studied . the conclusion that from both the chemically induced and the sporadic sarcomas a cell - free agent can be recovered , which on injection into appropriate mice is capable of starting the tumour afresh . 
during this period attempts had been made again and again to propagate the tumour with cell - fi - ee filtrates and with dry tissue , and all had previously failed . ( 2 ) a sarcoma of the strain r3 . 
the sarcoma which formed from the stromal connective tissue was separated from the mamm ry cancer by ludford by early transplantations by which the slower growmg carcinoma was lost . ( 3 ) a similar sarcoma derived from the stroma of a carcinoma of the strain all three tumours are soft , rapidly growing sarcomas . 
the ' ampouilie - - s were then transferred to a storage cabinet kept at - 79 ' c . after an appropriate interv - al of hours , days or months , an ampoule was taken out , thawed to room temperature , opened , and 0 - 05 c.c. 
or less of the contents inoculated subcutaneously in the right flank of mice of the appropriate strawhen these ampoules were opened it was always noted that a physical change ha - d taken place in their contents , which had become more homogeneous , softer and slightly sticky after freezing . 
twe - four experiments were made with inoculations at varying intervals with uniform ] ' good results ( table 1 )  . it wiu be noted that in the last experiment on the time frozen makes no difference to the results . the list the material had been frozen 201 days , and was inoculated not only into its mouse of origin , but also into four other atains . 
refrigeration in van ; ow media . from experiments with mouse embryo tissue ( mann , 1949 ) we know that treatment with glucose ( without refrigeration ) reduces the activity of normal mamm lian cells , that storage in glycerol at 4 ' c . them , and that a combination of dextrose and cystein severely damages them , while fiwzing alone for one mour cells using glycerol , hour ik - ill them all . dextrose , eystein , and distifed water with and without fiwzing were therefore performed . table ii shows that these procedures , known to kill normal cells , have no effect on the activity of tumour tissue , dextrose indeed appearing definitely beneficial ( cmigie , 1949 )  . it wiff be noted that the results in table i and table 11 are very similar , and are condstent with the presence of a t nour agent other than a living mamm lian cell . 
as an example of the amount of man pulation which such t aour mince will stand we can cite the protocol of an experimentwith the c48 t ilour , carried various experiments with t 262 w . 
the mortar was then taken out of the c02 snow and solid frozen tissue and buffer pounded vigorously with the pestle , to break up as far as possible any remaining whole t thirty c.c. 
of cold distilled water was added , and when the mixture was finally hquid it was spun for 15 minutes at 10 , 000 revolutions a minute . ' the supernatant fluid was reddi - sh in colour and was of a ph 6 - 0 ; the deposit , firmly . 
of sahne ( - 85 per cent nacl in distffled water ) and the emulsion injected into six c57 mice . the mice developed ical c48 tumours , which were large on the 14th day . fibns of the emulsion were examined under the microscope and ' m none could a whole cell be found ; all that could be seen consisted of naked nuclei and separated masses of cytoplasm . experiments such as this do not appear to lend any aours started bv fi - ozen tissues are transplants , support t - o the belief that the t i.e. 
even there are even fewer apparentl - v intact bv the mincer . cells , while after treatment with glucose or with distified water profound changes appear affecting both nucleus and cytoplasa detailed studv of these changes is being made by one of us and wir be pubhshed later . a series of sections of implants of tumour tissue ( both sarcoma and carcinoma ) frozen to 79 ' c . 
by the fourth day much of this tissue is beiing absorbed and removed by the host , and on the fifth and sixth days and subsequently , the begi of a new sarcoma can be seen in the connective tissues surrounding the ne ; brotic mass . 
the appearances do not suggest survival of any of the implanted tissue , but rather a fresh sarcomatous reaction of the encapsulating in the case of frozen carcinoma also no living subcutaneous tissues of the host . tumour ceus were seen , and by the seventh day the decrotic m was encapsulated and shrunken . 
they were successful , as was also a similar experiment with the or tumour which wasaxied fresh by craigie . it has not so far been possible to obtain active dxied material from the c48 without preliminary storage at - 79 ' c . a brief account of the metbod of drying must be given , though for details of the apparatus used craigie 's paper ( 1949 ) should be consulted . 
the tissue to oule or , if freshly miinced , diluted be dried is either taken from the storage a v ' n ' th an eqltal volume of 5 - 3 per cent dextrose and is run into the drying flask . it sbould be distributed as evenly as possible over the bottom of the flask in a thin layer . 
ofm / 150 eystein ph added after cirying to zero , pumping is then continued for a further half or one hour to be absolutely certain of the removal of the last traces of wate ' r . 
of which is injected subcutaneously in each mouse . before this method of drying was evolved , many unsuccessful experiments with these tumours , using the knox method of drying , had been cam ' ed out . even now a successful res - ult - is not obtained in every case , and - much further experimentation will be required to determine the optimum conditions . 
hall. from the hugh adam cancer research department of the medical school and the new zealand branch of the british empire cancer campaign , university of otago , dunedin . received for publication may 26 , 1953 . rats numerous attempts have been made to elucidate the role of the thyroid hormone in the development of chemically induced tumours . 
the relevant literature has been reviewed by miller and baumann ( 1951 ) who studied this problem in treated with dimethyl - amino - azobenzene . these authors mention some of the experimental difficulties encountered when the metabolic rate is altered by the administration of goitrogens or of thyroxthe action of thiourea , thiouracil and related compounds , however , is not limited to the thyroid ; they can affect also other organs , as for instance the liver ( fitzhugh complications of this kind can be avoided by means of and nelson , 1948 )  . surgery if hypothyroidism is the goal of the investigation . 
the effects of partial thyroidectomy are comparable with those obtained by goitrogens in doses too small to inhibit completely thyroxine synthesis . complete elimination of thyroid hormones resulting from either the administration of large doses of goitrogens or from the removal of the gland leads to a complex endocrine imbalance , the outstanding feature of which is a pronounced disturbance of growth . the pituitaries of such rats contain no acidophils and therefore no somatotrophin is formed . 
the thyrotrophic hormone , however , is secreted in elevated amounts . we have studied the action of aminofluorene ( a.f. ) and its acetyl derivative ( a.a.f. ) on the thyroidectomized rat , choosing these carcinogens because they induce tumours in more than one organ . 
one hundred and thirty were wistar rats ( 106 males , 9 of which were castrates , and 24 females ) and 17 were males of a piebald strain . forty - nine intact rats and 9 castrates served as controls ; eighty - nine were thyroidectomized . sixty - two of the 89 ( 70 per cent ) were considered to be completely thyroidectomized because their pituitaries were virtually free of acidophils . 
with the same the intact but not the thyroidamount of carcinogen during the remainder . after the 20th week the ectomized animals consumed all the food offered . in experiment 2 the milkanimals were maintained on ordinary stock diet . flour diet was given throughout , but for the first 20 weeks the diet was adjusted in such a way that 2 mg . 
in acetone 70 applications being given to the albino and 90 to the piebald rats . in this experiment rats which were obviously not completely thyroidectomized as indicated by their growth curve were kept separately from the other animals in in experiment 4 , 9 castrated order to avoid thyroxine intake from the excreta . and 30 intact rats were treated with a.a.f. 
daily. maintained on the wet diet supplemented by grain . in the 15th week 19 of the intact animals were thyroidectomized . liberal amounts of drinking water were available to all animals . in our experience thyroidectomized rats kept on a wet diet are in a better in long - term experiments state of nutrition than when they receive dry food . care has to be taken that excessive growth of the incisors does not interfere with their food consumption . the rats were weighed once weekly and examined for the presence of tumours . they were sacrificed when a neoplasm was suspected or when their general state of health made it advisable . 
the earliest hepatoma was found in the controls in the 18th week of the experiment , multiple benign cystic cholangiomata being already present in the partially thyroidectomized males the earliest hepatoma was at the 15th . discovered in the 28th week . 
no mammary cancers were seen in the thyroidectomized females , the majority of which were in anoestrus already in the third week of the experiment when vaginal smears were taken for the first time . table ii ( experiment 3 ) shows the results obtained with a.f. 
two appeared in intact , 3 in partial and 4 in completely thyroidectomized rats . in addition , one adenoma of the lung appeared in the latter group , and a cancer of the breast in the controls . in experiment 4 thyroidectomy was performed after a.a.f. 
had been fed all the controls developed hepatomas , the first being for a period of 13 weeks . found 7 weeks after the withdrawal of the carcinogen . thyroidectomy performed in the 15th week of the experiment was without the slightest influence on the development of these neoplasms , the first hepatoma appearing already in the 19th week . 
when at the 38th week the experiment was terminated each surviving animal was found to have hepatomatous and cholangiomatous lesions . these results are summarized in table iii , which includes also an additional table iii . - tumours induced by a.a.f. 
hall reduced , the glands containing little fluid whereas the weight of the testes was well maintained . in such animals an atrophy of the interstitial cells of the testis was found histologically , indicating inadequate stimulation by interstitial cell - stimulating hormone ( i.c.s.h. ) , and subsequent failure to produce androgens in amounts sufficient for the maintenance of the accessory sex organs . in this connection it might be remembered that none of the completely thyroidectomized females had a regular oestrous cycle . the adrenals did not show uniform changes . the width of the cortex tended to be less than normally ; the sudanophobic zone , present in intact males , was frequently absent , and the amount of lipoid stainable with oil red 0 was reduced considerably in some but little in other glands . 
some of the thyroidectomized animals were rather obese at the time they were sacrificed , which made correlation between organ and body weight unreliable . as in an earlier experiment ( bielschowsky , 1949 ) , neoplastic changes were found in the thyroid tissue of a.a.f. - treated animals in which a small fraction of the gland had escaped removal . in one instance a highly anaplastic tumour arising from such a thyroid rest was found at autopsy - worth mentioning because a tumour - cell embolus was seen in the periphery of this neoplasm , the only case in which evidence suggestive of malignancy was found in such a thyroid tumour . in the pituitary of this male wistar rat an adenoma was found . the tumour , the only one seen in these experiments , had the same morphology as the basophil adenomata present in aged rats which had received an iodinedeficient diet ( bielschowsky , 1953 )  . all the other pituitaries showed the typical picture of total or partial thyroidectomy , i.e. , absence of acidophils or marked reduction in their numbers with evidence of degranulation coupled with the appearance of " thyroidectomy ' " cells . the retro - bulbar tumours . the appearance of carcinomata in the orbital tissue of two rats of experiment 1 , a type of neoplasm only found in the thyroidectomized groups , led to a systematic search for lesions in the lacrymal glands . 
6 shows that they vary considerably in size and shape and have lost the pale foamy appearance of the normal glandular epithelium . their nuclei are hyperchromatic and dividing cells are present . since these changes were obviously of a neoplastic nature it seems justified to consider the retro - bulbar tumours as carcinomata arising from the epithelium of the lacrymal gland . in intact rats the lacrymal glands are of a brownish colour ; in many of the thyroidectomized animals they were more whitish and appeared also to be more moist than normally . sometimes these changes were bilateral , but not infrequently only one side was affected . histologically in thyroxine - deficient rats , treated with a.f. 
whether the absence of thyroxine is the main factor responsible for the results obtained so far we can only state that thyroidectomy prior remains to be investigated . to the administration of the fluorene derivatives renders the livers of rats unsusceptible to the carcinogenic action without preventing the induction of neothyroidectomy performed after the administration of plasms in other organs . effective amounts of these compounds did not retard the development of malignant it seems therefore that it is the early stage in carcinogenesis liver tumours . preliminary results of experiwhich is influenced by the removal of the thyroid . ments yet to be terminated point into the same direction . bielschowsky and hall , 1952 ; during 1952 evidence was obtained by three independent groups of workers griffin , ( moon , simpson and evans , 1952 ; rinfret and corsigilia , 1953 ) that the action of highly potent carcinogens can be " inhibited " by the removal of the pituitary or of the thyroid . 
the growth and development of most organs , with the possible exception of the brain , is dependent on hormonal stimulation , but there exist considerable differences in degree , as shown by the effects of hypophysectomy . 
the gonads , thyroid and adrenals atrophy always , but if the hypophysectomized rat receives adequate amounts of food the growth of the liver is proportional to body growth . thus neither liver nor the subcutaneous tissue can be compared with the target organs of specific trophic hormones , although crude pituitary extracts can induce disproportionate growth in the liver ( selye , 1949 ) and in acromegalics the soft tissue can increase in size . the role of the somatotrophic or growth hormone in normal or pathological growth is only imperfectly understood . 
growth can be promoted in hypophysectomized rats not only by somatotrophin but also by thyroxine , as shown by geschwind and li ( 1952 ) , or by insulin ( best , 1952 )  . 
a great variety of neoplasms has been obtained in intact but not in hypophysectomized rats injected for long periods with purified growth hormone ( moon , simpson , li and evans , .oestro1950a , 1950b , 1950c , 1951 ; koneff , moon , simpson , li and evans , 1951 ) gens can retard growth by inhibiting the production of somatotrophic hormone . this does not prevent carcinogenesis . 
 ( 1950a , 1950b , 1950c , 1951 ) as well as griffin and his collaborators ( 1953 ) used carcinogens which acted on one organ only . their results could be due to a failure of the hypophysectomized rat to metabolise the compound administered into the carcinogen proper . this possibility has to be considered since the discovery by bonser , clayson , jull and pyrah ( 1952 ) that 1 - hydroxy 2 - naphthylamine is the active agent when 2 - naphthylamine is administered . however in our case , the fact that extrahepatic tumours developed while the liver , the most susceptible of all organs in the intact male rat , failed to respond to the carcinogenic action of a.f. 
and its acetyl - derivative does not favour this interpretation . some of the neoplasms found in the thyroidectomized animals are so rare in our material that we cannot be quite sure that they were due to the action of a.a.f. neither in normal nor in experimental rats of our colony have we ever observed a papilloma of the stomach arising from the glandular mucosa or a tumour of the brain , but vasquez - lopez ( 1945 ) as well as hoch - ligeti and russell ( 1950 ) have described gliomas in rats treated with a.a.f. benign tumours arising in the mucosa of the uterus are not infrequent in aged rats of however , the tumour depicted in fig . 
3 differs from all others our colony . observed by its strong resemblance to a deciduoma . in dunedin carcinomata aiising in the lacrymal glands have been found only in four a.a.f. 
no account of the state of the endocrine organs was given . in our experience a low fat content of the diet per se does not favour the development of cancers of the lacrymal glands . 
the mice which survived to tumour age , though not themselves used for experiment , were not certainly representative of the whole colony and , owing to the concentration of tumours in famihes , the inclusion or omission of a single litter could greatly alter the tumour incidence calculated on the small nurabers available . 
the " effective total " ' is the number of mice which surv - ived to the age at which the earfiest tumour was detected in any of the groups , namely i 10 days in an f4 mouse of strain br4 ( blue label )  . 
many of the mice recorded as dead without tumour succumbed to epidemic infection about a year after the beginning of the experiment . tables 1 - 111 show that the incidence of tumours in the strains br4 ( pink label ) , br4 ( blue label ) , and br6 was unequivocally " high . " the three strains were substantially alike . 
tumours developed , indiscriminately as it seemed , in 11 wild , " 11 white , " and " black " mice in the f . - f4generations and continue to develop in " white " and " black " sublines up to the f6 generation . 
the later age - incidence of tumours in the f2 females was probably attributable to the breeding history ; the f2 females , in contrast with later generations , were not subjected to forced breeding . the incidence of tumours in the br16 strain ( table iv ) so far is extremely low . 
the great majority of the mice survive and more tumours may yet develop , but the average age is already higher than th ' e average age of tumour development in the other three strains . 
tumours were ground in - a mortar with sand and saline to make suspensions which ranged in strength , in different experiments , from 13 to 20 per cent w / v . 
the debris was removed by spinning in an ordinary centrifuge , and the supematant fluid was injected , at weekly intervals , into the dorsal subcutaneous tissues , each dose being 1 - 0 c.c. sixteen mice reeeived each 4 injections of extracts of bri tumours of the first or third transplanted generations , five received 3 injections and two received 2 injections . twelve mice received each i injection of an extract of a primary spontaneous c3h tumour and 3 injections of primary spontaneous r3 tumours , and seven mice received 3 injections of the r3 extracts only . all the extracts were similarly prepared . the females were caged with males and subjected to forced breeding , litters being removed within 48 hours ( usually within 24 hours ) of birth . table v summarizes the results at 89 weeks from the beginning of the experiment . 
the " effective totals " comprise the mice which survived to the age when the first tumour was detected ( 180 days in a mouse injected with bri extract )  . 
the average number of tumours per mouse was 2 - 5 , 24 mice having from 2 to 6 t aours each . aour was 25 weeks ; the earhest the average age at the first detection of a t tumour wa - s recorded at the age of 17 weeks , and the latest at 54 weeks . the 3 mice without tumours are alive at ages of 81 , 81 and 85 weeks respecit is noteworthy that although continuously with males they have had tively . no litters for about a year ; previoxwly litters were not recorded . 
no observations were made on virgin mice . incidence of tumours in ( c57 black ? x agew - free r3 ct ) f ] l hybri& . remale mice taken at random from the laboratorystock of c57 black were mated with males : firom a colony of r3 mice free from milk agent . i am indebted for the male mice to my coreague b . 
the f , , hybrids were subjected to forced breeding throughout their reproductive lives . one hundred female hybrids were reared ; 21 died without tumours at an average age of 21 weeks and 79 are alive at an average age of 57 weeks . 
the diversity of genetic constitution in the f2 and subsequent generations did not noticeably affect the incidence of tumours which developed indiscriminately in " black , " " white " and " wild " mice 4 ' white " and " w - fld " subhnes estabhshed by and in each of the " black , ly 31 selective breeding . 
the patemal contribution was not probably genetic for , as shown in this paper and in previous reports from this laboratory ( dmochowski , 1944 , 1945 ) , the c57 black x a hybrids developed tumours abundantly when tumour agent was supphed to them artificiafly . 
the uncompleted observ - ations on hybrids having fathers belonging to a subline of r3 mice deprived of tumour agent accorded with similar observations bv anderv - ont and dinnn ( 1948a , b , c ) in showing a substantial reduction in the incidence of tumours comparison with similar hybrids whose fathers derived from a line carrying the tumour agent . 
the experiment was open to the criticism , which anderv - ont and dunn apphed to their ow - n experiment , that the agent - fiee and agentbearing malet ; were separated by several generations of inbreeding and possibly differed geneticary . 
tumour agent was not demonstrable in the hybrids or in their tumours , and the offspring of tumour - bearing mice , whether obtained by back - crossing or by brother - sister matings , rarely developed tumours . 
two litters of - ( c x c3h ) f , were exceptional in that tumours developed in sisters at unusually early ages ( 4 - 10 months )  . the backcross offspring of two tumour - bearing females of one litter all developed tumours at average ages of 7 and 9 months respectively . 
the progeny of two tumour - bearing ( c57 black y x r3 ct ) - p . , females had a high incidence of mammary tuinours ( about 70 per cent ) through at least 5 generations . 
i fowl sarcoma material which had been subjected to repeated applications of cold possessed a greater tumour - producing activity than those prepared from similar material which had not been subjected to this ' treatment ( selbie and mcintosh , in explanation of this finding selbie and mcintosh ( 1939 ) suggested 1939 )  . that the freezing broke up the tissue fragments , ' and thus allowed a greater liberation of virus than would otherwise have been possible . in the course of some recent quantitative investigations into the effects of cold and desiccation on the rous no . 
the chromatographic study of riley ( 1950 ) in which adsorption of the rous virus was shown to take place on to a column of diatomaceous earth suggested that adsorption would also take place when this virus was passed through berkefold filters which are likewise composed of diatomaceous earth . in the present work an attempt has been made to assess the decrease in the tumour - producing activity of suspensions of the rous no . 
i fowl sarcoma virus experiments are also reported which which berkefeld filtration brings about . were designed to show whether or not this decrease was due to adsorption of the virus on to the berkefeld candle during filtration , and in addition , an investigation has been carried out to find whether repeated freezing and thawing affects the filterability of the rous virus . 
the tumour was removed , cut small with scissors , and disinte a mechanical blender ; the method ated in has been described in detail in a previous paper ( epstein and cook , 1951 )  . 
filtration was continued until the filter candle was quite empty , as shown by air bubbles being sucked through after the filtered after use each filter candle was tested by passing through it 80 c.c. 
a sample was taken from the resulting filtrate , part of it being set aside ready for inoculation and part being used to make dilutions in serial tenfold steps with sahne ; a portion of each dilution was kept for inoculation . experiment8 4 and 5 ( recovery of virus from filter )  . - these experiments were performed in their early part as a repetition of the foregoing in which samples of a virus suspension and the berkefeld filtrate derived from it were couected and diluted in serial tenfold steps with saline for inoculation . in addition , however , a sahne wash of the filter was prepared ; a sample was taken from the saline wash , part being set aside undiluted for inoculation and part being used to make dilutions in serial tenfold steps with saline . 
a portion of each dilution was kept for inoculation . experiment8 6 , 7 , 8 and 9 ( effect of freezing and thawing on filterability )  . disintegrated tumour was prepared and in ' each of these experiments was divided into two samples , one being left untreated and the other being subjected to repeated freezing and thawing . 
suspension berkefeld untreated frozen and thawed x 6 f : 61trate untreated frozenandthawed x 6 untreated frozen and thaweci x 6 - .10% virus suspension berkefeld filtrate - .10% virus suspension berkefeld filtrate 10% virus suspension berkefeld filtrate untreated frozenandthawed x 6 untreated frozenandthawed x 6 untreated frozenandthawed x 6 untreated frozenandthawed x 6 dilutions of preparation and number of tumours arising from 4 inoculatioins of each . 10 - 2 10 - 11 10 - 4 10 - 5 10 - 0 10 - 11 2 + ( 2 ) 0 3 + ( l ) filter test . - - - n prod . 
now , froni the results shown in table ii , it is clear that an appreciable amount of the tumour - producing activity losb when virus suspensions were passed through berkefeld candles could be recovered by washing saline through the latter after use . in the experiments shown in table ii the filters when tested retained in each case both chr . 
aureu8 making it impossible for the lost tumour - producing activity , recovered on washing the filters with saline , to have been due to cells . this recoverability of at least part of the tumour - producing activity lost on filtering rous virus suspensions gives further support for . 
epstein freezing and thawing were passed through a berkefeld candle to give a filtrate ' , as where those made from similar untreated disintegrated tumour were so filtered . the application of repeated freezing and thawing to rous sarcoirna material has thus been found not to increase the ability of the virus contained in it to pass berkefeld filters . 
the present work has also shown ( table 111 ) , in confirmation of that previously reported ( epstein , 1951 ) , that repeated freezing and thawing of mechanically disintegrat - ed rous tumour materizal did not release more virus on extraction than where no such treatment was applied . 
from these two facts it appears that repeated freezing and thawing has no action on the rous virus likely to make berkefeld filtrates of treated tumour material , prepared as described here , more active than those made from untreated material . 
the results shown in table iii demonstrate this clearly ; they are at variance with those reported by selbie and mcintosh ( 1939 )  . in attempting to relate the results of the present work to those of previous investigators with which they do not agree , two possible explanations require consideration : firstly , it must be remembered that selbie and mcintosh carried out their during that time the rous virus may possibly have investigations 13 years ago . undergone variations affecting its properties which might account for the variations in the experimental results obtained . secondly , the method of extracting the rous virus employed by selbie and mcintosh ( 1939 ) consisted of coarse mincing of the tumour material followed by repeated freezing and thawing was then applied to the grinding with sand . resulting preparation , and may well have contributed to the achieving of complete disintegration of the tumour and the maximum extraction of virus from it . 
in the present work , however , disintegration of the tumour material was brought about mechanicall before the cold was applied , and would seem to have been so complete that the repeated freezing and thawing could not have any further this is considered to be the most likely explanation of the contradiction effect . between the findings reported by selbie and mcintosh ( 1939 ) and those recorded in this communication . in consequence of the results of the present work it is thought that where in the future attempts are to be made to demonstrate the presence of viruses in tumours not hitherto known to possess them , borkefeld filtration , with or without previous repeated freezing and thawing of the material to be filtered , should be avoided . 
i fowl sarcoma virus are described . also described are experiments in which the effect of repeated freezing and thawing on the filterabihty of this virus has been investigated . susceptible inbred brown leghorn fowl have been used . the method used to disintegrate rous sarcoma tissue mechanically in order to make from it a virus suspension is set forth . 
the technique used to apply repeated freezing and thawing to disintegrated rous tumour is indicated , as is the technique used in berkefeld filtrat - ion of suspensions of the virus . a titration method has been employed in which preparations whose tumourproducing activities were to be compared have been diluted in serial tenfold steps with saline and inoculations made with each dilution . the results show that wben rous virus suspensions prepared by extracting disintegrated rous tumour with saline were passed through berkefeld filters , part of the virus became adsorbed on to the filter . 
1. received for publication march 25 , 1947 . tins investigation was undertaken to bring up to date the records of the middlesex hospital , and to attempt to discover whether there was any statistical evidence to support commonly held views on factors affecting prognosis and methods of treatment . 836 cases of carcinoma of the female breast treated during the years 1926 - 35 were analysed and a smaller group of 265 cases , treated during the years 1936 - 40 , were the subject of a further investigation to discover the effect of more modern methods of treatment . the hopes held at the start of this work have not been completely fulfilled . for such an investigation to give a clear cut statistical answer to all of the controversial questions , careful planning and equal distribution of cases , according to the extent of the disease and type of treatment used , would be necessary . this was not so in this series of cases . 
as a result the evidence on some points is too inconclusive to give an answer . careful analysis of results would seem to be essential if methods of treatment are to be guided by fact rather than by impressions . if , in the future , reliable figures on a large scale are to be obtained , co - ordination of policy in treatment and planning ahead in large medical formations will be necessary . whether this is acceptable or desirable may be open to argument , for it must inevitably mean some acceptance by each individual surgeon of the combined opinion of his colleagues . only thus , however , can the successful analysis of large series be substituted for the analysis of smaller series for individual surgeons . despite the difficulties that have arisen owing to the many variations in technique during the years covering this investigation , certain facts that may be of interest and value have emerged . 
some previously held views have been supported ; others appear to be incorrect . the histological findings in this series have been omitted owing to the impracticability of examining over 1000 sections in the time available . this in itself would make the basis of a separate inquiry . from the statistical point of view therefore it has been assumed that any variations in the histological grouping of the growths have been spread evenly throughout the series . the presentation of results of an investigation such as this is difficult . while the actual detailed figures and tables by which conclusions have been reached are of importance , their inclusion in the text would make reading wearisome . therefore simplified tables only have been included . * late surgical registrar ( demobilized )  . 
a case dying of other causes or of unknown causes cannot really be called a success or a failure . two ways of expressing the result seem to be possible - the survival rate or the mortality rate . 
the former has the advantage that it allows easier comparison with previous series that are almost universally expressed in terms of survival it would appear , however , that results expressed in terms of ( or cure ) rates . those actually dying of carcinoma of the breast will give a more accurate picture of effects of various factors influencing the prognosis . for these reasons the " mortality rate " has been used as a routine in this it has been expressed as the percentage of those cases dead from carcinoma paper . of the breast of all traced cases . 
when both ulceration and axillary gland involvement are present as in stage iiib cases the outlook is gloomy in the extreme . the survival rate for stage i cases is 51 - 4 per cent . this appears poor when compared with other series , but it should be remembered that this figure includes only those cases alive . 
any case dying of unknown cause or other cause in this method of calculating the survival rate would be counted as a failure . if such cases are excluded from the total the figure would be very close to 60 per cent . a point that is striking is the number of cases that die from a carcinoma of the breast during the 5 - 10 - year period . there is a consistent increase in mortality rates . 
a further subdivision was made of cases with slight attachment into those with attachment to the skin and those with attachment to deeper tissues . the mortality rate for these sub - groups was : deep attachment skin attachment percentage ~~~~~stage . 
when a growth lay between two quadrants it was described as lying in a " mid " position . thus there were the " mid - medial , " the " mid - inferior , " the " mid - lateral " and thbe " mid - superior " positions . 
the average age for stage i growths was 55 years , for stage ii growths 54 years , and for stage iii growths 59 years . the incidence of growths in decades was : decade . 0 - 39 40 - 49 50 - 59 60 - 69 70 - 79 80 - 89  . 
when associated with surgery the radiotherapy was either the implantation of radium into the wound at the time of operation according to the method described by sampson handley ( 1922 ) or prophylactic deep x - ray therapy . the latter was given in multiple intermittent sub - erythemal doses with medium voltage . 
as would be expected , this interval was increased with the stages of the disease . thus stage i cases had an average delay of 7 months , stage ii cases of 10 months and stage iii cases of 18 months . one can therefore reason that an average case has between 7 and 10 months ' unforgrace before the catastrophe of axillary gland involvement takes place . tunately there is too much variance in individual growths for this figure to be of anly real value . 
they should probably be called " insufficiently treated , " but have those under the heading " breast , been included in this analysis for convenience . contralateral " are those in which the evidence strongly suggests a true recurrence rather than a second primary . the low incidence of mediastinal recurrences is probably due to the fact that practitioners were likely to call any intrathoracic recurrences " pulmonary metathese figures , however , do not lend support to rowlands and turner stasis . " ( 1936 ) , who state that the mediastinum is the commonest site for a recurrence . this investigation has cleared up one point that has been debated in the past . there is a direct path of spread from the breast to the supraclavicular glands . of 91 cases that developed recurrences in those glands 17 were stage i cases , treated by radical mastectomy , and with axillary glands proved to be free from in view of the fact that the axillary glands were removed growth histologically . at operation and that path of spread permanently closed , the subsequent invasion of the supraclavicular glands must have been through a more direct path . relation of site of primary growth to site of recurrence . some quadrants of the breast have been considered to be more likely to metathus , inner quadrant growths have been held to give stasize in certain areas . secondaries in the chest more frequently . 
the ten to fifteen year period . no surgeon who has followed cases of carcinoma of the breast can have failed to see cases of recurrence occurring at long intervals after operation . these are often regarded as interesting rarities . 
the actual number that do develop these late recurrences is small owing to the cases that die early , and this has given the false impression that survival to five years means that the chance of recurrence is negligible . 
1. received for publication january 26 , 1949 . thie application of the methods of urinary cholesterol estimation given by burchell and maclagan ( 1949 ) to cancer and control cases will now be described . bmaterial urine specimens were obtained from 13 normal subjects and 116 patients admitted to westminster hospital or to westminster hospital ( .all saints ' ) twenty - five of these patients were suffering from diseases urological centre . thought to be unlikely to affect cholesterol excretion as shown in the tables , and 73 were suffering from various forms of cancer . 
sample of the mixed specimen , or a smaller aliquot in the case of a concentrated urine , was centrifuged , the deposit washed once with normal saline and extracted directly with three portions of boiling acetone . 
the acetone extract was evaporated to dryness , and the residue extracted with petroleum ether for the determination of cholesterol as above . the cholesterol content of the supernatant urine ( su ) was determined on 200 ml . 
the sex difference is mainly due to the larger number of epithelial cells and leucocytes in urinary deposits froimw omen . it appears likely from these figures that the total urinarcholesterol would not exceed 4 - 49 mg . 
maclagan it is evident from this that if albuminuria is present from whatever cause , the su may be expected to show an increase in cholesterol content , and such a rise is unlikely to be specifically related to cancer . 
of urine with phenol reagent as described by burchell and maclagan ( 1949 ) , where reasons are given for assuming that this method does , in fact , estimate a proteose - like substance . it will be seen from fig . 
the ud cholesterol is associated with the cells of the deposit and shows a marked sex difference , being much higher in women than in it appears to have no direct relation to cancer . 
the su cholesterol on men . the other hand is associated with a urinary proteose fraction , or with heatraised su cholesterol excretion in the coagulable protein when present . absence of albuminuria did occur in a small proportion of cases of cancer ( 5 out this gives a of 51 cases ) , although the increase was slight in 3 of these cases . total incidence of hypercholesteroluria in cancer of 9 - 8 per cent , which is somewhat lower than that reported by other workers . 
thus sobotka , bloch and rosenbloom ( 1940 ) found 20 - 6 per cent , and bruger and ehrlich ( 1943 ) 34 - 3 per cent of positive results . 
for example , bruger and ehrlich ( 1943 ) reported some rise of total urinary cholesterol in 11 out of 32 cases of cancer , but 5 of these had either albuminuria or excess of cells in the deposit or both , findings which , according to our experience , might have been sufficient to account for the hypercholesteroluria even in the absence of cancer . 
the exclusion of these cases would bring the incidence of positive results down to the figure of 22 - 2 per cent . , which does not differ significantly from our own finding of 9 - 8 per cent ( difference 12 - 4 , standard error of difference 8 - 2 )  . we must conclude , therefore , that hypercholesteroluria , while frequently due to associated albuminuria or pyuria , does occur in a small proportion of in such cases it is the supercases of cancer in the absence of these factors . natant urine cholesterol which is increased . 
the incidence is not high enough to make the estimation of any diagnostic value , but it is of theoretical interest , and presumably depends upon increased tissue breakdown as suggested by previous workers . it should be noted that the colorimetric method of estimation used is not entirely specific for cholesterol , and the possibility that other steroids might have given rise to the few positive results must be admitted . however , the earlier work of bloch and sobotka ( 1938 ) on pooled 100 litre specimens of urine suggests that the results probably were due to increase of cholesterol itself . the question of urinary proteose is of some interest in view of the work of winzler and burk ( 1943 ) on blood proteose and cancer in rats . 
bartholomew 's hospital , london , e.c.i. received for publication august 25 , 1949 . the question , whether the recent increase in deaths attributed to cancer of the lung is due to a real increase in the incidence of this disease , has been discussed the present paper , which brings forward no new data , is an by many writers . examination of some of the evidence offered on this subject . ( 1 ) the application of statistics from hospitals . the problem of finding the best index of a real increase in the incidence of lung cancer from hospital statistics is a difficult one . the relevant facts usually ascertainable at a hospital are : the annual total 1 . 
3 , 6 and 9 could be used as a measure 3 and 6 are open to objection on the grounds of the incidence of lung cancer . of possibly mistaken diagnosis , hence presumably 9 is the most accurate measure of what it is stated to be . as a measure of the population at risk , i.e. 
of becoming a case on which a post - mortem for cancer of the lung is done at a given hospital , we could use 1 , 2 , 4 , 5 , 7 or 8 . do these measures change in their quality of being representative of the the answer is that they all do so , because the cross - section general population ? of the population being admitted to hospital , whether of all cases or of cancer alone , changes , and therefore the cross - section of all deaths represented by deaths in hospital changes also . 
the assertion that the cross - section changes is based on knowledge that many people are now admitted to hospital who never would have been so admitted in the past - e.g. 
the same argument , that the cases would have been sufficiently serious to be admitted under any circumstances , could be applied , though with less certainty , to 2 , 4 , 5 , 7 , 8 . 
kennaway pathologists who deny that there is any satisfactory evidence of the suspicion is that where the incidence of lung cancer during recent years . such increase has appeared to have been conspicuous , there was formerly a low standard of accuracy of pathological diagnosis , and that the standard has improved with the passage of time . " real increase elusive disease has estimates of incidence , " for the foregoing reasons , comparisons of early and recent clinical or necropsy comparisons of the findings in different countries or in different hospitals , must be quite unreliable . so much depends on the personal experience of the clinicians and pathologists concerned , and current jotmrnals contain evidence enough that a uniformly high standard of diagnosis of this yet been attained by either . 
3 shows no steady rise , " which is perfectly true , and they conclude : " in our opinion the figures given illustrate that even a pronounced increase of the crude mortality rate for lung cancer among males , and almost only among males , does not necessarily mean an increase in incidence of that disease . 
presumably the more frequent detection of the disease is due to improvement of diagnostic procedures , and increased attention , and similar explanations may be correct in the case of occupations with an increased mortality from lung cancer . " one has difficulty in believing that any generalizations about cancer of the lung can be based upon such a peculiar succession of such small totals , drawn from a highly selected population , as those given in the third columnu of the table , namely 7 , 5 , 5 , 7 , 6 , 20 , 13 , 20 , 13 , 14 cases of pulmonary cancer . is one to believe that the totals 6 , 20 , 13 in the three consecutive years 1940 , 1941 and 1942 correspond to any similar annual incidence upon the whole population of denmark ? if there is no such parallelism , what is the value of these figures , and how can they provide a basis for the important generalization quoted above ? one may doubt whether the radiographic examination of any population for pulmonaty tuberculosis adds much to our knowledge of the total incidence of cancer of the lung . such a survey will , of course , reveal a number of cases , previously undetected , of such cancer . 
blackburn. from the department of haematology , the royal infirmary and ho8pital , sheffield . received for publication april 3 , 1954 . the forowing considerations led us to carry out a trial of stilboestrol in acute it is well know - n that the endocrine organs play an important part leukaemia . in the regulation of haemopoiesis , and many workers have reported the beneficial unfortunately , effects of acth and cortisone in some cases of this disease . however , remissions so induced are usuary only of short duration . re - treatment may again have a beneficl ' al effect , but eventually a refractory state is reached . on the other hand , " myelokentric " and " lymphokentric " acids , steroid - like principles extracted from urine ( afiller and turner , 1943 ) and from serum ( foster and miller , 1950 ) cause leukaemoid effects of myeloid and of lymphatic type furthermore , myelokentric respectively when injected into normal animals . acid has induced partial remissions in human cases of lymphoblastic leukaemia in the hands of miller , herbutt and jones ( 1947 )  . 
hence hormonal imbalance may play a part in the aetiology of leukaemia . finkelstein , gordon and charipper ( 1944 ) demonstrated that oestrogens have a depressant effect on the bone - marrow in certain conditions , but they did not study leukaemic states . burrows and horning ( 1952 ) have shown that not only are oestrogens careinoaenic , but they may be careinostatic , often restraining and causing regression of some human neoplasms such as carcinomata of the breast and prostate . 
they also state that the liabihty of mice to leucosis can be increased by oestrogens and reduced by androgens . furth ( 1952 ) , however , reminds us that in most strains of mice loukaemia is more common in the female , but in man it is more it is possible , therefore , that in human leukaemia oestrogens common in males . may have the opposite effect to that in niiee . dausset and schwarzman ( 1951 ) reported a study of 212 cases of acute leukaemia witb , regard to the influence of age and sex upon the frequency and cytological types . 
they also reviewed other series in the literature from these points of view , including both acute and chronic leukaemia . these authors state that between 18 and 50 years in both males and females there is an overwhelrning preponderance of myeloid leukaemia , while under the age of 18 and over the age of 50 lymphoid leukaemia occurs almost exclusively . 
as the former occurs most commonly during the period of maximal procreative activity , they suggest that there may be an endocrine effect on the lymphatic - mye - loid equilibrium , especially with regard to the sex hormones . stilb ' oestrol appears to have had a beneficial effect in single cases of chronic myeloid leukaemia ( loeper , lesourd and sterboul , 1947 ) and of chronic lymphatic leukaemia ( lemaire , loeper , housset and koupernik , 1947 )  . 
on admission to hospital in sheffield he was given 3 pints of packed cells and stilboestrol and penicillin therapy were begun . this was followed by an excellent clinical remission for 6 weeks , with complete disappearance of clinical splenomegaly and hepatomegaly , the latter organs having been palpable 2 below the respective costal margins at the commencement of treatment . 
no complete remissions occurred , but 3 patients had transient partial remissions . there is no evidence from this series to suggest that either the prognosis or the clinical course of acute leukaemia is altered by adding stilboestrol to other therapy . i am indebted to my medical and surgical colleagues in sheffield , to r . 
 data have been published which indicate that the average latent period for all methylcholanthrene - induced fibrosarcomas ( strong , 1948 ) and the survival time ( strong , 1950 ) of mice developing such tumours are both influenced by litter seriation . 
the latent period ( time between the subcutaneous injection of the carcinogen and the initiation or the initial growth of the ensuing fibrosarcoma ) was variouslv affected in the different strains bv the litter to which the mouse in mice of some strains ( prunt and f : of c67 x brs ) the latent period belonged . 
 ( strong , 1948 ) for the appearance of fibrosarcomas decreased in the successirn litters of the same pair of mice , while in other strains susceptibility to fibro sarcomas was relatively constant . 
the sunival time ( strong , 1950 ) of mice growing a chemically - induced fibrosarcoma ( time between the initial growth of the malig nancy and the death of the individual ) is also affected by litter seriation . 
 ' \\nen a mouse developed a fibrosarcoma in less than 100 days following the subcutaneous injection of methylcholanthrene it sunived an average of 52 days with the progressive growth of the tumour ifit belonged to a first litter . 
in the succeeding litters mice of the same age and latent i : eriod survived longer and longer with growing fibrosarcomas until , if they belonged to an eighth litter , they lived on an average of 130 days . 
these genes were derived from mice of the ancestral stocks jk and n which were used in the origin of the : xho stra the first three generations ( cba... " x jk ) were used for breeding purposes only . 
at the time of the separation of prunt from pbr descent , the average latent period for the appearance of methylcholanthrene - induced fibrosarcomas of the direct in the f 20 generation of the experimental maternal ancestry was 3695 days . 
these two untreated descents , prunt and 2prunt , should possess genetic similarity , and any biological differences that they have may be due either to divergent segregation from a residuum of genetic heterozygosity ( probably very small after 17 generations of brother - to - sistier matings ) or to new biological variability which has appeared in one or both untreated deseents following their separation from a common ancestry . 
the mice were fed a standard diet of nurishmix pellets ( pratt food company ) and water ad libitu a supplement of mixed grains ( oats , wheat and sunflower seeds ) and calf meal pellets were given to the mice once a week . 
the mice were examined periodically for tumoun ; , and the latent period taken to be the time at which a firm nodule at the site of the injection of the carcinogen began to increase progressively in si mice which developed tumours at sites other than the one at which the carcinogen had been injected were tabulated separately . 
 table i gives the percentage incidence of tumours in mice which developed in less than 100 days ( column e ) , together with the data obtained on all tumours at the site of injection of the carcinogen irrespective of latent period ( column d )  . 
 data on the total number of mice injected with methylcholanthrene ( column a ) , the number of mice which died without developing any tumour ( column b ) , and the total number of mice showing tumours irrespective of latent period and site are also given ( column c )  . 
since the two separate descents disclose similar genetic origin and show similar trends by which the females are consistently more susceptible to fibrosarcomas than the males , the data for both series may be added together . 
however , the present analysis of trends of susceptibility in s1icoessi.e litters discloses differences , and therefore the two strains , the prunt and the 2prunt , should be kept distinct . 
thus 27 2 pe : r cent of the females developed fi.brosa : rcomas within less than 100 days of latent period , whereas only 92 pe : r cent of the males developed similar tumou : rs during the same period , a difference of 180 pe : r cent in tumour susceptibility  . 
the trend for the percentage incidence of tumours developing in less than 100 days among females of successive litters was found to be y = 85 + 5 lz , with a standard deviation of the slope of 0 - 741 . 
comparison of this value with a no - trend or 0 - slope value by t test reveals that the observed trend is statistically significant ( since p = < 001 )  . 
 similarly the trend for the percentage incidence of tum.ours developing in less than 100 days among males of successive litters was found to be y = 132 - 12x , with a standard deviation of the slope c , f 0968 . 
 the analysis of the data thus discloses that there is an increasing sex differential in relation to chemically induced : fi.brosarcomas in successive litters in mice of the prunt descent . 
the difference between the mice of the two descents must , therefore , be associated with a mechanism which in the 2prunt descent changes in litter seriation at least in some strains of mice . 
 the maximal sexual differential is found in mice of the second litters ( 302 per cent ) , and this sex differential fluctuates in the succeeding litters and thus shows no significant trend . 
however , the data of the 2prunt descent are complicated by a high value for females of the second litter ( 382 per cent ) and a high value for males of the sixth litter ( 143 per cent )  . 
in one strain ( the chi ) males were more susceptible to chemically induced fibrosarcomas than were the females , whereas in the 15th inbred strain ( the cul ) the females were more susceptible to tumours than were the males . 
 the mechanism involved in the development of this new sexual differential in relation to chemically - induced fibrosarcomas in mice of the prunt descent gives a slight sex difference in mice belonging to the early litters of a breeding female , and apparently gradually increases in the succeeding litters . 
h the sex pattern of an individual is the reflection of the genetic constitution of the individua1 , then it is not clear whether mice of the early litters or mice of the later litters are to be considered the pattern for the species . 
but sex physiology is also under the influence of several hormones , and the sex pattern can be influenced significantly when a hormone is administered at an early age , preferably before birth . 
 the present investigation with methylcholanthrene has disclosed that several aspects of malignancy ( the average latent period , the survival time and the per centage susceptibility of tumours in mice of latent periods of less than 100 days ) are influenced by litter seriation . 
do hormones fluctuate in the female body with advancing litter frequency , pass the placental barrier and influence the subsequent physiology of the offspring at least in relation to malignancy and perhaps to other characteristics as well ? or are we to conclude that maturation phenomena in cytoplasm ( perhaps the mito chondria or other constituents ) are responsible for this transmission from mother to offspring which apparently is not through the genes ? perhaps another biological reason may eventually be found for the data at hand . 
these mice belonged to two separate descents derived from a common genetic orig in one descent , the prunt , there was an increasing sexual differential in relation to chemically - induced fibrosarcoma.s in the succeed320 l . 
strong. from the school of medicine , yale university , u.s.a. received for publication february 2 , 1949 . the idea of somatic mutation as an explanation for the origin of cancer has had a long and interesting history . its original use as an interpretation of cancer is usually associated with the names of murray and of boveri . these men arrived at the somatic mutation conclusion from different fields , murray from experimental cancer research , and boveri from his classical observations in experimental embryology . 
at that time he stated : " the existence of such tumours , the biological characters of which are retained through long periods of propagation , shows that the cellular transformation which initiates carcinomatous growth may take place in varying degrees . 
strong this biological alteration colours permanently their whole biological behaviour . is of such a kind that the cells are able to take up nourishment , increase in size and multiply indefinitely . 
they acquire an individuality and powers of resistance to injurious agencies superior to those of normal tissue elements . " boveri 's idea on the nature of the origin of cancer stemmed from his observations on atypical mitoses . 
he had suggested the possibility that by this means an unequal distribution of the chromosomes would ensue and thus lead to unconhe was loath to expand upon this concept until aichel trolled growth . ( 1911 ) , using boveri 's own observations on the development of the sea urchin egg , arrived at the conclusion that a malignant tumour was caused by a fusion boveri 's concept has not received wide recogof a tissue cell with a leucocyte . the essential feature of the idea is expressed in his own words ( boveri , nition . 1929 ) : " the essence of my theory is not abnormal mitosis , but in general , a however this may arise , the result definite abnormal chromosome - complex . beside the multipolar mitoses , which might would always be a definite tumour . depend either on a simultaneous multidivision of the centrosome or on distorting the parallelism between the division of the centrosome and the cell - division , asymmetrical mitosis should be chiefly considered for the origin of tumours . indeed , according to analogy with certain occurrences in sea urchins , these would depend on a lack in certain chromosomes of the power to divide ; this would result far more surely in tumour than would multipolar mitoses , which depend agencies which would act on chance for the distribution of their chromatin . most directly would be those that have the power of destroying definite chromoboveri , therefore , saw quite somes.of a cell , while leaving the others uninjured . " distinctly that irrespective of the mechanism of origin , the disturbed chromatin content is the essential feature in cellular physiology , including the abnormal condition of a tumorous growth . 
the pleomorphism of cancer cells and the irregular atypical mitoses so characteristic of their nuclei had been observed and carefully studied , however , since the middle of the nineteenth century . whitman ( 1919 ) wrote a critical review of von hansemann 's theory of aiiatyzzer ( 1916 ) expressed plasia in the origin of cancer in terms of modern genetics . the opinion that a somatic mutation may be involved in the origin of cancer . tyzzer stated , " there are marked differences in the behaviour of various tumours on transplantation in given classes of mice . 
even tumours arising in homogeneous races show such differences , and this may be attributed to the acquisition of new characteristics by the soma which are manifested in the development of the tumour . 
as a matter of fact some investigators express the opinion that tb.e two genetic phenomena are entirely antagonistic to each other . for example , a very recent article by bauer ( 1948 ) of heidelberg discusses at length that susceptibility to cancer in the human population is not inherited , and at the same time elaborates on the development of the concept that cancer arises as a somatic mutation . 
that a tendency to undergo somatic mutation may have a definite genetic basis in normal tissues has been discussed by east ( 1917 )  . this phenomenon has been encountered especially in plants , but also has been reported in the higher animals . an early example of a genetic influence on somatic mutations is discussed by whitman ( 1919 )  . 
a yellow chrysanthemum carrying white flowers on one branch . it is obvious that , since the colour of the flower is known to depend on definite factors , the development of a branch carrying flowers of a colour different from that of the flowers on the other branches could only arise through the failure of the factors to pass over , during mitosis , into the cell from which that branch developed . the branch , in other words , represents a somatic mutation due to asymmetrical mitosis . " many tumours have been described in a variety of genetic material . 
many of these tumnours are pigmented and occur especially in the early development of the individual , and some are associated with or are identical to lethal mutathese have been recorded by stark and bridges ( 1926 ) , wilson ( 1924 ) tions . and others in drosophila , by federly ( 1936 ) in lepidopterous larvae , and by many other investigators . most of these tumours are inherited by a multiple factor complex and , in the opinion of t . 
morgan , expressed to the author many years ago , at least one of the drosophila tumours arose as a mutation . time alone prevents the further discussion of this interesting genetic material and its bearing on the somatic mutation hypothesis . the next advance in the somatic mutation idea in relation to neoplastic lesions was undertaken by the present author , in 1918 . 
from that time onward until 1930 , a considerable amount of evidence was accumulated from the study of the transplantation of adenocarcinomata of the mammary gland in mice that eventually provided the first experimental evdence that a somatic mutation may be involved in the origin of cancer ( strong , 1926a , b )  . 
some of these data have been published , but a considerable amount has not . the development of this experimental approach to the nature of the origin of cancer was as follows : tyzzer and little investigated the transplantation of a sarcoma that originally arose in a japanese waltzing mouse . 
they found that the progressive growth of this transplantable tumour was dependent upon thesimultaneous presence of multiple mendelizing units strong and little , in 1920 , and later in 1924 , determined that two transplantable adenocarcinomata of the mammary gland in mice were dependent upon different genetic complexes for their continued the dbrb tumour was dependent upon the simultaneous presence of growth . two genes , whereas the dbra tumour was dependent upon the simultaneous presence of these same two genes , but required the presence , in the host , of a third gene . 
the presence of the third gene had no detectable influence on the growth of the dbrb tumnour . this geneticsimilarity and difference was obtained in the same series of mice in spite of the fact that the two tumours were histologically indistinguishable ( strong andlittle , 1920 ; little and strong , 1924 )  . the opinion of identical diagnosis was given by the late james ewing , as well as by francis carter wood . 
a third spontaneous tumour that arose in a dilute brown mouse ( given symbol dbrc ) provided evidence that six genes were involved originally in the growth of this tumour when transplanted into a series of suitable f2 individuals derived from a cross between mice of the dilute brown stock and mice of a totally unrelated stock , the well - known a stock . 
growth rates on the various transplantable tumours derived from the dbrc tumrour were determined . after several transplant generations a tumour was obtained that grew so rapidly that it was considered out of the normal range of growth for the original transplantable tumour . 
when the derived transplant was tested it was found that now its growth was dependent upon the simultaneous presence of only two genes . this sudden acquirement of growth capacity also resulted consequently in a new transplantability characteristic . 
a second sudden change subsequently took place , and a new derived ( mutant ) type of tumour would now grow in 75 per cent of all inoculated f2 mice ( observation 75 - 00 + : 23 - 002 - 79 )  . 
theprobable error difference between this newratio and the actual observation for the original dbrc tumour was 5813 per cent 3 ' 67 or 15 - 84 x p.e. this new derived transplantable tumnour continued to give a 3 : 1 ratio in a series of f2 mice for several transplant generations . eventually a new sudden change took place at which time the - new mutant type grew in practically all f2 mice ( observation 243 ' 00 + : 2 - 004 0 ' 94 )  . these new data in the f2 deviated from the ratio obtained with the original dbrc tumour as follows : 80 - 78 per cent 2 - 48 or 32 - 57 x p.e. there can be no doubt that sudden genetic changes are taking place somewhere in the relationship between the host and the transplanted tumour . since the mutant types and the original transplantable tumour were being compared in the same series of f2 mice , these sudden changes could not be taking place in the host but must be taking place within the tumour cell ( strong , 1926a , 1926b )  . another point that should also be emphasized is the fact that when the derived mutant transplantable tumour would grow in all f2 mice derived from a cross between the dilute brown and the a stocks , then , and not till then , would it grow in all mice irrespective of genetic relationship . in other words , it had lost all characteristics of tissue specificity . the transplanted tumour would grow invasively at a tremendous rate and would metastasize extensively . 
thus evidence was obtained that a highly specific , relatively benign , slow - growing transplantable tumour had progressively and suddenly acquired periodically new biological characteristics until finally it had been converted over into a carcinoma . 
toward the end of the series , however , especially at the terminal non - specific state , the tumour had become more cellular and had lost most of its original adenocarcinomatous arrangement . another series of investigations was performed on the transplantation of the 13 spontaneous tumours that mouse f1 79 gave rise to . this mouse was not only the common ancestor of the well known c , c3h , cba , c121 and chi strains of inbred mice , but also gave rise to a wealth of neoplastic tissue as follows : all 13 of her spontaneous tumours of mammary origin were tested out by trans102 l . 
and if one tumour tissue has deviated from the genetic constitution of the somatic tissue from which it arose , then possibly all tumours undergo the same process in their origin . the above phenomena especially of sudden changes taking place during the transplantability of spontaneous adenocarcinomas of the mammary gland in mice has been amply verified , not only by the investigation of the 11 other tumours derived from female f179 referred to previously , but also by many other all the phenomena of tumours derived from other mice in my laboratory . transplantation consistent with the genetic theory of transplantation were also verified by the independent work of bittner ( 1931 ) and by cloudman ( 1932a , b )  . eight years later little ( 1934 ) wrote a review on the genetic work on the transplanted tumour in mice in which he included a discussion of the somatic mutation in this paper little points out that he had outlined the genetic basis for idea . susceptibility and nonsusceptibility to transplanted tumours in 1914 ( little , 1914 )  . 
he states again in 1934 that " the bit of tumnour tissue to be transplanted has a genetic composition , which is determined by that of the animal in which it if this idea was exclusively true , then it is obvious that a somatic arises . " mutation could not have been involved in its origin since any mutational process it is indeed true that in the should alter or change the original genetic state . early transfer generations , the transplanted tumour derived originally from a spontaneous source does retain the characteristic of tissue specificity determined by the genetic constitution of the mouse , inthat it will grow only in individuals which are genetically related to the mouse that gave rise to the tumour . 
but it has now been definitely proven that this phenomenon of tissue specificity is periodically and progressively lost by sudden changes resulting in simpler and simpler mendelian ratios , until eventually the tumour retains no or very little tissue specificity which it originally had . these sudden or mutational changes have always produced more and more malignancy , as determined by percentage of takes , growth rate and eventual invasive and metastatic characteristics . has also been indicated that no two tumours , even though they be derived from the same mouse and present the same histological appearance , ever showed the same mendelian ratio when transplanted into a series of appropriate f2 individuals . 
when two transplantable tumours were derived from - the same mouse , some of the genetic complex involved in their successful transplantation is common to both tumours , whereas some are unique to either the one or the similarity of genetic transplantability complex indicates genetic other tumour . in this particular little 's concept of 1914 is relationship of origin for tumours . it is also evident that the more recently chemicallytherefore partially correct . induced tumours show , from their very origin , on an average , more malignancy , and thus less tissue specificity than do the spontaneous tumours that arise in it is probably true , although at present the same series of inbred or hybrid mice . impossible of proof , that there is some genetic mechanism within cells that determines to some extent normal cell relationship , but that under certain conditions this controlling genetic mechanism alters or changes , and by so doing permits an uncontrolled or cancerouscondition to arise somewhere in the biological this phenomenon of break of co - ordinating influence of system ( the organism )  . a definitive part is , i believe , the essential feature in the origin of cancer . 
strong following the experimental evidence reported by strong ( 1926a , b ) , and verified later by bittner ( 1931 ) and by cloudman ( 1932a , b )  . little ( 1941 ) stated the problem in different terms from those he used in 1914 . he now states : " in accepting as a working hypothesis the statement that cancer ilfils the requirements of a somatic mutation , namely , a sudden change which is perpetuated in succeeding cell generations , it must be noted that there are already on record two distinct general types of somatic mutation , either of which might be involved in the appearance of cancer . 
the other has a sporadic etiology dependent on various influences which affect individual cells or groups in the first category belong such cases as mutations in colour of cells directly . genes in rodents described by castle , pincus , bittner and others . 
one of castle 's cases is particularly interesting in that it shows several cases of mutations occurring in several generations of a single family of rabbits , thus indicating a definite cases of the second type have been recorded as the result of genetic basis . lockhart - mummery ( 1934 ) has published a exposure to radio - active agents . " book on mutations and cancer , and there have been several other articles dealing with this problem . these cannot be discussed at this time . the disovery that carcinogenic compounds can induce mutations when injected into suitable experimental animals has not originated the idea that cancer may arise by a process of somatic mutation , but rather has reopened interest in an old concept . 
some experimental evidence , although of an indirect nature , has been obtained , and it is clear that there is no evidence contrary to such a somatic mutation concept . 
on the other hand , the somatic - mutation theory hardly explains the embryonic features of malignant tumours ; and it does not explain why growths are more frequent in older than in younger individuals , because it has not been observed that mutations in cells of the gonads are more frequent in older than in younger persons . 
by this means he has reported many mutations ( notably sex - linked lethals ) with the carcinogenic hydrocarbons , and has stated that he has obtained a few chromosome breaks in his experimental material . it is an established fact that the term mutagenic is not an absolute characteristic for any physical or chemical agent . for example , it may be said that x - rays are a powerful mutagen . they are very effective in inducing mutations in drosophila and in neurospora , and to acertain extent in maize , but do so very poorly in mice . 
on the other hand , ultra - violet light appears to be more effective in inducing mutations in maize than in drosophila . again it must be taken into consideration that the different physical and chemical agentsbring about mutations in various ways . x - rays break chromosomes , thus leading to rearrangements of parts , transpositions and inversions , to deletions , etc . ultra - violet light , however , seems more effective in inducing point mutations in both corn and drosophila , possibly by a specific wave - length , 2600 , that is selectively absorbed by the nucleoproviding proteins of the chromosomes . 
strong sex ratios and in distorted mendelian ratios which may be later proven to be chromosomal , but so far all mutations induced by methylancholthrene in mice finally , the which have been tested have been proven to be point mutations . mustard gas derivatives , the fourth group of powerful mutagens seem very effective in breaking chromosomes , producing embryonic disturbances very similar to the effects of x - rays , but apparently may have another action on mutagenesis , since they appear to affect cells even before there be any evidence of cell division ( action therefore similar , but possibly different from that obtained with x - rays )  . carcinogenesis again is a relative rather than an absolute property . 
on the other hand , other species are extremely liable to great resistance to experimentally for example , the rhesus monkeys in new haven have been induced tumours . injected with methylcholanthrene for 15 years without producing a single neoone must conclude , therefore , that for this species methylplastic tumour . cholanthrene is not a carcinogen , or at least , if it is , the evidence for such a conclusion is still not available . 
the problem is even more complex than this , as will be indicated in the following discussion . not all of the biological variants obtained in mice with methylcholanthrene are genetic . 
the somatic mosaic may be due to a chromosomal aberration rather than a point mutation in somatic differentiation , although the actual mechanism involved in their origin cannot be determined . we have obtained , therefore , a multiplicity of biological effects when methylcholanthrene has been injected into a series of mice over many generations . whether all these phenomena are due to the original methylcholanthrene or to one or many of the derived metabolites is still not known . 
some of these effects are definite point mutations , some are non - genetic , some are non - genetic produced possibly by inhibitions of embryonic morphogenesis at critical periods , and other biological effects may have other mechanisms in their origin , such as abnormal distribution of chromosomes during morphogenesis . but these mice whose ancestry has been injected with methylcholanthrene for many generations are not only giving rise to a wealth of biological effects , but also to a great variety of cancers . 
the first genetic result was the production of germinal mutations which changed the rate at which fibrosarcomas appeared at the site of injection of methylcholanthrene ( a decreased latent period )  . 
the second genetic effect was the production of spontaneous lesions involving the mucus - secreting cells of the gastric mucosa following the induction of this same gastric lesion from the subcutaneous injection of methylcholanthrene . 
the appearance of this specific type of pathological lesion was brought about by a mutation on the " brown tagged " chromosome . several new sudden changes have appeared in the gastric lesion subline changing the latent period for the appearance of the lesion - a phenomenon that was obtained in the transplantation of adenocarcinomata of the mammary gland in mice and discussed in this same paper . 
bartholomew 's hospital , and the statistical research unit of the me - dical research council , london school of hygiene and tropical medicine . received for publication february 20 , 1951 further data on the arsenic content of cigarette - 3 . in an earher paper ( daff and kennaway , 1950 ) data were given for the arsenic content of 5 brands of cigarettes of british and american types , of 8 brands of turkish type , and of two - others ( french , rhodesian ) , and also for the amount of arsenic volatilized in smoking . 
and germany are those of saglam ( 1944 ) from turkey and of dungal ( 1950 ) from iceland . ( 4 ) population by aores . ( 2 ) number of autopsies showing cancer . the data which one would like to obtain from any given country are : a . 
from univer8ity clinic8 ( for men and women separatelv - preferably by ages )  . - ( i ) number of autopsies . number of autopsies showing cancer of lung . in the countries where a ( 5 ) is not available one must rely on b . 
le hastaliklari klinigi in 23 months ( among 2084 patients , 53 in a thousand )  . " cc all these statistics show that in our country , within the last 30 years , pulmonary carcinoma has increased in a great proportion . " saglam rejects cigarette - smoking as a factor in the incidence of bronchial " some authors believe that there exists a relationship between carcinoma . smoking tobacco and inhahng the smoke and pulmonary carcinoma . 
we do not hold for a long time , at least since 50 years , almost only cigarettes the same view . in spite of this the increase in pulmonary ca . 
kennaway data for 1934 and have removed 3 cases , all in men , described as benign tumours , from the total of primary lung tumours ( a chondroma in 1935 , another in 1939 , and a polyp in 1937 )  . 
the official figures for tobacco consumption ( hutson , 1937 ) and for population ( statesman 's year book , 1931 ) indicate an annual consumption of about 17 lb . 
he writes that the kinds of tobacco smoked in yugoslavia ( b ) type hercegovina ( cigarettes ) ; and are : " ( a ) type makedonija ( cigarettes ) ; ( c ) type vojcodina ( pipe and cigars )  . " these are of the oriental type , i.e. , they have a natural ' bouquet ' which gives the tobacco type makedonija . " only in the north - western part of yugoslavia there are smoked cigars and pipe . 
use of tobacco for snuffing is quite insignificant . before the last war the only import was some dutch tobacco for cigars - ; during the war some bulgarian and after it some american tobacco ( through u.n.r.r.a. ) was imported , but all these amounts were very small . we are also indebted to kosir and to f . 
among 20 , 681 autopsies , there were 3584 cases of cancer and 276 cases of cancer of the lung . these figures are compared with those from other countries in table ix . ( 3 ) it is , however , not necessary to be dependent on hospital statistics for an estimate of the incidence of cancer of the lung in switzerland , as vital statistics are available for - the whole country . 
the calculated death rates from cancer of the lung are shown in table x . the national figures are reasonably reliable ' as , according to jakobsen , " the doctor who has treated the deceased during his last illness is required to which must be fill in an official certificate stating the cause of death stated in medical terms as precisel.v as possible , and it is not permitted to use general diagnoses such as " disease of the heart " or " disease of the lungs . " the 16fl ravii i 12 . 
kennaway clearl no exact proportionaht however , reasonably close to the difference in cigarette consumption . in switzerland tobacco consumption was much the same as in england and wales , but death rates were only half as high . differences in cigarette consumption again agree with the differences in death rates better than do differences in total tobacco conthis is because a much smaher proportion of the tobacco is consumed sumption . in the form of cigarettes in switzerland than in england . has been found in the data so far available between the amount of tobacco smoked and the prevalence of cancer of the lung cancer of the lung appears to in different countries and at different periods . increase more rapidly than does the use of tobacco ; such a change in effect may occur at a certain level in the dosage of a drug . 
the figures from a single hospital in yugoslavia suggest an incidence of cancer of the lung comparable to the british figures of 30 years ago . we have no figures for the incidence of cancer of the lung upon the whole population of any east european country . 
the e - vidence available at the moment is against the direct carcinogenic importance of tobacco smoke , because it tends to exclude the two most obvious carcinogens , arsenic and benzpyrene , which one might expect to be present . 
known carcinogens is now so wide that one must consider other possibilities . ( 3 ) all the older data about cancer of the lung must be reviewed in the light of this connection with smoking ; one must reconsider the inverse relationship with sunhght ( stocks , 1947 ) and the very low incidence upon a rather curious - assortment of occupations , namely , agriculture , coal - niining , and mule - spinning ( kennaway and kennaway , 1947 )  . 
one cannot answer this question at present , but it is especiary important in countries where smokers buy the cheaper pipe tobacco to make their own cigarettes . norway one obtains in this way nearly twice as many cigarettes for a given sum ( jakobsen , personal communication )  . 
the incidence of cancer of the lung upon the two sexes is not very different ( death rate , male to female , i : 07 ) ; it is greater in urban than in rural districts , and this difference is greater in men than in women . 
the increase in mortality in the last 20 years has been greater in men than in women , and greater in the towns than in the country . ( 5 ) in sweden , snuff makes up a much larger fraction ( one - third in 1949 ) of the total tobacco products consumed than is recorded in other countries . ( 6 ) a comparison is made of the data available for the increases since 1931 in ( a ) deaths attributed to cancer of the lung , and ( b ) in the consumption of tobacco , in england and wales , norway and switzerland . 
the consumption of tobacco per head has been for the last 10 years rather higher in switzerland than in the united kingdom , and in norway has been about one - half that in the other two countries , while the crude death rates at the beginning and end of the period were m . 
kennaway rou - ahlv in the proportion of i 0 ( england and wales ) to 5 ( switzerland ) , and 2 cigarette consumption was approximately in the proportion of 4 ( norway )  . ( england and wales ) to 2 ( switzerland ) and i ( norway ) and was more in accord with the relative death rates . 
the increase in the number of deaths has been about the same ( twofold ) in all three countries , but the increase in consumption oftobacco and cigarettes has , been ' less . 
the differences in the incidence of cancer of the lung are therefore quite different in extent from those in the quantity of tobacco consumed ; they are m ' ore hke ( though stir different from ) those in the quantity of cigarettes consumed . 
the study of the relation between the national consumption of tobacco and the national incidence of cancer of , the lung has scarcely begun . we wish to thank saglam and prol p . 
causes an almost complete cessation ofepidermal mitotic activity , and the same effect is caused by phloridzin , which interferes with the phosphorylation of sugar . since insulin and phloridzin both exert their mitosis depressing effect bv reducing the availabifity of sugar , it appeared probable that a similar depression would also be induced bv starvation . 
12 hours of starvation the blood sugar level remained fairlv high , but the drop in the mitosis rate was pronounced . however , during the afternoon sleep period there was a marked rise in mitotic activitv at 14.00 hours to a figure of 4 - 1 0 - 35 , which is about half the figum reachc ; l by the well - fed controls . - after 24 hours ' starvation the blood sugar level had faren seriously , and mitotic .1gain , however , there was a slight activitv had reached a very low level . response to the period of rest , so that at 14.00 hours a figure of 2.0 0 - 21 was this is about a quarter of the figure for the control animals obtained . ..a&r 36 hours the blood sugar concentration had dropped to 100 mg . 
observations were next made on the effects of such diets on epidermal mitotic acti - %ity . in prehminary experiments determinati ' ons were made of the dailv food intake of threeor four - monthold strong cba males which were allowe ; 1 to feed ad libitum on a diet of rat cake . 
13ttllo ' ug14 66 per cent diet remaitied active aiid in excelleiit health , btit those which received a 50 per cent diet showed sigiis of ill - health , and it is dotibtftil whether they could have stirvived the treatnieiit for i - iiore tliaii another few ' weeks , at the end of the experiment at 20.00 hours all the niice wei - e killed and blood sugaiestimations were made . 
that middle age is characterized bv a generallv raised mitosis rate which must itself assist in the development of any latent tumour cells which may be present . ll ' hfle the reason for the increased initotic activitv is still obscure . 
this observation mav afford some explanation of the fact that middle age is characteristically the cancer age . since the development of latent tumour cells is assisted bv conditions of hyperplasia , it is reasonable to suppose that conditions of hypoplasia wih have the hyperplasia increases the chances that the latent tumour cells opposite effect . will be stimulated to multiplv . 
and so delavs the development of those tumours which do forand prevents altogether the appearance of manv which otherwise would forthus it is possible to provide a logical explanation of tannenbaum 's re - sults . 
and the fact that carbohvdrate - supply is the critical factor in both cell division and tumour genesis can be taken as corroborative evidence . at last it iis becoming possible to split the cancer problem into two separate parts : that which is coneemed with the formation of latent tumour cells . 
but it now appears possible that a practical control of cancer mav be developed from a thorough understanding of those factors which govern normal cell division . if hypoplasia can be maintained without damage to health . 
and reveal fewer pathological changes in the tissues . " this mav well be related to , the fact that hypoglyeaemia not onlv reduces the rate of cell division of a host . but also that of a parasite . 
a sin - iilar effect is produeed bv restricted diets . - kninials rationed to 66 per cent of what thev would eat if fed .4 , libitunt have an epidermal mitosis rate which is less than 40 per cent of that of well fed controls . if rationed to 50 per cent . 
the mitosis rate dxops to about 15 per cent of that of the controls . it is suggested that these observations provide an explanation of tannenbaum 's results on the prevention of tumour genesis bv diet restriction . 
mottram ( 1945 ) published the results of experiments designed to demonstrate a relationship between the tumour yield induced by benzpyrene and the number of epidermai mitoses present at the time of application of the carcinogen . painting the skin of rnice at midnight resulted in a higher yield ofpapillomata than did painting at midday . 
the concentration of aaf in the food was 0.03 per cent for 5 weeks and then 0 - 05 per cent for 20 weeks , the total period of administration extending over 25 weeks from october , 1945 , to april , 1946 . thereafter the mice received the standard diet of rat cubes for the rest of their lives and the control mice received that diet throughout . all mice had an the experiment was terminated in unrestricted - supply of food and water . the eighty - fourth week by killing the mice then surviving , namely 2 males which had eaten aaf and 3 control males . several attempts to control the localization of tumours were made on small during the third to sixth weeks of experiment 5 females of groups of mice . the aaf group and 5 control females were mated with vasectomized males and silk threads were tied in the pseudo - pregnant uterine horns to elicit deciduomata . in the twenty - second week of experiment , pellets containing 25 per cent of diethylstilboestrol ( b.d.h. ) in cholesterol and weighing 5 or 6 mg . 
the stilboestrol pellets shortened life , the testosterone tablets were harmless ; especially by causing pyometra . their possible effect on the induction of bladder tumours is mentioned below . granulation tissue , fibrosis and abscess were found in uterine horns where silk threads were tied but tumours were not recognized there . " spontaneous " tumours were found in 3 male control mice as follows : osteogenic sarcoma of the thigh , with secondary deposits in liver and lungs , in the forty - eighth week ; hepatoma in the sixty - third week ; and lymphoma in the sixty - eighth week . 
the average time was 50.7 weeks , but 8 of the tumours only 1 tumour occurred were found from the thirty - ninth to forty - sixth weeks . in an effective total of 8 mice carrying testosterone tablets ; 6 of the mice survived from 58 to 64 weeks without tumours . 
the incidence as a percentage of the effective total was 12.5 for aaf males with testosterone tablets , 73 - 3 for aaf males without testosterone , and 52 - 2 for all aaf males . most of the tumours were intravesical , pedunculated or sessile papilliferous tumours of transitionalor squamous - cell types , and conformed with the descriptions of armstrong and bonser ( 1944 ) , who distinguished benign and malignant forms but observed no extra - vesical extension . 
the distribution of tumours amongst these and less common sites depends on the species and on the strain of the animals used ( bielschowsky , 1946 , 1947 ; armstrong and bonser , 1947 ; dunning , curtis and madsen , 1947 ; harris , 1947 )  . harris concludes that " the site at which tumours may be induced by fluorene compounds is probably determined by the genetic constitution of the rat employed . " this statement , though true , is not a definitive explanation . 
the genetic constitution determines how particular cells react to particular stimuli ; remains to determine what reactions are iminportant in the production of tumours by acetylaminofluorene and how the reactions are variable in kind or degree . the importance of the genotype is accepted ; the urgent task is to elucidate its phenotypic expression . carcinogenic hydrocarbons affect tissues remote from the site of application and increase the incidence of certain tumours , notably tumours of the breast , long adenomas and leukaemia . 
many observations , summarized recently by enge ] breth - holm and rask - nielsen ( 1947 ) , suggest that the action is , essentially , an acceleration of spontaneous tumour development . 
some actions of acetylaminofluorene may be attributable to acceleration of a spontaneous disease as , for examnple , the increase of mammary tumours recorded by armstrong and in my experiment the evidence for an increase in the naturally high bonser . incidence of breast tumours in r3 mice was not decisive , and acetylaminofluorene did not accelerate or increase the incidence of lymphoma or sarcoma which occur , uncommonly , in normal r3 mice . 
the unequivocal effect of aminofluorene was the growth of tumours of the bladder which are not found in normal controls and which , as yet , have not been induced in mice by other carcinogens ( bonser , 1947 )  . in some strains of rats , acetylaminofluorene notably induces carcinoma of the external auditory canal , previously unrecorded . these and other comparable observations show that tumours induced by acetylaminofluorene do not grow preferentially in tissues prone to develop " spontaneous " tumours and are not accelerated " spontaneous " tumours . 
foulds hepatoma is much less frequent and , as my observations show , develops later than tumours of the bladder by about 20 weeks - a substantial fraction of the life of a mouse . consequently , intercurrent diseases , mammary tumours in females or bladder tumours in males kill most r3 mice before the response of the liver to aminofluorene is manifested by the growth of hepatoma . 
some geneticists maintain that the processes directed by allelomorphic genes differ only in speed . speed of reaction is probably one of the most important expressions of the genetic factors which govern the distribution of the tumours induced by acetylaminofluorene . 
the high and relatively early incidence of mammary tumours in female r3 mice presumably reduces the opportunities for other tumours to become evident , but it does not account for the complete absence of bladder tumours from the females in my experiment , or for the lower incidence in females compared with males in the 5 strains studied by armstrong and bonser . 
tumours of the bladder grew in about half the male mice which survived 9 months or longer from the beginning of the experiment . testosterone seemed to inhibit the induction of bladder tumours in males and none grew in females . 
jackson memorial laboratory , bar harbor , maine . received for publication april 20 , 1954 in a comparative study of alkaline phosphatase in tumors , greenstein ( 1942 ) noted that the mouse tumours studied possessed little or no alkahne phosphatase activity with the exception of spontaneous mammary tumors and lymphomas . comparative q values were calculated as the ratio of the percentage of disodiumphenyl phosphate hydrolyzed to milligrams of total n per cubic centimeters of several concentrations of n were taken and the q values remained tissue extract . remarkably constant in relation to nitrogen . 
the highest value for any tumor was the transplanted hepatoma 31 in rats with a q value of ' 542 . kabat and furth ( i 941 ) could not demonstrate alkahne phosphatase by means of gomori procedure in mammary adenocarcinoma spontaneously arising in c3h however , hard , pratt - thomas and belkin ( 1948 ) examined twenty - nine stock . spontaneous mammary carcinomas in c3h , a and dba mice and noted high alkaline phosphatase activity . 
the predominent picture in the mouse tumours examined was the high alkahne phosphatase activity shown by the cells of the acinar units while the stroma and vascular elements were free . 
descenden ' t of a mouse that was bom from a fertilized ovum transplanted into the uterug of a dba mouse this has gone through 320 transplant and was nursed by her dba mother . results from the jackson laboratory indicates 100 per cent transgenerations . plantability in c57bi , and their f , hybrids . 
the histological type is an adenocarcinoma ( sneh , cloudman and woodworth , 1948 )  . the tumor h2712 originated spontaneously in the mammarv gland of ' a c3h mouse in 1948 at the roscoe b . 
the histological type - is an adenocarcinoma . the tumor dbrb originated in the department of genetics , carnegie institute of wasliington in 1918 in the mammary gland of a dba / i mouse and has gone throuo ' h 845 transplant generations . refjults from the roscoe b . 
the histological type is an adenocarcinoma ( strong and little , 1920 ; ' little and strong , 1924 )  . the tumor 15091a originated in the laboratory of the university of a - fichigan in the mammary gland of an a albino mouse in 1928 ( cloudman , 1928 )  . this has gone through 341 transplant generations and shows 100 per cent transplantability in a albino and their f , hybrids . 
the histological type now is that of an anaplastic carcinoma . for the purposes of the experiment the animals were divided into four serieg . series 1 consisted of tumor e0771 transplanted into males and females of c57bl and their f , hybrids between c57bl and a / l . series ii consisted of tumor h2712 transplanted into c3h males and females . series iii consisted of tumor dbrb transplanted into dba male and female . series iv consisted of tumor 15091a transplanted into f , hybrids of c57bl and a / he male and female . the total number of animals of the four series were i 00 and 8 1 . all mice were transplanted with their respective tumor at the roscoe b . 
jackson memorial laboratory and sacrificed at intervals between 7 and 22 days or an average of 16 davs . the am - mals were kired by ether anesthesia and thin slices of the ever , spleen , adrenals , kidney , testes , ovary and the tumor were placed immediately into formoltissues placed in fo ' rmol - saline were stained saline and into ice - cold acetone . by the regular hemotoxyliin and eosin methods . tissues placed in ice - cold acetone were left for 24 hours with one change of acetone . 
they were then placed in cold absolute alcohol over night followed by two changes of benzol of 45 minutes e " ach . they were then imbedded and infiltrated in paraffin at 56 ' c . 
they were then dehydrated and mounted in permount . incubation times in the substrate were as follows : series 1 , ily ill : i minute , 5 miinutes , 15 minutes and 1 hour , and series iv : i.minute , 5 minutes , 15 minutes , i hour , ij hours , 2 hours and 21 hours . 
a total of 1124 shdes was prepared . the shdes were read and visually graded according to their intensities from i to 6 . in this way a relative assay could be arrived at which showed a distribution within each incubation time , and the earliest reaction or end point could be ascercontrols were run . 
at i minute incubation time 29 of the 39 reactions had a reaction intensity of one and hence this was considered as the " end point . " at 5 minutes there were no negati ' ve reactions , the majority of reactions being two or three . 
the distribution was , however , strikingly different from the other tumors and will be referred to in the subsequent section . series iii consisted of 48 transplanted dbrb mammary carcinomas . 
l. - tumor e0771 transplanted into c57bl and f , hydribs ( series i )  . alkaline phosphatase reaction at 15 minutes ' incubation tixne slides show irregular areas of activity . 
3. - tumor dbrb transplanted into dba ( series iii )  . alkaline phosphatase reaction at 15 minutes ' incubation tixne shows a tendency of the peripheral cells of islands to show more activity than central portion . 
7. - tumor h2712 transplanted into c3h ( series ii ) under high power magnification . note the peculiar and intense distribution at the borders of cells lining the lumens . in the center is also seen gomori positive acerular material in a very small lumen . 
4 , 5 , 6 at 60 minutes incubation period . it is quite evident that 1.5091 a exhibits no alkaline phosphatase activity and further extension of the incubation time to 2 - 1 hours showed no reaction except in the vessels . in series i tumor e0771 is composed of irregular islands of tumor tissue . these varv in size and shape . 
the islands are fairly solid with the exception of small areas where itimens can be seen . larger iiimens are lined by several rows and layers of cells . most of ' the lumen8 are very small . 
8 shows a photoreticular material with many large thin - walled vessels . micrograph of this tumor stained by hemotoxyl - in and eosin showing the islands of the interstitiat tumor tissue with an attempt at luminal and tubular forma - tion . tissue seen to the left is loose and reticular in nature . there seem to be attempts to form tubular structures . the phosphatase reaction began during the first minute of incubation time with a blackening or graying of the cytoplasm . this gave a gray appearance to the slide except in such areas where the blackening is much more pronounced . 
at this time enzyme activity large irregular can be demonstrated in many areas in the centres of the island . clefts can be distinguished and stand out prominently bv the activity of the enzyme of the cells lining the lumens . intraluminal material which is not apparent in the hemotoxylin and eosin preparation shows intense activity . small globules are present in the cytoplasm of many cells adjacent to lumens or attempted lumen formation . 
the connective tissue and vessels do not react . tumor dbrb iin series iii under hemotoxylin and eosin stain shows a more lobular distribution than either e0771 or h2712 and is shown in fig . 
the interstitial tissue is composed of loose connective tissue , connective tissue and large thin - walled vessels . with graded gomori stains the reaction has an " end point " at 15 minutes . here the areas of activity are irregular thoughout the tumor . 
some activity is there is a tendency for present in the small lumens but this is not marked . increased activity at the periphery of the lobule as shown in fig . 
the loose supporting connective tissue and vessels show no activity . in series iv tumor 15091a differs somewhat from the other 3 in that it is a fairly solid tumor not composed of islands or lobules . there are numerous mitosis and the cells vary more in size and shape . 
at i hour this showed rather marked activity . the cells of the peripheral portion of the nodules showed most intense activity . there is no essential difference between the various series . the liver shows , for the most part , two types of reaction . 
the ovary begins in most series with the 5 minute incubation time in the theca externa which shows progressive activity to i hour . ksmall stromal vessels also show activity . all the series were approximately the same . 
one difference that occurs in the e0771 is the presence of doubl - e layers of tumor cells in the attempt at acinar formation . in the 15091 a the tumor is solid with very few smar acini . the overall phosphatase reaction was very intense in e07 7 1 , very much less in this was evident in spite of the fact that h2712 and bdrb and none in 15091a . the rate of growth of the tumor was about the same in all the series . 
numerous hard , pratt - thoma 's and belkin ( 1948 ) mitoses were present in all the tumours . felt that there was a loss of alkahne phosphatase activity in the more anaplastic although it would seem in our series that the anaplastic tumor tumor cells . 15091a having no reaction would point toward such contention , the e0771 is a very rapidly growing tumor with numerous mitoses . 
we would prefer to delay our judgment on this issue until a larger variety and strains of tumors have been studied . an interesting and significant paper which has come to our attention as this work was completed was the observation of shelton ( 1952 )  . this investigator found a tumor in a 6 - month - old strain a female mouse that had been exposed to 400 r whole body x - radiation on the date of birth . 
lymphoma 1 was propagated in a mice from a large tumor mass located in the anterior mediastinum . lymphoma 2 was propagated by subcutaneous injection into strain a mice of fragments of lymph nodes from a mouse bearing the original tumor . strain i grew as a localized tumor with local invasion ' but no metastasis , but strain 2 reacted as an acute leukemia with rapid growth infiltration of the organs and high peripheral count . in determination of the alkahne phosphatase activity it was found the lymphoma 1 showed no reaction while lymphoma 2 showed a reaction as expressed by 5 mg . 
no detectable reaction could be seen by the gomori method in lymphoma i whereas lymphoma 2 was here we have a rapidly growing persumably anaplastic tumor strongly positive . showing an increase in alkahne phosphatase reaction . in our laboratory we have also determined the alkahne phosphatase activity as expressed in gamma of phosphorus per mflhgram of nitrogen at 30 minutes incubation time in a large series of these 4 mammary tumours . 
daab seemed to interfere with vitamin - a storage and hence to cut down food consumption , leading to a deficiency of some other kind , such as that suggested by zucker , berg and zucker ( 1945 ) , which was the immediate cause of the forestomach lesions . the present author ( 1947 ) was , however , unable to elicit such lesions in the stomachs of stock mice given daab orally , even up to 294 days , in either a balanced or one type of unbalanced diet . 
he concluded that otsuka 's diet ( which was grossly deficient in several essentials ) allowed a deficiency not existing in his own diets but necessary for eliciting such forestomach lesions in mice . he also found no tumours in similar mice injected subcutaneously with daab in arachis oil , on three occasions , up to 329 days after the first injection . 
daab thus appears not to be a carcinogen for the subcutaneous tissues of mice . hiowever , in another experiment which apparently broke fresh ground , the present author applied daab in acetone , in increasing strengths , to the nape of the neck of stock mice , and obtained two squamous carcinomas , one with spindlecell metaplasia , in 2 of 8 mice surviving 346 days or more . this seemed to be sufficient grounds for classing daab as a carcinogen for the skin of mice . a further series of experiments has now been carried out to check the previous findings with daab painted in acetone ; at the same time , the effect , if any , of added croton oil was investigated . 
at the commencement , all mice were painted with the solution containing croton oil ; after 15 days , the croton oil was withdrawn . croton oil was re - introduced for one - third of the mice after a further 167 days , and the experiments therefore fall into two groups : ( a ) initial croton oil , and ( b ) initial - plus - subsequent croton oil . applications were made to the nape of the neck , after clipping away hair , thrice weekly for a total period of 545 days . the basal diet throughout was rat - cake ( thomson , 1936 )  . ( a ) initial croton oil series . - the survival rates of mice surviving more than 200 days are shown in fig . 
kirby the commencement of painting ; of these , two showed acute yellow atrophy of hyperthe liver and one showed slight hyperkeratosis at the site of painting . keratosis , with or without necrosis or ulceration , was common after 200 days , but the first papilloma was found in a mouse killed after 320 days ' painting ; this papilloma grew as a coxcomb - shaped wart . 
the other mouse had a similar , though smaller , lesion which also proved to be a squamous carcinomna of low - grade malignancy . although a mouse dying after 435 days showed no gross abnormality at the site of painting , another dying after 449 days had two tumours in the painted area ; one of these was found to be a squamous carcinoma with invasion down to the depth of the muscle . 
a necrotic , warty outgrowth , showing parakeratosis beneath , and a multiple papillary growth , were found at the site of painting in mice painted for 495 and 545 days respectively , the latter dying at mice dying at 524 , 528 and 588 days , i.e. 
33 days after painting was stopped . 549 days respectively showed nothing more than a little hyperkeratosis at the the last survivor and one of the mice from group ( b ) unfortusites of painting . nately died at the same time ( 611 days ) and were examined post - mortem in the absence of the author . 
one of these mice showed early squamous carcinoma , and the other simple infected ulceration ; as there is no way of deciding which histological report belongs to which mouse , they will be excluded from consideration in comparing the results in the two groups . 
a mouse dying at 623 days showed no lesion at the site of injection . thus in 16 mice surviving more than 300 days ' painting with 5 per cent daab in acetone , 3 developed simple papilloma , and 3 others developed squamous hyperkeratosis and / or ulceration was found carcinoma , at the site of painting . in nearly all the rest . abdominal organs were only examined histologically when gross damage was visible , but the male dying at 449 days and one of the mice dying at 611 days showed amyloid changes in spleen , liver and kidney similar to those referred to in the previous paper ( kirby , 1947 )  . 
the mouse dying at 623 days showed pulmonary adenoma . ( b ) initial - plus - subsequent croton oil series . - survival rates , beyond 200 days , only slight are shown in fig . 
1. hyperkeratosis was found at the site of painting in 4 mice dying at 215 days and in 1 dying at 305 days . of 3 mice dying at 435 , 442 and 443 days respectively , all had papillomas , keratinized and partly necrotic , at the site of painting . 
of 17 mice painted for more than 400 days , 5 developed squamous carcinoma , and 5 others squamous papiiloma , at the site of painting , a tumour incidence of 60 per cent . 
the malignancy appears to have been low - grade in all cases , and metastases were never found . no evidence of spindle - cell metaplasia was seen in this series . 
as this substance is an intermediate in the manufacture of p - aminoazobenzene , the possible danger to workers handling it , especially in organic solvents , must not be overlooked . induction by daab of tumours at sites remote from that of application seems to be confined to the lung . 
no tumours were found in the liver of any mouse . co - carcinogen tests : neither the incidence of skin carcinomas - 3 / 11 ( surviving 400 days ) in group ( a ) and 1 / 5 in group ( b ) - nor the time of appearanceabout 400 days in either group - lends any weight to the view that croton oil acted as a co - carcinogen when applied concurrently with daab in group ( b )  . on the contrary , it would seem that croton oil had no effect either way on the induction of skin tumours . this suggests that the mechanism involved in carcinogenesis by daab differs from that of the polycyclic hydrocarbons . 
croton oil painted concurrently caused no increase in the incidence of the significance of the skin tumours , nor did it decrease the latent period ; absence of co - carcinogenic action of croton oil is discussed . the author is greatly indebted to p . 
kirby. from the research department , glasgow royal cancer hospital . received for publication , june 28 , 1984 . activity the carcinogenic of orally - administered 2 - acetylaminofluorene , aaf , for a number of tissues , mainly epithelial , was first demonstrated by wilson , deeds and cox ( 1941 ) for the rat , and subsequently confirmed for this species by bielschowsky ( 1944 , 1946 ) and others , for mice of various strains ( armstrong and bonser , 1944 , 1947 ; foulds , 1947 ) , for the fowl ( bielschowsky and green , 1945 ) and for the cat ( harding , quoted by bielschowsky , 1947 )  . recently wilson , deeds and cox ( 1947a ) have reported their own findings in mice of three pure strains . 
aaf has now been shown to be a carcinogen ( when given per os ) in eight strains of mice : cba , if , riii , white label , strong a , c57 black , c3h , and bagg albino . 
of the first five strains mentioned , cba were most prone to both liver and bladder tumours ; strong a were the least susceptible ( armstrong and bonser , 1947 )  . 
mark 's hospital , london . received for publication december 29 , 1949 . it is well known that different histological types of cancer vary in their rate of growth and liability to metastasise , but this relationship between histology cancer and spread is easier to study in some organs of the body than in others . of the intestine provides exceptionally good material for investigating this question becauise ; surgical operations are planned to remove a good stretch of bowel above and below the tumour as well as a wide field of lymphatic drainage in the mesentery . 
it happens , therefore , that the pathologist is presented not only with the tumour , but ' also with its surroundings , so he can first classify the tumour according to ' its histology , then measure the extent ' qf local spread by direct continuity , and finally record the number and position - of lymphatic metastases . when the histology and spread are compared in ' this way it soon becomes obvious that different histological ' varieties often behave differently , but a fairly big series of cases must be examined in order to express these differences quantiit takes a long time tatively and give them anything like precise significance . to collect many cases of cancer of one organ of the body such as the bowel at a general hospital admitting patients of all sorts , ' but this is done much more quickly in a special hospital like st . 
mark 's , which is intended primarily for in the last 21 years ( 1928 - 1948 inclusive ) diseases of the rectum and colon . 3 , 220 operation specimens of intestinal cancer were examined and dissected in the laboratory of st . 
mark 's hospital , the general distribution being as follows : 2 , 642 cases of cancer of the rectum , 429 of the colon , 51 of the anal canal , and 98 cases of multiple malignancy . in each case the records describe the extent of local spread and the n ' umber and position of all lymphatic metastases . preparation for this paper i ' have re - examined the sections of all these tumours and compared their histology ' and their local and lymphatic spread . since rectal cancer is much the largest of these groups , forming 82 per cent ' of all cases , it will be convenient to start with this . histological varieties of rectal cancer . the three main histological varieties of rectal cancer are adenocarcinoma , colloid ( or mucinous ) carcinoma and an undifferentiated variety which i shall call " anaplastic carcinoma simplex . " aden ? ocarcinoma is much the commonest variety , constituting 85 per cent its histological characteristics are well known , and it is sufficient of all cases . to point out that though the growth usually shows a tubular or acinar pattern there is as a rule very little evidence of mucous secretion , either in the cells or 60cuthbert e . 
the last two varieties were too few in number for analysis , but comparisons between adenocarcimoma and colloid showed once more that mucus thus , secreting or colloid tumours spread more rapidly than adenocarcinoma . at the time of operation , only 5 per cent of the colloid carcinomas were still stage 1 , i.e. 
dukes such relationship between histology and spread can only be established if the histological varieties are clearly distinct : as definitely different , let us say , as adenocarcinoma , colloid carcinoma and carcinoma simplex of the intestine . this is seldom the case . another handicap arises from the fact that in some organs of the body the injury caused by a malignant growth is much more attributable to its anatomical position than to any niceties of histology . this is particularly true of tumours of the kidney , ureter and bladder . 
lane. from the department of pathology , royal cancer ho8pital , london . received for publication june 7 , 1952 . scepticism is a healthy response to diagnosis of any tumour as angiosarcoin ' - 11 . to dispel all doubts , full necropsy and thorough histological exaniination of all lesions with indisputable evidence of the vasoformative natuire of the tumour and proof of metastasis are essential . 
many lymph nodes in the cervical regions , supraclavicular fossae and posterior triangles were enlarged ( up to 2 - 0 cmain diameter ) , soft , dark red and cystic . 
on the surface and in the substance of both lungs were numerous discrete , firm , red , spherical nodules ( up to 3 - 0 cdiameter ) whose cut surfaces resembled blood clot . other parts the paratracheal and mediastinal lymph nodes of the lungs were oedematous . were greyish - black and slightly enlarged . 
the common bile - duct was dilated and contained several small pigment calculi . the spleen was small , and on its surface was a cyst ( 1 - 2 cdiameter ) containing clear fluid.. 
the nuclei are pale - staining , vesicular and variable in size : in shape 's.pherical , ovoid or indented . mitotic figures are few , but within such aggregations capillary channels are forming by separation of the cells . 
he interpreted his case as a benign angioma producing metastases , and separated it sharply from a case described by theile ( 1904 ) in which all the lesions showed sarcomatous areas . his interpretation is not acceptable in pathology , and the histology of the lesions in shennan 's ( 1914 ) case was no less benign , yet penetration of a pulmonary vein shennan suggested that our criteria of malignancy were at fault in was found . regarding such lesions as histologically benign . 
livingstone and klemperer ( i 926 ) concluded that the lesions in these cases were not as benign histolozicallv as the authors believed , and they thought that all showed atypical areas suggestive of malignancy . stout ( i 943 ) confirmed this , and considered the same applied to the original lesion in robinson and castleman 's ( 1936 ) case . however , in this connection it is well know - n that other malignant tumours and their metastases sometimes show deceptively benign histological appearances ( e.g. , chondrosarcoma and carcinoma of prostate , thyroid or kidney )  . the present case seems to be of the same nature as these and , in addition , has fe ' atiires similar to some others in which the histological structure was more malignant . kettle ( 1918 ) described an angioblastic tumour of the leg with metastases in the inguinal lymph nodes , ulrich ( 1921 ) a similar tumour of the lumbar muscles with metastases in the lungs , and downing and manory ( 1930 ) multiple angiomatoid lesions appearing in successive crops over an area which had been bumt some months previously . in the last two cases intravascular extensions were present , which strongly suggests that the lesions other than the primary mallory ( 1 914 ) , however , states that some caverwere blood - bome metastases . nous haema - ngiomas - not only arise in veins but permeate their wa ' ils and , at various placeg , rupture them , infiltrate the tissues and produce lesions which , while simulating metastases , are , in reality , in contiguity . this must be unusual , and would clearly be an incorrect interpretation of some of the cases cited where the lesions are far apart and particularly where peripheral lesions are associated with pulmonary ones . 
on the other hand , if benign angiomatous lesions extend into the lumina of veins , it cannot be concluded from observing intravascular extension alone that multiple lesions are metastatic rather than multifocal . in the present case , however , tumour tissue , not in cont ' lguity with large masses , is demonstrable within pulmonary vessels and , of special interest , witbin perivascular lymphatics and afferents to cervical lymph nodes . 
the only acceptable explanation of such findings is that the lesions in the cervical lymph nodes and lungs are embolic metastases . this case does not appear to shed any new light on those angiomatous conditions having a " system " distrib - ution described by theile ( 1904 ) , jores ( 1908 ) , wright ( 1928 ) and de nava , squez ( 1936 )  . in all these the authors described primary angioblastic tumours of the spleen with metastases in the liver . in theile 's case there were additional lesions in the lungs and stomach . willis ( 1948 ) , while c ' onceding these authors ' interpretations may have been correct , points out the possibility of the lesions in their cases being multifocal in origin . evidence suggestive of such cases being malignant metastasizing neoplasms ( local infiltration , poor differentiation , mitoses and intravascular extensions ) is not sufficient to classify them , witb certainty , as such . 
tumour tissue was found within perineural lymphatics of the scalp , in the afferent lymphatics of cervical lymph nodes ' and within pulmonary blood - vessels . i am indebted to j . 
a. - ( 1934 ) ' the spread of tumours in the human body , ' london ( chiirchill ) , p 148 . - ( 1948 ) ' the pathology of tumours , ' london ( biitterworth ) , p . 
of 9 : 10dimethyl - 1 : 2 - benzanthracene into the lungs of mice of strain street formed a part of a comprehensive study ( rask - nielsen , 1948 ) carried out in an endeavour to confirin or repudiate the hypothesis that tumours can be induced only in tissues that produce tumours spontaneously , whereas tissues that do not produce tumours spontaneously are unable to develop tumours even following very powerful carcinogenic action . the experiments showed that direct application of 0 - 5 mg . 
to this end , 3 : 4 - benzpyrene , 1 : 2 : 5 : 6 dibenzanthracene , or 20 - methylcholanthrene , all in doses of 0 5 mg . , were introduced directly into the lungs of street mice . since application of 9 : 10 - dimethyl - 1 : 2 - benzanthracene to the lung had previously induced thymic tumours ( rask - nielsen , 1948 ) , the present experiments were presumed to afford information also about the tumours which the three hydrocarbons would be capable of inducing in the thymus . these experiments will be reported in the present paper , which also includes the previous experiments with 9 : 10 - dimethyl - 1 : 2 - benzanthracene for comparison . benzpyrene , dibenzanthracene , or methylcholanthrene , 0 5 mg . , was injected , suspended in 0 01 c.c. 
the technique was as follows : in order to make the path of the needle in the lung tissue as long as possible , the needlewas inserted through the abdominal wall , immediately below the right costal border , and plunged through the diaphragm and longitudinally upwards through about two - thirds of the chest , before the suspension was injected . the mice used were litter mates of strain street , aged 5 to 7 weeks , divided into four lots . one lot was left untreated as controls , whereas the other three were injected with the respective hydrocarbons . 
as far as possible , the litters were divided equally in all four lots , an equal number of males and females in each in the corresponding experiments carried out with 9 : 10 - dimethyl - 1 : 2lot . benzanthracene ( rask - nielsen , 1948 ) litters of street mice were divided into lots of equal number , one of which was set aside as a control group . 
the effective total is the number of mice living to be as old as the youngest tumour - bearing mouse , namely , 12 months . in the sections prepared for the study of the spindle - cell sarcomas and thymic tumours , small adenomas were observed in a few mice . 
as shown in table ii , this phenomenon ' occurred following injection of dibenzanthracene in 4 out of 21 mice aged 5 - 7 months , following injection of methylcholanthrene in 2 out of 29 mice aged 6 and 7 months , and following injection of 9 : 10 - dimethyl - 1 : 2 - benzanthracene in 1 out of 9 mice aged 5 months . these figures apply only to the adenomas , one , or at most two or three , in each mouse , observed in one section through the lungs . nothing is known about microscopically visible adenomas in the remaining part of the lungs '  . neither is it known whether a corresponding production of adenomas had taken place in the mice showing no grossly visible it is worth mentioning that untreated tumours , but it is considered probable . street mice do not usually exhibit microscopic adenomas . serial sections from 48 mice , ranging in age from four to twelve months ( rask - nielsen , 1948 ) , showed one adenoma , only microscopically visible , in a mouse aged 12 months . 
the present experiments , therefore , appear to indicate that 9 : 10 - dimethyl - 1 : 2 - benzanthracene , methylcholanthrene , and dibenzanthracene , and presumably also benzpyrene , have induced pulmonary adenomas . 
the explanation why these growths were in most cases visible only upon microscopic examination is probably afforded by the short survival time of the mice ( table i )  . although the experiments allow of but an estimate of the development of pulmonary adenoma , they appear to indicate that injection of dibenzanthracene has induced a more marked increase in the development of adenoma than did injection of benzpyrene , methylcholanthracene and 9 : 10 - dimethyl - 1 : 2 - benzanthracene . the macroscopic adenomas as well as those visible only upon microscopic examination were of the ordinary , typical sub - pleural variety . in addition to the adenomas of the right lung , adenomas were also present in the left lung of a 7 - month - old mouse injected with dibenzanthracene and in the two 12 - month - old mice injected with 9 : 10 - dimethyl - 1 : 2 - benzanthracene . 
1rask - nielsen sarcomas of varying differentiation . associated with lymphosarcomatous thymic tumours . in two mice the spindle - cell sarcomas were table ii shows that spindle - cell sarcomas were observed following injection of benzpyrene in 2 out of 81 ( 2 - 5 per cent ) , following injection of dibenzanthracene in 14 out of 73 ( 19 per cent ) , and following injection of methylcholanthrene in 19 out of 59 mice ( 32 per cent ) , the effective total of experimental mice being the number of mice living to be as old as the youngest tumour - bearing mouse of all 4 groups , namely four months . 
the effective total of experimental mice was calculated on the basis of the number of mice living to be as old as the youngest mouse exhibiting a thymic tumour within all four groups , namely three months . with the exception of one spindle - cell sarcoma of the thymus , observed in a mouse injected with methylcholanthrene , all the growths were typical lymphosarcomas , made up of stem cells and accompanied by a frequently highly pronounced perivascular infiltration , particularly in the central areas of the lungs . 
one mouse injected with 9 : 10 - dimethyl - 1 : 2benzanthracene exhibited such violent infiltration without actual thymic tumour . the latent period of thymic tumours is given in table iii , which shows that the average latent period following injection of benzpyrene , dibenzanthracene , methylcholanthrene , and 9 : 10 - dimethyl - 1 : 2 - benzanthracene was 20 , 16 , 10 and 16 weeks respectively . the experiments revealed that the carcinogenicity of the hydrocarbons used , in doses of 0 - 5 mg . 
none of the six cases of generalized leukaemia was affected with thymic tumour . on the whole , the experiments showed that the order of carcinogenicity of the four hydrocarbons , as estimated from the incidence of tumours induced , varied , when 0 5 mg . 
of the same four hydrocarbons ( rask - nielsen , 1950a ) which showed that the lung tissue of street mice was susceptible to this dose of dibenzanthracene , less so to this dose of 9 : 10 - dimethyl - 1 : 2 - benzanthracene , but failed to respond to benzpyrene and iiethylcholanthrene . these recent experiments also showed that the thymus was particularly susceptible to direct application of 0 - 02 mg . 
of 9 : 10 - dimethyl - 1 : 2 - benzanthracene and methylcholanthrene , less to the same dose of dibenzanthracene and least to the same dose of benzpyrene . the incidence of thymic tumours induced by the four hydrocarbons was therefore decreasing in the same order following injection of a small dose into the thymus ( rask - nielsen , 1950a ) and a large dose into the lung . the results cannot be compared with earlier experiments , since thymic tumours following a direct carcinogenic action on the lung or thymus have not been reported before , with the exception of a few instances of thymic lymuphosarcoma observed after injection of methylcholanthrene into the lung ( esmarch , 1940b )  . but such remote effect was not observed . it is evident that the carcinogenicity of 9 : 10 - dimethyl - 1 : 2 - benzanthracene for the interstitial connective tissue of the lungs differed from that of the other three hydrocarbons . 9 : 1 0 - dimethyl1 : 2 - benzanthracene proved to be non - carcinogenic for this tissue , whereas the carcinogenicity of the other three hydrocarbons increased in the same order as their carcinogenicity for the subcutaneous connective tissue following application of the same dose into this tissue ( rask - nielsen , 19501 ) , i.e. 
kemp. from ae university institute , for human genetics , copenhagen . given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . from ancient times it has been observed that cancer often occurs familially the inheritance of tumour 's in human beings has been studied and discussed for centuries , and it has especiallv been the object of extensive systematic investigations during the last decade , , about 6 years ago , a group of collaborators at the university institute for human genetics - in copenhagen decided to make an attempt to investigate the the ' investigation has partlybeen carried genetics of various aspects of cancer . out in co - operation with the danish cancer registry , under the direction of j . 
clemmesen , the university institute of pathological anatomy , and the radium centre in copenhagen and numerous danish hospitals and clinics . the work was supported by kong christian x 's fond , landsforenigen til kraeftens bekaempelse and anders hasselbalchs fond . these investigations include genetic experiments ' in mice and studies in man . twin examinations were also initiated in co - operation with the cancer registry . the statistical - genealogical investigations have been performecl as a series of surveys each concerning a considerable number , usually sevetal hundreds , of probands or propositi with cancer of a ce - rtain - type , e.g. 
canc - er mammae , cancer uteri , cancer oesophagi , multiple cancer or leukaemia , picked at random from the population . these investigations were made by a number of physicians , each being a ecialist in the field his study coneems . 
the families have been studied thoroughly , and every case of cancer in the relatives has , so far as possible , been verified ; in that respeot these investigations differ from earlier , more compre hensive statistical studies in the heredity of cancer . 
cancer of the breast , uterus , stomach and rectum , their occasionally familial occurrence has been know - n for in order to elucidate the etiologic significance of hered ' itary many years . factors in these tumours , proband investigation has been carried out . jacobsen 's ( 1946 ) study on the heredity in breast cancer includes ' 200 probands , ( 197 women and 3 men ) with cancer of the breast . 
the investigations of the probands ' families comprise the following categories of relationsi : parents , brothers and sisters , grandparents and brothers and sisters of the parents . videbaek ( 1947 ) has investigated ' edigrees of 20 ' 9 patients with leukaemia and 200 control probands , using the same methods as jacobsen . the propositi - method of investigation is , of course , rather complicated and leaves room for several sources of error . it must be realized that the diagnoses and the information on the whole as regards the older or more distant groups of relations are rather unreliable . 
some of the grandparents , for instance , died more than 50 years ago , and at that time , diaanostic skill was more limited and hospital records and death certificates were more insufficient than to - day . furthermore , it is difficult to procure a comparable control , material , because the probands are normally more interested in the occurrence of cancer in the family than normal control probands are ; probably that is the reason why the information conceming the distant groups of relations in jacobsen 's control material is insufficient . also it must be considered that during recent years many cancer patients , e.g. 
suffering from skin cancer , get cured without knowing they have had cancer , and thus escapenotice by the investigator . it should also be pointed out that the calculation of the morbidity risk is a complicated method , and cannot always be relied on . 
lip and skin cancer , are not represented in the families of the probands , but cancer of most other forms , sites and types occurs in the relatives in about the same proportion as among the r ' elations of the control probands , and is comprised under the collect ' lve term " endogenous cancer as a whole . " according to jacobsen ( 1946 ) , a study of the case histories of the probands gives no basis for supposing that extemal factors play any important role in the development of breast cancer . 
an excess incidence of breast cancer among the female relatives of the probands and likewise a significant excess incidence of " endogenous cancer as a whole " in all the categories of relatives , both male and female , were found . this indicates hereditary predisposition as the chief factor in the development of breast cancer . breast cancer is dependent o ' n hereditary factors , and the tendency to this particular form of the diseas.e is bound up with an inherited predisposition to endogenous cancers endogenous cancer in general . 
the localization of the tumour is determined either by hereditary or extemal factors . in many pedigrees the general hereditary predisposition appears to be inherited as a dominant character . in the leukaemia proband material , the familial incidence of the disease was found to be at least 8 per cent . several types of leukaemia ( acute or chronic lymphoge ' neous or myelogenous , monocytic or stem - cell leukaemia ) may occur in the same family -  . 
the multiple occurrence of leukaemia in an individual family is usually not confined to some particular type , which indicates that leukal - imia is probably genetically a morbid - entity . among members of a family there seems to be an age correlation as regards the onset of leukaemia.. 
according to videbaek ( 1947 ) leukaemia , as such , is not simple domininherited ; it is a question of inherited disposition to the disease . ance or recessivity may possibly be excluded ; it may have a genetical - basis with incomplete dominance or polymeria ( homologous polymeric factors )  . hereditary predisposition to disease is generally regarded as polymeric . 
the expression of the character is furthermore dependent on the interaction between the genotype and environment . the incidence of pemicious anaemia is significantly higher among the relatives of patients with leukaemia than among the relatives of the control probands . the relation is probably due . 
among the relatives of the leukaemia patients there is a significantly excessive incidence cancer , of cancer as a whole , due to a high incidence of all forms of cancer . including leukaemia , is piobably a disease entity genetically dependent on a dominant gene common for all the different forms of " endogenous cancer . " according to videbaek 's oplinlon the - development of leukaemia seems to depend on various conditions , among others , on a non - specific hereditary predisposition to cancer , which is believed to be present in at least 20 per cent of thepopulation in general , and partly on one or several genes , the activity of which plays a role for the localization of the cancer to the leukon ; leukaemia seems to constitute an entity genetically and environmental changes influence the type of the disease . there is evidence that extemal factors also ' play a role in the development of leukaemia . 
of several cases of leukaemia in the same family is rather slight ; but the risk of getting cancer in near relatives of a patient with leukaemia is as high as almost 50 per cent . table iii shows some of the data from the breast cancer and leuka6mia material in relation to cancer risk . 
among the relatives of probands with cancer of the body of the uterus the incidence of " endogenous cancer " was high , and the relatives of probands with cancer of the cervix show the same frequency as the relations in the families of the patients with cancor of the cervix , of control probands . on the other hand , a relatively increased frequency of cancer of the oesophagus in this connection it is worth while to draw attention to the simiwas present . larity of the histological structure of cancer of the cervix and cancer of the in the families of probands suffering from cancer of the oesophagus oesophagus . investigated by mogensen ( unpublished data ) , cancer of the oesophagus was often found in a close male relative , the father or a brother of the proband . in these families , both the proband and the relative with cancer of the oesophagus 148 t . 
the differences could , however , not be significantly proved , owing to the insufficiency of the material , and the differences may be due to the fact that - the old propositi generally have less detailed and exact information about their relatives in earlier generations than the young propositi have . still the accordance of the experimental results and the clinical observations makes the significance of the latter probable . our investigations into the heredity of various aspects of cancer , have show - n that hereditary disposition is an important tumour - causing factor . it was found that it is the chief factor in the development of breast cancer , and many other tumours are more or less dependent on hereditary factors . 
the general cancer - tendency " ( likely not a general blastoma tendency ) occurs with considerable frequency in the population ( it is probablv higher than 20 per cent ) , and is possibly inherited as a character showing incomplete dominance . 
our investigations have shown that the tendency of tumour - localization has also a hereditary basis . the development of leu ' kaemia and cancer is probably due to a common hereditary . 
koller. from the chester beatty research institute , the royal cancer hospital , london . given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . xeroderma pigmentosum ( hence referred to as x.p. ) , is marked by rougheniing , dryness , pigmentation and ulceration of the skthe condition is attributed to hypersensitivity of the skin , particularly that of the basal cell layer , to sunlight . the disease was first described adequately by kaposi in 1874 ( hebra and kaposi , 1874 )  . 
occurs in the first year of life , often within a few , months after birth . first , erythema develops on the skin , on those regions which are exposed to light , followed by freckling and frequently by telangiectasis . from these regions epithelioma or basal - cell carcinoma develops . 
the malignant change may take place not only in the skin , but also in the cornea and conjunctiva the genetical and chromosomal basis of x.p. the hereditary basis of x.p. 
was first investigated by siemens and kohn ( 1925 )  . they analysed 76 families , and cockayne ( 1933 ) brought this more up - to - date with a further 43 family histories . in both groups of data the incidence of consanguineous marriages was very high : 24 out of 76 and 16 out of 43 respectively , were first - cousin marriages . 
jv.7. reeeived for publication march 5 , 1949 . the pressure mincer described in this article was designed to facilitate the preparation of tumour suspensions for a quantitative studv of the survival of tumours in the frozen state . 
the choice of principle and design was determined by the foflowing requirements : ( a ) rapid reduction of tumour tissue to a suspension of fine fragments and separated ceus , ( b ) ease of aseptic manipulation , ( c ) mechanical simpficitv and ( d ) ease of cleaning . the essential feature of this instrument is a grooved plunger which operates in a closed eyfinder . 
the tissue is placed below ' the plunger , and on the application of pressure is forced through the grooves to the upper face . the niincer and its component parts are shown in fig . 
1. all parts are machined from brass and are chromium plated . * it is important to have the plungers ( c and d ) fitted to the body tube ( a ) with the minimum clearance consistent with smooth working . 
the nose of each plunger fits accumtely into the recess in the base plate ( b )  . similarlv the nose of the fine pliiin er fits accurately into the base of the coarse plunger . 
the diameter of the bodv tube is 1 - 6 cm . thus i mof displacement of the plung - er corresponds to a volume of 0 - 2 ml . cleaning is facilitated and design of the plungers is simplified bv the prov - ision of a detachable base plate . the operating screw ( f ) is of sufficient length to extrude the plungers from the lower end of the body tube . the various parts of the mincer are sterilized separately in glass tubes . 
the mincer is shaken immediately to disperse the mince if the coarse plunger has been used the cap and operating in the suspending fluid . screw are detached and the fine plunger is inserted . tlle cap and screw are replaced and mincing is completmed . the suspension may contain coarse fragments of fibrous tissue or portions of iiiince which have clotted before dispersal in the suspending fluid . these may be removed either by l ' ow speed centrifugation , or by filtering the suspension througli a column of glass ballotini of suitable diameter . the mincer , which is illustrated in fig . 
1 , is the sixth of a series desigiied to all models have proved most satisfactory for operate with grooved plungers . the purpose for which they are intended , and it might be suggested that this tvpe of mincer will , for special purposes , be suitable for preparing suspensions of manimalian tissue or fowl embryos infected with certain viruses or bacteria . i wish toacknowledge my indebtedness to w . 
powell. from the department of experimental pathology , mount vernon hospital , northwood , middlesex . received for publication april 30 , 1951 . in previous communications ( powell , 1944 , 1946a , 1946b , 1947 ) it has been suggested that many of the typical properties of tumour cells can be explained by the occurrence of a derangement of the submicroscopic protein framework it was also suggested that the growth of malignant cells of their protoplasm . might be inhibited by chemical compounds able to link together the ultrastructural protein fibrils of tumour cells , and thus simulate the arrangement of comparable fibrils in normal cells . disulphide bridges constitute one of the more important classes of interconnections between the fibrils of native proteins . 
the quinone was recovered from the fitered bisulphite solution by precipitation with sodium carbonate or sulphuric acid . further purification was carried out by repeated formation of the additive bisulphite compound and subsequent precipitation as above . 
the following transplantable tumours were used : carcinoma 63 , a fibrosarcoma ( ac ) of strong a mice , a spindle cell tumour and a squamous cell carcinoma of cba mice , a rapidly growing undifferentiated sarcoma ( rb ) and a mammary adenocarcinoma of riii mice . the tumours were grafted subcutaneously in the right flanks of the mice , in each experiment the implants being portions taken from one tumour . in each experiment mice with actively growing tumours were divided into groups , control and experimental . 
1. - effect of 1 per cent phenanthraquinone upon the growth of carcinoma 63 in a strain in all the text - figures the horizontally placed numbers indicate days after inoculation , mice . and the concentration of phenanthraquinone in the food is given as a percentage of the combined weights of flour and rat cake powder . the quinone used in this experiment possibly contained a trace of the hot tasting impurity , but treated mice showed no harmful effects from it . 
2 and 3 demonstrate the similarity of the results obtained in different experiments with a particular strain of tumour when the rate of tumour growth is approximately the same , and the concentration of the drug is constant . 
1 , in which the growth of the control tumours is appreciably faster . here , with a higher concentration of the drug , smaller initial sizes and slower intrinsic growth rates of the tumours , the inhibitory effects of the treatment are greater than in c ontrol a . 
the inhibition of several spontaneous mammary tumours of a and riii strains of mice by the quinone was comparable with that of the transplanted tumours . also , the growth of a transplantable mammary adeno . carcinoma of riii mice and a transplantable squamous cell carcinoma of cba mice was strongly inhibited . thus the growth of a variety of different tumours can be inhibited by phenanthraquinone under the conditions described . unpalatable cause loss of weight of the treated animals . samples of phenanthraquinone containing sufficient impurity to make it gastro - enteritis some270 a . 
the latter causes prepared food to be unpalatable , and can give rise , if present in sufficient amount , to gastrobut , by comparing the effects of samples of quinone of varying degrees enteritis . of palatability , it has been established that the purest samples are the least toxic this greater inhibition to mice and inhibit tumour growth to the greatest extent . of tumour growth may be due in part to a greater intake of food , and therefore detailed investigation of the effects of phenanthraquinone upon of quinone . tumours and of its possible value in tumour chemotherapy is dependent upon the preparation of the drug in a pure state . its further purification and alternative methods of synthesis are under investigation . the additive compound of phenanthraquinone with sodium metabisulphite is serial intraperitoneal very readily hydrolyzed in vivo to yield the free quinone . injections in mice of aqueous solutions of the quinone - bisulphite compound in sub - lethal doses at frequent intervals indicate that the liberated quinone is very rapidly metabolized in the body . in view of the rapid destruction of phenanthraquinone in vivo and of the rapid growth rates of many tumours , it is essential that continuous administration of the drug be maintained in order to expose the tumour cells to an effective concentration of the drug for sufficiently long periods . failure to achieve this could permit a proportion of the tumour cells to grow and in the early stages of an experiment cessation of treatment with the divide . quinone may result , in some instances , in stationary tumours recommencing growth . 
on the other hand , similar tumours continually subjected to the action of the quinone fail to grow and often regress completely . in these preliminary experiments the simplest method of ensuring an adequate and continual supply of the quinone , namely , by adding it to the food , was adopted . however , this method is not entirely satisfactory . adequately controlled experiments in which individual mice receive known amounts of quinone at definite times are necessary for detailed studies of the action of the drug . very little of the quinone in a concentration of 1 to 2 per cent in the food ingested by a mouse can be available for action on substrates in tumour cells , since there is a very great disparity between this amount and the lethal dose given as the bisulphite derivative by injection . 
much of the fed quinone may not be absorbed from the food , and the greater part of that reaching the body fluids may be metabolized . sodium salts of the phosphoric and sulphuric acid esters of 9 : 10 - phenanthrahydroquinone have been synthesized and tested for possible inhibitory effects on tumour growth , but they have been found to be unsuitable for the treatment of tumour - bearing mice . 
the former very readily hydrolyzes to give the hydroquinone , which readily oxidizes to the quinone , and the latter is extremely resistant to hydrolysis . preliminary experiments upon the administration of thiol compounds concurrently with phenanthraquinone to tumour - bearing mice appear to indicate that its inhibitory action on tumour the latter reacts with thiol groups in vivo . growth may be due , at least in part , to this effect . 
powell may enable phenanthraquinone to function as an oxidation - reduction catalyst and so disturb the natural oxidation - reduction balance of tumour cells , possibly to a greater extent than that of most normal cells . in view of its considerable chemical reactivity , as well as of its affinity for proteins , phenanthraquinone despite the possibility that would be expected to have multiple effects on cells . phenanthraquinone has in vivo the mode of action postulated earlier , that is , of forming cross - linkages between ultra - structural protein fibrils , it is emphasized that its actual mode of action remains a completely open question . 
the available evidence favours the view that over a certain range of concentration tumour cells are more sensitive than normal cells to the effects of phenanthraquinone . it has been reported by mitchell ( 1948 ) that the combined antimitotic effect of " synkavit " and x - rays is greater than the additive effects of both agents used singly . 
in giemsa preparations feulgen staining was the membrane appeared as a faintly stained pink line . completely ineffective , whilst after iron haematoxylin the membrane lost its stain early in the decolorising process . 
the mercuric bromphenol blue staining technique of mazia , brewer and alfert ( 1953 ) was tried and found to stain the membrane feebly . this method , however , proved useful for determining the relationship of the membrane to the spindle structure . examinat - ion of a large number of preparations has allowed the following description to be pieced together : 1 . 
telopha8e. - as the cytoplasm constricts to form two daughter cells , the membrane also constricts and finally , after fusion at the centre , separates into two complete membranes - - one to each daughter cell . 
7. examination of fresh smears of the tumour by phase contrast microscopy demonstrates the occurrence of this membrane within the living cell and dispels ' any possibility of its origin being that of a fixation artefact . 
the membrane , under these conditions , appears as a thin clear zone form ' mg a sharp line of demarcation between the karyoplasm and the cytoplasm . because of the overlying cytoplasm it is difficult to focus and photograph . in this particular tumour mitosis within a persistent membrane appears to be the standard type of division . 
3. other than that it was taken from a section of a hepatic metastasis of a carcinoma of the tonsil no further comment or information is it appears then that the phenomenon as described is not a completely given . isolated case but does occur occasionally in other material , but not with the regularity shown in this instance . it would not appear to be a fixation ar - tefact since it can be determined by phase contrast microscopy in living cells . further , the fact that such a wide variety of fixatives will preserve it is against such a view . 
on the basis of the evidence available it seems unhkely that such an event has taken place , and if so , takes no cogmsance of the fact that similar appearances do occur irregularly in other tumours . although no evidence can be adduced to show that this membrane arises de novo within each dividing cell it is possible to offer a fairly plausible explanation it has been demonstrated by chambers and of its appearance in this fashion . ludford ( 1932 ) that the cytoplasm of malignant cells has a ph value lying between 6 - 4 and 7 - 0 whilst the intranuclear ph is in excess of 7 - 2 . 
under such conditions a gradient of ph would occur . it is suggested that at some point on this gradi ' ent the isoelectric point of neighbouring cytoplasmic structural protein is reached . this would involve the localised precipitation of protein and the ensuing membrane appearance . if such an event does occur it suggests that intracytoplasmic disturbance during mitosis is minimal , otherwise one would expect distortion and breakage of the membrane . a mechanism of origin as suggested above would account for the isolated examples of similar appearances in other tumours . 
doniach. from the pathology department , postgraduate medical school of london , w.12. received for publication february 20 , 1953 . the increasing use of radioactive iodine in clinical medicine has made imperitive the experimental study of its possible carcinogenic action on the thyroid gland . radioactive iodine , like stable iodine , is rapidly concentrated by the thyroid , bound to protein in its colloid in the synthesis of thyroxine , and then gradually released from the gland as part of the normally secreted hormone . during these processes the follicular cells of the thyroid are submitted to a course of ionizing irradiation from the i131 , which gives out f and y rays . 
the duration of this irradiation is of the order of days , since the half - life of 1131 is 8 days and the effective half - life of intrafollicular hormone is also of the order of days in the normal human gland . the carcinogenic activity of ionizing radiations in general has been repeatedly demonstrated during the past 50 years . recently , the effects of ft irradiation from newly developed sources have been studied . raper , henshaw and snider ( 1951 ) reported that single doses of 4000 to 5000 rep ( roentgens equivalent physical ) of f rays from a p32 source induced skin tumours in rats . 
they also found that 13 , 400 rep given in daily exposures of 50 rep over a period of 348 days proved carcinogenic . glucksmann ( 1951 ) found epitheliomas in 4 out of 24 mice in an area of skin exposed 14 months previously to 7900 rep given in 30 seconds by an electron beam . it has proved necessary in the treatment of graves ' disease with radioactive iodine to administer an amount of 1131 calculated to give an overall irradiation to the thyroid gland of 8000 to 10 , 000 rep ( wayne , 1952 ) in order to produce a complete remission of the disease . this dosage lies within the range found to be carcinogenic to other animal tissues . but owing to the uneven distribution of radioactive iodine in the thyroid one cannot make a confident direct comparison , from the dosage point of view , between the animal experiments mentioned above and 1131 therapy of humans . the mechanism of the experimental production of tumours of the thyroid by goitrogens has been greatly clarified by the new zealand team of workers ( purves , griesbach and kennedy , 1951 )  . 
doniach in evaluating the possible danger of carcinogenesis by j131 one must consider not only the direct action of , rays on the cells , but also the indirect action of radiation damage , which leads to diminished thyroxine synthesis and a resultant increased thyrotrophic hormone production . 
a suitable dose of 1131 might therefore lead to a summation of two carcinogenic stimuli ; a short exposure to / 6 irradiation and a long exposure to stimulation by the pituitary . 
the latter could be ensured experimentally by giving a prolonged course of antithyroid drug subsequent to a dose of j131 . doniach ( 1950 ) found that 10 out of 16 rats , given 32 microcuries of 1131 ( 15 , 000 rep to the thyroid ) , developed adenomas . in a series of 5 rats given additional methylthiouracil for 14 months , the thyroids showed a striking increase in adenomas and i - n one instance a metastasizing carcinoma , in contrast to 16 rats tested for the same period with methylthiouracil alone , whose thyroids showed only occasional adenomas . this summating effect of a carcinogen with an antithyroid drug was first demonstrated by bielschowsky ( 1944 , 1045 ) , who used the carcinogen acetamidofluorene combined with allylgoldberg and chaikoff ( 1952 ) have recently reported the development thiourea . of poorly differentiated non - colloid - forming thyroid cancers in 7 out of 25 rats injected 12 to 2 years previously with 400 microcuries of i131 . 
the authors attributed the cancer development entirely to beta irradiation , and not to thyrotrophic hormone stimulation . the object of the experiments described below was to repeat previous investigations of the action of 30 microcuries of j131 alone and combined with methylthiouracil in a larger series of rats , and to compare these results with the effects of 5 and of 100 microcuries . 
the trachea and thyroid attached were fixed in helly 's fluid ; the thyroid was then dissected off and weighed to the nearest milligrathe thyroids were embedded in wax , all glands which weighed 30 mg . 
were secstained by haemalum and eosin . tioned at 6 levels , so arranged as to traverse the whole thickness of the thyroid three sections cut at each level at 5#t were mounted and at regular intervals . one of them stained by haemalum and eosthe spare sections in general were used for extra stains . 
followed 24 hours later by methylthiouracil in the drinking water until the end of the experiment . ( 8 ) 100 lsc 1131 , 30 , uc 1131 , 24 hours later methylthiouracil till end of experiment . 24 hours later methylthiouracil till end of experiment . 
they were taken off the drug for 4 weeks from the 6th to 7th month and were given the i131 in two doses , half at the beginning of the experiment and half 24 hours before commencing their second course of methylthiouracil . in july , 1951 , eight months after this experiment was begun , the thermostat broke down one night and the heating was unfortunately left full on . 
a further 40 rats died or were killed during the course of the experiment , wasted from interfortunately the differences in current infection . the findings between the major groups proved striking even with the reduced number of rats . 
the nuclei were paler than those of neighbouring follicle cells and showed occasional mitoses . the affected follicles had lost their original lining , and had tended to flatten out apart from the altered appearthe normally scalloped perifollicular reticulum . ance of the cells , these " solid " follicles were easily differentiated by serial section from the apparent solid follicles produced by tangential sections of normal ones . they occured both centrally and peripherally in the glands and varied in diameter from 50 to 200 , t . 
to this end , serial sections were examined of the growing thryoids of 5 rats aged .23 days and 5 rats aged 100 days . in none of them were any microadenomas seen . 
nor were there any structures seen resembling these microadenomas in sections of the developing thyroids of 15 - day and 19 - day rat embryos . the incidence of microadenomas in the 9 controls of the main experiment is there were no macroadenomas found similar to those seen in there was a great variation in rats ' and thyroids ' weights . 
the lining cells were about 15 , t tall with a voluminous eosinophilic cytoplasm and capillaries were prominent , and oval vesicular nuclei . did not indent the follicular cells as in the controls . in reticulin preparations the capillaries appeared pushed back by the hypertrophic follicular cells . 
the arrangement of the cells included solid sheets , packed solid trabeculae , closely packed tubules containing pale fluid , microand macrofollicles filled with watery or deeply eosinophilic colloid , and gross cystic follicles distended with colloid and lined by a papillary epitheium . engorged sinusoids were prominent in many of the adenomas . 
the majority , however , presented a closer affinity to those seen in the rats treated with 5 , uc 1131 alone except that the cells were a little taller . their nuclei varied in size and chromatisthe adenomas presented an exaggerated and bewildering variety of cell types and differentiation fundamentally similar to those produced by methylthiouracil alone . 
some of the very large adenomas appeared to be derived from the confluence of more than one tumour . colloid secretion was well marked in many tumours and a papillary arrangement was not rare . 
however , the major part of the bulk of even the large tumours was cellular rather than secretory in origin . solid areas of undifferentiated spheroidal cells were seen within some tumours as with methylthiouracil alone . rat 35b tongues of adenoma tissue were seen penetrating the capsule at one however , in the absence of evidence of permeation of extra - capsular point . veins or obvious morphology of cancer , malignancy was not diagnosed . 
the majority of the follicles appeared active but could be differentiated from those seen in rats treated with methylthiouracil alone , as they were smaller , less regular and showed a tendency to a syncytial arrangement of their lining cells . 
the rats ' weights were similar to those treated by methylthiouracil alone , but the thyroid weights were very considerably reduced to an average size comparable with that of the rats treated with 100 , uc 1131 alone . the pituitaries were similar to those of the rats treated with methylthiouracil alone . 
was injected with the same syringe into a glass vial containing a small pledgelet of cotton - wool . fifteen hours later in vivo measurements were made of the radioactivity of the rats ' thyroids and of the 1 ml . 
19.chart showing the average in vivo count - rates of radioactivity of the thyroid glands of 8 rats following an intraperitoneal injection of 9 - 2 yc 1131 . after 24 hours 4 of the rats ( lowermost curve ) were put on to methylthiouracil in their drinking water . 
the findings in the remaining 4 are charted in the middle curve ; the calculated loss of radio - activity due solely to the radioactive decay of il31 is charted in the uppermost curve . count - rate of standard at 15 hours was 10 , 700 . due to the normal radioactive decay of 1131 . 
the differential effects on these functions of varying doses of 1131 have been recently reported in 3 publications . skanse ( 1948 ) studied the effects of 1 , 10 and 50 , tc i131 on young cockerels primed with a short course of thyrotrophic hormone . 
a definite inhibition of normal thyroid growth was produced by 10 j#c and 50 , uc 1131 after 16 days but not of body growth . all the thyroids showed a growth response to thiouracil , less marked in the chicks given the higher dosages of 1131 . but this response to thiouracil was much more inskanse ( 1948 ) remarks that though hibited after 38 days than after 26 days . the major part of the radiation was received irn the first few days , it took a much longer time to produce the full biological effect on gland function . the calculated dosages of total radiation to the thyroid glands were 1700 rep in the 1 , uc group , 13 , 000 rep in the 10 , tc group and 60 , 000 rep in the 50 1c group . feller , chaikoff , taurog and jones ( 1949 ) studied the effects on adult male rats of single intraperitoneal doses of 24 , 300 and 875 4ac of 1131 . 
the average uptake of 1131 into the thyroids was 40 per cent between 23 and 48 hours after those treated with the two injection and was about equal in all the animals . higher doses showed an abnormally rapid depletion of the iodine that had been trapped during the first 24 to 48 hours . chemical analyses showed an increasing depletion of thyroid 1127 from 2 to 3 days onwards after the injection of the 300 and 875 jtc 1131 , whereas there was no loss after 6 days in animals given 24 1ac . the values of plasma protein bound iodine were not changed in the 24 , uc group but they fell steadily in the rats injected with 300 , uc from 3 - 6 , ug . 
the thyriod uptake in the 1 , uc reached 53 per cent 48 hours after receiving a tracer dose of i131 , but was significantly less in all the other groups . response to subsequent thiouracil was similarly impaired when tested . 
the thyroids of animals which had received 50 and 100 , tc 1131 demonstrated no capacity to increase in weight on a 30 - day course of thiouracil instituted 64 davs after the initial dose of radioactive iodine . even 5 jtc initial t131 impaired the capacity of the thyroid to increase in size ; the 194 i . 
in the animals given an initial dose of 1 iac 1131 . measurements of body weight showed no disturbance in animals which had received up to 20 , uc , a slight impairment in the 50 and 100 , uc groups and a marked disturbance in the 300 , uc group . 
the calculated maximum total irradiation doses delivered to the thyroid in rep were respectively 1800 in the 1 , uc , 5800 in the 5 , uc , 30 , 000 in the 20 , uc , 80 , 000 in the 50 1ac , 197 , 000 in the 100 , tc and 288 , 000 in the 300 / tc groups . effects of internal irradiation by j131 on thyroid histology . findlay and leblond ( 1948 ) studied the thyroid histology of two rats killed 6 days after a single injection of 1131 calculated to have given a total thyroid irradiation of about 20 , 000 rep . the rats had previously been maintained on a low iodine diet . most of the follicles , especially the central ones had lost their colloid , and were replaced by irregularly arranged solid groups of epithelial cells , some of which showed nuclear and cytoplasmic degeneration . interstitial oedema was pronounced , fibrosis and lymphocytic infiltration slight . 
many follicles in the periphery and isthmus were normal autoradiographs showed evidence of accumulation of iodine only in the surviving colloid containing follicles of the periphery . goldberg , chaikoff , lindsay and feller ( 1950 ) studied the progressive changes in the thyroids of adult rats given varied doses of 1131 and killed after intervals of 1 day , 2 days , 3 days , 1 week , 2 weeks , 3 weeks , 1 month , 6 months 875 1 , c ( 330 , 000 rep to gland centre at 72 hours ) produced a total and 8 months . destruction of the thyroids evident within 48 hours , associated with an intense fibrinous oedema and acute inflammatory changes , followed by fibroblastic proliferation and arterial thrombosis . 
by 3 weeks there was a total collagenous permeation of the original gland structure , which by 6 months was replaced by 525 itc produced essentially similar changes . a band of hyalinised collagen . there were , however , at 5 months a few surviving thyroid follicles at the poles , lined by bizarre granular cells thought to resemble the so - called hurthle cells of the human gland . at 8 months surviving thyroid cells appeared more numerous 300 / , tc ( 110 , 000 rep at and formed occasional colloid containing peripheral follicles . 72 hours ) produced extensive swelling and desquamation of thyroid cells within 24 hours , followed during the next 2 days by further degeneration and by interstitial inflammation which appeared resolved by 8 days . at 4 months the glands were lobular and made up of mixed disturbed and normal follicles . at 8 months the glands appeared essentially normal , though the follicle cell nuclei were hyperchromatic and interstitial tissue was increased . 
produced total thyroid cell necrosis in 2 days . following the administration of minimal thyroid - lethal doses , parenchymatous degenerative changes first appeared within a few days in the gland and were followed by an inflammatory picture . intrathyroidal doses of 2 , uc / mg . 
 ( 1952 ) described the histology of the thyroids at varying time intervals after administration to rats of i131 as a part of the study of functional changes quoted above . after 2 days parenchymal degeneration and interstitial inflammation were seen centrally in the 300 , #c group ; no significant changes after 48 days the 300 , #c group showed a virtual were seen in the other groups replacement by fibrous scar tissue . the other groups , including the rats given 1 / uc , all showed hypertrophy of follicle cells and diminution of colloid . 
from 30 , 000 rep upwards there is an increase in rate of loss of organically bound iodine from the thyroid , associated with radiation damage to the follicles . in general , functional disturbances precede histological changes . 
from 5000 rep upwards the thyroid cells are able to respond to activating stimuli by hypertrophy , but hyperplasia proves abnormally limited . bizarre thyroid cells are seen histologically up to 12 years and are accentuated by thiouracil treatment , increasing in number in thyroids submitted to 5800 rep upwards . 
even though irradiated thyroid cells are histologically abnormal , they appear by means of hypertrophy to synthesize enough thyroxine to maintain the animals ' health and growth after dosages up to 30 , 000 rep . calculation of dosage of radiation to the thyroid . calculations of radiation dosage to the thyroid following the administration of radioactive iodine can at the best only be rough estimates because of the con196 i . 
 ( 1949 ) pointed out that since the maximum range of the beta particles enmitted from i131 is comparable to the dimensions of the rat 's thyroid , a geometrical factor must be calculated to estimate the reduction in radiation dose at points near the surface of the gland as compared with the centre . 
by following the uptake of radioactive iodine into the thyroid and its sequential loss from the gland it is possible to estimate the total the conversion factor for rep was calculated by evans ( 1947 ) : radiation dose . 1 , kc per mg . 
the animals were all killed after 15 months and 5 , tc 1131 and methylthe rats on methylthiouracil alone showed no carcinomas . thiouracil led to the production of a greatly increased incidence of adenomas , these findings confirm the additive action of radiobut no definite carcinomas . active iodine in carcinogenesis by antithyroid drugs , and show that the range of 2270 to 16 , 200 rep was more effective than 380 to 2700 rep . in contrast , the methylthiouracil given to rats treated with 100 , tc i131 produced less adenomas this inhibition at a dosage level of 8400 to 60 , 000 than methylthiouracil alone . rep confirms the findings of maloof et al . 
 ( 1952 ) of the greatly diminished capacity adenomas of a similar of heavily irradiated thyroid cells to undergo hyperplasia . morphology to those present in the thyroids of rats treated with antithyroid drugs were found in the animals given 5 , uc and 30 , uc 1131 alone , the latter appearing more effective than the former . 
doniach and pituitaries of this group are significant . there are two points of evidence that a certain amount of thyroxine was being synthetized . firstly , the animals grew from an initial weight of 100 g . , when the radioactive iodine was first administered , to the full weight of 250 to 350 g . , at the end of the experiment . secondly , the pituitaries showed a good coinplement of well granulated alpha cells . purves and griesbach ( 1946 ) demonstrated that the iiaintenance of the alpha granulations is dependent upon a supply of thyroxine ; the pituitary alpha cells disappear after thyroidectomy and are restored by the daily administration of one - fifth or more of the normal thyroxine requirements . nevertheless , the beta cells showed the typical changes of a raised level of thyrotrophic hormone secretion described by griesbach and purves ( 1945 )  . this suggests that the irradiated thyroid glands were secreting at least one - fifth but less than the normal thyroxine requirements . the diminished capacity of the irradiated glands to regenerate prevented a return to quantitatively normal thyroxine synthesis , thus perpetuating an increased thyrotrophic hormone output and accounting for the hypertrophy of the thyroid cells . another possibility to be considered is that irradiated thyroid cells differ from normal in requiring a higher level of thyrotrophic hormone stimulation in order to secrete a normal quantity of thyroxine . similar changes in the thyroids and pituitaries were observed to a lesser extent in the 301 , c 1131 treated rats and to a slight extent in the thyroids only of the 5 , tc i131 group . the bizarre thyroid cell nuclei observed by maloof et al . 
 ( 1952 ) in 1131 treated rats were seen in the present experiment , and were also accentuated in rats treated by additional thiouracil . one cannot say whether the neoplastic cells arise from these or from their apparently normal neighbours . they do , however , constitute evidence of irreversible irradiation changes . 
we do not know the life span of thyroid cells , but it is almost inconceivable that they could survive and function without replacement for15 months , a period of nearly one half the animal 's life span . neoplasms in general arise in tissues made up of actively dividing cells . they do not arise from irreversibly differentiated cells such as neurones or keratinized epidermal cells . the chances of the development of neoplastic thyroid cells following irradiation are likely to vary directly with the number of post irradiation mitoses which take place . this number will be increased in the thyroid bythyrotrophic hormone stimulation . the proportion of cells which become neoplastic is presumably related to the dose of irradiation . the absence of adenomas in the100 , uc 1131 treated group is considered to be due to a lower incidence of dividing cells following irradiation than that which followed in the 30 and 5 1tc groups . adenomas might have been found after afurther 6 months since they were present , albeit in small number , in the 100 , tc treated rats submitted to additional thyrotrophic hormone stimulation by a prolonged course of methylthiouracil . 
one of these was a malignant anaplastic carcinoma transplantable into hosts without thyroxine deficiency . it was not dependent on a high level of thyrotrophic hormone , did not concentrate iodine , grew rapidly , and killed its hosts within a few weeks . the main difference between the action of antithyroid drugs alone and combined with the carcinogen acetamidofluorene is that with the latter treatment thyroid tumours appear considerably earlier and in greater number . bielschowsky ( 1949 ) found also that acetamidofluorene produced thyroid adenomas in the absence of a chemical goitrogen in the regenerating thyroids of subtotally thyroipurves et at . 
 ( 1951 ) have concluded that neoplastic cells arise dectomised rats . spontaneously in the normal rat thyroid and that the carcinogen acetamidofluorene seems to act by speeding up their formation , their further development and growth , however , being dependent upon a high thyrotrophic hormone level . this has been produced experimentally by the prolonged administeration of antithyroid drugs , or by prolonged iodine deficiency ( wegelin , 1927 )  . the results of the previous ( doniach , 1950 ) and the present experiments show that the action of radioactive iodine is comparable with that of the carcinogen acetamidofluorene . in addition , radioactive iodine on its own leads by partial inhibition of thyroxine synthesis to a prolonged rise in thyrotrophic hormone level . 
from the description of goldberg and chaikoff ( 1952 ) , the experimental thyroid carcinomas which resulted from an extremely intense irradiation with radioactive iodine appeared to be of a high grade malignancy . the possibility that a carcinoma of low grade malignancy might become anaplastic must be considered after the findings in the transplantation experiment of purves et al . 
 ( 195 1 ) ; the probability however is unknown . dangers of carcinogenesis to humans treated with j131 . after an exposure to 1800 rep from j131 , maloof et al . 
 ( 1952 ) found a normal thyroid histology in rats 11 years later . but following 5800 rep and upwards , hypertrophied thyroid cells and increasing numbers with abnormal nuclei were seen , accentuated by a short course of methylthiouracil . these cells persisted tracer doses of j131 in clinical medicine fall into the first category , up to 1 1 years . those therapy doses which lower thyroid function below normal into the second . from the experimental findings in rats it would appear that 9000 rep to the thyroid in graves ' disease must act chiefly by functional damage leading to diminished thyroxine production rather than by actual total destruction of the gland . this is borne out by the brief descriptions of chapman and evans ( 1949 ) , williams et al . 
 ( 1949 ) and shapiro ( 1950 ) , who noted no histological evidence of gross destruction in the thyroids of thyrotoxic patients after treatment with radioactive the hyperplastic thyroid of the thiouracil treated rat is not strictly iodine . comparable with the hyperplastic thyroid of thyrotoxicosis . there is certainly as yet no clear - cut evidence that the latter is associated with a high blood level 200 i . 
a raised production of thyrotrophic hormone . the conditions may thus be fulfilled for tumour production , i.e. , an initial radiation damage followed by prolonged thyrotrophic hormone stimulation . one would expect the latent period in humans to be many years , and it is possible that in time thyroxine production and thyrotrophic hormone levels might return to normal and the chances of tumour production be much diminished . this could be ensured by deliberate thyroxine medication . 
on present data therefore the carcinogenic danger of radioactive iodine appears to be the initiation of irreversible radiation damage to the thyroid . this renders a greater number of cells liable to tumour formation when later stimulated by thyrotrophic hormone than in the non - radiated gland . 
a proportion of the resultant tumours are likely to be carcinomas , probably of a low grade malignanoy . anaplastic carcinomas may develop , especially when very destructive doses of i131 are used . the dosage lies within the known carcinogenic range . in view of the experimental findings in rats ( of various strains and in different countries ) , it is probable that the present methods of clinical i131 therapy in thyrotoxicosis may eventually prove carcinogenic . 
the radioactive iodine was found to increase the formation of thyroid adenomas as compared with controls in the 5 and 30 1tc groups but not in the 100 , uc group . out of 20 rats treated with combined 30 , tc 1131 and methylthiouracil 5 developed thyroid carcinomas . 
the radiation dosage range to the thyroid of 2270 - 16 , 200 rep in the latter experiment is likely to include the dosage aimed at , of about 9000 rep , in the treatment of graves ' disease . 
the carcinogenic danger of radioiodine therapy is regarded as due to the production of irreversible cells changes in the thyroid , which render them more liable than normal cells to tumour formation when stimulated to undergo hyperplasia . at the same time , a dosage strong enough to interfere with thyroxine synthesis by radiation damage to the thyroid leads indirectly to a prolonged increased output of thyrotrophic hormone from the pituitary . this constitutes a perpetual stimulus to hyperplasia of the thyroid cells . it is thought that the present dosage of i131 used in the treatment of graves ' disease may eventually prove carcinogenic . i am grateful to drs . 
hall. from the cancer research laboratories of the new zealand branch of the british empire cancer campaign , medical school , university of otago , dunedin . received for publication january 1 , 1951 . kseveralmethods are available for studying the effect of prolonged stimu ( 1 ) repeated implanlation of the sex organs by gonadotropic hormones : tation of pituitaries obtained from castrates , ( 2 ) implantation of testis or ovary into the spleen of a castrated animal ( biskind and biskind , 1944 , 1945 ) and ( 3 ) parabiotic junction of a normal animal to a gonadectomized litter - mate . the first method is not very effective owing to the generally short survival of the grafted pituitaries . 
the ingenious method of the biskinds ( 1944 , 1945 ) , however , does not allow one to study how the increased hormonal output of the stimulated gonads affects the accessory sex organs , whereas in parabiotic animals this effect can be observed . 
we have fouiid in the literature only one reference to an investigation in which a similar experimental procedure has been used . strombeck and ekman ( 1949 ) fed a diet containing a.a.f. 
a breakdown of the junction is a rare event . operative mortality was practically nil , but in the second or third week after the operation a great number of pairs died due to incompatibility of the partners no pair sbowing signs of " parabiotic intoxication " survived longer than 5 weeks . the use of more closely inbred rats ( three generations of brother - sister mating ) did not lower the rate of mortality at this critical period . in most experiments acetylaminofluorene - vvas given by stomach tube to the normal partner , who received the carcinogen in doses of 4 mg . 
the treatment with the carcinogen was started 3 to 4 weeks after parabiosis had been established , when consistent gain in weight and the good condition of the animals showed that the operation had been successful . 
were given on 6 consecutive days by stomach tube and in another the carcinogen was given to the twins with their diet ( 4 mg . dailv to each rat for a period of 4 weeks )  . all the rats which received a.a.f. by stomach tube were fed the ordinary stock diet , consisting of meat meal , pollard , bran , maize meal , bone flour and whole wheat supplemented by cabbage . 
was administered with their food were kept for 4 w - eeks on a diet consisting of skimmed milk and whole meal flour supplemented by codliver oil and cabbage . subsequently they received the same food as the other pairs . the twins were killed when a tumour was suspected or when loss of weight indicated that they were in a precarious state of health . pituitary , testis , accessory sex organs , breast , suprarenal and liver of most pairs were studied histologically . 
the pituitaries were fixed in mercury - saline ( 6 per cent ) , the other organs in zenker or neutralized formol - saline . for staining of the pituitaries a modified papanicolau tecbnique was used ' the other organs were stained with ha , matoxylin - eosin or according to weigert - vail cxieson . the material presented consists of 20 r . , airs of parabiotic rats treated with a.a.f. 
the remaining 2 pairs consisted of litter - mates , none of which had been castrated . of the 6 pairs not treated with a.a.f. , 4 were combinations of a normal male joined to a castrated brother , while in the other 2 the male was joined to a spayed female . in addition , cc single " ' rats were treated with a.a.f. 
 - in a similar manner as the normal partners of parabiotic pairs . five young males received by stomach tube 15 doses of the carcinogen and to a group of 10 , 24 doses ofa.a.f. 
only benign lesions of the liver were seen when the animals were killed in the 52nd week of the experiment . in the group treated with 24 dosos malignant hepatomas were also found from the 37th week onwards . 
was seen in any of the rats joined to a partner to whom the carcinogen had been administered . these livers were macroscopically and histologically normal . in the pair of rats who had received a.a.f. 
the hyperplastic seminal vesicles contained macroscopically visible iiodules which were situated near the cephalad margin of the organ ; in addition a fifth neoplasm of microscopic size was found . in all 5 instances the fundamental histological change was the invasion of the muscular coat of the seminal vesicle by atypical glandular epithelium and therefore the lesion must be considered an adenocarcinoma . 
11 and 12 illustrate the appearance of the only tumour of the seminal vesicle found previously in our material . this large anaplastic carcinoma arose in a seminal vesicle of an albino rat of the " sheffield " strain which had received a diet containing a.a.f. 
and para - amino benzoic acid . in another case both seminal vesicles and prostates formed one large mass containing multiple cysts filled with yellowish creamy material . adhesions connected this mass with loops of the bowel . this - lesion was of inflammatory origin and no neoplastic changes were found in it although many sections were studied . as already mentioned not a single neoplastic lesion was found in the castrates joined to brothers treated with a.a.f. in the pair where both received the carcinogen with their food , the kidnev of the castrate showed a lesion of microscopic size which we have not found so far in rats treated with a.a.f. 
the whole picture resembled that of a benign tubular a4enoma of the kidney . morphological sign8 of increa8ed androgen secretion in the normal parabiotic male partner . the changes to be described were found in most cases and were i ' ndependent of the administration of a.a.f. 
the testes were frequently increased in size . their tubules were normal ; the leydig cers were large , having ample cytoplasm and in some animals their numbers h ' ad increased . 
the hyperplasia of prostate and in tlle latter the glandular part of seminal vesicle has already been mentioned . the organ was more stimulated than the outer mesenchyma ' l layer . this disproportionate growth led to compression of the epithehal folds lined by high cylindrical cells . 
the suprarenals of the intact partner differed histologically from those of the castrated twthe glomerulo 'sa of the former was denser and consisted mainly of cells the cytoplasm of which stained wen with eosin . however , thev - were not as frequent the fascicularis contained signet - ring cells . as in the example ifustrated by selye ( 1947 ) on p . 
133 of his text - book of endoin the castrate the glomerulosa was formed by large pale cells , the crinology . fascicularis was free of signet - ring cells and the reticularis was hyperaemic and better developed than in the normal twthe kidneys of the normal partner their surfa ' e was more granular and were larger than the ones of the castrate . histologically the tubular apparatus showed slight degenerative changes , the , connective tissue being more prominent , especially in the outer layer of the cortex . the pituitaries of the norma - i litter - mates were consistently smaher than in the castrates and were sometimes even ' below the weights normally found in our in these pituitaries the numbers of acidophils were increased rats of similar size . with a corresponding decrease in chromophobes . 
griesbach with faintl the periodate - feulgen technique , the cells gave a slight schiff reaction . this agglomeration of basophils was probably not a - permanent structure . zahler ( 1950 ) has described the appearance of nests of basophils in the pituitaries of rats treated with testosterone . the state of parabiosis did not seem to influence the growth of the rats , nor to increase their suseeptibihty tor a.a.f. in the two pairs where two normal males had been joined together , the animals treated with 24 doses of a.a.f. reached the same size as " single " rats , which had received the same dosage of the carcinogen . 
amounts as used in this investigation are administered . in this respect the carcinogen behaves like the steroid hormones which ass only from one to the other partner when extremely high concentrations are reached in the eirculat ' ion of one of them ( zeckwer , 1946 )  . the liver tumours obtained appeared after approximately the same interval as in " single " rats treated with similar amounts of a.a.f. metastases occurred in two instances , proving the malignant character of these hepatomas . 
although the hepatomatous liver was situated in the same peritoneal cavity as the hver of the untreated partner , the latter remained perfectly normal . there was not the slightest indication for an agent derived from the malignant hepatoma affecting the liver of the litter - mate . as mentioned in the introduction a constant flow of gonadotropins passes from the castrate to the normal partner stimulating its gonads . however , no tumours in this respect our experiments failed where of the testicles have been found . those of biskind and bisldnd ( 1945 ) and of twombly , meisel and stout ( 1949 ) these authors obtained tumours in testes which had been grafted succeeded . into the splee ' n . 
two further points should be considered . spermatogenesis is inhibited in testes situated in the abdomen and consequently the morphology of such gonads is considerably altered ; in additionil transplantation of a testis into the spleen involves traumatic injury . these two factors further studies seem are not present in the experimental procedure used by us . to be required to elucidate the conditions essential for tumour induction in the male gonad , a problem which has been recently reviewed by lipschutz ( 1950 )  . the androgens secreted by a testis transplanted into the spleen do not reach the general circulation and therefore the accessory sex organs of rats bearing such the normal parabiotic male joined to a gonadecgrafts are not stimulated . tomized litter - mate shows the effects of increased androgen secretion . in the pairs not treated with a.a.f. 
this stimulation did not lead to tumour formation in any of the target organs , but in the group treated with the carcinogen five tumours appeared in the seminal vesicles of normal partners . 
we have never found this type of tumour occurring spontaneously in the new zealand strain , nor have we seen it in " single " rats only once a carcinoma of the seminal vesicle was observed treated with a.a.f. in an albino rat of the " sheffield " strain treated with a.a.f. our combined material represents the post - mortem findings of nearly a thousand male rats treated with a.a.f. in the rat spontaneous tumours of the seminal vesicle are extremely rare and we have found in the literature only the case reported by flexner and jobling ( 1907 )  . in men , tumours of this organ are also of very lazarus ( 1946 ) has critically reviewed the cases reported , infrequent occurrence . and since then only a few additional observations have been recorded ( mccree , ] 1948 ; gee , 1948 )  . in the case of the thyroid , the carcinogenic action of a.a.f. 
the liyperplasia induced by increased amounts of androgens secreted by the stimulated testis enhances tlle susceptibflity of the seminal vesicle to the carcinogenic action of ' a.a.f. the ' fact that tumours of macroscopic size were found only in rats treated with 24 or more doses of 4 mg . 
and not in animals receiving less suggests that considerably more of the carcinogen is required to bring about tumour development in the stimulated seminal vesicle than was needed for the induction of adenomata in the hyperplastic thyroid ( hall , 1948 )  . that only one minute tumour of the seminal vesicle was found in an intact male joined to a spayed female is probably due to the lower dosage of a.a.f. given to this group and not to the nature of the gonadotropins of the spayed female . only 17 to 20 doses of the carcinogen were administered because great difficulties were encountered in keeping alive pairs of opposite sex for periods necessary for tumour development . however , our material is not large enough to decide this question . to summarize , we consider the tumours of the seminal vesicle to be due to a hormonal imbalance created by gonadotropins in the normal partner . these hormones , by stimulating the gonads of the ' normal partner , increase the androgen production of its testes and therefore are responsible for the hyperplasia of the although unable to promote tumour growth in the testes they , seminal vesicles . bring about conditions in which an accessory sex organ becomes susceptible to the . 
the possibility cannot be excluded that stin more prolonged androgenic stimulation may ultimately bring about tumour formation in the seminal vesicle without the aid of a.a.f. addend - um ( received for publication february 7 , 1951 )  . after the above paper had been submitted an additional pair of parabiotic rats had to be sacrificed . in this pair a normal male was joined in parabiosis to a castrate , the former having received 20 doses of a.a.f. 
left seminal vesicle with loops of small intestine and a hard small nodule was found embedded in these adhesions at approximately 3 mdistance from the upper margin of the fig . 
in normal male rats joined in parabiosis neoplasms were only to castrated or spayed litter - mates have been described . obtained in animals receiving the carcinogen and never in their untreated littermates . ( 2 ) benign or malignant tumours of the liver were found depending on the amounts of the carcinogen given and on the duration of the experiment . 
the mahgnant hepatomas metastasized to the lungs of the animal bearing the cancer of the liver , but never " infected " the other partner . these neoplasms are considered to be due to two factors ; tlle carcinogenic action of a.a.f. 
w.7. received for publication august 22 , 1949 . it has been suggested from studies of the action of inhibitors of s - metabolism on tumour induction by carcinogenic hydrocarbons that s - metabolism and the this association is process of carcinogenesis are closely linked ( crabtree , 1947 )  . emphasized when the properties of azo - carcinogens are considered . 
the products of their metabolism have been shown to inhibit the activities of urease ( potter , 1942 ) and succinoxidase ( elson and hoch - ligeti , 1944 ) , two enzymes dependent kensler , dexter and rhoads upon intact sh - groups for their proper functioning . ( 1942 ) studied the relative inhibitory action of a series of " split - products " on a standard diphospho - pyridine - nucleotide system , and sought to correlate this suggestive activity with the carcinogenic power of the parent azo - compound . parallelisms were found , but a clear - cut relationship did not emerge . much of this work has been based on the conception ( experimentally verified for p - dimethylaminoazobenzene by stevenson , dobriner and rhoads ( 1942 ) , and for azo - benzene by elson and warren ( 1944 ) ) that an azo - carcinogen is initially metabolized by the process of reductive fission with the liberation of an amine and a diamine , and that the latter is the effective carcinogen by virtue of its capacity to interfere with normal enzymic . 
a survey of the azo - compounds so far tested for carcinogenic activity makes it evident that in some cases the amine moiety of the reduced molecule plays a significant role in determining potency , e.g. 
on comparing p - dimethylaminoazobenzene with its p ' - methyl derivative , the introduction of the p ' - methyl group causes a change from a very strong to a very weak carcinogen . the present work was undertaken to probe this problem further . isomeric aminoazotoluenes were chosen as suitable for a study of the relative contributions made by the amines and diamines which would arise from their reduction . 3 ' - azotoluene ( o : o ) and the non - carcinogen well - known carcinogen 4 ' - amino - 2 2 ' - amino - 4 : 5 ' - azotoluene ( p : p ) served as bases of reference , and their behaviour was compared with that of the hybrid compounds o : p and p : o . 
water , at room temthe crystalline hydrochloride suspended in etoh perature ( mehner , 1902 )  . was neutralized with ammonia , and an equal volume of boiling water added . the free base was re - crystallized from aqueous etoh . 
the first characteristic abnormalities visible in the livers of rats differed from those commonly observed in the livers of mice , though the final pictures of tumour emergence were similar in both species . building up the general picture four grades of cellular change have been used . in rat livers perilobular necrosis constituted the primary change , and this was followed by regeneration of liver cells in the necrotic areas . in some regions of regeneration microscopic nodules of hepatoma made their appearance , and later these became visible to the eye as dull grey patches on the surface of the lobes . often a simultaneous intense proliferation of bile ducts was manifest in one or more lobes , and the early phases of cholangioma formation would coincide with the development of small hepatomas . 
no metastases were ever found . in mouse livers no preliminary necrosis was observed , but the cellular pattern became abnormal with great irregularity in the disposition and size of the nuclei . further distortion was accompanied by the formation of cells with giant vacuolated nuclei , which preceded the emergence of microscopic hepatomas . 
the fully developed hepatomas of mice were more malignant than those of rats as judged by the degree of atypical growth , though again no metastases were ever found . it will be noted that the earliest histological changes mentioned sometimes occurred in both rats and mice consuming the basal diet without dye - addition . this response of the liver to diets inadequate in several essential food factors may well reflect the basic disturbance of liver function which is a necessary precursor to the carcinogenic action of azo - compounds . ( b ) relative carcinogenic activity of the six isomeric amrnoazotoluene8 . considering the data summarized in table i from the point of view of the relative carcinogenic potency of these isomers , three points emerge : 1 . 
crabtree the possible influence of sex on the growth rate was assessed by keeping the rats of each series separated into three sub - groups containing respectively 10 males , 10 females and 5 males + 5 females . 
2. - growth curves of rats fed on the basal diet , with addition of 0 06 per cent oor p - toluidine . in the work reported here , the two carcinogenic isomers would yield o - toluidine , while 3 of the 4 growth - stimulating isomers would yield p - toluidine as the primary products of reductive fission . the possible effects on growth of these two toluidines were therefore tested . forty - five rats of the 52 strain , each weighing 110 - 130 g . , were divided into three groups of 15 . 
two groups were fed on the basal diet containing 0.06 per cent of either o - toluidine or p - toluidine , and the third group received the basal diet alone . 
each group of 15 rats was subdivided into three separated groups of 5 males , 5 females , and 5 containing males and females . in the latter group two litters were born during the first 3 months , but none subsequently . 
on the other ha ; nd , miller and miller ( 1947 ) emphasize the importance of the protein constitution of the host , and associate the likelihood of tumours developing with the degree to which an azo - compound is " bound " to the proteins of rat livers . in the case of rats consuming p - dimethylaminoazobenzene this fixation is prominent , but does not occur in species resistant to the carcinogenic action of the substance . these differences can only be attributed to the special properties of the proteins characteristic for each species . the " split - product " hypothesis . kirby ( 1945 ) has reviewed the work bearing on this hypothesis of the mechanism of carcinogenesis by azo - compounds , and throws doubt on its validity by citing several examples which fail to conform with its main premises . 
the experiments demonstrated that o : o and o : p were carcinogenic , while p : p and p : o were non - carcinogenic . reviewing these results in the light of the " split - product " hypothesis , no correlation between carcinogenic activity and the potential diamine fission products is found . 
p : p and o : p should yield the same " innocuous " o - toluylene diamine , while o : o and o : p should yield the same " active " .p - toluylene diamine , but the facts are otherwise , since each of these pairs contains one carcinogen and one non - carcinogen . by contrast , when the amine halves of the reduced molecule are considered , the results show that the two isomers which should yield p - toluidine on reductive fission - p : p and p : o - are non - carcinogenic , while the pair yielding o - toluidine in brief , if carcinogenic activity is indeed - o : o and o p - are carcinogenic . a consequence of the action of the " split - products , " it is the amine , and not the diamine , which determines the subsequent biological response . the literature contains several examples of the controlling influence which a methyl group exerts on carcinogenesis , when attached at a para position with miller and baumann ( 1945 ) compared the activities respect to the azo - group . of the o ' , m ' , and p ' methyl derivatives of p - dimethylaminoazobenzene with that of the parent compound . 
the m ' - compound was extremely active ( 7 rats out of 8 bearing liver tumours in 4 months ) , the o ' - compound moderately active ( 4 rats out of 9 bearing tumours in 8 months ) , and the p ' - compound was very weakly carcinogenic ( 1 rat out of 10 with a tumour in 10 months )  . 
nagao ( 1941 ) also found that p ' - methyl - p - dimethylaminoazobenzene was only a weak carcinogen , and that the substances formed by placing a further methyl group in the ring carrying the dimethylamino - group were entirely innocuous . sugiura ( 1948 ) tested a series of derivatives of p - methylaminoazobenzene containing an additional methyl group in the o ' , m ' or p ' positions . the order of potency - m ' > o ' > p ' was similar to that mentioned above . all these observations seem relevant to the present work . 
the common feature is the great loss of tumour - producing activity which results when a methyl group is added in the p ' position to the parent carcinogenic molecule , forming derivatives with the potentiality of liberating p - toluidine on reductive fission . a working hypothesis is put forward tentatively in an attempt to co - ordinate the data outlined above . 
when the structural features and the biological action of these six isomeric azotoluenes are considered , a correlation is evident between their growth - stimulating properties , their lack of carcinogenic power , and the presence of an additional methyl group in the position described . 
crabtree and hilbert ( 1888 ) fed the three isomers of acetotoluidine to dogs . o - acetotoluidine was oxidized to an aminophenol and isolated as methylbenzoxalone , while mand p - acetotoluidines were excreted as acetylated amino - benzoic acids . it may therefore be conjectured that all azo - compounds containing a methyl group at a position para to the azo - group will yield p - aminobenzoic acid as a final product of their metabolic degradation within the rat . the experiment pictured in fig . 
six isomeric aminoazotoluenes have been tested for their carcinogenic activity in the livers of mice and rats , when added at 0 06 per cent concentration to a standard , semi - synthetic diet . 
since the detoxication of p - toluidine can produce p - aminobenzoic acid , the possible relation between folic acid metabolism and the mechanism of cancer induction is considered . i am greatly indebted to e . 
bruzzone. from the department of experimental medicine , national health service of chile , santiago . received for publication june 9 , 1949 . abdominal fibroids induced in the guinea - pig by a - oestradiol ( nelson , 1937 ; lipschutz and iglesias , 1939 ) are prevented when progesterone or other 3 - ketosteroids are administered simultaneously with the oestrogen ( lipschutz and vargas , 1941 ; lipschutz , 1944 ; lipschutz , iglesias , bruzzone , fuenzalida and riesco , 1948 )  . 
he has shown that bacterial growth can be understood in terms of certain simple mathematical relationships , in spite of the fact that the molecular mechanisms involved are extraordinarily complicated . in this paper an attempt is made to see if such an approacli may help to throw a little light on the problem of tumour formation and growth . 
a brief review of carcinogene8i , 3 . the various ways of producing cancer have been reviewed in a recent paper by boyland ( 1952 )  . but it will help for the purposes of the present communicatioii to provide a summary of the results . ( a ) chemical carcinogens . - the first chemical compound to be identified as a carcinogenic agent was 1 : 2 : 5 : 6 - dibenzanthracene ( cook , hieger , kennaway and mayneord , 1932 )  . since that time , the polycyclic hydrocarbons have been investigated systematically from this point of view . 
a certain degree of specificity has been observed and it has been correlated to some extent with the presence of a region of high electron density ( k region , schmidt , 1939 ) , within the molecule . some recent work has suggested , however , that the carcinogenic activity of the polycyclic hydrocarbons may be related in part to their ability to undergo autooxidation and liberate free radicals ( alexander , 1952 )  . the second class of carcinogenic compounds includes mustard gas ( heston , 1950 ) , nitrogen mustards ( boyland and horning , 1949 ) , epoxides , mesyl glycols ( haddow and timmis , 19051 ) , etc . 
these compounds are all known to act as alkylating or esterifying agents and probably prodtice their effects on the body by this type of reaction . certain inorganic salts of beryllium and zinc can produce tumours ( bagg , 1936 )  . there are also a number of other chemical compoiinds , showing little relationship 260 f . 
urethane it might , from the ( nettleship and henshaw , 1943 ) is an example of this kind . nature of its amide group be expected to attack the secondary forces of cohesion known as hydrogen bonds , which play a most important part in maintaining the structure of biological organeres such as chromosomes ( ambrose and gopalayengar , 1952 )  . ( b ) physical methods of producing cancer . - it appears that the simple process of implanting an inert object such as a disc of bakelite ( tumer , 1941 ) or sheets of cellophane ( oppenheimer , oppenheimer and stout , 1948 ) into a tissue can produce a tumour , while an extreme change of temperature produced by the application of carbon dioxide snow ( berenblum , 1929 ) can have a similar effect . although the ionising radiations can induce cancer , it is probable that their mode of action is indirect and is due to the formation of free radicals ( butler and conway , 1952 )  . ( c ) biological carcinogens . - the well - known rous sarcoma can be propagated by virus particles ( rous , 1936 )  . certain parasitic organisms are able to cause a malignant change to occur in the tissue ( e.g. , phytomonas tumefaciens in plants , smith , 1916 )  . 
the spontaneous tumours , whose origins are unknown should perhaps be considered to be due to the action of a biological carcinogen . it was pointed out by haddow in 1938 that there was a striking multiplicity of tumour - producing agents and , as can be seen from the summary given above , the number has greatly increased since that time ( hartwell , 1951 )  . 
the agents appear to have one feature in common ; they may be expected to lead to a more random configuration of the molecular structure within the cell , than is present in the normal tissue . 
we will try , at a later stage , to see ff the models may have any bearing on the behaviour of real biological systems . ( a ) classical treatment . ( 1 ) simple growth . - we will consider the very simple system shown in fig . 
is considered to contain a dilute solution of metabolite molecules ; the molecules could , for example , be molecules of amino acids . it will be assumed that the solution is ideal ; i.e. , that the molecules are quite independent of each other and do not interact with the solvent molecules . 
the work in thermodynamic which has to be done is generally known as osmotic work . nomenclature the work which must be done on the system is in this case expressed as a change ofentropy . 
1. in real systems the energy required for the decrease of entropy is in some cases balanced by a change in the potential energy , e.g. , the formation of a chemical precipitate is accompanied by a decrease of potential energy , because forces of attraction occur betw ' een the molecules forming the precipitate and a closer approach of the molecules leads to a decrease of potential energy . ( 2 ) growth with differentiation . - we will at this stage consider the problem of growth with differentiation since it has been the subject of a beautiful study by tyler ( 1939 ) , using sea urchin eggs , although the treatment is not in this case strictly thermodynamic . the cells at the two cell stage of embryonic development are isolated . 
2d. it is possible to compare the energy requirements for growth in the case of the normal and dwarf embryos by making use of the principle of dimensional analysis ( galmeo , rayleigh , tolman , buckingham )  . these principles tell us that there is a relationsfii . 
the war thickness is d / a / 2 units and fie , 2 . ( in d the longer line between arrows indicates r . ) suppose that an invaginot d - umits as in the real dwarf embryo shown in fig . 
3b the molecules are shown to be arranged in a hnear sequence , inert gas . in the form of a polymer chain . in this case the chain is coiled in an irregular in fig . 
3a there are very few regtrictions upon they are called , in the three figures . it is only necessary that one molethe arrangement of the metabohte molecules . cule should not coincide with another and that they should an be confined within in fig . 
3b we see that the number of possible ways of the sphere of radius r . arranging the units is greatly reduced , because each unit must be maintained at a 264 e . 
a statistical thermodynamic treatment makes it possible to express the entropy ( s ) of the systems in terms of the number of conformations with the most probable energy . if n is the number of conformations with this energy : k log n , where k is a constant . the logarithmic form occurs because the result is obtained by integration over a large number of terms . it wffl be seen that the simple process of polymerization must lead to a decrease in the entropy of the systems and that any process leading to a preferred orientation of the polymer molecules must lead to a further decrease . let us now consider a case in which we have a number of the spherical units of the kind shown in fig . 
4b is shown a similar corection of units which are now arranged another . if we now consider the configurational entropy in a definite sequence or pattem . of the polymer chlains in an individual spherical unit , we see that in the case of fig . 
4b the restriction in the configurations of the spheres , produced by arranging them in a definite pattern , wfll also restrict the number of configurations of the polymer chain 's inside the spheres . quantitatively , the actual energy difference . 
the building up of biological structures takes place by a complicated process of cefl division in which metabohte molecules are constantly being absorbed from the surroundings , while energy sources are broken down to carbon dioxide and water and subsequently discarded ; i.e. , we are dealing with an open system in which there is a constant flow of material . 
4 and let us suppose for a moment that they represent living cells containin ' g protein , nucleic acid , polysaccharide and lipid molecules . in fig 4a they are considered : to be unicellular organisms which do not interact in any way ; in fig . 
now we may be able to produce a series of reversible processes whereby we could bring metabolite niolecules in dilute solution first within the semi - permeable membrane of the cell by a process of isothermal compression , then into an orientated configuration by a series of compressions and extensions in the sohd state . because no process can be more efficient than a reversible process the energy changes involved would be the least that could possibly occur in the formation of these structures . in the actual biological process of synthesis , where events take place at a finite rate , the energy requirements for synthesis must be greater than they would be in our hypothetical models , we have also neglected for a reversib - le process . changes of internal energy in the systethe formation of polymers involves the synthesis of chemical bonds between the nionomer units . 
the synthesis of the peptide bond - co - nh - from the free amino acids h2n - c - - cooh is an endothermic reaction which requires a supply of energy , and the same is true for other biological polymers . 
the two factors of irreversibility and internal energy changes will require that all growth processes will involve a continuous supply of energy in excess of that which would be required for the entropy component in our hypothetical models . 
but these factors do not prevent us from calculating the entropy term in a real biological system . fowler and guggenheim ( 1939 ) point out that material which is not in an equihbirum state still has a definite entropy . 
ambrose the cell , the number of units of which they are composed is very large and although the number of configurations is restricted it is still very large . in the succeeding sections in the present paper , we shafl attempt to make some purely quahtative observations on what we might expect to be the differences in energy requirements for the growth of normal cells and tumour cells , in so far as the entropy component is concerned . 
m corresponds , as before , to b corresponds to the entropy state of the materials incormetabohte molecules . porated within the embryonic cell . this is represented as a lower entropy than the cell a . 
because the cell contains within itself a molecular structure which is capable of interacting with other cers . b ' + d represents the cell when differentiated and fulfilling its proper function . 
an attack upon the cer which leads to a more randoni condition of its parts can lead to an increase of - entropy . we might expect to find therefore that such an attack would lead to a reversal of the sequence shown in fig . 
a cell ' vh11 remain in an equirbrium state , or wir grow , depending upon whether the rate of synthesis is equal to or greater than the rate of degradation of the striictural components . 
5c the cell is considered to be in an equihbrium state , i.e. , in the condition of dynamic equilibrium in a polypbase system suggested by gowland hopkins ( quoted by baldwin , 1947 )  . 
5c represents the energy requirement for the carrying out of the proper function of the cefl in its differentiated foras for the autosynthetic system , the synthesis of extra - cellular material is also associated with a condition of dynamic equilibriumany of the extracellular proteins , for example , represent unstable svstenis . insuhn exhibts a spontaneous denaturation jaqueous solution which appears to be due to an autocatalytic mechanism ( waugb , 1946 ; ambrose and elliott , 1951 )  . in order that a steady concentration of materials can be present in the organism , continual synthesis must occur to make up for the loss . 
the regionrrepresents the reservoir of energy producing material in the cell , together with its associated system of enzymes . in the normal differentiated cell all the levelsof a , dandrare steady . 
we note that the present simple theory accounts for the existence of the steady state in in any system in which a large number of chemical differentiated organisms . reactions occur in a stepwise sequence , the products of one reaction being utihzed by the next , as occurs in a series of enzymatic reactions in a unicerular orgamsm , the growth curve should obey an exponential law ( hinshelwood , 1952 ) , i.e. , the rate of growth is proportioned to the number of cers present . various possible explanations for this difference have been suggested as for example by rashevsky ( 1945 ) who proposes that each cer prodiices some substance which inhibits the growth of other cells . 
chromosome breakage may therefore be a gross manifestation of the kind of change in the synthetic mechanism which can produce cancer cells . gopal - ayengar ( 1953 ) has made an extensive studv of the chromosome abnormalities which are found in normal cells and in tumour cells , particularly ascites tumour cells . 
the ducts can be seen in cross section ; it will be seen that they are arranged in the form of honow cylinders with the cefls forming a single layer round the wall . in a case of carc ' moma it is the duct cers which become mahgnant . 
6. as in the analysis given in section iib , it would be possible to calculate the energy differences for the three forms of growth , if a reversible process for changing from one form to the other could be visuahzed . 
the increase in surface area associated with the formation of the new surface requires that work must be done to provide the surface energy . in addition , a posit ' lve pressure must be apphed witbin the bubble . 
6 will require still more . were an elastic solid much more work would have to be done to form the hollow it would be similar to blowing up a rubber tube . 
the successive stages in the loss of differentiation , in this case of duct carcinoma , do therefore appear to represe ' nt states of increasing entropy , at least in so far as the observed changes of form are concemed . phenomena of this kind are again clearly irustrated in the very complete account of the genetic origin and morphology of the melanomata by dawson it is generally agreed that the naevi are derived from cefls which would , ( 1925 )  . in normal tissue , be engaged in building up a layer or sheet type of tissue . 
the transition from a layer structure to a warty form and finafly to the smooth malignant tumour represent successive stages in the reduction of surface area / unit mass for the hquid case . 
as in the previous example , the elastic case would require successively increasing amounts of energy for the formation of the structure corresponding to a departure from the uniformly spherical form . we will now examine some changes associated with tumour growth , which are taking place on a molecular scale , and can be considered in terms of the analysis in fig . 
this is because the refractive index of the fibres measured parallel to their length is different from the refractive hidex measured perpendicular to their length ; the brightness of the fibres , for a given thickness , is in these pictures a measure of the degree of molecular orientation which has been achieved during the synthesis , e.g. , the fibres of fig . 
the fibroblast cells are still laying dow - n fibrous material , but it is much more wavy in appearance , as was the case for the tumour derived from the uterine wall . 
the tissue was stained for coragen , using the technique of van gieson . the tumour showed a considerable reduction in the amount of collagen present as compared with the nornial tissue . 
20 still gives a considerable colour due to - collagen . it will be seen that there is practically no detectable birefringence m the tumour region which is on the right of the field . this result suggests that there is a relative decrease in the amount of orientated fibrous material deposited in the tumour region . in fig 9 is shown a section of normal breast tissue , ' hotographed under the polarizing microscope . 
a fibrous structure can be seen but there is a complete absence of any birefringence . the experimental evidence given above therefore appears to be in agreemedt with the general conclusion of section iv , that tumour growth corresponds to disordered states of high entropy and that in so far as the forms of growth and molecular orientation of fibrous protein are concerned the successive stages of malignancy do in fact appear to be associated with successive increases of entropy . it may be pointed out that a number of other instances could be quoted in favour of the general picture . 
the principle may help a httle in understanding the relationship between carcinogenesis and growth inhibition , as first described by haddow ( 1947 )  . if the carcinogens do in fact have a disorganising effect upon the synthetic system , it is more likely that in a given instance they wir disorganise the structure to such an extent that it can no longer function at all . 
of the hydrocarbon suspended in arachis oil , by intraperitoneal injection , in no case , however , even with 100 mg . and the rats weighed daily as a group . average 1 : 2 : 5 : 6 - dibenzanthracene , after 9 days a second injection of growth rate of any group of rats observed . 1 : 2 : 5 : 6 - dibenzanthracene was given and again no the same amounts of constant effect was observed , but after several days considerable fluctuations in the growth rates occurred , and eventually 21 days after the first injection all the groups showed a lower average weight than the control group , which had although the average weight of the received injections of arachis oil alone . group which had received the two injections of 100 mg . 
1 : 2 : 5 : 6 - dibenzanthracene was slightly lower than that of the other group , there was no direct relation to the amount of hydrocarbon given , and the rats receiving 50 mg . had a higher average weight than the group which had been given 25 mg . 
of the other five left as controls . pituitary extract daily had practically no effect on the growth of the animals , but when the amount was increased to 1 c.c. 
1 : 2 : 5 : 6 - dibenzanthracene to rats maintained on a 20 per cent protein diet causes no immediate inhibition of growth , a delayed toxic effect occurs which ultimately results in death of all the animals . 
when the length of time survival of the dibenzanthracene - treated rats on the 20 per cent protein diet is compared with that of the similarly treated animals maintained on the 10 per cent protein diet , as shown in fig . 
6 , it is seen that although in the latter group there is an immediate growth inhibitory action , the animals survive for a longer period after treatment . ( mean survival time of 17 animals on the 20 per cent protein diet was 44 days after dibenzanthracene injection , whilst that of the same number of animals on the 10 per cent protein diet was 67 days . ) effect of 1 : 2 : 5 : 6 - dibenzanthracene on tail growth in rats maintained on a 20 per cent protein diet . an attempt was made to see if any growth - inhibiting action of 1 : 2 : 5 : 6 - dibenzanthracene could be demonstrated by measuring the increase in tail length of rats treated with this hydrocarbon . young male rats of about 50 g . 
the animals were lightly anaesthetized with ether , placed on x - ray film in the prone position , and skiagrams taken of the bones from the pelvic region to the tip of the tail . 
of stilboestrol was injected in all but two of the animals growth of the tail was markedly into each rat . inhibited , and in 3 rats tail growth practically ceased . 
the difference in the diets used is mainly of the protein content and , since the fat content of the diets is the same , their total it would thus appear that the procalorific values do not differ appreciably . tective action of the 20 per cent protein diet is directly attributable to its high this is confirmed by the protective action of the 25 per cent protein content . protein diet , which differs from the unprotective 10 per cent protein diet only in protein content . the growth - inhibitory action of the hydrocarbon does not appear to be the result of a direct action on the anterior lobe of the pituitary gland causing interadministration of growth hormone ference with growth hormone secretion . to rats maintained on the high protein diet and treated with dibenzanthracene does , in most cases , bring about some increase in growth , but appears at the same time to increase the toxic action of the hydrocarbon since this growth dibenzanthracene is increase is rapidly followed by the death of the animal . only very slightly soluble in body fluids , and when administered by intraperitoneal injection remains in the peritoneum for a considerable period of time . metabolism of the compound probably takes place by combination with since this combination is sulphydryl - containing substances as its first stage . not the direct cause of its growth - inhibiting action ( elson , goulden and warren , 1947 ) , it is probable that a later stage metabolic product is responsible for this effect . 
one of the effects of the growth hormone is to increase the total metabolism of the animal , and it seems likely that this results in increased absorption and metabolism of the dibenzanthracene from the peritoneum of the treated animal . 
of 1 : 2 : 5 : 6dibenzanthracene per rat usually results in very little growth inhibition for about 14 days , after which the animals , at varying intervals , lose weight rapidly and die . 
howatson. * from the cancer research department , royal beatson memorial hospital , glasgow . received for publication july 6 , 1953 . several attempts have been made to purify and isolate the particulate agent or virus associated with mammary carcinoma in certain strains of mice , with a view to its identification and the study of its morphology by electron microscopy . the conclusions reached by different observers , particularly about the average size of the particles identified with the active agent , have been considerably at variance . passey , dmochowski , reed and astbury ( 1950 ) and dmochowski , and passey ( 1952 ) have described an extensive investigation in which biological tests for the presence of the agent in extracts of tissues and milk from high and low cancer strain mice were correlated with the appearance of the same extracts in the electron microscope . 
they conclude that the tumour - inducing activity is associated with typical particles of diameter 200 - 300 a , preparations of high activity containing large numbers of these particles and inactive extracts containing none or very few . graff et al ( 1949 ) , using as starting material milk from riii and c57 mice , and c57 mice foster - nursed by riii mothers , claim to have isolated from infected milk , after treatment with chymotryspin and differential centrifugation , particles of diameter 750 - 1000 a which they regard as the mouse mammary carcinoma virus . 
 ( 1950 ) but this could hardly account for the large difference in size . porter and thompson ( 1948 ) carried out an electron microscope investigation of epithelial cells grown in tissue culture from spontaneous and transplanted adenocarcinoma in c3h mice . 
they found that in three out of six preparations spherical particles were present in the cytoplasm of the cells . these had a double structure - a dense centre portion of diameter about 750 a and a less dense outer zone of diameter approximately 1300 a . 
they conclude that their findings are consonant with the view that the particles represent the milk agent . huseby , barnum and bittner ( 1950 ) , on the other hand , observed particles of diameter 500 - 2500 a in extracts of milk and mammary gland tissue but were unable to distinguish in the active extracts any distinctive particles which could be identified with the virus . in view of the long period required to obtain results from biological tests for the agent , any method of assaying rapidly the activity would be of great value in experimental work . 
howatson it was thought that a new attack on the problem involving a direct comparison of extracts of tumours from agent - carrying and agent - free mice of the same strain might give a conclusive answer . for our experiments we were fortunate in having available , through the courtesy of b . 
a commercially produced atomiser or spray gun operated from the high - pressure air line was found to be satisfactory the specimen was deposited by directing a fine spray of droplets of the extract to be examined on to filmed specimen mounts . 
each droplet is a representative sample of the extract and most are small enough to be included in a single micrograph . thus to obtain information about a given extract it is necessary to photograph only a few droplet patterns instead of searching the whole field . 
as far as possible the treatment of agent - containing and agent - free tumours was identical . a weighed amount of tissue was homogenised for a few minutes in a glass homogeniser , the suspending fluid ( usually distilled water ) being added slowly to give a final tissue concentration of 2 per cent . 
both extracts contain much particulate material together with fragments of mitochondria , collagen fibres and other cellular debris . these larger fragments can be removed if desired by further centrifugation , leaving an array of macromolecular particles on a background composed of low molecular weight material , which is not generally resolved by the electron microscope . 
the particles are approximately spherical and , although covering a range of size , are predominantly of diameter 250 - 300 a , the size attributed to the milk agent by dmochowski and passey ( 1952 )  . the particle - size distribution based in each case on the measurement of 200 particles , is shown in histogram form in fig . 
the number of particles present in a given extract depends somewhat on the type of tumour , the efficiency of homogenisation and the centrifugal forces employed but provided the simple standard procedures outlined were adhered to , the variations were small . despite careful search , it was not found possible to detect any significant difference in the number , size or appearance of the particles present in the droplet patterns obtained from extracts of agent - containing and agent - free tumours . the failure to observe any difference in the tumour extracts is readily understood when similarly prepared extracts of normal organs are investigated . nearly every case examined , including spleen , liver , marrow , brain and lymph 396 a . 
14 ( c ) shows that diameter . in size - distribution , these presumably normal particles do not differ significantly from those derived from tumour tissue . it is apparent that simple fractionation procedures are not sufficient to effect a separation enabling the pathogenic particles to be distinguished from the numerous cell particulates of similar size and shape which are also present . 
14. - size distribution of particles in extracts of ( a ) agent - containing mouse m ary tumour , ( b ) agent - free mouse mammary tumour , and ( c ) normal mouse spleen . the field of the smaller animal viruses , where it has been satisfactorily overcome in only a few cases . 
an attempt was made to differentiate in this way between normal and infective particles by studying the action of the enzymes trypsin , chymotrypsin , ribonuclease and deoxyribonuclease on extracts of agent containing and agent - free tumours . 
the buffer salts , which crystallise on drying , can fairly readily be distinguished from organic material , but the latter shows a tendency to aggregate in the presence of electrolytes . this effect had previously 398 a . 
a comparison was made between extracts of the mill hill 2 endothelioma which has all the attributes of a " virus " tumour and the chemically - induced grch 16 tumour which in addition rous no . 
1 sarcoma and has no such " filterable " properties . biological tests for the infectivity normal chicken tissue extracts were examined . of the various preparations were carried out in many cases . the methods adopted were similar to those previously described , and need not fig . 
extracts of the first type regularly excite tamours on injection into susceptible birds , whereas the second type of extract , though apparently of similar particulate content , is these micrographs may be compared with fig . 
9 , which completely innocuous . shows an extract from a normal chicken spleen . it is again evident that the infective particles , if present , are masked by the large quantity of other particulate material which occurs in normal as well as tumour tissue extracts . 
the lower limit of size ( 600 a ) given by claude ( 1946 ) appears to be arbitrary . certainly most of the particles observed were smaller than this , though some shrinkage during desiccation may occur . 
the presence of still smaller cytoplasmic particles in cells fixed by osmium tetroxide has recently been demonstrated by high resolution electron micrographs of ultra - thin sections ( eaves , 1953 , private communication )  . there is no doubt that the particles observed in the present experiments are products of the disintegrated cells , the spraying method giving consistent and conclusive evidence of their presence in the extracts . 
the dissolving action of trypsin and chymotrypsin is a strong indication that they are protein in nature . the crystalline trypsin used was tested for the presence of particulate material by spraying on to coated specimen mounts a solution 100 times as strong as that used in the experiments . 
12 is shown a partially disintegrated mitochondrion with what appears to be an extrusion of particulate material . a group of mitochondrial membranes largely devoid of contents is illustrated in fig . 
it seems very probable that some of the particles observed in tissue extracts are derived from such disruptions . it is clear that the range of size of the normal cell particulates covers that of the smaller animal viruses . this is a major difficulty in the isolation of any presumed virus by purely physical methods such as differential centrifugation or in chemical composition also , the microsomes are found to be strikfiltration . ingly similar to certain known viruses , so that attempts at chemical or enzymatic separation may also fail . there remains the possibility of using the highly specific methods of serology , though even these may not distinguish normal from pathogenic particles unless the latter are of extraneous origin . the difficulty of identifying tumour - inciting particles , particularly the milk agent , by electron microscopy is enhanced by lack of independent data on size . estmates have been made by finding the centrifugal force required to clear the supernatant of infectious material . there have also been some attempts to deduce the approximate size of the milk agent by measurement of sedimentation rates in an ultracentrifuge ( kahler and bryan ( 1943 ) )  . 
the presence of normal particles and aggregates makes the interpretation of such experiments somewhat doubtful , but the evidence is in favour of the association of the infectivity with rather small particles of molecular weight 3 - 5 million , corresponding to a particle diameter of at most a few hundred angstroms . it is remarkable that no attempts appear to have been made to determine the approximate size of the milk factor particles by means of gradacol membranes , as has been done for example in the case of several fowl tumour viruses . 
by this method it should be possible to determine conclusively whether the infectivity is associated with particles in the large ( > 1000 a ) or small ( < 300 a ) size range . it is clear that further work is necessary to establish the elementary physical properties of the tumour viruses . 
the examination of tissue culture cells and of thin sections of fixed tissue may give valuable information , but is not likely to be conclusive because of the difficulty of relating the observations to biological tests . on the other hand , the method of extracting homogenised tissue , while convenient for biological testing , presents problems arising from the presence of much contaminating material which can only be satisfactorily solved by the discovery of some action highly specific to the virus , which can be used for its isolation . in conclusion it is emphasised that the presence in tumour extracts of particulate material in the size range of the smaller animal viruses is no indication that the particles are in any way related to virus activity , unless it can be conclusively 400 a . 
i fowl sarcoma material which had been subjected to repeated applications of cold possessed a greater tumour - producing activity than those prepared from similar material which had not been subjected to this ' treatment ( selbie and mcintosh , in explanation of this finding selbie and mcintosh ( 1939 ) suggested 1939 )  . that the freezing broke up the tissue fragments , ' and thus allowed a greater liberation of virus than would otherwise have been possible . in the course of some recent quantitative investigations into the effects of cold and desiccation on the rous no . 
the chromatographic study of riley ( 1950 ) in which adsorption of the rous virus was shown to take place on to a column of diatomaceous earth suggested that adsorption would also take place when this virus was passed through berkefold filters which are likewise composed of diatomaceous earth . in the present work an attempt has been made to assess the decrease in the tumour - producing activity of suspensions of the rous no . 
i fowl sarcoma virus experiments are also reported which which berkefeld filtration brings about . were designed to show whether or not this decrease was due to adsorption of the virus on to the berkefeld candle during filtration , and in addition , an investigation has been carried out to find whether repeated freezing and thawing affects the filterability of the rous virus . 
the tumour was removed , cut small with scissors , and disinte a mechanical blender ; the method ated in has been described in detail in a previous paper ( epstein and cook , 1951 )  . 
filtration was continued until the filter candle was quite empty , as shown by air bubbles being sucked through after the filtered after use each filter candle was tested by passing through it 80 c.c. 
a sample was taken from the resulting filtrate , part of it being set aside ready for inoculation and part being used to make dilutions in serial tenfold steps with sahne ; a portion of each dilution was kept for inoculation . experiment8 4 and 5 ( recovery of virus from filter )  . - these experiments were performed in their early part as a repetition of the foregoing in which samples of a virus suspension and the berkefeld filtrate derived from it were couected and diluted in serial tenfold steps with saline for inoculation . in addition , however , a sahne wash of the filter was prepared ; a sample was taken from the saline wash , part being set aside undiluted for inoculation and part being used to make dilutions in serial tenfold steps with saline . 
a portion of each dilution was kept for inoculation . experiment8 6 , 7 , 8 and 9 ( effect of freezing and thawing on filterability )  . disintegrated tumour was prepared and in ' each of these experiments was divided into two samples , one being left untreated and the other being subjected to repeated freezing and thawing . 
suspension berkefeld untreated frozen and thawed x 6 f : 61trate untreated frozenandthawed x 6 untreated frozen and thaweci x 6 - .10% virus suspension berkefeld filtrate - .10% virus suspension berkefeld filtrate 10% virus suspension berkefeld filtrate untreated frozenandthawed x 6 untreated frozenandthawed x 6 untreated frozenandthawed x 6 untreated frozenandthawed x 6 dilutions of preparation and number of tumours arising from 4 inoculatioins of each . 10 - 2 10 - 11 10 - 4 10 - 5 10 - 0 10 - 11 2 + ( 2 ) 0 3 + ( l ) filter test . - - - n prod . 
now , froni the results shown in table ii , it is clear that an appreciable amount of the tumour - producing activity losb when virus suspensions were passed through berkefeld candles could be recovered by washing saline through the latter after use . in the experiments shown in table ii the filters when tested retained in each case both chr . 
aureu8 making it impossible for the lost tumour - producing activity , recovered on washing the filters with saline , to have been due to cells . this recoverability of at least part of the tumour - producing activity lost on filtering rous virus suspensions gives further support for . 
epstein freezing and thawing were passed through a berkefeld candle to give a filtrate ' , as where those made from similar untreated disintegrated tumour were so filtered . the application of repeated freezing and thawing to rous sarcoirna material has thus been found not to increase the ability of the virus contained in it to pass berkefeld filters . 
the present work has also shown ( table 111 ) , in confirmation of that previously reported ( epstein , 1951 ) , that repeated freezing and thawing of mechanically disintegrat - ed rous tumour materizal did not release more virus on extraction than where no such treatment was applied . 
from these two facts it appears that repeated freezing and thawing has no action on the rous virus likely to make berkefeld filtrates of treated tumour material , prepared as described here , more active than those made from untreated material . 
the results shown in table iii demonstrate this clearly ; they are at variance with those reported by selbie and mcintosh ( 1939 )  . in attempting to relate the results of the present work to those of previous investigators with which they do not agree , two possible explanations require consideration : firstly , it must be remembered that selbie and mcintosh carried out their during that time the rous virus may possibly have investigations 13 years ago . undergone variations affecting its properties which might account for the variations in the experimental results obtained . secondly , the method of extracting the rous virus employed by selbie and mcintosh ( 1939 ) consisted of coarse mincing of the tumour material followed by repeated freezing and thawing was then applied to the grinding with sand . resulting preparation , and may well have contributed to the achieving of complete disintegration of the tumour and the maximum extraction of virus from it . 
in the present work , however , disintegration of the tumour material was brought about mechanicall before the cold was applied , and would seem to have been so complete that the repeated freezing and thawing could not have any further this is considered to be the most likely explanation of the contradiction effect . between the findings reported by selbie and mcintosh ( 1939 ) and those recorded in this communication . in consequence of the results of the present work it is thought that where in the future attempts are to be made to demonstrate the presence of viruses in tumours not hitherto known to possess them , borkefeld filtration , with or without previous repeated freezing and thawing of the material to be filtered , should be avoided . 
i fowl sarcoma virus are described . also described are experiments in which the effect of repeated freezing and thawing on the filterabihty of this virus has been investigated . susceptible inbred brown leghorn fowl have been used . the method used to disintegrate rous sarcoma tissue mechanically in order to make from it a virus suspension is set forth . 
the technique used to apply repeated freezing and thawing to disintegrated rous tumour is indicated , as is the technique used in berkefeld filtrat - ion of suspensions of the virus . a titration method has been employed in which preparations whose tumourproducing activities were to be compared have been diluted in serial tenfold steps with saline and inoculations made with each dilution . the results show that wben rous virus suspensions prepared by extracting disintegrated rous tumour with saline were passed through berkefeld filters , part of the virus became adsorbed on to the filter . 
some authors ( willis , 1948 ) believe that the association is mainly indirect , and that the nutritional features result chiefly from anorexia , ulceration and infection , to which , with cancers of the alimentary tract especially , are added disordered digestion and absorption of food . 
others consider that the connection may be morp , direct , perhaps through metabolic competition for circulating nutrients , and in - recent years animal experiments have lent some support for this hypothesis . a reduction in plasma albumin concentration has been noted both in cancer patients and in tumour - bearing animals ( abols , rekers , binkley , pack and rhoads , 1942 ; siebert , siebert , atno and campbell , i 947 ; huggins , 1949 )  . 
a - n early fall in the blood haemoglobin level ( greenstein , andervont and thompson , 11942 taylor and pollack , 1942 ; el mahairy , 1950 ) and marked reductions in tissue enzyme activity , e.g. , in liver catalase , in organs far d - istant from the growth have been similarly recorded ( greenstein , 1947 )  . while tb - ere is no doubt that a growing cancer often exerts widespread effects on general metabolism , no study has yet been made of any process common to both normal and tumour cells in whicb any such metabolic competition might be expected to disclose itself . 
with this in mind , therefore , it seemed profitable to niake a comparative study in normal and tumour - bearing animals of the occurrence of mitosis - a phase of the growth process which to the best of our present knowledge presents similar features in botb types of cell . 
in some experiments the protein content was doubled at the expense of the carbohydrate ; this " high - protein " diet contained 40 per cent of casewith the aid of a little water all the ingredients were mixed into a stiff paste which the mice ate readily . 
present work , bullough ( 1949b ) stated that changes in temperature within the range normally found indoors in this country had no noticeable effect on mitotic activitv in the skin of the ear . 
this anaplastic tumour grows readily in the strain of mouse used ; it has a high percentage of " takes , " and shows very little tendency to regress when once established . 
immediately after its removal the piece was trimmed all round in order to expose a raw edge which facilitated the penetration of the cold i per cent acetic acid solution in which it was at once immersed . 
after i to 2 hours in this solution ( the time depending on the size of the piece ) , it w ' as possible to slide the epithelium from both sides of the underlying connective tissues . 
after it had been covered with a similar piece of filter - paper the whole was moistened with fixative , compressed lightly between two coverslips held together with an elastic band , and immersed in fixative for at least an hour . the fixative used was a mixture of one part of glacial acetic acid with two parts of absolute alcohol ; exposure to this solul . - jon for several days did not greatly harm the cells , biit a period of not more than 24 hours gave optimal results . 
the " sandwich " was therefore removed intact from the coverslips , washed free from fixative in 33 per cent alcohol followed by water , and then soaked for a short time inn / 1 hci before it was replaced between the coverslipa and their rubber band . 
after about i hour the maximum degree of staining was reached , and a subsequent washing for a similar period in s02 water was needed to remove unchanged stain which would otherwise have become re - oxidized in the later treatment . 
changes in the mitotic activit qf ear epithelium in tumour - bearing m - ice . the results of three similar experiments which show the general effect of the growth of the tumour on the mitotic activity of the epidermal cells of the pinna are given in table i . 
the relation of the reduction in the mitotic activity of epidermal ce118 in tumoursince it is possible , as some authors have suggested , that systemic effects in persons or animals bearing tumours may be due more to depression of appetite and lowered food intake than to the direct competitive effect of the tumour itself , some experiments were performed to determine how far such indirect factors may be - the cause of the reduction in mitotic activity . 
since the tumours had reached about 10 per cent of the animal 's body weight at the time of death , the neoplastic tissue was resected before the final carcass weight was determined . from table ii it can be seen that even wlien the tumours had reached this lai - ge proportion of the body weight , the average carcass weight had not fallen from the time of implantation . 
although there was no sign of wasting in these tumour - bearing mice , and their behaviour continued to be typically active , the frequency of mitosis in the epidermal cells had fallen significantly . a further test of the inanition theory of reduction of mitosis lay in determining whetlier there was any correlation between the change in the weiglit of the mouse in the ' final 24 hours and the frequency of mitosis . 
since the mouse has a very higil food intake - 15 to 20 per cent of its body weight daily - any abst ' inence from eating is quickly revealed by a . 
in table iii the control and tumourbearing mic - e have been subdivided into two aroups on the basis of change of weight in the preceding 24 hours , and the mean number of mitoses per i 00 fields of the ear epidermis recorded . 
these general findings are in conformity with those of sherman , morton and mider ( i 9 , 50 ) , who observed that in tumour - bearing rats the nitrogen required for the nutrition of the tumour was derived from the diet until the growth reached about 10 per cent of the liost 's body weight , and that it was only after this proportion had been exceeded that this requirement was obtained at the expense of wasting of the body tissues . 
it is difficult , consequently , to attribute the decreased rate of multiplication of the epidermal cells in our mi - ce to any change ' comparable with ihe intoxication and cachexia often present in the later stages of neoplastic disease in man . an alternative explanatioii for our observations is that during the growth of such implanted . 
tumours nutrient substances essentia ' l for cell division may be diverted from normal to neoplastic cells to the detriment of the former . during periods of nutritional stress , such as severe malnutrition or pregnancy ( barcroft , 1946 ) , it is nowbelieved that a competition for circulating anabolites develops between cells , which be ' ars unequally on those of different types . 
should a coniparable contest for nutrients develop in neoplasia , the processes associated with the multiplication of ' normal cells might be amongst the first to reflect a deterioration in the metabolic state of the host . 
westlake avenue , l08angele8 , california . received for publication january 19 , 1951 . huggins , miller and jensen ( 1949 ) studied the thermocoagulability of the serum protein in health and disease in the presence of an extrinsic inhibitortheir results , however , while showing an abnormal behaviour iodoacetic acid . fo ' r mahgnant and certain non - mahgnant diseases , tend to establish a seeming relationship between cancer and tuberculosis . 
serum m micromoles of sodium iodoacetate in the nearest where 05 tube containing a firm clot preceding a tube in which the coagulum is a " shapeless viscous mass , .. 
or in the ori inal state before boiling . the serum protein determinations were carried out by the semi - micro kjeldahl procedure recommended by huggins , miller and jensen ( 1949 )  . every fifth determination was duplicated with an average agreement of 0 - 5 per cent . procedure was frequently checked for accuracy for the recovery of 5 - 0 mg . quantities of nitrogen by carrying out a complete determination with 23 - 6 mg . quantities of anhydrous ammonium sulfate with recovery in duplicate being 0 - 02 to 0 , 03 mg . 
the two myelomas were r - eceiving nitrogen mustards ; the leucaemias , x - rays and / or transfusions ; some of the advanced cases , polysaccharides or chymotrypsin ; eight of the breast cancer cases , testosterone . 
none of the patients in groups i and ii received any special diet , except that the most advanced , uncontrolled cases were getting either plasma transfusions or protein hydrolysates of one kind or another . all of them received an iron - vitamin preparation . 
for the series of cases in group iv merely reflects the ratio of concentration of iodoacetate as an inhibitor of coagulation for decreasing quantities of serum protein . there is no evidence that age distribution of the group tested played any significant role in the ratios obtained for the i.i. discussion analysis of the factors involved in the procedure showed that the coagulability of 52 per cent of the group i sera is abnormal for entirely different reasons from the sera ofgroups 11 and iii . 
evidence for this lack ofhomogeneity between the protein values and the degree of positive h.m.j. , obtained for tubercular sera and the sera from new untreated cancer soon became apparent , i.e. 
to the group i patients . in some the " positive " was altered to " negative , " andin there was good agreeothers the positive results became more or less positive . ment , however , for the i.i. 
values established by both kjeldahl and falhng - drop methods of protein estimations , for the remah - iing groups of cases . this led us to consider an " aberrant " or " combined - protein " as being responsible for the striking disagreement with the h.m.j. 
for the two types of disease . the possibihty that we were deahng with an increase in one of the normal fractions of the serum , the alpha globulins , reported to be increased in mahgnancy , led us to carry out the test on a sample of mucoprotein from cancerous serum , generously supphed by richard winzler , of the department of biochemistry , of the university of southem califomia , and a " bence jones " protein , isolated from the urine of one of the two myeloma cases in this series . 
neither ofthese substances yielded a positive i.i. if , as others have observed , the degree of increase of alpha globuhn is proportional to the severity of active tissue destruction , then apparently the alpha globulin plays no part in the huggins test , either for advanced tuberculosis , which certainly involves active tissue destruction , or in the early and new untreated cancer cases in our study . the question of the existence of modified serum protein molecules in various disease states , including mahgnancy , has been discussed recently by one of those whose published data estabhshed a close correlation between plasma viscosity , and the severity of the organic changes ( harkness , 1949 )  . 
added emphasis that we may be dealing with an altered protein in malignant tumours is given by the experimental observations ofhellwig ( 1949 ) , who found that as a rule the size ofspherical bodies in the form ofclusters , short - chains and aggregates from tumour extracts when examined by electron microscope was exceeffingly larger than that in benign or normal tissue . 
he concludes that these globules are not virus - like causative agents of cancer , but rather globular proteins , probable aggregates of the cytoplasmic globules due to an alteration in the cohoidal state of the cancer it is quite possible that this cytoplasmic material may be introduced into cell . the blood serum and may play a role in accounting for the positive i.i. 
duboff subjective element in the estiniation of the coaguluthe task for the future is the elimination of this factor , perhaps , by measuring the degree of coagulation by an appropriate electronic procedure characterized by the electrical resistance or potential of the coagulum . can one explain the paradox of the 22 negative h.m.j. 
tests have a pattern of deformation in one direction , it is not inconceivable that the former 22 cases have - a pattern of deformation in another direction that is not manifested in the test through the present combination ofreagents . 
furthermore , if the mechanism of action of this method is due to the increase of net negative charges on the protein molecule by the addition of sodium iodoacetate , then our results strongly suggest variations in the polar character of the proteiii molecule in the serum of certain phases of tumour development . the magnitude of relationship between absolute protein per cent and the ll demonstrated by group iii serum does not reflect the magnitude of the effect , when apphed to group i . 
the term " false positive " cannot be apphed ' there - t fore , to such tuberculosis sera , in the same sense as the term " positive " is applied to that of early cancerous sera , since the term " positive " itself is not tenableon the same basis . 
the serum of individuals undergoing some type of degenerative process , appears to possess an admixture of some elements , the source of which we do not yet comprehend , but the presence of which has been demonstrated by measurable function of the thermocoagulation of protein in the presence of an extrinsic inhibitor , iodoacetate . tentatively , we are inclined toward the conclusiozi that abnormality in the blood serum of some cancer patients , unhke that of some tuberculosis blood with a corresponding i.i. 
it is suggested that the use of the expression " false positive " or " pseudo positive " be elimiiaated from the terminology of cancer tests . the ' author wishes to tender his sincere thanks to clyde k . 
blackburn. from the department of haematology , the royal infirmary and ho8pital , sheffield . received for publication april 3 , 1954 . the forowing considerations led us to carry out a trial of stilboestrol in acute it is well know - n that the endocrine organs play an important part leukaemia . in the regulation of haemopoiesis , and many workers have reported the beneficial unfortunately , effects of acth and cortisone in some cases of this disease . however , remissions so induced are usuary only of short duration . re - treatment may again have a beneficl ' al effect , but eventually a refractory state is reached . on the other hand , " myelokentric " and " lymphokentric " acids , steroid - like principles extracted from urine ( afiller and turner , 1943 ) and from serum ( foster and miller , 1950 ) cause leukaemoid effects of myeloid and of lymphatic type furthermore , myelokentric respectively when injected into normal animals . acid has induced partial remissions in human cases of lymphoblastic leukaemia in the hands of miller , herbutt and jones ( 1947 )  . 
hence hormonal imbalance may play a part in the aetiology of leukaemia . finkelstein , gordon and charipper ( 1944 ) demonstrated that oestrogens have a depressant effect on the bone - marrow in certain conditions , but they did not study leukaemic states . burrows and horning ( 1952 ) have shown that not only are oestrogens careinoaenic , but they may be careinostatic , often restraining and causing regression of some human neoplasms such as carcinomata of the breast and prostate . 
they also state that the liabihty of mice to leucosis can be increased by oestrogens and reduced by androgens . furth ( 1952 ) , however , reminds us that in most strains of mice loukaemia is more common in the female , but in man it is more it is possible , therefore , that in human leukaemia oestrogens common in males . may have the opposite effect to that in niiee . dausset and schwarzman ( 1951 ) reported a study of 212 cases of acute leukaemia witb , regard to the influence of age and sex upon the frequency and cytological types . 
they also reviewed other series in the literature from these points of view , including both acute and chronic leukaemia . these authors state that between 18 and 50 years in both males and females there is an overwhelrning preponderance of myeloid leukaemia , while under the age of 18 and over the age of 50 lymphoid leukaemia occurs almost exclusively . 
as the former occurs most commonly during the period of maximal procreative activity , they suggest that there may be an endocrine effect on the lymphatic - mye - loid equilibrium , especially with regard to the sex hormones . stilb ' oestrol appears to have had a beneficial effect in single cases of chronic myeloid leukaemia ( loeper , lesourd and sterboul , 1947 ) and of chronic lymphatic leukaemia ( lemaire , loeper , housset and koupernik , 1947 )  . 
on admission to hospital in sheffield he was given 3 pints of packed cells and stilboestrol and penicillin therapy were begun . this was followed by an excellent clinical remission for 6 weeks , with complete disappearance of clinical splenomegaly and hepatomegaly , the latter organs having been palpable 2 below the respective costal margins at the commencement of treatment . 
no complete remissions occurred , but 3 patients had transient partial remissions . there is no evidence from this series to suggest that either the prognosis or the clinical course of acute leukaemia is altered by adding stilboestrol to other therapy . i am indebted to my medical and surgical colleagues in sheffield , to r . 
peacock. froni the research department , glavotr royal cancer hospital . received for publication april 6 , 1949 . it is a remark - able fact that while carcinogenic hydrocarbons readily , induce sarcoma in the connective tissues of fowls , the same agents have not vet been shown to induce epithehal tumours in this species . indeed until recentlv all attempts to induce epithelial tumours in fowls by any means seem to have f.iled. recently gottschalk ( 1948 ) mentioned iin uccessful attempts to induce hepatoma in 89 fowls fed with " massive " doses of p - dimethylaminoazobenzene ( da - ab ) , and maintained for over a year on a diet deficient in protein and riboflavine or enriched with biothe also quoted similar iin uccessu attempts to induce hepatoma with butter yerow by kinosita ( i940 ) , and other failures to induce epithelial tumours in general with various carcinogens . 
one case of hepatoma was observed by ober , guerin and boic ( 1933 ) in a bird inoculated 9 months previously with a mtrate of 3fill hill 2 endothelioma . this hepatoma was not transmissible , but birds grafted with fragments of hepatoma developed leucosis which was transmissible . 
the aetiolo&y of this hepatoma therefore is uncertain.induced epithehal tumours were amongst the teratomas obtained by michalowsky ( i 929 ) following the injection of 5 per cent zinc chloride into the testicles of cocks . duran - reynals ( 1946 ) , while trying to transmit primitive renal tumours in fowls , obtained sarcomas but no epithelial tumours in the inoculated birds . it may be that other workers have made extensive negative tests without them , but it seenis advisable to describe our ow - n mainlv negative recor attempts , which have now extended over a period of about 17 years , and have 3ielded only two carcinomas , apparently caused by 2 - acetylaminofluorene ( 2 - a - a - f )  . in contrast with these negative results , bielchowsky and green ( i945 ) obtained an anaplastic renal carcinoma in i of 5 rhode island red cocks maintained on a mixed diet to which was added 2aaf , at the rate of 3 - 30 mg . 
149 , previously recorded as bearing a tumour , was found to have a necrobiotic lesion in the region of the eneysted tar and a large haematoma in the breast muscle , but no sarcoma . ( 2 ) twenty - seven bpr fowls ' were painted on the right side of the comb witli tar and injected in the right breast muscle with the same sample of tar . while these experiments were running the first recognition of a pure hydrocarbon , 1 : 2 : 5 : 6 - dibenzanthracene ( db ) , as a carcinogen was reported by kennaway and hieger ( i 930 ) , and thereafter when the substance became available these birds were painted with a saturated solution of db in chloroform instead of tar . in view of the lack of obvious reaction to the previous tar painting , light cautery of the comb with a hot platinum wire was applied to survivors immediately before painting with db . 
pellet of 3 : 4 : 5 : 6dibenzearbazole ( dbc ) implanted into the right preening gland , followed by twice - weekly painting of the preening gland area with 0 - 1 per cent solution of croton oil in acetone . 
no tumours occurred in other birds in this experiment . ( 8 ) in preliminary experiments , 7 bl fowls were injected into the preeiiing gland with solutions of dmb in arachis oil . 
the fluorescent solution was expelled through the duct of the preening gland and , as there was no evidence of retention in the gland cavity after a few days , this type of experiment was abandoned . implants into the preening glands . tllree groups of 6 bl hens had the following pellets iniplanted into the left preening gland : 15 - 30 mg . 
the other birds had ar been implanted with similar perets of methylcholanthrene , n : n - dimethylami ioswbene , or 1 : 2 : 5 : 6 - dibenzanthracene into the right preening gland , without obvious effect . of the remaining 6 survivors after 17 months , 2 had palpable nodules in the wah of the crop . 
one of these , a bl hen , was kffled because it was rapidly losing weight and was found to have several ulcers in the crop varying in diameter from about i to 8 mm . , and partly covered by tenaceous slough . there were also numerous oily cysts in the connective tissues around the crop and in the muscular and submucous tissues of the crop itself representing renmant - 8 of previous injections of 2aaf in arachis oil . 
the re ndaer of the alimentarv tract was thoroughly examined but no other lesions were found . the hver and spleen were smafl and paler than normal but showed no locahzed lesions . 
the ovary was in a resting stage and the oviduct was involuted . sections of the ulcers in the crop show that it was the seat of at least 3 apparently separate squamous carcinomass . 
the low incidence of carcinoma in fowls n - iight be explained on these grounds . on the other hand , mammals differ in their susceptibility to the actioli of carcinogens . 
a case of keratinizing squamous carcinoma of the buccal mucosa in a fowl was described and well ifustrated bv puente - duany ( 1932 )  . the orilv case of spontaneous squamous carcinoma se ' en by us in over 3000 post - mortem examinations of fowls , over a period of 20 years , occurred in a bpr fowl that had been unsuccessfully graftedwith fragments firom a chemicauyinduced sarcoma . 
the absence of sebaceous follicles in fowls and the concentration of the sebaceous glands in the paired preening gland prompted us to implant potent carcinogens into the cavity of these glands . 
1. received for publication february 23 , 1948 . in a previous communication we reported that a strain of the shope rabbit papilloma had been transmitted for 10 serial passages in the domestic rabbit ( selbie and robinson , 1947 )  . 
the adaptation of this tumour virus as a transmissible infection in domestic rabbits occurred during experiments in which the simultaneous inoculation of shope papilloma virus and sheep dermatitis virus in domestic rabbits produced papillomas that proved infective to other rabbits ( selbie , 1946 )  . 
the further passage of this virus in domestic rabbits was materially helped by continuing the procedure of mixing a second virus suspension with the infective inoculum of papilloma virus , and also by treating the skin with croton oil before inoculation , but there was no evidence of an increase in the virulence of the virus suspensions during passage . in the present communication we shall show that carcinomatous transformation has occurred regularly in this transmissible papillomatosis , and in a mamner similar to that found in the non - infective papillomatosis that is produced in domestic rabbits by the inoculation of extracts of papillomas from the cotton - tail rabbit , the original host of the shope papilloma virus . 
thus in all rabbits frqm passage 4 in table i , area 1 represents normal skin inoculated with a 10 per cent extract of papillomas , area 2 , normal skin inoculated with a mixed extract of papillomas and sheep dermatitis lesions , area 3 , skin pre - treated with croton oil and inoculated with a 10 per cent extract of papillomas , and area 4 , skin pre - treated with croton oil and inoculated with the mixed virus suspensions . rabbit 2 , passage 2 was inoculated with papilloma extract on normal skin only , and in rabbit 4 , passage 3 , the procedures for areas 1 and 3 were omitted . the rabbits were examined at weekly intervals from the 7th week onwards . the tumours were charted and their size was measured by callipers at regular intervals . 
as was noted in the earlier communication ( selbie and robinson , 1947 ) , there was a tendency for individual papillomas to unite during development , and in some instances papillomas regressed completely . 
hence in this communication only those papillomas that were recognizable as single entities at 6 months in areas with confluent or semi - confluent papillomatosis will be considered . only a few papillomas could be distinguished as independent entities at 6 months , and no accurate count could be made , so that a fair estimate was given of 30 papillomas for confluent papillomatosis and 20 for semi - confluent papillomatosis . it became apparent early in this investigation that if the development of individual papillomas was to be studied over a prolonged period , cancers which arose in adjoining papillomas should be removed . 
1 are shown the times at which carcinomatous growths were first detected in passages 2 to 7 , passage 9 being omitted because the surviving rabbits have been observed for only 40 weeks after inoculation . it will be seen that the passage rabbit 2 __ to ~~ * : 6s weeks after inoculation fig . 
1. - times of observations of carcinomas in all rabbits from the 2nd to the 7th passage . each rabbit is represented by a line indicating the period of observation from the 40th week blocked circles indicate carcinomas arising from papillomas on skin treated onwards . with croton oil before inoculation , and open circles indicate carcinomas arising from papillomas on untreated skin . x = removal by operation of all remaining papillomas . 
the time at which glandular metastases were observed varied with the time at which the primary carcinomatous growth occurred , the limits being 33 weeks after inoculation in rabbit 2 , passage 9 , and 89 weeks in rabbit 2 , passage 2 , while the average time was 67 weeks for all the rabbits in the series . 
to draw any definite conclusions from this series concerning the incidence of glandular metastases as many of the primary growths were removed , and in consequence the opportunity for metastases to occur was reduced . 
2 , area 2 , the central papilloma with a concurrent thickening of the base . had slowly enlarged from the time of its original appearance to attain a diameter nine weeks later it measured 1 * 8 cin diameter and of 1 - 4 cat 53 weeks . the base had now thickened and was fleshy in appearance and consistency , in three weeks later , at 65 contras , t to the dry horny surface shown in fig . 
19. the secondary deposits in glands and lungs have all proved to be of the in many of squamous cell type showing varying degrees of differentiation . the lung metastases it can be seen that the infiltrating carcinoma cells form nodules within the alveolar spaces , so that the stroma of the deposit retains the architectural structure of the lung tissue . 
the time at which this change first occurs has tended to become shorter in successive passages , and has been reduced to 45 and 28 weeks in single rabbits of the 7th and 9th passages respecthis may indicate an increase in the virulence of the virus , but it is more tively . likely to be due to variations in the sensitivity of the rabbits . it has also been shown that the first occurrence of carcinomatous transformation in a rabbit is usually followed by others up to at least 5 in number and up to 85 weeks after indeed carcinomatous transformation occurs so regularly in this inoculation . transmissible infection that it would appear that all papillomas would show this change , provided that their evolution was not interrupted by the death of the host or by other factors such as spontaneous regression , infiltration from nearbv carcinomatous growth , or operative removal . carcinomatous transformation should then be regarded as a natural stage in the development of infective papillomatosis , and attributable only to the activity of the virus which initiates the infection . it is also clear from the work of kidd and rous ( 1938a , 1938b ) and mcintosh and selbie ( 1940 ) , who observed rapid transformation to malignancy in tar - induced papillomas infected with shope papilloma virus , that this virus is capable of causing immediate carcinomatous change . it is not therefore necessary to postulate an extraneous precipitating factor such as bacterial infection or an alteration in the virus itself to account for the change to malignancy , as suggested by rous , beard and kidd ( 1936 )  . although carcinomatous transformation would appear to be a natural stage in the development of this infection , it presents such a radical change in behaviour that it is difficult to see how it could be part of a continuous process . 
robinson cidence of tar tumours in mouse skin was increased at the site of injury by deep incision , numerous investigators have demonstrated the enhancing effect of unspecific irritation on the tumour - producing activity of carcinogenic substances . that chemical agents can also have this stimulating effect was shown by twort and twort ( 1928 ) , who accelerated the production of tar tumours in mice by pretreatment with oleic acid , and by berenblum ( 1941a , 1941b ) , who obtained a higher proportion of mice with tumours after painting with a dilute solution of benzpyrene by concurrent treatment with croton oil or by applying croton oil after a limited course of benzpyrene treatment . 
the possibility then had to be considered whether the hereditary condition acted through genetically transmitted differences in ovarian functional activity , or through an unequal response of mammary glands or genital tract to ovarian hormone . 
numerous and extensive observations were made with somewhat conflicting results , both on the nature of the oestrous cycle and on variations in organ sensitivity to hormonal stimulation , in strains of mice with differing mammary cancer incidence . when the existence of the third factor , i.e. 
the alveolar epithelium in this particular graft actually shows the various stages in the secretory cycle of the normal prostatic gland . it is interesting to compare the histology of these successfully vascularised grafts seen in fig . 
horning the prostatic it is relatively easy to detect by palpation non - vascularised grafts , as they are smaller and invariably softer when compared with successfully vascularised prostatic grafts , which are firmer on palpation , larger and invariably cystic in the earlier phases of growth , owing to active secretion of the glandular epithelium . non - vascularised graft fixed5fig . 
in this particular implant this was the only malignant focus within the entire graft . alveoli distended with secretion and with normal epithelium are seen adjacent to this exhausted alveolus from which this early malignant lesion arose . 
the proliferating epithelial components in all the early malignant foci break through the alveolar basement membrane , invariably forming tongue - like lesions which rapidly invade the fibromuscular stroma of the host as depicted in all prostatic grafts which have grown after treatment with 20in fig . 
10. methylcholanthrene , it was found possible to distinguish between three distinct types of early epithelial growth , all of which have been previously described in detail ( horning , 1949 )  . by studying the cytology and mode of growth in serial sections of these early invasive foci through later phases of development in other similar older grafts , it has been possible to predict the types of tumours which would have subsethere is one type which gives rise to a glandular carcinoma , quently arisen . both these types of and a second which develops into a squamous growth . lesions are easily recognised by their histological character , and both arise from the alveolar epithelium . the third type is the most uncommon variety , characterised by a stratified squamous metaplasia of the epithelium in sita , followed by a diffuse marginal growth into the stroma , which clearly arises from the duct occasionally some grafts show two or sometimes three types of epithelium . proliferating foci within the same implant . 
11. this shows a section of a graft treated with crystals of stilboestrol alone and fixed 4weeks after implantation . there is a complete inhibition of secretion after this period of grafting . every individual alveolus within the graft was entirely devoid of prostatic secretion , and the epithelial cells had undergone marked morphological changes . 
the prostatic epithelium at this period of treatment contrasts greatly with similar grafts treated with the female sex hormone after implantation in host mice for the same period of time . 
the alveolar epithelium not only maintains its glandular character , but in the majority of grafts testosterone propionate induces a pronounced stimulation of the prostatic secretions , invariably resulting in a cvstic dilatation of the alveoli . 
grafts combined with the carcinogen and sex hormones . 20 - methylcholanthrene and testosterone propionate . - three squamous - celled carcinomas developed from prostatic grafts impregnated with the carcinogen and the male sex hormone in strain a mice at intervals ranging from 7 to 9 weeks following implantation . one sarcoma arose in a c3h mouse after 8 weeks ( table i , group iii )  . 
the differences in histological structure between prostatic tumours derived from grafts treated with the carcinogen together with either the male or female sex hormone can best be appreciated by comparing fig . 
examination of pituitary and adrenal glands and gonads from host mice bearing grafts . examination of pituitaries from mice bearing grafts from all three groups of rodents treated either with the carcinogen alone or in combination with either of the sex hormones revealed no abnormal histological changes , with the exception of excessive amounts of colloid seen in the clefts of pituitaries from mice treated with stilboestrol and the carcinogen . there was no diminution of acidophiles or increase in chromophobe cells following implantation of the stilboestroltreated grafts . neither did the adrenal cortex reveal any histological abnormalities resulting from this treatment . 
horning of both lung and prostatic tissues have been previously described by the author in mice the survival of an homologous graft seems to be dependent ( 1949 , 1952 )  . upon the use of a closely inbred strain , since stock mice of indeterminate ancestry do not easily tolerate grafts of this kind . 
pan and gardner ( 1948 ) found an increased susceptibility to tumour formation from grafted uterine epithelium in pure line mice as compared with hybrids . recent experiments ( horning , 1952 ) have further indicated that the age of the donor providing the graft , and the age of the host mouse into which the grafts are implanted , also have an important bearing on the problem of graft other factors such as the reaction beween the graft and the host survival . mouse play an important role . it has been previously reported that more difficulty has been experienced in obtaining malignant tumours from lung grafts impregnated with 20 - methylcholanthrene than with similar grafts of other it was interesting to note that tissues which are normally under hormonal tissues . influence are much better tolerated as grafts in host mice of the same sex and strain than tissues which are to a greater extent ( such as the lung ) independent of hormone action ( horning , 1952 )  . nevertheless , a certain number of prostatic grafts , when implanted subcutaneously into host mice of the same strain as those of the donor , fail to survive irrespective as to whether the grafts were impregnated either with the carcinogen alone , or in combination with either the male or female sex hormone . 
the present experiments on the reaction between the implanted prostatic graft and the host bearing mouse have shown conclusively that the survival of the donor graft is dependent upon a rapid vascularisation within the subcutaneous tissues of the host mouse . 
the reason , however , why some grafts fail to become vascularised still remains obscure . the influence of steroid hormones on the behaviour and growth of prostatic cancer has been the subject of intensive investigation . 
1. - the skin of a c3h male mouse is cut and pinned back so as to expose an untreated homologous prostatic graft which had been growing subcutaneously for 6 weeks . 
6. - transverse section through an homologous prostatic graft which failed to become vascularised , fixed 6j weeks after subcutaneous implantation . the relationship of the grafted prostate to that of the skin of the host rodent is clearly shown . 
horning alveoli have completely broken down ; there is also evidence of lymphatic infiltration . the whole graft has been encapsulated and is slowly being absorbed by the tissues of the host mouse . 
 it has been found ( elson and lamerton , 1949 ) that the protein content of the diet has a profound influence on the response of the , valk.er rat carcinoma 256 to x - radiation . 
two effects were considered to be concerned in this response : ( a ) the initial inhibition of tumour growth and ( b ) the elimination of the inhibited tumour and cure of the animal . 
 although variations occur within a group of animals maintained on a standard diet of fixed composition , the general type of response to a suitably fractionated in animals maintained on the 5 per x - ray treatment is illustrated in fig . 
growth may be nearly stopped for a consider able time , but the tumour usually begins to grow again , at first slowly in animals maintained on a 20 and then more rapidly , finally causing death . 
the rate at which it decreases in size is at first fairly rapid , but gradually becomes slower , and in many cases the animal is then able to rid itself entirely of the tumour by a " shelling out " process or sometimes by the gradual complete regression of the tumour . 
 a detailed histological and c : ytological examination of tumours , particularly under conditions presented by a and b , was therefore undertaken in an attempt to elucidate the part played by diet in influencing the ultimate response of these tumours to radiation . 
they were kept in individual cages , and maintained on the 20 per cent or the 5 per cent protein diet for at lea.st 7 * r - arch fellow of the xorwegian defence research establishment . 
15 c under these conditions the dosage rate at the centre of the tumour is about 140 r / m h~ tuhnique . - - cross sections of the implanted tumours were made by means of a special cutter with parallel kni , es giving a section of about 5 m thickness . 
 the conventional method of propagation of the walker carcinoma consists of the insertion of small pieces of tumour ( approximately 7 x 5 x 5 mm . ) under the skin of rats . 
although factors such as age , sex , weight , etc . , and general condition may account for some of this variation , it is probably mainly an expression of genotypic differences present in the colony from which the rats used in these experiments were drawn . 
 with our present routine for breeding and selection of animals for tumour implantation , when the tumours of a group of ten rats are measured 8 days after implantation the measurements show a distribution of i or 2 large tumours , about 5 medium - sized tumours and the rest considerably smaller . 
that this is largely the result of genetical variation is shown by comparison of the growth rates of tumours in litter mates and by implantation of similar sized pieces of tumour in both flanks of the same animal . 
the animals were divided into 4 groups , each group consisting of 4 litter mates , 2 male and 2 female ; they were killed 14 days after implantation and the weights of their tumours recorded . 
it is seen that the 3 most rapidly growing and largest tumours ( over 35 g . ) all occurred in group 1 , whilst in group 3 none of the tumours grew well . 
the growth rates of both tum , ours were remarkably similar , and if a tumour implant in one flank failed to grow the corresponding tumour in the opposite flank also failed . 
the average daily weight increase of rat.s maintained on our 20 per cent protein diet is about 3 to 4 : g . , whilst that of those maintained on our 5 per cent protein diet is less than 05 g . 
 weight of tumours 14 : days after implantation taken from 85 animals maintained on the 20 per cent protein diet was 24 : g . , whilst that of 86 animals maintained on the 5 per cent protein diet was 20 g . 
thus the ratio between the tumour weight.s on 20 and on 5 per cent protein diet is 12 , and the average growth rate of tumours is only about 20 per cent higher in anima1s maintained on high prot.ein than in those receiving a low protein diet . 
la.merton for comparison of the histology of tumour grafts in animals maintained on high and low protiein diets transverse sections were made through the tumour implants and their surrounding tissue 4 , 7 , 8 , 10 and 15 days after implantation . 
 it was found that 4 days after grafting the int , ensity of the inflammatory reaction as estimated by the number of cells of the reticulo - endothelium accumulated around the graft was great.er in rats maintained on the 5 per cent than in those on the 20 per cent prot , ein diet . 
on the other hand , the capsule of connective tissue was much more distinct in rats on the 20 per cent than on the 5 per cent prot , ein diet . 
the boundary of the tumour is well defined in rats on the 20 per cent prot , ein diei , the migration of tumour cells is negligible and the tumour grows by the very high rat.a of cell proliferation at the periphery of the tumour parenchyma . 
on the 5 per cent prot , ein diet , how ever , there is no definit , e tumour boundary and many isolated tumour cells can be seen scatt , ered amongst the loose , primitive network of connective tissue , which included in this " incomplet , e " capsule are also many surrounds the tumour . 
 during the early stages of the " take " and growth of the implanted tumours the reactions of the surrounding tissues may be very complex and int , erpretation of the exact course of events is difficult , but in older tumours the histological features of the process are much more clearly defined . 
the cell - elements ofthe capsule undergo progressive : fibrous differentiation , which can be clearly seen in high prot , ein diet rats with 10 to 15 - days - old tumours . 
the : fibrous nature of the capsule is much less obvious in animals maintained on a low prot , ein diet , and its clear demarcation is obscured by the migrating tumour cells . 
 it should be emphasized that on both diets there may be a considerable variation in the " take " of the graft and in the spread of the implanted tumour in the " host , and instances have occasionally been encount , ered in which the tumour graft took and grew more rapidly in animals maintained on low than in those fed on high prot , ein diet . 
such cases were , however , rare , and the effect of the diet is so marked that the differences in capsule - formation and organization between animals maintained on high and low prot , eins diets can easily be det , ected in most cases . 
 in the study of the reaction of the host rat to the implantation of tumour grafts the fact that , the tumour graft is itself growing may very often obscure the observations of the organization of the newly formed connective tissue capsule . 
 it was felt that the introduction into the subcutaneous tissues of a " passive " non - growing hetierologous tissue fragment such as muscle inst , ead of the " active " tumour fragment , would make it easier to analyse the influence of environmental factors , etc . , on the host reaction . 
 pieces of muscle of about the sam~ size as the usual tumour implants , taken from the leg of a second rat were implanted under the skin of the host animal . 
 305 in the mbsequent reaction to this muscle implant two phases could be clearly distinguished : ( a ) the inflammatory tissue response , ( b ) the formation of a capsule around the implant . 
 the in : oammatory reaction ( a ) is intense 3 days after implantation in rats receiving either the 20 per cent or 5 per cent protein diet , but in those main tained on the 20 per cent protein diet it rapidly subsides and has almost dis appeared 5 days after grafting . 
 the 5 per cent protein diet rats , however , still show persistence of the inflam matory reaction at a time when the capsule is already well developed in 20 per cent protein diet animals . 
it appears that the prolonged persistence of the inflammatory reaction around the implant is related to the absence of the connective tissue capsu1e , the development of which is impaired or delayed in animals maintained on a diet deficient in prote the evidence derived from the histological investigation of muscle implants thus supports the conclusion reached from the tumour implant experiments that while maintenence of th~ animals on a low protein diet has no deleterious influence on the inflammatory reaction to the implant , it does interfere to a very large extent with the organization and development of the connective tissue capsule . 
 a quantitative analysis of radiation - induced injuries in cells of the walker carcinoma is difficult owing to the large number of chromosomes ( 2n = 40 ) in the relatively small tumour cell . 
 injuries to the chromosomes can be detected only in ana - telc : , phase during which the lagging of ' " acentric " chromosome fragments ( segments without the centromere ) can be seen . 
13. - dividing tumour cell in late anaphase with three acentric fragments , one lying out of focus , 6 hours after 300 r , 5 per cent protein diet . 
27. - the same tumour ( l . ) , showing the active tumour cell island which lies beyond the periphery of the tumour which occupies the bottom right hand corner . 
28. - the same tumour ( l . ) at a higher magnification to show that tumour cells which were scattered in the loose connective tissue capsule form foci of renewed activity after irradiation . 
 a comparison has been made of the frequency of tumour cells with various cytological abnormalities , such as chromosome fragments , bridges and micro nuclei at different times after irradiation of a six - day old walker tumour in rats kept on high and low protein diets . 
the data obtained ( table i ) do not represent the full extent of the radiation injury resulting from any particular treat ment , since an unknown proportion of injuries undergo restitution before the cytological examination is made and are thus lost for analysis . 
moreover , the acentric chromosome fragments can only be seen when they lie freely in the cytoplasm , and consequently those fragments which are mixed up with the normal chromosomes and moved to the pole escape detection . 
a similar difficulty arises when we attempt to estimate the true extent of the radiation injury to cells by the number of micro nuclei , since some of the chromosome fragments may be included in the daughter nuclei and some of the micronuclei can contain more than one fragment . 
on the assumption , however , that the chance for such events is the same in all the samples , it is justifiable to use the quantitative differences shown by the data as a basis for comparing differences in cell behaviour which may be induced by maintaining the animals on different diets . 
 the data of table i shows that the greatest amount of injury was found in tumour samples taken 12 hours after irradiation , suggesting that these samples contained cells which were , at the time of irradiation in the most sensitive period of the mitotic cycje . 
it is inferred on experimental evidence , derived from x - rayed pollen grains and root - tips cells ( darlington and lacour , 1945 ) that the most sensitive period coincides with the duplication of the chromosome filament , which process marks the beginning of prophase . 
since the injury in the cells is at a maximum at 12 hours after both 100 and 300 r , we may conclude that the duration of mitotic suppression does not differ over this range of dosage . 
 the number of cells with chromosome injuries and with micronuclei was found to be already quite high in the 6 hours ' sample , suggesting that the time interval between treatment and the first appearance of chromosome fragments may be shorter than 6 hours . 
therefore some rats were killed 4 hours after treatment , but although both chromosome fragments and bridges were observed in this early sample , their frequency could not be estimated because the so - called " physiological " effects of radiation grossly interfered with the analysis ( mar quardt , 1938 ; koller , 1943 )  . 
l. - uierton the number of resting cells with micronuclei increases up to 24 hours after treatment , but at 72 hours such cells have almost disappeared from the tumour . 
 comparison of the results obtained at different times after irradiation with both 100 rand 300 r ( table i ) , in all samples except those taken after 12 hours , shows no significant difference in the number of cells injured or the number of chromosome fragments per cell , between animals maintained on high or low protein diets . 
 the inconsistent behaviour observed in the 12 - hour samples may be related to the fact that all the mitotic cells in this sample represent those cells which were in their most sensitive stat , e when irradiated . 
it was , therefore , felt that the results obtained with the 12 - hour samples could not be relied upon and should be disregarded in view of the consistent results obtained with all the other samples . 
it is thus possible to compare the length of the inter - mitotic period on the two diets in order to find whether the duration of the mitotic cvcle is altered bv the diet or not . 
 the greatest amount of injury are those which at the time of irradiation were at in the x - rayed \\ ' alker carcinoma the the beginning of prophase of mitosis . 
 greatest amount of injury is found in the - 12 - hour sample and consequently the duration of mitosis from prophase to the end of telophase cannot be longer than 12 hours ; the cycle of the second division ( from prophase to telophase ) would occupy another 12 hours , thus lea , ing 20 hours for the duration of the inter mitotic period , if an allowance of 4 hours is made for the time lost by the radiation induced mitotic suppression . 
thus we may conclude that the active cells in the \\ ' alker carcinoma have a mitotic cycle of the order of 32 hours ( 20 hours spent in resting and 12 hours in mit - 0sis ) : this agrees with data obtained in similar experi ments in which irradiation is replaced by radiomimetic chemicals , such as the nitrogen mustards ( koller , unpublished communication )  . 
 from the observations of the frequency of cells in second mitosis 48 and 72 hours after irradiation with 100 or 300 r there was no indication that diet affects the duration of the mitotic cycle . 
at 6 days after the radiation treatment tissue fibrosis was much more developed , and the connective tissue capsule could be clearly distinguished from the actual tumour , since no tumour cells were present in this capsule zone . 
 it does , however , contain islands of active tumour cells , which , since the growth - inhibiting effect of a single dose of 1000 r is usually only temporary , no doubt represent centres from which the renewed growth of the tumour develops . 
 in the animals receiving the 5 per cent protein diet , the loosely organized con nective tissue capsule , 3 days after 1000 r , was not undergoing fibrosis to the same extent as the capsule in the 20 per cent protein diet animals . 
 six days after irradiation the tumour boundary which was previously very ill defi.ned , becomes more distinct , presumably owing to the fact that some of those tumour cells which were scattered in the connective tissue network and have survived the radiation treatment , have now undergone proliferation and brought about the formation of a new continuous tumour parenchyma . 
 w ' hile a single dose of 1000 r may cause considerable inhibition of growth of the walker rat carcinoma , and the inhibition may last for several days , complete regression of the tumour rarely occurs . 
the experiments with the single dose have shown , however , that at least in the 20 per cent protein diet animals , whilst the extent and rate of histological changes induced in the capsule or tumour bed is very favourable , it is insufficient to enable the animal to effect a cure . 
it is obvious , therefore , that the amount of radiation injury in the tumour cells must be increased without at the same time adversely affecting the favourable tissue response . 
it was therefore decided to increase the dose to 4000 r , which is of the order employed in the radiotherapy of human cancer , and a suitable method of fractionation for applying this dose was devised based on observations of the response of tumour capsule and tumour bed obtained in the single dose experi ments . 
the method employed can be illustrated as follows : 1000 r 500 r , 500 r 500 r , 500 r 500 r 250 r 250 r , in which represents two consecutive days on which no treatment was given . 
lamerton for the histological investigations 15 rats were treated with this fractionated dose , 7 of them being maintained on the 20 per cent and 8 on the 5 per cent protein diet . 
 two rats of each diet group h 1 , h ! ( high protein ) and l 1 , l 2 ( low protein ) were selected for histological examination ; of the remaining rats 4 out of the 5 maindied " 's e .. 
h 1 and h , indicate animals maintained on high protein diet and li and l , animals maintained on low protein diet at the time they were killed for histological examination . 
from similar results obtained in previous experiments ( elson and lamerton , 1949 ) and from a com parison of the growth curves of the tumours selected for histological investigation with those of the remainder of the groups shown in fig . 
22 it is reasonable to suppose that animals h 1 and h 2 on the high protein diet would have been cured , whereas li and l 2 maintained on the low protein diet would not . 
those of the high protein diet animals ( h1 and hz ) , however , had grown to a considerable maximum size and were then regressing , whilst those of the low protein diet animals ( l1 and lz ) had shown earlier retardation of growth , but were beginning to assume a more rapid growth rate . 
the animals h 1 and li had received a total dose of 3000 r ( in 5 fractions ) , whereas hz and lz had received the foll total dose of 4000 r ( in 8 fractions )  . 
 in assessing the response of tumours to radiation two effects must be con sidered : ( i ) the initial inhibition of tumour growth and ( 2 ) the elimination of the inhibited tumour and cure of the animal . 
in work with the walker rat carcinoma 256 it has previously been shown that the diet of the animal has a definite influence on each of these processes ( elson and lamerton , 1949 )  . 
 dealing first with process ( i ) , it has been shown that maintenance of the animal on a low protein diet favours the immediate growth inhibitory response of the implanted walker rat carcinoma 256 to radiation . 
it might be thought that this diet effect is related to an increased sensitivity of dividing tumour ce ] ls to radiation induced in some manner by the poor protein diet . 
furthermore , we do not feel that such differences as have been revealed in the course of the histological investigation of the tumour bed reported here offer any very satisfactory explanation of the greater immediat.e tumour response in the low protein diet animals , and we are inclined to believe that some more fundamental biochemical relation is involved . 
lamerton investigations with carcinogenic chemicals have suggest , ed that these sub stances may inhibit cellular growth by interfering with the normal processes of protein synthesis ( elson and warren , 1947 ; elson , 1949 )  . 
h this is the case it might be expected that animals in which the full capacity for protein synthesis is already restricted by a deficient protein diet would probably show an immediate growth inhibitory response of both animal and tumour to treatment . 
with ample protein to furnish more than suffi cient amino acid " building blocks ' for the full efficiency of protein synthesis would not be expected to show such an immediate reaction . 
it is not unlikely that x - radiation may act in a similar way and in order to throw more light on this problem the protein metabolism of irradiated animals maintained . 
 of the walker rat carcinoma a high protein diet has been shown to be of great assistance to the animal in the process of elimination of the inhibited tumour and the replacement of the tumour area by healthy tissue . 
that in the case of the implanted walker rat carcinoma 256 the main effect of the high protein diet is to ensure the development of a well organized . , extensive capsule of connective tissue around the growing tumour . 
response to radiation treat ment the ill - defined tissue capsule zone does not undergo fibrosis in the same way as does the firm capsule of the high protein diet animals . 
altho~h its main mass usually becomes necrotic , new tumour growth often develops in the incomplete capsule zone , from cells which have escaped the lethal action of the radiation . 
on a diet deficient in prote the effect of addition to the low protein diet of substances such as amino acids , particularly cystine and methionine , amino sugars , etc . , which may conceivably be involved . 
 313 taking into consideration the connective tissue reaction , devised treatment methods for epitheliomata , in which the total dose is of the order of 3000 r , far below the so - called " standard tumour lethal dose . " also it has been shown by jolles and koller ( 1950 ) that the tumour bed reaction can be influenced by a method of fractionation of the dose in space as well as time to give favourable therapeutic resu.jts. 
 the present animal experiments have shown that the character and organization of the tumour bed can be influenced by nutritional factors , and suggest that suitable regulation of nutritional conditions , such as , for instance , supplying a high protein diet , or supplementing the diet by protein hydrolysates , etc . , and by increasing protein anabolism by hormone administration , etc . , may jead to improvements in clinical response . 
 the effect of the protein content of the diet on the establishment and growth of implants of the walker rat carcinoma 256 , and on the response of this tumour to x - radiation , has been investigated . 
 the nature and extent of the inflammatory reaction has been found to be similar in animals maintained on both high ( 20 per cent ) and low ( 5 per cent ) protein diets , but in the low protein diet animals it persists for a longer period after implantation owing to slower and less complete development of the connec tive tissue capsule . 
 in assessing the response of the tumour to radiation two effects have been considered , ( a ) the initial inhibition of tumour growth , which is favoured by a low protein diet , and ( b ) the elimination of the inhibited tumour which is favoured by a high protein diet . 
 histological investigation has suggested that the favourable effect of a high protein diet on the elimination of the tumour is related to the development of the well organized capsule of connective tissue around the growing tumour . 
radia tion treatment , besides inhibiting division of the tumour cells , also increases the fibrosis in this capsu.je , which aids in the eventual elimination of the damaged tumour . 
 we wish to express our thanks for grants supporting this investigation from the british empire cancer campaign , the anna fuller fund , the jane coffin childs memorial fund for medical research and the u.s. 
may be important factors in the pathogenesis of cancer of deeply seated as well as of superficial organs indeed , early in the 19th century a careful local study of is hardly a new one . cancer deaths in verona was made by stern ( 1842 ) , the notion of cancer houses was looked into and fragmentary studies of cancer endemiology were made in the concept of cancer as a unitary disease switzerland , america and elsewhere . affecting organs rather at random has tended to discourage detailed research on site distributions . the general register office in london worked patiently upon the occupational aspects at successive census periods with increasing detail as death certification recently the danish cancer registry , initially of primary cancer sites improved . rather sceptical of some of the conclusions reached , has admitted that danish data tend to confirm them ( clemmesen , 1951 ) , and similar data have been pubin 1950 and 1951 the council for lished from the netherlands ( versluys , 1949 )  . co - ordination of international congresses of medical sciences arranged conferences on what was at first called " geographical pathology " , but has now become " endemiology " of cancer . very little has been written , however , on lung cancer from this aspect except with respect to tobacco , although it came under discussion at the first of the above - mentioned conferences ( c.c.i.c.m.s. , 1951 ; daff , doll and kennaway , 1951 )  . in the present paper the short term " lung cancer " is used to mean carcinoma and other malignant neoplasm of the bronchus , lung and pleura ; and when " mortality " is mentioned it is understood that whatever rate or index is used has been standardized for age . * the substance of this paper was communicated to the metropolitan brench of the society of medical officers of health on may 9 , 1952 , 100 p . 
at that time i still had to work upon the rates for 1921 - 30 , and failed to find any significant correlation either in the large towns or in the metropolitan boroughs between lung cancer mortality and the social class or overcrowding indices . recent research on correlation with tobacco smoking . between 1930 and 1946 the recorded rates in england and wales as a whole had been rising steadily , and to a surprising extent even when increasing use of x rays was considered . consequently the medical research council called a conference to advise whether any useful research could be undertaken to throw light on the causes . 
the result of that study was published in september , 1950 ( doll and hill , 1950 ) , and in my view it proved beyond doubt , in conjunction with american evidence of the same kind ( wynder and graham , 1950 ) that 102 p . 
stocks tobacco smoking is a very important factor in the causation of lung cancer . did not prove , however , that tobacco smoking is the only important factor in it has been suggested that the figures could mean that 90 per cent causation . of the lung cancer occurring in greater london is caused by tobacco ; but they do not necessarily mean that , and there are reasons for thinking that the proportion is - much lower , and that atmospheric pollution may be an additional factor . these reasons derive from some statistical facts not hitherto published . mortality in london in 1946 to 1949 . in the four years 1946 to 1949 the deaths of males attributed to lung cancer in in 1950 england and wales averaged 8183 annually , and of females 1774 . deaths of males exceeded 10 , 250 and those of females were just short of 2000 , and in 1951 the total reached 13 , 000 . 
the national rates at different ages are compared in table iv , which shows that at ages 35 - 45 the increase was 6 - fold , and at 55 - 75 it was more than 11 - fold . how much of this enormous increase has been due to more complete detection of the disease and how much to increased incidence we can only surmise . london county as a whole , allowing for age changes , male mortality increased about 8 - fold between the two periods ; and it is now about 60 per cent greater than in the country as a whole , and 21 times the rate for rural districts . 
1 shows the mortality distribution in 1946 - 49 . for 1921 - 30 there were two areas of specially high certified mortality , one in bermondsey , stepney , bethnal green , hackney and stoke newington , the other in hampstead , st . 
stocks wind direction as a possible factor . returning now to greater london , clark ( 1951 ) has shown that the logarithm of the population density per acre in successive zones diminishes in arithmetic progression on passing outwards from the business centre , and the same is true supposing domestic smoke to be an important factor in of other large cities . producing lung cancer , if there was no wind at all one would expect to find the highest incidence at the centre , diminishing in all directions outwards . if there is wind and it comes from all directions equally , a more diffused but still symmetrical distribution would be expected , with less contrast near the centre and more near the periphery . however , wind does not blow equally from all directions , and greenwich records over 12 - hour periods throughout the year 1950 show that the wind direction , when analysed into 8 sectors , was s.w. 
orr. from the department of experimental pathology and cancer research , medical school , university of leeds , and the de7partment of pathology , university of birmingham.. received for publication , april 23 , 1949 . the discovery of the milk factor or mammary tumour agent ( bittner , 1936 ) and the demonstration that three factors , namely , the genetic factor , the hormonal factor , and the milk factor , are essential for the development of spontaneous breast cancer in certain inbred strains of mice ( bittner , 1939 ) , was followed by investigations of the part played by the agent in the induction of breast cancer by oestrogenic hormone . 
lacassagne ( 1939a ) , lacassagne and danysz ( 1939 ) , twombly ( 1939 , 1940 ) , and later bittner ( 1940 , 1941 ) , gardner ( 1941a ) , shimnkin and andervont ( 1941 , 1942 ) , dmochowski and gye ( 19.44 ) , found that fosternursing of low - breast - cancer strain mice by high - breast - cancer strain females increases the incidence of breast cancer in the low - breast - cancer strain mice treated with oestrogenic hormones . 
tlhus male mice do not develop breast cancer , even if they are susceptible and have the mammary tumour agent , unless the hormonal or oestrogenic factor is supplied , and only few or no breast tumours are induced in low - breast - cancer strain mice , i.e. 
it is probable that a quantitative deficiency of any one factor can be overcome by a relative quantitative increase in the other two factors ( shimkin , 1945 )  . the accelerating influence of carcinogenic hydrocarbons on the appearance of breast cancer in mice of unmknow - n genetic constitution was first observed by maisin and coolen ( 1936 ) and perry and ginzton ( 1937 ) , while dobrovolskaiazavadskala and adamova ( 1938 , 1939 ) , were unable to observe a similar effect on inbred mice employed by theexperiments carried out by mider and morton ( 1939 ) , bonser and orr ( 1939 ) , strong and smith ( 1939 ) , bonser ( 1940 ) , strong and williams ( 1941 ) , engelbreth - holm ( 1941 ) , engelbreth - holm and lefevre ( 1941 ) , orr ( 1943 ) , kirschbaum , lawrason , kaplan and bittner ( 1944 ) , kirschbaum , williams and bittner ( 1946 ) , shimkin and bryan ( 1943 ) , strong ( 1945 ) , orr ( 1946 ) , demonstrated that carcinogenic hydrocarbons induce the appearance of breast cancer in females of low - cancer strains or accelerate the development of breast tumours in female mice of high - cancer strains . 
in these experiments it was also shown that oestrogenic influence is important in the induction of breast cancer by carcinogenic hydrocarbons , since mice developing these tumours in the absence of treatment with oestrogens were females , with one exception , a cba male ( orr , 1943 )  . 
the male mice of both strains , when 60 - 70 days old , were divided into three groups : mice of the first group received oestrone and methylcholanthrene , mice of the second group methylcholanthrene alone , and mice of the third group oestrone alone . 
c57 strain females , when 6 weeks old , were divided into two groups : mice of the first group were forcibly bred , mice of the second group were maintained as virgins ; both groups were treated with methylcholanthrene alone . oestrone was applied as a 0.02 per cent solution of keto - hydroxy - oestrone in alcohol , by painting the mice twice weekly for a period of 12 months . 
methylcholanthrene was administered according to the method described by orr ( 1946 )  . sixteen drops of 0 - 5 per cent solution in sweet almond oil were applied , at fortnightly intervals , to the skin of mice , four on each side of the dorsal and ventral surfaces of each animal . the c57 females of both groups received fortnightly applications of methylcholanthrene from the age of 6 weeks till death or the appearance of a tumour . in male mice the mammary gland is in a rudimentary state and they do not have nipples ( lacassagne , 1936 )  . 
administration of oestrogenic hormones leads to the progressive development of the mammary glands described in detail by turner and gomez ( 1934 ) , burrows ( 1935 , 1936 ) , gardner ( 1935 , 1939 , 1941b ) , lacassagne ( 1939b ) , loeb ( 1940 ) , loeb and suntzeff ( 1941 )  . 
because of these observations and the findings that breast cancer can be induced by methylcholanthrene in males treated simultaneously with oestrogens ( orr , 1943 ) , the c57 and c3h strain males were painted for 3 months with oestrone before the treatment with methylcholanthrene . 
after 3 months of painting with oestrone both the 057 and c3h males received their first application of methylcholanthrene which was then continued at fortnightly intervals until death or appearance of a tumour , except in the group of c3h males painted with oestrone which received only 14 applications of methylcholanthrene . 
two 057 males and one c57 female , which were treated with methylcholanthrene , developed general enlargement of lymphoid tissue , and in the c57 male which developed breast cancer a lymphosarcoma was found in the abdominal cavity . 
one c57 virgin female developed a spindle - celled sarcoma in the subcutaneous tissue . skin tumours were found in 10 out of 18 c57 males treated with methylcholanthrene alone and in 10 out of 35 c57 males treated with oestrone and methylcholanthrene . 
squamous metaplasia was observed in two tumours induced in 03h males by oestrone and .methylcholanthrene. the breast tumours induced in c57 mice , both male and female mice , showed a much greater degree of squamous metaplasia than the breast tumours induced in some of the induced tumours , as in the case of spontaneous in c3h male mice . tumours , variations in structure between different parts of the same tumour were found . 
sometimes the tumour cells were packed in solid sheets while elsewhere they occurred singly or - in small groups surrounded by an abundant stroma ; in some parts acinar structure could be clearly seen while in other parts it was lost entirely ; in places elongated cords of spindle - shaped tumour cells were found . 
thus the observations recorded by previous workers in this field were confirmed and the influence of hormonal factors on the development of carcinogen - induced breast cancer in mice shown . mammary tumours were also induced in c57 low - breast - cancer strain females , both breeding and non - breeding . 
orr results of other investigators . in mice , both males and females , which lack the mammary tumour agent . it is , therefore , possible to induce breast cancer in the present experiments breast cancer was induced in mice of a sub - line of c57 black low - breast - cancer strain which has a low susceptibility to spontaneous breast cancer . 
breast tumours developed in c57 black strain females , both breeding and non - breeding , after treatment with methylcholanthrene , and a c57 black strain male after combined treatment with oestrone and methylcholanthrene . it is not known whether the failure of kirschbaum , williams and bittner ( 1946 ) to induce breast cancer in breeding c57 black strain females with methylcholanthrene was due to the different method of application of methylcholanthrene or to differences in susceptibility of the two sub - lines of the c57 strano tumours were observed in either c3h or c57 black strain males treated with methylcholanthrene alone . this supports the findings of other workers that hormonal factors play a part in the development of tumours induced by methylcholanthrene . orr ( 1943 ) , however , reported the induction of breast cancer in one out of 16 cba low - breast - cancer strain males after intranasal administration of methylcholanthrene in the absence of treatment with oestrogen . 
he also obtained breast cancer in virgin mice of two low - breast - cancer strains ( if and cba ) after treatment with methylcholanthrene . in the present experiments tumours developed in two non - breeding c57 females treated with methylcholanthrene . mider and morton ( 1939 ) failed to induce breast cancer in non - breeding dba high - breast - cancer strain mice , while strong and smith ( 1939 ) , strong and williams ( 1941 ) and strong ( 1945 ) in their successful experiments on the induction of breast cancer in jk , nh and nho low - breast - cancer strain mice employed breeding females only . the present experiments confirm the findings of other workers that breast tumours can be induped in male mice of susceptible strains after treatment with oestrogenic hormones ( shimkin and andervont , 1941 , 1942 ; bittner , 1941 ; dmochowski and gye , 1944 ; bonser , 1944a , 1944b )  . the present findings also confirm the observation that mammary tumours develop in mice of resistant strains following treatment with methylcholanthrene . 
the c57 low - breastcancer strain mice of the sub - line used , when foster - nursed soon after their birth by riii high - breast - cancer strain females , develop an incidence of 11 per cent of breast tumours , but fail to develop breast cancer when given the tumour agent at the age of 4 - 6 weeks , even after large quantities of the agent and in spite of increased hormonal stimulation by repeated pregnancies ( dmochowski , thus mature mice of this strain though resistant to the mammary 1948 )  . tumour agent are susceptible to a different agent , i.e. 
methylcholanthrene. the treatment with oestrogenic hormones failed to induce breast cancer in c57 low - cancer strain males . this again confirms the previous findings ( twombly , 1939 , 1940 ; bittner , 1940 , 1941 ; shimkin and andervont , 1941 , 1942 ; dmochowski and gye , 1944 ) that an increased oestrogenic stimulation overcomes the threshold of lowor non - susceptibility and results in the development of breast tumours only in the presence of the mammary tumour agent . treatment with methylcholanthrene induced in c57 black low - cancer strain females , breast tumours which , although similar in their structure to spontaneous breast cancer of highand of low - breast - cancer strain mice , show an increased incidence of squamous metaplasia . 
1. received for publication january 21 , 1948 . surgery in the form of the so - called radical operation is in general at the present time the principal weapon in the treatment of carcinoma of the breast . what it can achieve and its limitations are now fairly well established . 
thus it is known that if a patient with carcinoma of the breast has microscopical invasion of the axillary glands , the strong probabilities are that the disease has passed beyond the local area and that the patient will sooner or later succumb it is to the disease , although there will be a minority of fortunate exceptions . also known that if the axillary glands are not invaded the chances that the patient will be cured of the disease are appreciably increased . 
the present article attempts to assess the comparative results of a personal series of cases in which various techniques of treatment were used . the technical surgical problem in carcinoma of the breast has been rendered clearer by modem work on lymphatic anatomy , and in particular by that of gray ( 1939 )  . this has shown , firstly , that the dermis is a plane rich in lvmphatics and hence a rich potential plane of spread of carcinoma , particularly if the growth has spread into or near the skin ; and secondly , that the deep fascia is a plane devoid of or very poor in lymphatics , and hence not an important potential plane of spread . the former fact suggests greater radicalism in removal of skin , and the latter removes the main pathological reason for the routine sacrifice of the pectoralis major muscle . accordingly , since the appearance of gray 's work we have modified our technique to remove more skin , in our more recent cases skin grafting in a high proportion , and we have gradually abandoned routine sacrifice of the pectoralis major , only removing it if actually invaded , which is rare and late . the argument is sometimes used that the removal of the pectoralis major is necessary for the adequate dissection of the axilla , and that in any case its nerve supply is bound to be cut . these technical arguments are with elevation of the arm , retraction of the pectoralis major and not valid . removal of the pectoralis minor it is easy to do a complete clearance of the axillary glands and fatty tissue right up to the apex of the axilla , and at the same time to preserve the lateral pectoral nerve , the main nerve supply to the pectoralis apart from this modified radical operation there are smaller series of major . cases which have been treated in other ways , e.g. 
the comparison between the figures of standard radical and the modified radical operation in this series is rendered more difficult in that the modified radical was evolved out of the standard radical , and hence has not been done for so long . 
survivals it would therefore seem fair to conclude that these figures bring no evidence to support the view that the addition of the removal of the pectoralis major muscle brings any increase in the survival figures , and to raise the question whether the routine removal of this muscle should remain an essential technical point . a further way in which the two operations can be compared is by the comparative incidence of recurrence in the operative area , i.e. 
skin , chest wall and out of 42 traced standard radical operations there were 10 cases with axilla . of these , 7 were recurrence in the operative area , chiefly skin recurrences . cases with original lymphatic glandular involvement and 3 without such involveout of 40 traced modified radical operations there were 7 local recurment . of these , 5 were cases with original lymphatic glandular involvement rences . while the incidence in both is too high , and and 2 without such involvement . approximates to the 22 per cent noted by truscott in the middlesex hospital series , the standard radical shows no lower incidence of local recurrence than the modified . the 3 other types of procedure which have been carried out were : ( 1 ) in the first place there were 10 cases in which partial mastectomy was combined with the usual full axillary dissection after the removal of the pectoralis minor . ( 2 ) secondly , there were 9 cases in which a simple removal of the breast was combined with irradiation to the axilla . ( 3 ) thirdly , there were 7 cases in which irradiation only of the breast was combined with axillary dissection . the last group comprised cases which were all advanced , the glands being invaded in all . 
one case died as a result of the operation ; the other 6 died of the disease at intervals varying from 6 months to 4 years after treatment . moreover , in a high proportion of cases - 4 out of 6 cases which survived operationthere was either failure of the irradiation to get rid of the original primary growth in the breast , or recurrence of the growth locally after apparent disappearance . in view of the advanced character of these cases , the ultimate fatal result was likely whatever form of treatment was adopted . 
of the 2 cases with invaded glands , 1 was alive with the disease at 5 years and 1 died of other causes at 2 of the 5 cases with glands not invaded , 3 were alive and well at 10 years , years . 9 years , and 5 years , 1 was alive with the disease at 8 years , and 1 had died of other causes 1 year after operation . there were also 3 cases in which no histological note was made of the condition of the axillary glands ; of these , 1 was alive and well at 6 years and 2 died of the disease . 
from the point of view of survival after operation , this group compares favourably with other groups . but the procedure has been abandoned because of the occasional further development of carcinoma in the portion of the breast remaining . this happened in 2 of the cases of this series , and it also happened in other similar cases in a private series , in spite of the fact that , as already noted , only small and apparently early cases were considered suitable for this procedure , that a wide area of breast tissue around the growth was removed , and that post - operative irradiation was it was concluded , therefore , that while in an occasional case given as a routine . partial mastectomy combined with a dissection of the axilla might be justifiable a conclusion supporting that of fitzwilliams ( 1940 ) and of mitchener , bailey and price ( 1937 ) - the danger of further development of carcinoma in the remaining breast tissue rendered the procedure an unwise routine . the last group was of 9 cases in which simple removal of the breast was the only surgery performed , the axilla being treated by irradiation . these cases wvere usually elderly patients with apparently early growths . 
the main routine removal is not necessary ? advantages of preserving the pectoralis major are three . firstly , there is the cosmetic consideration , mutilation being much less with the pectoralis major preserved as compared with the skeleton - like prominence of ribs and costal cartilages after - its removal . 
and if there is no curative advantage in removal of the muscle , cosmetic considerations , although secondary , are entitled to a place . secondly , there is less loss of blood if the muscle is not removed , and consequently less operative shock . while in most cases this does not amount to a point of much practical importance , in old or feeble patients it may . with preservation of the pectoralis major combined surgical treatment of the breast and axillary contents is , in our experience , much less upsetting than simple mastectomy combined with irradiation to the axilla , and we have as already stated for this reason largely abandoned the latter procedure . 
1. received for publication january 13 , 1948 . one of the implications underlying the work of huggins and hodges ( 1941 ) on the factors governing prostatic hypertrophy is that malignant prostatic cells are not fully , if at all , autonomous . that is , they are still controlled in some measure by the same factors that control normal prostatic cells . specifically , this is certainly true of their susceptibility to the influence of circulating androgens and oestrogens . carcinoma of the prostate is essentially a disease of old age . the possibility exists that the chief factor in deciding the individual 's susceptibility to this disease may be his androgenic state , that is , the level of circulating androgens in his blood , particularly during the later decades of life . the only practical method for ascertaining androgenic state is a determination of 17 - ketosteroids excreted in the urine of the subject . in the hands of many different workers ( barnett , henly , morris and warren , 1946 ) this method has revealed a very wide range of excretion of 17 - ketosteroids by normal men , at least in the lower age - groups . 
carr it is considered that the same proportion of the reproductive life of a - human female would bring her to 21 years of age , and that of a mouse to 41 moiiths , this emphasizes the very restricted nature of the sample usually studied . most of the data on spontaneous tumours comes from reports from poultry pathology laboratories , and is given as a percentage of all cases of deaths sent in for examination . this causes a serious under - estimate for several reasons for example , many of the deaths are due to s . 
pullorum and coccidial infections which strike the immature chick long before cancer age ; further , pathological investigations are usually only requested when a si - idden rise in mortality suggests an epizootic is beginning , while isolated deaths , which would include those due to cancer , are not always submitted for investigation post mortem . thirdly , there is little agreement among poultry pathologists upon the classification of cancer . 
the data available are thus no guide to " cancer incidence " in fowls , in the sense that this can be defined for mice and man , and are , for the reasons suggested above , usually a gross underestimate . 
although many such figures are available from various parts of the world it will perhaps be sufficient to compare the tumour incidence in the edinburgh flock , where all birds dying are examinedpodmortem , with data given bv goss ( 1940 ) , where a similar analysis has been made with flocks totalling 24 , 900 birds . 
out of 675 female birds dying since 1932 in the edinburgh flock at ages ranging from i to 9 years , only 50 , or 7 - 4per cent of the total adult mortality , gave evidence of neoplastic or allied conditions . 
the edinburgh flock in general is thus a " low cancer " line of considerable ' interest . at present it is sufficient to indicate that complications due to spontaneous cancer can be neglected in considering the resiilts which follow a preliminary 8tudy0f 8u8ceptibility . the first analysis was made upon a series of 429 birds inoculated with cell - free rous no . 
the susceptibility of each bird was graded a ' ceording to the following scheme : negative result ( 0 ) , regression of tumours ( r ) , tumour response to i to 4 , for increasing lasses in all . 
the largest size and malignancy of the induced sarcoma at death or after 28 days was used as an indicator of the susceptibility of the bird . the grading was ' done by inspection . weighing the tumour is unlikely to be more accurate because of the difficulty of clean dissection , and the neglect of such factors as the size and condition of the host , the invasiveness of the tumour , and the presence of large blood - clots and mucoid fluids within its substance . in addition to differences in susceptibility of the host other possible causes of variation were considered , such as sex , season , site of inoculation , number of inoculations , dose of virus , and presence of other factors , such as antibodies injected with the virus . 
no consistent sex difference in susceptibility was noted during the work , and , as was later seen in the analysis , sex does not seem to affect the susceptibility to any detectable extent . 
teratoma testis ( michaelowsky , 1928 ; falin and gromzewa , 1939 ; bagg , 1936 ) ; transplantable sarcoma ( peacock , 1935 ) ; chemical induction ( murphy and sturm , 1941 ) , an analysis of the susceptibility of these young chicks to the rous no . 
i sarcoma showed no seasonal trend ( carr , 1942 )  . that the site of inocul - ation is of little importance in influencing tumour growth was shown by gye and purdy ( 1 931 ) ; it was claimed by them that the resistance depends upon the dose of virus inoculation . no support for this claim could be found by carr ( 1942 , 1943a and b ) , and the results described below indicate that any such variation is inconsiderable . that the other factors mentioned , viz . 
number of inoculations - and presence of other materials , were probably of sl ' l ' ght importance was concluded from several experiments in which only one of these factors varied in an otherwise identically treated g ' roup . 
a large number of the experiinents found in the literature illustrate this point , which is seen to be confirmed in the analyses that follow . the birds comprising the first group of tested animals were from most - of the " all the year round " hatching had been necessary to maintain institute lines . an even supply of experimental chicks , but not many matingswere represented by more than a few tested chicks . in fact , the 429 birds were descended from 33 sires and 147 dams , and many matings had but one tested offspring . 
carr the various institute lines ; these were all selected for physiological characters associated with egg production . dividing each line into two clear classes , negatives and non - negatives , the difference between the breeding line ( with the largest number ) and non - moult line was examined . 
a similar test on the most conspicuous of the other negative tumour groups , intensity , gave a value of chisquare 1 - 23 , which is of no significance , being the variation that would occur in i out of 4 random samples . it was therefore unnecessary to test the rest of the since the regressio ' ns ( r ) are equal to the negatives in the breeding line , groups . but more frequent than the negatives in the non - moult line , the frequency of regressions in the non - moult line is therefore even more significantly disproportionate than the number of negatives , so that this line has an obviously high resistance to the production of fatal cancer by the rous no . 
at about the close of the period when the first analy 'sis was made , the breeding programme at edinburgh resulted in a large proportion of chicks provided for experimental inoculations being derived from a single pen of the breeding line . these chicks were all offspring of a single sire , f651 , and dams fairly closely related to him . .the annual population of the breeding line is larger than that in any of the other groups constituting the flock , and the method of selection used is such that they are also less inbred . in table iv the responses of the groups of progeny from 14 females mated to f651 are illustrated . it is obvious that there is considerably variation among the groups ; in the first three the responses are mainly restricted to the two highest types , and in the fourth the same tendency is only slightly less marked . in the next six progenies ( g196 - g866 ) they are distributed fairly evenly over all the classifications except the completely negative one . 
carr this tendency for the susceptibility of chicks from certain individual matings to fall into a rather narrow range has been encountered in other pens of birds , and knowledge of its existence has been made use of both in providing susceptible chicks for experimentation 4nd avo - iding the progeny of dams whose offspring were known to give a poor response . 
it had been noted that when the non - moult chicks included in the original analysis were grouped according to sires , one of the latter , h573 , stood out as producing particularly resistant progeny ( table v )  . fact , when the data from his chicks were omitted the difference between the responses of chicks from the breeding and non - moult lines were no longer signifiit seemed probable then that the disproportionate number of regressions cant . and negatives produced by h573 was not due to the character non - moult as such , but to some mechanism which can . 
on the other hand , to have carried out an experiment on a large number of birds in the hope that one of the rare resistant class was included would require labour and accommodation far beyond the facilities of any place engaged in fowl tumour work at this time.. the dams of the offspring of h573 analysed in table v were all fairly close relatives of the cock himself , and the way was thus opened for breeding a line of birds which would include a large proportion of resistant animals . it was arranged to carry out a small series of tests on the progeny of h573 , and almost all the birds tested gave either very ismall tumours , or more often , complete regressions . 
the slight shift towards greater resistance in the first 1941 test in both halves ofthe table suggests that some environmental factor leading t ' o a greater inhibitive effect on tumour growth may have been operative then . when the line had been thus established , a number of females , which had been tested and found to produce resistant offspring with a male of their own line , were then outcrossed to a non - moult cock , a descendant of the group from which the non - susceptible ( n - s ) line had been extracted . 
1 sarcoma virus varies from 40 per cent ( fischer , 1927 ) to 4 = 5 per cent ( gye and purdy , these figures may be complicated , however , by different rates of spon1931 )  . taneous sarcoma occurring in the population , either causing " natural " - or this complication is " induced " antibodies to be formed in the tested animal . probably absent at 6 weeks ( the age used here ) as no antibodies are present at this stage ( amies , 1937 ; andrewes , 1939 ; carr , 1943b ) , though they occur in resistance also varies with the age of the bird ( duranyounger and older chicks . reynals , 1940 ; rous and murphy , 1914 ; carr , 1943b ) , so a standard of age for testing is requisite for comparison . in the selection experiment for high and low susceptibility to jungherr sarcoma , carried out by cole ( 1941 ) for example , a resistant line with only 12 und impossible to raise the per cent susceptibility was obtained , but it was high incidence line much above the . 
original 70 per cent ; the cause of this may have been acquired r ' ather than a genetic immunity , since transmission from the immune female to the chick via the egg - yolk ( andrewes , 1939 ; carr , 1944 ) cannot be excluded , for progeny were tested at 7 - 18 days ,  . 
a time when yolk antibody is possibly still present in the chick . acquired immunity was ruled out as a cause of resistance of the n - s line . perhaps the most useful comparison which can be ' made is that between the present analysis of responses of the edinburgh flock to the filtrable tumour rous no . 
1 , and those of birds from the same stock to chemical tumour inducing material as demonstrated in the work of peacock , a report on which has been published ( greenwood and peacock , 1945 )  . 
the data , which cover the behaviour of 4 lines for the 10 - year period from 1935 - 1944 , are interesting both in their points of si ' milarity and dissimilarity to the material already discussed : they were classified only into positive and negative groups , but the non - moult line again showed itself the most resistant to cbemically induced tumours with a susceptibility of about 30 per cent , while breeding and large egg gave a figure close to 40 per cent and intensity had the highest susceptibility with 60 per cent since the early analysis of rous data places breeding ' as highest in posit ' lves . order of susceptibility , this difference in rank order might reflect inconsistency in the relative responses of the two lines to the different types of tumou ' r , but it appears more likely that with continued inbreeding the intensity line has chanced to become more susceptible . 
when most of the inbred lines were tested it was found that one in particular showed an obviously high resistance to the production of fatal cancer by the rous no . 
calcutt oestrogens : oestrone and diethystilboestrol . a group of arsenicals chosen in view of neubauer 's ( i947 ) implication of arsenicals as carcinogem ' c agents . three unrelated compounds : nitrogen mustard ( shown to be carcinogenic by boyland and homing , 1949 ) , urethane ( shown to be carcinogenic by jaff6 , 1947 ) and light green f.s. 
numbers represent duration of exposure in minutes to filtered ultraviolet radiatioxi required to cause death of 90 per cent of the cultures . with the exception of the oestrogens none of the agents tested compare in comparisons among the compounds tested effectiveness with 3 : 4 benzpyrene . suggest a certain degree of correlation with carcinogenicity . 
whether the effects achieved are a true photodynamic response as occurs with fluorescein and other dye stuffs , or ate a consequence of intracellular action by the radiation leading to increased susceptibility to a toxic agent , as calcutt ( i 950 ) has show - n to occur in the case of certain - sh inhibitors must remain in doubt . 
on the other hand , it appears possible that the radiation has brought about such an intracenular change that the chemical carcinogen is now able to attack a much greater amount of substrate than is normary the case . 
thus complete disruption of the intracellular functioning and death occurs , rather than ' a partial disruption which manifests itself as the carcinogenic change . certain evidence supporting such a view exists calcutt ( 1950 ) showed that irradiation with wave - lengths in the literature . greater than 3300 a increased the availabihty of intracellular - sh groups . such treatment prior to the addition of a photosensitising agent was show - n by calcutt ( 1951 ) to enhance the photodynamic response . 
with a stroke of 6 cat a rate of 90 per minute , and melanin formation estimated at appropriate intervals by observing the extinction coefficients of the solution with an e.e.l. 
on 15 occasions there was acceleration averaging approximately 0 - 3 per cent as compared with the control . on the assumption that the errors inevitably present will tend to operate equally in either direction , the probability of obtaining 15 results out of 17 in one direction is ( approximately ) only i in 500 , so that the results are statisticany significant . the possibility of the acceleration being due to change in ph was reconsidered . the ph of the various solutions employed was estimated , electrometrically , using a glass electrode . 
one of these paths is through the physiology of hormone production , a second is through the physiology of the propagation and transmission of the milk agent , and a third possible path is through the physiology within the mammary tissue cell governing its reaction to the hormonal stimulation or the milk agent . other paths may exist , and it is probable that these three are interwoven . proof of the effect of genic action in relation to the hormonal stimulation resulted from crosses between strain a in which the incidence in the virgin females is low althougb that of the breeders is high , and strain ch , in which the incidence in the virgin females as well as the breeders is high . results of such crosses were reported by bittner , h ' useby , visscber , ball and smith ( 1944 ) , heston and andervont ( i 944 ) , and bittner and huseby ( i 946 )  . 
heston incidence occurred among the virgin f , females resulting from mating strain a females to ' ch males as well as in the reciprocal f , virgins , together with evidence of segregation in the f2 generation , indicated that the difference between the strains was primarily a genetic difference . such a difference was to be regarded as either a difference in horinone metabolism , or a difference in the reaction of the mammary gland cell to horinone such evidence as the fact that added oestrogen stimulation yielded stimulation . tumours ' m ' strain a virgins , ' and the relationship of number of litters to tumour incidence in strain a reported by jones ( 1940 ) , strongly indicated a difference studies of the oestrus cycles of virgin females of strains in hormone stimulation . a and ch and their reciprocal f , hybrids reported by deringer , heston and andervont ( 1945 ) , however , failed to show any difference in the cycles , except that in the low - tumout group of virgin strain a females the onset of oestrus was these observations , therefore , suggested later than in the high - tubiour groups . that at least a part of the genetic difference could be manifested through the physiology within the mammary gland cell . in an attempt to determine the physiological mechanisms through which the genetic differences are manifest , huseby and bittner ( 1947 ) also have carried out a number of studies , including ovarian transplantation to f , hybrid mice , ovariectomy , vaginal smearing , and histologic examination of the genital tissues . these studies have indicated that more than ' one endocrine organ is affected . we have become more interested in the second path of gene action - that it has concemed with the propagation and transmission of the milk agent . been observed by a number of investigators that certain strains will propagate all the . 
high - mammary tumour strains the agent , whereas others will not . normally transmit the agent from generation to generation , and even low - tumour strains may have the same innate capacity as andervont ( 1945 ) has reported for strain c . 
on the other hand , bittner ( 1948 ) has found tumours in his descendants of strain c57 black females into which the agent was introduced , and fekete and little ( 1942 ) bave reported mammary tumours in descendants of c , , 7 black females that had developed in strain dba uteri from transferred ova . 
thus the situat ' lon m regard to the capacities of the various strains to propagate the agent is not clear . by crossing high - tumour strain c , h females with low tumour strain c57 black males , and comparing the two . 
types of backeross females produced by backcrossmg the f , females to both the ch and c.7black males , definite evidence of genetic differences in the ability to propagate and transmit the agent was revealed ( heston , deringer and andervont , 1945 )  . 
the possibility of an intracellular origin of the agent also enters into consideration . in some respects the picture is comparable to the gene - kappa relationship stuclied by sonneborn ( 1945 ) and co - workers in parameciuthe picture is not as , simple , however , in that there is . 
our attention has been directed toward tumours that arise in females in which the agent cannot be demonstrated ( heston , deringer and levillain , unpublished data )  . in order to obtain a line of strain ch friae of the milk agent a litter of 3 females and 3 males were removed from their c3h mother by cesarean section and fostered upon c57 black female . 
these females were later mated to their male littermates , and subsequently the line has been maintained by brother x sister matings , the young being nursed upon their own mothers . in this line ; designated as c3hb , mammary tumours are arising . in 194 of the females of this line 14 months of age and older and extending through the first 5 generations to date , 47 , or 24 per cent , have developed mammary tumours at an average age of 18 - 9 months ; seventy - eight have died without mammary tumours at an average age of 16 months , and 69 are still living at an average age of 18 months . study of the histologic section of 37 of these tumours has not revealed mucli dissimilarity between these and the usual ch mammary tumours . nine , however , ' have show - n areas of squamous metaplasia with pearl formation similar to that described by kirschbaum , williams and bittner ( 1946 ) in mammary tumours induced by methylcholanthrene in the absence of the milk agent such areas also occasionally occur in normal strain ch females of older age , suggesting that the occurrence of squamous areas may be characteristic of older age rather than specifically owing to the possible absence of the milk agent . three of the tumours arising in these c3hb females have been transplanted into other c3hb females , and in each case the transplant has grown . we have no evidence , however , of a milk agent in the etiology of these tumours . distribution of the tumour - bearing females in the pedigree chart does not show segregation of tumour families , as ' would be expected if the agent were present . furthermore , we have found no evidence of the agent in cell - free extracts of these these extracts varied from 2 to 5 per cent , and were prepared in 0 - 005 m tumours . .phosphate buffer ph 7 - 0 with a waring blendor ; cleared in a multispeed centrifuge at 18 , 000 g.. 
heston extracts premonths of age and older , and none has yet developed a tumour . pared with the same technique from tumours from c3h females produce tumours in c3hb test females in approximately 8 to 10 months . this is not presented as absolute proof that the agent is not present . if it is , however , it is not comparable to that in c3h females , nor is there any evidence of its being built up comparable to that of c3h . if the agent is not present we have proof that the accumulation of genetic factors and of non - genetic factors other than the milk agent can cause the tumour threshold to be surpassed . this light the milk agent would be comparable to the carcinogens in the induction of lung tumours , where the presence of the carcinogen appears to merely increase the probability that the tumour will appear and at an earlier age ( heston , 1942a , b )  . while all of these factors have been shown to alter the probability that the normal mammary tissue cell may be transformed into a malignant cell , these investigations have not answered the question as to what constitutes this change to malignancy . 
parsons. from the department of chemistry , the university , manchester , 13 , and the royal eye hospital , southwark , london . received for publication august 15 , 1949 . the effects of injection of the four nucleotides obtained from ribonucleic acid on the tissues of normal and tumour - bearing mice have been described ( barakan , barker , gulland and parsons , 1948 ; parsons , gulland and barker , further experiments have now been carried out to note the action 1947 )  . of purine and pyrimidine bases on sarcoma growth , and to correlate if possible the biological activities of the compounds with their chemical structure . these experiments fall into two groups : ( a ) those relating to the amino and hydroxy purines , ( b ) those relating to uracil and its derivatives . both pure - line and stock mice were grafted with homologous or heterologous sarcomas , including c57 and s37 and the crocker sarcoma , the latter known to be very refractory to inhibitory action ( badger , elson , haddow , hewett and robinsoir , 1942 )  . 
the technique for treatment of the grafted mice was similar to that previously described , but the standard dose ( 0025 g . ) was reduced in proportion to the toxicity of any particular compound , or where the condition of the mouse necessitated this . insoluble or relatively insoluble compounds such as xanthine , guanine , adenine or hypoxanthine were administered as fine suspensions in the same media as used for the soluble compounds . examination of the tissues at varying intervals showed that all compounds injected as suspensions were rapidly removed within 24 hours with the exception of guanine , the relative inactivity of which on tumour growth seems to be due to its marked insolubility . deposits of the injected guanine were found at the site of injection long after the last dose . table i summarizes the results obtained . 
the total number of control mice was in all cases the same as the total number of treated mice . the amino and hydroxy purines . it had been noted that adenylic acid or adenosine produced shock when injected into mice , and it seems probable that this effect was due to the adenine radical , of which the pyrimidine ring is in a lower state of oxidation than those of the other compounds used which do not induce shock . injection of adenine in doses comparable with those used for adenosine or adenylic acid has been found to cause immediate and profound shock with heavy mortality , the minimum sublethal dose being 0005 g . 
parsons radical , but may be connected with the presence of the amino group in position 6 in the adenine radical . xanthine injected in tumour - bearing mice was removed from the tissues injected into in 24 hours , but no inhibition of sarcoma growth was observed . normal mice in doses of from 0 - 02 - 0 * 025 g . 
2 , where it wir be seen that the addition to the medium of the drug in a concentration of 60 mg . / litre reduces mitosis to less than one half . 
the drug was given intraperitoneary in doses of 5 y twice daily over a 5 - day period . there was no difference between treated and untreated mice in the time of disposal . ar experimental animals have shown a temporary loss of weight after being given the drug . effed , 8 on human malignant di8ea8e . this report , which is prehminary in nature , describes the testing of the compound in 17 patients suffering from leukaemia , polycvthaemia vera , lymphadenoma , multiple myeloma and three types of carcinoma . cases were chosen because they were unfavourable or too advanced for other forms of treatment . this choice of the least favourable cases - makes it harder to compare the effects ofthe drug with effects obtained with other forms oftherapy . it is , however , of value to compare in any individual patient the effects of the drug against those of any other type of appropriate treatment given either prior or subsequent to 9500 . the drug was given intravenously in doses ranging from 0 - 09 mg . 
probably represents a working range within which toxic effects on the bone marrow are not serious . side effects were slight ; half the patients had anorexia or nausea , generary very shght . 
the dose used in this case resulted in a temporary fan in haemoglobin and a fall in the white blood count to 1000 per c.mm. , at which time she was given a simple blood transfusion . 
at the end of this remission she was suffering from lassitude and following treatment with abdominal discomfort , and a rising white blood , count . 9500 her well - being improved ; her white cell count fell and the haemoglobin , which was 86 per cent , rose slightlv . primitive cells fell from 26 per cent to 14 per cent within 4 davs . 
the siibsequent historyof this patient was complicated , 2 months - after treatment , by , ' hepatogenous jaundice with leukopenia . four months after treatment the ' haemoglobin began to fall and the white cell count commenced to rise , and sore throat appeared , primitive cells rising above the she was re - treated a month later with 9500 , no beneficial pre - treatment level . effects being observed in the week that elapsed between treatment and death . post - mortem examination showed a massive leukaemic infiltration of liver , kidneys and spleen . 
the bone - marrow from femur and sternum appeared active . multiple capillary haemorrhages were present in the myocardium and brain . is difficult to assess the effect of treatment in this case owing to the complicating factors . 
the i - emission , however , was not as satisfactory as that previously obtained with x - rays . myeloblastic leukaemia of naegeli type . - this was a terminal case which had been treated previously only with transfusions . within 7 days after treatment the haemoglobin rose and the white cell count fell from 145 , 000 to within normal limits . primitive cells fell from a pre - treatment level of 76 per cent to 37 per cent and the myeloblasts and premyelocytes disappeared completely . however , this remission , although dramatic , was short . 
an improvement in the leukaemic condition was prompt . five days after the first treatment the total white cell count was within normal limits and immature cells had been reduced from 40 per cent to ' 6 per cent of the total . 
a good remission was noted 20 davs after treatment , which compared well with her first and best remission fohowing splenic irradiation . the patient felt better within a few days . 
the remission as far as the blood count was concerned after 9500 a similar reduction in red cells and haemoglobin lasted one month . this las ' ted 4 months , after which the symptoms recurred . 
none ( transfusion ) ( bleeding from tumour ) ca8e ix . generalized lymphadenoma ( section proved )  . - this patient was nevertheless the enlarged nodes decreased given a rather low dose of 9500 . very temporarily and the skin itching from which he suffered disappeared for - about a week . 
a drill - biopsy of the mass did not yield conclusive proof of lymphadenoma , which was nevertheless following treatment the itching was reduced and probable on clinical grounds . the skin , which was dry and iethyotic , became more normal in texture . 
the relief of symptoms this patient showed an interesting initial rise in the lasted less than 3 months . polymorphonuclear count at 4 days , prior to the onset of leukopenia . 
on both these occasions improvement in well - being occurred and some reduction took place in the size of his enlarged liver , - the improvement after the second course of nitrogen mustard being much less . on admission for treatment with 9500 , the liver was enlarged and oedema of the legs was present . 
none of them responded to the drug . case xv was later given a second course of treatment simultaneously with x - ray therapy , both agents being given as weekly treatments for growth - restraint . 
a leukopenia is generary seen , especiary at higher dose levels . this is due to a fall in both granular and lymphocyte series . the fafl in the granular cells may be preceded by a transitory rise over 3 to 4 days , not seen unless blood counts are repeated soon after treatment . 
effect of 9500 on peripheral blood count in a non - leukaemic case . no initial rise has been seen in the lymphocyte count , which reaches the lowest level 1 to 7 days after the end of treatment . complete recovery is slow and would seem to take about 2 months . 
for these two reasons we have waited for several weeks before instituting a second course of treatment in any patient . an extended clinical trial of 9500 would , however , seem to be justified on several grounds : first because it may be found effective in diseases other than those quoted ; even among the cases described there would seem to be a place for secondly , it could be regarded as a it in the treatment of polycythaemia . pleasanter alternative to nitrogen mustard , since , unlike nitrogen mustard , 9500 does not produce gastric effects and there is no fear of local thrombosis . 
thirdly , the drug is active ifgiven by mouth ; we have so far not tried its clinical effectiveness by this method , since , animal experiments have demonstrated that by this route the haematological response was less predictable . there is one disadvantage to an easily given drug of such potency . it is possible that it might be used without the constant supervision and check of blood counts which is necessary . 
as with all cytotoxic agents , its use should be contemplated only by those with facilities for adequate laboratory examinations . the tissue culture work has been carried out by m . 
bartholomew 's hospital , london , e.c.i. received for publication august 25 , 1949 . the question , whether the recent increase in deaths attributed to cancer of the lung is due to a real increase in the incidence of this disease , has been discussed the present paper , which brings forward no new data , is an by many writers . examination of some of the evidence offered on this subject . ( 1 ) the application of statistics from hospitals . the problem of finding the best index of a real increase in the incidence of lung cancer from hospital statistics is a difficult one . the relevant facts usually ascertainable at a hospital are : the annual total 1 . 
3 , 6 and 9 could be used as a measure 3 and 6 are open to objection on the grounds of the incidence of lung cancer . of possibly mistaken diagnosis , hence presumably 9 is the most accurate measure of what it is stated to be . as a measure of the population at risk , i.e. 
of becoming a case on which a post - mortem for cancer of the lung is done at a given hospital , we could use 1 , 2 , 4 , 5 , 7 or 8 . do these measures change in their quality of being representative of the the answer is that they all do so , because the cross - section general population ? of the population being admitted to hospital , whether of all cases or of cancer alone , changes , and therefore the cross - section of all deaths represented by deaths in hospital changes also . 
the assertion that the cross - section changes is based on knowledge that many people are now admitted to hospital who never would have been so admitted in the past - e.g. 
the same argument , that the cases would have been sufficiently serious to be admitted under any circumstances , could be applied , though with less certainty , to 2 , 4 , 5 , 7 , 8 . 
kennaway pathologists who deny that there is any satisfactory evidence of the suspicion is that where the incidence of lung cancer during recent years . such increase has appeared to have been conspicuous , there was formerly a low standard of accuracy of pathological diagnosis , and that the standard has improved with the passage of time . " real increase elusive disease has estimates of incidence , " for the foregoing reasons , comparisons of early and recent clinical or necropsy comparisons of the findings in different countries or in different hospitals , must be quite unreliable . so much depends on the personal experience of the clinicians and pathologists concerned , and current jotmrnals contain evidence enough that a uniformly high standard of diagnosis of this yet been attained by either . 
3 shows no steady rise , " which is perfectly true , and they conclude : " in our opinion the figures given illustrate that even a pronounced increase of the crude mortality rate for lung cancer among males , and almost only among males , does not necessarily mean an increase in incidence of that disease . 
presumably the more frequent detection of the disease is due to improvement of diagnostic procedures , and increased attention , and similar explanations may be correct in the case of occupations with an increased mortality from lung cancer . " one has difficulty in believing that any generalizations about cancer of the lung can be based upon such a peculiar succession of such small totals , drawn from a highly selected population , as those given in the third columnu of the table , namely 7 , 5 , 5 , 7 , 6 , 20 , 13 , 20 , 13 , 14 cases of pulmonary cancer . is one to believe that the totals 6 , 20 , 13 in the three consecutive years 1940 , 1941 and 1942 correspond to any similar annual incidence upon the whole population of denmark ? if there is no such parallelism , what is the value of these figures , and how can they provide a basis for the important generalization quoted above ? one may doubt whether the radiographic examination of any population for pulmonaty tuberculosis adds much to our knowledge of the total incidence of cancer of the lung . such a survey will , of course , reveal a number of cases , previously undetected , of such cancer . 
7. received for publication february 3 , 1949 . one of the surprising elements in bittner 's discovery of the " milk factor " is the apparent entry of the virus into the bodies of the young mice via the it is , however , obviousthat during suckling a small amount alimentary canal . of milk may also enter the nostrils , and that the nasal mucosa may offer an alternative port of entry . it also seems possible that the milk factor might be destroyed by digestion in the stomach . if this were so it would be difficult to recover milk factor from the stomach contents of suckling mice . 
the unfortunately the experiresult was entirely negative in the 40 mice so treated ment was not adequately controlled , in that none of the mice were tested for susceptibility by means of extracts known to contain the virus . 
jackson memorial laboratory , bar harbor , me . received for publication december 16 , 1948 . it is known from the work of andervont and bryan ( 1944 ) and green , moosey and bittner ( 1946 ) that the milk agent is antigenic for the rabbit and the rat . if , as is probable , the agent , is a virus one would expect it to be antigenic for the susceptible species . 
the nature of the s35 substance . the presence of s35 in the cytoplasm of myeloid cells after culture in s35 sulphate was all the more interesting since most mammalian cells will not utilise experiments were designed to obtain information on the nature of sulphate . the s25 substance in the myeloid cells . 
the summarised findings are presented in table i . ( 1 ) since 2 - 3 hours ' washing of the slides in running water , or 1 hours ' incubation at 370 c . 
a search was therefore made for a sulphate compound in these cells . although most sulphates form an insoluble precipitate with barium , it has been reported that the barium salt of chondroitin sulphate is soluble in water it was thought that the s35 in the cells might ( bray , gregory and stacey , 1944 )  . be in a form related to the chondroitin sulphates , therefore experiments were carried out to test the barium solubility of the su compound in the cells . both 1 m and 0 - 1 m ba ( oh ) 2 removed all the cells from the slides , but this could be prevented by m.aking up the ba ( oh ) 2 solutions in 10 per cent formalin . the morphology of the cells on the slides thus treated was unaltered , and it was found that 15 minutes ' treatment with 1 m or 0 - 1 m ba ( oh ) 2 formalin at 20 c . removed all isotope from the cells . formalin alone did not appear to remove any s35 . it had to be ascertained whether the effect of ba ( oh ) 2 was a specific ba effect slides subjected to distilled water alkalinised with or due to the alkaline ph . nh3 to ph 9 - 10 for 15 minutes at 200 c . 
for one hour or longer . that the s35 substance is not closely related to hyaluronic acids . ( 6 ) no parallelism could be detected between the behaviour of the s35 substance and those polysaccharides and other substances which give a positive hotchkiss reaction ( periodic acid schiff reagent )  . 
for 1 hour , the s3 substance was not . all hotchkiss - positive substances ( fresh human saliva , 1 hour at 370 c . ) , but it did not affect the isotope content of the cells . 
the uptake of methionine s35 . at the beginning of the experiments it was considered to be unlikely that s35 administered as sulphate to the culture medium would be transformed by the cells to sh groups . 
oliver s35 sulphate administered in vivo to rats has been shown to be taken up into the chondroitin sulphate of the growing cartilage ( dziewiatkowsky , benesch and benesch , 1949 ; dziewiatkowsky , 1949 ; 1951a , 1951b , 1952 )  . irrespective of whether sulphate or sulphite s35 compounds were given to rats in vivo , activity this finding has was found in the bone marrow by singher and marinelli ( 1945 )  . been confirmed by dziewiatkowsky ( 1949 , 1952 ) using whole bone autoradiographs and it was thought not to be chondroitin sulphate , but to be inorganic sulphate this sulphur after the first 30 minutes sulphur which is precipitated by ba ( oh ) 2 . of administration , is incorporated into a more complex compound from which the sulphur is slowly removed . the s35 substance in the myeloid cells of the bone marrow cultured in vitro it is possible that the dose of s35 sulphate given is removed by barium salts . to the rats by dziewiatkowsky was too small to be detectable in whole bone autoradiographs in the acid formalin fixed bones ( 5 / tc . / 7 - day - old rat in one series and 1 - 25 , tc . / 7 - day - old - rat in the other series of experiments )  . at this stage of the work it is probably too speculative to attempt to correlate the specific s35 sulphate uptake of the myeloid cells with the specific action of myleran ( 1 - 4 dimethanesulphonyloxybutane ) on the myeloid cells . 
doll. from the statistical research unit of the medical research council , london school of hygiene , keppel stred , kondon , w.c.i. received for publication november 28 , 1953 . attempts to derive theoretical laws from changes in the death rate with age have a long history . 
they have not , in general , been very fruitful and there has - been some hesitation in applying the technique to the study of cancer . recently , however , two hypotheses about the mechanism of carcinogenesis have been put forward , which have been derived from analysis of cancer mortahty fisher and horomon ( 1951 ) used statistics from the united states statistics . for cancer of the stomach in women , and nordling ( i953 ) , classing all sites together , used statistics for cancer in men from britain , france , norway and the u.s.a. both found that , within the age group 25 - 74 years , the logarithm of the death rate increased in direct proportion to the logarithm of the age , but about six times as rapidly ; in other words , the death rate increased proportionany with the sixth power of the age . 
death rates in some age groups under 24 years were . higher than would be expected had this basis been a general law throughout iffe . rates for the age groups above 75 years were considered unreliable and were excluded . in interpreting these observations , both assumed that mortahty gave a good indication of incidence and treated the data as if they referred to age specific incidence rates . 
they considered that cancer in youth might be affected by special factors which were unhkely to operate at later ages and they , therefore , felt justified in basing their hypotheses on the data recorded for adults . fisher and horomon ( 1951 ) pointed out that the observed relationship between age and mortahty could result if a colony of six or seven cancer cells was a critical size below which independent growth was not sustained . thig hypothesis , however , also leads to the conclusion that cancer incidence should be proportional to the fifth or sixth power of the concentration of the effective carcinogen whereas experimental data suggest that , in general , tumour incidence and concentration of the carcinogen vary ' in arithmetical proportion . 
the hypothesis in its simple form is , therefore , untenable . nordling ( 1 - 953 ) , on the other hand , suggested that the observed relationship would be explained if a cancer cell was the end - result of sev ' en successive mutations . this hypothesis does not lead to the observed result in all circumstances . does so only if the probability of occurrence of each mutation remaihs constant throughout hfe . in this case and so long as the occurrence of each mutation p . 
the logarithm of the incidence rate will then be directly proportional to the logarithm of the age and will increase six times as rapidly , ix , log rate 6 log t + a constant . in contrast to fisher and hollomon 's ( 1951 ) hypothesis this leads to the conclusion that the final rate of tumour formation wir be directly proportional to the concentration of an applied carcinogen , so long as the probability of occurrence of a mutation is proportional to the concentration of the carcinogenic factor and different factors are required to effect different mutations . in his analysis , nordling ( 1953 ) grouped all types of cancer in men together and considered them as a whole . 
he gave no detailed figures for cancer in women , but stated that for cancer of the sex organs the increase in mortahty was fairly small above the age of 4.5 and that for other sites the mortality seemed " to increase according to the sixth power of the age both before and after the forties but not during the decade of the menopause when the increase is smaller . " he considered that the entire increase with age in cancer incidence among adults was not wholly explicable by a mechanism of multiple mutations but that hormonal control of growth might play an independent part . 
1 - 4 show the logarithms of the male and female death rates from cancer of the commonest sites in england and wales in 1950 and 1.951 ( registrar general , 19521 1953 ) , plotted against the logarithms of the mid - points of the age groups . for each sex , all those sites are shown for which more than 1000 deaths were recorded in each of the two years . 
are less reliable than those for the subsequent ones since they are based on much smaller numbers , and both standard errors and ( to a lesser extent ) arithmetical errors are much larger . for example , the standard error of the death rate from gastric cancer in women is 18 per cent of the rate for the first age group ( 25 - 29 years ) and .2 per cent of the rate for the last ( 70 - 74 years )  . that is to say , the logarithms of the true rates for these age groups are likely to lie within a range of - .0 - 13 and - .0 - 02 respectively of those showm in fig . 
i. - change in mortality with age for cancer of the oesophagus , stomach and pancreas in men and for cancer of the stomach and pancreas in women shown on a double logarithmic scale , that is , the logarithm of the death rate per million persons plotted against the logarithm of the mid - point of the age group . 
the straight line through the points has been drawn arbitrarily to give the best fit , subject to the gradient being 6 to 1 . cancer of the stomach , colon , rectum and pancreas in women - the observations fall fairly close to the lines . in some , however ( cancer of the colon in men , cancer of the stomach , colon and rectum in women ) , the observations fall closer to lines with less steep gradients . 
the relatively high rates at the younger ages could , however , result if the population contained a group of subjects speciahy susceptible to cancer of these sites in early life - and so far as cancer of the p . 
doll colon and rectum is concerned , such a group is in fact found in subjects of polyposis coli . the actual regression coefficients for all the first group of cancers are shown in all the mortahty rates for this group , table i ; they vary from 4 - 97 to 6 - 48 . therefore , show the same sort of association with age as was reported by fisher andhoromon ( 1951 ) andbynordling ( 1953 )  . 
3. - change in mortality with age for cancer of the lung , bladder and prostate in men and for cancer of the lung in women , shown as in fig . 
1. i 0 - 1 - 6 consists of cancers for which there is already reason to believe that the strengths of some of the factors responsible for their development are variable . cancers of the prostate , breast , ovary , and cervix and corpus uteri are all believed to be influenced by endocrine secretions , which vary 1 ' n each individual in the course of his life ; a proportion of the cases of cancer of tho lung is believed to be related to cigarette smoking which has become more prevalent in the last 50 years and a proportion of the cases of cancer of the bladder is due to occupational hazards , to which men have been exposed for various periods at various ages . 
on the hypothesis that carcinogenesis is a multi - stage process , therefore , cancer at these sites would not be expected to show a uniform relationship between death rates and any power of the age . 
doll the sort of irregularities to be expected will depend on the periods when the carcinogenic factors are most active and on which of the stages in the process of carcinogenesis are affected . 
an increased rate of production for a short time during early life will provide a larger number of altered cells to be acted upon by other factors in the future , and wfll therefore appreciably affect the incidence at , say , age 60 . 
the weight depends both on the time of operation of the carcinogenic factor concemed and on the order of the cellular change which it affects . if t is the age at which the cancer incidence is observed , to the age at which the subjects are exposed to the factor , and s takes a value between i and 6 according as the change effected is the first , or sixth , then the weight is proportional to second , tos - i ( t - to ) 6 - 8 . hence it can be show - n that the weight by which a varying probability of the first mutation would be multiplied is highest at age 0 and decreases rapidly throughout life . 
the weight for the probabihty of the second change reaches its maximum one - fifth of the way through the exposure period ; that for the third change , for factors effecting the seventh change ' , two - fifths of the way ; and so on . the cancer incidence will be directly proportional to the rate of production of this change at the time of observation , and the previous behaviour of this rate will be irrelevant ( i.e. , will have zero weight )  . since the weighted mean of the varying probabihty will in general depend on the age , t , the overall incidence at age t is no longer proportional to the sixth power of t . 
doll old ages , can be attributed to the fact that the populations concerned belong to if mortality generations which smoked fewer cigarettes than subsequent ones . at different ages is examined for cohorts born in different five - year periods , the effect disappears . the concept of carcinogenesis as a multi - stage process also makes it easy to understand the mechanism of the latent period which occurs after exposure to a for example , kennaway carcinogenic agent before the appearance of a tumour . ( 1947 ) has demonstrated that circumcision deferred until the fourteenth year of life fails to give the complete protection against cancer of the penis that it provides when carried out on the eighth day ; in other words some change must take place within the first 14 years of life which eventually leads to the development of the disease after a latent period which may be as long as 70 years . 
on the hypothesis that penile cancer is the end - result of a series of seven successive cellular changes - the first of which occurs only in the presence of the prepuce , it can be shown that at age 53 ( the average age * of all the patients referred to in table ii ) the relative risks of developing the disease when circumcision is carried out at the mid - points of shrek and lenowitz 's ( 1947 ) age periods are 0 - 30 , 0 - 77 , 0 - 98 and 1 - 00 ( see mathematical appendix )  . children circumcised during the first six years of life are , however , most likely to have been operated on within the first few weeks , in which case the theoretical risk for this group would be of the order of 0 - 01 . 
on the other hand , u the present data were held to be equally accurate at all ages ( including the oldest ) it would be possible to obtain a quite different mathematical relationship . thirdly , other mechanisms than that of a multi - stage process can also be postulated to account for the observed relationship - and one has , in fact , been suggested by fisher and hollomon ( 1951 )  . moreover , some other diseases show similar types of increase with age ( e.g. , cerebral haemorrhage , coronary thrombosis and gastric ulcer ) and it is difficult to believe that they should all be dependent on the same type of mechanism . bereilblum and shubik ( 1949 ) , in summarizing the results of their experiments , have , however , stated that " whatever interpretation is adopted as a base line for research , the recognition that careinogenesis is at least a two - stage process , should invariably be bome in mind "  . it is , therefore , natural to see whether a twostage process - or even a more complex multi - stage one can account for the human data . 
from the analysis that has been made here , it would seem that a complex process of perhaps six or seven stages could account for : sites , and ( 1 ) the rapid increase in mortahty with age observed in cancer of some ( 2 ) the irregularity in the increase in cancer of some other sites ( 3 ) the long latent period observed after exposure to a carc ' mogen ( 4 ) clinical observation . 
doll the mortality rates for cancer of the lung , bladder and prostate in men and for cancer of the lung , breast , ovary and cervix and corpus uteri in women also accord with the theory , if it is postulated that the carcinogenic factors responsible have varied in strength . a formula has been obtained which can be used to weight the strengths of the carcinogenic factors at different periods and it is shown that the time when the strength of the factors responsible for the individual changes is of greatest importance varies according to which change in the series is affected . 
the conclusion provides a possible explanation for the observation that circumcision exerts an important protective effect against the development of cancer of the penis only if it be carried out early in life . we are most grateful to r . 
more precisely , we assume that the probability that the sth change occurs in the small interval i changes have already occurred , is p , ( to ) dto where ( tw to + dto ) , given that s all the other p 's are assumed to be constant . 
then p , ( to ) is a function of to . the probability that the sth change occurs in the interval ( to , to + dto ) , and the rth in the interval ( t , t + dt ) , is the product of ( i ) the probability that exactly s i changes occur in the interval  . 
we shall ignore the variation in age amongst the patients , and consider the expected incidence of penile cancer at t = 53 years ( the average age of all the patients referred to in table ii )  . 
3. received for publication february 6 , 1947 . following the discovery by haddow and sexton ( 1946 ) of the effects of ethylcarbamate ( urethane ) on the growth and differentiation of the walker rat carcinoma , the action of this substance in human leukaemia was described by paterson , apthomas , haddow and watkinson ( 1946 )  . since the early experiments of warburg in 1910 , ethylphenylcarbamate ( phenylurethane ) had been known to arrest the division of the sea - urchin egg at metaphase . 
a similar effect in plant cells , closely resembling that of colchicine , was discovered in 1939 by lefevre and by simonet and guinochet ( 1939 ) , and was further studied by gavaudan ( 1943 ) and deysson ( 1944 )  . ludford ( 1936 ) treated tissue cultures with a 2 per cent solution of ethylcarbamate and noted abnormal metaphases and a considerable amount of nuclear and cellular degeneration . similar results were obtained by geiersbach ( 1939 ) , and ostergren ( 1944 ) also claimed that ethylcarbamate arrested mitosis at metaphase in alliutempleman and sexton ( 1945 ) , investigating the growth - inhibiting properties of numerous carbamic esters in plants , found , however , that ethylcarbamate was inactive in this respect , although marked activity was shown by ethyl phenylcarbamate and it was this work which led haddow and sexton isopropyl phenylcarbamate . to investigate the action of carbamic esters on tumour growth , and to the discovery that in this case ethylcarbamate itself is effective . these authors suggested that this substance might belong to the group of caryoclastic ( nuclear ) poisons studied by a . 
described by gorer ( 1946 ) was utilized , in which large stem - cells produced a diffuse infiltration of lymph nodes , spleen , liver and kidneys , leading to death of the mice in from 14 to 22 days after subcutaneous grafting of leukaemic tissue . enumeration of reticulocytes was made from dry blood smears , following postvital staining with * associe du fonds national belge de la recherche scientifique . 
the nucleus then disintegrates , the thymonucleic acid material forming shell - like structures covering an acidophil sphere , which also contains some ribonucleic acid ( by the further destruction ribonuclease method ) and the remnant of the nucleolus . leads to irregular fragments , with a positive or negative feulgen reaction . 
52 and within 3 days the number of reticulocytes was close to the initial value . no significant change in the numbers of red blood cells , or in the leucocytes , was observed . these results may be compared with those following the injection of colchicine , when the reticulocyte fall is marked and the minimum is also reached in 48 hours . 
the erythroblastic mitoses are arrested in metaphase by colchicine during the first hours following its injection , and the delayed decrease of the number of reticulocytes is a consequence of the time needed for the maturation of the erythroblasts . 
by comparing the histological pictures found in these last two groups it is possible to appreciate the specific effects of ethylcarbamate . ( ii ) blood - picture . - the principal changes were an increase in the percentage of granulocytes and a decrease in the numbers of reticulocytes . 
no evident atrophy of the mucosa , nor ulceration . leukaemic infiltrations : no evidence of increased cell - destruction nor mitotic abnormalities . in animals losing weight during the experiment , splenic and lymphoid atrophy is more pronounced . 
the bone - marrow remains very cellular , with a there is no evidence of aplasia . high proportion of immature granulocytes . in the thymus the atrophy of the cortical region is complete , the epithelial cells this picture of " inverted thymus " is found after the pycnotic only remaining . destruction of thymus cells by mitotic poisons ( dustin , 1929 ) or non - specific in this case it is probable that the stimuli ( selye , 1937 ) , and in inanition . ethylcarbamate injections and the loss of body weight have additive effects . no marked changes are found in other organs . * rosin and doljanski ( 1944 ) have described inclusions in liver cells of rats treated with urethane , which they believe are red blood cells engulfed in cytoplasm and even in the nucleus . 
for 5 days , there was an increase in leucocytes from 9200 to 22 , 800 , and in granulocytes from 33 to 58 per cent . the bone - marrow showed very active granulopoiesis . bring about any aplasia of the bone - marrow in the rabbit . these preliminary results indicate that large doses of ethylcarbamate do not 2 . 
the possible relations between mitotic poisoning and the therapeutic action of ethylcarbamate in human leukaemia are discussed . this work has been supported by grants to the royal cancer hospital ( free ) from the british empire cancer campaign , the anna fuller fund , and the jane coffin childs memorial fund . 
kirby , research department , glasgow royal cancer hospital . received for publication march 4 , 1948 . althoulgh the claims of roffo ( 1938 , 1939a , 1939b , 1941 ) to have induced adenocarcinoma of the stomach in rats given orally fats or cholesterol which had been heated to 3500 c . 
for half - an - hour have not been confirmed ( beck and peacock , 1941 ; kirby , 1943 , 1944 , 1945 ; morris , larsen and lippincott , 1943 ) , the positive results obtained by other routes in mice by other workers make a good case for regarding the products of such pyrolyses as carcinogenic . thus beck ( 1941 ) found sarcomas in 2 of 12 mice at the site of injection of cottonseed oil , previously heated to 340 - 360 c . 
for 1 hour , and steiner , steele and koch ( 1943 ) reported sarcomas in 3 of 9 mice injected with sesame oil heated to 3500 c . these latter workers found no tumours in similar mice injected with cholesterol that had been heated to 2000 c . 
for half - an - hour , and another sarcoma in 1 of 9 mice surviving more than 340 days after subcutaneous injection with a residue left after removing dicholesteryl ether and a4cholestenone from cholesterol heated in the same way ; these tumours were closely associated with the sites of injection . negative results were obtained by these workers when mice were injected subcutaneously with cholesterol that had been heated to 4300 c . 
of the remaining 14 , 5 died or were killed between 400 and 500 days , 1 was killed at 533 days , 1 died and 2 were killed between 500 and 600 days and 3 were killed at 735 days . bronchiectatic abscesses were found in most animals surviving 400 days ; there was no other frequent lesion . 
the stomachs were all normal , save for that of a rat dying after 616 days which had the haemorrhagic erosions of the glandular zone commonly found in rats in a state of inanition . 
shortly after the paintings had been commenced , 0 5 per cent of croton oil was added to the benzene solution in the hope of speeding up or enhancing the action of any carcinogen present in the c.o.h. after 258 days ' experimentation , a 15 per cent benzene solution of c.o.h , containing 0 5 per cent croton oil , replaced the original 10 per cent solution ; 2 males and 5 females survived long enough to be treated with the stronger three other male mice were painted with the 10 per cent - solution solution . for 168 days and then with the 15 per cent solution . atrophic changes at the site of painting were seen in mice dying at 80 days after the beginning of the experiment , but even in 2 females painted 258 days with the 10 per cent solution and then 374 days with the 15 per cent solution , and surviving totals of 640 and 656 days respectively , no further lesion was found . fourteen mice showed fatty changes in the liver , sometimes with extensive necrosis , but no hyperplastic changes were seen in this organ . 
the first mouse to die survived 80 days after the first injection ; 2 others died before 100 days , 10 survived 100 to 200 days , 2 from 200 to 300 days , 2 from 300 to 400 days , 5 from 400 to 500 days , and 1 survived 518 days . a mild histiocytic reaction was seen around sites of injection , but no sign of fatty changes in the liver were rare , but in mice any tumour was ever found . dying at 126 , 130 and 384 days after the first injection there were leukemic changes involving the liver and spleen in the first , the liver in the second , and the liver , spleen , kidney and lungs in the third mouse . ( c ) cholesteryl linoleate ( heated ) in mice . linoleic acid was prepared from cottonseed oil ( organic syntheses , 1942 ) and converted to the acid chloride by refluxing with oxalyl chloride . purified cholesterol was heated with linoleyl chloride on the water - bath for 3 hours , and purified as for the oleate . 
in the left flank , 31 days after the injection in the other flank . eight mice survived 100 to 200 days after the first injection , 1 for 247 days , 1 for 357 days , 2 for 470 days , 1 for 509 days , 1 for 558 days and 1 for 653 days . at the site of injection nothing beyond a mild histiocytic reaction was found in any mouse . 
and the time was 2 hours . as the esters were prepared by a non - pyrolytic method and were subsequently purified till non - fluorescent , any polycyclic hydrocarbon present in the residue after heating would have arisen as a direct result of the heating . 
no chemical experiments have been made to determine the nature of the components of the but the biological tests reported here gave no evidence of carcinoresidue . genicity in these residues for the skin or subcutaneous tissues of stock mice . such tumours as did arise in the mice used were almost certainly spontaneous . damage to the liver and the spleen was frequent , and the occurrence of leukemic infiltration was significant and probably attributable to the injections . it now seems that cholesterol and its naturally occurring esters may not be an important source of carcinogens in heated food . 
the results of the experiments described in this paper lend no support to the results reported by beck , kirby and peacock ( 1945 ) , who administered cholesterol heated to 3000 c . 
for the same period , as recorded by those authors , also of the negative findings of steiner , steele and koch ( 1943 ) with cholesterol heated to 2000 and to 300 c . 
it is concluded from this work and from other reports in the literature that cholesterol and its esters heated to reasonable dfooking temperatures for a reasonable cooking period have not been proved to yield carcinogenic residues . the author is indebted to p . 
no definite conclusions were obtained with regard to the influence of the thyroid gland , but the results showed a remarkable reduction of mammary cancer in one of the two strains used . preliminary experiments ( vazquez - lopez , 1946 ; morris , dubnik and dalton , 1946a , b ) showed that thiourea ingestion produced a continuous anoestrus due to ovarian atrophy and the consequent underdevelopment of the mammary glands . 
vazquez - lopez breeding females only were used from the two lines of r3 mice , and both breeders and virgins from the c3h strathe breeders were allowed to rear their first litters and , after weaning , the mothers were taken for the experiment . the litters of the c3h breeders provided the majority of the virgins employed . in all groups care was taken to divide animals of the same litters between the experimental and control groups . the animals were housed in metal boxes , 6 to 8 in each , and fed rat cubes breeders and virgins of the c3h strain were separated , but and water ' ad lib . the two lines of r3 breeders were mixed together . thiourea was administered in the drinking water , beginning with a 0 1 per cent solution and increasing the concentration as much as possible without endangering the survival of the animals . 
the approximate amount of fluid ingested was determined by dividing the total content of the water bottles by the number of animals in the box . all the animals were individually marked . 
the age of the breeders at the beginning of thiourea ingestion was between 3 and 4 months . the virgins were 3 months old , so that the mammary gland was fully developed before the action of the drug started . the period of administration for some of the r3 animals was a year , so that most of the animals with tumours died during the period of ingestion . for the c3h mice , both breeders and virgins , it was 6 months , so that the animals were under normal conditions again when 9 or 10 months old . 
even concentrations of 1 per cent could be used before the mice showed toxic signs such as loss of weight and reduction of water intake . if the solutions were kept between 0 - 2 per cent and 0 * 5 per cent the r3 mice could be maintained for most of their lives without any apparent difference from the controls . 
per animal per day , and if the concentration of the drug was not reduced , many it was possible to reach a concentration of 0 5 per cent in most animals died . boxes , but it could be maintained only for one or two weeks . solutions of 0 - 2 and 0.1 per cent were used most of the time , and even with these doses the intolerance to the drug and mounting mortality necessitated the withdrawal of the after some days without thiourea the mice recovered drug after 4 or 5 months . and the drug could then be administered again , but the recurrence of toxic it was preferable to maintain the 6 months ' period without symptoms was rapid . interruption , even at the risk of losing more animals in the last months of treatment . the doses administered were therefore variable for the different animals and for different periods for the same animal , but from the approximate estimate of water intake it may be assumed that the minimum of thiourea ingested per day was between 2 and 3 mg . 
daily. effects on the thyroid gland . in the r3 mice the ingestion of thiourea provoked the typical hypertrophy this effect was and hyperplasia with great vascularity , loss of colloid , etc . evident even with a 0 - 2 per cent solution , and became more pronounced in proportion to the length of treatment and increased concentration . 
the mice surviving after the withdrawal of the drug had thyroids of normal size and structure without any signs of the former hyperplasia . the effects of thiourea in c3h mice were observed in the experimental animals that died during the period of ingestion of the drug . 
as the great majority of these mice restricted their water intake to a minimum in the weeks before their death , it could be argued that they were not actually under the action of the for this reason additional mice not included in the experiment were used drug . to test the effect under identical conditions , and were killed when the actual ingestion of thiourea was known . in all the 03h mice of the experiment the results of the examination of the thyroid gland were similar . thiourea administered in the drinking water did there was clearly not produce hyperplasia or hypertrophy of the thyroid . congestion of the organ which might cause a slight increase in size , but the histological picture remained normal , showing follicles filled with colloid and lined by cubic epitheliunone of the mice that died during the period of thiourea ingestion showed changes in the microscopical picture . 
among 20 additional female adult mice killed after drinking solutions of 0 - 2 to 0 5 per cent for periods of a week to 2 months , with actual ingestion of about 5 mg . 
the small difference in favour of the thiourea group was not significant , for the small number of animals and the lower tumour age of this group was also against any inhibitory effect of thiourea . all the tumours appeared during the period of treatment when some of the mice had received thiourea for 10 months without interruption . mammary cancer in c3h breeders . - of 49 mice at the start of the experiment , only 29 ( effective number ) reached the age at which the earliest tumour appeared . the other 20 died as a consequence of thiourea ingestion . eight of the 29 table i . - lymphoma incidence in r3 breeding female8 ( mh subline )  . total effective mice with number . 
the earliest developed in a mouse 367 days old , 80 days after the withthis was only 10 days less than the average tumour age drawal of thiourea . these figures confirmed the assumpfor the control group , which was 377 days . tion that ovarian function and mammary development were normal in the experimental animals at the average tumour age of the normal mice . in spite of these normal conditions the percentage of mammary cancer in the thiourea - treated mice was 28 per cent compared with 54 per cent for the controls . 
vazquez - lopez mammary cancer in c3h virgins . - twenty - one animals died during the period of thiourea ingestion , and 64 mice survived the 6 months of treatment and five among the 64 reached the period of appearance of the first tumour . in the developed marmmary cancer ( 8 per cent ) , at the average age of 571 days . control group 40 out of 96 animals ( 43 per cent ) developed tumours at the average age of 432 days . 
the animals treated were 270 days old when thiourea was withdrawn , so that ovarian activity and mammary gland development were presumably normal not only long before the average tumour age of the controls , but also very nearly when the first tumour appeared among the normal mice . 
assuming that after the end of thiourea administration the mice needed a month for total recovery , they would be comparable with the normals when 300 days old . only 4 mammary cancers developed in the control group below this age . the decrease in cancer incidence in the thiourea - treated virgins was highly significant ( x2 = 20 9 ) , and confirms the results in c3h breeders . in this group the average tumour age was also later than in the controls . 
the average life span of mice without tumours was longer , but with smaller differences from the control mice than in the breeders . microscopical study of organs in thiourea - treated c3h mice . a detailed study of the organs of c3h mice after longer periods of thiouracil and thiourea ingestion was published by dalton et al . 
1 and 2 show condition and weight of the mouse rather than with its age . glands of control and experimental mice of approximately the same age . the two main differences between thiourea - treated and control mice are the number of nodules of alveolar hyperplasia and the width of the ducts . 
the alveolar nodules of one gland or even of all the glands in a single mouse cannot be used to distinguish a thiourea - treated from a control mouse ; in some thioureatreated animals the number and distribution of nodules is the same as in the controls . 
the thiourea - treated mice , as a whole , had considerably fewer nodules of alveolar hyperplasia than the control mice , and the average number of nodules per mouse was less than half the figure for the controls . there are no records of comparative counts in similar experiments with reduced mammary cancer incidence , but huseby and bittner ( 1946 ) report that less than 1 nodule per 4 glands was found in some animals more than 340 days old kept on the restricted diet in the experiments of huseby et al . 
vazquez - lopez to indiscriminate reduction affecting equally all the glands of each animal , but to a clear decrease of the number of glands with ' nodules . only 32 per cent of the glands in the thiourea - treated mice had nodules compared with 51 per cent in there were also significant differences in the distribution the control group . and the average number of nodules for glands in each group , but the main effect of the thiourea administration is to increase the number of glands without any nodules . 
as the hormonal and systemic influences are the same for all the mammary glands in each mouse the cause of these differences between glands in the same mouse must be the result of local factors whose activity is restricted to the fields represented by particular glands . thiourea acts systemically mainly by stopping ovarian function and oestrogen production . 
at the post - mortem examination the ducts appeared full of whitish material , which made them conspicuous to the naked eye . it seemed therefore to be due to engorgement by a secretory product , but it could exist in glands without any indication of alveolar proliferafig . 
some ovaries appeared in regressed condition without corpora lutea or follicular development ; this occurred with the same frequency in both groups . most ovaries had follicles in all stages of development , and some of the corpora lutea became hyalinized , forming large homogeneous masses with few cells . this formation of corpora albicantis is probably a strain characteristic , since fekete ( 1946 ) found them in the dba high cancer strain , but never in the c57 black strahuseby et al . ( 1945 ) found them only in one c311 mouse with mammary - cancer , and in none of the diet restricted animals of their experiment . that is probably another of the characteristics in which the c3h mice used here differ from those in american laboratories . 
the corpora albicantis were very prominent among the cancerbearing animals , and their ovaries owed their large size partly to these structures . apart from their size , however , the ovaries of mice with tumours showed mature follicles and abundant corpora lutea , which suggested normal activity . these findings were contrary to the results of allen , diddle , strong , burford and gardner ( 1935 ) in several strains of mice , including the c31 . in all groups the condition of the uterus reflects that of the ovaries having tall epithelium , extensive glandular development and loose cellular stroma in the animals with well functioning ovaries and signs of regression in those with ovarian atrophy . no differences were found in the adrenal glands of thiourea - treated mice compared with the controls . 
the most important change , affecting both groups equally , was the presence of amyloid degeneration , which appeared on the borderline between cortex and medulla and progressed towards the glomerular zone with advancing age . in the oldest animals amyloid invaded most of the cortex , and only two or three layers of cortical cells remained intact . 
the medulla was never affected . " brown degeneration " was , on the contrary , never found in c311 mice , but was almost constant in r3 animals of high or low mammary cancer lines . 
the possibility of a specific activity of thiourea on blood - forming tissues and their neoplasms suggested by the agranulocytic reactions in patients treated with goitrogenic substances is not supported by the results in the mh high lymphoma line which agree with clinical reports of treatment of leukaemic patients ( limarzi , kulasavage and pirani , 1946 ; hansen - pruss , 1947 )  . that lack of oestrogen is the effective factor is shown also by the normal incidence of cancer corresponding with regular oestrus in spite of thiourea administration in r3 mice . 
the action of progesterone is still a matter for discussion ( lacassagne , 1937 ; heiman , 1945 ; burrows and hoch - ligeti , 1946 )  . indirect ways of decreasing oestrogenic activity give , however , less clear results . although low cystine diets completely inhibit mammary cancer in c3h mice ( white and andervont , 1943 ) , the effects of stilboestrol pellets which restore the development of the mammary glands raise the incidence only to 44 per cent compared with 92 per cent in the controls ( white and white , 1944 )  . in similar experiments , ball , huseby and visscher ( 1946 ) found that in spite of continuous administration of stilboestrol the mammary glands of strain a mice maintained on restricted diets during 15 months were considerably less developed than those of control mice . 
the possibility of other hormonal factors influencing mammary cancer development is also suggested by the results of shimkin and wyman ( 1945 ) , who found greater reduction in adrenalectomized c3h mice than after ovariectomy , and by the observation of wallace , wallace and mills ( 1945 ) of persistence of effects of temperature in mammary cancer incidence after ovariectomy . the results of temporary lack of oestrogenic activity caused by thiourea show that reduction of mammary cancer is not due to defective glandular development , and therefore that the hormonal effects are not limited7 to the morphogenesis of tissue susceptible to carcinogenic agents . 
mammary glands of two groups of mice of the same strain with different cancer incidences have identical morphology , and only differ in the total number of nodules ofalveolar hyperplasia in each group . 
huseby and bittner ( 1946 ) have shown by the study of c3h and hybrid mice with or without the " milk influence " that this influence is necessary for the presence of nodules . 
the distension of ducts found also in the thioureatreated group seems to be a sign of excessive oestrogenic stimulation , and may indicate compensatory excess after thiourea withdrawal . if the hormonal influences are sufficient for the alveolar development the smaller number of glands with nodules , which is the main result of thiourea administration , must be due to local factors . 
the lack of oestrogens ( and its possible compensation afterwards ) affects all the glands of the mice equally . these results point to the possibility of effects on local factors and producing different consequences in each gland of the same animal . 
the local factor involved in the persistence of alveolar nodules is the milk factor , and the distribution of nodules suggests that it is affected by thiourea administration . there are no grounds for supposing that thiourea can directly influence the milk factor , but there are some in favour of an indirect action resulting from the lack of oestrogens . apart from the considerable number of experiments on the effects of oestrogen adninistration on cancer incidence , there is evidence even in normal circumstances of hormonal influences in breeding and forced breeding , increasing the number of tumours . 
the higher incideixce in mice born in later litters compared with the earliest from the same mother has been explained by the different concentration of the milk influence with advancing age ( bittner , 1942a )  . if the increased hormonal stimulation is the cause of increase in the amount or activity of the milk influence , it is probable that the converse holds true also , and that the lack of hormones acts in the opposite sense . the relative importance of the hormonal influence among the three factors necessary for the genesis of spontaneous mammary cancer in mice is not exactly opinions range * from that of shimkin and andervont ( 1941 ) , who known . consider the hormonal factors as necessary only for the creation of a substratum for the carcinogenic action of the factor transmitted by the milk , to that of warner , reinhard and goltz ( 1945 ) , who maintain the dominance in certain cases of the inherited susceptibility upon the concentration of the milk agent . loeb , suntzeff , burns and schenken ( 1944 ) believe that the oestrogens act by stimulating the growth of the mammary tissue and sensitizing it to all types of stimuli , and bonser ( 1945 ) as a developing factor acting upon breast tissues already sensitized by the milk factor . 
the only common feature of all these opinions is to consider the different factors as completely independent variables . on this basis the opinion of bittner ( 1942b ) that each influence must be considered as complementary to the others and all equally important is , besides being noncontroversial , nearest to the observed facts . 
but if there are some factors which are affected and increased , or even created de novo by interaction of the others , the relative significance of each would not be the same . the effect of genetic susceptibility on some aspects of the milk factor activities has been already proved by heston , deringer and andervont ( 1945 )  . 
w.3. received for publication july 16 , 1949 . in an investigation of the body growthand tumour growth - inhibiting action of carcinogenic substances , it was found that the nature of the inhibition is profoundly influenced by the protein content of the diet ( elson and warren , 1947 ; elson and haddow , 1947 ; elson , 1949 )  . in rats maintained on a 20 per cent protein diet injection of the carcinogen usually has little immediate effect on body growth although a delayed action , resulting in rapid loss of weight followed by death , may occur later . 
carcinogenic hydrocarbon 1 : 2 : 5 : 6 - dibenzanthracene 24 hours after implantation of walker carcinoma 256 in rats maintained on a 20 per cent protein diet results in very little difference in size and weight of the tumours from those of controls . if , however , the animals are fed a 5 per cent protein diet the tumours of those similarly treated with dibenzanthracene were found to be only one - quarter the weight of those of controls when the rats were killed 13 days after implantation ( elson and haddow , 1947 )  . 
hewitt. from the john burford carlill laboratories , westminster hospital , london . received for publication may 26 , 1953 . in a previous paper ( hewitt , 1953 ) a method for the preparation and titration of single - cell suspensions of sarcoma 37 was described . 
each mouse received 4 subcutaneous inoculations of the appropriate cell dose , one in each axilla and one in each grothe new - born mice were allowed to remain with their mothers throughout the experiments . 
1 the points plotted represent the percentage incidence of palpable tumours from inocula containing various numbers of viable tumour cells ( expressed logarithmically ) calculated from the cell count of a dense suspension and the dilution factor . the points shown were obtained from the data of various experiments . although the points are insufficient in number and accuracy to disclose the true character of the curve relating log . 
cell dose and tumour incidence , they serve to show the general trend of the relationship . litters born to mothers which had been rendered completely resistant to s37 by their having regressed a tumour before pregnancy ensued gave results which did not depart appreciably from those indicated in the graph . the td50 , read from the graph , is approximately 9 viable tumour cells . this figure is less than that obtained for the titration of c3h sarcoma in c3h mice over one year old , that is , 18 viable cells . certain technical difficulties have the effect of reducing the observed percentage of tumours obtained from specified cell doses below the maximum value . 
owing to the fact that the tumours which form in the suckling mice quickly reach a size which jeopardises health and movements , some animals die with tumours in 2 or 3 of the inoculated sites before all sites have been observed for a sufficiently long period to exclude tumour formation in thethe incidence of tumours recorded for any group thus tends to be submaximal . 
a further difficulty is that a proportion of each inoculum volume often exudes from the needle track after withdrawl of the needle . cell counts and volume measurements of the exuded fluid have shown that a quarter or more of the inoculated cells are lost in this way . if these known errors were to be eliminated , the points in the graph would be shifted slightly upward and to the left of their present positions , and the td50 would be rather lower than is obtained from the results as they stand . the effect of the diluent on the activity of sarcoma cells in dilute suspensions . craigie ( 1949 ) , discussing various technical requirements for the quantitative transplantation of tumour cells , drew attention to the inimical effects of various 386 h . 
hewitt in a later paper ( craigie , lind , diluents upon the viability of tumour cells . hayward , and begg , 1951 ) an account was given of certain atypical cells which composed about 5 per cent . 
the number of various mean doses of viable 837 cells . beside each point is the number of sites inoculated at that dose level . diluents in which inoculated cells were suspended : o tyrode solution . a , 0 5 per cent gelatine in tyrode solution . * 33 per cent mouse aacitic fluid in tyrode solution . a 10 per cent ascitic fluid in tyrode solution . the suspensions used to obtain the points shown in fig . 
the order of inoculation was so arranged that the interval elapsing between the time of preparation of the dilute suspension and the average time at which the mice of any litter were inoculated was equal for all the litters . 
hewitt quantitative transplantation of s37 in mature mice of different age - groups . a single - cell suspension of s37 was titrated in three series of mice , all from the same albino stock that provided the new - born mice and belonging to the following age - groups : 18 - 25 days ; 143 - 174 days ; 268 - 296 days . 
the incidence of palpable tumours in the inoculated sites was recorded for a period of 27 days following inoculation . in table i the td50 value given by each series is given together with its limits of error . 
the difference between the youngest and the oldest mice is not significant , but the td50 given by the oldest mice is considerably lower than that given by the mice of interthis last different , however , does not attain a significant level by mediate age . the irwin and cheeseman ( 1939 ) test . 
the systemic mechanisms by which resistance is manifested are cellular and humoral . in the case of resistance to tissue grafts , it appears that cells primarily are concerned in the resistance mnechanisms ( murphy , 1926 )  . 
young mice would therefore not be expected to possess resistance against a homologous tumour by virtue of maternal serum factors reaching them by the transplacental route before birth or by the colostrum after birth . in the course of the present studies it has been observed that a litter from a mother which was completely resistant to s37 , a tumour having regressed in her before pregnancy ensued , showed the gross developthe same high susceptibility as the litters of normal mothers . ment of the lymphatic system appears to be quite advanced in new - born animals as indicated by the presence of the thymus and by the existence of lymphocytes if it is assumed that the lymphocyte is responsible for tumour in the circulation . immunity , then the low resistance of new - born mice must be ascribed to functional immaturity of the lymphatic system . the td50 given by the titration of s37 in new - born mice , as will be seen fronm the trend of the points shown in the fig . 
1 , is about 9 viable sarcoma cells . graph suggests that the percentage of tumours to be expected from an average it is of interest to note that furth and inoculum of 1 viable cell is about 7 . kahn ( 1937 ) succeeded in transplanting leukaemia to about 5 per cent . 
kahn and furth ( 1938 ) obtained only about 20 per cent of tumours from subcutaneous sites inoculated with 50 - 100 viable sarcoma cells in circumstances where there was no genetic difference between the animal in which the tumour arose and those to which it was transplanted . 
a c3h sarcoma gave a td50 of 18 viable it is evident from tumour cells when titrated in old c3h mice ( hewitt , 1953 )  . these comparisons that the results of quantitative transplantation of s37 in newborn mice of a heterogeneous strain of which the adults exhibit some resistance to small inocula , are compared with those obtained for similar tumours within a genetically homogeneous tumour - host systethe use of new - born mice therefore provides a more uniform and more sensitive system for the detection or assay of viable tumour cells in circumstances where , owing to limited facilities or to the use of genetically unknown tumours , a genetically homogeneous system cannot be achieved . it will be seen from fig . 
2 , however , that quantitative resistance increases this being so , it is likely that very differperceptibly within the first week of life . ent quantitative results would be obtained from the titration in new - born mice of tumours with a slower growth rate than s37 . in these circumstances resistance factors could produce their effects before the tumours became palpable . 
a lymphoma which occurred spontaneously in a mouse of the albino strain used in these experiments gave a td50 of over 10 , 000 viable cells when titrated in newborn mice ; krebs ' carcinoma ascitic cells gave a td50 of over 2000 viable cells . it must be concluded from the results of titrating s37 in new - born mice that the high td50 levels obtained in adult mice of the same stock ( i.e. , about 1600 ) were due to immunity factors and not to intrinsic limitations of the proliferative power of the cells . an estimate can be made of the proportion of inoculated s37 cells which are capable of continued proliferation if it is assumed that such cells are not subject to any hazard affecting their proliferation once they have been successfully in these circumstances the dose - response curve deposited in the host tissues . would represent the chances of getting one or more " taking units " in random samples ( inocula ) taken from suspensions containing different mean densities of 390 h . 
1 , a suspension which gives 37 per cent of negative sites ( 63 per cent of takes ) contained a mean density of about 17 tumour cells per inoculum , and this evidently constitutes a single " taking unit . " thus , when it is assumed that there are no influences at the inoculated site which inhibit continued proliferation of a cell intrinsically capable of giving rise to a palpable tumour , then the results reported indicate that such cells are present in a proportion of about 1 : 17 ( 6 per cent ) of the population of cells designated as living s37 cells on the grounds of their resistance to staining with trypan blue the frequency with which abnormal mitoses and and their diameters ( > 9 , )  . cellular pleomorphy are found in rapidly growing tumours would seem to indicate that there may be a continuous production of cells which , though alive , are various observations have been intrinsically incapable of indefinite proliferation . fischer ( 1935 ) was unable to cultivate made which suggest that this is the case . solid s37 in vitro , and concluded that dying cells exceeded living cells in this lasnitzki ( 1952 ) found that the majority of s37 ascites cells failed to tumour . she found also that one - third to two - thirds of the mitoses seen divide in vitro . since it is unlikely that all mitotic in the ascitic cells in vivo were abnormal . abnormalities are visible , it is possible that the proportion of mitoses which are koller and smithers ( 1946 ) give experiabnormal in vivo may reach 50 per cent . mental proof that loss of chromosomes or of chromosome segments leads to the death of the cell . 
on the other hand , there is evidence that cells with a defective chromosome complement may survive and divide under conditions where contiguity between the cells is preserved , while being unable to do so when isolated these considerations favour the assumption from one another ( koller , 1947 )  . that a significant proportion of the s37 ascitic cells are unlikely to be capable of furthermore , if the cells should indefinite proliferation and tumour formation . be dependent upon one another for a stimulus to divide , dilution may reduce the proliferative power of an inoculum , not by damaging individual cells , but by abolishing essential mutual influences between the cells . 
quantitative transplantation of s37 in new - born mice of a heterogeneous strain gave results similar to those obtained for the transplantation of a c3h sarcoma in adult c3h mice . 
fodden. from the department of pathology , university of liverpool , and the liverpool cancer control organization . received for publication august 20 , 1948 . in his ' textbook of pathological anatomy ' rokitansky ( 1861 ) made his classical statement that pyloric cancer was exactly bounded by the pyloric ring , and that the growth never reached beyond into the duodenufrom this time it appears that the majority of observers , with the early exception of brinton ( 1864 ) , commented upon the integrity of the duodenum in cases of carcinoma of the stomach . 
many well - known surgical teachers spoke of the habitual immunity of the duodenum from invasion . kocher ( 1893 ) , mikulicz ( 1898 ) and most ( 1899 ) believed it to be always constant . kocher ventured that it was a problem of extreme interest to consider why gastric carcinoma grows in almost all cases towards the cardia , yet stops , on the contrary , at the duodenopyloric junction . it was brinton who first took especial exception to this proposition of rokitansky . 
he brought against it criticisms founded upon numerous personal caseobservations : " we may justifiably apply to it a criticism of unusual severitya criticism which , even if it weigh every word , will scarcely do more than the author 's ( rokitansky 's ) terse and weighty proposition really deserves . " from 125 cancers of the pylorus studied by brinton , there were no less than ten cases in which the disease was not bounded by the valve , but passed beyond it for a variable distance , often an inch or two inches , into the duodenuhe gave no information concerning the method of this spread . brinton concluded by saying the rules which rokitansky had the merit of laying down were , in this respect , like many others in pathology , of general though not of universal importance ; their value was not much affected by occasional exceptions . this question of duodenal invasion by pyloric cancer seemed to present a surgical problem of out of a large series of gastric carcinoma cun6o ( 1903 ) took 11 j . 
fodden some magnitude , and the frequency and degree of its occurrence has been the object of several subsequent researches . carle and fantino ( 1898 ) , discussing the pathology and treatment of carcinoma of the stomach , expressed disagreement with kocher . they mention three cases out of fourteen gastric resections in which neoplastic infiltration had spread under brunner 's glands for a distance of one to three centimetres . in addition , these authors stated that such a progression of the growth towards the duodenum necessitates an extension of the surgical resection as far as the second part of the duodenum . other later surgical authors , notably borrmann ( 1901 ) and cuneo ( 1903 ) , attached considerable importance to brinton 's " occasional exceptions " with reference to the spread of cancer into the duodenum . cuneo ( 1900 ) , during his classical work on the lymphatic propagation of pyloric cancer , was already investigating the accepted state of immunity of the duodenuhe remarked on the disaccord between the macroscopic aspect - the abrupt termination of a gastric cancer at the level of the pylorus and the results of the histological examination . this examination systematically diminished , he said , the percentage of the duodenal integrity . cancers of the pylorus , and subjected their distal extension to a critical macroscopic and microscopic examination . 
but , he goes on to say , it is impossible to ignore the fact that such a wide resection complicates the operation , and necessitates a dissection of the duodenum from the pancreas . the researches of fenwick and fenwick ( 1902 ) do not lend support to brinton 's findings , as they discovered out of a series of 87 pyloric cancers only 2 cases which demonstrated lymphatic spread into the duodenum . these authors remarked that though malignant growths of the pylorus may project into the lumen of the duodenum , they rarely implicated its walls . however , it is open to question whether their histological investigations were sufficiently careful to give reliable evidence , and the conclusions they advanced could scarcely have been founded upon their own results . moynihan ( 1926 ) was very familiar with the possibility of duodenal permeation in gastric cancer , and stressed that the duodenal integrity was more apparent than real . 
he quoted several of the authors already mentioned , and advocated the removal in all cases of pyloric cancer of the whole of the first portion of the duodenum . sherren ( 1932 ) , describing the methods of spread of gastric cancer , mentioned his own surgical familiarity with duodenal infiltration by carcinoma . 
a study of the microscopical anatomy of this region , in relation to the morbid process in situ , may help to clear up some components of each problem . to cun6o ( 1900 ) the apparent arrest of a cancer at the pylorus was due , for him it depended upon an absence of conabove all , to a mechanical cause . tinuity between the gastric and duodenal submucous spaces - a continuity broken by the annular muscle of the pylorus , and by a condensation of cellular tissue within the submucosa to form a dividing septum . his gelatine injections into the gastric submucosa near the pylorus met with an " invincible obstacle . " at the same time he admitted that though it would be ludicrous to consider such barriers as absolute in opposing gastric cancer , they still played the principal role in the protection of the duodenum against neoplastic invasion . for brinton ( 1864 ) and for fenwick and fenwick ( 1902 ) the reason was again anatomical . 
the muscular coats of the two viscera along the line of fusion were so distinct from one another that a direct continuity could scarcely be said to these authors made the analogy between the duodenal attachment to exist . the stomach and the manner in which the vagina embraces the neck of the uterus . to them a growth of the pylorus was more apt to infiltrate the contiguous walls of . 
in their opinion it was obvious that the duodenum may possibly be invaded from the pylorus by the in addition , extension of cancer along the submucous and subserous channels . both jamieson and dobson and cun6o emphasized the final possibility of retrograde lymphatic spread . 
as these nodes also receive vessels from the that this may be of some duodenum , an indirect communication also exists . importance was stressed by moynihan ( 1926 ) when he says that the implication of a single gland may so sufficiently disturb the direction of the lymph current that cancer cells from a primary growth may pursue an erratic course . horton ( 1928 ) working upon stomachs removed from young subjects one to three hours after death , injected 35 with coloured gelatine or indian ink . 
two of the present surgical specimens were small cancer - ulcers upon the lesser curvature of the pyloric antrum , and cancer had invaded on the gastric side only the submucosa , with extension in one case into the pyloric sphincter muscle . 
yet both these specimens showed cancer islands within duodenal lymphatics at a distance of almost 1 ' 5 cfrom the pyloro - duodenal junction . this was also instanced by a cabot case no . 
25472 ( 1939 ) of the massachusetts general hospital , in which specimen a small malignant ulcer of the pylorus was limited to the mucosa except for a small extension below the muscularis mucosae . 
the only structure apparently offering any kind of barrier action was the gland layer of brunner . it has been the experience of the majority of authors that the layer of brunner glands remains free , in spite of many , and long , extensions of gastric cancer beyond the pylorus . in addition , whatever the method of propagation , it is exceptional that the mucosal epithelium of the 246 j . 
a resection of almost 3 cof the duodenum was made . microscopically the duodenal growth was so proliferative in the muscular and serous layers that one could not avoid an impression that an even greater length of duodenal resection would have been advisable . 
davie for his help with the plan of this research during its initial stages , and those pathologists and surgeons of the liverpool regional hospitals who have so kindly supplied me with specimens and helpful data . 
1. received for publication june 9 , 1948 . recent reviews of the literature on the association between asbestosis and lung cancer reveal different opinions as to whether asbestosis has been an aetiological factor in these cases . homburger , in 1943 , reviewing nineteen known cases found no reliable answer to the problem , whereas wedler in the same year considered there was a significantly high incidence of lung cancers in autopsied cases of asbestosis . kennaway and kennaway ( 1947 ) , who examined this problem as part of their broader statistical study of lung cancer , also leave the question unanswered . the statistical difficulties are due to the small numbers of asbestos workers , and to paucity of published autopsy records of such cases . the following case is therefore presented : 294 a . 
croton oil painted concurrently caused no increase in the incidence of the significance of the skin tumours , nor did it decrease the latent period ; absence of co - carcinogenic action of croton oil is discussed . the author is greatly indebted to p . 
kirby. from the research department , glasgow royal cancer hospital . received for publication , june 28 , 1984 . activity the carcinogenic of orally - administered 2 - acetylaminofluorene , aaf , for a number of tissues , mainly epithelial , was first demonstrated by wilson , deeds and cox ( 1941 ) for the rat , and subsequently confirmed for this species by bielschowsky ( 1944 , 1946 ) and others , for mice of various strains ( armstrong and bonser , 1944 , 1947 ; foulds , 1947 ) , for the fowl ( bielschowsky and green , 1945 ) and for the cat ( harding , quoted by bielschowsky , 1947 )  . recently wilson , deeds and cox ( 1947a ) have reported their own findings in mice of three pure strains . 
aaf has now been shown to be a carcinogen ( when given per os ) in eight strains of mice : cba , if , riii , white label , strong a , c57 black , c3h , and bagg albino . 
of the first five strains mentioned , cba were most prone to both liver and bladder tumours ; strong a were the least susceptible ( armstrong and bonser , 1947 )  . 
 ( 1941 ) were at that time unable to elicit tumours in rats by subcutaneous implantation of aaf , and , in view of the probable hydrolysis of aaf in the intestine to 2 - aminofluorene , af , they suggested that aaf might be converted to af and that the latter product was the real carcinogen . might be expected that af would be more carcinogenic for rats than aaf in however , in a recent paper , wilson , deeds and similar feeding experiments . cox ( 1947b ) report tumours to have arisen more slowly and in fewer numbers in rats given af orally than in similar rats receiving aaf in the same basal diet . they suggest that af , being more soluble than aaf , may have been absorbed more rapidly and then excreted rapidly . 
af derived from aaf by hydrolysis in the intestine could only be absorbed as fast as it was liberated . however , against this view it must be said that af , once absorbed , should be excreted as fast in either case and that tissues in remote sites will stand the same chance of receiving af , whether af or aaf be given orally , provided the presentation it seems possible that the hydrolysis takes place after absorption ; is continuous . studies on the liver after ingestion of aaf , using the extraction technique of miller and baumann ( 1945 ) and the estimation method of westfall ( 1945 ) , should yield information to test this point . it is significant that wilson , deeds and cox ( 1947a ) report tumours and other changes after implantation of crystalline aaf for long periods . bielschowsky ( 1944 ) painted af in acetone thrice weekly on the epilated contrary to expectation , no tumours at the site of painting skin of wistar rats . occurred , but all 5 rats developed liver tumour by 280 days and one had a tumour of the ductus acusticus externus . 
at first , arachis oil was used as vehicle , but later , tricaprylin became available and was used in place of arachis oil . injections were made at intervals of approximately 4 weeks , in alternate flanks ; 0 5 ml . 
a female dying at 351 days showed no abnormality , but of 7 mice dying between 530 and 594 days , all showed one or more liver tumours , some solid , some diffuse , with three cases being notably vascular . six of these seven mice were females , the other mouse being the only male to survive more than 125 days . 
one female , killed at 582 days , showed a mammary adenocarcinoma , and another , killed the same day , had a granulosa - cell tumour of the ovary . ( c ) c3h mice . - thirteen mice survived 52 to 126 days ; the rest survived 206 days or more . three females , killed at 206 , 235 and 235 days respectively , had one or two mammary adenocarcinomas . 
a fourth female was killed at 242 days without any gross tumour . three males survived 409 , 426 and 506 days respectively ; the first and the last of these showed hepatoma ; the other had primary renal carcinoma . 
ten male and two female mice survived over 500 days , four of these dying between 602 and 616 days ; extensive leukaemic infiltration was seen in two of these mice , but no liver tumours ( or tumours in other sites ) were found in any . 
kidney and bladder tumours were not found , nor were mammary tumours . ( b ) cba mice . - only cysts with minimal tissue reaction were found at the sites of injection in these mice . 
a variety of degenerative changes were found in the liver in most mice ; a male , injected eight times and dying at 285 days , had hepatoma at the edge of the left lobe . 
hepatomas were also found in three mice injected 11 times ; in two males dying at 565 and 574 days respectively , and in a female dying at 630 days . 
no tumours were found in the bladder or kidney in any of these mice . ( c ) c3h mice . - no tumours were found at the sites of injection , only minimal tissue reaction . 
one injected male of this strain had hepatomas by 242 days ; two males of the painted group had hepatomas by 409 and 506 days respectively . burns and schenken ( 1943 ) record a spontaneous primary hepatoma in a c311 male 14.5 months ( 440 days ) old . thus the mouse dying at 506 days may well have borne spontaneous tumours , and this possibility may also apply to that dying at 409 days . 
the earliest case seems to be undoubtedly induced by af which is therefore probably carcinogenic for the liver when injected subcutaneously in male c3h mice . among 12 cba mice given 2 - acetylaminofluorene ( aaf ) orally by armstrong and bonser ( 1944 ) , which survived more than 12 weeks , four showed liver tumours , but two were females dying at 63 and 65 weeks respectively and therefore liable to bear spontaneous tumnours . armstrong and bonser ( 1947 ) record three malignant hepatomas in seven male mice and five benign hepatomas in eight females given aaf orally . 
the time required for these tumours to develop is not recorded , but the incidence is well above that for spontaneous hepatoma in this species and , moreover , the latter always appear to remain benign . 
hence aaf and af both appear to be carcinogenic for the liver of cba mice ; af is slightly active also in c3h and c57 black mice . armstrong and bonser ( 1944 , 1947 ) record a high incidence of bladder tumours , wilson et al . 
in the present series no bladder tumours were found in mice injected subcutaneously with af and only in one female cba mouse , painted with af and killed at 582 thus af seems to be a bladder carcinogen when given orally to c57 black days . mice , but not when painted or injected subcutaneously . 
c3h mice appear to be unaffected in this organ . the kidney was free from tumour in all three strains after injection of af . painted mice of the c57 black and cba strains likewise had no kidney tumours , but a probable renal carcinoma was found in a painted c3h mouse , a male dying wilson et al . 
 ( 1947b ) record a carcinoma of the kidney in one of at 426 days . their c57 black mice given af orally . armstrong and bonser do not record any kidney tumour in cba mice given aaf orally . neither compound is very active for mouse kidney . armstrong and bonser ( 1947 ) record one mammary cancer in 9 females given aaf orally and surviving more than 20 weeks . 
one cba female in the present series , painted with af , showed mammary adenocarcinoma at 582 days . spontaneous mammary tumours in this strain seem not to occur and the solitary tumour found in each series referred to above is therefore considered to be significant . 
1949. although convenient and entirely suitable for the purposes for which they are employed , the u8ual methods of maintaining and utilizing transplantable tumours are not well adapted to special needs in experimental oncology , particularly when quantitative refinements and the desirability of repeating experiments with aliquot portions of the same tumour suspension are important considerations . the basis of any method which might meet such requirements must necessarily be one in which a number of separate and sin - iilar portions of a tumour can be preserved with some assurance that different portions tested at different - times will exhibit sirnilar activities . this means that the tumour tissue must be reduced to a fine state of division and homogeneous suspension and subjected to a method of preservation whereby its capacity to induce tumours remains constant for a long time . it is well known that growth may be obtained on transplantation of frozen and thawed tu ' mour tissue . although breedis and furth ( 1938 ) demonstrated the feasibility of preserving neoplastic cells in the frozen state for periods up to 400 days , this useful method does not seem to have been adopted to the extent that might have been expected . 
most of the observations have been made using the c3h sarcoma referred to by gye , begg , mann and craigie ( 1949 )  . pr " ration of tumour mmpennow . - the tumours were reduced to the necessary - state of subdivision by means of the pressure mincer described by craigie ( 1949 ) and the mince dispersed immediately in diluting fluid . 
the diluting fluids were freshly prepared from reboiled sterile distilled water and concentrated preparations of the required solutes . in experiments in which different diluting fluids were compared , a low primary dilution was first made in fluid of suitable composition and from this the secondary dilutions in the other fluids were prepared . 
were given subcutaneously in the approexcept in limited experiments , four sites in the anterior priate strain of mice . abdominal wall were used , these being approximately the centres of the upper and lower right and left quadrants . the " rtival of tumour 8uspen8iow in sau 8olulion& when a solution of sodium chloride is frozen at a temperature above 21t . ice crystalhzes out and the salt is concentrated as a strong solution ( 30 per cent ) between the cryistals . if a cer suspension in 34 volumes of 0 - 85 per cent nacl 20 ' c . 
in the strong salt solutions , in others the material was held in the frozen state for several hours at selected temperatures above and below that of the eutectic of nacl ; the results showed that although complete inactivation might require several hours , the initial rate of inactivation referable to electrolyte concentration was undesirably high . survival of tumour 8u8pen8ion8 in dextro8e solutiom . in view of the difficulties that might be expected to arise in attempts to obtain adequate preservation of activity in tumour suspensions frozen in inorganic salt solutions , the possibihty of substituting a suitable non - electrolyte was considered . of water - soluble organic materials , accordingly it was planned to test a variet but dextrose , the first of these selected for test , was found to have such a favourabl . 
effect that no attempt has yet been made to find substances more suitable for the purpose . because of the varied nature of the comparative tests which have been carried out and the introduction of a number of improvements in technique , it is not possible to present a synopsis of the results on a quantitative basis . table i provides a brief summary which , at best , merely shows that there is no contraindication to the use of - dextrose solution as a suspending fluid . 
16 - 25 m / 150cysteinehcim / 15onaohm / l5ona2hpoi , s = 0 - 85pereentn&o ; r four compamtive injections were msule in each mouse and the figure given in the cohnnn headed mouse group " ] indicates the arrangement of injections , eg . 
1 : 300 n&c1 the tumour mince was first thoroughly mixed with an equal volume of 0 - 85 per cent nacl and the mi iture was diluted i in 15 in the stated diluents . 
the first test was made after the suspensions had been kept frozen for 4 days . the lag periods observed in this test are not comparable w ' ith those in the later tests because of changes in technique of thawing , diluting and injecting , and also because of the use of a eysteine - phosphate.saline mixture in the later tests . however , it would appear legitimate to compare the activity of the material frozen for 253 days in the mixture of dextrose and 40 per cent glycerol withthe average activity of fresh c3h sarcoma suspension in ringer . 
further investigation is desirable not onlv with a view to improvements that might facifitate studies in which the tise of preserved tumour material is preferable , but also with the object of dete - riiiining the mecha i ms of surv - ival of activity . 
the varietv of pkvsical and physico - chemical factors which may he involved and the potential coniplexitv of their interplav need not be detailed , but some of the points which require investigation from the practical point of view mav be mentioned . thev are as follows : 1 . 
the development of a suitable fluid for dilution of thawed material . tumour ' suspensions which have been frozen may show a definite drop in activitv in 15 to 30 minutes after thawing . 
on the degree to which the material is diluted . it will have been noted from tables 11 and iii that cysteine phosphate buffer mixtures have been used as diluting fluids . 
the experimental evidence for the use of dilutina fluid of this composition is contradictorv and suggests a different approach to this problem . another matter which merits further investigation is the effect of dextrose on the nuclei of tumour c , - - ils as viewed with the phase contrast microscope . 
after preservation in the frozen state , sarcoma and c#rcinoma suspensions in dextrose solution show a greater and more consistent retention of activity than suspensions prepared with saline and ringer.. 
bartholomew 's hospital , london . received for publication may 15 , 1951 the data given below have been collected in continuation of two earlier studies ( kennaway and kennaway , 1936 , 1947 )  . stocks ( 1936 , 1947 ) has compared the death rates from cancer of the lung in males during the years 1921 to 1930 in london , and in the county boroughs , other urban districts , and rural districts of england and wales , and has shown that the rate increased with increasing urban conditions . * the same method has now been applied to the data , for both sexes ( table ii ) , of a later period ( 1946 to 1949 ) , when a great increase in deaths attributed to cancer of the lung has taken place . 
as recently as 1929 the numbers of cancers of the larynx , and of the lung , in males , were about equal ( table i ) ; since then , deaths attributed to the latter have increased enormously . 
the crude death rate for males has been falling in recent years ( 1946 to 1949 ) and in view of the ageing population this indicates an actual decrease in the incidence ; the death rate for females has been remarkably constant since these considerations show that the pooling of data for the larynx and 1935 . lung under any such heading as " respiratory system " is very undesirable . the standardized death rates show no perceptible difference in either sex between the figures for the 14 years 1926 to 1939 immediately preceding the war , and the since 1942 standardized death rates have been means for the period 1921 to 1930 . superseded by comparative mortality indices , which compare the death rate in each year with that of 1938 . . 
22 - 5 = 1 - 0 100 ' 0 82 - 2 17 - 8 100.0 the data at present available on the social incidence of cancer of the larynx show a gradient which is steep in men and absent in women ( table vi )  . 
hence the increased consumption of tobacco in recent years has not affected this form of cancer . ( 5 ) thus cancer of the larynx in men , and in women , differs in geographical , anatomical and social distribution , and in changing prevalence at the present time , and should be regarded as constituting two separate diseases . ( 6 ) the sexual distribution of intrinsic and extrinsic cancer of the larynx appears to be changing . we are greatly indebted to w . 
phillips , of the general register office , for a large part of the material contained in this paper , for help in the preparation of it , and for advice upon many matters . 
oliver. from the radiotherapy and cancer research departments , london ho8piw , london , e.i. received for publication january 25 , 1951 . the distribution of radioactive phosphorus can be investigated in vivo using a thin - walled g.m. 
the maximum range for beta paitioles of p32 in soft tissue is 8 mm . , but the half - value thickness is only of the order of 0 - 6 to 0 - 8 mconsequently , measurements on the skin can only indicate the amount of phosphorus in the superficial layers of the body ( effectively some 3 mdeep )  . however , under favourable conditions , such measurements can be used to demonstrate the increased phosphorus up - take by malignant tumours . low - beer , glenn - bell , mccorkle and stone ( 1946 ) have used this method with some success , as a diagnostic procedure , but its value is reduced by the low penetration of the beta rays and by the increased up - take also found in inflammatory conditions . 
phosphate buffer at ph 7 - 35 . it has been found advisable to check , by counter measurements , that the radioactive material has actuary left the site of injection , as , even after taking more than ordinary care , some unsatisfactory injections have been detected . this is particularly important , of course , when giving the isotope therapeutically , as dose calculations assume that the whole of the material enters the blood streano side effects of the injection were observed . 
the tumour may have received 4 to 8 times the dose calculated above , a total which is in excess of the generally agreed " tolerance " level . this was , however , of no consequence as the site was to re ' ceive an x - ray dose of 3000 to 4000 r . a number of sites over the tumour , on the diseased breast , and at corresponding points on the normal breast , were marked with gentian violet , and measurements of the activities were made with a counter tube applied directly f . 
the background rate was in each case the complete set of measurechecked before and after the series . ments was repeated and a mean value for each site was used . it was not feasible to prolong the counting time at any site ab ' ove about 3 minutes . 
as the count rates with this tracer dose were rather low , the measurements were usually limited to 1000 counts ( 3 per cent standard deviation ) or , after several days and over normal tissue , to only 100 counts ( 10 per cent standard deviation )  . consequently , the ratios of count rates over tumour and normal skin were not obtained very accurately , but the results sufficed to demonstrate large changes . series of measurements were made before , during , and 4 to 6 weeks after the treatment , at which time the x - ray erythema had subsided but the skin of the irradiated breast remained highly pigmented . observations were made on altemate days during 2 to 3 weeks . after intravenous injection , the radioactive phosphorus could be demonstrated immediately ( 5 to 8 seconds ) in the tissues of the breast , but the concentrations continued to increase in both healthy and dis6ased breasts for 10 to considerably lower count rates were observed when the next series 15 minutes . of measurements were made after an interval of 2 days , a change probably due to the excretion of p32 ' thereafter the fall was found to follow a roughly exponential form , corresponding to a " biological half - hfe " of between 7 and 9 typical cases are ifustrated in fig . 
i. and over both axillae are also included . it is of interest to note that the readings , over the forehead ( indicating thep . , 2 content of the frontal bone ) increased to the level found over the tumour and then decreased more slowly than the latter . values of the ratio of count rates over the diseased breast to those over the healthy breast are presented in fig . 
a sudden drop in the ratio occurred after the third irradiation . four weeks after the end of the treatment a second tracer dose was given , and the results obtained are shown in fig . 
for the first three readings the skin was still very red . by the time the erythema had subsided the count rates were rather low , but they appeared to be higher than normal over the whole of the diseased breast , without any locahzed . 
at a later date the authors hope to continue the investigation , although working at different institutions . the countrate due to p..2 was fodnd to be very much higher ( between 3 and 8 times ) over malignant mammary tumours attached to the skin , than over the corresponding parts of the healthy breast . 
no such , change occurred during after erythema stilboestrol treatment in a case which did not respond clinically . had subsided the ratios of measurements over the two breasts were found to be similar to those before treatmentbut the primary growth was still present in all these cases . the authors wish to thank sykes , the chief pharmacist at the hos ital , for preparing the material for intravenous injections , and k . 
1. received for publication january 26 , 1949 . thie application of the methods of urinary cholesterol estimation given by burchell and maclagan ( 1949 ) to cancer and control cases will now be described . bmaterial urine specimens were obtained from 13 normal subjects and 116 patients admitted to westminster hospital or to westminster hospital ( .all saints ' ) twenty - five of these patients were suffering from diseases urological centre . thought to be unlikely to affect cholesterol excretion as shown in the tables , and 73 were suffering from various forms of cancer . 
sample of the mixed specimen , or a smaller aliquot in the case of a concentrated urine , was centrifuged , the deposit washed once with normal saline and extracted directly with three portions of boiling acetone . 
the acetone extract was evaporated to dryness , and the residue extracted with petroleum ether for the determination of cholesterol as above . the cholesterol content of the supernatant urine ( su ) was determined on 200 ml . 
the sex difference is mainly due to the larger number of epithelial cells and leucocytes in urinary deposits froimw omen . it appears likely from these figures that the total urinarcholesterol would not exceed 4 - 49 mg . 
maclagan it is evident from this that if albuminuria is present from whatever cause , the su may be expected to show an increase in cholesterol content , and such a rise is unlikely to be specifically related to cancer . 
of urine with phenol reagent as described by burchell and maclagan ( 1949 ) , where reasons are given for assuming that this method does , in fact , estimate a proteose - like substance . it will be seen from fig . 
the ud cholesterol is associated with the cells of the deposit and shows a marked sex difference , being much higher in women than in it appears to have no direct relation to cancer . 
the su cholesterol on men . the other hand is associated with a urinary proteose fraction , or with heatraised su cholesterol excretion in the coagulable protein when present . absence of albuminuria did occur in a small proportion of cases of cancer ( 5 out this gives a of 51 cases ) , although the increase was slight in 3 of these cases . total incidence of hypercholesteroluria in cancer of 9 - 8 per cent , which is somewhat lower than that reported by other workers . 
thus sobotka , bloch and rosenbloom ( 1940 ) found 20 - 6 per cent , and bruger and ehrlich ( 1943 ) 34 - 3 per cent of positive results . 
for example , bruger and ehrlich ( 1943 ) reported some rise of total urinary cholesterol in 11 out of 32 cases of cancer , but 5 of these had either albuminuria or excess of cells in the deposit or both , findings which , according to our experience , might have been sufficient to account for the hypercholesteroluria even in the absence of cancer . 
the exclusion of these cases would bring the incidence of positive results down to the figure of 22 - 2 per cent . , which does not differ significantly from our own finding of 9 - 8 per cent ( difference 12 - 4 , standard error of difference 8 - 2 )  . we must conclude , therefore , that hypercholesteroluria , while frequently due to associated albuminuria or pyuria , does occur in a small proportion of in such cases it is the supercases of cancer in the absence of these factors . natant urine cholesterol which is increased . 
the incidence is not high enough to make the estimation of any diagnostic value , but it is of theoretical interest , and presumably depends upon increased tissue breakdown as suggested by previous workers . it should be noted that the colorimetric method of estimation used is not entirely specific for cholesterol , and the possibility that other steroids might have given rise to the few positive results must be admitted . however , the earlier work of bloch and sobotka ( 1938 ) on pooled 100 litre specimens of urine suggests that the results probably were due to increase of cholesterol itself . the question of urinary proteose is of some interest in view of the work of winzler and burk ( 1943 ) on blood proteose and cancer in rats . 
the histology was typical of lymphosarcoma , the cells being chiefly of the " lymphoblastic " type . local spread had occurred upwards in the submucosa for a short distance , and there was infiltration of adjacent rectal muscle but no noticeable spread in perirectal fat . 
the tumour was softer in consistence than most carcinomas and the base of the ulcer was covered by a greyish slough . both " lymphoblastic " and " lymphocytic " cells present . extensive spread in rectal muscle and commencing invasion of perirectal fat . 
the adenocarcinoma had infiltrated more deeply , but there was no sign of venous spread and the lymphatic glands were all free from metastases . we have given careful consideration to the possibility that this might actually be only one tumour , exhibiting an undifferentiated pattern in one part and a differentiated glandular pattern in another , as was suggested by gr . 
tumour b is a melanoma with four lymphatic metastases containing melantumour a is an adenocarcinoma with two lymphatic metastases free from pigment . section of tumour a showing adenocarcinoma , fig . 
1945 , and found to have a fibrous nodular tumour on posterior quadrant of lower third of rectum . biopsy showed spindle cell sarcoma apparently of low grade malignancy . seven years previously patient had had the coccyx removed in another hospital , and three years later a small tumour had been found in the rectum which was reported as a mass of fibrous tissue . synchronous combined excision of rectum by o . 
more than 4 inches of healthy bowel both tumours had given separated the melanoma from the adenocarcinoma . rise to lymphatic metastases , those from the melanoma being pigmented , whereas those from the adenocarcinoma contained no melana unique pathological specimen . 
twenty years ago weeks ( 1927 ) found reference to 100 cases of rectal sarcoma , but he regarded many as of doubtful validity and remarked - " if the questionable cases were discarded it is certain that the number would be reduced by one half . " we agree with this comment and have formed the impression that many of the cases which until recent years were recorded in the literature as " sarcomas " were probably examples of anaplastic carcinoma . this criticism does not apply to the cases reported in the last 15 to 20 years . most authors who have written about this subject seeinm to take the view that all varieties of sarcoma tend to run a rapidly fatal course , but we think that each of the four varieties of malignancy has this attitude needs qualification . to some extent its own characteristics and peculiarities and the prognosis differs considerably in each . lymphosarcoma is the commonest form of sarcoma of the rectum . it may manifest itself either as a single localized tumour or give rise to a crop of tumours bensaude ( 1929 ) was one of the first to spread over a long stretch of the bowel . draw attention to the diffuse or generalized variety of lymphosarcoma . in one of our cases ( case 3 ) the disease affected the whole rectum and distal end of the pelvic colon , so that in describing the primary focus we could not speak of a particular site of origin , but only of a region of involvement . 
the largest tumour was in the lower third of the rectum , but the other smaller tumours at a higher level were certainly not secondary growths resulting from the transportation of tumour cells , but were obviously new foci of lymphosarcoma developing indein spite of this unfavourable feature of lymphosarcoma the prognosis pendently . after surgical treatment would seem to be fairly good on the whole . 
at present there is insufficient evidence on which to base a comparison because it is only within recent years that these tumours interest in the subject have been separated out from other varieties of sarcoma . of reticulosis , stimulated by the writings of robb - smith ( 1938 ) , has had considerreticulum cell sarcoma is certainly a rarer tumour able influence in this respect . in the rectum than lymphosarcoma , and we think that it will probably be found to have a worse prognosis . spindle cell sarcoma is also a rare form of malignancy in the rectum . it is the one most easy to distinguish clinically from carcinoma because it obviously arises from the tissues of the bowel wall and not from the surface mucous membrane . the firm solid structure and covering of intact mucosa are of special significance . it has already been mentioned that in their histology these tumours give the impression of being of a relatively benign character . all who have written about melanoma speak of it as the most malignant of all the tumours now being considered . 
we agree with the view expressed by linder and wood ( 1936 ) that melanomas arise from the skin of the anal canal and tend to grow up into the rectum and that they metastasize early . 
the lymphatic glands may contain more pigment than the primary tumour , but it is worth mentioning that the haemorrhoidal and paracolic lymphatic glands may contain melanin apart altogether from the presence of a melanotic tumour . 
bussey although the cases we have examined personally are only ten in number they do provide material for comparison with the much commoner tumour , carcinoma of the rectum . in the end , of course , these rarer forms of malignancy can only be identified by detailed study of their histology , but there are some features in their gross characters which would make an experienced surgeon suspect that he was dealing with something " out of the ordinary . " we have summarized these by setting them out in tabular form ( table i )  . finally we have left to the end what we consider to be the most interesting feature of these rare tumours , namely , the frequency of multiple foci of malignancy . in four of our ten cases more than one tumour was found in the rectum , and in two cases the second tumour was of a completely different histological type . 
this raises the question both of multiple malignancy and dissimilar primary tumours . multiple malignancy is commoner in the rectum than is usually supposed . it so happens that we are in a position to give facts about this question because for many years we have been making observations on the frequency of the occurrence of multiple primary carcinoma and have found that in operation specimens of rectal cancer removed by a combined operation more than one primary rectal carcinoma is present in 4 per cent of cases ( over 2 , 000 examined )  . moreover we have records of many additional cases in which a second carcinoma was present in the colon in association with rectal cancer , so we were not surprised to find multiple tumours in our cases of lymphosarcomas ; in fact , we expected this to be the case . the occurrence of dissimilar primary tumours was , however , a complete surprise , and as far as we have been able to find out has not been previously recorded as a characteristic of these rarer forms of malignancy . in any organ of the body the existence of two tumours of a different histological type is a rare phenomenon . it is indeed remarkable that this unusual combination should have occurred twice in a series of only ten cases . 
we have searched through the literature but found no reference to the simultaneous occurrence of carcinoma and sarcoma in the rectum , though kreibig ( 1929 ) mentions a case of carcinoma plus lymphosarcoma in the ileo - caecal region . as far as the general relationship between carcinoma and sarcoma is concerned we note that warren and gates ( 1932 ) in a study of this subject were only able to collect from the literature 25 instances of the co - occurrence of carcinoma and sarcoma as separate tumours in the same organ . three were cases of sarcoma and carcinoma of the breast , twelve were of the uterus , and ten were in miscellaneous organs , including four cases of the combination of carcinoma and 1ynphosarcoma of the stomach . they found no recorded case of carcinoma and sarcoma of the rectum or indeed of carcinoma of the rectum associated with sarcoma of any other part of the intestine . 
melanoma arises always from the ano - rectal region and spreads upwards into the rectum . it metastasizes early and runs a rapid course . in four of the ten cases we are reporting more than one malignant tumour was present in the rectuone of these was an example of multiple lymphosarcoma and another of multiple reticulum cell sarcoma . in these cases tlhe associated tumours were of the same histological type , but in two other cases the associated tumours were of completely different histological type . 
she complained of cough , haemoptyses and loss of weight for about four months , but since october , 1944 , she had been feelmg listless and noticing palpitations and dyspnoea on exertion . 
a little lymphatic permeation was traced to the base of this lobe and involved the diaphragthe lower part of the lower lobe showed a fairly marked collapse together with a slight fibrosis . the left upper lobe was well aerated , but showed a sharply defined , firm area of congestion ( suggesting venous infarction )  . 
adjacent broncho - pulmonary lymph nodes are almost completely infiltrated . section of a main pulmonary vein shows neoplastic cells infiltrating the wall of the vessel ; in places these have reached the lumen and lined the wall with a thin layer of squamous carcinoma . 
ante - mortem thrombi have resulted , and some regions show early organization of the thrombus , which itself contains typical neoplastic cells . section of the left lower lobe shows lymphatic permeation by neoplasm , marked collapse and areas of oedema and haemorrhage . 
intra - alveolar phagocytes containing iron and carbon are numerous , and foreign body giant cells are seen in relation to the typical golden - yellow asbestos bodies lying in the alveoli and the finer lung trabeculae . there is marked subpleural fibrosis not related to neoplastic infiltration , but a consequence of the asbestosis . 
one section of this lobe shows that the neoplastic cells , although preserving most of the features of squamous carcinoma , nevertheless show a definite tendency to acinus formation , and here there is much secretion of mucus into the intercellular 252 r . 
the period of exposure also was long ( seven years ) , and although this is usual , cases have been reported of a neop ] asm developing after considerably shorter exposures . 
1. received for publication july 1 , 1948 . in the course of experiments on a transplantable mouse sarcoma it was observed that fresh saline extracts of tumour tissue agglutinated mouse and rabbit red cells . 
when extract and red cell suspension were mixed on a slide agglutination became visible to the naked eye within 30 seconds and appeared to be complete within 2 minutes . a number of different mouse and rat tumours , and a wide range of normal tissues , have been examined for haemagglutinating activity . 
horning. from the chester beally research institute of the royal cancer hospital , london , s.w.3. received for publication december 12 , 1948 . recfnt experience has shown that many of the growth - inhibiting agents used in the palliative treatment of cancer are carcinogenic . 
they also cause specific damage to cell nuclei and chromosomes and are able to induce mutations . the association of these biological effects of ( 1 ) growth inhibition , ( 2 ) chromosome damage , ( 3 ) production of mutations and ( 4 ) induction of cancer suggests if the that they may have a common fimundamental biochemical mechanism . induction of cancer is indeed a somatic mutation , then cancer induction might be included as a special mutation . 
the fact that x - rays can produce cancer in man was published in 1902 ( frieben , 1902 ) , seven years after rontgen 's discovery muiiller ( 1928 ) found that x - rays are mutagenic , and later mather of x - rays . and stone ( 1933 ) and koller ( 1934 ) described the chromosome damage following irradiation . 
the idea that cancer arises as a somatic mutation was supported by the demonstration of an increased incidence of mutations occurring in mice this was shown with methylcholanthrene treated with chemical carcinogens . by strong ( 1945 ) and with 1 : 2 : 5 : 6 - dibenzanthracene by carr ( 1947 )  . carcinogenic hydrocarbons inhibit the growth of animals and the establishment and growth of transplanted tumours ( haddow , scott and scott , 1937 )  . 
body - weight of freshly made aqueous solutions of nitrogen the first group was given methyl di ( 2 - chloroethyl ) amine hydromustard . chloride ( hn2 ) in a concentration of 1 mg . 
the second group received the same dose of tri ( 2 - chloroethyl ) amine hydrochloride ( hn3 ) for 10 weeks , after which time only 4 mice remained alive and injections were stopped . post - mortem findings of the mice which died within a week of an injection usually showed congestion of the alimentary tract or lungs . 
with the doses used all the coloured mice showed the effect of no nervous symptoms were seen . greying of hair ( boyland et al . , 1948 ) similar to that produced by x - rays ( hance and murphy , 1926 ) or by subcutaneously implanted plutonium ( prosser , painter , lisco , brues , jacobson and swift , 1947 )  . 
hornin - g occur spontaneously , and the tumours were larger and more malignant than those generally encountered in untreated mice . the carcinogenic action of the nitrogen mustards is very slow , the earliest lung tumour and lymphosarcoma being seen at 284 and 347 days respectively . berenblum and shubik ( 1947 ) have shown that carcinogenesis can be divided into an initial stage in which cells are irreversibly changed into latent tumrnour cells , and a development stage which can be brought about by cocarcinogens such as croton oil . 
the incidence of tumours expressed as quantal response is probably dependent on the initiating action , but the rate of appearance of the tumours depends upon cocarcinogenic action . the present authors consider that the initial irreversible conversion to latent tumour cells is the change which is associated with chromosome damage and mutations . 
the nitrogen mustards are possibly active in initiating the carcinoin order to test this hypothesis genic change but poor in developing action . experiments are in hand in which mice are being treated with nitrogen mustard and croton oil . a possible explanation of the association between inhibition of growth and carcinogenic power is that the inhibition is due to interference with cell division in growing tissues . 
the drugs under discussion reduce the rate of growth by impeding mitosis . this interference with mitosis is likely to increase the chances of a somatic mutation from normal to malignant cells occurring . 
of 14 mice which survived more than 250 days , 10 had tumours including lung tumours ( 8 ) , lymphosarcomas ( 2 ) , a uterine fibromyoma and a spindle - celled sarcoma at site of injection . this investigation has been supported by grants to the royal cancer hospital from the british empire cancer campaign , the jane coffin childs memorial fund for medical research , the anna fuller fund , and the u.s. 
3. received for publication january 18 , 1949 . the investigation of carcinogenic substances which have been obtained from human tissues ( kleinenberg , neufach and shabad , 1940 ) and from other biological sources has been the subject of earlier publications from this institute ( hieger , 1940 , 1941 , 1946 , 1947 ) , reporting that : 1 . 
whittick. from the department of pathology , royal cancer hospital , london . received for publication december 13 , 1952 . there is an astonishing neglect in the literature of the benign cutaneous condition described by maccormac and scarff in 1936 , and named by them molluscum sebaceuan alternative name , kerato - acanthoma , has since been applied , and this has the merit of preventing confusion with molluscum contagiosum , an unrelated condition . it has been the subject of only 4 papers , all by british authors , maccormac and scarff ( 1936 ) , musso and gordon ( 1950 ) , rook and whimster ( 1950 ) and beare ( 1953 ) , and does not yet appear to have reached text - books of dermatology or pathology . that this in no way reflects the incidence of molluscum sebaceum is certain . indeed , it is not an uncommon condition , and is still very frequently misdiagnosed as squamous - cell carcinoma by both dermatologist and pathologist . smith , in 1934 , described the occurrence in a young man of multiple primary squamous - cell carcinomas of the skin which healed spontaneously and he was unable to find any comparable case in the literature . since that time there have been eight papers on the subject ( table ii ) with descriptions of similar lesions in 11 patients . comparison of the clinical and pathological descriptions of these cases with the natural history and the structure of molluscum sebaceum at once suggests the probability that all the recorded cases of spontaneously healing epithelioma of the skin are in fact instances of molluscum sebaceum . the following account is based on a series of 7 cases of molluscum sebaceum which after biopsy diagnosis were untreated and allowed to run their natural course to spontaneous regression . 
whittick that the lesion of molluscum sebaceum closely simulates highly differentiated squamous carcinoma will be apparent from the histological description and illustrations . there is no single feature which will allow differentiation . 
about this central plug of keratin is a broad , dull red , convex and smooth - surfaced epidermal riwith regression there is gradual flattening of the lesion , and finally the keratin plug falls off to leave a clean , slightly depressed scar . ordinary squamous carcinoma usually has a much slower rate of growth with progressive increase in size for several months . although rapid growth may occur , it is usually accompanied by early involvement of the regional lymph that rapidity of growth has not been accompanied by metastasis to the glands . local lymph nodes in the spontaneously regressing cases has indeed been stressed by some of the authors . 
whittick local and lymphatic spread . were the alleged cases of spontaneously healing epithelioma true instances of squamous carcinoma there should be good evidence of local destructive invasion and lymphatic extension , but this is lacking . in over half the recorded cases the regional glands are not mentioned and , in view of the long survival period in these cases , it may reasonably be presumed that they were not involved . example in smith 's ( 1948 ) case 1 there is a history of lesions for 21 years and in his case 2 for 29 years . 
lack of lymphatic involvement was mentioned by dunn and smith ( 1934 ) , and grzybowski ( 1950 ) comments on " the lack of involvement of lymph nodes in spite of the 9 years ' duration of in only one case , charteris ' second ( charteris , 1951 ) , is enlargethe disease . " ment of lymphatic glands mentioned , and these were found , histologically , to be tumour - free . local invasion , too , appears to have been very limited in the spontaneously healing cases , being restricted to local penetration into the dermis . thus , dunn and smith ( 1934 ) , " the invasive process at its height never appears to transgress this feature applies equally well to molluscum the limits of the true skin . " description by two authors of local tissue destruction in sponsebaceum . taneously healing epithelioma requires comment . smith ( 1948 ) , when recording the subsequent history of his case 1 , states that he was an irregular attender and " permitted much further destruction of the soft tissues of his face , ' and that he now wore a prosthesis to cover extensive destruction of his nose . this on first reading seems proof of an infiltrating and destructive malignant growth , but it must be pointed out that in the previous paragraph we are told that following treatment of a lesion of the right leg by radium , the same patient had to undergo amputation of his leg because of necrosis of the tibia . lesions of his face had been treated by contact x - ray therapy . if this extensive destruction of the nose , requiring a prosthesis , was indeed due to squamous carcinoma , then the lack of lymphatic extension and the ultimate spontaneous healing ( sic ) make this case even more remarkable . 
chadwin. since completion of this paper two other relevant communications have currie and smith ( 1952 ) , under the title " multiple primary sponappeared . taneous - healing squamous - cell carcinomata of the skin " , describe 2 new cases . they maintain that the lesions they describe are unrelated to molluscum sebaceum . whittle and lyell ( 1952 ) , in commenting on a case of molluscum 'sebaceum which f . 
7. received for publication february 3 , 1949 . one of the surprising elements in bittner 's discovery of the " milk factor " is the apparent entry of the virus into the bodies of the young mice via the it is , however , obviousthat during suckling a small amount alimentary canal . of milk may also enter the nostrils , and that the nasal mucosa may offer an alternative port of entry . it also seems possible that the milk factor might be destroyed by digestion in the stomach . if this were so it would be difficult to recover milk factor from the stomach contents of suckling mice . 
the unfortunately the experiresult was entirely negative in the 40 mice so treated ment was not adequately controlled , in that none of the mice were tested for susceptibility by means of extracts known to contain the virus . 
jackson memorial laboratory , bar harbor , me . received for publication december 16 , 1948 . it is known from the work of andervont and bryan ( 1944 ) and green , moosey and bittner ( 1946 ) that the milk agent is antigenic for the rabbit and the rat . if , as is probable , the agent , is a virus one would expect it to be antigenic for the susceptible species . 
modem technique - 3 of microscopy , particularly phase - contrast , by greatly facilitating the study of fiving cells have resulted in greater use being made of tissue cultures for morphological studies . 
when cells migrate from an explant in vitro , they tend to spread out so that around the periphery of an outgrowth they are considerably flattened . this is most obvious in fluid culture media where the cells furthest away from the explant may be reduced to little such cells are ideal for the study of cytoplasmic structure , more than a fine film . as many investigators have realised . 
the necessity of rapid fixation , in order to obtain perfect preservation of chromosome structure has also resulted in the common practice of using squash preparations and smears for the study of nuclear strucsuch procedures possess the further merit of eliminating the possibihty tures . of slight morphological alterations being brought about by the prolonged dehydrating , clearing and imbedding necessary for the preparation of sections . nevertheless ah these methods have the common disadvantage of rendering difficult the correct appreciation of the spatial arrangement of intranuclear structures . in this respect the older method of studying stained sections of fixed tissues presents definite advantages . 
by cutting sections of approximately the same thickness as the average diameter of the cell nuclei , one obtains preparations with whole nuclei and shces of nuclei of varying thickness . 
yet with all fixed preparations of cers there arise doubts as to the validity of the finest structural details which are detectable , especially when they approach the limits of mic ' roscopic resolution . so the results of the study of fixed and stained cells have been correlated with observations made by other techniques , which have included the microscopical examination of living cers immediately after their removal from the animal body . structural organization of the nucleus seen in sections . the ideal type of cell with which to commence an investigation of this kind would be one with a large nucleus , single nucleolus and well - defined chromatin granules . 
most nearly fulfilling these requirements were the cells of a mouse adenocareinoma ( t27 )  . its selection was further influenced by the fact that it had been employed in several previous researches ( ludford , 1924 , 1930 , 1932 a , 1932 b , 1933 , 1934 ) so that the structure of its cells , and their behaviour under a variety of experimental conditions , as well as their growth characteristics , both in vivo and in vitro , were already well known . the same methods of fixation and staining were adopted as were formerly used for the study of chromosomes ( ludford , 1930 ) , except that after iron - alumhaematoxylin staining the differentiation was not carried so far as is usual in making chromos ' ome preparations . frozen sections stained by the feulgen technique were also found most useful . for demonstrating the smallest structural details it is doubtful whether any method can excel the results obtained by flemming fixation followed by intense staining with iron - alum - haematox.ylin when the surface of a nucleus from such a preparation is examined there are to be seen numerous minute granules varying in size . 
4 depicts a slice through the membranes in the process of section cutting . here are seen the same peripheral granules , and also the centre of a nucleus . strands connecting the two nucleoli with the membrane . 
3 , which is another slice from in this one , some well - defined strings of granules are the surface of a nucleus . discernible , while in the adjacent fig . 
the latter obviously represent an early stage in the formation of the prophase ch - romosomes . actually in a section of a rapidly growing tumour it is impo - ssible to determine ex ' actly when the prophase does . 
8 they appear as single threads , and granules are perceptible on some of them . it thus seems that the nucleoli are connected with the periphery of the nucleus by chromosomes , and also by threads of a different nature . frequently the latter appear to be attached distally to a chromosome . examination of prophases at the stage when the nuclear membrane is undergoing dissolution will sometime reveal chromosomes connected with remnants of nucleoli . 
13 represents part of a cell of the same carcinoma on the edge of a 2 - days - old culture , which had been fixed in flemming 's fluid , and intensely stained with iron - alum - haematoxylthe fine peripheral granules beneath the nuclear membrane which are shown in fig . 
16 and 17 , these are projection drawings ( indicated by " p " in the legends )  . there are included in the plane of each drawing bodies distributed throughout the whole thus each drawing constitutes a diagrammatic representation of the nucleus . structures as if they were projected on to the equatorial plane . 
this does not mean none is present , because extremely thin threads might only be visible when coated with dyestuff , and this may have beeil removed in the early stage of differentiating with the iron - aluthe nucleus seen in fig . 
each of the nucleoli is surrounded by particles which appear to have been emitted froin its surface . if observation is now extended to feulgen - stained cultures it becomes evident that the nucleolus is of a dual nature . 
the latter constitutes what caspersson ( 1947 ) has termed " the nucleolar associated chromat - in " and to which he attributes a special role in it can be distinguished from the material of which the bulk protein synthesis . of the nucleolus is composed in the living condition , and in fixed preparations after a variety of staining methods . 
on the basis of the reaction of the two nucleolar components to staining by the feulgen and the pyronine - methyl - green techniques it is concluded that the chromatin is composed of appreciable amounts of deoxyribose nucleotides , and the remainder of the nucleolus of ribose nucleotides . there is considerable variation in the amount of nucleolar chromatin in different cers , as is demonstrated by fig . 
20. in one form of cellular degeneration as has previously been mentioned , particles are discharged from the surface of the nucleolus . comparison of cells stained by the iron - alum - haematoxylin technique , such as that show - n in fig . 
the latter are stained by haematoxylin , but are invisible in feulgen preparations . there are also distinguishable in nucleoli varying numbers of argentophile bodies . page , regan and maccarty ( i 938 ) found that both types of inclusions were more numerous in malignant than in non - malignant cells , and that " the more ma - lignant the neoplasm , the greater the number of intra - nucleolar bodies . " examination of nucleoh with the electron microscope has suggested the existence of a more comphcated structure . 
the hmit of resolution attained with the ultra - violet this is the best resolution represented in microscope is only about 0 - 1 micron . the figures ' illustrating this paper . occasionaby when a nucleolus is highly vacuolated its appearance is rather deceptive , and might erroneously be interpreted as being reticulate . 
one wonders whether the coniphcated internal structure which has been described may be the result of a slight distortion induced by the procedures involved in preparing sections for examination by the electron microscope . 
the vacuolated appearance is not an artefact of fixation as it is visible in hving cells . distribution of nucleic acids in the nucleus . as a matter of convenience , for the purpose of description , it is proposed to refer to the material of which the nucleolus is mainly composed as " plasmosomin "  . 
the nucleolar chromatin contains sufficient deoxyribose nucleotides to stain intensely by the feulgen techiiique , while plasmosomin this does not ehminate the possibility that in the latter deoxyribose does not . nucleotides may be present in such small quantities as to be below the limit of sensitivity of the feulgen techn ' ique , upon the vahdity of which as a tes t for deoxyribonucleic acid this only definitely known chemical difference between nucleolar chromatin and plasmosomin depends . cytogeneticists find it convenient to use the terms " euchromatin " and their usage is based upon the conclusion that at the heterochromatin "  . end of mitosis as the chromosomes uncoil deoxyribonucleic acid remains attached at certain loci ( heterochromatin ) , and is discharged from the rest of since it has been impossible to see whether the chromosome ( euchromatin )  . this actually happens in the cells employed in this investigation no attempt has been made to distinguish between two types of chromatthe term " chromatin " is used in this paper to describe nuclear material which gives a positive reaction with the feulgen test , and which is not visibly connected with the nucleolus . whether one studies fixed and stained feulgen preparations , or ultra - violet micrographs of living cells , it becomes obvious that there must be an increase of deoxyribonucleic acid at prophase , and a corresponding decrease during the evidence has already been adduced that the reconstruction phase of mitosis . chromosomes are represented in the " resting " nucleus by the fine granules at its surface , and on strands connecting the nucleolus with the nuclear membrane . these granules appear to be faintly stained by the feulgen technique . 
but it is doubtful whether any definite conclusion can be justifiably drawn from pale staining , as it has been pointed out , most of these minute particles are just ifthey fail to stain that does above , or below , the liniits ofmicroscopic resolution . not necessarily mean that they contain no deoxyribonucleic acid . it might well be that the amounts present in such small particles would be undetectable by the techni ' que . also very faint staining could result from absorption of traces of diffuse dyestuff . 
the latter usually appears to be very faintly coloured in feulgen preparations , and the nuclear periphery is always most clearly defined there is by comparison an even in the absence of cytoplasmic counterstaining . intense staining of larger peripheral particles , and of others on the filaments connecting the nucleoli with one another and with the nuclear membrane . 
30 is an ultra - violet micrograph of some flattened coagulated nuclei from another mouse carcinoma ( af )  . it will be noticed that the superficial structure is very similar to that draw - n in fig . 
they have been regarded as the chromosomes , or fragments of chromosomes , of the interphase nucleus , but this interpretation of their nature has been disputed by lamb ( 1950 )  . it is highly probable that they are identical with the strands of material exhibiting strong absorption of ultra - violet radiations of 2570 a wave - length , which are seen in fig . 
the absorbing material presumably comprises the fine granules , which for reasons already stated are regarded as representing the chromosomes of the interphase probably their volume is augmented at the time of nuclear coagulation nucleus . as the result of the accretion of additionaldeoxyribonucleic acid from the nuclear sap , by a piocess analogous to that which occurs at the prophase of mitosis . relationship between nucleolar chromatin and plasmosomin . on the basis of the preceding considerations it is concluded that the highest concentration of deoxyribonucleic acid is in the nucleolar chromatin and ' bodies deoxyribonucleic acid is also present in the minute granules derived from it . which represent the interphase chromosomes and in the nuclear sap . considerable evidence has been adduced by other investigators to prove that the plasmosome is particularly rich in oxyribonucleic acid . the functional relationship between nucleolar chromatin and plasmosomin requires furtber investigation . 
the observations that are reported in this paper are consistent with two possibifities . either nucleolar chromatin and plasmosomin are built up from the same simple precursors , and there are altemate pathways of metabolism the 122 r . 
ludford course of which can be deflected so as to lead to the formation of either oiie or or , there are contained in plasmosomin enzynie systems the other substance . responsible for the synthesis of the substances comprising nucleolar chromatin from certain of its chemicary simpler constituents ; or perhaps for breaking dow - n plasmosomin and converting it into nucleolar chromatthe latter notion finds support from the behaviour of the nucleolus during the formation of the chromofig . 
36 and 37 are ultra - violet micrographs of hving prophase somes at prophase . nuclei of a sarcoma ( rb )  . in the former the surface of the nucleus is in focus and the peripheral chromosomes are seen in process of formation . in the latter the nucleoli in the interior of the nucleus are in focus . 
the darker nucleolar chromatin is distinguishable from the lighter plasmosomthe nucleoli are in process of disintegration , and there is a considerable production of nucleolar after the chromosomes are fully formed , small spherical masses of chromatin . plasmosomin are often distinguishable devoid of any chromatat this stage all the feulgen positive material has been deposited on the chromosomes . 
the fonner was photograpbed from a 3 - days - old primary culture of mammary carcinoma ( ad ) in a strain mice ; the latter from a 5 - days - old primary culture of mammary gland from a low - cancerstrain mouse ( c57 )  . 
38 exhibited the greatest degree of polymorphisnote also in this figure the derangement of mitosis which has resulted in two daughter nuclei with numerous karyomeres instead of nuclei . despite the similaritv of the nuclear structure of the cers of fig . 
38 and 39 explants of carcinomata give rise to sheet their behaviour in vitro differs . growths of cells with greater reg - ularity and rapidity than do those of mammary glands . 
the cehs of other mammary cancers induced by carcinogenic compounds , independently of the milk factor , exhibit greater abnormalities , which distinguish them from normal mammary gland cells . there is corresponding variation in the extent to which sarcoma cells differ from fibroblasts with respect to nuclear structure . 
the former is a photomicrograph of a 7 - days - old primary culture of mouse - embryo - heart fibroblasts , and the latter of a 3 - days - old culture of a sarcoma ( aa ) which originated fig . 
34 and 35 mosomin and nucleolar chromatin are included in the drawing . are ultra - violet micrographs of a cell of the same sarcoma , which was the most rapidly growing of the tumours which have been studied in the course of this since all the fig . 
ludford of these the " simplest and rarest type " involved the formation reconstruction . of chromosomal vesicles , or karyomeres . this was " long ago described by biitschli and fol in the blastomeres of segmenting eggs and since observed in embryonic cells of many species.. in this process each chromosome is converted into a small vesicle exactly hke a minature nucleus , the whole group then fusing together progressively so as to form first an irregular , chambered structure and finally a smgyle nucleus . 
from the outer wall of this , apparently , arises the nuclear menibrane , while the inner walls of the vesicles break down irregularly to form the nuclear network "  . 
he considers that the interphase nucleus " probably consists of closely packed , firmly adherent , swollen chromosomes ( chromosomal vesicles or karyomeres )  . " this condition is brought about by the telophase chromosomes swelling to form vesicles whicli adhere to one another . 
each vesicle retains its identity throughout the interphase . " during prophase each chromosome sinks into a dense gel which is separated from its neighbours by the fluid which collects between them . " lewis ( 1947 ) points out that amitosis and nuclear fragmentation would be readily explicable as resulting from the loss of adhesion between chromosomal vesicles . since nuclear formation by the fusion of chromosomal vesicles is said to be speciary characteristic of embryonic cells , it would not be surprising to find the same process occurring in malignant cells which they resemble in many respects . karyomeres are often clearly distinguishable in dividing cancer cells . attention has already been directed to the example in the middle of fig . 
28 and 29 could originate from the cohesion of karyomeres , formed , not as lewis ( 1947 ) suggested , by the swelhng of chromosomes , but by their spreading out over the surface of after coalescence of the latter , the chromosomes would adhere to the vesicles . internal surface of the external walls of the vesicles , which form the nuclear membrane . 
instead of the inner walls of the vesicles breaking down irregularly to form a nuclear network as wilson ( 1925 ) suggested , they would constitute the strands which extend between the nucleoli and the cell membrane . 
to bring ' about such an arrangement it would be necessary to postulate the existence of some mutually repellant force between the chromosomes and the nucleolus at this stage , so that the latter was forced into the centre of the coherent vesicles . from a cursory examination which has been made of different tissues of a number of mammals , including man , it appears that their nuclear structure is essentially the same as that which has been described , but no comparable study has been made of the nuclei of lower animals , or plants . apparently there is a fundamental difference between the nuclear structure of the higher animals and the higher plants . according to the recent work of chayen , davies and miles ( 1953 ) the interphase nucleus of plants ( vicia , allium , zea , tradescantia ) consists of a peripheral zone containing the chromosomes , a middle zone with a protein content , the width of which varies with the state of mitotic activity , and an inner zone occupied by the nucleoli . 
the most striking morphological feature is the manner in which most of the chromosomes are spread out over a spherical area , therebv brinain - a about maximum exposure to the cytoplasm on the outside , and to subsiances emitted from the nucleolus on the inside . 
some of the mitochondria and other granules are applied to the surface of the nucleus , and only clearly , the thickness of its membrane separates them from the chromosomes . substances which penetrate the plasma membrane must pass through the cordon of mitochondria before reaching the nucleus , and between the chromosomes if they are to get to the nucleolar system . in pioneer studies on living cells in tissue cultures , lewis and lewis ( 1915 ) followed the movements of mitochondria backwards and forwards between the nucleus and the cell periphery , and recently pomerat ( 1953 ) has observed rotary movements of the nucleus in epithelial cells growing in vitro . reasons have already been adduced for believing that the mitochondria are involved in protein synthesis ( ludford , smiles and welch , 1948a , 1948b . ) , and attention has been directed to the correlation between . 
hypertrophy of the nucleolar system , proliferation of mitochondria and increased nucleotide content of the cytoplasm ( ludford , 1951 )  . such observations as these tend to emphasise that the cell as a whole is a unified functional system . it is reasonable to suppose that the performance of its fundamental vital processes necessitates intimate correlation of the functional activities of all its organerae . recently brenner ( 1953 ) has deduced from the appearance presented by the nucleus in phase - contrast photomicrographs and ultra - violet micrographs ( ludford and smiles , 1950a , 1950b . ) , and from the results of ultra - centrifugation experiments carried out by himself and others ( beams , 1948 ; claude , 1943 ) that segments of chromosomes are attached to the inner surface of the nuclear mem " these segments maintain their continuity with the remaining parts of brane . the chromosomes which , as uncoiled threads , occupy the inside of the interphase nucleus . " on the basis of caspersson 's ( 1950 ) hypothesis that heterochromatic regions of the chromosomes control protein synthesis brenner ( 1953 ) has proposed that " the nuclear membrane heterochromatin with its specific genes controls the synthesis of equally specific microsomes , each desoxyribonucleic acid directing the synthesis of a characteristic type of ribonucleoprotein "  . " protein synthesis is a two phase reaction " , according to haurowitz and crampton ( 1952 )  . in its first phase , an expanded peptide film acts as a template on which a layer of amino acids is absorbed . 
the peptide film is maintained in the expanded state , its only efficient form , by combining with nucleic acids to form a nucleoin the second phase of protein synthesis , the peptide chain is folded protein . to form ' a three - dimensional globular molecule . haurowitz and crampton ( 1952 ) suggest that the highly specific peptide chains are probably first formed in the nucleus , and that they pass out into the cytoplasm where they are specifically since antigens are deposited mainly in cytoplasmic granules in liver folded . cells it is concluded that the folding of the peptide chains takes place in these granules . 
as haurowitz and crampton ( 1952 ) admit , their evidence that the first phase occurs inside the nucleus is equivocal , while certain of their experimental results indicate that it takes place in the cytoplasmic granules . 
the peptide ffim which haurowitz and crampton ( i952 ) suggest acts as a template in protein synthesis might be locahsed on the surface of the occasionally fibroblasts migrating from explants in tissue cultures mitochondria . leave behind them small pieces of cytoplasm which contain mitochondria and other granules . 
one possible reason for this is that the function of mitochondria is controlled by substances produced by the chromosomes . certain observations also suggest that the latter are dependent upon substances supphed by the nucleolar system . in conclusion , it should be pointed out that cells may undergo malignant transformation without any alteration occurring in their nuclear structure that can be detected by the present methods of microscopy . this is understandable if as has been suggested ( lufford , 1952 ) the cytological basis of malignancy ie ; an imbalance in the genes brought about by processes which result in ( 1 ) the loss of some chromosomes , or parts of chromosomes , and ( 2 ) the intranuclear duphhcation of others . 
this implies that with improvements in microscopical technique it should be possible to distinguish differences between the chromosomes which are spread out on the inner surface of the nuclear membranes of malignant cells and their non - mahgnant prototypes . 
accorcling to caspersson and santesson ( 1942 ) the heterochromatin system of malignant cers is stimulated to abnormal activity . this leads to disturbances in the formation of cytoplasmic proteins and in the reproduction of gene protein , which result in abnormal growth . 
it would be more compatible with the results of the present study if hypertrophy of the nucleolar system and a hyperchromatinic condition of the nucleus were the morphological expression of an increased growth rate rather than being indicative of malignant growth . it has not been possible to identify the nucleolar organizer on the chromosomes of the cells examined during the course of this work . so it remains to be deter ' mined whether increase in size of the nucleolar system is the result of duplication of chromosomes bearing nucleolar orgadizers . there is , however , indirect evidence that this may be the case ( biesele , poyner and painter , 1942 )  . the studies with ultra - violet microscopy described in this paper were carried out in the national institute for medical research and constitute part of a wider 130 r . 
hall. from the hugh adam cancer research department of the medical school and the new zealand branch of the british empire cancer campaign , university of otago , dunedin . received for publication may 26 , 1953 . rats numerous attempts have been made to elucidate the role of the thyroid hormone in the development of chemically induced tumours . 
the relevant literature has been reviewed by miller and baumann ( 1951 ) who studied this problem in treated with dimethyl - amino - azobenzene . these authors mention some of the experimental difficulties encountered when the metabolic rate is altered by the administration of goitrogens or of thyroxthe action of thiourea , thiouracil and related compounds , however , is not limited to the thyroid ; they can affect also other organs , as for instance the liver ( fitzhugh complications of this kind can be avoided by means of and nelson , 1948 )  . surgery if hypothyroidism is the goal of the investigation . 
the effects of partial thyroidectomy are comparable with those obtained by goitrogens in doses too small to inhibit completely thyroxine synthesis . complete elimination of thyroid hormones resulting from either the administration of large doses of goitrogens or from the removal of the gland leads to a complex endocrine imbalance , the outstanding feature of which is a pronounced disturbance of growth . the pituitaries of such rats contain no acidophils and therefore no somatotrophin is formed . 
the thyrotrophic hormone , however , is secreted in elevated amounts . we have studied the action of aminofluorene ( a.f. ) and its acetyl derivative ( a.a.f. ) on the thyroidectomized rat , choosing these carcinogens because they induce tumours in more than one organ . 
one hundred and thirty were wistar rats ( 106 males , 9 of which were castrates , and 24 females ) and 17 were males of a piebald strain . forty - nine intact rats and 9 castrates served as controls ; eighty - nine were thyroidectomized . sixty - two of the 89 ( 70 per cent ) were considered to be completely thyroidectomized because their pituitaries were virtually free of acidophils . 
with the same the intact but not the thyroidamount of carcinogen during the remainder . after the 20th week the ectomized animals consumed all the food offered . in experiment 2 the milkanimals were maintained on ordinary stock diet . flour diet was given throughout , but for the first 20 weeks the diet was adjusted in such a way that 2 mg . 
in acetone 70 applications being given to the albino and 90 to the piebald rats . in this experiment rats which were obviously not completely thyroidectomized as indicated by their growth curve were kept separately from the other animals in in experiment 4 , 9 castrated order to avoid thyroxine intake from the excreta . and 30 intact rats were treated with a.a.f. 
daily. maintained on the wet diet supplemented by grain . in the 15th week 19 of the intact animals were thyroidectomized . liberal amounts of drinking water were available to all animals . in our experience thyroidectomized rats kept on a wet diet are in a better in long - term experiments state of nutrition than when they receive dry food . care has to be taken that excessive growth of the incisors does not interfere with their food consumption . the rats were weighed once weekly and examined for the presence of tumours . they were sacrificed when a neoplasm was suspected or when their general state of health made it advisable . 
the earliest hepatoma was found in the controls in the 18th week of the experiment , multiple benign cystic cholangiomata being already present in the partially thyroidectomized males the earliest hepatoma was at the 15th . discovered in the 28th week . 
no mammary cancers were seen in the thyroidectomized females , the majority of which were in anoestrus already in the third week of the experiment when vaginal smears were taken for the first time . table ii ( experiment 3 ) shows the results obtained with a.f. 
two appeared in intact , 3 in partial and 4 in completely thyroidectomized rats . in addition , one adenoma of the lung appeared in the latter group , and a cancer of the breast in the controls . in experiment 4 thyroidectomy was performed after a.a.f. 
had been fed all the controls developed hepatomas , the first being for a period of 13 weeks . found 7 weeks after the withdrawal of the carcinogen . thyroidectomy performed in the 15th week of the experiment was without the slightest influence on the development of these neoplasms , the first hepatoma appearing already in the 19th week . 
when at the 38th week the experiment was terminated each surviving animal was found to have hepatomatous and cholangiomatous lesions . these results are summarized in table iii , which includes also an additional table iii . - tumours induced by a.a.f. 
hall reduced , the glands containing little fluid whereas the weight of the testes was well maintained . in such animals an atrophy of the interstitial cells of the testis was found histologically , indicating inadequate stimulation by interstitial cell - stimulating hormone ( i.c.s.h. ) , and subsequent failure to produce androgens in amounts sufficient for the maintenance of the accessory sex organs . in this connection it might be remembered that none of the completely thyroidectomized females had a regular oestrous cycle . the adrenals did not show uniform changes . the width of the cortex tended to be less than normally ; the sudanophobic zone , present in intact males , was frequently absent , and the amount of lipoid stainable with oil red 0 was reduced considerably in some but little in other glands . 
some of the thyroidectomized animals were rather obese at the time they were sacrificed , which made correlation between organ and body weight unreliable . as in an earlier experiment ( bielschowsky , 1949 ) , neoplastic changes were found in the thyroid tissue of a.a.f. - treated animals in which a small fraction of the gland had escaped removal . in one instance a highly anaplastic tumour arising from such a thyroid rest was found at autopsy - worth mentioning because a tumour - cell embolus was seen in the periphery of this neoplasm , the only case in which evidence suggestive of malignancy was found in such a thyroid tumour . in the pituitary of this male wistar rat an adenoma was found . the tumour , the only one seen in these experiments , had the same morphology as the basophil adenomata present in aged rats which had received an iodinedeficient diet ( bielschowsky , 1953 )  . all the other pituitaries showed the typical picture of total or partial thyroidectomy , i.e. , absence of acidophils or marked reduction in their numbers with evidence of degranulation coupled with the appearance of " thyroidectomy ' " cells . the retro - bulbar tumours . the appearance of carcinomata in the orbital tissue of two rats of experiment 1 , a type of neoplasm only found in the thyroidectomized groups , led to a systematic search for lesions in the lacrymal glands . 
6 shows that they vary considerably in size and shape and have lost the pale foamy appearance of the normal glandular epithelium . their nuclei are hyperchromatic and dividing cells are present . since these changes were obviously of a neoplastic nature it seems justified to consider the retro - bulbar tumours as carcinomata arising from the epithelium of the lacrymal gland . in intact rats the lacrymal glands are of a brownish colour ; in many of the thyroidectomized animals they were more whitish and appeared also to be more moist than normally . sometimes these changes were bilateral , but not infrequently only one side was affected . histologically in thyroxine - deficient rats , treated with a.f. 
whether the absence of thyroxine is the main factor responsible for the results obtained so far we can only state that thyroidectomy prior remains to be investigated . to the administration of the fluorene derivatives renders the livers of rats unsusceptible to the carcinogenic action without preventing the induction of neothyroidectomy performed after the administration of plasms in other organs . effective amounts of these compounds did not retard the development of malignant it seems therefore that it is the early stage in carcinogenesis liver tumours . preliminary results of experiwhich is influenced by the removal of the thyroid . ments yet to be terminated point into the same direction . bielschowsky and hall , 1952 ; during 1952 evidence was obtained by three independent groups of workers griffin , ( moon , simpson and evans , 1952 ; rinfret and corsigilia , 1953 ) that the action of highly potent carcinogens can be " inhibited " by the removal of the pituitary or of the thyroid . 
the growth and development of most organs , with the possible exception of the brain , is dependent on hormonal stimulation , but there exist considerable differences in degree , as shown by the effects of hypophysectomy . 
the gonads , thyroid and adrenals atrophy always , but if the hypophysectomized rat receives adequate amounts of food the growth of the liver is proportional to body growth . thus neither liver nor the subcutaneous tissue can be compared with the target organs of specific trophic hormones , although crude pituitary extracts can induce disproportionate growth in the liver ( selye , 1949 ) and in acromegalics the soft tissue can increase in size . the role of the somatotrophic or growth hormone in normal or pathological growth is only imperfectly understood . 
growth can be promoted in hypophysectomized rats not only by somatotrophin but also by thyroxine , as shown by geschwind and li ( 1952 ) , or by insulin ( best , 1952 )  . 
a great variety of neoplasms has been obtained in intact but not in hypophysectomized rats injected for long periods with purified growth hormone ( moon , simpson , li and evans , .oestro1950a , 1950b , 1950c , 1951 ; koneff , moon , simpson , li and evans , 1951 ) gens can retard growth by inhibiting the production of somatotrophic hormone . this does not prevent carcinogenesis . 
 ( 1950a , 1950b , 1950c , 1951 ) as well as griffin and his collaborators ( 1953 ) used carcinogens which acted on one organ only . their results could be due to a failure of the hypophysectomized rat to metabolise the compound administered into the carcinogen proper . this possibility has to be considered since the discovery by bonser , clayson , jull and pyrah ( 1952 ) that 1 - hydroxy 2 - naphthylamine is the active agent when 2 - naphthylamine is administered . however in our case , the fact that extrahepatic tumours developed while the liver , the most susceptible of all organs in the intact male rat , failed to respond to the carcinogenic action of a.f. 
and its acetyl - derivative does not favour this interpretation . some of the neoplasms found in the thyroidectomized animals are so rare in our material that we cannot be quite sure that they were due to the action of a.a.f. neither in normal nor in experimental rats of our colony have we ever observed a papilloma of the stomach arising from the glandular mucosa or a tumour of the brain , but vasquez - lopez ( 1945 ) as well as hoch - ligeti and russell ( 1950 ) have described gliomas in rats treated with a.a.f. benign tumours arising in the mucosa of the uterus are not infrequent in aged rats of however , the tumour depicted in fig . 
3 differs from all others our colony . observed by its strong resemblance to a deciduoma . in dunedin carcinomata aiising in the lacrymal glands have been found only in four a.a.f. 
no account of the state of the endocrine organs was given . in our experience a low fat content of the diet per se does not favour the development of cancers of the lacrymal glands . 
of tissue was homogenised in a total volume of 50 ml . , more sucrose being added later to dilute the homogenate to this , as was shown by direct comparison , a final concentration of 10 per cent . had no effect on subsequent fractionation . it was found in preliminary experiments that maximal liberation of cytoplasmic proteins into the supernatant fluid was achieved by homogenisation lasting for 1 to 2 minutes . the separation of the cell components was carried out by means of differential centrifugation at a temperature of 20 to 40 c . 
in a " spinco " ( specialised instruments corporation , belmont , california ) model e ultracentrifuge , except for the preliminary removal of nuclei , unbroken cells , debris , etc . , which was carried out in an ordinary low - speed centrifuge . 
the centrifugal procedure with some modifications was based on those , originally described by claude ( 1943 , 1946 ) and developed by hogeboom , claude , and hotchkiss ( 1946 ) , hogeboom , schneider , and pallade ( 1948 ) , schneider ( 1946b )  . in some of the later experiments it was based on that of schneider and hogeboom ( 1950a )  . 
stickland oxidase activity of each fraction was determined by the method described by umbreit , burris and stauffer ( 1945 ) and the results expressed as q02 ( n )  . 
a concentration of the enzyme in this fraction was also found in normal lactating breast tissue of highand low - cancer - strain mice . while the microsomal fractions of rat and mouse liver showed only a small amount of the enzyme , the same fractions of breast tissue revealed an appreciable quantity of it . in order to establish that this activity was a true property of these fractions , on some occasions the activity was determined after washing and was found to be unaltered . these results expressed as specific activity of the enzyme in the respective fractions are shown in table iii . 
hogeboom and schneider ( 1950 ) observed partial inactivation of succinoxidase after sonic disintegration of mitochondria . the possibility of contamination with mitochondria could not be excluded by histological examination alone . mitochondria were invariably observed in smears of microsome fractions , but in considerably smaller numbers than in the mitochondrial fractions : it is , however , impossible to make such comparisons quantitative . 
the chief arguments against contamination with whole mitochondria are first , that in rat liver no succinoxidase was found in the microsomes ( or negligible quantity ) , and second , that in a number of experiments with mammary tumour tissues the same mitochondria were sedimented in two parallel ways , at 5000 times gravity and at 8000 times gravity , and in the latter case the activity of the microsomes subsequently obtained was only slightly lower than in the former . the possibility of contamination of microsome fractions by fragments of broken mitochondria cannot easily be disposed of . clearly such contamination was not observed in liver microsome fractions , obtained in similar manner as those from normal and malignant mammary tissues . 
the results showed clearly that mitochondria from normal and malignant mammary cells of mice are not more fragile than those from liver , and if anything rather less . it can therefore be deduced that mammary cell mitochondria are not appreciably fragmented during homogenisation , and that consequently the enzymic activity present in the microsome fractions from these cells is likely to be a true property of microsomes present in these fractions . 
the ready fragmentability of liver - cell mitochondria in the potter - elvehjen homogeniser after they have been separated from other cell components is at a first glance not easily reconciled with the fact that no damage appears to occur to these cellular components during the homogenisation of the tissue . this difference is presumably due to the protection of the mitochondria against friction and damage by the presence in the latter case of larger cell fragments . the enzyme activity of microsomal fractions of normal and malignant breast tissues remained practically unaltered in spite of washings in isotonic sucrose 258 l . 
stickland solution , which again points against the possibility of the enzyme being adsorbed on microsomes . from the experiments of price , miller and miller ( 1948 ) , price , miller , miller and weber ( 1949a , 1949b ) on rat hepatoma and of schneider and hogeboom ( 1950b ) on mouse hepatoma , it is now known that in both rat and mouse hepatoma , the protein content of the mitochondrial and microsomal fractions is much lower than that in similar fractions of normal rat and mouse liver . further , it has been shown in these studies that in rat and mouse hepatoma the succinoxidase content of the mitochondrial fraction is lower than that in normal rat and mouse liver , the total activity being 5 times lower and the specific activity 2 times lower , in spite of the same distribution of the enzyme in normal and malignant livers . in the present experiments no significant difference was observed between normal and malignant mammary cells , either in the total amount of succinoxidase or in its distribution between the various fractions of these cells . 
a curious point , so far unexplained , is that although in liver homogenates 100 per cent recovery of succinoxidase could be obtained in the fractions , in mammary tissues the recovery was much lower , amounting on the average to only 70 per cent . no difference was found between highand low - cancer - strain mice either in the protein and succinoxidase content or in the distribution of these in the various fractions , but the mammary tumour tissue in each case showed a reduced content of mitochondria , compared with the normal mammary tissue , which agrees with that found by schneider and hogeboom ( 1950b ) in mouse hepatomas . the reliability of the simplified centrifugal procedure used in the majority of the experiments has been tested by the use on three occasions of the more complicated procedure of schneider and hogeboom ( 1950a ) , which gave substanit may perhaps be of interest to mention that stern tially the same results . ( 1939 ) demonstrated the association of succinoxidase with particles of microsome size in beef heart muscle . a small point worthy of mention is that for greater convenience of making the enzyme assays , the samples of washed particles from rat and mouse liver and mammary tissues were kept overnight at - 79 c . 
at that time research into their pharmacological and toxicological properties showed that certain of their effects were strikingly similar to those of ionizing radiation ( gilman and philips , 1946 ; kamofsky , graef and smith , 1948 ; philips , 1950 )  . this led to further investigations of their action as mutagenic agents and as growth inhibitors and their importance in these connections quickly became apparent . 
tumours were selected for treatment on the 6th or 7th day after implantation . the standard dose level adapted as the basis for comparison of chemical and x - ray effects was 1 mg . 
casarini analyses showed that 200 r might be considered to be an " equivalent " dose , i.e. , it produced in tumour and bone marrow the same amount and the same type of cellular injuries up to 24 hours after administration ( koller , unpublished )  . 
body weight throughout the experiments . for cytological analysis small pieces of tumours were fixed in acetic alcohol mixture ( 1 : 3 ) , and squash preparations were made and stained with feulgen 's for histological study fragments were fixed in bouin 's solution , basic fuchsin . embedded in paraffin wax , and sections cut at 5 . 
were stained with haematoxylin and orange - g . cytological effects : method of analysis . the method used for estimating cellular injuries is the same as was employed by devik , elson , koller and lamerton ( 1950 )  . cells in post - metaphase stages were selected . 
the importance of some biological variables ( age , sex , diet , etc . ) has already been discussed ; devil and coworkers ( 1950 ) demonstrated by analysis that the data , obtained by the same method as used in the present study , do in fact provide a valid basis for comparison . but as will be seen , the effects observed 72 hours after treatment by x ray and hn2 were in any case qualitatively so different that they could not be compared on a quantitative basis . x - ray effects : localised treatment ( 200 r )  . the tumours only were irradiated and cytological effects were analysed at 6 , 12 , 24 , 48 , 72 , 96 , 120 , 144 and 168 hours after treatment . 
date when the animal was killed . this is also the sample in which the number of chromosome fragments per cell is at a maximuthe damage is less 24 hours after irradiation , and it diminishes further in the 48 and 72 - hour samples . in tumours 4 to 6 days after treatment the number of dividing tumour cells with abnormalities is very small . our observations indicate that 200 r is insufficient to inhibit the growth of the walker carcinoma ; the growth rate of treated tumours 6 days after irradiation was found this finding is in close agreement with that to be the same as that of the controls . of devik and co - workers ( 1950 )  . hn2 - induced cytological effects . it has already been reported by the senior author that a dose of 1 mg . 
by including such cells in the analysis , the true number of injured cells is reduced ( compare the 144 and 168 hour samples , table iib )  . ' 0060 - / time after treatment in hours fig . 
they are particularly evident in young tumour implants and are found in large numbers at the periphery of growalthough these cells are normally present in the walker carcinoma , ing tumours . they play a small part in the histological organisation of the tumour . 
the great and rapid increase in histiocytes after hn2 administration , which takes place in the cellular tumour parenchyma , has important consequences . table iib shows the drastic change which occurs in the cell composition of the tumour . 
the proliferation of those tumours cells which may recover from nuclear or cytoplasmic disturbances was found to be seriously hindered by the altered environmental conditions . establishment of a solid tumour parenchyma does occur occasionally in the region of the connective - tissue capsule . 
the histological structure of this particular region is known to differ greatly from that of the tumour , and the different type of reaction to hn2 is presumably correlated with this . in conclusion we wish to emphasize the important fact that 72 hours after hn2 administration a series of changes takes place which result in the complete transformation of the histological structure of the walker carcinoma . 
the effect is , however , temporary ; the animals were already gaining weight again 7 days after treatment . in this respect the effect of hn2 is much more drastic than was observed after  . 
body weight ( intraperitoneal injection )  . while the cellular injury in the femoral bone marrow induced by 500 r was much more drastic than that produced by the hn2 " ld50 - equivalent " this x - ray dose was found to be still insufficient to bring about by way of the systemic effects the same amount of cellular and the same kind of histological changes which have been observed after hn2 administration . 
the delay in the developmental cycle may last 12 hours . this is indicated by the fact that in the 12 - hour sample 6 cells with chromosome injuries were observed out of 27 in which the chromosome abnormality suggested that they were already the frequency distribution of the injured cells in mitosis during irradiation . observed in the various samples is a further indication that cell development has been retarded . while the highest proportion of injured cells is seen 12 hours after exposure to 300 r , the maximum damage was in the 48 - hour tumour sample 182 p . 
the true significance of this observation lies in the relationship which it has to the primary reactions induced by the two ' agents . the view is now widely accepted that during irradiation ionisation takes place in the cell , followed immediately by the formation of free radicals of the water molecules ( lea , 1946 )  . this event represents the first step in the complex similarly reaction - chain which in time leads to a definite biological phenomenon . there is no substantial delay between administration of hn2 and its primary reaction in the organisthe rate of hydrolysis of nitrogen mustard is extremely high in a biological system , and it has been shown recently by batemann , klopp and cromer ( 1951 ) that it remains " active " only for a few minutes after intravenous injection . this is the main reason which allows us to compare the cellular effects of x ray and hn2 in tumour samples taken at the same time after treatment and to determine the relative sensitivity of the various stages of the mitotic cycle . the measure of sensitivity of a cell is the frequency of chromosome injuries observed at a given time . 
the interval between treatment and fixing the cell for analysis indicates the cell stage during treatment . using this criterion the radiation sensitivity of the mitotic cycle has been determined in pollen grains of tradescantia ( koller , 1946 )  . if our present data are interpreted on the same criterion , then we must draw the conclusion that tumour cells of the walker carcinoma are most sensitive to x rays at the end ofthe resting stage or beginning of prophase , while they are least sensitive to hn2 at this stage . 
revell ( 1952 ) employed a new and critical method in the study of chromosome effects of several substances ( mustards , epoxides , etc . ) and came to the same conclusion . 
the fact that x ray ' and hn2 initiate their effects at different stages strongly suggests that their mode of action is different . thus , for instance , we may assume that x rays act directly on definite linkages of the already synthesized polypeptide structure of the prophase chromosome thread , while a chemical agent affects the ' resting stage chromosome - filament either before or during the process of synthesis . this aspect of the chemical effects is discussed in more detail by revell ( 1952 )  . the higher sensitivity of the resting stage to the chemical agents has another implication . 
the non - dividing ( intermitotic or " resting " ) cells are ' believed to be functionally the most active cells , and often referred to as cells in the " metait is not unlikely that the degeneration of many bolic phase " ( hughes , 1952 )  . non - dividing tumour cells which we observed after hn2 administration is due to a gross disturbance in this metabolic phase . if it is found that chemical agents act preferentially on cells in the resting stage , a new approach may possibly be opened to the chemotherapy of cancer . because the functional or morphological characteristics of cells are determined 184 p . 
our experiment demonstrated such difference between tumour cells and histiocytes . the study of the cytological behaviour of the walker carcinoma under x ray and hn2 treatment has accordingly led to the conclusion that the mode of action of these agents differs , and there is other experimental evidence of different kinds to support this conclusion . 
no such reduction was observed when nitrogen mustard in barley , mutations of particular genes was used ( auerbach and moser , 1951 )  . were induced with a higher frequency by hn2 than was expected , indicating some specificity of action ( gustafsson and mackey , 1948 )  . 
cytological analysis of the femoral bone marrow and implanted walker carcinoma of the rat has shown that up to 24 hours after treatment , 200 roentgens produce the same amount of chromosome injuries as 1 mg . 
the difference in behaviour shown by the walker carcinoma after irradiation and after nitrogen mustard treatment indicates that the primary basic reactions initiated by these agents are fundamentally different . this investigation has been supported by grants to the royal cancer hospital and chester beatty research institute from the british empire cancer campaign , the jane coffin childs memorial fund for medical research , the anna fuller fund , and the national cancer institute of the national institutes of health , u.s. 
the centrifugary - cleared extract was adjusted to i per cent with 20 per cent saline and the crude mucoprotein , etc . , precipitated by addition of two volumes of alcohol . 
the washed deposit was redissolved in 1 per cent saline , adjusted to ph 7 - 5 , and digested for 24 hours at room temperature with commercial trypsthe mixture was then filtered with the aid of hyflo super - cel and reprecipitated with 2 volumes of alcohol . 
the yield of protein - free , crude polysaccharide was about 2 per cent of the acetone - dried tumour tissue . the material contained polysaccharide and pentosenucleic acid , and the analytical data were : nitrogen ( kjeldahl ) varying between 5 - 2 and 6 - 3 per cent ; phosphorus ( fiske and subbarow ) 1 - 7 to 1 - 8 per cent ; and sulphur ( paulson ) 0 - 6 per cent . 
the ninhydrin reaction was negative . metachromatic sta ' m ' mg with azure a ( sylv6n and malmgren , 1952 ) revealed the presence of one orthochromatic component and another presenting alcohol - resistant metachromatic precipitates . 
d was also degraded by testis hyaluronidase at a similar rate to other such hyaluronates . glucose ( glucosamine ) could be demonstrated by following acid hvdrolvsis onl paper electrophoresis in borate buffer ( consden and stanier , 1952 )  . in addition , infra - red spectral measurements kindly performed by s . 
the two spectra were identical in the region 680 - 980 cm . - ' , showing the two materials to have the same molecular skeleton ( orr , 1954 )  . 
the only difference lay in the relative intensities of the bands due to the acid and amide carbonyl groups . however , the acid : amide ratio in sample d cannot be more than 5 per cent greater than that in the purified umbilical hyaluronate . nature of the_8uiphur - containing material . the metachromatic precipitate formed by azure a treatment of sample b was almost insoluble and further information about its composition could not be obtained . 
attempts were therefore made to remove the nucleic acid and the hyaluronate from the starting material by successive ribonuclease and hyaluroafter removal of the hydrolysis products by dialysis it was nidase treatments . thought possible to obtain the s - bearing material in a purified and soluble state . however , this latter material exhibited a powerful inhibitory effect on hyaluroin one experiment , when 30 mg . 
the s content of the remaining non - digested material was 0 - 49 per in the course of this long digestion , however , a small precipitate was cent . formed , which had a n content of 10 - 0 per cent and s content of 4 - 8 per cent . these figures suggested that a new product had been formed between the protein hyaluronidase and a s - containing polysaccharide . if the polysaccharide is assumed to contain 3 per cent nitrogen , the nitrogen value of the precipitate would fit a protein content of about 55 per cent , and hence a polybaccharide content of about 45 per cent . 
the intermediate protein - free material ( sample a ) contained about 70 per cent hyaluronate , 25 per cent nucleic acid , and about 5 per cent of a sulphur - be ' aring polysaccharide presenting some biological characteristics of heparthe largest part of the polysaccharide material present is thus beyond doubt hyaluronic acid with identical molar characteristics with that obtained from mammahan sources . 
the rous hyaluronate has probably a lower particle size than the hyaluronate from normal tissues , and the high viscosity of the intercellular rous material seems partly due to the protein components . the identification of heparin in the sulphur - bearing polysaccharide has not been fury established . 
the same study indicated that an excessive output of ethanolamine frequently occurred in the urine of patients with severe parenchymal liver disease , but that in such cases it was always accompanied by an excess of many other amino - acids . 
the presence in the urine of large quantities of ethanolamine , as an isolated abnormality , in the patient with primary carcinoma of the liver was therefore of some interest . its excretion might have resulted from a disorder of metabolism consequent on non - specific liver damage or from some peculiar form of neoplastic cell metabolism ; or it might even have represented a hitherto unrecognised error of metabolism which preceded and led to the development of a primary carcinoma in the liver . it has for some years been recognised , as a result mainly of animal experiment , that ethanolamine plays an important role as an intermediary in the metabolism of phospholipids and amino - acids . 
1. - some metabolic inter - relationships involving ethanolamine and other compounds . experimental dietary deficiency of choline has also been extensively studied . in rats it has been shown to lead to the production of fatty livers , followed after some time by cirrhosis . 
more recent work has shown that this may be followed eventually by the development of hepatoma - like tumours ( copeland and salmon , 1946 ; staub , viollier and werthemann , 1948 )  . furthermore , choline deficiency of a severity not sufficient to produce cirrhosis predisposes the liver to the carcinogenic action of butter yellow ( opie , 1944 ; buckley , buckley and snipes , 1951 )  . in the human hepatomata that occur in tropical countries there is ample evidence that dietary factors are concerned ( berman , 1951 )  . 
il - , a housewife , was aged 45 years at the time of her death in 1950 . apart from an attack of jaundice at the age of 7 she was in good health until april , 1939 , when , at the age of 34 , she had a febrile illness with delirium and a she had a second similar attack in october that year , and productive cough . this , like the first , lasted about 3 weeks . she suffered from similar attacks each in that year her illness was more severe and was associated winter until 1942 . with generalised aches and pains and vomiting . since that time she suffered from morning vomiting and also a burning pain , relieved by alkalis , behind the despite these regular winter illnesses and an increasing lower end of her sternum . sense of tiredness she continued with her work in a munitions factory until 1945 . from then until her death in 1950 she worked as a housewife . in january , 1947 , she first noticed a painless lump in her abdomen . in march of that year , following an illness with fever and malaise , she noticed that her abdomen was becoming tender to touch , her appetite was failing and the morning vomiting was becoming more severe . it was at this time that she was first seen and admitted for investigation at university college hospital . both parents and her 4 siblings were all alive and well . examination revealed a pale , slightly cyanosed , nonicteric woman of 8 st . 
the pulse was regular at 90 and the blood - pressure was 110 / 60 mhg . there were rhonchi over both lung fields and bilateral basal rales . in the nervous system the pupils were normal , the kneeand ankle - jerks were absent and the plantar responses were flexor . 
was normal , the circulation time was 17 seconds from arm to lung and 35 seconds from arm to tongue . the cyanosis was rapidly and completely relieved by breathing oxygen . whilst in hospital she ran a continuous low - grade fever . 
the metabolic studies carried out during this period are described later . opportunity was also taken at this time to examine chromatographically the urines of both parents and the other 4 siblings ; all showed a normal concentration and pattern of amino - acids . at the conclusion of the metabolic studies the patient went home much improved for her fortnight 's rest and largely pain - free for the first time for 2 years . she was instructed to take methionine 1 g . 
ethanolamine labelled with 50 per cent atom excess of 15n . on june 21 , 1950 , she was readmitted to hospital with an attack of bronchopneumonia . apart from her chest infection and gross oedema of the legs her general condition was unchanged . she volunteered the information that since taking choline her abdomen had felt looser and had been less painful . pneumonia was successfully treated with penicillin , and whilst on this her fever was . 
cameron reported on the histological sections of the liver as follows : " the tumour is undoubtedly a hepatoma ; it has not changed in character between 1947 , when the biopsy was taken , and 1950 . the most notable 170 c . 
each 24 - hour urine was analysed chromatographically for amino - acids and chemically for in addition blood specimens were drawn soon after the last dose ethanolamine . of each supplement had been taken and were analysed for amino - acids . further 24 - hour urine specimens were collected from her as an outpatient after she had been for 4 weeks on 10 g . 
7. apart from the very strong spot given by ethanolamine the remaining spots are of similar strength and pattern to those found in many normal urines . there was , however , an increase to a slightly abnormal level of the strength of the cystine spot during the patient 's last few months of life , and the unidentified weak " under proline " is not commonly found in urine whether normal or pathological . 
the only clearly identified gross abnormality was , nevertheless , the increased excretion of ethanolamine . these results clearly suggested that the ethanolamine was not the result of variable extrinsic factors ( such as diet ) , but was more likely to be due to a continuing metabolic abnormality . the plasma chromatograms were quite normal , except for a faint trace of ethanolamine . this indicated a definite , if slight , increase in the ethanolamine concentration of the plasma since the quantity ( 625 ' , d . ) of desalted plasma ultrafiltrate analysed was not sufficient to reveal the ethanolamine present in the plasma of a normal person . 
the urine to be analysed was placed at the right - hand bottom comer of the paper . phenol was run as the first solvent in the direction from right to left , followed by collidine - lutidine in an upward direction as the second solvent . 
8. - section of rat hepatoma kindly provided by copeland . hepatocellular type of tumour produced by prolonged feeding on a choline deficient diet ; ( no carcinogens added )  . note the difference from the histology shown in the illustrations of the tumour of c . 
9. it is seen that the output of " apparent ethanolamine " increased most on the day after the ethanolamine had been given and was probably still increased on the second day . 
the " increase " after serine was almost certainly due entirely to serine and not to additional ethanolamine , since the chromatograms showed a greatly increased serine output on this day and serine also gives rise , in the assay method , to its equivalent of " apparent ethanolamine " ( see above )  . 
walshe it has been involved were very small and the " spots " only just detectable . shown that many of the known amino - acidurias result mainly , if not entirely , from a raised renal clearance ( low threshold ) for amino - acids ( dent , 1951b )  . is not necessary to invoke this mechanism as a cause for the ethanolamine excretion here , since the normal subject who took ethanolamine by mouth produced a comparable urinary output at an induced blood level roughly similar to that of the patient . 
we suggest that it can best be understood in terms of an increased membrane permeability , on the part of the tumour cells , for ethanolamine . if such abnormnal permeability existed , a larger percentage of the loading dose of ethanolamine would pass from the extra - cellular fluid to the cells of the patient than it would in the case of the normal control . 
the urine from 7 cases of extensive secondary carcinoma of the liver ( appendix b ) was also investigated with negative results . it is not clear from the evidence at present available whether there is a specific , though rare , type of hepatic neoplasm characterised by an increased urinary excretion of ethanolamine or whether any tumour , if large enough , would give rise to a similar finding . 
ch3 + ch3 ch20h ( c . ) ch2n ( ch3 ) 2 + ch3 ch20h ( d . ) ch2n ( ch3 ) 3 ch20h ( choline )  . but , as has been mentioned already , we now prefer the theory of an increased membrane permeability for ethanolamine . 
the principal objection , perhaps , to the theory of a metabolic block is that if it were appreciable im extent it should have resulted in some degree of choline deficiency as well as in the accumulation of unmetabolisable ethanolamine . in experimental animals choline deficiency leads to fatty infiltration of the liver followed by fibrosis and , if sufficiently 176 c . 
we are , therefore , unable to accept the possibility that the large urinary excretion of ethanolamine resulted from a disturbance of the known metabolism of choline or one of its important intermediate metabolites . 
we prefer to believe that it was caused by some other property of this particular type of heptoma , such as a disturbance of " cell membrane permeability . " a further problem has been posed by the complete failure to increase the excretion of ethanolamine in the experiments in which large doses of its known biochemical precursors ( glycine and serine ) were given by mouth . this fact appears to underline further the difficulties encountered in attempting to interpret the results of this type of " classical " experiment . presumably the compounds failed to reach the cellular site where they would have been converted into ethanolamine and , therefore , that our patient did not provide a suitable opportunity to study normal ethanolamine metabolism by the rather crude methods at out disposal . 
the prospect of success would have been much greater using isotopically labelled precursors , since isolation from urine of pure samples of ethanolamine for isotype analysis presents no great difficulty . in conclusion we wish to thank sir harold himsworth for permission to publish this case , professor m . 
copeland for his very great kindness in lending us his original blocks of rat hepatoma tissue ; also the following for permission to investigate the cases referrred to in the appendices : a . 
a chemical method of analysis suggested that the output of ethanolamine this was confirmed by a rough quantitative was of the order of 0 - 5 to 1 - 0 g . 
as this substance is probably not readily taken up even in normals from the extracellular fluids by the tissue cells or from the glomerular filtrate by the renal tubule cells , it was therefore readily excreted into the urine . 
the presumed source of the ethanolamine was the neoplastic liver cell , and the enormous mass of these cells ( about a quarter of her body weight ) in the patient was probably sufficient to account for a greatly excessive production of this compound . 
he had a cholecystectomy for gall in 1950 he had an attack of renal colic and a ureteric calculus was stones in 1942 . in 1951 he was admitted to hospital with severe upper abdominal and demonstrated . laparotomy revealed a moderate enlargement of the liver , with left shoulder pain . very numerous white opaque nodules scattered throughout the liver substance . 
h - , aged 53 years . apart from a cholecysfectomy in 1950 he was in good health until shortly before admission to hospital when he was seized with a severe low back pain , greatly aggravated by movement . 
a diagnosis was made of carcinoma of the bronchus with secondaries in the lumbar spine and liver . his course was marked by very rapid enlargement of the liver and progressive cachexia . 
nielsen. from the danish cancer registry under the national anti - cancer league , copenhagen , denmark . received for publication april 30 , 1951 . in comparing mortality figures for cancer from various countries for the years around 1938 , clemmesen and busk ( 1947a , 1947b , 1948a , 1949a , 1949b ) found that while denmark ranked third for total mortality from cancer , her figures for females were highest among the countries investigated , surpassing even switzerland and england , for which countries the total mortality was higher . appeared that the high mortality figures for danish women were caused primarily by a high mortality from cancers of " accessible site . " from figures given in the series of articles quoted , it is seen that the incidence of cancer as expressed by the figures from the danish cancer registry for 1942 to 1944 was higher for copenhagen than for the provincial towns , which in their turn exceeded the incidence for rural areas . consequently , in the following paper the incidence of cancer in copenhagen will be studied separately , with particular reference to social conditions , social class being expressed in terms of annual house rent for various subdistricts of greater copenhagen . it is an established fact that expenditure on housing is a more reliable indicator of the social status of a family than the annual income of the breadwinner . 
the level of house rent in copenhagen was fixed during the period studied , and the wartime shortage of housing which prevailed during the entire period must have restricted the number of changes of residence . 
hence several factors working together tended to stabilize some of the variables which would be inclined to disturb a study such as this . the city of copenhagen , the capital of denmark , has one million inhabitants , another million live that is , about a quarter of the total for the whole country . in the provincial towns , and two millions in rural areas . medical facilities in copenhagen left very little to be desired during the period examined , in spite of current political and military events . 
nielsen the reference of cases of cancer to distinct geographical location has , however , inforin the present study been made without regard to the place of treatment . mation on cases was collected by the cancer registry , and each case was referred to the subdistrict corresponding to the address of the patient when first seen in hospital for cancer . 
the cancer registry collects notifications from hospitals all over denmark of all cases of cancer seen , and separately of all post - mortem examinations performed on cases of cancer . for patients not entering hospitals it is assumed that death certificates will give sufficient information , but it will appear that such cases amount to rather insignificant numbers in copenhagen . further details of the activities of the danish cancer registry have been given elsewhere by clemmesen and busk ( 1948b , 1948c ) clemmesen , busk and nielsen ( 1949 ) , and clemmesen ( 1950 , 1951 )  . the subdivision of the greater copenhagen area into 22 subdistricts used for the present study had originally been worked out for various administrative purposes , and proved useful because detailed figures for the distribution of the population by age were available for each subdistrict . 
the wealthy boroughs of frederiksberg and gentofte had , however , to be treated as separate entities because ot the absence of such information for their subdistricts . after a careful analysis of the figures for each site of cancer in each of the 22 subdistricts , it became possible to collect subdistricts within the same range of house rent into however , the average annual house rent of five major classes or " districts . " each subdistrict is given together with the relative values for cervical , mammary and pulmonary cancer in table v . in order to describe the material from a medical point of view table i has been worked out . 
we denote the observed number in subdistrict v comprising all age groups . by l we denote the total number of person - years of exposure , and by c the addition of the subscripts v computed number of persons developing cancer . and s to l and c has the same meaning as described for 0 . if the morbidity from cancer were identical for all subdistricts of the town , we would expect the number of cancer cases at a given age to be distributed on the subdistricts in the same manner as the number of person - years of exposure for a given age . consequently we obtain the computed number c , 8 from the equationlvs . and this number should be compared with the observed number ov.. now the number ov , , is usually small ; we therefore take this number for all age groups and make the comparison for all of them at the same time . 
nielsen pondingly the incidence of cervical , mammary and pulmonary cancer is given as a percentage of the values computed for each subdistrict , with full allowance for differences in age distribution of the population o.. 
nielsen between the social status of the various subdistricts and the incidence of cervical cancer demonstrated in table v is striking to anybody familiar with the city . the irregularity presented by district ii , which mainly consists of the borough of frederiksberg , showing an unexpectedly high frequency of cervical cancer , can by no means be ascribed to a particularly high birth rate in the borough , since this rate is lower than for gentofte , and also lower than the average for the - ~~~~~.... . 
from these areas patients with cervical cancer are centralized for treatment in the radiumstations of the anti - cancer league , situated in the towns of copenhagen , aarhus and odense . 
nielsen connection with the development of cancer , it should be pointed out that the cases have been puboccurrence of cervical cancer in virgins is questionable . lished of cervical cancer in children ( heckel , 1950 ) , but these have been adenocarcinomata , so that it may be desirable in statistical work to separate this group from squamous cell cancers of the cervix . 
paper the clinical aspects of androgen therapy in 70 cases of advanced mammary cancer in women are described , the literature surveyed , and the effect of androgens compared with that of castration carried out by surgery and by the prophylactic use of androgens in mammary cancer as x - irradiation . described by prudente ( 1945 ) is not discussed here . the investigation was begun in december , 1947 , and since then sufficient experience has been gained to permit an assessment to be made in general terms of the place of androgens in treatment . 
the pellets were inserted through a skin incision 1 clong in the lower abdominal quadrants , and pushed through radially to rest on the external oblique aponeurosis between 5 and 10 cfrom the incision , the pellets lying at points on a circle with the incision as centre . absorption from these pellets depends on a variety of local factors , including their relation to surrounding fat , adjacent fascia , lymphatic plexuses and encapin three patients who came to autopsy 55 , 82 and sulation by fibrous tissue . 93 days respectively after insertion , the pellets were removed , allowed to dry for 48 hours and weighed . 
per month for their evocation when injections of testosterone propionate are used ( geist , salmon and gaines , 19388 ; geist , salmon and walter , 1940 ) , representing daily doses of nearly double the maximal ( initial ) daily absorption from ten 100 meg . 
were given 3 - 7 times weekly , but later opinion came to advocate larger and larger doses of the order of 100 mg . daily , or even twice daily . the authors of the latest progress report of the council on pharmacy and chemistry ( 1949 ) , however , no longer favour heavy dosage , and consider that there is no advantage in exceeding 150 mg . 
daily for the first 2 - 4 weeks , given . to halve this dose if the patient is responding favourably , or to continue for another all 30 patients who derived month if there is no evidence of specific benefit . benefit from androgen therapy showed clear evidence of it within 8 weeks , and in most cases within the first month . 
no patient who failed to respond after 8 weeks of treatment did so after a longer interval , and it is probably not worth continuing treatment after 10 weeks in the absence of specific response . cannot be said from the small number of cases in this series whether such cases should be transferred to testosterone propionate injections , but the general impression gained has been that patients likely to benefit substantially from androgen therapy are extremnely sensitive to its action , in whichever form it is given , and do as well on doses just large enough to suppress inenstruation in the average premenopausal woman as they do on inuch larger doses . 
on the other hand , most cases which fail to respond to the smaller doses do no better when the dose is increased or the route of administration changed , but the minority which does so led to the tendency already mentioned to advocate heavy dosage . patients given methyltestosterone were advised to leave the tablets ( 50 mig . ) under the tongue , and to allow them to dissolve without chewing or swallowing . the time taken for solution varied between 10 and 30 minutes in different individuals , and a few patients disliked the bitter taste of the substance . duration of treatment in cases responding favourably . no definite recommendation can be given with regard to the length of time cutler and schlemenson , reporting 19 cases , administration should be continued . advised continuing injections until palliation was no longer being obtained the american council already referred to ( cutler and schlemenson , 1948 )  . found that the most favourable results were obtained when injections were conin this series it has frequently tinued until a total of 3 0 g . 
methyltestosterone daily , commenced 6 months after the second implantation and continued for a further 4 months . widespread evidence of fresh activity of the carcinoma then appeared , accompanied by recurrence of pain testosterone propionate injections 100 mg . 
as far as the effect on menstruation is concerned , it may therefore be said that the hormone absorbed from 50 mg . sublingual methyltestosterone is less potent than 10 mg . 
testosterone ( table marked facial hirsuties was present in the second of these cases at the ii )  . time of death , 12 weeks after implantation , not more than 250 mg . 
amenorrhoea persisted for weight gain , 4 months , and weight gain continued during the same period . and subjective benefit , occurred without other side - effects in patients who were it therefore appears that the given methyltestosterone in 40 mg . 
in the first month are bilitated patients is very striking . common , and with the other " tonic " effects are independent of the specific effect in case 65 for example 7 lb . 
in the second 4 months . clinical oedema , however , was seen in only 3 cases , and was always slight in in a few of the amount , and confined to the ankles and lower half of the legs . patients with post - mastectomy oedema of the arm , an increase in the swelling of the arm followed testosterone administration , but this was more closely related to spread of the disease than to the treatment , for when lesions regressed , the lymphoedema sometimes diminished , even when rapid increase in weight was during the initial period of weight gain , some of the occurring , as in case 44 . patients stated that they passed less water , but no measurements have been carried out . much of the initial rapid weight gain is due to the effects of electrolyte , and particularly sodium retention , which holds equivalent quantities of water in the body . this effect is a feature of the activity of many steroid hormones , notably progesterone , oestradiol , and of kendall 's compounds desoxycorticosterone , e and f . 
daily for 6 months and 1 year respectively . they were abundant across the back , and were also present on the cheeks and chin . the skin , which was heavily infected , had the lumpy appearance typical of adolescent pustular acne . in case 44 hirsuties was nminimal , consisting of a sparse growth of soft hairs on either side of the upper lip , but was much more developed in case 63 . in 7 cases facial hirsuties reached an extreme degree , the sides of the face being covered with thick downy hair between 1 and 2 clong ( cases 7 , 9 , 24 , 25 , 56 , 63 )  . in cases 9 and 56 , after 6 months ' treatment the hairs increased in calibre , and resembled the stiff bristles of the male beard . there was some variation in the changes in the scalp hair . 
when fully developed they form a characteristic syndrome , which has been observed in 3 patients in this series . it was first seen when 2 patients ( cases 10 and 45 ) were given 80 mg . 
testosterone. in case 10 , after taking the tablets for 2 days , the patient complained of extreme weakness , drowsiness , severe headache , nausea , and increase in the dyspnoea which was her initial complaint , and due to pulmonary and pleural metastases . 
the symptoms subsided rapidly after she stopped taking the tablets . in case 45 the patient took the tablets for 5 days , during the last 3 of which she felt generally ill , nauseated , and complained of increase in the severity of her backache . 
on the 5th day she was distressed by vomiting , abdominal distension and pain , and took no more tablets , after which her condition improved rapidly . one week later she again attempted to take the tablets , but with exactly the same result . case 30 was a woman of 45 who had been previously treated for 2 years by repeated doses of x - rays for widespread skeletal recurrences for a stage iv carcinoma , and had had induction of an d . 
daily for one month , towards the end of which she complained of increase in the severity of her pains , dizziness , nausea and vomiting , and general malaise . 
the symptoms abated within a day or two of discontinuing the drug . she was asked to resume the tablets a fortnight later , but the symptoms recurred after one week . case 47 , doses of 35 mg . 
by a " moderate success " is meant a case in which symptomatic relief was short - lived ( mostly less than 6 months ) , or not accompanied by considerable objective evidence of regression or by restoration of function . all other cases are classed as failures , even when non - specific benefit was marked and increased the patients ' range of activity . 
testosterone propionate three times weekly without benefit after 6 weeks . she is classed as a " moderate success " in view the remaining patients obtained less benefit , and 3 were of the initial response . never able to leave their beds , although all volunteered that their pains were improved . one of these declared that she had not felt so well nor had so little pain for 2 years , 20 months of which had been spent in bed . case 66 was a woman of 44 years with extensive metastasis to the spine which was responsible for gross collapse of the bodies of dorsal vertebrae 7 - 11 . had been unable to walk or stand for 6 weeks owing to severe proprioceptive loss in both legs , a short course of radiotherapy having conferred no benefit . 
a mild paraplegia was present , and there was some impairment of sensation to light touch , deep pressure and pinprick from the level of the umbilicus downwards . there was , in addition , a girdle of almost.complete anaesthesia to touch and pinprick over the distribution of dorsal segments 7 - 10 . curiously , sensation over the paravertebral regions of these segments was normal . there was no hyperalgesic zone , and no disturbance of sphincter control . the patient was given 28 daily injections of 100 mg . 
two other patients with dyspnoea as one in cases 3 , 10 , 24 of several symptoms obtained some relief after treatment . and 63 the rapidity with which the breathing improved was astonishing , and colnparable with the speedy relief of bone pain in the cases mentioned above . in case 25 , however , with dyspnoea at rest , no significant improvement had ten oz . 
galton with a conjugate defect of elevation , thought to indicate a lesion in the anterior this lesion could have been of vascular origin , part of the oculomotor nucleus . or might have been due to a metastasis . that the latter was the more likely is suggested by the development 6 months later of diminished sensation to touch this patient took methyland pinprick over the right trigeminal distribution . testosterone 50 - 100 mg . 
daily thereafter , also showed marked regression of axillary and supraclavicular lymph nodes , though some of these were still just palpable 4 months later . this patient relapsed 11 months later . case 35 was a stage iv growth in a woman of 36 years . she had nodes in both axillae . 
one year previously she had been given radiotherapy to the primary growth and to the axilla , and 6 months previously had been given pelvic five months later she was given irradiation for induction of the menopause . * methyltestosterone sublingually 80 nig . 
31 premenopausal cases with bone deposits . of the 31 cases , 3 received palliative radiotherapy to the affected bones ( cases 2 , 27 , 69 ) and are not included in the figures . 
of the remainder , 8 were premenopausal cases , all but 2 of whom received great benefit from androgen therapy . three of these were the bedridden patients referred to above , who were able to resume their normal activities within a few weeks of being treated with testosterone , and remained in good health for over 1 year in 2 cases ( cases 3 , 7 , 37 )  . 
the fourth was a housewife of 45 with extensive spinal , costal and pelvic deposits , whose activities were drastically curtailed by pain . she also secured complete relief from pain in the month after methyltestosterone therapy was d . 
galton begun and is well 6 months later ( case 26 )  . case 4 was a nulliparous woman of 49 complaining of pain in the left hip and right shoulder , the latter exacerbated by the smallest movements of the arm , active or passive . 
no deposits were seen radiologically to account for the hip pain , but the medial end of the right clavicle was swollen and tender , expanded by a tumour measuring 4 x 6 cm . after taking methyltestosterone 50 mg . 
daily for one month , the complete range of shoulder movements was restored , the patient was without pain , and the bony swelling no longer tender to percussion , this latter finding being a characteristic result of the treatment . six months later the swelling is slightly smaller , but intrathoracic metastases have appeared and the general condition has deteriorated . of the 3 remaining premenopausal cases , 1 ( case 47 ) , a para - 2 aged 38 , attended hospital 9 months after radical mastectomy with severe anaemia and pains in back , hips and shoulders . she was intolerant to doses of methyltestosterone greater than 30 mg . , and although the dose was increased in 5 mg . 
on 3 occasions the treatment had to be abandoned each time , owing to the sudden onset of headache , nausea , fever , drowsiness and increased intensity of the pains . these symptoms subsided within a day or two on withholding the hormone . case 66 , who presented with paraplegia of 6 weeks ' duration , has been referred to above . 
the last premenopausal case ( case 10 ) , a para - l aged 45 , had had a right radical mastectomy 2 years previously for a lump in the breast present for 1 year . she attended hospital with cough , dyspnoea , pain in the chest and stiff neck . 
methyltestosterone arising in submammary lymph nodes . daily , and in 3 months ' time was walking well and without pain . in addition surprisingly she the parasternal swelling had become greatly reduced in size . showed on examination 3 - 5 cof shortening of the right femur , but no impairment of movement about the hip - joint , within the limits imposed by the rigidity of her paralysis agitans , and the x - ray revealed an unusual subcapital fracture of the neck with downward and backward displacement of the capital fragment . the second case was of a nullipara aged 51 , who had been recurrence - free for 10 years after a radical mastectomy , when she developed backache , which persisted , increased in severity , eventually confining her to bed . multiple deposits of secondary carcinoma filled most of the dorsal and lumbar vertebrae , and all the pelvic bones . within a few days of the implantation of 500 mg . 
testosterone the pain abated , walking was quickly resumed , and she is pain - free 14 months later without further treatment . the nature of the pain is therefore open to question , but the patient gave no history of previous chronic backache , the onset of the pain coincided with the finding of multiple secondary deposits in ' the appropriate bones , and relief promptly followed implantation of a very small dose of testosterone . testosterone was also given to 5 young patients in whom an artificial menoin the first and last pause had been earlier induced ( cases 5 , 30 , 50 , 62 and 70 )  . of these cases pain was relieved , but in the first it was never severe , and in the last its recurrence followed the appearance of further metastases . the difference in response by premenopausal and postmenopausal cases just mentioned was not met with in the series of 70 cases described by adair , mellors , farrow , woodard , escher and urban ( 1949 ) , or in the larger series of 285 cases analysed by the council on pharmacy and chemistry ( 1949 )  . in adair 's series 9 out of 48 cases with skeletal metastases benefited from androgen therapy , but no difference in response was noted in premenopausal and postmenopausal cases ( 12 of the 48 cases were premenopausal , compared with 8 out of 28 in the present in the council 's series of 285 cases , the ages ranging from 25 - 79 years , series )  . favourable effects were evidently independent of age . 
no explanation is offered for this discrepancy . post - treatment changes in bone . it is difficult to account for the rapid relief of skeletal pain brought about by equally striking is the speedy disappearance of tenderness to testosterone . percussion of affected bones , and the later diminution in size of bony swellings radiologically all that can be observed are the due to subperiosteal deposits . changes in position and concentration of minerals in the neighbourhood of a deposit following testosterone administration , but these changes are characthey consist in the homogeneous deposition of minerals in the bone teristic . surrounding the deposit , so that at first the deposits acquire a more definite outline , and may appear more extensive in size and number . this is a common appearance 2 months after commencing treatment , but soon gives way to a more uniform but rather structureless appearance , as the areas of decalcification in the deposits become filled in from the periphery with deposited minerals . after 6 months this process usually comes to a standstill . it is questionable whether this confers increased strength on the bones in which it recalcification d . 
the same is true of cases with bone deposits , and in the illustrations in the first two papers just cited fresh osteolytic deposits are present in bones showing extensive recalcification round earlier deposits . several instances of this phenomenon have been met with in the present series . the paradox is probably to be explained by the fact that the metabolic processes accelerated by the androgen proceed most rapidly at the beginning of treatment , and then slow down to a stationary though raised level . 
he had learned of " the custom in certain countries to remove the ovaries of the cow after calving if it is wished to keep up the supply of milk , and that if this is done the cow will go on giving milk indefinitely , " and this " pointed to one organ holding the control over the secretion of another and separate organ , and thus explained the absence of that distinct nervous control that i pointed out as characteristic of the mamina . " beatson 's first case will be described in some detail , as it illustrates very well the kind of result that may be obtained by testosterone therapy as well as by castration . the patient was a married woman of 33 years , with 2 children , in fair general health , and menstruating regularly . three years previously she had noticed a lump in the breast while suckling her first child . this increased in size after the birth of her second child 20 months later . 
when first seen there was a mass 5 x 3 inches with ulceration of the skin of the breast over an area of 1 square inch and adjacent skin nodules . radical mastectomy was performed on 25 . 
95 , but 3 months later an ulcerated recurrence in the scar was found , and a fortnight later had increased in size to 21 x 31 inches . the whole chest wall was studded with skin nodules , but no lymph nodes were felt . 
95 the " largest area of disease " measured 1i x 2 inches . eight months later regression continued . the patient lived until april , 1899 , dying 46 months after castration , with fresh recurrences in skin and spine ( thomson , 1902 )  . seconid case was that of a woman aged 40 , with massive involvement of the whole breast by carcinoma , with extensive infiltration of skin , axillary and cervical nodes as far as the chin ; the opposite cervical nodes were also involved , and she complained of pain in neck and arm . after castration the pain was relieved , and regression and softening of the primary mass and of the nodes occurred , lasting 5 months . thus in the first 2 cases in which castration was used , shrinkage of the primary growth , regression of skin deposits and of involved nodes , general improvement , and relief of pain were described . six years later the method had been used extensively , and its value sunmmed up by beatson ( morris , 1902 ) as follows : " it is not easy to say beforehand if the operation will do good ; but from the cases recently published by abbe , of new york ( thomson , 1902 ; abbe , 1901 ) , it would seem that it is not contra - indicated after the menopause th ' value of an operation may be judged by statistics and by personal impressions . both may be deceptive , though both may contain in them the germs of truth . i believe that from both standpoints the evidence is in favour of the utility of oophorectomy in certain cases , in prolonging life and lessening pain . it has never , as far as i know , done any harm , and its mortality has been nil the treatment has been placed before the profession , because i felt that d . 
galton they should judge its effects themselves and if , in their opinion , after a fair trial , it is found wanting , then i hope it will be abandoned and forgotten . " oophorectomy passed out of fashion a few years later , and was slowly superseded by " x - ray castration , " and in the last 10 years by " medical castration " with androgens also . boyd ( 1900 ) reported the results of oophorectomy in 54 cases of mammary cancer . six cases out of 46 showed remissions lasting for 2 years or more , and 17 out of 46 derived some benefit , but in most of these , recurrences occurred between 6 and 12 months later . with one exception the treatment failed in all the patients who had passed the menopause , and the maximum benefit seemed to be derived by women over 40 and still menstruating . 
two years later thomson ( 1902 ) of edinburgh collected and published the results in 80 inoperable cases castrated by several surgeons . in 40 of the cases some imnprovement took place , although this was negligible in 11 , and only temporary in 11 others ( under 12 months ' survival following oophorectomy )  . 
in 18 , however , decided improvement was recorded , " remarkable " in 4 . thirteen cases survived more than 20 months , and 4 between 40 and 46 months . of the 29 more favourable cases , 6 had ceased menstruating , and 3 were 70 years of age , and of the 20 women still menstruating , 12 were between 40 and 50 years of age . 
thomson concluded that the value of castration , though limited , had been established beyond doubt , but , like beatson ( 1896 ) , stressed the unpredictability of the results . 
jessett " had operated on 5 cases , and admlitted that the patients complained of less pain , but in no single case was there any improvenment in the local disease . 
he also noted that beneficial results occurred sometimes after the menopause . bruce clarke recorded a case in which castration was followed within 6 weeks by rapid subsidence of pain and regression of a chest wall recurrence , the patient being in good health 5 years later . in this case unfortunately the age was not stated , but the original excision of the growth was performed 2 years before castration , and a recurrence excised 1 year before ( lett , 1905 )  . in spite of the well - documented reports described above , castration for mammary cancer fell out of favour , no doubt because of the absence of precise indications , the large number of poor results and the increasing success obtained by local radiotherapy . morris ( 1902 ) stressed the difficulty of assessing the results , and compared " beatson 's treatment " with the favourable results claimed for the injection of coley 's fluid ( heat killed broth cultures of streptococci obtained from erysipelas lesions , and of bacillus prodigiosus ) in cases of spindlecelled sarcoma . 
he found that in the cancer - bearing group the incidence of complete oophorectomy before carcinoma was diagnosed was 1.5 per cent , whereas in the non - cancer group it was 15 - 4 per cent , or ten times as great . these figures implied that castration might have protected a small number of women from the subsequent development of mammary carcinoma . arguing on these lines horsley , late in 1937 , performed bilateral oophorectomy on the first of a series of patients immediately after radical mastectomy , in the hope of deferring the onset of recurrence . 
by june , 1944 , recurrences had occurred in only 4 out of the 25 patients who had been observed for between 9 months and nearly 7 years . horsley felt that the appearance of metastases had been delayed by castration ( horsley , 1944 , 1945 )  . snapper castrated 22 patients after radical mastectomy , and in addition treated them for 3 months with injections of testosterone propionate 25 mg . 
of methyltestosterone was given on the day after oophorectomy , but in view of subsequent experience with this hormone , it is probable that the immediate effect was due to the oophorectomy alone . a case of intrathoracic recurrence in a woman of 40 years , 2 years after radical mastectomy for stage ii carcinoma , in whom relief of dyspnoea was observed 4 days after castration , and during the next 2 months absorption of nialignant pleural effusion and resolution of pulmonary parenchymal deposits took place , was reported by jochweds , baranowicz and horecki ( 1948 )  . the effects of surgical castration may be summarized by saying that it confers definite benefit on a small percentage of cases of carcinoma of the breast , even in an advanced stage , as shown by relief of severe bone pain , recalcification of areas of destroyed bone , regression of primary and secondary manifestations of disease , and conspicuous improvement in general health lasting uisually for several months only , but sometimes for several years . 
the most favourable cases are those in the premenopausal group in women , though striking improvement occasionally follows castration in older women , and in the small number of cases reported in males the best results are to be expected in elderly patients . in passing it should be noted that some of the remarkable spontaneous regressions which occur , and which may resemble in all respects those already described , are probably the result of " self - castration , " both ovaries being completely destroyed by metastases . 
ahlbom 1920 , and described his ( 1930 ) attempted a statistical analysis of his cases , but as 147 out of 163 cases received treatment to other sites as well as to the pelvis , and his series included very few instances of women in the most favourable age group , he failed very few clinical data are presented in to demonstrate any positive effects . ahlbom 's paper , and in spite of his obvious anxiety to attain statistical precision , it is perhaps surprising that he could quote no examples of undoubted success similar to those reported so often in cases subjected to surgical castration . 
clarkson and barker ( 1936 ) reported a case of mammary carcinoma in a woman of 41 with axillary node involvement , and deposits in a metacarpal , a tarsal phalanx , femur and ilium , in which recalcification of destroyed areas in the skeleton the patient was in followed ovarian irradiation and general body irradiation . good health 5 years later ; the ovarian irradiation was considered to be the main cause of the good result . dresser ( 1936 ) reported the results of ovarian irradiation in 59 cases with skeletal metastases , having selected this group for special observation because of the dramatic benefit conferred on two women with generalized skeletal and other deposits after they had received small doses ( 700 r ) of x - ravs directed to in both cases the periods ceased , general the pelvis for local relief of pain . regression of deposits occurred , with skeletal recalcification , and in the second case multiple pulmonary deposits disappeared ; the patient led an active life for of dresser 's 59 cases , 30 were under another 3 years , largely symptom - free . 45 years of age , and 29 had passed the menopause . 
the discrepancy between the results of the early surgical cases , in which castration was believed to be contra - indicated in the presence of bone metastasis , and the more recent results in which skeletal metastasis is regarded as one of the most favourable indications for ovarian irradiation , is probably adequately explained by the lack of satisfactory x - rays in the early work , so that the extent and incidence of bone metastasis was less accurately known . the effects of androgens in mammary carcinoma . among the first reports of the use of testosterone for mammary carcinoma were those of ulrich ( 1939a , b ) and of loeser ( 1941 )  . the latter used intramuscular injections and also subcutaneous implants of testosterone propionate . the first 3 cases were recurrence free at the time treatment was begun , but remained so for the next 3 to 4 years , whereas all had been treated by x - rays or surgery for recurrences shortly after the original operation ( 6 months , 8 months and 2 years respectively )  . farrow and woodard ( 1942 ) felt that there was a specially close relation between susceptibility to skeletal metastasis and ovarian activity , and were thus led to use testosterone for cases with bone deposits . 
doses gilven on alternate days . the case reports published by adair and herrmann ( 1946 ) illustrate very well the kind of result which may follow the use of testosterone therapy . weight gains , general improvement , rapid relief of pain previously intractable , regression of primary growths and secondary deposits , and increased calcification of bony deposits were recorded . the following are especially noteworthy : ( 1 ) the ages of the patients . - one of the best responses occurred in a woman of 63 years , though the other successes were in women of 47 , 44 and 42 years compare also taylor , slaughter , smejkal , fowler and preston ( 1948 ) , of age . who report andillustrate regression of pulmonary deposits in a woman of 70 , and the progress report of the american council on pharmacy and chemistry ( council on pharmacy and chemistry , 1949 ) , in which the response to androgens in 285 patients ranging from 25 - 79 years was evidently independent of age . in a later paper adair ( 1947 ) reports a dramatic result in a woman of 71 who had been confined to bed for 10 months with severe pain and pathological fractures due to widespread metastases in spine , pelvis and ribs . three weeks after commencing testosterone therapy she remarked on her improved sense of well being , and 4 weeks later left her bed . 
two years and 4 months after she was still up and about and in good health , the only untoward effect being irritation from the enlarged clitoris . this case recalls some of those treated by surgical castration ( thomson , 1902 )  . ( 2 ) the speed of response . - in case 4 relief of pain was evident within1 week of the first injection , and in case 3 regression in primary and metastatic growths was noted one month later . ( 3 ) the favourable effect on skeletal metastases . - the earlier results of adair ( 1947 ) suggested that the best results of androgen therapy were to be expected in cases with metastases in bone . however , equally dramatic results have been obtained in cases with pulmonary deposits and skin deposits , but no secondaries in bone of the common osteolytic type revealed by x - rays . these results are said to be much less common than in cases with bone deposits . thus adair stated : " in only a small percentage of cases is improvement to be anticipated where carcinoma involves soft tissues such as liver , lungs , brain and local skin recurrence . it is true that in a few instances there have been striking improvements where an advanced ulcerating breast cancer with surrounding skin nodules , axillary nodes and neck nodes were involved ; where masses were metastatic to the lungs ; and in one instance where the patient was having convulsions from metastatic disease in the brain receive no benefit  . 
in weight , her periods had not recurred , and the mass in the breast none of the nodes was more than 1 cacross , and the skin was no longer palpable . deposits had undergone complete regression , being visible only as brownish impalpable mottlings . 
48 signs of masculinization had appeared , namely slight hirsuties of chin , and lips , and a deepening of the voice . she complained of slight swelling of the feet her weight was 10 st . 
ii.49 she attended hospital complaining of severe backache , and on examination the nodes felt previously were found to be hard and much enlarged , numerous deposits not exceeding 3 min diameter were present in the skin of the left breast , and the liver edge , which was thickened , firm and somewhat irregular was felt in the right iliac fossa . 
47. - admitted to royal cancer hospital . on examination she was in good general condition , but bedridden with crippling pain . there were 3 ulcerated recurrences along the left mastectomy scar , the two larger measuring 2 x 4 and 5 x 3 crespectively , with irregular thickened raised edges , fixed bases and coarsely nodular floors . 
in 9 months , had recurrence of low back pain ; the mass in the upper inner quadrant of the right breast was larger , as were the axillary nodes ; the bony parasternal swelling in the left 4th interspace had ulcerated through the skin . there were numerous hemispherical bony projections on the vault of the skull , on the frontal bone , on the right clavicle . x - rays showed many fresh osteolytic deposits in spine , ribs and pelvis , side by side with old sclerosed deposits , which were less dense than before . injections of testosterone propionate 100 mg . 
viii.44. - left radical mastectomy for stage i carcinoma of breast . postremained well until april , 1948 , when first noticed breathlessness on effort , which june , 1948 . - signs of large left pleural effusion . 
49 the only abnormality found was slightly diminished radiologically there is now only obliteration of the left expansion at the left base . costophrenic sulcus , and the parenchymal opacities are no longer visible . she was last seen on 25 . 
48 the entire area was paler in colour , the young skin over it thicker , but there was still one small discharging area . this dried up during the next two months , dressings were discarded and therapy discontinued , but on 1 . 
bartholomew 's hospital , london , e.c.1. received for publication december 22 , 1948 . in an earlier publication ( robinson , 1948 ) the results obtained in a study of 17 - ketosteroid excretion in men of different age - groups were reported . special attention was paid to quantitative dlifferences in total ketosteroid excretion during the later decades of life . 
the method involves adsorption of the 17 - ketosteroids on a column of alumina from benzene solution followed by fractional elution with benzene of increasing ethanol content . the amount of 17 - ketosteroid present in each eluate is determined by the zimmerpreliminary experiments with the method described by these mann reaction . authors showed that separation and estimation of the steroid components could be achieved , but that reproducible results depended on careful standardization of the alumina and of the conditions of elution . 
of total 17 - ketosteroid was found to be the most suitable amount , and sufficient urine from a 24 or 48 - hour collection was hydrolysed and extracted to furnish a quantity in excess of 5 mg . 
was evaporated to dryness , and two successive portions of pure benzene ( 5 ml . ) added and distilled off to remove the last traces of alcohol prior to solution in benzene for adsorption on alumina . ( 2 ) stamndardization of alumina and adsorption columns . since merck 's alumina was not available , many samples of alumina were examined in order to obtain a product with the desired activity . spence " type h " alumina was finally selected for this purpose . 
when samples of this alumina are exposed to air ( by spreading the material in thin layers and exposing it to the moist air of the laboratory ) they gradually lose their adsorptive activity . this loss of activity is accompanied by a decrease in ph of aqueous suspensions of the alumina . suspensions prepared by mixing alumina with distilled water ( 1 part a1203 with 2 parts distilled water , by weight ) show an initial ph of 105 before deactivation and a gradual decrease to ph 9 - 3 after 2 to 3 days ' deactivation . the adsorptive power of the alumina was checked at intervals during the deactivating process by employing brockman 's method of standardization , using mixtures of dyes ( brockmann and schodder , 1941 )  . 
gurr , and further purified by solution in benzene and filtration through alumina followed by crystallization from benzene ( sudan red ) or alcohol ( sudan yellow )  . for standardization , 1 ml . of this dye solution is adsorbed on a column ( 50 x 14 mm . ) prepared from a suspension of alumina ( 7 g . ) in benzene - petroleum ether ( 1 : 4 ) and eluted with 30 ml . 
the alumina is considered to have suitable adsorptive power when the yellow band reaches the bottom of the column , while the red band moves down about 10 mm . the alumina columns for adsorption of the urine extracts are contained in glass tubes of 14 minternal diameter fitted with glass taps at the lower end . above the tap a plug of cotton - wool is inserted and the alnmina filling is added above the plug . 
fractions. these fractions are numbered successively 1 - 43 . ( 4 ) assay of eluted fractions . the solvent is removed from each fractional eluate and the residue dissolved in 2 ml . 
the observed colorimetric values are corrected when necessary for interfering chromogens ( robinson , 1948 )  . ( 5 ) recording of chronatograms and identification of con8tituents . the amount of 17 - ketosteroid in each eluate is plotted as the ordinate against an abscissa corresponding to the serial number of the eluate . 
with normal urine as many as nine peaks may occur , but in no case was the number less than seven . dingemanse , huis in ' t veld and de laat ( 1946 ) adopted a system of numbering in which successive peaks on the curve were indicated by roman numerals i - vi , the lowest numeral corresponding to the peak with the lowest eluate serial numbers . this system was used in the present work to facilitate comparison with the results of these authors . in some urines , however , small quantities of a component occurred which appeared to be different from any of the 8 main fractions , and this component was allocated to a sub - group hia . 
the substance originarlly referred to as " 17 - ketosteroid hi " ( dingemanse , huis in ' t veld and de laat , 1946 ; dingemanse , huis in ' t veld and hartogh - katz , 1948a ) was later shown to be i - androsten - 6 - ol - 17 - one ( dingemanse , huis in ' t veld and hartoghkatz , 1948b ; barton and klyne , 1948 )  . the dutch workers isolated this substance from the urine of a two - year - old girl with an adrenal tumour , but found only small quantities in the urine of normal persons . in the presence of hydrochloric acid this substance gives rise to chlorodehydroisoandrrone and dehydroisoandrosterone , and such a conversion may occur durijg the preliminary hydrolysis of the urine . in the present investigation confirmation of these identities for peaks mn , rv and v was obtained ( a ) by the isolation of the constituents of peaks rv and v and the preparation of crystalline acetates and benzoates therefrom , ( b ) by the urinary 17 - ketosteroids application of a colour reaction ( patterson , 1947 ) to the constituents of peak ni , ( c ) by identification of the peaks formed by authentic specimens of dehydroi&oandrosterone , androsterone and aetiocholan - 3 - ( x ) - ol - 17 - one . in order to study the reproducibility of the method and the type of curves to be expected , known amounts of dehydroisoandrosterone , androsterone and aetiocholan - 3 - ( x ) - ol - 17 - one were chromstographed alone and in mixtures containing varying proportions of these three steroids . 
the curves obtained in these experiments revealed immediately a problem that has to be faced in determining the amount of ketosteroid represented by any one peak . this is the complication introduced by the fact that the minimum values in the depressions between peaks are not always zero . 
the question therefore arises as to how the amount of ketosteroid corresponding to such minimum values should be partitioned between two peaks on either side of the minimuthree treatments were considered : ( i ) half of the minimum value might be allotted to each of the adjacent peaks ; ( ii ) the whole of the mimmum value might be added to the preceding peak ; ( iii ) the descending curve of the preceding peak might be projected down to the abscissa axis , and the additional values thus formed added to the preceding peak while corresponding deductions are made from the succeeding peak . these three methods were tested by their application to the model chromatograms obtained with the known mixtures of the three steroids menin general it was found that the projection method ( iii ) gave a tioned above . closer approximation to the actual amounts of the individual steroids than did either method ( i ) or method ( ii )  . this method was therefore adopted as standard for computing the amounts of 17 - ketosteroids represented by different peaks . a striking example of the reproducibility of the method has recently been encountered . 
1. the curve of case 0 was obtained on an extract from a 10 - day collection of urine from a boy aged three . in cases 1 and 2 it was necessary to collect urine for 2 to 3 days in order to obtain sufficient ketosteroid for analysis . 
the percentage distribution of the total ketosteroids among the different peaks is given in table i for all the normal males investigated with the exception of case 0 , in which the peaks normally present in adult curves could not be differentiated . urines from 2 cases of benign hyperplasia of the prostate and from 6 cases of carcinoma of this gland were also examined , and the corresponding curves and percentage compositions of these extracts are shown in fig . 
1. - 17 - ketosteroid content of eluates from chromatograms of urines from normal males case 0 ( not included in table i )  . three - year - old boy . incomplete differentiation into cases 1 , 8 , 17 represent the predominant type in which androsterone exceeds aetiocases 2 and 5 are unusual types in which aetiocholanolone exceeds androsterone . case 13 is a man , aged 50 ; 17 - ketosteroid output 20 mg . 
per day ; dehydroi / androcholanolone . sterone 34 per cent of total ketosteroid . .420 urinary 17 - ktogsrerrds case 20 case 19 ly~~~~~~~~~~~l case21 case22 case 23 case 24 case 25 case 26 eluate number fio . 
peak ii , average value 11 - 7 per cent , ranges from 2 to 34 per cent of the total , peak iv averages 30 per cent , with a range from 8 - 4 to 49 per cent , and peak v has a mean it will be seen that the of 24 - 2 per cent , with a range from 11 to 38 per cent . average output of androsterone exceeds that of actiocholanolone in the ratio only in three of the 18 cases shown in table i does the output of 1 - 25 : 1 - 0 . aetiocholanolone notably exceed that of androsterone ( cases 2 , 3 and 5 ) ; in the other 15 cases the amount of the former ketosteroid is either approximately equal to or less than the amount of androsterone . luriary 17 - ketosteroimds a comparison of the data shown in table ii with those of table i shows , however , what appears to be a significant change in pattern . in the urines from the cases of prostatic disease there is a consistent preponderance of peak v over peak iv . this is illustrated in fig . 
3. - relative excretions of dehydroisoandrosterone , androsterone and aetiocholanolone in normal males and in males with prostatic disease . cases 13 - 18 , normal . cases 19 - 20 , prostatic hyperplasia . cases 21 - 26 , prostatic carcinoma . amounts of dehydroisoandrosterone and aetiocholanolone are shown relative to the same amount ( 100ug . ) of androsterone in all cases . three major peaks are shown for extracts prepared from urines of six normal males over 50 years of age ( cases 13 - 18 ) , and from the urines of two subjects with prostatic hyperplasia ( cases 19 and 20 ) , and six patients with carcinoma of the prostate ( cases 21 - 26 )  . utrines from the carcinoma cases were obtained before oestrogen therapy was begun , and those from the benign hyperplasias before operation . 
for convenience of comparison , the amounts of peaks in and v are shown relative to the same amount ( 100lg . ) of peak rv ( androsterone ) in it will be seen that the most striking feature is the relative increase in all cases . peak v in the cases suffering from prostatic disease . in the normal males over 50 years of age the amount of this peak is equal to or less than the amount of androsterone , but in the 8 cases of prostatic disease it is present in considerable excess . as mentioned earlier , the preliminary experiments with known mixtures of 17 - ketosteroids showed that peak v normally indicates the presence of aetiocholanolone , and the total amount represented by this peak is a measure of the quantity of this steroid in the original mixture . it may be provisionally assumed , therefore , that the increased size of this peak in prostatic disease indicates a a . 
goulden relative increase in the output of aetiocholan - 3 - ( x ) - ol - 17 - one , although the possibility is not excluded that the incrased peak size is caused by the inclusion of a second substance ( of at present unknown nature ) which is eluted with the aetiocholanolone . 
a decision on this question can be made only when sufficient of the material present in this peak has been accumulated to allow of its identification , and experiments are in progress with this object in view . if it is assumed that aetiocholanolone is the substance that is increased in relative amount in prostatic disease , the cause of this increase is at the moment obscure . this ketosteroid has been recognized as a transformation product of more than one other steroid . callow ( 1939 ) isolated aetiocholanolone and androsterone in approximately equ%l amounts from the urine of normal men , and also much larger , but still approximately equal , amounts from the urine of a man treated with testosterone . these two isomers appear to be end - products of testosterone metabolism , and to be formed in about equal amounts from this hormone . 
an explanation of increased testosterone metabolism does.not appear to fit the present observations , however , since they show an increase in aetiocholanolone relative to androsterone . in this connection it may be remarked that callow 's finding of approximately equal amounts of these two ketosteroids in normal male urine was based on experiments with a pooled sample of urine , and an examination of table i shows that in individual urines the ratio between the amounts of androsterone and aetiocholanolone is not constant , although the average values are approximately equal . aetiocholan - 3 - ( c ) - ol - 17 - one has also been shown to be a metabolite of dehydrosoandrosterone . 
mason and kepler ( 1945 , 1947 ) found that administration of dehydroandsterone acetate by intramuscular injection in oil to patients with very low outputs of 17 - ketosteroids ( a man with anterior pituitary deficiency and a man and a woman with addison 's disease ) led to greatly increased excretion of both aetiocholanolone and androsterone . in the two male subjects the amount of androsterone excreted exceeded that of aetiocholanolone by approximately 75 per cent , while in the female patient the aetiocholanolone was about 4 - 5 times as much as the androsterone . the observed increase in the ratio of aetiocholanolone to androsterone in the cases of prostatic disease might be produced by an increase in the absolute amount of aetiocholanolone , or by a decrease in the absolute amount of androsterone excreted , or by both simultaneously . the amounts of 3 - ( , ) - hydroxyketosteroids , androsterone and aetiocholanolone excreted in 24 - hours were calculated for the cases illustrated in fig . 
3. normal males in the later decades have been examined . such a comparison will be possible when further in a study of z - ketosteroid excretion in patients with neoplastic disease dobriner , rhoads , lieberman , hill and fieser ( 1944 ) found abnormal patterns in in the one case of prostatic cancer reported in several cancerous conditions . their paper there was a great excess of aetiocholanolone as compared with androthey found a similar relation in a case of carcinoma of the larynx and sterone . a case of carcinoma of the breast . 
the presence of the agent in mammary tumour cells after 42 transplantations in c57 black low - cancer - strain mice cbaracterised by low susceptibility to mammary cancer and lack of the agent again raised the question what part is played by the mammary tumour inducing agent im the propagation of the transplanted breast tumour cells , and whether bho cells will continue to multiply in the same way should the agent disappear from the bittner , evans and green ( 1945 ) reported the presence of the agent after cells . 10 serial passages of high - cancer - strain tumour cells in ag - ent - free but susceptible mice , and concluded that the agent is continuously produced in the transplanted tumour cells . in an attempt to ascertain whether the presence of the agent is a permanent feature of the transplanted cells , the c57 x transplantable mammary tumour was again tested for the presence of the agent after 86 serial transplant 's in c57 black low - cancer - strain mice . 
the method of preparation of the tissue for testing and the test itself were the same as those employed for the 42nd transplant of the tumour in whicb the agent was found to be present . 
 ' they succeeded in the recovery of small amounts of papilloma v ' irus from the first and second transplant of carcinoma cells in now - bom rabbits , but failed to recover the virus from tumours of the third and fourth passage . 
the same authors reported positive neutralisation and complement - fixation test - s between papiroma vi ' lrug iand sera of adult rabbits in which a carcinoma originally derived from a v ' irus papilloma was transplanted for 22 the 46th and 50th tumour transsuccessive passages . plants failed to give a positi - ve compl - e ' ment - fixation test . 
they concluded that the " virus was - no longer present in the attempts tumour , although the morphology of - the tumour , remained unaltered . to reinfect the tumours by placing them in a v ' l ' r - us s - aspension or mjecting papiltransplants of the carcinoma loma virus int - ravenously into rabbits be - ar ' m ' yielded dubious results . serological tests m mice similar to those carried out in rabbits by smith , kidd ancl rous ( 1947 ) in order to ascertain the . 
presence of a lat - ent or masked mammary tumour induc ' mg agent in cells of the , 86th transplant could not be of any help in this respect . 
no atte - mpts were made to test the nells of the 86th transplant c57 xmanimar tumou ' r by grafting the tumour tisgue into susceptible mice in view of , the previous observations by various authors . 
thus transplantation of mammary tumour tissue with the agent into susceptible mice has either failed to transfer the agent ( andervont , 1949 ) , or has induced only a low incidence of tumours ( humniel and little , 1949 )  . it has also been found to be a poor source of the a ' gent ( barnum , ball and bittner , 1947 )  . it was therefore considered an unsatisfactory method for testing the transplantreinfection of the tumour able c57 x tumour for the presence of the agent . tissue wit - h the mammary tumour agent was not carried out because of the observations of smith , kidd and rous ( 1947 ) , although it is quite posgible that it would in connection with the negative result obtained have yielded unequivocal results . with the 86th transplant , another test for the presence of the agent in cells of the so far no tumours have been observed 101st tumour transplant was carried out . in an of the test mice , but it is too early to draw a conclusion , as all the mice bittner ( 1952 , personal comare still alive , havi ' n ' g lived now for 11 months . munication ) has found the agent present in the 15th transplant of a c3h mammary tumour in agent - free cm mice , while in t - he cells , of the 7th transplant , of the same tumour it was apparently difficult to detect . it is not known whether a similar situation may arise with the c57 x transplant ' able mammary tumour , although the agent could not be detected in the 86th s ' en ' al passage by the same means which revealed its presence in the 42nd transplant . 
the agent or bittner virus , as it is described by some authors , may have been altered in some unknown way , possibly by mutation . it may have become closely associated with one of the particulate this close integration of the agent with the tumour elements of the tumour cells . cers may have produced a state in which it was unable to survive the death of the tumour cells during extraction procedures preparatory to the test . 
kreyberg. from the institutt for generell og eksperimentell patologi , universitetet i oslo . received for publication april 24 , 1952 . the importance of using pure , inbred strains in cancer research is now generally accepted . strong ( 1942 ) , emphasizing this point , gave a survey of the origin of some of the best known strains , originating from his laboratory . 
the new informal document , mouse news letter , acts as a most valuable source of information as to a great many strains used all over the world . the characteristics of the different strains as to susceptibility to development of spontaneous tumours , reaction to carcingenic agents , as well as susceptibility to transplantation and other biological responses may be very marked and very different . it is therefore of great importance to have a great number of different strains with marked characteristics , in order to supply research workers with the best material possible for their special needs . the purpose of the present paper is to describe the origin , development and some of the features of our strain " white label "  . the first animals were obtained from a commercial dealer in oslo , a young man who used to supply a number of laboratories with albino mice , and who , from time to time , added animals of different origin to his stock . 
the figure shows , however , very clearly how two lines were developing with extreme differences as to the formation of spontaneous mammary carcinomas . " line 27 " produced a high , and an increasingly higher percentage of mammary tumours . 
from f2 and up to and including f14 , when this line became extinct , mammary tumours occurred in every generation , and from fg until f14 16 out of 18 females developed such tumours . from the sister of no . 
2 indicated by an arrow . in the 11 generations ( f5 - f15 ) in which there were 102 females , not a single instance of mammary carcinoma was observed . such was the situation in 1935 . in the following years the brother - to - sister matings were continued and many sidelines died out , or were not continued , but " line 426 " was kept until the war interrupted the experiment at f37 . in table i is given a complete record of the females of all the generations descending from pair ( 2 + 3 ) up to f37 . in the first column is indicated the generation . 
in the second column is given the total number of females in each generation . in the third column is given the number of females belonging to " line 426 " ; from f15 all the females belong to this line , as this is the only line surviving . in the fourth column is given the total number of mammary carcinomas occurring in each generation . finally the 24 columns show the number of animals dying in each month after birth , as well as the occurrence of mammary carcinomas , the figures in brackets . at the bottom of the table the sum total is recorded , as well as the reversed figures , indicating the number of females alive at the end of each month . table i gives a clear picture of the great number of mammary tumours occurring in the first heterozygous generations . 
from f12 it is only " line 27 " which still contributes to the presentation of mammary tumours , and from f14 when this line becomes extinct , only one single mammary tumour occurs ( that is in " line 426 " )  . 
an exception , indicated by a cross , will be mentioned later . this means that " line 426 " , during 34 generations , comprising 1595 females , presents one single case of mammary carcinoma . 
bonser , university of leeds , wrote me in august , 1949 , regarding this question as follows : " i still keep ' white label ' mice in this laboratory . 
by suckling ' white label ' babies on riii mothers i showed that they were susceptible to the riii milk factor , which i regard as a potent factor . never published these experiments , and they were only done on small numbers of mice , but they point strongly to the fact that ' white label ' mice in leeds have the milk factor , and are susceptible to it . " bonser wrote me a further letter in february , 1951 , stating that " mammary cancers still occur in our line and we regard the incidence as being about 30 per cent in mice which live to be 12 months of age or more , but i have not any recent accurate estimate of the mammary cancer incidence , because we have been very short of accommodation lately . addition : " now , in 1952 , we find that 7 of 33 females over 10 months of age have developed mammary cancer , usually rather late in life . but we make no attempt to assess the mammary cancer incidence accurately as we usually dispose of breeding females when they reach the age of one year . " i also had the following information , dated january , 1951 , from w . 
s. bullough : " my experienoe with these mice falls into two periods . first when i was working in the zoology department in leeds and when i obtained the mice from bonser . then mammary carcinoma was common in females of from 11 months of age onwards . i have no figures now , but my recollection is that it developed in not less than 40 per cent of the older females . also there was the odd occurrence in a male , though this was of course rare . " further : " this colony was allowed to die out in 1944 , but in 1946 i obtained two females and a male from bonser and started a new colony in the university of sheffield . since then a colony of some 200 - 300 mice has been maintained . . 
kreyberg further , in 1952 : " the final chapter in the story starts in 1949 when , in the autumn , i took the milk factor out by fostering one family with a strong 's cba female . 
my present colony is entirely derived from this one family , and now mammary cancer is exceedingly rare . so my present stock of your mice indeed i cannot remember when i last had a case . is without the milk factor , and i suppose it is no use for your own present purposes . " i regret very much that i cannot definitely state so , but i have strong reasons to believe that the animals sent to leeds in may , 1934 , were descendants from the pair ( 4 + 5 ) ofmy " white label "  . 
at present one " white label " strain ( leeds ) carries the milk factor and produces about 30 per cent of mammary carcinomas in females reaching the age of 12 months or more . this strain has the characteristics of the branch ( 4 + 5 ) of the original material . 
another " white label " strain ( oslo ) is devoid of the milk factor and produces practically no mammary carcinomas spontaneously . this strain is probably one of the lowest mammary for future references it is therefore of great imcancer strains in existence . therefore portance to distinguish between these two " white label " strains ; the leeds strain will be designated wll and the oslo strain wlo . 
the two strains most probably have only a heterozygous founding mother in common . this experiment has been carried out for more than twenty years . it would not have been possible without the assistance of my collaborator miss hj . 
aasland , who continued the experiment during the first years of the war , until illness and an untimely death ended her devoted and loyal partnership . during the remaining years of the war the strain was maintained at a reduced scale through the initiative and faithful service of e . 
koller. from the chester beatty research institute , the royal cancer hospital , london . given at the symposium on the genetics of cancer , london , june 24 and 25 , 1948 . xeroderma pigmentosum ( hence referred to as x.p. ) , is marked by rougheniing , dryness , pigmentation and ulceration of the skthe condition is attributed to hypersensitivity of the skin , particularly that of the basal cell layer , to sunlight . the disease was first described adequately by kaposi in 1874 ( hebra and kaposi , 1874 )  . 
occurs in the first year of life , often within a few , months after birth . first , erythema develops on the skin , on those regions which are exposed to light , followed by freckling and frequently by telangiectasis . from these regions epithelioma or basal - cell carcinoma develops . 
the malignant change may take place not only in the skin , but also in the cornea and conjunctiva the genetical and chromosomal basis of x.p. the hereditary basis of x.p. 
was first investigated by siemens and kohn ( 1925 )  . they analysed 76 families , and cockayne ( 1933 ) brought this more up - to - date with a further 43 family histories . in both groups of data the incidence of consanguineous marriages was very high : 24 out of 76 and 16 out of 43 respectively , were first - cousin marriages . 
is one of those which is borne in that particular region of the sex chromosome , common to x and y . it may not be superfluous to describe the cytological mechanism which underlies incomplete sex - linkage . the genetical basis of incomplete sex - linkage is clear , and is already described 150 p . 
they found thait during the first meiotic d ' ivi ' lsion of the male rat , one or two chiasmata are forined between the x and y chromosomes , and that the shape of the sex bivalent is determined by the position of chiasmata in relation to the centromere and differential segment . 
the structure of the bivalent is determined by the position of the chiasmata ; the frequencies of the various bivalents are shown by the number of percentages beside the figures . similar chromosome behaviour wak ; observed in man . 
the fact that the heteromorphic pair present in the chromosome set of the human male , forms a very easily observable unequal or asymmetrical bivalent during meiosis , is - well known from the - works of painter ( 1923 ) , evans and swezy ( 1929 )  . 
when a ' man inherits such incompletely sex - linked gene from his mother , it will be in his x chromosome , but occasionally it may cross - o ' ver to the y consequently the man will transmit the gene to most of his daughters and to a similarly , if he inherits it from his . 
father , he , will transmit it few of his sons . to most of his sons and to a few of his daughters . human genetics is greatly indebted to j . 
he analysed 82 family histories collected from various sources , and by using ingemoug statistical methods drew the conclusion thatthe incomplete sex - linkage of the g ' ene x.p. 
he made the suggestion tha ' t one of the 82 families on which analysis was based should be excluded on intemal evidence , which would further loosen linkage , and in this case the true position of x.p. 
the data obtained from these cases has given further information on the hereditary basis of this morbid condition , and has shown the 'significance of the pathological symptoms , including the degree of manifestation and the time of onset of the disease . 
of one year the whole skin of the left 'side of her face came up in red blisters after being exposed to the sun . she was treated with calomel lotion . 
the condition it was treated by " caustic pencl ' l , " and six months later , was diagnosed as x.p. in 1943 another lesion developed on the right when it recurred , by radium . cheek , which was similarly treated by radiuthe patient 's skin of the exposed both the pathological skin conparts ( face , hands , legs ) shows some scarring . ditions and the histological analysis of the lesions indicated x.p. 
the parents are unrelated ; father is of a dark complexion , mother has some slight freckling search through family histories on the cheek , which is of the common type . of near relatives and sibs did not reveal any similar conditions . 
at that time some of the freckles developed into small ulcers , which healed slowly . she found out , by experience , that strong sunlight accentuates the lesions , and since the age of 40 she has taken care not to expose herself to direct sunli.ght. 
the matemal and patemal grandfathers were brothers.. the eldest brother , frank , hischildren and grandchildren are all normal . isabel , the patient concemed , was the second child , but before her birth one ' miscarriage took place . 
her sister , dorothy , is one year younger ; she is afflicted with the dorothy shows freckling on the face disease in the same mild manner as isabel . and forearm , which is somewhat less severe than that of - her elder sister '  . a younger brother , thomas , was born after isabel . 
the high number of miscarriages and the death of the sixth child , a girl , on the day following birth , may , not unlikely , find its sidney died at the age of 25 explanation in the consanguineous marr ' lage . up to his death he appeared to be normal . there are two unusual features in the history of the hfamily which require explanation . 
in the hfamily does not conform closely to expectations ; the number of cross - overs is higher than could be expected . in order to explain the exceptional pathological features and the unexpected segregation of x.p. 
in the sexe - s in this particular family , the followina possibilities may be considered : ( a ) this condition - may be due to an independent autosomal gene , or ( b ) the gene x.p. 
the increased distance of the gene from the differential segment would loosen linkage with sex , and its altered position may also lead to a lower degree of malignancy . the data at our disposal from this one family , however , is too meagre to draw definite conclusions , whether we are dealing here with a distinct autosomal gene or with position effect . 
bather. from the poultry research centre , edinburgh , 9 . received for publication november 18 , 1953 . considerable interest has been centred lately on the use of ascites tumours in rats and mice for biological research . 
such tumours include , in mice , the ebrlich and krebs - 2 carcinomata ( loewenthal and jahn , 1932 ; klein and klein , 1951 ) and s - 37 and malignant lymphoma ( goldie and felix , 1951 ) and , in rats , the yoshida sarcoma , the mtk sarcomata i and 11 ( tanaka and kan ' o , 1951 ) and the hirosaki sarcoma ( makino and kano ' , 1953 )  . 
the growth of these tumours is characterized by the formation of large numbers of discrete tumour cers , living in a compatible medium ( peritoneal fluid ) which lend themselves admirably to various biochemical studies . during the course ofinvestigations into the nature of the virus of rous sarcoma , an ascitic variant of the tumour was estabhshed early in 1953 in order to see if infective virus could be obtained in a more pure forthe fact that rous no . 
of the suspension was no evidence of necrosis . inoculated into the abdominal cavity of each of 2 6 - week old chickens from a known rous susceptible strain maintained at this centre . 
most of the negative results can be attributed to failure of the inoculum to enter the abdomen and , usually , subcutaneous tumours grew at the site of entry of the needle . the rapid growth of the rous tissue and its invasion of the intemal organs has been observed in all subsequent passages . 
smar nodules of tumour tissue attach themselves to the surface of the organs and then proceed to infiltrate the membrane and musculature , often penetrating to the interior of the organ itsell of 30 birds from the ist to the 12th passages autopsied 9 - 19 days after the inoculation of the tumour , the following tissues showed invasion microscopically and grossly : gizzard ( 30 ) , pancreas ( 27 ) , duodenum ( 27 ) , spleen ( 25 ) , ovary ( 17 out of 20 ) , testis ( 5 out of 10 ) , liver ( 12 ) , heart ( 10 ) , adrenal ( 10 ) , kidney ( 7 ) , and lungs in the cases of the ovary and pancreas , invasion has frequently pr ' ogressed ( 1 )  . to such an extent that only a few fragments of normal tissue remain , almost the whole organ having been replaced by tumour tissue . 
pale yellow , viscous fluid present in a 6 - week - old bird after 10 - 1 2 days ' incubation , and , on occasion , as much as 60 ml . 
the cells account for approximately 10 per cent of the fluid volume . counts of the cells made from stained smears ( giemsa ) at variou 's times during the passaging of the tumour show that of over 2000 cells counted approximately 50 per cent are round tumour cehs , and the remainder leukocytes , lymphocytes and connective tissue cehs . this proportion has remained unchanged throughout the 18 passages so far maintained . 
on various occasions , virus or whole cells from ascitic fluid have been inoculated into the muscles of birds and , again , there is an immediate return to the normal histological picture of a solid rous no . 
1 shows the typical appearance of the fluid using iron haematoxylin stathe cytoplasmic and nuclear changes in rous sarcoma cells cultivated in vitro have been described ( tenenbaum and doljanski , 1941 ; doljanski and tenenbaum , 1943 ) and many of the descriptions apply to the appearance of the cells grown intraperitoneahy . 
the blue staining granule and thread network in the peripheral cytoplasm ( giemsa stain ) and the dense central zone can be seen , as well as the large cytoplasmic vacuoles and clubshaped pseudopodia . 
bather scattered at random and sometimes arranged round the periphery of the nucleus . the gathering of this granular material into the centre of the nucleus , leaving a clear zone between it and the nuclear membrane as described by tenenbaum and doljanski ( 1941 ) , is only rarely seen in these preparations . large round or rod - shaped , densely staining nucleoh are common and irregularly shaped ones are frequently found . multinucleate cells are present and many contain fragmented nuclei with as many as 15 - 20 fragments . measurement of the frequency of multinucleate cells is given in a later section . ascitic fluid examined microscopically by dark field illumination on the warm stage provides an excellent opportunity to observe the living cells for long periods of time . 
the lace - edge appearance of the .cells is typical of fresh preparations and the cell membrane tends to become more regular after a few hours . in the peripheral area of the cytoplasm , and in the pseudopodia , a very fine " mist " of minute particles can sometimes be seen , although this does not occur in every cell . 
the club - shaped pseudopodia described by doljanski and te ' nenbaum ( 1943 ) are a common feature and sometimes become detached , forming free spherical " bubbles " of protoplasm , usuary shimmering with the rapid brownian movement of very fine particles . similar vesicles are seen , especially in the clumps of aggregated cells in which either particles or an interwoven mass of fine filaments appears . 
they have been show - n to be present not only in pathological blood conditions , b ' ut also in normal blood after it has been kept for several hours under sterile conditions . in the rous ascitic tumour and in erythroleukaemic blood ( campbell , 1952 ) filamentous structures are present i fresh preparations and may be associated with the high rate of c ' ell degeneration . the possibility that they may represent a method of virus release from infected cells must be borne in mind . similar structures have been observed associated with cells infected with influenza virus by hoyle ( 1950 ) and wyckoff ( 1951 )  . a peculiar filamentous structure encountered , only occasionauy , is represented by the spear - shaped process shown in fig . 
the outer part of the thread becomes an external , free floating filament and the inner part withdraws to the cell , forming a small protrusion on the cell membrane . 
this type of structure usually occurs in cells having a poorly defined nucleas under conditions of dark - ground inumination . the fluid surrounding the cers also contains numerous tiny rapidly moving particles which scatter light . multinucleate cells show up very well and , occaascitic rous no . 
the samples were taken between the 10th and only cers containing morphologically 14th days after inoculation of the tumour . such cells arise because normal nuclei are included in the multinucleate forms . of failure of cell cleavage after nuclear division . 
the incidences of karyorrhexis and mitosis after 10 - 1 4 days ' tumour growth have not altered to any significant extent . number of nuclei per cell . karyorrhexis mitosis infective viru8 in ascite8 and 8olid rou8 8arcomata . the amounts of infective virus contained in either the cytoplasm of the washed cells or in the cell - free fluid have been determined by means of the day - old chick titration method first described by carr and harris ( 1951 )  . 
bather table ii shows the results for the first i 0 passages , along with two sets of figures obtained for solid rous sarcoma tissue and cell - free exudate . 
the virus titres of fluid and cell contents follow each other fairly closely . in 4 of the 7 pairs of results , however , the intracellular virus shows a higher titre by one logio dose . in this respect the ascitic tumour behaves in the same way as the sohd sarcoma and its exudate . edimation of total 4cpurified " viru8 material in cell8 and cell - free fluid of the rou8 in view of the differences encountered in the infectivity titres of the fluid and cehs , estimates have been made of the amounts of virus material obtainable from both sources . 
the " purified " virus was isolated by the fractional centrifugation method of carr and harris ( 1951 )  . ascites fluid was first treated with hyaluronidase to reduce the viscosity . 
the cells were then centrifuged off , washed in 0 - 85 per cent saline , lysed in distilled water and the cell debris discarded . both the cell extract and the fluid were then deposited in the servall s.s. 
the resulting pellets were treated with trypsin in m / 5 phosphate solution , clarified and deposited agathe amounts of virus material obtained in this way were estimated by the biuret method described in a previous pubhcation ( bather , 1953 )  . virus content is expressed as ml . 
dry weight per unit of tissue or fluid . during the washing of the cers , a cell count was done in a haemacytometer . table iii summarizes the results and includes an estimate of the virus per cell in solid rous sarcoma tissue . 
the figure is based on the average virus yields , as reported elsewhere ( bather , 1953 ) and an approximate cer count using the method of afizen and petermann ( 1952 )  . this method utifizes controlled homogenization in sucrose and acetic acid solutions containhag calcium chloride , ( 0 - 0023 m cac12 is used here )  . after suitable dilution , a count can be made of the whole cells and nuclei in a haemacytometer . 
an average figure for rous sarcoma , based on five different tumours was found to be 32 - 2 x 107 cells per g . with a standard deviation of + 2 - 8 . 
bather peritoneal fluid containing suspended round cehs . it seems probable that this type of conversion does not depend on the selection of round cells since when the tumour invades the abdominal organs , the secondary growth reverts to the normal spindle - cell form immediately . moreover , virus or whole fluids , from the ascites tumour , when inoculated intramuscularly , produces a typical solid rous doljanski and tenenbaum ( 1941 ) have described the reversible transsarcoma . formation of the one type olf rous cell to the other in tissue culture . 
thus the rous sarcoma , in chickens , closely resembles s37 sarcoma and t2146 tumour in mice , both of which have been shown to form ascites tumours by reversible adaptation ( lasnitzki , 1953 )  . results from the first ten passages of the ascites tumour indicate that the cell - free ascitic fluid contains infective virus to an extent , roughly , of that contained in a 10 per cent extract of the cehs . similar observations have been made on the liquid exudate from sohd rous sarcomata . there are various ways in the first place , of course , in which the virus may be released from the cells . virus might be released upon necrosis and disintegration of the cell . secondly , there is the possibihty that the filaments , which are abundant even in fresh preparations of rous ascites fluid , may represent a method of eiectinlr virus particles . 
of the sohd tumour growing in birds of the same age . in terms of dry weight of virus material , this infective titre was given by 0 - 30 m.g " purified " virus for the ascites tumour and 0 - 46 mg . 
the ascites produced is made up of cells ( 10 per cent ) and a pale yellow viscous fluid ( 90 per cent ) both of which contain infective virus . 
the cells are mostly dispersed singly but some are aggregated into small clumps . approximately 50 per cent of them are round tumour cells , the remainder lymphocytes , leucocytes and connective tissue cehs . 
after 10 - 14 days ' growth , 10 - 5 per cent of the tumour cells are multinucleate ( with 2 - 4 nuclei ) 1 - 8 per cent contain pyknotic , fragmentary nuclei ( karyorrhexis ) and 1 - 1 per cent are undergoing mitosis . 
no improvement in the yields of infective virus , as detectable by the day - old chick titration method , has been obtained from either washed cehs or cell - free fluid of the rous ascites tumour when compared with the sohd rous no . 
86 x 10 9 mg fluid exhibits the same , or more often , all expenses in connection with this work were borne by the british empire cancer campaign . i should fike to thank j . 
they reported an increase of urinary cholesterol in 19 out of 92 cancer patients . later , bruger and ehrlich ( 1943 ) , using a similar method of estimation , found a higher incidence of hypercholeshowever , the teroluria , 11 of their 32 cases giving results above normal limits . actual concentration of cholesterol reported by bruger and ehrlich in normal urine was approximately twice as great as that found by sobotka , bloch and rosenbloom . it appeared , therefore , that the methods used in these two studies must have been significantly different , and we felt that further work on the technique of estimation was needed . while admitting the possible non - specificity of the liebermann - burchard reaction , we felt that this type of method was the only one likely to find immediate clinical application , and it does appear to have given results consistent with the original work of bloch and sobotka . 
an attempt was made to correct for this error by the introduction of a blank reading , which was obtained by adding to half the urinary extract in 5 ml . 
the general procedure was to filter the urine slowly through a 3 - inch wide column of adsorbent from 1 to 3 inches in length , and after washing the column with water , to elute with 40 ml . 
maclagan treated with the liebermann - burchard reagent as described below under aluminthe final chloroform solution was invariably colourless , ium tungstate method . and gave negligible blank values when treated with 09 ml . 
the large tarry residue after alcohol - ether extraction was difficult to extract with petroleum ether and recovery of added cholesterol was poor , ranging from 30 to 70 per cent in various experiments . in this method a gross excess of sulphuric acid is used which is difficult to wash out of the precipitate and results in charring . 
for 20 minutes , and the green colour compared in the photoelectric absorptiometer ( king - gallenkamp ) with the ilford spectrum red filter ( maximum transmission 660mi . ) a preliminary calibration curve showed that beer 's law was well obeyed up to optical densities of 0 - 5 . 
proteose. the results given above under adsorption methods indicate a definite association between urinary cholesterol and protein when present , and suggest a possible association between cholesterol and proteose in normal urine . further evidence of the latter association was sought by estimating the nitrogen content of the aluminium tungstate precipitate obtained ( a ) after washing the precipitate with water as above , and ( b ) after dialysis of the original urine before precipitation . it was assumed that any nitrogen remaining in the precipitate after these procedures was attributable to urinary proteose . ( a ) washing of aluminium tungstate precipitate . - the relationship between cholesterol and proteose was investigated by serial washing of the aluminium in this experiment four identical samples of 100 ml . 
some support for this assumption was obtained by treating the washed precipitate with biuret reagent ( hiller , 1927 ) and also with the phenol reagent of folin and ciocalteu ( 1927 ) as follows : the precipitate from 5 ml . 
of normal urine with added cholesterol ( 0 - 5 mg . ) were duplicate dialysed in a collodion sac against running tap - water for 16 hours . samples were similarly dialysed for 40 and 64 hours . 
of the dialysed urine were precipitated with aluminium tungstate , and the nitrogen content of the precipitate determined by the kjeldahl method as above , without further washing . from the result of this experiment , as shown in table x , it can be seen that the m . 
the precipitated protein was removed by centrifugation , washed once with 0 - 5 per cent acetic acid and extracted three times with boiling acetone , the acetone extract evaporated to dryness and the residue extracted with petroleum ether for cholesterol determination as above . 
the experiments reported give some support to the hypothesis that the urinary cholesterol is normally associated with a urinary proteose fraction , and also with heat - coagulable protein if present . removal of heat coagulable protein does not , however , carry down the whole of the native cholesterol . urine is not a homogeneous substance , and the varying distribution of cholesterol between the deposit and supernatant urine which we have demonstrated is obviously relevant to clinical studies . this factor has been recognized previously , e.g. 
bruger in 1935 reported up to 40 per cent of the cholesterol in the urinary deposit in cases of nephritis . however , most authors have not recognized its importance , and have included the deposit even though pathological in certain cases ( sobotka , bloch and rosenbloom , 1940 ; bruger and ehrlich , 1943 )  . 
much or all of the urinary cholesterol may at times be found in the urinary deposit , or associated with heat - coagulable protein when present . this work was aided by a grant from the british empire cancer campaign to westminster hospital . 
pyrene per animal and the same procedure followed . 1 : 2 : 5 : 6 - dibenzanthracene . for the determination of lipid and nucleic acid phosphorus the animal was weighed and then killed by dislocation of the neck . the liver was removed immediately , weighed , and a sample of about 500 mg . 
from each of two lobes was analysed for lipid , " pentosenucleic acid " ( p.n.a.p. ) and " desoxypentosenucleic acid " ( d.n.a.p. ) phosphorus by a modification of the method of schmidt and thannhauser ( 1945 )  . in this modification the whole of the separation is carried out in a single centrifuge tube and transfer of material is thereby avoided . the tubes have a ground neck to which may be fitted a condenser for refiuxing the solvent in the lipid extraction stage . the dry weight of the liver was determined by drying a weighed sample of the organ in an air oven at 110 c . 
has occurred , and the decrease is due solely to an increase in the concentration of d.n.a.p. there was no significant difference in the liver weight : body weight ratio between the groups of animals on 5 per cent and 20 per cent protein diets . influence of 1 : 2 : 5 : 6 - dibenzanthracene . administration diet on either l : 2 : 5 : 6 - dibenzanthracene produced a marked increase in the p.n.a.p. 
1 , which shows the growth curves of six animals , three of which were killed before any very marked growth inhibitory effect of the carcinogen was observed , and three after considerable loss in weight had the p.n.a.p. 
concentration , and a decrease in p.n.a.p. concentration was also found in the low protein group . administration of the non - carcinogenic hydrocarbon pyrene to rats maintained on the 5 per cent protein diet produced no significant change in the nucleic acid balance . the increase which was found in the liver weight : body weight ratio in the carcinogen - treated rats did not appear to be a result of any increase in the water or neutral fat content of the livers . kennaway , kennaway and warren ( 1944 ) have observed a similarly - increased ratio in mice treated with carcinogenic hydrocarbons . non - carcinogenic substances gave only irregular results . 
no explanation of this increase in liver weight was advanced . the possibility that the increase is related to a differential inhibitory action of the carcinogen on the growth of the animal body as a whole compared with the growth of the liver is being investigated . in considering the possible relationship of the decrease in the d.n.a.p ; concentration in the livers of the carcinogen - treated rats to growth inhibition and carcinogenesis , it is of interest that gopal - ayengar ( 1947 ) has shown that in epidermal carcinogenesis , induced experimentally in mice by application of 20 - methylcholanthrene , the initial effect of the compound was to produce a decrease in the desoxyribosenucleic content of chromosome threads isolated from the epidermal cells . the low desoxyribosenucleic acid content persisted during.the precancerous ( hyperplastic ) stage and was then followed by an increase when malignancy ensued . stowell ( 1945 ) found that x - irradiation of transplanted mouse mammary carcinoma brought about an initial decrease in cellular desoxyribosenucleic acid content , and mitchell ( 1942 ) found a small increase in cytoplasmic pentose nucleotides in biopsied specimens of x - irradiated human tumours . 
w.3. received for publication march 1 , 1952 . some of the effects of ionising radiations are associated with formation of such effects are often reduced free hydroxyl radicals and possibly of peroxides . for these under anaerobic conditions and increased in the presence of oxygen . reasons it appeared possible that conditions which would increase the concentration of hydrogen peroxide in tissues might augment the biological effects of of the possible ways of increasing radiosensitivity by such a radiation . mechanism , two have been investigated . the first depends upon increasing the concentration of substrates , the metabolism of which is known to produce hydrogen peroxide . such substrates are those oxidised by flavine enzymes , including aldehydes , xanthine , hypoxanthine this method is being investigated by administration of and d - amino acids . suitable substrates and riboflavin , the latter in attempts to increase the concentration of flavin enzymes of the tumours . a second method of increasing hydrogen peroxide concentration of tissues depends upon the inhibition of the enzymes involved in hydrogen peroxide thus by inhibition of catalase and peroxidase of tissues this destrucdestruction . tion and utilisation of hydrogen peroxide might be prevented and its concentration in cells should increase . this was attempted without success in the present paper . many of the known catalase inhibitors including sodium azide and cyanide are also respiratory poisons . 
x 77 , 500 so , where ko = the reaction constant at zero time expressed in logarithms to base 10 and so = the h202 concentration at zero time . 
kat.f. qo21 - 29 x 10 - ' mxh20 activity of a tissue to the other metabolic processes expressed in q values . the tables of this catalase activity is expressed as qo21 29 x lothis expression makes it easier to relate the catalase the catalase activity is determined at 0 c . 
the testis probably oxidised primary carbohydrate . recent work has shown that ionising radiations ( taylor , greenstein and radiomimetic hollaender , smith , 1950 ) induce depolymerization of deoxyribonucleic acid . 
some of the biological effects of radiation may be due to such an action occurring in in the case of ionising radiations the effect is probably due to fr~e the cell . hydroxyl radicals which are produced in irradiated water . if increase in hydrogen peroxide concentration should increase sensitivity to x - rays , then addition of hydrogen peroxide to a solution of deoxyribonucleic acid should augment the depolymerising action of x - rays . such an effect has indeed been found by conway and butler ( 1952 )  . a large number of substances have been reported as catalase poisons , and some of these are listed in table i in order of potency . in the present work the activity of catalase poisons is compared with their toxicity to mice and an attempt made to measure the inhibition in vivo . 
the toxicities are also listed for some of the compounds , and the ratio of the concentrations causing inhibition of the enzyme to the toxicity expressed as the ld 50 ( calculated on a molar basis ) is given in table i . 
gallico the poisoning of catalase in vivo is difficult to estimate owing to the reversibility of the poisoning . blaschko ( 1935a ) showed that of a number of catalase poisons only potassium chlorate produced irreversible inhibition . 
the pretreatment with the catalase poisons tried did not modify the effect of radiation on the tumour . reduction of sensitivity of mice treated with sodium azide and hydroxylamine to x - radiation . groups of each stock of 10 mice were exposed to 700 r irradiation from an x - ray tube operated at 220 kvp . 
the result with azide is similar to that obtained by bacq ( 1951 ) , but the result with hydroxylamine appears to be a new finding in general agreement with bacq 's work . a4 [ 2 ' ' ' " 45 1 : contro . 2 : - inece wit 1.5per kg ; nan3 15m.bfoeradato. 
gallico the search for a poison more specific for catalase than is azide was also unsuccessful . sodium azide and hydroxylamine are much the most specific catalase poisons of the compounds examined . p - hydroxyphenylazide had only 1 / 50 of the activity of sodium azide ( calculated on a molar basis )  . this difference is exactiy the same as that found between hydroxylamine and phenylhydroxylamine . on the other hand , phenylhydrazine appears to be a more potent inhibitor than hydrazine ( blaschko , 1935a )  . 
the introduction of a methyl group into the hydroxylamine molecule ( o - methyl - hydroxylamine ) reduced the activity 400 fold . the inhibition produced by diethyl - p - phenylenediamine was of the same order as that described for dimethyl - p - phenylenediamine by homer and betzel ( 1950 )  . the figures on the ratio of catalase inhibiting concentration to lethal dose show that sodium azide is the most favourable catalase poison as well as the most active . 
the median lethal dose of sodium azide should give , a concentration of azide which is one thousand times that causing 50 per cent inhibition of catalase . thus , sodium azide is probably the safest catalase poison for use in vivo in spite of its high toxicity . the increase in poisoning activity of azide with decrease in the ph is similar to that found for the effect of formate and acetate by agner and theorell ( 1946 ) , who showed that anions in general combine with catalase and inactivate it to an extent increasing with decrease in ph . these authors explained this as due to the anion displacing the hydroxyl group of the catalase and it is possible that azide acts in the same way . 
the data of agner and theorell ( 1946 ) on poisoning of catalase with formate at different ph values are analogous to those found for azide ( table iv ) as shown in table xi . 
the replacement of a hydrogen of formic acid by ch3 as in acetic acid reduces the activity 800 fold ( agner and theorell , 1946 ) , which is the same order as that produced by the introduction of an o - methyl group in hydroxylamine . thus the poisons may combine readily with catalase , possibly replacing a hydroxyl group or hydrogen peroxide because of their shape and size . in testing this hypothesis , methylamine was found to be a poison ( of the same order as hydrazine ) , but ethyl formate and formamide , which it was hoped would be as effective as undissociated formic acid , were both found to be inactive . while the poisoning with hydrazoic acid and formic acid may depend upon the entire undissociated molecule , the poisoning with hydroxylamine and hlydrazine appeared to be independent of the ph . 
now in the cases of hydrazoic acid and formic acid , ionisation would result in loss of a hydrogen atom ( and gain of a charge ) , so that the ion would have less structural resemblance to hydrogen peroxide . 
gallico ( 3 ) poisoning of catalase of liver and tumour of rats was demonstrated after injection of azide . ( 4 ) injection of small doses of azide or hydroxylamine into rats immediately before x - irradiation did not increase the radiosensitivity of tumours . injection of large doses of azide or hydroxylamine into mice before irradiation reduced the mortality from x - irradiation of the whole body . ( 5 ) catalase activity could be expressed as a qo21 - 29 x 10h202 value in p11 . 
the investigation was supported by grants to the royal cancer hospital and chester beatty research institute from the british empire cancer campaign , the jane coffin childs memorial fund for medical research , the anna fuller fund , and the national cancer institute of the national institutes of health , u.s. 
cook. from the bland sutton institute of pathology and the barnato joel laboratories , middlesex hospital , london , w.1. received for publication april 5 , 1951 . the effects of high - frequency electric fields on living tissues , micro - organisms and viruses have been extensively investigated in the last fifty years . short - wave fields were first applied to animal tumours by schereschewsky ( 1928 ) using a transplantable mouse sarcoma , and by schereschewsky and andervont ( 1928 ) working with the rous fowl sarcoma ; these workers claimed to have produced complete regression of a number of the tumours following their results , together with those of subsesubjection to electric fields in vivo . quent workers who have reported investigations into the effects of short waves on animal tumour tissue in vivo and in vitro , were reviewed by dickens , evans and weil - malherbe ( 1936 )  . 
from a consideration of previous studies and as a result of their own extensive investigations performed with tumour tissue to which short - wave fields were applied both in vitro and in vivo , dickens , evans and weil - malherbe ( 1936 , 1937 ) concluded that , when heat effects were eliminated , ultra - short waves had no action on tumours or isolated tissues ; all the findings reported were , in their view , due to the action of heat . the many studies concerned with the effects of high - frequency electric fields on bacteria , bacterial toxins , viruses and unicellular protozoa which followed the original experiments of d ' arsonval and charrin ( 1896a , 1896b ) have been reviewed by burton ( 1949 )  . in commenting on the extreme variation of the results reported , burton ( 1949 ) emphasized that it still remained to be determined whether or not any action of the short waves existed apart from heating effects in the suspending medium . a recent claim was made by nyrop ( 1946 ) to have discovered a so - called specific effect distinct from heat effects , when pulsed fields at a frequency of 20 mc / s were applied to bacterium coli cultures in a fluid medium , the virus of foot and mouth disease , and unspecified tissue cultures . this claim was been criticized on the grounds that energy absorption by ionic and dipolar processes in the medium immediately surrounding the bacterium or virus might have resulted in a local temperature rise sufficiently high to destroy it , even though the general temperature of the test specimen were kept below the lethal level ( jackson , 1946 )  . 
they were between 8 and 14 weeks old when inoculated and belonged to two of the institute 's lines , which have been described by greenwood , blyth and carr ( 1948 ) , or crosses derived from them ( intensity , breeding , breeding hen x intensity cock , intensity hen x breeding cock )  . 
both the age at which the birds were inoculated and their distribution within the various line groups were determined by the exigencies of the supply . preparation of material for irradiation . for each experiment a bird with a large actively growing sarcoma of anything from 8 to 35 days ' growth from the date of inoculation was selected as a source of tumour material . 
the speed and the duration of the run of this machine was varied with each individual tumour , but was so arranged as to give homogenization of the resulting the tissue ; disintegrated material was placed in tubes and centrifuged very lightly ( in a horizontal centrifuge accelerated to 2000 - r.p.and at once decelerated ) , so as to bring all the contained air to the surface as a sharply demarcated layer of foam , which was then removed . 
the material obtained in this way , referred to in this paper as " disintegrated tumour , " was either mounted at once for irradiation , as described below , or was used in the preparation of other material for irradiation as follows : this was usually achieved in less than 1 minute . where a preparation of disintegrated tumour largely free of cells was to be irradiated , it was made by placing 4 c.c. 
the filings and tissue debris were separated off after the period of shaking by centrifuging in a horizontal centrifuge for 5 minutes at 2000 r.p.the supernatant fluid so obtained was drawn off and centrifuged again for 15 minutes at 6000 r.p.in order to throw down at least the greater part of any remaining cells . 
the supernatant fluid resulting from this final centrifugation , designated " virus suspension " , was drawn off ready for irradiation . water flow calorimeter c02 ice specimen standin wave icatoa from magnetron jacket for freezing mixture specimen at centre of waveguide ( maximum field strength ) 1 1 + 1 specimen at side of waveguide ( field of low intensity ) fig . 
with the experimental conditions used this peak field was given by1016v / a volts cm . - 1 , where a - average power in watts . where a section of the guide was filled with carbon dioxide ice the field at the central point of the section depended as well on the length of the section filled and on the dielectric constant of the carbon dioxide ice . this latter had been found ( in a separate experiment ) to be 1.6 at 3000 mc / s , and the length of the guide filled with the carbon dioxide ice was adjusted so as to be half the wave - length of the radiation in the ice . 
under suic ' h conditions the block of carbon dioxide ice was non - reflecting and the field strength at the central point was given bythe field in a frozen specimen at the central point of the carbon diqxide ice blbck differed from that in the ice in the absence of the specimen , since the dielectric constant of the frozen material differed from that of the ice . although the dielectric constant at 3000 mc / s of disintegrated tumour or virus suspension at room temperature is high ( between 40 and 80 ) , in the frozen state it drops to the region of 2 , not far removed from the dielectric constant of the carbon dioxide ice . 
the overall exposure time in each experiment was thirty minutes , corresponding to 9 x 105 pulses of 0.6 x 106 seconds ' duration each . the total time for which the field was operative was therefore 0.54 seconds in each thirty - minute exposure . 
cook surrounding the virus could take place ( experiment 1 , irradiation without cooling ) , and under conditions where precautions had been taken to prevent such energy absorption ( experiments 2 , 3 , 4 , 5 , irradiation with cooling )  . it is considered that the method of cooling employed was such as to keep the temperature of the material exposed to irradiation below 70 c . exposure to a temperature of below 70 c . 
on the other hand , absorption at a resonant frequency increases as the such absorption does not occur in water at microwave temperature falls . frequencies , but may possibly take place in large molecules or parts of them . thus , in the irradiation of frozen material , absorption by ionic conduction and dipolar orientation was avoided . 
at the frequency of 3000 mc / s . the results in table i can be attributed to energy absorption by ionic and dipolar processes in the medium surrounding the virus causing a general temperature rise well above the lethal level . thus the results of the present experiments show that any effects of the microwave field upon the rous no . 
where such heat production was eliminated , microwaves of a frequency of 3000 mc / s were found to have no demonstrable effect on the virus of the rous fowl sarcoma as judged by changes in its tumour - producing activity . 
where heat production was allowed to take place the tum6ur - producing activity was abolished . the expenses of this investigation were borne by the british empire cancer campaign . the authors wish to express their thanks to professor j . 
calcutt. from the department of cancer research , mount vernon hospital , and the radium institute , northwood , middlesex . received for publication april 16 , 194 ' the question of the involvement of the polycyclic hydrocarbons with sulphydryl groups during the process of carcinogenesis has been under discussion for a ceitain indirect evidence exists and has been reviewed by crabtree long time . ( 1947 ) , who concludes that the available evidence does not warrant any decision as to whether the involvement is direct or not . in other directions mueller and rusch ( 194t3 ) could obtain no effect upon the activity of urease - - this being dependent upon the integrity of the - sh groups - with benzpyrene solution . 
on the other hand rondoni and bassi ( 1948 ) have found benzpyrene to inhibit the papainaise digestion of gelatin and also the cathepsins of rat sarcoma and horse liver . 
the non - carcinogenic hydrocarbons , phenanthrene , anthracene and pyrene , had no action . calcutt and newhouse ( 1948 ) also found that the photodynamic action of benzpyrene was inhibited by cysteine hydrochloride . further work ( calcutt , 1948 ) showed that certain other - sh - containing compounds were also effective as inhibitors of photodynamic action . in the attempt to find the mechanism of this action it was found that after incubation of a colloidal suspension of benzpyrene in a solution of cysteine hydrochloride the characteristic yellow - green fluorescence colour of the benzpyrene changed to preliminary attempts to isolate this blue fluorescing compound were pale blue . unsuccessful , but it was found that the mixture no longer gave a positive test this implied the inhibition of the - sh group of the with sodium nitroprusside . cysteine and formed the starting - point for further work . the inhibition of - sh groups . using colloidal benzpyrene in distilled water , tests were carried out with a in each case the technique was the same . equal aniounts series of compounds . of a solution of the sulphydryl - containing compound were placed in each of two tubes . 
with slightly alkahne nlixtures , however , a small amount of blue fluorescing compound was obtained in solution . for all the cases mentioned above incubation has to be continued for upwards of 11 hours at a temperature of 65 ' c . 
before any inhibition became apparent . at lower temperatures the action was very slow , and at room temperature - 25 ' c . - there appeared to be no effect whatever . prelim ' mary trials having indicated that caffeine solutions had fittle effect upon - sh as detected by nitroprusside , some of the previous experiments were repeated , using a solution of benzpyrene in a 3 per cent caffeine solution . 
inhibition of - sh was achieved as before , but this time inuch more rapidly - 30 nlinutes at 650 c . isolation of the reaction products . under the conditions so far investigated the yield of the pale blue fluorescing product of the reactions has only been very small . nevertheless it has been found possible to isolate products from both the alkahne and acid reaction rmxtures . 
from the alkahne reaction raixture a water soluble compound was derived . this is soluble in organic reagents , strongly adsorb6d to silica , strongly adsorbed to alurnina , fluoresces a whitish - blue colour in ultra - violet light . 
on addition of dilute acid it changes to a blue fluorescing compound which is soluble in organic reagents insoluble in water but soluble in dilute alkali . from the acid reaction mixture a com ound has been separated wliich is soluble in organic reagents , soluble in dilute alka ' li , insoluble in water , adsorbed strongly to alumina and is weakly adsorbed to silica , moving down slowly as elution is continued . thus in their physical properties these compounds show a remarkable similarity to those separated from aninials and designated by weigert and mottrani ( 1946 ) as bpx . , , bpfj aind bpf21 this latter apparently being identical with the compound characterized by berenblum , crowfoot , holiday and schoental ( 1943 ) as 8 . 
furthermore the evidence suggests a considerable parallelism , even if not identity , with the course of events after the introduction of benzpyrene into the animal body . whilst the apparent identity of the fluorescence spectra does not - imply strict cheniical identity , it does mean the very close relationship of the substances concerned . from this it is reasonable to suggest that the interaction of benzpyrene with a - sh containing compound gives rise to a substance of the bpxj type . this , bowever , is in the presence of acid successively broken to compounds of bpfj and bpf2 type . it has already been suggested by crabtree ( 1947 ) that the detoxication of benzpyrene is by way of - sh compounds . in view of the evidence presented above this must now be regarded as being most probable . crabtree ( 1940 , 1944 , 1945 ) has as a result of his work with - sh inhibitors suggested the involvement of sulphur in carcinogenesis caused by the polycyclic hydrocarbons . this is further reinforced by the recent work of lusky , braun and woodard ( 1947 ) , who inhibited the carcinogenic action - of the benzpyrene by application of b.a.l. 
 ( 2 : 3 - dimercaptopropanol )  . additionally , evidence has been put forward by weigert , calcutt and powell ( 1947 ) to the effect that detoxication occurs at the site of careinogenesis . 
korteweg. victorieplein 45 , amskrdam , neturlands . received for publication february 12 , 1951 . most curves indicating the cancer death rates per million living in the different age - groups rise continuously with advancing age . 
the numbers of lung cancer deaths in the netherlands and in england and wales , 1125 and 5941 respectively , were large enough to exclude the possibility that these exceptions to the rule might be caused by chance only . is this decrease of the cancer death rate at a relatively early age a characteristic of lung cancer or is there some other explanation ? cia l%22 r . 
2 , which is derived from table l the age curves for males in all yeargroups given show the same early decline of the lung cancer death rate , with the .2 ^ ^ ^ 35 - 44 45 - 54 55 - 64 65 - 74 75 + years fig . 
3 , constructed from the numbers following one another vertically , show us , for each age - group separately , the increasing lung cancer mortahty in the course of years . 
3 we can derive , by interpolation , for each year and each age - group separately the approximate values of the cancer death rates . this has been done for the years 1915 , 1925 and 1935 . 
4 it is necessary to take the following points into consideration : cancer only develops after a long period of preparation , which in most cases can be counted in decades . those who at the age of 80 contract cancer , do so partly because of a tendency to cancer years before - let us say at the age of 70 . 
they are recruited from the group of the younger ones , whose tendency to cancer is also known , as it is given by the length of the ordinate belonging to that age in fig . 
4. it would be wrong , therefore , to compare in this respect persons who in ' 1945 were 80 years of age with persons surely this comparison should be who , in the same year , were 70 years of age . made with those who , ten years earlier , in 1935 , belonged to that age - group . 
we must presume , therefore , that it is not only the younger people who are endangered by these irritative factors . third : let us suppose that all males were exposed to these irritative factors to about the same degree , and that in each of the last 40 years the quantity of these factors had increased considerably . 
were this so , then really we would expect to find an " age curve " for lung cancer approximately such as we did if one fact proves the reality of the great increase of lung cancer in the find . last 40 years , it is the sbape of its " age curve , " now demoilstrated as being an illusion only . artificiary , with older age , the lung cancer age curve is pushed down . at this moment , the increase of lung cancer in the younger age - groups came to a halt , even then it would - progress in the older age - gr6ups up tir the moment when theillusionary age curve changed into the real one . 
we can ' ut it in another way : at this moment the lung cancer mortalitv is lower than corresponds mr - ith the tota - i amount of the causative irritative factors . the lung cancer " ceifing is lying at a far higher level than the mortality figures would suggest . up to the age - group 55 to 64 inclusive the shape of - the illusionarv age curve is about normal ; it is only after that , that the sharp dechne begins . in 1945 , in england and wales , in males less than 65 years of age , the number of deaths from lung cancer was 33 - 8 per cent of those from " all cancers with the exclusion of lung - cancer " ( registrar - general , 1947 )  . 
6 this also means that in this case the total number of cancer deaths in males would have ' been 30 , 835 + 10422 = 41 , 257 , and not 36 , 776 , which was the number given in the statistical review for 1945 ( registrar - general , 1947 )  . following the same reasoning it appears that in 1949 , in the netherlands , for males this " ceiling " was lying at a level of 1.710 , whereas the lung cancer deaths registered numbered 1125 only . it is clear that the " ceiling " will never be reached except after a long period of rest , with stationary lung cancer death rates in the younger age - groups . 
6. - balance of lung cancer mortality in males , england dnd wales , 1945 : - _ rears ..1 - - lung cancer deaths recorded deficit lung cancer risk lung cancer age curve after correction . ..... 
pseudo lung cancer age curve . 5941 4481 10 , 422 after having studied this table we no longer wonder that in england and wales . cancer in males is again on the increase . 
and still , if in 1948 the " ceiling " of lung cancer had been reached , the total number of cancer deaths would not have been 40 , 026 , but more than 46 , 000 ! in females lung cancer is also increasing . 
an analysis of its age curve , such as i have made in this paper for lung cancer in males , shows us that , just as formales , the real age curve for females closely resembles that for the " non - hormonal cancers " ( cancers other than those of breast and uterus ) in females . the problem which lung cancer presents to us is a very serious one , not only for england and wales , but also for the netherlands and many other countries . * in the review for 1948 ( registrar - general , 1950 ) the numbers for lung cancer are only given in combination with those for cancer of the mediastinum . during the last 40 years cancer of the mediastinum has not changed much in frequency . 
the number of cockerels was too in all examinations consmall to admit a proper comparison between sexes . cerning weight , ratio of cortex : medulla and cholesterol content , however , the examined cockerel adrenals did not differ from the female ones . 
each group in the following , control as well as lymphomatosis groups , thus included one to three cockerels . elliot and tuckett ( 1906 ) found no positive correlation between adrenal weight and body weight in adult birds , and our figures support their observations . if the normal birds are equally divided according to body weight into one light and one heavy group , the former had on the contrary some higher mean adrenal weight . 
the gland weights are therefore given in absolute figures . about half of each examined group was composed of white leghorn birds , while the other half consisted of rhode island reds , barred plymouth rocks and crossbreds . 
as was stated concerning sexes , this material did not indicate still , if such differences exist , which may very well differences between breeds . be demonstrated with bigger breed groups , their effect ought to have been fairly eliminated in this investigation by similar breed distribution within the compared groups of birds . control birds as well as lymphomatosis birds were collected during the same seasons - autumn and winter . 
in order to reduce the error produced by the uneven distribution of the cortex within the gland , part of the gland periphery was thus always made part of the picture . the picture was then enlarged to an area of about 300 cm.2 the total area , with the fibrous capsule and notable vessels excluded , and the area of the cortex were determined by planimetry . 
216.833 11273 1 - 61 the difference between gland weights of lymphomatosis birds and normal birds , 63 - 23811 - 334 , is highly significant ( t = 5579 * * * for df = 58 )  . 
the tumour size in neural lymphomatosis is always considerably less than in the visceral variety . the measurements of cortical and medullary areas clearly show that the enlargement is entirely caused by increase of the cortex . 
la than from comparison is in fact decreased . of the means , as two lymphocytosis cases showed exceptionally high cholesterol values and thus brought the mean to a higher level than that characteristic of the majority of cases . 
the impression was that lymphomatosis in a more or less advanced stage is characterised by a big adrenal cortex which in most cases is relatively poorer in cholesterol than normal cortex . the histological examination now and then showed cases of lymphomatosis with considerable disappearance of sudanophil substances but hardly revealed the slight but systematic deprivation as did the chemical determination . as will be shown in a later paper , acutely running experimental lymphoid tumours in chicks ( a transplantable strain of lymphoid tumour derived from a case of spontaneous lymphomatosis ) runs with a still more marked depletion of cholesterol than spontaneous , chronic lymphomatosis as well as with adrenal enlargement . this would correspond with sokoloff 's first stage of adrenal hyperactivity in rous sarcoma . 
he concluded that the second stage was one of hypoactivity and lipoid storage . the results of our investigation indicate , however , that in avian lymphomatosis the second stage is also characterised by hyperactivity . 
a less probable alternative explanation would be that the cortex does not grow as a consequence of increased demand of steroids but because of difficulties in producing theon the contrary , the parallel with selye 's ( 1950 ) general adaptation syndrome with increased adrenal activity first during the alarm reaction and later in the stage of resistance is obvious . 
3. received for publication april 21 , 1951 . studies of the electrophoretic behaviour of sera from patients bearing malignant and non - malignant tumours have been carried out by various workers with the view to their utilization as diagnostic or prognostic tests for cancer . 
polarography has also been investigated as a possible clinical tool for diagnosis or prognosis following brdicka 's ( 1933 ) discovery that proteins give a characteristic " catalytic double wave " in ammoniacal cobalt salt solutions . applications of this method to the study of sera have shown differences between specimens obtained from normal and pathological cases , and the results of an examination of over 100 sera from cancer subjects has been published by one of us ( butler , although good correlation between the wave height and the pathological 1951 )  . condition was obtained , a number of false positive and false negative results were included , the latter particularly in cases of cancer of the buccal cavity and skin . the electrophoretic behaviour of sera has been discussed by a number of workers ( seibert , seibert , atno and campbell , 1947 ; petermann and hogness , 1948a , 1948b ) , and the most consistent changes found in pathological conditions were increases in the c1and a2 - globulin levels . however , no specific changes have been found for any particular clinical conditions ( mides , alling and morton , 1950 )  . the work of winzler and his colleagues ( winzler , devor and mehl , 1947 ; winzler , devor , mehl and smyth , 1948 ; winzler and smyth , 1948 ; mehl , humphrey and winzler , 1949 ; mehl , golden and winzler , 1949 ; weimer , mehl and winzler , 1950 ) , has shown that a mucoprotein , isolated from normal plasma , appears to be responsible for the polarographic filtrate wave . this mucoprotein migrates in the al - globulin fraction , when examined electrophoretically at ph 8 ' 4 in barbiturate buffer . it has an isoelectric point at ph 1 ' 8 and still has a negative charge at ph 4 ' 5 . 
the acidic protein fraction demonstrated in cases of gastric cancers by petermann and hogness ( 1948b ) has been shown to be a similar mucoprotemayer ( 1942 ) had previously isolated a " mucoid - like substance " from horse serum which he claimed was the polarographically active seibert et al . 
 ( 1947 ) showed that increases in a2 - globulin were paralsubstance . leled by increases in the polysaccharide content of the serum . these results suggest that the polarographic and electrophoretic patterns of cancer sera may be due to mucoproteins which migrate in the a - globulin group . 
the results were expressed either as the height of the filtrate wave , or as the protein index of muller and davis ( 1947 ) or as the blood index ( butler , 1951 )  . the results of the electrophoretic measurements were expressed in terms of the areas ( measured by means of a planimeter ) under each of the peaks of the descending differential patterns ( longsworth and macinnes , 1940 ; svensson , 1943 )  . 
the relative composition was expressed by computing the percentage contribution of the area under any one peak with respect to the area under the whole pattern , excluding the boundary anomaly . 
a statistical evaluation of the results was therefore necessary in order for this an estimation of the change in per cent to draw any valid conclusions . and absolute composition from the normal to the pathological condition was made . 
no such increase in this fraction was observed in the two lymphosarcoma cases investigated here , nor in any of the three cases investigated by petermann , karnofsky and hogness ( 1948 )  . calculation of correlation coefficients between the component peak areas and the polarographic fitrate wave heights ( f ) and blood indices ( bi ) indicate significant relationships between the ocl - globulin area and the filtrate wave heights and blood indices factors in the pathological group . it is of interest and importance that no correlation between the ocl - globulin content and the polarographic factors was found in the normal group , and similarly none of significance if the normal and pathological groups are considered as one population . this is demonstrated in the correlation diagram shown in fig . 
3. correlations between the oq - globulin content and the polarographic indices were not found when percentage composition was considered , indicating that complications by other variable factors may occur . calculations of the correlation coefficients and inspection of the correlation diagrams shown in fig . 
correlations between the serum composition and polarographic factors are discussed , and their relation to the suggested anomalous mucoprotein content in the ocl - globulin fraction is indicated . we wish to thank d . 
the investigation has been supported by grants from the british empire cancer campaign , the jane coffin childs memorial fund for medical research , the anna fuller fund and the u.s. 
pda was all rats received greens once ' %eekly . added in a concentration of 0 - 06 per cent . the diet and water were given ad libitum . twenty of 50 rats r ' eceiving this diet served as controls , whilst the remaining 30 rats each received - a daily addition of 10 ml . 
in two of the tumours ( rat ca 801 on the pda control diet and rat cl 917 receiving fresh milk ) pancreatic tissue was recognized and the origin of the tuniours . 
he describes these extraas being similar to reticulo - sarcomas or " composed of spindleordinarv tiimours shaped cells and multi - nucleated giant cells . as another deviiation some tumour cells . 
grouped in bulbous masses , show an evident tendenev to concentiric arrangement of cells which are more or less keratinized towards the centre sin - tilar to ordinarv squamous cell epithelionias . " the hver of rat cl 912 was infiltrated , %ith tumour cells . over the two - vear period in which the semi - synthetic diet was used , some 150 rats receivmg this diet without the addition of pda have been dissected . during this period carcinomas of the no pancreatic tumour was encountered . small intestine were found in three piebald and in one albino rat . in the present series of 50 rats fed pda one gimilar intestinal carcinoma was found . 
tumours not localized in the liver have not been observed previously , partly because of the preference of the compound for the liver and partly because experiments in which the liver has been protected ' were terminated too early for ttimours to develop elsewhere . whether or not the substances in the diet which defer or prevent tlle produetion of hepatic tun ' iours by feeding pda are really anticarcinogenic or only protect the liver has been questioned . it seems that in the case of milk at any rate only the liver is protected from the carcinogenic activity of pda , and that tumour development generally is not prevented . it is remarkable that the extra - hepatic tumours should arise in an intestinal gland , and this seems to siiggest that organs which might be connected with the metabolism of pda are attacked . the conception that milk has a protective action on the liver only is interesting in ' view of the general plan of these experiments , the object of which was to find , if possible , an explanation of the prevalence of hepatic tumours in the natives in it might be assumed that the effect of a careinocertain parts of africa and asia . - genic factor which produces hepatic tumours , given a certain set of dietary habits and environmental conditions can be modified when the conditions differ . 
the usefulness of ultraviolet absorption in studying solutions of methylcholanthrene in different solvents and the physico - qhemical significance of spectroscopic data suggested an approach to the standardization problem . it has become necessary for us to discontinue work on the suspensions , and there is no likelihood of our returning to the matter . 
at the end of the exposure time the paste , which had been kept moist through the 42 - day period , was extracted for 24 hours with 35 ml . 
3. - ultraviolet absorption curves from a " protected " suspension of mnethylcholanthrene after 80 days ' exposure to diffuse davlight . curves a , b , c as in fig . 
4. - ultraviolet absorption curves from an " unprotected " suspension of methylcholanthrene after 80 days ' exposure to diffuse daylight . curves a , b , c as in fig . 
4 , a , the contribution of scatter predominates . it is evident that these solutions differ markedly in the type of suspended material , and this might well result in substantial differences in biological behaviour . in order to arrive at a representative selective absorption curve the scatter component must be subtracted from the observed data . 
5. - ultraviolet absorption curves of a saline extract of ground , moist methylcholanthrene crystals which had been exposed to diffuse daylight 42 days before the extract was prepared . curves a , b , c as in fig . 
the procedure for making these approximations is as follows : from an inspection of the curve plotted from the spectrophotometer data the contribution of scatter and turbidity is estimated and a smooth curve characteristic of scatter and turbidity is drawn to approximate this contribution . 
the ratio of the difference obtained as above and the density value at the same wave - length of a known concentration of the solute dissolved in a solvent in which scatter is absent is determined . 
the ratio so found is used as a multiplying factor which is applied to the density values of the reference curve ( known solute in true solution ) at several wave - lengths . this product is subtracted from the density values at corresponding wave - lengths of the mixed scatter - absorption curve to give a series of density values which are plotted - , and ' a smooth curve is fitted to the points so determined . 
the procedure as outlined gives a family of curves , with each member progressing towards a more accurate estimation and being uniquely related to the known absorption curve of the solute , and a mixed curve which is assumed to be composed of true absorption of the solute and a scatter - turbidity component . such a series of approximations can be made when one knows from other sources the true absorption spectrum of the solute , and can use this information as a standard of reference . the above procedure has been used to determine the scatter curve presented in the figures of this paper . 
5 shows the absorption of th3 extract from the ground methylcholanthrene there is a large scatter contribution , otherwise the curve bears little crystals . resemblance to the curves from the suspensions . 
belcher. from the wards and the bland - sutton institute of pathology , the middlesex hospital , london , w.1. received for publicatiod , may 16 , 1952 . adenocarcinoma in the oesophagus may arise in three ways : ( 1 ) as an upward extension of a carcinoma of the stomach , ( 2 ) as malignant change in the mucous glands that are normally found in the oesophageal submucosa , and ( 3 ) from ectopic gastric mucosa . the first is not uncommon ; the second is rare . the third , also rare , is the type that concerns us here . in 1950 carrie reported a case of adenocarcinoma of the oesophagus that had arisen in an area of ectopic gastric mucosa . 
he reviewed the literature and likened the lesion to the unicorn in that many authors had described it , but only one had ever seen it . although he makes no reference to the possibility of a hernia being present , the description of the specimen , coupled with the fact that the tumour was in the cricothyroid region ( where ectopic gastric mucous membrane is most frequently encountered - schridde ( 1904 ) ) , makes it certain that this was indeed a genuine example of this rare lesion . bosher and taylor ( 1951 ) describe a case in which ectopic gastric mucosa was present in the oesophagus . there was reflux oesophagitis and stricture formation at the level of the aortic arch . at first sight this would appear to be a case similar to the one about to be described , but it seems more likely that it is another example of hiatus hernia with extreme secondary shortening of the oesophagus , as the barium meal strongly suggests that only a small portion of the stomach is in the abdomen . although they state that at operation the hernia was small , it is notoriously difficult to distinguish stomach from gullet on external appearances alone . 
he had no difficulty with fluids . one week prior to admission food stuck in the same place and was regurgitated . since then solid food would not pass at all . and no glands were palpable . on examination the patient looked well . there was no wasting or dehydration on 14 . 
the oesophagus was removed from a point 2 inches above the tumour to the cardia and the free end implanted into the upper part of the stomach . post - operatively the patient had some fever and pylorospasm but was eventually discharged from hospital well on 4 . 
the mucous membrane above the tumour was entirely squamous in type and was normal for the oesophagus ; that below the growth was entirely glandular apart from a few islands of squamous epithelium about 2 inches above the lower limit of excision . 
the lower half of the oesophagus contains only smooth muscle in its muscular coat . in the case described , striated muscle was present down to and including the site of the adenocarcinoma . this represents a point about 5 inches above the junction of gullet and stomach . 
the normal gullet is about 10 inches long as meastired from the termination of the pharynx at the level of the cricoid cartilage to the cardiac orifice of the stomach . it would thus appear that the striated muscle in this case had a normal distribution . however , the circular muscle - fibres at the squamo - glandular junction appear to have thickened to form a sphincter . 
this constitutes a departure from normal , and it may have prevented regurgitation of acid secreted by the gastric mucosa lining the lower half of the gullet . this may well have prevented the development of oesophagitis earlier in the patient 's life . there was definite histological evidence in this case that the adenocarcinoma had arisen in the glandular mucous membrane immediately adjacent to the junction of the squamous and glandular epithelium . the emphasis is placed on " glandular " , for this showed varied degrees of chronic inflammatory and atrophic changes which obscured the normal picture of a gastric type of mucous membrane . these changes conform to those that may be seen in stomachs that are affected by chronic gastritis , including the atrophic type . 
thus we conclude that the lower half of the gullet in this case was lined by gastric mucous membrane that was the seat of chronic inflammatory and atrophic changes , which may well have been a factor in the development of carcinoma . the rare occurrence of adenocarcinoma in the oesophagus , other than those cases that have obviously spread from a gastric origin , has been put down in the 130 13 . 
of these large towns other than london 42 are located in the northern region of england and 41 in the remainder of england and wales , these groups being designated as " north " and " south " in this paper . 
the total deaths in all county boroughs , numbering 70 , 110 , were divided by the census population at the centre of the period , 1931 , and " expected ratios " of deaths to population obtained for each of the 16 sex - age groups . 
the census population of each town at each sex - age group was then multiplied by the corresponding " expected ratio , " giving the " expected " deaths in that group in 1921 - 39 . 
the actual deaths in a particular age group , or at all ages , divided by the expected deaths at the corresponding ages , or by the summation of them in all the age groups , and multiplied by 100 , gave for each sex comparative mortality ratios ( c.m.r. ) at each age group and at all ages . 
these are only approximate indices , since no correction has been made for irregular population trends in some of the towns ; but in no instance would the error from that cause exceed 5 per cent , and for only a few towns would it exceed 2 per cent . 
the values range from 130 to 55 , those which are greater than 100 by at least twice the standard deviation being denoted by a + sign , and those less than 100 by twice the standard deviation are denoted by a sign . 
stocks as a percentage of the expected deaths , giving a series of group c.m.r. 's , indi cating how the relative excess or deficiency of mortality characterizing the towns within the group changed according to age . 
 other digestive indices : similar ratios for deaths from cancer of the liver and causes included in the international groups for diseases of the diges tive system except appendicitis and peptic ulcer . 
 in table i 23 of the towns had c.m.r. 's exceeding 100 by twice their standard deviation or more , and 30 had c.m.r. 's less than 100 by that amount . 
no more than 4 out of the 83 would be expected to give values differing from 100 by as much as this on account of random variations due to a small number of deaths , so it is evident that the deviations for 49 of the towns cannot be so accounted for . 
was from 142 to 61 , with 27 towns showing significant excess over 100 and 27 a significant deficiency , so the deviations for 50 of the towns cannot be accollnted for by random variation . 
 and the social class index on the one hand and the age index on the other have been calculated separately for the 42 northern towns and 41 other towns , and also for the 83 together . 
the values given in table iii show that cancer of stomach mortality is positively correlated with the proportion of unskilled workers in the populapion , more so in south than north ; and in england and wales as a whole the coefficients are about + 5 for each sex . 
there is , however , a strong negative correlation between the social class index and age index , mean ing that towns with larger proportions of unskilled labour tend to have smaller proportions of their adult populations with ages over 55 . 
when the age index is kept constant cancer of stomach is still positively correlated with social class index to the extent of + 3 to + 4 , and this agrees with a similar result for the metropolitan boroughs of london in 1921 - 30 ( registrar - general , 1948 )  . 
 it is now seen that there is also a small negative correlation between cancer of stomach and the proportion of people of the same sex alive at ages 55 and over , and that this does not disappear when the social class index is made constant . 
 this is important , because it shows that the great variation in cancer of stomach rates in the towns is not due to mere differences in the correctness of certification of this cause of death by doctors . 
it has been suggested that the notable differences between cancer of stomach rates recorded in britain , denmark and switzerland may be due to excessive statement of this as cause of death in parts of those countries . 
if that were so it would be expected that the over - state ment would be greater amongst old than amongst young people , since the care devoted to diagnosis and correct statement of cause of death inevitably diminishes after about age 55 . 
consequently , if the great differences in standardized death rates in different towns , and in social grades , were due to excessive statement of cancer of stomach in certain of them , those with high rates would tend to have larger proportions of old people . 
for males the contrast is greatest at ages between 35 and 55 , when the death rates in the aggregate of 20 towns forming group ( a ) were just double those in the 18 towns forming group ( d ) ; and after 55 the relative excess diminishes to only 35 per cent at ages 75 and over . 
for females the excess in group ( a ) compared with group ( d ) was around 90 per cent up to age 65 , and then diminished to 45 per cent at 75 and over . 
 a pronounced decrease in the dispersion of death rates as age advances after 45 in males and after 55 in females is to be seen in table v , where rates for cancer of the stomach in the different social classes of the population of england and wales in 1930 - 32 are compared in the same way . 
for males the excess mor tality in class v , unskilled labourers , compared with that in classes i - ii , pro fessional and higher graded occupations , was as great as 86 per cent at 35 - 45 , and diminished progressively to only 8 per cent at 70 and over . 
for the wives of men in class v and single women in unskilled occupations the excess was 80 per cent at 45 - 55 , and fell to 37 per cent at 70 and over . 
 these relations with age are the reverse of what would be expected if the large differences in causes of stomach mortality between various large towns and between social classes arose from differing accuracy of death certification . 
 it will be seen from table 13 of that publication that the dispersion of rates according to a housing density grouping were much smaller at ages over 65 than at 45 - 65 . 
if the differences between cancer rates in groups ( a ) and ( d ) of table iv were due to more complete diagnosis , or excessive diagnosis , of stomach cancer in ( a ) than ( d ) , there must have been corresponding deficiencies in causes of death other than cancer in the towns of group ( a )  . 
the total deaths in all the county boroughs during 1931 - 39 from the various groups of causes concerned , and from cancer of the stomach , at ages 45 - 65 and at 65 and over were as shown in table vii . 
10 , 568 at the age - groups 45 - 65 , 65 and over , the totals of cancer of stomach deaths were not much different from the totals of digestive diseases excluding peptic ulcer and appendicitis , and since the great bulk of any transfer of deaths into or out of the cancer group by wrong certification must pass out of or into the " other digestive " group , this means that a strong negative correlation must exist between death rates attributed to the two groups if the large variation in stomach cancer rates arises from diagnostic differences amongst the towns . 
 indeed , if this were the main cause , doubling the stomach cancer rate ( as in group ( a ) compared with group ( d ) towns ) would involve almost abolishing the rate for other digestive diseases . 
transfer might also occur between stomach cancer and peptic ulcer as a result of mis taken diagnosis , but the numbers of deaths attributed to the latter group were small in comparison with those attributed to the former , except amongst males aged 45 - 65 . 
it follows that the transfer of the whole of them to cancer , if that were possible , in certain towns would fail to account for the excess of certified cancer in those towns ; and even large negative correlations between the two causes could not account for the variation in stomach cancer amongst males over 65 or amongst females of either age group . 
 table vi shows that for males of both age groups cancer of stomach death rates had no appreciable correlation with either peptic ulcer or other digestive diseases ; for females of both age groups there was a small but statistically insig nificant negative correlation with peptic ulcer , and a small just significant positive correlation with other digestive diseases in the country as a whole . 
 since none of the factors tested above can account for the differences in tables i and ii , the remaining explanation is that some extrinsic irritant factor is concerned which differed in intensity in the various towns . 
the curious contrasts in cancer of stomach mortality in london boroughs when grouped according to their source of water supply ( registrar - general , 1947 ) , correlation coefficients were calculated between the c.m.r. 
for cancer of the stomach during 1921 - 39 and the estimated total hardness of thewater supply of the 83 towns during 1911 - 31 , as shown in tables i and ii . 
it has to be remembered that the location of certain industries has been influenced to some extent in the past by the need for a soft water supply , and that in the north of england much of the industrial population is served by soft water . 
of the 42 northern towns 23 had water supplies with less than 7 degrees , or 10 parts per 100 , 000 of total hardness ; their average social class index was about 37 per cent unskilled and partly skilled workers . 
the 9 northern towns with water supplies of 20 or more parts per 100 , 000 total hardness had an average social class ' lildex about 42 per cent ; and the correlation between hardness and social index was in the 41 towns elsewhere there was no correlation between significantly positive . 
 the coefficients of correlation in table iii between cancer of stomach mor tality and hardness were positive in the north for males and negative in the south for each sex ; and when all the towns are grouped according to an ascending scale of water hardness , it appears that the c.m.r. 
no expla nation is offered for this , but the : figures are placed on record because they might at some future time fit in with other facts ( table viii )  . 
 102 100 llo 100 100 whatever irritant may be concerned in the production of gastric cancer , the effect of greater intensity of the irritant in one environment than in another might be ( 1 ) to increase the proportion of people developing cancer at each age without changing the latent period of develop : ment ; ( 2 ) to shorten the average latent period before cancer appears without changing the proportions of people affected , or ( 3 ) to increase the proportion affected and also shorten the latent period . 
if it is a long - continued mechanical or chemical injury to cells the predominant effect to be expected from increased intensity of action would be a shortening of the latent period , without necessarily increasing the proportion of people affected , although that might also occur . 
the ratios between death rates in groups of towns and between social groups would not be expected to fall with advancing age after 50 or 60 as in tables iv and v , and this suggests that the irritant is not an infective organis assuming some form of chemical or mechanical irritant is concerned , the latent period before cancer appears will vary rather widely about a mean value in the manner characteristic of most measurable vital factors ; and if the latent periods of gastric cancers in residents of all towns could be measured they would form a distribution approximating to the " normal " type . 
 it would be possible to reproduce the observed age - incidence of deaths from gastric cancer on the supposition that the irritant begins to affect a certain pro portion , p , of people who are susceptible to it at about 15 years of age , and th.at at each year of age thereafter an additional proportion p / k become susceptible to it and begin to be affected . 
 if , for example , the mean latent period before death were 20 years , with a standard deviation of 5 and normal distribution , k being a suitable constant , the numbers of deaths at successive quinquennial age periods from 25 - 29 to 65 - 69 would give a reasonably good fit to the deaths registered in england and wales in 194 7 . 
a shortening of the latent period in the towns where the irritant was most intense and a lengthening of it in those towns where it was least intense would then result in the ratio between the respective death rates diminishing with advancing age after about 40 , as in table iv . 
the observed facts appear to be consistent with such an explanation , but a careful study of the mathematics of the assumptions outlined above is needed before definite conclusions can be reached . 
 the death rates from cancer of the stomach in the 83 county boroughs of england and wales in the period 1921 - 39 show great differences , which cannot be explained either by chance variations , or by differing accuracy of certification of the cause of death . 
standardized mortality tends to be greater in the northern towns , in towns with a low proportion of people of advanced age and in towns with a high proportion of men in unskilled and partly skilled occupations . 
 cancer of the stomach death rates of men were unrelated with those for peptic ulcer or other digestive diseases ; but amongst women there was a small negative correlation with peptic ulcer and a positive correlation with other digestive diseases . 
shown bv irregular movements of the indicator between 3 ' and these bursts last for a few seconds , and are separated bv shghtlv longer after the indicator has re - ached zero . 
grow and form large embrvomas which persist for months or indefinitely . the following series of experiments was , undertaken to gain knowledge of the beha - viour of such embrvo grafts under various conditions . 
the teratomas consisted of a me ' lange of normal tissues arranged - in an irregular manner skin , cartilage , bone , around cysts containing hair , skin and various secretions . marrow , muscle , fat , cubical and columnar epithelium , lymphoid tissue , blood vessels , glandular structures and pigment were all found . 
no necrotic patches were present , nor was there evidence of any reaction on the part of the host . in order to discover the best time to transplant such a primary teratoma for passaging mice were killed once a week , beginning a week after inoculation , the ' teratoma removed , lightly minced with scissors and a small portion ( approximately 0 - 03 - 0 - 05 c.c. ) iiioculated with a grafting needle into the right flank of in this generation increase in six cm mice . size of the graft occurred in 66 per cent of transplants done before the 4th week , in 33 per cent or less in those between the 4th and the 8th week , while after the 10th week practically no increase in size took place , the small masses either persisting in practically the same state as when implanted , or actually disappearing . several of those passaged at the first and second weeks were again divided and a portion transplanted . these only increased to between 12 x 6 mand 4 x 4 this is the 2ageneration . 
the e of 8toring findy min - ed embryo tiww in glycerol at 40 c . - the embryo tissue was passed through the craigie pressure mi ioer and then stored in ampoules at 4 ' c . 
extremely active material capable of s " rdng fivsh t clours in less than two weeks has been obtained from both chemically induced and spontaneous sarcom firozen at - 790 c . 
almost all cases of leukaemia in 2955 days . the aka line are lymphogenous or of a very ' immature type - the so - called stem - cell leukaemia . the low leukaemia line which we have used for the cross - breediilg is called b . this line is also inbred . 
among hundreds of b mice we have previously observed , only one case of lymphogenous leukaemia was found . in the fl there was found 43 per cent of deaths due to leukaemia ; the age distribution of these mice dying from leukaemia is shown in fig . 
the age distribution is somewhat wider than in aka mice and the peak is displaced about 300 days later , so that in th ' is case the average lifetime for mice dying from leukaemia is 59119 - 6 days . in the f2 we encountered 37 per cent cases of leukaemia , and the distribution of age appears to be very wide , as shown by fig . 
the curve may be regarded as a combination of two distributions , one corresponding to the aka mice and one corresponding to the fl . the backcross derived from the fl crossed with aka shows a rise in the frequency of leukaemia to 49 per cent , and the curve concerning the distribut ' ion of age shows , as is seen from fig . 
l. - the age distribution of mice dying from leukaemia within the aka line - - - - - and the a ' ckli ii i t 60days ] days age lunit = 60daysj fig . 
the curve for the f2 shows a tendency to a bimodal form . you will see that , in spite of the genotypical uniformity of the mice of the aka line and the fl , leukaemia only develops.in respectively 63 per cent and 43 per cent of the animals . 
the percentages mentioned as is the case with the figures from previous experiments - calculated by comparing the number of mice dying from leukaemia with the total number of day , ; fig . 
4. - age distribution of leukaemia in backeross of fl mice to b line . dead mice , the animals dying before the first case of leukaemia occurs not being included . it is quite evident that this mode of calculation is entirely inadequate , because deaths caused by other reasons than leukaemia ( such as intercurrent diseases ) , will influence the result to a very high degree . 
the frequency in the fl is not so great as in the aka line , but this can be in part due to the fact that the cases appear at an average age of 300 days later , so that the deaths from other causes will decrease the frequency of leukaemia . the figures found in ' this material , as seen from the following considerations , support the view that the inheritance may depend on one single dominant gene . if we suppose that this is the case , we can calculate the expected numbers of leukaemic animals in the f2 and backcrosses to the parent lines on the basis of the observations in the aka and the fl mice . these results are calculated in table 1 , taking into account the deaths from other causes . these figures agree very well with the observations , but in the calculated mortality curves the agreement was not so evident . 
the difference is not significant . if we assume the presence of two genes the calculated number will only be 8 - 2 '  . these data thus support the hypothesis of a single domi ' nant gene . the variation in the average lifetime of the leukaemic animals has previously been described by macdowell , potter and tayl ' or ( 1945 ) , macdowell and richter ( 1935 ) , furth and barnes ( 1941 ) , kirschbaum ( 1944 ) , and cole and furth ( 1941 ) in similar crossbreeding experiments . 
the phenomenon seems therefore to be independent of the character of the lines used , which fact seems to refute the previous supposition that casual changes in the average lifetime of all the mice may influence the result . i would sooner say that the discovery confirms the supposition that the immature leukaemic cells arise by a somatic mutation in cells which possess a labile 112 g . 
where and how this mutation arises could be a if the animals are homozygous with regard to the labile gene , matter of chance . the mutation will naturally arise earlierand more frequently than ' is the case if the animals are only heterozygous . 
we spent more than eight months working on one such incidental problem , which had to be solved before we could proceed with the main work . this effort was well rewarded , however . 
we not only improved our experimental approach , but also acquired information that will further our understanding of the intricate role played by biological factors in regulating the influence of external agencies on living cells . in view of all this , i should like to point out that our investigations of the genetic potencies of carcinogens are still in an early stage . 
bartholomew 's hospital , london , e.c.1. received for publication february 11 , 1952 . radium and thorium are widely distributed throughout the earth 's crust , although , as yet , no large deposits have been found ( it has been estimated that uranium and thorium exist in as much as 4 and 12 parts per million respectively )  . both these materials decay through a radioactive series , and in each case one ' these two radioactive gases escape into the member of the series is gaseous . atmosphere where their respective decay products will be formed and these prothere are other sources ducts will probably attach themselves to dust particles . of naturally occurring radioactive material ( actinium , which decays to gaseous actinon ; potassium ; rubidium and carbon 14 ) , but these are insignificant compared to members of the radium and thorium series . the presence of radioactive material in the atmosphere was first observed by their observations were confirmed by other workers elster and geitel ( 1901 )  . most of these early determinations were made by in various parts of the world . the charged wire method , which gave quite good qualitative results . 
the rate of air flow depended on the length of tubing connecting the pump and filter - holder , but was always between 30 and 35 litres a minute . it was measured by a u - tube manometer , and controlled by a screw clip . 
the collecting time was 20 minutes , which does not necessarily give the maximum measurable activity , but allows the experiment to be completed in an hour . the efficiency of the filter was examined in a series of experiments in which one , two or three filter - papers of different types were used in series as a backing for the whatman no . 
as it was not possible to obtain standard samples of each member of the radium and thorium series , we determined the efficiency of the counter for measuring the ft particles from three radioactive these efficiencies were plotted against energy , and the efficiency for the isotopes . , f emitting members of the radium and thorium series were read from this graph . the efficiency of the counter for measuring a particles cannot be determined in the same way as for f8 particles ; since an unknown number of a particles will be absorbed in the filter - paper . in order to calculate the ac efficiency , a dust sample was taken and counted in the usual way ; a piece of aluminium foil ( thick enough to absorb all the ac particles ) was then inserted between the filter - paper and the geiger counter , and the count was continued . this experiment was the proportion of ac to ft particles emitted by repeated for a series of samples . the dust was deduced theoretically , and the proportion of a particles which were counted was determined from the drop in counting rate caused by interposing the aluminium foil . 
dawson the data for kew were taken unless there was a significant difference between these two sites , in which case that day was ignored or mean values were taken . 
dawson than those taken in the morning . rothamsted is a third - order station of the meteorological office and more accurate correlation of weather phenomena and activity were possible ; the conclusions drawn from the hospital results were confirmed . the activity was greater if the ground was dry or drying . 
the floor of the cellar was only 4 below street level ; there was some ventilation from a window and the door leading to the main part of the house . 
the cellar had not been used for several in order to avoid disturbing the dust , the pump and filter - holder were weeks . kept outside the cellar ( table iii )  . 
the proportion of radon atoms that will decay whilst passing through the filter - paper is negligible , but when one considers the very large absorptive capacity of carbon for radon , it is rather surprising that no radon was found . we have not investigated the proportion of the other members of the radioactive series that will adhere to dust particles . carmichael and tunniciffe ( 1948 ) removed the dust from a closed room by fanning the air across a greasy plate and noted the diminution of activity in an air sample . 
on introducing smoke there was an immediate increase in activity , showing that in a small room there is never an appreciable quantity of free radioactive atoms - they either become attached to dust or migrate to the walls . 
shelter tolerance ( maximum concentration ) 11 , 800 100 , 000 the figure for droitwich spa was taken from a brochure issued by the local council . our inquiries as to who made the original measurements were not very successful , but it is believed that they were made by professor h . 
schrodinger , at seeham ; 8 - 9 x 10 - 12 curie / metre3 of air . simpsoin ( 1905 ) made a very exacting survey of the air - borne radioactive material in lapland in 1904 , using the charged wire method ( table vi )  . 
read and mottram ( 1939 ) have published the results of some determinations of the radon concentration in the radium laboratories at mount vernon hospital , at the radium institute , and in the radium workshops of messrs . 
 ( all in or near london )  . in the last case a concentration of 22 x 10 - 7 curie / metre3 of air was observed . evans ( 1950 ) has calculated the dose received by joachimstal ( jachymov ) miners in a working day through inhaling radon . 
bartholomew 's hospital , in the city of london ; rothamsted , a semi - rural area , thirty miles from london ; chiswick , geographically in middlesex but actually a semi - industrial part of london ; and in the centre of manchester . 
the results were similar to those of other observers in different parts ofthe world ( table v )  . large day - to - day variations in activity were observed , but these can be explained by variations in the weather - the more stationary the air , the greater the concentration of active material . 
the slight differences between the four sites mentioned above may be seasonal . the experimental procedure was not sufficiently accurate to show whether thoron or its decay products were present . radon , absorbed on dust particles , was not detected . 
no variation with height up to 100 above ground level was observed . the results obtained in rooms with varying degrees of ventilation are of in the laboratory there was usually twice the activity found at the interest . open - air site on the roof . 
at chiswick , ventilating the room lowered the activity by as much as one - half , whilst in the coal - cellar the activity was 14 times greater than at the site in the garden . in the post office air - raid shelter the ventilation was as restricted as any found in habitable buildings ; the activity was 120 times as high as at the open - air control site , i.e. , nearly one - eighth the tolerance concentration of radon . in every case the large day - to - day variations were similar to those at the control site . these large amounts are due to the greater emanating area in a room ( compared with the open air ) from which the parent radon can escape , and to the restricted ventilation which will allow the radon concentration to build up . 
the amounts show no considerable difference between urban and rural districts , but there are large day - to - day variations which in situations out of doors are affected chiefly by the wind ; the more stationary the air , the greater in closed places , e.g. , an air - raid shelter underground , the amount the activity . may be 100 or more times greater than those in the street outside , but even these are small in comparison with the lowest amounts considered harmful to man . i wish to thank professor j . 
fisk , of the general post office , london , for their kindness in providing facilities for this investigation , which has been supported by grants from the medical research council , the british empire cancer cami wish to express my indebtedness to t . 
no definite conclusions were obtained with regard to the influence of the thyroid gland , but the results showed a remarkable reduction of mammary cancer in one of the two strains used . preliminary experiments ( vazquez - lopez , 1946 ; morris , dubnik and dalton , 1946a , b ) showed that thiourea ingestion produced a continuous anoestrus due to ovarian atrophy and the consequent underdevelopment of the mammary glands . 
vazquez - lopez breeding females only were used from the two lines of r3 mice , and both breeders and virgins from the c3h strathe breeders were allowed to rear their first litters and , after weaning , the mothers were taken for the experiment . the litters of the c3h breeders provided the majority of the virgins employed . in all groups care was taken to divide animals of the same litters between the experimental and control groups . the animals were housed in metal boxes , 6 to 8 in each , and fed rat cubes breeders and virgins of the c3h strain were separated , but and water ' ad lib . the two lines of r3 breeders were mixed together . thiourea was administered in the drinking water , beginning with a 0 1 per cent solution and increasing the concentration as much as possible without endangering the survival of the animals . 
the approximate amount of fluid ingested was determined by dividing the total content of the water bottles by the number of animals in the box . all the animals were individually marked . 
the age of the breeders at the beginning of thiourea ingestion was between 3 and 4 months . the virgins were 3 months old , so that the mammary gland was fully developed before the action of the drug started . the period of administration for some of the r3 animals was a year , so that most of the animals with tumours died during the period of ingestion . for the c3h mice , both breeders and virgins , it was 6 months , so that the animals were under normal conditions again when 9 or 10 months old . 
even concentrations of 1 per cent could be used before the mice showed toxic signs such as loss of weight and reduction of water intake . if the solutions were kept between 0 - 2 per cent and 0 * 5 per cent the r3 mice could be maintained for most of their lives without any apparent difference from the controls . 
per animal per day , and if the concentration of the drug was not reduced , many it was possible to reach a concentration of 0 5 per cent in most animals died . boxes , but it could be maintained only for one or two weeks . solutions of 0 - 2 and 0.1 per cent were used most of the time , and even with these doses the intolerance to the drug and mounting mortality necessitated the withdrawal of the after some days without thiourea the mice recovered drug after 4 or 5 months . and the drug could then be administered again , but the recurrence of toxic it was preferable to maintain the 6 months ' period without symptoms was rapid . interruption , even at the risk of losing more animals in the last months of treatment . the doses administered were therefore variable for the different animals and for different periods for the same animal , but from the approximate estimate of water intake it may be assumed that the minimum of thiourea ingested per day was between 2 and 3 mg . 
daily. effects on the thyroid gland . in the r3 mice the ingestion of thiourea provoked the typical hypertrophy this effect was and hyperplasia with great vascularity , loss of colloid , etc . evident even with a 0 - 2 per cent solution , and became more pronounced in proportion to the length of treatment and increased concentration . 
the mice surviving after the withdrawal of the drug had thyroids of normal size and structure without any signs of the former hyperplasia . the effects of thiourea in c3h mice were observed in the experimental animals that died during the period of ingestion of the drug . 
as the great majority of these mice restricted their water intake to a minimum in the weeks before their death , it could be argued that they were not actually under the action of the for this reason additional mice not included in the experiment were used drug . to test the effect under identical conditions , and were killed when the actual ingestion of thiourea was known . in all the 03h mice of the experiment the results of the examination of the thyroid gland were similar . thiourea administered in the drinking water did there was clearly not produce hyperplasia or hypertrophy of the thyroid . congestion of the organ which might cause a slight increase in size , but the histological picture remained normal , showing follicles filled with colloid and lined by cubic epitheliunone of the mice that died during the period of thiourea ingestion showed changes in the microscopical picture . 
among 20 additional female adult mice killed after drinking solutions of 0 - 2 to 0 5 per cent for periods of a week to 2 months , with actual ingestion of about 5 mg . 
the small difference in favour of the thiourea group was not significant , for the small number of animals and the lower tumour age of this group was also against any inhibitory effect of thiourea . all the tumours appeared during the period of treatment when some of the mice had received thiourea for 10 months without interruption . mammary cancer in c3h breeders . - of 49 mice at the start of the experiment , only 29 ( effective number ) reached the age at which the earliest tumour appeared . the other 20 died as a consequence of thiourea ingestion . eight of the 29 table i . - lymphoma incidence in r3 breeding female8 ( mh subline )  . total effective mice with number . 
the earliest developed in a mouse 367 days old , 80 days after the withthis was only 10 days less than the average tumour age drawal of thiourea . these figures confirmed the assumpfor the control group , which was 377 days . tion that ovarian function and mammary development were normal in the experimental animals at the average tumour age of the normal mice . in spite of these normal conditions the percentage of mammary cancer in the thiourea - treated mice was 28 per cent compared with 54 per cent for the controls . 
vazquez - lopez mammary cancer in c3h virgins . - twenty - one animals died during the period of thiourea ingestion , and 64 mice survived the 6 months of treatment and five among the 64 reached the period of appearance of the first tumour . in the developed marmmary cancer ( 8 per cent ) , at the average age of 571 days . control group 40 out of 96 animals ( 43 per cent ) developed tumours at the average age of 432 days . 
the animals treated were 270 days old when thiourea was withdrawn , so that ovarian activity and mammary gland development were presumably normal not only long before the average tumour age of the controls , but also very nearly when the first tumour appeared among the normal mice . 
assuming that after the end of thiourea administration the mice needed a month for total recovery , they would be comparable with the normals when 300 days old . only 4 mammary cancers developed in the control group below this age . the decrease in cancer incidence in the thiourea - treated virgins was highly significant ( x2 = 20 9 ) , and confirms the results in c3h breeders . in this group the average tumour age was also later than in the controls . 
the average life span of mice without tumours was longer , but with smaller differences from the control mice than in the breeders . microscopical study of organs in thiourea - treated c3h mice . a detailed study of the organs of c3h mice after longer periods of thiouracil and thiourea ingestion was published by dalton et al . 
1 and 2 show condition and weight of the mouse rather than with its age . glands of control and experimental mice of approximately the same age . the two main differences between thiourea - treated and control mice are the number of nodules of alveolar hyperplasia and the width of the ducts . 
the alveolar nodules of one gland or even of all the glands in a single mouse cannot be used to distinguish a thiourea - treated from a control mouse ; in some thioureatreated animals the number and distribution of nodules is the same as in the controls . 
the thiourea - treated mice , as a whole , had considerably fewer nodules of alveolar hyperplasia than the control mice , and the average number of nodules per mouse was less than half the figure for the controls . there are no records of comparative counts in similar experiments with reduced mammary cancer incidence , but huseby and bittner ( 1946 ) report that less than 1 nodule per 4 glands was found in some animals more than 340 days old kept on the restricted diet in the experiments of huseby et al . 
vazquez - lopez to indiscriminate reduction affecting equally all the glands of each animal , but to a clear decrease of the number of glands with ' nodules . only 32 per cent of the glands in the thiourea - treated mice had nodules compared with 51 per cent in there were also significant differences in the distribution the control group . and the average number of nodules for glands in each group , but the main effect of the thiourea administration is to increase the number of glands without any nodules . 
as the hormonal and systemic influences are the same for all the mammary glands in each mouse the cause of these differences between glands in the same mouse must be the result of local factors whose activity is restricted to the fields represented by particular glands . thiourea acts systemically mainly by stopping ovarian function and oestrogen production . 
at the post - mortem examination the ducts appeared full of whitish material , which made them conspicuous to the naked eye . it seemed therefore to be due to engorgement by a secretory product , but it could exist in glands without any indication of alveolar proliferafig . 
some ovaries appeared in regressed condition without corpora lutea or follicular development ; this occurred with the same frequency in both groups . most ovaries had follicles in all stages of development , and some of the corpora lutea became hyalinized , forming large homogeneous masses with few cells . this formation of corpora albicantis is probably a strain characteristic , since fekete ( 1946 ) found them in the dba high cancer strain , but never in the c57 black strahuseby et al . ( 1945 ) found them only in one c311 mouse with mammary - cancer , and in none of the diet restricted animals of their experiment . that is probably another of the characteristics in which the c3h mice used here differ from those in american laboratories . 
the corpora albicantis were very prominent among the cancerbearing animals , and their ovaries owed their large size partly to these structures . apart from their size , however , the ovaries of mice with tumours showed mature follicles and abundant corpora lutea , which suggested normal activity . these findings were contrary to the results of allen , diddle , strong , burford and gardner ( 1935 ) in several strains of mice , including the c31 . in all groups the condition of the uterus reflects that of the ovaries having tall epithelium , extensive glandular development and loose cellular stroma in the animals with well functioning ovaries and signs of regression in those with ovarian atrophy . no differences were found in the adrenal glands of thiourea - treated mice compared with the controls . 
the most important change , affecting both groups equally , was the presence of amyloid degeneration , which appeared on the borderline between cortex and medulla and progressed towards the glomerular zone with advancing age . in the oldest animals amyloid invaded most of the cortex , and only two or three layers of cortical cells remained intact . 
the medulla was never affected . " brown degeneration " was , on the contrary , never found in c311 mice , but was almost constant in r3 animals of high or low mammary cancer lines . 
the possibility of a specific activity of thiourea on blood - forming tissues and their neoplasms suggested by the agranulocytic reactions in patients treated with goitrogenic substances is not supported by the results in the mh high lymphoma line which agree with clinical reports of treatment of leukaemic patients ( limarzi , kulasavage and pirani , 1946 ; hansen - pruss , 1947 )  . that lack of oestrogen is the effective factor is shown also by the normal incidence of cancer corresponding with regular oestrus in spite of thiourea administration in r3 mice . 
the action of progesterone is still a matter for discussion ( lacassagne , 1937 ; heiman , 1945 ; burrows and hoch - ligeti , 1946 )  . indirect ways of decreasing oestrogenic activity give , however , less clear results . although low cystine diets completely inhibit mammary cancer in c3h mice ( white and andervont , 1943 ) , the effects of stilboestrol pellets which restore the development of the mammary glands raise the incidence only to 44 per cent compared with 92 per cent in the controls ( white and white , 1944 )  . in similar experiments , ball , huseby and visscher ( 1946 ) found that in spite of continuous administration of stilboestrol the mammary glands of strain a mice maintained on restricted diets during 15 months were considerably less developed than those of control mice . 
the possibility of other hormonal factors influencing mammary cancer development is also suggested by the results of shimkin and wyman ( 1945 ) , who found greater reduction in adrenalectomized c3h mice than after ovariectomy , and by the observation of wallace , wallace and mills ( 1945 ) of persistence of effects of temperature in mammary cancer incidence after ovariectomy . the results of temporary lack of oestrogenic activity caused by thiourea show that reduction of mammary cancer is not due to defective glandular development , and therefore that the hormonal effects are not limited7 to the morphogenesis of tissue susceptible to carcinogenic agents . 
mammary glands of two groups of mice of the same strain with different cancer incidences have identical morphology , and only differ in the total number of nodules ofalveolar hyperplasia in each group . 
huseby and bittner ( 1946 ) have shown by the study of c3h and hybrid mice with or without the " milk influence " that this influence is necessary for the presence of nodules . 
the distension of ducts found also in the thioureatreated group seems to be a sign of excessive oestrogenic stimulation , and may indicate compensatory excess after thiourea withdrawal . if the hormonal influences are sufficient for the alveolar development the smaller number of glands with nodules , which is the main result of thiourea administration , must be due to local factors . 
the lack of oestrogens ( and its possible compensation afterwards ) affects all the glands of the mice equally . these results point to the possibility of effects on local factors and producing different consequences in each gland of the same animal . 
the local factor involved in the persistence of alveolar nodules is the milk factor , and the distribution of nodules suggests that it is affected by thiourea administration . there are no grounds for supposing that thiourea can directly influence the milk factor , but there are some in favour of an indirect action resulting from the lack of oestrogens . apart from the considerable number of experiments on the effects of oestrogen adninistration on cancer incidence , there is evidence even in normal circumstances of hormonal influences in breeding and forced breeding , increasing the number of tumours . 
the higher incideixce in mice born in later litters compared with the earliest from the same mother has been explained by the different concentration of the milk influence with advancing age ( bittner , 1942a )  . if the increased hormonal stimulation is the cause of increase in the amount or activity of the milk influence , it is probable that the converse holds true also , and that the lack of hormones acts in the opposite sense . the relative importance of the hormonal influence among the three factors necessary for the genesis of spontaneous mammary cancer in mice is not exactly opinions range * from that of shimkin and andervont ( 1941 ) , who known . consider the hormonal factors as necessary only for the creation of a substratum for the carcinogenic action of the factor transmitted by the milk , to that of warner , reinhard and goltz ( 1945 ) , who maintain the dominance in certain cases of the inherited susceptibility upon the concentration of the milk agent . loeb , suntzeff , burns and schenken ( 1944 ) believe that the oestrogens act by stimulating the growth of the mammary tissue and sensitizing it to all types of stimuli , and bonser ( 1945 ) as a developing factor acting upon breast tissues already sensitized by the milk factor . 
the only common feature of all these opinions is to consider the different factors as completely independent variables . on this basis the opinion of bittner ( 1942b ) that each influence must be considered as complementary to the others and all equally important is , besides being noncontroversial , nearest to the observed facts . 
but if there are some factors which are affected and increased , or even created de novo by interaction of the others , the relative significance of each would not be the same . the effect of genetic susceptibility on some aspects of the milk factor activities has been already proved by heston , deringer and andervont ( 1945 )  . 
w.3. received for publication july 16 , 1949 . in an investigation of the body growthand tumour growth - inhibiting action of carcinogenic substances , it was found that the nature of the inhibition is profoundly influenced by the protein content of the diet ( elson and warren , 1947 ; elson and haddow , 1947 ; elson , 1949 )  . in rats maintained on a 20 per cent protein diet injection of the carcinogen usually has little immediate effect on body growth although a delayed action , resulting in rapid loss of weight followed by death , may occur later . 
carcinogenic hydrocarbon 1 : 2 : 5 : 6 - dibenzanthracene 24 hours after implantation of walker carcinoma 256 in rats maintained on a 20 per cent protein diet results in very little difference in size and weight of the tumours from those of controls . if , however , the animals are fed a 5 per cent protein diet the tumours of those similarly treated with dibenzanthracene were found to be only one - quarter the weight of those of controls when the rats were killed 13 days after implantation ( elson and haddow , 1947 )  . 
harris. * from the chester beatty research in.3titute , royal cancer hospital , london , s.w.3. received for publication january 11 , 1951 . themajor requirement for the investigation of the physical and chemical properties of the rous no . 
i sarcoma agent is a source of " purified " agent of a uniformly high infectivity , and which may be stored without loss of this activity , since it is not always convenient to compress the investigation to within the relatively short period in which the purified agent is stable in vitro . this paper describes a method for the production of concentrates of the vir ' us in a form suitable for storage , and the storage problem itself is considered in the following paper . a variety of chemical and physical methods have hitherto been used for separating and concentrating the agent from biologically - inactive tumour protein and nucleoproteins . claude ( 1935a ) found that such contaminants could be removed by adsorption , on to freshlv prepared aluminium bvdroxide gel , and the carbohydrate contaminant by precipitation with gelatthe agent was not directly affected , and a - combined chemical fractionation and dialysis gave a 25 - fold concentration in - terms of dry weight ( claude , 1935b )  . this method was also investigated by dmochowski ( 1948b ) , who recorded a 50 per cent increase in the activity of the agent compared with the startino , c5 filtrate . shemin , sproul and jobling ( 1940 ) , and shemin and sproul ( 1942 ) precipitated the agent from filtrates by addition of papain or of histone . 
nitrogen ( the range was 1 - 3 x 10 - 3 mg . to 7 x 10 - 7 mg . ) were found to be infective . concentration of the agent by centrifugation was first described by ledingham and gye in 1935 and fractional centrifugation methods have since been widely used ( mcintosh , 1935 ; amies , 1937 ; claude , 1937 , 1938 , 1939 , 1940 ; claude and rothen , 1940 ; pollard , 1938 , 1939 ; stern and duran - reynals , 1939 ; dmochowski , 1948a )  . claude ( 1938 , 1939 ) isolated a fraction corresponding to less than 3 - 5 per cent of the dry weight of the original extract which , in amounts corresponding to 4 - 0 x 10 - 10 mg . 
harris pollard ( 1939 ) and amies and carr ( 1939 ) were able to overcome the first difficulty by precipitat - ing the agent from a clarified extract by adjusting the ph to 5.0 to 5 - 5 . 
the isoelectric point of the purified agent is 3 - 5 , but tumour extracts deposit a mucoprotein at ph 5 - 0 , which takes down the agent with it . the aggregated agent , etc . , was then further concentrated by a low - speed centrithe aggregates were.broken down by tryptic digestion at fugation procedure . ph 8 - 8 to 9 - 0 and the released agent further " purified " by fractional centrifugation . alternatively , the ' mucoprotein may be hydrolysed by incubation of the extract - with hyaluronidase , which has no deleterious effect upon the agent ( siturm , and duran - reynals , 1932 ; shemin and sproul , 1942 )  . 
the agent may then be deposited directly from the non - viscous extract . bryan , riley , deihl and voorhees ( 1947 ) have avoided this difficulty by the simple , but uneconomical , procedure of rejecting the first , viscous extract . a further method which has been claimed to apply to the purification of small arrlounts of agent is that of chromatography on celite ( riley , 1948 )  . 
the agent is strongly adsorbed to celite from physiological salt solution , but may be eluted intermsoftheratio , biologicalactivity : withverydilute ( o - 001m ) saltsolution . nitrogen content , a 3to 7 - fold enrichment was obtained from a partially ' - purified three of the above methods , papain precipitation , fractional tumour extract . centrifugation after precipitation at ph 5 - 0 and direct deposition after treatment of the initial extract with hyaluronidase , have been reinvestigated from the point of view of the preparation of large quantities of agent in a form suitable for storage . 
a further method , methanol precipitation , hitherto applied to a number of other animal viruses , has also been investigated . cox , van der scheer , aiston and bohnel ( 1947 ) found that influenza virus ( from arantoic fluid ) could be precipitated in active form from suspensions in phosphate buffer at ph 7 - 0 to 8 - 0 by addition of methanol to a final concentration of 15 to 30 per cent . 
sharples air - driven supercentrifuge , stainles - 3 the rous agent becomes progressively more thermolabile as purification centrifuge b was used in a cold - room at o ' c . , and c was adjusted to the sharples centrifuge ( a ) was cooled with the centrifugal procedures used are listed below and steel bowls . - ( i ) clarifica ( ii ) deposition at ( iii ) deposition at ph 7 - 0 . 
the bulk of the tumour was extracted in the waring " blendor " with 10 volumes of water containing 1 : 10 , 000 hcn , and the small sample " minced with scissors " and extracted into a similar medium . suitable dilutions of these extracts were injected into fowls . 
0.005 m buffer at ph 7 - 2 containing hyaluronidase and 1 : 10 , 000 ticn . the tissue suspension was clarified ( b , i ) and the agent deposited from the supernatant ( b , ii )  . 
the control suspension ( a ) was ' assayed comparatively against ( b ) - resuspended papain - agent complex , etc . , ( b ) - supematant and ( c )  . 
and methanolic buffer solutions prepared , and cooled to these buffers contained ( a ) 40 per cent methancil , ( b ) 50 per cent methanol and ( c ) 60 per cent . methanol respectively in 0 - 005 m buffer . equal volumes of ( a ) , ( b ) , ( c ) or buffer ( d ) - control , were added to volumes of the supernatant . 
an equal volume of 50 per cent methanolic buffer was added under the same conditions as method 1 and the resuspended deposit assayed against the extract . the assay showed that the alcohol - deposited agent had only i per cent of the activity of the control suspension . ( 3 ) a similar clarified extract was prepared , cooled to o ' c . 
the deposit was resuspended in 0 - 005 m buffer at ph 7 - 2 , clarified ( b , i ) and the agent in the supernatant deposited ( b , ii )  . the pellet was finary resuspended in 0 - 005 m buffer , ph 7 - 2 , adjusted in concentration to the equivalent of i g . 
de osition after precipitation at ph 5 - 0 . a 10 per cent fresh tumour tissue suspension was prepared in the usual way , but with the omission of hyaluronidase . 
the air - driven sharples supereentrifuge with a maximum speed of rotation of 50 , 000 r.p. ( 62 , 000 g . ) was used by stanley in 1945 for the concentration of influenza virus for vaccine production . 
for deposition of the virus the stainlesssteel centrifuge bowl was invariably lined with cell ' ophane ( 7 / 1000 in . ) which may ( a ) be readily withdrawn and which ( b ) minimizes contact of the virus with metals . when unrolled this " liner " invariably showed a boundary , the distance of which from the bottom of the bowl , varied with the rate of flow of the virus in terms of total nitrogen distribution , the suspension through the centrifuge . deposit was uniformly distributed above and below the boundary , although the virus content of the top and bottom fractions differed considerably . 
the " liner was therefore divided at the boundary and the deposits removed and separately resuspended in 0 - 01 m buffer at ph 7 - 2 ( dband dt )  . 
per cent crystalline trypsin was added to each , the suspensions incubated at room temperature for 40 minutes and the virus re - deposited ( a , iii ) ( sb , st - supernatants and dbj , dt , - deposits )  . 
fresh tumour tissue was disintegrated with 10 volumes of 0 - 005 m phosphate buffer at ph 7 - 2 containing 1 : 10 , 000 hcn - but without hyaluronidase - and the suspension clarified ( a , i )  . 
88. the " liner " was removed and the total deposit ( d . , ) resuspended in 375 ml . 0 - 005 m phosphate buffer at ph 7 - 5 to 8 - 0 containing a few mg . 
harris constant standard could be obtained or that the subsequent comparison would be unaffected by such factors as the season of the year or the nature of the medium in which the virus was suspended . 
the first group ( " limiting dilution " methods ) had , also , the advantage of giving some degree of absolute measure corresponding to the actual number of tumour - producing particles . two injection routines have been favoured by prev - ious workers ; either the intradermal or the intramuscular . 
the former allows several different dilutions to be tested in the same bird , but accurate localization of the injection is rendered difficult by the paper - thin structure of the avian skin . moreover , the frequent occurrence of resistant fowls necessitates replication of the tests . intramuscular injections involve a more readily standardized reaction site , but 6 - week - old birds frequently show a variable and unpredictable resistance to the virus . such resistant birds may not react to 1000 times the virus dose which will produce tumours in susceptible birds , and if the end - point , ( m.i.d. ) is dependent , as may be shown , upon a poisson distribution of virus particles , then a large number of 6 - week - old fowls would be required for an accurate assay . it is inconvenient to use large numbers of birds of this age for a single assay and for such reasons the use of embryos or of day - old chicks , which may readily be obtained in large numbers , was investigated . 
the survivors are killed and examined at 28 - days . sudden deaths in the young birds as a result of the " haemorrhagic disease " described by milford and duranreynals ( 1943 ) did not occur when the inoculum was given into the leg muscle . irregular results were obtained in the assay when the . 
harris trypsin ( which removes more than 60 per cent of the non - virus proteins ) gives a product containing only 2 per cent of the nitrogen of the clarified tumour extracts , but possessing almost ar the virus activity . 
a biological assay method using very young chicks has beerr developed . these chicks are found to be more sensitive to the virus than older birds and are obviously more convenient to handle and maintain . the authors wish to express their thanks to professor a . 
the number of cockerels was too in all examinations consmall to admit a proper comparison between sexes . cerning weight , ratio of cortex : medulla and cholesterol content , however , the examined cockerel adrenals did not differ from the female ones . 
each group in the following , control as well as lymphomatosis groups , thus included one to three cockerels . elliot and tuckett ( 1906 ) found no positive correlation between adrenal weight and body weight in adult birds , and our figures support their observations . if the normal birds are equally divided according to body weight into one light and one heavy group , the former had on the contrary some higher mean adrenal weight . 
the gland weights are therefore given in absolute figures . about half of each examined group was composed of white leghorn birds , while the other half consisted of rhode island reds , barred plymouth rocks and crossbreds . 
as was stated concerning sexes , this material did not indicate still , if such differences exist , which may very well differences between breeds . be demonstrated with bigger breed groups , their effect ought to have been fairly eliminated in this investigation by similar breed distribution within the compared groups of birds . control birds as well as lymphomatosis birds were collected during the same seasons - autumn and winter . 
in order to reduce the error produced by the uneven distribution of the cortex within the gland , part of the gland periphery was thus always made part of the picture . the picture was then enlarged to an area of about 300 cm.2 the total area , with the fibrous capsule and notable vessels excluded , and the area of the cortex were determined by planimetry . 
216.833 11273 1 - 61 the difference between gland weights of lymphomatosis birds and normal birds , 63 - 23811 - 334 , is highly significant ( t = 5579 * * * for df = 58 )  . 
the tumour size in neural lymphomatosis is always considerably less than in the visceral variety . the measurements of cortical and medullary areas clearly show that the enlargement is entirely caused by increase of the cortex . 
la than from comparison is in fact decreased . of the means , as two lymphocytosis cases showed exceptionally high cholesterol values and thus brought the mean to a higher level than that characteristic of the majority of cases . 
the impression was that lymphomatosis in a more or less advanced stage is characterised by a big adrenal cortex which in most cases is relatively poorer in cholesterol than normal cortex . the histological examination now and then showed cases of lymphomatosis with considerable disappearance of sudanophil substances but hardly revealed the slight but systematic deprivation as did the chemical determination . as will be shown in a later paper , acutely running experimental lymphoid tumours in chicks ( a transplantable strain of lymphoid tumour derived from a case of spontaneous lymphomatosis ) runs with a still more marked depletion of cholesterol than spontaneous , chronic lymphomatosis as well as with adrenal enlargement . this would correspond with sokoloff 's first stage of adrenal hyperactivity in rous sarcoma . 
he concluded that the second stage was one of hypoactivity and lipoid storage . the results of our investigation indicate , however , that in avian lymphomatosis the second stage is also characterised by hyperactivity . 
a less probable alternative explanation would be that the cortex does not grow as a consequence of increased demand of steroids but because of difficulties in producing theon the contrary , the parallel with selye 's ( 1950 ) general adaptation syndrome with increased adrenal activity first during the alarm reaction and later in the stage of resistance is obvious . 
further , as cortisone fails to produce this effect when it is applied in the " pre - induction " and " induction " phase of carcinogenesis , no support is given to mottram 's hypothesis , that the inducing action of a carcinogen is exerted on a dividing cell . 
the number of cells in mitosis in the cortisone pre - treated animals during the period of " induction " would be few or none and on this hypothesis far less papillomata should have appeared whereas the numbers equalled that of the controls . baserga and shubik ( 1954 ) have obtained essentially siniilar results . 
the findings of green and savigear ( 1951 ) may bear on this point . they showed that after 4 applications of dmb , over 14 days , the epidermal cells of the mouse ear became relatively refractory to the antimitotic action of cortisone given systematically . this was confirmed by an in vitro technique ( green , 1952 )  . it might be expected therefore that chemical carcinogenesis would not be delayed or prevented by cortisone , whereas in fact the present results show that it was . 
a possible explanation of this apparent discrepancy is that the time of appearance of resistance to cortisone depends directly on the rate of mitosis . if this rate is strongly diminished ( by cortisone ) from the outset of carcinogen treatment resistance would then appear much more slowly . 
at the time of its appearing however the treated area of epidermis would be rich in cells in the " induction " phase of carcinogenesis since cortisone does not affect this process . such a mass of cells , after a long exposure to the carcinogen , could be envisaged , when finally they had become completely resistant to cortisone , as emerging as a frank carcinoma . 
doniach. from the pathology department , postgraduate medical school of london , w.12. received for publication february 20 , 1953 . the increasing use of radioactive iodine in clinical medicine has made imperitive the experimental study of its possible carcinogenic action on the thyroid gland . radioactive iodine , like stable iodine , is rapidly concentrated by the thyroid , bound to protein in its colloid in the synthesis of thyroxine , and then gradually released from the gland as part of the normally secreted hormone . during these processes the follicular cells of the thyroid are submitted to a course of ionizing irradiation from the i131 , which gives out f and y rays . 
the duration of this irradiation is of the order of days , since the half - life of 1131 is 8 days and the effective half - life of intrafollicular hormone is also of the order of days in the normal human gland . the carcinogenic activity of ionizing radiations in general has been repeatedly demonstrated during the past 50 years . recently , the effects of ft irradiation from newly developed sources have been studied . raper , henshaw and snider ( 1951 ) reported that single doses of 4000 to 5000 rep ( roentgens equivalent physical ) of f rays from a p32 source induced skin tumours in rats . 
they also found that 13 , 400 rep given in daily exposures of 50 rep over a period of 348 days proved carcinogenic . glucksmann ( 1951 ) found epitheliomas in 4 out of 24 mice in an area of skin exposed 14 months previously to 7900 rep given in 30 seconds by an electron beam . it has proved necessary in the treatment of graves ' disease with radioactive iodine to administer an amount of 1131 calculated to give an overall irradiation to the thyroid gland of 8000 to 10 , 000 rep ( wayne , 1952 ) in order to produce a complete remission of the disease . this dosage lies within the range found to be carcinogenic to other animal tissues . but owing to the uneven distribution of radioactive iodine in the thyroid one cannot make a confident direct comparison , from the dosage point of view , between the animal experiments mentioned above and 1131 therapy of humans . the mechanism of the experimental production of tumours of the thyroid by goitrogens has been greatly clarified by the new zealand team of workers ( purves , griesbach and kennedy , 1951 )  . 
doniach in evaluating the possible danger of carcinogenesis by j131 one must consider not only the direct action of , rays on the cells , but also the indirect action of radiation damage , which leads to diminished thyroxine synthesis and a resultant increased thyrotrophic hormone production . 
a suitable dose of 1131 might therefore lead to a summation of two carcinogenic stimuli ; a short exposure to / 6 irradiation and a long exposure to stimulation by the pituitary . 
the latter could be ensured experimentally by giving a prolonged course of antithyroid drug subsequent to a dose of j131 . doniach ( 1950 ) found that 10 out of 16 rats , given 32 microcuries of 1131 ( 15 , 000 rep to the thyroid ) , developed adenomas . in a series of 5 rats given additional methylthiouracil for 14 months , the thyroids showed a striking increase in adenomas and i - n one instance a metastasizing carcinoma , in contrast to 16 rats tested for the same period with methylthiouracil alone , whose thyroids showed only occasional adenomas . this summating effect of a carcinogen with an antithyroid drug was first demonstrated by bielschowsky ( 1944 , 1045 ) , who used the carcinogen acetamidofluorene combined with allylgoldberg and chaikoff ( 1952 ) have recently reported the development thiourea . of poorly differentiated non - colloid - forming thyroid cancers in 7 out of 25 rats injected 12 to 2 years previously with 400 microcuries of i131 . 
the authors attributed the cancer development entirely to beta irradiation , and not to thyrotrophic hormone stimulation . the object of the experiments described below was to repeat previous investigations of the action of 30 microcuries of j131 alone and combined with methylthiouracil in a larger series of rats , and to compare these results with the effects of 5 and of 100 microcuries . 
the trachea and thyroid attached were fixed in helly 's fluid ; the thyroid was then dissected off and weighed to the nearest milligrathe thyroids were embedded in wax , all glands which weighed 30 mg . 
were secstained by haemalum and eosin . tioned at 6 levels , so arranged as to traverse the whole thickness of the thyroid three sections cut at each level at 5#t were mounted and at regular intervals . one of them stained by haemalum and eosthe spare sections in general were used for extra stains . 
followed 24 hours later by methylthiouracil in the drinking water until the end of the experiment . ( 8 ) 100 lsc 1131 , 30 , uc 1131 , 24 hours later methylthiouracil till end of experiment . 24 hours later methylthiouracil till end of experiment . 
they were taken off the drug for 4 weeks from the 6th to 7th month and were given the i131 in two doses , half at the beginning of the experiment and half 24 hours before commencing their second course of methylthiouracil . in july , 1951 , eight months after this experiment was begun , the thermostat broke down one night and the heating was unfortunately left full on . 
a further 40 rats died or were killed during the course of the experiment , wasted from interfortunately the differences in current infection . the findings between the major groups proved striking even with the reduced number of rats . 
the nuclei were paler than those of neighbouring follicle cells and showed occasional mitoses . the affected follicles had lost their original lining , and had tended to flatten out apart from the altered appearthe normally scalloped perifollicular reticulum . ance of the cells , these " solid " follicles were easily differentiated by serial section from the apparent solid follicles produced by tangential sections of normal ones . they occured both centrally and peripherally in the glands and varied in diameter from 50 to 200 , t . 
to this end , serial sections were examined of the growing thryoids of 5 rats aged .23 days and 5 rats aged 100 days . in none of them were any microadenomas seen . 
nor were there any structures seen resembling these microadenomas in sections of the developing thyroids of 15 - day and 19 - day rat embryos . the incidence of microadenomas in the 9 controls of the main experiment is there were no macroadenomas found similar to those seen in there was a great variation in rats ' and thyroids ' weights . 
the lining cells were about 15 , t tall with a voluminous eosinophilic cytoplasm and capillaries were prominent , and oval vesicular nuclei . did not indent the follicular cells as in the controls . in reticulin preparations the capillaries appeared pushed back by the hypertrophic follicular cells . 
the arrangement of the cells included solid sheets , packed solid trabeculae , closely packed tubules containing pale fluid , microand macrofollicles filled with watery or deeply eosinophilic colloid , and gross cystic follicles distended with colloid and lined by a papillary epitheium . engorged sinusoids were prominent in many of the adenomas . 
the majority , however , presented a closer affinity to those seen in the rats treated with 5 , uc 1131 alone except that the cells were a little taller . their nuclei varied in size and chromatisthe adenomas presented an exaggerated and bewildering variety of cell types and differentiation fundamentally similar to those produced by methylthiouracil alone . 
some of the very large adenomas appeared to be derived from the confluence of more than one tumour . colloid secretion was well marked in many tumours and a papillary arrangement was not rare . 
however , the major part of the bulk of even the large tumours was cellular rather than secretory in origin . solid areas of undifferentiated spheroidal cells were seen within some tumours as with methylthiouracil alone . rat 35b tongues of adenoma tissue were seen penetrating the capsule at one however , in the absence of evidence of permeation of extra - capsular point . veins or obvious morphology of cancer , malignancy was not diagnosed . 
the majority of the follicles appeared active but could be differentiated from those seen in rats treated with methylthiouracil alone , as they were smaller , less regular and showed a tendency to a syncytial arrangement of their lining cells . 
the rats ' weights were similar to those treated by methylthiouracil alone , but the thyroid weights were very considerably reduced to an average size comparable with that of the rats treated with 100 , uc 1131 alone . the pituitaries were similar to those of the rats treated with methylthiouracil alone . 
was injected with the same syringe into a glass vial containing a small pledgelet of cotton - wool . fifteen hours later in vivo measurements were made of the radioactivity of the rats ' thyroids and of the 1 ml . 
19.chart showing the average in vivo count - rates of radioactivity of the thyroid glands of 8 rats following an intraperitoneal injection of 9 - 2 yc 1131 . after 24 hours 4 of the rats ( lowermost curve ) were put on to methylthiouracil in their drinking water . 
the findings in the remaining 4 are charted in the middle curve ; the calculated loss of radio - activity due solely to the radioactive decay of il31 is charted in the uppermost curve . count - rate of standard at 15 hours was 10 , 700 . due to the normal radioactive decay of 1131 . 
the differential effects on these functions of varying doses of 1131 have been recently reported in 3 publications . skanse ( 1948 ) studied the effects of 1 , 10 and 50 , tc i131 on young cockerels primed with a short course of thyrotrophic hormone . 
a definite inhibition of normal thyroid growth was produced by 10 j#c and 50 , uc 1131 after 16 days but not of body growth . all the thyroids showed a growth response to thiouracil , less marked in the chicks given the higher dosages of 1131 . but this response to thiouracil was much more inskanse ( 1948 ) remarks that though hibited after 38 days than after 26 days . the major part of the radiation was received irn the first few days , it took a much longer time to produce the full biological effect on gland function . the calculated dosages of total radiation to the thyroid glands were 1700 rep in the 1 , uc group , 13 , 000 rep in the 10 , tc group and 60 , 000 rep in the 50 1c group . feller , chaikoff , taurog and jones ( 1949 ) studied the effects on adult male rats of single intraperitoneal doses of 24 , 300 and 875 4ac of 1131 . 
the average uptake of 1131 into the thyroids was 40 per cent between 23 and 48 hours after those treated with the two injection and was about equal in all the animals . higher doses showed an abnormally rapid depletion of the iodine that had been trapped during the first 24 to 48 hours . chemical analyses showed an increasing depletion of thyroid 1127 from 2 to 3 days onwards after the injection of the 300 and 875 jtc 1131 , whereas there was no loss after 6 days in animals given 24 1ac . the values of plasma protein bound iodine were not changed in the 24 , uc group but they fell steadily in the rats injected with 300 , uc from 3 - 6 , ug . 
the thyriod uptake in the 1 , uc reached 53 per cent 48 hours after receiving a tracer dose of i131 , but was significantly less in all the other groups . response to subsequent thiouracil was similarly impaired when tested . 
the thyroids of animals which had received 50 and 100 , tc 1131 demonstrated no capacity to increase in weight on a 30 - day course of thiouracil instituted 64 davs after the initial dose of radioactive iodine . even 5 jtc initial t131 impaired the capacity of the thyroid to increase in size ; the 194 i . 
in the animals given an initial dose of 1 iac 1131 . measurements of body weight showed no disturbance in animals which had received up to 20 , uc , a slight impairment in the 50 and 100 , uc groups and a marked disturbance in the 300 , uc group . 
the calculated maximum total irradiation doses delivered to the thyroid in rep were respectively 1800 in the 1 , uc , 5800 in the 5 , uc , 30 , 000 in the 20 , uc , 80 , 000 in the 50 1ac , 197 , 000 in the 100 , tc and 288 , 000 in the 300 / tc groups . effects of internal irradiation by j131 on thyroid histology . findlay and leblond ( 1948 ) studied the thyroid histology of two rats killed 6 days after a single injection of 1131 calculated to have given a total thyroid irradiation of about 20 , 000 rep . the rats had previously been maintained on a low iodine diet . most of the follicles , especially the central ones had lost their colloid , and were replaced by irregularly arranged solid groups of epithelial cells , some of which showed nuclear and cytoplasmic degeneration . interstitial oedema was pronounced , fibrosis and lymphocytic infiltration slight . 
many follicles in the periphery and isthmus were normal autoradiographs showed evidence of accumulation of iodine only in the surviving colloid containing follicles of the periphery . goldberg , chaikoff , lindsay and feller ( 1950 ) studied the progressive changes in the thyroids of adult rats given varied doses of 1131 and killed after intervals of 1 day , 2 days , 3 days , 1 week , 2 weeks , 3 weeks , 1 month , 6 months 875 1 , c ( 330 , 000 rep to gland centre at 72 hours ) produced a total and 8 months . destruction of the thyroids evident within 48 hours , associated with an intense fibrinous oedema and acute inflammatory changes , followed by fibroblastic proliferation and arterial thrombosis . 
by 3 weeks there was a total collagenous permeation of the original gland structure , which by 6 months was replaced by 525 itc produced essentially similar changes . a band of hyalinised collagen . there were , however , at 5 months a few surviving thyroid follicles at the poles , lined by bizarre granular cells thought to resemble the so - called hurthle cells of the human gland . at 8 months surviving thyroid cells appeared more numerous 300 / , tc ( 110 , 000 rep at and formed occasional colloid containing peripheral follicles . 72 hours ) produced extensive swelling and desquamation of thyroid cells within 24 hours , followed during the next 2 days by further degeneration and by interstitial inflammation which appeared resolved by 8 days . at 4 months the glands were lobular and made up of mixed disturbed and normal follicles . at 8 months the glands appeared essentially normal , though the follicle cell nuclei were hyperchromatic and interstitial tissue was increased . 
produced total thyroid cell necrosis in 2 days . following the administration of minimal thyroid - lethal doses , parenchymatous degenerative changes first appeared within a few days in the gland and were followed by an inflammatory picture . intrathyroidal doses of 2 , uc / mg . 
 ( 1952 ) described the histology of the thyroids at varying time intervals after administration to rats of i131 as a part of the study of functional changes quoted above . after 2 days parenchymal degeneration and interstitial inflammation were seen centrally in the 300 , #c group ; no significant changes after 48 days the 300 , #c group showed a virtual were seen in the other groups replacement by fibrous scar tissue . the other groups , including the rats given 1 / uc , all showed hypertrophy of follicle cells and diminution of colloid . 
from 30 , 000 rep upwards there is an increase in rate of loss of organically bound iodine from the thyroid , associated with radiation damage to the follicles . in general , functional disturbances precede histological changes . 
from 5000 rep upwards the thyroid cells are able to respond to activating stimuli by hypertrophy , but hyperplasia proves abnormally limited . bizarre thyroid cells are seen histologically up to 12 years and are accentuated by thiouracil treatment , increasing in number in thyroids submitted to 5800 rep upwards . 
even though irradiated thyroid cells are histologically abnormal , they appear by means of hypertrophy to synthesize enough thyroxine to maintain the animals ' health and growth after dosages up to 30 , 000 rep . calculation of dosage of radiation to the thyroid . calculations of radiation dosage to the thyroid following the administration of radioactive iodine can at the best only be rough estimates because of the con196 i . 
 ( 1949 ) pointed out that since the maximum range of the beta particles enmitted from i131 is comparable to the dimensions of the rat 's thyroid , a geometrical factor must be calculated to estimate the reduction in radiation dose at points near the surface of the gland as compared with the centre . 
by following the uptake of radioactive iodine into the thyroid and its sequential loss from the gland it is possible to estimate the total the conversion factor for rep was calculated by evans ( 1947 ) : radiation dose . 1 , kc per mg . 
the animals were all killed after 15 months and 5 , tc 1131 and methylthe rats on methylthiouracil alone showed no carcinomas . thiouracil led to the production of a greatly increased incidence of adenomas , these findings confirm the additive action of radiobut no definite carcinomas . active iodine in carcinogenesis by antithyroid drugs , and show that the range of 2270 to 16 , 200 rep was more effective than 380 to 2700 rep . in contrast , the methylthiouracil given to rats treated with 100 , tc i131 produced less adenomas this inhibition at a dosage level of 8400 to 60 , 000 than methylthiouracil alone . rep confirms the findings of maloof et al . 
 ( 1952 ) of the greatly diminished capacity adenomas of a similar of heavily irradiated thyroid cells to undergo hyperplasia . morphology to those present in the thyroids of rats treated with antithyroid drugs were found in the animals given 5 , uc and 30 , uc 1131 alone , the latter appearing more effective than the former . 
doniach and pituitaries of this group are significant . there are two points of evidence that a certain amount of thyroxine was being synthetized . firstly , the animals grew from an initial weight of 100 g . , when the radioactive iodine was first administered , to the full weight of 250 to 350 g . , at the end of the experiment . secondly , the pituitaries showed a good coinplement of well granulated alpha cells . purves and griesbach ( 1946 ) demonstrated that the iiaintenance of the alpha granulations is dependent upon a supply of thyroxine ; the pituitary alpha cells disappear after thyroidectomy and are restored by the daily administration of one - fifth or more of the normal thyroxine requirements . nevertheless , the beta cells showed the typical changes of a raised level of thyrotrophic hormone secretion described by griesbach and purves ( 1945 )  . this suggests that the irradiated thyroid glands were secreting at least one - fifth but less than the normal thyroxine requirements . the diminished capacity of the irradiated glands to regenerate prevented a return to quantitatively normal thyroxine synthesis , thus perpetuating an increased thyrotrophic hormone output and accounting for the hypertrophy of the thyroid cells . another possibility to be considered is that irradiated thyroid cells differ from normal in requiring a higher level of thyrotrophic hormone stimulation in order to secrete a normal quantity of thyroxine . similar changes in the thyroids and pituitaries were observed to a lesser extent in the 301 , c 1131 treated rats and to a slight extent in the thyroids only of the 5 , tc i131 group . the bizarre thyroid cell nuclei observed by maloof et al . 
 ( 1952 ) in 1131 treated rats were seen in the present experiment , and were also accentuated in rats treated by additional thiouracil . one cannot say whether the neoplastic cells arise from these or from their apparently normal neighbours . they do , however , constitute evidence of irreversible irradiation changes . 
we do not know the life span of thyroid cells , but it is almost inconceivable that they could survive and function without replacement for15 months , a period of nearly one half the animal 's life span . neoplasms in general arise in tissues made up of actively dividing cells . they do not arise from irreversibly differentiated cells such as neurones or keratinized epidermal cells . the chances of the development of neoplastic thyroid cells following irradiation are likely to vary directly with the number of post irradiation mitoses which take place . this number will be increased in the thyroid bythyrotrophic hormone stimulation . the proportion of cells which become neoplastic is presumably related to the dose of irradiation . the absence of adenomas in the100 , uc 1131 treated group is considered to be due to a lower incidence of dividing cells following irradiation than that which followed in the 30 and 5 1tc groups . adenomas might have been found after afurther 6 months since they were present , albeit in small number , in the 100 , tc treated rats submitted to additional thyrotrophic hormone stimulation by a prolonged course of methylthiouracil . 
one of these was a malignant anaplastic carcinoma transplantable into hosts without thyroxine deficiency . it was not dependent on a high level of thyrotrophic hormone , did not concentrate iodine , grew rapidly , and killed its hosts within a few weeks . the main difference between the action of antithyroid drugs alone and combined with the carcinogen acetamidofluorene is that with the latter treatment thyroid tumours appear considerably earlier and in greater number . bielschowsky ( 1949 ) found also that acetamidofluorene produced thyroid adenomas in the absence of a chemical goitrogen in the regenerating thyroids of subtotally thyroipurves et at . 
 ( 1951 ) have concluded that neoplastic cells arise dectomised rats . spontaneously in the normal rat thyroid and that the carcinogen acetamidofluorene seems to act by speeding up their formation , their further development and growth , however , being dependent upon a high thyrotrophic hormone level . this has been produced experimentally by the prolonged administeration of antithyroid drugs , or by prolonged iodine deficiency ( wegelin , 1927 )  . the results of the previous ( doniach , 1950 ) and the present experiments show that the action of radioactive iodine is comparable with that of the carcinogen acetamidofluorene . in addition , radioactive iodine on its own leads by partial inhibition of thyroxine synthesis to a prolonged rise in thyrotrophic hormone level . 
from the description of goldberg and chaikoff ( 1952 ) , the experimental thyroid carcinomas which resulted from an extremely intense irradiation with radioactive iodine appeared to be of a high grade malignancy . the possibility that a carcinoma of low grade malignancy might become anaplastic must be considered after the findings in the transplantation experiment of purves et al . 
 ( 195 1 ) ; the probability however is unknown . dangers of carcinogenesis to humans treated with j131 . after an exposure to 1800 rep from j131 , maloof et al . 
 ( 1952 ) found a normal thyroid histology in rats 11 years later . but following 5800 rep and upwards , hypertrophied thyroid cells and increasing numbers with abnormal nuclei were seen , accentuated by a short course of methylthiouracil . these cells persisted tracer doses of j131 in clinical medicine fall into the first category , up to 1 1 years . those therapy doses which lower thyroid function below normal into the second . from the experimental findings in rats it would appear that 9000 rep to the thyroid in graves ' disease must act chiefly by functional damage leading to diminished thyroxine production rather than by actual total destruction of the gland . this is borne out by the brief descriptions of chapman and evans ( 1949 ) , williams et al . 
 ( 1949 ) and shapiro ( 1950 ) , who noted no histological evidence of gross destruction in the thyroids of thyrotoxic patients after treatment with radioactive the hyperplastic thyroid of the thiouracil treated rat is not strictly iodine . comparable with the hyperplastic thyroid of thyrotoxicosis . there is certainly as yet no clear - cut evidence that the latter is associated with a high blood level 200 i . 
a raised production of thyrotrophic hormone . the conditions may thus be fulfilled for tumour production , i.e. , an initial radiation damage followed by prolonged thyrotrophic hormone stimulation . one would expect the latent period in humans to be many years , and it is possible that in time thyroxine production and thyrotrophic hormone levels might return to normal and the chances of tumour production be much diminished . this could be ensured by deliberate thyroxine medication . 
on present data therefore the carcinogenic danger of radioactive iodine appears to be the initiation of irreversible radiation damage to the thyroid . this renders a greater number of cells liable to tumour formation when later stimulated by thyrotrophic hormone than in the non - radiated gland . 
a proportion of the resultant tumours are likely to be carcinomas , probably of a low grade malignanoy . anaplastic carcinomas may develop , especially when very destructive doses of i131 are used . the dosage lies within the known carcinogenic range . in view of the experimental findings in rats ( of various strains and in different countries ) , it is probable that the present methods of clinical i131 therapy in thyrotoxicosis may eventually prove carcinogenic . 
the radioactive iodine was found to increase the formation of thyroid adenomas as compared with controls in the 5 and 30 1tc groups but not in the 100 , uc group . out of 20 rats treated with combined 30 , tc 1131 and methylthiouracil 5 developed thyroid carcinomas . 
the radiation dosage range to the thyroid of 2270 - 16 , 200 rep in the latter experiment is likely to include the dosage aimed at , of about 9000 rep , in the treatment of graves ' disease . 
the carcinogenic danger of radioiodine therapy is regarded as due to the production of irreversible cells changes in the thyroid , which render them more liable than normal cells to tumour formation when stimulated to undergo hyperplasia . at the same time , a dosage strong enough to interfere with thyroxine synthesis by radiation damage to the thyroid leads indirectly to a prolonged increased output of thyrotrophic hormone from the pituitary . this constitutes a perpetual stimulus to hyperplasia of the thyroid cells . it is thought that the present dosage of i131 used in the treatment of graves ' disease may eventually prove carcinogenic . i am grateful to drs . 
of 9 : 10 : dimethyl - 1 : 2 - benzanthracene ; specific tumours of the thymus and lung , which are prone to spontaneous tulour developmnent in this strain , were induced by this treatment , whereas the mammary and subcutaneous tissues which are also prone to spontaneous tumour developmnent did not respond . the testis and kidney , in which spontaneous tumours have never been observed , and the spleen , lymph nodes and bone marrow , inl which tumours are not presumed to develop spontaneously , also proved insusceptible to this slight carcinogenic influence , and did not respond to a very powerful direct carcinogeniic influence . 
paraffin of the same batch as that used for analogous experiments performed with 9 : 10 - dimethyl - 1 : 2 - benzanthracene ( engelbreth - holm and rask - nielsen , 1947 ; rask - nielsen , 1948 )  . the technique of application was the same as that used in earlier experiments . 
rask - nielsen zanthracene - induced specific tumours , lymphosarcomatous hyperplasia of the thymic gland in 1i7 per cent , 6.7 per cent , 10 4 per cent and 13 per cent , the effective total being the number of mice whose survival time was at least that of the youngest tumour - bearing animal within all four groups , namely , three months . in addition a thymic spindle - cell sarcoma was found in one animal injected with methylcholanthrene . injections of three of the hydrocarbons into the lung induced thymic tumours in 2 , 2 and 4 per cent following the injection of benzpyrene , dibenzanthracene and 9 : 10 - dimethyl - 1 : 2 - benzanthracene respectively . 
the average latent period , the interval between injection and the death of the animal , following injections into the thymus of benzpyrene , dibenzanthracene , methylcholanthrene , and 9 : 10 - dimethyl - 1 : 2 - benzanthracene was 15 , 14 , 16 and 21 weeks , respectively ; the minimum latent period of the three latter hydrocarbons was 11 , 14 and 10 weeks , respectively . 
analogous negative results were observed by others , using mice from various strains ( esmarch , 1940 ; strong and smith , 1939 )  . experiments including the nho strain of mice , however , exhibit positive results ( strong and smith , 1939 ; strong and williams , 1941 ; strong , 1945 )  . 
from these experiments it is rather difficult to form an estimate of the susceptibility of the mammary tissue to carcinogenic hydrocarbons , since susceptibility is dependent on simultaneous hormonal stimulation as well as on the presence of the milk agent . since the present experinments included only non - breeding mice . 
the hormonal stimulation was at least not maximal . street mice have been used in all the experiments recorded here and the deductions made apply only to mice of that strain . the susceptibility to carcinogenic action of various tissues being genetically determined ( lefevre , 1945 ) it is necessary , in order to elucidate the susceptibility to a direct carcinogenic action of a particular tissue , to examine animals from different strains under identical experinmental conditions . such investigations , for the time being with application of 9 : 10 - dimethyl - 1 : 2 - benzanthracene to the thymus , lung and subcutaneous 116 r . 
moroney. from the leicester general h08pital and the colleges qf technology and commerce , leicester . received for publication juno 3 , 1952 . does chronic g " tric ulcer tend to undergo malignant change ? the importance of this question is beyond doubt , both because of the high incidence of gastric ulcer , and because many patients suffering from it are still treated medically over long periods before reference to a surgeon . 
the answer to it is also of considerable importance to the surgeon , owing to the great difficulty of assessing at laparotomy whether early malignant change may have already if the development of secondary carcinoma supervened on chronic ulceration . is really as common as some pathologists ' figures would appear to indicate , there might be a strong case for an e ' ven more radical gastrectomy than is usually carried out for such lesions . conflicting clinical and pathological evidence . the consensus of opinion in the medical literature is that gastric ulcer is a definite predisposing cause of carcinoma , but there is profound disagreement among pathologists about the percentage of cases ' which undergo such malignant change . 
udaondo ( 1939 ) , in an extensive survey of the world literatu ' re , found estimat - es of the incidence of " ulcer - cancer " which ranged frotn 0 to 100 per obviously , some statistical cent , with most intermediate values represented . uncertainty must be present in the majority of these assessmeints , yet there is usually quite inadequate evidence in the published data to lead to its detection . the only general conclusion that can be drawn from a study of these papers is that the authors ' confidence in the more extraordinary estimates seems to vary inversely with the amount of evidence at their disposal . since , according to the registrar - general 's returns , s ' omething like 14 , 000 people die each year from cancer of the stomach , it is not surprising that the heavy incidence of gastric ulcer in the population should have suggested that there was some connection between the two conditions . 
moroney as regards the stomach , and suggests that the incidence of mahgnant change must be very considerably lower than is suggested by most pathologists ' figures . livingstone and pack ( 1939 ) assess the average survival of patients admitted to hospital with inoperable cancer of the stomach at under 4 months , whilst the total duration of untreated primary gastric carcinoma is often under a year , from the onset of the first chnical symptoms until death supervenes . chronic gastric ulcer , on the other hand , appears to be relatively benign . 
the importance of this is amply justified in view of the gravity of the problem at issue ; particularly so since the ultimate verdict of malignancy must so frequently depend on a but , apart ' from the fact that no two histologists will microscopical opinion . whollv agree on the evidence which justifies such an opinion , the same pathologist not infrequently changes his criteria on reviewing the same series of specimens after a lapse of years . this , of course , is bound to occur as the result of increasing experience ; yet it means that the findings of an individual pathologist over a lengthy series of cases are only likely to be sufficiently homogeneous for statistical purposes if the time i - nvolved in the assessment of his data is not too lengthy , in terms of his tendency to change his pathological criteria . equally , it means that there is grave statistical objection to " averaging " blindly the data of until pathologists in general are able to come to an different pathologists . agreement to assess their findings on common criteria as to what constitutes malignant change in an ulcer and what differentiates this from a cancer arising de novo , each set of figures must be judged independently on internal statistical criteria , 1 4 . 
the only statistical task then remaining would be that of estimating the frequency of its occurrence . but no such agreement on the microscopical findings and their histological interpretation appears probable as ven amongst the most experienced workers . 
on the contrary , the conflicting evidence and extreme variations in the published pathological data make any attempt at mathematical analysis of them futile . as has already been suggested , the chnical evidence of malignant change in pre - existing gastric ulcer is a relative rarity in the collective experience of both thus , as soon as doubts are cast on the infauibility surgeons ' and pathologists . of histological diagnosis , it would seem that the only logical basis for continued belief in " ulcer - cancer " as a comparatively common entity is the analogy of the causative influence of chronic inflammation on subsequent development of carcinoma elsewhere in the body . such arguments by analogy may , however , be most fallacious , and can directly contravene the famous principle first enunciated by william of occam several centuries ago - " entia non sunt multiplicanda praeter nece88itatem . " the importance of this principle in scientific thought , of which it forms one of the main pillars , arises from the fact that it is impossible in practice to prove a universal negative by inductive processes . 
an example will make this clear . our ancestors , influenced by superstition rather than by scientific method , were constantly inventing ad hoc explanations for things in terms of witches , goblins , evil eyes , fairies and leprechauns which the whole weight of philosophy and christianity has not yet succeeded in completely eradicating . 
the survival of such hypotheses is understandable on the grounds that they are easy to conceive , better than no explanation at all and , once accepted , virtually impossible to disprove . 
and then we shall always be countered by the fellow who is positive he saw several the other night such hypotheses are by their very nature invulnerable . in contrast , the opposite scientific hypothesis that the genu8 leprechaun does not exist , except possibly as a freak of natural development in isolated instancesis extremely vulnerable . indisputable evidence of widespread loprechaun activity will entirely refute it , even if no actual leprechaun can be produced for examination . it is essential that this characteristic be present in any scientific hypothesis , so that the collection of sufficient opposition evidence can establish its falsity . adoption o the null hypothesi8 . applying these basic principles to the problem of " ulcer - cancer , " can it be said that this conception has really been adequately demonstrated clinically , 218 a . 
moroney or have we or even that it forms an essential entity in morbid anatomy perhaps accepted its existence with little more justification than our ancestors had in welcoming the leprechaun ? the very phraseology of the relevant literature - " the incidence of ulcer - cancer probably does not exceed x per cent suggests that this ma be the case , and that pathologists are themselves engaged in an attempt to depreciate its frequency of occurrence . it would seem , therefore , that a more profitable approach to the problem will be to attack it from the opposite direction , by the assumption of a " nufl hypothesis "  . in other words , to investigate the situation on the postulate that ulcer and cancer always occur independently in the stomach , in the aetiological sense , and that their occasional intimate association is entirply a matter of pure chance . failing the demonstration of " ulcer - cancer " as a necessary and unmistakable microscopical entity , such a null hypothesis could still be chanenged by statistical evidence that the relative frequency of occurrence of associated ulcer if the frequency and cancer is significantlv areater than the hypothesis predicts . of such associated lesions is so high that it is unlikely to have arisen by chance , the aetiological connection would be estabhshed , but even then this might only indicate that ulcer is a favoured site for the development of a carcinoma destined however , since it is on the relative frequencies of these to arise ' m any case . lesions that the null hypothesis must stand or fall , it is now important to survey the position statistically . 
and this is best carried out ' m general terms in order to avoid errors present in the conflicting pathological figures , and any profitless mathematical resolution of them . in - such an investigation , general figures of cancer incidence are obviously valueless . 
the only data worthy of consideration are thos ' e which have been verified pathologically , and the analysis must therefore be carried from the population at large up to the point where gastric specimens arrive at the patholoin the statistical model of the situation about to be constructed , it is gist . assumed that the specimens eventually arriving at the pathologist are drawn from a population ' whose characteristics with respect to both gastric ulcer and carcinoma may be regarded as stable over a period of time covered by any particular analysis ; and that , by virtue both of its size and its autogenerative properties , the composition of this population remains substantially unaffected by the withdrawal of the specimens . it will also be assumed that u1cer and cancer occur quite independently . development of the statistical model . in the population postulated therewill . 
i with the frequencies stated , the left - hand compartments of the diagrams representing the pyloric zone and the right - hand ones the lesser curvature ; the two lower sections showing the position with respect to the relative frequencies of double lesions occurring in the same region , compared with double lesions which are sited in separate regions of the stomach . 
we shall refer to such double lesions occurring in the same region of the stomacb as " amalgamated lesions " throughout the remainder of this paper , even if they are actually separate . within this limited selection of the whole data evidence might be found of a tendency for co - existing ulcer and cancer lesions to become amalgamated in this sense more frequently thad predicted by the null hypothesis , on wbicb basis the expected value of the ratio of amalgamat - ed lesions to non - amalgamated lesions is seen from fig . 
moroney the developmaint of a cancer , if not an actual aetiolooical factor in its developit will be - seen that this ratio has the statistical advantage that it does ment . not include the imponderable quantities u and c , but depends only on the site it wfll . 
i gives the expected distribution of an cases of if the null hypothesis is true , ulcer and carcinoma into the various categories . we should expect that a randoni collection of pathological specimens from the population at large would agree with the predicted ratios within the linlits of sampling error . but , of course , pathological specimens are not collected at random , and it is therefore essential to consider in some detail the effect of the selection chain along which patients have to pass before their stomachs become pathological specimens . the first group illustrated in the model shown in fig . 
1 , those cases which have neither ulcer nor cancer , will , of course , be entirely missing from the pathologist 's data . is it likely that ulcer of consider next the two categories of simple ulcer . the pyloric canal will have the same probability of pass ' mg along the reference chain to the pathologist as ulcer at the lesser curve ? prepyloric ulcer , in its uncomplicated form , is notoriously difficult to demonstrate radiologicany but , once diagnosed with certainty , is usually operated on without delay , since so many of these lesions prove eventually to have been malignant since their benign ulcers at this site tend to give rise to early symptoms from the onset . secondary pylorospasm which they cause ; yet they are often undetected until pyloric obstruction supervenes later , as the result of gradual stenosis , or unless acute perforation occurs . 
moroney hand , it is more often inoperable at laparotomy , and therefore is less hkely to reach the pathologist as a specimen . there is thus a complex selection mechanism operating along the whole of the reference chain , from the differential symptomatology in the patient through only after all the medical practitioner or consulting physician to the surgeon . this does the stomach become a potential pathological specimen , the likehhood of which is again govemed by the surgeon 's technical skill , and the influence of this on his decision whether to operate and finally to resect . moreover , still other factors may influence this selection of specimens , which will be different for each pathologist . 
the number of patients with gastric ulcer under observation by the medical side of the hospital wif be one of these , for associated " ulcercancer " lesions will have a better chance of being diagnosed early if the chronic ulcer lesions are kept under continual scrutiny by the consultant physician rather than being referred back to their own farnily doctors for routine treatment . further , the alteration in symptomatology resulting from the development of a secondary cancer in these cases is often relatively smah in the early stages . much depends on the vigilance and diagnostic skill of the physician concerned . the almost universal tendency among reporters is to ignore these very important weightings of their figures , and to hope that in the end they win cancel each other out over a large series . comparison of one series with another - or even the first and second part of the same observer 's series - soon demonstrates that this does not happen . 
the fact is that each pathologist 's series must be considered a unique set , not only because of his particular pathological criteria for " ulcer - cancer " but , perhaps even more , because his experience will include a unique set of weights in the data . introduction of the weighting factors . it is now evident that the simple model shown in fig . 
2 appropriate weighting factors have been introduced , which will take unique values for each individual pathologist in accordance with the varying nature of the particular selection chain along which his specimens have passed . 
the group of the population having neither ulcer nor cancer will be completely absent from such specimens and so will have weight zero . this is the only weight that the data of all pathologists will . 
and w6 , are the weightings for referability of associated ulcer and cancer losions in separate compartments of the same stomach - the pathological data for the frequency of wbich is scanty and its clinical recognition difficult . 
moroney if the individual pathologist could be given this ratio of the phcit values . weiahts concerned , and the values for u and c , he could then calculate the expected values for a and d3 , against which the observed values for these ratios in his own series could be tested . the evaluation of u and c from existin - a fi - aures is , however , rendered impossible by the effect of the reference chain and the different criteria adopted for the boundaries of the various stomach zones , nearly all the pubhshed work on stomach pathology conflicting in this respect . minor differences on the latter score between different series - may be partially taken up in the weights w7 and w8 , since all comparisons wif be intemal ones within each set of individual data , but the question is of academic interest only so long as marked reference chain separate independent research , deliberately designed to eliminate effects persist . moreover , the effect of reference chains , would be necessary for their estimation . even if the individual pathologist was supplied with values for u and c , he would still be unable to determine whether his observed values for a and a differed from the null hypothesis expectation for these ratios . 
the ratio w7 is inherently a property of his own series , representing the effect of the reference chain operating in his particular case . it can thorefore only be determined by using his own observed frequencies for u and c and checking them against those established manifestly , he cannot use his own data through such an independent survey . both to establish the expected value of the expressions d2 and d3 , and then to see if his observed values differ from expectation . 
the position , from the point of view of the individual pathologist , is that significant departures in this respect from the null hyporthesis predictions are inextricably confused with the effect of the reference chain ; and the same applies , even mo ' re , to the ratios a2 and a 35 which are under the influence of the reference chain to a greater extent . its implication is that no pathologist , however competent , and no consensus of opinion among pathologists , however widespread , can refute the null hypothesis on the basis of existing figures . it follows inevitably that the solution to this problem of the incidence of cc ulcer - cancer " must await a research project specifically designed to remove the insuperable obstacle facino , the individual pathologist - the effect of the reference chain represented by the weights w in our analysis . 
the results of such an investigation could then be evaluated by means of the ratios al and dl , which only involve the site ' distribution factors u and c . it would be essential for this purpose to establish some sufficiently accurate convention defining the admittedly , these are not boundaries between the various stomach zones . always easy to demarcate in practice , but the contrivance ot ' some effective standard in this respect ought not to present insuperable difficulties . the most hopeful way of establishing reliable values for u and c would appear to be by planned , clinical research at a number of large centres . the assiduous co - operation of all the local general practitioners surrounding these centres would be an essential , so that every case of a suspected stomach lesion reaches them at the earliest possible moment . 
the research would have to be based on several centres , whose respective findings could finally be checked for statistical homogeneity , and these cent - res ought to be carefully select ' ed . 
they should be situated at large - population centres which are growing , to minimise loss of patients during prolonged observation from drift to other areas ; and in provincial towns rather than in very large cities , where it might be difficult to trace their admission to , or treatment at , a large number of alternative hospitals for a gastric emergency or some intercurrent disease . if the establishment of such centres were practicable , it might be possible to eliminate the reference chain weigbts w for all practical purposes , and thus - trrive at reliable values for the site distribution factors u and c . there are , of course , dangers of leakage in the procedure outlined . in addition to the inevitable loss of some patients during the prolonged observation necessary , there is the certainty that some cases would not reach autopsy . moreover , there will be a fraction of the population who will not seek any medical advice until the disease has progressed too far for any statistical value to be elicited regarding the site diagnosis . this is not the place to discuss the details of such an investigation , but the essential problem regarding such losses is not so much their magnitude as whether they are likely to introduce bias in the determination of u and c . .if unbiased estimates of u and c could be ob - tained in this way , the data collected would suffice to test the null hypothesis expectations . cases with single lesions could be used to provide values for these quantities , and thus for the expected values of al and dl , of a high degree of accuracy deriving from the large number of observations on which they would be based . cases with double lesions would then give an adequate check on such results , using the incidence figures of ulcer and cancer in different stomach zones for this purpose in addition to those of amalgamated lesions in the sa - me zone . 
ghadially. from the department of pathology , the univemity of sheffleld . received for publication may 18 , 1951 . havingobserved that regeneration of hair in a clipped area on the back of a rabbit did not occur uniformly over the entire area , and occurred after varying intervals of time in different animals , it was decided to investigate the mode of hence , a band of hair , approximately regeneration of hair on the rabbit trunk . 4 inches wide , completely encircling the trunk , was removed from a few rabbits , and it was observed that the depilated skin was not uniformly pink , but that there were roughly linear bluish - black bilaterally symmetrical zones . besides these major dark zones , there were , in some animals , a few small irregular patches of a similar colour . 
the skin in these dark zones felt thicker than the rest of the after two days , when the animals were inspected , it was found that there skin . was prohfic hair growth in the dark zones , which continued until the hair reached its normal length , but during all this time no hair growth was observed in the pink zones . 
for a further period of 12 weeks with 2 per cent w / w , 9 : 10 - dimethyl - 1 : 2 - benzanthracene in lanohne , which was warmed to melting - point to facihtate apphcation . 
the animals were photographed at the beginning ofthe experiment and on numerous subsequent occasions . skin biopsies were taken at intervals from 3 experimental and 2 control animals . these were fixed in alcoholic bouin and sections were stained with haematoxylin 354 h . 
with the appearance of the papillomata changes began to occur in the surrounding skin ; the hair on the painted side now failed to show the vigorous growth it had shown so far . 
as already stated , by this time the whole of the skin on the painted side had attained a uniform appearance , and no distinction between active and quiescent zones was seen . 
the epidermis was much thicker than either the this change appeared to extend down active or quiescent skin before painting . into the folhcles , which were more prominent and deeply seated and showed the usual arrangement in groups . 
mottram ( 1944a , 1944b ) showed that the skin of mice can be sensitized by painting with croton oil , so that a subsequent single application of 3 : 4 - benzpyrene leads to an abundant production of tumours and a single application of carcinogen followed by repeated applications of croton oil also procluces tumours . 
hence mottram ( 1944b ) concluded that carcinogenesis was composed of three phases : ( 1 ) a " sensitizing factor " which could be brought about by preliminary treatment with croton oil , acting presumably in a non - specific manner by causina hyperplasia , on the supposition that proliferating cells were more responsive than resting cells to the specific ( 2 ) a " specific cellular reaction " induced carcinogenic action which followed . by the specific action ( even of a single application ) of a carcinogen , this representing the essential neoplastic change . ( 3 ) a " developing factor " responsible for the actual appearance of a visible wart , and produced by croton oil of by a carberenblum and shubik ( 1947a , 1947b ) confirmed the latter part of his cilnogen . observations , but found no evidence to suggest that a prelimi - nary croton oil hyperplasia had an augmenting influence on tumour production , which would 356 h . 
the depilated skin showed bila ' terally symmetrical dark zones which showed prolific hair growth within a few days , the rest of the skin remaining 9 : 10 - dimethyl - i : 2 - benzanthracene.in lanoline was painted on the left bald . half of 6 animals ; the remaining 4 were painted with lanoline . 
the following changes were observed : ( 1 ) within 30 days the painted area in the experimental animals became dark and showed prolific hair growth , while on the opposite side there was suppression of hair growth , the skin becoming bald . ( 2 ) in all animals initial papiromata appeared at the site of an originary active zone . 
the first papilloma appeared after 53 days . ( 3 ) after the appearance of papillomata hair growth ceased and zones of activity appeared on the opposite side . ( 4 ) the first carcinoma appeared after 81 days at the site of an originary active zone . it was concluded that ( a ) the naturally occurring zones of increased mitotic activity which continued for some time after the first painting must have acted as a promoting agent ; ( b ) it seems more probable that the tumours arose from hair folfcles , and not the epidermis , as the former are the site of increased mitotic activity during hair growth . it gives us great pleasure to express our thanks to professor h . 
eye hospital , southwark , london . received for publication december 15 , 1950 . in a previous communication ( barker , dhar and parsons , 1949 ) the effects on sarcoma growth in mice produced by treatment with some derivatives of purine and pyrimidine compounds were recorded . further experiments described below have extended the range of pyrimidine compounds studied , and an attempt has been made to correlate molecular structure with biological activity . 
the compounds employed fall into the forowing groups : ( 1 ) cytidyfic acid and alhed compounds . ( 2 ) barbituric acid , its imino derivatives and related compounds . ( 3 ) acyclic analogues of some of the above compounds . pure line or stock mice were grafted with the homologous c57 or the heterologous 837 or crocker sarcomas . 
the technique for treatment was similar to that previously described ( barakan , barker , guband and parsons , 1948 ) , the standard dose ( 0 - 025 g . ) being reclueed in proportion to the toxicity of the particular compound . 
table i summarizes the statistical analysis has shown that the differences in tumour results obtained . weights for treated and untreated grafted mice are significant for groups 2 , 10 , ii and 12 . 
cytosine and uracil , on the other hand , are not utilized in nucleic acid synthesis . this suggested that examination should be made of the action of cytosine when injected into grafted mice . 
the behaviour of 4 - aminouracil appears to be specifically associated with its chemical structure , since the introduction of the sulphur atom at position 2 to give 2 - thio - 4 - amino - 6oxypyrimidine is sufficient to abohsh its inhibitory action . it is considered possible that the marked difference in activity between 4 - aminouracil and 5aminouracil is due to the difference in the electron - availability at the free positions of the two molecules ( positions 4 and 5 respectively ) , which would influence the abihty of the tissues to metabohze the compounds by oxidation . since 4 - aminouracil may also be regarded in its tautomeric form as 4 - iminobarbituric acid , a systematic study was made of barbituric acid and its derivatives , all of which are characterized by high chemical reactivity at position 5 . 
parsons ( 3 ) the 8pecificity of the heterocyclic ring . in the above discussio ' n of the biological act ' ivity of the varl ' ous constituent groups in the pyrimidine derivati - ves the structures were considered only as part of the heterocychc system . in order to determi e whether the ring structure of the compounds is essential to the activities observed three acychc compounds in the first , carbamido - acetamidine , the whole of the structure have been tested . of 4 - aniinouracil is present except for the carbon atom at position 2 . in the second , acetyl urea , the configurations at positions 1 , 2 , 3 and 6 of 4 - aminouracil the third , acetamidine , possesses a similar grouping of atoms to are inaitated . that at position 4 of 4 - aminouracil . 
bartholomew 's hospital , london . received for publication february 3 , 1953 . this investigation was planned at a meeting on august 29 , 1949 , at the baggeridge colliery , staffordshire , ' arranged by a . 
some small series of cases suggest that silicosis may predispose to cancer of the ' lung , but the general indication of the literature is that silicosis is not active in this respect . 
hence the area where there is most pneumokoniosis shows a low incidence of lung cancer . the prevalence of pneumokoniosis in any area is indicated by the number of compensation cases arising under the workmen 's compensation acts and the industrial injuries scheme . 
4. - numbers of deaths from cancer of the lung in coal miners , and of wage - earners on colliery books , from 1921 . 1930 1940 face may be included , but this criterion must be difficult to apply to non - stationary however , in practice , the doubtful cases may not be very numerous , workers . and one must match the entry on the death certificate as nearly as possible with one of the official occupations . the increase in deaths from cancer of the lung . the increase in deaths per annum attributed to cancer of the lung from 1921 to 1950 is shown in tables iii and ix and in fig . 
some possible reasons for these differences are discussed . ( 2 ) in the last 30 years the mortality from cancer of the lung has increased in coal miners in the same way as in the general population ; in view of the peculiarities of the miner 's life , this fact calls for further inquiry . ( 3 ) the incidence of cancer of the lung differs ( a ) in face - workers and in other it is low coal miners , and ( b ) in the various coalfields of england and wales . in the south wales coalfield , where pneumokoniosis is most prevalent . we are indebted to the officers of the national coal board named at the begining of this paper for the suggestions which initiated this inquiry . 
w.7. received for publication august 22 , 1949 . it has been suggested from studies of the action of inhibitors of s - metabolism on tumour induction by carcinogenic hydrocarbons that s - metabolism and the this association is process of carcinogenesis are closely linked ( crabtree , 1947 )  . emphasized when the properties of azo - carcinogens are considered . 
the products of their metabolism have been shown to inhibit the activities of urease ( potter , 1942 ) and succinoxidase ( elson and hoch - ligeti , 1944 ) , two enzymes dependent kensler , dexter and rhoads upon intact sh - groups for their proper functioning . ( 1942 ) studied the relative inhibitory action of a series of " split - products " on a standard diphospho - pyridine - nucleotide system , and sought to correlate this suggestive activity with the carcinogenic power of the parent azo - compound . parallelisms were found , but a clear - cut relationship did not emerge . much of this work has been based on the conception ( experimentally verified for p - dimethylaminoazobenzene by stevenson , dobriner and rhoads ( 1942 ) , and for azo - benzene by elson and warren ( 1944 ) ) that an azo - carcinogen is initially metabolized by the process of reductive fission with the liberation of an amine and a diamine , and that the latter is the effective carcinogen by virtue of its capacity to interfere with normal enzymic . 
a survey of the azo - compounds so far tested for carcinogenic activity makes it evident that in some cases the amine moiety of the reduced molecule plays a significant role in determining potency , e.g. 
on comparing p - dimethylaminoazobenzene with its p ' - methyl derivative , the introduction of the p ' - methyl group causes a change from a very strong to a very weak carcinogen . the present work was undertaken to probe this problem further . isomeric aminoazotoluenes were chosen as suitable for a study of the relative contributions made by the amines and diamines which would arise from their reduction . 3 ' - azotoluene ( o : o ) and the non - carcinogen well - known carcinogen 4 ' - amino - 2 2 ' - amino - 4 : 5 ' - azotoluene ( p : p ) served as bases of reference , and their behaviour was compared with that of the hybrid compounds o : p and p : o . 
water , at room temthe crystalline hydrochloride suspended in etoh perature ( mehner , 1902 )  . was neutralized with ammonia , and an equal volume of boiling water added . the free base was re - crystallized from aqueous etoh . 
the first characteristic abnormalities visible in the livers of rats differed from those commonly observed in the livers of mice , though the final pictures of tumour emergence were similar in both species . building up the general picture four grades of cellular change have been used . in rat livers perilobular necrosis constituted the primary change , and this was followed by regeneration of liver cells in the necrotic areas . in some regions of regeneration microscopic nodules of hepatoma made their appearance , and later these became visible to the eye as dull grey patches on the surface of the lobes . often a simultaneous intense proliferation of bile ducts was manifest in one or more lobes , and the early phases of cholangioma formation would coincide with the development of small hepatomas . 
no metastases were ever found . in mouse livers no preliminary necrosis was observed , but the cellular pattern became abnormal with great irregularity in the disposition and size of the nuclei . further distortion was accompanied by the formation of cells with giant vacuolated nuclei , which preceded the emergence of microscopic hepatomas . 
the fully developed hepatomas of mice were more malignant than those of rats as judged by the degree of atypical growth , though again no metastases were ever found . it will be noted that the earliest histological changes mentioned sometimes occurred in both rats and mice consuming the basal diet without dye - addition . this response of the liver to diets inadequate in several essential food factors may well reflect the basic disturbance of liver function which is a necessary precursor to the carcinogenic action of azo - compounds . ( b ) relative carcinogenic activity of the six isomeric amrnoazotoluene8 . considering the data summarized in table i from the point of view of the relative carcinogenic potency of these isomers , three points emerge : 1 . 
crabtree the possible influence of sex on the growth rate was assessed by keeping the rats of each series separated into three sub - groups containing respectively 10 males , 10 females and 5 males + 5 females . 
2. - growth curves of rats fed on the basal diet , with addition of 0 06 per cent oor p - toluidine . in the work reported here , the two carcinogenic isomers would yield o - toluidine , while 3 of the 4 growth - stimulating isomers would yield p - toluidine as the primary products of reductive fission . the possible effects on growth of these two toluidines were therefore tested . forty - five rats of the 52 strain , each weighing 110 - 130 g . , were divided into three groups of 15 . 
two groups were fed on the basal diet containing 0.06 per cent of either o - toluidine or p - toluidine , and the third group received the basal diet alone . 
each group of 15 rats was subdivided into three separated groups of 5 males , 5 females , and 5 containing males and females . in the latter group two litters were born during the first 3 months , but none subsequently . 
on the other ha ; nd , miller and miller ( 1947 ) emphasize the importance of the protein constitution of the host , and associate the likelihood of tumours developing with the degree to which an azo - compound is " bound " to the proteins of rat livers . in the case of rats consuming p - dimethylaminoazobenzene this fixation is prominent , but does not occur in species resistant to the carcinogenic action of the substance . these differences can only be attributed to the special properties of the proteins characteristic for each species . the " split - product " hypothesis . kirby ( 1945 ) has reviewed the work bearing on this hypothesis of the mechanism of carcinogenesis by azo - compounds , and throws doubt on its validity by citing several examples which fail to conform with its main premises . 
the experiments demonstrated that o : o and o : p were carcinogenic , while p : p and p : o were non - carcinogenic . reviewing these results in the light of the " split - product " hypothesis , no correlation between carcinogenic activity and the potential diamine fission products is found . 
p : p and o : p should yield the same " innocuous " o - toluylene diamine , while o : o and o : p should yield the same " active " .p - toluylene diamine , but the facts are otherwise , since each of these pairs contains one carcinogen and one non - carcinogen . by contrast , when the amine halves of the reduced molecule are considered , the results show that the two isomers which should yield p - toluidine on reductive fission - p : p and p : o - are non - carcinogenic , while the pair yielding o - toluidine in brief , if carcinogenic activity is indeed - o : o and o p - are carcinogenic . a consequence of the action of the " split - products , " it is the amine , and not the diamine , which determines the subsequent biological response . the literature contains several examples of the controlling influence which a methyl group exerts on carcinogenesis , when attached at a para position with miller and baumann ( 1945 ) compared the activities respect to the azo - group . of the o ' , m ' , and p ' methyl derivatives of p - dimethylaminoazobenzene with that of the parent compound . 
the m ' - compound was extremely active ( 7 rats out of 8 bearing liver tumours in 4 months ) , the o ' - compound moderately active ( 4 rats out of 9 bearing tumours in 8 months ) , and the p ' - compound was very weakly carcinogenic ( 1 rat out of 10 with a tumour in 10 months )  . 
nagao ( 1941 ) also found that p ' - methyl - p - dimethylaminoazobenzene was only a weak carcinogen , and that the substances formed by placing a further methyl group in the ring carrying the dimethylamino - group were entirely innocuous . sugiura ( 1948 ) tested a series of derivatives of p - methylaminoazobenzene containing an additional methyl group in the o ' , m ' or p ' positions . the order of potency - m ' > o ' > p ' was similar to that mentioned above . all these observations seem relevant to the present work . 
the common feature is the great loss of tumour - producing activity which results when a methyl group is added in the p ' position to the parent carcinogenic molecule , forming derivatives with the potentiality of liberating p - toluidine on reductive fission . a working hypothesis is put forward tentatively in an attempt to co - ordinate the data outlined above . 
when the structural features and the biological action of these six isomeric azotoluenes are considered , a correlation is evident between their growth - stimulating properties , their lack of carcinogenic power , and the presence of an additional methyl group in the position described . 
crabtree and hilbert ( 1888 ) fed the three isomers of acetotoluidine to dogs . o - acetotoluidine was oxidized to an aminophenol and isolated as methylbenzoxalone , while mand p - acetotoluidines were excreted as acetylated amino - benzoic acids . it may therefore be conjectured that all azo - compounds containing a methyl group at a position para to the azo - group will yield p - aminobenzoic acid as a final product of their metabolic degradation within the rat . the experiment pictured in fig . 
six isomeric aminoazotoluenes have been tested for their carcinogenic activity in the livers of mice and rats , when added at 0 06 per cent concentration to a standard , semi - synthetic diet . 
since the detoxication of p - toluidine can produce p - aminobenzoic acid , the possible relation between folic acid metabolism and the mechanism of cancer induction is considered . i am greatly indebted to e . 
bruzzone. from the department of experimental medicine , national health service of chile , santiago . received for publication june 9 , 1949 . abdominal fibroids induced in the guinea - pig by a - oestradiol ( nelson , 1937 ; lipschutz and iglesias , 1939 ) are prevented when progesterone or other 3 - ketosteroids are administered simultaneously with the oestrogen ( lipschutz and vargas , 1941 ; lipschutz , 1944 ; lipschutz , iglesias , bruzzone , fuenzalida and riesco , 1948 )  . 
as a differerit method of approach , studies are in progress on the antagonisation of the antimitotic action of tetra - sodium 2 - methyl - 1 : 4 - naphthohyd - roquinone diphosphate ( compound 1 ) by nucleotides , nucleosides , purines and pyrimidines and some other compounds ( mitchell , 1950 , 1951 )  . this paper is an account of the cytological effects of a series of 29 selectect 318 j . 
marrian. details of preparations have been published as follows : compounds 11 and iv ( friedmann , marrian and simon - reuss , 1948a ) , compounds xx - xxiii ( friedmann , marrian and simon - reuss , 1948b ) , compounds viii , ix and xxiv ( friedmann , marrian and simon - reuss 1952a )  . we are indebted to f . 
the cytological effects are considered in relation to those of compound 1 . of especial interest from the point of view of the selection of compounds as possible therapeutic radiosensitisers is the appearance of mitotic abnormalities in concentrations which produce mitotic inhibition without gross toxic effects on the resting cells . 
the most important abnormalities studied are chromosome fragmentation , anaphase bridges , metaphase chromosomes with regions of impaired stainabihty and with irregular and beaded structure , metaphases with clumping of the chromosomes , arrested metaphases and other spindle abnormalities , of which many of the types described by barber and callan ( 1943 ) have been noted . 
as already discussed ( mitchell and simonreuss ' 1952 ) , it seems likely that the most relevant cytological abnormalities are chromosome fragmentation , anaphase bridges and chromosome aberrations in examples of the various types of cytological effects observed with this general . series of compounds are show - n in lb ' ig . 
on the basis of the concentration required to procluce 50 per cent mitotic inhibition , hydroquinone diphosphate ( xvi ) is about twice as active as hydroquinone ( xv ) , but the increase of mitotic inhibition with concentration is much more rapid in the case of the former than with the latter . 
somewhat sirnilarly , for 50 per cent mitotic inhibition , phenyl phosphate ( xviii ) is more active than phenol ( xvii ) by a factor of about 20 , but the increase of mitotic inhibition with concentration is much less rapid in the case of phenyl phosphate than for phenol . the only exception so far to the finding of increased antimitotic activity on phosphorylation is in the case of resorcinol . friedmann and simonreuss found that 50 per cent mitotic inhibition is produced by resorcinol at 6 x 10 - 8m concentration but by resoreinol diphosphate at about 5 x 10 - 7m . - kt these concentrations both compounds showed few abnormal mitoses . the usual increase in antimitotic activity on phosphorylation is not associated with any change in character of the cytological effects . 
the frequency of cytological abnormalities appears to be of the same order for the phospborylated and non - phosphorylated compound at concentrations which produce the same mitotic inhibition . ( 2 ) methyl group . the methyl group plays an important part in modifying the biological action , especially from the point of view of practical applications . moreover , among methyl derivatives , the limitations of the use of mitotic inhibition as a sorting test are emphasised . in the chick fibroblast cultures , compound 11 is more active than compound i as a mitotic inhibitor by a factor of about 1000 , and it also shows potentiation of mitotic inhibition in combination with x - rays . however , compound ii appears to be ineffective as a radiosensitiser for the jensen rat sarcoma , and did not produce a focal reaction in the tumour after intravenous injection in a few it may be mentioned that its use in man in high doses may be cases in man . undesirable because of the possibility of induction of cataract , analogous to the experimental naphthalene cataract in rabbits ( boume , 1937 )  . it is of interest to compare the methyl free compound 11 , tetra - sodium 1 : 4 - naphthohydroquinone diphosphate , with the 2 - methyl derivative , compound 1 , and with the 2 : 3 - dimethyl derivative , compound xxviii . 
no other compound examined showed this behaviour . compound 11 showed an increase in the number of metaphases by a factor of about 2 - 5 on increasing the concentration for 50 per cent mitotic inhibition by a factor of io . it bas been show - n in the preceding paper ( mitcher and simon - reuss , 1952 ) that compound i is somewhat unstable in aqileous solution in the absence of oxygen at 39 ' c . 
one may speculate and suggest the possibihty of combination of the phosphates with arginine side cha ' ms of histones the chromosome structure . biological action of 2 : 3 - dimethyl - 1 : 4 - naphthohydroquinone diphosphate , tetrasodium salt ( compound xxviii )  . - the biological action of compound xxviii differs considerably from that of ar the other c ' , ompounds . 
even in concentration 2 x 10 - 6m ( though not at 5 x 10 - 7m ) , which produces no detectable mitotic inhibition , there is metaphase accumulation by about 50 per cent above the fraction of metaphases in the controls , together with spindle abnormalities . as an example of the frequency of relevant chromosome aberrations , it will be seen from table iii that after application in concentration 4 x 10 - 6mfor 24 hours , out of 534 cells in mitosis , there were 74 cells in metaphase showing chromosome fragmentation and 17 anaphase bridges . it appears that with compound xxviii there is more rejoining of chromosome breaks than with moreover , compound xxviii only any of the other compounds examined . starts to show effects on resting cells in high con ' entrations associated with almost complete mitot - ic inhibition ; this behaviour suggests low toxicity . detailed studies of the combination of the effects of compound xxviii and x - radiation on the chick fibroblast cultures have been made by the summation method . 
the results of one experiment are given in table iii . it is concluded that under the experimental conditions , the combination of the action of compound xxviii and of x - radiation shows potentiation of mitotic inhibition and that the mechanism of action of the two agents is different . ( 3 ) carboxyl group . the compound most closely resembling compound xxviii in biological action is compound xi , the hexasodium salt of 1 : 4 - dihydroxy - 2 : 3 - naphthalene dicarboxylic acid diphosphate . replacement of the methyl groups in the 2 and 3 positions in compound xxviii by carboxyl groups slightly reduces the activity as measured by mitotic inhibition and considerably reduces the metaphase effects , so that at 50 per cent mitotic inhibition there is metaphase accumulation by about 25 per cent and sphadle abnormalities are much less frequent . 
compound vi produces clumped metaphases with some chromosome fragmentation and many undivided telophases , but practically no anaphase bridges . metaphase accumulation appears at a concentration which produces about 70 per cent mitotic inhibition ; this is associated with negligible effect on resting ceus . the weak metaphase arrest with compound vi is perhaps associated . 
simon - reuss 5 - methyl - 4 : 7 - thionaphthene quinone ( xxvii ) is active when tested in solution in cerosolve and gum ghatti , and produces 50 per cent rnitotic inhibiti ' on m approximately 5 x 10 - 6m concentration . 
mitotic inhibitor than compound ( xiv ) by a factor of 2 , but produces metaphase accumulation and abnormal mitoses , mainly clumped metaphases , chromosome fragmentation and multipolar cells , at all concentrations showing mitotic inhibition . the tetra - sodium salt of hydroquinone diphosphate ( xvi ) is a very active compound , and produces 50 per cent mitotic inhibition at almost exactly the same concentrat - ion , 3 x 10 - 9m , as does compound il in this region of concentratioin it also produces few abnormal mitoses . 
simon - reuss variably increased by phosphorylation , often by a factor of 5 or more . these results and those with dinitrophenol suggest that blockage of the entry of cells into mitosis depends upon inhibition of synthetic processes mvolving phosit is interesting that from the non - protein moiety of a phosphorylation . phorylase has been isolated a yellow pigment tentatively identified as 2 - methylit seems unlikely that at the concentrations 1 : 4 - naphthoquinone ( buell , 1952 )  . used there is interference with glycolysis ( gemmill , 1949 )  . the comparison of the cytological effects of different compounds of closely related chemical constitution and also of ionising radiations makes it possible to separate some of the main types of mechanism of action of these agents on proliferating cells as follows : ( a ) blockage of the entry of ceus into mitosis , without specification of the exact time of the action and presumably including pre - prophase and antephase this is the characteristic action of compound 1 , and in this case is inhibition . other less clearly associated with relatively few chromosomal abnormalities . defined examples are compounds x and xv . ( b ) metapbase arrest and disturbances of the spindle mechanism apparent - ly examples of this type of action in association similar to the effects of coichicine . with ( a ) are compounds 11 , vi , viii , xi , xxvii and xxx at the higher concentrations , compound ix at low concentrations and compound xxviii iso - naphthazarin produces metaphase over a wide range of concentrations . arrest with little or no inhibition of the entry of cells into mitosis . ( c ) chromosome , including chromatid , brea - kage - with varyina degrees of rejoinability of the broken ends . 
these experiments suggest that the tetra - godium salt of 2 : 3 - dimethyl - 1 : 4naphthohydroquinone diphosphate , compound xxviii , is the most suitable compound for further investigation as a possible therapeutic radiosensitiser . in addition to the help acknowledged in the text , e . 
paper the clinical aspects of androgen therapy in 70 cases of advanced mammary cancer in women are described , the literature surveyed , and the effect of androgens compared with that of castration carried out by surgery and by the prophylactic use of androgens in mammary cancer as x - irradiation . described by prudente ( 1945 ) is not discussed here . the investigation was begun in december , 1947 , and since then sufficient experience has been gained to permit an assessment to be made in general terms of the place of androgens in treatment . 
the pellets were inserted through a skin incision 1 clong in the lower abdominal quadrants , and pushed through radially to rest on the external oblique aponeurosis between 5 and 10 cfrom the incision , the pellets lying at points on a circle with the incision as centre . absorption from these pellets depends on a variety of local factors , including their relation to surrounding fat , adjacent fascia , lymphatic plexuses and encapin three patients who came to autopsy 55 , 82 and sulation by fibrous tissue . 93 days respectively after insertion , the pellets were removed , allowed to dry for 48 hours and weighed . 
per month for their evocation when injections of testosterone propionate are used ( geist , salmon and gaines , 19388 ; geist , salmon and walter , 1940 ) , representing daily doses of nearly double the maximal ( initial ) daily absorption from ten 100 meg . 
were given 3 - 7 times weekly , but later opinion came to advocate larger and larger doses of the order of 100 mg . daily , or even twice daily . the authors of the latest progress report of the council on pharmacy and chemistry ( 1949 ) , however , no longer favour heavy dosage , and consider that there is no advantage in exceeding 150 mg . 
daily for the first 2 - 4 weeks , given . to halve this dose if the patient is responding favourably , or to continue for another all 30 patients who derived month if there is no evidence of specific benefit . benefit from androgen therapy showed clear evidence of it within 8 weeks , and in most cases within the first month . 
no patient who failed to respond after 8 weeks of treatment did so after a longer interval , and it is probably not worth continuing treatment after 10 weeks in the absence of specific response . cannot be said from the small number of cases in this series whether such cases should be transferred to testosterone propionate injections , but the general impression gained has been that patients likely to benefit substantially from androgen therapy are extremnely sensitive to its action , in whichever form it is given , and do as well on doses just large enough to suppress inenstruation in the average premenopausal woman as they do on inuch larger doses . 
on the other hand , most cases which fail to respond to the smaller doses do no better when the dose is increased or the route of administration changed , but the minority which does so led to the tendency already mentioned to advocate heavy dosage . patients given methyltestosterone were advised to leave the tablets ( 50 mig . ) under the tongue , and to allow them to dissolve without chewing or swallowing . the time taken for solution varied between 10 and 30 minutes in different individuals , and a few patients disliked the bitter taste of the substance . duration of treatment in cases responding favourably . no definite recommendation can be given with regard to the length of time cutler and schlemenson , reporting 19 cases , administration should be continued . advised continuing injections until palliation was no longer being obtained the american council already referred to ( cutler and schlemenson , 1948 )  . found that the most favourable results were obtained when injections were conin this series it has frequently tinued until a total of 3 0 g . 
methyltestosterone daily , commenced 6 months after the second implantation and continued for a further 4 months . widespread evidence of fresh activity of the carcinoma then appeared , accompanied by recurrence of pain testosterone propionate injections 100 mg . 
as far as the effect on menstruation is concerned , it may therefore be said that the hormone absorbed from 50 mg . sublingual methyltestosterone is less potent than 10 mg . 
testosterone ( table marked facial hirsuties was present in the second of these cases at the ii )  . time of death , 12 weeks after implantation , not more than 250 mg . 
amenorrhoea persisted for weight gain , 4 months , and weight gain continued during the same period . and subjective benefit , occurred without other side - effects in patients who were it therefore appears that the given methyltestosterone in 40 mg . 
in the first month are bilitated patients is very striking . common , and with the other " tonic " effects are independent of the specific effect in case 65 for example 7 lb . 
in the second 4 months . clinical oedema , however , was seen in only 3 cases , and was always slight in in a few of the amount , and confined to the ankles and lower half of the legs . patients with post - mastectomy oedema of the arm , an increase in the swelling of the arm followed testosterone administration , but this was more closely related to spread of the disease than to the treatment , for when lesions regressed , the lymphoedema sometimes diminished , even when rapid increase in weight was during the initial period of weight gain , some of the occurring , as in case 44 . patients stated that they passed less water , but no measurements have been carried out . much of the initial rapid weight gain is due to the effects of electrolyte , and particularly sodium retention , which holds equivalent quantities of water in the body . this effect is a feature of the activity of many steroid hormones , notably progesterone , oestradiol , and of kendall 's compounds desoxycorticosterone , e and f . 
daily for 6 months and 1 year respectively . they were abundant across the back , and were also present on the cheeks and chin . the skin , which was heavily infected , had the lumpy appearance typical of adolescent pustular acne . in case 44 hirsuties was nminimal , consisting of a sparse growth of soft hairs on either side of the upper lip , but was much more developed in case 63 . in 7 cases facial hirsuties reached an extreme degree , the sides of the face being covered with thick downy hair between 1 and 2 clong ( cases 7 , 9 , 24 , 25 , 56 , 63 )  . in cases 9 and 56 , after 6 months ' treatment the hairs increased in calibre , and resembled the stiff bristles of the male beard . there was some variation in the changes in the scalp hair . 
when fully developed they form a characteristic syndrome , which has been observed in 3 patients in this series . it was first seen when 2 patients ( cases 10 and 45 ) were given 80 mg . 
testosterone. in case 10 , after taking the tablets for 2 days , the patient complained of extreme weakness , drowsiness , severe headache , nausea , and increase in the dyspnoea which was her initial complaint , and due to pulmonary and pleural metastases . 
the symptoms subsided rapidly after she stopped taking the tablets . in case 45 the patient took the tablets for 5 days , during the last 3 of which she felt generally ill , nauseated , and complained of increase in the severity of her backache . 
on the 5th day she was distressed by vomiting , abdominal distension and pain , and took no more tablets , after which her condition improved rapidly . one week later she again attempted to take the tablets , but with exactly the same result . case 30 was a woman of 45 who had been previously treated for 2 years by repeated doses of x - rays for widespread skeletal recurrences for a stage iv carcinoma , and had had induction of an d . 
daily for one month , towards the end of which she complained of increase in the severity of her pains , dizziness , nausea and vomiting , and general malaise . 
the symptoms abated within a day or two of discontinuing the drug . she was asked to resume the tablets a fortnight later , but the symptoms recurred after one week . case 47 , doses of 35 mg . 
by a " moderate success " is meant a case in which symptomatic relief was short - lived ( mostly less than 6 months ) , or not accompanied by considerable objective evidence of regression or by restoration of function . all other cases are classed as failures , even when non - specific benefit was marked and increased the patients ' range of activity . 
testosterone propionate three times weekly without benefit after 6 weeks . she is classed as a " moderate success " in view the remaining patients obtained less benefit , and 3 were of the initial response . never able to leave their beds , although all volunteered that their pains were improved . one of these declared that she had not felt so well nor had so little pain for 2 years , 20 months of which had been spent in bed . case 66 was a woman of 44 years with extensive metastasis to the spine which was responsible for gross collapse of the bodies of dorsal vertebrae 7 - 11 . had been unable to walk or stand for 6 weeks owing to severe proprioceptive loss in both legs , a short course of radiotherapy having conferred no benefit . 
a mild paraplegia was present , and there was some impairment of sensation to light touch , deep pressure and pinprick from the level of the umbilicus downwards . there was , in addition , a girdle of almost.complete anaesthesia to touch and pinprick over the distribution of dorsal segments 7 - 10 . curiously , sensation over the paravertebral regions of these segments was normal . there was no hyperalgesic zone , and no disturbance of sphincter control . the patient was given 28 daily injections of 100 mg . 
two other patients with dyspnoea as one in cases 3 , 10 , 24 of several symptoms obtained some relief after treatment . and 63 the rapidity with which the breathing improved was astonishing , and colnparable with the speedy relief of bone pain in the cases mentioned above . in case 25 , however , with dyspnoea at rest , no significant improvement had ten oz . 
galton with a conjugate defect of elevation , thought to indicate a lesion in the anterior this lesion could have been of vascular origin , part of the oculomotor nucleus . or might have been due to a metastasis . that the latter was the more likely is suggested by the development 6 months later of diminished sensation to touch this patient took methyland pinprick over the right trigeminal distribution . testosterone 50 - 100 mg . 
daily thereafter , also showed marked regression of axillary and supraclavicular lymph nodes , though some of these were still just palpable 4 months later . this patient relapsed 11 months later . case 35 was a stage iv growth in a woman of 36 years . she had nodes in both axillae . 
one year previously she had been given radiotherapy to the primary growth and to the axilla , and 6 months previously had been given pelvic five months later she was given irradiation for induction of the menopause . * methyltestosterone sublingually 80 nig . 
31 premenopausal cases with bone deposits . of the 31 cases , 3 received palliative radiotherapy to the affected bones ( cases 2 , 27 , 69 ) and are not included in the figures . 
of the remainder , 8 were premenopausal cases , all but 2 of whom received great benefit from androgen therapy . three of these were the bedridden patients referred to above , who were able to resume their normal activities within a few weeks of being treated with testosterone , and remained in good health for over 1 year in 2 cases ( cases 3 , 7 , 37 )  . 
the fourth was a housewife of 45 with extensive spinal , costal and pelvic deposits , whose activities were drastically curtailed by pain . she also secured complete relief from pain in the month after methyltestosterone therapy was d . 
galton begun and is well 6 months later ( case 26 )  . case 4 was a nulliparous woman of 49 complaining of pain in the left hip and right shoulder , the latter exacerbated by the smallest movements of the arm , active or passive . 
no deposits were seen radiologically to account for the hip pain , but the medial end of the right clavicle was swollen and tender , expanded by a tumour measuring 4 x 6 cm . after taking methyltestosterone 50 mg . 
daily for one month , the complete range of shoulder movements was restored , the patient was without pain , and the bony swelling no longer tender to percussion , this latter finding being a characteristic result of the treatment . six months later the swelling is slightly smaller , but intrathoracic metastases have appeared and the general condition has deteriorated . of the 3 remaining premenopausal cases , 1 ( case 47 ) , a para - 2 aged 38 , attended hospital 9 months after radical mastectomy with severe anaemia and pains in back , hips and shoulders . she was intolerant to doses of methyltestosterone greater than 30 mg . , and although the dose was increased in 5 mg . 
on 3 occasions the treatment had to be abandoned each time , owing to the sudden onset of headache , nausea , fever , drowsiness and increased intensity of the pains . these symptoms subsided within a day or two on withholding the hormone . case 66 , who presented with paraplegia of 6 weeks ' duration , has been referred to above . 
the last premenopausal case ( case 10 ) , a para - l aged 45 , had had a right radical mastectomy 2 years previously for a lump in the breast present for 1 year . she attended hospital with cough , dyspnoea , pain in the chest and stiff neck . 
methyltestosterone arising in submammary lymph nodes . daily , and in 3 months ' time was walking well and without pain . in addition surprisingly she the parasternal swelling had become greatly reduced in size . showed on examination 3 - 5 cof shortening of the right femur , but no impairment of movement about the hip - joint , within the limits imposed by the rigidity of her paralysis agitans , and the x - ray revealed an unusual subcapital fracture of the neck with downward and backward displacement of the capital fragment . the second case was of a nullipara aged 51 , who had been recurrence - free for 10 years after a radical mastectomy , when she developed backache , which persisted , increased in severity , eventually confining her to bed . multiple deposits of secondary carcinoma filled most of the dorsal and lumbar vertebrae , and all the pelvic bones . within a few days of the implantation of 500 mg . 
testosterone the pain abated , walking was quickly resumed , and she is pain - free 14 months later without further treatment . the nature of the pain is therefore open to question , but the patient gave no history of previous chronic backache , the onset of the pain coincided with the finding of multiple secondary deposits in ' the appropriate bones , and relief promptly followed implantation of a very small dose of testosterone . testosterone was also given to 5 young patients in whom an artificial menoin the first and last pause had been earlier induced ( cases 5 , 30 , 50 , 62 and 70 )  . of these cases pain was relieved , but in the first it was never severe , and in the last its recurrence followed the appearance of further metastases . the difference in response by premenopausal and postmenopausal cases just mentioned was not met with in the series of 70 cases described by adair , mellors , farrow , woodard , escher and urban ( 1949 ) , or in the larger series of 285 cases analysed by the council on pharmacy and chemistry ( 1949 )  . in adair 's series 9 out of 48 cases with skeletal metastases benefited from androgen therapy , but no difference in response was noted in premenopausal and postmenopausal cases ( 12 of the 48 cases were premenopausal , compared with 8 out of 28 in the present in the council 's series of 285 cases , the ages ranging from 25 - 79 years , series )  . favourable effects were evidently independent of age . 
no explanation is offered for this discrepancy . post - treatment changes in bone . it is difficult to account for the rapid relief of skeletal pain brought about by equally striking is the speedy disappearance of tenderness to testosterone . percussion of affected bones , and the later diminution in size of bony swellings radiologically all that can be observed are the due to subperiosteal deposits . changes in position and concentration of minerals in the neighbourhood of a deposit following testosterone administration , but these changes are characthey consist in the homogeneous deposition of minerals in the bone teristic . surrounding the deposit , so that at first the deposits acquire a more definite outline , and may appear more extensive in size and number . this is a common appearance 2 months after commencing treatment , but soon gives way to a more uniform but rather structureless appearance , as the areas of decalcification in the deposits become filled in from the periphery with deposited minerals . after 6 months this process usually comes to a standstill . it is questionable whether this confers increased strength on the bones in which it recalcification d . 
the same is true of cases with bone deposits , and in the illustrations in the first two papers just cited fresh osteolytic deposits are present in bones showing extensive recalcification round earlier deposits . several instances of this phenomenon have been met with in the present series . the paradox is probably to be explained by the fact that the metabolic processes accelerated by the androgen proceed most rapidly at the beginning of treatment , and then slow down to a stationary though raised level . 
he had learned of " the custom in certain countries to remove the ovaries of the cow after calving if it is wished to keep up the supply of milk , and that if this is done the cow will go on giving milk indefinitely , " and this " pointed to one organ holding the control over the secretion of another and separate organ , and thus explained the absence of that distinct nervous control that i pointed out as characteristic of the mamina . " beatson 's first case will be described in some detail , as it illustrates very well the kind of result that may be obtained by testosterone therapy as well as by castration . the patient was a married woman of 33 years , with 2 children , in fair general health , and menstruating regularly . three years previously she had noticed a lump in the breast while suckling her first child . this increased in size after the birth of her second child 20 months later . 
when first seen there was a mass 5 x 3 inches with ulceration of the skin of the breast over an area of 1 square inch and adjacent skin nodules . radical mastectomy was performed on 25 . 
95 , but 3 months later an ulcerated recurrence in the scar was found , and a fortnight later had increased in size to 21 x 31 inches . the whole chest wall was studded with skin nodules , but no lymph nodes were felt . 
95 the " largest area of disease " measured 1i x 2 inches . eight months later regression continued . the patient lived until april , 1899 , dying 46 months after castration , with fresh recurrences in skin and spine ( thomson , 1902 )  . seconid case was that of a woman aged 40 , with massive involvement of the whole breast by carcinoma , with extensive infiltration of skin , axillary and cervical nodes as far as the chin ; the opposite cervical nodes were also involved , and she complained of pain in neck and arm . after castration the pain was relieved , and regression and softening of the primary mass and of the nodes occurred , lasting 5 months . thus in the first 2 cases in which castration was used , shrinkage of the primary growth , regression of skin deposits and of involved nodes , general improvement , and relief of pain were described . six years later the method had been used extensively , and its value sunmmed up by beatson ( morris , 1902 ) as follows : " it is not easy to say beforehand if the operation will do good ; but from the cases recently published by abbe , of new york ( thomson , 1902 ; abbe , 1901 ) , it would seem that it is not contra - indicated after the menopause th ' value of an operation may be judged by statistics and by personal impressions . both may be deceptive , though both may contain in them the germs of truth . i believe that from both standpoints the evidence is in favour of the utility of oophorectomy in certain cases , in prolonging life and lessening pain . it has never , as far as i know , done any harm , and its mortality has been nil the treatment has been placed before the profession , because i felt that d . 
galton they should judge its effects themselves and if , in their opinion , after a fair trial , it is found wanting , then i hope it will be abandoned and forgotten . " oophorectomy passed out of fashion a few years later , and was slowly superseded by " x - ray castration , " and in the last 10 years by " medical castration " with androgens also . boyd ( 1900 ) reported the results of oophorectomy in 54 cases of mammary cancer . six cases out of 46 showed remissions lasting for 2 years or more , and 17 out of 46 derived some benefit , but in most of these , recurrences occurred between 6 and 12 months later . with one exception the treatment failed in all the patients who had passed the menopause , and the maximum benefit seemed to be derived by women over 40 and still menstruating . 
two years later thomson ( 1902 ) of edinburgh collected and published the results in 80 inoperable cases castrated by several surgeons . in 40 of the cases some imnprovement took place , although this was negligible in 11 , and only temporary in 11 others ( under 12 months ' survival following oophorectomy )  . 
in 18 , however , decided improvement was recorded , " remarkable " in 4 . thirteen cases survived more than 20 months , and 4 between 40 and 46 months . of the 29 more favourable cases , 6 had ceased menstruating , and 3 were 70 years of age , and of the 20 women still menstruating , 12 were between 40 and 50 years of age . 
thomson concluded that the value of castration , though limited , had been established beyond doubt , but , like beatson ( 1896 ) , stressed the unpredictability of the results . 
jessett " had operated on 5 cases , and admlitted that the patients complained of less pain , but in no single case was there any improvenment in the local disease . 
he also noted that beneficial results occurred sometimes after the menopause . bruce clarke recorded a case in which castration was followed within 6 weeks by rapid subsidence of pain and regression of a chest wall recurrence , the patient being in good health 5 years later . in this case unfortunately the age was not stated , but the original excision of the growth was performed 2 years before castration , and a recurrence excised 1 year before ( lett , 1905 )  . in spite of the well - documented reports described above , castration for mammary cancer fell out of favour , no doubt because of the absence of precise indications , the large number of poor results and the increasing success obtained by local radiotherapy . morris ( 1902 ) stressed the difficulty of assessing the results , and compared " beatson 's treatment " with the favourable results claimed for the injection of coley 's fluid ( heat killed broth cultures of streptococci obtained from erysipelas lesions , and of bacillus prodigiosus ) in cases of spindlecelled sarcoma . 
he found that in the cancer - bearing group the incidence of complete oophorectomy before carcinoma was diagnosed was 1.5 per cent , whereas in the non - cancer group it was 15 - 4 per cent , or ten times as great . these figures implied that castration might have protected a small number of women from the subsequent development of mammary carcinoma . arguing on these lines horsley , late in 1937 , performed bilateral oophorectomy on the first of a series of patients immediately after radical mastectomy , in the hope of deferring the onset of recurrence . 
by june , 1944 , recurrences had occurred in only 4 out of the 25 patients who had been observed for between 9 months and nearly 7 years . horsley felt that the appearance of metastases had been delayed by castration ( horsley , 1944 , 1945 )  . snapper castrated 22 patients after radical mastectomy , and in addition treated them for 3 months with injections of testosterone propionate 25 mg . 
of methyltestosterone was given on the day after oophorectomy , but in view of subsequent experience with this hormone , it is probable that the immediate effect was due to the oophorectomy alone . a case of intrathoracic recurrence in a woman of 40 years , 2 years after radical mastectomy for stage ii carcinoma , in whom relief of dyspnoea was observed 4 days after castration , and during the next 2 months absorption of nialignant pleural effusion and resolution of pulmonary parenchymal deposits took place , was reported by jochweds , baranowicz and horecki ( 1948 )  . the effects of surgical castration may be summarized by saying that it confers definite benefit on a small percentage of cases of carcinoma of the breast , even in an advanced stage , as shown by relief of severe bone pain , recalcification of areas of destroyed bone , regression of primary and secondary manifestations of disease , and conspicuous improvement in general health lasting uisually for several months only , but sometimes for several years . 
the most favourable cases are those in the premenopausal group in women , though striking improvement occasionally follows castration in older women , and in the small number of cases reported in males the best results are to be expected in elderly patients . in passing it should be noted that some of the remarkable spontaneous regressions which occur , and which may resemble in all respects those already described , are probably the result of " self - castration , " both ovaries being completely destroyed by metastases . 
ahlbom 1920 , and described his ( 1930 ) attempted a statistical analysis of his cases , but as 147 out of 163 cases received treatment to other sites as well as to the pelvis , and his series included very few instances of women in the most favourable age group , he failed very few clinical data are presented in to demonstrate any positive effects . ahlbom 's paper , and in spite of his obvious anxiety to attain statistical precision , it is perhaps surprising that he could quote no examples of undoubted success similar to those reported so often in cases subjected to surgical castration . 
clarkson and barker ( 1936 ) reported a case of mammary carcinoma in a woman of 41 with axillary node involvement , and deposits in a metacarpal , a tarsal phalanx , femur and ilium , in which recalcification of destroyed areas in the skeleton the patient was in followed ovarian irradiation and general body irradiation . good health 5 years later ; the ovarian irradiation was considered to be the main cause of the good result . dresser ( 1936 ) reported the results of ovarian irradiation in 59 cases with skeletal metastases , having selected this group for special observation because of the dramatic benefit conferred on two women with generalized skeletal and other deposits after they had received small doses ( 700 r ) of x - ravs directed to in both cases the periods ceased , general the pelvis for local relief of pain . regression of deposits occurred , with skeletal recalcification , and in the second case multiple pulmonary deposits disappeared ; the patient led an active life for of dresser 's 59 cases , 30 were under another 3 years , largely symptom - free . 45 years of age , and 29 had passed the menopause . 
the discrepancy between the results of the early surgical cases , in which castration was believed to be contra - indicated in the presence of bone metastasis , and the more recent results in which skeletal metastasis is regarded as one of the most favourable indications for ovarian irradiation , is probably adequately explained by the lack of satisfactory x - rays in the early work , so that the extent and incidence of bone metastasis was less accurately known . the effects of androgens in mammary carcinoma . among the first reports of the use of testosterone for mammary carcinoma were those of ulrich ( 1939a , b ) and of loeser ( 1941 )  . the latter used intramuscular injections and also subcutaneous implants of testosterone propionate . the first 3 cases were recurrence free at the time treatment was begun , but remained so for the next 3 to 4 years , whereas all had been treated by x - rays or surgery for recurrences shortly after the original operation ( 6 months , 8 months and 2 years respectively )  . farrow and woodard ( 1942 ) felt that there was a specially close relation between susceptibility to skeletal metastasis and ovarian activity , and were thus led to use testosterone for cases with bone deposits . 
doses gilven on alternate days . the case reports published by adair and herrmann ( 1946 ) illustrate very well the kind of result which may follow the use of testosterone therapy . weight gains , general improvement , rapid relief of pain previously intractable , regression of primary growths and secondary deposits , and increased calcification of bony deposits were recorded . the following are especially noteworthy : ( 1 ) the ages of the patients . - one of the best responses occurred in a woman of 63 years , though the other successes were in women of 47 , 44 and 42 years compare also taylor , slaughter , smejkal , fowler and preston ( 1948 ) , of age . who report andillustrate regression of pulmonary deposits in a woman of 70 , and the progress report of the american council on pharmacy and chemistry ( council on pharmacy and chemistry , 1949 ) , in which the response to androgens in 285 patients ranging from 25 - 79 years was evidently independent of age . in a later paper adair ( 1947 ) reports a dramatic result in a woman of 71 who had been confined to bed for 10 months with severe pain and pathological fractures due to widespread metastases in spine , pelvis and ribs . three weeks after commencing testosterone therapy she remarked on her improved sense of well being , and 4 weeks later left her bed . 
two years and 4 months after she was still up and about and in good health , the only untoward effect being irritation from the enlarged clitoris . this case recalls some of those treated by surgical castration ( thomson , 1902 )  . ( 2 ) the speed of response . - in case 4 relief of pain was evident within1 week of the first injection , and in case 3 regression in primary and metastatic growths was noted one month later . ( 3 ) the favourable effect on skeletal metastases . - the earlier results of adair ( 1947 ) suggested that the best results of androgen therapy were to be expected in cases with metastases in bone . however , equally dramatic results have been obtained in cases with pulmonary deposits and skin deposits , but no secondaries in bone of the common osteolytic type revealed by x - rays . these results are said to be much less common than in cases with bone deposits . thus adair stated : " in only a small percentage of cases is improvement to be anticipated where carcinoma involves soft tissues such as liver , lungs , brain and local skin recurrence . it is true that in a few instances there have been striking improvements where an advanced ulcerating breast cancer with surrounding skin nodules , axillary nodes and neck nodes were involved ; where masses were metastatic to the lungs ; and in one instance where the patient was having convulsions from metastatic disease in the brain receive no benefit  . 
in weight , her periods had not recurred , and the mass in the breast none of the nodes was more than 1 cacross , and the skin was no longer palpable . deposits had undergone complete regression , being visible only as brownish impalpable mottlings . 
48 signs of masculinization had appeared , namely slight hirsuties of chin , and lips , and a deepening of the voice . she complained of slight swelling of the feet her weight was 10 st . 
ii.49 she attended hospital complaining of severe backache , and on examination the nodes felt previously were found to be hard and much enlarged , numerous deposits not exceeding 3 min diameter were present in the skin of the left breast , and the liver edge , which was thickened , firm and somewhat irregular was felt in the right iliac fossa . 
47. - admitted to royal cancer hospital . on examination she was in good general condition , but bedridden with crippling pain . there were 3 ulcerated recurrences along the left mastectomy scar , the two larger measuring 2 x 4 and 5 x 3 crespectively , with irregular thickened raised edges , fixed bases and coarsely nodular floors . 
in 9 months , had recurrence of low back pain ; the mass in the upper inner quadrant of the right breast was larger , as were the axillary nodes ; the bony parasternal swelling in the left 4th interspace had ulcerated through the skin . there were numerous hemispherical bony projections on the vault of the skull , on the frontal bone , on the right clavicle . x - rays showed many fresh osteolytic deposits in spine , ribs and pelvis , side by side with old sclerosed deposits , which were less dense than before . injections of testosterone propionate 100 mg . 
viii.44. - left radical mastectomy for stage i carcinoma of breast . postremained well until april , 1948 , when first noticed breathlessness on effort , which june , 1948 . - signs of large left pleural effusion . 
49 the only abnormality found was slightly diminished radiologically there is now only obliteration of the left expansion at the left base . costophrenic sulcus , and the parenchymal opacities are no longer visible . she was last seen on 25 . 
48 the entire area was paler in colour , the young skin over it thicker , but there was still one small discharging area . this dried up during the next two months , dressings were discarded and therapy discontinued , but on 1 . 
minutes. this degree ' of hydrolysis was found to remove ar non - dna phosphorus as wer as all non - dna adenine from the cells , while leaving dna phosphorus and dna adenine in situ , thus obviating the necessity for the use of the einzyme ribonuclease ( lajtha , 1954b )  . adenine 14c administered to cells appears both as labelled adenine and labelled guanine in the dna ( hamilton , 1953 )  . no attempts to separate labelled adenine from labehed guanine have been made in the experiments ; the activity of the dna has been measured as a whole . the autoradiographs were exposed for 10 - 14 days . 
ar autoradiographs belonging to the same experiment were coated with the same batch of stripping film , exposed the same length of time at the same temperature ( 3 - 4 ' c . ) , and developed in one batch of developer simultaneously , and , finally , were stained simul368 l . 
on each slide 100 - 200 nuclei were counted , and grains were counted over - 5 - 10 showing the strongest autoradiographs . this was to obtain the values for the maximum degree of dna synthesis and was done after surveying repeated counts at different times were performed to tes - t the over 500 nuclei . reprodiicibihty of the counts , and it was found that with the above technique the same observer showed ain error not greater than 10 - 15 per cent . for the calculations on the quantity of isotope i ' ricorporated into the cell , it was assumed ( 1 ) that the geometry of the autoradiographs was such that only 50 per cent of the electrons emitted frothe disintegrations reached the photograpbic emulsion , and ( 2 ) that the 32p electrons produced 0 - 7 grains per incident electron , and the 14c electrons 2 gfai - ns per incident electron ( lamerton , 1950 )  . if the exposure tiine and the half life of the isotope in question as well as the grain count per nueleus and the grain yield per electron of the isotope are known , then the number of the atoms of the isotope withiii the nucleus can be calculated . the technique is sensitive enough to detect 20 - 30 32p atoms per cen . because of the very long half - hfe of 14c ( and because exposure times were limited to the relatively short period of 14 - 30 days ) the minimum necessary number of 14c atoms per nucleus to show an autoradiograph is of the order of 106 . in preliminary experiments the maximum dna 32p uptake per nucleus was found to be the order of 600 atoms ( > i 00 grains in 14 days ' exposure 07 grains / electron )  . 
the labelling obtained in vitro of the dna is therefore of the order of i : 3 x 107 . if the cehs cannot differentiate between 31p and 32p then the total phosphorus pool available for the cens must also have been labered in the ratio of the order of i : 3 x 107 . thus for 5 , ac . 
in seruthe addition of adeniine 14c into the culture medium does not represent the labelling of a pool , but the provision of adenine to the cers which would otherwise have to synthesise it . 
the marked ability of the cellsto take up adenine from the cultur - e medium is shown by the fact that the average maximum uptake per nucleus was of the order of 1 - 2 x 107 atoms dna 14c ( 50 grains in 10 , days ' expostire , 2 grains / electron )  . 
i ' he cell cycle . if the cells svnthesised dna throughout the entire intermitotic period , then , after a few hours of culture in the presence of the isotope all the cers should contain labelled dna . 
ellis fore the cells within a hours of the end of the period of dna synthesis at the start of the culture in isotope wif not be labered , and , similarly , cers enterina the period of dna synthesis during the culture time ( t ) and spending less than a hours 11111hours fig . 
the length of the s period is also indicat - ed by the gradual increase of the grain count / nuclei up to about 15 hours , then reaching the maximum average value for a given concentration of the isotope in the culture mediuthe length of the total cen cycle can also be calculated in a different way . 
3 illustrates the timing this timing , in of the cell cycle obtained with the methods outhned above . principle agrees well with that found in the bean root by howard and pelc ( 1951 )  . it can be seen from tables i and ii and fig . 
the reason for this difference is , firstly the differences betweein eilergy of the beta particles emitted by 14c and the higher energy32p electrons result in a greater scatter and , consequently , in a higher background , thus interfering with the accuracy of the c ' secondly , while the half hfe of 14c is a neghgible problem when the isotope dilutions are added to the culture medium , variations in volume to allow for the decay are a also , diie to the decay of 32p and to the fact that32p is so - lirce of error with32p  . supplied in3lpphosphate buffer , the ratioof 31 to 32pwill not be constant : the degree of labelling of the phosphorus pool will be variable . these factors all contribute to the lesser accuracy of the counts in cultures with 32plabelling as compared with the 14c labelling . 25 - 30hours 12 - 15 hours 3 - 4hours g2 m fig . 
rate , of dna synthe , 8i8 in different type8 of bone marrow cei18 . in these experiments only those types of cells were found to synthesise dna which are known to be capable of mitosis . 
3 represents the average values for the majority of the dividing cells of the bone marrow , i.e. , promyelocytes , myelocytes , pronormoblasts , basophihc normoblasts and early polychromatic normoblasts . in a few experiments , however , where it was found to be possible to count early normoblasts and early myeloid cells separately , a shorter cycle than in fig . 
the affect of x - ray irradiation on dna synthesis large doses of x - rays ( 5000 r in 15 minutes , 140 kv , i . / mal filter ) exerted a marked inhibition on dna synthesis . 
14. - post - irradiation dna synthesis . was not a recovery in the sense of ability to synthesise normal amounts of dna , but the entry of some 0 , cers into the s period for a hmited time . it appears that ar 01 cehs are damaged by a dose of 5000 r . the effect of irradiation on theg2 period is indicated by the fa ' ct that no mitoses were observed in any of the irradiated cultures . a dose of 5000 r produced a marked degeneration of the cers in the cultures , but over 6 hour culture times were needed to show the first morphological signs 378 l . 
in a sense this could be better defined , as the assembly of the dna experiments with the synthesis , of the purines and pyrimidines from molecule . labeued one carbon compound precursors ( e.g. , formate 14c ) are in progress . the timing of the cer cycle is , in principle , in good agreement with that it is expected that described in the bean root cell by howard and pelc ( 1951 )  . the timing of the cer cycle in different . 
the total cycle time was found to be of the order of 40 - 48 hours for the average dividing bone marrow cers ( promyelocytes , myelocytes , pronormoblasts , basophihc and polychromatic normoblasts )  . 
bowles , md2 , 3 introduction fibroblast growth factor receptors ( fgfrs ) 1 through 4 are transmembrane tyrosine kinase receptors that play a key role in cell survival , differentiation , migration , angiogenesis , and carcinogenesis.1 uncontrolled activation of fgfr signaling has been described in almost every type of malignancy and typically is the result of amplication or activating mutation affecting the fgfr genes.2 these aberrations can one of function as cancer drivers but also may inuence prognosis and sensitivity to treatment.3 - 5 only two fgfr - specic inhibitors , erdatinib and pemigatinib , have been approved by the us food and drug administration , though a number of multikinase kinase inhibitors ( mkis ) have some degree of fgfr2 inhibiting properties . although fgfr1 and fgfr3 mutations are more frequent overall , activation of fgfr2 is common in patients with endometrial adenocarcinoma , cholangiocarcinoma , gastric adenocarcinoma , and ameloblastoma , a rare and difcult - to - treat odontogenic tumor.2 , 6 - 9 here , we present the case of a patient with ameloblastoma harboring an activating fgfr2 y375c mutation who had signicant tumor regression in response to treatment with lenvatinib . 
a specimen from a repeated biopsy was sent for nextgeneration sequencing using cancerselect 125 ( personal genome diagnostics , baltimore , md )  . analysis revealed clonal fgfr2 y375c , an activating mutation in the transmembrane domain causing constitutive receptor dimerization through the formation of intermolecular disulde bonds.12 although fgfr2 aberrations are common in ameloblastoma , this point mutation appears not to have been identied previously . 
first characterized in bearestevenson cutis gyrate syndrome , y375c is rare in cancers but has been described in adenoid cystic carcinoma and endometrial carcinoma.13 , 14 a second potential driver mutation affecting the hedgehog signaling pathway , smo l412f , was also found at a lower allele fraction . 
frequently seen in ameloblastoma as well as basal cell carcinoma , medulloblastoma , and a subset of meningiomas , this activating mutation alters the drug - binding pocket of smo , conferring resistance to smo inhibitors such as vismodegib and itraconazole.6 a third mutation , palb2 h786y , was reported but is of unclear signicance , having never been associated with human disease . 
although sensitivity to fgfr inhibition varies by type of mutation , drug - binding site , and degree of fgfr specicity , 15 , 16 cancer cell lines harboring fgfr2 y375c mutations were previously found to be responsive to nonselective pan - fgfr inhibition in vitro.17 because the fgfr - specic inhibitors were not yet commercially available , lenvatinib was selected because of its favorable half maximal inhibitory concentration for fgfr2 ( 27 nmol / l ) and efcacy in other malignancies . the patient started receiving the standard dose for radioactive - iodine refractory thyroid cancer of 24 mg daily . his dose was reduced to 20 mg daily after 1 month , then 20 mg alternating with 10 mg daily after an additional month . 
he experienced typical adverse effects of grade 2 hypertension , grade 2 hypothyroidism , grade 1 diarrhea , and grade 2 weight loss . on follow - up imaging at 6 months , he experienced a partial response with 40% shrinkage , per recist , version 1.1 ( fig 1 )  . 
as of this writing , the patient has been receiving this treatment for 13 months and has a continued partial response . discussion ameloblastoma is a locally invasive , typically benign distransformation with ease that can undergo malignant regional or distant metastasis , sometimes occurring decades after initial presentation.18 - 23 surgery is the mainstay of treatment , although conservative enucleation and curettage are associated with recurrence rates as high as 60% to 90%.24 radical surgery with wide en bloc resection improves these rates but at the cost of disgurement and high morbidity . 
overall , guidance regarding use of adjuvant treatment modalities is lacking . given the dearth of effective and tolerable treatment options , reports of targetable mutations dominating the genetic landscape of ameloblastoma were met with great interest.6 - 8 a large percentage of ameloblastomas , especially those arising from the mandible , have braf v600e mutation . 
ameloblastomas harboring braf v600e mutation are sensitive to braf inhibition in vitro at clinically relevant drug concentrations.6 multiple case reports have demonstrated durable clinical response to braf inhibition or combined braf / mek inhibition , on the basis of braf status.26 - 30 a second , and typically mutually exclusive , molecular phenotype featured mutations in fgfr2 , including c382r and v395d in the transmembrane domain and n549k in the kinase domatogether , braf and fgfr2 mutations are present in approximately 80% of ameloblastomas , with additional somatic mutations in smo , cttnb1 , pik3ca , and smarcb1 . interestingly , the patient we report on here had nearly 90% allele frequency of fgfr2 y375c , suggesting this may have been the initial truncal mutation . 
the implications of clonality for fgfr point mutations and fusions are not well understood , but truncal amplication of fgfr2 is an important predictor of drug response in gastric cancer.31 in a small trial of the selective fgfr inhibitor azd4547 in fgfr2 - amplied gastroesophageal cancer , robust , singleagent clinical response was restricted to high copy - number amplication events with associated high expression of fgfr2 mrna and fgfr2 protethe mechanism for this fig 1 . 
 case report effect was thought to be transactivation of alternate receptor tyrosine kinases resulting in a distinct oncogene addiction phenotype characterized by fgfr - dependent phosphatidylinositol - 3 - kinase and mammalian target of rapamycin signaling . although prospective trials in ameloblastoma continue to insight into be limited by low case numbers , additional treatment of this disease may be drawn from other , molecularly similar cancer types , including endometrial and gastric adenocarcinomas . 
fgfr2 mutations occur in approximately 10% of both tumor types and are mutually exclusive , with kras mutations suggesting a shared signaling mechanism.9 , 32 , 33 in vitro data from endometrial cancer conrmed fgfr2 activation results in increased mapk signaling that is abrogated by fgfr inhibition.34 , 35 these preclinical data prompted a phase ii trial of lenvatinib in advanced endometrial cancer , which reported an objective response rate of 14% and a clinical benet rate of 37% without selection for fgfr status.36 conrmatory trials of fgfr inhibition in endometrial cancer , both alone and in combination with pembrolizumab or other checkpoint blockade , are ongoing.37 subgroup analysis of biomarkerselected populations in studies like these may be a helpful surrogate for utility in rare cancers such as ameloblastoma . complicating the generalization of fgfr inhibitor trial data are the multiple mechanisms of fgfr dysregulation . 
although studies typically lump together patients with any fgfr aberration , the type of mutationfgfr overexpression including gene amplication , increased ligandbinding afnity or ligand expression , or ligand - independent receptor activationresults in differential effects on fgfr signaling . 
using mkis as an example , brivanib primarily affects cells with fgfr1 amplication , dovitinib inhibits fgfr1 - 3 kinase activity in a predominately liganddependent fashion , and ponatinib targets fgfr1 - 4 with three patterns of signaling alteration we have described.17 although the exibility of some nonselective mkis in targeting multiple fgfr aberrations may broaden their use , it comes at the cost of increased toxicity . 
in contrast , early data from selective fgfr tkis demonstrated a favorable adverse effect prole but more limited efcacy.38 , 39 characterizing drug - susceptibility phenotypes will be crucial for the continued development of these agents and other approaches to fgfr inhibition , including monoclonal antibody or peptide inhibitors and fgf ligand traps . although lenvatinib was chosen in this case to target fgfr2 , it is possible the patients response was due to inhibition of other targets , such as vascular endothelial growth factor receptors , platelet - derived growth factors , or kit . 
assessment of phosphorylated - fgfr2 by immunohistochemistry before and after therapy could clarify if his response was due to on - target or off - target effects , but he declined a repeated biopsy procedure . 
lenvatinib has been effective in thyroid , renal , and hepatocellular carcinomas without identiable mutation or overexpression of these kinases , suggesting the mechanism underlying drug sensitivity is incompletely understood.37 most clinical trials of lenvatinib have been in solid tumor types selected on the basis of response to other inhibitors of angiogenesis , but these agents have not been investigated in ameloblastoma . more studies are needed to clarify the role of fgf signaling in drug response and identify predictive biomarkers for lenvatinib . 
clin cancer res 22 : 259 - 267 , 2016 donnem t , al - shibli k , al - saad s , et al : prognostic impact of broblast growth factor 2 in non - small cell lung cancer : coexpression with vegfr - 3 and pdgf - b predicts poor survival . 
j thorac oncol 4 : 578 - 585 , 2009 turner n , pearson a , sharpe r , et al : fgfr1 amplication drives endocrine therapy resistance and is a therapeutic target in breast cancer . 
ware ke , hinz tk , kleczko e , et al : a mechanism of resistance to getinib mediated by cellular reprogramming and the acquisition of an fgf2 - fgfr1 autocrine growth loop . 
nat genet 46 : 722 - 725 , 2014 [ erratum : nat genet 47 : 97 , 2015 ] kurppa kj , cat on j , morgan pr , et al : high frequency of braf v600e mutations in ameloblastoma . 
j pathol 232 : 492 - 498 , 2014 brown na , rolland d , mchugh jb , et al : activating fgfr2 - ras - braf mutations in ameloblastoma . 
clin cancer res 20 : 5517 - 5526 , 2014 dienstmann r , rodon j , prat a , et al : genomic aberrations in the fgfr pathway : opportunities for targeted therapies in solid tumors . 
byron sa , chen h , wortmann a , et al : the n550k / h mutations in fgfr2 confer differential resistance to pd173074 , dovitinib , and ponatinib atp - competitive 17 . 
gozgit jm , wong mj , moran l , et al : ponatinib ( ap24534 ) , a multitargeted pan - fgfr inhibitor with activity in multiple fgfr - amplied or mutated cancer 18 . 
brunet m , khalifa e , italiano a : enabling precision medicine for rare head and neck tumors : the example of braf / mek targeting in patients with metastatic 27 . 
faden dl , algazi a : durable treatment of ameloblastoma with single agent brafi re : clinical and radiographic response with combined braf - targeted therapy in stage 4 ameloblastoma . 
byron sa , gartside m powell ma , et al : fgfr2 point mutations in 466 endometrioid endometrial tumors : relationship with msi , kras , pik3ca , ctnnb1 mutations and clinicopathological features . 
plos one 7 : e30801 , 2012 [ erratum : plos one 7 , 2012 doi : 10.1371 / annotation / 0bfaecca - 0f87 - 43fe - 97ccf2ae3ddeb6d5 ] 33 . 
konecny ge , kolarova t , obrien na , et al : activity of the broblast growth factor receptor inhibitors dovitinib ( tki258 ) and nvp - bgj398 in human endometrial cancer cells . 
chae yk , ranganath k , hammerman ps , et al : inhibition of the broblast growth factor receptor ( fgfr ) pathway : the current landscape and barriers to clinical application . 
 o accurate rna sequencing from formalin - fixed cancer tissue to represent high - quality transcriptome from frozen tissue purpose accurate transcriptional sequencing ( rna - seq ) from formalin - fixed paraffin embedded ( ffpe ) tumor samples presents an important challenge for translational research and diagnostic development . 
we evaluated the accuracy of rna - seq data generated from ffpe samples in terms of expression profiling . methods we designed a biospecimen study to directly compare gene expression results from different protocols to prepare libraries for rna - seq from human breast cancer tissues , with randomization to fresh frozen ( ff ) or ffpe conditions . 
the protocols were compared using multiple computational methods to assess alignment of reads to a reference genome , the uniformity and continuity of coverage , the variance and correlation of overall gene expression , patterns of measuring coding sequence , phenotypic patterns of gene expression , and measurements from representative multigene signatures . results the principal determinant of variance in gene expression was use of exon capture probes , followed by the conditions of preservation ( ff v ffpe ) and phenotypic differences between breast cancers . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction although it is generally best to identify gene expression biomarkers from cancer tissues using the highest quality of rna purified from fresh frozen ( ff ) samples , any subsequent development toward diagnostic testing will require translation for use with formalin - fixed paraffin - embedded ( ffpe ) tissue samples . 
however , the variably fragmented and chemically modified rna derived from ffpe samples presents a challenge for accurate measurement of gene expression.1 , 2 in a different context , there is great interest to perform transcriptome sequencing ( rna - seq ) for biomarker discovery research using large cohorts of precious archival ffpe samples from completed clinical trials . 
different approaches to generating libraries for rna - seq include selection of mrna by targeting the poly ( a ) tail ( mrna protocol ) , depletion of more abundant ribosomal rna ( rrna depletion ) using jialu li chunxiao fu terence p . 
 a 80c freezer ( ff ) or 10% neutral buffered formalin and paraffin - embedded as an ffpe tissue block.10 phenotypically , the nine breast cancers were defined by the pathologic status of hormone receptors ( hrs ) and human epidermal growth factor receptor 2 ( her2 ) as hr - positive / her2 - negative in five , hr - positive / her2 positive in one , and triple receptor - negative in three . 
a dnase - i treatment step was included in both . construction of rna - seq library and sequencing full details of all methods for library construction and sequencing of rna samples are in the data supplement . 
an overview diagram of the different rna - seq library protocols is shown in figure 1 , and details of the number of libraries prepared , starting rna requirement , cost , and duration to perform each protocol are summarized in the data supplement . the mrna protocol ( ff only ) used oligo - dt beads for poly ( a ) + mrna enrichment , followed by standard procedures of truseq rna sample prep kit v2 ( illumina , san diego , ca )  . 
then the rrna - depleted srna was used as the template for the mrna protocol described above . the cr protocol was performed using truseq access rnaseq kit ( illumina ) , using random primers . 
no technical replicates could share the same sequencing lane . rna - seq data analysis full details of all data analysis methods and an overview diagram of the analysis plan are provided in the data supplement . 
scatter plot of the first three principal components for count per millionnormalized and variance stabilizing transformed counts of 20 , 381 coding region ( cr ) - targeted poly ( a ) + genes . 
the rnaseq data generated from the srna protocol had a significantly lower concordant pair alignment rate as compared with those from non - srna protocols ( p < .001 ; data supplement )  . 
using the cr protocol , mapping was highly concordant between ff and ffpe and the exonic mapping rate was increased compared with non - cr methods ( data supplement )  . 
overall , the number of genes with read coverage ( transcripts per million [ tpm ] > 0.1 ) was slightly higher in ffpe samples than in ff samples for both non - cr and cr protocols ( data supplement ) .12 uniformity and continuity of read coverage of transcripts uniformity of read coverage was measured by the mean coefficient of variation , and continuity of coverage as the percentage of gaps without read coverage , across the top 1 , 000 highly expressed transcripts ( data supplement )  . 
 principal component analysis of expression of a total of 20 , 381 cr protocol targeted poly ( a ) + genes for all libraries showed that the 26.5% of total variation captured by the first principal component was due to use of exon capture probes ( cr protocol ) , and 20.6% from the second and third components due to the combined effects of ffpe and biologic differences ( fig 2 )  . 
hierarchical clustering results , with high confidence ( average bootstrap probability , 0.93 ) , showed that the major tumor phenotypes ( hr - positive v hr - negative ) and the source tumor clustered together with ffpe samples ( data supplement )  . 
comparing ff and ffpe samples using the same totalrna protocol , the log ratio values were still centered around zero at different mean expression levels ( fig 3b )  . 
difference in measurements as log ratio ( m ) versus mean average expression ( a ) of each gene ( ma plot ) of 20 , 381 coding region ( cr ) targeted poly ( a ) + genes for tumor sample c when using ff.k.totalrna sample c library as the reference . 
m is the log2 - transformed expression of a gene from first library divided by that from the second library , and a is the mean log2 transformed expression of the gene . 
generally , the cr protocol tended to overly enrich the highly expressed genes and was more likely to not capture low expressed genes ( fig 3c ; data supplement )  . 
this was not improved by prior demodification of methylol adducts from ffpe tissuederived rna using heat and amines or the srna protocol ( fig 4 ; data supplement )  . 
although both approaches seemed to slightly increase concordance of expression , neither was statistically significant . after further investigating these protocol induced biases , we calculated the number of genes that would be considered as differentially expressed or false positives compared with each reference ff standard protocol ( data supplement )  . 
cr , coding region protocol ; dem , demodification ; ff , fresh frozen ; ffpe , formalin - fixed paraffin - embedded ; srna , sense rna protocol . and triple receptornegative breast cancers within each protocol . 
the p value from de analysis followed the ideal uniform distribution for non - de genes , with a spike close to zero for the de genes ( data supplement )  . 
 principal component analyses demonstrated the following order of variables influencing gene expression measurements from rna - sequencing : whether the library preparation protocol used exon capture for cr , whether the sample was from ff tissue or ffpe tissue , and the biologic phenotype of the breast cancer based on hrs and her2 receptor status ( fig 2 )  . 
nevertheless , we identified one protocol , ffpe.k.totalrna , with consistently good transcript coverage uniformity and continuity , most concordant expression , and least differential expression when compared with the different non - cr protocols with fresh tissue . 
it had a reasonable requirement of total rna input ( 100 ng ) for ffpe biopsy samples . the first translational research scenario that we posed , in the introduction section , considered the best pairing of protocols that would enable discovery using ff samples with intention to later translate for use with ffpe samples . 
this interpretation was supported by the quality of read coverage , pattern of coding sequence expression , and translation of overall or phenotype - related gene expression profiles and prognostic signatures . the cr protocols yielded concordant results , but different from all other ( non - cr ) protocols . 
 also , changes to the population of exon capture probes within a commercial kit over time could be a potential risk to this approach . the most generalizable results for discovery research from ffpe samples were obtained using the total.rna protocols without exon capture . 
for our second scenario , therefore , we prefer the k.totalrna protocol for best representation of the transcriptome in ffpe samples used for discovery researchaiming to represent the transcriptional information that ff samples would have provided . our third translational research scenario involves the translation of an existing gene expression signature that was previously developed using a different method ( eg , microarray ) or a particular rna - seq protocol . 
the inclusion of random and dt primers and the t7 promoter region ( srna protocol ) to simulate the ff.mrna protocol produced good concordance overall but introduced a high number of nonconcordant mapped reads , nonuniformity , and discontinuity of read coverage across the transcriptome . limitations to our study include small sample size ( although cancers were selected to represent biologic diversity ) ; optimally short time to fixation of tissues and possibly , as a result , a modest degree of degradation of ffpe samples ( dv200 ranges from 65% to 85% ) ; optimal amount of input rna used for non - cr protocols ( at least 100 ng ) ; and lack of generalizability ( single institution conditions of tissue processing )  . 
one tumor ( sample n ) seems to be compromised by unknown technical processing ; our study conclusions , whenever involving this sample , are based on robust point estimates ( eg , median estimates in fig 4 ) across all samples to avoid being driven by its outlier effect . 
for more information about asco 's conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . jialu li stock and other ownership interests : genomic health chunxiao fu no relationship to disclose terence p . 
fraser symmans stock and other ownership interests : ionis pharmaceuticals , nuvera biosciences , delphi diagnostics consulting or advisory role : genentech patents , royalties , other intellectual property : patent pending for gene expression measurement of sensitivity to endocrine therapy travel , accommodations , expenses : luminex supported by the cancer prevention research institute of texas grant no . 
masuda n , ohnishi t , kawamoto s , et al : analysis of chemical modification of rna from formalin - fixed samples and optimization of molecular biology applications for such samples . 
fumagalli d , blanchet - cohen a , brown d , et al : transfer of clinically relevant gene expression signatures in breast cancer : from affymetrix microarray to illumina rna - sequencing technology . 
zhao w , he x , hoadley ka , et al : comparison of rna - seq by poly ( a ) capture , ribosomal rna depletion , and dna microarray for expression profiling . 
loi s , haibe - kains b , majjaj s , et al : pik3ca mutations associated with gene signature of low mtorc1 signaling and better outcomes in estrogen receptor - positive breast cancer . 
the patient declined surgery , and the centrally located rll tumor was treated with stereotactic body radiation therapy ( sbrt ) at a dose of 50 gy in five fractions . 
 six months later , the patient presented with a 3 - month history of right hip and left shoulder pain , which was a biopsy - proven recurrence of the rll primary with diffuse bony metastases . 
 the patient consented to an institutional review boardapproved protocol ( #226210 at the university of california , davis )  . tumor and germline genomic sequencing assays plasma ctdna was isolated from 10 ml of whole blood drawn in streck cell - free dna tubes , enriched by hybrid capture to target exons of 70 genes and critical introns of six genes , and sequenced to an average depth of approximately 15 , 000 on an illumina nextseq500 ( guardant360 assay ; guardant health , redwood , ca ) at a clinical laboratory improvement actcertified commercial laboratory.6 germline dna was isolated from the patients pbmcs and enriched for targeted regions using a hybridization - based protocol and was sequenced with 50 or greater depth using illumina technology at a commercial genetic testing laboratory ( invitae , san francisco , ca )  . 
 methods for in vitro studies are described in detail in the data supplement . this patients family history suggests a hereditary predisposition to lung cancer : three other individuals across three generations had a history of lung cancer ( data supplement )  . 
tumor genomic profiling of plasma ctdna with the guardant360 assay identified concurrent low ( 0.3% ) mutation allelic frequency ( maf ) of egfr l858r and high ( 51% ) maf of egfr t790m ( fig 1a - 1b )  . 
 the patient was referred to a genetic counselor , and germline dna sequencing confirmed that this patient carried a germline egfr t790m mutation . we determined the in vitro cytotoxicity of egfr tkis on this patients pbmcs using a permanent epstein barr virustransformed lymphoblastoid cell line , a good surrogate for the patients germline genomic materials , 11 , 12 established before initiation of any systemic treatment . 
we found that the lymphoblastoid cells ( fig 2a ) and the patients pbmcs ( fig 2b ) were resistant to all first - , second - , and third - generation egfr tkis tested . 
consistent with previous reports , 13 h3255 cells ( which harbored only an oncogenic egfr l858r mutation ) were sensitive to all egfr tkis ( fig 2c ) , whereas h1975 cells ( which harbored both oncogenic egfr l858r and t790m mutations ) were resistant to erlotinib but sensitive to afatinib and osimertinib ( fig 2d )  . 
we found that the patients pbmcs and pbmc - derived cells did not express egfr and akt protein , the two key signaling molecules that mediate the downstream oncogenic signaling pathways in h3255 and h1975 cells , by western blots ( fig 2e )  . 
we also confirmed that there was no egfr mrna expression in patients pbmcs or in the patient - derived ebv - transformed lymphoblastoid cell line ( data supplement )  . 
thus , afatinib and osimertinib could be used safely in this patient with a germline egfr t790 mutation . during the in vitro studies , the patient first received palliative radiation for the right hip pa he had stable disease after first - line combination chemotherapy with carboplatin , pemetrexed , and bevacizumab for four cycles , and he experienced tumor progression at the primary rll and metastatic bony lesions after maintenance treatment with pemetrexed and bevacizumab for four cycles . 
a restaging positron emission tomography ( pet ) / ct scan 2 months after afatinib treatment began revealed extensive tumor progression in the primary rll tumor and multiple bone metastases . 
serial guardant360 assays revealed that the changes in the maf of egfr l858r in plasma ctdna correlated with the tumor responses to chemotherapy , afatinib , and osimertinib ; the maf of egfr t790m remained at approximately 50% ( fig 1a - 1b )  . 
time points ( ie , dates ) were selected on the basis of radiographic assessments at baseline and after each treatment according to recist version 1.1 during the entire disease course . 
targeted ngs of malignant cells in pleural effusion by foundationone ( foundation medicine , cambridge , ma ) revealed multiple genomic alterations , including egfr l858r and t790m ( fig 1c )  . 
similar to plasma ctdna analysis , egfr genotyping of the patients tumor dna by droplet digital polymerase chain reaction revealed that the maf of egfr l858r in tumor dna increased with tumor progression , whereas the maf of egfr t790m remained at approximately 60% ( fig 1c )  . 
the patient did not tolerate subsequent treatment on a clinical trial and died on hospice approximately 3 months later . discussion routine clinical application of multiplex tumor genomic profiling has increased the detection rate of rare germline mutations that may affect the patients clinical care and that raise concerns for cancer risks in family carriers.15 , 16 the maf of a somatic mutation in the plasma ctdna is usually much lower than that of archived tumor dna , whereas the maf of germline egfr t790m remains at 50% to 60% in plasma ctdna and tumor dna . 
results from the inherit study ( nct01754025 ) , which provided free genetic counseling and tested for lung cancer in patients and family members with an egfr t790m mutation at diagnosis and at longitudinal surveillance with serial ct scans , 17 are highly anticipated . patients with lung cancer who have a germline egfr t790m usually harbor a distinct spectrum of one or more concurrent oncogenic egfr mutations ( data supplement )  . 
the median progression - free survival was 4.9 months in the pooled analysis of patients ( the data supplement ) , whereas the median progression - free survival was 9 to 13 months in patients with egfr tkisensitive somatic egfr mutations.18 furthermore , data from our patient and the patients in the data supplement also revealed less clinical benefit from chemotherapy and radiation , which suggests tumor resistance to these standard therapeutic strategies . 
stollenwerk , tianhong li provision of study material or patients : debbie lewis , yanhong zhang , jamie hung , jenna welborn , tianhong li data analysis and interpretation : weijie ma , jidong shan , wenwu xiao , yanhong zhang , sixi wei , kit s . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . weijie ma no relationship to disclose jay gong no relationship to disclose jidong shan no relationship to disclose debbie lewis no relationship to disclose wenwu xiao no relationship to disclose elizabeth h . 
moore no relationship to disclose yanhong zhang no relationship to disclose jamie hung no relationship to disclose patents , royalties , other intellectual property : patent for device and method for assessing beam quality in computed tomography by measuring the half - value layer ( inst ) sixi wei no relationship to disclose jeanna welborn no relationship to disclose nicholas s . 
lam leadership : lamnotherapeutics stock and other ownership interests : lamnotherapeutics , lp therapeutics , cardioprobe patents , royalties , other intellectual property : i have more than 20 issued patents , many with the university of california , davis ( no royalty received in the past 2 years )  . tianhong li consulting or advisory role : foundation medicine , takeda , puma research funding : foundation medicine ( inst ) , pfizer ( inst ) , astrazeneca ( inst ) , hengrui ( inst ) acknowledgment we dedicate this work to the late jeannine h . 
mans stock and other ownership interests : concert pharmaceuticals , corcept therapeutics , varex imaging , verasterm , ziopharm oncology affiliations weijie ma , jay gong , wenwu xiao , elizabeth h . 
3 - ( 4 , 5 - dimethylthiazol - 2 - yl ) - 5 - ( 3 - carboxymethoxyphenyl ) - 2 - ( 4 - sulfophenyl ) - 2h - tetrazolium assay for ebv - transformed lymphoblastoid cell line established from ( a ) this patient , ( b ) the patients pbmcs , ( c ) h3255 , and ( d ) h1975 . 
 ( e ) the expression of egfr and downstream signaling molecules in the ebv - transformed lymphoblastoid cells and pbmcs from this patient , h3255 , and h1975 are shown . 
mulloy r , ferrand a , kim y , et al : epidermal growth factor receptor mutants from human lung cancers exhibit enhanced catalytic activity and increased sensitivity to gefitinib . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
ancevski hunter k , friedland dm , villaruz lc , et al : first - line osimertinib in patients with treatment - naive somatic or germline egfr t790m - mutant metastatic nsclc . 
hussain t , kotnis a , sarin r , et al : establishment and characterization of lymphoblastoid cell lines from patients with multiple primary neoplasms in the upper aero - digestive tract & healthy individuals . 
li d , shimamura t , ji h , et al : bronchial and peripheral murine lung carcinomas induced by t790m - l858r mutant egfr respond to hki - 272 and rapamycin combination therapy . 
meric - bernstam f , brusco l , daniels m , et al : incidental germline variants in 1 , 000 advanced cancers on a prospective somatic genomic profiling protocol . 
hu y , alden rs , odegaard ji , et al : discrimination of germline egfr t790m mutations in plasma cell - free dna allows study of prevalence across 31 , 414 cancer patients . 
oxnard gr , heng jc , root ej , et al : initial results of a prospective , multicenter trial to study inherited lung cancer risk associated with germline egfr t790m : inherit egfr . 
kris mg , natale rb , herbst rs , et al : efficacy of gefitinib , an inhibitor of the epidermal growth factor receptor tyrosine kinase , in symptomatic patients with non - small cell lung cancer : a randomized trial . 
 clinical observation of improved outcomes of patients with brca 1 / 2 breast cancer treated with everolimus prior to dna - damaging therapy introduction the mammalian target of rapamycin ( mtor ) pathway comprises mtor complexes 1 and 2 ( mtorc1 / 2 ) and is primarily responsible for maintaining cellular growth and proliferation . 
although it is known to act as a catalyst toward anabolic synthesis , mtorc1 also is responsible for mitigating cellular catabolic processes , namely autophagy.1 mtorc2 complements mtorc1 in anabolic processes.2 phosphoinositide 3 - kinase ( pi3k ) and phosphatase and tensin homolog ( pten ) genetic mutations frequently are found in many tumor types , including breast cancer.3 these genes are important in regulating the downstream activation of the mtor pathway . 
many drugs have been developed to target the mtor pathway , including the mtorc1 inhibitor ( mtori ) everolimus.2 , 4 poly ( adp - ribose ) polymerase ( parp ) is a family of enzymes that repairs damaged dna . 
treatment with a parp inhibitor ( parpi ) prevents dna repair in cancer cells during homologous recombination ( hr ) events in patients with brca1 / 2 mutations , which leads to cell death through synthetic lethality.5 brca1 / 2 germline mutations are found in approximately 5% of unselected breast cancers , with higher prevalence in high - risk cohorts.6 activation of the pi3k / mtor pathway is important in decreasing the effect of brca1 / 2 mutations by stabilizing hr.7 ibrahim et al8 showed that when pi3k is inhibited , hr is impaired , which results in sensitization of brca wild - type triple - negative breast cancer cells to parpis . 
the details of the cohort are listed in table 1 . our analysis revealed that six patients among the 14 treated in brocade had experienced exceptional responses to treatment ; these six had been treated with the mtori everolimus before commencing study therapy . 
the patient with the second - best response had a documented pten deletion and nearly the same pfs at 29.2 months but did not receive veliparib with her carboplatin doublet . discussion most of the preclinical data of the role of the pik3ca / mtor pathway in dna damage sensitivity was carried out with concurrent therapeutic combinations . 
 these adaptive changes can persist over time , which allows one to use a treatment to sensitize a patients tumors to a subsequent therapy.14 , 15 the observed difference in pfs between eve + and eve patients raises the possibility that prior mtori exposure may render brca1 / 2 mutated breast cancer more susceptible to subsequent dna - damaging therapy . the interaction between activating pik3ca mutations or pten deletions and this sensitizing effect by mtori cannot be fully evaluated in this limited case series . 
however , it seems that the presence of these genomic abnormalities did not preclude those patients from deriving a significant benefit from subsequent dna - damaging therapy . in addition to the usual limitations and biases of case series , our analysis was limited by the heterogeneity of patient characteristics , prior treatments received , and study treatment assigned . 
this precludes us from specifically concluding how much of the observed clinical benefit in the eve + group was a result of improving the activity of the cytotoxic chemotherapy or the veliparib ( if given )  . 
in addition , veliparib did not significantly increase the median pfs of the experimental arm compared with the control arm in the overall study , so the effect of the treatment assignment may be minor in explaining the difference between the eve + and eve arms . 
the pfs of these two patients was similar to the main study population , so including them in the analysis with the other estrogen receptor positive patients is unlikely to change the main analysis . 
future studies in patients with brca1 / 2 mutations also should examine sequential treatment strategies by administering pi3kcai / mtori agents for one to two cycles as a sensitizing therapy before switching to a parpi or dna - damaging cytotoxic chemotherapy . 
han research funding : abbvie ( inst ) , prescient therapeutics ( inst ) , tapimmune ( inst ) , seattle genetics ( inst ) , g1 therapeutics ( inst ) , bristol - myers squibb ( inst ) , pfizer ( inst ) , novartis ( inst ) hatem h . 
lee moffitt cancer center and research institute , tampa , fl . presented at the 2017 annual meeting of the american society of clinical oncology , chicago , il , june 2 - 6 , 2017 . prior presentation references 10 : 18 , 2018 2012 150 , 2009 2017 1 . 
ibrahim yh , garca - garca c , serra v , et al : pi3k inhibition impairs brca1 / 2 expression and sensitizes brca - proficient triple - negative breast cancer to parp inhibition . 
mo w , liu q , lin cc , et al : mtor inhibitors suppress homologous recombination repair and synergize with parp inhibitors via regulating suv39h1 in brca - proficient triple - negative breast cancer . 
juvekar a , burga ln , hu h , et al : combining a pi3k inhibitor with a parp inhibitor provides an effective therapy for brca1 - related breast cancer . 
beuvink i , boulay a , fumagalli s , et al : the mtor inhibitor rad001 sensitizes tumor cells to dna - damaged induced apoptosis through inhibition of p21 translation . 
oreilly t , mcsheehy pmj , wartmann m , et al : evaluation of the mtor inhibitor , everolimus , in combination with cytotoxic antitumor agents using human tumor models in vitro and in vivo . 
han hs , diras v , robson m , et al : veliparib with temozolomide or carboplatin / paclitaxel versus placebo with carboplatin / paclitaxel in patients with brca1 / 2 locally recurrent / metastatic breast cancer : randomized phase ii study . 
 c rare pediatric invasive gliobroma has braf v600e mutation and transiently responds to targeted therapy before progressive clonal evolution kristiyana kaneva , md1 ; kee kiat yeo , md1 ; debra hawes , md1 , 2 ; jianling ji , md1 , 2 ; jaclyn a . 
a ventriculo - peritoneal shunt was subsequently placed to relieve the hydrocephalus . temporal microscopically , the tumor showed a biphasic growth pattern with an astrocytic component arranged in nests and cords surrounded by anastomosing bands of mesenchymal tissue ( fig 2 )  . 
the astrocytic component expressed glial brillary acidic protein ( gfap ) and showed mild - to - moderate cellular atypia , mitotic rate , and ki - 67 labeling index of 6% to 7% without palisading necrosis or glomeruloid endothelial proliferation , which is consistent with a low - grade glial neoplasthe mesenchymal component was cytologically bland , showed no mitotic activity , and did not express gfap . 
these features were consistent with a diagnosis of gliobroma , which consists of both mesenchymal and glial elements and has a low proliferation index ( fig 2 )  . signicant clinical improvement followed the initiation of dexamethasone , partial resection , and relief of hydrocephalus ; however , two careful attempts to wean the corticosteroids were unsuccessful . 
magnetic resonance ( mr ) spectroscopy at that time demonstrated highgrade featuresfor example , prominent cholinein the residual mass ( fig 3 )  . molecular studies using the oncoscan ( thermo fisher scientic , waltham , ma ) chromosomal microarray platform , which has been validated for brafv600e , revealed brafv600e mutation and biallelic deletion of cdkn2a ( table 1 )  . 
in children , cdkn2a / b loss has been found in gliomas and ependymomas.1 - 3 immunohistochemistry revealed mutant brafv600e in the glial elements of the tumor and absence of p16 ( cdkn2a ) in both glial and mesenchymal components ( fig 2 ) , which is consistent with the molecular analysis that shows brafv600e mutation and biallelic deletion of cdkn2a / b ( table 1 )  . the inltrative nature of the tumor ; the inability to wean the patient off dexamethasone ; the molecular studies showing a brafv600e mutation and deletion of cdkn2a , which portend a worse prognosis in pediatric glioma patients1 , 2 , 4 ; and the high - grade features on mr spectroscopy raised concern for the presence of high - grade inltrative components in the residual tumor despite the low - grade appearance of its resected portions . 
furthermore , in view of the poor prognosis of incompletely resected gliobromas ( fig 4 and data supplement ) , absence of established therapy for them , and an actionable mutation found in the patients tumor , targeted therapy was initiated . reports of emerging resistance to braf inhibitor monotherapy in patients with melanoma and improved survival with combined therapy prompted us to recommend combining braf inhibitor with mitogenactivated protein kinase ( mapk ) kinase ( mek ) inhibitor.5 the patient thus received vemurafenib 550 mg / m2 per dose two times per day ( a braf inhibitor ) and trametinib 4 mg / m2 per day ( an mek inhibitor )  . 
 ( o and p ) mri at 15 months shows a newly emerging dominant enhancing mass involving the posterior left temporal lobe with associated mass effect and deviation of the midbrain . tumors , was at more moderate levels . 
spectroscopy continued to suggest a lower grade lesion compared with pretherapy spectroscopy , similar to that at 9.5 weeks on therapy ( fig 3 and not liver transaminases prompted shown )  . 
 ( a ) hematoxylin and eosin ( h&e ) stained section of tumor showing biphasic growth pattern with nests of glial cells ( black arrow ) surrounded by bands of mesenchymal tissue ( white arrow )  . 
nuclei not in g0 phase are positive for ki - 67 ( dab , brown ) , whereas nuclei in g0 phase are negative ( hematoxylin , blue )  . 
 ( e and f ) in contrast to the trichrome and reticulin stains , glial brillary acidic protein ( gfap ) is expressed in the glial component of the tumor ( dab , brown ) and not in the mesenchymal component ( hematoxylin , blue )  . 
 ( j and k ) large areas of the glial components of the tumor were positive for the brafv600e - mutated protein ( dab , brown , ihc ) , although some regions did not express it . 
 ( n - r ) at recurrence , the tumor showed high - grade regions that were invasive into the lower - grade regions ( n ) , were highly cellular ( p ) , and had abundant mitotic gures ( q ; panels correspond to the dotted regions in panel p )  . 
the tumor continued to express the mutant brafv600e in many of the high - grade regions , although as in the original low - grade tumor , there were areas that were negative for this mutation ( r )  . routine mri at 15 months while on braf inhibitor ( vemurafenib ) plus mek inhibitor therapy revealed new growth with imaging features of a high - grade tumor , similar to pretherapy mr spectroscopy ( figs 1 and 3 )  . 
reresection showed gliobroma with areas of low - grade morphology which was ( trametinib ) similar to the original tumor , along with extensive areas that now contained high - grade features ( fig 2 )  . 
high - grade features included increased cellularity , high mitotic rate , and regions with markedly elevated mitotic index ( ki - 67 positive in up to 70% to 80% of nuclei )  . 
mr spectroscopy also demonstrates metabolic heterogeneity with markedly different levels of glycine ( glyc ) in different parts of the lesion with unclear signicance . lactate ( lac ) is observed in both regions of interest at approximately equal levels . 
 ( i and j ) at 15 months of targeted therapy , the newly the spectrum in the region of emerging mass shows signicantly increased cho that is consistent with proliferative tumor . 
brafv600e is not shown but present on chromosomal microarray , which has been validated for this mutation . abbreviation : cnv , copy number variant . proceeded to receive intensity - modulated radiation therapy to the tumor . molecular studies of the recurrent tumor again revealed the brafv600e mutation ( oncokids next - generation sequencing panel and oncoscan chromosomal microarray ) and the cdkn2a biallelic deletion ( oncoscan )  . 
on the basis of author - reported histopathology , patients with highgrade tumors had signicantly worse overall survival compared with low - grade tumors ( p , .001 ; fig 4 ) , similar to prior assessment.15 survival of patients who underwent complete resection was not signicantly different from that of patients with partial resection or biopsy ( p = .108 ; fig 4 )  . to date , only two patients with gliobroma have been reported to have experienced a response to chemotherapy . both were infants with unresectable low - grade gliobromas . 
one experienced a response to carboplatin and vincristine36 and the other to metronomic vinblastine.32 analysis of the rare published cases presented here is limited in that it depends on individual case reports that mostly provide relatively short and incomplete follow - up and may represent an unbalanced cohort of patients . 
thus , although this summary , to our knowledge , is the most comprehensive review of published gliobroma cases to date , these limitations must be considered . our patient provides four novel ndings with regard to gliobroma : this is the rst report , to our knowledge , on gliobroma with a molecularly proven brafv600e mutation and cdkn2a biallelic deletion ; the rst patient with gliobroma reported to receive targeted therapy and to respond to braf plus mek inhibition , albeit only for limited time ; the rst gliobroma described to acquire pdgfra and ptpn11 mutations upon recurrence as a high - grade glioma resistant to braf and mek inhibition ; and a rare opportunity to view the rapid molecular evolution of this tumor , with ( rst recurrence ) and without ( second recurrence ) therapy with braf plus mek inhibition . 
of interest , the brafv600e mutation in our patient was only found in the glial elements of the tumor , which suggests that the mesenchymal regions may be reactive to the glial pathology rather than independently tumorigenic . 
survival status ( alive / dead ) was not reported for ve of the 44 published patients , and duration of survival was not reported for ve others for whom survival status was reported . 
 ( a ) age of distribution at diagnosis of gliobroma . ( b ) overall survival for the 35 patients with gliobroma for whom survival data were reported ( of a total of 45 )  . 
at risk : glial and mesenchymal components , it is not known if this is a result of the deletion of cdkn2a in both compartments or if cdkn2a is deleted in one compartment and its expression is merely low in the other . 
it is interesting that in another glial tumor , ganglioglioma , brafv600e - expressing cells were mostly those with neuronal capacity or both neuronal and glial capacity.39 in contrast to that shown in our patient , in those gangliogliomas , p16 was robustly expressed.39 brafv600e oncogenic mutation is reported in 17% of pediatric low - grade gliomas.1 , 4 , 40 - 42 to date , brafv600e mutation has not been reported in gliobromas . 
one case report stated that braf mutations are present in approximately 50% of gliobromas7 ; however , the authors case did not have a braf mutation and no reference was provided to support the statement . 
this can be achieved best by a next - generation sequencing panel that covers both molecular alterations , such as the childrens hospital los angelesdeveloped oncokids panel.46 considering the poor response of incompletely resected brafv600e - mutated and cdkn2a - deleted pediatric lowgrade gliomas to chemotherapy and irradiation , 4 as well as the aggressive behavior of our patients tumor at presentation , we elected to treat him with the brafv600e inhibitor vemurafenib combined with the mek inhibitor trametinib . 
oncokids next - generation sequencing cancer panel findings from second reresection ( second recurrence , third tumor sample ) classication gene name exon genomic position dna change protein change strong clinical signicance ( tier i ) braf chr7 : 140 , 453 , 136 c . 
the results of this nal sample demonstrate two variants of strong clinical signicance ( braf and pik3ca ) and three variants of potential clinical signicance ( ptpn11 , pdgfra , and tp53 )  . abbreviation : chr , chromosome . brafv600e tumors to braf inhibitor monotherapy include an increase in braf dimers , braf splice variants , neuroblastoma ras viral oncogene homolog ( nras ) and kirsten rat sarcoma viral oncogene homolog ( kras ) mutations , braf amplications , mek mutations , and others.49 , 51 , 52 these render extracellular regulated kinase signaling insensitive to braf inhibitors while the downstream mek remains a viable target . 
combining a braf inhibitor with an mek inhibitor is thought to help retain tumor responsiveness and to have lower adverse effects compared with braf monotherapy.47 , 49 , 50 , 53 - 55 trametinib and other mek inhibitors are in clinical trials for pediatric patients . 
whereas preclinical research suggested a benet to combining a braf inhibitor and / or mek inhibitor with a cyclin - dependent kinase 4 / 6 inhibitor in tumors with both brafv600e and cdkn2a deletion , 57 , 58 peer - reviewed clinical data on the safety and efcacy of such combination were not available when the patient presented . our patients tumor showed clinical and radiographic response to vemurafenib and trametinib for more than 12 months . 
although it is possible that intratumor heterogeneity was the basis for their absence in the sample tested from the original tumor , their coexistence with the brafv600e mutation and cdkn2a deletion in the recurrent sample suggests that they are newly acquired and may have played a role in the recurrence . 
it is not known if these new mutations are in tumor cells that express the mutant braf protein or in those that stain negative for it ( fig 2r )  . overexpression and mutations in pdgfra are frequent in pediatric gliomas and in other cancers , with mutations occurring in more than 20% of high - grade gliomas but only rarely in low - grade ones.59 - 61 the pdgfra mutation , in addition to the aggressive nature of our patients recurrent glioma , supported the use of radiation therapy for the recurrence.61 mutations in d842 , located in the activation loop of pdgfra , 62 comprise more than one third of pdgfra substitution mutations reported to date in the cosmic catalogue ( 610 of 1 , 705 ) , with the majority ( 95% ) being d842v.63 , 64 to imatinib pdgfra - d842v renders pdgfra resistant in vitro , 64 - 66 but it remains sensitive to crenolanib , a newer pdgfra inhibitor that is currently in clinical trials.63 , 64 however , pdgfra - d842y , the mutation in our patients tumor , is moderately resistant to imatinib in vitro and is not as sensitive to crenolanib as the d842v mutant or the wild - type receptor.65 ptpn11 ( shp - 2 ) is mutated in the germline in noonan and leopard syndromes as well as somatically in cancer.67 across cancers , ptpn11 is mutated in 1 , 320 ( 1.9% ) of 70 , 712 cancer samples reported in cosmic.64 ptpn11 mutations are observed in both low - grade and high - grade pediatric gliomas.61 the ptpn11 ala72val ( a72v ) mutation in our patients recurrent tumor is located in the n - terminal sh2 domain of ptpn11 and is predicted to be a gain - of - function mutation.68 of the total 1 , 285 ptpn11 substitution mutations reported in cosmic , 180 are in alanine 72 , the second most mutated amino acid after glutamine 76 . 
several ptpn11 inhibitors are now beginning clinical trials in cancer in adults.69 in this respect , it is interesting that in mice ptpn11 can mediate glioma formation downstream of pdgfra overexpression and ink4a ( cdkn2a ) loss.70 it is therefore conceivable that , with this gliobromas pdgfra - d842y and ptpn11 - a72v activating mutations and the biallelic deletion in cdkn2a , this pathway may have contributed to the recurrent tumor in our patient . the serial acquisition of new mutations on subsequent recurrent specimens is consistent with clonal evolution of disease . 
pik3ca mutations are found in 21% of pediatric gliomas compared with 17% in adult glioblastomas71 , 72 ( table 2 )  . in summary , to our knowledge , this is the rst published report of a gliobroma that harbors a brafv600e mutation , the rst report on a gliobroma that responded to targeted therapy , and a unique view of clonal evolution of this rare tumor . 
biegel provision of study material or patients : kristiyana kaneva , debra hawes collection and assembly of data : kristiyana kaneva , kee kiat yeo , debra hawes , stefan bluml , mark d . 
nelson patents , royalties , other intellectual property : royalties from a book published in 2012 expert testimony : mdnelson , jr no other potential conicts of interest were reported . acknowledgment the authors thank yang fusheng for the expert immunohistochemistry contribution . 
mistry m , zhukova n , merico d , et al : braf mutation and cdkn2a deletion dene a clinically distinct subgroup of childhood secondary high - grade glioma . j clin oncol 33 : 1015 - 1022 , 2015 2 . 
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j clin oncol 35 : 2934 - 2941 , 2017 johnson db , flaherty kt , weber js , et al : combined braf ( dabrafenib ) and mek inhibition ( trametinib ) in patients with brafv600 - mutant melanoma experiencing progression with single - agent braf inhibitor . 
j neuropathol exp neurol 37 : 300 , 1978 ahmad mu , barborie a , pizer b , et al : midbrain gliobroma presenting in adulthood following cure of a childhood intraventricular pilocytic astrocytoma . pediatr neurosurg 52 : 151 - 154 , 2017 altamirano e , jones mc , drut r : gliobroma . 
escalante abril pa , salazar mf , l opez garca nl , et al : who grade iv gliobroma : a grading label denoting malignancy for an otherwise commonly misinterpreted neoplasj pathol transl med 49 : 325 - 330 , 2015 [ erratum : j pathol transl med 49 : 538 , 2015 ] 16 . 
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schindler g , capper d , meyer j , et al : analysis of braf v600e mutation in 1 , 320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma , ganglioglioma and extra - cerebellar pilocytic astrocytoma . 
clin cancer res 17 : 4790 - 4798 , 2011 jones dt , kocialkowski s , liu l , et al : tandem duplication producing a novel oncogenic braf fusion gene denes the majority of pilocytic astrocytomas . cancer res 68 : 8673 - 8677 , 2008 45 . 
j neurooncol 131 : 495 - 505 , 2017 johanns tm , ferguson cj , grierson pm , et al : rapid clinical and radiographic response with combined dabrafenib and trametinib in adults with braf - mutated high - grade glioma . 
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falchook gs , lewis kd , infante jr , et al : activity of the oral mek inhibitor trametinib in patients with advanced melanoma : a phase 1 dose - escalation trial . lancet oncol 13 : 782 - 789 , 2012 54 . 
king aj , arnone mr , bleam mr , et al : dabrafenib ; preclinical characterization , increased efcacy when combined with trametinib , while braf / mek tool combination reduced skin lesions . 
huillard e , hashizume r , phillips jj , et al : cooperative interactions of brafv600e kinase and cdkn2a locus deciency in pediatric malignant astrocytoma as a basis for rational therapy . 
cancer res 73 : 6219 - 6229 , 2013 johnson a , severson e , gay l , et al : comprehensive genomic proling of 282 pediatric lowand high - grade gliomas reveals genomic drivers , tumor mutational burden , and hypermutation signatures . 
liang l , yan xe , yin y , et al : structural and biochemical studies of the pdgfra kinase domabiochem biophys res commun 477 : 667 - 672 , 2016 63 . 
lasota j , dansonka - mieszkowska a , sobin lh , et al : a great majority of gists with pdgfra mutations represent gastric tumors of low or no malignant potential . lab invest 84 : 874 - 883 , 2004 64 . 
dempke wcm , uciechowski p , fenchel k , et al : targeting shp - 1 , 2 and ship pathways : a novel strategy for cancer treatment ? oncology 95 : 257 - 269 , 2018 70 . 
liu kw , feng h , bachoo r , et al : shp - 2 / ptpn11 mediates gliomagenesis driven by pdgfra and ink4a / arf aberrations in mice and humans . 
we have previously reported preliminary results of our precision medicine prograhere , we present response and survival outcomes for 637 additional patients who were referred for phase i trials and were treated with matched targeted therapy ( mtt ) when available . patients and methods patients with advanced cancer who underwent tumor genomic analyses were treated with mtt when available . results overall , 1 , 179 ( 82.1% ) of 1 , 436 patients had one or more alterations ( median age , 59.7 years ; men , 41.2% ) ; 637 had one or more actionable aberrations and were treated with mtt ( n = 390 ) or non - mtt ( n = 247 )  . 
patients with phosphatidylinositol 3 - kinase ( pi3k ) and mitogenactivated protein kinase pathway alterations matched to pi3k / akt / mammalian target of rapamycin axis inhibitors alone demonstrated outcomes comparable to unmatched patients . conclusion our results support the use of genomic matching . 
in 2007 , we initiated the impact ( initiative for molecular profiling and advanced cancer therapy ) trial , a personalized medicine program for patients who were referred to the phase i clinical trials program at md anderson cancer center . 
personalized , or precision , medicine uses traditional and emerging concepts with regard to the genetic and environmental basis of disease to individualize prevention , diagnosis , and treatment , 1 , 2 and integrates tumor genetics of individual patients into medical practice.3 the aim of the personalized medicine program is to use tumor molecular profiling to optimize the selection of targeted therapies for patients who are considered for phase i clinical trial participation . 
 here , we present results for 1 , 436 additional patients who underwent clinical - grade molecular profiling before treatment in the phase i clinical trials prograwe demonstrate that matched targeted therapy ( mtt ) is associated with improved outcomes . 
group 2 : mtt with tp53 match and inclusion of vegf / vegfr therapy as targeted therapy against tp53 ; non - mtt ( all others ) .13 group 3 : negative matches include only mtt against an alteration in the pi3k axis in the presence of kras or braf or another mek pathway mutation ( the latter being a known resistance pathway ) and no mek / raf inhibitor ; negative matches do not include patients with additional positive matches ; positive mtt minus negative matches include patients who had mtt ( without considering tp53 mutations matched to vegf / vegfr inhibitors ) minus the negative matches ; non - mtt ( patients who had no mtt minus the negative matches )  . 
baseline characteristics of 637 patients with mutations by type of therapy nonmatched ( n = 247 ) matched ( n = 390 ) characteristic age , years , 60 > 60 female male no . 
patients whose tumors had a molecular aberration were preferably treated in a clinical trial with a matched targeted agent when available . if two or more molecular aberrations were present , patients were preferably treated in a trial of therapies that targeted both aberrations . 
if such a trial was unavailable , a physicians choice of a trial that targeted one aberration was permitted ( appendix )  . definition of matched therapy a patients tumor was considered matched with a targeted therapy if a drug was known to inhibit the aberration at low nanomolar concentrations or if an antibody targeted the alteration product , per literature data 9 - 12 ( appendix )  . 
subset analyses are listed in table 1 . end points and statistical methods statistical analyses were performed by a biostatistician ( k.h. ) using s - plus software for windows ( version 8.2 ; tibco software , palo alto , ca )  . 
by using response evaluation criteria in solid tumors ( recist ) guidelines , 14 , 15 we assessed objective response rates ( complete response [ cr ] and partial response [ pr ] ) , clinical benefit rates ( stable disease [ sd ] > 6 months plus pr plus cr ) , and rates of failure - free survival ( ffs ) and overall survival ( os )  . 
failure - free survival and overall survival in patient subgroups . group 1 : matched targeted therapy ( mtt ) versus overall , 390 patients ( 27% of 1 , 436 total patients ; or 61.2% of 637 patients ) received matched therapy and 247 ( 17.2% ) received nonmatched therapy . tumor types treated are listed in table 3 . 
group 2 : mtt with tp53 ( includes matching as previously published plus inclusion of antivascular endothelial growth factor [ vegf ] therapy as targeted therapy against tp53 ) versus nonmtt . 
group 3 : mtt minus negative matches ( includes patients who had matched targeted therapy , without considering tp53 mutations matched to vegf / vegf receptor p = .041 ; table 1 and figs 1a and 1b )  . 
group 4 : mtt with tp53 match minus negative matches ( includes tp53 mutation matches to vegf / vegfr inhibitors but excludes negative matches ) versus negative matches ( see definition for group 3 ; table 1 ) versus non - mtt . ( g ) failure - free survival . ( h ) overall survival . 
group 5 : non - mtt ( includes nontargeted therapy or targeted therapy against an alteration in the pi3k axis in the presence of kras or braf or other mek pathway mutation and no mek / raf inhibitor ) versus mtt ( includes all other groups , including tp53 matches )  . 
no difference was observed in the unmatched group . group 3 analysis the group 3 analysis was similar to that of group 1 , except that we isolated a group of 36 patients who had negative matches , a term applied when patients were matched by virtue of a pi3k pathway alteration to a pi3k / akt / mtor axis inhibitor , but a ras / raf / mek alteration was also present and not matched to a drug . 
these results showed that negative matches attained rates of cr / pr and sd > 6 months / cr / pr that were similar to those of unmatched patients , and that ffs and os were equivalent . 
these results are consistent with the hypothesis that matching patients who harbor a pi3k and a mapk pathway alteration to a pi3k pathway inhibitor alone does not improve outcomes.8 , 16 group 4 analysis the group 4 analysis was similar to that of group 2 , which included tp53 mutations matched to vegf / vegfr inhibitors , except that we again isolated the group of 36 patients who had negative matches . 
these results demonstrate that negative matches had rates of cr / pr and sd > 6 months / cr / pr that were similar to those of unmatched patients , and that ffs and os were similar . 
conversely , depending on the outcome parameter , negatively matched patients either had significantly worse outcomes or trended toward worse outcomes compared with positively matched patients . group 5 analysis the group 5 analysis compared positively matched patients , which included tp53 mutations matched to vegf / vegfr inhibitors , with negatively matched patientsas defined for group 4plus unmatched patients . 
these results demonstrate that , for all outcome parameters , matched patients , which included tp53 mutations matched to vegf / vegfr inhibitors , did significantly better than the combined group of unmatched and negatively matched patients . discussion the application of precision medicine using targeted agents to inhibit the function of tumor molecular alterations is a complex process . 
 ( f ) failurefree survival according to objective response ( cr + pr , yes ) versus no response ( no ) in patients who received in this retrospective analysis , we report 1 , 436 patients who underwent prospective molecular profiling with a clia - certified test . 
of 637 treated patients with at least one alteration , 61% ( n = 390 patients ; 27% of the total 1 , 436 patients ) received mtt and 39% received non - mtt . 
this number is similar to that of other studies ; tumors that were interrogated with larger panels of genes are more likely to have alterations.13 , 17 , 18 indeed , in some reports , approximately 95% of patients from whom adequate tissue is obtained are found to have molecular alterations.13 , 16 - 20 the complexity of using precision medicine is explained , in part , by the coexistence of multiple molecular alterations , as seen in our population , the lack of efficient targeted agents for all alterations , and the fact that some alterations are more difficult to target than others.19 this observation is consistent with previously published results that have demonstrated that a matching scorenumber of matches divided by number of alterationscorrelates well with all outcome parameters.13 a limitation of the current analysis relates to the fact that gene panels of different sizes were used for molecular profiling , and , hence , the impact of the number of alterations in each patient could be confounded by the panel size . several previous studies have been performed with variable results . 
for instance , the shiva triala randomized molecular navigation trialdid not achieve its endpoints16 ; however , approximately 80% of patients in shiva were matched to single - agent hormone modulators or mtor inhibitors.21 , 22 our current results confirm that single - agent pi3k pathway inhibitors are generally not effective , especially in patients with concomitant mapk pathway alterations . 
indeed , for these patients , targeting the pi3k pathway without targeting the mapk pathway was associated with outcomes that were indistinguishable from , or numerically worse than , those in unmatched patients ( table 1 and figs 1e to 1h )  . 
outcome parameters were favorable in groups that included patients who were matched in this way , and the strongest p values were observed in the group of patients that both included tp53 alterations matched to vegf / vegfr inhibitors and excluded patients who harbored both pi3k and mapk pathway alterations matched to only pi3k / akt / mtor axis inhibitors ( group 5 ; table 1 and fig 1 )  . 
these observations are supportive of previously published findings that tp53 may be a targetable alteration , and suggest that , in patients with pi3k pathway anomalies , the presence of mapk resistance alterations must be addressed . our study had several limitations . 
third , molecular analysis was performed by using archival tissue in most patients , tissue was sometimes inadequate for testing all available molecular aberrations , and the availability of clia - certified tests varied over the years and , thus , some molecular profiles became suboptimal by later standards . 
to address the limitations of this study , we have initiated the impact 2 trial , a phase ii randomized study evaluating molecular profiling and targeted therapy in metastatic cancer ( nct02152254 ) .6 , 7 multiple other nonrandomized trials of molecular profiling have been completed or are ongoing ( nct01771458 , nct01827384 , nct02154490 , nct01042379 , and nct01856296 ) .6 , 7 , 13 , 16 , 17 , 23 , 24 in conclusion , our analysis demonstrates that precision medicine is feasible in an academic setting and that it is associated with superior outcomes compared with non - mtt in patients with advanced cancer . 
the rate of matching was approximately 27% in our group of patients , which is consistent with some previous studies but higher than that observed in other trials.6 , 7 , 13 , 17 , 18 higher rates of matching may be associated with larger gene panels , which produce more potentially actionable alterations , 18 with more robust clinical trial portfolios and with molecular studies with flexible algorithms and eligibility criteria that are not overly restrictive . 
kies , russell broaddus , john mendelsohn , razelle kurzrock collection and assembly of data : apostolia - maria tsimberidou , yang ye , carrie cartwright data analysis and interpretation : apostolia - maria tsimberidou , kenneth r . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs. org / site / ifc . apostolia - maria tsimberidou research funding : emd serono ( inst ) , baxter ( inst ) , foundation medicine ( inst ) , onyx pharmaceuticals ( inst ) , bayer ( inst ) , boston biomedical ( inst ) , placon therapeutics ( inst ) david s . 
hong stock and other ownership interests : molecularmatch , oncorena honoraria : adaptimmune , baxter , merrimack pharmaceuticals , bayer consulting or advisory role : baxter , bayer research funding : novartis , genentech , eisai , astrazeneca , pfizer , mirna therapeutics , amgen , daiichi sankyo , merck , mirati therapeutics , eli lilly travel , accommodations , expenses : loxo , mirna therapeutics other relationship : oncorena the following represents disclosure information provided by authors of this manuscript . 
kies no relationship to disclose russell broaddus no relationship to disclose john mendelsohn leadership : merrimack pharmaceuticals stock and other ownership interests : merrimack pharmaceuticals patents , royalties , other intellectual property : royalty payments from university of california san diego travel , accommodations , expenses : merck kenneth r . 
hess , the university of texas md anderson cancer center , houston , tx ; and razelle kurzrock , university of california , san diego , san diego , ca . supported in part by the alberto barretto donor fund , the jamie hope donor fund , funding from and zane w . 
wheler j , tsimberidou am , hong d , et al : survival of 1 , 181 patients in a phase i clinic : the md anderson clinical center for targeted therapy experience . 
druker bj , talpaz m , resta dj , et al : efficacy and safety of a specific inhibitor of the bcr - abl tyrosine kinase in chronic myeloid leukemia . 
tsimberidou am , wen s , hong ds , et al : personalized medicine for patients with advanced cancer in the phase i program at md anderson : validation and landmark analyses . 
clin cancer res 20 : 4827 - 4836 , 2014 janku f , hong ds , fu s , et al : assessing pik3ca and pten in early - phase trials with pi3k / akt / mtor inhibitors . cell reports 6 : 377 - 387 , 2014 9 . 
said r , hong ds , warneke cl , et al : p53 mutations in advanced cancers : clinical characteristics , outcomes , and correlation between progression - free survival and bevacizumab - containing therapy . 
koehler k , liebner d , chen jl : tp53 mutational status is predictive of pazopanib response in advanced sarcomas . ann oncol 27 : 539 - 543 , 2016 11 . 
schwaederle m , lazar v , validire p , et al : vegf - a expression correlates with tp53 mutations in non - small cell lung cancer : implications for antiangiogenesis therapy . 
therasse p , arbuck sg , eisenhauer ea , et al : new guidelines to evaluate the response to treatment in solid tumors . european organization for research and treatment of cancer , national cancer institute of the united states , national cancer institute of canada . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
le tourneau c , kurzrock r : targeted therapies : what have we learned from shiva ? nat rev clin oncol oncotarget 5 : 3871 - 3879 , 2014 drugs . 
von hoff dd , stephenson jj jr , rosen p , et al : pilot study using molecular profiling of patients tumors to find potential targets and select treatments for their refractory cancers . 
mol cancer ther 16 : e579 - e580 , 2015 14 : 847 - 856 , 2015 patients methods in brief , patients with advanced or metastatic cancer for whom standard - of - care therapy had been exhausted or for whom no food and drug administrationapproved therapy was available for their indication were referred for participation in phase i clinical trials . 
all protocols required that participants have evidence of evaluable or measurable disease according to recist guidelines14 , 15 and an eastern cooperative oncology group performance status of 0 to 2 . 
additional eligibility criteria varied depending on which specific phase 1 clinical trial in which the patient enrolled . archival formalin - fixed , paraffin - embedded tissue blocks or tissue from fine - needle aspiration or surgical biopsies was used for profiling . 
molecular and / or immunohistochemistry assays were performed when tissue was available by using standard operating procedures and pcr - based sequencing technology . tumor molecular profiling for 637 patients was performed in the following laboratories : md anderson cancer center ( hotspot testing of 11 genes , n = 282 ; 46 genes , n = 145 ; and 50 genes , n = 20 ; patients , n = 447 ) , foundation medicine ( nextgeneration sequencing of 182 genes ; patients , n = 151 ) , knight diagnostics ( 48 genes ; patients , n = 20 ) , and other cliacertified laboratories ( patients , n = 19 )  . analysis of molecular aberrations dna was extracted from microdissected , paraffin - embedded tumor samples , and the coding regions for specific exons , depending on the test ordered , were analyzed for the following genes : pik3ca ( exon 9 : codons 532 to 554 ; exon 20 : codons 1 , 011 to 1 , 062 ) ; braf ( exon 15 : codons 595 to 600 ) ; kras and nras ( exon 2 : codons 12 , 13 , and 61 ) ; egfr ( exons 18 to 21 of the kinase domain ) ; kit ( exons 9 , 11 , 13 , and 17 ) ; gnaq ( exon 5 ) ; tp53 ( exons 4 to 9 ) ; met ( exon 2 : codon 375 ; exon 11 : codon 848 ; exon 14 : codons 988 and 1010 ; exon 16 : codons 1112 and 1124 ; exon 19 : codons 1 , 248 , 1 , 253 , and 1 , 268 ) ; and ret ( exon 10 : codons 609 , 611 , 618 , and 620 ; exon 11 : codon 634 ; exon 16 : codon 918 )  . 
the sensitivity of mutation assays for the detection of pik3ca , braf , kras , nras , gnaq , and met mutations was approximately one in 10 mutation - bearing cells in the microdissected area . 
loss of expression of the tumor suppressor nuclear protein , pten , was determined by using immunohistochemical staining with the monoclonal mouse anti - human pten clone 6h2 ( code m3627 ; dako , carpinteria , ca )  . 
with the availability of next - generation sequencing , some patient tumors were assessed by more extensive molecular profiling of 48 or more genes at md anderson or in other clia - certified laboratories . selected patients also had tumor molecular profiling using next - generation sequencing performed by foundation medicine ( cambridge , ma )  . 
genomic libraries were captured for 3 , 230 exons in 182 cancer - related genes plus 37 introns from 14 genes that are often rearranged in cancer and sequenced to an average median depth of 734 3 with 99% of bases covered  . 
alternatively , fluorescent in situ hybridization analysis was used to measure the copy number of her2 / neu . before the use of next - generation sequencing , the clia pathology laboratory prioritized the testing of molecular aberrations on the basis of their known frequency in cancer and / or whether they were perceived as actionable or as having other clinical relevance to patients . 
treating physicians requested all available molecular tests that were clia - certified at md anderson cancer center at the time a patient who was interested in receiving treatment in the phase i clinical trials program presented to the phase i clinic . 
if tissue available for analysis was limited , the treating physician prioritized molecular testing on the basis of tumor type and the availability of clinical trials that could impact specific targets . treatment treatment regimens included one or more drugs . 
allocation of patients to investigational treatment varied over time according to protocol availability , eligibility criteria , histologic diagnosis , the patients prior response to therapy , potential toxicity , insurance coverage , and patient preference or physician choice . 
physicians prioritized matched therapyversus nonmatched therapyon the basis of the following criteria : patients had an actionable molecular aberration ; matched targeted therapy ( mtt ) was available ; patients met the eligibility criteria ; insurance coverage was obtained ; and patients agreed to comply with study requirements . 
 sponsored studies ( further limiting the number of patients enrolled per institution ) , and restrictions associated with eligibility criteria , not all patients with an actionable aberration could be treated on a protocol with matched therapy . definition of matched therapy pik3ca mutations and pten loss could be targeted by inhibitors of akt and mammalian target of rapamycin , as well as phosphatidylinositol 3 - kinase ( pi3k ) , as akt and mammalian target of rapamycin are downstream of activated pik3ca and because both pik3ca mutations and pten loss , which usually reflects pten mutation , activate pi3k . 
braf mutations could also be targeted by braf inhibitors . other aberrations , such as ret , egfr , kit , and met mutations , were targeted by drugs that inhibit the respective activated kinase with 50% inhibitory concentration ( ic50 ) in the low nanomolar range . 
tp53 mutations were not initially considered actionable by drugs available in our trials ; however , with more recent emerging data , 8 - 11 tp53 mutations were considered actionable by vascular endothelial growth factor ( vegf ) / vegf receptor ( vegfr ) inhibitors , and an appropriate secondary analysis on this basis was included . 
we performed subset analyses for clinical outcome comparisons using the following ( table 1 , footnote ) : group 1 : mtt , as previously published6 , 7 ; non - mtt ( all others )  . 
group 3 : negative matches , which includes only mtt against an alteration in the pi3k axis in the presence of kras or braf or another mek pathway mutation ( the latter being a known resistance pathway ) and no mek / raf inhibitor.8 negative matches do not include patients with additional positive matches ; positive mtt minus negative matches , which includes patients who had mtt ( without considering tp53 mutations matched to vegf / vegfr inhibitors ) minus the negative matches ; non - mtt refers to patients who had no mtt minus the negative matches . 
group 5 : mtt with tp53 match minus negative matches refers to all matched groups , including tp53 matches to vegf / vegfr inhibitors , but excluding negative matches ( group 4 negative matches excluded ) ; non - mtt plus negative matches includes patients without a match as well as mtt against an alteration in the pi3k axis in the presence of kras or braf or other mek pathway mutation ( the latter being a known resistance pathway ) and no mek / raf inhibitor ( group 4 negative matches included )  . the rationale for subset analyses is as follows : group 1 is straightforward and consistent with our previously published analyses.6 , 7 group 2 included anti - vegf / vegfr agents matched to tp53 alterations . 
recent studies demonstrated that altered tp53 upregulates angiogenesis and vegf - a.11 in addition , published data from our group and other investigators showed that patients with tp53 alterations have improved outcomes if treated with anti - vegf / vegfr agents.9 - 12 therefore , we did a subset analysis ( group 2 ) to examine matched versus unmatched therapy , including tp53 alterations matched to anti - vegf / vegfr agents . 
negative matches pertained to individuals who had both a pi3k axis alteration and a kras or braf or other mek pathway mutationthe latter being a known resistance pathwayand were matched only with a pi3k axis inhibitor . 
several investigators suggest that these patients are resistant to a pi3k axis inhibitor , likely because the mapk pathway is not targeted.8 , 17 , 18 in groups 4 and 5 , we further explored analyses that included negative matches together with unmatched patients as well as tp53 alterations matched to vegf / vegfr inhibitors . end points and statistical methods matched versus unmatched and grouping was verified by one of the coauthors ( r.k. ) who was blinded to outcome data at the time that designation was assigned . overall survival ( os ) was measured from the initiation of participation in the phase i trial until death or last follow - up . failure - free survival ( ffs ) was measured from the first day of treatment on a clinical trial until the date of discontinuation of treatment of any reason , including disease progression , treatment toxicity , or death , whichever came first . 
the decision to discontinue treatment on protocol was made by the treating physician and was based on the patients history , clinical presentation , and imaging studies ( response assessment using recist criteria )  . 
all p values presented are two sided , and the statistical significance level was p < .05. we also performed landmark analyses for ffs and os at 2 months , as we previously published.7 we omitted patients with event times or censoring times of , 2 months and stratified outcome by overall response status . we also compared matched responders with nonmatched responders ( supplemental file )  . results objective response in patients who were treated with matched and nonmatched therapy by tumor type and molecular alterations is shown in appendix tables a1 and a2 . 
the small numbers of patients in the subgroups preclude robust analyses . we also performed a 2 - month landmark analysis that compared ffs and os between matched responders and nonmatched responders . 
the small numbers of patients in the subgroups preclude robust analyses for most comparisons . we then performed an analysis , excluding patients with estrogen receptor / progesterone receptor overexpression from the matched therapy group . 
 ( c ) analysis excluding patients with estrogen receptor ( er ) / progesterone receptor ( pr ) overexpression - based matching from the matched therapy group 1 : failurefree survival . 
 novel egfr v834l germline mutation associated with familial lung adenocarcinoma introduction lung cancer is a worldwide , known disease with a high mortality rate and is often related to nicotine abuse . 
this analysis revealed a well - known oncogenic l858r mutation along with a v834l variant , which are both egfr exon 21 substitutions and are present in the same allele ( in cis )  . 
 the v834l variant seemed to be present in the germline , because both normal adjacent lymph node tissue and lymphocyte - derived dna from whole blood of the index patient showed this variant . the patient was treated with the egfr tyrosine kinase inhibitor ( tki ) erlotinib . 
the patients father died at a young age as the result of massive hemoptysis , without a diagnosis . after written consent and permission were obtained from the index patient and family members , egfr mutation and germline variation analyses were performed on tumor samples and healthy tissue , respectively , of the three affected relatives . 
 v834l l858r v834l l858r t790m v834l l858r t790m c797s the deaths of the index patients brother , sister , and daughter , no blood was available ; only archival formalin - fixed paraffin - embedded normal and tumor tissues were available . like our index patient , all three affected family members had a heterozygous egfr v834l germline variant in normal tissue and the same somatic egfr l858r mutation in cis with the v834l variant in their respective tumor tissues . 
to our knowledge , this is the first report of a family with multiple cases of nsclc associated with germline transmission of an egfr v834l germline variant ; all tumors in the four family members additionally harbored a somatic egfr l858r mutation in cis . 
all except one of the known v834l carriers developed lung cancer during their lifespan , but so far none of the investigated noncarriers . there are only a few reports documenting families with proven inherited egfr variants over generations that probably conferred predisposition to lung cancer.1 , 2 in a family with egfr v843i mutations , four family members in two generations ( three of them with proven germline variants ) developed lung adenocarcinoma.1 as in the family we studied , all of these adenocarcinomas displayed a somatically - acquired l858r mutation in cis with v843i . 
interestingly , multiple distinct synchronous tumors of the index patient displayed different additional egfr mutations ( ie , deletions of 3 and 15 bp in exon 19 and l858r ) .2 a report by bell et al3 describes a family with five members affected by lung cancer . 
proven germline egfr t790m mutations were present in three patients in two generations.3 in distinct lesions in two of these patients , the mutation co - occurred with different egfr substitutions ( l858r , l747t751del , and g716a ) , all in cis with t790m . 
 because t790m is a well - known tki resistance mutation , no response was observed upon gefitinib treatment in the only tki - treated patient of this family , as expected . incidental cases of germline egfr variants in patients with lung cancer have been reported . 
 these have included variants such as v769m , r776h , and t790m substitutions in exon 20 and r831c , v843i , and p848l substitutions in exon 21.4 - 9 however , in the absence of family histories , it is unclear whether these variants predisposed these patients to lung cancer . 
although this is a relatively small cohort , this may indicate that the v834l variant is not or only weakly oncogenic and by itself is not sufficient to drive uncontrolled growth . 
however , when a second oncogenic mutation in cis is present , cell growth accelerates more than proportionally . this hypothesis is in line with eight described egfr v834l variants found in the catalogue of somatic mutations in cancer database.9 the v834l variant in the lung tumors of all of these patients , with unknown germline status , is accompanied by the l858r mutation ( seven times ) or by an exon 19 deletion.10 - 12 the reason for the apparent preference of v834l for l858r is unclear . 
this is possibly because the energy balance of the egfr v769msubstituted protein does not favor combination with l858r.9 structural and functional analysis of the v834l variant alone and / or in combination with strong oncogenic driver mutations can provide additional mechanistic information on its pathogenicity and preferred association with other oncogenic egfr mutations . until now , lung cancer surveillance programs have been mainly recommended for active smokers or patients with a smoking history of 30 pack years but who have stopped within the last 15 years . 
for more information about ascos conflict of interest policy , please refer to or ascopubs.org / po / author - center . cor van der leest honoraria : roche , boehringer ingelheim , bristol - myers squibb consulting or advisory role : roche , abbvie , boehringer ingelheim , bristol - myers squibb rute m . 
aerts stock and other ownership interests : amphera consulting or advisory role : eli lilly , genentech , bristol - myers squibb , msd oncology , boehringer ingelheim , amphera speakers bureau : astrazeneca patents , royalties , other intellectual property : an allogenic lysate for vaccination ( inst ) winand n.m. 
van noesel j , van der ven wh , van os ta , et al : activating germline r776h mutation in the epidermal growth factor receptor associated with lung cancer with squamous differentiation . 
prim n , legrain m , guerin e , et al : germ - line exon 21 egfr mutations , v843i and p848l , in nonsmall cell lung cancer patients . 
heon s , yeap by , britt gj , et al : development of central nervous system metastases in patients with advanced non - small cell lung cancer and somatic egfr mutations treated with gefitinib or erlotinib . 
wu sg , chang yl , hsu yc , et al : good response to gefitinib in lung adenocarcinoma of complex epidermal growth factor receptor ( egfr ) mutations with the classical mutation pattern . 
tam iy , chung lp , suen ws , et al : distinct epidermal growth factor receptor and kras mutation patterns in non - small cell lung cancer patients with different tobacco exposure and clinicopathologic features . 
 molecular testing in patients with castration - resistant prostate cancer and its impact on clinical decision making purpose metastatic castration - resistant prostate cancer ( crpc ) is the lethal form of the disease . many groups have performed mutational or immunohistochemistry ( ihc ) testing in metastatic crpc to identify treatment targets . 
we report our institutions experience with mutational and ihc testing in patients with metastatic crpc and its impact on clinical decision making and patient outcomes . methods between 2012 and 2015 , 59 patients with crpc underwent metastatic tissue biopsies and were genotyped with a 37cancer gene panel in a clinical laboratory improvement amendmentscertified laboratory . 
a retrospective chart review was performed to determine whether the genomic information was acted upon and the outcome of patients whose treatment was guided by molecular testing . results forty - six of 59 patients with crpc ( 78.0% ) had biopsies with adequate tumor for mutational testing . 
thirty - one of 46 subjects ( 67.4% ) had mutations identified by sequencing . of the 35 patients with crpc whose biopsies were evaluated for pten expression by ihc testing , 13 had pten loss . 
two patients had treatment on the basis of molecular testing , and one of these subjects had greater tumor control with molecularly guided therapy than his immediate prior therapy . conclusion targeted sequencing and ihc can identify clinically informative molecular abnormalities in crpc . 
actionability of abnormalities identified in metastatic crpc may be improved with access to clinical trials , insurance approval for unapproved uses of existing anticancer drugs , and larger gene sequencing panels that include more frequently mutated genes . introduction prostate cancer is the second - leading cause of cancer - related death in men in the united states , and more than 26 , 000 men are predicted to die as a result of prostate cancer in 2017.1 nearly all of these deaths occur as a result of metastatic tumors . the principal treatment of metastatic prostate cancer is androgen - deprivation therapy or other treatments that disrupt the function of the androgen receptor ( ar ) protedespite these treatments , progression to lethal castration - resistant prostate cancer ( crpc ) is nearly universal . 
once a tumor becomes resistant to ar - targeted therapy , few effective treatment options remathis underscores the urgent need to identify important molecular targets that may guide not only drug development but also clinical decision making for patients . technological advances in sequencing have led to the identification of molecular abnormalities present in cancers . 
 several cancer types , including melanoma and adenocarcinoma of the lung.2 - 4 hospitals across the country are increasingly implementing tumorsequencing platforms to help guide therapy for patients with advanced cancers . several studies have reported the frequency of abnormalities detected with mutational testing in advanced cancers.5 in crpc , recent work demonstrates that these tumors share many aberrations in oncogenic pathways found in other solid tumors.6 , 7 indeed , the majority of patients with crpc have alterations in pten or phosphatidylinositol 3 - kinase ( pi3k ) .8 , 9 importantly , both pi3k and akt inhibitors are in clinical testing . thus , there is a strong rationale to test for abnormalities in pten , pi3k , and other alterations that may be targeted with specific therapies . 
however , molecular testing is not routinely performed in patients with crpc , and the utility of targeted molecular testing in this disease is unknown . moreover , there is limited information on the frequency with which molecular testing guides therapy , as well as limited information on the outcomes of patients whose treatment was guided by molecular testing . in this article , we describe our experience with mutational testing and pten immunohistochemistry ( ihc ) in a clinical laboratory improvement amendments ( clia ) certified laboratory to guide therapy in men with metastatic crpc . 
finally , we present outcomes of the patients whose treatment was guided by molecular testing . methods biopsies and subsequent mutational profiling and ihc testing were performed under an oregon health & science university institutional review board ( irb ) approved metastatic tissue collection protocol . 
progression was defined as soft tissue progression by response evaluation criteria in solid tumors ( recist ) version 1.1 , bone scan progression by the presence of at least two new lesions , symptomatic progression in an area of radiologicalevident disease , or prostate - specific antigen progression defined as levels  . 
those who received first - generation oral antiandrogens ( flutamide , bicalutamide , nilutamide ) as their most recent systemic therapy before biopsy had to have progressed after at least 4 weeks of antiandrogen discontinuation . 
patients had to have an eastern cooperative oncology group performance status of 0 to 3 , a platelet count > 75 , 000 / ml , prothrombin time or international normalized ratio and activated partial thromboplastin time , 1.5 times the upper limit of normal , and the ability to discontinue anticoagulation for at least 1 week before tumor biopsy . 
patients without prior orchiectomy continued medical castration therapy while they were participating in the study . all patients signed an informed consent form before biopsy . metastatic tissue was collected by computed tomography or ultrasound - guided biopsies performed at oregon health & science university in accordance with the standard operating procedure and institutional standards , with the goal of minimizing patient risk . 
biopsies were fixed in 10% neutral - buffered formalin for 8 hours and then transferred to 70% ethanol , paraffin embedded , and subsequently stained with hematoxylin and eosall hematoxylin and eosinstained slides were examined by a pathologist ( g.v.t. ) , who reviewed tumor content and decided if the sample was adequate for molecular testing . 
generated clinical reports , which were given to the treating clinician within 6 weeks . these reports included a discussion of the clinical significance and potential actionability of the findings on the basis of type of aberration , existing agents , or ongoing clinical trials . 
actionable is defined on the basis of the 2017 joint association for molecular pathology , asco , and college of american pathologists guidelines.10 mutations were grouped according to evidence - based variant categorization into three tiers , ie , tier 1 : variants of strong clinical significance ; tier ii : variants of potential clinical significance ; and tier iii : variants of unknown clinical significance . 
in total , 46 of 59 patients with crpc ( 78.0% ) had biopsies with adequate tumor to submit for genetrails mutational testing ( fig 1c )  . of the 46 patients who had sufficient tumor for mutational testing , 31 ( 67.4% ) had one or more mutational abnormalities identified by sequencing ( fig 2 , appendix table a1 )  . 
of 31 patients , 18 had at least one potentially actionable mutation , seven had at least one mutation of uncertain significance , and six had only nonactionable mutations . we also determined the frequency with which the mutational abnormalities were present in the patient cohort . 
there was a trend toward pten mutations and loss of pten protein expression by ihc testing ( fishers exact test two - tailed p value .075 ; fig 3b )  . we also examined the frequency with which potentially actionable alterations were actually acted upon . 
in both cases , these patients had mutational abnormalities in the pten / pi3k pathway . patient 1 was 77 years of age , had metastatic crpc , and experienced clinical and disease progression after 14 weeks of enzalutamide therapy . a biopsy of his iliac bone was performed , and testing revealed a pten p.i33fs * 11 somatic mutation and loss of pten protein expression by ihc . 
however , he developed clinical and disease progression 8 weeks later ( fig 4 )  . patient 2 was 67 years of age and had metastatic crpc ; his prostate - specific antigen level was decreasing in the setting of disease progression . biopsy of a pelvic mass revealed somatic mutations of fgfr3 , notch1 , and rb1 ; pten loss was found by ihc testing . 
on the basis of pten loss , as shown by ihc testing of his biopsy specimen , he was enrolled in a clinical trial of a pi3k inhibitor . his symptoms of pain improved , and his scans showed stable disease for 19 weeks before clinical and radiographic progression was eventually determined ( fig 4 )  . discussion advances in next - generation sequencing have improved our ability to identify tumor genomic alterations . 
baseline characteristics of 59 patients with metastatic castration - resistant prostate cancer characteristic age ( years ) median range median interquartile range total gleason score ( % ) time from initial diagnosis of prostate adenocarcinoma to biopsy ( years ) ecog performance status ( % ) psa at time of biopsy ( ng / ml ) median interquartile range no . 
importantly , results of mutational testing were available within 6 weeks ( mean time , 14 days ) , and all results were shared with the patients and treating providers . sixty - seven percent ( 31 of 46 ) of patients were found to have mutations . 
pten was inactivated by loss of expression or mutation in 19 of 46 ( 41.3% ) patient biopsy specimens , which is consistent with other reports.8 , 14 preclinical studies suggest that rb1 - mutant tumors do not respond to cdk4 / 6 inhibitors , and all of the rb1 aberrations we found were predicted to be loss of function . 
however , these rb1 aberrations were reported as nonactionable because there are currently no effective strategies to target tumors with mutant retinoblastoma protetsc2 mutations were regarded as potentially actionable because of the predicted activation of mammalian target of rapamycin signaling and possible increased sensitivity to mammalian target of rapamycin inhibitors such as everolimus . 
forty - eight percent ( 22 of 46 ) of tested cases had mutations involving the pi3k pathway , 4.3% ( two of 46 ) involving raf kinases , and 4.3% ( two of 46 ) involving cdk inhibitors , which is similar to previous studies.7 other potentially actionable mutations identified included targets of existing anticancer therapies such as egfr , erbb2 , and kit . 
ras / raf pathway abnormalities and cdk mutations were also seen in a subset of patients ; there were agents targeting these pathways in clinical testing or approved for melanoma treatment at the time patients were enrolled in our biopsy protocol . 
despite 31 of 46 patients ( 58% ) having a theoretically actionable mutation , true actionability was limited because only two of 46 of our patients ( 4% ) were prescribed agents that could block the identified gene alteration . 
possible reasons include the lack of information on the importance of these mutations in crpc and the lack of insurance coverage to prescribe these medicines for off - label uses in patients with crpc . 
this lack of insurance coverage is probably because of the absence of positive basket trials showing a benefit of molecularly guided therapy versus standard therapies.15 moreover , lack of nearby or onsite clinical trials with these abbreviations : crpc , metastatic castration - resistant prostate cancer ; ecog , eastern cooperative oncology group ; psa , prostate - specific antigen . obtaining adequate tumor from patients with metastatic crpc , particularly those with bone metastases , has been notoriously difficult.11 - 13 however , nearly 80.0% of our biopsy specimens had sufficient dna for tumor sequencing , including 71.0% of bone biopsy specimens . 
finally , many of the patients in our study had not exhausted all of the approved therapies for metastatic crpc . this was also probably a contributing factor to why so few patients with theoretically actionable molecular alterations received molecularly guided therapy . pi3k / akt / pten pathway abnormalities were quite common in our cohort , and both subjects who received molecularly guided therapy received a pi3k inhibitor . 
the first patient progressed immediately ( ie , as of the first assessment ) , and his progression - free survival with molecularly guided therapy was nearly identical to that with his immediate prior therapy . 
moreover , we did not have ontreatment biopsies or progression biopsies during pi3k inhibitor treatment , so we are unable to determine whether the drugs adequately hit the fig 4 . 
swimmer plots illustrating patients time on treatment of the therapy given immediately before molecular testing and time on treatment with a phosphatidylinositol 3 - kinase ( pi3k ) inhibitor that was guided by molecular testing . 
radiographic progression was the reason for treatment discontinuation in all cases . 14 weeks 8 weeks enzalutamide 11 weeks docetaxel carboplatin 19 weeks prior therapy pi3k inhibitor radiographic progression time on therapy ( weeks ) target in each subject . 
finally , prior work has shown that pi3k inhibition leads to reciprocal feedback and activation of the ar pathway.16 indeed , the subject who did not respond to pi3k inhibition had an adenocarcinoma , whereas the patient who seemed to derive greater benefit from pi3k inhibitor treatment had an arnegative neuroendocrine tumor . 
thus , it is possible that the latter patient did not have adaptive ar activation in response to pi3k inhibition . finally , because our targeted panel only examined 37 genes for mutations , we cannot rule out the possibility that the first , nonresponding patients tumor had additional genomic abnormalities that were more consequential than his pik3ca mutation and pten loss . 
functional testing in tumor avatars from metastatic biopsies may help clarify the importance of molecular alterations found in tumor biopsy specimens , and we are currently developing these cultures from metastatic biopsy samples . 
finally , it is unclear if intrapatient tumor heterogeneity with clones that did not harbor a pik3ca mutation or pten loss also contributed to disease progression in the first subject . our panel included only 37 genes , which was another limitation of our study . 
since adoption of the panel used in this study , robinson et al7 have performed exome sequencing of metastatic crpc tumors and identified additional genes frequently altered in metastatic crpc . 
in that report , robinson et al7 reported that 89% of patients with crpc harbored a clinically actionable mutation . however , upon examination of the mutations that were identified , it is uncertain what proportion of these identified alterations could truly be used to guide therapy . 
indeed , 63% of patients had genetic alterations of the ar pathway ; however , it is unclear which therapies should be used in men who harbor these genetic alterations of the ar pathway . 
moreover , several of the other genetic alterations described in that report are not readily targetable ( ie , wnt pathway , cell cycle pathway , p53 alterations )  . 
similarly , we report finding a mutation in 67% ( 31 of 46 ) of our patients , with 58% ( 18 of 31 ) harboring at least one potentially actionable mutation ( ie , a drug was either approved or in clinical testing to block that genetic alteration )  . 
subsequent work has demonstrated a low frequency of ar mutations , including activating ar f877l mutations in this patient population.17 moreover , robinson et al reported that a high proportion of tumors among patients with crpc ( 19.3% ) harbored aberrations in dna repair pathway genes such as brca2 , brca1 , or atm . 
those patients whose tumors harbored mutations or copy number loss of these and other related dna repair genes seemed to have the greatest benefit.18 on the basis of the frequency with which these abnormalities are found in crpc , we anticipate that we would have identified 10 additional subjects with an actionable alteration if our panel had included mutational and copy number testing for genes in the dna repair pathway . 
recently , we expanded our mutational panel to include dna repair genes with the hope that we identify patients who may benefit from poly ( adp - ribose ) polymerase inhibitor or platinumbased therapy . importantly , even with more comprehensive sequencing panels , many hurdles related to access to therapy must be overcome to make molecular testing truly actionable . 
both of the patients in our cohort who had therapy guided by molecular testing results enrolled in clinical trials with investigational agents , and one patient traveled out of state several hundred miles to receive his therapy . 
one solution to this is single - patient investigational new drug applications for compassionate use , although that process is time consuming and requires dedicated staff to assist with these requests . tumors from other patients had molecular abnormalities that could have been targeted with an existing approved agent . 
insurance company disapproval was one reason ; thus a critical aspect of making molecular abnormalities actionable is insurance approval for unapproved uses of existing anticancer drugs . we are in a new era of cancer treatment , wherein molecular testing of tumors is commonplace . 
foster honoraria : civco medical solutions molecular testing in patients with castration - resistant prostate cancer and its impact on clinical decision making the following represents disclosure information provided by authors of this manuscript . 
tao no relationship to disclose shawna bailey research funding : janssen research & development ( inst ) , medivation ( inst ) , millennium ( inst ) , aragon pharmaceuticals ( inst ) , bristol - myers squibb ( inst ) , oncogenex ( inst ) , astellas scientific & medical affairs ( inst ) , bayer ( inst ) , myriad genetics ( inst ) , sotio ( inst ) , sanofi ( inst ) , zenith epigenetics ( inst ) , boehringer ingelheim pharmaceuticals ( inst ) , merck ( inst ) tomasz m . 
beer stock and other ownership interests : salarius pharmaceuticals consulting or advisory role : astrazeneca , churchill pharmaceuticals , dendreon , janssen biotech , janssen oncology , janssen research & development , johnson & johnson , janssen japan , roche research funding : astellas pharma ( inst ) , bristol - myers squibb ( inst ) , dendreon ( inst ) , janssen research & development ( inst ) , medivation ( inst ) , oncogenex ( inst ) , sotio ( inst ) , sotio , theraclone sciences ( inst ) , boehringer ingelheim ( inst ) erik foss research funding : coinvestigator for a research grant from moximed for magnetic resonance imaging of potential and postoperative patients undergoing kinespring knee implant surgery ( an orthopedic implant designed to alleviate degenerative joint pain )  . brooke beckett no relationship to disclose alice fung speakers bureau : guerbet affiliations alexander guimaraes honoraria : philips healthcare 2015 ( $1 , 500 ) ; siemens medical solutions 2014 ( $2 , 000 ) speakers bureau : siemens medical solutions 2014 travel , accommodations , expenses : siemens medical solutions 2014 , philips healthcare 2015 jeremy p . 
graff honoraria : bayer , astellas medivation , janssen oncology consulting or advisory role : exelixis research funding : sanofi ( inst ) , medivation ( inst ) , bristol - myers squibb ( inst ) , merck sharp & dohme ( inst ) , janssen oncology ( inst ) patents , royalties , other intellectual property : oncoresponse : exceptional responders travel , accommodations , expenses : bayer , merck sharp & dohme , clovis oncology kristine m . 
small stock and other ownership interests : fortis therapeutics , harpoon therapeutics honoraria : janssen - cilag consulting or advisory role : fortis therapeutics , gilead sciences research funding : janssen ( inst ) christopher l . 
alumkal consulting or advisory role : astellas pharma , bayer research funding : aragon pharmaceuticals ( inst ) , astellas pharma ( inst ) , millennium ( inst ) , novartis ( inst ) , zenith epigenetics ( inst ) derrick l . 
small , university of california , san francisco , san francisco , ca . support supported by a stand up to cancerprostate cancer foundation prostate dream team translational cancer research grant ( su2c - aacr - dt0409 )  . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors based on molecular profiles : early results from mypathway , an open - label , phase iia umbrella basket study . 
cheng hh , klemfuss n , montgomery b , et al : a pilot study of clinical targeted next generation sequencing for prostate cancer : consequences for treatment and genetic counseling . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
mckay rr , zukotynski ka , werner l , et al : imaging , procedural and clinical variables associated with tumor yield on bone biopsy in metastatic castration - resistant prostate cancer . 
spritzer ce , afonso pd , vinson en , et al : bone marrow biopsy : rna isolation with expression profiling in men with metastatic castration - resistant prostate cancerfactors affecting diagnostic success . 
ross rw , halabi s , ou ss , et al : predictors of prostate cancer tissue acquisition by an undirected core bone marrow biopsy in metastatic castration - resistant prostate cancera cancer and leukemia group b study . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
azad aa , volik sv , wyatt aw , et al : androgen receptor gene aberrations in circulating cell - free dna : biomarkers of therapeutic resistance in castration - resistant prostate cancer . 
specific molecular abnormalities identified and pten expression status ( continued ) patient no . biopsy site sample identifier loss of pten expression by ihc testing ( indicated by x ) bone bone dtb - oh - 181 dtb - oh - 187 lymph node dtb - oh - 190 bladder dtb - oh - 200 liver dtb - oh - 202 abbreviation : ihc , immunohistochemistry . abnormality rb1 copy number loss ( t3 )  . pten copy number loss ( t2 )  . 
staging magnetic resonance imaging ( mri ) of the pelvis conrmed a large inltrative tumor 6 cm from the anus ; no metastases were detected in the thorax plus abdominal computed tomography ( ct ) scans . 
at this point , all ras and brafv600e mutational analysis by quantitative polymerase chain reaction did not reveal alterations . the patient then started chemotherapy with modied uorouracil , folinic acid and oxaliplatin regimen plus panitumumab . 
cytoreductive surgery was undertaken in march 2017 and the pathologist reported a 2.5 - cm , mucinous adenocarcinoma extensively invading the mesorectum , with a focus of adenocarcinoma in the resection margin , regression grade 5 ( american joint committee on cancers ajcc cancer staging manual , 7th edition , classication : ypt4bn2amx , r1 )  . one year later , ct scans showed progressive disease with pelvic lymph node metastasis and a peritoneal lesion with secondary left uretero - hydronephrosis . second - line chemotherapy with folinic acid plus uorouracil plus irinotecan hydrochloride plus aibercept was started in july 2018 , but progressive disease was documented in october 2018 . 
he signed an informed consent form authorizing molecular prescreening and use of de - identied clinical and molecular data for research purposes and clinical trial matching . we conducted an in - house , targeted , amplicon - based , 60 - gene next - generation sequencing ( ngs ) test that covers the most frequently mutated genes in colorectal cancer.1 apc , ras , and braf genes were wild type , but we found mutations in tp53 ( p.k132r ) , rnf43 ( p.a19fs ) , and notch1 ( p.v1578del ; table 1 )  . 
but we complemented the genomics proling with a customized in - house fusion panel that was negative for translocations involving 19 genes , including clinically validated targets ( e.g. , alk , ros1 , ntrk ) as well as fusions in notch1 , notch2 , and wnt pathway genes rspo2 and rspo3 . 
the case was then discussed at the institutional molecular tumor board meeting to dene potential matched targeted therapeutic strategies for notch1 and / or rnf43 mutations . first , we assessed the functionality of the mutations , per oncokb ( a precision oncology knowledge database ) .2 both notch1 and tp53 variants have been reported as oncogenic in different databases , whereas the rnf43 mutation is a novel frameshift the coding sequence . event predicted to disrupt variant allele fractions ( vaf ) of notch1 , rnf43 , and tp53 were 3% , 53% , and 53% , respectively , suggesting that notch1 mutation might be a subclonal event , while rnf43 and tp53 mutations are clonal ( truncal ) events ( table 1 )  . 
it is known that in - frame mutations in the negative regulatory region , where p.v1578del is located , lead to ligand - independent notch1 activation through continuous cleavage of the receptor , allowing the migration of the notch intracellular domain to the nucleus and subsequent pathway activation.3 we decided to complement genomic analysis with notch1 immunohistochemistry to get additional insights on its functionality and subclonality . in line with ngs results showing low vaf of the mutation , less than 1% of cancer cells stained positive for notch1 ( table 1 )  . 
a previous study with transcriptomic and phosphoproteomic proling showed deregulation of the notch pathway in this context.4 whether a notch inhibitor has antitumor activity in the anti - egfr refractory setting with emerging notch1 subclonal mutations is unknown . 
we have previously shown no association between vafs of kras , braf , or pik3ca mutations and time to progression on targeted therapies matched to these alterations in metastatic colorectal cancer , potentially related to the limited antitumor activity of the used drugs.1 loss - of - function mutations in the ubiquitin ligases rnf43 are early drivers in colorectal cancer carcinogenesis ; they activate the wnt / - catenin pathway in a ligand - independent manner.5 rnf43 is a negative wnt feedback regulator , and preclinical models of rnf43 mutated cancers are sensitive to inhibitors of the wnt secretion porcupine.6 these mutations are strongly enriched but not restricted to hypermutated microsatellite instable tumors . 
notably , a patient with rnf43 - mutated colorectal cancer had partial response to the rst - in - man porcupine inhibitor wnt974 as monotherapy.7 this agent is not in clinical investigation in colorectal cancers any longer ; there has been a shift toward combination strategies with immuno - oncology drugs in tumors where wnt activation has been reported to induce immunosuppression and checkpoint inhibitor resistance , such as melanoma . 
other agents potentially active in a wnt hypersensitive context , characterized by mutations in rnf43 / znfr3 or gain - of - function chromosome translocations involving rspo2 and rspo3 ( and lacking downstream apc inactivating mutations ) , include antibodies targeting wnt receptor lrp5 / 6.8 , 9 given the clonality and predicted truncal driverness of rnf43 mutation and the absence of apc alterations , we decided to prioritize a phase i clinical trial with a rstin - class lrp5 / lrp6 antagonist bi 905677 ( clinicaltrials.gov identier : nct03604445 )  . 
at our institution , we consider novel therapies guided by molecular proling in patients with good performance status before the use of regimens known to have small response rates in unselected populations . 
clonality may be used to guide target prioritization when the level of evidence for actionability of the co - existing alterations is comparable ( clinical trial matching with investigational agents , as in this case )  . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . guillem argiles consulting or advisory role : hoffman la roche , bristol - myers squibb , roche , bayer , servier travel , accommodations , expenses : bayer , roche , amgen , servier analia azaro consulting or advisory role : orion , amcure elena garralda consulting or advisory role : roche , ellipses pharma , neomed therapeutics , janssen , boehringer ingelheim , astrazeneca / medimmune , seattle genetics , tfs speakers bureau : msd oncology , bristol - myers squibb research funding : novartis travel , accommodations , expenses : bristol - myers squibb , menarini , glycotope , merck sharp & dohme josep tabernero consulting or advisory role : bayer , boehringer ingelheim , eli lilly , msd oncology , merck serono , novartis , sano , taiho pharmaceutical , merrimack , peptomyc , rafael pharmaceuticals , symphogen , chugai pharma , ipsen , merus , pzer , seattle genetics , array biopharma , astrazeneca , beigene , genentech , genmab , halozyme , imugene , inection biosciences , kura , menarini , molecular partners , pharmacyclics , proteodesign , roche , seattle genetics , servier , vcn biosciences , biocartis , foundation medicine , haliodx sas , roche diagnostics paolo nuciforo honoraria : bayer , novartis , msd oncology consulting or advisory role : bayer , msd oncology travel , accommodations , expenses : novartis ana vivancos consulting or advisory role : guardant health , novartis , bayer health rodrigo dienstmann consulting or advisory role : roche speakers bureau : roche , ipsen , sano , symphogen , amgen , msd oncology , servier research funding : merck no other potential conicts of interest were reported . acknowledgment any views , opinions , ndings , conclusions , or recommendations expressed in this material are those solely of the author ( s ) and do not necessarily reect those of european society for medical oncology or any pharmaceutical company . references 11 : 1263 - 1272 , 2017 1 . 
brijwani n , jain m , dhandapani m , et al : rationally co - targeting divergent pathways in kras wild - type colorectal cancers by canscript technology reveals tumor dependence on notch and erbb2 . 
nat genet 46 : 1264 - 1266 , 2014 van de wetering m , francies he , francis jm , et al : prospective derivation of a living organoid biobank of colorectal cancer patients . 
cell 161 : 933 - 945 , 2015 janku f , connolly r , lorusso p , et al : phase i study of wnt974 , a rst - in - class porcupine inhibitor , in advanced solid tumors . 
 diamond - blackfan anemia predisposing to myelodysplastic syndrome in early adulthood diamond - blackfan anemia ( dba ) is an inherited bone marrow failure syndrome ( ibmfs ) , characterized by congenital pure red cell aplasia typically presenting within the first months of life.1 - 4 hematologic abnormalities in dba include macrocytic or normocytic anemia and normocellular bone marrow with scarce erythroid precursors , often associated with short stature and phenotypic anomalies including craniofacial , musculoskeletal , and cardiac malformations.1 - 5 the most common pattern of inheritance is autosomal dominant , observed in 40% to 45% of cases with a frequency of two to seven cases per one million live births.2 - 5 establishing the diagnosis of classic dba is based on criteria originally described by diamond et al , 5a which included age younger than 1 year and the aforementioned hematologic abnormalities , without other significant cytopenias.5 supportive criteria include a family history , an elevated erythrocyte adenosine deaminase level , and / or an elevated fetal hemoglobin level at diagnosis ; confirmation is now possible by molecular identification of ribosomal protein mutations associated with dba.1 - 5 the most frequent ribosomal protein gene mutation is rsp19 , accounting for almost 25% of cases.6 , 7 other heterozygous mutations identified include the rps24 , rps17 , rpl5 , rps10 , rpl11 , rpl35a , rpl15 , and rps26 genes.7 , 8 overall ,  . 
11 different ribosomal gene mutations are linked to dba , accounting for approximately 70% of cases.2 , 9 , 10 the preferential sensitivity of erythroid precursors to constitutional ribosomal mutations represents a complex and multifactorial pathophysiology , which remains incompletely understood . 
potential mechanisms have been recently reviewed in detail and include impaired erythroid differentiation and increased erythroid apoptosis.2 , 11 - 14 mutations in ribosomal proteins lead to indirect and excessive activation of p53 , which is implicated in increased erythroid precursor apoptosis2 - 4 via inhibition of the p53 - degradation protein ( the murine double minute 2 protein ) .15 , 16 other mechanisms include reduced levels of mrna translation , which affect the expression of critical erythroid proteins.1 , 3 current treatment options for dba include recurrent red - cell transfusions , corticosteroid therapy , and allogeneic stem - cell transplant in refractory cases . 
herein , we describe the cases of two adult patients referred to our institution at the time of early - onset mds and diagnosed with dba related to a constitutional disease - associated mutation or deletion of rps19 . case 1 a 44 - year - old woman was referred for pancytopenia and fatigue . 
dinardo author affiliations and support information ( if applicable ) appear at the end of this article . the views expressed in the article are the views of the authors and do not reflect an official position of the institution . corresponding author : courtney d . 
one month before presentation , a blood count showed a hemoglobin level of 5.6 g / dl , a platelet count of 91 3 103 / ml , and a wbc count of 3.0 3 103 / ml . 
she was referred to our institution , where a repeat hematology evaluation confirmed pancytopenia with mean corpuscular volume 110 fl , and anisocytosis and ovalocytes were identified on manual review . 
blasts were not increased , and no ring sideroblasts or reticulin fibrosis was detected ( fig 1 )  . findings fulfilled diagnostic criteria of mds with multilineage dysplasia.20a a 28 - gene mutation analysis was negative for somatic mutations , including aslx1 , idh1 , idh2 , jak2 , kit , n / kras , nmp1 , tet2 , and tp53 . 
conventional cytogenetics demonstrated a diploid female karyotype ; array comparative genomic hybridization was negative for copy number losses or gains in chromosomes 5 , 7 , 8 , 17 , or 20 . 
he did not require additional therapy , and his counts stabilized throughout adulthood without additional therapy . at age 30 years , he was reportedly diagnosed with hemochromatosis and treated with periodic phlebotomies . 
patient 1 : ( a ) the bone marrow trephine biopsy demonstrated a hypercellular marrow . ( b ) bone marrow aspirate smears demonstrated dysplasia involving the megakaryocytic and erythroid lineages predominantly . 
a 28 - gene somatic mutation analysis identified a tp53 missense mutation in exon 8 and an asxl1 frameshift mutation in exon 12 ; no additional mutations were detected ( fig 2 )  . 
he was given hypomethylating agent therapy with intravenous azacitidine 75 mg / m2 daily for days 1 to 5 , which he has received for three cycles . discussion the development of mds in patients with dba was summarized in 2010 by shimamura and alter.19 their systematic review of 970 patients found 13 cases of aml and / or mds . 
in their prospective cohort of 63 patients , no cases of leukemia were found after 6 years and 1 , 449 person - years of followup.19 in 2012 , vlachos et al20 reviewed the dbar , which included a cohort of  . 
in the dbar , the rate of mds / aml was approximately 1% ( six of 608 cases ) after 20 years and 9 , 458 person - years of follow - up.20 the median age was 28 years at the time of mds development ( range , 2 to 51 years ) and 44.5 years for aml ( range , 44 to 45 years )  . 
overall , these patients had a 287 - fold increased risk of developing mds and a 29.7 - fold risk of developing aml.20 an updated review of the dbar in 2016 revealed that of 702 patients with  . 
12 , 000 person - years of followup , three patients had developed aml and eight had developed mds.21 there is no evidence of specific association of any particular dba genotype with a cancer type or frequency.21 mds is typically a disease of advanced age , with a median age at mds diagnosis of 76 years.20a , 23 in fig 2 . 
 contrast , hereditary predisposition syndromes are associated with the development of mds at a younger age.19 recognizing a genetic predisposition to malignancy has major implications for risk stratification and treatment decisions , such as selection of appropriate transplant donors and reduced - intensity conditioning regimens . 
other advantages include augmented cancer surveillance and genetic testing for patients and at - risk family members.24 , 25 proposed guidelines to identify potential hereditary predisposition syndromes in mds include age at diagnosis of younger than 50 years with either a family history of aml / mds or a personal history of cytopenias ; bleeding tendencies ; skin , skeletal , or nail abnormalities ; unexplained liver disease ; or pulmonary fibrosis and / or alveolar proteinosis.24 the majority of patients with ibmfs , including dba , are diagnosed during childhood with hematologic abnormalities and recognizable phenotypes ; however , some patients with milder expression of disease have subtle extrahematopoietic findings that are ultimately only identified after diagnosis with mds , aml , or solid tumors.24 , 25 as somatic and germline genetic analysis becomes more widely available , we can better identify patients with ibmfss ( eg , fanconi anemia , dyskeratosis congenita , shwachman - diamond syndrome , and dba ) in those with hematologic malignancies at earlier ages , particularly using next - generation gene panel sequencing of predisposition genes.24 , 25 the cumulative malignancy risk of nearly 20% by the age of 40 years , and the 287 - fold increase in lifetime risk of mds with dba , approaches that of other ibmfss . 
bannon no relationship to disclose rashmi kanagal - shamanna no relationship to disclose jessica shafer foglesong no relationship to disclose yesid alvarado no relationship to disclose gautam borthakur no relationship to disclose courtney d . 
fumagalli s , di cara a , neb - gulati a , et al : absence of nucleolar disruption after impairment of 40s ribosome biogenesis reveals an rpl11 - translation - dependent mechanism of p53 induction . 
lee h , lyssikatos c , atsidaftos e , et al : remission in patients with diamond blackfan anemia ( dba ) appears to be unrestricted by phenotype or genotype . 
sj ogren se , siva k , soneji s , et al : glucocorticoids improve erythroid progenitor maintenance and dampen trp53 response in a mouse model of diamond - blackfan anaemia . 
vlachos a , rosenberg ps , kang j , et al : myelodysplastic syndrome and gastrointestinal carcinomas characterize the cancer risk in diamond blackfan anemia : a report from the diamond blackfan anemia registry . 
babushok dv , bessler m : genetic predisposition syndromes : when should they be considered in the work - up of mds ? best pract res clin haematol 28 : 55 - 68 , 2015 25 . 
kutler di , singh b , satagopan j , et al : a 20 - year perspective on the international fanconi anemia registry ( ifar )  . blood 101 : 1249 - 1256 , 2003 29 . 
dores gm , devesa ss , curtis re , et al : acute leukemia incidence and patient survival among children and adults in the united states , 2001 - 2007 . 
this is reflected in the current national comprehensive cancer network ( nccn ) guidelines , which state that genetic testing for brca1 / brca2 should be offered to patients with pdac with close relatives with a brca - associated cancer or to those of ashkenazi jewish ( aj ) ancestry.3 , 4 in the nccn guidelines that focus specifically on pdac , recommendations for genetic evaluation are broad , which suggests that those patients with early - onset pdac , a family history of cancer , or aj ancestry should be referred for genetic counseling.4 the rapidly evolving era of precision oncology , as well as the observation that some of these underlying germline variants are clinically actionable , raises the question of whether our current testing criteria are sufficiently inclusive . 
the three articles that accompany this editorial highlight the importance of germline testing and the need for a broader approach for genetic risk assessment in pdac . to better understand the prevalence of germline mutations in pdac , smith et al5 evaluated germline susceptibility in a high - risk group of patients with pdac who were known to harbor founder mutations . 
the authors assessed 150 patients with pdac with french canadian ( fc ) ancestry for both germline fc founder and nonfounder mutations in four dna - damage repair genesbrca1 , brca2 , palb2 , and atmcomparing mutation rates with 236 patients who were not selected for fc ancestry.5 among the fc group , a 5.3% founder mutation rate was observed . 
for nonaj or - fc patients , the authors suggest that full analysis of the aforementioned genes should be completed in patients with pdac who are diagnosed at age < 50 years or who have a strong family history of hrd - related cancers.5 these recommendations somewhat parallel the current expanded nccn guidelines , with the additional consideration of fc ancestry given the comparable germline mutation rates in fc and aj founder populations . alicia latham schwark zsofia k . 
 in contrast , in the article by reiss et al , 8 the authors suggest that a lower threshold for germline assessment of brca1 , brca2 , and palb2 in patients with pdac may be warranted . 
in this retrospective analysis , clinical outcomes , including overall survival ( os ) and response to chemotherapy , of 29 patients with advanced pdac with known pathogenic variants in brca1 , brca2 , or palb2 were compared with 58 stage , age at diagnosis , year of diagnosis , and gender matched controls.8 improved os was observed in hrd mutationpositive patients who received platinumbased therapy , specifically with 1 - year os in mutation - positive patients being 94% compared with 60% in the control group.8 of importance , mutation status did not predict an improved os in the nonplatinum group.8 this suggests that hrd mutation status may be predictive of the response to platinum therapy , but , by itself , may not be a prognostic marker , although additional studies are needed for clarification . 
interestingly , patients who were at higher risk of harboring a genetic mutation on the basis of clinical parametersthat is , young age , strong family history of cancer , or aj ancestrywere not the majority of patients who were found to have germline alterations , and , therefore , most mutation - positive patients with pdac did not meet current nccn guidelines for testing.8 this is similar to another study that suggested that the majority of brca mutationpositive patients with pdac do not meet traditional testing criteria.7 given the improved os in platinum - treated hrd mutationpositive patients , as well as the lack of clinical parameters that accurately identify hrd mutation carriers , either upfront germline testing or reflex germline assessment after suggestive tumor testing was recommended.8 a potential alternative to germline testing is the development of specific mutation signatures that predict for hrd . 
in the study by shahda et al , 11 91 tumor samples were assessed for an hrd score , with an immediate dropout of 34 samples that failed analysis as a result of low tumor cellularity , 11 which highlights the difficulty in assessing pancreatic tumor tissue for genetic information . 
70% of pdacs , respectively.14 , 18 , 19 given the lack of clinically actionable somatic driver mutations in pdac , it would then seem that an approximate 5% germline mutation prevalence in unselected , nonfounder populations in hrd - associated genes , with implications for treatment , 14 , 19 may be our most promising tool yet for pdac precision oncology . 
in light of the recent us food and drug administration approval of immune checkpoint inhibitors , such as pembrolizumab , for the treatment of all mismatch repair deficient solid tumors , 20 whether evaluation for mismatch repair deficient statuspresent in approximately 1% of pdacsis also warranted remains another important consideration.12 , 19 , 21 , 22 such genomic assessment may seem daunting . there may be concern that the overall burden of testing all pdacs for germline variants is unreasonable . 
 ovarian cancer for brca1 / 2 and colorectal cancer for lynch syndrome , recently updated nccn guidelines now also recommend that all patients with metastatic prostate cancer be referred for genetic evaluation and consideration of testing for brca and related genes.3 this is on the basis of the recent finding that 5% to 11% of such patients harbor underlying germline genetic mutations in dna repair genes , with both prognostic and predictive implications.23 in contrast to prostate cancer , the vast majority of patients with pdac succumb to their disease , with a 5 - year survival of only 8% , which highlights the critical need for identifying opportunities for precision oncology for all patients with pdac.24 , 25 given the dearth of clinically actionable somatic mutations in pdac , as well as our inability to accurately capture all hrdpdacs on the basis of clinical variables , universal germline genetic assessment to identify , at minimum , the 5% of hrdpdacs with emerging treatment and targeted therapy implicationsseems to be a clinically reasonable undertaking . 
beyond the opportunity for precision oncology , knowledge of the presence of an inherited cancer predisposition syndrome will allow for the incorporation of precision prevention oncology for at - risk family members . 
stadler consulting or advisory role : allergan ( i ) , genentech ( i ) , regeneron ( i ) , optos ( i ) , adverum ( i ) alicia latham schwark no relationship to disclose affiliations alicia latham schwark and zsofia k . 
smith al , wc , cuggia a , et al : reflex testing for germline brca1 , brca2 , palb2 and atm mutations in pancreatic cancer : mutation prevalence and clinical outcomes from two canadian research registries . 
telli ml , timms km , reid j , et al : homologous recombination deficiency ( hrd ) score predicts response to platinum - containing neoadjuvant chemotherapy in patients with triple - negative breast cancer . 
domchek sm , aghajanian c , shapira - frommer r , et al : efficacy and safety of olaparib monotherapy in germline brca1 / 2 mutation carriers with advanced ovarian cancer and three or more lines of prior therapy . 
 o duodenal - jejunal flexure gi stromal tumor frequently heralds somatic nf1 and notch pathway mutations purpose gi stromal tumors ( gists ) are commonly associated with somatic mutations in kit and pdgfra . 
we define the anatomic distribution of nf1 alterations in gist . methods we describe the demographic / clinicopathologic features of 177 patients from two institutions whose gists underwent next - generation sequencing of 315 cancer - related genes . results we initially identified six ( 9.7% ) of 62 gists with nf1 genomic alterations from the first cohort . 
of the five djf gists with any nf1 alteration , three ( 60% ) had kit mutations , and three ( 60% ) had notch pathway mutations ( notch2 , maml2 , cdc73 )  . 
we validated these findings in a second cohort of 115 gists , where two ( 40% ) of five unifocal nf1 - mutated gists arose at the djf , and one of these also had a notch pathway mutation ( ep300 )  . conclusion broad genomic profiling of adult gists has revealed that nf1 alterations are enriched in djf gists . 
 ( ie , kras , nras , hras ) by increasing the catalytic conversion of the active form ( ras - gtp ) to the inactive form ( ras - gdp ) .14 pathogenic nf1 variants disrupt the normal function of neurofibromin and result in constitutive ras activation.15 this activation increases downstream signaling through braf / craf - mediated activation of the mitogen - activated protein kinase pathway and hence , facilitates tumor initiation and progression.16 until recently , nf1 mutations in gist were believed to be primarily of germline origin , associated with clinical nf - 1 , and combined with a second somatic mutation in the tumor . 
thus , our understanding of nf1 in gist continues to evolve . approximately 30% of all gists occur in the small intestine , 1 which measures approximately 5 to 6 m in length.18 most small bowel gists are associated with somatic kit mutations , whereas a subset is associated with nf1 mutations.19 the small bowel is divided into three anatomically , histologically , and functionally distinct segments , namely the duodenum , jejunum , and ileum.18 although some studies distinguish duodenal gists from other small bowel gists , most combine them and do not characterize the biology of tumors on the basis of these distinct locations . 
moreover , the exact transition from jejunum to ileum is somewhat arbitrary , but the duodenal - jejunal flexure ( djf ) , also known as the ligament of treitz , represents a clear anatomic site that marks the transition from duodenum to jejunuto date , no study of gists has specifically characterized tumors that arise from the djf . 
the current study , however , started with presumably sporadic gists , which led to the unexpected finding that somatic nf1 mutations in gists are more prevalent than previously suspected and that they are enriched in tumors that arise from the djf . methods primary study population patient demographic and tumor clinicopathologic data were retrospectively collected in an unbiased fashion from every patient with pathologically confirmed gist seen within the university of california , san diego ( ucsd ) , health system from january 1 , 2000 , to april 30 , 2017 , under a ucsd institutional review boardapproved protocol . 
available operating notes or imaging reports were reviewed for these 165 patients to distinguish the primary site of origin of any small bowel gist as duodenal , djf , jejunal , or ileal . comprehensive genomic profiling of the 165 patients with gist in the cohort , 62 underwent next - generation sequencing ( ngs ) of coding regions of cancer - related genes . 
dna was extracted from formalin - fixed paraffin - embedded ( ffpe ) sections that contained a minimum of 20% tumor tissue and were used for comprehensive genomic profiling with hybridization - captured , adaptor ligationbased libraries . 
the number of genes in the foundationone panel has evolved over time as new data on cancer - related genes have been published and currently includes 315 cancerrelated genes plus select introns from 28 genes often rearranged or altered in solid tumor cancers . however , all versions of the assay simultaneously analyze the extracted dna for base substitutions , short insertions and deletions , amplifications and homozygous deletions , and gene rearrangements with > 99% sensitivity.20 specimens submitted to the ucsd clinical genomics laboratory underwent ngs of the exons of 397 genes involved in pathways that control growth or differentiation and are known to be frequently mutated in solid tumors . 
coverage depth ranged from 2833 to 8303 across all sequenced tumor samples from both the foundationone and the ucsd assays . nontumor tissue sequencing patients found to have nf1 mutations in their tumor tissue were also tested for germline nf1 mutation . four samples tested by using dna extracted from ffpe sections of adjacent normal tissue or peripheral blood underwent ngs cancer - related mutation panel testing by the ucsd clinical genomics laboratory as just described . 
one additional sample was tested by using commercially available targeted sequencing of the nf1 gene from peripheral blood by arup laboratories ( salt lake city , ut )  . secondary study population a confirmatory cohort of  . 
1 , 000 patients with pathologically confirmed gists from memorial sloan kettering cancer center ( mskcc ) with retrospectively collected patient demographic and tumor clinicopathologic data also were analyzed under an institutional review boardapproved protocol . 
data included age , sex , primary gist site , tumor size , and pathologic characteristics . one hundred fifteen patients with gists had ngs results available in the msk - impact ( integrated mutation profiling of actionable cancer targets ) platform , which characterized frozen or ffpe tumor specimens for somatic dna mutations , copy number alterations , and select rearrangements of 341 cancer - associated genes.21 statistical analysis all statistical analyses were performed with stata 9.0 software ( statacorp , college station , tx )  . comparisons between groups were performed by using the two - sample test of proportions . 
none of these patients had previously known clinical manifestations of nf - 1 ( eg , cafe au lait spots ; axillary / inguinal freckling ; dermal neurofibromas ; lisch nodules ; iris hamartomas ; nervous system tumors , including malignant peripheral nerve sheath tumors [ mpnsts ] , pancreatic neuroendocrine tumors , pheochromocytomas , or other sarcomas ] ) .10 therefore , the application of expanded ngs panels to characterize the mutational profile of gists identified an unappreciated subset of patients with nf1 genomic alterations in their tumors but no clinical evidence of nf - 1 . nf1 genomic alterations in gist frequently occur at the djf of the six patients with gist for whom we identified nf1 genomic alterations , five ( 83.3% ) had unifocal tumors at the djf , and one ( 16.7% ) had a unifocal tumor in the stomach ( fig 1a )  . 
the demographic , clinicopathologic , and genomic characteristics of these nine patients with djf tumors are listed in table 1 ( n = 7 ngs gists ) and the data supplement ( n = 2 non - ngs gists )  . 
given the infrequency of nf1 genomic alterations in gist , the djf appears to be an over - represented focus of nf1 mutant tumors . nf1 genomic alterations in gist are primarily somatic but can herald mild neurofibromatosis nf1 genomic alterations for each patient are listed in table 2 . 
although none of the patients had known prior clinical evidence of nf - 1 , we next obtained nontumor dna from blood or nontumor tissue to determine whether any of these patients had germline nf1 mutations . 
 ( a ) from a total of 165 patients ( university of california , san diego [ ucsd ] ) with pathologically confirmed gists , 62 had available next generation sequencing ( ngs )  . 
this finding is consistent with the mild phenotype / low penetrance reported in other patients with this germline nf1 mutation.22 nf1 genomic alterations in gists are associated with other cancer - related mutations at the genomic level , the ucsd cohort ngs data generally parallel the known distribution of driver mutations in gists ( fig 2 )  . 
of the 65 driver mutations identified in the 62 gists , oncogenic kit ( 63% ) , pdgfra ( 11% ) , and sdh subunit ( 11% ) mutations were most frequent , whereas braf and kras mutations were less common . moreover , nf1 mutations ( six of 65 ) represented 9% of total genomic alterations . 
the loss of the nf1 tumor suppressor gene often leads to additional chromosomal alterations , but second - hit mutations and / or loss of heterozygosity are necessary to promote tumorigenesis.23 therefore , we analyzed a cohort of five patients with nf1 mutant djf gists for additional cancer - related gene mutations . 
demographic and clinicopathologic characteristics of these two patients are listed in table 3 , with the characteristics of the remaining five patients without djf and / or multifocal gists listed in the data supplement . 
in addition to an nf1 mutation , one patient had a concomitant mutation in ep300 , which encodes a histone acetyltransferase / transcriptional coactivator that has been previously reported to interact with maml1 and maml2 to potentiate notch signaling.24 this supports our earlier observation that unifocal nf1 mutant djf gists also can have impaired notch signaling . 
in summary , 40% of unifocal nf1 mutant gists in the mskcc cohort were localized at the djf , which corroborates the high rate ( 83.3% ) of similar tumors in the ucsd cohort . 
we now show in two single - institution cohorts of patients with gists that 6.1% ( mskcc , seven of 115 ) to 9.7% ( ucsd , six of 62 ) have nf1 alterations . 
if we exclude the 17 patients who overlap with our previous study ( two of whom have nf1 mutations [ ucsd patients 3 and 5 in the current study ] ) , we show that 8.9% ( four of 45 ) of patients with gists have nf1 alterations . 
taken together , nf1 mutation frequencies range from 6% to 10% in gist and are higher than previously appreciated but similar to our recent analysis.7 for the first time in our knowledge , we show that broad genomic profiling of djf or ligament of treitz gists in adults reveals frequent nf1 alterations ( somatic and / or germline ) that occur even in the absence of clinical nf - 1 . 
 yes high high high high pathologic feature tumor resection yes tumor size , cm 13 mitotic index / 5 mm2 high cellular morphology mixed spindle / epithelioid histologic grade g2 immunostain positivity dog - 1 genomic alterations somatic kit ( af ) kit exon somatic nf1 ( af ) ( 30% ) none other somatic alterations ( af ) none table 2 . 
of the 62 patients with gi stromal tumors with available next generation sequencing , 65 known driver mutations were identified : 41 in kit , seven in pdgfra , six in nf1 , seven in succinate dehydrogenase ( sdh ) subunits ( a , b , c , or d ) , two in braf , and two in kras . awareness of the risk of additional tumors ( eg , dermal neurofibromas , nervous system tumors [ including mpnsts , pancreatic neuroendocrine tumors , pheochromocytomas , and other sarcomas ] ) 10 and allows for earlier screening of these nf - 1associated tumors , although whether different malignancies and gists have unique presentation patterns remains to be determined ; and diagnosis of nf - 1 allows for genetic counseling and testing of potentially affected family members . 
of note , individuals can have segmental mosaicism of nf - 1 , which occurs when an nf1 somatic mutation occurs early in embryonic development such that only the tissues derived from the nf1 - mutated cell carry the mutation while the remaining tissues are wild type.26 as with ucsd patient 2 , a clinical genetics work - up may be indicated for patients with nf1 mutations on tumor profiling that could indicate a germline rather than a somatic origin . tumor development in the setting of nf1 genomic alterations is associated with additional second - hit mutations.23 we now have shown that djf tumors with nf1 genomic alteration also can harbor concurrent kit mutations . 
in general , most kit mutations portend imatinib sensitivity , whereas nf1 mutations do not.3 , 27 consistent with this aforementioned drug sensitivity , tumors with a high risk of recurrence are recommended for adjuvant imatinib on the basis of the american college of surgeons oncology group z900128 and scandinavian sarcoma group xviii / aio29 trials where cumulative results demonstrated improved relapse - free survival ( rfs ) and overall survival with 36 months of adjuvant imatinib in patients with high - risk disease . 
in our cohort , three patients with djf gists had a high risk of recurrence by validated risk assessment models.30 , 31 ucsd patient 4 lacked a kit mutation and was predicted to not respond to imatinib therapy ; instead , this patient could experience toxicity . 
this approach was supported by eortc - 62005 and southwest oncology group s0033 / cancer and leukemia group b 150105 phase iii trials that collectively demonstrated improved response rates and rfs in patients with advanced gists and kit exon 9 mutations treated with high - dose imatinib ( 800 mg daily ) compared with standard - dose imatinib ( 400 mg daily ) .32 the patient also had a germline nf1 variant and ultimately developed a local recurrence after 5 years of adjuvant imatinib therapy . ucsd patient 3 had a known kit exon 11 mutation and was treated with dose - escalated imatinib followed by the multikinase inhibitor sunitinib for metastatic disease . 
a brief trial of the mammalian target of rapamycin inhibitor everolimus in combination with imatinib was attempted to block parallel signaling pathways , but the patient rapidly succumbed to the disease . much like patients with metastatic colorectal cancer who are tested for pan - ras mutations before treatment targeting upstream cell surface receptors ( ie , epidermal growth factor receptor ) , 33 , 34 we propose that patients with djf gists be tested for nf1 ( which would activate ras ) and braf v600e genomic alterations ( which two of the patients with djf gists also harbored ) before being offered imatinib therapy , which targets upstream kit . we have recently shown that notch1 can be mutated in both wild - type and nonwild - type gists.7 we now add evidence that implicates the notch signaling pathway in gists . 
we identified another tumor with a mutation in cdc73 , which encodes the rna polymerase iiassociated factor complex protein parafibromin and was recently reported to potentiate notch signaling by binding to the nicd.36 we also found an ep300 mutation in the validation cohort that could abrogate signaling of the notch transcriptional complex . of note , notch signaling was reported to have a tumor suppressor function in gist by downregulating kit mrna expression.37 this prior study also demonstrated that patients with gists with low hes1 expression had shorter rfs times than those with high hes1 expression . whether loss - of - function mutations in notch pathway genes that lead to decreased hes1 expression contribute to gist tumorigenesis remains to be determined . 
nf1 regulates mek / smad3 / jagged1 / hes1dependent glial and neuronal differentiation.38 notch has also been reported to mediate transformation of benign plexiform neurofibromas into mpnsts in the setting of nf - 1 , 39 which suggests an alternate oncogenic role of notch in nf - 1 . 
more studies are required to elucidate the biologic significance of notch pathway mutations in gist , including nf - 1associated gist . finally , the mechanism by which nf1 mutations lead to gists that specifically arise at the djf remains unclear . 
summary of duodenal - jejunal flexure genomic alterations . tumor size , mitotic index ( mi ) , and genomic alterations for patients with duodenal - jejunal flexure gi stromal tumors ( gists ) from both cohorts are presented and organized by known driver genes and notch pathway alterations . somatic and germline mutations are annotated by color for mutation type . snp , single nucleotide polymorphism . cdc73 ep300 notch2 maml2 asxl1 men1 erbb4 tsc2 bcor ( per 5 mm2 ) high unknown genomic alteration nonsense frameshift missense in - frame indel deletion splicing ( somatic ) ( germline ) ( somatic ) braf ( somatic ) arid1a ( somatic ) notch pathway other pacemaker activity.42 one could postulate that this region of chromatin on 17q is open and transcriptionally active at the djf . 
belinsky no relationship to disclose margaret von mehren consulting or advisory role : cytrx , blueprint medicines , janssen pharmaceuticals , deciphera pharmaceuticals research funding : arqule travel , accommodations , expenses : janssen pharmaceuticals , blueprint medicines , novartis , arog pharmaceuticals other relationship : national comprehensive cancer network john a . 
thorson no relationship to disclose lisa madlensky employment : janssen pharmaceuticals ( i ) patents , royalties , other intellectual property : husband has patents through his employment with janssen pharmaceuticals ; these are methods patents and not related to any therapeutics ( i ) timothy bowler employment : regenxbio ( i ) stock and other ownership interests : regenxbio ( i ) francesco dangelo no relationship to disclose dwayne g . 
sicklick , university of california , san diego , la jolla , ca ; martina de siena and francesco dangelo , sapienza e universit `a di roma , rome , italy ; benjamin medina , timothy bowler , and ronald p . 
ma gl , murphy jd , martinez me , et al : epidemiology of gastrointestinal stromal tumors in the era of histology codes : results of a population - based study . 
hechtman jf , zehir a , mitchell t , et al : novel oncogene and tumor suppressor mutations in kit and pdgfra wild type gastrointestinal stromal tumors revealed by next generation sequencing . 
burgoyne am , somaiah n , sicklick jk : gastrointestinal stromal tumors in the setting of multiple tumor syndromes . curr opin oncol 26 : 408 - 414 , 2014 11 . 
belinsky mg , rink l , cai kq , et al : somatic loss of function mutations in neurofibromin 1 and myc associated factor x genes identified by exome - wide sequencing in a wild - type gist case . 
miettinen m , fetsch jf , sobin lh , et al : gastrointestinal stromal tumors in patients with neurofibromatosis 1 : a clinicopathologic and molecular genetic study of 45 cases . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
messiaen l , vogt j , bengesser k , et al : mosaic type - 1 nf1 microdeletions as a cause of both generalized and segmental neurofibromatosis type - 1 ( nf1 )  . 
dematteo rp , ballman kv , antonescu cr , et al : adjuvant imatinib mesylate after resection of localised , primary gastrointestinal stromal tumour : a randomised , double - blind , placebo - controlled trial . 
gastrointestinal stromal tumor meta - analysis group ( metagist ) : comparison of two doses of imatinib for the treatment of unresectable or metastatic gastrointestinal stromal tumors : a meta - analysis of 1 , 640 patients . 
pietrantonio f , cremolini c , petrelli f , et al : first - line anti - egfr monoclonal antibodies in panras wild - type metastatic colorectal cancer : a systematic review and meta - analysis . 
sorich mj , wiese md , rowland a , et al : extended ras mutations and anti - egfr monoclonal antibody survival benefit in metastatic colorectal cancer : a meta - analysis of randomized , controlled trials . 
a 5 - hmc model predicting outcome in high - risk patients was established using linear discriminant analysis . results comparison of lowversus high - risk tumors in the discovery cohort revealed 577 genes with differential 5 - hmc . 
hierarchical clustering of tumors from the discovery and validation cohorts using these genes identied two main clusters highly associated with established prognostic markers , clinical risk group , and outcome . genes with increased 5 - hmc and expression in the favorable cluster were enriched for pathways of neuronal differentiation and kras activation , whereas genes involved in inammation and the prc2 complex were identied in the unfavorable cluster . 
this technology has shown dynamic alterations in 5 - hmc patterns in rare hematopoietic cell populations.3 , 4 furthermore , nano - hmc - seal requires as little as 100 ng of dna from frozen tissue , enabling analysis of clinical samples with limited available tissue . 
more recently , 5 - hmc signatures in cell - free dna have been generated and shown to be robust diagnostic biomarkers for adult human cancer.5 , 6 neuroblastoma is characterized by a broad spectrum of clinical behavior , reecting its biologic heterogeneity.7 a combination of clinical and biologic prognostic markers , including age , stage , mycn status , ploidy , and histology , is used to classify risk and stratify treatment of patients with neuroblastoma . 
 applebaum et al context key objective recently , 5 - hydroxymethylcytosine ( 5 - hmc ) proles have been developed as a diagnostic biomarker for adult malignancies . to investigate the prognostic value of this dna modication in neuroblastoma , we applied nano - hmc - seal , a revolutionary , low - cost , genome - wide technology that requires minimal input dna , to prole 5 - hmc in 109 tumors . knowledge generated grouping of all tumors using genes with differential 5 - hmc in the discovery cohort identied two clusters highly associated with established prognostic markers , clinical risk - group , and outcome . 
using 5 - hmc levels in high - risk patients from the discovery cohort , we developed an outcome prediction model that successfully identied events an independent cohort ( n = 24 ) of high - risk patients . relevance 5 - hmc proling could provide information on factors that determine the clinical behavior of neuroblastoma ( ie , mycn status , copy number alterations , and transcriptional networks ) using one simple assay . 
ongoing efforts include prospectively testing the prognostic value of 5 - hmc proles from tumors and cell - free dna . disease , outcomes remain poor for high - risk ( hr ) patients , with fewer than half achieving long - term survival.7 signicant heterogeneity exists within hr tumors , 7 and new biomarkers are needed to distinguish patients who will respond to standard treatments from those who may benet from alternative approaches . 
clinical data for the cog cohort were extracted from the international neuroblastoma risk group database.7 differences in clinical and tumor characteristics were evaluated with fishers exact tests and t tests for categorical and continuous variables , respectively . 
all protocols were approved by local institutional review boards . nano - 5 - hmc - seal library preparation and sequencing nano - hmc - seal libraries were constructed from 100 ng of genomic dna , as described.3 , 4 fifty base - pair , paired - end libraries were sequenced on an illumina nextseq 500 ( san diego , ca )  . 
differences between log - transformed quantity of 5 - hmc between risk groups according to genomic feature were assessed by paired t tests with benjamini - hochberg correction.12 cpg islands were downloaded from the university of california , santa cruz , genome browser , and cpg shores were dened as 2 - kb regions adjacent to each cpg island . 
enhancer regions were annotated from prior studies in neuroblastoma cell lines from the enhancer atlas13 and van groningen et al.14 chromosomal copy number evaluation dna from hr tumors was analyzed using the oncoscan affymetrix microarray platform , following the manufacturers instructions ( affymetrix , santa clara , ca )  . 
read counts of 5 - hmc for the entire gene body and rna sequencing ( rna - seq ) across exons were loaded into the deseq2 v1.20.018 package in r , version 3.5.0 ( r project , old / 3.5.0 / ) with a model that adjusted for sex and batch . 
sensitivity and specicity were determined for the validation cohort.22 matthews correlation coefcients ( mcc ) were generated to determine the t of the model.23 similar to other correlation statistics , a score of 1 denotes a perfect model . 
kaplan - meier curves and hazard ratios were generated according to predicted event and survival status in prism , version 6.0h ( graphpad software , san diego , ca )  . results patient characteristics and outcome the discovery cohort included 51 patients with lr ( n = 24 ) , ir ( n = 11 ) , and hr ( n = 16 ) tumors . 
for the 36 lr patients with evaluable outcomes , the 5 - year eventfree survival ( efs ) was 94.4% ( se , 3.8% ) and the overall survival ( os ) was 100% . 
because of the unique biology conferred by amplication of the mycn oncogene , 25 we compared the accumulation of gene body 5 - hmc in the eight mycn - amplied hr tumors with accumulation in the 24 lr tumors , and then analyzed 5 - hmc levels in the eight non - mycnamplied hr tumors compared with the same lr tumors ( data supplement )  . hierarchical clustering of the pooled discovery and validation tumors ( n = 89 lr , ir , and hr tumors ) using these 577 genes revealed two primary clusters that correlated highly with prognostic markers , clinical risk group , and outcome ( fig 2a )  . 
among the ve ir patients in cluster 2 , three had an event and two died , which highlights the potential for 5 - hmc proles to rene risk stratication . 
because chromosomal aberrations vary according to mycn status , we conducted copy number analysis on nine available hr tumors from the discovery cohort , ve of which were mycn amplied and four were non - mycn amplied . 
interestingly , 131 ( 88.5% ) of the 148 genes were located on chromosome 1p , suggesting nano - hmc - seal has the potential to identify copy number alterations within tumor subsets . 
chromosome 1p contains several genes with important biologic including chd5 , casz1 , functions in neuroblastoma , arid1a , and mtor ( fig 3b - 3e ) .27 increased 5 - hmc was also detected in several additional genes in tumors with chromosome 1p loss , including tumor - promoting genes abcg1 and s100b mapping to chromosome 21q . gene expression , 5 - hmc levels , and cellular pathway analysis to further explore the biology of cluster 1 and cluster 2 tumors , we examined the 3 , 320 genes with differential 5 - hmc between these tumors ( data supplement )  . 
genes with elevated 5 - hmc in cluster 1 , compared with cluster 2 tumors , showed enrichment for gene ontology ( go ) pathways of neuronal differentiation , similar to prior analysis of genes expressed in lr tumors.28 also identied were oncogenic signatures of activated kras signaling and suppression of bmi1 and mel18 , integral components of the polycomb repressive complex 1 ( prc1 )  . 
in contrast , cluster 2 tumors had elevated 5 - hmc compared with cluster 1 tumors in genes enriched for pathways of an inammatory response and oncogenic signatures including activation of ezh2 and the prc2 complex , transcriptional networks reported previously in hr tumors ( fig 4a and 4b ) .29 because 5 - hmc is a marker of transcriptional activation , we performed rna - seq on one hr , 11 ir , and 17 lr tumors from cluster 1 and 17 hr and three lr tumors from cluster 2 ( data supplement )  . 
genes with increased expression in cluster 1 were highly represented for neuronal differentiation . in cluster 2 , overexpressed genes were enriched for pathways of embryo development and morphogenesis ( data supplement )  . differential 5 - hmc was assessed in these same 48 tumors , identifying 2 , 722 genes that met signicance and ltering criteria ( data supplement )  . 
to estimate the degree of sharing between genes with differential 5 - hmc and expression , we extracted the rna - seq differential fold change and calculated the p value for each of the genes with signicantly differential 5 - hmc.30 using the 1 statistic , 24 we estimated that 62% of the 2 , 722 genes with signicantly differential 5 - hmc were also differentially expressed between the two clusters . 
 a applebaum et al cohort age at diagnosis event survival mycn risk cohort discovery validation age at diagnosis < 12 months 12 - 18 months 19 months to 5 years > 5 years event unknown survival alive deceased unknown mycn nonamplified amplified unknown risk cohort 1 cohort 2 p < .001 years since diagnosis cluster 1 cluster 2 p < .001 years since diagnosis no . 
 ( a ) clustering of nine hr tumors with known copy number at chromosome 1p . genes with differential 5 - hmc predominate on chromosome 1p , highlighting the underlying chromosomal aberration . 
 ( b - e ) genome browser views of 5 - hmc signals detected in four genes ( chd5 , casz1 , arid1a , mtor ) with known biologic functions in neuroblastoma , illustrating decreased 5 - hmc in tumors with chromosome 1p aberrations that likely modulate tumor biology . 
pathway enrichment analysis for differentially regulated genes from cluster 1 and cluster 2 tumors . genes with increased 5hmc in cluster 1 tumors were enriched for gene ontology ( go ) pathways of neuronal differentiation and oncogenic signatures of activated kras signaling and genes that are regulated by bmi1 and mel18 . ( b ) cluster 2 tumors had increased 5 - hmc in genes enriched for go pathways of an inammatory response . 
 5 - hydroxymethycytosine proling in neuroblastoma tumors and oncogenic signatures of activated kras signaling . cluster 2 tumors were enriched for go pathways of embryo development and morphogenesis and oncogenic signatures including activation of il - 2 , il - 15 , and the prc2 complex ( fig 4c and 4d )  . predicted event predicted no event 5 - hmc as a predictive biomarker of outcome in hr patients we performed an exploratory evaluation of 5 - hmc levels as a prognostic marker for patients classied as hr ( data supplement )  . 
the lda model trained on hr patients in the discovery cohort based on their os correctly predicted efs in 18 of 24 patients from the combined validation and cog cohorts . 
in contrast , cluster 2 contained the majority of tumors that were clinically classied as hr ( 27 of the 29 evaluated ) , ve ir tumors , and ve lr tumors . 
three of the ve ir patients in cluster 2 relapsed and two died , suggesting 5 - hmc proles may provide additional prognostic information in these non - highrisk cohorts . we also demonstrate 5 - hmc levels may be prognostic of efs but not os in hr patients . 
 ( a ) event - free survival was signicantly better for patients who were predicted not to have an event , although no signicant difference in ( b ) overall survival was observed in those predicted to be alive or not . esophageal , pancreatic , liver , and colon cancers.5 , 35 to our knowledge , this is the rst study to show that gene - specic 5 - hmc proles may also be prognostic of patient outcomes . chromosomal copy number variation is commonly detected in neuroblastoma , and patterns of chromosomal aberrations are strongly prognostic of survival.26 we show for 5 - hmc proling to identify segmental the potential chromosomal aberrations without the need for additional testing . 
 applebaum et al there is increasing evidence that 5 - hmc plays an important role in activating transcription , and that variation in 5 - hmc positively correlates with gene expression levels.4 we found many genes with both increased expression and 5 - hmc , which is consistent with other tumor types.3 , 4 in the favorable cluster 1 tumors , we found go enrichment of neuronal differentiation from the genes with increased in 5hmc and expression . 
neuronal differentiation is a hallmark of lr tumors , 38 and studies indicate hras expression drives this phenotype.39 pathway analysis also suggested a role for the prc1 complex , which promotes gene silencing by monoubiquitinating histone 2a.40 additional efforts are needed to conrm our ndings , which could provide additional evidence for targeting bmi1 in neuroblastoma.41 in the unfavorable cluster 2 tumors , we found that the genes with increased 5 - hmc and expression were enriched for prc2 complex target genes . 
upregulation of ezh2 , an integral part of leads to the transcriptional repression of differentiation genes and maintains stem - like cell properties.42 recent studies have established the prc2 complex has high - binding afnity for the prc2 complex , methylated cytosines , 43 making a direct connection between histone and dna epigenetics.44 in addition , ezh2 is integral to the biology of hr neuroblastoma and considered a potential therapeutic target in the disease.45 can rapidly nano - hmc - seal identify 5 - hmc levels genomewide , using a cost - effective sequencing approach that requires low sample input and no specialized biospecimen handling . 
although a number of prognostic gene expression signatures have been validated in neuroblastoma , 28 , 46 - 48 these biomarkers have not been integrated into the clinic , in part because of challenges obtaining high - quality rna from diagnostic tumor samples . moreover , neuroblastoma tumorigenesis and phenotype are thought to be driven by epigenetic reprogramming , 49 driving the need for new epigenetic biomarkers . 
5 - hmc proling could provide information on factors that determine the clinical behavior of neuroblastoma : mycn status , copy - number alterations , and transcriptional networks , using one simple assay . 
k12ca139160 and k08ca226237 ( m.a.a. ) , and national institutes of health grants 1ul1tr002389 - 01 and 5ul1tr002389 - 02 that fund the university of chicago institute for translational medicine . 
the international neuroblastoma risk group ( inrg ) database is supported in part by the william guy forbeck research foundation , st baldricks foundation , little heroes cancer research fund , childrens neuroblastoma cancer foundation , neuroblastoma childrens cancer foundation , super jake foundation , and alexs lemonade stand foundation . 
 5 - hydroxymethycytosine proling in neuroblastoma tumors robert grossman stock and other ownership interests : tempus health , healthseq consulting or advisory role : healthseq research funding : abbvie lucy a . 
godley honoraria : agios patents , royalties , other intellectual property : royalties from uptodate , inc . chuan he stock and other ownership interests : accent therapeutics , epican genetech consulting or advisory role : accent therapeutics patents , royalties , other intellectual property : wisegene licensed tab - seq from the university of chicago susan l . 
cohn stock and other ownership interests : united therapeutics ( i ) , varian medical systems ( i ) , united therapeutics , vermillion , resmed ( i ) , merck ( i ) , merck , stryker ( i ) , stryker , amgen ( i ) , pzer ( i ) , abbvie , amgen , jazz pharmaceuticals , eli lilly , sano , varex imaging , pzer , united therapeutics ( inst ) , merck ( inst ) no other potential conicts of interest were reported . acknowledgment we thank the center for research informatics of the university of chicago for use of the gardner high - performance computing cluster and the cancer center support grant ( p30 ca014599 ) for support of the genomics core facility . 
we thank the childrens oncology group ( cog ) neuroblastoma biobank for providing dna specimens and arlene naranjo at the cog statistics and data center for support . the contents are solely the responsibility of the authors and do not necessarily represent the ofcial views of the national institutes of health . references vasanthakumar a , godley la : 5 - hydroxymethylcytosine in cancer : signicance in diagnosis and therapy . 
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oberthuer a , juraeva d , hero b , et al : revised risk estimation and treatment stratication of lowand intermediate - risk neuroblastoma patients by integrating clinical and molecular prognostic markers . 
nature 431 : 873 - 878 , 2004 infante jr , bedard pl , shapiro g , et al : phase 1 results of ptc596 , a novel small molecule targeting cancer stem cells ( cscs ) by reducing levels of bmi1 protej clin oncol 35 : 2574 , 2017 ( 15_suppl ) 42 . 
oberthuer a , juraeva d , li l , et al : comparison of performance of one - color and two - color gene - expression analyses in predicting clinical endpoints of neuroblastoma patients . 
marachelian a , villablanca jg , liu cw , et al : expression of ve neuroblastoma genes in bone marrow or blood of patients with relapsed / refractory neuroblastoma provides a new biomarker for disease and prognosis . 
 novel alk fusion , ppfibp1 - alk , in pancreatic ductal adenocarcinoma responsive to alectinib and lorlatinib arjan gower , md , ms1 ; barry golestany , md2 ; jun gong , md3 ; aatur d . 
singhi , md , phd4 ; and andrew eugene hendifar , md , mph3 introduction pancreatic cancer is the seventh leading cause of cancer death among men and women worldwide , with  . 
430 , 000 deaths in 2018.1 despite advances in surgical techniques , radiation , and systemic treatment in the past few decades , overall survival for pancreatic cancer is extremely poor , with a 5 - year survival rate of 9%.2 current systemic treatment regimens for metastatic pancreatic cancer include folfirinox ( uorouracil , folic acid , oxaliplatin , and irinotecan ) , gemcitabine plus nab - paclitaxel , and liposomal irinotecan with uorouracil.3 - 6 approximately 88% - 95% of pancreatic adenocarcinomas harbor kras driver mutations . 
in row 1 , the circled hepatic lesion in the right lobe of the liver at baseline ( a ) before alectinib decreased in size ( b ) 2 months after alectinib . 
the patient experienced progression during alectinib treatment after 5 months and experienced progression during uorouracil , folic acid , oxaliplatin , and irinotecan treatment shortly thereafter . fluorescence in situ hybridization ( fish ) was negative . 
it has been shown that fish may have different sensitivities of detecting alk compared with next - generation sequencing or immunohistochemistry.27 of note , molecular proling was negative for kras , microsatellite instability , nf1 and p53 inactivating mutations , and cdkn2a / b loss . proling was positive for brca2 c.8007a.g ( silent ) and cdkn1b 407a.g , both of unknown clinical signicance . patient was started on folfirinox with radiographic response . 
however , because of the adverse effects of chemotherapy , including nausea and neuropathy , the patient was switched to alectinib 600 mg twice daily , given her alk fusion status . 
subsequent cell - free plasma ( guardant360 ; guardant health , redwood city , ca ) showed newly acquired alk mutations g1202r and v1180l in addition to the ppfibp1 - alk translocation . 
the patients disease has been stable on lorlatinib on 2 - month follow - up imaging , and she continues to be treated with lorlatinib ( figs 2 and 3 )  . discussion currently , there are no fda - approved targeted therapies for pancreatic cancer , and standard chemotherapy ( folfirinox ) for metastatic pancreatic cancer is quite toxic , with a median overall survival of approximately 11 months.3 to our knowledge , we present the seventh case of alk - positive metastatic pancreatic cancer with a novel ppfibp1 - alk fusion gene and the rst patient to be treated with alectinib as rst - line targeted therapy . 
the patient ultimately acquired alectinib - resistant alk mutations g1202r and v1180l ; however , the disease has been stable on the third - generation alk inhibitor lorlatinib . alk translocation in pancreatic cancer was rst described in 2017 , and there have been only 6 documented cases of alk - positive pancreatic adenocarcinoma through literature review ( table 1 )  . 
although the prevalence of the alk fusion gene is rare , at 0.16% , the prevalence increases to 1.3%.among patients , 50 years old12 the majority of alk fusion partners seen in nsclc includes eml4 , but other partners include strn , kcnq , klc1 , kif5b , ppm1b , and tgf genes.28 ppfibp1 - alk gene fusion was rst described in 2011 in a patient with pulmonary inammatory myobroblastic tumor29 and subsequently was described in epithelioid brous histiocytoma , 30 but it has never been described in nsclc or pancreatic cancer . 
of the 6 patients with alk - positive pancreatic cancer , 4 patients had eml4alk translocation , 1 patient had strn - alk , and 1 patient had a dctn1 - alk translocation ( table 1 )  . 
 case report 3 / 19 4 / 19 5 / 19 7 / 19 9 / 19 12 / 19 1 / 20 3 / 20 3w pain + bloating ; us , pet / ct : liver lesions folfirinox #2 - 6 ; dose reduction due to adverse effects ct imaging : stable disease biopsy with acquired resistance mutations ; progression on folfirinox #7 - 8 ; start lorlatinib biopsy : pancreatic adenocarcinoma ; folfirinox #1 ct imaging : stable disease ; start alectinib ct imaging : progressive disease ct imaging : stable disease inferior r hepatic lobe posterior r hepatic lobe folfirinox alectinib lorlatinib folfirinox 3 / 1 / 19 5 / 1 / 19 7 / 1 / 19 9 / 1 / 19 11 / 1 / 19 1 / 1 / 20 3 / 1 / 20 date fig 2 . 
# , dose number ; 3w , 3 - week ; ct , computed tomography ; folfirinox , uorouracil , folic acid , oxaliplatin , and irinotecan ; pet , positron emission tomography ; r , right ; us , ultrasound . and proliferative capabilities , ultimately leading to activation of different signaling pathways.28 in nsclc , alk fusion is an oncogenic driver that is generally mutually exclusive of kras mutation16 ; however , there are reports of concomitant kras and alk fusion double alteration that may confer worse prognosis.31 all 7 patients with alk - positive pancreatic cancer were kras wild type , suggesting , albeit in a small sample size , that alk translocation drives oncogenesis and that these 2 oncogenic drivers are mutually exclusive . 
this case demonstrates the rst time , to our knowledge , that alectinib has been used as the rst targeted therapy , and the development of known alectinib - resistant alk mutations suggests that alk - positive pancreatic cancer acquires resistance in a fashion similar to that of nsclc . there is a clear unmet medical need for novel therapeutic approaches in pancreatic cancer . 
a recent study in metastatic pancreatic cancer with germ - line brca mutations ( 7% prevalence ) regardless of kras mutation status showed increased pfs with the poly adp ribose polymerase inhibitor olaparib after rst - line platinum - based chemotherapy.4 in a recent phase ii basket study , 2 of 9 patients who had pancreatic cancer with her2 amplication / overexpression experienced responses to her2 - targeted therapies trastuzumab plus pertuzumab . 
additionally , a patient who had pancreatic cancer and braf gene fusion ( cux1 - braf ) had a partial response to the braf - targeted therapy vemurafenib.32 last , 3 patients who had pancreatic cancer with ntrk1 and ros1 gene fusions experienced responses to entrectinib , a targeted inhibitor of these genes.33 these studies are additional proof of concept that targeted therapies can be used successfully when paired with the right genomic alteration and that alk inhibitors may be of substantial clinical benet in the right patient . 
in row 1 , the circled hepatic lesion after disease progression on uorouracil , folic acid , oxaliplatin , and irinotecan ( a ) before lorlatinib was stable in size ( b ) 2 months after lorlatinib treatment . 
the patient currently continues to be treated with lorlatinib . 38% of kras wild - type tumors have genomic alterations that could activate the mapk signaling cascade.9 identication of molecular alterations in patients with kras wild - type status that may drive oncogenesis will help guide therapeutic intervention in a small but clinically relevant population . alk translocations are rare in pancreatic cancer . 
this is , to the rst report of ppfibp1 - alk rearour knowledge , rangement in pancreatic or nsclc , the rst reported patient with pancreatic cancer to be treated with alectinib as the rst targeted therapy , and the rst patient to be treated with lorlatinib after acquired resistance to alectinib with stabilization of disease . 
all 7 occurrences of alk - positive pancreatic cancer have been kras wild type ; given that 6 of the 7 patients were , age 50 years , there may be clinical benet to screen young patients with pancreatic cancer who are kras negative for the alk gene fusion , as they may benet from alk inhibitors , which may lead to improved overall survival . 
clinical characteristics of patients with alk - positive pancreatic cancer patient case age ( years ) alk rearrangement exon 13 eml4 - exon 20 alk ( 1 ) crizotinib ; ( 2 ) ceritinib ; ( 3 ) alectinib alk inhibitor ( in order of treatment ) ( 1 ) crizotinib ( 1 ) crizotinib unknown none ( 1 ) crizotinib ; ( 2 ) alectinib duration of survival ( months ) unknown unknown ppfibp1 - alk ( 1 ) alectinib ; ( 2 ) lorlatinib exon 6 eml4 - exon 20 alk exon 3 strn - exon 20 alk exon 6 eml4 - exon 20 alk exon 6 eml4 - exon 20 alk dctn1 - alk note . 
singhi , andrew eugene hendifar collection and assembly of data : arjan gower , barry golestany , andrew eugene hendifar data analysis and interpretation : arjan gower , jun gong , aatur d . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / authors / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . jun gong honoraria : amgen , astellas pharma , clinical congress consultants , qed therapeutics , exelixis , elsevier consulting or advisory role : amgen , astellas pharma , clinical congress consultants , qed therapeutics , exelixis , elsevier aatur d . 
singhi honoraria : foundation medicine andrew eugene hendifar consulting or advisory role : novartis , ipsen , perthera , celgene , abbvie research funding : ipsen travel , accommodations , expenses : halozyme no other potential conicts of interest were reported . references bray f , ferlay j , soerjomataram i , et al : global cancer statistics 2018 : globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries . 
ca cancer j clin 69 : 7 - 34 , 2019 conroy t , desseigne f , ychou m , et al : folfirinox versus gemcitabine for metastatic pancreatic cancer . 
n engl j med 364 : 1817 - 1825 , 2011 golan t , hammel p , reni m , et al : maintenance olaparib for germline brca - mutated metastatic pancreatic cancer . 
n engl j med 381 : 317 - 327 , 2019 von hoff dd , ervin t , arena fp , et al : increased survival in pancreatic cancer with nab - paclitaxel plus gemcitabine . 
wang - gillam a , li cp , bodoky g , et al : nanoliposomal irinotecan with uorouracil and folinic acid in metastatic pancreatic cancer after previous gemcitabinebased therapy ( napoli - 1 ) : a global , randomised , open - label , phase 3 trial . 
lancet 387 : 545 - 557 , 2016 biankin av , waddell n , kassahn ks , et al : pancreatic cancer genomes reveal aberrations in axon guidance pathway genes . 
nature 518 : 495 - 501 , 2015 singhi ad , george b , greenbowe jr , et al : real - time targeted genome prole analysis of pancreatic ductal adenocarcinomas identies genetic alterations that might be targeted with existing drugs or used as biomarkers . 
lin e , li l , guan y , et al : exon array proling detects eml4 - alk fusion in breast , colorectal , and nonsmall - cell lung cancers . 
kelly lm , barila g , liu p , et al : identication of the transforming strn - alk fusion as a potential therapeutic target in the aggressive forms of thyroid cancer . 
no e j , lovejoy a , ignatius ou , s - h , et al : alk mutation status before and after alectinib treatment in locally advanced or metastatic alk - positive nsclc : pooled analysis of two prospective trials . 
solomon bj , besse b , bauer tm , et al : lorlatinib in patients with alk - positive nonsmall - cell lung cancer : results from a global phase 2 study . 
zou hy , friboulet l , kodack dp , et al : pf - 06463922 , an alk / ros1 inhibitor , overcomes resistance to rst and second generation alk inhibitors in preclinical 27 . 
lin c , shi x , yang s , et al : comparison of alk detection by fish , ihc , and ngs to predict benet from crizotinib in advanced nonsmall - cell lung cancer . 
takeuchi k , soda m , togashi y , et al : pulmonary inammatory myobroblastic tumor expressing a novel fusion , ppfibp1 - alk : reappraisal of anti - alk immunohistochemistry as a tool for novel alk fusion identication . 
ulivi p , chiadini e , dazzi c , et al : nonsquamous , nonsmall - cell lung cancer patients who carry a double mutation of egfr , eml4 - alk or kras : frequency , clinical - pathological characteristics , and response to therapy . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors on the basis of molecular proles : results from mypathway , an open - label , phase iia multiple basket study . 
 identifying erbb2 activating mutations in her2 - negative breast cancer : clinical impact of institute - wide genomic testing and enrollment in matched therapy trials pedro exman , md1 ; ana c . 
our primary aim was to describe the proportion of patients with a qualifying erbb2 mutation identied by our institutional genomic panel ( oncomap or oncopanel ) who enrolled in the trial . 
associations were calculated using fishers exact test . results we identied a total of 1 , 045 patients with metastatic breast cancer without erbb2 amplication who had available genomic testing results . 
of these , 42 patients were found to have erbb2 mutation and 19 patients ( 1.8% ) were eligible for the trial on the basis of the presence of an activating mutation , 18 of which were identied by oncopanel testing . 
fifty - eight percent of potentially eligible patients were approached , and 33.3% of eligible patients enrolled in the trial guided exclusively by oncopanel testing . conclusion more than one half of eligible patients were approached for trial participation and , signicantly , one third of those were enrolled in nct01670877 . 
 exman et al context key objective although tumor genomic sequencing has become more accessible , bridging the gap between patients with targetable mutations and recruitment to specic trials remains a challenge . 
this phase ii study has been active at dfci since 2013 , with consistent slot availability during this time . patients we included 1 , 817 patients in the profile cohort with mbc who had available genomic proling results between august 1 , 2011 , and june 1 , 2017 . 
from the ccpm cohort , we included patients with metastatic her2 - negative ( per ihc and / or fish ) breast cancer in whom targeted genomic panel results were available between june 22 , 2015 , and june we collected data on those patients who consented to be in the profile cohort and who had available oncomap or oncopanel . 
clinical data abstraction was performed via medical record review and included age and stage at initial diagnosis , date of recurrence , histologic features , and date of death or last follow - up . 
clinical her2 status was dened by the 2013 asco / college of american pathologists guidelines.7 survival data were collected through a combination of medical record review and linkage to the national death index . 
all data are stored in a custom redcap database . genomic analysis genomic testing was performed in a clia - certied environment within the center for advanced molecular diagnostics at brigham and womens hospital . 
 excluded patients with her2 - positive disease ( n = 772 ) profile cohort patients with mbc with at least one dfci visit and available oncopanel results between 2011 and 2017 ( n = 1 , 817 ) patients with er - positive / her2negative mbc with oncopanel result ( n = 1 , 045 ) patients with erbb2 mutation previously detected by other genomic test modality ( n = 1 ) erbb2 activating mutations in her2 metastatic breast cancer ccpm cohort patients with mbc with at least one dfci visit and available oncopanel results between 2015 and 2017 ( n = 194 ) excluded patients with her2 - positive disease ( n = 28 ) patients with her2negative mbc with oncopanel result ( n = 166 ) patients with erbb2 mutations ( n = 42 ) patients with eligible erbb2 mutations diagnosed by profile ( n = 18 ) patients approached by trial team after oncopanel result ( n = 11 ) patients approached by trial team after ccpm result ( n = 1 ) patients with eligible erbb2 mutations diagnosed by ccpm ( n = 6 ) patients with erbb2 mutations ( n = 10 ) patients enrolled on the basis of oncopanel results ( n = 6 ) patients enrolled on the basis of ccpm result ( n = 1 ) fig 1 . 
dfci , danafarber cancer institute ; er , estrogen receptor ; her2 , human epidermal growth factor receptor 2 ; mbc , metastatic breast cancer . muther trial this multi - institutional phase ii clinical trial ( clinicaltrials.gov identier : nct 01670877 ) evaluates the efcacy of neratinib in patients with her2 - negative ( per ihc and / or fish ) , erbb2 - mutant mbc . 
the study opened to accrual at dfci on september 20 , 2013 , and it remains open as of december 18 , 2018 . trial matching in 2016 , we instituted several programs , such as the ending metastatic breast cancer for everyone ( embrace ) program for patients with mbc , 11 the matchminer platform , 12 and an interactive web - based trial - matching tool13 ( data supplement ) to assist physicians in trial selection . 
through these programs , key data from oncopanel ( derived either from the profile or ccpm protocols ) , as well as results from other research testing conducted in a clia - environment , were collated into a central database . the data supplement presents details of the embrace prograa custom report ( data supplement ) was shared with the medical oncologist by a designated research coordinator at the time of a patients visit to facilitate realtime trial matching . 
in addition , a custom query for erbb2 alterations was created within the matchminer platform and allowed for the identication of all patients with breast cancer with an erbb2 alteration at dfci . 
this list was used to reach out directly to treating physicians as an extra outreach method to capture patients who might not have a return visit to dfci scheduled . statistical analysis the primary objective of our study was to describe the proportion of patients in the prespecied profile cohort with eligible erbb2 mutations detected by genomic analysis who enrolled in the selected trial . 
secondary objectives included describing the proportion of patients approached for trial screening , the median time from genomic testing results to trial enrollment , the median time from genomic testing to death , and overall survival ( os )  . 
os was dened as the time from diagnosis of metastatic disease ( by imaging or biopsy ) to death from any cause and was estimated using the kaplan - meier method . 
between august 2011 and june 2017 , 1 , 045 patients with her2 - negative mbc consented to the profile protocol and had successful genomic testing . oncopanel was performed in most of the patients of this cohort ( 98.8% , n = 1 , 032 of 1 , 045 )  . 
forty - two patients ( 4.0% ) were found to have erbb2 mutations in their tumors , and of these , 19 ( 1.8% ) had mutations eligible for the trial ( 18 detected by oncopanel , none by oncomap , and one by commercial panel )  . 
between june 2015 and june 2017 , 166 patients with her2 - negative mbc underwent successful oncopanel testing of a fresh tumor biopsy as part of the ccpm initiative ( fig 1 )  . 
in terms of our prespecied primary end point , the proportion of patients with eligible erbb2 mutations who enrolled in the selected neratinib trial on the basis of only oncopanel results was 33.3% ( six of 18 ) , representing 0.5% of the total tested population ( six of 1 , 045 )  . 
one additional patient with a previously known erbb2 mutation detected through commercial testing enrolled in the neratinib trial ; another patient enrolled in the trial on the basis of testing of a fresh metastatic specimen collected in the ccpm protocol ( fig 1 )  . all patients who enrolled in the trial had other multiple mutations detected by oncopanel ( data supplement ) , but none received additional treatment guided by another specic mutation . 
for patients with eligible erbb2 mutations , the median os was not reached ( data supplement )  . among 1 , 045 patients with her2 - negative mbc with available genomic results between 2011 and 2017 and included in our profile cohort , we identied a total of 19 patients with erbb2 mutations that rendered them eligible for the neratinib trial . 
although the total number of enrolled patients ( n = 8 ) at dfci was low , our site was the second - highest accruing site nationally , reecting the low overall prevalence of erbb2 mutations in breast cancer . we observed that the conversion rate from genomic result to trial enrollment was numerically higher in the profile cohort compared with the ccpm cohort , although the patients in the ccpm cohort were not approached because they had stable disease on current therapy . 
patients who enrolled in the trial had received a median of four ( range , one to six ) lines of systemic treatment in the metastatic ( mets ) setting before oncopanel results were available . 
in addition , 75% ( six of eight ) of the registered patients were enrolled after the rst progression after the oncopanel result was available and 25% ( two of eight ) after the second progression . 
 erbb2 activating mutations in her2 metastatic breast cancer mutated and eligible mutated not mutated test technical lack of an adequate archival sample , insufcient archival material , or failure ( the last being uncommon )  . 
because patients were not randomly assigned to undergo testing , we cannot denitively comment on the impact of enterprise - wide testing versus an approach of more selective testing at the time of clinical need . 
the potential for referral bias , given the long median interval between initial metastatic diagnosis and oncopanel testing , and the possibility that only more t patients were able to travel to our site later in their disease course , could have diluted differences in survival by erbb2 mutation status . 
in addition , 20% of patients with eligible mutations were lost to follow - up . the rarity of the patient population meant that only 0.5% of the total tested population ultimately enrolled in the neratinib study . 
it is notable that the median time from metastatic diagnosis to oncopanel result in mutated patients was 7.0 months , and in large part , this reected patients being referred to our institution after having lived with a diagnosis of mbc for some time before their initial visit . our data illustrate the hurdles involved in identifying and enrolling patients with rare molecular alterations in clinical trials . 
we believe that our data suggest that broad - based testing , the development of systems with the ability to query genomic testing results across a clinic population , and robust strategies to approach patients will be critical to the success of studies enrolling rare subsets . 
1 , 000 women to enroll eight patients in the selected trial ; however , this testing simultaneously identied patients for multiple other trials , in a tissueand timeefcient manner . one half of the patients enrolled in the neratinib trial were also potentially eligible for other simultaneous trials on the basis of targetable mutations such as in palb2 or pik3ca ( data supplement )  . 
a direct assessment of the drug activity of neratinib ( with or without fulvestrant ) will be performed in the muther trial , and progression - free survival will be evaluated as a secondary end point.17 a strength of our study is the selection of a specic clinical trial with wide slot availability over an extended period to evaluate the conversion rate from biomarker result to trial enrollment . 
a slightly lower conversion rate was observed by wheler et al , 5 who prospectively evaluated 500 patients ( only 16% with breast cancer ) ; 22% of them received directly guided treatment after tumor molecular proling . 
meric - bernstam et al18 evaluated 2 , 000 patients with different cancer types ; 7% of patients with potentially actionable alterations were enrolled in a specic mutation - driven trial , and 4% were treated in a trial with a molecular rationale . 
the safir - 01 study enrolled 423 patients with advanced breast cancer , and 13% of them were considered eligible for a mutationguided clinical trial.3 our study has several limitations . 
although all new patients to dfci were approached for participation in the overarching protocol allowing for tumor testing , we did not prospectively collect the decline rate , nor did we collect reasons for decline . 
not all consented patients with mbc underwent tumor testing , for a variety of reasons , including affiliations 1dana - farber cancer institute , boston , ma 2washington university school of medicine , st . 
freedman research funding : puma biotechnology ( inst ) , eisai ( inst ) lorenzo trippa consulting or advisory role : galera therapeutics romualdo barroso - sousa consulting or advisory role : lilly speakers bureau : roche , bristol - myers squibb travel , accommodations , expenses : roche simona di lascio honoraria : roche / genentech ethan cerami stock and other ownership interests : blueprint medicines honoraria : merck travel , accommodations , expenses : merck margaret s . 
tolaney consulting or advisory role : novartis , pzer , merck , lilly , nektar , nanostring technologies , astrazeneca , puma biotechnology , genentech , eisai , immunomedics , sano , celldex , bristol - myers squibb , paxman , seattle genetics research funding : genentech / roche ( inst ) , merck ( inst ) , exelixis ( inst ) , pzer ( inst ) , lilly ( inst ) , novartis ( inst ) , bristol - myers squibb ( inst ) , eisai ( inst ) , astrazeneca ( inst ) , nanostring technologies ( inst ) , cyclacel ( inst ) , nektar ( inst ) , immunomedics ( inst ) travel , accommodations , expenses : astrazeneca , lilly , merck , nektar , novartis , pzer , genentech / roche , immunomedics , eisai , nanostring technologies , puma biotechnology , celldex ian e . 
krop employment : amag pharmaceuticals ( i ) leadership : amag pharmaceuticals ( i ) stock and other ownership interests : amag pharmaceuticals ( i ) honoraria : genentech / roche consulting or advisory role : genentech / roche , daiichi sankyo , context therapeutics , macrogenics , taiho pharmaceutical research funding : genentech / roche ( inst ) , seattle genetics ( inst ) , pzer ( inst ) , daiichi sankyo ( inst ) ron bose honoraria : genentech , foundation medicine consulting or advisory role : genentech research funding : puma biotechnology ( inst ) bruce e . 
johnson research funding : novartis ( inst ) , toshiba ( inst ) , novartis ( inst ) , novartis ( inst ) patents , royalties , other intellectual property : dana - farber cancer institute cynthia x . 
ma consulting or advisory role : pzer , novartis , syndax , lilly , biovica , tempus , seattle genetics , agendia , myriad genetics , lilly research funding : pzer ( inst ) , eisai ( inst ) , puma ( inst ) travel , accommodations , expenses : syndax , lilly , agendia , pzer deborah a . 
winer stock and other ownership interests : verastem honoraria : genentech / roche , tesaro , genomic health consulting or advisory role : leap therapeutics , seattle genetics , jounce therapeutics , glaxosmithkline , carrick therapeutics , lilly research funding : genentech ( inst ) , novartis ( inst ) , merck ( inst ) nikhil wagle stock and other ownership interests : foundation medicine honoraria : lilly consulting or advisory role : novartis , lilly research funding : novartis , puma biotechnology nancy u . 
 erbb2 activating mutations in her2 metastatic breast cancer acknowledgment we thank kaitlyn bifolck for her editorial support . references 5 , 2010 ] andre f , mardis e , salm m , et al : prioritizing targets for precision cancer medicine . 
gynecol oncol 148 : 585 - 590 , 2018 andr e f , bachelot t , commo f , et al : comparative genomic hybridisation array and dna sequencing to direct treatment of metastatic breast cancer : a multicentre , prospective trial ( safir01 / unicancer )  . 
lancet oncol 15 : 267 - 274 , 2014 schwaederle m , zhao m , lee jj , et al : impact of precision medicine in diverse cancers : a meta - analysis of phase ii clinical trials . 
cancer res 76 : 3690 - 3701 , 2016 sholl lm , do k , shivdasani p , et al : institutional implementation of clinical tumor proling on an unselected cancer population . 
wolff ac , hammond me , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
plos one 4 : e7887 , 2009 [ erratum : plos one garcia ep , minkovsky a , jia y , et al : validation of oncopanel : a targeted next - generation sequencing assay for the detection of somatic variants in cancer . 
ramkissoon sh , bandopadhayay p , hwang j , et al : clinical targeted exome - based sequencing in combination with genome - wide copy number proling : precision medicine analysis of 203 pediatric brain tumors . 
hughes me , frank es , merrill ms , et al : embrace ( ending metastatic breast cancer for everyone ) : a comprehensive approach to improve the care of patients with metastatic breast cancer . 
lindsay j , del vecchio fitz c , zwiesler z , et al : matchminer : an open source computational platform for real - time matching of cancer patients to precision medicine clinical trials using genomic and clinical criteria . 
fish , phd1 , 3 , 4 introduction immune checkpoint inhibitors ( icis ) have signicantly improved advanced cancer outcomes.1 , 2 although icis unleash antitumor immune responses , negating natural immune inhibitory mechanisms can result in the development of self - reactive immune cells and an array of immune - related adverse events ( iraes ) .3 although reports of serious myocarditis have emerged , 4 - 9 data on long - term outcomes following an episode of ici - associated myocarditis are limited and molecular analyses on surviving patients are lacking . 
here , we report a case of recurrent myocarditis in a patient previously treated with a programmed death ligand 1 ( pd - l1 ) inhibitor ( durvalumab ) , an ici that blocks the binding of pd - l1 to programmed cell death protein 1 and cd80.10 molecular investigations revealed cytokine modulations suggestive of a sustained t - helper 1 ( th1 ) - like cellular immune response as well as unique antigen - bound serum autoantibodies , highlighting a induction of potential autoantibody production in iraes . role for th1 - cell - dependent case initial presentation a 67 - year - old woman presented to the emergency department with severe sudden - onset chest pain 2 days after receiving the 12th cycle of pd - l1 inhibitor ( 48 weeks after starting ici ) for the treatment of metastatic urothelial carcinoma ( fig 1a )  . 
she was previously treated for remote triple - negative right - sided breast invasive ductal carcinoma with surgery ; cyclophosphamide , epirubicin , and 5 - uorouracil chemotherapy ; and radiation . 
her treatment was largely uneventful , with only a noted diagnosis of hypothyroidism after receiving the 7th cycle of pd - l1 inhibitor , which was treated with hormone replacement therapy without interruption of ici . although the patient was hemodynamically stable , cardiac laboratory investigations revealed high - sensitivity troponin i levels were elevated at 4 , 114 ng per l ( reference range , , 26 ng per l ) , and brain natriuretic peptide levels ( bnp ) were elevated at 2 , 275 pg per ml ( reference range , , 100 pg per ml ) ( figs 1b and 1c , data supplement )  . 
2d echocardiogram demonstrated severely decreased biventricular systolic function , with a left ventricular ejection fraction ( lvef ) of 20% , right ventricular ( rv ) fraction area change of 22% , and a left ventricular ( lv ) thrombus ( fig 1d and video 1 )  . 
with a history of ici treatment , together with clinical , ecg ( fig 2a ) , and imaging ndings , a diagnosis of ici - associated myocarditis was made . 
treatment with intravenous methylprednisolone ( 2 mg / kg / d ) , furosemide , betablocker , angiotensin - converting enzyme inhibitor , and low - molecular - weight heparin was initiated . 
cardiovascular magnetic resonance ( cmr ) imaging conrmed severe biventricular dysfunction , mild pericardial effusion , and demonstrated late gadolinium enhancement ( lge ) involving the basal and apical segments predominantly in a subepicardial distribution ( video 2 , data supplement )  . 
follow - up ecg remained abnormal with low - voltage qrs complex as well as persistent st elevation ( fig 2b )  . the patient was discharged home in stable condition on 90 mg oral prednisone with a plan to taper and discontinue in 8 weeks . 
 echocardiogram ( apical the patient demonstrating four chamber view ) of biventricular dysfunction as well as a focused apex view with contrast showing a thrombus ( admission )  . case report evidence of pulmonary emboli , whereas whole - body computed tomography scanning showed no evidence of metastatic disease . 
there was signicant clinical recovery 1 month after discharge , with echocardiography revealing partial recovery of lv function ( lvef = 40% ; global longitudinal strain = 17.1% ) ; however , rv systolic function remained severely reduced ( rv fraction area change 17% , data supplement )  . 
ecg remained abnormal with low - voltage qrs , however , now with right bundle branch block ( fig 2c )  . recurrent presentation four months after the initial event , and without resumption of ici treatment , the patient presented to the cardiology clinic with extreme fatigue . 
she was admitted and a subsequent cmr conrmed the severity of lv dysfunction , multiple new lv thrombi , larger pericardial effusion , and a mild interval worsening of nonischemic pattern lge ( data supplement )  . a cardiac biopsy ( figs 2e and 2f ) conrmed the diagnosis of myocarditis with predominantly t cells ( fig 2g ) and macrophages ( fig 2h ) ; polymerase chain reaction was negative for cardiotropic viruses.11 given the biopsy result , she was diagnosed with recurrence of ici - associated myocarditis . 
she deteriorated and subsequently succumbed to heart failure a year - and - a - half later . methods the patient was enrolled in this research ethics boardapproved case report at the university health network after obtaining informed written consent . 
next - generation sequencing ( immunoseq ) , cytokine multiplex assays , targeted microrna proling , and a mass spectrometry - based assay to identify serum autoantibody complexes with their cognate antigens were performed ( for a detailed description of the methods , see the data supplement )  . results analysis of the distribution , clonality , and diversity of t - cell receptors pre - recurrence ( january ) and during treatment for recurrence of myocarditis ( march onward ) with immunosuppressants revealed limited clonal modulation ( data supplement )  . 
multiplex analysis of cytokines ( luminex , austin , tx ) in serum revealed remarkably high concentrations of t - cellderived cytokines during recurrence despite withdrawal of ici therapy and initiation of immunosuppressants . 
substratifying by t - helper ( th ) cell subtype revealed that there was an apparent dominance of th1 - cellenriched cytokines over th2 - cellenriched cytokines ( figs 3a and 3b ; data supplement )  . quantication of 92 micrornas ( fluidigm , san francisco , ca ) with known inammatory and cardiac roles revealed two micrornas ( mir - 130a - 3p and mir - 122 - 5 ) that displayed consistent upregulation and downregulation , respectively , in parallel with circulating cytokine abundance . 
increases in mir - 130a - 3p ( figs 3b and 3c ) appeared to correlate with declines in interleukin ( il ) - 4 , 12 whereas decreases in mir122 - 5p , a noted negative regulator of il - 1 , appeared to be associated with elevations in il - 1 ( figs 3a and 3c ) .13 principal component and pathway analysis of differentially regulated micrornas ( cross - timepoint comparison ) revealed modulation during treatment course ( data supplement )  . to assess the downstream effects of a potentially aberrant th1 cell response , we examined antigen - bound serum autoantibodies . 
it was noteworthy that the patient experienced hypothyroidism after receiving ici as we identied antithyroglobulin autoantibody across all sample collection points ( roche electrochemiluminescent assay ) ( data supplement )  . 
a mass spectrometry - based assay14 - 16 to identify serum autoantibodies complexed with their cognate antigens detected thirteen autoantibodies related to cardiac and coagulative pathway ( fig 3d ; data supplement )  . 
by contrast , the recurrent presentation , although displaying low ejection fraction and elevated bnp ( c and d ) , did not display markedly elevated troponin levels ( b )  . 
follow - up ecg after initial presentation shows persistence of st elevation and development of low - voltage ecg ( b )  . ecg one month after initial presentation shows low - voltage ecg and development of right bundle branch block ( rbbb , c )  . 
myocardial biopsy ( e ) demonstrated mixed inammation ( yellow arrow ) with some associated myocyte damage ( orange arrow ) and some healing injury ( green star ) near relatively uninvolved myocardium ( cyan star ) ; higher - power image is also shown ( f ) ; hematoxylin and eosin staining ; digital acquisition ; scale bars as shown = 100 m . immunohistochemistry demonstrated that most of this inltrate was composed of cd3positive t cells ( g , stained cells in brown ) and cd68 - positive macrophages ( h , stained cells in brown )  . 
avr , augmented vector right ; avl , augmented vector left ; avf , augmented vector foot . cmr showing the presence of severe biventricular dysfunction , mild pericardial effusion , and a short axis late gadolinium enhancement ( lge ) stack showing lge in a subepicardial distribution involving the basal and apical segments ( admission )  . although iraes often develop within the rst few weeks to months after initiation of treatment , these events can present at any time , including after cessation of ici therapy . 
luminex - based analysis of circulating inammatory cytokines revealed consistently elevated responses enriched in th1 cell cytokines ( a ) in contrast to th2 cell cytokines ( b )  . two mirs related to cardiac function and inammation displayed consistent trends in expression with mir - 130 - 3p trending upward and mir - 122 - 5p trending downward ( c )  . 
serum autoantibody complexes were observed consistently in the programmed cell death protein - 1 ligand 1induced fulminant myocarditis patient compared to ageand sex - matched control patients ( n = 7 ) , and a patient with myocarditis from systemic lupus erythematosus ( n = 1 ; d )  . serum autoantibody complex data are displayed as relative expression ( average protein intensity ) with data presented as the mean 6 the standard deviation . 
 case report surveillance after an episode of myocarditis , and the necessity for rapid and effective immunosuppression.4 treatment with high - dose glucocorticoids and mmf appeared to improve symptomology ; however , it may have failed to affect the underlying etiology as bnp levels remained marginally abnormal while high levels of inammatory cytokines and autoantibodies persisted . 
it is possible that in addition to generating autoreactive t cells against cardiac epitopes ( a proposed cause of ici - mediated myocarditis ) , icis may also induce th1 - dependent pathogenic autoantibody production ( data supplement )  . patients presenting with symptoms suggestive of ici - related myocarditis present a signicant diagnostic challenge . cardiac troponins can be negative in cases of myocarditis.20 indeed , in this case , troponins were only intermittently and mildly elevated during myocarditis recurrence . 
the diagnostic value of echocardiography and ecg is often limited , with ndings suggestive of myocarditis either absent initially or lacking specicity.4 , 6 the addition of cmr may be to detect benecial owing to its potential inammation , edema , and brosis within myocardial tissue . 
however , lge alone has low sensitivity and accuracy in diagnosing chronic or recurrent myocarditis , and cmr is difcult in hemodynamically unstable patients.21 although the recovery of lv function and resolution of symptoms followed by a separate recurrence of symptoms consistent with acute myocarditis is suggestive of a distinct myocarditis event , one other possibility could include the persistence of subclinical inammation between episodes , leading to smouldering myocarditis and subsequent heart raises awareness that serious iraes can occur after ici discontinuation , and without re - exposure , reinforcing the importance of cardiovascular surveillance after an episode of ici - associated myocarditis along with detailed diagnostic work - ups . 
fish provision of study materials or patients : nazanin aghel , paaladinesh thavendiranathan collection and assembly of data : nazanin aghel , dakota gustafson , ashley di meo , milena music , michael a . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . support preparation of this paper used the resources of the toronto general hospital research institute , university health network , toronto , canada . 
hansen consulting or advisory role : merck , glaxosmithkline , bristol - myers squibb , eisai research funding : karyopharm therapeutics , merck , bristol - myers squibb , boehringer ingelheim , glaxosmithkline , roche / genentech , janssen , astrazeneca / medimmune , astellas pharma , macrogenics paaladinesh thavendiranathan honoraria : amgen no other potential conicts of interest were reported . acknowledgment the authors would like to thank the peter munk cardiac centre cardiovascular biobank for the assistance provided with the clinical sample storage , maintenance , and retrieval . references antonia sj , villegas a , daniel d , et al : durvalumab after chemoradiotherapy in stage iii non - small - cell lung cancer . 
n engl j med 377 : 1919 - 1929 , 2017 larkin j , chiarion - sileni v , gonzalez r , et al : combined nivolumab and ipilimumab or monotherapy in untreated melanoma . 
n engl j med 373 : 23 - 34 , 2015 puzanov i , diab a , abdallah k , et al : managing toxicities associated with immune checkpoint inhibitors : consensus recommendations from the society for immunotherapy of cancer ( sitc ) toxicity management working group . 
j am coll cardiol 71 : 1755 - 1764 , 2018 l aubli h , balmelli c , bossard m , et al : acute heart failure due to autoimmune myocarditis under pembrolizumab treatment for metastatic melanoma . j immunother cancer 3 : 11 , 2015 8 . 
mir h , alhussein m , alrashidi s , et al : cardiac complications associated with checkpoint inhibition : a systematic review of the literature in an important emerging area . 
hsu sh , wang b , kota j , et al : essential metabolic , anti - inammatory , and anti - tumorigenic functions of mir - 122 in liver . 
ohyama k , yoshimi h , aibara n , et al : immune complexome analysis reveals the specic and frequent presence of immune complex antigens in lung cancer patients : a pilot study . 
recent work has determined that 20% to 30% of mcrpc tumors harbor alterations that result in the loss of dna repair capacity , in particular , the homologous recombination pathway.1 , 2 in turn , these alterations may predict response to poly - adp ribosylase ( parp ) inhibitors and platinum chemotherapy , which provides opportunities for precision therapy using agents that are not currently standard or available for mcrpc.3 , 4 in ovarian canceralso frequently marked by homologous recombination deficiencyplatinum is the standard front - line treatment , and parp inhibitors olaparib , niraparib , and rucaparib are now us food and drug administrationapproved for brca1 / 2mutated tumors . 
reversion mutations inbrca1 / 2 , 5 - 8 and more recently in rad51c and rad51d , 9 have previously been described as a mechanism of resistance to platinum and parp inhibitors in ovarian cancer and thus also represent an important consideration in patients with prostate cancer . recently , reversion mutations to restore brca1 / 2 function have been observed in prostate tumors sampled by biopsy and in circulating tumor dna ( ctdna ) from patients who were treated with parp inhibitors.10 , 11 here , we describe a case of a patient with mcrpc who exhibited polyclonal brca2 reversion mutations at the time of disease progression when receiving platinum chemotherapy . 
to our knowledge , this is the first report of brca2 reversions occurring in a patient with prostate cancer in the setting of platinum treatment and mediating resistance to therapy . our patient presented with a prostate - specific antigen ( psa ) of 8.6 ng / ml and gleason 4 + 5 prostate adenocarcinoma in october 2011 at 65 years of age . 
he underwent a radical prostatectomy with bilateral pelvic lymph node dissection in january 2012 with pt3br1n1 ( american joint committee on cancer 7 ) , gleason 5 + 4 staging . 
postoperative psa in april 2012 was 2.7 ng / ml ; restaging scans demonstrated bone involvement in the left iliac wing and l4 vertebral body without lymph node or visceral involvement . he was initially treated with bicalutamide and leuprolide ( a luteinizing hormonereleasing hormone agonist ) , which resulted in undetectable psa by may 2013 . 
in june 2016 , he began treatment with enzalutamide 160 mg once daily , but was noted to have primary refractory disease with a psa increase to 370.3 ng / ml in 1 month . 
black arrow denotes time of circulating tumor dna ( ctdna ) collection . in october 2016 , carboplatin area under the curve 5 was administered based on the results of the metastatic tumor biopsy sequencing . 
after seven cycles , psa increased to 428.1 ng / ml on april 12 , 2017 , concurrent with ctdna draw . samples from the primary prostate tumor , germline dna , and a ctdna sample that was obtained at the time of progression on carboplatin ( march 2017 ) were sequenced by using a clinically validated tumor sequencing next - generation sequencing assay12 and compared with the prechemotherapy metastasis biopsy . 
a frameshift mutation in brca2 ( p.n2452mfs * 17 ) as well as in spop and tp53 mutations were identified in the primary tumor and metastasis ( table 1 )  . 
we did not observe evidence of a germline pathogenic variant . we performed targeted next - generation sequencing on ctdna at the time of progression to 1 , 6103 mean coverage , and identified 90 brca2 reads that carried the frameshift mutation and 1 , 181 wild - type brca2 reads . 
of importance , no reversion mutations were detected in the primary or germline samples . secondary somatic mutations of brca1 / 2 mutations that restore the wild - type open reading frame were initially described as a mechanism of resistance to platinum chemotherapy in ovarian cancer and have subsequently been reported in patients with prostate cancer who were treated with parp inhibitors.5 , 10 , 11 to our knowledge , this is the first example of reversion mutations in the ctdna of a patient with prostate cancer associated with resistance to platinum chemotherapy . our finding provides evidence that polyclonal reversion is a mechanism of resistance to platinum chemotherapy in patients with prostate cancer . prospective clinical trials of parp inhibitors and platinum to exploit the molecular vulnerabilities of prostate cancer with dna repair defects are now underway , and although there is great enthusiasm , resistance to these strategies is expected . we previously described a series of patients with prostate cancer with biallelic brca2 inactivation who had exceptional and sometimes extended responses to carboplatin - based therapy ( up to 3 years ) .4 in addition , a recent retrospective analysis of patients whose prostate cancer was refractory to taxanes found that six ( 75% ) of eight patients who were carriers of pathogenic germline brca2 variants had a psa50 response from 12 weeks of carboplatin plus docetaxel therapy compared with 23 ( 17% ) of 133 noncarriers.15 two prospective clinical trials that are investigating carboplatin table 1 . 
dashed outline represents second allele . sequencing reads with secondary mutations in the plasma circulating tumor dna ( ctdna ) sample taken at the time of progression on carboplatin ( bottom )  . 
images were rendered by using the integrative genomics viewer.13 , 14 with docetaxel for patients with mcrpc and dna repair defects are currently recruiting patients ( clinicaltrials.gov identifiers : nct02598895 and nct02985021 ) and may be opportunities to explore this and other potential resistance mechanisms . the recent finding of secondary reversion mutations in patients with prostate cancer after parp inhibitor therapy occurred within 8 to 10 months of initiating therapy.10 , 11 in our patient , resistance emerged after the same apparent mechanism over approximately 7 months , a more abbreviated benefit compared with the multiple years of response to platinum - based therapy that we have observed and on which we have previously reported.4 our patients case provides evidence that reversion mutations selected by platinum chemotherapies can occur at a similar pace as parp inhibitors and implies that additional factors that are yet to be characterized influence the chronology of resistance to dna - damaging agents . 
although we did not detect a second inactivating mutation in our patient , it is possible that an undetected methylation or a complex rearrangement could be present . nonetheless , evidence of reversion mutations that restore gene function and that temporally coincide with clinical progression during platinum therapy together make a strong case that brca2 gene function was disrupted in the primary and metastatic tumor . it is possible that the mechanism of initial gene inactivation affects resistance . 
for example , patients with biallelic copy loss may be less likely to restore functional copy and thus have a longer response duration compared with those with biallelic singlenucleotide substitutions and / or indels , which might restore gene function more readily to give rise to resistance . 
in addition , although platinum and parp inhibitors share reversion as a mechanism of resistance , patients with ovarian cancer resistant to parp inhibitors can still exhibit sensitivity to platinum , which demonstrates that there are differences as well.16 many unanswered questions remain regarding the noninvasive assessment of resistance . 
 only been identified after selective treatment pressure and not as de novo events , although the lack of detection may reflect mutation levels that are lower than the sensitivity of assays used to detect reversion events . 
in this case , we did not have matched tumor biopsy and ctdna for each clinically relevant time point , although the samples we collected demonstrated proof of an important mechanism of resistance to platinumany in the field are investigating concordance and representative sampling of tumor versus ctdna , which will be important to clinical practice . another critical issue is whether early reversion clones specifically and reliably predict the development and pace of clinical resistance , the differences between platinum and parp inhibitors , and what other potential relevant genetic , epigenetic , and microenvironmental modifying factors are at play . 
nelson consulting or advisory role : janssen oncology , astellas pharma , genentech bruce montgomery research funding : essa pharma , medivation , astellas pharma , janssen oncology , ferring pharmaceuticals colin c . 
pritchard no relationship to disclose acknowledgment we thank arul chinnaiyan , daniel robinson , hiep nguyen , and the stand up to cancer / prostate cancer foundation precision oncology team for their assistance with the collection and analysis of the metastatic biopsy . affiliations support references 417 - 425 , 2017 1697 - 1708 , 2015 all authors , university of washington ; heather h . 
kondrashova o , nguyen m , shield - artin k , et al : secondary somatic mutations restoring rad51c and rad51d associated with acquired resistance to the parp inhibitor rucaparib in high - grade ovarian carcinoma . 
quigley d , alumkal jj , wyatt aw , et al : analysis of circulating cell - free dna identifies multiclonal heterogeneity of brca2 reversion mutations associated with resistance to parp inhibitors . 
pritchard cc , salipante sj , koehler k , et al : validation and implementation of targeted capture and sequencing for the detection of actionable mutation , copy number variation , and gene rearrangement in clinical cancer specimens . j mol diagn 16 : 56 - 67 , 2014 13 . 
ang je , gourley c , powell cb , et al : efficacy of chemotherapy in brca1 / 2 mutation carrier ovarian cancer in the setting of parp inhibitor resistance : a multi - institutional study . 
 c brca2 - associated prostate cancer in a patient with spinal and bulbar muscular atrophy introduction spinal and bulbar muscular atrophy ( sbma ) , or kennedys disease , is a rare x - linked recessive disorder characterized by expanded cag trinucleotide repeats involving exon 1 of the androgen receptor ( ar ) gene and a variable adult onset of muscle weakness , atrophy , fasciculation , tremor , and hyporeflexia or areflexia involving the proximal limb and bulbar musculature , with usually a slowly progressive course , and findings of androgen insensitivity , including gynecomastia , impotence , reduced fertility from oligospermia , testicular atrophy , and normal or increased testosterone levels , which may precede the neuromuscular symptoms by a decade.1 , 2 because cag repeat length is inversely correlated with the transactivation function of the ar , a higher number of cags has been associated with a much lower incidence of developing prostate cancer.3 , 4 ar signaling is a critical driver of prostate cancer development and progression . 
during this time , his initial prostate biopsy was re - reviewed and classified as mixed gleason 5 + 5 adenocarcinoma and small - cell carcinoma ( fig 2a )  . 
on the basis of re - reading his pathology and his presentation with aggressive disease ( which included liver metastases ) , carboplatin chemotherapy was added to the last four cycles of docetaxel . 
 re - evaluation after chemotherapy showed a persistent undetectable psa , decreased size of liver lesions , and reduced enhancement of several of the bone lesions . during this follow - up time , the patient gave his informed consent to participate in molecular studies ( institutional review board #1305013903 )  . 
 capillary electrophoresis was performed to calculate the size of the ar gene trinucleotide repeat , and it confirmed a genetic diagnosis of sbma with approximately 49 repeats in both germline and tumor dna ( fig 2b )  . 
gene expression and protein analysis5 , 6 revealed relatively high ar as well as expression of ar - target genes such as psa ( klk3 ) and tmprss2 ( fig 2c ; appendix figs a1 and a2 )  . targeted deep sequencing7 was performed of both his tumor and germline dna . 
pritchard himisha beltran author affiliations and support information ( if applicable ) appear at the end of this article . corresponding author : himisha beltran , md , joan and sanford i . 
his serum psa decreased approximately 75% at 1 month after initiating talozoparib ( last psa , 0.5 ng / ml )  . discussion prostate cancer arises as an androgen - driven disease . 
even in castration - resistant prostate cancer , the ar remains a key driver.9 this has been the basis for the clinical use of potent ar targeted drugs ( eg , abiraterone and enzalutamide )  . prostate cancer risk and outcomes have been associated with serum androgen levels and polymorphisms involving the ar gene . 
loh favored variation type frameshift insertion nonframeshift deletion large deletion variation type frameshift insertion single copy loss on chr13 in a region that includes brca2 and 10 11 12 13141516 17 18 19202122 x fig 2 . 
 ( a ) prostate biopsy hematoxylin and eosin stained photomicrographs showing mixed gleason 5 + 5 prostate cancer adenocarcinoma with neuroendocrine differentiation ( top panel ) and small - cell carcinoma ( bottom panel )  . 
 ( c ) expression of ar signaling genes ( klk3 , tmprss2 ) was performed by using a nanostring assay with custom probe sets previously used for studies of primary prostate cancer , castration - resistant prostate cancer , and neuroendocrine prostate cancer formalin - fixed paraffin - embedded biopsy tissue.5 , 6 horizontal bars represent median values . 
 ( d ) genomic landscape identified by tumor sequencing displayed germline brca2 pathogenic frameshift mutation ( c.3975_3978dup ) , with clear loss of heterozygosity ( loh ) in the tumor . 
additional somatic ( tumor ) mutations were detected in the patients tumor , including an foxa1 inframe deletion and tp53 mutation as well as copy number alterations involving myc , rb1 , and brca2 genes . 
this occurs after the association of ar with its ligand , which results in the exposition of the polyglutamine tract eliciting an interaction with another polyglutamine ar or the production of aberrant ar conformational modifications.11 , 16 , 17 this knowledge has led to the investigation of adt in these individuals , suggesting that long - term adt might delay functional decline , 18 , 19 although our patient did not experience a significant improvement in his sbma symptoms after the initiation of adt . 
molecular pathogenesis of spinal and bulbar muscular atrophy ( sbma ) and potential risk of prostate cancer associated with altered polyglutamine ( polyq ) chain length in the androgen receptor ( ar )  . 
the ar contains a c - terminal ligand - binding domain ( lbd ) , a central dna - binding domain ( dbd ) that attaches to androgen - responsive target genes , and an n - terminal domain ( ntd ) that influences transcription efficiency . 
exon 1 of the ar gene contains a polymorphic sequence of cag repeats , which usually varies in number up to 40 ( normal ) , and encodes polyglutamine stretches of ar transactivation doma under normal conditions , the ar first interacts with its ligand dihydrotestosterone ( dht ) and then migrates to the cell nucleus , where it interacts with specific dna sequences inducing transcription of androgen target genes , including klk3 ( prostate - specific antigen [ psa ] ) and other genes . 
 polyglutamine ar ( polyq - ar ) also migrates to the nucleus after binding with testosterone , and once it is in the nucleus , it can no longer interact properly with ar target genes . 
 damage to motor neurons can result both from cell - autonomous and noncell - autonomous toxic processes , the latter arising in other cell types such as skeletal muscle cells . 
in the age of precision medicine , the molecular profile of our patients tumor also highlights critical opportunities to translate genomics into clinical care . although ar was expressed , it is likely that this ar - expressing tumor was less dependent on ar signaling . 
consistent with this , this patients tumor exhibited alterations in both rb1 and tp53 , genes previously associated with the development of nonar - driven prostate cancer , lineage plasticity , and the neuroendocrine prostate cancer phenotype.5 , 24 - 26 this might also explain the patients lack of durable response to enzalutamide and the observed mixed small - cell morphologic features of his tumor at diagnosis . 
 in addition , his chemotherapy responsiveness is a clinical hallmark of a recognizable subset , aggressive variant of prostate cancer , which shares clinical and molecular features with small - cell carcinomas.27 , 28 additional mechanisms that may be the drivers of disease initiation beyond the ar in this case include loss of dna repair through the presence of a pathogenetic brca2 mutation and tumor loss of heterozygosity , or they could be biologically interconnected , as shown in studies of breast cancer in which the length of the ar - cag repeat affected the timing of cancer diagnosis in brca1 mutation carriers , likely through modulation of hormonal responses of mammary epithelial cells.29 recent studies showed a relatively high incidence of both germline and somatic alterations involving dna repair genes such as brca2 in patients with advanced prostate cancer.30 - 32 these alterations likely play a critical role in the pathogenesis of more aggressive prostate cancers , leading to an increased probability of the development of lethal disease . 
a germline brca2 mutation confers an increased risk of developing prostate cancer ( 8.6 - fold in men age 65 years or younger ) 33 and has been demonstrated to be an independent poor prognostic factor associated with higher rates of developing metastatic disease and shorter survival.34 in patients with metastatic castration - resistant prostate cancer treated with standard therapies , evidence on the prognostic and predictive role of dna repair alterations is still conflicting , 35 - 37 although brca2 carriers have been reported to have poorer outcomes overall compared with those who do not carry brca2.38 in this patient , the presence of a brca2 mutation may have trumped or bypassed the need for high ar signaling during the development of prostate cancer . 
the influence of dna repair alterations on the development of small - cell neuroendocrine prostate cancer and therapy response also warrants further study . notably , the germline brca2 mutation was not suspected on the basis of family history ; thus , it had important implications for his family members ( ie , cancer screening ) and helped in selecting therapy for hi tumor cells that harbor dna repair gene alterations are more susceptible to drugs that induce dna damage such as platinum - based chemotherapy39 , 40 or those that impair alternative dna repair pathways , 30 as shown in our patient ( who had a response to platinum and is currently responding to parp inhibitor therapy )  . 
it may also be informative to re - evaluate this patient at the time of progression on telazoparib for acquired brca2 reversion41 , 42 or other mechanisms of resistance , given the multiple drivers present in this case . our case outlines the striking clinical course and favorable treatment response of a patient with a rare genetic neuromuscular disease that harbors clinical and molecular features of an aggressive variant of prostate cancer treated with a precision oncology approach . 
a germline brca2 mutation likely overcame the need for high ar signaling ( evidenced by sbma as well as the small - cell features ) , and the acquisition of tp53 and rb1 alterations further supported non - ar driven disease , contributing to its clinical aggressiveness and lack of durable response to enzalutamide . 
pritchard , himisha beltran manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors michael sigouros no relationship to disclose andrea sboner no relationship to disclose colin c . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . himisha beltran consulting or advisory role : janssen oncology , sanofi , genzyme , glaxosmithkline , abbvie research funding : astellas pharma ( inst ) , janssen oncology ( inst ) , abbvie / stemcentrx , eli lilly ( inst ) , millennium pharmaceuticals ( inst ) affiliations vincenza conteduca , michael sigouros , andrea sboner , and himisha beltran , weill cornell medicine ; andrea sboner and himisha beltran , weill cornell medicine - new york presbyterian hospital , new york , ny ; vincenza conteduca , istituto scientifico romagnolo per lo studio e la cura dei tumori , istituto di ricovero e cura a carattere scientifico , meldola , italy ; and colin c . 
kosaka t , miyajima a , kikuchi e , et al : a case of spinal and bulbar muscular atrophy with highstage and high - gleason score prostate cancer responded to maximal androgen blockade therapy . 
beltran h , wyatt aw , chedgy ec , et al : impact of therapy on genomics and transcriptomics in high - risk prostate cancer treated with neoadjuvant docetaxel and androgen deprivation therapy . 
pritchard cc , salipante sj , koehler k , et al : validation and implementation of targeted capture and sequencing for the detection of actionable mutation , copy number variation , and gene rearrangement in clinical cancer specimens . 
freedman ml , pearce cl , penney kl , et al : systematic evaluation of genetic variation at the androgen receptor locus and risk of prostate cancer in a multiethnic cohort study . 
lindstrm s , zheng sl , wiklund f , et al : systematic replication study of reported genetic associations in prostate cancer : strong support for genetic variation in the androgen pathway . 
kumar r , atamna h , zakharov mn , et al : role of the androgen receptor cag repeat polymorphism in prostate cancer , and spinal and bulbar muscular atrophy . 
hashizume a , katsuno m , suzuki k , et al : long - term treatment with leuprorelin for spinal and bulbar muscular atrophy : natural history - controlled study . 
yasui t , akita h , itoh y , et al : cag repeats in the androgen receptor : a case of spinal and bulbar muscular atrophy associated with prostate cancer . 
kote - jarai z , leongamornlert d , saunders e , et al : brca2 is a moderate penetrance gene contributing to young - onset prostate cancer : implications for genetic testing in prostate cancer patients . 
castro e , goh c , olmos d , et al : germline brca mutations are associated with higher risk of nodal involvement , distant metastasis , and poor survival outcomes in prostate cancer . 
mateo j , cheng hh , beltran h , et al : clinical outcome of prostate cancer patients with germline dna repair mutations : retrospective analysis from an international study . 
antonarakis es , lu c , luber b , et al : germline dna - repair gene mutations and outcomes in men with metastatic castration - resistant prostate cancer receiving first - line abiraterone and enzalutamide . 
castro marcos e , romero laorden n , piulats rodriguez jm , et al : prorepair - b : a prospective cohort study of dna repair defects in metastatic castration resistant prostate cancer ( mcrpc )  . 
pomerantz mm , spisk s , jia l , et al : the association between germline brca2 variants and sensitivity to platinum - based chemotherapy among men with metastatic prostate cancer . 
quigley d , alumkal jj , wyatt aw , et al : analysis of circulating cell - free dna identifies multiclonal heterogeneity of brca2 reversion mutations associated with resistance to parp inhibitors . 
jonkers j , meuwissen r , van der gulden h , et al : synergistic tumor suppressor activity of brca2 and p53 in a conditional mouse model for breast cancer . 
gene expression was performed by using the nanostring ncounter assay with custom probe sets previously used for studies of primary prostate cancer ( pca ) , castration - resistant prostate cancer ( crpc ) , and neuroendocrine prostate cancer ( nepc ) using formalin fixed paraffin - embedded tissue . 
henick , md1 ; matthew ingham , md1 ; maryam shirazi , md2 ; charles marboe , md2 ; andrew turk , md2 ; susan hsiao , md , phd2 ; and mahesh m . 
intervening surveillance scans were unremarkable ; however , the patient experienced a 10 - pound weight loss over 2 months that culminated in acute - onset exertional dyspnea and chest pain , prompting admission . 
the patient was evaluated by cardiothoracic surgery and deemed not to be a surgical candidate . positron emission tomography ( pet ) ct conrmed uorodeoxyglucose avidity in the hypermetabolic mass , scattered left lung nodules , and an intralumina soft tissue mass in the distal colon proximal to the anastomosis as well as a right thyroid lesion ( fig 1 )  . 
restaging ct of the chest after 2 cycles revealed decreasing peripheral opacities in the left upper lobe , a decrease in irregular left - sided lung nodules , and a decrease in the extent of tumor thrombus , with an increase in patency of the left pulmonary artery and interval resolution of cavitation within the tumor thrombus . 
in addition , the vaf of 67% indicated loss of heterozygosity in the tumor , raising the possibility of a germline alteration ( germline testing has not been performed )  . 
because the msi status was indeterminate by ngs , msi testing by polymerase chain reaction ( pcr ) fragment analysis using ve quasimonomorphic mononucleotide repeats ( msi analysis system , version 1.2 ; promega , madison , wi ) was performed but was indeterminate , because normal dna for comparison was unavailable . 
using dna extracted from peripheral blood for comparison , msi with an altered allele at four of ve tested loci ( nr - 21 ; bat - 26 ; bat - 25 ; mono - 27 ; fig 3 ) was detected . 
positron emission tomography / computed tomography results ( a ) at baseline , ( b ) after treatment with doxorubicin plus olaratumab before pembrolizumab administration , and ( c ) after 8 cycles of pembrolizumab . with chemotherapy , the patient was amenable to treatment with pembrolizumab and began therapy . 
after 8 cycles of treatment , intervening pet - ct results have shown an additional decrease in the extravascular component of the pulmonary artery tumor with evolving dystrophic calcication , no evidence of tumor thrombus , stable ndings in the lungs likely consistent with pulmonary infarcts , an avid hypermetabolic right thyroid nodule , and a decrease in soft tissue nodularity at the suture line . discussion intimal sarcoma of the pulmonary artery is a rare tumor ( incidence , approximately 0.001%2 , 3 ) , and the scant existing medical literature highlights individual cases of response to various combinations of surgery , 4 chemotherapy , 5 and radiation.6 the molecular landscape of such tumors is not well characterized . studies of immune checkpoint blockade have demonstrated mixed results in the treatment of sarcomas . 
the phase ii alliance a091401 study examined nivolumab monotherapy and nivolumab plus ipiliumumab in the treatment of metastatic sarcoma.7 two of 38 patients experienced conrmed responses with nivolumab treatment , and six of 38 experienced responses to the combination.7 in a single - arm , phase ii study of pembrolizumab in soft tissue sarcoma or bone sarcoma cohorts , the overall response endpoint was not met , though seven of 40 patients with soft tissue sarcoma and two of 10 patients with liposarcoma experienced responses.8 preliminary activity was seen ( 3 - month progression - free survival , 72.7% ) with the combination of axitinib and pembrolizumab in a single - arm , phase ii study in alveolar soft - palate sarcomas.9 on may 23 , 2017 , the us food and drug administration ( fda ) approved pembrolizumab for the second - line treatment fig 2 . 
penta c and penta d are polymorphic pentanucleotide markers that show identity between the tumor and normal samples . of msi - high ( msi - h ) or mmr - decient ( dmmr ) solid tumors on the basis of ndings in patients retrospectively identied ( keynote - 012 , - 028 , and - 158 ) and prospectively enrolled ( keynote - 016 , - 158 ) .10 , 11 in these trials , msi status was identied prospectively by local laboratory - developed pcr ( n = 60 of 149 patients ) , ihc ( n = 47 of 149 ) , or both ( n = 42 of 149 ) ; retrospectively , 14 of 149 patients were identied by a central laboratorydeveloped pcr test.12 in practice , dmmr status is established by ihc for expression of mmr proteins ( mlh1 , msh2 , msh6 , and pms2 ) , and msi - h is demonstrated by multiplexed pcr of either the national cancer institute panel of two mononucleotide and three dinucleotide the more widely used panel of ve quasimarkers or monomorphic mononucleotide markers ( promega , madison , wi )  . 
at columbia university irving medical center , both ihc and pcr are used , as either can miss individual cases.13 in addition , large ngs panels , such as the one performed in the case , can be used to evaluate for msi . 
the cccp assay interrogates 8 , 682 monoand di - nucleotide repeat loci a 1.27 - mb region of interest and classies tumors as microsatellite stable or unstable according to the number of altered microsatellite loci.14 cases that cannot be denitively classied as microsatellite stable or unstable by the cccp assay are rare and were not seen in the more than 200 cases used in validation , which encompassed a range of tumor types . although the efcacy of programmed death 1 blockade in microsatellite - unstable tumors is well established , 10 , 11 , 15 to our knowledge this is the rst report of a clinical outcome of an intimal sarcoma associated with treatment with immune checkpoint blockade . 
we present this case in part for that reason but also to highlight challenges in executing the mmr - deciency testing that is critical to identify patients who may benet this fdaapproved indication ( table 1 )  . 
assays to evaluate microsatellite instability / mismatch repair deciency at columbia university irving medical center technique pcr fragment analysis what is five quasi - monomorphic mononucleotide evaluated ? repeats ( promega msi evaluation system ; promega , madison , wi ) indel variant detection in microsatellite repeat tracts ( columbia combined cancer panel , a 468 - gene ngs panel ) loss of expression of mlh1 , mlh2 , msh6 , pms2 in tumor cells limitations ? tumor percentage ; tissue type ( eg , lower sensitivity for endometrial or prostate cancers16 ; cancers in constitutional mismatch repair deciency17 ) can affect sensitivity . 
small biopsies , reactive non - neoplastic cells expressing mmr proteins , and loss - of - function variants with retained epitopes can cause false - normal ihc results , while msi testing in noncolorectal cancers may be more difcult to interpret.18 despite their greater overall sensitivity , individual results of ngs panels may be affected by heterogeneity in rates of alterations of individual repeats across tumor types , 19 especially in noncolorectal and endometrial cancers , as highlighted by this case and in a survey of 15 , 045 tumors in which lynch syndrome was identied in 1.6% ( 13 of 699 ) of msi - indeterminate tumors , predominantly noncolon / endometrial cancers ( 10 of 13 ) all of which showed abnormal ihc ( 10 of 10 tested cases ) .20 furthermore , commonly used markers ( by the national cancer institute and promega ) are not among the most frequently altered repeats in the cancer genome atlas exome data , 21 implying that that tests that use limited numbers of markers yield false negatives . 
therefore , when faced with advanced cancers with few options , multimodality testing for mmr deciency may be warranted . affiliations 1department of medicine , medical oncology , columbia university irving medical center , new york city , ny 2department of pathology , molecular pathology , columbia university irving medical center , new york city , ny subject matter of this manuscript . 
henick stock and other ownership interests : abbvie consulting or advisory role : boehringer ingelheim matthew ingham consulting or advisory role : daiichi sankyo research funding : apexigen ( inst ) , mirati therapeutics ( inst ) , ptc therapeutics ( inst ) travel , accommodations , expenses : mirati therapeutics , genentech andrew turk employment : ventana medical systems , bristol - myers squibb , medarex , bayer consulting or advisory role : bayer , bristol - myers squibb , ventana medical systems susan hsiao honoraria : medscape , illumina consulting or advisory role : ventana medical systems , bristol - myers squibb , loxo research funding : bristol - myers squibb mahesh m . 
mansukhani travel , accommodations , expenses : bristol - myers squibb , promega , er squibb sons no other potential conicts of interest were reported . references colmbia university department of pathology and cell biology : columbia combined cancer panel ( cccp )  . 
specialties / personalized - genomic - medicine / oncology - testing / columbia - combined - cancer - panel choi ey , yoon yw , kwon hm , et al : a case of pulmonary artery intimal sarcoma diagnosed with multislice ct scan with 3d reconstruction . 
yonsei med j 45 : 547 - 551 , 2004 alsou b , slater m , smith pp , et al : pulmonary artery sarcoma mimicking massive pulmonary embolus : a case report . 
asian cardiovasc thorac ann 14 : e71 - e73 , 2006 akomea - agyin c , dussek je , anderson dr , et al : pulmonary artery sarcoma mimicking pulmonary embolism : successful surgical intervention . 
ann thorac surg 61 : 1536 - 1538 , 1996 cantaloube m , moureau - zabotto l , mescam l , et al : metastatic intimal sarcoma of the pulmonary artery sensitive to carboplatin - vinorelbine chemotherapy : case report and literature review . 
case rep oncol 11 : 21 - 28 , 2018 long hq , qin q , xie ch : response of pulmonary artery intimal sarcoma to surgery , radiotherapy and chemotherapy : a case report . 
j med case reports 2 : 217 , 2008 dangelo sp , mahoney mr , van tine ba , et al : nivolumab with or without ipilimumab treatment for metastatic sarcoma ( alliance a091401 ) : two open - label , non - comparative , randomised , phase 2 trials . 
lancet oncol 19 : 416 - 426 , 2018 tawbi ha , burgess m , bolejack v , et al : pembrolizumab in advanced soft - tissue sarcoma and bone sarcoma ( sarc028 ) : a multicentre , two - cohort , single - arm , open - label , phase 2 trial . 
wilky ba , trucco mm , subhawong tk , et al : axitinib plus pembrolizumab in patients with advanced sarcomas including alveolar soft - part sarcoma : a singlecentre , single - arm , phase 2 trial . 
waalkes a , smith n , penewit k , et al : accurate pan - cancer molecular diagnosis of microsatellite instability by single - molecule molecular inversion probe capture and high - throughput sequencing . 
bakry d , aronson m , durno c , et al : genetic and clinical determinants of constitutional mismatch repair deciency syndrome : report from the constitutional mismatch repair deciency consortiueur j cancer 50 : 987 - 996 , 2014 . 
 c active disclosure of secondary germline findings to deceased research participants personal representatives : process and outcomes introduction somatic mutation profiling of tumors is performed in both clinical and research settings , often to identify patients with cancer who are candidates for targeted therapies . 
profiling also can be performed through simultaneous analysis of tumor and normal ( germline ) dna , which allows for identification of mutations unique to the tumor by subtracting germline variants . 
both the american college of medical genetics and genomics ( acmg ) 1 , 2 and asco3 recommend that disclosure of actionable secondary germline findings from tumor - normal paired testing be offered to patients . although these guidelines address clinical testing , they are also a starting point in considering secondary germline findings identified upon research testing . 
we reported our experience with return of secondary germline findings to living patients with advanced cancer who participated in a study of tumor - normal paired research testing.4 by using the acmg gene list2 ( plus palb2 ) as the disclosure threshold , we found that all contacted patients agreed to genetic counseling and testing . given both the poor prognosis of patients with advanced cancer and the long turnaround times of research testing , an unfortunate reality is that secondary germline findings are discovered after the patient has died . 
chan et al5 described disclosure to a decedents family upon the decedents request and advocated for a policy of at least passive disclosure of clinically significant results to relatives who request thepassive disclosure is unlikely in the context of somatic mutation profiling studies because relatives are likely unaware of possible secondary germline findings . wolf et al6 reported consensus recommendations with regard to returning research participants genomicresultstorelatives , includingthatresearchers may return results to relatives but are not obligated to do so , and emphasized the key role of the deceased participants representative ( eg , his or her personal representative [ pr ] as defined by the health insurance portability and accountability act ) as the initial contact and decision maker about disposition of the results . 
amendola et al7 described active disclosure of a germline ( possibly mosaic ) tp53 mutation to a decedents spouse and subsequent testing of relatives . disclosure of secondary findings to families of deceased participants has a potential benefit . 
in our previous studies , participants reported wanting to receive clinically relevant genetic findings.4 , 8 secondary findings can occur in the absence of suggestive personal / family history , which makes it unlikely that relatives would otherwise learn about the mutation . 
in this article , we report our experience with active disclosure of secondary germline findings to deceased participants prs . methods patients with cancer under consideration for genomically informed clinical trials were enrolled in an institutional review board ( irb ) approved protocol for genomic profiling with optional germline testing ( clinicaltrials.gov identifier : nct01772771 )  . 
this study was activated february 2012 ; patients enrolled after october 9 , 2013 , were also offered a companion protocol that ascertained molly daniels chetna wathoo lauren brusco karen h . 
 their preferences for return of secondary results to their pr in the event of their death . to address secondary findings in deceased participants who consented before the companion protocol , we sought advice from irb , legal , and ethics services . 
consensus was reached that the benefit of return of results to families outweighed the risk . the irb - approved revised protocol allowed disclosure of results to families of patients who died before soliciting their preferences . secondary germline findings were identified as previously described.4 we limited results return to pathogenic variants with established clinical utility ( acmg list2 plus palb2 ) not already identified through clinical testing . 
we took a conservative approach of disclosure to the affidavit - verified pr ( fig 1 )  . results we identified 16 secondary findings in decedents ( table 1 )  . 
results indicated a germline pathogenic tp53 mutation , which was disclosed to his wife . case 3 : a 69 - year - old man of partially ashkenazi jewish ancestry with recently diagnosed metastatic pancreatic adenocarcinoma presented for treatment . 
results indicated the germline ashkenazi jewish founder brca2 mutation , which was disclosed to his daughter . consistent with clinical practice and our disclosure practice for living study participants , we did not contact other relatives . 
a relative with no personal history of cancer has subsequently received positive test results for the familial mutation . case 1 : a 58 - year - old woman with recurrent high - grade serous ovarian cancer presented for discussion of phase i trials . 
she died 4 months after study consent . results indicated a germline pathogenic brca1 mutation , which was disclosed to her son . case 2 : a 74 - year - old man with recurrent metastatic melanoma presented for treatment . discussion management of secondary genetic findings in research studies is controversial , even more so when the participant is deceased . 
a relative subsequently underwent predictive genetic testing and can now benefit from more - intensive screening and by taking preventive action . study participant confirmed deceased at time pathogenic germline secondary finding identified . 
study coordinator calls participants next of kin as listed in ehr , up to three times ( n = 16 )  . no response contact made study coordinator mails introductory letter to next of kin ( n = 6 )  . pr declined discussion of secondary finding ( n = 2 )  . next of kin either verbally confirms that he or she is the pr or provides contact information for the pr . 
study coordinator mails affidavit with return envelope to pr ( n = 8 )  . no response to contact , no additional attempts made ( n = 6 )  . affidavit not returned affidavit returned study coordinator calls to confirm that pr received affidavit letter . 
of participants mutation gene brca2 tp53 brca1 palb2 apc * msh6 pten tsc2 i2675dfsx6 , s1764kfsx3 , e1493vfsx10 , s1982rfsx22 r282q , m237i , f270l , d324h v1757a , g1809v g1121vfsx3 , s254ifsx3 g267efsx26 l164rfsx3 l164rfsx3 q1284sfsx39 * germline apc mutation consistent with clinical presentation , but because no documentation indicated that the causative mutation had previously been identified , the participant was considered to not have been already aware of this finding . the participants in case 1 and 3 met established brca1 / brca2 testing criteria ; however , germline testing had not occurred . 
identification of the familial mutation also has the benefit of refining risk assessment ( eg , 5% to 10% chance brca positive if mother had ovarian cancer v 50% chance of brca positive if mother was brca positive ) , and relatives who test negative can be reassured . the participant in case 2 did not meet tp53 testing criteria , thus that the relatives would otherwise have undergone genetic testing is unlikely . we are not certain that this is a true germline mutation because alternate explanations , such as clonal hematopoiesis , 9 have not been ruled out . nonetheless , the testing of relatives is reasonable because relatives who test negative can be reassured . 
if a relative tests positive , then a germline mutation would be confirmed , and the subsequent changes in medical management would be substantial , even in the absence of a significant family history.10 identification and disclosure of secondary germline findings is labor intensive and is even more so when the study participant has died . 
as the field evolves , situations will continue in which information discovered during the normal course of research or clinical care will be significant for relatives in the future , even if not known to be so today . 
one option , particularly for academic medical centers engaged in both clinical care and research , would be to elicit patients global preferences at the front door with regard to disclosure of secondary findings after their death . after initial contact was made , two prs actively declined additional information . 
even if the participant designates a contact person at the time of consent , that does not mean that this person will necessarily be aware of that designation , let alone be welcoming of contact . researchers engaged in results disclosure should continue to be aware of the importance of offering findings in a noncoercive manner . in conclusion , active disclosure of secondary germline research findings to the prs of deceased study participants can be done successfully and ethically and can be of direct benefit to relatives who were unlikely to receive these results any other way . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . molly daniels no relationship to disclose chetna wathoo no relationship to disclose lauren brusco employment : celgene karen h . 
litton consulting or advisory role : novartis , medivation , pfizer research funding : novartis , bristol - myers squibb ( inst ) , medivation , genentech , pfizer patents , royalties , other intellectual property : uptodate travel , accommodations , expenses : medivation karina eterovic no relationship to disclose ugur aytac no relationship to disclose john mendelsohn leadership : merrimack pharmaceuticals stock and other ownership interests : merrimack pharmaceuticals patents , royalties , other intellectual property : royalty payments from the university of california , san diego travel , accommodations , expenses : merck gordon b . 
mills stock and other ownership interests : catena pharmaceuticals , ptv sciences , spindletop ventures , myriad genetics , immunome honoraria : symphogen , nuevolution , astrazeneca , isis pharmaceuticals , eli lilly , novartis , allostery , immunome consulting or advisory role : adventis , astrazeneca , catena pharmaceuticals , critical outcome technologies , millennium pharmaceuticals , nuevolution , precision medicine research associates , provista diagnostics , signalchem , symphogen , allostery , isis pharmaceuticals , medimmune , eli lilly , novartis , tarveda therapeutics , taurx research funding : adelson medical research foundation , astrazeneca , critical outcome technologies , nanostring technologies , komipharm international , the breast cancer research foundation , karus therapeutics , illumina , millennium pharmaceuticals patents , royalties , other intellectual property : homologous recombination deficiency assay to myriad genetics travel , accommodations , expenses : astrazeneca , symphogen , isis pharmaceuticals , medimmune , eli lilly , novartis , immunome , allostery , pfizer ken chen no relationship to disclose funda meric - bernstam honoraria : dialecta , sumitomo group consulting or advisory role : genentech , inflection biosciences , pieris pharmaceuticals , clearlight diagnostics , darwinhealth , grail research funding : novartis , astrazeneca , taiho pharmaceutical , genentech , calithera biosciences , debiopharm group , bayer ag , aileron therapeutics , puma biotechnology , cytomx therapeutics , jounce therapeutics , zymeworks , effective pharmaceuticals , curis , pfizer affiliations all authors : the university of texas md anderson cancer center , houston , tx . support supported in part by the cancer prevention and research institute of texas ( rp150535 ) , sheikh khalifa bin zayed al nahyan institute for personalized cancer therapy grant no . 
national comprehensive cancer network : genetic / familial high - risk assessment : breast and ovarian ( version 2.2017 ) , nccn clinical practice guidelines in oncology ( nccn guidelines ) , 2017 . 
to inform targeted treatment strategies , 3 , 476 clinically advanced prostate tumors were analyzed by cgp for genomic alterations ( gas ) and signatures of genomic instability . methods prostate cancer samples ( 1 , 660 primary site and 1 , 816 metastatic site tumors from unmatched patients ) were prospectively analyzed by cgp ( foundationone assay ; foundation medicine , cambridge , ma ) for gas and genomic signatures ( genome - wide loss of heterozygosity [ gloh ] , microsatellite instability [ msi ] status , tumor mutational burden [ tmb ] )  . results frequently altered genes were tp53 ( 44% ) , pten ( 32% ) , tmprss2 - erg ( 31% ) , and ar ( 23% )  . 
potentially targetable gas were frequently identied in dna repair , phosphatidylinositol 3 - kinase , and ras / raf / mek pathways . dna repair pathway gas included homologous recombination repair ( 23% ) , fanconi anemia ( 5% ) , cdk12 ( 6% ) , and mismatch repair ( 4% ) gas . 
metastatic site tumors were enriched for the 11q13 amplicon ( ccnd1 / fgf19 / fgf4 / fgf3 ) and gas in ar , lyn , myc , ncor1 , pik3cb , and rb1 compared with primary tumors . conclusion routine clinical cgp in the real - world setting identied gas that are investigational biomarkers for targeted therapies in 57% of cases . 
2019 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction genomic alterations ( gas ) that drive prostate cancer have been elucidated by proling primary tumors.1 , 2 comprehensive genomic proling ( cgp ) is increasingly used for routine clinical management of patients with prostate cancer , 3 with accumulating evidence associating gas with responses to therapy . 
 chung et al context key objective in this study , we use real - world data from routine prospective clinical genomic proling to evaluate genomic alterations ( gas ) and genomic signatures in primary and metastatic prostate cancer . knowledge generated we evaluated the landscape of gas in prostate cancer and identied those that are enriched in metastatic site tumors . approximately 3% of cases had high microsatellite instability and / or high tumor mutational burden status . 
genomic signatures , including microsatellite instability , tumor mutational burden , and genome - wide loss of heterozygosity , may further rene biomarker development for poly ( adp - ribose ) polymerase inhibitors and immunotherapies . samples using a validated assay16 ( foundationone ; foundation medicine , cambridge , ma ; appendix table a1 )  . 
the pathologic diagnosis of each case was conrmed on routine hematoxylin and eosinstained slides . results were analyzed for gas and gene signatures ( tmb , msi , genome - wide loss of heterozygosity [ gloh ] )  . 
germline / somatic mutation calls were predicted without a matched normal ; in validation testing of 480 tumor - only sequencing calls against matched normal samples , accuracy was 95% for somatic and 99% for germline calls.17 enrichment was dened as the difference in ga frequency between metastatic and primary sites . 
activating braf or raf1 fusions / rearrangements were observed in 1.2% of cases ( 35 braf and seven raf1 ; appendix fig a2 )  . the pi3k / akt / mammalian target of rapamycin ( mtor ) pathway was frequently altered ( 40.8% ; figs 1b and 1c )  . as expected , pten gas were frequent , and we observed in pik3ca , activating mutations and amplications pik3cb , akt1 / 2 / 3 , mtor , and rictor and loss - offunction alterations in pik3r1 / 2 and tsc1 / 2 . 
ras / raf / mek pathway alterations ( 5.7% ; figs 1b and 1f ) included oncogenic braf mutations ( appendix fig a2 ) , braf / raf1 rearrangements , activating mutations in raf1 / araf , kras / nras / hras and map2k1 / 2 , and nf1 loss - of - function alterations . 
 chung et al cdk12 ga ( p , .001 ) , and braf rearrangements / mutations ( p , .001 ; fig 1b )  . to compare primary and metastatic site tumors , we assessed relative enrichment in gas ( fig 1g ; appendix table a3 )  . 
predicted germline mutations were identied in 57.8% of brca2 - , 25.0% of brca1 - , 35.8% of atm - , 80.0% of chek2 - , 52.2% of fanca - , 42.3% of msh2 - , 20.0% of msh6 - , 25.0% of mlh1 - , and 44.4% of pms2 - mutated cases ; only 9.2% of cdk12 - mutated cases harbored a predicted germline mutation ( fig 2c )  . genomic signatures : gloh in addition to gas in individual dna repair genes , genomic signatures represent the phenotypic readout of deleterious dna repair and are potential predictive biomarkers . 
we evaluated the association between gloh and dna repair gas ( fig 2d )  . consistent with the established relationship between brca1 / 2 and hrd , 21 brca1 / 2 - altered cases ( both predicted germline and somatic mutations ) were more frequently gloh - h compared with the overall data set ( fig 2d )  . to evaluate the potential relevance of non - brca1 / 2 dna repair genes as biomarkers for parp inhibition , we assessed their association with gloh . 
gloh - h frequency was comparable between the overall data set and cases with non - brca1 / 2 dna repair gas , although cases with homozygous deletions in non - brca1 / 2 hrr pathway or fa / icl pathway genes were more frequently gloh - h ( appendix fig a4b )  . 
 ( b ) frequency of major pathway alterations , including ets fusions , braf rearrangements / mutations , spop / cul3 mutations , cdk12 gas , idh1 / 2 mutations , ar gas , phosphatidylinositol 3 - kinase ( pi3k ) pathway gas , homologous recombination gas , g1 / s - cell cycle gas , wnt pathway gas , fanconi anemia / interstrand crosslink repair ( fa / icl ) repair pathway gas , ras / raf / mek pathway gas ( other than braf ) , mismatch repair gas , and pole mutations ( see figs 1c to 1f for details )  . 
 ( c to f ) gas identied in each pathway , including the ( c ) pi3k / akt / mammalian target of rapamycin ( mtor ) pathway , ( d ) g1 / s - cell cycle pathway , ( e ) wnt pathway , and ( f ) ras / raf / mek pathway ( nf1 mutation , rearrangement , or copy number loss ; braf or raf1 mutations or rearrangements ; araf , k / n / h - ras , or map2k1 / 2 mutations were included ) ; percent of altered cases is indicated . 
genes that were enriched ( difference between primary site v metastatic site frequency ) by at least 2% are indicated ( all p , .05 by fishers exact test [ two - tailed ] ) , with genes enriched at least twofold indicated in red ( all p , .001 by fishers exact test [ two - tailed ] )  . 
msi status was assessable for 3 , 326 cases , and overall , 87 of 3 , 326 of cases ( 2.6% ) were msi - h ( 2.0% primary site , 3.1% metastatic site tumors )  . 
for the remaining cases , tmb - h phenotype was not attributed to msi - h or pole . landscape of ets fusions and ar gas in total , 1 , 236 ets fusions were detected ( one sample harbored two ets fusions ; fig 1b )  . 
tmprss2 - erg comprised the majority ( 87.7% ) of ets fusions ( fig 3a ) , with the remaining consisting of etv1 ( 8.5% ) , etv4 ( 2.6% ) , and etv5 ( 1.2% ) with diverse fusion partners , including several not previously described ( fig 3b )  . 
we also identied breakpoints that juxtaposed tmprss2 with the intergenic region upstream of erg ( 0.1 to 75 kb upstream ; fig 3c ) ; similar upstream intergenic breakpoints were observed for tmprss2 fusions with etv4 ( 10 of 32 cases ) and etv5 ( two of 15 cases )  . ar gas are associated with castration - resistant disease and were most enriched in metastatic site tumors ( 39.7% of metastatic site and 3.7% of primary site tumors ; fig 1g )  . 
five hundred fty - ve of 557 cnas were amplications ( median copy number , 24 ; range , six to 366 ) ; two homozygous deletions that encompass exons 5 to 7 or exons 5 to 8 are likely activating.27 ar missense mutations were observed in 6.9% of cases ( 1.3% primary site and 11.9% metastatic site ) , and most were ligandbinding domain antiandrogen resistance mutations ( fig 4b ) .28 finally , ar rearrangements were observed in 1.3% of cases ( 0.4% primary site and 2.2% metastatic site ) ; however , because the sequencing strategy was not explicitly designed to detect ar rearrangements and does not fully cover ar intronic regions , the true frequency of ar rearrangements is difcult to establish . 
gas that co - occur with targetable gas ( fig 1b ) and their impact on response to therapy warrant consideration in biomarker - driven trials . msi - h and tmb - h have been associated with immunotherapy benet , 10 , 24 , 25 and gloh - h has been associated with parp inhibitor benet21 ; therefore , assessment of signatures of genomic instability potentially expands the population of patients addressable with immunotherapy or targeted therapy . we identied a subset of tmprss2 rearrangements fused to upstream intergenic regions of erg , etv4 , or etv5 as well as novel ets fusion partners . 
 cgp of primary and metastatic prostate tumors etv4 ( 32 ; 2.6% ) etv5 ( 15 ; 1.2% ) etv1 ( 105 ; 8.5% ) tmprss2 - erg ( 1 , 084 ; 87.7% ) etv1 etv4 etv5 intron 4 ( n = 38 ) erg gene ( nm_004449 chr21 : 40033704 - 39751950 ) intron 3 ( n = 940 ) intron 1 ( n = 58 ) upstream ( 108 bp - 75 kbp ) ( n = 39 ) intron 2 ( n = 9 ) erg exon 1 genomic coordinates : chr21 : 40020000 40040000 40060000 40080000 40100000 40120000 fig 3 . 
consistent with distinct molecular subsets of prostate cancer , 1 , 8 ets fusions were mutually exclusive with spop / cul3 , cdk12 mutations , or braf rearrangements / mutations that may potentially comprise a clinically relevant genomic subset.30 , 31 diverse ar gas can mediate androgen axis inhibitor resistance28 , 29 and were strongly enriched in metastatic site tumors . 
ar rearrangements that disrupted the ligand - binding domain can mediate resistance to current ar inhibitors.29 development of novel ar inhibitors that target the diverse spectrum of ar alterations is needed . 
 ( a ) ar genomic alterations ( gas ) were identied in 721 cases , including amplication ( red ) , mutation ( green ) , and rearrangements ( purple )  . 
of note , 11q13 amplication is associated with endocrine resistance in breast cancer and potentially targetable by broblast growth factor receptor inhibitors32 - 34 and , therefore , warrants investigation in prostate cancer . 
second , cdkn2a gas were enriched in metastatic site tumors ( 1.9 - fold ) , and although not previously described in primary versus metastatic studies , 1 - 3 acquired cdkn2a alterations were described in enzalutamide - resistant tumors.15 metastatic enrichment of cell cycle gas suggests that cdk4 / 6 inhibitors could be explored . 
 cgp of primary and metastatic prostate tumors certain hrr genes , mmr genes , or msi status ; identication of such dna repair ga by tumor sequencing warrants follow - up germline testing and genetic counseling . 
however , compared with a recent study , 3 we identify similar relative proportions of germline / somatic mutations for frequently mutated hrr genes ( brca2 , atm , chek2 )  . 
furthermore , another study of prostate cancer similarly identied germline mutations for many of the dna repair genes evaluated here.37 we also describe potential cdk12 germline mutations ( four of 12 were rs138292741 )  . 
although not identied in one recent study of cdk12 in prostate cancer , 8 germline truncating cdk12 mutations were described in other studies , cluding prostate cancer , 38 - 40 and in germline databases.40 although preclinical studies have identied non - brca1 / 2 hrr genes , their association with hrd phenotype in clinical samples is unclear . 
identication of gloh - h cases lacking dna repair gas ( appendix fig a4a ) may have clinical value , but further investigation in parp inhibitor trials is required to assess the clinical utility of gloh as a biomarker in prostate cancer . similarly , dna repair gas were associated with msi - h or tmb - h genomic signatures that are associated with benet from immunotherapy ( fig 2 ; appendix fig a5 )  . 
a subset of msi / tmb - h cases do not harbor corresponding dna repair gas potentially because gas may occur in genes involved in dna repair that have not yet been discovered , complex rearrangements with intronic breakpoints may be challenging to detect , or genomic instability can occur through mechanisms such as gene silencing or extrinsic dna damage . limitations of this study should be acknowledged . 
furthermore , because samples were collected for routine clinical testing , primary samples in this cohort may be biased toward patients who have received prior treatment or developed metastatic disease that resulted in a distinct genomic landscape from untreated primary tumors . 
chung employment : foundation medicine ninad dewal employment : foundation medicine stock and other ownership interests : foundation medicine ethan sokol employment : foundation medicine paul mathew honoraria : exelixis patents , royalties , other intellectual property : patent pending on new therapeutic invention travel , accommodations , expenses : exelixis robert whitehead employment : unitedhealthcare , cancer treatment centers of america sherri z . 
pal honoraria : novartis , medivation , astellas pharma consulting or advisory role : pzer , novartis , aveo , myriad pharmaceuticals , genentech , exelixis , bristol - myers squibb , astellas pharma , ipsen , eisai research funding : medivation richard j . 
lee consulting or advisory role : janssen pharmaceuticals , exelixis research funding : janssen pharmaceuticals andrea necchi employment : bayer ag ( i ) stock and other ownership interests : bayer ag ( i ) honoraria : roche , merck , astrazeneca , janssen pharmaceuticals , foundation medicine consulting or advisory role : merck sharp & dohme , roche , bayer ag , astrazeneca , clovis oncology , janssen pharmaceuticals , incyte , seattle genetics , astellas pharma , bristol - myers squibb , rainier therapeutics research funding : merck sharp & dohme ( inst ) , astrazeneca ( inst ) travel , accommodations , expenses : roche , merck sharp & dohme , astrazeneca , janssen pharmaceuticals other relationship : bayer ag ( i ) jeffrey p . 
gregg consulting or advisory role : astrazeneca , bristol - myers squibb , roche , foundation medicine speakers bureau : astrazeneca , foundation medicine , bristol - myers squibb primo lara jr honoraria : pzer consulting or advisory role : exelixis , pzer , astrazeneca , bayer ag , genentech , roche , janssen pharmaceuticals , bristol - myers squibb , abbvie , turnstone bio , foundation medicine , merck , cellmax life , nektar research funding : millennium pharmaceuticals ( inst ) , polaris ( inst ) , glaxosmithkline ( inst ) , genentech ( inst ) , aragon pharmaceuticals ( inst ) , janssen pharmaceuticals ( inst ) , heat biologics ( inst ) , tracon pharma ( inst ) , merck ( inst ) , pharmacyclics ( inst ) , incyte ( inst ) emmanuel s . 
antonarakis honoraria : sano , dendreon , medivation , janssen pharmaceuticals , essa , astellas pharma , merck , astrazeneca , clovis oncology consulting or advisory role : sano , dendreon , medivation , janssen pharmaceuticals , essa , astellas pharma , merck , astrazeneca , clovis oncology research funding : janssen pharmaceuticals ( inst ) , johnson & johnson ( inst ) , sano ( inst ) , dendreon ( inst ) , aragon pharmaceuticals ( inst ) , exelixis ( inst ) , millennium pharmaceuticals ( inst ) , genentech ( inst ) , novartis ( inst ) , astellas pharma ( inst ) , tokai pharmaceuticals ( inst ) , merck ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , constellation pharmaceuticals ( inst ) patents , royalties , other intellectual property : co - inventor of a biomarker technology that has been licensed to qiagen travel , accommodations , expenses : sano , dendreon , medivation vincent a . 
miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine consulting or advisory role : revolution medicines patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center jeffrey s . 
 cgp of primary and metastatic prostate tumors research funding : bayer ag ( inst ) , bristol - myers squibb ( inst ) , glaxosmithkline ( inst ) , medivation ( inst ) , takeda pharmaceuticals ( inst ) , novartis ( inst ) , pzer ( inst ) , bn immunotherapeutics ( inst ) , tracon pharma ( inst ) , rexahn pharmaceuticals ( inst ) , amgen ( inst ) , astrazeneca ( inst ) , active biotech ( inst ) , bavarian nordic ( inst ) , calithera biosciences ( inst ) , celldex ( inst ) , eisai ( inst ) , genentech ( inst ) , immunomedics ( inst ) , janssen pharmaceuticals ( inst ) , merck ( inst ) , newlink genetics ( inst ) , prometheus ( inst ) , sano ( inst ) no other potential conicts of interest were reported . references abeshouse a , ahn j , akbani r , et al : the molecular taxonomy of primary prostate cancer . 
nat genet 50 : 645 - 651 , 2018 abida w , armenia j , gopalan a , et al : prospective genomic proling of prostate cancer across disease states reveals germline and somatic alterations that may affect clinical decision making . 
n engl j med 373 : 1697 - 1708 , 2015 de bono js , de giorgi u , massard c , et al : pten loss as a predictive biomarker for the akt inhibitor ipatasertib combined with abiraterone acetate in patients with metastatic castration - resistant prostate cancer ( mcrpc )  . 
mateo j , ganji g , lemech c , et al : a rst - time - in - human study of gsk2636771 , a phosphoinositide 3 kinase beta - selective inhibitor , in patients with advanced 8 . 
clin cancer res 23 : 5981 - 5992 , 2017 2018 lee l , ali s , genega e , et al : aggressive - variant microsatellite - stable pole mutant prostate cancer with high mutation burden and durable response to immune checkpoint inhibitor therapy . 
cell 161 : 1215 - 1228 , 2015 [ erratum : cell 162 : 454 , 2015 ] joseph jd , lu n , qian j , et al : a clinically relevant androgen receptor mutation confers resistance to second - generation antiandrogens enzalutamide and arn509 . 
han gc , hwang j , wankowicz sam , et al : genomic resistance patterns to second - generation androgen blockade in paired tumor biopsies of metastatic castration - resistant prostate cancer . 
mateo j , chakravarty d , dienstmann r , et al : a framework to rank genomic alterations as targets for cancer precision medicine : the esmo scale for clinical actionability of molecular targets ( escat )  . 
bajrami i , frankum jr , konde a , et al : genome - wide proling of genetic synthetic lethality identies cdk12 as a novel determinant of parp1 / 2 inhibitor sensitivity . 
blazek d , kohoutek j , bartholomeeusen k , et al : the cyclin k / cdk12 complex maintains genomic stability via regulation of expression of dna damage response 21 . 
swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
coleman rl , oza am , lorusso d , et al : rucaparib maintenance treatment for recurrent ovarian carcinoma after response to platinum therapy ( ariel3 ) : a randomised , double - blind , placebo - controlled , phase 3 trial . 
carbone dp , reck m , paz - ares l , et al : first - line nivolumab in stage iv or recurrent non - small - cell lung cancer . 
giltnane jm , hutchinson ke , stricker tp , et al : genomic proling of er + breast cancers after short - term estrogen suppression reveals alterations associated with endocrine resistance . 
luen sj , asher r , lee ck , et al : association of somatic driver alterations with prognosis in postmenopausal , hormone receptor - positive , her2 - negative early breast cancer : a secondary analysis of the big 1 - 98 randomized clinical trial . 
soria j - c , debraud f , bahleda r , et al : phase i / iia study evaluating the safety , efcacy , pharmacokinetics , and pharmacodynamics of lucitanib in advanced solid tumors . 
brovkina oi , shigapova l , chudakova da , et al : the ethnic - specic spectrum of germline nucleotide variants in dna damage response and repair genes in hereditary breast and ovarian cancer patients of tatar descent . 
popova t , mani e e , boeva v , et al : ovarian cancers harboring inactivating mutations in cdk12 display a distinct genomic instability pattern characterized by large tandem duplications . 
abida w , bryce ah , vogelzang nj , et al : preliminary results from triton2 : a phase ii study of rucaparib in patients ( pts ) with metastatic castration - resistant prostate cancer ( mcrpc ) associated with homologous recombination repair ( hrr ) gene alterations . 
clarke n , wiechno p , alekseev b , et al : olaparib combined with abiraterone in patients with metastatic castration - resistant prostate cancer : a randomised , double - blind , placebo - controlled , phase 2 trial . 
 cgp of primary and metastatic prostate tumors appendix methods approval for this study , including a waiver of informed consent and health insurance portability and accountability act waiver of authorization , was obtained from the western institutional review board ( protocol 20152817 )  . 
comprehensive genomic proling ( cgp ) results were reported clinically by prospective sequencing of tissue samples from 3 , 476 unique patients with prostate cancer ( august 2014 to february 2018 ) using a validated hybrid capture - based cgp assay ( foundationone [ baitset version t7 was used during this period ] ) 16 in a clinical laboratory improvement amendmentscertied , college of american pathologistsaccredited , new york stateapproved laboratory ( foundation medicine , cambridge , ma )  . 
for patients with multiple submitted samples , only a single sample was included , and the sample with the highest sequencing quality metrics was included . age and site of specimen collection were abstracted from the accompanying pathology reports , clinical notes , and requisition forms submitted by the treating physician . 
specimens included 1 , 660 primary site tumors and 1 , 816 metastatic site tumors ( appendix fig a1 )  . the pathologic diagnosis of each case was conrmed on routine hematoxylin and eosinstained slides , and all samples forwarded for dna extraction contained a minimum of 20% tumor nuclei . 
cgp was performed on hybridization - captured , adaptor ligation - based libraries to a median coverage depth of 743 for 395 cancer - related genes plus select introns from 31 genes frequently rearranged in cancer ( appendix table a1 )  . 
for ets fusions , targeted regions were tmprss2 ( introns 1 to 3 ) , erg ( all exons ) , etv1 ( introns 3 to 4 ) , etv4 ( intron 8 ) , and etv5 ( introns 6 to 7 )  . insertions / deresults were analyzed for base substitutions , short letions , rearrangements , and copy number alterations ( amplication and homozygous deletion )  . 
genomic alterations ( gas ) were called as reportable if the specic variant was present in the catalog of somatic mutations in cancer database ( forbes et al : nucleic acids res 45 : d777 - d783 , 2017 ) , if the variant has been characterized as pathogenic , or if the variant had likely functional status ( disruptive alterations in tumor suppressor genes ) ; all other variants were classied as variants of unknown signicance . to compare relative enrichments in primary site and metastatic site tumors , genes altered at a 2% or greater frequency were assessed for enrichment . 
enrichment was dened as the difference in frequency of gene alteration between metastatic site and primary site samples . to determine microsatellite instability status , 114 intronic homopolymer repeat loci on the foundationone panel were analyzed for length variability and compiled into an overall microsatellite instability score through principal components analysis ( chalmers et al : genome med 9 : 34 , 2017 )  . 
tumor mutational burden was calculated as the number of somatic base substitutions or insertions / deletions per megabase of the coding region target territory of the test ( 1.1 mb ) after ltering to remove known somatic and deleterious mutations and extrapolating that value to the exome or genome as a whole ( chalmers et al : genome med 9 : 34 , 2017 )  . 
tumor mutational burden was categorized as low ( fewer than six mutations / mb ) , intermediate ( six to 20 mutations / mb ) , or high ( 20 mutations / mb or more ; chalmers et al : genome med 9 : 34 , 2017 )  . 
germline , somatic , and zygosity statuses for mutations were determined without matched normal tissue as previously described17 ; in validation testing of 480 germline / somatic calls from tumor - only sequencing with matched normal reference samples , accuracy was 95% for somatic calls and 99% for germline calls . 
biallelic inactivation was dened as mutations under loss of heterozygosity ( loh ) as determined by zygosity status.17 percent genome - wide loh ( gloh ) was used as a marker of homologous recombination deciency and calculated as described , and a gloh score of 14% or greater was dened as gloh - high.21 potentially targetable gas were dened as those that have been associated with response to targeted therapy in prostate cancer , homologous recombination repair gas that have been associated with responses to poly ( adp - ribose ) polymerase inhibitors , or gas associated with response to targeted therapy in multiple other tumor types and were ranked according to modied european society for medical oncology scale for clinical actionability of molecular targets criteria.18 ln ( unk ) ln ( d ) ln ( l ) liver bone lung other prostate ( n = 1 , 660 ) metastasis ( n = 1 , 816 ) neuroendocrine ( 4% ) undifferentiated ( 3% ) acinar ( 93% ) fig a1 . 
for fusion , exons ( e ) are annotated with the last exon included in the 5 ( cid : 3 ) partner and the rst exon included in the 3 ( cid : 3 ) partner . 
in addition , braf rearrangements at intron 9 ( n = 6 ) and intron 7 ( n = 1 ) and raf1 rearrangement at intron 7 with no clear fusion partner were identied ( data not shown )  . 
 ( a ) genome - wide loss of heterozygosity ( gloh ) score was assessable for 2 , 624 cases , and cases with 14% or more gloh were designated loh - high ( loh - h )  . 
loh - h cases were assessed for the presence ( green ) or absence ( yellow ) of a genomic alteration ( ga ) in the homologous recombination repair ( hrr ) or fanconi anemia / interstrand crosslink repair ( fa / icl ) pathway . 
 ( b ) genes were grouped together into hrr pathway ( excluding non - brca1 / 2 ) or fa / icl pathway ( fanc ) ; genes were categorized as in figure 2a . 
 ( a ) microsatellite instability ( msi ) status was assessable for 3 , 326 cases , and each case was designated as msi - high ( msi - h ) , msi - low ( msi - l ) , or microsatellite stable ( mss )  . 
the percent tmb - high ( tmb - h ) or tmb of 10 or more mutations ( mut ) / mb cases in primary site and metastatic site tumors is indicated . 
 cdkn2c - null leiomyosarcoma : a novel , genomically distinct class of tp53 / rb1wild - type tumor with frequent cic genomic alterations and 1p / 19q - codeletion erik a . 
here , we searched for recurrent actionable genomic alterations in lms that occur in the absence of common untreatable oncogenic drivers . methods tissues from 276 , 645 unique advanced cancers , including 2 , 570 uterine and soft tissue lms , were sequenced by hybrid - capturebased next - generation dna and rna sequencing / comprehensive genomic proling of up to 406 genes . 
we further validated our ndings in two publicly available datasets : the cancer genome atlas and the project genie initiative . conclusion cdkn2c - null lms denes a genomically distinct tumor that may have prognostic and / or therapeutic clinical implications , including possible use of specic cyclin - dependent kinase inhibitors . jco precis oncol 4 : 955 - 971 . 
 williams et al context key objective leiomyosarcoma ( lms ) , an aggressive tumor with limited curative options , shows frequent mutations in tp53 and rb1 but few actionable genomic alterations . 
here , we searched for recurrent actionable genetic alterations in lms . knowledge generated a novel , genomically distinct class of lms ( 3.0% ; 77 of 2 , 570 cases ) harbor homozygous loss of cdkn2c , which encodes the cyclin - dependent kinase inhibitor - 2c . 
cdkn2c - null lms lack typical tp53 and rb1 mutations ; show concurrent homozygous deletion of cic , cdkn2a , and rad51b ; and show frequent 1p / 19q - codeletion . relevance the nding of recurrent cdkn2c - null lms provides insight into tumor biology and raises the possibility for use of specic cyclin - dependent kinase inhibitors in this aggressive disease . from the uterus , with signicantly low frequency of tp53 and rb1 gas . methods cohort and genomic analyses comprehensive genomic proling was performed in a clinical laboratory improvement amendmentscertied , college of american pathologistsaccredited laboratory ( foundation medicine , cambridge , ma )  . 
20% estimated percent tumor nuclei in each case , for which the percent tumor nuclei equals 100 times the number of tumor cells divided by total number of nucleated cells . 
the samples were assayed by adaptor ligation hybrid capture , performed for all coding exons of 236 ( v1 ) , 315 ( v2 ) , or 405 ( v3 ) cancer - related genes plus select introns from 19 ( v1 ) , 28 ( v2 ) , or 31 ( v3 ) genes frequently rearranged in cancer ( appendix table a1 ) .11 , 12 for samples with available rna , targeted rna sequencing was performed for rearrangement analysis in 265 genes.12 sequencing of captured libraries was performed using the illumina hiseq 4000 system ( illumina , san diego , ca ) to a mean exon coverage depth of targeted regions of  . 500 , and sequences were analyzed for gas , including short variant alterations ( base substitutions , insertions , and deletions ) , copy number alterations ( focal amplications and homozygous deletions ) , and select gene fusions or rearrangements.11 , 13 , 14 to maximize mutation detection accuracy ( sensitivity and specicity ) in impure clinical specimens , the test was previously optimized and validated to detect base substitutions at a 5% mutant allele frequency , indels with a 10% mutant allele frequency with 99% accuracy , and fusions occurring within baited introns / exons with  . 
6 - 8 copies depending on tumor ploidy and homozygous deletions was performed as previously described.11 in brief , the aligned dna sequences of each tumor specimen were normalized against a process - matched normal , producing log - ratio and minor allele frequency data . 
a gibbs sampler tted copy number model and a grid - based model were tted to the segmented log - ratio and minor allele frequency data , producing genome - wide copy number estimates . 
finally , the degrees - of - t of candidate models returned by gibbs sampling and grid sampling were compared , and the optimal model was selected by an automated heuristic . signal - to - noise ratios for each genomic segment were used to determine gain or loss per chromosome arm on the basis of tumor purity and ploidy ; the sum of segment sizes determined the fraction of each arm gained or lost . chromosomes were assessed for arm - level aneuploidy , dened as positive if  . 
the cohort of cdkn2c - null lms comprised 77 cases , each from a different patient , that were submitted to foundation medicine for comprehensive genomic proling during routine clinical care . 
human investigations were performed after approval by a local human investigations committee and in accordance with an assurance led with and approved by the department of health and human services , when appropriate . 
clinicopathologic data , including patient age , sex , tumor site , and figo stage or ajcc ( 8th edition ) stage , were extracted from the accompanying pathology report.4 , 16 primary site data were not available for a subset of patient cases ( indeterminant primary )  . 
these patients were signicantly older than the remainder of the lms cohort ( median age , 61 v 57 years ; p = .0009 , mann - whitney u test )  . 
patients were enriched for female sex compared with the remainder of the lms cohort ( 99% [ 76 of 77 ] v 79% [ 1 , 968 of 2 , 493 ] ; p , .0001 , fishers exact test )  . 
six female patients had a prior history of leiomyomatosis ( n = 2 ) or uterine smooth muscle tumor of uncertain malignant potential ( stump ; n = 4 )  . 
the majority of cdkn2c - null lms patients showed clinically advanced / metastatic disease , with 62% of conrmed uterine primary occurrences documented at figo stage iv ( n = 34 of 55 ) and 86% of indeterminant or soft tissue primary cases documented at ajcc stage iv ( n = 19 / 22 ) , as summarized indeterminant figo staging ( uterine primary ) no . 
locations of the sequenced tumor specimens are summarized in appendix table a2 . comprehensive genomic proling of cdkn2c - null lms the distribution of gas in the 77 cdkn2c - null lms is displayed in figure 1 . 
the frequent gas were identied in cic at 19q13.2 , most cdkn2a , and rad51b ( table 2 ) , and unsupervised analysis showed signicant enrichment of these alterations in the cdkn2c - null cohort ( appendix fig a1 )  . 
no other signicant differences based on age were identied . comparison of patient cases on the basis of clinical stage , three patients had two separate tissue specimens analyzed ( appendix table a4 )  . 
additional recurrent chromosomal arm - level changes were identied in the 72 patient cases available , including most frequently aneuploidy of chromosomes 6p ( n = 35 ) , 9p ( n = 19 ) , 10q ( n = 28 ) , 11p ( n = 39 ) , 13q ( n = 46 ) , 14q ( n = 46 ) , and 16q ( n = 52 )  . a review of 1p / 19q - codeletion status in available lms patient cases without homozygous deletion of cdkn2c ( n = 1 , 212 ) revealed 62 1p / 19q - codeleted lms ( 5% ; fig 2c )  . 
all four occurred in uterine lms ( one of which with a history of stump )  . all non - lms sarcoma patient cases in the foundation medicine dataset ( n = 12 , 097 ) were evaluated for cdkn2c status . 
these included diverse sarcoma diagnoses , including six high - grade sarcomas not otherwise specied , two osteosarcomas , two malignant peripheral nerve sheath tumors , and two inammatory myobroblastic tumors . 
both were alk rearrangementpositive tumors in women ( ages , 63 and 72 years )  . we also searched our entire lms cohort ( n = 2 , 570 ) for cases with pathogenic alterations in cdkn2c other than homozygous deletion . 
 ( a ) cdkn2c - null lms with a truncating variant in cic ( p.q907 * ) and homozygous deletion of cdkn2a at chromosome 9p21.3 ( red arrow )  . 
 ( c ) cdkn2c - retained lms with deep deletion of rb1 at chromosome 13q14.2 ( red arrow ) and a cn plot of high complexity . and rad51b were identied ; 1p / 19q status was not available . histopathology histopathologic evaluation was performed on all available high - resolution digital pathology h&e slides of our cohort of cdkn2c - null lms ( n = 70 )  . 
histopathology of cdkn2c - null leiomyosarcoma ranged from ( a , b ) epithelioid to ( c ) spindled ( hematoxylin & eosin [ h&e ] stains , 200 )  . 
 ( d ) occasional cases showed focal myxoid histology ( h&e stain , 200 )  . progesterone receptor ( 24 of 26 ; remaining two with focal positivity )  . 
cdkn2c - null lms were also positive for desmin ( 30 of 33 ; remaining three with focal positivity ) , smooth muscle actin ( 25 of 25 ) , muscle - specic actin ( 11 of 11 ) , and caldesmon ( 6 of 6 )  . 
patient cases showed frequent homozygous loss of cic ( n = 5 [ 42% ] of 12 ) , cdkn2a ( n = 4 [ 33% ] of 12 ) , and rad51b ( n = 3 [ 25% ] of 12 ) , and all were wild type for tp53 , rb1 , and atrx ( n = 12 of 12 )  . discussion in 2 , 570 patient cases of lms , cdkn2c - null lms ( n = 77 ; 3.0% ) comprised a genomically distinct molecular subgroup . cdkn2c - null lms typically lacked mutations in tp53 , rb1 , and atrx but showed frequent 1p / 19q - codeletion ( 81% ) , and nearly half ( 40.3% ) showed homozygous deletion or inactivating truncations of cic . 
 cdkn2c loss denes class of tp53 / rb1wild - type leiomyosarcoma which enhances fas - mediated apoptosis , and its loss may contribute to tumor pathogenesis.29 the cic gene on chromosome 19q13.2 represses genes induced downstream to rtk pathway activation.30 in the absence of rtk signaling , cic blocks transcription of genes that have diverse effects on cellular proliferation , metabolism , and migration.31 along with single copy loss of cic on 19q , concurrent inactivating mutations in cic are identied in a high percentage of oligodendrogliomas.32 whole - arm 1p / 19q - codeletion , with concurrent mutation in idh1 or idh2 , is entity - dening for oligodendrogliomas.33 - 35 oligodendrogliomas are associated with relatively long overall survival , and treatment strategies are often stratied on the basis of 1p / 19q status.36 - 38 the codeletion is a result of unbalanced translocation between two chromosomes , with subsequent loss of der ( 1 ; 19 ) ( p10 ; q10 ) , likely because chromosomes 1 and 19 are near each other in the nonrandom organization of the nucleus.39 - 41 a large percentage of oligodendrogliomas also show cic and fubp1 mutations.31 our cohort of cdkn2c - null lms shows notable similarities and differences to oligodendroglioma ; 40% of our cohort showed an inactivating alteration in cic , most commonly homozygous deletion . 
rare sarcoma - like tumors originating from oligodendrogliomas have been reported , with documented idh1 mutation and 1p / 19q - codeletion , and have been termed oligosarcoma.42 - 44 rodriguez et al43 identied 6 of 7 patient cases of oligosarcoma with at least focal smooth muscle actin positivity of the sarcomatous component by immunohistochemistry and one patient case with smooth muscle differentiation by electron microscopy . 
these results indicate a similarity in differentiation to our cohort of lms . among all sarcomas with cdkn2c loss , 1p / 19q - codeletion appears to be nearly exclusive to lms . 
in our overall lms cohort , however , occasional cdkn2c - retained lms showed tp53 and rb1 alterations and were positive by the 1p / 19qcodeletion detection algorithwe speculate that , given the complexity of these genomically unstable occurrences , occasional cdkn2c - retained lms satisfy these criteria ( fig 2c )  . as such , identication of homozygous deletion of cdkn2c may be the most specic distinguishing feature . cytogenetic ndings in lms and leiomyoma have been previously reported , although without characterization of cdkn2c status . 
a greater frequency of 1p loss has been documented in metastasized lms.45 from a cytogenetics study of 800 uterine leiomyomata , nine diploid occurrences with 1p loss were identied , with other associated alterations , particularly chromosome 19 and / or chromosome 22 loss.46 transcriptional proling of two of the 1p - deleted leiomyomas in that study showed alignment with malignant lms in a hierarchical clustering analysis.46 in another study , 1p loss was identied in approximately one quarter of uterine cellular leiomyomata.47 three reports on a total of eight pulmonary - based benign metastasizing leiomyoma reported 19q and 22q terminal deletion in each case.48 - 50 rare uterine leiomyomas with gas in rad51b have also been identied.51 the overlap in gas between our cohort of cdkn2c - null lms and a subset of leiomyoma of uncertain cdkn2c status in the literature suggests a possible connection between these entities . evaluations of gas in epithelioid or myxoid uterine smooth muscle neoplasms are limited in the literature.52 - 56 although a high percentage of cdkn2c - null lms in our study demonstrated epithelioid features on histology , histology was also varied . 
immunohistochemistry results extracted from pathology reports were typical for uterine lms , with expression of characteristic smooth muscle markers.57 given the overall low response rate of lms to standard therapies , the identication of this targetable alteration in cdkn2c may be useful for treatment decisions . 
cdk4 / 6 inhibitors have previously shown effectiveness in a lms with a cdkn2a alteration.10 nearly half of the cdkn2c - null lms harbored loss of cdkn2a ; cdk4 / 6 inhibitors may be effective in replacing the loss of inhibition of cdk4 / 6 that results from cdkn2c and cdkn2a loss in these patient cases that recur after standard chemotherapy regimens . 
a minor subset of cdkn2c - null and / or 1p / 19q - codeleted lms harbored activating fusions in alk , braf , fgfr1 , and ntrk1 , for which targeted inhibitors may be of utility.9 limitations of this study include its retrospective nature and the enrichment for aggressive tumors , mostly metastatic to distant sites . 
the latter may be due to collection bias from submission of specimens later in the disease course . additional studies will be needed to correlate the nding of cdkn2c loss in lms with prognostic data and treatment outcomes . 
williams , md , 150 second st , cambridge , ma 02141 ; twitter : @erikawilliamsm1 ; e - mail : erwilliams@foundationmedicine.com. meagan montesion stock and other ownership interests : roche ethan s . 
hoffmann - la roche radwa sharaf employment : foundation medicine stock and other ownership interests : roche brennan decker stock and other ownership interests : avidea technologies consulting or advisory role : foundation medicine , avidea technologies kevin jon williams stock and other ownership interests : hygieia , gemphire therapeutics consulting or advisory role : gemphire therapeutics research funding : novo nordisk jeffrey m . 
venstrom employment : genentech , foundation medicine leadership : genentech stock and other ownership interests : genentech research funding : genentech , roche , foundation medicine travel , accommodations , expenses : genentech brian m . 
alexander employment : foundation medicine leadership : foundation medicine stock and other ownership interests : roche research funding : eli lilly ( inst ) , puma ( inst ) , celgene ( inst ) ( optional ) open payments link : physician / 854258 / summary jeffrey s . 
ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine consulting or advisory role : celsius therapeutics research funding : foundation medicine lee a . 
elvin employment : foundation medicine no other potential conicts of interest were reported . acknowledgment we thank the american association for cancer research and appreciate its nancial and material support in the development of the american association for cancer research project genie registry , and we thank members of the consortium for their commitment to data sharing . references roberts me , aynardi jt , chu cs : uterine leiomyosarcoma : a review of the literature and update on management options . 
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holzmann c , markowski dn , koczan d , et al : genome - wide acquired uniparental disomy as well as chromosomal gains and losses in a uterine epithelioid leiomyoma . 
reyes c , karamurzin y , frizzell n , et al : uterine smooth muscle tumors with features suggesting fumarate hydratase aberration : detailed morphologic analysis and correlation with s - ( 2 - succino ) - cysteine immunohistochemistry . 
 cdkn2c loss denes class of tp53 / rb1wild - type leiomyosarcoma appendix attribute negative not significant positive % frequency cdkn2c faf1 tp53 sex : male atrx chr2p_loss chr1p_loss cdkn2a cdkn2b rad51b sex : female chr19q_loss chr14q_loss log2 odds ratio fig a1 . 
red and blue indicate positive and negative correlation , respectively , in cdkn2c - null leiomyosarcoma ( n = 77 ) compared with the remainder of the leiomyosarcoma cohort ( n = 2 , 493 )  . 
 clinical use of precision oncology decision support purpose precision oncology is hindered by the lack of decision support for determining the functional and therapeutic significance of genomic alterations in tumors and relevant clinically available options . 
to bridge this knowledge gap , we established a precision oncology decision support team that provides annotations at the alteration level and subsequently determined whether clinical decision making was influenced . methods genomic alterations were annotated to determine actionability on the basis of a variants known or potential functional and / or therapeutic significance . 
a webbased survey was implemented to capture the reasons genotype - matched therapies were not pursued . results the precision oncology decision support team processed 1 , 669 requests for annotation of 4 , 084 alterations ( 2 , 254 unique ) across 49 tumor types for 1 , 197 patients . 
sixty - six percent of patients had at least one actionable / potentially actionable alteration , and 20.6% of patients ( 110 of 535 ) annotated enrolled in a genotype - matched trial . 
clinicians cited a variety of reasons that patients with actionable alterations did not enroll in trials . conclusion over half of alterations annotated were of unknown significance or nonactionable . physicians were more likely to enroll a patient in a genotype - matched trial when an annotation supported actionability . 
 research indicates that clinicians are unlikely to use sources that take longer than 30 seconds to retrieve data10 and that information presented in graphical summaries enhances interpretation , improving health care quality.11 , 12 second , limited information may be exposed because these web sites are public facing . 
third , novel alterations continue to be discovered , and information is rapidly evolving , generally outpacing the rate of updates . thus , there is a need for an on - demand , real - time clinical interpretation service that annotates all requested alterations , beyond those available within accessible knowledge bases , to determine their actionability . the precision oncology decision support system ( pods ) was established at the university of texas md anderson cancer center in recognition of these needs.9 herein , we report our experience with large - scale , institution - wide decision support and its initial clinical utility . 
all requestors indicating the same physician as the contact for correspondence were considered a teastatistical significance was evaluated by a x2 test . annotation process and return of reports pods scientists receive requests , access our knowledge base of variant annotations , and review and update content as applicable.9 , 13 reports consist of a data summary with references and alteration frequency from external ( cbioportal , cosmic [ catalogue of somatic mutations in cancer ] ) and internal databases . 
in 2015 , reports evolved to routinely contain a functional significance , gene , and alteration - level actionability call ( table 1 ) .7 , 12 all reports are reviewed by the medical director and returned via e - mail . 
starting in august 2015 , reports referencing clia - validated panels were deposited within md andersons electronic health record ( ehr )  . manual review of clinical data medical records of 539 patients annotated in 2015 through clinician - initiated requests or for treatment planning conferences were reviewed for the presence of a clinical trial entrance note in the patients ehr . 
potential matches between the annotated variants and targeted therapy used within the trial were manually recorded . clinical decision follow - up surveys a web - based survey was initiated in 2015 , and 236 surveys were sent to annotation requestors with a 1 - month postannotation delivery date to ascertain whether the patient matched to genomically guided therapy . 
for patients not enrolled to receive therapy on the basis of the annotation provided , the survey inquired as to the reasons why . results patient annotation requests the pods team processed 1 , 669 requests for 1 , 197 patients , most in 2014 and 2015 ( data supplement )  . 
multiple requests for the same patient may have been due to additional alterations requested for annotation , different clinicians requesting annotation of the same molecular report , or the same molecular report requested for different venues in which the patient was evaluated ( eg , clinic visit v treatment planning conference )  . 
clinician - initiated requests originated from treating physicians and team members for point - of - care decisions on the basis of molecular testing or from clinical trial teams screening for patients to enroll in genotype - matched trials . fifty - six physician teams initiated requests for 724 patients and ranged from one to 176 per team , with two requests being the median ( data supplement )  . 
because not all aspects of protein function can possibly be determined , the functional significance is likely benign there is peer - reviewed published literature showing that the alteration has an inactivating or inferred inactivating effect on an oncogene . there is peer - reviewed published literature showing that the alteration has an activating or inferred activating effect on a tumor suppressor that enhances its function as a tumor suppressor overall , 4 , 084 variant - level annotations were provided for 2 , 254 unique alterations . 
genes most commonly annotated as part of a full panel request included tp53 , kras , and pik3ca , representing the three most commonly mutated genes in the genome ( fig 1c )  . 
the percentage of requests is shown for ( a ) each indicated tumor type ( top 25 ) and ( b ) annotation type ( full panel or select alterations )  . 
genes with 20 or more annotations are shown . actionable genes and alterations a gene is actionable if there are clinically available therapies that directly or indirectly targe alterations in the gene and / or there are clinical trials selecting for alterations in the gene . of the 4 , 084 annotations , 3 , 166 ( 78% ) were within a gene defined as actionable at the time of annotation ( fig 2a ; data supplement )  . 
in 2015 , annotation reports evolved to contain an actionable variant call describing the alterations functional and therapeutic significance , with classification into four broad categories actionable ( further subcategorized by source : literature based , inferred , or functional genomics ) , potentially actionable , unknown , and nonactionable ( table 1 ) 9which was provided with 2 , 444 annotations delivered for 669 patients . 
a total of 794 ( 32.5% ) annotations were actionable , 230 ( 9.4% ) were potentially actionable , 725 ( 29.7% ) were unknown , and 697 ( 28.4% ) were not actionable ( fig 2b )  . 
the total number of annotations in 2015 and the subset that was actionable / potentially actionable are shown per the patients tumor type ( fig 2e and data supplement , respectively )  . 
the actionability of kras has been controversial , particularly in colorectal cancer.14 - 16 when excluding kras as actionable , most actionable annotations were provided for breast cancer patients ( n = 90 ; data supplement )  . clinical utility of annotations we then asked whether physicians acted more often on alterations with evidence of actionability ( data supplement )  . 
we manually recorded clinical follow - up data by accessing the ehrs of 539 patients requested for annotation by clinicians or for treatment planning conferences . four patients were excluded : two had mutations that were actionable only for resistance to therapy , and two because of insufficient follow - up since physician notification . 
for the remaining 535 patients , variant annotation data were filtered , such that each patient in the data set is represented by a single alteration ( fig 3a )  . 
the fraction of patients represented by the highest variant call ( yellow column ) and the corresponding fraction of patients with that call enrolled in a trial ( gray column )  . 
the fraction of patients represented by the highest variant call ( yellow column ) and the corresponding fraction of patients with that call enrolled in a trial ( gray column )  . 
of patients with variant call enrolled in a trial ( % ) yes : literature based 214 ( 40.0 ) 49 ( 22.9 ) yes : inferred 54 ( 10.1 ) 26 ( 48.1 ) potentially 65 ( 12.1 ) 17 ( 26.2 ) unknown 136 ( 25.4 ) 16 ( 11.8 ) no ( nonactionable ) 66 ( 12.3 ) 2 ( 3 ) p < .001 actionable / potentially actionable alterations unknown alterations nonactionable alterations actionable variant classification total no . 
alterations of 214 patients ( 40% ) were classified into the yes : literature based category ; 54 ( 10.1% ) were classified into the yes : inferred category ; 65 ( 12.1% ) were classified into the potentially category ; 136 ( 25.4% ) were classified into the unknown category ; and 66 ( 12.3% ) were classified into the no ( nonactionable ) category ( fig 3a , yellow column )  . 
patients with actionable alterations in pten ( n = 20 ) , pik3ca ( n = 11 ) , and erbb2 ( n = 10 ) most frequently enrolled in a trial ( fig 4 ) , paralleling the data showing a large number of actionable / potentially actionable annotations delivered for pik3ca and pten ( fig 2d ; data supplement )  . to understand why physicians may not have acted on potentially actionable alterations , we introduced a web - based survey in 2015 to accompany physician - initiated requests . 
a combination of survey responses and manual review revealed that 26.8% of annotations ( 59 of 221 ) led to a genotypematched trial ( fig 3c , gray column )  . 
the latter had another well - characterized oncogenic variant ( idh1 r132c , akt1 e17k , or fgf3 and fgf4 amplifications ) or an alteration clearly inferable as actionable ( early truncating ptch1 mutation ) , where either decision support was not needed or was previously supplied outside the survey group . finally , three patients ( 3.3% ) elected to be treated elsewhere . among 161 patients with variants classified as yes : literature based and yes : inferred , there were fig 4 . 
reasons patients did not enroll in genotype - matched trials after precision oncology decision support annotations , as stated by the survey respondents patients ( n = 161 ) patients with yes : literature based and yes : inferred variants ( n = 42 ) patients with variants annotated as potentially actionable ( n = 29 ) patients with variants annotated as unknown and no for actionability ( n = 90 ) 71 ( 44.1 ) 5 ( 11.9 ) 9 ( 31.0 ) 57 ( 63.3 ) reasons patient did not enroll in trial physician stated annotation does not support trial enrollment , no . 
reasons included another alteration pursued ( two patients ) , the patient was responding to current therapy ( six patients ) , and the patient elected treatment elsewhere ( nine patients )  . 
other reasons included nongenotype - matched treatment options pursued ( five patients ) and ineligibility ( 11 patients )  . in five instances , physicians indicated that the annotation did not support trial enrollment , which we investigated further . 
 toxicity - related deaths in patients treated with targeted therapies.17 - 20 however , few biomarkers have an indication for treatment with a food and drug administrationapproved drug specific to the patients tumor type , 21 - 27 and a limited number of patients with potentially actionable alterations receive genotype - matched therapies in experimental contexts.3 - 7 one contributing factor may be a lack of decision support , as suggested by the cancer genome evaluation committee , which found that physicians are often overwhelmed by data of uncertain significance and that sound guidelines are essential for determining clinical action.28 moreover , we previously reported modest differences in trial enrollment between patients with or without potentially actionable alterations.3 assessing a new population of patients where decision support was provided , we observed that physicians acted more often when the function of the alteration was known . the necessity for real - time decision support is clear from the large volume of requests received from numerous physicians ( data supplement ) treating patients with diverse tumor types for alterations in a range of genes . 
however , bias likely exists toward physicians leading targeted therapy trials and for genes targeted by those therapies . the pods team is frequently asked to provide an annotation for sequencing reports from commercial vendors that produce end - to - end reports , some already containing alteration - level annotations . distinguishing factors of pods reports are a clear call of functional effect9 ( eg , activating , inactivating , unknown ) , a range of variant - level actionability categories on the basis of experimental evidence , and inclusion of all md anderson genotype - matched clinical trials in current reports . previous studies reported high rates of alterations in actionable genes , 4 , 29 - 33 and we provided an annotation in at least one actionable gene for 97% of patients in our cohort . 
using the target ( tumor alterations relevant for genomics - driven therapy ) database and phial ( precision heuristics for interpreting the alteration landscape ) algorithm to rank alterations followed by manual annotation for only selected patients , a study showed that 90% of patients have clinically relevant alterations.34 conversely , we found that 66% of patients annotated have at least one potentially actionable alteration on the basis of manual curation of all aberrations . 
potential differences between the studies include ( 1 ) the inclusion of potentially actionable diagnostic or prognostic alterations by van allen et al34 and not in our study ; ( 2 ) we provided annotations for only requested alterations , and few annotation requests were obtained for well - established alterations ( eg , braf v600e ) ; and ( 3 ) the difference in the number of patients assessed by manual curation in the two studies . among all annotations with an actionable variant call , only 32.5% were classified as actionable , with another 9.4% classified as potentially actionable . even within genes that are considered actionable , 47.4% of the annotations either had no evidence to support actionability or were not actionable . moreover , 58% of the genomic alterations annotated and evaluable for frequency have not been reported in the cosmic database ( data supplement )  . 
these data highlight the need to define actionability at the variant , rather than gene , level . to determine whether physicians acted according to the evidence presented in the pods reports , we followed 535 patients . 
thus , physicians more often act on alterations when sufficient evidence is provided that they may be driver events . the most often - cited reason that patients did not enroll in genotype - matched trials was that the annotation did not support trial enrollment . 
mills shaw , john mendelsohn , funda meric - bernstam provision of study materials or patients : sarina piha - paul , vivek subbiah , david hong , kenna r . 
mills shaw , funda meric - bernstam data analysis and interpretation : amber johnson , yekaterinab . khotskaya , lauren brusco , jia zeng , vijakumar holla , ann m . bailey , md abu shufean , russell broaddus , gordon b . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . jia zeng stock and other ownership interests : amgen , mckesson , mylan , adamas pharmaceuticals amber johnson no relationship to disclose yekaterina b . 
s anchez no relationship to disclose md abu shufean no relationship to disclose sarina piha - paul consulting or advisory role : genentech research funding : glaxosmithkline , xuanzhu , puma biotechnology , novartis , merck sharp & dohme , curis , principa biopharma , biomarin , helix biopharma , bayer , abbvie , incyte , five prime therapeutics , cerulean pharma , medimmune , medivation vivek subbiah research funding : novartis , glaxosmithkline , nanocarrier , northwest biotherapeutics , roche / genentech , bergpharma , bayer , incyte , fujifilm , pharmamar , d3 , pfizer , amgen , abbvie , multivir , bluprint medicines travel , accommodations , expenses : novartis , pharmamar , fujifilm david hong stock and other ownership interests : molecularmatch , oncorena honoraria : adaptimmune , baxter , merrimack , bayer consulting or advisory role : baxter , bayer research funding : novartis , genentech , eisai , astrazeneca , pfizer , mirna therapeutics , amgen , daiichi sankyo , merck , mirati therapeutics , eli lilly travel , accommodations , expenses : loxo , mirna therapeutics other relationship : oncorena mark routbort no relationship to disclose russell broaddus no relationship to disclose kenna r . 
stockley tl , oza am , berman hk , et al : molecular profiling of advanced solid tumors and patient outcomes with genotype - matched clinical trials : the princess margaret impact / compact trial . 
j pers med 3 : 306 - 325 , 2013 johnson a , zeng j , bailey am , et al : the right drugs at the right time for the right patient : the md anderson precision oncology decision support platfordrug discov today 20 : 1433 - 1438 , 2015 10 . 
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bennouna j , lang i , valladares - ayerbes m , et al : a phase ii , open - label , randomised study to assess the efficacy and safety of the mek1 / 2 inhibitor azd6244 ( arry - 142886 ) versus capecitabine monotherapy in patients with colorectal cancer who have failed one or two prior chemotherapeutic regimens . 
schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
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van allen em , wagle n , stojanov p , et al : whole - exome sequencing and clinical interpretation of formalin - fixed , paraffin - embedded tumor samples to guide precision cancer medicine . 
lolkema1 introduction the vhl gene is a tumor suppressor gene located at chromosome 3p25 , 1 and its protein has multiple functions linked to multiple effector proteins.2 aberrations in the vhl gene are associated with sporadic clear cell renal cell carcinomas ( ccrccs ) , sporadic hemangioblastomas , pheochromocytomas , pancreatic islet cell tumors , endolymphatic sac tumors , and benign cysts affecting various organs.3 germline inthe vhl gene causes the autosomal activation of dominant von hippel - lindau ( vhl ) syndrome . 
vhl is rarely mutated outside of the context of rcc or vhl syndromeassociated malignancies.4 biallelic vhl inactivation caused by genetic and epigenetic alterations ( including dna methylation , histone modications , and coincidental loss of genes localized adjacent to the vhl chromosome locus ) has been described.2 in both hereditary and sporadic tumors , vhl mutations are heterogeneous.5 the encoded vhl protein ( pvhl ) plays an important role in ubiquitination and proteosomal degradation of hypoxia inducible factor - 1 ( hif - 1 )  . 
our aim was to investigate the ( epi ) genetic background of this patients disease and to determine whether the identied vhl mutation could be a driving mutation in this patients fdcs . methods case report in 2013 , a 29 - year - old female patient presented with a large abdominally located mass and multiple hepatic lesions . 
a histologic biopsy from a liver lesion showed an epithelioid and spindle cell malignant neoplasm with scattered lymphocytes . immunohistochemistry ( ihc ) results matched the pattern of fdcs with exfollicular dendritic cell markers cd21 , pression of cd23 , and cd359 ( fig 1a - c )  . 
on the basis of radiologic diagnosis , the pattern did not t an rcc . this was further supported by a negative paired box gene 8 ( pax8 ) ihc result ( fig 1d )  . the clinical course of our patients disease is outlined in figure 2 . 
the second - line treatment was pazopanib , which resulted in unexpected stable disease for 22 months . rendering additional the patient participated in the dutch national center for personalized cancer treatment ( cpct ) - 02 program , 10 tumor sampling for whole - genome sequencing ( wgs ) analysis before and after treatment with pazopanib . 
wgs was performed on the illumina hiseq x platform with 100 ng dna as input using standard protocols ( pairedend 2 150 base pairs ; illumina , san diego , ca )  . within the framework of cpct - 02 , all germline variants are ltered , which guarantees that only somatic variants are reported . 
hif - 1 ihc showed positive nuclear staining ( e ) , and glucose transporter 1 ( glut1 ) ihc showed positive staining ( f )  . results genomic landscape the identied c.119delc vhl frameshift mutation led to a truncated pvhl ( p.pro40fs ; fig 4 )  . 
both biopsies showed genome - wide aberrations , with large chromosomal copy number alterations , structural rearrangements , and mutations in a broad spectrum of loci ( fig 5 )  . 
the patient received eight cycles of chop ( cyclophosphamide , doxorubicin , vincristine , prednisone ) , which resulted 6 months later ( t6 ) in metabolic complete remission ( cr )  . 
after that ( t36 ) , the patient showed pd and participated in a phase 1 trial with immunotherapy ( cd40 agonistic monoclonal antibody ) in combination with vanucizumab ( anti - angiopoietin - 2 and anti - vegf [ vascular endothelial growth factor ] ) for 11 months . 
both results provide circumstantial evidence for functional loss of pvhl.7 , 10 discussion our analysis revealed a unique patient with fdcs harboring a somatic functional vhl aberration , which is the rst description of a vhl mutation in sarcoma.14 because fdcs is a rare mesenchymal neoplasm with a largely unknown and rather complex genetic landscape15 and is unknown for harboring vhl aberrations , we veried specic ihcbased markers to conrm the diagnosis and to reject metastatic rcc ( mrcc ) as an alternative diagnosis . 
the positive hif - 1 ihc result may indicate a functional loss of pvhl ; in this patient , it was a consequence of mutation and potential methylation - derived changes leading to biallelic vhl gene inactivation . 
although most evidence in fdcs has been found for the involvement of the ras / raf signaling pathway , 16 , 17 we did not identify mitogen - activated protein kinase ( mapk ) alterations in our patient . 
a mutant allele - specic imbalance as a consequence of allele - specic amplication has been described.18 , 19 however , this seems to be a more common aspect with activating mutations . 
recurrent vhl locus amplications have not been described in ccrcc ; therefore , it does not seem to be a common aberration . however , these reports did not specically investigate post - tyrosine kinase samples.2 , 20 , 21 numerous nonspecic chromosomal translocations were present in the pretreatment biopsy , with a remarkable decline in the number of structural variants ( svs ) following treatment with pazopanib . 
treatment of cancer may have an inuence on involved processes and subsequently on patterns and frequency of svs.22 alterations in the number of svs between time points in a patients malignancy may also be a consequence of tumor evolution and selective survival of subclonal populations as a result of the selective pressure of treatment , similar to variable somatic alterations that cause the emergence of drug resistance.23 the changes in the genomic landscape between the two biopsies obtained in our patient could be associated with the observed disease response during treatment with pazopanib.24 moreover , the fact that the mutated vhl gene has been amplied in our patients disease over time emphasizes its importance and potential as a driver mutation . upregulated vegf is associated with the hif pathway . 
pazopanib , a multikinase inhibitor with activity against the vegf receptor and platelet - derived growth factor and receptors , is effective in the treatment of mrcc25 and metastatic sarcoma.26 during treatment with pazopanib , our patient had stable disease for 22 months . 
in march 2016 , progressive disease was revealed on a conventional ct scan ( a ) and on a positron emission tomography scan ( b ) before the start of treatment with pazopanib . 
untranslated regions ( utr ) and exonic regions are depicted by rectangular boxes ( colored dark blue and dark gold , respectively ) , and intronic regions are depicted by black lines . 
 ( c ) protein sequences of wild - type ( wt ) pvhl and mutant pvhl , colored by protein domains ; ( gxeex ) 8 is shown in light blue , - domain in light gold , and - domain in red . 
 ( the vhl gene model is on the basis of ensembl transcript nm_000551.2 and the protein model is on the basis of the uniprotkb p40337 entry . ) red asterisk indicates stop codon . 
moreover , saygin et al27 performed a pooled analysis of data from 462 patients with fdcs , showing a median survival for patients with metastatic disease of 9 months ( range , 0.25 to 72 months ) and a 2 - year survival rate of 15.8% ; these results suggest that the long pfs is not the result of better prognostic features of fdcs . 
the relatively long pfs during our patients last treatment , which consisted of the combination of a cd40 agonistic monoclonal antibody and an antiangiopoietin - 2 and anti - vegf bispecic monoclonal antibody , could possibly be explained by the effect of the latter drug ( vanucizumab )  . 
these data imply that the vhl mutation in our patient may predict a biologic behavior more similar to mrcc than to a metastatic sarcoma in response to treatment with pazopanib . 
these ndings are not sufcient to make an argument for vegf - targeted therapies in fdcs , because this is the rst case of a vhl - mutated fdcs as far as we know . indirect evidence for pvhl functional although hif stabilization and glut1 accumulation are , at the most , loss , a limitation of this case report is the absence of methylation analysis looking further into epigenetic silencing of the vhl gene . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . florence atra travel , accommodations , expenses : abbvie , astellas pharma paul j . 
van diest consulting or advisory role : panterhei ( inst ) patents , royalties , other intellectual property : ddx3 as a biomarker for cancer and methods related thereto ( inst ) geert j.l.h. 
we thank hartwig medical foundation for providing wholegenome sequencing data and for performing analysis of these data . moreover , this patient was entered into the center for personalized cancer treatment ( cpct ) - 02 study . 
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the tumor was negative for programmed death ligand 1 ( pd - l1 ) expression ( tumor proportion score , 1% ) .24 postoperative imaging 6 weeks later revealed two lowdensity liver lesions measuring 1.5 cm ( segment 4a ) and 1.9 cm ( segment 2 ; fig 2a ) , which were hypermetabolic on an 18 - uorodeoxyglucose ( fdg ) positron emission tomography ( pet ) / computed tomography scan . 
after two cycles of carboplatin and etoposide , 10 new liver lesions were evident on imaging ( fig 2b )  . he subsequently enrolled in a phase ii study of pembrolizumab ( 10 mg / kg intravenously [ iv ] every 2 weeks ) for patients with msi - h tumors ( clinicaltrials.gov identier : nct01876511 )  . 
these required discontinuation of pembrolizumab after 10 months despite ongoing disease response to treatment ( fig 2d )  . hypothyroidism the patient underwent total proctocolectomy ; both tumors were t1n0 . 
msi testing ( internal ucsf500 assay ; ucsf clinical cancer genomics laboratory ) and mmr protein immunohistochemistry of the resected colon cancer sample conrmed msi - h status and loss of mlh1 expression in the tumor and normal samples . 
by cycle 11 of pembrolizumab , worsening hypothyroidism was evident , peaking at cycle 13 with thyroidstimulating hormone levels of 79.2 iu / ml ( free t4 , 0.6 ng / dl ; total t3 , 63 ng / dl )  . 
 new - onset diabetic ketoacidosis / insulin - dependent diabetes mellitus arthritis whitman et al at 10 months after starting pembrolizumab ( cycle 19 ) , the patient presented with diabetic ketoacidosis ( dka ) ; he had blood glucose levels  . 
given the lack of data supporting use in this setting , corticosteroids were not immediately started , although grade 4 autoimmune type 1 insulin - dependent diabetes mellitus was suspected.25 pembrolizumab was discontinued , and daily insulin therapy was initiated . myocarditis the patient experienced bradyarrhythmias with complete heart block while recovering from dka . 
the troponin level was elevated at 7.4 g / l ( normal , 0.05 g / l ) , the electrocardiogram showed new inferior q waves , and the echocardiogram revealed a depressed ejection fraction ( 30% - 35% ) and regional wall motion abnormalities consistent with coronary artery disease . 
he was monitored with serial echocardiograms , electrocardiograms , and troponin levels for 20 months without evidence of recurrent myocarditis . at 5 months after treatment initiation , the patient noticed grade 1 stiffness and discomfort in his knees , ankles , hands , and bilateral wrists . 
differential diagnosis favored an autoimmune process , with an elevated erythrocyte sedimentation rate of 77 mm / h ( normal , 10 mm / h ) and a c - reactive protein level of 98.0 mg / l ( normal , 6 mg / l )  . 
within 24 hours of initiating corticosteroids for myocarditis , he experienced near - complete resolution of arthritic symptoms . autoimmune hepatitis at 4 months after discontinuation of immunotherapy , and within 3 weeks of completing the initial corticosteroid taper for myocarditis and arthritis , the patient was admitted for asymptomatic grade 3 transaminitis , with an alt level of 990 u / l ( normal , 60 u / l ) and an ast level of 791 u / l ( normal , 42 u / l )  . he was treated with high - dose iv methylprednisolone followed by prednisone 1 mg / kg orally ( tapered over 10 months ) with rapid improvement in liver function tests . 
work - up was negative for cytomegalovirus ; epstein - barr virus ; and hepatitis a , b , c , d , and e infections . follow - up imaging revealed two residual 18f - fdgpost - treatment petnegative liver lesions ( 1 cm and 0.6 cm ; fig 4b )  . 
computed tomography ( ct ) scan of abdomen / pelvis ( a ) 6 weeks after resection of primary tumors ( two lesions , largest was 1.9 cm ) , prechemotherapy ; ( b ) after two cycles of carboplatin / etoposide ( at least 10 lesions , largest was 3 cm ) ; ( c ) after three cycles of pembrolizumab ; and ( d ) after 11 cycles ( 5 months ) of pembrolizumab . responses to checkpoint blockade in ls are hypothesized to be the result of higher mutational load as a result of mmr deciency , which is associated with more mutationassociated neoantigens . 
his arthritis is controlled with methotrexate , and he remains on insulin for type 1 diabetes mellitus and on levothyroxine for hypothyroidishis myocarditis and hepatitis are in remission . discussion presence of a germline mutation in the mmr gene mlh1 with loss of the wild - type allele in the nec component suggests that the nec developed as a consequence of ls in this patient . this case , coupled with data suggesting that approximately 10%26 - 29 of gi necs ( including mixed adenoneuroendocrine carcinomas ) are msi - h ( predominantly from mlh1 methylation ) , underscores the need to consider ls in patients with colorectal nec and the potential value of msi testing in these tumors . 
checkpoint inhibitor ( cpi ) therapy is compelling given the paucity of treatments for platinum - refractory colorectal nec , the tissue / site - agnostic approval of pembrolizumab for msi - h tumors , 30 and evidence suggesting that cpis have activity in mmr - decient / msi - h cancers whether associated with ls or developing sporadically ( eg , in the setting of mlh1 promoter methylation31 - 33 )  . our patient experienced ve iraes . 
regarding immunotherapy - related diabetes , one series suggested that gad65 autoantibodies were present in 60% of patients and that a small proportion harbored a cd8 + t - cell response to a t1dm antigen.34 , 35 gad65 autoantibodies were absent in our patient , suggesting a lower probability of undiagnosed autoimmune disease and underscoring the need for validated markers of response and toxicity for patients treated with cpis.36 the relationship between toxicity and efcacy remains ill dened37 ( appendix )  . 
whether underlying ls increased the risk for iraes is unclear , but a germline mlh1 mutation could theoretically predispose a patient to mutations in normal tissue that are immunologically recognized . 
however , previous reports of cpi therapy in patients with msi - h tumors have not suggested a higher incidence of iraes in this population.5 , 31 , 32 , 38 the patient was treated with a relatively high dose ( 10 mg / kg iv every 2 weeks ) of pembrolizumab compared with the us food and drug administration approved dose for msi - h cancers ( 200 mg iv every 3 weeks ) ; this difference also may have contributed to iraes . jco precision oncology fig 3 . 
n engl j med 363 : 711 - 723 , 2010 topalian sl , hodi fs , brahmer jr , et al : safety , activity , and immune correlates of antipd - 1 antibody in cancer . 
n engl j med 366 : 2443 - 2454 , 2012 brahmer j , reckamp kl , baas p , et al : nivolumab versus docetaxel in advanced squamous - cell nonsmall - cell lung cancer . 
n engl j med 373 : 123 - 135 , 2015 larkin j , chiarion - sileni v , gonzalez r , et al : combined nivolumab and ipilimumab or monotherapy in untreated melanoma . 
n engl j med 372 : 2509 - 2520 , 2015 le dt , uram jn , wang h , et al : pd - 1 blockade in mismatch repair decient non - colorectal gastrointestinal cancers . 
j clin oncol 34 , 2016 ( suppl ; abstr 195 ) kl oppel g , perren a , heitz pu : the gastroenteropancreatic neuroendocrine cell system and its tumors : the who classication . 
antonia sj , l opez - martin ja , bendell j , et al : nivolumab alone and nivolumab plus ipilimumab in recurrent small - cell lung cancer ( checkmate 032 ) : a multicentre , open - label , phase 1 / 2 trial . 
paraghamian se , longoria tc , eskander rn : metastatic small cell neuroendocrine carcinoma of the cervix treated with the pd - 1 inhibitor , nivolumab : a case report . 
vijayvergia n , dasari a , ross ea , et al : pembrolizumab ( p ) monotherapy in patients with previously treated metastatic high grade neuroendocrine neoplasms ( hg - nens )  . 
mulvey c , raj np , chan ja , et al : phase ii study of pembrolizumab - based therapy in previously treated extrapulmonary poorly differentiated neuroendocrine carcinomas : results of part a ( pembrolizumab alone )  . 
kaufman hl , russell js , hamid o , et al : updated efcacy of avelumab in patients with previously treated metastatic merkel cell carcinoma after 1 year of follow - up : javelin merkel 200 , a phase 2 clinical trial . 
kervarrec t , samimi m , gaboriaud p , et al : detection of the merkel cell polyomavirus in the neuroendocrine component of combined merkel cell carcinoma . virchows arch 472 : 825 - 837 , 2018 24 . 
j immunother cancer 5 : 40 , 2017 jesinghaus m , konukiewitz b , keller g , et al : colorectal mixed adenoneuroendocrine carcinomas and neuroendocrine carcinomas are genetically closely related to colorectal adenocarcinomas . 
la rosa s , marando a , furlan d , et al : colorectal poorly differentiated neuroendocrine carcinomas and mixed adenoneuroendocrine carcinomas : insights into the diagnostic immunophenotype , assessment of methylation prole , and search for prognostic markers . 
overman mj , mcdermott r , leach jl , et al : nivolumab in patients with metastatic dna mismatch repairdecient or microsatellite instabilityhigh colorectal cancer ( checkmate 142 ) : an open - label , multicentre , phase 2 study . 
puzanov i , diab a , abdallah k , et al : managing toxicities associated with immune checkpoint inhibitors : consensus recommendations from the society for immunotherapy of cancer ( sitc ) toxicity management working group . 
overman mj , lonardi s , wong kym , et al : durable clinical benet with nivolumab plus ipilimumab in dna mismatch repair - decient / microsatellite instabilityhigh metastatic colorectal cancer . 
galon j , costes a , sanchez - cabo f , et al : type , density , and location of immune cells within human colorectal tumors predict clinical outcome . 
llosa nj , cruise m , tam a , et al : the vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter - inhibitory 313 : 1960 - 1964 , 2006 checkpoints . 
strosberg jr , coppola d , klimstra ds , et al : the nanets consensus guidelines for the diagnosis and management of poorly differentiated ( high - grade ) extrapulmonary neuroendocrine carcinomas . 
 checkpoint blockade in msi - high neuroendocrine carcinoma appendix summary us food and drug administration approvals of immunotherapy in may 2017 , the us food and drug administration approved pembrolizumab for unresectable or metastatic microsatellite instability high ( msi - h ) or mismatch repairdecient solid tumors that have progressed on prior standard treatment . 
in august 2017 , nivolumab was approved for msi - h colorectal cancer . modest activity has been demonstrated in advanced small - cell lung cancer , leading to drug approvals in rst - line ( atezolizumab plus chemotherapy ) and platinum - refractory settings.12 , 13 , 20 - 22 checkpoint inhibitors have an established role in merkel cell carcinoma , which is a virally mediated disease.21 - 23 beyond that , the role of immunotherapy in extrapulmonary neuroendocrine carcinomas ( necs ) remains ill dened . 
however , three recent pilot studies suggest minimal activity ( response rate , 10% ) of singleagent checkpoint inhibitors in unselected patients with extrapulmonary necs.15 - 17 a growing body of literature has served to clarify the nature and optimal management of immune - related adverse events . 
some reports suggest that patients with grade 3 immune - related adverse events experience longer median time to progression and higher radiographic response . recent biomarker data suggest a high rate of programmed death 1 ( pd1 ) / programmed death ligand 1 expression in poorly differentiated necs of the gi tract ( roberts ja , et al : hum pathol 70 : 49 - 54 ) and a greater proportion of msi - h in high - grade necs , mixed adenoneuroendocrine carcinomas , and insulinomas than in low - grade gastroenteropancreatic neuroendocrine tumors ( weber mm , fottner c : oncol res treat 41 : 306312 , 2018 )  . 
 o egfr genotyping of matched urine , plasma , and tumor tissue in patients with nonsmall - cell lung cancer treated with rociletinib , an egfr tyrosine kinase inhibitor jonathan w . 
wakelee author affiliations and support information ( if applicable ) appear at the end of this article . ( continued ) purpose liquid biopsies represent an attractive alternative to tissue biopsies , particularly rebiopsies , in determining patient eligibility for targeted therapies . 
we evaluated epidermal growth factor receptor ( egfr ) t790m detection in matched urine , plasma , and tissue and the clinical outcomes of patients with advanced nonsmall - cell lung cancer treated with rociletinib . methods tissue ( n = 540 ) , plasma ( n = 482 ) , and urine ( n = 213 ) were collected from evaluable patients enrolled in tiger - x , a phase i / ii study . 
genotyping was performed by therascreen egfr testing in tissue , beaming in plasma , and a quantitative short footprint assay ( trovera ) in urine , which was used to further examine discordant samples . results positive percent agreement with tissue t790m results was similar for urine ( 82% ; 142 of 173 ) and plasma ( 81% ; 313 of 387 ) genotyping . 
the ability to identify mutations in plasma was strongly associated with m stage ( p < .001 ) ; rate of t790m detection for patients with m1a / m0 disease increased from 54% for plasma alone to 85% when urine and plasma were both examined . 
objective response rates of patients who were t790m positive were comparable between tumor ( 34% ) , plasma ( 32% ) , and urine ( 37% )  . conclusion clinical response to rociletinib was comparable irrespective of whether t790m status was identified by liquid or tissue biopsy . 
combined , urine and plasma identified a higher percentage of patients who were t790m positive than tumor genotyping alone and improved detection of t790m , particularly in the absence of distant metastases . 
2018 by american society of clinical oncology introduction although tyrosine kinase inhibitors ( tkis ) targeting the epidermal growth factor receptor ( egfr ) have transformed the treatment of patients with egfr - mutant nonsmall - cell lung cancer ( nsclc ) , clinical responses are frequently brief . 
acquired resistance often develops within 10 to 16 months of treatment initiation.1 - 4 emergence of the gatekeeper egfr t790m mutation , which increases the atp - binding affinity of the oncogenic receptor , accounts for resistance in most patients.5 , 6 several third generation , mutant - selective inhibitors have been developed to target t790m , including osimertinib , rociletinib , nazartinib ( egf816 ) , avitinib ( ac0010 ) , olmutinib ( hm61713 / bi1482694 ) , asp8273 , and pf06747775.7 - 11 these agents target egfr - activating and t790m - resistance mutations , while largely sparing the wild - type receptor and minimizing related on - target toxicities . 
wakelee , md , stanford university , thoracic oncology , 875 blake wilbur dr , rm 2233 mc 5826 , stanford , ca 94305 ; e - mail : hwakelee@stanford.edu. t790m mutationpositive nsclc has been impressive and led to the accelerated approval of osimertinib in the united states.12 given their predictive value , the ability to detect driver and resistance egfr mutations is paramount in improving patient outcomes . 
tumor tissue genotyping is the current standard of care ; however , tumor tissue genotyping can be associated with biopsy - related complications ( ie , pneumothorax , bleeding , and infection ) because of its invasive nature.13 , 14 it is estimated that 20% of tissue biopsies are uninformative as a result of tumor inaccessibility , inadequate sample collection , or intratumoral heterogeneity.15 , 16 these challenges are pronounced in patients who have developed egfr - tki resistance and require a rebiopsy . 
in a study of 120 eligible patients experiencing disease progression while receiving an egfr - tki , 45 ( 38% ) did not undergo rebiopsy because of inaccessible tumor sites , patient refusal , or decision of the treating physician.17 the noninvasive genotyping of circulating tumor dna ( ctdna ) in plasma can identify predictive biomarkers while improving the patient experience . 
accordingly , plasma - based egfr mutation tests have been approved for use as companion diagnostics by health authorities in europe and the united states.18 ctdna from blood undergoes glomerular filtration in the kidney and can be detected in urine . 
 the feasibility of using urine to detect somatic alterations identified in matched plasma and / or tumor has been demonstrated in proof - of concept studies , unveiling urine as an alternative specimen type that can be collected with fewer constraints than tissue or plasma.19 - 21 previously , we reported that egfr mutations can be detected with high sensitivity in urine of patients with advanced nsclc using a short footprint mutation enrichment next - generation sequencing assay ( trovera ; trovagene , san diego , ca ) .19 in the tiger - x phase i / ii study , we examined the clinical utility of t790m urine genotyping in a large cohort of patients with advanced nsclc who received rociletinib . methods patients and study design for this analysis of patients enrolled in the tiger - x trial ( clinicaltrials.gov identifier : nct01526928 ) , all patients initiating treatment with rociletinib ( 500 , 625 , or 750 mg twice a day hydrobromide formulation ) were evaluable . 
confirmed objective response rate ( orr ) , progression - free survival ( pfs ) , and duration of response ( dor ) were assessed per response evaluation criteria in solid tumors version 1.1 ( recist ) by the investigators . 
tiger - x was approved by the institutional review board at each study site and conducted in accordance with the declaration of helsinki and good clinical practice guidelines of the international conference on harmonization . 
additional details regarding sample collection and processing are described elsewhere.19 statistical analysis clopper - pearson methods were used to obtain percentage point estimates and the 95% ci for analysis of orr as well as for percent agreement between two sample types . 
 tiger - x safety population : all patients treated at 500 , 625 , or 750 mg twice a day ( n = 548 ) did not participate in urine collection ( n = 335 ) clinical site did not submit biopsy ( n = 8 ) clinical site did not submit plasma ( n = 66 ) patients with pretreatment urine submitted for egfr testing by trovera ( n = 213 ) patients with pretreatment tumor submitted for egfr testing by therascreen ( n = 500 ) or cobas ( n = 40 ) patients with pretreatment plasma submitted for egfr testing by beaming ( n = 482 ) patients with matched urine and tumor for diagnostic comparison ( n = 209 ) patients with matched plasma and tumor for diagnostic comparison ( n = 475 ) fig 1 . 
the tiger - x safety population included all patients who received at least one dose of rociletinib and who were treated at 500 , 625 , or 750 mg twice a day . 
egfr , epidermal growth factor receptor . patients with matched urine and plasma for diagnostic comparison ( n = 181 ) patients with matched urine , plasma , and tumor for diagnostic comparison ( n = 177 ) results patient population among 548 evaluable patients , 540 tissue , 482 plasma , and 213 urine pretreatment samples were submitted for central egfr testing ( fig 1 )  . 
the biomarker analysis population was enriched for patients who were t790m positive by tumor genotype ( 500 of 548 with local and / or central tumor t790m - positive result ; 91.2% ; data supplement ) relative to an unselected population with acquired resistance to egfr - tkis . egfr mutation detection in liquid and tissue biopsies to examine the clinical validity of urine and plasma testing , concordance with tissue egfr genotyping results was evaluated . 
these 23 patients were retested on a second plasma test platform ( trovera ) , and 22 ( 95.7% ) were confirmed to be t790m positive ( data supplement )  . next , we compared urine and tumor egfr results . 
 a negative / inadequate by all 7 ( 4.0% ) positive by urine positive by plasma positive by tissue positive by plasma or urine positive by tissue or plasma positive by tissue or urine positive by at least one fig 2 . 
among the 41 patients who were t790m discordant , we observed an even split between patients who were t790m urine positive / plasma negative ( n = 21 ) and urine negative / plasma positive ( n = 20 )  . 
urine af was generally lower than plasma af ( p < .001 ; data supplement ) independent of test platform ( data supplement ) , which may reflect postrenal shedding of nontumor cell - free dna from the bladder into urine , contributing to background.23 among 43 patients for whom urine af exceeded the matched plasma af , no target or nontarget lesions were identified in the urinary system ( data supplement )  . a three - way diagnostic comparison was conducted for the patients with matched tumor , plasma , and urine . 
in addition , genotyping of each sample type uniquely identified patients who were t790m positive , including eight by tumor , five by urine , and three by plasma ( fig 2b )  . clinical response in patients who are t790m positive and t790m negative identified by different sample types all patients with central t790m genotyping were considered evaluable for efficacy . 
at risk : plasma 120 116 tissue 150 143 urine 62 plasma 374 282 192 135 tissue 443 354 255 182 125 urine 169 132 censored plasma tissue urine fig 3 . 
the high relative response rate of patients who were tumor t790m centrally negative was likely because most patients ( 59.5% ; 25 of 42 ) had a known local t790m - positive tumor result . 
in urine , no significant correlation between t790m : act and depth of response was found ( data supplement )  . clinical characteristics and mutation detection in liquid biopsies next , we examined clinical characteristics potentially predictive of ability to identify mutations in urine and plasma . 
patients in whom t790m was identified in plasma also had a greater median baseline tumor burden than those without detectable t790m ( as measured by the sum of longest diameters , 65 v 43 mm , respectively ; p < .001 ; data supplement )  . 
 this complementarity reflects the differences in plasma and urine ctdna levels within patients , which may depend on the rates of tumor dna shedding , ctdna degradation in blood versus urine , renal filtration , and excretion of ctdna in urine.23 the ppa for t790m detection between urine and plasma with tumor as reference was 82% and 81% , respectively ( table 1 )  . 
the ppa for plasma in this study was somewhat higher than previously described for t790m ( 70% to 73% ) in a similar patient population using the same testing methodology ( beaming ) .22 , 27 this difference could be explained by the substantially higher median tumor burden and frequency of distant metastatic disease observed compared with our previous report.22 this observation illustrates that clinical characteristics should be appropriately considered when comparing plasma ctdna assay sensitivities across different studies . in contrast to the high sensitivity of detection , the npa for t790m in urine and plasma with tumor as the reference was 31% and 43% , respectively . 
 the low apparent specificity likely reflects the subclonal , heterogeneous nature of the t790m resistance mutation , which can give rise to a false - negative result in a tumor biopsy because of sampling error.24 , 28 notably , for the majority of patients who were discordant t790m plasma or urine positive / tumor negative , the mutation was detected by at least one other test platform , and those patients had a demonstrable response to rociletinib , suggesting that they are indeed tumor false negative and urine / plasma true positive . 
patients identified as t790m negative by central tumor testing displayed a lower orr when treated with rociletinib as compared with patients who were centrally genotyped t790m tumor positive ( 21% v 34% )  . 
however , a formal comparison was not performed , because 60% of centrally genotyped , t790m tumor - negative patients had a locally assessed t790m - positive result in the same biopsy , confounding analysis of clinical outcomes in this cohort . 
discordance between local and central tumor results occurred in < 5% of evaluable patients and may be attributable to molecular heterogeneity between tumor sections , low tumor cellularity within the specimen , differential test sensitivities , or a combination of these . 
when stratified on the basis of local tissue test results , patients who were centrally negative with a corresponding locally negative or locally unknown t790m result had an orr of 6% . 
consistent with our previous findings in smaller patient cohorts , 22 m stage was a strong predictor of the ability to identify mutations in plasma ; t790m was more readily detected in patients with m1b ( 89% ) versus m1a / m0 ( 54% ) disease . 
urine testing identified both patients with m1a / m0 and m1b disease with high sensitivity ( 74% and 83% , respectively ) , suggesting that all patients would be good candidates for a urine test or for a combination of urine and plasma tests , where the combined sensitivity ( 85% and 94% for m1a / m0 and m1b , respectively ) was even higher than for either specimen alone . the cobas egfr mutation test v2 using plasma specimens recently became the first liquid biopsy test approved by the us food and drug administration as a companion diagnostic to identify patients with t790m - positive metastatic nsclc eligible for treatment with osimertinib.29 if after disease progression while receiving firstor second - generation egfrtkis patients receive plasma - based t790m genotyping , with consideration of additional urine analysis to increase sensitivity , this would simultaneously circumvent the pitfalls associated with tissue rebiopsies and minimize the test turnaround time for patients who test t790m positive or who are not candidates for tumor rebiopsy . 
wakelee between a scheduled percutaneous tumor biopsy and receipt of an egfr result by the treating physician.30 , 31 in our study , all three sample types were found to identify unique patients who were t790m positive . 
on the basis of a recent pharmacoeconomic assessment , urine testing alone obviates the need for rebiopsy in 48% of patients progressing on first - generation egfr - tkis.32 this not only improves patient experience but also results in a twoto 10 - fold cost savings as a result of a reduction in expenses incurred for biopsy - related complications and tissue - based molecular tests.32 one limitation is that an enriched population of patients with known tumor t790m mutations was enrolled ; therefore , our study was not optimally positioned to investigate clinical outcomes of patients who were t790m positive by liquid biopsy alone . 
goldman consulting or advisory role : astrazeneca , bristol - myers squibb , clovis oncology , genentech , lilly , trovagene , vortex biosciences , amgen speakers ' bureau : merck research funding : eli lilly , genentech , astex pharmaceuticals , clovis oncology , bristol - myers squibb , astrazeneca / medimmune , threshold pharmaceuticals , array biopharma , celgene , abbvie , astellas pharma , corvus pharmaceuticals chris karlovich employment : clovis oncology stock and other ownership interests : clovis oncology lecia v . 
sequist honoraria : astrazeneca consulting or advisory role : astrazeneca , genentech , bristol - myers squibb , pfizer research funding : boehringer ingelheim ( inst ) , clovis oncology ( inst ) , genentech ( inst ) , merrimack ( inst ) , novartis ( inst ) , astrazeneca ( inst ) , johnson & johnson ( inst ) , merck ( inst ) , pfizer ( inst ) vlada melnikova employment : trovagene aleksandra franovic employment : guardant health , trovagene , ignyta shirish m . 
gadgeel consulting or advisory role : pfizer , boehringer ingelheim , genentech , ariad pharmaceuticals , astrazeneca , bristol - myers squibb speakers ' bureau : astrazeneca research funding : pfizer ( inst ) , clovis oncology ( inst ) , merck ( inst ) , genentech ( inst ) , incyte ( inst ) , millennium ( inst ) , astrazeneca / medimmune ( inst ) , bristol - myers squibb ( inst ) , halozyme ( inst ) , acerta pharma ( inst ) , acea biosciences ( inst ) , janssen oncology ( inst ) , novartis ( inst ) , five prime therapeutics ( inst ) , oncomed ( inst ) travel , accommodations , expenses : ariad pharmaceuticals / takeda , genentech karen l . 
reckamp consulting or advisory role : amgen , boehringer ingelheim , ariad pharmaceuticals , astellas pharma , nektar , euclises pharmaceuticals , tesaro research funding : bristol - myers squibb ( inst ) , pfizer ( inst ) , novartis ( inst ) , ariad pharmaceuticals ( inst ) , clovis oncology ( inst ) , xcovery ( inst ) , gilead sciences ( inst ) , pfizer ( inst ) , adaptimmune therapeutics ( inst ) , genentech ( inst ) , boehringer ingelheim , abbvie d . 
ross camidge honoraria : roche , g1 therapeutics , mersana therapeutics , takeda research funding : takeda maurice prol consulting or advisory role : eli lilly , genentech , pfizer , astrazeneca , boehringer ingelheim , merck sharp & dohme , bristol - myers squibb , novartis , amgen research funding : astrazeneca ( inst ) , roche ( inst ) travel , accommodations , expenses : astrazeneca , roche , bristol - myers squibb sai - hong ignatius ou honoraria : pfizer , roche pharma ag , genentech , araid / takeda , novartis , astrazeneca , foundation medicine consulting or advisory role : pfizer , roche / genentech , novartis , astrazeneca , takeda / ariad , foundation medicine speakers ' bureau : genentech , astrazeneca , takeda / araid research funding : pfizer ( inst ) , roche pharma ag ( inst ) , astrazeneca / medimmune ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , ariad pharmaceuticals ( inst ) , ignyta ( inst ) , peregrine pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , astellas pharma ( inst ) , chugai pharmaceutical ( inst ) stephen v . 
langer honoraria : bristol - myers squibb , genentech , eli lilly / imclone research funding : clovis oncology , astrazeneca , astellas pharma , incyte , eli lilly jean - charles soria employment : medimmune honoraria : roche , astrazeneca , sanofi , servier , pierre fabre mark a . 
socinski honoraria : genentech , bristol - myers squibb , celgene , astrazeneca consulting or advisory role : genentech speakers ' bureau : genentech , bristol - myers squibb , astrazeneca , boehringer ingelheim research funding : pfizer ( inst ) , genentech tarek m . 
mekhail honoraria : genentech , eli lilly , bristol - myers squibb , merck , boehringer ingelheim speakers ' bureau : genentech , eli lilly , bristol - myers squibb , merck benjamin j . 
solomon honoraria : bristol - myers squibb , astrazeneca consulting or advisory role : bristol - myers squibb ( inst ) , merck sharp & dohme ( inst ) , astrazeneca , pfizer ( inst ) , genentech ( inst ) , eisai research funding : pfizer ( inst ) patents , royalties , other intellectual property : royalties from veristrat ( biodesix ) travel , accommodations , expenses : astrazeneca , roche , merck , bristol - myers squibb , novartis consulting or advisory role : genentech , eli lilly / imclone , merck , abbott biotherapeutics , bayer healthcare pharmaceuticals / onyx , clarient , clovis oncology , celgene , cancer support community , bristol - myers squibb , ariad pharmaceuticals , takeda , astrazeneca research funding : merck ( inst ) , advantagene ( inst ) , clovis oncology ( inst ) , celgene ( inst ) , inovio pharmaceuticals ( inst ) , ariad pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , genentech ( inst ) , stemcentrx ( inst ) other relationship : eli lilly , amgen , peregrine pharmaceuticals , synta pharmaceuticals joel w . 
neal consulting or advisory role : clovis oncology , boehringer ingelheim , armo biosciences , nektar , ariad pharmaceuticals / takeda , caret , astrazeneca , eli lilly research funding : genentech , merck , arqule , novartis , boehringer ingelheim , nektar , exelixis , ariad pharmaceuticals / takeda darrin despain employment : clovis oncology stock and other ownership interests : clovis oncology sergey yurasov employment : clovis oncology , immune design leadership : immune design stock and other ownership interests : clovis oncology , immune design jason b . 
natale consulting or advisory role : astrazeneca , eli lilly travel , accommodations , expenses : clovis oncology , juno therapeutics research funding : amgen , astrazeneca , bristol - myers squibb ( inst ) gregory a . 
otterson consulting or advisory role : amgen ( inst ) , novartis ( inst ) , takeda ( inst ) , merck ( inst ) research funding : genentech ( inst ) , pfizer ( inst ) , bristolmyers squibb ( inst ) , clovis oncology ( inst ) , novartis ( inst ) , acerta pharma ( inst ) , ignyta ( inst ) , merck ( inst ) vassiliki papadimitrakopoulou consulting or advisory role : clovis oncology , genentech , merck , biothera pharmaceuticals , eli lilly , janssen , genentech , gensignia life sciences , astrazeneca , ariad pharmaceuticals , nektar research funding : merck ( inst ) , novartis ( inst ) , celgene ( inst ) , clovis oncology ( inst ) , bayer healthcare pharmaceuticals ( inst ) , bristol - myers squibb ( inst ) , astrazeneca ( inst ) , pfizer ( inst ) , janssen oncology ( inst ) , acea biosciences ( inst ) mark erlander employment : trovagene leadership : trovagene stock and other ownership interests : trovagene mitch raponi employment : clovis oncology , beigene stock and other ownership interests : clovis oncology , beigene heather a . 
natale , cedars - sinai medical center , los angeles ; vlada melnikova , aleksandra franovic , and mark erlander , trovagene , san diego ; karen reckamp , city of hope comprehensive cancer center , duarte ; sai - hong ignatius ou , university of california irvine school of medicine , orange ; joel w . 
gadgeel , karmanos cancer institute , wayne state university , detroit , mi ; maurice prol , centre lon brard , lyon ; jean - charles soria , institut gustave roussy , villejuif , france ; stephen v . 
rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as first - line treatment for european patients with advanced egfr mutation - positive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
yu ha , arcila me , rekhtman n , et al : analysis of tumor specimens at the time of acquired resistance to egfr - tki therapy in 155 patients with egfr - mutant lung cancers . 
park k , lee j , lee k , et al : updated safety and efficacy results from phase i / ii study of hm61713 in patients ( pts ) with egfr mutation positive non - small cell lung cancer ( nsclc ) who failed previous egfr - tyrosine kinase inhibitor ( tki )  . 
tan ds - w , yang j , leighl nb , et al : first - in - human phase i study of egf816 , a third generation , mutant - selective egfr tyrosine kinase inhibitor , in advanced nonsmall - cell lung cancer ( nsclc ) harboring t790m . 
douillard jy , ostoros g , cobo m , et al : gefitinib treatment in egfr mutated caucasian nsclc : circulating - free tumor dna as a surrogate for determination of egfr status . 
yoon hj , lee hy , lee ks , et al : repeat biopsy for mutational analysis of non - small cell lung cancers resistant to previous chemotherapy : adequacy and complications . 
kawamura t , kenmotsu h , taira t , et al : rebiopsy for patients with non - small - cell lung cancer after epidermal growth factor receptor - tyrosine kinase inhibitor failure . 
janku f , vibat cr , kosco k , et al : braf v600e mutations in urine and plasma cell - free dna from patients with erdheim - chester disease . 
hyman dm , diamond el , vibat cr , et al : prospective blinded study of brafv600e mutation detection in cell - free dna of patients with systemic histiocytic disorders . 
karlovich c , goldman jw , sun jm , et al : assessment of egfr mutation status in matched plasma and tumor tissue of nsclc patients from a phase i study of rociletinib ( co - 1686 )  . 
su yh , wang m , brenner de , et al : human urine contains small , 150 to 250 nucleotide - sized , soluble dna derived from the circulation and may be useful in the detection of colorectal cancer . 
piotrowska z , niederst mj , karlovich ca , et al : heterogeneity underlies the emergence of egfr t790 wild - type clones following treatment of t790m - positive cancers with a third generation egfr inhibitor . 
tseng js , yang ty , tsai cr , et al : dynamic plasma egfr mutation status as a predictor of egfr - tki efficacy in patients with egfr - mutant lung adenocarcinoma . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced non - small - cell lung cancer . 
suda k , murakami i , katayama t , et al : reciprocal and complementary role of met amplification and egfr t790m mutation in acquired resistance to kinase inhibitors in lung cancer . 
sacher ag , paweletz c , dahlberg se , et al : prospective validation of rapid plasma genotyping for the detection of egfr and kras mutations in advanced lung cancer . 
 new medicare coverage policy for next - generation tumor sequencing : a key shift in coverage criteria with broad implications beyond medicare the centers for medicare and medicaid services ( cms ) recently issued a national coverage determination on next - generation tumor sequencing ( ngts ) tests for patients with advanced cancer ( see appendix for definitions )  . 
the cms policy provides coverage for ngts tests that received a positive us food and drug administration ( fda ) review and that have fda - approved indications for patients with advanced solid cancers . 
 more than 300 comments were submitted to cms during the public comment period on the draft issuance , and numerous commentaries and news items have been published . in this commentary , we argue that the new cms policy1 presents a key shift in coverage criteria for ngts tests and has important implications beyond medicare for private payer and medicaid coverage policies.2 our commentary draws on our prior and ongoing studies of coverage policies.1 - 3 we also use insights from a meeting held on march 9 , 2018 , and an accompanying survey conducted with the ucsf center for translational and policy research on personalized medicine ( transpers ) payer advisory board . 
the board includes senior executives from the largest private health plans , a medicaid plan , and leading regional private plans , as well as other thought leaders with expertise in coverage and reimbursement by medicare and other payers.4 we also build on our prior work that examined the broader health policy implications of the new cms policy.5 , 6 although observers have commented on the importance of the policy for private payers , there has been no assessment of the policys implications for these payers and for medicaid plans . 
our previous work showed limited coverage of ngts tests by private payers , 7 and our more recent analyses of the largest private payers ( unpublished data ) confirmed that coverage of ngts tests specifically is variable . 
some payers cover ngts tests only for specific cancer sites ( eg , nonsmall - cell lung cancer ) , and others cover only panels with a limited number of genes ( eg , fewer than 50 genes )  . 
for state medicaid plans , we are unaware of any published analyses of coverage policies for ngts tests , but coverage has been described as limited and variable.8 why the cms policy represents a key shift in coverage criteria the new cms policy represents a departure from medicares previous criteria , as well as from criteria currently used for coverage of ngts tests by private payers and medicaid plans , in several ways ( table 1 )  . notably , some private payers use criteria that were not included in the cms policy ( eg , that sequential single - gene testing must be found to be impractical for a patient before they will cover a gene panel )  . 
douglas author affiliations and support information ( if applicable ) appear at the end of this article . the views expressed in this article are his or her own and not an official position of their respective institutions or funders . corresponding author : kathryn a . 
however , contrary to a common assumption , private payers and medicaid do not always follow medicare policies because medicare decisions are only one of many factors that they consider . 
 for example , a review of 47 medicare national coverage determinations for medical devices found that medicare policies were equivalent to the corresponding private payer policies only approximately half of the time.10 medicaid policies are different for each state and thus can be even more variable . in our discussions with payers , we found that many of them are unsure of their plans or do not plan to change their policies to match the cms policy , at least in the short ter payers stated that the new cms policy provides some benefits and a step forward . 
however , they also perceived that it creates concerns that have to be addressed as a result of the significant shift in criteria used , as described in the previous section ( why the cms policy represents a key shift in coverage criteria )  . 
private payers and medicaid plans that decide to change their coverage policies to match those of cms will need to make adaptations . one key difference in the situation faced by private payers versus cms is that private payers do not have the option of the second coverage pathway that is included in the cms policy namely , the use of medicare administrative contractors to make local coverage decisions for tests not already covered by the national policy . 
 the medicare program thus has the flexibility to adapt policies to local situations , whereas private payers typically do not have the flexibility to have differing policies at the national and local levels . 
 for example , although an fda positive review is required by the medicare national coverage policy , medicare local policies may not require this level of review , whereas private payers will have to either require an fda positive review or not require it . changes in coverage policies are also likely to have ripple effects on the laboratory and medical center industries that will require adaptation by private payers and medicaid plans . 
it is possible that the cms national coverage policy requirement for a positive fda review will encourage smaller laboratories to submit their tests for approval and thus increase the number of approved tests . 
the result may be that private payers and medicaid plans have fewer testing options for their patients . the criteria used in the cms policy will also require the refinement of definitions , such as how advanced cancer is measured ( eg , by incorporating the evolving concept of tumor mutational burden in solid tumors )  . 
of particular importance will be whether there are billing codes ( ie , current procedural terminology codes ) that capture the needed information and that are consistently adopted and implemented . 
however , we recently found that there remain large gaps in the codes used for multigene tests as a category and wide variation in whether and how they are implemented.11 we also found that some payers are considering or implementing other approaches to deal with coverage issues for ngts tests that differ from the approach used by cms . 
for example , some payers are providing broader coverage but doing so while also implementing utilization management programs ( eg , preauthorization requirements , contracting agreements , and / or use of laboratory benefit managers )  . 
in the medium to long term , we expect to see an evolution of coverage such that at least some private payers and medicaid plans will establish positive coverage policies for ngts tests in at least some clinical situations . by viewing the past development of ngts policies , we observe that they have evolved over time and that the cms policy emerged from the gaps in that evolution.3 a number of experts have called for adaptation of coverage frameworks for next - generation sequencing tests , 1 , 2 , 12 and several frameworks have been proposed.12 - 14 these approaches all have pros and cons , and their acceptance and implementation have varied.3 regardless , it is clear that the lack of positive coverage policies for ngts tests ( where payers agree to pay for the test provided that criteria are met ) was a gap , and cms determined that they needed to address that gap . we can speculate that private payer coverage will evolve over time with at least some policies that provide broad coverage for ngts tests . 
private plans servicing medicare advantage plans have a mandate to cover ngts tests for those enrolleesa dichotomy in their own policy that may move them toward consistent coverage for all enrollees . 
trosman jr , weldon cb , douglas mp , et al : payer coverage for hereditary cancer panels : barriers , opportunities , and implications for the precision medicine initiative . 
trosman jr , weldon cb , kelley rk , et al : challenges of coverage policy development for nextgeneration tumor sequencing panels : experts and payers weigh j natl compr canc netw 13 : 311 - 318 , 2015 3 . 
center for translational and policy research on personalized medicine ( transpers ) at the university of california , san francisco : transpers program on evidence and reimbursement for personalized medicine . 
phillips ka : evolving payer coverage policies on genomic sequencing tests : beginning of the end or end of the beginning ? jama 319 : 2379 - 2380 , 2018 6 . 
phillips ka , deverka pa , hooker gw , et al : genetic test availability and spending : where are we now ? where are we going ? health aff ( millwood ) 37 : 710 - 716 , 2018 12 . 
after receiving institutional review boardapproved informed consent , targeted next - generation sequencing was performed on 20 patients formalin - xed parafn embedded tumors to characterize genomic alterations across 287 cancer - related genes . 
reverse phase protein array ( rppa ) analysis was performed on both the baseline biopsy and rd specimens , when available . results two of 20 rd tissues were her2 negative per next - generation sequencing ; one sample had insufcient tissue . 
although prevalence of individual tp53 and pik3ca mutations was only modestly higher than published estimates for those in her2 + primary bcs ( 55% and 32% for tp53 and pik3ca , respectively ) , prevalence of these as comutations appeared higher ( 41% ) , compared with less than 10% in several series . 
however , the pcr rate with preoperative her2 - targeted therapy and chemotherapy is only 40% to 60%.4 determining molecular alterations in cancers that persist despite preoperative therapy may dene cancer subtypes for which alternative therapies may be required and may identify important targets . 
pi3k pathway alterations have been associated with poorer outcomes in most her2 + metastatic and neoadjuvant trials , although not with adjuvant her2 - based therapy.5 - 7 tumors with either pten loss or pik3ca mutations are less likely to achieve a pcr after neoadjuvant anthracyclinetaxanebased chemotherapy plus her2 - targeted treatment , even with dual her2 - targeted treatments . 
 holmes et al context key objective to determine if potential nonresponders to therapy can be identied at baseline biopsy before initiation of her2 therapy by using a combination of genomic and proteomic assays . knowledge generated patients with her2 - positive breast cancer who do not achieve pathologic complete response ( pcr ) while receiving her2 therapy may harbor mutations and genomic amplications before therapy , or mutations may develop during or after therapy . 
thus , the presence of pik3ca / tp53 comutations is critical clinical information for selecting the optimal therapy regimen . relevance pretreatment proteomic and genomic molecular signatures reveal potential drug targets and driver mutations , respectively . tumors with pik3ca / tp53 comutations are less likely to achieve pcr and are more likely require additional therapies . 
seventeen had an evaluable amount of rd tissue for ngs and / or rppa analyses ( fig 1 ; data supplement )  . methods and materials targeted ngs was performed on hybridization - captured , adaptor ligationbased libraries using dna extracted from four 10 - micron , formalin - xed parafn - embedded ( ffpe ) sections cut from patients rd specimens ( n = 17 ) and pretreatment biopsy specimens ( n = 6 )  . 
all specimens yielded the minimum required 50 ng of dna at extraction , with the required minimum of 20% nuclei derived from tumor . all coding exons ( n = 3 , 769 ) of 287 cancer - related genes and 47 introns from 19 genes that are frequently rearranged in cancer were sequenced on the illumina hiseq platform ( san diego , ca ) at an average depth exceeding 700 times . 
sequence data were analyzed for all classes of genomic alterations , including base substitutions , short insertions / deletions , focal amplications , homozygous deletions , and gene fusions / rearrangements , as previously described.12 mutation - detection accuracy ( sensitivity and specicity ) was optimized for commonly impure clinical specimens.13 phosphoprotein expression analysis was also performed on the ffpe samples using an rppa platform at a clinical laboratory improvement amendmentscertied laboratory ( theranostics health , gaithersburg , md )  . 
the distribution of tp53 and pik3ca mutations ( mut ) and cdk12 amplications ( amp ) in 17 patients with human epidermal growth factor receptor ( her2 ) - positive breast cancer with residual disease ( rd )  . 
amplication of cdk12 ( which regulates expression of the dna damage response [ ddr ] genes brca1 , rad51 , atr , fanc1 , fancd2 , fancf , and xrcc2 , and which promotes homologous recombination14 ) was observed in eight of the 17 ( 47% ) her2 + rd specimens . of the two originally her2 + cancers that were not her2 amplied on ngs of rd tissue , one was triple negative ( ie , er , progesterone receptor negative , her2 ; patient 20 ) with mutations in pik3ca and map3k1 as well as high - level amplication of fgfr1 and znf703 . 
the second her2 rd specimen was er and progesterone receptor positive ( patient 16 ) and had tp53 and apc mutations with highlevel amplication of mcl1 and egfr ( data supplement 1 )  . pretreatment biopsy specimens because of minute amounts of tissue in archival core needle biopsy specimens , only six pretreatment biopsy specimens were sufcient to be analyzed by ngs ( table 1 )  . 
of the ve patients with matched tissue ( pretreatment and rd tissue ) , all carried a tp53 mutation that was present before treatment and retained in the rd tissue . 
cdk12 amplication was observed in two pretreatment biopsy specimens ( and preserved in the rd tissue ) , and both samples also contained the pik3ca / tp53 comutation . rppa exploratory analyses the small number of patients with rd in this study set , coupled with the limited rd tissue samples , precluded the utility of deep sequencing for novel mutations . 
the clinical and molecular importance of the identied genomic alterations was conrmed by quantifying levels of the associated phosphorylated proteins , which are the targets of us food and drug administrationapproved therapies ( table 1 ; fig 3 )  . 
retrospective analysis of rppa data obtained from frozen pretreatment biopsy specimens coltrial10 revealed that eight of lected during the clinical 12 patients with her2 + bc who did not achieve pcr expressed either higher pi3k and / or pten ser380 protein levels before treatment , compared with their population mean levels ( fig 3 )  . 
of the six patients with comutations , three ( patients 1 , 3 , and 7 ) had higher pten ser380 activity than the population mean , and three ( patients 13 , 18 , and 21 ) had lower levels . 
in contrast , pretreatment biopsy specimens from patients whose rd tissue did not have comutations indicated signicant interactions between akt - phospho - akt and erbb family proteins , phospho - fox01 / 03 - pi3k - mtor pathway , and lc3b - akt , signifying activated growth , proliferation , and autophagy ( table 2 )  . microdissected ffpe tumor tissues were adequate for evaluation by rppa in 12 of the 17 her2 + rd specimens , and six of these had comutations . 
in pretreatment biopsy specimens from patients who did not achieve pathologic complete response ( pcr ) , higher pten ser380 levels ( representing lower overall pten phosphatase activity in the cancers ) were associated with activation of akt and mtor signaling . 
rppa analysis was also available on two specimens for comparison : ( 1 ) the pretreatment biopsy specimen of patient 8 , whose disease did not achieve pcr and who did not have adequate rd tissue for analysis ; and ( 2 ) patient 16 , who was her2 + by ngs ( data supplement )  . discussion in this study of preoperative her2 - directed therapy , we performed biopsies at baseline and after 14 days of treatment to correlate ngs and rppa ndings with the development of pcr versus no pcr at denitive surgery . 
in previously published results , we showed that development of pcr correlated with low expression of egfr tyr1068 , phospho - foxo , and autophagy signaling ( low lc3b ) and high expression of phospho - pten on the baseline biopsy specimens.10 in the current study , we evaluated 17 patients her2 - amplied treatment - refractory rd tissue by ngs and rppa . 
the main ndings from these analyses were that pik3ca mutations were present in the rd tissue only in the context of comutation with tp53 , that pik3ca and tp53 comutations occurred in seven of 17 of the rd samples ( 41% ) , and that the comutated rd tissue indicated activation of egfr , akt , and mtor signaling on rppa . the cancer gene atlas network identied three common somatic mutations in bc , including tp53 , mutated in 37% , pik3ca in 38% , and gata3 in 11%.15 mutations in pi3k and tp53 occurred in 45% and 12% of luminal a , 29% and 29% in luminal b , 39% and 72% of her2 + , and 9% and 80% of basal - like bcs , respectively . 
as noted by skoulidis et al , 21 in dening molecular subgroups of kras - mutant lung adenocarcinoma , cooccurring genetic events may be major determinants of signaling and may highlight dependencies that can be exploited therapeutically . some in vitro data support the hypothesis of cooperativity . croessmann et al22 created single and double mutants of pik3ca by knock - in h1047r or e454k and tp53 by knock - in r248w in the nontumorigenic mcf10a immortalized human breast epithelial cells . 
compared with the the double mutants had a signicant single mutants , growth advantage and displayed anchorage - independent growth , although they were not tumorigenic when injected into athymic nude mice . 
adams instability that et al23 also generated pik3ca and tp53 double - mutant mice and observed the development of mammary cancers with rapid kinetics leading to decreased survival compared with single - mutant animals . loss of tp53 and pik3ca mutations after bc neoadjuvant chemotherapy is associated with a favorable prognosis.18 in 242 patients with bcs comprising all subtypes , 206 had rd . 
combined analyses of both cohorts showed that only four pretreatment biopsy specimens contained comutations in tp53 and pik3ca , and only two rd samples contained the comutations after chemotherapy . kotoula et al19 evaluated whether the outcome of early bc in patients who were treated with adjuvant chemotherapy was affected by comutation of tp53 and pik3ca . 
the entire coding region of tp53 , as well as hot - spot exons of pik3ca ( namely , exons 9 and 20 ) were evaluated in 568 tumors from patients treated with anthracycline and taxane therapy before the availability of h , and in 1 , 093 cancers from the post - trastuzumab era . 
in the entire cohort , pik3ca - only mutation was signicantly associated with better outcome , independent of til density , compared with tp53 - only and none or both mutations . additional evidence supporting oncogene cooperativity comes from cancer cell line enrichment analysis , which correlates response to anticancer agents with multilevelomics data.24 kim et al24 reported that genotypic categories of the cell lines could be distinguished by assessing the effects of coordinate mutations in the context of tp53mutant versus wild - type cancers . 
pi3k inhibitors were more effective in cell lines with tp53 and pik3ca comutations than in those with wild - type tp53 . several trials have reported lower pcr rates with preoperative chemotherapy plus her2 - targeted therapies in patients whose her2 + cancers contain pik3ca mutations or in pten - low or phospho - 4ebp1 - high , pik3ca wild - type her2 + bcs.8 , 9 , 25 , 26 interestingly , in our trial , all the rd tissues that had been treated with preoperative chemotherapy with either h , l , or h + l , and those containing a pik3ca mutation , also contained a tp53 mutation . 
the therapeutic implications of these comutations in primary - refractory her2 + bc are not known ; however , kim et al24 have shown that pi3k inhibitors may be more effective in bc cell lines that have both tp53 and pik3ca mutations than in those that have only a pik3ca mutation . cdk12 amplication was observed in eight of the 17 rd specimens ( 47% ) , four of which also harbored tp53 and pik3ca comutations . 
cdk12 is located on chromosome 17q12 , just 165 to 267 kb proximal to erbb2 and is frequently coamplied with erbb2 in human bcs.27 , 28 cdk12 plays a critical role in the ddr and maintenance of genomic integrity , and also inuences atm levels and bc migratory capacity by regulating alternative last exon splicing.13 deletion or inhibition of cdk12 in cell lines or patient - derived xenografts impairs ddr and homologous recombination repair and decreases atm levels , thereby inducing brca - ness and sensitivity to parp inhibition.13 , 27 - 30 in 13% of her2 + bcs , the breakpoint of the her2 amplicon is within the cdk12 allele , likely leading to loss of the cdk12 tumor suppressor function.28 conversely , cdk12 can act as an oncogene , whereby cdk12 amplication and overexpression lead to tumor aggressiveness , increased migration and invasiveness , and poor clinical outcome.15 , 27 in the current study , the entire cdk12 gene was amplied in the rd tissues and there was no evidence of truncated cdk12 . 
it is also possible that cdk12 amplication was a passenger alteration in the her2 + rd tissues . the her2 + comutated rd tissue after preoperative h versus l versus h + l treatments showed activation on rppa of egfr and the pi3k / akt pathway . 
some pik3ca mutations in her2 - amplied models enhance her family signaling by the production of heregulin.31 resistance of her2 + cell lines to lapatinib is mediated by heregulindriven activation egfr / her3 / pi3k signaling.32 in addition , the egfr ligand amphiregulin is induced in the context of a pik3ca mutation in luminal as well as basal - like bcs , leading to egfr - driven proliferation.33 whether tp53 comutation inuences which egfr ligand ( or ligands ) is produced by pik3ca - mutant her2 + cells is not known . this study has some limitations . 
in addition , because ngs was done on the rd and a few available pretreatment biopsy specimens , this study cannot suggest whether the pcr rate in the comutant her2 + cancers is lower than the pcr rate in cancers with a single mutation and / or in cancers with wild type for both genes . 
 pik3ca tp53 comutation in her2 + breast cancer residual disease cell inltrates , and noncoding regulatory mechanisms associated with therapy resistance.34 - 36 shi et al8 conducted whole - exome sequencing of primary bcs in the neoaltto trial and showed that pathway - level alterations , not single gene mutations , were predictive of her2 - targeted therapy resistance . 
last , at the time the patients in our study were treated , the signicance of stromal til was not appreciated , and til were not assessed in this study.36 in conclusion , this study provides insights into the molecular alterations present within her2 + rd tissue after preoperative chemotherapy and h , l , or h + l treatment . 
levin , virginia espina , joyce oshaughnessy provision of study material or patients : frankie ann holmes , ying cao , lea krekow , kristi mcintyre , cynthia osborne , lance liotta collection and assembly of data : frankie ann holmes , maren k . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / author - center . frankie ann holmes consulting or advisory role : agendia , novartis , genomic health , myriad genetics , genentech , puma biotechnology , eli lilly , myriad genetics , novartis , amgen travel , accommodations , expenses : genomic health , puma biotechnology sohail balasubramanian employment : foundation medicine stock and other ownership interests : foundation medicine travel , accommodations , expenses : foundation medicine jeffrey s . 
j clin oncol 32 : 3744 - 3752 , 2014 slamon dj , eiermann w , robert nj , et al : ten year follow - up of bcirg - 006 comparing doxorubicin plus cyclophosphamide followed by docetaxel ( act ) with doxorubicin plus cyclophosphamide followed by docetaxel and trastuzumab ( acth ) with docetaxel , carboplatin and trastuzumab ( tch ) in her2 + early breast cancer . 
presented at san antonio breast cancer symposium , san antonio , tx , december 11 , 2015 chan a , delaloge s , holmes fa , et al : neratinib after trastuzumab - based adjuvant therapy in patients with her2 - positive breast cancer ( extenet ) : a multicentre , randomised , double - blind , placebo - controlled , phase 3 trial . 
lancet oncol 17 : 367 - 377 , 2016 cortazar p , zhang l , untch m , et al : pathological complete response and long - term clinical benet in breast cancer : the ctneobc pooled analysis . 
lancet 384 : 164 - 172 , 2014 gingras i , gebhart g , de azambuja e , et al : her2 - positive breast cancer is lost in translation : time for patient - centered research . 
nat rev clin oncol 14 : 669 - 681 , 2017 goel s , krop ie : pik3ca mutations in her2 - positive breast cancer : an ongoing conundruann oncol 27 : 1368 - 1372 , 2016 zardavas d , te marvelde l , milne rl , et al : tumor pik3ca genotype and prognosis in early - stage breast cancer : a pooled analysis of individual patient data . j clin oncol 36 : 981 - 990 , 2018 shi w , jiang t , nuciforo p , et al : pathway level alterations rather than mutations in single genes predict response to her2 - targeted therapies in the neo - altto trial . 
ann oncol 28 : 128 - 135 , 2017 loibl s , majewski i , guarneri v , et al : pik3ca mutations are associated with reduced pathological complete response rates in primary her2 - positive breast cancer : pooled analysis of 967 patients from ve prospective trials investigating lapatinib and trastuzumab . 
holmes fa , espina v , liotta la , et al : pathologic complete response after preoperative anti - her2 therapy correlates with alterations in pten , foxo , phosphorylated stat5 , and autophagy protein signaling . 
blazek d , kohoutek j , bartholomeeusen k , et al : the cyclin k / cdk12 complex maintains genomic stability via regulation of expression of dna damage response genes . 
boyault s , drouet y , navarro c , et al : mutational characterization of individual breast tumors : tp53 and pi3k pathway genes are frequently and distinctively mutated in different subtypes . 
nature 486 : 400 - 404 , 2012 jiang y - z , yu k - d , bao j , et al : favorable prognostic impact in loss of tp53 and pik3ca mutations after neoadjuvant chemotherapy in breast cancer . 
kotoula v , karavasilis v , zagouri f , et al : effects of tp53 and pik3ca mutations in early breast cancer : a matter of co - mutation and tumor - inltrating lymphocytes . 
luen sj , asher r , lee ck , et al : association of somatic driver alterations with prognosis in postmenopausal , hormone receptor - positive , her2 - negative early breast cancer . 
skoulidis f , byers la , diao l , et al : co - occurring genomic alterations dene major subsets of kras - mutant lung adenocarcinoma with distinct biology , immune proles , and therapeutic vulnerabilities . 
croessmann s , wong hy , zabransky dj , et al : pik3ca mutations and tp53 alterations cooperate to increase cancerous phenotypes and tumor heterogeneity . breast cancer res treat 162 : 451 - 464 , 2017 23 . 
rimawi mf , de angelis c , contreras a , et al : low pten levels and pik3ca mutations predict resistance to neoadjuvant lapatinib and trastuzumab without chemotherapy in patients with her2 over - expressing breast cancer . 
loibl s , darb - esfahani s , huober j , et al : integrated analysis of pten and p4ebp1 protein expression as predictors for pcr in her2 - positive breast cancer . 
cell division 12 : 7 , 2017 doi : 01.1186 / s13008 - 017 - 0033 - x johnson sf , cruz c , greifenberg ak , et al : cdk12 inhibition reverses de novo and acquired parp inhibitor resistance in brca wild - type and mutated models of triple - negative breast cancer . 
cell reports 17 : 2367 - 2381 , 2016 iniguez ab , stolte b , wang ej , et al : ews / fli confers tumor cell synthetic lethality to cdk12 inhibition in ewing sarcoma . 
perez ea , thompson ea , ballman kv , et al : genomic analysis reveals that immune function genes are strongly linked to clinical outcome in the north central cancer treatment group n9831 adjuvant trastuzumab trial . 
in this clinical trial , we applied these technologies to map the activity of signal pathway proteins in the human epidermal growth factor receptor ( her2 ) network , before and after patients received lapatinib ( l ) , or trastuzumab ( h ) , alone or in combination.10 because these two classes of molecular inhibitors target different parts the trial design provided a means to gather direct in vivo information about potential molecular mechanisms of additive or synergistic activity . the her2 / egfr signaling cascade , methods specimen collection and preservation we collected 16 - gauge core needle breast biopsy specimens before and after her2 targeted therapy and placed the specimens in a proprietary , ethanol - based xative for transport at 4c to george mason university ( mueller c , et al : plos one 6 : e23780 , 2011 )  . 
preand post - treatment core needle biopsy specimens were processed and analyzed at george mason university , center for applied proteomics and molecular medicine . archival formalin - xed parafn - embedded ( ffpe ) pretreatment and residual disease ( rd ) specimens were collected after informed consent was given . 
the specimen identication numbers are listed in appendix table a1 . laser capture microdissection frozen tissue sections from preand post - treatment core - needle biopsy specimens were xed in 70% ethanol , stained with mayers hematoxylin , dehydrated in graded alcohols ( 70% , 95% , 100% ) , and cleared in xylene . 
microdissected cells were stored at 80c before lysis reverse phase protein microarray printing . ffpe tissue sections for the pretreatment and rd specimens were subjected to laser capture microdissection of tumor tissue using an arcturusxt ( thermo fisher scientic ) in near - infrared capture mode . microdissected cells were stored at 80c before lysis and reverse phase protein microarray printing . reverse phase protein array construction / staining ( theralink assay ) the microdissected cells were subjected to lysis with a 10% ( volumeto - volume ratio ) solution of tris ( 2 - carboxyethyl ) phosphine ( tcep ; pierce , rockford , il ) or a 2.5% solution of 2 - mercaptioethanol ( sigmaaldrich , st louis , mo ) in tissue protein extraction reagent ( t - per ; pierce ) and tris - glycine 2x sds buffer ( thermo fisher scientic )  . 
cell lysates were stored at 80c before microarray construction . cellular lysates were printed in two - fold dilution series on glass - backed nitrocellulose array slides ( fast slides ; whatman , florham park , nj , or sartorius stedim biotech sa , germany ) using an aushon 2470 arrayer ( aushon biosystems , billerica , ma ) equipped with 350 - m pins . 
the negative control slide was incubated with antibody diluent ( agilent )  . secondary antibody was goat anti - rabbit igg h + l ( 1 : 7 , 500 ; vector labs , burlingame , ca ) or rabbit anti - mouse igg ( 1 : 10 ; agilent )  . subsequent signal detection was amplied via horseradish peroxidasemediated biotinyl tyramide deposition with chromogenic detection ( diaminobenzidine ) for the preand post - treatment specimens or uorescent dye ( cy5 ) for the rd specimens . total protein per microarray spot was determined with a sypro ruby protein stain ( thermo fisher scientic ) per manufacturers directions and imaged with a ccd camera ( novaray ; alpha innotech , san leandro , ca ) or genepix scanner ( molecular devices )  . reverse phase protein array analysis the frozen preand post - treatment study set was analyzed in four batches : june 2008 ( n = 23 intent - to - treatevaluable [ itt - e ] samples ) , october 2008 ( n = 12 itt - e samples ) , april 2009 ( n = 29 itt - e samples ) , and november 2010 ( n = 95 itt - e samples )  . 
on each array set , the undiluted spot was used to quantify protein levels for patient samples . array slides were scanned at 600 dpi on a atbed scanner ( umax powerlook ; umax technologies , taiwan ) and saved in a .tiff format ( adobe photoshop ; adobe , san jose , ca )  . 
if the coefcient of variance between replicates was greater than 20% , the spot was agged and a value of coefcient of variance too high was reported . signal intensity from the secondary antibody - alone ( negative control ) slide was subtracted from the signal intensity of the primary antibody slide . 
the scores are reported as a value from 0 to 3 on the basis of the population mean for each individual analyte . spearmans correlations and mann - whitney u tests were performed on one pretreatment biopsy specimen and 12 patients rd specimens to compare samples with pik3ca - tp53 comutation with those without comutation ( table a3 ; data supplement 2 )  . statistical analysis all biomarker analyses were conducted on the itt - e population . wilcoxon rank - sum test was used to calculate group differences if the data were not normally distributed ( espina v , et al : plos one 5 : e10240 , 2010 )  . 
student t test was used if the data were normally distributed ( r software , r project for statistical computing ; sas , sas institute , cary , nc )  . 
we gathered proteomic data on the state of the signal pathway networks directly or indirectly inuenced by her2 in the tumor cells at three distinct time points related to therapy : ( 1 ) before administration of h or l or h + l ; ( 2 ) after 14 days of l or h or h + l therapy ; and ( 3 ) in the rd tissue specimens after h or l or h + l and chemotherapy.10 we correlated the data for the before and after h or l or h + l therapy to the pcr . specimens 16 and 20 ( us oncology identication numbers 1 and 195 , respectively ) had equivocal her2 immunohistochemistry results in the post - treatment specimens . 
evaluation of specimen 20 by reverse phase protein array for egfr / her2 family activation conrmed that the her2 , her2 tyr1248 , and her3 levels were greater than 1 sd below the populations means , compared with the study population data . 
however , we discovered activation of egfr in the posttreatment tumor specimen , compared with the study population ( specimen 20 : egfr = 32.27 , population mean = 27.95 ; data supplement 1 )  . heterogeneous signal transduction pathway signaling in rd specimens autophagy - , inammation - , and proliferation - associated proteins were activated in the rd specimens , but there was a lack of concordance between protein activation and pik3ca / tp53 comutations ( data supplement 1 )  . 
compensatory or adaptive changes after therapy provide insights concerning whether that pathway is an essential or collateral driver of the cancer . integrated genomic and proteomic analysis can elucidate the interplay between potential driver mutations and signal transduction pathways . pten catalytic activity is necessary for its tumor suppressor function . most pten missense mutations affect the phosphatase domain and cause a loss in pten phosphatase activity ( torres j , et al : j biol chem 276 : 993 - 998 , 2001 )  . 
many pten nonsense or frame - shift mutations are targeted to the c - terminal domain of the protein , which harbors serine phosphorylation sites ( torres j , et al : j biol chem 276 : 993 - 998 , 2001 )  . 
increased phosphorylation at ser380 results in decreased pten activity ( ie , less tumor suppression through pi3 ; yang z , et al : oncotarget 6 : 31916 - 31926 , 2015 ; yu h , et al : nat rev cancer 4 : 91 - 105 , 2004 )  . 
pten is phosphorylated at ser 380 by casein kinase 2 ( ck2 ) , thus stabilizing pten but suppressing its phosphatase activity ( torres j , et al : j biol chem 276 : 993 - 998 , 2001 )  . 
pd - 1 was recently shown to inhibit this stabilizing phosphorylation of ser380 in the c - terminal domain of pten ( patsoukis n , et al : mol cell biol 33 : 3091 - 3098 , 2013 )  . 
ck2 is a target of pd - 1 and pd - 1 has been shown to decrease pten expression while increasing pten activity , which , in turn , inhibits the pi3k / akt pathway ( patsoukis n , et al : mol cell biol 33 : 3091 - 3098 , 2013 )  . 
thus , the higher phosphorylation levels of pten ser380 that we identied in patients with pik3ca / tp53 comutations ( fig 3 ) could contribute to tumor proliferation and thus a lack of pcr . 
the role of immune cell function and pd - 1 expression in these specimens could also modulate tumor proliferation . inammation and jak / stat signal pathways overexpression of her2 in breast cancer cell lines has been induced , in part , by jak2 and prolactin ( yamauchi t , et al : j bio chem 275 : 33937 - 33944 , 2000 )  . 
jak2 is required for phosphorylation of the signal transducers and activators of transcription ( stat ) proteins stat3 and stat5 , thus activating a survival / proliferation axis ( watson cj , et al : cell death differ 21 : 185 - 186 , 2014 )  . 
cd4 / cd8 dual immunohistochemistry using biocare medical ( pacheco , ca ) antibody cocktail was performed to assess the location , quantity , and type of t cells in the preand post - treatment specimens . 
murray leslie samuel paolo nuciforo jose jimenez guillem argiles rodrigo dienstmann josef tabernero lucia picariello luca messerini stefania nobili enrico mini kieran sheahan elizabeth ryan ( continued ) purpose transcriptomic profiling of colorectal cancer ( crc ) has led to the identification of four consensus molecular subtypes ( cms1 to 4 ) that have prognostic value in stage ii and iii disease . 
2018 by american society of clinical oncology introduction colorectal cancer ( crc ) has the third highest worldwide incidence.1 although 75% of patients present with operable diseasemainly stages ii and iiiapproximately 40% experience disease recurrence.2 compared with surgery alone , adjuvant chemotherapy improves survival in only approximately 3% of patients with stage ii disease , rising to 15% to 20% for those with stage iii disease . 
 contributed equally to this work . corresponding author : daniel longley , phd , centre for cancer research and cell biology , queens university belfast , 97 lisburn rd , belfast , bt9 7bl united kingdom ; e - mail : d.longley@qub.ac.uk. licensed under the creative commons attribution 4.0 license significant advances have been made in the molecular stratification of crc , leading to the identification of four consensus molecular subtypes ( cms1 to 4 ) .3 cms1 is enriched for microsatellite instability , is immune rich , and correlates with good prognosis , and cms4 is stromal rich , with high levels of cancer - associated fibroblasts , and has a relatively poor prognosis . 
 cms3 is defined by the activation of multiple metabolic pathways , potentially as a result of its enrichment for kras mutations.4 the epithelial - rich cms2 is the largest group , accounting for approximately 40% of all tumors . 
of these , 188 samples with > 50% tumor content passed quality control and were subjected to rna and dna analysis ( appendix )  . transcriptomics high - quality transcriptomics data were obtained for 156 of the 188 samples ( almac xcel array ; almac diagnostics , craigavon , united kingdom )  . 
residual technical batch effects were corrected using the combat method ( sva package ) , and data were deposited in the national center for biotechnology information gene expression omnibus repository ( gse103479 )  . 
the clinical pathologic details of this cohort are provided in table 1 . data analysis gse3958210 and gse1433311 crc data sets were downloaded from the national center for biotechnology information gene expression omnibus repository and their respective cel files uploaded into r . 
similar results were obtained in the larger gse39582 cohort for stage ii disease ( log - rank test p = .071 ) , whereas in stage iii disease , this correlation reached significance ( p = .001 ; data supplement )  . 
when we combined the taxonomy and gse39582 cohorts to increase statistical power , the benefit from adjuvant chemotherapy for cms2 was significant in both stage ii disease ( log - rank test p = .02 ; hazard ratio [ hr ] , 0.21 [ wald test p = 3.52 102 ] ; fig 1a ) and stage iii disease ( log - rank test p < .001 ; hr , 0.22 [ wald test p = 1.48 104 ] ; fig 1b )  . 
of note , no significant benefit from adjuvant chemotherapy was observed in cms1 or cms4 , although a nonsignificant trend was observed in cms4 stage iii disease ( log - rank test p = .089 ; data supplement )  . these results suggest that the more epithelial cms2 and cms3 subgroups benefit from adjuvant chemotherapy . 
in support of this , in a combined analysis of cms2 and cms3 , benefit from adjuvant chemotherapy was significant in both stage ii disease ( log - rank test p = .0042 ; hr , 0.16 [ wald test p = .012 ] ; fig 1c ) and stage iii disease ( log - rank test p < .001 ; hr , 0.20 [ wald test p = 1.14 106 ] ; fig 1d )  . 
in contrast , in a combined analysis of the cms1 and cms4 subgroups , no benefit from adjuvant chemotherapy was observed ( data supplement )  . when cms2 subgroup results for stage ii disease were adjusted for t stage ( t4 v t3 ) , age , and sex using cox proportional hazards regression analysis , the significance of the benefit from chemotherapy was lost ( hr , 0.31 ; wald test p = .121 ; log likelihood ratio , 7.0 105 ; fig 1e ) ; however , in stage iii disease , adjusting for t stage ( t4 or n2 v t1 to t3 / n1 ) , age , and sex , the significance of benefit from chemotherapy in cms2 was maintained ( hr , 0.27 ; wald test p = .007 ; log likelihood p = 3.25 105 ; fig 1f )  . 
in cox proportional hazards regression analyses of the combined cms2 and cms3 subgroups , the significance of benefit from chemotherapy was maintained in stage ii disease ( hr , 0.23 ; wald test p = .049 ; log likelihood ratio , 2.72 105 ; fig 1g ) and stage iii disease ( hr , 0.21 ; wald test p < .001 ; log likelihood p = 3.19 107 ; fig 1h )  . clinical implications of tumor - intrinsic stratification in cms2 as previously reported , 6 there are limited associations between cms and cris classifications for example , cms4 is distributed relatively evenly between the five cris subtypes ( data supplement ) ; however , some clear patterns were observed , with cms2 almost exclusively distributed between cris - c , - d , and - e ( fig 2a )  . 
 subsequently , we investigated whether substratification of cms2 into cris - c , - d , or - e could identify a more specific subset of patients with stage ii and iii disease who derive benefit from adjuvant chemotherapy . 
these results were confirmed in an additional independent cohort , gse14333 ( logrank test p = .02 ; hr , 0.12 [ wald test p = .05 ] ; data supplement )  . 
in the other 2 cris subgroups ( cris - a and - b ) , no significant benefit from adjuvant chemotherapy was observed in either stage ii or iii disease , although a nonsignificant trend was observed in cris - a for stage iii disease ( p = .057 ; data supplement ) , which is consistent with this subgroup being enriched for cms3 , where a significant benefit was observed ( data supplement )  . 
when cris - c results were adjusted for t stage , age , and sex using cox proportional hazards regression analyses , the benefit from chemotherapy maintained significance in stage ii disease ( hr , 0.12 ; wald test p = .045 ; log likelihood p = .0054 ; fig 2d ) and stage iii disease ( hr , 0.27 ; wald test p = .02 ; log likelihood p = 1.11 103 ; fig 2e ) ; furthermore , for combined stage ii and iii disease , adjusted hr was 0.20 ( wald test p < .001 ; log likelihood p = 7.5 106 ; data supplement )  . 
 ( a and b ) kaplan - meier plots of 5 - year overall survival ( os ) for consensus molecular subtype 2 ( cms2 ) patients who received adjuvant fluorouracil ( fu ) - based treatment ( gold ) and those who did not receive treatment ( surgery alone , blue ) , in the ( a ) stage ii combined ( taxonomy and gse39582 ) cohort and the ( b ) stage iii combined ( taxonomy and gse39582 ) cohort . 
 ( c and d ) kaplan - meier plots for 5 - year os for combined cms2 and cms3 patients in the ( c ) stage ii combined ( taxonomy and gse39582 ) cohort and the ( d ) stage iii combined ( taxonomy and gse39582 ) cohort . 
to account for potential intratumoral heterogeneity of putative biomarkers in specific tumor regions , tmas were generated from the taxonomy cohort to incorporate three cores each from ct , if , and tumor - adjacent sr regions . 
cd8 and cd3 levels were defined using immunohistochemistry , and patients were stratified into high and low groups using the median as cutoff ( representative cd8 and cd3 images ; figs 3a and 3b )  . 
we further assessed cd8 + lymphocytes in histologically normal tissue adjacent to the tumor and found no difference in survival between high and low cd8 levels ( log - rank test p = .72 ; data supplement )  . 
of note , no correlations were found between cd3 + lymphocyte levels and prognosis ( if log - rank test p = .55 ; sr log - rank test p = .75 ; ct log - rank test p = .8 ; normal log - rank test p = .82 ; data supplement )  . for each patient , cd8 scores in each tumor subregion correlated closely with one another ( p < .001 ; data supplement )  . 
 as expected on the basis of correlations for individual regions ( data supplement ) , applying this tumor average score to the combined stage ii and iii taxonomy cohort revealed that high levels of cd8 + lymphocytes identified cris - c patients with good prognosis ( log - rank p = .0031 ; hr , 12.18 [ wald test p = .0191 ] ; fig 4a )  . 
in contrast , average cd3 scores , which again did not correlate closely with average cd8 scores ( fig 3c ) , were not prognostic ( fig 4b )  . 
of note , cd8 mrna expression was unable to distinguish good and poor prognosis patients ( log - rank p = .83 ; data supplement ) , which indicates the need for immunohistochemistry in combination with transcriptomic profiling for effective prognostication . 
 ( e - h ) forest plots show the results from the adjusted cox proportional hazards regression analysis for the ( e ) stage ii cms2 combined cohort , the ( f ) stage iii cms2 combined cohort , the ( g ) stage ii cms2 and cms3 combined cohort , and the ( h ) stage iii cms2 and cms3 combined cohort . 
for adjusted analyses , data are stratified by treatment and adjusted for t stage , sex , and age in stage ii , and for age and sex in stage iii . 
 ( a ) caleydo plots display mapping of patient samples from the consensus molecular subtype 2 to the colorectal cancer intrinsic subtypes ( cris ) in the combined ( taxonomy and gse39582 ) data sets . 
 ( a and b ) representative images of samples display high and low ( a ) cd8 and ( b ) cd3 expression in each of the three tumor regions sampled : invasive front ( if ) , stromal rich ( sr ) , and central tumor ( ct )  . 
 ( a - c ) kaplan meier plots of patients with ( a ) cd8 - high versus cd8 - low stage ii and iii disease in the colorectal cancer intrinsic subtype ( cris ) - c surgery - only , taxonomy cohort ; ( b ) cd3 - high versus cd3 - low stage ii and iii disease in the cris - c surgery - only , taxonomy cohort ; and ( c ) cd8 - high versus cd8low stage ii disease in the cris - c surgery - only , taxonomy cohort . 
therapeutic benefit from adjuvant chemotherapy is modest for this group as a whole , with an absolute improvement in survival of approximately 3%.18 - 22 currently , additional pathologic characteristics , such as obstruction , perforation , extramural venous invasion , and t stage ( t4 ) , are used to identify poor prognostic stage ii disease and guide the decision of whether to start chemotherapy treatment.23 additional methods of assessing the risk of recurrence in the adjuvant disease setting have been the focus of many studies in recent years , 24 - 29 which has led to the development of the 12 - gene oncotype dx assay.30 this algorithm has been extensively clinically tested31 - 33 and demonstrated to identify patients with stage ii disease who are at higher risk of recurrence and patients with stage iii disease who are at lower risk of recurrence.34 , 35 additional signatures have been proposed , including the 18 - gene prognostic classifier , known as coloprint , 36 and the 634 - gene prognostic classifier , known as coldx.27 the current study indicates that the transcriptionally definable cms2 / cris - c patient subgroup may be the cohort of patients within stage ii disease that benefits from standard adjuvant fu - based chemotherapy . 
these data correlate well with our previous study on the prognostic significance of immune - derived programmed death ligand 1 mrna expression in crc , in which we postulated that patients with low immune infiltrates would significantly benefit from adjuvant fu - based chemotherapy after surgery.9 , 37 meta - analysis of the idea ( international duration evaluation of adjuvant chemotherapy ) collaboration examined whether a 3 - month duration of oxaliplatin containing adjuvant chemotherapyfolfox4 , modified folfox6 , or xeloxis as effective as a 6 - month schedule in patients with stage iii crc . 
this study found that the 3 - month treatment was almost as effective as the 6 - month treatment and reduced the risk of treatment associated toxicity , thus concluding that a 3 - month treatment would be more beneficial for patients with low - risk ( t1 to 3 / n1 tumors ) stage iii disease.38 our study suggests that levels of cd8 + lymphocytes could also be used to identify such low - risk patients , at least in the cris - c subgroup . 
of note , cris - c is enriched for mutant tp53 and wild - type kras tumors , 6 but neither of these established molecular markers provided additional information with regard to disease outcome within the cris - c subgroup . collectively , these results provide the first evidence of the predictive value of the now well established cms and more recently described cris transcription - based classification systems . 
 our results also emphasize the utility of combining cms and cris subtyping in a substratification strategy to maximize clinical benefit from adjuvant fu - based chemotherapy in patients with stage ii and iii crc . 
murray , leslie samuel , jose jimenez , guillem argiles , lucia picariello , luca messerini , stefania nobili , enrico mini , elizabeth ryan , sandra van schaeybroeck , daniel b . 
longley tumor - infiltrating lymphocytes , would potentially enable the prospective identification of the cris - c / cd8 - low stage ii patients who significantly benefit from adjuvant fu - based chemotherapy . 
however , we recognize that there are a number of limitations in the current study , which was conducted on a relatively small number of retrospective samples that were collected outside of clinical trials , and we realize that this hypothesis - generating study now requires validation in either larger patient cohorts or stratified trial cohorts enriched for the cris - c patient subtype . 
nonetheless , this study suggests that transcription - based classification systems , such as cms and cris , have the potential to be developed into patient stratification tools and , when used alone or alongside other molecular pathology approaches , such as immunohistochemistry , could enable the selection of patients with crc who are most likely to benefit from adjuvant chemotherapy , while at the same time sparing nonresponders the potentially harmful treatment related adverse events and sequelae of chemotherapy . 
of importance , the cris subtyping method uses gene expression from tumor epithelial cells only and is independent of stromal - derived signals ; therefore , the cris subgroups can be detected irrespective of the profiling technology used or the tissue source.15 such robustness and reproducibility are critical for clinical translation . 
murray , leslie samuel , paolo nuciforo , jose jimenez , guillem argiles , josef tabernero , lucia picariello , luca messerini , stefania nobili , enrico mini , kieran sheahan , elizabeth ryan , sandra van schaeybroeck , mark lawler , daniel b . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . richard wilson consulting or advisory role : merck serono , sirtex medical , amgen , servier , clovis oncology , halozyme , bristol - myers squibb research funding : almac group ( inst ) travel , accommodations , expenses : merck serono , amgen vicky m . 
murray research funding : vertebrate antibodies ( inst ) leslie samuel consulting or advisory role : mundipharma research funding : mologen ( inst ) , roche ( inst ) , merck ( inst ) , eli lilly ( inst ) , taiho pharmaceutical ( inst ) travel , accommodations , expenses : mologen wendy l . 
longley stock and other ownership interests : fusion antibodies consulting or advisory role : astex pharmaceuticals sandra van schaeybroeck no relationship to disclose mark lawler honoraria : pfizer research funding : astex pharmaceuticals patents , royalties , other intellectual property : inhibitors of the antiapoptotic protein flip travel , accommodations , expenses : astex pharmaceuticals affiliations wendy l . 
murray and leslie samuel , national health service grampian , aberdeen , united kingdom ; najeeb ashraf syed and puthen veettil jithesh , sidra medical and research center , qatar ; paolo nuciforo , jose jimenez , guillem argiles , rodrigo dienstmann , and josef tabernero , university hospital vall dhebron , barcelona , spain ; lucia picariello , luca messerini , stefania nobili , and enrico mini , university of florence , florence , italy ; and kieran sheahan and elizabeth ryan , university college dublin , dublin , ireland . funded by cancer research uk grants no . 
lenz h - j , ou f - s , venook ap , et al : impact of consensus molecular subtyping ( cms ) on overall survival ( os ) and progression free survival ( pfs ) in patients ( pts ) with metastatic colorectal cancer ( mcrc ) : analysis of calgb / swog 80405 ( alliance )  . 
dunne pd , mcart dg , bradley ca , et al : challenging the cancer molecular stratification dogma : intratumoral heterogeneity undermines consensus molecular subtypes and potential diagnostic value in colorectal cancer . 
marisa l , de reynis a , duval a , et al : gene expression classification of colon cancer into molecular subtypes : characterization , validation , and prognostic value . 
jorissen rn , gibbs p , christie m , et al : metastasis - associated gene expression changes predict poor outcomes in patients with dukes stage b and c colorectal cancer . 
galon j , costes a , sanchez - cabo f , et al : type , density , and location of immune cells within human colorectal tumors predict clinical outcome . 
hutchins gga , treanor d , wright a , et al : intratumoral stromal morphometry predicts disease recurrence but not response to 5 - fluorouracil : results from the quasar trial of colorectal cancer . 
roepman p , schlicker a , tabernero j , et al : colorectal cancer intrinsic subtypes predict chemotherapy benefit , deficient mismatch repair and epithelial - to - mesenchymal transition . 
figueredo a , charette ml , maroun j , et al : adjuvant therapy for stage ii colon cancer : a systematic review from the cancer care ontario program in evidence - based cares gastrointestinal cancer disease site group . 
kelley rk , venook ap : prognostic and predictive markers in stage ii colon cancer : is there a role for gene expression profiling ? clin colorectal cancer 10 : 73 - 80 , 2011 26 . 
kennedy rd , bylesjo m , kerr p , et al : development and independent validation of a prognostic assay for stage ii colon cancer using formalin - fixed paraffin - embedded tissue . 
tabernero j , salazar r , roepman r , et al : additional validation of a genomic signature ( coloprint ) for the risk stratification of stage ii colon cancer patients . 
oconnell mj , lavery i , yothers g , et al : relationship between tumor gene expression and recurrence in four independent studies of patients with stage ii / iii colon cancer treated with surgery alone or surgery plus adjuvant fluorouracil plus leucovorj clin oncol 28 : 3937 - 3944 , 2010 31 . 
gray rg , quirke p , handley k , et al : validation study of a quantitative multigene reverse transcriptase - polymerase chain reaction assay for assessment of recurrence risk in patients with stage ii colon cancer . 
venook ap , niedzwiecki d , lopatin m , et al : biologic determinants of tumor recurrence in stage ii colon cancer : validation study of the 12 - gene recurrence score in cancer and leukemia group b ( calgb ) 9581 . 
cartwright t , chao c , lee m , et al : effect of the 12 - gene colon cancer assay results on adjuvant treatment recommendations in patients with stage ii colon cancer . 
srivastava g , renfro la , behrens rj , et al : prospective multicenter study of the impact of oncotype dx colon cancer assay results on treatment recommendations in stage ii colon cancer patients . 
dunne pd , mcart dg , oreilly pg , et al : immune - derived pd - l1 gene expression defines a subgroup of stage ii / iii colorectal cancer patients with favorable prognosis who may be harmed by sdjuvant chemotherapy . 
shi q , sobrero af , shields af , et al : prospective pooled analysis of six phase iii trials investigating duration of adjuvant ( adjuv ) oxaliplatin - based therapy ( 3 vs 6 months ) for patients ( pts ) with stage iii colon cancer ( cc ) : the idea ( international duration evaluation of adjuvant chemotherapy ) collaboration . 
 appendix development of a multiomics patient cohort the taxonomy cohort was assembled from an initial cohort of 363 patients with stage ii and iii disease from four european centers ( vall dhebron institute of oncology , barcelona , spain ; st vincents university hospital , dublin , ireland ; university of florence , florence , italy ; and university of aberdeen , aberdeen , united kingdom )  . 
this work was approved by the medicine , dentistry , and biomedical sciences school ethics committee ( ref : 12 / 12v4 )  . samples from 194 patients with 50% or more tumor content as assessed by hematoxylin and eosin staining were subjected to rna extraction . 
 data for samples were preprocessed and aligned according to best practices ( broad institute : tute.org / gatk / discussion / 3060 / how - should - i - pre - process - data - from - multiplexed - sequencing - and - multi - library - designs ) using burrows - wheeler aligner for alignment and gatk 3.4 for realignment and recalibration . 
cd8 was detected using the anti - cd8 antibody c8 / 144b ( dako , carpinteria , ca ) using the leica bond max staining platform ( leica microsystems , wetzlar , germany )  . 
for each tumor core , cd3 + and cd8 + t - cell populations were scored using the open access image analysis software qupath ( bankhead p , et al : sci rep 7 : 16878 , 2017 )  . 
measurements of tissue area and positive cell counts for each core provided cell density measurements , which are expressed as the number of positive cells per square millimeter of tissue . 
high and low cd3 / cd8 levels were calculated using the median level as cutoff . statistical analyses for kaplan - meier survival analysis and cox proportional hazards regression analysis , significance was assessed using log - rank and wald tests , respectively . 
cox proportional hazards regression analysis was used to assess overall survival at 5 years for adjuvant chemotherapy before and after adjustment for age , t stage , and sex ( stage ii and iii ) and age , sex , and stage ( stage ii and iii combined )  . 
 c subgrouping of unfavorable histology neuroblastomas with immunohistochemistry toward precision prognosis and therapy stratication naohiko ikegaki , phd1 and hiroyuki shimada , md , phd2 , for the international neuroblastoma pathology committee introduction neuroblastoma , as well dened by willis , 1 is an embryonal tumor of neural crest origtumors of the neuroblastoma group include neuroblastoma , ganglioneuroblastoma , and ganglioneuroma . 
we believe that all ganglioneuromas were once neuroblastomas in the early stage of tumor development.1 , 2 they are collectively called peripheral neuroblastic tumors ( pnts ) and are known to present with a wide range of clinical behavior , from spontaneous regression and tumor maturation to aggressive progression that is refractory to intensive treatment . 
these observations have led us to seek additional immunohistochemical biomarkers that are tightly associated with aggressive behaviorsthat is , unresponsiveness or resistance to the current intensive multimodal therapyof certain uh tumors . 
to address this problem , we and others have sought and identied the expression of potentially drug - targetable proteins that are responsible for their aggressive progression in the uh group . 
when we seek such biomarkers , they are preferably actionable / druggable by existing pharmaceutical agents , us food and drug administration approved , or currently tested in human clinical trials . 
of note , these three types of highly aggressive neuroblastomasthat is , myc - driven neuroblastoma , alt phenotype neuroblastoma because of atrx loss , and tert - overexpressing neuroblastoma with gene rearrangementare mutually exclusive and seem to comprise the majority of therapy - resistant or refractory uh neuroblastomas . these three markersthat is , myc family proteins , tert , and atrx can be the subjects of immunohistochemical assay and potentially incorporated in future inpc . it should be noted that other than the above gene and expression alterations found in aggressive uh neuroblastomas , alk mutations and amplication are found in approximately 10% of sporadic neuroblastoma cases.20 , 21 it was also found that alk gene amplication and f1174 mutations are associated with mycn amplication.22 moreover , alk overexpression as a result of alk amplication / mutations seems to be responsible for its oncogenic activity23 ; however , the prognostic signicance of alk mutations / overexpression has been controversial , as some reports suggest an association of alk mutations / overexpression with fatal outcomes of the disease , whereas others suggest otherwise.22 , 23 in particular , regairaz et al24 showed immunohistochemically that expression of alk and its active form palk was observed in many neuroblastomas independent of alk mutation / amplication . 
in this regard , myc / mycn and tert overexpression26 - 29 has been linked to the stemness of tumor cells , and direct and indirect therapeutic targeting of these alterations suppresses the stemness of aggressive and therapy - resistant uh neuroblastomas . 
similarly , alt is linked to tumor stem cells.30 , 31 these observations allow us to incorporate the information into the inpc toward the goal of precision prognosis and therapy stratication . our plan is to rene the uh neuroblastoma stratication using immunohistopathologic analysis with antipan - myc antibody ( or anti - mycn and anti - myc ) , anti - tert antibody , and anti - atrx antibody . 
it is expected that the nonresponder to current high - risk therapymyc , tert , and atl subgroups could be separated from the possible respondernull subgroupwith a high probability before the initiation of therapy : myc subgroup : myc - driven neuroblastoma shows augmented expression of mycn protein and / or myc proteas myc family protein overexpression stimulates rrna synthesis and protein translation , neuroblastoma cells in this subgroup often exhibit prominent nucleolar formationnucleolar hypertrophyand hypertrophic cell morphology.13 , 14 because of the presence of one to a few prominent nucleoli , myc - driven neuroblastoma is cytologically distinguishable from the conventional small blue - cell neuroblastoma with salt - and - pepper nuclei . myc - driven neuroblastomas , which comprise more than 50% of high - risk neuroblastomas , includes rare and unique tumor , named large - cell neuroblastoma , characterized by the bulls eye appearance of enlarged and uniquely open euchromatin - rich nuclei that contain highly conspicuous nucleoli.33 most large - cell neuroblastomas overexpress higher levels of mycn or myc protein than other myc - driven neuroblastomas . 
their euchromatin - rich open nuclei suggest the stem celllike nature of the tumor cells.29 a considerable number of myc - driven neuroblastomas are also associated with tert overexpression , as tert is a direct transcriptional target of myc family proteins . 
tert can also stabilize myc protein via its noncanonical enzymatic function , creating a positive feedback loop between mycn / myc and tert expression.34 , 35 tert subgroup : tert is the protein component of telomerase , which also includes terc , the telomerase rna component . 
immunohistochemical grading of tert is performed in essentially the same way as that of myc family proteins . estimated survival of each subgroup is based on wang et al12 and valentijn et al.18 to exclude the possibility of overlooking alt cases with atrx point mutations with no atrx loss and / or rare daxx mutations , patients older than age 5 years in the null group can be subjected to additional screening by either rdna32 or telomeric uorescence in situ hybridization assay.32a likely a result of long - range genomic rearrangements but not to promoter mutations in neuroblastoma.36 tert promoter hypermethylation may also be involved in its activating mechanism.37 , 38 alt subgroup : atrx loss results in the alt phenotype . atrx mutation is rarely observed in the myc and tert subgroups , and loss of atrx is exclusively observed in patients with neuroblastoma older than age 5 years.17 because a normal atrx function is to insert the variant histone h3 , namely h3.3 , in concert with daxx , into chromatin to maintain transcriptionally active euchromatin , 39 atrx loss may result in a more transcriptionally inactive heterochromatic state . 
thus , histologic / cytologic appearance of the alt subgroup could be the conventional type with salt - and - pepper nuclei . null subgroup : there are still uh neuroblastomas without myc family protein overexpression , tert overexpression , and atrx loss . 
strategies under consideration include , but are not limited to , transcriptional repression of myc and mycn genes by g - quadruplex ( g4 ) stabilizers , 42 , 43 bromodomain and extraterminal protein inhibitors , 44 , 45 and inhibitors of a transcriptional cyclin - dependent kinase 746 , 47 ; destabilization of mycn by aurora kinase a inhibitors48 and of myc and mycn proteins by ras signaling pathway inhibitors49 - 51 ; and translational blockade of myc mrna by eif4f inhibition and stabilization of rna g4 by rna g4 ligands.52 , 53 we recognize that uh tumors with high - level myc family protein expression tend to exhibit hypertrophic nucleoli , 13 , 14 which indicates that they are highly active in rrna synthesis and protein translation . 
small - molecule inhibitors , including cx - 5461 , an rna polymerase i inhibitor , and halofuginone , a protein translation inhibitor , could therefore effectively target these pathophysiologic features . as we have shown , these inhibitors in fact downregulate myc family protein expression in neuroblastoma cells.13 for those patients with tert - overexpressing neuroblastoma , telomerase inhibitors can be considered . 
if so , is there any other way by which the alt phenotype can be suppressed ? to address this question , we need to understand the mechanism of how atrx loss leads to alt . 
it has become evident that the acquisition of the alt phenotype uses the dna replication stress response , 30 which involves a cascade of events , including the obligatory activation of atr ( ataxia telangiectasia and rad3 related ) kinase . azd6738 is a novel potent and selective inhibitor of atr kinase with ic50 values of less than 1 mm in cell - based assays58 and would effectively target alt tumor cells . conclusion prognosis and therapy stratication of neuroblastomas have steadily improved since the introduction of the evans staging system59 with age used as a prognostic factor60 ; however , 5 - year survival of patients with high - risk neuroblastoma , dened by the international neuroblastoma risk group system ( inrg ) , remains at approximately 40% or lower.61 , 62 the same is true for the uh neuroblastoma group , which essentially overlaps high - risk neuroblastomas.8 , 63 unfortunately , no additional renement for the inrg has been proposed since 2009.61 the proposed histologic subgrouping of uh neuroblastomas represents a practical and immediately implementable approach with which to create the category leading to the most devastating clinical outcome , namely extremely unfavorable histology ( euh ) neuroblastoma . 
we expect that conventional uh neuroblastoma tumorsthat is , the null subgroupwould , we hope , respond to the current multimodal high - intensity therapy designed for high - risk neuroblastoma , whereas euh tumorsthat is , those of the myc , tert , and alt subgroupswould not . 
for those euh tumors , appropriate therapy protocols will have to be designed and tested . on the basis of our ndings , 12 myc family protein immunohistochemistry is already incorporated in the childrens oncology group pathology analysis as an integrated biomarker of neuroblastoma . 
similar attempts are now underway for tert and atrx immunohistochemistry for precision prognosis and the stratication of neuroblastoma . furthermore , we are planning to perform correlative studies between tert protein expression and tert gene rearrangements and between atrx protein loss and atrx gene mutations / deletions . 
for more information about ascos conict of interest policy , please refer to or po.ascopubs.org / site / ifc . no potential conicts of interest were reported . references willis r : neuroblastoma and ganglioneuroma . 
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 elevated levels of brafv600 mutant circulating tumor dna and circulating hepatocyte growth factor are associated with poor prognosis in patients with metastatic melanoma purpose we performed a retrospective exploratory analysis to evaluate the prognostic and predictive effect of two circulating biomarkers , brafv600 mutant circulating tumor dna ( ctdna ) and circulating hepatocyte growth factor ( chgf ) , in metastatic melanoma . materials and methods this study evaluated patients from brim - 3 , a phase iii trial comparing vemurafenib and dacarbazine in 675 patients with brafv600 mutated advanced melanoma . 
the use of circulating biomarkers has been an attractive strategy , primarily because of the ease of sample collection , and in the case of circulating tumor dna ( ctdna ) william lu luciana burton james larkin paul b . 
 and circulating tumor cells , the possibility that a traditional tissue biopsy may not reflect the current mutational landscape of a patients cancer.4 - 6 in this study , we characterized two circulating biomarkers for brafv600 - positive metastatic melanoma with the goal of using a combined multibiomarker approach to assess patient risk . approximately half of melanomas harbor mutations in the braf gene , and over 90% of these braf mutations occur at a single site , valine 600 ( v600 ) .7 - 9 early ctdna studies demonstrated the ability to detect brafv600e ctdna in patient serum and plasma , and in addition , some studies showed that the presence of ctdna correlated with worse survival and stage.10 - 19 some studies performed longitudinally demonstrated that a decrease in ctdna was observed after treatment but increases at progression.20 - 24 these studies have suggested that ctdna may be a useful prognostic indicator and , given the rapid kinetics of ctdna , 25 , 26 may also be useful in monitoring patients to predict disease progression . the introduction of braf inhibitor therapies , such as vemurafenib , have dramatically prolonged the survival of patients with braf - mutated metastatic melanoma.2 , 3 , 8 , 27 however , patients treated with braf inhibitor therapy often relapse , and mechanisms by which tumors obtain resistance are of considerable interest.3 , 27 , 28 one mechanism that has been described is the activation of the met signaling pathway by hepatocyte growth factor ( hgf ) .29 , 30 like ctdna , hgf is also detectable in circulation and can potentially be used as a prognostic or predictive biomarker . 
 studies assessing hgf as a circulating cancer biomarker have supported the idea that high levels of this ligand can support tumor growth , and elevated circulating hgf ( chgf ) has been associated with poor survival and worse stage in a variety of cancers.29 , 31 - 33 here , we present a retrospective exploratory analysis characterizing brafv600 mutant ctdna and chgf . 
although the use of these biomarkers has been studied before , this is , to our knowledge , the largest study of this kind using patients from a single randomized clinical trial comparing a braf inhibitor with a chemotherapy control . 
this test has been reported to also detect the v600k and v600d mutations . a total of 675 patients were randomly assigned to the chemotherapeutic drug dacarbazine ( 1 , 000 mg / m2 of body surface area by intravenous infusion every 3 weeks ; n = 338 ) or the braf inhibitor vemurafenib ( 960 mg twice daily orally ; n = 337 )  . 
samples from 605 patients ( 1 , 164 samples ) were available for ctdna analysis , and samples from 563 patients ( 665 samples ) were available for chgf analysis . investigations were performed after approval by a local human investigations committee and in accordance with an assurance filed with and approved by the department of health and human services , where appropriate . 
data were anonymized to protect the identities of research participants . ctdna assessment cell - free dna ( cfdna ) was extracted from 500 l of plasma using the qiaamp circulating nucleic acid kit ( 55114 ; qiagen , hilden , germany ) and eluted in 50 l of supplied buffer . 
all ctdna counts in this study are presented as a fraction of v600 mutant ctdna to wildtype v600 cfdna . hgf assessment chgf levels were measured by enzyme - linked immunosorbent assay as previously described.35 lower limit of quantification was determined to be 210 pg / ml using serial dilution experiments with recombinant human hgf . gene expression analysis gene expression was analyzed using 200 ng of total rna using the nanostring ncounter gene expression assay ( nanostring technologies , seattle , wa ) following manufacturers instructions . 
for data normalization , raw counts were log transformed and standardized to the geometric mean of each sample . statistical analysis kaplan - meier analysis was used to estimate overall survival ( os ) , postprogression survival , and median survival . 
the relationship between ctdna fractions , chgf , ldh , eastern cooperative oncology group ( ecog ) status , stage , treatment , sum of longest diameters , total metastases , and time point was assessed using student t test and tukey honest significant difference test . 
partitioning analysis was performed using jmp genomics ( version 8.0 ; sas institute )  . results circulating biomarkers and patient survival to determine whether baseline brafv600 mutant ctdna could be a prognostic factor in the metastatic melanoma , droplet digital polymerase chain reaction was initially used to evaluate brafv600 mutant ctdna levels in patients in the two treatment arms in the brim - 3 clinical trial ( fig 1 ; appendix table a1 )  . 
 shorter os was observed in vemurafenib - treated patients with detectable baseline levels of v600 mutant ctdna fraction compared with patients having undetectable levels of ctdna ( fig 2a )  . 
 furthermore , when patients with detectable levels of baseline ctdna were categorized into high and low subgroups , patients with high baseline ctdna had significantly shorter os than did those with low baseline ctdna ( fig 2b )  . 
notably , high levels of v600 mutant ctdna fraction were also a poor prognostic factor among dacarbazine treated patients . with the goal of using a combined biomarker approach to assess patient risk , we also measured baseline chgf levels of brim - 3 patients . 
in both vemurafeniband dacarbazine treated patients , high levels of baseline chgf were associated with significantly shorter os ( fig 2c )  . no statistical interaction was observed between baseline chgf and the treatment arm , nor did we observe interaction between baseline brafv600 mutant ctdna and the treatment arm , suggesting that these two biomarkers are not predictive in this setting . 
together , these results suggest that high levels of baseline ctdna and chgf are poor prognostic factors in patients with metastatic melanoma , regardless of treatment . longitudinal tumor monitoring using circulating biomarkers in addition to baseline measurements , we also assessed the utility of circulating biomarkers for longitudinal monitoring . 
 randomly assigned ( n = 675 ) allocation allocated to vemurafenib received allocated intervention ( n = 337 ) ( n = 335 ) analysis allocated to dacarbazine received allocated intervention ( n = 338 ) ( n = 289 ) ctdna analysis analyzed patients baseline samples c2d1 samples pd samples excluded from analysis samples unavailable all samples available failed ddpcr ( n = 324 ) ( n = 293 ) ( n = 282 ) ( n = 71 ) ( n = 11 ) ( n = 8 ) ( n = 3 ) chgf analysis analyzed patients baseline samples pd samples excluded from analysis samples unavailable all samples available failed chgf elisa ( n = 308 ) ( n = 302 ) ( n = 72 ) ( n = 27 ) ( n = 16 ) ( n = 11 ) ctdna analysis analyzed patients baseline samples c2d1 samples pd samples excluded from analysis samples unavailable all samples available failed ddpcr ( n = 281 ) ( n = 258 ) ( n = 219 ) ( n = 41 ) ( n = 8 ) ( n = 1 ) ( n = 7 ) chgf analysis analyzed patients baseline samples pd samples excluded from analysis samples unavailable all samples available failed chgf elisa ( n = 255 ) ( n = 249 ) ( n = 42 ) ( n = 34 ) ( n = 14 ) ( n = 20 ) fig 1 . 
 c2d1 , cycle 2 day 1 ; chgf , circulating hepatocyte growth factor ; ctdna , circulating tumor dna ; ddpcr , droplet digital polymerase chain reaction ; elisa , enzyme - linked immunosorbent assay ; pd , progressive disease . 
 patients longitudinally have demonstrated a reduction in patient ctdna levels after drug treatment and an increase at disease progression , changes thought to be reflective of the tumor burden in patients responsive and refractory to treatment.10 , 16 - 24 although our study only analyzed three time points as opposed to the more frequent monitoring performed by others , a significant reduction of mutant ctdna while receiving treatment at c2d1 followed by a significant increase in ctdna at disease progression was observed in patients in both treatment arms ( appendix fig a1b )  . we did not observe correlations between ctdna levels at c2d1 and survival , nor did we observe that decreases in ctdna levels between baseline and c2d1 correlated with survival or response ( data not shown )  . 
to our knowledge , this is the first study demonstrating that ctdna measured at progression can predict postprogression survival in patients with melanoma . approximately half of patients ( 62 of 112 ) with samples at disease progression had corresponding baseline samples with detectable ctdna . 
 among the vemurafenib - treated patients , those with a greater than two - fold decrease of brafv600 ctdna fraction between the baseline and progression time point experienced significantly longer postprogression survival than did those who experienced either no change or a greater than two - fold increase ( fig 3b )  . 
a similar correlation was not observed for chgf collected at disease progression , nor did we find that change in chgf between baseline and progression correlated with survival or response . circulating hgf and met signaling met - dependent signaling is associated with proliferation , metastasis , and activation of signaling pathways , including the pi3k pathway and the mapk pathway ; all processes that could contribute to a worse prognosis.36 to establish whether elevated chgf detected in patients contributes to met receptor activation , parallel experiments were performed analyzing met tumor rna . 
 a vemurafenib - treated patients dacarbazine - treated patients baseline v600 mutant ctdna detectable , n = 188 , 11.4 mo undetectable , n = 105 , 21.4 mo baseline v600 mutant ctdna detectable , n = 172 , 7.9 mo undetectable , n = 86 , 21 mo time ( months ) no . 
median survival of patients is displayed in the legend . groups , including high chgf / high met , high chgf / low met , low chgf / high met , and low chgf / low met . 
in contrast , patients with low chgf / low met performed the best , with os median values of 14.5 months ( vemurafenib ) versus 17.2 months ( dacarbazine )  . 
patients with high levels of baseline plasma circulating hepatocyte growth factor ( chgf ) and high tumor met expression have significantly shorter overall survival ( os ) than patients in other categories in both treatment arms . 
all covariables modeled , with the exception of stage , were significant independent predictors for os , indicating that the biomarkers included in this analysis are capable of contributing independently valuable insight regarding patient prognosis ( table 2 )  . a combined biomarker approach to assessing risk in patients with melanoma next , we assessed combining these prognostic biomarkers in a stepwise approach to evaluate patient risk . 
incorporating both baseline ctdna and baseline chgf biomarker data generated in this study with prognostic factors ldh level , ecog performance status , and stage , we defined four risk groups for vemurafenib - treated patients using partitioning analysis ( fig 5a )  . 
unpaired two - tailed t test was used to determine significance between groups . abbreviations : ctdna , circulating tumor dna ; ecog , eastern cooperative oncology group ; ldh , lactate dehydrogenase . was the first decisive factor , splitting the population into patients with a baseline brafv600 mutant ctdna fraction greater than and less than 0.039. 
this created four nodes : group 1 ( ctdna fraction < 0.039 , ecog status = 0 ) , group 2 ( ctdna fraction < 0.039 , ecog status = 1 ) , group 3 ( ctdna fraction > 0.039 , chgf < 438 pg / ml ) , and group 4 ( ctdna fraction > 0.039 , chgf > 438 pg / ml )  . patients with high levels of ctdna and chgf in group 4 had significantly shorter os than patients in any other group , with a median survival of 7.3 months ( fig 5b )  . 
although braf plus mek inhibitor combinations have been approved , single - agent braf inhibition is still widely used ; the brim - 3 study design allowed us to analyze a uniquely large patient cohort and characterize our biomarkers of interest for predictive or prognostic potential . 
here , we show that both brafv600 mutant ctdna and chgf are independent prognostic factors for patients with brafv600 mutant metastatic melanoma . recent publications analyzing ctdna in patients with metastatic melanoma have detected baseline mutant ctdna in approximately 70% to 80% of patients with mutation - positive tumors.14 , 15 , 17 , 18 this study detected mutant ctdna at baseline table 2 . 
 b 288 patients median os : 14.2 months v600 mutant ctdna fraction < 0.039 v600 mutant ctdna fraction > 0.039 156 patients median os : 20.0 months 132 patients median os : 10.0 months ecog status ecog status baseline chgf < 438 pg / ml baseline chgf > 438 pg / ml 123 patients median os : 22.0 months 33 patients median os : 12.3 months 68 patients median os : 11.5 months 64 patients median os : 7.3 months group 1 group 2 group 3 group 4 group 1 , n = 123 group 2 , n = 33 group 3 , n = 68 group 4 , n = 64 no . 
vemurafenib treated patients with elevated levels of baseline v600 circulating tumor dna ( ctdna ) and elevated levels of baseline circulating hepatocyte growth factor ( chgf ) are a high - risk patient group . 
 ( a ) decision tree for risk stratification : vemurafenib treated patients with eastern cooperative oncology group ( ecog ) performance status , baseline v600 ctdna , and baseline chgf data were stratified into four risk groups using recursive partitioning analysis . 
these findings , along with others , suggest that the differences in ctdna seem to primarily reflect tumor burden rather than the fraction of brafv600 mutant tumor cells within the tumor . we hypothesized that chgf could serve as a marker of resistance to vemurafenib . 
hgf can signal to met to reactivate the mapk pathway ; therefore , high levels of hgf may act as a potential resistance mechanism in patients treated with braf inhibitors.29 , 30 although there have been two studies evaluating the use of chgf as a biomarker for metastatic melanoma , 43 , 44 to our knowledge , this study is the first to evaluate the use of chgf as a biomarker in patients treated with a braf inhibitor in comparison with patients in a chemotherapy treatment group . 
this result suggests that although chgf may play a role in vemurafenib resistance , it likely also contributes to more aggressive disease , even in the absence of mapk pathway inhibition . it has been reported that hgf originating from stromal cells in the tumor microenvironment could contribute to mapk and pi3k - akt pathway activation.30 our findings from this study focused on circulating hgf , and the origin of the growth factor remains unclear . 
 findings do suggest that the hgf relevant to patient survival may be secreted from outside of the tumor microenvironment . a single biomarker can reveal one aspect of disease and can help in assessing patient risk , but the integration of multiple biomarkers is likely to provide greater insight . 
using partitioning analysis , we were able to categorize patients into four risk groups , with the highest risk group consisting of patients with high brafv600 mutant ctdna and high chgf . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . william lu employment : genentech / roche research funding : genentech / roche luciana burton employment : genentech / roche james larkin honoraria : eisai , bristol - myers squibb , msd , glaxosmithkline , kyocera , pfizer , novartis , roche / genentech , secarna , pierre fabre , eusa pharma consulting or advisory role : eisai , bristol - myers squibb , msd , glaxosmithkline , kyocera , pfizer , novartis , roche / genentech , secarna , pierre fabre , eusa pharma research funding : pfizer ( inst ) , novartis ( inst ) travel , accommodations , expenses : bristol - myers squibb , pfizer , novartis , roche / genentech paul b . 
chapman honoraria : bristol - myers squibb , glaxosmithkline , genentech / roche , provectus , momenta pharmaceuticals , daiichi sankyo consulting or advisory role : bristol - myers squibb , glaxosmithkline , genentech / roche , daiichi sankyo , provectus , momenta pharmaceuticals research funding : glaxosmithkline , genentech / roche , bristol - myers squibb , pfizer travel , accommodations , expenses : bristol - myers squibb paolo a . 
sosman honoraria : amgen , merck , array biopharma , bristolmyers squibb , amgen , merck , array biopharma , bristolmyers squibb ivor caro employment : genentech / roche stock or other ownership : roche grant a . 
mcarthur research funding : celgene ( inst ) , genentech / roche ( inst ) , bristol - myers squibb ( inst ) , amgen ( inst ) , array biopharma ( inst ) , novartis / pfizer ( inst ) ilsung chang employment : genentech / roche stock and other ownership : roche / genentech honoraria : roche / genentech consulting or advisory role : roche / genentech elicia penuel employment : genentech / roche stock and other ownership : genentech / roche yibing yan employment : roche / genentech stock and other ownership : roche / genentech patents , royalties , other intellectual property : roche / genentech matthew j . 
wongchenko employment : genentech / roche travel , accommodations , expenses : roche / genentech stock and other ownership : genentech / roche other relationship : roche / genentech stock and other ownership : ariad affiliations william lu , luciana burton , ilsung chang , ivor caro , elicia penuel , yibing yan , and matthew j . 
wongchenko , genentech , south san francisco ; antoni ribas , the jonsson comprehensive cancer center at university of california , los angeles , ca ; james larkin , the royal marsden nhs foundation trust , london , united kingdom ; paul b . 
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santiago - walker a , gagnon r , mazumdar j , et al : correlation of braf mutation status in circulating - free dna and tumor and association with clinical outcome across four brafi and meki clinical trials . 
schreuer m , meersseman g , van den herrewegen s , et al : quantitative assessment of braf v600 mutant circulating cell - free tumor dna as a tool for therapeutic monitoring in metastatic melanoma patients treated with braf / mek inhibitors . 
xi l , pham tht , payabyab ec , et al : circulating tumor dna as an early indicator of response to t - cell transfer immunotherapy in metastatic melanoma . 
chang ga , tadepalli js , shao y , et al : sensitivity of plasma brafmutant and nrasmutant cell - free dna assays to detect metastatic melanoma in patients with low recist scores and non - recist disease progression . 
janku f , huang hj , claes b , et al : braf mutation testing in cell - free dna from the plasma of patients with advanced cancers using a rapid , automated molecular diagnostics systemol cancer ther 15 : 1397 - 1404 , 2016 20 . 
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gonzalez - cao m , mayo - de - las - casas c , molina - vila ma , et al : braf mutation analysis in circulating free tumor dna of melanoma patients treated with braf inhibitors . 
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mcarthur ga , chapman pb , robert c , et al : safety and efficacy of vemurafenib in braf ( v600e ) and braf ( v600k ) mutation - positive melanoma ( brim - 3 ) : extended follow - up of a phase 3 , randomised , open - label study . 
busam kj , hedvat c , pulitzer m , et al : immunohistochemical analysis of braf ( v600e ) expression of primary and metastatic melanoma and comparison with mutation status and melanocyte differentiation antigens of metastatic lesions . 
 ( a ) baseline v600 mutant ctdna fraction and baseline chgf levels were not significantly different between patients from either treatment ardata points measured below the lower limit of quantitation are shown in the gray shaded region . 
 ( b ) v600 mutant ctdna fraction decreased post - treatment at cycle 2 day 1 ( c2d1 ) but returned to elevated levels at progression in patients in both treatment arms . 
 ( a ) larger tumor burden as determined by the sum of the longest diameters of target lesions correlated with higher levels of baseline v600 mutant ctdna fraction as well as high levels of baseline chgf . 
 tukey honest significant difference ( hsd ) test was used to determine significance between patients categorized by ctdna , and unpaired two - tailed t test was used for patients categorized by chgf . 
brim - 3 patient characteristics overview vemurafenib treated ( n = 337 ) dacarbazine treated ( n = 338 ) 56 ( 21 - 86 ) 52.5 ( 17 - 86 ) characteristic age , years ( range ) median sex , no . 
 use of low - frequency driver mutations detected by cell - free circulating tumor dna to guide targeted therapy in nonsmall - cell lung cancer : a multicenter case series purpose to evaluate the clinical outcome of patients with nonsmall - cell lung cancer treated by targeting low variant allelic frequency ( vaf ) driver mutations identified through cell - free dna ( cfdna ) next - generation sequencing ( ngs )  . 
detection of driver mutations in cancer is critically important in the age of targeted therapy , where both tumor - based as well as cfdna sequencing methods have been used for therapeutic decision making . 
we hypothesized that vaf should not be predictive of response and that low vaf alterations detected by cfdna ngs can respond to targeted therapy . patients and methods a multicenter retrospective case review was performed to identify patients with nonsmall - cell lung cancer who received targeted molecular therapy on the basis of findings of low vaf alterations in cfdna ngs . 
mutations at low vaf were defined as < 0.2% mutated cfdna molecules in a background of wild - type cfdna . results one hundred seventy - two patients underwent cfdna ngs testing . 
2018 by american society of clinical oncology introduction advances in the sensitivity and accuracy of plasma cell - free dna ( cfdna ) sequencing now allow for somatic genomic tumor alterations to be detected by cfdna next - generation sequencing ( ngs ) at the level of 0.02% variant allelic frequency ( vaf ) .1 this raises the question of whether targetable driver mutations that are detected at low frequencies , defined herein as < 0.2% vaf , retain importance even in the setting of other genomic alterations at higher vaf . 
 specific cases spanning each of the three major types of mutationssingle nucleotide variants ( snvs ) , insertion - deletion mutations ( indels ) , and fusionsare discussed . approximately 20% to 30% of tumors from patients with lung adenocarcinoma have targetable miriam t . 
 oncogenic driver mutations in alk , braf , egfr , erbb2 ( her2 ) , met , ret , and ros1.2 , 3 lung cancer patients with nonsmall - cell ( nsclc ) treated with targeted molecular therapies against these oncogenic driver mutations have been found to have prolonged survival compared4 , 5 with patients treated with standard chemotherapy , with improved treatment - related toxicity and quality of life.6 - 8 given these results , the national comprehensive cancer network now recommends that patients with metastatic nonsquamous nsclc have broad molecular profiling of their tumor performed , with cfdna as a secondary option if repeat tissue biopsy is not feasible.9 as tumor cells become necrotic and undergo apoptosis , they release circulating fragments of dna . 
with advances in ngs , it is now possible to detect cfdna from blood samples with significant accuracy and precision.10 , 11 three major approaches exist for analyzing cfdna in peripheral blood . 
the first uses probes to capture hotspots , or regions commonly mutated in selected genes ; an example of this approach is digital droplet polymerase chain reaction.12 the second uses ngs but requires a tumor tissue sample to identify specific mutations , which are used as a reference to select probes to specific regions in cfdna.13 , 14 the third method uses ngs but targets a gene panel of interest using complete exon sequencing in a blinded approach.15 , 16 mutations are quantified and represented as a vaf calculated as the total number of molecules with a given mutation divided by the total number of mutant plus wild - type molecules . detection of cfdna allows for tumor monitoring at disease progression via serial peripheral blood samples and , in the future , may eliminate the need for tumor biopsies or repetitive biopsies in patients with advanced cancer when the initial biopsy is inadequate , unobtainable for genomic testing , or uninformative , or when the patients cancer has progressed despite treatment.17 - 19 the quantity of cfdna has been shown to correlate with tumor staging and prognosis.17 , 20 limitations of cfdna include testing in patients with low disease burden and in patients with bone or brain metastasis , who may have quantities of cfdna below the limit of detection ( lod ) for currently available assays . 
although these false - negative results limit the application of cfdna ngs to a rule - in test , more than a dozen nsclc studies have shown that targetable genomic alterations detected in plasma predict response similarly to those detected in tissue.21 - 24 however , it is unclear whether variants at low allele fractions are as responsive to targeted therapy as those at high allele fractions . 
 samples were sent to a clinical laboratory improvement amendments ( clia ) certified , college of american pathologistsaccredited , new york state department of healthapproved clinical laboratory ( guardant health , redwood city , ca ) for clinical cfdna ngs testing ( guardant360 ) from one of three institutions ( university of california , san diego [ ucsd ] , duke university , or northwestern university )  . 
for this project , a vaf 0.2% was defined as low vaf because this is the 25th percentile vaf detected on guardant360 ( the median or 50th percentile is 0.39% ) in > 5 , 000 patient samples.1 medical records were reviewed in accordance with the respective institutional review board guidelines of ucsd , duke university , and northwestern university . 
response evaluation criteria in solid tumors ( recist v1.1 ) were used to assess response to treatment . tissue ngs individuals had tumor tissue from primary or metastatic sites sent for targeted tumor tissue ngs before or at approximately the same time as their cfdna analysis . 
tissue - based ngs was done at either ucsd , duke , foundation medicine ( foundation one ; cambridge , ma ) , or caris ( phoenix , az )  . 
the lower lod for this assay was 5% , meaning that a mutant allele can be detected in a 95% wild - type allele background ( or 10% neoplastic cell content )  . 
coverage of at least 250 was required to detect a mutation at the 5% lod . egfr sequencing , ros1 and alk fluorescent in situ hybridization testing neogenomics laboratories ( fort meyers , fl ) performed egfr sequencing , which included bidirectional sequencing of exons 18 to 21 of the egfr gene for detection of egfr - activating mutations and tyrosine kinase inhibitor resistance mutations ( including t790m )  . 
tumor enrichment was performed before extraction ( detailed information can be found on the neogenomics web site ) .29 ros1 and eml4 - alk fluorescent in situ hybridization ( fish ) testing was performed by response genetics ( los angeles , ca ) , and a positive test on fish was considered to be > 15% probe binding . plasma cell - free circulating dna analysis the cfdna ngs analysis was performed at guardant health ( guardant360 )  . 
the exons of up to 73 cancer genes were captured using biotinylated custom bait oligonucleotides ( agilent , la jolla , ca ) , resulting in a 148 , 000 base pair ( 78 kb ) capture footprint . 
these algorithms quantify the absolute number of unique dna fragments at a given nucleotide position , thereby enabling circulating tumor dna to be quantitatively measured as a fraction of total cfdna . 
because patients were not enrolled in a prospective clinical trial , assessments were not standardized across the cohort . results between september 2015 and september 2017 , six clinicians from the three institutions ( ucsd , duke university , and northwestern university ) identified 192 orders from 172 unique patients with a diagnosis of nsclc and in whom cfdna was detected . 
although pfs was roughly as expected in this small cohort , the median os of < 15 months is somewhat shorter than expected for egfrand alk positive patients , especially because the median pfs of patients receiving targeted therapies was 12 months . 
this finding of lower than expected os may be because of sample size , or it is possible that patients with driver mutations present at low vaf have a worse prognosis compared with patients with driver mutations at more standard frequencies . 
there may also be other negative prognostic factors associated with low vaf that are not yet known . met exon 14 skipping mutations represent a clinically unique molecular subtype of nsclc and are found in approximately 3% of all nsclc cases.32 met exon 14 mutations can be detected via tumor ngs or with cfdna ngs , which represents a novel detection method because currently , no other alternative detection modalities ( eg , immunohistochemistry or fish ) exist for this mutation . 
thus the met exon 14 skipping mutation can lead to increased activation of c - met , resulting in oncogenic driver mutation potential.33 fusions and midsize indels ( > 50 bp ; eg , met exon 14 skipping variants ) are especially challenging to detect in cfdna . 
as illustrated by case 8 , detection of met exon 14 skipping mutations can significantly affect the use of existing agents ( eg , crizotinib ) or novel agents ( eg , glesatinib ) for treatment . it is now known that intratumoral heterogeneity is associated with an increased risk of recurrence or death . 
 possible , all targetable driver mutations , both early in treatment and as the tumor evolves.34 using interval liquid biopsies on the basis of radiographic progression of an otherwise nonbiopsiable lesion can provide early detection of drug resistance and thus lead to early treatment intervention . 
this was illustrated by patient 5 , who declined invasive tumor biopsy and underwent repeat cfdna testing at the time of radiologic progression , which was 16 months after his initial cfdna testing . 
 a modest - sized prospective clinical trial by kim et al21 used cfdna - guided matched therapy when tissue was insufficient or unobtainable for ngs in patients with metastatic gastric cancer , nsclc , or melanoma . 
additional prospective studies are needed to further examine the value of early detection of resistance mechanisms before clinical evidence of progression and to determine if early detection and intervention truly influences os and pfs . in this series , patients were selected on the basis of the presence of low vaf driver mutations detected in cfdna . 
it has been reported that a subgroup of patients does not release tumor cfdna into their circulation , whereas other patients convert from having circulating tumor cfdna to noncirculating tumor cfdna after treatment , for reasons that include low tumor burden , slow - growing or inactive tumor growth , or therapeutic response.35 if safe , a tissue biopsy should be performed in patients who initially had detectable mutations on cfdna who no longer have detectable mutations but have clear progression of disease . 
there are several ongoing large prospective trials , including the azd9291 first time in patients ascending dose study ( aura ) , which serially compared cfdna versus tumor ngs over time in patients with egfr t790m mutations who were receiving osimertinib , which may help to address these clinical scenarios.16 one patient ( patient 10 ) was noted to have both eml4 - alk and ros1 mutations detected on tissue fish testing at the time of diagnosis . 
 difficulty detecting the mutation because recurrence of disease was located in the brain or the possibility that ros1 was present below the lod for the cfdna assay after 1 year of treatment . 
the patient was transitioned to hospice shortly after cfdna testing was done , and no additional testing was performed to confirm the presence of ros1 rearrangement . to our knowledge , this is the first case series examining treatment outcomes for patients with nsclc with low vaf driver mutations detected by cfdna . 
most patients had other passenger mutations that were present at a higher proportion compared with the low - frequency driver mutation , and the therapeutic significance of relatively low vaf driver mutations has not been previously reported . 
patel manuscript writing : all authors final approval of manuscript : all authors in the sensitivity and specificity of liquid biopsy , the ability to detect and confer clinical benefit to patients on the basis of these results will continue to improve with technological advances in cfdna . 
from this small case series , it seems that even at low vaf , targetable driver mutations retain clinical importance , even in the setting of other genomic alterations at higher vaf . 
mohindra consulting or advisory role : astrazeneca , genentech travel , accommodations , expenses : astrazeneca stock and other ownership interests : pfizer ( i ) lindsey shantzer no relationship to disclose ingrid l . 
patel consulting or advisory role : ariad , abbvie , astrazeneca jeffrey clarke consulting or advisory role : inivata , premier speakers bureau : guardant health , merck stock and other ownership interests : guardant health , biolase research funding : guardant health research funding : medpacto , genentech , bristol - myers squibb , adaptimmune , spectrum pharmaceuticals , abbvie , moderna therapeutics james christensen employment : mirati therapeutics stock and other ownership interests : mirati therapeutics , bluebird bio patents , royalties , other intellectual property : multiple patents in last 2 years while employed at mirati therapeutics covering discovery of kras , lsd1 , and ezh2 inhibitors . 
patel consulting or advisory role : eli lilly , novartis , bristolmyers squibb , astrazeneca / medimmune speakers bureau : merck , boehringer ingelheim research funding : bristol - myers squibb ( inst ) , eli lilly ( inst ) , incyte ( inst ) , medimmune ( inst ) , pfizer ( inst ) , genentech ( inst ) , xcovery ( inst ) affiliations miriam t . 
patel , university of chicago school of medicine , chicago , il ; lindsey shantzer and jeffrey clarke , duke university medical center , durham , nc ; ingrid l . 
lanman , guardant health , redwood city ; and james christensen , mirati therapeutics , san diego , ca . references 77 : 5705 , 2017 ( suppl 13 : abstr ) 1 . 
paik pk , varghese am , sima cs , et al : response to erlotinib in patients with egfr mutant advanced non - small cell lung cancers with a squamous or squamous - like component . 
aisner dl , sholl lm , berry ld , et al : the impact of smoking and tp53 mutations in lung adenocarcinoma patients with targetable mutationsthe lung cancer mutation consortium ( lcmc2 )  . 
zhou c , wu yl , chen g , et al : erlotinib versus chemotherapy as first - line treatment for patients with advanced egfr mutation - positive non - small - cell lung cancer ( optimal , ctong - 0802 ) : a multicentre , open - label , randomised , phase 3 study . 
sequist lv , yang jc , yamamoto n , et al : phase iii study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with egfr mutations . 
thompson jc , yee ss , troxel ab , et al : detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next - generation sequencing of cell - free circulating tumor dna . 
sacher ag , paweletz c , dahlberg se , et al : prospective validation of rapid plasma genotyping for the detection of egfr and kras mutations in advanced lung cancer . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
oxnard gr , paweletz cp , kuang y , et al : noninvasive detection of response and resistance in egfr - mutant lung cancer using quantitative next - generation genotyping of cell - free plasma dna . 
valle a , audigier - valette c , herbreteau g , et al : rapid clearance of circulating tumor dna during treatment with azd9291 of a lung cancer patient presenting the resistance egfr t790m mutation . 
kim st , banks kc , lee s - h , et al : prospective feasibility study for using cell - free circulating tumor dnaguided therapy in refractory metastatic solid cancers : an interim analysis . 
thompson jc , yee ss , troxel ab , et al : detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next - generation sequencing of cell - free circulating tumor dna . 
rozenblum ab , ilouze m , dudnik e , et al : clinical impact of hybrid capture - based next - generation sequencing on changes in treatment decisions in lung cancer . 
camidge dr , bang yj , kwak el , et al : activity and safety of crizotinib in patients with alk - positive non - small - cell lung cancer : updated results from a phase 1 study . 
awad mm , oxnard gr , jackman dm , et al : met exon 14 mutations in non - small - cell lung cancer are associated with advanced age and stage - dependent met genomic amplification and c - met overexpression . 
peschard p , fournier tm , lamorte l , et al : mutation of the c - cbl tkb domain binding site on the met receptor tyrosine kinase converts it into a transforming protemol cell 8 : 995 - 1004 , 2001 34 . 
lin c - c , shih jy , yu cj , et al : outcomes in patients with non - small - cell lung cancer and acquired thr790met mutation treated with osimertinib : a genomic study . 
 smart cancer navigator : a framework for implementing asco workshop recommendations to enable precision cancer medicine purpose data standards and interoperability are critical for improving care for patients with cancer . 
to facilitate improved patient care , several recommendations for data sharing and standardization were made to the community . methods to address these recommendations , we developed smart cancer navigator , a web application that uses application programming interfaces to gather clinical and genomic data from 11 public knowledge bases ranging from basic to clinical content coverage ; three ( civic , clinvar , and oncokb ) explicitly linked genomic variants to clinical factors such as prognosis and treatment selection . 
we illustrated the utility of this application by selecting one of the monthly case studies presented by the asco university molecular oncology tumor board : ovarian cancer ( brca mutation )  . 
 results smart cancer navigator aggregates and contextualizes data from 11 different knowledge bases and stores user queries in a lightweight web application that can link into fast healthcare interoperability resourcesenabled electronic health records . 
a comparison of the three clinico - genomic knowledge bases indicated substantial differences in coverage at the gene and variant levels . conclusion smart cancer navigator has immediate relevance to practicing oncologists and others . 
2018 by american society of clinical oncology introduction precision medicine is a model for disease prevention and treatment that considers variability in genes , environment , and lifestyle across individuals.1 to expand and broaden this model , the federal government invested $215 million in establishing the precision medicine initiative in 2015 , with a large portion of the funding devoted to precision oncology.2 , 3 advances in molecular profiling to identify actionable genetic aberrations provided a compelling rationale for this initial emphasis.4 - 6 in may 2016 , asco convened a data standards and interoperability summit to address the critical data sharing and standardization issues that could impede the realization of precision oncology . in typical genomic workflows , oncologists request genomic analysis of a recurrent or metastatic tumor , and genomics laboratories return variably structured narrative reports of the genes and variants they identified , which are often scanned into the patients electronic health record ( ehr )  . 
 some reports are more than 20 pages long , including characterization of genetic variants , potential targeted therapies , and relevant clinical trial information , whereas others may be brief and may lack any interpretation . 
facing a lack of clear actionability ( ie , translation of genomic findings into treatment recommendations ) , potential bias in report curation , and potentially outdated information , oncologists often have to access additional knowledge bases to obtain more comprehensive , up - to - date disease - gene - variant information . 
inconsistencies among knowledge bases ( eg , differences in querying syntax and graphical user interfaces ) lead to inconvenience and inefficiency ; furthermore , oncologists must re - enter patient data every time they need to update their queries . because of these issues , attendees at the 2016 asco omics and precision oncology ( opo ) workshop called for the development of new software applications to help community oncologists interpret the ever - increasing volumes of genomic data critical for selecting treatment.7 here we describe smart cancer navigator , a software application ( app ) we developed to facilitate knowledge base queries and identify actionable information for practicing oncologists . 
the purpose of this article is to describe the development of and demonstrate the utility of the smart cancer navigator and to disseminate it to the oncology community for implementation and feedback . methods app overview smart cancer navigator securely links patient - specific data from ehr and genomics laboratories to multiple independent knowledge bases to facilitate efficient and effective querying of disease - gene - variant information . 
the web - based app was built by a team of software developers at the biomedical cybernetics laboratory at the boston childrens hospital computational health informatics program and harvard medical school who used an angular and bootstrap front - end framework . 
the app , prepopulated with relevant information from genomic laboratories and ehrs , provides an interface that oncologists can use to query evidence from several cancer - focused , genomic , clinico - genomic , and clinical trials knowledge bases . 
through built - in feedback functionality , meaningful responses regarding the usability and efficacy of the app can be collected for continuing improvement . smart cancer navigator is a modular app built by using substitutable medical apps reusable technology ( smart ) on fast healthcare interoperability resources ( fhir ) technology . 
 the smart on fhir platform was chosen to ensure interoperability with as many ehr systems as possible.8 - 10 smart on fhir facilitates the development of apps that can connect to health data systems ( eg , ehrs , personally controlled health records , health information exchanges ) without any custom configuration.8 , 11 , 12 through the development of application programming interfaces ( apis [ apps ] ) developed on the smart platform are substitutable : just as applications on a mobile technology platform such as apples operating system ( ios ) are separated from the core system and services ( eg , camera , geolocation ) , apps on the smart platform are separated from health data systems ( the equivalent of the core system and services on ios )  . login and authentication smart cancer navigator can be linked to a patients api - enabled ehr . 
because smart on fhir - enabled ehrs provides an oauth2based approach for secure login and authentication , the user can login and authorize the app for data access once the user has been authenticated with the ehr ( ie , has provided a username and password linked to appropriate credentials ) .9 once successfully authenticated and authorized , the app can be populated with relevant patient and practitioner information stored in the local environment . nomenclature , drop - down menus , data entry , and queries after logging in and optionally linking to a patients ehr , the user is presented with an interface for querying gene - variant combinations ( fig 1 )  . 
smart cancer navigator uses a hybrid data entry field that allows filtering via text entry followed by selection from a drop - down menu , which ensures efficient and consistent querying of gene - variant combinations . 
name and age are randomly generated ; any resemblance to an actual person is purely coincidental . field , the complementary drop - down list is populated in real time with suggested gene - variant combinations from the linked knowledge bases ( fig 1 )  . 
using this real - time service guarantees access to the latest available gene - variant information . the user begins a query by typing in the search bar , and the apps search algorithm efficiently sifts through thousands of known gene - variant combinations . 
this approach enables users to select gene - variant combinations without having to adhere to formal nomenclature or having to know the latest gene names . for executing queries , the app uses the following standards : genes are represented using hugo gene nomenclature committeeapproved symbols and national center for biotechnology information entrez gene ids , and variants are represented using the human genome variation society ( hgvs ) protein and genomic reference sequences.13 - 16 once the user has selected the appropriate gene variant combination , smart cancer navigator translates the combination into relevant api calls and populates an expandable view containing the results . 
these searches and results can then be saved to the patients ehr . knowledge bases and apis smart cancer navigator accesses 11 knowledge bases : ( 1 ) clinical interpretations of variants in cancer ( civic ) , ( 2 ) clinvar , ( 3 ) catalogue of somatic mutations in cancer ( cosmic ) , ( 4 ) combined annotation dependent depletion ( cadd ) , ( 5 ) database for nonsynonymous snps functional predictions ( dbnsfp ) , ( 6 ) database for single nucleotide polymorphisms ( dbsnp ) , ( 7 ) cancer genome ( 8 ) precision medicine interpreter knowledgebase ( pmkb ) , ( 9 ) oncokb , ( 10 ) drug - gene interaction database ( dgidb ) , and ( 11 ) clinicaltrials.gov.17 - 27 these knowledge bases were selected because at least some or all of their content was freely available through apis in mid to late 2017 . ( cgi ) , variant annotation information from civic is queried via standardized apis that use the hgvs genomic reference sequence id through the myvariant.info restful service28 and via a proprietary api format . 
this field often provides information on prognostic implications , potential drug treatment options , and context on previous or ongoing clinical research on the variant . summary pathways list of pathways associated with the normally functioning gene . functional prediction and characterization a numeric score , rank , boolean operator , or text string describing the characterization of a variant ( eg , benign , gain of function )  . subsection gene , variant gene variant coordinates and details series of fields describing the location of the gene or variant . 
usually includes chromosome number , reference or alternate allele , start and end position , codon position , and reference codon . gene , variant type variant tab : series of fields describing the variant type ( eg , single nucleotide polymorphisms , single nucleotide variation ) and predicted consequence of the mutation ( eg , missense , nonsense )  . 
 gene tab : series of fields describing the gene type ( eg , protein - coding )  . gene , variant aliases and ids series of fields containing aliases and ids ( eg , entrez ) for genes . origin of variant text field or boolean operator that characterizes the variant as somatic , germline , or both . known diseases cancer subtypes that the variant is known to be implicated in . suggested treatment ( effective drugs ) potential drug treatment options . 
drug suggestions are often accompanied with a text field containing the name of the implicated disease , as well as a short description of predictive implications for users of the drug . tumor site and traits a text field containing information regarding the tumor from which the variant was discovered . variant supporting evidence quality of evidence clinical trials pointers to publications , conference proceedings , or other evidence that support assertions , in particular , suggested treatments . subjective or validated scales that rate the quality of supporting evidence , such as the levels of evidence scale used by oncokb . table of values from clinicaltrials.gov api : trials are separated into two tables : variantassociated trials and gene - associated trials . 
each table has the following 5 columns : ( 1 ) clinical trial id , ( 2 ) phase , ( 3 ) therapies , ( 4 ) title of trial , and ( 5 ) recruitment status . clinical trials gene variant variant variant variant variant through 7 are queried with myvariant.info , and knowledge bases 8 through 11 are queried using their own api formats . 
if the knowledge bases fail to return data for a query , the app records a knowledge base gap . displaying results upon selecting a gene - variant combination , the app queries the knowledge bases and then returns results in an expandable view . 
for example , when interacting with the functional prediction ( clinical significance ) field in the variant subsection , users can view evidence from clinvar and relevant accession numbers . because smart cancer navigator compiles information from many different data sources , there may be instances in which sources present conflicting information . 
upon clicking the link , the user can view all interpretations and respective data sources in a tabular format . interface with ehrs and local servers smart cancer navigator follows smart on fhir specifications to connect to ehrs and interpret patient context.9 optionally , users can also manually enter patient data ; by default , only non - protected health information ( phi ) is exposed to the external apis . 
in accordance with smart on fhir specifications , the observation resource is separate from the patient resource , but includes a link to the patient resource id to ensure that this information remains linked . 
during development , the following test systems were used : the healthcare services platform consortium sandbox29 to simulate ehrs and the fhir genomics server30 server to simulate a genomics archive communications system . knowledge base analysis in addition to developing smart cancer navigator , we performed analyses to examine concordance among publicly available knowledge bases . 
briefly , a 62 - year - old woman underwent optimal debulking for advanced - stage hgsoc and remained in complete remission for 3 years after first - line platinum doublet therapy . 
she then relapsed with platinum - sensitive disease and attained a second complete remission ( see data supplement for full case description ; materials used with permission )  . under current national comprehensive cancer network clinical practice guidelines , 34 , 35 this patient with hgsoc should have received genetic counseling , regardless of family history . 
 because testing at diagnosis frequently does not occur , 36 , 37 we present the following hypothetical results from next - generation sequencing panel testing conducted at the time of relapse : ( 1 ) brca1 p.trp1815ter at 50% allele frequency , consistent with a germ line variant ; ( 2 ) brca1 p.cys61gly at 10% allele frequency , consistent with a somatic variant ; and ( 3 ) an etv6 - ntrk3 fusion ( data supplement )  . in the de facto workflow , medical oncologists and tumor board coordinators face significant obstacles when they attempt to corroborate and enrich these genetic testing reports that use multiple knowledge bases.38 for each search , data must be manually entered ; in addition , nonstandardized nomenclature between reports and knowledge bases can lead to inconvenience and the omission of crucial information . 
furthermore , search results are not captured in a format amenable to ehr integration , which may diminish rapid translation.7 , 39 the smart cancer navigator app removes these obstacles . to begin , the oncologist logs in , chooses the appropriate patient from the ehrs , and launches smart cancer navigator , with prepopulated patient data , including name , sex , age , and any previously queried gene - variant combinations . 
a separate query would be made for each gene - variant combination ; smart cancer navigator automatically saves each query , allowing for subsequent re - querying of knowledge bases at a later time without the need for redundant data entry . immediately after entering each query , the user can interact with the results for each gene - variant combination . 
in gene results ( fig 2 ) , the user is presented with relevant summaries and descriptions as well as important details ( gene symbols and ids , gene type , aliases , genomic position , and pathways )  . 
the variant results provide relevant descriptions , evidence items , and details , such as variant type , functional prediction , coordinates , tumor site , knowledge base ids , mutation frequency , and suggested treatments ( data supplement )  . 
a prose description of the gene and its function is provided on the left ; structured information from the knowledge bases is presented in tabular format on the right . site - agnostic trials , including the nci - match basket trial ( nct02465060 : nci - match : targeted therapy directed by genetic testing in treating patients with advanced refractory solid tumors , lymphomas , or multiple myeloma ) , which contains larotrectinib in arm z1e . 
comparing variants present in the two cancer - focused knowledge bases ( civic and oncokb ) , we found only 8.1% ( 432 of 5 , 317 ) unique variants in common ( data supplement )  . using the functional annotation tool database for annotation , visualization and integrated discovery ( david45 ) , we performed gene enrichment analysis on data from civic , clinvar , and oncokb . 
the kyoto encyclopedia of genes and genomes ( kegg46 ) pathways in cancer genes were identified in all three databases , with 4.1% of clinvar genes , 30.8% of civic genes , and 34.1% of oncokb genes associated with kegg pathways ( all p < .001 via davids ease score threshold , 47 a modified fishers exact p value )  . 
 a database analysis : comparison of genes in clinvar , civic , and oncokb database analysis : comparison of genes in clinvar , civic , and oncokb that are involved in kegg pathways in cancer1 6105 civic ( 90 ) clinvar ( 236 ) oncokb ( 93 ) civic ( 328 ) clinvar ( 6 , 425 ) oncokb ( 273 ) 1analysis performed using data available on august 5 , 2017 total = 6 , 548 1analysis performed using data available on august 5 , 2017 total = 393 fig 3 . 
venn diagrams for a comparison of genes in ( a ) clinvar , clinical interpretations of variants in cancer ( civic ) , and oncokb and ( b ) clinvar , civic , and oncokb that are involved in kyoto encyclopedia of genes and genomes ( kegg ) pathways in cancer . 
third , inconsistency in interpretation is addressed by providing multiple interpretations from numerous knowledge bases , with the aim of presenting a more holistic approach on the basis of multiple up - to - date knowledge sources . 
fourth , with simple drop - down interfaces in the search / query view and well - organized structured outputs in the results view , oncologists can readily access disease - gene - variant information without any formal training . 
in addition , smart cancer navigator links genomic information to relevant clinical trials , provided that they have the relevant hgvs protein or genomic reference sequences , thereby addressing another key opo workshop recommendation . analysis of existing knowledge bases revealed fairly disparate coverage at the gene and variant levels . 
 be enriched for germ line variants means that cancer - focused knowledge bases should also have substantial germ line information.52 , 53 whether differences in coverage of the cancer - focused knowledge bases reflect differing priorities versus incomplete curation is unclear ; active efforts such as the variant interpretation for cancer consortium54 are underway to coordinate efforts and maximize interoperability . 
it is expected that more cancer - specific knowledge base content will become available via public apis ; our analysis was limited to civic and oncokb because of their api availability . review of the clinvar clinical significance results , which were enriched with variants of unknown significance , makes apparent the ongoing need for the interpretation and discovery of actionable variants . 
after the recent advances in next generation sequencing technologies , it is clear that there is still much work to be done in studying new variants and providing relevant information regarding actionable variants to oncologists.55 - 57 in addition , differences between proprietary and standardized apis pose an obstacle to directly incorporating data from some knowledge bases . 
 they would need to familiarize themselves with the structure of the apis and would find that different combinations of operations and internal terminologies or ids are often needed to access data across knowledge bases . 
because data granularity , specificity , and nomenclature may differ , and evidence reliability ratings are not readily translatable across knowledge bases , this highlights a need for better representation of genomic data and adoption of uniform standards . 
indeed , there is much work to be done to facilitate data sharing between different organizations ; nevertheless , our application serves to mitigate these gaps in data sharing by linking multiple knowledge bases together through one convenient solution . in its current state , smart cancer navigator is focused on data aggregation , and it presents data in a simple tabular format . 
more sophisticated methods of data visualization are necessary , in particular to highlight the co - occurrence of mutations , which is increasingly clinically relevant.58 we have previously shown that standard visual tools such as the pie chart do not scale when dealing with large numbers of genes and variants.59 to address this challenge , the biovis community recently convened a challenge workshop and will soon be formulating recommendations.60 although smart cancer navigator allows users to select a relevant patient condition and then select a gene - variant doublet , true clinical decision support in oncology requires a triplet of disease - gene - variant information and , in many cases , further metadata such as prior treatments received . 
current knowledge base api architecture does not meet this need , because most provide only end points for the querying of genes and variants or provide end points for other relevant clinical information that may not presently return data . 
the clinical trials example shown in the data supplement illustrates this challenge , because only one of the identified trials is directly relevant to the patient with relapsed hgsoc . it must also be acknowledged that apps such as smart cancer navigator may be seen as a security risk by some organizations . 
although smart on fhir apps have a security structure that meets or exceeds the requirements set forth by the health insurance portability and accountability act , 9 genetic information is nonetheless highly sensitive . 
first , we designed the app to be simple to use , but we are mindful of the fact that clinicians are under intense time pressures that are not expected to abate anytime soon . 
second , we assume that the app ecosystem will evolve rapidly11 , 12 , 62 ; endorsement by large ehr vendors through activities such as epics app orchard63 and cerners participation in the hl7 argonaut project is encouraging . 
however , for the app ecosystem to reach its full potential , apps must be able to write to ehrs or to intermediary systems that subsequently communicate with ehrs , such as genomics archive computer system.10 , 39 , 64 finally , we assume that the regulatory environment for apps , which are considered software as a medical device by the us food and drug administration under certain circumstances , 65 , 66 will remain on the track that has recently been indicated ; expensive regulatory burdens would likely stifle the environment of open - source apps because costs could not be recouped . in light of recent discussions regarding data sharing , 67 we believe that our framework acts as a model for facilitating a symbiotic relationship between clinicians , clinical researchers , and bioinformatics data scientists . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . clinical application , it also holds value as a tool for clinical researchers and knowledge base curators . 
although concerns of policy , privacy , and practicality must still be worked out to ensure that data sharing is efficient , sustainable , and secure , smart cancer navigator illustrates the benefits that are conferred when parties work together to share resources and knowledge . finally , smart cancer navigator seeks to support oncologists in the interpretation of ever increasing volume of patient molecular data.68 - 70 by linking directly to patient ehr systems , genomic laboratories , and multiple authoritative knowledge bases , smart cancer navigator can serve as an educational resource for clinical oncologists , trainees , patients , and researchers . 
 furthermore , smart cancer navigators use of apis , which are mandated by the 21st century cures act , 71 provides for a truly modular application that can be easily integrated into many environments . 
warner stock and other ownership interests : hemonc.org ishaan prasad no relationship to disclose makiah bennett no relationship to disclose monica arniella no relationship to disclose alicia beeghly - fadiel no relationship to disclose kenneth d . 
mandl , and gil alterovitz , boston childrens hospital / harvard medical school , boston ; gil alterovitz , massachusetts institute of technology , cambridge , ma ; and monica arniella , duke university , durham , nc supported in part by grants no . 
hughes ks , ambinder ep , hess gp , et al : identifying health information technology needs of oncologists to facilitate the adoption of genomic medicine : recommendations from the 2016 american society of clinical oncology omics and precision oncology workshop . 
forbes sa , tang g , bindal n , et al : cosmic ( the catalogue of somatic mutations in cancer ) : a resource to investigate acquired mutations in human cancer . 
liu x , wu c , li c , et al : dbnsfp v3.0 : a one - stop database of functional predictions and annotations for human nonsynonymous and splice - site snvs . 
national center for biotechnology information : dbsnp : database of single nucleotide polymorphisms ( snps ) and multiple small - scale variations that include insertions / deletions , microsatellites , and non - polymorphic variants . 
ihnen m , zu eulenburg c , kolarova t , et al : therapeutic potential of the poly ( adp - ribose ) polymerase inhibitor rucaparib for the treatment of sporadic human ovarian cancer . 
mcneish ia , oza am , coleman rl , et al : results of ariel2 : a phase 2 trial to prospectively identify ovarian cancer patients likely to respond to rucaparib using tumor genetic analysis . 
swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
hyman dm , laetsch tw , kummar s , et al : the efficacy of larotrectinib ( loxo - 101 ) , a selective tropomyosin receptor kinase ( trk ) inhibitor , in adult and pediatric trk fusion cancers . 
huang dw , sherman bt , tan q , et al : the david gene functional classification tool : a novel biological module - centric algorithm to functionally analyze large gene lists . 
alexandrov lb , nik - zainal s , wedge dc , et al : signatures of mutational processes in human nat rev genet 14 : 703 - 718 , 2013 cancer . 
ghazani aa , oliver nm , st pierre jp , et al : assigning clinical meaning to somatic and germline whole - exome sequencing data in a prospective cancer precision medicine study . 
blakely cm , watkins tbk , wu w , et al : evolution and clinical impact of co - occurring genetic alterations in advanced - stage egfr - mutant lung cancers . 
 potentially curative targeted therapy for undifferentiated high - grade sarcoma developing after malignant transformation of a braf v600emutated ameloblastic fibroma nikolay zhukov , md , phd1 , 2 ; yulia mareeva , md1 ; dmitry konovalov , md1 ; alexander druy , md , phd1 ; nikolay grachev , md , phd1 ; and dmitry litvinov , md1 introduction ameloblastic broma ( af ) is a rare benign odontogenic tumor , consisting of odontogenic epithelium and mesenchymal tissues . 
most afs can be cured by radical enucleation or wide excision , but some tumors are initially unresectable or become unresectable after relapse because of locally invasive behavior or malignant transformation to ameloblastic brosarcoma ( afs ) .1 , 2 currently , there are no established curative or even effective palliative treatments for unresectable af or its malignant derivates . 
within 2 years of diagnosis , the patient had undergone three consecutive attempts at tumor resection with r2 to r1 margins in local low - volume hospitals , but the disease continued to recur locally . after the third recurrence , the patient was admitted to a high - volume federal oncology center , where a biopsy revealed the malignant transformation of af into afs . the tumor retained its biphasic morphology , but the cytologically benign epithelial component was covered by highly mitotic atypical mesenchymal tissue ( figs 1c and 1d )  . 
because the tumor was borderline resectable , the patient had experienced multiple recurrences , and malignant transformation of the tumor had been conrmed ; aggressive soft tissue sarcoma oriented chemotherapy aimed at tumor shrinkage was administered . 
disease remained stable after second - line therapy with dactinomycin , carboplatin , and etoposide and third - line therapy with irinotecan and cisplatwhy secondand third - line treatments were administered is unclear , but in our opinion , this conrmed tumor chemotherapy resistance at this point . after chemotherapy , the patient underwent a fourth attempt at potentially curative surgery , and r0 resection was achieved . 
after pathologic examination , the tumor still had biphasic afs morphologic features with benign epithelial and malignant mesenchymal components , both with weak signs of chemotherapy - induced pathologic response . nine months later , the tumor recurred , with massive extension into the pterygopalatine and infratemporal fossae , and the patient was admitted to our hospital for the next attempt at radical curative - intent surgery . pathologic examination of the resected tumor , which had retained its afs morphology , revealed r1 margins , which we treated with adjuvant radiotherapy , with target volume dosing of 59.4 gy to the tumor bed . because of the high chance of recurrence and chemotherapy resistance of the tumor , we performed an extensive search for potential drug targets for noncytotoxic therapy and found a braf v600e mutation in the tumor , indicating the tumor could respond to anti - braf / mek therapy . 
primary tumor , an ameloblastic broma ; magnication at ( a ) 40 and ( b ) 200 , showing a benign biphasic tumor with odontogenic epithelial glands and hypocellular mesenchymal component . third relapsed tumor morphology , diagnosed as ameloblastic brosarcoma , magnication at ( c ) 40 and ( d ) 200 , showing benign odontogenic epithelial glands surrounded by atypical mesenchymal tumor with numerous mitotic gures . 
 case report eleven months later , in january 2018 , the tumor recurred again and was unresectable because of its size and the involvement of major vessels and the base of the skull ( fig 2a )  . 
the benign epithelial component we previously observed was lost , and the tumor had aggressive small round cell morphology and was nearly 80% ki - 67 positive . there was no expression of epithelial or hematolymphoid markers and no precise immunophenotype , with cells negative for ck ae1 / ae3 , p63 , gfap , calponin , cd99 , cd45 , desmin , s100 , ck19 , ck18 , ema , and cd34 ini1 , p16 , and fli1 after and positive for vimentin , immunohistochemical staining . 
 ( a ) magnetic resonance imaging ( mri ) of the large soft tissue lesion in the right mandibular , with spreading into the pterygopalatine fossa and parapharyngeal space ; soft tissues of both the buccal region and neck , with involvement of major vessels and base of the skull , shown at right . 
radical curative - intent surgery was performed in september 2018 ( fig 2d ) , and pathologic examination revealed r0 margins and complete pathomorphologic and even complete molecular tumor regression , with no signs of vital tumor and no braf v600e in highly sensitive allele - specic polymerase chain reaction of resected tissue . 
 case report adjuvant vemurafenib and cobimetinib therapy was discontinued in august 2019 , and at the time of this report , the patient , now 16 years of age , has survived for 18 months since the start of targeted therapy and has remained disease free for 11 months since his last surgery . discussion the braf v600e mutation is a valid drug target in some tumors , including melanoma , nonsmall - cell lung cancer , and anaplastic thyroid cancer ; anti - braf / mek therapy has led to high objective response rates and prolonged survival in these tumors.11 - 13 however , this effect is not universal , and in other tumors ( eg , colorectal cancer ) , braf v600e does not predict high efcacy of targeted therapy.14 accordingly , the presence of a braf mutation in af does not automatically imply the high efcacy of anti - braf / mek therapy in these tumors , especially those with complete morphologic transformation , as occurred in our patient . here we demonstrate an intrinsic characteristic of a braf mutation in af and its malignant derivates ; the mutation was retained during all stages of disease transformation . 
there are some reports about the effectiveness of anti - braf or anti - braf / mek therapy for ab and malignant ab , but there are no data on its use for braf - positive af.7 , 8 this report conrms a class effect of braf v600e in odontogenic tumors , irrespective of denitive tumor type . the pattern of transformation is also of interest , because braf mutation in odontogenic tumors is usually associated with the epithelial component , which was completely lost during transformation in our patient.3 the mechanism underlying the transformation from the initially benign tumor to the aggressive sarcoma is unclear , and such a transformation has not been previously observed in this tumor type , to our knowledge ; therefore , we plan to perform wholegenome next - generation sequencing to evaluate genetic markers associated with this transformation . to our knowledge , at the time of submission , this is the rst published report of the stepwise transformation of af to a high - grade usrcs , with complete disappearance of the epithelial component . 
furthermore , again to our knowledge , this is the rst report of a complete pathologic and molecular response of braf v600emutated highgrade usrcs to anti - braf / mek therapy , with the possibility of long - term disease control or even a cure after subsequent radical surgery with adjuvant anti - braf / mek therapy . 
one limitation of this case report is that a longer follow - up period after the cessation of anti - braf / mek therapy is required to distinguish between cure and prolonged control of persistent subclinical residual tumor . in conclusion , the braf v600e mutation is one of the primary oncogenic drivers in odontogenic tumors , even during malignant transformation . 
anti - braf / mek therapy is highly effective in malignant odontogenic tumors with braf v600e and , in combination with subsequent surgery , can be curative in patients with otherwise incurable disease . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . nikolay zhukov honoraria : novartis , teva , biocad , nutricia , takeda , astrazeneca , pzer , bristol - myers squibb , servier consulting or advisory role : pzer research funding : takeda travel , accommodations , expenses : biocad no other potential conicts of interest were reported . acknowledgment we conrm that written informed consent was obtained from the patients legal guardian . 
j oral pathol med 47 : 315 - 325 , 2018 kobayashi k , murakami r , fujii t , et al : malignant transformation of ameloblastic broma to ameloblastic brosarcoma : case report and review of the literature . j craniomaxillofac surg 33 : 352 - 355 , 2005 brown na , rolland d , mchugh jb , et al : activating fgfr2 - ras - braf mutations in ameloblastoma . 
clin cancer res 20 : 5517 - 5526 , 2014 kurppa kj , cat on j , morgan pr , et al : high frequency of braf v600e mutations in ameloblastoma . 
j pathol 232 : 492 - 498 , 2014 sweeney rt , mcclary ac , myers br , et al : identication of recurrent smo and braf mutations in ameloblastomas . 
nat genet 46 : 722 - 725 , 2014 [ erratum : nat genet 47 : 97 , 2015 ] brunner p , bihl m , jundt g , et al : braf p.v600e mutations are not unique to ameloblastoma and are shared by other odontogenic tumors with ameloblastic morphology . 
oral oncol 51 : e77 - e78 , 2015 tan s , pollack jr , kaplan mj , et al : braf inhibitor treatment of primary braf - mutant ameloblastoma with pathologic assessment of response . 
oral surg oral med oral pathol oral radiol 122 : e5 - e7 , 2016 kaye fj , ivey am , drane we , et al : clinical and radiographic response with combined braf - targeted therapy in stage 4 ameloblastoma . 
j natl cancer inst 107 : 378 , 2014 diniz mg , gomes cc , de sousa sf , et al : oncogenic signalling pathways in benign odontogenic cysts and tumours . 
planchard d , smit ef , groen hjm , et al : dabrafenib plus trametinib in patients with previously untreated brafv600e - mutant metastatic non - small - cell lung cancer : an open - label , phase 2 trial . 
de moor author affiliations and support information ( if applicable ) appear at the end of this article . creative commons attribution non commercial no derivatives 4.0 license corresponding author : andrew n . 
freedman , phd , epidemiology and genomics research program , division of ( continued ) purpose there are no nationally representative data on oncologists use of next - generation sequencing ( ngs ) testing in practice . 
the purpose of this study was to investigate how oncologists in the united states use ngs tests to evaluate patients with cancer and to inform treatment recommendations . methods the study used data from the national survey of precision medicine in cancer treatment , which was mailed to a nationally representative sample of oncologists in 2017 ( n = 1 , 281 ; cooperation rate = 38% )  . 
of these oncologists , 34.0% used them often to guide treatment decisions for patients with advanced refractory disease , 29.1% to determine eligibility for clinical trials , and 17.5% to decide on off - label use of food and drug administrationapproved drugs . 
oncologists younger than 50 years of age , holding a faculty appointment , having genomics training , seeing more than 50 unique patients per month , and having access to a molecular tumor board were more likely to use ngs tests . conclusion in 2017 , most oncologists in the united states were using ngs tests to guide treatment decisions for their patients . 
multimarker panels also include dna and rna analysis through next - generation sequencing ( ngs ) technologies , including custom panels that profile multiple actionable driver genes and tumor characteristics that may guide the selection of targeted therapies . despite advances in precision oncology , comparatively little empirical research is available that assesses the expectations and experiences of oncologists in the united states who may use these genomic tools to care for patients with cancer . 
to date , most studies have evaluated physicians experiences with specific genomic tests for individual gene mutations.3 - 5 studies that have attempted to assess providers experiences with custom ngs tumor panels6 - 9 have focused on intended rather than actual use . 
furthermore , previous studies of physicians use of ngs tests were on the basis of small samples and were conducted in tertiary referral centers ; as a result , their findings may not represent how oncology is practiced in the united states . 
currently , there are no nationally representative data describing oncologists awareness , knowledge , and use of ngs testing to inform patient care , especially in community practice settings . with the increasing application of precision oncology , understanding how oncologists use genomic tests in their practice and the factors that affect their use is essential to ensure that patients who can benefit receive appropriate testing and follow - up . 
the purpose of this study was to investigate how oncologists in the united states use ngs tests to evaluate patients with cancer and inform treatment recommendations . methods data source this study used data from the national survey of precision medicine in cancer treatment , a nationally representative survey of hematologists , hematologists / oncologists , and oncologists sponsored by the national cancer institute ( nci ) , the national human genomic research institute , and the american cancer society . 
 of the remaining 3 , 378 oncologists with verified contact information , 1 , 281 completed the survey ( cooperation rate = 38%10 )  . a survey packet containing a personalized invitation letter from the nci , an endorsement letter from asco , a pen with the studys name printed on it , a self - administered paper questionnaire , and a business reply envelope was mailed to eligible oncologists . 
participants received a $50 honorarium for completing the survey . the questionnaire ( data supplement ) took 20 minutes to complete on average , and data were collected from february through may of 2017 . 
information was ascertained about oncologists demographics , training , academic affiliation , specialty , and patient volume , and their practice characteristics , including practice setting , structure , and resources to support genomic testing . 
 information about oncologists use of commercially available multimarker tumor panels and noncommercial tumor panels performed at academic medical centers was collected , as were data on the clinical scenarios in which ngs test were used . 
because of differences in how survey questions were worded ( eg , some explicitly stated the exclusion of oncotype dx ) , sensitivity analyses were conducted to examine whether oncologists who reported using other gene expression tests , including the breast cancer index , mammaprint , prosigna , and / or myplan lung cancer , differed in their use of ngs tests from those who did not use these gene expression tests . 
the survey weights were applied in all analyses of the data and provide statistical adjustment so that respondents are representative of the population of practicing oncologists in the united states . each oncologist was classified as a user of ngs tests if he or she reported using at least one specific multimarker panel with ngs technologies profiling multiple actionable driver genes for guiding treatment decisions in the past 12 months . 
multivariable models were estimated to examine the independent association between each physician demographic and practice characteristic and the likelihood of ngs testing , when adjusting for the other characteristics in the model . 
all analyses were conducted using sudaan release 11.0.1 ( rti international , research triangle park , nc )  . results characteristics of survey respondents characteristics of the 1 , 281 oncologists who completed the survey are listed in table 1 . 
 more than one half of respondents ( 56.9% ) spent at least 90% of their time in patient care and almost one half ( 49.3% ) spent less than 10% of their time teaching ( data not shown )  . prevalence of ngs test use and predictors of test use to guide treatment decisions overall , 75.6% of oncologists reported using ngs tests in the past 12 months to guide treatment decisions ; use differed according to the physicians demographic and practice characteristics ( table 2 )  . 
oncologists who treated only patients with hematologic malignancies were less likely to use ngs tests than were oncologists who treated both solid and hematologic malignancies and those who only treated patients with solid tumors . 
oncologists who held a faculty appointment , had some training in genomics , or were part of a practice that had a molecular tumor board were more likely to use ngs tests than were those who did not have these characteristics . 
in addition , those who treated fewer than 50 patients with cancer per month were more likely to use ngs tests than were oncologists who treated more than 50 patients with cancer per month . 
the likelihood of ngs use was not statistically significantly associated with other provider and practice characteristics assessed . in this survey , oncologists in the united states were asked how often they used 11 commercially available ngs tests or a noncommercial ngs test during the past 12 months ( fig 1 )  . 
moreover , among oncologists who ordered any of these ngs tests ( n = 959 ) , 28.2% ordered one test , 31.7% ordered two tests , 23.3% ordered three tests , and 16.7% ordered four or more tests in the past 12 months . ngs test use practice patterns oncologists use of ngs test results varied by clinical presentation ( fig 2 )  . 
characteristics of oncologist respondents in the united states to the 2017 national survey of precision medicine in cancer treatment ( continued ) total ( n = 1 , 281 ) unweighted percentage ( % ) * weighted percentage ( % ) * characteristic no . 
we found that oncologists who used the breast cancer index , mammaprint , prosigna , and / or myplan lung cancer did not differ in the way they answered these questions in nine out of 10 comparisons . 
when analyses were restricted to oncologists who treated a high volume of patients with breast cancer , there were no differences in test use between oncologists who used the breast cancer index , mammaprint , and prosigna and those who did not ( data not shown )  . respondents were explicitly directed to exclude oncotype dx when answering questions about ngs testing for different clinical purposes . 
use of ngs testing during the past 12 months among oncologists in the united states , by physician demographics and practice characteristics ( continued ) total ( n = 1 , 281 ) ngs user ( unadjusted % ) ngs user ( adjusted % ) 95% ci adjusted or ( 95% ci ) characteristic sees patients at academic center or medical school no . 
twenty - five percent ( n = 319 ) indicated that they referred their patients to another location or provider for ngs testing ; among these oncologists , 84.4% ( n = 257 ) referred to an academic medical center , 82.1% ( n = 240 ) to a clinical trial , and 18.2% ( n = 51 ) to an oncologist outside of their practice ( data not shown )  . discussion in this nationally representative study of 1 , 281 oncologists , we examined how ngs tests are currently used in clinical practice and whether use is influenced by physician and practice characteristics . 
 initial diagnosis rare cancers advanced refractory disease unknown origin never rarely sometimes often reported using ngs test results to guide patient care , and the use of ngs tests differed by oncologists demographic and practice characteristics . 
 for example , younger oncologists were more likely to be ngs test users , suggesting that they may have had more recent training in genomic testing or that they are more receptive to the incorporation of ngs testing in their practice . 
 having a faculty appointment and access to a molecular tumor board were also associated with ngs test use , perhaps reflecting greater access to in - house ngs tests in academic settings or more involvement in clinical research . 
 not surprisingly , oncologists who treated only patients with hematologic malignancies were less likely to use ngs tests than were oncologists who treated both solid and hematologic malignancies or those who treated only patients with solid tumors , suggesting that ngs tests may be ordered reflexively by pathologists rather than by oncologists . 
use of next generation sequencing tests by clinical purpose during the past 12 months among oncologists in the united states . molecular testing for patients with hematologic malignancies . oncologists reported using ngs most often for patients with advanced refractory disease , but also used these tests often for patients diagnosed with a rare cancer or cancers of unknown origin and / or for patients with an initial diagnosis of cancer . 
these results may reflect oncologists use of ngs testing to inform treatment strategies when established therapies have failed or when there is uncertainty about the usefulness of existing treatment guidelines for less common clinical situations . 
with the recent approval of multiple therapeutic agents targeting specific driver mutations , oncologists may send a patients tumors for sequencing with the hope of identifying treatments that are potentially efficacious for their patients particular molecular tumor subtype . 
in our survey , more than one third of oncologists reported using ngs test results often to guide the use of an fda approved therapy . advances in precision oncology pose challenges for oncologists . 
at the time of the survey in may 2017 , nonsmall - cell lung cancer was the only cancer type for which there was a consensus recommendation for ngs use.13 furthermore , no professional group other than the national comprehensive cancer network has yet issued evidence - based guidelines recommending the use of ngs testing . 
 however , as the number of single - gene or focused - indication ( eg , microsatellite instability ) tests that are components of the standardized evaluation of common malignancies has grown , oncologists are increasingly faced with decisions about whether to order several targeted tests or a single ngs panel . there has also been an effort by pharmaceutical companies to develop tissue - agnostic cancer drugs that target a specific genetic marker independent of tumor type.14 given the prospect of tissue - agnostic labeling , the use of ngs will only grow , and in the absence of clinical guidelines informing ngs use , it is likely that many providers will experience uncertainty about how to clearly integrate this information into clinical care decisions . 
 treatment recommendations , suggesting that oncologists are already confronted with a large volume of genomic information that they must interpret . that ngs test results are frequently ambiguous presents another challenge for oncologists . 
in addition , 25% indicated that they referred patients to other providers for ngs testing , possibly suggesting a lack of expertise or resources for ordering and interpreting ngs tests . 
several academic and commercial groups have responded to this need , developing online decision support resources for genomic medicine , such as personalized cancer therapy , my cancer genome , and oncokb , with the goal of providing an accessible platform to oncologists.15 however , more work is needed to ensure that patients who can benefit from these new technologies receive appropriate testing and follow - up . a third challenge for oncologists is the lack of clinical usefulness data for many of the available ngs tests . 
two major , ongoing clinical trials , ( national cancer institute nci - match molecular analysis for therapy choice ) , 16 and pediatric match ( national cancer institute childrens oncology group pediatric match trial ) , will generate evidence to establish clinical usefulness to fill some of these gaps . 
furthermore , ascos targeted agent and profiling utilization registry ( tapur ) study17 and cureone ( formerly called med - c ) are building registries of ngs testing data to enhance informed treatment recommendations difficulty in interpreting results never / rarely sometimes often understanding of how testing is being used and its impact on patient outcomes.18 insurers are also recognizing the cost efficiencies of ngs panel testing as opposed to multiple single - gene testing for specific cancer - site indications but are grappling with a lack of clinical usefulness data for testing cancers broadly . 
 effective march 16 , 2018 , the centers for medicare & medicaid services has allowed non fda - approved assays run in clinical laboratory improvement amendmentscertified laboratories to be reimbursed , dependent on decisions made by local medicare administrative contractors.19 it is unclear whether and under what circumstances commercial private payers will decide to reimburse for ngs testing broadly for cancer . 
our findings offer an important baseline that could be used to evaluate the impact of these coverage decisions on ngs test use because our data were collected before implementation of these key changes in coverage . our study has several limitations . 
first , the cooperation rate was lower than that of previous surveys of physicians on the topic of genomic and genetic test use , 7 , 20 and responders may have differed from nonresponders in terms of their genomic testing practices and other characteristics such as academic affiliation . 
however , respondents are representative of the population of practicing oncologists in the united states in terms of age , sex , and geographic location on the basis of statistical adjustment for nonresponse using data available on the survey 's sample frame . another limitation is that we were unable to assess how oncologists use the tests in specific clinical situations and for which cancer types they are ordering these tests . 
however , these limitations are offset by several strengths of the study , including the nationally representative sample of practicing oncologists in a wide variety of practice settings and the large sample size , which allowed us to analyze multiple factors associated with ngs use and to examine a variety of practice patterns . the majority of oncologists in the united states use ngs tests to guide patient care , even in the absence of evidence - based practice guidelines . 
schilsky research funding : astrazeneca ( inst ) , bayer ag ( inst ) , bristol - myers squibb ( inst ) , genentech / roche ( inst ) , eli lilly ( inst ) , merck ( inst ) , pfizer ( inst ) deborah schrag consulting or advisory role : journal of the american medical association research funding : pfizer other relationship : journal of the american medical association naoko i . 
simonds stock and other ownership interests : cvs health ( i ) , walgreens boots alliance , anthem foundation , anthem foundation ( i ) , celgene ( i ) , cvs health , johnson & johnson , johnson & johnson ( i ) , walgreens boots alliance ( i ) , labcorp , labcorp ( i ) , valeant pharmaceuticals international , valeant pharmaceuticals international ( i ) , endo pharmaceuticals ( i ) , aetna ( i ) helmneh m . 
miller fa , hayeems rz , bytautas jp , et al : testing personalized medicine : patient and physician expectations of next - generation genomic sequencing in late - stage cancer care . 
gray sw , park er , najita j , et al : oncologists and cancer patients views on whole - exome sequencing and incidental findings : results from the canseq study . 
johnson lm , valdez jm , quinn ea , et al : integrating next - generation sequencing into pediatric oncology practice : an assessment of physician confidence and understanding of clinical genomics . 
 appendix physician characteristics included age , sex , race or ethnicity , faculty appointment , training in genomic testing ( eg , instruction throughout residency and / or fellowship , professional lectures or seminars , symposiums , conferences , or continuing medical education )  . 
practice characteristics , such as region , urbanicity , type of practice ( solo , single specialty group , multispecialty group , other ) , academic affiliation , and number of unique patients with cancer seen per month were also collected . 
in addition , questions were asked about whether the oncologists practice setting has a genomic or molecular tumor board , policies for genomic testing use , and / or an electronic medical record that provides alerts when a genomic test is recommended . 
an oncologists specialty was defined using information on type ( s ) of patients with cancer seen : only patients with solid tumors , only those with hematologic malignancies , or both . 
age and demographic characteristics other than race or ethnicity were obtained from the american medical association masterfile . an oncologists was classified as a user of next - generation sequencing ( ngs ) tests if he or she reported use , in the past 12 months , of specific multimarker panels with ngs technologies profiling multiple actionable driver genes used to guide treatment decisions . 
the following tests were included in this definition : cancerselect or cancer complete , caris molecular intelligence or target now , cgi complete , foundationone , foundationoneheme , foundationact , gps cancer , guardant360 , omniseq comprehensive , onkosight tumor panels , solid tumor mutation panel ( arup laboratories ) , and noncommercial tumor panels . 
multimarker tests that use gene - expression profiling ( eg , oncotype dx for breast cancer ) , primarily used to predict prognosis and / or the likelihood of recurrence , were not classified as ngs tests . 
tan author affiliations and support information ( if applicable ) appear at the end of this article . creative commons attribution non commercial no derivatives 4.0 license corresponding author : david s.p. 
indeed , hrd - related genetic aberrations may confer a similar clinical phenotype to gboc , 7 , 8 including improved responses and longer progression - free survival ( pfs ) in patients receiving maintenance parpi treatment.9 - 11 few effective treatment options exist for patients with recurrent hgsoc ( rhgsoc ) , especially in the context of malignant bowel obstruction , where the use of bevacizumab is associated with a higher risk of bowel perforation.12 we present the case of a patient with a germline pathogenic rad51c mutation who received salvage treatment for terminal malignant bowel obstruction with sixth - line carboplatin and pegylated liposomal doxorubicin ( pld ) , followed by maintenance olaparib . a 57 - year - old chinese woman was referred to our institution after hgsoc relapse . 
she first received the hgsoc diagnosis at 53 years of age , with no family history of breast or ovarian cancer , and had received initial treatment elsewhere ( table 1 )  . 
at first relapse of hgsoc ( after a 28 - month pfi ) , she received carboplatin plus pld every 4 weeks for six cycles , followed by secondary debulking . 
she received four cycles of carboplatin every 3 weeks with dose - dense paclitaxel once per week , followed by two cycles of cisplatin substituted for carboplatin , owing to grade 2 carboplatin hypersensitivity . 
complete response was achieved with a nadir ca - 125 concentration of 90 u / ml . three months later , however , the patients ca - 125 level rose to 571 u / ml , at which point she transferred care to our institution . 
clinical presentation and treatment summary treatment received at another institution presentation and treatment date may 2010 july 2010 july - nov 2010 mar 2013 apr - june 2013 july 2013 sept - nov 2013 june 2014 july - nov 2014 jan 2015 feb 2015 apr - june 2015 june 2015 june - nov 2015 feb - june 2016 june 2016 july - oct 2016 nov 2014 ca - 125 level nadir of 90 u / ml . complete response determined from repeated ct scan . transfer of care to our institution genetic testing performed using invitae panel . hgsoc diagnosed at another institution . 
 switched to cisplatin ( 75 mg / m2 ) and paclitaxel ( 175 mg / m2 ) every 3 weeks for two more cycles in view of grade 2 hypersensitivity to carboplatin . biochemical recurrence ; ttp : 3 months ; ca - 125 level : 571 u / ml . 
no measurable disease seen on ct scan . genetic testing showed pathogenic grad51c mutation . symptomatic hgsoc relapse ; pfi now 5 months ; ca - 125 level : 3 , 145 u / ml . 
intestinal obstruction resolved with conservative management . switched to fourth - line carboplatin ( auc 5 ) with desensitization protocol and gemcitabine ( 1 , 000 mg / m2 , days 1 and 8 ) every 3 weeks . carboplatin ( auc 5 ) treatment continued with desensitization protocol and gemcitabine ( 1 , 000 mg / m2 , days 1 and 8 ) every 3 weeks ; total of eight cycles with partial response . 
ct scan shows residual 3.6 - cm pelvic implant . ct scans showed new ascites and increasing size of peritoneal implant . patient enrolled in phase i study of low - dose whole abdominal radiation therapy ( 60 cgy fractions , 2 d / wk , twice on each of those days ) , in combination with weekly paclitaxel ( 80 mg / m2 )  . 
pfs while receiving olaparib treatment : 7 months . underwent surgical exploration and defunctioning jejunostomy . july 2017 multiple levels of malignant small - bowel obstruction developed . patient referred to hospice care . 
she joined a phase i trial of kpt330 ( clinicaltrials.gov identifier : nct02078349 ) , but her disease progressed after 2 months , and she had an intestinal obstruction from worsening peritoneal metastases . 
after eight cycles , sustained partial response ( pr ) was achieved according to gynecologic cancer intergroup criteria.14 the ca - 125 concentration improved from 7 , 550 u / ml to 368 u / ml . 
the patient was given a chemotherapy holiday . unfortunately , after another short ttp of 2 months , there was symptomatic disease progression , and the patient was enrolled in a phase ii clinical trial of low - dose , whole - abdomen radiation combined with weekly paclitaxel treatment . 
this approach was based on the knowledge that genomic hrd could enable prediction of response to dna - damaging agents such as carboplatin and topoisomerase iia inhibitors.15 the patient chose carboplatin plus pld every 4 weeks , and her disease responded according to gynecologic cancer intergroup criteria . 
the patient was experiencing grade 2 fatigue and , in view of emerging data at that time on the efficacy of parpis in tumors with hrd , 16 a complete break from treatment , versus off - label maintenance therapy with olaparib capsules , was discussed . 
 she chose the latter option . because of financial constraints , the patient was prescribed olaparib capsules 200 mg twice daily ( rather than the 400 - mg twice - daily standard dosing ) , but her disease continued to respond , with a reduction in ca - 125 level from 1 , 405 u / ml to a nadir of 44.7 u / ml ( fig 2 )  . 
after 6 months of olaparib treatment , scans confirmed pr by response evaluation criteria in solid tumors ( recist ) , version 1.1 , with improved ascites and resolution of a perihepatic peritoneal deposit ( fig 1 )  . unfortunately , the patients disease relapsed a month later with small - bowel obstruction and required parenteral nutrition . 
 ( a and b ) june 2016 ( preplatinum rechallenge ) : left lung lesion ( curved arrow ) , ascites , and peritoneal thickening ( double arrow )  . 
 ( e and f ) april 2017 resolution of ascites and of the deposit at the hepatic hilum after 6 months of olaparib treatment ( originally seen in ( d ) , arrow )  . many to be a preterminal event.17 retrospective data show limited responses from chemotherapy.17 yet , this patient responded remarkably to sixthline carboplatin plus pld and , per the recent us food and drug administration approval for maintenance parpis , derived significant benefit from olaparib , even at an off - label dose of 200 mg twice daily . 
notably , biologically effective inhibition of parp by olaparib capsules has been demonstrated even at 100 mg twice daily.18 to our knowledge , this report is the first to describe the clinical effect of this specific grad51c variant on treatment selection and patient outcome in rhgsoc . the cancer genome atlas project showed approximately 30% of hgsoc cases harbor nonbrca1 / 2 mutation - related hrd . 
 more recently , olaparib , 7 - 9 niraparib , 10 and rucaparib16 were approved for use in patients who have received at least two prior treatments of platinum - based chemotherapy and are platinum responsive . 
of note , all patients eligible for the landmark phase iii trials , 9 - 11 leading to approval of parpi for this indication , had experienced a ttp after penultimate platinum exposure of 6 months , which was not the case in our patient . efficacy data on resensitization to platinum based chemotherapy and thresholds constituting an effective pfi are ambiguous . 
dramatic reduction of ca - 125 level from 17 , 256 u / ml to 867.7 u / ml was noted after the patient commenced receiving carboplatin plus pld treatment . 
analyses revealed a frameshift - truncating mutation of tp53bp1 , which is associated with olaparib resistance.33 reversion mutations in rad51c have also been described in acquired rucaparib resistance.34 unfortunately , at the time of disease progression , surgeons were unable to obtain an adequate tumor sample for analysis of resistance mechanisms in the patient in the current report . in conclusion , patients with ovarian cancer with grad51c mutations may benefit from similar treatment paradigms as brca1 / 2 - mut disease . 
 darwin tay no relationship to disclose valerie heong consulting or advisory role : roche ; astrazeneca patents , royalties , other intellectual property : royalties received for drug venetoclax travel , accommodations , expenses : astrazeneca soo chin lee honoraria : roche ; astrazeneca ; pfizer ; novartis consulting or advisory role : pfizer ; novartis ; astrazeneca ; roche ; eisai research funding : eisai ; taiho pharmaceutical ; bayer ; aslan pharmaceuticals expert testimony : novartis travel , accommodations , expenses : roche ; novartis ; pfizer ; act genomics yee liang thian no relationship to disclose pei yi ong no relationship to disclose samuel g.w. 
jeyasekharan honoraria : msd oncology yi wan lim no relationship to disclose siew eng lim no relationship to disclose research funding : astrazeneca ; perkin elmer travel , accommodations , expenses : perkin elmer joseph ng no relationship to disclose jeffrey j.h. 
tan honoraria : astrazeneca ; roche consulting or advisory role : astrazeneca ; roche research funding : astrazeneca ( inst ) ; karyopharm therapeutics ( inst ) ; bayer ( inst ) travel , accommodations , expenses : merck sharp & dohme ; merck serono ; astrazeneca ; bayer ; roche affiliations natalie y.l. 
alsop k , fereday s , meldrum c , et al : brca mutation frequency and patterns of treatment response in brca mutation - positive women with ovarian cancer : a report from the australian ovarian cancer study group . 
song h , dicks e , ramus sj , et al : contribution of germline mutations in the rad51b , rad51c , and rad51d genes to ovarian cancer in the population . 
swisher em , harrell mi , lin k , et al : brca1 and rad51c promoter hypermethylation confer sensitivity to the parp inhibitor rucaparib in patients with relapsed , platinum - sensitive ovarian carcinoma in ariel2 part 1 . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
pujade - lauraine e , ledermann ja , selle f , et al : olaparib tablets as maintenance therapy in patients with platinum - sensitive , relapsed ovarian cancer and a brca1 / 2 mutation ( solo2 / engot - ov21 ) : a double - blind , randomised , placebo - controlled , phase 3 trial . 
coleman rl , oza am , lorusso d , et al : rucaparib maintenance treatment for recurrent ovarian carcinoma after response to platinum therapy ( ariel3 ) : a randomised , double - blind , placebocontrolled , phase 3 trial . 
rustin gj , vergote i , eisenhauer e , et al : definitions for response and progression in ovarian cancer clinical trials incorporating recist 1.1 and ca 125 agreed by the gynecological cancer intergroup ( gcig )  . 
tan ds , kaye sb : chemotherapy for patients with brca1 and brca2 - mutated ovarian cancer : same or different ? am soc clin oncol educ book 35 : 114 - 121 , 2015 16 . 
swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
kaye sb , lubinski j , matulonis u , et al : phase ii , open - label , randomized , multicenter study comparing the efficacy and safety of olaparib , a poly ( adp - ribose ) polymerase inhibitor , and pegylated liposomal doxorubicin in patients with brca1 or brca2 mutations and recurrent ovarian cancer . 
gayarre j , martn - gimeno p , osorio a , et al : characterisation of the novel deleterious rad51c p.arg312trp variant and prioritisation criteria for functional analysis of rad51c missense changes . 
hatanaka a , yamazoe m , sale je , et al : similar effects of brca2 truncation and rad51 paralog deficiency on immunoglobulin v gene diversification in dt40 cells support an early role for rad51 paralogs in homologous recombination . 
yokoyama h , sarai n , kagawa w , et al : preferential binding to branched dna strands and strand - annealing activity of the human rad51b , rad51c , rad51d and xrcc2 protein complex . 
somyajit k , mishra a , jameei a , et al : enhanced non - homologous end joining contributes toward synthetic lethality of pathological rad51c mutants with poly ( adp - ribose ) polymerase . 
ghiringhelli f , richard c , chevrier s , et al : efficiency of olaparib in colorectal cancer patients with an alteration of the homologous repair proteworld j gastroenterol 22 : 10680 - 10686 , 2016 33 . 
kondrashova o , nguyen m , shield - artin k , et al : secondary somatic mutations restoring rad51c and rad51d associated with acquired resistance to the parp inhibitor rucaparib in high - grade ovarian carcinoma . 
 exceptional response to akt inhibition in patients with breast cancer and germline pten mutations belinda kingston , mbchb1 ; caroline bailleux , md2 ; suzette delaloge , md , msc2 ; gaia schiavon , md , phd3 ; veronique scott , phd2 ; magali lacroix - triki , md , phd2 ; t . 
hedley carr , phd3 ; iwanka kozarewa , phd3 ; heidrun gevensleben , md4 ; zoe kemp , phd5 ; alex pearson , phd1 ; nicholas turner , md , phd1 , 5 ; and fabrice andr e , md , phd2 introduction cowden syndrome is an autosomal dominant genetic disease with an estimated incidence of one in 200 , 000 . 
pip3 functions as a secondary messenger in the pi3k pathway that binds and activates proteins that have a pleckstrin homology domain , such as akt1 , and triggers their activation and localization to the plasma membrane , promoting cellular proliferation and survival.4 germline pten loss - of - function mutations may result in dominant akt activation as a driving oncogenic event in cowden - related breast tumors.5 preclinical evidence suggests that cancers with akt activation have increased sensitivity to akt inhibition.6 preliminary clinical evidence is derived from phase i and ii trials in patients with breast cancers bearing somatic mutations in the pi3k / akt / mtor pathway.7 - 11 inhibitor of all three isoforms of capivasertib ( azd5363 , astrazeneca ) is a potent and selective oral serine / threonine kinase akt ( ie , akt1 , 2 , 3 ) and has preclinical evidence of efcacy as monotherapy or in combination with cytotoxic and targeted therapies.12 - 15 despite the encouraging progression - free survival observed with capivasertib monotherapy in heavily pretreated patients with akt1 e17kmutant breast and gynecologic cancers , 9 recist response rates in phase i studies only reached 22% ( table 1 ) .8 , 9 , 16 because pten loss activates akt1 , we hypothesized that tumors from patients with cowden syndrome could be sensitive to this drug family . which was estrogen ( er ) and progesterone receptor negative , human epidermal growth factor receptor 2 ( her2 ) negative , and was designated grade iii invasive carcinoma of no special type ( nst )  . 
the patient received six cycles of neoadjuvant cyclophosphamide 600 mg / m2 , epirubicin 75 mg / m2 , and docetaxel 100 mg / m2 before a mastectomy with left axillary lymph node dissection ( revealing residual disease in 10 of 18 lymph nodes ) and adjuvant radiotherapy . eight months later , the patient experienced relapse with cutaneous disease and thoracic nodal volvement . 
after enrolling in safir02 ( clinicaltrials.gov identier : nct02299999 ) , targeted panel sequencing ( ion torrent pgm ; thermo fisher scientic , villebon , france ) of a fresh tumor biopsy sample revealed the presence of a heterozygous germline pten mutation ( c.389g.a , rs121909229 ) alongside other variants ( fig 1a ; data supplement )  . 
in the context of safir02 , the patient was randomly assigned to maintenance targeted therapy and received capivasertib 480 mg twice per day , 4 days on and 3 days off . 
on the right side , she presented with a t3 , erpositive and progesterone receptorpositive , her2negative , grade ii invasive carcinoma nst with 20 of 23 involved lymph nodes . 
in may 2010 , the patient received six cycles of chemotherapy every 3 weeks ( uorouracil 600 mg / m2 , epirubicin 75 mg / m2 , and cyclophosphamide 600 mg / m2 ) before starting maintenance tamoxifen . 
she received eight cycles of paclitaxel ( 90 mg / m2 on days 1 , 8 , and 15 of a 28 - day cycle ) combined with capivasertib ( 360 mg twice per day on days 2 - 5 , 9 - 12 , and 16 - 18 of each 28 - day in june 2013 , a computed tomography scan cycle )  . showed a complete response of the liver metastases . 
she had a conrmed maintained response in may 2014 until progression occurred in june 2014a progression - free survival of 19 monthsand a maintained complete response for 12 months on capivasertib alone ( fig 2c )  . 
plasma circulating tumor dna analysis from baseline and progression time points during the beech study showed no major changes ( data supplement )  . discussion the akt inhibitor capivasertib has been examined in early - phase trials ( table 1 ) , and no complete responses have been noted , yet both patients with cowden syndrome presented here had durable complete responses to capivasertib . 
cdc14 , phosphatase domaillustration from ( b ) immunohistochemistry demonstrating ( i ) absent pten staining in the tumor and ( ii ) cytoplasmic and membranous expression of pakt in the 40% of tumor cells . 
cdc14 , phosphatase domaillustration from ( b ) pten immunohistochemistry from ( i ) control tissue and ( ii ) noncancerous and ( iii and iv ) tumor - containing lymph node . 
 ( c ) computed tomography ( ct ) scans during the patients time on capivasertib , with white arrows indicating disease in the liver : ( i ) october 2012 , baseline ct demonstrating a liver deposit ; ( ii ) january 2013 , ct following two cycles of paclitaxel and capivasertib ; ( iii ) ct demonstrating continued complete response on maintenance capivasertib 7 months after cessation of paclitaxel ; and ( iv ) may 2014 , nal ct before progression june 2014 . nonmalignant cells carrying a germline pten mutation to pi3k - pathway inhibition . 
similar to the cases presented here , no severe toxicity was noted in the pilot study , 21 with just one patient experiencing a grade 3 adverse event ( hypophosphatemia / hypercholesterolemia )  . in recent years , drugs targeting the pi3k / akt / mtor pathway have been developed . 
in contrast , akt inhibitors in combination with paclitaxel have been shown to be more active in patients with triple - negative breast cancers harboring a pik3ca / pten / akt1 pathway alteration.7 , 10 similarly , pi3k inhibitors have demonstrated activity in patients with pik3ca mutations.22 , 23 of note in case 2 is the presence of the second - hit pten stop - gain mutation y88x , present with an af of 25.5%. however , the presence of a tp53 mutation at an af of 51.9% indicates that this second - hit mutation is subclonal rather than a truncal driver mutation . 
explanations for the pten phenotype in case 1 include undetected loh , pten promotor hypermethylation , 24 , 25 complex pten genomic rearrangements , 26 and post - translational modication.27 contrary to the two - hit model by knudson et al28 of tumorigenesis in tumor suppressor genes , pten aberrations appear to be protumorigenic in the absence of a second hit . in 2010 , alimonti et al29 demonstrated that pten hypermorphic mice ( with 80% of the normal pten protein level ) had a greater propensity to tumorigenesis than mice with two functional alleles but were less tumorigenic than pten heterozygous mice , supporting a haploinsufciency model of tumorigenesis in pten aberrations . a later in vivo study of pten knock - in mouse models suggested that the conformation of pten underlies the dominant - negative behavior of pten heterozygous mutants . 
this supports the rationale that pten heterozygous mutants act in a dominant - negative manner to promote tumorigenesis . the patients in the cases presented here were treated with combination chemotherapy and capivasertib followed by capivasertib monotherapy . 
the patient in case 1 had previously demonstrated tumor resistance to taxane therapy , with residual disease after neoadjuvant chemotherapy and a short progression - free survival after treatment of primary breast cancer . 
the second patient achieved a complete response with the combination of paclitaxel and capivasertib and maintained this complete response for a period of 12 months on capivasertib alone , suggesting that capivasertib was highly active in this patient . in summary , these two patients with breast cancer and different germline pten mutations both showed a dramatic response to capivasertib superior to that seen in early trials of the drug . 
 magali lacroix - triki leadership : mypl stock and other ownership interests : mypl honoraria : myriad genetics , genomic health consulting or advisory role : roche travel , accommodations , expenses : agendia t . 
hedley carr employment : astrazeneca stock and other ownership interests : astrazeneca iwanka kozarewa employment : astrazeneca stock and other ownership interests : astrazeneca kingston et al zoe kemp honoraria : lilly honoraria : astrazeneca nicholas turner consulting or advisory role : roche , novartis , astrazeneca , pzer , bicycle therapeutics , bristol - myers squibb , merck sharp & dohme , tesaro , lilly research funding : pzer ( inst ) , roche ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , bio - rad ( inst ) , guardant health ( inst ) fabrice andr e research funding : astrazeneca ( inst ) , novartis ( inst ) , pzer ( inst ) , lilly ( inst ) , roche ( inst ) , daiichi ( inst ) travel , accommodations , expenses : novartis , roche , glaxosmithkline , astrazeneca no other potential conicts of interest were reported . references tan mh , mester jl , ngeow j , et al : lifetime cancer risks in individuals with germline pten mutations . 
j med genet 37 : 828 - 830 , 2000 lee yr , chen m , pandol pp : the functions and regulation of the pten tumour suppressor : new modes and prospects . 
miller tw , p erez - torres m , narasanna a , et al : loss of phosphatase and tensin homologue deleted on chromosome 10 engages erbb3 and insulin - like growth factor - i receptor signaling to promote antiestrogen resistance in breast cancer . 
oncogene 24 : 7455 - 7464 , 2005 davies br , guan n , logie a , et al : tumors with akt1e17k mutations are rational targets for single agent or combination therapy with akt inhibitors . 
mol cancer ther 14 : 2441 - 2451 , 2015 kim sb , dent r , im sa , et al : ipatasertib plus paclitaxel versus placebo plus paclitaxel as rst - line therapy for metastatic triple - negative breast cancer ( lotus ) : a multicentre , randomised , double - blind , placebo - controlled , phase 2 trial . 
lancet oncol 18 : 1360 - 1372 , 2017 banerji u , dean ej , p erez - fidalgo ja , et al : a phase i open - label study to identify a dosing regimen of the pan - akt inhibitor azd5363 for evaluation in solid tumors and in pik3ca - mutated breast and gynecologic cancers . 
schmid p , abraham j , chan s , et al : azd5363 plus paclitaxel versus placebo plus paclitaxel as rst - line therapy for metastatic triple - negative breast cancer ( pakt ) : a randomised , double - blind , placebo - controlled , phase ii trial . 
patnaik a , appleman lj , tolcher aw , et al : first - in - human phase i study of copanlisib ( bay 80 - 6946 ) , an intravenous pan - class i phosphatidylinositol 3 - kinase inhibitor , in patients with advanced solid tumors and non - hodgkins lymphomas . 
zhang c , xu b , liu p : addition of the p110 inhibitor byl719 overcomes targeted therapy resistance in cells from her2 - positive - pten - loss breast cancer . tumour biol 37 : 14831 - 14839 , 2016 14 . 
yu y , savage re , eathiraj s , et al : targeting akt1 - e17k and the pi3k / akt pathway with an allosteric akt inhibitor , arq 092 . 
agarwal r , liebe s , turski ml , et al : targeted therapy for genetic cancer syndromes : von hippel - lindau disease , cowden syndrome , and proteus syndrome . discov med 19 : 109 - 116 , 2015 77 : 787 - 795 , 2016 68 : 7066 - 7072 , 2008 16 . 
pediatr res 75 : 527 - 534 , 2014 iacobas i , burrows pe , adams dm , et al : oral rapamycin in the treatment of patients with hamartoma syndromes and pten mutation . 
komiya t , blumenthal gm , ballas ms , et al : a pilot study of sirolimus ( s ) in subjects with cowden syndrome ( cs ) with germ - line mutations in pten . 
baselga j , dent sf , cort es j , et al : phase iii study of taselisib ( gdc - 0032 ) + fulvestrant ( fulv ) v fulv in patients ( pts ) with estrogen receptor ( er ) - positive , pik3ca - mutant ( mut ) , locally advanced or metastatic breast cancer ( mbc ) : primary analysis from sandpiper . 
a chest computed tomography scan coupled with an 18f - labeled uorodeoxyglucosepositron emission tomography scan identied a metabolically active lymphadenomegaly in the mediastinuflow cytometric analysis identied a bone marrow ( bm ) blast population ( fig 1a ) that was transferase , terminal deoxynucleotidyl negative for cd2 , cd4 , cd8 , and cd1a and positive for human leukocyte antigen ( hla - dr ) , cd38 , cd117 , cd33 , cd34 , cd56 , cd99 , cycd3 , cd5 , cd7 , cd10 , cd19 , and cd13 . because of a t - all immature phenotype9 , 10 and a myeloid interface , and despite cd5 expression , this patient was diagnosed with near - etp all.11 molecular cytogenetic studies showed the presence of ddx3xmllt10 fusion gene derived from a three - way t ( x ; 3 ; 10 ) ( p11 ; ? ; p13 ) translocation . 
molecular cytogenetic studies revealed an abnormal karyotype characterized by an isolated numerical abnormality ( ie , 47 , xx , + 4 [ 6 ] / 46xx [ 4 ] ) , a rearrangement of bcl11b , and an internal tandem duplication of flt3 ( fig 1a )  . because of advanced age and comorbidities , patient was treated with prednisone 0.5 mg / kg and vincristine 1.4 mg / m2 ( days 1 , 8 , 15 , and 22 ) , without achieving hematologic remission . 
scoring the response in relation to data for t - all on a drp platform , 8 the b - cell lymphoma 2 ( bcl2 ) inhibitor bh - 3 mimetic venetoclax ( abt - 199 ) and the proteasome inhibitor bortezomib ranked in all three cases among the most active drugs ( fig 1b )  . 
next , we validated the activity of venetoclax and bortezomib using an open microwell microuidic platform developed by cellply15 ( bologna , italy ; figs 1c and 1d ) and an atp - based cellular viability assay ( fig 1e ) on etp cells that , consistent with data reported in the literature , 16 , 17 express a high level of bcl2 ( fig 1af )  . on the basis of these ndings , individualized treatment with venetoclax and bortezomib ( vebo ) was then administered on an outpatient basis , sequentially or in combination , as off - label agents after approval by the relevant institutional review board . 
all patients received a cycle with venetoclax ( 800 mg per day 28 days ) by mouth and bortezomib ( 1.3 mg / m2 twice a week 2 [ upr1 , upr2 ] or 4 [ upg3 ] ) , with no evidence of major toxicities . 
upr1 and upr2 received antiviral prophylaxis with acyclovir , whereas upg3 received antimycotic treatment with micafungin . patients obtained a hematologic complete ( upr2 ) or partial remission ( upr1 and upg3 ) assessed a month after vebo initial treatment quantied by morphology , ow cytometry analysis , and uorescence in situ hybridization on bone marrow cells ( figs 1g and 1h )  . 
dose - response curves were generated as previously published using ve - point drug dilutions in duplicate.8 a ranked list of top hits was generated according to the criteria , emax ( ie , the % viability at the maximal drug concentration used ) of less than 15% and signicant ( p , .05 ) difference of half maximal inhibitory concentration ( ic50 ) value for each drug compared with the median ic50 for each particular drug in the reference data set.8 in bold : venetoclax and bortezomib ( vebo )  . 
 ( c ) effect of 24 hours of the vehicle ( dimethyl sulfoxide ) , 10 mm hydrogen peroxide ( h2o2 ) , venetoclax , and bortezomib on cellular viability as measured by a functional proling , on the basis of laboratory - on - a - chip technology , which allows testing the response of live tumor cells exposed to anticancer drugs in an automated process developed by cellply . 
high - content time - lapsed imaging was performed for 24 hours , every 12 hours , on cells in 480 microwells ( 75 m diameter ) at a 1 px - 1 m resolution at each time point / condition tested . 
 ( g ) bm cytology at relapse ( pre vebo ) and after completion of treatment with vebo ( post vebo ; may - gr unwald giemsa staining at 63 )  . 
the marrow contains 50% ( patient 1 [ upr1 ] ) , 90% ( patient 2 [ upr2 ] ) , and 28% ( patient 3 [ upg3 ] ) of lymphoid blasts before treatment , which decreased rapidly after vebo . 
a 500 - cell count was performed based on examination of multiple elds to assess hematologic remission ( complete remission indicates a blast percentage , 5% in the bm )  . 
 ( i ) percentage of ddx3x - mllt10 ( upr1 ) and etv6 / cdkn1bdel ( upg3 ) fish - positive bm cells during vebo treatment and after an allogeneic hematopoietic stem - cell transplantation . 
the analysis was performed on 500 to 1 , 000 interphase nuclei for each experiment ; the cutoff for ddx3x - mllt10 fusion was 2% and for etv6 / cdkn1bdel was 3% . 
this is , for example , the case of - secretase inhibitors in notch1mutated t - all.21 an alternative option is to develop individualized approaches on the basis of pattern of responses to small molecule inhibitors.22 a rst example was the development of a drp in primary acute myeloid leukemia samples . 
the ex vivo testing of 187 drugs in 28 consecutive acute myeloid leukemia cases22 led to the identication of ve major taxonomic drug - response subtypes with distinct genomic features . 
importantly , therapies on the basis of drp resulted in several clinical responses.22 more recently , frismantas et al8 extended this idea and reported a proof - of - concept study by testing 60 drugs in 68 all cases . 
interestingly li et al27 demonstrated that venetoclax acts synergistically with the mcl1 - specic inhibitor s63845 in a broad panel of t - all cell lines and in zebrash embryos undergoing transplantation with t - all cells . 
moreover , venetoclax showed clinical activity in patients with t - all both in combination with chemotherapy28 or decitabine , 29 suggesting that it may be safely included in different therapeutic regimens . in addition , bortezomib has recently emerged as a potential modulator of the oncogenic t - all driver notch1 . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / author - center . antonio pierini consulting or advisory role : pzer , biotest patents , royalties , other intellectual property : antibody to deliver cells for islet engraftment in diabetes jean - pierre bourquin travel , accommodations , expenses : servier , amgen cristina mecucci patents , royalties , other intellectual property : patent on npm1 mutations in acute myeloid leukemia with normal karyotype no other potential conicts of interest were reported . references deangelo dj , yu d , johnson jl , et al : nelarabine induces complete remissions in adults with relapsed or refractory t - lineage acute lymphoblastic leukemia or lymphoblastic lymphoma : cancer and leukemia group b study 19801 . 
blood 109 : 5136 - 5142 , 2007 bassan r , bourquin jp , deangelo dj , et al : new approaches to the management of adult acute lymphoblastic leukemia . 
blood 126 : 833 - 841 , 2015 inukai t , kiyokawa n , campana d , et al : clinical signicance of early t - cell precursor acute lymphoblastic leukaemia : results of the tokyo childrens cancer study group study l99 - 15 . 
front med 6 : 416 - 420 , 2012 jain n , lamb av , obrien s , et al : early t - cell precursor acute lymphoblastic leukemia / lymphoma ( etp - all / lbl ) in adolescents and adults : a high - risk subtype . 
blood 127 : 1863 - 1869 , 2016 brammer je , saliba rm , jorgensen jl , et al : multi - center analysis of the effect of t - cell acute lymphoblastic leukemia subtype and minimal residual disease on allogeneic stem cell transplantation outcomes . 
bone marrow transplant 52 : 20 - 27 , 2017 frismantas v , dobay mp , rinaldi a , et al : ex vivo drug response proling detects recurrent sensitivity patterns in drug - resistant acute lymphoblastic leukemia . blood 129 : e26 - e37 , 2017 chopra a , bakhshi s , pramanik sk , et al : immunophenotypic analysis of t - acute lymphoblastic leukemia . 
coustan - smith e , mullighan cg , onciu m , et al : early t - cell precursor leukaemia : a subtype of very high - risk acute lymphoblastic leukaemia . lancet oncol 10 : 147 - 156 , 2009 2017 11 . 
murphy sb , bowman wp , abromowitch m , et al : results of treatment of advanced - stage burkitts lymphoma and b cell ( sig + ) acute lymphoblastic leukemia with high - dose fractionated cyclophosphamide and coordinated high - dose methotrexate and cytarabine . 
kantarjian h , thomas d , obrien s , et al : long - term follow - up results of hyperfractionated cyclophosphamide , vincristine , doxorubicin , and dexamethasone ( hyper - cvad ) , a dose - intensive regimen , in adult acute lymphocytic leukemia . 
bassan r , masciulli a , intermesoli t , et al : final results of northern italy leukemia group ( nilg ) trial 10 / 07 combining pediatric - type therapy with minimal residual disease study and risk - oriented hematopoietic cell transplantation in adult acute lymphoblastic leukemia ( all )  . 
rocchi l , faenza a , rambelli l , et al : ex - vivo drug response proling for precision medicine approaches in acute myeloid leukemia with the open microwell microuidic platforblood 128 : 1675 , 2016 124 : 3738 - 3747 , 2014 16 . 
peirs s , matthijssens f , goossens s , et al : abt - 199 mediated inhibition of bcl - 2 as a novel therapeutic strategy in t - cell acute lymphoblastic leukemia . 
punnoose ea , leverson jd , peale f , et al : expression prole of bcl - 2 , bcl - xl , and mcl - 1 predicts pharmacological response to the bcl - 2 selective antagonist venetoclax in multiple myeloma models . 
chonghaile tn , roderick je , gleneld c , et al : maturation stage of t - cell acute lymphoblastic leukemia determines bcl - 2 versus bcl - xl dependence and cancer discov 3 : 1416 - 1429 , 2013 sensitivity to abt - 199 . 
li z , he s , look at : the mcl1 - specic inhibitor s63845 acts synergistically with venetoclax / abt - 199 to induce apoptosis in t - cell acute lymphoblastic 28 . 
rahmat lt , nguyen a , abdulhaq h , et al : venetoclax in combination with decitabine for relapsed t - cell acute lymphoblastic leukemia after allogeneic hematopoietic cell transplant . 
koyama d , kikuchi j , hiraoka n , et al : proteasome inhibitors exert cytotoxicity and increase chemosensitivity via transcriptional repression of notch1 in t - cell acute lymphoblastic leukemia . 
 clinical equipoise for trials of novel biologic therapies , therapeutic success rates , and predictors of success : a meta - analysis purpose the demand for more rapid access to novel biologic therapies than randomized controlled trials can deliver is a topic of ongoing study and debate . 
we aimed to inform this debate by estimating therapeutic success from phase iii trials comparing novel biologic therapies with standard of care and identifying predictors of success . methods this was a meta - analysis of phase iii trials evaluating novel biologic therapies in advanced breast , colorectal , lung , and prostate cancers . 
therapeutic success was defined as statistically significant results for the primary end point favoring novel biologic therapies . results of 119 included phase iii trials ( 76 , 726 patients ) , therapeutic success was 41% , with a statistically significant relative reduction in disease progression and death for novel biologic therapies over standard of care of 20% and 8% . 
therapeutic success of phase iii trials with and without a preceding phase ii trial were 43% and 30% , respectively conclusion therapeutic success of novel biologic therapies in phase iii trials , including therapies with a matching predictive biomarker , was modest and has not significantly improved over time . 
2017 by american society of clinical oncology introduction advancement in genomic medicine has led to rapid development of novel biologic therapies ( nbts ) that target aberrant pathways in cancer . the success of some of these agents , 1 - 5 whose efficacy was evident from early - phase trials , has led to calls for expediting drug development , evaluation , and approval . 
the introduction of accelerated access programs in europe and the united states recognizes the potential value of using nonrandomized early - phase trials to support new drug approvals.6 , 7 however , there are serious concerns about the risk of relying on lessrobust evidence , and the evidence requirements for nbts is a topic of ongoing debate among the research community.8 - 12 although the requirement for phase iii randomized controlled trials ( rcts ) for assessing the efficacy of conventional treatments is well established , 13 - 15 this issue has not been definitively addressed for targeted therapies . central to the argument for retaining phase iii rcts to generate evidence is the assumption of equipoise . 
thus , critical questions for the evaluation of nbts are : does the evidence to date support the belief that nbts are associated with superior therapeutic success ( ts ) over nontargeted standard - of - care ( soc ) doah cho felicia t . 
characteristics of included phase iii randomized controlled trials and proportion of treatment comparisons achieving therapeutic success ( 119 trials , 132 treatment comparisons ) ( continued ) randomized treatment comparisons therapeutic success 16 ( 33 ) 37 ( 44 ) note . 
this information is critical to the ethics of continuing to require phase iii rcts of nbt versus a control soc treatment after promising results from early - phase studies . this study aims to assess whether existing phase iii evidence demonstrates that , as a class , nbts are superior to soc . 
we also sought to identify predictors of ts and to specifically examine whether the rate of success was higher for trials that used a personalized approach by selecting a population defined by a predictive biomarker . methods we searched the medline and embase databases to identify rcts published in english from january 1 , 1990 to january 31 , 2014 ( data supplement )  . 
we also hand - searched published meta - analyses , conference proceedings , clinical trial registries , reference lists , and citations of included studies . trials were eligible if they evaluated the superiority of nbts over soc and reported overall survival ( os ) or pfs . 
soc treatments were chemotherapy , endocrine therapy , or best supportive care . rcts were excluded if they involved radiotherapy or vaccine therapy , included nbts in all arms , or were of noninferiority design . we identified prior studies referenced by the included rcts . 
we defined prior studies as providing enough matched evidence to inform phase iii rcts if they assessed the same monotherapy or combination in the same advanced tumor population as the included rct . 
if not reported , these were estimated where possible using the methods described by parmar and colleagues.19 we plotted the distributions of the hr point estimates for pfs and os from phase iii trials to describe the spread of ts . 
distribution of overall therapeutic success ( ts ) for novel versus standard therapies for : ( a ) overall survival ( os ) and ( b ) progression - free survival ( pfs ) for randomized controlled trials ( rcts ) overall , and ( c ) os and ( d ) pfs for rcts with a biomarkerselected population . 
trial characteristics are summarized in table 1 ( individual trial characteristics , data supplement )  . we used logistic regression to identify characteristics of the phase iii rcts and preliminary evidence studies that predicted ts in phase iii rcts . 
we examined the correlation between objective tumor responses ( otrs ) reported in the nbt arms of the phase iii and companion phase ii trials using weighted linear regression . results the search strategy identified 132 randomized comparisons from 119 eligible phase iii rcts , comprising 76 , 726 patients ( appendix fig a1 , online only ; data supplement )  . 
pfs was used as the primary end point in 22 ( 92% ) , 22 ( 37% ) , two ( 17% ) , and 12 ( 32% ) breast , lung , prostate , and colorectal comparisons , respectively . 
os was used as the primary end point in two ( 8% ) , 37 ( 63% ) , nine ( 75% ) , and 19 ( 51% ) breast , lung , prostate , and colorectal comparisons , respectively . 
ts was highest in breast ( 15 comparisons , 63% ) , followed by colorectal ( 15 comparisons , 41% ) , lung ( 21 comparisons , 36% ) and prostate cancer ( two comparisons , 17% ) rcts . 
of 70 rcts with nonstatistically significant findings , four ( 6% ) were inconclusive because of premature closure secondary to slow accrual . figures 1a and 1b show distributions of hrs for os and pfs for all phase iii rcts . 
nbts were associated with a 20% and 8% statistically significant relative reduction in the risk of disease progression and death , respectively , over soc ( fig 2a )  . 
a statistically significant relative advantage of nbt versus soc for pfs and os was also observed within each tumor group , although there was substantial heterogeneity between tumor groups ( fig 2a )  . thirty - four comparisons from 29 ( 24% ) rcts were conducted in biomarker - selected populations . 
the shapiro - wilk test indicated that the results deviated from normality . nbts were associated with a statistically significant 28% and 8% relative reduction in disease progression and death , respectively , over soc ( fig 2b )  . a biomarker - selected population was associated with a 4.74 - fold increase in ts compared with rcts with an unselected population ( table 2 )  . trials that used pfs as the primary end point were associated with a 5.22 - fold increase in ts compared with rcts using os ( table 2 )  . 
this evidence was derived from phase i , single - arm phase ii , randomized phase ii , and randomized phase iii trials for 23 ( 17% ) , 41 ( 31% ) , 10 ( 8% ) , and 27 ( 20% ) phase iii comparisons , respectively . 
alternative preliminary evidence used to justify the included phase iii rcts were : monotherapy studies when the phase iii comparison was combination therapy , combination therapy with a different conventional backbone treatment to the included phase iii comparison , evidence from other solid cancers , evidence from observational studies , and evidence from retrospective biomarker analyses or unselected populations for phase iii randomized comparisons in biomarker - selected populations ( data supplement )  . 
in this sample , otr in phase ii trials correlated poorly with otr in the subsequent phase iii trials ( r2 = 0.15 ; appendix fig a2 , online only )  . 
for example , 73% of the randomized arms in the national cancer institutesponsored cooperative oncology group trials were in the setting of early rather than advanced cancer . despite sobering overall ts rates , even in the current era of nbt , debate about the need for phase iii rcts after promising results in earlierphase studies has largely focused on biomarkerselected populations.11 our findings provide some support for the concern that equipoise may be lost for such treatments that progress to testing in phase iii rcts . 
however , later studies showed responses in patients with tumors lacking egfr expression , 26 , 27 whereas significantly lower responses were seen in patients with tumors harboring a mutation in the kirsten - rat sarcoma gene ( k - ras ) .28 , 29 supportive evidence of this was shown in retrospective correlative biomarker analyses of rcts30 , 31 and confirmed by meta - analyses.32 , 33 cetuximab and panitumumab efficacy was restricted to k - ras wild - type patients , and there was no additional benefit of adding egfr - monoclonal antibodies to chemotherapy over chemotherapy alone in patients harboring mutations . 
past rcts30 , 31 of egfr - monoclonal antibodies with chemotherapy against chemotherapy alone allowed for this qualitative interaction between k - ras status and randomized treatment to be examined in an unbiased manner . 
consistent with other studies , seruga et al20 reported that 32% of phase iii trials did not have an appropriate preceding phase ii trial , and more than half of these trials failed to show ts . 
single - arm phase ii trials are also increasingly considered as not sufficiently informative for the decision to proceed to a phase iii rct.34 , 35 although a review of predominantly chemotherapy trials reported limited advantage of randomized over single - arm phase ii studies , 36 randomized phase ii trials with concurrent controls have been considered to be more informative for nbts.34 , 35 in the current study , an association between the level of evidence of preliminary trials ( phase i , single - arm phase ii , randomized phase ii , and randomized phase iii ) and ts of subsequent phase iii rcts was not demonstrated . 
we also excluded certain classes of drugs , such as vaccines , as we considered the mechanism of action of these therapies differed from other biologics , so our findings may not be generalizable to these other classes . despite the advances in genomic medicine and examples of exceptional success for some new therapies , the evidence for their ts from our review remains modest . 
despite calls that nonrandomized evidence may be sufficient in this era of targeted therapy , our review indicates that equipoise remains and supports the ongoing ethical and scientific requirement for phase iii rcts to provide a more definitive and valid estimate of treatment efficacy and a predictive and prognostic value of potential biomarkers . 
links no relationship to disclose should be confirmed using a larger sample size of comparisons . randomized phase ii trials may also have an important role in addressing the problem of optimal dose finding of targeted therapies . 
the maximum tolerated dose defined by the traditional dose - finding strategy for cytotoxic therapies may not be the optimal dose for targeted therapies where interand intrapatient variability has a greater effect on treatment efficacy and toxicity.37 , 38 randomized phase ii trials should be used to a larger degree to compare the efficacy and toxicity of different doses , better define long - term toxicity , and identify targeted therapies amenable to dose escalation or reduction.37 - 40 the main strength of our study is that we have comprehensively reviewed all published rcts that compared nbts against soc providing a contemporary review of predictors of ts . 
 chee khoon lee honoraria : astrazeneca travel , accommodations , expenses : astrazeneca acknowledgment we thank rhana pike ( national health and medical research council clinical trials centre ) for editorial support . affiliations doah cho , felicia t . 
lord , the university of notre dame , darlinghurst , new south wales , australia . supported in part by national health and medical research council ( nhmrc ) postgraduate research scholarship ( medical / dental ) no . 
shaw at , kim dw , nakagawa k , et al : crizotinib versus chemotherapy in advanced alk - positive lung cancer . n engl j med 368 : 2385 - 2394 , 2013 3 . 
camidge dr , bang yj , kwak el , et al : activity and safety of crizotinib in patients with alk - positive non - small - cell lung cancer : updated results from a phase 1 study . 
kim dw , ahn mj , shi y , et al : results of a global phase ii study with crizotinib in advanced alk - positive nonsmall cell lung cancer ( nsclc )  . 
european medicines agency : draft guideline on the scientific application and the practical arrangements necessary to implement the procedure for accelerated assessment pursuant to article 14 ( 9 ) of regulation ( ec ) no 726 / 2004 ( revision 1 )  . 
avorn j , kesselheim as : the 21st century cures actwill it take us back in time ? n engl j med 372 : 2473 - 2475 , 2015 9 . 
kurzrock r : on the shoulders of giants : cooperative groups and clinical trial design - past , present , and future . presented at american society of clinical oncology annual meeting , chicago , il , june 1 - 5 , 2016 10 . 
yao jc , meric - bernstam f , lee jj , et al : accelerated approval and breakthrough therapy designation : oncology drug development on speed ? clin cancer res 19 : 4305 - 4308 , 2013 11 . 
menis j , hasan b , besse b : new clinical research strategies in thoracic oncology : clinical trial design , adaptive , basket and umbrella trials , new end - points and new evaluations of response . 
djulbegovic b , kumar a , glasziou pp , et al : new treatments compared to established treatments in randomized trials . cochrane database syst rev 10 : mr000024 , 2012 15 . 
djulbegovic b , kumar a , soares hp , et al : treatment success in cancer : new cancer treatment successes identified in phase 3 randomized controlled trials conducted by the national cancer institute - sponsored cooperative oncology groups , 1955 to 2006 . 
schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
herbst rs , gandara dr , hirsch fr , et al : lung master protocol ( lung - map ) - a biomarker - driven protocol for accelerating development of therapies for squamous cell lung cancer : swog s1400 . 
van cutsem e , peeters m , siena s , et al : open - label phase iii trial of panitumumab plus best supportive care compared with best supportive care alone in patients with chemotherapy - refractory metastatic colorectal cancer . 
saltz lb , rubin m , hochster h , et al : cetuximab ( imc - c225 ) plus irinotecan ( cpt - 11 ) is active in cpt - 11 refractory colorectal cancer ( crc ) that expresses epidermal growth factor receptor ( egfr )  . 
chung ky , shia j , kemeny ne , et al : cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry . 
adelstein ba , dobbins ta , harris ca , et al : a systematic review and meta - analysis of kras status as the determinant of response to anti - egfr antibodies and the impact of partner chemotherapy in metastatic colorectal cancer . 
liang rf , zheng ll : the efficacy and safety of panitumumab in the treatment of patients with metastatic colorectal cancer : a meta - analysis from five randomized controlled trials . 
fromer mj : oncology drug dosing : can an optimal dose be fine - tuned for each patient ? issues / december - 15 - 2013 / oncology - drug - dosing - can - an - optimal - dose - be - fine - tuned - for - each - patient / 39 . 
companion phase ii trials , testing the same novel biologic therapies as the phase iii trials , were identified for 54% ( n = 64 ) of included phase iii trials . 
 predisposition to lung adenocarcinoma in a family harboring the germline egfr v843i mutation kouki ohtsuka , md , phd1 ; hiroaki ohnishi , md , phd1 ; masachika fujiwara , md , phd1 ; takeshi morii , md , phd1 ; satsuki matsushima1 ; wataru ogura1 ; satoko yamasaki , md1 ; tomonori kishino , md , phd1 ; ryota tanaka , md , phd1 ; and takashi watanabe , md , phd1 introduction in patients with nonsmall - cell mutations in the tyrosine kinase domain of epidermal growth factor receptor ( egfr ) , most commonly a deletion in exon 19 or an l858r substitution in exon 21 , are frequent lung cancer . 
these egfr mutations are speculated to constitutively activate egfr through phosphorylation and impart tumorigenic properties.1 most egfr mutations occur in somatic tumor tissue , with germline egfr mutations being extremely rare.2 , 3 as a scarce the germline egfr t790m or germline example , v843i mutation has been identied in several families susceptible to lung cancer.2 - 6 to several treatments , we previously reported multiple cases of lung adenocarcinoma in a family with the germline egfr v843i mutation.5 the proband had advanced - stage cancer including egfr resistant tyrosine kinase inhibitors , resulting in poor therapeutic outcomes . 
however , tumors of the proband and other family members both harbored the same somatic egfr l858r mutation in addition to the germline v843i mutation , and it is unclear how these two genetic mutations affected the prognosis of lung cancer in those patients . 
no other family history of malignant disease was revealed on a detailed interview ( table 1 )  . sample preparation the study was approved by the ethics committee of the participating institutes , and written informed consent was obtained from the proband and two family members with lung cancer . 
peripheral blood mononuclear cells of the proband , cancerous pleural effusion from the proband , and formalin - xed parafnembedded tumor samples from her mother and younger brother were subjected to genetic analysis . genomic dna was extracted from these samples for next - generation sequencing ( ngs ) analysis using the dneasy blood & tissue kit ( qiagen , hilden , germany )  . wgs analysis we performed wgs using genomic dna extracted from cancer cells harvested from the cancerous pleural effusion and compared it to that of whole blood . paired - end sequencing was performed using illuminas ngs platforms hiseq x ten system ( illumina , san diego , ca )  . 
 egfr v843i mutation and lung adenocarcinoma we adopted driver gene mutations registered in the catalogue of somatic mutations in cancer ( cosmic ) database , predicted functional consequences using sift and polyphen - 2 software , or categorized them as pathogenic or likely pathogenic per the clinvar database . 
furthermore , we examined known causative germline mutations of hereditary cancers and somatic mutations of major cancerrelated genes among the gene alterations in the proband . ts analysis for ts analysis , we used the ion ampliseq custom panel and ion torrent pgm deep sequencing ion ampliseq cancer hotspot panel v2 ( thermo fisher scientic , waltham , ma )  . 
we examined mutations in 50 cancer - related genes in tumor dna derived from the proband and two of her family members . genomic study wgs revealed the previously reported germline egfr v843i mutation ; however , no other germline oncogenic mutations were observed in the peripheral blood and cancerous pleural effusion of the proband . 
in the clinvar database , the egfr v843i mutation has been registered as likely pathogenic , and the tp53 r248w mutation is pathogenic . other genetic abnormalities , including insertions / deletions , copy - number variations , and structural variations of cancer - related genes , were not detected in blood or cancer cells of the proband ( tables 1 and 2 )  . 
whole - genome sequencing analysis for the proband germline / somatic gene mutation pleural effusion blood egfr v843i l858r tp53 r248w germline somatic somatic via wgs analysis in families harboring the germline egfr mutation ( table 1 ) .4 - 6 wgs analysis of peripheral blood samples of the proband revealed no known genetic abnormalities for hereditary cancers other than the germline egfr v843i mutation , further supporting the possibility that this mutation causes familial lung adenocarcinoma ( tables 1 and 2 )  . 
according to the tohoku university tohoku medical megabank organization database , frequency of the egfr v843i germline mutation is low , amounting to one in 3 , 509 healthy japanese individuals ; however , it is considered an important germline mutation associated with the risk of lung carcinogenesis.8 , 9 furthermore , the somatic tp53 r248w mutation was detected only in cancer cells of the proband but not the other two family members ( tables 2 and 3 )  . 
the other 48 cancer - related oncogenes assessed via ts analysis were not mutated in tumors of any of the family members . although this is an anecdotal case , the present results suggest that the tp53 mutation may serve as a prognostic factor predicting worse drug sensitivity and poor therapeutic outcomes in lung cancer harboring a germline egfr mutation . 
recent studies have reported that lung cancers with both egfr and tp53 mutations are associated with a poor prognosis.10 - 12 the present ndings support our hypothesis that lung cancers harboring egfr and tp53 mutations are refractory and have a poor prognosis , suggesting that analysis of tumor - related oncogenes via wgs or ts may help predict the clinical course of familial lung cancer cases . furthermore , mutations in genes other than egfr are reportedly associated with familial accumulation of lung including germline rb1 , her2 , or tp53 mutacancer , tions.3 , 13 among these , li - fraumeni syndrome , characterized table 3 . 
 ohtsuka et al by germline tp53 mutations , is potentially the most frequent multiple cancer syndrome associated with an increased risk of lung cancer.3 high - throughput analysis of genes associated with multiple cancers , particularly tp53 , is therefore crucial to elucidate the genetic background of patients with familial lung cancer . 
considering the high prevalence of tp53 mutations , somatic or germline , in cases of solitary or hereditary lung cancer , tp53 mutations are apparently an equally prominent cause of lung cancer as egfr mutations . the genome of family in conclusion , ngs analysis of members with the germline egfr v843i mutation reinforced the hypothesis that this mutation predisposes individuals to familial lung adenocarcinoma . 
n engl j med 362 : 2380 - 2388 , 2010 bell dw , gore i , okimoto ra , et al : inherited susceptibility to lung cancer may be associated with the t790m drug resistance mutation in egfr . 
nat genet 37 : 1315 - 1316 , 2005 oxnard gr , nguyen ks , costa db : germline mutations in driver oncogenes and inherited lung cancer risk independent of smoking history . 
j natl cancer inst 106 : djt361 , 2014 ikeda k , nomori h , mori t , et al : novel germline mutation : egfr v843i in patient with multiple lung adenocarcinomas and family members with lung cancer . ann thorac surg 85 : 1430 - 1432 , 2008 ohtsuka k , ohnishi h , kurai d , et al : familial lung adenocarcinoma caused by the egfr v843i germ - line mutation . 
matsushima s , ohtsuka k , ohnishi h , et al : v843i , a lung cancer predisposing egfr mutation , is responsible for resistance to egfr tyrosine kinase inhibitors . j thorac oncol 9 : 1377 - 1384 , 2014 8 . 
nat commun 6 : 8018 , 2015 yasuda j , kinoshita k , katsuoka f , et al : genome analyses for the tohoku medical megabank project towards establishment of personalized healthcare . j biochem 165 : 139 - 158 , 2019 10 . 
labb e c , cabanero m , korpanty gj , et al : prognostic and predictive effects of tp53 co - mutation in patients with egfr - mutated non - small cell lung cancer 11 . 
aggarwal c , davis cw , mick r , et al : inuence of tp53 mutation on survival in patients with advanced egfr - mutant nonsmall - cell lung cancer . 
liu dh , zhao zr , lin yb , et al : prognostic effect of tp53 and pkd co - mutations in patients with resected epidermal growth factor receptor - mutated lung 13 . 
in her article , she cited win et al , 3 who similarly concluded ( after reviewing 15 studies of breast cancer risk in patients with ls ) that a correlation between ls and breast cancer remains inconclusive . 
predisposition to breast cancer has been difcult to prove ( on the basis of studies of ls populations ) , but it might be possible to identify members of ls families who carry a germline pathogenic mismatch repair ( mmr ) variant that is likely to predispose them to breast cancer . for example , the immunohistochemical ngerprint of ls - related cancers has decient mmr ( dmmr ) protein expression that is demonstrated in nearly all cancers related to ls . 
breast cancer specimens are now commonly tested for mmr protein expression , in part because the us food and drug administration approved checkpoint inhibitor therapy for dmmr metastatic breast cancer.4 however , sporadic breast cancers are rarely associated with dmmr.3 , 5 thus , it seems reasonable that identifying dmmr in a patient with ls and breast cancer strongly suggests that the germline pathogenic mmr variant predisposed that patient to breast cancer . 
but it is possible that the loss of mmr expression was not the gatekeeper or cancer - initiating event ; instead , a later driver event may have contributed to the cancer phenotype . 
the natural history of ls and responsiveness to checkpoint inhibitor therapies in patients who have dmmr breast cancers may or may not be similar to the response in patients with sporadic dmmr breast cancers . 
it is also unclear whether risk factors besides the germline mmr mutation are involved in transformation to dmmr breast cancer . family members who harbor the same germline pathogenic variant might be at similar risk for developing ls - related breast cancers . 
the magnitude of risk for developing a dmmr breast cancer in a member of an ls family that has 1 or more members who have also developed breast cancer would be known only after mmr assays of breast cancers from several members of that family . 
this could be quite difcult , given that breast cancer tissue from family members from past generations might not be available . counseling patients in ls families , it remains crucial to recognize that personal and family history of breast cancer remain the standard basis for predicting risk of developing breast cancer , regardless of whether an ls family member is found to have a dmmror a procient mmr - expressing breast cancer . 
the possibility of ascertainment bias would prevent concluding that the same lost mmr expression in breast cancers from 1 ls family would imply the same likelihood of patients developing dmmr breast cancers in another ls family . as win et al3 noted , individual tumor testing results suggest that mmr deciency is involved with breast cancers in some individuals with lynch syndrome . future studies might include assays to detect mmr expression in patients with ls and breast cancer . 
stoll j , rosenthal e , cummings s , et al : no evidence of increased risk of breast cancer in women with lynch syndrome identied by multigene panel testing . 
katz , md1 ; huamin wang , md , phd1 ; xuemei wang , ms1 ; laura prakash , md1 ; milind javle , md1 ; rachna shroff , md1 ; david fogelman , md1 ; santiago avila1 ; mohamed zaid , md1 ; dalia elganainy , md1 ; yeonju lee , phd1 ; christopher h . 
we prospectively evaluated uorouracil , leucovorin , irinotecan , and oxaliplatin ( folfirinox ) - based treatment and imaging - based biomarkers for borderline resectable pdac . methods eligible patients had treatment - nave , histology - conrmed pdac and one or more high - risk features : mesenteric vessel involvement , ca 19 - 9 level of 500 mg / dl or greater , and indeterminate metastatic lesions . patients received modied folfirinox and chemoradiation before anticipated pancreatectomy . 
overall survival ( os ) and progression - free survival ( pfs ) were estimated using the kaplan - meier method , and subgroups were compared using log - rank tests . results thirty - three patients initiated therapy ; 45% underwent pancreatectomy . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction personalized treatment of patients with pancreatic ductal adenocarcinoma ( pdac ) has been limited by the dearth of validated biomarkers . 
ca 19 - 9 is the only food and drug administrationapproved prognostic biomarker for pdac , but it is limited to patients with sialyl lewis apositive genotype . terpretation of this test can be unreliable.1 , 2 smad4 , 3 which transduces intracellular signaling of transforming growth factor beta , and human equilibrative nucleoside transporter 14 - 6 may be useful in guiding therapy , 3 , 7 but prospective validation has been unsuccessful , highlighting the need for robust biomarker integration for pdac clinical trials . 
in a disease that is at - large systemically disseminated on presentation with a propensity for early progression while receiving therapy , there is an unmet need for predictive bioindicate benet from local markers for pdac that therapies , such as radiation and surgical resection . we have identied ct - based biomarkers using morphologic characteristics of pdac , 8 , 9 each identifying distinct prognostic groups . 
 koay et al context key objective few effective therapies exist for pancreatic ductal adenocarcinoma ( pdac ) , and no biomarkers have been validated to personalize therapy for this aggressive disease . 
we report a phase ii clinical trial of preoperative modied uorouracil , leucovorin , irinotecan , and oxaliplatin ( folfirinox ) and chemoradiation for patients with high - risk borderline resectable pdac , powered to differentiate a resection rate of 60% from 40% . 
we examined whether computed tomography ( ct ) based biomarkers had prognostic association with outcomes . knowledge generated the preoperative regimen achieved a resection rate of 45% , lower than the desired 60% rate in this high - risk population . notably , both a baseline ct - based biomarker ( delta classication ) and post - treatment ct - based biomarker ( interface response ) were associated with outcomes . 
in retrospect , the trial was enriched for patients with poor prognostic biomarkers . relevance the validation of biomarkers derived from standard - of - care ct imaging supports the development of trials that use these as integral biomarkers and may lead to personalized therapeutic management for localized pdac . a standard - of - care pancreas protocol ct , high - delta pdac tumors exhibit an abrupt changeor deltain hounseld units ( hu ) between the visualized tumor and normal pancreas , and low - delta pdac tumors do not exhibit such a change . 
after preoperative therapy , tumors with a type i response remain or become well dened at the interface of tumor and parenchyma , whereas those with a type ii response become less dened at the interface . 
notably , these ct - based biomarkers associate with pathologic features of pdac , such as the extent of stromal reaction10 and pathologic response to therapy.9 we prospectively evaluated clinical associations of ctbased delta scores and ct - interface responses in patients who received preoperative modied uorouracil , leucovorin , irinotecan , and oxaliplatin ( folfirinox ) and gemcitabine - based chemoradiation for localized cancers at high risk for early metastatic progression . 
the primary objective was rate of resection after preoperative therapy , and secondary objectives included toxicity rates , overall survival ( os ) , progression - free survival , and correlation with the imaging - based biomarkers . artery ( sma ) , celiac artery measuring less than or equal to 180 of the arterys circumference , and / or the common hepatic artery11 ; a serum ca 19 - 9 level of 500 mg / dl or more in the presence of a bilirubin level of 2.0 mg / dl or less ; and radiographic ndings consistent with malignant peripancreatic lymphadenopathy outside the planned radiation or surgical eld or liver or peritoneal lesions concerning but not diagnostic for metastatic disease . patients were also required to have an eastern cooperative oncology group performance status ( ps ) of 0 to 1 , 12 an absolute neutrophil count of more than 1 , 500 cells / mm3 , a platelet count of at least 100 , 000 cells / mm3 , a serum creatinine level less than 2 mg / dl , a serum bilirubin level less than 2 mg / dl , and hepatic transaminases less than ve times the upper limits of normal . 
the severity of each patients comorbidity prole was graded as 0 ( none ) , 1 ( mild ) , 2 ( moderate ) , or 3 ( severe ) .13 methods patient eligibility and disease staging the institutional review board at the university of texas md anderson cancer center approved the study protocol ( clinicaltrials.gov identier : nct01560949 ) , and all patients provided written informed consent . 
modied folfirinox , consisting of oxaliplatin ( 75 mg / m2 ) delivered over a 2 - hour period , followed by irinotecan ( 150 mg / m2 ) given over a 90 - minute period , and a continuous infusion of uorouracil ( 2 , 000 mg / m2 ) over 46 hours , was administered once every two weeks for a total of six doses . 
3d conformal radiation therapy at a total dose of 50.4 gy in 28 fractions ( 1.8 gy / fraction ) to the gross tumor volume plus the 1.5to 2 - cm margin was prescribed . 
in addition , delayed gastric emptying and postpancreatectomy hemorrhage were graded using international study group of pancreatic surgery denitions.26 , 27 follow - up patients were evaluated every 4 months after treatment . 
secondary clinical end points included r0 resection rate , toxicity rates , progression - free survival ( date of diagnosis to date of disease progression , date of recurrence , or date of death , whichever came rst ) , os ( date of diagnosis to date of death ) , and patterns of local and distant failure . 
additional assessments included associations of the delta classication and interface response with survival . patient demographic and clinical characteristics were summarized using median ( range ) for continuous variables and frequency ( percentage ) for categorical variables . 
the associations between binary variables , such as between delta classication or margin status and interface response , were presented using bar plots and were assessed for signicance using fishers exact test . 
the probabilities of patients registered ( n = 34 ) initiated mfolfirinox ( n = 33 ) initiated chemoradiation ( n = 23 ) underwent pancreatectomy ( n = 15 ) withdrew consent ( n = 1 ) did not complete mfolfirinox ( n = 6 ) ps / toxicity ( n = 4 ) comorbidities ( n = 1 ) progression of disease with metastasis ( n = 1 ) completed mfolfirinox local progression of disease ( n = 2 ) progression of disease with metastasis ( n = 2 ) progression of disease with metastasis ( n = 5 ) local progression of disease ( n = 1 ) resection aborted due to local tumor ( n = 1 ) factors comorbidities ( n = 1 ) os and pfs were estimated using the kaplan - meier method.28 log - rank tests were used to assess the differences in os or pfs between subgroups of patients dened by delta classication , interface response , and clinical factors ( jmp , sas institute , cary , nc )  . 
adverse events were summarized by frequency , grade , and event type . the simons optimum two - stage design was applied for this study.29 a sample size of 33 was chosen to differentiate between a good resectability rate of 60% and a poor resectability rate of 40% with 80% power and at a signicance level of .10. 
by the end of the trial , lack of efcacy would have been claimed if 16 or fewer patients underwent surgery among the total 33 enrolled patients . results patients from august 2012 through november 2015 , 34 patients enrolled in the study . 
the tumor of 14 patients ( 42% ) had a radiographic interface with the smvportal vein , and that of 16 ( 48% ) had a radiographic interface between both the smvportal vein as well as with the sma , celiac trunk , or the hepatic artery . 
major adverse events within 90 days of pancreatectomy occurred in three of the 15 patients ( 20% ) who underwent pancreatectomy , including postpancreatectomy hemorrhage of grade b ( n = 1 ) and grade c ( n = 1 ) and delayed gastric emptying of grade c ( n = 2 )  . 
there was no perioperative ( 90 - day ) mortality ( appendix table a1 )  . pathologic response and margins the histopathologic characteristics of the 15 resected tumors are listed in table 3 . 
ten of the 15 specimens ( 67% ) had negative ( r0 ) surgical margins ; cancer cells were identied at or within 1 mm of the sma margin in four patients and at the sma and bile duct margin in one patient . 
 ( a ) percent change in size of target lesions for each patient who nished therapy on protocol and had images available for re - review at restaging on the basis of recist ( response evaluation criteria in solid tumor ; n = 23 )  . 
within the progressive disease ( pd ) group , one patient had local progression ( light blue ) , three had distant progression ( gray ) , and two had local and distant progression ( light red )  . 
 ( c ) delta classication from computed tomography ( ct ) scans , showing a low - delta tumor without a distinct interface ( arrow , left ) and a high - delta tumor with a distinct interface ( arrow , right )  . 
events are based on common terminology criteria for adverse events , version 4 . * elevated levels of alkaline phosphatase , alt , and / or ast . imaging biomarkers ct - based delta classication . 
patients who exhibited a type ii interface response were less likely to undergo pancreatectomy compared with those who exhibited a type i response ( figs 2b and 2d ; appendix fig a2 )  . 
the interface response is a visual classication whereby type i responses demonstrate that the tumor / pancreas interface remains or becomes well dened , whereas type ii responses show the interface becomes less dened after treatment.9 arterial and portal venous phases of the pancreas protocol ct scan are used for the delta and interface response metrics . 
surgical and pathologic findings in patients who underwent resection at mdacc ( n = 15 ) discussion variable procedure performed pancreatoduodenectomy distal pancreatectomy vascular resection and reconstruction * isolated venous isolated arterial combined venous and arterial surgical margins tumor differentiation moderate poor % viable tumor no . 
first , the ndings align with our previous observation that preoperative treatment sequencing accurately discriminates patients who are likely to achieve a survival benet from surgery from those who are not.19 the median os of the 15 resected patients was 42.1 months , and ve patients remain alive without evidence of disease recurrence . 
the median os of all patients , 24 months , was similar to that of the alliance for clinical trials in oncology ( clinicaltrials.gov identier : ( alliance ) a021101 trial nct01821612 ) .30 from the baseline and operative characteristics ( median ca 19 - 9 of 200 and 500 in eight patients ; 48% of patients with both arterial and venous involvement , and a high rate of vein resections / reconstructions [ 14 of 15 resected tumors ] ) , it is clear that this study population was enriched with cancers that were both anatomically and biologically advanced . 
indeed , we have previously found baseline ca 19 - 9 values to not be predictive of outcomes , 2 but have observed that changes in ca 19 - 9 are meaningful.32 the scientic basis of the delta classication and interface response seems to involve associations with degrees of stromal inltrate , biologic drivers of the disease , and pathologic response to therapy.8 , 9 a combined approach of baseline and post - treatment imagingbased biomarkers using an early interim look may help inform preoperative and adaptive treatment recommendations for medical and surgical therapies , given both the demonthe imaging - based biostrated prognostic associations of markers and the association of margin status with interface response . 
 ct - based biomarkers in a borderline resectable pancreatic cancer although positron emission tomographyct has shown promise as a diagnostic and prognostic marker for pdac , it has inherent limitations with false negatives ( eg , cold tumors ) , false positives ( eg , pancreatitis ) , and higher cost.33 - 37 our trial used aggressive cytotoxic regimens to treat patients with unfavorable clinical features without regard to the ct - based biomarkers . 
the data show that this unselected approach to therapy leads to rates of radiographic partial response ( 23% ) within the range of previous studies ( 12% to 44% ) .31 , 38 although this approach had reasonable toxicity rates ( table 2 ) , the rates of resection were not as favorable as anticipated . 
the alliance protocol a021501 ( clinicaltrials.gov identier : nct02839343 ) will evaluate the role of hypofractionated radiotherapy in the preoperative setting for patients with borderline resectable disease who were selected only by failure to experience progression on induction chemotherapy.39 however , treatment intensication or selection for new combination systemic agents may be most appropriately considered in patients with high - delta tumors or type ii responses . 
we gratefully acknowledge support from the andrew sabin family fellowship , the sheikh ahmed center for pancreatic cancer research , institutional funds from the university of texas md anderson cancer center , the khalifa foundation , equipment support by ge healthcare and the center of advanced biomedical imaging , philips healthcare , and cancer center support ( core ) grant no . 
was supported by national institutes of health ( u54ca210181 - 01 , u54ca143837 , 1u01ca196403 - 01 , 1u01ca200468 - 01 , 1u01ca214263 - 01a1 , 1r01ca218004 - 01 , and 1r01ca221971 - 01a1 ) , the pancreatic cancer action network ( 1420 - 25 - koay ) , and the radiological society of north america ( rsd1429 )  . our study also highlights the need for rational and relevant end points for a borderline resectable trial that can be translated to a target population . 
in planning prospective trials for borderline resectable pancreas cancer , our results indicate that resection rate alone as a primary end point is inadequate . until additional validation of our response readout available , os and disease - free survival need to be considered as coprimary or primary end points . in conclusion , our data suggest that modied folfirinox followed by chemoradiation has a resection rate similar to historical controls for patients with borderline the novel resectable pdac . 
katz , huamin wang , laura prakash , milind javle , rachna shroff , david fogelman , santiago avila , mohamed zaid , dalia elganainy , yeonju lee , prajnan das , eric p . 
katz consulting or advisory role : alcresta therapeutics , abbvie milind javle other relationship : rafael pharmaceuticals , incyte , pieris pharmaceuticals , merck , merck serono , novartis , seattle genetics , beigene , qed therapeutics , bayer rachna shroff consulting or advisory role : halozyme , seattle genetics , exelixis , merck , qed therapeutics , debio pharma research funding : lilly , celgene , agios , halozyme , pieris pharmaceuticals , taiho pharmaceutical david fogelman stock and other ownership interests : gtx consulting or advisory role : incyte dalia elganainy employment : agios ( i ) stock and other ownership interests : agios ( i ) christopher h . 
crane honoraria : celgene sunil krishnan research funding : celgene ( inst ) , elekta ( inst ) patents , royalties , other intellectual property : receive royalties from a nanotechnology book i co - edited for taylor and francis , md anderson invention disclosures led to patent lings on a number of topics related to nanoparticles and / or minibeam radiation ; some are licensed out or options to license given out travel , accommodations , expenses : tae life sciences prajnan das consulting or advisory role : adlai nortye jason b . 
pct / us2016 / 065763 , entitled polymeric drug delivery systems for treatment of disease , by chun li et al ; in the name of board of regents , the university of texas system eric p . 
lee honoraria : boston scientic , olympus consulting or advisory role : boston scientic research funding : olympus travel , accommodations , expenses : boston scientic anirban maitra honoraria : celgene patents , royalties , other intellectual property : royalties from hangzhou guangkeande ( cosmos ) biotechnology company for blood - based biomarkers of early pancreatic cancer ; i do not own stocks in the company nor do i have any research or grant funding from them robert a . 
varadhachary employment : fannin partners ( i ) leadership : fannin partners ( i ) , pulmotect ( i ) , stock and other ownership interests : fannin partners ( i ) honoraria : celgene , merrimack , rexahn pharmaceuticals , momenta pharmaceuticals , sobi , armo biosciences consulting or advisory role : celgene , merrimack , rexahn pharmaceuticals , momenta pharmaceuticals research funding : merck ( inst ) , pici patents , royalties , other intellectual property : spouse is inventor on a pending patent for an elisa diagnostic platform not related to my area of research ( i ) , spouse is an inventor of use patents relating to recombinant human lactoferrin ; the molecule is no longer in development and patents are being allowed to lapse as payments come due ( i ) travel , accommodations , expenses : celgene , merrimack , sobi no other potential conicts of interest were reported . acknowledgment the authors thank michiko iwasaki for research nurse support and data retrieval . references passerini r , cassatella mc , boveri s , et al : the pitfalls of ca19 - 9 : routine testing and comparison of two automated immunoassays in a reference oncology center . 
am j clin pathol 138 : 281 - 287 , 2012 katz mh , varadhachary gr , fleming jb , et al : serum ca 19 - 9 as a marker of resectability and survival in patients with potentially resectable pancreatic cancer treated with neoadjuvant chemoradiation . 
ann surg oncol 17 : 1794 - 1801 , 2010 iacobuzio - donahue ca , fu b , yachida s , et al : dpc4 gene status of the primary carcinoma correlates with patterns of failure in patients with pancreatic cancer . j clin oncol 27 : 1806 - 1813 , 2009 elebro j , ben dror l , heby m , et al : prognostic effect of hent1 , dck and hur expression by morphological type in periampullary adenocarcinoma , including pancreatic cancer . 
acta oncol 55 : 286 - 296 , 2016 greenhalf w , ghaneh p , neoptolemos jp , et al : pancreatic cancer hent1 expression and survival from gemcitabine in patients from the espac - 3 trial . 
world j gastroenterol 20 : 8482 - 8490 , 2014 loehrer sr pjl , powell me , cardenes hr , et al : a randomized phase iii study of gemcitabine in combination with radiation therapy versus gemcitabine alone in patients with localized , unresectable pancreatic cancer : e4201 . 
j clin oncol 26 : 4506 , 2008 ( 15 suppl ; abstr ) koay ej , lee y , cristini v , et al : a visually apparent and quantiable ct imaging feature identies biophysical subtypes of pancreatic ductal adenocarcinoma . clin cancer res 24 : 5883 - 5894 , 2018 amer am , zaid m , chaudhury b , et al : imaging - based biomarkers : changes in the tumor interface of pancreatic ductal adenocarcinoma on computed tomography scans indicate response to cytotoxic therapy . 
blazer m , wu c , goldberg rm , et al : neoadjuvant modied ( m ) folfirinox for locally advanced unresectable ( lapc ) and borderline resectable ( brpc ) adenocarcinoma of the pancreas . 
allen pj , kuk d , castillo cf , et al : multi - institutional validation study of the american joint commission on cancer ( 8th edition ) changes for t and n staging in patients with pancreatic adenocarcinoma . 
wente mn , bassi c , dervenis c , et al : delayed gastric emptying ( dge ) after pancreatic surgery : a suggested denition by the international study group of pancreatic surgery ( isgps )  . 
katz mh , shi q , ahmad sa , et al : preoperative modied folfirinox treatment followed by capecitabine - based chemoradiation for borderline resectable pancreatic cancer : alliance for clinical trials in oncology trial a021101 . 
murphy je , wo jy , ryan dp , et al : total neoadjuvant therapy with folfirinox followed by individualized chemoradiotherapy for borderline resectable pancreatic adenocarcinoma : a phase 2 clinical trial . 
tzeng cw , balachandran a , ahmad m , et al : serum carbohydrate antigen 19 - 9 represents a marker of response to neoadjuvant therapy in patients with borderline resectable pancreatic cancer . 
asagi a , ohta k , nasu j , et al : utility of contrast - enhanced fdg - pet / ct in the clinical management of pancreatic cancer : impact on diagnosis , staging , evaluation of treatment response , and detection of recurrence . 
katz mh , fleming jb , bhosale p , et al : response of borderline resectable pancreatic cancer to neoadjuvant therapy is not reected by radiographic indicators . j roentgenol 174 : 1005 - 1008 , 2000 cancer 118 : 5749 - 5756 , 2012 39 . 
 appendix koay et al mfolfirinox d ose 1 d ose 2 d ose 3 d ose 4 d ose 5 d ose 6 ebrt 5 weekly gemcitabine d ose 1 d ose 2 d ose 3 d ose 4 d ose 5 surgery weeks systemic phase chemoradiation phase 3 4 5 11 12 13 disease progression or decline in ps protocol fig a1 . 
after initiating mfolfirinox ( uorouracil , leucovorin , irinotecan , oxaliplatin ) , patients would have restaging to assess imaging - based response ( type i v type ii )  . 
patients who show a type ii response would change chemotherapy , while those who have a type i response would continue with mfolifirnox . selective radiation therapy ( rt ) would be delivered or not delivered based on clinical and anatomical factors . 
finally , we characterize the in vitro use of entrectinib against a common alk fusion gatekeeper resistance mutation . methods genomic profiling we performed broad , hybrid capturebased next - generation sequencing using the integrated mutational profiling of actionable cancer targets assay and hisequation 2500 ( illumina , san diego , ca ) .14 alk immunohistochemistry and fluorescence in situ hybridization alk immunohistochemistry was performed using the ventana alk ( d5f3 ) cdx assay ( ventana medical systems , tucson , az )  . 
response , assessed by response evaluation criteria in solid tumors ( recist ) version 1.1 , was monitored by computed tomography ( ct ) imaging performed at baseline , 4 weeks after treatment initiation , and approximately every 8 weeks thereafter.15 treatment was administered until the patient experienced disease progression or unacceptable toxicity . 
permission to publish the patients case was obtained from his health care proxy . generation of engineered ba / f3 cells expressing vcl - alk fusion proteins cdnas that encode vcl - alk and vcl - alk l1196m fusion proteins were inserted into lentiviral vector pvlpuro - ef1a - mcs ( biosettia , san diego , ca )  . 
 transduced ba / f3 cells were selected in puromycin in rpmi 1640 media that contained 10% fetal bovine serum and 10 ng / ml mouse il - 3 ( thermo fisher scientific , waltham , ma ) for 2 weeks . 
 antiproliferative activities of entrectinib and crizotinib in engineered ba / f3 cells results cells seeded at 5 , 000 cells per well in 96 - well plates ( corning , corning , ny ) were treated with serial dilutions of entrectinib ( ignyta , san diego , ca ) and crizotinib ( selleckchem , houston , tx ) added in duplicate to the wells . 
data are averaged from at least two independent experiments and with an r2 of more than 0.9. western blot analysis ten million cells were treated with drug at the indicated concentrations for 2 hours , washed with phosphate - buffered saline , and lysed in 1 radioimmunoprecipitation assay buffer ( emd millipore , billerica , ma ) with benzonase , protease inhibitor , and phosphatase inhibitor cocktails ( emd millipore )  . 
exons 1 to 16 of vcl are fused to exons 20 to 26 of alk , which include the kinase domain . tpm3 eml4 rad51ap2 eml4 22 exons 26 exons exon 1 exon 16 exon 20 - 26 identification of rccs harboring alk fusions a total of 561 samples from 517 patients with rcc were sequenced using broad , hybrid capture based next - generation sequencing ( integrated mutational profiling of actionable cancer targets assay ) .14 , 16 , 17 three ( 0.6% ) of 517 patients with renal cell cancer had tumors that harbored alk fusions . in renal cell cancer , several fusion partners with alk have been previously identified , including vcl , tpm3 , and eml412 , 18 , 19 ( fig 1a )  . 
 the three alk fusions identified in our data set are represented in figure 1b and consist of unclassified - type rcc harboring an vcl - alk fusion developing in a patient with sickle cell trait , chromophobe - type rcc harboring an rad51ap2 - alk fusion , and unclassified - type rcc with an eml4 - alk fusion ( table 1 )  . of these three patients , one was eligible for therapy with a targeted agent against alk , as described below . 
he underwent a right radical nephrectomy , revealing a stage iii ( pt3an1 ) high - grade ( nuclear grade iv ) rcc with rhabdoid and pleomorphic features and nine of 23 positive lymph nodes . 
ct scan 2 months later demonstrated no evidence of recurrence . next - generation sequencing of the primary tumor identified a vcl - alk translocation generating a fusion between exon 16 of vcl and exon 20 of alk ( fig 1c )  . 
clinicopathologic characteristics and therapeutic interventions in patients with alk fusionpositive renal cell carcinoma case age at diagnosis , years pathology fusion additional alterations systemic therapy received vcl - alk none entrectinib renal cell carcinoma , unclassified type renal cell carcinoma , chromophobe type alkrad51ap2 * tert promoter variant pazopanib malt1 h257r nivolumab + bevacizumab nivolumab + lenvatinib lenvatinib + everolimus eml4 - alk tp53 d257y everolimus + bevacizumab renal cell carcinoma , unclassified type erbb4 t194s mst1r f803s dot1l g1358v nivolumab cabozantib abbreviations : alk , anaplastic lymphoma kinase ; f , female ; m ; male . * next - generation sequencing performed on the sarcomatous component of a metastasis . tumor and the age of the patient , hemoglobin electrophoresis was performed , discovering the presence of sickle cell trait.20 entrectinib demonstrates superior in vitro target inhibition and antitumor effect compared with crizotinib twelve months after nephrectomy , surveillance imaging demonstrated confluent lymphadenopathy in the right retrocrural space and an enlarged left superior mediastinal metastatic node . 
given evidence that suggested activity of entrectinib , a multikinase inhibitor of alk , ros1 , trka , trkb , and trkc , against common alk resistance mutations and improved cns penetration compared with crizotinib , the patient enrolled in a clinical trial of entrectinib ( rxdx - 101 ; clinicaltrials.gov identifier : nct02097810 ) .21 four weeks after commencing entrectinib 600 mg per day , a ct scan demonstrated a 31.4% decrease in disease , or partial response . 
the patient maintained excellent performance status throughout treatment , tolerating entrectinib well with the exception of grade 1 peripheral lower extremity edema and grade 2 weight gahis response to entrectinib continued for 19 months , after which imaging revealed radiographic disease progression with increased mediastinal adenopathy , which led to the discontinuation of entrectinib . we interrogated the activity of entrectinib against alk fusions and its common resistance mutations with ba / f3 cell lines that were engineered to overexpress vcl - alk and vclalk l1196m fusion proteins . 
vcl - alk l1196m , a key gatekeeper mutation in the atp binding pocket , confers resistance to crizotinib.22 , 23 for ba / f3 cells that harbor the vcl - alk fusion , entrectinib was more potent than crizotinib ( ic50 = 90.8 nm for entrectinib ; ic50 = 253.0 nm for crizotinib ; fig 3a )  . 
entrectinib and crizotinib both resulted in reduced levels of phospho - alk and the inhibition of phosphorylation of downstream targets , phospholipase c , extracellular signalregulated kinase , and signal transducer and activator of transcription 3 ( fig 3b )  . 
the introduction of the alk l1196m mutation to the vcl - alk construct ( ba / f3 - vcl - alk l1196m ) conferred significantly reduced sensitivity to crizotinib , with an ic50 proliferation value of more than 1 , 000 nm ; however , ba / f3 - vcl - alk l1196m cells remained sensitive to entrectinib , with an ic50 value that was only modestly increased compared with that of ba / f3 - vcl - alk cells ( ic50 = 111.9 nm ; fig 3a )  . 
recently , response to alectinib was reported in patients with metastatic papillary rcc.24 the case detailed here of durable clinical response in a patient with rccadditionally underscores the utility of alk inhibition in alk - rearranged rcc and the potential of targeting alk fusions irrespective of histology . large - scale sequencing efforts have increased the ability to identify alk fusions . 
given their low frequency , single analyte testing for specific alterations is impractical ; however , the increasingly widespread availability of multiplexed next - generation sequencing enhances the feasibility of detecting targetable fusions . 
screening efforts can lead to the detection of other actionable mutations in rcc , such as alterations that involve met , pten , bap1 , mtor , pik3ca , and birc7.25 , 26 clinical outcomes for nonclear - cell rcc ( ncrcc ) remain poor compared with clear - cell rcc . 
despite the small proportion of patients with alk fusion positive rcc , failure to screen in this patient would have prevented the delivery of a well tolerated therapy with a durable response that lasted 19 months . 
 ( b ) treatment of wt ba / f3 vcl - alk and resistant baf3 vcl - alk l1196m cell lines with entrectinib leads to more complete inhibition of downstream targets phosphophospholipase c ( plc ) and phosphosignal transducer and activator of transcription 3 ( stat3 ) compared with crizotinib . 
erk , extracellular regulated kinase . observed in other fusions led to the opening of a multicenter , phase ii basket trial of entrectinib ( clinicaltrials.gov identifier : nct02568267 )  . ultimately , as with this patient , resistance to therapy almost inevitably develops . 
 arcila , hikmat al - ahmadie , gary li , alexander drilon roopal patel employment : ignyta provision of study materials or patients : chung - han lee stock and other ownership interests : ignyta collection and assembly of data : jessica j . 
tao no relationship to disclose ge wei stock and other ownership interests : ignyta , halozyme research funding : ignyta patents , royalties , other intellectual property : ignyta , halozyme travel , accommodations , expenses : ignyta patrick fagan employment : ignyta stock and other ownership interests : ignyta travel , accommodations , expenses : ignyta xueli hao no relationship to disclose julia a . 
arcila consulting or advisory role : astrazeneca travel , accommodations , expenses : astrazeneca , invivoscribe , raindance technologies hikmat al - ahmadie consulting or advisory role : bristol - myers squibb , emd serono chung - han lee consulting or advisory role : exelixis , eisai research funding : pfizer ( inst ) , eisai ( inst ) , bristol - myers squibb ( inst ) , calithera biosciences ( inst ) travel , accommodations , expenses : eisai gary li employment : ignyta alexander drilon consulting or advisory role : ignyta , loxo , tp therapeutics , astrazeneca , pfizer , blueprint medicines , genentech , takeda , helsinn therapeutics , beigene affiliations jessica j . 
arcila , hikmat al - ahmadie , chung - han lee , and alexander drilon , memorial sloan kettering cancer center ; alexander drilon , weill cornell medical center , new york , ny ; ge wei , roopal patel , patrick fagan , and gary li , ignyta , san diego , ca ; xueli hao , st louis pathology associates , mercy hospital , st louis , mo ; and julia a . 
cui jj , tran - dub m , shen h , et al : structure based drug design of crizotinib ( pf - 02341066 ) , a potent and selective dual inhibitor of mesenchymal - epithelial transition factor ( c - met ) kinase and anaplastic lymphoma kinase ( alk )  . 
lin e , li l , guan y , et al : exon array profiling detects eml4 - alk fusion in breast , colorectal , and nonsmall - cell lung cancers . 
sugawara e , togashi y , kuroda n , et al : identification of anaplastic lymphoma kinase fusions in renal cancer : large - scale immunohistochemical screening by the intercalated antibody - enhanced polymer method . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
hyman dm , solit db , arcila me , et al : precision medicine at memorial sloan kettering cancer center : clinical next - generation sequencing enabling next - generation targeted therapy trials . 
debelenko lv , raimondi sc , daw n , et al : renal cell carcinoma with novel vcl - alk fusion : new representative of alk - associated tumor spectrumod pathol 24 : 430 - 442 , 2011 19 . 
smith ne , deyrup at , mario - enriquez a , et al : vcl - alk renal cell carcinoma in children with sickle - cell trait : the eighth sickle - cell nephropathy ? am j surg pathol 38 : 858 - 863 , 2014 21 . 
drilon a , siena s , ou si , et al : safety and antitumor activity of the multitargeted pan - trk , ros1 , and alk inhibitor entrectinib : combined results from two phase i trials ( alka - 372 - 001 and startrk - 1 )  . 
lee j - l , ahn j - h , lim hy , et al : multicenter phase ii study of sunitinib in patients with non clear cell renal cell carcinoma . 
tannir nm , jonasch e , albiges l , et al : everolimus versus sunitinib prospective evaluation in metastatic nonclear cell renal cell carcinoma ( espn ) : a randomized multicenter phase 2 trial . 
ardini e , menichincheri m , banfi p , et al : entrectinib , a pan - trk , ros1 , and alk inhibitor with activity in multiple molecularly defined cancer indications . 
robson , md10 purpose screening and prevention decisions for women at increased risk of developing breast cancer depend on genetic and clinical factors to estimate risk and select appropriate interventions . 
a combined risk score ( crs ) of an 86single - nucleotide polymorphism polygenic risk score and the tyrer - cuzick v7.02 clinical risk estimator was generated with attenuation for confounding by family history . 
calibration and discriminatory accuracy of the crs were evaluated in two independent validation cohorts of women of european ancestry ( n = 1 , 615 and n = 518 )  . 
risk stratication with a 20% risk threshold was compared between crs and tyrer - cuzick in an independent clinical cohort ( n = 32 , 576 )  . results simulation studies conrmed that the fixed - stratied method produced accurate risk estimation across patients with different family history . 
in an analysis with both crs and tyrer - cuzick as predictors of breast cancer , crs added signicant discrimination independent of that captured by tyrer - cuzick ( p , 1011 in validation 1 ; p , 107 in validation 2 )  . 
in an independent cohort , 18% of women shifted breast cancer risk categories from their tyrer - cuzickbased risk compared with risk estimates by crs . conclusion integrating clinical and polygenic factors into a risk model offers more effective risk stratication and supports a personalized genomic approach to breast cancer screening and prevention . jco precis oncol 5 : 307 - 316 . 
2021 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction mammography and adjuvant treatment of early - stage disease are widely used for the mitigation of morbidity and mortality in invasive breast cancer.1 effective prevention requires the identication of higher - risk individuals , which has typically involved family history evaluation , assessment of clinical and lifestyle factors , and testing for the presence of pathogenic variants ( pvs ) in a limited number of highor moderate - risk breast cancer genes.2 although guidelines for pv carriers have long recommended enhanced screening and discussion of surgical prevention measures , they do not adequately address the increased risk for unaffected pv - negative women with a strong family history of breast cancer . 
we examined confounding and interaction between an 86 - snp polygenic risk score and each clinical factor in the tyrer - cuzick model to inform the development of a combined clinical and polygenic risk score . 
20% lifetime risk of developing breast cancer . improved risk stratication from the combined risk score can help target the use of more intense screening strategies for unaffected women with truly elevated risk based on clinical and genetic factors . here , we describe the development and validation of a strategy for integrating a snp score with a validated clinical risk model , with attenuation for correlated effects . 
clinical testing cohort data were subdivided according to successive time intervals to ensure the independence of development and validation activities ( fig 1 ) ; this included two development sets ( development 1 and 2 ) , one validation set ( validation 1 ) , and one clinical performance population . 
genetic testing for all patients was performed at a clinical laboratory improvement amendments - approved and college of american pathology - approved laboratory ( myriad genetics , inc . , salt lake city , ut ) by next - generation sequencing.34 hybridization probes for the 86 snps in the prs were included in the sequencing panel.33 women were eligible if they were of age 18 - 84 years , of european ancestry ( ashkenazi and non - ashkenazi ; selfreported ) , and negative for likely pvs or pvs in 11 breast cancerrelated genes ( brca1 , brca2 , tp53 , pten , stk11 , cdh1 , palb2 , chek2 , atm , nbn , and bard1 )  . patients were excluded as unaffected controls if they reported a history of ductal carcinoma in situ , lobular carcinoma in situ , hyperplasia , or unspecied breast disease . women were excluded from the clinical testing cohort if they were submitted from states that disallow the research use of samples after completion of genetic testing . 
all studies were conducted with institutional review board ( irb ) oversight ( quorum review irb #31713 , 32556 , 32608 )  . development 1 included women from the clinical testing cohort unaffected by cancer of any type accrued between june 1 , 2017 and august 11 , 2017 ( n = 5 , 489 )  . 
development 2 ( n = 141 , 160 ) included 112 , 232 women unaffected by breast cancer and 28 , 928 breast cancer cases from the clinical testing cohort accrued between august 11 , 2017 and january 11 , 2019 . 
clinical characteristics of this set were previously reported.33 validation 1 consisted of women in the clinical testing cohort accrued between june 1 , 2017 and august 10 , 2017 ( n = 1 , 615 )  . 
controls included unaffected women submitted for genetic testing because of possible hereditary nonpolyposis colorectal cancer syndrome as these patients have a breast cancer family history consistent with that of general population controls . for validation 2 , a consecutive series of women who presented to four breast cancer screening centers ( elizabeth wende breast care , the breast center of nwa , bethesda health , and cuda women 's health center / cape cod healthcare ) were prospectively recruited between february 6 , 2017 and november 11 , 2017 ( n = 518 )  . 
 integrating clinical and polygenic factors to predict cancer risk cohort description noisulcni noisulcxe accrual findings development n = 5 , 489 clinical testing cohort : 18 - 84 years old , european ancestry , pv - negative , from states that allow research use of data or samples post - testing history of breast cancer , dcis , lcis , hyperplasia , or unspecified breast disease ; eligible for validation set 1 development n = 141 , 160 clinical testing cohort : same inclusion criteria as development set 1 controls could not have a history of dcis , lcis , hyperplasia , or unspecified breast disease validation 1 clinical testing cohort : same basic inclusion or exclusion criteria as development set 2 cases : breast cancer diagnosis , submitted for testing due to suspicion of hboc within 1 year of diagnosis controls : unaffected women submitted for testing due to suspicion of hnpcc prospectively acquired case - control study enrolled from imaging centers cases : pathologically confirmed first diagnosis of invasive breast cancer 12 mos . preenrollment or incident breast cancer 6 mos . 
for each factor , we conducted a simple linear regression with prs as the dependent variable and the clinical factor as the independent variable . from these models , we examined regression coefcients , p values based on f - statistics , and pearson correlation coefcients . design of the combined risk score . 
to calculate absolute risk incorporating tyrer - cuzick and prs , we developed a fixed - stratied method ( data supplement ) to attenuate prs after xing the effects of confounded factors from the tyrer - cuzick model and to constrain risk separately within strata of the confounders . 
absolute risk for a woman in strata k was calculated as follows : ( cid : 3 ) exp { prs + ck } , ( cid : 1 ) 1 tc where represents the per - unit log ( or ) of the prs from a multivariable logistic regression model with the effect of breast cancer family history xed . 
the combined risk score ( crs ) was validated in two independent studies ( table 1 ) : a clinical testing set ( validation 1 ; n = 1 , 615 ) and the prospective case - control cohort ( validation 2 ; n = 518 )  . 
exploratory analyses tested calibration of the crs by comparing average absolute risk estimates with those from the tyrer - cuzick model ; with proper calibration , we expected to observe the same average risk for tyrer - cuzick as for the crs among unaffected controls . 
these analyses were conducted separately for rlr and 5 - year risk of developing breast cancer . weighted logistic regression was used with weights for unaffected controls calculated such that average tyrercuzick rlr matched general population rates ( data supplement )  . performance of the 86 - snp prs . 
in contrast , for women with family history , the fixed - stratied method matched risks from multivariable co - estimation , whereas the unadjusted model overestimated risk ( data supplement )  . clinical validations validation 1 included 988 ( 61% ) breast cancer cases and 627 ( 39% ) unaffected controls ( table 1 )  . 
overall , 362 ( 22% ) patients reported breast cancer in 1 rst - degree relative and 620 ( 38% ) in 1 second - degree relative . the prospectively collected case - control validation 2 included 256 ( 49% ) breast cancer cases and 262 ( 51% ) unaffected controls ( table 1 )  . 
nearly one third ( 29% ) of patients reported breast cancer in a rst - degree relative and 204 ( 39% ) patients in 1 second - degree relative . in both validations , rlr and 5 - year risk estimates of the crs and tyrer - cuzick were signicantly associated with breast cancer ( table 2 and fig 2 )  . 
in a model with both risk predictors , the crs added signicant discrimination independent of that captured by tyrer - cuzick for both rlr ( p , 1011 in validation 1 ; p , 107 in validation 2 ) and 5year risk ( p , 1011 in validation 1 ; p , 107 in validation 2 ; data supplement )  . although all patients in validation 2 provided complete tyrer - cuzick risk factor questionnaires , data on tyrercuzick variables were incomplete for some patients in validation 1 . 
analyses were repeated in the subset of patients with complete information for all tyrer - cuzick risk factors with results similar to those from the full data set ( data supplement )  . calibration of the crs was examined by comparing average risk estimates with those from tyrer - cuzick in the control samples from either validation . 
 hughes et al average remaining lifetime risk in validation cohort 1 ( n = 627 ) average 5 - year risk in validation cohort 1 ( n = 627 ) crs mean ( 95% ci ) tyrercuzick mean ( 95% ci ) fig 3 . 
estimates for average remaining lifetime ( a and c ) and 5 - year risk ( b and d ) for unaffected controls in validation 1 ( a - b ) and validation 2 ( c - d )  . patients were grouped into 5 - year age bins , and average risks were evaluated according to crs and tyrer - cuzick . 95% cis are also reported . 
addition of the 86 - snp score signicantly improved discrimination relative to the tyrer - cuzick model for predicting risk of breast cancer , and the crs model showed excellent calibration across age groups . 
20% - 25% lifetime risk of breast cancer to improve early - stage cancer detection.36 in a clinical testing population , we showed that for 33% of pv - negative women , the crs - estimated risk was  . 
distribution of crs risk estimates in unaffected women . remaining lifetime risk ( rlr ) in a population of unaffected women ( clinical performance population , n = 32 , 576 ; excluded women with ductal carcinoma in situ , lobular carcinoma in situ , hyperplasia , or unspecied breast disease ) according to crs with thresholds at 20% ( increased ) and 50% ( high ) rlr . 
blue squares indicate patients with discordance between the scores ( eg , the tyrer - cuzick model produced a score that indicated a patient had low rlr , but the same patient was determined to have increased rlr by the crs model )  . 
crs , combined risk score . performance integrating clinical and polygenic factors to predict cancer risk increased risk ( crs > 20% ) 33.1% high risk ( crs > 50% ) 0.6% 3000 2500 2000 1500 1000 rlr according to crs ( % ) remaining lifetime risk according to tyrer - cuzick ( 20% ) increased ( > 20% ) crs total 56.5% n = 18 , 398 10.5% n = 3 , 407 67.0% n = 21 , 805 8.0% n = 2 , 619 25.0% n = 8 , 152 33.0% n = 10 , 771 64.5% n = 21 , 017 35.5% n = 11 , 559 100% n = 32 , 576 tyrer - cuzick or crs - based risk discordant increased risk ( > 20% ) high risk ( > 50% ) tyrer - cuzick remaining lifetime risk ( % ) women ( 8% ) who would have been categorized as , 20% using tyrer - cuzickestimated risk alone . combinations of prss with the tyrer - cuzick model have been presented previously , generally without attenuation for confounding . 
an 88 - snp score was added to tyrercuzick under the assumption of independence in a nested case - control study.25 although the snp score added disit had poor crimination to the tyrer - cuzick prediction , calibration . 
first , previous examinations of confounding often evaluated the correlations between a prs and a model rather than individual clinical factors.21 , 25 , 26 this approach might have obscured associations between prs and clinical factors with a stronger genetic component . 
second , genetic overlap is more likely with a larger prs since casting a wider for cancer - associated snps is more likely to also the genetic component of capture a larger fraction of clinical risk . 
1 rst - degree relative or those with second - degree relatives . limitations of the present study include a potential ascertainment bias in the clinical testing population cohorts . qualication for genetic testing is often based on family history . 
it has been previously shown that this potential bias can be avoided by accounting for family history in a multivariable model.38 , 39 consequently , all analyses presented here were conducted by multivariable co - estimation . 
in support of this approach , results from validation 1 ( clinical testing samples ) were similar to results obtained in validation 2 , for which participants were prospectively collected and were unaffected by potential bias due to selection for hereditary cancer testing . 
evaluation of the crs in a prospectively collected , unselected populationbased sample is being pursued.40 future work to expand assessment tools such as tyrer - cuzick and the prs for non - european ancestries will be required to apply these tools to other ancestry populations . 
however , subanalysis of validation 1 in women with complete tyrer - cuzick information showed similar results as the whole data set , indicating that missing information did not substantially affect the analysis . this crs model is suitable for reporting age - specic risk of developing breast cancer for unaffected women of european descent with or without signicant family history . 
it is currently the only tyrer - cuzickbased model fully adjusted for the shared risk between snps and family history and is therefore less likely to overestimate risk in women with a family history of breast cancer . 
lanchbury data analysis and interpretation : elisha hughes , placede tshiaba , susanne wagner , eric rosenthal , benjamin roa , shannon gallagher , stephanie meek , wade hedegard , susan m . 
 integrating clinical and polygenic factors to predict cancer risk benjamin roa employment : myriad genetics leadership : myriad genetics stock and other ownership interests : myriad genetics research funding : myriad genetics patents , royalties , other intellectual property : intellectual property held by employer myriad genetics travel , accommodations , expenses : myriad genetics shannon gallagher employment : myriad genetics stock and other ownership interests : myriad genetics stephanie meek employment : myriad genetics stock and other ownership interests : myriad genetics kathryn dalton speakers bureau : myriad genetics danna f . 
domchek honoraria : astrazeneca , clovis oncology , bristol - myers squibb research funding : astrazeneca , clovis oncology judy garber consulting or advisory role : novartis , gtx , helix biopharma , konica minolta , aleta biotherapeutics , h3 biomedicine , kronos bio research funding : novartis , ambry genetics , invitae , myriad genetics other relationship : susan g . 
lanchbury employment : myriad genetics leadership : myriad genetics stock and other ownership interests : myriad genetics patents , royalties , other intellectual property : i am an inventor on multiple patents led by myriad genetics travel , accommodations , expenses : myriad genetics alexander gutin employment : myriad genetics stock and other ownership interests : myriad genetics , gilead sciences mark e . 
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br j cancer 98 : 1457 - 1466 , 2008 costantino jp , gail mh , pee d , et al : validation studies for models projecting the risk of invasive and total breast cancer incidence . 
taylor a , brady af , frayling im , et al : consensus for genes to be included on cancer panel tests offered by uk genetics services : guidelines of the uk cancer 11 . 
mccarthy am , armstrong k , handorf e , et al : incremental impact of breast cancer snp panel on risk classication in a screening population of white and african american women . 
zhang x , rice m , tworoger ss , et al : addition of a polygenic risk score , mammographic density , and endogenous hormones to existing breast cancer risk prediction models : a nested casecontrol study . 
lakeman imm , hilbers fs , rodrguez - girondo m , et al : addition of a 161 - snp polygenic risk score to family history - based risk prediction : impact on clinical management in non - brca1 / 2 breast cancer families . 
dite gs , macinnis rj , bickerstaffe a , et al : breast cancer risk prediction using clinical models and 77 independent risk - associated snps for women aged under 50 years : australian breast cancer family registry . 
cuzick j , brentnall ar , segal c , et al : impact of a panel of 88 single nucleotide polymorphisms on the risk of breast cancer in high - risk women : results from two randomized tamoxifen prevention trials . 
j clin oncol 35 : 743 - 750 , 2016 van veen em , brentnall ar , byers h , et al : use of single - nucleotide polymorphisms and mammographic density plus classic risk factors for breast cancer risk prediction . 
brentnall ar , van veen em , harkness ef , et al : a casecontrol evaluation of 143 single nucleotide polymorphisms for breast cancer risk stratication with classical factors and mammographic density . 
evans dg , brentnall a , byers h , et al : the impact of a panel of 18 snps on breast cancer risk in women attending a uk familial screening clinic : a casecontrol study . 
jco precis oncol 4 : 585 - 592 , 2020 judkins t , leclair b , bowles k , et al : development and analytical validation of a 25 - gene next generation sequencing panel that includes the brca1 and brca2 genes to assess hereditary cancer risk . 
carbone , md , phd4 immunotherapy , the advent of including immune checkpoint inhibitors ( icis ) , has contributed to improved survival in patients with lung cancer.1 three antiprogrammed death - 1 ( pd - 1 ) / programmed death ligand 1 ( pd - l1 ) therapies , nivolumab , pembrolizumab , and atezolizumab , are approved by the us food and drug administration ( fda ) for the treatment of patients with advanced nonsmall - cell lung cancer ( nsclc ) in the rst - line setting.2 - 4 despite the improvements in survival seen with icis , there remains a subset of patients who do not benet from ici monotherapy or combination treatments.5 as a result , many research efforts have been initiated to identify biomarkers predictive of response to treatment with icis . 
predictive biomarkers that have entered clinical practice include pd - l1 expression on tumor and immune cells , and tumor mutational burden.2 - 4 , 6 , 7 however , the accuracy of predictions of patient response based on current biomarkers leaves ample room for improvement . 
successful attempts will likely rely on integrated models combining multiple features . the scientic to tackle such complex challenges , community has become increasingly interested in the possibilities offered by data sharing and crowdsourcing as a framework for mobilizing groups of people with shared interests around the world and bringing forward augmented solutions to the existing problems while encouraging collaboration . 
in this context , data sharing can be executed according to two paradigms.8 in the data - to - modeler paradigm , both training and validation data sets are provided to participants . 
the model - to - data paradigm , where participants submit containerized models to organizers , allows validation data sets to remain hidden from participants , which both protects condential data and helps ensure unbiased assessment of predictions . 
these approaches are instrumental broadening our understanding of tumor biology and the tumor microenvironment , which will ultimately drive precision medicine with immunotherapy . to enable such collaboration among scientists and physicians , dialogue on reverse engineering and assessment methods ( dream ) challenges engage experts from around the world and offer novel solutions to biological problems.9 since the rst dream challenges were initiated in 2006 , more than 60 have completed , with analysis results generated through virtual collaborative interactions.10 in oncology , crowdsourcing approaches have been used in multiple tumor types since 2012 for augmenting the work of teams generating data , developing protocols for randomized controlled trials , developing prognostic models , and assessing healthcare behaviors on a large scale.11 more specically , three key dream challenges in oncology have demonstrated the value of crowdsourcing for the development of prognosis models or the renement of diagnostic methods . 
 vincent et al following on from these successful dream challenges , experts in lung cancer have shown interest in accessing primary clinical trial data to explore alternative biomarkers to pd - l1 expression and tumor mutational burden . 
the antipd - 1 response prediction challenge will be the rst dream challenge focused on the prediction of response to immuno - oncology therapies using data from a large phase iii clinical trial , with the goal of addressing the need for additional predictive biomarkers to use with icis in patients with nsclc . 
moreover , this initiative will also enhance the understanding of tumor biology and provide further insights into the potential mechanisms underpinning treatment resistance to icis . challenge objectives the antipd - 1 response prediction challenge will focus on developing models that are predictive of benet from ici treatment , specically nivolumab monotherapy , using patient samples obtained during the conduct of a clinical trial . clinical trial data related to nivolumab , including data related to pd - l1 expression , tumor mutational burden , and tumor transcriptomics , will be used to assess the performance of predictive models submitted in a crowdsourced challenge setting . 
the testing data set will use deidentied data from checkmate 026 ( clinicaltrials.gov identier : nct02041533 ) , a phase iii trial that compared nivolumab with platinum - based chemotherapy in patients with metastatic or recurrent nsclc and pd - l1 tumor expression 5%.15 critically , the data set from checkmate 026 is large , randomized , mature , and well annotated , allowing it to support robust testing of predictive models . 
this challenge also lays the foundation for potential future challenges as the immunooncology landscape is rapidly evolving . the antipd - 1 response prediction challenge aims to assess the accuracy of models for predicting clinical outcomes after nivolumab monotherapy , including efcacy end points such as overall survival , progression - free survival , and best overall response , applying clinical and genomic data from checkmate 026 . 
the challenge will comprise three subchallenges , and their objectives are summarized in figure 1 . challenge design framework has been developed to receive and evaluate dockerized models , which are in silico packages comitself plus necessary software compoprising the model nents to run the model as developed by the participant team . the challenge is open to anyone who will be able to construct models for submission , subject to specications in the challenge rules , including experts and innovators in genomics , computational biology , and translational biomarker development . 
participants can sign up on the synapse website.16 to facilitate data ingestion , participants will have access to a small synthetic data set with the same formatting as the validation data set . 
the full synthetic data set can be downloaded on the synapse website.17 in the rst phase , participants will construct models using any data available to thethe iatlas platform , 18 a database supporting the study of interactions between cancer and the immune microenvironment , hosts harmonized genomics data and associated clinical annotations from published immuno - oncology studies . 
participants can use these as training data sets or to augment other training data already available to them.19 participants could also use professor shirley lius labs tide resources , 20 a computational framework that integrated and modeled data from 189 human cancer studies , comprising 33 , 197 samples.21 more resources to consider are listed on the antipd - 1 response prediction challenge website.16 participants dockerized models will be applied to the checkmate 026 data and evaluated during the competitive phase . 
performance metrics , such as area under the receiver operating characteristic or precision - recall curves and harrells c - index , will be used to assess the predictive performance of participants models in each arm of the checkmate 026 trial . top - performing teams will be invited to participate in the collaborative model - development phase , during which data will be analyzed and interpreted to deepen the understanding of the submitted models . 
eventually , opportunities for additional subchallenges and potential projects will be dened based on the learnings from the current challenge , and teams submitting the best - performing models will have the opportunity to co - author the planned publication in accordance with medical publication standards and best practices . the challenge will rely on a partnership between sage the organization supporting the dream bionetworks , challenge initiatives , and bristol myers squibb and will comprise three phases : competitive , collaborative , and nal . 
the anticipated outcome and impact the identied models are expected to identify potential biomarkers of response to nivolumab monotherapy and may then be applied to predict which patients are more likely to derive clinical benet from icis . 
nsclc , nonsmallcell lung cancer ; os , overall survival ; pd , progressive disease ; pd - 1 , programmed death - 1 ; pfs , progression - free survival . dream challenge challenge question 1 predict response to nivolumab , in terms of pfs , via an immune checkpointspecific model using clinical , demographic , and gene expression data challenge question 2 predict response to nivolumab , in terms of os , via an immune checkpointspecific model using clinical , demographic , and gene expression data challenge question 3 predict which patients will have a best overall response of pd checkmate 026 mature data set from a large randomized phase iii clinical trial in patients with nsclc nivolumab chemotherapy dream challenge impact ( cid : 129 ) provide biomarkers with predictive value for patients via a transparent and collaborative approach ( cid : 129 ) lay the foundation for understanding the utility of biomarkers in the setting of combination therapy patients . 
it is anticipated that some of the models may also have prognostic value for patients with nsclc . phenotype , thereby increasing their responsiveness to ici therapies . the challenge results should also provide a deeper understanding of biological mechanisms resulting in tumor resistance to treatment and assist in understanding why patients with advanced nsclc receiving nivolumab had similar efcacy outcomes to patients receiving chemotherapy in checkmate 026 . 
an improved understanding of the biological mechanisms behind resistance could pave the way to therapeutic interventions that immunologically activate tumors that do not display an immune - inltrated successful completion of this dream challenge will provide a model for future collaborations among industry , academia , and citizen scientists to discover determinants of response or resistance to immunotherapy , facilitating rapid development of clinically actionable biomarkers . 
szustakowski employment : bristol myers squibb stock and other ownership interests : bristol myers squibb research funding : bristol myers squibb patents , royalties , other intellectual property : i am an employee of bristol myers squibb , and an inventor on one or more pending patent applications , including applications for tmb as a predictive biomarker of immunotherapy travel , accommodations , expenses : bristol myers squibb parul doshi employment : bristol myers squibb stock and other ownership interests : bristol myers squibb travel , accommodations , expenses : bristol myers squibb justin guinney consulting or advisory role : astrazeneca research funding : celgene , genentech / roche , bristol myers squibb david p . 
carbone manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors references american cancer society : facts & figures 2020 reports largest one - year drop in cancer mortality . 
sambi m , bagheri l , szewczuk mr : current challenges in cancer immunotherapy : multimodal approaches to improve efcacy and patient response rates . j oncol 2019 : 4508794 , 2019 bristol myers squibb : yervoy ( ipilimumab ) [ package insert ]  . 
nat biotechnol 36 : 391 - 392 , 2018 saez - rodriguez j , costello jc , friend sh , et al : crowdsourcing biomedical research : leveraging communities as innovation engines . 
guinney j , wang t , laajala td , et al : prediction of overall survival for patients with metastatic castration - resistant prostate cancer : development of a prognostic model through a crowdsourced challenge with open clinical trial data . 
carbone dp , reck m , paz - ares l , et al : first - line nivolumab in stage iv or recurrent non - small - cell lung cancer . 
immunity 48 : 812 - 830.e814 , 2018 jiang p , gu s , pan d , et al : signatures of t cell dysfunction and exclusion predict cancer immunotherapy response . 
 acquired met exon 14 alteration drives secondary resistance to epidermal growth factor receptor tyrosine kinase inhibitor in egfr - mutated lung cancer ken suzawa , md , phd1 ; michael ofn , md1 ; adam j . 
in this study , we used targeted ngs with memorial sloan kettering integrated mutation proling of actionable cancer targets ( msk - impact ) , 12 immunohistochemistry , cell - free dna testing , and uorescence in situ hybridization to evaluate acquired resistance mediated by metex14 . 
furthermore , we used in vitro functional studies to establish metex14 as a novel mechanism of acquired resistance to egfr tkis . we used msk - impact , a large - panel ngs assay , to detect mutations , copy - number alterations , and select gene fusions involving up to 468 cancer - associated genes.12 metex14 was introduced into pc9 and h1975 cells as follows . 
briey , full - length metex14 was polymerase chain reaction amplied and subcloned into plenti - cmv - blast lentiviral vector ( plasmid 17451 ; addgene , cambridge , ma )  . 
the patient restarted erlotinib ( 100 mg daily ) with clinical and radiologic response for 12.5 months , at which time computed tomography revealed an increase in the dominant right upper lobe mass . 
 ( b - d ) representative images showing ( b ) baseline scan ( at time of progression during osimertinib monotherapy ) , ( c ) response to crizotinib monotherapy , and ( d ) response to combined crizotinib and osimertinib therapy . 
 ( * ) the patient initially received 1.4 months of combination osimertinib and savolitinib in a clinical trial , but treatment was changed to monotherapy with osimertinib because of intolerable toxicity . 
 ( ) as of 10 months of ongoing treatment with osimertinib and crizotinib . l858r mutation , no evidence of egfr t790m , and a new metex14 ( c.2899g.a ) alteration and met amplication ( fold change , 2.5 ; fig 1a ; appendix table a1 )  . 
therapy was changed to osimertinib with savolitinib daily ( clinicaltrials . gov identier : nct02143466 ) for 1.4 months , after which savolitinib was stopped because of toxicity and single - agent osimertinib 80 mg daily was continued . 
the patient continued to receive combination therapy with durable clinical and radiographic benet for more than 9 months . to dene the role of metex14 in mediating resistance to egfr tkis , we generated two isogenic egfr - mutant nonsmall - cell lung cancer ( nsclc ) cell models using pc9 ( exon 19 deletion ) and h1975 cells ( l858r and t790m ) by transduction with lentiviral vectors driving expression of metex14 ( fig 2a )  . 
western blot analysis showed that phosphorylation of egfr and its downstream effectors akt and erk was inhibited by osimertinib in pc9 cells transduced with empty plasmids ( pc9 empty ) , but phosphorylation of egfr , metex14 , and downstream effectors remained unaffected by osimertinib treatment in pc9 metex14 cells ( fig 2b )  . 
each condition was assayed in triplicate determinations ; data were normalized for cell number by measuring cell viability and shown relative to the control group ( mean 6 standard deviation )  . 
combination treatment with epidermal growth factor receptor ( egfr ) and met inhibitors is effective against met exon 14 skipping alteration ( metex14 ) induced drug resistance in egfr - mutant nonsmall - cell lung cancer cells . 
 ( c ) cells were treated with osimertinib ( 1 mm ) , crizotinib ( 1 mm ) , or a combination of the two agents for 3 hours , and lysates were subjected to immunoblotting . 
 ( e ) caspase 3 / 7 activity was analyzed in pc9 metex14 cells that were treated with osimertinib ( 1 mm ) , crizotinib ( 200 nm ) , or a combination of osimertinib ( 1 mm ) and crizotinib ( 200 nm ) for 48 hours . 
each condition was assayed in triplicate determinations , and data were normalized for cell number by measuring cell viability and are shown relative to the control group ( mean 6 standard deviation )  . 
pc9 metex14 cells showed a reduction in osimertinib - induced caspase 3 / 7 activation compared with pc9 - empty cells ( p , .001 ; fig 2d )  . 
together , these results indicate that metex14 induces resistance to osimertinib in egfr - mutant nsclc cells . investigated whether metex14 - mediated rewe next sistance to egfr tkis could be overcome by combination therapy with egfr and met inhibitors . 
as expected , crizotinib inhibited metex14 phosphorylation in pc9 metex14 cells ; however , phosphorylation of egfr , akt , and erk remained largely unchanged ( fig 3a ) , suggesting that egfr is still signaling effectively in pc9 metex14 cells . similarly , crizotinib was ineffective at modulating growth of egfr - mutated cell lines , with or without metex14 expression ( fig 3b )  . 
in agreement with these results , inhibition of egfr and met caused signicantly dual higher activation of caspase 3 / 7 ( fig 3e )  . discussion our study highlights the importance of serial and diverse molecular analyses , including ngs , to evaluate acquired alterations in the post - tki setting . 
although the patient did not respond to met - targeted therapy alone , the patient continued to have a durable clinical response to combination osimertinib and crizotinib , with stable disease by recist criteria and without notable toxicity . 
crizotinib restored sensitivity to egfr tkis ; however , crizotinib alone was not enough to suppress growth . two previous reports have demonstrated co - occurrence of egfr and metex14 mutations . 
in the rst report , three ( 0.2% ) of 1 , 590 patients with nsclc harbored concomitant egfr and metex14 mutations , one of whom received combination treatment with met ( volitinib ) and egfrtargeted therapies ( getinib ) , yielding a partial response.16 the second report noted one patient case of sarcomatoid carcinoma with egfr and metex14 alterations.17 in our msk - impact testing experience of 866 patient cases of egfr - mutant lung adenocarcinomas ( as of october 15 , 2018 ) , only two patients showed this combination of alterations : the patient described here , with acquired resistance , and a patient separate primaries at diagnosis . in whom the alterations were present the clinical benet of the combination of metand egfrtargeted therapies in patients with nsclc with acquired met amplicationmediated resistance to egfr tkis has trials , with varying tolerability been explored in clinical dependent upon the agents being combined.18 our ndings provide a rationale for future clinical evaluation of this combination approach , given its tolerability and efcacy in this case , for patients with egfr and metex14 mutations . furthermore , given the recent reports of secondary - site mutations in the met kinase domain , such as d1228n / v and y1230c , as mechanisms of acquired resistance to crizotinib in patients with metex14 , 19 it is plausible that these secondary met mutations will also emerge as mechanisms of resistance to the combination of osimertinib and crizotinib . we found that expression of metex14 upregulated phosphorylation of egfr itself , presumably via crossphosphorylation , because met is known to interact with egfr and drive the activity of egfr , 20 which resulted in blunting of the inhibition of egfr phosphorylation by egfr tkis . 
yu , romel somwar , marc ladanyi data analysis and interpretation : ken suzawa , michael ofn , adam j . schoenfeld , igor odintsov , daniel lu , alexander drilon , helena a . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter michael ofn consulting or advisory role : pharmamar travel , accommodations , expenses : bristol - myers squibb , merck sharp & dohme maria e . 
rudin consulting or advisory role : bristol - myers squibb , abbvie , seattle genetics , harpoon therapeutics , genentech / roche , astrazeneca , ascentage pharma , bicycle therapeutics , celgene , daiichi sankyo , ipsen , loxo , pharmamar , elucida oncology , bridge medicines research funding : abbvie / stemcentrx ( inst ) , viralytics ( inst ) , merck ( inst ) , daiichi sankyo ( inst ) alexander drilon honoraria : medscape , onclive , peervoice , physicians education resources , targeted oncology , more health , research to practice , foundation medicine , peerview consulting or advisory role : ignyta , loxo , tp therapeutics , astrazeneca , pzer , blueprint medicines , genentech / roche , helsinn therapeutics , beigene , hengrui therapeutics , exelixis , bayer healthcare pharmaceuticals , tyra biosciences , verastem , takeda / ariad / millennium pharmaceuticals , bergenbio , more health patents , royalties , other intellectual property : wolters kluwer ( royalties for pocket oncology ) other relationship : merck , glaxosmithkline , teva pharmaceuticals industries , taiho pharmaceutical helena a . 
yu consulting or advisory role : astrazeneca , eli lilly research funding : clovis oncology ( inst ) , astrazeneca ( inst ) , astellas pharma ( inst ) , eli lilly ( inst ) , novartis ( inst ) , pzer ( inst ) , daiichi sankyo ( inst ) travel , accommodations , expenses : eli lilly other relationship : astellas pharma gregory j . 
 acquired met exon 14 alteration and resistance to egfr tkis references yu ha , suzawa k , jordan e , et al : concurrent alterations in egfr - mutant lung cancers associated with resistance to egfr kinase inhibitors and characterization of mtor as a mediator of resistance . 
clin cancer res 24 : 3108 - 3118 , 2018 ofn m , somwar r , rekhtman n , et al : acquired alk and ret gene fusions as mechanisms of resistance to osimertinib in egfr - mutant lung cancers . 
jama oncol 4 : 1527 - 1534 , 2018 bean j , brennan c , shih jy , et al : met amplication occurs with or without t790m mutations in egfr mutant lung tumors with acquired resistance to getinib or erlotinib . 
proc natl acad sci usa 104 : 20932 - 20937 , 2007 onozato r , kosaka t , kuwano h , et al : activation of met by gene amplication or by splice mutations deleting the juxtamembrane domain in primary resected lung cancers . 
j thorac oncol 4 : 5 - 11 , 2009 kong - beltran m , seshagiri s , zha j , et al : somatic mutations lead to an oncogenic deletion of met in lung cancer . 
cancer res 66 : 283 - 289 , 2006 paik pk , drilon a , fan pd , et al : response to met inhibitors in patients with stage iv lung adenocarcinomas harboring met mutations causing exon 14 skipping . 
cancer discov 5 : 842 - 849 , 2015 drilon ae , camidge dr , ou s - hi , et al : efcacy and safety of crizotinib in patients ( pts ) with advanced met exon 14 - altered non - small cell lung cancer ( nsclc )  . 34 , 2016 ( suppl ; abstr 108 ) drilon a , ou s - h , clark j , et al : antitumor activity and safety of crizotinib in patients with met exon 14 - altered advanced non - small cell lung cancer . 
felip e , horn l , patel jd , et al : tepotinib in patients with advanced non - small cell lung cancer ( nsclc ) harboring met exon 14 - skipping mutations : phase ii trial . 
wolf j , han j , nishio m , et al : geometry mono - 1 : phase ii , multicenter study of met inhibitor capmatinib ( inc280 ) in egfr wt , met - dysregulated advanced nsclc . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
pennell na , neal jw , chaft je , et al : select : a phase ii trial of adjuvant erlotinib in patients with resected epidermal growth factor receptormutant nonsmallcharacteristics . 
borsu l , intrieri j , thampi l , et al : clinical application of picodroplet digital pcr technology for rapid detection of egfr t790m in next - generation sequencing libraries and dna from limited tumor samples . 
ou si , govindan r , eaton kd , et al : phase i results from a study of crizotinib in combination with erlotinib in patients with advanced nonsquamous non - small cell lung cancer . 
 future of evidence synthesis in precision oncology : between systematic reviews and biocuration precision medicine refers to the tailoring of interventions to patients using approaches that go beyond traditional clinical characteristics ( eg , age , sex , disease , symptoms , and medical history ) by considering biomarkers consisting of genetic characteristics or molecular profiles.1 in particular , precision oncology ( po ) 2 includes both the development of novel cancer therapies targeting specific changes that occur in some individuals but not others ( eg , inherited mutations or somatic mutations that arise in the carcinogenesis process ) and the stratification of individuals according to existing interventions ( eg , via screening programs tiered by genetic risk or chemotherapies predicted to work for certain molecular profiles ) , which is expected to show improved outcomes in the resulting subgroups . 
 herein , we discuss two approaches for synthesizing evidence for or against the use of specific po interventions , namely , systematic reviews ( srs ) and biocuration , and argue that their engagement with each other could facilitate the timely delivery of appropriate po interventions . cancer is known to be highly heterogeneous , and clinical responses differ among patients . 
 po thus includes a wide variety of applications , including both the use of targeted therapies against particular changes occurring in individual tumorssuch imatinib against the bcr - abl fusion in chronic myeloid leukemia , 3 vemurafenib4 and dabrafenib5 against specific braf mutations in melanoma , and trastuzumab against her2 - postive breast cancer6and the assignment between existing therapy classes via biomarkers that are not direct drug targets . 
this can be based either on the activation of certain pathways , as in the case of egfr inhibitors in kras - wild type colon cancer , 7 , 8 or on gene expression signatures or other molecular characteristics ( ie , oncotype dx test for breast cancer , used to predict recurrence and likely benefit of chemotherapy ) .9 , 10 these biomarkers are well - established and have been approved by the us food and drug administration for specific cancer types . the biomarkers discussed in the previous paragraph are classified as having the highest level of evidence for clinical use in society guidelines ( eg , those jointly put forward by the association for molecular pathology , asco , and the college of american pathologists ) .11 to be meaningful for clinical decision making , biomarkers should have high predictive performance ( ie , they can be used to stratify patients into treatment groups with differential outcomes )  . 
most research to date , however , has assessed whether biomarkers are prognostic ( ie , are associated with a clinical outcome for untreated or standardof - care patients ) .12 it is often the case that the supporting literature includes few if any randomized control trials.13 as a result , lower levels of evidence may be assigned , eg , to biomarkers that predict response to a treatment based on well - powered studies evaluated by expert consensus or that predict response in a different tumor type than that being studied . many conceptual frameworks have been proposed to facilitate clinical decision making by helping end users ( especially patients and clinicians ) assess the evidence for or against the use of medical tests , including biomarkers.14 , 15 with minor variations , the different frameworks involve a stepwise approach that starts with analytic validity ( laboratory reproducibility ) , proceeds to clinical validity ( predictive accuracy , including sensitivity , specificity , and positive and negative predictive values ) and clinical utility ( use of the biomarker leads directly to improvements in outcomes like survival and quality of life ) , and eventually assesses the cost effectiveness of the biomarker . 
comparison of key features of systematic reviews and biocuration feature systematic reviews biocuration overall approach top down ; starts with a key scientific question and develops evidence base around it either top down or bottom up ; starts with a number of scientific questions or allows individual articles to be curated and slotted into categories evidence assessment exhaustive literature searches and systematic multiple frameworks exist assessment of evidence presence of bias systematic assessment of the risk of bias higher chance for bias by selecting studies with delivery approach peer - reviewed article or whitepaper research approach team usually assembled at the start of the project often relies on crowdsourcing and expert review of updating significant lag closer to real time , depending on availability of authors or curators multidisciplinary teams , often exclude or reduce role of primary study authors or subject matter experts due to risk of bias scientists and clinicians , who are frequently subject matter experts , from a variety of fields , and often with an expert panel to resolve conflicting interpretations significant findings database of entries may include summary of findings and links to original studies and other resources entries curators as with all tests in clinical practice , tests related to biomarkers are delivered within given health care settings , and currently , little structured evidence exists in regard to how po can successfully be implemented for particular sets of patients , with different degrees of access to health care , across diverse health care systems . 
the first is the traditional evidence appraisal that applies the well - established concepts of evidence - based medicine ( ebm ) .17 , 18 ebm integrates a physicians clinical experience with scientific evidence that has undergone an sr , which involves extensive surveying of the literature followed by a synthesis of the primary studies.19 the second is biocuration , which refers to the distillation and integration of biologic information from scientific literature and large data sets using databaseor research field specific , controlled vocabularies or ontologies ; it is a cornerstone of bioinformatics.20 both disciplines have the same overarching goal of bringing to patients only the interventions proven to be effective by carefully balancing benefits and harms , doing so in different ways ; srs start with a particular question and systematically develop the evidence base around it , whereas biocuration may be either top - down or bottom - up and usually involves more real - time updating . 
we compare key aspects of these two approaches in table 1 and summarize important feature of specific curated databases in appendix table a1 . the goal of srs is to allow a comprehensive and global view of the available evidence base on a particular question of interest by analyzing primary studies that meet prespecified eligibility criteria via explicit , reproducible methodologies that minimize bias . 
the typical steps involved in an sr include identification of the clinical question , specification of eligibility criteria , systematic search for all studies that meet these criteria , extraction of evidence from eligible studies and assessment of their methodologic rigor , and analysis and qualitative and / or quantitative synthesis ( meta - analysis ) of the extracted data.19 an sr is typically structured around the picots elements : the population toward which the findings will be applicable , the intervention , the comparisons being performed , the clinical outcomes , the timing of the eligible studies , and the clinical setting . 
during the course of decades , a toolbox of methods has been developed in srs and ebm to evaluate key elements of published studies including systematic biases , statistical precision , applicability to a target clinical setting , and others.21 - 23 evidence for specific interventions on the basis of supporting srs is generally considered strong . 
 clinical practice guidelines on the basis of srs that support the use of a genomic test as tier one ( ready to implement in clinical practice24 ) and those that are not based on srs as tier two ( may be useful in the context of informed clinical decision making24 )  . in general , sr teams are multidisciplinary , consisting of clinicians , content experts , methodologists , statisticians , and librarians . 
because researchers may often be influenced by their own investment in the field when interpreting the evidence base , 25 ideally , parties involved in a sr should have no personal vested interests in the topic of the sr . 
although content experts are critical for the clinical interpretation of primary studies and for putting them in context with the broader evidence , their role as authors in srs has been debated.26 however , since srs have various degrees of complexity , relevant sponsors often make decisions on a case - by - case basis by thoroughly reviewing the potential conflicts of individual researchers . 
for example , the agency for healthcare and research quality evidence - based practice centers program has a set of guidelines for how different types of conflicts should be handled and alleviated.27 given that precision medicine is a relatively new and rapidly evolving field with potentially more complex statistical analyses and contextual questions compared to traditional studies , including and relying on content experts is more critical . synthesis of evidence from po studies encounters a number of specific challenges . 
consider for instance a cochrane review of first - line treatments in individuals with egfr - mutated noncurable stage iiib to iv nonsquamous nonsmall - cell lung cancer , which included 19 rcts.28 the overall conclusion was that the tyrosine kinase inhibitor therapies ( erlotinib , gefitinib , and afatinib ) led to improved progression - free survival but not to improved overall survival , whereas the monoclonal antibody cetuximab did not show any improved outcomes . 
while this study included 19 rcts total , comparing targeted therapies to chemotherapy or best supportive care , the number of rcts conducted for each therapy was between two and eight and the study designs , outcome measures , and analyses performed and reported varied . 
srs have also identified existing evidence gaps for other prevision medicine interventions , such as the lack of robust evidence for tailoring smoking cessation interventions on the basis of germline genetic variation.29 many issues with the sr of evidence for po interventions will be solved in time , as more studies accumulate , but it is important to note that the desire for new therapies may be at a historical high given the stated promise of precision medicine and po in particular . 
however , another challenge that will linger is that the definition of precision medicine will continue to make it difficult to satisfy the picots framework as , for example , more drugs are tested separately in different subsets of patients ( eg , if the same biomarker is considered for different tumor types ) or molecular tests rapidly evolve to add more biomarkers or change how they are tested ( eg , protein expression , gene expression , or dna amplification ) .13 new rct designs such as umbrella and basket trials are now being implemented to meet some of these challenges . 
although other study designs may provide additional valuable information on specific treatments , the question of clinical utility , in particular , is difficult to answer in the absence of evidence from rcts . biocuration identifies and summarizes biomedical results , including potentially those from srs , into bioinformatic databases , often by using controlled vocabularies and prespecified standards.20 a text summary of the evidence related to specific scientific questions may also be included , along with links to the original studies or other resources . 
in particular , medical curation focuses on disease associations and genetic factors and includes efforts like the omim ( online mendelian inheritance in man ) database30 as well as specialized databases for particular genes , variants , and diseases . 
clinvar , a public archive of relationships among medically important germline and somatic variants and human phenotypes , was launched in april 2013.31 biocuration generally provides more immediate updating than formal srs , but the lack of a unified systematic framework may lead to only partial or inconsistent results . 
 curators , resulting in a wealth of information but also in issues such as contradictory interpretations and many variants of uncertain significance.32 the clingen ( clinical genome resource ) initiative aims to resolve some of these problems and answer the critical questions of clinical validity , disease causality ( pathogenicity ) , and clinical actionability by standardizing data collection and sharing and implementing an approach for consensus among expert curators.32 many other cancer - specific databases now exist for synthesizing evidence for po approaches , 33 - 37 leading clingen to recently develop recommendations and guidelines for defining cancer somatic variants on the basis of their diagnostic , prognostic , and predictive roles , using evidence of their significance and clinical utility.38 in a translational field such as po , where molecular genomics and clinical practice come together , a particular challenge for biocuration is that each content area has its own detailed terminology ( eg , specific diseases and syndromes can be described using icd - 10 codes39 and snomed terms40 )  . 
however , mapping these terminologies to specific causal mutations , as well as to biomarkers and tratments , can often be a challenge . biocurators come from a wide variety of backgrounds , usually in biology , biochemistry , or medical genetics , and increasingly have interdisciplinary training that includes computer and information sciences , with subject matter experts playing an important role in the field of biocuration.20 , 41 a growing number of clinical laboratories in hospitals and community clinics are now also conducting molecular diagnostic testing to identify sequence variants and inform treatment decisions for patients , in which case the curation is often performed by molecular pathologists as well as clinicians . 
the expert panels used by resources such as clingen to resolve conflicting interpretations and curate variants of unknown significance include medical professionals , medical geneticists , clinical laboratory diagnosticians and molecular pathologists that have a long standing scope of work in the disease gene in question . an example of a curated cancer database is the recently released civic ( clinical interpretation of variants in cancer ) database , 36 which provides associations between drugs and genes or variants in specific cancer types . 
however , the authors state the presence of a bias toward positive associations with treatment outcomes ( 91% of records show support for the use of a therapy ) , which illustrates the absence of a systematic , unbiased framework for identifying the evidence base . 
given that civic considers many different types of studies and that studies of cancer drugs may have many different designs , it is true that it would be extremely challenging to have a fixed list of terms that would be applicable to all studies and easily translatable into picots terminology . in general , literature searches for srs are typically more exhaustive and thorough than for curated databases , where eligible studies tend to be identified by experts in a given field . 
 although currently this may have little direct impact on identifying relevant studies for biocuration , given the small number of clinical po publications , this will eventually become a major issue in the next years , because the field will need to deal with increasing amounts of published and unpublished evidence . 
srs also tend to have more well - defined a priori eligibility criteria that a study has to meet to be considered in the evidence synthesis ; in contrast , biocuration approaches show more variability and rely more on individual experts in the field . 
although biocuration currently lacks a standardized and widely accepted framework to assess the quality of published evidence , several efforts are under way to change this.38 , 42 - 44 beyond trying to develop standardized frameworks for biocuration , a number of other approaches for improving curated databases have been considered , including the use of specific incentives . 
the emphasis on a systematic survey of the literature which includes non - significant results that is present in srs is also not an explicit part of the biocuration philosophy . 
for example , if the ebm community engages the biocuration community , they may find ways of incorporating more elements of the picots framework into existing and future databases . there is often a tension between systematic , unbiased syntheses and assessments versus timely and accessible curated information , and the highly promising field of po is a clear example that shows the difficulties in finding a good solution that considers both of these dimensions . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . approaches , and products , instead of continuing to proceed on parallel paths . 
curated databases will continue to be an initial first stop for many researchers and clinicians , especially because srs may lag years behind current studies , and decisions often need to be made in cases where srs are lacking . 
 however , more training is needed for users to understand that , for example , a higher risk of bias exists for finding more significant associations when consulting a curated po database than when using a sr . 
obrien sg , guilhot f , larson ra , et al ; iris investigators : imatinib compared with interferon and low - dose cytarabine for newly diagnosed chronic - phase chronic myeloid leukemia . 
hauschild a , grob j - j , demidov lv , et al : dabrafenib in braf - mutated metastatic melanoma : a multicentre , open - label , phase 3 randomised controlled trial . 
slamon dj , leyland - jones b , shak s , et al : use of chemotherapy plus a monoclonal antibody against her2 for metastatic breast cancer that overexpresses her2 . 
de roock w , piessevaux h , de schutter j , et al : kras wild - type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab . 
calonge n , klein rd , berg js , et al : recommendations from the egapp working group : does the use of oncotype dx tumor gene expression profiling to guide treatment decisions improve outcomes in patients with breast cancer ?  . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
panagiotou oa , ioannidis jp : primary study authors of significant studies are more likely to believe that a strong association exists in a heterogeneous meta - analysis compared with methodologists . 
greenhalgh j , dwan k , boland a , et al : first - line treatment of advanced epidermal growth factor receptor ( egfr ) mutation positive non - squamous non - small cell lung cancer . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
hunter je , irving sa , biesecker lg , et al : a standardized , evidence - based protocol to assess clinical actionability of genetic disorders associated with genomic variation . 
strande nt , riggs er , buchanan ah , et al : evaluating the clinical validity of gene - disease associations : an evidence - based framework developed by the clinical genome resource . 
uniprot is not disease focused ; omim , clinvar , and clingen are disease agnostic ; the remaining databases are specific to cancer . abbreviations : asco , american society of clinical oncology ; civic , clinical interpretation of variants in cancer ; clingen , clinical genome resource ; cosmic , catalogue of somatic mutations in cancer ; fda , us food and drug administration ; nccn , national comprehensive cancer network ; nih , national institutes of health ; omim , online mendelian inheritance in man ; po , precision oncology . 
nucleic acids res 43 : d204 - d212 , 2015 . landrum mj , lee jm , benson m , et al : clinvar : public archive of interpretations of clinically relevant variants . 
nucleic acids res 44 : d862 - d868 , 2016 forbes sa , beare d , boutselakis h , et al : cosmic : somatic cancer genetics at high - resolution . 
nucleic acids res 45 : d777 - d783 , 2017 . taylor ad , micheel cm , anderson ia , et al : the path ( way ) less traveled : a pathway - oriented approach to providing information about precision cancer medicine on my cancer genome . 
in the clinical pharmacogenetics implementation consortium guideline , fluoropyrimidine treatment in homozygous dpyd variant carriers is discouraged , which implies that a potentially effective anticancer therapy is withheld from these patients.5 in this article , six unique patients with a homozygous or compound heterozygous dpyd variant allele genotype who were treated with tailored fluoropyrimidine treatment are described . 
analyses were part of individual patient care , so institutional review board approval was not applicable . genotyping results showed a homozygous or compound heterozygous dpyd genotype , and the functional effects of these genotypes were uncertatherefore , pretreatment dpd activity was measured in peripheral blood mononuclear cells ( pbmcs )  . 
dpd activity was used to reach an individualized dose , in which the percentage of remaining dpd activity was used as guideline for the starting dose ( expressed as percentage of the originally planned dose )  . pharmacokinetic analyses in three of six patients were performed to investigate whether applied dose reductions were adequate . 
pretreatment plasma uracil , the endogenous dpd substrate , and dihydrouracil levels were quantified ; results were unknown before the start of treatment . results clinical course of treatment patient characteristics are listed in table 1.6 - 9 linda m . 
therefore , it was decided to drastically reduce the capecitabine dose from 2 , 300 mg ( 1 , 000 mg / m2 ) twice daily to 150 mg twice daily ( 6.5% of planned dose ) and to start with capecitabine monotherapy . 
the adverse events resolved within 1 week ( neutropenia ) to 2 months ( diarrhea , mucositis )  . after a 2 - month period without any anticancer therapy , the patient had fully recovered , and monotherapy with capecitabine was restarted . 
on the basis of the severe toxicity and the pharmacokinetic results of the first cycle , the dose was reduced more to 150 mg once every 5 days ( ie , 0.65% of originally planned dose )  . 
this female patient with locally advanced colorectal carcinoma had a planned treatment that consisted of neoadjuvant chemotherapy ( capecitabine 825 mg / m2 twice daily , or 1 , 500 mg ) combined with radiotherapy ( 5 - week schedule )  . pretreatment dpyd screening revealed that the patient was homozygous for c.2846a.t ; dpd activity was reduced to 29% . 
it was decided to reduce the capecitabine dose to 500 mg once daily ( ie , 17% of planned doseslightly lower than recommended dose of 29% on the basis of dpd activity , as decided by physician and patient )  . 
dpd activity was reduced to 45% . remaining dpd activity was more than 50% reduced , so it was considered likely that this patient was a compound heterozygous carrier ( variants present on different alleles )  . 
in the first cycle , capecitabine was reduced to 1 , 800 mg daily ( 51% of planned daily dose of 3 , 500 mg ; 1 , 750 mg / m2 ) , which was tolerated without toxicity . 
however , the patient developed grade 2 thrombocytopenia after 8 days , and the daily capecitabine dose was adjusted to 1 , 000 mg for the rest of the cycle . platelets increased again until normal values were reached . 
after three cycles , disease progression was established , and capecitabine treatment was discontinued . pharmacokinetic results in all three patients , additional pharmacokinetic measurements were performed ( fig 1 )  . 
for patient 1 , only levels of capecitabine and of the metabolites 59 - deoxy - 5 - fluorocytidine , 59 - deoxy5 - fluorouridine , and fu could be quantified ; other fu metabolites were not detectable . 
results of noncompartmental analysis of the pharmacokinetic results in plasma are listed in table 2 and include values normalized to control values.10 in patient 1 , fu exposure was highly increased : the mean area under the plasma concentration - time curve ( auc ) of fu was 4 , 024 ng 3 h / ml , which is 10 times higher than in other pharmacokinetic studies with capecitabine.10 - 13 these results were used for the decision to lower the dose 10 - fold in the second cycle . baseline uracil and dihydrouracil levels are listed in table 1 . 
results of urine analysis for patient 1 are shown in appendix figure a1 . discussion to our knowledge , this is the first report to describe prospectively identified patients , who are homozygous or compound heterozygous for dpyd variants , who could be treated safely with fluoropyrimidines . 
for all three patients , the results after the first intake of capecitabine are depicted . capecitabine patient 1 ( homozygous dpyd * 2a ) patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) 5 ' - dfcr patient 1 ( homozygous dpyd * 2a ) patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) time ( h ) 5 ' - dfur time ( h ) 5 - fu patient 1 ( homozygous dpyd * 2a ) patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) patient 1 ( homozygous dpyd * 2a ) patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) 5 , 000 4 , 000 3 , 000 2 , 000 1 , 000 4 , 000 3 , 000 2 , 000 1 , 000 2 , 500 2 , 000 1 , 500 1 , 000 2 , 500 2 , 000 1 , 500 1 , 000 1 , 200 time ( h ) fuh2 time ( h ) fupa patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) time ( h ) time ( h ) fbal patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) time ( h ) pretreatment identification of the patient homozygous for dpyd * 2a with complete dpd deficiency saved this patient from receipt of a full fluoropyrimidine dose , which most likely would have been fatal . 
in patients who are not dpd deficient , fluoro - b - alanine is the major urinary metabolite , 13 , 20 whereas this metabolite was not present in the urine analyzed in the case presented in this paper . 
for example , mir27a polymorphisms could play a role in variation of dpd activity , because these polymorphisms reduce dpd activity.26 , 27 in conclusion , we showed that fluoropyrimidine treatment in homozygous or compound heterozygous dpyd variant allele carriers is feasible and that therapy does not have to be withheld . 
van der veldt consulting or advisory role : ipsen ( inst ) , roche ( inst ) , bristol - myers squibb ( inst ) , pfizer ( inst ) , msd oncology ( inst ) travel , accommodations , expenses : roche paul hamberg no relationship to disclose andr e b.p. 
bioanalysis 7 : 519 - 529 , 2015 van kuilenburg ab , van lenthe h , tromp a , et al : pitfalls in the diagnosis of patients with a partial dihydropyrimidine dehydrogenase deficiency . 
deenen mj , meulendijks d , boot h , et al : phase 1a / 1b and pharmacogenetic study of docetaxel , oxaliplatin and capecitabine in patients with advanced cancer of the stomach or the gastroesophageal junction . 
judson ir , beale pj , trigo jm , et al : a human capecitabine excretion balance and pharmacokinetic study after administration of a single oral dose of 14c - labelled drug . 
van kuilenburg ab , muller ew , haasjes j , et al : lethal outcome of a patient with a complete dihydropyrimidine dehydrogenase ( dpd ) deficiency after administration of 5 - fluorouracil : frequency of the common ivs14 + 1g.a mutation causing dpd deficiency . 
meinsma r , fernandez - salguero p , van kuilenburg ab , et al : human polymorphism in drug metabolism : mutation in the dihydropyrimidine dehydrogenase gene results in exon skipping and thymine uracilurea . 
van kuilenburg ab , meijer j , mul an , et al : intragenic deletions and a deep intronic mutation affecting pre - mrna splicing in the dihydropyrimidine dehydrogenase gene as novel mechanisms causing 5 - fluorouracil toxicity . 
offer sm , butterfield gl , jerde cr , et al : micrornas mir - 27a and mir - 27b directly regulate liver dihydropyrimidine dehydrogenase expression through two conserved binding sites . 
froehlich tk , amstutz u , aebi s , et al : clinical importance of risk variants in the dihydropyrimidine dehydrogenase gene for the prediction of early - onset fluoropyrimidine toxicity . 
int j cancer 136 : 730 - 739 , 2015 patients were treated at three different institutes in the netherlands ( the netherlands cancer institute , amsterdam ; erasmus medical center , rotterdam ; fransciscus gasthuis & vlietland , rotterdam )  . 
toxicity was scored according to the common terminology criteria for adverse events , version 4.03. dpyd genotyping genomic dna was isolated from peripheral blood cells , and screening for the dpyd variants dpyd * 2a ( ivs14 + 1g.a , rs3918290 ) , c.2846a.t ( rs67376798 ) , c.1679t.g ( dpyd * 13 , rs55886062 ) , and c.1236g.a ( rs56038477 ) was performed by using standard operating procedures in two different institutes ( the netherlands cancer institute , amsterdam , and erasmus medical center , rotterdam )  . 
in the netherlands cancer institute , screening for dpyd variants was performed with the roche lightcycler 480ii platform ( roche diagnostics , almere , the netherlands ) by using commercially available probes and primers ( tib molbiol , berlin , germany ) , and results were confirmed by direct sequencing . 
at the erasmus medical center , each sample was genotyped on two different platformstaqman ( with predefined drug - metabolizing enzyme [ dme ] assays ) and pcrrestriction fragment length polymorphism ( rflp ) assaysto allow checks for potentially wrong genotyping . 
both laboratories participated during the study in the dutch national quality control program for dpyd proficiency testing , in which all four dpyd variants were included . dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells dihydropyrimidine dehydrogenase ( dpd ) activity was measured in peripheral blood mononuclear cells ( pbmcs ) , isolated from a baseline ( pretreatment ) peripheral blood sample . 
one of two comparable validated methods by van kuilenburg et al7 or pluim et al6 was used ; both used radiolabeled thymine ( 14c - labeled thymine or 3h - labeled thymine ) as a substrate and consisted of high - performance liquid chromatography ( hplc ) with online radioisotope detection and with liquid scintillation counting . 
analytes were extracted by protein precipitation ; chromatographic separation was performed on an acquity ultra - performance liquid chromatography hss t3 column ( acquity waters , milford , ma ) and were analyzed with ms / ms with an electrospray ionization source ( jacobs ba et al : j pharm biomed anal 126 : 75 - 82 , 2016 )  . 
all samples were measured at one institute ( the netherlands cancer institute )  . pharmacokinetic measurements peripheral blood samples for patient 1 were obtained for pharmacokinetic analysis on cycle 1 , day 1 ( c1d1 ) ; cycle 2 , day 1 ( c2d1 ) ; and cycle 2 , day 16 ( c2d16 )  . 
 results dihydrouracil - uracil levels capecitabine intake ( at predose , and at 15 minutes , 30 minutes , 1 hour , 2 hours , 3 hours , 4 hours , 6 hours , 8 hours , and 10 hours after capecitabine intake )  . 
for patients 2 and 3 , samples were collected only on c1d1 , on the same time points as for patient 1 , except for the latest time point ( the sample 10 hours after capecitabine intake was not collected for patient 2 , and the last sample was collected at 12 hours instead of at 10 hours for patient 3 )  . 
for patients 2 and 3 , pharmacokinetic results were not known during treatment , because samples were analyzed after treatment had finished . capecitabine and its metabolites , 59 - deoxy - 5 - fluorocytidine ( 59 - dfcr ) , 59 - deoxy - 5 - fluorouridine ( 59 - dfur ) , fluorouracil ( fu ) , dihydro - 5 - fluorouracil ( fuh2 ) , a - fluoro - ureidopropionic acid ( fupa ) , and fluoro - b - alanine ( fbal ) , were quantified by using hplc coupled to electrospray ms / ms . 
two individual validated assays were used : one for the simultaneous quantification of capecitabine , 59 - dfcr and 59 - dfur and another for fu , fuh2 , fupa , and fbal ( deenen mj et al : j chromatogr b analyt technol biomed life sci 913 - 914 : 30 - 40 , 2013 )  . 
all pharmacokinetic samples were measured at the same institute ( the netherlands cancer institute )  . together with the sample for dpd activity in pbmcs , a pretreatment plasma sample was taken to measure uracil and dihydrouracil levels ( table 1 )  . 
for patients 2 and 3 , uracil levels were increased compared with reference levels ( a value of 28.7 ng / ml for patient 2 and of 35.6 ng / ml for patient 3 ) , and both values were within the top 1% of reference values . 
dihydrouracil levels were in the normal range for patients 2 and 3 compared with reference levels . other homozygous dpyd variant allele carriers three additional patients with a homozygous dpyd variant genotype were identified during routine dpyd screening ( table 1 )  . 
it is unclear why there is a relatively high variation of the effect of this genotype on dpd phenotype in patients , and more research on this variant is advised . in addition , one patient with a homozygous c.2846a.t genotype ( patient 4 ) was identified . 
according to the calculated dpd activity score , as described by henricks et al , 4 it could be concluded that a 50% dose reduction would be appropriate for homozygous c.2846a.t carriers , because a 25% dose reduction is recommended for heterozygous carriers of this variant . 
with the recent increase in integrative clinical sequencing for pediatric patients with cancer , our understanding of the dicer1 syndrome continues to evolve , as new and rare pathogenic variants are reported . 
as the frequency of integrative clinical sequencing increases , discussions regarding challenges encountered in the interpretation of sequencing results are essential to continue to advance the eld of cancer predisposition . 
the purpose of this work was to identify patients with somatic and / or germline dicer1 variants in our patient population and to discuss sequencing interpretation and the clinical recommendations that result from the integrative clinical sequencing results . methods patients were enrolled in the peds - mioncoseq study . 
2019 by american society of clinical oncology introduction the dicer1 gene is located on chromosome 14 ( 14q32.13 ) and encodes the dicer protedicer is an rnase iii enzyme functioning in micro - rna ( mirna ) processing . 
mirnas are single - stranded , noncoding rnas that target mrnas by binding to complementary mrna sequences , increasing their degradation and suppressing translation.1 through its ability to regulate mirna processing , dicer1 plays a critical role in cellular mrna expression and in early development.1 , 2 with respect to human disease , more than 130 germline and 95 somatic dicer1 mutations have been reported.2 the majority of somatic dicer1 mutations are hotspot mutations in the rnase iiib domain ; however , somatic mutations are reported to be elsewhere , including the domain of unknown function.2 germline dicer1 mutations have been reported in all of the 10 major domains and spanning regions.3 dicer1 syndrome , caused by pathogenic variants in dicer1 , is an inherited condition associated with an increased risk of developing pleuropulmonary blastoma ( ppb ) , multinodular goiter , cystic nephroma , sertoli - leydig cell tumors of the ovary , and other rare tumors . 
 bailey et al context the goal of this work was to describe sequencing interpretation challenges and clinical recommendations in patients with dicer1 somatic and / or germline variants identied on integrative sequencing . this work suggests that postzygotic mosaicism may play a signicant role in the development of dicer1 - associated tumors . furthermore , we report a patient with a dicer1 variant of uncertain signicance whose clinical course and response to treatment was highly unusual , suggesting this variant could be clinically meaningful . the use of integrative clinical sequencing has signicantly expanded in pediatric oncology in the past 5 years . 
reports also include a dicer1 syndrome family with both somatic and germline pathogenic variants found in family members with different types of dicer1 - related cancers.15 to our knowledge , our group reported the rst real - time , integrative clinical sequencing ( ics ) study in the pediatric oncology population in 2015.16 since that time , the role of real - time ics of tumors and germline samples in the pediatric oncology population has continued to expand.17 - 19 however , the interpretation of genetic variants and their translation into the clinical setting remain challenging . when sequencing results identify germline dicer1 variants in pediatric oncology patients , counseling and screening implications for family members are affected . 
the purpose of this work was to highlight ( 1 ) somatic and / or germline dicer1 pathogenic variants identied in our pediatric oncology sequencing cohort , ( 2 ) challenges encountered with sequencing result interpretation with respect to dicer1 , and ( 3 ) current patient and family screening recommendations . methods patients the pediatric patients described were treated at c.s. 
patients were enrolled in a prospective , institutional review boardapproved ics trial ( peds - mioncoseq ) 16 and underwent paired tumor / normal whole - exome sequencing and tumor transcriptome sequencing . 
peds - mioncoseq is an ongoing precision oncology cohort and focused on enrolling highrisk , relapsed / refractory , and rare pediatric and young adult ( younger than 25 years of age ) patients with cancer . integrative clinical sequencing specics of the sequencing procedure and bioinformatics analyses have been previously described.16 briey , germline sequence and copy number variants , as well as somatic mutations , copy number alterations / variants , insertions and deletions , gene fusions , and gene expression , were identied.20 , 21 pathogenicity of germline variants was determined through a review of the published literature and publically available databases ( ie , clinvar )  . clinical relevance of somatic variants was investigated using an integrated approach incorporating technical considerations ( eg , recurrence , variant allele fraction , expression levels , and predictive algorithms for pathogenicity ) , variant - specic information ( ie , clinvar , published literature , and curated gene - specic resources ) , and published correlations of drug and variant sensitivity proles . results four patients with somatic and / or germline dicer1 pathogenic variants were identied among the rst 300 patients enrolled in peds - mioncoseq ( table 1 )  . 
we detail the patient presentations , diagnoses , variant terpretation , and clinical actions taken on the basis of these results . patient 1 : a patient with a hotspot somatic variant and a germline pathogenic dicer1 variant patient 1 presented at 3 years of age with a history of left arm and abdominal paan abdominal x - ray was performed , and a left - sided pleural effusion was incidentally noted . 
surveillance for dicer1 - related tumors was initiated for the patients sister ( 6 years old at time of genetic testing ) and included baseline chest ct ( to screen for ppb ) , with a chest x - ray at 8 years of age , annual ultrasound scans of the abdomen and neck ( to screen for ovarian and kidney tumors and thyroid tumors , spectively ) , and annual serum markers to screen for ovarian tumors . 
the father received an annual ultrasound scan of the thyroid gland only , because dicer1 - associated cancers affecting males primarily present in childhood . the bony metastatic lesions completely responded to chemotherapy , and the patient underwent primary mass resection followed by two more cycles of chemotherapy . the resected mass showed mostly viable ppb , with malignant cartilaginous nodules overrun by an anaplastic prostate cancer multinodular goiter s / p thyroidectomy prostate cancer fig 1 . 
patient 1 continued to have disease progression despite chemotherapy , and the decision was made to transition to comfort care . patient 2 : a patient with a germline dicer1 variant of unknown signicance patient 2 initially presented at 3 years of age with a left lower quadrant / pelvic mass . 
remarkably , the patient had an excellent response to therapy and was deemed disease free at age 5 years . at 19 years of age , patient 2 presented to our medical center with severe abdominal and back paimaging revealed concern for new / recurrent widely metastatic disease , because a pelvic mass was noted along with multiple spine and liver lesions . 
palliative radiation was initiated for pain control , and the patient enrolled in the peds - mioncoseq study . sequencing results revealed a germline dicer1 variant of uncertain signicance ( vus ) ( table 1 )  . 
sequencing of the original tumor ( from 3 years of age ) was attempted for comparison / to determine whether this was a metachronous tumor ; however , only sparse , poor - quality material was available , and sequencing could not be performed . 
although vuss are not disclosed per the pedsmioncoseq protocol , at times , a recommendation for clinical germline genetic testing may be made for patients with striking personal / family histories . 
the majority of vuss will be reclassied as likely benign / benign , 22 but for those that are reclassied as likely pathogenic / pathogenic , there can be signicant implications for patients and their relatives . 
patient 2 died at 20 years of age as a result of metastatic disease . patient 3 : a patient with low - level dicer1 mosaicism patient 3 initially presented at 22 months of age with a leftsided pneumonia and pneumothorax . 
a persistent air - lled cystic cavity was present in the left chest and observed with imaging for approximately 3 months before a diserial agnosis of ppb was made after resection of the left lower lobe of the lung . 
regardless , clinical germline genetic testing of dicer1 was recommended but not pursued because of the out - of - pocket cost . approximately 6 months later , patient 3 was enrolled in the peds - mioncoseq study . 
 ( a ) he image ( 40 ) of the original tumor showing mixed morphology with alveolar pattern in the top half and more abundant rhabdomyoblastic differentiation in the lower half . ( b ) he image ( 400 ) of the late recurrence tumor showing spindle - shaped and round cell tumor cells with focal rhabdomyoblastic differentiation . 
as such , no signicant somatic copy number variations , point mutations , indels , or gene fusions were able to be called . no germline variants were considered reportable at this time . this patient also enrolled in the national institutes of healths ( nihs ) pleuropulmonary blastoma dicer1 syndrome study , which includes germline and somatic sequencing , approximately 1 year after initial diagnosis . germline sequencing identied a mosaic germline dicer1 pathogenic variant ( table 1 ; fig 3 ) in 2% of the allelic reads in the patients blood sample . 
both parents enrolled in the study , and their germline testing was negative for the dicer1 pathogenic variant , consistent with the low - level mosaicism detected in the patients blood sample . 
this somatic variant is commonly seen in dicer1 syndromeassociated tumors and involves the rnase iiib domain of dicer1 . analysis of the raw data available from the peds - mioncoseq the germline dicer1 pathogenic study indicates that variant identied by the nih was present in 2% of germline variant reads in our study as well . 
this patient continues to receive annual chest ct scans with no evidence of disease and undergoes annual kidney ultrasound scans , thyroid examinations , and targeted physical examinations as screening for other potential tumors . patient 4 : a patient with a rare dicer1 - associated tumor patient 4 presented at 3 years of age with headaches , photophobia , and hydrocephalus . 
the patient received ve cycles of chemotherapy , consisting of vincristine , etoposide , cyclophosphamide , and cisplatin , with tumor resection performed after the second cycle because of an increase in tumor size . 
the patient also underwent autologous bone marrow transplantation . a year later , an mri scan revealed an enhancing mass overlying the left middle cerebellar peduncle , suggestive of recurrent disease . 
in a previous study , 25 low - level mosaicism in four children with multiple dicer1 - associated tumors in whom no germline dicer1 variant had been identied by conventional sequencing techniques was observed . 
patient 4 contributes to the literature on dicer1 syndrome and illustrates how ics can be useful in identifying germline variants in individuals with rare cancers . from a cancer treatment perspective , all four of these challenging patients revealed dicer1 variants ; however , to date , there are no there are no clinically available pediatric therapies targeting specic mirnas . 
the possibility of targeting mirnas and dicer function in the future is currently being explored.26 from a genetic counseling standpoint , once a germline dicer1 pathogenic variant is identied , germline genetic testing is recommended for rst - degree relatives . 
approximately 13% to 20% of individuals with detectable germline dicer1 pathogenic variants have de novo variants , whereas 80% to 87% are inherited.9 , 27 germline genetic testing can provide tumor risk information to probands and their relatives and provide information regarding recurrence risk for future pregnancies . 
in families where the dicer1 variant is inherited , reports indicate it is uncommon to have multiple individuals diagnosed with ppb ; however , there may be higher penetrance of other manifestations , including goiters and benign lung cysts . 
family members lacking the familial dicer1 mutation do not need surveillance , whereas those who have inherited this variant should be observed according to current surveillance recommendations . screening recommendations at our institution evolved over time and are based on literature review , expert opinion , and patient preferences . 
beyond this age , for female adolescent and adult patients , we discuss symptoms and signs of hormone excess related to sex cordstromal tumors , such as hirsutism , virilization , and menstrual abnormalities . 
for all other manifestations , we recommended a detailed physical examination . recently , consensus guidelines for surveillance were published by the international ppb registry , which are largely in accordance with our institutions age - specic recommendations . 
these guidelines were developed at the rst international dicer1 symposium in may 2016.7 recommendations for surveillance begin in the prenatal period , with a third - trimester ultrasound for detection of large lung cysts that require intervention at birth . 
these differences reect the natural uncertainty in screening approaches for a recently described hereditary tumor syndrome , which aim to integrate the morbidity and the unknown mortality of syndrome - associated tumors , incidence in the target population , and presumed cost effectiveness and patient burden . least 10 years of age and do not tumor and germline sequencing in the pediatric oncology patient population has rapidly expanded in recent years . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . no potential conicts of interest were reported . references peng y , croce cm : the role of micrornas in human cancer . 
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int j cancer 141 : 2030 - 2036 , 2017 slade i , bacchelli c , davies h , et al : dicer1 syndrome : clarifying the diagnosis , clinical features and management implications of a pleiotropic tumour predisposition syndrome . 
hum mutat 32 : 1381 - 1384 , 2011 schultz kap , williams gm , kamihara j , et al : dicer1 and associated conditions : identication of at - risk individuals and recommended surveillance strategies . clin cancer res 24 : 2251 - 2261 , 2018 8 . 
science 325 : 965 , 2009 brenneman m , field a , yang j , et al : temporal order of rnase iiib and loss - of - function mutations during development determines phenotype in pleuropulmonary blastoma / dicer1 syndrome : a unique variant of the two - hit tumor suppression model . 
schultz ka , pacheco mc , yang j , et al : ovarian sex cord - stromal tumors , pleuropulmonary blastoma and dicer1 mutations : a report from the international pleuropulmonary blastoma registry . 
schultz ka , yang j , doros l , et al : dicer1 - pleuropulmonary blastoma familial tumor predisposition syndrome : a unique constellation of neoplastic conditions . dev pathol 18 : 504 - 511 , 2015 pediatr blood cancer 61 : 1695 - 1697 , 2014 pathol case rev 19 : 90 - 100 , 2014 22 : 564 - 567 , 2014 14 . 
fern andez - martnez l , villegas ja , santamara i , et al : identication of somatic and germ - line dicer1 mutations in pleuropulmonary blastoma , cystic nephroma and rhabdomyosarcoma tumors within a dicer1 syndrome pedigree . 
kline cn , joseph nm , grenert jp , et al : targeted next - generation sequencing of pediatric neuro - oncology patients improves diagnosis , identies pathogenic germline mutations , and directs targeted therapy . 
marks lj , oberg ja , pendrick d , et al : precision medicine in children and young adults with hematologic malignancies and blood disorders : the columbia university experience . 
messinger yh , stewart dr , priest jr , et al : pleuropulmonary blastoma : a report on 350 central pathology - conrmed pleuropulmonary blastoma cases by the international pleuropulmonary blastoma registry . 
 clinical factors that affect the establishment of soft tissue sarcoma patient - derived orthotopic xenografts : a university of california , los angeles , sarcoma program prospective clinical trial tara a . 
eilber , md , division of surgical oncology , university of california , los angeles , 10833 leconte ave , room 54 - 140 chs , los angeles , ca 90095 - 1782 ; e - mail : fceilber@mednet.ucla.edu. purpose given the diverse and aggressive nature of soft tissue sarcomas ( stss ) , a need exists for more - precise therapy . 
this study assessed the feasibility of incorporating pdoxs into the clinical setting and identifying factors associated with pdox establishment . patients and methods from may 2015 to may 2016 , 107 patients with biopsy - proven or potential sts were enrolled . 
tumors exposed to radiation preoperatively did not establish ( zero of 11 [ 0% ] ) , and tumors exposed to neoadjuvant chemotherapy had a lower er ( 31.9% ) than untreated tumors . 
2017 by american society of clinical oncology introduction over the past decade , there has been an evolution toward a personalized approach to the treatment of cancer in which individual patient and tumor characteristics are used to guide therapy . 
although responders tend to have significantly improved outcomes , nonresponders endure the toxic adverse effects of treatment without survival benefit.2 , 3 collectively , the rarity , histologic diversity , high risk of metastasis , and poor response rates to systemic therapy highlight the rationale and immediate need for more personalized sarcoma therapy . 
pdxs maintain genetic similarity with and mimic the therapeutic responses of a patients tumor.4 within stss , multiple subcutaneous pdx models have been developed and have shown promise for replicating the tumor histology , clinical chemosensitivity , growth kinetics , and local disease progression of a patients tumor.5 - 8 patient - derived orthotopic xenograft ( pdox ) models in which the patient - derived tissue is implanted into the corresponding anatomic location by a technique called surgical orthotopic implantation ( soi ) have also been shown to produce patterns of invasion and metastatic spread that have not been demonstrated in the subcutaneous pdx models.8 - 15 as well , pdox models have been shown to faithfully reproduce the histology of the human tumor16 , 17 and to have unique clinical applicability . 
in particular , pdox models have been shown to produce tumor response and resistance that mirror that of the patient.18 smith et al19 demonstrated that xenograftability of liposarcomas correlate directly with patient outcomes . 
by building upon the results of these previous studies , pdox models seem to present the best opportunity to test multiple potentially active systemic agents ( chemotherapy , targeted therapy , etc ) in a preclinical model , which shields patients from the potential toxicity of inactive drugs and identifies effective therapies that improve outcomes . 
all patients elected to enroll and were consented preoperatively . mice athymic nu / nu female nude mice ( anticancer , san diego , ca ) 4 to 6 weeks old were used to xenograft human tissues . 
all animal studies were conducted with an anticancer institutional animal care and use committee protocol specifically approved for this study and in accordance with the principles and procedures outlined in the national institutes of health guide for the care and use of animals under assurance number a3873 - 1.8 , 18 all surgical experiments were done under anesthesia and with analgesia to minimize suffering . 
anesthesia was administered by subcutaneous injection of a 0.02 - ml solution of 20 mg / kg ketamine , 15.2 mg / kg xylazine , and 0.48 mg / kg acepromazine maleate . 
all animals were fed an autoclaved laboratory rodent diet and housed in a barrier facility on a high - efficiency particulate arrestancefiltered rack under standard conditions of 12 - hour light / dark cycles . 
these procedures are similar to the standard of care at our facility and have been previously documented.8 , 18 tumor acquisition and xenograft establishment intraoperatively at the time of tumor resection , a tissue sample was obtained by the operating surgeon ( f.c.e. ) and transported to anticancerpdox on ice for immediate soi in nude mice . each tumor sample was divided into 5 - mm fragments . 
tumor fragments were then implanted both subcutaneously and orthotopically into nude mice using a well - validated protocol.8 , 15 , 18 , 20 - 24 soi procedures were then followed for orthotopic models specific to each tumor site.8 , 11 , 15 , 18 , 20 - 27 for extremity tumors , 10 - mm skin incisions were made in the corresponding anatomic site , and then a single tumor fragment was placed orthotopically between muscle layers . 
wounds were closed with 6 - 0 nylon suture ( ethilon ; ethicon , bridgewater , nj )  . nude mice were monitored for tumor growth for up to 6 months after implantation . 
in every pdox , the histopathology of the established tumors was compared with the original patients tumor and verified for concordance by a specialized sarcoma pathologist ( s.m.d. ) ; this method for histopathologic validation has been previously published by our group.8 , 15 , 18 , 20 , 23 , 24 , 26 , 28 established pdoxs were maintained for future study of potential systemic agents . statistical methods for all patients enrolled in the trial , demographics and clinical data were prospectively collected , and xenograft data ( time to establishment ) were obtained in collaboration with anticancer . 
neoadjuvant treatment information was collected for all patients . only therapy that occurred in the neoadjuvant setting before tumor excision was included in this analysis because the tumor sample used for xenograft implantation was only exposed to therapy that occurred before resection . 
tumors exposed to neoadjuvant therapy were graded by their pathologic response on the basis of the percent response indicated by a specialized sarcoma pathologist . given that xenografts were established only from high - grade tumors , factors associated with establishment and time to establishment were analyzed among the high - grade cohort . 
the rate of establishment , which indicated that the tumor was successfully engrafted and serially passaged , was measured in days from the time of original engraftment to the time adequate growth was achieved from serial passage . 
factors associated with establishment time were analyzed with survival functions using log - rank tests for equality and nonparametric methods for distribution . results study population all 107 patients approached for study enrollment elected to enroll . 
similar numbers of male and female patients were enrolled ( 48% female )  . median patient age was 61 years ( range , 16 to 91 years ) , and median tumor size was 7.6 cm ( range , 0.9 to 38 cm )  . 
the majority of tumors were high grade ( 67% ) , and the most common subtypes were liposarcoma ( 25% ) and leiomyosarcoma ( 14% )  . tumors spanned 28 histologic subtypes ( appendix table a2 , online only )  . 
given that only high - grade tumors were established , the high - grade subpopulation is the focus of the remaining analysis . high - grade cohort pdox development was attempted for 72 patients with high - grade stss . 
for patients who underwent neoadjuvant therapy , the median time between completion of neoadjuvant therapy and tumor resection was 35 days ( range , 9 to 74 days )  . factors associated with establishment of sarcoma pdox model no low - grade stss were established as pdoxs ( zero of 32 )  . 
patient and tumor characteristics for all high - grade tumors and by xenograft success characteristic high grade only ( n = 72 ) successful xenografts only ( n = 32 ) unsuccessful xenografts only ( n = 40 ) female male grade high presentation primary location extremity size , cm , 10  . 
no difference in the likelihood of pdox establishment was found by sex , age , presentation ( primary , recurrent , or metastatic ) , location , subtype , or size . establishment rates varied by subtype ( appendix table a2 )  . 
factors associated with patient - derived orthotopic xenograft establishment among high - grade tumors establishment rate among high - grade tumors ( n = 72 ) univariable logistic regression ( n = 72 ) characteristic % ( no . ) female male grade high presentation primary location extremity size , cm , 10  . 
only one patient with osteosarcoma and one with rhabdomyosarcoma were enrolled , and both tissue samples led to a successful pdox . among high - grade primary stss , 45.5% ( 20 of 44 ) established . 
primary versus recurrent / metastatic tumors : factors associated with patient - derived orthotopic xenograft establishment all high - grade primary tumors ( n = 44 ) high - grade primary establishment rates ( n = 20 ) all high - grade metastatic / recurrent tumors ( n = 28 ) high - grade metastatic / recurrent establishment rates ( n = 12 ) characteristic abdomen / retroperitoneum female male location extremity size , cm , 10  . 
on the basis of establishment rates alone , neoadjuvant treatment seems to have a consistent effect on primary and metastatic / recurrent stss ( table 3 )  . among untreated stss , 61.9% ( 26 of 42 ) were established . 
untreated high - grade tumors : factors associated with establishment high - grade untreated tumors ( n = 42 ) high - grade untreated establishment - rate ( n = 26 ) characteristic female male presentation primary location extremity size , cm , 10  . 
compared with untreated patients , no difference in time to establishment was observed for patients who received neoadjuvant therapy ( p = .180 ; fig 2 )  . specifically , given that none of the grafts exposed to radiation established , no difference in time to establishment was found when untreated grafts were compared with those exposed to neoadjuvant chemotherapy alone . 
furthermore , the study indicates that patients who receive no treatment before tumor resection are most likely to benefit , such that 61.9% of those patients tumors were successfully xenografted . 
the champions oncology trial , which developed 29 subcutaneous pdx models , indicated that growth rates varied and documented an establishment rate of 76% but did not indicate which patient or tumor factors predicted establishment.29 to appropriately select patients for xenograft studies , we first sought to understand which tumors are most likely to grow as xenografts . in the current study , we prospectively enrolled all patients who underwent resection of an sts at a single institution over a 1 - year period . 
specifically , low - grade tumors universally failed to grow , whereas high - grade tumors had an overall establishment rate of 44.4% ( 32 of 72 ) , which no treatment ct only xrt only ct + xrt n = 42 n = 19 days n = 5 n = 6 fig 1 . 
specifically , we found no correlation between the time to establishment and previous treatment - , subtype - , or patient - related factors . the median time to establishment of a xenograft among those that established was 53 days , which indicates that xenografts can be developed over a short period . 
we acknowledge that this amount of time required for xenograft establishment and serial drug testing may take longer than the patients recovery from surgery ; therefore , at present we believe that the results of xenograft testing should be used to supplement care and potentially define second - line therapies rather than replace standard treatments . the current study is limited by the sample size and diverse study population . 
although statistical analyses of the overall group were feasible , smaller subgroup analyses were limited , and therefore , conclusions about individual subtypes or treatment factors could not be made with a high degree of certainty . as we move forward with the use of pdxs as a tool for personalizing sarcoma therapy , we must be cognizant of the patient population with the greatest potential to benefit : patients with high - grade tumors that have not been exposed to neoadjuvant radiation therapy or those without a significant treatment response . 
eilber provision of study materials or patients : kei kawaguchi , yungfeng li , nicholas bernthal , bartosz chmielowski , arun s . singh collection and assembly of data : tara a . 
elliott no relationship to disclose kei kawaguchi no relationship to disclose tasuku kiyuna no relationship to disclose kentaro igarashi no relationship to disclose yungfeng li no relationship to disclose joseph g . 
 nicholas bernthal honoraria : daiichi sankyo consulting or advisory role : onkos surgical , bonesupport research funding : onkos surgical speakers bureau : genentech , roche , janssen pharmaceuticals travel , accommodations , expenses : genentech , roche , bristol - myers squibb , janssen pharmaceuticals , merck jane yanagawa no relationship to disclose anusha kalbasi no relationship to disclose noah federman stock and other ownership interests : genmab , kite pharma consulting or advisory role : zelda therapeutics bartosz chmielowski consulting or advisory role : amgen , bristol - myers squibb , merck , genentech , roche , eisai , immunocore arun s . 
singh consulting or advisory role : eli lilly , daiichi sankyo speakers bureau : eli lilly , eisai travel , accommodations , expenses : eli lilly , daiichi sankyo , eisai stock and other ownership interests : anticancer - pdox robert m . 
hoffman , anticancer ; and takashi murakami , kei kawaguchi , tasuku kiyuna , kentaro igarashi , and robert m . hoffman , university of california , san diego , san diego , ca . supported by the robert wood johnson clinical scholars program and the west los angeles veteran affairs health services research and development center for innovation ( t.a.r. ) and national cancer institute grant no . 
donahue tr , kattan mw , nelson sd , et al : evaluation of neoadjuvant therapy and histopathologic response in primary , high - grade retroperitoneal sarcomas using the sarcoma nomogracancer 116 : 3883 - 3891 , 2010 3 . 
hiroshima y , zhang y , zhang n , et al : patient - derived orthotopic xenograft ( pdox ) nude mouse model of soft - tissue sarcoma more closely mimics the patient behavior in contrast to the subcutaneous ectopic model . 
furukawa t , kubota t , watanabe m , et al : orthotopic transplantation of histologically intact clinical specimens of stomach cancer to nude mice : correlation of metastatic sites in mouse and individual patient donors . 
igarashi k , kawaguchi k , kiyuna t , et al : patient - derived orthotopic xenograft ( pdox ) mouse model of adult rhabdomyosarcoma invades and recurs after resection in contrast to the subcutaneous ectopic model . 
murakami t , singh as , kiyuna t , et al : effective molecular targeting of cdk4 / 6 and igf - 1r in a rare fus - erg fusion cdkn2a - deletion doxorubicin - resistant ewings sarcoma patient - derived orthotopic xenograft ( pdox ) nude - mouse model . 
smith kb , tran lm , tam bm , et al : novel dedifferentiated liposarcoma xenograft models reveal pten downregulation as a malignant signature and response to pi3k pathway inhibition . 
kiyuna t , murakami t , tome y , et al : high efficacy of tumor - targeting salmonella typhimurium a1 - r on a doxorubicinand dactolisib - resistant follicular dendritic - cell sarcoma in a patient - derived orthotopic xenograft pdox nude mouse model . 
kiyuna t , murakami t , tome y , et al : labeling the stroma of a patient - derived orthotopic xenograft ( pdox ) mouse model of undifferentiated pleomorphic soft - tissue sarcoma with red fluorescent protein for rapid non - invasive imaging for drug screening . 
yamamoto m , zhao m , hiroshima y , et al : efficacy of tumor - targeting salmonella a1 - r on a melanoma patientderived orthotopic xenograft ( pdox ) nude - mouse model . 
kawaguchi k , igarashi k , murakami t , et al : tumor - targeting salmonella typhimurium a1 - r combined with temozolomide regresses malignant melanoma with a braf - v600e mutation in a patient - derived orthotopic xenograft ( pdox ) model . 
kawaguchi k , murakami t , chmielowski b , et al : vemurafenib - resistant braf - v600e - mutated melanoma is regressed by mek - targeting drug trametinib , but not cobimetinib in a patient - derived orthotopic xenograft ( pdox ) mouse model . 
murakami t , delong j , eilber fc , et al : tumor - targeting salmonella typhimurium a1 - r in combination with doxorubicin eradicate soft tissue sarcoma in a patient - derived orthotopic xenograft ( pdox ) model . 
murakami t , igarashi k , kawaguchi k , et al : tumor - targeting salmonella typhimurium a1 - r regresses an osteosarcoma in a patient - derived xenograft model resistant to a molecular - targeting drug . 
kawaguchi k , igarashi k , murakami t , et al : tumor - targeting salmonella typhimurium a1 - r sensitizes melanoma with a braf - v600e mutation to vemurafenib in a patient - derived orthotopic xenograft ( pdox ) nude mouse model . j cell biochem 118 : 2314 - 2319 , 2017 28 . 
igarashi k , kawaguchi k , murakami t , et al : intra - arterial administration of tumor - targeting salmonella typhimurium a1 - r regresses a cisplatin - resistant relapsed osteosarcoma in a patient - derived orthotopic xenograft ( pdox ) mouse model . 
patient and tumor characteristics ( high grade and low grade ) characteristic all xenografts ( n = 107 ) female male grade high presentation primary location trunk extremity size , cm , 10  . 
 association of germline genetic test type and results with patient cancer worry after diagnosis of breast cancer background there are concerns that multigene panel testing compared with brca1 / 2 - only testing after diagnosis of breast cancer may lead to unnecessary patient worry about cancer because of more ambiguous results . methods patients with breast cancer diagnosed from 2013 to 2015 and accrued from seer registries in georgia and los angeles were surveyed ( n = 5 , 080 ; response rate , 70% ) , and responses were merged with seer data and germline genetic testing and results . 
we examined patient reports of cancer worry by test type and results in 1 , 063 women who linked to a genetic test and reported undergoing testing . results more than half of the sample ( n = 640 ; 60.2% ) received brca1 / 2 - only testing versus 423 patients ( 39.8% ) who had a multigene panel . 
a minority of tested patients reported substantial cancer worry after treatment : 11.1% ( n = 130 ) reported higher impact of cancer worry , and 15.1% ( n = 162 ) reported a high frequency of cancer worry ( worrying often or almost always ) in the past month . 
2018 by american society of clinical oncology introduction multiple - gene panel ( mgp ) testing has rapidly replaced brca1 / 2 - only testing after diagnosis of breast cancer.1 however , genetic testing use in patients who need it seems to be low.2 , 3 one barrier to more robust uptake of mgp testing is uncertainty about its clinical utility . 
indeed , some have argued that mgp testing should not replace brca1 / 2 - only testing , even in patients with higher pretest risk of a pathogenic mutation , because of these concerns about patient reactions , 4 whereas others strongly disagree.5 current clinical guidelines that recommend genetic testing in women at elevated pretest risk of carrying a pathogenic variant do not specify how many genes should be tested . 
we sent surveys to 7 , 810 patients : 507 patients were ineligible because of the exclusions noted previously or were deceased , institutionalized , too ill , or unable to complete a survey in spanish or english , leaving 7 , 303 patients . 
virtually all patients ( 5 , 026 ) had complete information for the test linkage phase , of whom 1 , 272 ( 25.3% ) linked to a genetic test result , 1 , 063 of whom ( 21.2% ) reported receipt of genetic testing in the survey . survey responses were merged with seer clinical data and genetic testing information obtained from four laboratories ( ambry genetics , aliso viejo , ca ; genedx , gaithersburg , md ; invitae , san francisco , ca ; myriad genetics , salt lake city , ut ) that performed nearly all germline cancer genetic testing in the study regions . 
test type and results were merged to 5 , 026 patients with complete information on all variables for seer genetic testing linkage using a probabilistic matching strategy performed by information management services ( rockville , md )  . 
the safe harbor method was used to de - identify the data set before transfer to the university of michigan for analysis.9 the collaboration was covered under data use agreements between the university of michigan , genetic laboratories , and information management services . 
the research was approved by institutional review boards of the university of michigan , emory university , the university of southern california , the georgia department of public health , the california state committee for the protection of human subjects , and california cancer registry . 
waivers of informed consent and authorization were approved , given the use of a third - party honest broker to conduct the linkage and create a de - identified data set for analyses . measures we used two questions to evaluate patient worry about future cancer . 
forest plot ( adjusted odds ratios and 95% cis ) for results of a multivariable model regressing a binary variable indicating higher versus low impact of cancer worry on test type , controlling for selected predisposing , clinical , and treatment factors and weighted for survey design and nonresponse . 
er , estrogen receptor ; her2 , human epidermal growth factor receptor 2 ; hs , high school ; mgp , multigene panel ; pr , progesterone receptor ; ref , reference . made it difficult for you to carry out your usual daily activities at home or work ? ; made you feel distant from family or friends ? ( responses were based on 5 - point likert categories from almost never to almost always )  . 
we developed a scale by averaging the responses across the three items to create a continuous range from 1 to 5 , with higher values indicating greater impact of cancer worry . 
we then created a binary variable indicating higher impact of cancer worry versus low impact using a cut point of 3.0 , which corresponded to responses of sometimes , often , or almost always . 
we also measured frequency of cancer worry for validation , which was assessed using a single item : in the past month , how often have you worried about your cancer coming back ? ( almost never to almost always ; five point categories )  . 
genetic laboratories provided results at the level of the gene tested ( eg , brca1 ) and the clinical interpretation sent to the ordering clinician ( consisting of pathogenic , likely pathogenic , uncertain significance , likely benign , or benign )  . 
we categorized a test that assessed only brca1 / 2 as brca1 / 2 only and a test that assessed any additional gene ( eg , atm , chek2 ) as a multigene panel . 
if patients received both tests on separate occasions ( eg , a brca1 / 2 - only test and later mgp ) , they were coded as having received mgp . 
we assessed clinical factors related to future cancer risk ( tumor behavior , triple - negative disease , nodal status , bilateral disease ) , treatments received , age ( in 10 - year groups ) , education , and family history . 
 defined as having two or more first - degree relatives with breast cancer ; any relatives with ovarian cancer , sarcoma , or male breast cancer ; ashkenazi jewish ancestry ; or a family history of a mutation - conferring risk ( eg , brca1 / 2 )  . statistical analysis we first examined cancer worry variables by test type , test results , and selected covariables . 
a minority of tested patients reported substantial cancer worry after treatment : 11.1% ( n = 130 ) reported high impact of cancer worry , and 15.1% ( n = 162 ) reported high frequency of worry ( worrying often or almost always in the past month )  . 
we observed similar findings in a model of frequency of cancer worry ( data not shown )  . figure 1 shows the results of a multivariable model regressing a binary variable indicating higher versus low impact of cancer worry on test type , controlling for selected predisposing , clinical , and treatment factors . 
in sensitivity analyses , we examined different permutations of the cancer worry variables , including different cut points for higher worry , and using a continuous variable , and results were consistent . discussion we found that the type of genetic testing patients received ( mgp v brca1 / 2 only ) was not associated with their report of cancer - related worry ( impact or frequency )  . 
 as noted in our prior work , cancer - related worry was associated with younger age and lower educational level , but not clinical factors.10 , 11 strengths of this study include a large population based sample of patients recently diagnosed with breast cancer and accrued shortly after the advent of multigene panel testing in the community , genetic testing results from companies linked to seer clinical data , and granular information about patients , including their report of cancer - related worry . 
we excluded them from the analytic sample because we did not think it was valid to assess the association of a test type with attitudes about health risk among patients who did not recall that they had the test . 
finally , generalizability of the findings are limited to two large geographic regions of the united states . the results from this study and our prior work suggest that testing more genes versus brca1 / 2 alone does not seem to foment more negative patient reactions . 
our prior research in this cohort suggested that mgp testing ( v brca1 / 2only testing ) does not lead to inappropriate rates of contralateral prophylactic mastectomy.1 virtually all testers in this study received some form of genetic counseling , 6 and our results suggest these efforts may sufficiently frame results in a manner that did not alarm patients . 
kurian manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors paul abrahamse no relationship to disclose sarah t . 
dolinsky , cgc , and melissa pronold at ambry genetics , delores bowman and benjamin solomon at genedx , edward esplin and stephen lincoln at invitae , and johnathan lancaster and brian dechairo at myriad genetics for their collaboration on the genetic test data linkage to seer data . 
the collection of los angeles county cancer incidence data used in this study was supported by the california department of public health pursuant to california health and safety code section 103885 , centers for disease control and preventions ( cdcs ) national program of cancer registries , under cooperative agreement 5nu58dp003862 - 04 / dp003862 , the national cancer institutes seer program under contract hhsn261201000140c awarded to the cancer prevention institute of california , contract hhsn261201000035c awarded to the university of southern california , and contract hhsn261201000034c awarded to the public health institute . 
the collection of cancer incidence data in georgia was supported by contract hhsn261201300015i , task order hhsn26100006 from the national cancer institute and cooperative agreement 5nu58dp003875 - 04 - 00 from the cdc . 
us department of health & human services : guidance regarding methods for de - identification of protected health information in accordance with the health insurance portability and accountability act ( hipaa ) privacy rule . 
janz nk , li y , zikmund - fisher bj , et al : the impact of doctor - patient communication on patients perceptions of their risk of breast cancer recurrence . 
forest plot ( adjusted odds ratios and 95% cis ) showing results of multivariable logistic regression for higher impact of cancer worry ( complete case n = 1 , 044 )  . 
 alk testing trends and patterns among community practices in the united states purpose targeted therapy of alk in patients with metastatic nonsquamous nonsmall - cell lung cancer ( nsclc ) and alk rearrangements improves outcomes compared with chemotherapy . 
logistic regression was used to examine the association between demographic and clinical characteristics and testing for alk rearrangements . results of 31 , 483 patients analyzed from the database , 16 , 726 patients ( 53.1% ) were tested for alk rearrangements . 
median ( interquartile range ) time from laboratory receipt of sample to first alk test result was 7 ( 7 ) days ; median time from advanced diagnosis to first alk test result was 25 ( 29 ) days . 
patients who were older , male , had a history of smoking , lived in non - western us regions , and who had recurrent disease or squamous histology were less likely to be tested for alk . 
2018 by american society of clinical oncology introduction recent advances in molecular genetics have increased the opportunity for precision medicine in metastatic nonsquamous nonsmall - cell lung cancer ( nsclc )  . 
for instance , translocation of the anaplastic lymphoma kinase ( alk ) gene is found in approximately 2% to 8% of all patients with nsclc and occurs primarily in patients who are never - smokers or light ex - smokers with adenocarcinoma histology.1 - 4 in addition to representing a distinct molecular nsclc subtype , alk rearrangements are indicative of tumor cell susceptibility to alk inhibitors.5 targeted inhibition of alk in patients with alk rearrangements can decrease tumor burden and significantly increase progression - free survival compared with chemotherapy.3 , 6 the first alk inhibitor , crizotinib , along with the vysis alk break apart fish probe kit , was given accelerated approval by the us food and drug administration ( fda ) in 2011 . 
since then , other firstand second - generation alk inhibitors have been approved , including ceritinib in 2014 and alectinib in 2015 . the college of american pathologists , international association for the study of lung cancer , and association for molecular pathology ( cap / iaslc / amp ) molecular testing and national comprehensive cancer network ( nccn ) peter b . 
 nsclc treatment guidelines recommend testing for alk , egfr , and ros1 in lung adenocarcinomas and in nsclc where an adenocarcinoma component cannot be excluded , regardless of clinical characteristics.1 , 7 , 8 fluorescent in situ hybridization ( fish ) or immunohistochemistry ( ihc ) are assays recommended by cap / iaslc / amp guidelines for the detection of alk rearrangements before selection of targeted therapy.8 in addition to fish , the alk ( d5f3 ) cdx assay ( ventana medical systems , tucson , az ) and foundationone cdx ( foundation medicine , cambridge , ma ) are ihc and next - generation sequencing ( ngs ) assays , respectively , that are fda approved to identify patients eligible for alk inhibitor treatment . 
alk rearrangements could also be detected using polymerase chain reaction , but no fda - approved assay is available to date , and this method is not currently recommended in guidelines.8 numerous challenges associated with molecular testing of malignancies may affect adoption of molecular testing guidelines and performance of alk testing in clinical practice . 
a major challenge in molecular testing is inadequate tissue samples.9 , 10 for example , in a study that examined the workflow of molecular testing for 157 lung cancer specimens , successful testing of all molecular targets could not be completed for 19% of specimens.11 additional challenges may include tumor heterogeneity , complex results that require multidisciplinary input , education and training of laboratory and staff , availability of assays , and patient affordability.9 , 10 in a retrospective analysis of 814 patients in new jersey and maryland with nonsquamous nsclc who were identified using the cota database ( cota , new york , ny ) , which extracts and organizes data from electronic health records , only 65% underwent alk testing , indicating limited integration of molecular testing recommendations into clinical practice.12 given the clinical benefit of alk inhibitor therapy in patients with nsclc with alk rearrangements , evaluation of alk testing using large , real - world , geographically diverse data sources is warranted . 
the objective of this study was to assess real - world alk testing patterns among community practices in the united states in patients with advanced nsclc from january 2011 through may 2017 . methods study overview and database description this was a retrospective analysis using the flatiron health electronic health record ( ehr ) derived database , a longitudinal , demographically and geographically diverse database.13 the database includes > 265 cancer clinics ( approximately 800 sites of care ) representing more than 2 million active us patients with cancer available for analysis . 
the current analysis was limited to data from community practices and included 34 , 911 patients diagnosed with advanced nsclc ( stage iiib or iv by american joint committee on cancer 7th edition ) from january 2011 through may 2017 . 
insurance information was categorized into commercial ( including those with dual medicare or medicaid coverage ) , medicare ( including dual medicaid coverage ) , medicaid , and other / unknown . 
 ( % ) unless otherwise noted . abbreviations : alk , anaplastic lymphoma kinase ; iqr , interquartile range , q3 - q1 ; nos , not otherwise specified ; nsclc , nonsmall - cell lung cancer . date specimens were received by the laboratory to the test report date . data analysis statistical analyses were primarily descriptive , with frequencies calculated for categorical data and means , medians , interquartile ranges ( iqrs ) , and standard deviations ( sds ) calculated for continuous data . 
odds ratios ( ors ) and 95% cis were calculated by logistic regression to examine the association between demographic and clinical characteristics and testing for alk rearrangements . results patient characteristics overall , 34 , 911 patients with advanced nsclc were identified from the flatiron health ehr - derived database from january 2011 through may 2017 ; 31 , 483 met the selection criteria and were included in the analysis . 
most of the samples used for alk testing were tissue ( 93.7% ; 15 , 664 of 16 , 726 ) ; 2.6% ( 439 of 16 , 726 ) were blood , and the rest were unknown . 
of the total 21 , 639 patients with nonsquamous histology , 66.9% ( 14 , 478 of 21 , 639 ) were tested for alk , and of the 7 , 923 patients with squamous histology , 18.5% ( 1 , 466 of 7 , 923 ) were tested for alk . 
 among the patients who had nonsquamous histology and who were not tested for alk , 87.2% ( 6 , 025 of 6 , 913 ) had a history of smoking , and among those with a squamous histology and who were either tested or not tested for alk , 91.5% ( 1 , 320 of 1 , 443 ) and 95.6% ( 5 , 992 of 6 , 268 ) , respectively , had a history of smoking . 
overall , 16 , 726 of 31 , 483 ( 53.1% ) patients were tested for alk , with 633 of 16 , 726 ( 3.8% ) patients positive for alk ( 581 of 14 , 478 [ 4.0% ] nonsquamous histology , 24 of 1 , 466 [ 1.6% ] squamous histology , and 28 of 782 [ 3.6% ] not otherwise specified histology )  . alk testing patterns alk testing rates by year of advanced diagnosis steadily increased over time from an average of 32.4% ( 1 , 093 of 3 , 371 ) in 2011 to 62.1% ( 3 , 526 of 5 , 680 ) in 2016 for all patients with advanced nsclc , from 41.1% ( 935 of 2 , 273 ) in 2011 to 75.1% ( 2 , 947 of 3 , 924 ) in 2016 for patients with nonsquamous histology , and from 12.1% ( 101 of 832 ) in 2011 to 26.5% ( 372 of 1 , 403 ) in 2016 for patients with squamous histology ( fig 1a )  . 
with test 2012 2014 year 178 201 245 368 395 471 483 446 525 595 593 625 665 765 717 746 728 801 891 820 860 805 787 702 631 2015 2013 2016 2017 squamous , no . 
 age , years 55 - 64 v < 55 65 - 74 v < 55 75 - 84 v < 55 85 + v < 55 female v male race asian v white black or african american v white other race v white missing v white hispanic v non - hispanic ethnicity histology nos v non squamous squamous v non squamous disease type recurrent v de novo unknown v de novo smoking status history of smoking v no history unknown history v no history insurance medicaid v commercial medicare v commercial other v commercial region midwest v west northeast v west south v west missing v west fig 2 . 
the overall median ( iqr ) turnaround time from the date samples were received by the laboratory to the test report date was 7 ( 7 ) days and ranged from 6 days for fish to 12 days for ngs ( table 2 )  . 
the overall median ( iqr ) time from advanced diagnosis date to first alk test result date was 25 ( 29 ) days ( table 2 ) and differed by type of test and insurance type . 
 median ( iqr ) times from advanced diagnosis to first alk test result for fish were 20 ( 19 ) , 22 ( 31 ) , and 23 ( 27 ) days for commercial insurance , medicaid , and medicare , respectively , and for ngs were 37 ( 34 ) , 58 ( 159 ) , and 41 ( 105 ) days , respectively ( table 2 )  . 
on the basis of tests conducted by foundation medicine ( cambridge , ma ) laboratory , the overall agreement between ngs and fish was 97.6% ( 360 of 369 )  . 
the percentage of tests positive by ngs that were initially negative by fish was 1.9% ( 7 of 369 )  . treatment before alk test results among patients tested for alk who had valid tissue collection and test result dates , 21.5% ( 3 , 290 of 15 , 320 ) initiated therapy before receiving their first alk test results . 
percentage of patients initiating treatment before first anaplastic lymphoma kinase ( alk ) test result . discussion this retrospective analysis of patients with advanced nsclc treated at community practices between january 2011 and may 2017 found that 53.1% of all patients , 66.9% of patients with nonsquamous histology , and 18.5% of patients with squamous histology were tested for alk rearrangements . 
cap / iaslc / amp and nccn guidelines recommend alk testing be conducted for patients with nsclc that has an adenocarcinoma component.7 , 8 however , alk testing may also be considered for patients with nsclc who have mixed histology , who are of young age , or who are without a history of tobacco exposure.7 , 8 the current analysis confirms and extends the findings of a retrospective analysis of 814 patients with nonsquamous nsclc treated in community centers in the united states , which found an overall 65% alk testing rate for the years 2013 to 2015.12 alk testing rates increased over time , reaching 75% in 2016 in patients with nonsquamous histology , indicating improvement in adherence to alk testing recommendations as well as physician recognition of the clinical benefits of alk targeted therapy . 
 the reasons for lack of testing were not captured in the flatiron health database , although possible reasons may include evolution of best practices and guidelines across the study period , patient disease severity , and insufficient tissue ( as was observed in 16% to 21% of patients in other nsclc studies ) .4 , 14 , 15 blood - based assays could address the issue of insufficient tissue , and research in this field is emerging.16 patients with squamous histology were less likely to be alk tested , which is consistent with guidelines that state there is no evidence to recommend alk testing in patients with typical squamous histology who do not have other clinical features to prompt alk testing.7 , 8 in the current analysis , testing rates increased over time in patients with squamous histology , and , overall , 1.6% had an alk rearrangement . 
the increased testing rate for patients with squamous histology may be due to a general increase in the perceived benefit of alk inhibitors for alk - positive nsclc by physicians or to the release of cap / iaslc / amp and nccn guidelines that suggest selectively testing patients with squamous histology , such as patients who are never - smokers or former light smokers.1 , 17 the likelihood of alk testing decreased as age increased . 
patients with alk - positive disease in clinical studies tended to be younger , nonsmokers , and have adenocarcinoma , perhaps increasing the likelihood that these patients would have been tested for alk positivity despite guidelines recommending that all patients with nonsquamous disease be tested . 
in addition , the current analysis suggests there may also be socioeconomic barriers to alk testing ; medicaid patients were 40% less likely to be tested than patients with commercial insurance . 
collectively , these results suggest that additional education on alk testing in the community oncology setting is warranted . in our study , fish was the most commonly ordered genomic testing method , which was not unexpected , given that fish was the only recommended and fda - approved assay during the majority of the time period for our study . 
 updated 2018 cap / iaslc / amp and nccn guidelines now recommend fish or ihc for alk testing.7 , 8 with the rapidly changing landscape of fda - approved assays , such as the 2017 approval of the foundationone cdx ngs assay , the distribution of assays used will likely continue to evolve . 
indeed , in our study we observed a trend of declining use of fish and an increase in the use of ngs and , to a lesser extent , ihc . 
there is a trend in clinical practice to use ngs as the primary testing method to identify all targetable alterations.18 , 19 turnaround times for laboratory receipt of sample to test results were consistently less than 2 weeks across different test types , including ngs . 
the reasons for this variation are unknown ; however , potential reasons for longer times for some patients may include the ordering process for biomarker testing , sample shipment to the laboratory , and sample processing . 
for example , a previous study in metastatic colon cancer found that testing performed at a nonlocal or noncancer centeraffiliated laboratory was associated with greater likelihood of a testing delay.20 the current study was conducted in the community setting , and the majority of biomarker testing was done in commercial laboratories , which may be one of the possible explanations for the observed variance in times . 
in addition , the 14 - day rule may also have contributed to the variation in times for some medicare patients , because testing may have been delayed to avoid reimbursement complexities . numerically , ngs had a longer time from advanced diagnosis to alk test result for both medicare and medicaid patients . 
given that turnaround times for ngs were similar across insurance types and only accounted for a minor portion of the time from advanced diagnosis to alk test result , this may imply that delays in ordering ngs , which was not an approved test during the study period , may have existed . 
additional research is needed to determine if this trend will continue in the future , particularly given the 2017 simultaneous fda approval of ngs tests for tumor profiling and centers for medicare & medicaid services approval of coverage for fda - approved ngs tests , which may substantially improve the ability to provide more timely access to ngs testing.21 , 22 the lack of alk testing or delay in results may have serious clinical implications , given the efficacy of alk inhibitors in patients with alk rearrangements.3 , 6 , 23 , 24 in a head - to - head comparison , a second - generation alk inhibitor ( alectinib ) exhibited a median progression - free survival of 34.8 months , compared with 10.9 months with a first - generation alk inhibitor ( crizotinib ) .24 , 25 delays in alk testing may potentially lead to delayed treatment , 15 and , as is seen in other cancers , delays in treatment may lead to poorer outcomes.26 , 27 although additional research is needed to assess the impact of delays on outcomes , specifically in alk - positive nsclc , alk testing is needed to help facilitate timely treatment with an efficacious alk inhibitor . day - level granularity of biomarker testing ordered date is not always available in ehr data , which has limited our ability to interpret and determine reasons for observed delays in testing . 
 last , this study focused on community practices in the united states , and hence the results of this study may not be generalizable to all clinical practices . the results of the current analysis indicate that the frequency of alk testing in patients with advanced nsclc treated in community centers in the united states has increased over time . 
 however , certain subgroups of patients , such as patients receiving medicaid , were less likely to be tested , suggesting that additional education on genomic testing and investigation into socioeconomic barriers is warranted . 
 laura chu employment : genentech stock and other ownership interests : roche ravindra gupta employment : genentech katja schulze employment : genentech stock and other ownership interests : genentech matthew a . 
gubens consulting or advisory role : bristol - myers squibb , ariad pharmaceuticals , genentech , astrazeneca , abbvie , novartis , mersana therapeutics research funding : celgene ( inst ) , merck ( inst ) , novartis ( inst ) , genentech ( inst ) , oncomed ( inst ) acknowledgment medical writing and editorial assistance was provided by erin p . 
illei , johns hopkins university school of medicine , baltimore , md ; william wong , laura chu , ravindra gupta , and katja schulze , genentech , south san francisco ; matthew a . 
gubens , university of california , san francisco , ca ; and ning wu , pro unlimited , boca raton , fl . support supported by genentech , south san francisco , ca . prior presentation presented in part at the international association for the study of lung cancer 18th world conference on lung cancer , yokohama , japan , october 15 - 18 , 2017 . 
lindeman ni , cagle pt , beasley mb , et al : molecular testing guideline for selection of lung cancer patients for egfr and alk tyrosine kinase inhibitors : guideline from the college of american pathologists , international association for the study of lung cancer , and association for molecular pathology . 
korpanty gj , graham dm , vincent md , et al : biomarkers that currently affect clinical practice in lung cancer : egfr , alk , met , ros - 1 , and kras . 
lindeman ni , cagle pt , aisner dl , et al : updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors : guideline from the college of american pathologists , the international association for the study of lung cancer , and the association for molecular pathology . 
distasio m , chen y , rangachari d , et al : molecular testing turnaround time for non - small cell lung cancer in routine clinicalpractice confirms feasibility of cap / iaslc / amp guideline recommendations : a single - center analysis . 
mayo - de - las - casas c , garzn ibez m , jordana - ariza n , et al : an update on liquid biopsy analysis for diagnostic and monitoring applications in non - small cell lung cancer . 
suh jh , johnson a , albacker l , et al : comprehensive genomic profiling facilitates implementation of the national comprehensive cancer network guidelines for lung cancer biomarker testing and identifies patients who may benefit from enrollment in mechanism - driven clinical trials . 
jensen ts , chin j , baldwin j , et al : proposed decision memo for next generation sequencing ( ngs ) for medicare beneficiaries with advanced cancer ( cag - 00450n )  . 
soria jc , tan dsw , chiari r , et al : first - line ceritinib versus platinum - based chemotherapy in advanced alk - rearranged non - small - cell lung cancer ( ascend - 4 ) : a randomised , open - label , phase 3 study . 
 o addition of chemotherapy is associated with decreased survival in early - stage ( t1 - 2n0m0 ) glottic squamous cell carcinoma treated with definitive radiotherapy purpose the role of chemoradiation ( crt ) in treating patients with early - stage glottic squamous cell carcinoma ( scc ) , especially for t2n0m0 glottic scc with impaired vocal cord mobility , remains unexplored . 
we sought to evaluate the impact of crt on survival in early - stage glottic scc by using the seer database . patients and methods we included patients with localized ( t1 - 4n0m0 ) glottic scc ( n = 4 , 743 ) diagnosed between 2004 and 2014 and treated with definitive radiotherapy ( rt ) alone , crt , or laryngectomy alone in the seer database . 
disease - specific mortality ( dsm ) was evaluated via multivariable regression using a competing risk model that accounts for other - cause mortality as a competing risk event for dsm . 
2019 by american society of clinical oncology introduction definitive treatment with radiotherapy ( rt ) for t1 - 2n0m0 squamous cell carcinomas ( scc ) of the glottis remains a standard of care in the united states and can help to preserve voice quality . 
several studies have shown lc rates for t1 and t2 glottic cancer treated with transoral laser microsurgery to range from 77% to 92% and 66% to 88% , respectively , 1 - 5 whereas open surgical procedures for glottic scc demonstrated a lc rate of 86% to 98%.2 , 6 - 10 the lc rate for early - stage glottic cancer treated with definitive rt depends significantly on t stage , with studies showing lc rates of 82% to 94% for t1 disease11 - 17 and 61% to 80% for t2 disease.11 - 13 , 15 - 19 the decreased lc for t2 glottic cancers treated with rt alone was thought to be attributed to a subset of patients with impaired vocal cord chenyang wang amar u . 
 the radiation therapy oncology group 9111 trial established concurrent crt as the standard treatment of t3 glottic cancer.28 the lc benefit associated with crt in the setting of t3 glottic cancer has raised the question of whether the subset of patients with t2 glottic cancer with impaired vocal cord mobility should be treated with crt as well , given the suboptimal outcomes with rt alone . 
seer routinely collects data on patient demographics , primary tumor site , morphology , stage at diagnosis , first course of treatment , and follow up for vital status . for this study , we selected patients diagnosed with n0m0 scc of the glottic larynx diagnosed from january 1 , 2004 , to december 31 , 2014 , who underwent definitive rt , crt , or subtotal or total laryngectomy . 
we excluded patients who had non - scc histology , unknown tumor grade , unknown race , unknown surgical status , unknown t stage , more than one primary malignancy , no active follow - up , or age younger than 18 years . 
this study was reviewed and approved by the institutional review board of the university of california , los angeles . statistical analysis extracted variables included age at diagnosis , sex , race , year of diagnosis , t stage , and tumor grade . 
analysis of variance , kruskal - wallis rank sum tests , and 2 tests were used to characterize differences in variables of interest between different treatment cohorts for continuous , ordinal , and categorical variables , respectively . 
 follow - up time was calculated via the reverse kaplan - meier method described by schemper and smith , 30 in which alive at last follow - up was designated the event of interest while death from any cause was censored . 
seer determines death and cause of death via algorithms to process death certificates , taking into account causes of deaths in conjunction with tumor sequence , site of original cancer diagnosis , and comorbidities . 
 to account for other - cause mortality ( ocm ) as a competing risk for dsm , multivariable regression analyses on the basis of the competing risk model as described by fine and gray31 were applied to the survival analysis while adjusting for age , year of diagnosis , sex , race , t stage , definitive treatment modality , and tumor grade . 
for t2n0 disease , an additional subset analysis was carried out on the basis of vocal cord impairment status to investigate whether patients with t2n0 disease with vocal cord impairment selectively benefit from crt . 
the validity of proportional hazards assumption was evaluated via schoenfeld residuals . to adjust for covariables and determine whether the addition of chemotherapy to rt affected dsm in patients with t1 - 4n0m0 glottic scc , we adopted propensity score matching ( psm ) via a nearest neighbor approach to balance patient characteristics including age , year of diagnosis , sex , race , and tumor grade between the rt and crt cohorts for each t stage . 
propensity score , first defined by rosenbaum and rubin32 as the probability of treatment assignment on the basis of a set of observed baseline patient characteristics , provides the means to retrospectively balance distribution of patient characteristics and thereby minimize effects of unmeasured confounders on treatment outcomes . 
the cumulative incidences of dsm and ocm after psm were plotted to illustrate the difference between rt and crt cohorts for each t stage , and their distributions were evaluated via a k - sample test described by gray.33 all statistical analyses were carried out using r version 3.4.1 ( r foundation for statistical computing , vienna , austria ) with two - sided testing and a statistical significance threshold of p = .05. results patient characteristics patient characteristics , stratified by definite treatment modality consisting of rt , crt , and subtotal or total laryngectomy , are listed in table 1 . 
patients who underwent rt alone tended to be older , with a median age of 66 years , compared with patients who underwent crt and surgery , who had median ages of 62 and 63 years , respectively . 
these patients were also more likely to present with higher tumor grade . multivariable regression for dsm and the results of the multivariable regression for dsm according to the competing risk model , accounting for ocm as a competing risk , are listed in table 2 . 
 plots of schoenfeld residuals versus failure times are shown in appendix figure a2 . subgroup multivariable regression for dsm using a competing risk model stratified by t stage the results of subgroup multivariable regression for dsm stratified by t stage are listed in table 3 . 
more recent year of diagnosis was not associated with any significant difference in dsm . cumulative incidence of dsm and ocm for rt and crt after psm patient characteristics stratified by definitive treatment modality before and after psm are listed in appendix tables a1 and a2 , respectively . 
a recent phase ii trial of concurrent crt with a fluoropyrimidine agent for t2n0m0 scc of the larynx ( 70% ) , oropharynx ( 16% ) , and hypopharynx ( 14% ) demonstrated a 3 - year lc rate of 89.0% and a 3 - year overall survival rate of 97.2%.29 another study by bhateja et al25 demonstrated superior lc in patients receiving crt for t2b - 3n0 - 2 glottic cancers compared with patients receiving rt alone for t2n0m0 glottic cancer with impaired vocal cord mobility ( t2bn0 ) , suggesting a potential therapeutic benefit of crt in this setting . 
in this study , 51.6% of the t2bn0 patients treated with rt alone received altered fractionation ( > 2.1 gy per fraction or twice - a - day fractionation ) compared with 38.5% of patients in the crt cohort . 
of note , the crt cohort contained only one patient with t2bn0 and one patient with t2bn1 ; therefore , it is difficult to assess the impact of crt on patients with t2bn0 disease . 
in accordance with the results from bhateja et al , 25 our study also demonstrated that patients with t3n0 glottic scc had decreased dsm with crt compared with rt alone . 
cumulative incidence plots of disease specific mortality ( dsm ) and other - cause mortality ( ocm ) for glottic squamous cell carcinoma at each t stage after propensity score matching . 
to our knowledge , our data from this study are the first to question the utility of chemotherapy in addition to rt for t2bn0 glottic scc . one possible hypothesis for the increase in dsm for patients with t1 - 2n0 glottic scc treated with crt may be treatment prolongation as a result of increased toxicity . 
rt treatment time prolongation past 40 days has been shown to correlate with decreased lc from 95% to 100% to 79% to 84%.13 , 34 additional factors that can contribute to the observed survival detriment in crt may be lower total rt dose and dose per fraction as a result of the higher combined toxicity with chemotherapy , because it has been established that total rt dose less than 65 gy results in decreased lc.2 , 10 , 13 , 14 , 35 furthermore , a randomized prospective study has shown that 2.25 gy per fraction , as compared with 2 gy per fraction , results in significantly improved lc.36 meanwhile , the survival benefit associated with chemotherapy in locally advanced glottic scc may be attributed to improved lc secondary to chemotherapy sensitization and / or additional regional and distant control , which could compensate for the decrease in lc associated with potential treatment prolongation . this study had several important limitations inherent to the seer database , including lack of information on radiation dose , treating physician experience , the type of treatment institution , and sequence of chemotherapy relative to rt , which prevented us from distinguishing between concurrent versus induction chemotherapy . 
however , given the relatively rare use of induction chemotherapy in the modern era for t1 - 2n0 glottic scc , it is reasonable to assume that the patients in the crt cohort of this study predominantly received concurrent crt . 
steinberg honoraria : viewray consulting or advisory role : viewray research funding : viewray , accuray travel , accommodations , expenses : viewray robert chin travel , accommodations , expenses : varian medical systems , brainlab affiliations chenyang wang , amar u . 
spector jg , sessions dg , chao ksc , et al : stage i ( t1 n0 m0 ) squamous cell carcinoma of the laryngeal glottis : therapeutic results and voice preservation . 
laccourreye o , muscatello l , laccourreye l , et al : supracricoid partial laryngectomy with cricohyoidoepiglottopexy for early glottic carcinoma classified as t1 - t2n0 invading the anterior commissure . 
marshak g , brenner b , shvero j , et al : prognostic factors for local control of early glottic cancer : the rabin medical center retrospective study on 207 patients . 
le q - tx , fu kk , kroll s , et al : influence of fraction size , total dose , and overall time on local control of t1t2 glottic carcinoma . 
cellai e , frata p , magrini sm , et al : radical radiotherapy for early glottic cancer : results in a series of 1087 patients from two italian radiation oncology centersi . 
warde p , osullivan b , bristow rg , et al : t1 / t2 glottic cancer managed by external beam radiotherapy : the influence of pretreatment hemoglobin on local control . 
mendenhall wm , parsons jt , million rr , et al : t1t2 squamous cell carcinoma of the glottic larynx treated with radiation therapy : relationship of dose - fractionation factors to local control and complications . 
frata p , cellai e , magrini sm , et al : radical radiotherapy for early glottic cancer : results in a series of 1087 patients from two italian radiation oncology centersii . 
garden as , forster k , wong p - f , et al : results of radiotherapy for t2n0 glottic carcinoma : does the 2 stand for twice - daily treatment ? int j radiat oncol biol phys 55 : 322 - 328 , 2003 20 . 
compton cc , byrd dr , garcia - aguilar j ( eds ) : ajcc cancer staging atlas : a companion to the seventh editions of the ajcc cancer staging manual and handbook ( ed 2 )  . 
gorphe p , blanchard p , breuskin i , et al : vocal fold mobility as the main prognostic factor of treatment outcomes and survival in stage ii squamous cell carcinomas of the glottic larynx . 
forastiere aa , zhang q , weber rs , et al : long - term results of rtog 91 - 11 : a comparison of three nonsurgical treatment strategies to preserve the larynx in patients with locally advanced larynx cancer . 
taguchi t , takahashi m , nishimura g , et al : phase ii study of concurrent chemoradiotherapy with s - 1 in patients with stage ii ( t2n0m0 ) squamous cell carcinoma of the pharynx or larynx . 
van der voet jcm , keus rb , hart aam , et al : the impact of treatment time and smoking on local control and complications in t1 glottic cancer . 
yamazaki h , nishiyama k , tanaka e , et al : radiotherapy for early glottic carcinoma ( t1n0m0 ) : results of prospective randomized study of radiation fraction size and overall treatment time . 
in the clinical pharmacogenetics implementation consortium guideline , fluoropyrimidine treatment in homozygous dpyd variant carriers is discouraged , which implies that a potentially effective anticancer therapy is withheld from these patients.5 in this article , six unique patients with a homozygous or compound heterozygous dpyd variant allele genotype who were treated with tailored fluoropyrimidine treatment are described . 
analyses were part of individual patient care , so institutional review board approval was not applicable . genotyping results showed a homozygous or compound heterozygous dpyd genotype , and the functional effects of these genotypes were uncertatherefore , pretreatment dpd activity was measured in peripheral blood mononuclear cells ( pbmcs )  . 
dpd activity was used to reach an individualized dose , in which the percentage of remaining dpd activity was used as guideline for the starting dose ( expressed as percentage of the originally planned dose )  . pharmacokinetic analyses in three of six patients were performed to investigate whether applied dose reductions were adequate . 
pretreatment plasma uracil , the endogenous dpd substrate , and dihydrouracil levels were quantified ; results were unknown before the start of treatment . results clinical course of treatment patient characteristics are listed in table 1.6 - 9 linda m . 
therefore , it was decided to drastically reduce the capecitabine dose from 2 , 300 mg ( 1 , 000 mg / m2 ) twice daily to 150 mg twice daily ( 6.5% of planned dose ) and to start with capecitabine monotherapy . 
the adverse events resolved within 1 week ( neutropenia ) to 2 months ( diarrhea , mucositis )  . after a 2 - month period without any anticancer therapy , the patient had fully recovered , and monotherapy with capecitabine was restarted . 
on the basis of the severe toxicity and the pharmacokinetic results of the first cycle , the dose was reduced more to 150 mg once every 5 days ( ie , 0.65% of originally planned dose )  . 
this female patient with locally advanced colorectal carcinoma had a planned treatment that consisted of neoadjuvant chemotherapy ( capecitabine 825 mg / m2 twice daily , or 1 , 500 mg ) combined with radiotherapy ( 5 - week schedule )  . pretreatment dpyd screening revealed that the patient was homozygous for c.2846a.t ; dpd activity was reduced to 29% . 
it was decided to reduce the capecitabine dose to 500 mg once daily ( ie , 17% of planned doseslightly lower than recommended dose of 29% on the basis of dpd activity , as decided by physician and patient )  . 
dpd activity was reduced to 45% . remaining dpd activity was more than 50% reduced , so it was considered likely that this patient was a compound heterozygous carrier ( variants present on different alleles )  . 
in the first cycle , capecitabine was reduced to 1 , 800 mg daily ( 51% of planned daily dose of 3 , 500 mg ; 1 , 750 mg / m2 ) , which was tolerated without toxicity . 
however , the patient developed grade 2 thrombocytopenia after 8 days , and the daily capecitabine dose was adjusted to 1 , 000 mg for the rest of the cycle . platelets increased again until normal values were reached . 
after three cycles , disease progression was established , and capecitabine treatment was discontinued . pharmacokinetic results in all three patients , additional pharmacokinetic measurements were performed ( fig 1 )  . 
for patient 1 , only levels of capecitabine and of the metabolites 59 - deoxy - 5 - fluorocytidine , 59 - deoxy5 - fluorouridine , and fu could be quantified ; other fu metabolites were not detectable . 
results of noncompartmental analysis of the pharmacokinetic results in plasma are listed in table 2 and include values normalized to control values.10 in patient 1 , fu exposure was highly increased : the mean area under the plasma concentration - time curve ( auc ) of fu was 4 , 024 ng 3 h / ml , which is 10 times higher than in other pharmacokinetic studies with capecitabine.10 - 13 these results were used for the decision to lower the dose 10 - fold in the second cycle . baseline uracil and dihydrouracil levels are listed in table 1 . 
results of urine analysis for patient 1 are shown in appendix figure a1 . discussion to our knowledge , this is the first report to describe prospectively identified patients , who are homozygous or compound heterozygous for dpyd variants , who could be treated safely with fluoropyrimidines . 
for all three patients , the results after the first intake of capecitabine are depicted . capecitabine patient 1 ( homozygous dpyd * 2a ) patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) 5 ' - dfcr patient 1 ( homozygous dpyd * 2a ) patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) time ( h ) 5 ' - dfur time ( h ) 5 - fu patient 1 ( homozygous dpyd * 2a ) patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) patient 1 ( homozygous dpyd * 2a ) patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) 5 , 000 4 , 000 3 , 000 2 , 000 1 , 000 4 , 000 3 , 000 2 , 000 1 , 000 2 , 500 2 , 000 1 , 500 1 , 000 2 , 500 2 , 000 1 , 500 1 , 000 1 , 200 time ( h ) fuh2 time ( h ) fupa patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) time ( h ) time ( h ) fbal patient 2 ( homozygous c.2846a > t ) patient 3 ( c.2846a > t and c.1236g > a ) time ( h ) pretreatment identification of the patient homozygous for dpyd * 2a with complete dpd deficiency saved this patient from receipt of a full fluoropyrimidine dose , which most likely would have been fatal . 
in patients who are not dpd deficient , fluoro - b - alanine is the major urinary metabolite , 13 , 20 whereas this metabolite was not present in the urine analyzed in the case presented in this paper . 
for example , mir27a polymorphisms could play a role in variation of dpd activity , because these polymorphisms reduce dpd activity.26 , 27 in conclusion , we showed that fluoropyrimidine treatment in homozygous or compound heterozygous dpyd variant allele carriers is feasible and that therapy does not have to be withheld . 
van der veldt consulting or advisory role : ipsen ( inst ) , roche ( inst ) , bristol - myers squibb ( inst ) , pfizer ( inst ) , msd oncology ( inst ) travel , accommodations , expenses : roche paul hamberg no relationship to disclose andr e b.p. 
bioanalysis 7 : 519 - 529 , 2015 van kuilenburg ab , van lenthe h , tromp a , et al : pitfalls in the diagnosis of patients with a partial dihydropyrimidine dehydrogenase deficiency . 
deenen mj , meulendijks d , boot h , et al : phase 1a / 1b and pharmacogenetic study of docetaxel , oxaliplatin and capecitabine in patients with advanced cancer of the stomach or the gastroesophageal junction . 
judson ir , beale pj , trigo jm , et al : a human capecitabine excretion balance and pharmacokinetic study after administration of a single oral dose of 14c - labelled drug . 
van kuilenburg ab , muller ew , haasjes j , et al : lethal outcome of a patient with a complete dihydropyrimidine dehydrogenase ( dpd ) deficiency after administration of 5 - fluorouracil : frequency of the common ivs14 + 1g.a mutation causing dpd deficiency . 
meinsma r , fernandez - salguero p , van kuilenburg ab , et al : human polymorphism in drug metabolism : mutation in the dihydropyrimidine dehydrogenase gene results in exon skipping and thymine uracilurea . 
van kuilenburg ab , meijer j , mul an , et al : intragenic deletions and a deep intronic mutation affecting pre - mrna splicing in the dihydropyrimidine dehydrogenase gene as novel mechanisms causing 5 - fluorouracil toxicity . 
offer sm , butterfield gl , jerde cr , et al : micrornas mir - 27a and mir - 27b directly regulate liver dihydropyrimidine dehydrogenase expression through two conserved binding sites . 
froehlich tk , amstutz u , aebi s , et al : clinical importance of risk variants in the dihydropyrimidine dehydrogenase gene for the prediction of early - onset fluoropyrimidine toxicity . 
int j cancer 136 : 730 - 739 , 2015 patients were treated at three different institutes in the netherlands ( the netherlands cancer institute , amsterdam ; erasmus medical center , rotterdam ; fransciscus gasthuis & vlietland , rotterdam )  . 
toxicity was scored according to the common terminology criteria for adverse events , version 4.03. dpyd genotyping genomic dna was isolated from peripheral blood cells , and screening for the dpyd variants dpyd * 2a ( ivs14 + 1g.a , rs3918290 ) , c.2846a.t ( rs67376798 ) , c.1679t.g ( dpyd * 13 , rs55886062 ) , and c.1236g.a ( rs56038477 ) was performed by using standard operating procedures in two different institutes ( the netherlands cancer institute , amsterdam , and erasmus medical center , rotterdam )  . 
in the netherlands cancer institute , screening for dpyd variants was performed with the roche lightcycler 480ii platform ( roche diagnostics , almere , the netherlands ) by using commercially available probes and primers ( tib molbiol , berlin , germany ) , and results were confirmed by direct sequencing . 
at the erasmus medical center , each sample was genotyped on two different platformstaqman ( with predefined drug - metabolizing enzyme [ dme ] assays ) and pcrrestriction fragment length polymorphism ( rflp ) assaysto allow checks for potentially wrong genotyping . 
both laboratories participated during the study in the dutch national quality control program for dpyd proficiency testing , in which all four dpyd variants were included . dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells dihydropyrimidine dehydrogenase ( dpd ) activity was measured in peripheral blood mononuclear cells ( pbmcs ) , isolated from a baseline ( pretreatment ) peripheral blood sample . 
one of two comparable validated methods by van kuilenburg et al7 or pluim et al6 was used ; both used radiolabeled thymine ( 14c - labeled thymine or 3h - labeled thymine ) as a substrate and consisted of high - performance liquid chromatography ( hplc ) with online radioisotope detection and with liquid scintillation counting . 
analytes were extracted by protein precipitation ; chromatographic separation was performed on an acquity ultra - performance liquid chromatography hss t3 column ( acquity waters , milford , ma ) and were analyzed with ms / ms with an electrospray ionization source ( jacobs ba et al : j pharm biomed anal 126 : 75 - 82 , 2016 )  . 
all samples were measured at one institute ( the netherlands cancer institute )  . pharmacokinetic measurements peripheral blood samples for patient 1 were obtained for pharmacokinetic analysis on cycle 1 , day 1 ( c1d1 ) ; cycle 2 , day 1 ( c2d1 ) ; and cycle 2 , day 16 ( c2d16 )  . 
 results dihydrouracil - uracil levels capecitabine intake ( at predose , and at 15 minutes , 30 minutes , 1 hour , 2 hours , 3 hours , 4 hours , 6 hours , 8 hours , and 10 hours after capecitabine intake )  . 
for patients 2 and 3 , samples were collected only on c1d1 , on the same time points as for patient 1 , except for the latest time point ( the sample 10 hours after capecitabine intake was not collected for patient 2 , and the last sample was collected at 12 hours instead of at 10 hours for patient 3 )  . 
for patients 2 and 3 , pharmacokinetic results were not known during treatment , because samples were analyzed after treatment had finished . capecitabine and its metabolites , 59 - deoxy - 5 - fluorocytidine ( 59 - dfcr ) , 59 - deoxy - 5 - fluorouridine ( 59 - dfur ) , fluorouracil ( fu ) , dihydro - 5 - fluorouracil ( fuh2 ) , a - fluoro - ureidopropionic acid ( fupa ) , and fluoro - b - alanine ( fbal ) , were quantified by using hplc coupled to electrospray ms / ms . 
two individual validated assays were used : one for the simultaneous quantification of capecitabine , 59 - dfcr and 59 - dfur and another for fu , fuh2 , fupa , and fbal ( deenen mj et al : j chromatogr b analyt technol biomed life sci 913 - 914 : 30 - 40 , 2013 )  . 
all pharmacokinetic samples were measured at the same institute ( the netherlands cancer institute )  . together with the sample for dpd activity in pbmcs , a pretreatment plasma sample was taken to measure uracil and dihydrouracil levels ( table 1 )  . 
for patients 2 and 3 , uracil levels were increased compared with reference levels ( a value of 28.7 ng / ml for patient 2 and of 35.6 ng / ml for patient 3 ) , and both values were within the top 1% of reference values . 
dihydrouracil levels were in the normal range for patients 2 and 3 compared with reference levels . other homozygous dpyd variant allele carriers three additional patients with a homozygous dpyd variant genotype were identified during routine dpyd screening ( table 1 )  . 
it is unclear why there is a relatively high variation of the effect of this genotype on dpd phenotype in patients , and more research on this variant is advised . in addition , one patient with a homozygous c.2846a.t genotype ( patient 4 ) was identified . 
according to the calculated dpd activity score , as described by henricks et al , 4 it could be concluded that a 50% dose reduction would be appropriate for homozygous c.2846a.t carriers , because a 25% dose reduction is recommended for heterozygous carriers of this variant . 
recent work has determined that 20% to 30% of mcrpc tumors harbor alterations that result in the loss of dna repair capacity , in particular , the homologous recombination pathway.1 , 2 in turn , these alterations may predict response to poly - adp ribosylase ( parp ) inhibitors and platinum chemotherapy , which provides opportunities for precision therapy using agents that are not currently standard or available for mcrpc.3 , 4 in ovarian canceralso frequently marked by homologous recombination deficiencyplatinum is the standard front - line treatment , and parp inhibitors olaparib , niraparib , and rucaparib are now us food and drug administrationapproved for brca1 / 2mutated tumors . 
reversion mutations inbrca1 / 2 , 5 - 8 and more recently in rad51c and rad51d , 9 have previously been described as a mechanism of resistance to platinum and parp inhibitors in ovarian cancer and thus also represent an important consideration in patients with prostate cancer . recently , reversion mutations to restore brca1 / 2 function have been observed in prostate tumors sampled by biopsy and in circulating tumor dna ( ctdna ) from patients who were treated with parp inhibitors.10 , 11 here , we describe a case of a patient with mcrpc who exhibited polyclonal brca2 reversion mutations at the time of disease progression when receiving platinum chemotherapy . 
to our knowledge , this is the first report of brca2 reversions occurring in a patient with prostate cancer in the setting of platinum treatment and mediating resistance to therapy . our patient presented with a prostate - specific antigen ( psa ) of 8.6 ng / ml and gleason 4 + 5 prostate adenocarcinoma in october 2011 at 65 years of age . 
he underwent a radical prostatectomy with bilateral pelvic lymph node dissection in january 2012 with pt3br1n1 ( american joint committee on cancer 7 ) , gleason 5 + 4 staging . 
postoperative psa in april 2012 was 2.7 ng / ml ; restaging scans demonstrated bone involvement in the left iliac wing and l4 vertebral body without lymph node or visceral involvement . he was initially treated with bicalutamide and leuprolide ( a luteinizing hormonereleasing hormone agonist ) , which resulted in undetectable psa by may 2013 . 
in june 2016 , he began treatment with enzalutamide 160 mg once daily , but was noted to have primary refractory disease with a psa increase to 370.3 ng / ml in 1 month . 
black arrow denotes time of circulating tumor dna ( ctdna ) collection . in october 2016 , carboplatin area under the curve 5 was administered based on the results of the metastatic tumor biopsy sequencing . 
after seven cycles , psa increased to 428.1 ng / ml on april 12 , 2017 , concurrent with ctdna draw . samples from the primary prostate tumor , germline dna , and a ctdna sample that was obtained at the time of progression on carboplatin ( march 2017 ) were sequenced by using a clinically validated tumor sequencing next - generation sequencing assay12 and compared with the prechemotherapy metastasis biopsy . 
a frameshift mutation in brca2 ( p.n2452mfs * 17 ) as well as in spop and tp53 mutations were identified in the primary tumor and metastasis ( table 1 )  . 
we did not observe evidence of a germline pathogenic variant . we performed targeted next - generation sequencing on ctdna at the time of progression to 1 , 6103 mean coverage , and identified 90 brca2 reads that carried the frameshift mutation and 1 , 181 wild - type brca2 reads . 
of importance , no reversion mutations were detected in the primary or germline samples . secondary somatic mutations of brca1 / 2 mutations that restore the wild - type open reading frame were initially described as a mechanism of resistance to platinum chemotherapy in ovarian cancer and have subsequently been reported in patients with prostate cancer who were treated with parp inhibitors.5 , 10 , 11 to our knowledge , this is the first example of reversion mutations in the ctdna of a patient with prostate cancer associated with resistance to platinum chemotherapy . our finding provides evidence that polyclonal reversion is a mechanism of resistance to platinum chemotherapy in patients with prostate cancer . prospective clinical trials of parp inhibitors and platinum to exploit the molecular vulnerabilities of prostate cancer with dna repair defects are now underway , and although there is great enthusiasm , resistance to these strategies is expected . we previously described a series of patients with prostate cancer with biallelic brca2 inactivation who had exceptional and sometimes extended responses to carboplatin - based therapy ( up to 3 years ) .4 in addition , a recent retrospective analysis of patients whose prostate cancer was refractory to taxanes found that six ( 75% ) of eight patients who were carriers of pathogenic germline brca2 variants had a psa50 response from 12 weeks of carboplatin plus docetaxel therapy compared with 23 ( 17% ) of 133 noncarriers.15 two prospective clinical trials that are investigating carboplatin table 1 . 
dashed outline represents second allele . sequencing reads with secondary mutations in the plasma circulating tumor dna ( ctdna ) sample taken at the time of progression on carboplatin ( bottom )  . 
images were rendered by using the integrative genomics viewer.13 , 14 with docetaxel for patients with mcrpc and dna repair defects are currently recruiting patients ( clinicaltrials.gov identifiers : nct02598895 and nct02985021 ) and may be opportunities to explore this and other potential resistance mechanisms . the recent finding of secondary reversion mutations in patients with prostate cancer after parp inhibitor therapy occurred within 8 to 10 months of initiating therapy.10 , 11 in our patient , resistance emerged after the same apparent mechanism over approximately 7 months , a more abbreviated benefit compared with the multiple years of response to platinum - based therapy that we have observed and on which we have previously reported.4 our patients case provides evidence that reversion mutations selected by platinum chemotherapies can occur at a similar pace as parp inhibitors and implies that additional factors that are yet to be characterized influence the chronology of resistance to dna - damaging agents . 
although we did not detect a second inactivating mutation in our patient , it is possible that an undetected methylation or a complex rearrangement could be present . nonetheless , evidence of reversion mutations that restore gene function and that temporally coincide with clinical progression during platinum therapy together make a strong case that brca2 gene function was disrupted in the primary and metastatic tumor . it is possible that the mechanism of initial gene inactivation affects resistance . 
for example , patients with biallelic copy loss may be less likely to restore functional copy and thus have a longer response duration compared with those with biallelic singlenucleotide substitutions and / or indels , which might restore gene function more readily to give rise to resistance . 
in addition , although platinum and parp inhibitors share reversion as a mechanism of resistance , patients with ovarian cancer resistant to parp inhibitors can still exhibit sensitivity to platinum , which demonstrates that there are differences as well.16 many unanswered questions remain regarding the noninvasive assessment of resistance . 
 only been identified after selective treatment pressure and not as de novo events , although the lack of detection may reflect mutation levels that are lower than the sensitivity of assays used to detect reversion events . 
in this case , we did not have matched tumor biopsy and ctdna for each clinically relevant time point , although the samples we collected demonstrated proof of an important mechanism of resistance to platinumany in the field are investigating concordance and representative sampling of tumor versus ctdna , which will be important to clinical practice . another critical issue is whether early reversion clones specifically and reliably predict the development and pace of clinical resistance , the differences between platinum and parp inhibitors , and what other potential relevant genetic , epigenetic , and microenvironmental modifying factors are at play . 
nelson consulting or advisory role : janssen oncology , astellas pharma , genentech bruce montgomery research funding : essa pharma , medivation , astellas pharma , janssen oncology , ferring pharmaceuticals colin c . 
pritchard no relationship to disclose acknowledgment we thank arul chinnaiyan , daniel robinson , hiep nguyen , and the stand up to cancer / prostate cancer foundation precision oncology team for their assistance with the collection and analysis of the metastatic biopsy . affiliations support references 417 - 425 , 2017 1697 - 1708 , 2015 all authors , university of washington ; heather h . 
kondrashova o , nguyen m , shield - artin k , et al : secondary somatic mutations restoring rad51c and rad51d associated with acquired resistance to the parp inhibitor rucaparib in high - grade ovarian carcinoma . 
quigley d , alumkal jj , wyatt aw , et al : analysis of circulating cell - free dna identifies multiclonal heterogeneity of brca2 reversion mutations associated with resistance to parp inhibitors . 
pritchard cc , salipante sj , koehler k , et al : validation and implementation of targeted capture and sequencing for the detection of actionable mutation , copy number variation , and gene rearrangement in clinical cancer specimens . j mol diagn 16 : 56 - 67 , 2014 13 . 
ang je , gourley c , powell cb , et al : efficacy of chemotherapy in brca1 / 2 mutation carrier ovarian cancer in the setting of parp inhibitor resistance : a multi - institutional study . 
 concordance of genomic variants in matched primary breast cancer , metastatic tumor , and circulating tumor dna : the mirror study fernando moreno , md1 ; javier gayarre , phd1 ; sara l opez - tarruella , md , phd1 , 4 , 5 ; mara del monte - mill an1 ; antonio c . 
picornell , phd1 ; enrique alvarez1 ; jos e angel garca - saenz , md , phd1 ; yolanda jerez , md1 ; iv an m arquez - rodas , md , phd1 ; isabel echavarra , md , phd1 ; maribel palomero , md1 ; coralia bueno , md2 ; ana mara arag on bod3 ; marta sanchez muoz , phd3 ; ricardo gonz alez del val , md , phd1 ; oscar bueno , md4 ; mara cebollero - presmanes , md5 ; inmaculada ocaa1 ; ainhoa arias1 ; paula romero , msc1 ; tatiana massarrah1 ; roco ramos - medina , phd1 ; and miguel martn , md , phd1 , 4 , 5 purpose genetic heterogeneity between primary tumors and their metastatic lesions has been documented in several breast cancer studies . 
however , the selection of therapy for patients with metastatic breast cancer and the search for biomarkers for targeted therapy are often based on ndings from the primary tumor , mainly because of the difculty of distant metastasis core biopsies . 
in addition , we identied 13 variants in metastatic tissue and ctdna not present in primary tumor . conclusion we identied genomic variants present in metastatic biopsies and plasma ctdna that were not present in the primary tumor . 
2019 by american society of clinical oncology introduction breast cancer is a heterogeneous disease with different clinical characteristics , disease courses , and responses.1 traditional biomarkers ( estrogen receptor [ er ] , progesterone receptor [ pr ] , and human epidermal growth factor receptor 2 [ her2 ] ) and imaging techniques play important roles in tumor diagnosis and treatment selection.2 however , these biomarkers only offer partial insight into the biology of the tumors . 
in recent years , technologic advances in genomics and the implementation of multi - institutional initiatives have improved our understanding of the landscape of genomic alterations in breast cancer.3 biopsies of metastatic sites are considered the gold standard for both the characterization of genomic alterations in patients with metastatic breast cancer and the search for prognostic and predictive biomarkers . 
 moreno et al context key objective the objective of this study was to evaluate the concordance of genomic variants between primary tumor tissue , metastatic tissue , and circulating tumor dna ( ctdna ) in patients with metastatic breast cancer . knowledge generated next - generation sequencing of metastatic samples shows mutations not present in the primary tumor . 
ctdna analysis with ion torrent technology identied the majority ( 97.2% ) of variants present in primary and metastatic tumors . relevance genotyping of metastases offers additional information for primary tumor analysis . 
ctdna can be used to detect genomic alterations in patients with metastatic breast cancer and could be an alternative to metastatic tissue samples . to determine the concordance of genomic alterations among primary breast cancer , metastatic breast cancer lesions , and ctdna , we conducted the mirror study ( clinicaltrials.gov identier : nct02626039 )  . 
all patients provided written informed consent for the study . patients included in the study had histologically conrmed progressive metastatic breast cancer before starting a new line of treatment and were at least 18 years old . 
exclusion criteria were metastatic bone disease only , coagulation disorders , and eastern cooperative oncology group performance status of 3 or 4.10 study objectives the primary objective of this study was to assess the concordance of genomic variants between primary tumor tissue and metastatic tissue in patients with metastatic breast cancer . 
secondary objectives included determining ctdna mutation proles from peripheral blood and comparing them with those from the primary tumor and metastatic tissue . study procedures eligible patients underwent a biopsy of a metastatic site . blood samples were obtained within a window of 30 days , spanning 15 days before or after the biopsy procedure . parafn blocks of matched primary tumor and metastasis tissues , as well as plasma samples , were used for genomic studies . 
the mirror project was a pilot study , and the sample size was established empirically . a recruitment rate of two fully evaluable patients per month for up to 2 years was considered a reasonable target . 
for solid tumor and ctdna samples , a minimum mean coverage of 5 , 000 and 15 , 000 , respectively , was established . we focused on single - nucleotide substitutions and small indels . 
single - nucleotide substitutions were ltered as follows : ( 1 ) rare variants ( minor allele frequency , 0.01 ) , ( 2 ) variants in coding exons and splice junctions ; ( 3 ) variants predicted to produce synonymous amino acid changes were discarded ( we selected only high / mediumimpact variants : frameshift indels and nonsense , missense , and canonical splice - site variants ; appendix ) ; ( 4 ) we then discarded benign and probably benign classied variants , as dened in different databases , which are listed in table 2 . 
variants were classied according to the american college of medical genetics and genomics guidelines.16 identication of variants from ffpe samples and plasma for ffpe - derived dna , variant calls with a mutant allelic fraction of 2% or greater in the paired solid tumor ( primary and metastasis ) tissues were selected . 
for ctdna , variants with at least two reads in one strand supporting variant alleles with a minimum region - coverage depth of 20 were retained . three comparisons were made sequentially . 
finally , to identify potential mechanisms of resistance to specic therapies , we analyzed genetic variants present in both metastatic tissue and ctdna but not in primary tumors ( fig 1 )  . conrmation of selected variants selected variants identied by ion torrent technology was conrmed using ngs technology ( illumina , san diego , ca ) the genomic unit of university hospital gregorio mara on ( madrid , spain )  . 
we designed a customized truseq panel ( custom amplicon low input kit , illumina ) to sequence 116 amplicons . genomic dna ( 10 ng ) was used to prepare libraries from two pools of paired oligos , following the manufacturers recommendations ( truseq custom amplicon low input kit , procedure dual strand v04 ; illumina )  . 
the libraries were sequenced on a miseq platform ( illumina ) with miseq reagent v2 ( 2 150 bp )  . sequence data were analyzed using the miseq reporter 2.6.2 ( illumina ) and igv win 2.4.3 software ( broad institute and the regents of the university of california )  . patient characteristics and samples analyzed between november 2013 and march 2017 , 80 patients who met the inclusion criteria and provided informed consent were enrolled ( fig 2 )  . 
 moreno et al primary tumor customized 54 - cancer gene panel ( ion torrent technology ) metastatic tumor plasma ( ctdna ) common variants present in primary tumor , metastatic tumor , and ctdna . common variants present in metastatic tumor and ctdna , not present in primary tumor . genetic variant validation ( illumina techonology ) fig 1 . 
dna was obtained from primary tumors , metastases ( liver , lung , skin , brain , lymph nodes , local relapse in breast , and bone ) , and plasma samples . 
genomic variants were identied using ion torrent technology ( thermofisher scientic , waltham , ma ) and were validated using illumina technology ( illumina , san diego , ca )  . when we reached the intended sample size of 40 patients who met the inclusion criteria , and with valid samples from the three sources ( primary and metastatic tumor biopsies and plasma ) , we proceeded to genomic variant analyses . pathologic analysis of primary tumors revealed that 17 patients ( 42.5% ) had hormone receptor - positive / her2negative disease , 15 patients ( 37.5% ) had her2 - positive disease , and seven patients ( 17.5% ) had triple - negative disease . 
pathologic analysis of metastatic biopsies revealed that 14 patients ( 35% ) had hormone receptor - positive / her2 - negative disease , 16 ( 40% ) had her2 - positive disease , and 10 ( 25% ) had the triple - negative phenotype . all patients had received antitumor drug therapy in the adjuvant and / or metastatic setting . 
most patients ( n = 33 ; 82.5% ) had received drug therapy for advanced disease , and only seven patients ( 17.5% ) had not received treatment in the metastatic setting . 
thirty - ve patients were previously exposed to ( neo ) adjuvant therapy , whereas ve patients had not received drug treatment of early - stage breast cancer ( table 3 )  . 
missense variants were the most frequently observed ( 81 ; 76% ) , followed by nonsense variants ( 13 ; 12% ) , frameshift variants ( 10 ; 9% ) , and indels not leading to frameshift ( 3 ; 3% ; appendix fig a1 )  . twenty - two of the variants were considered pathogenic ( 21% ) , 10 were considered likely pathogenic ( 9% ) , and 75 were of uncertain signicance ( 70% ; fig 4b )  . 
erbb2 mutations ( val777_ gly777insglyserpro and val777leu ) were found only in her2 - positive tumors , with a mutation frequency in her2 - positive metastatic tumors of 12.5%. variant analysis using ion torrent technology the median read coverage was 6 , 930 ( range , 5 , 354 to 9 , 324 ) for primary and metastatic tumor tissues and 15 , 400 ( range , 10 , 942 to 24 , 090 ) for ctdna . 
most of the variants ( 107 of 110 ) we identied 13 variants in metastatic tumors and ctdna that were not detected in the corresponding primary tumors ( fig 4d )  . 
 ( % ) er positive / her2 negative er positive / her2 positive er negative / her2 positive er negative / her2 negative ki 67 primary , % ( range ) ki 67 metastasis , % ( range ) stage at diagnosis , no . 
 ( % ) abbreviations : er , estrogen receptor ; her2 , human epidermal growth factor receptor 2 ; n / a , not available . in the metastatic setting than in the adjuvant setting ( three v one patient , respectively )  . conrmation of selected variants using illumina technology material was available for independent validation using illumina technology for 38 of the 40 patients . 
of the 13 variants commonly detected in metastatic tumors and ctdna samples but not in the primary tumors using ion torrent , 12 ( 92.3% ) were conrmed by illumina data ( fig 3 )  . discussion tumor genotyping is an important and evolving area of research in metastatic breast cancer . 
in many metastatic breast cancer clinical trials involving predictive biomarker identication , primary tumors have been mainly used because metastatic samples are more difcult to obtathe precise bias introduced by this approach is unknown . various studies have shown substantial genomic differences between primary and metastatic breast cancer tissues , which probably arise as a consequence of tumor evolution under the selective pressure of therapies.4 , 5 in the primary tumors . we used two independent ngs platforms to identify and conrm single - nucleotide changes and indels in primary tumor and metastases from 40 patients with breast cancer . 
in addition , we identied 13 pathogenic or likely pathogenic variants that were present in metastatic tumors and plasma samples , but illumina technology conrmed 85.5% of primary and metastatic common variants originally detected by ion torrent . 
these ndings conrm those of previous studies suggesting that , although most variants present in the primary tumor were also present in metastatic tissue , mutations not present in the primary tumor can be found in metastases.4 , 5 , 17 therefore , metastatic tissue and , alternatively , ctdna , rather than primary tumor tissue could be the most appropriate source of dna material identifying biomarkers for new drugs in breast cancer clinical trials or for selecting patients for current targeted therapies . the detection of ctdna variants in patients with metastatic cancer primarily depends on variant allele frequencies in plasma.18 - 20 because of the extremely low ratio of ctdna / cell - free dna in the blood , ctdna detection largely depends on new technologies with high sensitivity and specicity . 
roth e et al26 used a similar approach to analyze 2 , 800 cosmic mutations distributed across 50 genes using ion ampliseq cancer hotspot panel v2 ( life technologies , carlsbad , ca ) in matched plasma and metastatic tumor samples collected simultaneously . 
the concordance between ctdna and tissue biopsy results in breast cancer suggests that ctdna assays could be condently used to molecularly stratify patients for prognostic and predictive purposes . taken together , our results show that ctdna can be used to detect genomic alterations in patients with metastatic breast cancer . 
if conrmed in larger studies , this nding might be of relevant practical interest , eliminate the need for many invasive biopsies , and allow monitoring of metastatic breast cancer over the course of the disease . one of the challenges in the treatment of metastatic breast cancer is the emergence of mechanisms of resistance . increased knowledge of resistance mechanisms may help to select targeted therapies at the time of progression . 
we identied six likely pathogenic variants in three different genes , esr1 , akt1 , and braf , that are present in metastatic tissues and plasma , but not in primary tumors . esr1 mutations are associated with resistance to aromatase inhibitors , but may be sensitive to fulvestrant , an estrogen receptor downregulator.27 we detected esr1 mutations in ctdna and metastatic biopsies of four patients . 
consistent with previous reports , esr1 mutations were present only in er - positive patients with breast cancer previously treated with aromatase inhibitors and were predominantly acquired during the treatment of metastatic disease.28 the strengths of this study include the availability of triple - matched samples from individual patients with breast cancer , concomitant extraction of blood and biopsy samples , and the rigorous analytic methodology . one of the limitations of our study is the lack of study of ctdna to identify variants of germinal orighowever , many variants can be considered somatic if they meet certain criteria ( a variant allele fraction substantially less than 50% , a recurrent somatic variant with clinical signicance in cancer , is not commonly observed in population databases )  . 
nearly all the variants ( 107 of 110 ) were also detected in plasma circulating tumor dna ( ctdna )  . thirteen variants were identied in metastatic tumor and ctdna samples , but not in the corresponding primary tumor . 
however , the main objective of the study was the correlation between ndings in primary tumor , the nature metastases , and ctdna regardless of ( inherited or acquired ) of the variant . 
other limitations include a low number of patients , a relatively large fraction of patients for whom the three determinations were not available , heterogeneity of breast cancer phenotypes , and differential drug exposure of the pathese biases , we are tients . 
 moreno et al tp53 kmt2c pik3ca ptpn14 brca2 lrp1b gata3 erbb2 tbx3 ptprd pik3r1 ncor1 herc1 ctnna1 ctcf cdh5 cdh1 araf tbx5 runx1 pten map3k1 kras foxp1 egfr cdkn1b ccnd3 brca1 akt1 aff2 fig 4 . 
characteristics of variants identied in ( a ) distrithis study . bution , per gene , of the 107 common variants in primary tumors , metastatic tumors , and cirtumor dna culating ( ctdna )  . 
 mirror study : breast cancer tumor and ctdna genomic concordance in conclusion , our study showed that mutations present in breast cancer metastases , but not in the primary tumor , can be identied using ngs technologies . 
 moreno et al ricardo gonz alez del val research funding : novartis ( inst ) travel , accommodations , expenses : daiichi sankyo oscar bueno research funding : novartis ( inst ) inmaculada ocaa research funding : novartis ( inst ) ainhoa arias research funding : novartis ( inst ) paula romero research funding : novartis ( inst ) tatiana massarrah consulting or advisory role : novartis , astrazeneca , seom , roche , novartis speakers bureau : roche research funding : novartis ( inst ) patents , royalties , other intellectual property : methods for selecting , manipulating , and isolating circulating tumor cells in body uids by laserassisted transfer ( inst ) travel , accommodations , expenses : novartis , astrazeneca , novartis roco ramos - medina research funding : novartis ( inst ) patents , royalties , other intellectual property : methods for selecting , manipulating , and isolating circulating tumor cells in body uids by laserassisted transfer ( inst ) miguel martn consulting or advisory role : genentech , novartis , pzer , eli lilly , astrazeneca , taiho pharmaceutical , pharmamar research funding : novartis ( inst ) , roche patents , royalties , other intellectual property : methods for selecting , manipulating , and isolating circulant tumor cells in body uids by lasser transfer no other potential conicts of interest were reported . acknowledgment the authors thank carmen flores for collaboration on this study , as well as the patients with breast cancer who agreed to participate . references balko jm , stricker tp , arteaga cl : the genomic map of breast cancer : which roads lead to better targeted therapies ? breast cancer res 15 : 209 , 2013 colomer r , aranda - l opez i , albanell j , et al : biomarkers in breast cancer : a consensus statement by the spanish society of medical oncology and the spanish society of pathology . 
clin transl oncol 20 : 815 - 826 , 2018 [ erratum : clin transl oncol 20 : 1093 - 1095 ] swanton c , soria j - c , bardelli a , et al : consensus on precision medicine for metastatic cancers : a report from the map conference . 
mol cancer ther 13 : 1382 - 1389 , 2014 goswami rs , patel kp , singh rr , et al : hotspot mutation panel testing reveals clonal evolution in a study of 265 paired primary and metastatic tumors . 
n engl j med 366 : 883 - 892 , 2012 russnes hg , navin n , hicks j , et al : insight into the heterogeneity of breast cancer through next - generation sequencing . 
j clin invest 121 : 3810 - 3818 , 2011 gonzalez - rivera m , picornell ac , alvarez el , et al : a cross - sectional comparison of druggable mutations in primary tumors , metastatic tissue , circulating tumor cells , and cell - free circulating dna in patients with metastatic breast cancer : the mirror study protocol . 
hammond meh , hayes df , dowsett m , et al : american society of clinical oncology / college of american pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer . 
wolff ac , hammond meh , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american pathologists joint review . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
sci transl med 6 : 224ra24 , 2014 jovelet c , ileana e , le deley m - c , et al : circulating cell - free tumor dna analysis of 50 genes by next - generation sequencing in the prospective moscato trial . clin cancer res 22 : 2960 - 2968 , 2016 20 . 
kim st , lee w - s , lanman rb , et al : prospective blinded study of somatic mutation detection in cell - free dna utilizing a targeted 54 - gene next generation sequencing panel in metastatic solid tumor patients . 
roth e f , laes j - f , lambrechts d , et al : plasma circulating tumor dna as an alternative to metastatic biopsies for mutational analysis in breast cancer . 
schiavon g , hrebien s , garcia - murillas i , et al : analysis of esr1 mutation in circulating tumor dna demonstrates evolution during therapy for metastatic breast oncol 25 : 1959 - 1965 , 2014 34 : 2961 - 2968 , 2016 cancer . 
brody , phd3 ; lola rahib , phd4 ; emily lyons , ma4 ; patricia de arbeloa , md2 ; andrew hendifar , md , mph5 ; sameh mikhail , md6 ; vincent chung , md7 ; davendra p.s. 
matrisian , phd , mba4 ; and emanuel petricoin iii , phd2 , 9 purpose up to 25% of pancreatic adenocarcinomas ( pdacs ) harbor mutations in the homologous recombination dna damage response ( hr - ddr ) pathway . 
although known to affect responsiveness to dnadamaging chemotherapy , the prognostic relevance of these mutations is unclear and outcomes in patients with pdac who harbor hr - ddr mutations beyond brca1 / 2 remain unexplored . methods we evaluated 820 patients with pdac enrolled in the know your tumor program for whom we had collected comprehensive genomic testing results and longitudinal clinical outcomes . 
mos in patients with mutations in all three hr - ddrmut groups was greater than that for phr - ddr patients , but this difference was lost in platinum - nave patients . conclusion patients with advanced hr - ddrmut have improved mos when treated with platinum - based therapy compared with phr - ddr patients . 
with a 5 - year median overall survival ( mos ) of only 9% , pdac is poised to become the second leading cause of cancer - related death in the united states.1 treatment of metastatic pdac has improved with the use of uorouracil , oxaliplatin , and irinotecan ( folfirinox ) , and gemcitabine plus nab - paclitaxel , but mos remains less than 1 year.2 , 3 however , there is a substantial group of outlier patients whose survival exceeds the median of less than 1 year . 
 pishvaian et al context key objective up to 25% of pancreatic adenocarcinomas ( pdacs ) harbor mutations in the homologous recombination dna damage response ( hr - ddr ) pathway . 
we evaluated 820 patients with pdac who were enrolled in the know your tumor program for whom we had collected comprehensive genomic proling data and longitudinal clinical outcomes to assess the prognostic and predictive value of hr - ddr mutations . knowledge generated patients with advanced pdac and the presence of a somatic or germline hr - ddr mutation have an improved median overall survival ( mos ) when treated with platinum - based therapy compared with patients with no hr - ddr mutation . 
in platinumnave patients , there is no mos difference , which suggests that hr - ddr status has no pure prognostic value . relevance these ndings support the need to test all patients with advanced pdac to ensure that patients whose tumors harbor hr - ddr mutations receive the benet of treatment with platinum - based therapy . prolonged stable disease of 15 patients with pdac who were treated with parpi - based therapy , whereas kaufman et al21 reported one complete response and four partial responses of 23 brca - mutated patients with pdac who were treated with olaparib . 
combinations with chemotherapy may be even more promising , as oreilly et al17 reported a 78% response to the combination of gemcitabine , cisplatin , and veliparib in patients with brcaor palb2mutated pdacs . the treatments received , or we evaluated whether patients with hr - ddrmutated tumors have a biologically better prognosis , irrespective if patients with hrddrmutated tumors have longer than average survival strictly because the underlying mutation predicts for a high degree of responsiveness to platinum - based therapies ( part of the standard of care )  . methods patient recruitment into the know your tumor registry as reported , 4 patients with pdac were identied through the pancreatic cancer action network patient central call center , referred to perthera , and enrolled via an institutional review boardapproved registry protocol . 
study protocol , amendments , and informed consent forms were approved by the new england institutional review board . vestigators obtained informed consent from each participant or participants guardian before enrollment . 
the research was conducted in accordance with recognized ethical guidelines , including the declaration of helsinki , for international organizations of medical scicouncil ences , belmont report , and us common rule , as described during training in good clinical practice guidelines ( collaborative institutional training initiative training )  . patients with biopsy - conrmed pdac were included in the analysis cohort . 
patients with pancreatic adenosquamous carcinoma or other histologic subtypes were excluded . next - generation dna sequencing and identication of hr - ddr status formalin - xed , parafn - embedded blocks and / or slides were collected by surgical resection ( eg , whipple procedure or distal pancreatectomy ) , core - needle biopsy , or ne - needle aspiration . 
coverage of hr - ddr genes tested was relatively uniform with 97% of genomic testing completed by foundation medicine ( cambridge , ma ) , 1% by caris life sciences ( irving , tx ) , and the remaining by including in - house pathology deother partments . 
genomic alterations reported by the testing laboratory included both somatic and germline mutations ; however , it is important to note that the germline versus somatic status of each mutation could not be determined on the basis of tumor proling without parallel genetic sequencing of matched normal blood or tissue . patient tumor genomic proles were reviewed and pathogenic mutations were conrmed by pertheras molecular tumor board participants on a case - by - case basis . 
tumors that harbored no hr - ddr mutations were labeled ddr procient ( phr - ddr )  . results patient history and outcomes enrolled patients signed a health insurance portability and accountability act waiver , and for patient history and longitudinal outcomes assessment , patient coordinators the patients requested updated medical records until death . 
to reduce variability as a result of the timing of enrollment and to establish a denitive starting point for the determination of mos , survival was calculated from the initial date of diagnosis of pdac until death . 
patients were categorized herein as those who underwent a resection of the primary tumor at any point during their treatment history ( resected ) or and as those who had advanced disease at presentation , including locally advanced , never resected patients , and patients with metastatic disease at presentation . 
furthermore , patients were designated as platinum exposed if they received any platinum - containing therapy at any point during the course of their pancreatic cancer survival . statistical analysis all statistical analyses were performed in an r / bioconductor programming environment.22 mos was measured from the date of initial diagnosis until the date of death or the last date the patient was known to be alive . median progression - free survival ( mpfs ) was measured from the start date to the last date the patient was known to be on treatment without progression of disease . 
differences between groups were evaluated for statistical signicance using cox proportional hazards regression models ( the coxph function in the r survival package )  . computed values reported include mpfs , mos , p values , hazard ratios , and 95% cis for hazard ratios . a multivariable approach was implemented to delineate prognostic versus predictive effects of hr - ddr status using the entire analysis cohort . 
given the limitations of interpreting several pairwise comparisons across small groups of patients , we accounted for three binary independent factors : platinum exposure , hr - drr status , and resectability . 
multivariable cox proportional hazards regression was performed on os analysis data by factoring in platinum exposure as one variable , the presence of an hr - ddr alteration as a second variable , interaction between the rst and second variable , and a third variable to account for the patients resection status , which is known to have a highly signicant prognostic impact on os . of 1 , 140 patients with pdac who were enrolled in the know your tumor program between january 2013 and the end of december 2018 , 820 patients with molecular testing results , collection of longitudinal outcomes , and adequate treatment history were included in the analysis cohort . median age at diagnosis was 62 years ( range , 28 to 90 years ) , 52% of patients were male , and 81% of patients were white ( table 1 )  . 
prevalence of somatic or germline hr - ddr mutations ( 16.5% ) and other baseline characteristics was not signicantly different within the resectable and advanced cohorts ( table 1 )  . 
at a median follow - up of 637 days and 386 days for the resected and advanced cohorts , respectively , 47.6% of patients were last known to be alive and 29.2% of patients had not received platinum - based therapy . 
twentyve percent of deceased patients never received any platinum - based therapy . assessing the prognostic value of hr - ddr mutations in platinum - nave patients to assess the prognostic value of the presence of an hrddr mutation , in the absence of the confounding variable of the known predictive value of response to platinum - based therapy , we chose to assess outcomes in patients who never received platinum - based therapy at any time during their pancreatic cancer survival . 
these patients were labeled platinum nave and included 108 resected and 133 advanced patients for analysis ( table 1 and figs 1a and 1b )  . differences in mos were not statistically signicant when comparing hr - ddrmut with phr - ddr patients whose disease was platinum nave in both the resected and advanced subgroups . 
thus , there was no apparent positive prognostic value associated with the presence of an hrddr mutation in platinum - nave patients with pdac . conrming the predictive value for platinum - based therapy of an hr - ddr mutation we also assessed the predictive value for platinum - based therapy of an hr - ddr mutation ( figs 1c and 1d and table 2 )  . 
percentages of patients on the basis of ethnicity / race were only calculated for those for whom ethnicity / abbreviations : hr - ddrmut , homologous recombination dna damage response mutated ; n / a , not applicable ( demographic percentages were calculated for patients for whom the ethnicity / race were known ) ; pdac , pancreatic adenocarcinoma ; phr - ddr , homologous recombination dna damage response procient . after the diagnosis of pdac was established , then the patient was considered platinum treated . 
platinum - based regimens typically included folfirinox ( 85% in rst line and 39% in second line ) ; infusional uorouracil , leucovorin , and oxaliplatin ( 10% in rst line and 52% in second line ) ; or cisplatin with or without gemcitabine ( 3% in rst line and 7% in second line )  . 
variables in the analysis included the main effect of hr - ddr mutations ( prognostic term ) , the main effect of platinum exposure ( treatment term ) , an interaction between these two variables ( predictive term ) , and surgical status ( resectability term )  . 
genomic mutations in homologous recombination dna damage response ( hr - ddr ) genes are not prognostically favorable in patients with pancreatic adenocarcinoma who did not receive platinum - based chemotherapy . 
median overall survival ( mos ) differences on the basis of hr - ddr status were not signicant in platinum - nave patients diagnosed with either ( a ) resectable or ( b ) advanced disease . 
for patients with advanced disease who received platinum - based therapy , there was also an increased progression - free survival for hr - ddrmutated ( hr - ddrmut ) versus hr - ddrprocient ( phr - ddr ) patients in both the ( e ) rst line and ( f ) second line and later setting . 
instead , this trend ( not signicant ) suggested a potentially unfavorable prognostic effect for patients with mutations in the hr - ddr pathway , as shown in figures 1a and 1b . 
when factoring these four variables into our analysis , this model captures the value of hr - ddr mutations as a predictive class of biomarkers for increased responsiveness to platinum agents ( table 3 ) and indicates that this benet is not likely associated with an independent prognostic effect of hr - ddr mutations . assessing which ddr mutation confers the most predictive value the ddr pathway encompasses a functionally diverse network of interacting components spanning more than 50 enzymes ( fig 2 ) , many of which are critical for different aspects of the ddr process across a wide range of biologic contexts . 
atm and atr are critical for sensing double - stranded dna breaks and initiating a series of processes that allow a cell to recognize dna damage and halt the cell cycle until the damage is repaired . 
finally , mrn complex proteins and fanconi anemia core proteins play secondary , yet critical roles in the hr - ddr process . to assess which hr - ddr mutation ( s ) conferred the strongest predictive value to platinum - based therapy , we grouped the hr - ddr mutations into three groups ( fig 2 )  . 
hr - ddr subclassications ( group 1 , group 2 , and group 3 ) are based on the strength of evidence supporting each genes association with increased responsiveness to poly ( adp - ribose ) polymerase inhibitors and / or platinum agents . outcomes for each of the three groups were compared with phr - ddr patient outcomes . 
the number of patients in each group was small , but analysis that was focused on advanced patients conrmed that presence of an hr - ddr mutation is not prognostic , but is predictive of response to platinum - based that therapy . 
advanced pancreatic adenocarcinoma tumors with genomic mutations in hr - ddr genes were compared with the hr - ddrprocient ( phr - ddr ) cohort separately for groups 1 , 2 , and 3 , dened as follows : group 1 ( those with known clinical evidence of predictive value ) ; group 2 ( other genes with strong preclinical evidence of a suspected predictive value ) ; and group 3 ( other genes with preclinical evidence of a role in hr - ddr )  . 
in fact , there may be a trend toward worse outcomes in platinum - nave hr - ddrmut patients compared with platinum - nave phr - ddr patients , although not statistically signicant . in contrast , patients with advanced pdac with hr - ddrmut tumors who received at least one platinum - based regimen had a signicantly longer mos ( by nearly a full year ) compared with platinum - treated patients with phr - ddr tumors . 
this trend was also observed in resected patients who received any platinum - based therapy with an mos difference of nearly 1.5 years that we anticipate will become signicant as this cohort grows and data mature . 
moreover , for hr - ddrmut patients , mos was three times greater in the platinum - exposed versus platinum - nave patients . these results suggest that optimal therapy for hr - ddrmut patients includes a platinum - containing regimen . 
it is currently estimated that 17% to 25% of pdacs harbor somatic or germline hr - ddr mutations , 5 - 7 , 9 - 13 but only 50% of advanced patients are treated with platinum - based therapy in the rstline setting , and less than 50% of patients treated in the rstline go on to receive second - line therapy at all.2 this suggests that up to 25% of patientsnumerically up to 1 , 500 patients with pdac annuallywith hr - ddr mutated tumors are not being optimally treated with platinum - based therapy during the course of their pancreatic cancer survival . 
in addition , it is possible that patients with hr - ddrmutated pdacs who have undergone resection would have a greater chance for cure with an adjuvant platinum - based regimen , such as folfirinox , 23 instead of a nonplatinum - based regimen , such as gemcitabine plus capecitabine.24 there are limitations to this registry study . 
although testing results and treatment considerations were provided to the treating oncologist in real time , there was no prospective intervention to ensure that patients were treated according to the molecular test results . 
thus , this was a retrospective analysis of patients prospectively captured into the registry . longitudinal outcomes data were not yet furthermore , mature or not available for approximately 28% ( 320 of 1 , 140 ) of patients enrolled . 
second , although parallel genetic sequencing results of normal tissue or blood samples were not available to assess the germline status of the hrddr mutations identied in this study , approximately 54% of alterations in this pathway are estimated to be of germline origin on the basis of next - generation sequencing of pdac tumors.25 the relative impact of somatic versus germline hr - ddr mutations on responsiveness to platinums or parpi remains an important question to be answered , and one recent study in pdac demonstrated that patients with conrmed somatic - only brca2 mutations did still respond to the parpi rucaparib.19 additional studies are warranted to investigate the scope of hr - ddr pathway genes / variants and the types of molecular alterations ( eg , germline mutations with or without loss of heterozygosity in the tumor ) that are of clinical relevance . nevertheless , universal adoption of molecular proling of patients with pdac is growing , despite being a controversial issue . 
in april 2019 , the national comprehensive cancer network ( nccn ) modied their language for germline testing for patients with pdac.26 recognizing that up to 40% of patients with germline hr - ddrmutated pdac do not have any signicant family history to suggest a predisposing syndrome , such as a breast / ovarian cancer syndrome , 27 the new nccn guidelines state that germline testing is recommended for all patients with pancreatic cancer . 
recognizing that even somatic hr - ddr mutations may be therapeutically relevant and that germline testing alone may miss patients who could potentially benet from platinum - based therapy , the nccn went on to state that tumor / somatic testing is also recommended for patients with advanced pdac to identify uncommon but actionable mutations . 
matrisian , emmanuel petricoin iii administrative support : kimberly mason provision of study materials or patients : patricia de arbeloa , andrew hendifar , kimberly mason collection and assembly of data : michael j . 
pishvaian stock and other ownership interests : perthera honoraria : caris life sciences , celgene , sirtex medical , merrimack pharmaceuticals consulting or advisory role : caris life sciences , perthera , celgene , sirtex medical , astrazeneca , medimmune , renovorx , armo biosciences , rafael pharmaceuticals , ignyta , merck research funding : genentech ( inst ) , celldex ( inst ) , merck ( inst ) , glaxosmithkline ( inst ) , medimmune ( inst ) , pzer ( inst ) , gilead sciences ( inst ) , regeneron ( inst ) , novartis ( inst ) , karyopharm therapeutics ( inst ) , pharmacyclics ( inst ) , bristol - myers squibb ( inst ) , bayer ( inst ) , curegenix ( inst ) , calithera biosciences ( inst ) , tesaro ( inst ) , bavarian nordic ( inst ) , halozyme ( inst ) , armo biosciences ( inst ) , fibrogen ( inst ) , celgene ( inst ) patents , royalties , other intellectual property : perthera patient matching algorithm travel , accommodations , expenses : caris life sciences , sirtex medical , perthera , astrazeneca , medimmune edik m . 
blais employment : astrazeneca ( i ) , perthera stock and other ownership interests : perthera patents , royalties , other intellectual property : inventor on patents led by perthera ( as an employee ) jonathan r . 
brody stock and other ownership interests : perthera consulting or advisory role : perthera travel , accommodations , expenses : perthera patricia de arbeloa employment : perthera andrew hendifar consulting or advisory role : novartis , ipsen , perthera , celgene , abbvie research funding : ipsen travel , accommodations , expenses : halozyme sameh mikhail consulting or advisory role : ipsen , perthera vincent chung consulting or advisory role : celgene , perthera , ipsen , five prime therapeutics speakers bureau : celgene , ipsen , astrazeneca davendra p.s. 
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n engl j med 369 : 1691 - 1703 , 2013 pishvaian mj , bender rj , halverson d , et al : molecular proling of patients with pancreatic cancer : initial results from the know your tumor initiative . 
clin cancer res 24 : 5018 - 5027 , 2018 aguirre aj , nowak ja , camarda nd , et al : real - time genomic characterization of advanced pancreatic cancer to enable precision medicine . 
cancer discov 8 : 1096 - 1111 , 2018 lowery ma , jordan ej , basturk o , et al : real - time genomic proling of pancreatic ductal adenocarcinoma : potential actionability and correlation with clinical phenotype . 
clin cancer res 23 : 6094 - 6100 , 2017 bailey p , chang dk , nones k , et al : genomic analyses identify molecular subtypes of pancreatic cancer . 
domchek sm , aghajanian c , shapira - frommer r , et al : efcacy and safety of olaparib monotherapy in germline brca1 / 2 mutation carriers with advanced ovarian cancer and three or more lines of prior therapy . 
lowery ma , kelsen dp , stadler zk , et al : an emerging entity : pancreatic adenocarcinoma associated with a known brca mutationclinical descriptors , treatment implications , and future directions . 
oreilly em , lee jw , lowery ma , et al : phase 1 trial evaluating cisplatin , gemcitabine , and veliparib in 2 patient cohorts : germline brca mutation carriers and wild - type brca pancreatic ductal adenocarcinoma . 
oreilly em , lowery ma , segal mf , et al : phase ib trial of cisplatin ( c ) , gemcitabine ( g ) , and veliparib ( v ) in patients with known or potential brca or palb2mutated pancreas adenocarcinoma ( pc )  . 
neoptolemos jp , palmer dh , ghaneh p , et al : comparison of adjuvant gemcitabine and capecitabine with gemcitabine monotherapy in patients with resected pancreatic cancer ( espac - 4 ) : a multicentre , open - label , randomised , phase 3 trial . 
singhi ad , george b , greenbowe jr , et al : real - time targeted genome prole analysis of pancreatic ductal adenocarcinomas identies genetic alterations that might be targeted with existing drugs or used as biomarkers . 
 precision oncology in sarcomas : divide and conquer roberto carmagnani pestana , md1 ; roman groisberg , md1 ; jason roszik , phd , mba1 ; and vivek subbiah , md1 sarcomas are a heterogeneous group of rare malignancies that exhibit remarkable heterogeneity , with more than 50 subtypes recognized . 
advances in next - generation sequencing technology have resulted in the discovery of genetic events in these mesenchymal tumors , which in addition to enhancing understanding of the biology , have opened up avenues for molecularly targeted therapy and immunotherapy . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction sarcomas are a heterogeneous group of mesenchymal malignancies that comprise less than 1% of adult and 12% of pediatric cancers.1 , 2 the who has dened more than 50 sarcoma subtypes , including a wide array of tumors that arise from adipose , muscular , tissues.3 treatment bone , cartilage , and vascular options for patients with advanced soft tissue sarcomas ( stss ) are limited , and a one - size - ts - all paradigm has prevailed despite the diverse nature of stss . for metastatic stss , anthracycline - based chemotherapy remains the backbone of rst - line treatment regimens.4 however , efcacy is limited , with a median progression - free survival ( mpfs ) of approximately 6 months , and the incidence of treatment - associated adverse events ( aes ) is high.5 therefore , more individualized treatment options are critical to improve the survival and quality of life of this patient population . reecting sts heterogeneity , multiple molecular pathways are implicated in the development and progression of these cancers . 
the characterization of specic genetic aberrations has led to the identication of novel diagnostic , prognostic , and predictive biomarkers.6 an understanding of the prevalence and function of specic genetic alterations in sts subtypes is critical to developing more - effective diagnostic tests and therapeutic approaches . 
targeted therapies have the potential to produce signicant tumor response by disrupting molecular pathways that drive oncogenesis , thus providing highly personalized treatment.7 a recent report by lucchesi et al8 analyzed 584 patients with sts in the american association for cancer research ( aacr ) project genomics evidence neoplasia information exchange ( genie ) database and identied that 41% of patients harbored a genetic to inuence therapy . 
acalteration with potential cordingly , a single - center report by boddu et al9 analyzed 114 patients with sarcoma and identied that 49% carried a mutation deemed actionable ; 15 patients had next - generation sequencing ( ngs ) guided therapies , with 26% of these achieving clinical benet ( table 1 )  . 
studies that evaluated ngs in patients with sarcoma first author population boddu9 adult , sts and bone cote10 adult , sts and bone lucchesi8 adult , sts bone bone no . 
molecular approaches to detect the protein products of these fusion genes , such as uorescence in situ hybridization and reverse transcriptase polymerase chain reaction , can aid in the diagnosis of these tumors . 
the majority of clinical trials that have assessed chemotherapy for advanced stss have included heterogeneous populations , which complicates the assessment of clinical activity in specic subtypes . anthracyclines remain the most - used agents in the rst - line treatment of metastatic sts . 
 precision oncology in sarcomas alveolar rhabdomyosarcoma alveolar soft part sarcoma angiosarcoma chondroblastic osteosarcoma chondrosarcoma clear cell sarcoma dedifferentiated chondrosarcoma dedifferentiated liposarcoma dermatofibrosarcoma protuberans desmoid / aggressive fibromatosis desmoplastic small - round - cell tumor embryonal rhabdomyosarcoma endometrial stromal sarcoma epithelioid hemangioendothelioma epithelioid sarcoma ewing sarcoma extraskeletal myxoid chondrosarcoma fibrosarcoma follicular dendritic cell sarcoma hemangioma histiocytic dendritic cell sarcoma inflammatory myofibroblastic tumor intimal sarcoma leiomyosarcoma liposarcoma low - grade fibromyxoid sarcoma mesenchymal chondrosarcoma metaplastic carcinosarcoma myxofibrosarcoma myxoid chondrosarcoma myxoid / round - cell liposarcoma ossifying fibromyxoid tumor osteoblastic osteosarcoma osteosarcoma ovarian carcinosarcoma / malignant mixed mesodermal tumor perivascular epithelioid cell tumor pleomorphic liposarcoma pleomorphic rhabdomyosarcoma rhabdomyosarcoma round cell sarcoma , nos sarcoma , nos sarcomatoid renal cell carcinoma sclerosing epithelioid fibrosarcoma secondary osteosarcoma small cell osteosarcoma solitary fibrous tumor / hemangiopericytoma spindle cell rhabdomyosarcoma synovial sarcoma tenosynovial giant cell tumor diffuse type undifferentiated pleomorphic sarcoma / malignant fibrous histiocytoma / high - grade spindle cell sarcoma undifferentiated uterine sarcoma uterine adenosarcoma uterine carcinosarcoma / uterine malignant mixed mllerian tumor uterine leiomyosarcoma uterine perivascular epithelioid cell tumor uterine sarcoma , other well - differentiated liposarcoma fig 1 . 
adapted and updated from subbiah et al.122 abbreviations : c , clinical evidence ; ctla - 4 , cytotoxic t - cell lymphocyte - 4 ; gist , gi stromal tumor ; her2 , human epidermal growth factor receptor 2 ; mpnst , malignant peripheral nerve sheath tumor ; p , preclinical evidence ; pd - 1 , programmed cell death 1 ; pd - li , programmed cell death ligand 1 ; pecoma , perivascular epithelioid cell tumor ; sts , soft tissue sarcoma . limited to patients with liposarcoma.128 trabectedin , a synthetic alkaloid derived from the caribbean tunicate ecteinascidia turbinata , was approved by the food and drug administration ( fda ) for patients with advanced liposarcoma and leiomyosarcoma who received prior anthracycline - based regimens on the basis of a phase ii clinical trial that demonstrated its superiority to dacarbazine.129 retrospective reports have shown particular benet of therapy with trabectedin for myxoid liposarcoma ( orr , 51% ; disease control rate [ dcr ] , 90% ) , which achieved an mpfs of 14 months in patients who received multiple lines treatment.130 pazopanib is a multitargeted , smallmolecule tyrosine kinase inhibitor ( tki )  . 
however , as has been recently demonstrated in other rare tumor types that have led to fda - approved therapies , it is possible to establish efcacy with low patient numbers . 
therefore , trial designs are innovative clinical required to determine clinical benet of new therapies in patients with rare sts subtypes . basket trials are an emerging model of trial design centered on the assumption that the existence of a specic molecular aberration or biomarker predicts the benet of targeted therapies , regardless of cancer tissue of origin.136 the success of basket trials predominantly depends on the strength of the evidence that demonstrates tumor dependence on the targeted pathways and on reliable inhibition of the target by the drug.136 one example is the basket trial of vemurafenib for tumors that harbor a brafv600 mutation.71 vemurafenib is an orally available tki of braf , with higher selectivity for the brafv600 mutant form , that had been approved previously for patients with brafv600e mutationpositive metastatic melanoma.137 in this trial with six prespecied cohorts , there was one anecdotal response in a patient with clearinstitute cell sarcoma . 
some investigators are moving beyond searching for driver mutations and instead asking whether ngs can predict response or resistance to therapy ; others are creating expression proles that go beyond individual genes . 
ngs has the potential to become , in the coming years , an established platform of choice for researchers who are studying sarcomas . to date , many investigators have attempted to identify recurrent aberrations using ngs . 
they may be responsible for accelerated growth in advanced disease but are unlikely to be the sole cause of the malignancy . identication of these mutations is important , and with so much diversity in involved pathways , an individualized approach is necessary for proper sarcoma treatment . fruit bowl hypothesis current paradigm future paradigm one treatment for sarcoma precision therapy on the basis of molecular subtype fig 2 . 
for patients with few treatment options , a rationally chosen clinical trial on the basis of ngs - derived data is the best chance for prolonged survival . to demonstrate the ability of ngs to detect gene fusions , qadir et al138 designed child - seq , a pilot platform that rapidly screens cancer tissue for particular gene fusions characteristic of specic sarcomas . 
results demonstrated that the technique could reliably identify gene fusions in their corresponding tumor type without false positives . although this platform was limited to four particular childhood sarcomas , it is legitimate to conceptualize a more robust tool that could identify dozens or even hundreds of fusions . 
in fact , currently there are commercially available tools for molecular tissue testing that include detection of fusions ; identication of pathognomonic genetic alterations could enable a clinical diagnosis that is based solely on sequencing data . 
shukla et al139 demonstrated the feasibility of detecting ewsr1 fusions in plasma derived cell - free dna from patients with ewing sarcoma and desmoplastic small round - cell tumor . in general , ngs has proven to be a highly reliable technology . 
however , sanger sequencing of the patients tumor as well as her fathers germline testing revealed that the mlh1 mutation was a variant of unknown signicance rather than a pathogenic mutation . 
although this is a single case report , it highlights that even very robust technologies are subject to error . ngs for prediction ngs has the potential to identify specic molecular subtypes with particular sensitivity or resistance to approved treatments . 
to date , however , there have been few examples of ngs being used as a predictive tool in sarcomas , and it important to recognize that the examples discussed herein are preliminary and should be interpreted with caution . 
undoubtedly , as more patients undergo sequencing as part of their clinical care , patterns of response to therapy will emerge . koehler et al141 retrospectively reviewed 19 patients treated with pazopanib who also received ngs of their tumors . 
the authors found that the mpfs was signicantly longer in patients with tp53 mutant advanced sts ( 208 days ) than in those with tp53 wild - type tumors ( 136 days )  . lim et al142 performed ngs - based comprehensive genomic proling on 39 paired samples from tumor and normal tissue from patients treated with everolimus . 
these hypothesis - generating data reinforce the possibility of using ngs as a predictive tool in response to therapy . designing clinical trials on the basis of ngs wang et al143 performed a retrospective study of 75 patients with sarcomas who were referred to the clinical center for targeted therapy at md anderson cancer center . 
the majority of these tumors were sarcomas . multiomics studies included whole - exome sequencing ; germline , whole - transcriptome sequencing ; and singlenucleotide polymorphism array analysis of the tumor . germline cancer - associated gene mutations were reported in seven patients . 
one patient had an epithelioid imt that was identied to contain an ranbp2 - anaplastic lymphoma kinase ( alk ) fusion ; he experienced a complete response to crizotinib for 8 months . harris et al13 applied the same approach as chang et al144 to 89 pediatric tumors . 
this lack of response may have been due to the lower - quality preclinical evidence on which two of the recommendations were based . worst et al145 conducted the rst true prospective ngsbased trial with therapeutic intent . 
in this pilot study , 57 patients were enrolled from 20 centers ; approximately one half had sarcomas . one half of the patients harbored a potentially druggable alteration , and 10 patients went on to receive targeted therapy . 
however , because this was a pilot study designed to assess feasibility , there was no follow - up of patients who were treated with personalized targeted therapy and outcomes were not reported . 
early results with the use of inhibitors , however , have not been encouraging across all subtypes . therefore , the development of immunotherapy for sarcomas also benets from a precision oncology approach both in identifying predictive biomarkers and in developing strategies targeted to specic antigens . in the phase ii sarc028 trial ( clinicaltrials.gov identier : nct02301039 ) of pembrolizumab , an orr of 18% was seen in stss , with a 12 - week pfs of 55%.92 , 147 of note , this trial enrolled 40 patients with sts , including four tumor types ; subgroup analysis of the results identied encouraging activity in ups and dedifferentiated liposarcoma but not in other cohorts . 
combination of nivolumab and ipilimumab has provided promising efcacy in certain sts subtypes , with orr and mpfs in the range of those produced by standard - of - care options ; responses were observed in patients with leiomyosarcoma , myxobrosarcoma , ups , and angiosarcoma.148 furthermore , activity in specic subtypes has been suggested by retrospective studies . 
in a case series , two patients with chordoma experienced responses to single - agent antiprogrammed cell death ligand 1 ( pd - l1 ) antibodies , 149 and a review of patients enrolled in early - phase trials demonstrated that alveolar soft - part sarcoma was the most responsive subgroup to checkpoint blockade.91 , 150 the heterogeneity in the benet of checkpoint inhibitors across sarcoma subtypes observed in these early - phase trials highlights the need for a precision approach to immunotherapy in these diverse tumors , and research to identify predictive biomarkers is warranted . one challenge is that at this time , the immune microenvironment of specic subtypes is not sufciently characthe immune terized , and detailed characterization of microenvironment in each subtype is a major task . examples of potential predictive biomarkers to allow for selection of sarcomas sensitive to immune checkpoint inhibition include microsatellite instability ( msi ) status and pd - l1 expression in the tumor and microenvironment . 
in addition , responses were long lasting , with 78% lasting for 6 months or more.151 a recently published pan - cancer analysis identied that 5.7% of 785 sts cases analyzed were msi high , which highlights the relevance of this indication for sts.152 with regard to pd - l1 status , a recent meta - analysis with data from 1 , 451 patients and 15 independent studies identied pd - l1 expression to be independently associated with poorer os and event - free survival in bone and stss.153 however , the predictive value of pd - l1 in selecting patients with sarcoma for immune checkpoint inhibition is still under evaluation , and clinical activity of checkpoint inhibitors in these trials were seen even in the absence of pd - l1 expression.92 furthermore , cancer / testis antigens have been explored as potential targets for immunotherapy in sts and provide the utmost example of precision immunotherapy by targeting specic antigens . 
patients with kit exon 11 mutations have been identied as sensitive to imatinib , whereas patients with a kit exon 9 mutation , identied in 10% to 20% , respond poorly to imatinib at standard doses.155 most patients , however , eventually will develop resistance to treatment . 
in spite of the exceptional advances with rstline imatinib , the results with targeted treatments for later lines of therapy have been disappointing , emphasizing the need for new therapeutic approaches . 
imatinib dose escalation is one option for patients who have experienced disease progression on imatinib , and approximately 20% remain progression free for 1 year at the higher dose , especially among those with exon 9 mutations.162 furthermore , additional tkis are under development to optimize the treatment of imatinib - resistant gist . 
in addition , regorafenib was recently approved by the fda as a third - line therapy and has shown promising activity in patients with exon 17 mutations , which confer resistance to imatinib and sunitinib . 
co - occurring alterations in kit - mutated gi stromal tumor in the association for cancer research project genomics evidence neoplasia information exchange database ( n = 31 )  . available evidence indicates that kit mutation is likely an early event in the development of gist as suggested by the presence of activating kit mutations in gastric precursor lesions . 
in a previous study , 74% of gists analyzed had at least one nondriver genetic abnormality.164 in that report , the most frequently mutated genes were tp53 , rb1 , setd2 , pten , max , pik3ca , and tsc1 , and the most prevalent copy number alteration in kit - mutated gist was cdkn2a deletion.164 in addition , activation of alternate receptor tyrosine kinases has been suggested as a mechanism of resistance to currently approved tkis . 
for illustration , previous studies have identied that mutations that involve the rb1 gene are associated with high - risk tumors and aggressive clinical behavior and that activating mutations in the ras and phosphatidylinositol 3 - kinase pathways contribute to tki resistance.166 , 167 figure 4 illustrates co - occurring genetic alterations in kit - mutated gist in the aacr project genie database . in conclusion , sarcomas are a rare group of mesenchymal tumors in which the integration of ngs for diagnosis and management has provided informative evidence for precision medicine . 
the challenge to evaluate these mutations across rare cancer subtypes requires the careful characterization of these genetic alterations to further dene compelling drivers with therapeutic implications as well as novel models of clinical trial design . 
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martin , md1 introduction we read with interest two recently published articles in jco precision oncology that provide valuable insight regarding stk11 , an emerging biomarker in nonsmall - cell lung cancer ( nsclc ) .1 , 2 initially , shen et al1 detailed their ndings with the statistical learning tool oncocast . 
they identied stk11 as one of several prognostic genes afliated with poor survival from 1 , 054 patients with advanced lung adenocarcinoma proled with msk - impact next - generation sequencing ( ngs )  . 
by dividing the cohort into genomic risk - stratication groups , the authors highlighted the complexity of stk11 co - mutation patterns with other high - risk genes , including keap1 , kras , tp53 , and smarca4 . 
in the article by bange et al , 2 the authors retrospectively demonstrated heterogeneity of outcomes among patients with stk11 - mutated , advanced nsclc on the basis of co - mutation status . they reported that kras co - mutated cases had worse progression - free and overall survival outcomes after rst - line chemotherapy compared with tp53 . taken together , these ndings and others3 - 5 indicate that further work is warranted to characterize stk11 as an nsclc biomarker and to identify unique therapeutic approaches for patients with high - risk stk11mutated nsclc . 
a lumpectomy was performed in april 2014 , and adjuvant treatment included 12 doses of weekly paclitaxel , 1 year of trastuzumab , a partial breast radiation , and continuation on letrozole . 
a biopsy showed lung adenocarcinoma , and positron emission tomography ( pet ) / ct showed no additional disease . she received a robotic lobectomy , and pathologic staging revealed iiia disease . 
postoperative pet / ct in august showed a mildly hypermetabolic 1 - cm focus in the right middle lobe of the lung without other disease ; magnetic resonance imaging of the brain was negative . after radiologic review , it was initially believed that this focus represented postsurgical hypermetabolism , and close follow - up was recommended . 
she received adjuvant cisplatin and pemetrexed on day 1 of 21 - day cycles ; ct scans in october , after two of four cycles , showed no new disease . 
pet / ct revealed hypermetabolic disease limited to the 4 - cm adrenal mass . the adrenal and right middle - lobe lung lesions were biopsied , and both conrmed lung adenocarcinoma . she was referred for surgical evaluation but was not a candidate . 
testing included immunohistochemistry ( ihc ) , tumor mutational burden ( tmb ) , and 592 - gene ngs including egfr , braf , met , and her2 , as well as the previously mentioned high - risk genes stk11 , keap1 , kras , and tp53 ( nextseq ; illumina [ san diego , ca ] ) .6 ros1 and alk fusions were detected using the archerdx fusion assay ( archer fusionplex solid tumor kit )  . 
proling showed no targetable mutations , programmed death - ligand 1 ( pd - l1 ) ihc expression 0% ( 22c3 ; dako [ santa clara , ca ] ) , and tmb 15 mutations / megabase . 
no keap1 or tp53 mutations were identied . intravenous nivolumab 3 mg every 2 weeks and 1 mg / kg ipilimumab every 6 weeks were initiated in june 2018 . both treatments were withheld in july when the patient developed symptomatic autoimmune hyperthyroidism that necessitated treatment with methimazole and atenolol . 
nivolumab was resumed in august with symptom resolution , when ct scans showed a small increase in size of her adrenal metastasis with reduction of the right middle - lobe lesion . 
 nivolumab / ipilimumab in stk11 - mutated nonsmall - cell lung cancer by bange et al.2 although characterized by caris life sciences as a pathogenic mutation , we recognize that explicit pathogenicity of r39fs cannot be conrmed by commercial ngs alone . 
however , it should be noted that several studies have shown that frameshift variants ( including those of exon 1 ) generally express little stk11 protein by ihc , which serves as a reliable marker for loss of stk11.15 , 16 we recognize the complexity of dening high - risk stk11 molecular subsets from commercial ngs . 
martin consulting or advisory role : seattle genetics no other potential conicts of interest were reported . references shen r , martin a , ni a , et al : harnessing clinical sequencing data for survival stratication of patients with metastatic lung adenocarcinomas . 
cancer discov 8 : 822 - 835 , 2018 arbour kc , jordan e , kim hr , et al : effects of co - occurring genomic alterations on outcomes in patients with kras - mutant non - small cell lung cancer . 
clin cancer res 24 : 334 - 340 , 2018 skoulidis f , arbour kc , hellmann md , et al : association of stk11 / lkb1 genomic alterations with lack of benet from the addition of pembrolizumab to platinum doublet chemotherapy in non - squamous non - small cell lung cancer . 
vanderwalde a , spetzler d , xiao n , et al : microsatellite instability status determined by next - generation sequencing and compared with pd - l1 and tumor mutational burden in 11 , 348 patients . 
cancer med 7 : 746 - 756 , 2018 rizvi h , sanchez - vega f , la k , et al : molecular determinants of response to anti - programmed cell death ( pd ) - 1 and anti - programmed death - ligand 1 ( pd - l1 ) blockade in patients with non - small - cell lung cancer proled with targeted next - generation sequencing . 
nat rev drug discov 18 : 197 - 218 , 2019 ready n , hellmann md , awad mm , et al : first - line nivolumab plus ipilimumab in advanced non - small - cell lung cancer ( checkmate 568 ) : outcomes by programmed death ligand 1 and tumor mutational burden as biomarkers . 
frank r , schefer m , merkelbach - bruse s , et al : clinical and pathological characteristics of keap1and nfe2l2 - mutated non - small cell lung carcinoma 13 . 
p ecuchet n , laurent - puig p , mansuet - lupo a , et al : different prognostic impact of stk11 mutations in non - squamous non - small - cell lung cancer . 
brody , phd3 ; lola rahib , phd4 ; emily lyons , ma4 ; patricia de arbeloa , md2 ; andrew hendifar , md , mph5 ; sameh mikhail , md6 ; vincent chung , md7 ; davendra p.s. 
matrisian , phd , mba4 ; and emanuel petricoin iii , phd2 , 9 purpose up to 25% of pancreatic adenocarcinomas ( pdacs ) harbor mutations in the homologous recombination dna damage response ( hr - ddr ) pathway . 
although known to affect responsiveness to dnadamaging chemotherapy , the prognostic relevance of these mutations is unclear and outcomes in patients with pdac who harbor hr - ddr mutations beyond brca1 / 2 remain unexplored . methods we evaluated 820 patients with pdac enrolled in the know your tumor program for whom we had collected comprehensive genomic testing results and longitudinal clinical outcomes . 
mos in patients with mutations in all three hr - ddrmut groups was greater than that for phr - ddr patients , but this difference was lost in platinum - nave patients . conclusion patients with advanced hr - ddrmut have improved mos when treated with platinum - based therapy compared with phr - ddr patients . 
with a 5 - year median overall survival ( mos ) of only 9% , pdac is poised to become the second leading cause of cancer - related death in the united states.1 treatment of metastatic pdac has improved with the use of uorouracil , oxaliplatin , and irinotecan ( folfirinox ) , and gemcitabine plus nab - paclitaxel , but mos remains less than 1 year.2 , 3 however , there is a substantial group of outlier patients whose survival exceeds the median of less than 1 year . 
 pishvaian et al context key objective up to 25% of pancreatic adenocarcinomas ( pdacs ) harbor mutations in the homologous recombination dna damage response ( hr - ddr ) pathway . 
we evaluated 820 patients with pdac who were enrolled in the know your tumor program for whom we had collected comprehensive genomic proling data and longitudinal clinical outcomes to assess the prognostic and predictive value of hr - ddr mutations . knowledge generated patients with advanced pdac and the presence of a somatic or germline hr - ddr mutation have an improved median overall survival ( mos ) when treated with platinum - based therapy compared with patients with no hr - ddr mutation . 
in platinumnave patients , there is no mos difference , which suggests that hr - ddr status has no pure prognostic value . relevance these ndings support the need to test all patients with advanced pdac to ensure that patients whose tumors harbor hr - ddr mutations receive the benet of treatment with platinum - based therapy . prolonged stable disease of 15 patients with pdac who were treated with parpi - based therapy , whereas kaufman et al21 reported one complete response and four partial responses of 23 brca - mutated patients with pdac who were treated with olaparib . 
combinations with chemotherapy may be even more promising , as oreilly et al17 reported a 78% response to the combination of gemcitabine , cisplatin , and veliparib in patients with brcaor palb2mutated pdacs . the treatments received , or we evaluated whether patients with hr - ddrmutated tumors have a biologically better prognosis , irrespective if patients with hrddrmutated tumors have longer than average survival strictly because the underlying mutation predicts for a high degree of responsiveness to platinum - based therapies ( part of the standard of care )  . methods patient recruitment into the know your tumor registry as reported , 4 patients with pdac were identied through the pancreatic cancer action network patient central call center , referred to perthera , and enrolled via an institutional review boardapproved registry protocol . 
study protocol , amendments , and informed consent forms were approved by the new england institutional review board . vestigators obtained informed consent from each participant or participants guardian before enrollment . 
the research was conducted in accordance with recognized ethical guidelines , including the declaration of helsinki , for international organizations of medical scicouncil ences , belmont report , and us common rule , as described during training in good clinical practice guidelines ( collaborative institutional training initiative training )  . patients with biopsy - conrmed pdac were included in the analysis cohort . 
patients with pancreatic adenosquamous carcinoma or other histologic subtypes were excluded . next - generation dna sequencing and identication of hr - ddr status formalin - xed , parafn - embedded blocks and / or slides were collected by surgical resection ( eg , whipple procedure or distal pancreatectomy ) , core - needle biopsy , or ne - needle aspiration . 
coverage of hr - ddr genes tested was relatively uniform with 97% of genomic testing completed by foundation medicine ( cambridge , ma ) , 1% by caris life sciences ( irving , tx ) , and the remaining by including in - house pathology deother partments . 
genomic alterations reported by the testing laboratory included both somatic and germline mutations ; however , it is important to note that the germline versus somatic status of each mutation could not be determined on the basis of tumor proling without parallel genetic sequencing of matched normal blood or tissue . patient tumor genomic proles were reviewed and pathogenic mutations were conrmed by pertheras molecular tumor board participants on a case - by - case basis . 
tumors that harbored no hr - ddr mutations were labeled ddr procient ( phr - ddr )  . results patient history and outcomes enrolled patients signed a health insurance portability and accountability act waiver , and for patient history and longitudinal outcomes assessment , patient coordinators the patients requested updated medical records until death . 
to reduce variability as a result of the timing of enrollment and to establish a denitive starting point for the determination of mos , survival was calculated from the initial date of diagnosis of pdac until death . 
patients were categorized herein as those who underwent a resection of the primary tumor at any point during their treatment history ( resected ) or and as those who had advanced disease at presentation , including locally advanced , never resected patients , and patients with metastatic disease at presentation . 
furthermore , patients were designated as platinum exposed if they received any platinum - containing therapy at any point during the course of their pancreatic cancer survival . statistical analysis all statistical analyses were performed in an r / bioconductor programming environment.22 mos was measured from the date of initial diagnosis until the date of death or the last date the patient was known to be alive . median progression - free survival ( mpfs ) was measured from the start date to the last date the patient was known to be on treatment without progression of disease . 
differences between groups were evaluated for statistical signicance using cox proportional hazards regression models ( the coxph function in the r survival package )  . computed values reported include mpfs , mos , p values , hazard ratios , and 95% cis for hazard ratios . a multivariable approach was implemented to delineate prognostic versus predictive effects of hr - ddr status using the entire analysis cohort . 
given the limitations of interpreting several pairwise comparisons across small groups of patients , we accounted for three binary independent factors : platinum exposure , hr - drr status , and resectability . 
multivariable cox proportional hazards regression was performed on os analysis data by factoring in platinum exposure as one variable , the presence of an hr - ddr alteration as a second variable , interaction between the rst and second variable , and a third variable to account for the patients resection status , which is known to have a highly signicant prognostic impact on os . of 1 , 140 patients with pdac who were enrolled in the know your tumor program between january 2013 and the end of december 2018 , 820 patients with molecular testing results , collection of longitudinal outcomes , and adequate treatment history were included in the analysis cohort . median age at diagnosis was 62 years ( range , 28 to 90 years ) , 52% of patients were male , and 81% of patients were white ( table 1 )  . 
prevalence of somatic or germline hr - ddr mutations ( 16.5% ) and other baseline characteristics was not signicantly different within the resectable and advanced cohorts ( table 1 )  . 
at a median follow - up of 637 days and 386 days for the resected and advanced cohorts , respectively , 47.6% of patients were last known to be alive and 29.2% of patients had not received platinum - based therapy . 
twentyve percent of deceased patients never received any platinum - based therapy . assessing the prognostic value of hr - ddr mutations in platinum - nave patients to assess the prognostic value of the presence of an hrddr mutation , in the absence of the confounding variable of the known predictive value of response to platinum - based therapy , we chose to assess outcomes in patients who never received platinum - based therapy at any time during their pancreatic cancer survival . 
these patients were labeled platinum nave and included 108 resected and 133 advanced patients for analysis ( table 1 and figs 1a and 1b )  . differences in mos were not statistically signicant when comparing hr - ddrmut with phr - ddr patients whose disease was platinum nave in both the resected and advanced subgroups . 
thus , there was no apparent positive prognostic value associated with the presence of an hrddr mutation in platinum - nave patients with pdac . conrming the predictive value for platinum - based therapy of an hr - ddr mutation we also assessed the predictive value for platinum - based therapy of an hr - ddr mutation ( figs 1c and 1d and table 2 )  . 
percentages of patients on the basis of ethnicity / race were only calculated for those for whom ethnicity / abbreviations : hr - ddrmut , homologous recombination dna damage response mutated ; n / a , not applicable ( demographic percentages were calculated for patients for whom the ethnicity / race were known ) ; pdac , pancreatic adenocarcinoma ; phr - ddr , homologous recombination dna damage response procient . after the diagnosis of pdac was established , then the patient was considered platinum treated . 
platinum - based regimens typically included folfirinox ( 85% in rst line and 39% in second line ) ; infusional uorouracil , leucovorin , and oxaliplatin ( 10% in rst line and 52% in second line ) ; or cisplatin with or without gemcitabine ( 3% in rst line and 7% in second line )  . 
variables in the analysis included the main effect of hr - ddr mutations ( prognostic term ) , the main effect of platinum exposure ( treatment term ) , an interaction between these two variables ( predictive term ) , and surgical status ( resectability term )  . 
genomic mutations in homologous recombination dna damage response ( hr - ddr ) genes are not prognostically favorable in patients with pancreatic adenocarcinoma who did not receive platinum - based chemotherapy . 
median overall survival ( mos ) differences on the basis of hr - ddr status were not signicant in platinum - nave patients diagnosed with either ( a ) resectable or ( b ) advanced disease . 
for patients with advanced disease who received platinum - based therapy , there was also an increased progression - free survival for hr - ddrmutated ( hr - ddrmut ) versus hr - ddrprocient ( phr - ddr ) patients in both the ( e ) rst line and ( f ) second line and later setting . 
instead , this trend ( not signicant ) suggested a potentially unfavorable prognostic effect for patients with mutations in the hr - ddr pathway , as shown in figures 1a and 1b . 
when factoring these four variables into our analysis , this model captures the value of hr - ddr mutations as a predictive class of biomarkers for increased responsiveness to platinum agents ( table 3 ) and indicates that this benet is not likely associated with an independent prognostic effect of hr - ddr mutations . assessing which ddr mutation confers the most predictive value the ddr pathway encompasses a functionally diverse network of interacting components spanning more than 50 enzymes ( fig 2 ) , many of which are critical for different aspects of the ddr process across a wide range of biologic contexts . 
atm and atr are critical for sensing double - stranded dna breaks and initiating a series of processes that allow a cell to recognize dna damage and halt the cell cycle until the damage is repaired . 
finally , mrn complex proteins and fanconi anemia core proteins play secondary , yet critical roles in the hr - ddr process . to assess which hr - ddr mutation ( s ) conferred the strongest predictive value to platinum - based therapy , we grouped the hr - ddr mutations into three groups ( fig 2 )  . 
hr - ddr subclassications ( group 1 , group 2 , and group 3 ) are based on the strength of evidence supporting each genes association with increased responsiveness to poly ( adp - ribose ) polymerase inhibitors and / or platinum agents . outcomes for each of the three groups were compared with phr - ddr patient outcomes . 
the number of patients in each group was small , but analysis that was focused on advanced patients conrmed that presence of an hr - ddr mutation is not prognostic , but is predictive of response to platinum - based that therapy . 
advanced pancreatic adenocarcinoma tumors with genomic mutations in hr - ddr genes were compared with the hr - ddrprocient ( phr - ddr ) cohort separately for groups 1 , 2 , and 3 , dened as follows : group 1 ( those with known clinical evidence of predictive value ) ; group 2 ( other genes with strong preclinical evidence of a suspected predictive value ) ; and group 3 ( other genes with preclinical evidence of a role in hr - ddr )  . 
in fact , there may be a trend toward worse outcomes in platinum - nave hr - ddrmut patients compared with platinum - nave phr - ddr patients , although not statistically signicant . in contrast , patients with advanced pdac with hr - ddrmut tumors who received at least one platinum - based regimen had a signicantly longer mos ( by nearly a full year ) compared with platinum - treated patients with phr - ddr tumors . 
this trend was also observed in resected patients who received any platinum - based therapy with an mos difference of nearly 1.5 years that we anticipate will become signicant as this cohort grows and data mature . 
moreover , for hr - ddrmut patients , mos was three times greater in the platinum - exposed versus platinum - nave patients . these results suggest that optimal therapy for hr - ddrmut patients includes a platinum - containing regimen . 
it is currently estimated that 17% to 25% of pdacs harbor somatic or germline hr - ddr mutations , 5 - 7 , 9 - 13 but only 50% of advanced patients are treated with platinum - based therapy in the rstline setting , and less than 50% of patients treated in the rstline go on to receive second - line therapy at all.2 this suggests that up to 25% of patientsnumerically up to 1 , 500 patients with pdac annuallywith hr - ddr mutated tumors are not being optimally treated with platinum - based therapy during the course of their pancreatic cancer survival . 
in addition , it is possible that patients with hr - ddrmutated pdacs who have undergone resection would have a greater chance for cure with an adjuvant platinum - based regimen , such as folfirinox , 23 instead of a nonplatinum - based regimen , such as gemcitabine plus capecitabine.24 there are limitations to this registry study . 
although testing results and treatment considerations were provided to the treating oncologist in real time , there was no prospective intervention to ensure that patients were treated according to the molecular test results . 
thus , this was a retrospective analysis of patients prospectively captured into the registry . longitudinal outcomes data were not yet furthermore , mature or not available for approximately 28% ( 320 of 1 , 140 ) of patients enrolled . 
second , although parallel genetic sequencing results of normal tissue or blood samples were not available to assess the germline status of the hrddr mutations identied in this study , approximately 54% of alterations in this pathway are estimated to be of germline origin on the basis of next - generation sequencing of pdac tumors.25 the relative impact of somatic versus germline hr - ddr mutations on responsiveness to platinums or parpi remains an important question to be answered , and one recent study in pdac demonstrated that patients with conrmed somatic - only brca2 mutations did still respond to the parpi rucaparib.19 additional studies are warranted to investigate the scope of hr - ddr pathway genes / variants and the types of molecular alterations ( eg , germline mutations with or without loss of heterozygosity in the tumor ) that are of clinical relevance . nevertheless , universal adoption of molecular proling of patients with pdac is growing , despite being a controversial issue . 
in april 2019 , the national comprehensive cancer network ( nccn ) modied their language for germline testing for patients with pdac.26 recognizing that up to 40% of patients with germline hr - ddrmutated pdac do not have any signicant family history to suggest a predisposing syndrome , such as a breast / ovarian cancer syndrome , 27 the new nccn guidelines state that germline testing is recommended for all patients with pancreatic cancer . 
recognizing that even somatic hr - ddr mutations may be therapeutically relevant and that germline testing alone may miss patients who could potentially benet from platinum - based therapy , the nccn went on to state that tumor / somatic testing is also recommended for patients with advanced pdac to identify uncommon but actionable mutations . 
matrisian , emmanuel petricoin iii administrative support : kimberly mason provision of study materials or patients : patricia de arbeloa , andrew hendifar , kimberly mason collection and assembly of data : michael j . 
pishvaian stock and other ownership interests : perthera honoraria : caris life sciences , celgene , sirtex medical , merrimack pharmaceuticals consulting or advisory role : caris life sciences , perthera , celgene , sirtex medical , astrazeneca , medimmune , renovorx , armo biosciences , rafael pharmaceuticals , ignyta , merck research funding : genentech ( inst ) , celldex ( inst ) , merck ( inst ) , glaxosmithkline ( inst ) , medimmune ( inst ) , pzer ( inst ) , gilead sciences ( inst ) , regeneron ( inst ) , novartis ( inst ) , karyopharm therapeutics ( inst ) , pharmacyclics ( inst ) , bristol - myers squibb ( inst ) , bayer ( inst ) , curegenix ( inst ) , calithera biosciences ( inst ) , tesaro ( inst ) , bavarian nordic ( inst ) , halozyme ( inst ) , armo biosciences ( inst ) , fibrogen ( inst ) , celgene ( inst ) patents , royalties , other intellectual property : perthera patient matching algorithm travel , accommodations , expenses : caris life sciences , sirtex medical , perthera , astrazeneca , medimmune edik m . 
blais employment : astrazeneca ( i ) , perthera stock and other ownership interests : perthera patents , royalties , other intellectual property : inventor on patents led by perthera ( as an employee ) jonathan r . 
brody stock and other ownership interests : perthera consulting or advisory role : perthera travel , accommodations , expenses : perthera patricia de arbeloa employment : perthera andrew hendifar consulting or advisory role : novartis , ipsen , perthera , celgene , abbvie research funding : ipsen travel , accommodations , expenses : halozyme sameh mikhail consulting or advisory role : ipsen , perthera vincent chung consulting or advisory role : celgene , perthera , ipsen , five prime therapeutics speakers bureau : celgene , ipsen , astrazeneca davendra p.s. 
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n engl j med 364 : 1817 - 1825 , 2011 von hoff dd , ervin t , arena fp , et al : increased survival in pancreatic cancer with nab - paclitaxel plus gemcitabine . 
n engl j med 369 : 1691 - 1703 , 2013 pishvaian mj , bender rj , halverson d , et al : molecular proling of patients with pancreatic cancer : initial results from the know your tumor initiative . 
clin cancer res 24 : 5018 - 5027 , 2018 aguirre aj , nowak ja , camarda nd , et al : real - time genomic characterization of advanced pancreatic cancer to enable precision medicine . 
cancer discov 8 : 1096 - 1111 , 2018 lowery ma , jordan ej , basturk o , et al : real - time genomic proling of pancreatic ductal adenocarcinoma : potential actionability and correlation with clinical phenotype . 
clin cancer res 23 : 6094 - 6100 , 2017 bailey p , chang dk , nones k , et al : genomic analyses identify molecular subtypes of pancreatic cancer . 
domchek sm , aghajanian c , shapira - frommer r , et al : efcacy and safety of olaparib monotherapy in germline brca1 / 2 mutation carriers with advanced ovarian cancer and three or more lines of prior therapy . 
lowery ma , kelsen dp , stadler zk , et al : an emerging entity : pancreatic adenocarcinoma associated with a known brca mutationclinical descriptors , treatment implications , and future directions . 
oreilly em , lee jw , lowery ma , et al : phase 1 trial evaluating cisplatin , gemcitabine , and veliparib in 2 patient cohorts : germline brca mutation carriers and wild - type brca pancreatic ductal adenocarcinoma . 
oreilly em , lowery ma , segal mf , et al : phase ib trial of cisplatin ( c ) , gemcitabine ( g ) , and veliparib ( v ) in patients with known or potential brca or palb2mutated pancreas adenocarcinoma ( pc )  . 
neoptolemos jp , palmer dh , ghaneh p , et al : comparison of adjuvant gemcitabine and capecitabine with gemcitabine monotherapy in patients with resected pancreatic cancer ( espac - 4 ) : a multicentre , open - label , randomised , phase 3 trial . 
singhi ad , george b , greenbowe jr , et al : real - time targeted genome prole analysis of pancreatic ductal adenocarcinomas identies genetic alterations that might be targeted with existing drugs or used as biomarkers . 
 biomarkers intratumoral transcriptome heterogeneity is associated with patient prognosis and sidedness in patients with colorectal cancer treated with anti - egfr therapy from the co.20 trial elisa fontana , md , phd1 , 2 ; gift nyamundanda , phd1 , 2 ; david cunningham , md3 ; dongsheng tu , phd4 ; maggie c.u. 
siu , md8 ; francesco sclafani , md3 , 9 ; katherine eason , phd1 ; chanthirika ragulan , msc1 , 2 ; maria antonietta bali , md , phd10 , 11 ; sanna hulkki - wilson , bsc1 ; jonathan m . 
waring , mbbs , phd13 ; mirella giordano , phd14 ; patrick lawrence , ms1 , 2 ; daniel nava rodrigues , md3 ; ruwaida begum , bsc3 ; jeremy d . 
price , md16 ; chiara cremolini , md17 , 18 ; naureen starling , mbbs , mrcp , md ( res ) 3 ; filippo pietrantonio , md19 , 20 ; livio trusolino , md , phd21 , 22 ; christopher j . 
ocallaghan , dvm , msc , phd4 ; and anguraj sadanandam , phd1 , 2 purpose metastatic colorectal cancers ( mcrcs ) assigned to the transit - amplifying ( ta ) crcassigner subtype are more sensitive to antiepidermal growth factor receptor ( egfr ) therapy . 
progression - free survival ( pfs ) , overall survival ( os ) , and disease control rate ( dcr ) according to ta - ness classication were assessed by univariate and multivariate analyses . results the ta - ness was measured in 772 samples from 712 patients . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction epidermal growth factor receptor ( egfr ) - targeting antibodies cetuximab and panitumumab are available treatment options for approximately 40% of patients with metastatic colorectal cancer ( mcrc ) .1 patient selection based on ras and braf wild - type status and sidedness has improved overall response rates and survival outcomes . 
nevertheless , 30% - 60% of eligible patients do not benet from these expensive drugs.2 - 4 as a shift from the traditional paradigm of negative molecular selection , we previously demonstrated that the transit - amplifying ( ta ) crcassigner ( crca ) subtype was enriched for cetuximab - responsive tumors , 5 a nding independently validated in a clinical study , 6 in a panel of crc xenografts5 and cell lines.5 , 7 however , responses were also seen in other groups , such as the poorly differentiated stem - like subtype , 5 , 7 albeit at a lower frequency . 
 intratumoral transcriptome heterogeneity and anti - egfr therapy context key objective to evaluate whether the presence of the transit - amplifying ( ta ) subtype gene signature ( dubbed as ta - ness classication ) representing the intratumoral transcriptome heterogeneity is associated with antiepidermal growth factor receptor ( egfr ) therapy outcomes . knowledge generated the ta - ness classication is an easily detectable biomarker of intratumoral transcriptome heterogeneity , which was retrospectively evaluated in 712 patient samples , including those from a clinical ( co.20 ) trial , which showed prognostic signicance in patients treated with anti - egfr therapy . 
this biomarker provides additional biologic insights for the association between ras / braf wild - type left - sided tumors ( enriched for ta - high ) and anti - egfr therapy benet . relevance with further validation , ta - ness may represent a positive selection biomarker for patients with ras / braf wild - type left - sided metastatic colorectal cancer who are most likely to benet from anti - egfr therapy . ta subtype tumors are characterized by gene signatures similar to normal ta cells of the colonic crypt , that is , those in transit between stem cells in the crypt base and differentiated cells at the top of the crypt.5 after asymmetric division , stem cells generate rapidly proliferating ta cells characterized by increased egfr expression that eventually differentiate into goblet cells and enterocytes.8 , 9 we evaluated a hypothesis that tumors with increased ta gene signature expression ( irrespective of ta or other subtypes ) may be associated with anti - egfr therapy outcomes . 
the discovery cohort included chemorefractory patients ( n = 84 ) who had received anti - egfr therapy as a single agent or in combination with chemotherapy after progression while receiving irinotecan ( during or within 3 months from the end of treatment ) as part of standard treatment at the royal marsden hospital ( rmh ; n = 59 ; united kingdom , ethics committee : 10 / h0308 / 28 ; and clinicaltrials.gov identier : nct02112357 ) or within the context of a case - control study in italian institutions ( pressing , n = 25 ; ethics committee area vasta nord ovest number 1333 / 173 )  . 
nineteen and 12 patients from the rmh cohort were treated before the implementation of kras testing ( august 2009 ) and extended ras testing ( december 2011 ) , respectively.11 , 12 all patient samples from the pressing study had extended ras / braf wildtype tumors . one of the clinical validation cohorts included 121 patients with kras exon 2 wild - type tumors who had received single - agent cetuximab within the control arm of the co.20 phase iii randomized clinical trial ( clinicaltrials.gov identier : nct00640471 ) .13 this correlative analysis was approved by the joint canadian cancer trial group and australasian gastrointestinal trial group ( cctg / agitg ) correlative sciences and tumor biology committee . two additional public gene expression datasets ( n = 397 ; not treated with anti - egfr therapy ) of primary crc samples ( gse39582 ; n = 328 ) and liver mcrc lesions ( gse73255 ; n = 69 ) were evaluated.14 , 15 only samples with known kras wild - type status were selected . nucleic acids extraction formalin - xed parafn - embedded ( ffpe ) tissues were least evaluated by a trained pathologist ; areas with at 30% of tumor content were marked on hematoxylin and eosin slides and macrodissected in unstained slides ( 7to 10 - m thickness )  . 
after deparafnization , total rna and dna were simultaneously isolated using the ambion recoverall kit ( discovery ) or qiaamp nucleic acid ffpe tissue kit ( validation ) and quantied with nanodrop 2000 spectrophotometer ( thermo fisher , waltham , ma ) according to the manufacturers instructions . 
 a fontana et al discovery cohort retrospective tissue collections validation cohort 1 prospectively enrolled in the co.20 study outcome cohort ta - ness no . median pfs ( months ) univariate analysis hazard ratio ( 95% ci ) log - rank multivariate analysis hazard ratio ( 95% ci ) cox regression rmh cohort ( n = 90 ) italian cohort ( n = 42 ) patients ( out of 374 in cetuximab arm ) ( n = 163 ) patients with quality rna ( n = 65 ) patients with quality rna ( n = 25 ) patients with quality rna ( n = 146 ) excluded because of low tumor content in the block or low rna conc . 
 after extraction ( n = 25 ) patients excluded because of qc flag after ncounter analysis ( n = 6 ) patients excluded for anti - egfr in refractory setting as re - challenge ( n = 17 ) patients excluded because of qc flag after ncounter analysis ( n = 0 ) patients for final analysis ( n = 59 ) patients for final analysis ( n = 25 ) patients for final analysis ( n = 121 ) patients excluded because of low tumor content in the block or low rna conc . 
covariates included in the validation cohort : eastern cooperative oncology group performance status , sex , age , baseline lactate dehydrogenase level , baseline alkaline phosphatase , baseline hemoglobin , number of disease sites , number of previous chemotherapy drug classes , prior vegfr target therapy , and presence of liver metastases . 
conc. , concentration ; egfr , epidermal growth factor receptor ; pdx , patient - derived xenograft ; qc , quality control ; rmh , royal marsden hospital . ve published crca - 38 centroids ( expression summary of each gene in each subtype ) 16 and gene expression , each sample was assigned either to ta - high ( increased expression of ta signature genes ) or ta - low ( reduced expression ) ta - ness classes . 
when gene expression proles were compared with the ve crca - 38 centroids , ve correlation coefcients ( one for each subtype - centroid ) were calculated for each sample . 
the coefcients were then ranked from highest to lowest ; ta - high samples were those with a correlation coefcient value for the ta centroid ranking within the rst three highest values ; ta - low samples were those with a correlation coefcient for the ta centroid , which is second to last or the lowest . 
therefore , the ta - ness classication represents a measure of transcriptome - based intratumoral heterogeneity in mcrc , based on the idea that each sample can contain more than one subtype . 
this best cut - off for ta - ness classication was established based on the highest accuracy in dening disease control , measured as area under the curve ( auc ) of a receiver operating characteristic ( roc ) curve ( appendix fig a1 )  . statistical analysis progression - free survival ( pfs ) was the primary endpoint . overall survival ( os ) , disease control rate ( dcr ) , and response rate were secondary endpoints . 
fishers exact test was used to compare categorical variables , and wilcoxon signed rank test with p , .05 was used to assess the association between ta - ness classes and percentage of tumor shrinkage ( using recist ) criteria in a subgroup of the discovery cohort . 
although the statistical analysis of discovery cohort was performed by the institute of cancer research statistician , the validation cohort was independently analyzed by cctg / agitg investigators blinded to the biomarker cut - off analysis . 
additional methods are available in the data supplement . results retrospective antiegfr - treated tumor samples from 205 patients were identied from the discovery and validation ( co.20 ) cohorts after clinical review and quality control of the tumor blocks and tumor - derived rna ( fig 1a )  . 
 intratumoral transcriptome heterogeneity and anti - egfr therapy cohort from 30 patients along with 30 patient - derived xenografts ( pdxs ; derived from the patient tumors ) that were treated with anti - egfr therapy or vehicle ( control ) were subjected to the same crca gene expression assay . 
in addition , publicly available gene expression microarray data for 80 patients with mcrc ( treated with anti - egfr therapy ) was included as an additional clinical validation cohort.17 this publicly available cohort also served to validate ta - ness classication using a different platform ( microarrays ; fig 1a )  . patient characteristics for discovery and validation co.20 cohorts are shown in the data supplement . 
with the exception of sex , there were no signicant differences in patients characteristics between the co.20 subgroup included in this analysis and the overall co.20 clinical trial cohort ( data supplement )  . using conventional subtyping , 15 of 84 samples belonged to the ta subtype in the discovery cohort . 
the association of ta - ness classication with both pfs and dcr remained signicant after adjusting for multiple variables in both the discovery and validation cohorts ( fig 1b )  . 
conversely , after adjusting for multiple variables , signicant association of ta - ness with os was only borderline ( or not signicant with p = .1 in the discovery cohort and p = .06 in the validation cohort ; data supplement ) ; postprogression treatment information was not available . in the discovery cohort , ta - high tumors ( 62% ; n = 52 ) were predominantly ras / braf wild - type ( 69% ; n = 36 ) and were found in the left side of the colon ( 79% ; n = 41 )  . 
this result was mirrored in the experimental cohort , 18 , 19 in which 30 ras / braf wild - type liver metastases were classied into ta - high ( n = 16 ) and ta - low ( n = 14 ) classes . 
the percentage of cetuximab - induced tumor volume change in the pdx - based mouse - propagated patient metastatic tumors was signicantly associated ( p , .042 ) with the ta - ness signature ( fig 3b )  . in the discovery cohort , the ta - ness classication was assessed using samples from primary tumors in 76% of patients and samples from metastatic sites in 24% of patients . 
to further conrm that the association was independent of the diagnostic sample of origin and to further validate the results , we examined the khambata - ford publicly available ( microarray ) dataset17 of mcrc samples from patients treated with cetuximab . 
to further conrm that the ta - ness can be assessed in both primary tumors and metastatic lesions , kras wild - type samples from two publicly available datasets14 , 15 were selected ; 328 primary tumors and 69 liver metastases were classied into ta - high and ta - low . 
similar distribution of the two classes was demonstrated ( fig 3c )  . beyond ras / braf mutational status , sidedness is a recognized selection factor for anti - egfr therapy benet : patients with left - sided tumors benet more than patients with right - sided tumors.4 however , the biology behind this association remains unclear . 
first , we further conrmed signicant association ( p , .001 ) between ta - ness classication and sidedness in kras wild - type primary tumors ( gse39582 ; fig 3d )  . 
then , we sought to discover whether the ta - ness classication could further rene the selection of patients in addition to ras / braf status and sidedness . within discovery and validation cohorts ( n = 205 ) , highsensitivity next - generation sequencing ras / braf mutational analysis was available for 118 patients : 71 were classied as ras / braf wild - type , of which 53 were assigned to ta - high ( 75% ) class . 
 ( a ) a waterfall plot showing a subgroup of patients within the discovery cohort ( n = 35 ) showing disease response ( treated with anti epidermal growth factor receptor [ egfr ] drug ) according to recist criteria and ta - ness classication . 
 ( b ) a waterfall plot showing change in tumor ( percent ) volume in anti - egfrtreated mouse - propagated patient tumor samples ( n = 30 ) compared with matched control treated ( baseline ; n = 30 ) mouse - propagated patient tumors . 
the bars in the graph show ta - ness classication for the matched patient metastatic liver samples ( n = 30 ) , and the bars below the graph show the same classication for matched mousepropagated patient tumors ( treated v control )  . 
this classication has the advantage of providing a qualitative assessment in all the samples , including the non - ta subtypes , overcoming the limitations posed by intratumoral heterogeneity when using the conventional molecular subtyping classication as a potential tool to assess benet from anti - egfr therapy . 
kaplan - meier survival curves of patients with transit - amplifying ( ta ) - high versus ta - low tumors treated with antiepidermal growth factor receptor ( egfr ) therapy . 
 fontana et al ( and potentially clinical benet ) in patients treated with anti - egfrbased therapy in our discovery cohort ; this was validated in a kras exon 2 wild - type trial cohort of cetuximab - onlytreated patients , 13 which has the advantage of properly assessing prognostic value in a homogeneously treated population and in the absence of confounding effect of chemotherapy . 
moreover , ta - low assignment was enriched for ras / braf - mutant tumors , providing a potential alternative method to estimate prognosis and may be a treatment benet from anti - egfr therapy when the mutational status is missing . 
this signature and its association with anti - egfr treatment outcomes were also conrmed in the publicly available samples from patients with mcrc17 and the preclinical pdx models treated with cetuximab.18 , 19 finally , the taness classication retained prognostic signicance when assessed in either archival primary tumors or metastatic samples in multiple cohorts . 
this is highly clinically relethe classication can be vant , because it means that assessed in metastatic lesions when the primary tumor sample is not available or of poor quality ; however , intrapatient concordance was not assessed ; therefore , additional validation is required . several studies have now evaluated the association between single genes or micrornas ( ereg / areg , her2 , her3 , epha2 , or mir - 31 - 3p ) and responses to anti - egfr therapy.20 in contrast , we evaluated a rened form of our previously published gene expression signature ( with multiple genes ) to identify biologically different crc subtypes with distinct cellular phenotypes.5 , 16 the subtypes summarize a complex network of pathways potentially associated with therapeutic responses , simplifying multiple levels of information derived from heterogeneous samples . hence , the deployment of subtypes and their signatures , instead of single genes , has the advantage of reducing the dimension of complexity without losing biologic information . 
although the crca and consensus molecular subtype ( cms ) classications are highly concordant , 16 , 21 cms classication was not assessed here because it is technically challenging to dichotomize samples into two groups based on the current cms classier ( with multiple centroids )  . this study has some limitations . 
the identication of such patients in the context of clinical trials is challenging ; in fact , the negative predictive value of ras / braf mutations was retrospectively demonstrated in multiple clinical trials , and to our knowledge , none of them was designed with an up - front prospective inclusion of extended ras / braf wild - type tumors . 
last , this was a proof - of - concept study and was retrospectively designed on preexisting tissue collections in the absence of a control group , limiting the assessment of a ta - ness biomarker as prognostic rather than predictive . 
shapiro , chiara cremolini , filippo pietrantonio , livio trusolino , anguraj sadanandam data analysis and interpretation : elisa fontana , gift nyamundanda , david cunningham , dongsheng tu , maggie c . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . elisa fontana consulting or advisory role : astellas pharma ( i ) , celgene ( i ) , servier ( i ) , bristol myers squibb ( i ) patents , royalties , other intellectual property : patent no : 1716712.3 pending ( i ) travel , accommodations , expenses : bristol myers squibb ( i ) , servier ( i ) gift nyamundanda patents , royalties , other intellectual property : prognostic and treatment response prediction in gastric cancer priority patent csc / bp7295892 , patient classication and prognostic method patent application number pct / ep2019 / 053845 , patent application number 2011213.2 david cunningham stock and other ownership interests : ovibio consulting or advisory role : ovibio research funding : astrazeneca ( inst ) , amgen ( inst ) , sano ( inst ) , merrimack ( inst ) , celgene ( inst ) , medimmune ( inst ) , bayer ( inst ) , 4sc ( inst ) , clovis oncology ( inst ) , eli lilly ( inst ) , janssen ( inst ) , merck ( inst ) maggie c . 
siu leadership : treadwell therapeutics ( i ) stock and other ownership interests : agios ( i ) consulting or advisory role : merck , astrazeneca / medimmune , morphosys , roche , loxo , voronoi , oncorus , symphogen , mirati therapeutics , gsk , seattle genetics , treadwell therapeutics , arvinas , navire research funding : bristol myers squibb ( inst ) , genentech ( inst ) , glaxosmithkline ( inst ) , merck ( inst ) , novartis ( inst ) , pzer ( inst ) , medimmune ( inst ) , astrazeneca ( inst ) , boehringer ingelheim ( inst ) , bayer ( inst ) , amgen ( inst ) , astellas pharma ( inst ) , shattuck labs ( inst ) , symphogen ( inst ) , avid ( inst ) , mirati therapeutics ( inst ) , intensity therapeutics ( inst ) , karyopharm therapeutics ( inst ) francesco sclafani consulting or advisory role : bayer research funding : bayer ( inst ) , astrazeneca ( inst ) , roche ( inst ) , bristol myers squibb ( inst ) travel , accommodations , expenses : bayer , eli lilly jonathan m . 
waring employment : astrazeneca leadership : pillar biosciences , xing technologies , ori healthcare stock and other ownership interests : genentech , roche ( i ) , pillar biosciences , xing technologies consulting or advisory role : pillar biosciences , xing technologies , ori healthcare travel , accommodations , expenses : pillar biosciences , xing technologies timothy j . 
 fontana et al travel , accommodations , expenses : roche , servier naureen starling honoraria : eli lilly , msd oncology , merck serono , pierre fabre , servier consulting or advisory role : servier , astra zeneca , pzer research funding : astra zeneca ( inst ) , pzer / emd serono ( inst ) , bms ( inst ) travel , accommodations , expenses : msd oncology filippo pietrantonio consulting or advisory role : amgen , merck serono , bayer , eli lilly , sano , roche , servier research funding : bristol myers squibb livio trusolino honoraria : eli lilly , astrazeneca , merck research funding : symphogen ( inst ) , merus ( inst ) , pzer ( inst ) , servier ( inst ) , menarini ( inst ) anguraj sadanandam consulting or advisory role : ploughshare innovations research funding : merck , pierre fabre , bristol myers squibb patents , royalties , other intellectual property : patent colorectal cancer classication with differential prognosis and personalized therapeutic responses ( patent number pct / ib2013 / 060416 ) , prognostic and treatment response prediction in gastric cancer priority patent csc / bp7295892 , patent patient classication and prognostic method ( gep - net ) priority patent ep18425009.0 , patent molecular predictors of therapeutic response to specic anti - cancer agents ( patent number us9506926b2 ) no other potential conicts of interest were reported . acknowledgment we thank all patients and families participating in the studies included in this article . 
tebbutt , hagen kennecke , gabriella fontanini , eugenia zanella , and andrea bertotti for their contributions in the studies included in this article . references van cutsem e , cervantes a , adam r , et al : esmo consensus guidelines for the management of patients with metastatic colorectal cancer . 
ann oncol 27 : 1386 - 1422 , 2016 de roock w , claes b , bernasconi d , et al : effects of kras , braf , nras , and pik3ca mutations on the efcacy of cetuximab plus chemotherapy in chemotherapy - refractory metastatic colorectal cancer : a retrospective consortium analysis . 
lancet oncol 11 : 753 - 762 , 2010 cremolini c , morano f , moretto r , et al : negative hyper - selection of metastatic colorectal cancer patients for anti - egfr monoclonal antibodies : the pressing case - control study . 
ann oncol 28 : 3009 - 3014 , 2017 tejpar s , stintzing s , ciardiello f , et al : prognostic and predictive relevance of primary tumor location in patients with ras wild - type metastatic colorectal cancer : retrospective analyses of the crystal and fire - 3 trials . 
jama oncol 3 : 194 - 201 , 2017 [ erratum : jama oncol 3 : 1742 , 2017 ] sadanandam a , lyssiotis ca , homicsko k , et al : a colorectal cancer classication system that associates cellular phenotype and responses to therapy . 
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stem cells int 2017 : 7602951 , 2017 yang yp , ma h , starchenko a , et al : a chimeric egfr protein reporter mouse reveals egfr localization and trafcking in vivo . 
national institute for health and care excellence : cetuximab for the rst - line treatment of metastatic colorectal cancer : technical appraisal guidance [ ta176 ]  . 30 : 520 - 527 , 2019 12 . 
national institute for health and care excellence : panitumumab in combination with chemotherapy for the treatment of metastatic colorectal cancer ( terminated appraisal ) : technical appraisal guidance [ ta240 ]  . 
marisa l , de reyni `es a , duval a , et al : gene expression classication of colon cancer into molecular subtypes : characterization , validation , and prognostic value . 
plos med 10 : e1001453 , 2013 isella c , brundu f , bellomo se , et al : selective analysis of cancer - cell intrinsic transcriptional traits denes novel clinically relevant subtypes of colorectal cancer . 
khambata - ford s , garrett cr , meropol nj , et al : expression of epiregulin and amphiregulin and k - ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab . 
bertotti a , migliardi g , galimi f , et al : a molecularly annotated platform of patient - derived xenografts ( xenopatients ) identies her2 as an effective therapeutic target in cetuximab - resistant colorectal cancer . 
zanella er , galimi f , sassi f , et al : igf2 is an actionable target that identies a distinct subpopulation of colorectal cancer patients with marginal response to 20 . 
receiver operating characteristic ( roc ) curve to determine the best cut - off to dene disease control rate in the discovery cohort . 1 represents samples classied into transit - amplifying ( ta ) tumor with highest ( rank ) correlation with crcassigner ( crca ) - 38 centroids . 
 ( a ) kaplan - meier survival curve of patients with transit - amplifying ( ta ) tumor versus non - ta tumor in the discovery cohort subtyped using conventional subtyping approach . 
 decision - making preferences about secondary germline findings that arise from tumor genomic profiling among patients with advanced cancers purpose in patients with advanced cancers , tumor genomic profiling ( tgp ) can reveal secondary germline findings ( sgfs ) with regard to inherited disease risks . 
this study examined the process by which patients with advanced cancers would decide about whether to learn these sgfs and their preferences about specific challenging decision scenarios , including whether patients should be required to receive sgfs and whether sgfs should be returned to the family after a patients death . patients and methods we conducted qualitative semistructured interviews with 40 patients with advanced breast , bladder , colorectal , or lung cancer who had undergone tgp . 
data were collected on participants perspectives about the hypothetical decision to learn their sgfs , including their anticipated approach to the decision - making process , and their preferences about challenging decision scenarios . 
data were evaluated by thematic content analysis . results we identified themes with regard to participants preferred degree of decisional autonomy , perceived vital role of doctors , information needs , and anticipated process of deliberation . 
most participants stated that patients should be able to make a choice about receiving actionable sgfs , and a majority stated that sgfs should be available to family after a patients death . conclusion these results provide insight into sgf decision - making processes of patients with advanced cancers , which can allow clinicians to provide patients with optimal decision support in this context . 
these germline variants are considered secondary findings when actively sought by researchers or clinicians ( or incidental findings when not ) because they arise outside the original purpose of tgp.1 , 2 secondary germline findings ( sgfs ) that indicate risks for various health conditions are likely to be detected in a sizable minority of patients who receive tgp ; for example , presumed pathogenic germline variants have been observed jada g . 
this decision is likely to be challenging , particularly for patients with advanced cancers who are currently the primary users of tgp ( because of its utility for identifying eligibility for clinical trials of novel therapeutics5 , 6 )  . 
these individuals must choose whether to learn information about their future disease risks and potential shared familial risks while facing a poor prognosis and the psychosocial challenges of a terminal diagnosis.7 although patients with varying stages of cancer have reported interest in receiving such information from tgp in real8 and hypothetical9 - 11 settings , how patients decide whether to learn sgfs is unclear . 
understanding the decision - making processes of patients with advanced cancers would allow clinicians to anticipate patient informational and decision support needs in this context . the current study describes processes by which patients with advanced cancers decide whether to learn sgfs that arise from tgp . 
we analyzed qualitative data collected through an investigation of attitudes about sgfs among patients who received tgp at our institution.12 these patients were informed about the possible incidental discovery of germline variants during tgp consent conducted by their primary medical oncologists ; however , because our institution did not routinely conduct secondary analyses at the time of this study , none of the patients had made a definitive decision about learning their sgfs . 
 we also assessed preferences with regard to specific challenging decision scenarios , including whether patients should be required to receive sgfs and whether sgfs should be returned to the family after a patients death . study methods are described in detail elsewhere.12 in brief , we recruited 40 adults with advanced breast , bladder , colorectal , or lung cancer who had undergone tgp with an institutional somatic sequencing panel ( msk - impact [ memorial sloan kettering - integrated mutation profiling of actionable targets ] 13 , 14 )  . 
 the memorial sloan kettering cancer center institutional review board approved this study . individual semistructured interviews15 - 18 were conducted with participants in person or by telephone on the basis of participant preference . 
we categorized participant responses into four key themes and relevant subthemes ( indicated by italicized text ) ; illustrative quotes appear in table 2 . theme 1 : degree of decisional autonomy as participants considered how they would decide whether to learn sgfs , a spectrum emerged with regard to participants preferred degree of decisional control and autonomy from close others . 
influential factors for this perspective included a view that the decision was my choice because it involved highly personal information fundamentally related to my body and a desire to avoid burdening others , particularly family , with potentially distressing information . a second group of participants preferred that close others play a consultative role in the decision making process . 
these participants anticipated communicating with close others about the option to learn sgfs and would consider their advice and opinions but would ultimately make the final decision on their own . 
some in this group noted that their familys views were highly valued but would not be determinants in their decision making . finally , a smaller group preferred that close others serve as active partners in decision making . 
others explained that their family members should be actively involved in this decision because sgfs may have direct health - related implications for them . participants who anticipated the involvement of others in their decision making also described their process of selecting close others for communication about the option of learning sgfs . 
decision - making process and preferences regarding secondary germline findings and illustrative participant quotes participant quote theme theme 1 : degree of decisional autonomy patient as an autonomous decision maker well , i think that would be up to me to decide , so i wouldnt be asking my family what they think about doing that . 
 ( f / cc ) close others play a well , id like their input , but ultimately , i make the decisions for what...kind of consultative role in the decision - making process treatment....i would make the final decision . 
 ( f / brc ) close others serve as active partners in decision making process of selecting close others for communication i would expect my wife to be involved....i trust her knowledge and judgment in these matters . 
i dont think my husband could deal with it , so i dont want him burdened with it , and i dont think my step - kids have enough...skin in the game , so to speak , that they should actually be involved in making the decision and i guess i feel similarly about sisters and brother that thats too distant . 
 ( f / cc ) theme 2 : vital role of doctors nature and quality of the doctor - patient relationship primary source of relevant and valuable information theme 3 : information needs clinical benefit meaning tainty testing procedure believe me , its been a rough road , and so like i said , my oncologist and i , we have a good understanding . 
he was the one that put me in the tumor profiling and also on this new research , and anything he decides with me , im okay with it because we have that doctor - patient trust . 
i mean , you know , if they tell me to go get this test , i go get that test....i do it because i think its in the interest of my health.you know , you would have to make a strong argument for that case , but if he was insistent , i would do it . 
 ( m / blc ) i think from a personal standpoint i would ask...how realistic do you think , or how probable do you think , something that came up as high risk is likely to happen , or is there anything i can do to prevent it ? i mean , its more so in the latter that i would care about more if theres anything i can do to prevent it , to minimize the risk . 
is it certain mutations , is it only certain diseases that were talking about , or...is it kind of open ended ? ...i guess i would want to learn more and hear more about the science of what the mutations might mean . 
 ( m / cc ) degree of scientific uncerwhat im trying to have connection with is that if this testing were predictive of something , they would be more interested than if [ with ] this testing , nobody understood or knew how to interpret the results . 
so i guess it would be depending [ on ] how far along the continuum we are in being able to use this information [ that ] would make a difference . 
 ( m / lc ) yes , and if its nothing invasive and they wont...poke me anymore and they wont do anything to me , its fine with me....i would like to know . 
decision - making process and preferences regarding secondary germline findings and illustrative participant quotes ( continued ) theme participant quote who will have access who would have that information , would health care providers...have to have access to that or insurance providers have to have access to that information ? ( f / brc ) yes , what are the possible ramifications , like everything ? like the question that i have now , like what havent i thought of that could be a possible ramification of knowing ? yeah . 
 ( f / brc ) negative implications or harms theme 4 : process of deliberation ing process deliberative decision - makthe only potential benefit i see is if it discovers something that could be dealt with and prevent serious illness or genetic problems in the future . 
 ( m / cc ) take time to decide i think i would want to think about it and talk about it a little more before i made that quick decision , yeah . 
but i dont think its something that we would shy away fro ( m / lc ) well , i think i would research mutations first and find out a little about it before i answered him , but my nature is to go ahead and find out as much information as i can . 
 ( f / blc ) no need to engage in i would say , great , where do i sign ? when i first got diagnosed , i offered to extensive deliberation have my dna sequenced , and the doctor said , why would you bother ? theres only 30 markers , and weve already looked at theso yeah , to me , it was like a no - brainer and required no thought . 
so for me , its really once i understand what exactly ill be getting out of the study or what benefit it can provide , thats enough of what i need to make a decision on . 
i wouldnt want to procrastinate...it would be my decision....id discuss with family members...my husband and my sister , but...it would be my decision ultimately , and id really want to make it quickly . 
the interviewer described secondary germline findings as follows : i mentioned that with tumor genomic profiling , sometimes the lab will also look for mutations in the genes in your normal cells . 
 abbreviations : blc , bladder cancer ; brc , breast cancer ; cc , colorectal cancer ; f , female ; lc , lung cancer ; m , male . theme 2 : vital role of doctors participants perceived their doctors ( ie , oncologists ) as a vital influence on their decision making . 
for example , several participants reported great trust in their doctors on the basis of a foundation of past experiences and certainty that their doctors will act in their best interests . 
several participants anticipated that their doctors would possess expertise with regard to a range of issues relevant to sgfs and thus could help them to acquire all essential information . theme 3 : information needs participants described a typology of information that they would require to make an educated decision about learning sgfs , including an explanation of whether sgfs would provide a clinical benefit to the patient , his or her family , or other cancer patients and whether these benefits would outweigh possible harms ; assistance in interpreting the meaning of sgfs , such as the degree of certainty of the results and meaning of specific mutations ; degree of scientific uncertainty of sgfs and confidence in their applicability to health decisions ; description of the testing procedure in terms of the invasiveness of sample acquisition ; information about who will have access to the findings ( eg , insurers , health care providers ) ; and negative implications or harms of learning sgfs for the patient and family , including unanticipated consequences . 
many participants stated that they would ask questions about these issues to feel adequately informed , yet a minority doubted that they would have any specific questions if presented with this decision primarily because of placing a high innate value on sgfs . theme 4 : process of deliberation two preferences emerged among participants with regard to the necessity to engage in an extensive decision - making process . 
 ( a detailed description of these perceived benefits and harms is provided elsewhere.12 ) participants described procedural aspects of their deliberation and expressed a preference to take time to decide , during which they would consider the option on their own and seek out information about the value of sgfs . 
furthermore , a few participants expressed a preference to conduct independent research to learn more about receiving sgfs and the meaning of potential mutations . a minority of the sample articulated no need to engage in an extensive deliberation to determine their interest in sgfs . 
 finally , some described a sense of urgency about learning sgfs and stated the necessity to gain and act upon this information quickly to benefit their current health directly . preferences with regard to decision scenarios during the interview , participants were presented with challenging scenarios and asked to describe their preferences for how clinicians should handle these situations . 
first , in response to the debate about the disclosure of sgfs , 26 - 29 we asked participants whether findings about diseases that have effective medical interventions or medication adverse effects ( ie , actionable sgfs ) should always be returned to patients . 
most participants ( 28 [ 70% ] of 40 ) stated that patients should be able to choose whether to receive this information , whereas a minority ( 12 [ 30% ] of 40 ) stated that such information should always be disclosed to patients . participants also were asked to decide whether they believed that if actionable sgfs were detected after a patients death , then these findings should be made available to a patients family or significant other . 
a subset of participants ( 16 [ 69.5% ] of 23 ) also expressed a belief that patients should be required to provide consent for this disclosure before their death , such as at the time of agreeing to tgp , whereas fewer ( seven [ 30.5% ] of 23 ) deemed patient consent unnecessary . 
preferences with regard to specific decision scenarios that involve secondary germline findings and illustrative participant quotes participant quote question should actionable secondary germline findings * always be returned to patients ? yes ( 30% ) yes , i agree with that because they may not want to know , but theyre still going to be affected by it . 
because at least theyd have the opportunity to know...whats going on with thethey may not want to know , and it may be painful , but i think that they should be told . 
i think of a very close friend that was diagnosed with cancer , and he was in his 20s , and he survived , but when i learned that i had cancer , i reached out to him , and he said that at his lowest point he begged , i dont want to know any more information . 
and that was part of the healing for him , so i always think about that because that was a poignant point that he made , and i think its so personal . 
 ( f / brc ) should actionable secondary germline findings be made available to a patients family or significant other if a patient has died ? yes ( 90% ) unsure ( 5% ) no ( 5% ) should nonactionable secondary germline findings be made available to a patients family or significant other if a patient has died ? yes . 
well , if it in any way could...impact the timing of treatment or care for someone else in the family , they should....i would want them to know about it....i guess at the end of the day that you should get consent from the patient...as to what youre going to do with anything...you take from the ( m / lc ) you got a coin ; you wanna flip a coin ? because the problem that comes to me is that my family is very tight , and it wouldnt be a problem with my family , but you always have a family that [ is ] ...on the outs , so to speak , and if you tell one , you got to tell all . 
 ( m / lc ) thats a good question because im thinking that if the spouse , for example , were told after the person passed away that we had discovered this , i guess the first reaction would be , how come we didnt discover it earlier while the person was still alive and there may have been time for some kind of treatment ? so it might cause some kind of anger . 
preferences with regard to specific decision scenarios that involve secondary germline findings and illustrative participant quotes ( continued ) question unsure ( 2.5% ) no ( 15% ) participant quote i can project how i might think in the future , but its hard for me to say at this time in a practical way how i would feel about , you know , releasing . 
if they want the information they should go and get it...and i think that if they get it just because it was available for me , like as part of my estate , heres her genetic testing , and again , ill use my brother because he has the kids . 
if...he sees in black and white that theres an indicator that we have a genei have a gene so that becomes a family gene - - so hes now gotten a worry he didnt ask for in his life . 
 abbreviations : blc , bladder cancer ; brc , breast cancer ; cc , colorectal cancer ; f , female ; lc , lung cancer ; m , male . * in the interview , actionable secondary germline findings were described as follows : there are different ways to think about the many kinds of mutations or disease risks that you could theoretically learn about . 
when doctors know that someone has one of these mutations , they can recommend ways to help prevent a disease from developing or help find it earlier when it is more likely to be treatable . 
the doctors may also change the kinds of medications that they prescribe . in the interview , nonactionable secondary germline findings were described as follows : it is also possible that the lab will find mutations for conditions that do not have recommended or effective medical interventions . 
 but when two carriers of the same recessive mutation have a child , then the child could have the disease . unsure about whether actionable sgfs should be available to a patients family after death ( two [ 5% ] of 40 ) or stated that such information should not be made available ( two [ 5% ] of 40 )  . 
 preferences against disclosure were due to concerns about negative emotional implications of such information for families . participants were similarly asked to decide whether they believed that sgfs about diseases without effective medical interventions or that indicate one is a healthy carrier for recessive diseases ( ie , nonactionable sgfs ) should be made available to a patients family after a patients death . 
most participants who provided an opinion regarding consent reported that patients should be required to consent to the disclosure of this information to their families ( 11 [ 92% ] of 12 )  . 
fewer ( six [ 15% ] of 40 ) stated that nonactionable sgfs should not be made available to family after a patients death because of concerns about negative emotional reactions and the limited ability to intervene with such diseases . 
 finally , when comparing the preferences of participants with regard to the return of actionable versus nonactionable sgfs to family after a patients death , 22.5% ( nine of 40 ) were discordant in their preferences across these scenarios . discussion this study clarifies the decision - making processes of patients with advanced cancer with regard to sgfs from tgp . 
however , consistent with other medical decision contexts , 30 - 34 variability existed in participants preferences for involving others , including spouses / partners , children , and siblings , in their decision making . 
consequently , when presenting the option of learning sgfs , clinicians must allow patients to solicit input from close others and help to navigate challenges inherent in decision making with multiple individuals.35 additional research should investigate how such interpersonal influences shape , hinder , or support patients sgfs decision making . participants anticipated that their doctors ( ie , oncologists ) would be the primary source of guidance for this decision . 
participants also anticipated that they would have extensive questions about the benefits , harms , interpretation , and process of obtaining sgfs and would expect their oncologists to provide answers . 
however , research has demonstrated that this may not be feasible because many oncologists have limited experience with germline testing and express concerns about their ability to address challenges presented by sgfs.8 several approaches may help to bridge this gap between patient expectations and oncologist preparedness , including oncologist - targeted communication training , novel patient education materials , and referral to genetic counselors to address patient questions . 
future research should evaluate which of these approaches are most effective at achieving the delicate balance between meeting patients information needs and practical challenges of cancer care delivery ( eg , time demands , workforce limitations )  . 
research should also examine how various models of patient education ( eg , oncologist led , genetics professional led ) influence patient sgfs decisions and how patients weigh the opinions of various care providers in this context . many participants anticipated a preference to undergo a thoughtful deliberation about the prospect of learning sgfs . 
research suggests that the adoption of a more intuitive decision - making approach can yield similar outcomes to deliberative decision making , 36 although both approaches have benefits and drawbacks.37 of note , some participants preferences for a quick decision were motivated by beliefs that sgfs would provide clinical utility or necessitate urgent action for them to reap health benefits . 
accordingly , clinicians must ensure that all patients , including those immediately enthusiastic or accepting of sgfs , accurately understand the limitations of this risk information . these results also provide insight into preferences of patients with advanced cancers with regard to challenging scenarios that involve the return of sgfs . 
consistent with american college of medical genetics and genomics recommendations4 and expert opinion , 38 most participants stated that patients should choose whether they want to receive actionable sgfs from tgp . 
participants generally were more supportive of the return of actionable sgfs to family after a patients death than nonactionable sgfs ; although , in both instances , a majority supported the sharing of this information with family largely because of perceived family health benefits . 
the qualitative design enabled an in - depth analysis of the decision - making preferences of a sample of patients with advanced cancers diverse in diagnosis , sex , and health status . 
however , the majority was well - educated ( 85% reporting at least some college ) ; decision - making preferences and processes of these individuals may differ from those with less formal education . 
additional limitations are that this sample was racially and ethnically homogenous , recruited from one institution , and assessed at a time when the decision about learning sgfs was hypothetical in nature ; thus , the findings may not be generalizable to the broader population of patients with advanced cancers treated in other care settings who are navigating this decision in real time . 
recommendations for developing precision oncology programs with regard to how to manage and support patient decision making about secondary germline findings from tumor genomic profiling develop educational materials about tumor genomic profiling ( tgp ) and secondary germline findings ( sgfs ) that can be easily disseminated to and understood by close others ( eg , siblings , children , spouses / partners ) who may play a role in a patients decision making . ensure that individuals who lead education and consent discussions about the return of sgfs are prepared to help patients with varying preferences for decisional autonomy from their close others . patients attribute high trust and expertise to their oncologists ; therefore , prepare oncologists to serve as a primary resource who can provide balanced advice to patients about sgf decisions . create patient educational materials that provide clear information about the potential benefits and harms of sgfs . 
 distinguish between potential outcomes of sgfs for patients ( with a consideration of their cancer stage and prognosis ) and their families . ensure that patients understand that the decision to undergo tgp is separate from the decision about return of sgfs and that varying potential benefits and harms of each choice exist . structure education and consent discussions about tgp and the return of sgfs to be temporally flexible and , therefore , capable of accommodating patients preferences to take time to deliberate , seek additional input from close others , and conduct independent research . give patients a choice about the return of actionable sgfs . 
either opt - in or opt - out models of germline variant management could allow such patient choice , but each has unique implications for resources to support informed patient decision making and subsequent uptake of sgfs . require patients to make decisions about the management of actionable and nonactionable sgfs in the event of their death at the time of consenting to tgp and the return of sgfs . in conclusion , this study provides important insight into how patients with advanced cancers approach the decision to learn sgfs and informs how developing precision oncology programs can manage the reporting of germline variants from tgp ( table 4 )  . 
patient preferences for involvement in this decision can be accommodated in both opt - in and opt - out models , although these models likely will differ in the resources necessary to support patient deliberation and in the number of patients who select to receive sgfs.42 , 43 although most patients likely will want to retain decisional control in this context , some will desire time and space to include family and other influential figures in their decision making . 
whereas the tgp decision may be time sensitive because of treatment implications , patients may benefit from efforts to ensure that the decision to receive sgfs can be pursued on a different temporal schedule that aligns with their preferences for information seeking and deliberation . 
thus , educational and communication interventions targeted to patients , their families , and oncologists are needed to provide clear information that contextualizes the meaning of sgfs in the advanced cancer setting , assist the weighing of benefits and harms , and allow patients to explore and express their preferences about specific categories of sgfs and management of this information in the event of their death . 
presidential commission for the study of bioethical issues : anticipate and communicate : ethical management of incidental and secondary findings in the clinical , research , and direct - to consumer contexts ( december 2013 report of the presidential commission for the study of bioethical issues )  . 
tripathy d , harnden k , blackwell k , et al : next generation sequencing and tumor mutation profiling : are we ready for routine use in the oncology clinic ? bmc med 12 : 140 , 2014 6 . 
parsons dw , roy a , plon se , et al : clinical tumor sequencing : an incidental casualty of the american college of medical genetics and genomics recommendations for reporting of incidental findings . 
gray sw , park er , najita j , et al : oncologists and cancer patients views on whole - exome sequencing and incidental findings : results from the canseq study . 
hamilton jg , shuk e , genoff mc , et al : interest and attitudes of patients with advanced cancer with regard to secondary germline findings from tumor genomic profiling . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
burke w , antommaria ah , bennett r , et al : recommendations for returning genomic incidental findings ? we need to talk ! genet med 15 : 854 - 859 , 2013 27 . 
stiggelbout am , jansen sj , otten w , et al : how important is the opinion of significant others to cancer patients adjuvant chemotherapy decision - making ? support care cancer 15 : 319 - 325 , 2007 35 . 
laidsaar - powell rc , butow pn , bu s , et al : physician - patient - companion communication and decision - making : a systematic review of triadic medical consultations . 
scheuner mt , peredo j , benkendorf j , et al : reporting genomic secondary findings : acmg members weigh genet med 17 : 27 - 35 , 2015 39 . 
kaphingst ka , ivanovich j , biesecker bb , et al : preferences for return of incidental findings from genome sequencing among women diagnosed with breast cancer at a young age . 
 megakaryocyte potentiating factor as a predictive biomarker for therapies against malignant mesothelioma purpose effective biomarkers for malignant mesothelioma ( mm ) are needed for clinical management and the development of mesothelin - targeted therapies . 
we evaluated serum megakaryocyte potentiating factor ( mpf ) as a biomarker predictive of treatment outcome in patients with mm and for developing mesothelin - targeted therapies . materials and methods serial serum samples from patients with mm in two clinical trials of an antimesothelin immunotoxin were tested with our clinically validated mpf assay . 
mpf was further evaluated for an association with response to an antimesothelin therapy and for disease monitoring . results there was a significant reduction of serum mpf in patients with elevated baseline and radiologic response , with an average change from 52% to 78% after one to six cycles . 
 potentiating factor ( mpf ) and membrane - bound mesothelin.15 , 16 previous studies showed correlations between the computed tomography ( ct ) response and arbitrarily chosen 10% to 25% reduced serum mesothelin after chemotherapy.17 - 21 such low cutoff values are incompatible with a 30% reduction in tumor determined with ct , would contribute to high degrees of false signals , and would make it more difficult to correlate with clinical outcomes . the currently available mesomark test ( fujirebio , malvern , pa ) detects serum mesothelin , which is subject to the interference by mesothelin - targeted antibody - based agents . 
we developed and validated an assay for mpf and further showed that elevated mpf is a worse prognostic biomarker in patients with mm.22 its effectiveness for monitoring therapies is not known . 
the first trial was a phase i study with a standard dose of pemetrexed and cisplatin , and the antimesothelin immunotoxin ss1p ( nct01445392 ) in chemotherapy - nave patients with pleural mesothelioma.19 twenty patients were evaluable , 12 with partial response ( pr ) , three with stable disease ( sd ) , and five with progressive disease ( pd )  . 
the patients had received one to six ( and an average of three ) previous therapies.8 all patients were evaluated for responses : three with pr , three with sd , and four with pd . 
tumor responses were determined on the basis of ct and calculated in accordance to modified response evaluation criteria in solid tumors ( mrecist ) criteria for mesothelioma . statistical analyses prism , version 7.0 ( graphpad , la jolla , ca ) was used for statistical analysis . 
 kaplan - meier survival analyses were performed to determine log - rank hazard ratio ( hr ) and p value of mpf response or ct response patients on pfs or os . 
on the basis of the linear regression analysis of corresponding changes in ct and mpf in these 20 patients with mm , a 30% change in ct was interpolated to a 47% change in mpf level ( fig 1b )  . 
thus , a rounded 50% change in serum mpf was selected as the cutoff for mpf response , which corresponded to approximately 30% change in ct using mrecist . the serum mpf test enables continuous and multiple measurements during treatment . 
among pr patients with an elevated mpf using the upper limit of normal at 1.2 ng / ml , 22 there was an average of 52% change in mpf after one cycle ( p < .001 ; n = 12 ; fig 1c )  . 
in contrast , for the patients who did not have a ct response , there was no significant reduction of mpf ( n = 14 ; fig 1d )  . 
thus , the treatment induced reduction of serum mpf may be an informative biomarker of objective response in patients with mm with elevated baseline mpf . kaplan - meier analyses were performed to compare pfs and os of both trials . 
thus , the data suggest that a 50% change in mpf after systemic therapies is strongly associated with improved pfs . reduction of mpf after systemic treatment as a potential biomarker for better os the mpf response was further correlated with os . 
landmark analysis of os for mpf and ct response was performed , with a landmark date at 91 days , at which time all patients had experienced a response but one . 
 ( a ) analysis of the changes in serum mpf in patients with partial response ( pr ) , stable disease ( sd ) , and progressive disease ( pd )  . 
all data are shown as mean standard deviation . depletion regimen of pentostatin and cyclophosphamide , major cancer regressions were observed and were attributed primarily to the activities of ss1p.8 the nonparametric mann - whitney test showed that the patients with pr ( by ct ) had elevated baseline serum mpf levels , which were higher than those in the nonresponse group ( p = .033 ; fig 3a )  . 
thus , data suggest that elevated baseline serum mpf may be associated with the response to an effective antimesothelin therapy in patients with refractory mm , whereas the mpf response is associated with improved survival in these patients ( p = .012 ; appendix fig a4 )  . 
 20 pfs since in - study date ( months ) os since in - study date ( months ) mpf response mpf nonresponse log - rank p < .001 hazard ratio = 0.279 ct response ct nonresponse log - rank p = .022 hazard ratio = 0.457 mpf response mpf nonresponse log - rank p = .004 hazard ratio = 0.361 ct response ct nonresponse log - rank p = .035 hazard ratio = 0.476 pfs since in - study date ( months ) os since in - study date ( months ) fig 2 . 
 ( a ) progression - free survival ( pfs ) and ( b ) overall survival ( os ) using mpf response at a threshold of 50% or more after therapies . 
 ( c ) progression - free survival and ( d ) os for patients with malignant mesothelioma using computed tomography ( ct ) response on the basis of modified response evaluation criteria in solid tumors . 
102 was a 50 - year - old man with extensive peritoneal involvement and received two cycles of ss1p because of ss1p antibody development.8 he had a delayed response with substantial tumor shrinkage at 7 months . 
103 , a 56 - year - old woman with pleural mesothelioma , had rapid disease progression before treatment and was the only patient who completed all six cycles of therapy . 
105 , pr response progression cutoff response progress response time since in - study date ( months ) time since in - study date ( months ) cutoff cutoff fig 3 . 
elevated baseline megakaryocyte potentiating factor ( mpf ) associated with response and long - term follow - up of individual patients with major tumor regression after antimesothelin and immune suppression . 
there was evidence of increased serum mpf at the time of disease progression for all . discussion our study indicates that the serum mpf test is effective in monitoring systemic therapies for patients with mm , because the patients with mpf responses have much better pfs and os than those without , regardless of baseline levels . 
in mm , recist needs to be modified because one - dimensional measurement of the nonspherical growth pattern was difficult.24 the mrecist requires repeated total tumor measurements on two occasions 4 weeks apart for pr determination.25 the quantitative changes of serum mpf may provide an alternative , lowcost , and nonradiative assessment of therapy response in patients with mm with elevated mpf . 
 we used a more stringent cutoff value of a 50% change in mpf to define the biomarker response compared with the previous reports17 - 21 ; this cutoff was supported by our correlative analysis of ct and mpf responses in patients with mm . 
such a stringent cutoff correlates with the mrecist criterion of a 30% reduction in the sum for all target lesions and thus leads to a significant reduction of false - positive reports during monitoring . 
thus , in addition to confirming the association between changes in mpf and patients response to systemic therapy , 18 our results further showed that an mpf biomarker response is associated with improved pfs and os . as the cleavage product during mesothelin maturation with a short half - life in blood , 22 , 26 serum mpf levels reflect the expression of target antigen mesothelin on tumor cells , without being bound by the therapeutic agents . 
in fact , in untreated mesothelioma patients , there is a strong correlation between mpf and soluble mesothelin.22 , 27 the mpf test could be valuable for the clinical development of mesothelin targeted therapies and for monitoring targeted therapies against mm . 
therefore , serum mpf could facilitate long - term disease monitoring and provide a noninvasive assessment of the drug - target mesothelin expression in mm at the time of disease progression , which can be critical for evaluating alternative therapies . one of the most important issues in the development of targeted therapies is the stratification of the intended patient population . 
mesothelin is ubiquitously detected in epithelioid mm by immunohistochemistry ( table 1 ) ; therefore , mesothelin immunohistochemistry is not predictive of response to mesothelin - targeted therapies in mm , and a different biomarker test is required . 
 there are many challenges for a serum - based test for patient selection , including the expression of the tumor antigen by nontumor cells and the variations in shedding of it by tumor cells . 
 in addition , it is possible that serum mpf may be influenced by renal dysfunction in patients receiving nephrotoxic chemotherapies because prior reports showed elevated serum mpf in patients with renal disease.28 , 29 however , the patients described in our study had normal renal functions at the time of enrollment . 
although our previous data suggested that patients with mm with elevated mpf have a worse prognosis , 22 the results here suggest that the reduction of serum mpf in patients with refractory mm is associated with improved clinical outcome . 
vogelzang nj , rusthoven jj , symanowski j , et al : phase iii study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma . 
hassan r , cohen sj , phillips m , et al : phase i clinical trial of the chimeric anti - mesothelin monoclonal antibody morab - 009 in patients with mesothelin - expressing cancers . 
golfier s , kopitz c , kahnert a , et al : anetumab ravtansine : a novel mesothelin - targeting antibody - drug conjugate cures tumors with heterogeneous target expression favored by bystander effect . 
le dt , brockstedt dg , nir - paz r , et al : a live - attenuated listeria vaccine ( anz - 100 ) and a live - attenuated listeria vaccine expressing mesothelin ( crs - 207 ) for advanced cancers : phase i studies of safety and immune induction . 
hollevoet k , nackaerts k , gosselin r , et al : soluble mesothelin , megakaryocyte potentiating factor , and osteopontin as markers of patient response and outcome in mesothelioma . 
hassan r , sharon e , thomas a , et al : phase 1 study of the antimesothelin immunotoxin ss1p in combination with pemetrexed and cisplatin for front - line therapy of pleural mesothelioma and correlation of tumor response with serum mesothelin , megakaryocyte potentiating factor , and cancer antigen 125 . 
creaney j , francis rj , dick im , et al : serum soluble mesothelin concentrations in malignant pleural mesothelioma : relationship to tumor volume , clinical stage and changes in tumor burden . 
hooper ce , lyburn id , searle j , et al : the south west area mesothelioma and pemetrexed trial : a multicentre prospective observational study evaluating novel markers of chemotherapy response and prognostication . 
onda m , nagata s , ho m , et al : megakaryocyte potentiation factor cleaved from mesothelin precursor is a useful tumor marker in the serum of patients with mesothelioma . 
iwahori k , osaki t , serada s , et al : megakaryocyte potentiating factor as a tumor marker of malignant pleural mesothelioma : evaluation in comparison with mesothel lung cancer 62 : 45 - 54 , 2008 28 . 
briefly , the mpf assay uses capture antibody mpf49 ( 2a , ) , which binds to mpf topographic epitope 3 , and detection antibody mpf25 ( 1 , ) , which binds to epitope 1.26 mpf49 was biotinylated , and mpf25 was conjugated with sulfo - tag nhs - ester ( meso - scale diagnostics , rockville , md )  . 
after incubation for 1 hour at room temperature , plates were washed , and the serially diluted mpf calibrator , 1 : 10 diluted sample serum , and reference samples were added and further incubated for 1 hour . 
the data were analyzed with workbench 4.0 software ( meso - scale diagnostic ) for determining the concentration of mpf in serum samples using the 4 parameter logistic nonlinear regression model . 
vokes , md1 , 2 ; david liu , md1 , 2 ; biagio ricciuti , md1 ; elizabeth jimenez - aguilar , md3 ; hira rizvi4 ; felix dietlein , md , phd1 , 2 ; meng xiao he5 ; claire a . 
elmarakeby , phd1 , 2 ; jeffrey girshman , md4 ; anika adeni1 ; francisco sanchez - vega , phd4 ; nikolaus schultz , phd4 ; suzanne dahlberg , phd1 ; ahmet zehir , phd4 ; pasi a . 
awad , md , phd1 purpose heterogeneity in tumor mutational burden ( tmb ) quantication across sequencing platforms limits the application and further study of this potential biomarker of response to immune checkpoint inhibitors ( icis )  . we hypothesized that harmonization of tmb across platforms would enable integration of distinct clinical data sets to better characterize the association between tmb and ici response . methods cohorts of patients with nonsmall - cell lung cancer sequenced by 1 of 3 targeted panels or by wholeexome sequencing ( wes ) were compared ( n = 7 , 297 )  . 
in subcohorts of patients treated with icis ( dana - farber cancer institute n = 272 ; memorial sloan kettering cancer center n = 227 ) , the association between tmb and outcome was assessed . 
receiver operating characteristic curves yielded an area under the curve of 0.614 , with no natural inection point . conclusion the z score conversion harmonizes tmb values and enables integration of data sets derived from different sequencing panels . 
tmb quantication from targeted next - generation sequencing ( ngs ) panels has been shown to correlate with whole - exome sequencing ( wes ) derived tmb13 , 18 - 20 and to associate with ici response , which makes the clinical assessment of tmb practically feasible.19 , 21 to date , the only approved biomarkers of ici response are mismatch repair deciency and , in nonsmall - cell lung cancer ( nsclc ) , programmed cell death - ligand 1 ( pd - l1 ) expression . 
 vokes et al context key objective it is not known how to account for differences in tumor mutational burden ( tmb ) generated by different sequencing assays . we sought to address assay heterogeneity in tmb quantication by developing a technique to harmonize tmb across assays and applied this technique to a pool of distinct clinical cohorts to better characterize the association between tmb and response to immune checkpoint inhibitors . knowledge generated tmb differs across sequencing assays because of differences in gene panels and sequencing pipelines . 
from this pooled analysis , we observed that tmb may not associate with response in patients who are never - smokers or harbor targetable oncogenes , and tmb thresholds yield signicant trade - offs in sensitivity and specicity . relevance standardization of z scores harmonizes tmb values across assays for pooled analysis . 
it is not known whether this threshold heterogeneity reects different tmb quantication that arises from different platforms , variation across patient cohorts , or unknown clinical or biologic effects on the association between tmb and response . given these questions , we sought to develop a strategy to harmonize tmb across ngs platforms . 
we applied this method to integrate multiple clinically annotated cohorts and to more fully characterize the relationship between tmb and ici response to add nuance and context to our current understanding . 
we focused on nsclc because of the early interest in applying tmb to clinical practice in this disease subtype24 - 26 and to avoid confounding of tmb by tumor type.27 methods study population three cohorts of patients with nsclc whose tumors had been proled by a targeted ngs panel were evaluated . these panel cohorts were compared with a fourth wes cohort from the cancer genome atlas ( tcga )  . dana - farber cancer institute cohort . 
patients at the danafarber cancer institute ( dfci ) whose tumors had undergone oncopanel ngs were included if they had advanced nsclc and had consented to institutional review boardapproved protocols . 
somatic wes data from nsclcs sequenced by tcga31 were downloaded from the cbioportal for cancer genomics . the dfci cohort was sequenced as previously described.32 , 33 in brief , tumor dna was extracted and used for customdesigned hybrid capture library preparation . 
all variants were reviewed for technical quality.34 finally , minimize inadvertent inclusion of germline variants , consistent with previous aggregation efforts , 35 an additional germline lter was applied to exclude events present in the exome aggregation consortium with an allele count  . 
foundation medicine uses an internal algorithm to lter putative germline events . tmb was uniformly calculated for each sample as the number of nonsynonymous mutations per megabase ( mb ) of genome covered . 
scans were reviewed by thoracic radiologists at each institution , and response was determined using recist version 1.1.37 progression - free survival was assessed from the start of ici treatment until the date of progression or death ; patients without progression were censored at last scan . 
6 months was dened as durable clinical benet ( dcb ) ; no durable benet ( ndb ) was dened as progressive disease ( pd ) or sd 6 months . 
patients censored before 6 months of follow - up were considered not evaluable . statistical analysis cohort - specic gene mutation averages were calculated by summing the number of mutations in each gene within a cohort and dividing the total by the number of patients in the cohort . 
the linear correlation between log average mutations per gene in the panel cohorts versus tcga was evaluated using pearsons correlation coefcient . power transformations were used to normalize cohortspecic tmb distributions ; tukeys ladder of powers38 in the rcompanion package39 was used to identify the optimal transformation coefcient . 
receiver operating characteristic ( roc ) curve analyses were performed using the proc and optimalcutpoints packages.41 , 42 exploratory cutoffs were selected to maximize the distance to the y = x line ( youdens index ) , maximize specicity with sensitivity  . 
human investigations were performed after approval by a local human investigations committee and in accordance with an assurance led with and approved by the department of health and human services , where appropriate . results comparison of tmb quantication across panel and wes platforms patients with nsclc whose tumors had undergone sequencing through oncopanel ( n = 1 , 157 ) , msk - impact ( n = 1 , 520 ) , foundation medicine ( n = 3 , 476 ) , or tcga ( n = 1 , 144 ) were included ( n = 7 , 297 ; see data supplement for cohort diagram and clinical characteristics )  . 
to determine whether tmb differed between platforms , we plotted the distribution of tmb within each cohort ( fig 1a )  . tmb distributions differed between cohorts , and targeted panels were associated with higher tmb values than wes . because targeted panels sequence fewer bases with focused inclusion of mutated cancer genes , we hypothesized that the higher tmb measurements associated with ngs panels were attributable to gene selection . 
we tested this by subsetting the wes data to include only those genes captured by the targeted panels ( downsampling ; data supplement ; fig 1b ) and found that downsampled distributions retained greater tmb counts than the unltered tcga distribution , which suggests that gene panel composition contributes to the observed difference in tmb distributions between cohorts . 
however , the relative differences were less pronounced than in the real - world cohort comparisons , which suggests that assay - specic differences , such as depth of sequencing and the absence of a paired germline sample , might also contribute to inter - test variation . to further examine assay - specic sources of variation in tmb across panels , we compared the average number of mutations per gene in each cohort against the tcga averages , surmising that this could reect differences in assay performance or mutation calling ( figs 1c - e )  . 
the left side shows the kernel density plot of unadjusted tmb values in each cohort , and the right side shows the transformed density plot of tmb z scores that demonstrate high overlap . 
use of a power transformation converted the right - skewed tmb distributions ( fig 1f ) to normal distributions ( skewness values 0.06 ; data supplement ) , and standardization to z scores brought the tmb distributions into good concordance ( fig 1f ) with  . 
sampling simulations ( data supplement ) suggest that this negative nding may be due to decreased power in this subset , although lower tmb values and distinct biology may also contribute ( data supplement )  . 
similar exploratory analyses of patients who harbor targetable oncogenic drivers did not demonstrate an association between tmb z score and dcb ( total n = 74 ) , although power in these small driver subgroups was also limited ( fig 2e )  . 
300 may be necessary to detect a difference in tmb between patients with dcb and ndb in groups with lower response rates or effect sizes ( data supplement )  . tmb thresholds and response given the heterogeneity in previously identied thresholds and the percentile cut points used to identify such thresholds , we used our pooled cohort to systematically explore the relationship between tmb and response to icis across the tmb distribution . 
we calculated the rate of dcb and cr / pr with increasing tmb thresholds in the pooled and separate ici cohorts ( fig 3a ; data supplement )  . table 2 lists the tmb z scores and values associated with each threshold . 
we noted , however , that this could arise from enriching for high tmb outliers and therefore calculated the rate of dcb within each tmb decile ( joint cohort shown in fig 3b ; separate cohorts shown in the data supplement )  . 
in this analysis , we noted high dcb rates in the highest deciles ( 40.4% in patients with tmb z scores between the 80th and 90th percentiles ; 53.1% in patients with tmb z scores 90th percentile ) and low rates in the lowest deciles ( 16.7% in patients with tmb z scores , 10th percentile )  . 
the pattern of association between pd - l1 and dcb was similar to tmb , with increasing rates of dcb with higher pd - l1 thresholds but more variability within pd - l1 score groupings ( data supplement )  . given the heterogeneity of response rates over the mid - tmb distribution , we used roc curve analysis to formally quantify how well tmb z scores discriminate between dcb and ndb . roc analysis yielded an area under the curve ( auc ) of 0.614. 
the youdens index cutoff was associated with a sensitivity of 61.8% and a specicity of 57.3% , which resulted in undertreatment of 12% of patients and overtreatment of 30% ( fig 3d ; table 3 )  . 
analysis of tmb thresholds with respect to progression - free survival , rather than response , demonstrated similar results ( data supplement )  . abbreviations : cr , complete response ; ctla - 4 , cytotoxic t - cell lymphocyte - 4 ; dcb , durable clinical benet ; dfci , dana - farber cancer institute ; mskcc , memorial sloan kettering cancer center ; ndb , no durable benet ; pd , progressive disease ; pd - ( l ) 1 , programmed cell death 1 or programmed cell death - ligand 1 ; pr , partial response ; sd , stable disease . discussion we present a pragmatic comparison of tmb calculated from targeted panels and wes and apply tmb z score conversion to enable harmonized analyses . 
in addition , our use of realworld data sets allows us to incorporate and account for sources of variation not captured by in silico downsampling analyses , such as differences in mutation / indel calling pipelines , depth of coverage , and germline ltration . 
tmb z score deciles were selected as cut points , and rate of dcb was calculated for patients in the joint cohorts whose tmb z scores were greater than or equal to the cut point . 
auc , area under the curve . efforts , 22 , 43 which focus on standardization of tmb denitions and reporting and eventually aim to generate gold standard cell lines for benchmarking . 
we anticipate that our approach will be of immediate use to both clinicians and researchers and anticipate that it can be easily applied to other platforms and relevant tumor types . although the association between tmb and response to icis in nsclc has been demonstrated , less is understood about how clinical and biologic features affect this association . 
equivalent tumor mutational burden ( tmb ) values in the dfci , mskcc , foundation medicine , and tcga cohorts are shown . abbreviations : dfci , dana - farber cancer institute ; mskcc , memorial sloan kettering cancer center ; tcga , the cancer genome atlas . underpowered to detect a difference , and our power simulations indicated that larger cohorts are needed and caution against denitive conclusions in these small subgroup analyses . 
however , we also observed that neversmokers who benetted from icis had markedly lower tmb values than ever - smokers who did not , which suggests that further study to identify tmb - independent predictors of response in never - smokers may be warranted and raises the important possibility that the clinical application of tmb as a biomarker will need to take clinical and biologic features into account . the importance of context is further emphasized by our analysis of tmb thresholds . 
prior analyses have generally focused on identifying a single threshold to dene tmbhigh and tmb - low subgroups , with variation in selected thresholds across studies.13 , 15 , 26 , 44 our systematic analysis of tmb thresholds illustrates additional nuances in the relationship between tmb and response . 
we observed enrichment in dcb with higher tmb thresholds , as expected , but weaker discrimination in the midrange of tmb values without a single , natural biologic inection point . these ndings may account for some of the observed heterogeneity among previously proposed thresholds , as there may be a range of values that discriminate similarly between responders and nonresponders . 
in addition , our data suggest that the choice of a given threshold must be decided within a goal - specic context that considers the relative efcacy of the alternative treatment ; a tmb threshold selected to enrich for response to rst - line therapy may be different than a threshold selected for second - line therapy . 
of note , tmb is independent of pdl1 expression , 12 , 13 with similar biomarker performance : increasing expression is associated with improved efcacy without a natural cut point , there is variability in dcb enrichment within deciles of expression , and distinct thresholds are appropriately applied on the basis of the specic treatment scenario ( ie , 50% , 1% , none ) .3 , 5 , 45 ultimately , these data do not answer whether and how tmb should be applied to clinical practice , as this must be examined through prospective clinical trials , but add nuance to our understanding of how tmb associates with response . one limitation of this study is that our comparison of tmbs assumes that the observed distinctions reect differences in platforms rather than in patients / samples . 
we were not able to account for clinical and tumor features because of inconsistent sample annotation but note that our large cohorts help to mitigate sampling bias , and the overall consistencies in shape of distribution are reassuring . whether tmb distributions should be more narrowly table 3 . 
 vokes et al dened by sample features , such as histology or stage , is an open question , and the normalization we describe here can be adjusted as more is learned . 
at present , however , tmb is compared across patients and biopsy specimens without reference to these sample characteristics , which makes this aggregated approach consistent with current clinical practice . in conclusion , we provide a practical approach to the challenge of standardizing tmb across platforms , and we apply this approach to integrate distinct data sets to better understand how tmb associates with response . 
is a damon runyon clinical investigator supported ( in part ) by the damon runyon cancer research foundation ( ci - 98 - 18 ) and is a member of the parker institute for cancer immunotherapy . 
margolis employment : verana health suzanne dahlberg consulting or advisory role : astrazeneca patents , royalties , other intellectual property : patent pending for a statistical model assessing tumor growth ( inst ) pasi a . 
vokes , biagio ricciuti , elizabeth jimenez - aguilar , hira rizvi , jeffrey girshman , anika adeni , suzanne dahlberg , ahmet zehir , mizuki nishino , renato umeton , lynette m . 
sholl honoraria : astrazeneca consulting or advisory role : foghorn therapeutics , loxo oncology research funding : genentech ( inst ) travel , accommodations , expenses : bristol - myers squibb eliezer m . 
van allen stock and other ownership interests : syapse , tango therapeutics , genome medical , microsoft , ervaxx consulting or advisory role : syapse , roche , third rock ventures , takeda pharmaceuticals , novartis , genome medical , invitae , illumina , tango therapeutics , ervaxx speakers bureau : illumina research funding : bristol - myers squibb , novartis patents , royalties , other intellectual property : patent on discovery of retained intron as source of cancer neoantigens ( inst ) , patent on discovery of chromatin regulators as biomarkers of response to cancer immunotherapy ( inst ) , patent on clinical interpretation algorithms using cancer molecular data ( inst ) travel , accommodations , expenses : roche , genentech matthew d . 
hellmann stock and other ownership interests : shattuck labs honoraria : astrazeneca , bristol - myers squibb consulting or advisory role : bristol - myers squibb , merck , genentech , astrazeneca , medimmune , novartis , janssen pharmaceuticals , nektar , syndax , mirati therapeutics , shattuck labs research funding : bristol - myers squibb ( inst ) patents , royalties , other intellectual property : a patent has been led by memorial sloan kettering ( pct / us2015 / 062208 ) for the use of tumor mutation burden for prediction of immunotherapy efcacy and which is licensed to personal genome diagnostics ( inst ) travel , accommodations , expenses : astrazeneca , bristol - myers squibb mark m . 
awad consulting or advisory role : genentech , merck , pzer , boehringer ingelheim , abbvie , astrazeneca , medimmune , clovis oncology , nektar , bristol - myers squibb , ariad pharmaceuticals , foundation medicine , syndax , novartis , blueprint medicines , maverick therapeutics research funding : bristol - myers squibb , genentech ( inst ) , roche ( inst ) , eli lilly ( inst ) , astrazeneca ( inst ) , bristol - myers squibb ( inst ) no other potential conicts of interest were reported . references garon eb , rizvi na , hui r , et al : pembrolizumab for the treatment of non - small - cell lung cancer . 
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hansen , md , phd1 , 2 ; lars henrik jensen , md , phd1 , 2 ; and anders jakobsen , dmsc1 , 2 purpose analysis of circulating tumor dna ( ctdna ) is a potential improvement in precision medicine . 
plasma samples represented healthy donors and localized and metastatic crcs . results the level of neuropeptide ymethylated dna in the tissue cohorts differed between nonmalignant and malignant / premalignant tissues with minimal overlap . 
furthermore , meth - ctdna was detected in plasma from 100% of patients with metastatic disease , compared with 67% of those with localized disease and 8% of healthy donors . 
2019 by american society of clinical oncology introduction circulating tumor - specic dna ( ctdna ) is an attractive biomarker for obvious reasons , such as easily repeated access , better reection of overall tumor biology , and timely correct tumor mutational status . 
consequently , the era is marked by enthusiasm , but it should be noted that its clinical utility , with few exceptions , remains to be proven.1 ctdna can be determined by the presence of tumorspecic genetic or epigenetic alterations . 
ngs allows identication of mutations of interest and is a relatively broad approach applicable in the identication of mutational patterns and quantication of specic mutations , but this method is time and resource consuming . 
different platforms may lead to contradictive results , as shown in a recent report , with only 60% agreement between analyses of the same mutations.2 the second method relies on pcr with analysis of specic mutations . 
compared with ngs , pcr , especially digital pcr , has a faster data turnaround time3 and lower costs , but direct pcr is only applicable in the fraction of patients with known specic mutations . in colorectal cancer ( crc ) , ras / raf mutations are of interest , because their presence in tumor tissue contraindicates treatment with monoclonal antibodies cetuximab and panitumumab . 
multiplex technology allows for screening of 27 mutations in the same analysis , 4 but even so , only approximately 60% of patients present with a ras / raf - mutated tumor . 
 thomsen et al context key objective can circulating tumor - specic methylated dna serve as a universal biomarker in colorectal cancer ? knowledge generated methylated dna was tumor specic in different cohorts including tissue as well as plasma . 
high correlation was found between mutated and methylated dna in both localized and metastatic disease . relevance circulating tumor - specic methylated dna holds promise as a universal colorectal cancer marker that can be analyzed in a simple , highly reproducible approach . most of these patients , the same tumor - specic mutation is detectable in ctdna ( mut - ctdna )  . 
dna was eluted in 50 ml of nuclease - free water and further diluted with nuclease - free water , if necessary . aberrant methylation is an early event in carcinogenesis and occurs in almost all malignant tumors as methylation of promoter regions causing inactivation of genes . 
this , along with the stability of methylation changes , makes it a relevant biomarker for early diagnosis.5 analysis of abnormally methylated dna in plasma ( meth - ctdna ) has been proposed for screening , 6 and a test for crc has been approved by the us food and drug administration.7 however , because of insufcient sensitivity and specicity , it has not been generally accepted . 
the neuropeptide y gene ( npy ) has been correlated with invasion and proliferation.8 , 9 exogenous npy has been shown to inuence the growth of malignant cholangiocarcinoma cells in vitro and inhibit invasion of crc cells . 
in this disease , hypermethylation of the npy promoter region has been correlated with inactivation of gene expression and therefore carcinogenesis , and currently , hypermethylation of the npy gene is suggested as a blood - based biomarker.10 - 12 the purpose of our study was to compare mutated dna with methylated dna in malignant and nonmalignant colorectal tissue and plasma with the aim of determining a method applicable in all patients with crc . plasma sampling and purication in edta tubes , 9 - ml blood samples were collected from donors and different cohorts of patients . 
plasma was centrifuged again at 10 , 000 g for 10 minutes before purication , and cysteine - rich polycomb - like protein 1 ( cpp1 ) dna fragments were added as exogenous internal control.13 dna from patients was puried from 2 2 or 2 4 ml of plasma on the qiasymphony sp instrument using the qiasymphony circulating nucleic acid kit ( qiagen , hilden , germany ) according to manufacturer instructions . dna from healthy donors was puried from 1 4 ml of plasma . dna was eluted in 60 ml of the supplied buffer and water added to 400 ml . 
three 15 - mm slices of ffpe tissue were subjected to 180 ml of incubation buffer and 20 ml of protein kinase k overnight at 70c ; 400 ml of lysis buffer adenomas and tumor tissue samples were screened for ras / raf mutations as previously described.14 samples from adjacent tissue and plasma were analyzed for the specic mutation by droplet digital pcr in two wells . positive controls for each mutation ( gblocks ; idt , carolville , ia ) or mutated fragments generated according to spindler et al , 15 genomic donor dna , and water were included in the analyses as controls . 
patients with mcrc had plasma analyzed both at baseline and after the rst treatment cycle . tissue methylation in 25 nonneoplastic specimens , all but one were nonmethylated ( 4% were npy positive )  . 
figure 1 shows the results of the tissue npy methylation analysis , with methylated npy presented as actual clearly shows that the level of npy methylation was different in malignant and nonmalignant tissues . 
the range of npy fractions . mutated and methylated dna in colorectal cancer methylation analysis tumor - specic methylated dna was dened as dna with methylation of the npy gene.12 before droplet digital pcr methylation analysis , bisulte conversion of circulating and ffpe - isolated dna was performed as recommended by the manufacturer ( zymo research , irvine , ca )  . 
water and a pool of lymphocyte dna from cancer - free individuals were included in each round of analyses as negative controls ; universal human methylated control dna ( zymo research ) and epitech control dna ( qiagen ) were included as positive controls . 
the upper limit of the 95% ci was 1.8% , and therefore , the cutoff value was set to 2% . limit of blank in plasma limit of blank ( lob ) in the mutation analysis was determined by donor controls . 
because fewer than two fampositive droplets were observed in all analyses of the genomic donor dna controls and plasma dna from healthy individuals as previously described , 16 plasma samples with more than two fam - positive droplets in two wells were classied as positive . lob in the methylation analysis was dened as the quantity of npy droplets counted in control dna samples from a test cohort of healthy individuals ( n = 50 )  . 
correlations are also illustrated in the scatter plots shown in figures 3 , 4a , and 4b , which represent correlation between meth - ctdna and mut - ctdna in the localized and metastatic settings at baseline and in the metastatic setting after the rst treatment cycle , respectively . discussion our study on tissue and plasma samples from donors and different stages of crc indicates that meth - ctdna correlated with mut - ctdna across the cohorts and that methctdna was detectable in cases without activating dna mutations . 
therefore , meth - ctdna may be a universal biomarker in crc from the perspectives of detection and monitoring . key challenges in cancer management are early diagnosis and close monitoring during treatment and follow - up with minimal harm to the patients . 
in the landscape of tumor markers , ctdna represents a relatively new approach with the potential for progress in cancer management , because it contains the same molecular alterations as the corresponding tumor and potential metastases . 
even so , clinical utility has been limited . recently , presence of ctdna was conrmed in wt patients using epigenetic modications such as hypermethylation.17 - 19 meth - ctdna seems to represent tumor - specic dna and is found in nearly all colorectal adenocarcinomas , 10 as opposed to ctdna based on ras / raf mutations . 
three recent studies have suggested that hypermethylation of the npy gene may serve as a biomarker for monitoring the treatment of crc , 10 , 11 , 20 but a detailed comparison of mutated and methylated dna motivated our study . the level of methylation varied among different cohorts with malignant and nonmalignant tissues , the latter in general being negative . 
fifty healthy donors were only analyzed for methylated ctdna ( methctdna ) , and 20 healthy donors were analyzed for kras g12d mutated ctdna ( mut - ctdna )  . 
plasma was drawn from patients with localized ras / raf - mutated tumors ( n = 27 ) and metastatic ras / rafmutated tumors ( n = 20 ) and analyzed for the same specic mutations and methylation . methylation in tumors did not overlap that of nonmalignant npy methylation , except in three cases ( 3% )  . plasma methylation figure 2 shows the plasma analysis of different cohorts . samples from healthy donors showed that four ( 8% ) of 50 had detectable meth - ctdna above the lob . 
furthermore , the analysis showed that 67% ( 18 of 27 ) of patients with localized crc had mut - ctdna , compared with 52% ( 14 of 27 ) with meth - ctdna . 
scatter plot showing correlation of methylated circulating tumor dna ( ctdna ) and mutated ctdna ( mut - ctdna ) in localized disease . almost complete separation of malignant and nonmalignant tissues based on the statistical variation of the observed frequencies in healthy individuals . 
adjacent tissue showed npy hypermethylation in one third of the samples , although they were just positive ; none were mutated , despite ras / raf mutations in the nearby tumor . 
scatter plots showing correlation of methylated circulating tumor dna ( ctdna ) and mutated ctdna ( mut - ctdna ) in metastatic colorectal cancer ( a ) at baseline and ( b ) after one cycle of treatment . furthermore methylation is an early event in carcinogenesis , 22 which may represent a potential universal biomarker.23 all tumor tissue samples were hypermethylated irrespective of mutational status and to the same extent as ras / raf - mutated adenomas . 
this indicates that aberrant methylation takes place as an early part of cancer progression , which can predispose to additional genetic alterations.24 possibly , these epigenetic changes give an important and early window for therapeutic interventions in patients with crc . plasma analyses showed that the frequency of patients with ctdna varied depending on disease stage . 
lob of two positive droplets per reaction determined in a test cohort of 50 healthy donors seems in good agreement with previous results reporting an lob of 3.30 on the basis of 17 control dna samples without hypermethylated npy.11 the two self - reported healthy donor cohorts with 4% ( two of 50 ) and 8% ( four of 50 ) of patients having more than two positive droplets per reaction indicate good agreement . 
therefore , occurrence of aberrant methylation with increasing age does not seem to be a major problethe false - positive rate may be acceptable for patients with advanced disease , and therefore , clinical application of meth - ctdna in metastatic disease seems encouraging . 
in localized tumors , two thirds of patients harbored mut - ctdna and meth - ctdna , which is in accordance with current literature.10 in the metastatic setting , all indicating its potential as patients had meth - ctdna , a general biomarker . 
they found that 77% had detectable ctdna at baseline , assessed by mut - ctdna and meth - ctdna . boeckx et al11 reported that 87.5% of all baseline plasma samples were positive for npy methylation in 24 patients with mcrc . 
varying methods of analysis , which lack standardization throughout the eld of research in ctdna , are one obvious reason for the lower frequency of patients with detectable ctdna compared with our ndings . correlation between mut - ctdna and meth - ctdna was investigated to compare the two potential biomarkers . 
this is in agreement with previous studies concluding that methylated markers in ctdna could replace tumor - specic mutations in the blood.10 this conclusion was based on a correlation coefcient of r = 0.94 between mut - ctdna ( kras , braf , and tp53 ) and meth - ctdna ( npy )  . 
we did not investigate the potential role of meth - ctdna in monitoring wt patients , but we found a strong correlation between mut - ctdna and meth - ctdna after one cycle of treatment , which emphasizes the role of meth - ctdna in treatment monitoring . 
taken together , the current literature indicates high agreement between mut - ctdna and meth - ctdna , 10 , 11 , 20 further supported by our results . a minor disadvantage in the methylation assays is the need for bisulte conversion , which can degrade dna and therefore requires additional material . 
however , the need for prior establishment of mutation status , as with mutctdna , is bypassed , and therefore , meth - ctdna is applicable in nearly all patients with crc . 
it should be noted that meth - ctdna is not relevant in the selection of patients for targeted treatment with epidermal growth factor receptor inhibitors . in conclusion , to our knowledge , our study represents the rst detailed comparison of methylated and mutated dna in normal tissue as well as in localized and metastatic tumors . 
this indicates that hypermethylated npy in plasma is applicable as a universal biomarker in all patients with mcrc , but further investigation of clinical utility is warranted . affiliations 1danish colorectal cancer center south , vejle hospital , vejle , denmark 2university of southern denmark , odense , denmark relationships are self - held unless noted . 
all relationships are considered compensated . lars henrik jensen research funding : merck sharp & dohme ( inst ) travel , accommodations , expenses : bayer healthcare pharmaceuticals no other potential conicts of interest were reported . acknowledgment we thank the study participants and the staff at vejle hospital who collected samples for this study . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american pathologists joint review . 
jama oncol 4 : 868 - 870 , 2018 thierry ar , el messaoudi s , mollevi c , et al : clinical utility of circulating dna analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti - egfr treatment . 
front mol biosci 2 : 13 , 2015 church tr , wandell m , lofton - day c , et al : prospective evaluation of methylated sept9 in plasma for detection of asymptomatic colorectal cancer . 
boeckx n , op de beeck k , beyens m , et al : mutation and methylation analysis of circulating tumor dna can be used for the follow - up of metastatic colorectal cancer patients . 
roperch jp , incitti r , forbin s , et al : aberrant methylation of npy , penk , and wif1 as a promising marker for blood - based diagnosis of colorectal cancer . 
spindler kl , pallisgaard n , vogelius i , et al : quantitative cell - free dna , kras , and braf mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan . 
thomsen cb , hansen tf , andersen rf , et al : monitoring the effect of rst line treatment in ras / raf mutated metastatic colorectal cancer by serial analysis of tumor specic dna in plasma . 
garlan f , laurent - puig p , sefrioui d , et al : early evaluation of circulating tumor dna as marker of therapeutic efcacy in metastatic colorectal cancer patients ( placol study )  . 
step two was 44 cycles at 95c for 15 seconds and 56c for 1 minute , with a 1.5c per second ramp rate . step three was at 98c for 10 minutes . 
carriers are predisposed to a higher risk for developing colorectal , endometrial , and extracolonic cancers , including cancers of the stomach , ovary , small bowel , hepatobiliary and urinary tracts , skin , pancreas , and prostate . 
primary brain tumors ( pbts ) are a rare feature of ls , with an estimated lifetime risk of 1% to 6%.1 , 2 here , we report the case of a patient with a who grade iii glioneuronal tumor and pathogenic germline msh2 variant . 
microscopic examination revealed a high - grade glioma with eosinophilic granular bodies , elevated mitotic activity ( approximately 10 / 10 high - power eld [ hpf ] ) , vascular proliferation , and necrosis consistent with an anaplastic glioneuronal tumor ( who grade iii )  . tumor cells were positive for glial brillary acidic protein ( gfap ) and synaptophysin ( fig 2 )  . 
the mib - 1 proliferative index ( ki - 67 ) was approximately 10% . the patients initial postoperative imaging revealed no residual disease ; therefore , no adjuvant therapy was offered . 
postoperative head ct ( c ) with and ( d ) without contrast at the patients most recent follow up , 105 months postinitial diagnosis , shows postoperative changes of the left frontal lobe without evidence of tumor recurrence . disease progression identied pathogenic genomic alterations in six genesnf1 , pdgfra , atm , tp53 , ctnna1 , and msh2and 18 variants of uncertain signicance ( table 1 )  . 
follow - up ihc studies identied a loss of msh2 and msh6 protein expression in tumor cells , which is consistent with an ls - associated tumor and underlying msh2 germline mutation . 
microscopic examination revealed a high - grade glial neoplasm with ( a ) scattered pleomorphic cells , ( b ) spindle cells , and mitotic activity . necrosis and vascular proliferation were observed . 
immunostains showed that the tumor is positive for ( c ) gfap and ( d ) synaptophys ( e ) ki - 67 was moderately elevated and a ( f ) neurolament stain was negative within the tumor , highlighting a compact growth pattern . 
immunohistochemistry identied loss of ( g ) msh2 and ( h ) msh6 protein expression in tumor cells , with retained nuclear expression in the normal endothelial cells in the blood vessels . magnication of all images is 200 . 
glioblastoma is the most common pbt associated with ls.3 astrocytoma , oligodendroglioma , gliosarcoma , medulloblastoma , and neuroblastoma have also been reported.4 individuals with msh2 mutations have higher rates of pbts compared with carriers of other mmr mutations.5 the average age of pbt diagnosis occurs a decade earlier in individuals with ls , 4 and the age range for msh2 carriers is 33 to 53 years.1 , 3 , 6 , 7 ls is one of several hereditary cancer syndromes that predispose to pbts and other systemic cancers . 
therefore , interrogation of personal and family history of malignancies is critical for patients with d 70s dx 47 d 52 d 50s d 50s d 30s d 50s d 40s d 60 dx 42 5 polyps 2 polyps throat abdominal cancer , other breast cancer colorectal cancer glioneuronal tumor fig 3 . 
the patients family history is notable for multiple maternal family members affected with early - onset colorectal cancer , including his mother and multiple maternal aunts , uncles , and cousins . 
next - generation sequencing of 236 genes in the patients tumor specimen ( solid tumor panel ; foundationone , cambridge , ma ) identied pathogenic genomic alterations in six genes and 18 variants of uncertain signicance . * follow - up sequencing of msh2 in the patients saliva sample ( ambry genetics , aliso viejo , ca ) also identied the msh2 mutation in the germline sample . pbt as for all patients with cancer . 
referral for genetic counseling and germline testing is recommended for any patient with pbt with multiple relatives affected with earlyonset cancers , individual ( s ) with multiple primary cancers , and / or rare or syndrome - specic patterns of malignancies.8 , 9 appropriate germline genetic testing may result in additional screening guidelines and the identication of other at - risk relatives.10 ls surveillance guidelines include colonoscopy every 1 to 2 years starting at age 20 to 25 years or 10 years before the earliest crc diagnosis in the family.11 histologic ndings in this patients pbt showed overlapping features between anaplastic pleomorphic xanthoastrocytoma , who grade iii , and glioblastoma . 
genetic alterations that are present in the tumor tissuethat is , nf1 , pdgfra , and tp53 mutationshave been described in glioblastomas12 , 13 ; however , survival of 8 years would be unusual for a patient with glioblastoma . one indicator that germline molecular testing was warranted for this patient was the identication of an msh2 mutation in his tumor specimen . 
studies that have evaluated the yield of germline variants after somatic testing indicate that a small but signicant number of tumors possess germline mutations related to hereditary cancer predisposition syndromes.14 - 17 currently , consideration of follow - up germline testing is recommended after identication of somatic egfr t790m mutations in lung cancer or brca1 / 2 mutations in any tumor.18 - 21 interpretation of somatic mmr mutations , however , is more complex . hampel et al22 used tumor sequencing to screen for ls in a cohort of patients with crc in which a positive screen for ls was given to any patient who had mutation ( s ) identied in at least one mmr gene at a variant allele fraction that was suggestive of a germline mutation , with msi and braf v600e status taken into account . 
therefore , whereas the identication of somatic mmr mutation ( s ) is not diagnostic of ls , it does suggest that follow - up germline evaluation is warranted in these patients . identifying germline and / or somatic mmr mutations may affect treatment offerings , such as consideration for immune checkpoint inhibitors ( icis ) , which target checkpoint receptors of mmr - decient cancers.23 - 25 early studies have demonstrated that icis can effectively treat high tmb cancers ; mmr - decient tumors can possess 10 to 100 times the tmb compared with mmr - procient cancers.26 - 28 glioblastoma specimens from patients with constitutional mmr deciency caused by biallelic germline mmr mutations have signicantly higher tmb compared with sporadic gliomas and other pbts.29 whereas icis have primarily been evaluated in crc and endometrial carcinomas , therapeutic potential has been demonstrated in other mmrdecient tumors . 
in patients with recurrent tumors , high tmb may indicate resistance to traditional chemotherapy and necessitate targeted treatment with icis or other immunotherapies.30 these results must be validated on a larger scale to inform clinicians of the potential therapeutic benets of ici in this subset of patients . in conclusion , we report the rst case of an individual with anaplastic glioneuronal tumor with atypical histologic features and paired somatic and germline msh2 mutations , indicating the diversity of pbts observed within ls . 
the presence of mmr gene mutation ( s ) in tumor specimens indicate that additional genetic evaluation is warranted . ihc , msi , and tmb analyses are also supportive of an ls diagnosis . 
furthermore , a family history evaluation for multiple relatives affected with early - onset cancers , dividual ( s ) with multiple primary cancers , and / or rare or syndrome - specic patterns of malignancies is imperative for all patients with pbt . 
in addition , identifying mmr deciency in the germline or tumor specimens may be an indication for targeted therapy via ici or other immunotherapies in the future . affiliations 1the university of texas md anderson cancer center , houston , tx 2university of texas health science center at houston , houston , tx 3foundation medicine , morrisville , nc 4wake forest school of medicine , winston - salem , nc relationships are self - held unless noted . 
qualmann provision of study materials or patients : jay - jiguang zhu , krista j . qualmann collection and assembly of data : sarah azam , jay - jiguang zhu , krista j . 
ramkissoon employment : foundation medicine stock and other ownership interests : foundation medicine sigmund hsu consulting or advisory role : abbvie , cns pharmaceuticals research funding : abbvie patents , royalties , other intellectual property : partial patent holder , royalties ; at the time of ling , was associated with md anderson cancer center jay - jiguang zhu consulting or advisory role : tocagen research funding : novocure ( inst ) , boston biomedical ( inst ) , five prime therapeutics ( inst ) , tocagen ( inst ) , immunocellular therapeutics ( inst ) travel , accommodations , expenses : zai lab krista j . 
qualmann employment : my gene counsel no other potential conicts of interest were reported . references vasen hfa , stormorken a , menko fh , et al : msh2 mutation carriers are at higher risk of cancer than mlh1 mutation carriers : a study of hereditary nonpolyposis colorectal cancer families . 
int j cancer 81 : 214 - 218 , 1999 therkildsen c , ladelund s , rambech e , et al : glioblastomas , astrocytomas and oligodendrogliomas linked to lynch syndrome . 
n engl j med 348 : 919 - 932 , 2003 vasen hfa , sanders eacm , taal bg , et al : the risk of brain tumours in hereditary non - polyposis colorectal cancer ( hnpcc )  . 
hampel h , bennett rl , buchanan a , et al : a practice guideline from the american college of medical genetics and genomics and the national society of genetic counselors : referral indications for cancer predisposition assessment . 
catenacci dv , amico al , nielsen sm , et al : tumor genome analysis includes germline genome : are we ready for surprises ? int j cancer 136 : 1559 - 1567 , 2015 jones s , anagnostou v , lytle k , et al : personalized genomic analyses for cancer mutation discovery and interpretation . 
b. , et al : molecular testing guideline for selection of lung cancer patients for egfr and alk tyrosine kinase inhibitors : guideline from the college of american pathologists , international association for the study of lung cancer , and association for molecular pathology . 
hampel h , pearlman r , beightol m , et al : assessment of tumor sequencing as a replacement for lynch syndrome screening and current molecular tests for patients with colorectal cancer . 
sloan ea , ring kl , willis bc , et al : pd - l1 expression in mismatch repair - decient endometrial carcinomas , including lynch syndrome - associated and mlh1 promoter hypermethylated tumors . 
science 357 : 409 - 413 , 2017 johnson a , severson e , gay l , et al : comprehensive genomic proling of 282 pediatric lowand high - grade gliomas reveals genomic drivers , tumor mutational burden , and hypermutation signatures . 
 genomic and immune proling of a patient with triple - negative breast cancer that progressed during neoadjuvant chemotherapy plus pd - l1 blockade david casadevall , md1 , 2 ; xiaotong li , phd1 ; ryan l . 
wali , phd1 ; natalia buza , md1 ; vasiliki pelekanou , md , phd1 ; arjun dhawan , md1 ; julia foldi , md , phd1 ; borbala szekely , md , phd1 , 3 ; francesc lopez - giraldez , phd4 ; christos hatzis , phd1 ; and lajos pusztai , md , dphil1 introduction preliminary results from neoadjuvant trials combining immune checkpoint blockade ( icb ) with standardof - care chemotherapy suggest high pathologic complete response rates that range between 50% and 80% in triple - negative breast cancer ( tnbc ) .1 - 3 adding atezolizumab to nab - paclitaxel for rst - line treatment of metastatic tnbc signicantly improved response rate and progression - free survival compared with nabpaclitaxel alone , suggesting synergy between icb and chemotherapy.4 however , not all patients respond to icb , and a minority exhibit rapid progression of their disease.5 patients who experience exceptionally favorable or unfavorable responses provide unique opportunities for studying disease biology and for identifying response markers . 
progression during neoadjuvant chemotherapy is a rare event in tnbc . here , we report results from the molecular analysis of a tnbc that rapidly progressed during neoadjuvant chemotherapy plus programmed death - ligand 1 ( pdl1 ) blockade in a clinical trial neoadjuvant medi4736 concomitant with weekly nab - paclitaxel and dosedense ac for stage i - iii triple negative breast cancer ( clinicaltrials.gov identier : nct02489448 )  . 
baseline tumor - inltrating lymphocyte ( til ) count was 10% ( cd4 , 5% ; cd8 , 5% ; and cd20 , 1% ) , macrophage ( cd68 ) was 1% , and tumor cellularity was 50% . 
the patient agreed to participate in a neoadjuvant phase i / ii clinical trial that combined durvalumab ( 10 mg / kg once every 2 weeks ) with once - per - week nabpaclitaxel ( 100 mg / m2 ) for 12 cycles and dosedense doxorubicin plus cyclophosphamide ( ddac ) for 4 cycles . 
after 8 weeks of nab - paclitaxel plus durvalumab , physical examination showed increased tumor size and new skin edema conrmed by repeat mammogram and ultrasonograrepeat cnb showed 60% tumor cellularity but also an increase in til count to 20% ( cd4 , 10% to 15% ; cd8 , 5% ; and cd20 , 0% ) and an increase in macrophages ( cd68 , 20% )  . 
nab - paclitaxel was stopped but because of the increased immune inltration , durvalumab was continued , and the patient was administered ddac , which led to disease stabilization after four courses of therapy . 
because of the apparent clinical benet , she received two additional courses of ddac without durvalumab off protocol and underwent right skin - sparing mastectomy and lymph node dissection . pathology showed extensive multifocal disease ( largest focus , 2.4 cm ; tumor cellularity , 40% ) with lymphovascular invasion in the breast and more than 10 positive axillary lymph nodes ( ypt2 , ypn3 )  . 
immune cell proportions in the mastectomy were tils , 20% ; cd4 , 10% ; cd8 , 10% ; cd20 , 1% ; and cd68 , 10% . dna and rna were extracted from formalin - xed parafn - embedded sections of tumor samples obtained at baseline , at week 8 , and from the mastectomy . 
til counts and immune cell subtypes were determined by routine pathology and immunohistochemistry . when we compared our patients baseline expression of 26 immune cell types and 100 immune function metagenes with those in the reference cohort , her neutrophil metagene expression was below the 2.5th percentile , whereas the cell cycle and immunosuppression metagenes were above the 97.5th percentile of the reference distribution ( fig 1a ) , indicating a highly proliferative tumor with an immunosuppressed microenvironment . 
the tumor also showed low expression of two previously validated immunotherapy predictive gene signatures , 8 , 9 indicating low probability of response to icb ( fig 1b )  . 
single - gene level analysis revealed signicantly low expression of 27 genes including pdcd1 , cd8a , and klrc2 ( fig 1c ) , suggesting impaired t - cell and natural killer ( nk ) cell activity . 
in addition , we found high exincluding tgfb , hla - g , cd63 , pression of 33 genes , ccl28 , and cxcl16 ( fig 1d ) , which are associated with an immune evasive phenotype , increased cell motility , and invasion . 
overall , greater changes were observed between week 8 and mastectomy ( ddac treatment ) than between baseline and week 8 ( nab - paclitaxel treatment )  . we observed upregulation of fos , abcb1 , kir3dl3 , gzma , and gzmk in the mastectomy ( single - gene and metagene expression changes are provided in the data supplement )  . 
to study this further , we next analyzed t - cell exclusion and dysfunction features using the tumor immune dysfunction and exclusion ( tide ) method ( data supplement ) .10 in concordance with our previous ndings , cytotoxic t - cell inltration increased during the second period . 
however , this was accompanied by an increase in t - cell dysfunction score , indicating an ineffective immune response ( fig 2b )  . we also performed whole - exome sequencing of baseline and mastectomy specimens . 
other mutations with the highest variant allele frequency at baseline included znf385c , cacna1e , nxpe1 , and dync1h1 , which have poorly understood functions in cancer ( fig 2c ; data supplement )  . 
we also detected amplications in fgfr1 , fgf2 , fgf3 , myc , mcl1 , ccnd1 , and tgfb2 and deletions in cdkn2a and cdkn2b ( data supplement )  . 
by using sciclone ( waltham , ma ) 11 and clonevol , 12 we identied four distinct tumor clones , all present at baseline and in the mastectomy specimen , with little evidence for clonal selection during therapy ( figs 2c - d )  . 
compound heterozygous inactivating mutations cause severe combined immune deciency.14 discussion this cancer showed primary resistance to nab - paclitaxel and ddac chemotherapy concurrent with an anti - pd - l1 agent . 
the tumor harbored several poor prognostic genomic alterations at diagnosis , including a p53 mutation and coamplication of myc and mcl1 , both of which are implicated in chemotherapy resistance.15 , 16 the expression of permeability glycoprotein ( p - glycoprotein ; abcb1 ) , a drugefux transporter that mediates taxane and anthracycline resistance in vitro , also increased during treatment.17 we observed a measurable increase in intratumor ammatory response by the end of the treatment ( data supplement ) , but unfortunately this did not translate into clinical antitumor activity . 
tgf activation in cancers has been linked to primary chemotherapy resistance and also to immune evasion and resistance to pd - l1 blockade in multiple experimental systems.18 - 21 furthermore , r175h p53 mutation that this cancer harbored has been shown to render cancers insensitive to the growth inhibitory effects of tgf.22 on the basis of these ndings , we hypothesize that the high expression of tgfb2 by this tumor may have contributed to its immune escape and its resistance to chemotherapy . 
another notable feature of this cancer was the low level of expression of programmed cell death protein 1 ( pd1 ) at the time of diagnosis and the high level of expression of hla - g . 
categories in which our patients score is above the 97.5th percentile ( red dots ) or below the 2.5th percentile ( teal dots ) are highlighted , are shown as bold numbers , and are described in the legend key . 
finally , genes for which our patient had expression values below the 2.5th ( panel c , teal squares ) or above the 97.5th ( panel d , red squares ) bootstrap distribution percentiles are shown . 
legend boxes and bold numbers show categories for which we observed an increase or decrease of 0.5 log2 fold change between either baseline and 8 weeks ( rst period ) and / or 8 weeks and mastectomy ( second period )  . 
 ( b ) tumor immune dysfunction and exclusion ( tide ) - based ( data supplement ) t - cell dysfunction and exclusion shown together with correlation coefcients of the gene expression values for our patient with cancer - associated broblast ( caf ) , myeloid - derived suppressor cell ( mdsc ) , and m2 tumor - associated macrophages ( tam m2 ) signatures . 
 ( c ) variant allele frequency ( vaf ) plot ( left ) and shplot ( right ) illustrating the clonal architecture and evolution ( based on sciclone [ waltham , ma ] and clonevol tools ; data supplement ) of the tumor between baseline and mastectomy time points . 
ifng , interferon gamma signature ; math , mutant allele tumor heterogeneity ; til , tumor - inltrating lymphocyte ; tlr , toll - like receptor . contributed to the poor outcome . 
however , the ndings pose at least one testable therapeutic hypothesis : tgf - targeting therapies ( eg , galunisertib , m7824 , tew7197 , ly - 3200882 , fresolimumab , and nis793 ) may have improved the efcacy of pd - l1 blockade in this particular individual . in summary , this case demonstrates the complexity of chemotherapy and immunotherapy resistance mechanisms and suggests that multiple different biologic processes may contribute to disease progression during treatment . we nd it reassuring that previously published icb response signatures predicted low sensitivity to pd1 / pd - l1 blockade for this patient . 
for more information about asco 's conict of interest lajos pusztai honoraria : merck , astrazeneca / medimmune , pzer , syndax pharmaceuticals , almac diagnostics , pieris pharmaceuticals , genentech , immunomedics , eisai , seattle genetics / astellas pharma , biotheranostics consulting or advisory role : h3 biomedicine , merck , novartis , pieriandx , seattle genetics , syndax pharmaceuticals , athenex research funding : merck , genentech , seattle genetics , astrazeneca no other potential conicts of interest were reported . acknowledgment we thank erika esteve , md , for her invaluable support throughout the study period ; xavier monzonis , md , and santiago balseiro , md , for their insightful suggestions ; tao qing and michal marczyk for their frequent inputs regarding methodologic and technical aspects of the manuscript ; and jeremia walah ( developer of structural variation and indel analysis by assembly ) and peng jiang ( developer of tumor immune dysfunction and exclusion score ) for their feedback regarding the use and interpretation of their tools . references pusztai l , hofstatter ew , chung gg , et al : durvalumab ( medi4736 ) concurrent with nab - paclitaxel and dose dense doxorubicin cyclophosphamide ( ddac ) as neoadjuvant therapy for triple negative breast cancer ( tnbc )  . 
j clin oncol 36 , 2018 ( suppl ; abstr 586 ) loibl s , untch m , burchardi n , et al : randomized phase ii neoadjuvant study ( geparnuevo ) to investigate the addition of durvalumab to a taxane - anthracycline containing chemotherapy in triple negative breast cancer ( tnbc )  . 
nanda r , liu mc , yau c , et al : pembrolizumab plus standard neoadjuvant therapy for high - risk breast cancer ( bc ) : results from i - spy 2 . 
j clin oncol 35 , 2017 ( suppl ; abstr 506 ) schmid p , adams s , rugo hs , et al : atezolizumab and nab - paclitaxel in advanced triple - negative breast cancer . 
n engl j med 379 : 2108 - 2121 , 2018 ferrara r , mezquita l , texier m , et al : hyperprogressive disease in patients with advanced non - small cell lung cancer treated with pd - 1 / pd - l1 inhibitors or with single - agent chemotherapy . 
ann oncol 29 : 2232 - 2239 , 2018 shi w , jiang t , nuciforo p , et al : pathway level alterations rather than mutations in single genes predict response to her2 - targeted therapies in the neo - altto trial . 
ann oncol 28 : 128 - 135 , 2017 ayers m , lunceford j , nebozhyn m , et al : ifn - - related mrna prole predicts clinical response to pd - 1 blockade . 
j clin invest 127 : 2930 - 2940 , 2017 fehrenbacher l , spira a , ballinger m , et al : atezolizumab versus docetaxel for patients with previously treated non - small - cell lung cancer ( poplar ) : a multicentre , open - label , phase 2 randomised controlled trial . 
lancet 387 : 1837 - 1846 , 2016 jiang p , gu s , pan d , et al : signatures of t cell dysfunction and exclusion predict cancer immunotherapy response . 
ann oncol 28 : 3076 - 3082 , 2017 imamura t , takase m , nishihara a , et al : smad6 inhibits signalling by the tgf - beta superfamily . 
lee k , giltnane jm , balko jm , et al : myc and mcl1 cooperatively promote chemotherapy - resistant breast cancer stem cells via regulation of mitochondrial oxidative phosphorylation . 
when the efcacy of a new therapy may be limited to a biomarker - dened subgroup , the choice of an appropriate randomized clinical trial design should be guided by the strength of the biomarkers credentials . 
if there is strong evidence that the treatment is likely to be more benecial in the biomarker - positive patients but a meaningful benet is also possible in the biomarker - negative patients , then a properly powered biomarker - stratied design ( eg , a subgroup - specic or marker sequential test strategy ) would provide the most rigorous determination of the sensitive populations . 
if the evidence supporting the predictive value of the biomarker is weak and the treatment is expected to work in the overall population , then a fallback design could be used . 
2019 by american society of clinical oncology introduction as a result of advances in biotechnology and improved understanding of cancer biology , therapeutic development in oncology has largely shifted from a focus on cytotoxic agents toward novel molecularly targeted and immune anticancer therapies . 
efcient development of these new anticancer drugs requires identication of predictive biomarkers that can reliably differentiate the patient population into a subgroup that benets from the therapy versus the remaining population , in whom the benet - to - risk ratio is not sufcient for the therapy to be recommended . 
in such settings , the traditional broad - eligibility randomized clinical trial ( rct ) that focuses on assessing the overall treatment effect in the entire study population may be suboptimal because this strategy may miss effective agents or result in recommending therapy to patients who do not benet from it . 
design efciency considerations are particularly important in rare population settings that require balancing feasibility and evidential requirements . relevance this article provides a practical guide for designing a biomarker - based phase iii rct . 
careful selection of an appropriate phase iii design strategy that integrates evaluation of a new anticancer therapy and its companion diagnostic can ensure proper use of resources and enhance the scientic rigor of trials in the era of precision oncology . probability of incorrectly recommending an ineffective therapy ; the power , which is the probability of correctly recommending an effective therapy ; and the target treatment effect , which is the treatment effect that the study has a desired power to identify . 
for a desired target treatment effect , the rct sample size will depend on the design type i error and power ; a smaller type i error and / or higher power require more precision in estimating the treatment effect and thus larger sample sizes . 
most phase iii trials in oncology use time - to - event end points ( eg , progression - free survival or overall survival [ os ] ) , with the target treatment effect expressed in terms of the hazard ratio ( hr )  . 
with a time - to - event end point , the precision of the hr estimate is a function of the number of observed events ; thus , the sample size will also depend on the event rates , accrual rates , and length of follow - up . biomarker with very strong credentials when there is sufciently strong evidence that the treatis limited to a specic biomarker - dened ment effect subgroup ( eg , biomarker - positive patients ) , then the socalled enrichment design is often used ( fig 1a )  . 
thus , the enrichment design avoids treating patients with an experimental therapy that is not expected to work for them . the sample size calculation for enrichment designs is the same as for traditional ( untargeted ) rcts that do not involve biomarkers . 
however , to identify biomarker - positive patients , an enrichment design must either include its own screening component or depend on outside screening ( eg , from next - generation sequencing )  . 
note that when biomarker positivity is low in the patient population , many patients will need to be screened to identify the number of biomarker - positive patients necessary to complete the study . 
for example , the brim3 trial ( clinicaltrials.gov identier : nct01006980 ) , 5 which evaluated a braf inhibitor vemurafenib in patients with brafv600e - mutated melanoma , had to screen 2 , 107 patients with melanoma to enroll 675 patients with the braf mutation . 
when the biomarker is indeed predictive in identifying patients who benet from the therapy , an enrichment design is quite efcient because it can dramatically reduce the number of randomly assigned patients required ( by focusing the trial on a population with an undiluted treatment effect ) .6 despite its advantages , the enrichment design has some limitations . 
if the targeted therapy indeed benets all patients regardless of biomarker status , then restricting the trial population to only biomarker - positive patients slows trial accrual and limits the patient population that can ultimately be helped by the therapy . biomarker with strong credentials at the time a denitive trial is being designed , there is often sufcient evidence to assume that the targeted therapy is more likely to benet biomarker - positive patients than the biomarker - negative patients , but a potential treatment benet in biomarker - negative patients cannot be ruled out . in this case , a design that randomly assigns patients to the experimental and control treatments within biomarkerdened subgroups , the so - called biomarker - stratied design , would be the most appropriate approach for a combined assessment of the therapy and the companion biomarker . 
a biomarker - stratied trial could provide sufciently reliable information on the distribution of the riskbenet ratio of the new therapy across biomarker subgroups to inform treatment efcacy and the need for the biomarker to guide treatment use . biomarker - positive and biomarker - negative ( subgroup - specic ) strategy . 
m , marker ; m + , marker positive ; m , marker negative ; rx , treatment ; sig , signicant . the biomarker - positive and biomarker - negative patient subgroups . 
ideally , this subgroup - specic strategy should be powered to detect clinically relevant treatment effects in each subgroup , albeit in practice , the biomarker - positive subgroup is often prioritized . 
because the biomarker - positive patients are typically expected to derive more benet from the targeted therapy than the biomarker - negative patients , the design could assume a larger treatment effect for the former . because the design involves testing of two subpopulations , the overall study - wise probability of recommending an ineffective therapy to any subpopulation ( overall type i error or overall signicance level of the design ) is inated if no the multiple testing is made . 
in a well - established approach , one allocates the overall between the biomarker - positive and biomarker - negative tests , as follows : signicance level 1 ( 1 ) to testing the biomarker - positive subgroup and signicance level 2 , calculated as 1 , to testing the biomarker - negative subgroup . 
then , one can use recycling techniques to optimize the design by specifying the sequence in which the subgroups are tested , with the level from an earlier signicance test redistributed to the subsequent test.7 - 9 for example , one can rst test the biomarker - positive subgroup at signicance level 1 . 
if that test is signicant , then 1 is redistributed to testing the biomarker - negative subgroup , resulting in an = 2 + 1 signicance level available for testing the biomarkernegative subgroup ; if the test of the biomarker - positive subgroup is not signicant , then an 2 signicance level is used for the biomarker - negative test ( fig 1b )  . 
this provides considerable exibility for adjusting the design to a particular setting ; at one extreme , one can allocate only half of the overall to the initial testing of the biomarkerpositive subgroup ( 1 = / 2 )  . 
more typically , a sequential subgroup - specic approach allocates the entire signicance level to rst test the biomarker - positive subgroup . this is appropriate because one believes it is unlikely that the treatment works in the biomarker - negative subgroup unless it also works in the biomarker - positive subgroup . 
if the biomarker - positive test is signicant , then is redistributed to testing the biomarker - negative subgroup ; if the biomarker - positive test is negative , all testing is stopped ( fig 1c )  . 
the trial was designed to rst test the kras wild - type subgroup at the 1 = .025 signicance level , with testing of the kras - mutated subgroup ( at the 2 = .025 signicance level ) conditional on observing a signicant result in the kras wild - type subgroup . biomarker - positive and overall strategy . 
to account for testing of the two populations , the biomarker - positive / overall designs use multiple testing procedures similar to those used in the subgroup - specic designs . 
other biomarker - positive / overall designs are implemented in a fully sequential manner , rst testing the biomarkerpositive population at the signicance level , and then , if signicant , the overall population at the signicance level . 
note that some trials apply this design in the reverse order ; they rst test the overall population , and only if this test is signicant do they then test the biomarker - positive population.14 requiring a signicant result in the overall population to test the biomarker - positive subgroup is not logical and can result in failing to detect benet limited to the biomarker - positive population . 
for example , the impassion130 trial ( clinicaltrials.gov identier : nct02425891 ) , which tested the addition of atezolizumab to chemotherapy in metastatic breast cancer , was not able to formally test os in the pd - l1positive population because the test in the overall population was not signicant.14 implementation , a major regardless of the particular concern with the biomarker - positive / overall testing approach is that when the benet of the treatment is limited to the biomarker - positive patients , the design may inappropriately recommend the treatment to the biomarkernegative subgroup.8 , 15 , 16 this is because a statistically signicant result in the overall population may be driven by a large treatment effect in the biomarker - positive patients even with no effect in the biomarker - negative subgroup . treatment efcacy is from the clinical perspective , demonstrated in the biomarker - positive patients , then the only relevant question is whether the treatment works in the biomarker - negative patients . 
a potential concern with the subgroup - specic testing strategy is that it has lower power for therapies with moderate treatment effects that are similar across the biomarker subgroups , relative to the designs based on testing the overall population . 
 ( indeed , this is often used to justify the biomarker - positive / overall strategy . ) this concern is addressed by the marker sequential test ( mast ) design17 ( fig 1e ) , which incorporates sequential tests of the treatment effect in the biomarkerpositive subgroup , biomarker - negative subgroup , and overall population ( while controlling the probability of recommending an ineffective therapy to either biomarkerpositive or biomarker - negative patients at signicance level )  . 
for example , a phase iii study of blinatumomab ( ecog e1910 ; clinicaltrials.gov identier : nct02003222 ) in acute lymphoblastic leukemia evaluated minimum residual disease as a predictive biomarker for blinatumomab15 using the mast design . the mast design requires a slightly larger sample size ( approximately 4% ) than the sequential subgroup - specic design ( because the biomarker - positive subgroup is tested at the reduced signicance level 1 )  . 
at the same time , it has considerably higher power in situations where the treatment effect is homogeneous across the biomarker subgroups.17 biomarker with weak credentials the treatment effect when the treatment is expected to be broadly effective ( ie , reliable evidence for predictive value of the biomarker is lacking ) , the most appropriate strategy would be a design that enrolls all patients regardless of their biomarker status and focuses on the treatment effect in the overall population . 
however , it is still possible to incorporate a prospectively specied , statistically rigorous contingency plan to accommodate evaluation of a biomarker - dened subgroup if the test in the overall population is negative.11 this is known as a fallback design . in a fallback design , the treatment effect is rst tested in the overall population using a slightly reduced signicance level of 1 . 
if the results are positive , then the treatment is recommended for the overall population ; if the results are negative , then the test of treatment effect is performed in the biomarker - positive subgroup at a signicance level of ( 1 ) ( fig 1f )  . 
because of the relatively small reduction in the signicance level for the overall population test , the fallback design results only in a marginal ination of sample size compared with a traditional rct that uses the entire signicance level on the overall test.11 statistically speaking , the fallback approach is identical to the biomarkerpositive / overall strategy ( described earlier ) , and thus , it does not control for the probability of incorrectly recommending treatment to biomarker - negative patients when they do not benet from it . 
therefore , use of this design should be limited to settings where the treatment is expected to be widely benecial and the biomarker prevalence is low ( , 20% )  . 
when the prevalence is low , overall results are unlikely to be driven by the biomarker - positive effect , and conducting a subgroup - specic design may be infeasible . 
even when the use of the fallback design is considered justied , one should always include estimation of the treatment effect in the biomarker - negative patients as a secondary goal . unlike the designs described earlier , the fallback design is initially focused on testing the overall population , so there is no need to know patient biomarker status up front . 
however , in some situations , it may be difcult to ascertain the biomarker status before randomization ( eg , when the biomarker assay turnaround time is not short )  . 
theoretically , one can still use the biomarkerstratied approaches described earlier by allowing biomarker assessment to be performed after randomization ; this is sometimes known as the all - comers approach . 
however , because some patients may not have their biomarker status determined ( eg , due to missing or tumor specimen or assay failure ) , for the subgroup - specic designs , the primary analysis is limited to patients with determined status ( for the mast and the biomarker - positive / overall designs , all patients can be used in the overall test )  . if the biomarker assessments are done without knowledge of the randomized treatment assignment and outcome , this approach provides an unbiased evaluation of the biomarker . 
unfortunately , use of the all - comers approach is generally not practical for biomarkers with strong credenthese designs prospectively specify intials : some of dependent sample size goals for the biomarker - positive and biomarker - negative subgroups , and most others include interim monitoring that may at some point restrict accrual to one of the subgroups ( see interim monitoring )  . 
an advantage of obtaining the biomarker assessment up front is that only patients used in the analyses will be enrolled . furthermore , in some settings , the requirement of having a biomarker assessment to enter the study may encourage more rigorous specimen handling and thus improve the biomarker ascertainment success rate . 
suppose one is evaluating a new treatment in a setting where the median os with the current standard of care is 12 months ( in both biomarker subgroups ) and the expected accrual rate is 30 patients per month . 
for each biomarker subgroup , the table lists the probability of recommending the new therapy for that subgroup along with sample sizes , accrual durations , and for practical reasons , biofollow - up periods ( note that marker trials are usually designed to have accrual open to both groups , with the overall sample size dened by the sample size required for the biomarker - positive subgroup )  . the rst step in the design is to determine the number of biomarker - positive patients needed to detect the target treatment effect for that subgroup . 
in the current setting , for biomarkers with 50% , 25% , and 10% prevalence , one needs to enroll 300 , 240 , and 150 patients , respectively , to have acceptable power for an hr of 0.6. 
implementation of the biomarker designs for a range of biomarker prevalences biomarker positive biomarker negative design power ( % ; probability recommending for the subgroup ) sample size , no . accrual + follow - up time ( months ) power ( % ; probability recommending for the subgroup ) sample size , no . accrual + follow - up time ( months ) 50% biomarker prevalence ; target effects : biomarker - positive hr , 0.6 ; biomarker - negative hr , 0.7 25% biomarker prevalence ; target effects : biomarker - positive hr , 0.6 ; biomarker - negative hr , 0.75 enrichment design subgroup specic with recycling : 50% / 50% split sequential subgroup specic mast design biomarker positive and overall : 50% / 50% split enrichment design subgroup specic with recycling : 76% / 24% split sequential subgroup specic mast design biomarker positive and overall : 76% / 24% split enrichment design subgroup specic with recycling : 80% / 20% split sequential subgroup specic mast design fallback : 80% ( overall ) / 20% ( biomarker positive ) split 20 + 9 20 + 14 20 + 9 20 + 10 20 + 14 32 + 12 32 + 16 32 + 12 32 + 14 32 + 16 50 + 18 50 + 26 50 + 18 50 + 22 20 + 9 10% biomarker prevalence ; target effects : biomarker - positive hr , 0.6 ; biomarker - negative hr , 0.75 1350 1350 1350 20 + 40 20 + 40 20 + 40 20 + 14 32 + 16 32 + 11 32 + 14 32 + 16 50 + 9 50 + 9 50 + 9 20 + 20 note . 
when biomarker - positive prevalence is relatively high ( close to 50% ) , additional ( longer ) follow - up will be required on the biomarker - negative patients to achieve acceptable power for detecting the desired target effect . conversely , when the biomarker prevalence is low , additional ( longer ) follow - up may be required for the data to mature in the biomarker - positive subgroup . for the mast , biomarker - positive / overall , and fallback designs in table 1 , the probabilities of recommending therapy for a given subgroup include recommendations that are based on both the subgroup - specic and the overall tests . 
one can see in table 1 that for the biomarkers in the 25% to 50% range , mast , biomarker - positive / overall , and subgroup - specic with recycling designs provide the best power in the situations where treatment is benecial in both subgroups . 
however , it is important to remember that , unlike the biomarker - positive / overall design , the mast approach is explicitly designed to control for the probability of an incorrect treatment recommendation for biomarker - negative patients . 
one can see that although the mast design controls the probability of recommending ineffective therapy to biomarker - negative patients at 2.5% , this probability could be high for the biomarker - positive / overall design ( eg , 69% and 40% in 50% and 25% prevalence cases , respectively )  . 
for example , in the clinical settings considered in table 1 , if the biomarker prevalence is 10% , then it takes 50 months to enroll 150 biomarker - positive patients ( 1 , 500 patient total ) to have 80% power for an hr of 0.6 ( after an additional 18 months of follow - up )  . 
in rare biomarker settings where the biomarker credentials are weak ( ie , the treatment is expected to have broad benet ) , one can use the fallback design ( eg , with 10% prevalence , a 600 - patient fallback design would have 82% power when there is some effect in both biomarker subgroups ; table 1 )  . 
note that in this rare biomarker setting , when the treatment effect is limited to the biomarker - positive patients , the probability of the fallback design incorrectly recommending ineffective therapy to the biomarker - negative patients is low ( 5% ; table 2 )  . 
in this case , one would need to design the study so that enrollment of biomarkernegative patients can be stopped ( after the required number of these patients are enrolled ) , with only biomarkerpositive enrollment allowed thereafter . 
similarly , if the biomarker prevalence is greater than 50% , then one may have to continue accrual to the biomarker - negative subgroup after the biomarker - positive subgroup nishes accrual . 
 phase iii precision medicine trial designs treatment is likely to be more benecial in the biomarkerpositive patients but a meaningful benet is also possible in the biomarker - negative subgroup , then a properly powered biomarker - stratied design using a subgroup - specic strategy with recycling or the mast strategy would provide the most accurate and efcient determination of the sensitive population . 
if the evidence supporting the predictive value of the biomarker is weak and the treatment is expected to work in the overall population , then a fallback design could be used . biomarker - driven randomized phase iii trials are resource intensive and often take a long time to complete . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . no potential conicts of interest were reported . references sargent dj , conley ba , allegra c , et al : clinical trial designs for predictive marker validation in cancer treatment trials . 
clin cancer res 14 : 4358 - 4367 , 2008 chapman pb , hauschild a , robert c , et al : improved survival with vemurafenib in melanoma with braf v600e mutation . 
stat med 24 : 329 - 339 , 2005 bretz f , maurer w , brannath w , et al : a graphical approach to sequentially rejective multiple test procedures . 
douillard jy , siena s , cassidy j , et al : randomized , phase iii trial of panitumumab with infusional uorouracil , leucovorin , and oxaliplatin ( folfox4 ) versus folfox4 alone as rst - line treatment in patients with previously untreated metastatic colorectal cancer : the prime study . 
rittmeyer a , barlesi f , waterkamp d , et al : atezolizumab versus docetaxel in patients with previously treated non - small - cell lung cancer ( oak ) : a phase 3 , open - label , multicentre randomised controlled trial . 
powles t , dur an i , van der heijden ms , et al : atezolizumab versus chemotherapy in patients with platinum - treated locally advanced or metastatic urothelial carcinoma ( imvigor211 ) : a multicentre , open - label , phase 3 randomised controlled trial . 
clin trials 11 : 19 - 27 , 2014 jiang w , freidlin b , simon r : biomarker - adaptive threshold design : a procedure for evaluating treatment with possible biomarker - dened subset effect . 
dreno b , thompson jf , smithers bm , et al : mage - a3 immunotherapeutic as adjuvant therapy for patients with resected , mage - a3 - positive , stage iii melanoma ( derma ) : a double - blind , randomised , placebo - controlled , phase 3 trial . 
genomic profiling was performed on 703 appendiceal cancer specimens to compare the mutation profiles of appendiceal subtypes to crc and other cancers , with the ultimate aim to identify potential biomarkers and novel therapeutic targets . methods tumor specimens were submitted to a clinical laboratory improvement amendmentscertified laboratory ( foundation medicine , cambridge , ma ) for hybrid - capturebased sequencing of 3 , 769 exons from 315 cancer - related genes and 47 introns of 28 genes commonly rearranged in cancer . 
kras ( 35% to 81% ) and gnas ( 8% to 72% ) were the most frequent alterations in epithelial cancers ; apc and tp53 mutations were significantly less frequent in appendiceal cancers relative to crc . 
the mutation status of gnas and tp53 strongly predicted os ( median , 37.1 months for tp53 mutant v 75.8 gnas - tp53 wild type v 115.5 gnas mutant ; log - rank p = .0031 ) and performed as well as grade in risk stratifying patients . conclusion epithelial appendiceal cancers and goblet cell carcinoids show differences in kras and gnas mutation frequencies and have mutation profiles distinct from crc . 
2018 by american society of clinical oncology introduction the rarity of appendiceal neoplasms has made it difficult to conduct prospective or randomized clinical trials to guide therapy for these tumors . 
 the small number of appendiceal tumors that are detected , in many cases as an incidental finding in < 1% of appendectomy specimens , 1 comprise multiple histopathologic subtypes , including noninvasive mucinous neoplasms , mucinous and nonmucinous adenocarcinomas , carcinoids , goblet cell carcinoids ( gccs , now also called goblet cell tumors ) , and signet ring cell carcinomas.2 early - stage cancers can be treated definitively with surgery , and selected patients derive long - term benefit from cytoreductive surgery and heated intraperitoneal chemotherapy ( hipec ) .3 however , there is no standard of care for the systemic treatment of advanced , unresectable disease . in the absence of randomized phase iii data , the majority of medical oncologists use colorectal cancer ( crc ) chemotherapy regimens for the treatment of unresectable epithelial appendiceal neoplasms , as is currently recommended by the national comprehensive cancer network guidelines . 
clinical and demographic patient characteristics by subtype median age ( years ) sex ratio m : f subtype mucinous adenocarcinoma adenocarcinoma goblet cell carcinoid pseudomyxoma peritonei signet ring carcinoma 43 : 57 42 : 58 36 : 64 46 : 54 49 : 51 mutation analysis revealed kras to be the most frequently mutated gene in mad ( 77% ) , ad ( 56% ) , and pmp ( 81% ) and the second most frequently mutated gene in srcc ( 35% )  . 
in contrast , kras mutations were significantly less frequent in gccs ( 13% ; 2 p < .001 ) , where tp53 ( 33% ) was the most frequently mutated gene ( fig 1b ; appendix fig a1a )  . 
braf , brca1 , cdkn1b , cdkn2a , myc , pten , and tgfbr2 mutations were present in < 10% of cases across all subtypes ( table 2 )  . 
gnas and kras were the only gene pair significantly comutated ( odds ratio , 6.8 ; bonferroni corrected p = 8.6x10 - 17 ) ; gnas and tp53 were the only gene pair significantly mutually exclusive ( odds ratio , 0.20 ; bonferroni corrected p = 6.7x10 - 13 ; fig 1c ; data supplement )  . pathway - based analysis of mutation profiles genetic aberrations were subsequently grouped by signaling pathway ( appendix table a1 )  . 
 components of the ras / raf signaling pathway ( ie , braf , hras , kras , and nras ) were the most frequently altered genes in epithelial appendix cancers , occurring in > 80% of mads and pmps , 60% of ads , but only 33% of gccs ( 2 p < .001 ; fig 1d )  . 
alterations in homologous recombination deficiency genes were observed in > 50% of all subtypes but were most prevalent in srcc ( 80% ) fluorouracil , leucovorin , and oxaliplatin ; fluorouracil , leucovorin , and irinotecan ; and targeted agents in appendiceal adenocarcinomas compared with crc , 8 , 9 it is known that appendiceal neoplasms have a better prognosis after cytoreductive surgery with hipec treatment.10 there is also a growing body of data showing that there are clear molecular differences between appendiceal and colorectal cancers.11 - 14 here we present a cohort of 703 molecularly profiled appendiceal neoplasms , the largest such cohort to date in this rare disease . 
comparing the mutational landscapes across histologic subtypes we find significant differences in kras , gnas , and fat3 mutation prevalence and confirm that mutational profiles of appendiceal neoplasms are distinct from crc and other gi cancers . 
 in addition , we identify that patients can be risk stratified using the combined mutation status of gnas and tp53 and that outcomes are favorable for patients with kras wild - type disease when treated with irinotecan . results mutation landscape of appendiceal neoplasms the 703 cases were categorized into four different histopathological subtypes consistent with the recently updated consensus classification from the peritoneal surface oncology group international ; in addition , cases of pseudomyxoma peritonei ( pmp ) were included , because this syndrome usually arises from the appendix.2 the majority were either mucinous adenocarcinomas ( mad , 46% ) or adenocarcinomas ( ad , 30% ) , with the rest being gccs ( 12% ) , pmps ( 7.7% ) , or signet ring cell carcinoma ( srcc , 5.3% ; appendix ; table 1 )  . 
the majority of specimens submitted for sequencing came from intraperitoneal metastatic deposits , although there were also primary appendiceal tumors and a small number of lung , liver , and bone metastases ( fig 1a )  . 
data presented as % frequency . abbreviations : ad , adenocarcinoma ; crc , colorectal cancer ; gcc , goblet cell carcinoid ; mad , mucinous adenocarcinoma ; msi - h , microsatellite instability - high ; pdac , pancreatic ductal adenocarcinoma ; pmp , pseudomyxoma peritonei . comparison of genomic aberrations in appendix , colorectal , and pancreas cancers given the clinical practice of treating metastatic appendiceal cancers with crc regimens , we compared genomic alteration profiles of the appendiceal subtypes with those of 10 , 000 crcs profiled by the same laboratory ( table 2 )  . 
the high frequency of kras mutations observed in multiple appendiceal subtypes prompted inquiry into possible parallels with ( pdac ) , pancreatic ductal adenocarcinoma which harbors kras mutations in up to 95% of cases.16 sequencing of 2 , 800 pancreatic tumors revealed kras mutations in 87% of pdacs . 
there tended to be a higher frequency of microsatellite unstable or tumor mutational burdenhigh srccs and ads compared with mads ( table 2 ; appendix fig a1e )  . histopathologic and molecular features predictive of survival to determine the influence of histologic and molecular features on clinical outcomes , a retrospective review of a single - institution case series was performed . 
similar to the full 703 - patient cohort , the majority of cases were either ads ( n = 17 ) or mads ( n = 33 ) , with fewer low - grade appendiceal mucinous neoplasms that manifested as pmps ( n = 13 ) and few srccs ( n = 9 )  . 
data for chemotherapy treatment were available for 60 patients , showing that the majority were treated with a fluoropyrimidine and either oxaliplatin or irinotecan ( appendix ; table 3 )  . overall survival ( os ) , determined from time of initial diagnosis , was similar for ad and mad ( logrank p = .29 ; appendix fig a2a ) , so these groups were combined in subsequent analyses . 
 ( % ) unless otherwise noted . abbreviations : egfr , epidermal growth factor receptor ; hipec , heated intraperitoneal chemotherapy ; lamn , low - grade appendiceal mucinous neoplasms ; pd1 , programmed cell death protein 1 ; pd - l1 , programmed death ligand 1 ; pmp , pseudomyxoma peritonei ; sd , standard deviation ; vegf , vascular endothelial growth factor . for srccs ( p = .11 ; fig 2a )  . 
 however , use of irinotecan in any line of therapy was associated with a survival advantage in kras wild - type tumors ( log - rank p = .041 ; fig 2f ) but not in kras mutant tumors ( log - rank p = .32 ; appendix fig a2b )  . 
low - grade tumors were enriched for gnas mutations ( 72% v 18% for high grade ; 2 p < .001 ; appendix fig a3a ) , consistent with our prior report , 14 whereas highgrade tumors were enriched for tp53 mutations ( 56% v 6.9% for low grade ; 2 p < .001 ; appendix fig a3b )  . to assess the relative contributions of mutation and grade to the observed os differences , a cox proportional hazard analysis was performed including age , sex , kras , gnas , tp53 , and grade as covariates . 
however , the effect of tp53 mutation on survival was independent of grade , with the rare low - grade , tp53 - mutant tumors having os similar to other tp53 - mutant tumors ( median , 24.6 months ; fig 3b )  . 
at risk : irinotecan 8 no irinotecan 15 time ( months ) the mutational profile of 703 appendix neoplasms provides insight into the molecular aberrations that differentiate histologic subtypes and identifies putative prognostic and predictive biomarkers that may help guide treatment in this rare malignancy . 
compared with ad , mad , and pmp , kras and gnas mutations were much less frequent in gccs , whereas mutations in fat3 and arid1a were more frequent ( figs 1b and 1c )  . 
 these differences were also seen in the pathway analysis , in which gccs had less - frequent alterations of the ras / raf pathway relative to epithelial appendiceal cancers ( fig 1d )  . 
at risk : gnas mut 28 tp53 mut 25 neither 18 high - gnas mutant moderate - gnas mutant p = .011 low - gnas mutant time ( months ) no . 
these data also show that all of the appendiceal subtypes are molecularly distinct from crc , with more frequent gnas mutation and significantly lower prevalence of apc and tp53 mutations , which are key pathogenic alterations in crc . 
 the mutation profiles of ad and srcc bore the most resemblance to crc , with ad and crc sharing similar frequencies of mutation in kras , smad4 , and arid1a ; of note , appendiceal ads have been referred to as colonic - type adenocarcinoma , given clinical behavior more similar to crc.20 , 27 in this series , srcc had the worst prognosis of the epithelial appendiceal tumors and also has a clinical course similar to crc.28 the frequent comutation of kras and gnas in mad , ad , and pmp parallels the molecular profile of intraductal papillary mucinous neoplasms.14 , 29 notably , both intraductal papillary mucinous neoplasms and , in particular , gnas mutant appendiceal cancers are characterized by mucin production and a generally indolent clinical course . 
the differences in mutation profiles between the epithelial appendiceal tumors can be at least partially explained by grade , with significant association of gnas mutation with low - grade tumors and tp53 mutation with highgrade tumors ( appendix figs a3a and a3b )  . 
 consistent with this is the higher incidence of tp53 mutation and lower incidence of gnas mutation in srccs , which are by definition all high grade . histologic grade was also a strong predictor of survival ( fig 2b ) , consistent with prior reports.17 , 30 we did not observe a significant difference in os between ad and mad ; however , the distribution of tumor grade was similar between these two subtypes in our study . 
 conversely , tp53 mutation was an independent predictor of poor survival , with low - grade , tp53 - mutant tumors having survival similar to that of high - grade tumors . 
in addition , because appendiceal tumors are so rare , they are difficult to diagnose pathologically and are frequently overinterpreted by community pathologists.31 although gnas mutation is not an independent predictor of survival , because gnas and tp53 mutations occur mutually exclusive of each other and are associated with lowand high - grade tumors , respectively , the two genes can be substituted for grade to predict survival . 
the survival stratification achieved with the gnas - tp53 biomarker is similar to grade , an important observation , given that it is now much easier to obtain a mutation profile than an expert pathology review in the community oncology setting . the absence of a gnas mutation in the majority of high - grade tumors and the mutual exclusivity of tp53 and gnas mutations both strongly suggest that most high - grade appendiceal tumors occur de novo , rather than progressing from low - grade tumors , confirming , on a larger scale , our previous observations.14 however , there were a minority of tumors with both tp53 and gnas mutation ( n = 41 ; 6.7% ) , suggesting that transformation from low grade to high grade can occur . 
serial biopsy or serial measurement of circulating tumor dna would be needed to confirm that these tp53 mutations did in fact occur after the formation of a low - grade , gnas mutant tumor . 
however , given the low propensity for appendiceal tumors to spread beyond the abdominal cavity , there may be limited tumor dna in circulation , potentially making bloodbased tumor detection difficult . 
indeed , three of the four university of california , san diego , patients who underwent circulating tumor dna sequencing had no reportable alterations . regarding predictive biomarkers , kras wildtype status was associated with better survival in the subset of patients treated with irinotecan . 
a retrospective study in metastatic crc reported better response to irinotecan in patients with wild - type versus mutant plasma kras.32 however , a larger prospective study found that mutant kras was associated with poor survival but not with response to irinotecan in crc.33 regarding targeted therapies , there are unfortunately few clinically actionable mutations in appendiceal cancers , although the ras / raf signaling pathway is frequently altered in epithelial appendiceal cancers . 
data on therapeutic targeting of the ras / raf cascade in appendiceal tumors are limited , although a recent case report described clinical benefit in a patient with appendiceal mad harboring a gnas r201h mutation who was treated with trametinib.34 because only eight patients in this cohort received an anti - egfr antibody , we were unable to assess interactions between kras mutation status and response to these agents . a major limitation of this study is its retrospective design . 
with regard to the 703 - patient cohort , clinical information such as precise tnm stage was not available , but the fact that > 80% of specimens submitted came from metastases indicates that the majority of patients had stage iv disease . 
although specimens were independently reviewed by pathologists to confirm the diagnosis before undergoing sequencing , subtype definitions are potentially subject to variability and overlap , because there was no consensus classification system for appendiceal neoplasms and pmp until recently.2 for example , pmp is an inherently imprecise term used to describe the clinical syndrome of mucinous peritoneal dissemination from an appendiceal neoplas pmp encompasses a spectrum of both highand low - grade lesions but does not reference the histopathologic characteristics of the appendiceal primary from which it arises . 
 newer classification schemes separate pmps with low - grade and high - grade features ( also known as disseminated peritoneal adenomucinosis and peritoneal mucinous carcinomatosis , respectively35 ) and pmp with signet ring cells.2 because this study did not distinguish between these subtypes , we are unable to report on genomic differences associated with grade in the larger 70 - patient cohort . 
in addition , analysis of interactions between genotype and specific drugs are confounded by the fact that most patients received multiple lines of therapy . this study of unprecedented size in this rare disease highlights important molecular differences between different subtypes of appendix cancer and identifies gnas and tp53 mutation status as a prognostic biomarker . 
this comprehensive portrait of the molecular landscape of appendix cancer will help with the design of future clinical studies to develop and test therapeutic strategies specific to this disease . in conclusion , appendiceal neoplasms have molecular profiles that are distinct from crc and are characterized by frequent gnas and kras mutations , especially in low - grade tumors . 
ross employment : foundation medicine leadership : foundation medicine financial support : john paul shen , paul fanta , trey ideker administrative support : john paul shen , paul fanta stock and other ownership interests : foundation medicine provision of study material or patients : john paul shen , jeffrey s . 
miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center miriam t . 
ali employment : foundation medicine leadership : incyte stock and other ownership interests : exelixis , blueprint medicines , agios pharmaceuticalas , genocea biosciences patents , royalties , other intellectual property : patents via foundation medicine and seres health joel baumgartner research funding : merck andrew lowy consulting or advisory role : halozyme , merck research funding : mitsubishi tanabe pharma , syros pharmaceuticals expert testimony : merck ascopubs.org / journal / po jco precision oncology 9 justin k . 
 paul fanta no relationship to disclose trey ideker leadership : data4cure consulting or advisory role : ideaya biosciences , genetic modifiers newco , merck , la jolla institute for allergy and immunology research funding : pfizer travel , accommodations , expenses : sage bionetworks sherri z . 
chen , david xu , joel baumgartner , andrew lowy , paul fanta , trey ideker , and olivier harismendy , university of california , san diego , la jolla , ca ; jeffrey s . 
carr nj , cecil td , mohamed f , et al : a consensus for classification and pathologic reporting of pseudomyxoma peritonei and associated appendiceal neoplasia : the results of the peritoneal surface oncology group international ( psogi ) modified delphi process . 
sugarbaker ph , bijelic l , chang d , et al : neoadjuvant folfox chemotherapy in 34 consecutive patients with mucinous peritoneal carcinomatosis of appendiceal origj surg oncol 102 : 576581 , 2010 5 . 
farquharson al , pranesh n , witham g , et al : a phase ii study evaluating the use of concurrent mitomycin c and capecitabine in patients with advanced unresectable pseudomyxoma peritonei . 
garin l , corbinais s , boucher e , et al : adenocarcinoid of the appendix vermiformis : complete and persistent remission after chemotherapy ( folfox ) of a metastatic case . 
tejani ma , ter veer a , milne d , et al : systemic therapy for advanced appendiceal adenocarcinoma : an analysis from the nccn oncology outcomes database for colorectal cancer . 
johncilla m , stachler m , misdraji j , et al : mutational landscape of goblet cell carcinoids and adenocarcinoma ex goblet cell carcinoids of the appendix is distinct from typical carcinoids and colorectal adenocarcinomas . 
borazanci e , millis sz , kimbrough j , et al : potential actionable targets in appendiceal cancer detected by immunohistochemistry , fluorescent in situ hybridization , and mutational analysis . 
alakus h , babicky ml , ghosh p , et al : genome - wide mutational landscape of mucinous carcinomatosis peritonei of appendiceal orig genome med 6 : 43 , 2014 [ erratum : genome med 6 : 53 , 2014 ] 15 . 
reid md , basturk o , shaib wl , et al : adenocarcinoma ex - goblet cell carcinoid ( appendicealtype crypt cell adenocarcinoma ) is a morphologically distinct entity with highly aggressive behavior and frequent association with peritoneal / intra - abdominal dissemination : an analysis of 77 cases . 
singhi ad , davison jm , choudry ha , et al : gnas is frequently mutated in both low - grade and high - grade disseminated appendiceal mucinous neoplasms but does not affect survival . 
mccusker me , cot tr , clegg lx , et al : primary malignant neoplasms of the appendix : a population - based study from the surveillance , epidemiology and end - results program , 19731998 . 
asare ea , compton cc , hanna nn , et al : the impact of stage , grade , and mucinous histology on the efficacy of systemic chemotherapy in adenocarcinomas of the appendix : analysis of the national cancer data base . 
 appendix tumor tissue from 703 patients with appendiceal cancer was submitted to a clinical laboratory improvement amendments certified laboratory ( foundation medicine , cambridge , ma ) for dna sequencing and variant calling . 
a minimum of 50 ng of dna was extracted and a hybrid - capture method used to capture 3 , 769 exons from 315 cancer - related genes and 47 introns of 28 genes commonly rearranged in cancer ; this material was then sequenced to high ( average , 756x ) uniform coverage allowing for evaluation of genomic alterations , including base substitutions , indels , amplifications , copy number alterations , and fusions / rearrangements . 
clinical characteristics and outcomes , including tumor histology , grade , stage , overall survival ( os ) , and chemotherapy given were determined from review of the electronic medical record . 
sequencing failed quality control in two cases , and two patients with only blood - based cell - free dna sequencing were excluded , leaving 76 patients available for analysis . 
because there were only four goblet cell carcinoids , these were removed from survival analysis . for mutual exclusivity and comutation analysis , goblet cell carcinoid tumors and microsatellite instability - high tumors were removed , and a fishers exact test was performed for all gene combinations , followed by bonferroni multiple hypothesis correction . 
 lynch syndrome and breast cancer risk : weighing the data wendy kohlmann , ms , cgc1 as we continue to expand our understanding of the genotype - phenotype correlations in lynch syndrome ( ls ) , we must be cautious in the design of studies and interpretation of ndings to responsibly serve our patients with ls with as accurate information as possible . 
although pathogenic variants ( pvs ) in the mismatch repair ( mmr ) genes mlh1 , msh2 / epcam , msh6 , and pms2 are all associated with ls , each gene is associated with a unique risk prole . 
mlh1 and msh2 have been associated with the highest risk for ls - associated cancers , whereas risk for colorectal cancer is signicantly lower for msh6 and pms2 pv carriers.1 in studies of high - risk colorectal cancer cohorts , msh6 and pms2 pvs are less commonly identied than mlh1 and msh2 , but use of clinical multigene panel testing is expanding the identication of carriers of pvs in these genes , often in individuals and families lacking the classic pattern of cancers associated with ls.2 , 3 the large data sets being generated by genetic testing laboratories can be assets for research and can allow for gene - specic risk analysis . however , analytical caution needs to be taken to account for the biases that arise from patient selection for clinical genetic testing , and family history and clinical data provided to commercial laboratories may be incorrect or inaccurate . in the article accompanying this editorial , stoll et al2 add data from a large cohort of mmr carriers and demonstrate a successful strategy for using commercial laboratory data to assess associations between pvs and cancer risk . 
however , to account for the study population having a more signicant cancer history than the general population , pv carriers were also compared with other subpopulations within the laboratory cohort , including women who were negative for a pv in any cancer predisposition gene when testing was performed for evaluation for hereditary breast or ovarian cancer ( hboc ) and / or ls , evaluation for hboc only , and evaluation for ls only . no excess in breast cancer risk was noted when compared with seer or any laboratory comparison group for any of the mmr genes . in the cohort in the study by stoll et al , 2 pvs in msh6 and pms2 were more common in women undergoing genetic testing for the indication of hboc than in women being tested as a result of suspicion of ls . other studies of clinical testing populations have also found a predominance of msh6 and pms2 pvs in individuals meeting hboc guidelines rather than ls.4 however , this pattern does not conrm that these genetic variants increased the risk for breast cancer . this distribution of pvs in laboratory cohorts likely reects that , other than msh6 - associated endometrial cancer risk , pvs in msh6 and pms2 confer only a modest risk for colorectal and other lsassociated cancers . 
stoll et al2 have provided a systematic examination of a large laboratory cohort using the proper comparison groups to put the frequency of msh6 and pms2 pvs by testing criteria into context and have provided strong evidence against the association of breast cancer with any of the mmr genes . before widespread panel testing , most analyses of breast cancer risk and ls had come from small studies.5 data from studies with larger samples are now becoming available . 
signicant breast cancer history may be a result of other factors , and genetic testing of other affected family members may be warranted to determine whether there is an additional pv segregating in the family and contributing to that cancer risk . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . wendy kohlmann employment : biofire diagnostics ( i ) no other potential conicts of interest were reported . references dominguez - valentin m , sampson jr , sepp al a tt , et al : cancer risks by gene , age , and gender in 6350 carriers of pathogenic mismatch repair variants : findings from the prospective lynch syndrome database . 
cancer epidemiol biomarkers prev 26 : 404 - 412 , 2017 espenschied cr , laduca h , li s , et al : multigene panel testing provides a new prospective on lynch syndrome . 
mller p , sepp al a tt , bernstein i , et al : cancer risk and survival in path_mmr carriers by gene and gender up to 75 years of age : a report from the prospective lynch syndrome database . 
gut 67 : 1306 - 1316 , 2018 ten broeke sw , van der klift hm , tops cmj , et al : cancer risks for pms2 - associated lynch syndrome . 
j clin oncol 36 : 2961 - 2968 , 2018 slavin tp , maxwell kn , lilyquist j , et al : the contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk . 
npj breast cancer 3 : 22 , 2017 therkildsen c , ladelund s , smith - hansen l , et al : towards geneand gender - based risk estimates in lynch syndrome ; age - specic incidences for 13 extracolorectal cancer types . 
evans dg , woodward er , lalloo f , et al : are women with pathogenic variants in pms2 and msh6 really at high lifetime risk of breast cancer ? genet med 13 . 
2018 : is breast cancer truly caused by msh6 and pms2 variants or is it simply due to a high prevalence of these variants in the population ? genet med 21 : 256 - 257 , 2019 14 . 
in this analysis the hypothesis , we explore with an unsupervised learning algorithm whether distinct subtypes of btcp exist and whether they can provide new insights into clinical practice . methods partitioning around a k - medoids algorithm on a large data set of patients with btcp , previously collected by the italian oncologic pain survey group , was used to identify possible subgroups of btcp . 
a free online tool was created for new patients cluster computation to validate these clusters in future studies and provide handy indications for personalized btcp therapy . conclusion this work proposes a classication for btcp and identies subgroups of patients with unique efcacy of different pain medications . 
this work supports the theory that the optimal dose of btcp opioids depends on the dose of basal opioids and identies novel values that are possibly useful for future trials . 
these results will allow us to target btcp therapy on the basis of patient characteristics and to dene a precision medicine strategy also for supportive care . jco precis oncol 4 : 1339 - 1349 . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction breakthrough cancer pain ( btcp ) is a common event that affects a considerable proportion of patients with cancer.1 a variety of denitions for btcp have been proposed2 , 3 . 
according to the italian oncologic pain survey ( iops ) study group , 4 btcp should be dened as a relevant change in pain intensity of severe intensity in patients who receive an effective treatment with opioids.4 ( p963 ) nevertheless , despite this unique denition , btcp encloses a wide range of manifestations that differ , among other features , in intensity , duration , frequency , and triggering events . btcp represents a clinically relevant condition with a negative impact on the patients quality of life . 
patients characteristics characteristic value ( n = 4 , 016 ) 2 , 202 ( 54.8 ) male sex interview setting oncology pain therapy palliative care radiotherapy treatment corticosteroids anticonvulsant drugs acetaminophen constipation drugs anxiolytic drugs antidepressant drugs antiemetic drugs nsaids cannabis treatment setting inpatient outpatient domicile day hospital hospice cancer type lung breast pancreas colon prostate rectum stomach bladder at present , several gaps exist in the knowledge of btcp management . 
these partially unanswered questions , among others , concern the optimal drug administration route , pharmacokinetics , the balance between rapid - onset and slow - onset opioids , and the eventual difference of btcp response deriving from clinical features , such as stage , primary site , or metastases . in this analysis , it was thus hypothesized that the unique btcp denition might actually enclose diverse pathologic entities , possibly with peculiar clinical needs and specic responses to drugs . 
these so - called unsupervised learning algorithms have already been extensively used , for example , for the identication of breast cancer subtypes.6 nevertheless , to our knowledge , no authors have yet tried to explore the issue of btcp management using these techniques . to fulll this purpose , we used data that were collected by the iops group in a large , multicentric national study5 , 7 , 8 that enrolled 4 , 056 patients from 32 centers with opioidcontrolled basal pain suffering from btcp . 
this work is therefore a secondary analysis of the iops group survey that aims to identify novel subtypes of btcp through the use of unsupervised learning algorithms . methods patients enrollment and data collection details concerning the enrollment of patients are extensively described in the main paper from the iops group.5 in brief , local ethical committees approved the protocol , and written informed consent was obtained from each patient . interviews were performed in different settings , in particular , oncology , pain therapy , palliative care , and radiotherapy . 
a comprehensive list of clinical variables was collected for each patient that consisted of basal pain and btcp site , duration , frequency , intensity , relieving factors , triggers , drugs , primary cancer site and stage , and concomitant symptoms for a total of 1 , 086 1340 2020 by american society of clinical oncology multiple primary average age , years ( range ) karnofsky performance status ( range ) baseline pain , nrs scale ( range ) note . 
 breakthrough cancer pain clinical features and opioids response features with less than 5% of missing data and associated with a corrected p value of , .1 and , for categorical features , a log2 odds ratio greater than 1 were used to build a multivariable logistic regression . 
to investigate the correlationsimultaneously for all patients and on the same scalebetween the amount of opioids used and btcp therapy satisfaction , all doses of opioid drugs were converted10 to equivalent intravenous morphine doses and expressed as a total daily dose : one for btcp - directed opioids and for basal pain opioids . 
doses were converted to oral morto intravenous morphine doses and not phine doses because intravenous morphine has been increasingly used in different clinical settings and would provide more interpretable graphics in results . 
moreover , to explore the interaction of fast - acting and long - acting opioid dosages , we calculated for each patient the btcp opioidstobasal pain opioids ratio ( opr )  . 
a polynomial logistic regression was used to catch nonlinear relationships between opioid doses and therapy satisfaction . cluster computation and visualization features dening the clinical characteristics of btcp were selected to perform clusters computation . 
the above - mentioned features were used to calculate a dissimilarity matrix using a cluster package11 ; presence of background pain pain intensity 4 of 10 four or fewer episodes per day breakthrough pain presence of peaks well distinguished from background pain intensity cluster fig 1 . 
 ( a ) algorithm used for the diagnosis of breakthrough cancer pain ( btcp ) during patients enrollment in the italian oncologic pain survey ( modied from mercadante et al8 )  . 
 ( b ) a two - dimensional t - distributed stochastic neighbor embedding projection of all patients , colored by their clusters , on the basis of the following btcp features : number of btcp episodes , btcp peaks duration , btcp type , btcp intensity , number of days since the beginning of btcp episodes , eventual benet from pharmacotherapy , eventual benet from rest , and whether btcp was enhanced by movements . each point represents a patient . 
silhouette statistics14 were calculated for each run , and the optimal number of clusters was manually picked as being that with the best trade - off between silhouette statistics and reasonable clinical interpretation . 
we used complete - linkage hierarchical clustering with 12 clusters , along with rand index calculation , to explore the replicability of clusters with a different algorithm . t - distributed stochastic neighbor embedding15 algorithm was used to project dissimilarities between patients in a bidimensional space , with closer points representing patients with more similar clinical btcp features . 
an online tool allows for the repetition of the performed classication on new sets of patients.16 clusters analysis t - test , mann - whitney test , and 2 test were used to assess the association of parametric , nonparametric , and categorical features , respectively , with each cluster . 
patients characteristics are listed in table 1 . cluster computation to investigate whether subtypes of btcp exist , we used btcp features to build an unsupervised clustering model . the number of btcp episodes , btcp peaks duration , btcp type , btcp intensity , number of days since the beginning of btcp episodes , the eventual benet from pharmacotherapy , the eventual benet from rest , and whether btcp was enhanced by movements were the eight btcp - dening variables selected for the nal model , which was built with the k - medoids algorithwe chose 12 as an optimal trade - off between the average width of clusters silhouette ( 0.45 ; appendix fig a1a ) and the interpretability of the clusters themselves . 
 pantano et al figure 1 shows the algorithm used to dene btp ( fig 1a ) t - distributed stochastic neighbor and a 2 - dimensional embedding projection of all patients , colored by their clusters ( fig 1b )  . 
an online tool is available for the classication of new patients according to our method.16 characteristics of btcp clusters we analyzed the enrichment of the eight btcp - dening variables and of other clinical features among the clusters . a description of each cluster is available in table 2 . 
a summary of btcp features according to cluster is presented in figure 2 . btcp therapy satisfaction finally , we tried to assess what inuenced patient - reported btcp therapy satisfaction . 
after converting the opioid dose to a unique scalecorresponding to the equivalent dose of intravenous morphinewe investigated using a nonlinear model the inuence of basal opioid dose , btcp opioid dose , and opr on patient - reported therapy satisfaction . 
of higher therapy satisfaction lower therapy satisfaction btcp opioids dose / basal pain opioids dose higher therapy satisfaction higher therapy satisfaction lower therapy satisfaction lower therapy satisfaction btcp opioids dose ( mg ) basal pain opioids dose ( mg ) fig 3 . 
this roughly corresponds to a daily dose of sublingual fentanyl 100 g for btcp and a daily dose of oral morphine 30 mg as the basal opioid dose . we separately performed the same analyses on previously dened clusters ( fig 3d )  . 
for clusters 1 , 6 , and 10 , the satisfaction seemed to grow indenitely with an increase of the opr opioids , whereas clusters 7 , 8 , and 11 seemed to have clear , optimal peaks of opr . 
despite the interpretation being challenged by some large cis , we can say from these data that , depending on the cluster , optimal opr ranges from 15% to 50% . discussion this paper demonstrates a novel approach for the investigation of btcp . 
we acknowledge that this study was not designed to perform this analysis and , moreover , that the large number of clusters might interfere with their interpretability and clinical utility . 
nevertheless , this study represents a proof - ofconcept for this investigational approach . cluster 1 cluster 2 cluster 3 higher therapy satisfaction higher therapy satisfaction higher therapy satisfaction lower therapy satisfaction lower therapy satisfaction lower therapy satisfaction cluster 4 cluster 5 cluster 6 higher therapy satisfaction higher therapy satisfaction higher therapy satisfaction lower therapy satisfaction lower therapy satisfaction lower therapy satisfaction cluster 7 cluster 8 cluster 9 higher therapy satisfaction higher therapy satisfaction higher therapy satisfaction lower therapy satisfaction lower therapy satisfaction lower therapy satisfaction cluster 10 cluster 11 cluster 12 higher therapy satisfaction higher therapy satisfaction higher therapy satisfaction lower therapy satisfaction lower therapy satisfaction lower therapy satisfaction fig 3 . 
using our same data , another group proposed 0.20 ( one fth ) as the optimal ratio.5 nevertheless , they used a frequentistic approach , as 0.20 is simply the most common ratio among the cohort . 
this possibly suggests that , instead of starting btcp opioid titration with the lowest possible dosage , as proposed previously , 28 titration could start immediately with an optimal opioid dosage . moreover , cluster analysis reveals that this ratio might not be the same for all patients : some patients might benet from a higher btcp opioid dosage ( cluster 2 and 7 ) , whereas others might benet from a lower one ( cluster 11 )  . 
finally , for some patients , we did not observe an upper threshold for this ratio ( cluster 1 , 6 , and 9 ) , perhaps pointing out patients for whom a strong btcp opioid dosage increase is required . the interpretation is made difcult by the multiple associations and , at this stage , is not mature enough to suggest any immediate change in clinical practice . 
however , we made available an online free tool16 that allows for the classication of new patients according to our algorithm and returns a proposed btcp therapy which depends on the patient cluster optimal opr and basal opioid dose . 
we suggest that this tool might be used in the future to prospectively validate the clinical importance of our clusters in clinical practice and to compare our proposed opioid dosages in settings different from ours . the presence of distinct btcp phenotypes , each one associated with specic clinical features , could also be a reection of diverse underlying pathophysiologic mechanisms : our work suggests that preclinical research might gain insight into these possible differences and help the development of a tailored therapy also for btcp . the main limitation of our study is the appropriateness of collected data for the scope of our work . 
we believe that a prospective study specically designed for the investigation of btcp clusterspossibly with long - term follow - up and therapy success outcomes and not limited to a single timepoint evaluationmight enable a clearer identication of distinct clusters . 
businco hospital , asl 8 , cagliari , italy 20department of biotechnological and applied clinical sciences , section of clinical epidemiology and environmental medicine , university of laquila , laquila , italy 21section of clinical pharmacology and oncology , department of health sciences , university of florence , florence , italy 22department of medicine and surgery sciences , university of bologna , bologna , italy 23paintherapy , s . 
croce e carle hospital , cuneo , italy 24pain therapy ics maugeri , irccs foundation , pavia , italy 25medical oncology , azienda sanitaria universitaria integrata di trieste , trieste , italy 26medical oncology , azienda sanitaria universitaria integrata di udine , udine , italy 27palliative care and oncologic pain service , s . 
pain 41 : 273 - 281 , 1990 portenoy rk , payne d , jacobsen p : breakthrough pain : characteristics and impact in patients with cancer papain 81 : 129 - 134 , 1999 4 . 
mercadante s , marchetti p , cuomo a , et al : breakthrough pain and its treatment : critical review and recommendations of iops ( italian oncologic pain survey ) expert group . 
mercadante s , marchetti p , cuomo a , et al : breakthrough cancer pain : preliminary data of the italian oncologic pain multisetting multicentric survey ( iopsms )  . 
mercadante s , lazzari m , reale c , et al : italian oncological pain survey ( iops ) : a multicentre italian study of breakthrough pain performed in different settings . benjamini y , hochberg y : controlling the false discovery rate : a practical and powerful approach to multiple testing . 
davies an , dickman a , reid c , et al : the management of cancer - related breakthrough pain : recommendations of a task group of the science committee of the association for palliative medicine of great britain and ireland . 
the appropriate number of clusters for additional analyses was found to be 12 , an optimal trade - off between the average width of clusters silhouette ( 0.45 ) and the interpretability of clusters themselves . ( b ) heatmap showing the concordance of pam clustering with complete - linkage hierarchical clustering . 
 circulating tumor dna predicts the response and prognosis in patients with early breast cancer receiving neoadjuvant chemotherapy shunying li , phd1 , 2 ; hongna lai , phd1 , 2 ; jieqiong liu , md1 , 2 ; yujie liu , phd1 , 2 ; liang jin , md1 , 2 ; yudong li , phd1 , 2 ; fengtao liu , md1 , 2 ; yuhua gong , ms3 ; yanfang guan , phd3 ; xin yi , phd3 ; qianfeng shi , md1 , 2 ; zijie cai , md1 , 2 ; qian li , md1 , 2 ; ying li , md1 ; mengdi zhu , md1 , 2 ; jingru wang , md1 , 2 ; yaping yang , md1 , 2 ; wei wei , md4 ; dong yin , phd1 , 2 ; erwei song , md , phd1 , 2 ; and qiang liu , md , phd1 , 2 purpose many patients with breast cancer still relapse after curative treatment . 
serial plasma samples before , during , and after nac and paired tumor biopsies were harvested and subjected to deep targeted sequencing using a large next - generation sequencing panel that covers 1 , 021 cancerrelated genes . results positive baseline ctdna was detected in 21 of 44 patients before nac . 
more importantly , positive baseline ctdna is signicantly associated with worse disease - free survival ( p = .011 ) and overall survival ( p = .004 ) in patients with early breast cancer , especially in estrogen receptornegative patients . conclusion our study demonstrated that ctdna can be used to predict tumor response to nac and prognosis in early breast cancer , providing information to tailor an individuals therapeutic regimen . jco precis oncol 4 : 244 - 257 . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women worldwide.1 although early breast cancer is a curable disease , up to 40% of such patients still relapse after surgery.2 it is believed that breast cancer is a systemic disease and that clinically undetectable micrometastases often happened before the diagnosis.3 , 4 how to distinguish patients with early breast cancer with high relapse risk from those with low risk remains a critical and challenging clinical question . neoadjuvant chemotherapy ( nac ) is often used in patients with locally advanced or triple - negative / her2positive early breast cancer before surgery to decrease tumor size and enable breast - conserving surgery . 
the current consensus to monitor response to nac is clinical examination backed up by radiologic and sonographic measures.5 however , these measures are quite subjective and have poor interand intraobserver reproducibility.6 moreover , given the heterogeneity of cancer , a reliable method to examine the overall response from local , regional , and micrometastatic lesions in patients with early breast cancer is still lacking . circulating tumor dnas ( ctdnas ) are mutated gene fragments that are exclusively shed by cancer cells into blood , 7 which can be detected by digital polymerase chain reaction ( dpcr ) 8 or sequencing . 
 ctdna predicts prognosis in breast cancer context key objective can the presence and dynamic change of circulating tumor dna ( ctdna ) predict the tumor response and prognosis in patients with breast cancer receiving neoadjuvant chemotherapy ( nac ) ? knowledge generated using a next - generation sequencing panel of 1 , 021 genes , positive baseline ctdna was identied in 21 of 44 patients with early breast cancer before nac . 
positive ctdna before nac was associated with signicantly worse disease - free survival and overall survival , especially in estrogen receptor ( er ) negative patients . relevance our study demonstrates that ctdna has signicant value to complement imaging to monitor tumor response and predict prognosis in patients with early breast cancer , providing information to tailor an individuals therapeutic regimen . 
the high relapse rate in er - negative patients with positive ctdna indicates that it may be necessary to escalate treatment in these high - risk patients . patients with early breast cancer after surgery , ctdna by dpcr predicted metastasis at 7.9 - 11 months earlier than clinical relapse.11 , 12 a few studies also explored the role of ctdna in patients with breast cancer who received nac . 
it was reported that ctdna change during nac was correlated with tumor response13 - 15 or relapse , 16 , 17 although two of them only used dpcr to detect a single gene ( metrassf1a14 or tp5316 ) , and the other study only correlated the ctdna disappearance after the rst cycle of nac with two patients who achieved pathologic complete response.13 advances in next - generation sequencing ( ngs ) 18 technology have enabled the rapid identication and broad coverage of tumor - specic genomic alterations in cell - free dna ( cfdna ) of individual patients.19 - 21 the ngs assay provides an opportunity to screen more genes at a single time point and avoid missing mutations absent in biopsy because of intratumor heterogenicity . 
for patient cohort , sample collection and processing , and ngs sequencing and data analysis , please see the data supplement . statistical analysis the primary end point of this study was to evaluate the clinical value of the presence and dynamic change of ctdna to predict the tumor response and prognosis in patients with breast cancer treated with neoadjuvant chemotherapy . 
all statistical analyses were performed with graphpad prism version 6.0 or r version 3.4.1. results clinicopathological features of patients a total of 52 patients with early breast cancer who received nac were prospectively enrolled in the study since 2013 . eight patients were excluded from further study because of insufcient cfdna in plasma before nac ( fig 1 )  . the clinicopathological features of the 44 patients in the study are listed in table 1 and the data supplement . 
after surgery , the patients were followed up every 6 months . during the follow - up ( median , 46 months ; range , 11 - 68 months ) , 11 patients ( 25% ) had distant metastasis , and 9 of them died of metastatic breast cancer . baseline mutated dnas in plasma and tumor all baseline plasma samples , matched blood cells , and 32 paired tumor biopsy samples underwent parallel targeted ngs to screen for point mutations and structural variants , using the ngs panel of 1 , 021 cancer - related genes that covers a region of 1.1 mb ( data supplement ) .24 after deduplication , the median depths of unique coverage were 984 for primary tumor and 1 , 225 for plasma dna . 
the analysts were blinded to clinical and follow - up information during the analysis of sequencing data . among the 44 patients , 21 patients were found to have positive baseline ctdna . 
a total of 72 mutations in 46 genes were found in these 21 samples , with 1 - 8 ( median , 3 ) mutations per sample ( data supplement )  . 
tp53 ( 20 / 32 ) , pi3kca ( 13 / 32 ) , nf1 ( 5 / 32 ) , and gata3 ( 4 / 32 ) were the most frequently mutated genes in tumor ( fig 2c )  . in the 32 patients with both tumor and plasma sequencing data , 14 had concordant / partially concordant mutations in plasma and tumor , 16 patients had mutations only in tumor with negative ctdna , and the other two patients ( p034 and p036 ) had completely different mutations in tumor and plasma ( fig 2b )  . 
in the 16 patients with positive ctdna , 14 ( 87.5% ) of them had one or more mutations conrmed independently in tumor sequencing data , indicating that most positive ctdna represents tumor - specic mutations in the primary tumors . 
among the 33 concordant mutations found in both tumor and plasma of the 14 patients , tp53 ( 12 / 14 ) and pik3ca ( 4 / 14 ) mutations were the most frequent ( fig 2d )  . changes of ctdna and tumor response during nac the current gold standard to monitor tumor response during nac is ultrasound or magnetic resonance imaging , although it is subjective and sometimes falls behind the histologic changes . 
to explore whether ctdna surpasses imaging in monitoring or predicting overall tumor response , all plasma samples collected during nac in the 20 patients ( 1 patient was excluded because of insufcient cfdna in subsequent plasma samples ) with positive baseline ctdna were sequenced and compared with baseline data to track the dynamic changes of ctdna during nac ( data supplement )  . in this study , tumor response was dened as cr / pr / sd / pd using recist1.1 criteria when the imaging data of local / regional tumor before surgery was compared with that before nac . 
of the 20 assessable patients , 11 did not respond to nac ( including a patient with pd ) , and 9 responded to nac ( response rate , 45% )  . however , patient 038 ( p038 ) , who was dened as pr according to imaging , had an increasing ctdna amount ( clonal variant allele frequency [ vaf ] ) during all cycles of nac , 25 and a persisting high ctdna even after surgery , although she did not have any clinical metastasis before surgery . 
follow - up showed that she had multiple distant metastases ( liver , lung , and bone ) at 21 months and died at 46 months after surgery ( appendix fig a2 )  . 
the increase of ctdna in this case indicated the early micrometastases undetected by imaging did not respond to nac well , although the primary tumor did , suggesting that ctdna is better than imaging of local tumor to monitor the overall response to nac . next we did a longitudinal analysis of ctdna changes during nac and correlated it with local tumor response . p038 was excluded from this analysis because of discrepant tumor and ctdna data . 
the ctdna amount ( clonal vaf ) in baseline plasma was set as 100% , and those in subsequent plasma from the same patient were normalized it was found that patients who to the baseline value . responded to nac had more decreasing ctdna than patients who did not respond to nac ( fig 3a )  . 
the difference was signicant after the second cycle ( p = .03 ) and all cycles of nac ( p = .02 ) but was not statistically signicant after the rst cycle of nac ( p = .290 ; figs 3b - 3d )  . next , we constructed roc22 curves to compare the efcacy of ctdna amount ( clonal vaf ) and ultrasound to predict the response to nac ( fig 4 )  . 
it was found that clonal vaf after 2 cycles , clonal vaf before surgery , and ultrasound after 2 cycles were all predictive of the response to nac before surgery . 
the optimal criteria of clonal vaf in predicting the response to nac , as determined by the roc plots , are 40% drop in clonal vaf after 2 cycles and 60% drop in clonal vaf before surgery , respectively . 
the optimal criterion of ultrasound after 2 cycles was 20% decrease in the longest diameter of tumor in our study . concordant mutations between tumor and plasma were also identied in two patients with smaller tumor and negative lymph nodes ( t2n0m0 )  . 
the er - negative patients with negative baseline ctdna had 100% dfs and os , whereas those with positive baseline ctdna had a dfs / os of only approximately 36% . among the 20 patients with positive baseline ctdna ( one patient ruled out due to lack of subsequent samples ) , 6 of them turned negative and 14 remained positive for the ctdna before surgery . 
six out of 8 ( 75% ) er - negative patients with persistent positive ctdna relapsed , whereas only 1 out of 3 ( 33% ) such patients with negative ctdna before surgery relapsed , although the difference was not statistically signicant because of small sample size ( appendix figure a4 )  . discussion in this study , we used targeted deep sequencing of cfdna with a panel of 1 , 021 cancer - related genes to screen for mutated ctdna in patients with early breast cancer and found that 21 out of 44 patients had positive ctdna . 
more importantly , positive ctdna before nac is signicantly associated with worse prognosis in patients with early breast cancer , with the signicance mainly derived from er - negative patients . many patients with early breast cancer still relapse after surgery because of clinically undetectable micrometastases . 
current imaging methods are not sensitive enough to detect micrometastatic lesions ; even the most sensitive positron emission tomographycomputed tomography has the resolution limit at 4 mm.26 recently , ctdna has been suggested to be an ideal tumor biomarker because of its specicity , stability , and sensitivity . 
it is shown to be better than traditional biomarkers and imaging in predicting the relapse or progression in both early and metastatic cancer.9 , 11 , 12 , 27 - 29 however , the best method to measure ctdna is uncertadpcr is cheaper and easier , but it only measures one or few known mutations . 
to our knowledge , this study used the largest ngs panel so far to look for ctdna in patients with early breast cancer . using the large 1 , 021 - gene panel , somatic mutations were found in all the tumor biopsies . 
more importantly , positive ctdna was found in 47% of patients with early breast cancer and 73% of er - negative patients , which was consistent with previous reports.12 , 16 , 17 , 30 , 31 the majority of the ctdna was conrmed in tumor mutation data , suggesting that ctdna can be used to detect tumor - specic fig 2 . 
 ( c , d ) clonal vaf in no - response patients was signicantly higher than that in response patient after ( c ) 2 , and ( d ) all cycles of nac . 
in the 21 patients with positive baseline ctdna , 16 patients had paired plasma and tumor biopsy samples for ngs analysis , and 14 / 16 had at least one mutation conrmed in primary tumors . 
with the high specicity , at 98.72% , the discrepancy between the mutations in plasma and the mutations in tumor may be explained by the small tumor biopsy specimen not representing the whole primary tumor or micrometastasis . 
but this study also identied 24% ( 5 / 21 ) of patients with positive ctdna who had mutations other than tp53 and pi3kca . interestingly , our data showed that dynamic change of ctdna can be used to monitor and predict the response to nac in early breast cancer . 
kaplan - meier curve compares the disease - free survival ( dfs ) and overall survival ( os ) in patients with breast cancer with positive or negative baseline circulating tumor dna ( ctdna )  . 
nbc , negative baseline ctdna ; pbc , positive baseline ctdna . the change of ctdna level after 2 cycles of nac predicted local tumor response to nac better than ultrasound , although this was not statistically signicant . 
interestingly , none of the four er - negative patients with negative baseline ctdna relapsed during the follow - up , which was strikingly different from the high relapse rate in er - negative patients with positive baseline ctdna . 
other limitations of this study include limited sample size and suboptimal nac cycles , which hinder further subgroup analysis . in conclusion , our study demonstrates the feasibility and validity of ctdna in patients with early breast cancer receiving nac . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . yuhua gong employment : geneplus - beijing yanfang guan employment : geneplus - beijing xin yi employment : geneplus - beijing qiang liu consulting or advisory role : novartis , astrazeneca speakers bureau : pzer , roche , novartis , astrazeneca , eisai no other potential conicts of interest were reported . references bray f , ferlay j , soerjomataram i , et al : global cancer statistics 2018 : globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries . 
ca cancer j clin 68 : 394 - 424 , 2018 early breast cancer trialists collaborative group ( ebctcg ) , davies c , godwin j , et al : relevance of breast cancer hormone receptors and other factors to the efcacy of adjuvant tamoxifen : patient - level meta - analysis of randomised trials . 
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schiavon g , hrebien s , garcia - murillas i , et al : analysis of esr1 mutation in circulating tumor dna demonstrates evolution during therapy for metastatic breast 11 . 
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robin x , turck n , hainard a , et al : proc : an open - source package for r and s + to analyze and compare roc curves . 
roth e f , silva mj , venet d , et al : circulating tumor dna in her2 - amplied breast cancer : a translational research substudy of the neoaltto phase iii trial . 
 li et al et * 4 2 weeks after surgery no liver metastasis after surgery liver metastasis after 1 cycle b aselin e after 2 cycles b efore surg ery after 3 cycles after surg ery cycles 24 months fig a2 . 
et * 4 , 4 cycles of epirubicin and docetaxel . p031 ( t2n0m0 ) p041 ( t2n0m0 ) tuba3c tp53 raf1 pik3ca notch2 tsc2 tp53 gab2 dnmt3a brd3 fig a3 . 
little is known about the experiences of individuals tested for cdh1 variants in the multigene panel testing era . methods participants recruited from the prospective registry of multiplex testing completed a cross - sectional self - report survey regarding cdh1 genetic testing experiences , medical management , and psychosocial adaptation . results discordance existed in interpretations of cdh1 results ; 13.3% of cases had disagreements in variant classications among commercial laboratories , and 21.4% had disagreements between participant self - report and clinvar classication . 
2019 by american society of clinical oncology introduction hereditary diffuse gastric cancer syndrome ( hdgc ) , attributed to germline cdh1 pathogenic variants ( pv ) , is associated with increased risk for diffuse gastric cancer ( dgc ) and invasive lobular breast cancer ( lbc )  . 
germline cdh1 pv are present in approximately 40% of hdgc families , 1 and 1% to 3% individuals with gastric cancer have a cdh1 pv.2 reported cumulative lifetime dgc risk ranges from 50% to 80%.1 , 3 - 5 prophylactic gastrectomy is a standard management recommendation for individuals with cdh1 pv , given the inability of endoscopic surveillance to reliably identify dgc.5 because of cumulative lifetime lbc risk estimates ranging from 39% to 52% , recommendations for those with cdh1 pv include breast cancer screening via annual mammography , with consideration of breast magnetic resonance imaging beginning at age 30 years ; risk - reducing mastectomy decisions may occur dependent on family history.3 , 6 before the advent of multigene panel testing ( mgpt ) for hereditary cancer susceptibility , candidates for cdh1 genetic testing were evaluated on criteria dened by the international gastric cancer linkage including a conrmed case of dgc consortium , younger than age 40 years , at least two cases of dgc at any age , or the presence of both dgc and lbc younger than age 50 years.5 , 7 thus , individuals undergoing cdh1 gene analysis had a signicant perfamily history of hdgc - associated sonal and / or cancers . 
 hamilton et al context key objective germline cdh1 variants that are associated with hereditary diffuse gastric cancer syndrome ( hdgc ) are rare , but growth in multigene panel testing is leading to patients with limited personal exposure to hdgc being confronted with such results . 
we addressed the question of what are the genetic testing experiences , medical management , and psychosocial adaptation of patients with cdh1 variants identied through multigene panel testing ? knowledge generated discordant interpretations of cdh1 results were observed across testing laboratories , as well as among participants reporting of their results . 
participants with pathogenic cdh1 variants were more likely than those with variants of uncertain signicance to receive recommendations for prophylactic gastrectomy , yet both groups perceived similar , moderate levels of risk for gastric cancer . relevance findings highlight the unique informational and psychosocial needs of patients living with cdh1 variants , suggesting that they may benet from support tailored to the challenges associated with their particular risk status . genetic testing services are increasingly offered outside the cancer genetics specialty , such as via primary care providers , surgeons , and direct - to - consumer companies.10 - 12 given the variability by which preand post - test genetic counseling and testing for cdh1 are currently provided , we sought to characterize the genetic testing experiences , medical management , and psychosocial adaptation of individuals living with cdh1 variants . methods participants participants were recruited from the prospective registry of multiplex testing ( prompt ) , an online genetic registry founded by investigators at memorial sloan kettering , the university of pennsylvania , dana - farber cancer institute , and mayo clinic for individuals age 18 years or older tested for cancer susceptibility genes as part of any commercial mgpt . 
on self - selected enrollment in prompt , individuals consent to participate in the registry ; complete a baseline questionnaire of personal / family history , genetic testing results , and demographic information ; and share their testing report with prompt staff . 
a centralized institutional review board ( chesapeake irb ) approved this research . procedure study data were collected using redcap software13 through an online cross - sectional survey e - mailed to prompt participants with a self - reported cdh1 variant . invitations including a link to the survey ( with two reminders for nonresponders ) were e - mailed to 135 eligible individuals from september to december , 2017 . 
participants reported their understanding of their cdh1 result ( response options : positive genetic test result , likely positive genetic test result , variant of uncertain signicance , negative genetic test result , my test results are not clear to me ) and the year when they learned their result . 
we assessed personal ( yes , no ) and family history ( yes , no , uncertain ) of breast and gastric cancer , and details ( eg , surgery date ) regarding personal cancer treatment . 
recommendation by a health care provider for risk - reducing mastectomy , meeting with a gastroenterologist , and prophylactic gastrectomy , as well as adoption of breast magnetic resonance imaging , risk - reducing mastectomy , meeting with a gastroenterologist , upper endoscopy , and prophylactic gastrectomy , were assessed ( yes , no , unsure )  . 
genetic knowledge was assessed with 21 items regarding basic genetic concepts and aspects of mgpt selected or modied from the university of north carolina genomic knowledge scale.15 cdh1 knowledge was assessed with six investigator - designed items regarding cancer risks and inheritance of cdh1 variants . item response options were true , false , and don ' t know . 
items adapted from the national cancer institute health information national trends survey assessed absolute perceived cancer risks ( how likely are you to get [ breast / gastric ] cancer in your lifetime ? ; 1 = very unlikely to 5 = very likely ) and affective perceived cancer risks ( i feel like i could easily get [ breast / gastric ] cancer in my lifetime ; 1 = i feel very strongly that this will not happen to 5 = i feel very strongly that this will happen ) .16 decision - making experiences . 
ten items selected or modied from quality of a published measure21 assessed positive ( example : my cdh1 test result allows me to take control of my health ; = 0.75 ) and negative ( example : because of my cdh1 test result , i feel awed and stigmatized ; = 0.79 ) effects of cdh1 genetic testing on quality of life . 
three investigator - designed items assessed whether patients shared their cdh1 results with family , why this information was not shared with family , and family interest in genetic testing . statistical analysis seventy - one individuals responded to the survey ( 53% response rate ; appendix figure a1 ) ; however , three respondents did not have a cdh1 result and were excluded from all analyses . 
conicting interpretation was dened as either disagreement among one or more commercial laboratories in variant classications ( evaluable participants : n = 60 ) , or disagreement between participant self - report and clinvar classication ( evaluable participants : n = 56 )  . 
 medical and psychosocial experiences after cdh1 variant detection second , to determine how individuals understanding of their cdh1 results were associated with their medical and psychosocial experiences , we evaluated the survey data of the 60 respondents who self - reported either a pv ( ie , positive genetic test result or likely positive genetic test result ) or variant of uncertain signicance ( vus )  . 
data were examined for missing values ; patients missing responses to more than 20% of variables were excluded ( n = 3 ) , and average values on the basis of existing items were computed for scales missing less than or equal to 20% of items . 
of concern , three of these cases ( 25% of cases with participant / clinvar disagreement ; 5.4% of evaluable sample ) involved participant self - report of pv ( ie , positive genetic test result or likely positive genetic test result ) but clinvar and test report classication of vus . 
dashes indicate that a p value was not computed because of inadequate cell size . ||individuals with a complete stomach at the time of survey completion only . abbreviations : pv , pathogenic variant ; vus , variant of uncertain signicance . * responses of uncertain or unsure were combined with responses of no for analyses . females with at least one healthy breast at the time of learning their cdh1 results only . females with at least one healthy breast at the time of survey completion only . individuals with a complete stomach at the time of learning their cdh1 results only . least one healthy breast at the time of learning their cdh1 results , a minority ( 25.0% ) reported that a health recommended risk - reducing care provider had ever mastectomy ; no differences in recommendation receipt were observed based on cdh1 result . 
experiences with genetic testing among participants with cdh1 variants experience pv ( n = 16 ) vus ( n = 41 ) total ( n = 57 ) ever offered cancer genetic counseling yes , pros and cons discussed but i didn ' t 0 ( 0 ) unsure ever received cancer genetic counseling was given options to have different numbers of genes tested for hereditary cancer don ' t know was sufciently informed about pros and cons of having different numbers of genes tested for hereditary cancer yes , given sufcient information yes , pros and cons discussed but more information would have been helpful understand no , pros and cons not discussed not sure i felt sufciently informed would have preferred more time to decide about how many genes to have tested don ' t know was informed about possibility of identifying vus when deciding whether to undergo testing yes , i was informed and i understood yes , i was informed but it was not clear what that meant informed after i received results no , i was never informed not sure i was informed would have preferred more time to discuss options with family , friends , or health care providers before having genetic testing don ' t know no . 
approximately 39% of the sample recalled being given options to have different numbers of genes tested for hereditary cancer ; of these they received sufcient participants , most ( 68.2% ) felt information about the pros and cons of having different numbers of genes tested . 
specically , those with pv and vus both reported mean scores for the measures of perceived gastric cancer risks consistent with scale midpoints ( ie , absolute perceived gastric cancer risk : neither unlikely nor likely ; affective perceived gastric cancer risk : i feel i am just as likely to get cancer as i am to not get cancer )  . 
regarding emotional outcomes , participants with pv reported greater distress ( p .001 ) and were more likely to be worried about future discrimination on the basis of their cdh1 result ( p = .05 ) than those with vus . participants reported low decisional regret ( mean , 13.13 , 0 to 100 scale ) regarding cdh1 testing , irrespective of their result . 
consistent with a separate analysis of laboratory interpretations of variants identied by hereditary cancer mgpt , 27 discrepancies were not 13.3% of cases , laboratories differed in their interpretation of cdh1 variant pathogenicity . 
dashes indicate that a p value was not computed because of inadequate cell size . abbreviations : pv , pathogenic variant ; vus , variant of uncertain signicance . * missing response ( n = 1 )  . asked only among those who had not shared cdh1 results with all family ( n = 19 ) and participants could select more than one response . free - text responses included i ' m adopted , so my results don ' t apply to them , and most family members would not nd meaningful . asked only among those who shared with ( some ) family , and responses of yes . 
future research should examine how and why such misunderstandings arise and their implications for patients and families . time , differential provider the survey data indicated that , consistent with clinical guidelines , 5 only participants with a pv reported ever receiving a recommendation to undergo prophylactic gastrectomy . 
however , fewer than half of these individuals recalled receiving this recommendation , and receipt of this recommendation was not associated with clinical factors relevant to the timing of this surgical intervention , such as family gastric cancer history or patient age . 
it is unclear why certain individuals with cdh1 pv have been advised to pursue prophylactic gastrectomy and others have not . although there is speculation that the lifetime risk of gastric cancer may be lower in individuals with cdh1 pv and no family history of gastric cancer ( compared with those with a family history ) , at the current time a discussion of gastrectomy remains standard of care.26 , 28 their despite growing access to genetic testing through nongenetics health care providers , most participants were offered and received genetic counseling . 
 medical and psychosocial experiences after cdh1 variant detection patients understand the risks and benets and feel prepared to make an informed decision about mgpt . results also demonstrate that individuals with cdh1 pv and vus each have unique informational and emotional needs . 
those with vus were also less satised with their health care providers knowledge about the result ; however , it is not possible to discern whether this reects a lack of provider familiarity or existing limitations in scientic knowledge about risks and appropriate management of these variants . 
furthermore , although those with pv accurately perceived themselves to be at greater risk for breast cancer than did those with vus , these individuals had similar perceived risks for gastric cancer , which on average reected scale midpoints . 
risk perceptions are important for promoting cancer prevention behaviors , 30 , 31 including management of hdgc8 ; research is needed to understand why some with cdh1 pv seem to hold inaccurate risk perceptions . 
participants with pv did experience greater distress ; however , levels of distress were modest in this sample , consistent with a recent analysis of patient experiences with mgpt for hereditary breast and ovarian cancer.14 this study has several limitations . 
consequently , these ndings may not be generalizable to the individuals with cdh1 variants . broader population of furthermore , analyses used self - reported data regarding individuals medical and genetic testing experiences . 
nonetheless , this research extends beyond past work , which has largely focused on cdh1 mutation carriers prophylactic surgical decision making and adjustment , 8 , 32 - 34 to provide novel insight into the experiences of individuals with cdh1 variants in the era of mgpt . in conclusion , although many with cdh1 pv or vus are satised with their decision to pursue genetic testing , these individuals have unique informational and psychosocial needs . 
low levels of reported recommendations for prophylactic gastrectomy and perceived gastric cancer risks among those with cdh1 pv are particularly worrisome , suggesting a need for strategies ( eg , innovative applications of risk communication principles , 35 - 37 enhanced provider genetics education38 ) to more effectively engage and educate patients and providers about relevant medical management and cancer risks . 
long employment : depuy synthes companies ( i ) stock and other ownership interests : depuy synthes companies ( i ) travel , accommodations , expenses : depuy synthes companies ( i ) fergus j . 
couch consulting or advisory role : astrazeneca research funding : grail travel , accommodations , expenses : grail , qiagen , ambry genetics research funding : novartis ( i ) , ambry genetics , susan g . 
komen for the cure ( i ) , aacr , diana helis henry medical foundation ( i ) , james p . wilmot foundation ( i ) , adrienne helis malvin medical research foundation ( i ) , global biological standards institute ( i ) , breast cancer research foundation mark e . 
robson honoraria : astrazeneca , pzer consulting or advisory role : mckesson , astrazeneca research funding : astrazeneca ( inst ) , myriad genetics ( inst ) , invitae ( inst ) , pzer ( inst ) , abbvie ( inst ) , tesaro ( inst ) , medivation ( inst ) travel , accommodations , expenses : astrazeneca susan m . 
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balmaa j , digiovanni l , gaddam p , et al : conicting interpretation of genetic variants and cancer risk by commercial laboratories as assessed by the prospective registry of multiplex testing . 
robson me , bradbury ar , arun b , et al : american society of clinical oncology policy statement update : genetic and genomic testing for cancer susceptibility . oncol genomic med 4 : 232 - 236 , 2016 j clin oncol 33 : 3660 - 3667 , 2015 30 . 
katapodi mc , lee ka , facione nc , et al : predictors of perceived breast cancer risk and the relation between perceived risk and breast cancer screening : a meta - analytic review . 
hallowell n , lawton j , badger s , et al : the psychosocial impact of undergoing prophylactic total gastrectomy ( ptg ) to manage the risk of hereditary diffuse gastric cancer ( hdgc )  . 
worster e , liu x , richardson s , et al : the impact of prophylactic total gastrectomy on health - related quality of life : a prospective cohort study . 
odoherty k , suthers gk : risky communication : pitfalls in counseling about risk , and how to avoid thej genet couns 16 : 409 - 417 , 2007 36 . 
wolfe cr , reyna vf , widmer cl , et al : efcacy of a web - based intelligent tutoring system for communicating genetic risk of breast cancer : a fuzzy - trace theory approach . 
 appendix hamilton et al sent survey invitation ( n = 135 ) survey respondents ( n = 71 ) excluded : test report indicated no cdh1 result ( n = 3 ) survey respondents with self - reported understanding of their cdh1 result ( n = 68 ) excluded : respondent did not provide site - specific variant or test report respondent did provide sitespecific variant but it could not be found within the cdh1 gene in clinvar ( n = 5 ) ( n = 3 ) excluded : respondent self - reported understanding of their cdh1 result as my test results are not clear to me respondent self - reported understanding of their cdh1 result as negative genetic test result respondent missing responses to greater than 20% of study variables ( n = 4 ) * ( n = 3 ) ( n = 3 ) evaluable sample for analysis of disagreement among commercial laboratories in variant classifications ( n = 60 ) evaluable sample for analysis of how pv or vus cdh1 results are associated with medical and psychosocial experiences ( n = 57 ) excluded : respondent self - reported understanding of their cdh1 result as my test results are not clear to me ( n = 4 ) * evaluable sample for analysis of disagreement between participant self - report and clinvar classification ( n = 56 ) fig a1 . 
study diagra ( * ) indicates that these excluded cases are the same ; these cases are repeated in the gure to more clearly explain the derivation of the samples used in the study analyses . 
 c association of germline palb2 mutation and response to platinumbased chemotherapy in metastatic breast cancer : a case series case 1 a 51 - year - old female was diagnosed with stage iiia ( ct3 cn2 m0 ) triple - negative invasive ductal carcinoma of the right - side breast . 
at the time of diagnosis , the patient underwent genetic evaluation that was negative for a germline mutation in brca1 and brca2 . six months after completion of her primary therapy , the patient developed recurrent disease in the right - side chest wall and breast . 
next generation sequencing ( foundationone ; foundation medicine , cambridge , ma ) performed on her biopsy specimens showed loss of pten , mutations in partner and localizer of brca2 ( palb2 ) r170fs * 14 , arid2 r890fs * 49 , atrx k108fs * 35 , and ctnna1 duplication of exons 4 to 7 . 
given the finding of a palb2 mutation in the tumor , repeat genetic counseling was advised with expanded germline testing , which showed a pathogenic mutation in palb2 3323dela ( fig 1 )  . 
the patient has remained disease free for 30 months . case 2 a 47 - year - old female was diagnosed with stage ia ( pt1c n0 m0 ) invasive ductal carcinoma of the right - side breast that was estrogen receptor and progesterone receptor positive and human epidermal growth factor receptor 2 ( her2 ) negative ( table 1 )  . 
 two years later , she developed metastatic disease to the lung and chest wall , and biopsy findings were consistent with invasive ductal carcinoma that was weakly estrogen receptor positive ( 20% ) , progesterone receptor negative , and her2 unamplified by fluorescent in situ hybridization . 
 she was treated with multiple lines of endocrine therapy ( fulvestrant , letrozole , and palbociclib ) and chemotherapy ( single agents nab - paclitaxel , eribulin , and navelbine and a combination cyclophosphamide , methotrexate , and fluorouracil regimen ) but progressed with an ulcerating leftside chest wall mass ( fig 3 )  . 
the patient was then treated with carboplatin with near - complete resolution , which was sustained for 2 months ( fig 3 ) ; however , her treatment was stopped because of recurrent thrombocytopenia . 
gene profiling results for each patient . in kdmsa , cdk6 , lyn , and myc ; a homozygous deletion in fat1 ; and a mutation in palb2 y1108fs * 16 were observed . 
509_510delga ( fig 1 )  . discussion palb2 is a tumor suppressor gene that interacts with brca2 and is required for some functions of brca2 in dna repair pathways.1 after interaction with brca2 , palb2 facilitates nuclear localization of the palb2 - brca2 complex , which is then involved in dna repair through homologous recombination.2 germline mutations in palb2 are associated with an increased risk of breast and pancreatic cancer.3 approximately 1% to 3% of women with familial breast cancer harbor a monoallelic mutation in palb2.4 in addition , approximately 1% of patients with triple - negative breast cancer carry such a mutation.5 the lifetime risk of developing breast cancer by age 70 years for a female palb2 mutation carrier depends on family history.6 for those with no family history , the risk of developing breast cancer is 33% , whereas for those with two or more family members with breast cancer , the risk is 58%.6 compared with age - matched cohorts , the relative risk of breast cancer development for women with a palb2 mutation who are younger than 40 years , 40 to 60 years , and older than 60 years is increased by eight - , six to eight - , and fivefold , respectively . 
the risk for development of breast cancer in women who carry a deleterious palb2 mutation seems to differ among ethnic groups.7 , 8 molecular profiling has led to the development of novel therapeutic strategies . 
such mutations in homologous recombination ( hr ) pathways have been shown to improve sensitivity to dna - targeting agents and represent an important therapeutic target . targeted agents have increased treatment efficacy in a variety of breast cancer subtypes . 
these include endocrine manipulation with or without the addition of cyclin - dependent kinase 4 and 6 inhibitors or mammalian target for rapamycin inhibitors for hormone - sensitive disease or her2 - targeted agents for her2 - overexpressed tumors . 
approximately 70% of individuals with germline mutations in brca1 or brca2 and 30% with germline palb2 mutations have triple - negative disease.6 , 9 current data on clinical outcomes in patients fig 2 . 
 in addition , the development of polyp ( adp ribosome ) polymerase ( parp ) inhibitors has introduced the synthetic lethality concept by targeting the base excision repair pathway , which provides a way for targeting both dna repair pathways in patients with a brca defect . 
thus , inactivating mutations in these genes lead to defective hr - mediated dna repair , which makes cancer cells more susceptible to dna - targeting agents.10 increased sensitivity to dna - targeted therapy is described in a variety of solid tumors that harbor palb2 mutations . 
 a previous report described a case of gemcitabine resistant pancreatic cancer with somatic and germline palb2 mutations with an excellent and durable response to platinum agents.11 furthermore , earlier studies have shown improved overall survival and platinum responsiveness in patients with germline or somatic mutations in hr genes and ovarian , fallopian tube , and peritoneal carcinomas.12 although previous case series have documented improved disease response to platinum agents with a variety of solid tumors that harbor palb2 mutations , we present the first documentation to our knowledge of metastatic palb2 - associated breast cancer . 
the patient with a palb2 mutation ( case 1 ) treated with carboplatin had not only a complete response but also a prolonged duration of response of 30 months , which is clinically meaningful . 
a possible explanation for a cancer escape mechanism and retained platinum sensitivity is tumor dormancy or transitory senescence.13 fungating breast lesions present a clinical challenge and can have a severe effect on quality of life . 
these case reports provide additional evidence that platinum agents seem active in metastatic breast cancers , particularly among patients with germline palb2 mutations . the patient with a germline palb2 mutation who had a durable response to carboplatin had several somatic mutations , including pten loss . 
the progect study also seeks to understand predictors of response to neoadjuvant and adjuvant chemotherapy in triple - negative breast cancer associated with a variety of germline mutations , including palb2 . 
ongoing trials are evaluating the efficacy of parp inhibitors ( clinicaltrials . gov identifier : nct02401347 ) and checkpoint kinase 1 inhibitors ( clinicaltrials.gov identifier : nct02873975 ) in women with germline or somatic mutations in hr pathway genes , including palb2 . 
the role of pten in hr is controversial ; however , pten loss and sensitivity to dna - damaging agents , including platinum and parp inhibitors , have been described.14 , 15 a recent study reported long - term responses to maintenance olaparib in patients with brca1 / 2 and pten mutated metastatic ovarian cancer.16 we hypothesize that the combination of germline palb2 mutation and alteration in pten are responsible for the response noted . 
the second patient had a somatic mutation in stag2 , which is a core subunit of a multifunctional cohesion complex , and its mutations have been associated with increased sensitivity to inhibitors of dna repair.17 , 18 whether somatic stag2 mutations contributed to increased platinum sensitivity is unclear . palb2 mutations are associated with higher mortality independent of tumor stage , type of chemotherapy , or hormone receptor status.19 additional studies are required to evaluate whether women with palb2 mutations are at an increased risk of death . 
couch fj , hart sn , sharma p , et al : inherited mutations in 17 breast cancer susceptibility genes among a large triple - negative breast cancer cohort unselected for family history of breast cancer . 
haanp m , pylks k , moilanen js , et al : evaluation of the need for routine clinical testing of palb2 c.1592delt mutation in brca negative northern finnish breast cancer families . 
villarroel mc , rajeshkumar nv , garrido - laguna i , et al : personalizing cancer treatment in the age of global genomic analyses : palb2 gene mutations and the response to dna damaging agents in pancreatic cancer . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
lheureux s , lai z , dougherty ba , et al : long - term responders on olaparib maintenance in high - grade serous ovarian cancer : clinical and molecular characterization . 
 epicapture : a urine dna methylation test for early detection of aggressive prostate cancer purpose liquid biopsies that noninvasively detect molecular correlates of aggressive prostate cancer ( pca ) could be used to triage patients , reducing the burdens of unnecessary invasive prostate biopsy and enabling early detection of high - risk disease . 
epicapture was performed by quantitative methylation - specific polymerase chain reaction in dna isolated from prebiopsy urine sediments and evaluated by receiver operating characteristic and decision curves ( clinical benefit )  . 
the epicapture score was developed and validated on a two thirds training set to one third test set . results higher methylation of epicapture genes was significantly associated with increasing aggressiveness in pca tissues . 
2019 by american society of clinical oncology introduction prostate cancer ( pca ) is the most common noncutaneous malignancy in men and the third leading cause of cancer - related deaths in men in the western world . 
 with an aging population , spread of westernized diet , and increasing use of prostate - specific antigen ( psa ) testing , this figure is predicted to double by 2035.1 pca demonstrates extreme clinical heterogeneity . 
overall , 10 - year survival rates are close to 100%2 ; thus , there is much interest in strategies to reduce overtreatment of low / intermediate risk tumors.3 however , more than 300 , 000 men die as a result of pca each year . 
this stark figure , together with the knowledge that overtreatment comes with the high price of prevalent life - changing side effects , illustrates the need for tools to selectively identify high - risk pca ( those tumors with a high propensity to metastasize ) at an early stage , while the disease is potentially curable . research is ongoing into methods to address this need and better risk - stratify patients . 
urinary analysis of molecular correlates of aggressive disease could be used to triage patients and identify those who actually require an invasive prostate biopsy to histologically diagnose and grade their disease . in 2012 , movember launched four collaborative global action plan ( gap ) initiatives , focusing on pca biomarkers in urine , plasma , serum , and exosomes . 
 underpinning this aim was the cooperative establishment of prebiopsy postdigital rectal examination ( dre ) urine biorepositories at each institution , with a confederated database housing accompanying clinical , pathologic , and lifestyle data . 
six centers from the united states ( emory healthcare , atlanta , ga ) , canada ( university health network and lunenfeld - tanenbaum research institute , sinai health systems , toronto ) , and europe ( st jamess hospital , mater misericordiae university hospital , and tallaght hospital , all in ireland ; norfolk and norwich university hospital , england ) , collected prebiopsy , postdre urine ( 50 ml ) between january 2012 and october 2015 . 
exclusion criteria included previous diagnosis of pca , active urinary tract infection , recent prostate biopsy or transurethral resection of the prostate ( less than 6 wk ) , and confirmed presence of metastatic disease ( by pelvic magnetic resonance imaging or bone scan )  . 
 in total , urine samples were collected from 503 men , with matched formalin - fixed paraffin embedded trus biopsy cores acquired , where available ( fig 1 )  . 
in addition to this prebiopsy population , a retrospective analysis of the epicapture gene loci was performed on two independent cohorts of prostate tissues ( fig a2 ; data supplement )  . sample collection , processing , and epicapture analysis urine samples were stored on ice and processed within 4 hours of collection , following a movember gap1 standard operating procedure . 
details of nucleic acid isolation and quantification and epicapture analysis are provided in appendix table a1 and the data supplement . statistical analysis statistical analyses were performed with r version 3.4.1 and graphpad prism version 6 . 
 clinical utility was assessed by decision curve analysis ( dca ) .16 results epicapture genes demonstrate prognostic utility in prostate tissues mindful that most potential biomarkers fail to translate to the clinic , 17 we first set out to verify the prognostic utility of each epicapture constituent gene by measuring its methylation in two independent cohorts of radical prostatectomy samples . 
there was strong evidence for differences in mean methylation - values between four different groups ( benign , low - risk , aggressive , and metastatic pca ) in a small cohort of prostate tissues ( subjected to epithelial cell enrichment ) measured using the infinium humanmethylation450 beadchip ( fig 2a )  . 
 extracting freely available data from the same analytical platform for a larger the cancer genome atlas cohort yielded similar results ; each epicapture gene demonstrated significantly higher levels of dna methylation in clinically significant and high - risk tumors , relative to histologically benign tissue ( fig 2b )  . 
logistic regression models were developed using a 319 - specimen training set ( corresponding to 70% of the total cohort ) and then evaluated for their ability to correctly predict cancer / high - grade cancer / high - risk cancer in the remaining test set . 
not forgetting the clinical significance of gleason 7 disease , we also applied the revised five - grade grouping system , 18 with a view to detecting group 3 ( gleason 4 and 3 ) and above . 
the combination of epicapture and psa gave the best model ( auc , 0.82 ; sensitivity , 0.73 ; specificity , 0.76 ; appendix fig a1 ; appendix table a2 )  . we next evaluated the training set data to select an epicapture score cutoff that could best inform the decision on whether to perform a biopsy . 
finally , dca showed that at this threshold , epicapture demonstrated a net benefit over psa in the decision on whether to perform a biopsy , enabling a 15% reduction in prostate biopsies ( figs 3i and 3j )  . epicapture genes show high reproducibility by independent analysis to examine the robustness of epicapture , a matched quantitative methylation specific pcr analysis of two of the panel ( g1 : gstp1 , and g5 : apc ) was carried out in two independent laboratories on 236 of the urine samples . 
 ( a ) laser capture microdissected enriched prostate epithelial cells from benign prostate tissue ( procured from men undergoing radical cystoprostatectomy for bladder cancer , with no clinical or histologic evidence of prostate cancer [ pca ] ) and low - risk , aggressive , and metastatic pca . 
 ( b ) data extracted from the tcga repository for matched - benign prostate tissue and low - risk , significant , and high - risk pca , all procured from men undergoing radical prostatectomy . 
the performance of epicapture compared with / in conjunction with prostate - specific antigen ( psa ; treated as a continuous variable ) in the test set ( n = 134 ) for predicting each end point was assessed by receiver operating characteristic curves : ( a ) cancer ( v no cancer detected on transrectal ultrasound biopsy ) , ( b ) high - grade cancer ( gleason score greater than or equal to 8 v all else ) , ( c ) damico high - risk cancer ( v all else ) , and ( d ) capra ( cancer of the prostate risk assesment ) high - risk cancer ( v all else )  . 
the distribution of epicapture scores in the whole study population ( n = 453 ) , viewed by ( e ) biopsy outcome , ( f ) gleason grade , ( g ) risk categorization by damico risk - classification systems , and ( h ) risk categorization by capra risk - classification systethe epicapture score was calculated for each sample by summing the normalized index of methylation ( data supplement ) for g1 to g6 . 
epicapture score distributions were analyzed by t test or one - way analysis of variance ( anova ) , followed by post hoc analyses using the tukey - kramer multiple comparisons test . 
furthermore , matched data were available for 18 of 30 high - risk / grade samples that were epicapture negative , of which 15 ( 83.33% ) and 16 ( 88.89% ) were also negative for g1 and g5 , respectively , in the independent analysis . parallel epicapture analysis in urine and matched biopsy cores highlights value of liquid biopsy pca is renowned for its multifocal nature . 
matched formalin fixed paraffin - embedded biopsy cores were available for a proportion of patients , enabling parallel epicapture analysis on multiple tumor foci and urine from individual men ( n = 15 )  . 
the most interesting insights came from the matched analysis of urine with multiple biopsy cores , of which two notable cases are highlighted . patient sjh149 was diagnosed with a minute focus of gleason score 6 adenocarcinoma . 
this finding could suggest that the source of the tumor dna analyzed in urine could have been the high - grade tumor , which was not sampled on the original biopsy . 
 in contrast , patient sjh189 was diagnosed with a high - percentage , high - grade ( intermixed patterns of grade 4 and 5 ) pca on both sides of his prostate . 
so , despite pathologic heterogeneity within the gland , the three tumor areas seemed to be epigenetically homogeneous . discussion in this urine pca biomarker study , we evaluated the performance of epicapture , a six - gene dna methylation panel , for noninvasive detection of pca and more specifically high - grade or high - risk pca in urine from patients referred for prostate biopsy on the basis of elevated psa and / or abnormal dre . 
complying with remark guidelines , we describe a new dna methylation - based urine test for early detection of aggressive pca . many prostate tumors display a protracted disease trajectory , almost indolent in behavior.19 the widespread use of the serum psa test in many countries has significantly increased the incidence and thus overtreatment of these lowrisk cancers with little likelihood of clinical manifestation.20 - 23 an important first consideration in developing epicapture was to therefore critically appraise the evidence for prognostic utility of each constituent gene . 
conversely , however , it is estimated that approximately two thirds of men undergoing invasive prostate biopsy have no tumor diagnosed , because of the high false positive rate of serum psa.23 , 24 as such , millions fig 3 . 
 ( i ) decision curve analysis demonstrated net clinical benefit of performing biopsy on patients in the test set on the basis of an epicapture score greater than 1.25 versus psa greater than or equal to 3 ng / ml across a range of clinically relevant threshold probabilities ( the risk of cancer , such that a patient would choose to undergo biopsy by weighing the relative harms of false - positive and false - negative predictions )  . 
 ( a ) two of the six epicapture assays ( g1 : gstp1 and g5 : apc ) were analyzed independently in laboratories in ireland ( perry ) and canada ( bapat ) by quantitative methylation specific pcr in urine sediments from 236 of 453 men in the study . 
 relevant subsets were then considered by applying the study end points : ( b ) men with biopsy - positive disease , ( c ) men with high - grade ( gleason score greater than or equal to 8 ) prostate cancer , and men with highrisk prostate cancer defined by ( d ) damico , and ( e ) capra ( cancer of the prostate risk assessment ) classification . 
 repeat biopsy ( 8 months later ) sjh149 urine biopsy core ( s ) sample type base apex base apex histologically benign gleason 6 gleason 7 gleason ( cid : 116 ) 8 urine tissue sjh189 urine biopsy core ( s ) sample type fig 5 . 
 tumor presence is denoted by pink ( gleason score 6 ) , red ( gleason score 7 ) , or brown ( gleason score greater than or equal to 8 ) circles , with size indicative of percentage tumor in each core . 
 ( a ) low - risk prostate cancer sjh149 ( 62 years of age ; prostate - specific antigen , 3.08 ng / ml ) demonstrated that urine as a liquid biopsy is a viable alternative to sampling bias inherent to tissue needle biopsies . 
 sophisticated imaging technologies , such as magnetic resonance imaging , may improve the accuracy of prostate biopsy , 30 their widespread application is hampered by high cost , access to specialist equipment and personnel , interobserver subjectivity , and requirement for anesthesia . 
alternatively , transperineal biopsy enables a more complete sampling of the gland , including the anterior prostate , but also requires anesthesia and is thus unsuitable as a population - based detection method for such a prevalent disease . the dearth of blood - based prognostic biomarkers as a screening tool for aggressive pca in conjunction with limitations of the prostate biopsy in its various forms has led to excitement around urine biomarkers . 
physical manipulation of the prostate by dre , followed by a first - void urinalysis , could provide a simple , holistic insight into molecular alterations occurring in the prostate gland . 
indeed , many studies have demonstrated significant value in detecting and noninvasively monitoring several genitourinary malignancies through urine.31 - 34 some other prognostic urine indicators of aggressive pca have already been described . 
early studies demonstrated detection of gstp1 methylation in urine from patients with pca , although sensitivity was poor ( less than 30% ) .39 follow - up work expanded the repertoire of markers ( gstp1 , apc , and rar2 ) , which improved sensitivity to approximately 60%.40 the epicapture panel represents five commonly perturbed pathways in pca : intracellular detoxification , wnt and igf axes , cell cycle , and prostaglandin biosynthesis . 
in contrast , applying an epicapture threshold ( greater than 0.73 ; developed on training set ) did not markedly alter its diagnostic performance ( auc , 0.68 ) and delivered a 79% improvement over the tumor specificity of psa . in an effort to address the prognostic utility of epicapture within the context of the clinically heterogeneous gleason 7 disease , we applied the revised five - grade grouping system , 18 in addition to two widely used risk - classification systems . 
the epicapture performance characteristics represent a potential improvement on existing methods for patient stratification and for determining the need to perform biopsy , detecting 85% of highgrade tumors , with an npv of 98% . 
furthermore , the overlap of gstp1 and apc in the two gap1 urine dna methylation biomarker panels revealed excellent correlation in detection of aggressive pca , illustrating the robustness of these biomarkers . 
in the context of the diagnostic gray zone , psa 4 to 10 ng / ml , epicapture demonstrated potential for further classifying patients , positive in 40% of men with highgrade pca . 
 although these data are promising , a large prospective clinical trial is needed to assess whether epicapture offers a survival advantage . a limitation of our study is that the cohort is young ; analysis was only possible using biopsy as the end point . 
we cannot rule out the possibility that a proportion of our epicapture - positive urine samples in men with low - grade or benign tissues were indeed tumor cases missed on biopsy . 
evidence actually shows that dna methylation detected in histologically benign tissue ( due to an epigenetic field effect from the cancer ) , can be used as a tool to rule out the possibility that the biopsy missed the tumor . 
 for example , the confirm mdx test , which measures dna methylation at three genes ( gstp1 , apc , and rassf1 ) in histologically benign biopsy tissue cores , is used to guide the decision to repeat a biopsy.41 conversely , 14 of 47 ( 29.79% ) high - grade tumors were epicapture negative . 
independent analysis revealed concordant negativity in more than 80% of these men , and lowering the epicapture threshold did alter this result ; they were negative across all six markers . 
we believe that the most likely reason is interoperator differences in dre ; these high - risk / grade cancers were epicapture negative because they had insufficient / undetectable prostate cells present in their urine . 
 relationships may not relate to the subject matter of this enable confirmation of the presence of prostate cells in the urine , validating its suitability for epicapture analysis and ultimately improving the performance of the test . 
future work will also investigate the intra - individual reproducibility of epicapture by repeated measures in men . finally , to the best of our knowledge , this is the first pca biomarker study to perform a matched analysis of tissue and liquid biopsies . 
a more extensive project is now warranted to fully assess the effects of the multifocal nature of pca on signatures derived from liquid biopsies . epicapture could be used in conjunction with existing tools ( ie , psa and risk calculators ) to help guide the decision to biopsy . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . eve o ' reilly no relationship to disclose alexandra v . 
dale no relationship to disclose rick brugman no relationship to disclose gavin clarke no relationship to disclose olivia schmidt no relationship to disclose shane o ' meachair no relationship to disclose dattatraya patil no relationship to disclose kathryn l . 
pellegrini no relationship to disclose julia garcia no relationship to disclose fang zhao no relationship to disclose stephen finn honoraria : roche robert mills no relationship to disclose marcelino y . 
gallagher employment : oncomark leadership : oncomark neil fleshner honoraria : amgen , janssen oncology , bayer , sanofi , abbvie , ferring pharmaceuticals , astellas medivation consulting or advisory role : hybridyne health research funding : ferring ( inst ) , astellas pharma ( inst ) , janssen oncology ( inst ) , amgen ( inst ) , nucleix ( inst ) , progenix ( inst ) , spectracure ab ( inst ) research funding : amgen ( inst ) travel , accommodations , expenses : pfizer stock and other ownership interests : oncomark consulting or advisory role : carrick therapeutics research funding : carrick therapeutics patents , royalties , other intellectual property : two patents which have been licensed to oncomark travel , accommodations , expenses : oncomark rustom p . 
brewer patents , royalties , other intellectual property : patents held in drug discovery with novartis ( i ) , patents pending concerning cancer subtype detection and biomarkers with university of east anglia bharati bapat no relationship to disclose martin g . 
perry patents , royalties , other intellectual property : university college dublin holds a patent that relates to this work acknowledgment we thank the urologists and their teams at participating hospitals and the health research boardfunded dublin centre for clinical research network , affiliated research nurses , and technical staff , in particular john mccourt . 
we thank investigators drs robert vessella , and colm morrissey , and the patients and families who participated in the prostate cancer donor program at the university of washington medical center . 
bristow , director , manchester cancer research centre , chief academic officer and honorary consultant , christie nhs trust , university professor of cancer studies , university of manchester , manchester cancer research centre , university of manchester , manchester , uk . chris parker , royal marsden hospital , sutton , uk . ian mills , queens university belfast , centre for cancer research and cell biology , movember / prostate cancer uk centre of excellence , school of medicine , dentistry , and biomedical sciences . 
sanda , emory university school of medicine , atlanta , ga ; neil fleshner , julia garcia , fang zhao , and bharati bapat , university of toronto , toronto , ontario , canada ; robert mills , norfolk and norwich university hospital ; marcelino y . 
brewer , earlham institute , norwich , england . supported by grant funding from movember ( gap1 urine biomarker award ) , us prostate cancer foundation ( young investigator award , a.s.p. ) , science foundation ireland grants no . 
bastian pj , ellinger j , heukamp lc , et al : prognostic value of cpg island hypermethylation at ptgs2 , rar - beta , ednrb , and other gene loci in patients undergoing radical prostatectomy . 
damico av , whittington r , malkowicz sb , et al : biochemical outcome after radical prostatectomy , external beam radiation therapy , or interstitial radiation therapy for clinically localized prostate cancer . 
cooperberg mr , pasta dj , elkin ep , et al : the university of california , san francisco cancer of the prostate risk assessment score : a straightforward and reliable preoperative predictor of disease recurrence after radical prostatectomy . 
vickers aj , cronin am , roobol mj , et al : a four - kallikrein panel predicts prostate cancer in men with recent screening : data from the european randomized study of screening for prostate cancer , rotterdaclin cancer res 16 : 3232 - 3239 , 2010 29 . 
siddiqui mm , rais - bahrami s , turkbey b , et al : comparison of mr / ultrasound fusion - guided biopsy with ultrasound - guided biopsy for the diagnosis of prostate cancer . 
costa vl , henrique r , danielsen sa , et al : three epigenetic biomarkers , gdf15 , tmeff2 , and vim , accurately predict bladder cancer from dna - based analyses of urine samples . 
torres - ferreira j , ramalho - carvalho j , gomez a , et al : mir - 193b promoter methylation accurately detects prostate cancer in urine sediments and mir - 34b / c or mir - 129 - 2 promoter methylation define subsets of clinically aggressive tumors . 
wei jt , feng z , partin aw , et al : can urinary pca3 supplement psa in the early detection of prostate cancer ? j clin oncol 32 : 4066 - 4072 , 2014 35 . 
leyten gh , hessels d , jannink sa , et al : prospective multicentre evaluation of pca3 and tmprss2 - erg gene fusions as diagnostic and prognostic urinary biomarkers for prostate cancer . 
van neste l , partin aw , stewart gd , et al : risk score predicts high - grade prostate cancer in dna - methylation positive , histopathologically negative biopsies . 
 ( a ) the performance of epicapture compared to and in conjunction with prostate - specific antigen ( psa ; treated as a continuous variable ) in the test set ( n = 134 ) for predicting isup group 3 , 4 and 5 pca on biopsy was assessed by receiver operator characteristic curves . 
 ( b ) the distribution of epicapture scores in biopsy - negative ( no cancer detected ) v biopsy positive patients , categorized by isup group , shows increasing methylation by group . 
epicapture was performed in urine and matched formalin - fixed paraffin embedded biopsy cores from randomly selected biopsy - positive ( n = 15 : low - risk [ capra and damico , gleason score 6 ; intermediate risk , gleason score 7 ; and high risk , gleason score 8 ] ) patients . 
dna methylation of each gene is indicated by the nim ( normalised index of methylation ) , and for the collective panel , by the epicapture score ( nim sum g1 - g6 )  . 
each point represents a single sample ( biopsy core or urine ) , with the mean for each group indicated by a horizontal line ( yellow , urine ; blue , tissue )  . 
 multigene hereditary cancer panels reveal high - risk pancreatic cancer susceptibility genes purpose the relevance of inherited pathogenic mutations in cancer predisposition genes in pancreatic cancer is not well understood . 
we aimed to assess the characteristics of patients with pancreatic cancer referred for hereditary cancer genetic testing and to estimate the risk of pancreatic cancer associated with mutations in panel - based cancer predisposition genes in this high - risk population . methods patients with pancreatic cancer ( n = 1 , 652 ) were identified from a 140 , 000 patient cohort undergoing multigene panel testing of predisposition genes between march 2012 and june 2016 . 
gene - level mutation frequencies relative to exome aggregation consortium and genome aggregation database reference controls were assessed . results the frequency of germline cancer predisposition gene mutations among patients with pancreatic cancer was 20.73%. 
mutations in atm , brca2 , cdkn2a , msh2 , msh6 , palb2 , and tp53 were associated with high pancreatic cancer risk ( odds ratio , > 5 ) , and mutations in brca1 were associated with moderate risk ( odds ratio , > 2 )  . 
in a logistic regression model adjusted for age at diagnosis and family history of cancer , atm and brca2 mutations were associated with personal history of breast or pancreatic cancer , whereas palb2 mutations were associated with family history of breast or pancreatic cancer . conclusion these findings provide insight into the spectrum of mutations expected in patients with pancreatic cancer referred for cancer predisposition testing . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction pancreatic cancer ( pc ) is the fourth most common cause of death resulting from cancer in the united states.1 epidemiologic studies have suggested that 10% to 20% of pcs are associated with an inherited component , with familial pc , defined as kindreds containing at least two affected first - degree relatives , as an established entity of inherited disease.2 pc is a component of hereditary breast - ovarian cancer syndrome , 3 , 4 lynch syndrome , 5 , 6 familial adenomatous polyposis , 7 familial atypical multiple mole melanoma syndrome , 8 hereditary pancreatitis , 9 peutz - jeghers syndrome , 10 and li - fraumeni syndrome.11 recent studies involving familial pc kindreds have further characterized the role of brca1 / 2 , cdkn2a , atm , and palb2 in pc susceptibility.12 - 14 until recently , germline studies of pcs have focused on single cancer predisposition genes.15 , 16 the first panel - based study of 13 cancer predisposition genes among patients with pc identified 11 mutations ( 3.8% ) in atm , brca1 / 2 , mlh1 , msh2 , msh6 , and tp53 , 17 whereas a 22 - gene panelbased study identified atm , brca1 / 2 , chek2 , and palb2 mutations in 13% of 96 sequentially collected pcs.18 a majority of these mutations were identified in pcs with a family history of pancreatic , breast , ovarian , or colorectal cancer , suggesting that a better understanding of pc risk will depend on evaluation of families with broad constellations of tumors.18 more recently , panel - based approaches identified germline chunling hu holly laduca hermela shimelis eric c . 
 mutations in 4% ( 33 of 854 ) of patients with apparently sporadic pc19 and in 25% ( 44 of 176 ) of patients with advanced pc.20 here , we report results from panel - based clinical testing of 1 , 652 patients with pc from a large cohort of > 140 , 000 patients evaluated by a single diagnostic laboratory and calculate gene - specific risks of pc by comparison with exome aggregation consortium ( exac ) and genome aggregation database ( gnomad ) reference controls.21 , 22 methods study population patients with pc ( n = 1 , 819 ) were identified from a large cohort of > 140 , 000 patients undergoing multigene panel testing of seven to 49 cancer predisposition genes between march 2012 and june 2016 at ambry genetics23 ( aliso viejo , ca ; appendix table a1 )  . 
exclusion criteria , including the presence of neuroendocrine tumors or intraductal papillary mucinous neoplasms , reduced the number of patients for analysis ( n = 1 , 652 ; appendix )  . 
 the study was approved by the western institutional review board . multigene panel testing mutation testing was performed by sequencing of targeted custom capture products from several multigene panels and targeted chromosomal microarray analysis , as previously described.24 genomic dna was isolated from each patients blood or saliva specimen using a standardized methodology ( qiagen , valencia , ca )  . 
sequence enrichment was performed by incorporating the genomic dna into microfluidics chip or microdroplets along with primer pairs or by a bait - capture methodology using long biotinylated oligonucleotide probes ( raindance technologies , billerica , ma ; integrated dna technologies , san diego , ca ) , followed by polymerase chain reaction and then next - generation sequencing analysis ( illumina , san diego , ca ) of all coding exons plus at least five bases into the 5 and 3 ends of all the introns and untranslated regions . 
all mutations identified by ambry genetics are submitted to the clinvar public database . statistical methods the observed frequency of all pathogenic mutations within each gene in white patients with pc was compared with the frequency of pathogenic mutations in the exac non - finnish european ( nfe ) nonthe cancer genome atlas ( tcga ) reference control after data cleaning and filtering ( appendix ) as previously described.23 copy number variants in all genes and mutations in pseudogene homology regions ( pms2 exons 9 and 11 to 15 ) were excluded from cases and controls for risk estimation , because these alterations were not individually validated in exac or gnomad controls . 
although these gnomad controls partially overlap with exac nfe non - tcga controls , the substantially increased number along with updated variant calling algorithms identified gnomad as an independent reference control data set . 
 ( % ) patients of all ethnicities ( n = 1 , 652 ) white patients ( n = 1 , 256 ) age at pc diagnosis were evaluated using the kolmogorov - smirnov test . 
 compared with a median age at pc diagnosis of 70 years in surveillance , epidemiology , and end results registries between 2010 and 2014 , 26 the median age at diagnosis was 63 years among patients with pc . 
among white patients with pc , 38.1% had a firstor second - degree relative with pc , and 48.8% had a family history of breast cancer ( table 1 )  . 
similar frequencies were observed for patients with pc of all races and ethnicities . pathogenic mutations among patients with pc the combined frequency of mutations in genes from all hereditary cancer testing panels was 20.73% for patients with pc of any race or ethnicity and 21.12% for white patients ( appendix table a2 )  . 
brca2 was the most frequently mutated predisposition gene ( 4.64% ) among patients diagnosed at age 63 years , and atm was most frequently mutated ( 4.03% ) in patients with pc diagnosed at age > 63 years ( appendix table a4 )  . 
association analyses using gnomad reference controls confirmed all significant associations , and gene - specific risk estimates were highly similar , except for slightly attenuated risk for palb2 mutations and increased risk for tp53 ( appendix table a6 )  . the same genes were associated with increased pc risk when considering patients of all races and ethnicities compared with exac all race and ethnicity controls ( appendix table a7 ) and after excluding those who had previously tested negative for brca1 / 2 mutations before panel testing ( appendix table a8 )  . 
risk estimates for most genes were slightly diminished when including only those patients with pc for whom pc was the first cancer diagnosis , although msh2 and tp53 mutations were no longer significantly associated with moderate risk of pc because of the decreased number of mutations in patients with pc , and the modest or associated with chek2 was marginally significant ( appendix table a9 )  . 
in contrast , analyses using only exac nfe non - tcga variants in the high - quality pass category marginally increased the ors for each gene ( appendix table a10 )  . 
mutations in atm , brca2 , and palb2 were also more frequent in patients with pc with a family history of breast cancer ( first or second - degree relative ; table 3 )  . 
in contrast , only palb2 and msh2 displayed a substantial increase in mutation frequency among patients with a family history of pc , and only chek2 , msh2 , and tp53 had increased frequencies of mutation among patients with pc with a family history of colorectal cancer ( table 3 )  . 
 family history of pc ( p = .029 ) or breast cancer ( p = .0056 ) and the association of chek2 mutations with family history of colorectal cancer ( p = .014 ; table 4 )  . performance of genetic testing criteria among mutation carriers consensus clinical genetic testing guidelines include pc as a component tumor for seven of the confirmed pc genes in this study ( brca1 / 2 , msh2 , msh6 , atm , palb2 , and cdkn2a ) .27 - 29 clinical histories of patients with mutations in these genes were evaluated to determine whether the respective genetic testing criteria were met ( table 5 )  . 
although a majority of brca1 / 2 and all msh2 mutation carriers displayed histories consistent with testing criteria , 50.0% of atm , cdkn2a , palb2 , and msh6 carriers met criteria . 
in addition , no cdkn2a families met diagnostic criteria for familial atypical multiple mole melanoma syndrome , 30 and 38.9% ( seven of 18 ) did not report any personal or family history of melanoma . discussion here we report a study of cancer predisposition gene mutations among patients with pc on the basis of a cohort of individuals undergoing hereditary cancer multigene panel testing from a single clinical laboratory . 
results from case control studies of the pc cases and exac reference controls identified six genes associated with high risk ( or , > 5 ) of pc ( atm , brca2 , cdkn2a , msh6 , palb2 , and tp53 ) , consistent with previous smaller studies and segregation studies from pc families . 
msh2 was also associated with a high risk of pc ; however , additional studies are needed to confirm these findings , because this association was based on a limited number of mutations detected among pc cases . 
 here we show that brca1 mutations are associated with a moderate risk ( or , > 2 ) of pc , even in a series of sensitivity analyses accounting for potential modifying effects of other cancers . 
 chek2 mutations were also associated with a moderate risk of pc ; however , this association was either diminished ( or , < 2 ) or nonsignificant in several sensitivity analyses . 
one likely explanation is that stk11 mutations are unlikely to occur in the absence of pathognomonic clinical characteristics associated with peutz - jeghers syndrome , and therefore , patients with suspected peutz - jeghers syndrome may be referred for single - gene testing more often than multigene testing . 
pathogenic mutations in other panel genes were still sufficiently uncommon to allow assessment of associations with risk ( eg , apc , mlh1 )  . the risk estimates for pc associated with each of these established predisposition genes will help improve clinical pc risk assessment . 
for some genes , these results offer more precise estimates than previously reported , whereas for others , such as palb2 and atm , we are the first to characterize the level of risk , to our knowledge . 
 it should be noted that the interpretation of the risks reported here is specific to patients referred for hereditary cancer genetic testing based on a personal or family history of cancer ( at least one diagnosis of pc in the family ) , and thus , these data may not be applicable to the general population or unselected pc cohorts . 
despite the enrichment for cases with personal or family history of cancer , these risks are derived from a broader clinical cancer testing cohort compared with previous studies selected for classic syndromic phenotypes such as fammm and therefore demonstrate that pc risk from syndromic genes remains high across a range of clinical histories . 
furthermore , this enrichment presented an opportunity to explore predictors of germline mutations . in total , 13% of patients had mutations in genes significantly associated with increased risk for pc across a range of sensitivity analyses ( atm , brca1 , brca2 , cdkn2a , msh6 , palb2 , and tp53 )  . 
family history of breast , pancreatic , or colorectal cancer was a significant predictor of positive results , suggesting that histories of these cancers should specifically be considered as genetic testing guidelines evolve for pc . 
the remaining 9% ( 15 of 173 ) of mutations were found in the approximately 65% of patients with pc without a family history of these cancers , suggesting a mutation rate of only 2.1% in white patients with pc without a family history of cancer ( 15 mutations in 698 ) in the clinically tested cohort . 
additional studies of population - based series of patients with pc are needed to determine whether clinical panel testing should be considered for patients with pc unselected for family history . in practice , patients with pc may not benefit directly from genetic testing because of the high mortality rate for this cancer . 
however , knowledge of mutation status for genes such as brca1 / 2 and palb2 with respect to clinical trial eligibility for targeted agents such as poly ( adp - ribose ) polymerase inhibitors may make genetic testing more appealing . 
in addition , mutation - positive family members can significantly benefit from knowledge of increased risk for a variety of cancers , including pc , and mutation - negative family members can also adjust their cancer screening protocols accordingly . 
in addition , the international cancer of the pancreas screening consortium and the american college of gastroenterology 29 , 31 recommend that pc surveillance , including annual endoscopic ultrasound and / or magnetic resonance imaging , be considered for individuals with > 5% lifetime or relative risk for pc . 
in addition , although brca1 mutation carriers with a firstor second - degree relative with pc are included in the list of patients for whom pc screening should be considered , the moderate pc risk categorization for brca1 in this study suggests this may not be clinically indicated . exac nfe non - tcga controls were used in this study because of the lack of a large series of matched controls . 
although the use of large reference data sets is not ideal , the large sample size allows precise estimation of the frequency of mutations in individuals without cancer and is likely reflective of the general population . 
in addition , we applied many data cleaning steps and used consistent criteria for selection of mutations in the clinical cohort of patients with pc and the exac controls to ensure that the data sets were adequately normalized for case - control association analyses . 
in a recent assessment of the quality of clinical history information for patients undergoing hereditary cancer panel testing , pedigrees and / or clinic notes were provided for 46% of randomly selected patient cases ( unpublished data )  . 
 when compared with pedigrees and clinic notes , a vast majority of proband cancers were reported completely ( 95% ) and accurately ( > 99% ) on test requisition forms . 
among family members , 76% of melanomas and > 80% of breast , ovarian , colorectal , endometrial , and pancreatic cancers were reported with 98% accuracy on test requisition forms . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . stock and other ownership interests : ambry genetics chunling hu no relationship to disclose holly laduca employment : ambry genetics hermela shimelis no relationship to disclose eric c . 
lee no relationship to disclose brigette tippin davis employment : ambry genetics stock and other ownership interests : ambry genetics research funding : ambry genetics ( inst ) travel , accommodations , expenses : ambry genetics mary helen black employment : ambry genetics research funding : ambry genetics travel , accommodations , expenses : ambry genetics tina pesaran employment : ambry genetics stock and other ownership interests : ambry genetics travel , accommodations , expenses : ambry genetics david e . 
rahib l , smith bd , aizenberg r , et al : projecting cancer incidence and deaths to 2030 : the unexpected burden of thyroid , liver , and pancreas cancers in the united states . 
risch ha , mclaughlin jr , cole de , et al : population brca1 and brca2 mutation frequencies and cancer penetrances : a kin - cohort study in ontario , canada . 
ruijs mw , verhoef s , rookus ma , et al : tp53 germline mutation testing in 180 families suspected of li - fraumeni syndrome : mutation detection rate and relative frequency of cancers in different familial phenotypes . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
laduca h , stuenkel aj , dolinsky js , et al : utilization of multigene panels in hereditary cancer predisposition testing : analysis of more than 2 , 000 patients . 
canto mi , harinck f , hruban rh , et al : international cancer of the pancreas screening ( caps ) consortium summit on the management of patients with increased risk for familial pancreatic cancer . 
 appendix patient cases of pancreatic cancer a total of 1 , 819 patients with pancreatic cancer were identified in a cohort of 140 , 449 individuals undergoing clinical germline cancer panel testing between march 2012 and june 2016 at a clinical testing laboratory ( ambry genetics , aliso viejo , ca )  . 
all variants classified by ambry were submitted to clinvar . exome aggregation consortium reference controls the exome aggregation consortium ( exac ) contains exome sequence data from 60 , 706 unrelated individuals sequenced as part of various disease - specific and population genetic studies . 
exclusion of sequence data from these patient cases yielded exac non - nfe non - tcga reference controls . genome aggregation database reference controls the genome aggregation database ( gnomad ) contains sequencing data of 123 , 136 exomes and 15 , 496 genomes from unrelated individuals sequenced as part of various disease - specific and population genetic studies . 
 gnomad data cleaning and filtering restricted to gnomad nfe exome data combined with gnomad ashkenazi jewish exome data . genomics viewer and by frequency in control data from dbsnp . pathogenic variant classification rules : same as in exac rules 1 to 8 . review variants with allele count 15 by integrative allele number was calculated as average of all variants within the coding region of a gene of interest . 
bekelman , md2 , 4 , 6 , 7 the senseless killings of george floyd , ahmaud arbery , breonna taylor , and countless others have led to a national reckoning on institutional racism . medicine , race has long been used as a heuristic , or mental shortcut , used by clinicians to process information and make decisions within the time and cognitive constraints of our healthcare system.1 however , using race as a heuristic in medical decision making has the potential to accentuate cognitive biases and lead to deviations from rational decision making , even among the most well - intentioned of clinicians.2 furthermore , it is well recognized that race is a social , not a biological , construct ; as a result , the broad race categories used in medicine are heterogeneously dened and may be incongruent with genetic ancestry , particularly among minority patients.3 , 4 because cancer is inherently a disease of genetic aberration , using these race categories to make decisions in oncology may lead to the delivery of ineffective , or even detrimental , patient care . the rapid advancement of precision oncology , the use of molecular and genetic testing to tailor cancer therapies to individual patients , has uniquely positioned the eld of oncology to move beyond racebased medicine . 
since the discovery of the rst targeted therapy , imatinib , in the 1990s , over 80 targeted agents have been approved by the us food and drug administration for the treatment of numerous solid organ and hematologic malignancies.5 , 6 some of these precision oncology applications correlate with patient race . 
for instance , epidermal growth factor receptor ( egfr ) mutations in nonsmall - cell lung cancer are found in over 60% of patients of asian descent and strongly predict sensitivity to egfr tyrosine kinase inhibitors.7 , 8 similarly , several founder mutations in brca1 / 2 have been observed in multiple ethnic groups , including patients with ashkenazi jewish ancestry , and can be used to predict sensitivity to poly ( adp - ribose ) polymerase inhibitors in patients with breast , ovarian , prostate , and pancreatic cancers.9 - 14 fortunately , the tools of precision oncology enable the administration of agents such as egfr and poly ( adpribose ) polymerase inhibitors to be guided by genetic testing rather than race or ancestry . however , differential use of precision oncology has the potential to widen the very racial disparities that the eld seeks to eliminate.15 indeed , multiple studies have demonstrated decreased rates of genetic counseling , germline and somatic genetic testing , targeted therapy administration , genomic database participation , and clinical trial enrollment among minorities compared with white patients.16 - 22 there are many barriers to achieving the promise of precision oncology , including lack of access to and payment for testing , targeted treatment , and counseling . 
additionally , one major challenge is simply a lack of awareness among minority patients and clinicians.23 the rapid advancement of precision oncology has made it difcult for many clinicians to have the time and expertise to select appropriate genetic testing , interpret and apply test results , and educate patients on their implications.24 this challenge may be especially pertinent among minority - serving physicians , who have been shown to be less likely to refer to genetics and order germline testing for diseases such as breast and colorectal cancer.25 the resultant use of race as a heuristic in the setting of these time and cognitive constraints may lead to decisions that are inuenced by implicit bias , unconscious stereotypes , or attitudes toward people that inuence ones actions and behaviors . 
much like the general population , physicians have been found to have implicit bias with a preference for white over black patients and to associate race with their assessments of patient intelligence , risk - taking behavior , and adherence to medical advice.26 - 29 in turn , these perceptions have been linked to poorer communication , biased treatment recommendations , and unequal access to quality healthcare for minority patients.30 , 31 in this paper , we propose three strategies to advance health equity in precision oncology ( table 1 )  . 
ehr - based strategies to advance health equity in precision oncology ehr strategy key features default orders preselected ehr orders that are automatically applied unless a clinician actively modies them behavioral economics principle nudge decision making by taking advantage of individual status quo bias , or the tendency to favor the current state of affairs automated patient dashboards programs that harness the automation and computing power of the ehr to identify candidate patients for a given diagnostic or therapeutic intervention minimize choice overload by searching through a clinicians full patient panel to identify the subset that is eligible for precision oncology care clinician - directed clinical integration of standardized clinical pathways into the ehr for minimize choice overload by simplifying and facilitating use at the point of care complex clinical decision making use of electronic patient portals to send educational materials prime patients to initiate conversations with their clinicians about precision oncology and communicate normative feedback on its uptake among other patients about precision oncology note . 
we propose that behavioral economic principles should be leveraged to promote health equity in precision oncology as well . to steer the ehr offers a scalable , cost - effective approach to harnessing behavioral economic principles to advance health equity in precision oncology . 
defaultspreselected choices that are applied unless an individual actively changes themare powerful tools that take advantage of the status quo bias to nudge decision making while minimizing cognitive effort and preserving choice . 
this approach has been shown to optimize cancer care delivery by increasing high - value medication prescribing and reducing unnecessary daily imaging during palliative radiotherapy.36 , 37 in the precision oncology space , default orders in the ehr can be used to make direct referrals to genetics for patients who meet guideline criteria on the basis of factors such as tumor type and age , clinical characteristics that can be easily identied in the ehr . 
 advancing health equity in precision oncology decision altogether.47 - 49 the ehr is well equipped to solve the problem of choice overload through automated patient dashboards , which can efciently search through all the patients cared for by a given clinician or at a particular practice to identify a more limited pool of individuals who are eligible for a given intervention . 
this approach has been leveraged by multiple clinical trial matching programs to match patient and tumor characteristics from the ehr to study eligibility criteria , thereby enabling oncologists to identify study opportunities for individual patients . 
these programs have been effective in facilitating increased clinical trial participation not only within academic institutions but also at community oncology practices , sites that are more likely to serve minority and underserved populations.50 , 51 similar automated patient dashboards should be leveraged to identify candidate patients for other precision oncology opportunities that remain underutilized by minorities , such as genetics referrals , germline and somatic testing , targeted therapy administration , and genomic database participation . 
because this strategy matches patients to precision oncology care using the automation and computing power of the ehr rather than the heuristics of human decision making , it is likely to be more effective than individual clinicians in advancing health equity . clinical decision support systems decision support also mitigates choice overload by guiding individuals toward more desirable decisions while preserving their freedom of choice . 
in precision oncology , clinical pathways have emerged as a decision support strategy for clinicians to handle the elds ever - expanding scope and complexity.52 however , these pathways are ineffective unless they are seamlessly integrated into routine clinical practice to maximize their usefulness . 
embedding clinical decision support systems into the ehr has been shown to facilitate decision making in other areas of cancer diagnosis , treatment , and supportive care , 53 and it has been associated with decreased racial disparities in the management such as hypertension.39 this approach should also be leveraged to standardize precision oncology care for all patients , regardless of race . of nononcologic conditions perhaps an even more compelling clinician decision aid is a patients own request for a given medical intervention . 
priming , the use of subtle stimuli to activate thoughts and ideas , 54 is a powerful tool that can be directly used by patients to support clinicians in overcoming the use of race as a heuristic . 
first , we recognize that our proposed strategies do not fully combat the lack of access , nancial language barriers , and challenges , health literacy or longstanding history of mistrust in the healthcare system that have also impeded the adoption of precision oncology among minority patients . 
to be successful , ehr - based strategies informed by the principles of behavioral economics must be implemented as part of a broader , multifaceted approach that addresses all these challenges . second , the application of behavioral economics in medicine is still in its nascency , so there is a paucity of data on the impact of choice architecture redesign on subgroups within the population . 
future studies should incorporate the emerging concept of precision nudging to tailor behavioral economics interventions to specic patient populations as a strategy to ensure equal effectiveness for all . finally , the implementation of ehr - based strategies may require initial investment of nancial and human resources that is not feasible at all institutions . 
as implementation efforts get underway , we must monitor for and address any signs of differential uptake so that our proposed strategies do not inadvertently widen existing inequities in care . in conclusion , in this article , we present three ehr - based strategies that leverage the principles of behavioral economics to nudge oncologists away from the use of race as a heuristic in their decision making . 
 lau - min et al 4abramson cancer center , perelman school of medicine , university of pennsylvania , philadelphia , pa 5division of translational medicine and human genetics , department of medicine , perelman school of medicine , university of pennsylvania , philadelphia , pa 6department of radiation oncology , perelman school of medicine , university of pennsylvania , philadelphia , pa 7department of medical ethics and health policy , perelman school of medicine , university of pennsylvania , philadelphia , pa otherwise noted . 
guerra leadership : freenome , guardant health , genentech stock and other ownership interests : crispr therapeutics , beam therapeutics , intellia therapeutics , editas medicine honoraria : lundbeck ( i ) consulting or advisory role : bristol - myers squibb ( i ) speakers bureau : janssen , pzer ( i ) research funding : bristol - myers squibb ( inst ) travel , accommodations , expenses : lundbeck , janssen ( i ) uncompensated relationships : tapestry networks justin e . 
bekelman honoraria : unitedhealthcare , national comprehensive cancer network , cvs health , optum , american journal of managed care research funding : pzer , united health group , north carolina blue cross blue shield , embedded healthcare no other potential conicts of interest were reported . acknowledgment we thank mitesh patel , md , mba , and callie scott , msc , both at the university of pennsylvania , for their helpful comments during the writing of this manuscript . 
new york , farrar , straus and giroux , 2011 zhang f , finkelstein j : inconsistency in race and ethnic classication in pharmacogenetics studies and its potential clinical implications . 
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 systemic and cns activity of selective ret inhibition with selpercatinib ( loxo - 292 ) in a patient with ret - mutant medullary thyroid cancer with extensive cns metastases alexander andreev - drakhlin , md1 ; maria cabanillas , md1 ; behrang amini , md , phd1 ; and vivek subbiah , md1 introduction medullary thyroid cancer ( mtc ) is a neuroendocrine tumor that originates from the parafollicular cells ( or c cells ) of the thyroid gland and accounts for 1% to 2% of thyroid cancers in the united states.1 the cns is a rare site of metastasis in mtc , with , 15 cases reported in the medical literature ; however , the prevalence may be underreported because of the infrequent use of cns imaging in patients with mtc.1 , 2 the optimal treatment approach for patients with mtc with cns involvement is currently unknown . 
although surgical resection , radiation therapy , or stereotactic radiosurgery ( srs ) may improve local disease control in some patients , most still succumb to their disease within months after the diagnosis of cns metastases.1 - 4 the proto - oncogene ret ( rearranged during transfection ) , located on chromosome 10q11.2 , is central to development of a majority of cases of mtc.5 germline gain - of - function ret point mutations result in multiple endocrine neoplasia 2 , a hereditary syndrome associated with mtc and pheochromacytoma.6 , 7 similar somatic mutations occur in approximately 50% of sporadic mtc , approximately 20% of sporadic pheochromocytomas , and rarely in other cancer types.8 - 10 recent identication of highly potent and selective ret inhibitors holds great promise in the management of these ret - driven cancers as well as other ret - altered malignancies.11 - 14 in 2018 , selpercatinib ( loxo - 292 ) received us food and drug administration breakthrough therapy designation for treatment of patients with previously treated metastatic ret - mutant mtc , ret fusion - positive nonsmall - cell lung cancer , and ret fusionpositive thyroid cancers . 
durable intracranial responses with selpercatinib have been described in patients with brain metastases with ret fusionpositive lung and thyroid cancers.15 , 17 however , the activity of selpercatinib in patients with ret - mutant mtc with brain metastases and / or leptomeningeal disease ( lmd ) has not been characterized , to our knowledge . 
here , we describe a patient with ret m918tmutant mtc with extensive cns metastases who had a clinically meaningful durable response to selective ret inhibition with selpercatinib . case report total a 67 - year - old woman presented to a local hospital with bilateral neck swelling . 
positron emission tomography imaging showed metabolically active , left - side , level v lymph nodes ; a 1.1 - cm , hypermetabolic , right lower lobe lung nodule ; several hypermetabolic liver lesions ; as well as lytic lesions in the c5 vertebral body , l4 left lamina , and left posterior iliac wing . 
magnetic resonance imaging ( mri ) of the brain revealed a 4 - mm lesion in the right caudate nucleus . the patient presented to the university of texas md anderson cancer center for a second opinion . 
she was then treated with standard - of - care cabozantinib but experienced multiple adverse effects , including handand - foot syndrome , mucositis , weight loss , nausea , and diarrhea , requiring several dose reductions ultimately to 40 mg daily . 
 ( a ) magnetic resonance imaging ( mri ) oblique axial cerebellar images of the patients metastatic brain disease ( left panel ) before and ( right panel ) 8 weeks after the patient initiated treatment with selpercatinib . 
 ( c ) mri axial cerebellar and ( d ) sagittal thoracic spine images of the patients metastatic disease ( left panels ) before and ( right panels ) 33 weeks after the patient initiated treatment with selpercatinib . 
 ( e ) mri axial brain images demonstrating a new caudate lesion after 17 months of therapy with selpercatinib ( left panel ) before and ( right panel ) after stereotactic radiosurgery ( srs )  . 
 andreev - drakhlin et al given the absence of any remaining standard therapies with established benet , the patient enrolled in a phase i / ii study of selpercatinib in patients with advanced solid tumors ( libretto - 001 ; clinicaltrials.gov identier : nct03157128 )  . the patient provided written informed consent before enrollment . 
baseline imaging at the time of the treatment initiation with selpercatinib revealed bilateral central and lateral neck disease ; lytic lesions in the c5 , t4 , t8 , l1 vertebrae and left posterior hilum ; , nodular opacities in bilateral lungs ; interlobular septal thickening suspicious for lymphangitic carcinomatosis ; numerous hepatic lesions ( largest , 7 cm in the dome of liver ) ; 25 lesions in the brain and cerebellum ( largest , 7 mm in the left cerebellar hemisphere ; fig 2a and 1c , left panels ) , and a 5 - mm leptomeningeal nodule at the level of t11 ( fig 2d , left panel )  . calcitonin and cea levels measured 2 , 180 pg / ml and 12 , 312 ng / ml , respectively ( fig 2b )  . the patient experienced clinical response within the rst month of therapy , with resolution of baseline diarrhea and nausea , and with appetite improvement . 
at 17 months , an mri of the brain showed an isolated , new , 5 - mm left caudate lesion that was treated with srs ( fig 2e , left and right panels ) with continuation of selpercatinib . 
at 20 months , imaging revealed non - cns multifocal progression with a new 6 - mm bony lesion in the c7 vertebral body , as well as several new , right - side liver lobe lesions ( the largest measured 2 cm )  . 
the patients calcitonin level rose to 235.9 pg / ml ; her cea level remained unchanged . peripheral blood cell - free dna analysis at the time of systemic progression revealed only the presence of the previously identied ret m918t missense mutation . 
the patient declined a liver biopsy . cns disease remained in response until 22 months , when several new brain lesions were found to have developed ; these were managed with srs ( fig 2 )  . 
her treatment course has been complicated by several hospitalizations unrelated to selpercatinib . discussion metastatic dissemination of mtc to the cns is a rare but devastating complication associated with poor prognosis and no well - established treatment strategies.1 although limited evidence from previous reports suggests that surgical resection and / or radiation therapy may be used in select cases , there remains a strong need for novel treatment modalities with durable therapeutic responses.1 - 3 cns efcacy of multikinase inhibitors such as vandetanib or cabozantinib has not been reported in mtc . here , we present a report of successful treatment of the brain and leptomeningeal metastases with systemic therapy in a patient with ret - mutated mtc , using the purpose - built selective and cns - penetrant ret inhibitor selpercatinib . 
treatment with this agent resulted in complete and durable resolution of the measurable brain and leptomeningeal lesions in a patient with previous disease progression while she received cabozantinib and radiation therapy . 
 case report notably , the patients tumor harbored a heterozygous deletion in the cdkn2c gene linked to an aggressive disease phenotype in mtc.19 cdkn2c is a member of the ink4 family of cyclin - dependent kinase inhibitors that block the cell cycle progression in the g1 phase through interaction with cyclin - dependent kinases 4 and 6.19 cdkn2c is lost in approximately 40% to 50% mtcs and is associated with a signicantly shorter time to distant metastasis and decreased overall survival in mtc , an effect further enhanced by concomitant ret m918t mutation.19 , 21 in addition , an earlier report showed that cdkn2c haploinsufciency is associated with a strong trend toward reduced progressionin ret - mutated mtc treated with systemic free survival therapy.21 however , this relationship has not been studied in patients receiving selective ret inhibitors , to our knowledge , and the signicance of cdkn2c loss in this setting remains to be determined . the patient is continuing selpercatinib therapy , with gradual disease progression in the liver . 
a recent study demonstrated that ret solvent - front mutations ( ret g810s / g810r / g810c ) can cause on - target resistance to selective and multikinase ret inhibitors.22 a better understanding of mechanisms of acquired resistance to selective ret inhibitors and frequency of resistant alterations would guide the development of novel agents and combination therapies directed towards further improvement of treatment outcomes for patients with ret - altered malignancies . in conclusion , we observed a radiographically conrmed durable response with selpercatinib in a patient with a heavily pretreated ret m918tmutated mtc with extensive cns metastases . 
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nature 367 : 375 - 376 , 1994 elisei r , cosci b , romei c , et al : prognostic signicance of somatic ret oncogene mutations in sporadic medullary thyroid cancer : a 10 - year follow - up study . j clin endocrinol metab 93 : 682 - 687 , 2008 beldjord c , desclaux - arramond f , rafn - sanson m , et al : the ret protooncogene in sporadic pheochromocytomas : frequent men 2 - like mutations and new molecular defects . 
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the patient subsequently underwent neoadjuvant radiation in addition to receiving weekly carboplatin and paclitaxel , followed by esophagogastrectomy.7 pathology revealed moderately differentiated adenocarcinoma invading the adventitia ( pt3 ) , but none of the 14 lymph nodes were involved ( pn0 )  . 
one and a half years after resection , surveillance imaging revealed new concerning hypodensity in the liver , accompanied by an increase in the carcinoembryonic antigen ( cea ) levels . 
next - generation sequencing ( ngs ) was performed on biopsies from the time of initial and recurrent liver metastasis using the commercially available comprehensive genomic proling ( cgp ) assay from foundation medicine ( cambridge , ma ; table 1 )  . 
serial blood collections were also obtained to determine the presence of ctdna using a tumorinformed multiplex polymerase chain reaction - ngs assay from natera ( san carlos , ca ) designed from tumor genomic alterations ( table 1 )  . 
at the time of disease recurrence in the liver , the tumor - informed assay with an established analytical sensitivity5 revealed elevated levels of ctdna , measured in mean tumor molecules / ml of plasma ( fig 1 )  . the patient underwent the patient approximately 760 days postdiagnosis , received 6 cycles ( c6 ) of uorouracil ( fu ) , leucovorin , and oxaliplatin ( mfolfox6 ) , with radiographic response in the liver lesion . 
after another year ( approximately 1 , 490 days postdiagnosis ) , the patient developed a new fdgavid mass in the ablation cavity , accompanied by an increase in cea level . 
the patient received 1 cycle ( c1 ) of fu and leucovorin ( fu / lv ; approximately 1 , 520 days postdiagnosis ) , complicated by oral mucositis , followed by resection of the liver mass ( approximately 1 , 580 days postdiagnosis ) , and pathologically conrmed to be metastatic eac . 
after metastasectomy , surveillance imaging showed no evidence of recurrence , ctdna levels became negative , and the patient remains in remission , currently 11 months ( approximately 320 days ) postmetastasectomy . 
the use of more sensitive cancer detection methods may help optimize treatment approaches ( escalation or de - escalation of therapies ) in the localized setting . in this case study , the patient experienced oligometastatic recurrence despite aggressive trimodality therapy . 
although we were unable to assess ctdna in the localized setting for this patient , a recent study investigated ctdna in the context of neoadjuvant chemoradiation for localized esophageal cancer and showed that detectable ctdna levels postchemoradiation , along with metabolic imaging analysis , predicted tumor progression with 100% sensitivity versus ctdna alone ( 71% ) and imaging alone ( 57% ) .15 thus , detectable , ctdna could help clarify ambiguous ndings ( as observed in this case study ) and could help identify high - risk patients with mrd who could be upper limit of normal cea 1 , 000 0.01 1 , 370 1 , 523 1 , 677 1 , 826 1 , 219 time since diagnosis ( days ) 1 , 066 no evidence of disease or disease response ctdna - positive ( mtm / ml ) liver metastasis suspicious finding , subsequently resolved or stable ctdna - negative ( mtm / ml ) mfolfox6 suspicious finding , subsequently confirmed cea ( ng / ml ) fluorouracil / leucovorin fig 1 . 
another study evaluated prognosis based on the presence of detectable ctdna after neoadjuvant chemotherapy prior to cystectomy in patients with bladder cancer.3 the preoperative ctdna status was 100% sensitive in predicting pathologic complete response , suggesting that trials could strategize testdeferred surgery in patients with undetectable ctdna postneoadjuvant therapy . in this case study , the initial plan was to proceed with chemotherapy ( mfolfox6 ) , followed by metastasectomy for removal of any residual disease ; however , the latter was delayed because of the ambiguous ndings that showed enlarged pulmonary / soft tissue nodules ( table 2 ; fig 1 ) , requiring further follow - up . 
retrospectively , serial ctdna analysis suggested that chemotherapy was more effective than rfa in reducing the tumor burden , and thus , consideration of consolidation metastasectomy in lieu ofor soon afterrfa could have prevented the second local recurrence . 
 ( * ) brd4 and smad4 have 3 missing data points between september 2017 and june 2018 . mfolfox6 , 6 cycles of uorouracil , leucovorin , and oxaliplatin . this case study illustrates the potential advantage of using ctdna as a conrmatory , adjunctive surveillance tool . 
the early and continued increase in ctdna post - rfa showed the sensitivity and specicity of the ctdna assay in predicting disease recurrence by a lead time of 174 days compared with cea and 350 days before radiographic conrmation . 
cea is known to be a lagging indicator of disease and is relatively insensitive , nonspecic , and can be confounded by acute or chronic inammation , smoking , liver disease , and diabetesall common comorbidities in this patient population.16 in this particular case , the multiple postresection complications confounded the interpretation of an increase in cea , an increase that ultimately attenuated and has not translated into relapse by imaging or ctdna with 11 months of follow - up . 
this could be a faster and more accurate measure than following the standard approach of using radiographic progression - free survival alone . it is important to note that the tumor - informed ctdna assay is a personalized method that requires separate whole - exome sequencing of the tumor tissue to develop patient - specic primers . 
the customized design of this assay targeting a set of clonal mutations known to be present in the specic tumor leads to its high sensitivity and specicity ; it is not intended to be a discovery assay for assessment of actionable genomic targets , but rather , a tool for detecting mrd . 
it was notable to observe that genomic alterations in the primary tumor were detectable with the tumor - informed ctdna assay at the time of relapse , and they all appeared to track in parallel ( fig 2 )  . 
we did not observe evidence of signicant selection for or against clones harboring any of the genomic targets tested during treatment , but we cannot rule out the acquisition of additional subclonal genomic alterations . our study has certain limitations . 
another aspect is how often and how long a patient should be monitored in this setting . although tumor burden may be undetectable by the ctdna assay , the patient is still at risk and needs surveillance . however , given the features described , it is reasonable to test this assays utility in multiple disease settings as part of clinical trials in esophageal and other cancers . 
einstein research funding : trovagene ( inst ) , bristol - myers squibb ( inst ) nathan liang employment : natera stock and other ownership interests : natera meenakshi malhotra employment : natera stock and other ownership interests : natera alexey aleshin employment : natera stock and other ownership interests : natera consulting or advisory role : mission bio solomon moshkevich employment : natera , evidation health ( i ) stock and other ownership interests : natera patents , royalties , other intellectual property : named as inventor on a patent led by natera travel , accommodations , expenses : natera paul r . 
billings employment : natera leadership : trovagene , biological dynamics , omniseq , alveo biotechnologies stock and other ownership interests : natera , trovagene , biological dynamics , omniseq , alveo biotechnologies consulting or advisory role : bethesda group , guidepoint global eirini pectasides consulting or advisory role : clovis oncology research funding : bristol - myers squibb no other potential conicts of interest were reported . references shah m , denlinger cs : optimal post - treatment surveillance in cancer survivors : is more really better ? oncology ( williston park ) 29 : 230 - 240 , 2015 2 . 
natl j maxillofac surg 7 : 17 - 20 , 2016 christensen e , birkenkamp - demtr oder k , sethi h , et al : early detection of metastatic relapse and monitoring of therapeutic efcacy by ultra - deep sequencing of plasma cell - free dna in patients with urothelial bladder carcinoma . 
j clin oncol 37 : 1547 - 1557 , 2019 abbosh c , birkbak nj , wilson ga , et al : phylogenetic ctdna analysis depicts early - stage lung cancer evolution . 
nature 545 : 446 - 451 , 2017 [ erratum : nature 554 : 264 , 2018 ] coombes c , page k , salari r , et al : personalized detection of circulating tumor dna antedates breast cancer metastatic recurrence . 
clin cancer res 25 : 14255 - 4263 , 2019 reinert t , henriksen tv , christensen e , et al : analysis of plasma cell - free dna by ultradeep sequencing in patients with stages i to iii colorectal cancer . 
jama oncol 5 : 1124 - 1131 , 2019 shapiro j , van lanschot jjb , hulshof mccm , et al : neoadjuvant chemoradiotherapy plus surgery versus surgery alone for oesophageal or junctional cancer ( cross ) : long - term results of a randomised controlled trial . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american pathologists joint review . 
arch pathol lab med 142 : 1242 - 1253 , 2018 araujo dv , bratman sv , siu ll : designing circulating tumor dna - based interventional clinical trials in oncology . 
bray f , ferlay j , soerjomataram i , et al : global cancer statistics 2018 : globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries . 
hagerty2 ; and amanda gammon , ms1 , 2 purpose to compare the classication of genetic variants reported on tumor genomic proling ( tgp ) reports with germline classications on clinical test results and clinvar . 
somatic variant interpretations were compared with classications from germline test results as well as with clinvar interpretations . results of the 623 variants identied on tgp , 353 had a denitive classication in clinvar , and 103 were assayed with a germline test , with 66 of the variants tested observed in germline . 
analysis of somatic variants of uncertain signicance listed on tgp reports determined that 22% had a different interpretation compared with clinvar and that 32% differed from the interpretation on a germline test result . 
pathogenic variants on tgp test results were found to differ 13% and 5% of the time compared with clinvar interpretations and germline test results , respectively . conclusion these results suggest that tgp variants are often classied differently in a germline context . differences may be due to different processes in variant interpretation between somatic and germline laboratories . 
2019 by american society of clinical oncology introduction tumor genomic proling ( tgp ) is the application of next - generation sequencing to tumor samples for the purpose of targeted cancer treatment.1 tgp can aid in informing clinicians of treatment options because it allows for the identication of molecular targets for clinical trials or nonconventional therapy options . 
for example , tumors with a brca1 or brca2 pathogenic variant are more likely to respond to pharmaceuticals , like poly ( adp - ribose ) polymerase inhibitors , so the identication of one of these variants on tgp can lead to a different treatment option.2 , 3 other pharmaceuticals have been shown to be effective in tumors with defective mismatch repair.4 in contrast , germline genetic testing analyzes blood or saliva samples for genes associated with hereditary cancer predisposition syndromes . 
tgp and germline tests overlap in their analysis of hereditary cancer genes , and variants identied in a tumor on tgp may also be present in the germline.5 , 6 studies have shown that between 3% and 16% of tgp samples carry a pathogenic germline variant.7 , 8 asco recommends that physicians communicate incidental germline ndings to patients , which may lead to secondary germline analysis.1 thus , germline variants may inform treatment , and somatic variants may inform germline testing . 
given this overlap , it is crucial to consider classication of the variants because they may differ depending on the test type . tgp and germline testing have different purposes and indications as well as different recommended guidelines for variant interpretation . 
 moody et al context key objective are differences in variant interpretation between somatic and germline cancer genetic testing observed in a clinical setting ? knowledge generated both pathogenic mutations and variants of uncertain signicance found on somatic tumor testing have different classications when considered in a germline context . 
variants of uncertain signicance found on somatic tumor testing are more likely to have a different classication in germline than in pathogenic mutations . relevance health care providers may carefully consider both pathogenic and uncertain ndings on somatic tumor testing when they are found in genes that have germline relevance . 
these genes were selected on the basis of the wellestablished cancer risks and clear management recommendations associated with germline pathogenic variants as illustrated in current national comprehensive cancer network guidelines.9 , 10 variants were excluded if a specic nucleotide change was not reported for the variant , such as for whole - exon rearrangements or full gene loss , because these variants are less likely to be present in germline and often follow a different classication schema . 
study protocols with regard to patient data were subject to institutional review board approval ( university of utah )  . comparison with clinvar classications clinvar is a public archive of genetic variation that facilitates comparisons of variant classications between laboratories and research initiatives . 
classication categories in clinvar are consistent with those listed in the american college of medical genetics recommendations11 and include benign , likely benign , variant of uncertain signicance ( vus ) , likely pathogenic , and pathogenic . 
this facilitated an easier comparison with somatic classications since the tgp results only listed variants as pathogenic or uncertadifferences in classication between tumor and germline testing were considered clinically signicant if medical management recommendations would be made on the basis of one classication but not the other . 
a variant that is vus on one result and benign on another would not be clinically signicant . comparison with germline classications a subset of patients who had tgp also underwent germline testing within the study time frame . 
when results did not indicate any variant and were considered negative , germline testing laboratories were contacted to determine whether the somatic variant found on tgp was present in the germline and to determine its classication at the time of testing . 
variants were identied in 31 different tumor types , the majority of which were found in brain ( 19% ) , colorectal ( 15% ) , and prostate ( 13% ) tumors . 
frameshifts , nonsense , and in - frame insertions / deletions made up , 3% of the remaining vus variants , and no vus splice variants were seen ( fig 1 )  . somatic classications compared with clinvar of the 623 somatic variants found on tgp , 353 ( 56.7% ) had a denitive germline classication in clinvar . 
the majority of these somatic vuss were also classied as vuss in clinvar ( 203 of 260 ; 78.1% ) , while 48 somatic vus variants ( 18.5% ) were classied as benign and nine ( 3.5% ) as pathogenic in clinvar ( fig 2 )  . the additional 93 somatic variants analyzed in clinvar were classied as pathogenic on the tgp test result . 
of these differences , 30.4% ( 21 of 69 ) would result in different management recommendations and so are considered to have a clinically signicant difference in interpretation for this study ( table 1 )  . somatic classications compared with classications on germline test results of the 623 somatic variants assessed , 103 ( 16.4% ) were assayed using a germline test , and 66 ( 64% ) were observed in germline . 
the majority of these somatic vuss were also classied as vuss in the germline test results ( 32 of 47 ; 68.1% ) , while 12 ( 25.5% ) were classied as benign and three ( 6.4% ) as pathogenic in the germline test results . 
comparison of pathogenic tgp variants with the germline test results found that 18 ( 94.7% ) of 19 were also reported as pathogenic in the germline and only one ( 5.3% ) was classied as vus in the germline ( fig 2 )  . 
clinically signicant differences in interpretation were seen with four ( 25% ) of 16 variants ( table 2 )  . we assessed how many of the germline test results were returned after somatic test results were obtained to investigate the impact of tgp variant classication on clinic ow . 
the majority of patients had at least one pathogenic somatic mutation . however , when the patients who had germline testing after somatic testing were subdivided by length of time , those who had germline testing  . 
90 days after somatic testing were more likely to have solely a vus somatic result , which suggests that patients in this data set were not referred for germline testing on the basis of a vus found on tgp . discussion the results of this study demonstrate distinct differences in the interpretation of variants found in tgp test results compared with clinvar and clinical germline test results . specically , 23% and 32% of somatic variants classied as vus and 13% and 5% of somatic variants classied as pathogenic on tgp had a different interpretation in clinvar and germline test results , respectively . 
the tgp report suggests that the variant is not associated with targeted cancer therapy , and thus , no this result . action was recommended on the basis of however , a germline result that shows a pathogenic mutation in mlh1 does warrant changes to medical management recommendations because this patient would meet national comprehensive cancer network criteria for a diagnosis of lynch syndrome and increased cancer screening , and consideration of risk - reducing surgeries associated with mlh1 pathogenic variants would be indicated.10 in addition , these germline test results indicate that this patients family members also may have signicant cancer risks , and genetic testing may now be indicated for them if it was not previously . 
if germline testing had not been pursued after reviewing the tgp results , this important risks to information about additional cancer the patient and his or her family would not have been realized , which highlights the importance of providers understanding differences in germline and somatic variant interpretation . 
these results show that clinvar is a source of information for tgp variants that may have different interpretations in germline . tgp and germline genetic tests have different indications and outcomes and , as such , have different published variant interpretation guidelines . 
thus , discrepancies in classications of specic variants between tgp and germline laboratories do not necessarily indicate that one classication is incorrect : the tests are being used for different purposes , and the classication models used reect this difference . 
clinvar listed this variant as benign , and no submitter referenced the variant being found in a tumor . this demonstrates the utility of somatic variant databases in the classication of variants found in tgp and highlights how using uniquely somatic sources of evidence can lead to a different classication than in germline . in contrast , the purpose of a germline genetic test is to diagnose hereditary cancer predisposition syndromes and aid in the prediction of the risk of cancer in a patient . professional guidelines published in 2015 by the american college of medical genetics and genomics and the association for molecular pathology list sources of evidence unique to germline interpretation.11 these include segregation information , in trans and de novo ndings , and patient phenotype data . 
these guidelines also include many sources of information that are similarly listed in somatic classication guidelines , such as mutation type , minor allele frequency , germline variant databases , published functional and population studies , and in silico prediction tools ( fig 3 )  . 
these lines of evidence are not currently used in somatic variant classication , which may lead to differing classications for this variant . it is possible that the difference in variant interpretation guidelines between somatic and germline do not account for all of the differences in interpretation found in this study . disagreement with regard to variant interpretation in a strictly germline context has been previously demonstrated.14 - 17 disagreement between germline testing laboratories has been reported to be as high as 26%15 and disagreement between germline databases as  . 
30%.16 in a study where four germline laboratories agreed to share internal data and discuss their interpretation rationales , there was still a 13% difference in interpretation because of variation in interpreting guidelines.17 the rates of disagreement found in the literature are consistent with the ndings of the current study . 
the subjective nature of guideline interpretation previously demonstrated in germline variant classication may also contribute to the differences seen in this study between somatic and germline variant interpretation . another known difculty in variant interpretation is classifying missense variants . 
the gathering of data to help to classify missense vuss is difcult for both somatic and germline classications and may further contribute to the differences seen in this study . this study had some limitations . it was performed at a single center and included tgp results from a single commercial laboratory . 
in addition , the majority of the tgp test results were gathered before the publication of the somatic variant interpretation guidelines.12 updated classications could not be obtained from the tgp testing identied the somatic mutations in this laboratory that study , and it is unclear whether the interpretations of the variants reported in this study are now different on the basis of these guidelines . 
additional research is required to determine whether the rate of difference found in this study is generalizable to a wider patient population . family history , referral in conclusion , although literature has been published on variant interpretation in both the somatic and the germline context , none has compared variant interpretations between the two methods . 
we found that somatic vus reported on a tgp test have a different interpretation in 57 ( 22% ) of 260 cases compared with clinvar and in 15 ( 32% ) of 47 cases compared with germline test results . 
pathogenic mutations from tgp test results were found to have a different interpretation in 12 ( 13% ) of 93 variants compared with clinvar and in one ( 5% ) of 19 variants compared with germline test results . understanding the observed differences in variant classications as well as the philosophies behind somatic and germline variant interpretation may inform clinicians as they order germline testing for patients . 
moody , jennie vagher , whitney espinel , david goldgar , amanda gammon administrative support : amanda gammon provision of study material or patients : jennie vagher , whitney espinel , amanda gammon collection and assembly of data : emily w . 
 moody et al open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . jennie vagher consulting or advisory role : erin mundt , cgc no other potential conicts of interest were reported . acknowledgment this article is based on a research project conducted by e.w.m. 
we thank the university of utah graduate program in genetic counseling . references robson me , bradbury ar , arun b , et al : american society of clinical oncology policy statement update : genetic and genomic testing for cancer susceptibility . j clin oncol 33 : 3660 - 3667 , 2015 2 . 
mccabe n , turner nc , lord cj , et al : deciency in the repair of dna damage by homologous recombination and sensitivity to poly ( adp - ribose ) polymerase inhibition . 
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lancet 376 : 235 - 244 , 2010 le dt , durham jn , smith kn , et al : mismatch repair deciency predicts response of solid tumors to pd - 1 blockade . 
science 357 : 409 - 413 , 2017 jain r , savage mj , forman ad , et al : the relevance of hereditary cancer risks to precision oncology : what should providers consider when conducting tumor genomic proling ? j natl compr canc netw 14 : 795 - 806 , 2016 6 . 
hall mj , daly mb , ross ea , et al : germline variants in cancer risk genes detected by ngs - based comprehensive tumor genomic proling ( cgp )  . 
j clin oncol oncol 27 : 795 - 800 , 2016 33 , 2015 ( suppl ; abstr 11084 ) schrader ka , cheng dt , joseph v , et al : germline variants in targeted tumor sequencing using matched normal dna . 
jama oncol 2 : 104 - 111 , 2016 [ erratum : jama oncol 2 : 279 , 2016 ] daly mb , pilarski r , berry mp , et al : genetic / familial high - risk assessment : breast and ovarian , 2019 . 
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 exceptional response to akt inhibition in patients with breast cancer and germline pten mutations belinda kingston , mbchb1 ; caroline bailleux , md2 ; suzette delaloge , md , msc2 ; gaia schiavon , md , phd3 ; veronique scott , phd2 ; magali lacroix - triki , md , phd2 ; t . 
hedley carr , phd3 ; iwanka kozarewa , phd3 ; heidrun gevensleben , md4 ; zoe kemp , phd5 ; alex pearson , phd1 ; nicholas turner , md , phd1 , 5 ; and fabrice andr e , md , phd2 introduction cowden syndrome is an autosomal dominant genetic disease with an estimated incidence of one in 200 , 000 . 
pip3 functions as a secondary messenger in the pi3k pathway that binds and activates proteins that have a pleckstrin homology domain , such as akt1 , and triggers their activation and localization to the plasma membrane , promoting cellular proliferation and survival.4 germline pten loss - of - function mutations may result in dominant akt activation as a driving oncogenic event in cowden - related breast tumors.5 preclinical evidence suggests that cancers with akt activation have increased sensitivity to akt inhibition.6 preliminary clinical evidence is derived from phase i and ii trials in patients with breast cancers bearing somatic mutations in the pi3k / akt / mtor pathway.7 - 11 inhibitor of all three isoforms of capivasertib ( azd5363 , astrazeneca ) is a potent and selective oral serine / threonine kinase akt ( ie , akt1 , 2 , 3 ) and has preclinical evidence of efcacy as monotherapy or in combination with cytotoxic and targeted therapies.12 - 15 despite the encouraging progression - free survival observed with capivasertib monotherapy in heavily pretreated patients with akt1 e17kmutant breast and gynecologic cancers , 9 recist response rates in phase i studies only reached 22% ( table 1 ) .8 , 9 , 16 because pten loss activates akt1 , we hypothesized that tumors from patients with cowden syndrome could be sensitive to this drug family . which was estrogen ( er ) and progesterone receptor negative , human epidermal growth factor receptor 2 ( her2 ) negative , and was designated grade iii invasive carcinoma of no special type ( nst )  . 
the patient received six cycles of neoadjuvant cyclophosphamide 600 mg / m2 , epirubicin 75 mg / m2 , and docetaxel 100 mg / m2 before a mastectomy with left axillary lymph node dissection ( revealing residual disease in 10 of 18 lymph nodes ) and adjuvant radiotherapy . eight months later , the patient experienced relapse with cutaneous disease and thoracic nodal volvement . 
after enrolling in safir02 ( clinicaltrials.gov identier : nct02299999 ) , targeted panel sequencing ( ion torrent pgm ; thermo fisher scientic , villebon , france ) of a fresh tumor biopsy sample revealed the presence of a heterozygous germline pten mutation ( c.389g.a , rs121909229 ) alongside other variants ( fig 1a ; data supplement )  . 
in the context of safir02 , the patient was randomly assigned to maintenance targeted therapy and received capivasertib 480 mg twice per day , 4 days on and 3 days off . 
on the right side , she presented with a t3 , erpositive and progesterone receptorpositive , her2negative , grade ii invasive carcinoma nst with 20 of 23 involved lymph nodes . 
in may 2010 , the patient received six cycles of chemotherapy every 3 weeks ( uorouracil 600 mg / m2 , epirubicin 75 mg / m2 , and cyclophosphamide 600 mg / m2 ) before starting maintenance tamoxifen . 
she received eight cycles of paclitaxel ( 90 mg / m2 on days 1 , 8 , and 15 of a 28 - day cycle ) combined with capivasertib ( 360 mg twice per day on days 2 - 5 , 9 - 12 , and 16 - 18 of each 28 - day in june 2013 , a computed tomography scan cycle )  . showed a complete response of the liver metastases . 
she had a conrmed maintained response in may 2014 until progression occurred in june 2014a progression - free survival of 19 monthsand a maintained complete response for 12 months on capivasertib alone ( fig 2c )  . 
plasma circulating tumor dna analysis from baseline and progression time points during the beech study showed no major changes ( data supplement )  . discussion the akt inhibitor capivasertib has been examined in early - phase trials ( table 1 ) , and no complete responses have been noted , yet both patients with cowden syndrome presented here had durable complete responses to capivasertib . 
cdc14 , phosphatase domaillustration from ( b ) immunohistochemistry demonstrating ( i ) absent pten staining in the tumor and ( ii ) cytoplasmic and membranous expression of pakt in the 40% of tumor cells . 
cdc14 , phosphatase domaillustration from ( b ) pten immunohistochemistry from ( i ) control tissue and ( ii ) noncancerous and ( iii and iv ) tumor - containing lymph node . 
 ( c ) computed tomography ( ct ) scans during the patients time on capivasertib , with white arrows indicating disease in the liver : ( i ) october 2012 , baseline ct demonstrating a liver deposit ; ( ii ) january 2013 , ct following two cycles of paclitaxel and capivasertib ; ( iii ) ct demonstrating continued complete response on maintenance capivasertib 7 months after cessation of paclitaxel ; and ( iv ) may 2014 , nal ct before progression june 2014 . nonmalignant cells carrying a germline pten mutation to pi3k - pathway inhibition . 
similar to the cases presented here , no severe toxicity was noted in the pilot study , 21 with just one patient experiencing a grade 3 adverse event ( hypophosphatemia / hypercholesterolemia )  . in recent years , drugs targeting the pi3k / akt / mtor pathway have been developed . 
in contrast , akt inhibitors in combination with paclitaxel have been shown to be more active in patients with triple - negative breast cancers harboring a pik3ca / pten / akt1 pathway alteration.7 , 10 similarly , pi3k inhibitors have demonstrated activity in patients with pik3ca mutations.22 , 23 of note in case 2 is the presence of the second - hit pten stop - gain mutation y88x , present with an af of 25.5%. however , the presence of a tp53 mutation at an af of 51.9% indicates that this second - hit mutation is subclonal rather than a truncal driver mutation . 
explanations for the pten phenotype in case 1 include undetected loh , pten promotor hypermethylation , 24 , 25 complex pten genomic rearrangements , 26 and post - translational modication.27 contrary to the two - hit model by knudson et al28 of tumorigenesis in tumor suppressor genes , pten aberrations appear to be protumorigenic in the absence of a second hit . in 2010 , alimonti et al29 demonstrated that pten hypermorphic mice ( with 80% of the normal pten protein level ) had a greater propensity to tumorigenesis than mice with two functional alleles but were less tumorigenic than pten heterozygous mice , supporting a haploinsufciency model of tumorigenesis in pten aberrations . a later in vivo study of pten knock - in mouse models suggested that the conformation of pten underlies the dominant - negative behavior of pten heterozygous mutants . 
this supports the rationale that pten heterozygous mutants act in a dominant - negative manner to promote tumorigenesis . the patients in the cases presented here were treated with combination chemotherapy and capivasertib followed by capivasertib monotherapy . 
the patient in case 1 had previously demonstrated tumor resistance to taxane therapy , with residual disease after neoadjuvant chemotherapy and a short progression - free survival after treatment of primary breast cancer . 
the second patient achieved a complete response with the combination of paclitaxel and capivasertib and maintained this complete response for a period of 12 months on capivasertib alone , suggesting that capivasertib was highly active in this patient . in summary , these two patients with breast cancer and different germline pten mutations both showed a dramatic response to capivasertib superior to that seen in early trials of the drug . 
 magali lacroix - triki leadership : mypl stock and other ownership interests : mypl honoraria : myriad genetics , genomic health consulting or advisory role : roche travel , accommodations , expenses : agendia t . 
hedley carr employment : astrazeneca stock and other ownership interests : astrazeneca iwanka kozarewa employment : astrazeneca stock and other ownership interests : astrazeneca kingston et al zoe kemp honoraria : lilly honoraria : astrazeneca nicholas turner consulting or advisory role : roche , novartis , astrazeneca , pzer , bicycle therapeutics , bristol - myers squibb , merck sharp & dohme , tesaro , lilly research funding : pzer ( inst ) , roche ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , bio - rad ( inst ) , guardant health ( inst ) fabrice andr e research funding : astrazeneca ( inst ) , novartis ( inst ) , pzer ( inst ) , lilly ( inst ) , roche ( inst ) , daiichi ( inst ) travel , accommodations , expenses : novartis , roche , glaxosmithkline , astrazeneca no other potential conicts of interest were reported . references tan mh , mester jl , ngeow j , et al : lifetime cancer risks in individuals with germline pten mutations . 
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baselga j , dent sf , cort es j , et al : phase iii study of taselisib ( gdc - 0032 ) + fulvestrant ( fulv ) v fulv in patients ( pts ) with estrogen receptor ( er ) - positive , pik3ca - mutant ( mut ) , locally advanced or metastatic breast cancer ( mbc ) : primary analysis from sandpiper . 
thall , phd1 purpose despite the fact that almost any sample of patients with a particular disease is heterogeneous , most clinical trial designs ignore the possibility that treatment or dose effects may differ between prognostic or biologically dened subgroups . 
the purpose is to illustrate the benets of accounting prospectively for treatment - subgroup interactions and how utilities may be used to quantify risk - benet trade - offs . methods two bayesian clinical trial designs that perform subgroup - specic decision making and inference based on elicited utilities of patient outcomes are reviewed . 
the second is a sequentially adaptive trial of natural killer cells for treating hematologic malignancies that is based on ve time - to - event outcomes and that performs safety monitoring and optimizes cell dose within six disease subgroups . 
similarly , qualitatively different disease subtypes or biologically dened subgroups typically are ignored by the decision rules and hypotheses of conventional trial designs , aside from possibly stratifying on subgroups when randomly assigning patients . 
this problem persists in all phases of conventional trial designs , from early - phase dose - nding to randomized comparative trials . to illustrate the problem , consider a prospective randomized trial in patients with relapsed or refractory leukemia , with the aim of evaluating the effect on overall survival ( os ) of adding a new targeted agent , x , to standard chemotherapy . 
suppose that the historical mean os with chemotherapy is old = 15 months in older patients ( age 60 years or older ) and young = 24 months in younger patients ( age younger than 60 years ) , with two thirds of patients being older than age 60 years and one third being younger . 
a group sequential design comparing chemotherapy + x with chemotherapy constructed on the basis of the average mean os , avg = ( 2 / 3 ) old + ( 1 / 3 ) young , would have null mean os avg 0 = ( 2 / 3 ) 15 + ( 1 / 3 ) 24 = 18 months . suppose a design is constructed to target an improvement of 33% from avg 0 = 18 to avg * = 24 months , equivalently , a targeted hazard ratio of .75 in favor of chemotherapy + x . 
 thall context key objective an important question in designing and conducting clinical trials is whether and how a trial design should account explicitly and prospectively for patient heterogeneity when making treatment assignment decisions and reaching nal conclusions . the two designs reviewed in this article illustrate how a bayesian utility - based framework can be used to construct practical designs that account for patient heterogeneity . knowledge generated each of the two designsone is a randomized comparative trial and the other is a complex dose - nding trialis a novel methodology that has been published in the statistical literature . 
an aim of this review is to make members of the medical community aware that such designs exist so that they may consider taking this sort of approach in their own trials . relevance the bayesian utility - based approach may be used to construct designs for a wide variety of clinical trials that account for patient heterogeneity by making subgroup - specic , personalized treatment decisions . 
in the presence of patient heterogeneity , this approach renes treatment development and evaluation and thus is more likely to protect safety and provide benet for both the patients enrolled in the trial and future patients . chemotherapy alone in younger patients . 
suppose that , in fact , adding x to chemotherapy decreases mean os in older patients by 10% to 13.5 months but increases mean os in younger patients by 30% to 31.2 months , that is , there is a treatment - age subgroup interaction . 
thus , a group sequential test based on average mean os would be likely to conclude that adding x to chemotherapy fails to improve os , thus making a false - negative conclusion in younger patients . although post - trial data analyses accounting for age may show that chemotherapy + x is superior in younger patients , such an inference may be dismissed as post hoc data dredging , that is , searching the data set for subgroups in which the x effect is nominally signicant . 
arguing for the future use of chemotherapy + x in younger patients thus would be problematic , and it probably would be necessary to repeat the trial , if possible . this problem , consider a trial similar problems arise in early - phase trials in which the goal is to identify an optimal dose , dose pair , or ( dose , schedule ) combination for a new agent or treatment combination at the start of clinical evaluation . 
it has been well established that choosing a dose in a phase i trial on the basis of toxicity alone is a highly awed convention that can easily lead to choosing an unsafe or ineffective dose.1 , 2 as a rst example to optimize the dose d among ve levels ( 1 , 2 , 3 , 4 , 5 )  . 
if the continual reassessment method ( crm ) 3 is used to choose a dose with pr ( toxicity ) closest to .30 , then d = 3 is most likely to be chosen , whereas a 3 + 3 algorithm is most likely to choose either d = 2 or d = 3 . 
in practice , the best and worst outcomes are assigned respective utilities of 100 and 0 , and numerical utilities between these two values for termediate outcomes are elicited from the investigators planning the trial . 
during trial conduct , the posterior mean of uavg ( d , g ) is computed for each d , and the dose giving the largest value is selected for the next good - prognosis patient enrolled . 
the outcome is y = clavien - dindo postoperative morbidity ( pom ) , 7 - 10 scored within 30 days of surgery , a six - level ordinal variable with possible integer values from 0 ( normal recovery ) to 5 ( death )  . 
moreover , because differences in utilities between successive pom levels are far from being equal , using the raw scores 0 , 1 , 2 , 3 , 4 , 5 would also be a misleading way to quantify pom . the design accounts for patient heterogeneity by allowing possibly different conclusions within the subgroups p and s when comparing treatment n to c . 
a key property of the model is that all treatment and subgroup effect parameters vary with the level of pom score , so it is a robust generalization of the conventional proportional odds model.11 the model allows the magnitudes of the n - versus - c effects on y to differ between subgroups and is formulated to borrow strength between subgroups , with prior location parameters determined from elicited information on pom score . 
details are provided in murray et al.6 the trial will enroll a maximum of 200 patients , with an interim comparative test of the n - versus - s effect on pom score after n = 100 patients have been treated and evaluated and possibly a nal test at n = 200 within each subgroup p and s . 
elicited numerical utilities of 30 - day pom scores after esophageal resection pom score utility abbreviation : pom , postoperative morbidity . the subgroup - specic comparative tests , computed under the bayesian model , conclude that n is superior to c in subgroup g = p or s if ( cid : 4 ) ( cid : 1 ) ( cid : 3 ) ( cid : 1 ) uavg n , g  . 
the numerical cutoffs pcut ( 100 ) = .997 and pcut ( 200 ) = .976 were chosen by conducting the overall preliminary computer simulations to control within - subgroup type i error probability of the group sequential test procedure to be .025. 
to ensure balance and comparability with either design , patients were randomly assigned in blocks of four , so for each block of four patients within each prognostic subgroup , two receive nuprehab and two receive the control . the operating characteristics of the two designs are summarized in table 2 . 
the goals are to perform safety monitoring and to identify an optimal nk cell dose from the set { 105 , 106 , 107 } cells per kg of body weight ( hereafter dose levels 1 , 2 , 3 ) within each of the subgroups , while borrowing strength between subgroups . 
this is an example of a sequentially adaptive precision or personalized clinical trial . the trial has ve coprimary outcomes : the times from nk cell infusion to d = death , and the four nonfatal events 4 2019 by american society of clinical oncology p = disease progression , r = response , t = severe toxicity , and c = severe cytokine release syndrome . 
each patients events are monitored for a 365 - day follow - up period . denoting the time to event j by yj for j = d , p , r , t , c for each patient , each yj is a potential outcome because it may or may not be observed ; yp and yr are competing risks because , at most , one of these two events can occur ; the ve potential event times are highly associated with each other ; d censors any events that have not occurred , but such censoring is not independent ; and the distribution of y = ( yd , yp , yr , yt , yc ) varies substantially with disease subgroup . three challenges in designing an nk cell dose - nding trial are ( 1 ) if one ignores the subgroups , then there are high probabilities of making incorrect decisions within subgroups ; ( 2 ) on the basis of current knowledge about nk cell biology , the rates of the ve outcomes are neither monotone increasing nor decreasing in nk cell dose ; and ( 3 ) small to moderate sample sizes in early - phase trials limit the reliability of subgroup - specic decisions . 
the model includes a hierarchical structure with latent ( unobserved ) patient frailties to induce association among the ve outcomes and account for variability not explained by dose and subgroup . 
to establish a prior probability distribution , hyper parameters characterizing location were computed from elicited probabilities of particular combinations of events occurring during the rst 100 days of follow - up , and prior hyper parameters characterizing dispersion were calibrated to reect vague prior knowledge about dose effects . 
details are given in lee et al.14 first assigning utility 0 to any outcome for which the patient dies before day 100 , utilities of the 12 possible combinations of nonfatal outcomes during the rst 100 days following nk cell infusion were elicited from the principal investigator for the trial ( table 3 )  . denote the vector of binary indicators of the 12 possible nonfatal outcomes within 100 days by = ( p , r , t , c )  . 
elicited utilities of the 12 possible 100 - day outcomes for patients in the nk cell dose - finding trial who survive beyond 100 days given that u ( death within 100 days ) = 0 progression , response cytokine storm toxicity yes , no no , no no , yes note . 
the cytokine storm outcome corresponds to a 30 - day evaluation period . abbreviation : nk , natural killer . weighted by the probability distribution of [ |d , g ]  . 
during the trial , for each disease type , patients are randomly assigned among the three doses in order of entry to the trial by randomly permuting the integers ( 1 , 2 , 3 ) , subject to being in accordance with the safety rule . 
at the end of the trial , for each disease subgroup g , the nk cell dose d that is safe and that maximizes u ( d , g|data ) is chosen , with no dose chosen if all three are unsafe within subgroup g . to establish its operating characteristics , the design was simulated under each of six different possible dose - subgroup - outcome scenarios . 
table 4 summarizes the operating characteristics of the design in one particular scenario that includes dose - subgroup interactions , with some doses unsafe for some subgroups but not for others . 
the results in table 4 show that the design is likely to stop accrual to doses in subgroups in which they are unsafe and otherwise is likely to select the truly optimal dose in each subgroup . figure 1 compares the dose - nding design that makes subgroup - specic decisions with a similar design that ignores the six disease subgroups when performing safety monitoring and dose selection . 
histograms are given for the differences between the empirical proportions of pr ( stop| subgroup , dose ) and pr ( stop|dose ) , for truly safe doses ( fig 1a ) and for truly unsafe doses ( fig 1b ) on the basis of computer simulations under the six dose - subgroup - outcome scenarios . 
histograms of differences between empirical proportions of pr ( stop|subgroup , dose ) pr ( stop|dose ) are given for ( a ) truly safe doses , ( b ) truly unsafe doses , and ( c ) pr ( select|subgroup , dose ) pr ( select| dose ) differences showing the probabilities of correctly selecting truly optimal doses . pr ( stop|subgroup , dose ) , pr ( stop|dose ) for truly safe doses , the design that ignores subgroups has a much larger probability of incorrectly stopping treatment in subgroups in which doses are safe . 
for truly unsafe doses , the two designs have similar stopping probabilities , with the important the design with subgroup - specic safety exception that monitoring is about .40 more likely to stop accrual to truly unsafe doses within subgroups . 
figure 1c shows that the subgroup - specic design is much more likely to select truly optimal doses within disease subgroups . additional computer simulations ( not shown ) evaluated the behavior of the nk cell dose - nding design in cases in which the true underlying time - to - event distributions were log - logistic and thus differed substantially from the assumed weibull dose - subgroup - outcome probability model . the results show that the model and design are extremely robust to the true underlying event time distribution model the design persist . and that details are provided in lee et al.14 the nkcelldosending computer program for implementing this design is available at the superior properties of utility - based clinical trial designs for a wide variety of other settings , assuming that patients are homogeneous , are given in houede et al , 15 thall et al , 4 , 16 - 18 lee et al , 19 hobbs et al , 20 murray et al , 21 and xu et al.22 an explanation of how trial design can be formulated as utility - based clinical a decision - making problem is provided by mueller et al.23 the simulation results show that the designs that make subgroup - specic decisions are greatly superior to comignore patient peting conventional methodologies that heterogeneity and treatment - subgroup or dose - subgroup interactions when such subgroup effects are present . 
in the randomized comparative trial , this is seen in terms of extremely large within - subgroup type i and type ii error probabilities of the conventional design in cases of treatment - subgroup interactions . 
these results strongly suggest if patient heterogeneity is that trial designs should be known , conventional clinical replaced by designs that account explicitly and prospectively for patient subgroups and treatment - subgroup or dose - subgroup effects in trial design , conduct , and decision making . 
an important caveat is that , if enrollment to a trial is limited to a homogeneous patient population , then a design with subgroup - specic rules would be an inappropriate elaboration . affiliation 1the university of texas md anderson cancer center , houston , tx preprint version available on biorxiv . support supported by core grant no . 
 bayesian utility - based designs for oncology clinical trials references yan f , thall pf , lu kh , et al : phase i - ii clinical trial design : a state - of - the - art paradigm for dose nding . 
new york , ny , chapman and hall / crc , 2011 thall pf , nguyen hq : adaptive randomization to improve utility - based dose - nding with bivariate ordinal outcomes . 
j biopharm stat 22 : 785 - 801 , 2012 braga m , gianotti l , nespoli l , et al : nutritional approach in malnourished surgical patients : a prospective randomized study . 
semin thorac cardiovasc surg 19 : 79 - 88 , 2007 clavien pa , sanabria jr , strasberg sm : proposed classication of complications of surgery with examples of utility in cholecystectomy . 
surgery 111 : 518 - 526 , 1992 dindo d , demartines n , clavien pa : classication of surgical complications : a new proposal with evaluation in a cohort of 6336 patients and results of a survey . ann surg 240 : 205 - 213 , 2004 10 . 
hoyos v , savoldo b , quintarelli c , et al : engineering cd19 - specic t lymphocytes with interleukin - 15 and a suicide gene to enhance their anti - lymphoma / leukemia effects and safety . 
houede n , thall pf , nguyen h , et al : utility - based optimization of combination therapy using ordinal toxicity and efcacy in phase i / ii trials . 
xu y , thall pf , m uller p , et al : a decision - theoretic comparison of treatments to resolve air leaks after lung surgery based on nonparametric modeling . 
triplenegative breast cancer ( tnbc ) is dened by the lack of expression of estrogen receptor ( er ) , progesterone receptor , and human epidermal growth factor receptor 2 ( her2 ) .1 , 2 this subtype represents 15% to 20% of all breast cancers and is associated with the worst outcome of all subtypes , with greater tendency to distant recurrence in general and visceral metastasis in particular , including brain metastasis.3 , 4 to date , chemotherapy remains the standard of care for tnbc.5 molecular stratication of tnbc will have treatment implications.6 for example , approximately 40% of tnbc patients have expression of programmed death - ligand 1 ( pd - l1 ) protein in immune cells , and this biomarker predicts survival benet from anti - pd - l1 therapy in combination with chemotherapy in the rst - line metastatic setting.7 in addition , approximately 10% of patients with tnbc harbor a germline brca1 / 2 mutation , which confers sensitivity to platinum and / or poly ( adpribose ) polymerase ( parp ) inhibitors.2 , 8 parp is involved in the repair of dna single - strand breaks via the base excision pathway . 
parp inhibitors such as olaparib or talazoparib lead to an accumulation of double - strand dna breaks , resulting in the activation of homologous recombination repair , which can compensate for the lack of activity of the base excision pathway and repair the dna damage.9 however , patients with defects in the homologous recombination dna repair pathway cannot repair dna damages caused by parp inhibitors , and the tumor cell eventually dies ( a term known as synthetic lethality )  . the patient is a 46 - year - old woman diagnosed in december 2015 with stage iib ( ct3cn1 ) moderately differentiated invasive papillary carcinoma with marked tumoral inltrating lymphocytes10 ( 60% ) and the presence of vascular invasion . 
she had a mastectomy and lymphadenectomy in june 2016 . analysis of the surgical specimen revealed extensive invasive residual disease ( ypt2ypn1 ) with a tnbc images of vascular inphenotype and abundant vasion . 
she then underwent adjuvant radiation to the breast and started adjuvant endocrine therapy with tamoxifen ( clinical decision based on baseline er positivity )  . in august 2017 , a positron emission tomography scan revealed multiple pathologic deposits in the bone , lung , and mediastinum and a prepectoral lesion . 
biopsy of the lesion conrmed recurrence of the disease ( gata3 positivity ) and a tnbc phenotype with a ki - 67 of 70% and androgen receptornegative and tumor inltrating lymphocytes around 10% ( fig 1 )  . 
no germline mutation was detected . here , we describe a heavily pretreated patient with tnbc brain metastasis and a brca1 somatic mutation with a remarkable and durable response to parp inhibitor therapy . 
to our knowledge , this is the rst case report to demonstrate disease response to parp inhibition in a tnbc without a germline brca1 or brca2 mutation . the patient was treated with carboplatin and gemcitabine for six cycles , and she achieved a partial response ( fig 2 )  . 
 ( a ) the tumor ( hematoxylin and eosin stain ) was composed of neoplastic inltrative nests with scattered stromal tumor - inltrating lymphocytes with many images of ( b ) lymphovascular invasion ( cd31 )  . 
 ( g ) the tumor had a high proliferation index ( ki - 67 ) , and ( h ) programmed death - ligand 1 ( pd - l1 ) was negative in both the neoplasm and the stromal cells . emergency department with progressively worsening headache . 
a brain magnetic resonance imaging scan showed multiple metastases , the largest being found in the right frontal lobe with surrounding edema ( fig 3a )  . the patient consented to participate in a research project in which tumor proling at the dna and rna level was performed with results discussed at a molecular tumor board . the prepectoral lesion was used for all the molecular analyses . 
the results revealed somatic mutations in brca1 ( s1253fs * 10 9435_9436delgt ) and tp53 ( s37fs * 6 * ) , a low tumor mutational burden ( four mutations per megabase ) , and a stable microsatellite status . 
at the rna level , an ncounter - based breast cancer 360 panel was performed.11 this assay includes 752 breast cancerrelated genes and 23 signatures , the tumor inammation signature , 12 the pam50 subtype predictor , 13 and the tnbctype classications14 , 15 ( fig 4 )  . 
results revealed a pam50 basal - like subtype with the following features : high expression of brca - ness and dna scar signatures , high expression of proliferationrelated genes , low expression of androgen receptorand estrogen - regulated genes , low expression of cd8 t cells and pd - l1 , high expression of immunosuppressive genes or signatures such as transforming growth factorand regulatory t - cell signatures , and a tnbctype mesenchymal subtype . on the basis of these results , off - label use of olaparib 300 mg twice per day was indicated . 
consideration was given to introducing corticosteroids or delivering whole - brain radiotherapy ( wbrt ) , but after discussion with the patient , a clinical decision was made to start olaparib under close observation and without the addition of either radiotherapy or corticosteroids . 
after 2 weeks of treatment , neurologic symptoms improved , and a restaging magnetic resonance imaging scan at week 8 demonstrated a signicant reduction in the size of the brain lesions and disappearance of associated cerebral edema ( fig 3b )  . 
brca proteins play a critical role in the homologous recombination dna repair pathway.16 in the presence of a brca germline mutation , one allele is affected , and the occurrence of a genetic alteration in the other allele ( eg , through methylation or loss of heterozygosity ) leads to a nonfunctional brca and the appearance of breast cancer , among other cancers.9 to date , two phase iii clinical trials have shown that parp inhibition with olaparib17 or talazoparib18 is superior to standard chemotherapy in terms of progression - free survival in her2 - negative advanced breast cancer harboring a brca germline mutation . 
 short title neoadjuvant doxorubicin + cyclophosphamide followed by paclitaxel radiation completed sep 2016 ; adjuvant tamoxifen carboplatin + gemcitabine with clinical response ; treatment stopped for toxicities palliative radiotherapy for bone metastasis ; progression on capecitabine + vinorelbine olaparib initiated dec 2015 jul 2016 aug 2017 feb 2018 may 2018 diagnosed with stage iib ( ct3n1 ) ; er , 50% ; pr , 0% ; her2 - ki - 67 , 80% right mastectomy and alnd ; ypt2n1 ; tnbc ; ki - 67 , 40% metastatic disease , bone , lymph node , subcutaneous tissue ; tnbc reappearance of pain and progression of bone metastases brain metastases fig 2 . 
alnd , axillary lymph node dissection ; er , estrogen receptor ; her2 , human epidermal growth factor receptor 2 ; pr , progesterone receptor ; tnbc , triple - negative breast cancer ; yp , pathologic staging after neoadjuvant therapy . patients with germline brca1 / 2 - mutated advanced breast cancer . 
brain magnetic resonance imaging before and after olaparib monotherapy . images before olaparib therapy show a cortical enhancing lesion on gadolinum - enhanced t1 - weighted imaging in the inferior frontal gyrus with perilesional edema visualized on uid - attenuated inversion recovery ( flair ) sequencing , as well as diffuse dural enhancement in the right hemisphere . 
selected signature scores are shown with the tumor inammation signature ( tis ) and pam50 signatures at the core ; the other signature scores are shown as bars around the riscores range from 0 to 1 mapped to quantiles of the population with invasive breast carcinoma in the cancer genome atlas ( tcga )  . 
apm , antigen processing machinery ; ar , androgen receptor ; brca - ness , brcaness signature ; cldl - ness , claudin - low subtype signature ; diffrn , differentiation ; dna scar , dna scar signature ; er , estrogen receptor ; her2 - e , her2 - enriched ; inm chmkn , inammatory chemokines ; pd - 1 , programmed cell death protein 1 ; pdl1 , programmed death - ligand 1 ; pgr , progesterone receptor ; tigit , t cell immunoreceptor and ig and itims domains ; treg , regulatory t cell . somatic mutations may also arise in genes involved in homologous recombination . 
early - phase clinical trials of parp inhibitors in metastatic tnbc and germline brca1 / 2 wild - type her2 - negative breast cancer with specic somatic genomic alterations such as brca1 / 2 mutations are underway ( eg , nct02401347 ; phase ii talazoparib in brca1 + brca2 wild - type & triple - neg / her2 - negative breast cancer / solid tumors and nct03330847 ; to assess safety and efcacy of agents targeting dna damage repair with olaparib versus olaparib monotherapy )  . the long - lasting response of breast cancer brain metastasis to olaparib in the absence of any other treatment is worth discussing . 
first , this suggests that olaparib , which had not previously been thought to cross the blood - brain barrier , 28 is able to get to the site of the tumor . 
concordant with this , other case reports with olaparib monotherapy have described regression of brain metastasis.29 , 30 second , olaparib , and other highly effective targeted systemic therapies , allow the delay of wbrt.31 - 35 this is important because wbrt can have a negative impact on quality of life and long - term neurocognitive functioning.36 thus , strategies to avoid , delay , or abrogate the effects of wbrt using systemic targeted therapies should be prioritized . in summary , comprehensive genomic alteration testing may provide novel clinical strategies for personalized therapy in advanced tnbc with improvement in overall survival and quality of life . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / author - center . tom as pascual consulting or advisory role : genentech research funding : genentech ( inst ) , msd oncology ( inst ) consulting or advisory role : bristol - myers squibb , diaceutics research funding : novartis travel , accommodations , expenses : pzer , boehringer ingelheim , menarini , msd , takeda pharmaceuticals , thermo fisher scientic maria vidal consulting or advisory role : novartis / pzer speakers bureau : novartis / pzer , genentech , eisai travel , accommodations , expenses : pzer montserrat muoz consulting or advisory role : eli lilly speakers bureau : roche travel , accommodations , expenses : roche , eli lilly aleix prat employment : novartis ( i ) honoraria : pzer , novartis , roche , msd oncology , eli lilly , daiichi sankyo consulting or advisory role : nanostring technologies ( inst ) , amgen , roche , novartis , pzer , bristol - myers squibb research funding : roche ( inst ) , novartis ( inst ) patents , royalties , other intellectual property : pct / ep2016 / 080056 : her2 as a predictor of response to dual her2 blockade in the absence of cytotoxic therapy travel , accommodations , expenses : daiichi sankyo other relationship : oncolytics biotech no other potential conicts of interest were reported . cristina teixid o honoraria : msd , pzer references bauer kr , brown m , cress rd , et al : descriptive analysis of estrogen receptor ( er ) - negative , progesterone receptor ( pr ) - negative , and her2 - negative invasive breast cancer , the so - called triple - negative phenotype : a population - based study from the california cancer registry . 
nature 490 : 61 - 70 , 2012 carey la , perou cm , livasy ca , et al : race , breast cancer subtypes , and survival in the carolina breast cancer study . 
jama 295 : 2492 - 2502 , 2006 dent r , trudeau m , pritchard ki , et al : triple - negative breast cancer : clinical features and patterns of recurrence . 
clin cancer res 13 : 4429 - 4434 , 2007 cardoso f , senkus e , costa a , et al : 4th eso - esmo international consensus guidelines for advanced breast cancer ( abc 4 )  . 
ann oncol 29 : 1634 - 1657 , 2018 prat a , adamo b , cheang mc , et al : molecular characterization of basal - like and non - basal - like triple - negative breast cancer . 
oncologist 18 : 123 - 133 , 2013 schmid p , adams s , rugo hs , et al : atezolizumab and nab - paclitaxel in advanced triple - negative breast cancer . 
n engl j med 379 : 2108 - 2121 , 2018 ahn sg , kim sj , kim c , et al : molecular classication of triple - negative breast cancer . 
j breast cancer 19 : 223 - 230 , 2016 ashworth a : a synthetic lethal therapeutic approach : poly ( adp ) ribose polymerase inhibitors for the treatment of cancers decient in dna double - strand break repair . 
salgado r , denkert c , demaria s , et al : the evaluation of tumor - inltrating lymphocytes ( tils ) in breast cancer : recommendations by an international tils 11 . 
lehmann bd , jovanovi c b , chen x , et al : renement of triple - negative breast cancer molecular subtypes : implications for neoadjuvant chemotherapy j clin invest 121 : 2750 - 2767 , 2011 selection . 
chandran ea , kennedy i : signicant tumor response to the poly ( adp - ribose ) polymerase inhibitor olaparib in heavily pretreated patient with ovarian carcinosarcoma harboring a germline rad51d mutation . 
ngoi nyl , tay d , heong v , et al : reversal of bowel obstruction with platinum - based chemotherapy and olaparib in recurrent , short platinum - free interval , rad51c germline mutationassociated ovarian cancer . 
winter c , nilsson mp , olsson e , et al : targeted sequencing of brca1 and brca2 across a large unselected breast cancer cohort suggests that one - third of mutations are somatic . 
sahgal a , soliman h , larson da : whole - brain radiation therapy of brain metastasis , in kim dg , lunsford ld ( eds ) : current and future management of brain metastasis . 
gaspar le , mehta mp , patchell ra , et al : the role of whole brain radiation therapy in the management of newly diagnosed brain metastases : a systematic review and evidence - based clinical practice guideline . 
bachelot t , romieu g , campone m , et al : lapatinib plus capecitabine in patients with previously untreated brain metastases from her2 - positive metastatic breast cancer ( landscape ) : a single - group phase 2 study . 
 outcomes of chemotherapy for microsatellite instablehigh metastatic colorectal cancers purpose microsatellite instable - high ( msi - h ) colorectal cancers ( crcs ) are known to carry better survival in the local disease stage even without treatment . 
the influence of types of treatment on survival of msi - h metastatic crcs ( mcrcs ) is still unclear and is evaluated in this study . materials and methods patients with msi - h mcrc treated with first - line chemotherapy , with or without bevacizumab , identified in the israeli population - based molecular epidemiology of colorectal cancer ( mecc ) study , were diagnosed between 1998 and 2013 and followed up until may 2017 ; msi status was determined by comparing 10 markers in tumor and normal tissue . 
dates of metastases and death and treatment details were extracted from oncology records . results among 590 patients treated for mcrc , 106 ( 18% ) had msi - h tumors . 
although treatment advances with modern protocols including antivascular endothelial growth factor and antiepidermal growth factor receptor ( egfr ) monoclonal antibodies1 - 3 prolonged median overall survival ( os ) of patients with crc to > 2 years , mcrc , unfortunately , is still an incurable disease in most cases . k - ras proto - oncogene ( kras ) or n - ras proto oncogene ( nras ) mutation ; the presence of a braf v600e mutation has a detrimental effect on overall survival ; the presence of human epidermal growth factor receptor 2 ( her2 ) in mcrc tumors suggests the possibility of benefit from anti - her2 biologic treatment ; and , more recently , microsatellite instable ( msi ) high tumors have been identified as candidates for immunotherapy.4 - 7 molecular heterogeneity in metastatic crcs influences treatment and outcome . 
anti - egfr agents are less effective in the presence of a loss of genomic stability is identified as an early step in colon cancer tumorigenesis and has pathologic , phenotypic , and clinical consequences.8 - 11 stephen b . 
 molecular analysis enables better understanding and separation of colorectal tumors now identified as carrying differences in clinical outcome.12 , 13 under this classification , tumors defined as consensus molecular subtype - 1 ( cms1 ) are characterized as microsatellite instable ( msi - h ) and hypermutated tumors.14 the clinical importance and exclusiveness of msi - h crcs have formerly been established.15 - 18 msi - h tumors have significantly better prognosis at early local stages of disease and rarely metastasize . 
cases identified in the mecc study were approached for risk - factors interview and contributed a venous blood sample after signing a consent form approved by carmel medical center in israel and the university of southern california in the united states . 
dna was extracted from both the peripheral blood and the tissue sample and was routinely studied for msi status , kras and braf mutations , as well as known founder mutations in mmr genes , mostly in ashkenazi jews . 
clinical follow - up included identification of treatment modalities used during the whole follow - up period and identification of changes in disease status , including identification of new metastases . 
details of treatments , as well as date of identification of metastases ( at diagnosis or during disease progression ) , were extracted from the medical files of the patients , which were extracted by experienced senior physicians . for this study , mecc cases that presented with metastatic disease at diagnosis or developed metastases during follow - up were sought . 
in addition to cases from the mecc population - based series , we recruited into the study other mcrc cases that were evaluated in our laboratory on the basis of their clinical presentation or suspicion of lynch syndrome to increase the size of the case series . 
detailed treatment options were grouped into three categories : fu or capecitabine only , combination chemotherapy ( oxaliplatin - based or irinotecan - based in combination with fluoropyrimidine ) , and combination chemotherapy with bevacizumab . 
 metastases with information on treatment details , and have available tissue and germline dna to analyze msi and braf status . laboratory assay dna was extracted using a commercial dna extraction kit . 
five loci of mononucleotide repeats ( bat25 , bat26 , bat40 , - catenin , and tgfbrii ) and five of dinucleotide repeats ( d2s123 , d5s346 , d10s197 , d17s250 , and d18s58 ) were amplified and tested . 
those include the five original bethesda panel markers and five additional monoand dinucleotide markers.33 results for each marker were registered as stable , unstable , equivocal , or suspected as loss of heterozygosity . 
most msi - h cases were also validated by immunohistochemistry tests of the mmr genes . mutations in codon 600 of braf were identified by direct sequencing of exon 15 of the braf gene . 
similarly , kras mutations in exon 12 , 13 , and 61 were identified using a taqman - based single - nucleotide polymorphism genotyping assay on the abi prism 7900ht sequence detection system ( applied biosystems ) in a 96 - well format . until the recorded date of death ( of any cause ) or the last date of available follow - up . 
overall survival was estimated with the use of kaplanmeier method and presented using medians and 2 - year and 5 - year survival probabilities . differences in os were summarized using hazard ratios ( hrs ) , estimated using cox proportional hazard modeling . 
the predictive effect of msi status on treatment effects was assessed using cox proportional hazard models with terms for treatment , msi status , and their interaction results patient population the study population consisted of 106 msi - h mcrc ( met / msi - h ) cases for which first - line chemotherapy - based treatment was identified . 
the metastatic msi - h group had mean age at treatment of 63 15 years , 54% female , 86% with jewish ethnicity , and 48% presenting at stage iv at diagnosis . 
generally , the met / mss group had comparable sex and ethnicity ( jewish / non - jewish ) distribution , as well as frequency of presenting at stage iv at diagnosis . 
this distribution reflects that fact that the patients with met / msi - h were younger at diagnosis ( proportion of patients younger than 50 years : 25% in met / msi - h group and only 10% in met / mss group )  . 
overall survival was calculated from the start date of first treatment of the first identified metastasis ( either at time of crc diagnosis or at time of disease progression ) and treatments for metastatic disease common first - line treatment chemotherapy for the metastatic disease in the msi - h group included fu + leucovorin ( lcv ) or capecitabine only in 21% ( n = 22 ) , combination chemotherapy protocols ( irinotecan based or oxaliplatin based ) in 33% ( n = 35 ) , and combination chemotherapy ( irinotecan based or oxaliplatin based ) and bevacizumab in 46% ( n = 49 ) of cases . 
demographic and disease characteristics of study participants with metastatic colorectal cancer by microsatellite instability status ( n = 590 ) characteristic metastatic msi - h ( n = 106 ) metastatic mss ( n = 484 ) age younger than 50 years at diagnosis female sex jewish ethnicity braf mutation positive stage iv at diagnosis right colon primary tumor location 26 ( 25 ) 57 ( 54 ) 88 * ( 86 ) 19 ( 18 ) 45 ( 48 ) 39 ( 38 ) note . 
 figure 1 presents frequency of the three classes across period years . second - line treatment was given to 55% of the patients treated before 2003 , 63% of those treated in 2003 to 2006 , and 68% of patients treated in 2007 and after . 
no difference in number of treatment lines was noticed between cases with msi - h and cases with mss tumors , probably reflecting the fact that in those years the treating oncologists were unaware of the msi status at time of treatment . in addition , in the msi - h population , the baseline characteristics of patients in the different treatment regimen were comparable ( data not shown )  . 
figure 4 presents os by treatment group , separately for patients with met / mss ( fig 4a ) and patients with met / msi - h ( fig 4b ) , in the homogenous population of braf - wt cases . 
 a sensitivity analysis adjusting for study period revealed similar results . prognostic effect of msi status in the braf - wt population the prognostic role of msi status in the metastatic disease setting was assessed in the population of patients who were indicated to receive an fu - only regimen as having the smallest or even no treatment effect , according to the literature . 
overall survival of patients with microsatellite instable , brafwild type metastatic colorectal cancer by treatment groups in the in the molecular epidemiology of colorectal cancer study , northern israel ( n = 87 )  . 
the mecc study reported here has recruited and collected materials over a time span of 17 years from close to 6 , 000 cases of crc and served as the source for the 590 cases who were diagnosed with , or later developed , metastases and for whom detailed treatment information was available . 
all cases participating in the final analysis were evaluated for their msi status , which was performed in only approximately two thirds of all cases recruited into mecc , with enrichment for suggestive phenotypes . in accordance with other published literature , cases with msi - high mcrc in our study were younger at diagnosis and had a higher proportion of braf mutations , 33 , 34 with a similar proportion of right - sided tumors . 
although msi - h tumors are described as having a much better prognosis than mss tumors for early - stage disease , the proportion of tumors diagnosed with metastases at the time of diagnosis was similar between msi - h cases and mss cases . 
the leading randomized controlled trials evaluating the effect of various chemotherapy regimens in mcrc were not randomized by msi status , and most have not provided subgroup analyses of the results according to their msi status . our study has three major findings . 
we verified that the survival of patients with mcrc indeed improved with the introduction of new chemotherapies but showed that the improvement in survival was limited to the met / mss , braf - wt cases and was not evident in the met / msi - h , braf - wt group of patients . 
we were unable to have a comparison with an observation - only group , and no future studies are likely to include such an arour observational data found no difference in os among metastatic msi - h cases between cases treated with a basic fu + lcv regimen only , compared with cases treated with any ( oxaliplatin or irinotecan ) combination chemotherapy with or without bevacizumab , whereas a significant os improvement was found with advanced chemotherapy in met / mss cases . 
similarly , we did not have any cases that were treated with immune checkpoint inhibitors at the time interval of the current analysis . few reports evaluated the effect of chemotherapy in met / msi - h cases . 
although early studies reported a positive effect of high - dose fu + lcv in patients with met / msi - h in comparison with patients with met / mss , 35 , 36 more recent studies of fu , in combination with irinotecan or oxaliplatin , did not show benefit.16 , 31 , 34 , 37 one study31 showed a trend toward benefit of oxaliplatin - based treatment in cases of msi - h mcrc . 
our data do not have enough power to evaluate specific therapeutic regimens . we chose to exclude from our study all metastatic cases that were not treated at all , because of the inability to assess the causes for no treatment and biases that could stem from it . 
comparison of os results in met / mss and met / msi - h groups supports biologic differences , with a baseline survival advantage for the msi - h group . 
effect of combination treatment on overall survival of patients with metastatic , brafwild type colorectal cancer by microsatellite instability status in the molecular epidemiology of colorectal cancer study , northern israel . 
 published data on the effect of braf mutation on prognosis in patients with met / msi - h were published on a group of crc stages ii to iv , 38 potentially underrepresenting the few cases with stage iv disease . 
extremely poor outcomes in this patient group support the use of the folfoxiri ( oxaliplatin , irinotecan , fluorouracil , leucovorin ) protocol in the unselected group of braf - mutated cases . 
given the much higher prevalence of braf mutations in the msi - h cases , 11 , 26 , 39 it is important to study the outcomes of folfoxiri in this subgroup of patients with met / msi - h . our study is limited by the relatively modest sample size of patients with msi - h mcrc . 
 advantages of our population - based sampling of incident cases with long - term follow - up include generalizability to community practice in a large and well - defined population . 
the representative , observational nature of the data from a large health system , in combination with molecular evaluation of all tumor tissues , offers a framework for understanding clinical practice over nearly two decades in a universally insured group of patients in a single country . msi status is becoming a cornerstone of individualized decision making with regard to a variety of potential oncological interventions , and this trend is likely to continue with the introduction of immunotherapy.40 , 41 routine mutational profiling of mcrc and specifically msi testing and other assays that designate deficient mismatch repair have already been recommended.39 , 42 , 43 the failure of conventional advanced treatments to improve survival in the meaningful subset of met / msi - h cases emphasizes the need to evaluate the role of the newly introduced immunotherapy as a first - line treatment in these cases with msi - h / braf - wt mcrc . in conclusion , therapeutic approaches to mcrc have changed dramatically during the past two decades , with increasing reliance on the molecular features of tumors to inform treatment decisions . 
gruber stock and other ownership interests : teva pharmaceuticals provision of study material or patients : flavio lejbkowicz collection and assembly of data : all authors data analysis and interpretation : katerina shulman , ofra barnett - griness , joel k . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . katerina shulman no relationship to disclose vered friedman no relationship to disclose joel k . 
gruber employment : brogent technologies leadership : brogent technologies stock and other ownership interests : brogent technologies , fulgent therapeutics consulting or advisory role : myriad genetics , fulgent therapeutics research funding : myriad genetics ( inst ) flavio lejbkowicz no relationship to disclose gad rennert no relationship to disclose affiliations katerina shulman , hillel yaffe medical center , hadera ; katerina shulman , ofra barnett - griness , vered friedman , flavio lejbkowicz , and gad rennert , carmel medical center , and technion , haifa , israel ; joel k . 
zhou sw , huang yy , wei y , et al : no survival benefit from adding cetuximab or panitumumab to oxaliplatin - based chemotherapy in the first - line treatment of metastatic colorectal cancer in kras wild type patients : a meta - analysis . 
chan dl , pavlakis n , shapiro j , et al : does the chemotherapy backbone impact on the efficacy of targeted agents in metastatic colorectal cancer ? a systematic review and meta - analysis of the literature . 
siena s , sartore - bianchi a , di nicolantonio f , et al : biomarkers predicting clinical outcome of epidermal growth factor receptor - targeted therapy in metastatic colorectal cancer . 
tol j , dijkstra jr , klomp m , et al : markers for egfr pathway activation as predictor of outcome in metastatic colorectal cancer patients treated with or without cetuximab . 
mller ci , schulmann k , reinacher - schick a , et al : predictive and prognostic value of microsatellite instability in patients with advanced colorectal cancer treated with a fluoropyrimidine and oxaliplatin containing first - line chemotherapy . 
sargent dj , marsoni s , monges g , et al : defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil - based adjuvant therapy in colon cancer . 
weitzel jn , blazer kr , macdonald dj , et al : genetics , genomics , and cancer risk assessment : state of the art and future directions in the era of personalized medicine . 
webber em , kauffman tl , oconnor e , et al : systematic review of the predictive effect of msi status in colorectal cancer patients undergoing 5fu - based chemotherapy . 
des guetz g , schischmanoff o , nicolas p , et al : does microsatellite instability predict the efficacy of adjuvant chemotherapy in colorectal cancer ? a systematic review with meta - analysis . 
ribic cm , sargent dj , moore mj , et al : tumor microsatellite - instability status as a predictor of benefit from fluorouracil - based adjuvant chemotherapy for colon cancer . 
cremolini c , loupakis f , masi g , et al : folfoxiri or folfoxiri plus bevacizumab as first - line treatment of metastatic colorectal cancer : a propensity score - adjusted analysis from two randomized clinical trials . 
van cutsem e , khne c - h , lng i , et al : cetuximab plus irinotecan , fluorouracil , and leucovorin as first - line treatment for metastatic colorectal cancer : updated analysis of overall survival according to tumor kras and braf mutation status . 
peeters m , price tj , cervantes a , et al : randomized phase iii study of panitumumab with fluorouracil , leucovorin , and irinotecan ( folfiri ) compared with folfiri alone as secondline treatment in patients with metastatic colorectal cancer . 
tran b , kopetz s , tie j , et al : impact of braf mutation and microsatellite instability on the pattern of metastatic spread and prognosis in metastatic colorectal cancer . 
goldstein j , tran b , ensor j , et al : multicenter retrospective analysis of metastatic colorectal cancer ( crc ) with high - level microsatellite instability ( msi - h )  . 
liang j - t , huang k - c , lai h - s , et al : high - frequency microsatellite instability predicts better chemosensitivity to high - dose 5 - fluorouracil plus leucovorin chemotherapy for stage iv sporadic colorectal cancer after palliative bowel resection . 
brueckl wm , moesch c , brabletz t , et al : relationship between microsatellite instability , response and survival in palliative patients with colorectal cancer undergoing first - line chemotherapy . 
kim je , hong ys , ryu m - h , et al : association between deficient mismatch repair system and efficacy to irinotecan - containing chemotherapy in metastatic colon cancer . 
overman mj , mcdermott r , leach jl , et al : nivolumab in patients with metastatic dna mismatch repair - deficient or microsatellite instability - high colorectal cancer ( checkmate 142 ) : an open - label , multicentre , phase 2 study . 
dunne pd , oreilly pg , coleman hg , et al : stratified analysis reveals chemokine - like factor ( cklf ) as a potential prognostic marker in the msi - immune consensus molecular subtype cms1 of colorectal cancer . 
 genomic profiling to expand management options for locally advanced esophagogastric cancers : a proof of principle case introduction the management of locally advanced esophageal and gastric cancer is an evolving field , but despite improvement in response rates and surgical techniques , recurrence rates for stage iii disease remain high.1 , 2 surgical resection is potentially curative and associated with established morbidity and mortality risk , limiting surgery to medically fit patients . 
emerging data suggest a role for positron emission tomography ( pet ) adaptive treatment strategies and possibly incorporation of trastuzumab in neoadjuvant therapy for patients with human epidermal growth factor receptor 2 ( her2 ) positive disease.3 , 4 however , additional tumor biologic features are not incorporated routinely in the management of locally advanced disease . 
large collaborative molecular characterization efforts , including the cancer genome atlas , the asian cancer research group , and commercial sequencing databases , have highlighted genomically defined subgroups.5 - 9 within gastric and esophageal cancers , 4% to 22% are reportedly microsatellite instable ( msi - h ) , a subgroup that has demonstrated significant responsiveness to immune checkpoint inhibitors ( icpis ) in small metastatic data sets.10 , 11 the potential to incorporate this approach in earlier - stage disease is understudied , and , to our knowledge , we report the first case describing a complete response to pembrolizumab in an msi - h stage iii gastroesophageal junction ( gej ) adenocarcinoma with persistent disease after definitive chemoradiotherapy ( crt )  . 
staging petcomputed tomography ( ct ) demonstrated a large and intensely avid mass at the gej ( standardized uptake value of 20.3 ) with no evidence of distant disease ( fig 2 )  . 
pet - ct scanning was repeated 10 weeks after crt completion , demonstrating partial response with residual pet - avid mass at the gej ( standardized uptake value of 7.1 ) , confirmed by endoscopic evidence of persistent disease and symptomatic dysphagia ( fig 2 )  . 
to explore additional therapeutic options , the original gej biopsy was submitted for comprehensive genomic profiling ( foundationone ) , which revealed msi - h , a high tumor mutational burden ( tmb ) of 36 mutations per dna megabase , an absence of erbb2 ( her2 ) alterations , and several additional genomic alterations ( appendix table a1 )  . 
orthogonal immunohistochemical mismatch repair testing confirmed loss of mlh1 and pms2 , and mlh1 promoter polymerase chain reaction testing confirmed mlh1 promoter hypermethylation , all consistent with a sporadic msi - h tumor ( fig 1 )  . 
 ( a ) scattered luminal necrosis as well as punctate single cell apoptoses and frequent mitoses are visible . mismatch repair immunohistochemistry demonstrates loss of ( b ) mlh1 and ( c ) pms2 , with intact ( d ) msh6 and ( e ) msh2 . 
 ( a ) scattered tumor infiltrating lymphocytes were present , at a density of approximately one lymphocyte per five to 10 tumor cells . response with resolution of pet - avid gej mass ( fig 2 )  . 
at the time of submission , he continues on pembrolizumab and remains clinically asymptomatic with ongoing complete response now over 8 months . discussion the ability to identify the optimal therapy for each patient is the hallmark of the precision medicine paradigm but has largely remained elusive in esophagogastric cancers to date . 
in the nonmetastatic setting , attempts to incorporate biologic agents , including the antiepidermal growth factor receptor monoclonal antibodies and antiangiogenesis agents , have not improved outcomes.12 , 13 the fully accrued radiation therapy oncology group ( rtog ) - 1010 trial examining a role for trastuzumab in combination with crt in locoregional esophageal / gej adenocarcinoma may ultimately be the first to identify a molecularly selected group to derive benefit from a biologic agent during neoadjuvant therapy for resectable disease . 
however , at the present time , patients with persistent disease after definitive crt who are unfit for resection represent a difficult clinical scenario with limited established approaches and 3 - year survival less than 10% in some series.14 , 15 although only a single case is presented herein , we suggest that in locally advanced unresectable esophagogastric cancer there may be clinical utility to molecular profiling to expand potential treatment options . 
the cancer genome atlas data sets for gastric and esophageal cancer are almost exclusively derived from nonmetastatic surgical samples and report rates of msi - h of 22% and 0% , respectively ( 0% for confirmed esophageal adenocarcinoma , although three of 36 among gej tumors not clearly of esophageal origin ) .5 , 6 this differs somewhat from the 6% rate of msi - h reported from the capecitabine and oxaliplatin adjuvant study in stomach cancer ( classic ) trial analysis of resected stage ii to iii gastric cancer and the 6.7% rate of msi - h identified in the european medical research council adjuvant gastric infusional chemotherapy ( magic ) trial of resectable gastroesophageal cancer.16 in both data sets , msi - h appears to confer a favorable prognosis and unclear benefit from adjuvant or perioperative chemotherapy , analogous to stage ii colon cancer.17 - 19 although analyses from ongoing metastatic and locoregional trials will refine the incidence of msi - h tumors , a portion will be msi - h and thus will be optimal candidates for immunotherapy consideration as supported by our case . 
complete resolution of [ 18f ] fluorodeoxyglucose activity was observed after three doses of pembrolizumab , and imaging is shown in coronal , sagittal , and axial planes for reference . pretreatment 10 weeks postchemoradiotherapy 10 weeks postpembrolizumab start strong biologic rationale , including in nonmetastatic locally advanced unresectable patients who have progressed on prior therapy.20 the ongoing checkmate - 577 trial ( nct02743494 ) will be important to more clearly establish the role of icpis in the adjuvant / locoregional setting after concurrent crt and surgery with persistent disease , and analyses for msi testing as well as tmb will be important.21 importantly , there is scientific rationale for moving these agents into the true neoadjuvant setting when the primary tumor remains.22 , 23 notably , our patient was not deemed a surgical candidate and would not be eligible for this trial . additional small studies incorporating programmed death - 1 inhibitors into concurrent crt will likely add more details , and there is strong scientific rationale for this approach ( nct03044613 ) .22 more broadly , predictive biomarkers for icpis are critical to identify patients who are most likely ( or least likely ) to benefit from icpi incorporation . not surprisingly , our patient harbored an elevated tmb at 36 mutations / dna megabase . 
however , a fraction of microsatellite stable gi cancers have also been shown to have high tmb.24 next - generation sequencingderived tmb has emerged as a robust biomarker for icpi benefit across several tumor types.25 - 27 in a small retrospective series of esophagogastric cancers , it was suggested that msi - h or tmb of > 14 mutations / megabase was associated with icpi benefit , although larger series will be critical to refine tmb in esophago - gastric cancers ( note that tmb determination was slightly different in this study ) .28 overall , our case highlights the potential role for genomic profiling in this nonmetastatic clinical situation and suggests a possible treatment approach . 
klempner consulting or advisory role : eli lilly , boston biomedical , celgene , astellas pharma speakers bureau : foundation medicine travel , accommodations , expenses : eli lilly the following represents disclosure information provided by authors of this manuscript . 
schrock employment : foundation medicine stock and other ownership interests : foundation medicine joseph chao consulting or advisory role : eli lilly , bayer , five prime therapeutics , boston biomedical , merck research funding : merck ( inst ) , novonco therapeutics ( inst ) siraj m . 
ali employment : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine , patents via seres health on microbiome stuff in non - neoplastic disease ( i ) affiliations samuel j . 
klempner , cedars - sinai medical center ; winnie wu , providence saint johns hospital , los angeles ; joseph chao , city of hope comprehensive cancer center , duarte , ca ; and alexa b . 
oppedijk v , van der gaast a , van lanschot jj , et al : patterns of recurrence after surgery alone versus preoperative chemoradiotherapy and surgery in the cross trials . 
smyth ec , fassan m , cunningham d , et al : effect of pathologic tumor response and nodal status on survival in the medical research council adjuvant gastric infusional chemotherapy trial . 
lordick f , ott k , krause bj , et al : pet to assess early metabolic response and to guide treatment of adenocarcinoma of the oesophagogastric junction : the municon phase ii trial . 
goodman ka niedzwiecki d , hall n , et al : initial results of calgb 80803 ( alliance ) : a randomized phase ii trial of pet scan - directed combined modality therapy for esophageal cancer . 
ali sm , sanford em , klempner sj , et al : prospective comprehensive genomic profiling of advanced gastric carcinoma cases reveals frequent clinically relevant genomic alterations and new routes for targeted therapies . 
gainor jf , tan ds , de pas t , et al : progression - free and overall survival in alk - positive nsclc patients treated with sequential crizotinib and ceritinib . 
le dt , durham jn , smith kn , et al : mismatch - repair deficiency predicts response of solid tumors to pd - 1 blockade . science 357 : 409 - 414 , 2017 12 . 
cunningham d , stenning sp , smyth ec , et al : peri - operative chemotherapy with or without bevacizumab in operable oesophagogastric adenocarcinoma ( uk medical research council st03 ) : primary analysis results of a multicentre , open - label , randomised phase 2 - 3 trial . 
intergroup phase iii trial of neo - adjuvant chemotherapy , followed by chemoradiation and surgery with and without cetuximab in locally advanced esophageal carcinoma : first results from the sakk 75 / 08 trial . 
smyth ec , wotherspoon a , peckitt c , et al : mismatch repair deficiency , microsatellite instability , and survival : an exploratory analysis of the medical research council adjuvant gastric infusional chemotherapy ( magic ) trial . jama oncol 3 : 1197 - 1203 , 2017 17 . 
bang yj , kim yw , yang hk , et al : adjuvant capecitabine and oxaliplatin for gastric cancer after d2 gastrectomy ( classic ) : a phase 3 open - label , randomised controlled trial . 
kelly rj , lockhart ac , jonker dj , et al : checkmate 577 : a randomized , double - blind , phase 3 study of adjuvant nivolumab ( nivo ) or placebo in pts with resected esophageal ( e ) or gastroesophageal junction ( gej ) cancer . 
rosenberg je , hoffman - censits j , powles t , et al : atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum - based chemotherapy : a single - arm , multicentre , phase 2 trial . 
ku gy , sanchez - vega f , chatila w , et al : correlation of benefit from immune checkpoint inhibitors with next gen sequencing ( ngs ) profiles in esophagogastric cancer ( egc ) patients . 
the tumor was negative for programmed death ligand 1 ( pd - l1 ) expression ( tumor proportion score , 1% ) .24 postoperative imaging 6 weeks later revealed two lowdensity liver lesions measuring 1.5 cm ( segment 4a ) and 1.9 cm ( segment 2 ; fig 2a ) , which were hypermetabolic on an 18 - uorodeoxyglucose ( fdg ) positron emission tomography ( pet ) / computed tomography scan . 
after two cycles of carboplatin and etoposide , 10 new liver lesions were evident on imaging ( fig 2b )  . he subsequently enrolled in a phase ii study of pembrolizumab ( 10 mg / kg intravenously [ iv ] every 2 weeks ) for patients with msi - h tumors ( clinicaltrials.gov identier : nct01876511 )  . 
these required discontinuation of pembrolizumab after 10 months despite ongoing disease response to treatment ( fig 2d )  . hypothyroidism the patient underwent total proctocolectomy ; both tumors were t1n0 . 
msi testing ( internal ucsf500 assay ; ucsf clinical cancer genomics laboratory ) and mmr protein immunohistochemistry of the resected colon cancer sample conrmed msi - h status and loss of mlh1 expression in the tumor and normal samples . 
by cycle 11 of pembrolizumab , worsening hypothyroidism was evident , peaking at cycle 13 with thyroidstimulating hormone levels of 79.2 iu / ml ( free t4 , 0.6 ng / dl ; total t3 , 63 ng / dl )  . 
 new - onset diabetic ketoacidosis / insulin - dependent diabetes mellitus arthritis whitman et al at 10 months after starting pembrolizumab ( cycle 19 ) , the patient presented with diabetic ketoacidosis ( dka ) ; he had blood glucose levels  . 
given the lack of data supporting use in this setting , corticosteroids were not immediately started , although grade 4 autoimmune type 1 insulin - dependent diabetes mellitus was suspected.25 pembrolizumab was discontinued , and daily insulin therapy was initiated . myocarditis the patient experienced bradyarrhythmias with complete heart block while recovering from dka . 
the troponin level was elevated at 7.4 g / l ( normal , 0.05 g / l ) , the electrocardiogram showed new inferior q waves , and the echocardiogram revealed a depressed ejection fraction ( 30% - 35% ) and regional wall motion abnormalities consistent with coronary artery disease . 
he was monitored with serial echocardiograms , electrocardiograms , and troponin levels for 20 months without evidence of recurrent myocarditis . at 5 months after treatment initiation , the patient noticed grade 1 stiffness and discomfort in his knees , ankles , hands , and bilateral wrists . 
differential diagnosis favored an autoimmune process , with an elevated erythrocyte sedimentation rate of 77 mm / h ( normal , 10 mm / h ) and a c - reactive protein level of 98.0 mg / l ( normal , 6 mg / l )  . 
within 24 hours of initiating corticosteroids for myocarditis , he experienced near - complete resolution of arthritic symptoms . autoimmune hepatitis at 4 months after discontinuation of immunotherapy , and within 3 weeks of completing the initial corticosteroid taper for myocarditis and arthritis , the patient was admitted for asymptomatic grade 3 transaminitis , with an alt level of 990 u / l ( normal , 60 u / l ) and an ast level of 791 u / l ( normal , 42 u / l )  . he was treated with high - dose iv methylprednisolone followed by prednisone 1 mg / kg orally ( tapered over 10 months ) with rapid improvement in liver function tests . 
work - up was negative for cytomegalovirus ; epstein - barr virus ; and hepatitis a , b , c , d , and e infections . follow - up imaging revealed two residual 18f - fdgpost - treatment petnegative liver lesions ( 1 cm and 0.6 cm ; fig 4b )  . 
computed tomography ( ct ) scan of abdomen / pelvis ( a ) 6 weeks after resection of primary tumors ( two lesions , largest was 1.9 cm ) , prechemotherapy ; ( b ) after two cycles of carboplatin / etoposide ( at least 10 lesions , largest was 3 cm ) ; ( c ) after three cycles of pembrolizumab ; and ( d ) after 11 cycles ( 5 months ) of pembrolizumab . responses to checkpoint blockade in ls are hypothesized to be the result of higher mutational load as a result of mmr deciency , which is associated with more mutationassociated neoantigens . 
his arthritis is controlled with methotrexate , and he remains on insulin for type 1 diabetes mellitus and on levothyroxine for hypothyroidishis myocarditis and hepatitis are in remission . discussion presence of a germline mutation in the mmr gene mlh1 with loss of the wild - type allele in the nec component suggests that the nec developed as a consequence of ls in this patient . this case , coupled with data suggesting that approximately 10%26 - 29 of gi necs ( including mixed adenoneuroendocrine carcinomas ) are msi - h ( predominantly from mlh1 methylation ) , underscores the need to consider ls in patients with colorectal nec and the potential value of msi testing in these tumors . 
checkpoint inhibitor ( cpi ) therapy is compelling given the paucity of treatments for platinum - refractory colorectal nec , the tissue / site - agnostic approval of pembrolizumab for msi - h tumors , 30 and evidence suggesting that cpis have activity in mmr - decient / msi - h cancers whether associated with ls or developing sporadically ( eg , in the setting of mlh1 promoter methylation31 - 33 )  . our patient experienced ve iraes . 
regarding immunotherapy - related diabetes , one series suggested that gad65 autoantibodies were present in 60% of patients and that a small proportion harbored a cd8 + t - cell response to a t1dm antigen.34 , 35 gad65 autoantibodies were absent in our patient , suggesting a lower probability of undiagnosed autoimmune disease and underscoring the need for validated markers of response and toxicity for patients treated with cpis.36 the relationship between toxicity and efcacy remains ill dened37 ( appendix )  . 
whether underlying ls increased the risk for iraes is unclear , but a germline mlh1 mutation could theoretically predispose a patient to mutations in normal tissue that are immunologically recognized . 
however , previous reports of cpi therapy in patients with msi - h tumors have not suggested a higher incidence of iraes in this population.5 , 31 , 32 , 38 the patient was treated with a relatively high dose ( 10 mg / kg iv every 2 weeks ) of pembrolizumab compared with the us food and drug administration approved dose for msi - h cancers ( 200 mg iv every 3 weeks ) ; this difference also may have contributed to iraes . jco precision oncology fig 3 . 
n engl j med 363 : 711 - 723 , 2010 topalian sl , hodi fs , brahmer jr , et al : safety , activity , and immune correlates of antipd - 1 antibody in cancer . 
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vijayvergia n , dasari a , ross ea , et al : pembrolizumab ( p ) monotherapy in patients with previously treated metastatic high grade neuroendocrine neoplasms ( hg - nens )  . 
mulvey c , raj np , chan ja , et al : phase ii study of pembrolizumab - based therapy in previously treated extrapulmonary poorly differentiated neuroendocrine carcinomas : results of part a ( pembrolizumab alone )  . 
kaufman hl , russell js , hamid o , et al : updated efcacy of avelumab in patients with previously treated metastatic merkel cell carcinoma after 1 year of follow - up : javelin merkel 200 , a phase 2 clinical trial . 
kervarrec t , samimi m , gaboriaud p , et al : detection of the merkel cell polyomavirus in the neuroendocrine component of combined merkel cell carcinoma . virchows arch 472 : 825 - 837 , 2018 24 . 
j immunother cancer 5 : 40 , 2017 jesinghaus m , konukiewitz b , keller g , et al : colorectal mixed adenoneuroendocrine carcinomas and neuroendocrine carcinomas are genetically closely related to colorectal adenocarcinomas . 
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galon j , costes a , sanchez - cabo f , et al : type , density , and location of immune cells within human colorectal tumors predict clinical outcome . 
llosa nj , cruise m , tam a , et al : the vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter - inhibitory 313 : 1960 - 1964 , 2006 checkpoints . 
strosberg jr , coppola d , klimstra ds , et al : the nanets consensus guidelines for the diagnosis and management of poorly differentiated ( high - grade ) extrapulmonary neuroendocrine carcinomas . 
 checkpoint blockade in msi - high neuroendocrine carcinoma appendix summary us food and drug administration approvals of immunotherapy in may 2017 , the us food and drug administration approved pembrolizumab for unresectable or metastatic microsatellite instability high ( msi - h ) or mismatch repairdecient solid tumors that have progressed on prior standard treatment . 
in august 2017 , nivolumab was approved for msi - h colorectal cancer . modest activity has been demonstrated in advanced small - cell lung cancer , leading to drug approvals in rst - line ( atezolizumab plus chemotherapy ) and platinum - refractory settings.12 , 13 , 20 - 22 checkpoint inhibitors have an established role in merkel cell carcinoma , which is a virally mediated disease.21 - 23 beyond that , the role of immunotherapy in extrapulmonary neuroendocrine carcinomas ( necs ) remains ill dened . 
however , three recent pilot studies suggest minimal activity ( response rate , 10% ) of singleagent checkpoint inhibitors in unselected patients with extrapulmonary necs.15 - 17 a growing body of literature has served to clarify the nature and optimal management of immune - related adverse events . 
some reports suggest that patients with grade 3 immune - related adverse events experience longer median time to progression and higher radiographic response . recent biomarker data suggest a high rate of programmed death 1 ( pd1 ) / programmed death ligand 1 expression in poorly differentiated necs of the gi tract ( roberts ja , et al : hum pathol 70 : 49 - 54 ) and a greater proportion of msi - h in high - grade necs , mixed adenoneuroendocrine carcinomas , and insulinomas than in low - grade gastroenteropancreatic neuroendocrine tumors ( weber mm , fottner c : oncol res treat 41 : 306312 , 2018 )  . 
biegel , phd1 , 2 introduction molecular alterations are increasingly becoming clinically useful adjuncts to histology and immunohistochemistry in the classication of pediatric cns tumors.1 , 2 in particular , mutations in idh , tp53 , braf , and histone h3 genes are diagnostic and prognostic in gliomas.2 , 3 however , because of the large and growing list of molecular alterations in brain tumors , targeted approaches to molecular testing , such as sanger sequencing , may fail to capture the driver alteration in a sample . 
despite recent advances in the classication of pediatric cns neoplasms , 2 integrated histopathologic and molecular diagnoses are challenging in many cases.1 powerful , genome - wide approaches used for molecular diagnosis include chromosomal microarray ( cma ) for the identication of copy - number alterations and next - generation sequencing ( ngs ) for single - nucleotide variants , insertions and deletions , and / or gene fusions . these methods benet from surveying a larger portion of the genome than single - gene testing approaches as well as an ability to identify novel alterations . 
recent studies have highlighted the clinical utility of routine proling of pediatric brain tumors with cma and / or ngs approaches.4 - 7 we present the case of a 7 - week - old girl found to have a large intraparenchymal mass in the right frontoparietal region of the brahistopathologic and cma analyses were not diagnostic for a specic subtype of pediatric brain tumor , although a breakpoint in met was identied with cma . 
further study of this tumor with an ngs panel designed for pediatric malignancies , oncokids ( oncomine childhood cancer research assay ; thermo fisher scientic , waltham , ma ) , identied a novel and targetable tripartite motif containing 24 ( trim24 ) - met gene fusion consistent with a diagnosis of high - grade glioma . 
computed tomography ( ct ) of the head revealed a large right parietal mass with central hemorrhage , associated 12 - mm midline shift , and obstructive hydrocephalus ( fig 1 )  . 
she was airlifted to the local childrens hospital , where she underwent emergent craniotomy for frontal external ventricular drain placement , partial evacuation of intracranial hemorrhage , and partial resection of the mass . 
intraoperative pathology consultation with frozen section interpretation showed a high - grade malignant neoplasextensive blood loss limited the extent of resection . postoperative course her immediate postoperative course was complicated by critically elevated intracranial pressures despite aggressive interventions . 
she died on postoperative day 5 . felt methods informed consent was obtained from the guardian before collection of tumor specimens for further analysis of dna and rna using cma and ngs . 
dna was extracted from frozen tumor tissue using the qiagen gentra puregene tissue kit , and rna was extracted with the qiagen rneasy mini rna extraction kit ( qiagen , valencia , ca )  . 
analysis of dna and rna was performed with the oncokids panel , a comprehensive dnaand rna - based ngs panel for pediatric malignancies , as described previously.8 cma analysis of tumor dna was performed according to manufacturer protocol ( oncoscan ; thermo fisher scientic ) .9 the trim24 - met fusion was conrmed by reverse transcription polymerase chain reaction and sanger sequencing . 
overall , these features were consistent with a high - grade neuroepithelial tumor . molecular findings chromosomal microarray analysis of the tumor specimen demonstrated gain of chromosomes 2 , 3 , 5 , 7 , 8 ( four copies ) , 9 , and 10 ( fig 3a )  . 
there was a 482 - kb area of high copy - number gain identied at 8q23.3 that overlapped the csmd3 gene ; the clinical signicance of this alteration was unclear . 
however , a small alteration at this locus could not be ruled out . oncokids analysis of the tumor sample revealed a gene fusion between exon 12 of trim24 ( enst00000415680 ) and exon 15 of met ( enst00000397752 ; fig 3c ) in 110 , 747 of 2 , 575 , 589 mapped reads . 
no variants of strong or potential clinical signicance were identied from the analysis of the dna data from oncokids , including variants within tp53 , braf , pik3ca , h3f3a , hist1h3b , and atrx . 
there is marked right cerebral hemisphere edema as well as mass effect with associated hydrocephalus and right to left midline shift . synthesized from 200 ng of total rna with the superscript iii rst - strand synthesis system ( thermo fisher scientic ) with oligo ( dt ) , followed by amplication with gene - specic primers . 
there was patchy expression of gfap ( fig 2c ) and olig2 and no immunoreactivity for neuron - specic enolase or synaptophysthe tumor was also immunonegative for ema , cytokeratin , smooth muscle actin , plap , immunoreactivity for smarcb1 and afp , and cd99 . smarca4 was retained in tumor cell nuclei . immunostaining for p53 ( d07 ) showed strong expression in a majority of tumor cells ; however , there was no corresponding tp53 deletion or point mutation identied with cma or the oncokids panel ( described in molecular findings )  . 
ki67labeling index exceeded 25% ( fig 2d )  . in this 7 - week - old patient , histopathologic review was consistent with a high - grade neuroepithelial tumor with an embryonal appearance and patchy expression of gfap and olig2 . 
treatment regimens for pediatric high - grade gliomas and embryonal tumors differ signicantly ; therefore , specic diagnosis is crucial for therapy.10 analysis with oncokids identied a novel trim24 - met gene fusion . 
 ( d ) ki - 67labeling index was high ( range , 25% to 60% )  . by the international cancer genome consortium identied gene fusions involving met in seven cases ( 12% ) .11 one met gene fusion observed in that study , ptprz1 - met , has in secondary also been reported as a recurrent event glioblastoma.12 in the context of cns malignancies , met gene fusions seem to be limited to glial neoplasms.11 , 12 met gene fusions , such as ptprz1 - met , carry an unfavorable prognosis in high - grade gliomas.14 trim24 encodes a protein that functions in mediating transcriptional control via the interaction with specic regions of nuclear receptors.15 trim24 has been observed as a 5 ( cid : 1 ) partner with several different driver fusion genes , including braf , fgfr1 , ntrk2 , and ret.16 - 19 the nding of a trim24 - met gene fusion expands the spectrum of met gene fusions in pediatric gliomas . 
this large sefusion has not been reported in several quencing efforts or in publicly available databases such as cosmic ( catalogue of somatic mutations in cancer ) .12 , 20 , 21 the ptprz1 - met fusion has been shown to increase met expression , 22 and several tyrosine kinase inhibitors have been or are currently in clinical trials for met - overexpressed or met - amplied cancers.23 - 25 one pediatric patient with ptprz1 - met fusionpositive glioblastoma experienced substantial tumor volume reduction after treatment with crizotinib.11 unfortunately , that child ultimately acquired resistance to met inhibition.11 it is possible that novel tyrosine kinase inhibitors or inhibition by multiple receptor tyrosine kinase inhibitors may be more effective for met inhibition than crizotinib.26 the patient described in our report died before met inhibition could be attempted ; however , routine molecular proling for met fusions should expand the therapeutic options for a signicant fraction of children with cns tumors . in this case , information from histopathology , ultrastructural analysis , and cma studies enabled the diagnosis of a high - grade neuroepithelial tumor with a few features suggestive of glial differentiation . 
 ( a ) whole - genome view from oncoscan chromosomal microarray ( cma ) in nexus copy number software ( version 9.0 ; biodiscovery , el segundo , ca ) demonstrating the copy - number gains of chromosomes 2 , 3 , 5 , 7 , 8 , 9 , and 10 . 
 novel trim24 - met fusion in a neonatal brain tumor such as ptprz1 - met , have been reported to demonstrate more aggressive biologic behavior , and unfortunately , our patient died soon after tumor resection.12 for future patients diagnosed with high - grade gliomas harboring the trim24 - met gene fusion , available therapeutic options would include inhibitors of met , such as crizotinib or cabozantinib.13 integration of the information from histopathologic , copynumber , and sequencing analyses enabled a substantial improvement in the diagnostic , prognostic , and therapeutic understanding of our patients tumor . 
hiemenz consulting or advisory role : loxo oncology travel , accommodations , expenses : thermo fisher scientic no other potential conicts of interest were reported . references aldape k , pster sm : next - generation molecular diagnostics , in berger ms , weller m ( eds ) : handbook of clinical neurology . 
cambridge , ma , elsevier , 2016 , pp 121 - 130 louis dn , perry a , reifenberger g , et al : the 2016 world health organization classication of tumors of the central nervous system : a summary . 
adv anat pathol 25 : 143 - 171 , 2018 roth jj , santi m , rorke - adams lb , et al : diagnostic application of high resolution single nucleotide polymorphism array analysis for children with brain tumors . cancer genet 207 : 111 - 123 , 2014 shao l , miller s , koschmann c , et al : clinical application of whole genome array improves the diagnosis of pediatric brain tumors . 
int j surg pathol 25 : 688 - 695 , 2017 sahm f , schrimpf d , jones dt , et al : next - generation sequencing in routine brain tumor diagnostics enables an integrated diagnosis and identies actionable targets . 
worst bc , van tilburg cm , balasubramanian gp , et al : next - generation personalised medicine for high - risk paediatric cancer patients : the inform pilot study . eur j cancer 65 : 91 - 101 , 2016 8 . 
j mol diagn 20 : 765 - 776 , 2018 foster jm , oumie a , togneri fs , et al : cross - laboratory validation of the oncoscan ffpe assay , a multiplex tool for whole genome tumour proling . 
j pediatr hematol oncol 38 : 249 - 260 , 2016 international cancer genome consortium pedbrain tumor project : recurrent met fusion genes represent a drug target in pediatric glioblastoma . 
bao zs , chen hm , yang my , et al : rna - seq of 272 gliomas revealed a novel , recurrent ptprz1 - met fusion transcript in secondary glioblastomas . 
belloni e , trubia m , gasparini p , et al : 8p11 myeloproliferative syndrome with a novel t ( 7 ; 8 ) translocation leading to fusion of the fgfr1 and tif1 genes . 
davies kd , ng tl , estrada - bernal a , et al : dramatic response to crizotinib in a patient with lung cancer positive for an hla - drb1 - met gene fusion . 
gazzaniga et al we are grateful for your comments on our recent publication1 that highlighted that nearly 50% of the patients with ras - mutated metastatic colorectal cancer and prior chemotherapy could display wildtype ras status in the circulating tumoral dna ( ctdna ) , and benet from the late introduction of an antiepidermal growth factor receptor ( egfr ) .2 we indeed acknowledge that your team had worked in a similar direction , and appropriately cited the report that your group published in 2019.3 this article also includes results from preliminary ndings in abstract forms . 
both of our works , as well as that from moati et al , 4 conrm the principle of expected efcacy from targeting egfr in the late stage of treatment of patients with ras - mutated colorectal cancer , if their liquid biopsy does not identify any ras mutation in the ctdna.2 - 4 however , there is a broad discrepancy in the rates of patients without ras mutation detection in plasma between these studies , ranging from 9 / 16 ( 56% ) , 2 through 5 / 11 ( 45% ) 3 to 8 / 36 ( 22% ) , 4 as also discussed in your opinion paper.5 a critical issue involves the conrmation of circulating tumoral dna in the absence of ras mutation , which could be reached through the detection of a persistent somatic mutation3 or the occurrence of wif1 / npy methylation.4 however , we concur and admit that these and other methods have intrinsic limitations , such that no standard one can currently ascertain the presence of circulating tumor dna in the absence of detected ras mutation.5 moreover , although their application drastically reduced the ras mutation conversion rate down to 6.6% - 14% , 3 , 4 this did not impair the fact that the introduction of an antiegfr apparently extended median progression - free survival to nearly 6 months in previously treated patients from both studies.2 , 3 therefore , we feel that two important issues need to be jointly addressed to advance the current guidelines for anti - egfr indication for metastatic colorectal cancer . 
gazzaniga p , nicolazzo c , caponnetto s , et al : about ras mutation clearance in plasma ctdna from ras - mutant colorectal cancer patients . jco precis oncol , 2021 2 . 
bouchahda m , saffroy r , karaboue a , et al : undetectable ras - mutant clones in plasma : possible implication for anti - egfr therapy and prognosis in patients with ras - mutant metastatic colorectal cancer . 
raimondi c , nicolazzo c , belardinilli f , et al : transient disappearance of ras mutant clones in plasma : a counterintuitive clinical use of egfr inhibitors in ras mutant metastatic colorectal cancer . 
nicolazzo c , belardinilli f , caponnetto s , et al : why the therapeutic impact of ras mutation clearance in plasma ctdna deserves to be further explored in metastatic colorectal cancer . 
in metastatic melanoma , the extent to which ctdna reflects changes in metabolic disease burden assessed by 18f - labeled fluorodeoxyglucose positron emission tomography ( fdg - pet ) is unknown . 
we assessed the role of ctdna analysis in combination with fdg - pet to monitor tumor burden and genomic heterogeneity throughout treatment . patients and methods we performed a comprehensive analysis of serial ctdna and fdg - pet in 52 patients who received systemic therapy for metastatic melanoma . 
next - generation sequencing and digital polymerase chain reaction were used to analyze plasma samples from the cohort . results ctdna levels were monitored across patients with mutant braf , nras , and braf / nras wild type disease . 
tert promoter mutant ctdna levels also paralleled changes in tumor burden , which provide an alternative marker for disease monitoring . of note , subcutaneous and cerebral disease sites were not well represented in plasma . 
the use of mitogen - activated protein kinase inhibitors ( mapki ) in patients with advanced braf mutant melanoma1 - 3 and , more immune checkpoint inhibitors4 , 5 has recently , led to improved overall survival . 
mcarthur shahneen sandhu sarah - jane dawson invasive and metastatic disease.8 moreover , clonal evolution of melanoma continues to occur during systemic therapy , with diverse resistance mechanisms observed at relapse.9 in patients with melanoma who receive systemic therapy , tumor burden and treatment responses currently are assessed through clinical evaluation , serial lactate dehydrogenase ( ldh ) levels , 10 and radiologic imaging with 18f - labeled fluorodeoxyglucose positron emission tomography ( fdgpet ) .11 fdg - pet provides unique functional information that enables an accurate assessment of changes in metabolic disease activity and tumor volume throughout treatment.11 although current imaging methods are effective at monitoring treatment response , complementary strategies are needed to assess molecular alterations and capture genomic evolution in the context of therapeutic resistance to guide treatment decisions . circulating tumor dna ( ctdna ) has emerged as a noninvasive biomarker to track tumor burden and allow monitoring of the cancer genome in blood across several malignancies.12 - 22 in melanoma , the relationship between ctdna levels and tumor burden as assessed by fdg - pet is unknown . 
three of the 52 patients also consented to multiregional tumor sampling after death as part of a rapid autopsy progratumor material and matched normal and plasma dna were retrospectively analyzed through targeted amplicon ( ta ) sequencing and digital polymerase chain reaction ( dpcr ) as well as through whole - exome sequencing ( wes ) and low - coverage wholein selected genome sequencing ( lc - wgs ) cases . 
the + symbol denotes the development of new sites of disease . statistics nonparametric spearman rank correlation was used to investigate associations between continuous variables , and the mann - whitney u test was used to assess associations between continuous and dichotomous variables . 
time to event was measured from the start of treatment to the first documentation of progression on fdg - pet , with the relative hazard ratio computed for each comparison . results ctdna is detectable across various molecular subtypes of metastatic melanoma fifty - two patients with a median age at diagnosis of 61 years ( range , 24 to 83 years ) were serially monitored with fdg - pet , ldh , and ctdna during therapy ( data supplement )  . 
on the left , columns correspond to patients and are ordered by mutational subtype and disease burden as assessed by 18f - labeled fluorodeoxyglucose positron emission tomography ( fdg - pet ) imaging . 
rows indicate the pretreatment fdg - pet disease volume , pretreatment lactate dehydrogenase ( ldh ) level , and pretreatment circulating tumor dna ( ctdna ) levels detected by dpcr ( both absolute and fractional concentrations shown )  . 
mutations are shown if they were found in a pretreatment tumor sample ( pink ) , in plasma ( green ) , or in both a pretreatment tumor sample and plasma ( gold )  . the type of pretreatment tumor material used for targeted amplicon sequencing in each patient is indicated . 
for patients mel - 70 , - 80 , and - 81 , pretreatment ctdna levels were not detectable ( left ) , but ctdna was detected at subsequent time points after treatment ( middle )  . 
 ( % ) primary metastatic mutation detected in : tumor plasma tumor only plasma only 2 - 10 10 - 50 50 - 100 site of disease present absent pretreatment ldh level ( ratio upper / normal ) ( 71% ) and seven patients ( 13% ) , respectively , and eight patients ( 15% ) were braf / nras wt . ctdna was detected in 39 ( 80% ) of 49 patients by ta sequencing and / or dpcr ( fig 1 )  . 
screening of matched plasma detected tert promoter mutations in 27 ( 75% ) of 36 patients . absolute levels of ctdna before treatment show variation according to sites of disease pretreatment plasma was available in 41 of 49 patients ( fig 1 )  . 
pretreatment ctdna levels varied among patients ( median , 1 , 112 copies / ml of plasma ; range , 63 to 97 , 000 copies / ml of plasma ) , which represented between 0.1% and 82% ( median , 5.4% ) of total circulating dna . 
of note , although a strong correlation existed between high metabolic disease burden as assessed by fdg - pet and high levels of ctdna before treatment , several discordant cases were observed ( fig 2 )  . 
we hypothesized that discordance may relate to a differential release of ctdna into the peripheral circulation dependent on specific anatomic sites of disease . consistent with this premise , patients with visceral , bone , or lymph node involvement often displayed high levels of ctdna despite at times low disease burden as detected by fdg - pet . 
in contrast , individuals with extensive subcutaneous disease consistently showed low levels of ctdna despite measurable levels of disease burden as assessed by fdg - pet ( data supplement )  . 
these findings are consistent with other studies that have shown low ctdna levels despite extensive cerebral disease.12 , 25 serial ctdna monitoring correlates with tumor burden and treatment response as assessed by fdg - pet we investigated whether ctdna levels correlate with changes in disease burden throughout treatment as assessed by ldh and fdg - pet . 
serial ctdna analysis was performed on 312 plasma samples by using dpcr for selected mutations identified from ta sequencing and compared with serial ldh measurements ( n = 329 ) and fdg - pet ( n = 244 ) across the series . 
of note , serial changes in mutant braf or nras ctdna levels closely reflected metabolic responses across multiple therapies , including mapki and immunotherapies ( figs 3b and 3c )  . 
by using a cox proportional hazards regression model , we identified that patients with an early decrease in ctdna levels ( between baseline and 1 to 4 weeks on treatment ) had improved pfs compared with patients where ctdna levels remained unchanged or increased over time ( fig 3e )  . 
similar findings were observed for patients who demonstrated an early decrease in tumor volume as assessed by fdg - pet ( fig 3e )  . although baseline levels of ldh were prognostic ( data supplement ) , no significant association between pfs and an early decline in ldh levels after treatment were found ( fig 3e )  . 
3 months were found to have a significantly greater log - fold reduction in ctdna levels at 1 to 4 weeks after treatment initiation compared with those who had a pfs , 3 months ( p = .03 ; fig 3f )  . 
rapid responses by ctdna were most notable after mapki therapy in keeping with the known response kinetics of these agents . tert promoter mutant ctdna provides an alternative marker for disease monitoring we next evaluated whether tert promoter mutations could be serially monitored in ctdna and whether levels correlated with disease burden . 
 ( a ) high ctdna levels detected in three patients with melanoma ( mel - 74 , mel - 2 , and mel - 111 ) , with 18f - labeled fluorodeoxyglucose positron emission tomography ( fdg - pet ) scans indicating only small - volume disease ( mel - 74 , small liver and adrenal deposits ; mel - 2 and mel - 111 , solitary liver deposit )  . 
 ( c ) low ctdna levels detected in three patients who presented with high - volume subcutaneous disease ( mel - 122 , mel - 92 , and mel - 4 ) as shown by fdg - pet imaging . 
 ( d ) low to undetectable levels of ctdna in two patients with isolated brain disease ( mel - 120 and mel - 5 ) as indicated by magnetic resonance imaging . 
all bar graphs are plotted to the same y - axis and on a logarithmic scale . mutations followed the same dynamic changes in ctdna as braf or nras mutations by analyzing plasma from all braf and nras mutant cases that were also tert promoter mutant ( n = 33 )  . 
paired plasma ( and tumor biopsy specimens where available ) before treatment and at disease progression were analyzed by using ta sequencing and lc - wgs / wes in selected cases . 
acquired nras mutations were the predominant genomic alteration identified through analysis of ctdna at the time of disease progression , as illustrated in patient mel - 76 ( fig 5a )  . 
circulating tumor dna ( ctdna ) levels correlate with the kinetics of 18f - labeled fluorodeoxyglucose positron emission tomography ( fdg - pet ) disease burden and patterns of response to therapy . 
 ( b ) dynamic changes in ctdna levels correlate with response to therapy as assessed by fdg - pet imaging in a patient with a braf v600k mutation ( mel - 97 ) who showed a complete metabolic response to dabrafenib + trametinib ( dab + tram ) treatment ; ( c ) a patient with an nras q61k mutation ( mel - 36 ) who displayed an initial response to several lines of therapy ( dedn6526a , radiotherapy [ rt ] / surgery [ sur ] , and temozolomide [ temo ] ) followed by disease progression before commencement of pembrolizumab ( pembro ) and then a mixed response to pembro treatment ; and ( d ) a patient with braf / nras mutant wild type ( mel - 39 ) with ctdna levels monitored through an rac1 p29s mutation during dedn6526a , rt , and abraxane treatment . 
wholebody fdg - pet scans are shown for each patient . before a routine fdg - pet scan that confirmed progressive metabolic disease , which highlights the ability of ctdna analysis to identify early treatment failure . 
for example , in patient mel - 31 , ta sequencing of plasma samples before and after vemurafenib + cobimetinib treatthe braf ment confirmed the presence of v600e mutation in addition to an increasing plasma abundance of map2k1 and pten mutations ( fig 5b )  . 
although the map2k1 e203k mutation was a likely cause for resistance in this individual , pten mutations also have been associated with diminished response to mapki therapy and may have contributed to the rapid emergence of the therapeutic resistance observed.26 in patient mel - 7 , ta sequencing and dpcr showed that changes in ctdna levels of two preexisting mutations ( braf and cdkn2a ) closely paralleled the dynamics of response and progression documented on fdg - pet ( fig 5c )  . 
 ( e ) global responses to therapy indicated by a cox proportional hazards regression model that assessed the relationship between progression - free survival ( pfs ) and the change in ctdna levels , fdg - pet volumes , or ldh levels between the pretreatment and early assessment time points . 
 + , development of new sites of disease ; cmr , complete metabolic response ; hr , hazard ratio ; mapki , mitogen - activated protein kinase inhibitor ; mmr , mixed metabolic response ; nivo , nivolumab ; pmd , progressive metabolic disease ; pmr , partial metabolic response ; smd , stable metabolic disease . akt1 mutation that represented a potential resistance mechanism to explain the short - lived response after recommencement of vemurafenib.27 finally , braf amplification is considered a common resistance mechanism to mapki therapy27 and may confound quantification of mutant braf ctdna levels for disease monitoring . 
therefore , we explored the ability of ctdna analysis to detect the presence of copy number alterations by performing plasma lc - wgs / wes in three patients treated with mapki therapy . 
these findings highlight the ability of plasma analysis to detect this mode of therapeutic resistance and emphasize the advantage of tracking tert mutations in parallel to reflect changes in tumor burden accurately . plasma dna captures spatial heterogeneity across multiple disease sites increasing appreciation of tumor heterogeneity has revealed the importance of assessing the full spectrum of molecular alterations present across multiple disease sites to appropriately guide treatment decisions . 
in contrast , plasma may overcome these limitations because it may represent a summation of molecular alterations across multiple disease sites.28 thus , we first sequenced multiple tumors collected at autopsy from patient mel - 99 with braf v600e mutant melanoma treated with vemurafenib ( data supplement )  . 
 ( b ) time course of a patient with braf / nras mutant wild type disease ( mel - 67 ) with ctdna levels tracked through a tert c . - 124 mutation during dedn6526a and pembrolizumab ( pembro ) treatment . 
 ( d ) time course of a patient with a braf v600e mutation ( mel - 28 ) with ctdna levels monitored through both a braf v600e and a tert c . - 124 mutation in plasma during vemurafenib ( vem ) treatment and radiotherapy ( rt )  . 
cmr , complete metabolic response ; pmd , progressive metabolic disease ; pmr , partial metabolic response ; smd , stable metabolic disease . subcutaneous metastasis , respectively , which were not detectable in either a bowel or mesenteric sample . 
mutant braf ctdna levels remained elevated throughout treatment , with targeted amplicon ( ta ) sequencing of plasma detecting the emergence of an acquired nras q61k mutation 18 weeks after starting treatment . 
 ( b ) ctdna analysis indicated acquired resistance to treatment in patient mel - 31 who presented with metastatic braf v600e positive disease that involved left - side pelvic lymph nodes , subcutaneous deposits , and solitary bone and lung metastases . 
the patient commenced combination therapy with vemurafenib and cobimetinib ( vem + cobi ) after disease progression in the pelvis with single - agent vem , which resulted in a short - lived ( 4 weeks ) partial metabolic response ( pmr ) ; the patient died 10 weeks after starting combination therapy as a result of rapid disease progression . 
 ( c ) this patient with braf v600k mutant disease ( mel - 7 ) initially presented with extensive disease over the left - side scapula and multiple pulmonary metastases and commenced vem for 2 months . 
 ( d ) patient mel - 1 initially presented with bulky disease of the bowel , lung , and lymph nodes and several sites of subcutaneous disease , which were braf v600e mutant positive . 
braf amplification was detected by whole - exome sequencing of a post - treatment bowel metastasis and low - coverage whole - genome sequencing of the plasma sample taken at disease progression . 
 disease sites but also demonstrates the ability of ctdna to capture genomic heterogeneity from multiple sites of disease that may be missed from a single tissue biopsy specimen . to comprehensively assess the extent to which ctdna was able to reflect spatial heterogeneity across various disease sites , we performed wes on multiregional tumor samples from two additional autopsy cases alongside matched plasma collected close to death ( fig 6 )  . 
for patient mel - 28 , 307 ubiquitous mutations ( present in all tumors ) , 36 shared mutations ( present in two or more tumors ) , and 36 private mutations ( present in only one tumor ) were detected ( fig 6a )  . 
for patient mel - 28 , the limited ability to detect private mutations in plasma was a result of the low mafs ( median maf , 9% for private mutations absent in plasma )  . 
for patient mel - 10 , the higher mafs observed for private mutations not detected in the plasma ( median maf , 38% ) compared with patient mel - 28 was likely a result of the limited ability to detect brain - specific private mutations ( median maf , 49% v 22% across other sites )  . discussion over the past decade , a major paradigm shift has occurred in the treatment of metastatic melanoma through clinical implementation of targeted therapeutics and immune checkpoint inhibitors . 
one potential avenue is through the use of ctdna analysis to provide patient - specific genomic information that can be monitored in real time during clinical management . mel - 28 ubiquitous shared private brain hilar lymph node liver pouch of douglas brain liver small bowel plasma hilar lymph node pouch of douglas liver brain plasma small bowel liver brain mel - 10 ubiquitous shared private 80% - 100% 60% - 80% 40% - 60% 20% - 40% 0% - 20% fig 6 . 
inference of the genomic architecture of disease as determined by whole - exome sequencing of multiregional tumor biopsy specimens and matched plasma from ( a ) patient mel - 28 and ( b ) patient mel - 10 . 
 previous reports16 - 22 have provided increasing evidence that ctdna has prognostic value in metastatic melanoma , with ctdna levels correlating with established measures of disease burden ( eg , ldh )  . 
this study provides the first correlative analysis to our knowledge that compares ctdna and fdg - pet imaging in this disease . fdg - pet has unique advantages in monitoring treatment responses in metastatic melanoma compared with other radiologic imaging techniques29 - 31 because it enables whole - body imaging with high sensitivity and specificity and can provide an early functional readout of response to targeted therapies.11 we reveal that ctdna levels correlate closely with changes in metabolic disease burden assessed through fdgpet and show that an early decline in ctdna levels provides an important alternative indicator of treatment response , which predicts patients with prolonged pfs . to fdgpet , a key strength of ctdna analysis is its ability to allow noninvasive sampling of the changing tumor genomic landscape under the selective pressure of therapy . 
in conjunction with other studies , 17 , 18 we highlight the ability of serial ctdna analysis to detect resistance mechanisms to mapki therapy to assist with the early identification of treatment failure and allow consideration of alternative therapeutic strategies . in contrast we monitored ctdna across all molecular subtypes of melanoma . 
previous reports have demonstrated the ability to measure ctdna levels through dpcr of braf and nras mutations ; however , the limitation of this approach in patients with copy number alterations that involve these genes has not been previously addressed . 
braf and nras amplifications are known to occur frequently in melanoma ( approximately 7% and 3% , respectively ) , 6 and in the current study , we show the advantage of plasma analysis in assessing the emergence of copy number alterations . 
the data highlight that tracking tert promoter mutations , in addition to braf and nras , offers an improved strategy for disease monitoring . in conclusion , ctdna can play a vital complementary role to functional imaging in metastatic melanoma because it can be used to monitor tumor response kinetics and can capture the mutational spectrum from many sites of disease . 
a significant limitation to ctdna monitoring is that subcutaneous and cerebral disease sites are not well represented in the plasma , and recognition of this limitation is pivotal if ctdna monitoring is to be integrated into routine clinical practice . 
 carleen cullinane no relationship to disclose damien kee no relationship to disclose benjamin brady consulting or advisory role : bristol - myers squibb , novartis , merck speakers bureau : bristol - myers squibb , merck travel , accommodations , expenses : bristol - myers squibb , merck fergal kelleher travel , accommodations , expenses : bristol - myers squibb mark a . 
mcarthur pfizer , celgene , ventana medical systems travel , accommodations , expenses : bristol - myers squibb , roche , novartis shahneen sandhu honoraria : bristol - myers squibb , amgen consulting or advisory role : bristol - myers squibb speakers bureau : bristol - myers squibb , merck sarah - jane dawson no relationship to disclose acknowledgment we thank the anatomical pathology department from the peter maccallum cancer centre and all other participating hospitals for assisting in the acquisition of tumor material . 
vergara stock and other ownership interests : genomedx patents , royalties , other intellectual property : co - inventor on three patents pending kenneth doig stock and other ownership interests : csl behring , healthscope jeanette m . 
colebatch no relationship to disclose jason li no relationship to disclose timothy semple no relationship to disclose christopher mintoff no relationship to disclose devbarna sinha no relationship to disclose paul yeh no relationship to disclose maria joao silva no relationship to disclose kathryn alsop no relationship to disclose heather thorne no relationship to disclose david e . 
papenfuss , walter eliza hall institute of medical research , melbourne , victoria , australia . supported by a peter maccallum cancer foundation grant , the national health and medical research council ( program grant app1053792 , project grant app1107126 ) , a pfizer study grant , a gundry perpetual endowment for melanoma research , and a viertel charitable foundation clinical investigator award . 
mcarthur ga , puzanov i , amaravadi r , et al : marked , homogeneous , and early [ 18f ] fluorodeoxyglucose - positron emission tomography responses to vemurafenib in braf - mutant advanced melanoma . 
lipson ej , velculescu ve , pritchard ts , et al : circulating tumor dna analysis as a real - time method for monitoring tumor burden in melanoma patients undergoing treatment with immune checkpoint blockade . 
schreuer m , meersseman g , van den herrewegen s , et al : quantitative assessment of braf v600 mutant circulating cell - free tumor dna as a tool for therapeutic monitoring in metastatic melanoma patients treated with braf / mek inhibitors . 
chang ga , tadepalli js , shao y , et al : sensitivity of plasma brafmutant and nrasmutant cell - free dna assays to detect metastatic melanoma in patients with low recist scores and non - recist disease progression . 
tsao sc , weiss j , hudson c , et al : monitoring response to therapy in melanoma by quantifying circulating tumour dna with droplet digital pcr for braf and nras mutations . 
xi l , pham th , payabyab ec , et al : circulating tumor dna as an early indicator of response to t - cell transfer immunotherapy in metastatic melanoma . 
santiago - walker a , gagnon r , mazumdar j , et al : correlation of braf mutation status in circulating - free dna and tumor and association with clinical outcome across four brafi and meki clinical trials . 
janku f , huang hj , claes b , et al : braf mutation testing in cell - free dna from the plasma of patients with advanced cancers using a rapid , automated molecular diagnostics systemol cancer ther 15 : 1397 - 1404 , 2016 23 . 
de mattos - arruda l , mayor r , ng ck , et al : cerebrospinal fluid - derived circulating tumour dna better represents the genomic alterations of brain tumours than plasma . 
long gv , fung c , menzies am , et al : increased mapk reactivation in early resistance to dabrafenib / trametinib combination therapy of braf - mutant metastatic melanoma . 
 olaparib monotherapy for brip1 - mutated high - grade serous endometrial cancer kohei nakamura , md , phd1 , 2 ; eriko aimono , md1 ; shigeki tanishima , md , phd3 ; mitsuho imai , md , phd1 ; akiko kawano nagatsuma , md , phd1 ; hideyuki hayashi , md , phd1 ; yuki yoshimura , md4 ; kentaro nakayama , md , phd4 ; satoru kyo , md , phd4 ; and hiroshi nishihara , md , phd1 introduction treatment the prognosis for women with high - grade endometrial cancers is poor , with little improvement in the last two decades.1 the mainstay of is surgery lymphadenectomy . ( hysterectomy ) with or without although adjuvant radiotherapy is considered standard for high - risk endometrial cancers , the added value of chemotherapy has been the subject of recent trials . 
recently , other mutations were reported in ovarian cancers , such as those in fanconi anemia genes ( eg , palb2 [ fancn ] , brip1 [ fancj ] , and rad51c ) and other genes involved in hrr ( eg , atm , bard1 , nbn , cdk12 , tp53 , and chek2 ) .2 , 3 poly ( adp - ribose ) polymerase ( parp1 ) is a dna repair enzyme involved in base excision and single - stranded break repair pathways.4 parp1 inhibition in hrrdecient cells blocks single - stranded dna break repair pathways , leaving only double - stranded dna break repair pathways functional . 
this results in synthetic lethality from the loss of homologous recombination and base excision repair , thus activating highly error - prone dna repair pathways , eventually causing cell death.5 , 6 parp inhibitors also trap parp - dna complexes at the replication fork , increase toxic nonhomologous end joining in parp1 - decient cells , and block parp1 / pol - mediated alternative end joining.7 the us food and drug administration ( fda ) has approved three parp inhibitors ( olaparib , rucaparib , and niraparib ) as monotherapies for breast and ovarian cancers . 
after surgery , the patient received six cycles of carboplatin and paclitaxel . there was also evidence of after rst - line chemotherapy , ct indicated stable disease based on recist 1.0 criteria , with only improvements in the cardiophrenic swollen lymph node ( fig 1 )  . 
after six cycles , ct revealed essentially stable disease without iliac in the para - aortic or left improvement swollen lymph nodes ( fig 1 )  . lateral targeted next - generation sequencing of the patients blood and resected specimen was performed using an in - house assay during treatment . 
a tp53 somatic point mutation ( p.r175h ) and somatic frameshift brip1 ( p.q554hfs * 35 ) alterations were detected as pathogenic variants in the tumor ; detailed information is provided in appendix figures a1 and a2 . 
secondary germ line examination found no american college of medical genetics and genomics recommended genes for testing . given her somatic brip1 mutation and ndings indicating loh high status , the potential efcacy of the parp inhibitor olaparib was discussed with the patient . 
9 months of olaparib and showed complete response on her most recent ct . discussion endometrial cancers are categorized into two main histologic types.8 type i endometrial tumors show endometrioid histology and typically express estrogen ( er ) and progesterone ( pr ) receptors . 
in contrast , type ii endometrial tumors do not exhibit endometrioid histology ( predominantly serous histology ) and are associated with poorer prognoses , with a 5 - year overall survival rate of 55%.1 type ii tumors neither express er / pr nor respond to endocrine therapy . 
meanwhile , type i tumors exhibit signicantly more pi3k pathway alterations ( primarily pten and pik3ca mutations ) .9 , 10 in our case , next - generation target panel sequencing results reected the molecular events observed in serous endometrial cancers , such as tp53 mutations . whereas brip1 is implicated in double - stranded dna break repair via hrr pathways , the frequency of brip1 mutations in type ii endometrial tumors remains unclear . in a previous study by heeke et al , 11 the prevalence of recombinationrelated gene mutations homologous across multiple cancer types was investigated in 52 , 426 malignant tumors . 
this has promoted clinical studies of parp inhibitors in contexts other than ovarian cancer . moreover , their potential therapeutic applications might be extended from germ line brca mutations to target a more diverse group of sporadic tumors , such as those with epigenetic disruption of brca1 / 2 function or genetically or epigenetically acquired aberrations in other important hrr pathway constituents.12 there is clinical evidence to support this theory , with olaparib approved by the fda as an alternative for patients with germ line or somatic brca1 / 2 mutations and as maintenance therapy after platinumbased chemotherapy in platinum - sensitive recurrent epithelial ovarian carcinoma , regardless of brca mutation status.13 hrr alterations are associated with brca1 / 2 mutations in high - grade serous endometrial cancers ; however , parp inhibitors await fda approval for this indication , and there are currently no trials of parp inhibitors in patients with endometrial cancers with brca or other hrr - associated genealterations . accumulating evidence indicates that patients with brip1 mutations have hrr deciency and are consequently hypersensitive to parp inhibition.14 brip1 is a brca1interacting protein ( the brip1 - brca1 interaction is important for hrr ) and associates with brca1 as cells progress through the s phase of the cell cycle.15 , 16 brip1 is a dna helicase that interacts with the cooh - terminal brct repeat of brca1 . 
brip1 is associated with the gm1 / 2 checkpoint , as well as the activation of chk1 , regulation of entry into the s phase , and maintenance of genomic stability.16 thus , if the complex involving brca1 and brip1 is involved in tumor suppression , mutations in the genes that encode these proteins should be associated with altered cancer risk . 
recently , three independent dnabased measures of genomic instability reecting underlying tumor homologous recombination dna repair deciency were developed based on loh , telomeric allelic imbalance , and large - scale state transitions.17 - 19 however , we could not determine the homologous recombination deciency score , because the relevant test is not covered by insurance in japan , and therefore , we could not prove this . 
however , we can hypothesize that the brip1 mutation results in hrr deciency and subsequently causes a high loh frequency and scattered allelic imbalance , potentially resulting in good response to parp inhibitors . in conclusion , we describe the case of a 70 - year - old woman exhibiting a durable clinical radiographic response to olaparib . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . support supported by the japan agency for medical research and development under grant no . 
lancet 387 : 1094 - 1108 , 2016 somyajit k , subramanya s , nagaraju g : rad51c : a novel cancer susceptibility gene is linked to fanconi anemia and breast cancer . 
carcinogenesis 31 : 2031 - 2038 , 2010 sigurdsson s , van komen s , bussen w , et al : mediator function of the human rad51b - rad51c complex in rad51 / rpa - catalyzed dna strand exchange . genes dev 15 : 3308 - 3318 , 2001 4 . 
morales j , li l , fattah fj , et al : review of poly ( adp - ribose ) polymerase ( parp ) mechanisms of action and rationale for targeting in cancer and other diseases . crit rev eukaryot gene expr 24 : 15 - 28 , 2014 farmer h , mccabe n , lord cj , et al : targeting the dna repair defect in brca mutant cells as a therapeutic strategy . 
nature 434 : 917 - 921 , 2005 ashworth a : a synthetic lethal therapeutic approach : poly ( adp ) ribose polymerase inhibitors for the treatment of cancers decient in dna double - strand break repair . 
j clin oncol 26 : 3785 - 3790 , 2008 konstantinopoulos pa , ceccaldi r , shapiro gi , et al : homologous recombination deciency : exploiting the fundamental vulnerability of ovarian cancer . 
gynecol oncol 15 : 10 - 17 , 1983 kandoth c , schultz n , cherniack ad , et al : cancer genome atlas research network : integrated genomic characterization of endometrial carcinoma . 
sato k , koyasu m , nomura s , et al : mutation status of rad51c , palb2 and brip1 in 100 japanese familial breast cancer cases without brca1 and brca2 oncogene 26 : 2157 - 2165 , 2007 mutations . 
dong y , hakimi ma , chen x , et al : regulation of brcc , a holoenzyme complex containing brca1 and brca2 , by a signalosome - like subunit and its role in 16 . 
genomic testing was performed on a plessision internal clinical sequencing apparatus ( keio university , tokyo , japan ) , which is used for all genome sequencingrelated analyses in our hospital ( keio university hospital )  . 
this apparatus was used to extract genomic dna from tumor samples and peripheral blood mononuclear cells extracted from patients with cancer , after the provision of consent to undergo comprehensive genomic testing . 
this study was conducted in accordance with the declaration of helsinki and title 45 , us code of federal regulations , part 46 , protection of human subjects , effective december 13 , 2001 . dna quality was checked by calculating the dna integrity number ( din ) using an agilent 2000 tapestation ( agilent technologies , waldbronn , germany ) before conducting targeted amplicon exome sequencing of the 160 genes implicated in cancer using the illumina miseq sequencing platform ( illumina , san diego , ca )  . 
cancer - specic changes in somatic genes , including single - nucleotide variants , insertions / deletions , and copynumber variations were detected and used to determine the tumor mutation burden . 
heterozygous germline mutations in brca1 or brca2 confer up to an 85% lifetime risk of breast and a 15% to 40% risk of ovarian cancer , and they significantly increase the risk of pancreatic , prostate , and male breast cancers.1 tumors in these patients have often lost the wild - type allele and therefore are hr deficient . 
in the absence of a functional copy of the brca gene that maintains dna integrity , the use of error - prone , nonhomologous end - joining repair mechanisms leads to an accumulation of genetic aberrations and promotes carcinogenesis.2 inhibition of poly ( adp - ribose ) polymerase ( parp ) induces synthetic lethality in cells with brca1 or brca2 deficiency , and clinical trials of parp inhibitors have demonstrated responses in brcamutated breast and ovarian cancers.3 the bestdescribed mechanisms of resistance to platinum agents and parp inhibitors in brca1 or brca2 carriers with ovarian cancer are secondary mutations that restore the open reading frame or reversion mutations that restore the wild - type protein.4 , 5 , 6 these mutations are present in more than 40% of recurrent platinum - resistant ovarian cancers and are more common in women with recurrent ovarian carcinoma who were previously treated for breast cancer3 , 6 . 
minimal data exist about resistance to single - agent parp inhibitor therapy in women with breast cancer.7 we report a case of a brca2 mutation carrier who received single - agent parp inhibitor therapy and had subsequent resistance conferred by a reversion mutation identified by targeted capture and massively parallel sequencing . carcinoma of the right breast with multiple positive regional lymph nodes , for which she underwent a mastectomy and axillary node dissection and received adjuvant chemotherapy with fluorouracil , epirubicin , andcyclophosphamidefollowed by tamoxifen for 5 years . 
additional details of her initial care are not available , and cancer surveillance was not performed . after presentation at age 72 with back pain , workup identified a left breast mass with extensive metastatic disease to the bone . 
biopsy of the breast mass confirmed a new primary invasive ductal carcinoma that stained positive for estrogen receptor and progesterone receptor expression and negative for her2 / neu . her disease was staged as ct4an2m1 , and she started anastrazole monotherapy . 
 genomic dna isolated from peripheral blood to evaluate for germline mutations in brca1 and brca2.8 the uw - oncoplex assay ( laboratory medicine , university of washington ) was used to perform whole - exome deep sequencing and copy number variation analysis to identify somatic mutations in dna isolated from formalin - fixed , paraffinembedded tumor tissue.8 , 9 neoplastic cellularity was assessed by estimating the fraction of neoplastic to nonneoplastic nuclei on a hematoxylin and eosin stained section by a trained pathologist . 
predicted allelic frequency for the retained alleles of heterozygous germline variant of brca2 ( and reference single nucleotide polymorphism [ snp ] 2126042 ) was calculated with the following formula : [ ( tumor percentage ) + ( nontumor percentage ) ] 4 [ ( tumor percentage ) + 2 ( nontumor percentage ) ]  . 
predicted allelic frequency for the wild - type brca2 ( and reference snps 1799943 , 1799955 , and 1801406 ) was calculated as 100 2 the predicted allelic frequency of the variant . the fred hutchinson cancer research center consortium institutional review board approved this study . 
the reference snps were mapped to separate brca2 alleles with a predicted germline allelic frequency of approximately 50% ( table 1 )  . the pre - veliparib sample ( a bony lesion ) had a lower neoplastic cellularity ( 25% ) than the postveliparib sample ( chest wall ; 78% )  . 
the variant allele frequency of rs2126042 , present on the same copy of the mutated brca2 allele , was 60% in the preand 92% in the post - treatment sample . 
the allelic frequency of mutated brca2 in the pre - treatment sample is consistent with the loss of heterozygosity of the wild - type allele ( table 1 ; fig 1 )  . 
in the posttreatment sample , the marked discrepancy in the allelic frequency of the mutated brca2 ( 50% ) and rs126042 ( 92% ; both expected to be on the same allele ) , along with the low allelic frequency of other heterozygous germline snps ( predicted frequency , approximately 18% ) present on different copies of the alleles , strongly supports the restoration of wildtype brca sequence on the mutated allele ( table 1 )  . these collective findings suggest a reversion of the brca2 frameshift deletion mutation on the mutant allele during treatment ( fig 1 )  . 
in the lymphocytes ( germline ) , the wild - type brca2 allele and the heterozygous reference single nucleotide polymorphisms ( snps ) rs1799943 , rs1799955 , and rs1801406 were detected , along with the brca2 c.9097dup and a heterozygous snp , 2126042 . 
this method greatly simplifies the detection of such events and has important implications for the clinical care of patients with brca - mutant disease . we predict that similar phenomena may be sensitively identified earlier , before disease progression , with new platforms that incorporate next - generation sequencing to circulating tumor dnaso - called liquid biopsies . we conclude that the somatic reversion mutation was the mechanism of resistance to parp inhibitor and was responsible for progression during therapy . 
in the germline sample , the wild - type brca2 allele and the heterozygous reference single nucleotide polymorphisms ( snps ) rs1799943 , rs1799955 , and rs1801406 were detected together , along with the brca2 c.9097dup and a heterozygous snp rs2126042 . 
gadi stock and other ownership interests : sengine precision medicine research funding : genentech / roche ( inst ) affiliations all authors , university of washington ; kalyan banda and v.k. 
pritchard cc , salipante sj , koehler k , et al : validation and implementation of targeted capture and sequencing for the detection of actionable mutation , copy number variation , and gene rearrangement in clinical cancer specimens . j mol diagn 16 : 56 - 67 , 2014 10 . 
computed tomography ( ct ) scan with ( a ) the index lesions in the breast ( arrows ) before parp inhibitor and ( b ) 5 months later , which demonstrates decreased volume of disease . 
the absolute numbers of variant alleles and total number of alleles are in parentheses . mutations in tp53 , depdc5 , plk2 , and tacc3 are present in both preand post - treatment biopsies but are enriched in the post - treatment sample . 
identification of biomarkers predictive of a benefit from trastuzumab will minimize overtreatment and lower health care costs . methods to identify possible single - gene biomarkers , an exploratory analysis of 3 , 669 gene probes not expected to be expressed in normal breast tissue was conducted . 
 disease - free survival ( dfs ) was used as the end point in a cox regression model , with the interaction term between c8a mrna and treatment as a categorical variable split on the cohort mean . results a significant interaction between c8a mrna and treatment was detected ( p < .001 ) , indicating a predictive response to trastuzumab treatment . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction breast cancer is the leading cause of cancer death among women worldwide.1 it is a heterogeneous disease , so to maximize benefit to the patient by individualizing treatment regimens , clinicians need a sound classification syste clinicopathologic variables , including the estrogen receptor 1 , progesterone receptor , and human epidermal growth factor receptor 2 ( her2 ) , are used to classify tumors as hormone positive , her2 positive ( her2 + ) , or triple negative and to guide treatment decisions . 
pam50 , oncotype dx , and mammaprint were developed using retrospective analysis of mrna gene expression data from breast cancer cohorts . identification of single genes or gene signatures that can be used to classify a breast cancer tumor as a subtype with known treatment strategies can improve outcomes and minimize unnecessary treatment . 
the identification that 15% to 25% of breast cancer tumors overexpress the her2 protein , a transmembrane tyrosine kinase that regulates growth and cell survival , resulted in the development of monoclonal antibody therapy that targets her2 . 
 the exploratory analysis identified c8a , a member of the membrane attack complex and part of the innate immune system , which was prognostic of outcome in the observation arm and predictive of benefit from trastuzumab . 
we also characterized c8a protein expression in stable cancer cell lines . methods study population the hera trial was an international , intergroup , open - label , phase iii randomized study.5 a total of 5 , 102 women with her2 + primary breast cancer ( after a minimum of four courses of standard chemotherapy ) were enrolled and assigned randomly to one of the following three treatment arms : observation ( no trastuzumab ) and 1 or 2 years of adjuvant trastuzumab administered intravenously every 3 weeks . 
an interim analysis5 showed that patients assigned to the 1 - year trastuzumab arm experienced a significantly lower hazard of a dfs event than those in the observation arthis finding resulted in a protocol amendment that allowed for observation patients to selectively crossover to trastuzumab treatment ( 1 or 2 years ) , with the restriction of being alive and disease - free as of may 16 , 2005 . 
the transhera tissue resource consisted of 1 , 203 blocks that originated from 15 countries and were processed at royal marsden hospital in the united kingdo in addition , an extra 600 - m core was taken from each block . 
sample rna was extracted using the expressart ffpe rnaready isolation kit ( amptec , hamburg , germany ) , which is optimized for isolation of total rna specifically from ffpe tissue samples , and eluted into a volume of 20 l . 
the biotinylated cdna is then immobilized to a streptavidin - coated solid support and annealed to a dasl assay pool of gene - specific oligonucleotides for extension and ligation followed by polymerase chain reaction amplification with a biotinylated and a fluorophore - labeled universal primer . 
 control ( liver and brain ) rna samples were included for each processed batch of 48 samples to ensure that rna processing was successful and for quality control and normalization of data between assay batches . 
cubic spline has been shown to combine the positive effects of quantile normalization and to avoid the drawbacks of discontinuous mapping of intensity values and no - rank preservation.11 analysis information from the hera database , with a clinical cutoff date of april 12 , 2012 , and 8 years of median follow - up , was used in the current study.7 the primary end point was dfs , defined as the time from random assignment to first occurrence of any of the following dfs events : recurrence of breast cancer at any site ; the development of ipsilateral or contralateral breast cancer , including ductal carcinoma in situ but not lobular carcinoma in situ ; second nonbreast malignant disease other than basal - cell or squamous - cell carcinoma of the skin or carcinoma in situ of the cervix ; or death as a result of any cause without documentation of a cancer related event . exploratory analysis of possible predictive biomarkers in this exploratory analysis , 3 , 669 gene mrna probes on the illumina humanht - 12 v3 array , which are based on normal breast tissue mrna expression profiles not expected to be expressed in breast tissue , 10 were used to conduct an exploratory analysis of possible predictive biomarkers.1 each probe was used in a cox proportional hazards regression predictive model as a categorical variable bifurcated on the mean of gene mrna expression . 
the list of probes ranked by interaction p value were then used to identify c8a as a gene of interest because it is not normally expressed in breast tissue and is a member of the membrane attack complex and part of the innate immune systea volcano plot of the exploratory analysis is shown in figure 1 . 
exploratory analysis of treatment interaction p value as a categorical variable split on probe mrna expression of 3 , 669 gene probes in the herceptin adjuvant cdna - mediated annealing , selection , extension , and ligation cohort that have zero expected expression in normal breast tissue . 
in the early events analysis , only early disease - defining events were taken into account ( ie , at 2.2 years of median follow - up time ) , when almost no patient had switched from observation to trastuzumab . 
 cox models were adjusted for several clinicopathologic factors : age , pathologic tumor size , progesterone receptor local , estrogen receptor local , tumor grade , menopausal status , nodal status , prior ( neo ) adjuvant chemotherapy , eastern cooperative oncology group performance status , race , and region . 
the weights for the ipw analysis were estimated using a time - varying covariate and the following two baseline covariates : age at study entry and prior ( neo ) adjuvant chemotherapy . 
follow - up time of each eligible patient was divided into 1 - month intervals , and the time - varying covariate was estimated as the within - interval cumulative time of eligibility to cross over . prognostic her2 + gastric cancer cohort we used the publicly available data from the hungarian academy of science ( has ) gastric cancer cohort to further explore c8a as a possible biomarker in her2 + gastric cancer.12 the has gastric cancer cohort is a collection of publicly available cohorts with outcome data profiled on the affymetrix platform ( santa clara , ca )  . 
molecular signature database ( version 4.0 ) gene sets tested included immunologic signatures ( n = 1 , 910 ) , canonical pathways ( n = 1 , 320 ) , molecular gene ontology terms ( n = 1 , 454 ) , microrna ( n = 221 ) , chromosome positions ( n = 326 ) , transcription factors ( n = 615 ) , chemical genetic perturbations ( n = 3 , 402 ) , and oncogenic signatures ( n = 189 )  . c8a mrna normal and cancer tissue expression profile the gtex web portal17 was used to generate a figure for the c8a mrna expression in normal tissues . 
figures that illustrate c8a mrna expression from cancer cell lines were generated using the cancer cell line portal.18 the cancer cell line encyclopedia was used to determine the c8a mrna mean and standard deviation in 1 , 036 cancer cell lines . 
tumor cell lines , hepatocellular carcinoma ( tma809 ) , lung cancer ( dms454 ) , triple negative breast cancer ( mda - 231 ) , her2 normal breast cancer ( mda - 175 ) , and her2 + breast cancer ( sk - br - 3 ) were used for c8a ihc staining . 
 the mrna expression profile suggests that for approximately 70% of the human epidermal growth factor 2positive cohort , c8a mrna was not expressed , and 30% had high expression of c8a mrna . 
kaplan - meier curve for disease - free survival ( dfs ) in the herceptin adjuvant cdna - mediated annealing , selection , extension , and ligation cohort , where the c8a mrna split on the mean represents c8a - low and c8a - high categories . 
the c8a phenotype is illustrated in the gsea histogram shown in figure 4 , with the top 50 genes enriched in the c8a - low group versus the top 50 genes enriched in the c8ahigh group . 
c8a is part of the innate immunity and membrane attack complex and clearly differentiated the enrichment of gene sets associated with immunologic response . c8a mrna expression the c8a mrna on / off expression profile in our analysis cohort suggests that c8a may be expressed in other cancer cell lines . 
on the basis of the results presented in the data supplement , we see that c8a is highly expressed in distinct ovarian , liver , stomach , pancreatic , lung , and hematopoietic / lymphoid cancer types . 
to verify that c8a mrna results in c8a protein expression , c8a antibody was used to stain available cancer cell lines , and the results are the data supplement . discussion identification of single genes or gene signatures that can be used to classify a breast cancer tumor as a subtype with known treatment strategies can improve outcomes and minimize unnecessary treatments . 
 the immune gene list from molecular signature database ( version 4.0 ) on the right represents the top 50 enriched immune genes in c8a - low versus c8ahigh samples and the top 50 enriched immune genes in c8a - high versus c8a - low samples . 
the cancer cell line encyclopedia data show that dms454_lung , ov90_ovary , and snu719_stomach are examples that have high expression of c8a and that c8a is a feature of those immortal cancer cell lines . c8a protein is a critical component of the membrane attack complex that inserts itself into the outer membrane of target cells and anchors the recruitment of c9 proteins to form a pore that leads to cell lysis and death.20 , 21 the membrane attack complex is a component in the complement system , which is part of the innate immune system found in plant and animals and evolved before adaptive immunity.22 the innate immune system can work independently of or be triggered by the adaptive immune system and plays a role in the monitoring of host cells that are damaged or have died and should be cleared.23 the complement system proteins are synthesized by the liver and normally circulate in the blood until stimulated by complement activation . 
molecular characterization of c8a high her2 + tumors may indicate that it is a new cancer phenotype that can escape the immune response and become an important mechanism in cancer that affects survival . 
goldhirsch a , gelber rd , piccart - gebhart mj , et al : 2 years versus 1 year of adjuvant trastuzumab for her2 - positive breast cancer ( hera ) : an open - label , randomised controlled trial . 
skedgel c , rayson d , younis t : is adjuvant trastuzumab a cost - effective therapy for her - 2 / neu - positive t1bn0 breast cancer ? ann oncol 24 : 1834 - 1840 , 2013 10 . 
benita y , cao z , giallourakis c , et al : gene enrichment profiles reveal t - cell development , differentiation , and lineage - specific transcription factors including zbtb25 as a novel nf - at repressor . 
gyrffy b , lanczky a , eklund ac , et al : an online survival analysis tool to rapidly assess the effect of 22 , 277 genes on breast cancer prognosis using microarray data of 1 , 809 patients . 
gyorffy b , lnczky a , szllsi z : implementing an online tool for genome - wide validation of survival - associated biomarkers in ovarian - cancer using microarray data from 1287 patients . 
gyrffy b , benke z , lnczky a , et al : recurrenceonline : an online analysis tool to determine breast cancer recurrence and hormone receptor status using microarray data . 
said ea , dupuy fp , trautmann l , et al : programmed death - 1 - induced interleukin - 10 production by monocytes impairs cd4 + t cell activation during hiv infection . 
to our knowledge , there are no previous reports of basal cell carcinoma of the prostate occurring in association with a germline brca2 mutation , as in the patient reported here ; however , brca2 mutations , which are a recognized risk factor for prostate adenocarcinoma , are increasingly recognized as a driver for more aggressive disease5 and as a potential therapeutic target . 
olaparib , a poly ( adp ribose ) polymerase ( parp ) inhibitor , has shown high response rates in patients with prostate cancer and defects in homologous repair.6 unfortunately , many patients receiving treatment with parp inhibitors may develop reversion mutations that restore brca function and reduce sensitivity to parp inhibition or confer resistance . 
to our knowledge , there are no reports of the successful treatment of patients with metastatic prostate cancer after brca reversion mutations . in this article , we present a patient with a germline brca2 mutation who developed de novo metastatic basal cell carcinoma of the prostate and experienced a complete clinical response with olaparib . 
 after a mixed response , the patient underwent metastasectomy and subsequently had minimal to no evidence of disease while continuing to receive olaparib maintenance . in january 2016 , a 77 - year - old man with a normal psa presented with urinary obstruction . 
a computed tomography ( ct ) scan confirmed dozens of soft tissue nodules in all lobes of both lungs , suggestive of metastatic disease , as well as an enlarged prostate with a hypodense lesion in the left lobe causing mass effect on the urethra , suggestive of malignancy . 
tumor cells stained diffusely positive for bcl2 and ttf1 ; focally positive for ck903 , ck20 , p63 , and ar ; and negative for ck7 , napsin a , chromogranin , synaptophysin , psa , erg , nkx3.1 , uroplakin , gata3 , and s100 . 
overall , the immunophenotype and morphology were compatible with basal cell carcinoma of the prostate , basaloid variant ( fig 1 )  . the patients lung metastases continued to grow rapidly , he was no longer ambulatory , and he joseph e . 
bhatt author affiliations and support information ( if applicable ) appear at the end of this article . the content is solely the responsibility of the authors and does not necessarily represent the official views of harvard catalyst , harvard university , and its affiliated academic health care centers or the national institutes of health . corresponding author : joseph e . 
 ( a ) x100 , ( b ) x400 , ( c ) x200 , and ( d ) x400 ; prostate biopsies demonstrated a high - grade carcinoma with basaloid morphology : monotonous cells with high nuclear to - cytoplasmic ratio , arranged in solid nests , with peripheral palisading , frequent comedonecrosis . 
we met him for an initial visit at this time , and targeted sequencing of his tumor was performed by foundationone ( foundation medicine , cambridge , ma ) , revealing a mutation in brca2 : s1982fs * 22 , also known as 6174delt , present at 70% allele frequency . 
after 2 months of treatment , he developed progressive macrocytic anemia , which is a known adverse effect of olaparib , and his dose was reduced to 300 mg twice daily . 
three months after initiating treatment , a ct scan of the torso revealed a dramatic response , with the innumerable pulmonary nodules and masses decreased in size and quantity throughout the lungs ( fig 2c ) and shrinkage of the hypodense lesion in the prostate . eight months after beginning treatment , the patient continued receiving olaparib , which was further dose reduced to 200 mg twice daily for anemia . 
the second mutation restored the open reading frame presumably restoring brca2 function , thereby mediating resistance to olaparib . because of frequent complicated urinary tract infections , we wished to avoid cytotoxic chemotherapy , and we continued giving olaparib while adding atezolizumab and androgen deprivation therapy ( adt ) with leuprolide acetate . 
we did not treat with adt initially because of what we thought would be a low likelihood of response for this type of tumor on the basis of the limited case series available , 8 but we added it at this point because of limited low - toxicity options . 
imaging again showed a mixed response : the four new lung nodules seen on the previous scan had disappeared , other nodules were stable to smaller , but the largest right subpleural nodule and one additional nodule continued to grow and cavitate . 
 the two growing lung nodules were resected , and the patient continues to receive olaparib monotherapy , now 28 months after diagnosis , with his most recent scan showing only a single 1.4 - cm nodule that continues to shrink . discussion we present a patient who was successfully treated for a rare disease that has no standard of care . 
the patient is notable for rationally selected targeted therapy with parp inhibition in a new tumor subtype and for further success in treatment after the development of a brca2 reversion mutation that seemed to confer resistance . 
parp inhibition is showing increasing promise across tumor types that harbor defects in homologous recombination , and mechanisms and patterns of resistance are being elucidated.9 in prostate cancer , the first case series6 reporting the successful treatment of tumors harboring defects in homologous repair with olaparib showed a response in 88% of patients with dna repair defects . 
long - term data are still evolving and have not yet been reported in this setting , but a recent abstract10 suggests that , at least in combination with adt , olaparib results in a median pfs of approximately 13 months in patients with metastatic prostate cancer . 
at the time of progression , various mechanisms of resistance have been reported , including brca2 reversion mutations , as in this patient , 11 which may be polyclonal , suggesting that these reversion mutations may be relatively common under the evolutionary pressure of parp inhibition . in the patient presented here , we elected to add atezolizumab in the hope that there could be a synergistic effect with parp inhibition . 
 preclinical rationale for this approach is based on cell culture and xenograft models where parp inhibitors upregulate pd - l1 expression , thereby attenuating anticancer immunity , and pd - l1 inhibitors resensitize tumors to t - cell killing , increasing therapeutic efficacy.12 clinical trials investigating these combinations are ongoing.13 , 14 after adding atezolizumab , this patient experienced a mixed response , and with only two lung nodules remaining , we elected to proceed with a metastasectomy . 
if reversion mutations are indeed common under the evolutionary pressure of parp inhibition , we hypothesize that the patient remains disease free , at least in part , as a result of continued efficacy of immune checkpoint blockade . 
in conclusion , to our knowledge , this is the first report of the successful treatment of basal cell prostate cancer with a parp inhibitor , and more importantly , the first report of the successful treatment of a brca2 - mutated tumor after a reversion mutation . 
 bhatt collection and assembly of data : all authors data analysis and interpretation : all authors manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors relationships may not relate to the subject matter of this manuscript . 
simper nb , jones cl , maclennan gt , et al : basal cell carcinoma of the prostate is an aggressive tumor with frequent loss of pten expression and overexpression of egfr . 
chang k , dai b , kong y , et al : basal cell carcinoma of the prostate : clinicopathologic analysis of three cases and a review of the literature . 
banda k , swisher em , wu d , et al : somatic reversion of germline brca2 mutation confers resistance to poly ( adp - ribose ) polymerase inhibitor therapy . 
clarke n , wiechno pj , alekseev b , et al : olaparib combined with abiraterone in patients ( pts ) with metastatic castration - resistant prostate cancer ( mcrpc ) : a randomized phase ii trial . 
quigley d , alumkal jj , wyatt aw , et al : analysis of circulating cell - free dna identifies multiclonal heterogeneity of brca2 reversion mutations associated with resistance to parp inhibitors . 
karzai f , madan ra , owens h , et al : a phase 2 study of olaparib and durvalumab in metastatic castrate - resistant prostate cancer ( mcrpc ) in an unselected population . 
friedlander m , meniawy t , markman b , et al : a phase 1b study of the anti - pd - 1 monoclonal antibody bgb - a317 ( a317 ) in combination with the parp inhibitor bgb - 290 ( 290 ) in advanced solid tumors . 
 minimally invasive real - time detection of actionable mutations in patients with metastatic solid tumors using fine - needle and liquid biopsies purpose fine - needle biopsy ( fnb ) and liquid biopsy are minimally invasive methods of tumor sampling that provide feasible means to assess tumor genotypes in real time . 
 however , more data are needed to establish the strength of these methods by benchmarking against the current gold standard methods , core - needle biopsy ( cnb ) or surgical excision of the tumor . patients and methods eligible patients with advanced solid tumors were prospectively recruited . 
we performed mutation profiling using matched tumor dna obtained by cnb , fnb and liquid biopsy , and matrix - assisted laser desorption / ionization time - offlight custom mass - spectrometry or targeted next - generation dna sequencing . 
median dna yield from cnb and fnb were 775 ng ( interquartile range , 240 to 347 4ng ) and 649 ng ( interquartile range , 180 to1350 ng ) , respectively . 
2018 by american society of clinical oncology next - generation dna sequencing ( ngs ) of archival tumors is now routinely performed to identify actionable somatic mutations to guide treatment decisions in patients with solid tumors with either standard of care therapy or genotype matched clinical trials.1 - 5 fresh tumor biospecimens that are needed to accurately assess tumor genotype in real time are usually obtained by a core - needle biopsy ( cnb ) or surgical excision of the tumor ; however , these procedures are invasive and carry inherent risks , including bleeding , infection , and injuries to nearby tissues and / or organs , and often are not suitable for serial tumor samplings . 
noninvasive methods that readily allow that sampling of tumor or tumor - derived dna are therefore needed to overcome these limitations . fine - needle biopsy ( fnb ) is a minimally invasive and safe technique for tumor sampling . 
 sample processing , sufficient tumor dna can be obtained for screening of actionable mutations using ngs , considering that the input dna requirement for targeted ngs has decreased.7 - 9 circulating tumor dna ( ctdna ) obtained by liquid biopsy can now be used for tumor mutation profiling , 10 - 12 and this approach will be useful , particularly for those who do not have tumor lesions that are easily accessible to biopsy . 
the us food and drug administration have recently approved ctdna - based companion diagnostic tests in nonsmall - cell lung cancer to detect egfr exon 19 deletions or l858 mutations and egfr p.t790m mutation to select patients for treatment with egfr tyrosine kinase inhibitors erlotinib and osimertinib , respectively.13 in the current study , we identified prospectively actionable somatic mutations in a cohort of patients with advanced solid tumors using matched cnb , fnb , and ctdna to compare the performance of these methods in a realworld clinical setting . 
the study was approved by the university health network research ethics board ( #12 - 0361 - c )  . tumor tissue and blood collection fresh tumor tissue was procured by an image ( ultrasound or computed tomography ) - guided percutaneous biopsy of a metastatic lesion . 
the most recent formalin - fixed , paraffin embedded ( ffpe ) archival tumor specimen from the date of consent , either surgical excision or cnb , was obtained . tumor sample processing , pathologic evaluation , and dna extraction archival tumor samples were processed as previously described.4 cell recovery from fnb samples was optimized using a rapid onsite evaluation algorithm by cytopathologists.14 with each pass , a small , representative portion of the sample was expressed from the needle and used to produce one to two air - dried , direct smears that were then stained using a rapid romanowsky method and examined microscopically to judge the adequacy of the sampling . 
tumor content of the sample was estimated by manual microscopic examination of the adequacy assessment slides and expressed as a percentage of tumor cells ( tumor cells / total nucleated cell content )  . 
cell - free dna that contained ctdna was isolated from plasma using the qiagen circulating nucleic acids kit ( qiagen ) according to manufacturer instructions , with the exception that carrier trna was not used . 
 germline dna was isolated from the wbc fraction using either the standard manual phenol / chloroform extraction method or automated extraction . sequencing reads were aligned to the human genome reference using the burrows - wheeler aligner.16 somatic variants were called from the resulting bam files using strelka17and annotated with annovar.18 a minimum read depth of 500 at the location of the variant was required for mutation reporting . 
only mutations with > 5% af in ctdna and < 1% af in germline dna were reported to avoid false - positive results . mutation profiling of tumor and germline tumor dna from fnb and cnb as well as germline dna were profiled using one of three platforms : matrix - assisted laser desorption / ionization time - of - flight ( maldi - tof ) custom mass spectrometry ( massarray ; agena bioscience , san diego , ca ) assay that detects 279 hotspot mutations in 23 genes ( appendix table a1 ) at a limit of detection of 5% ; and two ngs assays : the truseq amplicon cancer panel ( illumina , san diego , ca ) , which covers mutations in 48 genes ( appendix table a2 ) , on the miseq sequencer ( illumina ) ; or the ion ampliseq cancer panel ( thermo fisher scientific ) , which covers mutations in 50 genes ( appendix table a3 ) , on the ion proton sequencer ( thermo fisher scientific ) as previously described.4 somatic variants identified have to meet laboratory defined thresholds of > 500 read depth coverage and allele frequency ( af ) of > 10% ; however , these analyses were performed as research analyses in a molecular diagnostics laboratory , and mutation results were not formally reported in the patients electronic medical records . 
profiling results from fresh biopsies and plasma samples were captured and stored in a research database on the medidata rave ( new york , ny ) platform . mutation profiling of plasma - derived ctdna thunderbolts cancer panel ( raindance technologies , billerica , ma ) , which can detect mutation in 50 genes , 15 was used to profile ctdna and germline dna . 
mutations classified as levels 1 , 2a , 2b , 3a , 3b , and 4 were considered actionable . clinical data collection patient and tumor characteristics , cancer treatment ( s ) before study entry , and subsequent treatments after molecular profiling were collected . 
 adverse events related to study procedures were recorded and graded according to the common terminology criteria for adverse events version 4.0.19 patients were folllowed up to 7 days after tumor biopsy via telephone for assessment of biopsy - related adverse events ( aes )  . 
combined fnb and cnb results of each patient were considered as tumor mutation results and compared with ctdna mutation results . results patients from october 2012 to january 2015 , 45 patients were enrolled . 
 cnb , core - needle biopsy ; fnb , fine - needle biopsy . safety of fresh tumor biopsy of the 41 patients biopsied , 12 ( 29% ) experienced 14 aes . 
other biopsy - related aes were grade 1 or 2 abdominal pain ( n = 10 ) , vomiting ( n = 2 ) , bleeding ( n = 1 ) , and fever ( n = 1 )  . feasibility of mutation analysis from fnb and cnb in the 41 patients biopsied , a median of three fnbs ( range , two to five fnbs ) and four cnbs ( range , one to five cnbs ) were obtained . 
of the remaining patients , five did not have tumor in cnb and one did not have tumor in fnb , which yielded 30 cnbs and 34 fnbs with sufficient tumor and dna for mutation profiling ( table 2 )  . 
whereas the dna yield from cnbs was generally higher than that from fnbs , all 34 fnbs yielded enough dna for mutation profiling . comparison of cnb and fnb mutation results of the 30 cnbs analyzed , 27 were profiled by miseq and three by maldi - tof custom mass spectrometry . 
in the remaining 22 patients , eight ( 20% of 41 total evaluable patients for mutation results ) had nine actionable mutations ( table 2 and fig 2 )  . 
blue , mutation detected ; red , mutation not detected ; gold , sample not available . actionable mutation plasma directly comparablesame patients , same biopsy site , and same profiling platformmutation calls were identical in 23 of 25 pairs , which resulted in 92% agreement ( table 3 )  . comparison of mutations detected in archival and fresh tumors there were 32 archival and fresh tumoreither cnb or fnbpairs ; however , results were only directly comparable in 23 pairs because the use of sequencing platform was not uniform in all cases . 
more mutations were detected in plasma compared with tumors , and mutation results were concordant in six ( 19% ) of 32 pairs ( summarized in table 4 )  . 
in both cases , tumor sequencing was performed using the truseq panel , and reviewing the tumor sequencing data revealed that position cdkn2a p.p114s was not covered but that pten p.s10n position was covered with > 3 , 000 coverage . 
in contrast , three actionable pik3ca mutations that were found in the cnb / fnb pairs were not detectable in plasma ( fig 2 ) ; afs of two pik3ca mutations were high in tumors ( 78% and 49% ) but low in the remaining one ( 9% )  . 
as the cutoff for mutation calling in plasma in this study was conservative , we reviewed the plasma sequencing data in three cases in which pik3ca mutation results were discordant and found that all three pik3ca mutations could be found in corresponding plasma with low afs ( pik3ca p.h1047r : af 2% ; pik3ca p.n345k : af 1% ; and pik3ca p.e545k : af 1% )  . 
none of these three mutations was found in germline sequencing . discussion the feasibility of targeted ngs of cytology samples to identify actionable mutations has been demonstrated in multiple solid tumor types20 - 26 ; however , results from several real - world studies have demonstrated that 30% to 50% of fnb samples were not suitable for ngs analysis because of low tumor cell content.21 , 23 , 27 , 28 a large retrospective study reported a success rate of 54% , which , albeit low , was comparable to results that were achieved with cnb at the same institution.28 in contrast , a high success rate ( > 90% ) can be achieved by using rapid onsite evaluation algorithm combined with optimized sample processing.22 our results support the growing body of evidence that suggests that optimally processed fnbs can be successfully used for mutation profiling with ngs , yielding results that are comparable to those of cnb . 
comparison of tumor and plasma mutations detected ( n = 32 ) ( continued ) tumor type tumor sequencing platform tumor mutations detected , shown with allele frequency ( % ) plasma mutations detected using thunderbolts cancer panel , shown with allele frequency ( % ) concordant patient mat - 034 melanoma miseq atm p.e2744k ( 33 ) , idh1 p.r132c ( 28 ) , nras p.q61h ( 26 ) idh1 p.r132c ( 19 ) , nras p.q61h ( 19 ) , tp53 p.r116w ( 19 ) mat - 035 melanoma mat - 036 ovarian mat - 037 breast miseq miseq miseq kit p.e861k ( 30 ) tp53 p.p151h ( 22 ) none detected none detected none detected atm p.t1819a ( 27 ) , csf1r p.s946n ( 7 ) , erbb2 p.n850s ( 8 ) , erbb4 p.c234y ( 5 ) , pdgfra p.p567l ( 4 ) , smad4 p.q183r ( 7 ) mat - 038 breast miseq pik3ca p.h1047r ( 14 ) , pik3ca p.h1048r ( 14 ) pik3ca p.h1047r ( 39 ) , pik3ca p.h1048r ( 39 ) mat - 039 melanoma miseq braf p.v600k ( 77 ) , idh1 p.r132c ( 32 ) akt p.i19n ( 8 ) , braf p.v600k ( 19 ) , erbb2 p.i767t ( 9 ) , met p.y1253h ( 11 ) , mat - 040 breast miseq egfr p.v292m ( 16 ) pik3ca p.n345k ( 49 ) , tp53 p.e2q ( 45 ) mat - 041 mat - 042 mat - 044 mat - 045 breast ovary breast breast miseq miseq miseq miseq tp53 p.n210fs ( 18 ) none detected pik3ca p.e545k ( 9 ) tp53 p.v272m ( 70 ) none detected none detected none detected none detected note . 
the time interval between the collection of the archival tumor and fresh tumor biopsies was approximately 5 years , and , during that interval , the patient received an aromatase inhibitor for approximately 3 years in the adjuvant setting and capecitabine chemotherapy for 18 months in the first - line metastatic setting . 
one possible explanation is that the pik3ca p.h1047r mutation , in this case , is clonal and the atm p.e1971d subclonal and atm mutant subclone arose during metastasis and / or treatment resistance . we have also demonstrated that actionable mutations can be detected from plasma - derived ctdna using raindance thunderbolts gene panel and ngs . 
using a cutoff af of 5% , three actionable pik3ca mutations that were detectable in the tumors of three patients with breast cancer were not detectable in plasma ; however , all three pik3ca mutations were indeed present in plasma at low af ( 1% to 2% )  . 
considering that afs of tumor - derived mutations in plasma in patients with solid tumors can be low and one requires an assay with a limit of detection of 0.1% or less to pick up the majority of ctdna mutations , our criteria for mutation calling in plasma were too conservative and limited the sensitivity of our assay . 
our results reinforce the fact that ctdna mutation assays with low limits of detection can indeed miss actionable mutations , and , when ctdna results are negative , every effort should be made to profile tumors . 
in one case with the cdkn2a p.p114s mutation in plasma , tumor sequencing did not cover the mutation position , and this is likely to be the reason for the discordance between tumor and plasma results . 
taken together , our results demonstrate that mutation profiling of ctdna can be used as a primary tool to detect actionable mutations and can also be used to monitor tumor genetic evolution overtime . 
of importance , results from recent studies that assessed the efficacy of pan phosphatidylinositol 3 - kinase ( pi3k ) inhibitors in hormone receptorpositive advanced breast cancers demonstrated that patients derived benefit from pan - pi3k inhibitors regardless of tumor pik3ca mutation status , 29 , 30 but that patients with pik3ca mutation in ctdna derived better clinical benefit in terms of tumor response and progression - free survival.30 these findings support tumor agnostic mutation testing from ctdna in the right clinical context . 
 use of multiple platforms has inherent bias in the ability to detect alterations in different genomic regions , an important caveat in comparing mutation results between fnb and cnb as well as between plasma and tumor . 
 we have to acknowledge that tumor structural tumor microenvironment , architecture and including immune infiltrates , cannot be assessed using fnb , an important limitation . our results indicate that , with proper specimen handling , processing , and assessment , cytology samples , such as the direct smear and / or cell block , and liquid biopsies are suitable for molecular profiling using ngs . 
bedard research funding : bristol - myers squibb ( inst ) , sanofi ( inst ) , astrazeneca ( inst ) , genentech ( inst ) , servier ( inst ) , glaxosmithkline ( inst ) , novartis ( inst ) , signalchem ( inst ) , ptc therapeutics ( inst ) , cascadian therapeutics ( inst ) , nektar ( inst ) , merck ( inst ) eitan amir honoraria : apobiologix john d . 
stockley honoraria : astrazeneca , bristol - myers squibb consulting or advisory role : astrazeneca , janssen pharmaceuticals , novartis , bristol - myers squibb research funding : astrazeneca suzanne kamel - reid consulting or advisory role : novartis , astrazeneca consulting or advisory role : merck , astrazeneca , medimmune , morphosys , symphony evolution research funding : bristol - myers squibb ( inst ) , genentech ( inst ) , glaxosmithkline ( inst ) , merck ( inst ) , novartis ( inst ) , pfizer ( inst ) , medimmune ( inst ) , astrazeneca ( inst ) , boehringer ingelheim ( inst ) , bayer ( inst ) , amgen ( inst ) , symphony evolution ( inst ) , astellas pharma ( inst ) aaron r . 
hansen honoraria : merck , astrazeneca , medimmune , pfizer , glaxosmithkline , novartis , merck serono , boehringer ingelheim , bristol - myers squibb research funding : karyopharm therapeutics ( inst ) , merck ( inst ) , bristol - myers squibb ( inst ) , boehringer ingelheim , glaxosmithkline ( inst ) , novartis ( inst ) acknowledgment we thank william geddie , md , retired staff pathologist of laboratory medicine program , university health network ( toronto , on , canada ) , for his contributions to the study . research funding : astrazeneca ( inst ) , immunovaccine ( inst ) travel , accommodations , expenses : astrazeneca affiliations kyaw l . 
hyman dm , solit db , arcila me , et al : precision medicine at memorial sloan kettering cancer center : clinical next - generation sequencing enabling next - generation targeted therapy trials . 
stockley tl , oza am , berman hk , et al : molecular profiling of advanced solid tumors and patient outcomes with genotype - matched clinical trials : the princess margaret impact / compact trial . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
roy - chowdhuri s , chen h , singh rr , et al : concurrent fine needle aspirations and core needle biopsies : a comparative study of substrates for next - generation sequencing in solid organ malignancies . 
kanagal - shamanna r , portier bp , singh rr , et al : next - generation sequencing - based multigene mutation profiling of solid tumors using fine needle aspiration samples : promises and challenges for routine clinical diagnostics . 
li h , durbin r : fast and accurate short read alignment with burrows - wheeler transfor cancer - panel / bioinformatics 25 : 1754 - 1760 , 2009 17 . 
dibardino dm , rawson dw , saqi a , et al : next - generation sequencing of non - small cell lung cancer using a customized , targeted sequencing panel : emphasis on small biopsy and cytology . 
gleeson fc , kipp br , voss js , et al : endoscopic ultrasound fine - needle aspiration cytology mutation profiling using targeted next - generation sequencing : personalized care for rectal cancer . 
padmanabhan v , steinmetz hb , rizzo ej , et al : improving adequacy of small biopsy and fineneedle aspiration specimens for molecular testing by next - generation sequencing in patients with lung cancer : a quality improvement study at dartmouth - hitchcock medical center . 
sakai k , takeda h , nishijima n , et al : targeted dna and rna sequencing of fine - needle biopsy ffpe specimens in patients with unresectable hepatocellular carcinoma treated with sorafenib . 
zheng g , tsai h , tseng lh , et al : test feasibility of next - generation sequencing assays in clinical mutation detection of small biopsy and fine needle aspiration specimens . 
gleeson fc , kerr se , kipp br , et al : targeted next generation sequencing of endoscopic ultrasound acquired cytology from ampullary and pancreatic adenocarcinoma has the potential to aid patient stratification for optimal therapy selection . 
krop ie , mayer ia , ganju v , et al : pictilisib for oestrogen receptor - positive , aromatase inhibitorresistant , advanced or metastatic breast cancer ( fergi ) : a randomised , double - blind , placebocontrolled , phase 2 trial . 
baselga j , im sa , iwata h , et al : buparlisib plus fulvestrant versus placebo plus fulvestrant in postmenopausal , hormone receptor - positive , her2 - negative , advanced breast cancer ( belle - 2 ) : a randomised , double - blind , placebo - controlled , phase 3 trial . 
forty - eight genes included in the truseq amplicon cancer panel ( illumina ) , which includes 212 amplicons covering a total genomic region of 35.84 abl1 csf1r erbb4 fgfr3 gnaq jak2 kras akt1 braf cdh1 cdkn2a ctnnb1 egfr fbxw7 fgfr1 flt3 gna11 hnf1a hras jak3 mlh1 nras erbb2 fgfr2 gnas idh1 notch1 npm1 pdgfra pik3ca pten ptpn11 smad4 smarcb1 stk11 tp53 table a3 . 
responses to avelumab were observed regardless of pd - l1 expression , the presence or absence of tumor - inltrating therapy , and lymphocytes , multiple prior lines of somatic or germline origin of mmrd , which suggests that baseline clinical and pathologic characteristics could not predict response . 
ten of the 12 tumors were determined to be mmrd by oncopanel on the basis of mutational signature analysis using two independent algorithms , 4 , 5 which was consistent with the ihc determination . 
the remaining 2 tumors were determined to be mmrp by oncopanel and microsatellite stable using polymerase chain reactionthat is , both oncopanel and polymerase chain reaction were discordant with ihcand none of them responded to avelumab . 
 ( b ) frequency of common jak1 and b2m mutations found in our data set are compared with the frequency in the mismatch repairdecient ( mmrd ) endometrial cancers ( ecs ) from the cancer genome atlas ( tcga )  . 
fs , frameshift ; fsdel , frameshift deletion ; fsins , frameshift insertion ; nr , nonresponder ; or , objective response ; r , responder ; recur , recurrent . 
therefore , these 2 tumors were more likely to be mmrp and were excluded from the analysis . of the remaining 10 patients ( table 1 ) with tumors determined to be mmrd using both ihc and oncopanel , 3 exhibited an objective response to avelumab ( responders ) , whereas 7 did not ( nonresponders )  . 
all 7 nonresponders harbored either jak1 ( 6 tumors ) or b2m mutations ( 1 tumor ) , while only 1 of the 3 responders harbored a jak1 mutation ( fisher exact test , two - sided p = .067 ; fig 1a )  . 
in addition , of the 7 nonresponders , 4 harbored two mutations of jak1 ( 3 tumors ) or b2m ( 1 tumor ) , possibly reecting biallelic inactivation of these genes . 
all jak1 mutations were frameshift ( deletions or insertions ) , with the exception of two missense mutations that occurred together with frameshift mutations : a missense mutation q750r on the pseudokinase domain ( exon 16 ) and a missense mutation l1071p toward the end of the kinase domain ( exon 23 )  . 
frameshift jak1 mutations involved the hotspot position k860 / p861 ( deletions in 5 tumors and insertion in 1 tumor ) and the hotspot position p430 / l431 ( insertions in 2 tumors )  . 
 ( a ) number of indels , deletions , insertions , and tumor mutational burden , dened as the number of nonsynonymous mutations per mb for responders ( r ) and nonresponders ( nr ) in our data set . 
 ( b ) same as panel a comparing patients with endometrial cancer ( ec ) in the cancer genome atlas ( tcga ) data set with and without frameshift jak1 mutations . 
furthermore , in another data set ( msk - imp ) , which included 29 patients with recurrent mmrd ecs ( dened by ihc ) , 6 the overall frameshift jak1 mutations was 51.7% cidence of signicantly higher than that in the tcga data set , but comparable with that in our data set ( fig 1c )  . 
nonresponders had a signicantly higher number of total deletion mutations compared with responders ( p = .03 ) , but the number of total insertion mutations was not different ( fig 2a )  . 
 ( a ) exposure ( left ) and fraction ( right ) , dened as the exposure divided by the total number of single - nucleotide variants of signatures 20 . 
bottom : blue and red boxes and points indicate cases with and without frameshift ( fs ) jak1 mutations in whole - exome sequencing data from the cancer genome atlas ( tcga )  . 
to calculate the fraction , the exposure of each mmrd signature is divided by the sum of exposures of all mmrd signatures ( the contribution of the non - mmrd signatures is not shown as they did not vary signicantly across data sets )  . 
konstantinopoulos , md , phd , dana - farber cancer institute , 450 brookline ave , boston , ma 02115 ; e - mail : panagiotis_ konstantinopoulos@dfci.harvard.edu. support supported by the ludwig center at harvard and by a fund in memory of bina sareen . 
gulhan patents , royalties , other intellectual property : a provisional patent application is being drafted for an algorithm developed by the author for which a coversheet provisional has been led on september 24 , 2018 , titled computational method to identify mutational signatures from sequencing data ( inst ) joyce f . 
liu consulting or advisory role : tesaro , mersana , clovis oncology , genentech , glaxosmithkline research funding : genentech ( inst ) , astrazeneca ( inst ) , boston biomedical ( inst ) , atara biotherapeutics ( inst ) , acetylon ( inst ) , bristolmyers squibb ( inst ) , agenus ( inst ) , cytomx therapeutics ( inst ) , regeneron ( inst ) , tesaro ( inst ) , clovis oncology ( inst ) , surface oncology ( inst ) , 2x oncology ( inst ) , vigeo therapeutics ( inst ) , aravive ( inst ) , arch oncology ( inst ) travel , accommodations , expenses : astrazeneca , merck uncompensated relationships : merck , astrazeneca ursula a . 
konstantinopoulos consulting or advisory role : merck , vertex , astrazeneca , pzer , emd serono , tesaro , bayer research funding : pzer ( inst ) , eli lilly ( inst ) , tesaro ( inst ) , merck serono ( inst ) , astrazeneca ( inst ) , merck ( inst ) , bayer ( inst ) no other potential conicts of interest were reported . references aghajanian c , sill mw , darcy km , et al : phase ii trial of bevacizumab in recurrent or persistent endometrial cancer : a gynecologic oncology group study . 
j clin oncol 29 : 2259 - 2265 , 2011 alvarez ea , brady we , walker jl , et al : phase ii trial of combination bevacizumab and temsirolimus in the treatment of recurrent or persistent endometrial carcinoma : a gynecologic oncology group study . 
gynecol oncol 129 : 22 - 27 , 2013 konstantinopoulos pa , luo w , liu jf , et al : phase ii study of avelumab in patients with mismatch repair decient and mismatch repair procient recurrent / persistent endometrial cancer . 
j mol diagn 19 : 84 - 91 , 2017 gulhan dc , lee jj , melloni gem , et al : detecting the mutational signature of homologous recombination deciency in clinical samples . 
nat genet 51 : 912 - 919 , 2019 soumerai te , donoghue mta , bandlamudi c , et al : clinical utility of prospective molecular characterization in advanced endometrial cancer . 
clin cancer res 24 : 5939 - 5947 , 2018 alexandrov lb , nik - zainal s , wedge dc , et al : signatures of mutational processes in human cancer . 
gao j , shi lz , zhao h , et al : loss of ifn - gamma pathway genes in tumor cells as a mechanism of resistance to anti - ctla - 4 therapy . 
albacker la , wu j , smith p , et al : loss of function jak1 mutations occur at high frequency in cancers with microsatellite instability and are suggestive of 15 . 
howitt be , shukla sa , sholl lm , et al : association of polymerase e - mutated and microsatellite - instable endometrial cancers with neoantigen load , number of tumor - inltrating lymphocytes , and expression of pd - 1 and pd - l1 . 
aldubayan , md1 , 2 , 3 systematic tumor molecular proling of patients with cancer , using next - generation sequencing , has become an integrated part of current clinical oncology care , providing diagnostic , prognostic , therapeutic , and predictive utility and informing highly personalized molecular - based treatment decisions.1 - 3 until recently , most prospective tumor - proling programs of patients with cancer had primarily focused on exploring clinically actionable somatic alterations ( using tumor - only or paired tumor - normal sequencing ) and only used the concurrently generated or inferred germline data to achieve better variant ltration.3 , 4 however , during the past few years , there has been a growing interest in exploring the clinical utility of germline genetic variants in these clinical contexts to achieve a more comprehensive characterization of the genomic events driving tumor initiation , progression , and resistance . using whole - exome sequencing or selected gene panels , large tumor - normal sequencing efforts provided valuable insight into the prevalence of disruptive germline genomic alterations in various clinical settings . 
depending on the cancer type , the sequencing platform , and the variant calling pipeline , prior studies have shown a germline pathogenic variant prevalence between 3% in patients with primary cancer from earlier studies and up to 18% in selected high - risk advanced and metastatic cancer cohorts.5 - 7 building on these studies , and as reported in the article accompanying this editorial , dumbrava et al8 implemented a 201 - gene panel to capture the somatic and germline coding variants of 1 , 000 patients ( who had locally advanced or metastatic solid tumors and exhausted standard treatment options ) and subsequently deployed a tier - wise analysis approach to explore oncology - related clinically actionable germline alterations . 
for example , men with prostate cancer who carry pathogenic germline variants in brca2 and chek2 are , respectively , 27 times and ve times more likely to experience failure of initial hormonal therapy and progression to advanced metastatic disease.13 similarly , patients with colorectal cancer who have pathogenic germline alterations in atm tend to be more likely to have an advanced ( american joint committee on cancer stage 3 or stage 4 ) atm - decient tumors compared with patients with colorectal cancer carrying germline wild - type atm alleles.14 the predictive utility of such germline biomarkers can be clinically informative when weighing various treatment options where , selected clinical settings , a more interventional and aggressive treatment plan could be more appropriate than watchful waiting or active surveillance . systematic germline genomic analysis of patients with cancer can also pinpoint patients with certain inherited genomic defects whose tumors tend to be exquisitely sensitive to specic targeted treatment interventions , thus providing a therapeutic silver lining for these highly burdened patients . 
 editorial gene family , may also confer similar tumor sensitivity to poly ( adp - ribose ) polymerase inhibitors , potentially expanding the therapeutic utility of germline genomic characterization in patients with cancer.20 , 21 similarly , germline defects in the lynch syndrome genes ( mlh1 , msh2 , msh6 , and pms2 ) predispose to a whole array of dna mismatch repairdecient tumors that are selectively sensitive to immune checkpoint blockades ( such as monoclonal antibodies against programmed cell death protein 1 [ anti - pd - 1 ] and programmed death - ligand 1 [ anti - pd - l1 ] ) regardless of the cancers tissue of origin , underscoring the advantages of performing paired tumor - normal proling that simultaneously explores germline mismatch repair defects in conjunction with somatic hypermutation and microsatellite instability.22 identication of germline cancer - risk variants through tumor - focused molecular characterization also represents a unique opportunity to capture patients with inherited cancer predisposition syndromes , which has important ramications . 
first , almost all germline cancer predisposition syndromes confer susceptibility to multiple cancer types , some of which have gene - specic cancer surveillance recommendations aiming to improve survival through early detection and treatment of synchronous and metachronous primary tumors.23 , 24 so far , the national comprehensive cancer network has endorsed molecularspecic cancer screening and / or cancer risk reduction recommendations for patients with germline pathogenic variants in one or more of 31 different cancer predisposition genes.11 , 12 in addition , establishing the molecular diagnosis of a cancer predisposition syndrome in an individual triggers cascade testing of immediate family members whose risk of sharing the same pathogenic alteration could be as high as 50% and in whom implementing gene - specic cancer - preventive recommendations can be lifesaving . despite the clear clinical actionability of most germline cancer predisposition alterations , many patients discovered ( through tumor - focused molecular proling ) to carry such alterations do not receive formal clinical genetics evaluation to conrm the presence of germline mutation , establish the diagnosis , or receive appropriate counseling for cancer - risk management . 
as highlighted by another study accompanying this editorial , fishler et al25 retrospectively evaluated how their multidisciplinary genomic tumor board handled putative germline variants discovered from tumor - only sequencing of 34 women with advanced breast cancer . despite meeting the national comprehensive cancer network criteria for germline testing and having strong clinical and molecular features suggestive of an underlying germline cancer predisposition syndrome , approximately 40% of these patients , including some patients with suspected pathogenic germline alterations in pten and cdh1 ( where the overall lifetime cancer risk exceeds 80% ) , were not offered conrmatory germline testing or referred to clinical genetics , underscoring the importance of implementing a clear and easy - to - follow protocol to ensure full use of such results . 
ideally , all patients who are found to carry germline cancer - risk variants should be evaluated in the medical genetics or genetic counseling clinic , regardless of the clinical context . 
however , this approach may not be feasible in all clinical oncology settings , given the already overburdened genetic counseling system and the severe national and international shortage of clinical cancer geneticists and genetic counselors . 
to achieve this , however , educational programs in germline cancer genetics should be implemented through easily accessible didactic sessions ( during clinical oncology training ) , departmental seminars , hands - on workshops , online training modules , and conferencebased educational sessions . germline data generated as part of the tumor - focused genomic proling efforts provided a much - needed understanding of the diagnostic role and clinical utility of germline variant analysis in precision oncology . 
in addition , such programs provided large collections of thoroughly annotated clinical samples , which have been instrumental for exploring novel germline determinants of cancer risk , response to treatment , and disease progression.13 , 26 , 27 however , such programs face major technical , logistic , and clinical challenges that need to be addressed in a thorough and timely manner . 
technically , germline variant calling , using the matched normal sample , requires a distinct set of bioinformatics expertise and tools to minimize false - positive and false - negative results.28 furthermore , germline variant analysis should take into consideration the ancestral background of the examined patients , especially when conducting enrichment analysis to identify clinically informative molecular predictors . logistically , these programs should be multidisciplinary in nature , with a clear protocol for subsequent conrmatory germline testing and formal clinical genetics evaluation for all carriers of pathogenic germline variants . 
also , adequate pretest counseling for the possibility of identifying a germline alteration ( and its potential implications ) is critical to minimize post - test emotional stress.29 clinically , there is a substantial degree of uncertainty involving the clinical implications of some germline results . 
as reported by dumbrava et al8 and several other studies , almost all enrolled patients had germline variants of unknown clinical signicance , underscoring the importance of input from germline experts when evaluating such results . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . no potential conicts of interest were reported . references le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular proling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
j natl cancer inst 107 : djv193 , 2015 sholl lm , do k , shivdasani p , et al : institutional implementation of clinical tumor proling on an unselected cancer population . 
jci insight 1 : e87062 , 2016 zehir a , benayed r , shah rh , et al : mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10 , 000 patients . 
nat med 23 : 703 - 713 , 2017 [ erratum : nat med 23 : 1004 , 2017 ] jones s , anagnostou v , lytle k , et al : personalized genomic analyses for cancer mutation discovery and interpretation . 
sci transl med 7 : 283ra53 , 2015 schrader ka , cheng dt , joseph v , et al : germline variants in targeted tumor sequencing using matched normal dna . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
jama 318 : 825 - 835 , 2017 dumbrava ei , brusco l , daniels ms , et al : expanded analysis of secondary germline ndings from matched tumor / normal sequencing identies additional clinically signicant mutations . 
mccabe n , turner nc , lord cj , et al : deciency in the repair of dna damage by homologous recombination and sensitivity to poly ( adp - ribose ) polymerase inhibition . 
rebbeck tr , friebel t , lynch ht , et al : bilateral prophylactic mastectomy reduces breast cancer risk in brca1 and brca2 mutation carriers : the prose study group . 
fishler kp , breese eh , walters - sen l , et al : experiences of a multidisciplinary genomic tumor board interpreting risk for underlying germline variants in tumoronly sequencing results . 
aldubayan sh , pyle lc , gamulin m , et al : association of inherited pathogenic variants in checkpoint kinase 2 ( chek2 ) with susceptibility to testicular germ cell 27 . 
van der auwera ga , carneiro mo , hartl c , et al : from fastq data to high condence variant calls : the genome analysis toolkit best practices pipeline . 
next - generation sequencing ( ngs ) of the primary tumor site using the oncomine comprehensive assay ( oca ; thermofisher scientic , west sacramento , ca ) detected the following genomic events : loss of function in tp53 ( c.1024c.t ) and pcm1 - alk fusion . 
the bioinformatician on the board conrmed the presence of soft - clipped reads at the ends of exon 20 in alk , and the clipped sequence was consistent with exon 23 in pcm - 1 ( fig 1 )  . 
the mtb members next searched 2 large databases , cosmic ( catalogue of somatic mutations in cancer ) and msk - impact ( memorial sloan ketteringintegrated mutation proling of actionable cancer targets ) to determine whether the pcm1 - alk fusion had ever been previously observed , but it had not . the mtb discussed the possibility that the alk fusion was a false - positive detection . 
in the past , ampliconincluding oca , were based ngs assays of dna , considered to be unable to detect unknown fusion variants , and therefore a capture - based dna or rna approach is preferred for ngs testing of alk fusions . however , the limitations of the amplicon - based assay have since been circumvented . 
in the rst step , during the analysis of the sequenced reads , all reads that are initially unaligned to the reference sequence are split in half and allowed to realign . 
another method to detect fusions involving unknown partners is to assess the 3 ( cid : 1 ) / 5 ( cid : 1 ) imbalance value.1 this step analyzes the expression levels of the 3 ( cid : 1 ) and 5 ( cid : 1 ) ends of each driver gene . 
for genes involved in a fusion event , the 3 ( cid : 1 ) end of the gene is now under different regulatory control and shows overexpression relative to the 5 ( cid : 1 ) end of the gene . 
in contrast , chimeric pcm1 - alk fusion transcripts were conrmed by direct sanger sequencing of the product , which was formed through a reciprocal translocation that joined the 5 ( cid : 1 ) end of pcm1 with the 3 ( cid : 1 ) exons of alk ( figs 3b and 3c )  . 
as with echinoderm microtubule - associated protein - like 4 ( eml4 ) - alk fusion found in lung cancer , pcm1 has multiple coiled - coil domains that are hypothesized to promote receptor activation by dimerization . 
integrative genomics viewer screenshot showing the breakpoint of the pcm1 - alk rearrangement detected by the oncomine comprehensive assay v3 . fresh - frozen tissues because treatment of samples with formalin and parafn , along with sample storage and extraction procedures , can result in signicant fragmentation , denaturation , and chemical modications of nucleic acids.3 although oca does not have a distinguishing nucleic acid sequence barcode incorporated into each amplicon , the number of barcodes detected in chimeric reads may pcm1 forward primer halted extension following pcr cycle this product function as a primer extension occurs pcm1 formation of chimera reads pcm1 fig 2 . 
 ( c ) schematic of the translocation event that creates the fusion gene pcm1 - alk ( upper ) and the functional domain structures of the pcm1 - alk protein ( bottom )  . the blue regions signify coiled - coil domains of pcm1 , and the red region indicates the tyrosine kinase domain of alk . 
 hosono et al recommended that alk status should be assessed by using two techniques and a third one in discordant cases for maximal sensitivity and specicity.10 however , discordance of results still continues to be a challenge . 
the fact that positive results for fish , ngs , and direct sequencing of alk mrna were accompanied by negative ihc results suggests the presence of posttranscriptional dysfunction or post - translational events affecting pcm1 - alk , but the precise mechanism is still unknown . in conclusion , whereas fish and direct sequencing conrmed the presence of pcm1 - alk fusion that was newly identied by a cancer genomic proling assay , the role of this fusion in tumor growth is still unclear because of the absence of protein expression . 
in addition , on the basis of a series of reports suggesting that activating fusions are responsible for acquired resistance to anti - egfr therapy , 13 - 15 the mtb also recommended repeating the genome proling test after the patient became resistant to standard chemotherapy . 
despite this , the patient could present as a suitable candidate for alk tyrosine kinase inhibitor therapy if a japanese compassionate use program were to be established in the future . 
notably , our institute has a system to treat patients with off - label drugs at the expense of the institute after receiving approval from the institutional review board when a tumor is positive for biomarkers that can predict its response to therapies approved by the japanese government for different types of tumor . 
 evaluation of alk fusion in colon cancer references lira me , choi yl , lim sm , et al : a single - tube multiplexed assay for detecting alk , ros1 , and ret fusions in lung cancer . 
haile s , corbett rd , bilobram s , et al : sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic dna from formalin - xed parafn - embedded samples . 
nat med 20 : 1479 - 1484 , 2014 yakirevich e , resnick mb , mangray s , et al : oncogenic alk fusion in rare and aggressive subtype of colorectal adenocarcinoma as a potential therapeutic target . 
clin cancer res 22 : 3831 - 3840 , 2016 lindeman ni , cagle pt , aisner dl , et al : updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors : guideline from the college of american pathologists , the international association for the study of lung cancer , and the association for molecular pathology . 
j thorac oncol 10 : 1648 - 1652 , 2015 rogers tm , arnau gm , ryland gl , et al : multiplexed transcriptome analysis to detect alk , ros1 and ret rearrangements in lung cancer . 
sci rep 7 : 42259 , 2017 kalemkerian gp , narula n , kennedy eb , et al : molecular testing guideline for the selection of patients with lung cancer for treatment with targeted tyrosine kinase inhibitors : american society of clinical oncology endorsement of the college of american pathologists / international association for the study of lung cancer / association for molecular pathology clinical practice guideline update . 
letovanec i , finn s , zygoura p , et al : evaluation of ngs and rt - pcr methods for alk rearrangement in european nsclc patients : results from the european thoracic oncology platform lungscape project . 
marchetti a , di lorito a , pace mv , et al : alk protein analysis by ihc staining after recent regulatory changes : a comparison of two widely used approaches , revision of the literature , and a new testing algorithj thorac oncol 11 : 487 - 495 , 2016 13 . 
piotrowska z , isozaki h , lennerz jk , et al : landscape of acquired resistance to osimertinib in egfr - mutant nsclc and clinical validation of combined egfr and ret inhibition with osimertinib and blu - 667 for acquired ret fusion . 
liang w , he q , chen y , et al : metastatic eml4 - alk fusion detected by circulating dna genotyping in an egfr - mutated nsclc patient and successful management by adding alk inhibitors : a case report . 
daly c , castanaro c , zhang w , et al : fgfr3 - tacc3 fusion proteins act as naturally occurring drivers of tumor resistance by functionally substituting for egfr / erk signaling . 
clinical validation of ctdna using amplicon - based ngs analysis ( with a 36 - gene panel ) was performed against standard - of - care tissue molecular analysis in treatment - nave patients . the feasibility , utility , and prognostic value of ctdna as a dynamic marker of treatment efcacy was evaluated . results of tissue molecular prole were blinded during ctdna analysis . results of 214 patients with advanced nsclc who were recruited , 156 were treatment - nave patients and 58 were pretreated patients with unknown tissue molecular prole . 
for clinically relevant variants , concordance agreement between ctdna and tumor tissue analysis was 95% among 94 treatment - nave patients who had concurrent liquid and tumor biopsy molecular proles . sensitivity and specicity were 81% and 97% , respectively . 
 remon et al context key objective relevance to assess the feasibility and utility of circulating tumor dna ( ctdna ) by amplicon - based next - generation sequencing ( ngs ) analysis in a daily clinical setting in a cohort of patients with advanced nonsmall - cell lung cancer ( nsclc ) as an alternative approach to tissue molecular proling . knowledge generated these real - world data from a prospective study endorse ctdna molecular prole by amplicon - based ngs as an accurate and reliable tool to detect and monitor clinically relevant molecular alterations in patients with advanced nsclc . 
also , they endorse the theory that liquid biopsy may predict earlier than standard radiologic criteria the effectiveness of platinum - based chemotherapy . this large , real - world , prospective study in patients with advanced nsclc provides additional validation about ctdna for molecular proling and monitoring of alterations with high sensitivity and specicity to detect clinically relevant and actionable mutations when tissue biopsy is unavailable or uninformative . 
patients with advanced nsclc were eligible for the study if they were treatment nave for advanced disease and expected to receive rst - line platinum - based chemotherapy ( treatment - nave cohort ) or if the tissue - based molecular prole failed or was not performed on the primary tissue sample ( treated rescue cohort ; appendix fig a1 )  . 
all patients provided written informed consent for biomedical research approved by the institutional ethics committee under one of the two studies ( clinicaltrials . gov identier : nct02105168 ; or clinicaltrials.gov identier : nct02666612 ) to perform molecular analysis in tissue and plasma collections . 
in a subset of treatment - nave patients ( with or without tissue molecular proles ) who received standard platinum - based chemotherapy , additional blood specimens were collected at baseline and within 6 weeks of treatment initiation to match to the radiologic evaluation of the disease for clearance of baseline genomic alterations or emergence of new mutations after treatment . the study aimed to correlate detection of molecular abnormalities in tissue versus blood in treatment - nave patients , to evaluate the prognostic value of ctdna as a dynamic biomarker of treatment efcacy , and to assess the feasibility and utility of liquid biopsy in patients for whom tissue analysis failed . blood samples and ctdna isolation and sequencing blood for plasma preparation was collected into a single 10 - ml k2 edta tube before the start of platinum - based chemotherapy treatment or at the time of study enrollment for patients enrolled in the rescue cohort . in this study , plasma analysis was performed using invisionseq lung with an earlier 35 - gene panel8 ( n = 164 ) and a revised 36 - gene panel9 ( n = 50 , treatment - nave only ; appendix fig a2 )  . 
in this study , before analysis , we dened a core gene variant panel for clinically relevant gene mutation hotspots as follows : egfr exons 18 to 21 , braf v600 , met exon 14 , erbb2 ins 20 , and kras ; panel selections were based on recent recommendations of asco and international association for the study of lung cancer biomarker guidelines for ngs panels used in molecular proling.5 , 13 on the basis of emerging clinical interest , stk1114 also was included in the core gene panel . 
clinical validation ( concordance , sensitivity , specicity ) of invisionseq lung for snvs and indels was evaluated in patients with concurrent matched tissue analysis for these select gene variants . 
structural rearrangements ( alk , ros1 ) were not evaluated in this study , because this testing was unavailable until the end of the study ( as part of a revised plasma - based assay ) when insufcient plasma volume remained from the initial collection . all statistical analyses were performed using r version 3.2.5. in the concordance analysis , the data can be summarized as a two - by - two table , in which tissue data are the standard reference material . 
to evaluate the prognostic value of ctdna as a dynamic biomarker of treatment efcacy , all patients underwent tumor imaging , including computed tomography of the chest and abdomen and / or positron emission tomography scan at baseline . 
the obtained within 6 weeks after treatment senior radiologist ( c.c. ) centrally reviewed the response rate ( rr ) and determined the best response according to recist version 1.1.15 the objective rr was dened as the percentage of patients with response ( complete or partial ) at rst restaging after chemotherapy initiation . 
progressionfree survival ( pfs ) was calculated from the date of chemotherapy initiation until the date of progression by recist version 1.1 or death ( that occurred without recorded progression ) , and censoring was the date of last follow - up if the patient had not experienced progression . 
statistical methods for correlative and trend analyses are described in the data supplement . results from june 2015 through april 2017 , 214 patients with advanced nsclc were recruited ; 156 treatment - nave patients were included ( treatment - nave cohort ) , and patients with unknown molecular status were also enrolled ( n = 58 ; treated rescue cohort )  . 
characteristics of the patients at enrollment are provided in appendix table a1 . in the whole population , 41% were women and 17% were never smokers ; the population had predominantly stage iv disease ( 77% ) and the adenocarcinoma subtype ( 75% )  . molecular prole was unknown at the time of enrollment for 29% of treatment - nave patients and for 48% of the overall study population . tissue molecular proling among the 156 treatment - nave patients , 111 ( 71% ) had successful tissue molecular prole analyses . 
in these patients , somatic mutations were found in 78% of patients ( n = 87 ) ; the highest frequencies were in kras ( 27% ) , egfr ( 6% ) , and braf ( 5% )  . 
of the remaining 103 patients ( n = 45 , treatmentnave ; n = 58 treated rescue cohort ) without prior tissue molecular analyses , either molecular testing was not performed ( 44% ) or tissue biopsies had insufcient quality or cellularity for testing ( 51% ) or results were not reported in patient referral records . ctdna molecular proling liquid biopsy ctdna proling was successfully performed for 91% of all patients ( n = 194 ; appendix table a2 ) , from a median plasma volume of 3.7 ml for dna extraction ( range , 2.4 to 5 ml )  . 
the time to complete testing for this research study was 10 days from receipt of sample . in the treatment - nave cohort ( n = treatment - nave cohort . 156 ; appendix table a2 ) , liquid biopsy analysis was successful for 142 patients ( 91% ) and , of these , mutations were detected in 111 patients ( 78% ) ; the most frequent were tp53 ( 52% ) , kras ( 28% ) , stk11 ( 16% ) , egfr ( 9% ) , and braf ( 6% ; fig 1 )  . 
of treatment - nave patients without tissue molecular testing ( n = 45 ; appendix fig a1 ) , mutations were detected in 37 patients , of which clinically relevant mutations were detected in 16 patients ( n = 1 , erbb2 exon 20 ; n = 1 , egfr exons 18 to 21 ; n = 11 , kras with and without stk11 ; and n = 3 , stk11 )  . in the rescue cohort ( n = 58 ; treated rescue cohort . appendix table a2 ) , liquid biopsy analysis was successful in 52 patients ( 90% ) , of whom mutations were detected in 38 patients ( 73% ; fig 2 )  . 
mutations included egfr exon 20 / 21 mutation ( n = 3 patients ) , erbb2 exon 20 in - frame insertion ( n = 1 patient ) and amplication ( n = 1 patient ) , met amplication ( n = 1 patient ) ; braf p.v600e ( n = 1 patient ) , pik3ca ( n = 3 patients ) , fgfr1 amplication ( n = 3 patients ) , and idh1 ( n = 1 patient ) ; potentially actionable molecular alterations were identied in 27% of this cohort . kras was detected in an additional 10 patients ( 19% of the cohort , including one case with concurrent stk11 ) , and just the stk11 mutation was detected in one additional patient . overall , 48% of the rescue cohort had clinically relevant mutations detected with liquid biopsy , and , among those patients with mutations detected in the liquid biopsy , 10% received personalized treatment according to these results . the feasibility of the liquid biopsy in the pooled analysis for all patients with nsclc without tissue molecular prole regardless of treatment line and with a successful liquid biopsy ( n = 41 treatment - nave patients and n = 52 previously treated patients ) was 90% , and 73% of samples had a mutation detected . 
in this population , utility of liquid biopsy was 17% ( n = 16 patients with potentially actionable alterations : n = 2 treatment - nave patients and n = 14 pretreated patients ) , and an additional 25 patients ( 27% ) had clinically relevant molecular information , mainly kras mutation with or without stk11 mutation ( appendix table a3 )  . 
liquid biopsy circulating tumor dna ( ctdna ) molecular proles of the previously treated rescue cohort that had unknown tissue molecular proles from primary tissue , with successful plasma - based testing by invisionseq ( n = 52 )  . the median time between tissue biopsy and liquid biopsy collection was 34 days . 
overall , concordance for the broader panel in which concurrent tissue testing was performed was 95% ; sensitivity and specicity were 72% and 97% , respectively ( appendix table a4 )  . 
there was an increase in detection when plasma testing was used in addition to tissue testing ( appendix fig a3 )  . longitudinal analysis of liquid biopsy only : unselected chemotherapy first - line treatment within the treatment - nave cohort , 31 patients had successful dynamic collection of ctdna at baseline and at day 42 after treatment initiation ( rst radiologic evaluation )  . serial liquid biopsies among patients with positive liquid biopsy at baseline who had at least one somatic mutation to track ctdna ( n = 20 ) demonstrated that the mutation burden ratio was signicantly correlated with change in response per recist version 1.1 at clinical assessment on day 42 ( p = .008 ; fig 5 )  . 
in our population , 29% of treatment - nave patients had an unknown tissue molecular prole at the time of enrollment , similar to previous series8 ; however , in other series , the rate of failure had reached up to 46% of patients.16 by contrast , we have shown the successful feasibility of amplicon - based ngs ctdna plasma analysis from patients with lung cancer : only 9% of patients did not achieve a ctdna prole because of insufcient sequencing depth . 
true comparative analysis studies ( with both analyses centralized ) in an nsclc population have reported similar rates of concordance.16 , 18 other series in patients with nsclc have reported concordance rates that range from 60% to 90% for specic mutations ( egfr mutations ) .19 however , some recent data in the cancer population have reported signicant discordance between tissueand plasma - based ngs sequencing tests20 and between different liquid biopsy tests , 21 which highlights the importance of robust analytic and clinical validation data for the choice of tests used in clinical practice . as the numbers of targeted therapies available and approved in lung cancer increase over time , a need exists for companion diagnostics for real - time detection of therapeutically targetable genetic lesions . 
among those 103 tissue molecular proles regardless of patients without treatment line , the amplicon - based ctdna analysis was the only means for molecular prole in 90% of cases ; identied potential actionable molecular alterations in 17% of cases , which may have allowed an increased percentage of patients to get the benet of personalized treatment . although we did not perform tests for rearrangements in this study , because plasma volume collected was insufcient after the assay was available , the percentage of potential actionable alterations reported is similar to that of previous series3 and within the range of expected frequency of these mutations . 
liquid biopsy mutation correlation to response rate as calculated by change in radiologic response ( recist 1.1 ) versus change in mutation molecules , representative of tumor mutation burden , after rst - line platinum - based chemotherapy in an unselected cohort with advanced nonsmall - cell lung cancer ( n = 31 )  . 
in our cohort , 44% of patients without tissue biopsy had ctdnadetected clinically relevant mutations for personalized treatment as well as putative negative predictive markers of immunotherapy efcacy , such as stk11 mutation.14 , 26 also , ctdna testing demonstrated feasibility of use as complementary to tissue testing because detection of clinically relevant mutations increased when plasma testing was used . 
ctdna testing may have implications for monitoring treatment efcacy , clinical especially in patients with high afs or with decreased ctdna and stable radiologic disease by recist , as a potential earlier marker of response . 
all of these observations merit additional evaluation . it remains unknown what the appropriate number of genes to be screened in tumor type - specic gene panels or as universal tests irrespective of the tumor type will be . 
progression - free survival plot in unselected patients with advanced nonsmall - cell lung cancer treated with rst - line platinumbased chemotherapy ; plot compares circulating tumor dna ( ctdna ) mutations at baseline . 
plasma ctdna release is affected by many factors25 ; previous reports have identied tumor burden and metastatic sites as factors associated with ctdna release.26 these factors were not fully evaluated in this study but could explain lack of detected driver mutations in some patients . 
however , the ctdna blood analysis was centralized and , in most cases , provided expanded gene coverage for molecular proling . in conclusion , our data provide additional validation that ctdna with invisionseq lung , an amplicon - based technology , can be used for molecular proling and to monitor disease in patients with advanced nsclc with high sensitivity and specicity to detect clinically relevant and actionable mutations when tissue biopsy is unavailable or uninformative . 
n engl j med 377 : 849 - 861 , 2017 barlesi f , mazieres j , merlio j - p , et al : routine molecular proling of patients with advanced nonsmall - cell lung cancer : results of a 1 - year nationwide programme of the french cooperative thoracic intergroup ( ifct )  . 
novello s , barlesi f , califano r , et al : metastatic nonsmall - cell lung cancer : esmo clinical practice guidelines for diagnosis , treatment and follow - up . 
ann oncol 27 : v1 - v27 , 2016 kalemkerian gp , narula n , kennedy eb , et al : molecular testing guideline for the selection of patients with lung cancer for treatment with targeted tyrosine kinase inhibitors : american society of clinical oncology endorsement of the college of american pathologists / international association for the study of lung cancer / association for molecular pathology clinical practice guideline update . 
j clin oncol 36 : 911 - 919 , 2018 gutierrez me , choi k , lanman rb , et al : genomic proling of advanced nonsmall cell lung cancer in community settings : gaps and opportunities . 
clin lung cancer 18 : 651 - 659 , 2017 tredan o , corset v , wang q , et al : routine molecular screening of advanced refractory cancer patients : an analysis of the rst 2 , 490 patients of the proler study . 
cancer 121 : 631 - 639 , couraud s , vaca - paniagua f , villar s , et al : noninvasive diagnosis of actionable mutations by deep sequencing of circulating free dna in lung cancer from never - smokers : a proof - of - concept study from biocast / ifct - 1002 . 
gale d , lawson arj , howarth k , et al : development of a highly sensitive liquid biopsy platform to detect clinically relevant cancer mutations at low allele plos one 13 : e0193802 , 2018 fractions in cell - free dna . 
lindeman ni , cagle pt , aisner dl , et al : updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors : guideline from the college of american pathologists , the international association for the study of lung cancer , and the association for molecular pathology . 
thompson jc , yee ss , troxel ab , et al : detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next - generation sequencing of cell - free circulating tumor dna . 
kwapisz d : the rst liquid biopsy test approved : is it a new era of mutation testing for nonsmall - cell lung cancer ? ann transl med 5 : 46 , 2017 20 . 
guibert n , hu y , feeney n , et al : amplicon - based next - generation sequencing of plasma cell - free dna for detection of driver and resistance mutations in advanced nonsmall - cell lung cancer . 
mezquita l , jovelet l , lacroix l , et al : an amplicon - based liquid biopsy for detecting alk and ros1 fusions and resistance mutations in advanced nonsmallcell lung cancer ( nsclc ) patients . 
kim st , banks kc , lee s - h , et al : prospective feasibility study for using cell - free circulating tumor dnaguided therapy in refractory metastatic solid cancers : an interim analysis . 
skoulidis f , carter bw , zhang j , et al : association of stk11 / lkb1 mutations with primary resistance to pd - 1 / pd - l1 axis blockade in pd - l1positive nonsquamous nsclc . 
j clin oncol 36 , 2018 ( suppl ; abstr 9028 ) jordan ej , kim hr , arcila me , et al : prospective comprehensive molecular characterization of lung adenocarcinomas for efcient patient matching to approved and emerging therapies . 
 remon et al appendix blood samples and circulating tumor dna isolation and sequencing samples were considered acceptable for analysis if the dna extraction procedure could recover at least 2 , 000 input copies of the genome , as measured by droplet digital polymerase chain reaction . 
the revised 36 - gene panel required an average depth of 10 , 000 , and a minimum locus - specic read - depth requirement of 3 , 000 was applied for lung cancer stratication hotspots ( including egfr exon 19 deletions , l858r , t790m , braf v600e , and common kras activating mutations )  . 
individual variants were called using the inivata in - house analytic pipeline for somatic mutation detection . statistical methods all statistical analyses were performed using r version 3.2.5. clinical validation : concordance , sensitivity , specicity tissue positive , false negative ( fn ) ; and liquid negative and tissue negative , true negative ( tn )  . the following denitions were used : concordance = ( tp + tn ) / ( tp + tn + fp + fn )  . 
 ngs - based liquid biopsy analysis in advanced nsclc patients with advanced nsclc enrolled ( n = 214 ) liquid biopsy qc fail ( n = 6 ) treated rescue cohort : unknown tissue molecular profile ( n = 58 ) utility cohort treatment nave ( n = 156 ) liquid biopsy qc fail ( n = 4 ) treatment nave : unknown tissue molecular profile ( n = 45 ) liquid biopsy qc fail ( n = 10 ) biopsies after 100 days ( n = 7 ) patients with tissue and liquid biopsy molecular profile ( n = 101 ) evaluable for clinical validation ( n = 94 ) validation cohort fig a1 . 
flowchart of enrollment and cohorts for analysis : patients with advanced nonsmall - cell lung cancer ( nsclc ) were eligible for the study if they were treatment nave and expected to receive rst - line platinum - based chemotherapy ( treatment - nave cohort ) or were previously treated but did not have tissue - based molecular prole of a primary tissue sample available . 
invision ( inivata , research triangle park , nc , and cambridge , united kingdom ) gene panels : indicated is the coverage per gene , including hotspots , comprehensive or full coverage of coding regions ( 70% to 100% tiling coverage ) , and copy number variants ( cnvs )  . 
clinical characteristics of the patients observed for the overall population , treatment - nave cohort , and treated rescue cohort ngs - based liquid biopsy analysis in advanced nsclc no . 
ultrasound imaging identied a hypoechoic nodule in the right breast ; right axillary lymph node ne - needle aspiration biopsy performed at previous hospital on day 13 revealed invasive carcinoma with a focal micropapillary pattern ( fig 1 )  . immunostaining of estrogen receptor ( er ) and progesterone receptor ( pgr ) was evaluated by using the allred score and the allred scores were positive ( proportion score [ ps ] 1 ( , 1% ) , intensity score [ is ] 2 ) and negative ( ps 0 , is 0 ) , respectively.12 , 13 the tumor was human epidermal growth factor 2 ( her2 ) negative ( immunohistochemistry [ ihc ] 0 )  . 
given these results , she was diagnosed with stage iv breast cancer . targeted next - generation sequencing ( ngs ) analysis using the foundationone companion diagnostic panel ( foundation medicine , cambridge , ma ) was performed on the right axillary lymph node specimen . 
the result of ngs was reported on day 58 , and the ngs identied a ccdc6 - ret fusion ( c1 ; r12 ) with no other reported genomic alterations known to contribute to human breast tumorigenesis , including none in brca1 or brca2 . 
consistent with local standard - of - care guidelines , she received treatment with tamoxifen plus goserelin from day 14 to day 91 , but these were discontinued due to progression in the right breast and new lesions detected in the left lower lung . 
rebiopsy of the right breast tumor revealed the following results : er allred score 2 ( ps 1 [ , 1% ] , is 1 ) , pgr allred score 2 ( ps 1 , is 1 ) , her2 ihc 2 + , her2 uorescence in situ hybridization negative ( her2 / her2 / cep ( centromere ) 17 = 0.9 ) , and programmed death ligand 1 ( sp142 ) expression on tumor - inltrating immune cells of 1% - 4% . 
on day 126 , she was started on treatment with selpercatinib at the recommended phase 2 dose of 160 mg twice daily in the libretto - 001 study after providing written informed consent from the patient to publish information and images . 
 case report sorafenib and vandetanib , multikinase inhibitors with preclinical inhibitory activity against ret , have been used to treat unselected patients with breast cancer , but minimal clinical activity was observed.14 , 15 a patient with ncoa4 - retpositive breast cancer experienced a partial response to the multikinase inhibitor cabozantinib in combination with trastuzumab and exemestane although the cabozantinib dose was reduced for toxicity , the total time on treatment was short , and the relative contribution of each agent to the overall antitumor activity was not known.8 in addition , although cabozantinib has preclinical inhibitory activity against ret , its much stronger inhibition of other kinases ( eg , vegfr2 ) likely accounts for its clinical activity.16 , 17 in contrast , in the current case , the highly selective and potent ret inhibitor selpercatinib demonstrated a durable singleagent response in a patient with ret fusionpositive breast cancer . to our knowledge , this is the rst report of a breast cancer patient with a complete and sustained response to selective , ret - targeted therapy and adds to the diversity of ret fusionpositive tumor types that may benet from selective ret inhibition . 
rothenberg , kazuhiko nakagawa financial support : elizabeth olek administrative support : elizabeth olek provision of study materials or patients : elizabeth olek collection and assembly of data : satomi watanabe , tomoyuki otani , takeshi yoshida , kazuko sakai , elizabeth olek , s . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . masayuki takeda honoraria : astrazeneca kk , chugai pharma , bristol - myers squibb , novartis , ono pharmaceutical kazuko sakai honoraria : roche diagnostics , bio - rad , astrazeneca , chugai pharma elizabeth olek employment : loxo oncology inc stock and other ownership interests : loxo oncology , inc travel , accommodations , expenses : loxo oncology , inc s . 
michael rothenberg employment : loxo , pzer stock and other ownership interests : loxo , pzer jennifer kherani employment : loxo oncology at lilly , ajax health , aisling capital , summus global leadership : ajax health and aisling capital stock and other ownership interests : ajax health and aisling capital consulting or advisory role : ajax health and aisling capital travel , accommodations , expenses : ajax health and aisling capital , loxo pearl p . 
nature 511 : 543 - 550 , 2014 yoshihara k , wang q , torres - garcia w , et al : the landscape and therapeutic relevance of cancer - associated transcript fusions . 
nat med 18 : 378 - 381 , 2012 lipson d , capelletti m , yelensky r , et al : identication of new alk and ret gene fusions from colorectal and lung cancer biopsies . 
nat commun 9 : 4821 , 2018 drilon a , oxnard gr , tan dsw , et al : efcacy of selpercatinib in ret fusion - positive non - small - cell lung cancer . 
allison kh , hammond meh , dowsett m , et al : estrogen and progesterone receptor testing in breast cancer : american society of clinical oncology / college of american pathologists guideline update . 
miller kd , trigo jm , wheeler c , et al : a multicenter phase ii trial of zd6474 , a vascular endothelial growth factor receptor - 2 and epidermal growth factor receptor tyrosine kinase inhibitor , in patients with previously treated metastatic breast cancer . 
drilon a , rekhtman n , arcila m , et al : cabozantinib in patients with advanced ret - rearranged non - small - cell lung cancer : an open - label , single - centre , phase 17 . 
schlumberger m , elisei r , m uller s , et al : overall survival analysis of exam , a phase iii trial of cabozantinib in patients with radiographically progressive 2 , single - arm trial . 
 r precision oncology and the universal health coverage system in japan hiromichi ebi , md , phd1 , 2 , 3 and hideaki bando , md , phd4 although precision oncology is transforming clinical management of patients with cancer , many hospitals face challenges to effectively implement precision oncology . 
the most promising development is that tests to prole the genomes of select cancers are now fully covered by the national health insurance systein may 2019 , two gene panels were approved with reimbursement : foundationone cdx cancer genomic prole and oncoguide ncc oncopanel system , the latter of which was developed in japan . 
to make better use of scarce resources , the reimbursement is restricted to patients with solid tumors that have progressed on standard chemotherapy , rare tumors , or tumors of unknown primary . 
in addition , japans national cancer center launched a center for cancer genomics and advanced therapeutics ( c - cat ) that collects genomic information and clinical characteristics of patients who received genomic proling tests . 
the centralized system under the national health insurance system could be a double - edged sword . although tight regulation may make it hard to keep up with the rapid development of precision oncology , a federated ecosystem for sharing clinical and genomic data will be a precious asset and allow for shared access to data . 
high - cost medical expense benet also denes maximum out - of - pocket payment month according to household income of the patient . for example , the maximum is roughly $800 for a household income of approximately $34 , 000$70 , 000 . 
although for - prot insurance companies have voluntary health insurance programs , holding this type of insurance does not exempt an individual from mandatory enrollment in the universal health coverage scheme . 
the role of voluntary health insurance is supplemental and complements social health insurance benet packages . prices for all drugs and devices reimbursed by the universal health coverage system are the ofcial price set by the government ( japanese ministry of health , labor , and welfare [ mhlw ] )  . 
if a physician uses an unapproved device or off - label drug , any costs related to the patient , including but not limited to blood tests and physician fees , are not reimbursed . 
therefore , any cancer genomic proling tests must be examined and approved by the pharmaceuticals and medical devices agency ( pmda ) and mhlw before insurance reimbursement is set by the government , unless all the cost is to be paid by the patients . 
 ebi et al context key objective to summarize the framework of precision oncology recently arranged in japan . knowledge generated the japanese government designated core , hub , and liaison hospitals for cancer genomic medicine . 
also , reimbursement applies only to patients who nished standard chemotherapy , as a way to restrict potentially unnecessary investigations . relevance each country must design a precision oncology initiative under their distinct social care systejapan is an example of a centralized precision oncology under a universal health care coverage system . for clinical trials , even when gene proling testing identies actionable alterations . the japanese mhlw has a structured system to promote the development of drugs and devices under governmental regulations . 
although system a is designed for intervention with approved drugs / devices or minimally invasive intervention with unapproved drugs / devices , system b is designed for unapproved drugs or medical technologies . 
the patientrequested therapy system is also expected to be used as the japanese version of what the united states has hailed as the compassionate use prograto start off - label drug treatment using patient - requested therapy systems , it is necessary to pass several review processes equivalent to clinical trials , and the cost of the unapproved medical care should be fully paid by the patients . 
second , similar to the breakthrough therapy designation by the us food and drug administration , mhlw has the sakigake program , an accelerated inspection scheme for rapid authorization of unapproved drugs and devices . 
beginning around 2010 , pan - cancer genome screening started in japan by using researchuse only next - generation sequencing ( ngs ) panels.2 to promote genome - based clinical trials , the nationwide genome screening consortium for lung cancer was launched in february 2013 . 
the group , lc - scrum - japan , originally aimed to identify patients harboring ros1 and ret fusions originally discovered in japan.3 in february 2014 , gi - screen - japan multicenter screening project also started for gi cancer . 
the project began to screen for braf , nras , and pik3ca mutations in patients with metastatic colorectal cancer , using multiplex polymerase chain reaction ( pcr ) and luminex ( xmap ) technology.4 in february 2015 , these two groups merged into scrumjapan and started to use oncomine comprehensive assays as a screening platform.5 with the advent of plasma - based genome screening , scrum - japan subsequently started plasma - based ngs using guardant 360 . 
the screening and associated clinical trials have been research based , funded by government agencies through grant mechanisms ( the japan agency for medical research and development or the national cancer center research and development fund ) as well as by pharmaceutical companies . 
therefore , patients were not required to pay screening costs for any of these studies . from february 2013 to december 2018 , 6 , 860 and 6 , 391 patients were enrolled in lc - scrum - japan and gi - screenjapan , respectively . 
on the basis of this screening platform , 28 umbrella and 20 basket - type genome - based studies have been conducted.6 one of the notable accomplishments was a clinical trial of vandetanib , for patients with ret - rearranged nonsmall - cell lung cancer ( nsclc ) identied by the screening . 
 precision oncology in japan results of clinical trials conducted by lc - scrum japan led to the approval of crizotinib for the treatment of ros1translocated nsclc and the combination of trametinib and dabrafenib for use in patients with braf v600e - mutant nsclc . 
scrum - japan also contributed to the approval of in vitro diagnostic testing by pmda including simultaneous detection of ras and braf mutations in colorectal cancer , 8 oncomine dx target test cdx ngs panel for egfr and braf mutations and ros1 and alk translocations in nsclc , pcr - based microsatellite instability testing for solid tumors , 9 and plasma - based ras mutation testing for colorectal cancer.10 another notable genome screening is the top - gear ( trial of oncopanel for gene proling to estimate both adverse events and response ) project , which is a prospective cohort study conducted by japans national cancer center ( ncc ) to investigate the feasibility and utility of an ngsbased panel customized for japanese patients with cancer ( ncc oncopanel ) .11 this work was carried out within the context of the sakigake program and clinical utility was investigated within the advanced medical care b system . during the second stage of the top - gear project , 187 patients with advanced solid tumors obtained the gene proling data , and 25 ( 13.3% ) of them have received molecular - targeted therapy on the basis of their genome alterations.12 the achievements of this project led to governmental approval for the oncoguide ncc oncopanel system . some other medical university hospitals are also developing their own genome screening systems . 
these tests are performed under the advanced medical care b systekyoto university hospital ( oncoprime ) 13 , 14 and keio university hospital ( plessision - rapid ) offer their screening service outside of the national health care systerecently , keio university hospital started a whole - exon sequencing service ( plessision - exome )  . 
importantly , the report also concluded that cancer genome medicine should be accomplished under a universal health coverage systeafter the report , the japanese government designated 11 hospitals throughout japan to serve as core hospitals for cancer genomic medicine . 
the eligibility to be designed as a hub hospital is based on their ability to organize their resources and infrastructure akin to the core hospitals , which have their own molecular tumor board ( mtb ) and organic capabilities to run clinical trials . in addition , core hospitals have more responsibilities to train fellows and clinical coordinators and to be involved in translational research . 
liaison hospitals , on the other hand , are dependent on core and hub hospitals for their sequencing , reports , and mtb ( fig 1 )  . from the academia side , three major japanese cancerrelated societies ( the japanese society of medical oncology [ jsmo ] , the japanese society of clinical oncology [ jsco ] , and the japanese cancer association [ jca ] ) issued consensus clinical practice guidelines for ngs - based cancer tests in october 2017.18 the guideline dened the evidence level of each genomic alteration suitable for the japanese medical care system in harmony with classications of evidence levels set by regulators in the united states and european union ( eu ) .19 in june 2018 , japans national cancer center founded the center for cancer genomics and advanced therapeutics ( c - cat ) to coordinate an integrated network of core , hub , and liaison hospitals . 
c - cat is expected to aggregate and deploy cancer genomic medicine information to advance the quality of health care offered under the universal health coverage system and to devise new modalities of health care ( fig 2 )  . 
first , the center has been constructing a cancer knowledge database ( ckdb ) optimized for japan to assist in decision making by mtbs . c - cat will collect and share updated information on clinical trials , promoting and improving matching between patients genomic data and clinical trials . 
oncoguide ncc oncopanel system is a 114 - gene ngs panel for tumor and germline analysis developed by japans ncc and health instrument maker sysmex corp , and foundationone cdx cancer genomic prole ( foundation medicine ) sequences 324 genes and also can detect microsatellite instability . oncoguide ncc oncopanel system was approved as a device combining the oncoguide ncc oncopanel kit and the oncoguide ncc oncopanel analyzing systein contrast , although foundation medicine had to be reviewed 11 core hospitals 34 hub hospitals 122 liaison hospitals their quality for sample analysis by pmda for the approval , pmda could not perform assessment as an in vitro diagnostic because the analysis was done in the united states . 
first , physicians submit tumor samples for foundationone cdx analysis to foundation medicine , and foundation medicine performs sequencing and reports the variant calls in an xml le , uploaded to a portal site created by chugai , a subsidiary of roche . 
because this step is not subject to reimbursement , and to set up a procedure suitable for reimbursement , physicians need to download the xml le from the portal site and then send it back to foundation medicine . 
in addition , the japanese act on the protection information denes genomic sequence as of personal personal information that was amended in response to the general data protection regulation enacted by the eu . 
on comply with the law , written consent consent , samples are sent to third parties outside of japan to analyze personal information and sequence data . after approval by pmda , the mhlw granted the use of these two gene panel analyses and set the ofcial price of reimbursement at the end of may 2019 . 
the second reimbursement is 480 , 000 , applied after the physician explains the results to patients following train personnel for precision medicine play central role in running clinical trials based on molecular profiling translational research run molecular tumor board run clinical trials based on molecular profiling provide patients with genomic testing interpretation is supported by core and hub hospitals refer patients to core and hub hospitals for clinical trial fig 1 . 
the test is approved for patients with solid tumors that have progressed on standard chemotherapy , with rare tumors , or with cancers of unknown primary . foundationone cdx is also approved for the use of companion diagnostic tests , such as those for the detection of egfr , ras , and braf mutations during standard care . for these companion dihowever , agnostic tests is much cheaper . 
furthermore , the results obtained outside of companion diagnostics will not be taken the reimbursement into consideration for decision making about treatment even when the test analyzes a number of genes . 
if a physician uses a test result during standard care , the difference for companion between the amount of reimbursement diagnostics and the actual cost paid to foundation medicine will be a decit for the hospital . 
if the physician wants to use the test results when the patient experiences progression while receiving standard therapy , additional reimbursement can be submitted as a fee for mtb ( 480 , 000 )  . 
 precision oncology in japan patient dies during standard care or fails to follow - up , it is unlikely that genome proling would be used during standard therapy in japan under current reimbursement rules . although only a few hospitals run these genome panels inhouse , most hospitals outsource these genomic proling tests to a clinical testing company . 
laboratories in core and hub hospitals are required to be certied by independent organizations such as iso15189 to handle patient samples . clinical testing companies also have clinical laboratory improvement amendments certication . 
formalin - xed and parafn - embedded samples ( and also blood samples in case of oncoguide ncc oncopanel system ) are prepared in each hospital and sent to the company . 
turnaround time in general is 16 to 22 days . to fulll its function as a data repository and to facilitate access to clinical data , a requirement of test granted by the government is that physicians need to submit detailed clinical data from patients with cancer to c - cat , including diagnostics , treatment , and outcomes information , as well as the raw bam or fastq and vcf or xml les ( fig 2 ; in japan , each hospital has an appendix table a1 )  . electronic platform that contains individual health records for patients . 
test results are usually reported in pdf les that are incorporated into electronic medical records , making it difcult for physicians to match patients with ongoing clinical trials or to identify patients who are eligible for new drugs when they become available . thus , a central data repository at c - cat will make it easier to identify candidates for clinical trials in a timely manner . using the submitted data , c - cat also issues their own report . 
ckdb1 is further divided into four databases : marker database listing genomic abnormalities such as egfr mutation or brca1 germline mutation ; drug database listing approved drugs or drugs under clinical trials in and outside of japan and their targets ; evidence database curated from biologic , clinical , and therapeutic information in multiple public information resources , including civic ( clinical interpretation of variants in cancer ) , brca exchange , clinvar , and cosmic ( catalogue of somatic mutations in cancer ) ; and a clinical trial database generated from clinicaltrials.gov as well as several japanese clinical trial registries . 
for instance , a patient with a druggable mutation needs to be treated by the appropriate drug , even as an off - label indication in the absence of a clinical trial or current approval . 
patient - requested therapy system , a japanese compassionate use program , has to be initiated by the request from a patient to the government with required documentation from physician . because the preparation of documents is a heavy burden for physicians , the system has not been widely adopted . also , as mentioned above , the cost of the drug has to be fully paid by the patient . 
to enable large basket / umbrella trials , a core facility supporting the protocol creation , drug distribution , monitoring , and audit will be needed , similar to the cancer therapy evaluation program in the united states . the other big challenge facing japanese precision oncology is the timing of reimbursements for genome proling tests . 
in addition , it was difcult to grant the use of cancer genomic proling during standard care because clear evidence for the benet of testing did not exist when consensus clinical practice guidelines for ngs - based cancer testing were published . 
for example , french clinical trials suggested the use of molecularly targeted agents outside established indications do not improve progression - free survival for heavily pretreated patients with cancer when compared with oncologists preferred treatment regimen.20 however , patients who experienced progression while receiving standard chemotherapy tend to have poor performance status and may not have enough time to wait for the results of genomic testing ; even in the very best case scenarios , their tumors will continue to grow and their health worsen each day that they are required to wait . 
currently , uncovered genome screening services are the only option for patients without rare tumors and before disease progression with standard therapy . quality and sustainability of mtbs are also challenging . 
however , a problem that arises is that it is challenging to demand this level of participation of physicians , given their limited availibility.21 overall , the cost for precision oncology is a heavy burden for hospitals . 
to put this into global perspective , memorial sloan kettering cancer center has already achieved clinical sequencing of 10 , 000 patients in the united states.22 furthermore , the united kingdom nished sequencing for  . 
in september 2019 , the mhlv unveiled a project with the goal of sequencing whole genomes from 100 , 000 patients with cancer over 3 years , a number chosen by referring to the united kingdoms sequencing achievements . 
although details of the project have not emerged yet , it is expected to result in the analyses of fresh frozen samples in collaboration with biobanks at core and hub hospitals . 
as the primary aim is to accelerate the development of new diagnostics and treatments , the project should be research based and funded by the japanese government and private sources . 
although the primary purpose of this initiative in precision oncology is to harness articial telligence ( ai ) for improved genomic analyses and drug target identication , the use of ai could connect genomic data with other prioritized areas in this initiative , such as pathology and radiology . 
the relatively homogenous genetic background of the japanese population and the detailed clinical outcomes collected by c - cat will be an advantage when harnessing the power of genomic data to develop new therapies . 
also , the genomic and clinical data would be integrated with other japanese databases , such as the medical genomics japan variant database and the japanese multi omics reference panel.23 conclusion in conclusion , precision oncology covered by the health insurance system has just begun in japan . 
on the other hand , although the japanese health care system has so far achieved excellent health outcomes with a relatively low cost , 24 the centralized structure under the national health insurance system with its inherent tight regulation may cause difculty in keeping up with the rapid development of precision oncology . 
in the united kingdom , genomics england was founded as a subsidiary limited company , as it was the most effective way to ensure the project running as quickly as possible . 
in the united states , for instance , protocols and treatment decisions surrounding personalized medicine are largely decided by the institutions treating the patients , with governing bodies like the national cancer institute playing ancillary roles and the ability to pay for patient care largely dictated by individuals private insurance policies . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . hiromichi ebi honoraria : taiho pharmaceutical , astrazeneca consulting or advisory role : merck serono , eli lilly , astellas pharma hideaki bando honoraria : taiho pharmaceutical , lilly japan , takeda , chugai pharma , sano , yakult honsha research funding : astrazeneca , sysmex no other potential conicts of interest were reported . acknowledgment we thank anthony faber , md ( vcu massey cancer center ) , for critical reading of the manuscript . references 48 : 559 - 564 , 2018 sakamoto h , rahman m , nomura s , et al : japan health system review . 
nat med 18 : 375 - 377 , 2012 bando h , yoshino t , shinozaki e , et al : simultaneous identication of 36 mutations in kras codons 61 and 146 , braf , nras , and pik3ca in a single reaction by multiplex assay kit . 
hovelson dh , mcdaniel as , cani ak , et al : development and validation of a scalable next - generation sequencing system for assessing relevant somatic variants in solid tumors . 
yoh k , seto t , satouchi m , et al : vandetanib in patients with previously treated ret - rearranged advanced non - small - cell lung cancer ( luret ) : an open - label , multicentre phase 2 trial . 
lancet respir med 5 : 42 - 50 , 2017 taniguchi h , okamoto w , muro k , et al : clinical validation of newly developed multiplex kit using luminex xmap technology for detecting simultaneous ras and braf mutations in colorectal cancer : results of the rasket - b study . 
neoplasia 20 : 1219 - 1226 , 2018 bando h , okamoto w , fukui t , et al : utility of the quasi - monomorphic variation range in unresectable metastatic colorectal cancer patients . 
bando h , kagawa y , kato t , et al : a multicentre , prospective study of plasma circulating tumour dna test for detecting ras mutation in patients with metastatic colorectal cancer . 
sunami k , ichikawa h , kubo t , et al : feasibility and utility of a panel testing for 114 cancer - associated genes in a clinical setting : a hospital - based study . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular proling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
nucleic acids res 46 : d551 - d557 , 2018 ikegami n , yoo bk , hashimoto h , et al : japanese universal health coverage : evolution , achievements , and challenges . 
 o year 1 in the molecular era of pediatric brain tumor diagnosis : application of universal clinical targeted sequencing in an unselected cohort of children purpose next - generation sequencing is gaining acceptance as a clinical tool to aid diagnosis and guide treatment of pediatric cancer . 
here , we report an unselected prospective cohort study to evaluate the clinical use of universal targeted sequencing in pediatric patients with brain tumors . methods we applied a universal sequencing protocol for all tumors of the cns undergoing diagnostic workup at seattle childrens hospital during the study period of november 2015 to november 2016 . 
all tumors were sequenced using the uw - oncoplex platform , which is a multiplexed targeted deep gene sequencing panel that detects genetic alterations in 262 cancer - related genes performed in a college of american pathologists accredited clinical laboratory improvements amendmentscertified laboratory . results eighty - eight patients underwent diagnostic evaluation during the study period , of which 85 tumors ( 95% ) yielded sufficient dna for sequencing , including 59 newly diagnosed and 26 relapsed . 
of these , 57 ( 67% ) had disease - defining or disease - modifying mutations , 44 ( 52% ) had potentially targetable mutations , and 31 ( 36% ) had mutations requiring germline follow - up . 
2018 by american society of clinical oncology introduction high - throughput genomic and epigenomic technologies have recently been used to thoroughly describe the genomic landscape of pediatric brain tumors.1 - 3 this work has transformed our understanding of the molecular basis of these diseases , 4 which are the leading cause of pediatric cancer death.5 the current challenge for the field of pediatric neuro - oncology is to translate this knowledge into clinical practice . the 2016 revision of the who classification of cns tumors has begun to incorporate molecular findings into tumor diagnosis in addition to traditional histopathology.6 however , there is currently no consensus regarding best - practice diagnostics for routine clinical practice . 
recently , a few institutions have reported the use of clinically validated next generation sequencing ( ngs ) panels to identify molecular alterations in selected subsets of pediatric patients with brain tumors.7 - 10 these studies demonstrated that molecular profiling is bonnie l . 
atrt , atypical teratoid rhabdoid tumor ; dnet , dysembryoplastic neuroepithelial tumor ; etmr , embryonal tumor with multilayer rosettes ; gg , ganglioglioma ; nos , not otherwise specified ; pxa , pleomorphic xanthoastrocytoma . feasible and that clinically relevant genetic alterations may be identified . methods instability the uw - oncoplex version 5 test is a multiplex gene sequencing panel used to detect alterations in 262 cancer - related genes , including single nucleotide variants , small insertions or deletions , amplifications , selected translocations , and microsatellite ( washington.edu / tests / genetics / uw - oncoplex )  . 
in a retrospective pilot study using this test , sequencing identified a large proportion of targetable mutations in high - grade supratentorial tumors.11 we hypothesized that the use of a selected or convenience cohort in our retrospective pilot as well as all prior studies may have identified a higher proportion of actionable findings that would not necessarily be applicable to the wider population of all patients undergoing pathologic diagnosis of pediatric brain tumor.7 - 12 we designed this follow - up , prospective , unselected cohort study to evaluate the feasibility and impact of the universal application of molecular tumor testing on routine pediatric brain tumor diagnosis and to also assess how molecular results may alter medical management . all tumors of the cns undergoing diagnostic evaluation at seattle childrens hospital from november 2015 to november 2016 were included in this study . 
details of the laboratory workflow and methods are included in the data supplement . results demographics and sample characteristics we identified 88 patients who met the clinical and diagnostic inclusion criteria ( fig 1 )  . 
 of these , 97% ( 85 of 88 ) had sufficient dna obtained for sequencing , and only three patients did not have adequate tissue in formalin - fixed paraffin - embedded ( ffpe ) blocks to perform ngs . 
of patients ( % ) * targeted agent braf inhibitor mek inhibitor fgfr inhibitor braf v600e , brafpa598t599insv ganglioglioma , pleomorphic xanthoastrocytoma , pilocytic astrocytoma , desmoplastic infantile ganglioglioma / astrocytoma kiaa1549 - braf fusion , nf1 mutations and deletions , ptpn11 mutation pilocytic astrocytoma , anaplastic astrocytoma , pleomorphic xanthoastocytoma fgfr1 mutations and duplication , fgfr3 fusions pilocytic astrocytoma , dysembryoplastic neuroepithelial tumor , diffuse astrocytoma , ganglioglioma mtor pathway inhibitor mtor , fbxw7 diffuse midline glioma , medulloblastoma smo inhibitor ptch1 mutations medulloblastoma pd1 / pdl1 inhibitor biallelic msh2 loss , pms2 mutation with loh anaplastic astrocytoma , medulloblastoma pdgfra inhibitor pdgfra amplification diffuse midline glioma met inhibitor kit inhibitor met amplification kit amplification diffuse midline glioma diffuse midline glioma cdk4 / 6 inhibitor cdk4 amplification glioblastoma ezh2 inhibitor loss of smarcb1 atypical teratoid rhabdoid tumor * ten patients had more than one targetable mutation in their tumors . 10 ( 12 ) 18 ( 21 ) 10 ( 12 ) 3 ( 4 ) 3 ( 4 ) 2 ( 2 ) 2 ( 2 ) 2 ( 2 ) 2 ( 2 ) 1 ( 1 ) 1 ( 1 ) were sequenced , including 59 newly diagnosed tumors and 15 tumors from patients with a radiologic relapse during the time of this study . 
 eleven additional patients had sequencing of newly resected recurrent tumors . patients were a median age of 10 years ( range , 3 months to 22 years ) and 49% female ( 42 of 85 )  . 
medical history included only three patients with previously known cancer predisposition syndrome ; two patients had neurofibromatosis type 1 and one had neurofibromatosis type 2 . genetic results the average depth of sequencing coverage was 441 ( range , 102 to 1 , 112 )  . 
five reports with turnaround times of > 60 days were from patients with minimal tissue that required cutting additional ffpe sections with subsequent repeat dna extraction . genetic result details are shown in data supplement 1 , and criteria for tiers of alterations are listed in appendix table a1 . 
genetic alterations were found to be disease defining or modifying in 67% ( n = 57 ) , targetable with an available therapeutic drug in 52% ( n = 44 ; table 1 ) , and potentially germline associated with cancer predisposition in 36% ( n = 31 )  . 
as of the last follow - up , seven patients ( 8% of total , 16% of those with an identified molecular target ) had been prescribed targeted agents ; molecularly targeted agents were obtained through insurance - approved off - label use in five patients and through enrollment in a clinical trial in two patients . analysis of tumor subtypes tumors were analyzed separately as high - grade glioma ( hgg ) , low - grade glioma ( lgg ) , medulloblastoma , and other rare pediatric cns tumors . 
 medulloblastoma ngs results were correlated with traditional histology , immunohistochemistry , and fluorescence in situ hybridization studies.6 , 13 all 20 medulloblastomas were able to be classified into subgroups as defined by the who , 6 , 14 including a wingless ( wnt ; n = 1 ) , sonic hedgehog ( shh ; n = 7 ) , or non - wnt / non - shh group ( n = 12 ; fig 3 )  . 
for example , one patient with a wnt pathway medulloblastoma had only weak nuclear beta catenin staining ; in this patient , the finding of a ctnnb1 mutation and copy loss consistent with monosomy 6 by ngs helped place this tumor firmly in the wnt - activated subgroup . 
in another instance , an shh group medulloblastoma that was gab1 immunopositive displayed ambiguous p53 immunohistochemical staining of approximately 40% of tumor cell nuclei ; ngs not only confirmed the shh subgroup by finding sufu mutations , but also confirmed that this tumor contained a tp53 mutation . 
as of the last follow - up , two patients in the relapsed shh group with ptch1 mutations had been prescribed smo inhibitors , one of whom experienced a partial response to treatment . forty - four lggs were analyzed ( fig 4 )  . 
 a , high - grade astrocytoma ; aa , anaplastic astrocytoma ; apa , anaplastic pilocytic astrocytoma ; dipg , diffuse intrinsic pontine glioma ; gbm , glioblastoma ; hgg , high - grade glioma , not otherwise specified . pik3ca pten smarcb1 msh2 idh1 pdgfra h3f3a atrx cdk6 cdkn2a tp53 ptpn11 v600 mutations and 10 braf - kiaa translocations . 
 twenty - nine patients with lgg had targetable mutations , and at the last follow - up , three patients had been prescribed targeted therapy with a braf inhibitor for braf v600e mutation , two at the time of recurrence and one as initial therapy for unresectable brainstem tumor . 
 three patients with lgg were confirmed to have germline mutations , including the two known patients with nf1 and one patient with a recurrent pleomorphic xanthoastrocytoma who was confirmed to have a germline tp53 mutation in addition to a braf v600e mutation in the tumor ; the patient was treated with a braf inhibitor instead of radiation for recurrent tumor and continues without disease progression with > 12 months of receiving targeted therapy . 
molecular profiling did not alter treatment of the 17 patients in the lgg group with completely resected primary tumors . eleven other uncommon tumors were evaluated ( fig 5 ) , including six nonmedulloblastoma embryonal tumors , choroid plexus carcinoma , choroid plexus papilloma , vestibular schwannoma , rhabdoid meningioma , and chordoma . 
one patient had a known nf2 mutation , and the other two patients tested negative for smarcb1 ( atypical teratoid rhabdoid tumor ) and tp53 ( choroid plexus carcinoma )  . analysis of primary versus recurrent tumors considering newly diagnosed patients , 85% ( n = 50 of 59 ) had clinically relevant mutations , including disease - defining or disease - modifying mutations in 76% of tumors ( n = 45 ) , targetable mutations in 46% of tumors ( n = 27 ) , and potentially germline mutations in 41% of tumors ( n = 24 )  . of the 15 primary tumors sequenced at the time of radiologic relapse , 10 ( 67% ) were found to have clinically significant molecular alterations considered disease defining , and nine ( 60% ) identified molecular targets for therapy . 
 of 11 tumors sequenced using newly resected tumor obtained at the time of recurrence , clinically significant alterations were identified in eight ( 73% )  . discussion germline sequencing germline dna was not collected at the time of somatic sequencing in this study . 
however , somatic sequencing identified mutations considered to be potentially germline in 31 of 85 of tumor samples ( 36% ) on the basis of variant interpretation , which led to initiation of clinical follow - up and genetic counseling to recommend germline evaluation . 
in this population , germline sequencing has been performed on 13 patients , and germline mutations were confirmed in nine patients ( 75% of those tested ; 10.6% of the study population ) as of the last follow - up . 
 one patient had ctnnb1 mutation with monosomy of chromosome six diagnostic of wingless ( wnt ) subgroup , seven had either ptch1 or sufu mutations characteristic of sonic hedgehog ( shh ) subgroup , and the other 12 were non - shh / nonwnt group ; three of our seven shh group tumors were tp53 mutant . 
in the group of newly diagnosed patients , alterations were identified in 85% of patients ( n = 50 of 59 ) , a finding that was contrary to our hypothesis that selected cohorts previously reported by our group and others may have been limited by a selection bias increasing the likelihood of clinically relevant findings.7 , 8 , 11 , 12 targetable mutations were identified in 44 tumors ( 52% ) , and seven of these patients ( 8% ) have been prescribed targeted therapeutic agents to date . 
this number likely underestimates the longer - term use of targeted therapy because the study has a relatively short follow - up period and there are effective standard treatment regimens of cytotoxic therapy for most diagnoses . 
if successful , the routine use of targeted agents will greatly increase in patients with pediatric brain tumors . the main aims of cancer genomic profiling tests are to identify somatic mutations . 
it is crucial to include genetic counseling in oncology care , ideally with a geneticist or genetic counselor embedded into the pediatric oncology clinic.15 in this study , we identified 31 patients ( 36% ) with potential germline mutations . 
 be difficult to overstate in a patient for whom radiation therapy is being considered , because the long - term second malignancy rate in these patients who undergo therapeutic irradiation approaches 100%.16 molecular profiling is important for the diagnosis and management of pediatric patients with brain tumors , 6 although at present , there are several competing platforms and currently no diagnostic gold standard . 
whatever platform is used must be clinically validated and should balance cost , turnaround time , and ability to identify clinically relevant mutations in small samples , which is no small task . 
 alternatively , many research studies have argued the advantages of potentially subclassifying pediatric brain tumors primarily by rna - based gene expression17 or methylation analyses.18 however , these types of platforms are difficult to establish fig 4 . 
the majority of low - grade gliomas in our series had dysregulated braf signaling , which included 10 ( 26% ) with braf fusions and 10 ( 26% ) with codon 600 mutations ; these alterations were considered both targetable and either disease defining or modifying . 
ag , angiocentric glioma ; da , diffuse astrocytoma ; dig / dia , desmoplastic infantile ganglioglioma or desmoplastic infantile astrocytoma ; dnet , dysembryoplastic neuroepithelial tumor ; ep , ependymoma ; gg , ganglioglioma ; lgg , low - grade glioma , not otherwise specified ; o , oligodendroglioma ; pa , pilocytic astrocytoma ; pma , pilomyxoid astrocytoma ; pxa , pleomorphic xanthoastrocytoma . atrx cdk6 cdkn2a tp53 ptpn11 fgfr3 fgfr1 braf with the degree of reliability that clinical testing requires for use on a single - patient basis , and even if molecular classification was established , expression or methylation analysis might not identify candidates for targeted therapy . 
as an example , although shh pathway medulloblastoma may be reliably identified with immunohistochemistry , 13 nanostring , 17 or methylation profiling , 18 none of these platforms is specific for the site of mutation , which is required for consideration of therapy with a smoothened inhibitor that would be predicted to be effective in ptch1 mutant tumors but unlikely to be effective in the case of downstream alterations , such as gli1 amplification . 
 similarly , braf inhibitor therapy is indicated in braf v600 mutant tumors but contraindicated in braf fusion.19 the detection of fusions such as braf using a dna - based platform such as uw - oncoplex requires the specific inclusion of intronic regions in sequencing . 
alternatively , rna - based sequencing analysis may have an advantage in identifying fusions and splice variants that would be missed on a dnabased platform that does not include intronic sequencing . although 80% of our patients had clinically relevant mutations identified , this means that 20% of tumors sequenced did not have clinically relevant mutations . 
one limitation of this study and other similar studies is that not all known clinically relevant genetic alterations are yet able to be identified on any one clinically validated panel . 
for example , the angiocentric glioma would be expected to have an myb fusion , 20 embryonal tumor with multilayer rosettes would be expected to have mir c19 cluster amplification , 21 and a subset of midline gliomas would be expected to have acvr1 alterations , which are not yet included in this version of the test.22 one advantage of using a targeted sequencing panel is the ability to add and validate targets rapidly with minimal increase in cost . 
however , turnaround time can be shortened with experience , because it is most dependent on clinical interpretation and data generation may be relatively short . another question that our study did not address was the cost comparison between sequential single - gene sequencing in specific diagnostic entities . 
although single - gene testing for the most common variations costs less than a panel , it is not clear that the overall cost of sequential sequencing would be decreased for the population . 
if even a small minority of patients required three - gene testing or subsequent biopsy if tissue were depleted , then there may be a benefit to the population from an initial multiplexed approach . molecular profiling may be particularly useful in patients in whom the histologic diagnosis is unclear . 
 mutations in smarcb1 ( p.p374rfs * 100 ) , bap1 ( p.q392 * ) , pten ( p.g36e ) , nf2 ( p.q319 * ) , and tp53 ( p.r175h ) were all considered both disease defining and potentially germline . 
because only two of our h3f3a k27m mutant diffuse midline gliomas were classic radiologic diffuse intrinsic pontine gliomas , it is important to evaluate for this diagnostic entity whenever a diffuse midline glial neoplasm is encountered . this study provides rigorous evidence regarding the use of routine molecular profiling of an unselected cohort of pediatric brain tumors . 
 on the basis of the analysis presented here , we recommend incorporation of routine molecular profiling into standard clinical practice for all newly diagnosed pediatric hggs , medulloblastomas , and incompletely resected lggs . 
this may be especially important in patients who have undergone stereotactic biopsy in which tumor tissue is severely limited . there was one subgroup in which sequencing did not affect medical care . 
lockwood travel , accommodations , expenses : cambridge healthtech institute shannon stasi no relationship to disclose jeffrey stevens stock and other ownership interests : seattle genetics amy lee no relationship to disclose jeffrey g . 
leary no relationship to disclose acknowledgment we extend our deepest thanks to michael astion , md , medical director of seattle childrens hospital department of laboratories , for his tireless support of this project . 
kool m , korshunov a , remke m , et al : molecular subgroups of medulloblastoma : an international meta - analysis of transcriptome , genetic aberrations , and clinical data of wnt , shh , group 3 , and group 4 medulloblastomas . 
kline cn , joseph nm , grenert jp , et al : targeted next - generation sequencing of pediatric neuro - oncology patients improves diagnosis , identifies pathogenic germline mutations , and directs targeted therapy . 
ramkissoon sh , bandopadhayay p , hwang j , et al : clinical targeted exome - based sequencing in combination with genome - wide copy number profiling : precision medicine analysis of 203 pediatric brain tumors . 
bandopadhayay p , ramkissoon la , jain p , et al : myb - qki rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanisnat genet 48 : 273 - 282 , 2016 21 . 
patients with medulloblastoma were subgrouped on the basis of both histology and standard immunohistochemistry methods for beta - catenin ( dako 1 : 200 ) , gab1 ( santa cruz 1 : 100 ) , and p53 ( protein tech 1 : 50 ) .13 the study pathologist estimated the percentage of tumor and selected appropriate formalin - fixed paraffin - embedded ( ffpe ) blocks for testing . 
it was collected at a separate encounter only if a possible deleterious germline mutation was detected that needed to be investigated further . template preparation , gene capture , massively parallel sequencing , and bioinformatics . sequencing was preformed using a clinically validated method performed in a college of american pathologistsaccredited clinical laboratory improvements amendmentscertified laboratory as previously reported ( pritchard et al , j mol diagn 16 : 56 - 67 , 2014 )  . 
sequencing libraries were prepared from dna samples and hybridized to a custom set of complementary rna biotinylated oligonucleotides targeting the exons of 262 genes and select intronic regions , for a total of > 1.4 mb of targeted dna sequenced ( agilent sureselect ; agilent technologies , santa clara , ca )  . 
uw - oncoplex assay version 5 is a targeted , massively parallel gene sequencing assay that detects mutations in 262 cancer - related genes ( washington.edu / uw - oncoplex )  . 
the test uses next - generation deep sequencing to detect mutations , including single nucleotide variants ( snvs ) , insertions and deletions ( indels ) , copy number changes including gene amplifications , selected structural rearrangements such as gene fusions , and microsatellite instability . 
uw - oncoplex was performed on samples from the seattle childrens hospital cohort , as previously described ( pritchard et al , j mol diagn 16 : 56 - 67 , 2014 ; salipante et al , clin chem 60 : 1192 - 1199 , 2014 )  . dna was extracted from ffpe solid tumor tissue samples using the gentra puregene dna isolation kit ( catalog #158489 ; qiagen , valencia , ca )  . 
hematoxylin and eosinstained slides were reviewed before dna extraction for all ffpe samples , and when feasible , macrodissection of tumor - containing regions was performed to enrich tumor cellularity . 
sequencing libraries were constructed from a minimum of 200 ng of dna using kapa hyper prep kits ( kapa biosystems , wilmington , ma ) , and hybridization was performed with custom agilent sureselect probes ( agilent technologies , santa clara , ca )  . 
dna sequencing was performed on a hiseq2500 sequencing system ( illumina , san diego , ca ) with 2 101bp , paired - end reads , and on a nextsequation 500 ( illumina ) with 2 150bp , paired - end reads according to the manufacturers instructions . 
for copy number variation ( cnv ) analysis , copy number states for individual probes were initially called using contra version 2.0.5 ( files ) with reference to a cnv control comprising reads from two independent rounds of library preparation and sequencing of the hapmap individual na12878 . 
adjacent exons were merged into larger segments if the read depths of their component baits were not significantly different ( p > .001 ) by students t test , and read - depth statistics were recalculated for the larger segments . 
filters are applied to exclude variants extremely unlikely to be clinically significant , including variants in intergenic dna , and variants are removed that are present at > 1% frequency in general population databases ( as reported by the exome variant server , 1000 genomes , and the exome aggregation consortium )  . 
variants of interest are subsequently evaluated in conjunction with a combination of multiple external databases ( cosmic , iarc , insight , clinvar ) , literature review ( for newly described mutations ) , and algorithms ( splice prediction tools )  . three separate expert molecular oncology reviewers independently cull data to a list of potentially reportable variants and review quality of data for variant calls by examining genotype quality score metrics , evaluating base call quality , assessing mapping quality , and evaluating other quality assessments as indicated for specific alterations . 
 once the molecular oncology experts agree on a final list of reportable variants , one laboratory director composes a fully customized , formal report that is available in the electronic health record . 
the shh subgroup was initially identified by mutations ( snvs , indels , structural alterations , and copy number alterations ) in ptch1 , smo , sufu , and copy loss for genes on the long arm of chromosome 10 . 
each month , all new patients were systematically reviewed in a presentation format consisting of clinical features and magnetic resonance imaging presented by a neuro - oncologist ( s.e.s.l. ) , histologic findings and photomicrographs by a pediatric pathologist ( b.l.c. ) , molecular results by a molecular pathologist ( c.m.l. ) followed by group discussion . 
the molecular tumor board welcomed rotating trainees from all disciplines and included attendance of trainees from oncology , neurosurgery , pathology , laboratory medicine , medical genetics , and medical students . 
when applicable , a new final integrated diagnosis was reported in the pathology addendum . genetic alterations were considered to be clinically significant as defined by meeting criteria for at least one of the following three categories : ( 1 ) alterations that define a diagnosis or molecular subtype , for example , an activating beta - catenin mutation in wnt - subtype medulloblastoma ; ( 2 ) alterations that identify a proven therapeutic target , such as braf v600e ; and / or ( 3 ) alterations that identify possible deleterious germline mutation associated with cancer predisposition syndrome , such as tp53 . 
with consideration of the variant allele fraction as well as previously described genes associated with cancer predisposition , additional constitutional follow - up testing was recommended for the patients with potential germline mutations , which was coordinated by a genetic counselor . 
 efcacy of vemurafenib in patients with nonsmall - cell lung cancer with braf v600 mutation : an open - label , single - arm cohort of the histology - independent ve - basket study vivek subbiah , md1 ; radj gervais , md2 ; gregory riely , md , phd3 ; antoine hollebecque , md4 ; jean - yves blay , md , phd5 ; enriqueta felip , md , phd6 ; martin schuler , md7 ; anthony gonalves , md , phd8 ; antonio italiano , md , phd9 ; vicki keedy , md10 ; ian chau , md11 ; igor puzanov , md12 ; noopur s . 
raje , md13 ; funda meric - bernstam , md1 ; martina makrutzki , md14 ; todd riehl , pharmd15 ; bethany pitcher , mmath16 ; jose baselga , md , phd3 , 17 ; and david m . 
hyman , md3 , 17 purpose to study whether braf v600 mutations in nonsmall - cell lung cancer ( nsclc ) may indicate sensitivity to the braf inhibitor vemurafenib , we included a cohort of patients with nsclc in the vemurafenib basket ( ve - basket ) study . 
we present results from this cohort . methods this open - label , histology - independent , phase ii study included six prespecied cohorts , including patients with nsclc , and a seventh all - comers cohort . 
because the prespecied clinical benet endpoint was met in the initial nsclc cohort , the cohort was expanded . results sixty - two patients with braf v600mutant nsclc were enrolled and treated : 13% ( n = 8 ) had received no prior systemic therapy , and 87% ( n = 54 ) had received prior therapies . 
the safety prole of vemurafenib was similar to that observed in melanoma studies . conclusion vemurafenib showed promising activity in patients with nsclc harboring braf v600 mutations . the safety prole of vemurafenib was similar to previous observations in patients with melanoma . 
 subbiah et al context key objective to establish the efcacy and safety of vemurafenib in patients with braf v600 mutation - positive nsclc who were enrolled in the histology - independent vemurafenib basket ( ve - basket ) trial . knowledge generated vemurafenib has prolonged efcacy in patients with braf v600mutant nsclc ( n = 62 ) , as demonstrated by a 37% overall response rate . 
no new safety signals were observed in this expanded cohort of patients with nsclc . relevance single - agent vemurafenib has clinically meaningful and durable activity in patients with nsclc harboring braf v600 mutations . 
this analysis adds to the overall ndings of the ve - basket trial , which demonstrated clinically relevant activity of vemurafenib in a number of solid tumors . clinical study of patients with braf v600emutated nsclc.14 dual braf / mek inhibition has also been investigated as rstand second - line treatment of patients with nsclc.15 , 16 we present the results from the expanded nsclc cohort of the vemurafenib basket trial . 
this trial assessed the efcacy of vemurafenib in seven cohorts of patients with braf v600mutated malignancies.17 ( ve - basket ) methods study design the ve - basket study was a multicenter , single - arm , phase ii study of vemurafenib in patients with a variety of nonmelanoma cancers harboring braf v600 mutations . braf v600 mutations were identied by means of mutational analysis assays routinely performed at each participating site . 
six prespecied cohorts were recruited , consisting of patients with nsclc , ovarian cancer , colorectal cancer , cholangiocarcinoma , breast cancer , and multiple myeloma ; all patients with solid tumors other than those mentioned were included in a seventh cohort . 
the design of this study has been described in detail elsewhere.17 this trial was performed in accordance with the provisions of the declaration of helsinki and good clinical practice guidelines . 
all patients provided written informed consent . patients patients were eligible for inclusion in the study if they were 16 years of age or older and had histologically conrmed , measurable ( response evaluation criteria in solid tumors [ recist ] , version 1.1 ) , braf v600 mutation - positive cancers that were refractory to standard therapy or for which standard or curative therapy did not exist or was not considered appropriate by the investigator . 
assessments were performed using computed tomography or magnetic resonance imaging of the chest , abdomen , and pelvis at baseline and then every 8 weeks until disease progression , death , or withdrawal from the study . 
adverse events ( aes ) were graded by the investigators using national cancer institute common terminology criteria for adverse events ( version 4.0 ) until 28 days after discontinuation of study treatment . 
aes of special interest were cutaneous squamous cell carcinoma ( scc ; keratoacanthoma , squamous cell carcinoma of the skin , and bowen disease ) , fatigue ( fatigue and asthenia ) , liver injury ( increased alt , ast , blood alkaline phosphatase , blood bilirubin , and gamma - glutamyltransferase ; hyperbilirubinemia , hepatocellular injury , and cholestatic jaundice ) , and prolonged qt interval . 
patients were assessed for aes at each clinic visit and as necessary throughout the study . outcomes the primary objective of the study was to evaluate the efcacy of vemurafenib in patients with braf v600 mutation - positive cancers . 
the primary end point for the nal analysis in the nsclc cohort was objective response rate ( orr ) , dened as the proportion of patients with an objective response ( complete response [ cr ] or partial response [ pr ] ) conrmed on two consecutive occasions 4 or more weeks apart . 
 vemurafenib in nsclc with braf v600 mutation : the ve - basket study objectives included assessments of clinical benet rate ( dened as the overall proportion of patients with a cr , pr , or stable disease lasting 6 months ) , duration of response , progression - free survival ( pfs ) , overall survival ( os ) , and safety . 
efcacy data were analyzed separately for patients who had received no prior therapy and for those with prior therapies . statistical analysis this was a modied , two - stage simon design study . 
stage i was complete when seven patients with measurable disease were enrolled and had completed a minimum of 8 weeks of treatment , developed progressive disease , prematurely withdrew , or died . 
an additional six or 12 patients could be enrolled , to 13 or 19 patients , depending on the results for stage i ; if two , three , or four of the initial seven patients responded to treatment , an additional 12 patients could be enrolled in stage ii ; if ve or more of the initial seven patients responded to treatment , an additional six patients were recruited . 
recruitment into any cohort / indication could be further expanded up to 70 patients if a response rate was demonstrated in stage ii of that cohort , according to the stopping rules dened in the protocol or a clear clinical benet for patients was observed , as determined by the steering committee . 
for the nsclc cohort , with 50 treated patients , the study would have approximately 90% power for the lower bound of the twosided 95% ci to exclude 20% , given a true orr of 40% . the lower bound of the 95% ci was set at 20% because established therapy in the second and later lines had an orr of less than 20% when the study was designed . 
most patients had adenocarcinoma ( n = 58 ; 94% ) , three patients ( 4.8% ) had cns metastases , and most were former smokers ( n = 36 ; 58% )  . among previously treated patients , the median number of prior systemic regimens was two ( interquartile range [ iqr ] , 1 to 2 ) ; the most common prior chemotherapies were platinum agents ( 39 of 54 patients ; 72% ) , pemetrexed ( 33 of 54 patients ; 61% ) , and taxanes ( 22 of 54 patients ; 41% )  . all patients ( n = 62 ) previously untreated ( n = 8 ) previously treated ( n = 54 ) 65 ( 59 - 74 ) 73 ( 65 - 79 ) 64 ( 57 - 72 ) table 1 . 
overall , the investigator - determined orr was 37% ( 95% ci , 25% to 50% ) , and the clinical benet ( cr plus pr plus stable disease lasting 6 months ) rate was 48% ( 95% ci , 36% to 61% )  . 
the median relative dose intensity achieved was 78% ( iqr , 64% to 91% ) overall . all 62 patients experienced at least one any - cause ae ; grade 3 or 4 aes occurred in 48 patients ( 77% ) , and two patients had grade 5 aes ( 3% ; one patient with sepsis , one with a pulmonary embolism and respiratory failure ; both patients had been previously treated , and none of the events were considered to be related to vemurafenib )  . table 3 lists all - cause and grade 3 or greater aes occurring in 20% or more of patients . aes leading to treatment interruption occurred in 25 of 62 patients ( 40% )  . 
treatment outcomes in patients with nonsmall - cell lung cancer outcome all patients ( n = 62 ) previously untreated ( n = 8 ) previously treated ( n = 54 ) investigator - assessed best response , no . 
 vemurafenib in nsclc with braf v600 mutation : the ve - basket study - 100 previously untreated patients partial response stable disease progressive disease previously treated patients time to progression time to response died alive time ( months ) before treatment after 2 months of treatment fig 1 . 
 ( b ) waterfall plot of maximum percent decrease from baseline in the sum of diameters of target tumors on the basis of investigator assessment : best overall response in individual patients . ( c ) pretherapy and post - therapy 18f - uorodeoxyglucose positron emission tomography images of a chemotherapy - naive patient with braf v600e mutationpositive nsclc . 
at risk previously untreated ( n = 8 ) previously treated ( n = 54 ) overall ( n = 62 ) censored previously untreated ( n = 8 ) previously treated ( n = 54 ) overall ( n = 62 ) censored no . 
at risk 9 12 15 18 21 24 27 30 33 36 39 time ( months ) time ( months ) previously untreated previously treated 54 45 36 29 22 16 12 11 overall 62 52 42 34 26 18 14 12 previously untreated previously treated overall fig 2 . 
a total of 82 serious aes occurred in 39 patients ( 63% ) , the most common of which were scc of the skin ( nine patients ; 15% ) and keratoacanthoma ( nine patients ; 15% ) , which was dened as a serious ae . 
in total , 25 patients ( 40% ) had serious aes considered by the investigator to be caused by vemurafenib ( keratoacanthoma , n = 9 ; scc of the skin , n = 9 ; basal cell carcinoma , n = 1 ; bowen disease , n = 1 ; acute kidney injury , n = 4 ; pericarditis , n = 1 ; stomatitis , n = 1 ; pyrexia , n = 1 ; hypersensitivity , n = 1 ; sepsis , n = 1 ; and dehydration , n = 1 ) ; serious aes not considered to be related to vemurafenib included pneumonia ( n = 2 ) , bronchitis ( n = 2 ) , dyspnea ( n = 3 ) , pericardial effusion ( n = 1 ) , sepsis ( n = 3 ) , pulmonary embolism ( n = 2 ) , and lung infection ( n = 2 )  . targetable oncogenic drivers in nsclc with robust clinical validation include egfr mutations and alk and ros1 fusions , but identifying other targetable , clinically important subgroups of nsclc is a high priority . 
in this context , we found that patients with braf v600emutated nsclc treated with vemurafenib had an orr of 37% , with similar response rates in previously treated and untreated patients . median os was 15 months in the overall patient population , but had not been reached in the group of previously untreated patients after 12 months of follow - up . 
similarly , our previously untreated patients had a median pfs of 12.9 months , which was considerably longer than the 6.5 months observed in patients who had received prior therapies . 
the safety prole of vemurafenib in our group of patients with nsclc was similar to that seen in patients with melanoma.10 , 18 no new safety signals were observed in this population . 
we suggest that future studies should examine additional combinations in patients with braf mutation - positive nsclc . in conclusion , the results of the present cohort analysis suggest a role for braf inhibition in patients with nsclc with braf mutations . 
 vemurafenib in nsclc with braf v600 mutation : the ve - basket study affiliations 1university of texas md anderson cancer center , houston , tx 2centre franois baclesse , caen , france 3memorial sloan kettering cancer center , new york , ny 4institut gustave roussy , villejuif , france 5centre l eon b erard , lyon , france 6vall dhebron university hospital and vall dhebron institute of oncology , barcelona , spain 7university hospital essen , essen , germany 8aix - marseille universit e , marseille , france 9institut bergonie , bordeaux , france 10vanderbilt university , nashville , tn 11the royal marsden nhs foundation trust , london , united kingdom 12roswell park cancer institute , buffalo , ny 13massachusetts general hospital , boston , ma 14f . 
hyman administrative support : vivek subbiah provision of study materials or patients : vivek subbiah , gregory riely , antoine hollebecque , jean - yves blay , enriqueta felip , martin schuler , anthony gonalves , antonio italiano , igor puzanov , funda meric - bernstam collection and assembly of data : vivek subbiah , radj gervais , gregory riely , antoine hollebecque , jean - yves blay , martin schuler , antonio italiano , vicki keedy , igor puzanov , funda meric - bernstam , martina makrutzki , todd riehl , david m . 
hyman data analysis and interpretation : vivek subbiah , radj gervais , antoine hollebecque , jean - yves blay , enriqueta felip , martin schuler , anthony gonalves , antonio italiano , vicki keedy , ian chau , igor puzanov , noopur s . 
raje consulting or advisory role : amgen , celgene , takeda , novartis , bristolmyers squibb , merck , janssen oncology research funding : astrazeneca ( inst ) funda meric - bernstam honoraria : sumitomo group , dialectica consulting or advisory role : genentech , inection biosciences , pieris pharmaceuticals , clearlight diagnostics , darwin health , samsung bioepis , spectrum pharmaceuticals , aduro biotech , origimed , xencor , debiopharm group , mersana research funding : novartis , astrazeneca , taiho pharmaceutical , genentech , calithera biosciences , debiopharm group , bayer , aileron therapeutics , puma biotechnology , cytomx therapeutics , jounce therapeutics , zymeworks , curis , pzer , effector therapeutics , abbvie , boehringer ingelheim ( i ) , guardant health ( inst ) , daiichi sankyo , glaxosmithkline martina makrutzki employment : roche todd riehl employment : genentech stock and other ownership interests : genentech bethany pitcher employment : hoffmann - la roche jose baselga leadership : innity pharmaceuticals , varian medical systems , bristolmyers squibb , foghorn stock and other ownership interests : pmv pharma , juno therapeutics , grail , tango , venthera , northern biologics , apogen biotechnologies , aura biosciences consulting or advisory role : novartis patents , royalties , other intellectual property : combination therapy using pdk1 and pi3k inhibitors , use of phosphoinositide 3 - kinase inhibitors for treatment of vascular malformations , inhibition of kmt2d for the treatment of cancer travel , accommodations , expenses : roche , daiichi sankyo david m . 
n engl j med 361 : 947 - 957 , 2009 solomon bj , mok t , kim d - w , et al : first - line crizotinib versus chemotherapy in alk - positive lung cancer . 
n engl j med 371 : 1963 - 1971 , 2014 thomas a , liu sv , subramaniam ds , et al : rening the treatment of nsclc according to histological and molecular subtypes . 
medicine ( baltimore ) 96 : e6552 , 2017 paik pk , arcila me , fara m , et al : clinical characteristics of patients with lung adenocarcinomas harboring braf mutations . 
mcarthur ga , chapman pb , robert c , et al : safety and efcacy of vemurafenib in braf ( v600e ) and braf ( v600k ) mutation - positive melanoma ( brim - 3 ) : extended follow - up of a phase 3 , randomised , open - label study . 
long gv , stroyakovskiy d , gogas h , et al : dabrafenib and trametinib versus dabrafenib and placebo for val600 braf - mutant melanoma : a multicentre , double - blind , phase 3 randomised controlled trial . 
ascierto pa , mcarthur ga , dr eno b , et al : cobimetinib combined with vemurafenib in advanced braf ( v600 ) - mutant melanoma ( cobrim ) : updated efcacy results from a randomised , double - blind , phase 3 trial . 
planchard d , kim tm , mazieres j , et al : dabrafenib in patients with braf ( v600e ) - positive advanced non - small - cell lung cancer : a single - arm , multicentre , open - label , phase 2 trial . 
planchard d , besse b , groen hjm , et al : dabrafenib plus trametinib in patients with previously treated brafv600e - mutant metastatic non - small cell lung cancer : an open - label , multicentre phase 2 trial . 
planchard d , smit ef , groen hjm , et al : dabrafenib plus trametinib in patients with previously untreated brafv600e - mutant metastatic non - small - cell lung cancer : an open - label , phase 2 trial . 
reis - filho , md , phd1 ; diana mandelker , md , phd1 ; and britta weigelt , phd1 purpose endometrial cancer ( ec ) is not considered a component of the hereditary breast and ovarian cancer syndrome but can arise in patients with germline brca1 / 2 ( gbrca1 / 2 ) mutations . 
we sought to determine if ecs in gbrca1 / 2 mutation carriers harbor biallelic alterations and / or features of hrd . methods of 769 patients with ec who underwent germline panel testing , 10 pathogenic gbrca1 / 2 mutation carriers were identied , and their tumorand normal - derived dna was subjected to massively parallel sequencing targeting at least 410 cancer - related genes . 
somatic mutations , copy number alterations , loss of heterozygosity , microsatellite instability ( msi ) , and genomic hrd features were assessed . results of the 13 patients included who had ec , eight harbored pathogenic gbrca1 mutations and ve harbored gbrca2 mutations . 
 smith et al context key objective to determine if endometrial cancers ( ecs ) that arise in patients with pathogenic brca1 or brca2 germline mutations are sporadic cancers or display genomic features of homologous recombination dna repair deciency ( hrd ) , which may guide treatment strategies . knowledge generated ecs in patients with brca1 germline mutations showed biallelic brca1 alterations coupled with genomic features of hrd . 
in contrast , only a subset of ecs in brca2 germline mutation carriers harbored biallelic brca2 alterations ; the remaining cases were likely sporadic cancers . relevance knowledge about the brca1 / 2 allele status and / or hrd features of ecs in patients with germline brca1 / 2 alterations may guide therapeutic decision making , given that tumors with biallelic alterations in brca1 / 2 have been associated with increased response to hr - targeted therapies such as platinum salts and parp inhibitors . brca1 / 2 alterations and show features of hr deciency , including the catalog of somatic mutations in cancer mutational signature 3 , signature multivariate analysis ( sigma ) hr deciency signature sig3 , or large - scale chromosomal breaks in the form of large - scale state transitions ( lsts )  . 
in addition , we assessed the clinicopathologic data of ecs in gbrca1 / 2 mutation carriers and explored the repertoire of somatic mutations present in these tumors . methods case selection all patients with ec and gbrca1 / 2 mutations who consented to germline analysis under an institutional review boardapproved protocol at memorial sloan kettering cancer center and whose tumors were subjected to targeted massively parallel sequencing via memorial sloan ketteringintegrated mutation proling of actionable cancer targets ( msk - impact ) 10 , 11 from july 2015 to may 2019 were identied ( n = 769 sequenced ; n = 10 with pathogenic gbrca1 / 2 mutation )  . 
all gbrca1 / 2 variants were reviewed by a board - certied molecular pathologist ( d.m. ) and classied according to the american college of medical genetics and genomics criteria12 as pathogenic . clinicopathologic data , including age at diagnosis , cancer stage , and past cancer history , were obtained from medical records . 
mlh1 promoter methylation was assessed in the clinical setting on dna obtained from formalin - xed parafn - embedded tumor samples , as previously described.28 statistical analyses fishers exact and mann - whitney u tests were used for comparison of categorical and continuous variables , respectively . 
two - tailed p , 0.05 was considered statistically signicant . results clinicopathologic features of ecs arising in gbrca1 / 2 carriers ten ecs from patients with pathogenic gbrca1 / 2 mutations subjected to msk - impact sequencing ( n = 769 ecs ; 1.3% ) and three subjected to wes by tcga ( n = 232 ecs ; 1.3% ) 13 were included in this study . 
of these 13 gbrca1 / 2associated ecs , eight and ve patients with ec harbored gbrca1 and germline brca2 ( gbrca2 ) mutations , respectively ( table 1 )  . 
the majority of gbrca1 / 2 - associated ecs ( n = 6 of 8 ; 75% ) displayed an endometrioid histology ( n = 2 and 4 international federation of gynecology and obstetrics [ figo ] grades ii and iii , respectively ) but lacked a solid / pseudoendometrioid / transitional cell - like pattern of morphology , which has been associated with gbrca1 / 2 mutations in high - grade serous ovarian cancers.29 , 30 in contrast , ecs from patients with pathogenic gbrca2 mutations were more heterogeneous at the phenotypic level , and included endometrioid ( n = 1 ; figo grade ii ) , carcinosarcoma ( n = 2 ) , high - grade ec not otherwise specied ( nos ; n = 1 ) and serous ( n = 1 ) cancers ( tables 1 and 2 )  . 
of note , none of the gbrca1 / 2 - associated ecs included in this study were of serous histology . gbrca1 / 2 - associated ecs presented at different clinical figo stages ( 2009 staging system31 )  . 
taken together , our data suggest the clinicopathologic features associated with ecs occurring in patients with pathogenic gbrca1 or gbrca2 mutations may be heterogeneous . biallelic brca1 / 2 alterations allele - specic copy number analysis revealed that 77% ( n = 10 of 13 ) of the ecs in patients with pathogenic gbrca1 / 2 mutations displayed biallelic inactivation of brca1 / 2 uniformly through loh of the wild - type allele ( table 2 ; fig 1 )  . 
not all ecs occurring in pathogenic gbrca1 / 2 mutation carriers harbored biallelic brca1 / 2 alterations , which have previously been associated with hr deciency.8 although all ecs in pathogenic gbrca1 mutation carriers ( 100% ) harbored biallelic brca1 inactivation , only two of the ve ecs ( 40% ) from patients with pathogenic gbrca2 mutations displayed biallelic brca2 inactivation ( table 2 ; fig 1 )  . biallelic brca1 / 2 inactivation was found across all histologic subtypes and clinical stages of ec . 
ecs in brca2 mutation carriers lacking loh of the wild - type allele are likely sporadic tumors ; all three were stage iii at diagnosis and were endometrioid grade ii , serous , or high - grade ecs ( table 2 ; fig 1 )  . 
 ( % ) unless otherwise indicated . abbreviations : msk - impact , memorial sloan ketteringintegrated mutation proling of actionable cancer targets ; nos , not otherwise specied ; tcga , the cancer genome atlas ; wes , whole - exome sequencing . * fishers exact and mann - whitney u tests were used for comparison of categorical and continuous variables , respectively . staging information was performed according to the international federation of gynecology and obstetrics system.31 past cancer histories and information regarding radiation exposure were available for the 10 msk - impact cases and reect cancers diagnosed and treated prior to the diagnosis of ec . 13 gbrca1 / 2 - associated ecs was six ( range , 2 to 38 ) , with a median of ve ( range , 2 to 32 ) nonsynonymous mutations ( data supplement )  . 
we observed that all ecs analyzed , irrespective of the presence of monoor biallelic brca1 / 2 alterations , harbored somatic tp53 mutations , of which 12 ( 92% ) were hotspot mutations ( fig 1 ; data supplement )  . 
alterations affecting the pi3k pathway were 4 2019 by american society of clinical oncology common , with pik3ca mutations present in ve ecs ( 38% ) , pik3r1 mutations in two ecs ( 15% ) , and pten mutations / homozygous deletions in three ecs ( 23% ; figs 1 and 2 )  . 
other recurrently altered genes included fat1 , ptch1 , kras , and map3k1 ( each n = 2 ; fig 1 , data supplement )  . we noted that bec - 1 , a likely sporadic ec from a gbrca2 mutation carrier with a monoallelic brca2 alteration , had a high mutational burden with 32 nonsynonymous somatic mutations . 
the ecs with very low levels of gene cnas ( ie , bec - 1 , bec - 6 , and bec - 12 ) had monoallelic brca2 alterations and were likely sporadic , with one ( bec - 1 ) being msi high , as mentioned . 
in addition , we found an nf1 homozygous deletion in the biallelic brca1 grade ii endometrioid tcga - 2 and a smarca4 homozygous deletion in the biallelic brca2 carcinosarcoma bec - 2 . 
of note , smarca4 ( brg1 ) loss is commonly found in unthe endometrium , 32 , 33 and differentiated carcinoma of bec - 2 was a carcinosarcoma with heterologous elements , which , in addition to the carcinoma and sarcomatous components , also displayed an undifferentiated component . 
finally , amplication of bcl6 , tp63 , and eed was found in the biallelic brca1 grade iii endometrioid tcga - 1 ec ( fig 2 )  . taken together , we found that ecs in patients with a gbrca1 / 2 mutation are heterogeneous at the mutational and gene copy number levels . 
we observed that ecs with biallelic brca1 / 2 alterations ( n = 10 ) had high lst scores , whereas ecs with monoallelic brca1 / 2 alterations did not ( table 2 ; fig 3a )  . 
nonsynonymous somatic mutations identied in 410 cancer - related cancer genes in ecs from germline brca1 ( n = 8 ) and germline brca2 ( n = 5 ) mutation carriers ( left ) are shown . the mutation types are color coded according to the legend . 
information on the germline mutation status , somatic loss of heterozygosity of brca1 / 2 , histologic type , microsatellite instability ( msi ) status , and sequencing type is provided in the phenobar below the sample names . 
tcga , the cancer genome atlas . with hr deciency ( fig 3b ) .14 given the limited number of snvs and indels in the ecs subjected to msk - impact sequencing , other genomic hr - deciency features such as indel length , microhomology , or mutational signatures using decomposition algorithms could not be used . therefore , we used sigma , a recently described , likelihoodbased measure signature analysis combined with machinelearning techniques , which can be used to detect the mutational signature sig3 associated with hr deciency from targeted gene panels including msk - impact ( sensitivity range , 48% to 62% for uterine cancers ) .25 consistent with the lst analysis , all six biallelic , brca1 / 2associated ecs subjected to msk - impact and with at least ve snvs displayed either a dominant hr deciency - related sig3 ( n = 2 ) or a dominant clock signature with secondary hr - deciency signatures sig3 ( n = 3 ) or sig834 ( n = 1 ) , to the two sporadic ecs with monoallelic in contrast brca2 alterations , which displayed msi ( sigma ) / signature 6 ( deconstructsigs ) and apobec signatures ( table 2 ; fig 3c )  . 
previous reports of ecs in this population have acknowledged that the prevalence of ec in gbrca1 / 2 mutation carriers is low.5 here , through an in - depth analysis of ecs from patients with pathogenic gbrca1 / 2 mutations , we demonstrate that the majority of these cases ( 77% ) harbored biallelic brca1 / 2 alterations and displayed genomic features of hrd , providing evidence to suggest an etiological link between the pathogenic gbrca1 / 2 mutations and the development of these ecs . 
in fact , this phenomenon was uniformly observed in patients with pathogenic gbrca1 mutations ; conversely , only two of the ve patients with pathogenic gbrca2 mutations had somatic inactivation of the brca2 wild - type allele and genomics features of hr deciency . 
in ovarian cancer , patients with hr - decient tumors have improved overall survival with platinum - based therapy and have better responses to hr deciencydirected treatments such as parp inhibitors than their wild - type counterparts.35 , 36 knowledge of the allele status of brca1 / 2 alterations and / or hr - deciency features could be benecial in therapeutic decision making for patients with gbrca1 / 2 mutations and ec . 
our analysis has demonstrated that not all ecs arising in the context of pathogenic gbrca2 mutations harbor loh of the wild - type allele and genomics features of hr deciency . 
in addition , it revealed that one of these cases was msi high , likely representing a sporadic ( ie , non - brca1 / 2 ) ec arising in a gbrca2 mutation carrier . 
in this context , this patient would potentially benet from immune checkpoint inhibitors , which have been approved for the management of recurrent msi - high / dna mmrdecient cancers.37 in fact , a recent case report highlighted a complete remission after pd - 1 blockade in a patient with mmr - decient ec and a pathogenic monoallelic gbrca1 mutation.38 the majority ( 75% ) of biallelic gbrca1 / 2 - associated ecs were of intermediate / high - grade endometrioid as opposed to serous histology in previous reports.5 , 6 all six biallelic gbrca1 / 2 - associated endometrioid ecs harbored tp53 mutations , had high lst scores , and relatively high levels of cnas ( table 2 ; fig 2 ) , and would likely be of copy - number high ( serous - like ) molecular subtype , as described by tcga.13 it should be noted , however , that of these six endometrioid ecs with biallelic brca1 alterations , four harbored somatic mutations characteristic of endometrioid ecs , including pik3ca mutations , pik3r1 mutations , and pten mutations / homozygous deletions . 
conversely , two of these ecs lacked alterations affecting genes recurrently altered in endometrioid ecs ( fig 1 ) , suggesting that at the these latter two cases resembled serous genetic level , rather than endometrioid carcinomas , and that there is heterogeneity in gbrca1 / 2 - associated ecs . 
carcinosarcomas of the uterus are rare , representing less than 5% of all uterine tumors.39 we noted a high frequency ( 30% ) of carcinosarcomas in the 10 gbrca1 / 2 - associated ecs subjected to msk - impact sequencing , whereas overall , only 10% of the 769 ecs subjected to germline mskimpact sequencing were carcinosarcomas ( remaining cases : 55% endometrioid , 15% serous , 10% endometrial cancer nos , 4% mixed endometrial , 3% clear cell , 1% deor undifferentiated , and 1% other )  . 
interestingly , recurrent somatic brca1 / 2 mutations and brca2 deletions also have been reported in uterine carcinosarcomas.40 - 42 these ndings suggest that ecs arising in gbrca1 / 2 mutation carriers are heterogeneous at the histologic level and are potentially enriched for endometrioid tumors and carcinosarcomas . the data presented here do provide insight into the genomics of ecs in patients with pathogenic gbrca1 / 2 mutations , which may play a role in the tumorigenesis and / or progression of ec in some patients . 
this study has several limitations , however , primarily driven by the small sample size , and it does not resolve the controversy of ec as a feature of hboc syndrome . 
the low incidence of these cases ( 1.3% ; n = 10 of 769 ecs subjected to germline mskimpact testing ) not only limits the sample size of this study but also , on its own , presents a challenge in dening ec as part of the hboc syndrome spectrum of malignancies . 
 brca1 / 2 endometrial cancer lsts mutational signatures ( decontructsigs ) signature 1 ( aging ) signature 3 ( hr defect ) signature 15 ( mmr defect ) signature 19 signature 20 ( mmr defect ) signature 24 signature 25 unknown tcga - 1 tcga - 2 tcga - 3 mlh1 biallelic brca1 / 2 ( n = 10 ) monoallelic brca2 ( n = 3 ) pms2 msh2 msh6 signature 1 aging signature 6 mmr defect signature 21 mmr defect unknown mutational signatures bec - 1 fig 3 . 
 ( a ) large - scale state transition ( lst ) scores in germline ( gbrca1 / 2 ) - associated endometrial cancers with biallelic ( n = 10 ) and monoallelic ( n = 3 ) brca1 / 2 alterations . 
 ( b ) mutational signatures dened by deconstructsigs24 in three gbrca1 / 2 - associated endometrial cancers from the cancer genome atlas ( tcga ) subjected to whole - exome sequencing.13 , 14 mutational signatures are color coded according to the legend on the right . 
 ( c ) immunohistochemical analysis of the ( left ) dna mismatch repair ( mmr ) proteins mlh1 , pms2 , msh2 , and msh6 and ( right ) mutational signatures of case bec - 1 , a monoallelic / sporadic gbrca1 / 2 - associated endometrial cancer . 
bec - 1 shows loss of mlh1 and pms2 expression , mlh1 promoter hypermethylation ( not shown ) , and dominant mutational signatures 6 and 21 associated with defective dna mmr . mutations in patients with ec nor the impact of these mutations on the outcome of patients with ec could be fully dened . 
although we have assessed the genomics features of hr deciency , including lsts and sigma hr deciency signature sig3 in all cases , hr deciency is more accurately assessed with whole - genome sequencing.43 hence , additional studies testing ecs developing in the context of pathogenic gbrca1 / 2 mutations but lacking loss of the wildtype allele by whole - genome sequencing are warranted . despite these limitations , here we demonstrate the importance of germline and somatic genetic characterization of ecs . 
in fact , ecs developing in the context of pathogenic gbrca1 / 2 mutations may harbor genomics features of hr deciency and be causally linked to the loss of function of these tumor suppressor genes . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . nadeem riaz honoraria : peerview speakers bureau : illumina research funding : bristol - myers squibb , pzer travel , accommodations , expenses : varian medical systems mark e . 
robson honoraria : astrazeneca consulting or advisory role : mckesson , astrazeneca research funding : astrazeneca ( inst ) , myriad genetics ( inst ) , invitae ( inst ) , abbvie ( inst ) , tesaro ( inst ) , medivation ( inst ) travel , accommodations , expenses : astrazeneca , pzer other relationship : research to practice , clinical care options , physician education resource robert a . 
assignee : 2015 the regents of the university of california 8 / 18 / 2015 ( inst ) ; application : compositions and methods for the diagnosis and treatment of ovarian cancers that are associated with reduced smarca4 gene expression or protein function ( inst ) ; royalties from published books : cambridge university press and springer publishing jorge s . 
reis - filho consulting or advisory role : roche , invicro , ventana medical systems , volition rx , paige.ai , goldman sachs , novartis britta weigelt consulting or advisory role : roche ( i ) , invicro ( i ) , ventana medical systems ( i ) , volition rx ( i ) , paige.ai ( i ) , goldman sachs ( i ) , novartis ( i ) no other potential conicts of interest were reported . acknowlegement we thank the members of the molecular diagnostics service in the department of pathology , memorial sloan kettering cancer center . references gudmundsdottir k , ashworth a : the roles of brca1 and brca2 and associated proteins in the maintenance of genomic stability . 
oncogene 25 : 5864 - 5874 , 2006 king mc , marks jh , mandell jb : breast and ovarian cancer risks due to inherited mutations in brca1 and brca2 . 
nat rev clin oncol 13 : 41 - 54 , 2016 segev y , iqbal j , lubinski j , et al : the incidence of endometrial cancer in women with brca1 and brca2 mutations : an international prospective cohort study . gynecol oncol 130 : 127 - 131 , 2013 shu ca , pike mc , jotwani ar , et al : uterine cancer after risk - reducing salpingo - oophorectomy without hysterectomy in women with brca mutations . 
jama oncol 2 : 1434 - 1440 , 2016 pennington kp , walsh t , lee m , et al : brca1 , tp53 , and chek2 germline mutations in uterine serous carcinoma . 
cell 173 : 355 - 370.e14 , 2018 riaz n , blecua p , lim rs , et al : pan - cancer analysis of bi - allelic alterations in homologous recombination dna repair genes . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
ashley cw , da cruz paula a , kumar r , et al : analysis of mutational signatures in primary and metastatic endometrial cancer reveals distinct patterns of dna repair defects and shifts during tumor progression . 
geyer fc , li a , papanastasiou ad , et al : recurrent hotspot mutations in hras q61 and pi3k - akt pathway genes as drivers of breast adenomyoepitheliomas . nat commun 9 : 1816 , 2018 e131 , 2016 17 . 
karnezis an , hoang ln , coatham m , et al : loss of switch / sucrose non - fermenting complex protein expression is associated with dedifferentiation in endometrial carcinomas . 
ramalingam p , croce s , mccluggage wg : loss of expression of smarca4 ( brg1 ) , smarca2 ( brm ) and smarcb1 ( ini1 ) in undifferentiated carcinoma of the endometrium is not uncommon and is not always associated with rhabdoid morphology . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
dizon ds , dias - santagata d , bregar a , et al : complete remission following pembrolizumab in a woman with mismatch repair - decient endometrial cancer and a germline brca1 mutation . 
gynecol oncol 137 : 581 - 588 , 2015 jones s , stransky n , mccord cl , et al : genomic analyses of gynaecologic carcinosarcomas reveal frequent mutations in chromatin remodelling genes . 
zhao s , bellone s , lopez s , et al : mutational landscape of uterine and ovarian carcinosarcomas implicates histone genes in epithelial - mesenchymal transition . proc natl acad sci usa 113 : 12238 - 12243 , 2016 jones nl , xiu j , chatterjee - paer s , et al : distinct molecular landscapes between endometrioid and nonendometrioid uterine carcinomas . 
 c circulating cell - free tumor dna in the management of double primary tumors introduction synchronous lung and colorectal cancer ( crc ) occurrence in one individual is a rare event.1 - 3 the genomics of both malignancies have been extensively studied and found to be distinct . 
 broad molecular profiling in advanced non small - cell lung cancer is widely accepted and routinely used in selecting targeted therapy , because response rates ( rrs ) to single targeted agents are twoto three - fold higher than those to cytotoxic chemotherapy.4 - 11 in advanced crc , potentially targeted alterations are mostly detected in the braf gene ; however , the rr in crc is typically worse than in nonsmall - cell lung cancer ( rr range in crc , 10% to 15% )  . 
 in addition , in crc , expanded testing of the kras and nras genes is used as a negative predictor of antiepidermal growth factor receptor ( egfr ) antibody treatment.12 circulating cell - free tumor dna ( ctdna ) sequencing is a blood - based test , also known as liquid biopsy , which is currently widely used for tumor genomic profiling.13 in contrast to tissue biopsy , ctdna sequencing is a noninvasive approach and thus especially valuable where the tumor tissue obtained is insufficient for molecular testing . 
most importantly , liquid biopsy may provide a global summary of intraand intertumor heterogeneity.14 guardant 360 ( guardant health , redwood city , ca ) is a liquid biopsy test based on digital sequencing technology . 
 the clinical sensitivity and specificity of this test are comparable to those of tissue - based next - generation sequencing ( ngs ; analytic specificity of > 99.9999% at the per - nucleotide level ) .15 absolute variant allele fractions , including egfr and braf single - nucleotide variants , have been shown to tightly correlate with a well - validated orthogonal digital droplet polymerase chain reactionbased plasma analysis.16 we present a case of a patient with synchronous lung and colorectal cancers driven by two different oncogenic drivers ( egfr l858r and braf v600e for the lung and colorectal tumors , respectively )  . 
whereas the incidence of the abovementioned molecular aberrations in these tumor types ranges from 10% to 20% , 17 - 19 it is rare to discover both in the same individual . 
biopsy of the colonic mass confirmed a cdx2 - positive adenocarcinoma compatible with primary colonic orig positron emission tomographycomputed tomography scan showed the abovementioned tumor in the right colon and also demonstrated metastatic disease in the bones and a speculated right upper lobe ( rul ) lesion with an atelectatic component compatible with a second primary lung tumor ( fig 1a )  . 
a biopsy of both the rul lesion and the t9 lytic mass confirmed the diagnosis of metastatic lung adenocarcinoma in both tumor specimens ; tumor cells were positive for ttf1 and ck7 and negative for cdx2 and ck20 . because the rul lesion and the vertebral metastasis had insufficient tumor tissue for elizabeth dudnik tal twito iris faull addie dvir lior soussan - gutman ofer purim richard b . 
 ( a ) positron emission tomographycomputed tomography scan demonstrating metastatic disease in the spiculated right upper lobe ( rul ) lesion with an atelectatic component compatible with a second primary lung tumor . 
targeted ngs of the ctdna identified two driver mutations : egfr l858r at 2.4% mutant allele fraction ( maf ) and braf v600e mutation at 0.9% maf ( fig 2a )  . 
after the ctdna analysis , the tumor specimen obtained from the bone lesion was reviewed , and polymerase chain reaction analysis for egfr ( amoydx egfr 29 mutations detection ce - ivd kit ; xiamen , peoples republic of china ) was performed , confirming the presence of egfr l858r mutation in the bone metastasis . 
the origin of the braf v600e clone at that point remained unclear . when the patient was about to start treatment with anti - egfr therapy , she was diagnosed with acute large - bowel obstruction and underwent extended right hemicolectomy , which revealed moderately differentiated colonic adenocarcinoma infiltrating the pericolic fat with three involved regional lymph nodes and positive surgical margins ( pt3n2r1 )  . 
soon thereafter , the patient developed a large pericardial effusion , and pericardial window was performed , revealing no viable tumor in the pericardial tissue specimen . erlotinib was initiated , resulting in a significant shrinkage of the rul mass ; however , new liver metastases developed ( fig 1b )  . 
this time , braf testing as well as kras and nras testing was performed ( amoydx ce - ivd kit ) , revealing that the tumor was kras wild type and nras wild type and harbored a braf v600e mutation . after the diagnosis of metastatic crc , in march 2016 , capecitabine was added to erlotinib . 
although not known as driver mutations , these mutations are more common in crc than in lung cancer , and their mafs increased from the initial test to that performed after the patient received egfr inhibitors ( fig 2b )  . after the second ctdna testing , the treatment was switched to a combination of vemurafenib and erlotinib.20 - 22 however , the treatment was poorly tolerated ( severe diarrhea ) and was therefore discontinued after 1 month . 
previous genetic studies of crc have identified somatic mutations in four main candidate pathways : tgf - , wnt , p53 , and rtk - ras , all of which were identified as important contributors to crc development.29 among them , mutations in the smad4 , apc , fbxw7 , and braf genes were identified as recurrent mutations in patients with crc.29 - 31 in contrast , apc was suggested to be a gatekeeper , which regulates epithelial cells to develop into adenoma and even carcinoma cells.32 notably , not only did liquid biopsy detect both tumor driver mutations at the time of initial diagnosis , it also reflected the progression of the second primary colon tumor , early in the course of the disease , even before its growth was shown in imaging . 
a rise in the maf of braf v600e , a driver mutation , as well as other crc - associated mutations ( smad4 , apc , fbxw7 ) , alongside a drop in the maf of the egfr l858r mutation , suggested that the new retroperitoneal lesions were related to the crc . 
this finding is consistent with other reports showing how ctdna allele fractions serve as early indicators of response versus progression in earlyand late - stage crc.33 , 34 later on , liquid biopsy allowed simultaneous response monitoring of both tumors . 
however , it should be emphasized that for clinical application of monitoring with serial testing , the assay used must be validated not only for detecting mutations but also for accurate quantitation of variant allele fractions . 
our patient did not benefit from the braf inhibitor , possibly because single - agent braf inhibitor yields poor results in patients with crc with braf mutations.12 therefore , inhibition of multiple pathways and immunotherapy should have been considered.35 although tumor mutational burden was not assessed here , analysis of blood - derived ctdna demonstrated high mutation in november 2015 as well as in june 2016 ( fig 2 )  . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . elizabeth dudnik consulting or advisory role : boehringer ingelheim speakers bureau : boehringer ingelheim , roche , astrazeneca , pfizer , merck sharp & dohme , bristol - myers squibb , novartis research funding : boehringer ingelheim ofer purim no relationship to disclose richard b . 
lanman employment : guardant health , veracyte leadership : guardant health , biolase stock and other ownership interests : guardant health , biolase research funding : guardant health affiliations elizabeth dudnik and ofer purim , davidoff cancer center , rabin medical center , petach tikva ; tal twito , addie dvir , and lior soussan - gutman , teva pharmaceuticals industries , shoam , israel ; and iris faull and richard b . 
yun hr , yi lj , cho yk , et al : double primary malignancy in colorectal cancer patients : msi is the useful marker for predicting double primary tumors . 
chiang jm , yeh cy , changehien cr , et al : clinical features of second other - site primary cancers among sporadic colorectal cancer patients : a hospital - based study of 3 , 722 cases . 
zhou c , wu yl , chen g , et al : erlotinib versus chemotherapy as first - line treatment for patients with advanced egfr mutation - positive non - small - cell lung cancer ( optimal , ctong - 0802 ) : a multicentre , open - label , randomised , phase 3 study . 
sequist lv , yang jc , yamamoto n , et al : phase iii study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with egfr mutations . 
mazires j , barlesi f , filleron t , et al : lung cancer patients with her2 mutations treated with chemotherapy and her2 - targeted drugs : results from the european euher2 cohort . 
paik pk , drilon a , fan pd , et al : response to met inhibitors in patients with stage iv lung adenocarcinomas harboring met mutations causing exon 14 skipping . 
sarfaty m , moore a , neiman v , et al : ret fusion lung carcinoma : response to therapy and clinical features in a case series of 14 patients . 
prez - callejo d , romero a , provencio m , et al : liquid biopsy based biomarkers in non - small cell lung cancer for diagnosis and treatment monitoring . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
ulrich bc , nagy rj , odegaard ji , et al : cross - platform detection and quantification of actionable mutations in cell - free dna shows high concordance and correlation between next - generation sequencing and droplet digital pcr . 
pao w , miller va : epidermal growth factor receptor mutations , small - molecule kinase inhibitors , and non - small - cell lung cancer : current knowledge and future directions . 
sanz - garcia e , martinez am , elez e , et al : survival determinants with matched targeted therapies in braf mutant metastatic colorectal cancer ( mcrc )  . 
barlesi f , mazieres j , merlio jp , et al : routine molecular profiling of patients with advanced nonsmall - cell lung cancer : results of a 1 - year nationwide programme of the french cooperative thoracic intergroup ( ifct )  . 
ohashi k , sequist lv , arcila me , et al : lung cancers with acquired resistance to egfr inhibitors occasionally harbor braf gene mutations but lack mutations in kras , nras , or mek1 . 
blakely cm , watkins tbk , wu w , et al : evolution and clinical impact of co - occurring genetic alterations in advanced - stage egfr - mutant lung cancers . 
kim st , lee ws , lanman rb , et al : prospective blinded study of somatic mutation detection in cell - free dna utilizing a targeted 54 - gene next generation sequencing panel in metastatic solid tumor patients . 
tie j , wang y , tomasetti c , et al : circulating tumor dna analysis detects minimal residual disease and predicts recurrence in patients with stage ii colon cancer . 
 role for nucleotide excision repair gene variants in oxaliplatin - induced peripheral neuropathy purpose oxaliplatin forms part of routine treatment of advanced colorectal cancer ; however , it often causes severe peripheral neuropathy , resulting in treatment discontinuation . 
we sought to determine the molecular and cellular mechanism underlying this toxicity . patients and methods we exome resequenced blood dna samples from nine patients with advanced colorectal cancer who had severe peripheral neuropathy associated with oxaliplatin ( pnao ) within 12 weeks of treatment . 
we tested the functionality of ercc4 variants using viability and dna repair assays in schizosaccharomyces pombe and human cell lines after exposure to oxaliplatin and ultraviolet light . results exome resequencing identified one patient carrying a novel germline truncating mutation in the nucleotide excision repair ( ner ) gene ercc4 . 
we subsequently found that multiple rare ercc4 nonsynonymous variants were over - represented in affected individuals ( p = 7.7 103 ) and three of these were defective in the repair of ultraviolet lightinduced dna damage ( p < 1 103 )  . 
2018 by american society of clinical oncology introduction oxaliplatin , a third - generation platinum drug , in combination with fluorouracil and leucovorin or oral capecitabine is standard treatment of locally advanced and metastatic colorectal cancer ( crc ) ; it improves both response and progression - free survival.1 , 2 it also improves disease - free survival in the adjuvant treatment of patients with stage ii and iii colon cancer.3 in addition , oxaliplatin is widely used to treat other gi malignancies . 
platinum agents exert their effects by forming interand intrastrand dna cross - links , 4 which stall the cell cycle , inhibit dna synthesis , 5 and trigger apoptosis.6 oxaliplatin also induces oxidative dna damage.7 peripheral neuropathy is a well - recognized dose limiting toxicity of oxaliplatin.8 , 9 high cumulative doses of oxaliplatin are associated with chronic peripheral nerve damage causing sensory ataxia and functional impairment.10 chronic sensory neuropathy has been observed in approximately half of patients who received oxaliplatin with infusional fluorouracil and leucovorin.11 importantly , it is neurotoxicity , rather than tumor progression , that is often the cause of treatment discontinuation.12 because neurotoxicity is not correlated with response , 12 it is considered a potentially avoidable adverse effect . 
cheadle author affiliations and support information ( if applicable ) appear at the end of this article . none of the sponsors played a role in the study design or in the collection , analysis , and interpretation of the data . corresponding author : jeremy p . 
 although numerous genetic associations with peripheral neuropathy have been proposed ( gstp1 , 16 - 19 agxt , 20 ercc1 , 21 , 22 fars2 , 22 tac1 , 22 scn10a , 23 scn4a , 23 vac14 , 24 and several genome - wide associated loci25 , 26 ) , none have been independently validated and introduced into patient stratification . 
patients with grade 3 or 4 peripheral neuropathy or who had had an oxaliplatin dose reduction as a result of severe peripheral neuropathy were classified as suffering from peripheral neuropathy associated with oxaliplatin ( pnao )  . 
patients with grade 2 peripheral neuropathy were excluded to allow a better discrimination between the two patient groups . molecular analyses we excluded known inherited neuropathies by carrying out multiplex ligation - dependent probe amplification analysis of pmp22 ( approximately 75% of patients with charcot - marie - tooth syndrome , the most common form of inherited neuropathy , have a 1.4 - mb duplication ) and by examining the exome resequencing data for pmp22 and 65 other genes associated with rare inherited neuropathies ( appendix )  . library fragments containing exomic dna were collected using the roche nimblegen seqcap ez exome library ( roche , basel , switzerland ) solution - based method . 
on average , across the exome , we had 55% ( range , 46% to 60% ) coverage of the open reading frame ( orf ) at 20 - fold depth . 
fastq files were processed through a sequence analysis pipeline using burrows - wheeler aligner software for sequence alignment and modules from the broad institutes genome analysis toolkit ( cambridge , ma ) to recalibrate quality scores , refine alignments around potential insertions or deletions ( indels ) , eliminate duplicate reads , call indel and single nucleotide polymorphism ( snp ) genotypes , generate quality control metrics , and apply quality filters to the genotype calls . 
ercc4 and ercc6 nonsynonymous variants were genotyped using kaspar ( lgc , teddington , united kingdom ) or beadarray ( illumina ) technologies ( appendix )  . functional analyses production of a schizosaccharomyces pombe rad16 base strain and the rad16 wild - type vector , cre recombinase - mediated cassette exchange , transformation of the base strain , site - directed mutagenesis , and treatment with oxaliplatin and ultraviolet light were carried out as described in the appendix . 
four hundred eighty epsteinbarr virustransformed human lymphoblastoid cell lines established from healthy white individuals ( european collection of authenticated cell cultures , salisbury , united kingdom ) were assayed for the ercc4 variants pro379ser , arg576thr , and glu875gly using kaspar . 
pombe were normalized to wild type and analyzed using spss v.23 ( spss , chicago , il ) analysis of variance ( anova ) with dunnett correction ( after transformation using the arcsine function )  . 
linkage disequilibrium was obtained using haploview v.4.2 ( broad institute )  . results of the 2 , 445 patients with advanced crc in the coin trial , 23% of patients who received oxaliplatin and fluorouracilbased therapy and 16% of patients who received oxaliplatin and capecitabinebased therapy had severe ( grade 3 ) peripheral neuropathy over the course of the trial.27 we focused on patients with severe pnao within the first 12 weeks of treatment ( appendix and table 1 ) as a potentially enriched group for causal germline mutations ( 63 patients )  . 
 although fewer of these patients responded to treatment at 12 weeks ( 47% , 26 of 55 patients with response data ; table 1 ) compared with patients without pnao ( grade 1 , n = 1 , 763 ; 57% , 884 of 1 , 542 patients with response data ) , this was not statistically significant ( p = 1.4 x 10 - 1 )  . nine of the 63 patients with severe pnao had exome resequencing of their germline blood dna samples . 
we identified , on average , 48 stop gains ( range , 40 to 56 stop gains ) and 88 indels ( range , 73 to 111 indels ) predicted to result in frameshift mutations , per patient exome ( appendix table a1 )  . 
we excluded known inherited neuropathies in these patients by pmp22 dosage analysis and by examining the resequencing data for 66 candidate genes ( no stop gains or truncating indels were predicted )  . novel truncating mutation in ercc4 variants not present in dbsnp v.132 ( assigned as novel ; national center for biotechnology information , bethesda , md ) were considered most likely to cause pnao ; we identified on average eight novel stop gains ( range , two to 11 stop gains ) and 28 novel frameshifting indels ( range , 16 to 57 indels ) per patient ( appendix table a1 )  . 
 we also considered that germline truncating mutations in genes involved in oxaliplatin transport , metabolism , or the repair of its associated damage might be responsible for pnao ; we identified 104 such genes from literature reviews ( appendix )  . 
all nine patients carried truncating variants in these selected genes ( range , one to four variants ) ; however , only one of these variants , in a single patient , was novel ( appendix table a1 )  . 
patient 8 carried the novel stop gain ser613x in the nucleotide excision repair ( ner ) gene ercc4 , which was confirmed by sanger sequencing of an independent pcr product ( appendix table a1 )  . 
we did not find any other coding region variants in the second ercc4 allele in patient 8 after direct sequence analysis of the patients entire orf and flanking intronic sequences . 
 functional analysis of the ercc4 stop mutation we investigated whether the ercc4 nonsense mutation induced sensitivity to oxaliplatin and ultraviolet light ( which causes cpds that are repaired by ner )  . 
after oxaliplatin treatment , we observed decreased survival for rad16 - ser585x ( p = 3.5 102 ) in comparison with wild - type rad16 ( rad16 + ) and , in a similar range with a control rad16 - deleted mutant ( rad16 ; fig 1a )  . 
 similarly , we observed decreased survival of uve1 - rad16 - ser585x after treatment with ultraviolet light ( p < 1 103 ; fig 1b )  . multiple rare ercc4 variants associated with peripheral neuropathy we sought further evidence for a role of ercc4 in pnao and sanger sequenced the ercc4 orf and flanking intronic sequences in all 63 patients with pnao . 
we did not find any additional stop gains or truncating indels ; however , we did identify four rare ( minor allele frequencies < 5% in dbsnp ) nonsynonymous variants ( pro379ser , rs1799802 in three patients ; his466gln , rs372950439 in one patient ; arg576thr , rs1800068 in one patient ; and glu875gly , rs1800124 in four patients ; table 1 )  . 
the ercc4 nonsense mutation induced sensitivity to oxaliplatin and ultraviolet ( uv ) light in a schizosaccharomyces pombe ( rad16 ) model syste average percent survival from ( a ) four independent experiments after oxaliplatin treatment or ( b ) three independent experiments after treatment with uv light for a control rad16 gene deletion mutant ( rad16 ) and rad16 - ser585x ( mimics ser613x seen in a patient with peripheral neuropathy associated with oxaliplatin ) , normalized to rad16 +  . 
 ser613x was not included in the total because it was only assayed in patients with pnao ; one patient with pnao carried arg576thr and glu875gly , and another without pnao carried pro379ser and glu875gly . 
values reflect the number of patients successfully genotyped ( totals for all variants )  . functional analysis of ercc4 nonsynonymous variants we sought evidence for causal effects of pro379ser , arg576thr , and glu875gly using epstein - barr virustransformed human lymphoblastoid cell lines established from healthy individuals who carried each variant in a heterozygous state ( n = 3 for each variant and wild - type controls )  . 
although treatment with ultraviolet light reduced viability in all lines , we did not observe any differences between wild - type and variant cell lines ( data not shown )  . 
in terms of repair capacity after dna damage with ultraviolet light , all wild - type cell lines showed noticeable repair 24 hours after treatment , with the majority of cpds being repaired by 48 hours ( fig 2 )  . 
in contrast , all three sets of variant cell lines displayed reduced repair in the initial ( p < 1 103 at both 24 and 48 hours ) and validation ( p < 1 103 at both 24 and 48 hours ) experiments ( fig 2 )  . rare ( table 1 ) and five common nonsynonymous variants , two synonymous variants , and one 5utr variant ; we genotyped nonsynonymous variants in all available patients . 
seven rare nonsynonymous variants were predicted to be damaging ( c55 - c65 ) , three of which ( asp425ala , pro694leu , and ser797cys ) were individually over - represented in patients with pnao ( table 3 )  . 
 combined , rare ercc6 nonsynonymous variants were highly associated with peripheral neuropathy ( present in 20.6% of patients [ 13 of 63 patients ] with pnao v 4.7% of patients [ 82 of 1 , 749 patients ] without pnao ; p = 2.4 108 ; table 3 )  . no common ercc6 nonsynonymous variants were associated with pnao . 
we identified nine combined analyses of ercc4 and ercc6 because private variants may skew statistical associations , we considered only rare ercc4 and ercc6 nonsynonymous variants that were present in two or more patients with pnao . 
 average cyclobutane pyrimidine dimer ( cpd ) concentrations ( in nanograms per milliliter ) after ultraviolet c ( 70 j ) irradiation in wild - type cells and cells carrying ( a ) pro379ser , ( b ) arg576thr , and ( c ) glu875gly over a 48 - hour period , showing the initial ( top panels ) and validation ( lower panels ) experiments . 
cpd concentrations are plotted as an average of two duplicate samples from the same experiment run on separate enzyme - linked immunosorbent assay plates , which are shown against time with se bars ( data from the wild - type cells are shown in a , b , and c )  . 
 1 - 3 , different cell lines ; c , wild - type control cell line ; v , variant cell line . pro379ser initial experiment arg576thr glu875gly untreated ( c1 - 3 , v1 - 3 ) untreated ( c1 - 3 , v1 - 3 ) untreated ( c1 - 3 , v1 - 3 ) time ( hours ) time ( hours ) time ( hours ) validation experiment c1 - 3 c1 - 3 v1 - 3 c1 - 3 v1 - 3 c1 - 3 untreated ( c1 - 3 , v1 - 3 ) untreated ( c1 - 3 , v1 - 3 ) untreated ( c1 - 3 , v1 - 3 ) time ( hours ) time ( hours ) time ( hours ) c1 - 3 v1 - 3 c1 - 3 discussion the rare variant hypothesis predicts that individually rare but collectively common inherited variants play a significant role in disease susceptibility.32 for example , rare nonsynonymous variants in the genes encoding apolipoprotein a1 , the atp binding cassette transporter a1 and lecithin cholesterol acyltransferase , are over - represented in individuals with low plasma levels of high - density lipoprotein cholesterol , a major risk factor for coronary atherosclerosis.33 furthermore , multiple rare nonsynonymous variants in apc play a significant role in inherited predisposition to colorectal adenomas.34 here , after identifying a novel ercc4 truncating mutation in a patient with pnao , we found that multiple rare ercc4 and ercc6 nonsynonymous variants were over - represented in affected individuals . 
two rare variants in dpyd have been associated with severe toxicity in patients receiving fluorouracil , 44 and a polymorphism in ugt - 1a has been linked to a higher risk of developing irinotecan - associated neutropenia and diarrhea45 ; however , none of these biomarkers have been introduced into routine clinical practice because of their poor sensitivity and specificity . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . hannah west no relationship to disclose michelle coffey no relationship to disclose michael j . 
adams honoraria : merck serono , servier , bristol - myers squibb , amgen consulting or advisory role : merck serono , amgen , servier speakers ' bureau : merck serono travel , accommodations , expenses : servier , amgen , merck serono oliver fleck no relationship to disclose timothy s . 
cheadle patents , royalties , other intellectual property : hold ipr and patents on mutyh variants and have received royalties from myriad acknowledgment we thank simon reed and edgar hartsuiker for helpful advice and shelley idziaszczyk , rebecca williams , marc naven , and christopher smith for technical support . affiliations hannah west , michelle coffey , james p . 
mcleod , debartolo family personalized medicine institute , moffitt cancer center , tampa , fl . supported by tenovus , the kidani trust , a cancer research uk development award from the cardiff cancer research uk centre , cancer research wales , the wales gene park , a knowledge economy skills scholarship , north west cancer research , and the wales assembly government national institute of social care and health research cancer genetics biomedical research unit . 
goldberg rm , sargent dj , morton rf , et al : a randomized controlled trial of fluorouracil plus leucovorin , irinotecan , and oxaliplatin combinations in patients with previously untreated metastatic colorectal cancer . 
johnson np , hoeschele jd , rahn ro , et al : mutagenicity , cytotoxicity , and dna binding of platinum ( ii ) - chloroammines in chinese hamster ovary cells . 
afzal s , jensen sa , srensen jb , et al : oxidative damage to guanine nucleosides following combination chemotherapy with 5 - fluorouracil and oxaliplatcancer chemother pharmacol 69 : 301 - 307 , 2012 8 . 
di cesare mannelli l , zanardelli m , failli p , et al : oxaliplatin - induced oxidative stress in nervous system - derived cellular models : could it correlate with in vivo neuropathy ? free radic biol med 61 : 143 - 150 , 2013 14 . 
grothey a , mcleod hl , green em , et al : glutathione s - transferase p1 i105v ( gstp1 i105v ) polymorphism is associated with early onset of oxaliplatin - induced neurotoxicity . 
peng z , wang q , gao j , et al : association between gstp1 ile105val polymorphism and oxaliplatin - induced neuropathy : a systematic review and meta - analysis . 
lecomte t , landi b , beaune p , et al : glutathione s - transferase p1 polymorphism ( ile105val ) predicts cumulative neuropathy in patients receiving oxaliplatin - based chemotherapy . 
argyriou aa , cavaletti g , antonacopoulou a , et al : voltage - gated sodium channel polymorphisms play a pivotal role in the development of oxaliplatin - induced peripheral neurotoxicity : results from a prospective multicenter study . 
hertz dl , owzar k , lessans s , et al : pharmacogenetic discovery in calgb ( alliance ) 90401 and mechanistic validation of a vac14 polymorphism that increases risk of docetaxel - induced neuropathy . 
dolan me , el charif o , wheeler he , et al : clinical and genome - wide analysis of cisplatininduced peripheral neuropathy in survivors of adult - onset cancer . 
preston tj , henderson jt , mccallum gp , et al : base excision repair of reactive oxygen speciesinitiated 7 , 8 - dihydro - 8 - oxo - 2 - deoxyguanosine inhibits the cytotoxicity of platinum anticancer drugs . 
broyl a , corthals sl , jongen jl , et al : mechanisms of peripheral neuropathy associated with bortezomib and vincristine in patients with newly diagnosed multiple myeloma : a prospective analysis of data from the hovon - 65 / gmmg - hd4 trial . 
reardon jt , vaisman a , chaney sg , et al : efficient nucleotide excision repair of cisplatin , oxaliplatin , and bis - aceto - ammine - dichloro - cyclohexylamine - platinum ( iv ) ( jm216 ) platinum intrastrand dna diadducts . 
troelstra c , van gool a , de wit j , et al : ercc6 , a member of a subfamily of putative helicases , is involved in cockaynes syndrome and preferential repair of active genes . 
fousteri m , vermeulen w , van zeeland aa , et al : cockayne syndrome a and b proteins differentially regulate recruitment of chromatin remodeling and repair factors to stalled rna polymerase ii in vivo . 
schwab m , zanger um , marx c , et al : role of genetic and nongenetic factors for fluorouracil treatment - related severe toxicity : a prospective clinical trial by the german 5 - fu toxicity study group . 
 appendix clinical assessment clinical assessment of patients from the coin trial occurred every 3 weeks , and toxicity data were recorded on clinical research forms at weeks 0 , 6 , 12 , and 24 . 
a quality - of - life questionnaire ( european organisation for research and treatment of cancer quality of life questionnaire c30 ) was also completed at 0 , 6 , 12 , and 24 weeks . 
patients with severe peripheral neuropathy had a minimum of grade 3 toxicity within the first 12 weeks of treatment , which was backed up by quality - of - life data . molecular analyses and exome resequencing using the exome resequencing data , the following genes associated with rare inherited neuropathies were analyzed : pmp22 , aars , aifm1 , atl1 , atp7a , bicd2 , bscl2 , cct5 , ctdp1 , dctn1 , dhtkd1 , dnajb2 , dnm2 , dnmt1 , dync1h1 , egr2 , fam134b , fbln5 , fgd4 , gars , gdap1 , gjb1 , gnb4 , hars , hint1 , hk1 , hspb1 , hspb3 , hspb8 , ighmbp2 , ikbkap , kars , kif1a , kif5a , litaf , lmna , lrsam1 , mars , med25 , mfn2 , mpz , mtmr13 , mtmr2 , myh14 , ndrg1 , ngfb , ntrk1 , pdk3 , plekhg5 , prnp , prps1 , prx , rab7 , reep1 , sbf1 , scn9a , setx , sh3tc2 , slc5a7 , sptlc1 , sptlc2 , tfg , trim2 , trpv4 , wnk1 , and yars . genes potentially involved in the pharmacokinetics and mechanism of action of platinum compounds were identified from literature reviews . 
four genes were involved in drug influx ( oct1 , oct2 , ctr1 , and hmate1 ) , three in trafficking ( ccs , cox17 , and sod1 ) , seven in detoxification ( mt1a , mt2a , nqo1 , gstt1 , gstp1 , gstm1 , and mpo ) , two in oxalate metabolism ( agxt and grhpr ) , three in sequestration ( atp7a , atp7b , and hah1 ) , 32 in dna damage response and subsequent signaling pathways ( spt16 , ssrp1 , hmgb1 , rag1 , rag2 , abl1 , rb1 , p53 , p73 , aurka , ccng2 , p38mapk , msk1 , mkk3 , mkk6 , histone h3 , erk , mek1 , mek2 , jnk , mkk4 , mkk7 , mpk1 , akt , nf - kb , xiap , bax , apaf1 , cyc , casp3 , casp6 , and casp9 ) , 46 in dna damage repair and the associated response pathways ( polb , polh , polm , rev3l , fanca , fancb , fancc , fancd2 , fance , fancf , fancg , fanci , fancl , fancm , fancn , faap100 , rm1 , fan1 , mlh1 , msh2 , msh6 , pms2 , atm , atr , chek1 , chek2 , brca1 , brca2 , gadd45 , ddb2 , cdc25c , cdc2 , csa , hr23b , rnapolii , rpa1 , ercc1 , ercc2 , ercc3 , ercc4 , ercc5 , ercc6 , xpa , xrcc1 , xrcc3 , and mgmt ) , and seven in drug efflux ( abcc1 , abcc2 , abcc3 , abcc4 , abcc5 , abcb1 , and abcg2 )  . polymerase chain reaction and sanger sequencing thermal cycling conditions consisted of an initial denaturation at 94c for 10 minutes , followed by 32 cycles of 94c for 30 seconds , 57c for 30 seconds , and 72c for 30 seconds , followed by a final elongation stage of 72c for 10 minutes . 
 polymerase chain reaction ( pcr ) products were purified using exosap , in which 15 l of pcr product was incubated with 2 u of exonuclease i ( new england biolabs , ipswich , ma ) and 2 u of shrimp alkaline phosphatase ( ge healthcare , chicago , il )  . 
samples were run on an abi 3100 genetic analyzer ( applied biosystems ) , and sequence data were viewed using sequencher v4.6 ( gene codes , ann arbor , mi )  . single nucleotide polymorphism genotyping genotyping of arg415gln ( rs1800067 ) in ercc4 and gly399asp ( rs2228528 ) , arg1213gly ( rs2228527 ) , and gln1413arg ( rs2228529 ) in ercc6 was carried out using illuminas fast - track genotyping service using their high - throughput beadarray technology ( illumina , san diego , ca )  . 
the overall genotyping success rate was 99.9% ( 8 , 731 of 8 , 744 genotypes were called successfully ) , and the concordance rate for previously sequenced samples ( n = 63 ) was 100% ( 252 of 252 genotypes were concordant )  . 
genotyping of pro379ser ( rs1799802 ) , arg576thr ( rs1800068 ) , his466gln ( rs372950439 ) , glu875gly ( rs1800124 ) , and rs1799800 in ercc4 and of asp425ala ( rs4253046 ) , gly446asp ( rs4253047 ) , pro694leu ( rs114852424 ) , ser797cys ( rs146043988 ) , gly929arg ( novel ) , phe1217cys ( rs61760166 ) , arg1230pro ( rs4253211 ) , ala1296thr ( rs139509516 ) , thr1441ile ( rs4253230 ) , and phe1437ile ( rs758679804 ) in ercc6 was carried out by lgc ( teddington , united kingdom ) using kaspar technology . 
the overall genotyping success rate was 97.5% ( 33 , 415 of 34 , 320 genotypes were called successfully ) , and the concordance rate for previously sequenced samples ( n = 63 ) was 99.9% ( 881 of 882 genotypes were concordant )  . 
in addition , these primers carried sequences corresponding to regions upstream and downstream of ura4 in the plasmid paw1 ( shown in lowercase ; forward : 5 - tccatccaaattggaaaattttcgcatcaaagtatttaacagctttcagaaatcaaaattgcaaattggaaaatctctacgaataacaccaccattaaatcggatccccgggttaattaa - 3 ; reverse : 5 - ttattaattaggtgcgcttaacattctatatatggtgaaccaatatatatcagatgtagaagcaaaaattaaatatattacaaaattataaaaaaataaagaattcgagctcgtttaaac - 3 )  . 
amplification of ura4 in paw1 ( linearized with acci ; new england biolabs ) was achieved by pcr to produce a 2.1 - kb product consisting of ura4 flanked by loxp / loxm sites ( loxp - ura4 - loxm ) and rad16 gene sequences , as previously described ( watson at , et al : gene 407 : 63 - 74 , 2008 )  . 
transformants were grown on minimal medium lacking uracil and restreaked on plates containing the same mediua replica plate was irradiated with 150 j / m2 of ultraviolet light to identify those with ultraviolet sensitivity , a characteristic of rad16 loss . 
extracted dna was sequenced at 5 and 3 lox sites to ensure their integrity . rad16 wild - type pcr product with flanking lox sites . the creation of a rad16 wild - type pcr product with flanking lox sites ( loxp - rad16 - loxm3 ) was performed using primers designed with lox sites incorporated ( forward : 5 - gggataacttcgtatagcatacattatacgaagttatatgcatcatcatcatcatcatggaggaggagaaacaaaggttcatttgcc - 3 ; reverse : 5 - gggataacttcgtatataataccatatacgaagttatttactcatagtcctttaactgttttcgg - 3 )  . 
pombe dna was carried out using phusion polymerase ( new england biolabs ) , with thermal cycling conditions consisting of an initial denaturation of 98c for 2 minutes , followed by 30 cycles of 98c for 30 seconds , 47c for 30 seconds , and 72c for 3 minutes and 30 seconds , with a final elongation of 72c for 10 minutes . 
pcr products were cleaned using a nucleospin gel and pcr clean - up kit ( macherey - nagel , bethlehem , pa )  . cre recombinase - mediated cassette exchange . cre recombinase - mediated cassette exchange was carried out using a paw8 - ccdb plasmid and the loxp - rad16 - loxm3 pcr product ; 170 ng of paw8 - ccdb , 90 ng of loxp - rad16 - loxm3 pcr product , and 2 u of cre recombinase ( new england biolab ) were incubated at 37c for 30 minutes , followed by 70c for 10 minutes . 
 aliquots of the cultures were plated on yea plates with added 5 - fluoroorotic acid ( 1 g / l ) to allow for growth of ura4 cells , which were streaked on master plates . 
functionality of the strains transformed with the wild - type rad16 was performed to determine any potential detrimental effects associated with the incorporation of the lox sites flanking the rad16 region . 
thermocycling conditions consisted of an initial denaturation at 95c for 1 minute , followed by 18 cycles of 95c for 50 seconds , 60c for 50 seconds , and 68c for 6 minutes , and a final elongation at 68c for 7 minutes . 
strains were mixed on sporulation medium ( malt extract agar ) , incubated for 2 days at 30c , and treated for 30 minutes with 30% ethanol to kill vegetative cells . 
the resulting strains were ol2123 ( smt - 0 loxp - rad16 + - loxm uve1 : : leu2 leu1 - 32 ura4 - d18 ) and ol2131 ( smt - 0 loxp - rad16 - s585x - loxm uve1 : : leu2 leu1 - 32 ura4 - d18 )  . drug and ultraviolet light treatments . a primary culture of cells was grown overnight in yel with shaking at 30c ; 107 cells were cultured in yel with and without 1 mm of oxaliplatfor the untreated cultures , an equivalent volume of dimethyl sulfoxide was added . 
 for ultraviolet light treatment , appropriate numbers of cells were irradiated with ultraviolet light using a stratalinker ( agilent ; 5 j / m2 for ol2116 [ rad16 uve1 ] and ol2131 [ rad16 - s585x uve1 ] and 20 j / m2 for ol2112 [ uve1 ) ]  . 
plates were incubated at 30c for 4 days , and survival rates were determined by counting colonies of treated compared with untreated plates . functional studies in human cells survival analyses . before survival analyses , 3 106 cells / ml were serum starved for 1 hour before treatment with ultraviolet light ( 40 jm2s1 ) irradiation . 
cells were resuspended in normal tissue culture media containing fetal bovine serum and antibiotics , and aliquots were removed at 0 , 4 , 24 , and 48 hours after treatment for analysis . 
dna samples were probed for cyclobutane pyrimidine dimers ( cpds ) using an enzyme - linked immunosorbent assay ( elisa ) kit according to the manufacturers instructions ( cell biolabs , san diego , ca )  . 
briefly , dna samples and a dna standard conjugated to a known amount of cpds were converted to single - stranded dna by incubation at 96c for 10 minutes followed by rapid chilling on ice for 10 minutes . 
wells were washed twice with cold phosphate - buffered saline , followed by hour - long incubations at room temperature with assay diluent , primary cpd antibody , blocking reagent , and secondary horseradish peroxidaseconjugated antibody , with wash steps between each incubation . 
 biological validation of rna sequencing data from formalinfixed paraffin - embedded primary melanomas purpose initiatives such as the cancer genome atlas and international cancer genome consortium have generated high - quality , multiplatform molecular data from thousands of frozen tumor samples . 
although these initiatives have provided invaluable insight into cancer biology , a tremendous potential resource remains largely untapped in formalin - fixed , paraffin - embedded ( ffpe ) samples that are more readily available but which can present technical challenges because of crosslinking of fragile molecules such as rna . materials and methods we extracted rna from ffpe primary melanomas and assessed two gene expression platformsgenome - wide rna sequencing and targeted nanostringfor their ability to generate coherent biologic signals . 
we also make comparisons to the cancer genome atlas and other publicly available melanoma datasets . results the comparison of the gene expression patterns to each other , to established biologic modules , and to clinical and immunohistochemical data confirmed the fidelity of biologic signals from both platforms using ffpe samples to known biology . 
moreover , correlations with patient outcome data were consistent with previous frozen - tissue based studies . conclusion ffpe samples from previously difficult - to - access cancer types , such as small primary melanomas , represent a valuable and previously unexploited source of analyte for rna sequencing and nanostring platforms . 
2018 by american society of clinical oncology introduction whole transcriptome rna sequencing ( rnaseq ) has drastically improved the fidelity , resolution , and comprehensiveness of global rna assessment compared with gene expression microarray technology . 
in addition to providing higher - quality assessment , particularly of low - expressing genes , it is unbound by hybridization constraints , meaning that micrornas and noncoding rnas , as well as multiple gene isoforms , exon - exon junctions , and fusion genes can also be assessed . 
these advantages have been leveraged to great effect in largescale efforts such as the cancer genome atlas ( tcga ) 1 and international cancer genome consortium , 2 where thousands of samples have been assessed together with high confidence and accuracy . one of the limitations of implementing rna - seq technology thus far has been the requirement for analytes prepared from frozen tissues.3 formalin fixed , paraffin - embedded ( ffpe ) samples are lawrence n . 
 much more plentiful but are also more susceptible to nucleic acid fragmentation and general sample deterioration , making the extraction of well - preserved rna for sequencing a difficult challenge.4 if , however , ffpe - derived rnaseq data could be optimized and standardized , it would exponentially expand the number of biospecimens available and streamline sample processing . 
moreover , in many cancer types , limiting the analyses to only frozen tissues may result in skewing of samples toward , for example , surgically resectable and / or bulky tumors , resulting in a sample population biased by selection . 
in the melanoma tcga dataset , which used frozen samples only , primary melanomas were heavily skewed toward very thick tumors ( median tumor thickness , 11 mm ) that correspond to an american joint committee on cancer t4 primary tumor.1 , 5 , 6 in contrast , the vast majority of cutaneous melanomas diagnosed in the united states have a tumor thickness 4 mm , most of which are 1 mm , and all of which generally undergo ffpe processing for diagnostic purposes . 
tumor thickness is a well - established , independent predictor of melanoma clinical outcome and the principle primary tumor factor for treatment decision making6 ; therefore , the lack of thinner , more clinically relevant primary melanomas in the tcga dataset limits a full molecular understanding of the disease and optimal potential translation to the clinic . 
ffpe samples offer the opportunity to include tumors across all tumor thicknesses in rna - seq datasets , for which we demonstrate feasibility in this study . to date , only 10 studies have assessed the technological feasibility of transcriptome - scale rna seq from analytes prepared from human ffpe tumor tissue.7 - 17 although these studies conducted technical quality controls of the data , including direct comparison of frozen and ffpe from the same samples , 7 - 10 , 15 the question of whether the data accurately reflected the underlying biology relied mainly on detecting large differences between either normal and tumor tissues8 or between transcriptionally distinct tumor subtypes , 8 - 14 , 16 leaving open the question of fidelity for more subtle gene expression differences between tumors . 
for full patient information , see the data supplement . rna extraction the high pure mirna isolation kit ( roche , basel , switzerland ) was used to extract rna , and the zymo - spin column ( zymo research , irvine , ca ) was used to remove melanin from all samples , regardless of melanin content , for consistency . 
additional details , including rnaseq run metrics , are reported in the data supplement . pathology - specific assessment we conducted pathologic assessment to quantify the tumor surface area before rna extraction , and we used this to predict the required number of 10 m slides to yield the minimum amount of total rna per case required . 
 a sort by correlation ( ( cid : 85 ) ) to previous median median of all genes ( per sample ) median of top 20% genes ( per sample ) median of genes with > 0.6 ( per sample ) median of genes with ( > 0.6 per sample ) i . 
 ( e ) final melanocyte gene set , tcga data . defining pathway scores our iterative subsetting approach to generating pathway scores depends on the quality of the initial input gene set . 
it makes two assumptions : highly cocorrelating genes largely represent shared biologic functionality , and high - quality input gene sets contain sufficiently strong signal from true biology genes such that when the initial crude score is calculated ( fig 1a ) , the true biology signal will emerge from the noise ( figs 1b and 1c )  . 
patient disease characteristics stratified by disease recurrence status for corresponding study specimens ( n = 38 ) ( continued ) no recurrence recurrence overall cohort characteristic cd8 score of tumor center not scored ( range ) mart1 / phh3 score , median 5.5 ( 0 - 47 ) 14 ( 0 - 122 ) 8.5 ( 0 - 122 ) the basis of eventual clinical outcome ( table 1 )  . 
of 1 , 362 38 ffpe primary melanomas no recurrence ( n = 18 ) > 60 months recurrence ( n = 20 ) 3 - 18 months rna extraction rna - seq > 21 , 000 genes nanostring 1 , 432 genes nanostring v rna - seq fig 2 . 
 ( b ) spearman correlation values for all 1 , 362 genes shared by the rna - seq and nanostring datasets , as well as stratified by expression and dynamic range . 
 initially , this seemed to indicate a limited overall correlation between platforms , but we reasoned that certain genes may seem to correlate poorly for causes unrelated to cross - platform fidelity . 
genes with low absolute expression ( n = 221 genes , 16% of total ) showed poor correlation across platforms , suggesting the existence of a limit of resolution ( fig 2b )  . 
the best interplatform - correlating genes had a significantly higher mad / m compared with the poorest interplatform correlating genes ( p < .001 ; figs 2c and 2d )  . 
after removing low - expressing ( < 1.0 average reads per kilobase million ) and low - dynamicrange genes ( < 0.25 mad / m ; n = 251 genes , 18% of total ; fig 2b ) , the percentage of genes with a spearman correlation value of > 0.5 improved from 68% to 84% ( 746 of 890 )  . 
for example , bub1 and top2a , both cell cyclerelated genes , are consistently coexpressed across a wide variety of datasets , including that of the current study ( appendix fig a1 )  . 
when this gene - togene cocorrelation analysis is extended to a larger scale to identify cocorrelating pathways , the overall strength of the correlations can gauge how well the underlying biology is captured by the quality of the dataset . 
we chose to use tcga melanoma data as a reference ground truth for identifying cocorrelating core pathway genes , on the basis of the high quality and quantity of data available , as well as the high degree of uniformity of the tcga pipeline.1 to simplify pathway analyses , we sought to create a pathway score for each pathway that accurately describes its relative activation state in each sample . 
one obstacle to this approach is that genes within existing defined biologic gene sets ( eg , from molecular signatures database [ msigdb ] ; gsea / msigdb ) 19 are not necessarily all cocorrelated with one another , potentially leading to a noisy score . 
therefore , we developed a method that repetitively iterates the score calculation on the basis of gene subsetting , until the score converges on a subset of highly cocorrelated genes . 
figure 1a illustrates this iterative process using the example of starting with the go_immune_system_process from msigdb to create an immune score ; a detailed description of the steps is presented in the data supplement . 
 figures 1b and 1c show examples of how the tcga immune and melanocyte scores change during each score iteration , and their final subset gene lists are shown in figures 1d and 1e . this high degree of cocorrelation of the final gene subset implies functional similarity within a broad pathway or cell type ; indeed , the final immune subset of 407 genes ( of an initial 1 , 984 ) was highly enriched for known true biology genes , including cd3 , cd8 , and cd4 genes ( fig 1d ; data supplement )  . 
of the 407 core immune genes we identified , 190 were present on both platforms and included core canonical immune genes such as cd3d / e / g , cd4 , cd8a / b , and cd86 , again retaining strong cocorrelation within each platform ( figs 3a and 3b )  . 
the majority of genes ( 154 of 190 ; 81% ) had a correlation of > 0.5 on both platforms , which further increased ( 140 of 163 ; 86% ) after removal of low - expressing genes . 
direct comparison of the final immune scores showed strong concordance between rna - seq and nanostring ( = 0.91 ) on a per - sample basis ( fig 3c )  . 
these results suggest that , after controlling for expression level , both platforms were able to successfully capture true positive immune genes while preserving biologic fidelity . we generated scores for additional signatures : cell cycle , skin , pigment , and epithelial - mesenchymal transition ( emt ; the list of starting gene sets and final gene subsets is given in the data supplement )  . 
first , we performed a hierarchical clustering of our dataset with 21 tcga tumor types using the most variable pan - cancer tcga genes and showed that our ffpe dataset clustered tightly with both cutaneous and uveal melanoma , primarily by the high expression of melanocyte - specific genes ( appendix fig a4 )  . 
finally , we examined a known pathway correlation among our five gene sets , shown in multiple cell - line studies : the emt and pigment signatures anticorrelate.21 in vivo , multiple stromal cell types express the emt signature , weakening the correlation . 
nevertheless , in the tcga dataset , among primary melanoma samples ( n = 103 ) , the emt and pigment signatures anticorrelated ( = 0.47 ; fig 3f )  . 
the cell cycle signature from rna - seq correlated well with ihc for mart1 / phh322 ( ie , cells double positive for mart1 , a melanoma marker , and phh3 , a marker of dividing cells ; fig 4a ) and the observed mitotic rate ( fig 4b )  . 
emt , epithelial - mesenchymal transition ; rna - seq , rna sequencing ; tcga , the cancer genome atlas . correlations were also detected in the nanostring dataset ( appendix fig a6 )  . 
this correlation was stronger for positive staining in the center of the tumor ( figs 4c and 4d ) compared with the periphery ( appendix fig a6 )  . we next sought to determine how pathway scores correlated with other clinical data . 
in addition , the melanocyte score negatively correlated with the chronic sun damage score ( fig 5c ) , which is a measure of the degree of solar elastosis in normal skin adjacent to the primary melanoma lesions in hematoxylin and - eosinstained tissue sections.23 finally , we performed an unbiased gene - set enrichment analysis to identify pathways that correlate with patient outcome . 
consistent with findings of previous gene expression studies of primary melanoma , 24 - 26 an increase in cell cycle gene expression was significantly enriched in samples of patients in whom melanoma eventually recurred ( fig 5d )  . 
to further substantiate the biologic fidelity of our gene - set scores , we next compared the cell cycle score between primary tumors from the recurrence versus no recurrence cohorts . 
in this study , we created an approach to assessing pathway scores that subsets gene lists by enriching for genes that together form functionally related sets of well - correlated expression patterns . 
for example , the use of unbiased whole - transcriptome rnaseq allows for robust discovery of unanticipated pathways of interest , using a vastly expanded set of archival ffpe samples . to our knowledge , our study is the first to assess the biologic fidelity of continuous differences in gene expression in ffpe - derived rna - seq data rather than bulk differences between tumor subtypes and / or normal tissue.7 - 17 it is also the first to directly compare ffpe - derived rna - seq data with nanostring data and with quantitative ihc findings . 
in the case of primary melanoma , cell cycle scores may help stratify patients with early - stage melanoma into those likely to relapse or not ; more broadly , the unbiased nature of rna - seq will enable uncovering novel biomarkers in a variety of settings . 
 we further suggest that our approach can be widely used to more accurately calculate pathway scores , because current methods process all genes in a gene set regardless of degree of cocorrelation . 
davies consulting or advisory role : glaxosmithkline , roche , novartis , sanofi , vaccinex , bristol - myers squibb , syndax , nanostring technologies research funding : glaxosmithkline ( inst ) , roche ( inst ) , astrazeneca ( inst ) , merck ( inst ) , oncothyreon ( inst ) , myriad genetics ( inst ) , sanofi alexander j . 
lazar employment : ge healthcare ( i ) leadership : beta cat pharmaceuticals , archer biosciences stock and other ownership interests : archer biosciences , beta cat pharmaceuticals honoraria : novartis , bristol - myers squibb , janssen oncology , roche consulting or advisory role : novartis , illumina , ge healthcare research funding : medimmune , astrazeneca , roche , novartis patents , royalties , other intellectual property : elsevier travel , accommodations , expenses : bristol - myers squibb , novartis jeffrey e . 
camargo cancer center , sao paulo , brazil ; and man kam kwong , hong kong polytechnic university , hong kong , china . support supported in part by the national institutes of health ( nih ) specialized program of research excellence melanoma grant no . 
 p30ca016672 ; a melanoma research alliance team science award ; generous philanthropic contributions to the university of texas md anderson melanoma moon shots program ; the dr miriam and sheldon g . 
adelson medical research foundation ; the robert and lynne grossman family foundation ; the michael and patricia booker melanoma research endowment ; aim at melanoma ; the university of texas rising stars award and the melanoma research alliance young investigator award 508743 , both to l.n.k. references 1696 , 2015 1 . 
gershenwald je , scolyer ra , hess kr , et al : melanoma staging : evidence - based changes in the american joint committee on cancer eighth edition cancer staging manual . 
eikrem o , beisland c , hjelle k , et al : transcriptome sequencing ( rnaseq ) enables utilization of formalin - fixed , paraffin - embedded biopsies with clear cell renal cell carcinoma for exploration of disease biology and biomarker development . 
jovanovi b , sheng q , seitz rs , et al : comparison of triple - negative breast cancer molecular subtyping using rna from matched fresh - frozen versus formalin - fixed paraffin - embedded tissue . 
morton ml , bai x , merry cr , et al : identification of mrnas and lincrnas associated with lung cancer progression using next - generation rna sequencing from laser micro - dissected archival ffpe tissue specimens . 
sharron lin x , hu l , sandy k , et al : differentiating progressive from nonprogressive t1 bladder cancer by gene expression profiling : applying rna - sequencing analysis on archived specimens . 
esteve - codina a , arpi o , martinez - garca m , et al : a comparison of rna - seq results from paired formalin - fixed paraffin - embedded and fresh - frozen glioblastoma tissue samples . 
nikitina as , sharova ei , danilenko sa , et al : novel rna biomarkers of prostate cancer revealed by rna - seq analysis of formalin - fixed samples obtained from russian patients . 
li j , fu c , speed tp , et al : accurate rna sequencing from formalin - fixed cancer tissue to represent high - quality transcriptome from frozen tissue . 
 ( c ) top : heatmap of the 38 formalin - fixed paraffin - embedded ( ffpe ) rna - seq dataset for all 407 tcga - derived immune gene - set genes . 
bottom : the median absolute deviation divided by the median ( mad / m ) for all 407 immune genes for each sample , a measure of correlative discordance within the immune signature . 
heatmaps showing the final fixedpoint gene sets for ( a ) epithelial - mesenchymal transition ( emt ) , ( b ) cell cycle , and ( c ) skin for rna sequencing ( rna - seq ) and nanostring . 
each gene - set member is correlated with its respective overall gene - set score , for primary melanomas only in the ( a ) the cancer genome atlas ( tcga ) and current formalin - fixed paraffin embedded ( ffpe ) rna - seq and ( b ) gse7553 and gse15605 datasets . 
 we note that the mitf / melanocyte pathway was not recovered , because of the smaller dataset ( n = 38 samples here v n = 333 in tcga ) and the lower variability in melanocyte gene expression , resulting in those genes not being among the top 5% . 
 ( b ) average reads per kilobase million ( rpkms ) for all genes within the five gene sets used in this study and for three gene sets not expected to be highly expressed in melanoma . 
 evolving intersection between inherited cancer genetics and therapeutic clinical trials in prostate cancer : a white paper from the germline genetics working group of the prostate cancer clinical trials consortium purpose advances in germline genetics , and related therapeutic opportunities , present new opportunities and challenges in prostate cancer . 
the prostate cancer clinical trials consortium germline genetics working group was established to address genetic testing for men with prostate cancer , especially those with advanced disease undergoing testing for treatment - related objectives and clinical trials . methods the prostate cancer clinical trials consortium germline genetics working group met monthly to discuss the current state of genetic testing of men with prostate cancer for therapeutic or clinical trial purposes . 
we assessed current institutional practices , developed a framework to address unique challenges in this population , and identified areas of future research . results genetic testing practices in men with prostate cancer vary across institutions ; however , there were several areas of agreement . 
cheng , md , phd , division of medical oncology , university of washington , 825 eastlake ave e , seattle , wa 98109 ; e - mail : hhcheng@ uw.edu. introduction although the contribution of heredity to prostate cancer has long been known , the underlying genetic causes remained elusive . 
 clarify patient preferences in genetic testing to guide prostate cancer treatment / clinical trial * collect family history tumor testing tumor testing germline testing discuss potential to identify germline findings pretest education by genetic counselor and / or oncologist depending on resources recommend gc if tumor sequencing suggestive ( eg , brca1 / 2 ) for dedicated , confirmatory germline testing recommend gc if germline results concerning for mutation ( eg , brca1 / 2 ) recommend gc post - test if germline mutation positive : discuss additional cancer risks for patient cascade testing in family if germline mutation negative or variant of uncertain significance : discuss additional genetic testing in family as appropriate or as indicated by family history if family history suggestive ( see table 2 ) fig 1 . 
for prostate cancer , genetic counseling and testing practices are newly driven by a growing interest in identifying patients who are candidates for enrollment in biomarker - selected clinical trials . 
this treatment - driven ascertainment , where patients are referred for germline testing in part for therapy selection , presents unique opportunities and challenges for practitioners regarding appropriate delivery of elements of genetic counseling in a feasible manner . the prostate cancer clinical trials consortium is a group of researchers from 11 institutions working together to develop novel therapeutics and biomarkers , translating scientific discoveries to improve standards of care in prostate cancer . 
for example , the brcaaway trial ( clinicaltrials.gov identifier : nct03012321 ) is a phase ii randomized trial of the parp inhibitor olaparib versus abiraterone versus the combination of the two agents in men with germline or somatic homologous recombination deficiency mutations . 
additional trials are exploring the efficacy of rucaparib , niraparib , and olaparib as single agent for men with metastatic castration - resistant prostate cancer and a deleterious genomic alteration , either germline or somatic , in brca2 , brca1 , and other ddr genes ( clinicaltrials . gov identifiers : nct02952534 , nct02975934 , nct02854436 , nct02987543 )  . 
the triumph ( trial of rucaparib in patients with metastatic hormone - sensitive prostate cancer harboring germline dna repair gene mutations ) trial ( clinicaltrials.gov identifier : nct03413995 ) will enroll men with metastatic hormone - sensitive prostate cancer and a germline ddr gene pathogenic alteration , who will be treated with the parp inhibitor rucaparib in the absence of hormonal therapy . 
clinical trials testing novel approaches to genetic testing title novel approaches genetic evaluation of menthe gem registry gentlemen : genetic testing for men with metastatic prostate cancer evaluating an alternative clinical genetics cancer care delivery model : a pilot study of patient outcomes prospective research registry of men with prostate cancer or at increased risk web - based consent and pretest counseling web - based and phone - based post - test counseling pathogenic and likely pathogenic mutation results are delivered by a genetic counselor over the phone and invited for in - person counseling consenting by primary oncologist or urologist , same - day blood draw standard pretest education and video risk assessment and delivery of results by a genetic counselor by telephone clinicaltrials.gov identifier nct03076242 nct03503097 nct02987543 progen : genetic counseling processes and outcomes among males with prostate cancer comparison of standard genetic counseling v video - based counseling with in - person counseling only for patients with germline mutations nct03328091 trial possibilities , and , second , inherited cancer risk . 
for providers , it is worth discussing and confirming patient goals before offering testingfor example , asking whether patients are interested primarily in treatment options , familial risk assessment , or both . 
table 2 summarizes clinical criteria to consider for referral to genetic counseling . this panel agrees that men with prostate cancer who undergo germline genetic testing for therapeutic or clinical trial options should receive pretest education on the implications of a positive result for themselves and for their families . 
in several recent studies of research somatic sequencing , germline mutations with clinical implications were identified.32 , 33 tumor sequencing may even be more sensitive in detecting genetic syndromes , such as lynch , than the traditional molecular tests.34 most commercial tumor assays do not specifically report whether a mutation is present in the germline , and some subtract the germline component , but the presence of well - described founder mutations may be highly suggestive . 
 for example , the ashkenazi jewish brca1 / 2 founder mutations ( brca1 185delag ; brca1 5382insc ; brca2 6174delt ) are almost always germline , not somatic , events , and ordering providers should be familiar with the somatic tumor profiling can also identify increased mutation load and msi , which can be associated with germline mmr mutations ( lynch syndrome ) .35 although it seems that most patients are interested in knowing secondary germline findings , they also expect their providers to offer decision making guidance and clarify key information.36 , 37 this panel agrees that men with prostate cancer who undergo tumor - based genetic testing for therapeutic or clinical trial options should be educated about the potential for uncovering germline mutations , which may warrant referral to a cancer genetic specialist for confirmatory germline testing . 
the traditional clinical genetics cancer care delivery modelwhere patients are referred to a genetic counselor for in - person , pretest risk assessment and education and in - person post - test counselingcannot meet the projected demand for testing of patients with prostate cancer , some facing time - sensitive treatment decisions . 
challenges and new research directions challenge or direction defining which patients should be offered germline genetic testing and at what point ( s ) in the diagnosis / treatment timeline the role of less - studied germline dna - damage repair genes for therapeutic decision making and clinical trial eligibility best framework and workflows for rapid testing for time - sensitive therapeutic decisions integration of cascade education and testing for family members ensuring historically underserved populations have access to genetic testing cancer and lynch syndromes . 
these barriers include process issues ( referral to genetic counseling and testing , access , wait times , insurance coverage ) ; physician knowledge , comfort , and time ; patients lack of awareness and understanding ; and refusal of testing . 
for example , > 85% of patients with ovarian cancer reported willingness to be tested if there were therapeutic implications or benefit to family , and a majority believed that genetic testing should be offered before or at the time of diagnosis.38 current barriers to genetic testing have been described in the context of testing for cancer predisposition in hereditary breast and ovarian when testing for therapeutic decision making , the ability to undergo assessment and testing and receive results in a timely manner is of utmost table 5 . 
newer approaches in cancer risk genetics include videoor phone - based pretest counseling and mainstreaming , an approach in which trained individuals provide standardized consenting and counseling before testing and genetics referral.39 the engage ( evaluation of a streamlined oncologist - led brca mutation testing and counseling model for patients with ovarian cancer ) study of oncologist - led brca1 / 2 mutation testing in women with ovarian cancer showed that this process is feasible , with high patient satisfaction.40 these alternate genetic counseling delivery approaches need to be studied for provider feasibility and patient acceptability . 
although awareness of cascade testing is important in any situation where a germline mutation is identified , it can be particularly important when genetic testing is performed for therapeutic selection . 
in contrast to patients who pursue genetic testing because of familial cancer risk , those who undergo germline testing for therapy selection may be less aware of the potential implications to family members , because they were not necessarily tested because of an identified familial risk . 
moreover , men with prostate cancer are often diagnosed when their children are adults , can pursue testing , and , if positive , undergo enhanced cancer screening or risk - reduction strategies . there are several known barriers for a patients communication of results to family members . 
 in brca1 / 2 screening studies , factors associated with of lack of communication include high worry about genetic risks , low interest or understanding of genomic information , and negative family history.57 - 59 men and second - degree relatives are less likely to pursue genetic testing.58 thus , there is an opportunity through education to increase familial communication of results . 
 framework for genetic testing was developed for individuals believed to be at risk for inherited syndromes , but this model requires adaptation for men with mpc who are increasingly referred for genetic testing to aid in treatment decisions . 
alumkal consulting or advisory role : astellas pharma , bayer ag , janssen biotech research funding : aragon pharmaceuticals ( inst ) , astellas pharma ( inst ) , novartis ( inst ) , zenith epigenetics ( inst ) , gilead sciences ( inst ) tomasz m . 
beer stock and other ownership interests : salarius pharmaceuticals consulting or advisory role : abbvie , astrazeneca , astellas pharma , bayer ag , boehringer ingelheim , clovis oncology , janssen biotech , janssen oncology , janssen research & development , johnson & johnson , janssen japan , merck , pfizer research funding : boehringer ingelheim ( inst ) , bristol - myers squibb / medarex ( inst ) , janssen research & development ( inst ) , medivation / astellas ( inst ) , oncogenex ( inst ) , sotio ( inst ) , sotio , theraclone sciences ( inst ) himisha beltran consulting or advisory role : janssen oncology , genzyme , glaxosmithkline , abbvie research funding : astellas pharma ( inst ) , janssen ( inst ) , abbvie / stemcentrx , eli lilly ( inst ) , millennium pharmaceuticals ( inst ) daniel j . 
heath honoraria : bayer , dendreon , sanofi consulting or advisory role : agensys speakers ' bureau : sanofi research funding : tokai pharmaceuticals ( inst ) , seattle genetics ( inst ) , agensys ( inst ) , dendreon ( inst ) , genentech ( inst ) , millennium pharmaceuticals ( inst ) , celldex therapeutics ( inst ) , inovio pharmaceuticals ( inst ) , celgene ( inst ) celestia s . 
higano employment : cti biopharma ( i ) leadership : cti biopharma ( i ) stock and other ownership interests : cti biopharma honoraria : genentech consulting or advisory role : bayer ag , ferring pharmaceuticals , orion , astellas pharma , clovis oncology , asana biosciences , endocyte , blue earth diagnostics , myriad genetics , tolmar , janssen research funding : aragon pharmaceuticals ( inst ) , astrazeneca ( inst ) , dendreon ( inst ) , genentech ( inst ) , medivation ( inst ) , emergent biosolutions ( inst ) , bayer ( inst ) , pfizer ( inst ) , roche ( inst ) , astellas pharma ( inst ) travel , accommodations , expenses : bayer ag , astellas pharma , clovis oncology , blue earth diagnostics , endocyte , ferring pharmaceuticals , genentech , orion pharma , menarini , myriad genetics , pfizer rana r . 
morgans honoraria : genentech , janssen , johnson & johnson , sanofi , astrazeneca consulting or advisory role : genentech , astrazeneca , sanofi akash patnaik honoraria : clovis oncology , merck , prime oncology consulting or advisory role : janssen oncology research funding : bristol - myers squibb ( inst ) , progenics pharmaceuticals ( inst ) , janssen oncology ( inst ) , clovis oncology ( inst ) travel , accommodations , expenses : clovis oncology , bristol - myers squibb , merck , prime oncology , janssen oncology charles j . 
ryan honoraria : janssen oncology , astellas pharma , bayer consulting or advisory role : bayer ag , millennium pharmaceuticals , ferring pharmaceuticals research funding : bind biosciences , karyopharm therapeutics , novartis walter m . 
stadler honoraria : cvs caremark , sotio , astrazeneca , bristolmyers squibb consulting or advisory role : cvs caremark , sotio , astrazeneca , bristol - myers squibb , eisai , bayer ag , pfizer , clovis oncology , genentech research funding : bayer ag ( inst ) , bristol - myers squibb ( inst ) , boehringer ingelheim ( inst ) , exelixis ( inst ) , novartis ( inst ) , genentech ( inst ) , glaxosmithkline ( inst ) , medivation ( inst ) , pfizer ( inst ) , merck ( inst ) , millennium pharmaceuticals ( inst ) , janssen ( inst ) , johnson & johnson ( inst ) , astrazeneca ( inst ) , abbvie ( inst ) , x4 pharmaceuticals ( inst ) , calithera biosciences ( inst ) other relationship : uptodate , american cancer society mary - ellen taplin honoraria : janssen - ortho , clovis oncology , astellas pharma , incyte , uptodate , research to practice consulting or advisory role : janssen - ortho , bayer ag , guidepoint global , best doctors , uptodate , clovis oncology , research to practice , myovant sciences , incyte research funding : janssen - ortho ( inst ) , medivation ( inst ) , bayer ( inst ) , tokai pharmaceuticals ( inst ) travel , accommodations , expenses : medivation , janssen oncology , tokai pharmaceuticals , astellas pharma , incyte noah d . 
antonarakis honoraria : sanofi , dendreon , medivation , janssen biotech , essa pharma , astellas pharma , merck , astrazeneca , clovis oncology consulting or advisory role : sanofi , dendreon , medivation , janssen biotech , essa pharma , astellas pharma , merck , astrazeneca , clovis oncology research funding : janssen biotech ( inst ) , johnson & johnson ( inst ) , sanofi ( inst ) , dendreon ( inst ) , aragon pharmaceuticals ( inst ) , exelixis ( inst ) , millennium pharmaceuticals ( inst ) , genentech ( inst ) , novartis ( inst ) , astellas pharma ( inst ) , tokai pharmaceuticals ( inst ) , merck ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , constellation pharmaceuticals ( inst ) patents , royalties , other intellectual property : coinventor of a biomarker technology that has been licensed to qiagen travel , accommodations , expenses : sanofi , dendreon , medivation heather h . 
carlo and wassim abida , memorial sloan - kettering cancer center ; himisha beltran , weill cornell medical center ; jacob vinson , prostate cancer clinical trials consortium , new york , ny ; veda n . 
stadler , university of chicago , chicago , il ; and mary - ellen taplin , dana - farber cancer institute , boston , ma . supported by the office of the assistant secretary of defense for health affairs , through the prostate cancer research program under awards no . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
pomerantz mm , spisk s , jia l , et al : the association between germline brca2 variants and sensitivity to platinum - based chemotherapy among men with metastatic prostate cancer . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
antonarakis es , lu c , luber b , et al : germline dna - repair gene mutations and outcomes in men with metastatic castration - resistant prostate cancer receiving first - line abiraterone and enzalutamide . 
isaacsson velho p , silberstein jl , markowski mc , et al : intraductal / ductal histology and lymphovascular invasion are associated with germline dna - repair gene mutations in prostate cancer . 
castro e , goh c , olmos d , et al : germline brca mutations are associated with higher risk of nodal involvement , distant metastasis , and poor survival outcomes in prostate cancer . 
castro e , goh c , leongamornlert d , et al : effect of brca mutations on metastatic relapse and cause - specific survival after radical treatment for localised prostate cancer . 
na r , zheng sl , han m , et al : germline mutations in atm and brca1 / 2 distinguish risk for lethal and indolent prostate cancer and are associated with early age at death . 
aarnio m , sankila r , pukkala e , et al : cancer risk in mutation carriers of dna - mismatch - repair hum mutat 34 : 490 - 497 , 2013 genes . 
mller p , seppl tt , bernstein i , et al : cancer risk and survival in path_mmr carriers by gene and gender up to 75 years of age : a report from the prospective lynch syndrome database . 
abida w , armenia j , gopalan a , et al : prospective genomic profiling of prostate cancer across disease states reveals germline and somatic alterations that may affect clinical decision making . 
gillessen s , attard g , beer tm , et al : management of patients with advanced prostate cancer : the report of the advanced prostate cancer consensus conference apccc 2017 . 
cheng hh , klemfuss n , montgomery b , et al : a pilot study of clinical targeted next generation sequencing for prostate cancer : consequences for treatment and genetic counseling . 
abida w , armenia j , gopalan a , et al : prospective genomic profiling of prostate cancer across disease states reveals germline and somatic alterations that may affect clinical decision making . 
hampel h , pearlman r , beightol m , et al : assessment of tumor sequencing as a replacement for lynch syndrome screening and current molecular tests for patients with colorectal cancer . 
hamilton jg , shuk e , genoff mc , et al : interest and attitudes of patients with advanced cancer with regard to secondary germline findings from tumor genomic profiling . 
hamilton jg , shuk e , garzon mg , et al : decision - making preferences about secondary germline findings that arise from tumor genomic profiling among patients with advanced cancers . 
colombo n , huang g , scambia g , et al : evaluation of a streamlined oncologist - led brca mutation testing and counseling model for patients with ovarian cancer . 
jones t , mccarthy am , kim y , et al : predictors of brca1 / 2 genetic testing among black women with breast cancer : a population - based study . 
levy de , byfield sd , comstock cb , et al : underutilization of brca1 / 2 testing to guide breast cancer treatment : black and hispanic women particularly at risk . 
allford a , qureshi n , barwell j , et al : what hinders minority ethnic access to cancer genetics services and what may help ? eur j hum genet 22 : 866 - 874 , 2014 46 . 
vadaparampil st , wideroff l , breen n , et al : the impact of acculturation on awareness of genetic testing for increased cancer risk among hispanics in the year 2000 national health interview survey . 
moses ka , orom h , brasel a , et al : racial / ethnic disparity in treatment for prostate cancer : does cancer severity matter ? urology 99 : 76 - 83 , 2017 53 . 
elrick a , ashida s , ivanovich j , et al : psychosocial and clinical factors associated with family communication of cancer genetic test results among women diagnosed with breast cancer at a young age . 
 pten promoter variants are not associated with common cancers : implications for multigene panel testing purpose pten mutations are associated with breast , colon , endometrial , kidney , and thyroid cancers . 
most pten promoter alterations , however , are characterized as variants of unknown significance , and their contribution to cancer risk is unclear . materials and methods personal and family histories of 88 , 333 patients undergoing pten analysis as part of multigene panel testing ( mgpt ) were retrospectively reviewed . 
individuals were categorized as positive for one or more mutations ( patho ) , without mutations but carrying one or more promoter variant ( prom ) , or negative for alterations ( wt )  . 
a subsequent study by teresi et al identified promoter variants in five patients with cancer but observed no differences in pten transcription or transcription factor binding compared with wild - type controls.8 the authors did report , however , that altered mrna folding patterns predicted for two of the variants correlated with inhibition of pten translation , suggesting a role for nontraditional regulatory mechanisms in cs pathogenesis . 
demographic , family history , and clinical information ( sex , age , self - reported ethnicity , personal cancer history , age at diagnosis , and breast cancer pathology ) were collected from test requisition forms and clinic notes provided by ordering clinicians . 
demographic and clinical information relevant to the condition for which genetic testing was ordered , and consent to participate in research that included pten sequencing , was obtained for these individuals . 
because the presence of pathogenic and / or likely pathogenic mutations in other known cancer genes ( data supplement ) may influence pten variant classification ( ie , co - occurrence ) , we further excluded such carriers ( n = 12 , 560 )  . 
given that patients may be referred for genetic testing because of family history of disease in the absence of personal history , we performed three sensitivity analyses , comparing cases to controls with no firstdegree relatives with the cancer of interest , controls without any family history of the cancer of table 1 . 
 interest , and an independent collection of research controls who initially underwent genetic testing for inherited disorders unrelated to cancer . age of diagnosis for each phenotype among the patho , prom , and wt groups were tested using wilcoxon rank sum tests . molecular analysis molecular methods have been previously described.9 briefly , genomic dna was isolated from blood or saliva and quantified . 
initial data processing and base calling , including extraction of cluster intensities , was performed using rta 1.17.21.3 ( real time analysis , hiseq control software , version 2.0.10 ; illumina , san diego , ca )  . 
sequence reads were aligned to reference human genome grch37 using novoalign , version 3.02.07 ( novocraft technologies , selangor , malaysia ) and variant calls generated using gatk , version 3.2.2 ( broad institute , cambridge , ma )  . 
30 was required for variant calling . variants were annotated with the ambry variant analyzer ( ambry genetics , aliso viejo , ca ) and their clinical significance assessed using ambrys five - tier classification framework10 , 11 on the basis of guidelines published by the american college of medical genetics and genomics.12 sanger sequencing was performed to check regions with no or insufficient coverage and confirm or clear all variant calls except those known to be benign or likely benign . statistical analysis for each phenotype , we performed case / control association analyses using multivariate logistic regression , adjusted for race / ethnicity , age at testing , sex , and type of multigene panel ordered , as appropriate . 
we present adjusted odds ratios and 95% cis for two mutually exclusive groups : pten mutation ( pathogenic and / or likely pathogenic ) carriers ( pathos ) , and individuals without pten mutations but carrying one or more pten promoter variant ( proms ) , compared with those in whom no alteration was detected ( wts )  . 
differences in median results among the 88 , 333 patients eligible for analysis , we examined 52 , 517 female breast cancer cases , 4 , 099 colorectal cancer cases , 1 , 569 uterine / endometrial cancer cases , 643 thyroid cancer cases , and 956 kidney cancer cases , and 28 , 549 controls with no personal cancer history ( table 1 )  . 
compared with controls , breast and uterine / endometrial cancer cases tended to be slightly older , whereas colorectal , thyroid , and kidney cancer cases were of similar age at the time of genetic testing ( data supplement )  . 
the distribution of race / ethnicity was also relatively similar among cases and controls . a total of 268 unique pten variants were included in analyses : 219 proms , all of which were classified as vus , and 49 pathogenic variants in the coding region ( data supplement )  . 
there were no significant differences in the age of onset of any cancer for proms compared with wts . discussion limited data have suggested that at least some pten promoter variants may be associated with cs and increased cancer risks , particularly for breast , thyroid , and uterine cancers . 
however , this evidence is insufficient for clinical assessment ofriskassociatedwithptenpromotervariation.we therefore examined associations between promoter variants and cancer risks in a large cohort of patients tested at a clinical diagnostic laboratory using mgpt that included pten . 
we observed no difference between carriers of pten promoter variants and those with no pten alterations in either ptenassociated cancer risk or median age of cancer diagnosis . these observations were based on a large sample of nearly 90 , 000 patients for whom detailed clinical and family history information were available , which allowed us to account for potential confounders and bias in our analyses . 
moreover , our target - enrichment , highly sensitive assay and robust sequencing methods ensured adequate capture of true variants with high specificity and sensitivity . despite our large - scale systematic approach , our study has some limitations . 
although the primary phenotype of interest was breast cancer , analyses of patho associations with most other cancer types were underpowered because of low sample size , and we lack adequate data for analysis of nonmalignant features . 
we did not observe any patho carriers among kidney cancer cases or research controls , and only two to four carriers each among those with thyroid , colorectal , and uterine / endometrial cancers . 
our sensitivity analyses restricting controls to those without family history of the cancer of interest yielded results similar to the primary analysis , and we were able to replicate our findings using a modest - sized , independent sample of controls not enriched for family history of cancer . 
however , we acknowledge that some bias may still be present , particularly for analyses of colorectal cancer , where we did not observe a significant association with pten gene - region mutations . incomplete data on polyps and other clinical factors among controls may have influenced our findings . 
last , we note that although we did not observe any significant effects of individual promoter variants , it is possible that additional rare variation exists in the promoter region beyond what has been currently detected , which could be associated with cancer . promotor regions are not sequenced for most genes analyzed by clinical laboratories because of insufficient data regarding the pathogenicity of such variants , yet many clinical laboratories in the united states include the promoter in pten sequencing . however , the vast majority of identified promoter variants are classified as vus , which provides no direction for clinical management of patients carrying these variants . 
given that 10 times as many prom as patho carriers were observed in this cohort , the number of affected patients as well as the time and effort spent on promoter variant classification is substantial . 
pilarski r , stephens ja , noss r , et al : predicting pten mutations : an evaluation of cowden syndrome and bannayan - riley - ruvalcaba syndrome clinical features . 
tan mh , mester j , peterson c , et al : a clinical scoring system for selection of patients for pten mutation testing is proposed on the basis of a prospective study of 3042 probands . 
zhou xp , waite ka , pilarski r , et al : germline pten promoter mutations and deletions in cowden / bannayanriley - ruvalcaba syndrome result in aberrant pten protein and dysregulation of the phosphoinositol - 3 - kinase / akt pathway . 
landrum mj , lee jm , benson m , et al : clinvar : public archive of interpretations of clinically relevant variants . nucleic acids res 44 : d862 - d868 , 2016 8 . 
laduca h , stuenkel aj , dolinsky js , et al : utilization of multigene panels in hereditary cancer predisposition testing : analysis of more than 2 , 000 patients . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
 novel pde10a - braf fusion with concomitant nf1 mutation identified in an undifferentiated sarcoma of infancy with sustained response to trametinib introduction childhood cancers are relatively rare entities ( approximately 140 per million among children < 15 years of age ) .1 malignant solid tumors that occur in infants ( < 1 year of age ) constitute a uniquely heterogeneous subgroup . 
in general , they are driven by acquisition of potent oncogenes , including chimeric gene fusions , such as the etv6 - ntrk3 rearrangement in infantile fibrosarcoma , or loss of tumor suppressor genes , such as rb1 in retinoblastoma or smarcb1 in rhabdoid tumors.2 - 4 in addition , it is estimated that approximately 10% of pediatric cancers carry a mutation in a cancer predisposing gene.5 , 6 tumor of the cns , infantile fibrosarcoma , rhabdoid tumors , and undifferentiated sarcoma are among the most frequently encountered tumor types in infancy.7 - 9 many of the undifferentiated tumors are often considered orphan diseases , given the absence of distinctive histopathologic hallmarks , the small size of patient cohorts , and until recently , a limited molecular classification.10 consequently , standard - of - care guidelines for treatment of these pediatric patients are not soundly established . 
 in the past decade , the list of oncogenic chimeric gene fusions in cancers has grown exponentially.11 next - generation sequencing of the whole - transcriptome ( or tumor rna sequencing [ rnaseq ] ) now enables identification of cytogenetically silent gene fusions , as well as single nucleotide variants . 
abnormal tissue uptake was not documented by [ 125i ] metaiodobenzylguanidine scintigraphy . the patient underwent a surgical biopsy , and pathologic analysis of the specimen revealed an undifferentiated malignant sarcoma . 
histopathologic analysis showed a neoplasm composed of stphanie vairy loubna jouan mlanie bilodeau virginie dormoyraclet patrick gendron franoise couture france lveill frdrique tihy emmanuelle lemyre dorothe bouron - dal soglio nada jabado claudia l . 
circled area shows the mass with extension into the sacral medullary canal and foramen and compression of the left iliac ve ( b ) histologic analysis of the neoplasm composed of undifferentiated tumor cells and sparse collagenous stroma . 
cytogenetic analyses displayed a complex karyotype with multiple aberrations , including several trisomies ( of chromosomes 3 , 8 , 11 , and 17 ) , monosomy x , a balanced translocation t ( 1 ; 12 ) , and a 7q terminal deletion ( fig 1c )  . 
analyses by fluorescent in situ hybridization demonstrated no rearrangements of ss18 ( synovial sarcoma ) , etv6 ( infantile fibrosarcoma ) , dux4 , and ccnb3 ( ewing - like sarcoma ) but confirmed a 7q deletion using an internal probe . 
overall , karyotype and fish studies did not reveal a chromosomal anomaly specifically associated with pediatric sarcomas . chemotherapy ( vincristine , actinomycin , and cyclophosphamide regimen ) was urgently initiated while awaiting the final pathology report . 
surgical resection was declined , given the high risk of morbid neurologic sequelae , and the child was closely followed . progression of the residual mass was documented 18 months after the end of chemotherapy . 
given that pediatric sarcomas are frequently associated with chimeric gene fusions , and in the absence of a precise molecular diagnosis for this patient , tumor rnaseq was performed to uncover putative cancer driver alterations , including gene rearrangements . 
rnaseq analysis performed on the diagnostic tumor sample revealed an in - frame fusion between pde10a ( 6q27 ) exon 3 and braf ( 7q34 ) exon 9 ( fig 2a ; table a1 )  . 
 chimeric product pde10a - braf was confirmed by reverse transcriptase polymerase chain reaction using complementary dna from the tumor and subsequent direct sanger sequencing ( figs 2b and 2c )  . 
the pde10a and braf gene breakpoints were corroborated by array comparative genomic hybridization analysis of the diagnostic sample , and these alterations were present in a high proportion of cells ( 80% ; data supplement )  . the transcriptomic data set was also mined to predict damaging nucleotide alterations in a list of 2 , 604 cancer - related genes ( data supplement )  . 
 a pde10a chr6 ( q27 ) braf chr7 ( q34 ) 8 9 10 11 12 ras binding domain s365 kinase domain 11 12 14 15 pde10a - braf kinase domain ladder 500 bp pde10a - braf ( 300 bp ) pde10a exon 3 braf exon 9 breakpoint fig 2 . 
 ( b ) validation by reverse transcriptase polymerase chain reaction performed on tumor rna ( expected product size of 300 bp ) , normal control , and no rna template . 
the patient did not present any clinical stigmata of neurofibromatosis and tested negative for germline nf1 mutations ( the patient was referred to clinical genetics , physical evaluation , and sanger sequencing of peripheral blood cell dna ) , suggesting a somatic deletion of nf1 in neoplasm cells . 
the patient was also tested for constitutional tp53 gene alterations , but no pathogenic mutation was detected . because both braf and nf1 play converging roles in activating the rasmitogen - activated protein kinase ( mapk ) signaling pathway ( fig 3a ) , we evaluated the phosphorylation status of erk1 / 2 protein using immunohistochemistry . 
on the basis of genomic profiling of the diagnostic tumor sample , rescue treatment with an mek inhibitor ( trametinib 0.025mg / kg , equivalent to the recommended adult dose , 17 was proposed to the family )  . 
this rearrangement accordingly preserves the raf kinase domain while disrupting the upstream inhibitory cr2 domain encoded by exon 8 , removing the regulatory s365 phosphorylation site required for signal inhibition.25 , 26 in parallel , nf1 is a tumor suppressor that negatively regulates ras pathway . 
given that fulllength protein is 2819 amino acids , heterozygous loss of function of nf1 is predicted , leading to a sustained activation of the mapk pathway through ras phosphorylation . 
we therefore postulate that as previously hypothesized in melanomas , 28 nf1 mutation and pde10a - braf gene fusion could both converge to activate downstream mapk pathway in tumor cells , and functional interactions should be explored in pediatric sarcomas . in sum , we report a novel pde10a - braf chimeric gene fusion and somatic nf1 p . ( tyr80serfs * 26 ) deletion identified using rna sequencing in an infant diagnosed with undifferentiated pelvic sarcoma . 
 pediatric relapsed soft tissue sarcomas generally have a poor response to second - line chemotherapy.29 , 30 some studies of advanced soft tissue sarcomas showed minimal or no response to mek inhibitor , but correlation with mapk pathway alterations is lacking.31 , 32 this report underscores the critical impact of deep sequencing technologies on the molecular classification and clinical management of infant tumors . 
multicenter clinical trials will be critical to determine the frequency of braf fusions in infantile undifferentiated sarcoma and to test the therapeutic benefit of incorporating mek inhibitors or other agents targeting mapk pathway signaling into the chemotherapy backbone for these tumors . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . franoise couture no relationship to disclose france lveill no relationship to disclose frdrique tihy no relationship to disclose emmanuelle lemyre no relationship to disclose dorothe bouron - dal soglio no relationship to disclose nada jabado no relationship to disclose claudia l . 
kleinman no relationship to disclose monia marzouki no relationship to disclose sonia cellot no relationship to disclose acknowledgment stphanie vairy no relationship to disclose loubna jouan no relationship to disclose mlanie bilodeau no relationship to disclose virginie dormoy - raclet no relationship to disclose we sincerely thank the patients and their families for their outstanding support , as well as members of the pediatric hematology - oncology team of centre hospitalier universitaire sainte - justine . 
 affiliations stphanie vairy , mlanie bilodeau , monia marzouki , and sonia cellot , charles - bruneau cancer center ; stphanie vairy , loubna jouan , mlanie bilodeau , franoise couture , france lveill , frdrique tihy , emmanuelle lemyre , dorothe bouron - dal soglio , monia marzouki , and sonia cellot , centre hospitalier universitaire sainte - justine ; patrick gendron , emmanuelle lemyre , dorothe bouron - dal soglio , monia marzouki , and sonia cellot , universit de montral ; nada jabado and claudia l . 
jackson em , sievert aj , gai x , et al : genomic analysis using high - density single nucleotide polymorphism - based oligonucleotide arrays and multiplex ligation - dependent probe amplification provides a comprehensive analysis of ini1 / smarcb1 in malignant rhabdoid tumors . 
orbach d , rey a , oberlin o , et al : soft tissue sarcoma or malignant mesenchymal tumors in the first year of life : experience of the international society of pediatric oncology ( siop ) malignant mesenchymal tumor committee . 
hayes - jordan aa , spunt sl , poquette ca , et al : nonrhabdomyosarcoma soft tissue sarcomas in children : is age at diagnosis an important variable ? j pediatr surg 35 : 948 - 953 , 2000 ; discussion 953 - 954 9 . 
bishop aj , mcdonald mw , chang al , et al : infant brain tumors : incidence , survival , and the role of radiation based on surveillance , epidemiology , and end results ( seer ) data . 
turski ml , vidwans sj , janku f , et al : genomically driven tumors and actionability across histologies : braf - mutant cancers as a paradigmol cancer ther 15 : 533 - 547 , 2016 14 . 
shukla n , ameur n , yilmaz i , et al : oncogene mutation profiling of pediatric solid tumors reveals significant subsets of embryonal rhabdomyosarcoma and neuroblastoma with mutated genes in growth signaling pathways . 
kao yc , fletcher cdm , alaggio r , et al : recurrent braf gene fusions in a subset of pediatric spindle cell sarcomas : expanding the genetic spectrum of tumors with overlapping features with infantile fibrosarcoma . 
matallanas d , birtwistle m , romano d , et al : raf family kinases : old dogs have learned new biol 16 : 281 - 298 , 2015 tricks . 
subbiah v , meyer c , zinner r , et al : phase ib / ii study of the safety and efficacy of combination therapy with multikinase vegf inhibitor pazopanib and mek inhibitor trametinib in advanced soft tissue sarcoma . 
dembla v , groisberg r , hess k , et al : outcomes of patients with sarcoma enrolled in clinical trials of pazopanib combined with histone deacetylase , mtor , her2 , or mek inhibitors . 
list of candidate fusion genes detected by rna sequencing gene 1 ( 5 end fusion partner ) coordinates ( 5 end ) coordinates ( 3 end ) gene 2 ( 3 end fusion partner ) pde10a chr6 : 165895802 ensg00000250859 chr5 : 126847434 ensg00000268750 chr19 : 58331312 ensg00000236537 chr6 : 158733082 akr1c6p chr10 : 4951797 tvp23c - cdrt4 chr17 : 15343598 ensg00000236537 chr6 : 158703294 loc100507412 chrun_gl000220 : 114339 atp6v1f chr1 : 1271794 chr17 : 18707578 mmab chr12 : 110006325 tvp23c - cdrt4 chr17 : 15341516 ensg00000146426 chr6 : 155182131 scaf8 gli3 chr7 : 42129309 loc100507412 clasp2 chr3 : 33686248 ensg00000174444 chr15 : 66795841 kansl1 kansl1 tulp4 akr1e2 tvp23b rpl19 tulp4 tmem126b arglu1 rpl27 dvl1 tvp23b ccnd2 styx rasal2 tcf7l2 nav2 crybg3 nap1l1 plekha5 znf517 epas1 dzip1l mdm4 uba2 chr17 : 44171925 chr17 : 44171925 chr6 : 158735299 chr10 : 4872866 chr17 : 18707578 chr17 : 37360967 chr6 : 158735299 chr11 : 85339650 chr13 : 107211929 chr17 : 41154962 chr12 : 4411895 chr14 : 53197246 chr1 : 178136886 chr10 : 114861983 chr11 : 19389080 chr3 : 97594908 chr12 : 76470081 chr12 : 19408057 chr8 : 146029800 chr2 : 46537949 chr3 : 137782099 chr1 : 204502515 chr19 : 34945628 no . 
 frequent molecular subtype switching and gene expression alterations in lung and pleural metastasis from luminal atype breast cancer max klebe , ms1 ; carlo fremd , md2 ; mark kriegsmann , md1 ; katharina kriegsmann , md3 ; thomas albrecht , md1 ; verena thewes , phd2 , 6 ; martina kirchner , phd1 ; pornpimol charoentong , phd2 , 6 ; nadine volk , phd1 , 2 ; johannes haag , md4 ; ralph wirtz , phd5 ; thordur oskarsson , phd6 ; alexandra schulz , ms1 ; j org heil , md7 ; andreas schneeweiss , md8 ; hauke winter , md4 ; and peter sinn , md1 purpose conversion of tumor subtype frequently occurs in the course of metastatic breast cancer but is a poorly understood phenomenon . 
in luma cancers , 62 genes were identied with differential expression in metastatic versus primary disease , compared with only 10 differentially expressed genes in lumb , human epidermal growth factor receptor 2 ( her2 ) enriched , and basal subtypes combined . gene expression changes in luma cancers affected not only the repression of the estrogen receptor pathway and cell cyclerelated genes but also the wnt pathway , proteinases ( mme , mmp11 ) , and motility - associated cytoskeletal proteins ( ck5 , ck14 , ck17 )  . 
this involved 83 notable gene expression changes . conclusion our results indicate that gene expression changes and subsequent subtype conversion occur on a large scale in metastatic luminal atype breast cancer compared with other molecular subtypes . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction visceral metastases often occur as late events in the course of metastatic breast cancer and are generally followed by a rapidly fatal outcome . 
we examined changes in pam50 gene signatures in a matched - pair series of 57 breast cancer primaries and corresponding lung or pleural metastases to determine the nature and extent of tumor subtype conversion . knowledge generated subtype conversion to pam50 luminal b subtype was observed in the majority of luminal atype breast cancers . 
only few cases of primary luminal b , human epidermal growth factor receptor 2enriched , and basal - like subtypes converted to another pam50 category in metastasis . relevance pam50 - based subtype classication of breast cancer lung and pleural metastases may be different than that seen in the primary tumor , especially in primary luminal a cancers . 
this change in pam50 classication between primary and metastatic disease may affect response to systemic therapy and must be investigated . other three subtypes by tissue microarray analysis , 7 and basal subtypes in particular were more frequent than expected.9 in this study , we included patients with invasive breast cancer of all molecular subtypes and metachronous breast cancer with lung or pleural metastasis ( bclpm ) to provide insights into the upand downregulation of genes during the course of the disease . 
therefore , we retrospectively examined gene expression proles in a wellcharacterized , prospectively assembled group of patients with breast cancer and metachronous lung or pleural metastasis ( data supplement 2 , fig s1 )  . 
exclusion criteria included no availability or insufciency of tumor tissue from pbc or bclpm tumor tissue ( n = 11 ) , primary lung cancer after review of patient records ( n = 2 ) , and low rna content or not meeting quality control criteria of rna data ( n = 11 )  . gene expression analysis for the selection of tumor tissue for rna extraction , ffpe tissue blocks from the pbc and metastatic lesions were selected after reviewing all original tissue slides and were recut for hematoxylin & eosin sections , to be used for reference and to determine tumor cell content ( tumor surface area )  . 
microdissection was performed in most cases to avoid normal breast tissue contamination . a minimum of approximately 50 ng of total rna was used . hybridization time per cartridge was 16 hours before measurement . rna expression analysis was performed by measuring a custom panel of 269 breast cancerrelated genes and 11 housekeeping genes ( data supplement 2 , table s2 ) , using the ncounter platform ( nanostring technologies , seattle ca ) according to the manufacturers instructions . 
the gene panel cludes 25 published gene signatures with prognostic or predictive properties in luminal - type breast cancer with the aim to determine their role in metastatic disease ( data supplement 2 , table s3 )  . 
the selection of gene signatures covered by this list of genes includes nine proliferationrelated signatures , four estrogen receptorrelated signatures , one immune - related signature , and 11 signatures related to cancer pathways . 
for gene function , david bioinformatics resources were used ( version 6.8 ) , 17 , 18 and functional annotation analysis in the biological process category and kegg pathway enrichment were computed . 
all statistical calculations , except gene function analysis , were done using r , version 3.6.1.20 additional information is available in data supplement 1 . results patient characteristics a total of 57 patients with bclpm were included in this study . 
pam50 molecular subtype conversion occurred in the majority of luminal atype pbc ( n = 16 ; 57.1% ) , evolving mostly into luminal btype or her2 - type metastasis ( table 1 ; fig 1b )  . 
gene expression clustering of bclpm was clearly different from pbc in the low - risk category but not with cancers of high - risk subtypes ( figs 2a - 2b )  . this indicates a similar tumor biology of bclpm and pbc for tumors of high - risk subtypes but a different biology in the low - risk ( luminal a ) category . 
by tnm classication luma lumb her2 basal ( range ) initial metastatic site before the treatment of metastatic breast cancer lung or pleural metastasis endocrine therapy at time of metastatic other unknown biopsy yes , tamoxifen yes , aromatase inhibitor yes , gnrha no . 
similarly , heatmap clustering conrmed separate luminal a clusters for primary tumors and metastases ( fig 3 )  . pgr was signicantly downregulated ( p = .0015 , wilcoxon rank - sum test ) , and mki67 was not signicantly increased ( data supplement 2 , fig s5 )  . differential gene expression analysis to characterize which genes are involved in the process of bclpm , differential gene expression analysis was performed . 
these included 3 cytoskeletal proteins ( krt14 , krt17 , krt5 ) , 2 matrix metalloproteinases ( mmp11 , mme ) , and ankrd30a . the latter is a breast differentiation gene that is frequently expressed in the breast and in receptor - positive breast cancer.22 other molecular changes in metastases were linked to proliferation ( cdc6 , ccnb1 , mki67 , top2a , aurkb , pttg1 ) , cell cycle checkpoints ( brca2 , bub1 , bub1b , chek1 ) , and epithelial - mesenchymal transition ( emt ) genes . 
among the genes most relevant for therapy in breast cancer ( esr1 , pgr , her2 , mki67 ) , the expressions of esr1 and her2 were unaffected in bclpm , but subtype - specic changes the pam50 subtypes of pbc showed marked variation with regard to the extent of differential gene expression of this gene panel in bclpm . 
the greatest number of differentially expressed genes was seen in luminal atype breast cancer , with 47 and 11 downand upregulated genes , respectively , compared with fewer expression changes in metastatic high - risk tumors ( luminal b , her2 - enriched , basal - like ) , with six and four downand upregulated genes , respectively ( figs 4a - 4b )  . 
only 1 gene was downregulated in bclpm that was her2 enriched , and no genes reached signicance after adjustment for multiple testing in the basal - like subtype , which in part may be attributed to the comparably small sample sizes of 6 and 7 , respectively ( data supplement 2 , table s6 )  . 
of the genes repressed in luminal a metastases , several are associated with emt . four of these ( twist2 , twist1 , zeb1 , tgfb3 ) are key regulators of emt . 
multidimensional scaling ( umap ) of gene expression , showing ( a ) gene expression in primary versus metastatic cancers , and ( b ) pam50 subtypes of the primary and metastatic tumors . 
for luminal a ( luma ) subtype , two subtype clusters can be distinguished , ( bottom ) one for primary breast cancers , and ( top ) the smaller one representing lung metastases . 
lumb , luminal b ; her2 , human epidermal growth factor rector 2enriched ; basal , basal - like ; normal , normal - like subtype . with bclpm ( figs 2a - 2b ) , and the magnitude and type of gene expression changes in lung metastasis ( table 1 )  . 
thirty - four and 49 genes were signicantly upand downregulated , respectively , in bclpm from luminal a cancers with subtype switch ( adjusted p , .05 ) compared with only 7 genes downregulated and none upregulated in the nonsubtype switch group ( figs 4c - 4d )  . 
in luminal a cancers , subtype switch affected cell cycle and cell proliferation genes ( mitotic cell cycle checkpoint , sister chromatid cohesion , mitotic nuclear division , cell division ) and the p53 - signaling as well as progesterone - signaling pathways ( data supplement 2 , figs 4a and 4b )  . clustering of gene expression in luminal atype cancers to better characterize the phenomenon of subtype conversion in luminal a cancers , we set up a logistic model for the assessment of differential gene expression with and without subtype conversion . 
in the group of 28 luminal asubtype tumors , a subtype conversion was more likely to occur when genes involved in certain growth factors , nuclear proteins , and signaling molecules were upregulated ( gnaz , hoxb13 , vegfa ) and some genes involved in immune response and inammation ( foxp3 , cd8a , cd68 , csf1r ) were downregulated ( fig 5a )  . 
this included 10 genes of various functions with downregulation in metastases and 52 genes , also of diverse functions , that were upregulated after subtype switch ( fig 5b )  . 
this upregulation of a substantial number of genes suggests a common mechanism of gene regulation that is involved in subtype switching . comparison of gene signatures in luminal a and btype cancers for additional insight into the classes of genes involved in subtype switching , a couple of well - characterized gene signatures for estrogen receptor signaling , tumor proliferation , and hallmarks of cancer were analyzed in bclpm ( data supplement 2 , figs s6a and s6b )  . 
interestingly , and despite the different genes involved in these signatures , all 4 tumor proliferation signatures tested showed similar patterns of gene expression in metastases derived from luminal atype tumors with and without subtype switch . 
 klebe et al subtype luminal a luminal b basal - like her2 - enriched normal - like site breast primary lung metastasis expression subtype site brca2 diaph3 chek2 ccne2 aurka bub1 mki67 rrm2 cenpa exo1 mybl2 cep55 plk1 pttg1 cdc6 top2a tyms hif1a flvcr2 cd44 rragd uchl1 scrg1 qsox2 oxct1 krt7 cd24 fgfr4 phgdh cdca7 adgrg6 prkcb pim2 ptgds zeb2 tp53 il17rb rbbp8 krt5 krt14 krt17 tspyl5 elf5 vgll1 prom1 sfrp1 mmp12 mmp9 igfbp5 cxcl14 abl1 zeb1 twist2 twist1 snai2 aldh1a1 slc2a3 csnk1d fam213b fzr1 bbc3 rassf7 plau mmp11 tgfb3 ankrd30a tmem45b pik3ca col4a2 cdc42bpa gstm1 esm1 csnk1e znf703 ccnd1 ebf4 esr1 thsd4 tbc1d9 gata3 cxxc5 ca12 tff1 tff3 foxa1 spdef krt18 pak1 blvra cdk7 stk32b bcl2 stc2 greb1 mapt scube2 igf1r slc39a6 siah2 krt19 cldn4 ddr1 azgp1 fig 3 . 
tumors are discriminated according to their molecular subtype , with separation of metastatic luminal a tumors from primary luminal a cancers ( shown as blue bars across top )  . 
different patterns of gene expression changes are observed , with ( a ) signicantly more changes in the luminal a subtype compared with ( b ) high - risk tumors ( lumb , human epidermal growth factor receptor 2 ( her2 ) enriched , basal - like )  . 
within the luminal a subtype , gene expression changes were mostly conned to ( c ) tumors with subtype switch in lung metastasis compared with ( d ) nonswitched metastases . intrinsic tumor subtype as subtype conversion or subtype switch.26 our results indicate that evolution within cancer cell populations during metastasis leads to more transcriptomic and phenotypic changes than might be expected from the acquisition of genetic events . 
 molecular subtype switching in luminal atype breast cancer the pam50 subtype , subtype conversion only partly reects the changes occurring in tumor evolution , and our data indicate that , even the molecular without change of phenotype may still be different in the metastasis , especially in low - grade tumors . 
in multidimensional scaling , tumors of luminal a subtype in bclpm form a different cluster than luminal a subtype in the primary ( fig 2 ) , and , in the differential expression heatmap , they cluster with more aggressive subtypes ( fig 3 )  . 
along that line , relevant gene expression changes in brain metastases were found without change of pam50 subtype.28 when pam50 subtype conversion occurs , it can be regarded as an indicator of genetic remodeling in metastasis and as a risk factor of additional disease progression . 
of note , although estrogen receptor and her2 statuses as predictive clinical markers remained unchanged in this group , the proliferation rate greatly increased , and breast hormone receptor signaling declined . 
as such , although therapeutically relevant markers were unaltered upon subtype switch , remarkable changes occurred that are generally associated with a worsened malignant behavior and prognostic impact . the metastatic tumor cell the gene expression changes that were evident across all subtypes included evidence for increased mobility and invasive behavior of through downregulation of cytokeratins ( krt5 / 14 ) 29 ; tumor activation through downregulation of mmp1130 , 31 ; and deactivation of estrogen receptordependent pathways , as indicated by the downregulation of the progesterone receptor and the ankyrin repeat - domain gene ( ankrd30a )  . conversely , genes involved in emt and growth signaling were upregulated in all molecular subtypes ( data supplement 2 , fig s3 )  . 
as far as datasets are comparable , most changes found in bclpm have been reported to occur also in other metastatic sites , 27 , 32 with the exception of her2 , which was demonstrated to be upregulated in the brain28 ; however , it was not in our dataset . luminal a cancers undergoing a subtype switch were characterized by a much higher variability in gene expression in bclpm compared with high - risk carcinomas . this variability affected various molecular pathways . 
from gene expression data alone , it appears not possible to name a common denominator for the changes in metastasis of luminal a cancers that affected several dozens of genes . 
when trying to dissect the alterations that occur in subtype conversion , we found that primary tumors with low expression of inammation - related genes but increased expression of growth signalingassociated genes were more likely to undergo subtype conversion . 
in the metastatic tumor cell , the subtype switch was characterized by quite extensive gene expression changes of various molecular pathways , similar to the nding of important gene expression changes in  . 
100 genes in brain metastasis.29 no hypothesis about the root cause of changes for subtype conversion can be put forward , but the manifold changes of expression status in bclpm from luminal a cancers do indicate a major reprogramming of the tumor cell . limitations of this study include the use of a curated gene list . 
last but not least , although the discrimination of patients with breast cancer into specic cancer subtypes is a widely applied clinical concept with profound prognostic implications , luminal a and b cancers may much more represent a continuum rather than true distinct subtypes from a biologic point of view . in conclusion , we have shown that luminal atype breast cancer is most affected by subtype conversion and differential gene expression in matched metastases to lung or pleura involving but not limited to estrogen receptor signaling ( downregulation ) and tumor proliferation ( upregulation )  . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . carlo fremd honoraria : roche , pzer , celgene , astrazeneca , amgen consulting or advisory role : roche , pzer travel , accommodations , expenses : celgene , roche ralph wirtz employment : stratifyer molecular pathology gmbh stock and other ownership interests : stratifyer molecular pathology gmbh consulting or advisory role : janssen oncology ( inst ) , biontech ag ( inst ) research funding : janssen research & development ( inst ) patents , royalties , other intellectual property : royalties from biontech ( inst ) ; royalties from qiagen ( inst ) thordur oskarsson stock and other ownership interests : neovasc , cyclacel pharmaceuticals , windtree therapeutics , enzon pharmaceuticals patents , royalties , other intellectual property : migrastatins and uses thereof . 
description : the present invention provides compounds , pharmaceutically acceptable compositions thereof , and methods of using the same . other relationship : genentech j org heil honoraria : roche , siemens healthcare diagnostics , bard consulting or advisory role : roche , siemens healthcare diagnostics research funding : bard travel , accommodations , expenses : celgene andreas schneeweiss honoraria : roche pharma ag , celgene , astrazeneca , pzer , novartis , msd oncology , lilly , tesaro research funding : roche pharma ag ( inst ) , celgene ( inst ) , abbvie , molecular partners expert testimony : roche , astrazeneca travel , accommodations , expenses : roche , celgene hauke winter expert testimony : astrazeneca travel , accommodations , expenses : astrazeneca peter sinn honoraria : nanostring technologies , roche pharma ag research funding : roche pharma ag travel , accommodations , expenses : nanostring technologies no other potential conicts of interest were reported . references vanharanta s , massagu e j : origins of metastatic traits . 
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thompson am , jordan lb , quinlan p , et al : prospective comparison of switches in biomarker status between primary and recurrent breast cancer : the breast recurrence in tissues study ( brits )  . 
at resistance depends on activation of bypass pathways or development of secondary mutations in the driver oncogene.2 , 3 oncogenic fusions involving the ros1 gene are detected in approximately 1% to 2% of patients with nsclc and the majority of cases occur in tumors of adenocarcinoma histology.4 they are generated by the fusion of the 3 ( cid : 1 ) end of the ros1 gene containing the tyrosine kinase with a variety of partner genes , including gopc , slc34a2 , cd74 , tpm3 , sdc4 , ezr , lrig3 , kdelr2 , ccdc6 , among others.4 ros1 fusions function as primary oncogenic drivers following the oncogene addiction model.4 indeed , inhibition of ros1 with the small molecule tyrosine kinase inhibitor ( tki ) crizotinib induced durable responses in 72% of the patients with an ros1 - positive nsclc in the profile - 1001 study ( clinicaltrials.gov identier : nct00585195 ) , 5 leading to approval of crizotinib by regulatory agencies for this patient population . 
other tkis , including ceritinib , 6 lorlatinib , 7 and entrectinib , 8 have shown activity in this patient population as the eld evolves . our understanding of the landscape of resistance mechanisms for ros1 - positive nsclc is still evolving . although several tki options currently exist for patients with alk - positive nsclc and secondary alk mutations , effective tkis are limited for the ros1 - positive lung cancer population in the postcrizotinib setting.9 , 10 importantly , the molecular mechanisms of resistance are variable , which poses a barrier for the study of different drugs or drug combinations in molecularly dened cohorts of progressing patients in the form of clinical trials . 
here , we describe an unusual case of a ros1 - positive patient who developed a ros1 f2004v secondary mutation and whose cancer progressed in the brain and bone sites after 34 months of treatment with entrectinib , followed by dramatic response to lorlatinib . 
an ophthalmologic examination revealed a right amelanotic perifoveal choroidal tumor . subsequent evaluation with positron emission tomography / computed tomography scanning conrmed metastatic disease with multiple left lung nodules , left hilar lymphadenopathy , and bone lesions . 
interestingly , choroidal metastases have been reported in a number of cases of alk11 , 12 or other oncogene - driven lung cancer.13the patient was treated with radiation therapy to the metastatic lesion in the right eye , followed by rst - line crizotinib . 
the patient was initially treated at the recommended phase ii dose of 600 mg orally once daily for the rst eight 28 - day cycles , but she experienced asymptomatic progression in multiple , small brain metastases , which led to a decision to escalate the dose to 800 mg orally once daily , resulting in decrease in disease burden in the braafter another 18 cycles of entrectinib , an increase in the size of the brain lesions was noted . 
magnetic resonance images of brain ( t1 axial postcontrast series ) ( a ) before and ( b ) 1 month after treatment with lorlatinib , showing signiresponse of all cant metastatic brain lesions . progression in the brain developed with concomitant progression in osseous metastases . at the time of brain and bone progression , a secondary ros1 f2004v mutation was detected via circulating cellfree dna testing using a commercial next - generation sequencing assay . 
at that time , the patient was removed from the startrk - 1 study and therapy was begun with brigatinib , a compound approved for the treatment of lung cancers with alk fusions15 but also with activity against ros1.16 , 17 in addition , radiation of a metastatic choroidal lesion diagnosis with metastatic cd74 - ros1positive lung cancer entrectinib dose escalation ( 800 mg ) sar to progressing brain lesions progression in the brain and bones progression in the brain progression in the brain progression in the brain and bones crizotinib months entrectinib months brigatinib x1 month lorlatinib x6 months ( ongoing ) radiation to thoracic spine lesions detection of ros1 f2004v secondary mutation ( cfdna ) treatment x10 days fig 2 . 
secondary mutations in ros1 fusions reported in in vitro experiments in the literature and differential sensitivity to drugs with known ros1 activity . the numbers in the boxes correspond to the average concentration ( cave ) for the drug in steady state in patients , divided by the drug ic50 values calculated in ba / f3 cells expressing the mutant ros1 molecules . 
repotrectinib is another potent ros1 inhibitor with activity against many of these secondary mutations ; it is not shown in the gure because the rp2d is not known yet for this drug.29 cave values were calculated as the ratio of the area under the curve for 0 to 24 hours to 24 hours of the drug at steady state in patients.35 , 36 secondary mutations that have been reported in clinical cases are highlighted in red and the ros1 f2004v mutation described clinically for the rst time in this report is highlighted in purple . 
 ( * ) these alk mutations also are found in neuroblastoma . brigatinib had activity against cd74 - ros1 with f2004l in vitro.18 the patient also underwent radiation therapy for several thoracic spine progressing lesions . 
disease remains in control 6 months later , while the patient continues receiving lorlatinib ( fig 2 )  . although , to our knowledge , the ros1 f2004v has not been described before in patients , it is analogous to the alk f1174l mutation described in patients with neuroblastoma19 and confers paradoxic apoptosis to ba / f3 cells , a result of excessive oncogenic signaling through ros1 and p38mapk.20 also , a secondary f1174v mutation has been described in alk - positive lung cancer with resistance to crizotinib.21 instead , ba / f3 cells expressing ros1 f2004v low - dose ros1 inhibitor.20 survive in the presence of however , the patient in this report did not respond to withdrawal of brigatinib , and symptoms , namely headaches , developed from brain metastases . 
given that the concept of tki addiction is suggested in preclinical models only , 23 - 25 the approach of tki withdrawal cannot be recommended unless there is additional clinical validation . the molecular spectrum of secondary resistance to crizotinib and other ros1 inhibitors in ros1 fusionpositive patients with lung cancer is not yet well established . 
the two largest published cohorts include 16 patients from massachusetts general hospital26 and 12 patients from the university of colorado.27 in the massachusetts general hospital cohort , nine of 16 patients had a ros1 secondary mutation identied at the time of progression . 
seven of these patients harbored the g2032r mutation and none had the f2004v mutation . conversely , a ros1 compound secondary mutation ( l2026m / l1951r ) was identied in one of 12 patients in the university of colorado cohort . 
in addition , in this cohort , there was also activation of the her2 axis , an activating kit d816g mutation and a ctnnb1 s45f mutation , each case a possible mechanism of resistance to ros1 inhibition . 
figure 3 summarizes the in vitro10 , 16 , 20 , 28 - 33 and clinical10 , 27 , 30 , 34 data of ros1 secondary mutations reported in the literature . molecular testing during the course of treatment with a targeted therapy often identies molecular mechanisms of resistance to the inhibition of the driver oncogene.1 in the best - characterized cases of secondary resistance , there are data from large cohorts of patients and available effective tkis , as is the case with egfr t790m secondary mutation . nevertheless , predicting response to a tki in the presence of a secondary mutation in the driver oncogene might rely on less extensive clinical data or on preclinical data only . patient - derived cell lines , 27 ba / f3 cells engineered to express and depend on the driver oncogene of interest with and without the secondary mutation , 10 and xenograft tumors in mice29 serve as resistance models in many of these scenarios . 
in vitro development of resistance in cell - line models is possible by exposure to increasing doses of tkis for several months.37 alternatively , brief exposure to the mutagen n - ethyl - n - nitrosourea , followed by drug screening , can select for resistant clones.20 , 28 finally , it is possible for a clinician to encounter a case of resistance for which molecular testing might identify resistance mechanisms not described before . 
reports of such n of 1 cases , including outcomes , is an important resource that can guide treatment of cases with rare acquired resistance mutations . in conclusion , we show that ros1 f2004v secondary mutation confers resistance to entrectinib and brigatinib , and possibly other ros1 inhibitors , but is sensitive to lorlatinib . 
ou stock and other ownership interests : turning point therapeutics honoraria : pzer , roche pharma ag , roche , ariad / takeda , astrazeneca , foundation medicine , merck consulting or advisory role : pzer , roche , astrazeneca , takeda , foundation medicine , turning point therapeutics , ignyta speakers bureau : genentech , astrazeneca , takeda research funding : pzer ( inst ) , roche pharma ag ( inst ) , astrazeneca / medimmune ( inst ) , astrazeneca ( inst ) , ariad ( inst ) , ignyta ( inst ) , astellas pharma ( inst ) , chugai pharma ( inst ) , revolution medicines ( inst ) robert c . 
 lorlatinib for ros1 - positive lung cancer with f2004v mutation acknowledgment lorlatinib was obtained by the pzer expanded access program ( clinicaltrials.gov identier : nct03178071 )  . references pakkala s , ramalingam ss : personalized therapy for lung cancer : striking a moving target . 
jci insight 3 : e120858 , 2018 pao w , miller va , politi ka , et al : acquired resistance of lung adenocarcinomas to getinib or erlotinib is associated with a second mutation in the egfr kinase domaplos med 2 : e73 , 2005 turke ab , zejnullahu k , wu yl , et al : preexistence and clonal selection of met amplication in egfr mutant nsclc . 
clin cancer res 19 : 4040 - 4045 , 2013 shaw at , ou sh , bang yj , et al : crizotinib in ros1 - rearranged non - small - cell lung cancer . 
n engl j med 371 : 1963 - 1971 , 2014 lim sm , kim hr , lee js , et al : open - label , multicenter , phase ii study of ceritinib in patients with non - small - cell lung cancer harboring ros1 rearrangement . j clin oncol 35 : 2613 - 2618 , 2017 shaw at , felip e , bauer tm , et al : lorlatinib in non - small - cell lung cancer with alk or ros1 rearrangement : an international , multicentre , open - label , singlearm rst - in - man phase 1 trial . 
lancet oncol 18 : 1590 - 1599 , 2017 drilon a , siena s , ou si , et al : safety and antitumor activity of the multitargeted pan - trk , ros1 , and alk inhibitor entrectinib : combined results from two phase i trials ( alka - 372 - 001 and startrk - 1 )  . 
facchinetti f , loriot y , kuo ms , et al : crizotinib - resistant ros1 mutations reveal a predictive kinase inhibitor sensitivity model for ros1and alk - rearranged lung cancers . 
clin cancer res 22 : 5983 - 5991 , 2016 jiang k , brownstein s , sekhon hs , et al : ocular metastasis of lung adenocarcinoma with elm4 - alk translocation : a case report with a review of the literature . saudi j ophthalmol 27 : 187 - 192 , 2013 12 . 
weickhardt aj , scheier b , burke jm , et al : local ablative therapy of oligoprogressive disease prolongs disease control by tyrosine kinase inhibitors in oncogeneaddicted non - small - cell lung cancer . 
gettinger sn , bazhenova la , langer cj , et al : activity and safety of brigatinib in alk - rearranged non - small - cell lung cancer and other malignancies : a singlearm , open - label , phase 1 / 2 trial . 
anjum r , vodala s , kohlmann a , et al : an in vitro mutagenesis screen identies l1951r and g2032r as drug - resistant mutants of cd74 - ros1 . 
debruyne dn , bhatnagar n , sharma b , et al : alk inhibitor resistance in alk ( f1174l ) - driven neuroblastoma is associated with axl activation and induction of 20 . 
suda k , tomizawa k , osada h , et al : conversion from the oncogene addiction to drug addiction by intensive inhibition of the egfr and met in lung cancer with activating egfr mutation . 
gainor jf , tseng d , yoda s , et al : patterns of metastatic spread and mechanisms of resistance to crizotinib in ros1 - positive non - small - cell lung cancer . 
drilon a , ou si , cho bc , et al : repotrectinib ( tpx - 0005 ) is a next - generation ros1 / trk / alk inhibitor that potently inhibits ros1 / trk / alk solvent - front 30 . 
song a , kim tm , kim dw , et al : molecular changes associated with acquired resistance to crizotinib in ros1 - rearranged non - small cell lung cancer . 
zou hy , li q , engstrom ld , et al : pf - 06463922 is a potent and selective next - generation ros1 / alk inhibitor capable of blocking crizotinib - resistant ros1 mutations . 
lacy s , nielsen j , yang b , et al : population exposure - response analysis of cabozantinib efcacy and safety endpoints in patients with renal cell carcinoma . cancer chemother pharmacol 81 : 1061 - 1070 , 2018 36 . 
zhang s , anjum r , squillace r , et al : the potent alk inhibitor brigatinib ( ap26113 ) overcomes mechanisms of resistance to rstand second - generation alk inhibitors in preclinical models . 
nelson - taylor sk , le at , yoo m , et al : resistance to ret - inhibition in ret - rearranged nsclc is mediated by reactivation of ras / mapk signaling . 
 phase i study of the braf inhibitor vemurafenib in combination with the mammalian target of rapamycin inhibitor everolimus in patients with braf - mutated malignancies vivek subbiah shiraj sen kenneth r . 
are co - first authors and contributed equally to this work . the funders had no role in the design of the study ; the collection , analysis , and interpretation of the data ; the writing of the article ; and the decision to submit the article for publication . corresponding author : vivek subbiah , md , department of investigational cancer therapeutics ( phase i clinical trials program ) , unit ( continued ) purpose parallel activation of the phosphatidylinositol 3 - kinasemammalian target of rapamycin pathway represents a mechanism of primary and acquired resistance to braf - targeted therapy , but the two pathways have yet to be cotargeted in humans . 
we performed a phase i study to evaluate the safety and activity of the braf inhibitor vemurafenib in combination with the mammalian target of rapamycin inhibitor everolimus in braf - mutated advanced solid tumors . patients and methods we performed a 3 + 3 dose - escalation study with escalating doses of both oral ( po ) vemurafenib administered twice a day and po everolimus administered daily . results twenty patients with advanced cancers were enrolled . 
 patients were heavily pretreated with prior braf or mek inhibitors ( n = 11 ) , phase i clinical trial therapy ( n = 10 ) , surgery ( n = 18 ) , radiation therapy ( n = 11 ) , and chemotherapy ( n = 13 )  . 
one of the two pediatric patients initially experienced grade 3 rash , but after dermatologic intervention , the patient remains on trial with partial response and no dose reduction at time of analysis . 
one pediatric patient with pleomorphic xanthroastrocytoma remains on protocol with continued clinical response after 38 cycles . conclusion the combination of vemurafenib 720 mg po twice a day and everolimus 5 mg po daily is safe and well tolerated and has activity across histologies , with partial responses noted in advanced nonsmall - cell lung cancer , melanoma , optic nerve glioma , and xanthroastrocytoma , including patients who previously experienced progression on braf and / or mek inhibitor therapy . 
a recent basket study of vemurafenib demonstrated clinical activity in patients with advanced nonmelanoma cancers.8 unfortunately , many patients exhibited primary and acquired resistance to therapy . reactivation of mapk signaling and activation of alternative parallel signaling pathways such as the phosphatidylinositol 3 - kinase ( pi3k ) mammalian target of rapamycin ( mtor ) pathway have been hypothesized to contribute to primary and acquired resistance to braf - targeted therapy.9 , 10 specifically , akt mutation , 9 pten deficiency , 11 and downstream mtor activation have been implicated in resistance to both vemurafenib and dabrafenib in metastatic melanoma , both clinically and in preclinical models.12 , 13 preclinical studies have demonstrated efficacy in dual targeting of both the mapk and pi3kmtor pathways in various tumor types.14 , 15 we also recently identified a strong association between co - occurring pi3k - mtor pathway aberrations and primary resistance to braf targeted therapy . 
all patients were treated at the clinical center for targeted therapy at the university of texas md anderson cancer center from september 2013 to august 2015 and met all the eligibility criteria . 
patients who had a myocardial infarction within 3 months before starting treatment , who were pregnant or breastfeeding , or who had undergone a major surgical procedure within 28 days were excluded . 
palliative radiation therapy was allowed during the study treatment . study design and treatment this study was a nonrandomized , open - label , standard 3 + 3 dose - escalation study of oral ( po ) vemurafenib , starting at a dose of 720 mg daily , and po everolimus , starting at a dose of 5 mg daily , for 28 days . 
the primary objectives were to determine the safety , mtd , and dose - limiting toxicity ( dlt ) of the combination of vemurafenib and everolimus in patients with braf - mutated advanced cancer . 
 two pediatric patients were enrolled onto this study and were treated with vemurafenib po 480 mg daily and everolimus po 2.5 mg daily , per body surface areabased dose adjustment . if a patient experienced new grade 3 or greater toxicity , treatment was withheld until the condition was addressed and it recovered to grade 1 or baseline . 
the patients continued treatment until they experienced disease progression or intolerable toxicities , until the treating physician or patient felt that it was not in the patients best interest to continue for any reason , or until consent was withdrawn for any other reason . all patients were evaluated for dlts during the first 28 days and evaluated in clinic every 28 days before initiation of each subsequent cycle . 
dlts were treatment - related grade 3 or 4 nonhematologic toxicities , as defined by the national cancer institute common terminology criteria for adverse events version 3.0 , or grade 4 hematologic toxicities lasting > 7 days or accompanied by fever or bleeding during the first 4 weeks of therapy . 
clinical laboratory improvement amendments ( clia ) certified next generation sequencing data were collected for each patient . response to therapy was assessed using response evaluation criteria in solid tumors ( recist ) version 1.1. 
median time to disease progression and os duration were determined using the kaplan - meier method . circulating tumor dna three patients agreed to have an optional blood draw to measure circulating tumor dna ( ctdna ) at baseline and at each restaging . 
at least 8 ng of unamplified cell - free dna were tested with a droplet digital polymerase chain reaction brafv600e mutationspecific assay and / or multiplex brafv600 screening kit ( bio - rad , hercules , ca ) to distinguish the wild - type allele from the three most common mutations ( brafv600e , brafv600k , and brafv600r ) using the qx200 droplet digital pcr system ( bio - rad ) according to the manufacturers standard protocol . 
planned dose schedule in patients who received the combination of vemurafenib and everolimus in a phase i study dose level vemurafenib orally twice a day ( mg ) everolimus orally once a day ( mg ) * body surface area - based dose adjustment was made ( level 1 ) for pediatric patients dose level 1 was the recommended phase ii dose . frequency for the multiplexed screening assay and < 0.1% mutant allele frequency per single well for the mutation - specific assays . results patient characteristics the demographics and clinical characteristics of the 20 patients who received at least one dose of vemurafenib and everolimus and completed the dlt window are listed in table 2 . 
the most common tumor types were melanoma ( n = 7 ) , glioma ( n = 5 ) , and thyroid cancer ( n = 4 ) , and one patient each had appendiceal cancer , colorectal cancer , nsclc , and unknown primary cancer . 
patients were heavily pretreated with prior therapies , including braf or mek inhibitor therapy ( n = 11 ) , prior phase i clinical trial therapy ( n = 10 ) , surgery ( n = 18 ) , radiotherapy ( n = 11 ) , and chemotherapy ( n = 13 )  . 
one patient had measurable disease but not evaluable disease , and a second patient was not evaluable as a result of withdrawing consent before the first restaging , but both patients cleared the toxicity window and therefore are included in the toxicity evaluation but not in the response evaluation . safety all 20 patients were evaluated for dlts . 
 five patients ( 25% ) had everolimus dose reductions throughout their therapy as a result of toxicity , and one patient had the vemurafenib dose reduced as a result of renal insufficiency . 
two patients without dlts withdrew consent before disease progression . response of the 18 patients evaluable for response , 11 patients ( 61% ) had objective tumor volume reduction as best response . 
two patients were not evaluable for response ; one patient had measurable but not evaluable disease and a second patient completed the dlt window but withdrew consent before restaging . prs were seen in patients with nsclc , melanoma , optic nerve glioma , and pleomorphic xanthroastrocytoma . 
of the nine patients who had disease progression on braf or mek inhibitors before enrolling onto this study , two ( 22% ) had prs and five ( 56% ) had stable disease with vemurafenib and everolimus . at the time of analysis , 14 patients ( 78% ) had discontinued therapy because of disease progression . 
 ( b ) representative restaging images of a patient with metastatic melanoma and a pten ( p95s ) mutation who had a partial response on vemurafenib and everolimus after disease progression on vemurafenib and px866 , an investigational pi3k inhibitor . 
astro , anaplastic astrocytoma ; at , anaplastic thyroid ; crc , colorectal cancer ; cup , cancer of unknown primary ; glio , glioblastoma ; glioma , optic nerve glioma ; mel , melanoma ; pt , papillary thyroid . prior braf or mek inhibitor use no prior braf or mek inhibitor use still on trial without progression pleomorphic xanthroastrocytoma remains on protocol with continued clinical response after 38 cycles . genomic profiling all evaluable patients underwent clia - certified clinical next - generation sequencing of at least 50 genes before trial enrollment . 
among the four patients who achieved pr , a patient with metastatic melanoma who had previously experienced progression on vemurafenib plus an investigational pi3k inhibitor ( px866 ) had a concurrent pten ( p95s ) mutation . 
a patient with nsclc whose tumor harbored an idh1 ( r132c ) mutation , arid2 alteration ( splice site 92 + 1 g > a ) , and ppp1r2a mutation ( r183w ) achieved a pr after previously experiencing progression on dabrafenib . 
all concurrent molecular aberrations and best response to therapy for each patient are listed in table 4 . circulating tumor dna patients enrolled onto this study were offered an optional blood draw to measure ctdna throughout the course of the study . 
 a third patient with a brafv600e mutation had detectable ctdna levels , which were measured at four time points throughout therapy and are depicted in figure 2 alongside the patients scans . 
dlts included fatigue ( 20% ) and rash ( 15% ) , but both were present in only one patient at the mtd and recommended phase ii dose of vemurafenib 720 mg twice daily and everolimus 5 mg daily . 
 the patient with glioblastoma who experienced a grade 3 rash had a resolution of his rash after initial everolimus discontinuation and was able to resume everolimus at a dose of 2.5 mg daily without toxicity . 
 the use of nonpharmacologic approaches , such as physical and mindfulness - based exercise therapy , 20 , 21 and pharmacologic approaches , such as corticosteroids22 in treating cancerand therapy related fatigue , has recently demonstrated efficacy in randomized controlled trials and may benefit these patients moving forward . we observed only one metabolic adverse event ( hypertriglyceridemia and hyperglycemia ) , which is attributed to mtor inhibitors . 
overall , most patients at the recommended phase ii dose tolerated the treatment well . our trial also demonstrates that the addition of an mtor inhibitor to everolimus treatment is able to overcome resistance to braf and / or mek inhibition in a subset of patients with braf - mutant advanced cancers . 
graphical depiction of a patients circulating tumor dna level of brafv600e ( shown as copies per milliliter of plasma ) while on treatment with vemurafenib plus everolimus with representative restaging scans and response to therapy , per response evaluation criteria in solid tumors ( recist ) 1.1. 
pd , progression of disease ; pr , partial response . baseline baseline july 16 , 2014 october 14 , 2014 november 12 , 2014 december 10 , 2014 the 20 patients in this study had a pr to therapy . 
 next - generation sequencing performed at the time of initial diagnosis revealed idh1 ( r132c ) , arid2 ( splice site 92 + 1 g > a ) , and ppp1r2a ( r183w ) aberrations . 
unfortunately , no molecular profiling was available at the time the patient experienced progression on dabrafenib immediately before enrolling onto this trial . a second patient with metastatic melanoma and a pten ( p95s ) mutation who had recently had disease progression on vemurafenib and px866 , an investigational pi3k inhibitor , also achieved a pr on our trial ( fig 1b )  . 
in vivo , pten - deficient , braf - mutated mouse models treated with vemurafenib and pi3k inhibitors also demonstrate synergistic antitumor activity.23 interestingly , this patient experienced progression on the vemurafenib and investigational pi3k inhibitor combination but responded with a pr to vemurafenib and everolimus . 
a 78 - year - old patient with metastatic papillary thyroid cancer whose tumor harbored a pik3ca ( h1047r ) mutation had 7 months of stable disease with a 27% reduction in tumor burden before having any disease progression . 
both of these patients had no co occurring genomic alterations identified on their tumors other than the brafv600e mutation , illustrating the fact that although co - occurring alterations may lead to activation of parallel signaling pathways that increase resistance to therapy in some patients , they may render other tumors susceptible to therapy when actionable . we have previously reported our institutions experience with implementing multiplex hotspot testing and the need to enroll patients onto genotype - matched trials when feasible.24 a number of the responders on this trial further support the hypothesis that the identification of actionable alterations can help guide the right patient to the right targeted therapy . 
all nonresponders were either previously treated with braf or mek inhibitor or harbored non - v600 braf mutations . in the patient with metastatic melanoma with measurable ctdna , the level of mutated brafv600e copies per milliliter decreased with clinical response and increased before subsequent restaging that demonstrated disease progression . 
previous studies have identified an association between detection and levels of circulating tumor dna and disease burden and survival across braf and mek inhibitor trials.27 it is plausible that this may explain why only wildtype braf was detectable in the plasma of these two patients . vitro data suggest that the combination of mek and mtor inhibition may be more effective than braf and mtor inhibition in activating bax and caspase - 3 and inducing caspase - dependent apoptosis in melanoma cell lines.31 future studies to investigate the safety and effectiveness of combining everolimus with both braf and mek inhibitors together are being planned . our study has several limitations , including a small patient population and the variety of molecular profiling platforms used . 
although all patients underwent clia - certified next generation sequencing of at least 40 genes , including those in the pi3k - mtor pathway , some patients underwent more extensive 400gene profiling . 
ideally , all patients should have a broader panel of testing to identify all actionable aberrations , both at the time of study initiation and at disease progression , to better understand the clonal evolution of tumors treated with vemurafenib and everolimus . 
 sherman , patrick hwu manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors in conclusion , the combination of vemurafenib and everolimus is safe and well tolerated and has clinical activity across histologies , with prs noted in patients with advanced nsclc , melanoma , optic nerve glioma , and xanthroastrocytoma . 
our results do not support the addition of everolimus based solely on alterations in the pi3k - mtor pathway ; however , given the responders who did harbor concurrent mutations in this pathway , further investigation in a larger cohort of molecularly matched patients with uniform genomic profiling is warranted . 
hess no relationship to disclose filip janku stock and other ownership interests : trovagene consulting or advisory role : deciphera , trovagene , novartis , sequenom , foundation medicine , guardant health , immunome research funding : novartis , biomed valley discoveries , roche , agios , astellas pharma , deciphera , symphony evolution , plexxikon , piqur , fujifilm other relationship : bio - rad david s . 
hong stock and other ownership interests : molecularmatch , oncorena honoraria : adaptimmune , baxter , merrimack , bayer consulting or advisory role : baxter , bayer , guidepoint global , janssen speakers ' bureau : genentech , janssen oncology research funding : novartis , genentech , eisai , astrazeneca , pfizer , mirna therapeutics , amgen , daiichi sankyo , merck , mirati therapeutics , eli lilly , adaptimmune , abbvie , bayer , bristol - myers squibb , genmab , ignyta , infinity pharmaceuticals , kite pharma , kyowa hakko kirin , loxo , medimmune , molecular templates , takeda travel , accommodations , expenses : loxo , mirna therapeutics other relationship : oncorena , eisai soumen khatua no relationship to disclose research funding : phosplatin therapeutics travel , accommodations , expenses : phosplatin therapeutics javier munoz consulting or advisory role : kite pharma , pfizer , pharmacyclics , bayer , alexion pharmaceuticals , bristolmyers squibb , janssen , seattle genetics , gilead sciences , kyowa hakko kirin speakers ' bureau : kite pharma gerald s . 
falchook employment : sarah cannon research institute , healthone research funding : millennium , emd serono , celgene , medimmune , genmab , vegenics , novartis , astrazeneca , incyte , armo biosciences , kolltan pharmaceuticals , 3 - v biosciences , abbvie , aileron therapeutics , delmar pharmaceuticals , effector therapeutics , strategia therapeutics , fujifilm , hutchison medipharma , regeneron , biothera , curegenix , curis , eli lilly , jounce therapeutics , oncomed , precision oncology , syndax , taiho pharmaceutical , tesaro , takeda , beigene , ignyta , merck , rgenix , tarveda therapeutics , tocagen patents , royalties , other intellectual property : handbook of targeted cancer therapy travel , accommodations , expenses : millennium , sarah cannon research institute , emd serono , bristol - myers squibb roman groisberg no relationship to disclose apostolia m . 
tsimberidou consulting or advisory role : roche research funding : emd serono ( inst ) , baxter ( inst ) , foundation medicine ( inst ) , onyx ( inst ) , bayer ( inst ) , boston biomedical ( inst ) , placon ( inst ) , immatics ( inst ) , karus therapeutics ( inst ) , stem cells ( inst ) , obi pharma ( inst ) steven i . 
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penna i , molla a , grazia g , et al : primary cross - resistance to brafv600e - , mek1 / 2and pi3k / mtor - specific inhibitors in braf - mutant melanoma cells counteracted by dual pathway blockade . 
 clinical utility of clia - grade ar - v7 testing in patients with metastatic castration - resistant prostate cancer purpose a splice variant of the androgen receptor , ar - v7 , confers resistance to ar - targeted therapies ( atts ) but not taxane chemotherapies in patients with metastatic castrationresistant prostate cancer . 
since august 2015 , a clinical - grade assay to detect ar - v7 messenger rna expression in circulating tumors cells ( ctcs ) has been available to providers through a clinical laboratory improvement amendmentscertified laboratory at johns hopkins university . methods we contacted ordering providers of the first 150 consecutive tests by using a questionnaire - based survey to determine how the results of ar - v7 testing were used to influence clinical practice . results in all , 142 ( 95% ) of 150 questionnaires were completed by 38 providers from 29 sites across the united states and canada . 
prevalence of ar - v7 detection increased with prior exposure to atts ( abiraterone and enzalutamide nave , 22% ; after abiraterone or enzalutamide , 35% ; after abiraterone and enzalutamide , 43% )  . 
2017 by american society of clinical oncology introduction metastatic castration - resistant prostate cancer ( mcrpc ) remains dependent on androgen receptor ( ar ) signaling for survival in the presence low testosterone levels.1 two novel artargeted therapies ( atts ) , abiraterone and enzalutamide , induced high objective response rates and improved overall survival in patients with mcrpc.2 , 3 treatment with abiraterone or enzalutamide in the prechemotherapy setting led to a 50% decline in prostate - specific antigen ( psa50 ) response in 62% and 78% of these patients , respectively.2 , 3 however , sequential use of abiraterone and enzalutamide has resulted in much lower psa response rates with the use of the second att agent ( ie , abiraterone after enzalutamide , 10% to 15% ; enzalutamide after abiraterone , 20% to 30% ) .4 - 6 these data underscore the importance of identifying a predictive biomarker of resistance to att to prevent the use of subsequent futile therapy . potential mechanisms of resistance to att include ar amplification and mutation , as well as the expression of arsplicevariants.7a well - characterized mark c . 
eisenberger jun luo emmanuel s . antonarakis author affiliations and support information ( if applicable ) appear at the end of this article . the content is solely the responsibility of the authors and does not necessarily represent the official views of the national cancer institute or the national institutes of health . corresponding author : emmanuel s . 
 splice variant , ar - v7 , is a truncated form of fulllength ar ( ar - fl ) that lacks the ligand - binding domain but retains both the transactivation and dna - binding domains , allowing for constitutive ar signaling in the absence of androgen.8 - 10 in patients with mcrpc , detection of ar - v7 in circulating tumors cells ( ctcs ) was shown to predict resistance to novel atts.11 - 13 moreover , ar - v7 + patients had a significantly shorter overall survival , suggesting a prognostic value of ar - v7 in addition to its use as a predictive biomarker.11 , 13 chemotherapy is an alternative to att for arv7 + patients with mcrpc . 
detection of ar - v7 has been shown not to preclude response to taxanebased chemotherapy.14 , 15 further prospective investigation found a significant survival benefit with the use of taxanes versus atts in patients with ar - v7 + disease.16 interestingly , the presence of this splice variant is a dynamic feature with possible conversion from ar - v7 + to ar - v7 status after chemotherapy with taxanes.14 , 17 these data suggest that serial arv7 testing may guide the clinical treatment of patients with mcrpc . 
those patients with ar - v7 prostate cancer may continue to benefit from att , whereas chemotherapy may be more effective in patients with detectable ar - v7 transcript.18 if the clinical utility of ar - v7 testing can be confirmed , it may also have an economic benefit . in a recent study , we modeled the cost of treating all patients with mcrpc with abiraterone or enzalutamide versus using ar - v7 testing to direct treatment.19 by using a clinical scenario in which ar - v7 + patients were changed from treatment with abiraterone and / or enzalutamide to chemotherapy thus avoiding the cost of futile att therapy , ar - v7 testing resulted in a theoretical cost savings to the health care system of $150 million per year . 
to this end , since august 2015 , clinicalgrade ar - v7 testing performed in a clinical laboratory improvement amendments ( clia ) certified laboratory at johns hopkins university has been available to health care providers for clinical use.20 however , despite the commercial availability of this ar - v7 test , its clinical utility is unknown . 
here , we have retrospectively compiled questionnaire - based data on how ordering providers are applying the results of ar - v7 testing in their clinical practice to influence decision making . methods the analytical validation and test characteristics of our clia - grade ar - v7 assay have been described previously.20 our molecular pathology database was queried for all ar - v7 tests ordered by internal and external providers for clinical purposes . 
clinical results of this test were reported as ctc , ctc + / ar - v7 , or ctc + / ar - v7 + , because each of these categories is associated with different outcomes.12 a clinical utility questionnaire ( data supplement ) was generated for each ar - v7 test ordered and was mailed or e - mailed to each ordering provider . we defined a biomarker - based change in treatment as a confirmation of treatment choice or a change from one therapy to another after ar - v7 testing . 
the institutional review board at johns hopkins university approved this study and granted a waiver of consent to contact the provider of each ar - v7 test ordered because that was considered a clinical audit . 
the ordering provider was then contacted for participation and asked to complete a questionnaire pertaining to treatment decisions that were made on the basis of results of that specific ar - v7 test . 
if a provider did not wish to participate or the questionnaire was not returned after two attempts to contact the provider , data for that patient were not included in the analysis . one hundred fifty consecutive ar - v7 clinical test results were obtained between august 31 , 2015 , and august 31 , 2016 , representing the first 150 tests ordered . 
from these , 142 questionnaires ( 95% ) were completed and returned by 38 providers across 29 sites ( 28 in the united states [ in 22 states ] and one in canada )  . 
the significance level was set at p , .05 , and corrections were not performed for multiple comparisons . results information from 142 of 150 questionnaires sent ( 95% participant response rate ) were included in this analysis . 
the number of lines of additional systemic therapies for mcrpc among these patients was reported as follows : 24% ( n = 33 ) had received no other lines , 27% ( n = 39 ) had received one line , 27% ( n = 39 ) had received two lines , 13% ( n = 18 ) had received three lines , and 9% ( n = 13 ) had received four or more lines of systemic therapy before testing . 
of tests abiraterone enzalutamide abiraterone or enzalutamide abiraterone enzalutamide nave test result ctc 40 / 142 28.17 ctc + / ar - v7 42 / 142 29.58 8 / 40 20.0 11 / 40 27.5 21 / 40 52.5 8 / 42 19.0 20 / 42 47.62 14 / 42 33.33 ctc + / ar - v7 + 60 / 142 42.25 26 / 60 43.33 21 / 60 35.0 13 / 60 21.66 abbreviations : ar - v7 , androgen receptor splice variant 7 ; att , ar - targeted therapy ; ctc , circulating tumor cell ; n / n , number of patients in that category divided by total number of patients . overall , the prevalence of a ctc result was 28% , the prevalence of a ctc + / ar - v7 result was 30% , and the prevalence of a ctc + / ar - v7 + result was 42% . 
patients who were treated with abiraterone and / or enzalutamide resulted in a higher prevalence of ar - v7 detection compared with patients who were not treated with an att : 22% of treatment - nave patients were ar - v7 + ; after treatment with abiraterone or enzalutamide , 35% of patients were ar - v7 + ; and after treatment with abiraterone and enzalutamide , 43% of patients were ar - v7 +  . to assess the clinical utility of ar - v7 testing , providers were asked whether the ar - v7 status influenced their decision making . 
the majority of ar - v7 tests ( ctc or ctc + / ar - v7 ) did not change the clinical practice of the providers ( table 2 )  . 
however , almost two thirds ( 62% ) of ar - v7 + tests resulted in a change in management . in patients for whom treatment was changed , providers were then asked to specify the type of therapy selected on the basis of the test result . 
patients with an ar - v7 result ( ctc or ctc + / ar - v7 ) were preferentially treated with an att agent ( confirmed ar treatment , or changed from taxane to ar therapy )  . 
conversely , after an ar - v7 + result , most patients were changed from an att agent to taxane chemotherapy ( 43% ) or were enrolled in a clinical trial ( 43% )  . 
psa50 rr data were missing from 16% ( n = 12 ) and 22% ( n = 15 ) of questionnaires in which management was changed or not changed , respectively . we also investigated which systemic therapy was used in patients with mcrpc after progression on both abiraterone and enzalutamide , according to ar - v7 status ( table 5 )  . 
by contrast , ar - v7 + patients who had already received abiraterone and enzalutamide were more often treated on a clinical trial ( 54% ) compared with treatment using chemotherapy ( 19% )  . 
in the patients who were treated with docetaxel , those who were ar - v7 were commonly treated with standard chemotherapy ( ie , cabazitaxel ; 67% [ six of nine ] ) whereas ar - v7 + patients were more frequently placed on a clinical trial ( 58% [ 11 of 19 ] )  . finally , we examined provider treatment preferences for ar - v7 + patients irrespective of the prior therapies received ( table 6 )  . 
a minority of patients ( 7% ) received either enzalutamide or abiraterone despite an ar - v7 + result , whereas all ar - v7 + patients managed with observation ( 10% ) enrolled in hospice shortly thereafter . to summarize the data compiled from providers real - world experience with ar - v7 testing , we propose a hypothetical treatment algorithm for making decisions regarding patients with mcrpc using ar - v7 as a potential treatment - selection biomarker ( fig 1 )  . 
after first - line systemic therapy with abiraterone or enzalutamide , ar - v7 + patients would preferentially cross over to taxane - based therapy , whereas those who are ar - v7 may continue on a second att . 
because of occasional conversions from ar - v7 + to ar - v7 status , men progressing on taxane treatment can be retested and could potentially consider treatment with an att if the ar - v7 status reverts to negative . 
finally , even patients progressing after treatment with abiraterone and enzalutamide may be considered for ar - v7 testing if an ar - v7directed clinical trial is available . discussion the optimal sequencing of therapeutic agents in patients with mcrpc is unknown and remains a major challenge . 
to this end , the national comprehensive cancer network prostate cancer guidelines now suggest that ar - v7 testing can be considered and may play a role in guiding therapy selection in mcrpc , but at this time , these guidelines have not gone as far as recommending testing to determine treatment choice . 
in another recent consensus report , the majority of prostate cancer specialists polled ( 59% ) stated that ar - v7 testing would be useful for some ( majority or minority of ) patients with mcrpc.21 although further prospective validation of the predictive ability of ar - v7 is currently ongoing , here we investigated the clinical utility of ar - v7 testing in a real - world setting . we asked providers whether the result of the arv7 test influenced their clinical practice for that specific patient . 
although our findings are retrospective , they suggest that ar - v7 testing may possibly lead to improved clinical responses to treatment , at least in terms of psa50 rr . 
we did not assess for radiographic progression - free or overall survival , which may not have correlated with psa50 rr . the statistically significant psa50 rr difference between the change and no - change groups is largely driven by the ar - v7 + subgroup ( described in appendix table a3 )  . 
ar - v7 + patients for whom management did not change had a psa50 rr of 5% ( v 39% in ar - v71 patients for whom treatment was changed )  . 
we hypothesize that ar - v7 + patients in the change group were more commonly treated with chemotherapy compared with the no - change group , resulting in improved outcomes . 
we also acknowledge a high psa50 rr in ar - v7 + patients treated with chemotherapy and ar - v7 patients treated with att ( both in the change group )  . 
nonetheless , these data suggest that further prospective investigation is warranted to study biomarker - driven clinical outcomes . by using the treatment history captured by our questionnaires , we were able to observe an increasing prevalence of ar - v7 detection with prior exposure to atts , as expected . 
this finding is corroborated by the recent biomarker data from the armor3 - sv ( a study of galeterone compared with enzalutamide in men expressing androgen receptor splice variant - 7 mrna [ ar - v7 ] metastatic crpc ) trial , in which first - line patients with mcrpc who had previously received docetaxel for metastatic hormone - sensitive disease had a higher prevalence of ar - v7 detection compared with chemotherapy - nave patients.22 moreover , 79% of men with newly developed mcrpc had prior exposure to bicalutamide . 
the higher trend of ar - v7 + tests after treatment with docetaxel or bicalutamide , in the absence of a novel att , may suggest that total number of therapies ( ie , more advanced disease ) contributes to ar - v7 expression in addition to the known relationship with prior att exposure . 
another provocative hypothesis might be that docetaxel works as an ar - modulating therapy in prostate cancer , inhibiting microtubule - dependent nuclear transport of wild - type ar but not ar - v7 , thereby selecting for the emergence of ar - v7expressing clones during or after chemotherapy treatment . this study had some additional limitations . 
first , the prevalence of ar - v7 was probably overestimated compared with earlier reports because providers were more likely to order a test if the clinical scenario suggested that the test might be positive . other significant weaknesses of this analysis were its retrospective nature and reliance on selfreporting by providers . 
an audit of 14 randomly selected questionnaires determined that providers gave accurate responses to the questionnaire in most instances ( see appendix ) , suggesting that our data represent the true clinical course for each patient . 
finally , we concede that many patients will receive all atts approved by the us food and drug administration , regardless of ar - v7 status , because clinical responses to atts are sometimes observed in arv7 + patients.13 however , the clinical utility of arv7 testing may potentially be clearest in ar - v7 + patients who have additional atts still available for treatment . 
by identifying high - risk patients ( ie , arv7 + ) who have adequate performance status earlier in their mcrpc treatment , this would allow them to be treated with taxane therapy before the chemotherapy window closes . 
potential decision algorithm based on serial androgen receptor splice variant 7 ( ar - v7 ) testing across the castration - resistant prostate cancer landscape . after each line of therapy , we propose an algorithm for consideration of ar - v7 testing . 
after abiraterone and enzalutamide treatment , patients should be subject to ar - v7 testing only if an ar - v7directed clinical trial is available . ( * ) indicates limited clinical data supporting the use of atts in ar - v7 + patients that subsequently convert to ar - v7 . 
 ( ) denotes a clinical trial that does not require a positive ar - v7 test for ar - v7 patients , whereas ar - v7 + patients should consider an arv7directed trial , if available . first - line therapy clinical ar - v7 testing ? second - line therapy clinical ar - v7 testing ? third - line therapy abiraterone enzalutamide consider ar - v7directed clinical trial / taxane docetaxel taxane / trial enzalutamide abiraterone consider ar - v7directed clinical trial / taxane receive either chemotherapy or atts at their physicians discretion . we propose a decision algorithm using serial arv7 testing across the mcrpc landscape . 
this algorithm is based largely on published data suggesting that the presence of ar - v7 confers resistance to atts but does not influence response to taxane chemotherapy.11 , 13 , 14 , 16 we note that at this time , there is no clinical trial evidence to support the use of atts in ar - v7 + patients who convert to ar - v7 status after chemotherapy . 
in our study , 17 of 22 ar - v7 + patients were enrolled on a clinical trial that mandated ar - v7 detection as an entry criterion , suggesting that many of the tests in this study were ordered for the purpose of screening for a clinical trial . 
finally , there is ongoing debate in the prostate cancer community about whether ar - v7 detection is merely a proxy for ar amplification or overexpression and not an independent predictor of response to therapy or clinical outcomes.21 although several studies have shown that ar - v7 detection is indeed correlated with ar - fl expression , ar - v7 still remains independently prognostic in multivariable analysis after controlling for ar - fl levels.11 , 13 , 23 in conclusion , to our knowledge , this is the first study to examine the preliminary real - world clinical utility of clia - grade ar - v7 testing in patients with mcrpc . 
we show that ar - v7 testing influenced clinical decision making overall ( regardless of test results ) but that its utility was greatest in the setting of ar - v7 + results . 
 jun luo honoraria : gilead sciences , sanofi consulting or advisory role : tokai pharmaceuticals , sun pharmaceutical industries , janssen oncology research funding : sanofi ( inst ) , orion pharma ( inst ) , mirati therapeutics ( inst ) , gilead sciences ( inst ) , astellas pharma ( inst ) patents , royalties , other intellectual property : co - inventor of an ar - v7 biomarker technology assigned to johns hopkins university who licensed to tokai pharmaceuticals ; co - inventor of a technology licensed to qiagen emmanuel s . 
antonarakis honoraria : sanofi , dendreon , medivation , janssen biotech , essa , astellas pharma consulting or advisory role : sanofi , dendreon , medivation , janssen biotech , essa , astellas pharma research funding : janssen biotech ( inst ) , johnson & johnson ( inst ) , sanofi ( inst ) , dendreon ( inst ) , aragon pharmaceuticals ( inst ) , exelixis ( inst ) , millennium pharmaceuticals ( inst ) , genentech ( inst ) , novartis ( inst ) , astellas pharma ( inst ) , tokai pharmaceuticals ( inst ) travel , accommodations , expenses : sanofi , dendreon , medivation mark c . 
w81xwh - 15 - 2 - 0050 and w81xwh - 12 - 1 - 0605 ( j.l. ) , johns hopkins prostate specialized programs of research excellence grant p50 ca058236 ( j.l. ) , the patrick c . 
beer tm , armstrong aj , rathkopf de , et al : enzalutamide in metastatic prostate cancer before chemotherapy . n engl j med 368 : 138 - 148 , 2013 n engl j med 371 : 424 - 433 , 2014 4 . 
maughan bl , luber b , nadal r , et al : comparing sequencing of abiraterone and enzalutamide in men with metastatic castration - resistant prostate cancer : a retrospective study . 
dehm sm , schmidt lj , heemers hv , et al : splicing of a novel androgen receptor exon generates a constitutively active androgen receptor that mediates prostate cancer therapy resistance . 
hu r , dunn ta , wei s , et al : ligand - independent androgen receptor variants derived from splicing of cryptic exons signify hormone - refractory prostate cancer . 
antonarakis es , lu c , wang h , et al : ar - v7 and resistance to enzalutamide and abiraterone in prostate cancer . n engl j med 371 : 1028 - 1038 , 2014 12 . 
antonarakis es , lu c , luber b , et al : clinical significance of androgen receptor splice variant - 7 mrna detection in circulating tumor cells of men with metastatic castration - resistant prostate cancer treated with firstand second - line abiraterone and enzalutamide . 
antonarakis es , lu c , luber b , et al : androgen receptor splice variant 7 and efficacy of taxane chemotherapy in patients with metastatic castration - resistant prostate cancer . 
onstenk w , sieuwerts am , kraan j , et al : efficacy of cabazitaxel in castration - resistant prostate cancer is independent of the presence of ar - v7 in circulating tumor cells . 
scher hi , lu d , schreiber na , et al : association of ar - v7 on circulating tumor cells as a treatment - specific biomarker with outcomes and survival in castration - resistant prostate cancer . 
sprenger c , uo t , plymate s : androgen receptor splice variant v7 ( ar - v7 ) in circulating tumor cells : a coming of age for ar splice variants ? ann oncol 26 : 1805 - 1807 , 2015 19 . 
markowski mc , frick kd , eshleman jr , et al : cost - savings analysis of ar - v7 testing in patients with metastatic castration - resistant prostate cancer eligible for treatment with abiraterone or enzalutamide . 
lokhandwala pm , riel sl , haley l , et al : analytical validation of androgen receptor splice variant 7 detection in a clinical laboratory improvement amendments ( clia ) laboratory setting . 
gillessen s , attard g , beer tm , et al : management of patients with advanced prostate cancer : the report of the advanced prostate cancer consensus conference apccc 2017 . 
taplin me , antonarakis es , ferrante kj , et al : clinical factors associated with ar - v7 detection in armor3 - sv , a randomized trial of galeterone ( gal ) vs enzalutamide ( enz ) in men with ar - v7 + metastatic castration - resistant prostate cancer ( mcrpc )  . 
silberstein j , luber b , wang h , et al : clinical significance of ar mrna quantification from circulating tumor cells ( ctcs ) in men with metastatic castration - resistant prostate cancer ( mcrpc ) treated with abiraterone ( abi ) or enzalutamide ( enza )  . 
j clin oncol 35 , 2017 ( suppl ; abstr 132 ) we further queried our database to investigate the difference in the 50% decline in prostate - specific antigen ( psa50 ) response rate ( rr ) between the change and no - change groups on the basis of androgen receptor splice variant 7 ( ar - v7 ) status . 
for patients with ar - v7 + disease , the psa50 rr in those who did not change therapy was 5.3% ( one of 19 ) compared with 38.7% ( 12 of 31 ) in the change group ( table a3 )  . 
in the ar - v72 patients , the psa50 rr was 45.45% ( 15 of 33 ) in the no - change group versus 68.88% ( 22 of 32 ) in the change group . 
within both the ar - v7 + and ar - v7 groups , there was no discernible difference in number of lines of therapy between the change and no - change group . 
we also examined psa50 rr by treatment choice in those patients who had a change in treatment ( these data were not solicited for patients who did not have a treatment change in the questionnaire )  . 
in the 14 questionnaires audited , we found two minor discrepancies : ( 1 ) bicalutamide was listed in error as a prior therapy ( one time ) , and ( 2 ) the correct clinical trial was misidentified for a patient ( one time )  . subjective responses ( ie , clinical helpfulness and change in management ) were not assessed . 
the authors stated that there are currently no trials of parp inhibitors in endometrial carcinoma , mainly because mutations in brip1 , brca1 , brca2 , and other homologous recombination ( hr ) related genes are too rare , being reported overall in , 5% of cases.2 in fact , the french group gineco ( groupe dinvestigateurs nationaux pour letude des cancers ovariens ) is conducting a national randomized phase ii trial evaluating olaparib as maintenance therapy in platinumsensitive advanced uterine carcinoma ( utola trial ; clinicaltrials.gov identier : nct03745950 ; fig 1 ) versus placebo . 
we anticipate that patients with endometrial tumors might benet from a parp inhibitor , even in cases of no brca or related genetic mutations . the hr - decient ( hrd ) phenotype was initially characterized in approximately 50% of high - grade serous ovarian carcinomas , and 20% to 25% of these had no brca1 or brca2 mutations.3 importantly , this phenotype is associated with clinical activity of platinum and parp inhibitors in platinumsensitive ovarian carcinomas regardless of brca mutations.4 , 5 according to the cancer genome atlas data , the hrd phenotype was also recently reported in 25% of uterine endometrial carcinomas.6 it is almost exclusively found in the serous - like , tp53 - mutated subtype , in which it represents nearly 50% of cases.7 molecular alterations responsible for the hrd phenotype in endometrial carcinoma have not been largely explored and remain to be identied , because brca1 promoter methylation seems uncommon.7 finally , hr deciency could be induced by platinum - based chemotherapy ; a recent study showed decreased expression of rad51 protein , a key partner of hr , after neoadjuvant chemotherapy in a subset of patients with ovarian cancer.8 in tp53 wild - type endometrioid carcinoma , common molecular alterations such as pten or arid1a loss have been also associated in vitro with signicant parp inhibitor activity.9 , 10 according to these preliminary data , inclusion criteria in the utola trial are not restricted to specic molecular alterations ; rather , patients could be enrolled if their disease is platinum sensitive , a well - recognized landmark of the hrd phenotype . 
patients are stratied according to mrr and tp53 immuno - phenotypes , and a large screening of molecular alterations , including hrd prole , will be conducted to determine the best candidates for parp inhibitor maintenance . in conclusion , we agree with nakamura et al1 that parp inhibitors should be explored in patients with advanced endometrial carcinoma , beyond those with rare mutations in brca or related genes . 
the secondary end points are overall survival , response rate , quality of life , safety , time from random assignment to rst and second subsequent therapies , and time from random assignment to second disease progression or death . 
exploratory analyses included homologous recombinationdecient ( hrd ) phenotype and multiple - gene next - generation sequencing , including brca1 and brca2 and other hrd - related genes ( tp53 , pten , pole , and arid1a ) , and microsatellite instability status . 
nature 474 : 609 - 615 , 2011 [ erratum : nature 490 : 298 , 2012 ] ray - coquard i , pautier p , pignata s , et al : olaparib plus bevacizumab as rstline maintenance in ovarian cancer . 
marquard am , eklund ac , joshi t , et al : pan - cancer analysis of genomic scar signatures associated with homologous recombination deciency suggests novel indications for existing cancer drugs . 
biomark res 3 : 9 , 2015 de jonge mm , auguste a , van wijk lm , et al : frequent homologous recombination deciency in high - grade endometrial carcinomas . 
clin cancer res 25 : 1087 - 1097 , 2019 kubelac p , genestie c , auguste a , et al : changes in dna damage response markers with treatment in advanced ovarian cancer . 
cancers ( basel ) 12 : 707 - 719 , 2020 dedes kj , wetterskog d , mendes - pereira am , et al : pten deciency in endometrioid endometrial adenocarcinomas predicts sensitivity to parp inhibitors . 
 systemic and cns activity of selective ret inhibition with selpercatinib ( loxo - 292 ) in a patient with ret - mutant medullary thyroid cancer with extensive cns metastases alexander andreev - drakhlin , md1 ; maria cabanillas , md1 ; behrang amini , md , phd1 ; and vivek subbiah , md1 introduction medullary thyroid cancer ( mtc ) is a neuroendocrine tumor that originates from the parafollicular cells ( or c cells ) of the thyroid gland and accounts for 1% to 2% of thyroid cancers in the united states.1 the cns is a rare site of metastasis in mtc , with , 15 cases reported in the medical literature ; however , the prevalence may be underreported because of the infrequent use of cns imaging in patients with mtc.1 , 2 the optimal treatment approach for patients with mtc with cns involvement is currently unknown . 
although surgical resection , radiation therapy , or stereotactic radiosurgery ( srs ) may improve local disease control in some patients , most still succumb to their disease within months after the diagnosis of cns metastases.1 - 4 the proto - oncogene ret ( rearranged during transfection ) , located on chromosome 10q11.2 , is central to development of a majority of cases of mtc.5 germline gain - of - function ret point mutations result in multiple endocrine neoplasia 2 , a hereditary syndrome associated with mtc and pheochromacytoma.6 , 7 similar somatic mutations occur in approximately 50% of sporadic mtc , approximately 20% of sporadic pheochromocytomas , and rarely in other cancer types.8 - 10 recent identication of highly potent and selective ret inhibitors holds great promise in the management of these ret - driven cancers as well as other ret - altered malignancies.11 - 14 in 2018 , selpercatinib ( loxo - 292 ) received us food and drug administration breakthrough therapy designation for treatment of patients with previously treated metastatic ret - mutant mtc , ret fusion - positive nonsmall - cell lung cancer , and ret fusionpositive thyroid cancers . 
durable intracranial responses with selpercatinib have been described in patients with brain metastases with ret fusionpositive lung and thyroid cancers.15 , 17 however , the activity of selpercatinib in patients with ret - mutant mtc with brain metastases and / or leptomeningeal disease ( lmd ) has not been characterized , to our knowledge . 
here , we describe a patient with ret m918tmutant mtc with extensive cns metastases who had a clinically meaningful durable response to selective ret inhibition with selpercatinib . case report total a 67 - year - old woman presented to a local hospital with bilateral neck swelling . 
positron emission tomography imaging showed metabolically active , left - side , level v lymph nodes ; a 1.1 - cm , hypermetabolic , right lower lobe lung nodule ; several hypermetabolic liver lesions ; as well as lytic lesions in the c5 vertebral body , l4 left lamina , and left posterior iliac wing . 
magnetic resonance imaging ( mri ) of the brain revealed a 4 - mm lesion in the right caudate nucleus . the patient presented to the university of texas md anderson cancer center for a second opinion . 
she was then treated with standard - of - care cabozantinib but experienced multiple adverse effects , including handand - foot syndrome , mucositis , weight loss , nausea , and diarrhea , requiring several dose reductions ultimately to 40 mg daily . 
 ( a ) magnetic resonance imaging ( mri ) oblique axial cerebellar images of the patients metastatic brain disease ( left panel ) before and ( right panel ) 8 weeks after the patient initiated treatment with selpercatinib . 
 ( c ) mri axial cerebellar and ( d ) sagittal thoracic spine images of the patients metastatic disease ( left panels ) before and ( right panels ) 33 weeks after the patient initiated treatment with selpercatinib . 
 ( e ) mri axial brain images demonstrating a new caudate lesion after 17 months of therapy with selpercatinib ( left panel ) before and ( right panel ) after stereotactic radiosurgery ( srs )  . 
 andreev - drakhlin et al given the absence of any remaining standard therapies with established benet , the patient enrolled in a phase i / ii study of selpercatinib in patients with advanced solid tumors ( libretto - 001 ; clinicaltrials.gov identier : nct03157128 )  . the patient provided written informed consent before enrollment . 
baseline imaging at the time of the treatment initiation with selpercatinib revealed bilateral central and lateral neck disease ; lytic lesions in the c5 , t4 , t8 , l1 vertebrae and left posterior hilum ; , nodular opacities in bilateral lungs ; interlobular septal thickening suspicious for lymphangitic carcinomatosis ; numerous hepatic lesions ( largest , 7 cm in the dome of liver ) ; 25 lesions in the brain and cerebellum ( largest , 7 mm in the left cerebellar hemisphere ; fig 2a and 1c , left panels ) , and a 5 - mm leptomeningeal nodule at the level of t11 ( fig 2d , left panel )  . calcitonin and cea levels measured 2 , 180 pg / ml and 12 , 312 ng / ml , respectively ( fig 2b )  . the patient experienced clinical response within the rst month of therapy , with resolution of baseline diarrhea and nausea , and with appetite improvement . 
at 17 months , an mri of the brain showed an isolated , new , 5 - mm left caudate lesion that was treated with srs ( fig 2e , left and right panels ) with continuation of selpercatinib . 
at 20 months , imaging revealed non - cns multifocal progression with a new 6 - mm bony lesion in the c7 vertebral body , as well as several new , right - side liver lobe lesions ( the largest measured 2 cm )  . 
the patients calcitonin level rose to 235.9 pg / ml ; her cea level remained unchanged . peripheral blood cell - free dna analysis at the time of systemic progression revealed only the presence of the previously identied ret m918t missense mutation . 
the patient declined a liver biopsy . cns disease remained in response until 22 months , when several new brain lesions were found to have developed ; these were managed with srs ( fig 2 )  . 
her treatment course has been complicated by several hospitalizations unrelated to selpercatinib . discussion metastatic dissemination of mtc to the cns is a rare but devastating complication associated with poor prognosis and no well - established treatment strategies.1 although limited evidence from previous reports suggests that surgical resection and / or radiation therapy may be used in select cases , there remains a strong need for novel treatment modalities with durable therapeutic responses.1 - 3 cns efcacy of multikinase inhibitors such as vandetanib or cabozantinib has not been reported in mtc . here , we present a report of successful treatment of the brain and leptomeningeal metastases with systemic therapy in a patient with ret - mutated mtc , using the purpose - built selective and cns - penetrant ret inhibitor selpercatinib . 
treatment with this agent resulted in complete and durable resolution of the measurable brain and leptomeningeal lesions in a patient with previous disease progression while she received cabozantinib and radiation therapy . 
 case report notably , the patients tumor harbored a heterozygous deletion in the cdkn2c gene linked to an aggressive disease phenotype in mtc.19 cdkn2c is a member of the ink4 family of cyclin - dependent kinase inhibitors that block the cell cycle progression in the g1 phase through interaction with cyclin - dependent kinases 4 and 6.19 cdkn2c is lost in approximately 40% to 50% mtcs and is associated with a signicantly shorter time to distant metastasis and decreased overall survival in mtc , an effect further enhanced by concomitant ret m918t mutation.19 , 21 in addition , an earlier report showed that cdkn2c haploinsufciency is associated with a strong trend toward reduced progressionin ret - mutated mtc treated with systemic free survival therapy.21 however , this relationship has not been studied in patients receiving selective ret inhibitors , to our knowledge , and the signicance of cdkn2c loss in this setting remains to be determined . the patient is continuing selpercatinib therapy , with gradual disease progression in the liver . 
a recent study demonstrated that ret solvent - front mutations ( ret g810s / g810r / g810c ) can cause on - target resistance to selective and multikinase ret inhibitors.22 a better understanding of mechanisms of acquired resistance to selective ret inhibitors and frequency of resistant alterations would guide the development of novel agents and combination therapies directed towards further improvement of treatment outcomes for patients with ret - altered malignancies . in conclusion , we observed a radiographically conrmed durable response with selpercatinib in a patient with a heavily pretreated ret m918tmutated mtc with extensive cns metastases . 
mcwilliams rr , giannini c , hay id , et al : management of brain metastases from thyroid carcinoma : a study of 16 pathologically conrmed cases over 25 years . cancer 98 : 356 - 362 , 2003 drilon a , hu zi , lai ggy , et al : targeting ret - driven cancers : lessons from evolving preclinical and clinical landscapes . 
hofstra rmw , landsvater rm , ceccherini i , et al : a mutation in the ret proto - oncogene associated with multiple endocrine neoplasia type 2b and sporadic medullary thyroid carcinoma . 
nature 367 : 375 - 376 , 1994 elisei r , cosci b , romei c , et al : prognostic signicance of somatic ret oncogene mutations in sporadic medullary thyroid cancer : a 10 - year follow - up study . j clin endocrinol metab 93 : 682 - 687 , 2008 beldjord c , desclaux - arramond f , rafn - sanson m , et al : the ret protooncogene in sporadic pheochromocytomas : frequent men 2 - like mutations and new molecular defects . 
guo r , schreyer m , chang jc , et al : response to selective ret inhibition with loxo - 292 in a patient with ret fusion - positive lung cancer with leptomeningeal metastases . 
drilon a , oxnard gr , wirth l , et al : a phase 1 / 2 trial of loxo - 292 in patients with ret fusion - positive lung cancers . 
maxwell je , gule - monroe mk , subbiah v , et al : novel use of a clinical laboratory improvements amendments ( clia ) - certied cyclin - dependent kinase n2c ( cdkn2c ) loss assay in sporadic medullary thyroid carcinoma . 
 undetectable ras - mutant clones in plasma : possible implication for anti - egfr therapy and prognosis in patients with ras - mutant metastatic colorectal cancer mohamed bouchahda , md1 - 4 ; raphael saffroy , phd5 ; abdoulaye karabou e , md2 , 6 ; jocelyne hamelin , phd5 ; pasquale innominato , md , phd2 , 7 , 8 ; faouzi saliba , md , phd9 ; francis l evi , md , phd1 , 2 , 8 ; nelly bosselut , md5 ; and antoinette lemoine , pharmd , phd5 purpose combining cetuximab with chemotherapy provides clinical benet to 60% of the patients with ras wild - type ( ras - wt ) metastatic colorectal cancer ( mcrc )  . 
the patients with raswt ctdna received cetuximab + uorouracil , leucovorin , and irinotecan ( folfiri ) , whereas those with ras - mt ctdna were treated with the oncologists choice of therapy . results of 16 registered patients , 11 were male and ve female . 
they were age 48 to 81 years , and they had unresectable metastatic adenocarcinoma from the colon ( n = 11 ) or rectum ( n = 5 ) , with a median of two metastatic sites . 
in the patients with wt ctdna , objective tumor response rate was 50.0% , including one complete response and four partial responses after a median number of 6 courses of cetuximab + folfiri ( range , 1 to 16 courses )  . 
this was particularly the case for the combination of cetuximab with uorouracil , leucovorin , and irinotecan ( folfiri ) or uorouracil , leucovorin , and oxaliplatin ( folfox ) .1 , 2 however , several studies have shown that the survival of those patients with tumor ras mutations ( ras - mt ) was shorter than for those with ras - wild - type ( ras - wt ) tumors.3 , 4 since 2014 , it has been recommended that only patients with ras - wt mcrc should receive an anti - egfr targeted agent.5 ras mutations have been found in 30% to 50% of patients with mcrc , 6 - 9 which makes these patients ineligible for egfr - targeted therapies . recent studies have demonstrated that the analysis of circulating tumor dna ( ctdna ) in blood samples , through its ability to recapitulate tumor heterogeneity , is a remarkable surrogate of tumor biopsy for detecting mutations.10 - 12 this technique has the advantage of being less invasive than a tissue biopsy and can be easily repeated over time . 
 anti - egfr therapy for ctdna ras - wt despite ras - mt in tumor tissue context key objective to determine whether the lack of ras mutation in a liquid biopsy supports the administration of an antiepidermal growth factor receptor ( egfr ) antibody , despite earlier documentation of a pathogenic ras mutation in the primary colorectal cancer ( crc )  . knowledge generated nearly half the patients in this pilot study received cetuximab - based chemotherapy , because no ras mutation was detected in the liquid biopsy , despite such mutations having been found in the primary tumor . 
the progression - free survival and the overall survival of these heavily pretreated patients largely exceeded those in patients whose liquid biopsy revealed ras mutation and who received chemotherapy only . relevance tracking the gain or loss of ras - mutated cancer clones through liquid biopsies along the course of crc disease may have a profound impact on its therapeutic management . 
this is achieved through enabling the administration of anti - egfr that was initially rejected on the basis of previous molecular testing of tissue . in the characterization of the dynamics of acquired resistance to anti - egfr therapies.13 to date , studies with liquid biopsy have been selectively focused on the early detection of the appearance of ras - mt clones in tumor deposits by analyzing ctdna in blood samples from patients with ras - wt primary crc13 as biomarkers of an increasing resistance to anti - egfr agents . 
indeed , no specication in the european marketing authorisation for cetuximab mentions that tumor ras testing should be determined on solid tumor tissue in order to allow for anti - egfr administration . 
this is also the case for the recommendations by the french high health authority regarding cetuximab use . all consecutive patients treated between august 2017 and february 2019 at one of three participating centers in france were screened for inclusion in this pilot study . 
inclusion required histologic or cytologic proof of colorectal adenocarcinoma , with a kirsten rat sarcoma viral oncogene homolog ( kras ) or neuroblastoma rat sarcoma viral oncogene homolog ( nras ) mutation ( nras - mt ) from a tissue biopsy . 
the massarray ultraseek procedure involves a 3 - step process consisting of the initial polyinactivation of unincorporated merase chain reaction , nucleotides by shrimp alkaline phosphatase , and a singlebase primer extension according to the manufacturers protocol . 
the products were then nano - dispensed onto a matrix - loaded silicon chip ( spectrochipii , agena bioscience ) , and the mutations were detected by matrixassisted laser desorption - ionizationtime of ight mass spectrometry . 
for our study , the sensitivity for the detection of clinically relevant ras gene mutations in ctdna was 88% for patients with crc liver metastases , in good agreement with bettegowda et al.16 we currently use these highly sensitive mass spectrometry ctdna analyses for monitoring treatment of patients with nonsmall - cell lung cancer or breast cancer in routine oncology practice . study treatment all patients had ras - mt tissue biopsies . 
the study population was divided in two groups according to the results of the ctdna mutational analysis : group 1 included the patients with ras - mt also found in plasma ; group 2 included the patients with ras - wt ctdna . 
the sum of the largest diameters of the target lesions was computed on the inclusion imaging and used as baseline for the quantication of tumor downsizing and response categorization according to recist . 
response was classied as complete response , partial response , stable , or progressive disease.15 statistical consideration this pilot exploratory study included consecutive patients , and no sample size was dened a priori . 
the durations of progression - free survival ( pfs ) and overall survival ( os ) were measured from inclusion until the date of progression or death , respectively , or that of last follow up , with the database locked on may 25 , 2019 . 
all analyses were performed with intentto - treat using spss v18.0 software ( spss , chicago , il )  . results patients sixteen patients with unresectable mcrc and ras - mt in a cancer tissue were registered at one of three centers in initial ras mutational status was dethis pilot study . termined in the resected primary tumor for 11 patients ( 69% ) or in a tumor biopsy for 5 patients ( 31% )  . 
 anti - egfr therapy for ctdna ras - wt despite ras - mt in tumor tissue two or more chemotherapy regimens for metastatic disease . two groups of patients were identied according to the results of ctdna mutational analysis at inclusion . 
group 1 received the investigators choice of chemotherapy regimens , which included bevacizumab + folfox for three patients , aibercept + folfiri for three patients , and regorafenib for one patient . 
baseline characteristics were similar in the 2 groups ( table 1 )  . safety data for the nine patients with ctdna ras - wt ( group 2 ) overall , the cetuximab + folfiri regimen was well tolerated . 
the main grade 1 or 2 clinical toxicities were fatigue , diarrhea , nausea or vomiting , mucositis , acneiform rash , and alopecia ( 6 [ 67% ] of 9 for all )  . 
this fourth - line protocol was well tolerated , with the most severe toxicities being grade 2 leukopenia , neutropenia , fatigue , and acneiform rash . discussion the patients in this study had advanced and chemotherapy - resistant metastatic disease , without any imbalance in apparent overall tumor burden that could reasonably inuence the liquid biopsy results . 
thus , no evidence supports the possibility that the differences in detection of ctdna ras mutation would be related to between - patient variations in tumor burden . the identication of a mutation in kras or nras from a cancer tissue biopsy precludes the use of anti - egfr treatment in association with chemotherapy against mcrc , based on consistent evidence.5 our group rst reported the occurrence of acquired kras mutations along the progression of colorectal metastases in patients treated with cetuximab . 
we then hypothesized that the late acquisition of kras mutations could represent a possible mechanism of secondary resistance to anti - egfr antibodies.18 however , the assumption of persisting ras - mt genotype for patients who received chemotherapy has not been challenged before now because of the expected evolutionary advantage of ras - mt clones.19 yet in this pilot study , we found that nearly half the patients with mcrc displayed no detectable ras - mt in ctdna , although their cancer tissue genotyping had demonstrated ras - mt . 
this nding raised several questions . first , is there a concordance between the ras mutational status of tumor tissues and that of ctdna ? several studies have highlighted discrepant results in ras mutational status for 10% - 15% of the patients tested , depending on the method used.11 , 14 , 20 vidal et al explained such plasmaversus - tissue ras discrepancies with spiral and temporal heterogeneity in ras - mt tumor clones within the tumor tissue.11 according to thierry et al , 20 such discrepancies could relate to the use of biopsies . 
other discrepancies that seemed to affect concordance were long intervals between assessments of the ras status in tumor tissue and that in the blood sample , resection of the tumor at the time of blood draw , tissue analyzed . grasselli et al14 ascribed the discrepancies in ras status to differences in technical sensitivity of the methods used for analysis or to heterogeneity . tumor site , and type of second , is there a change in ras status over time and / or during treatment ? this question does not yet have a clear answer . 
we were the rst to report 1 case of acquired kras mutation in metastases after progression under combined anti - egfr and doublet chemotherapy in 12 patients with kras - wt mcrc.18 other authors have highlighted the concurrent detection of sensitive and resistant clones to anti - egfr antibodies within tumor deposits from different locations in the same patient.21 - 24 this multiclonality could explain the dissociated antitumor responses that are frequently encountered in clinical practice . 
such tumor heterogeneity assumes the existence of wt clones sensitive to anti - egfr treatment alongside resistant mutant clones . interestingly , the team of raimondi et al25 recently reported the disappearance of ras - mt clones in ctdna after tumor progression while receiving bevacizumab and chemotherapy in 4 patients with ras - mt mcrc . 
 anti - egfr therapy for ctdna ras - wt despite ras - mt in tumor tissue feb 19 , 2019 before cetux + folfiri may 29 , 2019 after 6 courses sept 19 , 2019 after 12 courses target lesion target lesion target lesion fig 4 . 
case report illustrating exceptional efcacy of cetuximab + uorouracil , leucovorin , and irinotecan ( cetux + folfiri ) as fourth - line chemotherapy received by a 72 - year - old patient with kras mutation in tumor tissue but kras wild - type in liquid biopsy , upon inclusion in the study . 
indeed , the observed median pfs and os in these patients were similar to those reported for patients with ras - wt tumor tissue who received this combination as rst - line treatment for mcrc . 
our internal steering committee proposed to stop the pilot study , once the information was adequate for designing a randomized trial for testing the hypothesis further on the basis of an apparent three - fold increase in the median pfs of the experimental treatment compared with cetuximab - free chemotherapy . the intriguing and encouraging results of this exploratory trial need to be conrmed in randomized clinical trials . such validation steps are particularly relevant because all our patients with ras - wt ctdna received cetuximab ; thus , we cannot differentiate between a prognostic and a predictive role of ras - wt ctdna for outcomes of patients receiving cetuximab - based chemotherapy . 
 bouchahda et al than prognostic of an aggressive tumor biology.34 such consideration would further support the hypothesis of an added benet from cetuximab in this subgroup of patients . yet recent evidence suggests that the proportion of ras - mt in ctdna was a prognostic indicator for both os and pfs in patients with mcrc.35 this nding raised the question of a potential divide on the clinical signicance between tumor and ctdna genotyping , which this study cannot respond to . 
the fact that orrs exceeded 40% and were similar in both treatment groups suggested that all the patients included in the study were not completely resistant to chemotherapy and that those with ras - mt in the liquid biopsy could have a worse prognosis , as proposed by elz et al.35 however , single - agent cetuximab achieved only a 10% orr in patients with chemotherapy refractory mcrc , 36 despite prolonging pfs by 4 months . 
taken together , the literature results support a much greater value for pfs prolongation compared with orr as an efcacy end point , which supports the hypothesis that the liquid biopsy results have a predictive value . the lack of a prespecied sample size and the low number of patients in each group constitute the main limitations of our pilot salvage study . 
the three - fold increase in median pfs in the absence of any apparent bias does suggest that antiegfrbased chemotherapy could represent a promising option for nearly half the patients with initially documented ras - mt on tumor tissue at a later stage of their disease . our study was in line with the chronos study ( clinicaltrials.gov identier : nct03227926 ) , 37 whose design is based on the concept that crc genome adapts dynamically to intermittent anti - egfr drug schedules . 
a follow - up on our study would involve a randomized clinical trial in which eligible patients with ras - mt on solid tumor tissue undergo ras mutational status assessment in ctdna upon progression on chemotherapy . 
 anti - egfr therapy for ctdna ras - wt despite ras - mt in tumor tissue references venook ap , niedzwiecki d , lenz hj , et al : effect of rst - line chemotherapy combined with cetuximab or bevacizumab on overall survival in patients with kras wild - type advanced or metastatic colorectal cancer : a randomized clinical trial . 
jama 317 : 2392 - 2401 , 2017 tejpar s , stintzing s , ciardiello f , et al : prognostic and predictive relevance of primary tumor location in patients with ras wild - type metastatic colorectal cancer : retrospective analyses of the crystal and fire - 3 trials . 
modest dp , ricard i , heinemann v , et al : outcome according to kras - , nrasand braf - mutation as well as kras mutation variants : pooled analysis of ve randomized trials in metastatic colorectal cancer by the aio colorectal cancer study group . 
ann oncol 27 : 1746 - 1753 , 2016 de roock w , claes b , bernasconi d , et al : effects of kras , braf , nras , and pik3ca mutations on the efcacy of cetuximab plus chemotherapy in chemotherapy - refractory metastatic colorectal cancer : a retrospective consortium analysis . 
lancet oncol 11 : 753 - 762 , 2010 van cutsem e , cervantes a , nordlinger b , et al : metastatic colorectal cancer : esmo clinical practice guidelines for diagnosis , treatment and follow - up . 
ann oncol 25 : iii1 - iii9 , 2014 douillard jy , oliner ks , siena s , et al : panitumumab - folfox4 treatment and ras mutations in colorectal cancer . 
heinemann v , von weikersthal lf , decker t , et al : folfiri plus cetuximab versus folfiri plus bevacizumab as rst - line treatment for patients with metastatic colorectal cancer ( fire - 3 ) : a randomised , open - label , phase 3 trial . 
lancet oncol 15 : 1065 - 1075 , 2014 van cutsem e , lenz hj , k ohne ch , et al : fluorouracil , leucovorin , and irinotecan plus cetuximab treatment and ras mutations in colorectal cancer . 
j clin oncol 33 : 692 - 700 , 2015 peeters m , kafatos g , taylor a , et al : prevalence of ras mutations and individual variation patterns among patients with metastatic colorectal cancer : a pooled analysis of randomised controlled trials . 
vidal j , muinelo l , dalmases a , et al : plasma ctdna ras mutation analysis for the diagnosis and treatment monitoring of metastatic colorectal cancer patients . ann oncol 28 : 1325 - 1332 , 2017 12 . 
grasselli j , elez e , carat `u g , et al : concordance of bloodand tumor - based detection of ras mutations to guide anti - egfr therapy in metastatic colorectal cancer . 
bouchahda m , macarulla t , liedo g , et al : feasibility of cetuximab given with a simplied schedule every 2 weeks in advanced colorectal cancer : a multicenter , retrospective analysis . 
lacina l , coma m , dvor ankov a b , et al : evolution of cancer progression in the context of darwinisanticancer res 39 : 1 - 16 , 2019 20 . 
thierry ar , el messaoudi s , mollevi c , et al : clinical utility of circulating dna analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti - egfr treatment . 
raimondi c , nicolazzo c , belardinilli f , et al : transient disappearance of ras mutant clones in plasma : a counterintuitive clinical use of egfr inhibitors in ras mutant metastatic colorectal cancer . 
zhou m , yu p , hou k , et al : effect of ras status on anti - egfr monoclonal antibodies + 5 - fu infusion - based chemotherapy in rst - line treatment of metastatic colorectal cancer : a meta - analysis . 
giantonio bj , catalano pj , meropol nj , et al : bevacizumab in combination with oxaliplatin , uorouracil , and leucovorin ( folfox4 ) for previously treated metastatic colorectal cancer : results from the eastern cooperative oncology group study e3200 . 
van cutsem e , tabernero j , lakomy r , et al : addition of aibercept to uorouracil , leucovorin , and irinotecan improves survival in a phase iii randomized trial in patients with metastatic colorectal cancer previously treated with an oxaliplatin - based regimen . 
tabernero j , yoshino t , cohn al , et al : ramucirumab versus placebo in combination with second - line folfiri in patients with metastatic colorectal carcinoma that progressed during or after rst - line therapy with bevacizumab , oxaliplatin , and a uoropyrimidine ( raise ) : a randomised , double - blind , multicentre , phase 3 study . 
sobrero af , maurel j , fehrenbacher l , et al : epic : phase iii trial of cetuximab plus irinotecan after uoropyrimidine and oxaliplatin failure in patients with metastatic colorectal cancer . 
peeters m , oliner ks , price tj , et al : analysis of kras / nras mutations in a phase iii study of panitumumab with folfiri compared with folfiri alone as second - line treatment for metastatic colorectal cancer . 
de stefano a , rosanova m , malapelle u , et al : discordance in ras mutations between primary colon tumor and metastases : a real event or a matter of methodology ? int j biol markers 32 : e474 - e477 , 2017 34 . 
elez e , chianese c , sanz - garca e , et al : impact of circulating tumor dna mutant allele fraction on prognosis in ras - mutant metastatic colorectal cancer . 
siena s , bardelli a , sartore - bianchi a , et al : exploiting clonal evolution and liquid biopsy to overcome resistance to anti - egfr treatment in metastatic colorectal cancer : the chronos trial . 
we show that kit inhibition markedly decreased cell viability in melanoma cell lines with distinct kit mutations ; however , this effect was countered in the presence of hepatocyte growth factor ( hgf ) , the ligand for met . 
a physical examination revealed a raised , erythematous , and well - circumscribed cutaneous lesion on her left great toe , which the patient attributed to trauma that occurred several months earlier . 
staging computed tomography ( ct ) and positron emission tomography scans revealed left inguinal and left external iliac lymphadenopathy ; two soft - tissue densities in the left breast ( one biopsy proven to be melanoma ) ; numerous subcentimeter , hypodense liver lesions ; and multiple subcentimeter , subcutaneous nodules , suggestive of metastatic disease . 
gene mutation analysis of the metastatic melanoma tissue did not reveal mutations in braf or nras . after two cycles of biochemotherapy ( data supplement ) , restaging ct scans revealed overall stable disease and a mixed tumor response in the lymph nodes . 
the patient received whole - brain radiation therapy ( 30 gy in 10 fractions ) , then stereotactic radiosurgery , followed by combination treatment with temozolomide , cisplatin , and vinblastine . subsequent restaging showed new mediastinal lymphadenopathy and lung nodules . 
the patient received two cycles of a carboplatin - paclitaxel regimen before evidence of disease progression in the left inguinal lymph nodes , liver , and bra the patient was then treated with ipilimumab , which had just received us food and drug administration approval for metastatic melanoma , 3 mg / kg for four cycles . 
there was evidence of stable disease response to ipilimumab , with tumor reduction of 30% by immune - related response criteria overall with disease response in the brain , liver , and lymph nodes . 
after 19 months of disease control with ipilimumab treatment , a brain magnetic resonance imaging demonstrated a left frontal operculum metastasis with perilesional edema ( fig 1a , upper panel ) and positron emission tomography / ct junna oba sun - hee kim wei - lien wang mariana p . 
 ( a ) representative brain magnetic resonance images show left frontal operculum metastasis with perilesional edema pretreatment ( top panel ) and the on - treatment combination dasatinib and crizotinib therapy effect ( lower panel )  . 
 ( b ) polymerase chain reactionbased dna sequencing chromatogram showing a kit nucleotide transition ( 2464 a > t ) at codon 822 ( aat to tat ) in exon 17 from the patients acral melanoma metastatic tumor . 
 ( c ) hematoxylin and eosin ( he ) and immunohistochemical staining for total kit and phosphorylated kit ( p936 ) and total met in the patients primary ( upper panel ) and metastatic ( lower panel ) acral melanoma tumors ( original magnification , 20 )  . scanning showed new hypermetabolic activity in the mediastinal lymph nodes , which was verified to be metastatic melanoma by interventional radiology - guided fine - needle aspiration ( data not shown )  . 
kit mutations and copy number alterations in 4q ( kit ) and 11q ( ccnd1 ) for the melanoma cell lines cell line name exon 4q ( kit ) 11q ( ccnd1 ) kit mutation copy number increase aa change l576p w556 - k558 n822y s476i m230 melms 2391 ma - mel - 144 histopathologic examination of the patients primary and metastatic melanoma tumors showed widespread expression of phosphorylated / total kit protein in melanoma cells ( fig 1c )  . 
given the response to combination dasatinib and crizotinib therapy , we also assessed the expression of the met , for which crizotinib has high binding affinity , and observed positive staining in both primary and metastatic tumors , with moderately higher intensity in the metastatic tumor . to better understand the mechanism by which combination therapy resulted in the observed clinical response , we performed a molecular analysis of the melanoma cell line we established from the patients metastatic tumor ( cell line 2391 ) , as well as three other melanoma cell lines shown to have endogenous kit mutations ( namely , melms , ma - mel - 144 , and m230 ) .17 - 19 mutation analysis confirmed the presence of the 2464 a > t nucleotide transition in kit with a resultant n822y amino acid change in the activation loop within 2391 , the patients melanoma cell line ( data supplement )  . 
we verified the presence of each specific kit mutation within each cell line using targeted next - generation dna sequencing ( table 1 ; data supplement ) .20 no braf or nras mutations , or secondary mutations in kit were detected in any of the cell lines . we also determined the global chromosomal copy number alteration status within each kit - mutant melanoma cell line ( fig 2a ; table 1 )  . 
cell lines 2391 and ma - mel - 144 had chromosome 4q copy number elevation , which corresponds with the location of the kit locus , consistent with initial observations that kit mutations co - occur with kit amplifications.1 , 2 three of the four cell lines also had chromosome 11q copy number elevation , which corresponds with the location of the ccnd1 locus , also well described to be amplified in am tumors.21 , 22 thus , the patients cell line 2391 and others in our panel had characteristic genetic features observed in am tumors . to determine the endogenous constitutive activity of each kit mutation , we assessed the total and phosphorylated levels of kit in each melanoma cell line . 
western blot analysis showed phosphorylated kit to be present in all the kit - mutant melanoma cell lines in the presence or absence of serum ( fig 2b ) ; this was not observed in those without a kit mutation ( data supplement )  . 
each of the cell lines showed sensitivity to gene - specific kit knockdown and to multiple tkis that share kit as a target but have many nonoverlapping targets ( eg , dasatinib targets src - family kinases ) , whereas imatinib does not ( data not shown )  . 
cell lines 2391 ( kitn822y ) , melms ( kit556 - 558del ) , and ma - mel - 144 ( kits476i ) show amplification at 11q ( location of ccnd1 allele )  . 
cell viability assay of m230 ( kitl576p ) , melms ( kit556 - 558del ) , 2391 ( kitn822y ) , and ma - mel - 144 ( kits476i ) melanoma cell lines : no treatment , dasatinib ( 50 nm ) , dasatinib plus hgf ( 100 ng / ml ) , dasatinib plus hgf plus crizotinib ( 5 m ) , and crizotinib alone . of met may enhance the effect of single - agent dasatinib in the tumor setting . 
thus , we tested if the presence of hgf , the only known ligand for met , in the extracellular environment can modulate the reduction in cell viability observed with dasatinib treatment ( fig 2c )  . 
analysis of the patients tumor and cell line ( and three other kit - mutant cell lines ) suggests that the hgf - met axis may be a mechanism of de novo resistance in kit - mutant melanomas . the emergence of secondary mutations in kit is a common mechanism of clinical resistance to kit inhibition in kit - mutant gist.29 however , nearly all resistance observed in kit mutant melanoma occurs in the initial treatment setting , before the clonal evolution of secondary mutations can emerge , 10 - 13 , 23 - 26 suggesting that the presence of coactivating pathways may play a role in this primary resistance . the observed efficacy of the combination dasatinib and crizotinib could , in part , be due to the inhibition of multiple kinases ; however , the melanoma cell line studies indicate the primacy of the kit and met receptors to explain the observed results . 
furthermore , crizotinib alone was only effective in the presence of extracellular hgf , the only known met ligand , indicating crizotinibs modulation of the well - described hgf - met axis , for which it was initially designed.30 these data are consistent with those from preclinical studies showing that met inhibition increases the effect of imatinib in kit - mutant gist models.31 it is intriguing to speculate that the response to combination dasatinib and crizotinib may have been immunologically enhanced , given the patients prior tumor response to ipilimumab therapy . 
hong collection and assembly of data : junna oba , sunhee kim , wei - lien wang , mariana macedo , fernando carapeto , meredith a mckean , john van arnam , agda k . 
woodman manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors junna oba no relationship to disclose sun - hee kim no relationship to disclose wei - lien wang no relationship to disclose mariana p . 
haydu no relationship to disclose honoraria : novartis , bristol - myers squibb , janssen oncology , roche consulting or advisory role : novartis , illumina , ge healthcare research funding : medimmune , astrazeneca , roche , novartis patents , royalties , other intellectual property : elsevier travel , accommodations , expenses : bristol - myers squibb , novartis elizabeth a . 
hong stock and other ownership interests : molecularmatch , oncorena honoraria : adaptimmune , baxter , merrimack , bayer consulting or advisory role : baxter , bayer , guidepoint global , janssen research funding : novartis , genentech , eisai , astrazeneca , pfizer , mirna therapeutics , amgen , daiichi sankyo , merck , mirati therapeutics , eli lilly , adaptimmune , abbvie , bayer , bristol - myers squibb , genmab , ignyta , infinity pharmaceuticals , kite pharma , kyowa hakko kirin , loxo , medimmune , molecular templates , takeda travel , accommodations , expenses : loxo , mirna therapeutics other relationship : oncorena chantale bernatchez stock and other ownership interests : lexicon ( i ) research funding : nektar , idera , iovance biotherapeutics , medimmune , pfizer , emd serono patents , royalties , other intellectual property : patent pending on btla as a marker for better cd8 t cells for adoptive immunotherapy alexander j . 
woodman no relationship to disclose acknowledgment leadership : beta cat pharmaceuticals , archer biosciences stock and other ownership interests : archer biosciences , beta cat pharmaceuticals we thank the center for environmental and molecular carcinogenesis anderson cancer center , smithville , tx , for antibody optimization . affiliations junna oba , sun - hee kim , wei - lien wang , mariana p . 
macedo , ac camargo cancer center , so paulo , brazil . supported by the university of texas system rising star award , national cancer institute ( nci ) specialized programs of research excellence ( spore ) in melanoma ( p50 ca093459 to d.s.h. ) ; developmental research project award , national institutes of health / nci cancer center support grant no . 
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woodman se , trent jc , stemke - hale k , et al : activity of dasatinib against l576p kit mutant melanoma : molecular , cellular , and clinical correlates . 
yang y , liu c , peng w , et al : antitumor t - cell responses contribute to the effects of dasatinib on c - kit mutant murine mastocytoma and are potentiated by anti - ox40 . 
hodi fs , corless cl , giobbie - hurder a , et al : imatinib for melanomas harboring mutationally activated or amplified kit arising on mucosal , acral , and chronically sun - damaged skj clin oncol 31 : 3182 - 3190 , 2013 11 . 
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kato s , jardim dl , johnson fm , et al : phase i study of the combination of crizotinib ( as a met inhibitor ) and dasatinib ( as a c - src inhibitor ) in patients with advanced cancer . 
todd jr , scurr ll , becker tm , et al : the mapk pathway functions as a redundant survival signal that reinforces the pi3k cascade in c - kit mutant melanoma . 
lee sj , kim tm , kim yj , et al : phase ii trial of nilotinib in patients with metastatic malignant melanoma harboring kit gene aberration : a multicenter trial of korean cancer study group ( un10 - 06 )  . 
kalinsky k , lee s , rubin km , et al : a phase 2 trial of dasatinib in patients with locally advanced or stage iv mucosal , acral , or vulvovaginal melanoma : a trial of the ecog - acrin cancer research group ( e2607 )  . 
 c response to mammalian target of rapamycinbased therapy and incidental finding of lynch syndrome in a patient with solid pseudopapillary neoplasm of the pancreas with akt1_e17k mutation introduction solid pseudopapillary neoplasms ( spns ) of the pancreas are exocrine neoplasms that predominantly affect young females and are considered to have low malignant potential . 
surgical resection offers patients an excellent chance of long - term survival , even in cases of local invasion , recurrence , and metastatic disease.1 - 3 recent studies have demonstrated that invasion of these neoplasms into muscular vessels , advanced tumor stage by european neuroendocrine tumors society classification , and distant metastasis correlated with poor prognosis.4 in such instances and especially when complete surgical resection is unattainableuse of salvage chemotherapy is needed.5 , 6 in this setting , some chemotherapy agents have offered favorable responses , 7 - 10 yet because of the scarce number of reported cases requiring this treatment modality , no regimen has been demonstrated as definitely superior.11 first described by frank in 1959 and histologically defined as spns by the who in 2010 , spns have been demonstrated to harbor somatic point mutations in exon 3 of ctnnb1 , the gene that encodes for - catenin , a downstream transcriptional activator in the wnt signaling pathway that is involved in cell growth regulation.12 , 13 spns have not previously been associated with genetic mutations linked to dna mismatch repair ( mmr ) syndromes , such as lynch syndrome and hereditary nonpolyposis colorectal cancer ( hnpcc ) syndrome . 
to clarify the likely oncologic diagnosis , additional workup was performed , including - fetoprotein , carcinoembryonic antigen , cancer antigen 125 , and cancer antigen 19 - 9 testing , with normal values . 
postoperatively , she developed acute gi bleeding from a gastric ulcer and remained admitted for more than 30 days . pathologic evaluation revealed a uniform grayish maroon , soft , necrotic , and hemorrhagic lesion macroscopically . 
herzog author affiliations and support information ( if applicable ) appear at the end of this article . creative commons attribution non - commercial no derivatives 4.0 license corresponding author : cynthia e . 
 ( c - f ) immunohistochemical staining showing that the liver metastasis is negative for pan - cytokeratin ( c ) and chromogranin ( d ) , but positive for nuclear expression of - catenin ( e ) and progesterone receptor ( f )  . 
hepatocytes , which are positive for pan - cytokeratin ( c ) and negative for nuclear expression of - catenin ( e ) and progesterone receptor ( f ) , serve as internal controls . 
magnification , 100 ( a - f )  . results were positive for vimentin , cd10 , - catenin , progesterone receptor , and synaptophysin , but negative for pan - cytokeratin and chromogranin ( fig 1 )  . 
these results were consistent with the diagnosis of an spn involving the pancreatic body and tail . on the patients first follow - up after 1 year , abdominal ultrasonography and computed tomography revealed multiple liver masses involving hepatic segments iii , vi , vii , and viii ; a tumoral thrombus in the spleno - portal venous confluence ; and enlargement of several lymph nodes that raised concern of metastases ( fig 2a )  . 
she was referred to the university of texas md anderson cancer center for additional management . at our institution , the pathologic findings of her primary and metastatic lesions were found to be identical to those obtained at the outside institution . 
she completed four cycles of chemotherapy with oxaliplatin , irinotecan , and fluorouracil , with subsequent computed tomography images that demonstrated mild progression of metastatic disease ( fig 2b )  . 
 ( a ) computed tomography ( ct ) scan of the liver dome showing metastatic solid pseudopapillary neoplasm in a 13 - year - old girl before arrival at our institution . 
 on the basis of the akt1 mutation , the patient was enrolled in a clinical trial ( clinicaltrials.gov identifier : nct01582191 ) , with each cycle consisting of 28 days of oral vandetanib ( 300 mg ) ; a multikinase inhibitor of epidermal growth factor receptor ( egfr ) , vascular endothelial growth factor receptor ( vegfr ) , and ret ; and oral everolimus ( 10 mg ) , a mammalian target of rapamycin ( mtor ) inhibitor . 
she was taken off the protocol and continued on single - agent everolimus at the same dosing schedule . the patient has remained on mtor - based therapy for more than 3 years with excellent performance status and stable disease ( 15% reduction ) from baseline tumor size ( fig 2c )  . 
she has had several episodes of mucositis that have been managed with topical corticosteroids and sucralfate or a brief interruption of therapy . fourteen months after the start of single - agent everolimus , additional genetic testing using targeted exome sequencing of 202 genes with tumor and matched normal dna resulted in the identification of a pathogenic germline msh6 mutation ( c.2147_2148delca ) , which is consistent with lynch syndrome / hnpcc syndrome and later confirmed in a clinical laboratory improvement amendmentscertified laboratory ( table 1 ) and via immunohistochemistry testing . 
 her mother underwent genetic testing and was found to be negative for the mutation . discussion to our knowledge , this is the first case reporting the use of genomic testing to guide the treatment of metastatic spn of the pancreas . 
pembrolizumab , a programmed death - 1 checkpoint inhibitor , recently demonstrated immune - related clinical benefit , which resulted in us food and drug administration approval for its use in mmr - deficient colorectal neoplasms.19 of importance , this genetic entity has also been reported in pancreatic neoplasms.20 geary et al21 investigated 130 families with mmr mutations that were comparable to that of our patient and reported 22 cases of early - onset pancreatic cancers . 
well - differentiated pancreatic neuroendocrine tumors , medullary carcinomas , and intraductal papillary mucinous neoplasms of the pancreas , among others , have been associated with microsatellite instability syndromes , 22 , 23 but spns have not been previously described in association with lynch syndrome . although a few case reports have demonstrated that chemotherapy agents offer variable degrees of activity toward this rare neoplasm , 7 - 11 we elected treatment with the well - known regimen for gi tumors , oxaliplatin , irinotecan , and fluorouracil ; however , the tumor progressed . 
recently , guo et al24 conducted whole genome sequencing analysis on nine patients spns and identified numerous genetic mutations , including usp9x , ep400 , pdk1 , med12 , htt and ar . 
met germline mutations have never been reported in association with spns , yet its presence in this case is of interest and could have also contributed , in part , to the development of this disease . 
molecular testing in this disease affords the opportunity of identifying a targetable mutation , as demonstrated in our case . targeting the phosphatidylinositol 3 - kinase / akt / mtor pathway , which is known for its participation in cell proliferation , apoptosis , and angiogenesis , 25 was effective in our patient with spn . 
analogous to the response achieved in cases of pancreatic neuroendocrine tumors , 26 treatment with everolimus , an oral signal transduction inhibitor that blocks mtor , resulted in stable disease . 
the patient continues to tolerate the treatment well , with most adverse events being managed successfully with medical treatment . although germline msh6 mutationassociated lynch syndrome / hnpcc syndrome and spn may have been separate entities arising coincidentally in our patient , suspicion of the genetic inherited disorder as an impending trigger for this neoplasm is warranted . 
obtaining longitudinal analysis of the mtor pathway activation posteverolimus as well as the akt_e17k mutation frequency in the tumor would have allowed us to obtain additional data ; however , we elected to keep research interventions in this pediatric patient at a minimum . spns should be considered in the differential diagnosis of pancreatic masses in patients with lynch syndrome / hnpcc syndrome . 
herzog financial support : vivek subbiah administrative support : vivek subbiah collection and assembly of data : branko cuglievan , vivek subbiah , huamin wang , ajaykumar morani , funda mericbernstam , cynthia e . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . branko cuglievan no relationship to disclose vivek subbiah consulting or advisory role : medimmune research funding : novartis ( inst ) , glaxosmithkline ( inst ) , nanocarrier ( inst ) , northwest biotherapeutics ( inst ) , genentech ( inst ) , berg pharma ( inst ) , bayer ( inst ) , incyte ( inst ) , fujifilm ( inst ) , pharmamar ( inst ) , d3 oncology solutions ( inst ) , pfizer ( inst ) , amgen ( inst ) , abbvie ( inst ) , multivir ( inst ) , blueprint medicines ( inst ) , loxo ( inst ) , vegenics ( inst ) , takeda ( inst ) , alfasigma ( inst ) , agensys ( inst ) , idera ( inst ) , boston biomedical ( inst ) , inhibrx ( inst ) , exelixis ( inst ) travel , accommodations , expenses : pharmamar , bayer ajaykumar morani no relationship to disclose funda meric - bernstam honoraria : sumitomo group , dialectica consulting or advisory role : genentech , inflection biosciences , pieris pharmaceuticals , clearlight diagnostics , darwin health , samsung bioepis , spectrum pharmaceuticals , aduro biotech , origimed , xencor , debiopharm group research funding : novartis , astrazeneca , taiho pharmaceutical , genentech , calithera biosciences , debiopharm group , bayer , aileron therapeutics , puma biotechnology , cytomx therapeutics , jounce therapeutics , zymeworks , curis , pfizer , effector therapeutics , abbvie , boehringer ingelheim ( i ) vijaykumar holla no relationship to disclose cynthia e . 
estrella js , li l , rashid a , et al : solid pseudopapillary neoplasm of the pancreas : clinicopathologic and survival analyses of 64 cases from a single institution . 
tang lh , aydin h , brennan mf , et al : clinically aggressive solid pseudopapillary tumors of the pancreas : a report of two cases with components of undifferentiated carcinoma and a comparative clinicopathologic analysis of 34 conventional cases . 
strauss jf , hirsch vj , rubey cn , et al : resection of a solid and papillary epithelial neoplasm of the pancreas following treatment with cis - platinum and 5 - fluorouracil : a case report . 
hofmann h , von haken r , werner j , et al : unresectable isolated hepatic metastases from solid pseudopapillary neoplasm of the pancreas : a case report of chemosaturation with high - dose melphalan . 
irtan s , galmiche - rolland l , elie c , et al : recurrence of solid pseudopapillary neoplasms of the pancreas : results of a nationwide study of risk factors and treatment modalities . 
abraham sc , klimstra ds , wilentz re , et al : solid - pseudopapillary tumors of the pancreas are genetically distinct from pancreatic ductal adenocarcinomas and almost always harbor betacatenin mutations . 
kastrinos f , mukherjee b , tayob n , et al : risk of pancreatic cancer in families with lynch engl j med 372 : 2509 - 2520 , 2015 syndrome . 
geary j , sasieni p , houlston r , et al : gene - related cancer spectrum in families with hereditary non - polyposis colorectal cancer ( hnpcc )  . 
sparr ja , bandipalliam p , redston ms , et al : intraductal papillary mucinous neoplasm of the pancreas with loss of mismatch repair in a patient with lynch syndrome . 
banville n , geraghty r , fox e , et al : medullary carcinoma of the pancreas in a man with hereditary nonpolyposis colorectal cancer due to a mutation of the msh2 mismatch repair gene . 
case series 2 and 3 included archival samples from men treated at johns hopkins hospital ( n = 21 ) and university of calgary ( n = 8 ) , respectively . 
the frequency of pathogenic / likely pathogenic mutations are reported . results overall , 25 patients ( 49% ) had at least one dna damage repair gene alteration , including seven ( 14% ) with a mismatch repair gene mutation and 16 ( 31% ) with a homologous repair mutation . 
activating mutations in the pi3k pathway ( n = 19 ; 37% ) , wnt pathway ( n = 16 ; 31% ) , and mapk pathway ( n = 8 ; 16% ) were common . conclusion this study strongly suggests that dpcs are enriched for actionable mutations , with approximately 50% of patients demonstrating dna damage repair pathway alteration ( s )  . 
2019 by american society of clinical oncology introduction ductal prostate cancer ( dpc ) is a rare prostate cancer variant characterized by large glands lined by tall , pseudostratied , columnar , neoplastic epithelial cells , typically arranged over brovascular cores or cribriform glands and associated with an aggressive clinical course.1 - 3 outcomes for dpc generally mirror those of gleason score 4 + 4 = 8 carcinomas , and tumors with at least 10% ductal morphology have been found to associate with a higher stage and suboptimal response to androgen deprivation.2 , 3 overall , approximately 3% of all prostate cancers have some component of ductal histology.2 , 4 although the histologic features of dpc are well described , there is relatively little information regarding the underlying molecular alterations associated with this prostate cancer subtype . 
fluorescence in situ hybridization studies have found that tmprss2 : erg fusions are present in 10% to 50% of patients with dpc , and erg protein expression ( consistent with tmprss2 : erg fusions ) is also present in this range.5 - 8 limited gene expression proling studies have found similarities between dpc and patients with acinar tumors , and there is molecular evidence that concurrent ductal and acinar tumors are clonally related.4 , 9 , 10 more recent immunohistochemical proling studies have demonstrated that positive phosphomammalian target of rapamycin staining correlated with risk of biochemical recurrence in patients with ductal carcinoma.11 in a separate study , it was found that loss of pten protein expression occurred more frequently in dpc compared with acinar adenocarcinoma , again , potentially implicating mammalian target of rapamycin signaling pathway in the pathobiology of dpc . 
 schweizer et al context key objective the key objective was to provide an overview of the genomic alterations associated with ductal prostate cancers . knowledge generated ductal prostate cancers are associated with a high incidence of mutations in dna repair genes . 
we previously reported the ngs results from a small series characterizing patients with dpc at our institution ( university of washington [ uw ] ) .12 in that preliminary study , we observed a high rate of dna damage repair ( ddr ) mutations , including loss - of - function mutations in mismatch repair ( mmr ) genes . 
building from our initial case series , we now report sequencing results from an expanded multiinstitutional collaborative cohort of 51 patients with dpc . methods study populations we assembled three case series comprising 51 patients with dpc from institutions in the united states and canada ( data supplement )  . 
all tumor tissue was sequenced on the targeted ngs assay uw - oncoplex according to previously published methods.12 , 13 case series 1 consisted of prostate cancer specimens ( radical prostatectomies and needle biopsies of prostate and metastatic tumors ) from 22 men actively receiving treatment at the university of washington / seattle cancer care alliance who were prospectively identied as having a diagnosis of dpc . 
preliminary sequencing results from this series have been previously published.12 all men provided informed consent to have their tissue sequenced as part of this study . case series 2 included 21 radical prostatectomy samples . 
a subset was obtained from a tissue microarray composed of primary prostatectomy specimens from men with either dpc ( n = 51 ) or gleason pattern 4 acinar prostatic adenocarcinoma ( n = 75 ) treated at johns hopkins hospital ( jhh ) between 1984 and 2004 . 
case series 3 included archival tissue from eight men treated by transurethral resection of the prostate at the university of calgary . blinded morphologic evaluation to reevaluate the morphologic classication of all patients in a blinded manner , an expert genitourinary pathologist ( j.i.e. ) examined scanned digital images of a representative slide from each patient corresponding to the formalin - xed parafn - embedded ( ffpe ) block that was macrodissected for sequencing . 
each patient was scored for percentage of the tumor that had ductal morphology overall , as well as the percentage of several described morphologic subtypes of ductal carcinoma : cribriform , papillary , gland - like , prostatic intraepithelial neoplasialike and solid . macrodissection of tumor tissue all tissue was previously ffpe . 
for patients with clinical follow - up ( uw and university of calgary , n = 28 ) , seven ( 25% ) were deceased , and 12 ( 43% ) had developed table 1 . 
demographics and characteristics characteristic median age ( range ) , years gleason * value 67.5 ( 47 - 94 ) metastatic disease at presentation source of tissue for sequencing turp prostatectomy prostate needle biopsy metastatic biopsy 4 ( 13 ) 7 ( 23 ) 18 ( 60 ) 1 ( 3 ) 11 ( 26 ) 8 ( 15 ) 29 ( 56 ) 11 ( 21 ) 3 ( 6 ) note . 
 ( % ) unless otherwise indicated . abbreviation : turp , transurethral resection of the prostate . * gleason score was only provided for patients with mixed ductalacinar histology ( n = 30 )  . disease state at time of initial presentation was only available for johns hopkins hospital and university of washington ( n = 43 )  . metastatic disease during long - term follow - up . 
additional demographics details are listed in table 1 . dpc genomics overall , our combined cohort of patients with dpc demonstrated a high number of recurrent genomic alterations ( fig 1 ; data supplement )  . 
twenty - ve ( 49% ) of 51 patients had at least one alteration in a ddr pathway gene . overall , seven of 51 patients ( 14% ) had evidence of mmr alterations , six of whom had evidence of hypermutation ( ie , 10 mutation per megabase ) , consistent with decient mmr ( one patient with monoallelic loss of msh2 was not hypermutated )  . 
compared with published genomic data from men with localized and castration - resistant prostate cancer ( crpc ) , our combined cohort of men with dpc was signicantly enriched for mutations in ddr genes ( both mmr and hr genes ) , as well as other genes of interest ( table 2 ) .20 , 21 additional recurrent genomic alterations . 
similar to localized and metastatic crpc , mutations in foxa1 were frequently observed ( n = 17 ; 33% ) .20 , 21 interestingly , mutations in genes involved in wnt signaling were also frequent ( n = 16 ; 31% )  . 
this includes activating / stabilizing mutations in ctnnb1 ( n = 4 ) , as well as inactivating mutations in apc ( n = 12 ) , a negative regulator of wnt pathway activation . 
pathogenic mutations were those predicted to either activate oncogenic signaling pathways ( eg , wntor pi3k - signaling ) or inactivate tumor suppressors ( eg , dna damage repair [ ddr ] genes , tp53 )  . 
hr , homologous recombination ; mmr , mismatch repair ; vus , variant of uncertain signicance . alterations co - occurred with pi3k - pathway alterations , including one patient with an apc and pik3ca mutation , and two patients with ctnnb1 and pik3ca mutations . 
compared with patients with crpc , there were fewer pten alterations and more pik3ca mutations in our dpc cohort , with an overall similar incidence of pi3k - pathway alterations ( table 2 )  . 
alterations in ar were infrequent ( n = 4 ; 8% ) ; however , the majority of tissue samples sequenced were primary prostate tissue that had not been exposed to hormonal therapies . 
although the quantity of dpc in each patient varied , there was no evidence that percentage of ductal involvement correlated with the underlying mutational prole , and a high frequency of ddr mutations was observed regardless of the overall quantity of dpc reported on secondary pathology review ( table 3 )  . discussion to our knowledge , this is the largest cohort of dpc to be examined by ngs . 
mutational frequencies for comparator data sets were derived from either the primary publications or extracted from cbioportal.20 - 23 cancer genome atlas . abbreviations : ddr , dna damage repair ; hr , homologous recombination ; mmr , mismatch repair ; rr , relative risk ; su2c , stand up 2 cancer ; tcga , the * the tcga data set comprises 333 men with localized prostate cancer.20 su2c - pcf international prostate cancer dream team discovery set comprises 150 men with metastatic castration - resistant prostate cancer . 
one patient classied as positive for ductal involvement did not provide an estimate on percent involvement . abbreviations : ddr , dna damage repair ; hrd , homologous recombination deciency ; mmr , mismatch repair . * three patients were indeterminate regarding whether the ddr alteration was germline versus somatic and were therefore excluded from this analysis . archival tissue , negating the need to obtain fresh metastatic tissue in men with more advanced disease . 
although we did not intentionally sequence the acinar carcinoma component in patients with mixed ductal - acinar tumors , the fact that concurrent ductal and acinar carcinomas share common erg rearrangements and other alterations suggests that the ddr alterations are likely shared between these components as well.4 , 9 this study adds to the literature suggesting that aggressive histologic subtypes of localized prostate cancer ( eg , primary gleason pattern 5 acinar carcinomas , small cell carcinomas , and now dpcs ) may be enriched for mmr defects.25 it is also notable that in addition to ddr alterations , there were a number of recurrently mutated genes , including those involved in wntand pi3k - signaling pathways . interestingly , and consistent with prior work from our group , we found that pi3k - signaling alterations occurred more commonly in ductal carcinoma via pik3ca mutations than by pten gene alterations , which is in contrast to unselected patients with metastatic crpc.6 , 21 these ndings are also consistent with a recent report examining genomic and transcriptomic differences between dpc and acinar prostate cancer foci from the same individual.9 however , in that report , it is worth noting that the authors did not observe enrichment for mmr alterations , and ddr alterations were relatively infrequent . 
we also conrmed that ets gene rearrangements were signicantly less common in patients with dpc compared with both primary ( the cancer genome atlas ) and metastatic ( stand up 2 cancerprostate cancer foundation ) patients with prostate cancer.4 importantly , because the ngs panel used in this study ( uw - oncoplex ) provides intronic gene coverage for rearrangement hotspot areas in tmprss2 and other recurrently rearranged genes , it has a higher degree of sensitivity for detecting ets fusions and other complex genomic rearrangements that could be missed by panels that only sequence exonic regions.13 , 26 these ndings indicate that alternative drivers may underlie dpc biology . another interesting observation was the apparent enrichment in patients with dpc for germline ddr gene alterations . 
in contrast , the uw cohort comprised prospectively identied men receiving care for prostate cancer in the clinic , and thus represented a real - world example of selecting patients for sequencing on the basis of histology . another key difference is that the jhh cohort only included patients who underwent prostatectomy , and no long - term follow - up data were available . 
given that all patients in the jhh cohort were considered to be prostatectomy candidates , this raises the possibility that these men had less aggressive disease , especially in light of the fact that 70% of men in the calgary and uw cohorts either died or developed metastatic disease . it is also notable that the uw cohort included a number of patients who were disputed in terms of whether dpc was present , and overall , 12 patients ( 26% ) included in this series who underwent secondary pathology review were felt to not contain signicant ductal features ( n = 11 from uw and n = 1 from jhh )  . 
 ductal prostate cancer genomics however , that all patients included in this analysis were believed to contain a component of ductal histology by at in addition , least one expert genitourinary pathologist . because slides from uw were not scanned before macrodissecting the ductal component , we cannot exclude the possibility that the slides sent for secondary review were not representative of the slides used for sequencing . 
in addition , the observed interpathologist variance is consistent with prior experience evaluating patients with dpc.28 importantly , there were no clear differences in mutational proles for patients felt to not possess a clear ductal component on secondary pathology review or between patients with pure versus mixed ductal histology ( table 3 )  . larger studies aimed at evaluating differences between patients with pure and mixed dpc and studies evaluating differences between ductal and acinar foci from the same patient are warranted but are beyond the scope of this study . in conclusion , despite the heterogeneity of our cohort , these results indicate that patients with any fraction of dpc should be offered ngs , given that the presence of dpc histology can serve as a rapid means to select patients enriched for actionable mutations , particularly in mmr and hr genes . 
schweizer consulting or advisory role : janssen research funding : janssen ( inst ) , astrazeneca ( inst ) , roche ( inst ) , pzer ( inst ) , zenith epigenetics ( inst ) , madison vaccines ( inst ) emmanuel s . 
antonarakis honoraria : sano , dendreon , medivation , janssen biotech , essa , astellas pharma , merck , astrazeneca , clovis oncology consulting or advisory role : sano , dendreon , medivation , janssen biotech , essa , astellas pharma , merck , astrazeneca , clovis oncology research funding : janssen biotech ( inst ) , johnson & johnson ( inst ) , sano ( inst ) , dendreon ( inst ) , aragon pharmaceuticals ( inst ) , exelixis ( inst ) , millennium ( inst ) , genentech ( inst ) , novartis ( inst ) , astellas pharma ( inst ) , tokai pharmaceuticals ( inst ) , merck ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , constellation pharmaceuticals ( inst ) patents , royalties , other intellectual property : co - inventor of a biomarker technology that has been licensed to qiagen travel , accommodations , expenses : sano , dendreon , medivation heather h . 
cheng research funding : inovio pharmaceuticals ( inst ) , sano ( inst ) , astellas medivation ( inst ) , janssen ( inst ) , clovis oncology ( inst ) , color foundation ( inst ) eric q . 
hsieh honoraria : hotspot therapeutics research funding : effector ( inst ) patents , royalties , other intellectual property : mtor modulators and uses thereofpatent number : 9629843 , use of translational proling to identify target molecules for therapeutic treatmentpublication number : 20140288097 peter s . 
nelson consulting or advisory role : janssen oncology , astellas pharma , genentech expert testimony : veneble - fitzpatrick law firm travel , accommodations , expenses : janssen oncology evan y . 
yu consulting or advisory role : janssen , bayer , merck , astrazeneca , emd serono , churchill pharmaceuticals , incyte , amgen , tolmar , qed , dendreon , seattle genetics , agensys ( inst ) , astellas pharma ( inst ) , dendreon ( inst ) , genentech ( inst ) , bayer ( inst ) , merck ( inst ) , seattle genetics ( inst ) r . 
true stock and other ownership interests : lightspeed micro research funding : ventana medical systems patents , royalties , other intellectual property : lens on an open - top lightsheet microscope jonathan i . 
histopathology 60 : 59 - 74 , 2012 brinker da , potter sr , epstein ji : ductal adenocarcinoma of the prostate diagnosed on needle biopsy : correlation with clinical and radical prostatectomy ndings and progression . 
am j surg pathol 23 : 1471 - 1479 , 1999 lotan tl , toubaji a , albadine r , et al : tmprss2 - erg gene fusions are infrequent in prostatic ductal adenocarcinomas . 
prostate 75 : 1610 - 1619 , 2015 vinceneux a , bruyere f , haillot o , et al : ductal adenocarcinoma of the prostate : clinical and biological proles . 
prole 77 : 1242 - 1250 , 2017 chaux a , albadine r , toubaji a , et al : immunohistochemistry for erg expression as a surrogate for tmprss2 - erg fusion detection in prostatic adenocarcinomas . 
am j surg pathol 35 : 1014 - 1020 , 2011 gillard m , lack j , pontier a , et al : integrative genomic analysis of coincident cancer foci implicates ctnnb1 and pten alterations in ductal prostate cancer . 
mod pathol 22 : 1273 - 1279 , 2009 jeong su , kekatpure ak , park jm , et al : diverse immunoprole of ductal adenocarcinoma of the prostate with an emphasis on the prognostic factors . 
pritchard cc , salipante sj , koehler k , et al : validation and implementation of targeted capture and sequencing for the detection of actionable mutation , copy number variation , and gene rearrangement in clinical cancer specimens . 
herawi m , epstein ji : immunohistochemical antibody cocktail staining ( p63 / hmwck / amacr ) of ductal adenocarcinoma and gleason pattern 4 cribriform and noncribriform acinar adenocarcinomas of the prostate . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
shlien a , campbell bb , de borja r , et al : combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultrahypermutated cancers . 
true l , gulati r , lange j , et al : histological patterns of ductal adenocarcinoma of prostate correlates with mutations in dna repair genes and may aid in selecting the type of systemic therapy for castration - resistant prostate carcinoma . 
 ductal prostate cancer genomics isaacsson velho p , silberstein jl , markowski mc , et al : intraductal / ductal histology and lymphovascular invasion are associated with germline dna - repair gene mutations in prostate cancer . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
 comprehensive analysis of the unfolded protein response in breast cancer subtypes purpose triple - negative breast cancers ( tnbcs ) are associated with a worse prognosis and patients with tnbc have fewer therapeutic options than patients with non - tnbc . 
recently , the ire1a - xbp1 branch of the unfolded protein response ( upr ) was implicated in tnbc prognosis on the basis of a relatively small patient population , suggesting the diagnostic and therapeutic value of this pathway in tnbcs . 
in addition , the ire1a - xbp1 and hypoxia - induced factor 1 a ( hif1a ) pathways have been identified as interacting partners in tnbc , suggesting a novel mechanism of regulation . 
to comprehensively evaluate and validate these findings , we investigated the relative activities and relevance to patient survival of the upr and hif1a pathways in different breast cancer subtypes in large populations of patients . materials and methods we performed a comprehensive analysis of gene expression and survival data from large cohorts of patients with breast cancer . 
the patients were stratified based on the average expression of the upr or hif1a gene signatures . results we identified a strong positive association between the xbp1 gene signature and estrogen receptorpositive status or the hif1a gene signature , as well as the predictive value of the xbp1 gene signature for survival of patients who are estrogen receptor negative , or have tnbc or her2 +  . 
2017 by american society of clinical oncology introduction triple - negative breast cancers ( tnbcs ) represent a breast cancer subpopulation lacking estrogen receptor ( er ) , progesterone receptor ( pr ) , and her2 expression.1 patients with tnbc have a worse prognosis than those with non - tnbc , suggesting more aggressive biologic behavior . 
the unfolded protein response ( upr ) is an adaptive process to alleviate endoplasmic reticulum stress during tumor growth and proliferation.2 one major upr signaling pathway , ire1a leading to activation of xbp1 , is implicated in multiple types of cancers , including breast cancer.3 a recent study reported that tnbc cell lines and primary tumor samples from patients with tnbc express higher levels of the activated form of xbp1 than non - tnbc.4 furthermore , this study also revealed that in tnbc cells , xbp1 coregulates hif1a target genes , and that both xbp1 and hif1a gene signatures were associated with poor prognosis in tnbc , but not in patients with er + breast cancer . 
for example , the expression of esr1 , gata3 , and xbp1 were highly correlated in breast cancer.5 , 6 in addition , xbp1 enhances the transactivation activity of era7 and was identified as an estrogenresponsive gene.8 recently , our laboratory identified that doxorubicin , a widely used chemotherapeutic agent in breast cancer treatment , is a potent inhibitor of xbp1 activation.9 therefore , clarifying the role of xbp1 in breast cancer prognosis would not only improve the application of existing treatment strategy but also would dadi jiang brandon turner jie song ruijiang li maximilian diehn quynh - thu le purvesh khatri albert c . 
using the same four luminal and six basal - like cell lines reported by chen et al , 4 ( a ) average expression of the xbp1 gene signature was higher in the luminal type , although not statistically significant , whereas ( b ) average expression of the atf4 / chop signature was significantly higher in the basal - like type . 
the cell line collection consists of 19 estrogen receptor ( er ) + and 32 er , or 25 triple - negative breast cancer ( tnbc ) and 26 non - tnbc cell lines defined on the basis of mrna / protein levels of er / progesterone receptor / her2 . 
 ( e ) the same analysis on microarray data of patients with breast cancer from the cancer genome atlas database ( n = 404 er + and n = 118 er , or n = 91 tnbc and n = 431 non - tnbc )  . 
 support the preclinical development of therapy on the basis of modulating xbp1 activity.10 , 11 in this study , we comprehensively investigated the activity of xbp1 , its relationship with the er status as well as the hif1a pathway , and its prognostic value in a large group of patients with breast cancer . 
the rnaseq ( n = 1 , 182 ; illumina hiseq ; illumina , san diego , ca ) and microarray ( n = 597 , a subset of the 1 , 182 patients ; agilentg4502a_07_3 array ; agilent technologies , santa clara , ca ) data with the clinical information of the patients with breast cancer were downloaded from the cancer genome atlas ( tcga ) database through the university of california santa cruz cancer browser . 
all signature and expression data were normalized as z - scores with a mean of 0 and standard deviation of 1 . differences in average expression ( microarray or rnaseq ) level of the gene signatures by er and tnbc status were assessed using the nonparametric wilcoxon rank - sum test . 
mrna expression levels of foxa1 , esr1 , and gata3 were first individually normalized ( mean , 0 ; standard deviation , 1 ) and then averaged together before comparing with either the xbp1 gene signature expression or xbp1 gene expression using regression . 
cox proportional hazards regression models were fit to breast cancer survival data , and the nonparametric log - rank test was used to test for significant differences in relapse - free survival ( rfs ) between subtypes of patients with high or low levels of average xbp1 , atf4 / chop , or hif1a gene signature expression . 
patient survival data up to 150 months were used for the analysis . cell culture and viability assay mda - mb - 231 , hs578t , mda - mb - 468 , mcf7 , t47d , and bt474 cells were purchased from american type culture collection ( manassas , va ) and maintained in dmem supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin , and cells were cultured at 37c with 5% co2 . 
after 72 hours of treatment , xtt reagent ( american type culture collection ) was added to the wells , then cells were incubated for 2 hours , and cell viability was calculated with the following formula : absorbance = a475nm ( test ) a475nm ( blank ) a660nm ( test ) using a biotek synergy h1 plate reader ( biotek , winooski , vt )  . results first , we tested the initial conclusion of the previous study4 that basal - like breast cancer cell lines , which consist primarily of tnbc cells , express higher levels of the spliced / activated form of xbp1 compared with those of the luminal type , which consist primarily of er + cells.4 we found the average expression of the same xbp1 gene signature used by the previous investigators to measure xbp1 activity4 was actually higher in the same set of luminal ( n = 4 ) than basal - like ( n = 6 ) cell lines , albeit without statistical significance ( fig 1a )  . 
 2 average expression of the xbp1 gene signature was significantly higher in er + compared with cell lines , consistent with previous reports ( fig 1c ) .5 - 8 however , there was no statistically significant difference in the xbp1 signature between tnbc and non - tnbc cell lines ( fig 1c )  . in contrast , expression of the atf4 / chop sigor tnbc than in er + or nature was higher in er non - tnbc cell lines , respectively ( fig 1c )  . in established cell these observations lines prompted us to evaluate these pathways in tumor samples from patients with breast cancer . 
we performed the same analyses on 1 , 182 patients with breast cancer from tcga , on the basis of rnaseq ( fig 1d ) , 5 or a subset of 597 patients on the basis of microarray ( fig 1e ) , as well as 1 , 809 curated microarray gene expression profiles ( fig 1f ) .14 in each comparison , we found expression of the xbp1 gene signature was significantly higher in er + or non - tnbc than in er or tnbc tumor samples , respectively . 
overall , we identified a strong correlation between the expression of xbp1 , which is also a transcriptional target of activated xbp1 itself , 4 and er pathway genes ( ie , esr1 , gata3 , and foxa1 ; fig 2a and 2b )  . 
this relationship was also significant when we correlated the average expression of the xbp1 gene signature with the average er pathway gene expression ( fig 2c and 2d )  . these data are consistent with a previous report of xbp1 playing a crucial role in 17 - b - estradiolinduced growth of er + breast cancer cells.15 to test the reported coregulation of target gene expression by activated xbp1 and hif1a4 in a large cohort of patients with breast cancer , we performed similar correlation analysis using the tcga dataset . 
overall , we found a strong correlation between the xbp1 and hif1a gene signatures in patients either er + or er , and with non - tnbc or tnbc , who were from tcga ( fig 2e and 2f )  . 
this result confirms the molecular - level interaction between the two pathways with large - scale gene - expression data from patients and suggests the xbp1 and hif1a pathways are highly correlated in breast cancer regardless of the subtypes . to assess the influence of the upr and hif1a pathways on survival of patients with breast cancer and extend the analysis of the previous study4 to larger patient cohorts , we performed survival analyses of the 1 , 809 - patient data set , using the same high - low expression cutoff ( 58th percentile ) as previously reported.4 surprisingly , the xbp1 signature was not associated with worse rfs in the whole population of patients with breast cancer ( fig 3a , left )  . 
however , when the patient population was further stratified into tnbc versus , or her2 + versus non - tnbc , er + versus er , higher expression of the xbp1 signature her2 was associated with worse rfs in tnbc ( fig 3b , ( fig 3c , left ) , or her2 + ( fig 3d , left ) left ) , er patients , but not in patients positive for er ( fig 3c , left ) or negative for her2 ( fig 3d , left )  . 
interestingly , the atf4 / chop signature was not associated with rfs ( fig 3a , middle ) , even after stratification into either tnbc / non - tnbc , er + / 2 her2 + / 2 status ( fig 3b - 3d , middle )  . 
in contrast , the hif1a signature was significantly associated with rfs regardless of how the patients were stratified ( fig 3a - 3d , right )  . to evaluate whether using gene signature expression cutoffs at the extremes would reveal stronger association , we applied quartile ( the highest 25% v the lowest 25% ) cutoff and performed the same survival analysis . 
the atf4 / chop signature became significantly associated with rfs in the whole population ( data supplement ) and the xbp1 signature became significantly associated ( data with rfs in patients who were her2 supplement )  . 
however , the positive association between the xbp1 signature and rfs in the patients with non - tnbc using the 58th percentile cutoff became negative when using the quartile cutoff , which likely was due to the reduced number of patients in the new categories ( data supplement )  . 
nevertheless , the major conclusions of the current study remain unchanged . discussion in summary , on the basis of a comprehensive analysis on large cohorts of patients with breast cancer , our findings confirm the initial observations from chen et al4 that xbp1 activity was associated with a worse prognosis in tnbc , but not er + patients , and extend this relationship to ( fig 3c , left )  . 
correlation of xbp1 mrna expression and the xbp1 gene signature with estrogen receptor ( er ) pathway gene expression and correlation of the xbp1 and hif1a gene signatures in the the cancer genome atlas ( tcga ) breast cancer data set . 
correlation analysis of expression of the xbp1 gene and the average of three er pathway genes ( esr1 , foxa1 , and gata3 ) using ( a ) rnaseq or ( b ) microarray data from 1 , 182 patients with breast cancer in tcga breast cancer database and the same correlation analysis of the average expression of the xbp1 gene signature and the three er pathway genes on the basis of ( c ) rnaseq or ( d ) microarray data in the tcga database . 
 ( e , f ) correlation of the average expression of the xbp1 and hif1a gene signatures in the groups of patients with breast cancer , on the basis of the rnaseq data in the tcga database : ( e ) er + ( n = 600 patients , gold ) and er ( n = 179 patients , blue ) ; and ( f ) triple - negative breast cancer ( tnbc ; n = 125 patients , blue ) and non - tnbc ( n = 647 patients , gold )  . 
kaplan - meier graphs showing relapse - free survival ( rfs ) of the different subtypes of patients with breast cancer stratified by the xbp1 , atf4 / chop , or hif1a gene signatures . 
kaplan - meier graphs of rfs of ( a ) the total of 1 , 640 patients , ( b ) 225 patients with triple - negative breast cancer ( tnbc ) and 1 , 415 with non - tnbc , ( c ) 1 , 286 who are estrogen receptor ( er ) + and 354 who are er with rfs < 150 months separated by high and low ( with the 58th percentile cutoff used previously4 ) levels of the average expression of the xbp1 ( left column ) , atf4 / chop ( middle column ) , or hif1a ( right column ) gene signature . 
this may partially explain those who are her2 why the xbp1 - rfs association is stronger in than in those with tnbc patients who are er ( fig 3b and 3c , left ) , because some of the patients / her2 + are included in the patients who are er but not in the patients with tnbc . who are er however , through analyzing large - scale gene expression profiles , we identified that average expression of the same xbp1 gene signature reported by chen et al4 was higher in er + or non - tnbc or tnbc cell lines or patients , respecthan er tively ( fig 1c - 1f ) , to correct the initial conclusion by chen et al.4 these data are consistent with other previous studies . 
our findings suggest that , albeit with higher xbp1 activities , patients who are er + , and other patients with non - tnbc ( excluding her2 + ) , may not benefit from therapeutic strategies targeting xbp1 activity . 
the design and aims of the current study are summarized in the schematic shown in the data supplement . recently , we have shown that doxorubicin blocks xbp1 activation through the inhibition of ire1a rnase activity.9 these data suggest that pa , and possibly tients with tnbc , who are er those who are her2 + , may benefit the most from anthracycline - based therapies . 
davies mp , barraclough dl , stewart c , et al : expression and splicing of the unfolded protein response gene xbp - 1 are significantly associated with clinical outcome of endocrine - treated breast cancer . 
finlin bs , gau cl , murphy ga , et al : rerg is a novel ras - related , estrogen - regulated and growth - inhibitory gene in breast cancer . 
j biol chem 276 : 42259 - 42267 , 2001 jiang d , lynch c , medeiros bc , et al : identification of doxorubicin as an inhibitor of the ire1a - xbp1 axis of the unfolded protein response . 
jiang d , niwa m , koong ac : targeting the ire1a - xbp1 branch of the unfolded protein response in human diseases . semin cancer biol 33 : 48 - 56 , 2015 11 . 
gy orffy b , lanczky a , eklund ac , et al : an online survival analysis tool to rapidly assess the effect of 22 , 277 genes on breast cancer prognosis using microarray data of 1 , 809 patients . 
sengupta s , sharma cg , jordan vc : estrogen regulation of x - box binding protein - 1 and its role in estrogen induced growth of breast and endometrial cancer cells . 
lacroix m , leclercq g : about gata3 , hnf3a , and xbp1 , three genes co - expressed with the oestrogen receptoralpha gene ( esr1 ) in breast cancer . 
tozlu s , girault i , vacher s , et al : identification of novel genes that co - cluster with estrogen receptor alpha in breast tumor biopsy specimens , using a large - scale real - time reverse transcription - pcr approach . 
andres sa , wittliff jl : relationships of esr1 and xbp1 expression in human breast carcinoma and stromal cells isolated by laser capture microdissection compared to intact breast cancer tissue . 
wang dy , fulthorpe r , liss sn , et al : identification of estrogen - responsive genes by complementary deoxyribonucleic acid microarray and characterization of a novel early estrogen - induced gene : eeig1 . 
karn t , metzler d , ruckhaberle e , et al : data - driven derivation of cutoffs from a pool of 3 , 030 affymetrix arrays to stratify distinct clinical types of breast cancer . 
 e role of community - based genomics programs see accompanying article doi : increasingly , genomic changes in patients tumors are used to inform individualized management , although the optimal approach and impact of tumor profiling on cancer care , especially in the community setting , remain important research questions.1 , 2 challenges in applying precision medicine approaches include cost and access to genomic testing and to indicated therapies , lack of actionable gene alterations , education of all stakeholders , patient expectations , complexity of interpretation of data , access to big data solutions , and expansion of physicians comfort zones . 
arguably , the latter represents the most formidable challenge . in the article accompanying this editorial , burkard et al3 address many of these challenges in their report , implementation and clinical utility of an integrated academic - community regional molecular tumor board . 
the implementation of a novel wisconsin state - wide model , including a precision - medicine molecular tumor board ( pmmtb ) , an institutional review boardapproved registry protocol , and a journal club , allows for addressing many of the challenges of precision medicine . 
the authors point out that in the united states , much of precision medicine is implemented in an academic - center context , often in conjunction with robust clinical trial platforms , to include big data analytic informatics solutions . conversely , most patients are cared for outside of such academic systems.4 the challenge is to provide patients access , through their community - based providers , to the potential benefits of precision medicine . 
the described model is an academic and community partnership that allows for nextgeneration sequencing ( ngs ) through any clinical laboratory improvement amendmentscertified laboratory , and uses as its cornerstone a twiceweekly web - based pmmtb teleconference , provided as a free service . 
as with most molecular tumor boards ( mtbs ) , the focus is on prioritizing labeled and off - label therapies , as well as clinical trial options , on the basis of biologic profiling of the patients tumor . 
the model addresses the increasingly dynamic nature of the molecular pathology report with its interpretation of a specific genotype and its clinical importance often becoming outdated in the short term . the presence of an institutional review board approved registration trial assists in observing the clinical course of patients to include relevant outcomes . 
patients may have molecular profiling performed early in their clinical course ; thus , long - term follow - up allows for evaluating the downstream impact of the original molecular profiling or that of any subsequent repeat molecular profiling . this type registry is especially important given the uncertainties regarding the value proposition of precision medicine in daily practice . 
the construct of this tripartite model seeks to address the challenges of precision medicine , though the results reported by burkard et al3 emphasize the remaining barriers to successful implementation of precision medicine platforms in community settings and beyond . over approximately 1 year , a modest number of patients with metastatic / incurable cancer ( n 5 74 ) were presented at the pmmtb ; only 38 evaluable patients were enrolled in the registration trial , and some of these were enrolled retrospectively . 
 actionable target ( ie , molecularly targeted clinical trial and / or off - trial treatment ) was identified in 32 patients , with treatment accepted in only nine patients ( 28% )  . 
only one patient was enrolled in an instate clinical trial , although a total of 14 patients had clinical trial options within the state , and no patients were enrolled in out - of - state clinical trials . 
during much of the reported period in the registry trial , the national cancer institute molecular analysis for therapy choice ( match ) basket trial was on hold , and there was no access to the asco targeted agent and profiling utilization registry ( tapur ) basket trial . 
clinical benefit was seen in three of eight ( 38% ) evaluable patients , with a response evaluation criteria in solid tumors ( recist ) - defined response in one patient . this novel precision - medicine model aims at networking academic and community providers to deliver genomically driven care to patients closest to where they live and under the care of their community physicians . 
what role does cost play with regard to accessing ngs testing and targeted drugs ? given the wide range of ngs testing platforms , how is quality assured ? this is especially important because clinical laboratory improvement amendments certification does not address many quality dimensions inherent to ngs testing . in this report , the mean number of prior therapies for patients was two . 
when is the most effective time to perform ngs testing during a patients clinical course : earlier or later ? similarly , a range of ngs panel sizes was used , with a not - surprising suggestion that larger comprehensive panels are more frequently associated with treatment options . where does cost effectiveness guide us in selecting ngs panel size ? with regard to actionable gene alterations , it is remarkable that by recent analysis , the us food and drug administration has only approved 22 gene alterations with associated biomarkers.7 , 8 the key to defining labeled indications for targeted therapies is participation in clinical trials such as match and tapur . additional clinical trial approaches include prospective registration - type trials that contain detailed data elements , including demographic data , clinical outcomes measures , and quality - of - life and economic metrics that allow for long - term follow - up and planned statistical analyses . one aspect not referenced in the burkard et al3 report is the underlying informatics platforthe scalability of such a platform , as well as its interfacing capabilities , along with its analytic functions , are key , especially across a multisite network . 
in particular , a clinical - trials matching function that allows for prioritizing variables such as geographic distance and relevance to an individual patients unique characteristics , and those of their tumor , will facilitate identification of the most promising clinical trials for a given patient . 
in addition , engaging busy clinicians is always a challenge , and one presumed solution is to have approaches and platforms that align with their daily work flow , including integration into existing informatics platforms , most especially the electronic medical record , along with automatic extraction of data elements from existing electronic databases , which limits the need for form completion , whether electronic or manual . 
for example , maximal use of high quality databases , such as tumor registries or the seer program database , can help overcome the challenges of relying on semistructured data within the electronic medical record . the demonstration of the value proposition for precision medicine is a work in progress . 
it will require clinical trial experience with genomically driven therapies across a broad population of patients with varied tumor types , cared for by a range of providers , and in varied settings , to include the broad variety of community practices . 
the newer formulation of clinical trial design , whether the basket or umbrella type design or that of sophisticated registry trials , such as those described by burkard et al , will substantially facilitate the access to targeted therapy trials in the community setting.9 , 10 these trials provide guidance for community physicians in identifying gene alterations or biomarkers with appropriate targeted therapies and provide access to off - label targeted therapies . 
brown td , tittel pd , gold pj , et al : impact of a personalized medicine research program ( pmrp ) , using targeted tumor profiling and a cloud based clinical trials matching platform , on clinical decision - making . 
of interest , previous reports have identified germline mutations in driver oncogenes that are associated with lung cancers , such as epidermal growth factor receptor ( egfr ) , 1 - 6 receptor tyrosine - protein kinase erbb - 2 ( erbb2 ) , 7 and possibly others , 8 which suggests that heritable predisposition to lung cancer is a contributor in some cases . 
in this work , we describe a novel germline egfr v769m variant in a patient with multiple lung cancers , each of which harbors co - occurring somatic mutations in egfr . 
to our knowledge , we also describe for the first time the activating and sensitizing characteristics of egfr v769m in preclinical and in silico structural models . a 57 - year - old man was found to have five distinct lung nodules and no mediastinal or hilar lymphadenopathy , which raised the possibility of multiple synchronous lung cancers . 
the patient was a remote former smoker ( 34 packyears ) , quitting 21 years before presentation . he underwent several procedures to resect the five distinct lung nodules , each of which was determined to be lung adenocarcinoma . 
molecular analysis was performed on the five tumors and each revealed an egfr v769m alteration . in addition , the presence of distinct g719 mutations in egfr in several tumors suggested that these tumors represented independent primary lung cancers ( table 1 and appendix fig a1 , online only )  . as v769m is predicted to have a significant functional impact ( polyphen9 ) , the possibility of a germline variant was considered . 
the co - occurring somatic variant in exon 20 ( egfr s768i ) was found on the same allele as the germline egfr v769m . it was not possible to determine conclusively whether somatic mutations in exon 18 were similarly in cis with the germline variant given the genomic distance between the variants , which were captured separately and sequenced on distinct short reads in the genomic dna - based assay that was performed . 
he has remained without evidence of progressive disease or new lesions for more than 3 years . the egfr v769 codon is in exon 20 and corresponds to the region just after the c - helix of the egfr kinase domain.10 this variant is uncommonly identified in public databases ( 2 of 120 , 770 alleles in exome aggregation consortium11 ; 1 of 13 , 005 alleles in exome variant server12 )  . 
cases of ( presumably ) somatic egfr v769m mutations have been reported13 - 16 ; one patient received treatment with egfr tyrosine kinase inhibitors with no clinical response.14 as the functional and sensitizing impact of egfr v769m has not been ascopubs.org / journal / po jco precision oncology matthew d . 
consistent with an activated phenotype , p - akt ( s473 ) / akt and p - erk ( t202 / y204 ) / erk were increased in egfr v769m mutant compared with wild - type cells in an egf ligand - dependent setting ( fig 3 )  . 
the percent activation , quantified by the ratio of pegfr ( y1068 ) to total egfr , was inhibited by 50% in egfr v769m cells at an erlotinib concentration of 0.4 mm compared with 0.01 mm in egfr l858r cells ; 50% inhibition was not reached in egfr t790m cells at the doses fig 1 . 
to explore the functional impact of comutations in egfr on egfr v769m , we also examined 293t cells that were transfected with egfr v769m + g719c . activation of downstream pathways and sensitivity to erlotinib were both enhanced in the setting of egfr v769m plus g719c compared with egfr v769m alone ( appendix figs a2 and a3 , online only )  . we also used qualitative in silico structural modeling to assess the predicted functional impact of v769m on egfr kinase activity . 
we examined the location and interactions of the original valine residue in the three - dimensional structure of egfr in both inactive and activated conformations ( fig 5 )  . 
in the inactive conformation , the v769 residue is completely buried in the hydrophobic core of the protein globule where it has multiple noncovalent interactions with atoms of surrounding residues ( fig 5a )  . 
a substitution of the val residue with a large met residue will result in the destruction of molecular interaction and the destabilization of the inactive conformation of the kinase domain the activated conformation , the molecular surrounding of v769 undergoes significant changes , the total number of noncovalent interactions is reduced , and structural rearrangements that involve the dfg loop region facilitate the penetration and stabilization of atp ( fig 5b )  . thus , it is reasonable to hypothesize that the structural accommodation of met residue in the activated conformation costs less energy than in the inactive conformation . 
the total effect of the v769m mutation is an activation of egfr caused by a shift of the energy balance between activated and inactivated conformations as a result of significant destabilization of the inactivated state of the molecule . 
moreover , the quantitative energy balance also favors the activated / open conformation in egfr v769m and each of the comutant scenarios identified in this case ( appendix table a1 , online only )  . 
by contrast , the energy balance does not favor that activated state in the context of egfr v769m plus l858r , which perhaps explains the absence of this combination in our patient or other reported cases . mutant egfr represents a paradigmatic targetable oncogene in lung cancers and is present in almost 20% of lung adenocarcinomas . 
egfr v769m mutation activates mitogen - activated protein kinase and akt signaling . 293t cells were transiently transfected with plasmids that encode wild type ( wt ) egfr or egfr mutants with the following changes : v769m , l858r , and t790m . 
quantification of the protein expression of phospho - epidermal growth factor receptor ( pegfr ) , total egfr , phosphoerk ( perk ) , total - erk , phospho - akt ( pakt ) , and total akt was analyzed by using imagej . 
 ( * ) p , .05 , students t test , each condition compared to pegfr / egfr perk / erk pakt / akt have been several reports of patients with germline egfr t790m mutations that have occurred both sporadically and in families.1 , 3 , 5 , 18 , 19 much like the case we present here , the majority of germline egfr variants that have been detected in lung cancers are associated with secondary , somatic in egfr ( with some activating mutations exceptions1 , 3 , 17 , 19 )  . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . mutations in egfr further contribute to lung cancer oncogenesis . in summary , we describe an egfr v769m germline variant found in a patient with five synchronous lung cancers that represented several distinct primaries . 
our structural modeling and functional studies demonstrate that egfr v769m is activating and intermediately sensitive to erlotinib and support the proposed causal role of this variant in the development of multiple lung cancers in this patient . 
hellmann consulting or advisory role : bristol - myers squibb , merck , genentech , astrazeneca , medimmune , novartis , janssen research funding : bristol - myers squibb , genentech takuo hayashi no relationship to disclose boris reva no relationship to disclose helena a . 
riely consulting or advisory role : novartis , genentech research funding : novartis ( inst ) , genentech ( inst ) , millennium pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , pfizer ( inst ) , infinity pharmaceuticals ( inst ) , ariad pharmaceuticals ( inst ) patents , royalties , other intellectual property : patent application submitted covering pulsatile use of erlotinib to treat or prevent brain metastases ( inst ) travel , accommodations , expenses : novartis prasad s . 
robson honoraria : astrazeneca consulting or advisory role : mckesson , astrazeneca research funding : astrazeneca ( inst ) , abbvie ( inst ) , biomarin ( inst ) , medivation ( inst ) , tesaro ( inst ) travel , accommodations , expenses : astrazeneca maria e . 
paik honoraria : celgene , bristol - myers squibb , lilly , ariad pharmaceuticals consulting or advisory role : celgene , lilly , ariad pharmaceuticals , bristol - myers squibb research funding : celgene , emd serono travel , accommodations , expenses : emd serono marc ladanyi honoraria : merck ( i ) consulting or advisory role : nccn / boehringer ingelheim afatinib targeted therapy advisory committee , nccn / astrazeneca tagrisso rfp advisory committee research funding : loxo pharmaceuticals ( inst ) affiliations matthew d . 
in the closed / inactive conformation , v769 is tightly packed in hydrophobic surroundings formed by the residues of two alpha helices ( 752 to 768 , 812 to 831 ) , betastrand ( 777 to 782 ) and two loop regions ( 769 to 776 and the conserved dfg loop region 855 to 857 , a key structural element that regulates kinase activity )  . 
in the open / active conformation , the three - dimensional ( 3d ) structure of the kinase domain changes drastically ( for example , see flipping of 3d position of glu865 shown in magenta in both panels ) and the molecular packing of v769 is relaxed . the total number of 4 atom - to - atom contacts is reduced from 34 in the closed conformation to 27 in the open conformation . the significant structural rearrangements involving the dfg loop region facilitate the penetration and stabilization of atp molecule ( b ; atp analog shown in brown )  . 
nat genet 37 : 1315 - 1316 , 2005 ikeda k , nomori h , mori t , et al : novel germline mutation : egfr v843i in patient with multiple lung adenocarcinomas and family members with lung cancer . 
ohtsuka k , ohnishi h , kurai d , et al : familial lung adenocarcinoma caused by the egfr v843i germ - line mutation . j thorac oncol 4 : 139 - 141 , 2009 j clin oncol 29 : e191 - e192 , 2011 5 . 
j thorac oncol 7 : 1049 - 1052 , 2012 van noesel j , van der ven wh , van os ta , et al : activating germline r776h mutation in the epidermal growth factor receptor associated with lung cancer with squamous differentiation . 
huang sf , liu hp , li lh , et al : high frequency of epidermal growth factor receptor mutations with complex patterns in non - small cell lung cancers related to gefitinib responsiveness in taiwan . 
wu jy , yu cj , chang yc , et al : effectiveness of tyrosine kinase inhibitors on uncommon epidermal growth factor receptor mutations of unknown clinical significance in non - small cell lung cancer . 
santis g , angell r , nickless g , et al : screening for egfr and kras mutations in endobronchial ultrasound derived transbronchial needle aspirates in non - small cell lung cancer using cold - pcr . 
girard n , lou e , azzoli cg , et al : analysis of genetic variants in never - smokers with lung cancer facilitated by an internet - based blood collection protocol : a preliminary report . 
jama oncol 2 : 104 - 111 , 2016 dnas from five tumor samples and blood were extracted and testing was performed by using a custom gene panel , mskampliseq , which covers 94 cancer - related genes . 
library construction was performed with 20 ng of formalin - fixed , paraffinembedded tumor and matched blood dna by multiplexed amplification of 891 regions tiled by 2 , 126 amplicons . sequencing was performed on an ion torrent pgm ( thermofisher , waltham , ma ) , and data were analyzed with a custom pipeline developed at memorial sloan kettering cancer center . 
aligned bam files generated by torrent suite ( thermofisher , waltham , ma ) were primer trimmed and variant calling was performed to detect single nucleotide and indel variants using varscan2 , which were annotated using annovar . 
for transient transfections , 293t human embryonic kidney cells were transfected ( 2 3 105 cells per well in sixwell plates ) by using fugene6 and 0.8 mg of plasmid dna . 
cells were grown in dmem with high glucose , 10% fetal bovine serum , 2 mm l - glutamine , 10 u / ml penicillin , and 10 mg / ml streptomycin at 37c and 5% co2 . 
after 36 hours , cells were serum starved in media that contained 0.1% serucells were treated with different concentrations of erlotinib ( selleckchem , houston , tx ) for 3 hours . 
three independent experiments were performed for all analyses . forward : 59 - cgaacgcaccggagcacagcactttgatctt - 39 , reverse : 59immunoblotting cells were lysed in 50 mm tris$hcl , ph 8.0 , 150 mm sodium chloride , 5 mm magnesium chloride , 1% triton x - 100 , 0.5% sodium deoxycholate , 0.1% sds , 40 mm sodium fluoride , 1 mm sodium orthovanadate , and complete protease inhibitors ( roche )  . 
after quantitation by bio - rad protein assays ( bio - rad , hercules , ca ) , approximately 50 mg of each sample was separated by gel electrophoresis on 4% to 12%bis - tris precast gels ( thermo fisher scientific life sciences , waltham , ma ) and transferred to 0.2 mm polyvinylidene difluoride membrane ( thermo fisher scientific life sciences ) by using wet / tank blotting systems ( bio - rad )  . 
specific proteins were detected by amersham ecl western blotting detection reagent ( thermo fisher scientific life sciences ) , and the following antibodies : antitotal egfr 1 : 2 , 500 ( bd transduction laboratories ) , antiphospho - egfr ( tyr - 1068 ) 1 : 1 , 000 ( cell signaling technology , danvers , ma ) , antitotal p44 / 42 mitogen - activated protein kinase ( erk1 / 2 ) 1 : 1000 ( cell signaling technology ) , antiphospho - p44 / 42 mitogen - activated protein kinase ( erk1 / 2 ; thr202 / tyr204 ; d13.14.4e ) 1 : 1000 ( cell signaling technology ) , antitotal akt 1 : 1000 ( cell signaling technology ) , antiphospho - akt ( ser473 ; d9e ) , antihorseradish peroxidaseconjugated anti - rabbit ig 1 : 10 , 000 ( r&d systems , minneapolis , mn ) , and horseradish peroxidaseconjugated anti - mouse igg 10 , 000 ( r&d systems )  . quantification of protein expression was analyzed by using imagej 2.0.0 - rc - 30 ( nih , bethesda , md )  . 
modeling of mutations in kinase domain of epidermal growth factor receptor ( egfr ) molecule was done for a chain region l703 - g983 using two template structures , 2gs6 for closed and 2gs7 for open conformations . 
this is a pooled analysis . competing interests the authors declare that they have no competing interests . publishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations . author details 1service dhmatologie et thrapie cellulaire , hpital saint antoine , ap - hp , 168 rue du faubourg saint antoine , 75012 paris , france . 
16division of hematology and bone marrow transplantation , the chaim sheba medical center , tel - hashomer , tel aviv university ( tau ) , tel aviv , israel . received : 6 february 2018 accepted : 2 march 2018 reply to s . 
romero - cordoba et al we appreciate the commentary and analysis by romero - cordoba et al1 in their response to our recent article in jco precision oncology.2 our research groups pursue the common goal of elucidating immunologic differences between breast cancers that could inform future immunotherapy strategies . 
all immune metrics , including counts of tumor inltrating lymphocytes and other immune cells , as well as immune gene signatures , represent a continuum and display substantial variations across samples . nevertheless , it is possible to dene more versus less immune - rich cancers by any of these metrics , and for some types of analyses , such dichotomization is necessary , even using an arbitrary threshold . our study focused on the question of whether immune - rich estrogen receptor ( er ) positive and triple - negative ( tn ) breast cancers ( bcs ) have similar or different immune microenvironments . 
to perform this comparison across multiple types of molecular data , we had to dene a shared denition of immunerichness ; we used the 75th percentile of a total tumor inltrating lymphocyte gene expression score , which encompasses the expression of 11 immune cell populations , calculated across all samples as the threshold . 
we were delighted to see that this admittedly arbitrary denition of immune - richness corresponded reasonably well with the tnbc immune classication developed by romero - cordoba et al3 : 62% of our immune - rich cancers were classied as ima ( t cell - inamed ) and 21% as imc ( immune warm )  . 
several tnbc classications have been proposed using this method.3 - 6 despite the high and mostly positive correlation in immune gene expression in breast cancer tissues , the different classication schemas yield discordant results in a substantial minority of cases , partly because of different design principles and partly because of methodology . 
however , which classication schema or molecular marker is most useful in the clinic is yet to be determined in clinical trials . the clear majority of immune biomarker studies in breast cancer focus on tnbc because of the higher prevalence of tumor inltrating lymphocytes in this subtype . 
we hope that our article draws attention to immunotherapy opportunities in er - positive cancers , because in absolute numbers , there are more immune - rich er - positive cancers than immune - rich tnbcs . 
omeara t , marczyk m , qing t , et al : immunological differences between immune - rich estrogen receptor - positive and immune - rich triple - negative breast cancers . 
prado - v azquez g , g amez - pozo a , trilla - fuertes l , et al : a novel approach to triple - negative breast cancer molecular classication reveals a luminal immune - positive subgroup with good prognoses . 
j ez equel p , loussouarn d , gu erin - charbonnel c , et al : gene - expression molecular subtyping of triple - negative breast cancer tumours : importance of immune response . 
 efcacy of lorlatinib in primary crizotinib - resistant adult neuroblastoma harboring alk y1278s mutation antoine vasseur , md1 ; luc cabel , md1 ; romain geiss , md1 ; gudrun schleiermacher , md , phd1 ; ga elle pierron , phd1 ; maud kamal , phd1 ; nina jehanno , md1 ; guillaume bataillon , md1 ; jean - marc guinebretiere , md1 ; and laurence bozec , md1 introduction anaplastic lymphoma kinase ( alk ) encodes a highly conserved receptor tyrosine kinase described as an oncogene in anaplastic large - cell lymphoma and other cancers.1 alk fusions ( mostly alk - eml4 ) represent 3% to 7% of all nonsmall - cell lung cancers ( nsclcs ) , and these tumors are generally sensitive to crizotinib ( a tyrosine kinase inhibitor [ tki ] targeting alk ) as rstline therapy in the metastatic setting.2 neuroblastoma harbors activating somatic alk mutations in 8% to 9% of patients ( up to 14% of high - risk neuroblastomas ) , with mutations occurring in the tyrosine kinase domain at three key positions ( f1174 , f1245 , and r1275 ) , which account for approximately 85% of all alk mutations in neuroblastoma.3 activation of alk may also occur by genomic amplication in 3% to 4% of patients , and it has been suggested that patients with neuroblastoma who have genetic alterations of alk could also be treated with crizotinib.4 in this article , we report , to our knowledge for the rst time , the case of an adult patient with ganglioneuroblastoma harboring a somatic alk y1278s mutation with primary resistance to crizotinib , but which was highly sensitive to lorlatinib , a third - generation tki targeting alk genomic alterations . 
computed tomography ( ct ) the chest , abdomen , and pelvis showed scan of disseminated lymph node and vertebral bone lesions . 123i - metaiodobenzylguanidine ( 123i - mibg ) scintigraphy was positive with bone and lymph node metastases . 
laboratory tests showed normal lactate dehydrogenase levels and increased urinary catecholamines ( normetanephrine , 40 , 825 nmol / l ; metanephrine , 572 nmol / l )  . the patient received rst - line induction chemotherapy according to the rapid cisplatin , vincristine , carboplatin , etoposide , and cyclophosphamide ( cojec ) regimen.5 after eight cycles , stable disease was observed in lymph nodes , and progression was observed in bone lesions . 
four months after chemotherapy , clinical disease progression was observed with bone pain , and urinary catecholamines were increased ( normetanephrine , 104 , 651 nmol / l ; metanephrine , 1 , 208 nmol / l ) despite stable mibg scintigraphy and ct scan . a lymphadenectomy ( left subclavicular lymph node ) was performed before second - line therapy with topotecan and cyclophosphamide was started for complementary molecular and immunohistochemistry analyses . immunohistochemistry revealed expression of alk by 100% of tumor cells . 
targeted next - generation in alk , sequencing found a pathogenic variant ptyr1278ser , with an allelic frequency of 23% ( fig 1 )  . the patient was included in the french acs e protocol ( nct02034981 ; phase 2 study assessing efcacy and safety of crizotinib in patients harboring an alteration on alk , met or ros1 ) , and crizotinib was initiated in june 2017 ( 250 mg taken orally twice per day )  . 
treatment was stopped in october 2017 after rapid progression of symptomatic bone metastasis conrmed by mibg scintigraphy , with marked general physical deterioration ( eastern cooperative oncology group [ ecog ] performance status 3 ) , asthenia , anorexia , and weight loss . 
lorlatinib was then obtained from a pzer compassionate - use program and was initiated ( 100 mg taken orally once per day ) in november 2017 , with a signicant clinical benet after 2 weeks of treatment . after 2 months , the patient was asymptomatic with marked improvement of performance status ( from ecog 3 to ecog 0 )  . 
alk positivity by ( a ) immunochemistry and ( b ) next - generation sequencing . partial reossication of bone metastases in favor of treatment response ( left subclavicular lymph node , largest lymph node target lesion ; fig 2 )  . 
after 10 months , 123i - mibg scintigraphy re - evaluation of treatment showed a good partial metabolic response of the left supraclavicular lymph node inltration as seen on the single photon emission computed tomography scan associated with partial response of bone inltration . 
international society of pediatric oncology europe neuroblastoma group ( siopen ) score for bone extension6 , 7 was 30 on baseline scintigraphy , which decreased to a siopen score of 15 on the re - evaluation scintigraphy ( 50% decrease ; relative mibg score of 0.5 ; fig 3 )  . 
overall response according to the international neuroblastoma response criteria consensus was assessed as a partial response.8 after 12 months ( last evaluation in november 2018 ) , the clinical benet was maintained ( ct scan showed a partial reduction of 33% in tumor size [ recist1.1 ] corresponding to the nadir ; left subclavicular lymph node ; fig 2 )  . 
no toxicity was reported during treatment with lorlatinib except for grade 1 hypertriglyceridemia and grade 1 edema of the lower limbs ( common terminology criteria for adverse events [ ctcae ] v5 )  . discussion to our this case report of adult neuroblastoma shows , knowledge for the rst time , that lorlatinib , unlike crizotinib , can be effective in patients harboring an alk y1278s mutation . 
the most common resistance mutations after crizotinib therapy are the l1196m ( 5% to 10% ) and g1269a ( , 5% ) mutations , 9 and many other mutations have also been reported . 
these mutations confer various mechanisms of resistance , such as catalytic domain alteration , which causes atp competitive inhibitor resistance , as t790m mutation in the epidermal growth factor receptor ( egfr ) mutation alters the conformation and / or the atp - binding afnity of the kinase.10 lorlatinib , a third - generation alk inhibitor that also targets ros1 , is an attractive tki in patients with nsclc pretreated with rstor second - generation alk inhibitors because of its activity against most alk resistance mutations , such as g1202r mutations11 ( fig 4b )  . 
lorlatinib has demonstrated high clinical activity in alkor ros1 - rearranged nsclc in treatment - naive or crizotinib pretreated patients.12 in nsclc , the y1278s alk mutation has not been described in the clinical setting , and the efcacy of lorlatinib in nsclc remains unknown . 
however , a different mutation at the same position , y1278h , has been reported in nsclc cell lines that have the alk - eml4 fusion exposed to accelerated mutagenesis , 13 which was resistant to crizotinib . neuroblastoma is the most common solid tumor in children , but adult patients are exceptional , because fewer than 0.3 patients are diagnosed per million people per year.14 crizotinib has been shown to be less effective in neuroblastoma than in nsclc , in which alk mutations , rather than alk fusions , are a major oncogenic event in nearly 10% of the patients , 15 and alk is the most common somatically mutated gene . 
in a phase i / ii trial ( nct00939770 ; crizotinib in treating younger patients with relapsed or refractory solid tumors or anaplastic large cell lymphoma ) , 11 patients with alk - mutated neuroblastoma were treated with crizotinib . 
crystal structure of ( a ) crizotinib / alk and ( b ) lorlatinib / alk . in neuroblastoma reported that y1278s is oncogenic ( ligand - independent activation ) and that crizotinib is inlines effective in vitro and in vivo in neuroblastoma cell harboring the alk y1278s mutation , whereas lorlatinib seemed to be highly effective.19 y1278s is located in the the alk activation loop . 1278 - yrasyy - 1283 motif of donella - deana et al20 demonstrated the important role of initial phosphorylation of tyrosine y1278 for the a - loop activation in alk - npm fusion . 
a study of the crystal structure of alk conrmed this role and highlighted a tight interaction in the inactive form of alk between the unphosphorylated y1278 and a cysteine at position 1097.21 in our patient , a y1278 mutation from tyrosine to serine the phosphorylation and thus the alk should inhibit activation , 20 but guan et al19 showed in a preclinical study that y1278 phosphorylation in neuroblastomas was not for alk activation ( unlike phosphorylation of essential y1283 , which is essential )  . 
however , this hypothesis was not conrmed , so the mechanism of alk activation with y1278s mutation is controversial.19 the y1278s mutation seems to be a rare mutation , but it has been reported in six neuroblastoma patients ( catalogue of somatic mutation in cancer [ cosmic ] database ) .17 , 22 - 24 no clinical data regarding the efcacy of alk inhibitors were reported in these six tumors harboring a y1278s alk mutation , but in two patients , preclinical data showed an in vitro resistance to crizotinib ( high concentration that inhibits 50% [ ic50 ] )  . 
note that a clinical trial of lorlatinib for patients with alk - driven relapsed or refractory neuroblastoma is ongoing as part of the new approaches to neuroblastoma therapy ( nant ) consortium ; this trial ( nct03107988 ; study of lorlatinib [ pf - 06463922 ] , an oral small molecule inhibitor of alk / ros 1 , for patients with alk - driven relapsed or refractory neuroblastoma ) is not yet recruiting in europe , and results are pending . in conclusion , we show that lorlatinib is effective against the alk y1278s mutation in neuroblastoma , whereas this mutation confers primary resistance to crizotinib . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . gudrun schleiermacher honoraria : bristol - myers squibb research funding : bristol - myers squibb ( inst ) , pzer ( inst ) , msdavenir ( inst ) , roche ( inst ) travel , accommodations , expenses : roche no other potential conicts of interest were reported . references chiarle r , voena c , ambrogio c , et al : the anaplastic lymphoma kinase in the pathogenesis of cancer . 
nat rev cancer 8 : 11 - 23 , 2008 solomon bj , mok t , kim dw , et al : first - line crizotinib versus chemotherapy in alk - positive lung cancer . 
moss e yp , lim ms , voss sd , et al : safety and activity of crizotinib for paediatric patients with refractory solid tumours or anaplastic large - cell lymphoma : a childrens oncology group phase 1 consortium study . 
lancet oncol 14 : 472 - 480 , 2013 pearson ad , pinkerton cr , lewis ij , et al : high - dose rapid and standard induction chemotherapy for patients aged over 1 year with stage 4 neuroblastoma : a randomised trial . 
lancet oncol 9 : 247 - 256 , 2008 lewington v , lambert b , poetschger u , et al : 123i - mibg scintigraphy in neuroblastoma : development of a siopen semi - quantitative reporting , method by an international panel . 
matthay kk , shulkin b , ladenstein r , et al : criteria for evaluation of disease extent by ( 123 ) i - metaiodobenzylguanidine scans in neuroblastoma : a report for the international neuroblastoma risk group ( inrg ) task force . 
br j cancer 102 : 1319 - 1326 , 2010 park jr , bagatell r , cohn sl , et al : revisions to the international neuroblastoma response criteria : a consensus statement from the national cancer institute clinical trials planning meeting . 
shaw at , felip e , bauer tm , et al : lorlatinib in non - small - cell lung cancer with alk or ros1 rearrangement : an international , multicentre , open - label , singlearm rst - in - man phase 1 trial . 
yoda s , lin jj , lawrence ms , et al : sequential alk inhibitors can select for lorlatinib - resistant compound alk mutations in alk - positive lung cancer . 
cancer discov 8 : 714 - 729 , 2018 3 : 108ra114 , 2011 cell 26 : 682 - 694 , 2014 infarinato nr , park jh , krytska k , et al : the alk / ros1 inhibitor pf - 06463922 overcomes primary resistance to crizotinib in alk - driven neuroblastoma . 
donella - deana a , marin o , cesaro l , et al : unique substrate specicity of anaplastic lymphoma kinase ( alk ) : development of phosphoacceptor peptides for the assay of alk activity . 
lee cc , jia y , li n , et al : crystal structure of the alk ( anaplastic lymphoma kinase ) catalytic domabiochem j 430 : 425 - 437 , 2010 22 . 
de brouwer s , de preter k , kumps c , et al : meta - analysis of neuroblastomas reveals a skewed alk mutation spectrum in tumors with mycn amplication . 
clin cancer res 16 : 4353 - 4362 , 2010 janoueix - lerosey i , lequin d , brugi `eres l , et al : somatic and germline activating mutations of the alk kinase receptor in neuroblastoma . 
cgp altered physician treatment selection in 25% of evaluable patients ( n = 7 of 28 ) and was associated with improved progression - free survival . conclusion to our knowledge , this is the largest technical evaluation of the performance of cgp in sarcoma . cgp was effectively performed in the vast majority of sarcoma samples and altered physician treatment selection . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction sarcomas are uncommon , highly morbid cancers that constitute approximately 1% of all adult malignancies . currently , more than 70 recognized histologic subtypes exist , which makes them extremely challenging to accurately diagnosis and treat.1 sarcomas have multiple genomic alterations , including copy number changes , point mutations , insertions and deletions , and fusions.2 - 4 thus , detailed rna and dna sequence analysis is key for diagnosis as well as for treatment decision - making.5 is not surprising , that comprehensive therefore , genomic proling ( cgp ) is increasingly being used in the evaluation and management of bone and soft tissue sarcomas . 
cgp is the sequencing of dna and rna from tumor samples , which enables the identication of known and novel alterations that may drive inherent oncogenicity.6 - 9 cgp may rene the histologic tumor diagnosis , with its management.10 , 11 indeed , in a study by groisberg et al1 specic to sarcomas , the authors found 61% of 102 patients in a retrospective cohort had a potentially actionable genomic target . ramications despite the potential role that cgp may have in the treatment of sarcoma , the impact of dnaand rnabased cgp on physician decision - making is poorly documented.12 , 13 similarly , it is equally unclear what factors may contribute to the success or failure of cgp in an outpatient clinical setting . 
 challenges in sarcoma comprehensive genomic proling as mentioned previously is unknown ; neither are the relative contributions of biopsy site ( bone v soft tissue ) and methods ( ie , computed tomography [ ct ] - guided core needle biopsy v open excisional biopsy )  . 
to answer these questions , we herein report the results of two studies : the rst is a prospective clinical trial examining the impact of dnaand rna - based cgp on physician decision - making . the second is a retrospective analysis of the success and failure rates of cgp in an even larger patient cohort , which includes the aforementioned patients . methods sarcoma decision impact clinical trial study design . 
to examine the effects of cgp on physician decision - making , a prospective clinical trial ( the ohio state university [ osu ] institutional review board approval no . osu - 12067 ) was performed in collaboration with foundation medicine . 
the primary objectives of the study were to assess the feasibility and logistics associated with a clinical trial using a commercially available cgp platform ( foundationoneheme ; foundation medicine , cambridge , ma ) in an academic clinical setting and to determine the proportion of patients who would receive a cancer - related therapy based on cgp results . 
progression - free survival ( pfs ) was summarized using kaplan - meier survival analysis . the ohio state retrospective sarcoma study data following : date of sample procurement , method of specimen procurement ( eg , excisional biopsy , needle core biopsy , ne needle aspiration ) , biopsy site , specimen source ( hospital ) , and pathology classication by osu . 
the foundation medicine records were interrogated for all sarcoma samples sent by osu and the following information was compiled : specimen type ( formalin - xed parafn - embedded tissue blocks v unstained slides ) , morphologic tumor purity , dna and rna extraction yield , sequencing metrics , and the nature of the released report ( ie , pass , fail , or qualied )  . 
a qualied report indicates a sample for which it was unable to complete the entire cgp process ( eg , rna extraction , low tumor purity ) but for which some genomic information was obtained . in contrast , a passed report indicates a sample for which dna and rna proling was completed successfully . 
2 analysis was used to calculate the signicance of intergroup alterations . results physician decision - making was prospectively altered for 25% of patients with sarcoma who underwent cgp as part of a decision impact trial , patients with sarcoma whose disease had not yet progressed on the current line of therapy were approached to undergo cgp as part of the study . 
treating oncologists were blinded to cgp results time of progression ( based on imaging results )  . until treating oncologists rst documented their treatment decision for the patient with sarcoma without cgp results . cgp data were then released for review and the treating oncologist would note whether their treatment decision had been altered by the cgp results . 
 a hay et al patients with sarcoma diagnosis ( n = 34 ) treatment recommendations chosen before cgp patients with liposarcoma ( n = 15 ) patients with nonliposarcomatous sarcoma ( n = 19 ) no data on whether recommendation changed ( n = 3 ) did not switch therapy on the basis of cgp results ( n = 8 ) switched therapy on the basis of cgp results ( n = 4 ) no data on whether recommendation changed ( n = 3 ) did not switch therapy on the basis of cgp results ( n = 13 ) switched therapy on the basis of cgp results ( n = 3 ) patients assessed for eligibility ( n = 493 ) excluded ( n = 31 ) misdiagnosed or miscategorized tumors excluded ( n = 16 ) duplicates or no clinical data available excluded ( n = 41 ) samples that failed initial cgp remaining individuals evaluated ( n = 408 ) patients included in analysis ( n = 392 ) patients with preliminary reports ( n = 351 ) total no . 
of patients for whom full cgp analysis was completed ( n = 297 ) qualified ( n = 54 ) samples for which cgp could not be completed fig 1 . 
 ( % ) male female mean age ; median ( range ) , years data 14 ( 50 ) 14 ( 50 ) 57 ; 62 ( 2480 ) no change ( n = 12 ) changed ( n = 5 ) p = .03 altered , six patients received the selected treatment ; one patient died before initiating therapy . 
as an exploratory end point , we noted that the median pfs in the cgp - selected group was 124 days versus 54 days in the noncgpselection group ( p = .03 ; fig 2 )  . cgp was performed on almost 400 patients with sarcoma a total of 392 patients representing 413 unique samples were proled over 3 years . 
l - type sarcomas ( ie , leiomyosarcoma , liposarcoma ) constituted the greatest proportion of cases , and this is indicative by the preponderance of tp53 , rb1 alterations and mdm2 and cdk4 amplications ( table 3 )  . fifty - six of the 413 samples ( 13.6% ) had reports qualied for low tumor purity , failed rna extraction , or suboptimal sequencing metrics , whereas 10.7% ( n = 44 of 413 ) samples failed testing . 
examining sarcoma subtypes individually , leiomyosarcomas ( 74.0% ; n = 57 of 77 ) and liposarcomas ( 79.4% ; n = 50 of 63 ) had proportionally higher pass rates than did bone - based sarcomas . 
chordomas had the lowest successful initial genomic proling rate , with a 43% initial pass rate ( table 3 )  . biopsy site and method inuence successful cgp completion biopsy sites . 
the other category encompassed an array of sites , including , but not limited to , brain , eye , epicardium , aorta , lymph node , submandibular gland , bladder , prostate , testes , ovary , cervix , and vagina . 
this was partially due to nding duplicate patients ( ie , some data were obtained with patients identities removed , because they were included in the decision impact trial , which was blinded ; in unblinding , we found some individuals had been included twice )  . 
other reasons included being unable to locate individuals within ihis ( ohio state universitys electronic medical record ) and data necessary for additional analysis ( including biopsy location and method ) were unavailable . 
breakdown of the most common sarcomas subtypes proled sarcoma subtype qualied passed failed total bone - based chondrosarcoma osteosarcoma ewing chordoma soft tissue based leiomyosarcoma liposarcoma spindle cell liposarcoma myxoid liposarcoma pleomorphic liposarcoma well - differentiated liposarcoma dedifferentiated liposarcoma gist synovial myxobrosarcoma angiosarcoma fibromatosis dscrt epithelioid epithelioid hemangioendothelioma solitary brous tumor abbreviations : dscrt , desmoplastic small round cell tumor ; gist , gi stromal tumor . 180 2020 by american society of clinical oncology in conclusion , to our knowledge , this study is the only prospective evaluation of dnaand rna - based cgp in sarcoma on physician decision - making and the largest indepth evaluation of success of cgp in sarcoma to date . 
we note in this cohort that cgp in sarcoma is successful in a large majority of patients and alters physician treatment decision - making in an estimated 25% of patients . 
furthermore , this percentage may have been driven by the fact that the study data were from the era when cdk4 inhibitors were just starting to be considered for use off - label in adipocytic tumors , and conrmation of cdk4 amplication was critical for this drug selection . 
we would anticipate , as time progresses , the types of tumor for which there are dened targetable alterations will increase and thus there would be an increase in physician treatment - decision changes . 
a classic example would be that of gi tumors for which kit / pdgfr sequencing is now considered standard of care for determining whether a patient has these alterations and , if so , whether the patient has resistance alterations.17 a developing example would be that in leiomyosarcoma , where we have previously reported that homologous recombination alterations are a common feature of uterine leiomyosarcomas and may be amenable to parp inhibition.18 tumor location and tissue subtype signicantly clearly , inuence proling success , likely secondary to preanalytic variables that inuence quality of dna and rna , such as decalcication , tumor cellularity , and available tissue volume . 
of note , of the 36 bone biopsy samples that were collected in this study , pathology reports for only 13 mentioned the sample underwent decalcication ; however , no specications regarding the decalcication agent used or the xation times were documented in the report . 
protocols to prevent decalcication during sample processing may aid in yielding higher cgp success rates.19 , 20 in addition , obtaining excisional biopsy specimens rather than imaged - guided biopsy specimens seems to lead to greater likelihood of cgp success . in these studies , it should be mentioned that germline aberrations were not evaluated , because foundation medicine does not test for these . 
 challenges in sarcoma comprehensive genomic proling causes of sample failure causes of sample qualification low cellularity rna failure dna and rna failure dna failure unknown contamination rna failure failed rna metrics low tumor purity noisy cna data contamination rna failure and noisy cna data failed rna metrics and contamination low dna coverage unknown n = 44 samples n = 56 samples fig 3 . 
also , many of our samples are often obtained at outside hospitals , and there is no standardization of pathology report contents . in summary , sarcoma cgp appears to alter physician decision - making regarding treatment and may affect the ultimate outcome of the patient . 
chen consulting or advisory role : novartis , immune design , syapse speakers bureau : novartis , foundation medicine , eisai patents , royalties , other intellectual property : matchtx . no other potential conicts of interest were reported . references groisberg r , hong ds , holla v , et al : clinical genomic proling to identify actionable alterations for investigational therapies in patients with diverse sarcomas . oncotarget 8 : 39254 - 39267 , 2017 abeshouse a , adebamowo c , adebamowo sn , et al : comprehensive and integrated genomic characterization of adult soft 171 : 950 - 965.e28 , 2017 yakirevich e , madison r , fridman e , et al : comprehensive genomic proling of adult renal sarcomas provides insight into disease biology and opportunities for targeted therapies . 
cancer res 76 : 3690 - 3701 , 2016 fang w , ma y , yin jc , et al : comprehensive genomic proling identies novel genetic predictors of response to anti - pd - ( l ) 1 therapies in non - small cell lung cancer . 
clin cancer res 25 : 5015 - 5026 , 2019 chung jh , dewal n , sokol e , et al : prospective comprehensive genomic proling of primary and metastatic prostate tumors . 
jco precis oncol 3 : 1 - 23 , 2019 al - rohil rn , tarasen aj , carlson ja , et al : evaluation of 122 advanced - stage cutaneous squamous cell carcinomas by comprehensive genomic proling opens the door for new routes to targeted therapies . 
ross js , gay lm , wang k , et al : comprehensive genomic proles of metastatic and relapsed salivary gland carcinomas are associated with tumor type and reveal new routes to targeted therapies . 
rodriguez - rodriguez l , hirsheld km , rojas v , et al : use of comprehensive genomic proling to direct point - of - care management of patients with gynecologic 14 . 
thway k , rockcliffe s , gonzalez d , et al : utility of sarcoma - specic fusion gene analysis in parafn - embedded material for routine diagnosis at a specialist 16 . 
thway k , wren d , lee j , et al : evaluation of the optimal provision of formalin - xed , parafn - embedded material for reverse transcription - pcr in soft - tissue 21 : 1315 - 1325 , 2016 cancers . 
singh vm , salunga rc , huang vj , et al : analysis of the effect of various decalcication agents on the quantity and quality of nucleic acid ( dna and rna ) recovered from bone biopsies . 
choi s - e , hong sw , yoon so : proposal of an appropriate decalcication method of bone marrow biopsy specimens in the era of expanding genetic molecular study . 
therefore , less is known about the prevalence and extent of msi among other types of cancer . methods using our recently published msi - calling software , mantis , we analyzed wholeexome data from 11 , 139 tumor - normal pairs from the cancer genome atlas and therapeutically applicable research to generate effective treatments projects and external data sources across 39 cancer types . 
within a subset of these cancer types , we assessed mutation burden , mutational signatures , and somatic variants associated with msi . results we identified msi in 3.8% of all cancers assessedpresent in 27 of tumor typesmost notably adrenocortical carcinoma ( acc ) , cervical cancer ( cesc ) , and mesothelioma , in which msi has not yet been well described . 
in addition , msi - high acc and cesc tumors were observed to have a higher average mutational burden than microsatellite - stable acc and cesc tumors . conclusion we provide evidence of as - yet - unappreciated msi in several types of cancer . 
2017 by american society of clinical oncology introduction large - scale sequencing projects of cancer genomes have opened the door to studies that have identified putative biomarkers with potential clinical and therapeutic value , among them the presence or absence of microsatellite instability ( msi )  . 
microsatellites are defined as 10 to 60 base pair regions that contain multiple repeats of 1 to 5 base pair motifs.1 microsatellites occur at microsatellite loci , which are widely dispersed throughout the human genome . 
in normal cells , repeat count of microsatellites is verified and maintained during cell division by the mismatch repair ( mmr ) system , 2 , 3 one of many cellular dna repair mechanisms . 
after multiple cycles of cell division , cells with an impaired mmr system will develop varying lengths in their microsatellite sequences . mismatch repair deficiency is known to occur in some tumors , 2 either by somatic hypermutation of mmr genes , most commonly , mlh14 , 5 ; an inherited germline mmr pathway mutation , such as in lynch syndrome6 , 7 ; or double somatic mutations in mmr genes . 
 durable responses and a statistically significant improvement in overall survival.12 msi polymerase chain reaction ( pcr ) and immunohistochemistry are two molecular biology based methods that are in routine use for clinical msi testing . 
msi - pcr analyzes the distribution of microsatellite lengths at five standardized loci ( bethesda panel ) , 14 and immunohistochemistry detects the presence or absence of four proteins that are involved in the mmr pathway ( msh2 , msh6 , mlh1 , and pms2 )  . 
examples of such software include msings , 15 msisensor , 16 and mantis.17 a recent study by our group17 demonstrated that mantis achieves high sensitivity ( 97% ) and specificity ( 99% ) across six cancer typestested using samples with known msi status by msipcrand provides stable performance with varying numbers of microsatellite loci . 
because of this , mantis is particularly well suited for application to a wider variety of cancer types . as clinical msi testing is routinely performed only on colorectal and endometrial tumors , 18 the prevalence of msi in many other cancer types has been less well described . 
in addition , evidence exists that msi - pcr may be less accurate in other cancer types.19 a recent study by hause et al20 developed and applied the msi detection tool , mosaic , to perform a detailed survey of msi across 18 cancer types ( n = 5 , 930 cases ) ; however , many other cancer types have yet to be analyzed for msi . 
the ability to detect msi in novel cancer types would permit the investigation of immuneenhancing therapies in these cancers , with the potential to benefit previously unknown subsets of patients with cancer with msi . to perform a more comprehensive assessment of msi across many additional cancer types than those analyzed by hause et al , our study determined the prevalence of msi in 39 distinct cancer types ( n = 11 , 139 tumors from 11 , 080 patients ) by using our previously published msi - calling tool , mantis . methods data preprocessingthe cancer genome atlas and therapeutically applicable research to generate effective treatments for analysis , 10 , 701 cases of paired tumor - normal whole - exome sequencing data were obtained from the cancer genome atlas ( tcga ) 21 - 44 and therapeutically applicable research to generate effective treatments ( target ) 45 , 46 projects . 
data from all of these cases , with the exception of diffuse large b - cell lymphoma ( dlbcl ) were processed via our in - house automated pipeline , l - map ( landscape microsatellite analysis pathway )  . 
l - map is implemented in python and mysql and was run on the oakley supercomputer at the ohio supercomputing center.47 first , the metadata for all dna whole - exome bam files were downloaded from the genomic data commons ( gdc ) 48 and were converted to sql database entries . 
premarked duplicate reads were removed as above . data preprocessingother sources four hundred thirty cases of paired tumor - normal whole - exome sequencing data were obtained from the sequence read archive51 : 338 chronic lymphocytic leukemia cases from 279 patients from landau et al , 52 32 cutaneous t - cell lymphoma cases from choi et al , 53 51 nasopharyngeal carcinoma cases from zheng h et al , 54 and 8 cholangiocarcinoma cases from ong et al.55 fifteen additional cholangiocarcinoma cases were obtained from the european nucleotide archive56 from chan - on et al.57 all sample identifiers used are available in the data supplement . these cases were processed via l - map . 
coordinates for 2 , 539 microsatellite loci within or near the exomeoriginally introduced by salipante et al15 and used by later studies17 were converted from hg19 to hg38 by using liftover.61 nine unlifted loci were discarded , which left 2 , 530 regions that were used for analysis with mantis in all cohorts , with the exception of dlbcl ( data supplement )  . 
mantis was run with authorrecommended settings for whole - exome data minimum read quality , 20 ; minimum locus quality , 25 ; minimum locus coverage , 20 ; minimum repeat reads , one ; all other settings left at defaults . eight samples were observed to have fewer than 10 loci sufficiently covered and were dropped . 
variant annotation was performed by using annovar ( version 201602 - 01 ) 63 and gnu parallel.64 somatic mutations in the repair genes msh2 , msh6 , mlh1 , pms2 , exo1 , pold1 , and pole were determined by filtering variants with a dann68 , 69 pathogenicity score greater than 0.96 ( included in annovar )  . this threshold for dann was chosen as it was previously shown to provide optimal sensitivity and specificity.69 mutational signature calling was performed by using the tool deconstructsigs70 with the nature 2013 signatures set , which contains 27 signatures , 71 and the exome2genome normalization method . 
of 2 , 530 loci , we identified 22 loci that , within at least five cohorts , had an msi - h versus mss difference score greater than 0.75 and were sufficiently covered by at least 50% of samples in the cohort ( appendix table a2 )  . 
note that for chronic lymphocytic leukemia ( cll ) , the listed msi prevalence in panel a is out of 279 patients , and all 338 tumors are shown in panel b . 
acc , adrenocortical carcinoma ; aml , pediatric acute myeloid leukemia ( target ) ; blca , bladder carcinoma ; brca , breast carcinoma ; cesc , cervical squamous cell carcinoma and endocervical adenocarcinoma ; chol , cholangiocarcinoma ; coad , colon adenocarcinoma ; ctcl , cutaneous t - cell lymphoma ; dlbc , diffuse large b - cell lymphoma ; esca , esophageal carcinoma ; gbm , glioblastoma multiforme ; hnsc , head and neck squamous cell carcinoma ; kich , kidney chromophobe ; kirc , kidney renal clear cell carcinoma ; kirp , kidney renal papillary cell carcinoma ; laml , acute myeloid leukemia ( tcga ) ; lgg , lower - grade glioma ; lihc , liver hepatocellular carcinoma ; luad , lung adenocarcinoma ; lusc , lung squamous cell carcinoma ; meso , mesothelioma ; nbl , pediatric neuroblastoma ; npc , nasopharyngeal carcinoma ; ov , ovarian serous cystadenocarcinoma ; paad , pancreatic adenocarcinoma ; pcpg , pheochromocytoma and paraganglioma ; prad , prostate adenocarcinoma ; read , rectal adenocarcinoma ; sarc , sarcoma ; skcm , skin cutaneous melanoma ; stad , stomach adenocarcinoma ; tcgt , testicular germ cell tumor ; thca , thyroid carcinoma ; thym , thymoma ; ucec , uterine corpus endometrial carcinoma ; ucs , uterine carcinosarcoma ; uvm , uveal melanoma ; wt , wilms tumor . were within the set of 22 top - performing loci . these results indicate a striking heterogeneity of msi patterns across various types of cancer . all four disease types with the highest rates of msi prevalence were lynch syndromeassociated tumor types that have been previously known to exhibit msi : endometrial carcinoma , colon adenocarcinoma , gastric adenocarcinoma , and rectal adenocarcinoma . 
somatic variant calling was performed on whole - exome samples from these four cancer types , and the mean absolute number of somatic mutationsboth nonsynonymous and synonymouswas found to be increased among msi - h versus mss tumors within their own cohorts ( fig 3 )  . 
p values were calculated by using two - sided fishers exact test ( using signature presence or absence ) , with benjamini correction for multiple hypotheses.78 mmr pathway alterations msi - h lynch syndromeassociated tumors are known to lack the expression or function of at least one mmr protein ; therefore , we analyzed somatic mutations that were predicted to be deleterious ( by dann68 ) in the mmr genes msh2 , msh6 , mlh1 , pms2 , and exo1 , and the proofreading dna polymerases pold1 and pole , among msi - h and mss samples within acc , cesc , and meso ( appendix table a3 ; data supplement )  . 
although pold and pole are not considered mmr proteins , mutations in these genes have been shown to lead to somatic hypermutation.22 , 79 within these cohorts , 64% of msi - h cases and 7% of mss cases were found to contain at least one predicted deleterious somatic mutation in at least one of these genes ; however , given that these samples were sequenced with potentially different exome captures , together with the increased mutational burden of msi - h tumors , we could not determine the statistical significance of this finding . discussion in this study , we have performed , to our knowledge , the largest analysis of msi in human cancer exomes to date , including 11 , 139 whole - exome tumor - normal pairs from 39 types of cancer . compared with a study by hause et al , 20 we mantis score mantis score mantis score ascopubs.org / journal / po jco precision oncology 5 fig 2 . 
kernel density plots of mantis scores within ( a ) adrenocortical carcinoma ( acc ) , ( b ) cervical squamous cell carcinoma and endocervical adenocarcinoma ( cesc ) , and ( c ) mesothelioma ( meso )  . 
somatic mutational burden correlates with microsatellite instability high ( msi - h ) status within adrenocortical carcinoma ( acc ) and cervical squamous cell carcinoma and endocervical adenocarcinoma ( cesc )  . mutational burden is listed for ( a ) acc , ( b ) cesc , and ( c ) mesothelioma ( meso )  . 
previous studies of msi in acc have implicated lynch syndrome as a risk factor for familial acc80 , 81 ; however , to our knowledge , ngs - based msi analysis has not yet been applied to acc . msi - h colorectal tumors have been previously shown to be exceptionally sensitive to therapy with pd - 1 immune checkpoint inhibitors.12 identification of msi in novel tumor types may lead to an expanded role for immunotherapy and a broader scope of clinical msi testing.82 in addition , msi is known to be prognostic within colorectal cancer , 83 which may apply in other cancer types as well . 
clinical trials of immune checkpoint inhibitors are beginning or are underway in acc ( clinicaltrials.gov identifier : nct02673333 ) , cesc ( clinicaltrials.gov identifier : nct02635360 ) , and meso ( clinicaltrials.gov identifiers : nct02784171 , nct02991482 , nct02707666 , and nct02399371 ) , and a previous study of dendritic cell immunotherapy in acc84 demonstrated tumor marker but not clinical response . these studies may benefit from the retrospective evaluation of msi - h as a biomarker . 
prospective expansion of clinical msi testing to other cancer types may enlighten the prognostic and predictive value of msi - h for noncolorectal cancers . mmr deficiency is well recognized as the predominant cause of msi within colorectal , endometrial , and gastric cancers . 
if future studies indicate that msi in acc , cesc , and / or meso is indeed a result of mmr deficiency , the findings of this study may implicate previously unappreciated cancer types as being part of lynch syndrome . 
compared with germline alterations in mmr genes , somatic events are most often a result of hypermethylation of cpg islands in the promoter region of mlh1.4 additional investigation is needed to elucidate other molecular mechanisms that can lead to msi , as well as the downstream effects of msi on tumor - specific biology . 
in addition , of 9 , 569 tumors assessed in this study not within colorectal , endometrial , or gastric cancer , 77 ( 0.8% ) were msi - h . 
only 14 of these were within acc , cesc , or meso , which compromised the statistical power of our mutational signature analysis . a larger cohort of msi - h tumors would permit more comprehensive studies , including correlation with clinical data . in summary , we have detected msi in multiple cancer types , including acc , cesc , and meso , which indicates that msi may affect nonlynch syndrome tumor types . 
kautto no relationship to disclose affiliations all authors : the ohio state university , columbus , oh . support sameek roychowdhury stock and other ownership interests : johnson & johnson ( i ) research funding : takeda , ignyta acknowledgment we thank current and past members of the roychowdhury laboratory for their helpful insight and discussion . 
the chronic lymphocytic leukemia sequencing data ( dbgap : phs000922.v1.p1 ) used in this work was supported by national human genome research institute large - scale sequencing program grant no . 
kane mf , loda m , gaida gm , et al : methylation of the hmlh1 promoter correlates with lack of expression of hmlh1 in sporadic colon tumors and mismatch repair - defective human tumor cell lines . 
study of two large midwestern kindreds . 812 - 816 , 1993 arch intern med 117 : 206 - 212 , 1966 imai k , yamamoto h : carcinogenesis and microsatellite instability : the interrelationship between genetics and epigenetics . 
boland cr , thibodeau sn , hamilton sr , et al : a national cancer institute workshop on microsatellite instability for cancer detection and familial predisposition : development of international criteria for the determination of microsatellite instability in colorectal cancer . 
niu b , ye k , zhang q , et al : msisensor : microsatellite instability detection using paired tumor - normal sequence data . chem 60 : 1192 - 1199 , 2014 bioinformatics 30 : 1015 - 1016 , 2014 17 . 
giardiello fm , allen ji , axilbund je , et al : guidelines on genetic evaluation and management of lynch syndrome : a consensus statement by the us multi - society task force on colorectal cancer . 
faulkner rd , seedhouse ch , das - gupta ep , et al : bat - 25 and bat - 26 , two mononucleotide microsatellites , are not sensitive markers of microsatellite instability in acute myeloid leukaemia . 
cancer genome atlas research network : comprehensive genomic characterization of squamous cell lung cancers . nature 489 : 519 - 525 , 2012 [ erratum : nature 491 : 288 , 2012 ] 24 . 
hoadley ka , yau c , wolf dm , et al : multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origcell 158 : 929 - 944 , 2014 33 . 
zheng s , cherniack ad , dewal n , et al : comprehensive pan - genomic characterization of adrenocortical carcinoma . cancer cell 29 : 723 - 736 , 2016 [ erratum : cancer cell 30 : 363 , 2016 ] 42 . 
zheng h , dai w , cheung akl , et al : whole - exome sequencing identifies multiple loss - of - function mutations of nf - kb pathway regulators in nasopharyngeal carcinoma . 
chan - on w , nairismagi m - l , ong ck , et al : exome sequencing identifies distinct mutational patterns in liver flukerelated and non - infection - related bile duct cancers . 
oleary na , wright mw , brister jr , et al : reference sequence ( refseq ) database at ncbi : current status , taxonomic expansion , and functional annotation . 
quang d , chen y , xie x : dann : a deep learning approach for annotating the pathogenicity of genetic variants . bioinformatics 31 : 761 - 763 , 2015 69 . 
bacher jw , flanagan la , smalley rl , et al : development of a fluorescent multiplex assay for detection of msi - high 1113 - 1120 , 2013 tumors . 
gatalica z , vranic s , xiu j , et al : high microsatellite instability ( msi - h ) colorectal carcinoma : a brief review of predictive biomarkers in the era of personalized medicine . 
challis bg , kandasamy n , powlson as , et al : familial adrenocortical carcinoma in association with lynch syndrome . j clin endocrinol metab 101 : 2269 - 2272 , 2016 81 . 
patterns of mutational signatures ( s ) across microsatellite instability cancers : ( a ) adrenocortical carcinoma ( acc ) , ( b ) cervical squamous cell carcinoma and endocervical adenocarcinoma ( cesc ) , and ( c ) mesothelioma ( meso )  . 
listed are the number of samples ( mss or msi - h ) with at least one predicted deleterious mutation in msh2 , msh6 , mlh1 , pms2 , exo1 , pold1 , and pole . mutations were called by using mutect ( variant calling in methods ) and included in this table if the dann pathogenicity score was  . 
idh1 / 2 mutations are associated with a more favorable prognosis among glioblastomas , whereas tumors with mgmt promoter hypermethylation are more likely to respond to treatment with alkylating agents.1 , 2 the pattern of genomic alterations can also provide insight into the origin and evolution of the tumor . 
for example , the presence of coexistent mutations in idh1 and atrx suggest a secondary glioma that has progressed from a lower - grade glioma.3 - 5 in addition to informing prognosis and diagnostic classification , some genomic alterations in glioma represent potential therapeutic targets . 
specifically , recurrent alterations in one of several receptor tyrosine kinases ( rtks ) , including egfr , pdgfra , met , and fgfr1 / 2 / 3 , have been observed in the majority of glioblastomas.6 unfortunately , efforts to develop genomically targeted therapy for the treatment of glioblastoma have been unsuccessful to date.7 it remains unknown the extent to which the lack of activity of targeted therapy may be attributed to so - called key alterations that do not represent true clonal drivers that are critical to tumor growth and progression compared with the simple inability of existing drugs to achieve therapeutic exposures within the cns . in addition to these more common genomic alterations , less common fusions that involve the kinase domain of tropomyosin receptor kinases ( trks ; a / b / c ) encoded by the genes , ntrk1 , ntrk2 , and ntrk3 have also been identified in gliomas.8 - 11 in other cancer types , selective inhibition of these trk fusions has resulted in remarkable efficacy12 ; however , it remains unknown whether this therapeutic strategy is effective in primary brain tumors . 
 disease progression nov 2011 may 2015 nov 2015 feb 2016 sept 2016 july 2016 primary resection resection resection and r temporal lobectomy adjuvant rt temozolomide lomustine + bevacizumab larotrectinib anaplastic astrocytoma grade iii temozolomide + ido inhibitor secondary gbm grade iv recurrent gbm temporal pole grade iii hippocampus grade iv central tumor grade iv mixed response fig 1 . 
three weeks into the treatment , a brain mri indicated a response to treatment , the degree of which was radiologically discrepant between disease sites ( fig 2 )  . 
 whereas there was significant periventricular response to larotrectinibtumor shrinkage of 67 52 mm to 8 4 mm , which corresponded to a partial response by response assessment in neuro - oncology criteria , and of 162 cm3 to 57 cm3 , which represented a 65% volumetric decreasethere was no shrinkage in the right supraorbital area and occipital lobe . 
she died of her disease shortly thereafter . we hypothesized that intratumoral heterogeneity of the fusion oncoprotein was the basis for the marked response to therapy observed in some areas of her tumor but not others . 
of interest , the ntrk fusion was not detected in either lesion , a finding that is consistent with the radiographic effects of larotrectinib and , overall , a subclonal sensitizing fusion . 
to better characterize the full extent of intratumoral heterogeneity , we subsequently performed whole - exome sequencing of her original tumor from the right temporal lobe and the three resected areas of her recurrence . 
only the hippocampal and central tumors contained a focal cdkn2a / b deletion and a mutational repertoire indicative of temozolomide - induced hypermutation ( figs 3b and 3c and data supplement )  . 
 moreover , the hippocampal and central tumors had distinct rtk alterations , including a pdgfra amplification in the hippocampal specimen and the eml4 - ntrk3 fusion in the central tumor . 
serial contrast t1 - weighted images at pretreatment ( left column ) show enhancing disease in the infundibulum ( green arrow #1 ) , basal ganglia ( green arrow #2 ) , frontal lobe ( green arrow #3 ) , subependyma , and corpus callosum ( green arrow #4 ) , which is improved by day 18 of treatment with larotrectinib ( middle column )  . 
at day 40 ( right column ) , there is worsening enhancing disease in the inferior frontal lobe ( red arrow #1 ) , occipital lobe ( red arrow #2 ) , leptomeninges ( red arrow #3 ) , and parietal lobe ( red arrow #4 )  . tumor , which suggests that multiple clonally related tumors underwent independent waves of therapy - associated mutagenesis . 
thus , our genomic characterization of the patients tumor confirmed profound intratumoral heterogeneity , including subclonality of the ntrk fusion . discussion gene fusions that involve the trk family of kinases have been identified in a wide array of adult and pediatric cancers in which they result in constitutive kinase activation and drive tumor growth.8 , 15 - 20 larotrectinib is a novel small - molecule inhibitor that specifically targets trk and has demonstrated unprecedented efficacy in trk fusionpositive cancers irrespective of tumor histology , with the majority of patients achieving rapid and durable clinical responses12 , 21 ; however , the efficacy of trk inhibition in gliomas is currently unknown . 
 ( b ) pattern of hallmark truncal mutations in idh1 , tp53 , and atrx across the initial tumor ( a ) and recurrent specimens ( b - d ) as well as private driver events in postprogression samples ( c and d , as indicated )  . 
 ( c ) overall somatic mutational burden ( top ) of all tumor samples is shown , which indicates hypermutation in lesions c and d , but not in the initial pretreatment tumor or post - tmz temporal pole tumor ( b )  . 
snv , single nucleotide variant . to therapeutic resistance and disease recurrence.22 , 23 this case highlights that molecular profiling of a single area may not capture the full genomic landscape of a tumor . 
kinase fusions , including trk fusions , are typically clonal when present in other diseases and thus represent some of the most vulnerable targets of novel therapeutics.8 the presence of the trk fusion in only one subclone suggests that distinct cell populations in this tumor relied on different driver alterations ; therefore , genomic heterogeneity limits , but does not preclude , clinical benefit from a targeted agent . 
another aspect of the profound intratumoral heterogeneity observed here was somatic hypermutation , a well - recognized consequence of temozolomide therapy that occurs in a subset of treated patients.24 when present , this may increase the risk of malignant progression via the introduction of novel driver mutations that activate pathways that are quiescent at diagnosis , but as evident here , does not preclude sensitivity to molecularly targeted therapy.25 the observation that hypermutation does not occur in all patients whose tumors are exposed to this therapy suggests that certain cancers are more vulnerable to the mutagenic effect of alkylating agents . 
our report demonstrates that susceptibility can differ even within a single tumor , resulting in hypermutation of some , but not all , subclones ; however , precisely what molecular context underlies these differences is not yet known . 
an improved understanding of what permits this phenotypic change and whether a biomarker of susceptibility to therapy induced hypermutation exists may ultimately lead to more informed treatment decisions for patients with glioma . in conclusion , these data suggest that trk fusions may represent potential therapeutic targets in glioblastoma , a disease with few effective therapies , and that larotrectinib has the potential for antitumor activity in cns tumors that harbor these fusions . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . alexander drilon consulting or advisory role : ignyta , loxo oncology consulting or advisory role : tp therapeutics , astrazeneca , pfizer , blueprint medicines , genentech , takeda pharmaceuticals , helsinn therapeutics , beigene thomas j . 
young stock and other ownership interests : agios , alexion pharmaceuticals , biogen , celgene , gilead sciences , karyopharm therapeutics , spark therapeutics , regeneron , stemline therapeutics , vertex consulting or advisory role : agios , puma biotechnology research funding : agios ( inst ) christian grommes consulting or advisory role : btg nora ku employment : loxo oncology stock and other ownership interests : loxo oncology , novartis ( i ) , biotheranostics ( i ) , pfizer ( i ) , amgen ( i ) , genentech ( i ) , astrazeneca ( i ) , macrogenics ( i ) , peregrine pharmaceuticals ( i ) , pierian bioscience ( i ) , cascadian therapeutics ( i ) travel , accommodations , expenses : novartis ( i ) , biotheranostics ( i ) , pfizer ( i ) , amgen ( i ) , genentech ( i ) , astrazeneca ( i ) , macrogenics ( i ) , peregrine pharmaceuticals ( i ) , pierian bioscience ( i ) david m . 
hyman consulting or advisory role : atara biotherapeutics , chugai pharma , cytomx therapeutics , boehringer ingelheim , astrazeneca , pfizer , bayer , debiopharm group , arqule , genentech research funding : astrazeneca , puma biotechnology , loxo oncology barry s . 
hyman , weill cornell medical college , new york , ny ; and nora ku , loxo oncology , stamford , ct . funded by the sontag foundation , american cancer society grant no . 
van den bent mj , baumert b , erridge sc , et al : interim results from the catnon trial ( eortc study 26053 - 22054 ) of treatment with concurrent and adjuvant temozolomide for 1p / 19q non - co - deleted anaplastic glioma : a phase 3 , randomised , open - label intergroup study . 
russell jp , powell dj , cunnane m , et al : the trk - t1 fusion protein induces neoplastic transformation of thyroid epitheliuoncogene 19 : 5729 - 5735 , 2000 16 . 
one year later , she presented with a recurrent ipsilateral breast mass and underwent a second lumpectomy , and her diagnosis was revised to secretory breast carcinoma on the basis of repeat histopathology evaluation . 
approximately 1 year later , her disease recurred locally , and she underwent a simple mastectomy with axillary lymph node dissection , followed by four cycles of carboplatin and docetaxel . 
less than a year later , she presented with a recurrent fungating mass in the left chest wall and was treated with two cycles of carboplatin and paclitaxel , with no clinical benefit . having exhausted all available options , her treating oncologist presented her case at a virtual multidisciplinary tumor board organized by the global cancer institute , a nonprofit organization focused on improving care of underserved patients with cancer . 
given the histologic diagnosis of secretory breast carcinoma , the board recommended molecular testing for an etv6 - ntrk3 fusion , the pathognomonic genomic alteration in this cancer type.1 to accomplish this , a formalin - fixed tumor block was paraffin - embedded ( ffpe ) located and analyzed at memorial sloan kettering cancer center ( mskcc ) , where the presence of an in - frame etv6 - ntrk3 fusion was confirmed by anchored multiplex rna sequencing ( figs 1a and 1b ) and separately by targeted hybridization capture - based dna and ultimately whole - genome sequencing ( wgs )  . 
immunohistochemistry with a pantropomyosin receptor kinase ( trk ) antibody confirmed overexpression in a perinuclear nuclear pattern , consistent with a fusion involving the nuclear transcription factor etv6 ( fig 1c )  . 
of note , both targeted capture - based sequencing and wgs also identified a tert promoter mutation , a common alteration in solid tumors that leads to increased expression of telomerase2 but to our knowledge has never been reported previously in secretory breast carcinoma . after confirmation of an expressed etv6 - ntrk3 fusion , which is known to result in constitutive trkc kinase activity , 1 treatment with a trk inhibitor was recommended by mskcc pediatric and medical oncology teams . 
ultimately , with approval from the us food and drug administration ( fda ) and loxo oncology ( stamford , ct ) , a single patient use protocol for larotrectinib ( loxo - 101 ) was written and opened for this patient at mskcc . methods anchored multiplex rna sequencing the tumor was analyzed using the msk - solid fusion assay , a targeted rna - based panel that uses the archer anchored multiplex polymerase chain reaction technology3 and next - generation sequencing to detect gene fusions . 
exons 1 to 5 of etv6 are fused to exons 15 to 18 of ntrk3 , which include the kinase domain . ( b ) representation of cdna sequencing reads supporting the etv6ntrk3 fusion transcript . red and blue rectangles represent bidirectional sequencing chimeric reads , where the exact location of the fusion breakpoint is indicated with a dotted vertical line and with an i symbol in the fusion transcript sequence . 
the median depth of coverage across all exons was 9723 . copy number and cellularity information for caveman were predicted with the battenberg algorithm6 using 1 , 000 genomes7 loci within the next - generation sequencing data . 
small somatic insertions and indels were identified using a modified version of pindel ( cgppindel ) .8 structural rearrangements were detected by an in - house algorithm , brass ( breakpoints via assembly ; brass )  . 
mutational signature analysis of the substitutions was performed using the r package deconstructsigs.9 small insertion / deletions were interrogated for the presence of either short tandem repeat or microhomology at the breakpoints , as described previously.10 data relating to this patient will be deposited in the european genome - phenome archive.11 wgs was performed using illumina x10 paired end sequencing standard methods under the auspices of the cancer alliance study in collaboration with the new york genome center . 
single - base substitutions were called using caveman ( cancer variants through expectation maximization ; github.io / caveman / ) , as described previously.6 trk immunohistochemistry expression of trkc , the protein product of ntrk3 , was detected via immunohistochemistry with antipan - trk abcam monoclonal antibody epr17341 in a 1 : 250 dilution . 
 expression of trk with at least weak ( 1 + ) intensity in a membranous , cytoplasmic , and / or nuclear pattern . larotrectinib treatment the treatment dose of the oral pan - trk inhibitor larotrectinib was 100 mg twice per day . 
significant and rapid reduction in size of the left chest mass was achieved within 1 week of starting therapy , with near - complete resolution after 2 months of therapy ( figs 2 and 3b )  . 
the patient tolerated larotrectinib well , with the only drug - related toxicities being two discrete episodes of dizziness ( grade 1 and 2 ) associated with postural change . she was provided with a 6 - month supply of larotrectinib and has since returned home for continued treatment by her oncologists in gopalganj , bangladesh , with plans to return to mskcc twice a year for follow - up . to further investigate the genomic landscape of this patients secretory breast carcinoma , a fresh pretreatment tumor biopsy was obtained and paired tumor - normal wgs performed . 
a total of 697 somatic alterations consisting of 530 singlenucleotide substitutions , 157 deletions ( indels ) , and 10 rearrangements were identified , although only two of these resulted in alteration of a coding region , and of these , only the etv6 - ntrk3 fusion was clonal . 
in addition , wgs also identified two reciprocal inversion events leading to a 9 , 500 - bp deletion containing the first exon of cdkn2a , followed later in this tumors evolution by a subclonal copy - neutral loss of heterozygosity as a result of loss of the wild - type allele and duplication of the mutated allele ( fig 4 )  . 
although the allele with focal deletion of cdkn2a seemed to be clonal , loss of the other allele was not . therefore , biallelic inactivation of cdkn2a was a subclonal event in this tumor . 
we will discuss this option with the family on their follow - up visit to mskcc . discussion ntrk1 , ntrk2 , and ntrk3 encode for the trka , trkb , and trkc receptor tyrosine kinases . oncogenic gene fusions involving the ntrk family of genes have been identified across numerous different tumor types and seem to result in constitutive activation of trk kinase activity.14 larotrectinib is an orally administered highly selective inhibitor , with nanomolar activity against all three trk protein isoforms.15 substantial tumor regressions with larotrectinib have been reported in a child with congenital fibrosarcoma bearing the etv6 - ntrk3 fusion , as well as an adult with a soft tissue sarcoma harboring an lmna - ntrk1 fusion.16 , 17 preliminary activity has also been described in multiple other ntrk fusionpositive cancer types with larotrectinib.18 in july 2016 , the fda granted breakthrough designation to larotrectinib for the treatment of unresectable or metastatic solid tumors bearing ntrk fusions.19 although targeted therapy of its extramammary counterpart has been reported , 20 this is the first report of a patient with secretory carcinoma of the breast treated with a trk inhibitor . 
secretory breast carcinoma is an extremely rare tumor type , accounting for , 0.02% of breast cancer cases.21 it is classically an indolent tumor , with primary management usually consisting of surgical resection . 
although initially believed to occur primarily in children , these tumors have subsequently been identified in both pediatric and adult - age patients.22 histologically , secretory breast carcinomas are characterized by their low - grade appearance , granular or vacuolated cytoplasm , and abundant intracellular and extracellular secretory material.19 furthermore , these tumors are generally triple negative for expression of estrogen receptor , progesterone receptor , and human epidermal growth factor receptor 2.22 although axillary involvement is seen in a significant proportion of patients , widely metastatic disease is uncommon.21 as a result , the role of radiation and chemotherapy is not established for patients with locally advanced or metastatic disease . secretory breast carcinomas are characterized by the etv6 - ntrk3 fusion . 
rearrangements are plotted as arcs inside the robust clinical response to a single - agent trk inhibitor , support previous data demonstrating etv6 - ntrk3 as a driver lesion.23 the 14 - yearold reported here , despite being heavily pretreated for 6 years and having a high burden of disease , achieved an almost immediate and complete response to larotrectinib that remains ongoing with minimal toxicity . 
1 year in duration , with the longest response approaching 2 years.24 whole - genome sequencing analysis reveals a simple mutation and clonal structure , predominantly defined by the fusion gene , mutation in the tert locus , and loss of cdkn2a . 
 ( e ) distribution of corrected variant allele frequency ( vaf ) identifying the etv6 - ntrk3 fusion and tert promoter mutation as clonal events . fusion as the primary mitogenic driver in this patients tumor from the initial presentation onward . along with being the first detailed genomic evaluation of secretory breast carcinoma to our knowledge , and establishing the extraordinary response of this subtype of breast cancer to larotrectinib , this report also describes a unique and dynamic collaboration between multiple international academic centers , global health initiatives , and the pharmaceutical industry . 
the global cancer institute began organizing virtual multidisciplinary tumor boards connecting oncology experts based in the united states and western europe with physicians from lowand middleincome countries in 2012 , with the goal of improving care of patients with cancer in underserved populations worldwide.28 although these tumor boards have typically focused on improving standard practice in the developing world , here we demonstrate how these boards can also lead to the implementation of investigational therapy with dramatic benefit . 
darnell no relationship to disclose elli papaemmanuil employment : tesaro ( i ) stock and other ownership interests : tesaro ( i ) honoraria : novartis , celgene speakers bureau : tesaro ( i ) research funding : celgene patents , royalties , other intellectual property : my husband receives royalties for azd5363 or azd5393 ( not sure ) , an akt inhibitor through the institute of cancer research in the united kingdom ( i ) travel , accommodations , expenses : celgene , novartis , tesaro ( i ) marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network / boehringer ingelheim afatinib targeted therapy advisory committee , national comprehensive cancer network / astrazeneca tagrisso request for proposals advisory committee research funding : loxo oncology ( inst ) nora ku employment : loxo oncology stock and other ownership interests : loxo oncology honoraria : novartis ( i ) , biotheranostics ( i ) , pfizer ( i ) , amgen ( i ) , genentech ( i ) , astrazeneca ( i ) , macrogenics ( i ) , peregrine pharmaceuticals ( i ) , pierian biosciences ( i ) consulting or advisory role : baylor college of medicine ( i ) travel , accommodations , expenses : novartis ( i ) , biotheranostics ( i ) , pfizer ( i ) , amgen ( i ) , genentech ( i ) , astrazeneca ( i ) , macrogenics ( i ) , peregrine pharmaceuticals ( i ) , pierian biosciences ( i ) andrew l . 
kung consulting or advisory role : darwin health , mi bioresearch , imago biosciences patents , royalties , other intellectual property : royalty from licensing agreements with mi bioresearch jos e baselga leadership : infinity pharmaceuticals , varian medical systems , grail , foghorn stock or other ownership : pmv pharma , juno therapeutics , infinity pharmaceuticals , grail , varian medical systems , tango , foghorn , aura biomedical , apogen , northern biologics honoraria : pmv pharma , juno therapeutics , infinity pharmaceuticals , grail , northern biologics consulting or advisory role : eli lilly , novartis , grail patents , royalties , other intellectual property : combination therapy using pdk1 and pi3k inhibitors . 
may 16 travel , accommodations , expenses : roche / genentech alexander drilon honoraria : ignyta , exelixis , genentech , astrazeneca , blueprint medicines , loxo oncology consulting or advisory role : ariad pharmaceuticals research funding : foundation medicine david m . 
hyman consulting or advisory role : atara biotherapeutics , chugai pharma , cytomx therapeutics , boehringer ingelheim , astrazeneca research funding : astrazeneca , puma biotechnology , loxo oncology acknowledgment we thank muhammed yunus for his significant assistance in facilitating the patients travel to the united states for treatment . 
darnell , howard hughes medical institute , chevy chase , md ; and nora ku , loxo oncology , south san francisco , ca . affiliations support supported by national institutes of health grants no . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
cell 149 : 994 - 1007 , 2012 [ erratum : cell 162 : 924 , 2015 ] 1000 genomes project consortium , abecasis gr , auton a , et al : an integrated map of genetic variation from 1 , 092 human genomes . 
nik - zainal s , davies h , staaf j , et al : landscape of somatic mutations in 560 breast cancer whole - genome sequences . nature 534 : 47 - 54 , 2016 11 . 
nagasubramanian r , wei j , gordon p , et al : infantile fibrosarcoma with ntrk3 - etv6 fusion successfully treated with the tropomyosin - related kinase inhibitor loxo - 101 . 
doebele rc , davis le , vaishnavi a , et al : an oncogenic ntrk fusion in a patient with soft - tissue sarcoma with response to the tropomyosin - related kinase inhibitor loxo - 101 . 
drilon a , li g , dogan s , et al : what hides behind the masc : clinical response and acquired resistance to entrectinib after etv6 - ntrk3 identification in a mammary analogue secretory carcinoma ( masc )  . 
li d , xiao x , yang w , et al : secretory breast carcinoma : a clinicopathological and immunophenotypic study of 15 cases with a review of the literature . 
hong ds , dowlati a , burris iha , et al : 150o clinical safety and activity from a phase 1 study of loxo - 101 , a selective trka / b / c inhibitor , in solid - tumor patients with ntrk gene fusions . 
shern jf , chen l , chmielecki j , et al : comprehensive genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic axis in fusion - positive and fusion - negative tumors . 
shukla n , ameur n , yilmaz i , et al : oncogene mutation profiling of pediatric solid tumors reveals significant subsets of embryonal rhabdomyosarcoma and neuroblastoma with mutated genes in growth signaling pathways . 
louis j , bukowski a , mendoza rer , et al : global cancer institute ( gci ) multi - disciplinary tumor boards ( mtbs ) as an educational tool to improve guideline - based cancer clinical practice in lowand middle - income countries ( lmics )  . 
garcia , md3 ; amir momeni - boroujeni , md1 ; qi gao , mls1 ; jeeyeon baik , ba1 ; dory londono , bs1 ; ryma benayed , phd1 ; allison sigler , ma1 ; michael haddadin , md2 ; alexander v . 
goldberg , md , phd2 ; yanming zhang , md1 ; wenbin xiao , md , phd1 ; and caleb ho , md1 introduction ighcytokine receptorlike factor 2 ( crlf2 ) rearrangement is the most common mechanism of overexpression of crlf2 in precursor b - cell acute lymphoblastic leukemia ( b - all ) ; however , p2ry8 - crlf2 rearrangement or alternative alterations leading to crlf2 overexpression have also been observed , which are associated with increased relapse rate and progression.1 although many myeloid neoplasms , particularly acute myeloid leukemia ( aml ) , are associated with various recurrent genetic rearrangements and fusions , 2 alterations leading to crlf2 overexpression are exceedingly rare in aml . 
one patient with myelodysplastic syndrome with excess blasts - 1 ( mds - eb1 ) with crlf2 overexpression via multiple copies of an isodicentric y chromosome , without evidence of crlf2 rearrangement , has been reported.3 to our knowledge , we report the rst patient with aml arising from an mds , with a p2ry8 - crlf2 fusion , which responded to a myeloid regimen in a clinical trial of hypomethylating agent and immune checkpoint blockade combination therapy . 
at mskcc , karyotype analysis of the aml bone marrow aspirate specimen showed a normal male karyotype , and fish analysis was negative for the presence of kmt2a ( mll ) , evi1 , and cbfb translocations , and tp53 deletion . 
a targeted nextgeneration sequencing ( nsg ) panel ( msk - impact heme panel ; mskcc , new york , ny ) was performed on the bone marrow specimen obtained at the time of the aml diagnoses , which showed the alterations including mutations in asxl1 , listed in table 1 , kmt2d , runx1 , sf3b1 , tet2 , phf6 , rad21 , and xbp1 . 
evaluation of this bone marrow sample using an anchored multiplex polymerase chain reaction based , targeted rna sequencing assay for detection of fusions ( archer fusionplex , archerdx , boulder , co ) revealed the presence of a fusion between exon 1 of p2ry8 and exon 1 of crlf2 ( fig 1c )  . 
cell sorting , molecular characterization , and immunostaining characterized the myeloid origin of this fusion and subsequent signaling effects . relevance this patient was treated with the novel combination of decitabine and ipilimumab with an outstanding response . 
immune cell inltrates in the bone marrow with treatment suggests the response was related to the immune checkpoint inhibition . additional study of p2ry8 - crlf2 fusions and response to immunotherapy is warranted and could have implications for fusion discovery in novel disease contexts . sorting to isolate the leukemic blasts , myelomonocytic cells , lymphocytes . 
fish using the crlf2 breakand normal apart probe revealed the crlf2 rearrangement in a subset of the myeloid blasts ( 27.5% ) and myelomonocytic cells ( 19.5% ) but not in b cells ( fig 2 )  . fish evaluation of the prior bone marrow biopsy with mdseb1 was also performed , which showed the presence of the p2ry8 - crlf2 rearrangement in 10% of the cells ( appendix fig a1c )  . 
oncoscan array ( thermofisher , waltham , ma ) of the mds - eb1 biopsy did not reveal any unbalanced genomic changes or copy - neutral loss of heterozygosity ( data not shown ) , consistent with the normal fish study observed by the outside institution a year prior . 
targeted ngs sequencing ( msk - impact heme panel ) of the mds - eb1 bone marrow specimen also showed overlapping mutations with the aml bone marrow sample ( table 1 ) , but with acquisition of new mutations ( phf6 , rad21 , xbp1 ) in the aml sample , suggesting a myeloid primed hematopoietic stem cell / progenitor origin and clonal evolution with expansion of the clone with the p2ry8 - crlf2 rearrangement , likely due to acquisition of a loss - of - function mutation in phf6 , which led to downregulation of genes involved in normal b - cell development.4 total rna sequencing of the aml bone marrow specimen further conrmed the presence of the p2ry8 - crlf2 fusion along with overexpression of crlf2 ( fig 3a )  . 
principal component analysis comparing our patient with cohorts of aml or b - all from the target study5 showed that the patients neoplasm clustered with other patients with aml , conrming the myeloid differentiation of the blasts ( fig 3b )  . gene ontology analysis showed transcriptome - wide increases in cytokine - related signaling pathway genes potentially related to the p2ry8 - crlf2 fusion , as well as genes involved in regulating hematopoietic progenitor cell differentiation and the spliceosome ( fig 3c )  . because it is known that stat5 is activated by p2ry8crlf2 fusion in b - all , we performed immunohistochemistry ( ihc ) using phosphorylation - specic antibodies that showed increased stat5 phosphorylation and partial erk phosphorylation but not stat3 phosphorylation ( fig 4a )  . 
gene expression and ihc were largely correlated ; however , the results are interpreted in the context of the importance of signaling in leukemia overall , and these are not necessarily specic for p2ry8 - crlf2 fusion . cell sorting myeloid blasts monocytes 27.5% ( 55 / 200 ) 19.5% ( 22 / 113 ) bone marrow aspirate fluorescence in - situ hybridization 0% ( 0 / 174 ) b lymphocytes crlf2 fusion positive fig 2 . 
 ( b ) principal component analysis plot showing 2 strongest principal components ( pc1 on x - axis and pc2 on y - axis ) from publicly available data from aml ( green dots ) and b - all ( red dots ) patient samples compared with our patient with p2ry8 - crlf2 fusion ( blue dot )  . 
he was consented and enrolled in a study regimen consisting of standard - dose decitabine plus investigational administration of the cytotoxic t - lymphocyte associated protein - 4 ( ctla - 4 ) antagonist , ipilimumab ( clinicaltrials.gov identier : nct02890329 )  . 
after 2 cycles of combination therapy , he achieved complete remission with incomplete count recovery and shortly thereafter a full complete remission ( table 2 ) with negative measurable residual disease ( mrd ) status by a ow cytometric assay with a detection limit of 1 in 2 , 500 cells . 
this was accompanied by a robust molecular response with undetectable levels of the previously detected phf6 , runx1 , tet2 , and rad21 mutations , and a marked reduction in the variant allele frequencies for the mutations asxl1 ( 0.4% ) and sf3b1 ( 1.4% ) , likely representing mrd below the detection limit of ow cytometry . 
ihc was performed on the checkpoint molecules including programmed death ( pd ) - 1 , pd - ligand 1 ( pd - l1 ) and pd - l2 ( fig 4b )  . 
 ( c ) using multiplex immunouorescence microscopy , with digital image analysis , we found that the patients bone marrow was highly enriched with cd8 plus granzyme b ( gzmb ) plus t cells . 
we further identied a population of cd3 + cd4 + t cells at baseline . discussion crlf2 is the protein product of a synonymous gene located in the par1 on the short arm of the x chromosome and the y chromosome . 
it forms a heterodimer with interleukin 7 receptor alpha and is involved in lymphoid signaling pathways . activation of this receptor leads to activation of janus family tyrosine kinases and stat5 . 
deregulated expression of this gene has been described as the characteristic alteration in philadelphia chromosomelike b - all in adolescent and adult patients.7 , 8 russell et al9 showed that this subgroup of b - all has either a translocation juxtaposing the crlf2 to the igh gene on chromosome 14 ( t ( x ; 14 ) ( p22 ; q32 ) or t ( y ; 14 ) ( p11 ; q32 ) ) or a deletion of par1 region , resulting in overexpression of the crlf2 gene . 
this has been observed in down syndromerelated all as well as nondown syndrome all , supporting the cooperation of crlf2 events with other genetic alterations in progression of disease.11 however , irrespective of the clone size , presence of this rearrangement is associated with a higher relapse rate.12 there have been suggestions that the increased relapse rate may be due to other concurrent alterations , such as ikzf1 deletion.13 however , other studies have shown the rearrangement as well as the crlf2 overexpression to be independent predictors of worse outcome in b - all.14 , 15 until now , only one patient with myeloid neoplasm with crlf2 overexpression has been reported . 
however , this patient was associated with multiple copies of an isodicentric y chromosome and not associated with p2ry8crlf2 rearrangement.3 to our knowledge , our patient represents the rst p2ry8 - crlf2 rearrangement myeloid neoplasms , which in our patient was an aml arising from the underlying mds . 
the ow - sorted fish the p2ry8 - crlf2 was not analysis has shown that present in the b lymphocytes of this patient but only in the myeloid - lineage cells ( myeloid blasts and myelomonocytic cells )  . 
in addition , based on the results from total rna sequencing , our patients neoplasm clustered with other patients with aml , providing additional evidence for the myeloid differentiation of the blasts . 
furthermore , the alterations observed in this patient , including the p2ry8affiliations 1department of pathology , memorial sloan kettering cancer center , new york , ny 2leukemia service , department of medicine , memorial sloan kettering cancer center , new york , ny 3department of medical oncology , dana - farber cancer institute , boston , 4department of pathology , brigham and womens hospital , boston , ma crlf2 rearrangement , were present at the mds stage , suggesting a stem cell origin with selective pressure leading to development of aml . phf6 mutations are rare in de novo aml but relatively common in aml - mrc and exceedingly rare in b - all.16 , 17 phf6 and dnmt3a are the most common mutations in mixed phenotype acute leukemias ( mpal ) with t - lineage differentiation.18 phf6 is a critical component in lineage determination of precursor cells , especially between t and b lineages , and it is believed that a functioning phf6 is required for b - cell differentiation leading to selective abundance of this mutation in t - all and mpal with t - lineage differentiation.18 , 19 this notion is further supported by the observation that phf6 suppression leads to impaired tumor progression in b - all.4 it has also been shown that pax5 - decient pro - b cells can undergo myeloid - lineage differentiation in the presence of transcription factors , such as gata or cebpa.20 in our patient , the immunophenotype and rna expression data unequivocally point toward myeloid differentiation . 
although it is difcult to ascertain the functional consequences of the p2ry8 - crlf2 rearrangement and the acquired phf6 mutation at the time of aml transformation , we hypothesize that the two may interact in a way that led to the development of aml instead of b - all . remarkably , this patient with aml - mrc and phf6 , runx1 , tet2 , and rad21 mutations had an mrdnegative complete response by ow cytometry after just 4 cycles of combination decitabine with ipilimumab . 
although the complete results from clinical trials of these agents are needed to evaluate the efcacy and safety of this this patients exceptional response could combination , suggest the unique molecular characteristics of his tumor the p2ry8 - crlf2 may have been important , namely , fusion . 
recent evidence in head and neck cancers with oncogenic chromosomal rearrangements suggests that gene fusions may be a source of immunogenic neoantigens and stimulate t - cell responses that could be unleashed after immune checkpoint blockade.21 this patients t cells were positive for pd - 1 , suggesting a relation to the response to ctla - 4 blockade . 
garcia , wenbin xiao , caleb ho administrative support : jeeyeon baik , allison sigler collection and assembly of data : umut aypar , justin taylor , jacqueline s . garcia , amir momeni - boroujeni , qi gao , dory londono , ryma benayed , michael haddadin , alexander v . 
goldberg , yanming zhang , wenbin xiao , caleb ho data analysis and interpretation : umut aypar , justin taylor , jacqueline s . garcia , amir momeni - boroujeni , alexander v . 
garcia consulting or advisory role : abbvie research funding : abbvie ( inst ) , pzer ( inst ) , genentech ( inst ) , eli lilly ( inst ) maria e . 
arcila honoraria : invivoscribe , biocartis references mikhail roshal honoraria : celgene consulting or advisory role : agios , auron research funding : agios , roche , boehringer ingelheim ana lako employment : bristol - myers squibb scott j . 
rodig honoraria : perkin elmer , bristol - myers squibb consulting or advisory role : bristol - myers squibb research funding : bristol - myers squibb , merck , afmed therapeutics , kite pharma patents , royalties , other intellectual property : patent pending for use of antigalectin - 1 antibodies for diagnostic use travel , accommodations , expenses : roche , bristol - myers squibb aaron d . 
goldberg honoraria : dava oncology consulting or advisory role : abbvie , celgene , daiichi sankyo research funding : abbvie ( inst ) , adc therapeutics ( inst ) , pzer ( inst ) , arog ( inst ) travel , accommodations , expenses : arog open payments link : 1195852 / summary wenbin xiao research funding : stemline therapeutics ( inst ) caleb ho honoraria : invivoscribe travel , accommodations , expenses : invivoscribe no other potential conicts of interest were reported . acknowledgment we acknowledge the members of the memorial sloan kettering cancer center diagnostic molecular laboratory , cytogenetics laboratory , ow cytometry laboratory , and immunohistochemical stain laboratory for their support in performing the relevant assays . schm ah j , fedders b , panzer - gr umayer r , et al : molecular characterization of acute lymphoblastic leukemia with high crlf2 gene expression in childhood . pediatr blood cancer 64 : e26539 , 2017 arber da , orazi a , hasserjian r , et al : the 2016 revision to the world health organization classication of myeloid neoplasms and acute leukemia . 
meacham ce , lawton ln , soto - feliciano ym , et al : a genome - scale in vivo loss - of - function screen identies phf6 as a lineage - specic regulator of leukemia cell therapeutic target . 
nature 555 : 371 - 376 , 2018 patel ss , weirather jl , lipschitz m , et al : the microenvironmental niche in classic hodgkin lymphoma is enriched for ctla - 4 - positive t cells that are pd - 1negative . 
curr opin pharmacol 8 : 249 - 254 , 2008 vesely c , frech c , eckert c , et al : genomic and transcriptional landscape of p2ry8 - crlf2 - positive childhood acute lymphoblastic leukemia . 
leukemia 31 : 1491 - 1501 , 2017 russell lj , capasso m , vater i , et al : deregulated expression of cytokine receptor gene , crlf2 , is involved in lymphoid transformation in b - cell precursor acute lymphoblastic leukemia . 
potter n , jones l , blair h , et al : single - cell analysis identies crlf2 rearrangements as both early and late events in down syndrome and non - down syndrome leukemia . 
morak m , attarbaschi a , fischer s , et al : small sizes and indolent evolutionary dynamics challenge the potential role of p2ry8 - crlf2 - harboring clones as main relapse - driving force in childhood all . 
dou h , chen x , huang y , et al : prognostic signicance of p2ry8 - crlf2 and crlf2 overexpression may vary across risk subgroups of childhood b - cell acute lymphoblastic leukemia . 
fang q , zhao x , li q , et al : ikzf1 alterations and expression of crlf2 predict prognosis in adult chinese patients with b - cell precursor acute lymphoblastic leukemia . 
xiao w , bharadwaj m , levine m , et al : phf6 and dnmt3a mutations are enriched in distinct subgroups of mixed phenotype acute leukemia with t - lineage differentiation . 
presence of the p2ry8 - crlf2 fusion in earlier bone marrow biopsy with myelodysplastic syndrome ( mds )  . ( a ) photomicrographs of the bone marrow biopsy showing mds with excess blast - 1 ( mds - eb1 )  . 
 c significant clinical response to a mek inhibitor therapy in a patient with metastatic melanoma harboring an raf1 fusion introduction systemic therapy for metastatic melanoma has changed dramatically since the development of targeted drugs for braf - mutant melanoma and immune checkpoint inhibitors . 
the overall survival duration of those with advanced melanoma has improved significantly with these novel drugs , with the median survival duration now reaching nearly 2 years.1 - 3 the checkpoint inhibitors ( anti cytotoxic t - cell lymphocyte - 4 antibodies and anti - programmed cell death - 1 [ pd - 1 ] antibodies ) produce antitumor response through their ability to activate cytotoxic t - lymphocytes . 
although many of these clinical responses are durable , the median progression - free survival durations are approximately 2 to 3 months and 5 to 6 months with anti - ctla4 antibody and ipilimumab , respectively.4 - 7 a combination of nivolumab and ipilimumab improves the overall response rate and progression - free survival compared with anti - ctla4 antibody alone.7 for patients with metastatic v600 braf - mutant melanoma , combinations of a braf inhibitor and a mek inhibitor have been established as a standard therapy because of a high response rate and superior survival over single - agent braf inhibitors.1 - 3 however , approximately 50% of melanomas do not harbor a v600 braf mutation and therefore do not benefit from braf inhibitor therapy.8 for these patients with wildtype braf , the identification of relevant and actionable genetic alterations and the development of effective matched therapies are urgently needed . 
recently , next - generation sequencing ( ngs ) analyses have revealed a multitude of less frequent and less well - characterized genetic alterations in melanoma , and these findings could lead to the development of effective treatments . 
in october 2014 , he had recurrent melanoma in the right cervical lymph node , and by december 2014 , his disease had progressed in the cervical and intrathoracic lymph nodes and the liver . 
in january 2015 , the patient started treatment with the combination of nivolumab and ipilimumab , but the treatment was discontinued after two doses because of the development of diabetic ketoacidosis . 
from august 2015 to march 2016 , he was treated with single - agent nivolumab and subsequently nivolumab in combination with intralesional talimogene laherparepvec injection to the right cervical nodes , but by march 2016 , the disease had progressed in the right neck nodes and liver . 
 in april 2016 , he underwent a debulking radical right neck lymph node dissection for a palliative purpose . in october 2016 , the patient developed a painful right neck mass , and his performance status worsened ( eastern cooperative oncology group performance status of 2 )  . 
he was found to have rapid disease progression in the right supraclavicular , mediastinal , gastrohepatic , and portocaval lymph nodes ; the liver ; the spleen ; and the t - 12 spine . 
 melanoma specimen from the right neck node was analyzed for genetic alterations using a hybrid - capturebased ngs assay ( foundationone ) .9 the results were wild type for braf , nras , and kit ; however , a number of genetic alterations , including a ano10 - raf1 fusion , flt1 r281q , notch3 splice site 4404 - 1g > a , arid2 q760fs * 7 , pbrm1 loss , spta1 q1720 * , and tert promoter - 146c > t , were detected . 
because of the development of significant skin toxicity , the dose was reduced to 1.5 mg / d after 4 weeks and further down to 1 mg / d in january 2017 . 
the patients pain in the right neck and shoulder improved dramatically with the treatment , his performance status improved , and a computed tomography scan performed at 10 weeks showed significant regression of the metastatic lesions in the right supraclavicular , mediastinal , gastrohepatic , and portocaval lymph nodes ; the liver ; and the spleen ( fig 1 )  . 
treatment with another mek inhibitor was discussed with the patient , but because of rapid clinical deterioration and the concern for significant skin toxicity , he decided on palliative care , and he died in june 2017 . discussion in this article , we describe , to our knowledge , the first case of clinical benefit from an mek inhibitor in a patient with metastatic melanoma harboring an ano10 - raf1 fusion . 
the patients tumor did not harbor a v600 braf mutation , and therefore , selective braf inhibitors or mek inhibitors were not clinically indicated at the time of the treatment . 
without the comprehensive genomic testing capable of detecting gene fusions and rearrangements in addition to mutations and copy number changes , we could have missed the great response witnessed with the mek inhibitor , underscoring the importance of ngs analysis . 
it is not clear whether the tumor progression was caused by true acquired resistance to mek inhibition or by suboptimal dosing of the treatment because of the intolerable cutaneous toxicity . although a single base substitution at codon 600 is the most common genetic alteration in raf genes in melanoma , other raf gene alterations have been observed , with various functional consequences.10 many of the non - v600 braf mutations do not activate the mitogen - activated protein kinase ( mapk ) signaling pathway in as robust a manner as do v600 mutations , and they are largely insensitive to the braf v600 specific inhibitors dabrafenib and vemurafenib.11 genetic mutations in the other raf genes , such as araf and craf ( raf1 ) , are relatively rare.12 abnormal rearrangements of raf1 , including raf1 fusions , occur in approximately 1% of cutaneous melanomas , 13 and their prognostic significance is not known . 
some of these genetic alterations , such as esrp1 - raf1 fusion , were found to activate the mapk pathway and are capable of transforming cells.10 palanisamy et al10 showed that prostate epithelial cells expressing an esrp1 - raf1 fusion were sensitive to a combination of a raf inhibitor ( sorafenib ) and a mek inhibitor ( u0126 ) in vitro . 
although the function of ano10 - raf1 fusion , as found in our case , has not been described , it is likely that this alteration activates the mapk pathway because mek protein inhibition caused significant tumor regression in our case . 
the breakpoint in this case is in raf1 exon 7 and in the fact that the resulting in - frame fusion retains the raf1 kinase domain while removing the n - terminal auto - inhibitory domain ( fig 2 )  . 
this case , together with ours , strongly suggests the clinical relevance of raf gene fusions in melanoma and offers mek inhibitors as a potential therapeutic option for patients with raf fusionpositive tumors . 
for patients with melanoma harboring a braf fusion , there is a phase ii study of trametinib ( the national cancer institute molecular analysis for therapy choice [ nci - match ] trial [ clinicaltrials . gov identifier : nct02465060 ] ) ; however , at this time there is no specific trial for patients with raf1 fusionpositive melanoma . 
in addition to the clinical investigation of mek inhibitors , clinical studies of pan - raf inhibitors , such as sorafenib and regorafenib , should be encouraged on the basis of their preclinical antitumor activity in raf1 fusionassociated cells . furthermore , a mek inhibitorbased combination approach may lead to more durable disease control . 
several groups have demonstrated potential mechanisms of resistance to mek inhibition , including induction of immunoglobulin transcription factor 2 , 15 induction of signal transducer and activator of transcription 3 , 16 and amplification of cyclin - dependent kinase 4 and 6 , 17 among others . 
 furthermore , there is increasing evidence that mek inhibition leads to antigen - specific cd8 + t cells proliferation within the tumor and pro motes the survival of tumor - infiltrating cd8 + t cells.18 these phenomena were corroborated by experiments in which the addition of an mek inhibitor to an anti pd - 1 ( or pro grammed death - ligand 1 [ pd - l1 ] ) antibody resulted in durable and synergistic tumor regression.18 several clinical trials are evaluating the clinical efficacy of an mek inhibitor in combination with the checkpoint inhibitors in patients with melanoma ( clinicaltrials.gov identifiers : nct03149029 , nct03178851 , and nct02910700 )  . in conclusion , the ngs analysis of the melanoma lesion in our patients tumor was essential in identifying the clinically relevant genetic alteration , ano10 - raf1 fusion , and in selecting a mek inhibitor treatment that resulted in brief , but significant , tumor regression and symptom improvement . 
kim honoraria : genentech , glaxosmithkline , novartis , foundation medicine consulting or advisory role : genentech , glaxosmithkline , novartis , foundation medicine speakers ' bureau : bristol - myers squibb , merck , novartis research funding : glaxosmithkline ( inst ) , novartis ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) , amgen , immune design ( inst ) travel , accommodations , expenses : genentech , bristol - myers squibb , merck , novartis thomas semrad consulting or advisory role : amgen , genentech , onyx pharmaceuticals , celgene , eisai , genzyme ( i ) research funding : eisai ( inst ) , millennium pharmaceuticals ( inst ) , novartis ( inst ) alexa b . 
ali employment : foundation medicine honoraria : emd merck serono research funding : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine ; patents via seres health on microbiome stuff in non - neoplastic disease ( i ) mark singer no relationship to disclose jeffrey s . 
ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine honoraria : pfizer mohammed kashani - sabet stock and other ownership interests : melanoma diagnostics , dnarx honoraria : cepheid consulting or advisory role : castle biosciences research funding : merck patents , royalties , other intellectual property : myriad genetics affiliations kevin b . 
kim , mark singer , and mohammed kashani - sabet , california pacific medical center research institute , san francisco ; thomas semrad , gene upshaw memorial tahoe forest cancer center , truckee , ca ; alexa b . 
long gv , stroyakovskiy d , gogas h , et al : dabrafenib and trametinib versus dabrafenib and placebo for val600 braf - mutant melanoma : a multicentre , double - blind , phase 3 randomised controlled trial . 
weber js , dangelo sp , minor d , et al : nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti - ctla - 4 treatment ( checkmate 037 ) : a randomised , controlled , open - label , phase 3 trial . 
bid hk , kibler a , phelps da , et al : development , characterization , and reversal of acquired resistance to the mek1 inhibitor selumetinib ( azd6244 ) in an in vivo model of childhood astrocytoma . 
ebert pjr , cheung j , yang y , et al : map kinase inhibition promotes t cell and anti - tumor activity in combination with pd - l1 checkpoint blockade . 
walsh , md1 , 2 ; rosalba sacca , phd1 ; temima wildman , ms1 ; kimberly amoroso , ms1 ; jennifer kennedy , ms1 ; liying zhang , md , phd1 ; ozge birsoy , phd1 ; diana mandelker , md , phd1 ; zoe steinsnyder1 ; alicia latham , md1 , 2 ; maria i . 
stadler , md1 , 2 ; and kenneth oft , md , mph1 , 2 introduction telomere length assessthe original dyskeratosis congenita , telomere syndrome , was clinically described more than 100 years ago on the basis of individuals who presented with a distinct rash , abnormal nails , and whitening of the tongue.1 from this rare syndrome , our clinical and molecular understanding of telomere syndromes has evolved and has now expanded to include aplastic anemia , myelodysplastic syndrome , and pulmonary brosis.2 - 5 the identication of patients with telomere syndromes is of signicant clinical importance because these patients are exquisitely sensitive to alkylating chemotherapeutic agents and ionizing radiation.6 - 8 precision medicine efforts that deploy tumor - normal sequencing have used various molecular assays and platforms to interrogate and annotate the cancer census genes.9 , 10 tert , a gene that encodes a key protein involved in telomere maintenance , has been analyzed predominantly to identify genomic alterations that occur in tumors.11 , 12 although constitutional telomere syndromes are recognized , they are rarely considered in the oncologists differential diagnosis unless a diagnosis of a telomere syndrome occurred before the patients cancer diagnosis.13 from january 2016 to may 2019 , unselected patients with advanced solid tumors were presented with the option to participate and consent to a memorial sloan kettering cancer center institutional review board approved protocol ( #12 - 245 ; clinicaltrials.gov identier : nct01775072 ) of tumor and germline dna sequencing . 
cancer types in our cohort included four female patients with breast cancer ( ages at diagnosis , 28 , 37 , 46 , and 58 years ) , one male patient with gall bladder adenocarcinoma ( age 33 years ) , one male patient with pancreatic ampullary adenocarcinoma ( age 45 years ) , and one male patient with two malignancies : lymphoma at age 58 years and prostate cancer at age 66 years . 
in the six patients with a solid tumor as their rst malignancy , all were younger than the median age of diagnosis in the general population for their type of cancer24 - 27 ( data supplement )  . clinical histories of three ( 42.8% ) of the seven patients were suggestive of telomere syndromes . 
individual i , diagnosed with breast cancer , endured therapy - induced radiation pneumonitis and refractory thrombocytopenia ; individual iv , diagnosed with ampulla of vater cancer , experienced premature graying and thrombocytopenia that preceded a cancer diagnosis at age 19 years ; and individual vii revealed a history of bone marrow failure in a family member with a segregating tert mutation and showed abnormal pulmonary function testing ( table 1 )  . an understanding of the genetic basis that contributes to therapeutic sensitivities is important for oncology patients who receive chemotherapy , radiation , and other biologic therapeutics . 
patients with telomere syndromes may manifest overt or subtle clinical ndings.5 moreover , for patients with both obvious and subtle telomere syndromes , exquisite treatment sensitivities have been reported.6 , 7 although only one in 1 , 571 individuals in this series of patients with advanced cancer showed a germline tert mutation , this could translate to more than 1 , 000 patients diagnosed with malignancies a year in the united states who may have increased therapeutic sensitivities . 
because telomere syndrome disorders are complex and exhibit anticipation , incomplete penetrance , multiple genes that underpin disease , recognized modiers , and both autosomal dominant and autosomal recessive patterns of inheritance , this estimate will be rened with time . however , until a clearer picture is possible , it seems reasonable for individuals with constitutional germline tert mutations to be considered for monitoring given the potential long - term sequalae from chemotherapy and radiation as well as increased organ - specic damage.7 , 8 , 31 for two individuals , telomere length testing through cytometric uorescence in situ hybridization identied telomere lengths in the 1st percentile or less ; in two additional individuals , telomere lengths were in the 10th percentile or telomere length less ; and in one additional testing failed because of low blood counts that did not recover after chemotherapy ( fig 1 )  . 
somatic mutation analysis of all ve individuals with germline tert mutations and tumor specimens available for analysis individual , integration of germline , somatic , and clinical data for patients in our cohort was also notable for somatic tp53 mutations and a younger age at cancer diagnosis compared with the general population . 
moreover , multiple tert single nucleotide polymorphisms have been shown to be associated with telomere length and breast and ovarian cancer risk.31 all this information together with telomere lengths may provide insights for stratifying patients with regard to age at presentation , outcome , and tumor evolution . lymphocytes granulocytes 99th percentile 50th percentile 90th percentile 99th percentile 50th percentile 90th percentile 1st percentile 10th percentile 1st percentile 10th percentile age ( years ) age ( years ) fig 1 . 
 e walsh et al e113rfs * 79 p308hfs * 43 l392vfs * 145 r486c v694m exon 4 deletion f1084 * telomerase rvt1 1 , 000 1 , 132 aa a86vfs * 55 y163 * m237i m273h / c truncating mutations missense mutations rvt1 i , v / vi fig 2 . 
walsh , md , memorial sloan kettering cancer center , 222 70th st , room 412 , new york , ny 10021 ; e - mail : walshm2@ makcc.org. support supported by the robert and kate niehaus center for inherited cancer genomics and members of the molecular diagnostics service in the department of pathology and the marie - jos ee and henry r . 
also receives grant support from the v foundation , corning fund , and crawford fund for pediatric genomics . all authors at memorial sloan kettering are supported by the memorial sloan kettering cancer center support grant / core grant through national cancer institute grant no . 
walsh , kimberly amoroso , liying zhang collection and assembly of data : rosalba sacca , temima wildman , kimberly amoroso , jennifer kennedy , ozge birsoy , zoe steinsnyder , alicia latham , maria i . 
carlo consulting or advisory role : pzer other relationship : prostate cancer foundation , robert wood johnson foundation karen cadoo research funding : astrazeneca ( inst ) , syndax ( inst ) travel , accommodations , expenses : astrazeneca mark robson honoraria : astrazeneca consulting or advisory role : mckesson , astrazeneca references research funding : astrazeneca ( inst ) , myriad genetics ( inst ) , invitae ( inst ) , abbvie ( inst ) , tesaro ( inst ) , medivation ( inst ) travel , accommodations , expenses : astrazeneca , pzer other relationship : research to practice , clinical care options , physician education resource zsoa k . 
stadler consulting or advisory role : allergan ( i ) , genentech ( i ) , roche ( i ) , regeneron pharmaceuticals ( inst ) , optos ( i ) , adverum ( i ) , biomarin ( i ) , alimera sciences ( i ) , novartis ( i ) , spark therapeutics ( i ) , fortress biotech ( i ) , regenxbio ( i ) no other potential conicts of interest were reported . zinsser f : atrophia cutis reticularis cum pigmentatione , dystrophia unguium et leukoplakia oris . 
n engl j med 361 : 2353 - 2365 , 2009 alder jk , guo n , kembou f , et al : telomere length is a determinant of emphysema susceptibility . 
am j respir crit care med 184 : 904 - 912 , 2011 alder jk , parry em , yegnasubramanian s , et al : telomere phenotypes in females with heterozygous mutations in the dyskeratosis congenita 1 ( dkc1 ) gene . hum mutat 34 : 1481 - 1485 , 2013 armanios m , blackburn eh : the telomere syndromes . 
nat rev genet 13 : 693 - 704 , 2012 [ erratum : nat rev genet 14 : 235 , 2013 ] agrusa je , bertuch aa , dinardo cd , et al : severe therapy - related toxicities after treatment for hodgkin lymphoma due to a pathogenic tert variant and shortened telomeres . 
pediatr blood cancer 66 : e27779 , 2019 de la fuente j , dokal i : dyskeratosis congenita : advances in the understanding of the telomerase defect and the role of stem cell transplantation . 
pediatr transplant 11 : 584 - 594 , 2007 dietz ac , orchard pj , baker ks , et al : disease - specic hematopoietic cell transplantation : nonmyeloablative conditioning regimen for dyskeratosis congenita . bone marrow transplant 46 : 98 - 104 , 2011 cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
rusch m , nakitandwe j , shurtleff s , et al : clinical cancer genomic proling by three - platform sequencing of whole genome , whole exome and transcriptome . nat commun 9 : 3962 , 2018 11 . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
hampel h , bennett rl , buchanan a , et al : a practice guideline from the american college of medical genetics and genomics and the national society of genetic counselors : referral indications for cancer predisposition assessment . 
weitzel jn , blazer kr , macdonald dj , et al : genetics , genomics , and cancer risk assessment : state of the art and future directions in the era of personalized medicine . 
noone am , cronin ka , altekruse sf , et al : cancer incidence and survival trends by subtype using data from the surveillance epidemiology and end results program , 1992 - 2013 . 
bojesen se , pooley ka , johnatty se , et al : multiple independent variants at the tert locus are associated with telomere length and risks of breast and ovarian cancer . 
 o stromal content is correlated with tissue site , contrast retention , and survival in pancreatic adenocarcinoma purpose desmoplastic stroma is a cardinal feature of primary pancreatic ductal adenocarcinoma ( pda ) , but its effects on the biology , prognosis , and therapeutic outcomes in the disease are not known . 
we developed an automated method to assess tumor stroma density ( tsd ) and investigated computed tomography ( ct ) correlates of stroma in pda . patients and methods we collected pda samples from rapid autopsy and resection series and digitally annotated samples to quantify tsd . 
2018 by american society of clinical oncology introduction primary pancreatic ductal adenocarcinoma ( pda ) tumors are composed of malignant epithelial cells infiltrating and surrounded by an abundant desmoplastic stroma ( ds ) that , in turn , typically composes most of the tumor volume.1 ds is a diverse matrix comprising many cell types ( eg , fibroblasts , pancreatic stellate cells , immune cells , blood vessels ) and extracellular matrix proteins such as collagen and hyaluronic acid.2 tumor epithelium and ds interact in important ways and mediate tumor growth , immune surveillance , metabolism , and , possibly , metastasis.3 - 6 the interest in therapeutically targeting ds in pda is increasing because of its purported role in impeding drug delivery and excluding immune cells from the tumor microenvironment.7 - 10 clinical trials targeting ds , however , have been disappointing , and more recent studies have found that reducing ds burden through various stroma - depletion strategies in mouse models can lead to more aggressive tumors.11 - 13 this underscores the complex role ds plays robert j . 
we applied this method to investigate the relationship between primary tumors and metastases in a cohort of patients with metastatic disease , and the relationship between tsd of primary tumors and survival in two cohorts of patients who had undergone tumor resection . another significant hurdle in the assessment of ds is the lack of noninvasive biomarkers that can inform us of the qualitative and quantitative aspects of the ds . 
 the university of california , san francisco ( ucsf ) , institutional pathology and imaging databases were queried to identify patients with pda who met the following inclusion and exclusion criteria : had undergone curative intent resection with pathology confirmation of pda , had standard preoperative , multiphase pancreatic protocol staging ct imaging within 60 days of surgery , did not have any neoadjuvant therapies , and had records of 12 months of imaging and clinical follow - up available for review . automated stroma quantification for the rapid autopsy cohort , tissue microarrays were used ; for the denmark and ucsf cohorts , whole - tissue sections were used . 
using definiens architect xd 2.4 and tissue studio 4.1 ( both definiens , munich , germany ) , the annotated images were segmented into different regions of interest ( rois ) : tumor epithelium , tumor desmoplasia , and glass . 
for each specimen , tumor stroma density ( tsd ) was calculated as follows : tsd = tumor desmoplasia area / total tumor area ct image acquisition and analysis standard , multiphase ct imaging was performed for staging purposes before surgery for all patients in the ucsf cohort . 
after intravenous injection of 120 ml of omnipaque 350 ct contrast material ( ge healthcare , chicago , il ) at a rate of 5 ml / second , a pancreatic parenchymal ( pp ) phase ct image and then a portovenous ( pv ) phase ct image were acquired . 
for each patient , the following normalized tumor enhancement ratios were calculated : pp phase = ( hutumor , pp hutumor , ne ) / ( huaorta , pp huaorta , ne ) pv phase = ( hutumor , pv hutumor , ne ) / ( huaorta , pv huaorta , ne )  . automated tsd was compared with manually quantified tsd and reviewed by a board - certified pathologist ( k.e.v. ) , blinded to clinical and pathologic data , in a set of 77 resected pda samples . 
overall survival ( os ) and recurrence - free survival ( rfs ) were compared in high versus low tsd groups using a cox proportional hazards model controlling for overall histologic tumor area , tumor differentiation , stage , american society of anesthesiologists classification , and smoking status . 
the likelihood ratio test was used to assess the relevance of variables relative to the reduced model with that variable removed . for the ucsf cohort , cox proportional hazards model as well as cox model with time - varying coefficients were used to analyze the rfs and os when stratified by high versus low tsd using the same threshold value as the denmark cohort , controlling for clinical and tumor pathology variables . 
for the tumor ct enhancement analysis of the ucsf cohort , the tumors were categorized as high versus low enhancement ratios using the median value , and a cox proportional hazards model was used to analyze the rfs and os controlling for clinical and tumor pathology variables . results correlation of automated and manual quantification of tsd automated quantification of tsd was optimized using whole - tissue sections of primary pda tumors . 
these results suggest that pda stroma is not uniformly abundant across metastatic sites and is least abundant in solid organ metastases . high tsd was a favorable prognostic factor in resectable pda tumors automated tsd quantification was performed on samples from 123 patients with available tissue from copenhagen , denmark.20 this included 63 men and 60 women , whose mean age was 62 years at the time of surgery ( range , 33 to 87 years )  . 
 ( b ) automated segmentation of tumor epithelium ( green ) and tumor stroma : fibrosis - high stroma ( yellow ) and fibrosis - low stroma ( pink ) ( magnification , 10 )  . 
this cohort included 20 men and 25 women undergoing resection at ucsf ( mean age , of 66.3 years [ range , 46 to 81 years ] ; table 1 )  . 
 ( a , b ) kaplan - meier curves of ( a ) rfs and ( b ) os for high versus low tsd in resected pancreatic adenocarcinoma ( pda ) specimens from the denmark patient cohort . 
 ( c , d ) kaplan - meier curves of rfs and os for high versus low tsd in resected pda specimens from the university of california , san francisco , patient cohort . 
these results suggest that patients in the ucsf cohort with high tsd had improved prognosis early on , but its protective effect diminished over time . preoperative ct enhancement patterns in patients with resectable pda tumors ct scanning of patients with pda classically demonstrates delayed enhancement after contrast administration.21 this pattern has been attributed to the presence of the abundant stroma.16 therefore , we investigated the association of ct enhancement in primary ( resected ) pda and tsd , as well as the association between ct enhancement and survival in the ucsf cohort . 
 ( a ) an example of a tumor with high normalized enhancement ratio at both pancreatic parenchymal ( pp ) and portovenous ( pv ) phases of the ct studies . 
 ( c , d ) kaplan - meier curves of recurrence - free survival ( rfs ) and overall survival ( os ) for high versus low pv tumor enhancement . 
tumor stromal density ( tsd ) is associated with portovenous ( pv ) computed tomography ( ct ) enhancement patterns in patients with resectable pancreatic ductal adenocarcinoma ( pda ) tumors . 
 box - and - whisker plots of tsd in tumors with high versus low normalized enhancement ratios in ( a ) the pancreatic parenchymal ( pp ) phase and ( b ) the pv phase . 
this algorithm was then used to demonstrate tsd varies across metastatic sites and that tsd of the primary tumor positively associates with survival . most pda patients die with metastases.22 although effective treatment of metastatic disease is the most important priority in pda , translational efforts and preclinical models have focused largely on primary tissues.23 we found significant differences in tsd across metastatic sites . 
prior work has demonstrated that metastatic pda tumors have limited driver - mutation heterogeneity when compared with the primary tumor and largely concordant tumor - specific gene expression patterns in samples across many primary and metastatic sites.17 , 24 however , metastatic sites may have different proteomic profiles.25 these important observations , coupled with our results , suggest the variation in tsd is a function of host organ - site physiology and not secondary to cancer genomic or epigenomic / transcriptional differences between the primary and metastatic tumors . 
different sites of pda likely react differently to tumor cells with respect to generation of ds that may have therapeutic implications . we found that tsd correlated with both rfs and os in a mature cohort of patients with resected pda tumors who did not undergo adjuvant therapy . 
our prognostic findings imply a protective role of stroma in pda pathogenesis , in agreement with prior studies that used subjective assessments of various stromal characteristics.3 , 7 , 26 in the ucsf cohort , where most patients received adjuvant therapy , a lower risk of recurrence or death was seen initially in patients with high tsd , but the risk for both increased over time , with the survival curves eventually crossing . 
the denmark cohort did not receive any adjuvant therapy.19 , 20 in contrast , 91% ( 41 of 45 ) of the patients in the ucsf cohort received adjuvant therapy soon after resection , which more recently has become the standard of care.27 interestingly , sinn et al8 showed that a qualitatively dense stroma in pda was associated with a significantly longer rfs and os in the conko - 001 trial , but this effect was restricted to patients in the observation group ( no adjuvant therapy until recurrence ) and not seen in patients randomly assigned to adjuvant gemcitabine.8 our findings , along with those of sinn et al , 8 suggest patients with tumors that have low tsd may benefit more from adjuvant chemotherapy , closing the survival gap between high tsd and low tsd seen in the denmark cohort . assessment of tsd is limited by the availability of adequate tissue samples . 
we showed that normalized tumor enhancement ratio at the pv phase on ct imaging was correlated with tsd and prognosis , in agreement with a prior study that demonstrated resected pda tumors with lower enhancement tended to be less fibrotic.16 the association between pda enhancement and tsd may be explained by the properties of the ct contrast materials that pass freely between the intravascular and extravascular extracellular space but do not cross intact cell membranes . 
another limitation of our study is that only one board - certified pathologist reviewed specimens for manual tsd quantification , from which we show good correlation between manual and automated tsd quantification . 
to limit any potential bias , manual quantification was done blinded to the results of automated quantification and all other clinical and pathologic data . in summary , we found tsd was lowest at solid organ metastases , and that higher tsd was positively associated with outcome in patients with resected pda tumors without adjuvant therapy . 
torphy no relationship to disclose zhen wang stock and other ownership interests : nextrast honoraria : ge healthcare ( inst ) research funding : guerbet ( inst ) patents , royalties , other intellectual property : nextrast ( inst ) travel , accommodations , expenses : ge healthcare ( inst ) aisha true - yasaki no relationship to disclose keith e . 
volmar no relationship to disclose rashid naim no relationship to disclose benjamin yeh stock and other ownership interests : nextrast honoraria : ge healthcare consulting or advisory role : ge healthcare research funding : ge healthcare , philips healthcare , guerbet patents , royalties , other intellectual property : patents related to imaging contrast materials travel , accommodations , expenses : ge healthcare julia s . 
hollingsworth stock and other ownership interests : sanguine therapeutics and diagnostics research funding : glycomimetics ( inst ) patents , royalties , other intellectual property : royalties on cell lines ( inst ) jen jen yeh no relationship to disclose eric a . 
sinn m , denkert c , striefler jk , et al : - smooth muscle actin expression and desmoplastic stromal reaction in pancreatic cancer : results from the conko - 001 study . 
catenacci dvt , junttila mr , karrison t , et al : randomized phase ib / ii study of gemcitabine plus placebo or vismodegib , a hedgehog pathway inhibitor , in patients with metastatic pancreatic cancer . 
zdemir bc , pentcheva - hoang t , carstens jl , et al : depletion of carcinoma - associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival . 
iacobuzio - donahue ca , fu b , yachida s , et al : dpc4 gene status of the primary carcinoma correlates with patterns of failure in patients with pancreatic cancer . 
makohon - moore ap , zhang m , reiter jg , et al : limited heterogeneity of known driver gene mutations among the metastases of individual patients with pancreatic cancer . 
diana a , wang lm , dcosta z , et al : prognostic value , localization and correlation of pd - 1 / pd - l1 , cd8 and foxp3 with the desmoplastic stroma in pancreatic ductal adenocarcinoma . 
stein , md1 ; manjari pandey , md1 ; joanne xiu , phd2 ; hongseok tae , phd2 ; jeff swensen , phd2 ; sandeep mittal , md3 , 4 ; andrew j . 
variability existed for other luad metastasis sites , with adrenal metastases most likely to meet the cutoff of 10 mutations / mb ( 51% ) and bone metastases least likely to meet the cutoff ( 19% )  . 
checkmate 227 showed a 43% 1 - year progression - free survival ( pfs ) rate for patients with advanced nsclc with samples of 10 mutations / mb or greater receiving rstline nivolumab and ipilimumab compared with a pfs of 13% in patients who received platinum - based chemotherapy . 
 stein et al context key objective although tumor mutational burden ( tmb ) is an emerging biomarker described to predict benet to immune checkpoint inhibition in nonsmall - cell lung cancer , little is known about whether any difference exists between tmb and sample site , histology , or other tumor proling characteristics . knowledge generated in both lung adenocarcinoma and squamous cell carcinoma , tmb was more likely to be 10 mutations per megabase ( mb ) or greater in metastases compared with primary tumors and highest in lung adenocarcinoma brain metastases . 
lung squamous cell carcinoma primary tumors more commonly met this tmb cutoff than lung adenocarcinoma primary tumors ; poorly differentiated disease was more likely to have a tmb of 10 mutations / mb or greater . 
site - specic differences in programmed death ligand 1 positivity , stk11 and kras mutation rate , and other markers were observed along this tmb cutoff . relevance tmb is a heterogeneous biomarker in nsclc with important spatial and histologic distinctions . 
further work is needed to identify unique therapeutic approaches for brain metastases and other sites with high tmb . although a high tmb may predict for benet to icis in nsclc , additional information is necessary to maximize its clinical utility . 
here , we detail the distribution of tmb in nsclc based on sample site , comparing primary lesion data to metastases for both lung adenocarcinoma ( luad ) and lung squamous cell carcinoma ( lusc )  . methods study design and patients we evaluated a database of patients with advanced nsclc who underwent tumor proling between 2015 and 2017 with caris life sciences ( irving , tx ) , a clinical laboratory improvement amendmentscertied laboratory . 
luad nonbrain metastases were further subdivided into the following six categories as demarcated by the submitting provider : regional lymph thoracic , and other extranode , thoracic metastases . liver , bone , adrenal , tumor proling caris tumor proling included ihc , ngs , and tmb assessment , for which the methods have been previously described.10 , 11 the genomic dna of 592 cancer - related genes isolated from formalin - xed , parafn - embedded tumor samples were sequenced using nextseq ( illumina , san diego , ca )  . 
variants were detected with greater than 99% condence based on allele frequency and amplicon coverage , with an average sequencing depth of coverage greater than 500 times and analytic sensitivity of 5% variant frequency . 
tmb ( mutations per mb ) was calculated in accordance with the tmb harmonization project , 13 adding nonsynonymous , nonsense , in - frame indel and frameshift variants after ltering out presumed germline variants determined from the genome aggregation database ( release 2.1 ) , the single nucleotide polymorphism database human build 151 , and the caris in - house benign database . 
although ngs obtained from bone metastases with conventional decalcication methods with strong acids leads to reduced success rates , 14 we nd similar reporting rates of ngs and tmb for bone specimens compared with other sites by using an edta - based decalcication approach or nondecalcied specimens . pd - l1 ihc was completed on slides of tumor sections using procedures in accordance with the college of american pathologists . 
sp142 ( spring bioscience , pleasonton , ca ) was the antibody used against pd - l1 before january 2016 ; thereafter , the primary antibody was 22c3 ( dako , santa clara , ca )  . 
 distribution of tmb by sample site in nsclc pattern.15 for ros1 and alk fusion detection , anchored multiplex polymerase chain reaction was performed for targeted rna sequencing using the archerdx ( boulder , co ) fusion assay ( archer fusionplex solid tumor panel )  . the formalin - xed , parafn - embedded samples were microdissected to enrich to 20% or more tumor nuclei , and mrna was isolated and reverse transcribed into gene - specic complementary dna . 
the sensitivity of the assay allows for fusion detection present in 10% or more of cells . statistical analysis the rate that tmb was reported as 10 mutations / mb or greater and frequency of other established nsclc biomarkers , including paths in egfr , kras , stk11 , braf ( v600e ) , alk , and ros1 rearrangements and pd - l1 ihc ( 1% and 50% ) , were compared between primary lesions and metastases . 
in addition , we compared the rate of selected molecular alterations along a tmb cutoff of 10 mutations / mb to identify any site - specic differences . statistical analysis was completed using spss version 20.0 ( spss , chicago , il )  . 
the median age of patients with luad with primary tumors sampled ( n = 1 , 102 ) was 69 years ( range , 25 to 90 years ) compared with 66 years ( range , 28 to 90 years ) for patients with metastases ( n = 1 , 249 )  . 
the median age of patients with lusc primary tumors ( n = 779 ) was 71 years ( range , 23 to 90 years ) compared with 67 years ( range , 24 to 90 years ) for patients with metastases ( n = 294 )  . 
a similar percentage of primary luad and lusc tumors were poorly differentiated ( 20% v 26% , respectively ) compared with metastases ( 21% and 29% , respectively )  . of 10 tumor proling characteristics evaluated , six luad and four lusc differences were observed between primary and metastatic lesions ( table 1 )  . 
figure 2 depicts a scatter plot of tmb and pd - l1 for luad and lusc , for which no correlation was found . tmb increases with metastases and is highest in luad brain metastases to further assess luad and lusc by location , we divided metastases into brain and nonbrain metastases and compared them to primary tumors . 
both brain and nonbrain luad metastatic lesions were associated with increased stk11 and stk11 plus kras comutated paths and tmb of 10 mutations / mb or greater compared with primary tumors ( table 2 )  . in addition , compared with primary luad , patients with nonbrain metastasis were more likely to be male and alk rearranged and have higher rates of pd - l1 expression of 1% or greater and 50% or greater . 
figure 3a shows the rate that tmb was reported as 10 mutations / mb or greater based on sample site , comparing not only primary tumor and brain metastasis , but also nonbrain metastasis and six subdivisions ( table 3 )  . 
samples from adrenal metastases ( 51% ) and other extrathoracic metastases ( 43% ) were more likely to have a tmb of 10 mutations / mb or greater , and bone metastases ( 19% ) were less likely to have a tmb of 10 mutations / mb or greater . of 294 lusc metastases , 19 were brain metastases and 275 were other metastases . 
for lusc , a reduced frequency of egfr ( p = .025 ) and pd - l1 expression of 1% or greater ( p = .008 ) was found for metastases when tmb was 10 mutations / mb or greater ( appendix table a1 )  . 
nine of 10 and four of 10 sample site divisions had reduced egfr paths and alk rearrangements in cases where tmb was 10 mutations / mb or greater , respectively . 
here , we detail ndings from a large nsclc cohort with tmb data , providing novel insights with respect to tmb and sample acquisition site and histology , while also comparing the interplay of tmb with established and other emerging biomarkers . 
although wes may be considered a gold standard given its breadth , high cost and prolonged result times have contributed to the use of tmb from ngs panels . although more efcient and commercially available , accurate quantitation of tmb from ngs introduces additional nuance , including the number of genes needed for extrapolation ( ideally 1 mb ) , type of enrichment ( hybrid capture or amplication ) , and read depth.8 , 25 , 26 a limitation of our ngs data is that tmb was not correlated with wes from a subset of samples , although it was calculated in accordance with the tmb harmonization project.13 although we found that tmb was more likely to be 10 mutations / mb or greater in metastases compared with primary tumors , a range existed based on anatomic site for both luad and lusc , with brain and adrenal subgroups more likely and bone and liver lesions less likely to meet this cutoff in luad . 
 ( a ) frequency of lung adenocarcinomas ( luads ) with tumor mutational burden ( tmb ) of 10 mutations per megabase ( mb ) or greater varies by sample site and is highest in brain metastases . ( b ) lung squamous cell carcinoma ( lusc ) metastases are more likely to have a tmb of 10 mutations / mb or greater compared with primary tumor samples . 
although retrospective studies from trial patients or clinical practice have shown an amalgamation of improved overall response rates , pfs , and os for different icis at various tmb cutoffs , 24 , 26 prospective os benet remains to be shown . 
this could be particularly important for brain metastases , which occur in 40% of nsclcs , 40 portend a poor prognosis , and have historically lacked viable treatments.41 because standard chemotherapy has reduced blood - brain penetrance , 42 dual checkpoint inhibition should be compared with chemoimmunotherapy in this high - risk subgroup . we found that less than 3% of primary and 6% of metastatic egfr - mutated luad samples and no alk - rearranged samples had a tmb of 10 mutations / mb or greater . 
a comparatively elevated tmb in other patients correlates with recently described smoking and apolipoprotein b mrna - editing enzyme , catalytic polypeptide - like ( apobec ) mutation signatures.45 , 46 we observed several site - specic molecular combinations with tmb . 
furthermore , anatomic sites in luad including adrenal and other extrathoracic specimens were more likely to meet a threshold of 10 mutations / mb and have 1% or greater pd - l1 expression . 
because samples that are both pd - l1 positive and tmb high may derive improved benet from pd - 1 / pd - l1 inhibition , 29 our data show anatomic differences in this biomarker combination that could be clinically informative for icis . we found anatomic differences between tmb and stk11 , including the nding that luad metastases were less likely to be stk11 and kras comutated if tmb was 10 mutations / mb or greater . 
an emerging biomarker afliated with resistance to pd - 1 / pd - l1 inhibition and reduced immunogenicity in mouse models and patients with nsclc , 44 , 47 , 48 sitespecic differences in stk11 with tmb may add complexity to treating this molecular subset with novel approaches . another limitation of our study includes the reduced number of lusc metastases available , making analysis less powerful . 
furthermore , ndings such as a high egfr mutation rate in luad liver metastases could skew tmb accuracy . in addition , we are unable to provide prior therapies received for patients and the stage at time of tissue acquisition for primary tumor samples . 
although we show that surgically obtained primary tumors have similar rates of tmb of 10 mutations / mb or greater as other primary tumors , further evaluation is needed to determine whether a difference exists between , for example , early stage i and locally advanced stage iii disease or between stage iii and iv lesions . overall , we demonstrate that tmb is a site - specic biomarker in nsclc with important spatial and histologic differences . 
in both luad and lusc , the frequency of specimens with a tmb of 10 mutations / mb or greater is increased in metastases and highest with brain metastases . poorly differentiated disease from either histology was more likely to have a tmb of 10 mutations / mb or greater compared with other primary tumors or metastases . 
michael korn employment : caris life sciences leadership : caris life sciences stock and other ownership interests : oncocyte consulting or advisory role : merck sharp & dohme patents , royalties , other intellectual property : exelixis travel , accommodations , expenses : merck sharp & dohme amy b . 
heimberger stock and other ownership interests : celldex , caris life sciences consulting or advisory role : caris life sciences research funding : merck patents , royalties , other intellectual property : celldex travel , accommodations , expenses : caris life sciences mike g . 
martin consulting or advisory role : seattle genetics no other potential conicts of interest were reported . references yarchoan m , hopkins a , jaffee em : tumor mutational burden and response to pd - 1 inhibition . 
n engl j med 377 : 2500 - 2501 , 2017 ramalingam ss , hellmann ad , awad mm , et al : tumor mutational burden ( tmb ) as a biomarker for clinical benet from dual immune checkpoint blockade with nivolumab ( nivo ) + ipilimumab ( ipi ) in rst - line ( 1l ) non - small cell lung cancer ( nsclc ) : identication of tmb cutoff from checkmate 568 . 
n engl j med 378 : 2093 - 2104 , 2018 fr uh m , peters s : genomic features of response to combination immunotherapy in patients with lung cancer . 
mutational landscape determines sensitivity to pd - 1 blockade in non - small cell lung cancer . science 348 : 124 - 128 , 2015 carbone dp , reck m , paz - ares l , et al : first - line nivolumab in stage iv or recurrent non - small - cell lung cancer . 
hellmann md , nathanson t , rizvi h , et al : genomic features of response to combination immunotherapy in patients with advanced non - small - cell lung cancer . cancer cell 33 : 843 - 852.e4 , 2018 chalmers zr , connelly cf , fabrizio d , et al : analysis of 100 , 000 human cancer genomes reveals the landscape of tumor mutational burden . 
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vanderwalde a , spetzler d , xiao n , et al : microsatellite instability status determined by next - generation sequencing and compared with pd - l1 and tumor mutational burden in 11 , 348 patients . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
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cappuzzo f , mccleod m , hussein m , et al : impower130 : progression - free survival ( pfs ) and safety analysis from a randomised phase iii study of carboplatin + nab - paclitaxel ( cnp ) with or without atezolizumab ( atezo ) as rst - line ( 1l ) therapy in advanced non - squamous nsclc . 
reck m , rodrguez - abreu d , robinson ag , et al : updated analysis of keynote - 024 : pembrolizumab versus platinum - based chemotherapy for advanced nonsmall - cell lung cancer with pd - l1 tumor proportion score of 50% or greater . 
allg auer m , budczies j , christopoulos p , et al : implementing tumor mutational burden ( tmb ) analysis in routine diagnostics : a primer for molecular pathologists and clinicians . 
transl lung cancer res 7 : 703 - 715 , 2018 jamal - hanjani m , wilson ga , mcgranahan n , et al : tracking the evolution of non - small - cell lung cancer . 
gandara dr , paul sm , kowanetz m , et al : blood - based tumor mutational burden as a predictor of clinical benet in non - small - cell lung cancer patients treated with atezolizumab . 
rizvi h , sanchez - vega f , la k , et al : molecular determinants of response to anti - programmed cell death ( pd ) - 1 and anti - programmed death - ligand 1 ( pd - l1 ) blockade in patients with non - small - cell lung cancer proled with targeted next - generation sequencing . 
kowanetz m , zou w , shames ds , et al : tumor mutation burden ( tmb ) is associated with improved efcacy of atezolizumab in 1l + and 2l + nsclc patients . 2019 j clin oncol 34 : 9017 , 2016 ( suppl 15 ; abstr 9017 ) j thorac oncol 12 : s321 - s322 , 2017 33 . 
gandara d , kowanetz m , mok t , et al : blood - based biomarkers for cancer immunotherapy : tumor mutational burden in blood ( btmb ) is associated with improved atezolizumab ( atezo ) efcacy in 2l + nsclc ( poplar and oak )  . 
ross j , goldberg m , albacker l , et al : immune checkpoint inhibitor ( icpi ) efcacy and resistance det5ected by comprehensive genomic proling ( cgp ) in nonsmall cell lung cancer ( nsclc )  . 
singal g , miller p , agarwala v , et al : analyzing biomarkers of cancer immunotherapy ( cit ) response using a real - world clinico - genomic database . 
park w , lopes g , kwon d , et al : correlating isend and tumor mutation burden ( tmb ) with clinical outcomes of advanced non - small cell lung cancer ( ansclc ) patients on nivolumab . 
ready n , hellmann md , awad mm , et al : first - line nivolumab plus ipilimumab in advanced non - small - cell lung cancer ( checkmate 568 ) : outcomes by programmed death ligand 1 and tumor mutational burden as biomarkers . 
bristol - meyers squibb : bristol - myers squibb provides update on the ongoing regulatory review of opdivo plus low - dose yervoy in rst - line lung cancer patients with tumor mutational burden 10 mut / mb . 
schrock a , sharma n , peled n , et al : updated dataset assessing tumor mutation burden ( tmb ) as a biomarker for response to pd - 1 / pd - l1 targeted therapies in lung cancer ( lc )  . 
koyama s , akbay ea , li yy , et al : stk11 / lkb1 deciency promotes neutrophil recruitment and proinammatory cytokine production to suppress t - cell activity in the lung tumor microenvironment . 
 association of germline genetic test type and results with patient cancer worry after diagnosis of breast cancer background there are concerns that multigene panel testing compared with brca1 / 2 - only testing after diagnosis of breast cancer may lead to unnecessary patient worry about cancer because of more ambiguous results . methods patients with breast cancer diagnosed from 2013 to 2015 and accrued from seer registries in georgia and los angeles were surveyed ( n = 5 , 080 ; response rate , 70% ) , and responses were merged with seer data and germline genetic testing and results . 
we examined patient reports of cancer worry by test type and results in 1 , 063 women who linked to a genetic test and reported undergoing testing . results more than half of the sample ( n = 640 ; 60.2% ) received brca1 / 2 - only testing versus 423 patients ( 39.8% ) who had a multigene panel . 
a minority of tested patients reported substantial cancer worry after treatment : 11.1% ( n = 130 ) reported higher impact of cancer worry , and 15.1% ( n = 162 ) reported a high frequency of cancer worry ( worrying often or almost always ) in the past month . 
2018 by american society of clinical oncology introduction multiple - gene panel ( mgp ) testing has rapidly replaced brca1 / 2 - only testing after diagnosis of breast cancer.1 however , genetic testing use in patients who need it seems to be low.2 , 3 one barrier to more robust uptake of mgp testing is uncertainty about its clinical utility . 
indeed , some have argued that mgp testing should not replace brca1 / 2 - only testing , even in patients with higher pretest risk of a pathogenic mutation , because of these concerns about patient reactions , 4 whereas others strongly disagree.5 current clinical guidelines that recommend genetic testing in women at elevated pretest risk of carrying a pathogenic variant do not specify how many genes should be tested . 
we sent surveys to 7 , 810 patients : 507 patients were ineligible because of the exclusions noted previously or were deceased , institutionalized , too ill , or unable to complete a survey in spanish or english , leaving 7 , 303 patients . 
virtually all patients ( 5 , 026 ) had complete information for the test linkage phase , of whom 1 , 272 ( 25.3% ) linked to a genetic test result , 1 , 063 of whom ( 21.2% ) reported receipt of genetic testing in the survey . survey responses were merged with seer clinical data and genetic testing information obtained from four laboratories ( ambry genetics , aliso viejo , ca ; genedx , gaithersburg , md ; invitae , san francisco , ca ; myriad genetics , salt lake city , ut ) that performed nearly all germline cancer genetic testing in the study regions . 
test type and results were merged to 5 , 026 patients with complete information on all variables for seer genetic testing linkage using a probabilistic matching strategy performed by information management services ( rockville , md )  . 
the safe harbor method was used to de - identify the data set before transfer to the university of michigan for analysis.9 the collaboration was covered under data use agreements between the university of michigan , genetic laboratories , and information management services . 
the research was approved by institutional review boards of the university of michigan , emory university , the university of southern california , the georgia department of public health , the california state committee for the protection of human subjects , and california cancer registry . 
waivers of informed consent and authorization were approved , given the use of a third - party honest broker to conduct the linkage and create a de - identified data set for analyses . measures we used two questions to evaluate patient worry about future cancer . 
forest plot ( adjusted odds ratios and 95% cis ) for results of a multivariable model regressing a binary variable indicating higher versus low impact of cancer worry on test type , controlling for selected predisposing , clinical , and treatment factors and weighted for survey design and nonresponse . 
er , estrogen receptor ; her2 , human epidermal growth factor receptor 2 ; hs , high school ; mgp , multigene panel ; pr , progesterone receptor ; ref , reference . made it difficult for you to carry out your usual daily activities at home or work ? ; made you feel distant from family or friends ? ( responses were based on 5 - point likert categories from almost never to almost always )  . 
we developed a scale by averaging the responses across the three items to create a continuous range from 1 to 5 , with higher values indicating greater impact of cancer worry . 
we then created a binary variable indicating higher impact of cancer worry versus low impact using a cut point of 3.0 , which corresponded to responses of sometimes , often , or almost always . 
we also measured frequency of cancer worry for validation , which was assessed using a single item : in the past month , how often have you worried about your cancer coming back ? ( almost never to almost always ; five point categories )  . 
genetic laboratories provided results at the level of the gene tested ( eg , brca1 ) and the clinical interpretation sent to the ordering clinician ( consisting of pathogenic , likely pathogenic , uncertain significance , likely benign , or benign )  . 
we categorized a test that assessed only brca1 / 2 as brca1 / 2 only and a test that assessed any additional gene ( eg , atm , chek2 ) as a multigene panel . 
if patients received both tests on separate occasions ( eg , a brca1 / 2 - only test and later mgp ) , they were coded as having received mgp . 
we assessed clinical factors related to future cancer risk ( tumor behavior , triple - negative disease , nodal status , bilateral disease ) , treatments received , age ( in 10 - year groups ) , education , and family history . 
 defined as having two or more first - degree relatives with breast cancer ; any relatives with ovarian cancer , sarcoma , or male breast cancer ; ashkenazi jewish ancestry ; or a family history of a mutation - conferring risk ( eg , brca1 / 2 )  . statistical analysis we first examined cancer worry variables by test type , test results , and selected covariables . 
a minority of tested patients reported substantial cancer worry after treatment : 11.1% ( n = 130 ) reported high impact of cancer worry , and 15.1% ( n = 162 ) reported high frequency of worry ( worrying often or almost always in the past month )  . 
we observed similar findings in a model of frequency of cancer worry ( data not shown )  . figure 1 shows the results of a multivariable model regressing a binary variable indicating higher versus low impact of cancer worry on test type , controlling for selected predisposing , clinical , and treatment factors . 
in sensitivity analyses , we examined different permutations of the cancer worry variables , including different cut points for higher worry , and using a continuous variable , and results were consistent . discussion we found that the type of genetic testing patients received ( mgp v brca1 / 2 only ) was not associated with their report of cancer - related worry ( impact or frequency )  . 
 as noted in our prior work , cancer - related worry was associated with younger age and lower educational level , but not clinical factors.10 , 11 strengths of this study include a large population based sample of patients recently diagnosed with breast cancer and accrued shortly after the advent of multigene panel testing in the community , genetic testing results from companies linked to seer clinical data , and granular information about patients , including their report of cancer - related worry . 
we excluded them from the analytic sample because we did not think it was valid to assess the association of a test type with attitudes about health risk among patients who did not recall that they had the test . 
finally , generalizability of the findings are limited to two large geographic regions of the united states . the results from this study and our prior work suggest that testing more genes versus brca1 / 2 alone does not seem to foment more negative patient reactions . 
our prior research in this cohort suggested that mgp testing ( v brca1 / 2only testing ) does not lead to inappropriate rates of contralateral prophylactic mastectomy.1 virtually all testers in this study received some form of genetic counseling , 6 and our results suggest these efforts may sufficiently frame results in a manner that did not alarm patients . 
kurian manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors paul abrahamse no relationship to disclose sarah t . 
dolinsky , cgc , and melissa pronold at ambry genetics , delores bowman and benjamin solomon at genedx , edward esplin and stephen lincoln at invitae , and johnathan lancaster and brian dechairo at myriad genetics for their collaboration on the genetic test data linkage to seer data . 
the collection of los angeles county cancer incidence data used in this study was supported by the california department of public health pursuant to california health and safety code section 103885 , centers for disease control and preventions ( cdcs ) national program of cancer registries , under cooperative agreement 5nu58dp003862 - 04 / dp003862 , the national cancer institutes seer program under contract hhsn261201000140c awarded to the cancer prevention institute of california , contract hhsn261201000035c awarded to the university of southern california , and contract hhsn261201000034c awarded to the public health institute . 
the collection of cancer incidence data in georgia was supported by contract hhsn261201300015i , task order hhsn26100006 from the national cancer institute and cooperative agreement 5nu58dp003875 - 04 - 00 from the cdc . 
us department of health & human services : guidance regarding methods for de - identification of protected health information in accordance with the health insurance portability and accountability act ( hipaa ) privacy rule . 
janz nk , li y , zikmund - fisher bj , et al : the impact of doctor - patient communication on patients perceptions of their risk of breast cancer recurrence . 
forest plot ( adjusted odds ratios and 95% cis ) showing results of multivariable logistic regression for higher impact of cancer worry ( complete case n = 1 , 044 )  . 
 genetic testing for prostate cancer in clinical practice see accompanying article doi : there is increasing recognition of the role of inherited single - gene high - penetrance mutations in prostate cancer predisposition . 
in some ways , we are now learning for prostate cancer what has been known in the breast and ovarian cancer communities for at least two decadesthat patients with inherited mutations in genes such as brca1 and brca2 represent a clinically meaningful subset and cause a sufficiently high proportion of lethal cancers to warrant consideration of genetic testing in the appropriate clinical context ( eg , early - onset diagnosis )  . 
10% of men with metastatic prostate cancer have inherited mutations in dna repair genes.1 - 4 mutations were also found in approximately 5% of men with highrisk localized disease , with far lower prevalence in low - risk indolent cancers.2 , 3 in parallel , there is strong emerging evidence that these mutations may predict response to poly - adp ribose polymerase ( parp ) inhibitors and platinum - based chemotherapy.5 - 7 these findings make genetic testing appealing for the purpose of treatment selection and risk stratification , and testing is readily accessible , with widespread commercial availability of clinical multigene cancer predisposition panels . 
however , we have incomplete knowledge about who is best to test , when to test , and , importantly , how to counsel patients on the results with regard to their personal and family cancer risks . 
for example , testing may be ordered for the primary purpose of treatment selection without adequate preand post - test counseling by oncologists or urologists who are unaccustomed to genetic testing . 
for these reasons , there is urgency to better understand issues related to genetic counseling of men with prostate cancer who undergo cancer predisposition genetic panel testing . in the article that accompanies this editorial , giri et al8 describe the results of the genetic evaluation of men ( gem ) study . 
mutations were detected in genes related to dna repair , including brca1 , brca2 , atm , brip1 , msh6 , and mutyh . the relatively common chek2 p.i157t lowpenetrance risk variant was excluded from the analysis . the study has several findings that are important to inform genetic counseling in men . 
first , there was a significant association of a family history of breast cancer and being a pathogenic mutation carrier , confirming findings from earlier studies and highlighting the importance of both asking men about family history of breast cancer and counseling men with a strong family history of breast cancer of potentially increased risk for inherited mutations also predisposing to prostate cancer . 
third , mutations may be detected in at - risk unaffected men in genes other than brca1 and brca2 for which there are no established prostate cancer risk estimates or screening guidelines . 
the two mutations detected in this context were in msh6 , a mismatch dna repair gene associated with lynch syndrome , and in brip1 , a homologous recombination dna repair gene best established in ovarian cancer predisposition . 
even for brca1 and brca2 mutation carriers , the guidelines have yet to be firmly established , with the national comprehensive cancer network guidelines committees for prostate cancer early detection and for breast / ovarian cancer genetic high - risk assessment currently providing slightly different screening recommendations.9 , 10 the ongoing international impact ( identification of men with a genetic predisposition to prostate cancer ) prostate cancer screening trial is expected to provide valuable data to inform screening guidelines for brca1 and brca2 mutation carriers and has begun to enroll men with mismatch dna repair gene mutations.11 however , management for at - risk , unaffected men with mutations in genes such as brip1 will need to rely on clinical findings and family history until more data are collected . large - panel genetic testing may be associated with a high rate of variants of uncertain significance ( vus )  . more than one third of men in the study ( 70 of 200 , 35% ) had a vus reported back . 
vus reports may be particularly problematic when communicated to a patient by a nongenetics expert , because of the risk that the result is misinterpreted as a positive or actionable mutation finding . 
it is generally accepted that patients with vus should be managed solely on the basis of clinical findings and family history , similar to the case of a negative genetic testing result . 
vus rates vary among laboratories performing multigene cancer predisposition testing , so it is important to learn the expected vus rate in the performing laboratory to appropriately counsel men undergoing testing . 
fortunately , vus rates in cancer predisposition panel reporting are decreasing as more information is learned about newer genes and variant classification procedures are updated . this study has important limitations . 
it is therefore difficult to draw conclusions on the prevalence of mutations in most of the subgroups , which do not approach statistical significance . a much larger cohort will be needed to answer some of these important questions . 
there are known prostate cancer predisposition variants such as hoxb13 p.g84e that were not tested , and the authors note that they plan to follow up with hoxb13 testing.12 in addition , mutyh heterozygous carriers were included among the 11 mutation - positive patients . 
a recent study did not find an association of mutyh carrier mutations and prostate cancer.13 we are beginning to see increased cancer predisposition panel testing for men in clinical practice , largely driven by the excitement of tailoring treatments for prostate cancer . 
hart sn , ellingson ms , schahl k , et al : determining the frequency of pathogenic germline variants from exome sequencing in patients with castrate - resistant prostate cancer . 
na r , zheng sl , han m , et al : germline mutations in atm and brca1 / 2 distinguish risk for lethal and indolent prostate cancer and are associated with early age at death . 
mateo j , carreira s , sandhu s , et al : dna - repair defects and olaparib in metastatic prostate cancer . n engl j med 373 : 1697 - 1708 , 2015 8 . 
bancroft ek , page ec , castro e , et al : targeted prostate cancer screening in brca1 and brca2 mutation carriers : results from the initial screening round of the impact study . 
shang z , zhu s , zhang h , et al : germline homeobox b13 ( hoxb13 ) g84e mutation and prostate cancer risk in european descendants : a meta - analysis of 24 , 213 cases and 73 , 631 controls . 
 characterization of the epidermal growth factor receptor t790m mutation in colorectal cancer introduction the epidermal growth factor receptor ( egfr ) t790m mutation is a commonly found mutation encountered in approximately 60% of patients with nonsmall - cell lung cancer ( nsclc ) .1 it is the most frequent cause of resistance to anti - egfr therapy in nsclc.1 the biology behind the resistance is that the egfr t790m mutation increases the affinity of egfr for atp and attenuates the binding affinity of first generation anti - egfr tyrosine kinase inhibitors ( tkis ; gefitinib ) as well as second - generation anti - egfr tkis ( dacomitinib ) .1 , 2 however , third - generation anti - egfr tkis ( osimertinib ) were found to be more potent against egfr t790m - mutant cancer1 , 3 and are currently approved for patients with egfr t790m mutationpositive nsclc.4 in contrast , the egfr t790m mutation is rarely found in colorectal cancer ( crc )  . 
in this study , we are the first to our knowledge to report and characterize a patient with an egfr t790m - mutated crc . adenocarcinoma , and pathology of the anorectal mass was intramucosal adenocarcinoma ( fig 1b )  . given the concern for potentially three primaries , the patient underwent a right lower lobe segmentectomy with pathology revealing poorly differentiated adenocarcinoma . 
finally , a right hemicolectomy was performed and demonstrated a low - grade mucinous adenocarcinoma , invading through the muscularis propria into pericolonic rectal tissues ; 0 / 25 lymph nodes did not contain cancer ( stage pt3n0 )  . 
biopsy of the colonic mass was consistent with moderately differentiated characterization of the egfr t790m mutant tumor our patient presented with four primary cancers ( breast , lung , colon , and rectal ) , and we were able to obtain samples on the breast , lung , and colon specimens for additional studies . 
 ( a ) positron emission tomography ( pet ) scan images showing ( i ) breast , ( ii ) colon , and ( iii ) lung tumors of the patient . 
photomicrographs of the colonic adenocarcinoma demonstrated infiltrative glands lined in some areas by pseudostratified dysplastic columnar epithelium consistent with colonic origclassic features of primary adenocarcinoma of the colon , including intraluminal necrosis and mucin production , were also evident . 
using our duke next - generation sequencing panel ( ionampliseq cancer hotspot panelv2 ) , which covers more than 2 , 800 cosmic ( catalogue of somatic mutations in cancer ) mutations from 50 cancer genes , all three primaries were found to have the egfr t790m mutation ( fig 1c ) , with allele frequencies of 42% to 52% , suggesting a possible germline origin for this mutation . 
in addition , the breast cancer was found to have a pik3ca ( n345k ) mutation ; the lung primary had erbb4 ( l933f ) , gnaq ( i226v ) , and tp53 ( n239s ) mutations ; and the colon primary had abl1 ( g259c ) , apc ( s1465wfs ) , and braf ( v600e ) mutations ( fig 1c )  . 
these results suggest that these tumors are biologically distinct . development of patient - derived models of cancer because the three tumors seemed biologically distinct , we decided to further characterize these tumors by generating patient - derived models of cancer ( patient - derived xenografts [ pdx ] and matched cell lines ) , as previously described.5 , 6 of the three primaries , we were only able to obtain patient - derived models of cancer from the colon cancer specimen . 
 ii oxaliplatin ( ic50 undefined ) irinotecan ( ic50 0.28 ( cid : 80 ) m ) fluorouracil ( ic50 4.3 ( cid : 80 ) m ) 1 , 000 log ( dose , nm ) control oxaliplatin control irinotecan control fluorouracil 1 , 500 1 , 000 1 , 500 1 , 000 treatment ( days ) treatment ( days ) treatment ( days ) fig 2 . 
 ( a ) histologic features of the patients ( i ) colon tumor , ( ii ) patient derived xenograft model , and ( iii ) matched cell line image . 
 ( b ) in vitro ic50 ( half maximal inhibitory concentration ) studies with oxaliplatin , irinotecan , and fluorouracil suggesting that patients colon tumor was resistant to oxaliplatin , sensitive to irinotecan , and indeterminate to fluorouracil . 
ic50 values of the three cytotoxic chemotherapeutic agents ( oxaliplatin , irinotecan , and fluorouracil ) were found to be 25 m , 0.2 m , and 4.3 m , respectively ( fig 2b )  . 
although our in vitro data were indeterminate for fluorouracil , our in vivo data suggest sensitivity to fluorouracil ( fig 2c )  . next , we performed in vitro ic50 studies with the three us food and drug administration approved egfr inhibitors ( cetuximab , erlotinib , osimertinib ) to determine the effect of antiegfr therapy in our egfr t790m mutant colon tumor . 
ic50 values were found to be 25 m ( cetuximab ) , 2 m ( erlotinib ) , and 0.38 m ( oximertinib ) , respectively ( fig 3a ) , suggesting that the tumor was resistant to cetuximab and erlotinib and sensitive to osimertinib . 
similar to nsclc , where egfr t790m mutation is resistant to erlotinib and sensitive to osimertinib , 7 our pdx tumor was also found to be resistant to erlotinib and sensitive to osimertinib ( fig 3b )  . the matched cell line was then used for high - throughput drug screening to identify drug sensitivity to 29 egfr inhibitors . 
 ( a ) in vitro ic50 studies ( half maximal inhibitory concentration ) with anti - egfr therapeutics suggesting that osimertinib , a third generation egfr inhibitor , caused more cell inhibition than cetuximab and erlotinib . 
her braf mutation indicated that she does not have lynch syndrome , but given the genetic disposition of the egfr t790m mutation in lung cancer9 and her family history ( patient has a brother with brain cancer , sister with brain and lung cancer , sister with lung cancer , and sister with throat cancer ) , it remains to be seen whether this mutation is acquired in crc . 
to date she remains disease free , although she has some indeterminate small lung nodules that are currently being followed . the ability to biobank her cancers allowed us to develop patient - derived models of cancer to investigate the biology of her cancer and to identify potential therapeutic agents . 
furthermore , her cancer allowed us to develop a precision medicine pipeline , where a patients cell lines can be used to perform in vitro drug screen with in vivo validation to identify the most beneficial therapy . 
specifically , our preclinical modeling suggests that her tumor is sensitive to irinotecan and fluorouracil but resistant to oxaliplatthis would suggest that if she were to experience recurrence with metastatic disease , we should consider an irinotecan - based therapy for her initial therapy . in addition , we identified osimertinib as an effective anti - egfr therapeutic for the treatment of her colon tumor . 
finally , high - throughput drug screening allowed us to test 29 different anti - egfr agents , including first - , second - , and third generation egfr inhibitors . 
similar to the studies on egfr t790m mutationpositive nsclc , 3 , 10 we observed sensitivity to the third - generation egfr inhibitors and resistance to the first - generation egfr inhibitors , suggesting that egfr t790m mutation in crc shows similar biologic features with nsclc . 
high - throughput drug screening using patient derived cell lines revealed sensitivity and resistance to a number of first - , second - , and third - generation epidermal growth factor receptor inhibitors . in conclusion , the development of a precision medicine pipeline using matched cell lines and pdx models can be a powerful tool to understand tumor biology and to test new therapies , and this approach can be used to quickly translate bench findings to the bedside and guide patient care . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . erdem altunel no relationship to disclose abed alhalim aljamal no relationship to disclose kristen deak consulting or advisory role : lineagen wayne glover no relationship to disclose shannon j . 
mccall no relationship to disclose michael datto no relationship to disclose john strickler stock and other ownership interests : oncomed consulting or advisory role : amgen , genentech , bayer , celgene , chugai / roche , astrazeneca , seattle genetics research funding : abbvie ( inst ) , gilead sciences ( inst ) , genentech ( inst ) , exelixis ( inst ) , oncomed ( inst ) , sanofi ( inst ) , regeneron ( inst ) , medimmune ( inst ) , cascadian therapeutics ( inst ) , seattle genetics , macrogenics , leap therapeutics , nektar therapeutics david s . 
saad n , poudel a , basnet a , et al : epidermal growth factor receptor t790m mutation - positive metastatic non - small - cell lung cancer : focus on osimertinib ( azd9291 )  . 
odogwu l , mathieu l , goldberg kb , et al : fda benefit - risk assessment of osimertinib for the treatment of metastatic non - small cell lung cancer harboring epidermal growth factor receptor t790m mutation . 
pao w , miller va , politi ka , et al : acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the egfr kinase domaplos med 2 : e73 , 2005 8 . 
hu y , alden rs , odegaard ji , et al : discrimination of germline egfr t790m mutations in plasma cell - free dna allows study of prevalence across 31 , 414 cancer patients . 
barnes ta , okane gm , vincent md , et al : third - generation tyrosine kinase inhibitors targeting epidermal growth factor receptor mutations in non - small cell lung cancer . 
 appendix generation of the patient - derived xenografts and cell line . tumor sample was obtained under a duke institutional review boardapproved protocol ( pro00002435 ) , and written informed consent was provided vy the patient to participate in the study . 
all mouse experiments were performed in accordance with the animal guidelines and with the approval of the institutional animal care and use committee at duke university medical center . patient - derived xenograft ( pdx ) models were generated by injecting tumor samples into the flanks of 8to 10 - week - old jax nod.cb17 - prkdcscid - j mice obtained from the duke university rodent genetic and breeding core . 
to develop matched pdx cell line , harvested pdx tumor was homogenized and grown in 10 - cm2 tissue culturetreated dishes in cell culture media ( dmem media , 10% fetal calf serum , 1% penicillin and streptomycin ) at 5% co2 . 
cell line was authenticated using the duke university dna analysis facility human cell line authentication service by analyzing dna samples from each individual cell line for polymorphic short - tandem repeat markers using the geneprint 10 kit from promega ( madison , wi )  . high - throughput drug screening and in vitro ic50 studies ( half maximal inhibitory concentration )  . to perform high - throughput drug screening , robotic systems ( echo acoustic dispenser [ labcyte , san jose , ca ] for drug addition , well mate [ thermo fisher , waltham , ma ] for cell plating , and clarioscan [ bmg labtech , cary , nc ] for plate reading ) were used in each step of the screening process . 
a total of 384 well plates were first coated with a total of 2 , 100 drug compounds at a final concentration of 1 m , and then 1 , 000 cells / well were plated in the drug - precoated wells . 
three replicates were used for each drug . to perform ic50 studies , the cell lines were cultured in dmem plus 10% fetal bovine serum plus 1% penicillin / streptomycin and plated in drug - free medium at a concentration of 3 , 000 cells / well in 96 - well plate . 
 ic50 values were calculated using graphpad prism software ( la jolla , ca )  . in vivo drug sensitivity assays . mouse experiments were carried out in accordance with the animal guidelines and with the approval of the institutional animal care and use committee at the duke university medical center . 
to test drug sensitivities for each drug , 150 l of homogenized pdx tissuephosphate - buffered saline suspensions at 150 mg / ml concentration were subcutaneously injected into right flanks of five female and five male mice ( jax nod.cb17 - prkdcscid - j , 10 weeks old )  . 
randomly selected mice were treated with erlotinib ( 25 mg / kg ) or osimertinib ( 10 mg / kg ) orally five times a week ; oxaliplatin ( 10 mg / kg intraperitoneally per week ) , irinotecan ( 10 mg / kg intraperitoneally per week ) , fluorouracil ( 50 mg / kg intraperitoneally twice a week ) , and cetuximab ( 40 mg / kg intraperitoneally five times a week )  . 
hyman , md1 , 2 purpose matching patients to investigational therapies requires new tools to support physician decision making . we designed and implemented precision insight support engine ( precise ) , an automated , just - in - time , clinical - grade informatics platform to identify and dynamically track patients on the basis of molecular and clinical criteria . 
median time from sequencing to enrollment was 163 days ( interquartile range , 66 to 357 days ) , and from precise identication to enrollment 87 days ( interquartile range , 37 to 180 days )  . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction genomic data are increasingly used to guide both routine and investigational treatment decisions for patients with cancer . 
simultaneously , clinically validated broad next - generation sequencing platforms that permit the detection of multiple potentially actionable alterations are now accessible at the point of care.1 , 2 these improvements , however , have not been accompanied by commensurate advancements in the systems that are available to help physicians interpret and act on these data . indeed , previous experience with patients with advanced solid tumor who undergo genome proling suggests that only a minority5% to 24% , depending on the breadth of sequencing and practice settingare subsequently enrolled in genome - matched trials.3 - 8 numerous challenges exist in identifying and enrolling patients in appropriate genome - driven clinical trials . clinicians must correctly interpret sequencing data not only at the time of the initial results , but also longitudinally , as our understanding of genomic biomarkers and associated clinical evidence continuously evolves.9 enrolling patients in trials also requires access to , and up - to - date knowledge of , a changing portfolio of studies , each with its own study - specic eligibility criteria and dynamic slot availability . 
finally , all of this information must be readily accessible to the clinician at critical decision points in a patients care . to address these challenges , multiple strategies have emerged to facilitate matching patients to genomedriven clinical trials ( appendix table a1 )  . 
 tao et al context key objective new strategies to facilitate matching patients to genome - driven trials are needed to support physician decision making . precision insight support engine ( precise ) is an automated , just - in - time , clinical - grade informatics platform that identies and dynamically tracks patients on the basis of molecular and clinical criteria . 
to our knowledge , this represents the rst effort to evaluate outcomes of a bioinformatics patienttrial matching platform . knowledge generated precise identied nearly one half ( 43% ) of all enrollment in early - phase , genome - driven studies across a wide variety of tumor types and molecular alterations , with a 5 - month median time from sequencing to enrollment . 
a major area for improving matching efcacy is better integration of nonstructured , clinical eligibility criteria . relevance use of automated bioinformatics platforms represents an important means by which to increase clinical trial accrual and deliver precision oncology care to patients . straightforward of these approaches involves directly annotating molecular sequencing reports at initial sign - out to indicate the clinical signicance of each alteration and list potentially suitable clinical trials . 
multiple initiatives are underway to harmonize the annotation of individual variants and incorporate multitiered levels of evidence for actionability.10 - 12 some commercial laboratories and academic institutions have created on - demand molecular tumor boards that review molecular sequencing reports to make treatment recommendations and manually curate existing clinical trials for alterations of interest.13 all of these approaches have potential limitations . 
similarly , molecular tumor boards are time consuming , difcult to scale , and potentially inuenced by participants knowledge base and anecdotal experience . in response to these limitations and to address the ongoing unmet need of matching patients to clinical trials at our center , we created precision insight support engine ( precise ) , an automated , just - in - time , clinical - grade informatics platform to identify and dynamically track patients on the basis of molecular and clinical criteria . precise was designed to empower clinical investigators to proactively identify and recruit optimal candidates to their genome - driven trials . 
initially , limited proling data were generated via a mass spectrometrybased hotspot assay covering eight genes ( massarray ; sequenom , san diego , ca ) or an amplicon - based next - generation sequencing test covering 48 genes ( truseq amplicon cancer panel ; illumina , san diego , ca ) .14 a customized hybrid capture - based next - generation sequencing assay ( mskimpact ) was later performed per previously published methods.15 msk - impact can identify all classes of genomic alterationssingle - nucleotide variants , indels , copy number alterations , and select structural rearrangementsin up to 468 genes , depending on the assay version . 
these data are used for clinical report sign - out and delivery to the data warehouse that precise uses for patient identication.16 , 17 sequenced patients had cancer types for which proling was considered routine at the time performed or underwent clinical testing under a prospective institutional review boardapproved protocol that was designed to evaluate the utility of proling in these cancer types ( clinicaltrials.gov identier : nct01775072 )  . precise platform the precise platform was available to any principal investigator of an irb - approved therapeutic study that included molecular eligibility criteria . 
 outcomes of a physician support system for genome - driven oncology the functionality of the precise platform evolved during the study period on the basis of user feedback ( fig 1 )  . 
beginning in 2014 , precise was enhanced to offer notications , customized per study , of potentially clinically important events that could prompt a treatment change , such as upcoming appointments or restaging scans . 
for most of the study duration , precise notications were provided directly to the research team , which empowered them to further screen potential patients for appropriateness and notify treating physicians accordingly . 
use of this direct - to - primary oncologist notication function was at the discretion of the research teaall notications to the research team and the primary oncologist were generated as emails and did not directly integrate with the electronic medical record . cohort eligibility , data collection , and statistical analysis all precise cohorts for early - phase studiespilot , phase i , and phase i and iithat included at least one genomic eligibility criterion and that were created after april 16 , 2014 , were included for analysis . 
an internally developed mpath application was used to obtain the date that sequencing data was signed out and entered into the medical record for each patient , as well as the specic qualifying genomic alteration present . the msk institutional review board evaluated and approved a retrospective research protocol to evaluate precise platform outcomes . 
primary outcome measure was to determine what proportion of patients who were enrolled in the evaluated genome - driven studies was facilitated by precise . for the purpose of this analysis , enrollment was considered to be facilitated if precise identied the patient as eligible and generated a notication to either the research team or the primary oncologist before the enrollment date . 
to identify areas for future precise functionality enhancement , reasons for the failure of precise identify eligible patients before study enrollment were also explored through manual record review . in the relevant results study characteristics a total of 41 therapeutic trials used precise for genomic matching during the study period ( table 1 )  . 
the initial iteration ( version 1 ) of precise involved generating cohorts on the basis of complex genetic and clinical criteria dened by the studys principal investigator ( pi ) , which could then be sent to the pi at dened intervals . 
the capability of precise was later enhanced ( version 2 ) to enable pi notications triggered by certain events of interest , such as an upcoming patient appointment or computed tomography scan . 
present day ( version 3 ) precise can also incorporate a patients prior treatment history and allows for direct notication of the patients treating oncologist that a patient may be eligible for a study , often prompting an exchange between the treating oncologist and pi that initiates the patients future enrollment . 
of investigational agents antibody ( nonimmunotherapy ) therapeutic class * small molecule immunotherapy tumor type eligibility multiple breast lung ( nonsmall cell ) lymphoma mesothelioma glioma sarcoma prostate thoracic breast 5 - 10 10 - 15  . 
eight of 41 trials used the direct - to - oncologist notication system . principal investigator characteristics the 41 therapeutic trials were led by 19 unique principal investigators ( pis ) who individually led between one and ve studies ( table 1 )  . 
median time since the completion of terminal subspecialty training for pis was 7 years ( range , 2 to 27 years )  . patient matching during the study period , a total of 755 patients who were treated primarily by 150 unique oncologists were enrolled in 41 trials . 
precise prospectively identied 43% ( 327 of 755 ) of cases before patient enrollment , successfully notifying study investigators and / or the primary oncologist of the potential match ( fig 2 )  . 
reecting eligibility criteria among the trials , breast cancer ( 76 [ 23% ] of 327 ) and lung cancer ( 55 [ 17% ] of 327 ) were the most common tumor types among enrolled patients identied by precise . 
accounting for patients with more than one eligible molecular alteration , erbb2 ( 71 [ 21% ] of 335 ) was the most common genomic alteration , followed by pik3ca ( 47 [ 14% ] of 335 )  . at the individual protocol level , the percent of patients identied by precise before enrollment ranged from 0% to 100% . 
patients in whom the genomic alteration that ultimately led to enrollment did not meet the pre - established precise molecular criteria , as dened by the investigator , accounted for 33% ( 140 of 428 ) of missed cases . 
a lack of internal sequencing at the time of accrual accounted for 30% ( 127 of 428 ) of missed cases , predominantly among patients who enrolled on the basis of genomic proling that was performed outside the institution and was therefore not available for parsing by precise . 
another 23% ( 100 of 428 ) of cases was missed because precise cohort criteria that would have included the patient were amended only after the time of patient enrollment . 
several other technical and clinical reasons accounted for the remaining 14% ( 61 of 428 ) of cases , the majority of which ( 50 of 61 ) consisted of patients who did not meet clinical criteria available for * the total adds up to more than 41 as studies may include more than one agent class . twelve principal investigators had dual afliations with a diseasespecic group and the phase i group . one half ( 22 [ 54% ] of 41 ) of studies were phase i , and 61% ( 25 of 41 ) included multiple tumor types . 
of diseasespecic studies , the most common tumor types included breast cancer ( ve [ 12% ] of 41 ) and nonsmall - cell lung cancer ( four [ 10% ] of 41 )  . 
 ( b ) each column depicts patient enrollments by study principal investigator , with patient enrollment facilitated by precise shaded in blue and patient enrollment not facilitated by precise shaded in red . 
 ( c ) each column represents a unique study , with the absolute number of patients in each category labeled above ( non - precise ) and below ( precise enrollment ) each column . capture by precise , such as presence of triple - negative breast cancer , which was not always readily documented in the medical record . to further understand how physicians and patients use tumor genomic data to guide investigational treatment decisions , we evaluated the timing of enrollment relative to two important milestones : the completion of sequencing and the initial precise identication . 
upon evaluation of the 327 patients who were identied by precise and successfully enrolled in a genome - driven study , there was signicant variability in time intervals between these three events at the individual patient level . 
median time from sequencing to therapeutic enrollment was 163 days ( interquartile range , 66 to 357 days ; range , 5 to 1 , 281 days ) and from precise identication to enrollment 87 days ( interquartile range , 37 to 180 days ; range , 1 to 850 days ; fig 3 )  . 
reasons for delay from sequencing and precise identication to study enrollment included the availability of alternative routine therapy or a lack of need for treatment among patients without evidence of active disease . causes of patient attrition before enrollment to better understand the reasons why patients who were identied by precise did not subsequently enroll in the relevant therapeutic study , a representative cohort involving a multitumor phase i and ii expansion study of a targeted small molecule was selected for manual record review . 
of these 98 patients , 22 were immediately determined to be permanently ineligible or excluded as a result of a static characteristic that rendered the patient ineligible for the trial indenitely . 
these included having a second primary cancer ( n = 6 ) , a nonqualifying malignancy ( n = 6 ) , being deceased but not listed as such in the medical record ( n = 5 ) , and other reasons ( n = 5 )  . 
another 45% ( 10 of 22 ) involved a data element that was sometimes availablesecond primary cancer , prohibited prior therapy , and hiv / aidsfor use but not under all circumstances . 
for example , exclusions on the basis of prior therapy will miss agents administered at other medical centers or some oral anticancer agents , especially if dispensed by third - party pharmacies . after permanent exclusions , 76 potentially eligible patients remained . 
upon manual review , 78% ( 59 of 76 ) of patients did not immediately qualify for treatment at the time of the initial precise identication on the basis of not requiring active therapy ( n = 44 ) or ongoing response to current therapy ( n = 15 )  . 
collectively , 84% ( 59 of 70 ) of these temporary exclusion criteria are not consistently available for use by precise , primarily because the current disease status of the patient is not captured as a structured data element in the medical record . 
 a 2 , 000 1 , 500 1 , 000 500 1 , 000 1 , 500 outcomes of a physician support system for genome - driven oncology 1 , 400 1 , 200 1 , 000 sequencing to enrollment identification to enrollment patient sequencing identified by precise enrollment fig 3 . 
each series of three dots on a single vertical axis represents a single patients course , from initial sequencing ( blue dot ) to identication by precise ( precision insight support engine ; red dot ) to enrollment in the study ( gray dot )  . 
 ( b ) box and whisker plot showing the interval of time from sequencing to enrollment in the study ( left ; median , 163 days ) and from identication by precise to enrollment in the study ( right ; median , 87 days )  . therapies , rapidly deteriorating , or deemed inappropriate because of other issues , such as prior nonadherence . 
reasons for permanent or temporary exclusion from study qualication are depicted in blue ( criteria that is available for use by precise ) , red ( criteria that is sometimes available and could potentially be captured as an extractible structured data element ) , or teal ( criteria that is not yet available for use by precise )  . 
to our knowledge , this report represents the rst effort to evaluate real - world outcomes of an automated , just - in - time , clinicalgrade informatics platform to facilitate patient matching . we also discovered that a signicant time interval often elapses between the initial generation of tumor genomic data and subsequent enrollment in a matched study . specically , among those who ultimately accrue to a matched study , median duration from data generation to enrollment was 5 months , with some outliers enrolling up to 42 months later . 
these data emphasize the importance of supporting physician decision making longitudinally through a patients entire treatment . although these pilot data are encouraging , our analysis also identied areas of ongoing challenge for such matching systems as precise . 
of importance , nearly three quarters of initially identied patients were not immediately eligible on the basis of clinical factors that were not readily available for use by precise , most commonly related to challenges in algorithmically dening patient disease status . 
the result is that precise had a high false - positive rate that may ultimately limit the utility of this system for some indications , particularly for recruiting patients with more prevalent genomic alterations . 
indeed , a previous analysis of overall match rates at our institution that was conducted during roughly the same time period found a match rate of only 11% , despite 37% of patients harboring a potentially actionable alteration.5 taken together , these data demonstrate that even with the use of a sophisticated decision support system , there is an ongoing need for additional improvement in the methodologies to match patients to clinical trials on the basis of tumor genomic and clinical information . 
this requires developing agreed - upon standards , including discrete , structured criteria , for extracting clinical data from the electronic medical record . in the future , leveraging natural language processing and information extraction technologies may also enhance the ability to accurately capture unstructured data . this analysis has some important limitations . 
foremost , we considered any patient about whom precise successfully notied the pi or treating physician of potential eligibility before enrollment as an accrual that was potentially facilitated by the systehowever , we cannot determine exactly how many of these enrollments might have occurred without the use of this systeindeed , our study was retrospective in design and we cannot denitively make conclusions on the incremental value of precise . 
moreover , this analysis represents real - world use of precise by each pi , who were responsible for setting his or her own cohort criteria and using the results as he or she felt best complimented the practice . 
additional evaluation is needed to determine how the utility of this system is affected by recent features , such as full automation of direct - to - treating physician alerts triggered by critical events , like scans that show progression or rising tumor markers . precise is not the only system designed to address the emerging need of matching patients to relevant clinical trials on the basis of the patients genomic prole . 
several other centers have developed strategies by which to achieve this goal that range from on - site or virtual molecular tumor boards to automated matching platforms13 , 18 - 23 ( appendix table a1 )  . 
potential advantages of an automated informatics approach include scalability and the ability to track patients longitudinally and respond to changing molecular and clinical data . in summary , this pilot study of real - world use of precise to guide enrollment in genome - driven studies suggests that this type of real - time decision support system can meaningfully facilitate patient enrollment . 
harding consulting or advisory role : bristol - myers squibb , cytomx therapeutics , eli lilly , eisai research funding : bristol - myers squibb ( inst ) , pzer ( inst ) , eli lilly ( inst ) , novartis ( inst ) , incyte ( inst ) , calithera biosciences ( inst ) , polaris ( inst ) lillian m . 
smyth honoraria : astrazeneca , pzer consulting or advisory role : astrazeneca , genentech research funding : astrazeneca ( inst ) , genentech ( inst ) , puma biotechnology ( inst ) travel , accommodations , expenses : pzer , genentech , puma biotechnology research funding : novartis ( inst ) , genentech ( inst ) , debio pharmaceuticals ( inst ) , adc therapeutics ( inst ) , pzer ( inst ) , novita pharmaceuticals ( inst ) , clovis oncology ( inst ) , eli lilly ( inst ) , zymeworks ( inst ) travel , accommodations , expenses : taiho pharmaceutical , jounce therapeutics , pzer , astrazeneca other relationship : novartis , pzer , taiho pharmaceutical , jounce therapeutics alexander drilon honoraria : medscape , onclive , peervoice , physicians education resources , targeted oncology , more health , research to practice , foundation medicine , peerview consulting or advisory role : ignyta , loxo , tp therapeutics , astrazeneca , pzer , blueprint medicines , genentech , helsinn therapeutics , beigene , hengrui therapeutics , exelixis , bayer , tyra biosciences , verastem , takeda , ariad pharmaceuticals , millennium pharmaceuticals , bergenbio , more health , eli lilly research funding : foundation medicine patents , royalties , other intellectual property : wolters kluwer ( royalties for pocket oncology ) other relationship : merck , glaxosmithkline , teva pharmaceuticals , taiho pharmaceutical , pzer , pharmamar , puma biotechnology marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network / astrazeneca tagrisso rfp advisory committee , takeda , bristol - myers squibb , bayer , merck ( i ) research funding : loxo ( inst ) , helsinn therapeutics david b . 
solit stock and other ownership interests : loxo consulting or advisory role : pzer , loxo , illumina , intezyne technologies , vivideon therapeutics travel , accommodations , expenses : merck michael f . 
jhaveri consulting or advisory role : novartis , pzer , spectrum pharmaceuticals , astrazeneca , taiho pharmaceutical , jounce therapeutics , adc therapeutics , synthon , intellisphere , bristol - myers squibb acknowledgment the authors thank alisa pinkhasik for assistance with study data . 
for providing inspiration for this report . references 33 : 2753 - 2762 , 2015 schram am , berger mf , hyman dm : precision oncology : charting a path forward to broader deployment of genomic proling . 
plos med 14 : e1002242 , 2017 cheng ml , berger mf , hyman dm , et al : clinical tumour sequencing for precision oncology : time for a universal strategy . 
nat rev cancer 18 : 527 - 528 , 2018 jordan ej , kim hr , arcila me , et al : prospective comprehensive molecular characterization of lung adenocarcinomas for efcient patient matching to approved and emerging therapies . 
j clin oncol zehir a , benayed r , shah rh , et al : mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10 , 000 patients . 
 tao et al stockley tl , oza am , berman hk , et al : molecular proling of advanced solid tumors and patient outcomes with genotype - matched clinical trials : the princess margaret impact / compact trial . 
mantripragada kc , olszewski aj , schumacher a , et al : clinical trial accrual targeting genomic alterations after next - generation sequencing at a non - national cancer institute - designated cancer prograj oncol pract 12 : e396 - e404 , 2016 8 . 
massard c , michiels s , fert e c , et al : high - throughput genomics and clinical outcome in hard - to - treat advanced cancers : results of the moscato 01 trial . cancer discov 7 : 586 - 595 , 2017 schram am , reales d , galle j , et al : oncologist use and perception of large panel next - generation tumor sequencing . 
taylor ad , micheel cm , anderson ia , et al : the path ( way ) less traveled : a pathway - oriented approach to providing information about precision cancer medicine on my cancer genome . 
mateo j , chakravarty d , dienstmann r , et al : a framework to rank genomic alterations as targets for cancer precision medicine : the esmo scale for clinical actionability of molecular targets ( escat )  . 
ann oncol 29 : 1895 - 1902 , 2018 johnson a , zeng j , bailey am , et al : the right drugs at the right time for the right patient : the md anderson precision oncology decision support platfordrug discov today 20 : 1433 - 1438 , 2015 14 . 
rekhtman n , paik pk , arcila me , et al : clarifying the spectrum of driver oncogene mutations in biomarker - veried squamous carcinoma of lung : lack of egfr / kras and presence of pik3ca / akt1 mutations . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
lindsay j , fitz cdv , zwiesler z , et al : matchminer : an open source computational platform for real - time matching of cancer patients to precision medicine clinical trials using genomic and clinical criteria . 
tao j , eubank mh , pamer e , et al : precise : a clinical - grade automated molecular eligibility screening and just - in - time ( jit ) physician decision support solution for molecularly - selected oncology trials . 
notications are generated from scheduled queries and use an open - source java e - mail package implemented as a database user - dened function to send emails from the sql statement . 
a web application using open - source javascript libraries allows system administrators to capture cohort logic and settings and provides end users the ability to annotate patient status for their study . 
 ( a ) each column depicts patient enrollments by study pi , with pis with the most years in practice on the left and the fewest years in practice on the right . 
it also re - examines some of the conventional wisdom that previously dominated clinical trial design and discusses development and internal validation of a predictive biomarker as a new paradigm for optimizing the intended - use subset for a treatment . 
some previous paradigms for clinical trial design can limit the development of more effective methods on the basis of prospectively planned adaptive methods , but useful new methods have been developed for analysis of genome - wide data and for the design of adaptively enriched studies . 
in many cases , the heterogeneity of populations eligible for clinical trials as traditionally dened makes it unlikely that molecularly targeted treatments will be effective for a majority of the eligible patients . 
with the completion of the human genome project and the initiation of large dna sequencing of human tumors , somatic mutations were discovered that accounted for the invasion and progression of cancers . 
it became evident that the traditional tumor classications on the basis of site and histologic type were inadequate for developing therapeutics . the discovery of somatic mutations led to major changes in the development of oncology drugs . 
the focus of oncology drug development switched to developing inhibitors of mutated protein kinases and blocking the protein products of amplied receptors . the new strategies of drug discovery required new strategies for clinical trial design . 
the traditional assumption that qualitative interactions are unlikely was frequently shown to be wrong . at about the same time , new whole - genome assays such as rna transcript microarrays , copy number microarrays , and methylation arrays were becoming available . 
the resulting statistical methods were based on classication and prediction , which enabled the development of classiers on the basis of high - dimensional assays for predicting prognosis and response to therapy for individual patients.3 , 4 this article will highlight some of the important developments in clinical trial design and classication analysis that have been stimulated by the dramatic changes in oncology drug development . 
 simon context key objective to describe new and innovative methods for discovering and using predictive biomarkers to develop effective treatments in the presence of patient heterogeneity . knowledge generated a method was developed for discovering and validating therapeutically relevant patient subsets using a new paradigm on the basis of predictive classication rather than multiple hypothesis testing . 
with the development of drugs targeted to specic genomic alterations , attention has shifted to phase ii studies with eligibility determined by specic genomic alterations rather than histologic type , the so - called basket clinical trial.5 - 9 large basket trials include multiple drugs , each with a targeted genomic alteration . 
patients have their tumors sequenced , and then they are assigned the drug appropriate to the genomic alteration found in their tumor . basket trials have generally been sized by using standard methods either for inference on the basis of all patients with the genomic alteration histologic type or for separate inference for each histologic type . 
recently , bayesian designs have been developed that attempt to share response information among histology baskets for patients whose tumor contains the same genomic alteration.10 some of these are based on hierarchical models of treatment effects . 
however , cunanan11 has shown that the prior distributions for such designs must be chosen carefully or the false - positive error for some histology baskets may be highly inated . 
if the accumulating information indicates homogeneity of response probabilities , then the baskets are essentially pooled and a small sample size sufces . cunanan12 developed a frequentist design on the basis of two - stage sampling . 
otherwise , sampling and inference are conducted independently for the baskets . the platform design is for settings with multiple treatments and multiple candidate biomarker strata.13 the design is based on the assumption that the candidate biomarkers are the same for each drug , which somewhat limits the realm of applications . 
most platform designs are bayesian designs and the term promising is interpreted to mean that the posterior probability of a positive phase iii trial is at least 80% . in a platform design , a patient is initially randomly assigned with equal probability to receive any of the drugs . 
the objective is to nd candidates for phase iii trials with a smaller total sample size than would be required for conducting a separate simon two - stage optimal trial in each of the strata.14 the bayesian models used are generally based on hierarchical modeling.13 , 15 freidlin et al16 developed a simpler phase ii trial design for a single drug with a single binary biomarker . 
 statistical methods for biomarker - driven clinical trials simon and maitournam17 , 18 studied the number of patients needed to screen and randomize for the enrichment design compared with a standard design , in which the assay is not performed . 
they found that the enrichment design generally requires many fewer randomly assigned patients . they showed that the ratio of number of required randomly assigned patients was approximately ( cid : 1 ) nenrichment nstandard ( cid : 1 ) te + + ( 1 ) te ( cid : 3 ) 2 where te + and te are the treatment effects in mutationpositive and mutation - negative patients , respectively , and denotes the proportion of mutation - positive patients . 
this formula indicates that the randomization ratio equals the reciprocal of the square of the treatment effects in the two trial designs . if the treatment effect in mutation - negative patients is zero , then the randomization ratio equals 2 . 
if the treatment effect in the mutation - negative patients equals half that in mutationpositive patients , then the randomization ratio becomes { + ( 1 ) / 2 } 2 , so if = 0.5 , then the randomization ratio equals 9 : 16 , and the enrichment design requires slightly more than one half the number of patients as the standard design . adaptive determination of the intended - use population the efciency of the enrichment design highlights the critical relationship between the required size of a phase iii clinical trial and the target treatment effect . 
simon et al19 developed a focused approach for using such archived tissues called the prospective - retrospective study design , which was used for establishing that patients with advanced colorectal cancer do not benet from anti - egfr antibodies if their tumors contain a mutation in the kras gene.20 this approach was also used in a structured manner in the development of the oncotype dx recurrence score as a prognostic and predictive signature for patients with stage i and stage ii breast cancer.21 in addition to using archived samples from a failed randomized controlled trial ( rct ) , adaptive methods have been developed for prospective design of an rct in a statistically rigorous way to adaptively identify the subset of patients who benet from the test treatment . 
some of these adaptive methods will be discussed , but their use has required regulators and clinical trialists to accept that the intended - use population need not be the full eligible population . by prospectively allocating some of the type i error rate to a planned subset analysis , 22 we do not need to use the convention that subset analysis should be performed only if the all - comers analysis is signicant or if there is a statistically signicant interaction . 
statisticians and clinical investigators have sometimes been slow to shed some of the conventional wisdom of the previous paradigm , such as the requirement that the intended - use population must be the eligible population . adaptive enrichment single binary candidate biomarker . 
the most common type of adaptive enrichment in phase iii trials involves whether to use a binary candidate biomarker to restrict eligibility . initially patients are characterized with regard to the biomarker and then randomly assigned to the test treatment or control , but the biomarker is not used to restrict eligibility . 
at some point during the trial an interim analysis is performed and one of three decisions is made : ( 1 ) the entire trial is closed for futility , ( 2 ) the trial continues accruing without any restriction on eligibility until its originally planned sample size is reached , or ( 3 ) accrual is continued only for biomarker - positive patients . 
in the third case , the total target accrual may be increased to adequately power the nal analysis for biomarker - positive patients . several authors have proposed designs for the binary biomarker case described previously.23 - 32 they contain multiplicity adjustments to account for the fact that inferences are being made for the treatment effect overall and for biomarker - positive patients . 
in many cases , there is a quantitative or semiquantitative biomarker that is thought to be predictive of which patients are most likely to benet . often , previous studies have not reliably established the relationship between biomarker value and likelihood of benet , and there is no adequately determined cut point for positivity . 
a recent example is the programmed deathligand 1 ( pd - l1 ) level of expression on nonsmall - cell lung tumors as a predictor of response to t - cell checkpoint therapy . because of regulators requirements that sponsors demonstrate statistically signicant effectiveness of the treatment of an intended - use population , most sponsors want to have the intended - use population dened at the outset . this may require using a cut point selected on the basis of inadequate phase ii trials . 
 simon of the relationship between biomarker level and treatment effectiveness . biomarkers , not for settings in which whole - genome data are used for classier development . there are only a few cases in which investigators use an adaptive design to determine an optimal cut point on the basis of interim data , perhaps because they are unaware of the new statistical designs that may achieve this in a statistically valid manner . jiang et al34 showed how one could test the null hypothesis of no treatment effect using a minimal p value statistic for which a p value for treatment effect is computed for the cases above each of the candidate cut points . 
their proposed analysis is performed at the end of the trial using the full data set and is based on the null distribution of the min - p - value statistic . 
this approach is easy to implement because it does not require any changes during the trial . the intended - use population is adaptively determined at the end of the trial . is adaptive in that simon and simon35 illustrated the use of their adaptive enrichment approach for modifying eligibility during the trial on the basis of an interim analysis of the relationship of biomarker value to treatment effect . 
the signicance test preserves the type i error , regardless of the strategy used for modifying eligibility . although the method is frequentist , they advocate using a bayesian model for selecting features and building the classier at interim analyses.33 the bayesian model can also be used for selecting an intended - use population . one of the questions about using adaptive enrichment is how to estimate the treatment effect in the adaptively selected intended - use population in an unbiased manner . simon and simon36 described how to use bootstrap resampling in an unbiased manner to estimate treatment effects for the intended - use population using data from periods that also determined how to modify eligibility . 
one approach involves developing separate prognostic scores for the active treatment group ( eg , h ( x ; a ) for the log hazard ratio relative to baseline hazard for a patient with covariate vector x receiving the active treatment ) and the control group ( h ( x ; c ) )  . 
that is , if the hazard of a failure event on treatment a is less than the hazard of that event on the control by a tolerance of at least c for a patient with baseline covariate vector x , then the predictive biomarker is considered to have value 1 , and otherwise to have value 0 . 
instead of using proportional hazards modeling as suggested here , one could use other methods such as random forest modeling37 or any other method of prognostic modeling . a major feature of the new paradigm for subset analysis introduced by the adaptive signature design ( asd ) is that nding an appropriate intent - to - treat subpopulation should be treated as a classication problem , not as a hypothesistesting problefinding a subset with a large apparent treatment effect is of little value because the resubstitution estimate of treatment effect is so often optimistically biased . the asd and cross - validated asd ( cv - asd ) provide unbiased estimates and tests of such treatment effects . simon38 described sensitivity , specicity , positive predictive value ( ppv ) , and negative predictive value ( npv ) for predictive classiers with survival time outcome . 
when the survival time for active treatment versus control has proportional hazards for classier - positive and classier - negative patients , then the ppv can be written38 ppv ( cid : 1 ) 1 + + where + denotes the hazard ratio of control versus active treatment of classier - positive patients . 
us food and drug administration ( fda ) guidance on the use of enrichment strategies describes the asd in a positive manner.42 practical experience in designing a clinical trial using the asd is described by sher et al , 43 simon , 44 and mi.45 where + denotes the prevalence of classier - positive patients . 
the asd and cv - asd to be described in a later section were originally described in terms of a specic kind of classier for gene expression data , but the concept is quite general and will be described here in its generality . 
it is not limited to high - dimension genomic data or signatures of gene expression measurements ; is best used with a moderate number of candidate predictive biomarkers . in fact , at the nal analysis , the patients randomly assigned in the clinical trial are randomly partitioned into a training set and a validation set . 
a predictive classier is developed by using only training set data . with the asd , however , one is permitted to develop only a single predictive classier , and it must be completely specied before the validation set data are used in any way . finally , the patients in the validation set are classied as likely to benet from the active treatment ( f ( x ) = 1 ) or not ( f ( x ) = 0 )  . 
the set of covariate vectors for which f ( x ) = 1 is considered the intended - use subset s : s = { x : f ( x ) = 1 }  . although the asd provides a new paradigm for subset analysis of rcts , its statistical power is limited because of the sample splitting . 
freidlin et al46 introduced an improved method called the cv - asd with improved power establishing the signicance of the treatment effect in the adaptively determined intended - use subset . 
we denote the resulting predictive classier as c ( a , d ) where d denotes the complete data set . the resubstitution estimator of treatment effect is denoted ( c ( a , d ) , d ) where the operator computes the empirical treatment effect on the subset of d for which classier c is positive . 
this new training starts from scratch ; that is , it does not use the feature selection steps of development of the full sample classier c . let ck ( a , dk ) denote the classier developed on dk . 
i is the prevalidation classication of patient i . having developed a single predictive classier using training data , the next step is to evaluate the treatment effect for patients in the validation set with covariate vectors in s . 
outcomes of those who received active treatment are compared with outcomes of those who received the control . because the validation set was not used to develop the classier , this comparison of outcomes is unbiased . after completing all k - folds of the cross - validation , all of the cases have been classied once . 
these { i } are called prevalidated indicators of the intended - use subset.47 let iu = { i : i = 1 } , the intended - use subset on the basis of the prevalidated predictive classications . 
 simon empirical treatment effect in iu , and take that as an approximation for the treatment effect in using the full sample classier c ( a , d ) with new cases in the future . 
simulation studies have shown that this estimator is much better than the resubstitution estimator but somewhat conservative.46 a statistical signicance test for treatment effect among future patients classied positive using c ( a , d ) is approximated by computing the permutation distribution of ( iu )  . for each permutation of the treatment labels , the crossvalidation procedure is repeated giving a new iu , say iu ( cid : 4 ) , and a new value of ( iu )  . 
with many permutations of the treatment labels , one can approximate the null distribution . the null hypothesis here is that there is no subset for which there is a positive treatment effect . 
zhang et al48 describe an alternative approach to de - biasing the resubstitution estimator ( c ( a , d ) , d ) using bootstrap sampling instead of using prevalidated classication . other phase iii designs zhao et al49 describe a method for using the results of a previous clinical trial of a treatment and control to select a subset of patients for a new rct . 
for higher - dimensional data , the authors recommend sample splitting or cross - validation to separate the development of predictive scores from the evaluation of the outcome difference in the selected patients . 
the authors assume that feature selection does not use the clinical trial data because a partial cross - validation approach in which feature selection is not repeated for each resampling is described and can be highly biased when feature selection uses the data.50 foster et al37 describe a method very similar to the cv - asd for binary outcome data in which the random forest method is used for developing prognostic prediction function in the active treatment and control groups . 
they also investigate bootstrap resampling to estimate the treatment effect in the selected subset . cv - asds are quite general with regard to the type of models used for predicting difference in expected outcome between treatment groups as a function of the covariate vector . 
these treatment effect estimates may be biased if the assumed parametric model is misspecied , however . outcome weighted learning is an approach to developing approximately optimum individualized treatment rules that are robust to model misspecication.51 , 52 sequentially adaptive interventions are multistage treatto tailor a sequence of ment strategies that attempt treatment interventions to the characteristics of individual patients and their responses to previous treatments . 
such a score can be based on the difference between a prognostic risk function ft ( x ) trained on the treatment group and a prognostic risk function fc ( x ) trained on the control group , where x denotes the vector of measured covariates . 
using the k - fold cross - validation approach of the cv - asd , these functions can be developed on dk training sets and used to compute prevalidated predictive scores ft ( xi ; dk ) fc ( xi ; dk ) for the cases in the withheld sets dk . 
these prevalidated predictive scores si being quantiles are thus in the range ( 0 , 1 )  . matsui et al55 suggest that these prevalidated scores be used as the independent variable in a proportional hazards model containing a main effect of the treatment and an interaction between treatment and prevalidated predictive score quantile . 
for any test statistic involving the estimated treatment effect scores , the null hypothesis of no treatment effect can be performed by permuting the treatment labels and repeating the cross - validation procedure . 
predictions for future patients require rst computing the predictive index quantile score for the new patient using quantiles of the full data set and then plugging that value into the tted proportional hazards model . cai et al56 described a two - stage procedure for estimating a predictive score . 
for high dimensional their approach is probably best used with covariates , prevalidated predictive scores . discussion the development of personalized oncology has been a consequence of the discovery of somatic driver mutations in tumors . 
development of drugs and companion diagnostics for inhibiting driver mutations has provided many two decades . therapeutic successes during the past complex statistical methods have not been necessary for developing predictive biomarkers in these cases because the presence of is the biomarker . the mutation itself however , most somatic mutations are not driver mutations , and identication of predictive biomarkers in those cases can be much more difcult . 
consequently , the fda developed a regulatory pathway in which the diagnostic was not labeled as distinguishing patients who are likely to benet from the drug from those who are not , but rather for identifying patients who have a tumor containing the mutation that makes them eligible for the approved drug . 
during the past two decades , numerous drugs and companion diagnostics for patients with cancer have been approved on the basis of the enrichment design . the question of whether to restrict eligibility to a phase iii trial on the basis of a binary biomarker or on the basis of the value of a quantitative biomarker frequently arises in the design of pivotal trials . 
having larger phase ii trials would provide better evidence for designing phase iii trials , but often the phase iii design decisions are made on the basis of insufcient phase ii data . 
the adaptive designs described in which eligibility is not initially restricted but may subsequently be restricted to biomarker - positive patients on the basis of a prespecied interim analysis are widely applicable . 
more general adaptive enrichment designs with multiple candidate biomarkers can be effective if markers are sufciently powerful their effects on outcome can be discovered at interim analyses . that the asd and cv - asd are easy to use because they do not require any eligibility changes during the course of the trial . they are adaptive in the sense that the intended - use population is adaptively determined by the trial data . however , because they do not adapt eligibility , the opportunity for power gains is limited . 
the cv - asd has much better power and is appropriate for settings in which strong candidate biomarkers are not known at the start of the trial . analyzing phase iii trials by using a continuous score biomarker that reects relative treatment effectiveness can provide medically useful information without the arbitrariness of specifying a cut point . 
it is important , however , to internally validate the calibration of the scoring function by using resampling methods as described here . trials for nding optimal dynamic regimes for clinical multistage decision making have not yet found much application in oncology but have been used effectively in other areas . some of the conventional wisdom that has guided the design and analysis of phase iii trials is undergoing reevaluation . 
for example , the paradigm of broad eligibility and the use of subset analysis on the basis of multiple hypothesis testing require re - evaluation . is not always for molecularly targeted treatments , reasonable to expect a signicant treatment effect for all eligible patients dened by using conventional histology and stage criteria . 
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new york , ny , chapman and hall / crc , 2015 antonijevic z , beckman ra ( eds ) : platform trial designs in drug development : umbrella trials and basket trials . 
new york , ny , chapman and hall / crc , 2018 berry sm , broglio kr , groshen s , et al : bayesian hierarchical modeling of patient subpopulations : efcient designs of phase ii oncology clinical trials . 
clin trials 10 : 720 - 734 , 2013 simon rm : primary site independent clinical trials in oncology , in antonijevic z , beckman ra : platform trial designs in drug development : umbrella trials and basket trials . 
bokemeyer c , kohne c , rougier p , et al : cetuximab with chemotherapy ( ct ) as rst - line treatment for metastatic colorectal cancer ( mcrc ) : analysis of the crystal and opus studies according to kras and braf mutation status . 
paik s , tang g , shak s , et al : gene expression and benet of chemotherapy in women with node - negative , estrogen receptor - positive breast cancer . 
biom j 51 : 358 - 374 , 2009 jenniso c , turnbull bw : conrmatory seamless phase ii / iii clinical trials with hypotheses selection at interim : opportunities and limitations . 
stat med 32 : 2695 - 2714 , 2013 jenkins m , stone a , jennison c : an adaptive seamless phase ii / iii design for oncology trials with subpopulation selection using correlated survival endpoints . pharm stat 10 : 347 - 356 , 2011 30 . 
biostatistics 19 : 27 - 41 , 2018 jiang w , freidlin b , simon r : biomarker - adaptive threshold design : a procedure for evaluating treatment with possible biomarker - dened subset effect . 
bai x , tsiatis aa , lu w , et al : optimal treatment regimes for survival endpoints using a locally - efcient doubly - robust estimator from a classication perspective . 2003 lifetime data anal 23 : 585 - 604 , 2017 52 . 
 o use of low - frequency driver mutations detected by cell - free circulating tumor dna to guide targeted therapy in nonsmall - cell lung cancer : a multicenter case series purpose to evaluate the clinical outcome of patients with nonsmall - cell lung cancer treated by targeting low variant allelic frequency ( vaf ) driver mutations identified through cell - free dna ( cfdna ) next - generation sequencing ( ngs )  . 
detection of driver mutations in cancer is critically important in the age of targeted therapy , where both tumor - based as well as cfdna sequencing methods have been used for therapeutic decision making . 
we hypothesized that vaf should not be predictive of response and that low vaf alterations detected by cfdna ngs can respond to targeted therapy . patients and methods a multicenter retrospective case review was performed to identify patients with nonsmall - cell lung cancer who received targeted molecular therapy on the basis of findings of low vaf alterations in cfdna ngs . 
mutations at low vaf were defined as < 0.2% mutated cfdna molecules in a background of wild - type cfdna . results one hundred seventy - two patients underwent cfdna ngs testing . 
2018 by american society of clinical oncology introduction advances in the sensitivity and accuracy of plasma cell - free dna ( cfdna ) sequencing now allow for somatic genomic tumor alterations to be detected by cfdna next - generation sequencing ( ngs ) at the level of 0.02% variant allelic frequency ( vaf ) .1 this raises the question of whether targetable driver mutations that are detected at low frequencies , defined herein as < 0.2% vaf , retain importance even in the setting of other genomic alterations at higher vaf . 
 specific cases spanning each of the three major types of mutationssingle nucleotide variants ( snvs ) , insertion - deletion mutations ( indels ) , and fusionsare discussed . approximately 20% to 30% of tumors from patients with lung adenocarcinoma have targetable miriam t . 
 oncogenic driver mutations in alk , braf , egfr , erbb2 ( her2 ) , met , ret , and ros1.2 , 3 lung cancer patients with nonsmall - cell ( nsclc ) treated with targeted molecular therapies against these oncogenic driver mutations have been found to have prolonged survival compared4 , 5 with patients treated with standard chemotherapy , with improved treatment - related toxicity and quality of life.6 - 8 given these results , the national comprehensive cancer network now recommends that patients with metastatic nonsquamous nsclc have broad molecular profiling of their tumor performed , with cfdna as a secondary option if repeat tissue biopsy is not feasible.9 as tumor cells become necrotic and undergo apoptosis , they release circulating fragments of dna . 
with advances in ngs , it is now possible to detect cfdna from blood samples with significant accuracy and precision.10 , 11 three major approaches exist for analyzing cfdna in peripheral blood . 
the first uses probes to capture hotspots , or regions commonly mutated in selected genes ; an example of this approach is digital droplet polymerase chain reaction.12 the second uses ngs but requires a tumor tissue sample to identify specific mutations , which are used as a reference to select probes to specific regions in cfdna.13 , 14 the third method uses ngs but targets a gene panel of interest using complete exon sequencing in a blinded approach.15 , 16 mutations are quantified and represented as a vaf calculated as the total number of molecules with a given mutation divided by the total number of mutant plus wild - type molecules . detection of cfdna allows for tumor monitoring at disease progression via serial peripheral blood samples and , in the future , may eliminate the need for tumor biopsies or repetitive biopsies in patients with advanced cancer when the initial biopsy is inadequate , unobtainable for genomic testing , or uninformative , or when the patients cancer has progressed despite treatment.17 - 19 the quantity of cfdna has been shown to correlate with tumor staging and prognosis.17 , 20 limitations of cfdna include testing in patients with low disease burden and in patients with bone or brain metastasis , who may have quantities of cfdna below the limit of detection ( lod ) for currently available assays . 
although these false - negative results limit the application of cfdna ngs to a rule - in test , more than a dozen nsclc studies have shown that targetable genomic alterations detected in plasma predict response similarly to those detected in tissue.21 - 24 however , it is unclear whether variants at low allele fractions are as responsive to targeted therapy as those at high allele fractions . 
 samples were sent to a clinical laboratory improvement amendments ( clia ) certified , college of american pathologistsaccredited , new york state department of healthapproved clinical laboratory ( guardant health , redwood city , ca ) for clinical cfdna ngs testing ( guardant360 ) from one of three institutions ( university of california , san diego [ ucsd ] , duke university , or northwestern university )  . 
for this project , a vaf 0.2% was defined as low vaf because this is the 25th percentile vaf detected on guardant360 ( the median or 50th percentile is 0.39% ) in > 5 , 000 patient samples.1 medical records were reviewed in accordance with the respective institutional review board guidelines of ucsd , duke university , and northwestern university . 
response evaluation criteria in solid tumors ( recist v1.1 ) were used to assess response to treatment . tissue ngs individuals had tumor tissue from primary or metastatic sites sent for targeted tumor tissue ngs before or at approximately the same time as their cfdna analysis . 
tissue - based ngs was done at either ucsd , duke , foundation medicine ( foundation one ; cambridge , ma ) , or caris ( phoenix , az )  . 
the lower lod for this assay was 5% , meaning that a mutant allele can be detected in a 95% wild - type allele background ( or 10% neoplastic cell content )  . 
coverage of at least 250 was required to detect a mutation at the 5% lod . egfr sequencing , ros1 and alk fluorescent in situ hybridization testing neogenomics laboratories ( fort meyers , fl ) performed egfr sequencing , which included bidirectional sequencing of exons 18 to 21 of the egfr gene for detection of egfr - activating mutations and tyrosine kinase inhibitor resistance mutations ( including t790m )  . 
tumor enrichment was performed before extraction ( detailed information can be found on the neogenomics web site ) .29 ros1 and eml4 - alk fluorescent in situ hybridization ( fish ) testing was performed by response genetics ( los angeles , ca ) , and a positive test on fish was considered to be > 15% probe binding . plasma cell - free circulating dna analysis the cfdna ngs analysis was performed at guardant health ( guardant360 )  . 
the exons of up to 73 cancer genes were captured using biotinylated custom bait oligonucleotides ( agilent , la jolla , ca ) , resulting in a 148 , 000 base pair ( 78 kb ) capture footprint . 
these algorithms quantify the absolute number of unique dna fragments at a given nucleotide position , thereby enabling circulating tumor dna to be quantitatively measured as a fraction of total cfdna . 
because patients were not enrolled in a prospective clinical trial , assessments were not standardized across the cohort . results between september 2015 and september 2017 , six clinicians from the three institutions ( ucsd , duke university , and northwestern university ) identified 192 orders from 172 unique patients with a diagnosis of nsclc and in whom cfdna was detected . 
although pfs was roughly as expected in this small cohort , the median os of < 15 months is somewhat shorter than expected for egfrand alk positive patients , especially because the median pfs of patients receiving targeted therapies was 12 months . 
this finding of lower than expected os may be because of sample size , or it is possible that patients with driver mutations present at low vaf have a worse prognosis compared with patients with driver mutations at more standard frequencies . 
there may also be other negative prognostic factors associated with low vaf that are not yet known . met exon 14 skipping mutations represent a clinically unique molecular subtype of nsclc and are found in approximately 3% of all nsclc cases.32 met exon 14 mutations can be detected via tumor ngs or with cfdna ngs , which represents a novel detection method because currently , no other alternative detection modalities ( eg , immunohistochemistry or fish ) exist for this mutation . 
thus the met exon 14 skipping mutation can lead to increased activation of c - met , resulting in oncogenic driver mutation potential.33 fusions and midsize indels ( > 50 bp ; eg , met exon 14 skipping variants ) are especially challenging to detect in cfdna . 
as illustrated by case 8 , detection of met exon 14 skipping mutations can significantly affect the use of existing agents ( eg , crizotinib ) or novel agents ( eg , glesatinib ) for treatment . it is now known that intratumoral heterogeneity is associated with an increased risk of recurrence or death . 
 possible , all targetable driver mutations , both early in treatment and as the tumor evolves.34 using interval liquid biopsies on the basis of radiographic progression of an otherwise nonbiopsiable lesion can provide early detection of drug resistance and thus lead to early treatment intervention . 
this was illustrated by patient 5 , who declined invasive tumor biopsy and underwent repeat cfdna testing at the time of radiologic progression , which was 16 months after his initial cfdna testing . 
 a modest - sized prospective clinical trial by kim et al21 used cfdna - guided matched therapy when tissue was insufficient or unobtainable for ngs in patients with metastatic gastric cancer , nsclc , or melanoma . 
additional prospective studies are needed to further examine the value of early detection of resistance mechanisms before clinical evidence of progression and to determine if early detection and intervention truly influences os and pfs . in this series , patients were selected on the basis of the presence of low vaf driver mutations detected in cfdna . 
it has been reported that a subgroup of patients does not release tumor cfdna into their circulation , whereas other patients convert from having circulating tumor cfdna to noncirculating tumor cfdna after treatment , for reasons that include low tumor burden , slow - growing or inactive tumor growth , or therapeutic response.35 if safe , a tissue biopsy should be performed in patients who initially had detectable mutations on cfdna who no longer have detectable mutations but have clear progression of disease . 
there are several ongoing large prospective trials , including the azd9291 first time in patients ascending dose study ( aura ) , which serially compared cfdna versus tumor ngs over time in patients with egfr t790m mutations who were receiving osimertinib , which may help to address these clinical scenarios.16 one patient ( patient 10 ) was noted to have both eml4 - alk and ros1 mutations detected on tissue fish testing at the time of diagnosis . 
 difficulty detecting the mutation because recurrence of disease was located in the brain or the possibility that ros1 was present below the lod for the cfdna assay after 1 year of treatment . 
the patient was transitioned to hospice shortly after cfdna testing was done , and no additional testing was performed to confirm the presence of ros1 rearrangement . to our knowledge , this is the first case series examining treatment outcomes for patients with nsclc with low vaf driver mutations detected by cfdna . 
most patients had other passenger mutations that were present at a higher proportion compared with the low - frequency driver mutation , and the therapeutic significance of relatively low vaf driver mutations has not been previously reported . 
patel manuscript writing : all authors final approval of manuscript : all authors in the sensitivity and specificity of liquid biopsy , the ability to detect and confer clinical benefit to patients on the basis of these results will continue to improve with technological advances in cfdna . 
from this small case series , it seems that even at low vaf , targetable driver mutations retain clinical importance , even in the setting of other genomic alterations at higher vaf . 
mohindra consulting or advisory role : astrazeneca , genentech travel , accommodations , expenses : astrazeneca stock and other ownership interests : pfizer ( i ) lindsey shantzer no relationship to disclose ingrid l . 
patel consulting or advisory role : ariad , abbvie , astrazeneca jeffrey clarke consulting or advisory role : inivata , premier speakers bureau : guardant health , merck stock and other ownership interests : guardant health , biolase research funding : guardant health research funding : medpacto , genentech , bristol - myers squibb , adaptimmune , spectrum pharmaceuticals , abbvie , moderna therapeutics james christensen employment : mirati therapeutics stock and other ownership interests : mirati therapeutics , bluebird bio patents , royalties , other intellectual property : multiple patents in last 2 years while employed at mirati therapeutics covering discovery of kras , lsd1 , and ezh2 inhibitors . 
patel consulting or advisory role : eli lilly , novartis , bristolmyers squibb , astrazeneca / medimmune speakers bureau : merck , boehringer ingelheim research funding : bristol - myers squibb ( inst ) , eli lilly ( inst ) , incyte ( inst ) , medimmune ( inst ) , pfizer ( inst ) , genentech ( inst ) , xcovery ( inst ) affiliations miriam t . 
patel , university of chicago school of medicine , chicago , il ; lindsey shantzer and jeffrey clarke , duke university medical center , durham , nc ; ingrid l . 
lanman , guardant health , redwood city ; and james christensen , mirati therapeutics , san diego , ca . references 77 : 5705 , 2017 ( suppl 13 : abstr ) 1 . 
paik pk , varghese am , sima cs , et al : response to erlotinib in patients with egfr mutant advanced non - small cell lung cancers with a squamous or squamous - like component . 
aisner dl , sholl lm , berry ld , et al : the impact of smoking and tp53 mutations in lung adenocarcinoma patients with targetable mutationsthe lung cancer mutation consortium ( lcmc2 )  . 
zhou c , wu yl , chen g , et al : erlotinib versus chemotherapy as first - line treatment for patients with advanced egfr mutation - positive non - small - cell lung cancer ( optimal , ctong - 0802 ) : a multicentre , open - label , randomised , phase 3 study . 
sequist lv , yang jc , yamamoto n , et al : phase iii study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with egfr mutations . 
thompson jc , yee ss , troxel ab , et al : detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next - generation sequencing of cell - free circulating tumor dna . 
sacher ag , paweletz c , dahlberg se , et al : prospective validation of rapid plasma genotyping for the detection of egfr and kras mutations in advanced lung cancer . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
oxnard gr , paweletz cp , kuang y , et al : noninvasive detection of response and resistance in egfr - mutant lung cancer using quantitative next - generation genotyping of cell - free plasma dna . 
valle a , audigier - valette c , herbreteau g , et al : rapid clearance of circulating tumor dna during treatment with azd9291 of a lung cancer patient presenting the resistance egfr t790m mutation . 
kim st , banks kc , lee s - h , et al : prospective feasibility study for using cell - free circulating tumor dnaguided therapy in refractory metastatic solid cancers : an interim analysis . 
thompson jc , yee ss , troxel ab , et al : detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next - generation sequencing of cell - free circulating tumor dna . 
rozenblum ab , ilouze m , dudnik e , et al : clinical impact of hybrid capture - based next - generation sequencing on changes in treatment decisions in lung cancer . 
camidge dr , bang yj , kwak el , et al : activity and safety of crizotinib in patients with alk - positive non - small - cell lung cancer : updated results from a phase 1 study . 
awad mm , oxnard gr , jackman dm , et al : met exon 14 mutations in non - small - cell lung cancer are associated with advanced age and stage - dependent met genomic amplification and c - met overexpression . 
peschard p , fournier tm , lamorte l , et al : mutation of the c - cbl tkb domain binding site on the met receptor tyrosine kinase converts it into a transforming protemol cell 8 : 995 - 1004 , 2001 34 . 
lin c - c , shih jy , yu cj , et al : outcomes in patients with non - small - cell lung cancer and acquired thr790met mutation treated with osimertinib : a genomic study . 
 c clinical activity of olaparib in urothelial bladder cancer with dna damage response gene mutations introduction metastatic urothelial cancer has limited treatment options , especially when refractory to chemotherapy and immunotherapy . 
however , therapeutically targeting certain molecular alterations in the fgfr and erbb gene families has recently shown promise.1 - 5 comprehensive genomic sequencing efforts have also uncovered the presence of dna damage response ( ddr ) gene alterations , including ercc2 , brca1 , brca2 , and atm in up to 25% of patients with urothelial carcinoma.6 indeed , these alterations have been associated with response to platinum - based chemotherapy , as well as antiprogrammed death - 1 immunotherapy.7 - 11 inhibiting poly ( adp - ribose ) polymerase ( parp ) prevents repair of single strand dna breaks . 
thus , in the presence of homologous recombination gene mutations , parp inhibition leads to the accumulation of excessive dna damage , which becomes lethal to tumor cells.12 , 13 olaparib is a parp inhibitor with established efficacy in ovarian and breast cancers , and with emerging data in prostate cancer.14 - 16 however , there are no reported data on the clinical activity of this molecularly targeted therapy in urothelial bladder cancer . 
in this series , we present two patients with urothelial cancer with ddr mutations who both responded to therapy with olaparib . patient 1 in june 2013 , a 67 - year - old man presented with urinary obstructive symptoms , and a computed tomography ( ct ) scan demonstrated a lobular , asymmetric prostate gland . 
 his pathologic staging was pt4n2 , and from september to november of 2013 , he received adjuvant chemotherapy with gemcitabine and cisplatin , completing four cycles . the patient had no evidence of disease until june 2015 , when he was found to have a 1.1 - cm , left lower lobe lung nodule with prominent periaortic nodes , which were hypermetabolic on the basis of positron emission tomographyct scanning , consistent with metastatic disease . 
foundationone ( foundation medicine , cambridge , ma ) testing was performed on his primary tumor obtained from the prostatic urethral biopsy , which showed the tumor was microsatellite stable and had a low tumor mutational burden of four mutations per megabase . 
 one of these nodes was biopsied , and the tumor was sequenced using oncoplus , the university of chicago clinical laboratory improvement amendmentscertified next - generation sequencing platform.18 the brca1 n1018fs * 8 mutation was again detected . 
the chek2 mutation was indeed seen on the foundationone test from the original biopsy ; however , at that time , it was listed as a variant of unknown significance . 
this alteration has now been established as a germline founder mutation.19 by oncoplus , this new specimen was found to be microsatellite stable and had an intermediate tumor mutational burden of 12 mutations per megabase . 
he was found to have progression patient 2 in december 2013 , a 67 - year - old man underwent a cystoscopy for painless hematuria and was found to have a large friable area in the left bladder wall . 
the patient underwent chemotherapy with six cycles of gemcitabine and cisplatin for stage iv disease , with a complete response on the basis of ct scan , bone scan , and cystoscopy . 
he completed a course of radiotherapy ( 55 gy with 2.2 gy / fraction ) with concurrent capecitabine ( 825 mg / m2 twice per day ) in april 2017 for definitive local management . in september 2017 , a ct scan showed new hepatic lesions that were consistent with metastasis ( fig 1 )  . 
 next - generation sequencing was performed on the turbt specimen from january 2017 using oncoplus , which again identified a brca2 mutation ( c.7436 - 294_7567del ; table 1 )  . 
the mutyh g396d mutation ( previously reported as g382d ) found on his tumor testing was confirmed to be germline in orig the patient began receiving gemcitabine and cisplatin , and achieved another significant partial response after three cycles . 
he required a dose reduction to 300 mg twice per day because of thrombocytopenia , but continues receiving treatment as of september 2018 . discussion the parp enzyme is involved in base excision repair and is pivotal in repairing dna double strand breaks . 
mutation in brca1 / 2 predisposes cells to the effects of parp inhibition , which causes chromosomal instability , cell cycle arrest , and subsequent apoptosis.12 in cells with wild - type brca1 / 2 , dna repair by these proteins preserves the integrity of the dna . 
this functional buffering allows the cell to survive a variety of dna insults , but brca1 / 2 mutation leads to a loss of this compensation and renders the cell vulnerable to parp inhibition 's accumulation of dna defects , eventually leading to cell demise in the form of synthetic lethality.20 this synthetic lethality effect has formed the basis for clinical trials that led to approval of parp inhibitors in brca1 / 2 mutated ovarian and breast cancer.14 , 15 parp inhibitors have also been shown to have significant activity in patients with metastatic prostate cancer who harbor germline or somatic mutations in brca1 / 2.16 in bladder cancer , mutations in ercc2 , atm , and mmr genes are the most commonly mutated table 2 . 
 ddr and repair genes , but brca1 / 2 mutations are also found in up to 20% of patients ( fig 2 ) .21 , 22 here , we present the case reports of two patients with urothelial cancer with brca mutations who had clinical responses to parp inhibition with olaparib . 
these patients support the rationale for several recently initiated clinical trials investigating parp inhibitors in urothelial bladder cancer with ddr gene mutations ( table 2 )  . the first patient had a response lasting longer than a year , whereas the second patient has had an ongoing response lasting longer than 6 months . 
secondary resistance to parp inhibition can involve restoration of brca1 / 2 function , 53bp1 loss , or mdr1 upregulation.23 the rb1 loss observed in patient 1s tumor was not detected in the postolaparib biopsy . 
thus , the mechanism of acquired resistance in this patient may have been avoidance of synthetic lethality via rb1 reactivation , resulting in restoration of cell cycle arrest in the setting of dna damage.24 interestingly , neither of these patients responded to antiprogrammed death - 1 immunotherapy , which is unexpected , given data supporting a higher rate of clinical benefit with immunotherapy for patients with ddr alterations.11 this finding might be explained by the low tumor mutational burden observed in each patients initial tumor specimen ( four mutations per megabase ) and the finding that both tumors were microsatellite stable . 
sweis honoraria : bristol - myers squibb , exelixis , medscape brian heiss no relationship to disclose jeremy segal honoraria : bristol - myers squibb , abbvie research funding : abbvie lauren ritterhouse honoraria : bristol - myers squibb , abbvie research funding : abbvie travel , accommodations , expenses : bristol - myers squibb sabah kadri no relationship to disclose jane e . 
churpek honoraria : uptodate kenisha allen no relationship to disclose dawn conway no relationship to disclose carolyn marinier no relationship to disclose consulting or advisory role : bristol - myers squibb , exelixis , eisai norm d . 
stadler honoraria : cvs caremark , sotio , astrazeneca , bristolmyers squibb consulting or advisory role : cvs caremark , sotio , astrazeneca , bristol - myers squibb , eisai , bayer , pfizer , clovis oncology , genentech research funding : bayer ( inst ) , bristol - myers squibb ( inst ) , boehringer ingelheim ( inst ) , exelixis ( inst ) , novartis ( inst ) , genentech ( inst ) , glaxosmithkline ( inst ) , medivation ( inst ) , pfizer ( inst ) , merck ( inst ) , millennium ( inst ) , janssen ( inst ) , johnson & johnson ( inst ) , astrazeneca ( inst ) , abbvie ( inst ) , x4 pharma ( inst ) , calithera biosciences ( inst ) other relationship : uptodate , american cancer society affiliations all authors : university of chicago , chicago , il . support supported by national institutes of health grant no . 
hahn nm , bivalacqua tj , ross ae , et al : a phase ii trial of dovitinib in bcg - unresponsive urothelial carcinoma with fgfr3 mutations or overexpression : hoosier cancer research network trial hcrn 12 - 157 . 
nogova l , sequist lv , perez garcia jm , et al : evaluation of bgj398 , a fibroblast growth factor receptor 1 - 3 kinase inhibitor , in patients with advanced solid tumors harboring genetic alterations in fibroblast growth factor receptors : results of a global phase i , dose - escalation and dose - expansion study . 
loriot y , necchi a , park sh , et al : erdafitinib ( erda ; jnj - 42756493 ) , a pan - fibroblast growth factor receptor ( fgfr ) inhibitor , in patients ( pts ) with metastatic or unresectable urothelial carcinoma ( muc ) and fgfr alterations ( fgfra ) : phase 2 continuous versus intermittent dosing . 
plimack er , dunbrack rl , brennan ta , et al : defects in dna repair genes predict response to neoadjuvant cisplatin - based chemotherapy in muscle - invasive bladder cancer . 
iyer g , balar av , milowsky mi , et al : multicenter prospective phase ii trial of neoadjuvant dosedense gemcitabine plus cisplatin in patients with muscle - invasive bladder cancer . 
teo my , seier k , ostrovnaya i , et al : alterations in dna damage response and repair genes as potential marker of clinical benefit from pd - 1 / pd - l1 blockade in advanced urothelial cancers . 
bergman a , einbeigi z , olofsson u , et al : the western swedish brca1 founder mutation 3171ins5 ; a 3.7 cm conserved haplotype of today is a reminiscence of a 1500 - year - old mutation . 
kadri s , long bc , mujacic i , et al : clinical validation of a next - generation sequencing genomic oncology panel via cross - platform benchmarking against established amplicon sequencing assays . 
dhillon kk , taniguchi t : resistance to parp inhibitors mediated by secondary brca1 / 2 mutations , in curtin nj , sharma ra ( eds ) : parp inhibitors for cancer therapy . 
garsed dw , alsop k , fereday s , et al : homologous recombination dna repair pathway disruption and retinoblastoma protein loss are associated with exceptional survival in high - grade serous ovarian cancer . 
 mechanistic learning for combinatorial strategies with immuno - oncology drugs : can model - informed designs help investigators ? joseph ciccolini , pharmd , phd1 ; dominique barbolosi , phd1 ; nicolas andr e , md , phd1 , 2 ; fabrice barlesi , md , phd3 ; and s ebastien benzekry , phd4 the past couple of years have seen an unprecedented number of failures of clinical trials investigating combinatorial strategies with immuno - oncology drugs ( iods )  . beyond the highly publicized crashes of the mystic study1 ( antipd - l1 plus anti - ctla4 in nonsmall - cell lung cancer [ nsclc ] ) or the echo - 301 trial2 ( antipd - 1 and anti - ido in melanoma ) , many other attempts to combine immunotherapy with radiation therapy ( rt ) , 3 chemotherapy , 4 metronomic chemotherapy , 5 or antiangiogenics6 have similarly led to disappointing results . 
because successful immunotherapy is restricted today to a limited number of patients in a limited number of cancers ( melanoma , lung cancer , head and neck cancer , and kidney cancer , with 5 - year survival rates , 40% ) , developing appropriate strategies to stretch efcacy remains critical ( eg , by turning cold tumors [ noninammatory with lack of inltrating t cells ] into hot ones [ inltrated by t cells ] )  . 
several studies successfully associated conventional treatments with iods in comparative phase iii trials.7 , 8 however , these trials should not hide the high attrition rate of too many other studies . 
one of the common characteristics of the trials is the lack of computational pharmacology support , plus the lack of prior knowledge regarding the pharmacokinetics ( pk ) / pharmacodynamics ( pd ) relationships of the combined treatments . 
actually , comes from the fact that , sometimes , some combinatorial clinical trials manage to be successful . author affiliations and support information ( if applicable ) appear at the end of this article . accepted on march 20 , 2020 and published at ascopubs.org / journal / po on may 8 , 2020 : doi 1200 / po.19.00381 to improve the design of such combinatorial trials beyond trial - and - error methods , we propose an innovative strategy termed mechanistic learning ( fig 1 )  . we dene it as the combination of mechanistic modelingsimulation of the kinetics of pathophysiologic processesand statistical ( machine ) learning . using data generated from previous clinical trials and preclinical experiments , it consists in building computational models able to simulate and predict the toxicity and efcacy outcomes of candidate regimens . the optimal scheduling is then selected for clinical trial testing . 
limited options when performing combinatorial trials are therefore a major caveat , possibly explaining many failures , or at least limiting the conclusions regarding the real intrinsic potential of a given combination . 
for instance , in the checkmate032 study , the antipd - 1 nivolumab and the antictla4 ipilimumab were administered to patients with nsclc using only 2 dosing modes ( ie , 1 and 3 mg / kg and vice versa )  . 
these doses were chosen because they were already combined in a previous phase i study.9 in dose - nding trials , up to 5 dose levels ranging from 0.1 to 20 mg / kg have been tested for nivolumab and ipilimumab used as single agents without reaching dose - limiting toxicities.10 , 11 consequently , at least 25 different combinations in dosing could have been explored in checkmate - 032 , not to mention the countless variations in sequencing and scheduling . 
in the subsequent checkmate - 143 study , the same strategy failed to improve survival in patients with glioblastoma.12 another example of a poorly designed study is the modul umbrella trial , which tested a triple combination of uoropyrimidines , the antiangiogenic bevacizumab , and the antipd - l1 atezolizumab in patients with metastatic colorectal cancer ( mcrc )  . 
baseline data can be composed of demographic , clinical , pathologic ( eg , histologic type ) , molecular ( eg , genetic mutations ) , or biologic ( eg , blood counts ) variables . 
survival data ( eg , progression - free or overall survival ) can also be modeled with a mechanistic basis ( instead of biologically agnostic survival analysis based on , eg , cox regression ) , using adapted , survival learning statistical methods . 
ct1 , rst chemotherapy ; ct2 , second chemotherapy ; mtki , maintenance tyrosine kinase inhibitor ; ps , performance status ; tgi , tumor growth inhibition ; tki , tyrosine kinase inhibitor . surprisingly , in this study , all drugs were administered concomitantly at xed dosing . 
this combination failed to exhibit signicant efcacy in terms of progression - free survival , and thus , the conclusion was that adding atezolizumab to standard of care for maintenance in mcrc showed no benet.13 actually , this conclusion may sound peremptory because it is not possible to know whether or not different dosing or scheduling with exactly the same drugs would have performed better . 
for instance , trial of nivolumab , 23 the impact of dosing ( ie , 0.1 - 10 mg / kg ) on pd - 1 receptor occupancy was investigated . 
similar target engagement ( ie , 64% - 70% ) was achieved regardless of the dosing , thus prompting several observers to conclude that pk / pd relationships were at with iods and , therefore , that interpatient variability was not an issue . 
however , in this seminal study , pd - 1 inhibition was measured only on circulating t lymphocytes extracted from peripheral blood and not on inltrated t lymphocytes in the tumor microenvironment . 
importantly , the pharmacokinetics of most therapeutic monoclonal antibodies is characterized by large interindividual variability and reduced ability to diffuse the vascular space and reach solid tumors.24 out of therefore , to what extent differences in nivolumab dosing could affect or not target engagement at the tumor levels , and not only in circulating t cells , remains to be fully investigated . 
critically , it would also be highly informative to analyze the data generated using the initial model to close the mechanistic learning loop . using similar principles for tumor heterogeneity , others have proposed the concept of adaptive therapy , suggesting individuals be treated only upon disease progression.38 this was further successfully translated at bedside in patients with metastatic castration - resistant prostate cancer.39 such a strategy could help to personalize combinations of iods and aromatase inhibitors in the neoadjuvant treatment of breast cancer.40 despite their scarcity , these studies highlight how application of mathematical modeling in clinical trials in oncology is now feasible and can rationalize study design . these innovative methods are yet to be extended to iod combinations . 
modeling immunotherapy has recently been applied to gain insights on optimal modes for combinations with rt.41 - 43 for instance , kosinsky et al42 were able to simulate multiple sequences of antipd - 1 treatment in combination with rt and validated their results using preclinical data . 
 commentary the general modeling methodology that could be undertakenmechanistic learningis a combination of mechanistic modeling and statistical learning , either from machine learning , mixed - effects learning , 45 or survival learning , 46 for integration of the multimodal data arising from clinical trials or routine management ( fig 1 )  . 
as output , it would provide informative simulations of the effect of various doses and schedules to aid decisions in early clinical studies . these would consist of predicted probabilities of graded toxicities , tumor growth kinetics in response to treatment , and survival outcomes . 
of note , there is a major trend by health regulatory agencies such as the us food and drug administration to call for incorporating extensive modeling and simulation into clinical research.47 in this respect , and regarding the current challenges with iods , 48 we believe that mechanistic learning could be a valuable tool for decision making when setting up combinatorial strategies . conclusion after a rst phase of enthusiasm , success stories with immunotherapy seem to have reached a glass ceiling because many studies now fail to further stretch either response rates or survival.49 consequently , combining immune checkpoint inhibitors with other treatments likely to boost tumor immunity is a rising strategy in clinical oncology . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . joseph ciccolini honoraria : pierre fabre , pzer , roche , novartis research funding : roche , merck serono travel , accommodations , expenses : astrazeneca nicolas andr e research funding : bristol - myers squibb ( inst ) travel , accommodations , expenses : bristol - myers squibb fabrice barlesi honoraria : genentech , pzer , pierre fabre , astrazeneca , bristol - myers squibb , boehringer ingelheim , eli lilly , novartis , merck serono , msd oncology , takeda , bayer consulting or advisory role : genentech , pzer , novartis , pierre fabre , bristol - myers squibb , astrazeneca / medimmune , boehringer ingelheim , eli lilly , merck serono , msd oncology , takeda , bayer research funding : genentech ( inst ) , astrazeneca / medimmune ( inst ) , bristol - myers squibb ( inst ) , pierre fabre ( inst ) , abbvie ( inst ) , amgen ( inst ) , bayer ( inst ) , boehringer ingelheim ( inst ) , eisai ( inst ) , eli lilly ( inst ) , ipsen ( inst ) , innate pharma ( inst ) , novartis ( inst ) , merck serono ( inst ) , msd oncology ( inst ) , pzer ( inst ) , sano - aventis ( inst ) , takeda ( inst ) travel , accommodations , expenses : genentech , bristol - myers squibb , astrazeneca / medimmune , msd oncology no other potential conicts of interest were reported . acknowledgment we thank elena ivanchenko for graphic assistance . 
in memory of marie - christine masini . references 41 : 41 - 48 , 2019 rizvi na , chul cho b , reinmuth n , et al : durvalumab with or without tremelimumab vs platinum - based chemotherapy as rst - line treatment for metastatic nonsmall cell lung cancer : mystic . 
mcbride sm , sherman ej , tsai cj , et al : a phase ii randomized trial of nivolumab with stereotactic body radiotherapy ( sbrt ) versus nivolumab alone in metastatic ( m1 ) head and neck squamous cell carcinoma . 
 ciccolini et al rothschild s , zippelius a , savic s , et al : sakk 16 / 14 : anti - pd - l1 antibody durvalumab ( medi4736 ) in addition to neoadjuvant chemotherapy in patients with stage iiia ( n2 ) non - small cell lung cancer ( nsclc ) a multicenter single - arm phase ii trial . 
j clin oncol 36 , 2018 ( suppl 15 ; abstr tps8584 ) 1200 / jco.2018.36.15_suppl.tps8584 toulmonde m , penel n , adam j , et al : use of pd - 1 targeting , macrophage inltration , and ido pathway activation in sarcomas : a phase 2 clinical trial . 
jama oncol 4 : 93 - 97 , 2018 gao j , karam ja , tannir nm , et al : a pilot randomized study evaluating nivolumab ( nivo ) or nivo + bevacizumab ( bev ) or nivo + ipilimumab ( ipi ) in patients with metastatic renal cell carcinoma ( mrcc ) eligible for cytoreductive nephrectomy ( cn ) , metastasectomy ( ms ) or post - treatment biopsy ( bx )  . 
j clin oncol 36 , 2018 ( suppl 15 ; abstr 4520 ) socinski ma , jotte rm , cappuzzo f , et al : atezolizumab for rst - line treatment of metastatic nonsquamous nsclc . 
hellmann md , rizvi na , goldman jw , et al : nivolumab plus ipilimumab as rst - line treatment for advanced non - small - cell lung cancer ( checkmate 012 ) : results of an open - label , phase 1 , multicohort study . 
ribas a , camacho lh , lopez - berestein g , et al : antitumor activity in melanoma and anti - self responses in a phase i trial with the anti - cytotoxic t lymphocyteassociated antigen 4 monoclonal antibody cp - 675 , 206 . 
grothey a , tabernero j , arnold d , et al : fluoropyrimidine and bevacizumab plus or minus atezolizumab as rst - line treatment for braf wild type metastatic colorectal cancer : findings from the modul trial of biomarker - driven maintenance . 
schaer da , beckmann rp , dempsey ja , et al : the cdk4 / 6 inhibitor abemaciclib induces a t cell inamed tumor microenvironment and enhances the efcacy of pd - l1 checkpoint blockade . 
heery cr , osullivan - coyne g , madan ra , et al : avelumab for metastatic or locally advanced previously treated solid tumours ( javelin solid tumor ) : a phase 1a , multicohort , dose - escalation trial . 
lindauer a , valiathan cr , mehta k , et al : translational pharmacokinetic / pharmacodynamic modeling of tumor growth inhibition supports dose - range selection of the antipd - 1 antibody pembrolizumab . 
long gv , tykodi ss , schneider jg , et al : assessment of nivolumab exposure and clinical safety of 480 mg every 4 weeks at - dosing schedule in patients with cancer . 
h enin e , meille c , barbolosi d , et al : revisiting dosing regimen using pk / pd modeling : the model1 phase i / ii trial of docetaxel plus epirubicin in metastatic breast cancer patients . 
barlesi f , imbs d - c , tomasini p , et al : mathematical modeling for phase i cancer trials : a study of metronomic vinorelbine for advanced non - small cell lung cancer ( nsclc ) and mesothelioma patients . 
yu ha , sima c , feldman d , et al : phase 1 study of twice weekly pulse dose and daily low - dose erlotinib as initial treatment for patients with egfr - mutant lung cancers . 
poleszczuk jt , luddy ka , prokopiou s , et al : abscopal benets of localized radiotherapy depend on activated t cell trafcking and distribution between j immunother cancer 6 : 17 , 2018 metastatic lesions . 
claret l , girard p , hoff pm , et al : model - based prediction of phase iii overall survival in colorectal cancer on the basis of phase ii tumor dynamics . 
coosemans a , vankerckhoven a , baert t , et al : combining conventional therapy with immunotherapy : a risky business ? eur j cancer 113 : 41 - 44 , 2019 49 . 
komen big data for breast cancer initiative : how patient advocacy organizations can facilitate using big data to improve patient outcomes jerome jourquin , phd , ms1 ; stephanie birkey reffey , phd1 ; cheryl jernigan2 ; mia levy , md , phd3 ; glendon zinser , phd1 ; kimberly sabelko , phd1 ; jennifer pietenpol , phd4 ; and george sledge jr , md5 integrating different types of data , including electronic health records , imaging data , administrative and claims databases , large data repositories , the internet of things , genomics , and other omics data , is both a challenge and an opportunity that must be tackled head on . 
komen envisions a world with a seamless web of health care information , where patients are informed and empowered to use their data to improve their health care ; electronic health records ( ehrs ) are connected to other data sources to provide evidence - based support for clinical decision making ; many , if not all , patients participate in clinical research ; data systems are linked , secure , and easily accessible ; genomics and other omics are universally available and user friendly ; researchers can mine enhanced data sets to address questions ; and most importantly , fewer people die of breast cancer and quality of life improves for those living with the disease . 
komen convened three big data for breast cancer ( bd4bc ) meetings to foster open dialog among experts and strategically invited a wide array of participants ( patient advocates , oncologists , bioethicists , laboratory researchers , genomicand proteomics - based companies , big datafocused pharmaceutical companies , and data software companies ) to discuss the status of , challenges in , and barriers to optimizing big data and the opportunities big data can provide to advance health care ( fig 1 )  . the rst meeting ( new york , ny , october 2015 ) focused on the barriers , opportunities , needs , priorities , and solutions concerning the challenge of improving breast cancer research and care through big data . 
 jourquin et al bd4bc ( 2015 ) bd4bc\wc ( 2017 ) bd4bc3 ( 2018 ) all bd4bc meetings bioethics and policy data analytics and learning systems data integration and management funders omics government and insurance providers oncology and patient care patient advocate patient - centered data and systems fig 1 . 
omics experts were the most represented in the rst two meetings ( 35% and 29% , respectively )  . a more equal distribution of expertise was reached at the 2018 meeting among patient - centered data and systems , data analytics and learning systems , and data integration and management ( 15% to 23% each ) , highlighting the intent to work on solutions to big data challenges in breast cancer and implementation . 
looking at all 148 unique participants who attended komens bd4bc meetings , the most represented areas of expertise were omics ( 27% ) , data analytics and learning systems ( 19% ) , data integration and management ( 17% ) , and oncology and patient care ( 11% )  . 
bd4bc\wc , west coast ( second bd4bc meeting ) ; bd4bc3 , third bd4bc meeting . claims databases , large data repositories ( registries and cohorts ) , and genomics and other omics data . 
we share the insights gained from these meetings and komens planned actions to leverage big data to reduce the number of breast cancer deaths . as recently as 15 years ago , individual patient data were only available in paper charts located at a single institution and inaccessible to others outside that institution . 
data from epidemiologic studies , cooperative group trials , and individual laboratories were often quantitatively modest , dispersed , and not open to sharing except through manuscripts and public presentations.1 the world of data is rapidly changing , and the amount of data related to breast cancer has exploded . 
within the past decade , various types of data are now routinely collected : personal and family history ; breast density ; patient - reported outcomes ; imaging data ; clinical trial data ; genomics and other omics ; annotated mutations ; biospecimens ; and social , environmental , and behavioral data . 
one of us ( m.l. ) reported at the third bd4bc meeting that in 2015 , one health care systems ehr was accessed by 11 , 000 people per day , creating 6.8 million clinic notes that year , and was associated with 1.7 million outpatient electronic prescriptions and 15 million clinical communications that year . furthermore , genomics , proteomics , metabolomics , radiomics , and other emerging omics platforms are expanding the data generated in health care.3 decreasing costs and increasing availability of such tests allow clinicians to evaluate tumors via whole exome , genome , or deep sequencing . these are becoming part of the standard of care . 
afliations are at the time of each meeting . abbreviations : bd4bc\wc , west coast ( second bd4bc meeting ) ; bd4bc3 , third bd4bc meeting ; cloud , consortium for local ownership and use of data . area with a rapidly growing platform generating large amounts of individual - specic data is the internet of things ( iot ) , which includes all devices attached to the internet that generate data.4 wearable devices ( eg , tness trackers ) individual - specic generate continuous , multiparameter , data . 
by 2020 , an estimated 200 billion devices will be connected to the internet and generating continuous data , with approximately 30% of these data predicted to have medical applications.4 with rapid technologic advances and vast amounts of data come challenges that must be overcome to make efcient use of the technology and data being collected . 
big data technologies , such as ehrs , omics , and iot , provide great potential , but these technologies remain siloed and not interoperable.5 , 6 data are often limited geographically and by age group and type of care . 
the use of these technologies is also limited by the inability of different platforms to communicate with each other , hospital rewalls that create barriers to sharing data , privacy issues ( real and perceived ) , and the lack of nancial models to incentivize storage , sharing , and integration . 
 jourquin et al internet of things patient - reported outcomes and registries genomics other omics imaging data electronic health records integration big data for breast cancer research improved outcomes for patients with breast cancer fig 2 . 
the integration of various types of data ( patient - reported outcomes ; imaging data ; electronic health record data ; genomics and other omics ; and so on ) can fuel scientic discoveries and lead to improved outcomes for patients with breast cancer . multiple processing engines ; and a strong , unied security model . 
this includes the technology to support development of these systems and applications and harness the power of these enhanced data sets in big data projects . although data storage remains a challenge , many companies are creating scalable solutions for data storage , including health data . technologic innovation brings opportunities to accelerate research discovery and improve patient care . 
precision medicine can be achieved by using big data technologies to combine different types and sources of data to identify patterns , determine optimal treatments for individuals , and improve their outcomes.8 imaging is another technologic opportunity where big data can be leveraged in breast cancer . 
approximately 75 million mammograms are performed annually worldwide , currently requiring a large amount of personnel time to read and analyze the results of each test . emerging advances in articial intelligence ( ai ) methods ( eg , convolutional neural networks and deep learning ) are being used in mammography to distinguish patients with benign , malignant , and negative disease , 9 and objectively assess breast density.10 similar advances are taking place in magnetic resonance imaging , where machine learning is being used to predict patients response to therapy and outcomes.11 big data imaging applications can be used to automate image reading and generate reports without human interaction , allowing radiologists to only review images not easily interpreted by the algorithms . 
although these data are often not standardized , natural language processing and other advanced technologies such as ai can help simplify extraction of unstructured data.16 another challenge is that big data are less well curated than other classic data sets ( eg , prospective clinical trials and epidemiologic registries )  . 
perhaps the biggest barrier in big data research application is that basic laboratory and clinical translational researchers often have no expertise in the multidimensional analytics and visualization tools that big data requires . 
research funding organizations ( public and private ) often do not provide support to cover costs of data annotation , curation , and sharing , and they rarely support training programs focused on big data . applying big data to research applications offers enormous possibilities , with the potential to solve questions currently unanswerable in traditional research laboratories . 
 advancing big data for better breast cancer care and outcomes machine learning was successfully used on those data to identify mirna biomarkers in breast cancer.19 pros , rwd , and rwe represent other areas where big data can be generated and harnessed for research applications.15 pros include data collected directly from patients ( eg , quality of life and functional status ) that are not interpreted by physicians or others.20 because only approximately 3% of patients with cancer participate in clinical trials , big data approaches to pros and rwd / rwe may represent realistic ways of integrating information about under - represented populations and nding solutions to previously intractable clinical problems . 
these data are linked to deep , next - generation sequence proling across hundreds of cancer - related genes for each patients tumor , as assessed by foundation medicine ( cambridge , ma ) .23 pharmaceutical companies and the us food and drug administration are also active in the eld and understand the value of rwd / rwe in developing and regulating medical products.24 , 25 komens bd4bc meetings offered valuable insights into the great and unmet need to advance training in and improve access to big data . 
breast cancer researchers without a data science focus could be trained in the eld of data science by participating in workshops and conferences to learn new methods , skills , and techniques to advance their research projects using big data . 
effective use of big data to improve breast cancer care can also be facilitated by bringing together data set owners , both nonprot and forprot , with data scientists , with the goal of creating access to breast cancer data sets for research . 
several thousand patients with metastatic breast cancer have registered to share their data for research.27 a similar participation response was recently obtained by the national institutes of health ( nih ) with their all of us study.28 patients desire for new knowledge about their disease often outweighs their privacy concerns . 
still , many patients have questions about what control they have over their data , electronic accessibility , 26 privacy , and data security , and why data sharing is important . government laws and regulations make medical data one of the few remaining bastions of privacy . 
in addition to the health insurance portability and accountability act and ofce of human research protection , siloed data have additional barriers , including business or proprietary interests , privacy concerns , transaction costs associated with data infrastructure and data sharing , and the nature of local legacy systems . although data sharing has become more common , it is often inefcient because data must be standardized , deidentied , and possibly shared via an institutional review boardapproved study with appropriate participant consent . 
this presents another challenge because most patients only agree to one study at a time , meaning re - consent is needed for each use.29 the growing number of companies that view patient data as a commercial asset is particularly concerning . 
what a patient originally agreed to share with his or her initial consent may be unknown . whether data can be shared for additional investigations and commercial purposes is unknown.6 other challenges to data sharing include interface glitches , moving data between formats , and questions of whether the data depositor is legally liable if public data are misinterpreted by another user.7 some companies do not want their data placed in a centralized , accessible location because they want to retain control of them , retain exclusive rights to their analyses , and / or monetize the data and their usage . overall , little incentive exists to share or make data readily and easily accessible . patient advocacy organizations such as komen can help address some of the problems associated with data sharing , starting with educating and advocating for patient needs and concerns regarding privacy and data sharing . 
financial support for infrastructure , curation , de - identication , sustainability , and appropriate guidelines is necessary to implement this requirement.1 another highlight from komens bd4bc meetings is that motivation for sharing data may increase if patients learn the value of sharing information for big data - driven research and if the study results are shared with patients . 
this highlights the important role patient advocates can play in demanding that data be shared , such as pressuring different stakeholders to work together toward a common goal , urging sustainability of good ideas , and requesting that study results be shared with patients . 
such data can provide guidance on the likelihood a given patient with breast cancer will benet from chemotherapy.34 this type of testing has also stratied more than 15 subtypes of breast cancer , allowing omics data to inform prognoses and treatments.3 , 35 , 36 cases showing the actual value of big data in patient care are currently lacking . 
a few anecdotal examples of how big data are being integrated in the patient care workow exist . for example , one of us ( m.l. ) created an analytic dashboard to leverage patient data and show patients care status in nearreal time . 
the dashboard ultimately led to changing the policy and to women undergoing biopsy sooner . for millions of patients with breast cancer , ehrs represent an important , comprehensive resource for patient care . because patients continuously receive care , ehrs allow longitudinal tracking of long - term outcomes ( eg , time receiving therapy and safety events )  . 
ehr - based treatment plans are also being used to reduce medication errors compared with prior paper - based approaches , increase standardization , and allow retrieval of data for quality measures within institutions . challenges remain with ehr systems . 
with the integration of omics analyses in ehrs to drive clinical decision , ehr systems have become large enterprises , requiring large infrastructure and computational power many clinics cannot afford . 
current ehrs frequently decrease clinical efciency , demoralize physicians by turning them into data entry specialists , and decrease time of direct patient - physician interaction . physicians feel ehr systems exist primarily to aid hospital billing , rather than facilitate patient care . 
one of us ( g.s. ) highlighted what was at stake in improving how physicians interact with ehr systems to optimize patient care : save a doctors time , save a patients life . an identied opportunity for ehr systems is their ability to revolutionize how quality of care is assessed . 
including genomic and other omics data directly in ehrs should promote high - level clinical decision support and improved access to clinical trials to realize the full potential of precision medis higher - quality , more costicine . 
this trial used an ehr system to create a real - time registry of patients that alerted the health care team when participants missed appointments or had an unmet care milestone . 
the intervention imnavigation and clinical proved treatment completion and helped reduce racial disparities in treatment of these patients.38 accure exemplies the power of harnessing big data to address health disparities in breast cancer care . 
the patient populations at health systems with sophisticated ehrs , data sharing systems , and capabilities to collect and store biospecimens for later analysis are likely not representative of the true diversity of patients across all settings.37 thus , any advancement resulting from big data should be designed and implemented for the maximum number of individuals who can benet from it . 
minority populations should be adequately represented in data sets so that safe and effective treatment strategies for all patients can be drawn from the results of studies using them . increasing efforts to better classify breast cancer are driving wide use of molecularly based precision medicine in oncology practice . 
access to big data can supercharge these types of data - fueled studies by providing an ever - growing number of data points and characteristics about everyone within a population.37 ai may be able to identify patterns within subpopulations that predict the risk of occurrences , recurrences , and generally worse outcomes , as well as optimal , tailored treatment plans . 
these studies may allow more efcient access to care , better treatment adherence by patients within the continuum of care , and more precise identication and management of at - risk populations . 
big data may truly facilitate personalized medicine , regardless of race , ethnicity , or other characteristics . discussion with the growing availability of pros and rwd , ai will likely affect clinical practice and clinical trial design in the near term , and increase our understanding of patients response to therapy . yet , most of the general public and many patients are unaware of or have concerns about big data and its application to cancer research and treatment . 
developing and implementing educational resources ( eg , fact sheets , web portal on big data , patient advocate training ) will be critical to making data sharing understandable and easy . 
patients want to control the use of their data.4 an educated , engaged advocacy community is crucial for bd4bc to succeed . komen will start by developing an online knowledge portal designed as a hub of information for visitors to advance their knowledge about big data . meeting participants repeatedly mentioned the lack of both data scientists working in breast cancer and laboratory and clinical researchers who are uent in big data analytics . 
komen and similar organizations can support researcher education by providing funding opportunities to train breast cancer researchers in big data and attract data scientists to apply big data to solve remaining challenges in breast cancer.39 a central directory of existing breast cancer data sets available to big data scientists to use in breast cancer research is essential . in addition , support must exist for projects that will accelerate the technologic advancement and innovative thinking necessary to discover novel targets for precision medicine and provide earlier detection that will affect empower the public with information and tools to make data sharing understandable and easy to do big data for patients bd4bc communications bd4bc knowledge portal using big data to fuel scientific discoveries and accelerate the delivery of equitable , patient - focused care address challenges of incorporating big data applications into breast cancer research and clinical care big data travel scholarships bd4bc hackathons breast cancer data directory bd4bc meetings use big data to fuel scientific discoveries and accelerate the delivery of equitable , patient - focused care big data research projects bd4bc workshops fig 3 . 
komen is following a three - pronged approach ( white boxes ) in developing several programs ( pink boxes ) that will specically address some of the challenges identied during komens bd4bc meetings and detailed in this article . 
organizations such as komen can leverage their grant - making capabilities to identify and support big data research resources and projects that put the patient at the center of innovation to inform and accelerate the pace of breast cancer research and improve patient care . funding is needed to support data science projects and infrastructure for aggregation , visualization , and modeling of patientand laboratory - derived big data . 
public advocacy is needed so initiatives can leverage big data to support adherence to treatment and participation in clinical trials , and enforce safety , security , data standards , accessibility , and sharing requirements . 
protection of minorities and underserved populations in the big data revolution is needed to ensure these groups are included in the progress big data will make toward better breast cancer outcomes . in conclusion , many opportunities were identied at komens bd4bc meetings to harness big data to benet patients with breast cancer . 
strategies include educating the public to make data sharing understandable and easy , addressing challenges of incorporating big data applications into breast cancer research and clinical care , and funding data science projects . 
komen will continue to advocate for putting the patient at the center of innovation and support efforts using big data to fuel scientic discoveries and accelerate the delivery of improved , equitable , and patientfocused care . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . stephanie birkey reffey travel , accommodations , expenses : genentech mia levy employment : seqtech diagnostics ( i ) leadership : personalis stock and other ownership interests : personalis , genomoncology honoraria : roche consulting or advisory role : personalis , genomoncology , roche research funding : genomoncology patents , royalties , other intellectual property : royalties from genomoncology for licensing of mycancergenome content travel , accommodations , expenses : roche jennifer pietenpol stock and other ownership interests : bluebird bio , johnson & johnson , roche , gilead sciences ( i ) , quest diagnostics , illumina , kite pharma , novartis ( i ) research funding : incyte corporation ( inst ) patents , royalties , other intellectual property : jennifer pietenpol is an inventor ( pct / us2012 / 065724 ) of intellectual property ( tnbctype ) licensed by insight genetics other relationship : susan g . 
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denny j : aacr modernizing population sciences in the digital age , early progress on the all of us research prograpresented at the american association for cancer research , san diego , ca , february 19 - 22 , 2019 29 . 
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 beyond the androgen receptor : targeting actionable drivers of prostate cancer see accompanying article doi : more than 70 years ago , huggins et al1 exploited the exquisite and unique dependence of prostate cancer ( pc ) on androgenic hormones for survival and growth using a treatment strategy that foreshadowed the clinical benefits underlying the application of precision oncology . 
today , variations of androgen receptor ( ar ) pathway targeting remain the cornerstone of the initial management of men with metastatic pc , usually with gratifying responsesalthough unfortunately few cures . 
however , the advent and application of technologies capable of deep assessments of the full spectrum of molecular aberrations that underlie the development and progression of malignant neoplasms now forecast the near - term potential of new therapeutic approaches poised to change the near - exclusive reliance on ardirected interventions as initial treatment for advanced pc . studies using whole - exome sequencing and comprehensive rna sequencing , supported through the cancer genome atlas2 and stand up to cancer / prostate cancer foundation3 initiatives , have profiled hundreds of localized and castration - resistant metastatic pcs ( crpcs ) , resulting in the identification of numerous recurrent driver aberrations that comprise signaling programs and pathways for which there are pharmacologic inhibitors . 
lacking in these previous studies was an assessment of untreated metastatic tumors . in the article accompanying this editorial , abida et al4 have taken the first step in filling this knowledge gap . using a predefined gene panel , abida et al4 evaluated mutations and copy losses in key oncogenes and tumor suppressors across a spectrum of localized pc , untreated metastatic disease , and crpc . 
the tumor assays were conducted in a clinical laboratory improvement amendments environment using real - world clinical samples , which demonstrates the feasibility of molecular profiling to guide therapeutics , even in challenging situations such as evaluating bone metastases . overall , a high frequency of potentially actionable alterations were identified that included components of the phosphatidylinositol 3 - kinase ( pi3k ) pathway ( 24% ) , mitogen - activated protein kinase pathway ( 5% ) , and b - catenin program ( 15% )  . this study also confirmed previous reports that aberrations in genes involved in dna repair are common , particularly those involved in the process of homologous recombination repair.5 overall , 22% of tumors had somatic aberrations in a dna repair gene , and 19% were found to have a putative deleterious germ line mutation . 
this pc subtype is notable for therapeutics such as poly ( adp - ribose ) polymerase inhibitors and platinum - based chemotherapy that could exploit a dna repair vulnerability.6 , 7 however , it is important to recognize that it is currently unclear whether each gene comprising the dna repair machinery will exhibit enhanced responses to these agents , nor is it clear whether each type of aberration within a particular dna repair gene will confer congruent outcomes with respect to treatment response . 
preclinical studies using functional readouts of dna repair proficiency and clinical trials coupled with careful assessments of each dna damage response gene aberration will be required to confirm predictive associations . overall , there was substantial heterogeneity in the composition of putative cancer drivers between individuals , a finding that supports avoiding a onesize - fits - all approach to therapy . 
 number of matched tumors from the same patient demonstrated consistent truncal aberrations in tumor suppressors such as tp53 and brca2 and amplification of ar across each tumor evaluated . these results indicate that sampling a single metastatic site provides a reasonable assessment of the major molecular alterations that may influence treatment decisions . 
however , analyses of tumors across intervals of time and treatment indicated that molecular evolution does occur , with higher mutation counts in tumors sampled at later time points . consequently , it is not clear whether sampling a primary tumor or a metastatic tumor early in the course of disease will accurately reflect driver or resistance mechanisms that are operating later in the course of cancer progression . a distinguishing feature of the abida et al4 study involves the acquisition and assessment of metastatic tumors that had not been exposed to ar - directed therapy or other systemic treatment . comparing the features of these metastases with those of localized tumors serves to identify drivers of the metastatic process , and comparing their molecular composition with that of tumors withstanding ar - directed therapy serves to identify mechanisms contributing to treatment resistance . in the first comparison , it is notable that aberrations in ar did not occur , emphasizing that ar mutation and copy - number gain , frequent events in crpc , occur in response to pressures exerted by ar pathway inhibition and rarely or never arise as de novo drivers of pc . 
in addition to ar , the frequency of aberrations in tp53 , rb1 , pten , and atm was significantly increased in crpc compared with metastatic noncastrate disease , indicating potential roles in mediating castration resistance . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . interactions with the ar or ar program or if they promote pc growth through mechanisms that bypass ar requirements . 
although enriched in crpc , a substantial number of treatment - nave tumors did exhibit mutations or structural alterations in these genes , suggesting that cotargeting the aberrant pathways concurrently with the ar may serve to circumvent predestined pathways of resistance . that drug combinations current therapy for metastatic pc generally follows a paradigm of sequentially deploying a given therapeutic such as androgen deprivation to the point of resistance , switching to another agent such as abiraterone or enzalutamide , and treating again to resistance , until options are exhausted . 
nelson consulting or advisory role : janssen oncology , astellas pharma , genentech acknowledgment supported by national institutes of health grants no . p30ca15704 and p50ca097186 , us department of defense grant no . 
cancer genome atlas research network : the molecular taxonomy of primary prostate cancer . arch surg 43 : 209 - 223 , 1941 cell 163 : 1011 - 1025 , 2015 3 . 
mateo j , carreira s , sandhu s , et al : dna - repair defects and olaparib in metastatic prostate cancer . n engl j med 373 : 1697 - 1708 , 2015 7 . 
schweizer mt , zhou xc , wang h , et al : the influence of prior abiraterone treatment on the clinical activity of docetaxel in men with metastatic castration - resistant prostate cancer . 
smith mr , saad f , rathkopf de , et al : clinical outcomes from androgen signaling - directed therapy after treatment with abiraterone acetate and prednisone in patients with metastatic castration - resistant prostate cancer : post hoc analysis of cou - aa - 302 . 
 case of metastatic extramammary paget disease of the vulva treated successfully with trastuzumab emtansine introduction extramammary paget disease ( empd ) is a rare adenocarcinoma in situ that originates in skin that contains apocrine glands . 
empd is associated with invasive cancer in 4% to 20% of patients.1 in a series of 1 , 439 patients with invasive empd , 80.4% had localized disease , 17.1% had locoregional spread , and only 2.5% presented with distant disease.2 treatment is primarily surgical , but empd frequently is multicentric , and relapses are common regardless of treatment modality . 
since 2002 , she underwent six excisional procedures , three laser ablations , several courses of topical imiquimod treatment , external beam vulvar radiation to a dose of 56 gy to the perineum and the left - side inguinal nodes , and interstitial brachytherapy to 30 gy . 
her treatment was complicated by urethral swelling and eventually obstruction , requiring placement of a temporary suprapubic catheter and urethral reconstruction in late 2014 . in early 2016 , the patient noted worsening urinary retention , and a computed tomography ( ct ) urogram demonstrated two centrally hypodense liver lesions that measured up to 2.2 cm , multiple enlarged retroperitoneal lymph nodes , and a circumferential soft tissue mass that infiltrated the cervix and upper vagina . 
on examination under anesthesia , a paget lesion obliterated the perineum , including the urethra , vulva , and perianal area , and vaginoscopy was required to visualize the cervix . 
vulvar biopsy specimens confirmed invasive , poorly differentiated adenocarcinoma that had arisen from empd , with extensive lymphovascular space invasion and 3 + her2 immunostaining in > 10% of cells ( fig 1 )  . 
on the basis of high concordance between dna sequencingbased methods and fluorescent in situ hybridization ( fish ) in detecting erbb2 amplification , 5 fish was foregone . the patient underwent six cycles of carboplatin area under the curve of 5 mg ml / min and paclitaxel 175 mg / m2 chemotherapy every 21 days , gillian l . 
 ( a ) nests of extramammary paget disease in the epithelium ( black arrows ) with underlying invasive adenocarcinoma in the dermis ( red arrow ; hematoxylin - eosin stain magnification , 10 )  . 
 ( b ) strong , circumferential membranous staining of human epidermal growth factor receptor 2 ( her2 ) in extramammary paget disease and invasive tumor ( her2 immunohistochemical stain magnification , 10 )  . 
 ( c ) invasive adenocarcinoma ( hematoxylin and eosin stain magnification , 10 ) with ( d ) 3 + membranous staining of her2 ( her2 immunohistochemical stain magnification , 10 )  . with trastuzumab added from cycles 2 to 6 . 
repeat pet / ct scanning after 3 months of maintenance therapy revealed an intensely fdg - avid recurrent cervical and vaginal tumor and mildly enlarged and fdg - avid left - side external iliac nodes . 
treatment was well tolerated , with toxicity limited to grade 1 fatigue and grade 1 bleeding , including intermittent vaginal bleeding , hematuria , epistaxis , and gum bleeding , which started after cycle 2 . 
follow - up pet / ct scans were notable for markedly decreased size and metabolic activity of the primary pelvic lesion after cycle 3 and for complete resolution of disease on imaging after cycle 6 ( fig 2 )  . 
positron emission tomography / computed tomography ( a ) axial fusion , ( b ) sagittal fusion , and ( c ) maximum intensity projection images before ( top ) and after treatment ( bottom )  . 
 a hypermetabolic cervical mass ( arrow ) was seen with associated hydrometra , which had complete treatment response to chemotherapy . and hormonal suppression with letrozole.6 , 7 although insufficient data exist on the application of the current breast cancer asco - cap her2 testing guidelines8 to other cancer types , the amplification of her2 has been described in many cases of invasive empd ( table 1 )  . 
 multiple large , multicenter trials have firmly established the benefit of trastuzumab in the treatment of metastatic her2 - positive breast cancer , 9 which has led to consideration of trastuzumab in the treatment of invasive metastatic her2 - amplified empd . retrospective data have suggested that her2 overexpression is higher in patients with invasive empd than with noninvasive empd.19 furthermore , a significant correlation has been demonstrated among the presence of invasion , lymph node metastasis , and her2 overexpression.24 several case reports have demonstrated partial or complete response of widely metastatic empd on trastuzumab with or without paclitaxel ( table 2 )  . the mechanisms that underlie trastuzumabs clinical activity have not been completely table 1 . 
although tdm1 has been shown to be efficacious in patients with breast cancer with her2 - overexpressed malignancy unresponsive to trastuzumab , disease response has not been consistently demonstrated with other tumor types . 
 in her2 - positive unresectable gastric or gastroesophageal junction cancers with prior her2 - targeted therapy.36 the current patients favorable response indicates that in her2 positive invasive empd , tdm1 may be considered a viable treatment option . 
in addition , in hormone receptor and her2 - positive breast cancer , dual targeting of both signaling pathways may improve outcomes significantly.37 in breast cancer , bidirectional cross talk between the estrogen receptor and her2 pathways likely contributes to anti - her2 therapy resistance . 
 preclinical models with breast cancer cell lines have demonstrated a benefit to simultaneous hormone and her2 therapy over her2 - targeted therapy alone , but no phase ii or iii trials have compared the two.38 the tandem trial demonstrated a 2.4 - month survival benefit in postmenopausal patients with metastatic breast cancer who received anastrazole and trastuzumab versus hormonal therapy with anastrazole alone.39 hormone receptor immunohistochemistry was not examined in the current patient , but dual inhibition may be a theoretically superior strategy for treating hormone receptor and her2 - positive invasive empd . one limitation to this study is that the asco - cap her2 testing guidelines have not been routinely adopted or validated for tissues other than breast carcinoma ; thus , the her2 immunohistochemistry results may not be consistent or reliable in this tumor type . 
molecular subtyping has been used increasingly to find appropriate targeted therapies and may be more reliable than immunohistochemistry.23 vornicova et al7 reported a partial response that lasted 12 months in a male with adenocarcinoma with paget disease and negative her2 testing by both immunohistochemistry and fish , but received anti - her2 therapy on the basis of tumor somatic mutation testing alone ( table 2 )  . in light of the poor prognosis with metastatic epmd , paucity of treatment options , high incidence of her2 amplifications in empd , and favorable adverse effect profile of tdm1 , we suggest her2 testing and consideration of tdm1 treatment in patients with her2 overexpressed invasive empd who experience treatment failure with trastuzumab . 
ohara k , fujisawa y , yoshino k , et al : a proposal for a tnm staging system for extramammary paget disease : retrospective analysis of 301 patients with invasive primary tumors . 
vornicova o , hershkovitz d , yablonski - peretz t , et al : treatment of metastatic extramammary pagets disease associated with adnexal adenocarcinoma , with anti - her2 drugs based on genomic alteration erbb2 s310f . 
tchrakian n , flanagan l , harford j , et al : new asco / cap guideline recommendations for her2 testing increase the proportion of reflex in situ hybridization tests and of her2 positive breast cancers . 
ogawa t , nagashima y , wada h , et al : extramammary pagets disease : analysis of growth signal pathway from the human epidermal growth factor receptor 2 prote hum pathol 36 : 12731280 , 2005 16 . 
plaza ja , torres - cabala c , ivan d , et al : her - 2 / neu expression in extramammary paget disease : a clinicopathologic and immunohistochemistry study of 47 cases with and without underlying malignancy . 
villada g , farooq u , yu w , et al : extramammary paget disease of the vulva with underlying mammary - like lobular carcinoma : a case report and review of the literature . 
tanaka r , sasajima y , tsuda h , et al : concordance of the her2 protein and gene status between primary and corresponding lymph node metastatic sites of extramammary paget disease . 
tessier - cloutier b , asleh - aburaya k , shah v , et al : molecular subtyping of mammary - like adenocarcinoma of the vulva shows molecular similarity to breast carcinomas . 
barth p , dulaimi al - saleem e , edwards kw , et al : metastatic extramammary pagets disease of scrotum responds completely to single agent trastuzumab in a hemodialysis patient : case report , molecular profiling and brief review of the literature . 
arnould l , gelly m , penault - llorca f , et al : trastuzumab - based treatment of her2 - positive breast cancer : an antibody - dependent cellular cytotoxicity mechanism ? br j cancer 94 : 259 - 267 , 2006 31 . 
lewis phillips gd , li g , dugger dl , et al : targeting her2 - positive breast cancer with trastuzumab - dm1 , an antibody - cytotoxic drug conjugate . 
krop ie , kim sb , martin ag , et al : trastuzumab emtansine versus treatment of physicians choice in patients with previously treated her2 - positive metastatic breast cancer ( th3resa ) : final overall survival results from a randomised open - label phase 3 trial . 
thuss - patience pc , shah ma , ohtsu a , et al : trastuzumab emtansine versus taxane use for previously treated her2 - positive locally advanced or metastatic gastric or gastro - oesophageal junction adenocarcinoma ( gatsby ) : an international randomised , open - label , adaptive , phase 2 / 3 study . 
lousberg l , collignon j , jerusalem g : resistance to therapy in estrogen receptor positive and human epidermal growth factor 2 positive breast cancers : progress with latest therapeutic strategies . 
kaufman b , mackey jr , clemens mr , et al : trastuzumab plus anastrozole versus anastrozole alone for the treatment of postmenopausal women with human epidermal growth factor receptor 2 - positive , hormone receptor - positive metastatic breast cancer : results from the randomized phase iii tandem study . 
 r predictive and prognostic properties of human equilibrative nucleoside transporter 1 expression in gemcitabine - treated pancreatobiliary cancer : a metaanalysis purpose gemcitabine , the primary drug for the treatment of pancreatobiliary cancer ( pbc ) , requires human equilibrative nucleoside transporter 1 ( hent1 ) to enter cells . 
 high tumoral hent1 expression has been linked with improved survival among patients with pbc treated with gemcitabine ; however , this finding has been inconsistent , and studies used different expression assays . methods databases were reviewed for studies that examined hent1 and clinical outcome in pbc . 
five studies were excluded for redundancy , and 29 studies underwent detailed review . results on average , 51% of tumor samples had high hent1 expression ( range , 7% to 92% )  . 
among studies that examined hent1 expression and overall survival ( os ) , 58% ( 15 of 26 studies ) showed an association between high tumoral hent1 and improved os for gemcitabine - treated patients . 
an association between high tumoral hent1 expression and improved disease - free / progression - free survival ( dfs / pfs ) was demonstrated in 71% of studies ( 15 of 21 studies )  . 
2018 by american society of clinical oncology introduction the human equilibrative nucleoside transporter 1 ( hent1 ) is a member of the ent family of a sodium - independent nucleoside transporters encoded by the slc29 gene family . 
this meta - analysis examined whether immunohistochemistry of human equilibrative nucleoside transporter 1 ( hent1 ) had prognostic and / or predictive correlation with outcomes in gemcitabine - treated patients with pancreatobiliary cancer . 
it contains 29 studies and , to our knowledge , is the largest such meta - analysis to date . knowledge generated the pooled hazard ratio analyses demonstrated a link between high hent1 tumor expression and improved overall survival and disease - free / progression - free survival . 
this relationship is antibody dependent . relevance use of a validated hent1 immunohistochemistry assay before treatment could allow stratification of patients who would respond to gemcitabine and those who would not . 
the nonresponders could then be treated with another regimen , which would unnecessary toxicity and delay of effective treatment . the hent family of nucleoside transporters and hent1 in particularis expressed in many different types of malignant tumors , including colorectal cancer , 3 gastric cancer , 4 and pancreatobiliary cancer ( pbc ) .5 immunohistochemistry ( ihc ) analyses from various studies have shown that , on average , 51% of human pbcs display strong hent1 expression , although the rates of high or positive expression vary wildly , from as low as 7% to as high as 92% ( table 1 ) .6 - 34 interest in the hent family of transporters increased after it was found that they play a crucial role in facilitating the entry of cytotoxic nucleoside analogs , such as gemcitabine , cytarabine , and fluorouracil , into malignant cells . 
 gemcitabine relies almost entirely on hent1 and hent2 to gain entry into cells , and early in vitro studies showed that lack of hent1 expression confers high - level resistance to gemcitabine in gi tumor cell lines.35 this makes hent1 expression particularly important in pancreatic and biliary tract cancers , for which gemcitabine based treatments are commonly used.36 , 37 the biology of hent1 and its interactions with key chemotherapy drugs in pancreatic , gastric , colorectal , and other cancers has led to the interest in using hent1 tumor expression as a biomarker to predict the efficacy of gemcitabine and other cytotoxic nucleoside analogs , such as cladribine , cytarabine , fludarabine , fluorouracil , and the fluorouracil prodrug capecitabine . 
 since the late 1990s , at least five antihent1 antibodybased assays were developed to facilitate hent1 tumor expression testing via ihc ; development started with 10d7g2 in edmonton , canada . 
this antibody was first tested in cells of the cns , 38 breast cancer , 39 and hodgkin lymphoma40 and is now commercially developed by angam scientific ( hamden , ct ) and due north ( edmonton , alberta , canada )  . 
subsequently , commercial antibodies were developed by various providers , including the following : 11337 - 1 - ap by proteintech group ( rosemont , il ) , hent1 antibody by mbl international [ hereafter called mbl - hent1 ( woburn , ma ) ] , pab2255 by abnova corporation ( taipei city , taiwan ) , and sp120 by ventana medical systems ( oro valley , az )  . although early studies that used the 10d7g2 antibody assay consistently showed an association between high tumoral hent1 expression and improved outcomes among gemcitabine - treated patients with pbc , 6 - 9 later studies that used a variety of other assays and other antibodies did not consistently demonstrate this link . 
 methods statistical analysis we followed prisma ( preferred reporting items for systematic reviews and meta - analyses ) guidance to perform this study . search strategy we performed a review of the literature using keyword searches of the pubmed and embase databases for articles related to hent1 expression and its impact on pbc outcomes and / or response to treatment . 
the search terms were as follows : human ent1 and its synonyms ( ent1 , hent1 , slc29a1 , human equilibrative nucleoside transporter - 1 ) along with pbc and its synonyms ( pancreatic cancer , biliary tract cancer )  . 
study inclusion criteria included retrospective and prospective studies of patients with pbc , at least one subpopulation treated with gemcitabine , expression of hent1 reported and related to patient outcome , published in english , and full text available with a cutoff date of march 1 , 2018 . 
to determine if there were studies with redundant populations , studies by the same authors were examined to determine the type of cancer , the source of the patient sample , the study location ( s ) , the dates of patient inclusion , and the expression assay used . 
if there was consensus overlap in all of these criteria , then the older or smaller study was excluded unless the newer study was a subpopulation analysis . data extraction the data were extracted from the relevant studies into premade spreadsheets by one author and included the following : author ( s ) and affiliation ( s ) ; publication date ; journal ; cancer type ; treatment setting ; number of patients ; type of clinical sample ; methodology to determine hent1 expression ( if ihc , the antibody used ) ; hent1 scoring ; distribution of high and low hent1 ; and median overall survival ( os ) , disease - free survival ( dfs ) / relapse - free survival ( rfs ) , and progression - free survival ( pfs ) along with hazard ratios ( hrs ) , cis , and p values . 
single - arm studies that demonstrated association between hent1 expression and patient outcome were classified as positive for correlation . detailed analyses were only performed on ihcbased studies because of the low number of studies using other methods . 
the 2 test was performed with excel ( version 2013 ; microsoft , redmond , wa ) to determine whether the choice of antibody was associated with a significant difference in the likelihood of demonstrating a relationship between hent1 and clinical outcomes . 
forest plots were produced with code written by author d.y. each evaluable ihc study was examined using the remark ( reporting recommendations for tumor marker prognostic studies ) recommendations for completeness of biomarker reporting.41 this 20 - point checklist was completed with the corresponding page number for each study ; a point was awarded for each guideline completed , and half of a point was awarded for guidelines that were partially completed . 
 because the majority of studies were retrospective , sample sizes were not calculated as with a prospective study ; the rationale for sample size guideline was examined for effect size or power calculation on the basis of the available sample size . 
seventeen were removed because they were letters , editorials , or comments on studies ; 57 were reviews or meta - analyses ; 39 were animal models or laboratory experiments ; seven were in other cancer types ; one was a cost analysis ; one was a trial design ; and one was a pathology methodology paper . there were 51 studies identified for detailed analysis . 
among these , 34 examined hent1 expression relationship with clinical outcome using antihent1 antibodies , 6 - 34 , 42 - 46 whereas 10 used reverse transcriptase polymerase chain reaction , 47 - 56 and one used liquid chromatography / tandem mass spectrometry quantitative proteomics of hent1.57 there were two sets of studies that used the same patient population but different antibody assays , and both studies in each set were included in the analysis.11 , 12 , 19 , 28 of the 34 ihc studies , five were removed for redundant populations and assays.42 - 46 the remaining studies were excluded because of the following : the study examined small nucleotide polymorphisms of hent1 rather than expression ( n = 4 ) , 58 - 61 the study was a clinical trial exclusively in patients with low / negative hent1 ( n = 1 ) , 62 or the study did not correlate hent1 expression with survival ( n = 1 ; correlation with gemcitabine incorporation into dna63 )  . there was no consensus among studies that examined hent1 expression by reverse transcriptase polymerase chain reaction and improved survival . 
of the nine evaluable studies , five showed a correlation between hent1 mrna levels and improved survival.47 - 50 , 55 this may be in part to the differences in primers ( only two studies used the same set of primers ) , the gene ( s ) used for normalization of expression ( eg , gapdh ) , and the source of the mrna ( frozen , fresh , formalin - fixed , paraffin - embedded [ ffpe ] samples )  . 
 high hent1 and improved survival , 47 , 49 , 50 , 55 including one that used both ffpe and fresh biopsy samples in rna later.55 there was good concordance between hent1 expression from both preservation methods . 
the one study that was not evaluable performed survival analysis with multiple genes ( including hent1 ) but also examined the consistency of mrna and protein expression patterns of hent1.56 this analysis showed a reverse concordance : samples that had high mrna had low protein , and vice versa . the study that used liquid chromatography / tandem mass spectrometry to quantify hent1 expression was a pilot study and only examined eight patient samples . 
the study did not find a significant correlation between expression as a continuous variable and pfs.57 among the 29 ihc studies , a total of 3 , 979 patients were examined for hent1 expression , of whom 2 , 285 were treated with gemcitabine chemotherapy . 
more than half of these studies ( 18 of 29 , or 62% ) demonstrated that high hent1 tumor expression was significantly associated with improved outcomes ( at least one of the survival outcomes [ os , dfs , or pfs ] and / or time to progression [ ttp ] in gemcitabine - treated patients with pbc ; table 26 - 34 )  . 
 two of the studies found the opposite association : high hent1 was associated with shorter dfs or ttp ; eight studies showed no relationship between hent1 expression and survival outcomes . 
one study did not report p values for the survival analysis , although os was longer in the high - hent1 subpopulation . the first study to examine the relationship between hent1 expression and clinical outcome used the 10d7g2 mouse monoclonal antihent1 antibody.6 each study that used the 10d7g2 antihent1 antibody demonstrated a significant association between high hent1 tumor expression and improved dfs or pfs outcomes in gemcitabine - treated patients ( six of six studies , or 100% )  . 
in addition , all but one study that used the 10d7g2 anti - hent1 antibody demonstrated a significant association between high hent1 tumor expression and improved os outcomes in gemcitabine - treated patients ( seven of eight studies , or 88% )  . 
studies that used the mbl - hent1 antibody showed a correlation between hent1 expression and improved os ( two of two studies , or 100% ) and dfs / pfs ( one of one , or 100% )  . 
of the studies that used the pab2255 antibody , 67% showed a statistically significant and favorable association between high tumoral hent1 expression and improved outcomes ( two of three studies for both os and dfs / pfs )  . 
only 50% of studies that used the 11337 - 1 - ap antibody showed a statistically significant link ( one of two studies for each of os and dfs / pfs )  . 
studies using the sp120 antibody had the worst correlation rate : only two ( 22% ) of nine studies demonstrated significant os associations , and three ( 43% ) of seven demonstrated a dfs / pfs association . 
these antibody - dependent differences in prognostic abilities had a 2 test p of .024 for os and a p of .086 for dfs / pfs for the three most commonly used antibodies ( 10d7g2 [ n = 8 for os and 6 for dfs / pfs ] , sp120 [ n = 9 for os and 7 for dfs / pfs ] , and pab2255 [ n = 3 for os and 3 for dfs / pfs ] ; figs 2a and 2b )  . we also examined whether the lack of consistency among studies was due to treatment intent and found no difference in the proportion of studies in each setting ( 2 p = .18 ; fig 2c )  . 
among the studies that found a positive association between high hent1 and improved survival , 26% ( five of 19 studies ) were from the advanced setting , whereas 63% ( 12 of 19 studies ) and 11% ( two of 19 studies ) were adjuvant and neoadjuvant , respectively . 
the treatment setting for the negative association studies was 20% advanced ( two of 10 studies ) , 60% adjuvent ( six of 10 studies ) , and 20% neoadjuvant ( two of 10 studies )  . 
2 analysis for the positive association rates of studies that was based on the three most commonly used antibodies ( 10d7g2 , sp120 and pab2255 ) reveals a p value of .0237 for ( a ) overall survival and of .855 for ( b ) disease - free / progression - free survival , which suggests that antibody choice influences the likelihood of finding a statistically significant relationship between high tumoral human equilibrative nucleoside transporter 1 ( hent1 ) expression and improved outcomes . 
in the funnel plot , only four studies had an hr : sample size value outside the normalized mean shape , and they did not alter the overall funnel shape ( fig 4b )  . discussion protein abundance of hent1 in malignant cells is a promising predictive and prognostic biomarker for pbc tumors treated with gemcitabine chemotherapy . 
in the radiation therapy oncology group prospective , randomized , adjuvant clinical trial rtog 9704 that compared gemcitabine with fluorouracil , high hent1 expression assessed with the 10d7g2 antibody was associated with improved dfs and os in patients treated with gemcitabine but not in those treated with fluorouracil.7 this established hent1 as a predictive , not simply a prognostic , biomarker , because this effect was gemcitabine specific and the interaction between treatment allocation and hent1 status was significant . 
 randomized phase ii trial ( total enrollment , n = 367 ) investigated the prognostic role of hent1 in patients treated with co - 101 ( a lipid - drug conjugate of gemcitabine ) versus gemcitabine in metastatic pancreatic cancer . 
this study , which used the sp120 antibody , did not demonstrate a correlation of hent1 expression with outcomes in either treatment group.25 another sp120 study from the aio - pk0104 ( arbeitsgemeinschaft internistische onkologie ) randomized phase iii trial demonstrated that hent1 overexpression lacked a prognostic relationship with survival for patients treated with gemcitabine - based chemotherapy compared with surgery alone.27 all of the studies included in this meta - analysis examined the relationship between hent1 protein levels and outcomes in gemcitabine treated patients with pbc . 
although the pooled hr analyses for all studies and adjuvant alone demonstrated relationships between hent1 expression and os and dfs / pfs , this was not seen in the advanced / metastatic setting . 
this is likely the result of the small number of studies included in the analyses ( n = 4 and n = 3 , respectively ) and the antibodies used . 
the 10d7g2 studies had positive associations between high hent1 expression and clinical outcomes , 9 , 10 whereas the sp120 studies had no relationship with os and a negative association between high hent1 expression and pfs.25 , 26 the effect of hent1 expression on patient outcomes , regardless of therapy , has also been investigated . 
in this study , untreated patients with a high expression of hent1 had shorter median survival than patients with low hent1 expression , which indicated that high hent1 expression may be an independent negative prognostic factor . 
this relationship was not found in other studies.11 , 13 , 30 our analyses demonstrate that the likelihood of a positive prognostic study may be influenced by the anti - hent1 antibody used . 
whereas 100% of studies that used the 10d7g2 antibody found a relationship between high hent1 expression and improved os and / or dfs / pfs in patients with pbc who were treated with gemcitabine , signals from other antibodies were less consistent . during the past decade , at least eight different antibodies against ent1 have been developed by biotechnology firms in canada , taiwan , and the united states . 
the first of such antibodies , named 10d7g2 , was developed in edmonton , canada , at the cross cancer institute.38 approximately one third of all published studies about hent1 expression in pbc have used this murine monoclonal antibody . 
subsequent to the development of 10d7g2 , several rabbit - derived antibodies against hent1 for the purposes of ihc were separately developed and commercialized : 11337 - 1 - ap , mbl - hent1 , pab2255 , and sp120 . 
this antibody is an igg , rabbit - derived monoclonal antibody distributed by ventana medical systems in the united states for laboratory use on ffpe tissue stained with the benchmark ihc stainer ( ventana )  . as seen in figure 2 , there is a striking difference in the outcome correlation rates of studies that used the 10d7g2 antibody compared with those that used other antibodies . 
whereas studies that used the 10d7g2 antibody had an 88% to 100% rate of association between high hent1 and improved os and dfs / pfs , the correlation rates with other antibodies were lower . 
the set stained with the leica bond polymer refine detection kit ( leica biosystems , wetzlar , germany ) had very few highly stained cells compared with set 2 , which was stained using the recommended iview dab detection kit ( ventana ) .28 the same cut point was used for each pair of these comparisons , so this did not add an additional potential confounding factor . 
 another study compared cut point methodology for the same antibody and patient population.27 there were similar proportions of high and low hent1 expressers with each cut point , and neither resulted in a significant association between sp120 - determined hent1 expression and survival . both of the studies that performed direct antibody comparisons between sp120 and 10d7g2 showed that a high proportion of cells had discordant staining . 
the cluster with high staining with both antibodies was the most sensitive , the cluster with high 10d7g2 and low sp120 had an intermediate response , and the double - low cluster had a poor response . 
the high 10d7g2 and low sp120 discordant staining pattern occurred in 31% of the samples ( 70 of 227 samples ) .14 within set 2 of the study by svrcek et al28 , only 45% of high hent1 samples ( 25 of 55 samples ) , and 59% of low hent1 samples ( 49 of 83 samples ) were matched between the sp120 and 10d7g2 antibodies . 
monoclonal antibodies differ in target epitope , method of generation , and species of origconsequently , they may bind to different sites on the hent1 glycoprotein , or theoretically different isoforms , in a manner that influences the staining pattern , intensity , or specificity for formalin - fixed hent1 . 
studies have shown that differential expression of gemcitabine metabolism proteins , including deoxycytidine kinase , ribonucleoside reductases m1 and m2 , and hu antigen r , as well as other signaling pathways can affect clinical outcomes to gemcitabine treatment.11 , 64 , 65 in conclusion , on the basis of a comprehensive review of the literature , the likelihood of finding a predictive , prognostic , or correlative link between hent1 tumor expression and outcomes for gemcitabine - treated patients with pbc is linked to the anti - hent1 antibody used . 
 the ability to predict response to gemcitabine therapy according to the hent1 expression profile would allow stratification of patients with high hent1 to gemcitabine monotherapy or gemcitabine - containing regimens and patients with low hent1 to other regimens , including the combination chemotherapy folfirinox . 
spratlin honoraria : celgene , sanofi , lilly , imclone consulting or advisory role : celgene , sanofi , lilly , imclone , taiho pharmaceutical , astellas pharma research funding : sanofi travel , accommodations , expenses : astellas pharma john r . 
park j : human equilibrative nucleoside transporter subtype 1 : structure - function analysis using cysteine mutagenesis and thiol modifying techniques , pharmacology and toxicology [ doctoral thesis ]  . 
spratlin j , sangha r , glubrecht d , et al : the absence of human equilibrative nucleoside transporter 1 is associated with reduced survival in patients with gemcitabine - treated pancreas adenocarcinoma . 
marchal r , mackey jr , lai r , et al : human equilibrative nucleoside transporter 1 and human concentrative nucleoside transporter 3 predict survival after adjuvant gemcitabine therapy in resected pancreatic adenocarcinoma . 
santini d , schiavon g , vincenzi b , et al : human equilibrative nucleoside transporter 1 ( hent1 ) levels predict response to gemcitabine in patients with biliary tract cancer ( btc )  . 
borbath i , verbrugghe l , lai r , et al : human equilibrative nucleoside transporter 1 ( hent1 ) expression is a potential predictive tool for response to gemcitabine in patients with advanced cholangiocarcinoma . 
marechal r , bachet jb , mackey jr , et al : levels of gemcitabine transport and metabolism proteins predict survival times of patients treated with gemcitabine for pancreatic adenocarcinoma . 
murata y , hamada t , kishiwada m , et al : human equilibrative nucleoside transporter 1 expression is a strong independent prognostic factor in uicc t3 - t4 pancreatic cancer patients treated with preoperative gemcitabine - based chemoradiotherapy . 
kalloger se , riazy m , tessier - cloutier b , et al : a predictive analysis of the sp120 and 10d7g2 antibodies for human equilibrative nucleoside transporter 1 ( hent1 ) in pancreatic ductal adenocarcinoma treated with adjuvant gemcitabine . 
kawada n , uehara h , katayama k , et al : human equilibrative nucleoside transporter 1 level does not predict prognosis in pancreatic cancer patients treated with neoadjuvant chemoradiation including gemcitabine . 
murata a , amano r , yamada n , et al : prognostic predictive values of gemcitabine sensitivityrelated gene products for unresectable or recurrent biliary tract cancer treated with gemcitabine alone . 
woo sm , yoon ka , hong ek , et al : dck expression , a potential predictive biomarker in the adjuvant gemcitabine chemotherapy for biliary tract cancer after surgical resection : results from a phase ii study . 
aoyama t , kazama k , miyagi y , et al : predictive role of human equilibrative nucleoside transporter 1 in patients with pancreatic cancer treated by curative resection and gemcitabineonly adjuvant chemotherapy . 
yamada r , mizuno s , uchida k , et al : human equilibrative nucleoside transporter 1 expression in endoscopic ultrasonography - guided fine - needle aspiration biopsy samples is a strong predictor of clinical response and survival in the patients with pancreatic ductal adenocarcinoma undergoing gemcitabine - based chemoradiotherapy . 
nakagawa n , murakami y , uemura k , et al : combined analysis of intratumoral human equilibrative nucleoside transporter 1 ( hent1 ) and ribonucleotide reductase regulatory subunit m1 ( rrm1 ) expression is a powerful predictor of survival in patients with pancreatic carcinoma treated with adjuvant gemcitabine - based chemotherapy after operative resection . 
sasaki h , murakami y , uemura k , et al : concurrent analysis of human equilibrative nucleoside transporter 1 and ribonucleotide reductase subunit 1 expression increases predictive value for prognosis in cholangiocarcinoma patients treated with adjuvant gemcitabine - based chemotherapy . 
fisher sb , fisher ke , patel sh , et al : excision repair cross - complementing gene - 1 , ribonucleotide reductase subunit m1 , ribonucleotide reductase subunit m2 , and human equilibrative nucleoside transporter - 1 expression and prognostic value in biliary tract malignancy . 
fisher sb , patel sh , bagci p , et al : an analysis of human equilibrative nucleoside transporter - 1 , ribonucleoside reductase subunit m1 , ribonucleoside reductase subunit m2 , and excision repair cross - complementing gene - 1 expression in patients with resected pancreas adenocarcinoma : implications for adjuvant treatment . 
poplin e , wasan h , rolfe l , et al : randomized , multicenter , phase ii study of co - 101 versus gemcitabine in patients with metastatic pancreatic ductal adenocarcinoma : including a prospective evaluation of the role of hent1 in gemcitabine or co - 101 sensitivity . 
ormanns s , heinemann v , raponi m , et al : human equilibrative nucleoside transporter 1 is not predictive for gemcitabine efficacy in advanced pancreatic cancer : translational results from the aio - pk0104 phase iii study with the clone sp120 rabbit antibody . 
sinn m , riess h , sinn bv , et al : human equilibrative nucleoside transporter 1 expression analysed by the clone sp 120 rabbit antibody is not predictive in patients with pancreatic cancer treated with adjuvant gemcitabine : results from the conko - 001 trial . 
svrcek m , cros j , marchal r , et al : human equilibrative nucleoside transporter 1 testing in pancreatic ductal adenocarcinoma : a comparison between murine and rabbit antibodies . 
elebro j , ben dror l , heby m , et al : prognostic effect of hent1 , dck and hur expression by morphological type in periampullary adenocarcinoma , including pancreatic cancer . 
brandi g , deserti m , vasuri f , et al : membrane localization of human equilibrative nucleoside transporter 1 in tumor cells may predict response to adjuvant gemcitabine in resected cholangiocarcinoma patients . 
xiao jc , zhang tp , zhao yp : human equilibrative nucleoside transporter 1 ( hent1 ) predicts the asian patient response to gemcitabine - based chemotherapy in pancreatic cancer . 
neoptolemos jp , palmer dh , ghaneh p , et al : comparison of adjuvant gemcitabine and capecitabine with gemcitabine monotherapy in patients with resected pancreatic cancer ( espac - 4 ) : a multicentre , open - label , randomised , phase 3 trial . 
jennings ll , hao c , cabrita ma , et al : distinct regional distribution of human equilibrative nucleoside transporter proteins 1 and 2 ( hent1 and hent2 ) in the central nervous syste neuropharmacology 40 : 722 - 731 , 2001 39 . 
reiman t , clarke ml , dabbagh l , et al : differential expression of human equilibrative nucleoside transporter 1 ( hent1 ) protein in the reed - sternberg cells of hodgkins disease . 
kondo n , murakami y , uemura k , et al : combined analysis of dihydropyrimidine dehydrogenase and human equilibrative nucleoside transporter 1 expression predicts survival of pancreatic carcinoma patients treated with adjuvant gemcitabine plus s - 1 chemotherapy after surgical resection . 
morinaga s , nakamura y , watanabe t , et al : immunohistochemical analysis of human equilibrative nucleoside transporter - 1 ( hent1 ) predicts survival in resected pancreatic cancer patients treated with adjuvant gemcitabine monotherapy . 
kobayashi h , murakami y , uemura k , et al : human equilibrative nucleoside transporter 1 expression predicts survival of advanced cholangiocarcinoma patients treated with gemcitabinebased adjuvant chemotherapy after surgical resection . 
kobayashi m , mizuno s , murata y , et al : gemcitabine - based chemoradiotherapy followed by surgery for borderline resectable and locally unresectable pancreatic ductal adenocarcinoma : significance of the ca19 - 9 reduction rate and intratumoral human equilibrative nucleoside transporter 1 expression . 
giovannetti e , del tacca m , mey v , et al : transcription analysis of human equilibrative nucleoside transporter - 1 predicts survival in pancreas cancer patients treated with gemcitabine . 
kim r , tan a , lai kk , et al : prognostic roles of human equilibrative transporter 1 ( hent - 1 ) and ribonucleoside reductase subunit m1 ( rrm1 ) in resected pancreatic cancer . 
orlandi a , calegari ma , martini m , et al : gemcitabine versus folfirinox in patients with advanced pancreatic adenocarcinoma hent1 - positive : everything was not too bad back when everything seemed worse . 
ashida r , nakata b , shigekawa m , et al : gemcitabine sensitivity - related mrna expression in endoscopic ultrasound - guided fine - needle aspiration biopsy of unresectable pancreatic cancer . 
sierzega m , pach r , kulig p , et al : prognostic implications of expression profiling for gemcitabine - related genes ( hent1 , dck , rrm1 , rrm2 ) in patients with resectable pancreatic adenocarcinoma receiving adjuvant chemotherapy . 
ohmine k , kawaguchi k , ohtsuki s , et al : quantitative targeted proteomics of pancreatic cancer : deoxycytidine kinase protein level correlates to progression - free survival of patients receiving gemcitabine treatment . 
tanaka m , javle m , dong x , et al : gemcitabine metabolic and transporter gene polymorphisms are associated with drug toxicity and efficacy in patients with locally advanced pancreatic cancer . 
li d , pant s , ryan dp , et al : a phase ii , open - label , multicenter study to evaluate the antitumor efficacy of co - 1.01 as second - line therapy for gemcitabine - refractory patients with stage iv pancreatic adenocarcinoma and negative tumor hent1 expression . 
costantino cl , witkiewicz ak , kuwano y , et al : the role of hur in gemcitabine efficacy in pancreatic cancer : hur up - regulates the expression of the gemcitabine metabolizing enzyme deoxycytidine kinase . 
ferguson , mbbs , mrcp , frcr4 ; alessandra curioni - fontecedro , md , pd1 ; martin zoche , phd1 ; holger moch , md1 ; and bart vrugt , md , phd1 introduction malignant mesothelioma ( mm ) is a rare neoplasm arising from the mesothelial cell lining of the pleura ( 80% to 90% ) , but it can also develop in the peritoneum ( 10% to 15% ) , pericardium , and tunica vaginalis.1 , 2 because of the long latency of the disease , the age at diagnosis ranges from 54 to 65 years3 - 5 ; thus , mm rarely occurs in young patients and children . the frequency of both pleural and peritoneal mm is almost equal in patients younger than age 40 years . 
in addition , previous asbestos exposure is less common in peritoneal compared with pleural mm.6 peritoneal mesothelioma commonly presents with vague and unspecic symptoms , including abdominal distension and weight loss.7 consequently , the diagnosis of peritoneal mesothelioma generally is made at an advanced stage of the disease , 8 which partly explains the poor clinical outcome , even after optimal oncologic and surgical treatment . 
the median survival is less than 12 months in the majority of patients.9 , 10 patients with mm are not routinely analyzed for the presence of potentially drug - targetable gene mutations , including anaplastic lymphoma tyrosine kinase ( alk )  . 
by using targeted next - generation sequencing of tumor dna and rna , hung et al11 recently identied alk rearrangements with three novel alk fusion partners in a small subgroup of patients with peritoneal mm . 
the alk breakpoint was mapped to intron 19 in all three cases , the strn breakpoint to intron 3 , the atg16l1 breakpoint to intron 2 , and the tpmi1 breakpoint to the beginning of exon 9 . here we report a 13 - year - old girl with peritoneal mesothelioma harboring an strn - alk gene fusion , which was identied by comprehensive genomic proling ( cgp )  . 
images before treatment showed large - volume ascites , bilateral hydrotissue nephrosis , and widespread peritoneal soft deposits particularly within the pelvis and around the liver with scalloping of the liver margin ( fig 1 )  . 
she had no history of asbestos or radiation exposure . peritoneal biopsies were obtained by an explorative laparoscopy and submitted for a histologic and molecular work - up . pathology multiple peritoneal biopsies revealed a tumor with a mixed tubulopapillary and solid growth pattern . immunohistochemistry with positivity for ck5 / 6 and calretinin in the absence of expression of the epithelial marker berep - 4 and claudin - 4 conrmed the mesothelial origin of the tumor . 
even though necrosis and mitotic activity ( less than 1 mitosis per 10 high - power elds ) were not evident , growth pattern , cytonuclear atypia of the tumor cells , and immune prole fullled the criteria for malignant epithelioid mesothelioma ( figs 2a and 2b )  . 
images before treatment demonstrate large - volume ascites , bilateral hydronephrosis , and widespread peritoneal soft tissue deposits particularly within the pelvis ( all ndings marked by green arrows )  . 
 ( b ) schematic representation of the strn - alk fusion showing the 700 - amino - acid ( aa ) - long fusion gene , with 137 aas originating from the n terminus of strn and the remaining 562 aas originating from the c terminus of alk . 
 ( a ) immunohistochemical staining showing strong expression of alk protein ( original magnication 40 )  . ( b ) alk uorescent in situ hybridization break - apart probes showing one fused red - green signal and one red signal in 96% of the tumor cells compatible with 5 ( cid : 1 ) alk deletion ( original magnication 100 )  . for a transient and self - limiting rise in alanine aminotransferase ( alt ) , no adverse effects were noted . discussion we report a dramatic tumor regression after the rst cycle of ceritinib in a 13 - year - old girl with alk - rearranged peritoneal mm . 
the alk receptor can be translocated , mutated , or overexpressed in non - squamous nonsmall - cell lung cancer , inammatory myobroblastic tumors ( imts ) , and anaplastic large - cell lymphomas ( alcls ) .14 it is well known that pediatric patients with imts or alcl can show strong and sustained clinical response to the inhibitory effects of the alk - targeting drug crizotinib.15 peritoneal mm is an extremely rare tumor in children , but an almost complete response to an alk - targeting drug has never been described before . 
recently , loharamtaweethong et al16 reported a 10 - year - old girl with an alk - translocated peritoneal mm detected by fish and immunohistochemistry , but the exact fusion partner was not determined . 
genetic alterations frequently seen in pleural and peritoneal mesothelioma , including cdkn2a , bap - 1 , nf2 , and setd2 , are not detected in patients with alk - rearranged disease.11 even though the prevalence of alk - rearranged peritoneal mesotheliomas is low , alk alterations seem to be restricted to peritoneal mesothelioma only and do not occur in pleural mm . 
this is consistent with our observation , because we were unable to demonstrate alk expression in a tissue microarray containing tumor samples from 221 patients with pleural mesothelioma , including 22 females of whom three were younger than age 40 years ( unpublished data )  . in our patients tumor sample , we identied a rare alk fusion by cgp . 
the platform used in this study can detect all alk alterations ( including atg16l1 and tpm1 fusions ) and has been extensively validated.12 even in alk fishnegative patients , alk rearrangements could be identied by using this method.18 in our patient , we detected an strn - alk fusion with similar breakpoints but with a fusion in the alk - rearranged patient protein identical described by hung et al.11 the strn - alk fusion was an inframe alteration with breakpoints for strn in intron 3 and alk in intron 19 . to that patients with alk - rearranged mm cannot be distinguished from their conventional counterparts , but the differences in clinical presentation ( young patients with no history of asbestos exposure ) and the detection of isolated alk mutations would justify a separate tumor entity of alk - rearranged mesothelioma . 
in the absence of genetic alterations of cdkn2a , bap - 1 , nf2 , and setd2 , it is tempting to speculate that fusions involving the alk gene may represent the predominant oncogenic driver in peritoneal mm in young female patients . 
on the basis of the success of therapeutic alk inhibition in our patient , alk fusion screening by cgp is highly advised in peritoneal mms , especially in young patients . 
the promising response in our patient also stresses the need for investigations involving a larger cohort of young female patients with alkrearranged peritoneal mm to determine the efcacy and durability of alk inhibition . 
r uschoff , elise gradhand , alessandra curioni - fontecedro , martin zoche , holger moch , bart vrugt administrative support : holger moch provision of study materials or patients : elise gradhand , helen rees , martin zoche , bart vrugt collection and assembly of data : elise gradhand , abdullah kahraman , helen rees , jane l . 
hoffmann - la roche holger moch honoraria : roche consulting or advisory role : roche , deniens research funding : roche ( inst ) travel , accommodations , expenses : roche pharma travel , accommodations , expenses : deniens no other potential conicts of interest were reported . references 20 : 935 - 944 , 2009 ray m , kindler hl : malignant pleural mesothelioma : an update on biomarkers and treatment . 
cancer causes control beebe - dimmer jl , fryzek jp , yee cl , et al : mesothelioma in the united states : a surveillance , epidemiology , and end results ( seer ) - medicare investigation of treatment patterns and overall survival . 
eur j gynaecol oncol 18 : 141 - 143 , 1997 asensio ja , goldblatt p , thomford nr : primary malignant peritoneal mesothelioma : a report of seven cases and a review of the literature . 
arch surg 125 : 1477 - 1481 , 1990 thomas a , chen y , yu t , et al : distinctive clinical characteristics of malignant mesothelioma in young patients . 
tumori 89 : 269 - 273 , 2003 kaya h , sezgi c , tanrikulu ac , et al : prognostic factors inuencing survival in 35 patients with malignant peritoneal mesothelioma . 
fabrizio da , george tj jr , dunne rf , et al : beyond microsatellite testing : assessment of tumor mutational burden identies subsets of colorectal cancer who may respond to immune checkpoint inhibition . 
moss e yp , voss sd , lim ms , et al : targeting alk with crizotinib in pediatric anaplastic large cell lymphoma and inammatory myobroblastic tumor : a childrens oncology group study . 
loharamtaweethong k , puripat n , aoonjai n , et al : anaplastic lymphoma kinase ( alk ) translocation in paediatric malignant peritoneal mesothelioma : a case report of novel alk - related tumour spectruhistopathology 68 : 603 - 607 , 2016 17 . 
ali sm , ou shi , he j , et al : identifying alk rearrangements that are not detected by fish with targeted next - generation sequencing of lung carcinoma . 
brodeur , md1 , 5 , 7 purpose the diagnosis of cancer predisposition in pediatric patients with cancer is vital for treatment decisions , surveillance , and management of at - risk family members . 
characteristics of patients who underwent constitutional testing , including family history and variant loss of heterozygosity , were tracked . results from 1 , 023 patients who underwent somatic tumor sequencing in a 28 - month period , 210 variants were identied in 141 patients ( 13.8% ) that were concerning for cancer predisposition syndromes requiring intervention . 
among patients tested , 23 ( 56.1% ) of 41 total patients were diagnosed with a cancer predisposition syndrome . conclusion our data demonstrate that more than one third of variants in tumor somatic sequencing that were concerning for underlying cancer predisposition were constitutionally conrmed . 
2019 by american society of clinical oncology introduction the incidence of constitutional genetic aberrations leading to cancer predisposition in pediatric patients with cancer is estimated at 8% to 12%.1 - 4 results from large somatic sequencing studies conrm that this rate of constitutional aberrations can be inferred from somatic sequencing results.5 , 6 the timely diagnosis of a cancer predisposition syndrome can have signicant implications not only for the patient , but also for affected and unaffected family members . 
follow - up of any features of a cancer predisposition syndrome is essential for appropriate management , surveillance , and family planning for these individuals . clinical and historical features that suggest a pediatric patient with cancer may have a predisposition syndrome include a family history of the same cancer or related cancers , clinical features of a predisposition syndrome , bilateral / multifocal primary cancer or multiple cancers , certain types of cancer that rarely occur except in a predisposed patient , and a much earlier than expected age at diagnosis . 
 macfarland et al context key objective when a child is diagnosed with cancer , most families are uncertain why their child developed a rare and devastating disease . for an important minority , an underlying cancer predisposition syndrome as a result of a constitutional pathogenic variant may underlie malignancy ; however , these can be difcult to identify in patients who lack syndromic features and / or a family history of cancer . knowledge generated this study demonstrates that pathogenic variants identied through somatic next - generation sequencing can indicate the presence of a constitutional cancer - predisposing variant . 
as tumor sequencing becomes more common and comprehensive , it is important for both patients and family members that appropriate measures are in place for genetic counseling and subsequent constitutional testing . 
in this report , we describe our experience in conducting cancer predisposition evaluation on the basis of somatic sequencing ndings in cancer samples identied using in - house next - generation sequencing testing panels . 
these targeted panels provided comprehensive analysis of hematologic and solid tumors for fusion genes , single - nucleotide variants , insertion / deletion variants , and copy number variants for 117 to 238 genes13 , 14 ( appendix table a1 )  . 
once identied in tumor tissue , the potential for suspicious variants to be of constitutional origin was described in the somatic tumor report issued to the ordering physician , usually an oncologist . testing , these suspected constitutional variants were tagged by the genomic diagnostics laboratory for referral to the cancer predisposition program ( cpp )  . 
somatic panel testing was not used as a substitute for clinical judgment , and a patient with concerning features of cancer predisposition would always be referred to the cpp regardless of somatic panel results . 
 pediatric somatic sequencing identies cancer predisposition available , but a complete family history review was not conducted by an oncologist , geneticist , or genetic counselor for patients not seen by the cpp . 
factors included in the analysis of patients seen by the cpp included family history of cancer , presence of multifocal or bilateral disease , loss of heterozygosity in the tumor , and clinical features of diagnosis , such as clinical diagnostic criteria for neurobromatosis type 1 ( nf1 ) or tuberous sclerosis , in addition to the aforementioned referral criteria . 
tumor type association with the presence of a constitutional variant for example , rhabdoid tumor and smarcb1 pathogenic variantwas also tracked.7 data also included age at diagnosis and turnaround time of constitutional testing ( days between the result report of somatic testing and the date of constitutional testing )  . 
as detailed in results , some variants had been previously constitutionally conrmed , and some were conrmed on the basis of clinical features alone and did not have testing completed . 
constitutional testing was listed as incomplete in all cases in which the cpp did not meet with the family to discuss the variant , even if testing may not ultimately have been suggested . 
finally , results were deemed conclusive when the genetic testing was interpretable and inconclusive when additional sample would be required to make a denitive diagnosis . data were analyzed using stata and r statistical programming languages , and statistical signicance was determined using wilcoxon rank - sum test and 2 test , when appropriate . 
visualizations were generated using the ggplot2 package.16 the human investigations performed in this study were completed after approval by the childrens hospital of philadelphia institutional review board and in accordance with the requirements of the department of health and human services , where appropriate . results population demographics records were reviewed for 1 , 023 patients who underwent somatic panel testing between february 2016 and june 2018 . 
a total of 210 somatic variants in 141 patients were suspected to be constitutional pathogenic or likely pathogenic variants , which is 13.8% of patients who underwent somatic panel testing during this time period . 
vaf met criteria for referral ( 0.4 ) in 78% of cases ( n = 164 ) , including those with copy number variant in a potential cancer predisposition gene . 
the remainder of variants were referred for the presence of a founder mutation ( 2.9% ; n = 6 ) or a tumor typespecic variant concerning for cancer predisposition ( 55.2% ; n = 116 ) , and some patients were referred for multiple reasons . 
demographics demographic age , years , mean female male race / ethnicity white black other hispanic / latino primary v relapse primary relapse / recurrent somatic findings in predisposition genes a total of 210 variants were identied in a total of 66 genes . a heatmap of genes with a suspected constitutional variant identied in 2 or more individuals is shown in figure 1 ( full variant list , including tumor type , in appendix table a2 )  . 
the second most frequent gene with potential constitutional variants was nf1 ( 17% ; n = 27 , including 6 individuals with an identied tp53 variant ) , followed by smarcb1 , mutyh , apc , atm , msh6 , wt1 , and dicer1 . 
founder mutations were identied in 6 patientsfor example , 4 of the 7 variants identied in apc were i1307k , a known founder risk allele in the ashkenazi jewish population that does not lead to familial adenomatous polyposis , but confers increased cancer risk in adulthood.17 some variants identied had implications for immediate treatment decisions , such as tp53 variants that were identied in patients with anticipated radiation therapy , and an attempt was made to prioritize these patients for testing and counseling . 
frequency of concerning somatic variants identied , as described by frequency , tumor type ( liquid , non - cns solid , and cns ) , primary versus relapsed / refractory specimen , and specic cancer diagnosis . 
aml , acute myeloblastic leukemia ; at / rt , atypical teratoid / rhabdoid tumor ; b - all , b - acute lymphoblastic leukemia ; dnet , dysembryoplastic neuroepithelial tumor ; gist , gi stromal tumor ; jmml , juvenile myelomonocytic leukemia ; mpnst , malignant peripheral nerve sheath tumor ; t - all , t - acute lymphoblastic leukemia . dicer1 , wt1 , 11p loss / aberrations , and non - i1307k apc variantsand in these cases screening was initiated during ongoing cancer treatment after a constitutional diagnosis was made . of note , several heterozygous variants identied had implications for adult cancer risk , but did not change management in the pediatric age range ( msh6 , mutyh , atm , brca1 , and brca2 )  . 
testing decisions on these variants were made on a case - by - case basis , in discussion with the family and the oncologist , and in most cases , it was recommended that constitutional testing of the proband be deferred until age 18 years or older . 
occasionally in adolescent patients , preferred to complete testing . the patient constitutional testing a total of 210 variants were determined to warrant follow - up for potential constitutional alterations on the basis of initial somatic panel review ( fig 2 ; appendix table a2 )  . 
schematic representation of patients referred for constitutional includes patients who did not have testing performed for clinical reasons , patients who still require referral and genetic testing , and results of testing for the patients for whom testing was conducted . 
patients were presumed positive if they met clinical criteria for a diagnosis without the need for genetic testing , and were presumed negative when they did not meet clinical criteria for a diagnosis . 
of the total number of patients with a somatic tp53 variant ( n = 46 ) , 19.6% were constitutionally affected ( n = 9 ; includes those patients who previously tested positive )  . 
this was based on known familial mutations in apc i1307l ( n = 2 ) , brca1 ( n = 1 ) , and brca2 ( n = 1 ) , or on the basis of an existing clinical diagnosis in seven nf1 patients already being observed by the neurobromatosis clinic . in six variants in ve individuals , testing was presumed negative , as individuals did not meet criteria for a syndrome that was not present clinically . 
in these cases , variants were not included in the tested category in subsequent analyses ; however , it is important to note that constitutional testing is not always required to conrm or rule out a diagnosis . 
in some cases , constitutional testing was not performed after discussion with the family , per family preference and / or clinician recommendation . constitutional testing was deferred in a total of 4 variants associated with adult - onset cancer risk until adulthood after family history discussion with family and review of ( apc i1307l [ n = 2 ] , blm [ n = 1 ] , mutyh [ n = 1 ] )  . 
this would likely be the recommendation for other somatic variants in adult - onset predisposition syndromes included in the genetic testing not done category ; however , genetic counseling and family history review need to take place before constitutional testing is deferred completely . 
a total of 8 families8 patients and 12 variantspreferred not to pursue constitutional testing , in one case because of a lack of insurance approval for testing . referral still required finally , a large number of patients66 patients and 91 variantsstill require referral to the cpp before constitutional testing can be completed . 
our program is actively working to ensure that all families with a concerning somatic variant receive appropriate genetic counseling . characteristics of tested patients historically , the suspicion for constitutional cancer predisposition is based on clinical characteristics , including family history , physical features of a predisposition syndrome , presence of bilateral / multifocal disease , and / or a specic tumor type strongly associated with a predisposition syndrome . 
there was no signicant difference between those conrmed and not conrmed in vaf 0.4 or in tumor loss of heterozygosity . discussion somatic findings concerning for cancer predisposition of the more than 1 , 000 children , adolescents , and young adults who underwent somatic tumor testing in the 28 months included within this study period , 13.9% had somatic results that were concerning for a constitutional cancer predisposition . 
0.4 , although in 22.2% of variants vaf was lower than this threshold , and clinical suspicion based on the specic variant prompted cpp referral . as expected , given the frequency of both constitutional and somatic mutations , 2 , 18 tp53 was the most frequently mutated gene that prompted referral to the cpp . 
of the 21 patients with a tp53 mutation in tumor tissue who were referred for testing , 6 ( 28.6% ) were diagnosed with lfs on the basis of a constitutional nding of a known tp53 pathogenic / likely pathogenic variant , three of whom would not have been referred based on accepted lfs testing guidelines.15 , 19 even including all somatic variants , at least 19.6% of patients with somatic lfs variants are constitutionally affected , a proportion higher than that observed in the adult population . 
all of these patients are now being observed by the cpp and are receiving recommended surveillance.20 the next most frequent mutation found was in nf1 , but decisions about constitutional testing were sometimes made on the basis of the presence or absence of characteristic clinical features of nf1 . 
of note , for some variants clinical guidelines are not yet established , and in these cases families received genetic counseling regarding the lack of accepted tumor surveillance practices and / or additional study needed regarding the constitutional presence of a variant . 
in cases in which constitutional testing would not affect clinical managementfor example , no additional childhood surveillance would be required families were informed of the result and adult relatives were counseled that testing for themselves , but testing was usually not sent for the child . finally , a discussion of somatic mosaicism occurred in the setting of inconclusive or negative results , or in cases in which families decided not to pursue genetic testing because of lack of clinical features of a disorder . they could consider additional study is required to analyze the power of somatic panel testing to identify pediatric patients with constitutional cancer predisposition , with and without the incorporation of clinical data , such as multifocality , tumor type , syndromic features , and family history . 
in addition , many patients have not yet been referred to the cpp to discuss constitutional testing , and qualitative review would suggest a variety of reasons for this , including emotional stress on the family at the time of cancer diagnosis and the additional stress of predisposition evaluation during treatment , although this has not been systematically studied at our institution . 
we plan to continue offering genetic testing and counseling to patients not yet referred to ensure an eventual 100% referral rate . interpretation of somatic results concerning for cancer predisposition the most signicant differences in those constitutionally conrmed were known clinical risk factors , namely , a signicant / concerning family history of cancer and bilateral or multifocal disease . 
this suggests that vaf and loh alone are not sufcient to rule out a cancer predisposition syndrome . although it may seem surprising that tumor type was not predictive , this is consistent with the known prevalence of a cancer predisposition syndrome in these tumor types . thus , clinical predictors of cancer predisposition remain important in the interpretation of somatic results . more than one half of patients who received constitutional testing were found to have an underlying cancer predisposition syndrome , often requiring changes in treatment plan , ongoing surveillance , and familial testing . 
some of these patients could have been referred for testing on the basis of patient age , type of cancer , and / or family history of cancer ; however , others would not have been suspected as a result of a lack of these features . 
thus , the incorporation of somatic testing results proves to be an important supjudgment in identifying cancer preplement to clinical disposition syndromes . in conclusion , somatic tumor sequencing is a powerful tool in pediatric cancer to risk - stratify patients and identify appropriate therapy , and its use is becoming increasingly widespread . 
 macfarland et al 6department of biomedical and health informatics , childrens hospital of philadelphia , philadelphia , pa 7department of pediatrics , the perelman school of medicine at the university of pennsylvania , philadelphia , pa data analysis and interpretation : suzanne p . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . pichai raman consulting or advisory role : scholar rock travel , accommodations , expenses : scholar rock gerald wertheim employment : johnson & johnson ( i ) stock and other ownership interests : johnson & johnson ( i ) stephen p . 
hunger stock and other ownership interests : amgen , merck ( i ) , amgen ( i ) , pzer ( i ) honoraria : amgen consulting or advisory role : novartis no other potential conicts of interest were reported . references 2018 1990 2017 brodeur gm , nichols ke , plon se , et al : pediatric cancer predisposition and surveillance : an overview , and a tribute to alfred g . 
clin cancer res 23 : e1 - e5 , 2017 zhang j , walsh mf , wu g , et al : germline mutations in predisposition genes in pediatric cancer . 
n engl j med 373 : 2336 - 2346 , 2015 parsons dw , roy a , yang y , et al : diagnostic yield of clinical tumor and germline whole - exome sequencing for children with solid tumors . 
nature 555 : 371 - 376 , postema fam , hopman smj , aalfs cm , et al : childhood tumours with a high probability of being part of a tumour predisposition syndrome ; reason for referral for genetic consultation . 
science 250 : 1233 - 1238 , jongmans mc , loeffen jl , waanders e , et al : recognition of genetic predisposition in pediatric cancer patients : an easy - to - use selection tool . 
goudie c , cullinan n , villani a , et al : retrospective evaluation of a decision - support algorithm ( mipogg ) for genetic referrals for children with neuroblastic tumors . 
genome med 11 : 32 , 2019 jain p , surrey lf , straka j , et al : novel fgfr2 - ina fusion identied in two low - grade mixed neuronal - glial tumors drives oncogenesis via mapk and pi3k / mtor pathway activation . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
achatz mi , porter cc , brugi `eres l , et al : cancer screening recommendations and clinical management of inherited gastrointestinal cancer syndromes in 49 : 3680 - 3685 , 2013 37 : 865 - 876 , 2016 cancer res 77 : 1250 - 1260 , 2017 childhood . 
the recommendation process in mtbs , however , has not been well defined , which limits applicability to larger clinical trials and patient populations . methods we created four fictional patients on the basis of recent real cases with genomic information on mutations , fusions , copy numbers , and gene expression . 
there was heterogeneity in the interpretation of tumor and germline aberrations as well as in standards of prioritization . conclusion differences in the interpretation and recommendation process contribute to heterogeneity in mtb recommendations . 
however , a randomized phase ii trial comparing therapy based on tumor molecular profiling versus conventional therapy in patients with refractory cancer ( shiva ) is the only prospective trial to have investigated a personalized therapy with negative results.8 in addition , many reported case studies have been criticized for incompleteness of data.9 thus , precision medicine has been called an unproven hypothesis.10 several potential explanations for the negative results have been suggested , including the limitations of predefined treatment strategies such as those used in the shiva trial.11 , 12 personalized oncology relies on several steps . 
the complexity of molecular aberrations , tumor heterogeneity , and technical and clinical difficulties have been addressed.13 , 14 in addition , the resulting data have to be interpreted and transformed into a treatment recommendation by identifying and prioritizing predictive biomarkers . 
several proposals have been made on how to arrive at clearly reportable recommendations.8 , 16 , 17 most rely on the definition of evidence levels attributed to every single aberration and the interdisciplinary discussion of the aberrations with regard to patient situation , availability of drugs , and clinical trials in a molecular tumor board ( mtb )  . 
 although this process is largely unstandardized , it is critical to facilitate the transformation of patient benefit from case studies to larger populations . to assess standards and differences in the interpretation process , we asked mtbs worldwide to make treatment recommendations for four fictional patients presented with different profiles of genomic aberrations . methods creation of sample patients four fictional patients were created on the basis of recent real cases . 
the possibility that selected aberrations could have co - occurred was validated in cbioportal for cancer genomics.18 data provided for every patient included age , cancer type , time of initial diagnosis , location and time of diagnosis of metastases , prior therapies , eastern cooperative oncology group performance status , and mutational profile ( one - letter amino acid code )  . 
molecular data provided for some of the patients included allele frequency , gene fusions , copy number alterations , gene expression data ( rna sequencing , reads per kilobase per million mapped [ rpkm ] ) , and germline aberrations . 
we asked for the percentage of patients with actionable alterations presented at the mtb , as well as for methods of trial recruitment practices and variant of unknown significance ( vus ) interpretation strategies after compilation of the data . identification of molecular tumor boards mtbs worldwide were identified through literature search , personal contacts , and online representations . 
mtbs were asked to participate in the survey under the prerequisite that a connection between recommendations and the participating board could not be made . data analysis and interpretation completed questionnaires and treatment recommendations from the mtbs were collected , and an analysis was performed . 
allele frequency data were requested by another two tumor boards ( mtb 3 and 5 ) but were not provided . results identification of molecular tumor boards and response we were able to identify and contact 29 mtbs in nine countries . 
26 - yearold patient mean coverage > 500 panel sequencing : kras g12v , bcorl1 r1332 * , tp53 h214fs * 7 , cdkn2c l65f , ctnna1 k577_ l578 > tkl , map3k1 t949_e950inst , mycn e47fs * 8 p365a , jak1 i597m , fancl t367fs * 12 + pik3ca amplification ( > 6 copies ) , myc amplification ( > 6 copies ) , mycl1 amplification ( > 6 copies ) , sox2 amplification ( > 6 copies ) , mutyh amplification ( > 6 copies ) 3 . 
58 - yearold patient gene fusions : tmem111tdrd3 gene fusion , prkdccdh17 gene fusion , ext1magi2 gene fusion whole - exome sequencing ( aug 2014 ) : germline : brca2 k3326 * ( 1n )  . 
sample patients as provided to the mtbs ( continued ) patient clinical information sequencing fusion genes copy no . gene expression additional information tumor biopsy from pulmonary metastasis april 2016 after progression on chemotherapy / cetuximab . 
 tumor purity 60% , coverage > 500 pik3ca e545k ( 25% allele frequency ) , mapk1 e322k ( 10% allele frequency ) , fgfr3 d786n ( 30% allele frequency ) 4 . 
 one mtb also requested information on specific patient factors ( willingness to travel , tolerance of treatment )  . comparison of treatment recommendations mechanistically similar treatment strategies ( eg , sorafenib or pan - raf inhibitor ) were interpreted as identical strategies . 
 there was a maximum overlap of three identical treatment strategies ( case 4 ) , whereas the other three cases ( cases 1 to 3 ) had a maximum of two identical top treatment recommendations ( tables 2 - 5 )  . 
patient cases had been designed with variable numbers of genetic aberrations ( case 1 , five aberrations ; case 2 , 14 aberrations ; case 3 , 18 aberrations ; case 4 , three aberrations )  . 
overall , treatment recommendations were more similar for the case with the least genomic information ( case 4 ) than for cases with more and more complex information ( case 2 and case 3 )  . 
one tumor board required additional confirmation by loss - of - heterozygosity testing.20 another tumor board stated that , according to an internal guidance document , vuss were generally not considered . potential germline aberrations , underlying tumor sequencing results , were considered by two mtbs . 
 germline variants triggering genetic counseling recommendations included a cdh1 mutation in patient 1 ( considered by two mtbs ) and tp53 mutations in patients 1 and 2 ( considered by one mtb )  . in the two cases with aberrations of copy number ( case 2 ) or gene expression and gene fusion ( case 3 ) data provided in addition to the mutations , there was no recommendation bias toward any one of these aberration types . 
recommendations were made on the basis of mutation and gene expression ( by two tumor boards each in patient 3 ) and amplification data ( three tumor boards in patient 2 )  . as included in the clinical information on the cases , checkpoint inhibitors had been tried unsuccessfully in two of the patients ( patients 1 and 4 )  . 
immune checkpoint inhibition was considered as a treatment option by two mtbs in one of the remaining two patients ( patient 2 ) and was the only recommendation by one of these tumor boards . 
the overall make - up of the mtbs was comparable , with similar numbers of individuals and specialties ( 15 to 30 individuals representing medical oncology , pathology , and additional specialties including radiology , informatics , and biology )  . 
in addition , whole - exome sequencing was used by two tumor boards , and copy number analyses ( comparative genomic hybridization or nanostring ) were used by another two . 
one tumor board reported using rna sequencing and germline controls in their routine diagnostics pipeline . another measure for comparing the functionality of an mtb was the rate of successful translation of recommendations into treatments . 
in contrast , the percentage of patients with potential targetable alterations , as reported by the mtbs , was between 20% and 90% . to analyze the prioritization process , we assessed the evidence base used for the interpretation of aberrations . 
two mtbs considered allele frequencies , and one tumor board reported a specific cutoff for considering allele frequency measurements ( > 50% deviation from that which was expected from other mutations )  . 
participants specialties medical oncology , pathology , molecular pathology , and pharmacy molecular pathology laboratory screening program , phase i unit , lung unit , head and neck unit , gi unit , gynecologic unit , sarcoma unit , and genitourinary unit tumor board pharmacy , medical oncology , pathology , genetic counseling , pediatric oncology , informatics , surgical oncology , biochemistry , hematology / oncology , pharmaceutical sciences , toxicology , pharmacology , and interventional radiology medical oncology , pathology , molecular biology , radiology , and interventional radiology medical oncology , pathology , molecular pathology , bioinformatics , biology , and treating physician no . 
results of questionnaire assessing participating tumor boards ( continued ) question evidence level patients with actionable alterations , % translated into treatment , % patients in structured clinical followup , % internal document ( including journal club with up to three studies and at least one clinical trial for offlabel therapy ) registration in clinical trials of all patients possible tumor board clinical evidence in humans needed published evidence levels published evidence levels data not yet collected data not yet collected 40 - 45 80 - 90 note . 
ranking of aberrations according to standardized evidence levels was performed regularly by two tumor boards , whereas an internal evidence document was used by another tumor board . discussion mtbs were established to provide a platform for precision oncology implementation . 
 studies in human genetics have shown differences among laboratories when analyzing identical variants.21 to our knowledge , this is the first study to analyze heterogeneities in the recommendation process of mtbs worldwide . currently , no standards for workflows and setups of mtbs exist . 
treatment recommendations as provided by the respective molecular tumor boards for patient 1 tumor board recommendation provided rationale additional recommendation pan - raf inhibitor no targeted therapy clinical trial for kras mutation downstream effect kras mutation kras mutation sorafenib clinical trial kras mutation met and ret testing genetic counseling for cdh1 and tp53 mutations ( potential germline mutation ) docetaxel and selumetinib kras mutation ( data from phase ii clinical trial ) genetic counseling ( cdh1 mutation ; potential germline mutation ) n / a because of missing information note . 
treatment recommendations as provided by the respective molecular tumor boards for patient 2 tumor board recommendation provided rationale additional recommendation pik3ca , akt , or mtor pik3ca mutation jak1 allele frequency testing ( infiltrating blood cells ? ) and if consistent with tumor : ruxolitinib jak1 mutation ( after considering allele frequency ) immunotherapy , atezolizumab independent of biomarker phase i trial with bromodomain inhibitor myc amplification pik3ca or mtor inhibitor pik3ca amplification ( data from case study ) consider genetic counseling and potential germline testing ( mutation burden , tp53 mutation , patient age ) everolimus clinical trial ( pik3ca amplification ) , sorafenib ( kras mutation ) mek inhibitor ( tp53 and kras mutation ) or checkpoint inhibition ( independent of biomarker , despite potential jak1 resistance ) n / a because of missing information note . 
rationales were provided for only some of the recommendations and asked for if missing initially . abbreviation : n / a , not available . and workflow of mtbs , the availability of clinical studies , the interpretation of vuss and potential germline aberrations , and the resulting recommendations . 
however , even this limited study was sufficient to uncover fundamental differences and identify problems in the recommendation process , the identification and discussion of which can be expected to help inform existing mtbs and the design of future studies . 
thus , we limited our analysis to identified heterogeneities and their potential effect on the precision oncology initiative . the heterogeneity in treatment recommendations presented here could be viewed , at first glance , as disturbing . 
treatment recommendations as provided by the respective molecular tumor boards for patient 3 tumor board recommendation provided rationale additional recommendation parp inhibitor brca2 mutation clinical trial for met amplification met amplification her2 inhibitor ( erbb2 overexpression ) , fgfr inhibitor ( fgfr3 overexpression ) or met inhibitor ( met overexpression ) off - label trastuzumab and pertuzumab , trastuzumab and lapatinib ( erbb2 overexpression ) or off - label crizotinib ( met overexpression ) , or olaparib if brca2 loh verified pan - her inhibitor olaparib n / a because of missing information erbb2 or erbb3 overexpression fgfr - inhibitor phase i trial ( fgfr3 overexpression ) brca2 mutation ( vus with potential effect ) erbb2 ihc validation and inhibition ( erbb overexpression ) note . 
treatment recommendations as provided by the respective molecular tumor boards for patient 4 tumor board recommendation provided rationale additional recommendation fgfr inhibitor fgfr3 mutation pik3ca inhibitor ( pik3ca mutation ) , combination treatment pik3ca and fgfr inhibitor in phase i trial ( pik3ca and fgfr3 mutation ) clinical trial for pik3ca or fgfr3 mutation pik3ca and fgfr3 mutation if ineligible , off - label everolimus ( pik3ca mutation ) cetuximab and pik3ca inhibitor clinical trial pik3ca or mtor inhibition n / a because of missing information pik3ca mutation pik3ca mutation ( case report ) fgfr inhibitor clinical trial ( fgfr3 mutation ) note . 
rationales were provided for only some of the recommendations and asked for if missing initially . abbreviation : n / a , not available . based on clear diagnostic results , are heterogeneous , especially in the context of complex molecular data . 
this uncertainty was further underlined by the fact that , across most studies , only approximately 20% of patients entering molecular diagnostic programs were reported to receive treatment , despite the identification of potentially targetable alterations in many more patients , as reported by the centers . 
these numbers have been validated recently in a large precision oncology study using comprehensive sequencing data.22 the presence of well - defined genomic aberrations such as braf v600e mutations or the absence of aberrations in known genes could be expected to improve the concordance among centers . 
 yet the discordance among identified targetable alterations , initiated treatments , and treatment effects highlights that most patients discussed at mtbs are found between those more well defined ends of the spectrum , as represented by the sample patients . 
currently , only 1% to 10% of evidence items ( 64 of 5 , 100 and 25 of 477 ) in the clinical interpretations of variants in cancer ( civic ) 23 and oncokb databases24 have us food and drug administrationapproved or standard - of - care evidence level . 
a single - center predefinition of biomarkers as performed in the shiva trial may be unlikely to identify the best treatment option given the amount of heterogeneity among different centers.8 this argument is further supported by positive results from meta - analyses incorporating data from several centers.25 this might be caused by swarm intelligence when considering multiple biomarker - treatment associations . 
however , we believe that the issue of recommendation is more important in the precision medicine context because the biomarker - based choice of treatment is unique to this concept and was expected to be reproducible . in this study , differences were observed on several levels . 
after analyzing the results , we hypothesized that recommendation heterogeneity may have originated from the availability of clinical trials , from shortcomings in the identification of targets , or from differences within the prioritization workflow . 
therefore , it seemed unlikely that the identification of predictive biomarkers was a relevant factor ( even though a jak1 mutation was considered as a predictive biomarker by only one tumor board , in patient 2 )  . 
additional differences were observed regarding the interpretation of vuss , germline variants , potential germline events detected in tumor - only sequencing , and the recommendation of treatments outside of biomarker - driven indications . the provided brca2 vus highlighted differences in the interpretation of unclear variants . 
yet working principles such as the consideration of allele frequencies , vus prediction tools , hierarchies within identified aberrations , structured evidence bases for the identification of biomarker information and prioritization , and the use of evidence levels differed significantly . the interpretation and prioritization of genomic information is not a simple task . 
mutations identified in tumor - only sequencing might sometimes represent a germline event and should therefore be considered for validation.4 information on allele frequency might help in identifying these cases . 
metzeler , serge leyvraz , ulrich keilholz manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors the following represents disclosure information provided by authors of this manuscript . 
 marissa schuh no relationship to disclose christophe le tourneau honoraria : novartis , bristol - myers squibb consulting or advisory role : novartis , celgene research funding : celgene travel , accommodations , expenses : janssen - cilag , celgene consulting or advisory role : amgen , msd , bristolmyers squibb , merck serono , astrazeneca travel , accommodations , expenses : msd , bristol - myers squibb , astrazeneca serge leyvraz consulting or advisory role : loxo consulting or advisory role : loxo travel , accommodations , expenses : ipsen neus bast consulting or advisory role : bristol - myers squibb , nanobiotix mark e . 
metzeler , university hospital , lmu munich , munich , germany ; marissa schuh , markey cancer center , university of kentucky , lexington , ky ; christophe le tourneau , institut curie and inserm u900 research unit , saint - cloud ; christophe le tourneau , institut curie , paris ; christophe le tourneau , versailles - saint - quentin - en - yvelines university , montigny - le - bretonneux , france ; neus bast , vall dhebron institute of oncology , barcelona , spain ; and mark e . 
is a participant in the berlin institute of healthcharit clinical scientist program funded by the charituniversittsmedizin berlin and the berlin institute of health . presented in part at the annual meeting of the european society of medical oncology , madrid , spain , september 8 - 12 , 2017 . support prior presentation references 1 . 
von hoff dd , stephenson jj jr , rosen p , et al : pilot study using molecular profiling of patients tumors to find potential targets and select treatments for their refractory cancers . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
soria jc , flp a , maciel c , et al : select - 2 : a phase ii , double - blind , randomized , placebocontrolled study to assess the efficacy of selumetinib plus docetaxel as a second - line treatment of patients with advanced or metastatic non - small - cell lung cancer . 
amendola lm , jarvik gp , leo mc , et al : performance of acmg - amp variant - interpretation guidelines among nine laboratories in the clinical sequencing exploratory research consortiu am j hum genet 98 : 1067 - 1076 , 2016 [ erratum : am j hum genet 99 : 247 , 2016 ] 22 . 
massard c , michiels s , fert c , et al : high - throughput genomics and clinical outcome in hardto - treat advanced cancers : results of the moscato 01 trial . 
schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
 precision trial drawer , a computational tool to assist planning of genomics - driven trials in oncology purpose trials that accrue participants on the basis of genetic biomarkers are a powerful means of testing targeted drugs , but they are often complicated by the rarity of the biomarker - positive population . 
however , bigger trials have higher chances of conflicting treatment allocations because of the coexistence of multiple actionable alterations ; allocation strategies greatly affect the efficiency of enrollment and should be carefully planned on the basis of relative mutation frequencies , leveraging information from large sequencing projects . methods we developed software named precision trial drawer ( ptd ) to estimate parameters that are useful for designing precision trials , most importantly , the number of patients needed to molecularly screen ( nnms ) and the allocation rule that maximizes patient accrual on the basis of mutation frequency , systematically assigning patients with conflicting allocations to the drug associated with the rarer mutation . 
we used data from the cancer genome atlas to show their potential in a 10 - arm imaginary trial of multiple cancers on the basis of genetic alterations suggested by the past molecular analysis for personalised therapy ( map ) conference . 
2018 by american society of clinical oncology introduction the availability of sequenced cancer genomes and the generation of new targeted drugs offer unprecedented opportunities for personalized treatment for patients with cancer . 
indeed , treatment within genomic - based clinical trials significantly improves clinical outcome.1 however , identification of the best biomarker - drug combination for patient stratification and treatment remains a critical research and ethical challenge . large multi - institutional projects such as the cancer genome atlas ( tcga ) revealed extreme heterogeneity of cancer mutational landscapes , with a prevalence of low - frequency mutations , giorgio e.m. 
are co - first authors . corresponding author : luca mazzarella , md , phd , istituto europeo di oncologia , via ripamonti 435 , 20141 milano , italy ; e - mail : luca . 
 including those critical for tumor growth ( driver mutations ) and those that might be targetable by specific drugs ( actionable mutations ) .2 , 3 this scenario renders the conduction of adequately powered genome - based clinical trials prohibitive in many cases : the biomarker - positive population is often rare , and identifying it requires screening large numbers of patients . strategies to circumvent this issue are proposed through novel trial designs in which multiple drugs and / or their matched biomarkers are tested simultaneously . 
these novel designs , usually defined as basket ( one biomarker - drug pair , multiple histologies ) , umbrella ( multiple biomarker - drug pairs , one histology ) , or platform ( multiple biomarker - drug pairs , multiple histologies ) , 4 - 6 may allow reducing the number of patients needed to molecularly screen ( nnms ) to achieve the desired statistical power for multiple treatment subgroups . 
however , these approaches have potential disadvantages : first is the occurrence of a large fraction of patients with potential allocation conflicts resulting from the concomitance of genetic alterations associated with different drugs , 2 and second is the high costs and turnaround time of the molecular screening , which must include sequencing enough genomic space to accommodate the studys need . 
the latter will become more critical in the near future as more complex , polygenic genomic signatures are used as stratification biomarkers ( eg , multiple homologous recombination genes to establish brcaness as a predictor of response to poly [ adp - ribose ] polymerase [ parp ] inhibitors7 )  . 
 for these reasons , patient allocation to individual trial arms significantly deviates from expectations in multigenic umbrella trials , which leads to inadequate statistical power8 , 9 and the inability to reach conclusions . here , we present precision trial drawer ( ptd ) , a comprehensive bioinformatics tool that takes advantage of patient - level genomic information from tcga or alternative sources to simulate genomically characterized cohorts and provide critical parameters for designing genetic biomarker - driven trials . 
df is used to calculate the nnms ( ie , the minimum number of patients that should be tested to obtain the desired sample size as calculated on the expected treatment effect by using conventional power analyses )  . 
 actionable mutations refers to those genetic alterations that dictate a therapeutic choice within the trial . description of the package ptd includes a series of functions in the r programming language to ( 1 ) translate genomic information into genomic coordinates and optimize coordinates for the design of a targeted sequencing panel , ( 2 ) retrieve single or grouped genetic alteration frequencies from public databases ( by default tcga , but other sources can be used ) that maintain patient - level associations , ( 3 ) simulate genomically defined patient cohorts by extracting frequencies of co - occurrence of single or grouped mutations , and ( 4 ) calculate nnms for defined power and / or sample size ( fig 1a )  . 
in our imaginary trial , patients are allocated to 10 possible drugs , including inhibitors of parp , notch , met , her2 , fgfr , egfr , braf , alk , akt , and immune checkpoints , and covering all the allocations linked to the map biomarkers . 
allocation to checkpoint inhibitors was based on the detection of inactivating mutations in mlh or msh genes , regardless of tumor type , which are highly correlated with microsatellite instability , a now - established predictor of immune checkpoint response.11 , 12 ptd was then used to design a dedicated custom targeted sequencing panel , identify the optimal allocation rule , and estimate sample size and statistical power . the list of genomic features associated with each drug is provided in table 1 . 
illustrations of the workflow , the codes used for generating data , and detailed ptd functionalities are available at shinyapp.html. the first step involves designing the sequencing panel , which is imported as a standard excel table with official gene symbols , type and exact alteration to be considered , and two possible grouping variables ( gene - linked drug and , if desired , a generic group of any sort )  . 
 the panel can also be expanded to include genes or genomic positions without therapeutic information , which may nevertheless be relevant for prognostic stratification , prediction of drug toxicity , or a generic biologic interest ( grouped as driver genes in the example )  . four classes of genetic alterations can be explored : single nucleotide variants ( snvs ) , copy number alterations ( cnas ) , translocation - associated fusion transcripts , and gene expression . 
for panels that contain multiple alteration types ( eg , snvs and cnas ) , ptd will construct a reference data set with the intersection of the relative data sets in the source database ( not all patients in tcga have data from all sequencing platforms )  . in projects that allow inclusion of multiple tumor histologies , their frequency of occurrence in the real world is likely to differ significantly from the relative representation in the reference data set , and this can be corrected by ptd by weighting the probability of sample extraction . 
in this example , we used relative frequencies obtained from the national cancer institute seer program ( fig 2a shows the comparison between the tcga and the example populations )  . output the percentage of patients with at least one actionable alteration ( detected fraction ; df ) is probably the most crucial estimate that can be obtained through ptd , which is 65.4% in this example ( fig 2b )  . 
ptd allows breakdown of the df by drug or by disease histology ( fig 2c - d ) or any other prespecified group . to optimize panel design , one can evaluate the trade - off between the feature of interest ( eg , available drugs or the inclusion of genes with uncertain predictive power ) and the size of the genomic space to be sequenced . 
this trade - off can be visualized with a saturationplot that expresses the mean number of alterations per patient or the number of patients with at least x alterations as a cumulative function of the genomic space , with alterations ranked in descending frequency order ( absolute or gene lengthnormalized ; fig 2e )  . 
 the same plot can be obtained by using drugs or other grouping variables ( fig 2f )  . the coocmutexplot indicates the degree of co - occurrence of grouped genetic alterations , ascopubs.org / journal / po jco precision oncology 5 accepted formats . 
for instance , the braf v600e mutation can be coded using amino acid notation ( v600e ) , the single nucleotide polymorphism database ( dbsnp ) identifier ( rs113488022 ) , a set of genomic coordinates ( eg , chr7 : 140753336 140753337 ) , or as genomic variants ( 7 : 140753336 a > c )  . 
mutations of interest can be batch filtered with any set of genomic coordinates to eliminate variants likely to be passengers.3 , 13 in this example , we selected only variants listed as probably pathogenic in the catalogue of somatic mutations in cancer ( cosmic ) database , 14 thus eliminating 33.5% of the initial variants . 
alternatively , individual genes can be visually inspected and edited by using an interactive interface built on the lowmaca ( low frequency mutations analysis via consensus alignment ) algorithm15 ( manual filtering ; fig 1b )  . 
in this example , we manually excluded egfr t790m and pik3ca exon 20 mutations associated with resistance to first - generation egfr inhibitors.16 , 17 specific mutations and / or drugs can be associated with specific tumors and excluded from others . 
 ( b ) percentage of patients with at least one ( detection fraction ) or more actionable alterations ; detection fraction is broken down by ( c ) tumor type or ( d ) drug . 
this plot can be used to decide which drugs should be combined in the same trial because prioritizing those associated with highly mutually exclusive mutations will maximize the biomarker - positive population . power analysis and calculation of the nnms multidrug trials can be powered to test the experimental arm as a whole , ignoring the number of patients allocated to each individual drug , in which case the nnms is a simple linear function of the target sample size divided by the detection power ( in our example , if the given target sample size = n , the nnms will be n / 0.654 ; no allocation rule design )  . 
if this is a desired end point , the trial can be divided into a series of drug - specific subtrials , each having its own target sample size.9 probability of allocation can vary significantly if the associated biomarkers have very different prevalence . 
furthermore , increasing the number of competing arms also increases the likelihood of conflicting allocations ; rules to follow in such cases should be specified in advance to avoid allocation biases . 
immcheckp , immune checkpoint ; inh , inhibitor . general a priori considerations that do not take into account mutation frequencies ( eg , allocation to endocrine therapy only if no mutation is present8 ; manual allocation design )  . 
if information regarding the co - occurrence of mutations is not considered , proper nnms - based sample size calculation is not possible because considering each subtrial independently leads to gross overestimation . 
with ptd , arms can be ranked in order of expected frequency of allocation , so that if a patient bears mutations associated with two drugs , he or she will be systematically assigned to the one with the lowest frequency . 
in nonoptimal designs , allocation does not take into consideration the relative rarity of c , so that when all patients have been allocated , drug z has been allocated to fewer patients than statistically needed . 
 ( c ) comparison of percentages of patients that can be allocated to one or two of the molecular pathway - based subgroups ( real values for shiva and mean values for precision trial drawer [ ptd ] 95% ci are plotted )  . 
 ( d ) power calculations showing numbers needed to sequence for the overall ( bottom panel ) and pathway - specific subgroup trials ( top and middle panels )  . 
cna , copy number alteration ; snv , single nucleotide variant ; tcga , the cancer genome atlas . nnms based on the rule , and simulate patient allocation so that each drug - specific arm reaches at least the desired level of power ( optimal allocation design ; fig 3a )  . 
we can thus precisely quantify the advantage of an optimized umbrella design : for the given example , if one were to run 10 independent one - arm studies with a target sample size of 13 to show a response rate of 40% against historical controls of 10% ( for = .05 and power = 0.8 ) , a total of 4 , 276 patients would need to be sequenced to obtain 130 ( ie , 10 13 ) biomarker - positive patients . 
 the researcher to specify different types of end points ( time to event , progression - free survival , overall survival , time to progression , or proportions such as response rate and survival rate ) , number of arms , allocation ratios ( symmetric or asymmetric ) , and other conventional trial parameters . 
simulation results for oneor twoarm studies with time - to - event or proportion end points and with no , manual , or optimal allocation rules are provided in fig 3b . 
simulation results across a range of powers and target proportions are provided in fig 3c . retrospective evaluation of the shiva trial to test ptd in a real - world scenario , we retrospectively evaluated its ability to correctly predict population composition and treatment allocation in the recent shiva trial , a phase ii randomized trial comparing conventional therapy to multiple targeted agents on the basis of tumor molecular profiling . 
in shiva , 741 patients were subjected to a triad of molecular tests , including sequencing with a targeted enrichment panel ( ampliseq cancer panel , covering 45 tumor - associated genes or mutational hotspots ) , gene copy number analysis by cytoscan hd , and immunohistochemistry for three endocrine receptors . 
although ptd can include information on gene expression in the simulation , we removed endocrine receptors from the analyzed parameters because the correlation between rna - based and immunohistochemistry based quantification of endocrine receptors is poor ( appendix fig a1 )  . 
after data cleanup , 417 of 741 patients were left for analysis ( fig 4a )  . we constructed a ptd panel by using the actionable alterations in shiva and ran 100 trial simulations , extracting tumor histologies from tcga with probabilities equal to their frequencies in shiva and with allocation rules reproducing those adopted in the actual trial . 
alteration frequencies for individual genes were within 95% cis in most cases ( appendix fig a1a - b ) , except for low - frequency genes , for which 95% cis can be expected to be wide . 
consequently , observed versus predicted nnms was also quite similar ( fig 4d ; appendix fig a1d ) , demonstrating that ptd can correctly predict the number of patients needed to be subjected to molecular screening . discussion we report a versatile bioinformatics platform that is based on mutational databases to simulate genetic biomarker - driven clinical trials . 
in a retrospective analysis of the shiva trial , we demonstrated that our tool is able to predict the actual number of patients to be sequenced and has the power to detect overall and arm - specific efficacy signals . 
we believe that our tool will be instrumental in overcoming emerging issues in the design of precision oncology trials . the conceptual framework of precision medicine implies an increased fragmentation of disease entities into distinct subgroups identified by the presence of biomarkers that predict response to targeted agents . 
as previously highlighted by several authors , 6 , 20 for trials aimed at demonstrating efficacy of targeted drugs , 20 more biomarkers means rarer biomarkers , which in turn implies higher and more variable numbers of patients needed to be screened . 
 match screening trial ( targeted therapy directed by genetic testing in treating patients with advanced refractory solid tumors , lymphomas , or multiple myeloma ) , the largest trial of this type to date , an interim analysis after 795 patients were screened showed that only 2.5% of patients could be allocated to seven of the 10 targeted agents on trial.21 the possibility of better predicting accrual rate afforded by ptd would allow trialists to drop inefficient arms and / or modify trial design to maximize patient allocation . an unavoidable limitation of our methodology is that simulations are affected by the prevalence of mutations in available databases , which may not be representative of what is found in study populations . 
instead , most trials with targeted drugs are initially aimed at the metastatic setting , and the mutational profile of metastatic tumors can be significantly different from that of primary tumors . 
an ideal setting for ptd - based simulations with current tcga data may be the neoadjuvant / preoperative setting , which is increasingly used to evaluate novel drugs.23 the availability of clinical metadata , such as tumor size or nodal involvement , can be used to strengthen the representativeness of database samples . finally , some disease tumor types are under represented in mutational databases , which results in high variability and low confidence of corresponding simulations . 
melloni , alessandro guida , giuseppe curigliano , edoardo botteri , ruggero de maria , piergiuseppe pelicci , luca mazzarella financial support : giuseppe curigliano , piergiuseppe pelicci administrative support : giuseppe curigliano provision of study materials or patients : giuseppe curigliano , angela esposito , maud kamal , christoph le tourneau collection and assembly of data : giorgio e.m. 
 edoardo botteri no relationship to disclose angela esposito no relationship to disclose maud kamal no relationship to disclose laura riva employment : philips healthcare ( i ) alberto magi no relationship to disclose ruggero de maria no relationship to disclose christophe le tourneau honoraria : novartis , bristol - myers squibb piergiuseppe pelicci stock and other ownership : genextra consulting or advisory role : amgen , msd , bristolmyers squibb , merck serono , astrazeneca travel , accommodations , expenses : msd , bristol - myers squibb , astrazeneca luca mazzarella no relationship to disclose affiliations giorgio e.m. 
schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
le tourneau c , delord jp , gonalves a et al : molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
lopez - chavez a , thomas a , rajan a , et al : molecular profiling and targeted therapy for advanced thoracic malignancies : a biomarker - derived , multiarm , multihistology phase ii basket trial . 
pao w , miller va , politi ka , et al : acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the egfr kinase domaplos med 2 : e73 , 2005 17 . 
mao c , yang zy , hu xf , et al : pik3ca exon 20 mutations as a potential biomarker for resistance to anti - egfr monoclonal antibodies in kras wild - type metastatic colorectal cancer : a systematic review and meta - analysis . 
the utility of ultra - deep genomic testing to predict and the impact of conditioning intensity to prevent mds relapse are unknown . methods targeted error - corrected dna sequencing was performed on preconditioning blood samples from patients with mds ( n = 48 ) from the blood and marrow transplant clinical trials network 0901 phase iii randomized clinical trial , which compared outcomes by allogeneic hematopoietic cell transplantation conditioning intensity in adult patients with , 5% marrow myeloblasts and no leukemic myeloblasts in blood on morphological analysis at the time of pretransplant assessment . 
clinical end points ( 53 - month median follow - up ) included transplant - related mortality ( trm ) , relapse , relapse - free survival ( rfs ) , and overall survival ( os )  . 
of the 48 patients examined , 14 experienced trm , 23 are relapse - free , and 11 relapsed , of which 7 died . results using a previously described set of 10 gene regions , 42% of patients ( n = 20 ) had mutations detectable before random assignment to reduced intensity conditioning ( ric ) or myeloablative conditioning ( mac )  . 
testing additional genes , including those associated with mds , did not improve prognostication . conclusion this study provides evidence that targeted dna sequencing in patients with mds before transplant can identify those with highest post - transplant relapse rates . 
2021 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction myelodysplastic syndrome ( mds ) , one of the most common hematologic disorders , is a collection of clinically and genetically heterogeneous diseases . 
 dillon et al context key objective allogeneic hematopoetic cell transplantation ( allohct ) is the only curative therapy for myelodysplastic syndrome ( mds ) but is associated with suboptimal rates of transplant - related mortality ( trm )  . 
using samples from a randomized phase iii clinical trial comparing allohct conditioning intensity in patients with mds , this study examined the prognostic signicance of pretransplant genetic markers on post - transplant outcomes . knowledge generated the presence of mutations within a previously identied set of 10 gene regions before allohct was associated with increased rates of relapse and decreased relapse - free survival in patients with mds who received reduced intensity versus myeloablative conditioning . 
library preparation and pairedend 150 - bp sequencing were performed using unique dualsample indices on an hiseq 2500 ( rapid run mode ; illumina , san diego , ca ) as previously described.10 an average of 43 million paired - end reads were acquired per sample ( data supplement )  . 
details are provided in the data supplement . bioinformatics consensus sequences based on umi read families were mapped to human genome version hg19 ( build grch37 )  . de novo variant calls were made using a minimum allele frequency of 0.001 and were ltered using information regarding umi read families , background error rate models , unique start sites , strand - specic priming , and homopolymer runs . 
alternative approaches were used for insertional mutations in npm1 and flt3 internal tandem duplication . details are provided in the data supplement . statistical analysis kaplan - meier estimation and log - rank test were used for analysis of os and relapse - free survival ( rfs ) and grays test for competing risks of trm and relapse . 
details are provided in the data supplement . results patient cohort a total of 54 patients with mds participated in the bmtctn 0901 clinical trial , of which 48 had blood collected after study enrollment before random assignment to mac ( n = 25 ) or ric ( n = 23 ) conditioning regimens , which was used for genomic analysis in this study . 
this subset of patients was well - matched for baseline characteristics ( table 1 ) , and clinical outcomes were aligned with those previously reported ( data supplement )  . 
ngsg10 mutational status served as a strong predictor of relapse in this cohort of patients with mds , predicting 73% of relapses at 24 months ( 100% for those receiving mac ) , with a specicity of 78% ( 100% for those receiving ric ) ( data supplement )  . to ngsg10 mutational status , no signicant in contrast difference in rates of relapse or os was observed when stratifying patients by disease classication , disease risk group , or cytogenetic prognostic group ( data supplement )  . rates of relapse trended higher in patients with poor cytogenetics or categorized as high risk . 
inclusion of patients with poor cytogenetics before transplant with ngsg10 mutational status improved prediction of relapse at 24 months from 73% to 91% ( data supplement ) and was associated with signicantly higher rates of relapse compared with ngsg10 - negative patients ( data supplement )  . presence of mutations pretransplant predicts relapse by conditioning intensity the impact of conditioning intensity and ngsg10 next , mutational status was examined ( data supplement )  . 
inclusion of patients with poor cytogenetics before transplant with ngsg10 mutational status improved prediction of relapse at 24 months in the ric group to 89% , with a specicity of 89% , and is associated with signicantly increased rates of relapse and decreased rfs compared with patients receiving mac ( data supplement )  . screening for additional genes does not improve test performance the mutational spectrum of mds is complex , with many additional genes recurrently mutated beyond the 10 amlassociated genes described above.7 , 11 , 12 therefore , we expanded our testing to also cover regions in an additional 19 genes including those commonly found in mds at diagnosis ( asxl1 , bcor , cbl , cux1 , dnmt3a , etv6 , ezh2 , gata2 , kras , phf6 , ppm1d , ptpn11 , setbp1 , srsf2 , stag2 , tet2 , u2af1 , wt1 , and zrsr2 )  . 
 ( c ) 10 - gene ngspositive patients had signicantly decreased rfs ( 3 - year rfs , 34% v 71% , p = .006 ) and os ( 3year os , 55% v 79% , p = .045 ) compared with ngs - negative patients . 
the inclusion of dta ( dnmt3a , tet2 , and asxl1 ) genes , known to be associated with age - related clonal hematopoiesis , 13 , 14 resulted in reclassication of 21% ( n = 6 ) of ngsg10negative patients to positive . 
similar to what we observed in patients with aml , 10 samples testing positive for only dta mutations showed no difference in relapse rates or os compared with those testing negative . 
no relapses were observed in patients testing positive only for these 16 genes regardless of conditioning intensity , and only one death was observed ( trm in the mac group )  . 
limiting analysis to those testing positive for an ngsg10 mutation at or above 2.5% vaf reduced the sensitivity at 24 months from 73% to 45% ( data supplement )  . 
overall p values ( in bold ) : grays test for transplant - related mortality and relapse ; log - rank test for rfs and os . abbreviations : ngsg10 , next - generation sequencing 10 - gene ; pos , positive ; neg , negative ; mac , myeloablative conditioning ; ric , reduced intensity conditioning ; rfs , relapse - free survival ; freq , frequency ; os , overall survival ; ci , condence interval . on patient outcomes and found no signicant difference in os based on the mutation number ( data supplement ) or number of mutated genes ( data supplement )  . patients with mds with excess blasts although the requirement for inclusion in the bmt - ctn 0901 study was , 5% marrow myeloblasts and no leukemic myeloblasts in blood on morphological analysis at the time of pretransplant assessment , a total of 22 of the 54 patients with mds ( 41% ) treated on the bmt - ctn 0901 trial had an initial diagnosis of refractory anemia with excess blasts ( raeb ) i or ii ( ie , between 5% and 19% blasts )  . 
seven of these samples tested positive for ngs10g , for which survival was 0% for ric ( n = 2 , relapse ) and 60% for mac - treated patients ( with one death from each relapse and transplantrelated mortality )  . 
ngs10g - positive patients accounted for four of six relapses observed in the raeb cohort , and the other two patients had high - risk cytogenetics with a monosomal karyotype that would not be detectable using this ngs assay . discussion here , we present the impact of genetic mutations detected before random assignment to ric or mac , identied without knowledge of the mutations originally present at diagnosis , on transplant outcomes in patients with mds . 
detection of mutations using a dna - sequencing panel covering regions of 10 genes , previously shown to have utility at the same timepoint in patients with aml , 10 was associated with increased rates of relapse and decreased rfs and os . 
 ( a ) differences in rates of relapse were identied between subgroups dened by conditioning intensity ( ric or mac ) and mutational status ( p , .001 ) , with the highest rate occurring in 10 - gene ngspositive ( ngsg10 - positive ) patients with mds receiving ric . 
the prognostic signicance of the remaining mutations on relapse was dependent on conditioning intensity , with higher rates of relapse and lower rfs observed in patients randomly assigned to receive ric compared with mac . this study provides evidence that the benet of mac versus ric in reducing relapse is found in patients with mds with detectable mutations using a 10 - gene region dnasequencing panel before allohct . 
patients are displayed in columns and grouped by conditioning intensity ( ric or mac ] ) and clinical outcome ( relapse , no relapse , or transplant - related mortality [ trm ] )  . 
genes are displayed in rows and sorted by diagnostic panel groupings , including 10 - gene ( prognostic in aml ) , dta ( associated with age - related clonal hematopoiesis ) , and 16 - gene ( commonly mutated in mds )  . 
although the size of this cohort limits extrapolation , it is possible that the prognostic implications of tp53 mutation detection in patients with mds before allohct and the impact of conditioning intensity differ from those reported previously when those variants are found at a level below the limit of detection of routinely used ngs assays . 
in addition to vaf , other factors such as persistence since initial diagnosis , development during therapy , mutation type , functional classication , and zygosity are likely to be important for determining the relapse risk associated with detecting a tp53 mutation.15 - 22 although mutation detection by targeted dna sequencing before allohct was strongly associated with increased relapse and decreased rfs and these outcomes could be improved with mac , unlike in patients10 with aml , we did not detect an os advantage for conditioning intensication in those testing positive . 
this might have been due to limited sample size , predominately nonrelapse causes of death in this mds cohort ( 67% of deaths , compared with 39% in the previously reported aml cohort ) , or other factors . mds is a genetically heterogenous disease.1 , 11 , 12 , 23 as was observed previously , 8 we found that inclusion of a large number of genes resulted in the majority of patients having mutations present before transplant . 
furthermore , mutations in genes associated with clonal hematopoiesis ( dta ) or 16 other mds - associated genes outside of 10 - gene improve test performance . mutational status did not reanalysis of pretransplant mutational status using only the 10 genes reported here , from another study of patients with mds with , 5% myeloblasts before allohct , 8 conrms our nding of signicantly increased relapse rates in patients testing positive and receiving ric versus mac . 
the modest there are several sample size here offers only a partial sampling of the heterogenous genetics associated with mds , whereas differences in pretransplantation disease burden , biology , and prior treatment history further limit exact comparisons . the signicance of mutation detection before allohct might be inuenced by history of prior treatment , and previous work using the same testing in patients with aml included only those treated to cytomorphological remission before transplant . 
 mds relapse after allohct allohct.9 two randomized studies of conditioning intensity in mds have failed to show a difference in survival between mac and ric , 4 , 6 , 25 although we show here a trend toward improved survival with increased conditioning intensity for those with detectable mutations particularly in the raeb group ( ie , biology and treatment history most analogous to aml )  . 
prior work had shown a benet for increased conditioning intensity in patients with mds with ras pathway mutations ( nras , kras , ptpn11 , cbl , nf1 , rit1 , flt3 , and kit ) undergoing allohct.9 as relapse is not primary cause of death in patients with mds undergoing allohct , the choice of conditioning intensity is currently dependent on the estimated ability of a patient to tolerate transplant - related toxicity.26 allohct remains the only curative therapy for mds ; however , given the median age at diagnosis , many patients will not be eligible for myeloablative approaches.27 , 28 as it is now possible to determine the genomic basis of disease before and after transplantation , personalized approaches for allohct of patients with mds are now conceivable with targeted strategies for those with detectable tp53 , ras pathway ( including flt3 ) , immune augmentation , or other therapies potentially able to supplement the impact of chosen conditioning intensity to minimize relapse risk.29 - 31 idh and jak2 mutations , in conclusion , we show that in adult patients with mds with , 5% marrow myeloblasts and no leukemic myeloblasts in blood before allohct , ultra - deep dna sequencing for mutations in 10 gene regions previously shown to be high risk in patients with aml could identify a subset of 42% of patients who experienced the majority of post - transplant relapses . 
the cibmtr registry is supported primarily by the u24 - ca76518 from nhlbi , nci , and the national institute of allergy and infectious diseases and from hhsh234200637015c ( hrsa / dhhs ) to the center for international blood and marrow transplant research . 
blood 122 : 2943 - 2964 , 2013 luger sm , ringden o , zhang mj , et al : similar outcomes using myeloablative vs reduced - intensity allogeneic transplant preparative regimens for aml or mds . bone marrow transpl 47 : 203 - 211 , 2012 shimoni a , hardan i , shem - tov n , et al : allogeneic hematopoietic stem - cell transplantation in aml and mds using myeloablative versus reduced - intensity conditioning : the role of dose intensity . 
leukemia 20 : 322 - 328 , 2006 kroger n , iacobelli s , franke gn , et al : dose - reduced versus standard conditioning followed by allogeneic stem - cell transplantation for patients with myelodysplastic syndrome : a prospective randomized phase iii study of the ebmt ( ricmac trial )  . 
j clin oncol 35 : 2157 - 2164 , 2017 pulsipher ma : reduced intensity for myelodysplastic syndrome : worth the gamble ? j clin oncol 35 : 2106 - 2108 , 2017 scott bl , pasquini mc , logan br , et al : myeloablative versus reduced - intensity hematopoietic cell transplantation for acute myeloid leukemia and myelodysplastic syndromes . 
j clin oncol 35 : 1154 - 1161 , 2017 bejar r , stevenson ke , caughey b , et al : somatic mutations predict poor outcome in patients with myelodysplastic syndrome after hematopoietic stem - cell transplantation . 
n engl j med 379 : 1028 - 1041 , 2018 lindsley rc , saber w , mar bg , et al : prognostic mutations in myelodysplastic syndrome after stem - cell transplantation . 
n engl j med 371 : 2477 - 2487 , jaiswal s , fontanillas p , flannick j , et al : age - related clonal hematopoiesis associated with adverse outcomes . 
montalban - bravo g , kanagal - shamanna r , benton cb , et al : genomic context and tp53 allele frequency dene clinical outcomes in tp53 - mutated 16 . 
coombs cc , zehir a , devlin sm , et al : therapy - related clonal hematopoiesis in patients with non - hematologic cancers is common and associated with adverse 18 . 
al - kali a , zblewski d , foran jm , et al : outcome of myelodysplastic syndromes over time in the united states : a national cancer data base study from 20042013 . 
sorror ml , sandmaier bm , storer be , et al : long - term outcomes among older patients following nonmyeloablative conditioning and allogeneic hematopoietic cell transplantation for advanced hematologic malignancies . 
maslah n , salomao n , drevon l , et al : synergistic effects of prima - 1met ( apr - 246 ) and azacitidine in tp53 - mutated myelodysplastic syndromes and acute myeloid leukemia . 
wei , md1 cdk12 is a kinase that regulates several key cellular processes , including transcription and maintenance of genomic stability.1 the prevalence of cdk12 alterations is higher in metastatic castration - resistant prostate cancer ( mcrpc ; 7% ) compared with primary prostate cancer ( 1% ) , which indicates an association with an aggressive phenotype.2 in the articles accompanying this editorial in jco precision oncology , schweizer et al3 and antonarakis et al4 report on the clinical features and outcomes of patients with cdk12 - altered prostate cancer in two independent retrospective multicenter series . 
in both studies , the source of tumor dna included primary prostate , metastatic sites , and circulating tumor dna sequenced for cdk12 alteration status through a variety of commercial and inhouse sequencing platforms . schweizer et al3 identied 52 patients who harbored cdk12 mutations , and antonarakis et al4 identied 60 . in both studies , there was a roughly even split incidence of monoallelic or biallelic cdk12 inactivation . because of limitations in the diverse sequencing platforms used , neither series required biallelic mutations . 
in the schweizer series , median time from initiation of androgen deprivation therapy ( adt ) to development of crpc was 1.4 years , and in the antonarakis series , median progression - free survival ( pfs ) after initiation of adt was 12.3 months , which indicates short responses to primary adt . 
median overall survival ( os ) from time of metastasis was 3.9 years in the schweizer series , and median os from initiation of adt was 40.8 months in the antonarakis data set , which suggests poor prognosis . 
in the schweizer cohort , the unconrmed 50% prostate - specic antigen decline ( psa50 ) response rate to rst - line abiraterone and enzalutamide for crpc was approximately 70% ; however , median pfs was short at approximately 9 months . 
first , antonarakis et al required a conrmatory psa at least 4 weeks after the initial psa50 response , which was not required in the schweizer series and likely contributed to the lower psa response rate in the antonarakis study . 
de novo metastatic presentation is a poor prognostic factor that likely contributed to the shorter pfs with rst - line abiraterone and enzalutamide in the antonarakis study . these data are consistent with another recently published retrospective series by reimers et al5 that included 46 patients with advanced prostate cancer with cdk12 mutations . 
of note , the patients with cdk12 loss - of - function alterations had worse clinical outcomes compared with those with other mutations , including atm , brca1 / 2 , and tp53 . 
taken together , all three studies have consistently shown that cdk12 alterations portend aggressive clinical features and relative resistance to second - generation androgen signaling inhibitors . while the antonarakis and schweizer series represent the largest cdk12 - altered cohorts to date , they are both limited by their retrospective nature . 
clinical assessments were variable and not standardized , and there were no comparisons of cdk12 alteration with other genomically dened cohorts ( as in the reimers et al5 study )  . 
regardless , the authors should be commended for assembling these large data sets that help to inform on the prognostic implications of cdk12 - mutated prostate cancer . furthermore , these series include the largest cohort to date of outcomes in cdk12 - mutated prostate cancer after immune checkpoint blockade , shedding light on its predictive potential . 
two phase iii , randomized , placebocontrolled trials of ipilimumab in the postdocetaxel and predocetaxel mcrpc settings failed to meet their primary end point of os.6 , 7 pembrolizumab alone in mcrpc leads to low rates of either psa or radiographic response.8 while the prevalence of mismatch repair ( mmr ) deciency in crpc is low ( , 5% ) , 9 deciencies in mmr proteins are associated with higher psa and objective responses to pd - 1 / pd - l1 inhibitors.10 the response rate to pd - 1 / pd - l1 inhibitors in crpc is higher than the prevalence of mmr deciency , and some patients achieve responses without harboring mmr - decient tumors , which suggests that there are other currently undetermined biomarkers of clinical benet . cdk12 biallelic inactivating mutations lead to the formation of ftd , which results in increased prevalence of gene fusions , elevated antigen load , and inltration of t cells into the tumor microenvironment.2 these ndings suggest that tumors that harbor cdk12 loss may be immunogenic and susceptible to immunotherapy . in a small cohort of 4 pretreated patients with cdk12 - mutant mcrpc treated with an anti - pd - 1 immune checkpoint inhibitor , 2 experienced a marked psa decline.2 this suggests the potential of cdk12 as a predictive biomarker for immunotherapy , a hypothesis that can be scrutinized further with new data from the schweizer and antonarakis series . in the antonarakis series , 9 heavily pretreated men with mcrpc received a pd - 1 inhibitor , of whom 3 ( 33% ) achieved a conrmed psa50 response . 
in the schweizer series , 19 patients received diverse immunotherapy regimens , including pembrolizumab , atezolizumab , nivolumab and ipilimumab , durvalumab and tremelimumab , and atezolizumab and radium - 223 . 
objective responses were not assessed in this series . heterogeneity in patient population , prior therapies , and treatment regimens likely account for the differences in clinical outcomes to immune checkpoint inhibitors observed in these two studies . 
furthermore , the rate of objective responses and duration of disease control remain unclear . ultimately , prospective data are needed to further dene the role of cdk12 as a potential biomarker to predict response and benet to immune checkpoint inhibitors in advanced prostate cancer . 
toward this end , multiple prospective clinical trials are currently under way to explore outcomes to immune checkpoint inhibition in patients with prostate cancer who harbor cdk12 alterations or other dna damage repair defects ( table 1 )  . 
of note , these studies are prospectively enriching for genomic alterations of interest and cover a spectrum of disease states , including biochemically recurrent prostate cancer , metastatic hormone - sensitive prostate cancer , and mcrpc . 
for example , cdk12 loss is associated with amplication of cell cycle genes ccn1 and cdk4 , which suggests that cdk12 - mutated tumors may be susceptible to cdk4 / 6 inhibition.11 the combination of atezolizumab and abemaciclib is being studied in cdk12 - altered mcrpc ( clinicaltrials.gov identier : nct04272645 )  . optimal patient selection using clinical and genomic / molecular characteristics along with effective combination strategies are likely to move the needle for immunotherapy strategies in prostate cancer . 
despite the limitations of retrospective analysis , these data with rich clinical annotation suggest that patients with prostate cancer with cdk12 mutations have a poor prognosis and that novel therapies are needed . 
wei honoraria : onclive research funding : bristol - myers squibb travel , accommodations , expenses : corvus pharmaceuticals no other potential conicts of interest were reported . references blazek d , kohoutek j , bartholomeeusen k , et al : the cyclin k / cdk12 complex maintains genomic stability via regulation of expression of dna damage response genes . 
cell 173 : 1770 - 1782.e14 , 2018 schweizer mt , ha g , gulati r , et al : cdk12 - mutated prostate cancer : clinical outcomes with standard therapies and immune checkpoint blockade . 
eur urol 77 : 333 - 341 , 2020 kwon ed , drake cg , scher hi , et al : ipilimumab versus placebo after radiotherapy in patients with metastatic castration - resistant prostate cancer that had progressed after docetaxel chemotherapy ( ca184 - 043 ) : a multicentre , randomised , double - blind , phase 3 trial . 
lancet oncol 15 : 700 - 712 , 2014 beer tm , kwon ed , drake cg , et al : randomized , double - blind , phase iii trial of ipilimumab versus placebo in asymptomatic or minimally symptomatic patients with metastatic chemotherapy - naive castration - resistant prostate cancer . 
j clin oncol 38 : 395 - 405 , 2020 abida w , cheng ml , armenia j , et al : analysis of the prevalence of microsatellite instability in prostate cancer and response to immune checkpoint blockade . jama oncol 5 : 471 - 478 , 2019 10 . 
agarwal n , loriot y , mcgregor ba , et al : cabozantinib ( c ) in combination with atezolizumab ( a ) in patients ( pts ) with metastatic castration - resistant prostate cancer ( mcrpc ) : results of cohort 6 of the cosmic - 021 study . 
because poly ( adp - ribose ) polymerase inhibitors have significant activity in brca1 / 2 - mutant ovarian and breast cancers , rucapanc investigated the efficacy and safety of rucaparib in brca1 / 2 - mutant pancreatic cancer . patients and methods rucapanc enrolled patients with measurable locally advanced / metastatic pancreatic cancer who had received one to two prior chemotherapy regimens . 
four patients achieved a response ; two partial responses and one complete response ( cr ) were confirmed ( objective response rate , 15.8% ; 3 of 19 ) , with an additional cr unconfirmed . 
the disease control rate ( cr , partial response , or stable disease for 12 weeks ) was 31.6% ( 6 of 19 ) in all patients and 44.4% ( 4 of 9 ) in those who had received one prior chemotherapy regimen . 
these mutations are predicted to lead to the reversion of a somatic not germlinemutation . conclusion rucaparib provided clinical benefit to patients with advanced pancreatic cancer and a brca1 / 2 mutation , and demonstrated an acceptable safety profile . 
2018 by american society of clinical oncology introduction pancreatic cancer ( pc ) is projected to become the second leading cause of cancer death by 2020.1 the majority of pcs are diagnosed at an advanced stage , precluding a surgical , curative approach . 
in the setting of advanced disease , folfirinox treatment ( folinic acid [ leucovorin ] , fluorouracil , irinotecan , and oxaliplatin ) and gemcitabine in combination with nab - paclitaxel are the standards of care , with both demonstrating an improvement in overall survival compared with gemcitabine alone.2 , 3 in the second - line setting , prospective data have shown modest results , with response rates to chemotherapy generally less than 20% , 4 including second - line folfirinox or fluorouracil with nanoliposomal irinotecan.4 - 8 additional therapeutic options are needed . approximately 9% of unselected pcs are associated with a germline or somatic mutation in brca1 or brca2 ( brca1 / 2 ) .9 , 10 cells with a deleterious brca1 / 2 mutation and resultant homologous recombination deficiency are unable to repair dna double - strand breaks reliably11 , 12 and are thus sensitive to poly ( adp - ribose ) polymerase ( parp ) inhibition.13 - 16 studies have demonstrated clinical benefit with parp inhibitors in clinical trials of germline brca1 / 2 mutant breast and ovarian cancer . 
the study design was approved by the independent review board at each participating site and was conducted according to the provisions of the declaration of helsinki and the good clinical practice guidelines of the international conference on harmonization of technical requirements for registration of pharmaceuticals for human use . 
all patients provided written informed consent before participating in the trial . enrolled patients were men and women at least 18 years of age with histologically confirmed locally advanced or metastatic pancreatic adenocarcinoma with measureable disease and a known deleterious germline or somatic brca1 / 2 mutation by local testing . 
patients could have received up to two prior lines of chemotherapy in the locally advanced / metastatic setting , but could not have received prior therapy with a parp inhibitor . 
to infer loss of heterozygosity ( loh ) , the log - ratio profile of sequencing data was segmented , and the allele frequencies of sequenced genome - wide single nucleotide polymorphisms were used to estimate the copy and minor allele frequency for each segment . 
pretreatment plasma specimens were collected on day 1 of cycle 1 for circulating tumor dna ( ctdna ) analysis by foundation medicine , which is a hybrid capture - based next - generation sequencing assay that sequences the coding regions of 62 cancer - related genes , including brca1 / 2 . 
safety results were reported by analysis of adverse events ( aes ) and graded according to the common terminology criteria for adverse events ( version 4 )  . the safety analysis included all patients who received at least one dose of rucaparib . 
for inclusion in the objective response rate ( orr ) , a complete response ( cr ) or partial response ( pr ) per recist had to have been confirmed at least 28 days after the initial response was observed . 
the median age was 57 ( range , 41 to 75 ) years , 57.9% of patients ( 11 of 19 ) were male , the median number of prior chemotherapy regimens for locally advanced / metastatic disease ( excludes adjuvant treatment where progression occurred > 6 months after completion of treatment ) was two ( range , 1 to 3 ) , 78.9% of patients ( 15 of 19 ) had an eastern cooperative oncology group performance status of 1 , and 78.9% ( 15 of 19 ) had a brca2 mutation . 
as prespecified in the protocol , enrollment was stopped because of lack of responses in the first 15 patients evaluated ; the three confirmed responses occurred in the last four patients enrolled . after study closure , one patient continued therapy on an individual patient ind application . 
thirteen patients discontinued the study because of radiologic or clinical progression , two discontinued therapy within a week of beginning ( one because of investigator decision , the other for an unknown reason ) , one discontinued because of aes with simultaneous radiologic progression confirmed at the end of treatment , one discontinued because of an ae with ongoing sd , and one withdrew consent with an ongoing pr . 
patients received rucaparib for a median of 57 days in the study ( range , 2 to 504 days )  . brca1 and brca2 mutation status sixteen of 19 patients had a germline brca1 / 2 mutation . 
of the 16 tumors associated with a germline mutation , paired somatic sequencing was available in eight ; the allelic frequency of the known germline mutant allele was between 41% and 53% . 
this was counted as three separate chemotherapy treatment regimens per the protocol - specified criteria . circulating tumor dna samples for ctdna analyses at cycle 1 , day 1 ( before treatment with rucaparib ) were available for 16 patients , and somatic mutations were detected in 11 patients ( 10 of whom had kras mutations ; appendix table a1 )  . 
investigator - assessed responses ( recist ) in patients with pancreatic cancer and a brca mutation ( n = 19 ) unconfirmed cr / confirmed sd response not evaluable confirmed response rate ( cr or pr ) confirmed response rate ( cr or pr ) in patients with only one prior chemotherapy for locally advanced / metastatic disease disease control rate ( cr , pr , or sd 12 weeks ) all patients patients with only one prior chemotherapy regimen for locally advanced / metastatic disease no . 
in the first patient ( patient 5 ) , ctdna analysis detected the germline brca2 mutation c.7060c > t ( allele frequency , 48% ) , the somatic alteration c.1499_1499delg ( allele frequency , 22% ) , and the secondary somatic mutation c.1416_1420delgcatc ( allele frequency , 19% ) that restored the open reading frame of the brca2 gene . 
in the second patient ( patient 14 ) , ctdna analysis detected the germline brca2 mutation c.5946deit ( allele frequency , 46% ) , the somatic alteration c.1387dela ( allele frequency , 13% ) , and the secondary somatic mutation c.1355_1380deltaccaaaatcagagaagccattaaat , which also restored the open reading frame , but seemed to be subclonal ( with an allelic frequency of only 1% )  . 
this patient , who had also previously received folfirinox , developed significant clinical progression at day 18 , although target lesions were stable . assessment of allele - specific loh tumor was available for analysis in 10 patients . 
 rucaparib treatment stable disease complete response progressive disease partial response progressive disease reported on last day of treatment < 1 weekd patient of prior regimens received prior platinum platinum refractory brca mutation brca2 somatic brca1 germline brca2 germline brca1 germline brca2 germline brca2 germline brca1 germline brca2 germline brca2 germline brca1 germline brca2 germline brca2 germlineg brca2 germline brca2 germline brca2 germline brca2 germline brca2 germline brca2 somatic brca2 somatic sd 72 weeks sd 20 weeks sd 24 weeks pr 36 weeks pr 25 weeks cr 19 weeks pr 5 weeksi cycle fig 2 . 
 ( f ) patient discontinued because of adverse events ( aes ; grade 3 fatigue and grade 4 thrombocytopenia ) ; best response of stable disease ( sd ) was ongoing after the last day of treatment . 
the second patient had a somatic brca2 mutation ( c.1748t > a ) and had a cr as best response ( patient 19 )  . safety all patients had at least one treatment - emergent ae ( table 3 )  . 
two other patients died as a result of disease progression . discussion this study tested the efficacy of a single - agent parp inhibitor , rucaparib , in patients with advanced pc with a known deleterious brca1 / 2 mutation . 
adverse events were graded according to the national cancer institutes common terminology criteria for adverse events ( version 4 )  . * includes adverse events of thrombocytopenia and decreased platelet count . with a confirmed orr of 16% and an observed disease control rate of 32% . 
our findings suggest that there may be a role for parp inhibition in patients with a brca1 / 2 mutation , particularly in those whose disease has not progressed while taking prior platinum therapy . although this was a single - arm study , making comparisons to other agents difficult , the clinical relevance of our results merits comparison with other current standards of care for this patient population . 
 importantly , the only approved chemotherapy combination in the second - line setting , fluorouracil with nanoliposomal irinotecan , had an overall response rate of 7.7% in the pivotal registration study.8 other small single - center studies investigating folfirinox or other chemotherapy combinations in a refractory population show similarly low response rates , reflecting the known chemoresistance of this disease . 
 furthermore , as treatment options improve for pc , patients are even able to move beyond second - line therapy , for which there are no standard of care options . 
this underscores the importance of looking outside of chemotherapy options , particularly in patients with potentially targetable mutations . previous small studies investigating parp inhibition in brca - mutated pc have shown similar efficacy . 
the response rate of single - agent olaparib in patients with metastatic pc harboring a germline brca1 / 2 mutation who had received prior chemotherapy was 22%.20 a phase ii study of veliparib alone in 16 previously treated patients with brca - mutated pc demonstrated single - agent activity , with 25% of patients having sd for at least 4 months . 
analysis is ongoing to understand the role platinum sensitivity played in these four patients.21 when considered with the prior studies , these trials should provide insight into the clinical utility of single - agent parp inhibition in patients with pc and a known brca1 / 2 mutation . in this study , responses to rucaparib were seen in individuals harboring a germline or somatic brca1 / 2 mutation . 
because of significant stromal infiltration in pc , assessing allele frequency of somatic tumor alterations may be challenging . none of the four patients with a confirmed or unconfirmed response had experienced disease progression on prior platinum therapy ( one had never received platinum ) , and three of the four patients had only received one prior chemotherapy regimen for locally advanced or metastatic disease . 
this finding highlights the important question of the role of platinum sensitivity in the setting of advanced / metastatic pc and underscores a potential role for rucaparib as a treatment for patients whose tumors are not platinum refractory . 
similar findings were noted in patients with advanced ovarian cancer treated with olaparib , because the highest response rates were noted in patients who were deemed platinum sensitive rather than resistant or refractory.22 this will need to be investigated in a larger clinical study . 
 secondary mutations that likely confer resistance have been observed in patients with ovarian or prostate cancer who harbor a brca1 / 2 mutation and whose disease has progressed while receiving platinum chemotherapy or parp inhibitors ; however , these reports have demonstrated reversion mutations that have restored the open reading frame in the vicinity of the germline mutation . 
the majority ( but not all ) of brca1 / 2 - mutant breast and ovarian cancers have allele - specific loh23 ; however , the loss of the second allele is most commonly due to copy neutral loh and rarely due to a somatic mutation . 
both of these patients had previously been treated with oxaliplat several studies have demonstrated the presence of reversion mutations in patients previously treated with chemotherapy , particularly platinum based.24 - 27 our study has several limitations , including a small sample size and lack of corresponding somatic sequencing and ctdna analysis for some patients . rucaparib is a well - tolerated parp inhibitor that could be considered in patients with advanced pc with known brca1 / 2 mutations who have received prior chemotherapy . 
domchek research funding : prism biolab ( inst ) , genentech ( inst ) , novartis ( inst ) , newlink genetics ( inst ) , eli lilly ( inst ) , aduro biotech ( inst ) , pfizer ( inst ) , sanofi ( inst ) data analysis and interpretation : rachna t . 
shroff consulting or advisory role : celgene , codiak biosciences , agios , halozyme travel , accommodations , expenses : astrazeneca ravit geva honoraria : bristol - myers squibb , msd , eli lilly , medison , roche , novartis , janssen , pfizer , teva travel , accommodations , expenses : bristol - myers squibb , roche ron epelbaum no relationship to disclose lindsey rolfe employment : clovis oncology leadership : clovis oncology stock and other ownership interests : clovis oncology , celgene , biomarin travel , accommodations , expenses : clovis oncology sandra goble employment : clovis oncology stock and other ownership interests : clovis oncology research funding : eli lilly , celgene , agios , halozyme travel , accommodations , expenses : celgene , codiak biosciences , agios , halozyme kevin k . 
domchek honoraria : astrazeneca , clovis oncology , bristol - myers squibb heidi giordano employment : clovis oncology stock and other ownership interests : clovis oncology acknowledgment research funding : astrazeneca ( inst ) , clovis oncology ( inst ) , pharmamar ( inst ) robert h . 
vonderheide honoraria : genentech , celgene consulting or advisory role : eli lilly , celldex research funding : eli lilly ( inst ) the authors thank lawrence leichman for his helpful input while the study was being designed . 
shroff , the university of texas md anderson cancer center , houston , tx ; andrew hendifar , cedars - sinai medical center , los angeles , ca ; robert r . 
mcwilliams , mayo clinic , rochester , mn ; ravit geva , tel aviv university , tel aviv ; ron epelbaum , rambam health care campus , haifa , israel ; lindsey rolfe , sandra goble , kevin k . 
rahib l , smith bd , aizenberg r , et al : projecting cancer incidence and deaths to 2030 : the unexpected burden of thyroid , liver , and pancreas cancers in the united states . 
assaf e , verlinde - carvalho m , delbaldo c , et al : 5 - fluorouracil / leucovorin combined with irinotecan and oxaliplatin ( folfirinox ) as second - line chemotherapy in patients with metastatic pancreatic adenocarcinoma . 
lee mg , lee sh , lee sj , et al : 5 - fluorouracil / leucovorin combined with irinotecan and oxaliplatin ( folfirinox ) as second - line chemotherapy in patients with advanced pancreatic cancer who have progressed on gemcitabine - based therapy . 
nagrial am , chin vt , sjoquist km , et al : second - line treatment in inoperable pancreatic adenocarcinoma : a systematic review and synthesis of all clinical trials . 
wang - gillam a , li cp , bodoky g , et al : nanoliposomal irinotecan with fluorouracil and folinic acid in metastatic pancreatic cancer after previous gemcitabine - based therapy ( napoli - 1 ) : a global , randomised , open - label , phase 3 trial . 
kristeleit r , shapiro gi , burris ha , et al : a phase i - ii study of the oral parp inhibitor rucaparib in patients with germline brca1 / 2 - mutated ovarian carcinoma or other solid tumors . 
swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
lowery ma , kelsen dp , smith sc , et al : phase ii trial of veliparib ( v ) in patients ( pts ) with previously treated brca or palb2 - mutated ( mut ) pancreas adenocarcinoma ( pc )  . 
domchek sm , aghajanian c , shapira - frommer r , et al : efficacy and safety of olaparib monotherapy in germline brca1 / 2 mutation carriers with advanced ovarian cancer and three or more lines of prior therapy . 
afghahi a , timms km , vinayak s , et al : tumor brca1 reversion mutation arising during neoadjuvant platinum - based chemotherapy in triple - negative breast cancer is associated with therapy resistance . 
weigelt b , comino - mndez i , de bruijn i , et al : diverse brca1 and brca2 reversion mutations in circulating cell - free dna of therapy - resistant breast or ovarian cancer . 
schrock , phd4 ; giovanni randon , md1 ; sandra romero - cordoba , phd1 , 5 ; daniele rossini , md6 , 7 ; giovanni fuc `a , md1 ; jeffrey s . 
ross , md4 , 8 ; daisuke kotani , md9 ; russell madison , phd4 ; seung tae kim , md10 ; lisa salvatore , md11 ; alessandra raimondi , md1 ; filippo pagani , md1 ; beatrice borelli , md6 , 7 ; federica perrone1 ; maria di bartolomeo , md1 ; vincent a . 
clinical data were available for 27 at - raspositive and 467 negative cases from the italian collaboration , memorial sloan kettering cancer center , samsung medical center , and the breac study . results at - ras mutations were identied in 163 ( 0.9% ) of 18 , 270 mcrcs . 
pole exonuclease domain mutations had higher frequency ( 7% ) than expected and were found only in microsatellite - stable tumors with high tumor mutational burden ( tmb )  . 
on the basis of these ndings , the use of anti - egfr monoclonal antibodies cetuximab and panitumumab in metastatic colorectal cancer ( mcrc ) is now restricted to patients with ras wild - type tumors . several studies also showed that patients with rasmutated mcrc have poorer survival as compared with those with ras wild - type disease , also when treated with bevacizumab - containing rst - line regimens.5 hotspot mutations affecting codons other than 12 , 13 , 59 , 61 , 117 , and 146 account for a small subset of ras mutations . 
a retrospective , next - generation sequencing ( ngs ) based study of 177 patients6 showed that up to 2.8% of mcrcs bear atypical ras ( at - ras ) alterations . 
 pietrantonio et al context key objective to provide molecular and clinical characterization of atypical ras mutations ( ie , those occurring outside major hotspots in codon 12 , 13 , 59 , 61 , 117 , and 146 ) in metastatic colorectal cancer . knowledge generated we provide the rst evidence , to our knowledge , that the prognostic role of atypical ras mutations is superimposable to that of typical ras and that tumors bearing these alterations are characterized by a high prevalence of pole exonuclease domain mutations and high tumor mutational burden . relevance our results suggest the clinical meaning of atypical ras mutations as markers of activation of ras pathway and highlight the potential clinical relevance of testing at - rasmutated tumors for the presence of pole exonuclease domain mutations to identify a niche of patients who may be candidates for immunotherapy approaches . reveal mutations otherwise missed by hotspot - targeted realtime polymerase chain reaction or multiplex - hotspot maldi - tof technologies , which are included in recommendations by current guidelines.7 because of the widespread use of ngs and other comprehensive genomic proling techniques that allow sequencing of the entire coding regions of ras genes , at - ras mutations are being detected with increasing frequency . 
extended ras sequencing data for each screening platform are detailed in the data supplement . we were able to retrieve complete clinical data for 33 patients with at - rasmutated mcrc , and we compared them with a cohort of 467 at - rasnegative cases retrieved by the italian institutions and the breac study in terms of baseline characteristics and overall survival ( os )  . 
six patients were excluded because of the co - occurrence of typical ras or braf v600e mutations in their specimens . extended molecular data were available for patients from the memorial sloan kettering cancer center database and the foundation medicine database . 
functional at - ras mutation characterization , namely mapk pathway activation compared with ras wild - type status , was reported according to loree et al.9 mutational frequency of recurrently mutated genes was compared with historical data reported by yaeger et al10 in the setting of mcrc . 
the study was approved by the fondazione irccs istituto nazionale dei tumori di milano institutional review board ( study id : int 117 / 15 ) and conducted according to the ethical principles for medical research involving human subjects adopted in the declaration of helsinki . 
these included sex , age , eastern cooperative oncology group performance status , primary tumor location ( rightv left - sided ) , mucinous histology , primary tumor resection , time to metastases ( synchronous v metachronous ) , number of metastatic sites ( one v more than one ) , and microsatellite instability ( msi ) status . assessment of primary resistance to anti - egfr treatment to assess primary resistance to anti - egfr antibody therapies , we evaluated the response to anti - egfrs for patients with tumors bearing any at - ras mutation but no co - occurring typical ras or braf mutations . 
only patients receiving cetuximab or panitumumab monotherapy , or in combination with irinotecan if previous irinotecan refractoriness was clearly demonstrated , were included , as previously described.11 molecular analyses the type of at - ras mutations , the presence of nonsilent mutations , and comutated genes were analyzed for each case . 
this comprehensive analysis can overcome numerous biases that confound the analyses to nd driver mutations from the passenger ones . statistical analysis the fishers exact test , v2 test , or mann - whitney test were used when appropriate to assess the associations of at - ras mutations with investigated characteristics . 
we investigated the impact of at - ras mutations on os , dened as the time from diagnosis of metastatic disease to death or last followup for patients who were alive . 
the association of at - ras mutations and clinicopathological characteristics with os was assessed in univariate analyses , and a multivariable cox proportional hazard model was built , including as covariates variables associated with survival with a p value , .10 in the univariate analyses . 
 pietrantonio et al results clinicopathological and molecular features of patients bearing at - rasmutated mcrc as listed in table 1 , when focusing on the 27 patients with mcrc with clinical information available and isolated atras mutations , this population was not signicantly associated with any specic clinical or pathologic feature , as compared with braf , typical ras , or ras / braf wild - type subgroups . 
the list of specic at - ras mutations with functional characterization , co - occurring typical ras / braf v600 mutations , and msi status is detailed in the data supplement . in the foundation medicine ( from august 2012 to january 2018 ) and memorial sloan kettering cancer center ( from january 2014 to january 2018 ) data sets , 142 and 21 cases with at - ras mutations were retrieved out of a total of 16 , 324 and 1 , 946 mcrc samples analyzed from unique patients , respectively , with an overall incidence of 0.9%. a total of 56 unique at - ras mutations were detected ( fig 2a ) , of which 30 kras and three nras were known oncogenic missense mutations or indels as dened by the oncokb database . 
overall , at - kras and - nras mutations occur primarily in the gtpase domain and consequently may affect the ability of ras gtpase - activating proteins , such as nf1 , to bind ras and regulate its signaling ( data supplement )  . 
in our data set , a specic at - ras mutation could be possibly recognized as driver in 76% of cases , suggesting a functional effect of a subset of at - ras alterations in mcrc tumorigenesis . 
in 24% of cases with at - ras mutations possibly recognized as passengers , we then explored the cooccurrence with other mutational events to dene possible cooperative mechanisms involved in tumor growth . some unique frequently comutated genes with at - ras passenger mutations were involved in critical tumorigenic pathways such as mapk , atm , and ras positive regulation of phosphorylation signaling ( data supplement )  . recurrently mutated genes identied by ngs for memorial sloan kettering cancer center database and foundations medicine database patient screening sources ( n = 163 ) are outlined in the data supplement . 
regarding msi status from 133 at - ras tumors including most with available data , 8% of tumors were classied as msihigh ( msi - h ) and 92% as mss , in agreement with the frequency observed in typical ras - mutated tumors10 ( n = 354 ; msi - h , 11% ; mss , 89% )  . 
furthermore , typical ras and / or braf v600e mutations were more common in the tmb - high group ( 43% ) than in tmb low ( 28% )  . 
patient characteristics according to the presence of at - ras , braf , and typical ras mutations and ras / braf wild - type status at - ras mutated ( n = 27 ) braf mutated ( n = 17 ) ras mutated ( n = 176 ) ras / braf wild - type ( n = 274 ) characteristics female male age , years median ecog performance status primary tumor location right colon left colon rectum mucinous histology primary tumor resection time to metastases synchronous metachronous no . 
summary of mutagenesis and molecular features associated with atypical ras mutations in patients with extensive next - generation sequencing ( ngs ) data from screening sources of memorial sloan kettering cancer center database and foundation medicine database including 163 patients . ( a ) at - ras mutations oncoplot . 
the upper bar plot indicates the number of mutations per sample , and the right bar plot describes the mutational frequency among the overall population , indicating the variant classication by colors . 
kaplan - meier curves for overall survival ( os ) according to ras , atypical ras ( at - ras ) , and braf v600e mutational ( mut ) status . 
hr , hazard ratio ; na , non - assessable ; ref , reference . the primary resistance cohort ( data supplement ; table 3 )  . one patient experienced a partial response to panitumumab monotherapy , two patients had stable disease lasting less than 6 months , and three patients had progressive disease . 
all samples were assessed using the primary resistance in ras and braf wild - type metastatic colorectal cancer patients treated with anti - egfr monoclonal antibodies ( pressing ) panel , 11 which groups together several genomic alterations associated with anti - egfr primary resistance beyond ras and braf mutations . 
none of the resistant patients with at - ras mutations and primary resistance showed a concomitant genomic alteration known to be associated with anti - egfr primary resistance . discussion recent advances in the depiction of the molecular landscape of mcrc provides evidence for the potential impact of several biomarkers other than ras and braf mutational status and msi status for improving the clinical management of affected patients . 
here we focus on atras mutations to identify the similarities and differences of cases with at - ras versus those with typical ras mutations and their independent weight on patients outcomes . although we found that at - ras mutations are not associated with specic clinicopathological characteristics in mcrc , the prognostic role of isolated at - ras mutations typical ras seems highly superimposable with that of mutations , with a worse prognosis when compared with ras and braf v600e wild type and a clearly better outcome than the braf v600e mutant subgroup . 
supporting this , in a preliminary report loree et al9 functionally characterized a wide set of at - ras mutations by means of an ectopic expression assay , and although the median mapk activity of at - ras mutations was lower than that of typical ras mutations , it was higher than ras wild type . 
bold text indicates signicant p values . abbreviations : hr , hazard ratio ; msi , microsatellite instability ; mss , microsatellite stable ; ref , reference . intriguingly , a high proportion of cases with at - ras mutations ( 30% ) displayed co - occurring typical ras and / or braf v600e mutations . 
given that 92% of cases with at - ras mutations were mss and 83% were tmb low , the occurrence of at - ras mutations was not limited to hyperor ultramutated tumors enriched with passenger mutations . 
it is arguable that the mild hyperactivation of the oncogenic mapk pathway driven by some at - ras mutations may require additional hits ( eg , a co - occurring ras or nf1 mutation ) to efciently drive tumor progression . 
because our results on the negative predictive role of at - ras mutations on the efcacy of anti - egfrs are descriptive and could be biased by several confounding factors , they cannot be used to drive decision making in clinical practice . 
however , even if validation studies of our preliminary ndings are challenging , retrospective or pooled analyses of randomized clinical trials should be ideally carried out , possibly by means of targeted genomic panels including at - ras mutations combined with other multiple , albeit uncommon , primary resistance alterations , as recently performed by our group.26 we highly emphasize that a prospective international data set and preclinical collaborations should be established to better assess the individual role of specic at - ras mutations as predictive biomarkers . the main limitations of our study include the small subset of patients with at - rasmutated tumors with available data about clinical outcome , especially about response to antiegfr agents ; the lack of comprehensive genomic sequencing data for all patients included in the study ; and the heterogeneity of ngs platforms adopted . 
however , our data suggest a close phenotypic similarity between isolated at - ras and typical ras mutations that is reasonably driven by at - ras mutations with established increased mapk activity or by the cooperation of multiple activating signals in the mapk pathway . 
schrock , giovanni randon , daniele rossini , giovanni fuc `a , jeffrey s . ross , daisuke kotani , russell madison , seung tae kim , lisa salvatore , filippo pagani , beatrice borelli , federica perrone , jeeyun lee , takayuki yoshino , filippo de braud , alfredo falcone , jaclyn f . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . filippo pietrantonio consulting or advisory role : amgen , merck serono , bayer , eli lilly , sano , roche , servier rona yaeger research funding : array biopharma , glaxosmithkline , novartis , boehringer ingelheim travel , accommodations , expenses : array biopharma alexa b . 
ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine research funding : foundation medicine daisuke kotani honoraria : merck serono , chugai pharmaceutical , takeda , eli lilly japan consulting or advisory role : merck serono russell madison employment : foundation medicine stock and other ownership interests : foundation medicine lisa salvatore honoraria : roche , merck serono , servier , bayer consulting or advisory role : merck serono , servier , bayer travel , accommodations , expenses : sano , merck serono , bayer maria di bartolomeo honoraria : eli lilly , msd oncology , servier consulting or advisory role : eli lilly , msd oncology research funding : eli lilly ( inst ) travel , accommodations , expenses : roche , sano vincent a . 
miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine , mirati therapeutics consulting or advisory role : revolution medicines patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center siraj m . 
ali employment : foundation medicine leadership : incysus stock and other ownership interests : exelixis , blueprint medicines , agios pharmaceuticals , genocea biosciences consulting or advisory role : revolution medicines , azitra ( i ) , princepx tx ( i ) patents , royalties , other intellectual property : patents via foundation medicine ; patents via seres health on microbiome stuff in nonneoplastic disease ( i ) takayuki yoshino research funding : chugai pharmaceutical ( inst ) , sano ( inst ) , sumitomo dainippon pharma ( inst ) , glaxosmithkline ( inst ) filippo de braud consulting or advisory role : ignyta , pzer , amgen , novartis , daiichi sankyo , bristol - myers squibb , domp `e , pierre fabre , roche , octimet oncology , incyte , teofarma , emd serono , pharm research associates speakers bureau : msd , novartis , bristol - myers squibb , roche , pzer , menarini research funding : novartis ( inst ) , roche ( inst ) , msd ( inst ) , ignyta ( inst ) , medimmune ( inst ) , nektar ( inst ) , bristol - myers squibb ( inst ) , merck serono ( inst ) , bayer ( inst ) , celgene ( inst ) , glaxosmithkline ( inst ) , boehringer ingelheim ( inst ) , eli lilly ( inst ) , pzer ( inst ) , servier ( inst ) alfredo falcone honoraria : eli lilly , roche , merck , servier , amgen consulting or advisory role : amgen , bayer , bristol - myers squibb , eli lilly , merck , roche , servier research funding : amgen ( inst ) , bayer ( inst ) , merck ( inst ) , roche ( inst ) , sano ( inst ) , msd ( inst ) , servier ( inst ) travel , accommodations , expenses : amgen , bayer , roche , merck , servier jaclyn f . 
nat rev cancer 11 : 761 - 774 , 2011 janakiraman m , vakiani e , zeng z , et al : genomic and biological characterization of exon 4 kras mutations in human cancer . 
cancer res 70 : 5901 - 5911 , 2010 douillard jy , oliner ks , siena s , et al : panitumumab - folfox4 treatment and ras mutations in colorectal cancer . 
n engl j med 369 : 1023 - 1034 , 2013 pietrantonio f , cremolini c , petrelli f , et al : first - line anti - egfr monoclonal antibodies in panras wild - type metastatic colorectal cancer : a systematic review and meta - analysis . 
crit rev oncol hematol 96 : 156 - 166 , 2015 cremolini c , loupakis f , antoniotti c , et al : folfoxiri plus bevacizumab versus folfiri plus bevacizumab as rst - line treatment of patients with metastatic colorectal cancer : updated overall survival and molecular subgroup analyses of the open - label , phase 3 tribe study . 
harl e a , filhine - tresarrieu p , husson m , et al : rare ras mutations in metastatic colorectal cancer detected during routine ras genotyping using next generation sequencing . 
ann oncol 29 : 44 - 70 , 2018 shinozaki e , yoshino t , yamazaki k , et al : clinical signicance of braf non - v600e mutations on the therapeutic effects of anti - egfr monoclonal antibody treatment in patients with pretreated metastatic colorectal cancer : the biomarker research for anti - egfr monoclonal antibodies by comprehensive cancer genomics ( breac ) study . 
br j cancer 117 : 1450 - 1458 , 2017 loree jm , miron b , holla v , et al : not all ras mutations created equal : functional and clinical characterization of 80 different kras and nras mutations . 
cremolini c , morano f , moretto r , et al : negative hyper - selection of metastatic colorectal cancer patients for anti - egfr monoclonal antibodies : the pressing case - control study . 
fabrizio da , george tj jr , dunne rf , et al : beyond microsatellite testing : assessment of tumor mutational burden identies subsets of colorectal cancer who may respond to immune checkpoint inhibition . 
kumar p , henikoff s , ng pc : predicting the effects of coding non - synonymous variants on protein function using the sift algorithnat protoc 4 : 1073 - 1081 , 9 : 34 , 2017 2009 19 . 
domingo e , freeman - mills l , rayner e , et al : somatic pole proofreading domain mutation , immune response , and prognosis in colorectal cancer : a retrospective , pooled biomarker study . 
cremolini c , di bartolomeo m , amatu a , et al : braf codons 594 and 596 mutations identify a new molecular subtype of metastatic colorectal cancer at favorable prognosis . 
morano f , corallo s , lonardi s , et al : negative hyper - selection of ras and braf wild - type metastatic colorectal cancer patients receiving panitumumab - based maintenance therapy : a pre - specied exploratory analysis of the valentino study . 
elvin , md , phd1 purpose vulvar squamous cell carcinoma ( vscc ) encompasses two predominant variants : one associated with detectable high - risk strains of human papillomavirus ( hrhpv ) and a second form often occurring in the context of chronic dermatitis in postmenopausal women . 
sixty - one percent of hpv + vsccs had a pathogenic alteration in the pi3k / mtor pathway , whereas hpv vsccs showed alterations in tp53 , tertp , cdkn2a , ccnd1 , and egfr , and biomarkers associated with responsiveness to immunotherapy . jco precis oncol 4 : 647 - 661 . 
90% of vulvar cancers and nearly 5% of all gynecologic cancers.1 , 2 radical excision imposes high morbidity , and one third of patients have been shown to experience recurrence after primary treatment.3 recent reports have shown durable responses with denitive or neoadjuvant chemoradiation for unresectable cancers.4 , 5 for patients with recurrence or distant metastasis , prognosis is poor , with an overall 2 - year survival rate of less than 15%.6 there is a critical need to improve our understanding of the molecular pathogenesis of vscc to provide insights that may guide more effective therapies . vscc develops through two distinct oncogenic pathways . 
this subgroup often associates with usual - type vulvar intraepithelial neoplasia ( vin ) , also known as high - grade squamous intraepithelial lesion.7 although other anogenital squamous cell neoplasms have  . 
 williams et al context key objective previous analyses have suggested genetic differences in vulvar squamous cell carcinoma based on human papillomavirus ( hpv ) status , but restricted sample volume and testing platforms have limited comprehensive identication of statistically signicant differences . 
in a large - scale comparative genomic study , what undiscovered genomic alterations distinguish high - risk hpv - driven versus dystrophic / inammatory - associated vulvar squamous cell carcinoma ? knowledge generated we identify signicantly different molecular proles based on hpv status . 
for genomic analyses , 60 ng of dna was extracted from 40 - m sections of 255 , 008 tumor samples , including 280 vscc specimens and 1 , 031 cervical squamous cell carcinoma ( cscc ) , each from a different patient , in formalin - xed parafnembedded ( ffpe ) tissue blocks . 
hpv genome sequences were detected by de novo assembly of nonhuman sequencing reads and nucleotide basic local alignment search tool ( blastn ) comparison against all viral nucleotide sequences in the national center for biotechnology information refseq database . 
hpv types identied in this study were stratied according to the hpv classication described by muoz et al , 8 with hpv 16 , 18 , 31 , 33 , and 58 labeled hrhpv + and hpv 6 labeled low risk . 
sequencing was performed on the primary tumor in 200 patients and on metastases in 80 ( 57 regional lymph nodes and 23 distant sites )  . hpv status and typing were determined on all 280 patient samples ; 102 / 280 vsccs ( 36% ) contained hrhpv sequences , predominantly hpv 16 ( 88% ; table 1 )  . 
for the 177 hpv vsccs , 137 were sequenced using the original primary tumors and 40 from metastatic site biopsies ( 10 distant , including seven lung , one pleural , one abdominal , and one to bone )  . comprehensive genomic proling figure 1 displays the distribution of gas by hpv status . 
the only specic point mutation with a signicant difference between hpv + and hpv was pik3ca e545k , an activating mutation that was signicantly enriched in hpv + vsccs ( table 2 )  . 
of vsccs cell - free circulating tumor dna ( ctdna ) was evaluated from blood specimens collected from 10 patients with vscc ( liquid biopsy ) using the hybrid capture - based illumina hi - seq ( illumina , san diego , ca ) technology . 
maximum somatic allele frequency was used to estimate the fraction of ctdna per methods previously described.30 , 31 mutational signatures mutational signatures were assessed for all tumor samples with at least 20 nondriver somatic missense alterations . signatures were given by analysis of the trinucleotide context and proled using the sanger cosmic signatures of mutational processes in human cancer.32 a positive signature required a sample to have at least a 40% t to a characterized mutational process , including apobec overexpression , exposure to ultraviolet light , hypofunction of the brca tumor suppressor , and defects in mismatch repair.32 immunohistochemistry programmed death - ligand 1 ( pd - l1 ) immunohistochemistry ( ihc ) was performed regularly in tandem with cgp to guide patient selection for immunotherapy . 
pd - l1 protein expression was assessed by ihc on 5 - micron ffpe tissue sections using the dako pd - l1 ihc22c3 pharmdx assay ( agilent ; santa clara , ca ; n = 52 vsccs ) or the ventana ( oro valley , az ) pd - l1 ( sp142 ) assay ( n = 21 vsccs ) , following each manufacturers instructions . 
dako pd - l1 expression was reported as a tumor proportion score , and ventana pd - l1 was reported as percent tumor area covered by positively staining tumor cells and immune cells . less than 1% staining was dened as negative , 1% - 49% was dened as low positive , and 50% was dened as high positive . clinicopathologic analysis of the vscc cohort a total of 280 vsccs were assayed with cgp ( foundation medicine ) , using material sent from treating institutions , from 2014 to 2019 . 
human investigations were performed after approval by a local human investigations committee and in accordance with an assurance led with and approved by the department of health and human services , where appropriate . 
amp , amplication ; mb , megabase ; msi , microsatellite instability ; msi - h , microsatellite instability - high ; mss , microsatellite stable ; mut , mutation ; pd - l1 , programmed death - ligand 1 ; tmb , tumor mutation burden . of each ga in the hpv and hpv + cohorts is included in appendix figures a2a and a2b , respectively . in the entire cohort : an hpv 16 ( + ) vscc with an mlh1 splice site mutation . frequencies of specic biomarkers associated with responsiveness to immunotherapy differed between the vscc subgroups ( fig 2 )  . 
all other gas , as well as age and pd - l1 ihc staining , showed no signicant differences between primary versus metastatic samples controlled for hpv status . comparison of hpv 16 with other hrhpv subtypes revealed no signicant differences in demographics , tmb , or sequenced site . 
thirty - three ( 12.6% ) were identied with an apobec ( apolipoprotein b mrna - editing enzyme , catalytic polypeptide - like ) signature ( 12 hpv + and 21 hpv ) , two with brca signature ( one hpv + , one hpv ) , seven with mismatch repair ( two hpv + , ve hpv ) , and a single tumor with ultraviolet signature ( hpv )  . 
three of four liquid biopsies showed at least one pathogenic ga present in the associated tissue biopsy ( appendix table a2 )  . separate from our 280 patients in the vscc cohort , ctdna was evaluated on six patients with known vscc but without tissue biopsy sequencing data . 
gas were detected in ve of six of these patients ( appendix table a2 )  . hpv + cscc ( n = 864 ) showed gas that were largely similar to what we found in hpv + vscc ( n = 103 ; appendix table a3 )  . 
although low in frequency , gas in kdm6a , ar , and cdk12 were signicantly higher in vscc versus cscc ( appendix table a3 )  . discussion in this study , hybrid capture - based dna sequencing was applied to a large series of patient tumors to better characterize the genomic landscape of vscc and to identify important genetic differences between hpv + and hpv disease . 
consistent with prior studies , a high rate of mutation was identied overall , with 98% of tumors in the analysis containing one or more known oncogenic mutations.17 , 18 mutational proles sharply differentiated hpv + and hpv disease . 
hpv + vscc showed mutations in the pi3k / mtor pathway , with 61% of tumors containing gas in the pathway , with the majority of gas showing signicant association with hpv + status ( table 2 )  . 
choschzick et al22 specically examined ccnd1 copy number changes in 183 vsccs and identied amplications in 22% , with a signicant association with hpv tumors.23 growdon pd - l1 ihc negative low positive high positive genomic alterations rearrangement short variant deletion not performed amplification fig 2 . 
of the 73 vulvar squamous cell carcinoma for which pd - l1 ihc was performed , 33% of hpv - negative and 9% of hpv - positive were pd - l1 tumor high - positive ; p = .04. 
histopathology of vulvar squamous cell carcinoma ranged from ( a ) well differentiated with abundant keratin to ( b ) poorly differentiated ( hematoxylin and eosin stains , 400 )  . 
 ( c ) programmed death - ligand 1 ( pd - l1 ) staining of human papillomavirus ( hpv ) negative vsccs showed signicantly higher frequency of high - positive tumors , whereas ( d ) hpvpositive disease was largely negative for pd - l1 stain ( pd - l1 immunohistochemistries , 400 )  . et al24 evaluated egfr amplication in 51 vsccs , and identied amplication in 12% of tumors , with signicant association with poor prognosis and hpv status . 
zie ba et al19 performed sequencing of 81 vsccs with a 50 - gene panel , and the results differed from other studies , most strikingly in the absence of clear genomic differences between hpv + and hpv disease . 
the authors reported tp53 and cdkn2a mutations in both hpv + and hpv vscc , whereas mutations in pik3ca , fbxw7 , hras , fgfr3 , stk11 , akt1 , smad4 , and pten were found at low frequencies in both types of vscc.19 zie ba et al19 noted , however , that the 2 hpv tests that they used gave highly inconsistent results and that those hpv tests had not been developed for analyzing tissue - derived dna.19 these difculties in identifying hpv + and hpv disease may account for the divergence of their results from several prior studies.16 - 18 , 20 in our study , hpv status clearly divided our large cohort into two signicantly different genomic - dened diseases . pi3k / mtor pathway mutations , including stk11 , a negative regulator of mtor signaling , have been described in a wide range of hpv - driven cancers.33 , 34 in our cohort , a signicantly higher rate of stk11 gas was observed in hpv + tumors sequenced from metastases , compared with hpv + tumors sequenced from the primary site . stk11 has been previously correlated with poor response to antiprogrammed death - 1 therapy in kras - mutant lung adenocarcinoma.35 it is conceivable that a similar role could exist in hpv + vscc as a putative tumor immuneescape mechanism , but additional studies are needed . a minority of vsccs showed distinctive mutational signatures . 
this signature reects apobec cytidine deaminase dna - editing activity36 , 37 and has been noted to be important in development of thoracic cancers , with possible implications for predicting response to immunotherapy.38 , 39 several of the gas observed to be signicantly enriched in the hpv cohort are in pathways functionally relevant to hpv pathogenesis . 
tp53 , tert , and cdkn2a are deregulated by hpv e6 and / or e7 , whereas egfr recycling is altered by hpv e5.40 - 42 beyond specic gas , key differences were identied in tmb and pd - l1 ihc staining patterns . 
hpv induces genomic instability , which may account for the increased tmb in the primary hpv + cohort.43 in addition , hpv infection reduces the cellular immune response by decreasing the interferon antiviral response.44 cgp may reveal opportunities for targeted therapies to be tested in clinical trials . 
kmt2d , an epigenetic modier , and transcription factor sox2 can activate and interact with the pi3k pathway.49 - 51 gas in both are enriched in hpv + vscc ( table 2 )  . 
tumors with gas in kmt2d may be sensitive to aurora kinase inhibition.52 in light of the many recent successes of immune checkpoint inhibitors , a careful approach to patient selection for clinical trials of these agents may be valuable . 
in our cohort , hpv vsccs showed a signicantly higher rate of pd - l1 ihc highpositive tumor staining , a higher rate of pdl1 amplication , and signicantly lower rates of stk11 alterations . 
vsccs in this category may benet from novel targeted therapeutics.53 other identied potential therapeutic targets include gas in receptor tyrosine kinases , cell cycle regulation , and the mapk pathway . 
early work in gas that affect epigenetic regulation indicates ezh2 inhibitors may be a viable therapeutic strategy.54 , 55 our study also provides a proof of concept that liquid biopsy detects ctdna in vscc , with three of four demonstrating at least one pathogenic ga detected in the tissue biopsy from the same patient . 
liquid biopsy may be a valuable method in vscc , and additional investigation is warranted . limitations in the study include the distinct patient population . tumor samples undergoing cgp are usually sent by clinicians seeking targeted therapy for patients with advanced disease . an additional limitation is the inadequate data on treatment history of the patients before tumor sequencing ; controls for tmb and resistance gas that may have arisen from local radiation or systemic treatment were not available . 
future work is needed to correlate genetic ndings with treatment exposure and follow - up data , which are not included in this study . in this study , we provided evidence that hpv + and hpv vscc are two distinct diseases , each with a characteristic molecular prole . 
 genomics of hpv + versus hpv vulvar squamous cell carcinoma rachel erlich employment : foundation medicine stock and other ownership interests : foundation medicine kevin jon williams stock and other ownership interests : hygieia stock and other ownership interests : gemphire therapeutics consulting or advisory role : gemphire therapeutics , inc . research funding : novo nordisk jeff m . 
venstrom employment : genentech , foundation medicine leadership : genentech stock and other ownership interests : genentech research funding : genentech , foundation medicine travel , accommodations , expenses : genentech brian m . 
alexander employment : foundation medicine leadership : foundation medicine stock and other ownership interests : roche research funding : eli lilly ( inst ) , puma ( inst ) , celgene ( inst ) open payments link : 854258 / summary nikunj shah employment : foundation medicine natalie danziger employment : foundation medicine eric a . 
severson employment : foundation medicine , partners healthcare stock and other ownership interests : foundation medicine jonathan keith killian employment : foundation medicine stock and other ownership interests : foundation medicine douglas i . 
ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine consulting or advisory role : celsius therapeutics research funding : foundation medicine julie y . 
hemmerich employment : foundation medicine stock and other ownership interests : foundation medicine ( inst ) acknowledgment we thank our colleagues for their helpful comments during the preparation of this article . references judson pl , habermann eb , baxter nn , et al : trends in the incidence of invasive and in situ vulvar carcinoma . 
moore dh , ali s , koh wj , et al : a phase ii trial of radiation therapy and weekly cisplatin chemotherapy for the treatment of locally - advanced squamous cell carcinoma of the vulva : a gynecologic oncology group study . 
n engl j med 348 : 518 - 527 , 2003 alemany l , saunier m , alvarado - cabrero i , et al : human papillomavirus dna prevalence and type distribution in anal carcinomas worldwide . 
adv anat pathol 24 : 201 - 214 , 2017 van der avoort ia , shirango h , hoevenaars bm , et al : vulvar squamous cell carcinoma is a multifactorial disease following two separate and independent pathways . 
virgili a , borghi a , toni g , et al : prospective clinical and epidemiologic study of vulvar lichen sclerosus : analysis of prevalence and severity of clinical features , together with historical and demographic associations . 
weberpals ji , lo b , duciaume mm , et al : vulvar squamous cell carcinoma ( vscc ) as two diseases : hpv status identies distinct mutational proles including oncogenic broblast growth factor receptor 3 . 
trietsch md , nooij ls , gaarenstroom kn , et al : genetic and epigenetic changes in vulvar squamous cell carcinoma and its precursor lesions : a review of the current literature . 
swarts dra , voorham qjm , van splunter ap , et al : molecular heterogeneity in human papillomavirus - dependent and - independent vulvar carcinogenesis . cancer med 7 : 4542 - 4553 , 2018 24 . 
halec g , alemany l , lloveras b , et al : pathogenic role of the eight probably / possibly carcinogenic hpv types 26 , 53 , 66 , 67 , 68 , 70 , 73 and 82 in cervical 9 : 34 , 2017 cancer . 
sakamoto j , kamiura s , okayama k , et al : single type infection of human papillomavirus as a cause for high - grade cervical intraepithelial neoplasia and invasive cancer in japan . 
clark ta , chung jh , kennedy m , et al : analytical validation of a hybrid capturebased next - generation sequencing clinical assay for genomic proling of cell - free circulating tumor dna . 
gregg jp , li g , pavlick d , et al : comprehensive genomic proling of ctdna in patients with colon cancer and its delity to the genomics of the tumor biopsy . j clin oncol 36 : 569 , 2018 ( 4 ; suppl ) 32 . 
wang s , jia m , he z , et al : apobec3b and apobec mutational signature as potential predictive markers for immunotherapy response in non - small cell lung cancer . 
gameiro sf , kolendowski b , zhang a , et al : human papillomavirus dysregulates the cellular apparatus controlling the methylation status of h3k27 in different human cancers to consistently alter gene expression regardless of tissue of origoncotarget 8 : 72564 - 72576 , 2017 42 . 
zhang b , srirangam a , potter da , et al : hpv16 e5 protein disrupts the c - cbl - egfr interaction and egfr ubiquitination in human foreskin keratinocytes . oncogene 24 : 2585 - 2588 , 2005 43 . 
song d , li h , li h , et al : effect of human papillomavirus infection on the immune system and its role in the course of cervical cancer . 
tinker av , ellard s , welch s , et al : phase ii study of temsirolimus ( cci - 779 ) in women with recurrent , unresectable , locally advanced or metastatic carcinoma of the cervix . 
oncol lett 12 : 4107 - 4116 , jung k , kang h , mehra r : targeting phosphoinositide 3 - kinase ( pi3k ) in head and neck squamous cell carcinoma ( hnscc )  . 
pi3k pathway regulates er - dependent transcription in breast cancer through the epigenetic regulator kmt2d . oncogene 37 : 1354 - 1368 , 2018 science 355 : 1324 - 1330 , 2017 51 . 
kalu nn , mazumdar t , peng s , et al : comprehensive pharmacogenomic proling of human papillomavirus - positive and - negative squamous cell carcinoma identies sensitivity to aurora kinase inhibition in kmt2d mutants . 
strauss j , heery cr , schlom j , et al : phase i trial of m7824 ( msb0011359c ) , a bifunctional fusion protein targeting pd - l1 and tgfb , in advanced solid tumors . clin cancer res 24 : 1287 - 1295 , 2018 idris s , lindsay c , kostiuk m , et al : investigation of ezh2 pathways for novel epigenetic treatment strategies in oropharyngeal cancer . 
lindsay cd , kostiuk ma , harris j , et al : efcacy of ezh2 inhibitory drugs in human papillomavirus - positive and human papillomavirus - negative oropharyngeal squamous cell carcinomas . 
 hpv negative ( n = 177 ) hpv positive ( n = 103 ) rearrangement short variant deletion amplification multiple rearrangement short variant deletion amplification multiple williams et al fig a2 . 
 systematic assessment of tumor purity and its clinical implications syed haider , phd1 , 2 ; svitlana tyekucheva , phd3 , 4 ; davide prandi , phd5 ; natalie s . 
laird , phd10 ; chris sander11 , 12 ; wenyi wang , phd13 ; francesca demichelis , phd5 , 14 ; massimo loda , md15 , 16 ; and paul c . 
as molecular proles are frequently generated using bulk tissue sections , they represent an admixture of multiple cell types ( including immune , stromal , and cancer cells ) interacting with each other . 
bulk mrna and microrna proles were subject to in silico deconvolution to estimate cancer cellspecic mrna and microrna proles . results we present a systematic comparison of 10 tumor purity estimation methods on a cohort of 333 prostate tumors . 
limited concordance between dnaand mrna - derived purity estimates remained a general pan - cancer phenomenon when tested in an additional 4 , 497 tumors spanning 12 cancer types . conclusion the choice of tumor purity estimation method may have a profound impact on the interpretation of genomic assays . 
taken together , these data highlight the need for improved assessment of tumor purity and quantitation of its inuences on the molecular hallmarks of cancers . jco precis oncol 4 : 995 - 1005 . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction the tumor microenvironment represents an admixture of multiple cell types and complex interactions between bona de cancer cells and surrounding stromal and immune cells.1 because a majority of highthroughput experiments are performed on bulk tissue samples , the resulting signal is usually confounded by nonmalignant tumor - adjacent cells ( tacs )  . variable tumor content and variable tac composition can impinge upon interpretations of molecular data and subsequent clinical decisions.2 - 4 to delineate true residual signal representing individual cell populations , it is crucial to accurately estimate tumor purity . 
tumor purity represents the fraction of cancer cells in a tumor and can be estimated either by expert pathologists reviewing tumor sections5 or in silico ( using epigenomic , genomic , or transcriptomic proles ) .6 pathologic estimates can be inconsistent5 and pragmatically may not always represent the region of tumor that is subject to molecular proling . 
although in silico estimates could circumvent these problems , it remains unclear to what extent these estimates vary across purity calling methods and with the underlying type of biomolecule ( eg , dna v rna )  . 
previous studies have quantied the pan - cancer purity landscape2 , 7 and compared a panel of tools for estimating tumor purity.6 however , systematic benchmarking of in silico tumor purity against matched pathologic estimates and its association with multimodal clinico - genomic proles remains to be elucidated . 
herein , we present systematic benchmarking of 10 purity estimation methods using dna , mrna , and microrna ( mirna ) proles in a 333patient clinically - coherent cohort8 with matched multiobserver pathologic estimates of purity . 
to determine the context specicity of these algorithms , we compared tumor purity estimates from multiobserver pathology to those from multiple algorithms working on different biomolecules ( eg , dna , rna )  . knowledge generated tumor purity estimates from in silico tools varied signicantly from pathology estimates . 
we recommend parameterizing genomic analyses with tumor purity estimated from the matched molecular analyte being analyzed . relevance tumor purity is a key criterion for sample inclusion in clinico - genomic studies and subsequent interpretation of molecular results . 
computational tools often require purity estimates ; we show that these are inuenced by the selected purity estimator . both molecularly driven clinical trials , as well as therapeutic and theranostic decisions , may be affected by these choices . quantify how molecular correlates of tumor purity can skew clinico - genomic interpretations as the result of variable estimates of cancer cell fraction . 
integer infers purity , ploidy , and subclonality from paired tumor and normal samples using the following principles : ( 1 ) models the relationship between the observed allelic frequencies and the underlying copy number changes , and the possible existences and impacts of multiple subclones that may often mislead inferences if not explicitly modeled ; ( 2 ) simultaneous statistic inference on the basis of both copy number changes and major allelic frequencies ; ( 3 ) restoration of information lost as a result of the guanine - cytosine content and actual sizes of each library insert and other specic biases of each genomic location ; ( 4 ) avoid making inferences when the signal - to - noise ratio is not ideal because of technical artifacts ; ( 5 ) an explicit modeling of whole - genome duplication events and whole - chromosome duplication events , which are common in cancer genomics and have huge impacts on the accurate inference of purity and ploidy ; the cancer genome atlas pan - cancer purity estimates were generated using processed rna - seq data ( for isopure ) downloaded from ( download version 2015 ) and snp6 array level - 1 data ( for ascat ) downloaded from gdc data portal . consensus pathology , dna , and mrna purity estimates multiobserver pathology reviews yielded purity ranges , 8 which were further collapsed into single - point estimates using the median value of purity range in deciles . 
dna ( absolute , ascat , clonet , integer , oncosnp ) and mrna ( demix and isopure - r ) based purity estimates were aggregated using median dna and mrna estimates , respectively . availability of data and materials all processed data are available either in the data supplement or uploaded to as speciﬁed in the data supplement . 
 haider et al ethics approval and consent to participate tissue contributing sites followed appropriate consent documentation and approved submission of cases to the cancer genome atlas , as detailed in the original publication.8 results prostate cancer presents complex intraand interpatient heterogeneity . 
it is an ideal model to study heterogeneity because of frequent surgical management via radical prostatectomy of the whole gland , allowing spatio - genomic studies.9 , 10 we collated pathologic , molecular , and clinical data sets from the cancer genome atlas ( tcga ) prostate marker study , which comprised 333 patients.8 purity estimates from multiple pathologists were consolidated , resulting in point estimates as previously described8 ( see methods )  . 
for a subset of cases , both top and bottom tissue block slides ( with sections acquired for molecular analysis in between these ) were assessed by multiple pathologists , demonstrating moderate correlation between pathologists ( top sections : pearsons r = 0.64 , p = 6.23 107 ; bottom sections : pearsons r = 0.53 , p = 8.93 103 ; data supplement fig 1a - b )  . 
a similar trend was observed between the pathology estimates of top and bottom sections ( pearsons r = 0.59 , p = 2.03 1012 ; data supplement fig 1c ) , highlighting potential inuence of spatial heterogeneity . 
in silico estimates of tumor purity were generated using nine methods11 - 18 that leverage dna ( methylation or copy number data ) , mrna , or mirna proles ( data supplement tables 1 and 2 ; methods )  . 
of note , leuc estimates on the basis of dna methylation data were right skewed , with a median purity of 0.9 ( leuc - other = 0.33 , p = 1.44 1095 , wilcoxon rank sum test )  . 
interestingly , all these methods were based on dna proles ( genomic or epigenomic ) , suggesting intrinsic limitations in estimating tumor purity from dna - based assays in this setting . 
these limitations could be explained by the dna prole itself , because samples with failed purity estimates exhibited quiet genomes with low numbers of somatic single nucleotide variants ( snvs ; data supplement fig 2a - b )  . 
pathology calls did not show clear evidence of low purity ; however , rnabased methods predicted a trend toward low purity for a subset of samples ( data supplement fig 2c )  . 
however , it is probable that some may also represent quiet cancer genomes , which are now increasingly recognized as a real phenomenon , particularly in prostate cancer.8 , 19 inspection of the complete sample set revealed no association with histologic heterogeneity ( rationalized as gleason score10 ; fig 1b , data supplement fig 3a - b )  . 
hence , we preclude spatial heterogeneity as the primary factor underlying this lack of concordance . these data highlight that variation and error proles among the intraplatform estimates are probably correlated and suffer from similar intrinsic limitations , independent of the specic algorithm used . 
therefore , we created consensus dna and mrna purity estimates using the median for each class of methods , hereafter referred to as dna and mrna estimates ( see methods )  . 
the differences between pathology estimates and either dna or mrna estimates were strongly correlated ( pearsons r = 0.81 , p = 4.68 1079 ; fig 2 ) , with 29.13% of cases demonstrating agreement ( within 15% purity of each other )  . 
 ( a ) distribution of tcga prostate tumor purity estimates ( n = 333 ) using in silico methods and consolidated multiobserver pathology reviews ; ( b ) patient - wise purity estimates grouped by gleason score . 
data from integer were available for 107 samples using the low - pass dna sequencing data ; ( c ) pearson correlation between purity estimates inferred using in silico methods and pathology reviews . 
of the dnaand mrna - based estimates , only two samples displayed discordant directions of effect relative to pathologic estimates ( purple and yellow dots in fig 2 ) , highlighting overall similarity in error proles of the underlying biomolecules . next , we assessed whether the key transcriptional and genomic biomarkers that underpin prostate cancer biology are dependent on tumor purity . 
to further delineate the relationship between tumor purity and somatic mutations , we stratied purity estimates by the mutation status of a panel of recurrently altered genes in prostate cancer.8 tumor purity determined by at least one prole was associated with six genes , including erg fusions and spop , foxa1 , and tp53 point mutations ( false discovery rate [ fdr ] adjusted p , .25 , wilcoxon rank sum test ; fig 3d , data supplement table 3 )  . 
for these six genes , tumor purity was moderately higher in mutant samples . to characterize this association between driver gene status and tumor purity , we evaluated the associations between tumor purity and the variant allele frequency ( vaf ) in samples carrying mutations ( fig 3e )  . 
however , pathology estimates of tumor purity were unable to accurately capture the vaf of these recurrently altered genes . next , we evaluated whether pathology , dna , mrna , and mirna purity estimates vary in their associations with individual genes or mirnas and to what extent these can be overcome by using in silico deconvolution.15 each of the four consensus purity estimators was individually correlated with ve molecular proles ( bulk / nave and deconvolved mrna abundance , bulk / nave and deconvolved mirna abundance , and bulk copy number data ; deconvolved proles were generated using isopure )  . 
nave mrna and mirna proles exhibited the greatest proportion of features correlated with tumor purity , which diminished after in silico deconvolution , highlighting potentially confounding tacs . with the exception of nave mirna proles , purity estimates were inversely correlated with molecular proles regardless of the underlying purity estimation prole ( data supplement fig 5a - f )  . 
these data suggest that the presence of genomic and transcriptomic correlates of tumor purity are likely to confound biologic and clinical interpretations . because dnaand mrna - based assays are most commonly used in cancer genomics , we asked if the purity estimates from these two analytes are comparable in other cancers . 
given the strong intra - analyte correlation ( fig 1c ) , we considered a representative dna - based method ( ascat ) and an mrna - based method ( isopure ) to estimate tumor purity for an additional 12 cancer types ( 4 , 497 tumor samples ) from tcga project ( fig 4b , prostate cancer data discussed above is shown for reference only )  . overall , all cancer types showed an average purity of at least 0.56. 
these data further underscore the importance of using analyte - matched purity estimates for bioinformatics analysis and subsequent interpretation . discussion herein , we provide evidence that tumor purity estimates manifest intrinsic properties of the underlying information used for purity estimation and exhibit only modest interprole concordance . 
however , interpathologist variation observed in our study , as well as previous studies , suggests that there are probably some inaccuracies in these estimates because of their subjectivity / qualitative nature.5 , 21 these discrepancies may also be a result of the lack of full spatial heterogeneity of the pathologic slide . 
genomic correlates of tumor purity as summarized using androgen receptor ( ar ) signature score ( a ) , percent genome altered ( [ pga ] , b ) , and mutation burden ( c )  . 
statistical tests were performed for genes with more than three mutant samples . therefore , idh1 , rb1 , akt1 , and chd1 ( displayed with x ) were deemed inappropriate for statistical testing . 
 ( a ) correlation between purity estimates derived using pathology , dna , mrna , and microrna ( mirna ) proles and molecular proles ( mrna.naive = bulk mrna abundance , mrna.isopure = deconvolved mrna abundance , mirna.naive = bulk mirna abundance , mirna.isopure = deconvolved mirna abundance , and cna = bulk copy number data ; deconvolved rna proles were generated using isopure )  . 
each feature ( genes for mrna and copy number aberration [ cna ] proles , mirnas for mirna proles ) was correlated with tumor purity estimators ( pathology , dna , rna , mirna ) separately . 
 ( b ) distribution of tumor purity estimates across 13 tcga tumor types ( 4 , 830 tumors ) using an in silico dna - based ( ascat ) and mrna - based ( isopure ) method . 
for clinico - genomic sequencing studies requiring a minimum purity threshold for inclusion in the study , an alternative to pathology estimates is to infer purity directly from the analyte by performing low - pass dna sequencing to lter low - purity samples.22 in addition to poor concordance between pathology and dna / rna - based tumor purity in prostate cancer , our pan - cancer data reported herein suggest that the purity estimates from dna and mrna proles also show limited concordance . 
the concordance between purity estimators also varies depending upon the tumor type and patterns of somatic changes it exhibits ( eg , dna - based methods rely on the presence of copy number aberrations )  . 
furthermore , previous studies have reported varying levels of concordance in purity estimates inferred from dnaand rna - based methods.2 , 23 for instance , aran et al2 show much stronger concordance between estimate24 ( rna - based purity estimator ) and absolute13 ( dna - based purity estimator ) compared with the rnaand dna - based methods in our study . 
because purity estimates vary across methods , consensus estimates on the basis of matched analyte type may further improve purity estimates and may also overcome missing values and normalize outlier estimates . 
after condent purity estimates have been created , one way to account for these is to adjust bioinformatics and statistical analyses for tumor purity , as stressed in previous studies.2 , 7 , 15 because bulk tumor proles are heterogeneous compositions of tumor cells and tacs featuring complex interplay , it is crucial to interpret the clinico - genomic proles in the context of the underlying heterogeneity.25 many in silico deconvolution techniques have been developed to estimate relative abundance of different cell types , 24 , 26 , 27 as well as techniques that transcriptomic11 , 12 , 15 , 18 , 23 and explicitly generate residual genomic14 proles of tumor - only and stromal - only cells . 
herein , we recommend researchers to consider deconvolution of bulk proles into individual component proles ( e.g. , cancer and stromal proles ) to improve sensitivity and specicity of downstream analyses.4 , 15 affiliations 1ontario institute for cancer research , toronto , ontario , canada 2the breast cancer now toby robins research centre , the institute of cancer research , london , united kingdom 3department of data sciences , dana - farber cancer institute , boston , 4department of biostatistics , harvard t.h. 
boutros provision of study material or patients : massimo loda collection and assembly of data : syed haider , svitlana tyekucheva , davide prandi , andrew wei xu , peter w . 
park honoraria : pzer consulting or advisory role : neuroinammation newco patents , royalties , other intellectual property : patent on mutational signature - based detection of homologous recombination deciency peter w . 
laird consulting or advisory role : progenity , anchordx patents , royalties , other intellectual property : received royalties annually through 2018 for inventions licensed to epigenomics ag by usc travel , accommodations , expenses : anchordx wenyi wang stock and other ownership interests : genomic health francesca demichelis patents , royalties , other intellectual property : co - inventor on a patent led by the university of michigan and the brigham and womens hospital covering the diagnostic and therapeutic elds for ets fusions in prostate cancer . 
boutros consulting or advisory role : biosymetrics patents , royalties , other intellectual property : holds patents on multiple biomarkers no other potential conicts of interest were reported . acknowledgment the results published herein are based , in part , upon data generated by tcga pilot project established by the national cancer institute and the national human genome research institute . 
nature 501 : 346 - 354 , 2013 fox ns , haider s , harris al , et al : landscape of transcriptomic interactions between breast cancer and its microenvironment . 
nat commun 10 : 3116 , 2019 smits aj , kummer ja , de bruin pc , et al : the estimation of tumor cell percentage for molecular testing by pathologists is not accurate . 
mod pathol 27 : 168 - 174 , 2014 yadav vk , de s : an assessment of computational methods for estimating purity and clonality using genomic data derived from heterogeneous tumor tissue samples . 
brief bioinform 16 : 232 - 241 , 2015 zheng x , zhang n , wu hj , et al : estimating and accounting for tumor purity in the analysis of dna methylation data from cancer studies . 
wang l , sebra rp , sfakianos jp , et al : a reference prole - free deconvolution method to infer cancer cell - intrinsic subtypes and tumor - type - specic stromal 24 . 
 impact of kras and tp53 co - mutations on outcomes after first - line systemic therapy among patients with stk11 - mutated advanced nonsmall - cell lung cancer erin bange , md1 ; melina e . 
langer , md1 ; erica carpenter , mba , phd1 ; and charu aggarwal , md , mph1 purpose the stk11 gene encodes a serine / threonine protein kinase that regulates cell polarity and functions as a tumor suppressor . 
we evaluated the impact of kras and tp53 co - mutations on outcomes after rst - line systemic therapy for patients with metastatic or recurrent nsclc that harbors stk11 mutations . methods we conducted a retrospective review of patients with metastatic nsclc and stk11 mutations treated at the university of pennsylvania . 
the kaplan - meier method was used to estimate overall survival ( os ) and progression - free survival ( pfs )  . results from february 2013 to december 2016 , samples from 1 , 385 patients with nsclc were analyzed by ngs ; of these , 77 patients ( 6% ) harbored an stk11 mutation ( n = 56 , tissue ; n = 21 , plasma )  . 
of the 62 patients included , 18 had an stk11 mutation alone , 19 had stk11 / kras , 18 had stk11 / tp53 , and seven had stk11 / kras / tp53 . 
the majority of the mutations found during routine testing are not actionable currently , but their presence likely has predictive and prognostic relevance . stk11 , also known as liver kinase b1 ( lkb1 ) , is a tumor suppressor and a negative regulator of mammalian target for rapamycin signaling . 
loss - offunction mutations in germline stk11 are associated with peutz - jeghers hereditary cancer syndrome . stk11 mutations are estimated to be present in 8% to 39% of all nsclc , with increased prevalence in smokers and patients with kras mutations.2 , 3 animal that stk11 mutations are critical studies suggest tumorigenesis , and in lung cancer differentiation , metastasis.4 , 5 mutations in stk11 have emerged as a potential prognostic and predictive marker in nsclc . 
 bange et al context key objective how do kras and tp53 co - mutations affect outcomes after rst - line systemic therapy in patients with nonsmall - cell lung cancer and stk11 mutations ? knowledge generated among patients with metastatic nsclc and tumor - associated stk11 mutations , co - mutation with tp53 conferred better progression - free survival ( pfs ) and overall survival ( os ) after rst - line therapy compared with patients who had a kras comutation.tp53 mutation in the presence of an stk11 / kras co - mutation also conferred better pfs and os compared with patients who had only the stk11 / kras co - mutation . relevance stk11 / kras co - mutation has been associated with worse pfs after chemotherapy , but co - mutation with tp53 may modulate outcomes after rst - line chemotherapy in this group and among patients with stk11 mutations without kras mutations . nsclc , which suggests that stk11 mutations may be a more heterogeneous group than previously thought.6 , 7 methods patient population co - mutation status may be another source of heterogeneity among patients with stk11 mutations . 
patients who received treatment outside the institution or had another concurrent malignancy were excluded . mutational analysis the center plasma was analyzed by guardant health ( redwood city , ca ) as described previously.16 solid tumor sequencing was performed at for personalized diagnostics clinical laboratory at the university of pennsylvania ( data supplement )  . 
one kras amplication ; one kras variant of unknown signicance ( vus ) , q61h ; and one tp53 vus ( a161s ) were not considered mutations in the respective genes . 
 outcomes in nsclc by stk11 co - mutations status statistical analysis led to discontinuation of descriptive statistics , including mean , median , and proportions , were used to summarize patient demographics and tumor characteristics . 
os was calculated from the start of systemic treatment of metastatic or recurrent disease to the date of death or last follow - up . patient data were censored at the last follow - up visit or on september 1 , 2017 , if still alive . 2 and kruskal - wallis analyses were used to assess differences in baseline characteristics between the mutation groups for categoric and continuous variables , respectively . cox proportional hazard models were used to determine the relationship of stk11 co - mutations to survival . 
the effect of co - mutation status on pfs and os was investigated by looking at four mutation groups ( stk11 alone , stk11 / kras , stk11 / tp53 , and stk11 / kras / tp53 ) separately as well as at individual mutation effects and interactions in a cox regression model . 
hrs from the cox model were reported . results baseline characteristics during a 42 - month period , 1 , 385 unique patients had sequencing of a lung neoplasm in either tissue ( n = 1 , 526 samples ) or plasma ( n = 245 samples )  . 
the majority ( 51 of 62 , or 82% ) of patients received platinum doubletbased therapy as the rst - line regimen ( table 1 )  . nine patients had a driver mutation and received targeted therapy at some point during treatment ( data supplement )  . five patients received immunotherapy as rst - line systemic therapy ( data supplement )  . 
the baseline characteristics were well balanced among these co - mutation groups , except that patients in the stk11 alone or stk11 / kras / tp53 group were slightly older ( p = .015 ; table 1 )  . 
the most common stk11 mutation was p.l282afs * 3 , which resulted in a frameshift mutation in exon 6 ( fig 2 )  . there was no correlation between the position of the stk11 mutation and co - mutation status . 
kras alterations occurred at codon positions 12 , 13 , 22 , and 61 , and each was considered disease associated by pathology report using pubfrequent mutation licly available databases . 
 ( a ) columns represent individual patients with mutation type specied by color ; missense mutations in stk11 were found in six patients , but specic point mutations were not identied . 
median progression - free survival ( pfs ) and median overall survival ( os ) for co - mutation groups among patients with stk11 mutation ( all mutations reported ) were determined using kaplan - meier methodology . 
all p values are compared with stk11 / kras . patients with tp53 / da - stk11 compared with tp53 / nonda - stk11 . patients with stk11 / kras mutations experienced shorter median os ( 7.1 months ) compared with stk11 alone ( 16.1 months ; log - rank p , .001 ) , stk11 / tp53 ( 28.3 months ; log - rank p , .001 ) , and stk11 / kras / tp53 ( 22 months ; log - rank p = .025 ; table 2 ; fig 3c )  . 
interestingly , we did not nd that stk11ex1 - 2 mutations were associated with an increased risk of progression or death compared with stk11ex3 - 9 mutations , in time ( months ) no . 
kaplan - meier curves of ( b ) pfs and ( d ) os of patients with stage iv or recurrent disease and tumors with disease - associated stk11 mutation . 
other co - factors considered in the analysis but found not to be independent predictors in the univariable model or signicant contributors to the multivariable model were smoking status , performance status at diagnosis , age at diagnosis , stage at presentation , and race / ethnicity ( tables 3 and 4 )  . 
analyses that excluded patients who received immunotherapy or targeted therapy still showed superior outcomes for patients with stk11 / tp53 co - mutations , even in the presence of a kras mutation ( data supplement )  . relevance of disease - associated variants of stk11 as part of an exploratory subset analysis , we repeated the pfs and os kaplan - meier analyses using only the stk11 mutations characterized as disease associated ( da - stk11 )  . when only da - stk11 mutations were included , there was no difference in median pfs between co - mutation groups of interest ( fig 3b )  . 
there remained a signicant difference in median os when stk11 / kras and stk11 / tp53 were compared ( 7.1 months v 39 months ; log - rank p = .01 ; fig 3d )  . discussion in the era of precision medicine , it has become increasingly important to understand the full implications of an everincreasing quantity of tumor genetic information obtained as part of routine sequencing of nsclcs . 
this study includes one of the largest cohorts of patients with stk11 mutations ( n = 62 ) , and to our knowledge it is the only study to specically evaluate stk11 co - mutations with tp53 and kras and their relationship to response to rst - line systemic therapy in patients with metastatic or recurrent disease . 
the results show that stk11 / kras co - mutation is associated with a worse median pfs and os after front - line chemotherapy compared with patients who had stk11 mutation alone , whereas patients who had the stk11 / tp53 co - mutation had improved outcomes . we found that , in the context of an stk11 mutation , tp53 mutation is associated with better outcomes even in the presence of mutant kras . 
of interest , when examined outside the context of stk11 , tp53 mutations reportedly have a deleterious effect on os and response to platinum - based therapy , especially in early - stage disease.19 - 22 tp53 has not previously been found to be predictive or prognostic in the presence of a kras mutation.9 , 10 mutations in the tp53 binding sites of the stk11 promoter have been associated with decreased stk11 expression in endometrial cancer.15 in nsclc , 82% of tp53 mutations are in the dna binding region ; therefore , a mutation in tp53 in nsclc could lead to decreased expression of a deleterious stk11 protein , such as a gain - of - function stk11 mutation in exons 1 to 2.6 , 23 however , we observed a survival benet for tp53 co - mutation with stk11 that was independent of the location of the stk11 mutation . 
univariable and multivariable cox regression models of the effect of mutation status and covariables on the risk of death ( ie , overall survival )  . reference for stk11 exon 1 / 2 is a mutation in stk11 exon 3 - 9 . 
the proportion of patients with stk11 mutations in exons 1 and 2 in our cohort was similar to that of the cohort described by p ecuchet et al6 ( 35% v 25% ) , who came to a different conclusion . 
although some patients in our study had earlystage progression ( 37.1% ) , patients with early - stage nonprogressive disease and patients who never received any systemic therapy were formally excluded ( fig 1 )  . 
therefore , if stk11ex1 - 2 mutations do confer a higher risk of recurrence after early - stage disease , we would not have been able to identify this risk , given the study design . in the context of an stk11 mutation , we found that kras mutation in the absence of tp53 co - mutation conferred a signicantly worse pfs and os after rst - line systemic therapy for metastatic or recurrent disease . 
facchinetti et al3 also found that stk11 / kras co - mutated tumors had a higher metastatic burden and a trend toward worse os . this deleterious interaction between stk11 and kras may be explained by previous data showing that stk11 mutations enhance kras mutationassociated gene expression.11 in theory , this interaction would lead to augmentation of downstream kras signaling driving tumorigenesis . 
this is also supported by the observation that an acceleration of kras - induced tumorigenesis and metastasis has been found in stk11 - null mice as well as in humans who lack stk11 expression.4 , 15 in a separate study , arbour et al9 found that kras / stk11 co - mutations were associated with shorter os in univariable analysis but not in multivariable analysis . 
in their cohort , stk11 co - mutation status with keap1 or nfe2l2 could have contributed to a shorter os.9 keap1 / nfe2l2 comutation occurred in 63% ( 60 of 95 ) of stk11 mutations in their cohort and was highly correlated with the kras / stk11 subgroup in another study.9 , 10 they did not report a correlation between keap1 / nfe2l2 and tp53 mutations . 
the tumor and plasma ngs panels reported in our study did not include keap1 or nfe2l2 mutations , so we were unable to assess the effect of these mutations on outcome . detection of kras and tp53 mutations via plasma or tissue raises the possibility that the detected mutations may be due to clonal hematopoiesis ( ch ) in the blood . 
in another series , ve of 33 tp53 mutations detected by plasma ngs were found in peripheral - blood cells but not in the tumor.24 the same series reported that most jak2 , some tp53 , and rare kras mutations detected in cell - free dna are from ch and not from the tumor . 
in our cohort , there was no signicant difference in the proportion of tp53 or kras mutations detected in tumor versus plasma ( table 1 )  . according to the series by hu et al , 24 it is possible that approximately one of the seven tp53 mutations detected by plasma testing was from ch ; even if true , this small proportion is unlikely to change our results . 
in addition , ch is associated with worse outcome after therapy , and we report better outcomes with a tp53 mutation.25 therefore , this possible misclassication would bias our result toward the null and imply that the observed association may be stronger than reported.26 limitations that must be our study has additional addressed . 
 outcomes in nsclc by stk11 co - mutations status study , we used a real - world measurement of pfs dened as time from the start of treatment until radiologic progression , clinical deterioration , death , or change of therapy . this has been shown to be an appropriate surrogate for response evaluation criteria in solid tumors ( recist ) based pfs used in clinical trials.27 in addition , studies that many commercially available assays do not disclose how they determine whether an alteration is disease associated ; thus , there may be variation among vendors in how they categorize mutations . look to characterize the prognostic or predictive signicance of mutations in a specic gene have used different denitions of mutation ( eg , nonsynonymous , pathogenic )  . 
we initially used all nonsynonymous mutations in stk11 but then performed a subset analysis using only disease - associated stk11 mutations ( ie , mutations classied as disease associated or pathogenic on the molecular report )  . 
this analysis is limited by the small sample sizeonly ve patients were in the da - stk11 / kras / tp53 co - mutation groupand so should be considered exploratory . 
future work must be done to standardize how classication of molecular alterations and identication inuence response to therapy and of mutations that prognosis . in summary , this study shows that the co - mutation status of stk11 - mutated nsclc contributes to the heterogeneity of this molecular subgroup . 
marmarelis stock and other ownership interests : gilead sciences , portola pharmaceuticals , merck , bluebird bio consulting or advisory role : boehringer ingelheim other relationship : novartis research funding : lilly ( inst ) travel , accommodations , expenses : trizell wei - ting hwang research funding : janssen ( i ) jeffrey c . 
thompson consulting or advisory role : oncocyte jason rosenbaum consulting or advisory role : genentech , roche , abbvie , bristol - myers squibb travel , accommodations , expenses : agilent , bristol - myers squibb , takeda , abbvie , genentech , roche joshua m . 
bauml consulting or advisory role : bristol - myers squibb , merck , astrazeneca , genentech , celgene , boehringer ingelheim , guardant health , takeda research funding : merck ( inst ) , carevive systems ( inst ) , novartis ( inst ) , incyte ( inst ) , bayer ( inst ) , janssen ( inst ) , astrazeneca ( inst ) , takeda ( inst ) christine ciunci research funding : celgene ( inst ) , merck ( inst ) roger b . 
cohen consulting or advisory role : heat biologics , takeda , alkermes , kyn therapeutics , innate pharma , cantargia ab , genocea biosciences research funding : heat biologics ( inst ) , merck ( inst ) , celldex ( inst ) , innate pharma ( inst ) , kyn therapeutics ( inst ) , xencor ( inst ) , genocea biosciences ( inst ) travel , accommodations , expenses : heat biologics , takeda , innate pharma , kyn therapeutics , alkermes corey j . 
 bange et al consulting or advisory role : genentech , roche , lilly , imclone , merck , abbott biotherapeutics , bayer , onyx , clarient , clovis oncology , celgene , cancer support community , bristol - myers squibb , ariad , takeda , astrazeneca , pzer , novocure research funding : merck ( inst ) , advantagene ( inst ) , clovis oncology ( inst ) , celegene ( inst ) , inovio pharmaceuticals ( inst ) , ariad ( inst ) , glaxosmithkline ( inst ) , genentech ( inst ) , roche ( inst ) , stem centrx ( inst ) , lilly ( inst ) other relationship : lilly , amgen , peregrine pharmaceuticals , synta erica carpenter honoraria : astrazeneca research funding : merck , janssen , becton dickinson patents , royalties , other intellectual property : invention disclosure entitled , methods and compositions for treating neuroblastoma ; invention disclosure entitled , methods and compositions for identifying , diagnosing and treating neuroblastoma travel , accommodations , expenses : astrazeneca , foundation medicine charu aggarwal consulting or advisory role : genentech , bristol - myers squibb , lilly , celgene , medimmune research funding : genentech ( inst ) , roche ( inst ) , incyte ( inst ) , macrogenics ( inst ) , merck sharp & dohme ( inst ) , astrazeneca ( inst ) , medimmune ( inst ) no other potential conicts of interest were reported . references cheng tyd , cramb sm , baade pd , et al : the international epidemiology of lung cancer : latest trends , disparities , and tumor characteristics . 
oncogene 26 : 5911 - 5918 , 2007 facchinetti f , bluthgen mv , tergemina - clain g , et al : lkb1 / stk11 mutations in nonsmall - cell lung cancer patients : descriptive analysis and prognostic value . lung cancer 112 : 62 - 68 , 2017 gill rk , yang sh , meerzaman d , et al : frequent homozygous deletion of the lkb1 / stk11 gene in nonsmall - cell lung cancer . 
nature 448 : 807 - 810 , 2007 p ecuchet n , laurent - puig p , mansuet - lupo a , et al : different prognostic impact of stk11 mutations in non - squamous nonsmall - cell lung cancer . 
oncotarget 8 : 23831 - 23840 , 2017 dahmani r , just pa , delay a , et al : a novel lkb1 isoform enhances ampk metabolic activity and displays oncogenic properties . 
oncogene 34 : 2337 - 2346 , 2015 chen z , cheng k , walton z , et al : a murine lung cancer co - clinical trial identies genetic modiers of therapeutic response . 
nature 483 : 613 - 617 , 2012 arbour kc , jordan e , kim hr , et al : effects of co - occurring genomic alterations on outcomes in patients with kras - mutant nonsmall - cell lung cancer . 
skoulidis f , byers la , diao l , et al : co - occurring genomic alterations dene major subsets of kras - mutant lung adenocarcinoma with distinct biology , immune proles , and therapeutic vulnerabilities . 
schabath mb , welsh ea , fulp wj , et al : differential association of stk11 and tp53 with kras mutationassociated gene expression , proliferation , and immune surveillance in lung adenocarcinoma . 
co nn , iglesias d , celestino j , et al : loss of lkb1 in high - grade endometrial carcinoma : lkb1 is a novel transcriptional target of p53 . 
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deben c , deschoolmeester v , lardon f , et al : tp53 and mdm2 genetic alterations in nonsmall - cell lung cancer : evaluating their prognostic and predictive value . 
ahrendt sa , hu y , buta m , et al : p53 mutations and survival in stage i nonsmall - cell lung cancer : results of a prospective study . 
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kandioler d , stamatis g , eberhardt w , et al : growing clinical evidence for the interaction of the p53 genotype and response to induction chemotherapy in advanced nonsmall - cell lung cancer . 
shajani - yi z , de abreu fb , peterson jd , et al : frequency of somatic tp53 mutations in combination with known pathogenic mutations in colon adenocarcinoma , nonsmall - cell lung carcinoma , and gliomas as identied by next - generation sequencing . 
coombs cc , zehir a , devlin sm , et al : therapy - related clonal hematopoiesis in patients with non - hematologic cancers is common and associated with adverse clinical outcomes . 
bartlett c , mardekian j , cotter m , et al : concordance of real - world progression - free survival ( pfs ) on endocrine therapy as rst line treatment for metastatic breast cancer using electronic health record with proper quality control versus conventional pfs from a phase 3 trial . 
a positron emission tomography computed tomography ( pet - ct ) scan in april 2012 demonstrated extensive malignancy in the oral cavity with bilateral level ib node metastases and a suggestive fluorodeoxyglucose - avid subcutaneous lesion posterior to the sixth cervical spinous process . 
he had a smoking history of 60 pack - years and denied alcohol use , and he had no family history of cancer . the patient received induction chemotherapy with docetaxel , cisplatin , and fluorouracil . 
the patient was enrolled in a randomized , placebo - controlled , phase ii study that consisted of docetaxel and cisplatin or carboplatin with or without erlotinib ( clinicaltrials . gov identifier : nct01064479 )  . 
unblinding of the study revealed that he received erlotinib . the patient was referred to the phase i clinic and participated in the impact2 trial , a randomized trial in precision medicine ( clinicaltrials . gov identifier : nct02152254 )  . 
he was randomly assigned to receive targeted therapy and was enrolled in a study with a selective panfgfr inhibitor that began in september 2015 . ecaterina ileana dumbrava rasha alfattal vincent a . 
next - generation sequencing genomic profile by foundation one ( august 2015 ) genomic profile genomic alterations fgf19 amplification ccnd1 amplification fgf4 amplification ccnd2 amplification fgf23 amplification cdkn2a / b loss fgf3 amplification chd2 d213n fgf6 amplification crebbp r1392 * emsy amplification kdm5a amplification kras amplification myc duplication exons 2 and 3 tp53 e204 * variants of unknown significance akt2 e323q atm e322d igf1r s909c klhl6 amplification atr amplification notch2 t239s bcl6 amplification pik3ca amplification blm r543h pik3cb amplification card11 truncation pik3r2 r559h cdkn1b amplification prdm1 k188e chd4 amplification ranbp2 amplification dicer1 a948t sox2 amplification ephb1 amplification terc amplification grm3 amplification prkci amplification note . 
other adverse events included grade 1 hand - foot syndrome , xerostomia , and mucositis . in march 2016 , ct imaging after six cycles demonstrated a complete radiologic response ( fig 3 )  . 
he then underwent whole - brain radiation therapy , and 5 weeks after the completion of this treatment , he died of acute myocardial infarction . discussion to our knowledge , this is the first case of a complete response to an fgfr inhibitor in a patient with hnscc harboring several fibroblast growth factor ( fgf ) amplifications . 
alterations of the fgf / fgfr signaling pathways play important roles in carcinogenesis , stimulating cancer cell proliferation and angiogenesis.8 deregulation of the fgfr pathway occurs in fgf ligands ( fgfs ) or receptors via gene mutations , translocations , fusions , or amplifications that lead to protein overexpression that results in the activation of the downstream signaling pathway.9 the fgfr family has four highly conserved transmembrane receptor tyrosine kinases ( fgfr1 to fgfr4 ) that differ in their ligand affinity and tissue distribution . 
selected genomic alterations are indicated by an asterisk ( * ) : fibroblast growth factor receptor ( fgfr ) pathway ( fgf19 , fgf4 , fgf23 , fgf3 , and fgf6 amplifications )  . 
selected variants of unknown significance are indicated by a filled square ( ) : kras amplification , pik3ca and pik3cb amplifications , and pik3r2 and protein kinase b ( akt ) mutations . 
binding of fgfs to fgfrs induces dimerization of the intracellular domain of the receptor and downstream activation of signaling.10 with the advent of next - generation sequencing , aberrations in fgfrs have been better characterized than fgf alterations . 
the most common mechanisms of fgf activation are gene amplification that leads to overexpression and mutations that lead to increased affinity for fgfrs.9 fgf amplifications have been observed in several tumor types.12 in hnscc , fgfr1 overexpression has been reported in > 75% of both human papillomavirus ( hpv ) positive and negative hnscc , and it is associated with poor survival.13 fgfr1 , fgfr2 , and fgfr3 amplifications , as well as fgfr3 - tacc3 fusions , have been identified in hpv - positive tumors.14 fgf3 , fgf4 , fgf19 , and ccnd1 , also amplified in this patient , colocalize on the same amplicon of 11q13 and are frequently coamplified . 
 in addition , 11q13 amplification is prevalent in hnsccparticularly hpv unrelated and tobacco inducedand has been reported to be associated with poor prognosis.15 , 16 although the hpv status of our patient was unknown , he was a heavy smoker throughout the course of his disease . there has been exponential progress in the field of fgfr targeting , owing to the development of novel agents that inhibit fgf ligands and receptors . 
because they simultaneously target vascular endothelial growth factor receptor , platelet - derived growth factor receptor , and fgfr signaling pathways , these compounds are being developed primarily as antiangiogenic agents . 
azd4547 has been investigated in fgfr2 - amplified gastric cancers and in fgfr1 - amplified breast cancer and nsclc.24 - 26 bgj398 induced a 40% overall response rate in patients with urothelial cancer and fgfr alterations . 
21 debio 1347 may inhibit the fgfr2 mutation v564f , which causes resistance to other drugs.27 monoclonal antibodies that target fgf / fgfr signaling by blocking ligand binding or preventing receptor dimerization are in clinical development.28 adverse effects associated with nonselective fgfr inhibitors include vascular endothelial growth factor receptorrelated toxicities , and with selective fgfr inhibitors , hyperphosphatemia as a result of fgf23 signaling.17 , 29 in our patient , hyperphosphatemia was managed with a low - phosphate diet , phosphate binders , and a dose reduction . 
use of a selective pan - fgfr inhibitor that targets multiple fgf amplificationsfgf3 , fgf4 , fgf6 , fgf19 , and fgf23was associated with a durable complete response in a patient with hnscc . 
borad mj , champion md , egan jb , et al : integrated genomic characterization reveals novel , therapeutically relevant drug targets in fgfr and egfr pathways in sporadic intrahepatic cholangiocarcinoma . 
smeets sj , braakhuis bjm , abbas s , et al : genome - wide dna copy number alterations in head and neck squamous cell carcinomas with or without oncogene - expressing human papillomavirus . 
chae yk , ranganath k , hammerman ps , et al : inhibition of the fibroblast growth factor receptor ( fgfr ) pathway : the current landscape and barriers to clinical application . 
gozgit jm , wong mj , moran l , et al : ponatinib ( ap24534 ) , a multitargeted pan - fgfr inhibitor with activity in multiple fgfr - amplified or mutated cancer models . 
soria j - c , debraud f , bahleda r , et al : phase i / iia study evaluating the safety , efficacy , pharmacokinetics , and pharmacodynamics of lucitanib in advanced solid tumors . 
milowsky mi , dittrich c , durn i , et al : phase 2 trial of dovitinib in patients with progressive fgfr3 - mutated or fgfr3 wild - type advanced urothelial carcinoma . 
nogova l , sequist lv , perez garcia jm , et al : evaluation of bgj398 , a fibroblast growth factor receptor 1 - 3 kinase inhibitor , in patients with advanced solid tumors harboring genetic alterations in fibroblast growth factor receptors : results of a global phase i , dose - escalation and dose - expansion study . 
tabernero j , bahleda r , dienstmann r , et al : phase i dose - escalation study of jnj - 42756493 , an oral pan - fibroblast growth factor receptor inhibitor , in patients with advanced solid tumors . 
saka h , kitagawa c , kogure y , et al : safety , tolerability and pharmacokinetics of the fibroblast growth factor receptor inhibitor azd4547 in japanese patients with advanced solid tumours : a phase i study . 
paik pk , shen r , berger mf , et al : a phase ib open - label multicenter study of azd4547 in patients with advanced squamous cell lung cancers . 
van cutsem e , bang y - j , mansoor w , et al : a randomized , open - label study of the efficacy and safety of azd4547 monotherapy versus paclitaxel for the treatment of advanced gastric adenocarcinoma with fgfr2 polysomy or gene amplification . 
seckl m , badman pd , liu x , et al : radical trial : a phase ib / iia study to assess the safety and efficacy of azd4547 in combination with either anastrozole or letrozole in er positive breast cancer patients progressing on these aromatase inhibitors ( ais )  . 
henner voss m , hierro c , heist rs , et al : debio 1347 , an oral fgfr inhibitor : results from a first - in - human , phase i dose - escalation study in patients with fgfr genomically activated advanced solid tumors . 
hierro c , rodon j , tabernero j : fibroblast growth factor ( fgf ) receptor / fgf inhibitors : novel targets and strategies for optimization of response of solid tumors . 
kono sa , marshall me , ware ke , et al : the fibroblast growth factor receptor signaling pathway as a mediator of intrinsic resistance to egfr - specific tyrosine kinase inhibitors in non - small cell lung cancer . 
terai h , soejima k , yasuda h , et al : activation of the fgf2 - fgfr1 autocrine pathway : a novel mechanism of acquired resistance to gefitinib in nsclc . 
ware ke , marshall me , heasley lr , et al : rapidly acquired resistance to egfr tyrosine kinase inhibitors in nsclc cell lines through de - repression of fgfr2 and fgfr3 expression . 
mizukami t , togashi y , naruki s , et al : significance of fgf9 gene in resistance to antiegfr therapies targeting colorectal cancer : a subset of colorectal cancer patients with fgf9 upregulation may be resistant to anti - egfr therapies . 
george , md1 ; and susan halabi , phd8 purpose androgen receptor splice variant 7 ( ar - v7 ) detection in circulating tumor cells ( ctcs ) is associated with a low probability of response and short progression - free ( pfs ) and overall survival ( os ) in men with metastatic castration - resistant prostate cancer ( mcrpc ) treated with enzalutamide or abiraterone . 
os , conrmed prostate - specic antigen ( psa ) , and objective radiologic responses were secondary end points . results we enrolled 118 men with mcrpc treated with abiraterone or enzalutamide , 51 of whom received subsequent docetaxel or cabazitaxel . 
all patients provided written informed consent under stitutional review board approval at all participating centers within the department of defensefunded prostate cancer clinical trial consortium.16 study design and assessments prophecy is a prospective multicenter study evaluating the ability of baseline ( pretreatment ) ar - v7 status in ctcs to predict treatment outcomes with abiraterone or enzalutamide as well as subsequent taxane chemotherapy upon disease progression . 
cellsearch ( menarini silicon biosystems , huntington valley , pa ) ctc enumeration was performed at these time points for all patients and processed in a college of american physicians / clinical laboratory improvement amendmentsapproved central laboratory at memorial sloan kettering cancer center.17 , 18 treatment selection was at the discretion of the treating physician without knowledge of ar - v7 status . 
all data sets were separately sent to the study statistician ( s.h. ) , who unblinded the data after database lock . analysis of ctcs ctcs were analyzed in two central laboratories , each blinded to the results of the other . 
baseline pretaxane characteristics of patients enrolled according to epic sciences ar - v7 status based on ar - v7 detection after abiraterone or enzalutamide and before taxane epic sciences ar - v7 status ( % ) positive ( n = 8 ) negative ( n = 42 ) 62 - 79 45 - 87 6 - 881 96.5 1 - 850 0 - 325 characteristic age , years median range race white black other gleason sum 8 - 10 kps 90 poor - risk feature psa , ng / ml median range taxane used docetaxel cabazitaxel months median range hemoglobin , 12 g / dl elevated alp elevated baseline serum ldh presence of liver metastasis presence of clinically signicant pain requiring cellsearch ctcs , cells per 7.5 ml opiates range median , % ( n = 32 ) psadt , 3 months prior docetaxel for mhspc  . 
20 bone metastases time from ar - v7 sample to chemotherapy start , 0 - 17 0 - 33 abbreviations : alp , alkaline phosphatase ; ar - v7 , androgen receptor splice variant 7 ; ctc , circulating tumor cell ; kps , karnofsky performance score ; ldh , lactate dehydrogenase ; mhspc , metastatic hormone - sensitive prostate cancer ; psa , prostate - specic antigen ; psadt , prostate - specic antigen doubling time . registration to clinical or radiographic progression or death , whichever occurred rst . 
radiographic progression was assessed at each center using pcwg3 - modied recist ( version 1.1 ) soft tissue and bone scan criteria.22 clinical progression was dened as a composite end point including death , escalating pain or other symptomatic progression , initiation of new systemic therapy , or a skeletal - related event . secondary clinical end points included conrmed 50% prostate - specic antigen ( psa ) decline , radiographic response per recist ( verson 1.1 ) , 23 and os , and these same outcomes for subsequent taxane chemotherapy . data analysis the primary objective of prophecy was to validate that patients with pretreatment ar - v7negative ctcs have prolonged pfs with abiraterone or enzalutamide compared with ar - v7positive patients . 
with longer follow - up , analyses of the time - toevent end points ( pfs and os ) were also updated , and patients who received taxane chemotherapy were observed for response , radiographic progression , and death . 
details regarding study design have been published elsewhere.9 patients with no evaluable ctcs were considered ar - v7 negative , and all patients with sufcient blood collection were analyzed regardless of their evaluable ctcs . 
in secondary analyses , the proportional hazards model was used for assessing the prognostic value of ar - v7 status for pfs and os after adjusting for validated prognostic factors ( risk score ) .14 , 15 , 24 for taxane outcomes , pfs was dened from the date of starting chemotherapy to clinical or radiographic progression or death , whichever occurred rst . 
no power or sample size analysis is provided for this secondary descriptive analysis , because prophecy was powered around the primary objective and previously reported . between may 2015 and january 2017 , we prospectively enrolled 118 men with high - risk mcrpc who were initiating treatment with abiraterone , enzalutamide , or both at one of ve academic medical centers . 
baseline characteristics of the cohort were previously published and included 36 men who had received prior enzalutamide or abiraterone therapy.9 of these 118 men treated with subsequent ar inhibitor therapy , 51 experienced progression and were treated with taxane chemotherapy , including docetaxel ( n = 42 ) or cabazitaxel ( n = 9 ; consort diagram provided in appendix fig a1 )  . 
baseline characteristics of these patients at the time of taxane chemotherapy are listed in table 1 based on epic sciences ar - v7 status and appendix table a1 based on johns hopkins ar - v7 status . 
as of the nal database lock on october 15 , 2019 , median follow - up times from study registration ( before abiraterone or enzalutamide treatment ) and taxane initiation were 35 and 23 months , respectively . 
table 2 and figure 1 summarize the results of pfs and os by baseline ar - v7 status for each ctc assay . conrmed 50% psa declines were observed in 11% and 30% of patients with ar - v7positive and ar - v7negative disease by johns hopkins assay , respectively , and radiographic responses ( complete or partial responses by recist [ version 1.1 ] criteria ) were seen in 7% and 9% of patients , respectively . 
results were unchanged when two patients with psa - only progression were considered censored ( appendix table a3 )  . os did not differ from the start of taxane chemotherapy by ar - v7 status . 
table 3 and figure 2 summarize the results of pfs and os by baseline ar - v7 status for each ctc assay . conrmed 50% psa responses with taxane therapy were observed in 36% and 32% of patients with ar - v7positive versus ar - v7negative disease by johns hopkins assay , respectively , and radiographic responses ( complete or partial responses by recist [ version 1.1 ] ) were observed in 10% and 20% of patients , respectively . 
conrmed 50% psa responses were observed in 40% and 33% of those with ar - v7positive versus ar - v7negative disease by epic sciences nuclear assay , respectively , and radiographic responses ( complete or partial responses by recist [ version 1.1 ] ) were observed in 13% and 17% of patients , respectively ( appendix fig a2 )  . 
 a pre - abi / enza at progression on abi / enza at progression on taxane nuclear localized ar - v7 ctc total ctc nuclear localized ar - v7 ctc nuclear localized ar - v7 ctc total ctc total ctc 123456789 2345789 * * * * * * * * patient id patient id patient id armstrong et al 123456789 2345789 patient id patient id patient id 123456789 2345789 patient id patient id patient id * * * * * * * fig 3 . 
bar plot demonstrating heterogeneity of androgen receptor splice variant 7 ( ar - v7 ) expression in circulating tumor cells ( ctcs ) by either the johns hopkins ( jhu ) messenger rna assay or the epic sciences nuclear protein assay collected from men in the prophecy study at one of three time points : ( a ) before abiraterone ( abi ) or enzalutamide ( enza ) therapy , ( b ) at progression during abiraterone or enzalutamide treatment , and ( c ) at progression during subsequent taxane chemotherapy . 
for the epic sciences nuclear protein assay , approximately 1 ml of blood is analyzed , and total and nuclear - localized ar - v7positive ctcs are expressed per 1 ml of blood . 
 ( * ) no data . disease pretreatment who received abiraterone or enzalutamide and pretaxane therapy . finally , we examined how ar - v7 changes during the treatment course from before abiraterone or enzalutamide to progression and then after progression during taxane chemotherapy . 
at baseline , ar - v7 positivity was 10% versus 24% by epic sciences and johns hopkins assays , respectively , with a majority of men with ar - v7positive disease by epic sciences ( nine of 11 ; 82% positive agreement ) also testing positive by johns hopkins assay . johns hopkinspositive , epic sciencesnegative results were observed , particularly in those men with low epic sciences ctcs or nonnuclear ar - v7 protein expression . although a majority of ctcs in men with mcrpc were ar - v7 negative , even in those with ar - v7positive disease , the proportion of ar - v7positive cells ranged from 1% to 100% ( median , 20% ; fig 3 )  . 
at progression during taxane chemotherapy , 33% ( four of 12 ) and 48% ( 12 of 25 ) of evaluable men had ar - v7 detected by epic sciences and johns hopkins criteria , respectively ( appendix table a4 )  . 
these results conrm previously published data showing ar - v7positive disease remains sensitive to taxane chemotherapy.11 - 13 in the secondor third - line mcrpc setting , cabazitaxel improves os and should be considered a reasonable third - line treatment option , 5 and docetaxel or cabazitaxel are effective treatments after progression with abiraterone or enzalutamide , with pfs estimates of 4 to 6 months in these poor - risk men.14 , 24 , 26 , 27 the results are particularly important with regard to current practice , given the recent proven benet and use of these same ar inhibitors in the metastatic hormone - sensitive pc and nonmetastatic crpc settings , where ar therapy cross - resistance concerns remain when a patient experiences progression to mcrpc during therapy.28 - 32 a majority of men with ar - v7positive disease by epic sciences assay ( nine [ 82% ] of 11 ) were also positive by johns hopkins assay ; however , 17 ( 60% ) of 28 patients with positive disease by johns hopkins assay were negative by epic sciences assay at baseline . 
these differences largely reect the differential sensitivities of the assays for ar - v7 detection in ctc - based rna expression versus protein and the distinction between nuclear versus cytoplasmic localization . 
some patients developed ar - v7positive disease at progression during abiraterone or enzalutamide treatment , despite being ar - v7 negative at baseline , whereas some men with ar - v7positive disease converted to arv7negative status at progression , illustrating the need to assess ctc biomarker status at each management decision point ( fig 3 )  . 
a limitation of our study includes the lack of random assignment to taxane or ar therapy based on arv7 test results , and as such , our results show a prognostic rather than predictive utility . 
 armstrong et al support supported by a grant from the prostate cancer foundation and movember ; by the department of defense prostate cancer clinical trials consortium , which provided infrastructural support ; by the national institutes of health and grant 1r01ca233585 - 01 , duke cancer institute ( dci ) grant no . 
p30 ca014236 , and dci shared resources for biostatistics , ow cytometry , and sequencing and genomic technologies ( a.j.a. ) ; in part by department of defense grants no . 
george , susan halabi manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors consulting or advisory role : bayer , sano , dendreon , medivation , janssen biotech , pzer , astellas scientic and medical affairs , clovis oncology , astrazeneca speakers bureau : dendreon , bayer research funding : dendreon ( inst ) , sano ( inst ) , bayer ( inst ) , pzer ( inst ) , novartis ( inst ) , janssen oncology ( inst ) , medivation ( inst ) , astellas pharma ( inst ) , gilead sciences ( inst ) , roche / genentech ( inst ) , active biotech ( inst ) , bristol myers squibb ( inst ) , constellation pharmaceuticals ( inst ) , merck ( inst ) patents , royalties , other intellectual property : circulating tumor cell novel capture technology ( inst ) travel , accommodations , expenses : dendreon , janssen biotech , bayer , astellas scientic and medical affairs jun luo consulting or advisory role : sun pharma , janssen oncology , tolero pharmaceuticals research funding : sano ( inst ) , orion pharma ( inst ) , mirati therapeutics ( inst ) , gilead sciences ( inst ) , astellas pharma ( inst ) , constellation pharmaceuticals ( inst ) , calibr ( inst ) , cardiff oncology ( inst ) patents , royalties , other intellectual property : coinventor of a technology assigned to johns hopkins university , which licensed to tokai pharmaceuticals ( inst ) ; coinventor of a technology licensed to qiagen ( inst ) ; coinventor of a technology licensed to a&g pharmaceuticals ( inst ) david m . 
nanus consulting or advisory role : roche / genentech research funding : novartis ( inst ) , boehringer ingelheim ( inst ) , zenith epigenetics ( inst ) , astrazeneca ( inst ) , immumedics ( inst ) , janssen ( inst ) , clovis oncology ( inst ) , pzer ( inst ) paraskevi giannakakou employment : novartis ( i ) stock and other ownership interests : novartis ( i ) patents , royalties , other intellectual property : coinventor on international patent application docket no . 
danila honoraria : angle , bayer , screencell , janssen oncology , pzer , aximmune , pzer , clovis oncology , astellas pharma consulting or advisory role : angle , bayer , sanador , aximmune , pzer , clovis oncology , astellas pharma , janssen scientic affairs research funding : prostate cancer foundation , genentech , janssen research & development ( inst ) patents , royalties , other intellectual property : gene expression prole associated with prostate cancer travel , accommodations , expenses : cambridge healthtech institute , prostate cancer foundation , screencell , stopcancer , american austrian open medical institute , janssen biotech , genzyme , pzer , janssen scientic affairs , astellas pharma andrew j . 
berry honoraria : pzer , merck , genomic health , janssen oncology consulting or advisory role : merck , pzer , genomic health , janssen oncology research funding : merck ( inst ) travel , accommodations , expenses : merck , pzer , genomic health , janssen oncology tian zhang leadership : capio biosciences ( i ) , archimmune therapeutics ( i ) stock and other ownership interests : capio biosciences ( i ) , archimmune therapeutics ( i ) , nanorobotics ( i ) honoraria : exelixis , genentech / roche , mjh life sciences , pacic genuity consulting or advisory role : janssen , genentech / roche , sano , exelixis , astrazeneca , pzer , bristol myers squibb , foundation medicine , pharmacyclics , amgen , merck , seattle genetics speakers bureau : exelixis , genentech / roche , genomic health , sano research funding : janssen ( inst ) , acerta pharma ( inst ) , pzer ( inst ) , merrimack ( inst ) , stem centrx ( inst ) , novartis ( inst ) , omniseq ( inst ) , personal genome diagnostics ( inst ) , regeneron ( inst ) , merck ( inst ) , mirati therapeutics ( inst ) , astellas pharma patents , royalties , other intellectual property : circulating tumor cell novel capture by c - met technology ( inst ) ; prochelators as targeted prodrugs for prostate cancer ( inst ) travel , accommodations , expenses : acerta pharma , genomic health , astrazeneca michael r . 
harrison consulting or advisory role : bayer , sano , exelixis , genentech , argos therapeutics , fujilm , janssen oncology , astrazeneca , pzer , bristol myers squibb speakers bureau : genentech , exelixis research funding : argos therapeutics ( inst ) , bristol myers squibb ( inst ) , genentech ( inst ) , pzer ( inst ) , medivation / astellas pharma ( inst ) , merck ( inst ) , clovis oncology ( inst ) , acerta pharma ( inst ) , astrazeneca ( inst ) changxue lu patents , royalties , other intellectual property : inventor of the patent that was licensed to qiagen and received royalty joseph d . 
scher leadership : asterias biotherapeutics stock and other ownership interests : asterias biotherapeutics honoraria : research to practice consulting or advisory role : janssen biotech , amgen , janssen research & development , menarini silicon biosystems , wirb - copernicus group , essa , sano , ambry genetics , konica minolta , pzer , bayer research funding : janssen ( inst ) , illumina ( inst ) , epic sciences ( inst ) , menarini silicon biosystems ( inst ) , thermosher scientic biomarkers ( inst ) travel , accommodations , expenses : asterias biotherapeutics , menarini silicon biosystems , amgen , wirb - copernicus group , konica minolta , essa , prostate cancer foundation , sano , bayer , phosplatin therapeutics richard wenstrup employment : epic sciences leadership : epic sciences stock and other ownership interests : epic sciences consulting or advisory role : blueprint genetics , resolys patents , royalties , other intellectual property : patent royalties from assurexhealth travel , accommodations , expenses : epic systems scott t . 
tagawa consulting or advisory role : medivation , astellas pharma , dendreon , janssen , bayer , genentech , endocyte , immunomedics , karyopharm therapeutics , abbvie , tolmar , qed , amgen , sano , pzer , clovis oncology , novartis , genomic health , point biopharma research funding : lilly ( inst ) , sano ( inst ) , janssen ( inst ) , astellas pharma ( inst ) , progenics ( inst ) , millennium pharmaceuticals ( inst ) , amgen ( inst ) , bristol myers squibb ( inst ) , dendreon ( inst ) , rexahn pharmaceuticals ( inst ) , bayer ( inst ) , genentech ( inst ) , newlink genetics ( inst ) , inovio pharmaceuticals ( inst ) , astrazeneca ( inst ) , immunomedics ( inst ) , novartis ( inst ) , aveo ( inst ) , boehringer ingelheim ( inst ) , merck ( inst ) , stem centrx ( inst ) , karyopharm therapeutics ( inst ) , abbvie ( inst ) , medivation ( inst ) , endocyte ( inst ) , exelixis ( inst ) , clovis oncology ( inst ) travel , accommodations , expenses : sano , immunomedics , amgen uncompensated relationships : telix pharmaceuticals , atlab pharma , phosplatin therapeutics emmanuel s . 
antonarakis honoraria : sano , dendreon , medivation , janssen biotech , essa , astellas pharma , merck , astrazeneca , clovis oncology consulting or advisory role : sano , dendreon , janssen biotech , essa , merck , astrazeneca , clovis oncology , lilly , bayer research funding : janssen biotech ( inst ) , johnson & johnson ( inst ) , sano ( inst ) , dendreon ( inst ) , aragon pharmaceuticals ( inst ) , exelixis ( inst ) , millennium pharmaceuticals ( inst ) , genentech ( inst ) , novartis ( inst ) , astellas pharma ( inst ) , tokai pharmaceuticals ( inst ) , merck ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , constellation pharmaceuticals ( inst ) patents , royalties , other intellectual property : coinventor of a biomarker technology that has been licensed to qiagen travel , accommodations , expenses : sano , dendreon , medivation daniel j . 
george leadership : capio biosciences honoraria : sano , bayer , exelixis , emd serono , onclive , pzer , urotoday , acceleron pharma , american association for cancer research , axess oncology , janssen oncology , millennium medical publishing consulting or advisory role : bayer , exelixis , pzer , sano , astellas pharma , innocrin pharma , bristol myers squibb , genentech , janssen , merck sharp & dohme , myovant sciences , astrazeneca , michael j . 
n engl j med 371 : 424 - 433 , 2014 de bono js , oudard s , ozguroglu m , et al : prednisone plus cabazitaxel or mitoxantrone for metastatic castration - resistant prostate cancer progressing after docetaxel treatment : a randomised open - label trial . 
n engl j med 368 : 138 - 148 , 2013 [ erratum : n engl j med 368 : 584 , 2013 ] tannock if , de wit r , berry wr , et al : docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer . 
n engl j med 351 : 1502 - 1512 , 2004 de wit r , de bono j , sternberg cn , et al : cabazitaxel versus abiraterone or enzalutamide in metastatic prostate cancer . 
n engl j med 381 : 2506 - 2518 , 2019 khalaf dj , annala m , taavitsainen s , et al : optimal sequencing of enzalutamide and abiraterone acetate plus prednisone in metastatic castration - resistant prostate cancer : a multicentre , randomised , open - label , phase 2 , crossover trial . 
ann oncol 24 : 1802 - 1807 , 2013 schrader aj , boegemann m , ohlmann ch , et al : enzalutamide in castration - resistant prostate cancer patients progressing after docetaxel and abiraterone . 
eur urol 65 : 30 - 36 , 2014 armstrong aj , halabi s , luo j , et al : prospective multicenter validation of androgen receptor splice variant 7 and hormone therapy resistance in high - risk castration - resistant prostate cancer : the prophecy study . 
scher hi , graf rp , schreiber na , et al : assessment of the validity of nuclear - localized androgen receptor splice variant 7 in circulating tumor cells as a predictive biomarker for castration - resistant prostate cancer . 
scher hi , lu d , schreiber na , et al : association of ar - v7 on circulating tumor cells as a treatment - specic biomarker with outcomes and survival in castrationresistant prostate cancer . 
antonarakis es , lu c , luber b , et al : androgen receptor splice variant 7 and efcacy of taxane chemotherapy in patients with metastatic castration - resistant 14 . 
de bono js , scher hi , montgomery rb , et al : circulating tumor cells predict survival benet from treatment in metastatic castration - resistant prostate cancer . clin cancer res 14 : 6302 - 6309 , 2008 [ erratum : clin cancer res 15 : 1506 , 2009 ] 18 . 
scher hi , heller g , molina a , et al : circulating tumor cell biomarker panel as an individual - level surrogate for survival in metastatic castration - resistant prostate cancer . 
antonarakis es , lu c , luber b , et al : clinical signicance of androgen receptor splice variant - 7 mrna detection in circulating tumor cells of men with metastatic castration - resistant prostate cancer treated with rstand second - line abiraterone and enzalutamide . 
markowski mc , silberstein jl , eshleman jr , et al : clinical utility of clia - grade ar - v7 testing in patients with metastatic castration - resistant prostate cancer . ( clia ) laboratory setting . 
scher hi , morris mj , stadler wm , et al : trial design and objectives for castration - resistant prostate cancer : updated recommendations from the prostate cancer clinical trials working group 3 . 
halabi s , lin cy , small ej , et al : prognostic model predicting metastatic castration - resistant prostate cancer survival in men treated with second - line chemotherapy . 
scher hi , graf rp , schreiber na , et al : phenotypic heterogeneity of circulating tumor cells informs clinical decisions between ar signaling inhibitors and taxanes in metastatic prostate cancer . 
eisenberger m , hardy - bessard ac , kim cs , et al : phase iii study comparing a reduced dose of cabazitaxel ( 20 mg / m2 ) and the currently approved dose ( 25 mg / m2 ) in postdocetaxel patients with metastatic castration - resistant prostate cancer : proselica . 
oudard s , fizazi k , sengelv l , et al : cabazitaxel versus docetaxel as rst - line therapy for patients with metastatic castration - resistant prostate cancer : a randomized phase iii trialfirstana . 
armstrong aj , szmulewitz rz , petrylak dp , et al : arches : a randomized , phase iii study of androgen deprivation therapy with enzalutamide or placebo in men with metastatic hormone - sensitive prostate cancer . 
aggarwal r , huang j , alumkal jj , et al : clinical and genomic characterization of treatment - emergent small - cell neuroendocrine prostate cancer : a multiinstitutional prospective study . 
de laere b , van dam pj , whitington t , et al : comprehensive proling of the androgen receptor in liquid biopsies from castration - resistant prostate cancer reveals novel intra - ar structural variation and splice variant expression patterns . 
kohli m , ho y , hillman dw , et al : androgen receptor variant ar - v9 is coexpressed with ar - v7 in prostate cancer metastases and predicts abiraterone commun 7 : 13668 , 2016 resistance . 
de laere b , oeyen s , mayrhofer m , et al : tp53 outperforms other androgen receptor biomarkers to predict abiraterone or enzalutamide outcome in metastatic castration - resistant prostate cancer . 
 armstrong et al appendix assessed for eligibility ( n = 131 ) did not meet inclusion criteria ( n = 13 ) received hormone therapy abiraterone enzalutamide both ( n = 118 ) ( n = 55 ) ( n = 58 ) ( n = 5 ) progression during treatment death during treatment ( n = 105 ) ( n = 4 ) received subsequent taxane therapy docetaxel cabazitaxel ( n = 51 ) ( n = 42 ) ( n = 9 ) progression during treatment death during treatment ( n = 46 ) ( n = 3 ) fig a1 . 
waterfall plots of best overall prostate - specic antigen ( psa ) decline from pretaxane baseline during therapy with docetaxel or cabazitaxel according to ( a ) epic sciences and ( b ) johns hopkins androgen receptor splice variant 7 ( ar - v7 ) status . 
baseline pretaxane characteristics of patients enrolled according to johns hopkins ar - v7 status johns hopkins ar - v7 status ( % ) positive ( n = 20 ) 48 - 82 negative ( n = 31 ) 45 - 87 elevated baseline serum ldh presence of liver metastasis presence of clinically signicant pain requiring opiates cellsearch ctcs , cells per 7.5 ml baseline pretaxane characteristic age , years median range race white black other gleason sum 8 - 10 poor - risk feature hemoglobin , 12 g / dl elevated alp range median , % ( n = 37 ) psadt , 3 months prior docetaxel for mhspc  . 
long terra lucas shreshtha madaan kristin mattie danielle mckenna susan montgomery sarah nielsen jacquelyn powers kim rainey christina rybak michelle savage christina seelaus ( continued ) purpose multigene panels ( mgps ) are increasingly being used despite questions regarding their clinical utility and no standard approach to genetic counseling . 
how frequently genetic providers use mgp testing and how patient - reported outcomes ( pros ) differ from targeted testing ( eg , brca1 / 2 only ) are unknown . methods we evaluated use of mgp testing and pros in participants undergoing cancer genetic testing in the multicenter communication of genetic test results by telephone study ( clinicaltrials.gov identifier : nct01736345 ) , a randomized study of telephone versus in - person disclosure of genetic test results . 
genetic providers offered targeted or mgp testing based on clinical assessment . results since the inclusion of mgp testing in 2014 , 395 patients ( 66% ) were offered mgp testing . 
being offered mgp testing was significantly associated with not having ashkenazi jewish ancestry , having a history of cancer , not having a mutation in the family , not having made a treatment decision , and study site . 
after demographic adjustment , patients offered mgp testing had lower general anxiety ( p = .04 ) , state anxiety ( p = .03 ) , depression ( p = .04 ) , and uncertainty ( p = .05 ) pre - disclosure compared with patients offered targeted testing . 
there was a greater increase in change in uncertainty ( p = .04 ) among patients who underwent mgp testing . conclusion mgp testing was more frequently offered to patients with lower anxiety , depression , and uncertainty and was associated with favorable outcomes , with the exception of a greater increase in uncertainty compared with patients who had targeted testing . 
delivery of genetic testing results by telephone is one of several adaptations to the traditional genetic counseling practice model that have been studied in an effort to improve counseling access through the more efficient use of resources.3 - 5 beginning in 2012 , as part of the national cancer institute funded multicenter communication of genetic test results by telephone ( cogent ) clinical trial , patients eligible for brca1 / 2 genetic testing for hereditary breast or ovarian cancer risk assessment were recruited and randomly assigned to in - person or telephone disclosure of genetic test results . 
primary outcomes of the cogent study6 have demonstrated that telephone disclosure is noninferior to in - person counseling across all primary and secondary short term outcomes . a robust literature on patient - reported outcomes ( pros ) of brca1 / 2 and targeted genetic testing exists , including two randomized studies of telephone delivery of both pretesting and post - testing genetic services for patients considering genetic testing for these genes.3 , 5 cogent is , to our knowledge , the first randomized study to examine uptake of mgp tests in the population of patients presenting for risk assessment to a tertiary high - risk clinic , as well as to report on pros among patients receiving traditional targeted gene counseling and testing compared with patients receiving counseling and testing adapted to mgps . 
 in the current study , trends in testing choices with the advent of mgp testing and across the cogent study time period and participating sites are reported , and pros previously reported in aggregate are analyzed by counseling and testing modality selected for the patient . methods study design , setting , and participants the cogent study was a multicenter , randomized , noninferiority trial comparing the psychosocial and behavioral outcomes of phone versus in - person disclosure of genetic test results . 
 initially designed to include targeted brca1 / 2 testing only , the study eligibility was adapted in may 2014 to add all clinical germline genetic testing for hereditary breast , gynecologic , and / or gi cancer syndromes , allowing individuals receiving either targeted testing or mgp testing . 
participants were recruited after in - person pretest counseling with a genetic counselor and completed pros at the time of enrollment ( after pretest counseling [ t0 ] ) and after results disclosure ( t1 )  . random assignment after completion of the survey given after pretest counseling , participants were assigned to either the in - person arm or telephone ar random assignment was stratified by study site and sex only . 
participants who did not want to receive results by telephone and thus were not willing to be randomly assigned were permitted to self - select in - person disclosure and were analyzed separately . all 22 genetic counseling professionals used a tiered and binned counseling model for pretest sessions where mgp testing was offered.7 , 8 all disclosures were made using standardized communication protocols and visual aids.7 - 9 both a genetic counseling professional and a medical provider were present when results were provided in person , whereas individuals who received results by telephone were recommended to return to their institution to meet with a medical provider . 
patients responded on a seven - item likert scale and could mark not applicable . statistical analysis for baseline analyses , we characterized variables using means , proportions , standard deviations , and ranges for age . 
variables included in the tables and regressions included age , sex , race ( white v nonwhite ) , education ( college graduate v less than a college degree ) , jewish ethnicity ( no or missing v yes ) , number of firstand second - degree relatives with cancer , known cancer mutation , treatment decision , random assignment or self - selecting for in - person disclosure arm , and study site . 
we did not include in the imputation analyses individuals missing both t0 and t1 response data because we felt that such individuals had too much missing data to reliably impute data . 
the criterion for statistical significance was p < .05. results use and predictors of mgp testing versus targeted testing in total , 600 participants were offered genetic testing after inclusion of mgp testing ; 62.7% of participants ( 376 of 600 participants ) underwent mgp testing , whereas 37.3% of participants underwent targeted testing ( fig 1 )  . 
 targeted testing was most frequently offered to patients with a known familial mutation ( 86% ) and to patients of ashkenazi jewish ancestry ( 67% ) , a population in which three founder mutations in brca1 / 2 are found at high frequency ( prevalence of approximately 1 : 40 )  . 
of note , of 395 patients offered mgp testing by the genetic counselor , a small fraction ( 19 [ 4.8% ] of 395 patients ) declined mgp testing and chose targeted testing . 
characteristics associated with being more likely to be offered mgp testing were older age , female sex , nonashkenazi jewish heritage , personal history of cancer , no known familial mutation , nonrandomization status ( ie , preference for in - person disclosure ) , and study site . the percentage of patients offered mgp testing increased over time ( from 57% in 2014 to 66% in 2015 ; p = .02 ) and was found to vary significantly by study site ( range , 46% to 81% of all tests offered ; fig 2 )  . 
identification of carriers and true - negative results were higher in the targeted testing group , which is likely secondary to a higher number of patients with a known familial mutation ( table 1 )  . 
the frequency of uncertain variants was higher in the mgp group ( 25% v 2% with targeted testing )  . pros assessed by offer of targeted testing versus mgp testing in full models , adjusting for baseline variables , pros were assessed and compared by whether patients were offered mgp testing or targeted testing . 
characteristics of participants in the cogent study after adaptation for mgp by type of testing offered offered targeted testing only ( n = 205 ) offered multigene panel testing ( n = 395 ) characteristic had mgp testing , no . 
finally , there were no significant differences in knowledge at any time point . discussion in the largest study to date of pros after mgp testing , we found that patients eligible for hereditary cancer risk assessment and offered mgp , compared with targeted testing , had similar predisclosure and postdisclosure knowledge levels but lower predisclosure anxiety , depression , and uncertainty . 
these results demonstrate that genetic counselors can incorporate mgp testing into their practice without compromising patient understanding , even when results are delivered by telephone.6 one of the concerns commonly raised with mgp testing is the possibility of knowledge deficits as a result of the complexity of information communicated . 
 the potential negative impact of information overload from mgp counseling or testing on psychological outcomes is not supported by our findings , because we found that postdisclosure anxiety and depression were similar between the mgp and targeted testing groups . 
when the lower levels of predisclosure anxiety and depression among participants offered mgp are considered , our results also suggest that counselors may be more likely to offer complex mgp testing to patients they feel are at lower risk of negative psychological outcomes . 
in this way , these findings suggest that counselors are more than just gatekeepers to genetic testing and that comprehensive counseling includes both knowledge exchange and shared decision making with patients about concerns and preferences for genetic evaluation . 
 in the higher rates of selection of in - person disclosure among patients offered mgp testing , we observe counselors and patients counterbalancing a patients ability to manage the complexity and uncertainty of mgp testing with the standard approach of in - person results disclosure . these findings suggest that telephone disclosure is readily scalable to mgp testing and thus has an important role in the future of genomic medicine . 
although other studies have previously supported the efficacy of alternative telephone - based modalities of genetics services delivery , 3 , 5 these studies have not included mgp testing and have , notably , not reported detailed pros . 
one study recruited women at increased risk of hereditary breast and / or ovarian cancer and randomly assigned participants to in - person or telephone counseling , whereas the second study randomly assigned women with at least a 10% risk of having a brca1 / 2 mutation but without cancer to telephone counseling versus usual care . 
in both studies , telephone counseling was noninferior to in - person counseling for the selected outcomes ( which included anxiety , cancer - specific distress , perceived control , and decisional conflict in one study5 and satisfaction , distress , physical functioning , decisional conflict , and knowledge at 3 months and 1 year in the other study3 ) , although these results are specific to brca1 / 2 testing . 
although pros were not ascertained , the rate of preference for and receipt of mastectomy was similar regardless of whether a brca1 / 2 pathogenic variant or a pathogenic variant in a gene other than brca1 / 2 was identified by mgp , arguing that mgp may lead to more mastectomies over time . 
pros have not been explored in other areas of medicine where mgp testing has entered clinical care . lower postdisclosure anxiety and distress among patients offered mgp testing is consistent with differences seen pre - disclosure and reflect counselorand patient - driven selection of less anxious patients toward mgp testing . 
for others , high pretest risk was secondary to a known familial mutation ; 50% of the targeted testing population had a known familial mutation compared with 4% of the mgp testing population . 
similarly , patients tested by mgp had lower pretest likelihood of a pathogenic mutation , although the risk of identifying uncertain variants was higher as a result of the larger number of genes sequenced . although no other randomized studies have been conducted involving mgp , lumish et al19 performed a post - test survey measuring pros in patients undergoing testing for hereditary breast or ovarian cancer and in whom mgp testing was performed . 
they found older age , nonwhite race or non - hispanic ethnicity , lower educational attainment , and lower knowledge to be associated with higher anxiety and adverse psychological effects from counseling . 
analyses by cancer status ( affected or unaffected ) and stratified by result also found differential impact on psychological outcomes.19 nonetheless , it remains uncertain how the differences would have changed had mgp testing been replaced by targeted testing . 
 intolerance related to genomic testing21 have shown that uncertainty intolerance is associated with impaired decision making , feelings of vulnerability , and other adverse psychological outcomes.22 overall , we found that use of mgp testing increased over the study period.23 several factors are likely driving this change , including the need for more efficient delivery of genetic services . 
 the declining costs and the competitive marketplace for commercial testing established by the us supreme courts decision against myriad genetic laboratories and gene patenting have improved access to genetic testing . 
regional and community hospitals may be particularly disadvantaged when it comes to access to genetic counseling services and thus could be the earliest beneficiaries of models that incorporate remote counseling and testing options . 
however , after accounting for nonrandom differences between the targeted and mgp testing groups , short - term postdisclosure pros were generally similar , suggesting that the benefits of counseling rest in the live conversation between the counselor and patient and not in their proximity . 
time and resource limitations have undermined the traditional rigorous preand post - test counseling model used in cogent ; for example , katz et al25 found that 47% of patients did not receive counseling . 
in addition , few cogent participants were tested to guide a breast cancer treatment decision , and a recent population - based study found that mgp testing was associated with lower rates of testing among patients with breast cancer who had preoperative testing.18 finally , it also possible that patients choosing an mgp test versus targeted testing do so with family and family cancer risks in mind , so there may be additional information on testing selection gained from studying patterns of communication of results with relatives . in summary , the world of clinical genetic testing is changing rapidly , and use of mgp testing has increased over time . 
post - test knowledge , general anxiety , and depression were no worse and state anxiety was lower among patients who received mgp testing compared with those who received targeted testing . 
 bradbury manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors the following represents disclosure information provided by authors of this manuscript . 
 stock and other ownership interests : canceriq , tempus travel , accommodations , expenses : hospital of the university of pennsylvania research funding : novartis ( inst ) other relationship : tempus , color genomics , genentech , myriad genetics , bio ventures for global health kim rainey no relationship to disclose linda j . 
domchek honoraria : astrazeneca , clovis oncology , bristol - myers squibb research funding : astrazeneca ( inst ) , clovis oncology ( inst ) , pharmamar ( inst ) kristin mattie no relationship to disclose danielle mckenna no relationship to disclose susan montgomery no relationship to disclose sarah nielsen no relationship to disclose jacquelyn powers employment : carevive systems honoraria : cureconnect consulting or advisory role : carevive systems christina rybak no relationship to disclose michelle savage no relationship to disclose christina seelaus no relationship to disclose jessica stoll no relationship to disclose jill e . 
olopade employment : canceriq ( i ) leadership : canceriq dominique fetzer no relationship to disclose amanda brandt no relationship to disclose rachelle chambers no relationship to disclose dana f . 
clark no relationship to disclose rikki gaber honoraria : canceriq cassandra gulden no relationship to disclose janice horte no relationship to disclose andrea forman consulting or advisory role : invitae , astrazeneca , ambry speakers ' bureau : astrazeneca , ambry genetics , invitae jessica m . 
daly , andrea forman , susan montgomery , kim rainey , christina rybak , and michelle savage , fox chase cancer center , temple university health system ; susan m . 
olopade , center for clinical cancer genetics and global health , the university of chicago ; pamela ganschow , rikki gaber , terra lucas , and christina seelaus , the john h . 
hospital of cook county ; rachelle chambers , cassandra gulden , shreshtha madaan , sarah nielsen , and jessica stoll , the university of chicago , chicago , il ; and generosa grana , dana f . 
clark , janice horte , kristin mattie , and xinxin ( shirley ) yao , md anderson cancer center at cooper , camden , nj . support supported by national cancer institute grant no . 
kinney ay , steffen le , brumbach bh , et al : randomized noninferiority trial of telephone delivery of brca1 / 2 genetic counseling compared with in - person counseling : 1 - year follow - up . 
bradbury ar , patrick - miller lj , egleston bl , et al : randomized noninferiority trial of telephone vs in - person disclosure of germline cancer genetic test results . 
bradbury ar , patrick - miller lj , egleston bl , et al : patient feedback and early outcome data with a novel tiered - binned model for multiplex breast cancer susceptibility testing . 
bradbury ar , patrick - miller l , long j , et al : development of a tiered and binned genetic counseling model for informed consent in the era of multiplex testing for cancer susceptibility . 
kelly k , leventhal h , marvin m , et al : cancer genetics knowledge and beliefs and receipt of results in ashkenazi jewish individuals receiving counseling for brca1 / 2 mutations . 
cella d , hughes c , peterman a , et al : a brief assessment of concerns associated with genetic testing for cancer : the multidimensional impact of cancer risk assessment ( micra ) questionnaire . 
pieterse ah , van dulmen am , beemer fa , et al : cancer genetic counseling : communication and counselees post - visit satisfaction , cognitions , anxiety , and needs fulfillment . 
biesecker bb , klein w , lewis kl , et al : how do research participants perceive uncertainty in genome sequencing ? genet med 16 : 977 - 980 , 2014 22 . 
 mendel goldnger ramaswamy govindan jhanelle gray stacy gray samantha greenberg malachi grifth * roman groisberg * torsten haferlach michael hall * anis hamid * heather hampel * fei han sigurdis haraldsdottir * derrick haslem * eric haura masanori hayashi brian henick * , * * daniel herchenhorn * kenneth hess j . 
kevin hicks kim hirsheld brian hobbs howard hochster * daanish hoda * ahmed hosny david hsu wenhui huang yi huang * hatim husain maha hussain * david hyman * alexia iasonos * meredith irwin * steven isakoff katherine janeway * yelena janjigian lingyun ji xuemei ji * * yuchao jiang markus joerger steven joffe * thomas kaley mohammed kashanisabet steve katz kevin kim david kirsch kedar kirtane eric klein samuel klempner * todd knepper wendy kohlmann * jill kolesar * ian krop * priti kumari shivaani kummar ava kwong andrea laghi * jacky wai - kei lam angela lamarca jean - michel lavoie rachel layperson * christophe le tourneau * amy leblanc j . 
jack lee * jochen lennerz * antonio lerario benjamin levy mark lewis * jessica lin * steven lipkin jingyi liu * minetta liu * stephen liu * christine lovly ying lu * luke maese * david malkin diana mandelker sharon manne aaron manseld veronica mariotti thomas martin carlotta masciocchi joaquin mateo * shigeyuki matsui * joan maurel * shannon mccall jeannine mccune * rose mcgee * funda meric - bernstam everett meyer * ropa mhlanga * * luca mologni clara montagut * lucas moreno * daniel morgenstern * motomi mori * kent mouw anwesha nag katherine nathanson daniel nava rodrigues stewart neill kim nichols donna niedzwiecki nicola normanno * david norris paolo nuciforo jennifer oberg * stephen oh * coral omene stephen opat eileen oreilly * piet ost geoffrey oxnard * sukhmani padda tuya pal jose palacios phillip palmbos wei pan nickolas papadopoulos * soo park jai patel shiven patel * cloud paweletz andrew pearson raquel perez - lopez * thomas permutt * emanuel petricoin * gina petroni * david piccioni gaelle pierron filippo pietrantonio zoa piotrowska sharon plon eric polley mei - yin polley * erqi pollom mark pomerantz steven powell * colin pritchard * kanwal raghav * nitya raj w . 
we demonstrated previously that pemetrexed - based chemotherapy can achieve an objective response rate of 45% and a median progression - free survival ( pfs ) of 19 months.3 furthermore , the activity of targeted therapy has improved dramatically with the introduction of selective ret inhibitors to the clinic . 
in early - phase testing , objective response rates with loxo - 2924 and blu - 6675 are 68% ( 26 of 38 ) and 50% ( seven of 14 ) , respectively . 
these outcomes exceed the modest activity observed previously with multikinase inhibitors such as cabozantinib6 and vandetanib.7 in contrast , the activity of immunotherapy in retrearranged lung cancers has not been well characterized . 
this represents a clear unmet need , given that all prior regulatory approvals of immune checkpoint inhibitors , either alone or in combination with chemotherapy , and in stage iii or iv disease , have technically included patients with ret - rearranged lung cancers.8 , 9 furthermore , although increasing levels of programmed death - ligand 1 ( pd - l1 ) expression and high tumor mutational burden ( tmb ) have been associated with benet from immune checkpoint blockade , 10 the immunophenotype of ret - rearranged lung cancers and the role of pd - l1 and tmb status in relation to benet with immunotherapy remain poorly described . 
we set out to characterize these factors . methods in this retrospective study , patients from memorial sloan kettering cancer center with ret - rearranged lung cancers diagnosed between 2009 and 2017 were identied under an institutional review board approved waiver . 
patients who received immunotherapy , dened as a monoclonal antibody against programmed cell death protein 1 ( pd - 1 ) or pd - l1 , were included in an analysis of treatment history . ret rearrangements were identied using targeted next - generation sequencing of dna ( memorial sloan ketteringintegrated mutation proling of actionable cancer targets [ msk - impact ] or foundation one ) or rna ( anchored multiplex polymerase chain reaction [ pcr ] ; memorial sloan kettering solid fusion panel ) in more contemporary samples , and uorescence in situ hybridization ( 10q11 and 6q22 break apart probe , reversemetasystems , altussheim , germany ) or transcriptase pcr in older samples.11 , 12 pd - l1 immunohistochemistry was evaluated using the e1l3n antibody ( cell signaling technology , danvers , ma ) , our institutional standard , which has been validated against a 22c3 kit performed in a commercial laboratory with comparable results.13 for uniformity , tmb ( reported as the number of nonsynonymous mutations per megabase ) was analyzed only for samples sequenced using msk - impact.14 , 15 the median tmb of ret - rearranged was compared with that of ret wildtype nsclcs ( mann - whitney u test )  . in patients with both baseline and serial on - treatment imaging , the best objective response to therapy ( response evaluation criteria in solid tumors [ recist ] v1.1 ) was determined by a study radiologist . 
time to treatment discontinuation ( ttd ) was dened as the time from therapy initiation to the last dose.16 pfs was dened as the time from therapy initiation to radiologic progression or death . 
ret rearrangement was identied as follows : dna - based next - generation sequencing ( 80% [ n = 59 ] ) , rnabased anchored multiplex pcr ( 1% [ n = 1 ] ) , uorescence in situ hybridization ( 15% [ n = 11 ] ) , and reverse - transcriptase pcr ( 4% [ n = 3 ] )  . 
clinicopathologic features of ret - rearranged lung cancers : summary of demographics and tumor molecular features of 74 patients with ret - rearranged lung cancers feature all patients ( n = 74 ) age , years median range sex , no . 
in 44 patients with sufcient tissue for tmb analysis , the median tmb was 1.75 mutations / mb ( range , 0 to 9.65 mutations / mb ) , signicantly lower ( p , .0001 ) than the median tmb of 5.27 mutations / mb ( range , 0 to 164.20 mutations / mb ) in 3 , 631 patients with ret wild - type nsclcs ( fig 1b )  . the clinical outcomes of immunotherapy in patients with advanced ret - rearranged lung cancers are summarized in table 2 . 
 ( a ) the programmed death - ligand 1 ( pd - l1 ) expression ( e1l3n , cell signaling ) of 26 ret - rearranged lung cancers with sufcient tissue for testing is shown . 
 ( b ) the tumor mutational burden ( tmb ) of 44 ret - rearranged lung cancers is displayed ( left ) relative to the tmb of 3 , 631 ret wild - type lung cancers ( right )  . 
for ease of representation , three outlier ret wild - type lung cancer samples with tmb greater than 75 mutations / mb that were included in the statistical analysis were excluded in this plot . nivolumab ( n = 6 ) , atezolizumab ( n = 2 ) , durvalumab ( n = 1 ) , or ipilimumab plus nivolumab ( n = 1 )  . 
dashes represent tumor samples in which tissue was insufcient for pd - l1 or tmb testing . abbreviations : cr , complete response ; pd , progressive disease ; pd - l1 , programmed death - ligand 1 ; pfs , progression - free survival ; sd , stable disease ; non - cr / non - pd , not cr and not pd in nontarget lesions ; tmb , tumor mutational burden . * treatment discontinued for toxicity . a total of 13 patients with ret - rearranged lung cancers were assessed for clinical and / or radiologic response . response to immunotherapy was not observed . 
progression of disease was observed in 62% of cases ( n = eight of 13 ; table 2 ) ; disease progression involved new or worsening brain metastases in three patients ( cases 4 , 6 , and 15 )  . 
stable disease was achieved in 23% ( three of 13 ) and non - cr / non - pd ( not complete reponse and not progressive disease in nontarget lesions ) in 15% ( two of 13 )  . 
 ret - rearranged nsclc : immunophenotype and immunotherapy response these ndings are consistent with a growing body of evidence uncovering poor outcomes with immune checkpoint inhibition in select oncogene - addicted lung in egfr - mutant and alk - rearranged lung cancers . cancers , early data on the decreased activity ( compared with unselected cancers ) of immunotherapy resulted in the exclusion of patients with these tumors in registrationenabling studies . 
furthermore , we showed previously that met exon 14altered nsclcs had low tmb and poor outcomes with immunotherapy.17 finally , an ongoing global registry ( immunotarget ) and independent series have shown similarly low response rates and short median pfs with single - agent immune checkpoint inhibition in lung cancers with oncogenic drivers.18 immunotarget had 16 patients in the ret - rearranged cohort and reported a response rate of 6% and median pfs of 2.1 months , comparable to the ndings in our study . the clinical implications of these observations relate to the sequence with which immunotherapy is used and the type of immunotherapy strategy selected . 
regarding the former , it is becoming increasingly clear that specic , targeted therapies ( selective ret inhibitors ) 2 , 4 , 19 and chemotherapy agents ( pemetrexed - containing regimens ) 3 can achieve superior outcomes compared with immunotherapy in this series . 
thus , is reasonable to consider the use of checkpoint inhibition only after select targeted therapies and platinum doublet - containing chemotherapy have been administered . note that high pd - l1 expression ( 50% or more ) was uncommon in ret - rearranged lung cancers in this study . only one case treated with immunotherapy highly expressed pd - l1 ( 50% ) and , despite this , still responded poorly to dual immune checkpoint inhibitor therapy ( case 16 )  . 
a recommendation regarding the use of pembrolizumab in treatment - nave , advanced ret - rearranged lung cancers with high pd - l1 expression cannot be made on the basis of our ndings , although its use should be approached with caution . 
in met exon 14altered lung cancers , tmb is similarly low ; although a higher proportion of tumors have high pd - l1 expression , this was not associated with increased benet with single - agent immunotherapy.17 finally , no patients in this series received the combination chemotherapy and immunotherapy that is currently approved for the treatment of egfr and alk wild - type advanced nsclc . 
prospective trials of immunotherapy combinations should incorporate comprehensive molecular proling to establish prospective data in driver - positive subpopulations of patients . in summary , most ret - rearranged lung cancers have low pd - l1 expression and low tmb , and response to immunotherapy was not observed in this series . 
although this study is limited by a small sample size , given the rarity of ret - rearranged nsclcs , these ndings remain meaningful and support evolving literature showing that outcomes with immune checkpoint inhibition are poor in select oncogene - addicted nsclcs . 
contributed equally to this work . support supported in part by the national cancer institute of the national institutes of health ( t32 ca009207 , p30 ca008748 )  . provision of study material or patients : gregory j . 
rudin consulting or advisory role : bristol - myers squibb , abbvie , seattle genetics , harpoon therapeutics , genentech / roche , astrazeneca , ascentage pharma , bicycle therapeutics , celgene , daiichi sankyo , ipsen , loxo , pharmamar , elucida oncology research funding : abbvie / stemcentrx ( inst ) , viralytics ( inst ) , merck ( inst ) , daiichi sankyo ( inst ) gregory j . 
riely research funding : novartis ( inst ) , roche / genentech ( inst ) , millennium pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , pzer ( inst ) , innity pharmaceuticals ( inst ) , ariad pharmaceuticals ( inst ) , mirati therapeutics ( inst ) , merck ( inst ) patents , royalties , other intellectual property : patent application submitted covering pulsatile use of erlotinib to treat or prevent brain metastases ( inst ) travel , accommodations , expenses : merck sharp & dohme matthew d . 
hellmann stock and other ownership interests : shattuck labs honoraria : astrazeneca , bristol - myers squibb consulting or advisory role : bristol - myers squibb , merck , genentech , astrazeneca / medimmune , novartis , janssen pharmaceuticals , nektar , syndax pharmaceuticals , mirati therapeutics , shattuck labs research funding : bristol - myers squibb ( inst ) patents , royalties , other intellectual property : a patent has been led by memorial sloan kettering ( pct / us2015 / 062208 ) for the use of tumor mutation burden for prediction of immunotherapy efcacy , and which is licensed to personal genome diagnostics ( inst ) travel , accommodations , expenses : astrazeneca , bristol - myers squibb maria arcila honoraria : invivoscribe consulting or advisory role : astrazeneca travel , accommodations , expenses : astrazeneca , invivoscribe , raindance technologies mark g . 
li consulting or advisory role : roche , biosceptre international , thermo fisher scientic , mersana , guardant health , hengrui therapeutics research funding : roche / genentech ( inst ) , illumina ( inst ) , biomed valley discoveries ( inst ) , astrazeneca ( inst ) , grail ( inst ) , daiichi sankyo ( inst ) , hengrui therapeutics ( inst ) , guardant health ( inst ) marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network / astrazeneca tagrisso request for proposal advisory committee , takeda pharmaceuticals , bristol - myers squibb , bayer ag , merck ( i ) research funding : loxo ( inst ) , helsinn therapeutics alexander drilon honoraria : medscape , onclive , peervoice , physicians education resources , targeted oncology , more health , research to practice , foundation medicine , peerview consulting or advisory role : ignyta , loxo , tp therapeutics , astrazeneca , pzer , blueprint medicines , genentech / roche , helsinn therapeutics , beigene , hengrui therapeutics , exelixis , bayer ag , tyra biosciences , verastem , takeda pharmaceuticals / ariad pharmaceuticals / millenium pharmaceuticals , bergenbio , more health patents , royalties , other intellectual property : wolters kluwer ( royalties for pocket oncology ) other relationship : merck , glaxosmithkline , teva pharmaceuticals industries , taiho pharmaceutical no other potential conicts of interest were reported . references stransky n , cerami e , schalm s , et al : the landscape of kinase fusions in cancer . 
nat commun 5 : 4846 , 2014 drilon a , hu zi , lai ggy , et al : targeting ret - driven cancers : lessons from evolving preclinical and clinical landscapes . 
nat rev clin oncol 15 : 151 - 167 , 2018 drilon a , bergagnini i , delasos l , et al : clinical outcomes with pemetrexed - based systemic therapies in ret - rearranged lung cancers . 
ann oncol 27 : 1286 - 1291 , 2016 oxnard gr , subbiah v , park k , et al : clinical activity of loxo - 292 , a highly selective ret inhibitor , in patients with ret fusion + non - small cell lung cancer : an update from asco 2018 . 
toronto , canada , september 23 - 26 , 2018 subbiah v , taylor m , lin j , et al : abstract ct043 : highly potent and selective ret inhibitor , blu - 667 , achieves proof of concept in a phase i study of advanced , ret - altered solid tumors . 
drilon a , rekhtman n , arcila m , et al : cabozantinib in patients with advanced ret - rearranged non - small - cell lung cancer : an open - label , single - centre , phase 2 , single - arm trial . 
lancet oncol 17 : 1653 - 1660 , 2016 lee sh , lee jk , ahn mj , et al : vandetanib in pretreated patients with advanced non - small cell lung cancer - harboring ret rearrangement : a phase ii clinical trial . 
ann oncol 28 : 292 - 297 , 2017 gandhi l , rodrguez - abreu d , gadgeel s , et al : pembrolizumab plus chemotherapy in metastatic non - small - cell lung cancer . 
n engl j med 378 : 2078 - 2092 , 2018 reck m , rodrguez - abreu d , robinson ag , et al : pembrolizumab versus chemotherapy for pd - l1 - positive non - small - cell 375 : 1823 - 1833 , 2016 lung cancer . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
rizvi h , sanchez - vega f , la k , et al : molecular determinants of response to anti - programmed cell death ( pd ) - 1 and anti - programmed death - ligand 1 ( pd - l1 ) blockade in patients with non - small - cell lung cancer proled with targeted next - generation sequencing . 
ofn m , rizvi h , tenet m , et al : tumor mutation burden and efcacy of egfr - tyrosine kinase inhibitors in patients with egfr - mutant lung cancers . 
sabari jk , leonardi gc , shu ca , et al : pd - l1 expression , tumor mutational burden , and response to immunotherapy in patients with met exon 14 altered lung 18 . 
mazieres j , drilon ae , mhanna l , et al : efcacy of immune - checkpoint inhibitors ( ici ) in non - small cell lung cancer ( nsclc ) patients harboring activating molecular alterations ( immunotarget )  . 
 ofn et al immunotherapy naive ( n = 46 ) immunotherapy administered ( n = 16 ) log - rank p = .35 time since metastasis diagnosis ( years ) no . 
overall survival from the diagnosis of metastatic disease was compared between patients who received an immune checkpoint inhibitor ( n = 16 ) and those who did not ( n = 46 )  . 
hung , md , phd1 ; dora dias - santagata , phd1 ; maristela onozato , md1 ; nikunj shah , bs2 ; eric severson , md2 ; daniel duncan , md2 ; brendan j . 
we characterized molecular and clinicopathologic features of the pertinent fusion - positive patient cases . results we identied 11 patients with thymomas harboring a gene fusion of kmt2a and mastermind - like transcriptional coactivator 2 ( maml2 )  . 
the frequency of kmt2a - maml2 fusion was 4% of all thymomas ( 10 of 242 ) and 6% of thymomas of b2 or b3 histology ( 10 of 169 )  . conclusion kmt2a - maml2 represents the rst recurrent fusion described in type b thymoma . 
the identication of this fusion offers insights into the biology of thymoma and may have clinical relevance for patients with disease refractory to conventional therapeutic modalities . jco precis oncol 4 : 109 - 115 . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction thymoma , a neoplasm that arises from or exhibits thymic epithelial differentiation , is the most common tumor of the adult thymus.1 thymoma has a strong association with autoimmune diseases , particularly myasthenia gravis , which typically resolve upon successful tumor resection . 
we aim to identify recurrent genetic alterations present in this tumor . knowledge generated a subset of type b2 and b3 thymomas harbor recurrent gene fusions between lysine methyltransferase 2a ( kmt2a ) and mastermind - like transcriptional coactivator 2 ( maml2 )  . 
aside from rare case reports of hematologic neoplasms in the literature , the kmt2a - maml2 rearrangement seems specic to type b2 and b3 thymomas among human tumors . relevance the nding of recurrent kmt2a - maml2 fusion provides insights into tumor biology and potential therapeutic targets in type b2 and b3 thymomas , which are clinically aggressive with limited curative options . solid fusion assay analysis ( mgh solid fusion assay ; mgh , boston , ma ; data supplement provides list of gene targets ) and single - nucleotide variant and insertion / deletion analysis ( snapshot dna - based assay ; thermo fisher scientic , waltham , ma ; data supplement provides list of gene targets ) .7 the solid fusion assay used rna - based fusiontargeted anchored multiplex polymerase chain reaction ( archerdx primers , boulder , co ) and illumina ( san diego , ca ) sequencing . 
in brief , 60 ng of dna was extracted from 255 , 008 cancer specimens , including 242 thymoma specimens , in 40 m of formalin - xed , parafn - embedded tissue blocks . 
the samples were assayed by cgp using adaptor ligation , and hybrid capture was performed for all coding exons from 287 ( version 1 ) to 315 ( version 2 ) cancer - related genes plus select introns from 19 ( version 1 ) to 28 ( version 2 ) genes frequently rearranged in cancer ( data supplement )  . 
gtf2i included on any list of cancer - related genes . was not various samples were similarly assayed but performed in dna on 406 genes and selected introns of 31 genes involved in rearrangements ( data supplement ) and in rna on 265 genes commonly rearranged in cancer . 
sequences were analyzed for all classes of genomic alterations , including short variant alterations ( base substitutions , insertions , and deletions ) , copy - number alterations ( focal amplications and homozygous deletions ) , and select gene fusions or rearrangements by methods previously described.8 - 10 thymomas harboring a kmt2a - maml2 the cohort of fusion comprised 10 patient cases assayed with cgp ( foundation medicine ) during clinical care at other institutions . 
seven years later , the patient developed recurrent thymoma , with direct invasion of the lung , pericardium , and distal left main pulmonary artery , as well as metastasis to paratracheal lymph nodes ( fig 1a )  . the patient received induction chemotherapy and underwent surgical resection ( radical thymectomy , radical pericardiectomy , and left pneumonectomy ) , followed by paratracheal lymph node excision and radiotherapy . 
 ( a ) computed tomography of the chest from the patients rst recurrence demonstrates a 7.8 - cm anterior mediastinal mass ( arrow ) in contact with the pericardium , aorta , and portions of the main and left pulmonary arteries . 
 ( b ) histopathologic examination of the tumor shows sheets of large epithelioid cells lacking signicant lymphocytic inltration , consistent with type b3 thymoma ( hematoxylin and eosin stain ; magnication 400 )  . 
 ( d ) ihc for terminal deoxynucleotidyl lineage thymocytes associated with the tumor ( magnication 400 )  . transferase highlights scattered nuclei of t - cell recurrence of myasthenia gravis . 
the patient died as a result of complications of her thymoma 15 years after initial diagnosis . given the aggressive nature of this thymoma , molecular genetic assays were performed on the initial recurrence to lung , in an attempt to identify potentially targetable genetic alterations . 
this fusion assay result was reproduced on the patients subsequent paratracheal lymph node excision . kmt2a - maml2 fusion occurs in aggressive histologic subtypes of thymoma to determine the frequency of kmt2a - maml2 fusion in a cohort specically enriched in clinically more aggressive cases of thymoma , we reviewed a set of 242 patient cases of thymoma from the foundation medicine archives . 
the patient case was reviewed , and the original diagnosis was conrmed . the kmt2a - maml2 fusion was reamong thymomas , stricted to the most aggressive histologic thymoma subtypes , being present in 10 ( 5.9% ) of 169 thymomas that included b2 or b3 components , but 0 ( 0% ) of 64 of the remaining thymomas ( types a , ab , and b1 )  . 
no other known or likely pathogenic alterations were identied in 7 of 10 patient cases with a kmt2a - maml2 fusion , whereas a concurrent mutation in tp53 , arid1a , or sf3b1 was identied in 1 patient case each . 
of patients age at diagnosis , years final staging ( modied masaoka ) median range male female unknown histology b2 + b3 b3 + c 112 2020 by american society of clinical oncology discussion in the 243 patient cases of thymoma evaluated , we discovered a recurrent fusion of kmt2a and maml2 in 11 . this fusion seems to be highly specic to thymomas with aggressive histologic features , because all fusion - positive patient cases contained type b2 and / or b3 histology . 
the striking restriction of kmt2a - maml2 fusion to thymomas was underscored by the absence of the fusion in approximately 255 , 000 patient cases of diverse tumor types , including 366 thymic carcinomas , with the exception of a single patient case of plasmacytoma . kmt2a , rst described in 1991 , was initially termed mixedlineage leukemia - 1 because of its frequent appearance as a translocation partner in acute myeloid and lymphoid leukemias.12 the 36 - exon gene is located on chromosome 11q23 . 
the encoded protein binds dna and methylates histone h3 at lysine - 4 to regulate other genes , including several homeobox ( hox ) genes.13 kmt2a has been found to be a promiscuous translocation partner , with  . 
80 unique fusion partners identied.14 currently , there is no unifying theory on how kmt2a rearrangements lead to neoplasia.14 maml2 is a 5 - exon gene residing on chromosome 11q21 . 
maml2 and other maml family proteins are involved in notch pathwaymediated transcriptional activation.15 recurrent gene rearrangements involving maml2 have been described in mucoepidermoid carcinoma , in which fusion of the rst exon of the camp response element - binding proteinregulated transcription coactivator - 1 ( crtc1 ) with maml2 exons 2 to 5 leads to notch pathway disruption.16 the kmt2a - maml2 gene fusion results from inv ( 11 ) ( q21q23 ) , a cytogenetic abnormality rst reported in 1998 by obama et al17 in a patient with therapy - related acute myeloid leukemia . subsequent reports of the fusion are exceptionally rare and no . 
to our knowledge , only 8 patient cases have been reported , including 2 of acute myeloid leukemia , 2 of myelodysplastic syndrome , and 4 of acute lymphoblastic leukemia.17 - 22 reported fusion proteins in these patient cases involved regions similar to those identied in our cohort , with the exception of 2 patient cases reported by metlzer et al21 with maml2 breakpoints in introns 2 and 3 . prior functional studies of the kmt2a - maml2 construct have shown evidence of disrupted notch pathway signaling . 
in addition to describing the fusion in patient cases therapy - related myeloid neoplasms , nemoto et al19 the kmt2ademonstrated via luciferase assay that maml2 fusion suppresses promoter activation of the notch1 target gene hes1.19 gene expression proles from 2 patient cases of kmt2a - maml2positive t - cell acute lymphoblastic leukemia showed differential expression patterns relative to controls , suggesting activation of genes downstream of notch1.21 another study demonstrated oncogenic activity by kmt2a - maml2 fusion inserted into cell lines with sleeping - beauty vectors.23 the sum of these studies has demonstrated oncogenic function of kmt2a - maml2 fusion , likely via disruption of notch signaling . 
further study is warranted to examine the impact of this fusion on notch signaling . in patients with malignancies not amenable to traditional surgical or chemoradiotherapy protocols , targeted therapy offers an additional potential opportunity for disease control . 
early data suggest disease refractory to initial that maml2 fusionpositive mucoepidermoid carcinoma may respond to targeted therapeutics , including epidermal growth factor receptor inhibitors such as getinib.24 - 26 given the low tumor mutational burden seen in thymoma , identication of this small but genomically distinctive subset of kmt2a - maml2rearranged tumors may provide a therapeutic target in patients not responsive to traditional therapy . 
hasserjian , abner louissaint jr , erik a . williams manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors nikunj shah employment : foundation medicine eric severson employment : foundation medicine , partners healthcare stock and other ownership interests : foundation medicine daniel duncan employment : foundation medicine jeffrey s . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . dora dias - santagata consulting or advisory role : rarecyte ( i ) maristela onozato patents , royalties , other intellectual property : patent pending for multiplex fish assay , led on october 25 , 2019 ( no direct compensation related to this patent ) jo - anne vergilio employment : foundation medicine stock and other ownership interests : foundation medicine , roche valentina nardi consulting or advisory role : thermo fisher scientic ( i ) , cell signaling technology ( i ) robert p . 
hasserjian stock and other ownership interests : avanos medical , danaher , henry schein , hologic , idexx laboratories , johnson & johnson , kimberly clary , medtronic , stryker , cooper companies , thermo fisher scientic consulting or advisory role : jazz pharmaceuticals , promedior erik a . 
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ziemin - van der poel s , mccabe nr , gill hj , et al : identication of a gene , mll , that spans the breakpoint in 11q23 translocations associated with human leukemias . 
k ochert k , ullrich k , kreher s , et al : high - level expression of mastermind - like 2 contributes to aberrant activation of the notch signaling pathway in human lymphomas . 
tonon g , modi s , wu l , et al : t ( 11 ; 19 ) ( q21 ; p13 ) translocation in mucoepidermoid carcinoma creates a novel fusion product that disrupts a notch signaling pathway . 
obama k , furukawa y , tara m , et al : secondary monocytic leukemia with rearrangement of the mll gene occurring during the course of adult t - cell leukemia . int j hematol 68 : 323 - 326 , 1998 18 . 
mariani ra , silva m , caparelli e , et al : inv ( 11 ) ( q21q23 ) : kmt2a - maml2 , a recurrent genetic abnormality in t - cell therapy - related acute lymphoblastic leukemia . 
nemoto n , suzukawa k , shimizu s , et al : identication of a novel fusion gene mll - maml2 in secondary acute myelogenous leukemia and myelodysplastic syndrome with inv ( 11 ) ( q21q23 )  . 
tang g , lu x , wang sa , et al : homozygous inv ( 11 ) ( q21q23 ) and mll gene rearrangement in two patients with myeloid neoplasms . 
metzler m , staege ms , harder l , et al : inv ( 11 ) ( q21q23 ) fuses mll to the notch co - activator mastermind - like 2 in secondary t - cell acute lymphoblastic leukemia . 
chen z , chen j , gu y , et al : aberrantly activated areg - egfr signaling is required for the growth and survival of crtc1 - maml2 fusion - positive mucoe26 . 
li s , zhang z , tang h , et al : pathological complete response to getinib in a 10 - year - old boy with egfr - negative pulmonary mucoepidermoid carcinoma : a pidermoid carcinoma cells . 
 c sequential response to fgfr3 inhibition with subsequent exceptional response to atezolizumab in a patient with fgfr3 - tacc3 fusionpositive metastatic urothelial carcinoma introduction recent work has pointed to alterations in fibroblast growth factor receptors ( fgfrs ) as potential drivers of colder metastatic urothelial carcinoma ( muc ) microenvironments.1 , 2 fgfrs mediate cell proliferation , migration , differentiation , and survival.3 activating genomic alterations in fgfr3 are frequent in bladder cancer.4 - 6 in the muscle - invasive bladder cancer data set of the cancer genome atlas , fgfr3 alterations were found to be enriched in the luminal papillary subtype.7 clinical benefit from immune checkpoint inhibitors ( icis ) has been a major advance for many cancer types in recent years , including muc , leading to us food and drug administration approval.5 although durable clinical benefit is seen in multiple cancer types , objective response rates in muc are only 15% to 24%.8 mechanisms contributing to ici resistance and low response rates are poorly understood . a phase ii trial comparing the association between the cancer genome atlas mrna subtypes and response to atezolizumab , an antiprogrammed cell death ligand 1 ( pd - l1 ) monoclonal immunoglobulin g1 antibody , showed that the luminal cluster ( enriched for fgfr genomic alteration ) was associated with lower expression levels of cd8 + genes and lower response rates ( 10% ) .1 another study has shown that higher fgfr3 expression and fgfr3 pathway mutations are strongly associated with immune exclusion in bladder cancer.9 this association was also found in patients with bladder tumors harboring fgfr3 - tacc3 fusions.2 these observations suggest that patients harboring fgfr3 alterations are relatively poor candidates for ici therapy . herein , we report a patient with metastatic bladder cancer harboring an fgfr3 - tacc3 fusion and exhibiting an exceptional response to sequential fgfr3 inhibition and antipd - l1 blockade . 
as part of the standard care , the patient received three cycles of adjuvant cisplatin and gemcitabine chemotherapy . the patient demonstrated recurrent disease 5 months later when a computed tomography ( ct ) scan of the chest , abdomen , and pelvis ( cap ) showed two enlarged retroperitoneal lns , amin h . 
genomic profiling by next - generation sequencing ( oncopanel ; brigham and women 's hospital , boston , ma ) was requested on an archival formalin - fixed , paraffin - embedded block derived from the patients primary tumor , which showed an fgfr3 - tacc3 fusion involving intron 17 of fgfr3 and intron 10 of tacc3 . 
 the fgfr3 - tacc3 fusion breakpoints were confirmed by manual review using integrative genomics viewer ( fig 1 ) .10 the patient was then enrolled onto a clinical trial evaluating the combination of pazopanib and everolimus in may 2015 . 
however , the biopsy was insufficient for next - generation tumor sequencing but was sufficient for rnascope ( advanced cell diagnostics , newark , ca ) , a multiplex fluorescent in situ hybridization ( fish ) assay . 
 however , fgfr3 overexpression was seen by the rnascope assay ( fig 2 )  . the patient was enrolled onto a protocol in which he received treatment with both docetaxel and b - 701 ( a monoclonal antibody against fgfr3 ) and was found to have achieved complete remission ( cr ) 5 months later . 
 ( b ) patients lymph node ( after treatment with pazopanib and everolimus ) sample stained with hs - tacc3 - e11 - e16 - c1 ( green ) and hs - fgfr3 - e2e13 - c2 ( red )  . and the patient continues to be in cr 6 months later . discussion in this study , we describe a patient with an fgfr3 - altered muc that showed dramatic response to immunotherapy after receiving sequential treatment with an fgfr tyrosine kinase inhibitor and a monoclonal antibody against fgfr3 . 
next - generation sequencing showed an fgfr3 - tacc3 fusion , msh2 and msh6 homozygous deletions , and a tumor mutational burden of 22.9 mutations / mb ( data supplement )  . 
on the basis of the reported data in the literature , the question arises of what could be driving this unusual response to icis in the setting of an fgfr3 - altered cold tumor . several points of interest regarding this patient deserve to be highlighted and mentioned . 
first , interestingly , rnascope f luorescent in situ hybridization conducted on the ln site showing progression to docetaxel and b - 701 , was negative for the fgfr3 - tacc3 fusion . 
depletion of fgfr3 - tacc3 positive cells possibly molded the tumor microenvironment into a simple overexpressed fgfr tumor microenvironment , which can explain the subsequent cr obtained with the use of the different fgfr3 inhibitor b - 701 . 
however , this sequential response to two fgfr inhibitors is of interest and points to the possibility that sequential use of tyrosine kinase inhibitors and a monoclonal antibody might have different potency and nonoverlapping mechanisms of action for fgfr inhibition . the second intriguing point is the response seen with atezolizumab in this cold tumor . 
 ( mmr - d ) is an established biomarker to icis and has been shown to be associated with clinical benefit in several cancers.14 , 15 to determine whether our patients tumor was mmr - d and whether mmr - d could be driving the response to atezolizumab , we used two methods to assess mmr - d status . 
using a bayesian non - negative matrix factorization algorithm , we determined that our patients tumor has low cosmic 6 activity and thus lacks an mmr - d mutation signature.7 moreover , a recent study by the oncopanel team identified a cutoff value of > 40 mutations / mb or > 5 indels / mb in repeat regions to categorize a patients tumor as mmr - d , which was not the case in our patient ( 22.9 mutations / mb ; 4.8 indels / mb ) .16 in conclusion , on the basis of the present observation , this patients case provides evidence of a sequential response observed with the use of two different fgfr inhibitors with different mechanisms of action . 
however , high tumor mutational burden or the microsatellite instability mutant status can also explain this unexpected excellent response to the ici . one might envision combinatorial trials in patients with fgfr3 alterations involving fgfr3 inhibitors along with icis or studies of the use of these agents in the optimal sequence , which is probably fgfr3 inhibitors followed by a pd - 1 / pd - l1 inhibitor . 
choueiri honoraria : national comprehensive cancer network , uptodate consulting or advisory role : pfizer , bayer , novartis , glaxosmithkline , merck , bristol - myers squibb , genentech , eisai , foundation medicine , cerulean pharma , astrazeneca , peloton therapeutics , exelixis , prometheus laboratories , alligent , ipsen , corvus pharmaceuticals research funding : pfizer ( inst ) , novartis ( inst ) , merck ( inst ) , exelixis ( inst ) , tracon pharma ( inst ) , glaxosmithkline ( inst ) , bristol - myers squibb ( inst ) , astrazeneca ( inst ) , peloton therapeutics ( inst ) , genentech ( inst ) , celldex ( inst ) , agensys ( inst ) , eisai ( inst ) guru p . 
kwiatkowski consulting or advisory role : astrazeneca , genentech joaquim bellmunt honoraria : uptodate research funding : aadi consulting or advisory role : pierre fabre , astellas pharma , pfizer , merck , genentech , novartis , astrazeneca / medimmune , bristol - myers squibb research funding : millennium ( inst ) , sanofi ( inst ) travel , accommodations , expenses : pfizer , msd oncology affiliations amin h . 
rosenberg je , hoffman - censits j , powles t , et al : atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinumbased chemotherapy : a single - arm , multicentre , phase 2 trial . 
massard c , gordon ms , sharma s , et al : safety and efficacy of durvalumab ( medi4736 ) , an anti - programmed cell death ligand - 1 immune checkpoint inhibitor , in patients with advanced urothelial bladder cancer . 
necchi a , joseph rw , loriot y , et al : atezolizumab in platinum - treated locally advanced or metastatic urothelial carcinoma : post - progression outcomes from the phase ii imvigor210 study . 
sharma p , callahan mk , bono p , et al : nivolumab monotherapy in recurrent metastatic urothelial carcinoma ( checkmate 032 ) : a multicentre , open - label , two - stage , multi - arm , phase 1 / 2 trial . 
siefker - radtke ao , necchi a , rosenbaum e , et al : efficacy of programmed death 1 ( pd - 1 ) and programmed death 1 ligand ( pd - l1 ) inhibitors in patients with fgfr alterations : results from a data analysis of an ongoing phase 2 study of erdafitinib ( jnj - 42756493 ) , in patients ( pts ) with advanced urothelial cancer ( uc )  . 
 genetic testing and clinical management practices for variants in non - brca1 / 2 breast ( and breast / ovarian ) cancer susceptibility genes : an international survey by the evidence - based network for the interpretation of germline mutant alleles ( enigma ) clinical working group sarah m . 
domchek ros eeles anna efremidis ( continued ) purpose to describe a snapshot of international genetic testing practices , specifically regarding the use of multigene panels , for hereditary breast / ovarian cancers . 
we conducted a survey through the evidence - based network for the interpretation of germline mutant alleles ( enigma ) consortium , covering questions about 16 non - brca1 / 2 genes . methods data were collected via in - person and paper / electronic surveys . 
additional information was collected via country networks in the united kingdom and in italy . results responses from 61 cancer genetics practices across 20 countries showed that 16 genes were tested by > 50% of the centers , but only six ( palb2 , tp53 , pten , chek2 , atm , and brip1 ) were tested regularly . 
us centers tested the genes most often , whereas united kingdom and italian centers with no direct enigma affiliation at the time of the survey were the least likely to regularly test the most centers tested the 16 genes through multigene panels ; some centers tested tp53 , pten , and other cancer syndromeassociated genes individually . 
gene - specific guidelines for breast and ovarian cancer risk management were limited and differed among countries , especially with regard to starting age and type of imaging and risk - reducing surgery recommendations . conclusion currently , a small number of genes beyond brca1 / 2 are routinely analyzed worldwide , and management guidelines are limited and largely based on expert opinion . 
pedersen maria piane marianna puzzo mark robson maria rossing maria christina sini angela solano jana soukupova gianluca tedaldi manuel teixeira mads thomassen maria grazia tibiletti amanda toland therese trngren erica vaccari liliana varesco ana vega yvonne wallis barbara wappenschmidt jeffrey weitzel amanda b . 
currently , several multigene panels are available that include from < 10 to > 100 known or candidate cancer susceptibility genes , which are tested for diagnostic or research purposes . 
some panels are targeted at diverse cancers ( pan - cancer panels ) , whereas others target specific cancers only ( disease specific panels )  . the ability to run multigene panels at affordable prices has expanded the eligibility criteria and increased the demand for testing.1 - 5 however , the rapid pace at which candidate risk genes are moving from research based to clinical diagnostic testing has its drawbacks . 
the rate of variants of uncertain significance ( vus ) has increased proportionally to the extent of the sequenced genome.5 - 7 moreover , many genes currently included on multigene panels have imprecise cancer risk estimates , and there is no consensus on when to test for a given gene or how to manage a reported ( likely ) pathogenic variant.8 , 9 the aim of this study was to describe a snapshot of the landscape of international genetic testing practices and risk management approaches for bc and boc susceptibility genes beyond brca1 and brca2 . 
a survey was conducted among members of the evidence - based network for the interpretation of germline mutant alleles ( enigma ) , an international consortium focused on determining the clinical significance of variants in brca1 , brca2 , and other ( ascertained or suspected ) bc and boc susceptibility genes , providing expertise to global database and classification initiatives , and exploring optimal avenues of communication of such information at the provider and patient levels . 
additional information was collected via country networks in the united kingdom and in italy , from centers that were not directly involved in enigma research at the time of study initiation . in total , respondents represented cancer genetic experts from 61 centers across 20 countries . 
 to our knowledge , this is the first study to describe international testing practices and risk management guidelines for non - brca1 / 2 genes implicated in bc and boc susceptibility . methods this study was submitted for approval to the ethics committees of the two coordinating sites , the university of chicago and maastricht university . 
a survey about genetic testing practices for nonbrca1 / 2 bc and boc susceptibility genes was developed by enigma clinical working group ( cwg ) leaders during 2016 ( appendix table a1 )  . 
enigma members were invited to complete the survey if they had a clinical genetic testing or diagnostic laboratory affiliation and were involved in ordering , performing , or interpreting dna tests for inherited susceptibility to bc / boc at their center . 
an enigma member is currently defined as a researcher or research group ( consortium ) who is willing to work collaboratively toward classification of variants by contributing data from families and / or conducting statistical analysis or laboratory - based assays within a working group framework . 
there is no requirement for enigma members to state their primary role ( clinician , genetic counselor , laboratory scientist , basic researcher ) , but all members by definition have a research interest in the topic of gene / variant classification . individuals from the same center could work on the survey together or choose a designated representative to complete it , so only one survey per center was counted . 
in brief , an in - person survey of members of the cwg , consisting mainly of laboratory and clinical scientists from academic centers , was conducted during the enigma consortium meeting in limassol , cyprus , in january 2017 . 
 a total of 30 centers from 17 countries participated . a more detailed version of the survey was then distributed by e - mail ( paper / electronic survey ) to the same 30 centers that participated in the in - person survey and to additional enigma affiliated centers worldwide . 
participants were sent a copy of their answers and asked to verify them or to clarify any discrepancies . notably , in italy and in the united kingdom , the paper / electronic version of the survey was also distributed , via country networks , to centers that were not actively involved in enigma research . 
the effort comprised both the enigma - affiliated fondazione istituto di ricovero e cura a carattere scientifico istituto nazionale dei tumori ( milan ) and the santa chiara university hospital ( pisa ) , which were counted among the 38 participating enigma centers , and 14 additional centers , which were not directly affiliated with enigma at the time of the survey ( henceforth referred to as non - enigma ; fig 1 , lower right )  . 
of the 14 italian non - enigma centers , five were dedicated to diagnostic testing only , and nine were dedicated to both diagnostics and research ; moreover , half of them were university affiliated , and half were not . 
 completed the survey for her own enigma affiliated center ( one of the 38 participating enigma centers ) and also coordinated , with the assistance of y.w. , the distribution of the survey through surveymonkey via the association for clinical genetic science mailing list to cancer genetic leads from diagnostic laboratories providing genetic testing for the publicly funded national health service ( nhs )  . 
the recruitment flowchart and the global distribution of participants are illustrated in figure 1 . clinical utility to get a preliminary idea of the participants opinions about the clinical utility of the 16 genes on which the survey focused , the cwg members present at the 2017 enigma meeting in cyprus were asked to answer the following questions relating to each of them : should every patient with bc / oc who qualifies for ( brca1 / 2 ) genetic testing ( by criteria that we recognize may differ by country / center ) be tested for the gene ? and do you agree that the cancer risk associated with ( pathogenic variants in ) the gene is high enough to inform clinical management ? all participants ( n = 23 at this specific session ) stated that they would test every qualifying patient with bc ( as defined in the question ) for palb2 and every qualifying patient with oc ( as defined ) for brip1 , rad51c , and rad51d . 
results for the other nine genes were variable ( appendix fig a1a )  . with regard to clinical management , all participants agreed that palb2 , tp53 , cdh1 , pten , and stk11 along with brip1 , rad51c , and rad51d were associated with high enough ( bc or oc ) risk to alter clinical management . 
please note that 95% cis for this figure and for all the following figures are provided in appendix ( tables a2 - a10 )  . testing practices participants were also asked ( via in - person and / or paper / electronic surveys ) if and how frequently they tested each gene , the method ( single gene v gene panel ) and purpose of testing ( clinical v research ) , and the practices of reporting ( likely ) pathogenic variants and vus to patients . 
of centers that responded to the question varied by gene ( range , 29 to 38 centers ) .regularly was defined as ordered for > 50% of eligible patients ( ie , those who qualified for genetic testing , by criteria that we recognize may differ by center / country )  . 
even though each gene was tested by > 50% of the centers ( range , 52% to 100% ) , only palb2 , tp53 , pten , chek2 , atm , and brip1 were tested regularly by > 50% of centers . testing in a research setting in addition to the clinical setting was common for enigma centers ( appendix fig a2 )  . 
the genes that were most frequently tested ( ie , tested by at least > 30% of centers ) for research purposes only were : nbn , bard1 , rad50 , and mre11a . 
 single - gene testing was performed by a number of centers , varying from one to 21 , for : tp53 , pten , cdh1 , stk11 , palb2 , chek2 , nf1 , atm , men1 , and nbn ( in decreasing order of frequency ) , often based on a specific phenotype ( eg , pten hamartoma syndrome or neurofibromatosis type 1 ) , or these genes were tested as a reflex only when brca1 / 2 testing was noninformative . 
seven centers from four countries ( belgium , brazil , the netherlands , and spain ) testing chek2 only tested for the 1100delc variant . regarding the types of gene panels used , us respondents typically ordered broad cancer panels from commercial laboratories , although the specific panels varied depending on patient preferences , insurance considerations , and clinical scenarios . 
separately , the nine united kingdom nhs laboratories were asked , if you currently only report brca genes but might report broader panels in the future , what issues are major barriers / problems to overcome ? responses were chosen from a menu of nine options plus other , and the four main reasons selected ( by half or more of respondents ) were no request by the oncologists ( of note , nhs oncologists can ask directly for brca1 and brca2 testing but not for multigene panels ) , lengthy and laborious process of variant interpretation , lack of standardization of reporting , and lack of demand for testing . reporting practices and cascade testing . 
for genes analyzed through clinical testing , > 90% of enigma centers reported ( likely ) pathogenic variants to patients ( for chek2 and nbn , the percentages were slightly lower , at 88% and 71% , respectively ; fig 4 )  . 
some centers reported these variants only if the patient met criteria for the associated syndrome ( eg , hereditary diffuse gastric cancer for cdh1 , neurofibromatosis type 1 for nf1 )  . 
almost all centers ( 67% to 81% for nbn , rad50 , mre11a , and bard1 and > 90% for the other genes ) offered cascade testing to family members if a ( likely ) pathogenic variant was identified ( data not shown )  . 
notably , participants from the netherlands reported that they only tested first - degree relatives for chek2 1100delc variant when the estimated risk based on family history was lower than the risk conferred by having the variant , so testing for the variant had clinical utility because it would change surveillance recommendations.11 a high percentage ( 50% to 82% ) of enigma centers reported vus to patients ( fig 4 )  . 
 variant classification systems all respondents reported using the international agency for research on cancer five - tier classification system , 12 and many also used american college of medical genetics and genomics13 classification criteria . 
 the three methods are not mutually exclusive ; notably , the center in kuwait performs whole - genome sequencing for all cases , which is not represented in the figure . 
enigma , evidence - based network for the interpretation of germline mutant alleles . clinical management practices and guidelines most enigma centers ( 80% ) had risk management guidelines for a majority of non - brca1 / 2 genes considered reportable to patients ( fig 5 )  . 
 exceptions were bard1 , rad50 , and mre11a , for which 30% of centers had guidelines . although most enigma centers reported having some type of management guidelines for all genes except bard1 , rad50 , and mre11a , after review , only 10 of 20 countries had national guidelines for ( some of ) these genes ( table 1 )  . 
 furthermore , in some countries ( denmark and germany ) , the national guidelines were not gene specific ( ie , they were broken down by highand moderate - risk categories rather than by specific gene )  . 
ten countries had national guidelines for high - risk cancer syndromeassociated genes such as tp53 , cdh1 , and pten ( with the exception of belgium not having guidelines for cdh1 )  . 
the primary differences between countries were the starting age and type of diagnostic imaging ( mammography v magnetic resonance imaging [ mri ] v sonography ) and the policy on risk - reducing mastectomy . 
for instance , there was no consensus on the age to begin mammograms / mri for carriers of pathogenic variants in nf1 , men1 , palb2 ( age 25 v 30 years ) , or tp53 ( age 20 v 25 years )  . 
 the united kingdom guidelines differed from all others in that breast mri was not the standard imaging technique for carriers of pathogenic variants in other gene carriers ( except for tp53 )  . 
guidelines for risk - reducing mastectomy in carriers of palb2 pathogenic variants ranged among accepted ( n = 1 ) , consider depending on personal / family history ( n = 5 ) , and not enough evidence to recommend ( n = 1 )  . 
for pten and cdh1 , the guidelines that commented on preventive surgery ( four of the seven and five of the eight national guidelines , respectively ) mentioned risk - reducing mastectomy as a possible option . 
 subanalyses : enigma - us versus enigma - other centers and versus non - enigma centers discussion responses from the seven enigma centers in the united states ( enigma - us ) were compared with those of the other 31 enigma centers ( enigma - other )  . 
in addition , responses from 14 non - enigma centers in italy and nine non - enigma laboratories in the united kingdom were compared with those from 38 enigma centers across all countries . results of these comparisons are summarized in appendix figs a3 and a4 . 
a much smaller proportion of non - enigma centers from italy and the united kingdom tested each gene compared with enigma - affiliated centers ( appendix fig a3 )  . management guidelines were more likely to be available in the us - based enigma centers compared with the other enigma centers for all genes except bard1 , rad50 , mre11a , and men1 . 
of centers that responded varied by gene ( range , 12 to 36 centers responding about reporting pathogenic variants ; range , four to 20 centers responding about reporting vus )  . 
our global survey demonstrated that only a few genes are routinely analyzed beyond brca1 / 2 ; most centers clinically test them through multigene panels and report ( likely ) pathogenic variants ( and vus , to a slightly lesser extent ) to patients ; and gene - specific guidelines for bc and oc risk management are limited and differ between countries , especially in regard to starting age and type of imaging and risk - reducing surgery recommendations . with falling costs of sequencing and more genes being identified that are associated with increased bc and boc risk , multigene ( panel ) testing is becoming the northe results of our survey confirm this trend , showing that genes that are commonly offered on commercial panels were tested by > 50% of the surveyed centers . nevertheless , the value of multigene panel testing continues to be debated in the context of three main areas : limited additional yield of pathogenic variants in genes other than brca1 / 2 coupled with significantly increased interpretation workload , reliability of penetrance estimates for moderateor uncertain risk genes ( clinical validity ) , and evidence for informing management recommendations to improve patient outcomes ( clinical utility ) .9 our international survey demonstrates that the use of panel testing varies widely among countries . 
moreover , differences were observed when comparing enigma - affiliated centers with non - enigma italian and united kingdom centers ( with the latter testing nonbrca1 / 2 genes less than one third of the time )  . 
if management guidelines were available , centers were asked to specify the source of such guidelines ( local , national , or international , such as national comprehensive cancer network or national institute for health and care excellence )  . the genes included on panels was the most common concern raised by the participating centers . 
easton et al8 asserted that a genomic test should not be offered until its clinical validity is established8 ( p2 ) ; however , the utility of a gene needs to be continuously reconsidered as more data become available , and this can only be done by analyzing results from large cohorts of individuals who have been tested . 
concerns about the rates of vus were frequently expressed by the study participants , but just as variant rates have significantly decreased over the years for brca1 / 2 as a result of concerted classification efforts , the same trend will likely occur for other susceptibility genes , arguably at a faster pace as ( and provided that ) more laboratories worldwide contribute their testing data to population and peer - reviewed databases.5 , 28 , 29 despite the establishment of such databases , survey participants felt that robust , constantly updated international databases and global data sharing are still lacking . 
this is a worthy goal , but expert judgment in variant classification methods is still required , because fully automated approaches to variant classification that apply guidelines are not ready for clinical practice.6 at a basic level , some centers reported validation of the testing method as a barrier . 
therefore , it is important to recognize the technologic barriers in certain countries , although the transition to massively parallel sequencing is ultimately expected to increase throughput and optimize diagnosis without significantly elevating costs.30 there were also nonmedical barriers to implementing routine testing of many of these surveyed genes . 
insurance can be a major barrier in the united states , where , for example , medicare ( a us federal health insurance program for people who are age 65 years and for certain younger people with disabilities ) will only cover testing for individuals with a bc or oc diagnosis , and many insurers will not cover multigene panel testing if the patient has already had prior genetic testing . 
in many other countries , particularly those with national ( ie , universal ) health care , testing is approved on a gene - by - gene basis or as a package if research - derived evidence is considered robust enough to change clinical management . in terms of risk magnitudes , palb2 and tp53 are the only bc genes , in addition to brca1 / 2 , that consistently fall into the high - risk category across studies ( ie , confer levels of risk greater than four times that in the general population ) 8 ; the remainder have conflicting evidence regarding the risk category into which they fit.8 , 9 , 31 - 33 our survey confirmed that enigma centers test palb2 and tp53 relatively frequently and regard them as clinically actionable genes . 
 these two genes were tested much less consistently by non - enigma centers , evidencing the lack of consensus , even for genes that are generally regarded as high risk . 
 brip1 , rad51c , and rad51d are ever more accepted as oc but not bc risk predisposition genes ( two to five times the risk compared with the general population ) .15 , 32 notably , many respondents agreed that every patient with oc should be tested for these three genes ( in addition to brca1 / 2 )  . 
although there is currently no indication that oc treatment for a carrier of a pathogenic variant in one of these three genes would differ from that for a noncarrier , carriers may benefit from rrso at menopause . the uncertainties and inconsistencies regarding risk and testing practices are magnified when it comes to syndromic cancer genes like pten , cdh1 , stk11 , nf1 , nbn , and men1 , as well as genes conferring an uncertain risk such as bard1 , rad50 , and mre11a . 
although there is significant evidence for elevated bc risk and lobular bc risk in carriers of pathogenic variants in pten and in cdh1 , respectively , 34 - 36 it is likely that these bc risks ( and those from the other syndromic genes ) are overestimated and therefore unreliable , because they were derived from patients whose histories were consistent with these rare syndromes rather than from unselected patients.8 more robust and replicable penetrance estimates from large - cohort and population studies are certainly needed to further define risks . 
however , on the basis of both the evidence available from the literature and the results of our survey , which incorporate an international clinical perspective , the 16 genes can be grouped into five categories : high bc risk : palb2 , tp53 , pten , and cdh1 ; moderate bc risk : atm and chek2 ; bc risk of unclear magnitude ( but established risk for other cancer types ) : stk11 , nf1 , nbn , and men1 ; moderate oc risk : brip1 , rad51c , and rad51d ; and insufficient evidence for bc or oc risk : bard1 , rad50 , and mre11a . the clinical utility of multigene panel testing is assessed based on the improved outcomes of those managed by evidence - based surveillance or prevention approaches . 
a framework for management of moderate - risk bc / boc genes has been extensively reviewed by tung et al9 and includes a comparison of surveillance guidelines among the united states , united kingdom , and germany . 
there are limited national guidelines available even for genes such as palb2 , brip1 , rad51c , and rad51d , which most participants felt should always be tested because they importantly , are clinically actionable . 
this explains why the guidelines often differ in important aspects such as indication for risk - reducing surgery and type of diagnostic imaging recommendations . our study was initiated to provide a snapshot of enigma clinical practice for non - brca1 / 2 genes . 
because panel testing is currently being implemented in large regions of the world like asia , africa , and south america , similar surveys will need to be redistributed once more countries have established testing protocols . 
even at the time of the survey , testing protocols and surveillance recommendations were in flux in some countries , and broader gene panels were expected to be offered within a short time . 
 we acknowledge that our sampling of nonenigma centers was limited , and we aim to survey a more diverse collection of us , canadian , and other worldwide regional or community practices in future studies . massively parallel sequencing represents a transformational technology that we must learn to apply appropriately in health care . 
although the number of genes , other than brca1 / 2 , associated with bc / boc risk is growing , only a small subset of them have clinical utility at the moment . 
the goal of this study was to highlight the differences across countries and to determine what additional information and infrastructure are still needed to move toward more uniform testing practices and management guidelines internationally . our collected evidence suggests that the clinical usefulness of multigene panel testing for bc / boc predisposition can be improved by a better definition of the cancer risks associated with genetic variation in cancer susceptibility genes and by the availability of evidence - based management guidelines . 
to this end , it is key that clinicians share clinical and genetic data , through enigma and / or other international consortia focused on the clarification of the bc and oc risk associated with genetic variation , and that tested individuals are encouraged to participate in initiatives that collate genetic testing data and in long - term follow - up studies that evaluate intervention strategies . 
palmero , maria piane , marianna puzzo , maria christina sini , angela solano , manuel teixeira , mads thomassen , amanda toland , therese trngren , liliana varesco , jeffrey weitzel , encarna b . 
pedersen , maria piane , marianna puzzo , mark robson , maria rossing , maria christina sini , angela solano , jana soukupova , gianluca tedaldi , manuel teixeira , mads thomassen , maria grazia tibiletti , amanda toland , therese trngren , erica vaccari , liliana varesco , ana vega , barbara wappenschmidt , jeffrey weitzel , arcangela de nicolo , encarna b . 
 rien blok no relationship to disclose akira hirasawa research funding : astrazeneca maria adelaide caligo travel , accommodations , expenses : technogenetics mariarosaria calvello no relationship to disclose gabriele lorenzo capone no relationship to disclose pietro cavalli no relationship to disclose t.l. 
couch consulting or advisory role : astrazeneca research funding : grail other relationship : ambry genetics miguel de la hoya no relationship to disclose simona de toffol no relationship to disclose orland diez no relationship to disclose susan m . 
domchek honoraria : astrazeneca , clovis oncology , bristol - myers squibb research funding : astrazeneca ( inst ) , clovis oncology ( inst ) , pharmamar ( inst ) ros eeles honoraria : janssen - cilag speakers bureau : janssen - cilag anna efremidis no relationship to disclose florentia fostira no relationship to disclose david goldgar no relationship to disclose andreas hadjisavvas no relationship to disclose thomas v.o. 
hansen no relationship to disclose claude houdayer no relationship to disclose petra kleiblova no relationship to disclose sophie krieger no relationship to disclose conxi lzaro no relationship to disclose maria loizidou no relationship to disclose siranoush manoukian no relationship to disclose arjen r . 
 mark robson honoraria : astrazeneca erica vaccari consulting or advisory role : color genomics consulting or advisory role : mckesson , astrazeneca research funding : astrazeneca ( inst ) , myriad genetics ( inst ) , invitae ( inst ) , pfizer ( inst ) travel , accommodations , expenses : astrazeneca liliana varesco consulting or advisory role : pfizer maria rossing no relationship to disclose maria christina sini no relationship to disclose angela solano no relationship to disclose jana soukupova no relationship to disclose gianluca tedaldi no relationship to disclose manuel teixeira no relationship to disclose mads thomassen no relationship to disclose maria grazia tibiletti no relationship to disclose amanda toland no relationship to disclose therese trngren honoraria : pfizer , astrazeneca ana vega no relationship to disclose yvonne wallis no relationship to disclose barbara wappenschmidt no relationship to disclose jeffrey weitzel no relationship to disclose amanda b . 
domchek , university of pennsylvania , philadelphia , pa ; anna efremidis , athens medical center ; florentia fostira , national centre for scientific research demokritos , athens , greece ; david goldgar , university of utah school of medicine , salt lake city , ut ; andreas hadjisavvas and maria loizidou , cyprus institute of neurology and genetics , nicosia , cyprus ; thomas v.o. 
pedersen , aalborg university hospital , aalborg , denmark ; akira hirasawa , keio university school of medicine , tokyo , japan ; claude houdayer , universit paris descartes and unicancer genetic group , paris ; sophie krieger , normandy university , cancer center f . 
palmero , barretos cancer hospital , barretos , so paulo , brazil ; mark robson , memorial sloan kettering cancer center , new york , ny ; angela solano , university of buenos aires , buenos aires , argentina ; manuel teixeira , instituto portugus de oncologia do porto francisco gentil , porto , portugal ; amanda toland , ohio state university , columbus , oh ; therese trngren , lund university , lund , sweden ; erica vaccari , dana - farber cancer institute , boston , ma ; barbara wappenschmidt , university hospital cologne and german consortium of hereditary breast and ovarian cancer , cologne , germany ; and jeffrey weitzel , city of hope , duarte , ca . support supported by grant no . 
couch fj , hart sn , sharma p , et al : inherited mutations in 17 breast cancer susceptibility genes among a large triple - negative breast cancer cohort unselected for family history of breast cancer . 
laduca h , stuenkel aj , dolinsky js , et al : utilization of multigene panels in hereditary cancer predisposition testing : analysis of more than 2 , 000 patients . 
maxwell kn , hart sn , vijai j , et al : evaluation of acmg - guideline - based variant classification of cancer susceptibility and non - cancer - associated genes in families affected by breast cancer . 
domchek sm , bradbury a , garber je , et al : multiplex genetic testing for cancer susceptibility : out on the high wire without a net ? j clin oncol 31 : 1267 - 1270 , 2013 8 . 
richards s , aziz n , bale s , et al : acmg laboratory quality assurance committee : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
janatov m , boreck m , soukupov j , et al : palb2 as another candidate gene for genetic testing in patients with hereditary breast cancer in czech republic [ in czech ]  . 
robays j , stordeur s , hulstaert f , et al : oncogenetic testing and follow - up for women with hereditary breast / ovarian cancer , li fraumeni syndrome and cowden syndrome . 
robays j , stordeur s , hulstaert f , et al : oncogenetic testing , diagnosis and follow - up in birt - hoggdub syndrome , familial atypical multiple mole melanoma syndrome and neurofibromatosis 1 and 2 . 
national institute for health and care excellence : familial breast cancer : classification , care and managing breast cancer and related risks in people with a family history of breast cancer . 
lincoln se , kobayashi y , anderson mj , et al : a systematic comparison of traditional and multigene panel testing for hereditary breast and ovarian cancer genes in more than 1000 patients . 
castra l , krieger s , rousselin a , et al : next - generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes . 
pharoah pd , guilford p , caldas c : incidence of gastric cancer and breast cancer in cdh1 ( e - cadherin ) mutation carriers from hereditary diffuse gastric cancer families . 
questions included in the surveys ( by mode of distribution ) ( continued ) question testing methods and setting which method is used to test for gene x ? in - person survey only which genes do you agree should be tested for every bc or oc patient eligible for genetic testing ? describe the gene panels currently used ( if any ) and if they are used in the diagnostic or research setting if you are not currently using gene panels but may in the future , what do you think is required before starting to use them ? ii variant classification classification system ii - 1 ii - 2 reporting and cascade testing of variants clinical i . 
questions included in the surveys ( by mode of distribution ) ( continued ) in - person survey only question ii - 3 variant interpretation in - person and paper surveys paper survey only surveymonkey * who takes responsibility for interpreting the clinical significance of the identified variants ? for cancer susceptibility genes : who takes responsibility for variant interpretation and reporting ? clinical scientist clinical geneticist genetic counselor oncologist ( medical / surgical ) other ( specify ) who takes responsibility for discussing the clinical significance / utility of an identified variant ? ( same choices as above ) are there clinical guidelines for managing patients who carry a pathogenic or likely pathogenic variant in a bc susceptibility gene ? iii risk management guidelines for which genes do you agree that the cancer - associated risks are high enough to alter clinical practice / management ? are management guidelines available at your center for patients with ( likely ) pathogenic variants in these genes ? yes , national guidelines yes , local guidelines or local adaptations of national guidelines no , guidelines are not currently available if clinical management guidelines are available at your center for the specified genes , please provide digital copy , reference , or web site link note . 
questions i - 4 and iii of the in - person survey were asked at a different time compared with the remainder of the survey ; therefore , answers were collected from only 23 centers . 
 these two questions were asked at a different time ( during evidence - based network for the interpretation of germline mutant alleles meeting in cyprus in january 2017 compared with survey questionnaire )  . 
the centers that tested each gene through research only were compared with the proportion of centers that tested the gene only clinically and proportion of those that tested the gene for both clinical and research purposes . 
genes tested regularly by evidence based network for the interpretation of germline mutant alleles ( enigma ) centers in the united states ( enigma - us ) versus other enigma centers ( enigma - other ) versus italian and united kingdom non - enigma centers . 
 of centers testing given gene regularly ( defined as ordered for > 50% of patients eligible for genetic testing , by criteria that we recognize may differ by center / country ) is shown above each bar . 
of centers that answered this question was four to seven , depending on the gene ; of the 31 enigma - other centers , a range of 22 to 30 centers answered this question . 
most preceding studies have retrospectively sought to identify high - risk criteria among patients who are at low risk for nodal metastasis.2 such studies are inherently awed , because patients were offered sln biopsy ( slnb ) only if the surgeon believed the pretest probability of sln positivity was sufciently high to justify the procedure . 
bellomo et al1 approached this problem differently by including only patients in whom slnb would be considered on the basis of mayo clinic clinicopathologic ( cp ) criteria , and then they identied those at sufciently low risk to forgo slnb . 
a few previous studies have also identied lowrisk subsets among such patients , but the criteria for selecting those patients have not been widely adopted in clinical practice.3 , 4 perhaps the most signicant contribution of the bellomo et al1 study to melanoma risk prediction is the incorporation of a gene - expression prole ( gep ) not as a stand - alone risk predictor , but a continuous risk model combined with cp risk factors ( cp - gep )  . 
this approach permits better transparency of the additive predictive value of the gep , use of continuous variables , and individualized risk prediction ( as opposed to classifying patients into discrete groups )  . 
this facilitates a discussion of the clinically relevant risk threshold for the individual patient.5 development of all future predictive markers should adhere to this standard . future studies must more clearly demonstrate clinical utility . 
a proportion of sln - positive patients would therapy.6 , 7 be eligible to receive effective adjuvant furthermore , by omitting slnb , patients would not benet from the improved recurrence - free survival associated with the procedure or the potential diseasespecic survival advantage seen in the subset of patients with a positive slnb.8 finally , sln positivity as an end point is itself inaccurate . 
the mslt - 1 ( clinicaltrials.gov identier : nct00275496 ) trial demonstrated a 27% false - negative rate for slnb in patients with sufcient long - term follow - up , and recurrences are frequently delayed in thin melanoma.8 any statements about the performance characteristics of slnb and slnb rrs in patients with thin melanomas and inadequate follow - up should be made with caution . we explored the potential clinical utility of the models by using decision curve analysis ( fig 1 ) and found the cp - gep model only incrementally superior to the cp model when offering slnb above a threshold of 5% . net benet for the cp - gep model increases with increasing risk threshold . 
this suggests that a clinician with an anxious , t patient ( ie , a low threshold , 5%10% , for slnb ) should perform the procedure regardless of the models . 
however , in a patient with comorbidities in whom a threshold of 20% is more appropriate , cp - gep would maximize the relative probability of identifying positive nodes while limiting unnecessary procedures . clearly , the clinical validity of combining gep with an effective cp - based risk score requires further study . as the authors appropriately acknowledge , external validation will be critical . 
for example , a net benet of 0.1 ( 10% ) for a model indicates that if it is used to guide sentinel lymph node ( sln ) biopsy , it is equivalent to performing an additional 10 positive sln biopsies per 100 patients treated . threshold probability refers to the level of predictive certainty at which a treatment would be chosen by a patient and physician . 
in this case , the threshold probability could be considered the sln positivity rate at which a surgeon would perform the procedure for a given patient . this decision reects the relative value a surgeon and / or patient ascribes to a positive versus a negative sln biopsy . 
a guide to interpreting decision curve analysis is available in vickers et al.10 sln biopsy for none ( solid black ) , sln biopsy for all ( solid gray ) , sln biopsy based on cp model ( dashed red ) , sln biopsy based on gep model ( dashed black ) , sln biopsy based on cp - gep model ( dashed green )  . 
reprinted with permission from bellomo et al.1 we laud the efforts of bellomo et al1 to reduce the frequency of slnb , a costly , imperfect , and occasionally morbid procedure . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . no potential conicts of interest were reported . references bellomo d , arias - mejias sm , ramana c , et al : model combining tumor molecular and clinicopathologic risk factors predicts sentinel lymph node metastasis in primary cutaneous melanoma . 
j clin oncol 31 : 4387 - 4393 , 2013 bartlett ek , peters mg , blair a , et al : identication of patients with intermediate thickness melanoma at low risk for sentinel lymph node positivity . 
ann surg oncol 23 : 250 - 256 , 2016 sinnamon aj , neuwirth mg , yalamanchi p , et al : association between patient age and lymph node positivity in thin melanoma . 
mulder p , dwarkasing jt , hollestein lm , et al : validation of a clinicopathologic and gene expression prole ( cp - gep ) model for sentinel lymph node metastasis in primary cutaneous melanoma . 
 clinical activity of pembrolizumab in a patient with metastatic triplenegative breast cancer without tumor programmed death - ligand 1 expression : a case report and correlative biomarker analysis sajjad bhatti jaimie heldstab carolyn lehn ossama tawfik ryan m . 
godwin priyanka sharma author affiliations appear at the end of this article . supported by the university of kansas research career award and the university of kansas cancer centers cancer center support grant ( p30 ca168524 ) biospecimen repository core facility . clinical trial information : nct02302742 . corresponding author : priyanka sharma , md , university of kansas medical center , 2330 shawnee mission parkway , suite 210 , westwood , ks 66205 ; e - mail : psharma2@ kumc.edu. introduction triple - negative breast cancer ( tnbc ) accounts for 10% to 20% of all breast cancers and is associated with poor prognosis.1 compared with hormone - positive breast cancer , tnbc is characterized by a higher proportion of visceral relapse and a short survival after development of metastatic disease , highlighting the pressing need for identification of more effective systemic therapies for this subgroup.2 , 3 cancers use multiple mechanisms to evade the immune response . 
pd - 1 and its ligands , programmed death - ligands 1 and 2 ( pd - l1 and pd - l2 ) , play a major role in maintenance of immune evasion.4 , 5 pd - l1 is aberrantly expressed in many malignancies , including tnbc.6 , 7 binding of pd - 1 to its ligands results in the downregulation of lymphocyte activation , and inhibition of the interaction between pd - 1 and its ligands promotes immune responses . 
in support of preclinical findings , there is emerging evidence of the clinical efficacy of agents targeting pd - 1 / pd - l1 in tnbc.8 - 10 pembrolizumab is a humanized monoclonal antibody that binds to pd - 1 . 
a recent study demonstrated that in pd - l1expressing advanced tnbc , pembrolizumab produced response rates of 18%.8 several studies are investigating pembrolizumab in earlyand advanced - stage tnbc.11 - 14 here we describe the case of a 48 - year - old woman with metastatic tnbc who , after exhausting all effective chemotherapeutic options , demonstrated excellent clinical and radiologic response to pembrolizumab ( tumor pd - l1 negative )  . 
six months after finishing adjuvant radiation , she developed recurrence in left axillary lymph nodes and received carboplatin plus gemcitabine plus iniparib in a clinical trial . after five cycles of treatment she was switched to ixabepilone ( because of an allergic reaction to carboplatin )  . 
1 year and then developed metastatic disease in the left cervical lymph nodes , which was rendered into complete radiologic response with cisplatin . after a 4 - month break from cisplatin , there was progression of left cervical adenopathy , which was treated with radiation . 
 neoadjuvant docetaxel + carboplatin + erlotinib ( clinical trial ) adjuvant adriamycin + cytoxan followed by radiation completed aug 2010 carboplatin + gemcitabine + iniparib ( clinical trial ) with clinical response ; treatment stopped for toxicities progression ixebepilone cisplatin 90 mg / m2 x five cycles with cr left supraclavicular radiation cisplatin ; stopped for toxicity progression on capecitabine + doxil pembrolizumab initiated eribulin sep 2009 feb 2010 jan 2011 aug 2011 nov 2012 jul 2013 jul 2014 may 2015 mar 2016 diagnosed with stage iii ( ct3n3 ) tnbc left lumpectomy and alnd ypt2n3a metastatic disease , left axilla left alnd ; all lymph nodes negative for cancer metastatic disease , left supraclavicular lymph nodes re - appearance of left supraclavicular and cervical adenopathy pulmonary metastasis left cervical ln biopsy ; grade 3 metastatic tnbc cisplatin with good clinical response ( cisplatin was discontinued because of nephrotoxicity )  . 
she then received various sequential single - agent chemotherapies with temporary disease stabilization as best response and eventually developed progressive left cervical and occipital adenopathy . she continued to maintain an excellent performance status and did not qualify for any available clinical trials . 
pd - l immunohistochemical testing ( details provided in data supplement ) of the cervical lymph nodes ( specimen obtained in may 2015 ) showed no ( 0% ) pd - l1 expression ( fig 2 )  . given the lack of other available treatment options and the presence of other tumor biomarkers , the decision was made to offer treatment with pembrolizumab ( 2 mg / kg ) , which was started in march 2016 . 
hypermethylation of the brca1 promoter has been proposed as one of the mechanisms for functionally inactivating the brca1 gene.16 - 18 brca1 mutationassociated cancers are known to demonstrate a high degree of genomic instability , a characteristic that is also shared by tumors with brca1 pm.19 genetically complex tumors exhibit a high nonsynchronous fig 1 . 
in 2011 , lehmann et al22 used a 2188 gene expression signature to stratify ( tnbc ) into six subtypes : basal - like 1 ( bl1 ) , basal - like 2 , mesenchymal , mesenchymal stem celllike , immunomodulatory ( im ) , and luminal androgen receptorlike ( lar )  . 
 of 10% to 19% in patients with pd - l1positive tnbc ( although different pd - l1 antibodies and / or cut points and laboratories were used to define pd - l1 positivity in trials ) .8 , 9 , 10 , 24a on the basis of encouraging phase i data , larger studies are evaluating pd - 1 / pd - l1 antibodies in unselected tnbc . 
for efficient evaluation of this new class of agents , there is a critical need to develop biomarkers of response that can be used to successfully select patients who are most likely to benefit . we report a case study of a patient with heavily pretreated ( six prior lines of chemotherapy ) metastatic tnbc who demonstrated clinical response to pembrolizumab . 
the metastatic tumor did not demonstrate pd - l1 expression ; however , other biomarkers were detected , which provide some insight into the clinical response noted in this case . tnbc is a molecularly heterogeneous entity for which additional subclassifications have been proposed . 
using the insight tnbctype assay , our patients tumor was classified into the bl1 subclass and also demonstrated positive im status . many of the genes involved in this im signature are associated with natural killer cells and activated t cells , implying an active immune response to the tumor ( unpublished data )  . 
in support of this hypothesis , a recent study of atezolizumab plus nab - paclitaxel in metastatic tnbc demonstrated that responders had higher baseline tils compared with nonresponders.25 a recent study also demonstrated that the correlation between tils and pd - l1 expression may not be linear in breast cancer.26 expression of pd - l1 on tumor cells is of interest as a biomarker of response to pd - 1 / pd - l1 inhibitors . 
however , there are several variables that affect the analytical validity of pd - l1 testing ; to date , there is no single validated antibody for this testing . pd - l1 is expressed in approximately 20% to 58% of tnbc , and it is not clear whether pd - l1 expression is predictive of response to immune therapy in tnbc.7 , 8 , 27 our patients tumor demonstrated lack of pd - l1 staining . 
given the concerns surrounding the analytic and clinical validity of pd - l1 testing , it is possible that this was a falsenegative test and that the tumor may show a different outcome if tested using a different assay and antibody . pd - l1 expression may be associated with clinical response to antipd - 1 / pd - l1 antibodies in patients with malignant melanoma or nonsquamous nsclc.28 however , a small proportion of patients with pd - l1negative cancers also benefit from antipd - 1 therapy.10 , 27 - 29 this indicates that using pd - l1 alone as a marker of selection and response to these agents might exclude some patients who could potentially benefit . translational studies to identify reliable biomarkers that would aid patient selection for immune therapy and further improve the riskbenefit ratio for these drugs are needed . 
 sajjad bhatti no relationship to disclose jaimie heldstab consulting or advisory role : novartis travel , accommodations , expenses : novartis carolyn lehn no relationship to disclose ossama tawfik stock and other ownership interests : radpath solutions consulting or advisory role : radpath solutions patents , royalties , other intellectual property : trays for tissue biopsy samples ; methods of making and methods of using thereof . 
hout employment : insight genetics leadership : insight genetics stock and other ownership interests : insight genetics patents , royalties , other intellectual property : i hold patents with insight genetics , but do not receive royalties . travel , accommodations , expenses : insight genetics rob s . 
seitz employment : insight genetics , bethesda group leadership : insight genetics , bethesda group stock and other ownership interests : insight genetics , bethesda group consulting or advisory role : foundation medicine , agendia , celsee diagnostics daniel b . 
godwin no relationship to disclose priyanka sharma consulting or advisory role : abbvie , myriad genetics research funding : novartis ( inst ) , celgene ( inst ) , bristolmyers squibb ( inst ) , cosmo biosciences ( inst ) travel , accommodations , expenses : abbvie , celgene , myriad genetics affiliations sajjad bhatti , jaimie heldstab , carolyn lehn , ossama tawfik , ryan m . 
kohler ba , sherman rl , howlader n , et al : annual report to the nation on the status of cancer , 1975 - 2011 , featuring incidence of breast cancer subtypes by race / ethnicity , poverty , and state . 
ghebeh h , mohammed s , al - omair a , et al : the b7 - h1 ( pd - l1 ) t lymphocyte - inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma : correlation with important high - risk prognostic factors . neoplasia 8 : 190 - 198 , 2006 8 . 
emens la , braiteh fs , cassier p , et al : inhibition of pd - l1 by mpdl3280a leads to clinical activity in patients with metastatic triple - negative breast cancer . 
dirix l , takacs i , nikolinakos p : avelumab ( msb0010718c ) , an anti - pd - l1 antibody , in patients with locally advanced or metastatic breast cancer : a phase 1b javelin solid tumor trial . 
loi s , sirtaine n , piette f , et al : prognostic and predictive value of tumor - infiltrating lymphocytes in a phase iii randomized adjuvant breast cancer trial in node - positive breast cancer comparing the addition of docetaxel to doxorubicin with doxorubicin - based chemotherapy : big 02 - 98 . 
wei m , grushko ta , dignam j , et al : brca1 promoter methylation in sporadic breast cancer is associated with reduced brca1 copy number and chromosome 17 aneusomy . 
adams s , diamond jr , hamilton ep , et al : phase ib trial of atezolizumab in combination with nab - paclitaxel in patients with metastatic triple - negative breast cancer ( mtnbc )  . 
tung n , garber je , hacker mr , et al : prevalence and predictors of androgen receptor and programmed death - ligand 1 in brca1 - associated and sporadic triple - negative breast cancer . 
gandini s , massi d , mandal`a m : pd - l1 expression in cancer patients receiving anti pd - 1 / pd - l1 antibodies : a systematic review and meta - analysis . 
spigel dr , reckamp kl , rizvi na , et al : a phase iii study ( checkmate 017 ) of nivolumab ( nivo ; anti - programmed death - 1 [ pd - 1 ] ) vs docetaxel ( doc ) in previously treated advanced or metastatic squamous ( sq ) cell non - small cell lung cancer ( nsclc )  . 
brock , mbbs , phd1 , 2 purpose the 21 - gene recurrence score ( rs ) is used to identify patients with hormone receptorpositive earlystage breast cancer who may benet from the addition of chemotherapy to endocrine therapy . 
 iorgulescu et al context key objective although grade 3 is considered a poor prognostic factor in estrogen receptorpositive , human epidermal growth factor receptor 2negative breast cancer , the predictive benet of multigene recurrence score ( rs ) testing in this subgroup remains unclear . knowledge generated our analysis of the national cancer database that comprises more than 70% of all newly diagnosed cancers in the united states reveals that rs results in grade 3 t1c / t2 n0 and n1 breast cancer provide important discriminatory information with regard to chemotherapy benet . 
in addition , our ndings reveal signicant variability in national patterns of rs testing and chemotherapy use for grade 3 tumors . relevance expanding national clinical guidelines with regard to the value of rs testing and increasing use of rs testing in grade 3 tumors may facilitate de - escalation of therapy in those with a low rs . tumor grade is prognostic and independently associated with risk for recurrence ; however , histopathologic high grade may not correlate well with the risk provided by in the planb trial ( clinicaltrials.gov identier : the rs . nct01049425 ) , approximately 50% of grade 3 tumors had an rs less than 31.13 the correlation of histologic grade with the predictive benet of rs has not been assessed comprehensively . 
the proportion of grade 3 tumors in the tailorx trial , although low , reects the typical grade distribution , which accounts for 17.8% of all included tumors across scores , and it was 22% in the original cohort for the development of the rs.1 as a result , the predictive benet of rs and its potential for preventing overtreatment in grade 3 invasive breast carcinomas may be underappreciated . 
rs risk groups were categorized as low ( less than 18 ) , intermediate ( 18 to 30 ) , and high ( 31 or higher ) as originally dened by paik et al.1 variables were coded according to the facility oncology registry data standards manual revised for 2013.20 variables of interest our primary outcomes of interest were receipt of rs testing ( yes / no ; with no dened as multigene signature testing neither ordered nor performed ) and overall survival ( os ) using national death index data provided by the ncdb and dened as the time from date of diagnosis to death , with patients censored at the date of last follow - up available in the ncdb ( december 31 , 2015 )  . 
er , estrogen receptor ; her2 , human epidermal growth factor receptor 2 ; pr , progesterone receptor ; rs , recurrence score . multigene rs for grade 3 breast cancer women newly diagnosed with pt1c - 2 pn0 - 1 breast cancer from 2010 to 2015 excluded prior diagnosis of cancer metastatic disease at diagnosis unknown / negative er status unknown / negative pr status unknown / positive her2 status unknown nottingham grade unknown whether received multigene prediction assay multigene prediction assay other than rs treated entirely at an institution other than the initial diagnosing institution unknown / no breast surgery unknown / no endocrine therapy unknown / had neoadjuvant systemic therapy unknown whether received adjuvant chemotherapy excluded grade 1 and 2 invasive breast cancer study population : n0 - 1 t1c - 2 m0 er - positive / her2 - negative breast cancer treated with definitive surgery and endocrine therapy without neoadjuvant chemotherapy ( n = 172 , 937 ) grade 3 breast cancers for analysis ( n = 30 , 864 ) pn0 grade 3 ( n = 21 , 440 ) pn1 grade 3 ( n = 9 , 720 ) treatments received beyond chemotherapy ( ie , surgery type [ breast conserving surgery / mastectomy ] , adjuvant radiotherapy [ yes / no ] )  . statistical analyses rs testing by clinicopathologic characteristics for all patients was compared using 2 tests and t tests , as appropriate . 
multivariable logistic regression for receipt of rs testing was then performed , with stratication by pn status . to examine unadjusted differences in os by chemotherapy receipt and rs , kaplan - meier method and log - rank test were used . 
potential interaction effects of rs testing with adjuvant chemotherapy were explored using a previously described methodology.22 to evaluate the accuracy of rs testing and nottingham / bloom - richardson grade encoding , patients treated for breast cancer from 2010 to 2015 were queried from the cancer registry submitted data from brigham and womens hospital and dana - farber cancer institute . 
for patients with low rs , 9.1% received chemotherapy , which was not associated with a signicantly increased 5 - year unadjusted os rate ( p = .07 ; table 3 ; fig 2a )  . 
for patients with low rs , 25.9% received chemotherapy , and this was not associated with a signicantly increased unadjusted 5 - year os ( p = .27 ; table 3 ; fig 2b )  . 
of 351 adults with invasive breast carcinoma who had rs testing between 2010 and 2015 , 74.7% ( n = 259 ) were encoded and submitted for inclusion into cancer registries . 
nottingham / bloom - richardson grade was missing or incorrectly encoded in 6.6% of patients ( n = 17 ) , including 8.1% ( n = 3 ) with grade 3 disease . 
in particular , of the 37 grade 3 registry - submitted institutional patients with rs testing , 16.2% ( n = 6 ) had missing scores in registry data , and only 3.2% ( n = 1 ) were incorrectly encoded ( as no rs instead of an actual rs of 22 )  . discussion in this large national cohort , we evaluated the use of rs , use of adjuvant chemotherapy , and os for women with breast cancer , with a focus on high tumor grade . 
of note , chemotherapy was associated with signicant os improvements in grade 3 invasive breast cancers with high rs , and although associated with improved os in univariable analyses , insignicant termediate rs was not predictive of chemotherapy os benet when risk adjusted for clinicopathologic characteristics . 
to our knowledge , these ndings represent the largest analysis to date of the potential impact of rs on the outcomes and management of grade 3 tumors and suggest that the assumption that all pt1c / 2 pn0 / 1 , erpositive histopathologic grade 3 tumors are high risk and will consequently benet from adjuvant chemotherapy may be unmerited . although we await the rxponder trial nal results , the data presented here suggest that patients with a pt1c / 2 n0 / 1 grade 3 tumor with high rs derive benet from chemotherapy ( 92% 5 - year os with chemotherapy v 67% to 79% without ) , whereas grade 3 tumors with low and intermediate rs do not . 
our results further reinforce the utility of broad rs testing in grade 3 tumors and suggest that rs can help to distinguish the anticipated chemotherapy benet among this heterogeneous group of tumors . the rate of rs testing is less in pn1 disease than in pn0 disease ( 36% in 2015 v 72% in pn0 ) but is expanding in both groups because the importance of tumor biology is increasingly recognized . 
in our study , the increased use of rs testing in pn0 grade 3 disease ( 64.1% ) and lower rate of chemotherapy ( 9.1% ) in patients with low rs suggest that clinical practice is increasingly incorporating rs results into decision trees for pn0 grade 3 disease . 
however , our ndings also suggest that a signicant proportion of patients with pn1 grade 3 disease who do not undergo rs testing may be overtreated and receive chemotherapy with no added os benet . 
at risk no rs , no chemotherapy no rs , chemotherapy low rs , no chemotherapy low rs , chemotherapy intermediate rs , no chemotherapy 1 , 838 intermediate rs , chemotherapy high rs , no chemotherapy high rs , chemotherapy 3 , 058 3 , 446 2 , 569 2 , 335 3 , 081 2 , 961 3 , 401 2 , 501 1 , 790 2 , 300 3 , 026 2 , 545 3 , 034 2 , 133 1 , 508 1 , 987 2 , 605 1 , 832 2 , 298 1 , 504 1 , 028 1 , 406 1 , 812 1 , 193 1 , 538 1 , 147 no rs , no chemotherapy low rs , no chemotherapy no rs , chemotherapy low rs , chemotherapy intermediate rs , no chemotherapy intermediate rs , chemotherapy high rs , no chemotherapy high rs , chemotherapy time ( months ) no . 
at risk no rs , no chemotherapy no rs , chemotherapy low rs , no chemotherapy low rs , chemotherapy intermediate rs , no chemotherapy intermediate rs , chemotherapy high rs , no chemotherapy high rs , chemotherapy 1 , 005 4 , 803 4 , 742 4 , 231 3 , 127 2 , 045 1 , 145 no rs , no chemotherapy low rs , no chemotherapy no rs , chemotherapy low rs , chemotherapy intermediate rs , no chemotherapy intermediate rs , chemotherapy high rs , no chemotherapy high rs , chemotherapy fig 2 . 
overall survival ( os ) in patients with grade 3 invasive breast cancer , stratied by recurrence score ( rs )  . adjuvant chemotherapy os estimated by kaplan - meier method for patients with ( a ) pn0 and ( b ) pn1 grade 3 invasive breast cancer , stratied by rs and adjuvant chemotherapy , with an underlying number at risk table . adjuvant chemotherapy ( solid lines ) was associated with signicantly better median os than no adjuvant chemotherapy ( dashed lines ) for intermediate , high , and no rs . are notably higher than those reported in the planb trial ( 18% of grade 3 tumors ) .13 however , our ndings are in keeping with those reported in the microarray in nodenegative ( or 1 - 3 positive lymph node ) disease may avoid trial , where patients were chemotherapy ( mindact ) stratied by both anatomic and genomic risk using a 70gene signature . 
boldface indicates signicance at p , .05. abbreviations : ajcc , american joint committee on cancer ; hr , hazard ratio ; idc , invasive ductal carcinoma ; ilc , invasive lobular carcinoma ; imc , invasive mixed carcinoma ; pr , progesterone receptor ; ref , reference ; rs , recurrence score ; rt , radiotherapy . registryin our multi - institutional cohort of however , submitted patients , 92% with grade 3 disease demonstrated accurate grade coding of whom only 3% had an incorrectly encoded rs , which suggests that key breast cancerspecic factors are encoded into national registries with encouraging accuracy . 
in addition , the ncdb only includes os data , which precludes assessment of breast cancerspecic , progression - free , or recurrence - free survival , end points that may be of greater relevance in the clinical setting . 
as such , our median follow - up is only 41.3 months and the event rate only 6.2% ; nevertheless , we incorporated a large sample size that powered the detection of clinically relevant differences in rs predictive value . 
finally , the ncdb lacks detailed information about type , dose , and duration of chemotherapeutic and hormonal treatments and , in particular , has limited granular inuence clinical decision data about making about when to administer adjuvant chemotherapy . the factors that for example , across all patients with grade 3 disease who were not administered chemotherapy , the ncdb only encoded a reason in 27% ( 21% because chemotherapy was contraindicated and 7% because chemotherapy was recommended but refused by the patient )  . 
to help to address the lack of data about patient contraindications , we included charlson - deyo comorbidity index data in our riskadjusted analyses , and higher indices were associated with reduced chemotherapy use . 
furthermore , our ndings show signicant variability in national patterns of rs testing and chemotherapy use for grade 3 disease , which suggests opportunities for more comprehensive national guidelines for rs testing in high - grade tumors . 
lester employment : novartis institutes for biomedical research ( i ) , restorbio ( i ) leadership : blade therapeutics ( i ) stock and other ownership interests : novartis patents , royalties , other intellectual property : more than 20 patents issued and pending related to health care therapies ( i ) travel , accommodations , expenses : novartis ( i ) , blade therapeutics ( i ) elizabeth a . 
mittendorf honoraria : physician education resource consulting or advisory role : peregrine pharmaceuticals , tapimmune , sellas life sciences , merck , genomic health research funding : galena biopharma ( inst ) , genentech ( inst ) , roche ( inst ) , astrazeneca ( inst ) , emd serono ( inst ) no other potential conicts of interest were reported . acknowledgment we thank tari king , md , for helpful input and the cancer registrars at brigham and womens hospital and dana - farber cancer institute for invaluable assistance in data acquisition . references paik s , shak s , tang g , et al : a multigene assay to predict recurrence of tamoxifen - treated , node - negative breast cancer . 
n engl j med 351 : 2817 - 2826 , 2004 paik s , tang g , shak s , et al : gene expression and benet of chemotherapy in women with node - negative , estrogen receptor - positive breast cancer . 
j clin oncol 24 : 3726 - 3734 , 2006 albain ks , barlow we , shak s , et al : prognostic and predictive value of the 21 - gene recurrence score assay in postmenopausal women with node - positive , oestrogen - receptor - positive breast cancer on chemotherapy : a retrospective analysis of a randomised trial . 
lancet oncol 11 : 55 - 65 , 2010 dowsett m , cuzick j , wale c , et al : prediction of risk of distant recurrence using the 21 - gene recurrence score in node - negative and node - positive postmenopausal patients with breast cancer treated with anastrozole or tamoxifen : a transatac study . 
harris ln , ismaila n , mcshane lm , et al : use of biomarkers to guide decisions on adjuvant systemic therapy for women with early - stage invasive breast cancer : american society of clinical oncology clinical practice guideline . 
new york , ny , springer , 2017 sanders ma , wong sm , iorgulescu jb , et al : changes and clarications in the eighth edition of the ajcc cancer staging system for breast cancer . 
 multigene rs for grade 3 breast cancer giuliano ae , connolly jl , edge sb , et al : breast cancer - major changes in the american joint committee on cancer eighth edition cancer staging manual . 
wong wb , ramsey sd , barlow we , et al : the value of comparative effectiveness research : projected return on investment of the rxponder trial ( swog s1007 )  . 
gluz o , nitz ua , christgen m , et al : west german study group phase iii planb trial : first prospective outcome data for the 21 - gene recurrence score assay and concordance of prognostic markers by central and local pathology assessment . 
roberts mc , miller dp , shak s , et al : breast cancer - specic survival in patients with lymph node - positive hormone receptor - positive invasive breast cancer and oncotype dx recurrence score results in the seer database . 
j clin epidemiol 45 : 613 - 619 , 1992 iorgulescu jb , harary m , zogg ck , et al : improved risk - adjusted survival for melanoma brain metastases in the era of checkpoint blockade immunotherapies : results from a national cohort . 
yee martin kaiser heather landau jean - marie michot antoine hollebecque luisa veronese martina makrutzki bethany pitcher igor puzanov jose baselga author affiliations and support information ( if applicable ) appear at the end of this article . clinical trial information : nct01524978 . corresponding author : noopur raje , md , professional office building 216 , harvard medical school , 55 fruit st , boston , ma 02114 ; e - mail : nraje@mgh.harvard.edu. introduction multiple myeloma ( mm ) is a genetically heterogeneous , complex disease that arises as a result of a variety of mutations in pathways deregulating the intrinsic biology of the plasma cell.1 although immunomodulatory drugs and proteasome inhibitors have improved outcomes in patients with mm , 2 , 3 patients with relapsed or refractory disease have a poor prognosis.4 despite the identification of many of the genetic events involved in the development of mm , no treatments specifically targeting genetic mutations have been developed to date . 
additional study design details are described in the data supplement . patients patients in the mm cohort had to have previously treated , measurable , relapsed or refractory mm with confirmed brafv600 mutation . 
the presence of brafv600 mutations was confirmed retrospectively in a central laboratory using the roche companion diagnostic cobas 4800 braf v600 test or other standard methodology . assessments the following assessments for mm were performed 8 weeks after the start of therapy and every 4 weeks thereafter : serum protein electrophoresis with quantitation of monoclonal protein level ; urine protein electrophoresis using 24 - hour urine protein electrophoresis ; and serum levels of free light chains , lactate dehydrogenase , and - 2 microglobulbone marrow analysis was performed only to confirm complete response ( cr ) after two consecutive immunofixation analyses were negative . efficacy was evaluated using international myeloma working group uniform response criteria.17 , 18 evaluations were performed at baseline , 8 weeks after the start of vemurafenib administration , every 4 weeks thereafter during treatment , and at the end of treatment . 
responses were classed as cr , stringent cr , very good partial response ( vgpr ) , partial response ( pr ) , stable disease ( sd ) , progressive disease , and clinical relapse . 
responders were defined as those patients with pr or better ( ie , cr , stringent cr , pr , or vgpr )  . statistical analysis the primary efficacy end point of ve - basket was the response rate at week 8 . 
for the purposes of this analysis , two response assessments were required with no specified time between these assessments , although the protocol required that these two assessments be 4 weeks apart . an adaptive simon two - stage design was used to minimize the number of patients treated if vemurafenib was deemed ineffective for a specific tumor type . 
assuming response rates as specified in the hypothesis testing , a power of 80% for a high desirable response and 70% for a low desirable response , and a two - sided of 0.1 , the number of patients required in each cohort was seven for stage 1 , and 13 or 19 for stage 2 , depending on the results in stage 1 . 
however , if a clear clinical benefit was observed for patients in a cohort ( eg , the majority of patients recorded sd at week 8 and no cr or pr was recorded ) , then enrollment in stage 2 might be allowed for the cohort after discussion with the sponsor and study steering committee . 
the data presented here are the results of the final analysis . seven patients entered stage 1 of the mm cohort of the ve - basket study , followed by enrollment of an additional two patients because clear clinical benefit was observed , even though the prespecified minimal response rate was not achieved . 
the patient received palliative radiotherapy and eight cycles of lenalidomide , bortezomib , and dexamethasone before undergoing high - dose melphalan therapy with autologous stem - cell transplantation in november 2008 . 
baseline characteristics of patients with brafv600 mutationpositive multiple myeloma treated with vemurafenib patients with multiple myeloma ( n = 9 ) characteristic male female age , years , median ( range ) ecog performance status brafv600 mutation type * monoclonal protein v600e v600k iga kappa iga lambda igg kappa igg lambda risk stratification high standard lines of prior systemic therapies prior systemic therapies * immunomodulators ( lenalidomide , thalidomide , pomalidomide ) corticosteroids ( dexamethasone ) alkylating agents ( cyclophosphamide , melphalan , bendamustine ) proteasome inhibitors ( bortezomib , carfilzomib ) cytotoxic agents ( doxorubicin , idarubicin ) platinum agent ( cisplatin ) topoisomerase inhibitors ( etoposide ) novel antineoplastic agents ( acy - 1215 , cc - 223 ) note . 
 ( % ) unless indicated otherwise . abbreviations : ecog , eastern cooperative oncology group performance status ; ig , immunoglobulin . * on the basis of a manual review of the data . 
 testing was by snapshot ( n = 2 ) , capillary electrophoresis single - strand conformation analysis ( n = 2 ) , direct ( sanger ) sequencing ( n = 1 ) , sequenom ( n = 1 ) , single - strand conformation analysis ( n = 1 ) , 3730 dna analyzer ( n = 1 ) , and immunohistochemistry ( n = 1 )  . 
at the time of closure of the ve - basket study , the patient was still receiving treatment in the extension study . the second patient was diagnosed with smoldering mm in may 2006 and progressed to stage ii active mm in december 2009 , at which time he was treated with lenalidomide , bortezomib , and dexamethasone . 
 ( b ) change from baseline in serum m protein according to best overall response in individual patients with brafv600 mutation positive multiple myeloma treated with vemurafenib ( n = 9 )  . 
one patient with a substantial reduction in serum m protein level had an unconfirmed pr but was considered to have sd because the patient had pd at the subsequent assessment . 
 subsequent relapses were treated with cyclophosphamide , lenalidomide , and dexamethasone ( april 2013 ) ; carfilzomib , thalidomide , and dexamethasone ( august 2013 ) ; pomalidomide , bortezomib , and dexamethasone ( march 2014 ) ; and carfilzomib , pomalidomide , and dexamethasone ( august 2014 )  . 
snapshot testing of bone marrow aspirate identified an additional nras mutation at the time of relapse that had not been seen at study entry and was reported after study closure ; the original brafv600g mutation was also present at this time . one other patient , who had been treated previously with bortezomib and dexamethasone in the first line , the mammalian target of rapamycin inhibitor cc - 223 on relapse , and lenalidomide in the third line , had a vgpr after treatment with vemurafenib . 
one patient achieved an 83% reduction in serum monoclonal protein level and had a pr at one assessment but progressive disease at the next assessment , for a best overall response of sd . 
time to event details and change from baseline in serum monoclonal protein for individual patients are shown in figure 1 . at the time of study closure , disease progression was observed in six patients . 
median investigator assessed pfs was 4.63 months ( 95% ci , 2.89 months to not estimable ) ; the 6 - month pfs rate was 40% ( 95% ci , 6% to 74% ) , and the median time to progression was 4.63 months ( 95% ci , 2.89 months to not estimable )  . 
kaplan - meier plots of pfs and os are shown in the data supplement . safety the median vemurafenib dose intensity was 79% ( range , 51% to 100% )  . 
adverse events occurring in 20% of patients with brafv600 mutationpositive multiple myeloma treated with vemurafenib ( n = 9 ) any grade grade 3 or 4 event alopecia melanocytic nevus anemia hypokalemia keratosis pilaris skin papilloma sunburn acne back pain constipation diarrhea dysphonia fatigue * hyperkeratosis keratosis follicular leukoplakia oral rash papular seborrheic keratosis skin lesion xerosis upper respiratory tract infection blood alkaline phosphatase increase increased alanine aminotransferase note . 
one patient had grade 2 hypercalcemia , which was judged unrelated to vemurafenib treatment and resolved without treatment interruption , and a grade 3 chest infection , also unrelated to treatment , during which treatment was interrupted temporarily . 
the second patient had grade 4 diabetes that was judged unrelated to the study drug but resulted in dose reduction and grade 3 cellulitis that was also unrelated to treatment but required treatment interruption . 
 the final patient had treatment - related grade 4 sepsis that resolved with dose reduction and treatment - related grade 3 skin lesion and grade 2 upper respiratory infection that were not considered serious but resulted in permanent discontinuation of treatment . 
seven patients had at least one adverse event leading to dose reduction or interruption ( infections [ n = 4 ] , skin disorders [ n = 2 ] , anemia [ n = 1 ] , blood alkaline phosphatase increased [ n = 1 ] , keratosis follicular [ n = 1 ] , diabetes mellitus [ n = 1 ] , and acute kidney injury [ n = 1 ] ) ; one patient had a dose interruption for cataract surgery . qt prolongation , squamous cell carcinoma of the skin , and keratoacanthoma were not observed . 
however , identification of the brafv600 mutation as a driver mutation in mm may lead to patients with this mutation benefitting from targeted treatment with brafv600 inhibitors.1 , 7 , 20 in this study , we have shown that vemurafenib has promising efficacy in patients with brafv600 mutationpositive mm : two patients of nine had encouraging and long - lasting responses to treatment that were ongoing at the time of study closure , and an additional patient had a shorter response . 
these patients had been treated previously with bortezomib and lenalidomide , among other agents , suggesting that resistance mechanisms in those patients did not prevent response to vemurafenib . in this study , not all patients with the brafv600 mutation responded to therapy , providing us with the opportunity to study mechanisms of resistance in this patient population in the future . 
clonal evolution of mm cells and changes in the bone marrow environment have been implicated in resistance , 21 and alterations in the erk pathway are present in almost one half of patients with mm . 
interestingly , the patient in our study who relapsed after achieving a pr had acquired an nras mutation , suggesting an escape mechanism and possible resistance via this molecular pathway . 
 hyman , jean - yves blay , josep tabernero , jrgen wolf , igor puzanov , jose baselga provision of study material or patients : all authors collection and assembly of data : all authors data analysis and interpretation : noopur raje , martina makrutzki , bethany pitcher manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors mechanisms , including driver mutations within subclonal populations , may also account for differential responses . 
understanding resistance mechanisms could lead to the development of new therapies acting on the ras / raf pathway , either alone or in combination , or to the use of mek or pan - raf inhibitors . 
this subject is being addressed in an ongoing clinical trial using a braf inhibitor and a mek inhibitor ( dabrafenib and trametinib , respectively ; clinicaltrials.gov identifier : nct03091257 )  . the safety profile of vemurafenib in this patient cohort was broadly similar to that reported previously , 8 , 9 and no new safety signals were identified . 
cutaneous squamous cell carcinoma , which has been reported in vemurafenib treated patients in earlier studies , 8 , 9 was not observed in our patients . in conclusion , vemurafenib may be an appropriate and effective choice for patients with mm in whom a brafv600 mutation has been identified . 
because the ve - basket study included few patients with mm , additional studies are required to establish the role of vemurafenib , whether as monotherapy or in combination with other agents , for earlyor later - stage disease in the treatment of this poor - prognosis population . 
 ian chau honoraria : eli lilly , gilead sciences consulting or advisory role : eli lilly , bristol - myers squibb , msd , merck serono research funding : janssen - cilag ( inst ) , sanofi ( inst ) , merck serono ( inst ) , eli lilly ( inst ) travel , accommodations , expenses : msd , merck serono , sanofi , eli lilly , bristol - myers squibb martin kaiser honoraria : takeda pharmaceuticals , celgene , amgen , janssen oncology consulting or advisory role : janssen oncology , celgene , bristol - myers squibb , takeda pharmaceuticals , amgen , chugai pharmaceutical research funding : celgene travel , accommodations , expenses : takeda pharmaceuticals david m . 
yee consulting or advisory role : takeda pharmaceuticals , dexcel pharma , adaptive biotechnologies , celgene , janssen pharmaceuticals research funding : bristol - myers squibb ( inst ) , celgene ( inst ) bethany pitcher employment : f . 
hoffmann - la roche igor puzanov consulting or advisory role : amgen , roche / genentech , bristol - myers squibb travel , accommodations , expenses : amgen , merck jose baselga leadership : infinity pharmaceuticals , varian medical systems , grail , bristol - myers squibb stock or other ownership : pmv pharma , juno therapeutics , infinity pharmaceuticals , grail , varian medical systems , tango , foghorn , aura biomedical , apogen , northern biologics , bristol - myers squibb honoraria : pmv pharma , juno therapeutics , infinity pharmaceuticals , grail , northern biologics consulting or advisory role : eli lilly , novartis , grail patents , royalties , other intellectual property : combination therapy using pdk1 and pi3k inhibitors . 
hyman , heather landau , and jose baselga , memorial sloan kettering cancer center , new york ; igor puzanov , roswell park cancer institute , buffalo , ny ; vincent ribrag , jean - marie michot , and antoine hollebecque , institut gustave roussy , villejuif ; jean - yves blay , centre leonberard , lyon , france ; josep tabernero and elena elez , vall dhebron university hospital and institute of oncology , universitat autnoma de barcelona , barcelona , spain ; jrgen wolf , university hospital kln , kln , germany ; luisa veronese and martina makrutzki , f . 
hoffmann - la roche funded third - party writing and editorial support , which was provided by miller medical communications . presented in part at the 57th annual meeting of the american society of hematology , orlando , fl , december 5 - 8 , 2015 . support prior presentation references 1 . 
dimopoulos ma , swern as , li js , et al : efficacy and safety of long - term treatment with lenalidomide and dexamethasone in patients with relapsed / refractory multiple myeloma . 
kumar sk , lee jh , lahuerta jj , et al : risk of progression and survival in multiple myeloma relapsing after therapy with imids and bortezomib : a multicenter international myeloma working group study . 
walker ba , boyle em , wardell cp , et al : mutational spectrum , copy number changes , and outcome : results of a sequencing study of patients with newly diagnosed myeloma . 
mcarthur ga , chapman pb , robert c , et al : safety and efficacy of vemurafenib in braf ( v600e ) and braf ( v600k ) mutation - positive melanoma ( brim - 3 ) : extended follow - up of a phase 3 , randomised , open - label study . 
larkin j , del vecchio m , ascierto pa , et al : vemurafenib in patients with braf ( v600 ) mutated metastatic melanoma : an open - label , multicentre , safety study . 
subbiah v , gervais r , riely gj , et al : efficacy of vemurafenib in patients ( pts ) with nonsmall cell lung cancer ( nsclc ) with brafv600 mutation . 
hyman dm , kaley tj , hollebecque a , et al : vemurafenib in patients with brafv600 mutant glioma : a cohort of the histology - independent ve - basket study . 
diamond el , subbiah v , lockhart ac , et al : vemurafenib for brafv600 - mutant erdheimchester disease and langerhans cell histiocytosis : analysis of data from the histology - independent phase 2 open - label ve - basket study . 
rajkumar sv , harousseau jl , durie b , et al : consensus recommendations for the uniform reporting of clinical trials : report of the international myeloma workshop consensus panel 1 . 
kim , md , lineberger comprehensive cancer center , university of north carolina , 450 west dr , cb# 7295 , chapel hill , nc 27599 - 7295 ; e - mail : wykim@med.unc.edu. purpose urachal adenocarcinoma is a rare type of primary bladder adenocarcinoma that comprises less than 1% of all bladder cancers . 
we hypothesized that an in - depth molecular understanding of urachal adenocarcinoma would uncover rational therapeutic strategies . patients and methods we performed targeted exon sequencing and global transcriptome profiling of 12 urachal tumors to generate a comprehensive molecular portrait of urachal adenocarcinoma . 
a single patient with an msh6 mutation was treated with the antiprogrammed death - ligand 1 antibody , atezolizumab . results urachal adenocarcinoma closely resembles colorectal cancer at the level of rna expression , which extends previous observations that urachal tumors harbor genomic alterations that are found in colorectal adenocarcinoma . 
a patient with an msh6 mutation was treated with immune checkpoint blockade , which resulted in stable disease . conclusion because clinical trials are next to impossible for patients with rare tumors , precision oncology may be an important adjunct for treatment decisions . 
2017 by american society of clinical oncology introduction in the united states , bladder carcinoma is the 4th most common malignancy in men and the 9th most common in women.1 overall , an estimated 16 , 000 people died of bladder cancer in 2015 . bladder cancer takes on a spectrum of histomorphologic appearances . 
urachal carcinomas are a subtype of bladder adenocarcinoma that arises from the urachus , an embryologic remnant that connects the bladder and the allantois during fetal development.2 postnatally , fuses to become a fibrous cord known as the median umbilical ligament . 
 because of their frequent presentation at the dome of the bladder , urachal adenocarcinomas are clinically and pathologically grouped with bladder neoplasms and are typically seen by urologic oncologists and genitourinary pathologists . 
evidencebased treatment of this disease is hindered by its rarity ; thus , current treatment paradigms for urachal adenocarcinoma are primarily anecdotal in nature . as a result of the complex embryology of the allantois and cloaca , the cellular origin and molecular pathogenesis that drive the development of urachal adenocarcinoma are speculative.2 intestinal metaplasia or enteric rests are hypothesized as the histogenetic precursor of these tumors . 
although the genetics of urachal adenocarcinoma have been investigated by multiple groups , no reports on the global gene expression patterns of urachal adenocarcinoma have been published . one study selectively examined the prevalence of kras and braf mutations in high - stage urachal adenocarcinomas and , although they found no braf mutations , 42% harbored kras mutations.5 the study also noted a high rate of loss of protein expression for a number of genes that are correlated with microsatellite instability . 
a more recent study found a high rate of nf1 mutations via whole - exome sequencing of seven urachal adenocarcinomas.6 finally , another recent study showed that urachal adenocarcinomas harbor mutations in mitogen - activated protein kinase ( mapk ) pathways , similar to colorectal adenocarcinoma , and showed the potential for treatment with the antiepidermal growth factor receptor antibody cetuximab.7 herein , we report , to our knowledge , the first transcriptome analysis of urachal adenocarcinoma using whole - transcriptome profiling by rna sequencing . 
a pan - cancer transcriptomic analysis of urachal tumors comparing them with 12 cancers of different tissue origins suggest that their rna expression patterns most closely resemble colorectal adenocarcinoma and glioblastoma ( gbm )  . our work also validates reports that urachal adenocarcinomas harbor alterations that are typically found in colorectal carcinomathat is , apc , smad4 , and kras mutationbut extends those observations to show that a subset of urachal cancers has inactivation of genes that are involved in microsatellite instability ( msh2 , msh6 ) or hypermutation ( pole ) , and that all urachal tumors invariably have mutations of tp53 ( 100% )  . one patient with an msh6 mutation was treated with the antiprogrammed death - ligand 1 ( pdl1 ) antibody atezolizumab , which resulted in stable disease . 
thirteen primary urachal adenocarcinomas with formalin - fixed , paraffin - embedded ( ffpe ) tissue available at university of north carolina , chapel hill , were identified by using copath natural language search ( cerner corporation , kansas city , mo )  . 
hematoxylin and eosinstained slides and clinical history were reviewed by a board - certified pathologist ( s.e.w. ) to confirm the diagnosis on the basis of the following criteria : tumor in the dome or posterior wall of the bladder , sharp demarcation between tumor and surface epithelium , and exclusion of primary adenocarcinoma located elsewhere . 
the surgical procedure , tumor location within the bladder , histologic subtype , and tumor stage were all recorded from the accompanying pathology reports . rna expression for rna sequencing , rna was extracted from 10 - mm - thick unstained sections of ffpe blocks that were isolated from urachal tumors . 
extracted rna was converted to double - stranded cdna , and sequencing adapters were ligated by using the illumina rna access library prep protocol ( illumina , san diego , ca )  . samples were sequenced by paired - end , 100 - bp sequencing on an illumina hisequation 2000 at the high throughput sequencing core facility at the university of north carolina . 
sequence reads were aligned to the human reference transcriptome , and gene expression was generated as reads per kilobase of exon model per million mapped reads per gene by using mapsplice ( university of kentucky bioinformatics labs , lexington , ky )  . 
 clustering with the combined urachal ( sequenced urachal tumors ) and pancan ( tcga pan - cancer dataset13 ) data set was performed by using average linkage clustering with a centered correlation similarity metric with cluster 3.0 ( human genome center , tokyo , japan ) software on the top 10% most differentially expressed genes ( as determined by standard deviation ) across the combined pancan and urachal data set . 
a pearson correlation was calculated between each of the pancan tumor type centroids and each urachal tumor . clustering between the cancer genome atlas ( tcga ) bladder urothelial carcinoma ( blca ) , tcga colorectal adenocarcinoma ( coadread ) , and urachal samples was performed using average linkage clustering with a centered correlation similarity metric with cluster 3.0 software on the top 10% most differentially expressed genes after adjusting for batch effects as described above . targeted exon sequencing targeted exon sequencing was conducted through the uncseq pipeline as previously described.8 twelve of 13 urachal samples had both tumor and tumor - adjacent normal tissue submitted to the uncseq pipeline that passed quality control standards and were included in the dna analysis . 
analysis to identify significantly mutated genes , altered pathways , and clustering was confined to mutations that were classified as either having a moderate or high impact on protein function through uncseq . 
copy number alteration on a cohort level was derived by running genomic identification of significant targets in cancer ( gistic ) 2.0 on the gene pattern online platform.11 the dna mismatch repair ( mmr ) pathway was represented by the mlh1 , mlh3 , msh2 , msh3 , msh6 , and pms2 genes , with the pole gene included and separately identified . 
the insertion - to - deletion ratio was calculated by identifying the mutations that were identified as either nucleotide insertion or deletion events and dividing it by the total number of insertion and deletion events and single - nucleotide variant events ( total mutations ) in each sample . statistics a pearson correlation was performed in r between the pancan transcriptome centroids and the urachal sample expression values as detailed above . 
copy number alteration significance values were calculated using the gistic q - value metric . results urachal adenocarcinomas molecularly resemble colorectal adenocarcinoma and glioblastoma in a pan - cancer analysis thirteen urachal adenocarcinomas were identified from a search of the university of north carolina surgical pathology database ( table 1 )  . all were confirmed to be urachal adenocarcinomas on the basis of standard criteria ( patients and methods )  . 
in aggregate , these results support the notion that subsets of urachal tumors are molecularly similar to either colorectal cancer or gbm . urachal adenocarcinomas arise from an embryologic remnant of the allantois that is formed when the cloaca divides into an anterior and posterior portion . 
apc mutations were of particular interest , given it is uniquely mutated in colorectal cancers.13 although none of the mtor mutations has been previously reported , of interest , two of four of these mutations occur in the focal adhesion kinase targeting domain , where activating mutations have been previously described and shown to impart sensitivity to rapamycin.15 moreover , colorectal cancers seem to have one of the highest rate of mtor mutations across the published tcga data sets ( appendix fig a1 )  . 
 ( b ) unsupervised clustering of urachal , colorectal , and bladder mutation frequency across blca and coadread significantly mutated genes as defined by the the cancer genome atlas ( tcga )  . 
 ( c ) oncoprints of transforming growth factor ( tgf ) - b , mitogen - activated protein kinase ( mapk ) , and b - catenin pathway mutations in colorectal ( tcga ) , bladder ( tcga ) , and urachal ( uncseq ) tumors . 
 ( d ) a supervised heatmap of the frequency of tfg - b , mapk , and b - catenin pathway mutations in colorectal ( tcga ) , bladder ( tcga ) , and urachal ( uncseq ) tumors . 
 mlh1 msh2 msh6 pms2 pole 1% 1 , 000 urachal uncseq mutated mmr nonmutated mmr mutated pole colorectal uncseq bladder uncseq mutated mmr nonmutated mmr mutated pole bladder colorectal urachal bladder colorectal urachal fig 3 . 
 ( b ) supervised bar plot of the mutations / megabase ( mb ) across uncseq bladder , colorectal , and urachal samples , with samples that contain mutations in the dna mismatch repair ( mmr ) pathway indicated . ( c ) supervised bar plot of the ratio of insertions to deletions ( indel ) / mutation for uncseq bladder , colorectal , and urachal samples , with samples that contain mutations in the dna mismatch repair pathway indicated . loss , and tgfb activation by smad2 , smad3 , or smad4 mutations ( fig 2c )  . 
evaluation of a patient with urachal adenocarcinoma who was treated with atezolizumab . ( a ) spider plot of individual lesions and combined tumor burden of the atezolizumab - treated patient . 
 atezolizumab treatment results in stable disease programmed death - 1 ( pd - 1 ) blockade has resulted in significant responses in tumors with mmr deficiency.19 one of the patients whose urachal tumor was found to harbor an msh6 mutation was treated with the antipd - l1 antibody , atezolizumab . the patient received atezolizumab every 3 weeks with initial progression in target lesions ( two lung nodules and one hilar lymph node ) , followed by regression in the two lung nodules and an increase in the left hilar node associated with necrosis at second assessment ( figs 4a and 4b )  . 
grilley - olson consulting or advisory role : sanofi research funding : bayer ( inst ) , novartis ( inst ) , peregrine pharmaceuticals ( inst ) , nanocarrier ( inst ) , genentech ( inst ) , seattle genetics ( inst ) , medimmune ( inst ) , glaxosmithkline ( inst ) , pfizer ( inst ) nirali m . 
weck no relationship to disclose peter black consulting or advisory role : abbvie , astellas pharma , janssen oncology , amgen , novartis , biocancell , sitka , cubist , bayer , merck , sanofi , biosyent , ferring , eli lilly , roche , spectrum pharmaceuticals , allergan speakers bureau : ferring , red leaf medical , new b innovation , iprogen patents , royalties , other intellectual property : 1 . 
milowsky research funding : mirati therapeutics ( inst ) , pfizer ( inst ) , cerulean pharma ( inst ) , merck ( inst ) , seattle genetics ( inst ) , acerta pharma ( inst ) , bioclin therapeutics ( inst ) , genentech ( inst ) , bristol - myers squibb ( inst ) , x4 pharma ( inst ) , medimmune ( inst ) , incyte ( inst ) , innocrin pharma ( inst ) , inovio pharmaceuticals ( inst ) travel , accommodations , expenses : genentech d . 
neil hayes leadership : genecentric stock and other ownership interests : genecentric consulting or advisory role : genecentric patents , royalties , other intellectual property : i hold several diagnostic patents or pending patents in the area of solid tumor diagnostics william y . 
kim stock and other ownership interests : johnson & johnson , bristol - myers squibb , medivation , agios patents , royalties , other intellectual property : base47 bladder cancer subtype classifier acknowledgment we thank the members of the kim laboratory for useful discussions . 
singh h , liu y , xiao x , et al : whole exome sequencing of urachal adenocarcinoma reveals recurrent nf1 mutations . oncotarget 7 : 29211 - 29215 , 2016 7 . 
collazo - lorduy a , castillo - martin m , wang l , et al : urachal carcinoma shares genomic alterations with colorectal carcinoma and may respond to epidermal growth factor inhibition . 
zhao x , wang a , walter v , et al : combined targeted dna sequencing in non - small cell lung cancer ( nsclc ) using uncseq and ngscopy , and rna sequencing using uncqer for the detection of genetic aberrations in nsclc . plos one 10 : e0129280 , 2015 9 . 
mermel ch , schumacher se , hill b , et al : gistic2.0 facilitates sensitive and confident localization of the targets of focal somatic copy - number alteration in human cancers . 
li b , dewey cn : rsem : accurate transcript quantification from rna - seq data with or without a reference genome . bmc bioinformatics 12 : 323 , 2011 13 . 
hoadley ka , yau c , wolf dm , et al : multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origcell 158 : 929 - 944 , 2014 14 . 
hodi fs , hwu w - j , kefford r , et al : evaluation of immune - related response criteria and recist v1.1 in patients with advanced melanoma treated with pembrolizumab . 
rosenberg je , hoffman - censits j , powles t , et al : atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum - based chemotherapy : a single - arm , multicentre , phase 2 trial . 
van allen em , miao d , schilling b , et al : genomic correlates of response to ctla - 4 blockade in metastatic jci insight 1 : e85902 , 2016 melanoma . 
verhaak rgw , hoadley ka , purdom e , et al : integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in pdgfra , idh1 , egfr , and nf1 . 
this model combines the expression of eight genes in primary melanoma with breslow thickness and patient age to identify patients who may forgo a sentinel lymph node biopsy ( slnb ) because of their low risk of nodal metastasis . 
cp - gep can guide physicians on slnb referrals and reduce the frequency of slnb surgeries.4 the authors of both commentaries acknowledge the need for a tool to reduce the frequency of slnb procedures . 
bartlett et al1 appreciated our approach of incorporating cp variables into our model because it ensures transparency on the added value of the gep , and they nd the comparison of cp - gep and cp models appropriate . 
the authors of both commentaries reected on the evidence that supports the utility of the cp - gep model in clinical practice and elaborated on some methodologic considerations . both commentaries rightfully emphasized the need for adequately sized external validation and additional clinical utility studies before widespread clinical adoption of cp - gep . 
we agree that extensive validation in diverse patient populations and different geographical contexts is critical . for example , we recently validated the cp - gep model in a dutch retrospective patient cohort ( n = 211 ) 5 in which patient selection , surgical procedures , and pathologic assessment of slnb are signicantly different from clinical practice in the united states . 
cp - gep achieved an slnb reduction rate ( rr ) of 41% in t1 / t2 melanoma at a negative predictive value ( npv ) of 90.7%.5 additional studies are underway to elucidate cp - gep as an slnb reduction tool . 
slnb recommendations were made on the basis of individualized considerations and within the framework of institutional practice guidelines . we , in turn , derive institutional practice guidelines from established national and international guidelines . 
moreover , in terms of net benet , the cp - gep model is consistently superior to the cp model across a range of clinically reasonable risk thresholds with a more substantial gain for higher risk thresholds . varey et al2 rightfully observe that the npv is a function of the prevalence of slnb positivity in the population and , in particular , that npv decreases as the prevalence increases . 
second , the risk of metastasis to the sentinel lymph node ( sln ) ; as reected by npv and positive predictive value ( ppv ) upon taking a test is a clinically relevant metric in the process of making a decision for or against an slnb , as indicated by national comprehensive cancer network guidelines . 
furthermore , in a previous publication by mocellin et al , 9 team members thompson and scolyer themselves recommended building predictive models for sln metastasis with the aim of maximizing the npv and reducing the rate of slnb procedures through minimizing the error rate . 
in a consensus statement on the development of gep in cutaneous melanoma , grossman et al10 recently commented that gep - based tests should add to the npv of current models while minimizing false - negative predictions . bartlett et al discuss the stratication of the results by t categories . 
after consulting with dermatopathologists at mayo clinic , we concluded that the 140 excluded patients could results stratied by t category , and we hope that our data will enable physicians to critically assess the potential of cp - gep . 
knowing model performance within each t category ( as opposed to overall performance ) is likely to gain importance because the distribution of melanoma t categories is expected to change over time with more melanomas diagnosed at earlier t categories . 
we further sought to limit variability in our data by requiring slnb to be performed within 90 days of the diagnosis of primary melanoma.3 from our point of view , combining end points such as slnb status and regional recurrence can be problematic , because they occur on different time scales and vary in their methods of detection . 
the relatively high rate of patients who develop regional recurrences after negative slnb , however , highlights the need for better , more sensitive staging tools , such as the cp - gep model . 
accordingly , and to avoid missing stage iii disease , too many patients undergo invasive and costly slnb , which highlights the need for an slnb reduction tool . varey et al2 from the melanoma institute australia ( mia ) recently published a cp nomogram to predict the risk of sln metastasis.12 the mia nomogram is based on six cp variables : patient age , breslow thickness , ulceration , histologic subtype , mitotic rate , and lymphovascular invasion . in their commentary , the authors speculate that the mia nomogram outperforms the cp - gep model . 
therefore , since the mia nomogram is available online , we sought to assess the performance of the mia nomogram in our cohort.3 we found that the mia nomogram risk score could not be calculated for approximately 19% of the patients ( 143 of 754 ) in our cohort . 
of the 143 patients excluded , 140 were not suitable for the nomogram because of histologic type , and three patients were excluded because they were younger than age 18 years at biopsy . 
shown are models based on the mia nomogram , clinicopathologic variables ( cp model ) , and cp variables and a gene expression prole ( cp - gep )  . 
 correspondence not be consistently reclassied into the ve histologic types available for the mia nomograit is likely that in clinical practice , we would encounter a similar number of patients who are difcult to classify for the mia nomograit is important to note that 16.4% ( 23 ) of the 140 excluded patients showed sln metastasis . 
more data are needed to characterize our model fully , and to this end , we are currently implementing a comprehensive validation plan . domenico bellomo , phd skylinedx , rotterdam , the netherlands alina g . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . domenico bellomo employment : skylinedx stock and other ownership interests : synlogic , skylinedx patents , royalties , other intellectual property : gene signatures for predicting metastasis of melanoma tina j . 
hieken research funding : genentech ( inst ) alexander meves research funding : skylinedx ( inst ) patents , royalties , other intellectual property : mayo clinic has led several patent applications on which i am listed as an inventor . 
mulder eeap , dwarkasing jt , hollestein d , et al : validation of a clinicopathological and gene expression prole ( cp - gep ) model for sentinel lymph node metastasis in primary cutaneous melanoma . 
bioinformatics 21 : 3755 - 3762 , 2005 steyerberg ew , harrell fe jr , borsboom gj , et al : internal validation of predictive models : efciency of some procedures for logistic regression analysis . 
mocellin s , thompson jf , pasquali s , et al : sentinel node status prediction by four statistical models : results from a large bi - institutional series ( n = 1132 )  . 
mdm2 has increasing clinical relevance because inhibitors are under evaluation in clinical trials , and mdm2 amplification is a possible genomic correlate of accelerated progression , known as hyperprogression , after antipd - 1 / pd - l1 immunotherapy . 
we used next - generation sequencing ( ngs ) to ascertain mdm2 amplification status across a large number of diverse cancers . methods we interrogated the molecular profiles of 102 , 878 patients with diverse malignancies for mdm2 amplification and co - altered genes using clinical - grade ngs ( 182 to 465 genes )  . results mdm2 amplification occurred in 3.5% of patients ( 3 , 650 of 102 , 878 )  . 
an illustrative patient who harbored mdm2 amplification and experienced hyperprogression with an immune checkpoint inhibitor is presented . conclusion mdm2 amplification was found in 3.5% of 102 , 878 patients , 97.6% of whom harbored genomic co - alterations that were potentially targetable . 
the core function of mdm2 is to inhibit the tumor suppressor p53 , which is critical for regulating genes involved in dna repair , cell cycle , senescence , and apoptosis . 
when amplified , mdm2 facilitates proteasomal degradation of p53 , which promotes tumorigenesis.1 - 3 mdm2 amplification has been reported in multiple tumor types4 - 6 and is a hallmark of tumorigenesis.3 in certain tumor types , such as glioblastoma and well differentiated liposarcoma , mdm2 amplification and tp53 alterations are mutually exclusive , 4 , 5 which is consistent with the inhibitory function of mdm2 . 
however , in other tumors ( eg , osteosarcoma , esophageal cancer ) , mdm2 amplification and tp53 alterations co - occur.5 , 6 preclinical studies have suggested noncanonical p53 - independent roles for mdm2 . 
 for instance , an in vitro study that used an mdm2 - overexpressed / tp53 wild - type cancer cell line revealed a potential role for mdm2 in suppressing senescence in a tp53 - independent fashion.7 moreover , mdm2 - amplified / tp53null mice have a higher incidence of spontaneous tumorigenesis than tp53 - null mice shumei kato jeffrey s . 
 ( without mdm2 amplification ) .8 among several potential noncanonical roles for mdm2 , a functional angiogenesis effect has been proposed.9 indeed , a preclinical study showed that under hypoxic conditions , mdm2 - overexpressed / tp53 - null cancer cells produce vascular endothelial growth factor ( vegf ) mrna at higher levels than mdm2 - negative / tp53 - null cells . 
 hypoxia induces translocation of mdm2 from the nucleus to the cytoplasm , and subsequent binding of the mdm2 c - terminal domain to the 3untranslated region of vegf mrna increases mrna stability and translation.10 in addition , suppression of mdm2 activity with a small - molecule inhibitor leads to decreased hypoxia - inducible factor 1 and vegf expression , which support a role for mdm2 in angiogenesis.11 tp53 mutations also lead to increased vegf - a expression12 , 13 and have been associated with increased responsiveness to vegf / vegf receptor inhibitor therapy.14 - 16 taken together , these data suggest that mdm2 promotes angiogenesis through either inhibition of p53 or mechanisms independent of p53 . an understanding of mdm2 amplification status has clinical relevance for patients with cancer because mdm2 inhibitors are in early phase clinical development ( data supplemental )  . 
although preliminary results demonstrated no responses to mdm2 inhibitors among unselected patients , 17 - 19 responses occurred in wildtype tp53 liposarcoma ( mk - 8242 ; response rate [ rr ] , 11.1% [ three of 27 patients ] ) or melanoma ( amg232 ; rr , 28.6% [ six of 21 patients ] ) .20 , 21 mdm2 amplification also has been implicated as a potential marker for accelerated tumor growth with receipt of immune checkpoint inhibitors.22 this phenomenon is called hyperprogression and affects approximately 9% of patients who receive pd - 1 / pd - l1 inhibitors.23 hyperprogression has been defined as a time to treatment failure < 2 months from checkpoint inhibitor initiation , a > 50% increase in tumor burden compared with pre - immunotherapy imaging , and a more than two - fold increase in progression pace.22 to date , accelerated progression after antipd - 1 / pd - l1 agents has been reported by at least four groups.22 - 25 although the mechanisms that mediate this phenomenon remain unclear , we and others have demonstrated that mdm2 family gene amplifications and egfr alterations correlate with hyperprogression.22 , 25 given the clinical importance of mdm2 , we describe the landscape of cancer types that harbor mdm2 amplification and evaluate the comprehensive genomic profiles of 102 , 878 tumors from patients with malignancies . 
an illustrative patient with mdm2 amplification that demonstrated hyperprogression after checkpoint blockade is presented . methods patients we explored the mdm2 amplification status of patients with diverse malignances who were referred for comprehensive next - generation sequencing ( ngs ) from june 2012 through december 2016 ( n = 102 , 878 ; table 1 ; data supplement )  . 
this study was performed in accordance with the guidelines of the university of california , san diego , institutional review board with regard to analysis and consent . tissue samples and mutational analysis tumors were provided as formalin - fixed , paraffin embedded samples and evaluated by ngs in a clinical laboratory improvement amendments certified laboratory ( foundation medicine , cambridge , ma )  . 
the methods used for ngs have been validated and previously reported.26 - 29 dna was adaptor ligated , and hybrid capture was performed for all coding exons of 182 to 465 cancer - related genes plus select introns from 14 to 31 genes frequently rearranged in cancer ( data supplement )  . 
the median number of potentially targetable genomic co - alterations with either food and drug administrationapproved or investigational agents was three ( range , zero to 17 )  . analyses except those for tumor mutation burden ( tmb )  . cancer genomic data through publicly available data sets tmb was calculated on the basis of the total number of mutations counted per megabase.30 noncoding alterations , alterations reported as known somatic alterations in catalog of somatic mutations in cancer , truncations in tumor suppressor genes , and alterations predicted to be germline were not counted . 
high tmb was defined as 20 mutations / megabase . mdm2 amplification status was also curated from the genomics evidence neoplasia information exchange ( genie ) accessed in july 2017.31 - 33 end points , statistical methods , and case study descriptive statistics were used to summarize the cancer diagnoses and genomic alterations identified in the data set . 
comparison of rate of mdm2 amplification in the current report ( n = 102 , 878 samples ) versus in genomics evidence neoplasia information exchange ( genie ; n = 13 , 473 samples ) .31 data from genie were obtained as previously described.31 the 10 most common diagnoses that harbor mdm2 amplification from the current report were selected for the comparison . 
mdm2 amplifications were seen in 3.5% ( 3 , 650 of 102 , 878 ) of patients in the current report versus 5.5% ( 744 of 13 , 473 ) from genie . 
mdm2 amplification was most commonly seen among liposarcoma ( 63.6% [ 332 of 522 ] ) followed by gallbladder , adenocarcinoma ( 11.1% [ 62 of 554 ] ) ; sarcoma , not otherwise specified ( 10.7% [ 103 of 955 ] ) ; and urothelial carcinoma ( 10.4% [ 198 of 1 , 898 ] ; table 1 )  . 
 mdm2 amplification was not found among anaplastic and papillary carcinoma of thyroid ( zero of 166 and 376 , respectively ) and uncommonly among adenocarcinoma of the appendix , rectum , and colon ( 0.23% [ one of 440 ] , 0.28% [ four of 1 , 448 ] , and 0.33% [ 28 of 8 , 562 ] , respectively ; data supplement )  . 
in comparison , according to the genie database ( total , 13 , 473 samples ) , mdm2 amplification has been reported in 5.5% genomic co - alterations associated with mdm2 amplification among 3 , 650 patients with mdm2 amplification , 99.0% ( 3 , 613 ) were found to have genomic co - alterations ( data supplement )  . 
although uncommon , mismatch repair genes and pd - l1 amplification were co - altered in 2.2% ( 79 of 3 , 650 ) of patients ( table 2 )  . among patients with mdm2 amplification ( n = 3 , 650 ) , tp53 was co - altered in 733 . 
these differences in patient subsets were not seen in the genie data set perhaps because genie had considerably fewer patients ( approximately 13% of the patients in our data set )  . we have previously reported that mdm2 amplification can be associated with hyperprogression after treatment with antipd - 1 / pd - l1 agents.22 we describe herein a 36 - year - old woman ( not previously reported ) with adenocarcinoma of the gastro - esophageal junction who had stable disease ( sd ) while receiving second - line therapy with fluorouracil , oxaliplatin , and panitumumab ( fig 3 , left and middle )  . 
 the patient had rapid progression in the mediastinal and retroperitoneal lymph nodes as well as emergent massive ascites ( time to treatment failure , 3 weeks ; pace of progression increased by 6.4 - fold compared with the 4 months before the start of checkpoint blockade , and tumor burden increased 460% compared with pre immunotherapy imaging ; fig 3 )  . 
molecular profiling of the primary tumor revealed alterations , including amplifications in mdm2 , erbb3 , araf , cdk4 , and egfr and alterations in pik3ca , frs2 , gli1 , and ikzf1 . 
pd - 1 and pd - l1 immunohistochemistry was not evaluated . discussion we and others recently demonstrated that approximately 9% of patients treated with pd - 1 / pd - l1 checkpoint blockade exhibit a paradoxical acceleration in tumor progression ( designated as hyperprogression )  . 
this phenomenon associates with mdm2 amplification.22 - 24 therefore , caution is needed in treating patients who harbor mdm2 amplification with checkpoint inhibitors , and a thorough understanding of the mdm2 alteration landscape is clinically important . 
selected co - alterations that accompany mdm2 amplification and potential targeted therapies ( continued ) co - alteration examples of potential targeted therapies * tp53 - associated genes mismatch repair genes and pd - l1 amplification cd274 ( pd - l1 ) cdk4 cdk6 ccne1 tp53 mdm4 mlh1 msh2 msh6 pms2 no . 
mdm2 amplification most commonly has been seen in patients with liposarcoma ( 63.6% [ 332 of 522 ] ) 5 but discerned in a subset of most tumor types , albeit at different frequencies ( table 1 ; data supplement )  . 
certain diagnoses ( eg , anaplastic and papillary thyroid cancer ) were not associated with mdm2 amplification , and this anomaly was rare in acute myelocytic leukemia ( one of 1 , 006 patients ; data supplement )  . an understanding of the comprehensive landscape of mdm2 amplification also is therapeutically relevant because mdm2 inhibitors are in clinical development ( data supplement )  . 
clinical activity of mdm2 inhibitors among unselected diverse cancers has been limited17 - 19 ; however , occasional responses have been observed in individuals selected for wild - type tp53.20 , 21 the low response rate with single - agent mdm2 inhibitors may be due to the lack of patient selection for mdm2 amplification or to co - altered genes ( data supplement )  . 
accumulating evidence has suggested that the matched targeted therapy approach can demonstrate better clinical outcomes than a nonmatched approach , but this implies the need to select patients for the relevant aberration.34 - 37 wagner et al21 showed that responses with mk - 8242 ( mdm2 inhibitor ) were exclusively observed in patients with liposarcoma ( rr , 11.1% [ three or 27 ] ) whose molecular hallmark includes mdm2 amplification5 ( nonliposarcoma ; rr , 0% [ zero of 14 ] )  . 
on the other hand , even in a disease such as liposarcoma where > 60% of patients have mdm2 amplification , the rr is relatively low , which may be due to , as mentioned previously , the presence of co - alterations . 
indeed , the 12q13 - 15 amplicon on which mdm2 resides is large ( but discontinuous ) ; cdk4 and frs2 reside on the amplicon and frequently are co - amplified with mdm238 but are rarely abnormal in patients without mdm2 amplification ( data supplement )  . in keeping with the notion that co - alterations are important , we also assessed the alterations that co - occurred with mdm2 amplification . 
 the majority of mdm2 - amplified tumors harbored co - alterations ( 99% [ 3 , 613 of 3 , 650 ] ) ; the median number of alterations per patient was six ( range , one to 25 ; data supplement )  . 
among patients with mdm2 amplification ( n = 3 , 650 ) , 2.9% ( 105 ) had high tmb , 23.3% ( 852 ) had intermediate tmb , and 73.8% ( 2 , 693 ) had low tmb . 
in the current data set , certain cancers with mdm2 amplification were significantly less associated with high tmb than with mdm2 wild type ( sarcoma , not otherwise specified , 0% [ zero of 103 ] v 4.2% [ 36 of 852 ] , respectively [ p = .03 ] ; urothelial carcinoma , 3.5% [ seven of 198 ] v 11.8% [ 200 of 1 , 700 ] , respectively [ p < .001 ] ; glioblastoma , 0.4% [ one of 244 ] v 4.4% [ 120 of 2 , 725 ] , respectively [ p < .001 ] ) ; these subsets did not show significant differences in genie , but the number of patient samples in genie was considerably smaller ( data supplement )  . 
because frs2 and cdk4 are on the mdm2 amplicon , the targeting of them may warrant specific study . of note , we also observed that tp53 alterations were not mutually exclusive with mdm2 amplification , as previously reported.5 , 6 although tp53 alterations were less commonly seen in patients with mdm2 amplification compared with wild type ( data supplement ) , tp53 alterations were observed in 20.1% ( 733 of 3 , 650 ; fig 2 ; data supplement )  . 
one of the proposed noncanonical roles of mdm2 is to facilitate angiogenesis.39 zhou et al10 reported that mdm2 - overexpressed / tp53 - null cancer cells are associated with increased vegf mrna expression compared with mdm2 negative / tp53 - null cells . 
within 3 weeks , the patient showed marked clinical deterioration , and imaging showed rapid progression in mediastinal and retroperitoneal lymph nodes as well as emerging massive ascites ( right )  . 
pace of progression increased by 6.4 - fold , tumor burden increased by 460% compared with pre - immunotherapy imaging , and time to treatment failure was 3 weeks ( hyperprogression after immunotherapy previously defined as more than a two - fold increase in progression pace , a > 50% increase in tumor burden compared with pre - immunotherapy imaging , and a time to treatment failure < 2 months22 )  . 
the most common co - alterations associated with mdm2 amplification were cdk4 ( 43.6% [ 1 , 591 of 3 , 650 ] ) , frs2 ( 40.8% [ 1 , 491 of 3 , 650 ] ) , tp53 ( 20.1% [ 733 of 3 , 650 ] ) , cdkn2a ( 18.2% [ 665 of 3 , 650 ] ) , and egfr ( 12.7% [ 462 of 3 , 650 ] ; data supplement )  . cdk4 frs2 tp53 cdkn2a egfr ccnd1 cdkn2b kras fgf19 gli1 fgf4 pik3ca fgf3 tert fgfr1 pten erbb3 znf217 znf703 gnas arid1a erbb2 nkx2 - 1 frequency of mdm2 amplification ( % ) amplification mutation deletion fusion / rearrangement multiple alterations tmb . 
in the current study , we depict an individual with gastric cancer that harbored egfr as well as mdm2 amplification who had indolent disease ; the patient , however , showed explosive progression after being given the antipd - 1 inhibitor nivolumab ( fig 4 )  . 
whether patients who have both pd - l1 amplification ( a marker of sensitivity to checkpoint inhibitors in hodgkin disease43 ) and mdm2 amplification would respond to checkpoint blockade is unclear . 
because high tmb correlates with checkpoint blockade responsiveness , 44 , 45 this observation may partially explain resistance to pd - 1 / pd - l1 inhibitors in mdm2 - amplified tumors but does not clarify the mechanism that underlies the correlation between mdm2 amplification and hyperprogression . 
furthermore , how patients whose tumors have high tmb as well as mdm2 amplification would fare on checkpoint inhibitors is unclear ; however , one of our previously reported patients with mdm2 amplification who demonstrated hyperprogression with antipd - l1 immunotherapy had a high tmb.22 finally , an issue that merits prospective exploration is how a combination of mdm2 and checkpoint inhibitors would affect the risk of hyperprogression in patients whose cancers bear an mdm2 amplification . the current study has several limitations . 
first , the data set was de - identified , which limited the analyzable correlates ; thus , clinical questions such as the frequencies of mdm2 amplification that depend on the disease state ( early stage v metastatic and recurrent disease ) could not be evaluated . 
second , because the number of patients in each cancer type was based on the number of samples sent for ngs by the treating physicians , sample size bias is possible . 
despite these limitations , the current study provides , to our knowledge , the largest and most comprehensive analysis of mdm2 amplification in diverse malignancies to date . in summary , we have interrogated 102 , 878 patients with diverse cancers and demonstrated that amplification of mdm2 is found in 3.5% ( 3 , 650 ) of tumors . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center  . shumei kato no relationship to disclose jeffrey s . 
ito m , barys l , oreilly t , et al : comprehensive mapping of p53 pathway alterations reveals an apparent role for both snp309 and mdm2 amplification in sarcomagenesis . 
shibagaki i , tanaka h , shimada y , et al : p53 mutation , murine double minute 2 amplification , and human papillomavirus infection are frequently involved but not associated with each other in esophageal squamous cell carcinoma . 
kovatcheva m , liu dd , dickson ma , et al : mdm2 turnover and expression of atrx determine the choice between quiescence and senescence in response to cdk4 inhibition . 
lakoma a , barbieri e , agarwal s , et al : the mdm2 small - molecule inhibitor rg7388 leads to potent tumor inhibition in p53 wild - type neuroblastoma . 
schwaederl m , lazar v , validire p , et al : vegf - a expression correlates with tp53 mutations in non - small cell lung cancer : implications for antiangiogenesis therapy . 
said r , hong ds , warneke cl , et al : p53 mutations in advanced cancers : clinical characteristics , outcomes , and correlation between progression - free survival and bevacizumab - containing therapy . 
de jonge m , de weger va , dickson ma , et al : a phase i study of sar405838 , a novel human double minute 2 ( hdm2 ) antagonist , in patients with solid tumours . 
gounder mm , bauer tm , schwartz gk , et al : a phase 1 study of the mdm2 inhibitor ds - 3032b in patients ( pts ) with advanced solid tumors and lymphomas . 
kurzrock r , blay j - y , bui nguyen b , et al : a phase i study of mdm2 antagonist rg7112 in patients ( pts ) with relapsed / refractory solid tumors . 
moschos sj , sandhu sk , lewis kd , et al : phase 1 study of the p53 - mdm2 inhibitor amg 232 combined with trametinib plus dabrafenib or trametinib in patients ( pts ) with tp53 wild type ( tp53wt ) metastatic cutaneous melanoma ( mcm )  . 
wagner aj , banerji u , mahipal a , et al : phase i trial of the human double minute 2 inhibitor mk - 8242 in patients with advanced solid tumors . 
champiat s , dercle l , ammari s , et al : hyperprogressive disease is a new pattern of progression in cancer patients treated by anti - pd - 1 / pd - l1 . 
sada - bouzid e , defaucheux c , karabajakian a , et al : hyperprogression during anti - pd - 1 / pdl1 therapy in patients with recurrent and / or metastatic head and neck squamous cell carcinoma . 
singavi ak , menon s , kilari d , et al : predictive biomarkers for hyper - progression ( hp ) in response to immune checkpoint inhibitors ( ici ) analysis of somatic alterations ( sas )  . 
wagle n , berger mf , davis mj , et al : high - throughput detection of actionable genomic alterations in clinical tumor samples by targeted , massively parallel sequencing . 
roszik j , haydu le , hess kr , et al : novel algorithmic approach predicts tumor mutation load and correlates with immunotherapy clinical outcomes using a defined gene mutation set . 
jardim dl , schwaederle m , wei c , et al : impact of a biomarker - based strategy on oncology drug development : a meta - analysis of clinical trials leading to fda approval . 
schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
mejia - guerrero s , quejada m , gokgoz n , et al : characterization of the 12q15 mdm2 and 12q13 - 14 cdk4 amplicons and clinical correlations in osteosarcoma . 
 computed tomographyderived radiomic metrics can identify responders to immunotherapy in ovarian cancer yuki himoto , md , phd1 , 2 ; harini veeraraghavan , phd1 ; junting zheng , ms1 ; dmitriy zamarin , md , phd1 , 3 ; alexandra snyder , md1 , 3 , 4 ; marinela capanu , phd1 ; stephanie nougaret , md , phd1 , 5 , 6 , 7 , 8 ; hebert a . 
disease burden ( total tumor volume , number of disease sites ) , radiomic measures of intertumor heterogeneity ( clustersite entropy , cluster - site dissimilarity ) , and intratumor heterogeneity of the largest lesion ( haralick texture features ) were computed . 
2019 by american society of clinical oncology introduction ( oc ) ovarian cancer is the deadliest gynecologic malignancy.1 , 2 most patients respond to cytoreductive surgery and platinum - based chemotherapy but eventually experience recurrence and develop chemotherapyresistant disease.3 poor outcomes relate to the loss in dna repair mechanisms , resulting in highly heterogeneous tumors that exhibit early clonal evolution and rapid onset of chemotherapy resistance.3 - 6 immunotherapy is a novel treatment paradigm that has emerged with improved understanding of the cancerimmunity cycle.7 immune checkpoint inhibitors are effective in multiple solid tumors . 
in ovarian cancer , early multiregion dissemination and profound tumor heterogeneity ( th ) limit the discovery of predictive biomarkers on the basis of the tissue samples from few individual tumors . 
we aimed to explore the potential of quantitative analysis of standard - of - care computed tomography ( ct ) to noninvasively capture spatial th and predict response to immunotherapy . knowledge generated quantitative analysis of pretreatment ct including radiomic measures of th may facilitate the delivery of precision medicine by identifying patients who derive most benet from immunotherapy . 
indeed , fewer disease sites and lower image - based intrath were two independent indicators of durable clinical benet ; higher inter - th was a single independent indicator of shorter time to off - treatment . relevance number of disease sites and radiomic measures of th derived from baseline ct are potential noninvasive predictive imaging biomarkers of response that require additional exploration in the context of multimodal approaches to ovarian cancer . medical decisions , including the selection of patients appropriate for treatment with chemoimmunotherapy . in patients with oc , contrast - enhanced computed tois the standard - of - care imaging to mography ( ce - ct ) evaluate disease extent at all phases of patient management , including initial diagnosis , post - treatment surveillance , disease recurrence , and assessment of response . although the response evaluation criteria in solid tumors ( recist ) version 1.1 and immune - related response criteria are applied to assess response to immunotherapies , there is a paucity of predictive biomarkers of response to chemotherapy , targeted therapy , and immunotherapy to facilitate the selection of rational individualized combination therapies.17 - 19 the potential of radiomics to model intraand inter - th on imaging to predict the response of oc to immunotherapy is an unexplored area . 
progression was dened as clinical deterioration and / or imaging progression according to immune - related recist.19 time to off - treatment was dened as the number of days between the rst treatment date and the off - study date . 
durable clinical benet was dened as progression - free survival of 24 or more weeks.20 - 22 ct analysis baseline ce - ct acquisition parameters , segmentation of the entire tumor burden , and computation of volumes of interest ( vois ) are described in the appendix . 
all locations were mutually exclusive . extraction of radiomic measures from baseline ct scans texture features were extracted directly from ct images without additional preprocessing to avoid imaging blur and spurious features . 
five intra - th measures ( haralick texture features ) derived from the lesion with the largest volume and two inter - th measures ( cluster - site entropy , clustersite dissimilarity ) were computed following the steps described here ( fig 1 ) .23 step 1 : per - voxel haralick texture features . 
of disease sites , locations with disease , total tumor volume intratumor heterogeneity computed from the lesion with the largest volume contrast correlation energy entropy homogeneity intertumor heterogeneity cluster - site entropy cluster - site dissimilarity clinical variables histology figo stage cytoreductive outcome platinum sensitivity status prior lines of chemotherapy fig 1 . 
the above per - voxel haralick texture measures were then used to compute intra - th measures ( step 2 ) and inter - th measures ( step 3 )  . step 2 : intratumor heterogeneity measures . 
we chose the lesion with the largest volume on the basis of the results of prior radiomic studies.26 , 27 intra - th measures were summarized as the mean of all per - voxel haralick texture features within voilargest - lesion . 
first , per - voxel haralick textures within all segmented tumors were clustered together to produce subregions within all lesions using the kernel k - means clustering method , where each subregion represents a group of voxels with highly similar texture values . 
we then described each subregion by the mean of all per - voxel haralick texture values within that subregion . second , pairwise subregion dissimilarities were computed as the euclidean distance between the subregion pairs , where each subregion was represented by the mean textural values inside that subregion . 
a highly textured dissimilarity map points to greater frequency of dissimilarities between subregions , whereas more a homogeneous map suggests less frequent dissimilarities between subregions . third , a group dissimilarity matrix was computed from the dissimilarity map for each patient . 
group dissimilarity matrix is a two - dimensional histogram that captures the frequency of dissimilarities between subregions and the number of neighboring dissimilar subregions . cluster - site entropy ( cse ) was computed directly from the all subregion pair dissimilarities . 
it captures the variability in dissimilarities between subregions and is calculated using the shannon entropy formula : cse ( cid : 3 ) 1 ( cid : 1 ) n n ( cid : 3 ) 1 ( cid : 1 ) ( cid : 3 ) log2 ( p ( dn ) ) , where n is the number of subregion pairs . cluster - site dissimilarity ( cludiss ) was derived from the group dissimilarity matrix . 
a sensitivity analysis was performed using crossvalidated least absolute shrinkage and selection operator ( lasso ) regression including all variables with 200 random repetitions.29 the variables in nal multivariable models were also selected more than 90% of the time by lasso regression . 
model discriminability was assessed with the receiver operating characteristic analysis and internally validated concordance statistics ( c - index ) .30 in multivariable logistic regression , bias - correct c - index was estimated using the bootstrapping technique . 
the r code used in the analysis is available for download through com / harveerar / jco - po - ovaryimmunotherapy . the correlations between the measures signicant at multivariable analyses ( number of disease sites , intra - th [ energylargest - lesion ] and inter - th [ cluster - site dissimilarity ] ) were tested with spearman correlation coefcient ( rs )  . 
 ( % ) unless otherwise noted . abbreviations : ce - ct , contrast - enhanced computed tomography ; figo , international federation of gynecology and obstetrics ; ln , abdominopelvic lymphadenopathy ; pd , peritoneal disease ; sd , standard deviation . results patients patient clinical characteristics are listed in table 1 . 
axial baseline computed tomography ( ct ) images from two different patients with energy ( a measure of intratumor heterogeneity ) overlaid on segmented tumors ( arrow points to the largest lesion )  . 
two box plots illustrate the univariable associations of baseline ct - derived number of disease sites and energylargest - lesion ( a measure of intratumor heterogeneity ) with durable clinical benet ( progression - free survival 24 weeks )  . 
ideally , these biomarkers should be based on noninvasive or minimally invasive approaches to capture / sample th and enable individualized treatment decisions in oc . our study highlights a novel noninvasive method to model spatial th via radiomic analysis of ct and demonstrates that quantitative image - based measures relate to clinical outcomesspecically , the prediction of response to immunotherapy . 
we found that fewer disease sites and lower intra - th of the largest lesion ( higher energylargest - lesion ) were the only indicators of durable clinical benet at multivariable analysis . 
combined fewer disease sites and lower intra - th ( higher energylargest - lesion ) were a composite indicator of durable clinical benet , with a c - index of 0.821. notably , none of the patients with more than seven disease sites experienced durable clinical benet . 
higher inter - th ( higher cluster - site dissimilarity ) was the only indicator of shorter time to off - treatment at multivariable analysis . clinical variables , ttv , and locations of disease on ce - ct were not predictive of response . 
plots of receiver operating characteristic curves demonstrating the discriminatory ability of the model to identify patients with durable clinical benet on the basis of number of disease sites , energylargest lesion , or both . delivery of precision medicine to patients with oc by identifying patients who may benet from immunotherapy . likely require additional immunotherapy has transformed the treatment of several solid and hematologic malignancies , but the efcacy varies across tumor types and patients.10 oc exhibits poor response to immune checkpoint inhibitors , with the biomarkers of response and resistance remaining unexplored . effective implementation will renement of sights into cancer - immune interactions , immune - response criteria , and development of multifactorial predictive biomarkers ( eg , cancer immunogram ) to personalize the selection of immunotherapy.32 , 33 at present , predictive biomarkers of response to immunotherapy include pd - 1 expression , tumor mutational / neoantigen burden assessment , tumor - inltrating lymphocyte evaluation , and immune gene expression assays.34 nevertheless , composite multimodal multifaceted biomarkers that noninvasively capture spatiotemporal th will likely be necessary to comprehensively assess immune tme and serve as clinical decision support for prognosis inference and prediction of response . th is an important element to consider and quantify in any oc cancer immunogram , because lower th has been associated with improved prognosis and better response to chemotherapy and immunotherapy.5 , 11 , 12 two recent articles highlight the importance of spatial th for accurate characterization of immune tme in oc . 
the major nding was that the immune tme varied in space and potentially shaped clonal selection in patients with newly diagnosed hgsoc . ce - ct is the standard - of - care imaging approach that is widely used for the evaluation of patients with oc . 
although baseline ce - ctderived predictive biomarkers of response to immunotherapy would be useful as clinical decision support tools , the interplay of radiomic measures with clinical outcomes in oc remains largely unexplored . 
few studies to date have evaluated the role of radiomics to model spatial th and capture immune tme.33 , 35 vargas et al35 proposed a novel method to compute interth on ce - ct and found higher inter - th to associate with shorter overall survival and greater risk of incomplete surgical resection in 38 patients with newly diagnosed advanced - stage hgsoc . 
in contrast to the approach used by vargas et al , 35 we computed inter - th between lesions rather than anatomic sites of disease for more comprehensive assessment of th . 
similar to vargas et al , 35 we demonstrated that higher inter - th measured on the baseline ce - ct was associated with worse outcomes , as indicated by shorter time to offtreatment . although the assessment of image - based inter - th is a promising approach , at present , the translation to routine trials is limited by laborclinical practice and clinical intensive multiregion tumor segmentation and complex computational analysis across multiple tumors . 
in the future , deep learning techniques may enable accurate semiautomated tumor delineation reducing manual effort . in the meantime , we also examined the value of such readily accessible quantitative measures as number of disease sites , ttv , and intra - th of lesion measured from the baseline ce - ct . 
we found fewer disease sites and lower intra - th of the largest lesion ( indicated by higher energylargest - lesion ) to be signicant indicators of durable clinical benet at multivariable analysis . 
this nding is potentially explained by a positive relationship between the number of disease sites and inter - th ( rs , 0.842 ) , with more disease sites increasing the probability of inter - th and reducing the likelihood of durable clinical benet . 
our study suggests that the number of disease sites is a measure that deserves additional investigation and potentially indirectly reects spatial th in oc . ttv on baseline ce - ct was not predictive of response to immunotherapy in our cohort . 
recognizing the substantial limitation of this measure in assessing tumor burden , peritoneal bulky disease was not predictive of chemotherapy response.36 , 37 the role of tumor volume / burden as a prognostic measure and a predictor of benet from immunotherapy requires additional investigation . 
in contrast , sun et al33 reported that tumor volume was not predictive of overall survival in patients with various cancer types treated with antipd - 1 and antipdl1 immunotherapy . only one prior study examined the interplay between radiomic analysis , effector t cells , tumor immune phenotypes , and response to immunotherapy.33 in this study , sun et al33 developed a ce - ctderived radiomic signature of cd8 cells by segmenting and extracting image - based features from either biopsied or largest lesion in patients with one of ve advanced solid malignant tumors . 
they then proceeded to validate their radiomic signature of cd8 cells in two independent cohorts and demonstrate the associations of radiomic signature of cd8 cells with tumor immune phenotype and response to immunotherapy , respectively . 
second , given the hypothesisgenerating nature of our study , a single experienced radiologist segmented all tumor on each baseline scan . automated or semi - automated methods to segment oc implants are currently absent . 
third , a small set of wellestablished image features ( ie , haralick textures ) were extracted because of the relatively small sample size and focus on image - based inter - th , a measure computed on the basis of haralick features.35 larger numbers of features , including new features ( eg , co - occurence of local anisotropic gradient orientations [ coliage ] ) , should be explored further , but require a large data set ( independent training and validation cohorts ) to reliably estimate the models performance . this will in conclusion , the number of disease sites and radiomic measures of th from baseline ce - ct are potential predictive imaging biomarkers of responsiveness to immunotherapy . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . dmitriy zamarin employment : acorda therapeutics ( i ) consulting or advisory role : biomed valley discoveries , merck , psioxus therapeutics , synlogic , western oncolytics , tesaro , agenus , trieza therapeutics , acm biolabs research funding : merck patents , royalties , other intellectual property : i hold a patent regarding the use of recombinant newcastle disease virus ( ndv ) for cancer therapy ( inst ) travel , accommodations , expenses : roche alexandra snyder employment : adaptive biotechnologies , merck stock and other ownership interests : merck consulting or advisory role : driver group research funding : bristol - myers squibb travel , accommodations , expenses : genentech , bristol - myers squibb margaret callahan employment : bristol - myers squibb ( i ) , celgene ( i ) , kleo pharmaceuticals ( i ) , bristol - myers squibb ( i ) consulting or advisory role : astrazeneca , moderna therapeutics , merck research funding : bristol - myers squibb ( inst ) other relationship : clinical care options , potomac center for medical education evis sala honoraria : siemens healthineers speakers bureau : siemens healthineers travel , accommodations , expenses : siemens healthineers yulia lakhman stock and other ownership interests : y - mabs therapeutics no other potential conicts of interest were reported . acknowledgment we thank joanne chin , mfa , els , for her editorial assistance with this manuscript . references torre la , trabert b , desantis ce , et al : ovarian cancer statistics , 2018 . 
nature 474 : 609 - 615 , 2011 [ erratum : nature 490 : 298 , 2012 ] schwarz rf , ng ck , cooke sl , et al : spatial and temporal heterogeneity in high - grade serous ovarian cancer : a phylogenetic analysis . 
plos med 12 : e1001789 , 2015 bashashati a , ha g , tone a , et al : distinct evolutionary trajectories of primary high - grade serous ovarian cancers revealed through spatial mutational proling . j pathol 231 : 21 - 34 , 2013 chen ds , mellman i : elements of cancer immunity and the cancer - immune set point . 
science 359 : 1350 - 1355 , 2018 jim enez - s anchez a , memon d , pourpe s , et al : heterogeneous tumor - immune microenvironments among differentially growing metastases in an ovarian cancer patient . 
shukuya t , mori k , amann jm , et al : relationship between overall survival and response or progression - free survival in advanced non - small cell lung cancer patients treated with anti - pd - 1 / pd - l1 antibodies . 
yoo ts , ackerman mj , lorensen we , et al : engineering and algorithm design for an image processing api : a technical report on itk - - the insight toolkit . 
rao sx , lambregts dm , schnerr rs , et al : ct texture analysis in colorectal liver metastases : a better way than size and volume measurements to assess response to chemotherapy ? united european gastroenterol j 4 : 257 - 263 , 2016 27 . 
lubner mg , stabo n , lubner sj , et al : ct textural analysis of hepatic metastatic colorectal cancer : pre - treatment tumor heterogeneity correlates with pathology and clinical outcomes . 
apte ap , iyer a , crispin - ortuzar m , et al : technical note : extension of cerr for computational radiomics : a comprehensive matlab platform for reproducible radiomics research . 
sun r , limkin ej , vakalopoulou m , et al : a radiomics approach to assess tumour - inltrating cd8 cells and response to anti - pd - 1 or anti - pd - l1 immunotherapy : an imaging biomarker , retrospective multicohort study . 
vargas ha , veeraraghavan h , micco m , et al : a novel representation of inter - site tumour heterogeneity from pre - treatment computed tomography textures classies ovarian cancers by clinical outcome . 
gordon an , tonda m , sun s , et al : long - term survival advantage for women treated with pegylated liposomal doxorubicin compared with topotecan in a phase 3 randomized study of recurrent and refractory epithelial ovarian cancer . 
gynecol oncol 95 : 1 - 8 , 2004 joseph rw , elassaiss - schaap j , kefford r , et al : baseline tumor size is an independent prognostic factor for overall survival in patients with melanoma treated with pembrolizumab . 
all baseline contrast - enhanced computed tomography scans ( ce - cts ) were acquired using multidetector ct scanners with 16 to 256 rows of detectors ( ge healthcare , chicago , il )  . 
five of 75 scans were performed at outside institutions and digitized into the institutional picture archiving and communication system ( centricity pacs ; ge healthcare , chicago , il )  . 
using the insight segmentation and registration toolkitsnap version 3.4 software , the radiologist traced the outer contour of each lesion on every axial slice with tumor ( yushkevich , et al : neuroimage 31 : 1116 - 1128 , 2006 )  . 
regions of interest ( rois ) belonging to the same lesion ( roilesion ) and originating from the set of contiguous craniocaudal images in the same anatomic location were summed to create the volume of interest for that lesion ( voilesion )  . 
volumes of interest for lesions with noncontiguous rois were obtained by summing the volumes of various groups of contiguous rois in the same ( cid : 3 ) ( cid : 1 ) n ( cid : 3 ) 1 anatomic location as follows : voitotal lesion voin lesion , where n is the number of groups of contiguous rois belonging to the same lesion . the radiomic features for the lesions were represented as a matrix of features with the row elements corresponding to the voxels enclosed within all the rois belonging to that lesion and the columns corresponding to the individual haralick textures . description of haralick texture features energy captures the average frequency of co - occurring ct intensities between neighboring voxels within a tumor , such that tumors with more homogeneous appearance have higher energy . 
correlation measures the frequency of positive or negative correlation of ct intensities between neighboring voxels within a tumor . initial patient cohort patients with recurrent oc who received pd - 1 or pd - l1 targeting antibodies alone or in combination with other immunotherapy ( n = 91 ) patients enrolled in ongoing single - arm prospective trials ( n = 90 ) patient treated off - label ( n = 1 ) exclusions ( n = 16 ) patients with absent baseline ce - ct ( n = 10 ) patients with image artifacts from orthopedic hardware ( n = 2 ) patients discontinued trial because of immune - mediated toxicity ( n = 4 ) final patient cohort patients included in each analysis ( n = 75 ) durable clinical benefit ( pfs 24 weeks ) time to off - treatment fig a1 . 
 radiomic predictors of response to immunotherapy baseline ct cluster - site dissimilarity , 1 , 648 ; time to off - treatment , 569 days baseline ct cluster - site dissimilarity 568 , 399 ; time to off - treatment , 42 days fig a2 . 
 pathogenic variants in less familiar cancer susceptibility genes : what happens after genetic testing ? purpose as genetic testing expands , patients are increasingly found to carry pathogenic variants in cancer susceptibility genes that are less familiar to most clinicians , specifically genes other than those causing hereditary breast ovarian cancer syndrome ( brca1 and brca2 ) and lynch syndrome . 
little is known about the subsequent behaviors of such patients in terms of managing cancer risks and informing relatives . methods all adult patients who were counseled and tested at the stanford cancer genetics clinic from january 2013 to july 2015 and had a pathogenic variant in a nonbrca1 / 2 , nonlynch syndrome gene were invited to participate in a telephone interview about adherence to risk - reducing recommendations , genetic testing by relatives , and new cancer incidence . results fifty - seven ( 40% ) of 142 eligible patients were successfully contacted , and all 57 patients participated ; median follow - up was 677 days ( range , 247 to 1 , 401 days )  . 
most patients ( 82% ; 95% ci , 70% to 90% ) recalled that a risk - reducing intervention ( screening , medication , or surgery ) was recommended , and most patients ( 85% ; 95% ci , 72% to 93% ) adhered to the recommendation . 
nearly all patients ( 91% ; 95% ci , 81% to 97% ) shared results with relatives , and most patients ( 78% ; 95% ci , 64% to 88% ) reported that a relative was subsequently tested . 
during the follow - up period , 9% of patients ( 95% ci , 3% to 19% ) developed second cancers , and in 14% of patients ( 95% ci , 7% to 26% ) , a first - degree relative developed cancer , some of which were detected by recommended screening . conclusion patients with a pathogenic variant in a less familiar cancer susceptibility gene report high adherence to risk - reducing interventions . 
furthermore , in the 57 carriers and subsequently tested relatives with two years of follow - up , a total of three cancers ( one in a proband and two in relatives ) were detected through interventions recommended on the basis of the pathogenic variant . 
 germline genetic testing is increasingly used to explain unusual or early - onset cancers in patients and to identify at - risk family members for targeted risk - reducing interventions . 
expert societies such as the national comprehensive cancer network and asco have developed guidelines to assist clinicians in determining which patients should have germline genetic testing.1 - 3 as guidelines have become more inclusive and the cost of genetic testing has decreased , pathogenic variants are increasingly discovered in those without a personal history of cancer , which can present a challenge for clinicians . 
 germline pathogenic variants in less familiar cancer genes ( eg , non - brca1 / 2 , nonlynch syndrome genes [ non - bl genes ] ) are increasingly recognized as determinants of cancer susceptibility . 
however , lack of clinician and patient familiarity with these lesser known genes may result in lower patient adherence to cancer risk reduction recommendations than with better characterized genes.4 in addition , little is known about how patients with mutations in less familiar genes disseminate and share results with their families . we aimed to assess the real - world outcomes and downstream impact of germline genetic testing on patients found to have pathogenic variants in non - bl genes . 
a cohort of such patients was interviewed about their understanding of cancer risks ; adherence to risk - reducing recommendations such as medications , surgery , or screening ; and downstream effects , including results communication to relatives and second cancer occurrence . methods study population we identified all adult ( 18 years old ) patients seen at the stanford cancer genetics clinic ( stanford , ca ) between january 2013 and july 2015 who had a pathogenic or likely pathogenic variant in a non - bl gene ( with lynch syndrome genes defined as mlh1 , msh2 , msh6 , pms2 , and epcam ) on germline clinical genetic testing . 
 variants were defined according to the report of clinical testing provided by clinical laboratory improvements amendmentcertified laboratories , according to guidelines of the american college of medical genetics.5 most patients presented to the clinic because they met national comprehensive cancer network guidelines for cancer genetics referral.1 survey design a short ( 10to 15 - minute ) phone survey was designed to address the following three major areas : first , did patients accurately recall and adhere to specific risk - reducing recommendations made by the cancer genetics clinic ? second , did family members complete recommended genetic testing ? third , did either the patient or any first - degree family members develop new cancers after genetics consultation ? if so , were these new cancers identified as a result of intensified screening as recommended by the clinic on the basis of genetic testing results ? patients were also interviewed about obstacles they encountered in completing risk - reducing recommendations or sharing results with their relatives . 
if he or she was not reached within three phone calls , he or she was sent a secure message through the electronic medical record patient portal with the contact information of the research teainformed consent was obtained by telephone before administration of the survey . 
this included race or ethnicity ( classified as non - hispanic [ nh ] white , hispanic , asian , ashkenazi jewish , native american , african american , or other ) , for which patients could elect multiple races or ethnicities if applicable . 
 information regarding personal and family history of cancer and recommendations made by the cancer genetics clinic were also collected from the electronic medical record . analysis patient demographic characteristics and personal and family history of cancer were tabulated . 
of the 57 patients contacted , 38 ( 67% ) were nh white , 11 ( 19% ) were hispanic , seven ( 12% ) were nh asian , three ( 5% ) were nh ashkenazi jewish , two ( 4% ) were nh native american , one ( 2% ) was nh african american , and two ( 4% ) were other . 
among the three patients with nbn mutations , one had the founder mutation c657_661del ( p.lys219asnfs * 16 ) , whereas the other two patients had other variants . overall , 47 interviewed patients ( 82% ; 95% ci , 70% to 90% ) recalled having any intervention recommended during their genetics consultation visit . 
clinical recommendations included changes in cancer screening ( 79% ; 95% ci , 67% to 88% ) , preventive surgery ( 14% ; 95% ci , 7% to 26% ) , and / or preventive medication ( 11% ; 95% ci , 5% to 21% ; fig 1a )  . 
forty - six patients ( 81% ; 95% ci , 68% to 89% ) recalled being recommended to undergo an intervention that was not already clearly indicated by their personal risk and / or family history of cancer . the most common changes recommended are listed in table 3 . 
the most frequently recommended surveillance strategies included increased frequency of colonoscopy ( n = 33 ; 58% [ 95% ci , 45% to 70% ] ) , annual breast magnetic resonance imaging ( mri ; n = 25 ; 44% [ 95% ci , 32% to 57% ] ) , upper endoscopy at regular intervals ( n = 9 ; 16% [ 95% ci , 8% to 28% ] ) , and annual full - body mri ( n = 4 ; 7% [ 95% ci , 2% to 17% ] )  . 
of these , five patients ( 9% ) were recommended to undergo mastectomy , five patients ( 9% ) were recommended to undergo bilateral salpingo - oophorectomy , and one patient was recommended to undergo hysterectomy . 
 ( b ) patient - reported adherence to recommendations , rate of disclosure to any family members , and reported further testing by at - risk family members . basis of personal or family cancer history in the absence of genetic testing results . most patients ( 85% ; 95% ci , 72% to 93% ) who were recommended to undergo the risk reduction measures listed in table 3 reported adhering to these recommendations ( fig 1b )  . 
reasons patients gave for not completing risk - reducing recommendations included difficulty scheduling procedures , pursuing a holistic approach , and contemplating surgical risks . analysis of patient behavior as it pertained to penetrance of the genetic variant is shown in table 4 . 
genes were categorized as high penetrance ( myh [ biallelic ] , palb2 , apc , tp53 , cdh1 , pten , sdhb , cdkn2a , and flcn ) , moderate penetrance ( chek2 , atm , nbn founder mutation , rad51c , and rad51d ) , and low penetrance ( myh [ monoallelic ] , mre11a , rad50 , apc i1307k variant , other nbn mutations , and chek2 i157t variant ) , as shown in appendix table a1 . 
adherence was high in all penetrance groups , without significant differences . nine percent ( 95% ci , 3% to 19% ) of patients reported a new incident cancer during the follow - up period , all of whom had reported adherence to risk reduction recommendations . 
 among these patients , one of the incident cancers , a breast cancer in a patient with a palb2 pathogenic variant , was detected because of screening recommendations ( breast mri ) made during genetics consultation . 
the other four cancers were detected incidentally and included the following : meningioma in a patient with a pten pathogenic variant , basal cell skin cancer in a patient with an nbn pathogenic variant ( nonfounder mutation ) , renal cell carcinoma in a patient with a monoallelic myh pathogenic variant , and chronic lymphocytic leukemia in a patient with an nbn pathogenic variant ( nonfounder mutation )  . 
no new cancers were reported among participants who were not undergoing recommended screening . seventy - nine percent ( 95% ci , 67% to 88% ) of patients recalled being counseled to advise family members to undergo genetic testing . 
 although most patients ( 91% ; 95% ci , 81% to 97% ) shared the testing results with their family members , some probands did not share their genetic testing results with any family members despite being advised to do so . 
of patients ( n = 57 ) % ( 95% ci ) increased surveillance increased frequency of colonoscopy screening annual breast magnetic resonance imaging upper endoscopy whole - body magnetic resonance imaging risk - reducing surgery bilateral mastectomy bilateral salpingooophorectomy risk - reducing medication tamoxifen aromatase inhibitor 58 ( 45 to 70 ) 44 ( 32 to 57 ) 16 ( 8 to 28 ) 7 ( 2 to 17 ) 9 ( 3 to 19 ) 9 ( 3 to 19 ) 9 ( 3 to 19 ) 2 ( 0 to 10 ) family member undergo subsequent genetic testing ( fig 1b )  . 
reported reasons why family members chose not to undergo genetic testing included elderly age , difficulty with insurance coverage , planning for testing in the future , and unable to get genetic testing overseas . 
 four probands reported that some relatives either explicitly refused to hear the probands genetic testing results or dismissed and ignored the results immediately upon being notified . in the time between their genetics clinic visit and study participation , eight patients ( 14% ; 95% ci , 7% to 26% ) reported that first - degree relatives were newly diagnosed with cancer . 
among the group of eight newly diagnosed relatives , two ( 25% ; 95% ci , 6% to 60% ) had cancers that were detected in screening recommended after identification of the familial pathogenic variant in the relative ( one with colon cancer who was undergoing recommended screening after an apc pathogenic variant [ high penetrance ] was detected and one with breast cancer who was undergoing intensified screening including breast mri after identification of a nbn pathogenic variant [ founder mutation ] )  . 
in the case of the apc carrier who developed colon cancer , the genetic testing results were instrumental to her receiving the colonoscopy that detected the cancer ; she would not have qualified for colonoscopy without the detected pathogenic variant because of her young age ( mid - 30s )  . 
 % ( 95% ci ) shared genetic testing results with family members at least one member of family received cascade genetic high penetrance moderate penetrance low penetrance high penetrance moderate penetrance low penetrance testing high penetrance moderate penetrance low penetrance 18 / 20 14 / 16 8 / 11 23 / 23 16 / 19 13 / 15 17 / 21 10 / 16 10 / 12 90 ( 69 to 98 ) 88 ( 63 to 98 ) 73 ( 43 to 91 ) 100 ( 83 to 100 ) 84 ( 62 to 95 ) 87 ( 61 to 98 ) 81 ( 59 to 92 ) 63 ( 39 to 82 ) 83 ( 54 to 97 ) note . 
thus , understanding how patients and their families react to identification of such less familiar , non - bl pathogenic variants is increasingly important for oncology practice . a limitation of our study is the relatively low participation rate ( 40% ) , which was a function of difficulty in reaching the study population after a median of 677 days since their clinical evaluation . 
it is reassuring , however , that all patients who were successfully contacted ( n = 57 ; 40% of the 142 eligible patients ) agreed to participate in the study , which may somewhat reduce selection bias . 
although generalizability may be limited , several striking results of this study suggest a potential benefit for counseling and testing of non - bl genes and warrant confirmation in larger studies . most notably , at 2 years after genetics consultation , we observed high patient adherence to gene - specific recommendations for cancer riskreducing interventions and dissemination of results to relatives . 
this is encouraging because it suggests that genetic testing not only had its intended effect in patients seen in the clinic , but may also have benefited their relatives in a cascade effect . 
other studies of women at high breast cancer risk suggest that self - reported adherence to recommended interventions are in line with our results ( 59% reported full adherence ) .6 within a relatively short follow - up interval , nearly 10% of patients in our small sample developed a second malignancy . 
 participants reported several new cancers in first - degree relatives , some identified because of screening interventions that were prompted by detection of the familial pathogenic variant after recommended cascade genetic testing . 
this emphasizes the potential benefits of cascade testing among relatives and subsequent risk reduction interventions . nearly one quarter of participants reported that relatives did not have genetic testing , despite clinicians recommendations and patients reported sharing of genetic information . 
this represents a significant opportunity for improvement in cascade genetic testing of relatives , which is an essential component of population - wide cancer prevention.7 - 9 patients offered several reasons for the lack of subsequent testing , including incomplete dissemination ( some patients did not share genetic information with all at - risk family members , in some cases because of their own fear of stigma or limited understanding ) , lack of patient contact with family members , lack of access to genetic counseling or testing by family members , 10 cost or insurance barriers to testing by family members , and a perceived stigma associated with pathogenic variants . 
it is also possible that family members may have been tested but did not share this information with the proband , because our contact was exclusively with the proband in this study . the observed dissemination of genetic testing results to at - risk family members in this study is concordant with prior research on brca1 / 2 and lynch syndrome genes . 
healey et al11 found that most patients disclosed their results to at least one at - risk family member ; larger family size , higher psychological distress score in the carrier , and broader geographic distribution of at - risk family members were associated with lower rates of fully informed families . 
earlier studies have suggested that patients are more likely to contact first - degree family members than more distant relatives ( eg , cousins ) with brca1 / 2 results.12 we found that the reported genetic testing rate among relatives was notably less than the reported rate of these relatives being informed of the results , consistent with studies of brca1 / 2 and lynch syndrome gene pathogenic variants.13 , 14 our finding of a similar conundrum among patients with less familiar pathogenic variants suggests the need for more effective , widely accessible approaches to cascade genetic testing of family members . our study has some limitations , in addition to the previously noted participation rate of 40% . 
 patients who could be successfully contacted and offered participation ( all of whom agreed to participate ) may have been more likely to adhere to recommendations than those who could not be contacted , so these results may overestimate adherence and the downstream impact of testing . 
for some patients , we could not verify adherence to recommended interventions directly because they received their longitudinal care in a different medical system than the tertiary center at which they had genetic testing , so it is possible that the true rates of adherence are less than reported here . large population - based studies will be crucial to understand the real - world outcomes of germline multiple - gene panel testing and its contribution to precision oncology . 
as more is learned about the prevalence , penetrance , and cancer phenotype of the less familiar pathogenic variants studied here , a richer evidence base will develop to inform practice guidelines for gene - specific cancer risk reduction . 
novel care delivery models , such as dedicated follow - up programs for pathogenic variant carriers to assess adherence , barriers to risk - reducing interventions , and cascade testing of relatives , will be essential to fulfill the promise of germline genetic testing . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . divya parikh no relationship to disclose jennifer l . 
daly mb , pilarski r , berry m , et al : nccn guidelines insights : genetic / familial high - risk assessment : breast and ovarian , version 2.2017. 
desjardin jt , dhawan ms , blanco a , et al : effects of gene penetrance on adherence to breast cancer screening recommendations ( bcsr ) among high - risk women . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
vetter l , keller m , bruckner t , et al : adherence to the breast cancer surveillance program for women at risk for familial breast and ovarian cancer versus overscreening : a monocenter study in germany . 
samimi g , bernardini mq , brody lc , et al : traceback : a proposed framework to increase identification and genetic counseling of brca1 and brca2 mutation carriers through familybased outreach . 
caswell - jin j , zimmer a , stedden w , et al : cascade genetic testing of relatives for hereditary cancer risk : first year of a low - cost , accessible family testing program.presented at the american college of medical genetics and genomics annual meeting , charlotte , nc , april 13 , 2018 10 . 
fehniger j , lin f , beattie ms , et al : family communication of brca1 / 2 results and family uptake of brca1 / 2 testing in a diverse population of brca1 / 2 carriers . 
 revisiting epidermal growth factor receptor ( egfr ) amplification as a target for anti - egfr therapy : analysis of cell - free circulating tumor dna in patients with advanced malignancies shumei kato ryosuke okamura manvita mareboina suzanna lee aaron goodman sandip p . 
contributed equally to this work . corresponding author : shumei kato , md , center for personalized cancer therapy and division of hematology and oncology , department of medicine , uc san diego moores cancer center , 3855 health sciences dr , la jolla , ca 92093 ; e - mail : smkato@ucsd.edu. licensed under the creative commons attribution 4.0 license purpose to date , evidence for tissue epidermal growth factor receptor ( egfr ) overexpression as a biomarker for anti - egfr therapies has been weak . 
we investigated the genomic landscape of egfr amplification in blood - derived cell - free tumor dna ( cfdna ) across diverse cancers and the role of anti - egfr therapies in achieving response . methods we assessed egfr amplification status among 28 , 584 patients with malignancies evaluated by clinical - grade next - generation sequencing ( ngs ) of blood - derived cfdna ( 54to 73 - gene panel )  . 
furthermore , we curated the clinical characteristics of 1 , 434 patients at the university of california san diego who had cfdna testing by this ngs test . results overall , egfr amplification was detected in cfdna from 8.5% of patients ( 2 , 423 of 28 , 584 ) , most commonly in colorectal ( 16.3% [ 458 of 2 , 807 ] ) , nonsmall - cell lung ( 9.0% [ 1 , 096 of 12 , 197 ] ) , and genitourinary cancers ( 8.1% [ 170 of 2 , 104 ] )  . 
most patients had genomic coalterations ( 96.9% [ 95 of 98 ] ) , frequently involving genes affecting other tyrosine kinases ( 72.4% [ 71 of 98 ] ) , mitogen - activated protein kinase cascades ( 56.1% [ 55 of 98 ] ) , cell - cycleassociated signals ( 52.0% [ 51 of 98 ] ) , and the phosphoinositide 3 - kinase pathway ( 35.7% [ 35 of 98 ] )  . 
egfr amplification emerged in serial cfdna after various anticancer therapies ( n = 6 ) , including checkpoint inhibitors ( n = 4 ) , suggesting a possible role for these amplifications in acquired resistance . 
nine evaluable patients with egfr amplification were treated with anti - egfrbased regimens ; five ( 55.6% ) achieved partial responses , including three patients whose tissue ngs lacked egfr amplification . conclusion egfr amplification was detected in cfdna among 8.5% of 28 , 584 diverse cancers . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction epidermal growth factor receptor ( egfr ) , also known as human epidermal growth factor receptor 1 ( her1 ) or erbb1 , is a receptor tyrosine kinase that belongs to the erbb family proteins . 
 phosphorylation of tyrosine residues in the egfr intracellular domaactivation of egfr leads to multiple downstream signals , including mitogen - activated protein kinase and phosphoinositide 3 - kinase pathways , which enhances cell proliferation and survival.1 , 2 functional activation of egfr via mutation or amplification / overexpression has been identified in many tumor types , including lung , head and neck , gastroesophageal , and colorectal cancers , and has been associated with proliferation , invasion , and metastasis.3 , 4 alterations in egfr have also been linked to primary resistance and accelerated tumor growth ( designated as hyperprogression ) from immune checkpoint inhibitors.5 - 7 because of its critical role in tumor aggressiveness , egfr has been an attractive target for anticancer therapy.1 to date , there are various anti - egfr therapies that are us food and drug administration approved , including erlotinib , gefitinib , afatinib , and osimertinib for nonsmall - cell lung cancer ( nsclc ) with specific activating egfr mutations , 8 cetuximab and panitumumab for colorectal cancer without kras or nras mutations , 9 cetuximab for head and neck cancer , 10 and necitumumab for squamous cell carcinoma of lung.11 biomarkers to predict response to anti - egfr therapies have been studied extensively . 
egfr and kras mutation status are widely used in lung and colorectal cancer , respectively.8 , 9 , 12 , 13 in contrast , egfr amplification and overexpression in tissue have not been well established as reliable biomarkers for anti - egfr agents , ( selected studies that investigated egfr amplification / overexpression as a predictive marker for anti - egfr therapies are summarized in the data supplement ) 11 , 14 - 19 . 
overall , a meta - analysis concluded that tissue egfr amplification status could not be demonstrated to be a consistent biomarker to predict the outcome from antiegfr therapies in colorectal cancer.20 although it is somewhat surprising that tissue egfr amplification / expression status has not been established as a reliable biomarker for anti - egfr therapies , potential reasons include heterogeneity between primary and metastatic lesions , dynamic changes in genomic alterations that may emerge along with therapeutic pressure or progression , presence of genomic coalterations associated with resistance , and potential differences in response to copy number gain due to aneuploidy versus focal egfr amplification.21 - 23 use of plasma - derived cell - free tumor dna ( cfdna ) to assess egfr status by next - generation sequencing ( ngs ) could conceivably overcome some of these limitations by detecting tumor - specific alterations that are shed into the bloodstream from multiple metastatic sites as well as the primary cancer.23 - 29 herein , we examined the genomic landscape of egfr amplification by interrogating blood derived cfdna from 28 , 584 patients with diverse malignancies using clinical - grade ngs . 
furthermore , we investigated the clinical characteristics , concordance between tissue ngs and cfdna , and therapeutic outcome after antiegfr therapies among a subset of 1 , 434 clinically annotated patients at the university of california , san diego ( ucsd ) , moores cancer center . methods patients the genomic landscape of egfr amplification among 28 , 584 diverse solid cancers that were referred to guardant health from march 2014 to february 2017 , were evaluated . 
furthermore , we have curated the clinical characteristics of 1 , 434 evaluable patients with diverse cancers at ucsd who had cfdna testing at guardant health starting in march 2014 . 
all investigations followed the guidelines of the ucsd institutional review board for data collection ( profile related evidence determining individualized cancer therapy ; clinicaltrials.gov identifier : nct02478931 ) and for any investigational therapies for which the patients consented ( data supplement )  . ngs for cfdna and tissue all cfdna analyses were performed at guardant health as previously described ( data supplement ) .26 tissue ngs was performed at foundation medicine , as previously described30 ( data supplement )  . end points and statistical methods patient characteristics , prevalence of egfr amplification , and genomic coalterations were summarized by descriptive statistics . 
the most common cancer harboring egfr amplification was colorectal cancer ( 16.3% [ 458 of 2 , 807 ] ) , followed by nonsmall - cell lung cancer ( 9.0% [ 1 , 096 of 12 , 197 ] ) and genitourinary cancers ( 8.1% [ 170 of 2 , 104 ] )  . 
 ( b ) prevalence of egfr amplification by cfdna among diverse cancer from university of california , san diego ( ucsd ) , cohort ( n = 1 , 434 )  . 
the most common cancer harboring egfr amplification was small - cell lung cancer ( 30.0% [ three of 10 ] ) , followed by breast cancer ( 14.7% [ 16 of 109 ] ) and colorectal cancer ( 12.6% [ 16 of 127 ] )  . 
 includes four patients with carcinoma of unknown primary ( + [ n = 1 ] , + + [ n = 2 ] , + + + [ n = 1 ] ) and two patients with adrenocortical carcinoma ( + + [ n = 2 ] )  . concordances between cfdna and tissue dna were described by percentage of concordance and value with standard error . 
on the other hand , coalterations in these genes were found significantly less frequently among patients without egfr amplification : coalterations in tp53 in 32.1% of patients , braf ( 4.9% ) , met ( 2.5% ) , cdk6 ( 1.4% ) , and pik3ca ( 8.8% ; all p < .001 ; fig 2 )  . 
when the genes were categorized according to their oncogenic roles , 72.4% ( 71 of 98 ) of patients with egfr amplification had at least one characterized coalteration potential targeted therapies for coalterations associated with egfr amplification in the ucsd cohort of 98 patients positive for egfr amplification , 96.9% ( 95 of 98 ) of patient tumors had at least one characterized coalteration . 
all these 95 malignancies harbored at least one characterized coalteration potentially targetable with us food and drug administration approved agents as onor off - label use . clinical characteristics of patients who had emerging egfr amplification with serial cfdna analyses six patients who initially tested negative for egfr amplification in both tissue ngs and cfdna were found to have emerging egfr amplification with serial cfdna analyses . 
 diagnosis timeline of emerging egfr amplification detected by cfdna analysis along with systemic therapy patient age ( y ) / 71 / m lung adenocarcinoma 50 / f breast cancer 69 / m hepatocellular carcinoma 44 / f breast cancer 56 / m anal squamous cell carcinoma 49 / m rectal adenocarcinoma fig 3 . 
six patients who initially tested negative for egfr amplification on tissue next - generation sequencing as well as cfdna were found to have egfr amplification with serial cfdna analyses after various treatments ; in four patients , treatment regimens included immune checkpoint inhibitors ( n = 4 ; patients 30 , 33 , 43 , and 46 )  . 
among 72 patients with egfr amplification ( without coexisting egfr mutations ) , nine received treatment regimens that included anti - egfr agents after cfdna testing ( appendix fig a3 )  . 
types of anti egfrbased regimens were as follows : monotherapy with anti - egfr antibody ( n = 1 ) , anti - egfr antibody plus another targeted agent ( n = 1 ) , egfr tyrosine kinase inhibitor plus another targeted agent ( n = 1 ) , anti - egfr antibody plus cytotoxic agents ( n = 2 ) , and dual antiegfr therapybased regimens ( combination of anti - egfr antibody plus egfr tyrosine kinase inhibitor ; n = 4 )  . 
overall , tumor reduction was seen in six of nine patients ( 66.7% ) , including five ( 55.6% ) who attained a partial response ( pr ) per recist 1.1 ( fig 4 )  . 
illustrative responders are depicted in figure 5.28 discussion we describe the comprehensive landscape of egfr amplification in cfdna among 28 , 584 patients with varied malignancies whose liquid biopsy was evaluated at a central , clinical - grade laboratory . 
overall , tumor reduction was seen in six of nine ( 66.7% ) , including five of nine ( 55.6% ) patients with partial response per response evaluation criteria in solid tumors 1.1. 
 egfr amplification ( 3 + ) mtor l387l ( 1.0% ) apc d2696y ( 2.4% ) arid1a p469l ( 3.5% ) ntrk1 a500v ( 4.5% ) arid1a s2269 * ( 4.6% ) brca2 d3260h ( 10.8% ) brca1 l30f ( 19.6% ) nf1 d2482y ( 20.7% ) brca1 g211e ( 26.7% ) akt1 e17k ( 66.5% ) egfr amplification ( ) esr1 e380q ( 0.1% ) tp53 y220c ( 0.1% ) brca1 g211e ( 0.3% ) egfr amplification ( ) esr1 e380q ( 0.2% ) akt1 e17k ( 0.3% ) cfdna months from the initiation of anti - egfr therapies cfdna before treatment imaging shown in figure 5b pembrolizumab cetuximab erlotinib before adding anti - egfr therapies cetuximab / erlotinib x 2 months cetuximab / erlotinib x ( cid : 3 ) 4 months pretreatment 9 weeks post cetuximab monotherapy at diagnosis cetuximab / erlotinib x 2 months cetuximab / erlotinib x 17 months fig 5 . 
a 55 - year - old woman with metastatic triple - negative breast cancer to bone and lung was treated with pembrolizumab with initial disease stability for more than 1 year , but then progression in the bones and deteriorating performance status requiring a wheelchair . 
 correlate better with response than protein expression , perhaps because of technical limitations associated with assessment of immunohistochemistry staining.31 - 34 prior studies looking at the relationship between egfr amplification and therapeutic response to egfr inhibitors showed inconsistent results ( data supplement )  . 
 indeed , pectasides et al23 demonstrated that , among patients with treatment - naive metastatic gastroesophageal cancers , discordant gene alterations between primary and metastatic tissue were common , being seen in 42% of patients . 
the concordance rate documented by pectasides et al23 is similar to that in the current report that showed an 89.3% concordance rate for egfr amplification between cfdna and tissue ngs ( data supplement )  . 
these results suggest that biopsy of a limited tumor focus can misrepresent the overall genomic condition of disease and , thus , may not be a completely accurate guide for targeted treatment . 
consistent with this concept , among our nine evaluable patients who harbored egfr amplification by cfdna analysis , anti - egfrbased therapies led to tumor reduction in 66.7% ( six of nine ) including 55.6% ( five of nine ) who achieved a pr ( fig 4 )  . 
our data are comparable to those of maron et al , 35 who showed a 58% ( four of seven ) objective response rate among patients with egfr - amplified gastric cancer ( all seven patients were positive for egfr amplification by tissue ngs , and six were positive by cfdna analysis )  . 
 importantly , dual inhibition with both an antibody and a small molecule targeting the same receptor has been investigated among patients with her2 - positive breast cancer and reported to have significantly higher response rates when compared with either drug alone.37 efficacy of dual - targeted therapy was also seen in patients with her2 - positive colon cancer that showed a 30% response rate with trastuzumab / lapatinib combination.38 similarly , early - phase clinical trials with dual - egfr inhibition ( cetuximab / afatinibor cetuximab / erlotinib - based therapy ) showed favorable clinical outcomes among patients with refractory nsclc and colorectal cancer.39 - 43 the mechanism by which dual inhibition operates is not fully elucidated , but preclinical studies suggest that kinase receptors may function via kinase - dependent and - independent mechanisms.44 , 45 although responses were seen in more than half of the patients with egfr amplification fig 5 . 
after starting anti - egfr agents , the patient achieved 16% tumor shrinkage per response evaluation criteria in solid tumors ( recist ) 1.1 ( b , left to right ) , with symptomatic improvement allowing the patient to ambulate without narcotics for pain control . 
a 53 - year - old woman with metastatic rectal adenocarcinoma to the liver and lungs presented after experiencing disease progression while receiving two lines of therapies ( infusional fluorouracil , leucovorin , and oxaliplatin with bevacizumab and fluorouracil , leucovorin , and irinotecan with bevacizumab )  . 
therapy was started with single - agent cetuximab , and a 44% reduction in tumor burden by recist 1.1 was seen ( progression - free survival , 6.0 months ; c , left to right )  . 
therapy with dual anti - egfr therapy ( cetuximab and erlotinib ) was started ( patient was also administered one dose of nivolumab on the basis of programmed death ligand 1 positive by immunohistochemistry ; however , held because of severe rash )  . 
indeed , as seen in figure 4 , of the nine patients with cfdna egfr amplification treated with egfr targeting agents , the four nonresponders had six to nine genomic coalterations , whereas the five responders had only zero to five coalterations per patient . 
furthermore , the patient with the greatest tumor regression and most durable response ( fig 4 , patient 25 ; progression - free survival , 18 months ) demonstrated no genomic coalterations on cfdna . 
of interest , patients who failed to achieve prolonged responses had coalterations in specific oncogenic pathways , including cdk4 / 6 , met , pdgfra , erbb2 , fgfr1 , pik3ca , akt1 , kras , and braf , some of which are known to be associated with resistance to anti - egfr therapies.46 , 47 considering that patients with egfr amplification had frequent potentially tractable coalterations ( fig 2 ; appendix figs a1and a2 ) , a customized combination strategy may be required.48 , 49 although current findings do not provide definitive proof of antitumor activity , these observations suggest that studies of appropriate combinations of drugs that target both the egfr amplification and the coalterations would be of interest . 
investigation of such an approach is currently ongoing ( clinicaltrial.gov identifier : nct02534675 ; i - predict [ study of molecular profile - related evidence to determine individualized therapy for advanced or poor prognosis cancers ] )  . interestingly , oxnard et al50 and abbosh et al51 have shown that more extensive disease burden corresponds to higher rates of cfdna detection . 
the finding of higher numbers of comutations in patients harboring egfr amplification events could therefore be a possible effect of increased aggressiveness and higher tumor burden ( with more extensive disease shedding more cfdna and thus permitting detection of more alterations ) , or , alternatively , higher numbers of comutations could be a cause of increased aggressiveness . 
this observation suggests that the association between survival and number of alterations is independent of the percent cfdna ( with the latter correlating with disease burden ; unpublished data )  . in the current report , we also identified patients whose egfr amplifications emerged in their liquid biopsy with serial testing after a variety of anticancer therapies ( n = 6 ; fig 3 )  . 
for instance , one patient who was treated with the egfr tyrosine kinase inhibitor erlotinib showed emergence of blood - derived egfr amplification after disease progression ( fig 4 ) , consistent with a previous report demonstrating tumor evolution with egfr amplification as a potential resistance mechanism to egfr tyrosine kinase inhibitor administration.52 perhaps relevant in this regard , all of our responders had an egfr antibody included in their regimen . 
although egfr alterations are reported to be associated with primary resistance and hyperprogression after immune checkpoint blockade , 6 , 7 the current observation may suggest that egfr amplification can also be a possible mechanism for acquired resistance after checkpoint blockades . 
 this is suggested by our representative patient who was treated with pembrolizumab , had a mixed response , and then received erlotinib and cetuximab ( in addition to ongoing pembrolizumab ) and showed reduction in egfr cfdna copy number as well as regression of tumor foci and improvement in pain and performance status ( fig 4 , patient 26 ; figs 5a and 5b ) .39 , 40 additional investigation is required to understand the complex interplay of response and resistance associated with egfr amplifications , egfr - targeting pharmaceuticals , and checkpoint blockade . there were several limitations to the current study . 
 yet , despite these limitations , the study provides a comprehensive analysis of egfr amplification detected from plasma - derived cfdna in a wide range of malignancies . in conclusion , among patients with diverse cancers ( n = 28 , 584 from a central laboratory ) , cfdna interrogated by clinical - grade ngs revealed that 8.5% of patients with solid cancers harbored egfr amplification . 
most patients found to have egfr amplification also had genomic coalterations that are , in theory , pharmacologically tractable ( 96.9% [ 95 of 98 ] ) by available drugs . 
anti - egfr based therapies among patients found to have egfr amplification by cfdna analysis achieved responses in 55.6% of patients ( five of nine ) , including in three individuals who failed to show egfr amplification on tissue ngs . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . shumei kato no relationship to disclose ryosuke okamura no relationship to disclose manvita mareboina no relationship to disclose aaron goodman consulting or advisory role : mitsubishi tanabe pharma , gerson lehrman group sandip p . 
patel consulting or advisory role : eli lilly , novartis , bristol - myers squibb , astrazeneca / medimmune , nektar , compugen speakers ' bureau : merck , boehringer ingelheim research funding : bristol - myers squibb ( inst ) , eli lilly ( inst ) , incyte ( inst ) , medimmune ( inst ) , pfizer ( inst ) , genentech ( inst ) , xcovery ( inst ) , amgen ( inst ) , astrazeneca / medimmune ( inst ) paul t . 
schwab stock and other ownership interests : samumed ( i ) patents , royalties , other intellectual property : the patent covers sialylated glycans and antibodies that specifically bind to them for early detection and diagnosis of cancer . 
raymond employment : trovagene , guardant health stock and other ownership interests : trovagene , guardant health suzanna lee stock and other ownership interests : invitae , gilead sciences , merck , juno therapeutics , sequenom richard b . 
lippman no relationship to disclose stock and other ownership interests : curematch , idbydna consulting or advisory role : actuate therapeutics , loxo oncology , xbiotech , neomed , roche speakers ' bureau : roche research funding : guardant health ( inst ) , sequenom ( inst ) , merck serono ( inst ) , genentech ( inst ) , pfizer ( inst ) , foundation medicine ( inst ) , incyte ( inst ) , konica minolta ( inst ) affiliations shumei kato , ryosuke okamura , manvita mareboina , suzanna lee , aaron goodman , sandip p . 
lanman , guardant health , redwood city , ca . supported in part by the joan and irwin jacobs philanthropic fund and by national cancer institute at the national institutes of health grant no . 
lu z , jiang g , blume - jensen p , et al : epidermal growth factor - induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase . 
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rizvi h , sanchez - vega f , la k , et al : molecular determinants of response to anti - programmed cell death ( pd ) - 1 and anti - programmed death - ligand 1 ( pd - l1 ) blockade in patients with nonsmall - cell lung cancer profiled with targeted next - generation sequencing . 
allegra cj , jessup jm , somerfield mr , et al : american society of clinical oncology provisional clinical opinion : testing for kras gene mutations in patients with metastatic colorectal carcinoma to predict response to anti - epidermal growth factor receptor monoclonal antibody therapy . 
thatcher n , hirsch fr , luft av , et al : necitumumab plus gemcitabine and cisplatin versus gemcitabine and cisplatin alone as first - line therapy in patients with stage iv squamous nonsmall - cell lung cancer ( squire ) : an open - label , randomised , controlled phase 3 trial . 
tsigelny if , wheler jj , greenberg jp , et al : molecular determinants of drug - specific sensitivity for epidermal growth factor receptor ( egfr ) exon 19 and 20 mutants in non - small cell lung cancer . 
wheler jj , falchook gs , tsimberidou am , et al : aberrations in the epidermal growth factor receptor gene in 958 patients with diverse advanced tumors : implications for therapy . 
bokemeyer c , bondarenko i , hartmann jt , et al : efficacy according to biomarker status of cetuximab plus folfox - 4 as first - line treatment for metastatic colorectal cancer : the opus study . 
herbst rs , redman mw , kim es , et al : cetuximab plus carboplatin and paclitaxel with or without bevacizumab versus carboplatin and paclitaxel with or without bevacizumab in advanced nsclc ( swog s0819 ) : a randomised , phase 3 study . 
licitra l , mesia r , rivera f , et al : evaluation of egfr gene copy number as a predictive biomarker for the efficacy of cetuximab in combination with chemotherapy in the first - line treatment of recurrent and / or metastatic squamous cell carcinoma of the head and neck : extreme study . 
lordick f , kang yk , chung hc , et al : capecitabine and cisplatin with or without cetuximab for patients with previously untreated advanced gastric cancer ( expand ) : a randomised , openlabel phase 3 trial . 
yang zy , shen wx , hu xf , et al : egfr gene copy number as a predictive biomarker for the treatment of metastatic colorectal cancer with anti - egfr monoclonal antibodies : a metaanalysis . 
misale s , di nicolantonio f , sartore - bianchi a , et al : resistance to anti - egfr therapy in colorectal cancer : from heterogeneity to convergent evolution . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
schwaederl mc , patel sp , husain h , et al : utility of genomic assessment of blood - derived circulating tumor dna ( ctdna ) in patients with advanced lung adenocarcinoma . 
schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
jardim dl , schwaederle m , wei c , et al : impact of a biomarker - based strategy on oncology drug development : a meta - analysis of clinical trials leading to fda approval . 
kim st , banks kc , pectasides e , et al : impact of genomic alterations on lapatinib treatment outcome and cell - free genomic landscape during her2 therapy in her2 + gastric cancer patients . 
baselga j , bradbury i , eidtmann h , et al : lapatinib with trastuzumab for her2 - positive early breast cancer ( neoaltto ) : a randomised , open - label , multicentre , phase 3 trial . 
sartore - bianchi a , trusolino l , martino c , et al : dual - targeted therapy with trastuzumab and lapatinib in treatment - refractory , kras codon 12 / 13 wild - type , her2 - positive metastatic colorectal cancer ( heracles ) : a proof - of - concept , multicentre , open - label , phase 2 trial . 
falchook gs , naing a , hong ds , et al : dual egfr inhibition in combination with anti - vegf treatment : a phase i clinical trial in non - small cell lung cancer . 
janjigian yy , smit ef , groen hj , et al : dual inhibition of egfr with afatinib and cetuximab in kinase inhibitor - resistant egfr - mutant lung cancer with and without t790m mutations . 
wheler jj , tsimberidou am , falchook gs , et al : combining erlotinib and cetuximab is associated with activity in patients with non - small cell lung cancer ( including squamous cell carcinomas ) and wild - type egfr or resistant mutations . 
wheler j , falchook g , tsimberidou am , et al : revisiting clinical trials using egfr inhibitorbased regimens in patients with advanced non - small cell lung cancer : a retrospective analysis of an md anderson cancer center phase i population . 
janku f , huang hj , angelo ls , et al : a kinase - independent biological activity for insulin growth factor - 1 receptor ( igf - 1r ) : implications for inhibition of the igf - 1r signal . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced non - small - cell lung cancer . 
mandelker , md1 ; semanti mukherjee , phd1 ; carolyn stewart1 ; deborah delair , md1 ; vignesh ravichandran1 ; preethi srinivasan , phd1 ; daire hurley3 ; yelena kemel1 ; angela g . 
abu - rustum , md1 , 2 ; and zsoa stadler , md1 , 2 purpose mutations in dna mismatch repair genes and pten , diagnostic of lynch and cowden syndromes , respectively , represent the only established inherited predisposition genes in endometrial cancer to date . 
per institutional standards , all tumors underwent lynch syndrome screening via immunohistochemistry ( ihc ) for mismatch repair proteins . results of 156 patients who consented to germline genetic testing , 118 ( 76% ) had stage i disease . 
twenty - four pathogenic germline variants were identied in 22 patients ( 14% ) : seven ( 4.5% ) had highly penetrant cancer syndromes and 15 ( 9.6% ) had variants in low - penetrance , moderate - penetrance , or recessive genes . 
the remaining 17 variants ( 71% ) were incremental ndings in lowand moderate - penetrance variants or genes associated with recessive disease . conclusion in unselected patients with predominantly low - risk , early - stage endometrial cancer , germline multigene panel testing identied cancer predisposition gene variants in 14% . 
they are associated with a signicant lifetime risk of ec , but they account for less than 5% of diagnoses.2 data regarding inherited risk in ec have been generated from selected patient populations , often ascertained from cancer genetics clinics from patients who have been referred because of a personal or family cancer history . 
for women with ls , ec is often the sentinel event , 3 and universal tumor screening of ec via mmr protein staining or microsatellite instability ( msi ) assessment is recommended.4 data regarding the breadth of inherited genetic mutations in an unselected ec patient population are limited . 
 cadoo et al context key objective to determine the prevalence of pathogenic germline variants in unselected patients with endometrial cancer . knowledge generated a custom next - generation sequencing panel , the memorial sloan ketteringintegrated mutation proling of actionable cancer targetsmsk - impactidentied 24 pathogenic germline variants in 22 patients ( 14% ) , including 4.5% who had pathogenic variants in high - penetrance genes . 
mismatch repair protein immunohistochemistry ( ihc ) screening identied all patients with lynch syndrome ( ls ; 3% of the overall cohort ) , representing the majority of high - penetrance variants . relevance in patients with predominantly low - risk endometrial cancer , identication of pathogenic germline variants via a multigene panel test may have implications in altering cancer screening and risk - reduction recommendations . 
electronic medical records were reviewed for demographic and clinical variables , including family history . sequencing , variant calling , and reporting of results memorial sloan ketteringintegrated mutation proling of actionable cancer targets ( msk - impact ) , a 468 - gene targeted capture panel , was used for tumor sequencing . germline analysis initially included a 76 - gene hereditary predisposition panel which was later revised to an 88 - gene panel ( appendix table a1 ) .6 - 8 for patients sequenced on the original panel , targeted review was performed for mutations in pole and pold . 
clinical variables in subsets dened by mutation status were compared by analysis of variance using graphpad prism version 7.01 ( graphpad , san diego , ca ) test tumor analysis for mmr deciency and ls screening institutional standards , all ecs undergo universal screening for ls via mmr protein immunohistochemistry ( ihc ) staining for mlh1 , msh2 , msh6 , and pms2.15 if mlh1 or pms2 are absent , reex mlh1 promoter methylation analysis is performed . 
in this study , msi status was also assessed via msi sensor , a computational algorithm that analyzes sequencing reads at designated microsatellite regions in tumor - normal pairs and reports the percentage of unstable loci as a cumulative score.16 an msi sensor score of 10 or greater indicates msi - high status ( msi - h ) , a score of 3 to 9 indicates msi - intermediate status ( msi - i ) , and a score of less than 3 indicates microsatellite stable status ( mss )  . results patient characteristics germline genetic testing was performed in 156 women ( fig 1 )  . 
the majority self - identied as white ( 83% ) , and 24% were ashkenazi jews . median ( range ) overweight or obese age , years median ( range ) 50 self - identied race asian black white aj heritage unknown stage histology carcinoma nos carcinosarcoma clear cell mixed serous endometrioid grade hysterectomies performed for endometrial cancer from april 2016 to may 2017 signed consent for somatic sequencing signed consent for germline sequencing pathogenic germline mutations identified ( n = 435 ) ( n = 179 ) ( n = 156 ) ( n = 24 ) fig 1 . 
one patient had a sister with ec in her 30s and no other ls - associated cancers in her family , and one patient had limited family history available ( table 2 )  . beyond the mmr genes , two other high - penetrance germline mutations were identied in brca1 and smarca4 ( table 2 )  . the brca1 mutation carrier had previously tested positive for this known familial mutation and underwent riskreducing salpingo - oophorectomy ( rrso ) without hysterectomy . 
a 72 - year - old woman who had clear cell ec lobular breast cancer when she was in her 40s and a family history of breast and prostate cancer had a deletion of smarca4 exon 17 - 18 identied with smarca4 ihc loss in her tumor . of these seven patients with high - penetrance mutations , six ( 86% ) met criteria for testing for the implicated gene betheir personal or family cancer history . 
the cause of smarca4 deletion carrier had no personal or family history of small cell ovarian tumors or rhabdoid tumor predisposition syndrome ( table 2 )  . lowand moderate - penetrance or recessive - only genes the other 17 ( 71% ) pathogenic germline mutations ( in 15 patients ) were in lowand moderate - penetrance or abbreviations : aj , ashkenazi jewish ; bmi , body mass index ; nos , not otherwise specied . recessive - only genes ( table 2 )  . 
the remaining mutations were in chek2 ( missense variant i157t ) , mre11a , mutyh , and reql4 . seven ( 47% ) of the 15 patients with variants in lowor moderate - penetrance or recessive genes met national comprehensive cancer network ( nccn ) criteria for brca testing on the basis of personal or family history . 
 inherited risk in the development of endometrial cancer there was no association of pathogenic germline mutations in these lowor moderate - penetrance or recessive - only genes compared with population controls ; however , the analysis is limited by the small size of the cohort . 
the remaining two patients were discordant , with each having a single mmr gene somatic mutation in a gene that does not correspond to the ihc loss . msi sensor score was concordant with ihc in 140 patients ( 90% ; 112 mss / ihc retained and 28 msi - h / mmrdecient ; fig 2b )  . 
however , only two ls - associated tumors ( 40% ) had an msi - h tumor phenotypetwo were msi - i and one was mss ( table 4 )  . 
her tumor had 46 somatic mutations , including dual mlh1 mutations , possibly driving the msi phenotype . discussion although next - generation sequencing has facilitated the widespread adoption of multigene panel germline testing , its clinical utility remains unclear.17 , 18 studies of unselected patients with colorectal , 19 ovarian , 20 and breast cancers21 have helped dene the range and prevalence of germline pathogenic cancer predisposition genes in these diseases . in addition , with broader genetic testing of patients with cancer , it has become evident that cancer stage may inuence the prevalence of germline mutations . 
for example , dna repair gene mutations are enriched in advanced - stage prostate cancer.8 conversely , early - stage colon cancer is enriched for germline mutations in the mmr genes.22 data regarding the spectrum of inherited risk in ec remains table 3 . 
 ( * ) biopsy at outside hospital ; no tumor remaining at hysterectomy . limited ; only one prior study5 has explored the role of germline multigene panel testing in a clinical cohort of patients with ec , testing for 25 genes using banked samples . 
ls mutations were identied in 6% of patients with ec , and another 3% had mutations in other genes . this 6% ls prevalence is on the upper end of what might be expected , with 2% to 6% reported in the literature and a generally accepted rate of approximately 3% , 23 - 27 raising the possibility of an ascertainment bias in the banked samples that were analyzed.5 applying a broad gene panel , analyses of selected patients with ec from tcga suggested that 16% of patients with ec harbor cancer predisposition mutations.28 we sought to determine the prevalence of cancer germline predisposition mutations in an unselected real - world clinical cohort of newly diagnosed patients with ec presenting for hysterectomy by using a large research multigene panel test . 
this reects the experience with traditional polymerase chain reactionbased msi testing in which lsassociated ecs have a lower proportion of unstable markers per tumor compared with colon cancer.34 in addition , unlike next - generation sequencingbased msi analysis , ihc testing is not vulnerable to low tumor purity . patients with brca mutations may be at increased risk of developing ec , although data are conicting and confounded by tamoxifen use.28 , 35 potential ec risk has important implications for counseling , because these women routinely undergo rrso without hysterectomy . 
similar to prior data , the patient with a brca mutation in our study had a high - grade ec.28 , 35 her tumor demonstrated loh at brca1 , suggesting the contribution of the brca1 mutation to development of ec . 
although a discussion of potential ec risk with brca mutation carriers undergoing rrso is reasonable , routine hysterectomy in this population is not currently warranted . smarca4 germline mutations result in rhabdoid tumor predisposition syndrome type 2 , an autosomal dominant cancer syndrome associated with aggressive soft tissue rhabdoid tumors , commonly in the brain ( atypical teratoid / rhabdoid tumors )  . 
for women , germline smarca4 mutations are associated with small cell carcinoma of the ovary , hypercalcemic type ; however , association with ec is unknown.36 median age at presentation is in the 20s ; no patients older than age 60 years have been reported.36 , 37 interestingly , ihc loss was demonstrated in our patient with ec and germline smarca4 mutation , raising the possibility that the mutation had a role in tumor development . the majority ( 71% ) of identied germline mutations were in lowor moderate - penetrance or autosomal recessive - only genes and most likely represent incidental ndings . 
given our relatively young population , of whom the majority had curable stage i cancer , the identication of incidental germline mutations may have signicant clinical implications for future risk reduction and identication of atrisk family members . although a number of these patients met current nccn criteria for brca testing , 38 the majority did not have prior breast or ovarian cancer gene testing . 
nationally reported rates of genetic testing are poor ; less than a third of eligible patients with breast cancer and 15% of patients with ovarian cancer have discussions about genetic testing.39 we have shown that genetic testing is acceptable to newly diagnosed ec patients if they are approached and offered testing . atm is not known to be associated with ec . 
although somatic pole mutations are relatively common , it is not clear that germline mutations predispose to ec.52 germline pold1 mutations have been associated with ec , but they are rare.52 in conclusion , we report a pilot study of large multigene panel germline testing for patients with newly diagnosed ec presenting for surgery . 
although our study is limited by a small sample size , predominantly white cohort , and overrepresentation of ashkenazi jewish heritage , it reects a prospective unselected cohort of patients with earlystage ec . we demonstrated the feasibility , acceptability , and yield of multigene germline testing in unselected patients with early - stage ec , and we found that the 3% incidence of ls in our study is comparable to that in the literature . 
cadoo , md , memorial sloan kettering cancer center , 300 east 66th st , 13th floor , new york , ny 10065 ; e - mail : cadook@mskcc.org. support supported in part by the romeo milio lynch syndrome foundation , the robert and kate niehaus center for inherited cancer genomics , the marie - jos ee and henry r . 
cadoo research funding : astrazeneca ( inst ) , syndax pharmaceuticals ( inst ) travel , accommodations , expenses : astrazeneca semanti mukherjee employment : regeneron pharmaceuticals stock and other ownership interests : regeneron pharmaceuticals research funding : regeneron pharmaceuticals dennis s . 
chi leadership : csurgeries stock and other ownership interests : bovie medical , verthermia , intuitive surgical , transenterix consulting or advisory role : bovie medical , verthermia elizabeth l . 
leitao jr honoraria : intuitive surgical research funding : kci travel , accommodations , expenses : intuitive surgical kara long roche travel , accommodations , expenses : intuitive surgical yukio sonoda patents , royalties , other intellectual property : patent pending for a surgical instrument ( uterine manipulator ) maria i . 
berger consulting or advisory role : roche research funding : illumina references david hyman consulting or advisory role : atara biotherapeutics , chugai pharmaceutical , cytomx therapeutics , boehringer ingelheim , astrazeneca , pzer , bayer , genentech research funding : astrazeneca , puma biotechnology , loxo oncology travel , accommodations , expenses : genentech , chugai pharmaceutical liying zhang employment : shanghai genome center ( i ) leadership : shanghai genome center ( i ) honoraria : future technology research travel , accommodations , expenses : shanghai genome center ( i ) , roche diagnostics asia pacic mark e . 
robson honoraria : astrazeneca consulting or advisory role : mckesson , astrazeneca , merck , pzer research funding : astrazeneca ( inst ) , myriad genetics ( inst ) , invitae ( inst ) , abbvie ( inst ) , tesaro ( inst ) , medivation ( inst ) travel , accommodations , expenses : astrazeneca carol aghajanian consulting or advisory role : clovis oncology , tesaro , mateon therapeutics , immunogen research funding : genentech ( inst ) , abbvie ( inst ) , clovis oncology ( inst ) , astrazeneca ( inst ) nadeem r . 
abu - rustum honoraria : prime oncology research funding : stryker / novadaq ( inst ) , olympus ( inst ) , grail ( inst ) travel , accommodations , expenses : prime oncology zsoa stadler allergan ( i ) , genentech ( i ) , regeneron pharmaceuticals ( i ) , optos ( i ) , adverum biotechnologies ( i ) , biomarin pharmaceuticals ( i ) , alimera sciences ( i ) , novartis ( i ) , spark therapeutics ( i ) , fortress biotech ( i ) , regenxbio ( i ) no other potential conicts of interest were reported . siegel rl , miller kd , jemal a : cancer statistics , 2017 . 
ca cancer j clin 67 : 7 - 30 , 2017 bruegl as , djordjevic b , batte b , et al : evaluation of clinical criteria for the identication of lynch syndrome among unselected patients with endometrial cancer . cancer prev res ( phila ) 7 : 686 - 697 , 2014 lu kh , dinh m , kohlmann w , et al : gynecologic cancer as a sentinel cancer for women with hereditary nonpolyposis colorectal cancer syndrome . 
obstet gynecol 105 : 569 - 574 , 2005 bruegl as , kernberg a , broaddus rr : importance of pcr - based tumor testing in the evaluation of lynch syndrome - associated endometrial cancer . 
adv anat pathol 24 : 372 - 378 , 2017 ring kl , bruegl as , allen ba , et al : germline multi - gene hereditary cancer panel testing in an unselected endometrial cancer cohort . 
mod pathol 29 : 1381 - 1389 , 2016 cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
j mol diagn 17 : 251 - 264 , 2015 zehir a , benayed r , shah rh , et al : mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10 , 000 patients . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
jama 318 : 825 - 835 , 2017 richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
hampel h , bennett rl , buchanan a , et al : a practice guideline from the american college of medical genetics and genomics and the national society of genetic counselors : referral indications for cancer predisposition assessment . 
weitzel jn , blazer kr , macdonald dj , et al : genetics , genomics , and cancer risk assessment : state of the art and future directions in the era of personalized medicine . 
domchek sm , bradbury a , garber je , et al : multiplex genetic testing for cancer susceptibility : out on the high wire without a net ? j clin oncol 31 : 1267 - 1270 , 18 . 
tung n , lin nu , kidd j , et al : frequency of germline mutations in 25 cancer susceptibility genes in a sequential series of patients with breast cancer . 
pearlman r , frankel wl , swanson b , et al : prevalence and spectrum of germline cancer susceptibility gene mutations among patients with early - onset j clin oncol 29 : 3008 - 3015 , 2011 oncol 34 : 1460 - 1468 , 2016 colorectal cancer . 
ferguson se , aronson m , pollett a , et al : performance characteristics of screening strategies for lynch syndrome in unselected women with newly diagnosed endometrial cancer who have undergone universal germline mutation testing . 
buchanan dd , tan yy , walsh md , et al : tumor mismatch repair immunohistochemistry and dna mlh1 methylation testing of patients with endometrial cancer diagnosed at age younger than 60 years optimizes triage for population - level germline mismatch repair gene mutation testing . 
ryan naj , morris j , green k , et al : association of mismatch repair mutation with age at cancer onset in lynch syndrome : implications for stratied surveillance netw 15 : 1465 - 1475 , 2017 instability . 
cosgrove cm , cohn de , hampel h , et al : epigenetic silencing of mlh1 in endometrial cancers is associated with larger tumor volume , increased rate of lymph node positivity and reduced recurrence - free survival . 
haraldsdottir s , hampel h , tomsic j , et al : colon and endometrial cancers with mismatch repair deciency can arise from somatic , rather than germline , mutations . 
abida w , armenia j , gopalan a , et al : prospective genomic proling of prostate cancer across disease states reveals germline and somatic alterations that may affect clinical decision making . 
balmaa j , digiovanni l , gaddam p , et al : conicting interpretation of genetic variants and cancer risk by commercial laboratories as assessed by the prospective registry of multiplex testing . 
chek2 breast cancer case - control consortium : chek2 * 1100delc and susceptibility to breast cancer : a collaborative analysis involving 10 , 860 breast cancer cases and 9 , 065 controls from 10 studies . 
weischer m , nordestgaard bg , pharoah p , et al : chek2 * 1100delc heterozygosity in women with breast cancer associated with early death , breast cancerspecic death , and increased risk of a second breast cancer . 
stoffel em , mangu pb , gruber sb , et al : hereditary colorectal cancer syndromes : american society of clinical oncology clinical practice guideline endorsement of the familial risk - colorectal cancer : european society for medical oncology clinical practice guidelines . 
edwards jg , feldman g , goldberg j , et al : expanded carrier screening in reproductive medicine : points to considera joint statement of the american college of medical genetics and genomics , american college of obstetricians and gynecologists , national society of genetic counselors , perinatal quality foundation , and society for maternal - fetal medicine . 
bellido f , pineda m , aiza g , et al : pole and pold1 mutations in 529 kindred with familial colorectal cancer and / or polyposis : review of reported cases and recommendations for genetic testing and surveillance . 
egfr tyrosine kinase inhibitors ( tkis ) markedly increase progression - free survival in advanced disease.17 , 18 this discovery opened a new era of precision medicine leading to accelerated development and subsequent approval of drugs for several actionable genomic alterations in nsclc . 
while the molecular proling of nsclc is considered a standard of care in the advanced setting , the utility of molecular testing and targeted therapies in the adjuvant setting in early - stage nsclc has yet to be established . 
the br19 trial compared getinib versus placebo in a similar population.21 the trial closed prematurely after results of another trial showed detrimental effect of getinib after chemoradiation in stage iii nsclc.23 however , egfrmutant nsclc accounted for only 4% ( n = 15 ) of the patients . 
patients with resected stage ib - iiia ( american joint committee on cancer [ ajcc ] 7th edition ) adenocarcinoma with sensitizing egfr mutation were randomly assigned 1 : 1 to adjuvant osimertinib at the standard dose of 80 mg / d or matching placebo . 
random assignment was stratied by stage ( ib v ii v iiia ) , type of egfr mutation ( exon 19 del v l858r ) , and race ( asian v non - asian ) , but not based on receipt of adjuvant chemotherapy . 
patients received assigned treatment for a planned 3 - year duration or until disease recurrence , completion they met predetermined criteria for discontinuation . treatment , or if six - hundred eighty - two patients were randomly assigned to osimertinib ( n = 339 ) or placebo ( n = 343 )  . 
the published data are an unplanned interim analysis with the dfs data being only 33% mature ( 11% in osimertinib and 55% in placebo ) at the data cutoff date of january 17 , 2020 . 
similarly , dfs in the overall population ( including stage ib ) was also signicantly longer with a consistent benet seen across all predened subgroups ( age , smoking , race , stage , and type of egfr mutation )  . 
first , positron emission tomography - computed tomography ( pet - ct ) scan and brain mri were not mandated at screening in the study protocol.27 in the united states , pet - ct scan and brain mri are considered the standard of care for the complete staging of early - stage nsclc.28 data on proportion of patients with complete staging information ( pet - ct and brain mri ) in either arm are not available . 
in the adura trial , the median dfs and 2 - year dfs rate in the placebo arm are signicantly lower than those reported in historical trials , suggesting that suboptimal staging at baseline could have contributed to higher proportion of patients being understaged.29 , 30 compared to control arm , osimertinib showed an 82% reduction in risk for cns recurrence or death . 
however , this should be interpreted with caution , given limited follow - up and patients with occult intracranial metastatic disease missed on brain ct scans who get osimertinib are at an advantage because of potent cns activity . 
data regarding the recurrence rates adjusted by country or region of enrollment may be important as petct and brain mri are not readily available worldwide in resource - limited settings . second , about 40% of the patients did not get adjuvant chemotherapy . 
to accurately assess the benet of adjuvant osimertinib , adjuvant chemotherapy should have been offered to all patients in the control arm and not just left to the discretion of the attending physician . 
 commentary enrollment , port was considered a valid treatment option for resected stage iiia n2 disease . in retrospect , not allowing port and not mandating standard of care adjuvant chemotherapy with proven os benet among participants in the placebo arm may be more of an ethical consideration for patients and investigators rather than a confounding variable . 
in the adjuvant setting , in an asymptomatic context , daily treatment with osimertinib for up to 3 years can lead to considerable toxicity , especially if we consider that some patients have been already cured with surgery with or without adjuvant chemotherapy . in addition to some of the limitations mentioned above , several important aspects related to study outcomes remain to be dened . 
in the adjuvant setting , it is not known whether the 2 - year dfs superiority with egfr directed therapy will translate into an os benet with osimertinib . data from adjuvant / ctong 1104 trial suggest that the substantial dfs benet did not translate into os , a goldstandard measure of patient impact in the adjuvant setting . although pfs may be a reasonable surrogate for os in the metastatic setting , higher level of evidence is needed before advocating for dfs as a proxy for os benet in patients receiving adjuvant cancer therapies . 
unlike adaura , the alchemist trial is testing adjuvant egfrand alkdirected therapy using os as the primary end point . furthermore , it has been suggested that targeted therapies as cytostatic agents suppress micrometastatic disease progression , thereby delaying recurrence but not eliminating the disease completely . 
despite multiple studies across solid tumors , to our knowledge , imatinib is the only tki that has shown improved os when given in the adjuvant setting of high - risk gi stromal malignancies , 31 a disease with completely different molecular background compared with lung adenocarcinomas . 
of note , in the osimertinib arm of the adaura trial , about 15% of the patients who stopped the drug at 3 years experienced dfs event in the 6 - 9 months after drug discontinuation . 
this might suggest that recurrence - free survival is directly related to the length of drug exposure , and therefore adjuvant osimertinib might only delay recurrence and not cure the disease . 
in adaura , at the data cutoff , approximately 46% of patients in the placebo arm experienced disease reto know the post - protocol currence . treatment patterns , specically the proportion of patients in the placebo arm who received osimertinib at progression . if this percentage is lower than expected , then the dfs benet seen in adaura might only reect the suboptimal treatment of control arm upon progression . 
if similar it will be critical os can be achieved in the placebo arm with osimertinib treatment on recurrence , one can speculate that osimertinib has minimal effect on the natural history of the disease and is only delaying the inevitable relapsed disease . 
one can anticipate that having a nonngsbased molecular testing at the time of initial detection of the egfr mutation will make it hard to tease out the acquired , potentially actionable mechanisms of resistance at the dfs event . 
furthermore , development of acquired resistance mechanisms at relapse on osimertinib in the adjuvant setting could result in these patients missing the window of opportunity to receive osimertinib in the metastatic setting . robust biomarkers to predict durable clinical benet to adjuvant therapy are lacking . 
thus , it might not be as cost - effective to justify the use of osimertinib in the absence of an os benet , an issue that is contentious but is an important consideration from a societal healthcare perspective . 
furthermore , a proportion of patients who are probably cured of lung cancer with surgery alone may have unnecessary exposure to osimertinib and its toxicities with added hospitalizations . lowand middle - income countries might be affected the most because of lack of access to testing and drugs , as well as prohibitive costs . 
assessment of the quality of life ( qol ) while on therapy and beyond will also be a crucial parameter to consider the overall benet of treatment . in conclusion , os is the most important and clinically meaningful end point in the adjuvant setting . 
given the lack of evidence to demonstrate dfs as a validated surrogate end point with targeted therapy in the adjuvant setting , mature os data are recommended before a denitive statement on adjuvant osimertinib in egfr - mutant nsclc . we also worry that wide adoption of adura in the community might lead to lower use of adjuvant chemotherapy in favor of osimertinib . 
with the idmc decision to unblind and terminate the study early based on the perceived dfs benet , we might never know whether adjuvant osimertinib leads to a superior os versus treatment with osimertinib upon rst evidence of relapse . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . sonam puri consulting or advisory role : astrazeneca rodrigo dienstmann consulting or advisory role : roche , boehringer ingelheim speakers bureau : roche , ipsen , sano , msd oncology , servier , amgen research funding : merck no other potential conicts of interest were reported . references siegel rl , miller kd , jemal a : cancer statistics , 2020 . 
ca cancer j clin 70 : 7 - 30 , 2020 besse b , le chevalier t : developments in the treatment of early nsclc : when to use chemotherapy . 
ann thorac surg 83 : 409 - 418 , 2007 pignon jp , tribodet h , scagliotti gv , et al : lung adjuvant cisplatin evaluation : a pooled analysis by the lace collaborative group . 
j clin oncol 26 : 3552 - 3559 , 2008 kris mg , gaspar le , chaft je , et al : adjuvant systemic therapy and adjuvant radiation therapy for stage i to iiia completely resected non - small - cell lung cancers : american society of clinical oncology / cancer care ontario clinical practice guideline update . 
j clin oncol 35 : 2960 - 2974 , 2017 arriagada r , auperin a , burdett s , et al : adjuvant chemotherapy , with or without postoperative radiotherapy , in operable non - small - cell lung cancer : two metaanalyses of individual patient data . 
lancet 375 : 1267 - 1277 , 2010 strauss gm , herndon je , maddaus ma , et al : adjuvant paclitaxel plus carboplatin compared with observation in stage ib non - small - cell lung cancer : calgb 9633 with the cancer and leukemia group b , radiation therapy oncology group , and north central cancer treatment group study groups . 
j clin oncol 26 : 5043 - 5051 , 2008 burdett s , rydzewska l , tierney jf , et al : a closer look at the effects of postoperative radiotherapy by stage and nodal status : updated results of an individual participant data meta - analysis in non - small - cell lung cancer . 
lung cancer 80 : 350 - 352 , 2013 burdett s , stewart l : postoperative radiotherapy in non - small - cell lung cancer : update of an individual patient data meta - analysis . 
robinson cg , patel ap , bradley jd , et al : postoperative radiotherapy for pathologic n2 non - small - cell lung cancer treated with adjuvant chemotherapy : a review of the national cancer data base . 
mikell jl , gillespie tw , hall wa , et al : postoperative radiotherapy is associated with better survival in non - small cell lung cancer with involved n2 lymph nodes : results of an analysis of the national cancer data base . 
corso cd , rutter ce , wilson ld , et al : re - evaluation of the role of postoperative radiotherapy and the impact of radiation dose for non - small - cell lung cancer using the national cancer database . 
fukuoka m , wu yl , thongprasert s , et al : biomarker analyses and nal overall survival results from a phase iii , randomized , open - label , rst - line study of getinib versus carboplatin / paclitaxel in clinically selected patients with advanced non - small - cell lung cancer in asia ( ipass ) , j clin oncol 29 : 2866 - 2874 , 2011 inoue a , kobayashi k , maemondo m , et al : updated overall survival results from a randomized phase iii trial comparing getinib with carboplatin - paclitaxel for chemo - nave non - small cell lung cancer with sensitive egfr gene mutations ( nej002 )  . 
zhong wz , wang q , mao wm , et al : getinib versus vinorelbine plus cisplatin as adjuvant treatment for stage ii - iiia ( n1 - n2 ) egfr - mutant nsclc ( adjuvant / ctong1104 ) : a randomised , open - label , phase 3 study . 
kelly k , altorki nk , eberhardt wee , et al : adjuvant erlotinib versus placebo in patients with stage ib - iiia nonsmall - cell lung cancer ( radiant ) : a randomized , double - blind , phase iii trial . 
wu yl , zhong w , wang q , et al : ctong1104 : adjuvant getinib versus chemotherapy for resected n1 - n2 nsclc with egfr mutationnal overall survival analysis of the randomized phase iii trial 1 analysis of the randomized phase iii trial . 
douillard jy , rosell r , de lena m , et al : adjuvant vinorelbine plus cisplatin versus observation in patients with completely resected stage ib - iiia non - small - cell lung cancer ( adjuvant navelbine international trialist association [ anita ] ) : a randomised controlled trial . 
arriagada r , de radiomedic - ina i , santiago c , et al : cisplatin - based adjuvant chemotherapy in patients with completely resected nonsmall - cell lung cancer . n engl j med 350 : 351 - 360 , 2004 joensuu h , eriksson m , sundby hall k , et al : survival outcomes associated with 3 years vs 1 year of adjuvant imatinib for patients with high - risk gastrointestinal stromal tumors : an analysis of a randomized clinical trial after 10 - year follow - up . 
abbosh c , frankell a , garnett a , et al : phylogenetic tracking and minimal residual disease detection using ctdna in early - stage nsclc : a lung tracerx study . cancer res 80 , 2020 ( suppl ; abstr ct023 ) 36 . 
li n , ou w , ye x , et al : pemetrexed - carboplatin adjuvant chemotherapy with or without getinib in resected stage iiia - n2 non - small cell lung cancer harbouring egfr mutations : a randomized , phase ii study . 
zhong wz , chen kn , chen c , et al : erlotinib versus gemcitabine plus cisplatin as neoadjuvant treatment of stage iiia - n2 egfr - mutant non - small - cell lung cancer ( emerging - ctong 1103 ) : a randomized phase ii study . 
pennell na , neal jw , chaft je , et al : select : a phase ii trial of adjuvant erlotinib in patients with resected epidermal growth factor receptor - mutant non - smallcell lung cancer . 
 comparative analysis of public knowledge bases for precision oncology steffen pallarz , phd1 ; manuela benary , phd1 , 2 ; mario lamping , md2 ; damian rieke , md2 , 3 ; johannes starlinger , md2 ; christine sers , phd2 , 4 ; david luis wiegandt1 ; marc seibert1 ; jurica seva , phd1 ; reinhold sch afer , phd2 , 4 ; ulrich keilholz , md2 ; and ulf leser , phd1 purpose precision oncology depends on the availability of up - to - date , comprehensive , and accurate information about associations between genetic variants and therapeutic options . 
we performed a quantitative and qualitative comparison of clinical interpretations of variants in cancer , oncokb , cancer gene census , database of curated mutations , cgi biomarkers ( the cancer genome interpreter biomarker database ) , tumor alterations relevant for genomics - driven therapy , and the precision medicine knowledge base . methods we downloaded each kb and restructured their content to describe variants , genes , drugs , and genedrug associations in a common format . 
for the analysis of clinically relevant gene - drug associations , we obtained lists of genes affected by genetic alterations and putative drug therapies for 113 patients with cancer whose cases were presented at the molecular tumor board ( mtb ) of the charit e comprehensive cancer center . results our analysis revealed that the kbs are largely overlapping but also that each source harbors a notable amount of unique information . 
the relative importance of a kb in terms of cancer genes was assessed in more detail by logistic regression , which revealed that all but one source had a notable impact on result quality . 
we conrmed these ndings using a second data set obtained from an independent mtb . conclusion to date , none of the existing publicly available kbs on gene - drug associations in precision oncology fully subsumes the others , but all of them exhibit specic strengths and weaknesses . 
2019 by american society of clinical oncology introduction precision oncology ( po ) is based on the molecular characterization of tumors and the integration of these data into clinical decision making.1 molecular characterizations typically are based on the identication of genomic variants by genomic sequencing , which results in patient - specic variant proles of single nucleotide variants , small insertions or deletions , copy number variations , and gene fusion load of the events . 
this bottleneck of clinical interpretation in po currently is based mostly on manual work and still lacks standardization.2 clinical variant interpretation must be based on published information.3 however , such information currently is dispersed across a number of databases , repositories , and web sites . 
 pallarz et al context key objective several knowledge bases that support the clinical evaluation of genetic variants in patients with cancer have emerged during the past years , but their mutual overlaps and extents are unknown . knowledge generated we compared seven knowledge bases and found that they share information only to a moderate extent and that they altogether lack important data . 
also , none of the knowledge bases is clearly more comprehensive than the others . relevance the creation of structured and easy - to - use knowledge bases to support precision oncology has made notable progress in the past years , yet the current systems remain incomplete and inhomogeneous . 
our results underline the necessity that , to obtain comprehensive information , oncologists must interrogate multiple knowledge bases and search relevant literature directly . we compared these kbs by measuring their mutual overlap of genes , drugs , and gene - drug associations and , if possible , by comparing each kbs content to actual treatment recommendations for 113 patients with cancer whose cases were discussed at the molecular tumor board ( mtb ) of the charit e comprehensive cancer center . 
results were conrmed using a second , independent set of recommendations for 10 patients with cancer . methods we performed pubmed searches and expert interviews to obtain a list of seven publicly available knowledge bases that aim at supporting medical decision making in precision oncology ( po - kb ) by providing structured information regarding the relationship between genes , variants , cancer entities , and therapies . 
specically , we used ( 1 ) civic ( clinical interpretations of variants in cancer ) , ( 2 ) oncokb , ( 3 ) cgc ( cancer gene census ) , ( 4 ) docm ( database of curated mutations ) , ( 5 ) cgi biomarkers ( the cancer genome interpreter biomarker database ) , ( 6 ) target ( tumor alterations relevant for genomics - driven therapy ) , and ( 7 ) pmkb ( the precision medicine knowledge base )  . 
the technical data integration procedure and normalization methods we developed for genes , variants , and drugs can be found in the data supplement . overlap comparison for a quantitative comparison , we computed the mutual overlap of all sources with regard to the set of genes , variants , drugs , and gene - drug associations they contain and visualized them using upset diagrams.12 for clustering of po - kbs , we rst computed pairwise distances using jaccards formula for set similarity and then applied hierarchical clustering ( data supplement )  . comparison of clinically relevant gene - drug associations we compared the content of each po - kb alone and in combinations versus recommendations of the charit e comprehensive cancer centers mtb ( n = 113 patient cases ) , at which results of comprehensive molecular table 1 . 
diagrams show the overlap between different precision oncology knowledge bases ( po - kbs ; identied by colors ) the matrices in the lower panels indicate po - kbs involved in an intersection , and the bar plots show the number of elements in this intersection . 
cgi , cgi biomarkers ( the cancer genome interpreter biomarker database ) ; civic , clinical interpretations of variants in cancer ; db , database ; docm , database of curated mutations ; pmkb , precision medicine knowledge base ; target , tumor alterations relevant for genomics - driven therapy . analysis ( whole - exome or panel sequencing , rna sequencing , immunohistochemical validations ) from patients with advanced cancer are discussed on a weekly basis . clinical interpretation of molecular alterations in the mtb is performed by a trained physician3 , 13 ; the data supplement contains details on this process . 
we measured the overlap between this reference set and different combinations of po - kbs for each patient using the metrics precision ( percentage of associations of a [ set of ] po - kb that is also contained in the reference ) , recall ( percentage of associations in the reference that is also contained in a [ set of ] po - kb ) and f1 measure ( harmonic mean between precision and recall )  . 
these measures are visually displayed later in the results , and more details can be found in the data supplement . to assess the predictive power of the different po - kbs , we proceeded as follows : for each association present in any of the po - kbs , a vector representation was constructed to encode its coverage by civic , cgi biomarkers , target , and oncokb , respectively . 
every gene - drug association that also was listed in the mtb associations was considered positive ( association relevant ; n = 345 cases ) , and all others were considered negative ( association not relevant ; n = 808 cases )  . 
 ( a ) percentage of patients for which any gene - drug associations were found when their presence was required in one to four po - kbs ( y - axis )  . 
each dot represents one patient , and only patients for whom at least one gene - drug association had been found were used . a 10 - fold cross - validation scheme to compute the out - ofsample error . 
eventually , a new model was trained on the entire training set to take this error into account ( using r package caret ) ; we report the results of the second model when applied on the test set . to validate our ndings , we used a second data set , which described treatment recommendations for patients with cancer , as derived at the molecularly aided stratication for tumor eradication ( masters ) 14 program at the national center for tumor diseases in heidelberg ( n = 10 ) and obtained from perera - bel et al.15 the normalization of gene and drug names for both data sets is described in the data supplement . 
to make results comparable to our rst data set , we removed all affected genes for which we did not have a single association in any of the po - kbs or for which no drug recommendation had been provided . 
to compensate for the positive bias in this set , we then added twice the number of genes chosen randomly from those we removed before . results overlap of studied kbs we downloaded and normalized seven publicly available databases that aim to support clinical decision making in po . we found that all databases overlap to a certain degree in the information they provide but also that each provided unique information . 
civic ( n = 89 genes ) and oncokb ( n = 86 genes ) have the second largest number of unique genes not mentioned in any other po - kb ; 49 genes were contained in all seven databases . 
the data supplement shows a hierarchical clustering of po - kbs on the basis of their overlaps in genes , variants , and gene - drug associations . although civic , oncokb , and pmkb share a highly similar goal and creation process and also obtain their data from the same origin ( scientic publications ) , each of them contains a substantial amount of unique genes . 
the most probable reason for this observation is that all po - kbs are small compared with the huge amounts of published data . the data supplement lists all genes contained in one or more of the databases . 
in addition , the four sources that contain drug information provide both shared and unique information ( fig 1b ) : 17 drugs are mentioned in all sources , and civic has the largest number of unique drugs ( n = 223 )  . 
similarly , civic shows the largest number of unique gene - drug associations ( n = 698 ; fig 1c )  . comparison of clinically relevant gene - drug associations we next compared the content of each po - kb and combinations thereof versus expert considerations for the 113 patients discussed at the charit e mtb at the level of genedrug associations . 
the number of drugs is high , because , for the analysis , drug classes ( eg , mammalian target of rapamycin inhibitors ) were expanded to all drugs of the respective class to match the granularity of information in different data sets . 
we analyzed which patients for whom the associations presented to the mtb also were contained in different po - kbs ( fig 2 ) and which fractions of gene - drug associations presented in the mtbs also would be found by specic combinations of po - kbs ( fig 3 )  . overall , the mtb discussed 203 different genes and 245 different drugs , of which 152 and 176 , respectively , were found in at least one po - kb . 
this implies that 25% of genes and 28% of drugs discussed in the mtb could not be found by our software in any of the po - kbs we considered . 
next , we computed the set of prioritized genes per patient and checked for how many of those had associations with drugs in at least one , two , three , or four po - kbs . 
 comparison of knowledge bases for precision oncology precision true positives ( tp ) false negatives ( fn ) recall false positives ( tn ) true negatives ( tn ) in po - kbs precision recall no . 
 ( b ) median precision ( blue ) , recall ( red ) and f1 ( harmonic mean between precision and recall ) score ( gray ) when considering an increasing number of precision oncology knowledge bases ( po - kbs ) to support gene - drug associations discussed by the molecular tumor board ( mtb ) experts . 
 ( c ) precision and ( d ) recall obtained by using different combination of po - kbs as boxplots ( median , 25th and 75th percentiles ; whiskers extend to the maximum values within 1.5 the interquartile range ; dots represent outliers )  . 
cgi , cgi biomarkers ( the cancer genome interpreter biomarker database ) ; civic , clinical interpretations of variants in cancer ; pr , precision and recall ; target , tumor alterations relevant for genomics - driven therapy . gene - drug association , the number of patients decreases to 42 . 
figure 2b shows that the number of potential suggestions per patient drops sharply with the required number of supporting po - kbs : when support by any two po - kbs was required , we obtained a median of ve associations per patient . 
the median increased to 12 if only one po - kb was required and decreased to two for three po - kbs and zero for four po - kbs . next , we considered the mtb associations as reference and the contents of the four po - kbs that contained genedrug associations as predictors . 
when the mtb reference gene - drug association was required to be contained in only one po - kbsthat is , the union of all po - kbs was used as predictorthe median recall for all patients reached approximately 46% ( fig 3b , blue line ) , whereas the median precision was low because of the large number of associations found in the po - kbs that were not discussed in the mtb ( fig 3b , orange line ; data supplement contains evaluation metrics )  . 
intuitively , this means that searching in more than one po - kb in our evaluation always led to nding more relevant data but at the cost of also nding more data that are irrelevant for the concrete patient according to the mtb . finally , we trained a logistic regression classier on evidence from all po - kbs using mtb associations as ground truth . 
on our withheld test set ( n = 288 gene - drug associations ) , this model reached an f1 score of 43% ( precision , 64% ; recall , 33% )  . 
on the nct data set , this model reached a precision of 100% at a recall of 38% , which resulted in a higher f1 score of 56% . the data supplement contains an interactive html page with the complete list of gene - drug associations of the berlin and the heidelberg data sets together with the information about which of the po - kbs contains which reference associations . discussion we performed a quantitative and qualitative comparison of the current content ( february 2019 ) of seven knowledge bases specialized in po . 
our results indicate that the pokbs have partly overlapping and partly unique results . compared with associations discussed for 113 patients in an mtb of a comprehensive cancer center at a university clinic , we found that po - kbs contain relevant information not present in any of the others . 
however , we also want to point out some limitations of our work that warrant additional studies . even after careful normalization of gene and drug names in all po - kbs ( data supplement ) , 809 of the 1 , 154 gene - drug associations of the mtb data set were not found in any of the po - kbs . 
possible reasons for their absence are that these genes are simply not yet curated , are grossly misspelled , or designate gene families rather than individual genes and so could not be matched by our normalization procedure . 
similarly , 157 associations with 69 unique drugs were not found because these drugs are not contained in any of the kbs ; 383 associations were missing because none of the po - kbs reported this particular association , although both the genes and the drugs were found in principle . our analysis showed that civic has the highest recall and f1 score compared with mtb associations when data from only one po - kb are considered ( ie , no combinations were allowed )  . 
these submissions could be the reason for the higher recall of po - kb combinations contained in civic ( fig 3d ) ; however , civic also simply contains the highest number of gene - drug associations among all po - kbs considered , which also positively inuences recall . 
separation of these two effects is difcult , but we note that regression results on the heidelberg data set , for which no bias toward civic exists , are even better than for the mtb data set . 
on both data sets , removal of civic from the training data left the precision of the learned model unchanged but induced a notable decrease in recall ( mtb , 33% decreased to 19% ; heidelberg , 38% decreased to 21% ) and , thus , in the f1 score ( mtb , 43% decreased to 29% ; heidelberg , 56% decreased to 35% )  . 
the fact that the decrease in recall was comparable in both data sets is an argument against a strong bias of the charit e mtb analysis toward civic ; the overall strong decrease originates from the large number of gene - drug associations only contained in civic . as reported , we trained a classier on combinations of the different po - kbs using the mtb associations as ground truth . 
however , we note that the reported results in terms of prediction accuracy cannot be considered as the ability to predict treatments in a clinical decision support setting for multiple reasons . 
second , we considered all associations in the mtb data as relevant without taking into account the nal decision of the mtb , which could be the recommendation of a single drug , could be a combination of drugs , or could disregard all discussed associations . 
fifth , the patient cases presented in the mtb represent a large variety of different tumor types , such as breast cancer , pancreatic cancer , leiomyosarcoma , ovarian cancer , liposarcoma , and neuroendocrine tumors ( data supplement )  . our analysis ignored the information on cancer types , because this level of detail frequently is missing in the po - kbs and , when present , uses a highly idiosyncratic nomenclature . 
note that analysis of recommendations derived from genomic information across different cancer types is commonplace in current studies about po.16 , 17 in conclusion , we here report a quantitative and qualitative comparison of precision oncology knowledge databases . our analysis shows that each of the databases contains , besides a rather small common core , a relevant amount of unique information . 
when we compared po - kb contents with gene - drug associations discussed in two mtbs , we found that a considerable fraction of information indeed can be found in some po - kbs but also that many presumably clinically relevant associations are not yet contained in any of them and that relevant information often is dispersed across different po - kbs . 
n engl j med 372 : 793 - 795 , 2015 good bm , ainscough bj , mcmichael jf , et al : organizing knowledge to enable personalization of medicine in cancer . 
nat rev cancer 4 : 177 - 183 , 2004 van allen em , wagle n , stojanov p , et al : whole - exome sequencing and clinical interpretation of formalin - xed , parafn - embedded tumor samples to guide precision cancer medicine . 
rieke dt , lamping m , klauschen f , et al : efcacy of a structured workow for the interpretation of comprehensive genomic analysis data in clinical routine . j clin oncol 36 , 2018 ( suppl ; abstr e24164 ) 14 . 
perera - bel j , hutter b , heining c , et al : from somatic variants towards precision oncology : evidence - driven reporting of treatment options in molecular tumor boards . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular proling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
massard c , michiels s , fert e c , et al : high - throughput genomics and clinical outcome in hard - to - treat advanced cancers : results of the moscato 01 trial . cancer discov 7 : 586 - 595 , 2017 18 . 
 postmortem somatic sequencing of tumors from patients with suspected lynch syndrome has clinical utility for surviving relatives introduction after a patient dies , the medical history stops unfolding and genetic evaluation typically ceases . 
 however , newer techniques may resolve uncertain diagnoses for patients and families , even years after a cancer diagnosis and including after the death of the affected individual . lynch syndrome is a highly penetrant , autosomal dominant , cancer predisposition syndrome caused by germline pathogenic variants in one of the following mismatch repair ( mmr ) genes : msh6 , msh2 , mlh1 , pms2 , or epcam . 
individuals with lynch syndrome have a high lifetime risk of cancer , including colorectal cancer ( crc ; risk range , 10% to 80% ) , endometrial cancer ( risk range , 16% to 60% ) , and others.1 clinical suggestion for lynch syndrome is subjectively based on personal and family history and objectively on tumor screening.2 - 6 tumor screening assesses mmr deficiency by identifying dna replication errors in microsatellite repeats ( microsatellite instability - high ) or by immunohistochemical ( ihc ) staining with decreased or absent expression in one or more mmr proteins.1 , 5 , 6 greater than 85% of lynch - associated tumors have a high level of microsatellite instability or abnormal findings by ihc staining.3 , 4 however , tumor screening is not diagnostic : 10% to 15% of sporadic crc tumors are also mmr deficient.2 , 4 , 7 , 8 screening of abnormal tumors should include assessment of sporadic causes of tumor mmr deficiency , including mlh1 hypermethylation or braf codon 600 mutations , and germline testing.1 , 5 , 8 , 9 probands with an mmr - deficient tumor but negative somatic or germline testing present a diagnostic and clinical management challenge.4 preventive recommendations for these individuals are not well defined and vary widely in practice.3 , 4 sequencing mmr - deficient tumors in individuals with unexplained , abnormal tumor screening is a powerful tool for refining the risk of lynch syndrome . 
probands personal and family histories and relevant information about the consultand characteristic consultand ; age , years probands age at diagnosis , years case a son ; 61 case b case c case d daughter ; 53 maternal first cousin ; 57 daughter ; 33 cancer site ( proband ) sigmoid colon sigmoid colon crc ; duodenal crc : 45 and 47 . 
maternal aunt : crc ( age , 53 years ) yes ; 2001 ( research , sequencing only ) ; negative ; and 2008 ; ( clinical , mlpa only ) ; negative uterus yes ; 2007 ; negative yes ; 2015 ; negative yes ; 2015 ; negative 5 fdr 4 fdr 3 fdr 3 fdr family history : lynchassociated cancer ( proband ) fulfilled amsterdam i criteria ( proband ) fulfilled revised bethesda criteria ( proband ) prior germline testing of proband ; year ; result no . 
of known relatives clinically affected by result insurance coverage no , self - pay obtained by consultand ? yes ( ministry of health , canada ) preauthorized , then denied . 
 appealed and approved at 50% coverage ( aetna ) yes ( blue cross federal ) abbreviations : crc , colorectal cancer ; fdr , first - degree relatives ; mlpa , multiplex ligationdependent probe amplification . improvement amendmentscertified university of washington genetics and solid tumors laboratory . 
coloseq tumor was ordered on behalf of a surviving relative to clarify an uncertain diagnosis of lynch syndrome and for an associated need for preventive surveillance and / or prophylactic surgery . 
table 1 describes the personal and family history of each proband and relevant information about the consultand . patient a was diagnosed with crc at age 78 years and died at age 82 years . 
the recommendation for early , high - risk surveillance for the consultands children was rescinded . patient b was diagnosed with colorectal cancer at age 82 years and died at age 87 years . 
coloseq tumor testing revealed two likely pathogenic somatic mutations in the tumor , msh2 c.1760 - 1g > a and msh2 p.c693r.16 the cancer surveillance plan was reassessed for the consultand and her siblings ; recommendation for colonoscopy was reduced to every 5 years . patient c was diagnosed with metachronous crc at ages 45 and 47 years and duodenal cancer at age 55 years . 
 considering prophylactic total hysterectomy with bso and currently await cascade testing results to guide this decision . patient d was diagnosed with endometrial cancer at age 58 years and died within a year . 
prophylactic hysterectomy and bso were no longer recommended for probands daughter . discussion clinical judgment often supports high - risk surveillance on the basis of an mmr - deficient tumor and / or personal and family history , even in the absence of a pathogenic germline variant . 
indeed , individuals with an mmr - deficient tumor and negative germline testing have a higher recurrence rate of crc compared with individuals with known sporadic cancer , leading to concern for a missed germline variant.4 , 7 , 8 despite universal tumor screening recommendations , compliance is inconsistent and may be preferentially performed with high clinical suspicion of lynch syndrome.17 , 18 preventive cancer screening carries cost and risk ; clarification of an uncertain diagnosis should be sought when clinically feasible.2 genetic testing is unique ; a result potentially provides information about a proband and relatives . 
however , surrogate testing is not equivalent to testing the affected person and carries its own risks.19 whenever possible , it is preferred to test the affected individual , even postmortem . genetic testing of deceased patients warrants special consideration and regulation.20 to our knowledge , this is the first report of clinical , postmortem , somatic sequencing used to actively change surviving relative management . 
 information considered ethical to disclose during a patients lifetime , such as imminent harm to identifiable individuals and / or potential benefit for at - risk individuals or public health , is considered ethical to disclose postmortem with the familys consent.20 resolving an uncertain but clinically suspected diagnosis of lynch syndrome falls within this paradigm . despite the clinical utility of resolving an uncertain diagnosis , several factors may hinder clinical uptake . 
patients may misunderstand the uncertainty of this result and erroneously believe they have lynch syndrome or not understand the results.20 coloseq tumor will likely only be offered if diagnosis is recognized as uncertain.5 , 21 , 22 next , test implementation requires significant genetic literacy , clinical judgment , and expertise that is not universally available.17 clinical pathology laboratories are required by the college of american pathologists to retain patient - tissue blocks for 10 years . 
last , insurance coverage for postmortem testing is challenging and , if denied , out - of - pocket cost is a deterrent.5 in this report , insurance coverage was obtained for three of four patients . 
these numbers are small and may reflect ascertainment bias of those who expected coverage or could self - pay . this case series has several important limitations , including comprising few cases and that patients receiving tumor screening may be a selected group , which could skew the high diagnostic rate and significant downstream benefits for surviving family members.18 nevertheless , this case series sufficiently demonstrates feasibility and clinical utility of resolving an uncertain lynch syndrome diagnosis in a deceased proband . in conclusion , postmortem paired tumor and germline sequencing is feasible , reliable , and offers a high diagnostic yield . 
lockwood administrative support : angela jacobson provision of study material or patients : angela jacobson , anna newlin , amanda hamblett , brian shirts , stephanie more , eric q . 
stanich patents , royalties , other intellectual property : i am an author for uptodate and receive royalties . stephanie more no relationship to disclose amanda hamblett stock and other ownership interests : edge therapeutics , achillion pharmaceuticals , cerecor , delcath systems , opko health , sonoma pharmaceuticals , sierra oncology , synthetic biologics , trevena , oncogenix , corbus pharmaceuticals , waveguide , osiris therapeutics , bristolmyers squibb , gilead sciences , cellectar biosciences , chimerix , contravir , stellar biotechnologies , innovate biopharmaceuticals , verastem jonathan f . 
tait stock and other ownership interests : merck ( i ) , amgen ( i ) , glaxosmithkline ( i ) research funding : evicore ( inst ) patents , royalties , other intellectual property : evicore copyrights ( inst ) brian shirts no relationship to disclose colin c . 
 stanich , ohio state university , columbus , oh ; stephanie more , sarah lawrence college , calgary , alberta , canada ; and amanda hamblett , middlesex hospital cancer center , middletown , ct . support supported by grant nih5t32gm007454 to h.m.b. 
weissman sm , burt r , church j , et al : identification of individuals at risk for lynch syndrome using targeted evaluations and genetic testing : national society of genetic counselors and the collaborative group of the americas on inherited colorectal cancer joint practice guideline . 
evaluation of genomic applications in practice and prevention ( egapp ) working group : recommendations from the egapp working group : genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from lynch syndrome in relatives . 
haraldsdottir s , hampel h , tomsic j , et al : colon and endometrial cancers with mismatch repair deficiency can arise from somatic , rather than germline , mutations . 
mensenkamp ar , vogelaar ip , van zelst - stams wa , et al : somatic mutations in mlh1 and msh2 are a frequent cause of mismatch - repair deficiency in lynch syndrome - like tumors . 
thompson ba , spurdle ab , plazzer jp , et al : application of a 5 - tiered scheme for standardized classification of 2 , 360 unique mismatch repair gene variants in the insight locus - specific database . 
beamer lc , grant ml , espenschied cr , et al : reflex immunohistochemistry and microsatellite instability testing of colorectal tumors for lynch syndrome among us cancer programs and follow - up of abnormal results . 
gallego cj , perez ml , burt a , et al : next generation sequencing in the clinic : a patterns of care study in a retrospective cohort of subjects referred to a genetic medicine clinic for suspected lynch syndrome . 
 breast and ovarian cancer penetrance estimates derived from germline multiple - gene sequencing results in women purpose multiple - gene , next - generation sequencing panels are increasingly used to assess hereditary cancer risks of patients with diverse personal and family cancer histories . 
the magnitude of breast and ovarian cancer risk associated with many clinically tested genes , and independent of family cancer history , remains to be quantified . methods we queried a commercial laboratory database of 95 , 561 women tested clinically for hereditary cancer risk with a 25 - gene ( apc , atm , bard1 , bmpr1a , brca1 , brca2 , brip1 , cdh1 , cdk4 , chek2 , mlh2 , msh2 , msh6 , mutyh , nbn , p14arf , p16 , palb2 , pms2 , pten , rad51c , rad51d , smad4 , stk11 , and tp53 ) next - generation sequencing panel . multivariable logistic regression models accounting for family history were used to examine the association between pathogenic mutations and breast or ovarian cancer . 
as a confirmatory approach , a matched case - control analysis was conducted , defining cases as patients with breast or ovarian cancer and controls as women without cancer . results one or more pathogenic mutations were detected in 6 , 775 ( 7% ) of 95 , 561 women . 
multivariable models and matched case - control analyses yielded similar results . conclusion among nearly 100 , 000 clinically tested women , 7% carried a pathogenic mutation in one or more cancer - associated genes . 
2017 by american society of clinical oncology introduction next - generation sequencing has dramatically expanded the scope and speed of genetic testing for hereditary cancer risk while simultaneously reducing cost.1 , 2 these advances are most evident in the approach to suspected hereditary breast and ovarian cancer ( hboc ) syndrome . 
this has begun to broaden our understanding of gene - specific phenotype , as mutations are found unexpectedly among patients with a cancer not thought to be associated with a particular gene.8 , 15 despite this progress , questions have arisen about the clinical validity and utility of multiple - gene panel testing . 
perhaps the greatest question pertains to the magnitude of cancer risk , or penetrance , associated with mutations in less widely tested genes included on many panels , such as allison w . 
questions regarding cancer risk have become especially pressing as decreasing costs have increased testing access for patients with a wider range of personal and family histories than previously required.16 with some exceptions , 17 - 19 studies estimating cancer risks have been small and underpowered , yielding imprecise estimates . 
this is problematic because our understanding of a patients cancer risk guides treatment recommendations for enhanced breast screening and preventive surgeries.20 - 24 for example , risk - reducing salpingo - oophorectomy is indicated with a mutation that confers a 10to 20fold increase over the average ovarian cancer risk ( an absolute risk of 20% to 40% ) , but may be excessive with a mutation causing only a two - fold elevation ( an absolute risk of 2% to 4% ) .20 penetrance estimates adjusted for family history represent the magnitude of risk that is independently genetic . 
adjusted penetrance may be estimated in an unbiased manner from clinical populations where factors related to ascertainment bias are well captured , and represent the magnitude of risk that is independently genetic.25 these adjusted penetrance estimates are applicable to women with no family history of cancer and may inform personalized assessments that combine genetic risks with environmental and lifestyle risk factors . with clinical uptake of multiple - gene panel testing increasing rapidly , cancer risk estimates that control for diverse family histories are urgently needed to inform genetic counseling . 
in this study , we analyzed a testing laboratorys data on germline sequencing with a 25 - gene panel in a large , real - world cohort using two complementary methods to determine novel estimates of mutation penetrance adjusted for family history . methods patients and hereditary cancer testing female patients who underwent testing with a 25gene hereditary cancer panel between september 2013 and september 2015 were evaluated . 
testing was performed at a clinical laboratory improvement amendments and college of american pathologyapproved laboratory ( myriad genetic laboratories ) for a panel of 25 genes ( apc , atm , bard1 , bmpr1a , brca1 , brca2 , brip1 , cdh1 , cdk4 , chek2 , mlh2 , msh2 , msh6 , mutyh , nbn , p14arf , p16 , palb2 , pms2 , pten , rad51c , rad51d , smad4 , stk11 , and tp53 ) .26 informed consent for clinical testing was obtained and clinical information was collected on the test requisition forms ( trfs )  . patient data were deidentified and no additional information was obtained from patients or providers . 
as such , this analysis was not subject to ethics board review . variants were classified using american college of medical genetics and genomics recommendations , with supporting linkage , biochemical , clinical , functional , and statistical data used for specific missense and intronic alterations.27 - 29 pathogenic variants are those that receive a laboratory classification of deleterious or suspected deleterious . 
classifications used in the analysis matched those reported clinically . clinical information from provider - completed trfs included ancestry , personal and family cancer history , cancer type ( s ) , and age ( s ) at diagnosis . patients were excluded from analysis if they had an incomplete trf , if they had testing using the 25gene panel after receiving negative test results from a single / limited gene panel , or if results suggested mosaicisno patient was excluded on the basis of race / ethnicity , age , or other characteristics . statistical methods all analyses were conducted using r version 3.0.2.30 two - sided p values are reported . 
given that we had prior hypotheses about the sequenced genes , no adjustments were made for multiple testing . we used two different statistical methods to quantify adjusted risks of invasive ductal carcinoma of the breast ( bc ) and invasive epithelial ovarian cancer ( oc ) associated with mutations in panel genes . 
for genes associated with childhood mortality ( eg , tp53 and stk11 ) , the penetrance estimates represent bc and oc risks among mutation carriers who survive to adulthood . multivariable logistic regression models for each gene , we constructed two multivariable models to estimate ( 1 ) bc risk and ( 2 ) oc risk . 
our hypothesis was that family histories may be over - reported among unaffected controls to meet insurance coverage criteria for genetic testing and / or under - reported among affected cases . 
using brca1 and palb2 as widely studied examples , we modified family histories by adding one fdr diagnosis of bc to cases and subtracting one fdr diagnosis from controls , selected uniformly at randorandom patient selections were repeated 1 , 000 times and results were reported as medians of 1 , 000 iterations . matched case - control analyses results cases were defined as female patients with a single diagnosis of bc or oc . 
cases and controls were matched 1 : 1 according to age ( 6 3 years ) , ancestry ( exact match ) , and family cancer history ( breast , ovarian , colon , uterine )  . 
p values , ors , and cis were calculated with the exact mcnemars test . mutation status , age , and ancestry were coded as for models ( data supplement )  . 
for tests of oc associations , we included an extra three - level matching variable for familial colon and uterine cancer : ( 1 ) at least one fdr with colon or uterine cancer before the age of 50 years ; ( 2 ) at least one fdr with colon or uterine cancer at the age of 50 years or older ; and ( 3 ) none of the above . sensitivity analysis age - specific cumulative bc risks on the basis of adjusted penetrance were compared with estimates for brca1 and brca2 on the basis of literature.31 - 35 cumulative risks were calculated according to the product - limit method , 36 with agespecific incidences estimated as the product of agespecific ors and general population incidences . age - specific ors were calculated by subsetting to the age categories reported by satagopan et al35 ( , 40 , 40 to 49 , and > 50 years ) , and were combined with age - specific seer37 incidence rates from 2009 to 2013 in 5 - year intervals . the magnitude of potential bias that could result from differential underor over - reporting of family we identified 95 , 561 eligible patients ( table 1 )  . 
this includes 2 , 771 mutations detected in 2 , 701 ( 10% ) bcaffected patients , and 715 mutations in 701 ( 14% ) oc - affectedpatients.overall , 3 , 007 ( 44% ) mutations were in brca1 / 2 and 3 , 768 ( 56% ) were in other genes . multivariable logistic regression models eight genes were significantly associated with bc : atm , bard1 , brca1 , brca2 , chek2 , palb2 , pten , and tp53 ( table 3 and fig 1 )  . 
as an exploratory analysis , we constructed a multivariable model ( as described in the methods section ) with lobular breast cancer as the dependent variable , which showed a strong association of cdh1 with lobular breast cancer ( table 3 )  . eleven genes were significantly associated with oc : atm , brca1 , brca2 , brip1 , mlh1 , msh6 , msh2 , nbn , rad51c , rad51d , and stk11 ( table 3 and fig 1 )  . matched case - control analyses overall , 19 , 056 patients were eligible as bc cases , 3 , 695 patients as oc cases , and 51 , 200 patients as cancer - free controls . 
we identified matched controls for 15 , 826 bc and 2 , 731 oc cases . risk estimates from matched case - control analyses and multivariable models were consistent ( table 3 , table 4 , and fig 1 )  . 
patient clinical characteristics age at hereditary cancer testing , years variable total patients * range median % < 50 ancestry western / northern european central / eastern european latin american / caribbean african native american asian ashkenazi near / middle eastern family cancer history no bc or oc 1 bc , no oc 1 oc , no bc > 2 bc , no oc > 2 oc , no bc > 2 bc or oc all patients no . 
accounting for family history resulted in a slightly lower adjusted bc risk compared with the unadjusted published estimates . we also investigated the impact of overor underreporting family cancer history for brca1 and palb2 . 
sensitivity analyses showed that risk estimates will be inflated if family history is overreported by controls and / or under - reported by cases , which would cause more severe cases to be matched to less severe controls . 
we used multivariable modeling and matched case - control approaches to estimate mutation penetrance for 25 cancer - associated genes , adjusting for age , race / ethnicity , and family cancer history . 
we did not observe increased bc risk with mutations in nbn , which was surprising because nbn is generally considered to be a moderate - penetrance breast cancer susceptibility gene.16 reports of bc risks associated with brip1 , rad51c , and rad51d mutations are mixed ; 38 , 39 our results suggest no greater than average risk . 
in addition , there have been some reports of increased bc incidence among individuals with mutations in the lynch syndrome genes msh6 and pms2 ; 40 - 43 however , our analysis showed no increased risk of bc for individuals with mutations in these genes . we found 11 genes significantly associated with increased oc risk : atm , brca1 , brca2 , brip1 , mlh1 , msh6 , msh2 , nbn , rad51c , rad51d , and stk11 . 
these results are consistent with prior studies of brca1 / 2 , brip1 , rad51c , and rad51d and the mismatch repair genes in oc susceptibility.17 , 43 - 52 for nbn , there has been prior speculation but no definite evidence of an oc association.50 to our knowledge , this is the first report of increased oc risk associated with a germline atm mutation . 
given estimates that as many as 1% of bc patients and 0.5% of the general population may carry an atm mutation , 53 - 55 with many having no family history of oc , defining their risk of developing oc is an urgent research priority . 
significant associations are in bold . abbreviation : na , not applicable . * no mutations were observed in cdk4 and analysis could not be performed . genes associated with increased risk of breast and ovarian cancer . genes associated with an increased risk of breast cancer only . genes associated with an increased risk of ovarian cancer only . kadditional analysis with analogous models to predict lobular breast cancer risk in cdh1 . with bard1 mutations , in contrast with some recent studies.56 the estimated oc risk calculated for palb2 was  . 
in light of a recent study reporting increased incidence of palb2 mutations in women with oc , 56 additional studies will be essential to understand the contribution of palb2 mutations to oc susceptibility . previous studies of bc and oc gene penetrance have used various designs , including kin cohort and case control.17 , 18 , 31 , 33 , 57 - 62 all study designs are subject to bias . 
odds ratios and 95% cis from multivariable logistic regression models ( blue ) and exact mcnemars tests ( gold ) of ( a ) breast cancer associations and ( b ) ovarian cancer associations . 
only genes with results from both models are shown . genes with a significant association with breast or ovarian cancer by multivariable regression are shown in bold . bard1 brca1 brca2 brip1 cdh1 cdkn2a ( p16 ) chek2 mlh1 msh2 msh6 mutyh palb2 pms2 rad51c rad51d tp53 cdkn2a ( p16 ) bard1 brca1 brca2 brip1 chek2 mlh1 msh6 palb2 pms2 rad51c rad51d odds ratio ( 95% ci ) 0.5 1 odds ratio ( 95% ci ) multivariable regression matched case / control ( exact mcnemar 's test ) result of more comprehensive sequencing of clinical samples . 
the clinical testing data set we used did not include related individuals ( required for kin - cohort analysis ) ; thus we used the two other penetrance study designs that are feasible with data of this kind , ie , multivariable logistic regression modeling and an analysis of cases matched to comparable controls from the same patient population.66 we controlled for ascertainment bias using standard methods for confounding variables , 25 which yielded adjusted ors that estimate the relative risk conferred by a gene mutation after accounting for family cancer history , race / ethnicity , and age . the concordant results of the two analytic methods we used ( logistic regression and matched case / control ) suggest that both adequately accounted for ascertainment bias . 
for example , patients with cancer often meet guidelines for genetic testing regardless of their family history ; thus it is possible that family history was reported less completely by ordering clinicians for cases than for controls ( who generally meet guidelines for insurance coverage of testing only because of their relatives cancer histories )  . 
significant associations are in bold . abbreviation : na , not applicable . * there were too few mutation carriers to generate an odds ratio for this gene . genes associated with increased risk of breast and ovarian cancer . genes associated with an increased risk of breast cancer only . genes associated with an increased risk of ovarian cancer only . the limitations of this study are balanced by its considerable strengths , most notably its size ( n = 95 , 561 ) and representation of patients who receive multiple - gene panel testing under the routine conditions of clinical practice . 
the results cannot replace controlled studies of mutation penetrance in unselected patients with bc , who lack early age at onset or strong family history ; such research will require analysis of population - based cohorts , which is underway . 
if confirmed , the current results may justify a case - by - case discussion of oc risk - reduction approaches with women who carry these mutations , considering family history as well as these genespecific risk estimates . because prophylactic surgery is invasive and irreversible , the burden of proof should be more stringent than for a screening intervention . 
if confirmed , the three - fold or greater oc risk estimates presented here for brip1 and rad51c / d would support their recent addition to guidelines criteria for risk - reducing salpingo - oophorectomy.20 however , the lower estimates of oc risk with atm and nbnareless clearly an indication for prophylactic in conclusion , we observed and quantified significant bc risks associated with eight genes and oc risks associated with 11 genes that are commonly sequenced on multiple - gene panels . 
kurian research funding : myriad genetics ( inst ) , invitae ( inst ) , ambry genetics ( inst ) , genomic health ( inst ) , genedx / bioreference ( inst ) ; genentech ( inst ) elisha hughes employment : myriad genetics stock and other ownership interests : myriad genetics elizabeth a . 
hall research funding : myriad genetics ( inst ) patents , royalties , other intellectual property : i share a patent with several fox chase investigators for a novel method to investigate hereditary colorectal cancer genes . 
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meijers - heijboer h , van den ouweland a , klijn j , et al : low - penetrance susceptibility to breast cancer due to chek2 ( * ) 1100delc in noncarriers of brca1 or brca2 mutations . 
evans dg , shenton a , woodward e , et al : penetrance estimates for brca1 and brca2 based on genetic testing in a clinical cancer genetics service setting : risks of breast / ovarian cancer quoted should reflect the cancer burden in the family . 
risch ha , mclaughlin jr , cole de , et al : population brca1 and brca2 mutation frequencies and cancer penetrances : a kin - cohort study in ontario , canada . 
song h , dicks e , ramus sj , et al : contribution of germline mutations in the rad51b , rad51c , and rad51d genes to ovarian cancer in the population . 
because poly ( adp - ribose ) polymerase inhibitors have significant activity in brca1 / 2 - mutant ovarian and breast cancers , rucapanc investigated the efficacy and safety of rucaparib in brca1 / 2 - mutant pancreatic cancer . patients and methods rucapanc enrolled patients with measurable locally advanced / metastatic pancreatic cancer who had received one to two prior chemotherapy regimens . 
four patients achieved a response ; two partial responses and one complete response ( cr ) were confirmed ( objective response rate , 15.8% ; 3 of 19 ) , with an additional cr unconfirmed . 
the disease control rate ( cr , partial response , or stable disease for 12 weeks ) was 31.6% ( 6 of 19 ) in all patients and 44.4% ( 4 of 9 ) in those who had received one prior chemotherapy regimen . 
these mutations are predicted to lead to the reversion of a somatic not germlinemutation . conclusion rucaparib provided clinical benefit to patients with advanced pancreatic cancer and a brca1 / 2 mutation , and demonstrated an acceptable safety profile . 
2018 by american society of clinical oncology introduction pancreatic cancer ( pc ) is projected to become the second leading cause of cancer death by 2020.1 the majority of pcs are diagnosed at an advanced stage , precluding a surgical , curative approach . 
in the setting of advanced disease , folfirinox treatment ( folinic acid [ leucovorin ] , fluorouracil , irinotecan , and oxaliplatin ) and gemcitabine in combination with nab - paclitaxel are the standards of care , with both demonstrating an improvement in overall survival compared with gemcitabine alone.2 , 3 in the second - line setting , prospective data have shown modest results , with response rates to chemotherapy generally less than 20% , 4 including second - line folfirinox or fluorouracil with nanoliposomal irinotecan.4 - 8 additional therapeutic options are needed . approximately 9% of unselected pcs are associated with a germline or somatic mutation in brca1 or brca2 ( brca1 / 2 ) .9 , 10 cells with a deleterious brca1 / 2 mutation and resultant homologous recombination deficiency are unable to repair dna double - strand breaks reliably11 , 12 and are thus sensitive to poly ( adp - ribose ) polymerase ( parp ) inhibition.13 - 16 studies have demonstrated clinical benefit with parp inhibitors in clinical trials of germline brca1 / 2 mutant breast and ovarian cancer . 
the study design was approved by the independent review board at each participating site and was conducted according to the provisions of the declaration of helsinki and the good clinical practice guidelines of the international conference on harmonization of technical requirements for registration of pharmaceuticals for human use . 
all patients provided written informed consent before participating in the trial . enrolled patients were men and women at least 18 years of age with histologically confirmed locally advanced or metastatic pancreatic adenocarcinoma with measureable disease and a known deleterious germline or somatic brca1 / 2 mutation by local testing . 
patients could have received up to two prior lines of chemotherapy in the locally advanced / metastatic setting , but could not have received prior therapy with a parp inhibitor . 
to infer loss of heterozygosity ( loh ) , the log - ratio profile of sequencing data was segmented , and the allele frequencies of sequenced genome - wide single nucleotide polymorphisms were used to estimate the copy and minor allele frequency for each segment . 
pretreatment plasma specimens were collected on day 1 of cycle 1 for circulating tumor dna ( ctdna ) analysis by foundation medicine , which is a hybrid capture - based next - generation sequencing assay that sequences the coding regions of 62 cancer - related genes , including brca1 / 2 . 
safety results were reported by analysis of adverse events ( aes ) and graded according to the common terminology criteria for adverse events ( version 4 )  . the safety analysis included all patients who received at least one dose of rucaparib . 
for inclusion in the objective response rate ( orr ) , a complete response ( cr ) or partial response ( pr ) per recist had to have been confirmed at least 28 days after the initial response was observed . 
the median age was 57 ( range , 41 to 75 ) years , 57.9% of patients ( 11 of 19 ) were male , the median number of prior chemotherapy regimens for locally advanced / metastatic disease ( excludes adjuvant treatment where progression occurred > 6 months after completion of treatment ) was two ( range , 1 to 3 ) , 78.9% of patients ( 15 of 19 ) had an eastern cooperative oncology group performance status of 1 , and 78.9% ( 15 of 19 ) had a brca2 mutation . 
as prespecified in the protocol , enrollment was stopped because of lack of responses in the first 15 patients evaluated ; the three confirmed responses occurred in the last four patients enrolled . after study closure , one patient continued therapy on an individual patient ind application . 
thirteen patients discontinued the study because of radiologic or clinical progression , two discontinued therapy within a week of beginning ( one because of investigator decision , the other for an unknown reason ) , one discontinued because of aes with simultaneous radiologic progression confirmed at the end of treatment , one discontinued because of an ae with ongoing sd , and one withdrew consent with an ongoing pr . 
patients received rucaparib for a median of 57 days in the study ( range , 2 to 504 days )  . brca1 and brca2 mutation status sixteen of 19 patients had a germline brca1 / 2 mutation . 
of the 16 tumors associated with a germline mutation , paired somatic sequencing was available in eight ; the allelic frequency of the known germline mutant allele was between 41% and 53% . 
this was counted as three separate chemotherapy treatment regimens per the protocol - specified criteria . circulating tumor dna samples for ctdna analyses at cycle 1 , day 1 ( before treatment with rucaparib ) were available for 16 patients , and somatic mutations were detected in 11 patients ( 10 of whom had kras mutations ; appendix table a1 )  . 
investigator - assessed responses ( recist ) in patients with pancreatic cancer and a brca mutation ( n = 19 ) unconfirmed cr / confirmed sd response not evaluable confirmed response rate ( cr or pr ) confirmed response rate ( cr or pr ) in patients with only one prior chemotherapy for locally advanced / metastatic disease disease control rate ( cr , pr , or sd 12 weeks ) all patients patients with only one prior chemotherapy regimen for locally advanced / metastatic disease no . 
in the first patient ( patient 5 ) , ctdna analysis detected the germline brca2 mutation c.7060c > t ( allele frequency , 48% ) , the somatic alteration c.1499_1499delg ( allele frequency , 22% ) , and the secondary somatic mutation c.1416_1420delgcatc ( allele frequency , 19% ) that restored the open reading frame of the brca2 gene . 
in the second patient ( patient 14 ) , ctdna analysis detected the germline brca2 mutation c.5946deit ( allele frequency , 46% ) , the somatic alteration c.1387dela ( allele frequency , 13% ) , and the secondary somatic mutation c.1355_1380deltaccaaaatcagagaagccattaaat , which also restored the open reading frame , but seemed to be subclonal ( with an allelic frequency of only 1% )  . 
this patient , who had also previously received folfirinox , developed significant clinical progression at day 18 , although target lesions were stable . assessment of allele - specific loh tumor was available for analysis in 10 patients . 
 rucaparib treatment stable disease complete response progressive disease partial response progressive disease reported on last day of treatment < 1 weekd patient of prior regimens received prior platinum platinum refractory brca mutation brca2 somatic brca1 germline brca2 germline brca1 germline brca2 germline brca2 germline brca1 germline brca2 germline brca2 germline brca1 germline brca2 germline brca2 germlineg brca2 germline brca2 germline brca2 germline brca2 germline brca2 germline brca2 somatic brca2 somatic sd 72 weeks sd 20 weeks sd 24 weeks pr 36 weeks pr 25 weeks cr 19 weeks pr 5 weeksi cycle fig 2 . 
 ( f ) patient discontinued because of adverse events ( aes ; grade 3 fatigue and grade 4 thrombocytopenia ) ; best response of stable disease ( sd ) was ongoing after the last day of treatment . 
the second patient had a somatic brca2 mutation ( c.1748t > a ) and had a cr as best response ( patient 19 )  . safety all patients had at least one treatment - emergent ae ( table 3 )  . 
two other patients died as a result of disease progression . discussion this study tested the efficacy of a single - agent parp inhibitor , rucaparib , in patients with advanced pc with a known deleterious brca1 / 2 mutation . 
adverse events were graded according to the national cancer institutes common terminology criteria for adverse events ( version 4 )  . * includes adverse events of thrombocytopenia and decreased platelet count . with a confirmed orr of 16% and an observed disease control rate of 32% . 
our findings suggest that there may be a role for parp inhibition in patients with a brca1 / 2 mutation , particularly in those whose disease has not progressed while taking prior platinum therapy . although this was a single - arm study , making comparisons to other agents difficult , the clinical relevance of our results merits comparison with other current standards of care for this patient population . 
 importantly , the only approved chemotherapy combination in the second - line setting , fluorouracil with nanoliposomal irinotecan , had an overall response rate of 7.7% in the pivotal registration study.8 other small single - center studies investigating folfirinox or other chemotherapy combinations in a refractory population show similarly low response rates , reflecting the known chemoresistance of this disease . 
 furthermore , as treatment options improve for pc , patients are even able to move beyond second - line therapy , for which there are no standard of care options . 
this underscores the importance of looking outside of chemotherapy options , particularly in patients with potentially targetable mutations . previous small studies investigating parp inhibition in brca - mutated pc have shown similar efficacy . 
the response rate of single - agent olaparib in patients with metastatic pc harboring a germline brca1 / 2 mutation who had received prior chemotherapy was 22%.20 a phase ii study of veliparib alone in 16 previously treated patients with brca - mutated pc demonstrated single - agent activity , with 25% of patients having sd for at least 4 months . 
analysis is ongoing to understand the role platinum sensitivity played in these four patients.21 when considered with the prior studies , these trials should provide insight into the clinical utility of single - agent parp inhibition in patients with pc and a known brca1 / 2 mutation . in this study , responses to rucaparib were seen in individuals harboring a germline or somatic brca1 / 2 mutation . 
because of significant stromal infiltration in pc , assessing allele frequency of somatic tumor alterations may be challenging . none of the four patients with a confirmed or unconfirmed response had experienced disease progression on prior platinum therapy ( one had never received platinum ) , and three of the four patients had only received one prior chemotherapy regimen for locally advanced or metastatic disease . 
this finding highlights the important question of the role of platinum sensitivity in the setting of advanced / metastatic pc and underscores a potential role for rucaparib as a treatment for patients whose tumors are not platinum refractory . 
similar findings were noted in patients with advanced ovarian cancer treated with olaparib , because the highest response rates were noted in patients who were deemed platinum sensitive rather than resistant or refractory.22 this will need to be investigated in a larger clinical study . 
 secondary mutations that likely confer resistance have been observed in patients with ovarian or prostate cancer who harbor a brca1 / 2 mutation and whose disease has progressed while receiving platinum chemotherapy or parp inhibitors ; however , these reports have demonstrated reversion mutations that have restored the open reading frame in the vicinity of the germline mutation . 
the majority ( but not all ) of brca1 / 2 - mutant breast and ovarian cancers have allele - specific loh23 ; however , the loss of the second allele is most commonly due to copy neutral loh and rarely due to a somatic mutation . 
both of these patients had previously been treated with oxaliplat several studies have demonstrated the presence of reversion mutations in patients previously treated with chemotherapy , particularly platinum based.24 - 27 our study has several limitations , including a small sample size and lack of corresponding somatic sequencing and ctdna analysis for some patients . rucaparib is a well - tolerated parp inhibitor that could be considered in patients with advanced pc with known brca1 / 2 mutations who have received prior chemotherapy . 
domchek research funding : prism biolab ( inst ) , genentech ( inst ) , novartis ( inst ) , newlink genetics ( inst ) , eli lilly ( inst ) , aduro biotech ( inst ) , pfizer ( inst ) , sanofi ( inst ) data analysis and interpretation : rachna t . 
shroff consulting or advisory role : celgene , codiak biosciences , agios , halozyme travel , accommodations , expenses : astrazeneca ravit geva honoraria : bristol - myers squibb , msd , eli lilly , medison , roche , novartis , janssen , pfizer , teva travel , accommodations , expenses : bristol - myers squibb , roche ron epelbaum no relationship to disclose lindsey rolfe employment : clovis oncology leadership : clovis oncology stock and other ownership interests : clovis oncology , celgene , biomarin travel , accommodations , expenses : clovis oncology sandra goble employment : clovis oncology stock and other ownership interests : clovis oncology research funding : eli lilly , celgene , agios , halozyme travel , accommodations , expenses : celgene , codiak biosciences , agios , halozyme kevin k . 
domchek honoraria : astrazeneca , clovis oncology , bristol - myers squibb heidi giordano employment : clovis oncology stock and other ownership interests : clovis oncology acknowledgment research funding : astrazeneca ( inst ) , clovis oncology ( inst ) , pharmamar ( inst ) robert h . 
vonderheide honoraria : genentech , celgene consulting or advisory role : eli lilly , celldex research funding : eli lilly ( inst ) the authors thank lawrence leichman for his helpful input while the study was being designed . 
shroff , the university of texas md anderson cancer center , houston , tx ; andrew hendifar , cedars - sinai medical center , los angeles , ca ; robert r . 
mcwilliams , mayo clinic , rochester , mn ; ravit geva , tel aviv university , tel aviv ; ron epelbaum , rambam health care campus , haifa , israel ; lindsey rolfe , sandra goble , kevin k . 
rahib l , smith bd , aizenberg r , et al : projecting cancer incidence and deaths to 2030 : the unexpected burden of thyroid , liver , and pancreas cancers in the united states . 
assaf e , verlinde - carvalho m , delbaldo c , et al : 5 - fluorouracil / leucovorin combined with irinotecan and oxaliplatin ( folfirinox ) as second - line chemotherapy in patients with metastatic pancreatic adenocarcinoma . 
lee mg , lee sh , lee sj , et al : 5 - fluorouracil / leucovorin combined with irinotecan and oxaliplatin ( folfirinox ) as second - line chemotherapy in patients with advanced pancreatic cancer who have progressed on gemcitabine - based therapy . 
nagrial am , chin vt , sjoquist km , et al : second - line treatment in inoperable pancreatic adenocarcinoma : a systematic review and synthesis of all clinical trials . 
wang - gillam a , li cp , bodoky g , et al : nanoliposomal irinotecan with fluorouracil and folinic acid in metastatic pancreatic cancer after previous gemcitabine - based therapy ( napoli - 1 ) : a global , randomised , open - label , phase 3 trial . 
kristeleit r , shapiro gi , burris ha , et al : a phase i - ii study of the oral parp inhibitor rucaparib in patients with germline brca1 / 2 - mutated ovarian carcinoma or other solid tumors . 
swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
lowery ma , kelsen dp , smith sc , et al : phase ii trial of veliparib ( v ) in patients ( pts ) with previously treated brca or palb2 - mutated ( mut ) pancreas adenocarcinoma ( pc )  . 
domchek sm , aghajanian c , shapira - frommer r , et al : efficacy and safety of olaparib monotherapy in germline brca1 / 2 mutation carriers with advanced ovarian cancer and three or more lines of prior therapy . 
afghahi a , timms km , vinayak s , et al : tumor brca1 reversion mutation arising during neoadjuvant platinum - based chemotherapy in triple - negative breast cancer is associated with therapy resistance . 
weigelt b , comino - mndez i , de bruijn i , et al : diverse brca1 and brca2 reversion mutations in circulating cell - free dna of therapy - resistant breast or ovarian cancer . 
post - test surveys on distress , uncertainty , and positive experiences were administered at 3 months ( 69% response rate ) and 1 year ( 57% response rate )  . results of 2 , 000 participants , 81% were female , 41% were hispanic , 26% were spanish speaking only , and 30% completed high school or less education . 
a total of 242 participants ( 12% ) carried one or more pathogenic variant ( positive ) , 689 ( 34% ) carried one or more variant of uncertain signicance ( vus ) , and 1 , 069 ( 53% ) carried no pathogenic variants or vus ( negative )  . 
after testing , few patients ( 4% ) had prophylactic surgery , most ( 92% ) never regretted testing , and most ( 80% ) wanted to know all results , even those of uncertain signicance . 
positive patients were twice as likely as negative / vus patients ( 83% v 41% ; p , .001 ) to encourage their relatives to be tested . conclusion in a racially / ethnically and socioeconomically diverse cohort , mgpt increased diagnostic yield . 
recent advances in massively parallel sequencing have expanded germline testing for hereditary cancer risk assessment.1 - 3 multiplex gene panel testing ( mgpt ) simultaneously analyzes multiple genes and may provide an advantage over sequential single - gene testing in terms of cost , speed , and clinical utility . 
 idos et al context what are the benets , harms , and patient experiences after germline multiplex gene panel testing for cancer susceptibility ? among 2 , 000 participants in this fully accrued , multicenter , prospective cohort study of hereditary cancer testing with a 25or 28 - gene panel , more than one quarter of the pathogenic variants identied were not clinically anticipated . 
reassuringly , at a median follow - up of 13 months , patients did not regret having undergone multiplex testing , did not overuse preventive surgery , and encouraged their family members to be tested in accordance with practice guidelines . multiplex genetic panel testing enhances the diagnostic yield of genetic testing without discernible harm to patients . 
this study also demonstrates that testing at lower predicted levels of pathogenic variant carriagea probability threshold of 2.5% which reects recent changes in clinical practice and guidelines , is effective and safe . 
overall , this study offers signicant and novel results on the performance of multiplex genetic testing and has broad implications for its clinical implementation and acceptance . cancer risk assessment , and it is crucial to learn more as more comprehensive sequencing approaches on the horizon . we designed a prospective cohort study of hereditary cancer testing with a multigene panel to measure the benets , harms , and patient experiences of mgpt . 
participants were consecutively recruited between july 2014 and november 2016 at three medical centersusc norris comprehensive cancer center , los angeles county and usc medical center , and stanford university cancer institute . 
there were 1 , 664 participants who underwent 25 - gene panel testing and 336 who underwent 28 - gene panel testing . sequencing and large rearrangement analysis were performed for all genes , except pold1 and pole ( sequencing only ) and epcam and grem1 ( large rearrangement only )  . sample preparation for ngs was performed from frozen dna using the raindance microdroplet polymerase chain reaction ( pcr ) system ( raindance technologies , billerica , ma ) .17 in brief , pcr products that represented exons and proximal splicing elements of patient dna were amplied in merged droplets that consisted of fragmented patient dna and select target enrichment primers . 
to circumvent highly homologous pseudogenes , we used modied sample preparation with longrange and nested pcr , followed by ngs on the illumina miseq platform , for portions of chek2 and pms2 . 
all clinically actionable variants identied by ngs , as well as regions that did not meet preset ngs quality metrics , were independently conrmed with orthogonal site - specic sanger sequencing . 
 multicenter study of multiplex panel testing supporting linkage , biochemical , clinical , functional , and statistical data used for specic missense and intronic alterations.18 - 20 gene variants classied as deleterious or that were suspected to be deleterious were considered pathogenic . 
variants classied as polymorphism or that favored polymorphism were considered benign . undergone mgpt , participants desired amount of mgpt results information , participants notication of relatives about mgpt results , and relatives genetic testing behaviors . 
participants were also asked if they had undergone specic surgical procedures ( mastectomy , salpingooophorectomy , or hysterectomy ) and the reason for these treatment , cancer prevention , or procedures ( cancer other ) .23 differential diagnoses statistical analysis differential diagnoses were generated for each participant after expert clinical genetics assessment , in which up to eight inherited cancer syndromes were ranked by estimated likelihood using such factors as personal and family cancer history , tumor characteristics , and physical examination21 ( data supplement )  . 
the genetics clinician then claried a level of suspicion for each syndrome in the differential diagnosis by stating whether she or he would for that syndrome specically if mgpt were not test available . questionnaire procedures participants were invited to answer a baseline questionnaire at the time of their genetics evaluation , with followup questionnaires 3 , 6 and 12 months after disclosure of mgpt results . 
e - mail participants who did not respond to the initial invitation link received two e - mail reminders over a course of 2 weeks before receiving a reminder phone call from study personnel . 
using a standardized script , all participants who received a reminder phone call were given the option to receive another e - mail invitation , another mailed paper questionnaire , or to complete the questionnaire over the phone . 
participants were considered nonresponders if they had not completed their questionnaire 2 months after the initial send date . patient - reported experiences we used the validated multidimensional impact of cancer risk assessment ( micra ) 22 instrument , which contains subscales that measure distress , uncertainty , and positive experiences in relation to genetic testing . 
additional questions evaluated intrusive thoughts about cancer and regret about having the primary aim of this study was to test the impact of genetic test resultspositive , vus , or negativeon patient experience . 
micra subscale scores were used for this aim . patients with one or more pathogenic variant were considered positive , whereas those with only benign or uncertain variants were considered vus and those with only benign variants were considered negative . 
assuming a pathogenic variant prevalence of 10% , a vus prevalence of 35% , and a negative test prevalence of 55% , with the standard deviation of micra scores being 4 , a sample size of 2 , 000 patients was needed to achieve more than 80% power to detect a difference of 1 in micra scores . data analysis was based on information gathered as of march 19 , 2018 . 
we used the pearson 2 test to assess the association between genetic test results and survey responses . negative binomial regression with a log link was used to analyze the association of genetic test results with micra subscale scores while adjusting for covariates that included clinical site , age , gender , ethnicity , education level , and personal history of cancer . 
common reasons for genetics referral include the following : cancer diagnosis at age 50 years or younger ( 39% ) , two or more rstor second - degree relatives with cancer ( 64% ) , and one or more family members diagnosed with cancer at younger than age 50 years ( 63% )  . 
participants were diverse both racially / ethnically and sociodemographically39% were hispanic ( primarily of mexican or central american ancestry ) , 40% non - hispanic white , 12% asian ( primarily of chinese and filipino ancestry ) , and 4% black . 
seventy - six patients31% of all pathogenic variant carriershad a germline mutation in brca1 and / or brca2 , and 39 ( 16% ) had a pathogenic variant in a mismatch repair gene conferring a diagnosis of lynch syndrome . 
of pathogenic mutations brca1 and brca2 genes associated with breast cancer genes associated with ovarian cancer lynch syndrome genes genes associated with colon , pancreatic , or gastric cancer tp53 ( 2% )  . 
among patients without pathogenic variants , 689 ( 34% ) had at least one vus , with up to four per patient ( table 2 )  . differential diagnosis versus mgpt results of 242 participants with pathogenic variants , 160 ( 66% ) had pathogenic variants in genes related to syndromes that were included in the pretest differential diagnosis . 
eightytwo patients ( 34% ) had pathogenic variants that were not clinically anticipatedmonoallelic mutyh ( n = 32 ; 39% ) , apc i1307k ( n = 13 ; 16% ) , chek2 ( n = 10 ; 12% ) , palb2 ( n = 8 ; 10% ) , atm ( n = 7 ; 9% ) , brip1 ( n = 4 ; 5% ) , brca2 ( n = 3 ; 4% ) , brca1 ( n = 2 ; 2% ) , pms2 ( n = 2 ; 2% ) , and tp53 ( n = 1 ; 1% )  . use of surgery after mgpt at a median follow - up of 13 months , 198 ( 13% ) of 1 , 573 returning surveys reported undergoing surgery after mgpt . surgery rates and stated reasons were as follows : mastectomy ( n = 162 [ 11.3% ] : 90% for cancer treatment , 30% for cancer prevention , and 2% for benign breast disease ) , salpingo - oophorectomy ( n = 43 [ 3% ] : 27% for cancer treatment and 56% for cancer prevention ) , and hysterectomy ( n = 23 [ 2% ] : 50% for cancer treatment , 18% for cancer prevention , and 9% for benign disease )  . 
to illustrate , there were 10 patients identied as having highpenetrance founder mutations in brca1 or brca2 , six of whom were found to have undergone or were considering risk - reducing mastectomy or oophorectomy . 
in comparison , there were 16 patients identied as carriers of the lowpenetrance apc i1307k allele , none of whom had undergone an inappropriate surgery ( data supplement )  . patient experiences with mgpt at a median follow - up of 4 months , the response rate was 69% . 
mean scores for each micra subscaledistress , uncertainty , or positive experiencesall differed signicantly between patients with positive test results compared with the group of patients with a negative or vus result ( table 3 )  . 
sociodemographic and clinical characteristics by genetic test result characteristic positive ( n = 242 ) negative ( n = 1 , 069 ) vus ( n = 689 ) total ( n = 2 , 000 ) idos et al clinical site , no . 
also , a higher proportion of patients testing negative as compared to positive reported that they did not regret learning their test results ( 95% v 85% ; p , .001 ; fig 2 )  . 
we found little evidence of patient harm and substantial evidence that patients understood the meaning of their mgpt results , as patients who tested positive for a pathogenic variant were more likely to encourage appropriate genetic testing in relatives compared with those who tested with vus or negative results . 
these results suggest an important contribution of mgpt to clinical cancer risk assessment of probands and their families . a striking result was that one in three identied pathogenic variants was missed in the differential diagnoses generated by expert clinicians . 
of note , few of the missed variants were in genes associated with well - known syndromesfor example , lynch syndrome , hereditary breast / ovarian cancerwhich likely reects clinicians greater familiarity with their presentation . instead , most were in moderate - penetrance genes that confer a twoto four - fold increase in cancer risk ( eg , atm and chek2 ) as well as low - penetrance mutations ( eg , monoallelic mutyh and apc i1307k )  . given the short history of widespread clinical testing of these genes , 24 - 27 their phenotype is less well characterized and more difcult for clinicians to recognize . 
some questions remain about the clinical benet to moderate - penetrance pathogenic of detecting lowvariants ; however , these variants meet criteria for intensied cancer screening according to current practice guidelines , 28 , 29 which makes them relevant to patient care . by identifying unexpected pathogenic variants , mgpt can broaden our understanding of genotype - phenotype correlations and the spectrum of associated cancer risks . concerns have been raised about high rates of identifying vus in mgpt . 
consistent with prior studies , we report a vus rate of 34%.5 - 8 recent studies found limited genomic condence among oncologists and that few ( less than 15% ) community physicians who order brca1 and brca2 tests understand the correct management of vus.27 , 31 , 32 we recently published that patients who receive vus results from genetic testing are substantially more likely to seek a second opinion from a new medical oncologist.33 a related concern is that mgpt results of unknown clinical relevance , whether vus or a pathogenic variant whose cancer risk is insufciently characterized , might prompt operations.34 - 36 invasive data from our prospective cohort study do not support this hypothetical concern . 
reassuringly , we found that prophylactic operations were not overusedonly 4% underwent prophylactic mastectomy , hysterectomy , or prophylactic irreversible salpingo - oophorectomy , and these procedures were no more frequent among patients with vus results than those with negative results . 
with a median follow - up time of these results do not 13 months after genetic testing , suggest an overuse of surgery after mgpt in this study . of note , overall yield of pathogenic variants in this study is 12% , which is similar to other studies . 
whereas panels may vary in the number of genes tested , all large panels , including the one used in this study , include genes that are associated with known syndromesthat is , hereditary breast / ovarian cancer and lynch syndromewhich account for the majority of pathogenic mutations identied in most multigene panel studies . 
our group previously examined the additional yield of mutations identied via multigene panel testing and found that by increasing the the frequency of mutations number of genes tested , identied also increased.37 , 38 independently , a recent study by mandelker et al39 demonstrated a 17.5% yield of pathogenic variants after sequencing 1 , 040 patients with advanced cancer using the memorial sloan kettering impact panel of 410 genes . patient - reported experiences were also reassuring . 
 multicenter study of multiplex panel testing genetic test results in a multitude of settings.40 we found that micra subscale scores varied signicantly between patients who tested positive compared with those who had a negative or vus test result , but scores did not vary between patients with a vus versus negative except in the category of uncertainty . 
these ndings suggest that patients rarely misinterpret vus with appropriate pretest genetic counseling as most vuss that are reclassied are ultimately reclassied to benign.15 , 41 on the contrary , these ndings suggest that patients understood the implications of their mgpt results , which is particularly encouraging in our population as approximately one third received high school education or less . 
the present results contrast with our recent nding of gaps in the understanding and management of vus in community practice , and serve to demonstrate the value of preand post - test counseling with proper anticipatory guidance by a trained professional.42 - 44 there are limited published data on pathogenic mutation rates among hispanics after multiplex gene panel testing , as hispanics are underrepresented in a majority of clinical studies . 
in our previous usc - based paper by ricker et al , 37 in which hispanic patients made up 47.6% ( n = 228 ) of the cohort , there was a pathogenic mutation frequency of 14.1% ( n = 33 )  . 
in a study by rosenthal et al , 11 among the 8% ( n = 19 , 795 ) of hispanic patients who underwent multigene panel testing , 6.7% ( n = 1 , 326 ) were found to have a pathogenic mutation . 
its considerable strengths include a prospective , multicenter design ; a diverse population in terms of race / ethnicity , language , testing at and education ; a high survey response rate ; and uniform pretest assessment by experienced genetic counselors . the participation rate of hispanic / latino , african american , and asian patients was high compared with other published studies . 
to our knowledge , it is the rst study to demonstrate that lower predicted levels of pathogenic variant carriage , which reects recent changes in clinical practice and guidelines , is effective and safe . 
this study offers signicant and novel results on the performance of multiplex genetic testing and has broad impliimplementation and acceptance . cations for its clinical looking forward , interim analysis of our data suggests that patients who are found to have a pathogenic variant in a highor moderate - risk gene are more likely to undergo appropriate screening and surveillance compared with those who tested negative or with a vus within 1 year of mgpt testing . for example , patients who were identied as carrying a pathogenic variant in a gene associated with lynch syndrome were four times as likely ( p , .001 ) to undergo colonoscopy compared with patients with a vus or negative genetic test result . encouragingly , this trend is consistent when we stratify by ethnicity in our hispanic cohort . its limitations include 13 months of follow - up time to date . in addition , our survey response rate decreased to 57% at 12 months from 69% at 3 months , as we primarily recruited from a clinical cohort , most of whom were affected with cancer and undergoing active treatment . 
contributed equally to this work . employees of myriad genetic laboratories served as coinvestigators in this study and provided material support , including germline testing and interpretation , as described in the manuscript . 
kurian research funding : myriad genetics ( inst ) other relationship : ambry genetics , color genomics , genedx , bioreference , invitae , genentech charit e ricker research funding : myriad genetics ( inst ) peter levonian consulting or advisory role : pwnhealth katrina lowstuter research funding : myriad genetics ( inst ) meredith a . 
mills research funding : myriad genetics ( inst ) , natera ( inst ) anne - renee hartman employment : myriad genetics , grail stock and other ownership interests : myriad genetics , grail richard j . 
wenstrup employment : oxford immunotec , myriad genetics leadership : oxford immunotec , myriad genetics stock and other ownership interests : oxford immunotec , myriad genetics patents , royalties , other intellectual property : founding patent holder , assurexhealth , since 2016 , a wholly owned subsidiary of myriad genetics johnathan m . 
lancaster employment : myriad genetics leadership : myriad genetics stock and other ownership interests : myriad genetics consulting or advisory role : protean biodiagnostics travel , accommodations , expenses : myriad genetics krystal brown employment : myriad genetics john kidd employment : myriad genetics consulting or advisory role : myriad genetics brent evans employment : myriad genetics stock and other ownership interests : myriad genetics travel , accommodations , expenses : myriad genetics kevin j . 
mcdonnell consulting or advisory role : brogent international research funding : myriad genetics uri ladabaum stock and other ownership interests : universal dx , lean medical consulting or advisory role : motus gi , medtronic / covidien ( given ) , quorum consulting , clinical genomics james m . 
gruber employment : brogent international leadership : brogent international stock and other ownership interests : brogent international , fulgent therapeutics consulting or advisory role : myriad genetics , fulgent therapeutics research funding : myriad genetics ( inst ) no other potential conicts of interest were reported . acknowledgment the authors thank blanca ovalle , serina ovalle , and angelica mora for their tremendous help in the recruitment and observation of study participants . references easton df , pharoah pd , antoniou ac , et al : gene - panel sequencing and the prediction of breast - cancer risk . 
walsh t , casadei s , lee mk , et al : mutations in 12 genes for inherited ovarian , fallopian tube , and peritoneal carcinoma identied by massively parallel sequencing . 
 multicenter study of multiplex panel testing clin genet 86 : 510 - 520 , 2014 oncol 36 : 414 - 424 , 2018 j clin oncol 33 : 3660 - 3667 , 2015 110 : 1059 - 1066 , 2018 3 . 
walsh t , lee mk , casadei s , et al : detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing . proc natl acad sci usa 107 : 12629 - 12633 , 2010 yurgelun mb , allen b , kaldate rr , et al : identication of a variety of mutations in cancer predisposition genes in patients with suspected lynch syndrome . gastroenterology 149 : 604 - 613.e20 , 2015 yurgelun mb , kulke mh , fuchs cs , et al : cancer susceptibility gene mutations in individuals with colorectal cancer . 
j clin oncol 35 : 1086 - 1095 , 2017 tung n , lin nu , kidd j , et al : frequency of germline mutations in 25 cancer susceptibility genes in a sequential series of patients with breast cancer . 
j clin oncol 34 : 1460 - 1468 , 2016 thompson er , rowley sm , li n , et al : panel testing for familial breast cancer : calibrating the tension between research and clinical care . 
j clin oncol 34 : 1455 - 1459 , 2016 kurian aw , hare ee , mills ma , et al : clinical evaluation of a multiple - gene sequencing panel for hereditary cancer risk assessment . 
j clin oncol 32 : 2001 - 2009 , 2014 desmond a , kurian aw , gabree m , et al : clinical actionability of multigene panel testing for hereditary breast and ovarian cancer risk assessment . 
rosenthal et , bernhisel r , brown k , et al : clinical testing with a panel of 25 genes associated with increased cancer risk results in a signicant increase in clinically signicant ndings across a broad range of cancer histories . 
balmaa j , digiovanni l , gaddam p , et al : conicting interpretation of genetic variants and cancer risk by commercial laboratories as assessed by the prospective registry of multiplex testing . 
j clin oncol 34 : 4071 - 4078 , 2016 judkins t , leclair b , bowles k , et al : development and analytical validation of a 25 - gene next generation sequencing panel that includes the brca1 and brca2 genes to assess hereditary cancer risk . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
pruss d , morris b , hughes e , et al : development and validation of a new algorithm for the reclassication of genetic variants identied in the brca1 and brca2 20 . 
hampel h , bennett rl , buchanan a , et al : a practice guideline from the american college of medical genetics and genomics and the national society of genetic counselors : referral indications for cancer predisposition assessment . 
cella d , hughes c , peterman a , et al : a brief assessment of concerns associated with genetic testing for cancer : the multidimensional impact of cancer risk assessment ( micra ) questionnaire . 
yurgelun mb , mercado r , rosenblatt m , et al : impact of genetic testing on endometrial cancer risk - reducing practices in women at risk for lynch syndrome . gynecol oncol 127 : 544 - 551 , 2012 2013 netw 15 : 9 - 20 , 2017 canc netw 15 : 1465 - 1475 , 2017 prev res ( phila ) 4 : 9 - 22 , 2011 24 . 
j clin oncol genet test mol biomarkers 17 : 367 - 375 , 2013 35 : 2232 - 2239 , 2017 jama oncol 3 : 391 - 397 , 2017 susceptibility testing . 
bradbury ar , patrick - miller lj , egleston bl , et al : patient feedback and early outcome data with a novel tiered - binned model for multiplex breast cancer 35 . 
ricker c , culver jo , lowstuter k , et al : increased yield of actionable mutations using multi - gene panels to assess hereditary cancer susceptibility in an ethnically diverse clinical cohort . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
maxwell kn , hart sn , vijai j , et al : evaluation of acmg - guideline - based variant classication of cancer susceptibility and non - cancer - associated genes in families affected by breast cancer . 
 about ras mutation clearance in plasma ctdna from ras - mutant colorectal cancer patients bouchahda et al1 recently published in your journal . authors conducted a pilot study aimed to investigate the efcacy and safety of antiegfr - targeted therapy added to chemotherapy in patients with unresectable metastatic colorectal cancer with raswild - type circulating tumor dna ( ctdna ) but ras - mutant primary tumor . 
we would like to emphasize the following . in 2017 , our group for the rst time started to investigate the clearance of ras - mutant clones in plasma , supporting an unexpected negative selection of rasmutant clones during the clonal evolution of rasmutant metastatic colorectal cancer ( mcrc ) .2 one year later , we reported that ve patients with rasmutant ras ( ras - mt ) mcrc who switched to wildtype ras ( wt - ras ) in plasma ctdna received egfr inhibitors as a second - line treatment , achieving a durable clinical benet , 3 thus suggesting that ctdna analysis might reveal a therapeutically exploitable window of opportunity , characterized by the prevalence of wt - ras clones , which can be converted in a clinically meaningful benet for patients . 
we published a detailed case report4 in which we described the study of a patient with a primary n - ras - mutant colorectal cancer , who received second - line treatment with antiegfr , after failure of rst - line triplet chemotherapy plus bevacizumab , according to the absence of any clinically relevant mutation of ras genes in blood , achieving a partial response . 
with this background , we designed the currently ongoing kairos trial ( eudract number : 2019 - 001328 - 36 ) , aimed to investigate the efcacy and safety of second - line cetuximab plus chemotherapy treatment in initially ras - mt mcrc patients who converted to raswt at the time of rst progression.3 , 4 we regret having to note that bouchahda et al1 have not cited neither our publications nor have they named the kairos trial in their entire discussion . 
in fact , the authors state that the team of raimondi et al5 recently reported the disappearance of ras - mt clones in ctdna after tumor progression while receiving bevacizumab and chemotherapy in four patients with ras - mt mcrc . 
this last information is incorrect in that we prospectively enrolled mcrc patients at the time of progression of disease and the mutational status of ctdna was assessed before initiating a further line of therapy ( as far as we underthe same inclusion criteria as reported by stand , bouchahda et al1 in their study )  . 
furthermore , bouchahda et al strongly limited the discussion of our data to the number of patients who converted to wt - ras in plasma , omitting to specify that these patients were then treated with egfr blockade based on the results obtained in ctdna . 
in these patients , the introduction of anti - egfr agents , which would have not been supported by international guidelines , allowed to achieve a 12 - month , 10 - month , and 6 - month pfs in three patients treated in second - line setting and a pfs of 4 months in patients treated in fourth - line.5 these data should have been included and discussed . 
finally , the authors state that studies with liquid biopsy have been to date selectively focused on the early detection of the appearance of ras - mt clones in tumor deposits by analyzing ctdna in blood samples from patients with raswt primary tumors . 
bouchahda m , saffroy r , karaboue a , et al : undetectable rasmutant clones in plasma : possible implication for anti - egfr therapy and prognosis in patients with ras - mutant metastatic colorectal cancer . 
gazzaniga p , raimondi c , urbano f , et al : second line egfr - inhibitors in ras mutant metastatic colorectal cancer : the plasma ras wild type window of opportunity . 
gazzaniga p , raimondi c , urbano f , et al : egfr inhibitor as second - line therapy in a patient with mutant ras metastatic colorectal cancer : circulating tumor dna to personalize treatment . 
raimondi c , nicolazzo c , belardinilli f , et al : transient disappearance of ras mutant clones in plasma : a counterintuitive clinical use of egfr inhibitors in ras mutant metastatic colorectal cancer . 
nicolazzo c , belardinilli f , caponnetto s , et al : why the therapeutic impact of ras mutation clearance in plasma ctdna deserves to be further explored in metastatic colorectal cancer . 
 clinical utility of genomic profiling in the treatment of advanced sarcomas : a single - center experience see accompanying editorial doi : purpose sarcomas are a diverse group of malignant tumors that arise from soft tissues or bone . 
 in this study , we review our institutional experience with next - generation sequencing ( ngs ) based molecular profiling for nongi stromal tumors sarcomas , with a focus on the clinical utility of the results . patients and methods we retrospectively analyzed results of ngs performed on tumors from 114 patients with a diagnosis of sarcoma . 
for therapeutic trials and treatment guidelines , treatment options for sarcomas have historically frequently been lumped into broad categories ( eg , soft tissue sarcoma ) , despite highly divergent underlying disease biology and clinical behavior . 
furthermore , current treatment options for patients with advanced disease are limited and generally not curative , which highlights the need for novel approaches and greater understanding of disease biology . in recent years , there has been an increasing desire to personalize treatment of patients with advanced cancer via molecularly guided therapeutic selection . 
to a degree , sarcoma was on the forefront of successful molecularly targeted therapy with the clinical development of tyrosine kinase inhibitors against kit or pdgfra mutations for advanced gi stromal tumors ( gist ) in the early 2000s.1 for most other sarcoma subtypes , however , targetable mutations have not been widely described , and the clinical utility of routine spandana boddu christine m . 
 molecular profiling in sarcoma has not been established . in this study , we review our institutional experience with next - generation sequencing ( ngs ) based molecular profiling for non - gist sarcomas . 
 we review > 100 sarcoma cases that underwent comprehensive molecular profiling , with a focus on the clinical utility of the results , including molecular target identification , diagnostic clarification , and treatment outcomes . 
to our knowledge , this is one of the largest sarcoma specific analyses of this type to date . methods we used our personalized medicine clinical service ( pmcs ) database to identify all patients seen at the moffitt cancer center with a diagnosis of non - gist sarcoma who underwent commercial genomic testing between may 2013 and march 2017 . 
genomic findings from each patient were manually curated in the clinical genomic action committee database along with demographic , clinical , and histologic information , in addition to type and date of genomic test and date and source of biopsy specimen . 
all patients are reviewed either by the core members of the pmcs group weekly or by the moffitt molecular tumor board ( clinical genomic action committee ) on the basis of the complexity of the genetic results . 
 as previously described , clinical actionability is based on available literature , and recommendations are documented in the patients electronic medical record as a resource for the treating physicians.2 though tumor - only sequencing was performed , for findings that were felt to be potentially germline ( eg , previously reported pathogenic mutation in clinvar [ ncbi.nlm.nih.gov / clinvar / ] that could be either somatic or germline ) , a recommendation was given to the treating provider to refer the patient for genetic counseling and assessment . performed on identified patients to retrospectively collect treatments and outcomes data . 
genetic alterations were defined as clinically actionable if they had been documented in the literature to provide diagnostic or prognostic information or to predict response or resistance to commercially available or investigational agents . 
actionability was classified as predicting response to approved drugs available for the patients diagnosis ( on - label ) , for another diagnosis ( off - label ) , or investigational drugs being studied in humans for whom the genetic alteration has been shown to serve as a suggested biomarker for response . 
assessment of clinical actionability was limited to mutations previously reported or likely to be driver - mutations based on available literature and functional classification and did not consider analysis of variants of unknown significance . 
the study was approved by the university of south florida institutional review board . results we identified 114 patients with a diagnosis of non - gist sarcoma who underwent molecular profiling during the specified period . 
there were 51 men ( 45% ) and 63 women ( 55% ) in the cohort , with the median age at diagnosis of 55 ( interquartile range , 38 to 65 ) years . 
the most common disease histologies paralleled the most common sarcoma subtypes , such as leiomyosarcoma ( 16.7% ) , well - differentiated / dedifferentiated liposarcoma ( 12.2% ) , and undifferentiated pleomorphic sarcoma ( 10.5% ) as the most frequent soft tissue tumors . 
of small variants , truncating mutation types such as nonsense , frameshift , and splice site altogether ( n = 99 ) outnumbered missense mutations ( n = 65 )  . 
no patients had evidence of microsatellite instability . for five patients ( 4.4% ) , the genomic findings were felt to be diagnosis changing or diagnosis modifying by the treating physician . 
 one case of poorly differentiated sarcoma , favor neurogenic tumor / mpnst [ malignant peripheral nerve sheath tumor ] was diagnosed as synovial sarcoma after a typical ss18 - ssx2 fusion was detected by ngs and confirmed by conventional testing . 
in the remaining three cases , novel or seminovel fusions that were felt to be disease defining were detected in cases of small round cell sarcoma , not otherwise specified , including one each of ewsr1 - patz1 , bcor - zc3h7b , and phf1 - tfe3 . eighty - eight patients ( 77.2% ) had a therapeutic option recommended by the commercial ngs testing report . 
after review of the genomic findings by our institutional pmcs , 56 patients ( 49.1% ) were classified as having an actionable result ; the significant majority of these had both off - label targeted therapy and molecularly matched clinical trial options ( table 2 )  . 
of note , this number includes those in whom a standard treatment option was selected over an alternative ( eg , selection of pazopanib instead of an alternate , second - line agent because of molecular matching of this target ) and cases in which therapy selection was altered as a result of change in diagnosis . 
four of 15 ( 26.7% ) ngs - influenced therapies evaluable for outcome resulted in clinical benefit ( partial response or stable disease > 6 months )  . discussion in the past several years , there has been significant enthusiasm from patients and clinicians alike to personalize cancer therapy by fig 1 . 
more broadly , there have been indications that the use of molecular - based trial selection criteria leads to higher success rates within the context of early phase clinical trials.4 , 5 sarcomas , given their rarity , diversity , and lack of highly effective therapies in the majority of advanced cases , would seem a natural choice for such a personalized treatment approach . 
the genes most commonly affected by pathogenic mutations in our cohort mirror those most widely reported in sarcomas to date.6 our results are remarkably consistent with the limited published literature of the experience of clinical genomic profiling in sarcoma to date . 
in a separate study of 102 patients with sarcoma , groisberg et al8 reported a similar high rate of potentially actionable mutations , and furthermore that 16% of their cohort went on to receive molecularly guided therapy . 
this number , however , includes those who received therapy for mutations that could be assumed or tested without the need for ngs , such as mdm2 amplification in well differentiated / dedifferentiated liposarcoma . 
in a third study of 107 patients with sarcoma , ngs resulted in diagnosis change in 5% of patients and led to matched trials in 30%.9 this latest study has been presented in abstract form only ; therefore , details of the trial matching , outcomes , and whether ngs would truly have been necessary to detect the alterations of interest are not currently available . our study adds significantly to the existing literature on the clinical utility of ngs in the treatment of advanced sarcomas . 
furthermore , we report and account for the frequent situation where commercial ngs findings overlap with already known recurrent alterations , such as mdm2 / cdk4 amplification in well - differentiated / dedifferentiated liposarcoma . 
most critically , we describe the end point of clinical benefit , which is often omitted from molecular profiling reports . it is important for patients and clinicians to have a sense of expectations for clinical ngs . 
furthermore , without a true control comparator , it is impossible to say whether the patients who had treatment influenced by ngs did better than they would have done without this information . 
it is possible , for example , that the chondrosarcoma treated with prolonged stable disease was an indolent version of this malignancy that may have been stable for this period anyway . 
 to answer the important question of true clinical benefit of molecular profiling . despite the cited limitations , a minority of patients with sarcoma have their course profoundly influenced by clinical genomic profiling . 
looking forward , we expect the number of patients who have actionable ngs findings to steadily increase as more molecularly targeted therapies become available and as genomics becomes increasingly used as a biomarker for immunotherapy response . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . spandana boddu no relationship to disclose christine m . 
tsimberidou am , wen s , hong ds , et al : personalized medicine for patients with advanced cancer in the phase i program at md anderson : validation and landmark analyses . 
gounder mm , ali sm , robinson v , et al : impact of next - generation sequencing ( ngs ) on diagnostic and therapeutic options in soft - tissue and bone sarcoma . 
 aggressive - variant microsatellitestable pole mutant prostate cancer with high mutation burden and durable response to immune checkpoint inhibitor therapy the increasing application of next - generation tumor dna sequencing may define molecular subsets of prostate cancer for which precision medicine approaches are enabled . 
transrectal ultrasound guided needle biopsy demonstrated gleason 4 + 5 = 9 ( grade group 5 ) adenocarcinoma in 12 of 12 cores involving 95% to 100% of each core with perineural invasion . 
tumor cells stained positive for psa and negative for chromogranin and synaptophys radiologic staging studies indicated bilateral external iliac , common iliac , and retroperitoneal adenopathy without definitive evidence of bone metastases . 
with persistent bulky disease in the prostate on digital rectal examination at 12 weeks from the start of hormonal therapy , a repeat biopsy was performed , which demonstrated gleason 4 + 5 = 9 adenocarcinoma with focal synaptophysin expression . 
restaging studies showed only modest regression in pelvic adenopathy discordant with the brisk decline in psa , with tumor infiltrating the bladder base and seminal vesicles , and abutting the rectum with a preserved fat plane on magnetic resonance imaging . 
pathologic examination indicated a high - grade tumor with morphologic and immunohistochemical evidence of neuroendocrine differentiation ( synaptophysin and chromogranin expression ) with lymphocytic infiltration ( fig 1 ; appendix fig a1 )  . 
seminal vesicle involvement was absent , but there was extensive extraprostatic extension and multiple positive surgical margins including the bladder neck , and one of eight lymph nodes were involved.the patient did well in the postoperative period , with early recovery of continence ; however , restaging studies showed progressive retroperitoneal and pelvic adenopathy . 
repeat radiologic imaging after an interval on observation again demonstrated progressive pelvic adenopathy ( fig 2a )  . methodology genomic sequencing studies on the primary tumor were performed with the foundationone assay1 ( foundation medicine , cambridge , ma ) , and the tumor mutation burden was estimated as previously described.2 mutations of the androgen receptor , pten , tp53 , and atm , which have been previously implicated in the genomic landscape of prostate cancer , 3 , 4 were identified , along with a pole v511l mutation ( appendix table a1 )  . 
using all nondriver and nongermline missense mutations , we found that 77% of the alterations in the sample were attributable to the pole signature , making this the dominant signature over other lesions identified in the sample that have been implicated in dna repair , including tp53 , atm , or pten ( appendix table a1 )  . 
germline screens for inherited mutations included a screen for brca1 and brca2 ( ambry genetics , aliso viejo , ca ) and a breast - ovarian panel ( genedx , gaithersburg , md )  . 
infiltrating cd4 + and cd8 + lymphocytes were observed in a three to one ratio.the patients family history raised concerns of an autosomal - dominant inherited risk of prostate and breast cancer . 
his father had been diagnosed with high - grade prostate cancer at 76 years of age , and his two brothers had screen - detected low - risk prostate cancer at 54 and 48 years of age , respectively . 
one sister had died of triple - negative metastatic breast cancer at 45 years of age , and another was diagnosed with ductal carcinoma in situ at 55 years of age . 
 was identified in the father in brip1 ( c2392c > t [ p . arg798ter ] ) , along with a brip1 variant of uncertain significance ( c.3710c > a [ p.ser1237tyr ] ) ; the latter alone was found in the patient and two living siblings ( appendix fig a2 )  . 
given the early castration - resistant and chemotherapy - resistant progression of the patients disease and the high tumor mutation burden identified , he was started on immune checkpoint inhibitor blockade with pembrolizumab therapy 200 mg every 3 weeks intravenously . 
sixteen months later , he has remained without evidence of disease progression after 24 cycles of therapy , with residual subcentimeter pelvic nodes , a normal testosterone level without hormonal therapy , and an eastern cooperative oncology group performance score of zero . discussion to our knowledge , this first report in prostate cancer indicates the presence of a mutation in the highly conserved exonuclease domain of dna polymerase ( pole ) 7 in an aggressive - variant metastatic prostate cancer8 that presented with a bulky primary tumor , early castration , and chemotherapy resistance but with an excellent and durable subsequent response to immune checkpoint blockade . 
pole mutant cancers constitute a unique genomic subset of cancers described previously in colorectal and endometrial cancers.9 the somatic v411l mutation of pole has been associated with impaired dna proofreading repair and the generation of ultramutated tumor phenotypes that are microsatellite stable.9 , 10 mutations that could contribute to castration resistance ( androgen receptor , pten ) or dna damage repair insufficiency ( atm , tp53 ) were also identified , consistent with the known genomic landscape of prostate cancer.3 however , two methods of analyzing genomic signatures were used that implicate the pole lesion as the dominant source of the hypermutated phenotype in this case . 
 hypermutated phenotypes in prostate cancer are uncommon and were first identified in 12% of tumors ( 7 of 60 ) sourced from patient - derived xenografts and a rapid autopsy series.11 in this study set , hypermutated tumors , defined as > 300 somatic protein - altering mutations , were universally associated with micro satellite instability largely secondary to msh2 and msh6 structural rearrangements and not mlh1 epigenetic silencing , as is more commonly seen in colorectal and endometrial cancers with dna mismatch repair deficiency . 
 our case report suggests that microsatellite stable pole mutant prostate cancer represents a distinct , albeit exceedingly rare , genomic subset of highmutation - burden prostate cancers with demonstrable potential for high - quality and durable response to immune checkpoint blockade , similar to that reported in endometrial and colorectal pole mutant cancers.12 , 13although responses to immune checkpoint inhibitors in castration resistant prostate cancer have been uncommon to date , 14 - 16 in one study , three of 10 enzalutamide resistant tumors responded to pembrolizumab therapy.17 one evaluable tumor was found to be microsatellite unstable , and a second was microsatellite stable . 
to our knowledge , other that this case report , there are no data that link the control of prostate cancer with immune checkpoint inhibitors to genomically defined highmutation - burden subsets of prostate cancers , whether microsatellite stable or unstable . 
more studies will be required to establish whether high - quality and durable responses to immune checkpoint inhibitors as single agents will be confined to prostate tumors with high mutation burdens . 
hotspot germline genomic sequencing using commercially available assays via clinical genetics consultation did not identify a pathogenic lesion in the patient and two of the living siblings with cancer diagnoses , but his father was identified with a pathogenic brip1 variant along with the brip1 variant of uncertain significance found in his children . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center  . lisa lee no relationship to disclose elizabeth genega no relationship to disclose dallas reed no relationship to disclose ethan sokol no relationship to disclose paul mathew honoraria : exelixis patents , royalties , other intellectual property : patent pending on new therapeutic invention travel , accommodations , expenses : exelixis affiliations lisa lee , elizabeth genega , dallas reed , and paul mathew , tufts medical center , boston ; siraj ali and ethan sokol , foundation medicine , cambridge , references 1 . 
graff jn , puri s , bifulco cb , et al : sustained complete response to ctla - 4 blockade in a patient with metastatic , castration - resistant prostate cancer . 
kwon ed , drake cg , scher hi , et al : ipilimumab versus placebo after radiotherapy in patients with metastatic castration - resistant prostate cancer that had progressed after docetaxel chemotherapy ( ca184 - 043 ) : a multicentre , randomised , double - blind , phase 3 trial . 
kote - jarai z , jugurnauth s , mulholland s , et al : a recurrent truncating germline mutation in the brip1 / fancj gene and susceptibility to prostate cancer . 
 legend prostate cancer squamous cell carcinoma colorectal cancer thyroid cancer melanoma basal cell cancer breast cancer 61 years brip1 c.3710c > a ( p.ser1237tyr ) ; likely benign variant prostate cancer , 59 years gleason 9 60 years breast cancer ( unilateral ) , 54 years brip1 c.3710c > a , p.ser1237tyr ; likely benign variant 53 years prostate cancer , 52 years melanoma 49 years prostate cancer , 48 years basal cell cancer d . 
87 years colorectal cancer , 87 years 85 years brip1 c.2392c > t ( p.arg798ter ) ; pathogenic prostate cancer , 79 years squamous cell carcinoma 83 years thyroid cancer , 40s fig a2 . 
 epicapture : a urine dna methylation test for early detection of aggressive prostate cancer purpose liquid biopsies that noninvasively detect molecular correlates of aggressive prostate cancer ( pca ) could be used to triage patients , reducing the burdens of unnecessary invasive prostate biopsy and enabling early detection of high - risk disease . 
epicapture was performed by quantitative methylation - specific polymerase chain reaction in dna isolated from prebiopsy urine sediments and evaluated by receiver operating characteristic and decision curves ( clinical benefit )  . 
the epicapture score was developed and validated on a two thirds training set to one third test set . results higher methylation of epicapture genes was significantly associated with increasing aggressiveness in pca tissues . 
2019 by american society of clinical oncology introduction prostate cancer ( pca ) is the most common noncutaneous malignancy in men and the third leading cause of cancer - related deaths in men in the western world . 
 with an aging population , spread of westernized diet , and increasing use of prostate - specific antigen ( psa ) testing , this figure is predicted to double by 2035.1 pca demonstrates extreme clinical heterogeneity . 
overall , 10 - year survival rates are close to 100%2 ; thus , there is much interest in strategies to reduce overtreatment of low / intermediate risk tumors.3 however , more than 300 , 000 men die as a result of pca each year . 
this stark figure , together with the knowledge that overtreatment comes with the high price of prevalent life - changing side effects , illustrates the need for tools to selectively identify high - risk pca ( those tumors with a high propensity to metastasize ) at an early stage , while the disease is potentially curable . research is ongoing into methods to address this need and better risk - stratify patients . 
urinary analysis of molecular correlates of aggressive disease could be used to triage patients and identify those who actually require an invasive prostate biopsy to histologically diagnose and grade their disease . in 2012 , movember launched four collaborative global action plan ( gap ) initiatives , focusing on pca biomarkers in urine , plasma , serum , and exosomes . 
 underpinning this aim was the cooperative establishment of prebiopsy postdigital rectal examination ( dre ) urine biorepositories at each institution , with a confederated database housing accompanying clinical , pathologic , and lifestyle data . 
six centers from the united states ( emory healthcare , atlanta , ga ) , canada ( university health network and lunenfeld - tanenbaum research institute , sinai health systems , toronto ) , and europe ( st jamess hospital , mater misericordiae university hospital , and tallaght hospital , all in ireland ; norfolk and norwich university hospital , england ) , collected prebiopsy , postdre urine ( 50 ml ) between january 2012 and october 2015 . 
exclusion criteria included previous diagnosis of pca , active urinary tract infection , recent prostate biopsy or transurethral resection of the prostate ( less than 6 wk ) , and confirmed presence of metastatic disease ( by pelvic magnetic resonance imaging or bone scan )  . 
 in total , urine samples were collected from 503 men , with matched formalin - fixed paraffin embedded trus biopsy cores acquired , where available ( fig 1 )  . 
in addition to this prebiopsy population , a retrospective analysis of the epicapture gene loci was performed on two independent cohorts of prostate tissues ( fig a2 ; data supplement )  . sample collection , processing , and epicapture analysis urine samples were stored on ice and processed within 4 hours of collection , following a movember gap1 standard operating procedure . 
details of nucleic acid isolation and quantification and epicapture analysis are provided in appendix table a1 and the data supplement . statistical analysis statistical analyses were performed with r version 3.4.1 and graphpad prism version 6 . 
 clinical utility was assessed by decision curve analysis ( dca ) .16 results epicapture genes demonstrate prognostic utility in prostate tissues mindful that most potential biomarkers fail to translate to the clinic , 17 we first set out to verify the prognostic utility of each epicapture constituent gene by measuring its methylation in two independent cohorts of radical prostatectomy samples . 
there was strong evidence for differences in mean methylation - values between four different groups ( benign , low - risk , aggressive , and metastatic pca ) in a small cohort of prostate tissues ( subjected to epithelial cell enrichment ) measured using the infinium humanmethylation450 beadchip ( fig 2a )  . 
 extracting freely available data from the same analytical platform for a larger the cancer genome atlas cohort yielded similar results ; each epicapture gene demonstrated significantly higher levels of dna methylation in clinically significant and high - risk tumors , relative to histologically benign tissue ( fig 2b )  . 
logistic regression models were developed using a 319 - specimen training set ( corresponding to 70% of the total cohort ) and then evaluated for their ability to correctly predict cancer / high - grade cancer / high - risk cancer in the remaining test set . 
not forgetting the clinical significance of gleason 7 disease , we also applied the revised five - grade grouping system , 18 with a view to detecting group 3 ( gleason 4 and 3 ) and above . 
the combination of epicapture and psa gave the best model ( auc , 0.82 ; sensitivity , 0.73 ; specificity , 0.76 ; appendix fig a1 ; appendix table a2 )  . we next evaluated the training set data to select an epicapture score cutoff that could best inform the decision on whether to perform a biopsy . 
finally , dca showed that at this threshold , epicapture demonstrated a net benefit over psa in the decision on whether to perform a biopsy , enabling a 15% reduction in prostate biopsies ( figs 3i and 3j )  . epicapture genes show high reproducibility by independent analysis to examine the robustness of epicapture , a matched quantitative methylation specific pcr analysis of two of the panel ( g1 : gstp1 , and g5 : apc ) was carried out in two independent laboratories on 236 of the urine samples . 
 ( a ) laser capture microdissected enriched prostate epithelial cells from benign prostate tissue ( procured from men undergoing radical cystoprostatectomy for bladder cancer , with no clinical or histologic evidence of prostate cancer [ pca ] ) and low - risk , aggressive , and metastatic pca . 
 ( b ) data extracted from the tcga repository for matched - benign prostate tissue and low - risk , significant , and high - risk pca , all procured from men undergoing radical prostatectomy . 
the performance of epicapture compared with / in conjunction with prostate - specific antigen ( psa ; treated as a continuous variable ) in the test set ( n = 134 ) for predicting each end point was assessed by receiver operating characteristic curves : ( a ) cancer ( v no cancer detected on transrectal ultrasound biopsy ) , ( b ) high - grade cancer ( gleason score greater than or equal to 8 v all else ) , ( c ) damico high - risk cancer ( v all else ) , and ( d ) capra ( cancer of the prostate risk assesment ) high - risk cancer ( v all else )  . 
the distribution of epicapture scores in the whole study population ( n = 453 ) , viewed by ( e ) biopsy outcome , ( f ) gleason grade , ( g ) risk categorization by damico risk - classification systems , and ( h ) risk categorization by capra risk - classification systethe epicapture score was calculated for each sample by summing the normalized index of methylation ( data supplement ) for g1 to g6 . 
epicapture score distributions were analyzed by t test or one - way analysis of variance ( anova ) , followed by post hoc analyses using the tukey - kramer multiple comparisons test . 
furthermore , matched data were available for 18 of 30 high - risk / grade samples that were epicapture negative , of which 15 ( 83.33% ) and 16 ( 88.89% ) were also negative for g1 and g5 , respectively , in the independent analysis . parallel epicapture analysis in urine and matched biopsy cores highlights value of liquid biopsy pca is renowned for its multifocal nature . 
matched formalin fixed paraffin - embedded biopsy cores were available for a proportion of patients , enabling parallel epicapture analysis on multiple tumor foci and urine from individual men ( n = 15 )  . 
the most interesting insights came from the matched analysis of urine with multiple biopsy cores , of which two notable cases are highlighted . patient sjh149 was diagnosed with a minute focus of gleason score 6 adenocarcinoma . 
this finding could suggest that the source of the tumor dna analyzed in urine could have been the high - grade tumor , which was not sampled on the original biopsy . 
 in contrast , patient sjh189 was diagnosed with a high - percentage , high - grade ( intermixed patterns of grade 4 and 5 ) pca on both sides of his prostate . 
so , despite pathologic heterogeneity within the gland , the three tumor areas seemed to be epigenetically homogeneous . discussion in this urine pca biomarker study , we evaluated the performance of epicapture , a six - gene dna methylation panel , for noninvasive detection of pca and more specifically high - grade or high - risk pca in urine from patients referred for prostate biopsy on the basis of elevated psa and / or abnormal dre . 
complying with remark guidelines , we describe a new dna methylation - based urine test for early detection of aggressive pca . many prostate tumors display a protracted disease trajectory , almost indolent in behavior.19 the widespread use of the serum psa test in many countries has significantly increased the incidence and thus overtreatment of these lowrisk cancers with little likelihood of clinical manifestation.20 - 23 an important first consideration in developing epicapture was to therefore critically appraise the evidence for prognostic utility of each constituent gene . 
conversely , however , it is estimated that approximately two thirds of men undergoing invasive prostate biopsy have no tumor diagnosed , because of the high false positive rate of serum psa.23 , 24 as such , millions fig 3 . 
 ( i ) decision curve analysis demonstrated net clinical benefit of performing biopsy on patients in the test set on the basis of an epicapture score greater than 1.25 versus psa greater than or equal to 3 ng / ml across a range of clinically relevant threshold probabilities ( the risk of cancer , such that a patient would choose to undergo biopsy by weighing the relative harms of false - positive and false - negative predictions )  . 
 ( a ) two of the six epicapture assays ( g1 : gstp1 and g5 : apc ) were analyzed independently in laboratories in ireland ( perry ) and canada ( bapat ) by quantitative methylation specific pcr in urine sediments from 236 of 453 men in the study . 
 relevant subsets were then considered by applying the study end points : ( b ) men with biopsy - positive disease , ( c ) men with high - grade ( gleason score greater than or equal to 8 ) prostate cancer , and men with highrisk prostate cancer defined by ( d ) damico , and ( e ) capra ( cancer of the prostate risk assessment ) classification . 
 repeat biopsy ( 8 months later ) sjh149 urine biopsy core ( s ) sample type base apex base apex histologically benign gleason 6 gleason 7 gleason ( cid : 116 ) 8 urine tissue sjh189 urine biopsy core ( s ) sample type fig 5 . 
 tumor presence is denoted by pink ( gleason score 6 ) , red ( gleason score 7 ) , or brown ( gleason score greater than or equal to 8 ) circles , with size indicative of percentage tumor in each core . 
 ( a ) low - risk prostate cancer sjh149 ( 62 years of age ; prostate - specific antigen , 3.08 ng / ml ) demonstrated that urine as a liquid biopsy is a viable alternative to sampling bias inherent to tissue needle biopsies . 
 sophisticated imaging technologies , such as magnetic resonance imaging , may improve the accuracy of prostate biopsy , 30 their widespread application is hampered by high cost , access to specialist equipment and personnel , interobserver subjectivity , and requirement for anesthesia . 
alternatively , transperineal biopsy enables a more complete sampling of the gland , including the anterior prostate , but also requires anesthesia and is thus unsuitable as a population - based detection method for such a prevalent disease . the dearth of blood - based prognostic biomarkers as a screening tool for aggressive pca in conjunction with limitations of the prostate biopsy in its various forms has led to excitement around urine biomarkers . 
physical manipulation of the prostate by dre , followed by a first - void urinalysis , could provide a simple , holistic insight into molecular alterations occurring in the prostate gland . 
indeed , many studies have demonstrated significant value in detecting and noninvasively monitoring several genitourinary malignancies through urine.31 - 34 some other prognostic urine indicators of aggressive pca have already been described . 
early studies demonstrated detection of gstp1 methylation in urine from patients with pca , although sensitivity was poor ( less than 30% ) .39 follow - up work expanded the repertoire of markers ( gstp1 , apc , and rar2 ) , which improved sensitivity to approximately 60%.40 the epicapture panel represents five commonly perturbed pathways in pca : intracellular detoxification , wnt and igf axes , cell cycle , and prostaglandin biosynthesis . 
in contrast , applying an epicapture threshold ( greater than 0.73 ; developed on training set ) did not markedly alter its diagnostic performance ( auc , 0.68 ) and delivered a 79% improvement over the tumor specificity of psa . in an effort to address the prognostic utility of epicapture within the context of the clinically heterogeneous gleason 7 disease , we applied the revised five - grade grouping system , 18 in addition to two widely used risk - classification systems . 
the epicapture performance characteristics represent a potential improvement on existing methods for patient stratification and for determining the need to perform biopsy , detecting 85% of highgrade tumors , with an npv of 98% . 
furthermore , the overlap of gstp1 and apc in the two gap1 urine dna methylation biomarker panels revealed excellent correlation in detection of aggressive pca , illustrating the robustness of these biomarkers . 
in the context of the diagnostic gray zone , psa 4 to 10 ng / ml , epicapture demonstrated potential for further classifying patients , positive in 40% of men with highgrade pca . 
 although these data are promising , a large prospective clinical trial is needed to assess whether epicapture offers a survival advantage . a limitation of our study is that the cohort is young ; analysis was only possible using biopsy as the end point . 
we cannot rule out the possibility that a proportion of our epicapture - positive urine samples in men with low - grade or benign tissues were indeed tumor cases missed on biopsy . 
evidence actually shows that dna methylation detected in histologically benign tissue ( due to an epigenetic field effect from the cancer ) , can be used as a tool to rule out the possibility that the biopsy missed the tumor . 
 for example , the confirm mdx test , which measures dna methylation at three genes ( gstp1 , apc , and rassf1 ) in histologically benign biopsy tissue cores , is used to guide the decision to repeat a biopsy.41 conversely , 14 of 47 ( 29.79% ) high - grade tumors were epicapture negative . 
independent analysis revealed concordant negativity in more than 80% of these men , and lowering the epicapture threshold did alter this result ; they were negative across all six markers . 
we believe that the most likely reason is interoperator differences in dre ; these high - risk / grade cancers were epicapture negative because they had insufficient / undetectable prostate cells present in their urine . 
 relationships may not relate to the subject matter of this enable confirmation of the presence of prostate cells in the urine , validating its suitability for epicapture analysis and ultimately improving the performance of the test . 
future work will also investigate the intra - individual reproducibility of epicapture by repeated measures in men . finally , to the best of our knowledge , this is the first pca biomarker study to perform a matched analysis of tissue and liquid biopsies . 
a more extensive project is now warranted to fully assess the effects of the multifocal nature of pca on signatures derived from liquid biopsies . epicapture could be used in conjunction with existing tools ( ie , psa and risk calculators ) to help guide the decision to biopsy . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . eve o ' reilly no relationship to disclose alexandra v . 
dale no relationship to disclose rick brugman no relationship to disclose gavin clarke no relationship to disclose olivia schmidt no relationship to disclose shane o ' meachair no relationship to disclose dattatraya patil no relationship to disclose kathryn l . 
pellegrini no relationship to disclose julia garcia no relationship to disclose fang zhao no relationship to disclose stephen finn honoraria : roche robert mills no relationship to disclose marcelino y . 
gallagher employment : oncomark leadership : oncomark neil fleshner honoraria : amgen , janssen oncology , bayer , sanofi , abbvie , ferring pharmaceuticals , astellas medivation consulting or advisory role : hybridyne health research funding : ferring ( inst ) , astellas pharma ( inst ) , janssen oncology ( inst ) , amgen ( inst ) , nucleix ( inst ) , progenix ( inst ) , spectracure ab ( inst ) research funding : amgen ( inst ) travel , accommodations , expenses : pfizer stock and other ownership interests : oncomark consulting or advisory role : carrick therapeutics research funding : carrick therapeutics patents , royalties , other intellectual property : two patents which have been licensed to oncomark travel , accommodations , expenses : oncomark rustom p . 
brewer patents , royalties , other intellectual property : patents held in drug discovery with novartis ( i ) , patents pending concerning cancer subtype detection and biomarkers with university of east anglia bharati bapat no relationship to disclose martin g . 
perry patents , royalties , other intellectual property : university college dublin holds a patent that relates to this work acknowledgment we thank the urologists and their teams at participating hospitals and the health research boardfunded dublin centre for clinical research network , affiliated research nurses , and technical staff , in particular john mccourt . 
we thank investigators drs robert vessella , and colm morrissey , and the patients and families who participated in the prostate cancer donor program at the university of washington medical center . 
bristow , director , manchester cancer research centre , chief academic officer and honorary consultant , christie nhs trust , university professor of cancer studies , university of manchester , manchester cancer research centre , university of manchester , manchester , uk . chris parker , royal marsden hospital , sutton , uk . ian mills , queens university belfast , centre for cancer research and cell biology , movember / prostate cancer uk centre of excellence , school of medicine , dentistry , and biomedical sciences . 
sanda , emory university school of medicine , atlanta , ga ; neil fleshner , julia garcia , fang zhao , and bharati bapat , university of toronto , toronto , ontario , canada ; robert mills , norfolk and norwich university hospital ; marcelino y . 
brewer , earlham institute , norwich , england . supported by grant funding from movember ( gap1 urine biomarker award ) , us prostate cancer foundation ( young investigator award , a.s.p. ) , science foundation ireland grants no . 
bastian pj , ellinger j , heukamp lc , et al : prognostic value of cpg island hypermethylation at ptgs2 , rar - beta , ednrb , and other gene loci in patients undergoing radical prostatectomy . 
damico av , whittington r , malkowicz sb , et al : biochemical outcome after radical prostatectomy , external beam radiation therapy , or interstitial radiation therapy for clinically localized prostate cancer . 
cooperberg mr , pasta dj , elkin ep , et al : the university of california , san francisco cancer of the prostate risk assessment score : a straightforward and reliable preoperative predictor of disease recurrence after radical prostatectomy . 
vickers aj , cronin am , roobol mj , et al : a four - kallikrein panel predicts prostate cancer in men with recent screening : data from the european randomized study of screening for prostate cancer , rotterdaclin cancer res 16 : 3232 - 3239 , 2010 29 . 
siddiqui mm , rais - bahrami s , turkbey b , et al : comparison of mr / ultrasound fusion - guided biopsy with ultrasound - guided biopsy for the diagnosis of prostate cancer . 
costa vl , henrique r , danielsen sa , et al : three epigenetic biomarkers , gdf15 , tmeff2 , and vim , accurately predict bladder cancer from dna - based analyses of urine samples . 
torres - ferreira j , ramalho - carvalho j , gomez a , et al : mir - 193b promoter methylation accurately detects prostate cancer in urine sediments and mir - 34b / c or mir - 129 - 2 promoter methylation define subsets of clinically aggressive tumors . 
wei jt , feng z , partin aw , et al : can urinary pca3 supplement psa in the early detection of prostate cancer ? j clin oncol 32 : 4066 - 4072 , 2014 35 . 
leyten gh , hessels d , jannink sa , et al : prospective multicentre evaluation of pca3 and tmprss2 - erg gene fusions as diagnostic and prognostic urinary biomarkers for prostate cancer . 
van neste l , partin aw , stewart gd , et al : risk score predicts high - grade prostate cancer in dna - methylation positive , histopathologically negative biopsies . 
 ( a ) the performance of epicapture compared to and in conjunction with prostate - specific antigen ( psa ; treated as a continuous variable ) in the test set ( n = 134 ) for predicting isup group 3 , 4 and 5 pca on biopsy was assessed by receiver operator characteristic curves . 
 ( b ) the distribution of epicapture scores in biopsy - negative ( no cancer detected ) v biopsy positive patients , categorized by isup group , shows increasing methylation by group . 
epicapture was performed in urine and matched formalin - fixed paraffin embedded biopsy cores from randomly selected biopsy - positive ( n = 15 : low - risk [ capra and damico , gleason score 6 ; intermediate risk , gleason score 7 ; and high risk , gleason score 8 ] ) patients . 
dna methylation of each gene is indicated by the nim ( normalised index of methylation ) , and for the collective panel , by the epicapture score ( nim sum g1 - g6 )  . 
each point represents a single sample ( biopsy core or urine ) , with the mean for each group indicated by a horizontal line ( yellow , urine ; blue , tissue )  . 
 c wide and cystic brain metastases reveal ret - rearranged nonsmall - cell lung cancers francesco facchinetti , md , msc1 , 2 ; francesca bozzetti , md1 ; letizia gnetti , md1 ; roberta minari , phd1 ; pellegrino crafa , md1 ; sara elena rebuzzi , md1 ; roberto ferrara , md3 ; elisa gruppioni , phd4 ; elisa capizzi , phd4 ; ermanno giombelli , md1 ; girolamo crisi , md1 ; annalisa altimari , phd4 ; and marcello tiseo , md , phd1 , 5 introduction the detection of specic molecular activations ( eg , egfr , alk , ros1 , braf , met , ret ) is the basis of effective targeted treatments in nonsmall - cell lung cancer ( nsclc ) .1 in this eld , novel selective inhibitors extend survival outcomes , coupling global prolonged disease control and meaningful activity against metastases developing in the cns.2 lung tumors harboring egfr mutations or alk , ros1 , or ret rearrangements have a tendency toward more frequent cns spreading compared with wild - type ( wt ) nsclc.3 - 5 peculiar neuroradiologic characteristics have been reported for brain metastases from alkpositive nsclc , in terms of cystic aspects acquired after crizotinib treatment.6 - 8 such features can even be misleading , because intracranial cystic lesions from alk - rearranged tumors may be attributed to infectious diseases.9 here we report the histories of two patients presenting with wide cystic brain lesions that were neurosurgically removed and discovered to be metastases from ret - rearranged nsclc . 
brain computed tomography ( ct ) scan and magnetic resonance imaging ( mri ) were performed , documenting a unique bulky cystic - like lesion in the frontal region of the left hemisphere , the morphologic and functional characteristics of which were pathognomonic for an anaplastic astrocytoma ( fig 1 )  . 
a chest x - ray revealed a parahilar lesion of the left lung . diagnosis and staging neurosurgery was then performed with the radical removal of the wide cerebral lesion , the intraoperative macroscopic aspects of which were still in keeping with an anaplastic astrocytoma . 
the postoperative course was uneventful , with a rapid neurologic improvement . the histologic examination revealed a large ( 40 25 20 mm ) lesion , conrming the cystic structure that was evident at sampling ( fig 2a )  . 
ct and pet scans revealed a mass in the left pulmonary hilum and ipsilateral and contralateral lymph nodes ( ct4n3m1b according to tnm staging classication [ eighth edition ] ; fig 3 )  . 
considering the radiologic , pathologic , and molecular elements , which were clearly in line with a diagnosis of advanced nsclc , biopsies of the lung and nodal lesions were not performed . molecular analyses neither the molecular analyses nor ihc performed on the brain metastasis detected egfr , braf , or kras mutations or alk or ros1 rearrangements ; programmed death - 1 ligand ( clone sp263 ) was expressed by 10% of tumor cells . 
next - generation sequencing with a 52 - gene panel ( ion torrent oncomine focus assay kit ; thermo fisher scientic , waltham , ma ; gene list provided in data supplement ) was performed , and a kif5b - ret fusion was found ( figs 4a and 4b )  . 
ret rearrangement was conrmed by uorescence in situ hybridization analysis in 52% of tumor cells ( fig 4c )  . treatment received rst - line chemotherapy with the patient cisplatin and pemetrexed . 
postoperative stereotactic brain radiotherapy ( sbrt ) to the resection cavity was not performed , because available evidence supporting sbrt for better local control has not yet indicated a survival benet.10 , 11 brain mri after 4 months revealed normal surgical outcomes , with no residual disease . 
ct scan after four courses of cisplatin plus pemetrexed showed partial disease response , so the patient was switched to pemetrexed maintenance , four cycles of which led to further reduction of lymph node size , with continuing absence of brain disease on mri . 
no evidence is available concerning ret - rearranged nsclc , an entity of emerging interest given the current development of selective ret inhibitors , also active against cns disease . knowledge generated here we report the similar clinical histories of two patients with ret - positive nsclc , whose diagnoses were obtained on pathologic and molecular analyses performed on wide , cystic , and symptomatic brain metastases with a peculiar neuroradiologic presentation . relevance a characteristic behavior of cns lesions from ret - rearranged nsclc was recognized . 
the current or upcoming availability of selective , cns - active ret inhibitors strongly reinforces the importance of wide molecular screening of cns lung metastases , even more so in the presence of the reported neuroradiologic and pathologic features of brain lesions . the abrupt onset of dizziness , worsening headache , difculty with handgrip , and right homonymous hemianopsia . the observation of a large and cystic - like brain lesion in the left parietal - occipital region was followed by a contrastenhanced mri ( fig 5 )  . 
the brain imaging was suggestive for metastases from an extracerebral tumor . diagnosis and staging the whole - body ct scan documented a mass in the upper left lung lobe , with ipsilateral and contralateral nodal volvement in the hilar - mediastinal and retro - clavicular regions ( fig 6 ) , with clinical staging determined as ct2an3m1c . given the persisting neurologic symptoms , the patient underwent neurosurgery . the pathologic examination conrmed the radiologic hypothesis of a cavitated cystic ( fig 2f ) brain metastasis ( 40 30 20 mm ) from a lung tumor ( figs 2g to 2i )  . 
 ( a ) t1 weighted imaging ( wi ) and ( b ) t2 wi sequences show a well - delineated ( a ) hypointense and hyperintense mass in left frontal lobe cortex involving the subcortical area and deep white matter , ( c ) without dense dystrophic calcication or hemosiderin staining on t2 * gre . 
 ( d ) ttf - 1 ( 40 ) and ( e ) napsin a ( 40 ) immunohistochemistry showed diffuse ( d ) nuclear and ( e ) cytoplasmic positivity . 
 ( a ) a left parahilar lesion conditioning atelectasis was detected , together with ( b ) ipsilateral and ( c ) contralateral pathologic lymph nodes . elements supporting the diagnosis of nsclc , biopsies of the primary tumor and lymph nodes were not proposed . molecular analyses status of egfr , alk , ros1 , kras , braf , and met was found to wt , and ihc showed programmed death - 1 ligand ( clone sp263 ) negative status . 
again , targeted ngs was performed on the brain metastasis ( ion torrent oncomine focus assay kit ; thermo fisher scientic ; data supplement ) , and kif5b - ret fusion was revealed , conrmed by uorescence in situ hybridization analysis in 40% of tumor cells ( figs 4a to 4b and 4d )  . treatment because of the lack of trials with ret inhibitors in the rstline setting and the substantial benet potentially driven by pemetrexed , 12 pemetrexed was combined with cisplatin and administered as a rst - line treatment , once postsurgical recovery was complete . 
again , sbrt to the resection cavity was discussed but not performed , for the same reasons reported in the rst patient case15 , 16 and because of the presence of multiple brain metastases . 
 ( b ) next - generation sequencing analysis report showing the ret fusion with kif5b gene ( ion reporter software and irgv visualization [ thermo fisher scientic , waltham , ma ] ) detected in brain samples from patient cases 1 and 2 . 
t1 weighted imaging ( wi ) and t2 wi sequences show a well - delineated ( a ) hypointense and ( b ) hyperintense mass in left occipital lobe cortex , with a linear internal septum , involving the subcortical area and deep white matter , ( c ) without dense dystrophic calcication or hemosiderin staining on t2 * gre . 
 ( d ) axial t1 postcontrast imaging shows a large bilobed cystic - like supratentorial mass with ring enhancement , sharply delineated borders , and well - dened internal septu ( e ) the content is liquid , with no intralesional levels and with high diffusivity ( apparent diffusion coefcient , 0.00269 mm2 per second v csf , 0.00290 mm2 per second )  . 
similar radiologic appearance is shown by the following : ( g ) t1 wi before gadolinium administration , ( h ) t1 wi after gadolinium administration , and ( i ) t2 wi . imaging documented , in addition to the regular outcomes of the surgical intervention , signs of response in the two residual lesions ( data not shown )  . 
nevertheless , this was not conrmed in another series of ros1 - rearranged lung cancer with baseline brain involvement ; in that series , the ros1 partner gene was known in 17 patients for whom mri results were available.14 in ret - driven lung cancer , the most frequent fusion partner gene is kif5b , as observed in the two patient cases reported here . 
although no associations between ret partner genes and brain metastasis incidence have been anticipated , drilon et al5 reported that intracranial disease response to multitarget kinase inhibitors seemed to occur specically in nonkif5b - ret patient cases ( n = 2 of 5 ) , with no responses in the kif5bret patient cases ( n = 0 of 9 ) .5 at least ve cns responses to the selective ret inhibitor loxo - 292 were observed in ret - rearranged lung tumors ( n = 4 of 4 in the phase i study ) 15 , 16 harboring either kif5b ( n = 2 ) or non - kif5b ( clip - 1 ; n = 1 ) ret partner genes.15 - 17 because of the descriptive nature of our patient cases , no conclusion can be drawn with regard to a novel specic pattern of ret - rearranged lung tumors , potentially associated with large and cystic - like brain metastases at diagnosis . nevertheless , besides providing atypical and intriguing radiologic ndings , the surprising similarity between these two reports , together with the rarity of ret rearrangements in nsclc , suggests a characteristic behavior of cns lesions from this molecular subclass of tumors . 
these observations reinforce the importance of screening for nsclc molecular activations in addition to the canonical and frequent ones , with particular reference to ret fusions in the presence of atypical features of brain metastases . 
 ( a - c ) the lesion was not dissociable and was not completely distinguishable from an obstructive inammatory / atelectasic consolidation involving the entire lingula and part of the upper left lobe . 
 ( c , d ) bilateral hilar , mediastinal , and retro - clavicular lymph nodes were reported ( white arrows )  . involvement , with special regard to novel , specic agents , such as loxo - 292 and blu - 667.5 , 15 - 18 we hope that our two patients will not require further systemic treatment , meaning that their disease is controlled with the current chemotherapy and potential administration of radiotherapy . 
 large and cystic brain metastases from ret - rearranged nsclcs references reck m , rabe kf : precision diagnosis and treatment for advanced non - small - cell lung cancer . 
n engl j med 377 : 849 - 861 , 2017 remon j , besse b : brain metastases in oncogene - addicted non - small cell lung cancer patients : incidence and treatment . 
front oncol 8 : 88 , 2018 tan l , wu y , ma x , et al : a comprehensive meta - analysis of association between egfr mutation status and brain metastases in nsclc . 
pathol oncol res 25 : 791 - 799 , 2019 gainor jf , tseng d , yoda s , et al : patterns of metastatic spread and mechanisms of resistance to crizotinib in ros1 - positive nonsmall - cell lung cancer . 
j clin oncol 32 : e122 - e124 , 2014 saraceni c , li pm , gainor jf , et al : cystic brain metastases in nsclc harboring the eml4 - alk translocation after treatment with crizotinib . 
narayanan v , honce mj , mehrotra s , et al : cystic brain metastases occurring in anaplastic lymphoma kinase gene rearranged non - small - cell lung cancer patients receiving crizotinib . 
clin lung cancer 17 : 85 - 90 , 2016 kim sh , hyun jw , kim hj , et al : de novo cystic brain lesions mimicking neurocysticercosis in alk - positive lung cancer . 
mahajan a , ahmed s , mcaleer mf , et al : post - operative stereotactic radiosurgery versus observation for completely resected brain metastases : a single - centre , randomised , controlled , phase 3 trial . 
patil t , smith de , bunn pa , et al : the incidence of brain metastases in stage iv ros1 - rearranged non - small cell lung cancer and rate of central nervous system progression on crizotinib . 
drilon a , subbiah v , oxnard gr , et al : a phase 1 study of loxo - 292 , a potent and highly selective ret inhibitor , in patients with ret - altered cancers . 
oxnard g , subbiah v , park k , et al : clinical activity of loxo - 292 , a highly selective ret inhibitor , in patients with ret fusion + non - small cell lung cancer . 326 , 2013 27 : 1286 - 1291 , 2016 j thorac oncol 13 : 987 - 995 , 2018 oncol 36 , 2018 ( suppl ; abstr 102 ) j thorac oncol 13 : s349 - s350 , 2018 17 . 
 circulating tumor dna in nonsmall - cell lung cancer : a primer for the clinician circulating tumor dna ( ctdna ) consists of short , double - stranded dna fragments that are released into the circulation by tumor cells . 
the assays noninvasive nature , ability to reflect intratumoral heterogeneity , short turnaround time , and ability to obtain serial samples make it an attractive option compared with traditional tissue biopsy tumor sequencing . 
currently , this technology is mostly being used for the detection of egfr mutations in patients with advanced nonsmall - cell lung cancer where tissue is inadequate to detect egfr mutations that drive acquired resistance , most notably egfr t790m . 
emerging uses include the incorporation of ctdna testing into primary diagnosis , treatment monitoring , detection of minimal residual disease , and detection of early - stage disease in screening populations . 
2017 by american society of clinical oncology introduction including tumors , shed short , several tissues , double - stranded dna fragments called cell - free dna ( cfdna ) into the circulation.1 - 3 certain conditions that cause tissue damage , such as myocardial infarction , trauma , and cancer , can result in increased concentrations of cfdna.3 the cfdna derived from tumors , known as circulating tumor dna ( ctdna ) , is a product of apoptosis and necrosis and is distinguished from other cfdna by the presence of somatic mutations representative of tumor biology absent in normal cells . 
90% of all dna present in the plasma.4 , 5 in patients with advanced nonsmall - cell lung cancer ( nsclc ) , ctdna detection rates that range from 80% to  . 
unlike acquired t790m resistance to egfr tkis , one dominant secondary mutation appears not to exist , and second - generation alk inhibitors have shown high response rates in patients previously treated with crizotinib regardless of the presence of secondary alk mutations , which makes the role of a repeat biopsy less clear.27 however , emerging data suggest that specific secondary resistance alk mutations have important therapeutic implications . 
in a study that detected egfr mutations by an ngs assay , the sensitivity for urine was 93% for t790m , 80% for l858r , and 83% for exon 19 deletions . 
in nine patients monitored while receiving the third - generation egfr inhibitor rociletinib , a rapid decrease in urine ascopubs.org / journal / po jco precision oncology 7 t790m was 70% , and the objective response rate and median progression - free survival ( pfs ) with osimertinib were similar in patients with t790mpositive plasma or tumors.10 this finding suggests that with the use of ctdna testing , many patients can avoid a biopsy for t790m genotyping . 
however , because of the 30% false - negative rate of plasma genotyping , those with t790m - negative plasma still need to undergo tumor biopsy to determine the presence or absence of t790m.10 in addition to cases where ctdna is not detectable , tissue testing retains value in certain resistance mechanisms , such as small - cell transformation . thress et al24 performed ngs of cfdna from seven patients with t790m - mutated advanced lung cancer who had developed resistance to osimertinib and detected an acquired egfr c797s mutation . 
 t790m levels was observed by day 21.31 in a study of patients with t790m positivity , response was similar whether t790m status was identified by tissue , plasma ( through beaming ) , or urine ( through ngs ) .32 these data suggest that plasma and urine egfr analyses complement tissue biopsy specimen analysis in nsclc . brain metastasis is found in up to 40% patients with nsclc , 33 and ctdna analysis of patients with brain tumors has revealed either the absence or very low levels of tumor dna possibly as a result of the blood - brain barrier.34 one study used hybridization capturebased sequencing and ddpcr to characterize ctdna in csf of patients with brain lesions ( including brain metastasis from lung cancer ) and compared it with plasma ctdna.35 the authors concluded that csf ctdna has a significantly higher sensitivity than plasma for cns genomic alterations like egfr , pten , esr1 , idh1 , erbb2 , and fgfr2 and can be used to detect brain tumor mutations and monitor brain tumor progression ( sensitivity of ctdna was 58% v 0% in csf and plasma , respectively , in cns - restricted disease )  . 
hata et al37 investigated the response to osimertinib in 10 patients with leptomeningeal disease and showed that the drug might be more effective in csf of patients with t790m positivity than in those without csf t790m positivity . multigene testing as the number of genomic targets with matched therapies grows , the national comprehensive cancer network guidelines for nsclc recommend expanded genomic testing , including for egfr , alk , erbb2 , braf , met , ros1 , and ret.5 , 15 , 38 testing for all these alterations in addition to histopathologic and immune biomarker assessment pose a challenge when tissue biopsy samples are insufficient . 
in a study of patients with advanced nsclc , a tissue biopsy specimen with sufficient quality and quantity of dna for ngs was unobtainable for 52 ( 51% ) of 102 patients.7 two studies used ngs - based ctdna assays to evaluate , with high overall accuracy , somatic genomic profiles in patients with advanced nsclc  . 
15 , 000 patients with advanced - stage cancer with a concordance of 87% with matched tumor samples that increased to 98% when plasma and tissue testing was done , 6 months apart . 
the study detected actionable mutations in biopsy specimens with insufficient tissue ( alk fusion , egfr or braf activating mutations ) and with actionable resistance mutations at the time of progression ( met amplification or egfr t790m ) as well as in undergenotyped tumors ( braf v600e , or erbb2 indel )  . identification of nontargetable oncogenic drivers , such as kras mutations , that preclude the presence of other targetable alterations also guides clinicians to rapidly initiate alternative therapies , such as chemotherapy or immunotherapy.5 the detection of variants in other genes may lead to the off - label use of fda - approved therapies or enrollment in clinical trials of new therapeutic agents.7 several combinations of targeted therapies are based on resistance mechanisms and are being evaluated in lung cancer ( eg , mek and pi3k inhibitors in combination with egfr tkis ) , 41 which again underscores the need for real - time tumor genotyping to assess for resistance pathways . 
chabon et al43 identified several resistance mechanisms , including met , egfr , pik3ca , errb2 , kras , and rb1 , in patients with t790mmutated nsclc who received rociletinib . 
the study used cancer personalized profiling by deep sequencing ctdna analysis and found that 46% of patients ( 19 of 41 ) with t790m mutations had additional potential resistance mutations in the pretreatment plasma . 
this coexistence of different resistance mechanisms in the same patient may affect response to subsequent egfr tkis like rociletinib . treatment monitoring ctdna offers the possibility of serial testing for molecular monitoring of disease . 
 studies have demonstrated the predictive value of quantitative changes in egfr mutations in ctdna at various time points during tki treatment.1 a prospective analysis to validate ddpcr for the detection of common egfr and kras mutations in patients with advanced nsclc demonstrated that patients with complete resolution of ctdna at either 2 or 6 weeks after treatment exhibited a treatment discontinuation rate of 0% at the initial and 4% at the second imaging assessment . 
patients without complete resolution exhibited a treatment discontinuation rate of 33% at initial and 56% at second imaging . these patterns were postulated to correlate with radiographic response and emergence of acquired resistance.4 marchetti et al44 performed pcr and ultradeep ngs on serial plasma samples from 20 patients with advanced nsclc and known tissue and plasma egfr mutations before tki treatment . 
rapid responders who had at least a 50% decrease in plasma egfr copy number at 14 days had a greater mean percentage of tumor shrinkage than slow responders ( n = 6 ; 70% v 30% )  . 
a phase iii study evaluated egfr mutations in cfdna of patients randomly assigned to receive gemcitabine and platinum plus sequential erlotinib or placebo by using a cobas test.46 for patients treated in the erlotinib arm who were cfdna egfr positive at baseline , the disappearance of cfdna at cycle 3 was associated with significantly improved pfs ( hr , 0.38 ) and longer os ( hr , 0.45 ) compared with patients with persistence of cfdna egfr . the prognostic value of dynamic changes in ctdna has also been evaluated . 
additional studies are needed to evaluate end points such as pfs , os , and quality of life as a result of early detection of resistance . monitoring for minimal residual disease another potential application of ctdna is to evaluate for minimal residual disease ( mrd ) after curativeintent therapy , such as surgery and radiation.8 chaudhuri et al48 applied cancer personalized profiling by deep sequencing to detect ctdna in preand post - treatment blood samples from 41 patients with nonmetastatic lung cancer treated with chemoradiation , radiation , or surgery . 
these patients positive for mrd had a significantly worse outcome than patients without detectable ctdna mrd within 4 months of definitive treatment ( 3 - year freedom from progression , 0% v 92% [ hr , 38 ] ; 3 - year os , 8% v 75% [ hr , 12 ] )  . these data suggest that patients with residual ctdna after definitive treatment will be enriched for those who will ultimately develop recurrence . 
ctdna detection might facilitate the development of clinical trials that evaluate the escalation of therapy for patients at high risk for recurrence and the de - escalation of therapy for patients without residual disease . role in early diagnosis / screening the role of ctdna in early - stage disease also is being studied . 
bettegowda et al34 evaluated the ability of ctdna to detect tumors in patients with various malignancies at various clinical stages . forty - seven percent of patients with stage i cancers ( n = 49 ) had detectable ctdna , with the fraction of patients with detectable ctdna being 55% , 69% , and 82% for those with stage ii , iii , and iv cancers , respectively.34 trials are under way to assess the feasibility and utility of ngsbased ctdna to detect early - stage cancers in high - risk patients ( clinicaltrials.gov identifier : nct02612350 ; also project lunar48a )  . 
 mutation of interest in the patients tumor needs to be known to send for appropriate genomic panels . ctdna may be detectable before radiographically detectable disease , which raises the issue of managing such patients . 
nevertheless , early cancer detection remains a promising new avenue for ctdna that needs additional research . ctdna in the era of immune checkpoint therapy in the era of immune therapy for advanced nsclc , a key issue is the elucidation of a biomarker that can predict response and monitor disease . 
tumor dna sequencing has been shown to be a surrogate for total mutation burden , which predicts response and pfs with antiprogrammed death 1 inhibitors like pembrolizumab.49 however , total mutation burden analysis has been investigated only in tumor tissue , and a better understanding of ctdna is required before it can be applied for this purpose . lipson et al50 analyzed the ctdna of 12 patients with metastatic melanoma who underwent treatment with checkpoint inhibitors for the presence of hotspot somatic mutations in braf , ckit , nras , and tert by using beaming . 
hence , a negative result could signify that the tumor is not actively shedding ctdna , the patients disease is adequately controlled by therapy , or somatic variants either were not covered or were below the detection limit for the assay.7 in addition , any negative test result will need to be viewed in the overall context of ctdna and other molecular results . 
for example , in a patient with an egfr - mutated nsclc in the setting of acquired resistance , a negative egfr t790m test result is more likely to be a true negative if the original egfr mutation was detected with a significant allele frequency versus if the ctdna test did not identify either mutation . confidence in a positive test result extremely high analytic specificity and a positive predictive value that approaches 100% make false positives unlikely , so if a mutation is detected , it can be relied on as a true result . 
as expected , the allelic fraction of variants detected in ctdna is usually much smaller than that in tissue dna given that tumor - derived dna may be more diluted in the blood than in the tissue.7 the rare false positive result ( particularly t790m ) in plasma may be indicative of tissue heterogeneity in which the mutation was not identified but is present in tissue , which may be due to the timing of the biopsy and the location of the biopsy needle in the tumor . 
the only definitive method to clinically validate that the mutations detected in plasma are an accurate molecular proxy of disease biology in the context of it being absent in tissue is to correlate them with response to targeted inhibition . in the case of mutational events that are exclusive to certain types of cancers ( eg , egfr exon 19 deletion to nsclc ) , confidence of a positive result is higher than in genes that can be mutated in a variety of settings ( eg , tp53 )  . 
for instance , a small allelic fraction tp53 mutation in ctdna can originate from many sources ( eg , myelodysplastic syndrome ) , and in such cases , confidence is increased if the same mutation also has been detected in tumor . detection of concurrent malignancy / germline alterations the testing of cfdna will detect all dna that sheds into the circulation , and some findings may not be expected in the tumor of origin , may not match tumor tissue dna results , and may affect genes that are mutated in different tumor types ( eg , tp53 )  . 
a potential for detection of incidental hematologic malignancies exists , which , naturally , have high tumor burden in the blood , and results can be confounded by foreign dna , such as after transfusion or organ transplantation . another issue similar to what is seen with tissue genotyping is that one needs to be cautious about distinguishing somatic ( tumor ) versus germline alterations because germline dna is not analyzed in parallel . 
any mutation that affects genes implicated in familial syndromes should be considered specifically ( eg , brca ) , and mutations with high allele frequency ( between 30% and 70% ) should raise the possibility of being potentially associated with heterozygous germline mutation syndromes . in conclusion , tissue biopsy remains essential at least for primary diagnosis because tissue yields information about morphology , tumor type , site of origin , and the immune microenvironment . 
for instance , erbb2 ( her2 ) copy number amplification in nsclc does not predict tki response like it does in breast cancer.5 currently best validated in egfr - mutated lung cancer for primary molecular diagnosis and for egfr t790m testing , ctdna has proven to have excellent utility as a complement to tissue - based testing . 
singh no relationship to disclose xiaoling guo no relationship to disclose haiying cheng honoraria : astrazeneca , boehringer ingelheim , clovis oncology benjamin levy honoraria : genentech consulting or advisory role : eli lilly , boehringer ingelheim , genentech , roche , astrazeneca , celgene , pfizer , merck speakers bureau : eli lilly , genentech , roche balazs halmos consulting or advisory role : astrazeneca , boehringer ingelheim , celgene , genentech , roche , pfizer , eli lilly , foundation medicine research funding : merck , astrazeneca , mirati therapeutics , boehringer ingelheim , roche , genentech , ariad pharmaceuticals affiliations aditi p . 
singh , haiying cheng , xiaoling guo , and balazs halmos , montefiore medical center , bronx , ny ; and benjamin levy , johns hopkins sidney kimmel comprehensive cancer center at sibley memorial hospital , washington , dc . references oncologist 21 : 1121 - 1130 , 2016 1 . 
sacher ag , paweletz c , dahlberg se , et al : prospective validation of rapid plasma genotyping for the detection of egfr and kras mutations in advanced lung cancer . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
thompson jc , yee ss , troxel ab , et al : detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next - generation sequencing of cell - free circulating tumor dna . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced non - small - cell lung cancer . 
li t , kung hj , mack pc , et al : genotyping and genomic profiling of non - small - cell lung cancer : implications for current and future therapies . 
luo j , shen l , zheng d : diagnostic value of circulating free dna for the detection of egfr mutation status in nsclc : a systematic review and meta - analysis . 
kimura h , suminoe m , kasahara k , et al : evaluation of epidermal growth factor receptor mutation status in serum dna as a predictor of response to gefitinib ( iressa )  . 
douillard jy , ostoros g , cobo m , et al : gefitinib treatment in egfr mutated caucasian nsclc : circulating - free tumor dna as a surrogate for determination of egfr status . 
mao c , yuan jq , yang zy , et al : blood as a substitute for tumor tissue in detecting egfr mutations for guiding egfr tkis treatment of nonsmall cell lung cancer : a systematic review and meta - analysis . 
liu y , liu b , li xy , et al : a comparison of arms and direct sequencing for egfr mutation analysis and tyrosine kinase inhibitors treatment prediction in body fluid samples of non - small - cell lung cancer patients . 
wang j , wang b , chu h , et al : intrinsic resistance to egfr tyrosine kinase inhibitors in advanced non - small - cell lung cancer with activating egfr mutations . 
sequist lv , goldman jw , wakelee , ha , et al : efficacy of rociletinib ( co - 1686 ) in plasma - genotyped t790mpositive non - small cell lung cancer ( nsclc ) patients ( pts )  . 
zheng d , ye x , zhang mz , et al : plasma egfr t790m ctdna status is associated with clinical outcome in advanced nsclc patients with acquired egfr - tki resistance . 
thress ks , paweletz cp , felip e , et al : acquired egfr c797s mutation mediates resistance to azd9291 in non - small cell lung cancer harboring egfr t790m . 
niederst mj , hu h , mulvey he , et al : the allelic context of the c797s mutation acquired upon treatment with thirdgeneration egfr inhibitors impacts sensitivity to subsequent treatment strategies . 
ihuegbu n , banks kc , fairclough sr , et al : non - invasive detection of crizotinib resistance in alk - rearranged lung adenocarcinoma directs treatment with next - generation alk inhibitors . 
wakelee ha , gadgeel sm , goldman jw , et al : epidermal growth factor receptor ( egfr ) genotyping of matched urine , plasma and tumor tissue from non - small cell lung cancer ( nsclc ) patients ( pts ) treated with rociletinib . 
barlesi f , gervais r , lena h , et al : pemetrexed and cisplatin as first - line chemotherapy for advanced non - small - cell lung cancer ( nsclc ) with asymptomatic inoperable brain metastases : a multicenter phase ii trial ( gfpc 07 - 01 )  . ann oncol 22 : 2466 - 2470 , 2011 34 . 
de mattos - arruda l , mayor r , ng ck , et al : cerebrospinal fluid - derived circulating tumour dna better represents the genomic alterations of brain tumours than plasma . 
sasaki s , yoshioka y , ko r , et al : diagnostic significance of cerebrospinal fluid egfr mutation analysis for leptomeningeal metastasis in non - small - cell lung cancer patients harboring an active egfr mutation following gefitinib therapy failure . 
hata a , nanjo s , okuda c , et al : osimertinib at 80 mg for refractory leptomeningeal metastases in t790m - positive egfr - mutant non - small cell lung cancer . 
zill oa , mortimer s , banks kc : somatic genomic landscape of over 15 , 000 patients with advanced - stage cancer from clinical next - generation sequencing analysis of circulating tumor dna . 
canale m , petracci e , delmonte a , et al : impact of tp53 mutations on outcome in egfr - mutated patients treated with first - line tyrosine kinase inhibitors . 
marchetti a , palma jf , felicioni l , et al : early prediction of response to tyrosine kinase inhibitors by quantification of egfr mutations in plasma of nsclc patients . 
tseng js , yang ty , tsai cr , et al : dynamic plasma egfr mutation status as a predictor of egfr - tki efficacy in patients with egfr - mutant lung adenocarcinoma . 
mok t , wu yl , lee js , et al : detection and dynamic changes of egfr mutations from circulating tumor dna as a predictor of survival outcomes in nsclc patients treated with first - line intercalated erlotinib and chemotherapy . clin cancer res 21 : 3196 - 3203 , 2015 47 . 
karachaliou n , mayo - de las casas c , queralt c , et al : association of egfr l858r mutation in circulating free dna with survival in the eurtac trial . 
lipson ej , velculescu ve , pritchard ts , et al : circulating tumor dna analysis as a real - time method for monitoring tumor burden in melanoma patients undergoing treatment with immune checkpoint blockade . 
 specifying the trueand falsepositive rates in basket trials in the clinical evaluation of anticancer therapies , after identification of a recommended dose , investigators typically seek evidence of drug efficacy in populations of patients hypothesized to benefit from it . 
the purpose of these signal - finding studies is to prioritize additional development and to determine whether drugs should be tested in randomized phase iii trials and , if so , in which populations of patients . 
because these identified populations usually are small and efficacy often is substantially better than that for available therapies , conclusions from single - arm trials of this nature can be used to inform off - label treatment decisions and sometimes lead to inclusion in treatment guidelines or regulatory approvals . 
an understanding of the performance characteristics of the novel clinical trial designs commonly used in this setting is critical to avoid inappropriate clinical decision making on the basis of false - positive clinical trial results . the group of patients in whom a therapy is hypothesized to have activity can be identified in a number of ways , including genomic and proteomic profiles , and activity of the drug can be influenced by histology or the primary tumor site . 
the combination of a hypothesized mechanism of action and uncertainty around the populations of patients who would likely benefit has encouraged a trend toward more - complex earlyphase trials.1 a major challenge is the desire to study the effects of the drug simultaneously in patients with different primary sites of disease or histologies with the goal of evaluating the efficacy of the drug in these contexts , often referred to as baskets.2 - 9 a related recent trend in clinical trial design has been increasing enthusiasm for the use of adaptive designs , whereby design parameters can be changed dynamically as the trial progresses and evidence about efficacy gradually emerges.10 a final important trend has been the use of bayesian design as a tool to evaluate the emerging evidence in a formal statistical model.11 - 15 many of these methods are predicated on the underlying expectation of broadly similar efficacy across baskets because the statistical model allows the sharing of information across baskets with the purpose of completing the trial in a shorter time frame with fewer patients than a traditional strategy whereby each basket is regarded as an independent phase ii trial . 
this combination of design complexity with sophisticated statistical modeling has led to situations in which important clinical trials are being launched without a broad understanding of the implications of the novel methods used , an issue that has been identified previously in the context of novel phase i study designs.16 in this commentary , we examine the properties of a particular bayesian adaptive design that increasingly is being used in the context of basket trials , and we use the clinical setting of a completed basket trial to demonstrate that the design is heavily tilted toward positive conclusions about the efficacy of the drug.17 the complexity of these modern methods makes having a clear understanding of the properties of design , characterized by easily interpretable measures , essential when planning a new clinical trial . although bayesian statistical analyses generally focus on reporting the probability that an individual drug works ( the so - called posterior probability ) , we believe that the evaluation of properties of designs in terms of the familiar metrics used in the context of clinical trials is important . 
these properties are the true - positive rate ( the probability that a truly effective agent will be shown to be effective [ often referred to as power ] ) and the falsepositive rate ( the probability that an ineffective agent is erroneously judged to be effective [ often referred to as the type i error ] )  . 
we generally like to keep the false - positive rate low because we do not want to encourage additional study of a drug that does not work , and we want the true - positive rate to be high because we want to be confident that if the drug truly works , its effectiveness will be recognized . 
therefore , to fully understand the implications of these data - sharing methods , we must calculate a more - elaborate set of trueand false - positive rates , specifically when the drug does not work at all , when it only works in one kristen m . 
 basket , when it only works in two baskets , and so forth . as an illustration of the clinical setting , we refer to the findings of a recent basket trial that investigated the effects of vemurafenib , a selective oral inhibitor of the braf kinase.2 the trial assessed efficacy in five disease - specific baskets : nonsmallcell lung cancer , colorectal cancer , cholangiocarcinoma , erdheim - chester disease or langerhans cell histiocytosis , and anaplastic thyroid cancer . 
the investigators concluded that the drug shows promising activity in the nonsmall - cell lung cancer basket and the erdheim - chester disease or langerhans cell histiocytosis basket but that it is inactive in colorectal cancer . 
although this trial did not use the bayesian hierarchical design proposed by berry et al , 17 we use the clinical setting of the vemurafenib trial to investigate the performance of the berry design . the bayesian hierarchical model is structured to capture the correlation between the anticipated efficacies of the drug across various baskets . 
this aggregation of evidence is rooted in a key feature of bayesian methods , the prior distribution of the between - basket variability , an entity that is prespecified by the analyst . 
in the context of a basket trial , this prior distribution essentially titrates the extent to which emerging evidence of drug efficacy from one basket can be used to bolster the evidence in other baskets . 
however , they presented a sensitivity analysis that concluded that the properties are actually relatively insensitive to this choice . we evaluated the false - positive and false - negative error rates for a hypothetical trial similar in structure to the vemurafenib trial , with five baskets and null and alternative response rates of 15% and 45% , respectively . 
we simulated trials by following the bayesian hierarchical design and conducted interim analyses after the first 10 patients were accrued and then after every additional five patients until a maximum of 19 patients per basket were enrolled . 
early stopping rules terminated accrual to individual baskets if the basket - specific posterior probability that the true response rate is greater than a midlevel response rate of 30% was , 5% ( stop for futility ) or  . 
at the end of the trial , the treatment was declared efficacious in a basket if the posterior probability that the response rate that exceeded the null value of 15% was  . 
for example , in the second row ( one active basket ) , basket 1 is the one in which the drug is truly active , whereas in baskets 2 to 5 , the drug is not active . 
in this scenario , we see that a 79% chance for observing a true - positive result exists in the active basket , whereas for each nonactive basket , the false - positive rate is 18% . 
the next column on the right captures the probability that one or more of the nonactive baskets is a false - positive finding , a term usually referred to as the family - wise error rate , a key criterion for evaluating multiple hypotheses in clinical trials.18 , 19 we see that this overall false - positive rate is 37% when the drug truly works in only one basket and rises to 57% when the drug does not work in only one basket . 
although the maximum trial size was set to 95 patients , the expected trial size ranged from 59 to 84 patients , depending on the true efficacy configuration of the baskets . 
the table also lists the family - wise error rates for the setting in which each basket is regarded as an entirely independent clinical trial wherein the false - positive rate is 10% . 
each basket displays the observed response rate ( rr ) from the vemurafenib trial for a particular disease ( sample sizes in parentheses )  . ( * ) patients received combination therapy ( vemurafenib 1 cetuximab )  . 
statistical properties of bayesian hierarchical design probability drug will be declared efficacious , % family - wise error rate , % * basket 1 basket 2 basket 3 basket 4 basket 5 hierarchical independent no . 
bold represents baskets in which the drug is active ; no bold represents baskets in which the drug is not active . * the family - wise error rate represents the probability that the drug has false - positive efficacy in any one or more of the nonactive baskets ( ie , the overall false - positive rate of the design given the number of baskets in which the drug is truly active )  . 
this is shown for both the bayesian hierarchical design and the setting in which all five baskets are considered to be independent clinical trials . close examination of table 1 demonstrates two complementary results from using the bayesian design . 
although the targeted overall false - positive rate of 10% has been achieved for the setting in which the drug has no efficacy ( ie , the drug works in none of the baskets as shown in the first row ) , this standard rapidly erodes as we investigate settings in which the drug works in some baskets but not in others . for example , if we look at the two active baskets row of the table , we see a high probability ( 94% ) of declaring each of the two active baskets to be efficacious . 
consider the most extreme case ( the four active baskets row of the table ) where we see that the nonactive basket has a false - positive rate of 57% . 
in this setting , the evidence typically is dominated by strong positive results from the four baskets in which the drug is truly active , and the imposition of the prior distribution encourages the model to interpret the results from all five baskets as strongly correlated ( ie , similar in efficacy ) , which leads to a high probability that the inactive basket will be classified incorrectly as active . 
in related work , we explored in depth options for modifying the bayesian design used in our simulations by examining various choices of prior distributions and of decision rules used , demonstrating that trials can be designed on the basis of hierarchical modeling with much better control of the familywise error rates.20 we believe that straightforward reporting of the various trueand false - positive rates , as listed in table 1 , are fundamental to understanding the merits of any proposed basket study design . seemingly innocuous choices , such as the use of a prior distribution that berry et al17 characterized as weak , which allows the data to shape the amount of borrowing , can actually have a substantial influence on the real risks of obtaining false - positive results . 
hyman consulting or advisory role : atara biotherapeutics , chugai pharmaceutical , cytomx therapeutics , boehringer ingelheim , astrazeneca research funding : astrazeneca , puma biotechnology , loxo oncology gregory j . 
riely consulting or advisory role : genentech research funding : novartis ( inst ) , roche ( inst ) , genentech ( inst ) , millennium pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , pfizer ( inst ) , infinity pharmaceuticals ( inst ) , ariad pharmaceuticals ( inst ) patents , royalties , other intellectual property : patent application submitted covering pulsatile use of erlotinib to treat or prevent brain metastases ( inst ) travel , accommodations , expenses : novartis , merck sharp & dohme kristen m . 
begg no relationship to disclose affiliations all authors : memorial sloan kettering cancer center , new york , ny . supported in part through national cancer institute awards ca008748 and ca163251 . support references 33 : 975 - 977 , 2015 1 . 
hyman dm , puzanov i , subbiah v , et al : vemurafenib in multiple nonmelanoma cancers with braf v600 mutations . n engl j med 373 : 726 - 736 , 2015 3 . 
eortc network of core institutions : cross - tumoral phase 2 clinical trial exploring crizotinib ( pf - 02341066 ) in patients with advanced tumors induced by causal alterations of alk and / or met ( create ) , 2013 . 
lopez - chavez a , thomas a , rajan a , et al : molecular profiling and targeted therapy for advanced thoracic malignancies : a biomarker - derived , multiarm , multihistology phase ii basket trial . 
burris ha , hurwitz h , perez ea , et al : mypathway : an open - label phase iia study of trastuzumab / pertuzumab , erlotinib , vemurafenib , and vismodegib in patients who have advanced solid tumors with mutations or gene expression abnormalities targeted by these agents . 
iasonos a , g onen m , bosl gj : scientific review of phase i protocols with novel dose - escalation designs : how much information is needed ? j clin oncol 33 : 2221 - 2225 , 2015 17 . 
chen c , li n , shentu y , et al : adaptive informational design of confirmatory phase iii trials with an uncertain biomarker effect to improve the probability of success . 
klein , md2 purpose to assess the association between the oncotype dx genomic prostate score ( gps ) result and longterm oncological outcomes following radical prostatectomy ( rp )  . methods we evaluated the association of the gps result assayed from the index lesion from rp tissue with the risk of distant metastases ( dm ) and prostate cancerspecic mortality ( pcsm ) over the 20 years following rp in a stratied cohort sample of 428 patients from 2 , 641 treated between 1987 and 2004 . 
exploratory analysis using presurgical parameters and the gps test as prognostic variables was performed to assess the additional value of the gps test on 20 - year risk of dm and pcsm . 
model discrimination was measured using the area under the receiver operating characteristic curve . results the gps test appears to be independently associated with both 20 - year risk of dm and pcsm with a low false discovery rate . 
accuracy of models including clinical risk factors alone appeared to improve when including the gps test in assessing risk of both end points . conclusion the results suggest that the gps test provides information on the risk for the meaningful long - term outcomes of dm and pcsm . jco precis oncol 5 : 442 - 449 . 
2021 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction long - term cancer outcomes are an important consideration when deciding between active surveillance ( as ) and immediate treatment for newly diagnosed prostate cancer . 
multiple prospective as studies that predominantly include patients at the lowest risk of progression have demonstrated a low risk of distant metastasis ( dm ) and prostate cancerspecic mortality ( pcsm ) with extended follow - up.1 , 2 however , these excellent outcomes with as require strict selection criteria and stringent follow - up with frequent repeat prostate biopsies . 
based on these studies , the current use of as for newly diagnosed men is increasing and now includes expanded selection criteria , including younger men with longer life expectancy and those with biopsy - dened pathologic features that fall outside eligibility criteria of older studies.3 it is long - term as outunknown whether the excellent comes observed for low - risk disease can be maintained in this expanded group using only traditional clinical variables and existing surveillance strategies . for example , although both pivot and protect showed no statistically signicant difference in pcsm between observation and immediate treatment intermediate - risk patients with up to a 12.7 - year followup , 4 , 5 the condence intervals for the hazard ratio ( hr ) do not exclude substantial benet of radical prostatectomy ( rp )  . 
furthermore , the long - term results of scandinavian prostate cancer group trial 4 demonstrated an 11.7% absolute risk reduction in pcsm with rp , corresponding to a relative risk of 0.55 , compared with conservative management.6 with up to a 29 - year follow - up , the results of this study are more reective of the lifetime risk of cancer recurrence and death in men with intermediateor high - risk features . 
 gps assay associated with long - term prostate cancer outcomes context key objective localized prostate cancer has an extended natural history , making immediate treatment decisions difcult without the understanding of long - term cancer risks . 
we sought to evaluate the association between the genomic prostate score ( gps ) test and long - term prostate cancer outcomes : distant metastasis and prostate cancerspecic mortality following radical prostatectomy . knowledge generated the gps test appeared to be associated with both distant metastasis and prostate cancerspecic mortality at a 20 - year followup in both univariable and multivariable models , where model discrimination was improved when the gps results were included . relevance the use of the gps test can provide risk assessment of long - term prostate cancer outcomes , beyond clinical factors alone , for patients with localized prostate cancer . traditionally used clinical information in assessing risk of adverse pathology ( ap ) at rp.7 , 8 van den eeden et al9 established the association of the gps test with risk of dm and pcsm over 10 years . 
in this report , we sought to evaluate whether the gps test is also associated with 20 - year risk of dm and pcsm and whether its use improves risk assessment for these end points compared with clinical variables alone . methods using a stratied cohort sampling design , so that a weighted analysis of the study cohort is representative of all patients ( n = 2 , 641 ) who underwent rp between 1987 and 2004 at our institution , we selected 501 patients for inclusion . 
among the remaining 441 patients , the nal analysis cohort comprised the 428 patients with evaluable primary gleason pattern in prostatectomy tissue ( fig 1 )  . all patients were selected from our prospective institutional review board ( irb ) approved database that includes clinical staging , pathology from biopsy and rp , and followup information . 
follow - up and outcome data were obtained through subsequent clinic visits , telephone calls , and semiannual follow - up letters obtained through august 1 , 2019 , approximately 10 years after the previously reported cutoff.7 multiple data reviews and quality checks were performed to ensure delity of the data set . high grade at rp was dened as grade group 3 or above ( gleason score 4 + 3 or higher )  . 
the time to event in patients who died without an event or who were alive without an event at the end of follow - up was considered censored in these analyses . 
each patient was weighted using the inverse sampling fraction in his stratulin and weis robust variance estimate was used.12 in the multivariable analysis , the gps result , preoperative prostatespecic antigen ( psa ) , clinical stage , and biopsy grade were used as covariables . 
the hr for continuous gps results was reported per 20 - gps unit increase , which approximated the difference between the average gps value in the highest 25% and the average gps value in the lowest 25% of patients based on the gps value distribution from previous studies.7 , 13 preoperative psa value was included in the model as log2 ( psa value )  . 
characteristics of the patients in the cohort sample biopsy gleason grade , % brooks et al 61 ( 6 ) 62 ( 57 - 66 ) mean ( sd ) median ( iqr ) race , % white black / afro - caribbean asian / hispanic / others year of surgery , % surgical gleason grade results clinical tumor stage , % pathologic tumor stage % 3 + 4 3 + 5 4 + 3 4 + 5 5 , 5 + 4 low / very low intermediate high 1987 - 1989 1990 - 1994 1995 - 1999 2000 - 2004  . 
summary statistics and percentages are estimated using cohort sampling weights so they estimate the distribution of characteristics in the full cohort of 2 , 641 patients from which the cohort sample ( n = 428 ) was drawn . abbreviations : aua , american urological association ; psa , prostate - specic antigen . for overoptimism of effect estimates involving the gps test . therefore , all estimates using the gps test as a covariable were corrected for regression to the mean ( rm ) 14 and false discovery rates ( fdrs ) were reported rather than p values ( data supplement )  . 
applying the rm correction for gps test , the absolute risk estimates were computed as the weighted average of the absolute risk estimates over the study population patient age for each ap status , with cis derived using the delta method . 
receiver operating characteristic ( roc ) curve estimates were based on these absolute risk estimates , correcting for bias 444 2021 by american society of clinical oncology inherent in roc curves ( data supplement ) , with and without the gps results as a covariable . 
differences between models in the roc area under the curve ( auc ) were tested for signicance using the bootstrap . the overall cohort consisted of predominantly american urological association ( aua ) lowand intermediate - risk patients with prostate cancer , 55% and 35% , respectively ( table 1 )  . 
the distribution of gps results , accounting for the stratied cohort sampling weighting , in the overall population and by biopsy gleason grade is shown in figure 2 . the gps result appeared to be highly associated with both dm and pcsm at 20 years of follow - up . 
 gps assay associated with long - term prostate cancer outcomes total cohort 2 , 641 patients with prostatectomy 19872004 501 patients selected 127 with clinical recurrence 374 without clinical recurrence 441 patients evaluable for clinical / pathology / gene expression 428 patients with evaluable primary gleason pattern in prostatectomy tissue 51 excluded for insufficient tumor 7 did not meet inclusion criteria 2 outlier gene profile fig 1 . 
of 2 , 641 patients who underwent rp between 1987 and 2004 , a cohort of 501 patients was selected using a 1 : 3 sampling design , which included all 127 patients who did and 374 patients who did not have a clinical recurrence . 
rm - corrected estimates of the 20 - year absolute risk of dm and pcsm as a function of gps result among aua low - risk patients and favorable intermediaterisk patients have functional form similar to the estimates for the overall population , with a large increase in risk for the gps result  . 
the number of events in this subgroup was insufcient for multivariable analysis . discussion previous validation studies have clearly shown the gps test and other prostate biopsybased gene expression proles to predict the presence of ap ( grade group 3 or higher or extraprostatic disease ) in rp specimens or serial needle biopsies in men on as or post - rp.7 , 8 , 15 although ap on biopsy or rp is prognostic regarding risk of recurrence , dm , and pcsm , 16 its use as a surrogate end point for these outcomes in men considering starting or staying on surveillance has been questioned with no clear short - term alternative.17 in this study , we sought to assess whether the gps result , based on pathologic evaluation of the index lesion on rp , in the original discovery cohort of this biomarker appeared to be associated with the more clinically meaningful outcomes of dm and pcsm at long - term followup . 
absolute risks of these outcomes , considering death from causes other than prostate cancer as a competing risk , showed a lowslope relationship with the gps result from 1 to 29 , with an inection point evident at a score 30 , above which the risk of dm or pcsm increased substantially , reaching an absolute risk of 38% for dm and 13% for pcsm at a score of 60 ( fig 3 )  . 
furthermore , we found that including the gps results in models assessing risk of dm and pcsm at 20 years improved discrimination compared with using clinical variables alone ( tables 2 and 3 ; fig 4a and 4b )  . 
other commercially available gene expression proles have reported similar ndings , albeit at earlier time points.18 - 21 treatment because key drivers of biological progression in patients on as are yet to be elucidated , an open question is what aspect of tumor biology commercially available gene expression proles may be measuring . 
detailed pathological analysis of the canary - pass study , a large , well - characterized as cohort , identied the presence of cribriform or stromagenic histology as the strongest predictor of cancer recurrence after in men who progressed to requiring therapy.22 the presence of cribriform glands has been shown in other studies to be associated with higher rates of biochemical recurrence after rp or radiation , as well as both dm and pcsm , 23 , 24 likely driven by the observation that these glands exhibit genomic features characteristic of aggressive disease.25 in a recent study of 194 men with national comprehensive cancer network very low - , low - , or intermediate - risk disease considering as , we observed that only those with the gps results above 29 had cribriform histology present on biopsy ( falzarano et al , manuscript submitted for publication )  . 
gps , genomic prostate score . observed that a gps result 29 was associated with grade reclassication on subsequent biopsy in a cohort of lowand intermediate - risk men on as , whereas in an underpowered study using a cohort of lower - risk men whose mean gps results were 21 , no association with grade progression was observed.17 together , these table 2 . 
however , a number of published observations support a role for decision making in men with higher scores : ( 1 ) multiple biopsy - based studies have validated that higher gps scores are associated with traditional histologic measures of ap ( dened as grade group 3 or extraprostatic disease , both of which are associated with worse outcomes ) 7 - 9 ; ( 2 ) the association of higher gps scores with histologic variants ( cribriform [ falzarano et al , manuscript submitted for publication ] and stromagenic26 histology ) shown to be associated with higher rates of recurrence in an as cohort ; 22 and ( 3 ) long - term data from the current study that link higher gps scores to the risk of meaningful clinical end points ( dm and pcsm )  . 
rm - corrected estimates with 95% condence intervals of the 20 - year absolute risk of distant metastasis ( a ) and prostate cancerspecic mortality ( b ) as a function of the gps result . 
 a 100% brooks et al 100% gps grade stage spline ( psa ) grade stage spline ( psa ) reference gps grade stage spline ( psa ) grade stage spline ( psa ) reference 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% fig 4 . 
model - based rm - corrected , bias - corrected roc curve for dm ( a ) and pcsm ( b ) within 20 years of surgery with covariables : the gps result , high stage , high grade , and log2 psa . 
auc , area under the curve ; dm , distant metastases ; fpr , false positive rate ; gps , genomic prostate score ; pcsm , prostate cancerspecic mortality ; psa , prostate - specic antigen ; rm , regression to the mean ; roc , receiver operating characteristic ; tpr , true positive rate . strengths of this study include that the data were sourced from a prospectively maintained patient registry with locked baseline clinical , pathologic , and gene expression information ; centralized expert pathology review ; 20 - year followup , the longest reported in any similar study ; and independent verication of the statistical analysis . 
there are two limitations : ( 1 ) changes in standardized biopsy grading and stage migration since initial patient enrollment , although we note that all rp specimens were reviewed by an expert gu pathologist after the majority of substantive changes in prostate cancer grading occurred and ( 2 ) that this should be considered an exploratory analysis since the gps test was developed in this cohort . 
although the study by van den eeden et al9 largely replicates the results of this analysis , we believe that further validation in other cohorts is important to rmly establish our conclusions . in conclusion , with long - term follow - up , the gps test appears to be associated with both dm and pcsm and improves the accuracy of models containing clinical variables alone . 
these ndings suggest that genomic changes in the tumor tissue , quantied by the gps test , provide additional biological insight into the long - term risk of dm and pcsm . this information may be valuable to those considering as . prospective studies should be pursued to validate these results . affiliations 1scott department of urology , baylor college of medicine , houston , tx 2glickman urological and kidney institute , cleveland clinic , cleveland , 3department of anatomic pathology , university of alabama at birmingham , birmingham , al 4department of quantitative health sciences , cleveland clinic , cleveland , oh 5genomic health inc , an exact sciences corporation , redwood city , ca administrative support : tamer aboushwareb , eric a . 
 gps assay associated with long - term prostate cancer outcomes patents , royalties , other intellectual property : patent co - inventor on 17gene prostate assay with genomic health , inc conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . michael crager employment : exact sciences stock and other ownership interests : exact sciences travel , accommodations , expenses : genomic health ruixiao lu employment : genomic health , exact sciences stock and other ownership interests : genomic health , exact sciences consulting or advisory role : mission bio john abran employment : exact sciences stock and other ownership interests : exact sciences tamer aboushwareb employment : exact sciences stock and other ownership interests : exact sciences eric a . 
klein consulting or advisory role : grail no other potential conicts of interest were reported . references 2016 klotz l , vesprini d , sethukavalan p , et al : long - term follow - up of a large active surveillance cohort of patients with prostate cancer . 
j clin oncol 33 : 272 - 277 , 2015 tosoian jj , mamawala m , epstein ji , et al : intermediate and longer - term outcomes from a prospective active - surveillance program for favorable - risk prostate cancer . 
mahal ar , butler s , franco i , et al : conservative management of low - risk prostate cancer among young versus older men in the united states : trends and outcomes from a novel national database . 
n engl j med 377 : 132 - 142 , 2017 bill - axelson a , holmberg l , garmo h , et al : radical prostatectomy or watchful waiting in prostate cancer29 - year follow - up . 
n engl j med 379 : 2319 - 2329 , 2018 klein ea , cooperberg mr , magi - galluzzi c , et al : a 17 - gene assay to predict prostate cancer aggressiveness in the context of gleason grade heterogeneity , tumor multifocality , and biopsy undersampling . 
eur urol 66 : 550 - 560 , 2014 cullen j , rosner il , brand tc , et al : a biopsy - based 17 - gene genomic prostate score predicts recurrence after radical prostatectomy and adverse surgical pathology in a racially diverse population of men with clinically lowand intermediate - risk prostate cancer . 
eur urol 68 : 123 - 131 , 2015 van den eeden sk , lu r , zhang n , et al : a biopsy - based 17 - gene genomic prostate score as a predictor of metastases and prostate cancer death in surgically treated men with clinically localized disease . 
lin dw , zheng y , mckenney jk , et al : 17 - gene genomic prostate score test results in the canary prostate active surveillance study ( pass ) cohort . 
spratt de , youse k , deheshi s , et al : individual patient - level meta - analysis of the performance of the decipher genomic classier in high - risk men after prostatectomy to predict development of metastatic disease . 
klein ea , youse k , haddad z , et al : a genomic classier improves prediction of metastatic disease within 5 years after surgery in node - negative high - risk prostate cancer patients managed by radical prostatectomy without adjuvant therapy . 
cooperberg mr , davicioni e , crisan a , et al : combined value of validated clinical and genomic risk stratication tools for predicting prostate cancer mortality in a high - risk prostatectomy cohort . 
canter dj , freedland s , rajamani s , et al : analysis of the prognostic utility of the cell cycle progression ( ccp ) score generated from needle biopsy in men treated with denitive therapy . 
mckenney jk , wei w , hawley s , et al : histologic grading of prostatic adenocarcinoma can be further optimized : analysis of the relative prognostic strength of individual architectural patterns in 1275 patients from the canary retrospective cohort . 
kweldam cf , wildhagen mf , steyerberg ew , et al : cribriform growth is highly predictive for postoperative metastasis and disease - specic death in gleason score 7 prostate cancer . 
greenland ny , cowan je , chan e , et al : prostate biopsy histopathologic features correlate with a commercial gene expression assays reclassication of patient eur j cancer 66 : 26 - 33 , 2016 alterations . 
kornberg z , cooperberg mr , cowan je , et al : a 17 - gene genomic prostate score as a predictor of adverse pathology in men on active surveillance . 
 multi - institutional , prospective clinical utility study evaluating the impact of the 92 - gene assay ( cancertype id ) on final diagnosis and treatment planning in patients with metastatic cancer with an unknown or unclear diagnosis purpose metastatic cancers of unknown primary or with unclear diagnoses pose diagnostic and management challenges , often leading to poor outcomes . 
the current study assessed the clinical impact of the 92 - gene assay on diagnostic and treatment decisions for patients with unknown or uncertain diagnoses . methods patients in this prospective , multi - institutional , decision - impact study included those for whom the 92 - gene assay was ordered as part of routine care . 
the key study objective of clinical impact was calculated on the basis of changes in final diagnosis and treatment after testing . results data collection included 444 patients , 107 physicians ( 73 oncologists and 34 pathologists ) , and 28 sites . 
 physicians reported that the 92 - gene assay was used broadly for diagnostic dilemmas that ranged from single suspected tumor type ( 29% ) to a differential diagnosis of two or more suspected tumor types ( 30% ) or cancers of unknown primary ( 41% )  . 
integration of 92 - gene assay results led to a change in the recommended treatment in 47% of patients . conclusion findings from this clinical utility study demonstrate that the 92 - gene assay led to a change in treatment decisions in every other patient case . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction metastatic cancers that are initially characterized as unknown primary ( cup ) , or with other features that may lead to diagnostic uncertainty with respect to tumor type diagnosis , can prolong the clinicopathologic workup and result in delays in the initiation of treatment , additional costs , and relatively poor patient outcomes.1 - 3 sachdev p . 
schnabel fadi braiteh author affiliations and support information ( if applicable ) appear at the end of this article . licensed under the creative commons attribution 4.0 license corresponding author : sachdev p . 
numerous diagnostic and treatment strategies , such as enhanced pathologic techniques , molecular classification of tumor type and subtype , comprehensive mutational profiling , and novel combination regimens of cytotoxics plus biolo gics , have been proposed without universal consensus.4 - 10 investigational approaches to personalize the systemic treatment of metastatic cancer solely on the basis of the patients somatic genomic alterations , disregarding histologic context , have had mixed and disappointing results.11 - 14 results from several recent phase ii studies that evaluated targeted agents for specific genomic alterations across a variety of tumor types have provided evidence that mutations are not targetable in similar manners across tumor types.11 , 12 thus , the clinical utility of this approach is unclear . 
 whereas driver mutations matter , the integration of tumor type and subtype remains critical when considering the efficacy of a targeted therapy aimed at a putative driver mutation.15 given that empirical chemotherapy approaches are associated with poor prognosis in patients with cup or uncertain diagnoses , it might be more effective to refocus on methods with which to identify patient tumor type and subtype to guide therapy.1 , 16 gene expression profilingbased molecular classification of tumors might prove efficacious by helping to identify the primary tumor type and histologic subtype for patients with unknown or uncertain diagnoses . 
the primary objective was to assess clinical impact on the basis of changes in diagnostic and treatment decisions after the incorporation of 92 - gene assay results . methods study design observational in nature , the current study was prospectively defined to evaluate multidisciplinary clinical utility in patients for whom physicians ordered the 92 - gene assay as a clinical workup tool to help identify or narrow the tumor type and subtype diagnosis . 
prespecified objectives were to examine the diagnostic and clinical utility of the 92 - gene assay in oncology and pathology practice to characterize indications of use , and to evaluate its potential integration and impact on patient management by comparing changes in diagnosis and treatment selection after testing . data collection physicians were required to complete standard ized , discipline - specific questionnairespathology and oncologyafter receiving a 92 - gene assay test report . 
 the pathology ecrf consisted of 11 multiple choice questions and four questions that required written responses ( biopsy quality , number and types of immunohistochemistry [ ihc ] performed , and 92 - gene assay result )  . 
the medical oncologist ecrf consisted of 11 multiple choice questions and five questions that required written responses ( time between biopsy and treatment , clinical diagnosis for first - line treatment , name of first - line treatment , other diagnostic or clinical considerations for choosing first - line therapy , and 92 - gene assay result )  . 
 rna quality determined to be quantity not sufficient , in which a molecular prediction could not be determined , physicians completed quantity not sufficient ecrfs that consisted of four multiple choice questions and two questions that required written response ( biopsy quality and number of ihc performed )  . 
ecrf forms are included in the data supplement . physicians were instructed on the web portal and ecrf via a standardized training progra for the pathology questionnaire ( data supplement : pathology ecrf question 7 and oncology ecrf question 8 , respectively ) , physicians were instructed to align terminology with the 50 tumor types and subtypes classified by the 92 - gene assay . 
the testing laboratory was blinded to the working clinical diagnosis from the ecrf . 92 - gene assay the 92 - gene assay was performed as previously described.17 , 20 in brief , the assayreal - time rt pcrwas performed on isolated total rna from tumor cells that were enriched by either microdissection or laser microdissection . 
a prespecified computational algorithm generated probabilities for primary tumor type and subtype on the basis of the degree of similarity of the submitted sample to a reference database of gene expression information from more than 2 , 000 tumors of known tumor type . 
the test report provided a prediction of the main tumor type and subtype on the basis of the highest relative probability and any additional tumor types that cannot be ruled out . 
the report also provided a list of tumor types that could be excluded with 95% confidence . statistical analysis statistical considerations of study size were based on an anticipated treatment recommendation change rate of 35% , targeting a sample size of at least 156 patients to ensure a 95% two - sided ci width of 15% . 
the primary objective of the study was to measure the clinical impact of the 92 - gene assay results on the basis of changes in patient treatment , the narrowing of treatment options , or the elimination of a treatment option ( a , b , or c on question 15 of the oncology questionnaire in the data supplement )  . 
patients were considered eligible for a targeted agent if the physician responded a , " " b , " or c in question 15 and there was a us food and drug administrationapproved targeted therapy that was recommended by the national comprehensive cancer network for the molecular diagnosis provided by the 92 - gene assay . results patient enrollment began in february 2013 and closed october 2014 after enrolling 444 patients with 107 physicians73 medical oncologists and 34 pathologistsfrom 28 sites21 oncology sites and seven pathology sitesacross the united states . 
of the 444 patients enrolled , 397 had sufficient rna for analysis , which corresponded to an overall analytical success rate of 89% , taking into account samples that were determined to have insufficient tissue on pathology review before testing . 
a patient flow diagram is shown in figure 1 . biopsy and tumor characteristics the most common metastatic biopsy sites included the liver ( 23% ) , lymph nodes ( 17% ) , and lung ( 14% ; fig 2a )  . 
patient flow diagram showing the disposition of 92 - gene assay testing . patients submitted for testing ( n = 444 ) not reportable insufficient tissue or rna ( n = 47 ) reportable results ( n = 397 ) resulted to oncologist ( n = 271 ) resulted to pathologist ( n = 126 ) 92 - gene assay results ( n = 271 ) 92 - gene assay results ( n = 126 ) indeterminate ( n = 12 ) indeterminate ( n = 6 ) molecular diagnosis ( n = 259 ) molecular diagnosis ( n = 120 ) excluded did not receive anticancer therapy performance deaths patient choice other ( n = 56 ) ( n = 16 ) ( n = 8 ) ( n = 7 ) ( n = 25 ) patient received anticancer therapy ( n = 203 ) diagnostic testing and preassay diagnosis factors that contributed to an oncologists decision to order the 92 - gene assay were multidisciplinary and included the following : no primary site of origin after clinical review and imaging ( 42% ) , a pathology report that indicated a differential diagnosis ( 21% ) or that indicated an unknown primary site ( 20% ) , and distinguishing between new cancer versus recurrence ( 16% )  . 
 data that were collected to better characterize the sequence of diagnostic testing demonstrated that 72% of physicians responded that patients had pathology and ihc studies performed before the 92 - gene assay , 14% of samples were submitted for pathology and ihc evaluation and the 92 - gene assay concurrently , and approximately 14% of samples were submitted without an indication of the diagnostic sequence . 
the most common imaging tests were computed tomography scans ( 86% ) , fusion positron emission tomography / computed tomography scans ( 57% ) , magnetic resonance imaging ( 29% ) , ultrasound ( 28% ) , regular film radiographs ( 11% ) , or mammogram ( 11% ; data not shown )  . for pathologists , inconclusive ihc ( 50% ) was the most common reason for ordering the 92 - gene assay . 
the mean number of ihc stains performed before the molecular assay was ordered was 10 ( median , nine ; range , zero to 23 ; data not shown )  . 92 - gene assay results and impact on diagnosis of the 397 patients with sufficient tissue and rna for a reportable result , the 92 - gene assay provided a molecular - based tumor type and histologic subtype diagnosis in 379 patients ( 95.5% ) , whereas 4.5% had an indeterminate molecular diagnosis ( fig 1 )  . 
 ( * ) other indicates biopsy sites with fewer than three cases , encompassing uterus , gallbladder , gastroesophageal junction , spine , spleen , thyroid , and salivary gland . 
fna , fine - needle aspiration . liver lymph node lung peritoneal cavity or omentum soft tissue bone / marrow pleural cavity / pleura colon brain rectum skin breast bladder stomach pelvic mass retroperitoneum pericardial cavity adrenal gland pancreas mediastinum esophagus ovary duodenum other * core excision endoscopic other percentage percentage types . 
the most common diagnoses were pancreaticobiliary ( 21.9% ) , squamous cell carcinoma ( 10.1% ) , lung adenocarcinoma ( 9.3% ) , and intestinal ( 8.6% ) type tumors ( fig 3 )  . working diagnoses before the 92 - gene assay were assessed in the medical oncology subset ( n = 271 )  . 
preassay working diagnoses were reported as a single suspected site in 79 patients ( 29% ) , a differential diagnosis of two or more suspected sites in 80 patients ( 30% ) , and cup in 112 patients ( 41% ; fig 4a )  . 
comparison of the 92 - gene assay molecular diagnoses with preassay working diagnoses demonstrated that the assay provided a tumor type diagnosis that was not initially suspected in a large proportion of patients ( fig 4b )  . 
in patients with a single suspected primary site ( n = 79 ) , the 92 - gene assay confirmed the suspected diagnosis in 60% of patients but provided a tumor type result that was not initially suspected in 39% of patients . 
in patients with a differential diagnosis ( n = 80 ) , the 92 - gene assay narrowed the diagnosis in 66% of patients and provided a tumor type result that was not initially suspected in 27% of patients . 
in patients for whom the pathology report indicated cup site ( n = 112 ) , the assay provided a molecular tumor type prediction in 97% of patients . impact on treatment planning of the 271 oncologist - submitted cases , 203 patients ( 75% ) received anticancer treatment after the 92 - gene assay results were made available . 
the most common reasons for patients not receiving treatment were a rapid deterioration in the patients performance status ( 29% ) , patient death ( 14% ) , and a patient declination of treatment ( 13% ; fig 1 )  . 
molecular cancer classification predictions from submitted cases using the 92 - gene assay ( n = 397 )  . pancreaticobiliary squamous cell carcinoma lung adenocarcinoma intestine gastroesophageal adenocarcinoma urinary bladder neuroendocrine ovary breast adenocarcinoma liver hepatocellular carcinoma sarcoma head and neck salivary gland carcinoma prostate adenocarcinoma endometrial adenocarcinoma cervix adenocarcinoma melanoma kidney mesothelioma germ cell thyroid brain indeterminate skin basal cell carcinoma percentage ( fig 5a )  . 
the assay resulted in similar changes in treatment recommendations in all three scenarios of the original working diagnosis48% of cases with a single suspected primary ( n = 79 ) , 49% of differential diagnoses with two or more primaries ( n = 80 ) , and 42% of cases with an unknown primary site ( n = 112 )  . subset analysis within the most commonly predicted tumor type classesthose with 20 casesdemonstrated that physicians changed their recommended treatment in 58% of gi cancers ( n = 81 ) , 54% of gynecologic and breast cancers ( n = 28 ) , and 44% of lung cancers ( n = 27 ; fig 5b )  . 
mutational biomarker testing was most commonly ordered after the 92 - gene assay rendered a diagnosis of lung ( 81% ) or colorectal cancer ( 67% )  . discussion results from this large , multisite study have demonstrated that the 92 - gene assay was used across a spectrum of diagnostic uncertainty , beyond cups , and included cases with differential diagnoses and those with a suspected diagnosis for confirmatory testing . 
in addition , the assay provided a diagnosis that was not initially suspected in a substantial proportion of patients who had either a single suspected diagnosis or differential diagnosis before testing with the 92 - gene assay . 
 this clinical impact was more pronounced in gi and gynecologic and breast tumor types , which suggests that the 92 - gene assay may have additional utility in particular metastatic presentations in which standard approaches may be limited . 
 distribution of 92 - gene assay diagnostic results within the preassay working diagnosis classifications . differential diagnosis ( 30% ) unknown primary ( 41% ) single suspected site ( 29% ) indeterminate indeterminate indeterminate provided new diagnosis provided new diagnosis confirmed suspected diagnosis confirmed suspected diagnosis provided new diagnosis single suspected site differential diagnosis unknown primary approaches , what is both the current and future relevance of establishing the tumor type or cellular context of a metastatic cancer ? recent results from a number of basket trials have demonstrated that knowledge of tumor type and cellular context remains fundamental for the interpretation of potentially targetable dna mutations and the recommendation of treatment approaches in metastatic cancer . 
whereas molecularly targeted agents have been demonstrated to be effective in tumors that harbor a matching molecular alteration , a growing understanding of the importance of molecular heterogeneity and cellular context is emerging . 
for example , the efficacy of the targeted braf inhibitor , vemurafenib , has been shown to be mixed across a diverse set of nonmelanoma cancers with a braf v600 mutation11 and is well known to have poor efficacy in braf - mutated colorectal cancers.11 , 13 similarly , early results from the mypathway basket trial have demonstrated variable response rates in patients with identical mutations across different tumor types.14 finally , in a phase ii study in which patients with a specific molecular alteration were randomly assigned to receive treatment with a molecularly targeted agent or physicians choice of treatment , there was no difference in median progression - free survival between the two treatment groups.12 these data suggest that the effectiveness of targeting putative driver mutations with molecularly targeted agents may be dependent on the specific cellular context or tumor type . 
 no , treatment regimen was not changed ( 53% ) yes , treatment regimen was changed ( 47% ) gi cancers gynecological and breast cancers lung cancers treatment regimen was not changed ( 42% ) yes , treatment regimen was changed ( 58% ) treatment regimen was not changed ( 46% ) yes , treatment regimen was changed ( 54% ) treatment regimen was not changed ( 56% ) yes , treatment regimen was changed ( 44% ) fig 5 . 
 ( b ) impact of the 92 - gene assay on treatment decisions within tumor type subgroups that received therapy : gi cancers ( n = 81 ) , gynecologic and breast cancers ( n = 28 ) , and lung cancers ( n = 27 )  . breast adenocarcinoma , and lung adenocarcinoma.23 - 25 results presented here reinforce the continued relevance of tumor type diagnosis in optimizing treatment strategies that can potentially affect patient outcomes . 
with regard to pancreaticobiliary tumors , the 92 - gene assay also reports additional subtyping into gallbladder adenocarcinoma , pancreatic adenocarcinoma , or cholangiocarcinoma , which may inform treatment decisions for surgery type , neoadjuvant chemotherapy , or site - directed and targetable agents . 
in addition , a significant proportion of tumor types predicted by the 92 - gene assay , including lung adenocarcinoma , lung squamous , colorectal , gastroesophageal , urinary bladder , and neuroendocrine tumors , have not only specific chemotherapy approaches , but also us food and drug administrationapproved molecularly targeted therapies or immunotherapies . 
 decision making , with similar overall results , which supports the clinical utility of the assay.34 , 35 strengths of this study include the large number of patients and contributing physicians , which contributed to the generalizability of the study results . 
first , this was an observational study and no data on outcomes were collected ; however , previous studies have demonstrated that the use of the 92 - gene assay improved survival in patients with cup who were treated on the basis of assay results.16 , 34 although multiple aspects of the study design were prespecified and carefully planned , such as study physicians independently completing questionnaires , a blinded design such that the testing laboratory did not have knowledge of working diagnoses , and a preplanned statistical analysis , bias is an inherent feature of observational studies . 
this aspect of the cross - functional integration of molecular diagnostic results warrants investigation in future studies . this study demonstrated that the 92 - gene assay affected diagnosis and treatment selection in a significant proportion of patients , which supports the clinical utility of the assay as a standardized molecular approach to help streamline additional diagnostic testing in patients with metastatic cancer with unknown or uncertain diagnoses . 
schnabel data analysis and interpretation : all authors manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors lauren e . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . stock and other ownership interests : biotheranostics brock e . 
 fadi braiteh stock and other ownership interests : bristol - myers squibb , insys therapeutics , agios , clovis oncology , tesaro honoraria : genentech , insys therapeutics , bristol - myers squibb , amgen , boehringer ingelheim , astrazeneca , bayer , celgene , ipsen , incyte , taiho pharmaceutical , lexicon , ariad pharmaceuticals , eli lilly , abbott nutrition , heron consulting or advisory role : amgen , bristol - myers squibb , clovis oncology , boehringer ingelheim , ipsen , insys therapeutics , ambry genetics , genentech , eli lilly , incyte , bayer , sanofi , regeneron , astrazeneca , merck , celgene , lexicon , pfizer , merrimack pharmaceuticals speakers ' bureau : amgen , bristol - myers squibb , genentech , merck , pfizer , eli lilly , ipsen , insys therapeutics , incyte , boehringer ingelheim , astrazeneca , celgene , taiho pharmaceutical , merrimack pharmaceuticals , biotheranostics travel , accommodations , expenses : genentech , bristolmyers squibb , amgen , celgene , clovis oncology , tesaro , ipsen , insys therapeutics , incyte , heron , astrazeneca , medimmune , novartis , boehringer ingelheim , lexicon , taiho pharmaceutical , bayer , bayer , onyx pharmaceuticals , exelixis , regeneron , sanofi , merrimack pharmaceuticals , pfizer research funding : biotheranostics acknowledgment we thank andrea c . 
grschel s , bommer m , hutter b , et al : integration of genomics and histology revises diagnosis and enables effective therapy of refractory cancer of unknown primary with pdl1 amplification . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors based on molecular profiles : early results from mypathway , an open - label , phase iia umbrella basket study . 
hainsworth jd , rubin ms , spigel dr , et al : molecular gene expression profiling to predict the tissue of origin and direct site - specific therapy in patients with carcinoma of unknown primary site : a prospective trial of the sarah cannon research institute . 
weiss lm , chu p , schroeder be , et al : blinded comparator study of immunohistochemical analysis versus a 92 - gene cancer classifier in the diagnosis of the primary site in metastatic tumors . 
kim b , schroeder b , schnabel ca , et al : physician - reported clinical utility of the 92 - gene molecular classifier in tumors with uncertain diagnosis following standard clinicopathologic evaluation . 
ahmed ka , caudell jj , el - haddad g , et al : radiosensitivity differences between liver metastases based on primary histology suggest implications for clinical outcomes after stereotactic body radiation therapy  . 
spetzler d , xiao n , burnett k , et al : multi - platform molecular profiling of 1 , 180 patients increases median overall survival and influences treatment decision in 53% of cases . 
nystrom sj , hornberger jc , varadhachary gr , et al : clinical utility of gene - expression profiling for tumor - site origin in patients with metastatic or poorly differentiated cancer : impact on diagnosis , treatment , and survival . 
raghav k , mhadgut h , mcquade jl , et al : cancer of unknown primary in adolescents and young adults : clinicopathological features , prognostic factors and survival outcomes . 
hainsworth jd , schnabel ca , erlander mg , et al : a retrospective study of treatment outcomes in patients with carcinoma of unknown primary site and a colorectal cancer molecular profile . 
j nat cancer inst 105 : 782 - 790 , 2013 appendix we thank the following principal investigators of the clinical study sites who participated in the study : paul adams , md ( genesys hurley cancer institute ) , solhail akbani , md ( baptist hospitals of southeast texas ) , stephen anthony , do ( evergreen hematology - oncology ) , andrew j . 
brenner , md ( the university of texas health science center at san antonio ) , david campbell , md ( grass valley hematology - oncology medical group ) , arvind chaudhry , md , phd ( medical oncology associates ) , william gilles , md ( dignity health ) , h . 
carriers are predisposed to a higher risk for developing colorectal , endometrial , and extracolonic cancers , including cancers of the stomach , ovary , small bowel , hepatobiliary and urinary tracts , skin , pancreas , and prostate . 
primary brain tumors ( pbts ) are a rare feature of ls , with an estimated lifetime risk of 1% to 6%.1 , 2 here , we report the case of a patient with a who grade iii glioneuronal tumor and pathogenic germline msh2 variant . 
microscopic examination revealed a high - grade glioma with eosinophilic granular bodies , elevated mitotic activity ( approximately 10 / 10 high - power eld [ hpf ] ) , vascular proliferation , and necrosis consistent with an anaplastic glioneuronal tumor ( who grade iii )  . tumor cells were positive for glial brillary acidic protein ( gfap ) and synaptophysin ( fig 2 )  . 
the mib - 1 proliferative index ( ki - 67 ) was approximately 10% . the patients initial postoperative imaging revealed no residual disease ; therefore , no adjuvant therapy was offered . 
postoperative head ct ( c ) with and ( d ) without contrast at the patients most recent follow up , 105 months postinitial diagnosis , shows postoperative changes of the left frontal lobe without evidence of tumor recurrence . disease progression identied pathogenic genomic alterations in six genesnf1 , pdgfra , atm , tp53 , ctnna1 , and msh2and 18 variants of uncertain signicance ( table 1 )  . 
follow - up ihc studies identied a loss of msh2 and msh6 protein expression in tumor cells , which is consistent with an ls - associated tumor and underlying msh2 germline mutation . 
microscopic examination revealed a high - grade glial neoplasm with ( a ) scattered pleomorphic cells , ( b ) spindle cells , and mitotic activity . necrosis and vascular proliferation were observed . 
immunostains showed that the tumor is positive for ( c ) gfap and ( d ) synaptophys ( e ) ki - 67 was moderately elevated and a ( f ) neurolament stain was negative within the tumor , highlighting a compact growth pattern . 
immunohistochemistry identied loss of ( g ) msh2 and ( h ) msh6 protein expression in tumor cells , with retained nuclear expression in the normal endothelial cells in the blood vessels . magnication of all images is 200 . 
glioblastoma is the most common pbt associated with ls.3 astrocytoma , oligodendroglioma , gliosarcoma , medulloblastoma , and neuroblastoma have also been reported.4 individuals with msh2 mutations have higher rates of pbts compared with carriers of other mmr mutations.5 the average age of pbt diagnosis occurs a decade earlier in individuals with ls , 4 and the age range for msh2 carriers is 33 to 53 years.1 , 3 , 6 , 7 ls is one of several hereditary cancer syndromes that predispose to pbts and other systemic cancers . 
therefore , interrogation of personal and family history of malignancies is critical for patients with d 70s dx 47 d 52 d 50s d 50s d 30s d 50s d 40s d 60 dx 42 5 polyps 2 polyps throat abdominal cancer , other breast cancer colorectal cancer glioneuronal tumor fig 3 . 
the patients family history is notable for multiple maternal family members affected with early - onset colorectal cancer , including his mother and multiple maternal aunts , uncles , and cousins . 
next - generation sequencing of 236 genes in the patients tumor specimen ( solid tumor panel ; foundationone , cambridge , ma ) identied pathogenic genomic alterations in six genes and 18 variants of uncertain signicance . * follow - up sequencing of msh2 in the patients saliva sample ( ambry genetics , aliso viejo , ca ) also identied the msh2 mutation in the germline sample . pbt as for all patients with cancer . 
referral for genetic counseling and germline testing is recommended for any patient with pbt with multiple relatives affected with earlyonset cancers , individual ( s ) with multiple primary cancers , and / or rare or syndrome - specic patterns of malignancies.8 , 9 appropriate germline genetic testing may result in additional screening guidelines and the identication of other at - risk relatives.10 ls surveillance guidelines include colonoscopy every 1 to 2 years starting at age 20 to 25 years or 10 years before the earliest crc diagnosis in the family.11 histologic ndings in this patients pbt showed overlapping features between anaplastic pleomorphic xanthoastrocytoma , who grade iii , and glioblastoma . 
genetic alterations that are present in the tumor tissuethat is , nf1 , pdgfra , and tp53 mutationshave been described in glioblastomas12 , 13 ; however , survival of 8 years would be unusual for a patient with glioblastoma . one indicator that germline molecular testing was warranted for this patient was the identication of an msh2 mutation in his tumor specimen . 
studies that have evaluated the yield of germline variants after somatic testing indicate that a small but signicant number of tumors possess germline mutations related to hereditary cancer predisposition syndromes.14 - 17 currently , consideration of follow - up germline testing is recommended after identication of somatic egfr t790m mutations in lung cancer or brca1 / 2 mutations in any tumor.18 - 21 interpretation of somatic mmr mutations , however , is more complex . hampel et al22 used tumor sequencing to screen for ls in a cohort of patients with crc in which a positive screen for ls was given to any patient who had mutation ( s ) identied in at least one mmr gene at a variant allele fraction that was suggestive of a germline mutation , with msi and braf v600e status taken into account . 
therefore , whereas the identication of somatic mmr mutation ( s ) is not diagnostic of ls , it does suggest that follow - up germline evaluation is warranted in these patients . identifying germline and / or somatic mmr mutations may affect treatment offerings , such as consideration for immune checkpoint inhibitors ( icis ) , which target checkpoint receptors of mmr - decient cancers.23 - 25 early studies have demonstrated that icis can effectively treat high tmb cancers ; mmr - decient tumors can possess 10 to 100 times the tmb compared with mmr - procient cancers.26 - 28 glioblastoma specimens from patients with constitutional mmr deciency caused by biallelic germline mmr mutations have signicantly higher tmb compared with sporadic gliomas and other pbts.29 whereas icis have primarily been evaluated in crc and endometrial carcinomas , therapeutic potential has been demonstrated in other mmrdecient tumors . 
in patients with recurrent tumors , high tmb may indicate resistance to traditional chemotherapy and necessitate targeted treatment with icis or other immunotherapies.30 these results must be validated on a larger scale to inform clinicians of the potential therapeutic benets of ici in this subset of patients . in conclusion , we report the rst case of an individual with anaplastic glioneuronal tumor with atypical histologic features and paired somatic and germline msh2 mutations , indicating the diversity of pbts observed within ls . 
the presence of mmr gene mutation ( s ) in tumor specimens indicate that additional genetic evaluation is warranted . ihc , msi , and tmb analyses are also supportive of an ls diagnosis . 
furthermore , a family history evaluation for multiple relatives affected with early - onset cancers , dividual ( s ) with multiple primary cancers , and / or rare or syndrome - specic patterns of malignancies is imperative for all patients with pbt . 
in addition , identifying mmr deciency in the germline or tumor specimens may be an indication for targeted therapy via ici or other immunotherapies in the future . affiliations 1the university of texas md anderson cancer center , houston , tx 2university of texas health science center at houston , houston , tx 3foundation medicine , morrisville , nc 4wake forest school of medicine , winston - salem , nc relationships are self - held unless noted . 
qualmann provision of study materials or patients : jay - jiguang zhu , krista j . qualmann collection and assembly of data : sarah azam , jay - jiguang zhu , krista j . 
ramkissoon employment : foundation medicine stock and other ownership interests : foundation medicine sigmund hsu consulting or advisory role : abbvie , cns pharmaceuticals research funding : abbvie patents , royalties , other intellectual property : partial patent holder , royalties ; at the time of ling , was associated with md anderson cancer center jay - jiguang zhu consulting or advisory role : tocagen research funding : novocure ( inst ) , boston biomedical ( inst ) , five prime therapeutics ( inst ) , tocagen ( inst ) , immunocellular therapeutics ( inst ) travel , accommodations , expenses : zai lab krista j . 
qualmann employment : my gene counsel no other potential conicts of interest were reported . references vasen hfa , stormorken a , menko fh , et al : msh2 mutation carriers are at higher risk of cancer than mlh1 mutation carriers : a study of hereditary nonpolyposis colorectal cancer families . 
int j cancer 81 : 214 - 218 , 1999 therkildsen c , ladelund s , rambech e , et al : glioblastomas , astrocytomas and oligodendrogliomas linked to lynch syndrome . 
n engl j med 348 : 919 - 932 , 2003 vasen hfa , sanders eacm , taal bg , et al : the risk of brain tumours in hereditary non - polyposis colorectal cancer ( hnpcc )  . 
hampel h , bennett rl , buchanan a , et al : a practice guideline from the american college of medical genetics and genomics and the national society of genetic counselors : referral indications for cancer predisposition assessment . 
catenacci dv , amico al , nielsen sm , et al : tumor genome analysis includes germline genome : are we ready for surprises ? int j cancer 136 : 1559 - 1567 , 2015 jones s , anagnostou v , lytle k , et al : personalized genomic analyses for cancer mutation discovery and interpretation . 
b. , et al : molecular testing guideline for selection of lung cancer patients for egfr and alk tyrosine kinase inhibitors : guideline from the college of american pathologists , international association for the study of lung cancer , and association for molecular pathology . 
hampel h , pearlman r , beightol m , et al : assessment of tumor sequencing as a replacement for lynch syndrome screening and current molecular tests for patients with colorectal cancer . 
sloan ea , ring kl , willis bc , et al : pd - l1 expression in mismatch repair - decient endometrial carcinomas , including lynch syndrome - associated and mlh1 promoter hypermethylated tumors . 
science 357 : 409 - 413 , 2017 johnson a , severson e , gay l , et al : comprehensive genomic proling of 282 pediatric lowand high - grade gliomas reveals genomic drivers , tumor mutational burden , and hypermutation signatures . 
 genomic proling identies outcome - relevant mechanisms of innate and acquired resistance to third - generation epidermal growth factor receptor tyrosine kinase inhibitor therapy in lung cancer sebastian michels , md1 ; carina heydt , phd1 ; bianca van veggel , md2 ; barbara deschler - baier , md3 ; nuria pardo , md4 ; kim monkhorst , md2 ; vanessa r usseler , md1 ; jan stratmann , md5 ; frank griesinger , md6 ; susanne steinhauser , phd7 ; anna kostenko , msc1 ; joachim diebold , md8 ; jana fassunke , phd1 ; rieke fischer , md1 ; walburga engel - riedel , md9 ; oliver gautschi , md8 ; eva geissinger , md10 ; stefan haneder , md1 ; michaela a . 
de langen , md2 ; alex martinez - marti , md4 ; lucia nogova , md1 ; thorsten persigehl , md1 ; dennis plenker , phd1 ; michael puesken , md1 ; ernst rodermann , md12 ; andreas rosenwald , md10 ; andreas h . 
scheel , md1 ; matthias schefer , md1 ; werner spengler , md13 ; ruth seggewiss - bernhardt , md14 ; johannes br agelmann , md1 , 7 ; martin sebastian , md5 ; bart vrugt , md15 ; martin hellmich , phd7 ; martin l . 
smit , md2 ; reinhard b uttner , md1 ; and j urgen wolf , md1 purpose third - generation epidermal growth factor receptor ( egfr ) tyrosine kinase inhibitors ( tkis ) are effective in acquired resistance ( ar ) to early - generation egfr tkis in egfr - mutant lung cancer . 
we present the molecular proles of pretreatment and post - treatment samples from patients treated with third - generation egfr tkis and their impact on treatment outcomes . methods using the databases of two lung cancer networks and two lung cancer centers , we molecularly characterized 124 patients with egfr p.t790m - positive ar to early - generation egfr tkis . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction treatment with selective early - generation epidermal growth factor receptor ( egfr ) tyrosine kinase inhibitors ( tkis ) has demonstrated high efcacy in patients with lung cancer harboring activating egfr mutations . 
we present a comprehensive analysis of cooccurring genetic aberrations in pretreatment and posttreatment tumor tissue and their contribution to innate resistance ( ir ) and ar to three third - generation egfr tkis . methods study design , patient selection , and tumor tissue collection to determine the frequency of co - occurring genetic aberrations in samples of egfr p.t790m - mediated resistance to early - generation egfr tkis , we systematically searched the databases of the network genomic medicine , the nowel network , the department of thoracic oncology of the netherlands cancer institute , and the institute of oncology at the vall dhebron university hospital for patients with nonsmall - cell lung cancer ( nsclc ) who fullled the following selection criteria ( cohort a ; patients a1 to a68 / b1 to b56 ; fig 1 ; data supplement ) : ( 1 ) presence of cohort a ( n = 124 ; 100% ) 1 . 
cnv , copy number variation ; egfr , epidermal growth factor receptor ; erbb2 , human epidermal receptor 2 growth factor gene ; met , mesenchymalepithelial transition factor ; mps , massively parallel sequencing ; nsclc , non small - cell lung cancer ; recist , response evaluation criteria in solid tumors ; seq , sequencing ; tki , tyrosine kinase inhibitor . excluded ( n = 68 ) excluded ( n = 27 ) cohort b ( n = 56 ; 45% ) 1 . 
patients were treated in the aura 1 / 3 trials ( osimertinib ; nct01802632 / nct02151981 ) , tiger - 2 / - 3 trials ( rociletinib ; nct02147990 / nct02322281 ) , cegf816x2101 trial ( nazartinib ; nct 02108964 ) , osimertinib compassionate use program ( cup ) , or clinical routine . 
patients treated in trials or the cup were selected according to the specic eligibility criteria . in a subset of patients from cohort b , a rebiopsy was performed at disease progression for identication of mechanisms of ar . 
these patients were grouped in cohort c ( fig 1 )  . in all patients , tumor tissue was collected in growing lesions by aspiration biopsy , core needle biopsy , or excisional biopsy ( data supplement )  . 
in patients screened within network genomic medicine ( a1 to a68 / b1 to b31 / b37 to b43 ) , mps was performed with an ion ampliseq custom dna panel ( thermo fisher scientic , waltham , ma ) and a miseq benchtop sequencer ( illumina , san diego , ca ) or with a generead dnaseq custom panel v2 ( qiagen , santa clarita , ca ) consisting of 205 amplicons.31 in patients screened within the nowel network ( a33 to a36 ) , sequencing was performed using the neoplus hybrid - capturebased approach ( neo new oncology , cologne , germany )  . 
in the post - treatment samples ( cohort c ) of b41 to b56 , met and erbb2 status was assessed by uorescence in situ hybridization or chromogen in situ hybridization only if cnvs were detected by mps . small - cell lung cancer transformation was assessed using microscopy by experienced pathologists . 
progression - free survival ( pfs ) indicated the time from treatment start until pd or death . overall survival ( os ) was dened as the time from rst diagnosis until death . 
differences in time - to - event distribution were evaluated by the log - rank test , and statistical association between any two categorical variables was assessed by fishers exact test ; 95% cis for proportions were calculated using the clopper - pearson ( binominal ) formula . 
map of genetic aberrations detected by sequencing ( single nucleotide variant [ snv ] and insertion / deletion [ indel ] ) and copy number variation ( cnv ) analyses in biopsy specimens of epidermal growth factor receptor ( egfr ) p.t790m - positive patients before treatment with a third - generation egfr tyrosine kinase inhibitor ( tki ; ie , osimertinib , rociletinib , nazartinib ; upper block ; cohort b ; n = 56 ) and at progression to the specic treatment ( lower block ; cohort c ; n = 29 )  . 
the change in the frequency of specic aberrations during the course of treatment in matched samples is indicated in the lower block on the far right ( matched )  . 
 ( a ) waterfall plot showing the best change in percent of the target lesions according to response evaluation criteria in solid tumors ( recist ) 1.1 per patient during treatment with a third - generation epidermal growth factor ceptor tyrosine ( egfr ) kinase inhibitor ( tki ; n = 56 ; cohort b )  . 
kaplan - meier graphs displaying ( b ) progressionfree survival and ( c ) overfor patients with egfr p.t790m - posinonsmall - cell lung tive cancer with ( nsclc ) and without mesenchymaltransition factor epithelial ( met ) amplication ( ampl ) , who received treatment with third - generation egfr tkis . both median overall survival and progression - free survival are dramatically reduced in the presence of met amplications . 
erbb2 , human epidermal growth factor receptor 2 gene ; wt , wild type . survival time ( months ) 144 168 192 72 96 120 time ( months ) no . 
at risk ampl ampl tumor stage , gender , smoking status , and the initial number of prior egfr tkis had no signicant impact on treatment outcomes ( table 1 )  . 
the patient received local radiotherapy and died approximately 1.5 months timeline showing the course of treatment of a 76 - year - old female patient initially diagnosed at stage ii . 
fu , follow - up ; pd , progressive disease . percentage of samples in which we detected acquired changes in the molecular pattern was 89% ( n = 23 )  . 
the third most common genetic changes in cohort c were acquisition of egfr p.c797s ( n = 3 of 29 ; 10% ) , of which two were in cis and one in trans position , and loss of p.t790m with acquisition of p.g724s ( n = 3 of 28 ; 11% )  . 
acquired mutations in braf ( p.v600e ) , tp53 ( p.e180 * ) , and pten ( p.s229 * ) were detected in one sample each ( 4% )  . 
 ( a ) map of genetic aberrations detected by sequencing ( single nucleotide variant [ snv ] and insertion / deletions [ indels ] ) and copy number variation ( cnv ) analyses in biopsy specimens collected after treatment with a third - generation epidermal growth factor receptor ( egfr ) tyrosine kinase inhibitor ( tki ; cohort c ; n = 29 )  . 
patients were clustered in four groups : ( i ) changes outside of egfr only , ( ii ) changes in egfr and outside of egfr , ( iii ) changes in egfr only , and ( iv ) no changes found . 
the change in the frequency of specic aberrations during the course of treatment in matched samples is indicated in the lower block on the far right ( matched )  . 
erbb2 amplications were all found in samples that also harbored amplications of met . we therefore clustered the patients in four groups : ( i ) changes outside of egfr only , ( ii ) changes in egfr and outside of egfr , ( iii ) changes in egfr only , and ( iv ) no changes found ( fig 5a )  . 
in patients treated with osimertinib , a larger fraction belonged to cluster iii than cluster i or ii ( n = 10 ; 47 . 6% for iii v n = 5 ; 23.8% for i and ii )  . 
in patients treated with rociletinib , this trend was inversed ( changes in egfr , n = 0 ; 0% v no changes found , n = 4 ; 100% )  . 
however , most of these reports were not statistically signicant , and similarly , os , rr , and pfs were only numerically reduced in patients with tp53 mutations in our study.32 - 38 patient numbers with aberrations in pten , pik3ca , and erbb2 were low , and the differences in treatment efcacy were not statistically signicant . 
erbb2 , human epidermal growth factor receptor 2 gene ; freq , frequencies ; met , mesenchymal - epithelial transition factor ; pr , partial response ; sclc , small - cell lung cancer ; sd , stable disease ; wt , wild type . 
 michels et al met amplication was the second most frequent event associated with ar to third - generation egfr inhibition , and similar frequencies have been described in the literature.19 , 23 the high prevalence of met amplication in ir and ar points out the crucial role of met in egfr inhibitor resistance . 
in contrast , no patient treated with rociletinib displayed changes in egfr , and other studies have conrmed the absence of secondary egfr mutations in patients with progression while taking rociletinib.19 , 43 it is conceivable that this effect may be caused by a lower selection pressure of rociletinib on cells with on - target aberrations . 
however , differences in pfs or os after pd were not signicant . in summary , our study rst shows that molecular heterogeneity of p.t790m - mutant lung cancer with ar to earlygeneration egfr tkis inuences efcacy of thirdgeneration inhibitors . 
our observations also show the need to integrate information on co - occurring alterations in the design of clinical trials , aiming at a more precise identication of patients who benet from combined targeted treatment . 
but mechanisms of resistance to rst - line osimertinib have not been well characterized , and it is conceivable that recurrent mechanisms of resistance to egfr inhibition such as met amplication , met activation , and egfr p.c797s may also play a major role in this setting . affiliations 1university hospital of cologne , cologne , germany 2netherlands cancer institute , amsterdam , the netherlands 3university hospital of w urzburg and comprehensive cancer center mainfranken , w urzburg , germany 4vall d ' hebron university hospital , barcelona , spain 5university hospital of frankfurt , frankfurt , germany 6pius hospital oldenburg and lung cancer network nowel , oldenburg , germany 7university of cologne , cologne , germany 8cantonal hospital lucerne , lucerne , switzerland 9lung clinic merheim and hospitals of cologne , cologne , germany 10university of w urzburg and comprehensive cancer center mainfranken , w urzburg , germany 11robert bosch centrum f ur tumorerkrankungen , stuttgart , germany 12private practice in hematology and oncology , troisdorf , germany 13university hospital of t ubingen . 
smit , reinhard b uttner , juergen wolf data analysis and interpretation : sebastian michels , carina heydt , bianca van veggel , nuria pardo , kim monkhorst , frank griesinger , susanne steinhauser , michaela a . 
ihle , alex martinez - marti , lucia nogova , thorsten persigehl , dennis plenker , michael puesken , andreas rosenwald , werner spengler , martin sebastian , martin hellmich , enriqueta felip , sabine merkelbach - bruse , egbert f . 
de langen consulting or advisory role : astrazeneca ( inst ) , bristol - myers squibb ( inst ) , msd oncology ( inst ) , roche ( inst ) , boehringer ingelheim ( inst ) , pzer ( inst ) research funding : astrazeneca ( inst ) , bristol - myers squibb ( inst ) , merck serono ( inst ) , msd oncology ( inst ) , roche ( inst ) alex martinez - marti honoraria : roche , bristol - myers squibb , merck sharp & dohme , pzer , boehringer ingelheim consulting or advisory role : bristol - myers squibb , f . 
scheel honoraria : msd , bristol - myers squibb , roche , dako / agilent technologies consulting or advisory role : msd , bristol - myers squibb , roche , dako / agilent technologies matthias schefer honoraria : healthcare consulting cologne , boehringer ingelheim , takeda consulting or advisory role : boehringer ingelheim , takeda travel , accommodations , expenses : boehringer ingelheim ruth seggewiss - bernhardt honoraria : novartis , celgene , roche , bristol - myers squibb , ipsen , pzer , astrazeneca consulting or advisory role : msd , pzer travel , accommodations , expenses : astellas pharma , celgene , ipsen martin sebastian honoraria : astrazeneca , novartis , pzer / emd serono , msd , takeda , bristol - myers squibb , eli lilly , genentech , boehringer ingelheim , abbvie consulting or advisory role : genentech , msd , astrazeneca , abbvie , takeda , eli lilly , boehringer ingelheim , novartis , bristol - myers squibb , pzer , celgene travel , accommodations , expenses : pzer , takeda martin l . 
heukamp employment : neo new oncology , h amatopathologie hamburg honoraria : roche pharma , astrazeneca , bristol - myers squibb , boehringer ingelheim consulting or advisory role : roche pharma , bristol - myers squibb , novartis enriqueta felip consulting or advisory role : pzer , roche , boehringer ingelheim , astrazeneca , bristol - myers squibb , celgene , guardant health , novartis , takeda , abbvie , blueprint medicines , eli lilly , merck kgaa , merck sharp & dohme speakers ' bureau : astrazeneca , bristol - myers squibb , novartis , boehringer ingelheim , merck sharp & dohme , roche , pzer , abbvie , eli lilly , merck kgaa , takeda research funding : fundaci on merck salud ( inst ) , emd serono ( inst ) sabine merkelbach - bruse honoraria : astrazeneca , bristol - myers squibb , novartis , pzer , roche pharma consulting or advisory role : bristol - myers squibb , novartis , pzer egbert f . 
han jy , park k , kim sw , et al : first - signal : first - line single - agent iressa versus gemcitabine and cisplatin trial in never - smokers with adenocarcinoma of the lung . 
j clin oncol 30 : 1122 - 1128 , 2012 rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as rst - line treatment for european patients with advanced egfr mutationpositive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
lancet oncol 13 : 239 - 246 , 2012 zhou c , wu yl , chen g , et al : erlotinib versus chemotherapy as rst - line treatment for patients with advanced egfr mutation - positive non - small - cell lung cancer ( optimal , ctong - 0802 ) : a multicentre , open - label , randomised , phase 3 study . 
lancet oncol 12 : 735 - 742 , 2011 sequist lv , yang jch , yamamoto n , et al : phase iii study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with egfr mutations . 
j clin oncol 31 : 3327 - 3334 , 2013 sequist lv , waltman ba , dias - santagata d , et al : genotypic and histological evolution of lung cancers acquiring resistance to egfr inhibitors . 
sci transl med 3 : 75ra26 , 2011 yu ha , arcila me , rekhtman n , et al : analysis of tumor specimens at the time of acquired resistance to egfr - tki therapy in 155 patients with egfr - mutant lung cancers . 
kasibhatla s , li j , tompkins c , et al : egf816 , a novel covalent inhibitor of mutant - selective epidermal growth factor receptor , overcomes t790m - mediated resistance in nsclc . 
cancer res 74 , 2014 ( suppl ; abstr 1733 ) j anne pa , yang jch , kim dw , et al : azd9291 in egfr inhibitor - resistant non - small - cell lung cancer . 
tan dsw , seto t , leighl nb , et al : first - in - human phase i study of egf816 , a third - generation , mutant - selective egfr tyrosine kinase inhibitor , in advanced non - small cell lung cancer ( nsclc ) harboring t790m . 
bean j , brennan c , shih jy , et al : met amplication occurs with or without t790m mutations in egfr mutant lung tumors with acquired resistance to getinib or erlotinib . 
schefer m , merkelbach - bruse s , bos m , et al : spatial tumor heterogeneity in lung cancer with acquired epidermal growth factor receptor - tyrosine kinase inhibitor resistance : targeting high - level met - amplication and egfr t790m mutation occurring at different sites in the same patient . 
blakely cm , watkins tbk , wu w , et al : evolution and clinical impact of co - occurring genetic alterations in advanced - stage egfr - mutant lung cancers . 
yang z , yang n , ou q , et al : investigating novel resistance mechanisms to third - generation egfr tyrosine kinase inhibitor osimertinib in non - small cell lung 26 . 
eberlein ca , stetson d , markovets aa , et al : acquired resistance to mutant - selective egfr inhibitor azd9291 is associated with increased dependence on ras cancer patients . 
k onig k , peifer m , fassunke j , et al : implementation of amplicon parallel sequencing leads to improvement of diagnosis and therapy of lung cancer patients . j thorac oncol 10 : 1049 - 1057 , 2015 32 . 
molina - vila ma , bertran - alamillo j , gasc o a , et al : nondisruptive p53 mutations are associated with shorter survival in patients with advanced non - small cell lung cancer . 
aisner dl , sholl lm , berry l , et al : the impact of smoking and tp53 mutations in lung adenocarcinoma patients with targetable mutations - the lung cancer mutation consortium ( lcmc2 )  . 
labb e c , cabanero m , korpanty gj , et al : prognostic and predictive effects of tp53 co - mutation in patients with egfr - mutated non - small cell lung cancer 36 . 
vanderlaan pa , rangachari d , mockus sm , et al : mutations in tp53 , pik3ca , pten and other genes in egfr mutated lung cancers : correlation with clinical ( nsclc )  . 
shepherd fa , lacas b , le teuff g , et al : pooled analysis of the prognostic and predictive effects of tp53 comutation status combined with kras or egfr mutation in early - stage resected non - small - cell lung cancer in four trials of adjuvant chemotherapy . 
canale m , petracci e , delmonte a , et al : impact of tp53 mutations on outcome in egfr - mutated patients treated with rst - line tyrosine kinase inhibitors . 
piotrowska z , niederst mj , karlovich ca , et al : heterogeneity underlies the emergence of egfrt790 wild - type clones following treatment of t790m - positive cancers with a third - generation egfr inhibitor . 
thirty - one patients ( 38% ) and 17 patients ( 46% ) underwent germline testing from the automatic pipeline and other referrals , respectively , and of these patients , 23 ( 72% ) and four ( 24% ) had conrmed germline pathogenic variants ( gpvs ) , respectively . 
the majority of conrmed gpvs were in automatic referral genes , with brca2 being most common ( conrmed gpvs in 11 [ 85% ] of 13 patients tested ) , followed by palb2 ( ve [ 67% ] of six patients ) , brca1 ( two [ 40% ] of ve patients ) , msh6 ( two of three patients ) , and mlh1 ( two of two patients )  . 
germline testing was not performed in 50 ( 62% ) of 81 patients identied by automatic referral as a result of poor patient health or death ( 30% ) , lack of follow - up ( 30% ) , and patient refusal ( 30% )  . conclusion of patients undergoing tgp , 5% had somatic ndings triggering referral , and implementation of an automatic referral pipeline based solely on gene versus other clinical or molecular features resulted in a 74% germline conrmation . 
use of such testing has rapidly increased , with further growth anticipated.1 mutations identied on tumor sequencing may be acquired somatically , caused by postzygotic mosaicism , or identication of inherited through the germline . germline mutations potentially inuences treatment of the current cancer , may provide prognostic information about the potential development of future cancers , and has important implications for family members in terms of risk and prevention of disease.2 however , guidelines on both how to determine which somatic ndings may be potentially germline and the optimal clinical practice for referral and germline testing of patients undergoing tumor sequencing are not widely available , and there is no established standard of care.3 genomic testing of tumor tissue can be performed in tandem with germline testing ( via blood or sequence matched normal tissue ) 4 - 6 ; such tumor - germline paired sequencing allows determination of mutation etiology ( inherited v acquired )  . 
 clark et al context key objective as the use of somatic tumor genomic testing rapidly increases , a pipeline for referral of potential germline genetic ndings is critically needed . knowledge generated more than 70% of known cancer predisposition gene mutations identied in patients tumor genomic testing were conrmed to be germline . 
however , less than half of eligible patients underwent germline genetic testing as a result of poor patient health or death , lack of follow - up , and / or patient refusal . relevance our ndings indicate that patients with pathogenic mutations in well - characterized cancer predisposition genes should be referred for consideration of germline genetic testing , although signicant implementation challenges remain . be associated with hereditary cancer syndromes for both the patients and their family members . 
however , potential germline variants on tgp may be under - recognized.8 national comprehensive cancer network ( nccn ) guidelines are evolving ; they currently state that pathogenic variants in cancer susceptibility genes identied on tumor proling of any tumor type should undergo germline conrmation.9 however , the logistics of identifying potentially actionable germline mutations , such as those in brca1 and brca2 , from tgp can be complex , in large part as a result of the coordination of germline genetic testing for ill patients who may be unaware that their tumor testing could reveal inherited mutations . 
somatic pathogenic mutations as dened in cbioportal were downloaded from the provisional data sets of multiple tumor types in july 2015 ( data supplement ) and updated in july 2018 ( table 1 )  . 
original germline mutation rates were averaged from two large studies of patients with metastatic cancer5 , 6 and updated with tcga data , 11 and mutations were dened as reported in the original studies.5 , 6 , 11 the ratio of the germline mutation rate to total mutation rate was determined ( table 1 ; appendix fig a1 )  . 
genes were also annotated with the number of tumor types with somatic mutation rates greater than 10% ( data supplement )  . delineation of criteria for automatic referral for germline testing criteria for automatic referral for germline testing after tgp from june 2016 through june 2018 , the center for personalized diagnostics ( cpd ; college of american pathologists and clinical laboratory improvement amendments approved ) at penn medicine evaluated 2 , 308 unique tgp tests with either a 153 - gene panel ( n = 2 , 087 ) or a 47 - gene panel ( n = 221 ) , depending on the version of the panel available ( data supplement )  . 
a multidisciplinary team , including genetic counselors , medical oncologists , medical geneticists , and molecular pathologists , was established to determine which somatic results warranted automatic referral for germline genetic testing . 
 clark et al was not included in our analysis because of controversy over screening guidelines and its relatively high prevalence.12 implementation of germline testing referral pipeline solid tumor sequencing reports were signed out per clinical practice standards , and variants in the automatic referral pipeline were identied manually by the reviewing cpd attendings . 
language for clinical reports was created stating that additional testing would be necessary to determine whether the identied pathogenic variant on somatic testing was a germline pathogenic variant ( gpv )  . 
an e - mail was sent from the cpd to the ordering oncologist and a designated genetic counselor ( gc ) when a variant meeting automatic referral criteria was identied . 
initial disclosure of the somatic test results , including the possibility of a germline nding , was the responsibility of the ordering provider.13 after patients were made aware of somatic ndings by their oncologist , patients were contacted by the gc for coordination of care . 
molecular pathologists and oncologists also were encouraged to refer patients outside the automatic pipeline on the basis of genotype - phenotype correlation . data collection , chart review , and statistical analysis a university of pennsylvania institutional review board approved retrospective chart review was conducted , with data collected for all referrals within the automatic pipeline and other referrals based on tgp by the academic laboratory between june 2016 and june 2018 ( 81 referrals by automatic pipeline and 37 referrals outside the pipeline )  . data were collected on patient demographics , tumor type tested ( data supplement ) , and somatic and germline testing results . 
continuous variables were compared using the t test , and contingency tables were analyzed using fishers exact test . results referral characteristics from june 2016 to june 2018 , 2 , 308 patients underwent tgp at penn medicines cpd . 
of those patients , 118 ( 5.1% ) were referred for clinical germline genetic testing based on tgp performed at the cpd . referral of 81 patients was triggered by the automatic algorithm , and 37 patients were referred from either the molecular pathologist ( n = 21 ) or the patients physician ( n = 16 ; fig 2a )  . 
the majority of patients were white ( 88% ) and female ( 52% ) , and the mean age at tgp was tumor types 58 years ( table 2 )  . 
thirteen patients were referred for variants with a high likelihood in chek2 of being germline ( founder mutations ) ( n = 11 ) and one each for variants in atm and egfr ( fig 2a ; data supplement )  . 
direct referrals for germline genetic testing by the molecular pathologist and primary oncologists after a variant was found on tgp were largely based on genotype - phenotype concerns ( eg , tp53 mutations in patients with cancer age 45 years or younger or patients with brain tumors or sarcomas ; cdkn2a / cdk4 mutations in patients with melanoma )  . 
referrals of 21 patients by the molecular pathologist were initiated by e - mail to the gc and primary oncologist and included ndings in nine genes , most commonly tp53 ( fig 2a ; data supplement )  . treating physicians referred 16 patients with variants in 12 different genes ( fig 2a , data supplement )  . results of germline conrmation of 118 patients with referrals , 48 ( 41% ) underwent clinical genetic counseling and genetic testing ( fig 2a )  . 
genetic testing rates were similar for referrals from the automatic referral pipeline and those sent by the molecular pathologist ( 38% and 33% , respectively ) , whereas 63% of patients referred by their treating physician underwent genetic testing ( fig 2a )  . 
a majority of patients ( 27 [ 57% ] of 48 patients ) had variants that were conrmed gpvs , with 74% of automatic referrals being conrmed gpvs and 29% and 20% of molecular pathologist and treating physician referrals being conrmed gpvs ( fig 2a )  . 
the majority of patients ( 60% ) had multiplex panel testing ; however , 19% underwent a small custom panel , 17% were only tested for the variant by single - site testing , and 4% had ashkenazi jewish founder testing ( data supplement )  . the 81 patients referred by the automatic referral pipeline , the majority ( n = 59 ) were referred for variants in high - risk genes ( 73% ; fig 2b )  . 
genetic testing rates were highest for high - risk genes ( 28 referrals [ 47% ] underwent genetic testing compared with three referrals [ 14% ] in the other groups )  . 
mutations in the automatic referral pipeline were agged by the tumor testing laboratory , and an e - mail was sent from molecular pathologist to the ordering physician ( md ) and genetic counselor ( gc )  . 
results not included in the automatic referral pipeline but of concern to the molecular pathologist or ordering oncologist to be possibly germline were also e - mailed to the gc . 
the gc called the patient only after the somatic results were discussed with the patient by the ordering md , the patient agreed to be contacted , and the oncologist contacted the gc . 
an inperson cancer risk evaluation appointment was scheduled , and the appropriate germline testing modality was determined by gc at time of visit ( panel or single - site testing )  . 
results were provided by telephone by the gc , and patients with positive testing results along with their relatives were offered in - person follow - up visits for medical management . 
one hundred eighteen patients were referred for germline testing83 by the automatic pipeline , 20 by the testing laboratory at the discretion of the molecular pathologist , and 15 directly from the ordering physician . 
 ( b ) of the 83 patients referred by the automatic pipeline , 61 had mutations in high - risk genes ( brca1 , brca2 , palb2 , mlh1 , msh2 , and msh6 ) , nine had mutations in moderate - risk genes ( brip1 , rad51c , and rad51d ) , and 13 had mutations with high prior probability of being germline ( likely germline )  . 
 ( c ) in the 51 patients referred by the automatic pipeline but who did not undergo testing , patients were not seen as a result of death before scheduling an appointment , refusal of the consult by the patient , absence of referral placed by ordering physician , or other reason . or both ( fig 2c )  . 
 characteristic age , years mean range male race tumor type white black other lung brain breast other melanoma 526 ( 23 ) 676 ( 29 ) 519 ( 22 ) 125 ( 5 ) 59 ( 3 ) 25 ( 1 ) 122 ( 5 ) germline results in tumor - only genomic testing table 2 . 
 ( a ) in patients who presented for conrmatory germline testing , the age at diagnosis for patients with a conrmed germline pathogenic variant ( gpv ) versus somatic pathogenic variants ( spvs ) not conrmed to be in the germline . 
 ( c ) percentage of patients with a conrmed gpv versus an spv for whom the gene is known to be associated with the tumor where the variant was found . 
of 2 , 308 patients who underwent tgp at penn medicines cpd , 149 ( 6.6% ) had variants in the automatic referral genes , and 118 ( 5% ) were referred for clinical germline genetic testing . 
a majority of the variants tested were conrmed gpvs ( 27 [ 54% ] of 50 variants ) , with the highest germline conrmation rate in high - risk genes ( 21 [ 72% ] of 29 genes )  . 
thus , we have demonstrated that an automatic referral algorithm results in a high yield of conrmed germline mutations . in total , 27 ( 1.1% ) of 2 , 308 patients undergoing tgp had a conrmed gpv . 
germline ndings have been found by retrospective analysis in 4% to 12% of prior clinical tumor sequencing cohorts5 , 6 , 14 and in 8% of the tcga cohort , 11 with 2.8% of the tcga cohort having germline mutations in the genes selected for automatic referral in this study . 
if all 149 patients with mutations in automatic referral genes in our cohort had undergone germline testing , we would have expected 107 to have a germline mutation ( based on our 72% germline conrmation rate )  . 
a limitation of this study is that sequencing was not performed on all patients with metastatic disease at the university of pennsylvania ; in addition , germline sequencing was not performed in all dividuals with somatic sequencing . 
thus , we do not know the exact prevalence of germline mutations in our population . genes were chosen for automatic referral based on several factors , but the major determinant was the ratio of germline mutation rate to total ( germline plus somatic ) mutation rate calculated from public databases . 
thus , referring all individuals with mutations identied in such genes ( eg , tp53 and pten ) identied by tgp in the absence of other factors rarely results in a positive germline nding . 
however , other genes have higher mutation rates in the germline compared with somatic only ( eg , brca1 / 2 ) , and known founder mutations would likely always be germline . 
for example , in our study , all three ashkenazi jewish founder mutations in brca1 / 2 that were found on tgp were conrmed gpvs . the automatic referral algorithm did not rely on clinical characteristics or variant af or exclude patients with likely hypermutated tumors . 
the nding that germline af can be lower than the expected range of 50% or greater is consistent with prior data.15 multiple patients had germline mutations identied despite somatic testing done on tumors not generally considered part of the hereditary syndrome . 
the high germline conrmation rate for the automatic referral genes validates our approach of prioritizing the ratio of germline to total mutations of a gene , with lesser emphasis placed on af or tumor type . 
we have updated both germline and somatic mutation rates with current11 data ( table 1 ) , demonstrating that most of these genes have a ratio of germline mutation rate to total mutation rate of greater than 0.25. 
the only genes not included on our original list that t these criteria are atm and chek2 . we are adding all mutations in these genes to our referral list ( rather than restricting to certain mutations with high prior probability of being germline )  . other approaches for gene selection exist ; however , we wished to prioritize the highest impact genes related to prognosis or therapy , clinical trial enrollment ( eg , brca1 / 2 mutations and poly [ adp - ribose ] polymerase inhibitors ) , or effect on family members.16 - 19 in addition , we wished to assess the success of this approach and potential burdens before widening the scope . 
curation of the list of genes in which mutations prompt referral for germline genetic testing will be an ongoing process and will need to reect updates in guidelines from the american college of medical genetics and nccn . 
the automated referral algorithm had a high specicity , as 72% of ndings were conrmed gpvs . in patients for whom somatic sequencing triggered a referral by their oncologist without meeting criteria on the algorithm , only two patients ( 20% ) had conrmed gpvs ( atm and chek2 )  . 
as mentioned earlier , both genes are being added to our automated referral algorithm . criteria for germline genetic testing are rapidly evolving , and routine germline multiplex panel testing may soon become standard for all patients with metastatic disease . 
in our study , of 28 patients who underwent multiplex panels , either testing was negative ( n = 13 ) or only the somatic variant was found ( n = 15 ) , indicating that there may be limited incremental benet to expanded germline testing in patients with metastatic cancer who have undergone tumor testing . 
however , individuals with a high likelihood of having a germline mutation in a cancer susceptibility gene should be considered for germline testing even in the setting of a negative tgp . 
for example , in individuals with a strong family history of breast and ovarian cancer , approximately 10% of brca1 mutations are large genomic rearrangement , which may be more difcult to detect with current tgp assays . the majority of patients ( 61% ) with a somatic nding identied by the automatic referral pipeline were not seen by clinical cancer genetics services . 
published data suggest that patients express great interest in wishing to learn their germline status20 ; therefore , it is likely that patient and provider education and improved infrastructure for referrals are needed . 
 germline results in tumor - only genomic testing cancer therapy and symptom management , simplifying the process by which patients become aware of the possibility of germline ndings arising from tgp and have access to germline conrmation is critical . 
the ongoing communication and education in tumor proling ( comet ) trial ( clinicaltrials.gov identier : nct02823652 ) , a companion study to the national cancer institute molecular analysis for therapy choice ( nci - match ) trial , will provide additional information regarding best practices . genetic counseling challenges in this population of patients with advanced cancer included the substantial burden of patient illness ( including both symptom and appointment burdens , although considerable efforts were made to coordinate appointments with other visits )  . 
there was an appreciable time commitment by the gc that was not billable ( eg , discussions by phone ) , and a number of patients tested did not have insurance coverage because their tumor type did not meet published criteria for germline testing . 
nccn criteria for further genetic risk evaluation ( version 2.2019 ) now include individuals at any age with a known pathogenic variant or likely pathogenic variant in a cancer susceptibility gene found on tumor testing ; however , during the dates of our study , existing criteria included brca1 / 2 only.9 we performed a single - institution study and only evaluated ndings from penn medicines cpd ; thus , generalizability of this approach is not yet known . 
in addition , full somatic sequencing for all of the automatic referral genes was not performed on every patient as a result of availability of specic somatic sequencing panels at different points in time . 
we did not implement approaches such as telegenetics , which may further reduce patient burden ( although germline samples must still be obtained )  . we have demonstrated that an automated referral process for patients with mutations detected in tgp can lead to the identication of germline mutations in cancer susceptibility genes with implications for both patients and their family members . 
 clark et al jacquelyn powers employment : carevive systems honoraria : cureconnect consulting or advisory role : carevive systems travel , accommodations , expenses : hospital of the university of pennsylvania jessica m . 
long employment : depuy companies ( i ) stock and other ownership interests : depuy companies ( i ) travel , accommodations , expenses : depuy companies ( i ) payal shah stock and other ownership interests : johnson & johnson ( i ) , novartis ( i ) , novo nordisk ( i ) , pzer ( i ) , merck ( i ) , amgen , johnson & johnson , novo nordisk consulting or advisory role : tmunity therapeutics jennifer j.d. 
domchek honoraria : astrazeneca , clovis oncology , bristol - myers squibb research funding : astrazeneca ( inst ) , clovis oncology ( inst ) , pharmamar ( inst ) no other potential conicts of interest were reported . references raymond vm , gray sw , roychowdhury s , et al : germline ndings in tumor - only sequencing : points to consider for clinicians and laboratories . 
j pathol 244 : 610 - 615 , 2018 arango np , brusco l , mills shaw kr , et al : a feasibility study of returning clinically actionable somatic genomic alterations identied in a research laboratory . oncotarget 8 : 41806 - 41814 , 2017 4 . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
ann oncol 27 : 795 - 800 , 2016 schrader ka , cheng dt , joseph v , et al : germline variants in targeted tumor sequencing using matched normal dna . 
jama oncol 2 : 104 - 111 , 2016 robson me , bradbury ar , arun b , et al : american society of clinical oncology policy statement update : genetic and genomic testing for cancer susceptibility . j clin oncol 33 : 3660 - 3667 , 2015 catenacci dv , amico al , nielsen sm , et al : tumor genome analysis includes germline genome : are we ready for surprises ? int j cancer 136 : 1559 - 1567 , 2015 9 . 
yurgelun mb , chittenden ab , morales - oyarvide v , et al : germline cancer susceptibility gene variants , somatic second hits , and survival outcomes in patients with resected pancreatic cancer . 
bolton kl , chenevix - trench g , goh c , et al : association between brca1 and brca2 mutations and survival in women with invasive epithelial ovarian cancer . 2014 res 22 : 4087 - 4094 , 2016 jama 307 : 382 - 390 , 2012 33 : 244 - 250 , 2015 18 . 
hamilton jg , shuk e , garzon mg , et al : decision - making preferences about secondary germline ndings that arise from tumor genomic proling among patients with advanced cancers . 
 germline results in tumor - only genomic testing appendix brca1 brca2 palb2 mlh1 msh2 msh6 brip1 rad51c rad51d chek2 tp53 bap1 cdh1 cdkn2a men1 pten smad4 smarca4 stk11 tsc1 tsc2 original updated ratio of germline mutation rate to overall mutation rate fig a1 . 
 o prevalence of homologous recombinationrelated gene mutations across multiple cancer types purpose the prevalence of homologous recombination dna damage repair ( hr - ddr ) deficiencies among all tumor lineages is not well characterized . 
therapy directed toward homologous recombination ddr deficiency ( hrd ) is now approved in ovarian and breast cancer , and there may be additional opportunities for benefit for patients with other cancers . 
comprehensive evaluations for hrd are limited in part by the lack of a uniform , cost - effective method for testing and defining hrd . methods molecular profiles of 52 , 426 tumors were reviewed to identify pathogenic mutations in the hr - ddr genes arid1a , atm , atrx , bap1 , bard1 , blm , brca1 / 2 , brip1 , chek1 / 2 , fanca / c / d2 / e / f / g / l , mre11a , nbn , palb2 , rad50 , rad51 , rad51b , or wrn . 
 a total of 17 , 566 tumors were sequenced with ngs600 ( n = 592 genes ) , and 34 , 860 tumors underwent hotspot illumina miseq platform testing ( n = 47 genes )  . results of the tumors that underwent ngs600 testing , the overall frequency of hrddr mutations detected was 17.4% , and the most commonly mutated lineages were endometrial ( 34.4% ; n = 1 , 475 ) , biliary tract ( 28.9% ; n = 343 ) , bladder ( 23.9% ; n = 201 ) , hepatocellular ( 20.9% ; n = 115 ) , gastroesophageal ( 20.8% ; n = 619 ) , and ovarian ( 20.0% ; n = 2 , 489 )  . 
germline mutations in one or both of these genes place patients at heightened risk for development of breast , 1 - 6 ovarian , 1 - 6 prostate , 7 - 9 melanoma , 7 , 10 and pancreatic cancers7 , 10 - 12 during their lifetime . 
it has become apparent that brca interacts with a number of other dna repair proteins to form a complex system for ddr , including atm , rad51 , palb2 , mre11 , rad50 , nbn , and the fanconi anemia proteins.13 , 14 recent evidence suggests mutations in palb2 , atm , and the genes responsible for the mrn complex , rad50 , mre11 , and nbn , play a role in hereditary cancers.15 , 16 for example , palb2 mutation carriers have a lifetime risk of breast cancer development of approximately 50% , 17 , 18 and atm mutation carriers are at higher risk for development of breast , 19 , 20 pancreatic , 21 , 22 and prostate cancers.23 , 24 arielle l . 
in germline brca1 / 2 mutation carriers , exposure to platinum chemotherapy led to improved objective response rates in advanced triple - negative breast cancer versus taxanes ( 68% v 33% ) , 25 and overall survival in pancreatic cancer versus other nonplatinum chemotherapy ( 22 months v 9 months ) .26 mychoice hr - ddr deficiency ( hrd ) score - high triple - negative breast cancer responded better to platinum - based neoadjuvant therapy , with pathologic complete response ( cr ) rates of 27.5% versus 0% in the hr - ddrproficient cohort.27 the mychoice hrd score is frequently used to identify patients with hrd . 
it is a proprietary diagnostic test to assess a hrd phenotype , including an evaluation of loss of heterozygosity , telomeric allelic imbalance , and large - scale state transitions . on exposure to another class of dna - damaging agents , poly - adp ribose polymerase ( parp ) inhibitors , patients with germline or somatic deleterious mutations in the hr - ddr pathway have also achieved favorable responses . 
in this study , three patients had germline brca2 mutations , three patients had germline atm mutations , and the remaining responders had tumor expression of a deleterious mutation ( including palb2 , brca2 , brca1 , chek2 , fanca , and atm ) .35 all germline mutation carriers except for one patient with atm mutation responded to therapy . despite the exciting therapeutic potential of dna - damaging agents in patients with broader evidence of hrd , the prevalence of hrd among all tumors is largely unknown . 
the aim of our study was to determine the prevalence of hr - ddr pathogenic or presumed pathogenic mutations detected on tumor next - generation sequencing ( ngs ) testing across multiple cancer lineages , using commercially available dna sequencing ( ngs or sanger sequencing panel testing , multiplatform profiling ; caris life sciences [ caris ] , irving , tx ) to better define the proportion of patients who may benefit from such therapy . methods study design approval for this study was obtained from the georgetown university institutional review board . 
we defined hrd on tumor ngs testing as a mutation in the following genes , each of which has some activity within the hr - ddr pathway36 - 45 and has been included previously in hr - ddr biomarker clinical trials : arid1a , atm , atrx , bap1 , bard1 , blm , brca1 / 2 , brip1 , chek1 / 2 , fanca / c / d2 / e / f / g / l , mre11a , nbn , palb2 , rad50 , rad51 , rad51b , or wrn . 
frequencies of each mutation were determined for the total cohort , as well as for each cancer lineage ( biliary tract , bladder , breast , cervix , colorectal [ crc ] , endometrial , gastro esophageal [ ge ] , gastrointestinal stromal [ gist ] , glioma , head and neck , hepatocellular [ hcc ] , melanoma , neuroendocrine / small cell lung , nonsmall - cell lung [ nsclc ] , ovarian , pancreas , prostate , renal , sarcoma , thyroid , and unknown primary )  . ngs testing platforms hr - ddr mutation analysis from solid tumor biopsy specimens was determined by ngs at caris , a clinical laboratory improvement amendmentscertified laboratory . 
while 17 , 566 tumors were sequenced with ngs600 , 34 , 860 tumors underwent hotspot illumina miseq platform testing ( illumina truseq amplicon cancer hotspot panel , evaluating 47 genes including the hr - ddr genes atm , brca1 , and brca2 ; illumina , san diego , ca )  . 
tumor enrichment was achieved by harvesting targeted tissue by manual microdissection performed on all cases before molecular testing . the pathogenicity of gene variants identified were interpreted by board - certified molecular geneticists and categorized as pathogenic , presumed pathogenic , variant of unknown significance , presumed benign , or benign , according to american college of medical genetics and genomics standards on the basis of the level of evidence of published studies on the identified variants.46 , 47 only pathogenic or presumed pathogenic mutations were considered deleterious ; variants of unknown significance and variants that have not been previously reported in individuals affected by cancer in the literature were excluded . 
 deleterious mutations reported included frameshift mutations , premature stop codons , mutations shown to disrupt natural splicing , as well as point mutations ; deleterious mutations included those that have and have not been reported as causal for hereditary cancers . statistical analysis the proportion of pathogenic or presumed pathogenic mutations identified from all tumor specimens tested for each specific mutation were calculated for the total cohort and for each cancer lineage investigated . 
the total frequency of hr - ddr mutations in the complete cohort and per cancer lineage was calculated by dividing the number of tumors carrying at least one mutation by the total number of tumors tested , to avoid counting tumors carrying more than one hr - ddr mutation multiple times . 
the 95% cis were computed using the pearson - klopper exact method using r ( org / ) , and the graphics were generated by spss statistics , version 24 ( ibm , armonk , ny )  . results we evaluated 52 , 426 solid tumor pathologic specimens that underwent extended ngs for hrd . 
molecular profiling was performed on the primary tumor in 47.9% of cases ( n = 25 , 102 ) , and on a metastatic site of disease in 41.9% ( n = 21 , 956 )  . 
 within the tumor lineages , the frequencies of mutations varied ( tables 2 and 3 ; fig 2 )  . hr gene mutation frequency overall , pathogenic mutations within the homologous recombination pathway were seen in 17.4% of the 17 , 566 tumors tested with ngs600 , and 8.3% of the 52 , 426 solid tumors overall ( including 34 , 860 tumors that were evaluated for atm , brca1 , and brca2 mutations only on the hotspot panel )  . 
 brca1 / 2 mutations were seen predominately in ovarian and breast cancers , though pathogenic brca2 mutations were seen in high frequencies among gi and nonovarian genitourinary malignancies , as well . 
although palb2 mutations were less common overall and appreciated in only 0.6% of tumors tested , a significant proportion of palb2 mutations was found in bladder , breast , and gi malignancies . 
no pathogenic mutations were identified in bard1 , chek1 , fanca / d2 / e / f / g / l , rad51 , or rad51b ( table 4 )  . hr - ddr gene mutations were appreciated in both primary and metastatic lesions , though with different patterns . 
the remaining mutated hr - ddr genes ( blm , brip1 , chek2 , fancc , mre11a , nbn , palb2 , rad50 , and wrn ) had similar frequencies among primary and metastatic tissue evaluations . tumors with multiple hr gene mutations of the 17 , 566 tumors that underwent extended molecular profiling with the ngs600 platform , 362 were found to carry more than one hr - ddr pathway mutation , including 112 endometrial ( 7.6% ; n = 1 , 475 ) , 75 crc ( 3.1% ; n = 2 , 454 ) , 34 ovarian ( 1.4% ; n = 2 , 489 ) , 29 nsclc ( 0.9% ; n = 3 , 245 ) , 23 ge ( 3.7% ; n = 619 ) , 20 breast ( 1.2% ; n = 1 , 625 ) , and 15 biliary tract ( 4.4% ; n = 343 ) tumors . 
total bar height represents the overall frequency of hr - ddr deficient tumors within each lineage ; colored bar length represents the relative mutation frequency of individual genes in each cancer type . 
summary of homologous recombination dna damage repair mutations identified * overall , % ngs600 , % hotspot , % mutation arid1a brca2 brca1 atrx chek2 bap1 palb2 brip1 fancc rad50 mre11a abbreviation : n / a , not applicable . * no mutations identified : bard1 , chek1 , fanca / d2 / e / f / g / l , rad51 , rad51b . in this large - scale study of ngs molecular profiling of solid tumor samples across multiple cancer lineages , we confirmed hrd is common and was observed in 17.4% of the solid tumors evaluated by the ngs600 platform ( spanning 21 cancer lineages )  . 
in a recent evaluation of brca1 / 2 mutational patterns , for example , a model to detect brca1 / 2 deficiency failed to correlate brca1 / 2 mutational patterns with pathogenic mutations in several additional hr - ddr pathway genes , including atm , atr , chek2 , the fanc group of genes , palb2 , pten rad50 , and rad51c.48 the unique genetic signatures across the breadth of hr - ddr pathway genes are not well characterized , and , as such , it is unknown if the lack of a brca - like mutational pattern is associated with altered responses to parp inhibitors and dna - damaging chemotherapy . our reported frequencies of hrd are similar to previously published work , though a range of frequencies is appreciated ( table 5 )  . 
because there is not yet an established way to measure hrd , and previous studies have measured hrd by an hrd assay assessing large - scale transition scores and telomere allelic imbalances , germline mutations , somatic mutations , brca - like genetic signatures , or a combination of these methods , the differences in hrd prevalence may be related to nonuniformity of assessment . 
in addition , significant variation will occur between whole - exome sequencing versus use of hotspot panels , with the identification of hrd more common among studies that used whole - exome sequencing . 
within our study , 17 , 566 tumors ( 33.5% of the total 52 , 426 tumors tested ) underwent sequencing with the targeted whole - exome sequencing platform ngs600 , which evaluated tumors for 592 genes , assuming dna was sufficient . 
consequently , studies including an evaluation for pten mutations are expected to report higher frequencies of hrd . it is also possible frequencies of hrd may differ across studies as a result of differences in patient population . 
patients with tumors harboring hr - ddr pathway mutations may be more sensitive to dna - damaging chemotherapy and therefore less likely to recur after initial treatment of localized disease . 
although the clinical stage of patients included in our analysis at the time of tissue acquisition ( and if tissue was obtained at presentation ) was unknown , it is possible our population was enriched with patients with metastatic disease without hrd who were less likely to respond favorably to frontline treatment and , therefore , recurred . looking forward , classifying tumors as hr - ddr deficient will likely become increasingly important , as we now appreciate that hrd is common and has been associated with improved outcomes in some patients treated with dna - damaging therapies . 
several clinical trials are assessing in part the role of hrd in response to anticancer therapies and outcomes , including treatment with parp inhibitor therapy alone , but also in combinations with chemotherapy , radiation therapy , or immunotherapy to enhance antitumor efficacy . 
in tandem , it will be important to generate a consensus regarding uniform testing to identify appropriate patients for these tailored therapies . there are some limitations of this study to note . 
given the nature of the study , we evaluated patients tumor tissue for the presence of hr - ddr mutations and thus are unable to distinguish whether a given mutation was a somatic or germline mutation . 
we were also unable to assess tumors for epigenetic modifications such as dna methylation , which could also lead to significant and clinically relevant alterations in gene expression affecting functions of the hr pathway.57 , 58 in addition , hrd is defined by currently available literature regarding the suspected pathogenicity of each mutation . 
it is certainly possible that mutations labeled as a variant of unknown significance may prove important in the future , although the majority are reclassified as benign polymorphisms.59 - 61 we also recognize our results are influenced by the overrepresentation of certain cancer lineages , including ovarian ( n = 9 , 630 ) , nsclc ( n = 8 , 119 ) , crc ( n = 6 , 650 ) , breast ( n = 5 , 910 ) , and endometrial ( n = 5 , 540 ) cancers in the caris database . 
baker employment : caris life sciences , roche travel , accommodations , expenses : caris life sciences , roche honoraria : roche , amgen , bayer / onyx , taiho pharmaceutical , caris life sciences , celgene consulting or advisory role : roche , amgen , bayer / onyx , taiho pharmaceutical , caris life sciences , celgene speakers ' bureau : roche , amgen , bayer / onyx , celgene , taiho pharmaceutical research funding : bayer / onyx ( inst ) , roche ( inst ) , pfizer ( inst ) , amgen ( inst ) , boehringer ingelheim ( inst ) , medimmune ( inst ) claudine isaacs honoraria : roche , astrazeneca , pfizer , novartis , syndax , nanostring technologies consulting or advisory role : pfizer , roche , novartis , astrazeneca , medivation , nanostring technologies , syndax speakers ' bureau : genentech , pfizer , astrazeneca research funding : novartis ( inst ) , pfizer ( inst ) , genentech ( inst ) , tesaro patents , royalties , other intellectual property : uptodate , mcgraw hill publishing affiliations arielle l . 
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we show that kit inhibition markedly decreased cell viability in melanoma cell lines with distinct kit mutations ; however , this effect was countered in the presence of hepatocyte growth factor ( hgf ) , the ligand for met . 
a physical examination revealed a raised , erythematous , and well - circumscribed cutaneous lesion on her left great toe , which the patient attributed to trauma that occurred several months earlier . 
staging computed tomography ( ct ) and positron emission tomography scans revealed left inguinal and left external iliac lymphadenopathy ; two soft - tissue densities in the left breast ( one biopsy proven to be melanoma ) ; numerous subcentimeter , hypodense liver lesions ; and multiple subcentimeter , subcutaneous nodules , suggestive of metastatic disease . 
gene mutation analysis of the metastatic melanoma tissue did not reveal mutations in braf or nras . after two cycles of biochemotherapy ( data supplement ) , restaging ct scans revealed overall stable disease and a mixed tumor response in the lymph nodes . 
the patient received whole - brain radiation therapy ( 30 gy in 10 fractions ) , then stereotactic radiosurgery , followed by combination treatment with temozolomide , cisplatin , and vinblastine . subsequent restaging showed new mediastinal lymphadenopathy and lung nodules . 
the patient received two cycles of a carboplatin - paclitaxel regimen before evidence of disease progression in the left inguinal lymph nodes , liver , and bra the patient was then treated with ipilimumab , which had just received us food and drug administration approval for metastatic melanoma , 3 mg / kg for four cycles . 
there was evidence of stable disease response to ipilimumab , with tumor reduction of 30% by immune - related response criteria overall with disease response in the brain , liver , and lymph nodes . 
after 19 months of disease control with ipilimumab treatment , a brain magnetic resonance imaging demonstrated a left frontal operculum metastasis with perilesional edema ( fig 1a , upper panel ) and positron emission tomography / ct junna oba sun - hee kim wei - lien wang mariana p . 
 ( a ) representative brain magnetic resonance images show left frontal operculum metastasis with perilesional edema pretreatment ( top panel ) and the on - treatment combination dasatinib and crizotinib therapy effect ( lower panel )  . 
 ( b ) polymerase chain reactionbased dna sequencing chromatogram showing a kit nucleotide transition ( 2464 a > t ) at codon 822 ( aat to tat ) in exon 17 from the patients acral melanoma metastatic tumor . 
 ( c ) hematoxylin and eosin ( he ) and immunohistochemical staining for total kit and phosphorylated kit ( p936 ) and total met in the patients primary ( upper panel ) and metastatic ( lower panel ) acral melanoma tumors ( original magnification , 20 )  . scanning showed new hypermetabolic activity in the mediastinal lymph nodes , which was verified to be metastatic melanoma by interventional radiology - guided fine - needle aspiration ( data not shown )  . 
kit mutations and copy number alterations in 4q ( kit ) and 11q ( ccnd1 ) for the melanoma cell lines cell line name exon 4q ( kit ) 11q ( ccnd1 ) kit mutation copy number increase aa change l576p w556 - k558 n822y s476i m230 melms 2391 ma - mel - 144 histopathologic examination of the patients primary and metastatic melanoma tumors showed widespread expression of phosphorylated / total kit protein in melanoma cells ( fig 1c )  . 
given the response to combination dasatinib and crizotinib therapy , we also assessed the expression of the met , for which crizotinib has high binding affinity , and observed positive staining in both primary and metastatic tumors , with moderately higher intensity in the metastatic tumor . to better understand the mechanism by which combination therapy resulted in the observed clinical response , we performed a molecular analysis of the melanoma cell line we established from the patients metastatic tumor ( cell line 2391 ) , as well as three other melanoma cell lines shown to have endogenous kit mutations ( namely , melms , ma - mel - 144 , and m230 ) .17 - 19 mutation analysis confirmed the presence of the 2464 a > t nucleotide transition in kit with a resultant n822y amino acid change in the activation loop within 2391 , the patients melanoma cell line ( data supplement )  . 
we verified the presence of each specific kit mutation within each cell line using targeted next - generation dna sequencing ( table 1 ; data supplement ) .20 no braf or nras mutations , or secondary mutations in kit were detected in any of the cell lines . we also determined the global chromosomal copy number alteration status within each kit - mutant melanoma cell line ( fig 2a ; table 1 )  . 
cell lines 2391 and ma - mel - 144 had chromosome 4q copy number elevation , which corresponds with the location of the kit locus , consistent with initial observations that kit mutations co - occur with kit amplifications.1 , 2 three of the four cell lines also had chromosome 11q copy number elevation , which corresponds with the location of the ccnd1 locus , also well described to be amplified in am tumors.21 , 22 thus , the patients cell line 2391 and others in our panel had characteristic genetic features observed in am tumors . to determine the endogenous constitutive activity of each kit mutation , we assessed the total and phosphorylated levels of kit in each melanoma cell line . 
western blot analysis showed phosphorylated kit to be present in all the kit - mutant melanoma cell lines in the presence or absence of serum ( fig 2b ) ; this was not observed in those without a kit mutation ( data supplement )  . 
each of the cell lines showed sensitivity to gene - specific kit knockdown and to multiple tkis that share kit as a target but have many nonoverlapping targets ( eg , dasatinib targets src - family kinases ) , whereas imatinib does not ( data not shown )  . 
cell lines 2391 ( kitn822y ) , melms ( kit556 - 558del ) , and ma - mel - 144 ( kits476i ) show amplification at 11q ( location of ccnd1 allele )  . 
cell viability assay of m230 ( kitl576p ) , melms ( kit556 - 558del ) , 2391 ( kitn822y ) , and ma - mel - 144 ( kits476i ) melanoma cell lines : no treatment , dasatinib ( 50 nm ) , dasatinib plus hgf ( 100 ng / ml ) , dasatinib plus hgf plus crizotinib ( 5 m ) , and crizotinib alone . of met may enhance the effect of single - agent dasatinib in the tumor setting . 
thus , we tested if the presence of hgf , the only known ligand for met , in the extracellular environment can modulate the reduction in cell viability observed with dasatinib treatment ( fig 2c )  . 
analysis of the patients tumor and cell line ( and three other kit - mutant cell lines ) suggests that the hgf - met axis may be a mechanism of de novo resistance in kit - mutant melanomas . the emergence of secondary mutations in kit is a common mechanism of clinical resistance to kit inhibition in kit - mutant gist.29 however , nearly all resistance observed in kit mutant melanoma occurs in the initial treatment setting , before the clonal evolution of secondary mutations can emerge , 10 - 13 , 23 - 26 suggesting that the presence of coactivating pathways may play a role in this primary resistance . the observed efficacy of the combination dasatinib and crizotinib could , in part , be due to the inhibition of multiple kinases ; however , the melanoma cell line studies indicate the primacy of the kit and met receptors to explain the observed results . 
furthermore , crizotinib alone was only effective in the presence of extracellular hgf , the only known met ligand , indicating crizotinibs modulation of the well - described hgf - met axis , for which it was initially designed.30 these data are consistent with those from preclinical studies showing that met inhibition increases the effect of imatinib in kit - mutant gist models.31 it is intriguing to speculate that the response to combination dasatinib and crizotinib may have been immunologically enhanced , given the patients prior tumor response to ipilimumab therapy . 
hong collection and assembly of data : junna oba , sunhee kim , wei - lien wang , mariana macedo , fernando carapeto , meredith a mckean , john van arnam , agda k . 
woodman manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors junna oba no relationship to disclose sun - hee kim no relationship to disclose wei - lien wang no relationship to disclose mariana p . 
haydu no relationship to disclose honoraria : novartis , bristol - myers squibb , janssen oncology , roche consulting or advisory role : novartis , illumina , ge healthcare research funding : medimmune , astrazeneca , roche , novartis patents , royalties , other intellectual property : elsevier travel , accommodations , expenses : bristol - myers squibb , novartis elizabeth a . 
hong stock and other ownership interests : molecularmatch , oncorena honoraria : adaptimmune , baxter , merrimack , bayer consulting or advisory role : baxter , bayer , guidepoint global , janssen research funding : novartis , genentech , eisai , astrazeneca , pfizer , mirna therapeutics , amgen , daiichi sankyo , merck , mirati therapeutics , eli lilly , adaptimmune , abbvie , bayer , bristol - myers squibb , genmab , ignyta , infinity pharmaceuticals , kite pharma , kyowa hakko kirin , loxo , medimmune , molecular templates , takeda travel , accommodations , expenses : loxo , mirna therapeutics other relationship : oncorena chantale bernatchez stock and other ownership interests : lexicon ( i ) research funding : nektar , idera , iovance biotherapeutics , medimmune , pfizer , emd serono patents , royalties , other intellectual property : patent pending on btla as a marker for better cd8 t cells for adoptive immunotherapy alexander j . 
woodman no relationship to disclose acknowledgment leadership : beta cat pharmaceuticals , archer biosciences stock and other ownership interests : archer biosciences , beta cat pharmaceuticals we thank the center for environmental and molecular carcinogenesis anderson cancer center , smithville , tx , for antibody optimization . affiliations junna oba , sun - hee kim , wei - lien wang , mariana p . 
macedo , ac camargo cancer center , so paulo , brazil . supported by the university of texas system rising star award , national cancer institute ( nci ) specialized programs of research excellence ( spore ) in melanoma ( p50 ca093459 to d.s.h. ) ; developmental research project award , national institutes of health / nci cancer center support grant no . 
torres - cabala ca , wang wl , trent j , et al : correlation between kit expression and kit mutation in melanoma : a study of 173 cases with emphasis on the acral - lentiginous / mucosal type . 
demetri gd , van oosterom at , garrett cr , et al : efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib : a randomised controlled trial . 
woodman se , trent jc , stemke - hale k , et al : activity of dasatinib against l576p kit mutant melanoma : molecular , cellular , and clinical correlates . 
yang y , liu c , peng w , et al : antitumor t - cell responses contribute to the effects of dasatinib on c - kit mutant murine mastocytoma and are potentiated by anti - ox40 . 
hodi fs , corless cl , giobbie - hurder a , et al : imatinib for melanomas harboring mutationally activated or amplified kit arising on mucosal , acral , and chronically sun - damaged skj clin oncol 31 : 3182 - 3190 , 2013 11 . 
guo j , si l , kong y , et al : phase ii , open - label , single - arm trial of imatinib mesylate in patients with metastatic melanoma harboring c - kit mutation or amplification . 
schittenhelm mm , shiraga s , schroeder a , et al : dasatinib ( bms - 354825 ) , a dual src / abl kinase inhibitor , inhibits the kinase activity of wild - type , juxtamembrane , and activation loop mutant kit isoforms associated with human malignancies . 
kato s , jardim dl , johnson fm , et al : phase i study of the combination of crizotinib ( as a met inhibitor ) and dasatinib ( as a c - src inhibitor ) in patients with advanced cancer . 
todd jr , scurr ll , becker tm , et al : the mapk pathway functions as a redundant survival signal that reinforces the pi3k cascade in c - kit mutant melanoma . 
lee sj , kim tm , kim yj , et al : phase ii trial of nilotinib in patients with metastatic malignant melanoma harboring kit gene aberration : a multicenter trial of korean cancer study group ( un10 - 06 )  . 
kalinsky k , lee s , rubin km , et al : a phase 2 trial of dasatinib in patients with locally advanced or stage iv mucosal , acral , or vulvovaginal melanoma : a trial of the ecog - acrin cancer research group ( e2607 )  . 
 multimodal approach to outcome prediction in metastatic castration - resistant prostate cancer by integrating functional imaging and plasma dna analysis vincenza conteduca , md , phd1 ; emanuela scarpi , phd1 ; federica matteucci , md1 ; paola caroli , md1 ; giorgia ravaglia , msc1 ; lorenzo fantini , md1 ; giorgia gurioli , msc1 ; giuseppe schepisi , md1 ; daniel wetterskog , phd2 ; cecilia menna , md , phd1 ; salvatore luca burgio , md1 ; cristian lolli , md1 ; giovanni paganelli , md1 ; gerhardt attard , md , phd2 ; and ugo de giorgi , md1 purpose biomarkers for treatment personalization in metastatic castration - resistant prostate cancer ( mcrpc ) could help improve patient outcomes . 
multiple tests on blood have reported associations with poorer outcome , including serum lactate dehydrogenase ( ldh ) , chromogranin a ( cga ) , neutrophil : lymphocyte ratio ( nlr ) , and , recently , copy number ( cn ) of androgen receptor ( ar ) in plasma dna . 
fchpet / ct scan was performed at baseline , and maximum standardized uptake value ( suvmax ) , total lesion activity ( tla ) , and metabolic tumor volume ( mtv ) were calculated . 
kaplan - meier curves showed signicantly worse progressionfree survival and overall survival in patients with plasma ar gain and higher suvmax , tla , and mtv values ( p , .001 in each prognostic group )  . 
it is now widely accepted that the majority of crpcs are still dependent on the androgen receptor ( ar ) signaling pathway , 4 , 5 and new ar pathway inhibitors such as enzalutamide and abiraterone have shown efcacy against crpc.6 - 9 biomarkers for personalizing treatment in metastatic crpc ( mcrpc ) could improve patient outcomes . 
multiple tests have revealed associations with a poorer outcome , including serum lactate dehydrogenase ( ldh ) , 10 visceral metastasis ( especially liver involvement10 - 12 ) , chromogranin a ( cga ) , 13 , 14 and neutrophil : lymphocyte ratio ( nlr ) .15 , 16 during the past few years , the potential for plasma dna analysis to improve clinical practice has substantially increased , with real - time molecular characterization that permits patient stratication and thus facilitates treatment decision making . 
 conteduca et al context key objective in the context of multiple biomarkers strategy , we assessed the role of plasma dna analysis in combination with functional imaging and other routinely obtained circulating biomarkers to perform a better prognostication of metastatic castrationresistant prostate cancer ( mcrpc ) in patients . knowledge generated androgen receptor copy number detected in plasma , choline - positron emission tomography parameters , and other clinical factors can be considered as independent predictors of overall survival . 
our nal parsimonious prognostic model from the multivariable analysis of overall survival permitted classication of patients with mcrpc into three risk groups according to the independent prognostic role of these biomarkers . relevance our results suggested the potential usefulness of integrating functional imaging with plasma dna analysis and other noninvasive biomarkers as a tool to improve treatment selection for patients with mcrpc . is now acknowledged as a therapy - guiding predictive and prognostic biomarker.17 - 21 , 22 furthermore , recent biologic data suggest an association between choline uptake and ar copy number ( cn ) .23 increased ar protein expression and / or signaling has been indirectly related to the upregulation of choline kinase alpha ( chka ) , which can bind directly to the ar ligandbinding domachka could be considered a sort of chaperone for ar , resulting in the ubiquitination and transcription and displayactivation of ar - dependent ing different functions involved in tumor growth and progression.23 the overexpression of chka protein in multiple tumors , including prostate cancer , leads to an increased uptake of choline and provides the rationale for using 18f - uorocholine positron emission tomography / computed tomography ( fch - pet / ct ) in this patient setting . 
in addition , early fch - pet / ct has been associated the decline in with clinical outcome , prostate - specic antigen ( psa ) in patients with mcrpc treated with abiraterone or enzalutamide.24 , 25 independently of our aim was to integrate functional imaging , represented as choline uptake in fch - pet / ct , with plasma ar status , clinical data , and other routinely obtained circulating biomarkers and to evaluate their association with outcome . 
twenty - six ( 24.8% ) were chemotherapy naive and 79 ( 75.2% ) had previously undergone chemotherapy . all had a histologically conrmed diagnosis of prostate adenocarcinoma treated with abiraterone or enzalutamide in preor postchemotherapy settings . 
the study included patients participating in a protocol approved by the institutional review board of istituto scientico romagnolo per lo studio e la cura dei tumori ( irst ) irccs , meldola , italy ( rec 2192 / 2013 )  . patients were treated with abiraterone 1 , 000 mg plus prednisone 5 mg twice per day or enzalutamide 160 mg alone once per day . 
in addition , serum ldh , alkaline phosphatase , complete blood count ( used to determine nlr ) , and serum cga were also measured within 1 week of starting therapy . 
in this study , fch - pet / ct was performed after 12 6 4 weeks of treatment . peripheral blood samples were collected from each patient at baseline and at the rst radiologic evaluation . 
total extracted plasma dna was quantied using the quant - it high sensitivity picogreen doublestranded dna assay kit ( invitrogen ) or by spectrophotometric evaluation ( nanodrop nd - 1000 ; celbio , milan , italy )  . 
semiquantitative criteria based on the maximum standardized uptake value ( suvmax ) and the target : background ratio were used to aid the visual analysis.26 the metabolic tumor volume ( mtv ) was calculated as the sum of each 3 - dimensional volume of interest . 
mtv is a purely volumetric entity , whereas tla also takes into account the metabolic activity of the lesion leading to an estimate of tumor activity ( data supplement )  . statistical analysis data were summarized by frequency for categorical variables and by cutoff using receiver operating characteristic ( roc ) curves and median and range for continuous variables . 
a comparison between baseline imaging and clinical , laboratory , and molecular features in patients with or without ar gain was performed using the median test ( wilcoxon )  . 
roc curve analysis was performed to estimate the best cutoff value of fch - pet / ct parameters ( tla , mtv , and suvmax ) and serum cga level considered as continuous variables . progression - free survival ( pfs ) was measured as the time between the rst day of abiraterone or enzalutamide treatment and the date of disease progression or death ( whichever came rst ) or last tumor evaluation . 
statistical analyses were performed with sas 9.4 software ( sas institute , cary , nc )  . a weibull multiple regression model was adopted to evaluate the combined impact of molecular , laboratory , and imaging features on survival . 
 ( c ) linear regression ( diagonal line ) between maximum standardized uptake value ( suvmax ) , total lesion activity ( tla ) , and metabolic tumor volume ( mtv )  . 
a box plot is shown with the thick horizontal line depicting the median value of suvmax , tla , and mtv in overall , pre - , and postchemotherapy patients . 
a , ar copy number amplied ; cga , chromogranin a ; cht , chemotherapy , ldh , lactate dehydrogenase ; n , ar copy number normal ; nci , nanocurie ; nlr , neutrophil : lymphocyte ratio . ability of functional imaging and plasma ar status to predict clinical outcomes at the time of the analysis , 90 ( 85.7% ) of the overall 105 patients ( 15 [ 57.7% ] of 26 before chemotherapy and 75 [ 92.4% ] of 79 patients after chemotherapy ) had progressive disease and 82 patients ( 78.1% ) had died ( 11 [ 42.3% ] before chemotherapy and 71 [ 88.6% ] after chemotherapy )  . 
chemotherapytreated patients were divided into four prognostic groups according to baseline ar status and tla , mtv , and suvmax values : ar normal , tla , mtv , and suvmax low ; ar gain , tla , mtv , and suvmax low ; ar normal , tla , mtv , and suvmax high ; and ar gain , tla , mtv , and suvmax high . they were characterized by a signicant difference in pfs ( p , .001 for tla , p = .003 for mtv , and p , .001 for suvmax ) and os ( p , .001 for tla , p , .001 for mtv , and p , .001 for suvmax ; data supplement )  . identication of imaging , molecular , and clinical features as independent predictors of treatment outcome we performed univariable and multivariable cox proportional hazards regression analysis of os to evaluate the prognostic impact of different factors . 
it is possible that the strong correlation among all pet / ct parameters abrogated the statistical signicance of tla and mtv in this analysis . coefcient by the smallest one as described in statistical analysis . 
the choice of time and number of groups with different prognoses was made in terms of clinical consideration , such as time at 15 months ( group i , 15 months : os was greater than 70% ; group ii , 15 months : os was 30% to 70% ; group iii , 15 months : os was less than 30% )  . 
in the training set , 27 patients ( 39.1% ) were attributed to risk group i , 13 ( 18.8% ) to risk group ii , and 29 ( 42.0% ) to risk group iii . the survival experience of the three patient groups was identied by the score . 
the larger two - thirds group of patients was used as a training set to construct the score , and the remaining one third was used as a validation set . table 2 shows the partial score value for each category , which was obtained by subdividing each regression this study examined the feasibility of using functional imaging to assess tumor burden and also to evaluate its predictive and prognostic role in crpc treated with abiraterone or enzalutamide . 
progression - free survival ( pfs ) and overall survival ( os ) according to copy number ( cn ) of androgen receptor ( ar ) and functional imaging in overall population of patients with castration - resistant prostate cancer who were treated with abiraterone or enzalutamide . 
of events median os ( months ) 95% ci 95% ci , 112 112 suvmax , 50 50 , 12 12 total lesion activity , 563 , 979 563 , 979 median no . 
kaplan - meier curves for overall survival ( os ) and relative survival ( months ) by risk group in the training set ( a ) and validation set ( b )  . 
in both treatment groups , survival experience of the three groups of patients was identied by the score . however , the identication of biomarkers could facilitate personalized next - line systemic therapy in a more accurately selected population . 
during the past decade , molecular stratication from tissue biopsy has provided several therapeutic targets and prognostic markers , but numerous factors can limit the usefulness of tissue biomarkers ( from both primary and secondary tumors )  . 
this clone may not necessarily be from the largest lesion or the lesion with the highest gleason grade , as conrmed by the lack of signicance of the gleason score with clinical outcome in our univariable analysis . 
moreover , a polyclonal metastasis - to - metastasis seeding may occur during disease progression.27 given the complex tumor heterogeneity , assessment of the archival primary tumor or metastatic biopsies for the clinical management of patients with crpc may not be adequate for or entirely representative of current tumor status . 
 a functional imaging and plasma dna analysis in crpc baseline ar normal ; dsdna 27 ng / ml re - evaluation ar normal ; dsdna 11 ng / ml baseline ar normal ; dsdna 51 ng / ml re - evaluation ar normal ; dsdna 31 ng / ml baseline ar normal ; dsdna 122 ng / ml re - evaluation ar normal ; dsdna 136 ng / ml baseline ar normal ; dsdna 69 ng / ml re - evaluation ar normal ; dsdna146 ng / ml fig 4 . 
impact of baseline androgen receptor ( ar ) copy number , cell - free dna , and choline uptake by 18f - uorocholine positron emission tomography / computed tomography on clinical outcome in patients with castration - resistant prostate cancer who were treated with abiraterone or enzalutamide . 
 ( a ) patient 1 ; 78 years of age ; gleason score 9 ( 4 + 5 ) ; 10 sites of nodal localization ; baseline ar normal ; tla = 290576.3 , mtv = 50.26 ; suvmax = 68.52 : treatment with abiraterone postdocetaxel for 7 months . 
 ( b ) patient 2 ; 80 years of age ; gleason score 7 ( 3 + 4 ) ; four sites of nodal localization ; baseline ar gain ; tla = 23745.7 ; mtv = 14.67 ; suvmax = 12.97 ; treatment with abiraterone postdocetaxel for 30 months . 
 ( c ) patient 3 ; 85 years of age ; gleason score 8 ( 4 + 4 ) ; 26 sites of bone localization ; baseline ar normal ; tla = 1292423.8 ; mtv = 279.85 ; suvmax = 206.49 ; treatment with abiraterone postdocetaxel for 12 months . 
 ( d ) patient 4 ; 87 years of age ; gleason score 8 ( 4 + 4 ) ; 10 sites of bone localization ; baseline ar gain ; tla = 599060 ; mtv = 142 ; suvmax = 49.25 ; treatment with enzalutamide postdocetaxel for 5 months . 
re - evaluation after 40 days of therapy : progressive disease . addition , this study was a retrospective single - center work . additional limitations are the small sample size , especially in the prechemotherapy group , and the presence of pet / ct indices as nonabsolute measures , which can be affected by technical issues such as pet scanner calibration , imaging reconstruction , image acquisition , and timing of tracer administration . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . cristian lolli travel , accommodations , expenses : janssen oncology gerhardt attard honoraria : janssen , astellas pharma , janssen ( i ) consulting or advisory role : janssen - cilag , veridex , ventana medical systems , astellas pharma , medivation , novartis , millennium pharmaceuticals , abbott laboratories , essa , bayer , pzer speakers ' bureau : janssen , astellas pharma , takeda , sano , ventana medical systems research funding : janssen ( inst ) , arno therapeutics ( inst ) , innocrin pharma ( inst ) patents , royalties , other intellectual property : i am on the icr rewards to inventors list for abiraterone acetate . travel , accommodations , expenses : janssen , astellas pharma , medivation , ventana medical systems , abbott laboratories , bayer , essa , janssen ( i ) , astellas pharma ( i ) , pzer other relationship : institute of cancer research ugo de giorgi consulting or advisory role : pzer , janssen , astellas pharma , sano , bristol - myers squibb , bayer , ipsen , merck , research funding : sano ( inst ) , astrazeneca ( inst ) , roche ( inst ) travel , accommodations , expenses : bristol - myers squibb , ipsen , janssen , pzer no other potential conicts of interest were reported . acknowledgment we thank gr ainne tierney for editorial assistance . references siegel rl , miller kd , jemal a : cancer statistics , 2018 . 
ca cancer j clin 68 : 7 - 30 , 2018 ferlay j , soerjomataram i , dikshit r , et al : cancer incidence and mortality worldwide : sources , methods and major patterns in globocan 2012 . 
int j cancer 136 : e359 - e386 , 2015 gillessen s , omlin a , attard g , et al : management of patients with advanced prostate cancer : recommendations of the st gallen advanced prostate cancer consensus conference ( apccc ) 2015 . 
ann oncol 26 : 1589 - 1604 , 2015 kobayashi t , inoue t , kamba t , et al : experimental evidence of persistent androgen - receptor - dependency in castration - resistant prostate cancer . 
n engl j med 371 : 424 - 433 , 2014 scher hi , fizazi k , saad f , et al : increased survival with enzalutamide in prostate cancer after chemotherapy . 
n engl j med 367 : 1187 - 1197 , 2012 de bono js , logothetis cj , molina a , et al : abiraterone and increased survival in metastatic prostate cancer . 
n engl j med 364 : 1995 - 2005 , 2011 ryan cj , smith mr , de bono js , et al : abiraterone in metastatic prostate cancer without previous chemotherapy . 
chi kn , kheoh t , ryan cj , et al : a prognostic index model for predicting overall survival in patients with metastatic castration - resistant prostate cancer treated with abiraterone acetate after docetaxel . 
halabi s , lin cy , kelly wk , et al : updated prognostic model for predicting overall survival in rst - line chemotherapy for patients with metastatic castrationresistant prostate cancer . 
conteduca v , caffo o , fratino l , et al : impact of visceral metastases on outcome to abiraterone after docetaxel in castration - resistant prostate cancer patients . future oncol 11 : 2881 - 2891 , 2015 74 : 1691 - 1696 , 2014 21 : 487 - 493 , 2014 13 . 
conteduca v , crabb sj , jones rj , et al : persistent neutrophil to lymphocyte ratio .3 during treatment with enzalutamide and clinical outcome in patients with castration - resistant prostate cancer . 
salvi s , casadio v , conteduca v , et al : circulating cell - free ar and cyp17a1 copy number variations may associate with outcome of metastatic castrationresistant prostate cancer patients treated with abiraterone . 
salvi s , casadio v , conteduca v , et al : circulating ar copy number and outcome to enzalutamide in docetaxel - treated metastatic castration - resistant prostate cancer . 
conteduca v , wetterskog d , sharabiani mta , et al : androgen receptor gene status in plasma dna associates with worse outcome on enzalutamide or abiraterone for castration - resistant prostate cancer : a multi - institution correlative biomarker study . 
conteduca v , scarpi e , caroli p , et al : circulating androgen receptor combined with 18f - uorocholine pet / ct metabolic activity and outcome to androgen receptor signalling - directed therapies in castration - resistant prostate cancer . 
de giorgi u , caroli p , burgio sl , et al : early outcome prediction on 18f - uorocholine pet / ct in metastatic castration - resistant prostate cancer patients treated with abiraterone . 
de giorgi u , caroli p , scarpi e , et al : erratum to : ( 18 ) f - fluorocholine pet / ct for early response assessment in patients with metastatic castration - resistant prostate cancer treated with enzalutamide . 
mawlawi o , podoloff da , kohlmyer s , et al : performance characteristics of a newly developed pet / ct scanner using nema standards in 2d and 3d modes . j nucl med 45 : 1734 - 1742 , 2004 27 . 
conteduca v , caffo o , lolli c , et al : long - term clinical impact of psa surge in castration - resistant prostate cancer patients treated with abiraterone . 
science 355 : 84 - 88 , jahr s , hentze h , englisch s , et al : dna fragments in the blood plasma of cancer patients : quantitations and evidence for their origin from apoptotic and necrotic cells . 
khan k , rata m , cunningham d , et al : functional imaging and circulating biomarkers of response to regorafenib in treatment - refractory metastatic colorectal cancer patients in a prospective phase ii study . 
 cdkn2c - null leiomyosarcoma : a novel , genomically distinct class of tp53 / rb1wild - type tumor with frequent cic genomic alterations and 1p / 19q - codeletion erik a . 
here , we searched for recurrent actionable genomic alterations in lms that occur in the absence of common untreatable oncogenic drivers . methods tissues from 276 , 645 unique advanced cancers , including 2 , 570 uterine and soft tissue lms , were sequenced by hybrid - capturebased next - generation dna and rna sequencing / comprehensive genomic proling of up to 406 genes . 
we further validated our ndings in two publicly available datasets : the cancer genome atlas and the project genie initiative . conclusion cdkn2c - null lms denes a genomically distinct tumor that may have prognostic and / or therapeutic clinical implications , including possible use of specic cyclin - dependent kinase inhibitors . jco precis oncol 4 : 955 - 971 . 
 williams et al context key objective leiomyosarcoma ( lms ) , an aggressive tumor with limited curative options , shows frequent mutations in tp53 and rb1 but few actionable genomic alterations . 
here , we searched for recurrent actionable genetic alterations in lms . knowledge generated a novel , genomically distinct class of lms ( 3.0% ; 77 of 2 , 570 cases ) harbor homozygous loss of cdkn2c , which encodes the cyclin - dependent kinase inhibitor - 2c . 
cdkn2c - null lms lack typical tp53 and rb1 mutations ; show concurrent homozygous deletion of cic , cdkn2a , and rad51b ; and show frequent 1p / 19q - codeletion . relevance the nding of recurrent cdkn2c - null lms provides insight into tumor biology and raises the possibility for use of specic cyclin - dependent kinase inhibitors in this aggressive disease . from the uterus , with signicantly low frequency of tp53 and rb1 gas . methods cohort and genomic analyses comprehensive genomic proling was performed in a clinical laboratory improvement amendmentscertied , college of american pathologistsaccredited laboratory ( foundation medicine , cambridge , ma )  . 
20% estimated percent tumor nuclei in each case , for which the percent tumor nuclei equals 100 times the number of tumor cells divided by total number of nucleated cells . 
the samples were assayed by adaptor ligation hybrid capture , performed for all coding exons of 236 ( v1 ) , 315 ( v2 ) , or 405 ( v3 ) cancer - related genes plus select introns from 19 ( v1 ) , 28 ( v2 ) , or 31 ( v3 ) genes frequently rearranged in cancer ( appendix table a1 ) .11 , 12 for samples with available rna , targeted rna sequencing was performed for rearrangement analysis in 265 genes.12 sequencing of captured libraries was performed using the illumina hiseq 4000 system ( illumina , san diego , ca ) to a mean exon coverage depth of targeted regions of  . 500 , and sequences were analyzed for gas , including short variant alterations ( base substitutions , insertions , and deletions ) , copy number alterations ( focal amplications and homozygous deletions ) , and select gene fusions or rearrangements.11 , 13 , 14 to maximize mutation detection accuracy ( sensitivity and specicity ) in impure clinical specimens , the test was previously optimized and validated to detect base substitutions at a 5% mutant allele frequency , indels with a 10% mutant allele frequency with 99% accuracy , and fusions occurring within baited introns / exons with  . 
6 - 8 copies depending on tumor ploidy and homozygous deletions was performed as previously described.11 in brief , the aligned dna sequences of each tumor specimen were normalized against a process - matched normal , producing log - ratio and minor allele frequency data . 
a gibbs sampler tted copy number model and a grid - based model were tted to the segmented log - ratio and minor allele frequency data , producing genome - wide copy number estimates . 
finally , the degrees - of - t of candidate models returned by gibbs sampling and grid sampling were compared , and the optimal model was selected by an automated heuristic . signal - to - noise ratios for each genomic segment were used to determine gain or loss per chromosome arm on the basis of tumor purity and ploidy ; the sum of segment sizes determined the fraction of each arm gained or lost . chromosomes were assessed for arm - level aneuploidy , dened as positive if  . 
the cohort of cdkn2c - null lms comprised 77 cases , each from a different patient , that were submitted to foundation medicine for comprehensive genomic proling during routine clinical care . 
human investigations were performed after approval by a local human investigations committee and in accordance with an assurance led with and approved by the department of health and human services , when appropriate . 
clinicopathologic data , including patient age , sex , tumor site , and figo stage or ajcc ( 8th edition ) stage , were extracted from the accompanying pathology report.4 , 16 primary site data were not available for a subset of patient cases ( indeterminant primary )  . 
these patients were signicantly older than the remainder of the lms cohort ( median age , 61 v 57 years ; p = .0009 , mann - whitney u test )  . 
patients were enriched for female sex compared with the remainder of the lms cohort ( 99% [ 76 of 77 ] v 79% [ 1 , 968 of 2 , 493 ] ; p , .0001 , fishers exact test )  . 
six female patients had a prior history of leiomyomatosis ( n = 2 ) or uterine smooth muscle tumor of uncertain malignant potential ( stump ; n = 4 )  . 
the majority of cdkn2c - null lms patients showed clinically advanced / metastatic disease , with 62% of conrmed uterine primary occurrences documented at figo stage iv ( n = 34 of 55 ) and 86% of indeterminant or soft tissue primary cases documented at ajcc stage iv ( n = 19 / 22 ) , as summarized indeterminant figo staging ( uterine primary ) no . 
locations of the sequenced tumor specimens are summarized in appendix table a2 . comprehensive genomic proling of cdkn2c - null lms the distribution of gas in the 77 cdkn2c - null lms is displayed in figure 1 . 
the frequent gas were identied in cic at 19q13.2 , most cdkn2a , and rad51b ( table 2 ) , and unsupervised analysis showed signicant enrichment of these alterations in the cdkn2c - null cohort ( appendix fig a1 )  . 
no other signicant differences based on age were identied . comparison of patient cases on the basis of clinical stage , three patients had two separate tissue specimens analyzed ( appendix table a4 )  . 
additional recurrent chromosomal arm - level changes were identied in the 72 patient cases available , including most frequently aneuploidy of chromosomes 6p ( n = 35 ) , 9p ( n = 19 ) , 10q ( n = 28 ) , 11p ( n = 39 ) , 13q ( n = 46 ) , 14q ( n = 46 ) , and 16q ( n = 52 )  . a review of 1p / 19q - codeletion status in available lms patient cases without homozygous deletion of cdkn2c ( n = 1 , 212 ) revealed 62 1p / 19q - codeleted lms ( 5% ; fig 2c )  . 
all four occurred in uterine lms ( one of which with a history of stump )  . all non - lms sarcoma patient cases in the foundation medicine dataset ( n = 12 , 097 ) were evaluated for cdkn2c status . 
these included diverse sarcoma diagnoses , including six high - grade sarcomas not otherwise specied , two osteosarcomas , two malignant peripheral nerve sheath tumors , and two inammatory myobroblastic tumors . 
both were alk rearrangementpositive tumors in women ( ages , 63 and 72 years )  . we also searched our entire lms cohort ( n = 2 , 570 ) for cases with pathogenic alterations in cdkn2c other than homozygous deletion . 
 ( a ) cdkn2c - null lms with a truncating variant in cic ( p.q907 * ) and homozygous deletion of cdkn2a at chromosome 9p21.3 ( red arrow )  . 
 ( c ) cdkn2c - retained lms with deep deletion of rb1 at chromosome 13q14.2 ( red arrow ) and a cn plot of high complexity . and rad51b were identied ; 1p / 19q status was not available . histopathology histopathologic evaluation was performed on all available high - resolution digital pathology h&e slides of our cohort of cdkn2c - null lms ( n = 70 )  . 
histopathology of cdkn2c - null leiomyosarcoma ranged from ( a , b ) epithelioid to ( c ) spindled ( hematoxylin & eosin [ h&e ] stains , 200 )  . 
 ( d ) occasional cases showed focal myxoid histology ( h&e stain , 200 )  . progesterone receptor ( 24 of 26 ; remaining two with focal positivity )  . 
cdkn2c - null lms were also positive for desmin ( 30 of 33 ; remaining three with focal positivity ) , smooth muscle actin ( 25 of 25 ) , muscle - specic actin ( 11 of 11 ) , and caldesmon ( 6 of 6 )  . 
patient cases showed frequent homozygous loss of cic ( n = 5 [ 42% ] of 12 ) , cdkn2a ( n = 4 [ 33% ] of 12 ) , and rad51b ( n = 3 [ 25% ] of 12 ) , and all were wild type for tp53 , rb1 , and atrx ( n = 12 of 12 )  . discussion in 2 , 570 patient cases of lms , cdkn2c - null lms ( n = 77 ; 3.0% ) comprised a genomically distinct molecular subgroup . cdkn2c - null lms typically lacked mutations in tp53 , rb1 , and atrx but showed frequent 1p / 19q - codeletion ( 81% ) , and nearly half ( 40.3% ) showed homozygous deletion or inactivating truncations of cic . 
 cdkn2c loss denes class of tp53 / rb1wild - type leiomyosarcoma which enhances fas - mediated apoptosis , and its loss may contribute to tumor pathogenesis.29 the cic gene on chromosome 19q13.2 represses genes induced downstream to rtk pathway activation.30 in the absence of rtk signaling , cic blocks transcription of genes that have diverse effects on cellular proliferation , metabolism , and migration.31 along with single copy loss of cic on 19q , concurrent inactivating mutations in cic are identied in a high percentage of oligodendrogliomas.32 whole - arm 1p / 19q - codeletion , with concurrent mutation in idh1 or idh2 , is entity - dening for oligodendrogliomas.33 - 35 oligodendrogliomas are associated with relatively long overall survival , and treatment strategies are often stratied on the basis of 1p / 19q status.36 - 38 the codeletion is a result of unbalanced translocation between two chromosomes , with subsequent loss of der ( 1 ; 19 ) ( p10 ; q10 ) , likely because chromosomes 1 and 19 are near each other in the nonrandom organization of the nucleus.39 - 41 a large percentage of oligodendrogliomas also show cic and fubp1 mutations.31 our cohort of cdkn2c - null lms shows notable similarities and differences to oligodendroglioma ; 40% of our cohort showed an inactivating alteration in cic , most commonly homozygous deletion . 
rare sarcoma - like tumors originating from oligodendrogliomas have been reported , with documented idh1 mutation and 1p / 19q - codeletion , and have been termed oligosarcoma.42 - 44 rodriguez et al43 identied 6 of 7 patient cases of oligosarcoma with at least focal smooth muscle actin positivity of the sarcomatous component by immunohistochemistry and one patient case with smooth muscle differentiation by electron microscopy . 
these results indicate a similarity in differentiation to our cohort of lms . among all sarcomas with cdkn2c loss , 1p / 19q - codeletion appears to be nearly exclusive to lms . 
in our overall lms cohort , however , occasional cdkn2c - retained lms showed tp53 and rb1 alterations and were positive by the 1p / 19qcodeletion detection algorithwe speculate that , given the complexity of these genomically unstable occurrences , occasional cdkn2c - retained lms satisfy these criteria ( fig 2c )  . as such , identication of homozygous deletion of cdkn2c may be the most specic distinguishing feature . cytogenetic ndings in lms and leiomyoma have been previously reported , although without characterization of cdkn2c status . 
a greater frequency of 1p loss has been documented in metastasized lms.45 from a cytogenetics study of 800 uterine leiomyomata , nine diploid occurrences with 1p loss were identied , with other associated alterations , particularly chromosome 19 and / or chromosome 22 loss.46 transcriptional proling of two of the 1p - deleted leiomyomas in that study showed alignment with malignant lms in a hierarchical clustering analysis.46 in another study , 1p loss was identied in approximately one quarter of uterine cellular leiomyomata.47 three reports on a total of eight pulmonary - based benign metastasizing leiomyoma reported 19q and 22q terminal deletion in each case.48 - 50 rare uterine leiomyomas with gas in rad51b have also been identied.51 the overlap in gas between our cohort of cdkn2c - null lms and a subset of leiomyoma of uncertain cdkn2c status in the literature suggests a possible connection between these entities . evaluations of gas in epithelioid or myxoid uterine smooth muscle neoplasms are limited in the literature.52 - 56 although a high percentage of cdkn2c - null lms in our study demonstrated epithelioid features on histology , histology was also varied . 
immunohistochemistry results extracted from pathology reports were typical for uterine lms , with expression of characteristic smooth muscle markers.57 given the overall low response rate of lms to standard therapies , the identication of this targetable alteration in cdkn2c may be useful for treatment decisions . 
cdk4 / 6 inhibitors have previously shown effectiveness in a lms with a cdkn2a alteration.10 nearly half of the cdkn2c - null lms harbored loss of cdkn2a ; cdk4 / 6 inhibitors may be effective in replacing the loss of inhibition of cdk4 / 6 that results from cdkn2c and cdkn2a loss in these patient cases that recur after standard chemotherapy regimens . 
a minor subset of cdkn2c - null and / or 1p / 19q - codeleted lms harbored activating fusions in alk , braf , fgfr1 , and ntrk1 , for which targeted inhibitors may be of utility.9 limitations of this study include its retrospective nature and the enrichment for aggressive tumors , mostly metastatic to distant sites . 
the latter may be due to collection bias from submission of specimens later in the disease course . additional studies will be needed to correlate the nding of cdkn2c loss in lms with prognostic data and treatment outcomes . 
williams , md , 150 second st , cambridge , ma 02141 ; twitter : @erikawilliamsm1 ; e - mail : erwilliams@foundationmedicine.com. meagan montesion stock and other ownership interests : roche ethan s . 
hoffmann - la roche radwa sharaf employment : foundation medicine stock and other ownership interests : roche brennan decker stock and other ownership interests : avidea technologies consulting or advisory role : foundation medicine , avidea technologies kevin jon williams stock and other ownership interests : hygieia , gemphire therapeutics consulting or advisory role : gemphire therapeutics research funding : novo nordisk jeffrey m . 
venstrom employment : genentech , foundation medicine leadership : genentech stock and other ownership interests : genentech research funding : genentech , roche , foundation medicine travel , accommodations , expenses : genentech brian m . 
alexander employment : foundation medicine leadership : foundation medicine stock and other ownership interests : roche research funding : eli lilly ( inst ) , puma ( inst ) , celgene ( inst ) ( optional ) open payments link : physician / 854258 / summary jeffrey s . 
ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine consulting or advisory role : celsius therapeutics research funding : foundation medicine lee a . 
elvin employment : foundation medicine no other potential conicts of interest were reported . acknowledgment we thank the american association for cancer research and appreciate its nancial and material support in the development of the american association for cancer research project genie registry , and we thank members of the consortium for their commitment to data sharing . references roberts me , aynardi jt , chu cs : uterine leiomyosarcoma : a review of the literature and update on management options . 
gynecol oncol 151 : 562 - 572 , 2018 seagle bll , sobecki - rausch j , strohl ae , et al : prognosis and treatment of uterine leiomyosarcoma : a national cancer database study . 
moore kn , gunderson c , ramkissoon s , et al : comprehensive genomic proling of uterine leiomyosarcomas identies opportunities for personalized therapies . ann oncol 27 : vi309 , 2016 davis le , nusser kd , przybyl j , et al : discovery and characterization of recurrent , targetable alk fusions in leiomyosarcoma . 
frampton gm , fichtenholtz a , otto ga , et al : development and validation of a clinical cancer genomic proling test based on massively parallel dna seoncologist 22 : 416 - 421 , 2017 quencing . 
trabucco se , gowen k , maund sl , et al : a novel next - generation sequencing approach to detecting microsatellite instability and pan - tumor characterization of 1000 microsatellite instability - high cases in 67 , 000 patient samples . 
sweeney sm , cerami e , baras a , et al : aacr project genie : powering precision medicine through an international consortiucancer discov 7 : 818 - 831 , 2017 19 . 
guan kl , jenkins cw , li y , et al : growth suppression by p18 , a p16ink4 / mts1and p14ink4b / mts2 - related cdk6 inhibitor , correlates with wild - type prb function . 
genes dev 8 : 2939 - 2952 , 1994 jeffrey pd , tong l , pavletich np : structural basis of inhibition of cdk - cyclin complexes by ink4 inhibitors . 
kim kj , park mc , choi sj , et al : determination of three - dimensional structure and residues of the novel tumor suppressor aimp3 / p18 required for the interaction with atm . 
pohl u , cairncross jg , louis dn : homozygous deletions of the cdkn2c / p18ink4c gene on the short arm of chromosome 1 in anaplastic oligodendrogliomas . brain pathol 9 : 639 - 643 , 1999 28 . 
husemann k , wolter m , b uschges r , et al : identication of two distinct deleted regions on the short arm of chromosome 1 and rare mutation of the cdkn2c gene from 1p32 in oligodendroglial tumors . 
cell cycle 8 : 2528 - 2534 , 2009 jim enez g , shvartsman sy , paroush z : the capicua repressor : a general sensor of rtk signaling in development and disease . 
cairncross jg , ueki k , zlatescu mc , et al : specic genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodenneuropathol 131 : 803 - 820 , 2016 145 : 1175 - 1190 , 1994 j neuropathol exp neurol 54 : 91 - 95 , 1995 drogliomas . 
taal w , van der rijt ccd , dinjens wnm , et al : treatment of large low - grade oligodendroglial tumors with upfront procarbazine , lomustine , and vincristine chemotherapy with long follow - up : a retrospective cohort study with growth kinetics . 
grifn ca , burger p , morsberger l , et al : identication of der ( 1 ; 19 ) ( q10 ; p10 ) in ve oligodendrogliomas suggests mechanism of concurrent 1p and 19q loss . j neuropathol exp neurol 65 : 988 - 994 , 2006 jenkins rb , blair h , ballman k v . , et al : a t ( 1 ; 19 ) ( q10 ; p10 ) mediates the combined deletions of 1p and 19q and predicts a better prognosis of patients with oligodendroglioma . 
mandahl n , fletcher cdm , dal cin p , et al : comparative cytogenetic study of spindle cell and pleomorphic leiomyosarcomas of soft tissues : a report from the 46 . 
christacos nc , quade bj , dal cin p , et al : uterine leiomyomata with deletions of 1p represent a distinct cytogenetic subgroup associated with unusual histologic champ study group . 
bowen jm , cates jm , kash s , et al : genomic imbalances in benign metastasizing leiomyoma : characterization by conventional karyotypic , uorescence in situ hybridization , and whole genome snp array analysis . 
holzmann c , markowski dn , koczan d , et al : genome - wide acquired uniparental disomy as well as chromosomal gains and losses in a uterine epithelioid leiomyoma . 
reyes c , karamurzin y , frizzell n , et al : uterine smooth muscle tumors with features suggesting fumarate hydratase aberration : detailed morphologic analysis and correlation with s - ( 2 - succino ) - cysteine immunohistochemistry . 
 cdkn2c loss denes class of tp53 / rb1wild - type leiomyosarcoma appendix attribute negative not significant positive % frequency cdkn2c faf1 tp53 sex : male atrx chr2p_loss chr1p_loss cdkn2a cdkn2b rad51b sex : female chr19q_loss chr14q_loss log2 odds ratio fig a1 . 
red and blue indicate positive and negative correlation , respectively , in cdkn2c - null leiomyosarcoma ( n = 77 ) compared with the remainder of the leiomyosarcoma cohort ( n = 2 , 493 )  . 
henick , md1 ; matthew ingham , md1 ; maryam shirazi , md2 ; charles marboe , md2 ; andrew turk , md2 ; susan hsiao , md , phd2 ; and mahesh m . 
intervening surveillance scans were unremarkable ; however , the patient experienced a 10 - pound weight loss over 2 months that culminated in acute - onset exertional dyspnea and chest pain , prompting admission . 
the patient was evaluated by cardiothoracic surgery and deemed not to be a surgical candidate . positron emission tomography ( pet ) ct conrmed uorodeoxyglucose avidity in the hypermetabolic mass , scattered left lung nodules , and an intralumina soft tissue mass in the distal colon proximal to the anastomosis as well as a right thyroid lesion ( fig 1 )  . 
restaging ct of the chest after 2 cycles revealed decreasing peripheral opacities in the left upper lobe , a decrease in irregular left - sided lung nodules , and a decrease in the extent of tumor thrombus , with an increase in patency of the left pulmonary artery and interval resolution of cavitation within the tumor thrombus . 
in addition , the vaf of 67% indicated loss of heterozygosity in the tumor , raising the possibility of a germline alteration ( germline testing has not been performed )  . 
because the msi status was indeterminate by ngs , msi testing by polymerase chain reaction ( pcr ) fragment analysis using ve quasimonomorphic mononucleotide repeats ( msi analysis system , version 1.2 ; promega , madison , wi ) was performed but was indeterminate , because normal dna for comparison was unavailable . 
using dna extracted from peripheral blood for comparison , msi with an altered allele at four of ve tested loci ( nr - 21 ; bat - 26 ; bat - 25 ; mono - 27 ; fig 3 ) was detected . 
positron emission tomography / computed tomography results ( a ) at baseline , ( b ) after treatment with doxorubicin plus olaratumab before pembrolizumab administration , and ( c ) after 8 cycles of pembrolizumab . with chemotherapy , the patient was amenable to treatment with pembrolizumab and began therapy . 
after 8 cycles of treatment , intervening pet - ct results have shown an additional decrease in the extravascular component of the pulmonary artery tumor with evolving dystrophic calcication , no evidence of tumor thrombus , stable ndings in the lungs likely consistent with pulmonary infarcts , an avid hypermetabolic right thyroid nodule , and a decrease in soft tissue nodularity at the suture line . discussion intimal sarcoma of the pulmonary artery is a rare tumor ( incidence , approximately 0.001%2 , 3 ) , and the scant existing medical literature highlights individual cases of response to various combinations of surgery , 4 chemotherapy , 5 and radiation.6 the molecular landscape of such tumors is not well characterized . studies of immune checkpoint blockade have demonstrated mixed results in the treatment of sarcomas . 
the phase ii alliance a091401 study examined nivolumab monotherapy and nivolumab plus ipiliumumab in the treatment of metastatic sarcoma.7 two of 38 patients experienced conrmed responses with nivolumab treatment , and six of 38 experienced responses to the combination.7 in a single - arm , phase ii study of pembrolizumab in soft tissue sarcoma or bone sarcoma cohorts , the overall response endpoint was not met , though seven of 40 patients with soft tissue sarcoma and two of 10 patients with liposarcoma experienced responses.8 preliminary activity was seen ( 3 - month progression - free survival , 72.7% ) with the combination of axitinib and pembrolizumab in a single - arm , phase ii study in alveolar soft - palate sarcomas.9 on may 23 , 2017 , the us food and drug administration ( fda ) approved pembrolizumab for the second - line treatment fig 2 . 
penta c and penta d are polymorphic pentanucleotide markers that show identity between the tumor and normal samples . of msi - high ( msi - h ) or mmr - decient ( dmmr ) solid tumors on the basis of ndings in patients retrospectively identied ( keynote - 012 , - 028 , and - 158 ) and prospectively enrolled ( keynote - 016 , - 158 ) .10 , 11 in these trials , msi status was identied prospectively by local laboratory - developed pcr ( n = 60 of 149 patients ) , ihc ( n = 47 of 149 ) , or both ( n = 42 of 149 ) ; retrospectively , 14 of 149 patients were identied by a central laboratorydeveloped pcr test.12 in practice , dmmr status is established by ihc for expression of mmr proteins ( mlh1 , msh2 , msh6 , and pms2 ) , and msi - h is demonstrated by multiplexed pcr of either the national cancer institute panel of two mononucleotide and three dinucleotide the more widely used panel of ve quasimarkers or monomorphic mononucleotide markers ( promega , madison , wi )  . 
at columbia university irving medical center , both ihc and pcr are used , as either can miss individual cases.13 in addition , large ngs panels , such as the one performed in the case , can be used to evaluate for msi . 
the cccp assay interrogates 8 , 682 monoand di - nucleotide repeat loci a 1.27 - mb region of interest and classies tumors as microsatellite stable or unstable according to the number of altered microsatellite loci.14 cases that cannot be denitively classied as microsatellite stable or unstable by the cccp assay are rare and were not seen in the more than 200 cases used in validation , which encompassed a range of tumor types . although the efcacy of programmed death 1 blockade in microsatellite - unstable tumors is well established , 10 , 11 , 15 to our knowledge this is the rst report of a clinical outcome of an intimal sarcoma associated with treatment with immune checkpoint blockade . 
we present this case in part for that reason but also to highlight challenges in executing the mmr - deciency testing that is critical to identify patients who may benet this fdaapproved indication ( table 1 )  . 
assays to evaluate microsatellite instability / mismatch repair deciency at columbia university irving medical center technique pcr fragment analysis what is five quasi - monomorphic mononucleotide evaluated ? repeats ( promega msi evaluation system ; promega , madison , wi ) indel variant detection in microsatellite repeat tracts ( columbia combined cancer panel , a 468 - gene ngs panel ) loss of expression of mlh1 , mlh2 , msh6 , pms2 in tumor cells limitations ? tumor percentage ; tissue type ( eg , lower sensitivity for endometrial or prostate cancers16 ; cancers in constitutional mismatch repair deciency17 ) can affect sensitivity . 
small biopsies , reactive non - neoplastic cells expressing mmr proteins , and loss - of - function variants with retained epitopes can cause false - normal ihc results , while msi testing in noncolorectal cancers may be more difcult to interpret.18 despite their greater overall sensitivity , individual results of ngs panels may be affected by heterogeneity in rates of alterations of individual repeats across tumor types , 19 especially in noncolorectal and endometrial cancers , as highlighted by this case and in a survey of 15 , 045 tumors in which lynch syndrome was identied in 1.6% ( 13 of 699 ) of msi - indeterminate tumors , predominantly noncolon / endometrial cancers ( 10 of 13 ) all of which showed abnormal ihc ( 10 of 10 tested cases ) .20 furthermore , commonly used markers ( by the national cancer institute and promega ) are not among the most frequently altered repeats in the cancer genome atlas exome data , 21 implying that that tests that use limited numbers of markers yield false negatives . 
therefore , when faced with advanced cancers with few options , multimodality testing for mmr deciency may be warranted . affiliations 1department of medicine , medical oncology , columbia university irving medical center , new york city , ny 2department of pathology , molecular pathology , columbia university irving medical center , new york city , ny subject matter of this manuscript . 
henick stock and other ownership interests : abbvie consulting or advisory role : boehringer ingelheim matthew ingham consulting or advisory role : daiichi sankyo research funding : apexigen ( inst ) , mirati therapeutics ( inst ) , ptc therapeutics ( inst ) travel , accommodations , expenses : mirati therapeutics , genentech andrew turk employment : ventana medical systems , bristol - myers squibb , medarex , bayer consulting or advisory role : bayer , bristol - myers squibb , ventana medical systems susan hsiao honoraria : medscape , illumina consulting or advisory role : ventana medical systems , bristol - myers squibb , loxo research funding : bristol - myers squibb mahesh m . 
mansukhani travel , accommodations , expenses : bristol - myers squibb , promega , er squibb sons no other potential conicts of interest were reported . references colmbia university department of pathology and cell biology : columbia combined cancer panel ( cccp )  . 
specialties / personalized - genomic - medicine / oncology - testing / columbia - combined - cancer - panel choi ey , yoon yw , kwon hm , et al : a case of pulmonary artery intimal sarcoma diagnosed with multislice ct scan with 3d reconstruction . 
yonsei med j 45 : 547 - 551 , 2004 alsou b , slater m , smith pp , et al : pulmonary artery sarcoma mimicking massive pulmonary embolus : a case report . 
asian cardiovasc thorac ann 14 : e71 - e73 , 2006 akomea - agyin c , dussek je , anderson dr , et al : pulmonary artery sarcoma mimicking pulmonary embolism : successful surgical intervention . 
ann thorac surg 61 : 1536 - 1538 , 1996 cantaloube m , moureau - zabotto l , mescam l , et al : metastatic intimal sarcoma of the pulmonary artery sensitive to carboplatin - vinorelbine chemotherapy : case report and literature review . 
case rep oncol 11 : 21 - 28 , 2018 long hq , qin q , xie ch : response of pulmonary artery intimal sarcoma to surgery , radiotherapy and chemotherapy : a case report . 
j med case reports 2 : 217 , 2008 dangelo sp , mahoney mr , van tine ba , et al : nivolumab with or without ipilimumab treatment for metastatic sarcoma ( alliance a091401 ) : two open - label , non - comparative , randomised , phase 2 trials . 
lancet oncol 19 : 416 - 426 , 2018 tawbi ha , burgess m , bolejack v , et al : pembrolizumab in advanced soft - tissue sarcoma and bone sarcoma ( sarc028 ) : a multicentre , two - cohort , single - arm , open - label , phase 2 trial . 
wilky ba , trucco mm , subhawong tk , et al : axitinib plus pembrolizumab in patients with advanced sarcomas including alveolar soft - part sarcoma : a singlecentre , single - arm , phase 2 trial . 
waalkes a , smith n , penewit k , et al : accurate pan - cancer molecular diagnosis of microsatellite instability by single - molecule molecular inversion probe capture and high - throughput sequencing . 
bakry d , aronson m , durno c , et al : genetic and clinical determinants of constitutional mismatch repair deciency syndrome : report from the constitutional mismatch repair deciency consortiueur j cancer 50 : 987 - 996 , 2014 . 
this is reflected in the current national comprehensive cancer network ( nccn ) guidelines , which state that genetic testing for brca1 / brca2 should be offered to patients with pdac with close relatives with a brca - associated cancer or to those of ashkenazi jewish ( aj ) ancestry.3 , 4 in the nccn guidelines that focus specifically on pdac , recommendations for genetic evaluation are broad , which suggests that those patients with early - onset pdac , a family history of cancer , or aj ancestry should be referred for genetic counseling.4 the rapidly evolving era of precision oncology , as well as the observation that some of these underlying germline variants are clinically actionable , raises the question of whether our current testing criteria are sufficiently inclusive . 
the three articles that accompany this editorial highlight the importance of germline testing and the need for a broader approach for genetic risk assessment in pdac . to better understand the prevalence of germline mutations in pdac , smith et al5 evaluated germline susceptibility in a high - risk group of patients with pdac who were known to harbor founder mutations . 
the authors assessed 150 patients with pdac with french canadian ( fc ) ancestry for both germline fc founder and nonfounder mutations in four dna - damage repair genesbrca1 , brca2 , palb2 , and atmcomparing mutation rates with 236 patients who were not selected for fc ancestry.5 among the fc group , a 5.3% founder mutation rate was observed . 
for nonaj or - fc patients , the authors suggest that full analysis of the aforementioned genes should be completed in patients with pdac who are diagnosed at age < 50 years or who have a strong family history of hrd - related cancers.5 these recommendations somewhat parallel the current expanded nccn guidelines , with the additional consideration of fc ancestry given the comparable germline mutation rates in fc and aj founder populations . alicia latham schwark zsofia k . 
 in contrast , in the article by reiss et al , 8 the authors suggest that a lower threshold for germline assessment of brca1 , brca2 , and palb2 in patients with pdac may be warranted . 
in this retrospective analysis , clinical outcomes , including overall survival ( os ) and response to chemotherapy , of 29 patients with advanced pdac with known pathogenic variants in brca1 , brca2 , or palb2 were compared with 58 stage , age at diagnosis , year of diagnosis , and gender matched controls.8 improved os was observed in hrd mutationpositive patients who received platinumbased therapy , specifically with 1 - year os in mutation - positive patients being 94% compared with 60% in the control group.8 of importance , mutation status did not predict an improved os in the nonplatinum group.8 this suggests that hrd mutation status may be predictive of the response to platinum therapy , but , by itself , may not be a prognostic marker , although additional studies are needed for clarification . 
interestingly , patients who were at higher risk of harboring a genetic mutation on the basis of clinical parametersthat is , young age , strong family history of cancer , or aj ancestrywere not the majority of patients who were found to have germline alterations , and , therefore , most mutation - positive patients with pdac did not meet current nccn guidelines for testing.8 this is similar to another study that suggested that the majority of brca mutationpositive patients with pdac do not meet traditional testing criteria.7 given the improved os in platinum - treated hrd mutationpositive patients , as well as the lack of clinical parameters that accurately identify hrd mutation carriers , either upfront germline testing or reflex germline assessment after suggestive tumor testing was recommended.8 a potential alternative to germline testing is the development of specific mutation signatures that predict for hrd . 
in the study by shahda et al , 11 91 tumor samples were assessed for an hrd score , with an immediate dropout of 34 samples that failed analysis as a result of low tumor cellularity , 11 which highlights the difficulty in assessing pancreatic tumor tissue for genetic information . 
70% of pdacs , respectively.14 , 18 , 19 given the lack of clinically actionable somatic driver mutations in pdac , it would then seem that an approximate 5% germline mutation prevalence in unselected , nonfounder populations in hrd - associated genes , with implications for treatment , 14 , 19 may be our most promising tool yet for pdac precision oncology . 
in light of the recent us food and drug administration approval of immune checkpoint inhibitors , such as pembrolizumab , for the treatment of all mismatch repair deficient solid tumors , 20 whether evaluation for mismatch repair deficient statuspresent in approximately 1% of pdacsis also warranted remains another important consideration.12 , 19 , 21 , 22 such genomic assessment may seem daunting . there may be concern that the overall burden of testing all pdacs for germline variants is unreasonable . 
 ovarian cancer for brca1 / 2 and colorectal cancer for lynch syndrome , recently updated nccn guidelines now also recommend that all patients with metastatic prostate cancer be referred for genetic evaluation and consideration of testing for brca and related genes.3 this is on the basis of the recent finding that 5% to 11% of such patients harbor underlying germline genetic mutations in dna repair genes , with both prognostic and predictive implications.23 in contrast to prostate cancer , the vast majority of patients with pdac succumb to their disease , with a 5 - year survival of only 8% , which highlights the critical need for identifying opportunities for precision oncology for all patients with pdac.24 , 25 given the dearth of clinically actionable somatic mutations in pdac , as well as our inability to accurately capture all hrdpdacs on the basis of clinical variables , universal germline genetic assessment to identify , at minimum , the 5% of hrdpdacs with emerging treatment and targeted therapy implicationsseems to be a clinically reasonable undertaking . 
beyond the opportunity for precision oncology , knowledge of the presence of an inherited cancer predisposition syndrome will allow for the incorporation of precision prevention oncology for at - risk family members . 
stadler consulting or advisory role : allergan ( i ) , genentech ( i ) , regeneron ( i ) , optos ( i ) , adverum ( i ) alicia latham schwark no relationship to disclose affiliations alicia latham schwark and zsofia k . 
smith al , wc , cuggia a , et al : reflex testing for germline brca1 , brca2 , palb2 and atm mutations in pancreatic cancer : mutation prevalence and clinical outcomes from two canadian research registries . 
telli ml , timms km , reid j , et al : homologous recombination deficiency ( hrd ) score predicts response to platinum - containing neoadjuvant chemotherapy in patients with triple - negative breast cancer . 
domchek sm , aghajanian c , shapira - frommer r , et al : efficacy and safety of olaparib monotherapy in germline brca1 / 2 mutation carriers with advanced ovarian cancer and three or more lines of prior therapy . 
a major barrier is that many conventional designs are inadequate for modern drug development , yet most novel adaptive designs are difcult to understand , require complicated statistical modeling , demand complex computation , and need expensive infrastructure for implementation . 
the objective of this article is to introduce and review a class of novel adaptive designs , known as model - assisted designs , to remove this barrier and increase the use of novel adaptive designs . 
model - assisted designs enjoy superior performance comparable to more complicated , model - based adaptive designs , but their decision rule can be pretabulated and included in the protocolthus implemented as simply as the conventional designs . 
we review state - of - the - art model - assisted designs for phase i clinical trials for single - agent , drug - combination and late - onset toxicity scenarios . 
as a result , conventional designs ( eg , the 3 + 3 design ) are still dominantly used despite relatively poor performance . there is an urgent need to increase the adoption of novel adaptive designs . the objective of this article is to introduce and review a class of novel phase i and ii designs , known as model - assisted designs , 10 - 12 to overcome this quandary of simplicity versus performance . 
model - assisted designs yield superior performance compared with the conventional algorithm - based designs and are comparable to more complicated ( model - based ) designs . with the model - assisted designs , the decision rule can be pretabulated and included in the protocol and , thus , implemented in as simple a way as the conventional designs . 
 yuan et al context key objective is there any novel adaptive design that is simple to implement ? knowledge generated this article introduces and reviews a class of novel phase i and ii designs , known as model - assisted designs , to provide a stateof - the - art approach to overcome the quandary of simplicity versus performance that hinders the adoption of novel adaptive designs . 
model - assisted designs yield superior performance compared with more complicated model - based designs , but the decision rule can be pretabulated and included in the protocol and , thus , implemented in as simple a way as the conventional designs . relevance model - assisted designs are easy to implement and have great potential to improve the efciency and success rate of earlyphase trials . estimate the dlt rate , and has a greater tendency to underdose patients ( ie , it treats patients at the doses lower than the mtd )  . 
despite decades of advocacy by statisticians , the use of the crm , however , is still limited because of statistical and computational complexity of the design.8 , 9 for appropriate use , the crm requires specialized expertise to choose and calibrate the dose - toxicity model and to re - estimate the model at each decision of dose escalation / de - escalation . 
examples of model - assisted designs include the modied toxicity probability interval ( mtpi ) design23 and its variation , mtpi - 224 ; bayesian optimal interval ( boin ) design25 , 26 ; and keyboard design22 ( fig 1 )  . 
given the data observed at current dose , mtpi makes the decision of dose escalation and de - escalation on the basis of the unit probability mass ( upm ) of the three intervals . 
the upm of an interval is dened as the posterior probability that p is within the interval divided by the length of the interval , calculated according to a statistical model known as the beta - binomial model . 
comparison of design characteristics among algorithm - based , model - based , and model - assisted phase i designs algorithm based model assisted model based design characteristic transparency and simplicity in the protocol dose escalation / de - escalation rule can be predetermined and included avoids computation - intensive , repeated estimation of the dose - toxicity curve model to make interim decisions flexibility size targets any prespecied dlt rate allows decision making when the cohort size deviates from the planned no . 
6 sample size can be calibrated to ensure good operating characteristics performance identies the mtd accurately allocates a high percentage of patients to the mtd provides good overdose control abbreviations : dlt , dose - limiting toxicity ; mtd , maximum - tolerated dose . the dose stays at the current dose . 
one deciency of mtpi is that it overly downweighs the overdosing probability , because the overdosing interval is typically wider than the proper dosing interval , which leads to a high risk of overdose of patients ( ie , treatment of a high percentage of patients at doses greater than the mtd ) .22 the keyboard design addresses the overdosing issue of mtpi by dening a series of equal - width dosing intervals ( or keys ) to represent the potential locations of p.22 the proper dosing interval is called the target key . 
the design makes the decision of dose escalation and de - escalation by examining the relative position between the target key and the strongest key , in which the strongest key is dened as the interval that the true dlt is most likely located . 
if the strongest key is on the left ( or right ) side of the target key , the observed data suggest that the current dose is most likely underdosing ( or overdosing ) ; thus , the design escalates ( or de - escalate ) the dose ; otherwise , the dose stays at the current dose . 
the keyboard design outperforms the mtpi with substantially lower risk of overdosing patients and better accuracy to identify the mtd.11 , 22 the variation of the mtpi ( ie , mtpi - 224 ) adopts the same dose escalation / de - escalation rule as the keyboard design but is less transparent . 
the mtpi - 2 relies on complicated procedures , such as occams razor and model selection . compared with the mtpi / mtpi - 2 and keyboard designs , the boin design is more straightforward and transparent ( fig 1 )  . 
let p denote the observed dlt rate at the current dose , dened as the number of patients experiencing dlt at the current dose divided by the total number of dltevaluable patients treated at the current dose . the boin design makes dose escalation / de - escalation recommendations simply by comparing p with prespecied dose escalation ( e ) and de - escalation ( d ) boundaries , as illustrated in figure 2 and described as follows : 1 . 
repeat step 2 until the prespecied maximum sample size is reached or the number of patients treated on a single level reaches a certain number ( eg , 12 )  . 
at that point , select the mtd as the dose at which the dlt estimate is closest to the target.25 , 26 figure 2 provides the default , optimal dose escalation and deescalation boundaries ( e , d ) for commonly used target dlt rates ,  . 
for patient safety , boin imposes an overdose control rule : if the observed data indicate that there is more than a 95% chance that the current dose is higher than and at least three patients have been treated , the current and higher doses are eliminated from the trial . 
to determine the next dose , the modied toxicity probability interval design calculates and compares the values of the three unit probability masses ( upms ) , whereas the keyboard design compares the location of the strongest key with respect to the target key . 
bayesian optimal interval compares the observed dlt rate at the current dose with the optimized dose escalation boundary , e , and de - escalation boundary , d . the simple and intuitive structure of boin gives it several unique advantages . 
because boin guarantees deescalation of the dose when the observed toxicity rate p is higher than the de - escalation boundary d , it is particularly easy for clinicians and regulatory agents to assess the safety of a trial with boin . 
for example , given a target dlt rate of = 0.25 , we know a priori that a phase i trial using boin guarantees de - escalation of the dose if the observed dlt rate is higher than 0.298. 
suppose that , for a phase i trial with a new compound , the regulatory agency mandates that the dose must be de - escalated if the observed toxicity rate is higher than 0.25. 
we can easily fulll that requirement by setting the target dlt rate at = 0.21 , such that boin guarantees de - escalation of the dose if the p 0.25. 
such exibility and observed toxicity rate of transparency are the important advantages of boin compared with the other model - assisted designs , such as mtpi / mtpi - 2 and keyboard designs . because model - assisted designs are built upon rigorous statistical theory , like model - based designs , they also enjoy the same exibility as the model - based designs . 
in addition , unlike the 3 + 3 design , for which the dose escalation and de - escalation decisions can be made only when we have three or six evaluable patients , boin allows decision making with incomplete cohorts in the face of dropouts as a result of dlt inevaluability . 
boin design has been used for various treatment agents , including chemotherapy ( clinicaltrials . gov identier : nct02942264 ) , radiotherapy ( clinicaltrials.gov identier : nct03114462 ) , checkpoint inhibitor ( clinicaltrials . gov identier : nct03600155 ) , monoclonal antibody ( clinicaltrials.gov identier : nct03577704 ) , oncolytic virus ( clinicaltrials.gov identier : nct02705196 ) , and t - cell immunotherapy ( clinicaltrials.gov identier : nct03318900 )  . boin also has also been used in nononcology trials , such as stem cell therapy for stroke patients with stroke.35 we used an ongoing national cancer institute phase i to ii trial ( clinicaltrials.gov identier : nct02942264 ) to illustrate the design . 
one of the trials objectives of was to identify the mtd of tg02 , a pyrimidine - based multikinase inhibitor combined with temozolomide in adult patients with recurrent anaplastic astrocytoma or glioblastoma . because of lack of effective treatments for this patient population , the target dlt rate was set at a relatively high value of 0.35. 
according to the toxicity prole of tg02 in other patients with cancer , the principal vestigator chose 200 mg of tg02 as the starting dose . according to the boin design , the dose will be escalated if the observed dlt rate at the current dose is lower than e = 0.276 , and it will be deescalated if the observed dlt rate is greater than d = 0.419 ( fig 2 )  . 
for example , if the dlt takes up to 8 weeks to evaluate and the accrual rate is one patient per week , on average , then ve new patients could be accrued while investigators wait to evaluate the previous three patients outcomes . 
the question is this : how can new patients receive timely treatment when the previous patients outcomes are pending ? a few model - based designs have been proposed to address this logistic issue , including the time - to - event crm ( titecrm ) 38 and data - argumentation crm ( da - crm ) .39 these designs support continuous accrual and yield superior performance in nding the mtd.39 , 40 however , like the crm , they are statistically and computationally complex and require repeated model tting after each cohort , which limits their use . model - assisted designs , including time - to - event boin ( tite - boin ) 29 and time - to - event keyboard ( tite - keyboard ) designs , 41 provide a well - performing and yet easy - toimplement approach to address late - onset toxicity . 
table 2 shows the tite - boin decision rule with a cohort size of three patients , which can be generated easily using the software described in the software section . 
during the trial , at the current dose , we counted the number of patients , the number of patients who experienced dlt , and the number of pending patients and their standard total follow - up times ( stft ) ; we then used the table to make the dose escalation / de - escalation decision . 
to illustrate the use of the tite - boin decision table , suppose that three patients have been treated at the current dose : one had a dlt , one had no dlt , and one has dlt data pending . 
according to table 2 , if the stft of the pending patient is greater than 0.88 , we treat the next cohort at the same dose ; otherwise , we de - escalate the dose . 
suppose that the next cohort of three patients is treated at the same dose and that , among the six treated patients , one patient had a dlt , two had no dlt , and three have dlt data pending . 
unlike single - agent trials with a string of ordered doses , combinations in the dose matrix are only partially ordered in toxicity , and multiple mtds ( ie , the mtd contour ) may exist in the dose matrix ( appendix fig a1 )  . 
numerous designs have been proposed to nd an mtd or the mtd contour for combination trials.28 , 42 - 52 almost all are model - based designs using a strategy similar to that of a crm : devise a model to describe the dose - toxicity surface and then , on the basis of accumulating data , continuously update the model estimate to select a dose for the new patient . 
because of their statistical and computational complexity , despite good performance , these model - based designs are rarely used for conducting trials . model - assisted designs provide a simple and robust approach to phase i combination trials.43 one example is the boin combination design , 28 which makes the decision of dose escalation / de - escalation according to the same rule as the single - agent boin design described in the modelassisted designs section , and thereby inherits the singleagent designs simplicity and good performance . 
the only difference is that , in combination trials , when we decide to escalate or de - escalate the dose , there is more than one neighboring dose to which we can move . 
dose escalation and de - escalation rule for tite - boin with a target dlt rate of 0.3 and a cohort size of three patients , with up to nine patients treated at a dose no . 
for example , simons optimal ( or minimax ) two - stage design53 could be classied as a model - assisted design in the sense that it is derived from a statistical model ( ie , a binomial model for response ) , but its go / no - go decision rule can be predetermined when the design parameters are specied . 
simons optimal two - stage designs are appropriate for the simple setting in which the end point is binary and quickly ascertainable , and these designs allow only one interim look . phase ii trials sometimes are more complicated and have more than one end point . 
table 3 provides four trial examples with different types of end points ( eg , ordinal end point and coprimary end points ) .54 - 56 in addition , multiple interim looks are useful to improve the exibility and efcacy of the trial , especially in basket and platform trials.57 - 59 the bayesian optimal phase ii ( bop2 ) design60 provides a simple , exible , and efcient model - assisted design to allow multiple interims and handle different types of phase ii trials under a unied framework . the key feature of bop2 is that , although the end points in the four examples are clinically different , they all can be represented using a variable y with k distinct categories , statistically known as a multinomial random variable . 
as an example , consider a treatment that is deemed ineffective if the objective response rate ( orr ) is , in which is a threshold prespecied by clinicians ( eg , = 20% )  . 
at each interim , the go / no - go decision is made according to the following bayesian stopping criteria : stop the trial if pr ( orr | data )  . 
given the response rate deemed ineffective ( ie , the null hypothesis ) and the response rate deemed desirable ( ie , the alternative hypothesis ) , the value of cn is chosen such that the type i error rate is controlled at a prespecied level and the statistical power is maximized . 
 ( see zhou et al60 for details . ) similar bayesian stopping criteria can be applied to other types of end points to determine whether the treatment promising.60 in the equation , as a model - assisted design , one important advantage of the bop2 design is that its stopping boundary can be enumerated and included in the trial protocol before the onset of the trial . 
when they conduct the trial , clinicians simply count the number of relevant events and make the go / no - go decision according to whether that count exceeds the boundary or not . 
if the end point requires a long time to be scored , clinicians may have to suspend the accrual and wait for the interim data mature to make interim decisions . 
use the design decision table to conduct the trial and make adaptive decisions ( eg , dose escalation / stay / deescalation or go / no - go )  . discussion one major barrier for the adaptation of novel adaptive designs is that these designs often are complicated to implement . 
model - assisted designs offer the superior performance compared with the more complicated , model - based adaptive designs but , once designed , can be implemented in as simple a way as the conventional designs . 
the approach establishes a new kiss principle : keep it simple and smart ! as in all trial designs , the design parameters for modelassisted designs must be carefully chosen to reect the clinical setting and the study objective . 
for the boin design , the target dlt rate can vary with the type of phase i studies . the maximum sample size must be realistic and attainable . for the bop2 design , the choice of using a binary end point or coprimary end point , and their null and target rates , depends on which disease type is studied and how effective the standard of care is . 
the number of interim analyses should account for both design efciency and the logistic complexity . the choice of designs parameters should be validated and carefully calibrated through extensive computer simulation to ensure that the design has desirable operating characteristics under a variety of scenarios . 
jack lee consulting or advisory role : abbvie no other potential conicts of interest were reported . references kessel m : the problems with todays pharmaceutical business : an outsiders view . 
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clin cancer res 23 : 3994 - 4003 , 2017 ji y , liu p , li y , et al : a modied toxicity probability interval method for dose - nding trials . 
ruppert as , shoben ab : overall success rate of a safe and efcacious drug : results using six phase 1 designs , each followed by standard phase 2 and 3 designs . 
mu r , yuan y , xu j , et al : gboin : a unied model - assisted phase i trial design accounting for toxicity grades , and binary or continuous end points . 
bernhardt mb , de guzman mm , grimes a , et al : rapid infusion of rituximab is well tolerated in children with hematologic , oncologic , and rheumatologic 34 . 
place ae , goldsmith k , bourquin j - p , et al : accelerating drug development in pediatric cancer : a novel phase i study design of venetoclax in relapsed / refractory 35 . 
postel - vinay s , gomez - roca c , molife lr , et al : phase i trials of molecularly targeted agents : should we pay more attention to late toxicities ? j clin oncol 29 : 1728 - 1735 , 2011 june ch , warshauer jt , bluestone ja : is autoimmunity the achilles heel of cancer immunotherapy ? nat med 23 : 540 - 547 , 2017 38 . 
zhang l , yuan y : designing early - phase drug combination trials , in oquigley et al ( eds ) : handbook of methods for designing , monitoring , and analyzing dose - finding trials . 
ying y , heng z , yanhong z : phase i cancer clinical trial design : single and combination agents , in peace et al ( eds ) : biopharmaceutical applied statistics symposium , volume 1 . 
sacchi s , marcheselli r , bari a , et al : safety and efcacy of lenalidomide in combination with rituximab in recurrent indolent non - follicular lymphoma : final results of a phase ii study conducted by the fondazione italiana linfomi . 
moore kn , sill mw , tenney me , et al : a phase ii trial of trebananib ( amg 386 ; ind#111071 ) , a selective angiopoietin 1 / 2 neutralizing peptibody , in patients with persistent / recurrent carcinoma of the endometrium : an nrg / gynecologic oncology group trial . 
 programmed cell death 1 ( pd - 1 ) ligand ( pd - l1 ) expression in solid tumors as a predictive biomarker of benefit from pd - 1 / pd - l1 axis inhibitors : a systematic review and meta - analysis monica khunger adrian v . 
pennell james stevenson sanjay mukhopadhyay kurt schalper vamsidhar velcheti monica khunger , sagar rakshit , sanjay mukhopadhyay , cleveland clinic ; adrian v . hernandez , university of connecticut / hartford hospital , universidad peruana de ciencias aplicades ; vinay pasupuleti , case western reserve university school of medicine ; nathan a . pennell , james stevenson and vamsidhar velcheti , taussig cancer center , cleveland , oh ; and kurt schalper , yale university school of medicine , new haven , ct . corresponding author : vamsidhar velcheti , md , facp , center for immuno - oncology research , cleveland clinic , cleveland , oh 44195 ; e - mail : velchev@ ccf.org. purpose drugs targeting the programmed cell death 1 ( pd - 1 ) / programmed cell death ligand 1 ( pd - l1 ) pathway show significant clinical activity across several tumor types . 
use of biomarker assays to predict response to these agents is an active area of research ; however , the predictive value of pd - l1 immunohistochemistry ( ihc ) assays is largely inconsistent across clinical trials . 
in this meta - analysis of clinical trials of pd - 1 / pd - l1targeted agents , we evaluate the predictive value of a tumor and tumor - infiltrating immune cell pd - l1 ihc assay as a biomarker for objective response to pd - 1 / pd - l1 inhibitors . methods we searched databases ( pubmed , medline , asco abstracts , european society for medical oncology abstracts , and scopus ) up until december 2016 for clinical trials using pd - 1 / pd - l1 inhibitors with reported pd - l1 biomarker data . 
objective response rates ( primary end point ) from all phase i to iii trials investigating nivolumab , pembrolizumab , atezolizumab , durvalumab , and avelumab in advanced solid tumors were collected . 
odds ratios ( ors ) for response in pd - l1positive patients compared with pd - l1negative patients were calculated using the dersimonian - laird random effects model to combine trials . 
we performed metaanalysis as per the preferred reporting items for systematic reviews and meta - analyses guidelines . results forty - one distinct trials with 6 , 664 patients were identified . 
2017 by american society of clinical oncology introduction tumors often evade the immune system by either ineffective antigen presentation or by using mechanisms to thwart an effector response.1 - 3 programmed cell death 1 ( pd - 1 ) is a negative regulator or checkpoint receptor on t cells that is a key determinant of induction of immune tolerance in the tumor microenvironment . 
pd - l1 and pd - l2 expression in the normal tissues is a major mechanism of physiologic peripheral immune tolerance to dampen tissue autoimmune responses after a sustained inflammatory response to tissue damage . 
pdl1 is upregulated in various tumor types including melanoma , nonsmall - cell lung cancer ( nsclc ) , and squamous cell head and neck carcinomas and is a major mechanism of immune evasion . 
in early trials with these agents , pd - l1 expression on tumor cells ( tcs ) , measured by immunohistochemistry ( ihc ) assay , was thought to be an important biomarker for predicting response to these agents.4 however , subsequent trials , particularly atezolizuincorporated both tc and tumormab trials , infiltrating immune cell ( ic ) pd - l1 expression , and this combined pd - l1 expression was also associated with significantly higher objective response rates ( orrs ) in clinical trials . 
pharmaceutical companies have developed different pd - l1 ihc assays , and their comparability and equivalence are the subject of active research.5 , 6 however , most such studies have included relatively small numbers of patients and have not used response or outcome as an end point . 
the databases searched included pubmed , medline , embase , asco abstracts , european society for medical oncology abstracts , google scholar , and scopus . the dates searched were from the inception of each database to december 4 , 2016 . 
the search terms included the following keywords : pd - 1 , pd - l1 , cd274 , programmed cell death receptor 1 , programmed cell death receptor ligand , immune checkpoint inhibitor , nivolumab , bms936558 , pembrolizumab , mk - 3475 , mpdl3280a , atezolizumab , avelumab , msb0010718c , durvalumab , medi - 4736 , predictive biomarker , and cancer immunotherapy . 
the following information was extracted : study design , study population and setting , mean patient age , type of cancer , type of treatment , pd - l1 expression cutoff values , and follow - up time . 
pd - l1 clones used for the evaluation of pd - l1 expression varied across different trials ; 16 studies used 28 - 8 , seven studies used 22c3 , 10 studies used sp142 , three studies used sp263 , and five studies used dako clone 73 - 10 ( dako , carpinteria , ca )  . 
among all of the included studies , 10 were randomized clinical trials , whereas the rest were single - arm trials . data synthesis and analysis study quality analysis some degree of heterogeneity was expected , and random - effects meta - analyses were performed using dersimonian - laird random effects models.10 to consider the sources of heterogeneity , the following two subgroup meta - analyses were prespecified : type of cancer and type of pd - l1 ihc assay . we used the inverse variance method to calculate pooled odds ratios ( ors ) and their 95% cis . 
i2 values of 30% to 60% represented a moderate level of heterogeneity . we used review manager ( revman 5.3 ; cochrane collaboration , copenhagen , denmark ) for our statistical analyses . results eligible studies our search retrieved 3 , 542 publications . 
the majority of the studies had high risk of bias on performance bias and detection bias . meta - analyses of the association between pd - l1 expression and orr no evidence of publication bias was observed in the funnel plot ( eggers test p = .4 ; appendix fig a2 , online only )  . 
there was low heterogeneity of effects across studies in all categories ( fig 2 )  . we performed a subgroup analysis across all nsclc trials simultaneously assessing cutoff values of 1% , 5% , and 10% for pd - l1positive tcs to identify the optimal ihc cutoff threshold . this analysis included the following four trials : brahmer et al , 14 borghaei et al , 15 rizvi et al , 17 and gettinger et al.20 all of these trials included patients with nsclc ( both squamous and nonsquamous histology ) treated with nivolumab and used the dako 28 - 8 antibody for pd - l1 ihc . 
there was limited heterogeneity of effects across studies in all subgroups ( figs 3a - c )  . an additional subgroup analysis was performed across different pd - l1 ihc assays ( fig 4 )  . 
forest plots representing odds ratios of objective response in programmed cell death ligand 1 ( pd - l1 ) positive patients compared with pd - l1negative patients across different cancer types . 
however , because of the paucity of data and power limitations , we could not compare tc and ic subgroups separately . a major objective of our study was to identify the optimal pd - l1 cutoff value associated with maximum objective response . 
this was achieved by performing a subgroup analysis of four trials simultaneously reporting objective response data for three different cut points ( 1% , 5% , and 10% ) in nsclc.14 , 15 , 17 , 20 we restricted the analysis to these four trials to remove heterogeneity associated with tumor type , treatment agents , and pdl1 antibody used for ihc assay . 
all four trials assessed patients with nsclc ( both squamous and nonsquamous histology ) treated with nivolumab and used the 28 - 8 ( dako ) clone for pd - l1 ihc . 
unfortunately , because of power limitations , we were unable to assess the optimal ihc cutoff points of the us food and drug administrationapproved pd - l1 antibodies 22c3 and sp142 . 
these issues may be significant and require additional study . a standardized biomarker such as tc and ic pdl1 expression can identify patients who will derive maximum benefit from these novel agents , sparing others from potentially harmful immune - related toxicities and elevated costs . 
 a vexing issue is the finding that , in some studies , patients respond to pd - 1 / pd - l1 axis inhibitors despite negative tumor pd - l1 expression . 
this assumes special importance in the current scenario of synergistic treatment combinations to enhance therapeutic response to antipd - 1 therapy . some other limitations to using tumor pd - l1 as a standardized biomarker include different collection times of the pathology specimen and the dynamic nature of the tumor microenvironment . 
pd - l1 staining in most cases is performed on archival formalin - fixed , paraffinembedded tissue , because fresh biopsies just before or close to treatment are often not available . 
it has been suggested that previous lines of therapy might alter tumor pd - l1 expression and may explain some of the confounding results across different trials.11 however , we believe this may not have been a significant confounder in our meta - analysis because , more recently , pd - 1 / pd - l1 inhibitor trials have included fresh biopsy specimens ( within 6 months of starting treatment ) , especially many of the recent trials with pd - 1 / pd - l1 inhibitors in the front - line setting.58 tumor pd - l1 expression  . 
the response rate and progressionfree survival ( primary end point of the study ) were higher in the pembrolizumab group than in the chemotherapy group ( orr , 44.8% in pembrolizumab arm v 27.8% in chemotherapy arm )  . 
this is in stark contrast to the results of the checkmate - 26 trial ( nivolumab v platinum - based chemotherapy in the front - line setting ) , which included treatment - nave patients with advanced nsclc and allowed a more liberal inclusion criterion of  . 
these studies further highlight the importance of incorporating pd - l1 expression into future trials of pd - 1 / pd - l1 moabs , especially in the front - line setting where patients have multiple treatment options . we used objective response as the primary end point of our analysis to ensure inclusion of maximum patients , because mature survival data were not available for many of the recent trials . 
for instance , in the checkmate 17 , checkmate 57 , poplar , and oak trials , objective response did not improve as much as the os in the pd - 1 / pd - l1 arm , suggesting postprogression survival advantage.14 , 15 , 49 , 50 however , this should not significantly affect our aim of establishing the validity of pd - l1 expression as a biomarker because orr is commonly used as a surrogate biomarker for clinical benefit in oncology . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . vinay pasupuleti no relationship to disclose sagar rakshit no relationship to disclose nathan a . 
pennell consulting or advisory role : boehringer ingelheim , astrazeneca , eli lilly , regeneron research funding : genentech ( inst ) , newlink genetics ( inst ) , clovis oncology ( inst ) , astex pharmaceuticals ( inst ) , celgene ( inst ) , astrazeneca ( inst ) , pfizer ( inst ) , merck james stevenson research funding : merck sharp & dohme ( inst ) , bayer ( inst ) , bristol - myers squibb ( inst ) sanjay mukhopadhyay research funding : philips healthcare other relationship : phillips kurt schalper honoraria : takeda research funding : vasculox , tesaro , onkaido therapeutics , genoptix , takeda vamsidhar velcheti honoraria : novartis , foundation medicine , merck , bristolmyers squibb , genentech consulting or advisory role : clovis oncology , genentech , bristol - myers squibb , merck , celgene , foundation medicine , astrazeneca / medimmune , genoptix research funding : genentech ( inst ) , trovagene ( inst ) , eisai ( inst ) , oncoplex diagnostics ( inst ) , alkermes ( inst ) , nantomics ( inst ) , genoptix ( inst ) , altor bioscience ( inst ) , merck ( inst ) , bristol - myers squibb ( inst ) , atreca ( inst ) , heat biologics ( inst ) , leap therapeutics ( inst ) travel , accommodations , expenses : astrazeneca / medimmune , eisai 1 . 
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hamid o , sosman ja , lawrence dp , et al : clinical activity , safety , and biomarkers of mpdl3280a , an engineered pd - l1 antibody in patients with locally advanced or metastatic melanoma ( mm )  . 
hodi fs , kluger h , sznol m , et al : long - term survival of ipilimumab - nave patients ( pts ) with advanced melanoma ( mel ) treated with nivolumab ( anti - pd - 1 ; bms - 936558 , ono - 4538 ) in a phase 1 trial . 
ribas a , puzanov i , dummer r , et al : pembrolizumab versus investigator - choice chemotherapy for ipilimumabrefractory melanoma ( keynote - 002 ) : a randomised , controlled , phase 2 trial . 
daud ai , wolchok jd , robert c , et al : programmed death - ligand 1 expression and response to the anti - programmed death 1 antibody pembrolizumab in melanoma . 
topalian sl , hodi fs , brahmer jr , et al : safety , activity , and immune correlates of anti - pd - 1 antibody in cancer . 2521 - 2532 , 2015 n engl j med 366 : 2443 - 2454 , 2012 30 . 
choueiri tk , fishman mn , escudier bj , et al : immunomodulatory activity of nivolumab in previously treated and untreated metastatic renal cell carcinoma ( mrcc ) : biomarker - based results from a randomized clinical trial . 
mcdermott df , sosman ja , sznol m , et al : atezolizumab , an antiprogrammed death - ligand 1 antibody , in metastatic renal cell carcinoma : long - term safety , clinical activity , and immune correlates from a phase ia study . 
plimack e , bellmunt j , gupta s , et al : pembrolizumab ( mk - 3475 ) for advanced urothelial cancer : updated results and biomarker analysis from keynote - 012 . 
apolo ab , infante jr , hamid o , et al : safety , clinical activity , and pd - l1 expression of avelumab ( msb0010718c ) , an anti - pd - l1 antibody , in patients with metastatic urothelial carcinoma from the javelin solid tumor phase ib trial . 
massard c , gordon ms , sharma s , et al : safety and efficacy of durvalumab ( medi4736 ) , an anti - programmed cell death ligand - 1 immune checkpoint inhibitor , in patients with advanced urothelial bladder cancer . 
rosenberg je , hoffman - censits j , powles t , et al : atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum - based chemotherapy : a single - arm , multicentre , phase 2 trial . 
balar av , galsky md , rosenberg je , et al : atezolizumab as first - line treatment in cisplatin - ineligible patients with locally advanced and metastatic urothelial carcinoma : a single - arm , multicentre , phase 2 trial . 
sharma p , callahan mk , bono p , et al : nivolumab monotherapy in recurrent metastatic urothelial carcinoma ( checkmate 032 ) : a multicentre , open - label , two - stage , multi - arm , phase 1 / 2 trial . 
segal n , ou s , balmanoukian a , et al : safety and efficacy of medi4736 , an anti - pd - l1 antibody , in patients from a squamous cell carcinoma of the head and neck ( scchn ) expansion cohort . 
le dt , bendell jc , calvo e , et al : safety and activity of nivolumab monotherapy in advanced and metastatic ( a / m ) gastric or gastroesophageal junction cancer ( gc / gec ) : results from the checkmate - 032 study j clin oncol 34 , 2016 ( suppl 4s ; abstr 6 ) 43 . 
nishina t , shitara k , iwasa s , et al : safety , pd - l1 expression , and clinical activity of avelumab ( msb0010718c ) , an anti - pd - l1 antibody , in japanese patients with advanced gastric or gastroesophageal junction cancer . 
chung hc , arkenau h - t , wyrwicz l , et al : safety , pd - l1 expression , and clinical activity of avelumab ( msb0010718c ) , an anti - pd - l1 antibody , in patients with advanced gastric or gastroesophageal junction cancer . 
kaufman hl , russell j , hamid o , et al : avelumab in patients with chemotherapy - refractory metastatic merkel cell carcinoma : a multicentre , single - group , open - label , phase 2 trial . 
nghiem pt , bhatia s , lipson ej , et al : pd - 1 blockade with pembrolizumab in advanced merkel - cell carcinoma . n engl j med 374 : 2542 - 2552 , 2016 47 . 
antonia sj , l opez - martin ja , bendell j , et al : nivolumab alone and nivolumab plus ipilimumab in recurrent small - cell lung cancer ( checkmate 032 ) : a multicentre , open - label , phase 1 / 2 trial . 
wakelee h , patel jd , heist r , et al : phase ii trial of atezolizumab for patients with pd - l1 - selected advanced nsclc ( birch ) : updated efficacy and exploratory biomarker results : topic : medical oncology . 
rittmeyer a , barlesi f , waterkamp d , et al : atezolizumab versus docetaxel in patients with previously treated nonsmall - cell lung cancer ( oak ) : a phase 3 , open - label , multicentre randomised controlled trial . 
fehrenbacher l , spira a , ballinger m , et al : atezolizumab versus docetaxel for patients with previously treated nonsmall - cell lung cancer ( poplar ) : a multicentre , open - label , phase 2 randomised controlled trial . 
spiegel dr , chaft je , gettinger sn , et al : clinical activity and safety from a phase ii study ( fir ) of mpdl3280a ( anti - pdl1 ) in pd - l1selected patients with non - small cell lung cancer ( nsclc )  . 
carbognin l , pilotto s , milella m , et al : differential activity of nivolumab , pembrolizumab and mpdl3280a according to the tumor expression of programmed death - ligand - 1 ( pd - l1 ) : sensitivity analysis of trials in melanoma , lung and genitourinary cancers . 
aguiar pn jr , santoro il , tadokoro h , et al : the role of pd - l1 expression as a predictive biomarker in advanced nonsmall - cell lung cancer : a network meta - analysis . 
socinski m , creelan b , horn l , et al : nsclc , metastaticcheckmate 026 : a phase 3 trial of nivolumab vs investigators choice ( ic ) of platinum - based doublet chemotherapy ( pt - dc ) as first - line therapy for stage iv / recurrent programmed death ligand 1 ( pd - l1 ) - positive nsclc . 
poly ( adp - ribose ) polymerase inhibitors ( parpi ) represent one approach , as investigated in previous studies using olaparib.5 - 7 we describe a patient with an unresectable mgmt unmethylated , idh wild - type gbm . 
the patient was enrolled in an observational clinical study ( sponsored by strata through which wholeoncology , ann arbor , mi ) , exome and rna sequencing on formalin - xed parafn - embedded tumor tissue revealed loss - offunction mutations in atm and tumor protein 53 ( tp53 )  . 
the tumor was classied as tumor mutational burden ( tmb ) high , programmed death - ligand 1 ( pd - l1 ) low , and microsatellite stable ( mss ; table 1 )  . 
after completion of therapy , additional review of patients records revealed a germline msh6 loss - offunction mutation , which conrmed lynch syndrome . the genomic ndings and high likelihood of lynch syndrome , along with early - phase data suggesting safety and brain penetration , provided rationale for the treating oncologist to initiate olaparib with standard chemoradiation . 
she was also treated with alternating tumor - treating elds ( optune ; novocure , st helier , jersey ) , an externally applied , low - intensity electromagnetic eld treatment shown to improve survival by 4.9 months over maintenance temozolomide alone when used 18 hours per day.8 device compliance was limited , and treatment was discontinued after 1 month . 
outside hospital records included this nding on germline testing performed by ambry genetics ( aliso viejo , ca )  . msh6 is involved in dna mismatch repair ( appendix table a1 )  . 
most mutations are somatic , found almost exclusively after temozolomide therapy.11 in this setting , they are associated with resistance to alkylating agents , hypothesized to be the dominant mechanism of acquired temozolomide resistance after therapy , likely because of failure of temozolomideinduced dna damage to result in apoptosis in mismatch repairdecient ( dmmr ) cells.12 - 24 when msh6 mutations in treatment - nave patients with mgmt are present methylated , otherwise chemosensitive tumors , treatment response is markedly attenuated.13 beyond treatment resistance , ongoing temozolomide exposure after msh6 inactivation leads to a hypermutator phenotype and tumor progression.14 - 16 parpi restore sensitivity to temozolomide in dmmr cells.25 dmmr cells may also have increased resistance to radiotherapy.26 - 28 lynch syndrome was not identied by somatic testing . often , lynch syndrome is discovered after a tumor is found to be microsatellite instability high ( msi - h ) or dmmr on immunohistochemistry . 
msh6 - mutated brain tumors , however , are often not msi - h by standard polymerase chain reaction testing , and immunohistochemistry is not standardly performed.29 in this case , mss status was determined from next - generation sequencing ( ngs ) on the basis of length variant allele counts at multiple microsatellite loci . 
while alternate ngs methods have demonstrated sensitivity and specicity in brain tumors , this specic methodology in gbm is performance of unknown.30 , 31 while decient msh6 immunohistochemical staining would conrm a pathogenic mutation , intact staining would not rule out dmmr because some mutations result in intact expression of dysfunctional protein.32 - 35 the msh6 mutation was not reported by somatic testing because of its presence in a stretch of repeating cytosines , known as a homopolymer region . 
380 cns tumors associated with protein loss of function.45 as previously mentioned , p53 - decent cells may be particularly vulnerable to dna - damaging treatment when an atm mutation is present . 
conversely , the combination of dmmr and p53 - decient cells worsens response because of failed phosphorylation of p53 and , thus , failed cell arrest after treatment - induced dna damage.46 in general , cancers with mutant p53 have reduced sensitivity to chemotherapy and radiation ; however , there are many instances where mutant p53 has no effect or even enhances treatment effect.47 tmb high tmb was determined from ngs and included noncoding and coding , synonymous and nonsynonymous , and singlenucleotide and multinucleotide variants present at  . 
while frequently performed through immunohistochemistry , classication was based on sequencing results in this patient , using a score derived from the percent of maximum pd - l1 expression across tested tumor samples . 
this method is validated in a lung cancer cohort , but accuracy in gbm is less certapd - l1 is expressed on the surface of most glioma cells , with increased frequency in high - grade gliomas such as gbm , and variable detection is based on technique.52 - 54 of note , several studies demonstrated high pd - l1 association with worse survival gbm.55 there are no currently approved drugs that target pd - l1 in gbm , although several trials are ongoing . 
given the efcacy of programmed death 1 ( pd - 1 ) / pd - l1 blockade in dmmr tumors , immunotherapy could be considered.56 the pd - 1 inhibitor pembrolizumab is approved for all msi - h tumors , making it a treatment option for patients with lynch syndromeassociated cancers , which are typically msi - h . rationale for olaparib parp is involved in single - strand dna break and base excision repair . 
 file , morgan , and khagi expression in gbm.57 olaparib is a parpi that impairs the ddr , increasing treatment - induced chromosomal instability , cell cycle arrest , and apoptosis.58 in patients with germline brca1 / 2 mutations that impair double - strand dna break repair by homologous recombination , parpi cause synthetic lethality with signicant clinical benet.59 - 62 parpi also have efcacy in tumors with mutations in ddr including atm , and in patients with neither genes , a germline brca mutation nor other homologous recombination deciency.63 , 64 parpi have been used as monotherapy and in combination with radiation and chemotherapy to prevent the repair of treatment - induced dna breaks , thereby promoting tumor cell death . 
parpi increase sensitivity to temozolomide in cell and xenograft models of gbm.65 - 67 this effect persists in mgmt unmethylated tumors.68 parpi also restore sensitivity to temozolomide in dmmr cells.25 , 69 in addition , exposure to temozolomide before or concurrently with a parpi increases the magnitude of dna damage and led to complete regression of gbm cells in one study.70 this treatment - sensitizing effect is not present in patients with temozolomide resistance , which suggests optimal corporation in newly diagnosed gbm.71 there have been 3 phase i trials of olaparib with temozolomide and / or radiation in gbm . 
the oparatic trial conrmed tumor penetration and dosing schedule , with promising early results.5 , 7 paradigm - 2 investigated olaparib plus radiotherapy with or without temozolomide.6 , 72 these studies support the addition of olaparib to temozolomide and radiation as safe , well tolerated , and potentially radiosensitizing.5 discussion this patient had an excellent , durable response despite many factors that predict a poor prognosis . 
in addition , somatic msh6 loss - of - function mutations contribute to temozolomide resistance , glioma recurrence , and tumor progression , with similar effects expected from a deleterious germline mutation . 
while atm mutations improve treatment sensitivity , particularly with concurrent tp53 mutations , it is unlikely that this would result in a sustained , near - complete response with standard chemoradiation alone . 
it is also unlikely that tumor - treating elds improved clinical outcome given short duration of use . in addition to the general chemoand radiosensitizing properties of parpi , the ability for parpi to restore sensitivity to temozolomide in dmmr and mgmt unmethylated tumors , as well as efcacy of parpi in ddr - decient tumors , strongly suggests that olaparib was an essential component of the treatment regimen . 
the likelihood of olaparib - induced synthetic lethality is high , through impairment of single - stranded dna break repairs in a tumor already decient in base - base substitution , single - base insertion , and single - base deletion mismatch repair as well as double - stranded dna break repair . genomic sequencing allows identication of patients with targetable mutations who may benet from currently available treatments , which are increasing rapidly.73 this is particularly important for patients predicted to have poor outcomes with standard treatment and limited access to clinical trials . novel treatment approaches in the rst - line setting are needed . 
morgan honoraria : medscape , pharmacy times continuing education travel , accommodations , expenses : medscape , pharmacy times continuing education simon khagi research funding : lambda technologies no other potential conicts of interest were reported . acknowledgment we acknowledge jason merker , md , phd ; william kim , md ; shetal patel md , phd ; amber cipriani , pharmd ; and ashlynn messmore , ms , for their contributions to the university of north carolina multidisciplinary mtb . we also acknowledge carlos zamora , md , phd , for review of the radiographic images and scott tomlins , md , phd , co - founder and chief medical ofcer of strata oncology , for providing descriptions of the sequencing methodology . 
we thank the patient for allowing us to share a part of her story . references stupp r , mason wp , van den bent mj , et al : radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma . 
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j clin oncol 36 , 2018 ( suppl ; abstr 2018 ) fulton b , short sc , james a , et al : paradigm - 2 : two parallel phase i studies of olaparib and radiotherapy or olaparib and radiotherapy plus temozolomide in patients with newly diagnosed glioblastoma , with treatment stratied by mgmt status . 
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jama 318 : 2306 - 2316 , 2017 edelmann w , yang k , umar a , et al : mutation in the mismatch repair gene msh6 causes cancer susceptibility . 
cahill dp , levine kk , betensky ra , et al : loss of the mismatch repair protein msh6 in human glioblastomas is associated with tumor progression during temozolomide treatment . 
nguyen sa , stechishin od , luchman ha , et al : novel msh6 mutations in treatment - nave glioblastoma and anaplastic oligodendroglioma contribute to temozolomide resistance independently of mgmt promoter methylation . 
liu l , markowitz s , gerson sl : mismatch repair mutations override alkyltransferase in conferring resistance to temozolomide but not to 1 , 3 - bis ( 2 - chloroethyl ) 1068 , 2008 [ erratum : nature 494 : 506 , 2013 ] nitrosourea . 
pepponi r , marra g , fuggetta mp , et al : the effect of o6 - alkylguanine - dna alkyltransferase and mismatch repair activities on the sensitivity of human melanoma cells to temozolomide , 1 , 3 - bis ( 2 - chloroethyl ) 1 - nitrosourea , and cisplatj pharmacol exp ther 304 : 661 - 668 , 2003 19 . 
hochhauser d , glynne - jones r , potter v , et al : a phase ii study of temozolomide in patients with advanced aerodigestive tract and colorectal cancers and methylation of the o6 - methylguanine - dna methyltransferase promoter . 
aquilina g , ceccotti s , martinelli s , et al : n - ( 2 - chloroethyl ) - n ( cid : 2 ) - cyclohexyl - n - nitrosourea sensitivity in mismatch repair - defective human cells . 
stark am , doukas a , hugo hh , et al : the expression of mismatch repair proteins mlh1 , msh2 and msh6 correlates with the ki67 proliferation index and survival in patients with recurrent glioblastoma . 
koi m , umar a , chauhan dp , et al : human chromosome 3 corrects mismatch repair deciency and microsatellite instability and reduces n - methyl - n ( cid : 2 ) - nitro - nnitrosoguanidine tolerance in colon tumor cells with homozygous hmlh1 mutation . 
curtin nj , wang lz , yiakouvaki a , et al : novel poly ( adp - ribose ) polymerase - 1 inhibitor , ag14361 , restores sensitivity to temozolomide in mismatch repairdecient cells . 
yan t , schupp je , hwang hs , et al : loss of dna mismatch repair imparts defective cdc2 signaling and g ( 2 ) arrest responses without altering survival after ionizing radiation . 
gylling ah , nieminen tt , abdel - rahman wm , et al : differential cancer predisposition in lynch syndrome : insights from molecular analysis of brain and urinary 30 . 
trabucco se , gowen k , maund sl , et al : a novel next - generation sequencing approach to detecting microsatellite instability and pan - tumor characterization of 1000 microsatellite instability - high cases in 67 , 000 patient samples . 
dudley b , brand re , thull d , et al : germline mlh1 mutations are frequently identied in lynch syndrome patients with colorectal and endometrial carcinoma demonstrating isolated loss of pms2 immunohistochemical expression . 
am j surg pathol 39 : 1114 - 1120 , 2015 iv ady g , madar l , dzsudzs ak e , et al : analytical parameters and validation of homopolymer detection in a pyrosequencing - based next generation sequencing systebmc genomics 19 : 158 , 2018 36 . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
int j radiat oncol biol phys 50 : 511 - 523 , 2001 19 : 3189 - 3200 , 2013 18 : 1899 - 1908 , 2019 jiang h , reinhardt hc , bartkova j , et al : the combined status of atm and p53 link tumor development with therapeutic response . 
tchelebi l , ashamalla h , graves p : mutant p53 and the response to chemotherapy and radiation , in deb s , deb s ( eds ) : mutant p53 and mdm2 in cancer . subcellular biochemistry , volume 85 . 
salem m , grothey a , kim e , et al : impact of mlh1 , pms2 , msh2 , and msh6 alterations on tumor mutation burden ( tmb ) and pd - l1 expression in 1 , 057 microsatellite instability - high ( msi - h ) tumors . 
tentori l , leonetti c , scarsella m , et al : systemic administration of gpi 15427 , a novel poly ( adp - ribose ) polymerase - 1 inhibitor , increases the antitumor activity of temozolomide against intracranial melanoma , glioma , lymphoma . 
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miknyoczki sj , jones - bolin s , pritchard s , et al : chemopotentiation of temozolomide , irinotecan , and cisplatin activity by cep - 6800 , a poly ( adp - ribose ) base excision repair . 
clarke mj , mulligan ea , grogan pt , et al : effective sensitization of temozolomide by abt - 888 is lost with development of temozolomide resistance in glioblastoma xenograft lines . 
lesueur p , lequesne j , grellard jm , et al : phase i / iia study of concomitant radiotherapy with olaparib and temozolomide in unresectable or partially resectable glioblastoma : ola - tmz - rte - 01 trial protocol . 
ahluwalia , md5 ; howard colman , md , phd6 ; geoffrey fell , ms1 ; evanthia galanis , md7 ; john de groot , md8 ; jan drappatz , md9 ; andrew b . 
wen , md1 purpose adequately prioritizing the numerous therapies and biomarkers available in late - stage testing for patients with glioblastoma ( gbm ) requires an efcient clinical testing platforwe developed and implemented insight ( individualized screening trial of innovative glioblastoma therapy ) as a novel adaptive platform trial ( apt ) to develop precision medicine approaches in gbm . methods insight compares experimental arms with a common control of standard concurrent temozolomide and radiation therapy followed by adjuvant temozolomide . 
at the initiation of insight , three experimental arms ( neratinib , abemaciclib , and cc - 115 ) , each with a proposed genomic biomarker , are tested simultaneously . 
as the trial progresses , randomization probabilities adapt on the basis of accumulating results using bayesian estimation of the biomarker - specic probability of treatment impact on progression - free survival . treatment arms may drop because of low probability of treatment impact on overall survival , and new arms may be added . 
detailed information on the statistical model and randomization algorithm is provided to stimulate discussion on trial design choices more generally and provide an example for other investigators developing apts . conclusion insight ( nct02977780 ) is an ongoing novel biomarker - based , bayesian apt for patients with newly diagnosed unmethylated gbm . 
clinical trials in the era of precision medicine must consider how to develop biomarker data during the trials , make efcient use of multiplexed biomarker screening , and develop assignment algorithms for patients positive for more than one biomarker . 
 alexander et al master protocols and adaptive platform trials ( apts ) have been proposed as attractive solutions to efciently address multiple therapeutic and biomarker hypotheses.5 - 7 we developed insight ( individualized screening trial of innovative glioblastoma therapy ) , a multisite investigator - initiated phase ii screening apt , as a solution to precision medicine development challenges for patients with gbm . 
insight was specically designed to enable efcient use of randomly assigned controls , generate information to support genomic biomarker development , and leverage the xed cost of trial development across more experimental therapies . methods eligibility patients are eligible for insight if they have newly diagnosed gbm with unmethylated o6 - methylguanine dna methyltransferase ( mgmt ) gene promoters and negative idh1 r132h mutationspecic immunohistochemistry . 
the marginal benet of temozolomide ( tmz ) in patients with unmethylated mgmt promoters8 offers the opportunity to test experimental therapies without combinations with tmz.9 , 10 this potentially accelerates the overall time for drug development ( by not needing prior separate phase i studies with tmz ) and eliminates the potential for subtherapeutic dose of the experimental agent as a result of overlapping toxicity with tmz.2 , 3 insight therefore can support experimental arms with tmz combinations or with the experimental agent alone . 
experimental therapies may also replace tmz in the concurrent radiation therapy ( rt ) portion ( if there is a compelling radiosensitizing hypothesis ) , the adjuvant portion , or both . a pure radiosensitizing agent or experimental rt regimen could theoretically replace standard rt / tmz while keeping the adjuvant tmz intact . eligibility for insight requires sufcient genotyping data to dene the predetermined biomarker categories for arms currently in the trial . 
additional details regarding the initial experimental arms and biomarkers are included in the data supplement . statistical considerations operating characteristics , and secondary analyses use the proportional hazards ( ph ) model for both pfs and os . there are several arguments to support the use of different outcomes for adaptive randomization ( pfs ) and nal efcacy evaluation ( os )  . 
the relationship between accrual rate and event timing is crucial for response - adaptive trials , because effective variation of randomization probabilities requires rapid generation of treatment effect estimates on the basis of an adequate number of individual outcomes . 
in addition , treatment effects may have a stronger signal on pfs , a relationship illustrated previously.12 finally , potential issues with pseudoprogression and pseudoresponse13 , 14 are mitigated by preserving os as the foundation for stopping rules and nal efcacy analyses . that is , promising early results of an experimental treatment accelerate the accrual rate of the corresponding arm without reducing the nal sample size of the other experimental arms . group - specic adaptive randomization probabilities . 
patients are randomly assigned using an adaptive algorithm that updates randomization probabilities for the various arms in each biomarker group monthly.15 the algorithm uses available information generated by insight ( the individual biomarker groups combined with individual pfs ) to determine the randomization probabilities that will be used to allocate patients for the subsequent month . 
the hazard function for each patient assigned to an experimental arm is modeled , rescaling the baseline by a factor that depends on treatment ( main effect ) , biomarker - specic coefcients , and biomarker - treatment interaction terms . 
we previously described the use of ph models for the computation of randomization probabilities.17 we indicate the individual prole withx ( cid : 1 ) ( x1 , x2 , x3 )  . 
the hazard function x , a ( t ) for the timeto - event outcome of a patient with biomarker prole x , randomly assigned to experimental arm a , is proportional to the baseline ( t )  . 
insight will randomly assign a maximum of 70 patients to each experimental arthe sample size for the initial set of arms is 70 patients per arm across four arms , for a total of 280 patients , but there are early stopping rules for futility in each arm , and other arms may be added as the trial is ongoing . 
such an increase will depend on the number of experimental arms added and the time at which they will start enrollment . the later new arms start enrolling , the larger the corresponding sample size expansion will be . 
indeed , when a new experimental arm is added , the design increases the control - specic sample size to guarantee enrollment of 70 patients in the control after the addition of a novel arwe previously described the adjustment of the control sample size with the addition of novel arms and potential futility early stopping of experimental arms in platforms.18 operating characteristics of outcome adaptive randomization are related to the accrual rate relative to the event rate , because information from events is used to alter probability of random assignment for newer patients . 
biomarker frequencies were assumed on the basis of data from prior genomic proling.19 monthly updates of the randomization probabilities are combined with a sequential decision rule that drops experimental arms when there is insufcient preliminary evidence to warrant additional investigation of the treatment based on the primary end point ( os )  . 
we describe a linear boundary that provides thresholds for predictive probabilities to dene the decision rule . 2 of the main effect prior normal distributions were used for the regression parameters  . 
they are a priori independent , and the variance a a is set to have the 80th and 20th percentiles that correspond to ( log [ 2 ] ; duplication of the hazard ) and ( log [ 1 / 2 ] )  . 
 alexander et al whereas for a hypothetic arm added later during the study , it increases with the number of patients randomly assigned to the novel arm and the concomitant random assignments to the control aradaptive randomization intervenes only after the initial burn - in period . as we discussed previously , 17 , 20 to preserve the power of detecting treatment effects , it is important to guarantee sufcient enrollment in the control arrandomization probabilities for the control are obtained by matching ( under the hypothesis that all the active arms will complete accrual and will not be stopped for futility ) the expected number of patients randomly assigned to the control and the planned sample size specic to the control . 
for example , if after 10 enrollments two patients have been assigned to the control , then the 11th patient is assigned to the control with a probability of ( 70 2 ) / ( 280 10 )  . 
the same approach is used if one experimental arm is dropped for futility or a novel arm is added , with the consequence of an expansion of the control sample size.18 burn - in randomization . 
this is an important difference with respect to other adaptive designs , which include a component of competition and negative correlation with the nal number of patients enrolled by different experimental arms . 
the accrual can be stopped earlier for an experimental arm ( before this maximum is reached ) only when the likelihood of a positive nal result becomes insufcient and triggers the early stopping on the basis of futility . 
as a consequence , the adaptive algorithm has a substantial impact on the duration the arm - specic accrual period , which tends to be shortened for the arms with positive treatment effects , with little effect on other operating characteristics . 
bayesian randomization can only modify the enrollment rate and accelerate accrual to the most promising arms , particularly for the patients who are more likely to benet from these treatments . 
we also predict future biomarker proles x using a standard dirichlet conjugate model . monthly , bayesian sampling is used to generate nal trial data from the predictive distribution , including the enrollment of future patients , and pfs and os outcomes , both for patients previously enrolled and for those who have not yet been enrolled . 
using bayesian terminology , we sample multiple times from the predictive distribution every month . these data sets , including the actual data generated up to a time point by the trial and a complementary component of probabilistically imputed data , describe expectation and uncertainty on how we predict the data to look at completion of the study ( including censored data points ) on the the available information . 
these computations basis of assume that the open arms will reach the nal sample size of 70 patients and allow us to derive a single predictive probability of interest for each experimental arm , at each interim analysis , of a well - dened event . 
prediction ( the probability that the arm - specic result will be signicant ) is used monthly to decide either to continue or to discontinue the study of the experimental ara key step of the process is the simulation of the baseline and the ph model parameters from the posterior at each interim analysis . 
these steps are iterated so that the relative frequency of generated arm - specic p values below the signicance threshold approximates the targeted prediction probability . the arm is stopped if the prediction probability of a positive result becomes small . 
a linear boundary that increases with the number of enrollments from 0 at the onset of the study up to 0.1 is used to dene monthly the cutoff for discontinuing or proceeding with the study of the experimental treatment . 
in the comparison of these simulations with a balanced design , keeping the described early stopping mechanism for futility , enrollment in arms with positive treatment effects was completed faster , with an average time reduction between 16% and 27% across simulation scenarios . a similar power analysis like that for pfs was repeated for os . 
although the power to reject the null was of course considerably the power to detect a signicant biomarker / reduced , treatment interaction ( null hypothesis : no treatment effect in the cdk - positive group ) in the secondary analyses was higher ( 0.66 ; table 2 )  . 
the noncompeting maximum number of patients ( ie , 70 ) per arm maintained the power of detecting a positive treatment effect for each experimental arm stable with respect to the presence of treatment effects on the remaining arms . sensitivity to pfs and os correlation seemed limited . 
we considered both different magnitudes of pfs and os hrs contrasting an experimental treatment and a control ( appendix table a2 ) and various pfs - os correlation degrees at the individual level . 
additional sensitivity analyses , including the probability of reporting a positive treatment effect for the biomarker - positive group under selected scenarios ( appendix table a4 ) , the power for arms added later during the course of the platform trial ( appendix table a5 ) , and the power assuming different possible accrual rates ( appendix table a6 ) , are included in the appendix . 
in contrast to alternative response - adaptive designs , the low correlation of accrual rate with nal armspecic sample size ( which , as described in methods , can be fewer than 70 patients only as a result of early stopping for futility ) induces little variations of power estimates across table 2 . 
in the middle column , only a single arm has positive treatment effects restricted to a single biomarker - positive subpopulation ( prevalence , 0.5 ) and hazard ratios ( hrs ) of 1 for the rest of the patients . 
accrual rate therefore correlates with time required to complete the arm evaluation , but it does not correlate with power . discussion clinical trials under master protocols have been proposed as a methodologic innovation to more efciently answer therapeutic development questions.6 , 7 , 22 such innovations are particularly important in the era of precision medicine , where biomarker testing adds complexity by increasing the testable hypotheses . 
platform trials under number of master protocols are intended to study multiple targeted therapies in the context of a single disease in a perpetual manner , with therapies allowed to enter or leave the platform on the basis of a decision algorithm.7 ( p63 ) potential efciencies include the conservation of control arms , multiplexed biomarker screening data , and reduced downtime because the trial infrastructure is maintained as treatment arms enter or leave the trial . 
platforms also enable innovative statistical approaches to increase efciency.23 - 27 insight is the rst biomarker - based apt designed to apply these general solutions to specic problems in therapeutic development for gbm.28 late - stage clinical trial failures are a major issue for therapeutic development in general1 and in gbm specically.2 , 3 failure in phase iii may be linked to erroneous go / no - go decisions on the basis of phase ii results that have different end points than the desired pivotal trial , overestimate treatment effects on the basis of comparisons with historical controls , or both . 
prior data have shown that effects on imaging - based end points such as response rate and pfs may not translate to effects on os.4 , 13 , 29 - 31 furthermore , comparison of end points like pfs and os with historical controls may overestimate treatment effects through selection bias , temporal drift , and failure to account for control variability.4 for these reasons , we chose to use os as the primary end point of in unmethylated gbm , the trial . survival postprogression is generally short , and there are no proven effective therapies at recurrence that increase the chance of detecting a true therapeutic impact on os.12 we included a randomly assigned control arm to avoid the pitfalls associated with comparison with historical controls for os in gbm.4 the platform design affords considerable efciency by using a single control arm for comparison against multiple therapies and offers patients a higher probability of being randomly assigned to an experimental arfurthermore , we use bayesian rar17 based on accumulating pfs results to increase the probability of random assignment to arms that showed more promise.28 , 32 we had previously shown that rar using an os end point was possible for recurrent gbm17 and additionally supported this approach in our simulations during the development of insight . 
however , to increase efciency , we opted to use pfs , because earlier end point assessment can more rapidly inuence randomization.16 if we were to nd a discordance between therapeutic impact on pfs and os , randomization probability would be altered , but decision making on the primary end point would remain unchanged . another advantage of platform trials is the efcient use of multiplexed genomic biomarker data for treatment assignments . 
gbm is characterized by redundant and overlapping alterations in several molecular pathways rather than mutually exclusive driver mutations.33 as such , patients can be positive for multiple genomic biomarkers . some platform trials like nci - match ( national cancer institute molecular analysis for therapy choice ) , 34 lung map ( lung cancer master protocol ) , 35 battle ( biomarkerintegrated approaches of targeted therapy for lung cancer elimination ) , 36 and n2m2 ( national center for tumor diseases neuro master match ) 37 generally deal with this situation through an assignment algorithm on the basis of relative evidence of biomarker - specic accrual and / or therapeutic efcacy . i - spy 2 ( investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2 ) , in contrast , uses biomarker subgroupspecic randomization probabilities to allow data generated during the trial to drive the biomarker specicity of arm assignments.38 insight does the latter , starting with equal randomization among the experimental arms and allowing the rar procedure to prioritize randomization in a biomarker - specic way . 
for example , if only patients with egfr amplication in the neratinib arm were living longer than those in the control , the randomization algorithm would increase the probability of egfr - amplied patients assigned to neratinib ( regardless of their other biomarkers ) while potentially reducing assignment to neratinib for egfr wild - type patients . in fact , this situation occurred in i - spy 2 , where the adaptive randomization algorithm stopped assigning patients with human epidermal growth factor receptor 2 ( her2 ) negative / hormone receptorpositive cancer and those with her2negative / hormone receptornegative cancer to neratinib during the course of the trial , even as it reached the prespecied efcacy threshold in the her2 - positive / hormone receptor negative signature.39 this strategy may be optimal when there are limited pretrial data or a weak hypothesis suggesting a biomarker - specic effect . 
in these situations , more biomarker specicity may be desired from the start of the trial , and we have suggested ways to integrate this into a platform design.40 more generally , adding future biomarkers specic to additional experimental arms increases design complexity and requires appropriate denitions of the randomization probabilities . 
even though insight used randomization , we queried clinically annotated genomic data33 , 43 to investigate the possibility that the biomarkers we used had prognostic signicance or a variable relationship between pfs and os and found no such associations.19 although the perpetual clinical provided by the apt framework can provide signicant efciencies , it can create additional challenges as well . maintaining ongoing operations requires both nancial framework that trial models that support ongoing concerns and active pipeline development to maintain a regular ow of experimental arms . 
more complex bayesian designs require engaged clinical and statistical investigators and new ways of determining operating characteristics through simulation.44 reporting of trial results is complicated because of the separation between the master protocol and the armspecic data . 
arms that leave the trial because of success or failure need to be reported while the overall trial is still ongoing , which does not lend itself to traditional trial reporting best practices like consort . 
two recent publications from i - spy 2 are examples.39 , 45 conversely , reporting on the overall master protocol does not have a natural time point , although most groups publish a general description of the overall trial structure without results.35 , 36 , 38 , 41 in conclusion , insight is an ongoing novel biomarkerbased bayesian apt for patients with newly diagnosed unmethylated gbm . 
ahluwalia stock and other ownership interests : mimivax honoraria : prime oncology , elsevier , itamar medical ( i ) consulting or advisory role : monteris medical , astrazeneca , bristol - myers squibb , abbvie , cbt pharmaceuticals , kadmon , vbi vaccines research funding : novartis , tracon pharma , novocure , spectrum pharmaceuticals , eli lilly / imclone , boehringer ingelheim , astrazeneca howard colman honoraria : merck sharp & dohme consulting or advisory role : genentech , roche , novocure , omniox , insys therapeutics , abbvie research funding : newlink genetics , plexxikon , kadmon , orbus therapeutics , merck , dnatrix , abbvie , beigene evanthia galanis consulting or advisory role : genentech / roche ( inst ) , abbvie ( inst ) , oncorus research funding : genentech / roche ( inst ) , merck ( inst ) john de groot employment : helsinn therapeutics ( i ) , ziopharm oncology ( i ) leadership : ziopharm oncology ( i ) stock and other ownership interests : gilead sciences , ziopharm oncology ( i ) consulting or advisory role : celldex , deciphera , vascular biogenics , foundation medicine , genentech / roche , omniox , oxigene , abbvie , novogen , kadmon , merck , five prime therapeutics , insys therapeutics , astrazeneca , boston biomedical , gw pharmaceuticals , carthera research funding : deciphera , novartis , eli lilly , sano , emd serono , mundipharma patents , royalties , other intellectual property : sano , research support ; astrazeneca , research support ; emd serono , research support ; eli lilly , research support ; novartis , research support ; deciphera pharmaceuticals , research support jan drappatz employment : via oncology stock and other ownership interests : exelixis , bristol - myers squibb honoraria : uptodate , via oncology consulting or advisory role : oncorus , immunomix patents , royalties , other intellectual property : uptodate andrew b . 
burt nabors consulting or advisory role : bristol - myers squibb speakers bureau : merck / schering plough sandro santagata consulting or advisory role : rarecyte david schiff consulting or advisory role : orbus therapeutics , monteris medical , cavion ( inst ) research funding : bayer healthcare pharmaceuticals ( inst ) mary r . 
wen consulting or advisory role : abbvie , genentech / roche , agios , astrazeneca , karyopharm therapeutics , vivus , monteris medical , aurora biopharma , vascular biogenics , kadmon , ziopharm oncology , gw pharmaceuticals , eli lilly , immunomic therapeutics speakers bureau : merck , agios ( inst ) , abbvie ( inst ) , angiochem ( inst ) , genentech / roche ( inst ) , glaxosmithkline ( inst ) , astrazeneca ( inst ) , immunocellular therapeutics ( inst ) , karyopharm therapeutics ( inst ) , merck ( inst ) , novartis ( inst ) , oncoceutics ( inst ) , sano ( inst ) , ariad pharmaceuticals ( inst ) , vascular biogenics ( inst ) , eli lilly no other potential conicts of interest were reported references thomas dw , burns j , audette j , et al : clinical development success rates 2006 - 2015 . 
j clin oncol 35 : 2402 - 2409 , 2017 vanderbeek am , rahman r , fell g , et al : the clinical trials landscape for glioblastoma : is it adequate to develop new treatments ? neuro oncol 20 : 1034 - 1043 , 2018 grossman sa , schreck kc , ballman k , et al : point / counterpoint : randomized versus single - arm phase ii clinical trials for patients with newly diagnosed glioblastoma . 
nat rev clin oncol 9 : 199 - 207 , 2011 berry sm , connor jt , lewis rj : the platform trial : an efcient strategy for evaluating multiple treatments . 
n engl j med 377 : 62 - 70 , 2017 stupp r , hegi me , mason wp , et al : effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase iii study : 5 - year analysis of the eortc - ncic trial . 
alexander bm , trippa l : getting it rst versus getting it right : weighing the value of and evidence for progression - free survival as a surrogate endpoint for overall survival in glioblastoma . 
han k , ren m , wick w , et al : progression - free survival as a surrogate endpoint for overall survival in glioblastoma : a literature - based meta - analysis from 91 trials . neuro - oncol 16 : 696 - 706 , 2014 30 . 
herbst rs , gandara dr , hirsch fr , et al : lung master protocol ( lung - map ) : a biomarker - driven protocol for accelerating development of therapies for squamous cell lung cancerswog s1400 . 
pfaff e , kessler t , balasubramanian gp , et al : feasibility of real - time molecular proling for patients with newly diagnosed glioblastoma without mgmt promoter hypermethylation : the nct neuro master match ( n2m2 ) pilot study . 
the phosphatidylinositol 3 - kinase ( pi3k ) / akt / mtor signaling axis plays a central role in cell growth , survival , motility , and metabolism in a variety of cancers ( fruman da , et al : nat rev drug discov 13 : 140 - 156 , 2014 ; engelman ja : nat rev cancer 9 : 550 - 562 , 2009 ) , including glioblastoma ( gbm ; brennan cw , et al : cell 155 : 462 - 477 , 2013 )  . 
dna - dependent protein kinase is a serine / threonine kinase involved in the repair of dna double - strand breaks ( collis sj , et al : oncogene 24 : 949 - 961 , 2005 ) , which are considered to be the most lethal dna lesions and the main driver of cellular death after treatment with ionizing radiation therapy ( rt )  . 
therefore , beyond its hypothesized growth - inhibitory effect as monotherapy , cc - 115 has the potential to be a radiation - sensitizing agent in the treatment of gbm ( zhao y , et al : cancer res 66 : 5354 - 5362 , 2006 )  . 
one phase ia / ib multicenter open - label clinical study established 10 mg twice per day as the recommended dose for cohort expansion and phase ii with near - maximal inhibition of phosphorylated akt and partial inhibition of phosphorylated 4ebp ( munster pn , et al : j clin oncol 34 , 2016 [ suppl ; abstr 2505 ] )  . 
because this dose has never been combined with rt , the treatment arm will start with a safety lead - in with the combination before expansion to the full phase ii setting . abemaciclib retinoblastoma ( rb ) protein is a key tumor suppressor that inhibits progression through the g1 checkpoint ( sherr cj : science 274 : 16721677 , 1996 )  . 
abemaciclib is a highly specic atp - competitive cdk4 / 6 inhibitor that induces reversible g1 phase cell - cycle arrest in rb - procient tumor models and is approved for hormone receptorpositive , human epidermal growth factor receptor 2 ( her2 ) negative advanced or metastatic breast cancer . orally dosed abemaciclib achieved brain exposures in excess of the concentrations required for cdk4 / 6 inhibition in an orthotopic rat gbm model and signicantly increased survival alone or in combination with tmz ( raub tj , et al : drug metab dispos 43 : 1360 - 1371 , 2015 )  . 
neratinib , an orally available potent irreversible small - molecule panerbb family tyrosine kinase inhibitor that targets the intracellular tyrosine kinase domains in egfr , is approved for her2 - positive breast cancer ( martin m , et al : lancet oncol 18 : 1688 - 1700 , 2017 ; cancer discov 7 : of1 , 2017 ) 39 and has shown activity in controlling and delaying cns progression of breast cancer metastases ( awada a , et al : jama oncol 2 : 1557 - 1564 , 2016 )  . 
in preclinical gbm studies , neratinib was shown to selectively cause cell death in cell lines harboring genetic activation of egfr and was more effective than other egfr inhibitors in lines harboring the extracellular domain mutations seen in gbm ( vivanco i , et al : cancer discov 2 : 458 - 471 , 2012 )  . 
neratinib has also been shown to exhibit potential for potent inhibition of amplied egfrvii and egfrviii in gbm patient - derived cell - line models ( francis jm , et al : cancer discov 4 : 956 - 971 , 2014 )  . 
neratinib is signicantly more potent than lapatinib in limiting the growth of primary gbm cell lines , and this increased potency is an attractive feature , given that negative clinical trials for lapatinib have been attributed to inadequate tumor concentrations of the drug ( vivanco i , et al )  . 
neratinib will be administered in place of tmz after standard concurrent rt / tmz . biomarkers prospective patients may have biomarker data already available from academic or commercial sources , or they may take advantage of a companion consortium ( abc2 allele consortium ) that generates free portable genotyping data using whole - exome sequencing and copy array testing performed in a clinical laboratory improvement amendmentscertied clinical laboratory for patient use in clinical trials . 
the biomarkers determined are as follows : egfr positive dened as patients with egfr amplication or mutation ; pi3k positive dened as patients with pik3ca mutation / amplication , pik3r1 mutation , akt3 amplication , pik3c2b  . 
one copy gain , or pten dual allele inactivation through either homozygous deletion or deletion plus an inactivating mutation ; and cdk positive dened as patients with rb1 wild type and cdk4 amplication , cdk6 amplication , or cdkn2a or cdkn2b  . 
one copy loss , or cdkn2a or cdkn2b one copy loss plus an inactivating mutation . for amplications listed , the genotyping report must state clear gene amplication and not gain , which is typically greater than a log2 ratio of + 2.0. 
copy number losses would be values of , 0.3 , and more than single copy deletions are inferred relative to baseline for the chromosome on which they are located ( eg , single copy chromosome 9 loss with additional loss of cdkn2a / b below this level in focal region )  . 
ve prior events reported in cosmic or are well - established hotspots known to be activating or inactivating mutations through experimental data or novel mutations that are predicted to be clearly inactivating , such as nonsense and frameshift mutations . 
heterozygous germline mutations in brca1 or brca2 confer up to an 85% lifetime risk of breast and a 15% to 40% risk of ovarian cancer , and they significantly increase the risk of pancreatic , prostate , and male breast cancers.1 tumors in these patients have often lost the wild - type allele and therefore are hr deficient . 
in the absence of a functional copy of the brca gene that maintains dna integrity , the use of error - prone , nonhomologous end - joining repair mechanisms leads to an accumulation of genetic aberrations and promotes carcinogenesis.2 inhibition of poly ( adp - ribose ) polymerase ( parp ) induces synthetic lethality in cells with brca1 or brca2 deficiency , and clinical trials of parp inhibitors have demonstrated responses in brcamutated breast and ovarian cancers.3 the bestdescribed mechanisms of resistance to platinum agents and parp inhibitors in brca1 or brca2 carriers with ovarian cancer are secondary mutations that restore the open reading frame or reversion mutations that restore the wild - type protein.4 , 5 , 6 these mutations are present in more than 40% of recurrent platinum - resistant ovarian cancers and are more common in women with recurrent ovarian carcinoma who were previously treated for breast cancer3 , 6 . 
minimal data exist about resistance to single - agent parp inhibitor therapy in women with breast cancer.7 we report a case of a brca2 mutation carrier who received single - agent parp inhibitor therapy and had subsequent resistance conferred by a reversion mutation identified by targeted capture and massively parallel sequencing . carcinoma of the right breast with multiple positive regional lymph nodes , for which she underwent a mastectomy and axillary node dissection and received adjuvant chemotherapy with fluorouracil , epirubicin , andcyclophosphamidefollowed by tamoxifen for 5 years . 
additional details of her initial care are not available , and cancer surveillance was not performed . after presentation at age 72 with back pain , workup identified a left breast mass with extensive metastatic disease to the bone . 
biopsy of the breast mass confirmed a new primary invasive ductal carcinoma that stained positive for estrogen receptor and progesterone receptor expression and negative for her2 / neu . her disease was staged as ct4an2m1 , and she started anastrazole monotherapy . 
 genomic dna isolated from peripheral blood to evaluate for germline mutations in brca1 and brca2.8 the uw - oncoplex assay ( laboratory medicine , university of washington ) was used to perform whole - exome deep sequencing and copy number variation analysis to identify somatic mutations in dna isolated from formalin - fixed , paraffinembedded tumor tissue.8 , 9 neoplastic cellularity was assessed by estimating the fraction of neoplastic to nonneoplastic nuclei on a hematoxylin and eosin stained section by a trained pathologist . 
predicted allelic frequency for the retained alleles of heterozygous germline variant of brca2 ( and reference single nucleotide polymorphism [ snp ] 2126042 ) was calculated with the following formula : [ ( tumor percentage ) + ( nontumor percentage ) ] 4 [ ( tumor percentage ) + 2 ( nontumor percentage ) ]  . 
predicted allelic frequency for the wild - type brca2 ( and reference snps 1799943 , 1799955 , and 1801406 ) was calculated as 100 2 the predicted allelic frequency of the variant . the fred hutchinson cancer research center consortium institutional review board approved this study . 
the reference snps were mapped to separate brca2 alleles with a predicted germline allelic frequency of approximately 50% ( table 1 )  . the pre - veliparib sample ( a bony lesion ) had a lower neoplastic cellularity ( 25% ) than the postveliparib sample ( chest wall ; 78% )  . 
the variant allele frequency of rs2126042 , present on the same copy of the mutated brca2 allele , was 60% in the preand 92% in the post - treatment sample . 
the allelic frequency of mutated brca2 in the pre - treatment sample is consistent with the loss of heterozygosity of the wild - type allele ( table 1 ; fig 1 )  . 
in the posttreatment sample , the marked discrepancy in the allelic frequency of the mutated brca2 ( 50% ) and rs126042 ( 92% ; both expected to be on the same allele ) , along with the low allelic frequency of other heterozygous germline snps ( predicted frequency , approximately 18% ) present on different copies of the alleles , strongly supports the restoration of wildtype brca sequence on the mutated allele ( table 1 )  . these collective findings suggest a reversion of the brca2 frameshift deletion mutation on the mutant allele during treatment ( fig 1 )  . 
in the lymphocytes ( germline ) , the wild - type brca2 allele and the heterozygous reference single nucleotide polymorphisms ( snps ) rs1799943 , rs1799955 , and rs1801406 were detected , along with the brca2 c.9097dup and a heterozygous snp , 2126042 . 
this method greatly simplifies the detection of such events and has important implications for the clinical care of patients with brca - mutant disease . we predict that similar phenomena may be sensitively identified earlier , before disease progression , with new platforms that incorporate next - generation sequencing to circulating tumor dnaso - called liquid biopsies . we conclude that the somatic reversion mutation was the mechanism of resistance to parp inhibitor and was responsible for progression during therapy . 
in the germline sample , the wild - type brca2 allele and the heterozygous reference single nucleotide polymorphisms ( snps ) rs1799943 , rs1799955 , and rs1801406 were detected together , along with the brca2 c.9097dup and a heterozygous snp rs2126042 . 
gadi stock and other ownership interests : sengine precision medicine research funding : genentech / roche ( inst ) affiliations all authors , university of washington ; kalyan banda and v.k. 
pritchard cc , salipante sj , koehler k , et al : validation and implementation of targeted capture and sequencing for the detection of actionable mutation , copy number variation , and gene rearrangement in clinical cancer specimens . j mol diagn 16 : 56 - 67 , 2014 10 . 
computed tomography ( ct ) scan with ( a ) the index lesions in the breast ( arrows ) before parp inhibitor and ( b ) 5 months later , which demonstrates decreased volume of disease . 
the absolute numbers of variant alleles and total number of alleles are in parentheses . mutations in tp53 , depdc5 , plk2 , and tacc3 are present in both preand post - treatment biopsies but are enriched in the post - treatment sample . 
 high response to cetuximab in a patient with egfr - amplied heavily pretreated metastatic triple - negative breast cancer renaud sabatier , md , phd1 , 2 ; marc lopez , phd1 ; arnaud guille , msc1 ; emilien billon , md1 ; nadine carbuccia , msc1 ; s everine garnier , phd1 ; jos e adelaide , msc1 ; jean - marc extra , md2 ; maria - antonietta cappiello , md2 ; emmanuelle charafe - jauffret , md , phd1 ; jihane pakradouni , phd2 ; patrice viens , md1 ; anthony gonalves , md , phd1 ; max chaffanet , phd1 ; daniel birnbaum , md , phd1 ; and franois bertucci , md , phd1 introduction represent triple - negative breast cancers ( tnbcs ) 15% of breast cancers , 1 and patients with tnbc have an increased likelihood of distant recurrence and death compared with women with estrogen receptor and / or human epidermal growth factor receptor 2 positive tumors.2 although early tnbcs display frequent epidermal growth factor ( egfr ) overexpression , 3 incidence of egfr gene alteration is not clearly known in metastatic samples , and egfrtargeted agent results have been disappointing for this subtype , whether used alone or with chemotherapy.4 , 5 receptor cetuximab is a chimeric monoclonal antibody ( mab ) specic for egfr ; it interacts with egfr and blocks the downstream pathway , which interferes with the proliferation of cancer cells . 
however , their place in tnbc treatment is unclear , because early clinical studies showed contradictory results.4 , 6 we identied a patient with progressive metastatic tnbc resistant to chemotherapy and immunotherapy . 
however , disease progression occurred 8 months after treatment initiation . we explored all the tumor samples we had from this patient to identify resistance mechanisms both at the genomic and protein levels . 
she received anthracyclines and taxane - based neoadjuvant chemotherapy and underwent mastectomy , which revealed a residual 6cm triple - negative inltrating ductal carcinoma as well as residual axillary lymph node involvement ( 9n + / 14 )  . treatment was completed by chest wall and lymph node irradiation . 
whole genomic proles were established by the array comparative genomic hybridization approach for the precetuximab biopsy ( blue line ) , postcetuximab biopsy ( brown line ) , and patient - derived xenograft ( pdx ) sample ( red line )  . 
from left to right , chromosome 7 ideogram with its genomic prole showing 7p11.2 amplication , with focus on the 52 , 072 , 97957 , 560 , 519 ( 5.48 mb ) amplied region that includes egfr gene . 
from left to right , chromosome 19 ideogram with its genomic prole showing 19q12 amplication , with focus on the 27 , 571 , 467 - 33 , 059 , 007 ( 5.48 mb ) amplied region that includes ccne1 gene . 
a new subcutaneous tumor biopsy was performed and analyzed by whole - genome array comparative genomic hybridization ( cgh ; highresolution 4 180 , 000 cgh microarray ; agilent technologies , massy , france ) and targeted next - generation sequencing ( nextseq500 ; illumina , san diego , ca ) as previously described.7 array cgh analysis identied focal amplication within the 7p12 region ( fig 2 ) , with  . 
before genomic results were known , the patient received additional therapies , including weekly paclitaxel , vinorelbine , and a programmed death - 1 inhibitor ( cpdr001x2101 trial ) , with subsequent visceral and cutaneous progression . in february 2017 , we proposed initiation of cetuximab therapy ( 250 mg / m2 per week ) as seventh - line therapy for metastatic disease . 
however , one of the main tyrosine kinase domains of egfr ( y1068 ) had lost its phosphorylation after progression , suggesting that egfr was no longer activated ( figs 4a and 4b )  . 
in contrast , akt phosphorylation was not modied after cetuximab treatment , suggesting no downstream activation of the phosphatidylinositol 3 - kinase in addition , sequencing ( pi3k ) / akt pathway ( fig 4a )  . showed a loss - of - function mutation of nf1 , absent in the precetuximab sample , suggesting that the mitogen - activated protein kinase ( mapk ) pathway might have been involved in cetuximab resistance . because she consented to participate in our permed - 01 trial , the patient also provided her consent to publication of data related to this prospective study . 
after a molecular tumor board decision , all clinical procedures were performed as routine clinical management . discussion we report here the case of a patient with heavily pretreated metastatic tnbc with egfr amplication who experienced a dramatic response to cetuximab as seventh - line treatment for metastatic disease . 
failure of egfr inhibitors in patients with tnbc may result not only from a lack of molecular data in the metastatic setting , but also from the misidentication of the right predictive molecular alteration . investigations exploring egfr mabs in breast clinical cancer have only correlated treatment efcacy with protein expression . 
first , immunohistochemistry is not excellent ( specicity , 78% ; sensitivity , 97% ) when compared with in situ hybridization.10 second , most cases of breast cancer with egfr overexpression involved gene polysomy instead of real gene amplication . 11 , 12 the biologic impact of these two alterations could be different , and amplied cases have been described as having better prognoses , 13 whereas polysomic cases seem to have poor outcomes.11 this prognostic impact could be correlated with treatment efcacy and higher sensitivity to egfr inhibitors.14 to evaluate how many patients with breast cancer could be candidates for this treatment , we explored egfr amplication incidence in primary breast tumors as well as in metastatic samples from the cancer genome atlas15 and genomics evidence neoplasia information exchange16 databases . 
cell lysates were separated on 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis . loading charge was ve times more in the postcetuximab sample , taking into account tumor cellularity ( 20% )  . 
p - egfr ( y1068 ) expression was detectable in the precetuximab ( here is shown a sample from a patient - derived xenograft [ pdx ] model developed from the precetuximab biopsy ) and not in the postcetuximab sample , even after extended exposure time . 
this suggests that this subtype is more likely to benet from egfr inhibitors . most randomized clinical trials assessing egfr mab efcacy in breast cancer were not designed to explore treatment impact according to biologic features . 
in the tbcrc001 phase ii trial comparing cetuximab monotherapy with a combination of carboplatin and cetuximab for metastatic tnbc , survival was poor in both arms.17 gene expression analyses showed that cetuximab was active in the egfr pathway in a few cases . 
another phase ii trial , which compared cisplatin with cisplatin plus cetuximab in the same setting and did not include biomarker analysis , did not show overall response improvement with egfr inhibition.4 in a cohort of patients with egfr - overexpressing tnbc treated with a taxane and cetuximab doublet , median overall survival was 12 months.6 nevertheless , no correlation between response and egfr amplication status was investigated . in phase ii trials exploring the impact of cetuximabor panitumumab - based combinations in the neoadjuvant setting , 18 , 19 treatment table 1 . 
other alterations favoring secondary resistance to egfr inhibitors , such as downstream alterations in the pi3k / akt / mammalian target of rapamycin pathway , including kras , pi3kca , and akt mutations and pten deletions , were not observed in our patients tumor sample.31 , 32 other mechanisms of resistance were not fully explored in our work , such as amplication of met , which is involved in resistance to egfr inhibitors.33 another point we could not explore was whether the combination of egfr inhibitors and cytotoxic chemotherapies may be more efcient than monotherapies , as recommended for metastatic colorectal and head and neck cancers . 
efcacy of cetuximab and panitumumab as monotherapies is indeed poor in these contexts.34 , 35 regarding tnbc , platinum compounds are presumably efcient partners that may deserve to be explored , because tnbc tumors harbor frequent dna repair alterations.36 in conclusion , most clinical studies exploring anti - egfr mabs have been disappointing . 
our case of tnbc and recent data in cancers other than breast cancer suggest we should give more consideration to egfr amplication than single immunohistochemical protein expression in selecting patients with metastatic tnbc for clinical trials evaluating anti - egfr agents . 
mechanisms of resistance remain unclear in responders to egfr inhibitors . efcacy seemed to be linked not only to egfr protein expression but also to a high cd8 + / foxp3 - positive tumorinltrating lymphocyte ratio , suggesting a potential impact of antibody - dependent cellular cytotoxicity activation.20 , 21 egfr amplication was associated with clinical response to cetuximab in colon cancer , squamous cell lung cancer , and refractory gastroesophageal adenocarcinoma.22 - 24 egfr copy number was also described as correlating with egfr tyrosine kinase inhibitor ( tki ) efcacy in a metaanalysis including  . 
however , such a combination is unlikely to be developed in the clinical setting because of skin and digestive toxicities . results that would improve our understanding of mechanisms underlying secondary resistance to cetuximab are lacking . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . anthony gonalves research funding : merck sharp & dohme ( inst ) , bristol - myers squibb ( inst ) , novartis ( inst ) , cascadian therapeutics ( inst ) , nektar ( inst ) , boehringer ingelheim ( inst ) , eli lilly ( inst ) , abbvie ( inst ) , roche / genentech ( inst ) , astrazeneca ( inst ) , roche ( inst ) travel , accommodations , expenses : pzer , novartis , roche / genentech , celgene no other potential conicts of interest were reported . acknowledgment we thank the datacenter it and scientic computing of the centre de recherche en canc erologie de marseille , which provided computing resources for this study . references foulkes wd , smith ie , reis - filho js : triple - negative breast cancer . 
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levva s , kotoula v , kostopoulos i , et al : prognostic evaluation of epidermal growth factor receptor ( egfr ) genotype and phenotype parameters in triple - negative breast cancers . 
el guerrab a , bamdad m , kwiatkowski f , et al : anti - egfr monoclonal antibodies and egfr tyrosine kinase inhibitors as combination therapy for triple - negative breast cancer . 
carey la , rugo hs , marcom pk , et al : tbcrc 001 : randomized phase ii study of cetuximab in combination with carboplatin in stage iv triple - negative breast cancer . 
multicentric neoadjuvant phase ii study of panitumumab combined with an anthracycline / taxane based chemotherapy in operable triple negative breast cancer : identication of biologically - dened signatures predicting treatment impact . 
roberti mp , barrio mm , bravo ai , et al : il - 15 and il - 2 increase cetuximab - mediated cellular cytotoxicity against triple negative breast cancer cell lines 21 . 
roberti mp , juli a ep , rocca ys , et al : overexpression of cd85j in tnbc patients inhibits cetuximab - mediated nk - cell adcc but can be restored with cd85j 22 . 
moroni m , veronese s , benvenuti s , et al : gene copy number for epidermal growth factor receptor ( egfr ) and clinical response to antiegfr treatment in 23 . 
herbst rs , davies am , natale rb , et al : efcacy and safety of single - agent pertuzumab , a human epidermal receptor dimerization inhibitor , in patients with non 24 . 
zhang x , zhang y , tang h , et al : egfr gene copy number as a predictive / biomarker for patients with non - small - cell lung cancer receiving tyrosine kinase inhibitor treatment : a systematic review and meta - analysis . 
mei z , shao yw , lin p , et al : smad4 and nf1 mutations as potential biomarkers for poor prognosis to cetuximab - based therapy in chinese metastatic colorectal i10 - i19 , 2018 ( suppl ) cancer patients . 
personeni n , hendlisz a , gallez j , et al : correlation between the response to cetuximab alone or in combination with irinotecan and the activated / phosphorylated epidermal growth factor receptor in metastatic colorectal cancer . 
li `evre a , ouine b , canet j , et al : protein biomarkers predictive for response to anti - egfr treatment in ras wild - type metastatic colorectal carcinoma . 
therkildsen c , bergmann tk , henrichsen - schnack t , et al : the predictive value of kras , nras , braf , pik3ca and pten for anti - egfr treatment in metastatic colorectal cancer : a systematic review and meta - analysis . 
yang z , yang n , ou q , et al : investigating novel resistance mechanisms to third - generation egfr tyrosine kinase inhibitor osimertinib in non - small cell lung 33 . 
bean j , brennan c , shih j - y , et al : met amplication occurs with or without t790m mutations in egfr mutant lung tumors with acquired resistance to getinib cancer patients . 
segelov e , thavaneswaran s , waring pm , et al : response to cetuximab with or without irinotecan in patients with refractory metastatic colorectal cancer harboring the kras g13d mutation : australasian gastro - intestinal trials group icecream study . 
couch fj , hart sn , sharma p , et al : inherited mutations in 17 breast cancer susceptibility genes among a large triple - negative breast cancer cohort unselected for family history of breast cancer . 
however , the clinical application of these targeted drugs and diagnostics often remains unclear . however , this approach is not newdoctors have been trying to provide personalized care with high precision for centuries . 
for example , during the last 150 years , virchow developed microscopic pathology , roentgen introduced radiographic imaging , and bloom and richardson proposed a tumor grading scale ; all represented major leaps forward in personalized medicine that we still apply.1a - 1e more recent examples include tests for estrogen receptor in breast cancer to guide endocrine therapy and evaluation of the cd20 antigen on b - cell lymphomas to guide the use of anti - cd20 monoclonal antibodies . so , whats new ? we have new and exciting tools that rival those giants on whose shoulders we stand . 
however , to deliver personalized medicine as precisely as possible , we need high levels of evidence that use of these new therapies and diagnostic tests does , in fact , improve patient care . we agree with the skeptics in oncology who question the benefit of precision oncology , but we maintain that the answer is not to declare it all hype and walk away but to continue to learn how best to use it . 
rather than hope for luck , clinicians should continue to use the scientific method to make evidence - based decisions . in may 2013 , journal of clinical oncology published a special series on precision oncology . the success of this special series led asco to recognize both the plethora of research that is occurring in this field and the need to disseminate this information effectively to the oncology community . 
the special issue of jco featured articles that outlined the current and emerging technologies for tumor genomic profiling , 1f ways to build personalized medicine infrastructure at major cancer centers , 2 and various applications of genomic profiling for different cancer diagnoses . 
it will also provide researchers and clinicians with a reliable source of high - quality precision oncology research , reviews , and commentary essential to real - world cancer care . 
in addition to original clinical research , the journal will publish articles about key care delivery issues , such as value , quality , and cost - effectiveness , and about ethical , legal , and social considerations to inform clinical practice and shape this rapidly evolving field . we are thrilled to have james m . 
ford is a professor of medicine ( oncology ) , pediatrics ( medical genetics ) , and genetics and is the director of the stanford cancer genetics clinic and the cancer genomics program at the stanford cancer institute . 
hayes , university of michigan comprehensive cancer center , ann arbor , mi ; and julie vose , university of nebraska medical center , omaha , ne corresponding author : daniel f . 
 genetic targets for personalized targeted therapies in breast and gastrointestinal cancer as well as in examination of the role of dna repair in the treatment and prevention of cancer in families and populations . 
with the commencement of the targeted agent and profiling utilization registry ( tapur ) study , asco aims to describe both the safety and the efficacy of targeted anticancer drugs to catalog genomic profiling tests and to learn about the utility of registry data . 
in addition , with the launch by asco of cancerlinq , oncologists will have access to a massive database of treatment options and results of those treatments from millions of patient records . 
vose consulting or advisory role : bio connections research funding : celgene ( inst ) , genentech ( inst ) , incyte ( inst ) , janssen biotech ( inst ) , acerta pharma ( inst ) , kite pharma ( inst ) , seattle genetics ( inst ) , novartis ( inst ) , amgen ( inst ) , bristol - myers squibb ( inst ) , allos therapeutics ( inst ) references 1a . 
hammond me , hayes df , dowsett m , et al : american society of clinical oncology / college of american pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer . 
wolff ac , hammond me , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
 circulating tumor dna in nonsmall - cell lung cancer : a primer for the clinician circulating tumor dna ( ctdna ) consists of short , double - stranded dna fragments that are released into the circulation by tumor cells . 
the assays noninvasive nature , ability to reflect intratumoral heterogeneity , short turnaround time , and ability to obtain serial samples make it an attractive option compared with traditional tissue biopsy tumor sequencing . 
currently , this technology is mostly being used for the detection of egfr mutations in patients with advanced nonsmall - cell lung cancer where tissue is inadequate to detect egfr mutations that drive acquired resistance , most notably egfr t790m . 
emerging uses include the incorporation of ctdna testing into primary diagnosis , treatment monitoring , detection of minimal residual disease , and detection of early - stage disease in screening populations . 
2017 by american society of clinical oncology introduction including tumors , shed short , several tissues , double - stranded dna fragments called cell - free dna ( cfdna ) into the circulation.1 - 3 certain conditions that cause tissue damage , such as myocardial infarction , trauma , and cancer , can result in increased concentrations of cfdna.3 the cfdna derived from tumors , known as circulating tumor dna ( ctdna ) , is a product of apoptosis and necrosis and is distinguished from other cfdna by the presence of somatic mutations representative of tumor biology absent in normal cells . 
90% of all dna present in the plasma.4 , 5 in patients with advanced nonsmall - cell lung cancer ( nsclc ) , ctdna detection rates that range from 80% to  . 
unlike acquired t790m resistance to egfr tkis , one dominant secondary mutation appears not to exist , and second - generation alk inhibitors have shown high response rates in patients previously treated with crizotinib regardless of the presence of secondary alk mutations , which makes the role of a repeat biopsy less clear.27 however , emerging data suggest that specific secondary resistance alk mutations have important therapeutic implications . 
in a study that detected egfr mutations by an ngs assay , the sensitivity for urine was 93% for t790m , 80% for l858r , and 83% for exon 19 deletions . 
in nine patients monitored while receiving the third - generation egfr inhibitor rociletinib , a rapid decrease in urine ascopubs.org / journal / po jco precision oncology 7 t790m was 70% , and the objective response rate and median progression - free survival ( pfs ) with osimertinib were similar in patients with t790mpositive plasma or tumors.10 this finding suggests that with the use of ctdna testing , many patients can avoid a biopsy for t790m genotyping . 
however , because of the 30% false - negative rate of plasma genotyping , those with t790m - negative plasma still need to undergo tumor biopsy to determine the presence or absence of t790m.10 in addition to cases where ctdna is not detectable , tissue testing retains value in certain resistance mechanisms , such as small - cell transformation . thress et al24 performed ngs of cfdna from seven patients with t790m - mutated advanced lung cancer who had developed resistance to osimertinib and detected an acquired egfr c797s mutation . 
 t790m levels was observed by day 21.31 in a study of patients with t790m positivity , response was similar whether t790m status was identified by tissue , plasma ( through beaming ) , or urine ( through ngs ) .32 these data suggest that plasma and urine egfr analyses complement tissue biopsy specimen analysis in nsclc . brain metastasis is found in up to 40% patients with nsclc , 33 and ctdna analysis of patients with brain tumors has revealed either the absence or very low levels of tumor dna possibly as a result of the blood - brain barrier.34 one study used hybridization capturebased sequencing and ddpcr to characterize ctdna in csf of patients with brain lesions ( including brain metastasis from lung cancer ) and compared it with plasma ctdna.35 the authors concluded that csf ctdna has a significantly higher sensitivity than plasma for cns genomic alterations like egfr , pten , esr1 , idh1 , erbb2 , and fgfr2 and can be used to detect brain tumor mutations and monitor brain tumor progression ( sensitivity of ctdna was 58% v 0% in csf and plasma , respectively , in cns - restricted disease )  . 
hata et al37 investigated the response to osimertinib in 10 patients with leptomeningeal disease and showed that the drug might be more effective in csf of patients with t790m positivity than in those without csf t790m positivity . multigene testing as the number of genomic targets with matched therapies grows , the national comprehensive cancer network guidelines for nsclc recommend expanded genomic testing , including for egfr , alk , erbb2 , braf , met , ros1 , and ret.5 , 15 , 38 testing for all these alterations in addition to histopathologic and immune biomarker assessment pose a challenge when tissue biopsy samples are insufficient . 
in a study of patients with advanced nsclc , a tissue biopsy specimen with sufficient quality and quantity of dna for ngs was unobtainable for 52 ( 51% ) of 102 patients.7 two studies used ngs - based ctdna assays to evaluate , with high overall accuracy , somatic genomic profiles in patients with advanced nsclc  . 
15 , 000 patients with advanced - stage cancer with a concordance of 87% with matched tumor samples that increased to 98% when plasma and tissue testing was done , 6 months apart . 
the study detected actionable mutations in biopsy specimens with insufficient tissue ( alk fusion , egfr or braf activating mutations ) and with actionable resistance mutations at the time of progression ( met amplification or egfr t790m ) as well as in undergenotyped tumors ( braf v600e , or erbb2 indel )  . identification of nontargetable oncogenic drivers , such as kras mutations , that preclude the presence of other targetable alterations also guides clinicians to rapidly initiate alternative therapies , such as chemotherapy or immunotherapy.5 the detection of variants in other genes may lead to the off - label use of fda - approved therapies or enrollment in clinical trials of new therapeutic agents.7 several combinations of targeted therapies are based on resistance mechanisms and are being evaluated in lung cancer ( eg , mek and pi3k inhibitors in combination with egfr tkis ) , 41 which again underscores the need for real - time tumor genotyping to assess for resistance pathways . 
chabon et al43 identified several resistance mechanisms , including met , egfr , pik3ca , errb2 , kras , and rb1 , in patients with t790mmutated nsclc who received rociletinib . 
the study used cancer personalized profiling by deep sequencing ctdna analysis and found that 46% of patients ( 19 of 41 ) with t790m mutations had additional potential resistance mutations in the pretreatment plasma . 
this coexistence of different resistance mechanisms in the same patient may affect response to subsequent egfr tkis like rociletinib . treatment monitoring ctdna offers the possibility of serial testing for molecular monitoring of disease . 
 studies have demonstrated the predictive value of quantitative changes in egfr mutations in ctdna at various time points during tki treatment.1 a prospective analysis to validate ddpcr for the detection of common egfr and kras mutations in patients with advanced nsclc demonstrated that patients with complete resolution of ctdna at either 2 or 6 weeks after treatment exhibited a treatment discontinuation rate of 0% at the initial and 4% at the second imaging assessment . 
patients without complete resolution exhibited a treatment discontinuation rate of 33% at initial and 56% at second imaging . these patterns were postulated to correlate with radiographic response and emergence of acquired resistance.4 marchetti et al44 performed pcr and ultradeep ngs on serial plasma samples from 20 patients with advanced nsclc and known tissue and plasma egfr mutations before tki treatment . 
rapid responders who had at least a 50% decrease in plasma egfr copy number at 14 days had a greater mean percentage of tumor shrinkage than slow responders ( n = 6 ; 70% v 30% )  . 
a phase iii study evaluated egfr mutations in cfdna of patients randomly assigned to receive gemcitabine and platinum plus sequential erlotinib or placebo by using a cobas test.46 for patients treated in the erlotinib arm who were cfdna egfr positive at baseline , the disappearance of cfdna at cycle 3 was associated with significantly improved pfs ( hr , 0.38 ) and longer os ( hr , 0.45 ) compared with patients with persistence of cfdna egfr . the prognostic value of dynamic changes in ctdna has also been evaluated . 
additional studies are needed to evaluate end points such as pfs , os , and quality of life as a result of early detection of resistance . monitoring for minimal residual disease another potential application of ctdna is to evaluate for minimal residual disease ( mrd ) after curativeintent therapy , such as surgery and radiation.8 chaudhuri et al48 applied cancer personalized profiling by deep sequencing to detect ctdna in preand post - treatment blood samples from 41 patients with nonmetastatic lung cancer treated with chemoradiation , radiation , or surgery . 
these patients positive for mrd had a significantly worse outcome than patients without detectable ctdna mrd within 4 months of definitive treatment ( 3 - year freedom from progression , 0% v 92% [ hr , 38 ] ; 3 - year os , 8% v 75% [ hr , 12 ] )  . these data suggest that patients with residual ctdna after definitive treatment will be enriched for those who will ultimately develop recurrence . 
ctdna detection might facilitate the development of clinical trials that evaluate the escalation of therapy for patients at high risk for recurrence and the de - escalation of therapy for patients without residual disease . role in early diagnosis / screening the role of ctdna in early - stage disease also is being studied . 
bettegowda et al34 evaluated the ability of ctdna to detect tumors in patients with various malignancies at various clinical stages . forty - seven percent of patients with stage i cancers ( n = 49 ) had detectable ctdna , with the fraction of patients with detectable ctdna being 55% , 69% , and 82% for those with stage ii , iii , and iv cancers , respectively.34 trials are under way to assess the feasibility and utility of ngsbased ctdna to detect early - stage cancers in high - risk patients ( clinicaltrials.gov identifier : nct02612350 ; also project lunar48a )  . 
 mutation of interest in the patients tumor needs to be known to send for appropriate genomic panels . ctdna may be detectable before radiographically detectable disease , which raises the issue of managing such patients . 
nevertheless , early cancer detection remains a promising new avenue for ctdna that needs additional research . ctdna in the era of immune checkpoint therapy in the era of immune therapy for advanced nsclc , a key issue is the elucidation of a biomarker that can predict response and monitor disease . 
tumor dna sequencing has been shown to be a surrogate for total mutation burden , which predicts response and pfs with antiprogrammed death 1 inhibitors like pembrolizumab.49 however , total mutation burden analysis has been investigated only in tumor tissue , and a better understanding of ctdna is required before it can be applied for this purpose . lipson et al50 analyzed the ctdna of 12 patients with metastatic melanoma who underwent treatment with checkpoint inhibitors for the presence of hotspot somatic mutations in braf , ckit , nras , and tert by using beaming . 
hence , a negative result could signify that the tumor is not actively shedding ctdna , the patients disease is adequately controlled by therapy , or somatic variants either were not covered or were below the detection limit for the assay.7 in addition , any negative test result will need to be viewed in the overall context of ctdna and other molecular results . 
for example , in a patient with an egfr - mutated nsclc in the setting of acquired resistance , a negative egfr t790m test result is more likely to be a true negative if the original egfr mutation was detected with a significant allele frequency versus if the ctdna test did not identify either mutation . confidence in a positive test result extremely high analytic specificity and a positive predictive value that approaches 100% make false positives unlikely , so if a mutation is detected , it can be relied on as a true result . 
as expected , the allelic fraction of variants detected in ctdna is usually much smaller than that in tissue dna given that tumor - derived dna may be more diluted in the blood than in the tissue.7 the rare false positive result ( particularly t790m ) in plasma may be indicative of tissue heterogeneity in which the mutation was not identified but is present in tissue , which may be due to the timing of the biopsy and the location of the biopsy needle in the tumor . 
the only definitive method to clinically validate that the mutations detected in plasma are an accurate molecular proxy of disease biology in the context of it being absent in tissue is to correlate them with response to targeted inhibition . in the case of mutational events that are exclusive to certain types of cancers ( eg , egfr exon 19 deletion to nsclc ) , confidence of a positive result is higher than in genes that can be mutated in a variety of settings ( eg , tp53 )  . 
for instance , a small allelic fraction tp53 mutation in ctdna can originate from many sources ( eg , myelodysplastic syndrome ) , and in such cases , confidence is increased if the same mutation also has been detected in tumor . detection of concurrent malignancy / germline alterations the testing of cfdna will detect all dna that sheds into the circulation , and some findings may not be expected in the tumor of origin , may not match tumor tissue dna results , and may affect genes that are mutated in different tumor types ( eg , tp53 )  . 
a potential for detection of incidental hematologic malignancies exists , which , naturally , have high tumor burden in the blood , and results can be confounded by foreign dna , such as after transfusion or organ transplantation . another issue similar to what is seen with tissue genotyping is that one needs to be cautious about distinguishing somatic ( tumor ) versus germline alterations because germline dna is not analyzed in parallel . 
any mutation that affects genes implicated in familial syndromes should be considered specifically ( eg , brca ) , and mutations with high allele frequency ( between 30% and 70% ) should raise the possibility of being potentially associated with heterozygous germline mutation syndromes . in conclusion , tissue biopsy remains essential at least for primary diagnosis because tissue yields information about morphology , tumor type , site of origin , and the immune microenvironment . 
for instance , erbb2 ( her2 ) copy number amplification in nsclc does not predict tki response like it does in breast cancer.5 currently best validated in egfr - mutated lung cancer for primary molecular diagnosis and for egfr t790m testing , ctdna has proven to have excellent utility as a complement to tissue - based testing . 
singh no relationship to disclose xiaoling guo no relationship to disclose haiying cheng honoraria : astrazeneca , boehringer ingelheim , clovis oncology benjamin levy honoraria : genentech consulting or advisory role : eli lilly , boehringer ingelheim , genentech , roche , astrazeneca , celgene , pfizer , merck speakers bureau : eli lilly , genentech , roche balazs halmos consulting or advisory role : astrazeneca , boehringer ingelheim , celgene , genentech , roche , pfizer , eli lilly , foundation medicine research funding : merck , astrazeneca , mirati therapeutics , boehringer ingelheim , roche , genentech , ariad pharmaceuticals affiliations aditi p . 
singh , haiying cheng , xiaoling guo , and balazs halmos , montefiore medical center , bronx , ny ; and benjamin levy , johns hopkins sidney kimmel comprehensive cancer center at sibley memorial hospital , washington , dc . references oncologist 21 : 1121 - 1130 , 2016 1 . 
sacher ag , paweletz c , dahlberg se , et al : prospective validation of rapid plasma genotyping for the detection of egfr and kras mutations in advanced lung cancer . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
thompson jc , yee ss , troxel ab , et al : detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next - generation sequencing of cell - free circulating tumor dna . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced non - small - cell lung cancer . 
li t , kung hj , mack pc , et al : genotyping and genomic profiling of non - small - cell lung cancer : implications for current and future therapies . 
luo j , shen l , zheng d : diagnostic value of circulating free dna for the detection of egfr mutation status in nsclc : a systematic review and meta - analysis . 
kimura h , suminoe m , kasahara k , et al : evaluation of epidermal growth factor receptor mutation status in serum dna as a predictor of response to gefitinib ( iressa )  . 
douillard jy , ostoros g , cobo m , et al : gefitinib treatment in egfr mutated caucasian nsclc : circulating - free tumor dna as a surrogate for determination of egfr status . 
mao c , yuan jq , yang zy , et al : blood as a substitute for tumor tissue in detecting egfr mutations for guiding egfr tkis treatment of nonsmall cell lung cancer : a systematic review and meta - analysis . 
liu y , liu b , li xy , et al : a comparison of arms and direct sequencing for egfr mutation analysis and tyrosine kinase inhibitors treatment prediction in body fluid samples of non - small - cell lung cancer patients . 
wang j , wang b , chu h , et al : intrinsic resistance to egfr tyrosine kinase inhibitors in advanced non - small - cell lung cancer with activating egfr mutations . 
sequist lv , goldman jw , wakelee , ha , et al : efficacy of rociletinib ( co - 1686 ) in plasma - genotyped t790mpositive non - small cell lung cancer ( nsclc ) patients ( pts )  . 
zheng d , ye x , zhang mz , et al : plasma egfr t790m ctdna status is associated with clinical outcome in advanced nsclc patients with acquired egfr - tki resistance . 
thress ks , paweletz cp , felip e , et al : acquired egfr c797s mutation mediates resistance to azd9291 in non - small cell lung cancer harboring egfr t790m . 
niederst mj , hu h , mulvey he , et al : the allelic context of the c797s mutation acquired upon treatment with thirdgeneration egfr inhibitors impacts sensitivity to subsequent treatment strategies . 
ihuegbu n , banks kc , fairclough sr , et al : non - invasive detection of crizotinib resistance in alk - rearranged lung adenocarcinoma directs treatment with next - generation alk inhibitors . 
wakelee ha , gadgeel sm , goldman jw , et al : epidermal growth factor receptor ( egfr ) genotyping of matched urine , plasma and tumor tissue from non - small cell lung cancer ( nsclc ) patients ( pts ) treated with rociletinib . 
barlesi f , gervais r , lena h , et al : pemetrexed and cisplatin as first - line chemotherapy for advanced non - small - cell lung cancer ( nsclc ) with asymptomatic inoperable brain metastases : a multicenter phase ii trial ( gfpc 07 - 01 )  . ann oncol 22 : 2466 - 2470 , 2011 34 . 
de mattos - arruda l , mayor r , ng ck , et al : cerebrospinal fluid - derived circulating tumour dna better represents the genomic alterations of brain tumours than plasma . 
sasaki s , yoshioka y , ko r , et al : diagnostic significance of cerebrospinal fluid egfr mutation analysis for leptomeningeal metastasis in non - small - cell lung cancer patients harboring an active egfr mutation following gefitinib therapy failure . 
hata a , nanjo s , okuda c , et al : osimertinib at 80 mg for refractory leptomeningeal metastases in t790m - positive egfr - mutant non - small cell lung cancer . 
zill oa , mortimer s , banks kc : somatic genomic landscape of over 15 , 000 patients with advanced - stage cancer from clinical next - generation sequencing analysis of circulating tumor dna . 
canale m , petracci e , delmonte a , et al : impact of tp53 mutations on outcome in egfr - mutated patients treated with first - line tyrosine kinase inhibitors . 
marchetti a , palma jf , felicioni l , et al : early prediction of response to tyrosine kinase inhibitors by quantification of egfr mutations in plasma of nsclc patients . 
tseng js , yang ty , tsai cr , et al : dynamic plasma egfr mutation status as a predictor of egfr - tki efficacy in patients with egfr - mutant lung adenocarcinoma . 
mok t , wu yl , lee js , et al : detection and dynamic changes of egfr mutations from circulating tumor dna as a predictor of survival outcomes in nsclc patients treated with first - line intercalated erlotinib and chemotherapy . clin cancer res 21 : 3196 - 3203 , 2015 47 . 
karachaliou n , mayo - de las casas c , queralt c , et al : association of egfr l858r mutation in circulating free dna with survival in the eurtac trial . 
lipson ej , velculescu ve , pritchard ts , et al : circulating tumor dna analysis as a real - time method for monitoring tumor burden in melanoma patients undergoing treatment with immune checkpoint blockade . 
pediatric methylation data were also compared systematically with the adult acc cohort from the cancer genome atlas ( tcga )  . results two pediatric act methylation groups were identied and showed differences in selected clinicopathologic and outcome characteristics . 
the a2 group was enriched for tp53 germline variants , younger age at onset , and favorable outcome . pediatric act methylation groups were maintained when international pediatric adrenocortical tumor registry cohort data were combined with tcga cohort data . 
when methylome ndings were combined with independent histopathologic review using the wieneke criteria , a high - risk population was identied with uniform fatal outcome . conclusion our results indicate dna methylation analysis can enhance current diagnostic algorithms . 
 clay et al context key objective risk stratication in pediatric adrenocortical tumors ( acts ) is challenging , because individual gene mutations and histomorphology do not reliably predict clinical risk . 
this study examined whether molecular subtypes of act could be detected using dna methylation proling and whether common act mutations differentially segregate into molecular groups . furthermore , we evaluated the prognostic signicance of molecular groups alone and in combination with histomorphologic risk models . knowledge generated two methylation groups were detected , distinct from adult act and enriched for specic mutations . 
methylation group may serve to highlight a high - risk treatment population in future risk - adaptive clinical trial designs . interplay of multiple genetic lesions that accumulate during tumorigenesis.7 recent advances in adult adrenocortical neoplasia studies have revealed distinct methylation patterns in non - neoplastic , benign , and malignant act samples.12 aggressive behavior has been associated with both global hypomethylation13 and cpg - island methylator phenotype ( cimp ) .9 this was expanded on by zheng et al14 in a comprehensive analysis of adult accs as part of the cancer genome atlas ( tcga ) , where levels of cpg - island methylation ( designated cimp - low , - intermediate , or - high ) were associated with an increasingly poor outcome . 
studies on methylation patterns in the pediatric population are lacking . we performed dna methylation analyses on 48 newly diagnosed acts from the international pediatric adrenocortical tumor registry ( ipactr ) to evaluate for distinct groups with prognostic signicance . 
methods of registry accrual and the sampling process have been described previously.4 histopathologic and clinical data were tabulated from existing databases . the outside diagnosis was recorded as initial clinical diagnosis for the purposes of this study . 
pathology central review was performed blinded to analysis data and included evaluation of wieneke criteria5 in all patients , with a subsequently assigned wieneke tumor classication ( scores : 0 - 2 , aca ; 3 , act with uncertain malignant potential [ actump ] ; 4 - 9 , acc )  . 
a summary of all clinical data can be seen in the data supplement . normal adrenal samples were obtained from non - neoplastic surgical specimens and autopsy tissues . immunohistochemical and molecular analysis immunohistochemical staining was performed for p53 ( diluted 1 : 200 ; #z2029m ; zeta corp , sierra madre , ca ) , - catenin ( undiluted ; #760 - 4242 ; ventana , tucson , az ) , and atrx ( 1 : 200 ; #hpa001906 ; sigma , st louis , mo ) and scored as either wild - type pattern ( ie , scattered nuclear positivity for p53 , membranous or cytoplasmic staining for - catenin , and intact nuclear staining for atrx ) or mutant pattern ( ie , absent or diffuse nuclear positivity for p53 , nuclear positivity for - catenin , and loss of nuclear stain for atrx )  . 
ki - 67 staining ( 1 : 200 ; #m7240 ; dako , santa clara , ca ) was scored manually as the raw percentage of ki67positive tumor cells and as an estimate rounded to the nearest 10th percentile . 
mutational status of tp53 , ctnnb1 , and atrx was determined using a combination of whole - genome sequencing ( wgs ) , whole - exome sequencing ( wes ) , sanger sequencing , and multiplex ligationdependent probe amplication ( mlpa )  . 
 dna methylation proling of pediatric adrenocortical tumors instances , multimodality testing was performed , and overall testing included the following genes : tp53 mutational ( sanger , n = 48 ; wes , n = 9 ; wgs , n = 1 ) , ctnnb1 ( sanger , n = 48 ; wes , n = 9 ; wgs , n = 1 ) , and atrx ( mlpa , n = 28 ; wes , n = 1 ; wgs , n = 1 )  . methylation proling and copy number analysis methylome analysis was performed using the human innium methylationepic beadchip array ( illumina , san diego , ca ) on 250 - 500 ng of dna extracted from formalinxed parafn - embedded ( ffpe ) tissues . 
samples were handled in accordance with the illumina innium hd methylation assay protocol , as published.15 a detailed description of data processing and methylation analysis is presented in the appendix . 
the group - wise methylation states of cpg - island sites were analyzed by mapping the top 20 , 000 most variably methylated probes from the combined tcga and ipactr datasets . gene set enrichment analysis gene set enrichment analysis ( gsea ) was performed using the top 20 , 000 most variably methylated probes between the two pediatric act groups . 
differential methylation patterns were mapped to specic gene loci , which were then secondarily cross - referenced to published gene sets by using established protocols.17 , 18 for a detailed description , see the appendix . clinicopathologic correlation summary statistics were calculated for values by the methylation group . 
survival analysis was performed to investigate the association of overall survival with histologic diagnosis , wieneke classication , and act methylation group using the log - rank test and cox regression models ( appendix )  . 
all p values reported were 2 - tailed and considered signicant if p .05. results demographics the demographic and clinical characteristics of the 48 pediatric act samples are presented in table 1 . 
the entire histologic spectrum of pediatric acts was present as classied by the wieneke criteria : aca ( n = 23 ) , act - ump ( n = 5 ) , and acc ( n = 19 ) , with 18 ( 38% ) stage i tumors , 10 ( 21% ) stage ii tumors , 15 ( 31% ) stage iii tumors , and ve ( 10% ) stage iv tumors . methylation and chromosomal copy number analysis we performed methylation proling on 48 acts and 12 normal control pediatric adrenal samples . 
by both unsupervised t - distributed stochastic neighbor embedding ( t - sne ) analysis ( fig 1a ) and hierarchical clustering ( fig 1b ) , we identied three distinct clusters of samples . 
these two tumor groups were 94.26% reproducible in a thorough bootstrap evaluation19 , 20 ( appendix )  . we observed several recurrent segmental chromosomal cnvs in both groups , including 4q and + 9q as previously reported ( figs 1c and 1d ) .7 we also identied distinct differences among groups ( fig 1e ) , with fewer recurrent chromosomal - level gains in the a1 group ( appendix fig a3 )  . 
all tumors in the tcga cohort were carcinomas , and median age at diagnosis was 49 years.14 visualization with t - sne highlighted the previously appreciated a1 , a2 , and pediatric control groups . 
tcga samples roughly clustered into groups that correlated with the published cimp - l , cimp - i , and cimp - h groups , although these designations are somewhat ambiguous , and several overlapping patients can be seen in the t - sne plot ( fig 3a )  . 
copy number analysis highlighted distinct copy number proles in the cimp groups ( appendix fig a4 )  . to further evaluate the relationship between pediatric acts and adult acts , we evaluated the methylation state at cpg islands across the tumor groups . 
in the tcga cohort , we reafrmed the presence of the cimp groups with the following mean beta values ( listed as % - methylated probes [ cimp - h , 69% ; cimp - i , 50% ; cimp - l , 29% ] )  . 
 ( a ) unsupervised analysis of dna methylation data visualized by t - distributed stochastic neighbor embedding ( t - sne ) plot identied 2 methylation groups , a1 and a2 . 
ihc , immunohistochemistry ; ump , uncertain malignant potential . the ipactr cohort , the a1 group showed greater cpgisland methylation when compared with the a2 group and the control samples ( fig 3b ) but not to the level of the cimp - h phenotypic patients ( a1 , 38% ; a2 , 23% ; control , 21% )  . 
unlike the adult tumors , noncpg - island methylation did not follow the same pattern as cpg islands , and overall , the a1 group was globally hypomethylated . clinicopathologic correlation occurred primarily in children younger than 4 years of age , whereas the a1 group showed an even age distribution ( fig 1b ; appendix fig a2b )  . 
no association was seen for sex , gross features ( ie , the size and weight of the tumor ) , or histologic diagnosis . the two methylation groups showed differences in selected clinicopathologic characteristics ( table 2 )  . 
cpgisland hypermethylator phenotypes ( cimps ) are noted as high ( cimp - h ) , intermediate ( cimp - i ) , or low ( cimp - l )  . 
p values are based on computed means for each patient . however , after strictly applying the wieneke criteria at central pathology review , we found that overall survival differed signicantly according to diagnosis ( p = .013 ; fig 4 )  . methylation grouping strongly correlated with overall survival ( p , .0001 ) , and except for one child in the a2 group who died of a separate primary brain tumor , all deaths directly attributable to acts occurred in the a1 group ( fig 4 )  . 
overall , bic indicates there is a 99.2% probability that the best predictive model for survival includes methylation class as one of its predictor variables . discussion our study demonstrates the potential of dna methylation proling to enhance current classication schemas by identifying prognostically relevant molecular groups in the pediatric population . 
clinical , histologic , immunophenotypic , and molecular characteristics by methylation group ( continued ) methylation group group a1 group a2 vascular invasion , present vascular invasion , absent soft - tissue invasion , present soft - tissue invasion , absent necrosis , any , present necrosis , any , absent mitoses  . 
clinical , histologic , immunophenotypic , and molecular characteristics by methylation group ( continued ) methylation group group a1 group a2 atrx wild - type pattern mutant pattern ( loss of staining ) note . 
bold type indicates statistical signicance . abbreviations : hpf , high power elds ; ipactr , international pediatric adrenocortical tumor registry . * local stage ( i / ii ) compared against nonlocalized stages ( iii / iv )  . virilization alone compared with all other categories . .321 clay et al because a proportion of tumors yield an intermediate score ( wieneke score = 3 ) , and the criteria can be difcult to apply outside of specialized centers . 
these limitations make the search for alternative predictive biomarkers a priority , because this ambiguity represents a major hurdle to the implementation of risk - adapted therapies . there are several advantages dna methylation proling offers as a clinical assay . 
for example , relevant prognostic groups have been extracted from dna methylation data in osteosarcoma , a tumor type characterized by heterogeneous driver mutations and genomic instability.31 because dna methylation marks are inherently linked to cell of origin , methylation class may represent a more biologically relevant and stable biomarker of disease than dna - sequence variation alone . between our two methylation groups , a1 tumors were associated with a worse prognosis . 
the aggressive nature of a1 tumors was also evident in other clinical variables , with patients from this group having a more advanced clinical stage , greater age , and a tendency to be treated with adjuvant chemotherapy . 
the discrepancy between p53 staining and tp53 mutational status is curious but represents a known shortcoming of interrogating the tp53 locus by immunohistochemical means34 ; additional factors , such as tissue xation , likely affected this analysis . our study identied several genes and key pathways involved in biologic processes and molecular functions . signicant genes included igf2 , gnas , and cldn1 , supporting a role of regulatory pathways such as signal transduction , cell growth , and cell signaling . 
this supports results from previous studies suggesting a role for these pathways in pediatric acts.26 - 29 , 35 gsea also identied differential methylation of genes involved in the prc2 pathway . 
this is particularly interesting , given the distribution of ctnnb1 alterations in this cohort , as the prc2 complex is involved in epigenetic regulation and is a known modulator of the wnt - signaling pathway.36 one limitation of the current study is our inability to validate our ndings in an independent pediatric clinical cohort . 
in addition to the unsupervised clustering features , analysis of cpg - island methylation highlighted distinct distributions of methylation marks , with an absence of hypermethylator phenotype in the pediatric samples . adult and pediatric acts also had unique copy number proles , with pediatric samples tending to show greater evidence of chromosomal losses . 
the pediatric tumors retained their methylation groups when combined with the adult patients , providing evidence for the stability of group assignments and suggesting that adult trial cohorts their pediatric are not appropriate comparisons for counterparts . 
validation of our molecular grouping in independent pediatric clinical cohorts will be a central focus of future studies , and international collaboration with carefully annotated data will be necessary to advance our knowledge of these rare tumors . in addition to methylation - based classication representing a signicant predictor of risk in our cohort , we also validated the effectiveness of the wieneke classication schema . 
at risk : high - risk low - risk combined histologic - and molecular - risk stratification high risk low risk 2 , 000 time ( days ) fig 4 . 
kaplan - meier plots of overall survival from disease based on ( a ) initial clinical diagnosis , ( b ) diagnosis rendered at the time of central review , ( c ) dna methylation group , and ( d ) combined risk stratication using central review and methylation - classication group . 
ump , uncertain malignant potential . risk stratication is to use a combinatorial system of risk assessment followed by risk - adapted or targeted therapy . this approach may optimize the exposure to therapy for patients with standard - risk disease , while maximizing the effectiveness of intervention in patients with high - risk disease . affiliation 1st jude childrens research hospital , memphis , tn b.a.o. 
clay , md , department of pathology , ms 250 , 262 danny thomas pl , memphis tn 38105 - 2794 ; michael.clay@stjude.org. support supported by the national institutes of health grant no . 
i receive small royalty on an annual basis patent pending for genotyping assays to identify mutations in xaf1 provisional application #62 / 659 , 427 ; foreign ling april 18 , 2019 no other potential conicts of interest were reported . acknowledgment the authors thank david w . 
endocr rev 25 : 309 - 340 , 2004 gulack bc , rialon kl , englum br , et al : factors associated with survival in pediatric adrenocortical carcinoma : an analysis of the national cancer data base ( ncdb )  . 
michalkiewicz e , sandrini r , figueiredo b , et al : clinical and outcome characteristics of children with adrenocortical tumors : a report from the international pediatric adrenocortical tumor registry . 
am j surg pathol 27 : 867 - 881 , 2003 gr obner sn , worst bc , weischenfeldt j , et al : the landscape of genomic alterations across childhood cancers . 
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nat commun 6 : 6302 , 2015 kebebew e , reiff e , duh qy , et al : extent of disease at presentation and outcome for adrenocortical carcinoma : have we made progress ? world j surg 30 : 872 - 878 , 2006 barreau o , assi e g , wilmot - roussel h , et al : identication of a cpg island methylator phenotype in adrenocortical carcinomas . 
pinto em , rodriguez - galindo c , pounds sb , et al : identication of clinical and biologic correlates associated with outcome in children with adrenocortical tumors without germline tp53 mutations : a st jude adrenocortical tumor registry and childrens oncology group study . 
zheng s , cherniack ad , dewal n , et al : cancer genome atlas research network : comprehensive pan - genomic characterization of adrenocortical carcinoma . cancer cell 30 : 363 , 2016 [ erratum : cancer cell 30 : 363 , 2016 ] 15 . 
moran s , arribas c , esteller m : validation of a dna methylation microarray for 850 , 000 cpg sites of the human genome enriched in enhancer sequences . epigenomics 8 : 389 - 399 , 2016 proc natl acad sci usa 102 : 15545 - 15550 , 2005 diabetes . 
albertin g , carraro g , petrelli l , et al : endothelin - 1 and adrenomedullin enhance the growth of human adrenocortical carcinoma - derived sw - 13 cell line by stimulating proliferation and inhibiting apoptosis . 
scholl ui , st olting g , nelson - williams c , et al : recurrent gain of function mutation in calcium channel cacna1h causes early - onset hypertension with primary aldosteroniselife 4 : e06315 , 2015 24 . 
swisshelm k , machl a , planitzer s , et al : semp1 , a senescence - associated cdna isolated from human mammary epithelial cells , is a member of an epithelial membrane protein superfamily . 
almeida mq , azevedo mf , xekouki p , et al : activation of cyclic amp signaling leads to different pathway alterations in lesions of the adrenal cortex caused by germline prkar1a defects versus those due to somatic gnas mutations . 
gao zh , suppola s , liu j , et al : association of h19 promoter methylation with the expression of h19 and igf - ii genes in adrenocortical tumors . 
 clay et al appendix processing of samples and methylation data the study cohort was derived from the international pediatric adrenocortical tumor registry database ( n = 110 total entries ) to include samples that satised two requirements , including those that contained sufcient dna for methylation analysis and those that had tumor sections for histologic review ( initial cohort , n = 73 )  . 
the resultant 48 patients were included in the study . all dna samples were quantied by the uorometric method ( qbit dsdna br assay , thermo fisher scientic , waltham , ma )  . 
the detection p value for each sample was computed , and only samples with a mean detection p value less than .05 were carried forward for subsequent analysis . additional quality control was performed by calculating the median log ( base 2 ) intensities for methylated and unmethylated signals for each array and excluding samples with unmethylated and methylated median intensity values below a cutoff of 9.5. functional normalization ( fortin jp , et al : genome biol 15 : 503 , 2014 ) with noob background correction and dye - bias normalization ( troche tj , et al : nucleic acids res 41 : e90 , 2013 ) was performed . 
specically , probes located on sex chromosomes , probes containing a nucleotide polymorphism ( dbsnp132 common ) within ve base pairs of and including the targeted cpg - site , probes mapping to multiple sites on hg19 ( allowing for one mismatch ) , and any cross - reactive probes were removed from the analysis ( pidsley r , et al : genome biol 17 : 208 , 2016 )  . 
the pearson correlation was calculated as the distance measured between samples , and the clustering was performed using the complete - linkage agglomerative method ( clifford h , et al : front genet 2 : 88 , 2011 )  . in brief , the result of the hierarchical clustering analysis previously mentioned was also recapitulated in a complementary analysis that exhaustively evaluated 16 , 000 euclidean - distance hierarchical clustering procedures ( hcps ) that used four different probe - selection methods ( choosing probes with the greatest standard deviation , median absolute deviation , most informative spacing statistic [ pawlikowska i , et al : bioinformatics 30 : 1400 - 1408 , 2014 ] , and the dip statistic21 modied to measure both evidence bimodality and distance between modes ) , selection of 1 - 1 , 000 probes , and dening two to ve groups with a post hoc evaluation of reproducibility of subgroup group assignments by exact k = 5 nearest neighbor exact bootstrap procedure ( steele bm , et al : mach learn 74 : 235 - 255 , 2009 ; four probeselection methods selection of up to 1 , 000 probes denition of two to ve groups = 4 1 , 000 4 = 16 , 000 hcps )  . 
the modied dip statistic was dened as 4 times the product of the hartigan and hartigan dip statistic and the difference between the data value dening the dip and the quantile of the unimodal distribution function corresponding to the value of the empirical distribution function of that value . 
in this way , the modied dip statistic considered both the strength of statistical evidence in favor of multimodality and the size of the gap between the two primary modes in the data . 
hcp 1141 selected the 286 probes with the greatest value of this modied dip statistic and dened two subgroups with 96.4% exact k = 5 nearest neighbor bootstrap assignment reproducibility . 
the two subgroups dened by hcp 1141 strongly associated with two subgroups dened by t - sne and complete - linkage agglomeration ( p = 1.5 108 ; table a3 )  . 
the wilcoxon rank - sum test was used to perform a post hoc comparison of the methylation values of all probe sets across the two subgroups dened by hcp 1141 . 
to adjust for multiplicity , we computed storeys q value ( storey jd , et al : proc natl acad sci usa ) using the pounds - cheng estimator of the proportion of tests with a true null hypothesis ( pounds s , et al : comput biol 12 : 482 - 495 , 2005 )  . 
the data supplement provides the modied dip statistic and wilcoxon results for the 286 probes selected by hcp 1141 . copy number analysis dna copy number variation ( cnv ) was inferred from the methylation data by using the conumee bioconductor package in r with the default settings.16 the combined intensities of all available cpg probes were normalized against that of pediatric non - neoplastic adrenal gland controls ( n = 12 ) and using a linear - regression approach . 
we used bic ews to evaluate the following six models of the association of survival with the wieneke criterion , the clinical diagnosis , and / or methylation group : ( 1 ) the null model , ( 2 ) the wieneke criterion as the sole predictor of survival , ( 3 ) the clinical diagnosis as the sole predictor of survival , ( 4 ) the methylation group as the sole predictor of survival , ( 5 ) the wieneke criterion and methylation group as predictors of survival , and ( 6 ) the clinical diagnosis and methylation group as predictors of survival . 
the bic ews for a specic statistical model range from 0 ( the data do not support the specic model ) to 1 ( the data overwhelmingly support the specic model ) , and the bic ews for a set of candidate models sum to 1 . 
 dna methylation proling of pediatric adrenocortical tumors cimp - high 13 14 15 16 17 18 19 2021 22 cimp - intermediate 13 14 15 16 17 18 19 2021 22 cimp - low 13 14 15 16 17 18 19 2021 22 chromosome fig a4 . 
modied disease staging system used by ipactr stage description tumor completely excised with negative margins , tumor weight 200 g , absence of metastatic disease tumor completely excised with negative margins , tumor weight  . 
15 mits / 20 hpf ( yes : no ) adjusted for atypical mitoses methylation group atypical mitoses ( yes : no ) adjusted for ki67 above 15% methylation group ki67  . 
lennerz author affiliations and support information ( if applicable ) appear at the end of this article . creative commons attribution non commercial no derivatives 4.0 license ( continued ) purpose targeted therapy is the cornerstone of treatment of advanced egfr - mutant nonsmall - cell lung cancer ( nsclc )  . 
here , we assessed workflows designed to rapidly identify patients with actionable egfr mutations and reduce time to initiation ( tti ) of epidermal growth factor receptor ( egfr ) directed therapy . patients and methods we performed rapid testing for egfr l858r mutations and exon 19 deletions on paraffin - embedded or frozen section biopsy specimens from newly diagnosed patients with metastatic nsclc by using an egfr - specific assay ( rapid test )  . 
to determine clinical utility , we assessed concordance with ngs results , turnaround time , and tti of egfr therapy , and we evaluated reimbursement data . results between january 2015 and september 2017 , we performed 243 rapid egfr tests and identified egfr mutations in 43 patients ( 18% )  . 
escalation of the initiative into an interdisciplinary ultra - rapid next - day frozen - section workflow for highly symptomatic patients ( n = 8 ) resulted in a reduction in the median ( standard deviation ) turnaround time to 1 0.4 days and allowed several patients to initiate therapy within 1 week of biopsy . 
2018 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license : introduction nonsmall - cell lung cancer ( nsclc ) is a heterogeneous disease composed of unique molecular subsets with distinct clinical outcomes.1 , 2 multiple randomized studies have established the superiority of molecularly targeted therapies versus chemotherapy for the treatment of egfr - mutant and alk - positive nsclc.3 - 6 in other molecular subsets , single - arm studies confirm that treatment with targeted therapies can induce durable responses.7 , 8 as drugs that target these molecular drivers are approved for first - line treatment , genotyping in newly diagnosed nsclc is considered the standard of care.9 because of the need to interrogate a growing number of genes , next - generation sequencing ( ngs ) has largely replaced traditional single - gene assays.10 guidelines endorsed by oncology and pathology societies recommend that molecular testing turnaround times not exceed 10 working days.9 genotyping by ngs requires complex bioinformatics that can create treatment delays . 
we selected egfr mutant nsclc on the basis of its relatively high prevalence ( 10% to 15% ) among patients with nsclc , 12 the lack of overlap between egfr mutations and other clinically relevant molecular alterations , 13 and the fact that egfr inhibitors were readily available for hospitalized patients . 
in the first phase ( january 2015 to may 2017 ) , rapid egfr testing was performed on tissue specimens from consecutive patients diagnosed with metastatic nsclc who were never smokers / minimal smokers ( 10 pack years ) or who were hospitalized at the time of diagnosis ( data supplement )  . 
we also identified a comparator population composed of 121 patients with egfr - mutant nsclc who were diagnosed before implementation of the rapid testing ( ie , historical cohort ; data supplement )  . 
isolated nucleic acids from tumor specimens were analyzed with our ngs assay that uses anchored multiplex pcr to detect single - nucleotide variants and insertions / deletions in a target set of cancer - related genes.15 in addition , we examined fusion transcripts and met exon 14 skipping by using an rna - based ngs solid - fusion assay.15 statistical and economic analysis for contingency analyses , we used t tests , fishers exact tests , and 2 statistics . 
we defined turnaround - time from the date of accessioning to the date of reporting , and we defined tti from the date of the diagnostic biopsy to the date of ingestion of an egfr tyrosine kinase inhibitor ( tki )  . 
 for economic assessment , we reviewed line items of reimbursement data ( january 3 , 2017 , to february 8 , 2018 ) and extracted ( by payor ) the number of encounters and claim adjustment codes , and defined reimbursed as a payment greater than 0 . 
note that there is a ( variable ) delay from reporting to treatment decision and initiation of therapy because of cost coverage determination , preauthorization requirements , etc.the ultra - rapid egfr testing pathway ( pathway c ) is a multidisciplinary workflow designed to improve turnaround time using fresh tissue ( frozen sections ) to extract nucleic acids . 
among the remaining 234 patients , 23 ( 10% ) did not have ngs results because of insufficient tissue or technical failure of ngs ( data supplement )  . diagnostic utility of the rapid egfr assay improvement in turnaround time . 
first , we compared the turnaround times of the rapid and ngs assays by reviewing all specimens submitted for genotyping between july 2014 and january 2017 ( fig 2b )  . 
through ngs , we identified one additional low - level ( allelic fraction , approximately 4% ) p.l858r case , which was missed by rapid egfr testing ( fig 3 )  . 
the mutation spectrum of the tumors that underwent rapid testing is depicted in figure 3 , and the data supplement shows probabilities of therapeutically actionable variants . clinical utility of rapid egfr testing tti of tki therapy . 
p values result from t test ( age ) , fishers exact test ( sex , histology ) , or 2 test ( ethnicity , smoking )  . * includes the patient with a false - negative egfr - positive result . tki as the primary measure of clinical utility of the rapid egfr test . 
we reviewed the medical records of the 39 patients with egfr - mutant disease who underwent both rapid testing and ngs to determine whether tki therapy was initiated before ngs results . 
 validation implementation of rapid egfr testing ngs panel rapid wt rapid mut rapid egfr assay rapid egfr assay exon 19 exon 20 exon 21 del / ins t790m l858r variant fig 2 . 
scatter plots portray turnaround times of all 243 rapid egfr samples ( jan 2015 to may 2017 ; black , egfr wild type [ wt ] ; red , egfr mutation [ mut ] detected ) and all specimens that underwent ngs ( gray dots ) during this period . 
a case report of a patient who benefitted from this effort and a summary of the experience to date are described in the next section . therapeutic utility of ultra - rapid testing . 
osimertinib was selected on the basis of its activity against leptomeningeal disease.19 within days , the cough resolved , and the patient experienced marked improvement in his swallowing and regained the ability to walk . 
figure 4d illustrates the difference in turnaround time for the rapid and ultra - rapid tests and the time to initiation of tki therapy for patients tested through each workflow ( fig 4d inset )  . 
the sites of disease and presenting symptoms of these patients are detailed in the data supplement , which also summarizes our ultra - rapid workflow . rapid testing is operationally and financially sustainable . 
the heatmap portrays clinicopathologic features ( top three rows ) , rapid egfr results , and key molecular drivers along with the results of next generation sequencing ( ngs ) based fusion detection , fluorescence in - situ hybridization ( fish ) , and ngs panel results . 
key findings include ( 1 ) an isolated false - negative rapid egfr result ( arrow ) , ( 2 ) the inability of the rapid egfr test to detect egfr mutations at other residues , ( 3 ) identification of at least one underlying driver mutation in more than 50% of all tested cases by using the integrated molecular diagnostic approach , and ( 4 ) the association between clinicopathologic features and certain key drivers ( eg , never - smoking women with adenocarcinoma and egfr v > 10 pack - year smoking history and kras )  . 
 review of 214 clinical encounters with 1 , 475 line items of reimbursement data showed large variability between payors ( range , 0% to 100% ) and that , overall , 63% of encounters received reimbursement ( fig 4f )  . 
these data indicate that rapid egfr testing in our practice is operationally and financially sustainable . discussion here , we report the results of a quality improvement initiative to explore parallel testing with a rapid egfr assay and ngs in newly diagnosed nsclc . 
our findings emphasize the diagnostic , clinical , and therapeutic utility of rapid egfr testing . metastatic nsclc is a devastating and incurable disease for which the prognosis is highly dependent on identification of actionable molecular drivers.2 the established efficacy of us food and drug administrationapproved targeted therapies in specific nsclc subsets and the increasing number of investigational compounds underscore the necessity of identification of molecular alterations in a timely fashion . 
 ( a ) event curve that shows rapid egfr test reporting times and a comparison of the tyrosine kinase inhibitor ( tki ) initiation times ( relative to date of diagnosis ) for patients in the rapid and historical cohorts . 
the second block shows the 49% of patients ( n = 17 of 35 patients ) with egfr - mutant disease who started a tki before next - generation sequencing ( ngs ) results were available . 
 ( c ) response to the egfr inhibitor osimertinib in a patient with nonsmall - cell lung cancer who underwent ultra - rapid egfr testing : ( left ) pretreatment image and ( right ) response after 3 weeks ; arrow indicates primary tumor . 
 ( d ) comparison of rapid test times ( average ) and the eight patients tested with the ultra - rapid protocol ( fig 1c ) ; inset shows event curve comparison of time to initiation of tki between the rapid and ultra - rapid subsets . 
 ( e ) turnaround times for rapid ( gray ) and ultra - rapid ( blue ) workflows in a 9 - month extended standardof - care evaluation phase ; red , cases with an egfr mutation . 
several studies suggest that genotyping with multiple single - gene assays may reduce reporting time20 , 21 ; however , selective interrogation of the ever - expanding set of targets is impractical and may be limited by tissue availability.22 at the time our initiative began , consensus guidelines supported testing newly diagnosed patients with nsclc for egfr mutations and rearrangements in alk and ros1.9 these mutually exclusive genetic alterations are predominantly found in neveror light - smokers.13 , 23 testing these alterations in two stages ( ie , restriction of testing for rearrangements to patients whose disease is wild type for egfr mutations ) may lead to unnecessary delays between diagnosis and initiation of treatment for a significant proportion of patients and potentially steer symptomatic patients away from molecularly targeted treatments . 
through co - testing , we detected alk and ros1 rearrangements in 11 and six patients , respectively , and identified molecular alterations with promising investigational therapies in more than 40 additional patients ( fig 3 )  . 
for example , in a recent retrospective analysis that involved 15 community oncology centers , the median turnaround time for egfr testing and ngs was 23 days and 30 days , respectively.24 although this practice is likely more common in the community setting , where in - house testing is infrequent , one study demonstrated that 19% of patients with egfr mutations or alk rearrangements at an academic medical center in canada initiated first - line chemotherapy before testing results became available.11 the sequence of therapies may be particularly important for those patients initially treated with immunotherapy , because recent studies demonstrate that immunotherapy is seldom effective for egfr - mutant nsclc and that treatment with immunotherapy before targeted therapy increases the likelihood of the development of significant toxicities.25 - 27 given these data , we believe that rapid testing should be performed in parallel with full ngs genotyping . with a parallel - testing approach , we successfully performed rapid testing and ngs in 90% of patients in our initiative with material from a single biopsy . 
because half of the patients with tissue / dna that was insufficient for the full ngs were effectively tested for both alk and ros1 rearrangements using either fluorescence in - situ hybridization or the fusion assay , 95% of the patients in the rapid cohort met the minimum molecular testing requirement proposed by current asco guidelines . 
although pcr - based plasma genotyping is an expedient alternative to ngsbased tissue genotyping , the sensitivity of liquid biopsy for detection of egfr mutations is lower than what we report here with the rapid assay.29 , 30 despite the high sensitivity and specificity of the rapid assay , half of the patients in our study did not start tki therapy before ngs results were available ( fig 4b )  . 
 because tti was assessed retrospectively for the historical cohort , the median of 37 days between diagnosis and tki therapy might be an overestimate ; however , a similar delay was observed in another study.31 it also is conceivable that the difference in tti might be explained by process improvements over time that have shortened the interval between requests for and receipt of a drug from specialty pharmacies . 
because tki use predated ngs results in approximately 50% of patients , the reduction in time to tki initiation in the rapid group relative to the historical cohort might also reflect faster ngs reporting times . 
the scale of ngs panels currently precludes significant reduction , and the median and mean turnaround times for ngs results in the ultrarapid cohort were 9 and 10 days , respectively . 
 as a result of the short duration of follow - up of our rapid cohort and imbalances between the rapid and historical groups ( including enrollment of patients in the rapid cohort into a consolidation radiation protocol ) , we were unable to evaluate differences in progression - free survival on egfr - directed therapy . 
lennerz the potential to defer brain radiation for these patients underscore the importance of rapid identification of egfr mutations.41 , 42 the small number of patients with egfr - mutant disease in our study also limits the potential impact of our findings . 
however , many laboratories are proficient in performing targeted egfr genotyping , 43 and our extended standard of care evaluation ( fig 4e ) and reimbursement analysis ( fig 4f ) indicate operational and financial sustainability . in summary , we demonstrate that expedited egfr genotyping enables early intervention with targeted therapies and allows symptomatic patients to access effective treatments . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . ibiayi dagogo - jack honoraria : foundation medicine consulting or advisory role : boehringer ingelheim administrative support : christopher g . 
farago honoraria : foundation medicine vania nose no relationship to disclose consulting or advisory role : pharmamar , takeda , abbvie , loxo , stemcentrx research funding : pharmamar ( inst ) , abbvie ( inst ) , astrazeneca ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) , loxo ( inst ) , ignyta ( inst ) miguel rivera consulting or advisory role : loxom asubio ( i ) speakers ' bureau : pfizer ( i ) research funding : advanced cell diagnostics , affymetrix travel , accommodations , expenses : pharmamar , abbvie , stemcentrx patents , royalties , other intellectual property : patents with affymetrix justin f . 
gainor honoraria : merck , incyte , ariad , novartis , pfizer consulting or advisory role : genentech , bristol - myers squibb , theravance , loxo , takeda , array biopharma , amgen research funding : merck ( inst ) , novartis ( inst ) , genentech , bristol - myers squibb ( inst ) , adaptimmune ( inst ) , astrazeneca ( inst ) , ariad , jounce therapeutics ( inst ) , blueprint medicines ( inst ) , moderna therapeutics ( inst ) , tesaro ( inst ) travel , accommodations , expenses : affymetrix ginger jiang employment : novartis ( i ) zofia piotrowska consulting or advisory role : boehringer ingelheim , astrazeneca , ariad , takeda , superdimension , guardant health , novartis , abbvie research funding : novartis ( inst ) , ariad ( inst ) , takeda ( inst ) , guardant health ( inst ) rebecca s . 
heist consulting or advisory role : boehringer ingelheim research funding : abbvie ( inst ) , novartis ( inst ) , roche ( inst ) , incyte ( inst ) , celgene ( inst ) , mirati therapeutics ( inst ) , peregrine pharmaceuticals ( inst ) , exelixis ( inst ) , millenium ( inst ) , debiopharm group ( inst ) , corvus pharmaceuticals ( inst ) valentina nardi stock and other ownership interests : ksq therapeutics ( i ) , navicor ( i ) consulting or advisory role : thermo fisher scientific ( i ) , cell signaling technology ( i ) , raze ( i ) , caloric tests ( i ) dora dias - santagata no relationship to disclose long p . 
le stock and other ownership interests : archer biosciences consulting or advisory role : archer biosciences patents , royalties , other intellectual property : i am a co - inventor of the anchored multiplex pcr technology which is licensed to archerdx . 
sequist honoraria : astrazeneca consulting or advisory role : astrazeneca , genentech , roche , bristol - myers squibb , pfizer research funding : boehringer ingelheim ( inst ) , clovis oncology ( inst ) , genentech ( inst ) , merrimack ( inst ) , novartis ( inst ) , astrazeneca ( inst ) , johnson & johnson ( inst ) , merck ( inst ) , pfizer ( inst ) , guardant health ( inst ) , incyte ( inst ) martha pitman consulting or advisory role : medtronic inga t . 
shaw honoraria : pfizer , novartis , roche , genentech , foundation medicine consulting or advisory role : pfizer , novartis , genentech , roche , ariad , ignyta , blueprint medicines , daiichi sankyo , emd serono , taiho pharmaceutical , kso therapeutics , natera , loxo , takeda research funding : pfizer , novartis , roche , genentech , a . 
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garassino mc , cho bc , kim jh , et al : durvalumab as third - line or later treatment for advanced nonsmall - cell lung cancer ( atlantic ) : an open - label , single - arm , phase 2 study . 
lee ck , man j , lord s , et al : clinical and molecular characteristics associated with survival among patients treated with checkpoint inhibitors for advanced nonsmall - cell lung carcinoma : a systematic review and meta - analysis . 
sholl lm , aisner dl , varella - garcia m , et al : multi - institutional oncogenic driver mutation analysis in lung adenocarcinoma : the lung cancer mutation consortium experience . 
sacher ag , paweletz c , dahlberg se , et al : prospective validation of rapid plasma genotyping for the detection of egfr and kras mutations in advanced lung cancer . 
lee ck , davies l , wu yl , et al : gefitinib or erlotinib vs chemotherapy for egfr mutation positive lung cancer : individual patient data meta - analysis of overall survival . 
yang jc , wu yl , schuler m , et al : afatinib versus cisplatin - based chemotherapy for egfr mutationpositive lung adenocarcinoma ( lux - lung 3 and lux - lung 6 ) : analysis of overall survival data from two randomised , phase 3 trials . 
vergnenegre a , massuti b , de marinis f , et al : economic analysis of first - line treatment with erlotinib in an egfr - mutated population with advanced nsclc . 
ting j , tien ho p , xiang p , et al : cost - effectiveness and value of information of erlotinib , afatinib , and cisplatin - pemetrexed for first - line treatment of advanced egfr mutationpositive nonsmall - cell lung cancer in the united states . 
lu s , ye m , ding l , et al : cost - effectiveness of gefitinib , icotinib , and pemetrexed - based chemotherapy as first - line treatments for advanced nonsmall - cell lung cancer in china . 
handorf ea , mcelligott s , vachani a , et al : cost effectiveness of personalized therapy for firstline treatment of stage iv and recurrent incurable adenocarcinoma of the lung . 
isla d , de castro j , juan o , et al : costs of adverse events associated with erlotinib or afatinib in first - line treatment of advanced egfr - positive nonsmall - cell lung cancer . 
park k , tan eh , obyrne k , et al : afatinib versus gefitinib as first - line treatment of patients with egfr mutationpositive nonsmall - cell lung cancer ( lux - lung 7 ) : a phase 2b , open - label , randomised controlled trial . 
schuler m , wu yl , hirsh v , et al : first - line afatinib versus chemotherapy in patients with non small - cell lung cancer and common epidermal growth factor receptor gene mutations and brain metastases . 
goss g , tsai cm , shepherd fa , et al : cns response to osimertinib in patients with t790mpositive advanced nsclc : pooled data from two phase ii trials . 
 patterns of metastatic spread and mechanisms of resistance to crizotinib in ros1 - positive nonsmall - cell lung cancer purpose the ros1 tyrosine kinase is activated through ros1 gene rearrangements in 1% to 2% of nonsmall - cell lung cancers ( nsclcs ) , which confer sensitivity to treatment with the anaplastic lymphoma kinase ( alk ) / ros1 / mesenchymal - epithelial transition factor inhibitor crizotinib . 
currently , insights into patterns of metastatic spread and mechanisms of crizotinib resistance among patients with ros1 - positive disease are limited . patients and methods we reviewed clinical and radiographic imaging data of patients with ros1and alk - positive nsclc to compare patterns of metastatic spread at initial metastatic diagnosis . 
to determine molecular mechanisms of crizotinib resistance , we analyzed repeat biopsy specimens from a cohort of patients with ros1 - positive disease who progressed on crizotinib . results we identified 39 and 196 patients with advanced ros1and alk - positive nsclc , respectively . 
ros1 mutations included g2032r ( 41% ) , d2033n ( 6% ) , and s1986f ( 6% )  . conclusion compared with alk rearrangements , ros1 rearrangements are associated with lower rates of extrathoracic metastases , including fewer brain metastases , at initial metastatic diagnosis . 
2017 by american society of clinical oncology introduction treatment paradigms for advanced nonsmallcell lung cancer ( nsclc ) continue to evolve and reflect a growing understanding of the genetic underpinnings of the disease . 
subsequent work has shown that such rearrangements are present in 1% to 2% of nsclcs and define a distinct molecular subgroup.2 - 4 of note , ros1 is phylogenetically related to the anaplastic lymphoma kinase ( alk ) receptor tyrosine kinase.5 thus , like alk rearrangements in nsclc , 6 , 7 ros1 fusions confer sensitivity to the alk / ros1 / mesenchymal - epithelial transition factor inhibitor crizotinib.3 in profile 1001 ( study of oral pf - 02341066 , a c - met / hepatocyte growth factor tyrosine kinase inhibitor , in patients with advanced cancer ) , crizotinib produced an objective response rate justin f . 
shaw author affiliations and support information ( if applicable ) appear at the end of this article . presented at the 2016 american society of clinical oncology annual meeting , chicago , il , june 3 - 7 , 2016 . corresponding author : alice t . 
on the basis of this activity , the us food and drug administration expanded crizotinibs approval to include patients with advanced , ros1 - positive nsclc.12 consistent with these reports , we observed several prolonged responses to crizotinib among patients with ros1 - positive nsclc at our institution . specifically , we observed particularly durable responses among patients with intrathoracic - only disease ( ie , m1a disease ) , 3 which led us to hypothesize that differences in malignant phenotypes between ros1 and alk rearrangements , including differences in patterns of metastatic spread , affect therapeutic outcomes . 
in one early study , doebele et al13 reported that alk rearrangements are associated with pericardial , pleural , and liver metastases compared with an egfr / kras / alk wild - type cohort . 
outside this report , however , few studies have evaluated patterns of metastatic spread according to molecular genotype in nsclc , and none have focused on ros1.14 , 15 in this study , we investigated the distribution of malignant disease at initial metastatic diagnosis and evaluated associations with clinical outcomes in ros1 - positive nsclc . 
data were updated as of january 2016 . all studies were performed under an institutional review boardapproved protocol . pfs was measured from the time of crizotinib initiation to investigator - assessed , radiographic progression or death in the absence of documented progression . 
overall survival ( os ) was measured from initiation of crizotinib until death . patients without a known date of death were censored at the time of last follow - up . in addition , patients with ros1 - positive nsclc who underwent repeat biopsies / resection of progressing lesions on crizotinib between july 2012 and december 2016 were identified . 
age and lines of therapy were analyzed by wilcoxon rank sum test . the kaplan - meier method was used to estimate pfs and os medians and probabilities , and the log - rank test was used to compare their differences between groups . 
no significant differences in baseline clinicopathologic characteristics were observed between groups ( data supplement ) , including no difference in the median number of prior lines of therapy before crizotinib ( median , 1 ; data supplement )  . 
to address whether this difference was confounded by prior therapy , we examined pfs among patients who received second - line crizotinib ( ie , after one prior therapy ) because this was the most common line of crizotinib in both cohorts ( ros1 , 60% ; alk , 42% )  . 
of note , 101 patients with alk - positive nsclc ( 58% ) received secondor thirdgeneration alk inhibitors after crizotinib , whereas only seven patients with ros1 - positive nsclc ( 23% ) received additional ros1 inhibitors ( p , .001 ; data supplement )  . 
thus , the longer median pfs with crizotinib among patients with ros1 - positive disease may have been partly offset by greater access to next - generation targeted therapies among patients with alk - positive disease . sites of metastatic disease to determine whether these clinical outcomes may have also been affected by distribution of metastatic disease , we compared sites of disease at initial metastatic diagnosis among patients with alkand ros1 - positive nsclc . 
metastatic involvement of a given anatomic site was determined on the basis of clinical staging ( tnm seventh edition ) .30 positron emission tomography - computed tomography ( ct ) and ct scans of the chest , abdomen , and pelvis were performed in 72% and 26% of patients with alk - positive nsclc and 82% and 18% of patients with ros1 - positive nsclc , respectively ( table 1 )  . 
brain magnetic resonance imaging ( alk , 76% ; ros1 , 82% ) or a head ct scan with intravenous contrast ( alk , 9% ; ros1 , 10% ) were also performed in most patients . 
in addition to having a lower rate of brain metastases at initial diagnosis , fewer patients with ros1 - positive nsclc developed brain metastases over time compared with those with alk - positive nsclc . 
the cumulative incidence of brain metastasis among patients with alkand ros1 - positive disease was 73% and 34% , respectively , at 5 years after initial metastatic diagnosis ( p , .001 ; fig 3a )  . 
percentages may not add to 100 because of rounding . abbreviations : ct , computed tomography ; ecog , eastern cooperative oncology group ; mri , magnetic resonance imaging ; pet , positron emission tomography . * includes one patient with adenosquamous histology . on the basis of performance status at initial metastatic presentation or at the first documented assessment . 
performance status was not available in four patients with alk - positive disease and one patient with ros1 - positive disease . systemic staging and neuroimaging studies were not done or were not available for review in five and 35 patients with alk - positive disease , respectively . 
four patients with alk - positive disease had both a brain mri and a head ct scan . neuroimaging studies were not done or not available for review in three patients with ros1 - positive disease . time was also greater among those with alkpositive disease ( 56% v 22% at 5 years ; p = .001 ; fig 3b )  . 
of note , 101 patients with alk - positive nsclc ( 58% ) received next - generation alk inhibitors ( ceritinib , 40% ; alectinib , 27% ; brigatinib , 3% ) after crizotinib ( data supplement ) ; however , 93% of these patients received such agents after the initial development of brain metastases . 
collectively , these findings suggest that alk - positive cancers have an increased tropism for the brain compared with ros1 - positive nsclcs . next , we evaluated outcomes of crizotinib treatment according to the presence of extrathoracic metastases ( ie , m1a v m1b disease )  . 
thus , this difference in pfs for crizotinib treatment outcomes between patients with alkand ros1 - positive nsclc in the overall study population may have been partly driven by the higher frequency of m1a disease among those with ros1 - positive disease because this group appears to derive greater benefit with crizotinib . ros1 : crizotinib resistance despite the effectiveness of crizotinib in alkand ros1 - positive nsclc , most patients acquire resistance . 
to investigate potential mechanisms of resistance to crizotinib in ros1 - positive nsclc , we analyzed postprogression biopsy specimens from patients with ros1 - positive disease who progressed on crizotinib treatment . 
all patients underwent tissue sampling within 7 days of crizotinib discontinuation . persistence of the original ros1 rearrangement was observed in nine ( 100% ) of nine postcrizotinib specimens available for testing ( repeat fish [ n = 2 ] , rt - pcr [ n = 5 ] , or ngs [ n = 2 ] )  . 
 ( d ) os as measured from the time of crizotinib initiation in the second - line setting . time since crizotinib initiation ( months ) time since crizotinib initiation ( months ) no . 
thus , ros1 resistance mutations were detected in a majority ( 10 [ 62.5% ] of 16 ) of patients with crizotinib resistance . because crizotinib has limited blood - brain barrier penetration , 31 we analyzed crizotinib - resistant samples by site of disease . 
among non - cns specimens ( n = 14 ) , the frequency of ros1 resistance mutations was 64% ( appendix fig a2 ) , with 50% of specimens harboring the ros1 g2032r mutation . 
among six specimens that underwent targeted ngs with the mgh snapshot platform , none was found to have high - level copy number changes in any analyzed gene ( data supplement )  . 
 a mgh047 - 5 ros1 g2032r mgh9018 - 1 ros1 wt biopsy ros1 wt ros1 wt ros1 g2032r ros1 g2032r ros1 g2032r ros1 s1986f ros1 g2032r * ros1 wt ros1 wt ros1 wt * mgh070 - 1 mgh096 - 1 mgh077 - 1 mgh080 - 2 mgh998 - 1 mgh508 - 1 mgh095 - 1 mgh996 - 1 mgh943 - 1 mgh995 - 1 mgh933 - 1 mgh081 - 1 mgh968 - 1 mgh986 - 1 fig 4 . 
mgh , massachusetts general hospital ; wt , wild type . g2032r ros1 d2033n ros1 wt ( 2 sites ) ros1 g2032r s1986f d2033n progression - free survival ( months ) ros1 g2032r discussion we observed that despite shared clinical features between ros1and alk - positive nsclcs , these genetic alterations were associated with different patterns of metastatic spread . 
although the shorter pfs with crizotinib treatment among patients with alk - positive nsclc may have been an influence in this series , we observed significantly higher rates of brain metastases at initial diagnosis ( ie , before exposure to crizotinib ) in this group . together , these observations suggest that alkrearranged nsclcs have a greater tropism for the cns . 
the majority of patients with alkpositive disease who developed brain metastases did so before receiving more cns penetrable , next - generation alk inhibitors , which provides an additional rationale for investigating the upfront use of next - generation alk inhibitors to prevent or delay the emergence of brain metastases . 
several ongoing first - line studies are comparing crizotinib with second - generation alk inhibitors ( eg , alex [ study comparing alectinib with crizotinib in treatment - naive anaplastic lymphoma kinasepositive advanced nonsmall cell lung cancer participants ] , alta - 1l [ phase iii study of brigatinib versus crizotinib in alk - positive advanced non small - cell lung cancer patients ] ) and have generally incorporated secondary end points focused on intracranial disease activity . one limitation of this analysis is that we observed a relatively higher frequency of baseline brain metastases in the alk - positive cohort than had other studies.6 , 13 although this observation may reflect differences in local imaging practices or referral patterns , such factors would have likely affected both alk and ros1 cohorts . 
nonetheless , several other studies have shown a comparable pfs to our series.8 - 11 of note , although patients with ros1 - positive disease had a longer median pfs with crizotinib treatment than patients with alk - positive disease , no difference in os was found between cohorts . 
we cannot exclude the possibility that this was due to unaccounted - for differences in patient characteristics , although the higher rates of next - generation targeted therapy use among patients with alk - positive disease may have offset the longer pfs with crizotinib treatment among patients with ros1positive disease . despite the significant activity of crizotinib , acquired resistance remains a challenge for patients with alkand ros1 - positive nsclc alike.16 , 43 - 45 thus far , insights into the mechanisms of resistance to crizotinib among those with ros1 - positive disease have been limited and generally consist of isolated case reports , small series , and preclinical evaluations.16 - 19 , 46 to date , seven different ros1 resistance mutations have been described.16 - 19 , 46 in addition , upregulation of bypass signaling pathways ( eg , egfr , ras , kit ) have also been reported.20 - 22 , 47 to our knowledge , we present the largest study of crizotinib resistance in ros1 - positive nsclc to date , which has found ros1 resistance mutations in a majority of specimens . 
 emerged through selection of pre - existing clones or through genetic evolution of drugtolerant cells.48 despite similarities between alk and ros1 , each has a different frequency and spectrum of on - target mechanisms of crizotinib resistance . 
indeed , patients with ros1 - positive nsclc have a much higher frequency of on - target resistance mutations , but such mutations are concentrated in a narrower segment of the kinase , which perhaps reflects the greater potency of crizotinib for ros1 than for alk . potential limitations of this analysis are that several different genotyping platforms were used . 
thus , we may have overlooked ros1 mutations outside this region . however , all specimens that underwent sanger sequencing and / or deep sequencing ( n = 8 ) were evaluated for mutations across the entire ros1 kinase domaanother important limitation is that we were unable to identify a mechanism of crizotinib resistance in all patients . 
thus , additional studies are necessary to elucidate other potentially target - independent mechanisms of crizotinib resistance . in alk - positive nsclc , second - generation alk inhibitors demonstrate significant activity in the crizotinib - resistant setting largely as a result of their greater potency against alk compared with crizotinib . 
beyond cabozantinib and tpx - 0005 , several other agents with ros1 activity are being evaluated , including entrectinib ( clinicaltrials.gov identifier nct02097810 ) , 53 ds - 6051b ( clinicaltrials.gov identifier nct02279433 ) , and lorlatinib ( clinicaltrials.gov identifier nct01970865 ) , 50 , 54 but these agents have not demonstrated clinical activity against ros1 g2032r to date ( data supplement )  . in summary , despite a shared susceptibility to crizotinib , alk and ros1 rearrangements differ in distributions of metastatic disease . 
nonetheless , acquired resistance to crizotinib remains a significant challenge for both molecular subsets . among patients with ros1 - positive nsclc , ros1 mutations ( most notably g2032r ) are the predominant mechanisms of resistance to crizotinib , which underscores the need for clinical development of next - generation ros1 inhibitors with activity against this mutation . 
provisional application to the us patent and trademark office ( application number 61817229 ) satoshi yoda no relationship to disclose ibiayi dagogo - jack no relationship to disclose luc friboulet no relationship to disclose jessica j . 
hubbeling no relationship to disclose leila dardaei no relationship to disclose james hardwick employment : pfizer stock and other ownership interests : pfizer travel , accommodations , expenses : pfizer donghui huang employment : pfizer stock and other ownership interests : pfizer travel , accommodations , expenses : pfizer mari mino - kenudson consulting or advisory role : merrimack , h3 biomedicine , acd pharmaceuticals , roche , genentech a . 
john iafrate stock and other ownership interests : archer biosciences consulting or advisory role : debiopharm group , constellation pharmaceuticals , chugai pharma , roche research funding : blueprint medicines patents , royalties , other intellectual property : archerdx exclusive license to amp technology anna f . 
farago honoraria : foundation medicine consulting or advisory role : pharmamar , merrimack , takeda pharmaceuticals , abbvie , intervention insights travel , accommodations , expenses : pharmamar , abbvie , stemcentrx aaron n . 
ferris no relationship to disclose zofia piotrowska consulting or advisory role : boehringer ingelheim , astrazeneca , ariad pharmaceuticals , takeda pharmaceuticals , superdimension research funding : guardant health ( inst ) alice t . 
shaw honoraria : pfizer , novartis , roche , genentech , foundation medicine consulting or advisory role : pfizer , novartis , genentech , roche , ariad pharmaceuticals , ignyta , blueprint medicines , daiichi sankyo , emd serono , taiho pharmaceutical , ksq therapeutics research funding : pfizer , novartis , roche , genentech acknowledgment we thank cyril h . 
shaw at , kim dw , nakagawa k , et al : crizotinib versus chemotherapy in advanced alk - positive lung cancer . med 371 : 2167 - 2177 , 2014 n engl j med 368 : 2385 - 2394 , 2013 8 . 
moro - sibilot d , faivre l , zalcman g , et al : crizotinib in patients with advanced ros1 - rearranged nonsmall - cell lung cancer ( nsclc ) : preliminary results of the acse phase ii trial . 
goto k , yang j , kim d , et al : phase ii study of crizotinib in east asian patients ( pts ) with ros1 - positive advanced nonsmall - cell lung cancer ( nsclc )  . 
russo a , franchina t , picone a , et al : different metastatic pattern according to the egfr mutational status in a cohort of lung adenocarcinomas ( adcs ) : a single - institution report . 
facchinetti f , loriot y , kuo ms , et al : crizotinib - resistant ros1 mutations reveal a predictive kinase inhibitor sensitivity model for ros1and alk - rearranged lung cancers . 
drilon a , somwar r , wagner jp , et al : a novel crizotinib - resistant solvent - front mutation responsive to cabozantinib therapy in a patient with ros1 - rearranged lung cancer . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
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gainor jf , ou sh , logan j , et al : the central nervous system as a sanctuary site in alk - positive nonsmall - cell lung j clin oncol 29 : e443 - e445 , 2011 cancer . 
soria jc , tan ds , chiari r , et al : first - line ceritinib versus platinum - based chemotherapy in advanced alkrearranged nonsmall - cell lung cancer ( ascend - 4 ) : a randomised , open - label , phase 3 study . 
gadgeel sm , shaw at , govindan r , et al : pooled analysis of cns response to alectinib in two studies of pretreated patients with alk - positive nonsmall - cell lung cancer . 
crin `o l , ahn mj , de marinis f , et al : multicenter phase ii study of whole - body and intracranial activity with ceritinib in patients with alk - rearranged nonsmall - cell lung cancer previously treated with chemotherapy and crizotinib : results from ascend - 2 . 
woo cg , seo s , kim sw , et al : differential protein stability and clinical responses of eml4 - alk fusion variants to various alk inhibitors in advanced alk - rearranged nonsmall cell lung cancer . 
huber kv , salah e , radic b , et al : stereospecific targeting of mth1 by ( s ) - crizotinib as an anticancer strategy . nature 508 : 222 - 227 , 2014 42 . 
shaw a , camidge d , engelman j , et al : clinical activity of crizotinib in advanced nonsmall - cell lung cancer ( nsclc ) harboring ros1 gene rearrangement . 
zou hy , li q , engstrom ld , et al : pf - 06463922 is a potent and selective next - generation ros1 / alk inhibitor capable of blocking crizotinib - resistant ros1 mutations . 
cui j , rogers e , zhai d , et al : ending the endless acquired tyrosine kinase resistance mutationsdesign of tpx0005 , a multi - target alk / ros1 / trk inhibitor with broad spectrum activity against wild - type and mutants including alk g1202r , ros1 g2032r , and trka g595r . 
drilon a , siena s , ou si , et al : safety and antitumor activity of the multi - targeted pan - trk , ros1 , and alk inhibitor entrectinib ( rxdx - 101 ) : combined results from two phase i trials ( alka - 372 - 001 and startrk - 1 )  . cancer discov 7 : 400 - 409 , 2017 54 . 
solomon b , bauer t , felip e , et al : safety and efficacy of lorlatinib ( pf - 06463922 ) from the dose - escalation component of a study in patients with advanced alk + or ros1 + nonsmall - cell lung cancer ( nsclc )  . 
progressionfree survival ( pfs ) of alkand ros1 - positive patients with ( a ) m1a and ( b ) m1b disease . time since crizotinib initiation ( months ) time since crizotinib initiation ( months ) no . 
 c targeted next - generation sequencing reveals clinically actionable braf and esr1 mutations in low - grade serous ovarian carcinoma introduction low - grade serous ovarian cancer ( lgsoc ) is a rare ( < 5% ) subset of epithelial ovarian cancer with unique biologic , clinical , and genetic features.1 compared with those with high - grade soc ( hgsoc ) , the most common histologic subtype of ovarian cancer , patients with lgsoc are diagnosed at a younger age and have a better prognosis.2 , 3 standard - of - care treatment of advanced - stage lgsoc and hgsoc is similar : surgical cytoreduction plus platinum and taxane chemotherapy.4 however , lgsoc is less responsive to platinum - based chemotherapy compared with hgsoc , 5 , 6 possibly because of slower proliferation and fewer abnormalities in the homologous recombination repair pathway ( including brca1 or brca2 mutations ) in lgsoc.7 in contrast , lgsoc may benefit more from endocrine or hormonal therapy ( aromatase inhibitors [ ais ] or tamoxifen ) , because a greater proportion of lgsocs express estrogen and progesterone receptors.8 endocrine therapy is a common treatment of recurrent lgsoc , 9 and in retrospective studies , it provided benefit as maintenance therapy in the adjuvant setting.10 although recurrent lgsoc can follow a chronic , indolent course , it is incurable with current treatments , and patients often die as a result of their disease , highlighting the need for novel therapies . we describe two patients with lgsoc whose clinical management was informed by targeted panel next - generation sequencing ( ngs ) performed in our institution . 
the oncopanel test at dana - farber cancer institute consists of targeted ngs of formalin - fixed tumor samples covering exons of > 300 cancer - associated genes , plus intronic regions of genes involved in somatic rearrangements.11 - 13 oncopanel tests report mutations , insertions and deletions , copy number variations , and structural variants . 
we present clinically relevant alterations identified by oncopanel in two patients with recurrent lgsoc : a patient with a braf v600e mutation who derived clinical benefit from braf inhibitor vemurafenib , and a patient with progressive disease after durable response to hormonal therapy whose recurrent tumor harbored an esr1 mutation associated with resistance to antiestrogen therapy . 
 konstantinopoulos author affiliations and support information ( if applicable ) appear at the end of this article . licensed under the creative commons attribution 4.0 license corresponding author : panagiotis a . 
 b time since diagnosis ( years ) time since diagnosis ( years ) ca - 125 surgery carboplatin plus paclitaxel ca - 125 surgery carboplatin plus paclitaxel anastrozole bevacizumab vemurafenib carboplatin - based chemotherapy anastrozole plus leuprolide basis of oncopanel testing of her tumor from her recurrence surgery , which revealed a braf c.1799t > a ( p.v600e ) mutation ( appendix ) , she started vemurafenib at 480 mg twice daily , which was dose reduced to 240 mg twice daily because of cutaneous toxicity . 
two years later , she developed recurrent disease and underwent secondary cytoreduction of metastatic serous carcinoma at multiple sites in the abdomen and pelvis , followed by platinum - based chemotherapy . 
she underwent complete surgical cytoreduction , with pathology showing stage iii invasive serous borderline tumor and one area suggestive for lg serous discussion recent genomic profiling of lgsoc suggested several potential targetable pathways and highlighted dramatic differences in the genomes of lgsoc and hgsoc . 
braf v600e mutations are present in 35% of serous borderline cancers or lgsocs18 and are associated with better prognosis and decreased likelihood of requiring systemic therapy.18 despite the low prevalence of braf v600e mutations in patients with recurrent disease requiring treatment , this case highlights that this mutation may correlate with sensitivity to braf inhibitors , such as vemurafenib . 
two previous patient cases of lgsoc with braf v600e mutations and sustained response to vemurafenib were described.26 , 27 one patient was treated in a basket trial of solid tumors with braf v600e mutations and responded for at least 12 months.26 a second patient , who had experienced progression throughout chemotherapy , hormonal therapy , and antiangiogenic therapy , had a braf v600e mutation on a recurrence biopsy and was treated with vemurafenib , achieving clinical and radiographic responses.27 despite dose reduction because of skin rash , the patient continued to receive vemurafenib with an ongoing partial response for nearly 2 years.27 it is notable that both this patient and our patient seemed to respond to significantly lower doses of vemurafenib than the us food and drug administration approved dose in melanoma ( 960 mg orally twice daily ) , suggesting that lgsoc tumors with braf v600e mutations may be more sensitive to raf inhibition . 
given that not all solid tumors with braf v600e mutations respond to raf inhibition ( eg , braf - mutant colon cancers do not benefit from vemurafenib28 ) , these cases illustrate that recurrent lgsoc with a braf v600e mutation may derive clinical benefit from treatment with vemurafenib or other raf inhibitors , arguing for routine assessment of these mutations in recurrent lgsoc . 
selumetinib showed a 15% response rate in recurrent lgsoc in a phase ii trial.25 an exceptional responder in this trial had a response to selumetinib of > 5 years and had a deletion in map2k1 ( encoding mek1 ) , which has oncogenic activity.22 another patient with lgsoc and a kras g12d mutation had a response of > 7 years to selumetinib.29 our second case of recurrent lgsoc highlights that targeted ngs can elucidate the mechanism of resistance in a patient with excellent response to hormonal therapy and may help tailor future therapy . 
although esr1 mutations in the ligand - binding domain ( most commonly d538g and y537c / s / n ) have been reported in breast cancers treated with hormonal therapy , this is to our knowledge the first esr1 mutation reported in lgsoc.30 - 35 esr1 mutations are rare in primary , untreated breast cancers but prevalent in metastatic breast cancers resistant to hormonal therapy.30 , 33 , 35 , 36 structural alterations conferred by these activating mutations , including y537s , result in ligand - independent activation of the estrogen receptor and resistance to endocrine antagonists.30 , 36 , 37 it is reasonable to surmise that this mutation contributed to tumor progression during ai therapy . 
 of action downstream of the activated estrogen receptor and bypass the resistance mechanis for instance , the oral selective estrogen receptor degrader ( serd ) azd9496 potently binds and downregulates d538g and y537s esr1 proteins in vitro and was effective against breast cancer xenografts with y537s and other esr1 mutations , whereas the us food and drug administrationapproved serd fulvestrant had only a partial effect against y537s.38 , 39 combinations of antiestrogen agents such as ais or serds with targeted therapies such as mammalian target of rapamycin inhibitors ( eg , everolimus ) and cdk4 / 6 inhibitors ( eg , palbociclib and abemaciclib ) may also overcome resistance associated with esr1 mutations . 
in the paloma3 ( palbociclib ongoing trials in the management of breast cancer 3 ) trial of fulvestrant plus palbociclib versus placebo in ai - resistant patients , the benefit of addition of palbociclib to fulvestrant was seen irrespective of specific esr1 mutation.40 in bolero2 ( breast cancer trials of oral everolimus 2 ) trial of exemestane plus everolimus , the benefit of everolimus was evident in tumors with an esr1 d538g mutation but was not clear in y537s esr1mutated tumors because of low numbers.41 , 42 esr1 mutations can be detected by targeted sequencing in tumors and cell - free dna , indicating that clinical testing for these resistance mutations is feasible.35 , 36 , 42 tumor heterogeneity is an important challenge in interpreting targetable mutations . 
on a practical level , both patients maintained stable low - volume disease with the selected targeted therapy , and identifying different mutations in other lesions might not necessitate a change in management as long as the patients remain asymptomatic . in conclusion , our first case highlights the potential utility of testing for braf v600e by targeted sequencing in lgsoc , as well as the possibility of meaningful clinical response to even low doses of vemurafenib in patients with lgsoc with a braf v600e mutation . 
in the second case , targeted sequencing helped elucidate the mechanism of resistance in a patient with lgsoc with prolonged response to ai therapy , which could be relevant for selection of additional therapy to overcome resistance . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . collection and assembly of data : elizabeth h . 
matulonis consulting or advisory role : merck , eli lilly , geneos , 2x oncology , myriad genetics , clovis oncology , fujifilm research funding : merck , novartis panagiotis a . 
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garcia ep , minkovsky a , jia y , et al : validation of oncopanel : a targeted next - generation sequencing assay for the detection of somatic variants in cancer . 
grisham rn , iyer g , garg k , et al : braf mutation is associated with early stage disease and improved outcome in patients with low - grade serous ovarian cancer . 
tsang yt , deavers mt , sun cc , et al : kras ( but not braf ) mutations in ovarian serous borderline tumour are associated with recurrent low - grade serous carcinoma . 
gershenson dm , sun cc , wong kk : impact of mutational status on survival in low - grade serous carcinoma of the ovary or peritoneubr j cancer 113 : 1254 - 1258 , 2015 22 . 
grisham rn , sylvester be , won h , et al : extreme outlier analysis identifies occult mitogenactivated protein kinase pathway mutations in patients with low - grade serous ovarian cancer . 
farley j , brady we , vathipadiekal v , et al : selumetinib in women with recurrent low - grade serous carcinoma of the ovary or peritoneum : an open - label , single - arm , phase 2 study . 
combe p , chauvenet l , lefrre - belda ma , et al : sustained response to vemurafenib in a low grade serous ovarian cancer with a braf v600e mutation . 
takekuma m , wong kk , coleman rl : a long - term surviving patient with recurrent low - grade serous ovarian carcinoma treated with the mek1 / 2 inhibitor , selumetinib . 
weir hm , bradbury rh , lawson m , et al : azd9496 : an oral estrogen receptor inhibitor that blocks the growth of er - positive and esr1 - mutant breast tumors in preclinical models . 
chandarlapaty s , chen d , he w , et al : prevalence of esr1 mutations in cell - free dna and outcomes in metastatic breast cancer : a secondary analysis of the bolero - 2 clinical trial . 
 appendix description of test the oncopanel assay surveys exonic dna sequences of 300 cancer genes and 113 introns across 35 genes for rearrangement detection.11 dna is isolated from tissue containing at least 20% tumor and analyzed by massively parallel sequencing using a solution - phase agilent sureselect hybrid capture kit ( santa clara , ca ) and an illumina 2500 sequencer ( san diego , ca )  . 
 detection of somatic structural variants enables quantification and characterization of circulating tumor dna in children with solid tumors objective liquid biopsies are being rapidly used in adult cancers as new biomarkers of disease . 
therefore , we developed a next - generation sequencing approach to detect and quantify ctdna in the blood of patients with the most common pediatric solid tumors . methods detection of ctdna requires assays sensitive to somatic events typically observed in the cancer type being studied . 
we adapted an ultralow passage whole - genome sequencing approach to capture copy number variants and a hybrid capture sequencing assay to detect translocations in liquid biopsy samples from pediatric patients . results copy number changes seen by ultralow passage whole - genome sequencing enabled detection of ctdna in patients with osteosarcoma , neuroblastoma , alveolar rhabdomyosarcoma , and wilms tumor . 
we also found that disease - specific genomic biomarkers of prognosis were detectable in ctdna . conclusion this study demonstrates that liquid biopsy approaches that detect somatic structural variants are well suited to pediatric solid tumors . 
we show that children with the most common solid tumor malignancies have detectable levels of ctdna , which may be used to track disease response and identify genomic subclassifiers of disease . 
2018 by american society of clinical oncology introduction cancer remains one of the most common childhood causes of disease - related death in developed countries.1 patients with pediatric solid tumor malignancies are commonly treated with regimens that combine intensive chemotherapy , surgery , and radiation therapy.2 this approach has led to tremendous improvements in outcome , with overall cure rates for pediatric solid tumors now > 80%.3 however , these cures come at the cost of exposing patients to a high risk of long - term toxicities resulting in significant morbidity.4 in some diseases , such as wilms tumor , efforts to de - escalate therapy have resulted in stable cure rates for low - risk patients accompanied by reduced treatment toxicity.5 , 6 in other diseases , such as ewing sarcoma , a lack of prognostic biomarkers limit the ability to risk stratify therapy.7 , 8 in both cases , the development of new and more precise assays for risk stratification could improve outcomes by facilitating patient - specific treatment modifications . kelly klega alma imamovic - tuco gavin ha andrea n . 
crompton author affiliations and support information ( if applicable ) appear at the end of this article . the contents are solely the responsibility of the authors and do not necessarily represent the official views of the national institutes of health or other funding agencies . corresponding author : brian d . 
numerous studies have now demonstrated that circulating tumor dna ( ctdna ) levels in adults with cancer correlate with disease burden and track with treatment responses over time.9 - 14 the development of ctdna assays rely on identification and quantification of somatic mutations.9 , 15 many ctdna assays have been developed to detect highly recurrent hot - spot mutations that frequently drive adult malignancies.9 , 12 , 13 , 16 - 18 however , recent comprehensive sequencing efforts in pediatric cancers show that pediatric solid tumors are rarely driven by highly recurrent single - nucleotide variants.19 instead , structural variants , including chromosomal copy number changes and dna translocations , are common somatic events in these tumor types.20 - 26 to determine whether patients with childhood solid tumor malignancies express detectable levels of ctdna , we adapted two next - generation sequencing strategies to detect and quantify structural variants in the plasma of these patients . 
 we applied these assays to five of the most common types of pediatric solid tumors , excluding cns tumors , to quantify ctdna levels , identify genomic subtypes , and confirm the feasibility of using ctdna assays to track disease response to therapy . methods patients and tissue samples patients at the dana - farber cancer institute and boston childrens hospital were consented to an institutional review boardapproved protocol . 
patients were included if an initial peripheral blood , pleural effusion , or bone marrow sample was collected within 3 days from the start of chemotherapy and before a surgical resection . 
additional details are available in the data supplement . sequencing library preparation , sequencing alignment , and ultralow passage whole - genome sequencing coverage for all experiments , sequencing library preparation and sequencing data alignment were performed using standard techniques . 
additional details are available in the data supplement . translocation - specific sarcoma sequencing assay development and sequencing to detect pediatric sarcoma - specific translocations in cell - free dna , we developed a unique hybrid capture sequencing assay . 
first , we reviewed the literature to identify the genomic introns involved in oncogenic translocations in the ewsr1 , fus , cic , ccnb3 , pax3 , pax7 , and tp53 genes.20 , 21 , 25 - 33 second , we used the sureselect advanced design wizard ( agilent technologies , santa clara , ca ) to create capture probes targeting these regions and the coding regions of tp53 and stag2 with the following design options : sense strand , 3 tiling density , least stringent masking , balanced boosting , and region extensions of 10 bases from the 3 and 5 ends ( data supplement )  . 
the average measured coverage at enrichment sites for all samples tested by transs - seq was 655 ( range , 31 to 1 , 464 )  . sequencing analysis ulp - wgs analysis was performed using the ichorcna algorithm ( dinstitute / ichorcna ) , with manual curation of results.34 in brief , ulp - wgs uses relative sequencing coverage of whole - genome data and computational correction of sequencing bias to detect segmental copy number changes . 
 wild - type reads detected in each sample , we developed a custom algorithm designed to realign all sequencing reads to either the reference human genome or the patient - specific translocation positive reference sequence ( vanallenlab / peds_ctdna )  . 
the algorithm then reported the number of reads aligned at the patient - specific translocation breakpoint and the number of wild - type reads at the equivalent genomic base - pair location within the human reference genome . 
because each cancer genome contains one translocated and one wild - type allele , whereas germline genomes contain two wild - type alleles , we used the following formula to calculate the percentage of ctdna , where t equals the number of translocation reads and w equals the number of wild - type reads : % ctdna = t / { [ ( w - t ) / 2 ] + t }  . cell lines and digital droplet polymerase chain reaction the ew8 ewing sarcoma cell line dilution experiments , digital droplet polymerase chain reaction ( ddpcr ) methods , and ddpcr primers are listed in the data supplement . highest values originating from patients with neuroblastoma ( fig 1a )  . 
ulp - wgs was performed for each sample to detect and quantify ctdna by measuring segmental copy number changes as recently described.34 ctdna was detected in 50% of pretreatment plasma samples from patients with osteosarcoma , neuroblastoma , wilms tumor , and alveolar rhabdomyosarcoma . 
in patients with detectable ctdna , the pattern of segmental chromosomal copy number changes in cellfree dna matched the copy number pattern observed in the tumor biopsy from the same patient ( figs 1c and 1d ; data supplement )  . 
in four of the seven patients with ewing sarcoma with no detectable ctdna , ulp - wgs analysis of tumor biopsy dna material demonstrated no discernable segmental copy number changes ( fig 2 )  . 
this finding is consistent with previous reports that ewing sarcoma tumors have few highly recurrent somatic events other than the ewsr1 / ets translocations and suggests that an alternative method for detection of ctdna is necessary for this disease.21 , 26 results patients and samples a liquid biopsy sample was collected before the initiation of therapy from 45 pediatric patients with cancer with a confirmed diagnosis of ewing sarcoma ( n = 11 ) , osteosarcoma ( n = 10 ) , neuroblastoma ( n = 10 ) , wilms tumor ( n = 8 ) , or alveolar rhabdomyosarcoma ( n = 7 )  . 
dna from tumor biopsy material from patients with neuroblastoma and wilms tumor were not readily available . circulating tumor dna is detectable in patients with pediatric solid tumor malignancies total cell - free dna levels ranged from 2 ng to 3.5 g of dna per 1 ml of plasma , with the translocation detection outperforms copy number detection for ewing sarcoma to detect ctdna in translocation - positive tumors , we developed a novel hybrid - capture assay , termed transs - seq , designed to sequence intronic regions involved in genomic rearrangements in pediatric sarcomas . 
first , we confirmed that the assay could detect the expected translocations in a panel of ewing and ewing - like sarcomas and alveolar rhabdomyosarcomas ( data supplement )  . 
ddpcr in these samples confirmed the ability to detect and quantify the ew8 - specific ewsr1 / fli translocation across the range of dilutions ( fig 3a ; data supplement )  . 
 ( a ) quantity of cell - free dna extracted per ml of plasma or body fluid in patients with osteosarcoma ( ost ) , alveolar rhabdomyosarcoma ( arms ) , ewing sarcoma ( ews ) , wilms tumor ( wilms ) , and neuroblastoma ( nb )  . 
the color for each data point corresponds to the relative copy number change from baseline , with blue equal to two copies of the genomic location , green equal to copy number loss , and red equal to copy number gains . 
the same pattern of copy number changes is observed in dna sequenced from a tumor biopsy ( top ) and a pretreatment blood sample ( bottom ) in ( c ) a patient with osteosarcoma and ( d ) alveolar rhabdomyosarcoma . and detected ew8 dna at a concentration of 1.56% ( figs 3b and 3c ; data supplement )  . identical to the breakpoint observed from the tumor biopsy sample . transs - seq was then applied to cell - free dna samples from patients with ewing sarcoma and alveolar rhabdomyosarcoma . 
for five patients with ewing sarcoma and three patients with alveolar rhabdomyosarcoma , dna from tumor biopsy samples was also profiled by transs - seq ( data supplement )  . 
in all cases , the unique genomic breakpoint in the ctdna was previous studies in ewing sarcoma have used ddpcr assays that amplify patient - specific ews / ets translocations to measure ctdna levels in patients.36 - 38 to compare transs - seq with this previously validated approach , patient specific polymerase chain reaction primers were developed for a subset of ewing sarcoma and alveolar rhabdomyosarcoma samples ( data supplement )  . 
 mrd0006 ewing sarcoma mrd0019 ewing sarcoma 108765 14 16 18 20 23 x 108765 14 16 18 20 23 x 14 16 18 20 23 x 108765 14 16 18 20 23 x chromosome mrd0023 ewing sarcoma chromosome mrd0046 ewing sarcoma chromosome chromosome mrd0007 ewing sarcoma 108765 chromosome mrd0041 ewing sarcoma 108765 chromosome mrd0047 ewing sarcoma 14 16 18 20 23 x 108765 14 16 18 20 23 x 14 16 18 20 23 x 108765 chromosome fig 2 . 
genome - wide copy number plots from seven ewing sarcoma tumor biopsy samples detected by ultralow passage whole - genome sequencing in patients for which circulating tumor dna could not be detected from peripheral blood . 
in three of the four patients with ewing sarcoma for which no copy number change could be detected by ulp - wgs in the tumor ( mrd0006 , mrd0041 , mrd0047 ) , transsseq was able to detect an ewsr1 - fusion in the tumor and cell - free dna ( data supplement )  . 
in the fourth patient ( mrd0007 ) , transs - seq detected an ewsr1 / fli fusion in the tumor but not in cell - free dna ( data supplement )  . 
however , four patients with tumor necrosis < 70% ( mrd0031 , mrd0036 , mrd0040 , mrd0054 ) had detectable ctdna in at least one sample collected after initiation of chemotherapy ( fig 4d ; data supplement )  . 
 finally , in patients whose ctdna levels became undetectable with therapy but then experienced a clinical relapse or progression , ctdna was again detectable before the initiation of relapsed therapy ( fig 4b ; data supplement )  . genomic markers of poor outcome are detectable in ctdna we also examined whether disease - specific genomic markers of prognosis could be detected in ctdna . 
recent studies demonstrate that mutations in stag2 and tp53 may be associated with a worse outcome in ewing sarcoma.21 , 26 a frame - shift mutation in stag2 was detected by ctdna from one patient ( mrd0023 ) , and a mutation in tp53 was detected in another patient ( mrd0003 ; data supplement )  . 
in alveolar rhabdomyosarcoma , studies have demonstrated that patients with pax3 / foxo1 have a worse prognosis than patients with pax7 / foxo1 translocation.41 the foxo1 fluorescent in situ hybridization probe , which is used in the diagnostic work - up of alveolar rhabdomyosarcoma , confirms a foxo1 rearrangement but not the fusion partner . 
in osteosarcoma , copy number gains of chromosome arm 8q are associated with a poor outcome.42 - 44 ulp - wgs detected copy number gains in 8q in seven of nine osteosarcoma samples with detectable ctdna ( fig 1c ; data supplement )  . 
amplification of mycn is a well - established marker of poor prognosis in neuroblastoma.45 , 46 mycn amplification was detectable in the ctdna of two patients by ulp - wgs ( fig 5a ; data supplement )  . 
finally , in wilms tumor , copy number gains of 1q were associated with poor prognosis in favorable histology tumors.47 ulp - wgs detected 1q gain in copy number change but ulp - wgs was unable to detect ctdna from the same patient ( mrd0019 , mrd0023 , mrd0046 ) , transsseq detected low levels of ctdna , suggesting that transs - seq may have greater sensitivity for ctdna than ulp - wgs in ewing sarcoma ( data supplement )  . ctdna levels track with disease burden in pediatric solid tumors when comparing ctdna levels across cancer types , neuroblastoma demonstrated a significantly higher percentage of ctdna ( median , 55% ; range , 13% to 98% ) than other cancer types ( data supplement )  . 
in patients with newly diagnosed ewing sarcoma and alveolar rhabdomyosarcoma , ctdna levels declined rapidly after initiation of chemotherapy , often becoming undetectable by the start of the second cycle ( figs 4a and 4b ; data supplement )  . 
in osteosarcoma , studies have shown that a high percentage of tumor necrosis observed in the primary tumor after neoadjuvant chemotherapy is associated with a better prognosis.39 , 40 in five patients with newly diagnosed osteosarcoma , the percentage of tumor necrosis was available . 
the translocation - specific sarcoma sequencing assay ( transs - seq ) detects circulating tumor dna ( ctdna ) in ewing sarcoma ( ews ) and alveolar rhabdomyosarcoma ( arms )  . 
 samples are plotted on the x - axis by the percentage of ew8 by experimental dilution and on the y - axis by the percentage of ew8 dna detected by digital droplet polymerase chain reaction ( ddpcr )  . 
 ( d ) percentage of ctdna levels in cell - free dna samples from patients with ewing sarcoma and arms ( same samples as in figure 1b ) determined by transsseq . 
 percentage of ctdna levels from serial cell - free dna samples collected from four patients with ( a ) ewing sarcoma , ( b ) alveolar rhabdomyosarcoma , ( c - d ) osteosarcoma . 
blue dots indicate peripheral blood samples , and the red dot ( b ) indicates a bone marrow sample collected simultaneously with a blood sample . mrd0019 ewing sarcoma mrd0045 alveolar rhabdomyosarcoma bone marrow clinical course clinical course mrd0061 osteosarcoma mrd0036 osteosarcoma clinical course clinical course had detectable levels of ctdna . 
patients with neuroblastoma had the highest levels of ctdna , including two patients with > 1 g of cell - free dna per ml of plasma , which was nearly 100% composed of tumor dna . 
however , one obvious limitation to the use of ulp - wgs was in tumor types with low rates of copy number alterations , such as ewing sarcoma . recent liquid biopsy studies in ewing sarcoma have used the development of patient - specific ddpcr assays.36 - 38 although this approach has proven to be highly sensitive and quantitative , the development of each assay requires genomic profiling of large amounts of tumor biopsy material to establish the patient - specific oncogenic translocation . 
one recent study used a combination of ddpcr and hybrid capture sequencing to detect ewsr1 translocations in ewing sarcoma and desmoplastic small roundcell tumors.38 results showed that it is feasible to detect ewsr1 translocations directly from the plasma of these patients without first sequencing the tumor sample . 
despite the larger genomic region targeted for hybrid capture , the transs - seq assay detected ctdna in 10 of 11 pretreatment samples obtained from ewing sarcoma , similar to rates observed by other groups . 
we also demonstrated that quantification of ctdna levels was similar using either transs - seq or ddpcr . in this study , changes in ctdna levels were observed during a patients clinical course and corresponded to changes in disease burden . 
with next - generation sequencing becoming increasingly available for clinical decision making , we anticipate that our ctdna assays could also be adapted to clinical laboratory testing if they demonstrate significant improvements to risk stratification for pediatric solid tumors . 
 ( a ) sequencing coverage ( read counts ) across the mycn gene ( top ) and c - myc gene ( bottom ) from four cell - free dna samples from patients with neuroblastoma . 
therefore , prospective studies should be designed to obtain frequent samples throughout treatment so that the time points most predictive of outcome can be appropriately identified . finally , our study demonstrates that next generation sequencing of ctdna can detect existing genomic biomarkers of outcome and may provide another modality for genomic profiling of tumors in patients . 
furthermore , it remains unclear whether these genomic biomarkers are primarily clonal or heterogeneous events within a tumor or across tumors and whether the clonality of these genomic events changes in patients who relapse after therapy . 
recent studies demonstrate that ctdna can detect intratumor and multitumor heterogeneity and detect complex patterns of treatment resistance.52 - 55 with the emergence of new sequencing modifications that improve sensitivity and decrease sequencing errors , we believe that ctdna profiling by next - generation sequencing approaches will improve our understanding of tumor heterogeneity and patterns of somatic evolution in pediatric solid tumors.12 , 16 in addition , the assays described in this study could facilitate broader profiling of ctdna , such as deep whole - exome sequencing , by providing a mechanism to screen samples for the presence of sufficiently abundant ctdna , allowing selection of samples most likely to yield informative data . in summary , our study demonstrates that patients with pediatric solid tumors have detectable levels of ctdna , which can be measured with next generation sequencing approaches that do not require profiling of tumor biopsy material or patient - specific assays . 
disease - specific genomic hallmarks of pediatric solid tumors can also be identified in liquid biopsy samples , and ctdna levels correlate with disease burden and response to therapy over time . 
crompton manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors anwesha nag no relationship to disclose eliezer van allen stock and other ownership interests : syapse , tango therapeutics , genome medical consulting or advisory role : syapse , roche , third rock ventures , takeda , novartis , genome medical , invitae speakers ' bureau : illumina research funding : bristol - myers squibb , novartis elizabeth mullen no relationship to disclose steven g . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . katherine janeway no relationship to disclose matthew meyerson stock and other ownership interests : origimed consulting or advisory role : origimed research funding : bayer ( inst ) patents , royalties , other intellectual property : patent on egfr mutations for lung cancer diagnosis aaron r . 
crompton employment : mersana ( i ) , shire ( i ) stock and other ownership interests : mersana ( i ) , shire ( i ) acknowledgment we thank the boston childrens hospital biorepository for processing a portion of the blood samples . 
pediatr blood cancer 60 : 1083 - 1094 , 2013 kelly klega no relationship to disclose alma imamovic - tuco no relationship to disclose gavin ha no relationship to disclose andrea n . 
bhakta n , liu q , ness kk , et al : the cumulative burden of surviving childhood cancer : an initial report from the st jude lifetime cohort study ( sjlife )  . 
fernandez cv , perlman ej , mullen ea , et al : clinical outcome and biological predictors of relapse after nephrectomy only for very low - risk wilms tumor : a report from childrens oncology group aren0532 . 
oxnard gr , paweletz cp , kuang y , et al : noninvasive detection of response and resistance in egfr - mutant lung cancer using quantitative next - generation genotyping of cell - free plasma dna . 
parkinson ca , gale d , piskorz am , et al : exploratory analysis of tp53 mutations in circulating tumour dna as biomarkers of treatment response for patients with relapsed high - grade serous ovarian carcinoma : a retrospective study . 
sacher ag , paweletz c , dahlberg se , et al : prospective validation of rapid plasma genotyping for the detection of egfr and kras mutations in advanced lung cancer . 
yung tk , chan kc , mok ts , et al : single - molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital pcr in non - small cell lung cancer patients . 
shern jf , chen l , chmielecki j , et al : comprehensive genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic axis in fusion - positive and fusionnegative tumors . 
tirode f , surdez d , ma x , et al : genomic landscape of ewing sarcoma defines an aggressive subtype with co - association of stag2 and tp53 mutations . 
kawamura - saito m , yamazaki y , kaneko k , et al : fusion between cic and dux4 up - regulates pea3 family genes in ewing - like sarcomas with t ( 4 ; 19 ) ( q35 ; q13 ) translocation . 
ng tl , osullivan mj , pallen cj , et al : ewing sarcoma with novel translocation t ( 2 ; 16 ) producing an in - frame fusion of fus and fev . 
sugita s , arai y , tonooka a , et al : a novel cic - foxo4 gene fusion in undifferentiated small round cell sarcoma : a genetically distinct variant of ewing - like sarcoma . 
yoshimoto m , graham c , chilton - macneill s , et al : detailed cytogenetic and array analysis of pediatric primitive sarcomas reveals a recurrent cic - dux4 fusion gene event . 
shukla nn , patel ja , magnan h , et al : plasma dna - based molecular diagnosis , prognostication , and monitoring of patients with ewsr1 fusion - positive sarcomas . 
meyers pa , schwartz cl , krailo m , et al : osteosarcoma : a randomized , prospective trial of the addition of ifosfamide and / or muramyl tripeptide to cisplatin , doxorubicin , and high - dose methotrexate . 
sorensen ph , lynch jc , qualman sj , et al : pax3 - fkhr and pax7 - fkhr gene fusions are prognostic indicators in alveolar rhabdomyosarcoma : a report from the childrens oncology group . 
tarkkanen m , elomaa i , blomqvist c , et al : dna sequence copy number increase at 8q : a potential new prognostic marker in high - grade osteosarcoma . 
gratias ej , dome js , jennings lj , et al : association of chromosome 1q gain with inferior survival in favorable - histology wilms tumor : a report from the childrens oncology group . 
lecomte t , berger a , zinzindohou f , et al : detection of free - circulating tumor - associated dna in plasma of colorectal cancer patients and its association with prognosis . 
tie j , wang y , tomasetti c , et al : circulating tumor dna analysis detects minimal residual disease and predicts recurrence in patients with stage ii colon cancer . 
vora a , goulden n , mitchell c , et al : augmented post - remission therapy for a minimal residual disease - defined high - risk subgroup of children and young people with clinical standard - risk and intermediate - risk acute lymphoblastic leukaemia ( ukall 2003 ) : a randomised controlled trial . 
de mattos - arruda l , weigelt b , cortes j , et al : capturing intra - tumor genetic heterogeneity by de novo mutation profiling of circulating cell - free tumor dna : a proof - of - principle . 
thress ks , paweletz cp , felip e , et al : acquired egfr c797s mutation mediates resistance to azd9291 in non - small cell lung cancer harboring egfr t790m . 
 c smo syndrome : a unifying molecular diagnosis that suggests therapeutic opportunities introduction classically , diseases have been given eponymous or descriptive labels , but , as dna sequencing has revealed the same causal variants in ostensibly different disorders , 1 this inevitably has led to reappraisal of whether entities are better referred to by their underlying genetics or by their apparently distinct phenotypic appearance . 
moreover , the identification of common molecular mechanisms that underlie seemingly distinct syndromic entities can have therapeutic implications . the sonic hedgehog ( shh ) pathway is an important signaling cascade in development and cancer . 
germline pathogenic variants in ptch1 are responsible for 67% to 85%2 of occurrences of nevoid basal cell carcinoma syndrome ( nbccs ) , a condition characterized by multiple basal cell carcinomas ( bccs ) , calcification of the falx cerebri , palmoplantar pitting , odontogenic keratocysts , and other rare features . 
smaller contributions from ptch2 have been reported in patients with nbccs , 3 and germline mutations in sufu are described in approximately 5% of nbccs cases with a strong association to medulloblastoma development.4 ptch1 is a tumor suppressor gene that downregulates smoothened ( smo ) oncogenic activity in the shh pathway . 
recently , a unique mosaic activating mutation in smo c.1234c > t [ p.leu412phe ] has been reported as the only known cause of curry - jones syndrome ( crjs ) .5 crjs is a rare multisystem disorder that features iris colobomas , asymmetric facies , unilateral craniosynostosis , polydactyly , cutaneous syndactyly , intellectual disability , cerebral malformation , areas of hypopigmentation , ectopic hair growth , and structural and functional gi anomalies.5 cancer has not been reported in crjs . mosaicism occurs during postzygotic development with the gain of a mutation that results in localized or less severe phenotypes than congenital alterations.6 in 1987 , happle7 postulated that some conditions with a mosaic presentation of their phenotype are the result of mosaic activating mutations in oncogenes that are embryonically lethal . lack of confirmation of a clinical diagnosis after genetic testing of blood dna may suggest the presence of a mosaic alteration , but assessment of mosaicism is challenging , because only comprehensive studies that include testing of several tissue and high - sensitivity assays can provide conclusive answers . 
in addition , he has had one bcc on the right side of the upper chest , two on the back ( left and right ) , four on the scalp ( two left sided ) , and one on each side of the sternuhe was born with his left eyelid closed , and visual acuity in this eye was markedly reduced by the age 40 years . on examination , he had facial asymmetry , left iris coloboma , microphthalmia of the left eye , and an ectopic patch of hair under this eye ( figs 1a and 1b )  . 
short thin gold arrow , left frontal sinus ; long thin gold arrow , left maxillary sinus ; short thick blue arrow , right frontal sinus ; long thick blue arrow , right maxillary sinus . 
 magnetic resonance imaging , fluid attenuation inversion recovery acquisition of ( d ) axial , ( e ) coronal , and ( f ) left parasagittal contrast enhanced 1.5 - t , t1 weighted magnetic resonance images that show extensive polymicrogyria lining and deep , irregular sulci within the left middle frontal gyrus ( white arrows )  . 
contrast magnetic resonance imaging with gadolinium showed marked left - sided polymicrogyria that involved the left frontal lobe and anterior parietal region as well as cranial asymmetry with a smaller left hemisphere ( figs 1d - 1f ; data supplement )  . 
only one likely causal variant was common to all bccs and absent in blood dna : c.1234c > t ( p.leu412phe ) in smo ( figs 1g and 1h ; data supplement )  . 
the list of genes found to be altered in one or more of the bccs and the complete list of variants that passed our filters are provided in the data supplement . 
histogram shows the fractional abundance of mutant allele smo c.1234c > t ; p.leu412phe ( mut ) compared with the wild - type ( wt ) allele c.1234c by high - sensitivity ddpcr in a total of 29 samples from the proband and germline dna from nine healthy controls ( healthy c )  . 
moreover , we tested a total of 11 extra samples from the proband in the assay , including dna from a fibroblasts cell line derived from normal tissue adjacent to a tumor , dna from the tonsils that were removed at 5 years of age ( tonsil ) , dna from a swab of the right mouth epithelia ( m - epi - r ) and the left mouth epithelia ( m - epi - l ) , two different samples of saliva ( slv and slv - 2 ) , germline dna from blood ( blood ) , dna from a lymphoblastic cell line ( lcls ) , and dna from hair from the right and left side ( hair - r and hair - l , respectively )  . 
the nine healthy controls show fractional abundances of 0 to 0.02 , with the maximum total of positive droplets for the mutant allele in a healthy control of four of 21 , 307 wt droplets . 
moreover , somatic mosaicism for the identical smo mutation has been identified as the cause of crjs.5 recently the same oncogenic mutation has been identified in a case reported as mosaic nbccs but considered by others to represent happle - tinschert syndrome ( hts ) .8 , 9 it has also been found in a patient definitively diagnosed with hts , the main feature of which is the development of basaloid follicular hamartomas.10 we then quantified the mutation in 29 samples from the patient , including 15 normal tissues and 14 bccs , using high - resolution digital droplet polymerase chain reaction . 
 basal follicular hamartomas optic glioma hyperlipidemia macroglossia dysplastic ears cutaneous ossification lipodermoidof the conjunctiva ntal b r pits etin al d ntal a rtric alie nbccs basal cell carcinoma calcification of falx epidermal cysts ovarian fibromas benign tumours cutaneous syndactyly iris coloboma microphthalmia polymicrogyria * medulloblastoma cognitive anomalies polydactyly skeletal / cranial / cerebral anomalies pigmentation cataracts intestinal anomalies hemimegancephaly ectopic hair naevi cleft lip and palate okc macrocephaly cjrs asymmetric face heart anomalies trichoblastoma lymphangiomas instestinal hamartomas glaucoma fig 3 . 
comparison of the major reported features of nevoid basal cell carcinoma syndrome ( nbccs ) , curry - jones syndrome ( crjs ) , happle - tinschert syndrome ( hts ) , and the proband phenotype . 
in contrast , the mutation was detectable in fibroblast dna , in dna from normal tissue adjacent to bccs , in the left mouth epithelium ; a very low level was detected in the tonsils ( fig 2 ) , which confirmed its mosaic nature . our patient showed substantial phenotypic overlap between the reported features of crjs , hts , and nbccs ( fig 3 )  . 
the collection of driver mutations found in our patients bccs exhibited similarities with the altered pathways in medulloblastoma , in accordance with the catalog of driver mutations in recently described bccs ( data supplement )  . malfunctions of shh pathway members early in development lead to a wide range of neoplastic and non - neoplastic complications , which reflects the importance of the signaling levels in the outcome phenotype . 
in our patient , the presence of palmar pits and multiple bccs is shared with nbccs , 2 and polymicrogyria and facial asymmetry is shared with crjs and hts ( fig 3 ) .5 , 11 polymicrogyria , a disorder that affects the development of the cortex that features epilepsy and developmental delay , 12 has been reported in two of 11 occurrences of crjs.5 there is only one occurrence of polymicrogyria reported in the more commonly occurring nbccs , and this case was not confirmed molecularly.13 the mild presentation of disease in our patient compared with those of other reported patients with crjs , and the large phenotypic overlap shown in figure 3 , could suggest that some patients with negative clinical testing for ptch1 , sufu , and ptch2 may have been misdiagnosed with nbccs but may unknowingly have , genotypically , crjs . currently , vismodegib , an smo antagonist , can be used to treat patients with bccs . 
 our findings confirm that widely distributed somatic variants in genes other than ptch1 can cause bcc and that people with mosaic smo mutations might be at substantial lifetime risk for bcc . 
thus , molecular screening of several tissues , including bccs , is required for patients with suspected nbccs and no identified germline ptch1 pathogenic variant , because the presence of this smo mutation could allow more precise management . 
foulkes no relationship to disclose unsuspected smo activating mutations.15 this case highlights the difficulty faced in diagnosis of a rare mosaic condition and the need for molecular genetic testing to aid this process . identification of the causal variant has affected medical management . 
smo mutations have been reported in meningiomas , 16 medulloblastoma , 17 and ameloblastomas18 ; thus , we have initiated tumor surveillance with annual head and brain magnetic resonance imaging . 
also , the identification of a mosaic mutation means that the patient 's antecedents and siblings are not at risk for bcc or other nbccs - related disorders . in the current era , integration of molecular data is becoming fundamental to clarify clinical diagnosis and improve patient management . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . barbara rivera no relationship to disclose acknowledgment we thank the proband of this study for his tireless support along the course of the study . 
we thank the lablink facility in the centre for applied genomics ( toronto ) for their support with the digital - droplet polymerase chain reaction experiments . affiliations barbara rivera , a . 
foulkes wd , clarke ba , hasselblatt m , et al : no small surprise : small cell carcinoma of the ovary , hypercalcemic type , is a malignant rhabdoid tumor . 
twigg srf , hufnagel rb , miller ka , et al : a recurrent mosaic mutation in smo , encoding the hedgehog signal transducer smoothened , is the major cause of curry - jones syndrome . 
happle r : lethal genes surviving by mosaicism : a possible explanation for sporadic birth defects involving the skj am acad dermatol 16 : 899 - 906 , 1987 8 . 
happle r , tinschert s : happle - tinschert syndrome can be caused by a mosaic smo mutation and is suggested to be a variant of curry - jones syndrome . 
spector , md1 , 2 , 4 purpose the veterans health administration ( vha ) is the largest cancer care provider in the united states , with the added challenge of serving more than twice the percentage of patients with cancer in rural areas than the national average . 
the vha established the national precision oncology program in 2016 to implement and standardize the practice of precision oncology across the diverse vha system . methods tumor or peripheral blood specimens from veterans with advanced solid tumors who were eligible for treatment were submitted for next - generation sequencing ( ngs ) at two commercial laboratories . 
levelsof - evidence treatment recommendations were based on oncokb criteria . results from july 2016 to june 2018 , 3 , 698 samples from 72 vha facilities were submitted for ngs testing , of which 3 , 182 samples ( 86% ) were successfully sequenced . 
the most frequent therapies prescribed in response to ngs testing were immune checkpoint inhibitors , egfr kinase inhibitors , and parp inhibitors . conclusion clinical implementation of precision oncology is feasible across the vha health care system , including rural sites . 
the use of us food and drug administration ( fda ) approved therapies that target specic tumor mutations requires genomic analysis to demonstrate mutation expression in tumor tissue or circulating tumor dna ( ctdna )  . 
the development of scalable , cost - effective , high - throughput next - generation sequencing ( ngs ) technologies has made tumor molecular proling practical for clinical care . the veterans health administration ( vha ) is the largest integrated health care system in the united states and the largest provider of cancer care in the country . 
approximately 50 , 000 new cancer cases are reported annually through the veterans affairs central cancer registry.1 veterans receive cancer care in vha facilities across 152 hospitals and 1 , 400 clinics nationwide . with a common , systemwide electronic medical record system , the vha represents an ideal health care system to establish a nationwide , clinical - genomic precision oncology program and database . 
 poonnen et al context key objective what were the ndings of molecular testing of advanced solid - tumor malignancies performed through the veterans health administration national precision oncology program ? knowledge generated from july 2016 to june 2018 , 3 , 182 samples were sequenced , of which 34% were from patients in rural areas . 
mutations in genes associated with a neuroendocrine prostate cancer phenotype were expressed at increased frequency among veterans than in the general population . relevance our ndings demonstrate the feasibility of implementing a large - scale precision oncology program across heterogenous and geographically dispersed systems such as the vha , including rural settings with limited pathology infrastructure . veterans remain a unique population in terms of demographics and exposure history ; comparison between veteran and civilian tumor mutational proles may shed light on tumorigenesis and potential etiologies for unidentied mutational signatures . in addition , 36% of vha patients with cancer live in rural areas , compared with the national average of 14%.2 the vha established the national precision oncology program ( npop ) in 2016 to address these challenges.3 the npop was tasked with creating guidelines for genomic testing of clinical tumor samples across the vha health care system ; a particular emphasis was placed on making ngs technologies available to veterans in rural areas , facilitating access to fda - approved targeted therapies and immune checkpoint inhibitors , and increasing participation in clinical trials . 
here , we discuss the genomic prole of the 3 , 182 samples that were successfully sequenced through the npop and the targeted or immunotherapeutic drugs administered on the basis of molecular testing . methods patients genomic testing was recommended for patients with advanced solid tumors who were medically eligible to receive additional treatment with a targeted or immunotherapeutic drug . 
veterans with rural - urban commuting area codes greater than one , based on their zip code , received a rural designation , as dened by the vha ofce of rural health . tumor sequencing formalin - xed , parafn - embedded tumor sections or peripheral blood specimens for ctdna testing were sent two clinical laboratory improvement amendmentscertied genomic laboratories : personalis ( menlo park , ca ) or personal genome diagnostics ( pgdx ; to one of baltimore , md )  . 
molecular testing was performed using one of ve panels ( pgdx cancerselect 88 , 125 , and 203 ; personalis ace cancerplus ; and pgdx plasmaselect 64 ) included between 64 and 203 cancer - associated that genes with a large degree of overlap among the panels . samples submitted to personalis were sequenced at 500 - fold coverage , and those submitted to pgdx were sequenced between 1 , 000and 1 , 500 - fold coverage to improve the sensitivity of testing and identication of lowfrequency mutated alleles . 
tumor mutational burden ( tmb ) was dened and calculated using ibm watson for genomics ( wfg ) ( armonk , ny ) methodology ( appendix ) and was only available to providers through vha precision oncology consultation . hematoxylin and eosinstained slides were analyzed by a pathologist at the testing laboratories to verify tumor histology and the percent tumor content in each sample . additional details of these commercial tests can be found on the pgdx and personalis websites . levels of evidence for actionable mutations annotated clinical reports of mutations identied by ngs testing were provided by pgdx and personalis . 
on the basis of data in the variant call les , ibm wfg provided a second annotated report that included fda - approved targeted therapies and immunotherapies and available trials . 
of the samples submitted for ngs testing , 35% were from patients in rural areas , which is consistent with the higher percentage of vha patients with cancer who live in rural areas , compared with the rest of the nation.2 the demographics of the patients tested in the npop were consistent with a recent report from the va central cancer registry , 5 with men comprising 97% of vha patients with cancer and a median age of approximately 72 years ( fig 1b )  . 
adenocarcinomas of the lung , colon , and prostate were the three most frequent tumor types tested ( fig 1c ) , at 18% , 8% , and 29% , respectively , which is consistent with their prevalence among vha patients with cancer.5 molecular analysis the quantity and quality of dna obtained from tumor biopsy specimens can vary depending on the tumor type , biopsy site , and type of biopsy performed . 
fine - needle biopsies are more likely to yield inadequate dna content compared with biopsy specimens from larger - bore core needles.6 , 7 needle biopsy specimens of lung cancer , which often have signicant stromal tissue , and bone metastases are more likely to be associated with a low yield of tumor dna , often insufcient for ngs testing.8 , 9 of the 3 , 698 samples submitted for molecular analysis , 3 , 182 ( 86% ) had sufcient quantity and quality of tumor dna to be successfully sequenced . 
the ctdna assay ( plasmaselect 64 ) comprised 4% of the total samples submitted for sequencing ( appendix table a1 )  . the average turnaround time was 19 days . the most frequently mutated gene in our patient population , regardless of tumor type , was tp53 . 
mutations in tp53 were identied in 66% of all sequenced samples . atm was the second most frequently mutated gene , occurring in 28% of samples , followed by kras mutations ( 27% )  . 
these ndings were consistent with reports in other genomic databases ( eg , the cancer genome atlas , memorial sloan kettering integrated mutation proling of actionable cancer targets ) where there was a high frequency of tp53 mutations in lung , esophageal , colorectal , and breast cancers.10 , 11 unexpectedly , in prostate cancers sequenced through the npop , mutations in smarca4 , crebbp , tsc2 , kmt2a , and notch1 were more prevalent than mutations in ar , pik3ca , and kras.12 - 15 other tumor types had expected mutation frequencies , including glioblastoma , in which egfr mutations , notably the egfrviii mutation , are common.16 we also identied 10 ntrk gene rearrangements . 
these included etv6 - ntrk3 fusions in patients with nonsmallcell lung cancer , papillary thyroid cancer , and adenocarcinomas of the colon and breast ( appendix table a3 )  . ntrk gene rearrangements are clinically relevant with the recent fda approval of larotrectinib , an ntrk inhibtumors expressing ntrk gene itor for treatment of rearrangements.17 treatment recommended and prescribed clinical annotations of ngs results were provided by pgdx and personalis , in addition to a separate wfg analysis . 
the breakdown of treatment recommendations on the basis of levels of evidence in each of the most frequent tumor types is shown in fig 3b . overall , an actionable mutation was identied in 70% of tumors the samples sequenced . 
a level 2b treatment recommendation for off - label use of a targeted drug or immunotherapy in a different tumor type from the fda - approved indication was made in 9% of the cases . 
most samples ( approximately 52% ) expressed one or more actionable mutations for which there was no fda - approved therapy , either on or off label . under these circumstances , a clinical trial was the recommended treatment option ( levels of evidence 3a and b )  . not surprisingly , we found that tumors such as melanoma , for which there are several fda - approved targeted therapies on the basis of a relatively high frequency of braf mutations , had the highest percentage of level 1 treatment recommendations ( fig 3b )  . a total of 136 patients received the fda - approved targeted therapy or immunotherapy recommended in the annotated clinical report , either onor off - label , on the basis of the ngs results . 
programmed cell death protein 1 ( pd - 1 ) / programmed death - ligand 1 ( pd - l1 ) targeted immune checkpoint inhibitors were the most frequently prescribed treatment after ngs results . 
many of the patients who received immune inhibitors had advanced - stage lung adenocheckpoint carcinoma , for which checkpoint inhibitors are fda approved as a rstor second - line therapy without requiring ngs testing . 
twenty of these patients received an fdaapproved egfr tyrosine kinase inhibitor ( eg , erlotinib , osimertinib ) on the basis of the presence of exon 19 deletions or an exon 21 missense mutation ( l858r ) that leads to constitutive activation of egfr . 
nine patients were treated with crizotinib or alectinib , including ve with an emla4 - alk translocation , and two each for a ros1 translocation and met splice variants , respectively . 
two patients with braf v600e mutations received braf - mek inhibitors ( fig 4 )  . thirteen patients with advanced castration - resistant prostate cancer were treated with targeted therapies or immune checkpoint inhibitors on the basis of ngs ndings . 
seven patients with pathogenic mutations in dna repair genes were treated off - label with an fda - approved parp inhibitor , including four patients with brca1 and brca2 frameshift mutations and three patients with pathogenic atm and atr mutations . 
four patients with msi - high status , high tmb , or pathogenic mutations in specic dna mismatch repair gene ( eg , mlh1 ) were treated with an immune checkpoint inhibitor ( fig 5 )  . the 27 patients with colorectal cancer whose treatment was selected according to the ngs results , four were treated with a pd - 1 checkpoint inhibitor , on the basis of msi - high status or high tmb . 
 a highest level of watson evidence across all patients other : 29.3% level 3b : 40.8% level 1 : 8.4% level 2a : 1.1% level 2b : 8.9% level 3a : 11.5% poonnen et al lung cancers ( 1 , 461 ) colon adenocarcinoma ( 327 ) prostate cancers ( 320 ) unknown primary origin ( 251 ) melanoma ( 162 ) h & n squamous cell carcinoma ( 78 ) rectal adenocarcinoma ( 70 ) pancreatic adenocarcinoma ( 67 ) esophageal adenocarcinoma ( 56 ) urothelial carcinoma ( 48 ) renal cell carcinoma ( 42 ) gastric adenocarcinoma ( 40 ) hepatocellular carcinoma ( 36 ) breast adenocarcinoma ( 21 ) other ( 409 ) fig 3 . 
 ( a ) actionable alterations were annotated using watson for genomics ( wfg ) according to oncokb levels of evidence : food and drug administration ( fda ) - recognized predictive biomarkers ( level 1 ) , standard - ofcare biomarkers predictive of response to fda - approved therapies ( level 2 ) , and biomarkers predictive of response to therapeutic agents currently under investigation ( level 3 ) , with levels 2 and 3 further categorized by whether evidence existed for the particular tumor type in question ( 2a , 3a ) or a different tumor type ( 2b , 3b )  . 
recent published data from the centers for disease control and prevention indicate rural areas in the united states have higher cancer mortality rates than do urban areas , despite lower adjusted cancer incidence rates.18 moreover , although adjusted death rates for all cancer sites are decreasing , the decrease is at a slower pace in rural areas , further widening the rural - urban disparity in cancer - related mortality.18 one factor contributing to the widening gap in cancer death rates seems to be limited access to health care and cutting - edge technologies in rural areas . 
we made a concerted effort to enroll rural vha facilities in the npop and worked closely with pathologists at rural sites to improve the selection of samples for ngs testing to improve testing success rates . 
the result of these efforts was an 86% success rate for sequencing samples across the vha system , comparable to rates reported in other largescale characterization studies performed largely at academic centers.11 , 19 - 22 we implemented blood - based ctdna assays to enable rural sites with limited pathology infrastructure to participate in the program through a simple blood collection . 
the npop experience , therefore , may inform strategies to maximize participation of patients with cancer who live in rural areas in precision medicine programs in other health care systems . we found numerous similarities in the overall mutational pattern among the veteran population to previous molecular proling studies performed in the general population . for example , tp53 was one of the most commonly mutated genes in the vha cancer population , consistent with previous large - scale molecular proling efforts outside the vha.11 , 19 , 23 - 25 kras mutations were identied in more than one - fth of our patients , with the majority occurring in gi malignancies . 
these ndings are clinically signicant , because pathogenic kras , nras , and braf mutations are associated with therapeutic resistance to fdaapproved antibodies and small molecules targeting egfr in colorectal and nonsmall - cell lung cancers , respectively.26 mutations in apc , pik3ca , crebbp , and several other genes have been associated with oncogenesis in more common malignancies such as colorectal , lung , and breast carcinomas . 
although no targeted therapies are currently approved for these mutations , pi3k inhibitors are showing promising clinical activity in breast cancers that express activating pik3ca mutations.27 similarly , therapies are in development that specically target colorectal cancers that express mutant apc.28 mutations in alk , ros1 , and ret already have corresponding targeted therapies that are fda approved in several malignancies.29 analysis of our data showed several differences in both overall and specic frequency of gene variants . 
 ( a ) distribution of actionable mutations ( ie , eligible for onor off - label use of a food and drug administrationapproved targeted therapy or immune checkpoint inhibitor on the basis of ngs test results ) demonstrated by ngs . 
msi - h , microsatellite instabilityhigh ; tmb , tumor mutational burden . mutations were identied in almost one - quarter of all patient samples sequenced through the npop , which seems to be a higher rate than those reported in other non - vha studies.11 , 24 atm plays a key role in repair of dna doublestrand breaks . 
however , the relatively increased rate of pathogenic atm mutations seen in our patient population , particularly those with gastric or neuroendocrine tumors , may provide a potential therapeutic target , because preliminary clinical data suggest benet from the use of parp inhibitors in atm - decient cancers.30 atm and mutations in other dna mismatch repair genes have been linked to germline mutations . 
although we did not sequence normal tissue as part of the npop , patients whose tumors expressed potential germline mutations were offered genetic counseling . the molecular architecture of prostate cancers sequenced through the npop seems to differ from that previously reported in large , non - vha , prostate cancer genomic databases . 
 ( a ) distribution of actionable mutations ( ie , eligible for onor off - label use of an food and drug administrationapproved targeted therapy or immune checkpoint inhibitor on the basis of ngs test results ) demonstrated by ngs . 
tmb , tumor mutational burden . however , the frein neuroendocrine prostate cancers , quency of kmt2a mutations has been reported to be 14% , with similar ndings for dnmt2 and crebbp.13 transformation from prostate adenocarcinoma to a neuroendocrine phenotype has been reported in approximately 17% of patients , coincident with disease progression.32 that process is thought to be multifactorial , including specic gene rearrangements and epigenetic changes that were not tested here.13 although patients whose tumors were sequenced almost universally had advanced prostate cancer , the majority of samples sent for sequencing were from the original archived prostate biopsy specimens rather than metastatic sites of disease . 
therefore , we speculate that these genomic features suggestive of a neuroendocrine phenotype may have been present before initiation of androgen - deprivation therapy . overall differences in the genomic signatures in our patient population compared with previous non - vha studies are likely to be multifactorial in etiology . 
as previously posited , veterans represent a demographically unique population ; the vast majority of patients reported here were men , which skews ndings toward proles associated with common male cancers ( ie , lung , prostate , colorectal )  . 
veterans also differ from the general population in their occupational exposure history ( eg , agent orange ) , which increases risk of specic tumor types and may lead to a distinct mutational prole , compared with sporadic cases of similar histology . additional investigation directly comparing the mutational signatures of tumors sequenced through the npop to those of patients in the general population may be crucial to link particular occupational or environmental exposures to mutations expressed in their tumors . importantly , we have shown that implementation of ngs testing inuenced the treatment of veterans across the country . 
 ( a ) distribution of actionable mutations ( ie , eligible for onor off - label use of food and drug administrationapproved targeted therapy or immune checkpoint inhibitor on the basis of ngs test results ) demonstrated by ngs . 
kelley consulting or advisory role : astrazeneca , eisai , ibm japan research funding : bavarian nordic , novartis , astrazeneca , bristol - myers squibb other relationship : ibm neil l . 
spector stock and other ownership interests : eydis bio , bessor pharma research funding : immunolight patents , royalties , other intellectual property : i am on a patent related to my work with the company immunolight , and i am listed on a patent through eydis bio no other potential conicts of interest were reported . acknowledgment this project is a clinical / operational endeavor under the heading of the veterans health administration ( vha ) national precision oncology program , and our manuscript is a reporting of the results ( at 2 years ) of this clinical prograsimilar to funding for other aspects of veteran clinical care through the vha ( eg , laboratory testing , imaging , drugs / therapies ) , funding for the program is provided by national congressional budgeting ( either as a separate program or under general veterans health administration hematology / oncology funding ) , and no outside ( eg , industry , pharmaceutical ) funding sources are used for the program . details of this funding are publicly available as a matter of congressional budgeting . 
us department of veterans affairs ofce of research and development : precision oncology prograchakravarty d , gao j , phillips sm , et al : oncokb : a precision oncology knowledge base . 
mil med 182 : e1883 - e1891 , 2017 schneider f , smith ma , lane mc , et al : adequacy of core needle biopsy specimens and ne - needle aspirates for molecular testing of lung adenocarcinomas . am j clin pathol 143 : 193 - 200 , quiz 306 , 2015 ukasiewicz e , ziemiecka a , jakubowski w , et al : fine - needle versus core - needle biopsy which one to choose in preoperative assessment of focal lesions in the breasts ? literature review . 
j ultrason 17 : 267 - 274 , 2017 richter pd , ilacqua j : correlation between biopsy type and insufcient tissue availability for biomarker testing in ve solid cancer types . 
wu js , goldsmith jd , horwich pj , et al : bone and soft - tissue lesions : what factors affect diagnostic yield of image - guided core - needle biopsy ? radiology 248 : 962 - 970 , 2008 10 . 
henley sj , anderson rn , thomas cc , et al : invasive cancer incidence , 2004 - 2013 , and deaths , 2006 - 2015 , in nonmetropolitan and metropolitan counties united states . 
harris mh , dubois sg , glade bender jl , et al : multicenter feasibility study of tumor molecular proling to inform therapeutic decisions in advanced pediatric solid tumors : the individualized cancer therapy ( icat ) study . 
aggarwal r , huang j , alumkal jj , et al : clinical and genomic characterization of treatment - emergent small - cell neuroendocrine prostate cancer : a multiinstitutional prospective study . 
 poonnen et al appendix methods : assessment of tumor mutational burden tumor mutational burden ( tmb ) was dened as the number of coding , nonsynonymous mutations divided by the target region , which is estimated as the size of the genome ( in mbps ) covered by the assay . 
of samples ( % ) ( n = 111 ) tumor type lung prostate carcinoma of unknown primary pancreatic adenocarcinoma colon adenocarcinoma hepatocellular carcinoma esophageal adenocarcinoma duodenal adenocarcinoma cholangiocarcinoma follicular lymphoma 42 ( 38 ) 27 ( 24 ) 14 ( 13 ) 7 ( 6 ) 4 ( 4 ) 2 ( 2 ) 2 ( 2 ) 1 ( 1 ) 1 ( 1 ) 1 ( 1 ) table a2 . 
 fundamental concepts in the application of plasma genotyping ( liquid biopsy ) to egfr mutation detection in nonsmall - cell lung cancer plasma genotyping has rapidly evolved from an investigational technology into a standard - of - care tool used to direct therapy in metastatic nonsmall - cell lung cancer ( nsclc )  . 
the optimal use and interpretation of plasma genotyping requires understanding of cell - free dna biology , the assay characteristics of the available testing technologies , and the application of testing in each clinical scenario . 
 current recommendations for plasma genotyping in metastatic nsclc are limited to patients with newly diagnosed disease and those with acquired resistance to targeted therapy , in particular , epidermal growth factor receptor ( egfr ) kinase inhibitors . 
in newly diagnosed metastatic nsclc , under certain circumstances , plasma genotyping is useful for the detection of targetable genomic alterations or nontargetable driver alterations ( eg , kras ) that are mutually exclusive with targetable alterations . 
in patients with acquired resistance to therapy , such as egfr t790m - mediated acquired resistance to egfr kinase inhibitors , plasma genotyping may detect resistance mutations missed by standard tissue genotyping because of tumor heterogeneity . 
in both scenarios , the high specificity and positive predictive value of validated plasma genotyping assays allow for the reliable selection of therapy on the basis of a positive plasma genotyping result . 
this technology has become commonplace in prenatal research as well as cancer diagnostics and biomarker research.1 - 6 liquid biopsy has emerged as the colloquial term for the specific use of cfdna genotyping analysis to interrogate plasma , urine , and csf.7 , 8 this technology has been piloted in multiple solid tumor types to detect specific genomic alterations.2 , 4 , 5 its potential utility has been best demonstrated in lung cancer , where the rapid detection of targetable genomic alterations is key to selecting optimal systemic therapy.9 , 10 liquid biopsy technology has inarguably entered the realm of standard care in the treatment of nonsmall - cell lung cancer ( nsclc ) .11 , 12 multiple platforms for performing liquid biopsy have been evaluated for the detection of targetable genomic alterations in metastatic nsclc.9 , 10 , 12 - 20 the most rigorous of these studies have used either prospective sample testing or batched testing of samples collected from prospective clinical trials compared with standard tissue genotyping . 
 ( fda ) approval of a liquid biopsy assay using the cobas epidermal growth factor receptor ( egfr ) mutation test v2 ( roche molecular systems ) platform for the detection of egfr exon 19 del / l858r mutations as well as the egfr t790m resistance mutation constitutes the best example of the rapid transition of this technology from a research assay to a tool for guiding clinical care.21 furthermore , the commercial availability of multiple liquid biopsy assays for the detection of increasingly large panels of genomic alterations underscores the growing clinical role of this technology.22 , 23 the reliability , interpretation , and optimal use of liquid biopsy technology in lung cancer are potentially confusing , given the long history of the field and the number of testing platforms available . 
this hypotheses was confirmed by the detection of known mutations from a patients tumor in cfdna isolated from matched patient plasma.31 tumor - derived cfdna constitutes only a minor and potentially variable fraction of cfdna in a patient with an active malignancy , with the majority of the cfdna originating from normal tissue and blood cells.1 , 30 , 32 the careful isolation of cfdna from plasma as opposed to serum can potentially limit additional contamination with nontumor dna derived from the lysis of white blood cells during specimen processing.33 technical aspects of cfdna isolation , including prompt plasma processing and the use of dna - preservation tubes , may also reduce contamination with nontumor dna ( see specimen type and handling section ) .34 however , the relatively low frequency of tumor - derived cfdna present in the blood as well the short fragment length historically constituted key technical barriers to the reliable detection of tumor genomic alterations in cfdna . the advent of highly sensitive genotyping technologies now allows for the reliable genotyping of the tumor - derived component of plasma cfdna . 
a variety of highly sensitive validated platforms can detect specific genomic alterations in the small fraction of plasma cfdna that is derived from tumor cells ( see technology section ) .9 , 10 , 12 - 20 however , the fundamental limitation of any highly sensitive plasma genotyping assay is the degree to which a given tumor sheds cfdna into the peripheral circulation . 
the determinants of tumor cfdna shed are incompletely understood at present but are hypothesized to relate to both tumor burden and site of disease with tumor characteristics , which include histology , mitotic rate , and tumor vascularization.27 , 28 , 30 , 35 in studies of quantitative plasma genotyping of patients with metastatic nsclc , increasing disease burden , as measured by an increase in metastatic sites , as well as the presence of bone or liver metastases correlate with the concentration of tumor - derived cfdna.35 in all cases , the fraction of tumor - derived cfdna relative to total cfdna , and thus the degree of tumor cfdna shed , must be inferred from the detection of mutant cfdna relative to wild - type cfdna at a given genetic locus . 
the distinction between a shedding and nonshedding tumor is key to the understanding and interpretation of the results of any plasma genotyping assay in nsclc . technology the development of highly sensitive genotyping platforms propelled the analysis of the tumor - derived portion of cfdna from a laboratory curiosity to a potentially reliable clinical tool . 
however , both the presence and rate of cfdna shed vary among patients and directly affect the diagnostic sensitivity of plasma genotyping assays , which can contribute to false - negative results with these assays . circulating cell - free normal dna circulating tumor dna bloodstream normal tissue metastatic nsclc . 
however , the ability of individual plasma genotyping assays to detect specific types of genomic alterations is highly dependent on the testing platform , with the identification of complex rearrangements and copy number alterations being more difficult than the detection of point mutations . 
 analytical sensitivity and specificity refer to the technical ability of a genotyping assay to detect low levels of a given genomic alteration and distinguish it from other alterationsa metric that is most relevant for the development of laboratory tests and procedures . 
this review focuses primarily on clinical diagnostic sensitivity and specificity , which refer to the ability of an assay to accurately identify patients who have or do not have a given genomic alteration compared with a reference standardthe key criteria for using a test for making treatment decisions . cobas egfr mutation test and polymerase chain reactionbased approaches the cobas egfr mutation test is a real - time polymerase chain reaction ( pcr ) based assay originally developed for the purpose of detecting egfr sensitizing and t790m resistance mutations in tumor tissue . 
it is presently the only plasma genotyping assay with an fda indication for the detection of egfr mutations in metastatic nsclc for the purpose of initiating treatment with an egfr kinase inhibitor in both the initial and acquired resistance setting.21 the cobas assay was evaluated for these indications through retrospective testing of prospectively collected plasma samples from the ensure trial compared with standard tissue genotyping . 
 tissue biopsy negative t790m test metastatic site 1 metastatic site 2 blood draw positive t790m test egfrm - expressing tumor cell egfr - t790m - expressing tumor cell circulating cell - free normal dna t790m - negative ctdna t790m - positive ctdna circulating tumor dna circulating cell - free normal dna circulating tumor dna fig 2 . 
the presence of egfr t790m has been demonstrated to exhibit heterogeneity across metastatic sites , potentially resulting in false - negative results when performing tissue genotyping on a rebiopsy specimen taken from a nonrepresentative metastatic site . 
pooled data from these studies demonstrated a sensitivity of 61% ( 95% ci , 57% to 66% ) and specificity of 79% ( 95% ci , 70% to 85% ) for the detection of the egfr t790m mutation.38 a similar comparison was performed using prospectively collected samples from the aura3 study that randomly assigned patients with metastatic nsclc with egfr t790m mediated acquired resistance to egfr kinase inhibitors to either standard platinum - based chemotherapy or osimertinib.39 the study of the plasma cobas assay for egfr t790m in aura3 demonstrated a lower sensitivity of 51% ( 95% ci , 46% to 57% ) but similar specificity of 77% ( 95% ci , 71% to 83% ) .40 interestingly , both studies demonstrated near - perfect specificity for the detection of egfr exon 19 del and l858r , leading to suspicion that the lower specificity was secondary to heterogeneity of resistance mechanisms between metastatic sites within the same patient , thus rendering tumor tissue genotyping a nondefinitive reference standard . 
this hypothesis is supported by the finding that patients who are plasma - positive for egfr t790m exhibit clinical benefit similar to those who are tissue genotypingpositive and that repeat tissue biopsy of patients with acquired resistance led to discordant tissue genotyping results for egfr t790m but not egfr sensitizing mutations ( fig 2 ) .20 , 35 the cobas assay is the best - validated example of the use of pcr - based plasma genotyping assay to assess specific genetic hotspots corresponding to potentially targetable genomic alterations . 
 multiple other plasma genotyping assays have been evaluated for the detection of genomic alterations in metastatic nsclc in studies ranging in scope from analysis of prospectively collected banked plasma samples from large clinical trials to small retrospective series . 
these studies have demonstrated a consistent trend of high specificity but more modest sensitivity.9 , 41 , 42 plasma genotyping using pcr - based approaches is readily able to detect point mutations as well as defined stereotypical insertions and deletions . 
 however , the detection of complex rearrangements and copy number alterations using these platforms is challenging . droplet digital pcr and beads , emulsion , amplification , and magnetics genotyping emulsion digital pcr - based assays for plasma genotyping afford the ability to detect mutant cfdna in a highly sensitive but also quantitative manner . 
 study demonstrated that this assay was able to detect egfr exon 19 del and l858r mutations with a sensitivity of 82% and 74% , respectively , and a specificity of 100% . 
similar assay characteristics were reported for the detection of kras codon 12 mutations ( sensitivity 64% , specificity 100% ) .35 interestingly , similar sensitivity ( 77% ) but more modest specificity ( 63% ) was noted for the detection of egfr t790m . 
a subset of five patients initially reported as being falsely positive for egfr t790m by plasma genotyping were found to be truly positive on repeat tissue biopsy and genotyping of an alternative metastatic sitea finding not observed for any of the aforementioned driver mutations.35 this finding underscores the potentially heterogeneous nature of resistance mutations such as egfr t790m , whereby only growing sites of disease may possess the resistance mutation of interest . 
 tissue genotyping thus has the potential to be falsely negative when testing for an acquired resistance mutation , in contrast to plasma genotyping , which allows for the detection across metastatic sites within the same patient . beads , emulsion , amplification , and magnetics ( beaming ) based plasma genotyping assays have similarly been evaluated for the detection of egfr sensitizing and resistance mutations in patients with metastatic nsclc with acquired resistance to egfr kinase inhibitors . 
despite the rapid and quantitative nature of emulsion - based technologies , they are somewhat limited in terms of panel size and ability to multiplex . next - generation sequencing multiple next - generation sequencing ( ngs ) based platforms for plasma genotyping have been evaluated for the detection of genomic in nsclc.16 , 17 , 22 , 43 ngs - based alterations plasma genotyping assays have a greater ability to detect complex genomic alterations , such as complex rearrangements and high copy number alterations , that may be difficult to identify using other methods . 
a recent retrospective study of plasma samples collected in the biocast / ifct - 1002 study compared an ngsbased ( iontorrent , thermo fisher scientific , waltham , ma ) plasma genotyping assay to standard tissue genotyping in 68 matched samples of never - smokers with metastatic nsclc . 
this study demonstrated a sensitivity of 58% ( 95% ci , 43% to 71% ) and specificity of 87% ( 95% ci , 62% to 96% ) for the detection of mutations in key genes , including egfr , her2 , kras , braf , and pik3ca.16 a more recent retrospective study of 48 patients with metastatic nsclc comparing an ngs - based plasma genotyping assay performed on a miseq platform ( resolution bioscience ) to both ddpcr - based plasma genotyping and standard tissue genotyping revealed comparable sensitivity ( 77% , 48 of 62 positive cases detected ) and specificity ( 100% ; 95% ci , 90% to 100% ) , as well as the ability to reliably detect complex alterations , including alk and ros1 rearrangements.43 commercial plasma genotyping assays that use ngs - based platforms with expansive gene panels have been compared with tissue genotyping reference standard across multiple tumor types , and concordance rates between the two sample types are approximately 85% ( guardant360 , foundationact ) .22 , 23 other methods a multitude of other testing platforms have been used to develop plasma genotyping assays . 
 testing platforms are beyond the scope of this article and have been recently reviewed elsewhere . specimen type and handling careful and standardized methods for the isolation of cfdna from plasma are essential to optimize assay sensitivity and reproducibility . 
the collection of blood in a dna preservation tube , rapid sample processing , and the careful isolation of plasma in a fashion that minimizes cellular contamination ( centrifugation with double spin and disabled brake ) have been previously demonstrated to optimize sample quality for subsequent plasma genotyping.35 the use of plasma is preferable to serum , because the level of nontumor cfdna contamination is lower and less variable in plasma . 
the total cfdna level is frequently higher in serum samples , presumably because of white blood cell rupture during standard serum isolation , which requires allowing the blood sample to clot . 
plasma genotyping results can be available significantly faster compared with standard tissue genotyping for metastatic nsclc in both patients with newly diagnosed disease and those with disease progression while receiving initial targeted therapy.35 however , the optimal use and interpretation of plasma genotyping results hinges on specific details of the clinical scenario in which it is used . clinical actionability plasma genotyping assays consistently demonstrate high specificity and positive predictive value but modest sensitivity . 
the detection of a targetable genomic alteration in genes , including egfr , alk , ros1 , braf , met , ret , and her2 , provides useful information that may be directly used to select therapy . 
for instance , the detection of an egfr exon 19 del mutation in a patient with newly diagnosed metastatic nsclc may be used to initiate therapy with a firstor second - generation egfr kinase inhibitor ( eg , erlotinib , gefitinib , or afatinib )  . 
as a corollary , the detection of mutually exclusive , nontargetable driver genomic alterations ( eg , kras mutation ) is also useful because it provides reassurance that one of the aforementioned targetable genomic alterations is not present and that targeted therapy is not a therapeutic option.48 , 49 thus , plasma genotyping panels must be designed to detect clinically actionable genomic alterations as well as known nontargetable driver mutations . treatment status the established utility of plasma genotyping for the purpose of treatment selection in metastatic nsclc is limited to a small number of clinical scenarios . 
the best - evaluated use of plasma genotyping is for the selection of therapy in patients with newly diagnosed metastatic nsclc.15 , 37 validated plasma genotyping assays have been demonstrated to yield high sensitivity as well as near - perfect specificity for the detection of targetable genomic alterations in patients with newly diagnosed disease . 
plasma genotyping also has the capability to yield genotyping results with improved turnaround time and obviates the need for repeat biopsies in patients with newly diagnosed disease whose initial diagnostic biopsy samples are insufficient for tissue genotyping . patients with a targetable genomic alteration and acquired resistance to initial targeted therapy represent an additional clinical scenario in which plasma genotyping can guide therapy . 
the best - characterized example is the egfr t790m resistance mutation in metastatic nsclc that confers acquired resistance to firstand second - generation egfr kinase inhibitors but sensitivity to third - generation egfr kinase inhibitors . 
potentially targetable acquired resistance mutations , such as egfr t790m , emerge in a heterogeneous fashion in metastatic nsclc ; as a result , not all sites of disease may harbor a given resistance mutation , and thus the detection of such genomic alterations is inherently complex . plasma genotyping has also been evaluated with regard to monitoring the emergence of treatment resistance in patients already receiving therapy.50 , 51 the use of plasma genotyping in this context to detect early disease progression , in advance of clinical or radiographic evidence of progression , is presently investigational and should not be used to change treatment in the absence of objective evidence of disease progression . clinical stage the use of plasma genotyping has demonstrated utility for guiding therapy in metastatic nsclc but is investigational in nonmetastatic disease . 
the optimal use of this technology and interpretation of its results hinges on careful synthesis of three key concepts : cfdna biology , specific assay technology , and the individual clinical oncology of the individual patient ( fig 3 )  . patients with newly diagnosed disease plasma genotyping in a patient with newly diagnosed metastatic nsclc is most informative when a targetable genomic alteration ( eg , egfr , alk , ros1 , braf , met ) or a nontargetable mutually exclusive driver alteration ( eg , kras ) is found . 
the use of an fda - approved and rigorously validated plasma genotyping assay allows for immediate initiation of appropriate targeted or systemic therapy in the instance of a positive result , given the high specificity and positive predictive value of these assays . 
negative plasma genotyping results where no driver alteration is detected are not useful in guiding therapy , given the modest sensitivity of such assays , and should prompt appropriate tissue genotyping if not already initiated . 
importantly , caution must be exercised in interpreting low - level positive results near the limit of detection , particularly of nondriver alterations detected on poorly validated panels , as there exists the risk of assay artifact and a false - positive result . acquired resistance plasma genotyping should be used as the initial genotyping method in the setting of acquired resistance to targeted therapy in metastatic nsclc . 
this approach has the ability to obviate the need for repeat biopsy in many instances but also detects resistance mechanisms that may be missed on repeat tissue genotyping because of heterogeneity of resistance mechanisms . 
the absence of an identifiable resistance mutation should prompt repeat tissue biopsy and genotyping to rule out a false - negative result , as well as examine for alternative resistance mechanisms that are not easily identified through plasma genotyping , such as small - cell transformation or met amplification . 
as a corollary , a plasma genotyping result that fails to detect the original targetable alteration in a patient with acquired resistance should be considered indicative of the absence of tumor cfdna shed , and the result invariably a false negative . 
importantly , plasma testing for acquired resistance as well as repeat biopsy should only be initiated in patients with clinically or radiographically significant disease progression , to avoid inadvertently labeling a patients disease t790m negative because of testing before the emergence of clinical resistance . 
a recent retrospective study demonstrated improved sensitivity with the use of combined urine and plasma genotyping in a cohort of patients with metastatic resistant egfr - mutant nsclc.52 this finding supports the hypothesis that the use of multiple orthogonal testing strategies may be effective in enhancing overall diagnostic sensitivity . 
furthermore , the combination of plasma and tissue genotyping alone has the potential to surmount the modest sensitivity in the context of acquired resistance . the potential utility of serial quantitative plasma genotyping for the purpose of monitoring response to therapy , as well as the evolution of acquired resistance , constitutes another promising area of research . 
 therapeutic response in clinical trials of novel agents may allow for real - time monitoring of response to therapy as well as early prediction of potential mechanisms of acquired resistance . 
sacher , division of medical oncology and hematology , princess margaret cancer centre , university of toronto , toronto , ontario . references med 14 : 985 - 990 , 2008 1 . 
botezatu i , serdyuk o , potapova g , et al : genetic analysis of dna excreted in urine : a new approach for detecting specific genomic dna sequences from cells dying in an organisclin chem 46 : 1078 - 1084 , 2000 8 . 
chen ww , balaj l , liau lm , et al : beaming and droplet digital pcr analysis of mutant idh1 mrna in glioma patient serum and cerebrospinal fluid extracellular vesicles . 
karachaliou n , mayo - de las casas c , queralt c , et al : association of egfr l858r mutation in circulating free dna with survival in the eurtac trial . 
kimura h , suminoe m , kasahara k , et al : evaluation of epidermal growth factor receptor mutation status in serum dna as a predictor of response to gefitinib ( iressa )  . 
tan ds , yom ss , tsao ms , et al : the international association for the study of lung cancer consensus statement on optimizing management of egfr mutation - positive non - small cell lung cancer : status in 2016 . 
li bt , janku f , janne pa , et al : ultra - deep next generation sequencing ( ngs ) of plasma cell - free dna ( cfdna ) from patients with advanced lung cancers : results from the actionable genome consortiucancer res 76 , 2016 ( abstr 4342 ) 14 . 
karlovich c , goldman jw , sun jm , et al : assessment of egfr mutation status in matched plasma and tumor tissue of nsclc patients from a phase i study of rociletinib ( co - 1686 )  . 
mok t , wu yl , lee js , et al : detection and dynamic changes of egfr mutations from circulating tumor dna as a predictor of survival outcomes in nsclc patients treated with first - line intercalated erlotinib and chemotherapy . 
couraud s , vaca - paniagua f , villar s , et al : noninvasive diagnosis of actionable mutations by deep sequencing of circulating free dna in lung cancer from never - smokers : a proof - of - concept study from biocast / ifct - 1002 . 
goto k , ichinose y , ohe y , et al : epidermal growth factor receptor mutation status in circulating free dna in serum : from ipass , a phase iii study of gefitinib or carboplatin / paclitaxel in nonsmall cell lung cancer . 
douillard jy , ostoros g , cobo m , et al : gefitinib treatment in egfr mutated caucasian nsclc : circulating - free tumor dna as a surrogate for determination of egfr status . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced non - small - cell lung cancer . 
kim st , lee ws , lanman rb , et al : prospective blinded study of somatic mutation detection in cell - free dna utilizing a targeted 54 - gene next generation sequencing panel in metastatic solid tumor patients . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
jahr s , hentze h , englisch s , et al : dna fragments in the blood plasma of cancer patients : quantitations and evidence for their origin from apoptotic and necrotic cells . 
sacher a , dahlberg s , paweletz c , et al : prospective validation of rapid plasma genotyping for the detection of egfr and kras mutations in advanced lung cancer . 
reck m , hagiwara k , han b , et al : ctdna determination of egfr mutation status in european and japanese patients with advanced nsclc : the assess study . 
jenkins s , yang jc , ramalingam ss , et al : plasma ctdna analysis for detection of the egfrt790m mutation in patients with advanced non - smallcell lung cancer . 
weber b , meldgaard p , hager h , et al : detection of egfr mutations in plasma and biopsies from non - small cell lung cancer patients by allele - specific pcr assays . 
yung tk , chan kc , mok ts , et al : single - molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital pcr in non - small cell lung cancer patients . 
paweletz cp , sacher ag , raymond ck , et al : bias - corrected targeted next - generation sequencing for rapid , multiplexed detection of actionable alterations in cell - free dna from advanced lung cancer patients . 
hu c , liu x , chen y , et al : direct serum and tissue assay for egfr mutation in non - small cell lung cancer by high - resolution melting analysis . 
brevet m , johnson ml , azzoli cg , et al : detection of egfr mutations in plasma dna from lung cancer patients by mass spectrometry genotyping is predictive of tumor egfr status and response to egfr inhibitors . 
zhao j , ye x , xu y , et al : egfr mutation status of paired cerebrospinal fluid and plasma samples in egfrmutant non - small cell lung cancer with leptomeningeal metastases . 
sholl lm , aisner dl , varella - garcia m , et al : multi - institutional oncogenic driver mutation analysis in lung adenocarcinoma : the lung cancer mutation consortium experience . 
gainor jf , varghese am , ou sh , et al : alk rearrangements are mutually exclusive with mutations in egfr or kras : an analysis of 1 , 683 patients with non - small cell lung cancer . 
thress ks , paweletz cp , felip e , et al : acquired egfr c797s mutation mediates resistance to azd9291 in non - small cell lung cancer harboring egfr t790m . 
sacher a , alden r , oconnell a , et al : a prospective study of rapid plasma genotyping utilizing sequential ddpcr and ngs in newly diagnosed advanced nsclc patients . 
 majority of b2m - mutant and - decient colorectal carcinomas achieve clinical benet from immune checkpoint inhibitor therapy and are microsatellite instability - high sumit middha , phd1 ; rona yaeger , md1 ; jinru shia , md1 ; zsoa k . 
hechtman , md1 purpose microsatellite instability - high ( msi - h ) colorectal carcinomas ( crcs ) show high rates of response to immune checkpoint inhibitors ( ios )  . 
all b2m - mutant crcs were assessed for expression of b2m , major histocompatibility complex class i , and programmed death - 1 ligand ( pd - l1 ) via immunohistochemistry and average cd3 + and cd8 + tumor - inltrating lymphocyte counts against a control group of msi - h b2m wild - type crcs . results fifty - nine ( 3.4% ) of 1 , 751 patients with crc harbored b2m mutations , with 84% ( 77 of 92 ) of the mutations predicted to be truncating . 
b2m mutation status was not associated with major histocompatibility complex class i expression , kras or braf mutation , tumor - inltrating lymphocyte level , or pd - l1 expression after adjustment for msi status . 
of 13 patients with b2m - mutant crc who received programmed death - 1 or pd - l1 ios , 11 ( 85% ) achieved clinical benet , dened as stable disease or partial response using response evaluation criteria in solid tumors criteria . conclusion b2m mutations occur in approximately 24% of msi - h crcs and are usually associated with loss of b2m expression . 
 middha et al context do b2m - mutant and decient colorectal carcinomas ( crcs ) respond to immune checkpoint inhibitors ? we found that b2m mutations are enriched in microsatellite instability - high crc , that these mutations commonly occur at coding microsatellite loci , and that these b2m mutations are truncating and associated with loss of b2m expression . 
of 13 patients with b2m - mutant crc and available recist data , six achieved stable disease , ve achieved partial response , and one experienced pseudoprogression . b2m mutations are not predictive of primary resistance to immune checkpoint inhibition in crc . administrationcleared , next - generation sequencing assay that interrogates  . 
presence of loss of heterozygosity ( loh ) was assessed via allelespecic copy - number analysis using the fraction and allelespecic copy number estimates from tumor sequencing ( facets ) algorithm14 and , in cases of low tumor content , via comparison of b2m mutation variant allele frequency against median variant allele frequency . 
clinical parameters , clinical response to immune checkpoint inhibitor therapy all patients with crc with msk - impact data and b2m mutations who underwent therapy with immune checkinhibitors ( ios ; durvalumab , nivolumab , or pempoint brolizumab ) before july 2018 were assessed for b2m expression ( ihc ) , response , stable disease ( sd ) , and progressive disease ( pd )  . 
formal response evaluation criteria in solid tumors ( recist ) scores were assessed via radiologic data as follows : complete response ( cr ) , disappearance of all target lesions , conrmed at 4 weeks ; partial response ( pr ) , 30% decrease , conrmed at 4 weeks ; pd , 20% increase over smallest sum observed ; and sd , meeting none of the other criteria . patients were deemed to have experienced clinical benet from ios if recist results were sd , pr , or cr . ihc staining for b2m using a polyclonal antibody with concentration of 1 : 6 , 000 ( catalog #a0072 ; dako , santa clara , ca ) , mhc class i using a monoclonal antibody with concentration of 1 : 200 ( catalog #14 - 9958 ; e - bioscience , carlsbad , ca ) , cd3 using a monoclonal antibody with concentration of 1 : 200 ( catalog #ncl - l - cd3 - 565 ; leica , lincolnshire , il ) , cd8 using a monoclonal antibody with concentration of 1 : 100 ( catalog #m7103 ; dako ) , and pd - l1 using a monoclonal antibody with concentration of 1 : 100 ( catalog #13684 ; cell signaling , danvers , ma ) was performed on all crcs with b2m mutations with available tissue as well as a set of 26 randomly selected wild - type ( wt ) crcs with in - house resection specimens and mskimpact testing ( performed between january 1 , 2014 , and october 31 , 2017 ) that were matched to the b2m - mutant group for prevalence of msi status . 
complete loss of b2m on ihc ( 0% of tumor cells with b2m expression ) was interpreted as loss of b2m expression . statistical analyses associations were assessed using pearsons 2 test with simulated p value based on 2 , 000 replicates for low count data . 
a cox proportional hazards model was tted to the data to calculate survival using the covariates of b2m mutation status , age at diagnosis , pathologic stage , msi status , proximal versus distal status , and kras , nras , braf , and pik3ca mutation status . 
these were each assessed through both univariable and multivariable cox regressions . r survival and survminer software packages were used to perform this analysis ( r foundation , vienna , austria )  . results molecular findings we rst sought to determine the spectrum of b2m mutations in a cohort of patients with crc ( n = 1 , 751 ) with msk - impact data ( appendix fig a1 )  . 
next , we classied the samples on the basis of whether they were microsatellite stable ( mss ) or unstable ( msi - h ) on the basis of genomic data . 
a medullary carcinoma of the colon shows immune - cell expression of programmed death - 1 ligand at the tumor - stroma interface , loss of b2m expression in tumor cells , retention of strong diffuse mhc class i expression in tumor cells ,  . 
twelve samples showed either loh or copy - neutral loh along with a clonal mutation , suggesting biallelic loss . b2m expression , mhc class i expression , and til level to evaluate the functional outcome of b2m mutations , we examined protein expression in samples with available tissue ( figs 1b and 1c ; appendix table a2 )  . 
because pdl1 status has been used as a predictive marker of ios and linked to expression of mhc class 1 , we performed pd - l1 ihc in available b2m - mutant and wt patient cases . 
thirtytwo ( 73% ) of 44 b2m - mutant and 13 ( 50% ) of 26 b2m wt crcs were positive for pd - l1 expression ( immune cells in tumor - stroma interface )  . 
average median cd3 + count per hpf was 22.3 in b2m - mutant and 37.3 in b2m wt crcs , whereas average median cd8 + count per hpf was 15.1 and 49 for b2m - mutant and b2m wt crcs , respectively . 
in comparison with b2m wt crcs matched for msi status , b2m - mutant crcs tended to have lower average levels of cd3 and cd8 per patient case ( fig 2a ) , these differences did not reach statistical significance . 
age , stage , and kras , nras , and braf status were associated with os , whereas pik3ca mutation status , msi status , and proximal versus distal location were not associated with os ( fig 2b ; appendix table a2 )  . response to immunotherapy because b2m mutations were identied as a possible resistance mechanism for checkpoint inhibition , we identied 13 ( msi - h , n = 11 ; mss , n = 2 ) patients with crc with b2m mutations who subsequently received io therapy , and we evaluated their response to treatment . 
 ( a ) box plot graphs of average cd3 ( left ) and cd8 ( right ) counts in b2m - mutant vs wild - type ( wt ) colorectal carcinoma ( crc ) show that median average cd3 and cd8 trend toward higher values in b2m wt carcinoma . ( b ) multivariable model showing the clinical variables associated with os of patients with crc in our cohort . 
analysis of their tumor response by recist criteria demonstrated pr in ve patients , sd in six patients , and pd in one patient , likely pseudoprogression ( fig 3a ; appendix table a3 )  . 
this response rate is in line with recent publications of ios in msi - h crc.8 , 9 of the two patients with mss tumors , one had an ultramutated pole hotspot mutated tumor and experienced tumor growth with io treatment but was able to continue treatment for 1 year , likely to be pseudoprogbecause growth was thought ression , and the other patient had sd during treatment with a combination of io treatment and targeted therapy ( after progression with targeted therapy alone )  . 
the association of b2m mutations with complete loss of expression on whole sections of pole s459f best response pseudoprogression partial response stable disease treatment stopped ongoing 40% 80% msi status io therapy b2m expression fig 3 . 
immune checkinhibitor ( io ) repoint sponse in b2m - mutant colorectal cancer ( crc )  . waterfall plot of io response in b2m - mutant crc ( colored by microsatellite instability [ msi ] status , type of b2m mutation , and type of io received )  . 
indeed , in the 42 b2m - mutant crcs analyzed by facets , 60% had evidence of either two clonal b2m mutations or one clonal b2m mutation and loh ( appendix table a1 )  . 
it is possible that the remaining b2m - mutant crcs have epigenetic modications as a mechanism of b2m silencing . unlike previous studies , 3 we found that b2m protein loss is not correlated with loss of mhc class i expression . 
we show that b2m mutation and loss do not correlate with mhc class i loss of expression , although the effect of b2m loss on the functional competence of mhc class i is known to be deleterious.16 limitations to our study include the retrospective analysis and relatively small number of patients treated with ios , as well as limitations in molecular testing for epigenetic issues and allele specicity of b2m mutations . most importantly , our study shows that b2m mutation and loss in immunotherapy - naive crc do not predict primary resistance to ios . 
we saw that most patients had some degree of regression with treatment ; larger , prospective studies are needed to clarify if the response rate and duration of response vary by b2m mutation status . however , our data indicate that patients with crc whose tumors harbor b2m mutations should not be excluded from io treatment . 
the clinical benet demonstrated in patients with b2m - decient crc who received ios may have resulted from the fact that despite b2m loss , there was still evidence of functional neoantigen recognition , as indicated by the high number of tils ( median , 22.3 per hpf ) still present in b2m - decient msi - h crc . 
 middha et al leonard saltz consulting or advisory role : mcneil ( i ) research funding : taiho pharmaceutical neil segal consulting or advisory role : bristol - myers squibb , pzer , astrazeneca / medimmune , imugene , roche / genentech , pieris pharmaceuticals , synlogic , aduro biotech , kyn therapeutics , boehringer ingelheim , merck , puretech , horizon pharma , emd serono , gritstone oncology , chugai pharma , trm oncology , ifm therapeutics , psioxus therapeutics research funding : medimmune , bristol - myers squibb , pzer , roche / genentech , merck , incyte marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network / astrazeneca tagrisso rfp advisory committee , takeda pharmaceuticals , bristol - myers squibb , bayer healthcare pharmaceuticals , merck ( i ) research funding : loxo ( inst ) , helsinn therapeutics ahmet zehir no relationship to disclose jaclyn f . 
hechtman honoraria : medscape consulting or advisory role : navigant consulting , axiom biotechnologies research funding : bayer no other potential conicts of interest were reported . references roberts ad , ordway dj , orme im : listeria monocytogenes infection in beta 2 microglobulin - decient mice . 
infect immun 61 : 1113 - 1116 , 1993 schaible ue , collins hl , priem f , et al : correction of the iron overload defect in beta - 2 - microglobulin knockout mice by lactoferrin abolishes their increased susceptibility to tuberculosis . 
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nat commun 8 : 1136 , 2017 janikovits j , m uller m , krzykalla j , et al : high numbers of pdcd1 ( pd - 1 ) - positive t cells and b2m mutations in microsatellite - unstable colorectal cancer . oncoimmunology 7 : e1390640 , 2017 grasso cs , giannakis m , wells dk , et al : genetic mechanisms of immune evasion in colorectal cancer . 
cancer discov 8 : 730 - 749 , 2018 le dt , uram jn , wang h , et al : pd - 1 blockade in tumors with mismatch - repair deciency . 
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cell 171 : 934 - 949.e16 , 2017 le dt , durham jn , smith kn , et al : mismatch repair deciency predicts response of solid tumors to pd - 1 blockade . 
science 357 : 409 - 413 , 2017 overman mj , mcdermott r , leach jl , et al : nivolumab in patients with metastatic dna mismatch repair - decient or microsatellite instability - high colorectal cancer ( checkmate 142 ) : an open - label , multicentre , phase 2 study . 
clendenning m , huang a , jayasekara h , et al : somatic mutations of the coding microsatellites within the beta - 2 - microglobulin gene in mismatch repairdecient colorectal cancers and adenomas . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
consort diagramutation and immunohistochemistry analyses included data from 1 , 751 patients with colorectal cancer ( crc ) with memorial sloan kettering integrated mutation proling of actionable cancer targets testing . 
 o precision trial drawer , a computational tool to assist planning of genomics - driven trials in oncology purpose trials that accrue participants on the basis of genetic biomarkers are a powerful means of testing targeted drugs , but they are often complicated by the rarity of the biomarker - positive population . 
however , bigger trials have higher chances of conflicting treatment allocations because of the coexistence of multiple actionable alterations ; allocation strategies greatly affect the efficiency of enrollment and should be carefully planned on the basis of relative mutation frequencies , leveraging information from large sequencing projects . methods we developed software named precision trial drawer ( ptd ) to estimate parameters that are useful for designing precision trials , most importantly , the number of patients needed to molecularly screen ( nnms ) and the allocation rule that maximizes patient accrual on the basis of mutation frequency , systematically assigning patients with conflicting allocations to the drug associated with the rarer mutation . 
we used data from the cancer genome atlas to show their potential in a 10 - arm imaginary trial of multiple cancers on the basis of genetic alterations suggested by the past molecular analysis for personalised therapy ( map ) conference . 
2018 by american society of clinical oncology introduction the availability of sequenced cancer genomes and the generation of new targeted drugs offer unprecedented opportunities for personalized treatment for patients with cancer . 
indeed , treatment within genomic - based clinical trials significantly improves clinical outcome.1 however , identification of the best biomarker - drug combination for patient stratification and treatment remains a critical research and ethical challenge . large multi - institutional projects such as the cancer genome atlas ( tcga ) revealed extreme heterogeneity of cancer mutational landscapes , with a prevalence of low - frequency mutations , giorgio e.m. 
are co - first authors . corresponding author : luca mazzarella , md , phd , istituto europeo di oncologia , via ripamonti 435 , 20141 milano , italy ; e - mail : luca . 
 including those critical for tumor growth ( driver mutations ) and those that might be targetable by specific drugs ( actionable mutations ) .2 , 3 this scenario renders the conduction of adequately powered genome - based clinical trials prohibitive in many cases : the biomarker - positive population is often rare , and identifying it requires screening large numbers of patients . strategies to circumvent this issue are proposed through novel trial designs in which multiple drugs and / or their matched biomarkers are tested simultaneously . 
these novel designs , usually defined as basket ( one biomarker - drug pair , multiple histologies ) , umbrella ( multiple biomarker - drug pairs , one histology ) , or platform ( multiple biomarker - drug pairs , multiple histologies ) , 4 - 6 may allow reducing the number of patients needed to molecularly screen ( nnms ) to achieve the desired statistical power for multiple treatment subgroups . 
however , these approaches have potential disadvantages : first is the occurrence of a large fraction of patients with potential allocation conflicts resulting from the concomitance of genetic alterations associated with different drugs , 2 and second is the high costs and turnaround time of the molecular screening , which must include sequencing enough genomic space to accommodate the studys need . 
the latter will become more critical in the near future as more complex , polygenic genomic signatures are used as stratification biomarkers ( eg , multiple homologous recombination genes to establish brcaness as a predictor of response to poly [ adp - ribose ] polymerase [ parp ] inhibitors7 )  . 
 for these reasons , patient allocation to individual trial arms significantly deviates from expectations in multigenic umbrella trials , which leads to inadequate statistical power8 , 9 and the inability to reach conclusions . here , we present precision trial drawer ( ptd ) , a comprehensive bioinformatics tool that takes advantage of patient - level genomic information from tcga or alternative sources to simulate genomically characterized cohorts and provide critical parameters for designing genetic biomarker - driven trials . 
df is used to calculate the nnms ( ie , the minimum number of patients that should be tested to obtain the desired sample size as calculated on the expected treatment effect by using conventional power analyses )  . 
 actionable mutations refers to those genetic alterations that dictate a therapeutic choice within the trial . description of the package ptd includes a series of functions in the r programming language to ( 1 ) translate genomic information into genomic coordinates and optimize coordinates for the design of a targeted sequencing panel , ( 2 ) retrieve single or grouped genetic alteration frequencies from public databases ( by default tcga , but other sources can be used ) that maintain patient - level associations , ( 3 ) simulate genomically defined patient cohorts by extracting frequencies of co - occurrence of single or grouped mutations , and ( 4 ) calculate nnms for defined power and / or sample size ( fig 1a )  . 
in our imaginary trial , patients are allocated to 10 possible drugs , including inhibitors of parp , notch , met , her2 , fgfr , egfr , braf , alk , akt , and immune checkpoints , and covering all the allocations linked to the map biomarkers . 
allocation to checkpoint inhibitors was based on the detection of inactivating mutations in mlh or msh genes , regardless of tumor type , which are highly correlated with microsatellite instability , a now - established predictor of immune checkpoint response.11 , 12 ptd was then used to design a dedicated custom targeted sequencing panel , identify the optimal allocation rule , and estimate sample size and statistical power . the list of genomic features associated with each drug is provided in table 1 . 
illustrations of the workflow , the codes used for generating data , and detailed ptd functionalities are available at shinyapp.html. the first step involves designing the sequencing panel , which is imported as a standard excel table with official gene symbols , type and exact alteration to be considered , and two possible grouping variables ( gene - linked drug and , if desired , a generic group of any sort )  . 
 the panel can also be expanded to include genes or genomic positions without therapeutic information , which may nevertheless be relevant for prognostic stratification , prediction of drug toxicity , or a generic biologic interest ( grouped as driver genes in the example )  . four classes of genetic alterations can be explored : single nucleotide variants ( snvs ) , copy number alterations ( cnas ) , translocation - associated fusion transcripts , and gene expression . 
for panels that contain multiple alteration types ( eg , snvs and cnas ) , ptd will construct a reference data set with the intersection of the relative data sets in the source database ( not all patients in tcga have data from all sequencing platforms )  . in projects that allow inclusion of multiple tumor histologies , their frequency of occurrence in the real world is likely to differ significantly from the relative representation in the reference data set , and this can be corrected by ptd by weighting the probability of sample extraction . 
in this example , we used relative frequencies obtained from the national cancer institute seer program ( fig 2a shows the comparison between the tcga and the example populations )  . output the percentage of patients with at least one actionable alteration ( detected fraction ; df ) is probably the most crucial estimate that can be obtained through ptd , which is 65.4% in this example ( fig 2b )  . 
ptd allows breakdown of the df by drug or by disease histology ( fig 2c - d ) or any other prespecified group . to optimize panel design , one can evaluate the trade - off between the feature of interest ( eg , available drugs or the inclusion of genes with uncertain predictive power ) and the size of the genomic space to be sequenced . 
this trade - off can be visualized with a saturationplot that expresses the mean number of alterations per patient or the number of patients with at least x alterations as a cumulative function of the genomic space , with alterations ranked in descending frequency order ( absolute or gene lengthnormalized ; fig 2e )  . 
 the same plot can be obtained by using drugs or other grouping variables ( fig 2f )  . the coocmutexplot indicates the degree of co - occurrence of grouped genetic alterations , ascopubs.org / journal / po jco precision oncology 5 accepted formats . 
for instance , the braf v600e mutation can be coded using amino acid notation ( v600e ) , the single nucleotide polymorphism database ( dbsnp ) identifier ( rs113488022 ) , a set of genomic coordinates ( eg , chr7 : 140753336 140753337 ) , or as genomic variants ( 7 : 140753336 a > c )  . 
mutations of interest can be batch filtered with any set of genomic coordinates to eliminate variants likely to be passengers.3 , 13 in this example , we selected only variants listed as probably pathogenic in the catalogue of somatic mutations in cancer ( cosmic ) database , 14 thus eliminating 33.5% of the initial variants . 
alternatively , individual genes can be visually inspected and edited by using an interactive interface built on the lowmaca ( low frequency mutations analysis via consensus alignment ) algorithm15 ( manual filtering ; fig 1b )  . 
in this example , we manually excluded egfr t790m and pik3ca exon 20 mutations associated with resistance to first - generation egfr inhibitors.16 , 17 specific mutations and / or drugs can be associated with specific tumors and excluded from others . 
 ( b ) percentage of patients with at least one ( detection fraction ) or more actionable alterations ; detection fraction is broken down by ( c ) tumor type or ( d ) drug . 
this plot can be used to decide which drugs should be combined in the same trial because prioritizing those associated with highly mutually exclusive mutations will maximize the biomarker - positive population . power analysis and calculation of the nnms multidrug trials can be powered to test the experimental arm as a whole , ignoring the number of patients allocated to each individual drug , in which case the nnms is a simple linear function of the target sample size divided by the detection power ( in our example , if the given target sample size = n , the nnms will be n / 0.654 ; no allocation rule design )  . 
if this is a desired end point , the trial can be divided into a series of drug - specific subtrials , each having its own target sample size.9 probability of allocation can vary significantly if the associated biomarkers have very different prevalence . 
furthermore , increasing the number of competing arms also increases the likelihood of conflicting allocations ; rules to follow in such cases should be specified in advance to avoid allocation biases . 
immcheckp , immune checkpoint ; inh , inhibitor . general a priori considerations that do not take into account mutation frequencies ( eg , allocation to endocrine therapy only if no mutation is present8 ; manual allocation design )  . 
if information regarding the co - occurrence of mutations is not considered , proper nnms - based sample size calculation is not possible because considering each subtrial independently leads to gross overestimation . 
with ptd , arms can be ranked in order of expected frequency of allocation , so that if a patient bears mutations associated with two drugs , he or she will be systematically assigned to the one with the lowest frequency . 
in nonoptimal designs , allocation does not take into consideration the relative rarity of c , so that when all patients have been allocated , drug z has been allocated to fewer patients than statistically needed . 
 ( c ) comparison of percentages of patients that can be allocated to one or two of the molecular pathway - based subgroups ( real values for shiva and mean values for precision trial drawer [ ptd ] 95% ci are plotted )  . 
 ( d ) power calculations showing numbers needed to sequence for the overall ( bottom panel ) and pathway - specific subgroup trials ( top and middle panels )  . 
cna , copy number alteration ; snv , single nucleotide variant ; tcga , the cancer genome atlas . nnms based on the rule , and simulate patient allocation so that each drug - specific arm reaches at least the desired level of power ( optimal allocation design ; fig 3a )  . 
we can thus precisely quantify the advantage of an optimized umbrella design : for the given example , if one were to run 10 independent one - arm studies with a target sample size of 13 to show a response rate of 40% against historical controls of 10% ( for = .05 and power = 0.8 ) , a total of 4 , 276 patients would need to be sequenced to obtain 130 ( ie , 10 13 ) biomarker - positive patients . 
 the researcher to specify different types of end points ( time to event , progression - free survival , overall survival , time to progression , or proportions such as response rate and survival rate ) , number of arms , allocation ratios ( symmetric or asymmetric ) , and other conventional trial parameters . 
simulation results for oneor twoarm studies with time - to - event or proportion end points and with no , manual , or optimal allocation rules are provided in fig 3b . 
simulation results across a range of powers and target proportions are provided in fig 3c . retrospective evaluation of the shiva trial to test ptd in a real - world scenario , we retrospectively evaluated its ability to correctly predict population composition and treatment allocation in the recent shiva trial , a phase ii randomized trial comparing conventional therapy to multiple targeted agents on the basis of tumor molecular profiling . 
in shiva , 741 patients were subjected to a triad of molecular tests , including sequencing with a targeted enrichment panel ( ampliseq cancer panel , covering 45 tumor - associated genes or mutational hotspots ) , gene copy number analysis by cytoscan hd , and immunohistochemistry for three endocrine receptors . 
although ptd can include information on gene expression in the simulation , we removed endocrine receptors from the analyzed parameters because the correlation between rna - based and immunohistochemistry based quantification of endocrine receptors is poor ( appendix fig a1 )  . 
after data cleanup , 417 of 741 patients were left for analysis ( fig 4a )  . we constructed a ptd panel by using the actionable alterations in shiva and ran 100 trial simulations , extracting tumor histologies from tcga with probabilities equal to their frequencies in shiva and with allocation rules reproducing those adopted in the actual trial . 
alteration frequencies for individual genes were within 95% cis in most cases ( appendix fig a1a - b ) , except for low - frequency genes , for which 95% cis can be expected to be wide . 
consequently , observed versus predicted nnms was also quite similar ( fig 4d ; appendix fig a1d ) , demonstrating that ptd can correctly predict the number of patients needed to be subjected to molecular screening . discussion we report a versatile bioinformatics platform that is based on mutational databases to simulate genetic biomarker - driven clinical trials . 
in a retrospective analysis of the shiva trial , we demonstrated that our tool is able to predict the actual number of patients to be sequenced and has the power to detect overall and arm - specific efficacy signals . 
we believe that our tool will be instrumental in overcoming emerging issues in the design of precision oncology trials . the conceptual framework of precision medicine implies an increased fragmentation of disease entities into distinct subgroups identified by the presence of biomarkers that predict response to targeted agents . 
as previously highlighted by several authors , 6 , 20 for trials aimed at demonstrating efficacy of targeted drugs , 20 more biomarkers means rarer biomarkers , which in turn implies higher and more variable numbers of patients needed to be screened . 
 match screening trial ( targeted therapy directed by genetic testing in treating patients with advanced refractory solid tumors , lymphomas , or multiple myeloma ) , the largest trial of this type to date , an interim analysis after 795 patients were screened showed that only 2.5% of patients could be allocated to seven of the 10 targeted agents on trial.21 the possibility of better predicting accrual rate afforded by ptd would allow trialists to drop inefficient arms and / or modify trial design to maximize patient allocation . an unavoidable limitation of our methodology is that simulations are affected by the prevalence of mutations in available databases , which may not be representative of what is found in study populations . 
instead , most trials with targeted drugs are initially aimed at the metastatic setting , and the mutational profile of metastatic tumors can be significantly different from that of primary tumors . 
an ideal setting for ptd - based simulations with current tcga data may be the neoadjuvant / preoperative setting , which is increasingly used to evaluate novel drugs.23 the availability of clinical metadata , such as tumor size or nodal involvement , can be used to strengthen the representativeness of database samples . finally , some disease tumor types are under represented in mutational databases , which results in high variability and low confidence of corresponding simulations . 
melloni , alessandro guida , giuseppe curigliano , edoardo botteri , ruggero de maria , piergiuseppe pelicci , luca mazzarella financial support : giuseppe curigliano , piergiuseppe pelicci administrative support : giuseppe curigliano provision of study materials or patients : giuseppe curigliano , angela esposito , maud kamal , christoph le tourneau collection and assembly of data : giorgio e.m. 
 edoardo botteri no relationship to disclose angela esposito no relationship to disclose maud kamal no relationship to disclose laura riva employment : philips healthcare ( i ) alberto magi no relationship to disclose ruggero de maria no relationship to disclose christophe le tourneau honoraria : novartis , bristol - myers squibb piergiuseppe pelicci stock and other ownership : genextra consulting or advisory role : amgen , msd , bristolmyers squibb , merck serono , astrazeneca travel , accommodations , expenses : msd , bristol - myers squibb , astrazeneca luca mazzarella no relationship to disclose affiliations giorgio e.m. 
schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
le tourneau c , delord jp , gonalves a et al : molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
lopez - chavez a , thomas a , rajan a , et al : molecular profiling and targeted therapy for advanced thoracic malignancies : a biomarker - derived , multiarm , multihistology phase ii basket trial . 
pao w , miller va , politi ka , et al : acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the egfr kinase domaplos med 2 : e73 , 2005 17 . 
mao c , yang zy , hu xf , et al : pik3ca exon 20 mutations as a potential biomarker for resistance to anti - egfr monoclonal antibodies in kras wild - type metastatic colorectal cancer : a systematic review and meta - analysis . 
 o accurate rna sequencing from formalin - fixed cancer tissue to represent high - quality transcriptome from frozen tissue purpose accurate transcriptional sequencing ( rna - seq ) from formalin - fixed paraffin embedded ( ffpe ) tumor samples presents an important challenge for translational research and diagnostic development . 
we evaluated the accuracy of rna - seq data generated from ffpe samples in terms of expression profiling . methods we designed a biospecimen study to directly compare gene expression results from different protocols to prepare libraries for rna - seq from human breast cancer tissues , with randomization to fresh frozen ( ff ) or ffpe conditions . 
the protocols were compared using multiple computational methods to assess alignment of reads to a reference genome , the uniformity and continuity of coverage , the variance and correlation of overall gene expression , patterns of measuring coding sequence , phenotypic patterns of gene expression , and measurements from representative multigene signatures . results the principal determinant of variance in gene expression was use of exon capture probes , followed by the conditions of preservation ( ff v ffpe ) and phenotypic differences between breast cancers . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction although it is generally best to identify gene expression biomarkers from cancer tissues using the highest quality of rna purified from fresh frozen ( ff ) samples , any subsequent development toward diagnostic testing will require translation for use with formalin - fixed paraffin - embedded ( ffpe ) tissue samples . 
however , the variably fragmented and chemically modified rna derived from ffpe samples presents a challenge for accurate measurement of gene expression.1 , 2 in a different context , there is great interest to perform transcriptome sequencing ( rna - seq ) for biomarker discovery research using large cohorts of precious archival ffpe samples from completed clinical trials . 
different approaches to generating libraries for rna - seq include selection of mrna by targeting the poly ( a ) tail ( mrna protocol ) , depletion of more abundant ribosomal rna ( rrna depletion ) using jialu li chunxiao fu terence p . 
 a 80c freezer ( ff ) or 10% neutral buffered formalin and paraffin - embedded as an ffpe tissue block.10 phenotypically , the nine breast cancers were defined by the pathologic status of hormone receptors ( hrs ) and human epidermal growth factor receptor 2 ( her2 ) as hr - positive / her2 - negative in five , hr - positive / her2 positive in one , and triple receptor - negative in three . 
a dnase - i treatment step was included in both . construction of rna - seq library and sequencing full details of all methods for library construction and sequencing of rna samples are in the data supplement . 
an overview diagram of the different rna - seq library protocols is shown in figure 1 , and details of the number of libraries prepared , starting rna requirement , cost , and duration to perform each protocol are summarized in the data supplement . the mrna protocol ( ff only ) used oligo - dt beads for poly ( a ) + mrna enrichment , followed by standard procedures of truseq rna sample prep kit v2 ( illumina , san diego , ca )  . 
then the rrna - depleted srna was used as the template for the mrna protocol described above . the cr protocol was performed using truseq access rnaseq kit ( illumina ) , using random primers . 
no technical replicates could share the same sequencing lane . rna - seq data analysis full details of all data analysis methods and an overview diagram of the analysis plan are provided in the data supplement . 
scatter plot of the first three principal components for count per millionnormalized and variance stabilizing transformed counts of 20 , 381 coding region ( cr ) - targeted poly ( a ) + genes . 
the rnaseq data generated from the srna protocol had a significantly lower concordant pair alignment rate as compared with those from non - srna protocols ( p < .001 ; data supplement )  . 
using the cr protocol , mapping was highly concordant between ff and ffpe and the exonic mapping rate was increased compared with non - cr methods ( data supplement )  . 
overall , the number of genes with read coverage ( transcripts per million [ tpm ] > 0.1 ) was slightly higher in ffpe samples than in ff samples for both non - cr and cr protocols ( data supplement ) .12 uniformity and continuity of read coverage of transcripts uniformity of read coverage was measured by the mean coefficient of variation , and continuity of coverage as the percentage of gaps without read coverage , across the top 1 , 000 highly expressed transcripts ( data supplement )  . 
 principal component analysis of expression of a total of 20 , 381 cr protocol targeted poly ( a ) + genes for all libraries showed that the 26.5% of total variation captured by the first principal component was due to use of exon capture probes ( cr protocol ) , and 20.6% from the second and third components due to the combined effects of ffpe and biologic differences ( fig 2 )  . 
hierarchical clustering results , with high confidence ( average bootstrap probability , 0.93 ) , showed that the major tumor phenotypes ( hr - positive v hr - negative ) and the source tumor clustered together with ffpe samples ( data supplement )  . 
comparing ff and ffpe samples using the same totalrna protocol , the log ratio values were still centered around zero at different mean expression levels ( fig 3b )  . 
difference in measurements as log ratio ( m ) versus mean average expression ( a ) of each gene ( ma plot ) of 20 , 381 coding region ( cr ) targeted poly ( a ) + genes for tumor sample c when using ff.k.totalrna sample c library as the reference . 
m is the log2 - transformed expression of a gene from first library divided by that from the second library , and a is the mean log2 transformed expression of the gene . 
generally , the cr protocol tended to overly enrich the highly expressed genes and was more likely to not capture low expressed genes ( fig 3c ; data supplement )  . 
this was not improved by prior demodification of methylol adducts from ffpe tissuederived rna using heat and amines or the srna protocol ( fig 4 ; data supplement )  . 
although both approaches seemed to slightly increase concordance of expression , neither was statistically significant . after further investigating these protocol induced biases , we calculated the number of genes that would be considered as differentially expressed or false positives compared with each reference ff standard protocol ( data supplement )  . 
cr , coding region protocol ; dem , demodification ; ff , fresh frozen ; ffpe , formalin - fixed paraffin - embedded ; srna , sense rna protocol . and triple receptornegative breast cancers within each protocol . 
the p value from de analysis followed the ideal uniform distribution for non - de genes , with a spike close to zero for the de genes ( data supplement )  . 
 principal component analyses demonstrated the following order of variables influencing gene expression measurements from rna - sequencing : whether the library preparation protocol used exon capture for cr , whether the sample was from ff tissue or ffpe tissue , and the biologic phenotype of the breast cancer based on hrs and her2 receptor status ( fig 2 )  . 
nevertheless , we identified one protocol , ffpe.k.totalrna , with consistently good transcript coverage uniformity and continuity , most concordant expression , and least differential expression when compared with the different non - cr protocols with fresh tissue . 
it had a reasonable requirement of total rna input ( 100 ng ) for ffpe biopsy samples . the first translational research scenario that we posed , in the introduction section , considered the best pairing of protocols that would enable discovery using ff samples with intention to later translate for use with ffpe samples . 
this interpretation was supported by the quality of read coverage , pattern of coding sequence expression , and translation of overall or phenotype - related gene expression profiles and prognostic signatures . the cr protocols yielded concordant results , but different from all other ( non - cr ) protocols . 
 also , changes to the population of exon capture probes within a commercial kit over time could be a potential risk to this approach . the most generalizable results for discovery research from ffpe samples were obtained using the total.rna protocols without exon capture . 
for our second scenario , therefore , we prefer the k.totalrna protocol for best representation of the transcriptome in ffpe samples used for discovery researchaiming to represent the transcriptional information that ff samples would have provided . our third translational research scenario involves the translation of an existing gene expression signature that was previously developed using a different method ( eg , microarray ) or a particular rna - seq protocol . 
the inclusion of random and dt primers and the t7 promoter region ( srna protocol ) to simulate the ff.mrna protocol produced good concordance overall but introduced a high number of nonconcordant mapped reads , nonuniformity , and discontinuity of read coverage across the transcriptome . limitations to our study include small sample size ( although cancers were selected to represent biologic diversity ) ; optimally short time to fixation of tissues and possibly , as a result , a modest degree of degradation of ffpe samples ( dv200 ranges from 65% to 85% ) ; optimal amount of input rna used for non - cr protocols ( at least 100 ng ) ; and lack of generalizability ( single institution conditions of tissue processing )  . 
one tumor ( sample n ) seems to be compromised by unknown technical processing ; our study conclusions , whenever involving this sample , are based on robust point estimates ( eg , median estimates in fig 4 ) across all samples to avoid being driven by its outlier effect . 
for more information about asco 's conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . jialu li stock and other ownership interests : genomic health chunxiao fu no relationship to disclose terence p . 
fraser symmans stock and other ownership interests : ionis pharmaceuticals , nuvera biosciences , delphi diagnostics consulting or advisory role : genentech patents , royalties , other intellectual property : patent pending for gene expression measurement of sensitivity to endocrine therapy travel , accommodations , expenses : luminex supported by the cancer prevention research institute of texas grant no . 
masuda n , ohnishi t , kawamoto s , et al : analysis of chemical modification of rna from formalin - fixed samples and optimization of molecular biology applications for such samples . 
fumagalli d , blanchet - cohen a , brown d , et al : transfer of clinically relevant gene expression signatures in breast cancer : from affymetrix microarray to illumina rna - sequencing technology . 
zhao w , he x , hoadley ka , et al : comparison of rna - seq by poly ( a ) capture , ribosomal rna depletion , and dna microarray for expression profiling . 
loi s , haibe - kains b , majjaj s , et al : pik3ca mutations associated with gene signature of low mtorc1 signaling and better outcomes in estrogen receptor - positive breast cancer . 
in this analysis the hypothesis , we explore with an unsupervised learning algorithm whether distinct subtypes of btcp exist and whether they can provide new insights into clinical practice . methods partitioning around a k - medoids algorithm on a large data set of patients with btcp , previously collected by the italian oncologic pain survey group , was used to identify possible subgroups of btcp . 
a free online tool was created for new patients cluster computation to validate these clusters in future studies and provide handy indications for personalized btcp therapy . conclusion this work proposes a classication for btcp and identies subgroups of patients with unique efcacy of different pain medications . 
this work supports the theory that the optimal dose of btcp opioids depends on the dose of basal opioids and identies novel values that are possibly useful for future trials . 
these results will allow us to target btcp therapy on the basis of patient characteristics and to dene a precision medicine strategy also for supportive care . jco precis oncol 4 : 1339 - 1349 . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction breakthrough cancer pain ( btcp ) is a common event that affects a considerable proportion of patients with cancer.1 a variety of denitions for btcp have been proposed2 , 3 . 
according to the italian oncologic pain survey ( iops ) study group , 4 btcp should be dened as a relevant change in pain intensity of severe intensity in patients who receive an effective treatment with opioids.4 ( p963 ) nevertheless , despite this unique denition , btcp encloses a wide range of manifestations that differ , among other features , in intensity , duration , frequency , and triggering events . btcp represents a clinically relevant condition with a negative impact on the patients quality of life . 
patients characteristics characteristic value ( n = 4 , 016 ) 2 , 202 ( 54.8 ) male sex interview setting oncology pain therapy palliative care radiotherapy treatment corticosteroids anticonvulsant drugs acetaminophen constipation drugs anxiolytic drugs antidepressant drugs antiemetic drugs nsaids cannabis treatment setting inpatient outpatient domicile day hospital hospice cancer type lung breast pancreas colon prostate rectum stomach bladder at present , several gaps exist in the knowledge of btcp management . 
these partially unanswered questions , among others , concern the optimal drug administration route , pharmacokinetics , the balance between rapid - onset and slow - onset opioids , and the eventual difference of btcp response deriving from clinical features , such as stage , primary site , or metastases . in this analysis , it was thus hypothesized that the unique btcp denition might actually enclose diverse pathologic entities , possibly with peculiar clinical needs and specic responses to drugs . 
these so - called unsupervised learning algorithms have already been extensively used , for example , for the identication of breast cancer subtypes.6 nevertheless , to our knowledge , no authors have yet tried to explore the issue of btcp management using these techniques . to fulll this purpose , we used data that were collected by the iops group in a large , multicentric national study5 , 7 , 8 that enrolled 4 , 056 patients from 32 centers with opioidcontrolled basal pain suffering from btcp . 
this work is therefore a secondary analysis of the iops group survey that aims to identify novel subtypes of btcp through the use of unsupervised learning algorithms . methods patients enrollment and data collection details concerning the enrollment of patients are extensively described in the main paper from the iops group.5 in brief , local ethical committees approved the protocol , and written informed consent was obtained from each patient . interviews were performed in different settings , in particular , oncology , pain therapy , palliative care , and radiotherapy . 
a comprehensive list of clinical variables was collected for each patient that consisted of basal pain and btcp site , duration , frequency , intensity , relieving factors , triggers , drugs , primary cancer site and stage , and concomitant symptoms for a total of 1 , 086 1340 2020 by american society of clinical oncology multiple primary average age , years ( range ) karnofsky performance status ( range ) baseline pain , nrs scale ( range ) note . 
 breakthrough cancer pain clinical features and opioids response features with less than 5% of missing data and associated with a corrected p value of , .1 and , for categorical features , a log2 odds ratio greater than 1 were used to build a multivariable logistic regression . 
to investigate the correlationsimultaneously for all patients and on the same scalebetween the amount of opioids used and btcp therapy satisfaction , all doses of opioid drugs were converted10 to equivalent intravenous morphine doses and expressed as a total daily dose : one for btcp - directed opioids and for basal pain opioids . 
doses were converted to oral morto intravenous morphine doses and not phine doses because intravenous morphine has been increasingly used in different clinical settings and would provide more interpretable graphics in results . 
moreover , to explore the interaction of fast - acting and long - acting opioid dosages , we calculated for each patient the btcp opioidstobasal pain opioids ratio ( opr )  . 
a polynomial logistic regression was used to catch nonlinear relationships between opioid doses and therapy satisfaction . cluster computation and visualization features dening the clinical characteristics of btcp were selected to perform clusters computation . 
the above - mentioned features were used to calculate a dissimilarity matrix using a cluster package11 ; presence of background pain pain intensity 4 of 10 four or fewer episodes per day breakthrough pain presence of peaks well distinguished from background pain intensity cluster fig 1 . 
 ( a ) algorithm used for the diagnosis of breakthrough cancer pain ( btcp ) during patients enrollment in the italian oncologic pain survey ( modied from mercadante et al8 )  . 
 ( b ) a two - dimensional t - distributed stochastic neighbor embedding projection of all patients , colored by their clusters , on the basis of the following btcp features : number of btcp episodes , btcp peaks duration , btcp type , btcp intensity , number of days since the beginning of btcp episodes , eventual benet from pharmacotherapy , eventual benet from rest , and whether btcp was enhanced by movements . each point represents a patient . 
silhouette statistics14 were calculated for each run , and the optimal number of clusters was manually picked as being that with the best trade - off between silhouette statistics and reasonable clinical interpretation . 
we used complete - linkage hierarchical clustering with 12 clusters , along with rand index calculation , to explore the replicability of clusters with a different algorithm . t - distributed stochastic neighbor embedding15 algorithm was used to project dissimilarities between patients in a bidimensional space , with closer points representing patients with more similar clinical btcp features . 
an online tool allows for the repetition of the performed classication on new sets of patients.16 clusters analysis t - test , mann - whitney test , and 2 test were used to assess the association of parametric , nonparametric , and categorical features , respectively , with each cluster . 
patients characteristics are listed in table 1 . cluster computation to investigate whether subtypes of btcp exist , we used btcp features to build an unsupervised clustering model . the number of btcp episodes , btcp peaks duration , btcp type , btcp intensity , number of days since the beginning of btcp episodes , the eventual benet from pharmacotherapy , the eventual benet from rest , and whether btcp was enhanced by movements were the eight btcp - dening variables selected for the nal model , which was built with the k - medoids algorithwe chose 12 as an optimal trade - off between the average width of clusters silhouette ( 0.45 ; appendix fig a1a ) and the interpretability of the clusters themselves . 
 pantano et al figure 1 shows the algorithm used to dene btp ( fig 1a ) t - distributed stochastic neighbor and a 2 - dimensional embedding projection of all patients , colored by their clusters ( fig 1b )  . 
an online tool is available for the classication of new patients according to our method.16 characteristics of btcp clusters we analyzed the enrichment of the eight btcp - dening variables and of other clinical features among the clusters . a description of each cluster is available in table 2 . 
a summary of btcp features according to cluster is presented in figure 2 . btcp therapy satisfaction finally , we tried to assess what inuenced patient - reported btcp therapy satisfaction . 
after converting the opioid dose to a unique scalecorresponding to the equivalent dose of intravenous morphinewe investigated using a nonlinear model the inuence of basal opioid dose , btcp opioid dose , and opr on patient - reported therapy satisfaction . 
of higher therapy satisfaction lower therapy satisfaction btcp opioids dose / basal pain opioids dose higher therapy satisfaction higher therapy satisfaction lower therapy satisfaction lower therapy satisfaction btcp opioids dose ( mg ) basal pain opioids dose ( mg ) fig 3 . 
this roughly corresponds to a daily dose of sublingual fentanyl 100 g for btcp and a daily dose of oral morphine 30 mg as the basal opioid dose . we separately performed the same analyses on previously dened clusters ( fig 3d )  . 
for clusters 1 , 6 , and 10 , the satisfaction seemed to grow indenitely with an increase of the opr opioids , whereas clusters 7 , 8 , and 11 seemed to have clear , optimal peaks of opr . 
despite the interpretation being challenged by some large cis , we can say from these data that , depending on the cluster , optimal opr ranges from 15% to 50% . discussion this paper demonstrates a novel approach for the investigation of btcp . 
we acknowledge that this study was not designed to perform this analysis and , moreover , that the large number of clusters might interfere with their interpretability and clinical utility . 
nevertheless , this study represents a proof - ofconcept for this investigational approach . cluster 1 cluster 2 cluster 3 higher therapy satisfaction higher therapy satisfaction higher therapy satisfaction lower therapy satisfaction lower therapy satisfaction lower therapy satisfaction cluster 4 cluster 5 cluster 6 higher therapy satisfaction higher therapy satisfaction higher therapy satisfaction lower therapy satisfaction lower therapy satisfaction lower therapy satisfaction cluster 7 cluster 8 cluster 9 higher therapy satisfaction higher therapy satisfaction higher therapy satisfaction lower therapy satisfaction lower therapy satisfaction lower therapy satisfaction cluster 10 cluster 11 cluster 12 higher therapy satisfaction higher therapy satisfaction higher therapy satisfaction lower therapy satisfaction lower therapy satisfaction lower therapy satisfaction fig 3 . 
using our same data , another group proposed 0.20 ( one fth ) as the optimal ratio.5 nevertheless , they used a frequentistic approach , as 0.20 is simply the most common ratio among the cohort . 
this possibly suggests that , instead of starting btcp opioid titration with the lowest possible dosage , as proposed previously , 28 titration could start immediately with an optimal opioid dosage . moreover , cluster analysis reveals that this ratio might not be the same for all patients : some patients might benet from a higher btcp opioid dosage ( cluster 2 and 7 ) , whereas others might benet from a lower one ( cluster 11 )  . 
finally , for some patients , we did not observe an upper threshold for this ratio ( cluster 1 , 6 , and 9 ) , perhaps pointing out patients for whom a strong btcp opioid dosage increase is required . the interpretation is made difcult by the multiple associations and , at this stage , is not mature enough to suggest any immediate change in clinical practice . 
however , we made available an online free tool16 that allows for the classication of new patients according to our algorithm and returns a proposed btcp therapy which depends on the patient cluster optimal opr and basal opioid dose . 
we suggest that this tool might be used in the future to prospectively validate the clinical importance of our clusters in clinical practice and to compare our proposed opioid dosages in settings different from ours . the presence of distinct btcp phenotypes , each one associated with specic clinical features , could also be a reection of diverse underlying pathophysiologic mechanisms : our work suggests that preclinical research might gain insight into these possible differences and help the development of a tailored therapy also for btcp . the main limitation of our study is the appropriateness of collected data for the scope of our work . 
we believe that a prospective study specically designed for the investigation of btcp clusterspossibly with long - term follow - up and therapy success outcomes and not limited to a single timepoint evaluationmight enable a clearer identication of distinct clusters . 
businco hospital , asl 8 , cagliari , italy 20department of biotechnological and applied clinical sciences , section of clinical epidemiology and environmental medicine , university of laquila , laquila , italy 21section of clinical pharmacology and oncology , department of health sciences , university of florence , florence , italy 22department of medicine and surgery sciences , university of bologna , bologna , italy 23paintherapy , s . 
croce e carle hospital , cuneo , italy 24pain therapy ics maugeri , irccs foundation , pavia , italy 25medical oncology , azienda sanitaria universitaria integrata di trieste , trieste , italy 26medical oncology , azienda sanitaria universitaria integrata di udine , udine , italy 27palliative care and oncologic pain service , s . 
pain 41 : 273 - 281 , 1990 portenoy rk , payne d , jacobsen p : breakthrough pain : characteristics and impact in patients with cancer papain 81 : 129 - 134 , 1999 4 . 
mercadante s , marchetti p , cuomo a , et al : breakthrough pain and its treatment : critical review and recommendations of iops ( italian oncologic pain survey ) expert group . 
mercadante s , marchetti p , cuomo a , et al : breakthrough cancer pain : preliminary data of the italian oncologic pain multisetting multicentric survey ( iopsms )  . 
mercadante s , lazzari m , reale c , et al : italian oncological pain survey ( iops ) : a multicentre italian study of breakthrough pain performed in different settings . benjamini y , hochberg y : controlling the false discovery rate : a practical and powerful approach to multiple testing . 
davies an , dickman a , reid c , et al : the management of cancer - related breakthrough pain : recommendations of a task group of the science committee of the association for palliative medicine of great britain and ireland . 
the appropriate number of clusters for additional analyses was found to be 12 , an optimal trade - off between the average width of clusters silhouette ( 0.45 ) and the interpretability of clusters themselves . ( b ) heatmap showing the concordance of pam clustering with complete - linkage hierarchical clustering . 
survival curves , number of samples classied as tmb high , and predicted neoantigens were used to evaluate the differences between thresholds . results the distribution of tmb varied dramatically among cancer types . 
2019 by american society of clinical oncology introduction immunotherapy ; immunotherapy , especially checkpoint inhibition , has been added recently as a successful treatment option for some patients with cancer . 
it is based on the presence of neoantigens eliciting a response from t cells by recognizing these antigens as foreign and inltrating the tumor microenvironment.1 , 2 however , only a subset of patients respond to checkpoint inhibitor therefore , predictive biomarkers are critical to guide the selection of patients for these therapies . 
although high - level programmed death - ligand 1 expression , microsatellite instability , and mismatch - repair deciency have been deemed including tumor clinically relevant , other markers , mutational burden ( tmb ) , interferon gamma prole , and human leukocyte antigen ( hla ) genotype , have also exhibited promising results.3 tmb is commonly dened as the total number of somatic mutations in a tumor genome , divided by the number of bases sequenced in megabases ( mb ) .4 , 5 previously , patients with higher tmb ( tmb high ) demonstrated increased response to immunotherapy compared with patients with lower tmb ( tmb low ) , especially in nonsmallcell lung cancer.6 , 7 however , no consensus has been established to dene a standard threshold for tmb high . 
last , previous studies have adopted panel target sequencing with small regions of genomic dna , possibly excluding neoantigen candidates.8 therefore , exploring the tmb distribution with more extensive sequencing platforms , such as whole exome sequencing ( wes ) , can provide a more comprehensive view of tmb . 
 fernandez et al context threshold ? key objective can a cancer - specic tumor mutational burden ( tmb ) provide more useful information compared with a xed pan - cancer knowledge generated a cancer - specic tmb threshold can dynamically adjust to identify dramatically more patients as tmb high compared with a restrictive pan - cancer threshold . 
the additional patients do not have a signicantly different outcome compared with the other tmb - high patients . relevance when interpreting a patients tmb , the overall tmb seen in the specic cancer type should be taken into consideration to give the tmb context . reect tmb in each cancer type , the wcm threshold uses interquartile range ( iqr ) : mean ( tmb ) + 1.25 iqr ( tmb ) applied within each cancer , meaning each cancer type will have its own threshold reective of the distribution of mutations within that cancer type . 
predicted neoantigens for tcga samples were obtained through the cancer immunome atlas.23 tmb calculation tmb was calculated as the total number of nonsynonymous mutated bases in the tumor genome divided by the mb of the genome covered . 
the wcm advanced cohort was sequenced using our exact - 1 wes haloplex pipeline using tumor - normal pairs , 24 which covers 37 mb of the genome . samples were collected under a protocol approved by the institutional review board of weill cornell medical college , and written informed consent was obtained . 
because tmb is divided by the total size of genome sequenced to help adjust for technical batch effects , the size of sequenced regions was determined for each tcga cohort according to their respective publications.11 - 22 samples with purity of less than 50% according to absolute ( tcga ) or clonality estimate in tumors ( exact - 1 ; weill cornell medine clinical genomics lab , new york , ny ) were removed.25 , 26 germline variants and variants appearing in the exome aggregation consortium database with a frequency of at least 1% were removed to lter them out.27 variants with a variant allele frequency ( vaf ) of less than 10% were ltered out after correcting vaf for tumor purity by dividing vaf by purity . 
a summary of ltering steps can be seen in the data supplement . mutational signatures mutational signatures were determined using the deconstructsigs r package.28 for each sample , a matrix containing the frequency a mutation appears within a trinucleotide context was generated . 
this matrix was compared with the signatures published previously in the catalogue of somatic mutations in cancer , 29 resulting in signature contributions . statistical methods the wilcoxon rank sum test was used to compare sample distributions . 
for example , the tmb for prostate cancer in tcga ranged from 0.03 mutations / mb to 14.13 distribution of tmb for tcga samples cancer type distribution of tmb for wcm advanced samples threshold chalmers et al threshold chalmers et al cancer type fig 1 . 
however , the tmb for bladder cancer in tcga ranged from 0.04 mutations / mb to 99.68 mutations / mb , with a mean of 6.92 mutations / mb ( n = 375 )  . because of the higher frequency of metastatic samples in the wcm advanced cohort , we sought to understand how the distribution of tmb changes between primary and metastatic samples . 
the chalmers et al threshold is a constant cutoff of 20 mutations / mb for all cancer types , 4 whereas our custom wcm threshold is a formula of mean ( tmb ) + 1.25 iqr ( tmb ) , adapted from a study by zehir et al.5 the wcm threshold was applied per cancer to account for the variation in tmb among cancer types . data for patients treated with immunotherapy were not available for either cohort ; however , tcga survival outcomes were investigated to understand the differences in survival between tmb high and tmb low using both thresholds . 
tmb - high patients showed signicantly improved survival compared with tmb - low patients for blca using both the wcm threshold ( log - rank p value = .0014 ) and the chalmers et al threshold ( log - rank p value = .009 ; data supplement )  . 
lusc showed improved survival for tmb - high patients compared with tmb - low patients when using the wcm cutoff ( log - rank p value = .036 ) but not when using the chalmers et al cutoff ( log - rank p value = .11 ; data supplement )  . 
ucec also showed signicantly improved survival for tmb - high patients compared with tmb - low patients when using chalmers et al ( log - rank p value = .023 ) but did not reach signicance when using wcm ( log - rank p value = .055 ; data supplement )  . 
to further compare the performances of the two thresholds , timedependent roc curves were created at the 5 - year time point ( fig 2d ; data supplement )  . 
to explore the difference between the patients selected by the thresholds , we split the samples into three groups : tmb high , wcm - tmb high , and tmb low . 
the wcm - tmbhigh group contained samples classied as tmb high by wcm and tmb low by chalmers et al ( when chalmers et al had more tmb high classications [ ucec ] than wcm , we used chalmers - tmb high [ chm - tmb high ] )  . 
the tmb - low group contained samples classied as tmb low by both methods ( fig 2a )  . the wcm - tmbhigh group did not attain signicantly improved survival compared with the tmb - low group for blca ( log - rank p value = .072 ; fig 2b )  . 
the chalmers et al threshold did not classify any patients with kirc as tmb high . although the survival trends were not signicantly different between the cutoffs , the number of samples classied as tmb high differed between thresholds . 
out of 14 tcga cancer types investigated , eight cancers had a statistically larger tmb - high group for wcm compared with chalmers et al ( blca [ fold change = 1.9 ] , brca [ fold change = 6.7 ] , gbm [ fold change = 2.7 ] , kirc [ no high by chalmers ] , lgg [ fold change = 13.0 ] , ov [ fold change = 5.67 ] , prad [ no high by chalmers ] , and thca [ no high by chalmers ] ) ; ve cancers had no statistically signicant change in the number of tmb - high classications ( coad , kich , luad , lusc , and read ) ; and only ucec had a large number of tmb high classied by chalmers et al compared with wcm ( 2 test with yatess continuity correction ; p value , .05 ; fig 3a )  . 
predicted neoantigens from the cancer immunome atlas compared the tmbhigh , the wcm - tmbhigh , and the tmb - low groups for tcga samples ( fig 4a ) .23 the tmb - low group had signicantly fewer neoantigens than both the tmb - high ( wilcoxon p value , 2.3e - 16 ) and wcm - tmbhigh ( wilcoxon p value = 2.1e - 14 ) group . 
kaplan - meier survival curves comparing the tmb classications between the weill cornell medicine ( wcm ) threshold and the chalmers et al threshold for ( b ) blca and ( c ) kirc . 
for kirc , the chalmers et al threshold was too high to classify any samples as tmb high , resulting in only two groups : wcm - tmb high and tmb low . 
 ( d ) time - dependent receiver operating characteristic ( roc ) curve comparing the true positive ( tpr ) and false positive ( fpr ) rates for blca using both the chalmers et al threshold and the wcm threshold shows the area under the curve ( auc ) for both cutoffs at 5 years from diagnosis . 
because of the observed variation in tmb among cancer types , neoantigens were also compared between the tmb groups for each cancer ( figs 4b and 4c ; data supplement )  . 
prostate adenocarcinoma ( prad ) and kidney renal clear cell carcinoma ( kirc ) are excluded from the plot because the chalmers et al threshold did not classify any samples as tmb high . 
blue bars represent cancer types with a higher or equal number of tmb - high classications for the weill cornell medicine ( wcm ) threshold than the chalmers et al threshold . 
overall , the tmb - high and wcm - tmbhigh groups showed increased neoantigen counts compared with the tmb - low group . finally , mutational signatures were compared among tmb groups . 
distribution of predicted neoantigens for tmb high ( blue ) , tmb high weill cornell medicine ( wcm ; red ) , and tmb low ( teal ) for the cancer genome atlas ( tcga ) shown for ( a ) all cancers pooled together , ( b ) lung adenocarcinoma ( luad ) , and ( c ) bladder urothelial carcinoma ( blca )  . 
 ( a ) violin plots showing the distribution of the contribution of signature 10 ( defective polymerase - ) in tumor mutational burden ( tmb ) high , chalmers ( chm ) - tmb high , and tmb low . 
 ( b ) pca showing the signature contributions for uterine corpus endemetrial carcinoma ( ucec ) with tmb - high samples in blue , wcm - tmbhigh samples in red , and tmb - low samples in teal . 
only signatures 2 , 4 , 6 , 10 , 13 , 15 , 20 , 21 , and 26 were considered , to focus on signatures with the most relevant interpretations . 
for example , the chalmers et al cutoff of 20 mutations / mb is higher than the maximum tmb seen within tcga prostate cancer ( 14.13 mutations / mb ) ; however , using a lower threshold might be too lenient for tcga bladder cancer ( maximum tmb of 99.68 mutations / mb )  . 
the dramatic difference in tmb among cancer types led us to explore a tmb threshold applied in a cancer - specic manner . to a cancer - specic when comparing a pan - cancer threshold , we found we were able to maintain differences in survival trends between tmb high and tmb low , often while including more patients in the tmb - high group by using the cancer - specic threshold . 
using a tmb threshold that has a larger number of tmb - high classications while not having signicantly different survival trends than a more stringent threshold can potentially open treatment options to patients if they can be shown to respond well . 
hr , hazard ratio ; os , overall survival ; wcm , weill cornell medicine . seen in recent publications suggests that tmb alone is not a critical predictor of survival for nonimmunotherapy treatment response.6 , 37 , 38 differences in tmb distribution between the tcga and wcm advanced cohorts could be a result of biologic or technical differences . 
tcga contains primary tumor samples , and wcm advanced contains mostly metastatic tumor samples . when comparing different cohorts of patients , many technical differences can inuence tmb , making a static threshold potentially less accurate . 
after sequencing , the exact - 1 pipeline and tcga pipeline use different mutation callers and apply different quality lters . in addition , when comparing tmb among studies , it is important to understand the variants that were considered . 
 fernandez et al calculating tmb.6 , 38 , 39 targeted panels cover different genes and sizes of the genome and at a higher sequencing depth than wes , inuencing the estimated distribution of tmb . 
the panel must cover at least 2 mb of the genome to have high sensitivity and specicity when measuring tmb.8 a static threshold would not be able to adjust to the differing levels of tmb resulting from different panels . 
however , a dynamic threshold would be able to adjust for technical differences . the cancer - specic threshold adjusted for the variation in tmb ; however , the pan - cancer threshold classied more patients as tmb high for ucec . 
mutational signatures for these samples showed a cluster of patients with pole deciency , which led to a large number of mutations and a tmb distribution with a large iqr , 22 causing the cancerspecic threshold to be strict in this case . the survival analyses in this study were limited by a lack of patients undergoing immunotherapy treatment . 
in addition , the number of predicted neoantigens was compared per cancer , showing that a cancer - specic threshold identies patients with a higher number of neoantigens compared with tmb - low patients . moreover , tmb might depict only one factor in the success of immunotherapy . 
a recent study has shown that patients with melanoma and nonsmall - cell lung cancer with heterozygous hla class i exhibit improved survival compared with patients who are homozygous.40 because of the limitation of tcga data , we could not examine the role of hla class i in the current study . 
fernandez employment : celgene travel , accommodations , expenses : foundation medicine himisha beltran consulting or advisory role : janssen oncology , genzyme , glaxosmithkline , abbvie , astellas pharma , astrazeneca research funding : janssen ( inst ) , abbvie / stemcentrx ( inst ) , eli lilly ( inst ) travel , accommodations , expenses : janssen oncology bishoy m . 
faltas honoraria : digital science press publications research funding : eli lilly juan miguel mosquera research funding : personal genome diagnostics travel , accommodations , expenses : personal genome diagnostics david m . 
hoffmann la roche ag , novartis , astellas pharma research funding : eli lilly , janssen , millenium pharmaceuticals , sano patents , royalties , other intellectual property : us patent ( 7 , 767 , 393 and 7 , 229 , 774 ) , expression prole of prostate cancer , 2007 ; us patent ( 7 , 332 , 290 ) , detection of amacr cancer markers in urine , 2008 ; us patent ( 7 , 718 , 369 ) , recurrent gene fusions in prostate cancer , 2010 ; us patent ( 7 , 803 , 552 and 7 , 666 , 595 ) , biomarkers for predicting prostate cancer progression , 2010 ; us patent ( 7 , 981 , 609 b2 ) , methods for identifying and using snp panels , 2011 ; us patent ( 8 , 106 , 037 b2 ) , identication and treatment of estrogen responsive pca , 2012 ; us patent ( 9 , 090 , 899 b2 ) , methods of diagnosing and treating prostate cancer characterized by ndrg1 - erg fusion , 2015 ; us patent ( 9 , 458 , 213 b2 ) , compositions and methods for diagnosing prostate cancer based on detection of slc45a3 - elk4 fusion transcript , 2016 ; us patent ( 9 , 568 , 483 b2 ) , molecular subtyping , prognosis and treatment of prostate cancer , 2017 ; us patent ( 9 , 678 , 077 b2 ) , erg / tff3 / hmwck triple immunostain for detection of prostate cancer , 2017 ; us patent ( 61 , 408 , 341 ) , exploration of novel gene fusion in prostate cancer by rna - seq travel , accommodations , expenses : f . 
shah consulting or advisory role : astellas pharma , eli lilly japan research funding : gilead sciences ( inst ) , merck ( inst ) , boston biomedical ( inst ) , oncolys biopharma ( inst ) , bristol - myers squibb ( inst ) wei song employment : genentech ( i ) employment : cytokinetics ( i ) honoraria : foundation medicine , loxo consulting or advisory role : foundation medicine , loxo no other potential conicts of interest were reported . acknowledgment we thank juan sebastian andrade martinez for his previous work with the wcm advanced cohort and bhavneet bhinder for her advice on predicted neoantigens . references 2018 topalian sl , weiner gj , pardoll dm : cancer immunotherapy comes of age . 
j clin oncol 29 : 4828 - 4836 , 2011 topalian sl , hodi fs , brahmer jr , et al : safety , activity , and immune correlates of anti - pd - 1 antibody in cancer . 
n engl j med 366 : 2443 - 2454 , 2012 tray n , weber js , adams s : predictive biomarkers for checkpoint immunotherapy : current status and challenges for clinical application . 
cancer immunol res 6 : 1122 - 1128 , 2018 chalmers zr , connelly cf , fabrizio d , et al : analysis of 100 , 000 human cancer genomes reveals the landscape of tumor mutational burden . 
genome med 9 : 34 , 2017 zehir a , benayed r , shah rh , et al : mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10 , 000 patients . 
n engl j med 378 : 2093 - 2104 , rosenberg je , hoffman - censits j , powles t , et al : atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum - based chemotherapy : a single - arm , multicentre , phase 2 trial . 
lancet 387 : 1909 - 1920 , 2016 buchhalter i , rempel e , endris v , et al : size matters : dissecting key parameters for panel - based tumor mutational burden analysis . 
plimack er , dunbrack rl , brennan ta , et al : defects in dna repair genes predict response to neoadjuvant cisplatin - based chemotherapy in muscle - invasive bladder cancer . 
biometrics 56 : 337 - 344 , 2000 johnson db , frampton gm , rioth mj , et al : targeted next generation sequencing identies markers of response to pd - 1 blockade . 
rizvi h , sanchez - vega f , la k , et al : molecular determinants of response to anti - programmed cell death ( pd ) - 1 and anti - programmed death - ligand 1 ( pd - l1 ) blockade in patients with non - small - cell lung cancer proled with targeted next - generation sequencing . 
 megakaryocyte potentiating factor as a predictive biomarker for therapies against malignant mesothelioma purpose effective biomarkers for malignant mesothelioma ( mm ) are needed for clinical management and the development of mesothelin - targeted therapies . 
we evaluated serum megakaryocyte potentiating factor ( mpf ) as a biomarker predictive of treatment outcome in patients with mm and for developing mesothelin - targeted therapies . materials and methods serial serum samples from patients with mm in two clinical trials of an antimesothelin immunotoxin were tested with our clinically validated mpf assay . 
mpf was further evaluated for an association with response to an antimesothelin therapy and for disease monitoring . results there was a significant reduction of serum mpf in patients with elevated baseline and radiologic response , with an average change from 52% to 78% after one to six cycles . 
 potentiating factor ( mpf ) and membrane - bound mesothelin.15 , 16 previous studies showed correlations between the computed tomography ( ct ) response and arbitrarily chosen 10% to 25% reduced serum mesothelin after chemotherapy.17 - 21 such low cutoff values are incompatible with a 30% reduction in tumor determined with ct , would contribute to high degrees of false signals , and would make it more difficult to correlate with clinical outcomes . the currently available mesomark test ( fujirebio , malvern , pa ) detects serum mesothelin , which is subject to the interference by mesothelin - targeted antibody - based agents . 
we developed and validated an assay for mpf and further showed that elevated mpf is a worse prognostic biomarker in patients with mm.22 its effectiveness for monitoring therapies is not known . 
the first trial was a phase i study with a standard dose of pemetrexed and cisplatin , and the antimesothelin immunotoxin ss1p ( nct01445392 ) in chemotherapy - nave patients with pleural mesothelioma.19 twenty patients were evaluable , 12 with partial response ( pr ) , three with stable disease ( sd ) , and five with progressive disease ( pd )  . 
the patients had received one to six ( and an average of three ) previous therapies.8 all patients were evaluated for responses : three with pr , three with sd , and four with pd . 
tumor responses were determined on the basis of ct and calculated in accordance to modified response evaluation criteria in solid tumors ( mrecist ) criteria for mesothelioma . statistical analyses prism , version 7.0 ( graphpad , la jolla , ca ) was used for statistical analysis . 
 kaplan - meier survival analyses were performed to determine log - rank hazard ratio ( hr ) and p value of mpf response or ct response patients on pfs or os . 
on the basis of the linear regression analysis of corresponding changes in ct and mpf in these 20 patients with mm , a 30% change in ct was interpolated to a 47% change in mpf level ( fig 1b )  . 
thus , a rounded 50% change in serum mpf was selected as the cutoff for mpf response , which corresponded to approximately 30% change in ct using mrecist . the serum mpf test enables continuous and multiple measurements during treatment . 
among pr patients with an elevated mpf using the upper limit of normal at 1.2 ng / ml , 22 there was an average of 52% change in mpf after one cycle ( p < .001 ; n = 12 ; fig 1c )  . 
in contrast , for the patients who did not have a ct response , there was no significant reduction of mpf ( n = 14 ; fig 1d )  . 
thus , the treatment induced reduction of serum mpf may be an informative biomarker of objective response in patients with mm with elevated baseline mpf . kaplan - meier analyses were performed to compare pfs and os of both trials . 
thus , the data suggest that a 50% change in mpf after systemic therapies is strongly associated with improved pfs . reduction of mpf after systemic treatment as a potential biomarker for better os the mpf response was further correlated with os . 
landmark analysis of os for mpf and ct response was performed , with a landmark date at 91 days , at which time all patients had experienced a response but one . 
 ( a ) analysis of the changes in serum mpf in patients with partial response ( pr ) , stable disease ( sd ) , and progressive disease ( pd )  . 
all data are shown as mean standard deviation . depletion regimen of pentostatin and cyclophosphamide , major cancer regressions were observed and were attributed primarily to the activities of ss1p.8 the nonparametric mann - whitney test showed that the patients with pr ( by ct ) had elevated baseline serum mpf levels , which were higher than those in the nonresponse group ( p = .033 ; fig 3a )  . 
thus , data suggest that elevated baseline serum mpf may be associated with the response to an effective antimesothelin therapy in patients with refractory mm , whereas the mpf response is associated with improved survival in these patients ( p = .012 ; appendix fig a4 )  . 
 20 pfs since in - study date ( months ) os since in - study date ( months ) mpf response mpf nonresponse log - rank p < .001 hazard ratio = 0.279 ct response ct nonresponse log - rank p = .022 hazard ratio = 0.457 mpf response mpf nonresponse log - rank p = .004 hazard ratio = 0.361 ct response ct nonresponse log - rank p = .035 hazard ratio = 0.476 pfs since in - study date ( months ) os since in - study date ( months ) fig 2 . 
 ( a ) progression - free survival ( pfs ) and ( b ) overall survival ( os ) using mpf response at a threshold of 50% or more after therapies . 
 ( c ) progression - free survival and ( d ) os for patients with malignant mesothelioma using computed tomography ( ct ) response on the basis of modified response evaluation criteria in solid tumors . 
102 was a 50 - year - old man with extensive peritoneal involvement and received two cycles of ss1p because of ss1p antibody development.8 he had a delayed response with substantial tumor shrinkage at 7 months . 
103 , a 56 - year - old woman with pleural mesothelioma , had rapid disease progression before treatment and was the only patient who completed all six cycles of therapy . 
105 , pr response progression cutoff response progress response time since in - study date ( months ) time since in - study date ( months ) cutoff cutoff fig 3 . 
elevated baseline megakaryocyte potentiating factor ( mpf ) associated with response and long - term follow - up of individual patients with major tumor regression after antimesothelin and immune suppression . 
there was evidence of increased serum mpf at the time of disease progression for all . discussion our study indicates that the serum mpf test is effective in monitoring systemic therapies for patients with mm , because the patients with mpf responses have much better pfs and os than those without , regardless of baseline levels . 
in mm , recist needs to be modified because one - dimensional measurement of the nonspherical growth pattern was difficult.24 the mrecist requires repeated total tumor measurements on two occasions 4 weeks apart for pr determination.25 the quantitative changes of serum mpf may provide an alternative , lowcost , and nonradiative assessment of therapy response in patients with mm with elevated mpf . 
 we used a more stringent cutoff value of a 50% change in mpf to define the biomarker response compared with the previous reports17 - 21 ; this cutoff was supported by our correlative analysis of ct and mpf responses in patients with mm . 
such a stringent cutoff correlates with the mrecist criterion of a 30% reduction in the sum for all target lesions and thus leads to a significant reduction of false - positive reports during monitoring . 
thus , in addition to confirming the association between changes in mpf and patients response to systemic therapy , 18 our results further showed that an mpf biomarker response is associated with improved pfs and os . as the cleavage product during mesothelin maturation with a short half - life in blood , 22 , 26 serum mpf levels reflect the expression of target antigen mesothelin on tumor cells , without being bound by the therapeutic agents . 
in fact , in untreated mesothelioma patients , there is a strong correlation between mpf and soluble mesothelin.22 , 27 the mpf test could be valuable for the clinical development of mesothelin targeted therapies and for monitoring targeted therapies against mm . 
therefore , serum mpf could facilitate long - term disease monitoring and provide a noninvasive assessment of the drug - target mesothelin expression in mm at the time of disease progression , which can be critical for evaluating alternative therapies . one of the most important issues in the development of targeted therapies is the stratification of the intended patient population . 
mesothelin is ubiquitously detected in epithelioid mm by immunohistochemistry ( table 1 ) ; therefore , mesothelin immunohistochemistry is not predictive of response to mesothelin - targeted therapies in mm , and a different biomarker test is required . 
 there are many challenges for a serum - based test for patient selection , including the expression of the tumor antigen by nontumor cells and the variations in shedding of it by tumor cells . 
 in addition , it is possible that serum mpf may be influenced by renal dysfunction in patients receiving nephrotoxic chemotherapies because prior reports showed elevated serum mpf in patients with renal disease.28 , 29 however , the patients described in our study had normal renal functions at the time of enrollment . 
although our previous data suggested that patients with mm with elevated mpf have a worse prognosis , 22 the results here suggest that the reduction of serum mpf in patients with refractory mm is associated with improved clinical outcome . 
vogelzang nj , rusthoven jj , symanowski j , et al : phase iii study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma . 
hassan r , cohen sj , phillips m , et al : phase i clinical trial of the chimeric anti - mesothelin monoclonal antibody morab - 009 in patients with mesothelin - expressing cancers . 
golfier s , kopitz c , kahnert a , et al : anetumab ravtansine : a novel mesothelin - targeting antibody - drug conjugate cures tumors with heterogeneous target expression favored by bystander effect . 
le dt , brockstedt dg , nir - paz r , et al : a live - attenuated listeria vaccine ( anz - 100 ) and a live - attenuated listeria vaccine expressing mesothelin ( crs - 207 ) for advanced cancers : phase i studies of safety and immune induction . 
hollevoet k , nackaerts k , gosselin r , et al : soluble mesothelin , megakaryocyte potentiating factor , and osteopontin as markers of patient response and outcome in mesothelioma . 
hassan r , sharon e , thomas a , et al : phase 1 study of the antimesothelin immunotoxin ss1p in combination with pemetrexed and cisplatin for front - line therapy of pleural mesothelioma and correlation of tumor response with serum mesothelin , megakaryocyte potentiating factor , and cancer antigen 125 . 
creaney j , francis rj , dick im , et al : serum soluble mesothelin concentrations in malignant pleural mesothelioma : relationship to tumor volume , clinical stage and changes in tumor burden . 
hooper ce , lyburn id , searle j , et al : the south west area mesothelioma and pemetrexed trial : a multicentre prospective observational study evaluating novel markers of chemotherapy response and prognostication . 
onda m , nagata s , ho m , et al : megakaryocyte potentiation factor cleaved from mesothelin precursor is a useful tumor marker in the serum of patients with mesothelioma . 
iwahori k , osaki t , serada s , et al : megakaryocyte potentiating factor as a tumor marker of malignant pleural mesothelioma : evaluation in comparison with mesothel lung cancer 62 : 45 - 54 , 2008 28 . 
briefly , the mpf assay uses capture antibody mpf49 ( 2a , ) , which binds to mpf topographic epitope 3 , and detection antibody mpf25 ( 1 , ) , which binds to epitope 1.26 mpf49 was biotinylated , and mpf25 was conjugated with sulfo - tag nhs - ester ( meso - scale diagnostics , rockville , md )  . 
after incubation for 1 hour at room temperature , plates were washed , and the serially diluted mpf calibrator , 1 : 10 diluted sample serum , and reference samples were added and further incubated for 1 hour . 
the data were analyzed with workbench 4.0 software ( meso - scale diagnostic ) for determining the concentration of mpf in serum samples using the 4 parameter logistic nonlinear regression model . 
 efcacy of vemurafenib in patients with nonsmall - cell lung cancer with braf v600 mutation : an open - label , single - arm cohort of the histology - independent ve - basket study vivek subbiah , md1 ; radj gervais , md2 ; gregory riely , md , phd3 ; antoine hollebecque , md4 ; jean - yves blay , md , phd5 ; enriqueta felip , md , phd6 ; martin schuler , md7 ; anthony gonalves , md , phd8 ; antonio italiano , md , phd9 ; vicki keedy , md10 ; ian chau , md11 ; igor puzanov , md12 ; noopur s . 
raje , md13 ; funda meric - bernstam , md1 ; martina makrutzki , md14 ; todd riehl , pharmd15 ; bethany pitcher , mmath16 ; jose baselga , md , phd3 , 17 ; and david m . 
hyman , md3 , 17 purpose to study whether braf v600 mutations in nonsmall - cell lung cancer ( nsclc ) may indicate sensitivity to the braf inhibitor vemurafenib , we included a cohort of patients with nsclc in the vemurafenib basket ( ve - basket ) study . 
we present results from this cohort . methods this open - label , histology - independent , phase ii study included six prespecied cohorts , including patients with nsclc , and a seventh all - comers cohort . 
because the prespecied clinical benet endpoint was met in the initial nsclc cohort , the cohort was expanded . results sixty - two patients with braf v600mutant nsclc were enrolled and treated : 13% ( n = 8 ) had received no prior systemic therapy , and 87% ( n = 54 ) had received prior therapies . 
the safety prole of vemurafenib was similar to that observed in melanoma studies . conclusion vemurafenib showed promising activity in patients with nsclc harboring braf v600 mutations . the safety prole of vemurafenib was similar to previous observations in patients with melanoma . 
 subbiah et al context key objective to establish the efcacy and safety of vemurafenib in patients with braf v600 mutation - positive nsclc who were enrolled in the histology - independent vemurafenib basket ( ve - basket ) trial . knowledge generated vemurafenib has prolonged efcacy in patients with braf v600mutant nsclc ( n = 62 ) , as demonstrated by a 37% overall response rate . 
no new safety signals were observed in this expanded cohort of patients with nsclc . relevance single - agent vemurafenib has clinically meaningful and durable activity in patients with nsclc harboring braf v600 mutations . 
this analysis adds to the overall ndings of the ve - basket trial , which demonstrated clinically relevant activity of vemurafenib in a number of solid tumors . clinical study of patients with braf v600emutated nsclc.14 dual braf / mek inhibition has also been investigated as rstand second - line treatment of patients with nsclc.15 , 16 we present the results from the expanded nsclc cohort of the vemurafenib basket trial . 
this trial assessed the efcacy of vemurafenib in seven cohorts of patients with braf v600mutated malignancies.17 ( ve - basket ) methods study design the ve - basket study was a multicenter , single - arm , phase ii study of vemurafenib in patients with a variety of nonmelanoma cancers harboring braf v600 mutations . braf v600 mutations were identied by means of mutational analysis assays routinely performed at each participating site . 
six prespecied cohorts were recruited , consisting of patients with nsclc , ovarian cancer , colorectal cancer , cholangiocarcinoma , breast cancer , and multiple myeloma ; all patients with solid tumors other than those mentioned were included in a seventh cohort . 
the design of this study has been described in detail elsewhere.17 this trial was performed in accordance with the provisions of the declaration of helsinki and good clinical practice guidelines . 
all patients provided written informed consent . patients patients were eligible for inclusion in the study if they were 16 years of age or older and had histologically conrmed , measurable ( response evaluation criteria in solid tumors [ recist ] , version 1.1 ) , braf v600 mutation - positive cancers that were refractory to standard therapy or for which standard or curative therapy did not exist or was not considered appropriate by the investigator . 
assessments were performed using computed tomography or magnetic resonance imaging of the chest , abdomen , and pelvis at baseline and then every 8 weeks until disease progression , death , or withdrawal from the study . 
adverse events ( aes ) were graded by the investigators using national cancer institute common terminology criteria for adverse events ( version 4.0 ) until 28 days after discontinuation of study treatment . 
aes of special interest were cutaneous squamous cell carcinoma ( scc ; keratoacanthoma , squamous cell carcinoma of the skin , and bowen disease ) , fatigue ( fatigue and asthenia ) , liver injury ( increased alt , ast , blood alkaline phosphatase , blood bilirubin , and gamma - glutamyltransferase ; hyperbilirubinemia , hepatocellular injury , and cholestatic jaundice ) , and prolonged qt interval . 
patients were assessed for aes at each clinic visit and as necessary throughout the study . outcomes the primary objective of the study was to evaluate the efcacy of vemurafenib in patients with braf v600 mutation - positive cancers . 
the primary end point for the nal analysis in the nsclc cohort was objective response rate ( orr ) , dened as the proportion of patients with an objective response ( complete response [ cr ] or partial response [ pr ] ) conrmed on two consecutive occasions 4 or more weeks apart . 
 vemurafenib in nsclc with braf v600 mutation : the ve - basket study objectives included assessments of clinical benet rate ( dened as the overall proportion of patients with a cr , pr , or stable disease lasting 6 months ) , duration of response , progression - free survival ( pfs ) , overall survival ( os ) , and safety . 
efcacy data were analyzed separately for patients who had received no prior therapy and for those with prior therapies . statistical analysis this was a modied , two - stage simon design study . 
stage i was complete when seven patients with measurable disease were enrolled and had completed a minimum of 8 weeks of treatment , developed progressive disease , prematurely withdrew , or died . 
an additional six or 12 patients could be enrolled , to 13 or 19 patients , depending on the results for stage i ; if two , three , or four of the initial seven patients responded to treatment , an additional 12 patients could be enrolled in stage ii ; if ve or more of the initial seven patients responded to treatment , an additional six patients were recruited . 
recruitment into any cohort / indication could be further expanded up to 70 patients if a response rate was demonstrated in stage ii of that cohort , according to the stopping rules dened in the protocol or a clear clinical benet for patients was observed , as determined by the steering committee . 
for the nsclc cohort , with 50 treated patients , the study would have approximately 90% power for the lower bound of the twosided 95% ci to exclude 20% , given a true orr of 40% . the lower bound of the 95% ci was set at 20% because established therapy in the second and later lines had an orr of less than 20% when the study was designed . 
most patients had adenocarcinoma ( n = 58 ; 94% ) , three patients ( 4.8% ) had cns metastases , and most were former smokers ( n = 36 ; 58% )  . among previously treated patients , the median number of prior systemic regimens was two ( interquartile range [ iqr ] , 1 to 2 ) ; the most common prior chemotherapies were platinum agents ( 39 of 54 patients ; 72% ) , pemetrexed ( 33 of 54 patients ; 61% ) , and taxanes ( 22 of 54 patients ; 41% )  . all patients ( n = 62 ) previously untreated ( n = 8 ) previously treated ( n = 54 ) 65 ( 59 - 74 ) 73 ( 65 - 79 ) 64 ( 57 - 72 ) table 1 . 
overall , the investigator - determined orr was 37% ( 95% ci , 25% to 50% ) , and the clinical benet ( cr plus pr plus stable disease lasting 6 months ) rate was 48% ( 95% ci , 36% to 61% )  . 
the median relative dose intensity achieved was 78% ( iqr , 64% to 91% ) overall . all 62 patients experienced at least one any - cause ae ; grade 3 or 4 aes occurred in 48 patients ( 77% ) , and two patients had grade 5 aes ( 3% ; one patient with sepsis , one with a pulmonary embolism and respiratory failure ; both patients had been previously treated , and none of the events were considered to be related to vemurafenib )  . table 3 lists all - cause and grade 3 or greater aes occurring in 20% or more of patients . aes leading to treatment interruption occurred in 25 of 62 patients ( 40% )  . 
treatment outcomes in patients with nonsmall - cell lung cancer outcome all patients ( n = 62 ) previously untreated ( n = 8 ) previously treated ( n = 54 ) investigator - assessed best response , no . 
 vemurafenib in nsclc with braf v600 mutation : the ve - basket study - 100 previously untreated patients partial response stable disease progressive disease previously treated patients time to progression time to response died alive time ( months ) before treatment after 2 months of treatment fig 1 . 
 ( b ) waterfall plot of maximum percent decrease from baseline in the sum of diameters of target tumors on the basis of investigator assessment : best overall response in individual patients . ( c ) pretherapy and post - therapy 18f - uorodeoxyglucose positron emission tomography images of a chemotherapy - naive patient with braf v600e mutationpositive nsclc . 
at risk previously untreated ( n = 8 ) previously treated ( n = 54 ) overall ( n = 62 ) censored previously untreated ( n = 8 ) previously treated ( n = 54 ) overall ( n = 62 ) censored no . 
at risk 9 12 15 18 21 24 27 30 33 36 39 time ( months ) time ( months ) previously untreated previously treated 54 45 36 29 22 16 12 11 overall 62 52 42 34 26 18 14 12 previously untreated previously treated overall fig 2 . 
a total of 82 serious aes occurred in 39 patients ( 63% ) , the most common of which were scc of the skin ( nine patients ; 15% ) and keratoacanthoma ( nine patients ; 15% ) , which was dened as a serious ae . 
in total , 25 patients ( 40% ) had serious aes considered by the investigator to be caused by vemurafenib ( keratoacanthoma , n = 9 ; scc of the skin , n = 9 ; basal cell carcinoma , n = 1 ; bowen disease , n = 1 ; acute kidney injury , n = 4 ; pericarditis , n = 1 ; stomatitis , n = 1 ; pyrexia , n = 1 ; hypersensitivity , n = 1 ; sepsis , n = 1 ; and dehydration , n = 1 ) ; serious aes not considered to be related to vemurafenib included pneumonia ( n = 2 ) , bronchitis ( n = 2 ) , dyspnea ( n = 3 ) , pericardial effusion ( n = 1 ) , sepsis ( n = 3 ) , pulmonary embolism ( n = 2 ) , and lung infection ( n = 2 )  . targetable oncogenic drivers in nsclc with robust clinical validation include egfr mutations and alk and ros1 fusions , but identifying other targetable , clinically important subgroups of nsclc is a high priority . 
in this context , we found that patients with braf v600emutated nsclc treated with vemurafenib had an orr of 37% , with similar response rates in previously treated and untreated patients . median os was 15 months in the overall patient population , but had not been reached in the group of previously untreated patients after 12 months of follow - up . 
similarly , our previously untreated patients had a median pfs of 12.9 months , which was considerably longer than the 6.5 months observed in patients who had received prior therapies . 
the safety prole of vemurafenib in our group of patients with nsclc was similar to that seen in patients with melanoma.10 , 18 no new safety signals were observed in this population . 
we suggest that future studies should examine additional combinations in patients with braf mutation - positive nsclc . in conclusion , the results of the present cohort analysis suggest a role for braf inhibition in patients with nsclc with braf mutations . 
 vemurafenib in nsclc with braf v600 mutation : the ve - basket study affiliations 1university of texas md anderson cancer center , houston , tx 2centre franois baclesse , caen , france 3memorial sloan kettering cancer center , new york , ny 4institut gustave roussy , villejuif , france 5centre l eon b erard , lyon , france 6vall dhebron university hospital and vall dhebron institute of oncology , barcelona , spain 7university hospital essen , essen , germany 8aix - marseille universit e , marseille , france 9institut bergonie , bordeaux , france 10vanderbilt university , nashville , tn 11the royal marsden nhs foundation trust , london , united kingdom 12roswell park cancer institute , buffalo , ny 13massachusetts general hospital , boston , ma 14f . 
hyman administrative support : vivek subbiah provision of study materials or patients : vivek subbiah , gregory riely , antoine hollebecque , jean - yves blay , enriqueta felip , martin schuler , anthony gonalves , antonio italiano , igor puzanov , funda meric - bernstam collection and assembly of data : vivek subbiah , radj gervais , gregory riely , antoine hollebecque , jean - yves blay , martin schuler , antonio italiano , vicki keedy , igor puzanov , funda meric - bernstam , martina makrutzki , todd riehl , david m . 
hyman data analysis and interpretation : vivek subbiah , radj gervais , antoine hollebecque , jean - yves blay , enriqueta felip , martin schuler , anthony gonalves , antonio italiano , vicki keedy , ian chau , igor puzanov , noopur s . 
raje consulting or advisory role : amgen , celgene , takeda , novartis , bristolmyers squibb , merck , janssen oncology research funding : astrazeneca ( inst ) funda meric - bernstam honoraria : sumitomo group , dialectica consulting or advisory role : genentech , inection biosciences , pieris pharmaceuticals , clearlight diagnostics , darwin health , samsung bioepis , spectrum pharmaceuticals , aduro biotech , origimed , xencor , debiopharm group , mersana research funding : novartis , astrazeneca , taiho pharmaceutical , genentech , calithera biosciences , debiopharm group , bayer , aileron therapeutics , puma biotechnology , cytomx therapeutics , jounce therapeutics , zymeworks , curis , pzer , effector therapeutics , abbvie , boehringer ingelheim ( i ) , guardant health ( inst ) , daiichi sankyo , glaxosmithkline martina makrutzki employment : roche todd riehl employment : genentech stock and other ownership interests : genentech bethany pitcher employment : hoffmann - la roche jose baselga leadership : innity pharmaceuticals , varian medical systems , bristolmyers squibb , foghorn stock and other ownership interests : pmv pharma , juno therapeutics , grail , tango , venthera , northern biologics , apogen biotechnologies , aura biosciences consulting or advisory role : novartis patents , royalties , other intellectual property : combination therapy using pdk1 and pi3k inhibitors , use of phosphoinositide 3 - kinase inhibitors for treatment of vascular malformations , inhibition of kmt2d for the treatment of cancer travel , accommodations , expenses : roche , daiichi sankyo david m . 
n engl j med 361 : 947 - 957 , 2009 solomon bj , mok t , kim d - w , et al : first - line crizotinib versus chemotherapy in alk - positive lung cancer . 
n engl j med 371 : 1963 - 1971 , 2014 thomas a , liu sv , subramaniam ds , et al : rening the treatment of nsclc according to histological and molecular subtypes . 
medicine ( baltimore ) 96 : e6552 , 2017 paik pk , arcila me , fara m , et al : clinical characteristics of patients with lung adenocarcinomas harboring braf mutations . 
mcarthur ga , chapman pb , robert c , et al : safety and efcacy of vemurafenib in braf ( v600e ) and braf ( v600k ) mutation - positive melanoma ( brim - 3 ) : extended follow - up of a phase 3 , randomised , open - label study . 
long gv , stroyakovskiy d , gogas h , et al : dabrafenib and trametinib versus dabrafenib and placebo for val600 braf - mutant melanoma : a multicentre , double - blind , phase 3 randomised controlled trial . 
ascierto pa , mcarthur ga , dr eno b , et al : cobimetinib combined with vemurafenib in advanced braf ( v600 ) - mutant melanoma ( cobrim ) : updated efcacy results from a randomised , double - blind , phase 3 trial . 
planchard d , kim tm , mazieres j , et al : dabrafenib in patients with braf ( v600e ) - positive advanced non - small - cell lung cancer : a single - arm , multicentre , open - label , phase 2 trial . 
planchard d , besse b , groen hjm , et al : dabrafenib plus trametinib in patients with previously treated brafv600e - mutant metastatic non - small cell lung cancer : an open - label , multicentre phase 2 trial . 
planchard d , smit ef , groen hjm , et al : dabrafenib plus trametinib in patients with previously untreated brafv600e - mutant metastatic non - small - cell lung cancer : an open - label , phase 2 trial . 
 programmed death - ligand 1 expression in a large cohort of pediatric patients with solid tumor and association with clinicopathologic features in neuroblastoma purpose programmed death - ligand 1 ( pd - l1 ) expression represents a potential predictive biomarker of immune checkpoint blockade response . 
however , literature about the prevalence of pd - l1 expression in the pediatric cancer setting is discordant . methods pd - l1 expression was analyzed using immunohistochemistry in 500 pediatric tumors ( including neuroblastoma , sarcomas , and brain cancers )  . 
positive cases were further characterized , with cases with weak intensity pd - l1 staining reported as having low pd - l1 expression and cases with a moderate or strong intensity of staining considered to have high pd - l1 expression . results pd - l1positive staining was identified in 13% of cases , whereas high pd - l1 expression was found in 3% of cases . 
2017 by american society of clinical oncology introduction in recent years , the ability of tumor cells to exert an immunosuppressive action in their local microenvironment has been well described.1 escaping the host immune system occurs via multiple strategies , including loss of tumor antigens or reduced expression of major histocompatibility complex molecules , as well as via recruitment of immunosuppressive inflammatory cells or inadequate costimulation of t cells.2 as a consequence , the ability to evade host immunity has been recognized as one of the hallmarks of cancer.3 the expression of immune inhibitory ligands such as programmed death - ligand 1 ( pd - l1 ) is another strategy used by cancer cells to inhibit the immunologic response.4 pd - l1 is a trans - membrane surface glycoprotein overexpressed in many solid tumors , including carcinomas5 - 8 and melanoma.9 moreover , pd - l1 is one of the two known ligands for programmed death ( pd ) - 1 , a receptor expressed by immune system cells ( including activated t cells , regulatory t cells , b cells , natural killer cells , activated monocytes , and dendritic cells ) that negatively regulates their proliferation federica saletta ricardo e . 
antibody - mediated blockade of pd - l1 induced objective response rates in 6% to 17% and prolonged disease stabilization in 12% to 41% of patients with advanced cancers , including nonsmall - cell lung cancer , melanoma , and renal cell cancer.16 similarly , cumulative response rates for pd - 1 mab immunotherapy were achieved in 18% of patients with nonsmall - cell lung cancer , 28% of patients with melanoma , and 27% of patients with renal - cell cancer.13 despite these promising results achieved in adult cancers , the prevalence of pd - l1 expression in the pediatric setting and the potential use of mabs directed against pd - 1 have only started to be explored . 
the 53 analyzed cases included 18 neuroblastomas , eight extracranial germ - cell tumors , seven germinomas , seven hepatoblastomas , four medulloblatomas , four renal tumors , three rhabdomyosarcomas , and two atypical teratoid / rhabdoid tumors . 
in contrast , chowdhury et al20 observed 66 of 115 pd - l1positive samples ( 57% , ranging from 47% to 86% , depending on tumor type ) in a multitumor study including neuroblastoma , rhabdomyosarcoma , ewing sarcoma , and osteosarcoma . 
alveolar rhabdomyosarcoma showed the highest prevalence of pd - l1positive cases ( 12 of 14 [ 86% ] ) and patients with high - risk neuroblastoma were commonly pd - l1 positive ( 31 of 43 [ 72% ] )  . no previous study has reported associations between pd - l1 staining and the features of pediatric patients with cancer . 
here we present the clinicopathologic features of and survival analysis associated with pd - l1 expression in patients with neuroblastoma , with particular focus on the potential relationship between pd - l1 expression and the risk of cancer relapse . 
we identified a subcohort of patients with pd - l1positive staining and poor outcome , suggesting that pd - l1 may be a negative prognostic factor associated with an elevated risk of cancer recurrence . methods patient cohort the retrospective pediatric cohort consisted of 500 patients who underwent diagnostic biopsy or surgical resection at the childrens hospital at westmead , new south wales , australia . 
formalin - fixed paraffin - embedded hematoxylin and eosinstained tumor samples were reviewed by a histopathologist , and approved samples were used to construct tissue microarrays ( tmas ) using a manual tissue arrayer ( beecher mta1 , diagnostic technology , belrose , new south wales , australia ) and a 1 - mm unitma premade recipient block ( ub06 - 1 , diagnostic technology , belrose , new south wales , australia )  . 
 immunohistochemistry each tma was sectioned at 4 mm , and immunohistochemistry was performed using a bondrx automatic stainer ( leica biosystems , macquarie park , new south wales , australia )  . 
slide deparaffinization was performed with bond dewax solution , followed by alcohol dehydration . epitope retrieval was performed using hier2 buffer ( bond , ar9540 ) , and slides were incubated for 60 minutes at room temperature using a rabbit monoclonal antipd - l1 primary antibody ( e1l3n , no.13684 , cell signaling technology , genesearch , arundel , queensland , australia ) at 1 : 500 dilution in primary antibody diluent ( bond , ar9352 )  . 
staining was visualized using a polymer refine detection kit ( bond , ds9800 ) , and nuclei were counterstained with hematoxylin . tumors with > 1% of cells showing pd - l1 membrane staining ( or a combination of membrane and cytoplasmic staining ) of any intensity were considered pd - l1 positive . 
pd - l1 intensity was scored as weak , moderate , or strong by a histopathologist ( r.e.v. ) with specific expertise in pd - l1 staining assessment in patients with melanoma . 
similarly , fibrillary and stromal pd - l1 staining was also observed occasionally but was not scored positively because the significance of nonmembranous pd - l1 staining remains unclear . statistical analyses statistical analyses were undertaken using spss version 20.0 ( spss , chicago , il )  . 
bivariate relationships between pd - l1positive cases and patient features were analyzed with cross - tabulation , and significance was tested with fishers exact test . overall survival and relapse or progression - free survival were assessed using the kaplan - meier method , and significance was established using a log - rank ( mantel - cox ) test . 
statistical significance was set at p , .05 for two - sided tests . results prevalence of pd - l1positive pediatric patients with cancer as summarized in table 1 , in the overall cohort , 65 ( 13.0% ) of 500 cases were found to be pd - l1 positive . 
of these , 50 ( 76.9% ) of 65 showed weak pd - l1 intensity , whereas 15 ( 23.1% ) of 65 showed moderate to strong pd - l1 intensity . 
as such , in the overall cohort , only 15 ( 3.0% ) of 500 cases had moderate or strong pd - l1 positivity . neuroblastoma cases showed pd - l1 expression more commonly ( 48 of 254 [ 18.9% ] ) than did sarcomas or brain tumors . 
of the 48 positive cases , 40 ( 83.3% ) of 48 were weakly positive for pd - l1 , whereas eight ( 16.7% ) of 48 were moderately to strongly pd - l1 positive . 
overall , the distribution of weakly versus moderately or strongly positive pd - l1 staining in the neuroblastoma cohort was 15.7% ( 40 of 254 ) and 3.1% ( eight of 254 ) , respectively . 
in the brain tumor cohort , six ( 4.4% ) of 136 cases stained positively for pd - l1 , of which three ( 50.0% ) of six showed moderate to strong pd - l1 positivity . 
of the 96 sarcoma samples , seven ( 7.3% ) of 96 stained positively for pd - l1 , with only one of 96 cases ( an undifferentiated sarcoma ) being moderately positive . 
of the lymphoma cases , three ( 50% ) of six were pd - l1 positive ; two of these ( two of three [ 66.7% ] ) were moderately or strongly positive . 
one nasopharyngeal carcinoma showed moderate to strong pd - l1 staining . pd - l1 immunohistochemistry staining pattern observed although in this study only membranous pd - l1 staining was considered positive , together with the occasional combination of both membranous and cytoplasmic positive staining ( n = 5 ) , a range of other staining patterns was observed . 
an example of each pattern is presented in figure 1 ; they included membranous staining in the placenta as a positive control ( fig 1a ) , membranous tumor staining of weak , moderate , and strong intensity ( figs 1b , 1c , and 1d , respectively ) , as well as the combination of both membranous and cytoplasmic tumor pd - l1positive staining ( fig 1e )  . 
pd - l1 positive cytoplasmic ( fig 1f ) , fibrillary ( fig 1g ) , and schwannian stromal staining ( fig 1h ) were also observed , together with pd - l1positive macrophage ( fig 1i ) , but they were scored as negative . cytoplasmic pd - l1 staining was detected in 30 ( 11.8% ) of 254 neuroblastoma cases , ranging from weak ( n = 24 [ 80.0% ] ) to moderate or strong ( n = 6 [ 20.0% ] ) ; however , these cases were not considered pd - l1 positive . 
exclusive macrophage pd - l1 staining ( without pd - l1 positivity in tumor cells ) was detected in one ( 5.6% ) of 18 rhabdomyosarcomas , one ( 3.3% ) of 30 osteosarcomas , one ( 0.4% ) of 254 neuroblastomas , and one ( 2.6% ) of 38 lowgrade gliomas . 
the presence of macrophages was verified via hematoxylin and eosin staining observation . association with clinicopathologic features in pd - l1positive neuroblastoma cases information concerning a number of clinicopathologic features of the broader pediatric cohort was collated and tested ( using a two - sided fishers exact test ) for nonrandom associations with pd - l1 staining . 
interestingly , no differences in pd - l1 staining were found between biopsy specimens obtained before and after radiotherapy ( p = .702 ) , but a significant association between pd - l1 positivity and biopsy specimens obtained before chemotherapy was observed ( p = .044 ) , contrary to the suggestion of uehara et al.18 other clinicopathologic features that define patient prognosis in the neuroblastoma population at diagnosis include the mitosis - karyorrhexis index , shimada classification , mycn status , and overall patient risk ( table 2 )  . 
next , we sought to analyze whether pd - l1positive neuroblastoma cases were associated with cancer recurrence or progression ; however , no significant ( p = .939 ) association was observed ( fig 2b )  . 
although we did not find an association ( p = .508 ) between moderate to strong pd - l1 staining and overall survival ( fig 2c ) , the association with poorer relapse - free survival in this subcohort was significant ( p = .002 , fig 2d )  . 
programmed death - ligand 1 ( pd - l1 ) immunohistochemistry staining patterns . representative images of pd - l1 staining ( in brown ) and counterstained nuclei ( in blue )  . 
black arrows highlight examples of ( a ) pd - l1 staining in placenta used as positive control tissue ; pd - l1 membranous staining of ( b ) weak , ( c ) moderate , and ( d ) strong intensity in lymphoma ; and ( e ) pd - l1positive staining in both membrane and cytoplasm in neuroblastoma . 
 ( f ) exclusive cytoplasmic pdl1 staining and ( g ) pdl1positive schwannian stroma in neuroblastoma were considered negative . ( h ) pd - l1positive fibrillary staining in medulloblastoma and ( i ) pd - l1positive macrophages in osteosarcoma were also scored as negative . 
there were no differences in overall survival ( p = .298 ) or relapse - free survival ( p = .507 ) according to pd - l1 positivity of any intensity ( figs 3a and 3b )  . 
when restricting the analysis to the lowand intermediate - risk neuroblastoma cohort with moderate to strong pd - l1 staining , we found no association ( p = .524 ) with overall survival ( fig 3c ) but significantly ( p , .001 ) poorer relapse - free survival ( fig 3d )  . discussion since discovering that pd - 1 / pd - l1 binding promotes cancer tolerance by blocking the t - cell antigen receptor - induced stop signal , 22 substantial research effort has been aimed at targeting this interaction . 
a list of the pd - l1 antibodies and the staining and scoring methods used by other research groups is summarized in table 3 , which highlights the need for standardized immunohistochemistry methodology and a standardized scoring system for pd - l1positive cases . 
 ( a ) and ( b ) show overall survival and relapsefree survival ( in months ) in association with pd - l1 positivity ( of any intensity ) , respectively . 
 ( c ) and ( d ) show overall survival and relapse - free survival ( in months ) in association with moderate or strong pd - l1 staining intensity , respectively . 
for example , in a glioblastoma study , there was no association between fibrillary pd - l1 detection and overall survival in patients with glioblastoma.31 in our cohort , pd - l1 fibrillary staining was observed in only one of 38 medulloblastoma biopsy specimens , whereas pd - l1 schwannian stromal staining was detected in twelve of 254 patients with neuroblastoma . 
associations between programmed death - ligand 1 ( pd - l1 ) status and survival of patients with neuroblastoma ( nb ) with low and intermediate risk . kaplan - meier plots compare patient survival ( in months ) in the lowand intermediate - risk nb cohort . 
 ( a ) and ( b ) show overall survival and relapsefree survival ( in months ) in association with pd - l1 positivity ( of any intensity ) , respectively . 
 ( c ) and ( d ) show overall survival and relapse - free survival ( in months ) in association with moderate or strong pd - l1 staining intensity , respectively . 
previous reports have shown a discordant association between myc oncogene status and pd - l1 expression . in fact , casey et al36 stated that myc regulates the antitumor immune response through cd47 and pd - l1 in melanoma and nonsmall - cell lung cancer cell lines , whereas dondero et al37 showed that pd - l1 expression in neuroblastoma was interferon - gamma dependent and mycn independent . 
because strong pdl1 staining has been shown to correlate with an elevated mutation burden , 26 we can speculate that these cases may share an underlying dna repair deficit that results in an increased presence of neoantigens and particularly high pd - l1 expression . 
more studies are necessary to verify this hypothesis and to correlate mutation burden in children with stronger pd - l1 staining and a higher likelihood of cancer relapse despite favorable clinical features . pd - l1 may represent a novel predictive marker of cancer recurrence that could be used to identify the proportion of children who may benefit from pd - 1 / pd - l1 mab therapy . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . geoffrey mccowage research funding : novartis ( inst ) , merck ( inst ) acknowledgment we thank the patients and families who agreed to donate samples to the childrens hospital at westmead tumour bank . 
we also thank michael krivanek , md , of childrens hospital at westmead for his help with the histologic review of patient samples before tissue coring for tma construction , and jason madore of royal prince alfred hospital for his help in optimizing the immunohistochemistry protocol and staining interpretation . federica saletta no relationship to disclose ricardo e . 
blank c , gajewski tf , mackensen a : interaction of pd - l1 on tumor cells with pd - 1 on tumor - specific t cells as a mechanism of immune evasion : implications for tumor immunotherapy . 
karim r , jordanova es , piersma sj , et al : tumor - expressed b7 - h1 and b7 - dc in relation to pd - 1 + t - cell infiltration and survival of patients with cervical carcinoma . 
thompson rh , kuntz sm , leibovich bc , et al : tumor b7 - h1 is associated with poor prognosis in renal cell carcinoma patients with long - term follow - up . 
madore j , vilain re , menzies am , et al : pd - l1 expression in melanoma shows marked heterogeneity within and between patients : implications for anti - pd - 1 / pd - l1 clinical trials . 
freeman gj , long aj , iwai y , et al : engagement of the pd - 1 immunoinhibitory receptor by a novel b7 family member leads to negative regulation of lymphocyte activation . 
topalian sl , hodi fs , brahmer jr , et al : safety , activity , and immune correlates of anti - pd - 1 antibody in cancer . n engl j med 366 : 2443 - 2454 , 2012 14 . 
chowdhury f , dunn s , mitchell s , et al : pd - l1 and cd8 + pd1 + lymphocytes exist as targets in the pediatric tumor microenvironment for immunomodulatory therapy . 
fife bt , pauken ke , eagar tn , et al : interactions between pd - 1 and pd - l1 promote tolerance by blocking the tcr - induced stop signal . 
saletta f , wadham c , ziegler ds , et al : molecular profiling of childhood cancer : biomarkers and novel therapies . 703 - 718 , 2013 bba clin 1 : 59 - 77 , 2014 26 . 
madore j , strbenac d , vilain r , et al : pd - l1 negative status is associated with lower mutation burden , differential expression of immune - related genes , and worse survival in stage iii melanoma . 
mu cy , huang ja , chen y , et al : high expression of pd - l1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation . 
yang cy , lin mw , chang yl , et al : programmed cell death - ligand 1 expression in surgically resected stage i pulmonary adenocarcinoma and its correlation with driver mutations and clinical outcomes . 
preusser m , berghoff as , wick w , et al : clinical neuropathology mini - review 6 - 2015 : pd - l1 : emerging biomarker in glioblastoma ? clin neuropathol 34 : 313 - 321 , 2015 32 . 
taube jm , klein a , brahmer jr , et al : association of pd - 1 , pd - 1 ligands , and other features of the tumor immune microenvironment with response to anti - pd - 1 therapy . 
 exploitation of precision medicine trials data : examples of long responders from the shiva01 trial clmence basse claire morel cline callens galle pierron vincent servois anne vincentsalomon aude jobard marie alt francesco ricci delphine loirat marie - paule sablin marie bretagne mathilde saintghislain sgolne hescot anthony gonalves olivier tredan coraline dubot cline gavoille jean - pierre delord mario campone nicolas isambert lisa belin ivan bieche maud kamal christophe le tourneau author affiliations and support information ( if applicable ) appear at the end of this article . clinical trials information : nct01771458 . licensed under the creative commons attribution 4.0 license ( continued ) purpose precision medicine trials constitute a precious source of molecular data with prospective clinical annotations allowing the exploration of patients subpopulations according to specific clinical or biological questions . 
using the shiva01the first randomized trial comparing molecularly targeted therapy on the basis of tumor molecular profiling versus conventional chemotherapy in metastatic cancer patients who failed standard of care therapyannotated database , we report cases of patients treated in the trial with targeted therapy who experienced an objective response or prolonged disease stabilization in light of patients molecular alterations . patients and methods we selected all patients included in shiva01 treated with a molecularly targeted agent ( mta ) who experienced an objective response or disease stabilization that lasted longer than 6 months according to response evaluation criteria in solid tumors version 1.1. results among the 170 patients who received mtas in the shiva01 trial , 15 patients ( 9% ) experienced an objective response ( n = 3 ) or disease stabilization that lasted longer than 6 months ( n = 12 )  . 
the remaining four patients were treated with tamoxifen , erlotinib , imatinib , and vemurafenib on the basis of progesterone receptor expression , egfr amplification , kit mutation , and braf mutation , respectively . 
2018 by american society of clinical oncology introduction some molecularly targeted agents ( mtas ) have been demonstrated to dramatically improve the outcome of patients whose tumors harbor a matching molecular alteration.1 we know from the cancer genome atlas data that most of the druggable molecular alterations , including gene mutations and gene copy number alterations , exist across various tumor types , although their prevalence and functional significance may vary.2 on the basis of the latter observation , a key question has emerged : should patients with cancer be treated according to their molecular profile in a histology - agnostic way instead of by tumor type and histology , at least in the metastatic setting ? whereas nonrandomized and retrospective studies have suggested the histology - agnostic approach might be valid , 3 - 5 the shiva01 trialthe first randomized precision medicine trialdid not demonstrate a significant difference in progression - free survival ( pfs ) between matched targeted therapy and conventional treatment in patients who eventually experienced progression after standard - ofcare therapy.6 some patients who were treated in the shiva01 trial , however , seemed to benefit from targeted therapy . 
all patients who were enrolled in shiva01 previously received the standard treatment approved for their indication , including mtas , and had an eastern cooperative oncology group performance status of 0 or 1.8 within the shiva01 trial , mtas were administered according to a prespecified treatment algorithm and each mta was administered according to a matched molecular biomarker ( data supplement )  . 
techniques used included next generation sequencing ( ampliseq cancer panel on an ion torrent / pgm system ; thermo fisher scientific , waltham , ma ) for detecting mutations , cytoscan hd ( affymetrix , santa clara , ca ) for gene copy number alterations , and immunohistochemistry for hormone receptor protein expression assessment . 
we also recorded the total number of patients in shiva01 with the same tumor type treated with the same drug on the basis of the same molecular alteration . we assessed relationships between genomic alteration and clinical response using fishers exact test . 
of previous lines of treatment 24 - 71 gender male female age , years ecog performance status tumor type breast cancer cervical cancer colorectal cancer hepatocellular carcinoma nonsmall - cell lung cancer bladder carcinoma germline tumor ependymoma 6 ( 40 ) 9 ( 60 ) 2 ( 13 ) 7 ( 47 ) 3 ( 20 ) 3 ( 20 ) 4 ( 27 ) 11 ( 73 ) 4 ( 27 ) 3 ( 20 ) 1 ( 6.6 ) 1 ( 6.6 ) 1 ( 6.6 ) 1 ( 6.6 ) 1 ( 6.6 ) 1 ( 6.6 ) 1 ( 6.6 ) 1 ( 6.6 ) head and neck squamous cell carcinoma gastroesophageal carcinoma abbreviation : ecog , eastern cooperative oncology group . longer than 6 months , seven patients ( 47% ) had a molecular alteration that involved the hormone receptor pathway ( table 2 )  . 
six patients who were treated with abiraterone had androgen receptor ( ar ) protein expression that ranged from 40% to 100% , including three patients with breast cancer , one patient with hepatocellular carcinoma , and one patient with bladder cancer ( table 2 )  . 
the remaining patient with gastroesophageal cancer and a 30% progesterone receptor expression was treated with tamoxifen . five patients ( 33% ) who were treated with everolimus had a molecular alteration involving the phosphatidylinositol 3 - kinase ( pi3k ) / akt / mammalian target of rapamycin ( mtor ) pathway . 
among these patients , three patients cervical cancer , head and neck , and germline cancerhad a phosphatase and tensin homolog ( pten ) heterozygous deletion associated with a loss of protein expression . 
the two remaining patients consisted of one patient with cervical cancer with a pik3ca mutation ( glu542lys ) and another with breast cancer with both pten loss and pik3ca mutation ( glu545lys )  . 
proportion of patients treated with molecularly targeted therapy in shiva01 who achieved an objective response or disease stabilization lasting longer than 6 months molecularly targeted agent tumor type abiraterone breast cancer no . 
of patients in shiva01 with the same tumor type treated with the same drug bladder cancer ependymoma breast cancer cervical cancer germline tumor hnscc cervical cancer colorectal cancer gastroesophageal cancer nsclc everolimus erlotinib vemurafenib tamoxifen imatinib total abbreviations : hcc , hepatocellular carcinoma ; hnscc , head and neck squamous cell carcinoma ; nsclc , nonsmall - cell lung cancer . than a single patient with the same tumor type and were treated with the same mta matching an alteration in a specific signaling pathway . 
in total , 22 patients with breast cancer were treated with abiraterone , 12 patients with breast cancer with everolimus , and 10 patients with cervical cancer with everolimus ( table 3 )  . 
 within these three larger cohorts , the proportion of patients with an objective response or disease stabilization that lasted longer than 6 months varied from 8% to 20% ( table 3 )  . 
 taken together , 15 ( 28% ) of the 53 patients in these cohorts achieved an objective response or disease stabilization that lasted longer than 6 months ( table 3 )  . focus on patients treated with everolimus . 
theonly patient with breast cancer treated with everolimus who experienced a complete response had a pik3ca mutation ( glu545lys ) , as well as a kras mutation ( ala146thr ) , associated with heterozygous deletions in the atm , brca2 , cdh1 , pten , rb1 , smad4 , and tp53 genes ( table 2 and data supplement )  . 
of the 11 nonresponder patients with breast cancer , seven received everolimus on the basis of a pik3ca mutation ( 3 his1047arg , 2 glu545lys , and 2 glu542lys ) and four received everolimus on the basis of pten inactivation . 
one patient had a kras mutation ( gly12val ) , and five patients had a tp53 mutation ( data supplement )  . the two patients with cervical cancer treated with everolimus who achieved disease stabilization that lasted longer than 6 months had either a pik3ca mutation ( glu542lys ) or a pten heterozygous deletion with a loss of protein expression ( table 2 and data supplement )  . 
among the eight nonresponder patients with cervical cancer , four received everolimus on the basis of pik3ca mutation ( glu545lys ) , two on the basis of pten inactivation , and two on the basis of akt1 mutation ( glu17lys )  . 
in total , among the three patients who achieved an objective response or disease stabilization that lasted longer than 6 months , all had a double alteration affecting the pi3k / akt / mtor pathway . 
none of the four patients who experienced an objective response or disease stabilization that lasted longer than 6 months and for whom a complete molecular profile was available had a mutation in the tp53 gene compared with 10 ( 31% ) of 32 nonresponders with a complete molecular profile ( p = .47 ; data supplement )  . impact of molecular alteration burden in resistance to treatment . 
a median of five molecular alterations ( range , three to eight ) were detected in three patients treated with everolimus who experienced an objective response or disease stabilization that lasted longer than 6 months compared with a median of six alterations ( range , two to 21 ) in 19 nonresponder patients . 
a median of three molecular alterations ( range , one to eight ) were found in three patients treated with abiraterone who experienced an objective response or disease stabilization that lasted longer than 6 months compared with a median of five alterations ( range , one to 17 ) in 19 nonresponder patients . 
 the total number of molecular alterations was not significantly associated with resistance to treatment ( p = .41 ; data supplement )  . discussion using the shiva01 trials annotated database , we here report 15 patients ( 9% ) who experienced an objective response or prolonged disease stabilization among the 170 patients with any kind of cancer treated with an mta outside its indication in the shiva01 trial . 
the total number of molecular alterations was not significantly associated with resistance to treatment , which suggests that other mechanisms that require additional investigation are involved . patients experiencing an objective response or a disease stabilization that lasted longer than 6 months were less heavily pretreated than the entire patient population that was included in the shiva01 trial.6 this result is in agreement with several reports in the literature that suggest that the sooner mtas are administered in the course of the disease , the higher the efficacy.1 , 10 in the two largest cohorts in the shiva01 trial treated with everolimus or abiraterone , patients who experienced an objective response or disease stabilization that lasted longer than 6 months had fewer molecular alterations than nonresponder patients . 
efficacy of mtas administered in a manner similar to that of shiva01 correlated with the matching score.11 , 12 in agreement with a recent report , three of 22 patients with breast cancer expressing ar and treated with abiraterone experienced disease stabilization that lasted longer than 6 months.13 one patient with hepatocellular carcinoma , one with bladder carcinoma , and one with ependymoma treated with abiraterone in shiva01 experienced disease stabilization that lasted longer than 6 months ; however , no evidence of efficacy of antiandrogens in these cancer types has been reported in the literature.14 - 17 one patient with gastroesophageal carcinoma treated with tamoxifen on the basis of progesterone receptor expression had an 18 - month disease stabilization in the shiva01 trial . 
antitumor activity of tamoxifen in hormone receptors expressing gastric cancer cells has been reported.14 to date , no clinical trials have been reported , to our knowledge , evaluating this strategy in the clinic . all three patients treated with everolimus who achieved an objective response or disease stabilization that lasted longer than 6 months had a double alteration affecting the pi3k / akt / mtor pathway , whereas less than one half of nonresponder patients with breast and cervical cancer had a double alteration involving that pathway . 
thirty - five percent of patients with breast cancer with an alteration in the pi3k / akt / mtor pathway who were treated with various therapies that inhibited the pi3k / akt / mtor pathway in the safir01 study had an objective response or pfs that lasted longer than 16 weeks.16 the difference in terms of efficacy observed in safir01 might be related to the 16 - week threshold used for prolonged disease stabilizationinstead of the 6 months in our studyand that various drugs used sometimes in combination , including direct pi3k inhibitors in safir01 . 
in a phase ii trial that evaluated everolimus in unselected patients with refractory testicular germcell tumors , no objective response was observed with a pfs rate at 3 months of 40%.18 finally , three patients in shiva01 seemed to benefit from mtas that targeted epidermal growth factor receptor , kit , and braf mutations , which are clinically validated targets in other tumor types.19 - 22 of interest , the patient with braf v600emutated colorectal cancer experienced an unusual response to vemurafenib . 
partial responses to vemurafenib in patients with braf v600emutated colorectal cancer have previously been reported in the literature.23 the absence of tp53 mutations and pi3k pathway alterations in these patients may explain the partial response to vemurafenib , 24 although this remains speculative . in conclusion , the design of the shiva01 trial has several limitations . 
second , the treatment algorithm was unidimensional and did not account for resistance mechanisms.9 third , heavily pretreated patients were included in the trial , which reduced the likelihood that mtas might be effective . 
despite these caveats , shiva01 allowed for the integration of clinically annotated molecular data that were used to analyze patients with unusual responses to mtas.6 the exploitation of clinically annotated molecular data from precision medicine trials is clearly useful to pinpoint potential biomarkers of interest in assessing sensitivity or resistance to mtas . 
whereas an objective response with a single - agent mta clearly indicates treatment efficacy , using prolonged disease stabilization as a criterion is questionable.25 the example of the long - responder patients analysis , although bearing the above limitations , highlights the precious information that could be inferred from precision medicine trials data analyses . 
 many lessons could definitely be learned using this approach : the possibility to focus on patients subpopulation with specific clinical or molecular questions within the same prospective study ( in our case , patients experiencing an objective response or prolonged disease stabilization following treatment within the shiva01 trial ) ; the accessibility of centralized molecular data obtained using the same techniques and bioinformatics pipelines and thus avoiding multiple sites biases ; the identification of potential biomarkers of interest depending on the question asked ( in our case , global number of molecular alterations , several alterations in the same pathways , or tp53 mutations ) that required additional validation in independent cohorts ; and the importance of sharing data with other precision medicine clinical trials to enlarge specific subpopulation or to validate results . 
tumor mutational burden and microsatellite instability have been suggested as potential biomarkers of efficacy for immune checkpoint inhibitors.26 - 28 it remains to be determined how the incorporation of immunotherapy in shiva01 would have affected the results . 
anr - 10 - eqpx - 03 from the agence nationale de le recherche ( investissements davenir ) and site de recherche intgr contre le cancer inca - dgos - 4654 . 
slamon dj , leyland - jones b , shak s , et al : use of chemotherapy plus a monoclonal antibody against her2 for metastatic breast cancer that overexpresses her2 . 
von hoff dd , stephenson jj jr , rosen p , et al : pilot study using molecular profiling of patients tumors to find potential targets and select treatments for their refractory cancers . 
tsimberidou am , wen s , hong ds , et al : personalized medicine for patients with advanced cancer in the phase i program at md anderson : validation and landmark analyses . 
massard c , michiels s , fert c , et al : high - throughput genomics and clinical outcome in hardto - treat advanced cancers : results of the moscato 01 trial . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
belin l , kamal m , mauborgne c , et al : randomized phase ii trial comparing molecularly targeted therapy based on tumor molecular profiling versus conventional therapy in patients with refractory cancer : cross - over analysis from the shiva trial . 
migliardi g , sassi f , torti d , et al : inhibition of mek and pi3k / mtor suppresses tumor growth but does not cause tumor regression in patient - derived xenografts of ras - mutant colorectal carcinomas . 
bonnefoi h , grellety t , tredan o , et al : a phase ii trial of abiraterone acetate plus prednisone in patients with triple - negative androgen receptor positive locally advanced or metastatic breast cancer ( ucbg 12 - 1 )  . 
kameda c , nakamura m , tanaka h , et al : oestrogen receptor - alpha contributes to the regulation of the hedgehog signalling pathway in eralpha - positive gastric cancer . 
geiger jl , bauman je , gibson mk , et al : phase ii trial of everolimus in patients with previously treated recurrent or metastatic head and neck squamous cell carcinoma . 
andr f , bachelot t , commo f , et al : comparative genomic hybridisation array and dna sequencing to direct treatment of metastatic breast cancer : a multicentre , prospective trial ( safir01 / unicancer )  . 
andr f , hurvitz s , fasolo a , et al : molecular alterations and everolimus efficacy in human epidermal growth factor receptor 2 - overexpressing metastatic breast cancers : combined exploratory biomarker analysis from bolero - 1 and bolero - 3 . 
schilder rj , sill mw , lee yc , et al : a phase ii trial of erlotinib in recurrent squamous cell carcinoma of the cervix : a gynecologic oncology group study . 
santin ad , sill mw , mcmeekin ds , et al : phase ii trial of cetuximab in the treatment of persistent or recurrent squamous or non - squamous cell carcinoma of the cervix : a gynecologic oncology group study . 
goncalves a , fabbro m , lhomm c , et al : a phase ii trial to evaluate gefitinib as secondor third - line treatment in patients with recurring locoregionally advanced or metastatic cervical cancer . 
lu hy , zhang g , cheng qy , et al : expression and mutation of the c - kit gene and correlation with prognosis of small - cell lung cancer . 
mao m , tian f , mariadason jm , et al : resistance to braf inhibition in braf - mutant colon cancer can be overcome with pi3k inhibition or demethylating agents . 
le tourneau c , paoletti x , coquan e , et al : critical evaluation of disease stabilization as a measure of activity of systemic therapy : lessons from trials with arms in which patients do not receive active treatment . 
 mendel goldnger ramaswamy govindan jhanelle gray stacy gray samantha greenberg malachi grifth * roman groisberg * torsten haferlach michael hall * anis hamid * heather hampel * fei han sigurdis haraldsdottir * derrick haslem * eric haura masanori hayashi brian henick * , * * daniel herchenhorn * kenneth hess j . 
kevin hicks kim hirsheld brian hobbs howard hochster * daanish hoda * ahmed hosny david hsu wenhui huang yi huang * hatim husain maha hussain * david hyman * alexia iasonos * meredith irwin * steven isakoff katherine janeway * yelena janjigian lingyun ji xuemei ji * * yuchao jiang markus joerger steven joffe * thomas kaley mohammed kashanisabet steve katz kevin kim david kirsch kedar kirtane eric klein samuel klempner * todd knepper wendy kohlmann * jill kolesar * ian krop * priti kumari shivaani kummar ava kwong andrea laghi * jacky wai - kei lam angela lamarca jean - michel lavoie rachel layperson * christophe le tourneau * amy leblanc j . 
jack lee * jochen lennerz * antonio lerario benjamin levy mark lewis * jessica lin * steven lipkin jingyi liu * minetta liu * stephen liu * christine lovly ying lu * luke maese * david malkin diana mandelker sharon manne aaron manseld veronica mariotti thomas martin carlotta masciocchi joaquin mateo * shigeyuki matsui * joan maurel * shannon mccall jeannine mccune * rose mcgee * funda meric - bernstam everett meyer * ropa mhlanga * * luca mologni clara montagut * lucas moreno * daniel morgenstern * motomi mori * kent mouw anwesha nag katherine nathanson daniel nava rodrigues stewart neill kim nichols donna niedzwiecki nicola normanno * david norris paolo nuciforo jennifer oberg * stephen oh * coral omene stephen opat eileen oreilly * piet ost geoffrey oxnard * sukhmani padda tuya pal jose palacios phillip palmbos wei pan nickolas papadopoulos * soo park jai patel shiven patel * cloud paweletz andrew pearson raquel perez - lopez * thomas permutt * emanuel petricoin * gina petroni * david piccioni gaelle pierron filippo pietrantonio zoa piotrowska sharon plon eric polley mei - yin polley * erqi pollom mark pomerantz steven powell * colin pritchard * kanwal raghav * nitya raj w . 
 o genomic profiling of t - cell neoplasms reveals frequent jak1 and jak3 mutations with clonal evasion from targeted therapies purpose the promise of precision oncology is that identification of genomic alterations will direct the rational use of molecularly targeted therapy . 
in this study , we tested the feasibility of targeted next - generation sequencing in profiles of diverse t - cell neoplasms and focused on the therapeutic utility of targeting activated jak1 and jak3 in an index case . patients and methods using foundation one and foundation one heme assays , we performed genomic profiling on 91 consecutive t - cell neoplasms for alterations in 405 genes . 
the samples were sequenced to high uniform coverage with an illumina hiseq and averaged a coverage depth of greater than 500 for dna and more than 8m total pairs for rna . 
t - pll cells were analyzed by rna - seq , in vitro drug testing , mass cytometry , and phospho - flow . results one third of the samples had genomic aberrations in the jak - stat pathway , most often composed of jak1 and jak3 gain - of - function mutations . 
adult patients with precursor neoplasms , such as acute t - cell lymphoblastic leukemia ( t - all ) , or with mature neoplasms , such as t - cell non - hodgkin lymphoma ( t - nhl ) , have a 5 - year survival rate of 20% to 30% even after intensive multiagent chemotherapy.1 - 6 there are rare exceptions to these dismal outcomes , such as children and adolescents who have t - all or anaplastic large - cell lymphoma ( alcl ) with unique gene rearrangements ( ie , alk positivity or dusp22 positivity ) in whom 5 - year survival rates are greater than 70% to 80% with similar chemotherapy regimens.5 , 7 , 8 however , relapsed disease is challenging to cure . 
clearly , novel therapeutic approaches are needed , and the development of commercially available next - generation sequencing has raised the possibility that genomically directed therapy may be applied to t - cell leukemias and lymphomas . 
genomic profiling has been performed on several histopathologic subtypes of t - cell leukemias and lymphomas to better characterize the molecular genetics.9 - 13 interestingly , recent genomic profiling has discovered frequent allison greenplate kai wang rati m . 
 aberrations within the janus kinase ( jak ) signal transducer and activator of transcription ( stat ) pathway in both precursor ( t - all ) and mature ( t - nhl ) t - cell neoplasms , which suggests that jak kinase inhibition may be important therapeutically.14 jaks are encoded by four paralogous genes , jak1 , jak2 , jak3 , and tyk2 . 
jak1 mutations have been found in 10% of childhood t - alls.15 our laboratory and others have found jak3 mutations in cutaneous t - cell lymphoma ( ctcl ) , adult t - cell leukemia / lymphoma ( atll ) , t - cell prolymphocytic leukemia ( t - pll ) , and natural killer / t - cell lymphoma ( nktl ) .16 - 20 analyses of human leukemia lines and mouse models show that jak mutations typically are activating and cause constitutive signal transduction , which may be blocked by tyrosine kinase inhibitors . 
ruxolitinb is approved for use in myeloproliferative neoplasms , and tofacitinib is approved for rheumatoid arthritis.21 , 22 in this study , we deployed a commercially available hybrid - capture / next - generation sequencing platform to characterize major recurrent oncogene and tumor suppressor aberrations in 91 t - cell neoplasms . 
this targeted approach found that 33% of samples had jak - stat abnormalities , which included missense mutations in jak1 and jak3 , rearrangements in jak2 and jak3 , and missense mutations and amplifications of stat3 and stat5 . 
we analyzed an index case of t - pll , a deadly mature t - cell neoplasm with both jak1 and jak3 gain - of - function missense mutations.23 this patient with t - pll had experienced progression during multiple lines of chemotherapy but experienced disease response with ruxolitinib , a jak1 / 2 inhibitor . 
adaptor ligated libraries were created from dna and cdna as described.24 libraries were sequenced on illumina hiseq2500 ( illumina , san diego , ca ) to > 500 coverage depth for dna and > 8m total pairs for rna . 
dna and rna sequence data were processed with a customized analysis pipeline designed to accurately detect multiple classes of genomic alterationsspecifically , base substitutions , indels , focal gene amplifications , homozygous gene deletions , gene fusions , and genomic rearrangements ( data supplement ) .24 gene expression the rna sequencing workf low has been described previously . 
a description also is in the data supplement.26 phosphatase enzyme assay and inhibitor studies frozen preand post - treatment t - pll cells and jurkat cells were lysed in hypotonic buffer and prepared with cytosol , as described.27 ruxolitinib and tofacitinib were purchased from selleck chemicals ( houston , tx ) , and working solutions were prepared in dimethyl sufoxide ( dmso )  . 
cell viability was quantified with a cyquant assay ( invitrogen , carlsbad , ca ) , as described.17 , 27 statistical analyses were performed with graphpad prism ( la jolla , ca )  . results genomic profiling of t - cell leukemias and lymphomas we analyzed 91 occurrences of diverse t - cell leukemias and lymphomas for alterations in 405 cancer - causing genes by comprehensive hybrid capture of genomic dna followed by next generation sequencing ( data supplement )  . 
the cohort showed a marked male predominance for both immature and mature t - cell neoplasms ( 4 : 1 and 1.75 : 1 , respectively ; data supplement ) consistent with the epidemiology of these cancers.28 , 29 samples were sequenced at a mean exon depth of 489 by illumina hiseq . 
stat3 mutations ( 0% v 18% , p = .032 ) were more common in mature t - cell neoplasms ( table 1 ) ; 31.8% ( 29 of 91 samples ) had alterations in the jak - stat pathway . 
 jak3 , which involved 13% ( 12 of 91 samples ) of samples , was the most commonly mutated kinase , followed by jak1 , which was mutated in 7.7% ( seven of 91 samples )  . 
jak3 mutations were present with a similar frequency in t - all and in mature t - cell neoplasms ( fisher 's exact test , p = .19 ) but were highly frequent in certain mature t - cell neoplasms , such as t - pll ( five [ 71% ] of seven samples )  . 
interestingly , all five occurrences of t - pll had the m511i jak3 mutation ; all of the jak3 and jak1 mutations were in the catalogue of somatic mutations in cancer ( cosmic ; ac.uk / cosmic ) database , frequently within the pseudokinase domains of the proteins ( fig 2 ) .30 jak3 missense mutations have been identified previously in ctcl and atl.16 focal stat3 , stat5a , and stat5b amplifications were identified in two occurrences ( n = 1 each in ctcl and alcl ) , in addition to missense mutations within the sh2 domains of stat3 and stat5b , such as stat3 d661y / v in t - lgl ( fig 2 )  . 
jak1 and jak3 mutations were observed in the same tumor in five occurrences , but concordant mutations in the same cell could not be confirmed ( data supplement )  . 
 jak3 mutations were concordant with mutations in tp53 in four samples ; notch1 mutations , in four samples ; and cdkn2a deletions , in three samples ( data supplement )  . exceptional response of a jak1 - mutant t - pll we evaluated a 62 - year - old woman in our clinic with relapsed t - pll . 
she presented with constitutional symptoms , splenomegaly , and leukemic blood counts that had increased to greater than 150 , 000 / l and progressed through alemtuzumab ; cyclophosphamide , doxorubicin , vincristine , and prednisone ( chop ) ; romidepsin ; and pralatrexate . 
the t - pll cells were cd2 + cd3 + cd4 + cd7 + cd8cd56cd57 ( fig 3a ) , had clonal t - cell receptor rearrangement ( data not shown ) , and had infiltration of bone marrow ( figs 3b through 3e )  . 
 a cdkn2a / b substitution / indel gene amplification gene homozygous deletion truncation gene fusion / rearrangement tet2 jak3 tp53 stat3 jak1 stat5b stat5a cdkn2a / b tet2 notch1 jak3 tp53 stat3 nras dnmt3a jak1 rhoa mll2 arid1a stat5b fbxw7 abl1 traf3 stat5a stag2 runx1 pik3r1 cd36 tnfaip3 smarcb1 smarca4 setd2 pten pik3ca phf6 pask nup214 ncor1 kras jak2 idh2 hist1h1d etv6 crebbp1 ciita cdkn1b ccnd3 card11 brca2 bcor asxl1 genetic aberration t - all cdkn2a / b tet2 notch1 jak3 tp53 stat3 nras dnmt3a jak1 rhoa mll2 arid1a stat5b fbxw7 abl1 traf3 stat5a stag2 runx1 pik3r1 cd36 tnfaip3 smarcb1 smarca4 setd2 pten pik3ca phf6 pask nup214 ncor1 kras jak2 idh2 hist1h1d etv6 crebbp1 ciita cdkn1b ccnd3 card11 brca2 bcor asxl1 t - cell neoplasm subtype fig 1 . 
 , alpha helical domain , a dna binding domain ; ferm , the conserved domain named for its founding members ( band 4.2 , ezrin , radixin , and moesin ) ; jh1 , jak homology domain 1 , the functional kinase domain ; jh2 , jak homology domain 2 ( also known as the pseudokinase domain ) ; sh2 , src homology domain 2 ; stat - int , stat interaction domain . jak3 ferm 1000 1125 jak1 ferm 1000 1155 stat3 stat - int ( cid : 68 ) dna - binding 11 1213 14 15 18 19 22 23 stat5b stat - int dna - binding 17 18 ( cid : 68 ) and 78% , respectively , which suggests loss of heterozygosity ( data supplement )  . 
both jak1 and jak3 mutations had been described in hematologic malignancies and have proven to be oncogenic in various assays.20 , 30 - 32 the clonal jak1 mutation could be targeted by ruxolitinib , a kinase inhibitor with activity against jak1 / 2.21 the patient was agreeable to this off - label therapy and received 20 mg twice daily ; her peripheral count ( 90% t - pll ) declined from 142 , 000 / l to 85 , 000 / l within 5 days . 
during the 100 days before ruxolitinib therapy , the patient required 10 apheresis units of platelets and 7 units of packed red blood cells ; conversely , during ruxolitinib therapy , she received 3 apheresis units of platelets and 2 units of packed red blood cells . 
unfortunately , by day 116 , the leukemic blood count increased to 116 , 000 / l , and a bone marrow biopsy confirmed relapsed disease ( fig 3g )  . 
subtype of each individual sample : angioimmunoblastic t - cell lymphoma ( aitl ) ; anaplastic large - cell lymphoma ( alcl ) ; cutaneous t - cell lymphoma ( ctcl ) ; natural killer / t - cell ( nkt ) lymphoma ; peripheral t - cell lymphoma ( ptcl ) ; t - all ; t - cell large granular leukemia ( t - lgl ) ; and t - cell prolymphocytic leukemia ( t - pll )  . 
 genetic and immunophenotypic analysis of resistance to ruxolitinib therapy at relapse , the jak3 m511i allele frequency had increased from 5% to 28% , whereas the allele frequency of jak1 v658f had decreased from 40% to 18% in the t - pll cells ( fig 3f )  . 
the pre and post - ruxolitinib t - pll cells were analyzed by rna sequencing , in which the steady state abundance of mutant mrnas approximated the allele frequencies ( data supplement )  . 
the multidimensional staining pattern was analyzed by visne , an algorithm that maps cells on to a two - dimensional plot.33 peripheral - blood mononuclear cells from healthy donors were stratified into distinct cell populations that corresponded to specific lineages : cd4 + / cd8 + t cells , natural killer cells , macrophages , and b cells ( fig 4a )  . 
the t - pll cells from the patient case were clustered into a unique island composed of 95.3% total peripheral cells and few nonmalignant cell types ( fig 4a )  . 
 the cd45lo / population showed increased cd27 ( tnfrsf7 ) , cd44 ( h - cam ) , ccr4 , and ccr7 and reduced cd43 ( leukosialin ) compared with preor post - treatment cd45 + cells ( figs 4c through 4d )  . cd45 is a receptor tyrosine phosphatase encoded by the ptprc gene that negatively regulates jak - stat and t - cell receptor signaling.34 , 35 cd45 downregulation coincident with clinical relapse on ruxolitinib implied that it may be a mechanism for ruxolitinib resistance . 
the jak1 mutation was not detectable by sanger sequencing , but the jak3 mutation was clonal and present in cd45hi , cd45intermediate , and cd45 cells ( figs 5a and 5b )  . 
whole - transcriptome analysis on preand post - treatment samples found that ptprc mrna abundance was reduced significantly in the relapsed sample by 1.95 - fold ( p = 3.18e74 ; fig 5d ) , which approximates the 50% reduction in protein levels observed by flow cytometry . 
 jak1 and jak3 were probed by western blot analysis of whole - cell lysates and showed lower protein abundance in the relapsed sample compared with lysates before ruxolitinib treatment ( fig 5c )  . next , the tyrosine phosphatase activity was analyzed in lysates prepared from t - pll cells pre and post - ruxolitinib / relapse therapy . 
immunodepletion with a specific antibody against cd45 reduced total tyrosine phosphatase activity to 20% to 22% of normal , which confirmed that cd45 was the major enzyme to contribute to this enzyme activity in t - pll cells ( fig 5e )  . 
 ( a ) dot plots show the immunophenotype of the t - pll cells at presentation as analyzed by flow cytometry ; t - pll cells were interpreted as tdt cd1 cd3 + cd5 + cd7 + cd4 +  . 
 ( g ) plot of the total leukocytes for the patient ; y - axis shows cells number ( 103 ) ; gray brackets show the normal range of peripheral leukocyte number . 
dark blue arrows , time points of physical exams that show a palpable spleen 4 cm below costal margin before ruxolitinib therapy ; spleen mass was not palpable after treatment . 
 ( h ) bar graphs of t - pll cells treated in vitro with varying concentrations of ruxolitinib ( left panel ) or tofacitinib ( right panel ) in molar ( m ) quantities ; the y - axis shows cell viability . 
 peripheral - blood samples from a healthy donor and the patient with t - pll , before ruxolitinib ( pretreatment ) and at relapse ( post - treatment ) were analyzed by mass cytometry after staining for 28 cell surface markers . 
 ( a ) visne analysis performed on the data to allow grouping of peripheral leukocytes into distinct islands that correspond to ( b ) cell lineages ; iridium ( dna intercalator ) - positive ( ir + ) events show those cells that were intact . 
 ( a ) the visne map shows cell density for all ir + ( nucleic dna intercalator ) cells from a healthy donor and in pretreatment and post - treatment / relapse cells . 
 ( a - c ) flow cytometry dot plots show ( a ) staining patterns of t - cell prolymphocytic leukemia ( t - pll ) cells before ( pre - rx ) and after ( post - rx ) ruxolitinib treatment / relapse for forward scatter ( fsc ) and side scatter ( ssc ) , 7 - aminoactinomycin d ( 7 - aad ) , and anti - cd45 . 
three distinct populations were noted on the basis of cd45 expression ; ( b ) cells were flow sorted , extracted for genomic dna , subjected to polymerase chain reaction for relevant exons , and sequenced . 
left panel , rpkm for t - pll , reads per kilobase of gene per million reads ; right panel , rpkm normalized to pre - rx reads ( set at 100% )  . 
 ( e ) bar graphs show measured in vitro phosphatase activity from cytosolic lysates prepared from t - pll cells before ( blue ) and after ( gold ) ruxolitinib . 
the third set of assays was done after incubation with anti - cd45 / protein a / g ; values were normalized to cytosolic lysates before immunodepletion ( set at 100% ) ; error bars show standard error of the mean from quadruplicates . 
 time of disease relapse after treatment with combination cyclophosphamide , doxorubicin , vincristine , and prednisone chemotherapy ( data supplement )  . enhanced phosphorylations downstream of jak1 / jak3 at relapse cd45 has been shown to directly dephosphorylate jak1 and jak3 , which suggests that its loss of function should show increased activity of jak3 or jak1 . 
to assess whether t - pll cells had increased sensitivity to cytokine stimulation in addition to increased basal signaling , we stimulated each sample with 20 ng / ml of cytokine for 15 minutes . 
increased phosphorylation of stat5 was seen after in vitro stimulation with il - 2 , il - 4 , il - 7 , il - 21 , and interferon gamma , but not with il - 9 , in both preand post - ruxolitinib / relapsetreated samples compared with healthy t cells ( fig 6d )  . 
a comparison between preand post - ruxolitinib / relapse t - pll cells showed similar p - stat5 levels at baseline and after stimulation by cytokines ( fig 6d )  . 
for example , there was an il - 2induced 1.49 - fold change in p - stat5 for pre - ruxolitinib samples and 1.44 - fold ( comparison of arcsine transformed raw values ) change for post - ruxolitinib / relapse samples . 
to understand the effects of cd45 expression , we gated on cd45hi , cd45lo , and cd45 post - ruxolitinib / relapse t - pll cells and analyzed p - stat5 and p - stat6 basally and in response to stimuli . 
 cd45hi had the lowest p - stat5 response to il - 2 ( 0.23 - fold ) , followed by cd45lo ( 0.61 - fold ) and cd45 ( 1.24 - fold ; fig 6d ) ; this pattern was seen for all common gamma chain cytokines compared with control ( p - stat6 ; figs 6d and 6e )  . 
in summary , t - pll cells were hyper responsive to common gamma chain cytokines , and cd45 expression was negatively correlated with p - stat5 at relapse . discussion in this study , diverse t - cell neoplasms were profiled by targeted next - generation sequencing of the exomes from approximately 400 known tumor suppressors and oncogenes . 
the cohort was composed of occurrences submitted to foundation medicine as a result of relapsed or resistant disease , so the observed genetic alterations may be specific to advanced - stage disease or therapeutic resistance . 
nevertheless , the mutation frequencies for jak3 ( 13% of occurrences ) and for jak1 ( 8.7% ) were consistent with genomic profiling studies focused on specific disease subtypes ( ie , t - all , atll , t - pll , and ctcl15 - 19 ; less often , aitl and ptcl ) .38 , 39 the jak mutations in this study were mutually exclusive with stat3 and stat5 gene alterations , as expected , because stat3 and stat5 proteins are downstream of il2rg ( common gamma chain ) / jak1 / jak3restricted cytokines . 
interestingly , recent data in cell lines and mouse models suggest that jak1 enzyme activity is required for mutant jak3 effects.40 - 42 however , as in the index case , the mutations were co - occurring but not present in the same cell ; allele frequencies approached 50% for jak1 and jak3 mutations in the same tumor . most important , the jak1 and jak3 mutations were functionally significant , because they induced constitutive phosphorylations of downstream stat5 proteins . 
p - stat3 was not observed in the t - pll cells ( data not shown ) , although it is an important downstream substrate in other t - cell neoplasms , such as alcl.43 thus , signaling patterns downstream of jak1 / jak3 may be unique to disease subtype . 
stat1 and stat5 phosphorylations in t - pll could be inhibited by the specific jak inhibitors ruxolitinib and tofacitinib.44 in the index case , treatment with ruxolitinib induced an impressive clinical response . 
intracellular signaling responses , monitored by using phospho - flow cytometry , of peripheral - blood mononuclear cells ( pbmcs ) from healthy donors , and from the patient with t - pll before ( pretreatment ) and after ( post - treatment ) relapse experienced during ruxolitinib treatment . 
 ( d ) graphs show p - stat5 and p - stat6 for health donor t cells , pretreatment t - pll , and post - treatment t - pll unstimulated or after 15 minutes of cytokine stimulation at 20 ng / ml . ( e ) similar graphs of cytokine - stimulated p - stat5 and p - stat6 for post - treatment t - pll cells with cd45hi , cd45lo , or cd45neg gating . 
furthermore , this oral drug worked when intensive parenteral therapies had failed to control the disease . this study showed two cell - intrinsic mechanisms to account for resistance to ruxolitinib : expansion of the mutant jak3 t - pll clone and downregulation of cd45 . 
in in vitro studies , the ic50 of ruxolitinib for jak1 is 3.3 nm ; for jak2 , it is 2.8 nm45 ; and for jak3 , it is 428 nm.45 this diminished potency against jak3 probably accounted for the expansion of the mutant jak3 clone from 10% before ruxolitinib to 56% at relapse . 
the t - pll cells showed cross - resistance to tofacitinib , which has a nanomolar ic50 for jak3.46 , 47 the downregulation of cd45 protein appears to be an additional resistance mechanism at relapse . 
the expression pattern of cd45 in a clonal t - pll population resembles position effect variegation , an epigenetic phenomenon.48 , 49 because the primary leukemia samples were consumed , we were unable to directly transduce ptprc cdna to test if jak inhibitor sensitivity could be restored . 
 reddy , xueyan chen administrative support : siraj ali , utpal dav financial support : utpal dav data analysis and interpretation : allison greenplate , kai wang , rati m . 
irish , utpal dav m511i mutation that had similarly downregulated cd45 after disease relapse during chop chemotherapy , which suggests that downregulation of the cd45 protein may play a role in chemotherapy resistance . 
notably , porcu et al50 discovered deletion , missense , and nonsense mutations in ptprc in t - allevidence that supports a tumor suppressor role for ptprc in this disease . 
furthermore , porcu et al50 also showed concordant loss of function in ptprc and gain of function in jak1 or il7r , which suggests that these two hits cooperate in t - all pathogenesis . 
our studies on the index case are similar to these findings , because we also observed an inverse correlation between cd45 levels and p - stat5 , albeit in t - pll . 
our studies do suggest that ptprc may be a tumor suppressor in more mature t - cell neoplasms in addition to precursor t - all and that its loss of function may be an important resistance mechanism to ruxolitinib . 
finally , the data presented in this study support the design of larger phase i / ii clinical trials to test ruxolitinib on its own or in combination with cytotoxic therapies in t - cell neoplasms . 
ali employment : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine ; patents via seres health on microbiome stuff in non - neoplastic disease ( i ) phil j . 
miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center yu shyr consulting or advisory role : aduro biotech , janssen research and development , novartis , roche , genentech yan guo no relationship to disclose nishitha m . 
reddy consulting or advisory role : celgene , abbvie , gilead sciences , bristol - myers squibb lina kozhaya no relationship to disclose derya unutmaz no relationship to disclose xueyan chen no relationship to disclose jonathan m . 
irish research funding : janssen ( inst ) , incyte ( inst ) , pharmacyclics ( inst ) patents , royalties , other intellectual property : methods and compositions for risk stratification , us patents ( us 7 , 393 , 656 , us 7 , 939 , 278 , us 8 , 206 , 939 , us 8 , 309 , 316 , us 8 , 394 , 599 ) other relationship : cytobank utpal dav no relationship to disclose acknowledgment we thank stephen brandt , md , scott hiebert , phd , carlos arteaga , md , and justin balko , phd , for helpful discussions and kevin weller , phd , for his expert advice . the content is solely the responsibility of the authors and does not represent the official views of the national institutes of health . affiliations allison greenplate , rati m . 
miller , foundation medicine , cambridge , ma ; kai wang , origimed , shanghai , china ; lina kozhaya and derva unutmaz , jackson laboratory , farmington , ct ; xueyan chen , university of washington medical center , seattle , wa ; and utpal p . 
coustan - smith e , mullighan cg , onciu m , et al : early t - cell precursor leukemia : a subtype of very high - risk acute lymphoblastic leukemia . 
patrick k , wade r , goulden n , et al : outcome for children and young people with early t - cell precursor acute lymphoblastic leukemia treated on a contemporary protocol , ukall 2003 . 
chiaretti s , li x , gentleman r , et al : gene expression profile of adult t - cell acute lymphocytic leukemia identifies distinct subsets of patients with different response to therapy and survival . 
weisenburger dd , savage kj , harris nl , et al : peripheral t - cell lymphoma , not otherwise specified : a report of 340 cases from the international peripheral t - cell lymphoma project . 
parrilla castellar er , jaffe es , said jw , et al : alk - negative anaplastic large - cell lymphoma is a genetically heterogeneous disease with widely disparate clinical outcomes . 
wood bl , winter ss , dunsmore kp , et al : t - lymphoblastic leukemia ( t - all ) shows excellent outcome , lack of significance of the early thymic precursor ( etp ) immunophenotype , and validation of the prognostic value of end - induction minimal residual disease ( mrd ) in childrens oncology group ( cog ) study aall0434 . 
boucheix c , david b , sebban c , et al : immunophenotype of adult acute lymphoblastic leukemia , clinical parameters , and outcome : an analysis of a prospective trial including 562 tested patients ( lala87 )  . 
koretzky ga , picus j , schultz t , et al : tyrosine phosphatase cd45 is required for t - cell antigen receptor and cd2 - mediated activation of a protein tyrosine kinase and interleukin 2 production . 
irish jm , myklebust jh , alizadeh aa , et al : b - cell signaling networks reveal a negative prognostic human lymphoma cell subset that emerges during tumor progression . 
vallois d , dobay mpd , morin rd , et al : activating mutations in genes related to tcr signaling in angioimmunoblastic and other follicular helper t - cell - derived lymphomas . 
boddicker rl , razidlo gl , dasari s , et al : integrated mate - pair and rna sequencing identifies novel , targetable gene fusions in peripheral t - cell lymphoma . 
degryse s , de bock ce , cox l , et al : jak3 mutants transform hematopoietic cells through jak1 activation , causing t - cell acute lymphoblastic leukemia in a mouse model . 
losdyck e , hornakova t , springuel l , et al : distinct acute lymphoblastic leukemia ( all ) associated janus kinase 3 ( jak3 ) mutants exhibit different cytokine - receptor requirements and jak inhibitor specificities . 
flanagan me , blumenkopf ta , brissette wh , et al : discovery of cp - 690 , 550 : a potent and selective janus kinase ( jak ) inhibitor for the treatment of autoimmune diseases and organ transplant rejection . 
quints - cardama a , vaddi k , liu p , et al : preclinical characterization of the selective jak1 / 2 inhibitor incb018424 : therapeutic implications for the treatment of myeloproliferative neoplasms . 
 detection of somatic structural variants enables quantification and characterization of circulating tumor dna in children with solid tumors objective liquid biopsies are being rapidly used in adult cancers as new biomarkers of disease . 
therefore , we developed a next - generation sequencing approach to detect and quantify ctdna in the blood of patients with the most common pediatric solid tumors . methods detection of ctdna requires assays sensitive to somatic events typically observed in the cancer type being studied . 
we adapted an ultralow passage whole - genome sequencing approach to capture copy number variants and a hybrid capture sequencing assay to detect translocations in liquid biopsy samples from pediatric patients . results copy number changes seen by ultralow passage whole - genome sequencing enabled detection of ctdna in patients with osteosarcoma , neuroblastoma , alveolar rhabdomyosarcoma , and wilms tumor . 
we also found that disease - specific genomic biomarkers of prognosis were detectable in ctdna . conclusion this study demonstrates that liquid biopsy approaches that detect somatic structural variants are well suited to pediatric solid tumors . 
we show that children with the most common solid tumor malignancies have detectable levels of ctdna , which may be used to track disease response and identify genomic subclassifiers of disease . 
2018 by american society of clinical oncology introduction cancer remains one of the most common childhood causes of disease - related death in developed countries.1 patients with pediatric solid tumor malignancies are commonly treated with regimens that combine intensive chemotherapy , surgery , and radiation therapy.2 this approach has led to tremendous improvements in outcome , with overall cure rates for pediatric solid tumors now > 80%.3 however , these cures come at the cost of exposing patients to a high risk of long - term toxicities resulting in significant morbidity.4 in some diseases , such as wilms tumor , efforts to de - escalate therapy have resulted in stable cure rates for low - risk patients accompanied by reduced treatment toxicity.5 , 6 in other diseases , such as ewing sarcoma , a lack of prognostic biomarkers limit the ability to risk stratify therapy.7 , 8 in both cases , the development of new and more precise assays for risk stratification could improve outcomes by facilitating patient - specific treatment modifications . kelly klega alma imamovic - tuco gavin ha andrea n . 
crompton author affiliations and support information ( if applicable ) appear at the end of this article . the contents are solely the responsibility of the authors and do not necessarily represent the official views of the national institutes of health or other funding agencies . corresponding author : brian d . 
numerous studies have now demonstrated that circulating tumor dna ( ctdna ) levels in adults with cancer correlate with disease burden and track with treatment responses over time.9 - 14 the development of ctdna assays rely on identification and quantification of somatic mutations.9 , 15 many ctdna assays have been developed to detect highly recurrent hot - spot mutations that frequently drive adult malignancies.9 , 12 , 13 , 16 - 18 however , recent comprehensive sequencing efforts in pediatric cancers show that pediatric solid tumors are rarely driven by highly recurrent single - nucleotide variants.19 instead , structural variants , including chromosomal copy number changes and dna translocations , are common somatic events in these tumor types.20 - 26 to determine whether patients with childhood solid tumor malignancies express detectable levels of ctdna , we adapted two next - generation sequencing strategies to detect and quantify structural variants in the plasma of these patients . 
 we applied these assays to five of the most common types of pediatric solid tumors , excluding cns tumors , to quantify ctdna levels , identify genomic subtypes , and confirm the feasibility of using ctdna assays to track disease response to therapy . methods patients and tissue samples patients at the dana - farber cancer institute and boston childrens hospital were consented to an institutional review boardapproved protocol . 
patients were included if an initial peripheral blood , pleural effusion , or bone marrow sample was collected within 3 days from the start of chemotherapy and before a surgical resection . 
additional details are available in the data supplement . sequencing library preparation , sequencing alignment , and ultralow passage whole - genome sequencing coverage for all experiments , sequencing library preparation and sequencing data alignment were performed using standard techniques . 
additional details are available in the data supplement . translocation - specific sarcoma sequencing assay development and sequencing to detect pediatric sarcoma - specific translocations in cell - free dna , we developed a unique hybrid capture sequencing assay . 
first , we reviewed the literature to identify the genomic introns involved in oncogenic translocations in the ewsr1 , fus , cic , ccnb3 , pax3 , pax7 , and tp53 genes.20 , 21 , 25 - 33 second , we used the sureselect advanced design wizard ( agilent technologies , santa clara , ca ) to create capture probes targeting these regions and the coding regions of tp53 and stag2 with the following design options : sense strand , 3 tiling density , least stringent masking , balanced boosting , and region extensions of 10 bases from the 3 and 5 ends ( data supplement )  . 
the average measured coverage at enrichment sites for all samples tested by transs - seq was 655 ( range , 31 to 1 , 464 )  . sequencing analysis ulp - wgs analysis was performed using the ichorcna algorithm ( dinstitute / ichorcna ) , with manual curation of results.34 in brief , ulp - wgs uses relative sequencing coverage of whole - genome data and computational correction of sequencing bias to detect segmental copy number changes . 
 wild - type reads detected in each sample , we developed a custom algorithm designed to realign all sequencing reads to either the reference human genome or the patient - specific translocation positive reference sequence ( vanallenlab / peds_ctdna )  . 
the algorithm then reported the number of reads aligned at the patient - specific translocation breakpoint and the number of wild - type reads at the equivalent genomic base - pair location within the human reference genome . 
because each cancer genome contains one translocated and one wild - type allele , whereas germline genomes contain two wild - type alleles , we used the following formula to calculate the percentage of ctdna , where t equals the number of translocation reads and w equals the number of wild - type reads : % ctdna = t / { [ ( w - t ) / 2 ] + t }  . cell lines and digital droplet polymerase chain reaction the ew8 ewing sarcoma cell line dilution experiments , digital droplet polymerase chain reaction ( ddpcr ) methods , and ddpcr primers are listed in the data supplement . highest values originating from patients with neuroblastoma ( fig 1a )  . 
ulp - wgs was performed for each sample to detect and quantify ctdna by measuring segmental copy number changes as recently described.34 ctdna was detected in 50% of pretreatment plasma samples from patients with osteosarcoma , neuroblastoma , wilms tumor , and alveolar rhabdomyosarcoma . 
in patients with detectable ctdna , the pattern of segmental chromosomal copy number changes in cellfree dna matched the copy number pattern observed in the tumor biopsy from the same patient ( figs 1c and 1d ; data supplement )  . 
in four of the seven patients with ewing sarcoma with no detectable ctdna , ulp - wgs analysis of tumor biopsy dna material demonstrated no discernable segmental copy number changes ( fig 2 )  . 
this finding is consistent with previous reports that ewing sarcoma tumors have few highly recurrent somatic events other than the ewsr1 / ets translocations and suggests that an alternative method for detection of ctdna is necessary for this disease.21 , 26 results patients and samples a liquid biopsy sample was collected before the initiation of therapy from 45 pediatric patients with cancer with a confirmed diagnosis of ewing sarcoma ( n = 11 ) , osteosarcoma ( n = 10 ) , neuroblastoma ( n = 10 ) , wilms tumor ( n = 8 ) , or alveolar rhabdomyosarcoma ( n = 7 )  . 
dna from tumor biopsy material from patients with neuroblastoma and wilms tumor were not readily available . circulating tumor dna is detectable in patients with pediatric solid tumor malignancies total cell - free dna levels ranged from 2 ng to 3.5 g of dna per 1 ml of plasma , with the translocation detection outperforms copy number detection for ewing sarcoma to detect ctdna in translocation - positive tumors , we developed a novel hybrid - capture assay , termed transs - seq , designed to sequence intronic regions involved in genomic rearrangements in pediatric sarcomas . 
first , we confirmed that the assay could detect the expected translocations in a panel of ewing and ewing - like sarcomas and alveolar rhabdomyosarcomas ( data supplement )  . 
ddpcr in these samples confirmed the ability to detect and quantify the ew8 - specific ewsr1 / fli translocation across the range of dilutions ( fig 3a ; data supplement )  . 
 ( a ) quantity of cell - free dna extracted per ml of plasma or body fluid in patients with osteosarcoma ( ost ) , alveolar rhabdomyosarcoma ( arms ) , ewing sarcoma ( ews ) , wilms tumor ( wilms ) , and neuroblastoma ( nb )  . 
the color for each data point corresponds to the relative copy number change from baseline , with blue equal to two copies of the genomic location , green equal to copy number loss , and red equal to copy number gains . 
the same pattern of copy number changes is observed in dna sequenced from a tumor biopsy ( top ) and a pretreatment blood sample ( bottom ) in ( c ) a patient with osteosarcoma and ( d ) alveolar rhabdomyosarcoma . and detected ew8 dna at a concentration of 1.56% ( figs 3b and 3c ; data supplement )  . identical to the breakpoint observed from the tumor biopsy sample . transs - seq was then applied to cell - free dna samples from patients with ewing sarcoma and alveolar rhabdomyosarcoma . 
for five patients with ewing sarcoma and three patients with alveolar rhabdomyosarcoma , dna from tumor biopsy samples was also profiled by transs - seq ( data supplement )  . 
in all cases , the unique genomic breakpoint in the ctdna was previous studies in ewing sarcoma have used ddpcr assays that amplify patient - specific ews / ets translocations to measure ctdna levels in patients.36 - 38 to compare transs - seq with this previously validated approach , patient specific polymerase chain reaction primers were developed for a subset of ewing sarcoma and alveolar rhabdomyosarcoma samples ( data supplement )  . 
 mrd0006 ewing sarcoma mrd0019 ewing sarcoma 108765 14 16 18 20 23 x 108765 14 16 18 20 23 x 14 16 18 20 23 x 108765 14 16 18 20 23 x chromosome mrd0023 ewing sarcoma chromosome mrd0046 ewing sarcoma chromosome chromosome mrd0007 ewing sarcoma 108765 chromosome mrd0041 ewing sarcoma 108765 chromosome mrd0047 ewing sarcoma 14 16 18 20 23 x 108765 14 16 18 20 23 x 14 16 18 20 23 x 108765 chromosome fig 2 . 
genome - wide copy number plots from seven ewing sarcoma tumor biopsy samples detected by ultralow passage whole - genome sequencing in patients for which circulating tumor dna could not be detected from peripheral blood . 
in three of the four patients with ewing sarcoma for which no copy number change could be detected by ulp - wgs in the tumor ( mrd0006 , mrd0041 , mrd0047 ) , transsseq was able to detect an ewsr1 - fusion in the tumor and cell - free dna ( data supplement )  . 
in the fourth patient ( mrd0007 ) , transs - seq detected an ewsr1 / fli fusion in the tumor but not in cell - free dna ( data supplement )  . 
however , four patients with tumor necrosis < 70% ( mrd0031 , mrd0036 , mrd0040 , mrd0054 ) had detectable ctdna in at least one sample collected after initiation of chemotherapy ( fig 4d ; data supplement )  . 
 finally , in patients whose ctdna levels became undetectable with therapy but then experienced a clinical relapse or progression , ctdna was again detectable before the initiation of relapsed therapy ( fig 4b ; data supplement )  . genomic markers of poor outcome are detectable in ctdna we also examined whether disease - specific genomic markers of prognosis could be detected in ctdna . 
recent studies demonstrate that mutations in stag2 and tp53 may be associated with a worse outcome in ewing sarcoma.21 , 26 a frame - shift mutation in stag2 was detected by ctdna from one patient ( mrd0023 ) , and a mutation in tp53 was detected in another patient ( mrd0003 ; data supplement )  . 
in alveolar rhabdomyosarcoma , studies have demonstrated that patients with pax3 / foxo1 have a worse prognosis than patients with pax7 / foxo1 translocation.41 the foxo1 fluorescent in situ hybridization probe , which is used in the diagnostic work - up of alveolar rhabdomyosarcoma , confirms a foxo1 rearrangement but not the fusion partner . 
in osteosarcoma , copy number gains of chromosome arm 8q are associated with a poor outcome.42 - 44 ulp - wgs detected copy number gains in 8q in seven of nine osteosarcoma samples with detectable ctdna ( fig 1c ; data supplement )  . 
amplification of mycn is a well - established marker of poor prognosis in neuroblastoma.45 , 46 mycn amplification was detectable in the ctdna of two patients by ulp - wgs ( fig 5a ; data supplement )  . 
finally , in wilms tumor , copy number gains of 1q were associated with poor prognosis in favorable histology tumors.47 ulp - wgs detected 1q gain in copy number change but ulp - wgs was unable to detect ctdna from the same patient ( mrd0019 , mrd0023 , mrd0046 ) , transsseq detected low levels of ctdna , suggesting that transs - seq may have greater sensitivity for ctdna than ulp - wgs in ewing sarcoma ( data supplement )  . ctdna levels track with disease burden in pediatric solid tumors when comparing ctdna levels across cancer types , neuroblastoma demonstrated a significantly higher percentage of ctdna ( median , 55% ; range , 13% to 98% ) than other cancer types ( data supplement )  . 
in patients with newly diagnosed ewing sarcoma and alveolar rhabdomyosarcoma , ctdna levels declined rapidly after initiation of chemotherapy , often becoming undetectable by the start of the second cycle ( figs 4a and 4b ; data supplement )  . 
in osteosarcoma , studies have shown that a high percentage of tumor necrosis observed in the primary tumor after neoadjuvant chemotherapy is associated with a better prognosis.39 , 40 in five patients with newly diagnosed osteosarcoma , the percentage of tumor necrosis was available . 
the translocation - specific sarcoma sequencing assay ( transs - seq ) detects circulating tumor dna ( ctdna ) in ewing sarcoma ( ews ) and alveolar rhabdomyosarcoma ( arms )  . 
 samples are plotted on the x - axis by the percentage of ew8 by experimental dilution and on the y - axis by the percentage of ew8 dna detected by digital droplet polymerase chain reaction ( ddpcr )  . 
 ( d ) percentage of ctdna levels in cell - free dna samples from patients with ewing sarcoma and arms ( same samples as in figure 1b ) determined by transsseq . 
 percentage of ctdna levels from serial cell - free dna samples collected from four patients with ( a ) ewing sarcoma , ( b ) alveolar rhabdomyosarcoma , ( c - d ) osteosarcoma . 
blue dots indicate peripheral blood samples , and the red dot ( b ) indicates a bone marrow sample collected simultaneously with a blood sample . mrd0019 ewing sarcoma mrd0045 alveolar rhabdomyosarcoma bone marrow clinical course clinical course mrd0061 osteosarcoma mrd0036 osteosarcoma clinical course clinical course had detectable levels of ctdna . 
patients with neuroblastoma had the highest levels of ctdna , including two patients with > 1 g of cell - free dna per ml of plasma , which was nearly 100% composed of tumor dna . 
however , one obvious limitation to the use of ulp - wgs was in tumor types with low rates of copy number alterations , such as ewing sarcoma . recent liquid biopsy studies in ewing sarcoma have used the development of patient - specific ddpcr assays.36 - 38 although this approach has proven to be highly sensitive and quantitative , the development of each assay requires genomic profiling of large amounts of tumor biopsy material to establish the patient - specific oncogenic translocation . 
one recent study used a combination of ddpcr and hybrid capture sequencing to detect ewsr1 translocations in ewing sarcoma and desmoplastic small roundcell tumors.38 results showed that it is feasible to detect ewsr1 translocations directly from the plasma of these patients without first sequencing the tumor sample . 
despite the larger genomic region targeted for hybrid capture , the transs - seq assay detected ctdna in 10 of 11 pretreatment samples obtained from ewing sarcoma , similar to rates observed by other groups . 
we also demonstrated that quantification of ctdna levels was similar using either transs - seq or ddpcr . in this study , changes in ctdna levels were observed during a patients clinical course and corresponded to changes in disease burden . 
with next - generation sequencing becoming increasingly available for clinical decision making , we anticipate that our ctdna assays could also be adapted to clinical laboratory testing if they demonstrate significant improvements to risk stratification for pediatric solid tumors . 
 ( a ) sequencing coverage ( read counts ) across the mycn gene ( top ) and c - myc gene ( bottom ) from four cell - free dna samples from patients with neuroblastoma . 
therefore , prospective studies should be designed to obtain frequent samples throughout treatment so that the time points most predictive of outcome can be appropriately identified . finally , our study demonstrates that next generation sequencing of ctdna can detect existing genomic biomarkers of outcome and may provide another modality for genomic profiling of tumors in patients . 
furthermore , it remains unclear whether these genomic biomarkers are primarily clonal or heterogeneous events within a tumor or across tumors and whether the clonality of these genomic events changes in patients who relapse after therapy . 
recent studies demonstrate that ctdna can detect intratumor and multitumor heterogeneity and detect complex patterns of treatment resistance.52 - 55 with the emergence of new sequencing modifications that improve sensitivity and decrease sequencing errors , we believe that ctdna profiling by next - generation sequencing approaches will improve our understanding of tumor heterogeneity and patterns of somatic evolution in pediatric solid tumors.12 , 16 in addition , the assays described in this study could facilitate broader profiling of ctdna , such as deep whole - exome sequencing , by providing a mechanism to screen samples for the presence of sufficiently abundant ctdna , allowing selection of samples most likely to yield informative data . in summary , our study demonstrates that patients with pediatric solid tumors have detectable levels of ctdna , which can be measured with next generation sequencing approaches that do not require profiling of tumor biopsy material or patient - specific assays . 
disease - specific genomic hallmarks of pediatric solid tumors can also be identified in liquid biopsy samples , and ctdna levels correlate with disease burden and response to therapy over time . 
crompton manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors anwesha nag no relationship to disclose eliezer van allen stock and other ownership interests : syapse , tango therapeutics , genome medical consulting or advisory role : syapse , roche , third rock ventures , takeda , novartis , genome medical , invitae speakers ' bureau : illumina research funding : bristol - myers squibb , novartis elizabeth mullen no relationship to disclose steven g . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . katherine janeway no relationship to disclose matthew meyerson stock and other ownership interests : origimed consulting or advisory role : origimed research funding : bayer ( inst ) patents , royalties , other intellectual property : patent on egfr mutations for lung cancer diagnosis aaron r . 
crompton employment : mersana ( i ) , shire ( i ) stock and other ownership interests : mersana ( i ) , shire ( i ) acknowledgment we thank the boston childrens hospital biorepository for processing a portion of the blood samples . 
pediatr blood cancer 60 : 1083 - 1094 , 2013 kelly klega no relationship to disclose alma imamovic - tuco no relationship to disclose gavin ha no relationship to disclose andrea n . 
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yung tk , chan kc , mok ts , et al : single - molecule detection of epidermal growth factor receptor mutations in plasma by microfluidics digital pcr in non - small cell lung cancer patients . 
shern jf , chen l , chmielecki j , et al : comprehensive genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic axis in fusion - positive and fusionnegative tumors . 
tirode f , surdez d , ma x , et al : genomic landscape of ewing sarcoma defines an aggressive subtype with co - association of stag2 and tp53 mutations . 
kawamura - saito m , yamazaki y , kaneko k , et al : fusion between cic and dux4 up - regulates pea3 family genes in ewing - like sarcomas with t ( 4 ; 19 ) ( q35 ; q13 ) translocation . 
ng tl , osullivan mj , pallen cj , et al : ewing sarcoma with novel translocation t ( 2 ; 16 ) producing an in - frame fusion of fus and fev . 
sugita s , arai y , tonooka a , et al : a novel cic - foxo4 gene fusion in undifferentiated small round cell sarcoma : a genetically distinct variant of ewing - like sarcoma . 
yoshimoto m , graham c , chilton - macneill s , et al : detailed cytogenetic and array analysis of pediatric primitive sarcomas reveals a recurrent cic - dux4 fusion gene event . 
shukla nn , patel ja , magnan h , et al : plasma dna - based molecular diagnosis , prognostication , and monitoring of patients with ewsr1 fusion - positive sarcomas . 
meyers pa , schwartz cl , krailo m , et al : osteosarcoma : a randomized , prospective trial of the addition of ifosfamide and / or muramyl tripeptide to cisplatin , doxorubicin , and high - dose methotrexate . 
sorensen ph , lynch jc , qualman sj , et al : pax3 - fkhr and pax7 - fkhr gene fusions are prognostic indicators in alveolar rhabdomyosarcoma : a report from the childrens oncology group . 
tarkkanen m , elomaa i , blomqvist c , et al : dna sequence copy number increase at 8q : a potential new prognostic marker in high - grade osteosarcoma . 
gratias ej , dome js , jennings lj , et al : association of chromosome 1q gain with inferior survival in favorable - histology wilms tumor : a report from the childrens oncology group . 
lecomte t , berger a , zinzindohou f , et al : detection of free - circulating tumor - associated dna in plasma of colorectal cancer patients and its association with prognosis . 
tie j , wang y , tomasetti c , et al : circulating tumor dna analysis detects minimal residual disease and predicts recurrence in patients with stage ii colon cancer . 
vora a , goulden n , mitchell c , et al : augmented post - remission therapy for a minimal residual disease - defined high - risk subgroup of children and young people with clinical standard - risk and intermediate - risk acute lymphoblastic leukaemia ( ukall 2003 ) : a randomised controlled trial . 
de mattos - arruda l , weigelt b , cortes j , et al : capturing intra - tumor genetic heterogeneity by de novo mutation profiling of circulating cell - free tumor dna : a proof - of - principle . 
thress ks , paweletz cp , felip e , et al : acquired egfr c797s mutation mediates resistance to azd9291 in non - small cell lung cancer harboring egfr t790m . 
 clinical value of rna sequencing based classifiers for prediction of the five conventional breast cancer biomarkers : a report from the population - based multicenter sweden cancerome analysis networkbreast initiative purpose in early breast cancer ( bc ) , five conventional biomarkersestrogen receptor ( er ) , progesterone receptor ( pgr ) , human epidermal growth factor receptor 2 ( her2 ) , ki67 , and nottingham histologic grade ( nhg ) are used to determine prognosis and treatment . 
we aimed to develop classifiers for these biomarkers that were based on tumor mrna sequencing ( rna - seq ) , compare classification performance , and test whether such predictors could add value for risk stratification . methods in total , 3 , 678 patients with bc were studied . 
patients with discordant classifications , predicted as hormone responsive by histopathology but non hormone responsive by mgc , had significantly inferior overall survival compared with patients who had concordant results . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license christian brueffer johan vallonchristersson dorthe grabau anna ehinger jari hkkinen cecilia hegardt janne malina yilun chen pr - ola bendahl jonas manjer martin malmberg christer larsson niklas loman lisa rydn ke borg lao h . 
immunohistochemistry ( ihc ) is the principal method for er , pgr , her2 , and ki67 measurement , and in situ hybridization ( ish ) methods are used to refine her2 ihc . 
 accuracy and reproducibility are concerns , with up to 20% false - positive or false - negative er / pgr ihc determinations.3 varying discordance has been reported for her2 ihc and fluorescent ish ( fish ) .4 - 7 accordingly , consensus guidelines emphasize standardization and validation of analytic performance.1 , 2 , 8 lack of standardization has slowed the entrance of ki67 into clinical routines.9 for example , ki67 status was only moderately concordant in an interlaboratory reproducibility analysis.10 thresholds for ki67 positivity are evolving ; cutoffs between 20% and 29% were recommended by the 2015 st gallen / vienna panel for laboratories with a quality assurance program.11 swedish quality assurance program guidelines recommend that each laboratory calibrate a cutoff yearly such that one third of 100 consecutive occurrences are ki67 - high . 
in 2010 , toward implementation of molecular profiling in the clinical routine , we launched the sweden cancerome analysis network breast initiative ( scan - b ; clinicaltrials.gov identifier : nct02306096 ) , an ongoing population - based multicenter observational study covering a wide geography of sweden that prospectively invites all patients with bc to participate.17 to date , approximately 85% of the eligible catchment population are included , more than 11 , 000 patients have enrolled , and blood and fresh tumor tissues are sampled for molecular research . 
thus , for each bc , it will be possible to report a multitude of biomarker tests simultaneously on the basis of its rna - sequencing data and within a clinically actionable time frame . herein , we aimed to validate the scan - b multicenter infrastructure and provide molecular analyses of clinical value by developing rnaseqderived classifiers for the conventional histopathologic bc biomarkers er , pgr , her2 , ki67 , and nhg . 
for this purpose , both single gene classifiers ( sgcs ) and multigene classifiers ( mgcs ) were developed by using a training cohort , the prediction accuracy was compared against current clinical practice across a large independent prospective cohort , and the classifier predictions and their discrepancies to histopathology were evaluated with respect to patient survival . methods patients the study ( fig 1 ) was approved by the regional ethical review board of lund at lund university and the swedish data inspection group . 
 diagnostic reading diagnostic stains : er , pgr , her2 , nhg two independent readings histopathology performance evaluation training set ( n = 405 ) five biomarkers : er , pgr , her2 , nhg , ki67 new stains : er , pgr , her2 , ki67 three independent readings rna sequencing train sgc / mgc consensus pathology scores independent validation set ( n = 3 , 273 ) rna sequencing apply trained sgc / mgc classifier performance evaluation and survival analysis five biomarkers : er , pgr , her2 , nhg , ki67 diagnostic stains : er , pgr , her2 , nhg , ki67 diagnostic reading fig 1 . 
study design flow diagraer , estrogen receptor ; her2 , human epidermal growth factor receptor 2 ; ki67 , proliferation antigen ki67 ; mgc , multigene classifier ; nhg , nottingham histologic grade ; pgr , progesterone receptor ; sgc , single - gene classifier . and er - negative tumors ( training cohort ; data supplement )  . 
for classifier testing , an independent , prospective , and population - based modern cohort of 3 , 273 patients with early bc was assembled from the ongoing scan - b study17 ( validation cohort ; appendix fig a1 ; data supplement )  . histopathology for the training cohort , all biomarkers with the exception of ki67 were evaluated at time of diagnosis . 
in addition , new formalin - fixed paraffin - embedded slides were analyzed for er , pgr , and ki67 ihc and for her2 silver ish , all performed at a central laboratory ( helsingborg hospital )  . 
the diagnostic slides and newly stained slides were each scored in total by three pathologists independently by using 1% or greater tumor cell staining threshold for hormone receptor positivity , standard her2 herceptest ( agilent / dako , santa clara , ca ) and ish criteria ( roche / ventana , tucson , az ) , greater than 20% positive nuclei for ki67high status , and the nhg scoring system ( data supplement )  . 
the biologic functional annotation clusters of each mgc signature were evaluated with the david bioinformatics resource.20 statistical analysis histopathology evaluations and single - gene and multigene predictions were compared with agreement statistics21 ( defined in the data supplement ) and balanced statisticscohens and matthews correlation coefficient ( mcc ) and were interpreted according to viera and garrett.22 the and mcc values were comparable ( data supplement ) , so we focused on  . 
 multivariable cox models included the variables age at diagnosis , lymph node status , tumor size , er , pgr , her2 , and nhg as covariates , as relevant ( data supplement )  . 
as expected with minimization of technical and heterogeneity factors , within - slide concordances were slightly better than betweenslide concordances ( data supplement )  . classifier training whole - transcriptome expression profiles were generated for the 405 training samples using rna - seq . 
for the sgcs , optimal thresholds were determined for esr1 ( which encodes the er protein ) , pgr ( pgr ) , erbb2 ( her2 ) , and mki67 ( ki67 ) ( data supplement )  . 
the constituent biologic themes for each mgc classifier were investigated with functional annotation clustering ( data supplement )  . performance on independent data to evaluate the classifiers , we tested them on rna - seq data generated for 3 , 273 independent tumors from the prospective population - based multicenter scan - b study ( n = 136 tumors were analyzed in technical replicates )  . 
for rna - seq replicates , 534 ( 98.2% ) of 544 sgc classifications and 675 ( 99.3% ) of 680 mgc classifications were concordant ( data supplement )  . 
within a biomarker staining group ( left - most column headings ) , all comparisons presented are the reference evaluation ( the diagnostic reading made in the clinical routine , or reader 1 in the case of ki67 ) versus each specified reader number . 
 abbreviations : er , estrogen receptor ; h&e , hematoxylin and eosin ; her2 , human epidermal growth factor receptor 2 ; ihc , immunohistochemistry ; nhg , nottingham histologic grade ; pgr , progesterone receptor ; sish , silver in situ hybridization . survival analysis to evaluate the possible clinical utility of our classifiers , we analyzed our classifier predictions within the validation cohort with respect to overall survival . 
kaplan - meier analysis revealed comparable patient stratification for both diagnostic histopathology and sgcs for the five biomarkers across the entire validation cohort , whereas the mgcs had a noticeably richer stratification , particularly for the hormone receptors and the hormone - responsive group , defined by er positivity and pgr positivity ( appendix figs a4 and a5 )  . 
after adjusting for important covariates in multivariable cox analyses , the mgc prediction for hormone nonresponsiveness was a significant stratifier among patients with histopathologic hormone - responsive disease who were treated with endocrine therapy , as were the mgc predictions discordant for her2 - negative or ki67 - high status in patients who received chemotherapy with or without trastuzumab and / or endocrine therapy . 
previously , several gene expressionbased approaches for determination of known treatment - predictive biomarkers have been developed16 , 23 - 26 ; however , they are not widely used clinically in most countries . 
 ( a ) forest plots of concordance statistics for histopathologic evaluation in the training set ( blue square markers ) , and single - gene classifiers ( sgcs ; gold circles ) and multigene classifiers ( mgcs ; gray diamonds ) in the validation cohort , which plots overall agreement with 95% cis , specific agreements ( positive and negative agreements for estrogen receptor [ er ] , progesterone receptor [ pgr ] , human epidermal growth factor receptor 2 [ her2 ] , and ki67 ) and nottingham histologic grade ( nhg ) category agreements ( grade [ g ] 1 , g2 , and g3 ) , and values with 95% cis . 
scan - b , sweden cancerome analysis networkbreast . demonstrated that accurate classifiers for er , pgr , her2 , ki67 and nhg can be built with rna - seq data , can provide a valuable complement to traditional histopathology , and represent the first of many potential clinical reports that can be delivered from a single rna - seq measurement . 
in the future , we foresee the development , validation , and clinical implementation of a multitude of signatures , classifiers , and mutational profiles within the scan - b population - based infrastructure and rna - seq platform.17 , 18 we also aim to use rna - seq analyses in the performance of interventional clinical trials.29 the quality of machine - learned classifiers is crucially dependent on the quality of the labels on which they have been trained . 
matched against routine histopathologic evaluation , repeated er , pgr , and her2 readings showed good concordance , whereas ki67 and nhg had notably lower concordance between pathologists ( table 1 )  . 
 ( a ) histopathologically hormone responsive ( defined as estrogen receptor [ er ] positive and progesterone receptor [ pgr ] positive ) group stratified by mgc hormone responsive classification ( concordant [ blue curve ] or discordant [ gold curve ] to histopathology ) within the subgroup of patients who received ( left ) no adjuvant systemic therapy , ( middle ) endocrine therapy alone , or ( right ) chemotherapy with or without trastuzumab or endocrine therapy . 
 it is unlikely that a classifier would perform better than the quality of training labels ; therefore , it is not surprising that our classifiers had the worst performance for ki67 and nhg . 
moreover , because we benchmarked our biomarker predictions in the validation cohort to the clinical diagnostic histopathology results that contained this inherent variability , we could not expect our classifiers to have higher accuracy than what is achievable within histopathology . generally , sgcs performed comparably to clinical diagnostic pathology . 
earlier work on mrna - based classifiers for er , pgr , and her2 has been performed with microarrays , quantitative reverse - transcriptase polymerase chain reaction , and , recently , with rna - seq and mainly has been restricted to signatures of either one16 , 31 or few23 , 24 , 26 , 32 , 33 genes . 
in each kaplan - meier plot , the histopathology to mgc concordant tumor cases are plotted in blue , the discordant tumor cases are plotted in gold , the log - rank p value is given , and the hazard ratio ( hr ) for discordant - versus - concordant result is given with a 95% ci and after multivariable ( mv ) cox regression adjustment . 
covariables included in the mv analysis were age at diagnosis , lymph node status , tumor size , and the variables denoted by the following symbols : , er , pgr , and nhg ; , er , pgr , her2 , and nhg ; , her2 and nhg ; # , er , pgr , and her2 . discrepancies between rna - seqbased classifications and histopathology may be a result of staining and reader variations , as discussed in this paper . 
 the consequence is that a mismatch between mrna biomarker prediction and histopathology may be influenced by various mechanisms active between these layers , for example rna silencing / interference / translation , protein stability and epitope availability , or tumor heterogeneity . despite these possible explanations for discrepancies , when benchmarked against patient outcome , our classifiers exemplified enhanced stratification of patients with significant differences in overall survival ( fig 3 ; appendix figs a4 and a5 )  . 
the fact that mgcs performed best overall suggests that a multigene signature captures the biologic signaling upand downstream of the biomarker in question in a more consequential way than the expression of the single gene or protein alone . 
another approach is to use clinical outcome as the training labels to develop new prognostic / predictive signatures.13 , 34 the scan - b material is excellently suited to evaluate previously published signatures ; as we accrue longer follow - up , we aim to develop rna - seq signatures trained on clinical outcomes . ki67 has been introduced relatively recently in international guidelines.11 to our knowledge , this study is the first to develop a validated predictor for ki67 status . 
the lower concordance between our ki67 predictions compared with the clinical reference is related to the relatively larger ki67 interrater disagreement seen within our consensus pathology evaluation , which is likely a consequence of the continuous nature of ki67 expression and of the spectrum of proliferation activity and pathways in bc . nhg is distinct from the other biomarkers . 
moreover , nhg prediction is a three - class proble even for pathologists , nhg can be difficult to determine , as evidenced by the moderate and oa results within clinical pathology , in line with the literature.12 most misclassified tumor cases in this study were histologically grade 1 ( g1 ) or grade 3 that were misclassified as grade 2 ( g2 ) by our predictor . 
all histologic g1 occurrences were misclassified , which may have been a result of the imbalanced composition of the training set for nhg ( 48 of 405 samples consensus - scored g1 ) , or may have occurred because g1 is not a discrete entity but rather the lower end of an underlying continuous scale . 
this approach has been suggested by others as a viable gene for expressionbased alternative to nhg translation into a clinical setting35 , 36 and essentially is what our nhg predictor has become . an important question when building classifiers is how many genes to use . 
when compared with clinical pathology , sgcs have slightly better concordance than mgcs for er and her2 , whereas the sgc and mgc performances were comparable for pgr and ki67 . 
mgcs may be more robust than sgcs , because they are able to classify tumors correctly even when the main gene that underlies a biomarker is poorly measured in a particular analysis . 
when clinical outcome was considered , the survival analyses indicated that our mgcs generally contained greater potential clinical utility than sgcs to complement histopathology . in summary , we have performed a systematic pathologic evaluation of 405 bc tumors , which resulted in consensus scores for the five conventional bc biomarkers and estimated a well - controlled bestcase scenario for the inherent uncertainty within clinical histopathology . 
classifiers based on the expression of single genes performed slightly better than mgcs for concordance to histopathology , but mgcs performed significantly better for stratification of patients into groups with clinically meaningful differences in survival , in particular for histopathologic hormone - responsive bcs . 
in conclusion , rna - seqbased classifiers may be suitable complementary diagnostics for bc , in particular for difficult diagnoses in which the classifier can add an additional vote toward the therapeutic choice . 
saal collection and assembly of data : christian brueffer , johan vallon - christersson , dorthe grabau , anna ehinger , jari hkkinen , cecilia hegardt , janne malina , jonas manjer , christer larsson , niklas loman , lisa rydn , ke borg , lao h . 
saal data analysis and interpretation : christian brueffer , johan vallon - christersson , dorthe grabau , anna ehinger , jari hkkinen , yilun chen , pr - ola bendahl , jonas manjer , martin malmberg , lisa rydn , ke borg , lao h . 
saal manuscript writing : all authors final approval of manuscript : all authors jonas manjer no relationship to disclose martin malmberg no relationship to disclose christer larsson honoraria : lilly ( i ) research funding : diamyd medical ab ( i ) travel , accommodations , expenses : lilly ( i ) niklas loman honoraria : astrazeneca consulting or advisory role : amgen lisa rydn research funding : roche ke borg honoraria : roche , astrazeneca travel , accommodations , expenses : roche , astrazeneca agree to be accountable for all aspects of the work : all authors lao h . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . christian brueffer employment : saga diagnostics ab johan vallon - christersson no relationship to disclose dorthe grabau no relationship to disclose anna ehinger no relationship to disclose jari hkkinen no relationship to disclose cecilia hegardt no relationship to disclose janne malina employment : unilabs honoraria : astrazeneca yilun chen no relationship to disclose pr - ola bendahl no relationship to disclose stock and other ownership interests : saga diagnostics patents , royalties , other intellectual property : patent filed for methods related to ultrasensitive quantification of nucleotide sequence variants . acknowledgment we thank the patients who were part of this study and the scan - b study . 
we also thank karin annersten , minerva li , inger remse , ralph schulz , jeanette valcich , cecilia wahlstrm , olle mnsson , nicklas nordborg , anna karlsson , christel reuterswrd , frida rosengren , and ingrid wilson of the scan - b laboratory and the division of oncology and pathology , lund university , for handling samples , genomic analyses , and database and administrative support , as well as cristina ciornei - karlsson for retrieving pathology slides and patient reports , daniel filipazzi and anders kvist for help with computing infrastructure , and johan staaf for help with classifier development . 
we thank the south sweden breast cancer group and all scan - b collaborators at hallands hospital halmstad , helsingborg hospital , blekinge county hospital , central hospital kristianstad , skne university hospital lund / malm , central hospital vxj , for inclusion of patients and sampling of tissue for this study , and we thank the swedish national breast cancer registry and regional cancer center south for clinical data . 
 this work is dedicated to the memory of dorthe grabau , who sadly passed away during the writing of this paper . affiliations christian brueffer , johan vallon - christersson , anna ehinger , jari hkkinen , cecilia hegardt , yilun chen , pr - ola bendahl , jonas manjer , christer larsson , niklas loman , lisa rydn , ke borg , and lao h . 
 support supported by the berta kamprad foundation and funded in part by the swedish foundation for strategic research , swedish research council , swedish cancer society , knut and alice wallenberg foundation , vinnova , governmental funding of clinical research within national health service , scientific committee of blekinge county council , crafoord foundation , lund university medical faculty , gunnar nilsson cancer foundation , skne university hospital foundation , biocare research program , king gustav vth jubilee foundation , maggie stephens foundation , royal physiographic society in lund , and the krapperup foundation . references netw 12 : 542 - 590 , 2014 1 . 
hammond meh , hayes df , dowsett m , et al : american society of clinical oncology / college of american pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer . 
press mf , sauter g , bernstein l , et al : diagnostic evaluation of her - 2 as a molecular target : an assessment of accuracy and reproducibility of laboratory testing in large , prospective , randomized clinical trials . 
perez ea , suman vj , davidson ne , et al : her2 testing by local , central , and reference laboratories in specimens from the north central cancer treatment group n9831 intergroup adjuvant trial . 
rydn l , haglund m , bendahl p - o , et al : reproducibility of human epidermal growth factor receptor 2 analysis in primary breast cancer : a national survey performed at pathology departments in sweden . 
ekholm m , grabau d , bendahl p - o , et al : highly reproducible results of breast cancer biomarkers when analysed in accordance with national guidelines : a swedish survey with central re - assessment . 
wolff ac , hammond meh , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
saal lh , johansson p , holm k , et al : poor prognosis in carcinoma is associated with a gene expression signature of aberrant pten tumor suppressor pathway activity . 
roepman p , horlings hm , krijgsman o , et al : microarray - based determination of estrogen receptor , progesterone receptor , and her2 receptor status in breast cancer . 
saal lh , vallon - christersson j , hkkinen j , et al : the sweden cancerome analysis network breast ( scan - b ) initiative : a large - scale multicenter infrastructure towards implementation of breast cancer genomic analyses in the clinical routine . 
hkkinen j , nordborg n , mnsson o , et al : implementation of an open source software solution for laboratory information management and automated rnaseq data analysis in a large - scale cancer genomics initiative using base with extension package reggie . 
kun y , how lc , hoon tp , et al : classifying the estrogen receptor status of breast cancers by expression profiles reveals a poor prognosis subpopulation exhibiting high expression of the erbb2 receptor . 
viale g , slaets l , bogaerts j , et al : high concordance of protein ( by ihc ) , gene ( by fish ; her2 only ) , and microarray readout ( by targetprint ) of er , pgr , and her2 : results from the eortc 10041 / big 03 - 04 mindact trial . 
wilson tr , xiao y , spoerke jm , et al : development of a robust rna - based classifier to accurately determine er , pr , and her2 status in breast cancer clinical samples . 
cieslik m , chugh r , wu ym , et al : the use of exome capture rna - seq for highly degraded rna with application to clinical cancer sequencing . 
 appendix all patients with breast cancer in the south sweden health care region diagnosed between september 1 , 2010 , and march 31 , 2015 who underwent operation for invasive primary * breast cancer ( n = 5 , 892 ) not enrolled ( n = 791 ) patients enrolled in scan - b ( n = 5 , 101 : 87% ) no tumor tissue for study ( n = 1 , 205 ) or tumor tissue obtained after neoadjuvant therapy or previous biopsy ( n = 49 ) enrolled patients with tumor biopsy sample for analysis ( n = 3 , 847 : 75% ) validation cohort with quality controlled rna - seq data ( n = 3 , 273 : 85% ) insufficient or poor quality rna and rna - seq not performed or failed ( n = 464 ) ; rna - seq data failed qc ( n = 110 ) fig a1 . 
for estrogen receptor ( er ) , progesterone receptor ( pgr ) , human epidermal growth factor receptor 2 ( her2 ) , and ki67 clinical histopathology diagnostic results ( y - axis ) , the single - gene classifier ( sgc ) gene expression ( x - axis ) ( a ) or the transformed multigene classifier ( mgc ) score ( x - axis ) ( b ) is plotted for the validation cohort ( circles )  . 
transformed multigene classifier ( mgc ) score ( x - axis ) versus single gene classifier ( sgc ) gene expression ( y - axis ) in the 3 , 273 samples of the independent validation cohort ( circles ) for ( a ) estrogen receptor ( er ) , ( b ) progesterone receptor ( pgr ) , ( c ) human epidermal growth factor receptor 2 ( her2 ) , and ( d ) ki67 . 
vertical dashed lines are drawn at the mgc score threshold of 0 to distinguish the classes , and horizontal dotted lines are drawn at the sgc gene expression thresholds determined from the training cohort . 
kaplan - meier overall survival estimates for histopathology , single - gene classifiers ( sgcs ) , and multigene classifiers ( mgcs ) within the validation cohort ( neg , classified as negative ; pos , classified as positive ; grade [ g ] 1 , g2 , or g3 )  . 
the biomarker is indicated at the far left , and the number of tumor cases with complete data across pathology , sgc , and mgc for a given biomarker is shown below each biomarker name . 
in columns are plotted the kaplan - meier survival curves for each classification : ( left ) pathology , ( middle ) sgc , and ( right column ) mgc . 
the log - rank p value is displayed , and horizontal dashed lines are drawn to aid identification of kaplan - meier estimates with the poorest outcome classification group within each row . 
at risk : concordant hormone nonresponsive concordant hormone responsive path hormone nonresponsive / mgc hormone responsive path hormone responsive / mgc hormone nonresponsive log - rank p < .001 concordant hormone nonresponsive concordant hormone responsive pathology hormone nonresponsive / mgc hormone responsive pathology hormone responsive / mgc hormone nonresponsive time ( years ) 2 , 310 2 , 302 2 , 262 1 , 946 1 , 413 fig a5 . 
kaplan - meier overall survival estimates for groups defined by pathology ( path ) versus multigene classifiers ( mgcs ) within the validation cohort ; the log - rank p value is given . 
survival curves , number of samples classied as tmb high , and predicted neoantigens were used to evaluate the differences between thresholds . results the distribution of tmb varied dramatically among cancer types . 
2019 by american society of clinical oncology introduction immunotherapy ; immunotherapy , especially checkpoint inhibition , has been added recently as a successful treatment option for some patients with cancer . 
it is based on the presence of neoantigens eliciting a response from t cells by recognizing these antigens as foreign and inltrating the tumor microenvironment.1 , 2 however , only a subset of patients respond to checkpoint inhibitor therefore , predictive biomarkers are critical to guide the selection of patients for these therapies . 
although high - level programmed death - ligand 1 expression , microsatellite instability , and mismatch - repair deciency have been deemed including tumor clinically relevant , other markers , mutational burden ( tmb ) , interferon gamma prole , and human leukocyte antigen ( hla ) genotype , have also exhibited promising results.3 tmb is commonly dened as the total number of somatic mutations in a tumor genome , divided by the number of bases sequenced in megabases ( mb ) .4 , 5 previously , patients with higher tmb ( tmb high ) demonstrated increased response to immunotherapy compared with patients with lower tmb ( tmb low ) , especially in nonsmallcell lung cancer.6 , 7 however , no consensus has been established to dene a standard threshold for tmb high . 
last , previous studies have adopted panel target sequencing with small regions of genomic dna , possibly excluding neoantigen candidates.8 therefore , exploring the tmb distribution with more extensive sequencing platforms , such as whole exome sequencing ( wes ) , can provide a more comprehensive view of tmb . 
 fernandez et al context threshold ? key objective can a cancer - specic tumor mutational burden ( tmb ) provide more useful information compared with a xed pan - cancer knowledge generated a cancer - specic tmb threshold can dynamically adjust to identify dramatically more patients as tmb high compared with a restrictive pan - cancer threshold . 
the additional patients do not have a signicantly different outcome compared with the other tmb - high patients . relevance when interpreting a patients tmb , the overall tmb seen in the specic cancer type should be taken into consideration to give the tmb context . reect tmb in each cancer type , the wcm threshold uses interquartile range ( iqr ) : mean ( tmb ) + 1.25 iqr ( tmb ) applied within each cancer , meaning each cancer type will have its own threshold reective of the distribution of mutations within that cancer type . 
predicted neoantigens for tcga samples were obtained through the cancer immunome atlas.23 tmb calculation tmb was calculated as the total number of nonsynonymous mutated bases in the tumor genome divided by the mb of the genome covered . 
the wcm advanced cohort was sequenced using our exact - 1 wes haloplex pipeline using tumor - normal pairs , 24 which covers 37 mb of the genome . samples were collected under a protocol approved by the institutional review board of weill cornell medical college , and written informed consent was obtained . 
because tmb is divided by the total size of genome sequenced to help adjust for technical batch effects , the size of sequenced regions was determined for each tcga cohort according to their respective publications.11 - 22 samples with purity of less than 50% according to absolute ( tcga ) or clonality estimate in tumors ( exact - 1 ; weill cornell medine clinical genomics lab , new york , ny ) were removed.25 , 26 germline variants and variants appearing in the exome aggregation consortium database with a frequency of at least 1% were removed to lter them out.27 variants with a variant allele frequency ( vaf ) of less than 10% were ltered out after correcting vaf for tumor purity by dividing vaf by purity . 
a summary of ltering steps can be seen in the data supplement . mutational signatures mutational signatures were determined using the deconstructsigs r package.28 for each sample , a matrix containing the frequency a mutation appears within a trinucleotide context was generated . 
this matrix was compared with the signatures published previously in the catalogue of somatic mutations in cancer , 29 resulting in signature contributions . statistical methods the wilcoxon rank sum test was used to compare sample distributions . 
for example , the tmb for prostate cancer in tcga ranged from 0.03 mutations / mb to 14.13 distribution of tmb for tcga samples cancer type distribution of tmb for wcm advanced samples threshold chalmers et al threshold chalmers et al cancer type fig 1 . 
however , the tmb for bladder cancer in tcga ranged from 0.04 mutations / mb to 99.68 mutations / mb , with a mean of 6.92 mutations / mb ( n = 375 )  . because of the higher frequency of metastatic samples in the wcm advanced cohort , we sought to understand how the distribution of tmb changes between primary and metastatic samples . 
the chalmers et al threshold is a constant cutoff of 20 mutations / mb for all cancer types , 4 whereas our custom wcm threshold is a formula of mean ( tmb ) + 1.25 iqr ( tmb ) , adapted from a study by zehir et al.5 the wcm threshold was applied per cancer to account for the variation in tmb among cancer types . data for patients treated with immunotherapy were not available for either cohort ; however , tcga survival outcomes were investigated to understand the differences in survival between tmb high and tmb low using both thresholds . 
tmb - high patients showed signicantly improved survival compared with tmb - low patients for blca using both the wcm threshold ( log - rank p value = .0014 ) and the chalmers et al threshold ( log - rank p value = .009 ; data supplement )  . 
lusc showed improved survival for tmb - high patients compared with tmb - low patients when using the wcm cutoff ( log - rank p value = .036 ) but not when using the chalmers et al cutoff ( log - rank p value = .11 ; data supplement )  . 
ucec also showed signicantly improved survival for tmb - high patients compared with tmb - low patients when using chalmers et al ( log - rank p value = .023 ) but did not reach signicance when using wcm ( log - rank p value = .055 ; data supplement )  . 
to further compare the performances of the two thresholds , timedependent roc curves were created at the 5 - year time point ( fig 2d ; data supplement )  . 
to explore the difference between the patients selected by the thresholds , we split the samples into three groups : tmb high , wcm - tmb high , and tmb low . 
the wcm - tmbhigh group contained samples classied as tmb high by wcm and tmb low by chalmers et al ( when chalmers et al had more tmb high classications [ ucec ] than wcm , we used chalmers - tmb high [ chm - tmb high ] )  . 
the tmb - low group contained samples classied as tmb low by both methods ( fig 2a )  . the wcm - tmbhigh group did not attain signicantly improved survival compared with the tmb - low group for blca ( log - rank p value = .072 ; fig 2b )  . 
the chalmers et al threshold did not classify any patients with kirc as tmb high . although the survival trends were not signicantly different between the cutoffs , the number of samples classied as tmb high differed between thresholds . 
out of 14 tcga cancer types investigated , eight cancers had a statistically larger tmb - high group for wcm compared with chalmers et al ( blca [ fold change = 1.9 ] , brca [ fold change = 6.7 ] , gbm [ fold change = 2.7 ] , kirc [ no high by chalmers ] , lgg [ fold change = 13.0 ] , ov [ fold change = 5.67 ] , prad [ no high by chalmers ] , and thca [ no high by chalmers ] ) ; ve cancers had no statistically signicant change in the number of tmb - high classications ( coad , kich , luad , lusc , and read ) ; and only ucec had a large number of tmb high classied by chalmers et al compared with wcm ( 2 test with yatess continuity correction ; p value , .05 ; fig 3a )  . 
predicted neoantigens from the cancer immunome atlas compared the tmbhigh , the wcm - tmbhigh , and the tmb - low groups for tcga samples ( fig 4a ) .23 the tmb - low group had signicantly fewer neoantigens than both the tmb - high ( wilcoxon p value , 2.3e - 16 ) and wcm - tmbhigh ( wilcoxon p value = 2.1e - 14 ) group . 
kaplan - meier survival curves comparing the tmb classications between the weill cornell medicine ( wcm ) threshold and the chalmers et al threshold for ( b ) blca and ( c ) kirc . 
for kirc , the chalmers et al threshold was too high to classify any samples as tmb high , resulting in only two groups : wcm - tmb high and tmb low . 
 ( d ) time - dependent receiver operating characteristic ( roc ) curve comparing the true positive ( tpr ) and false positive ( fpr ) rates for blca using both the chalmers et al threshold and the wcm threshold shows the area under the curve ( auc ) for both cutoffs at 5 years from diagnosis . 
because of the observed variation in tmb among cancer types , neoantigens were also compared between the tmb groups for each cancer ( figs 4b and 4c ; data supplement )  . 
prostate adenocarcinoma ( prad ) and kidney renal clear cell carcinoma ( kirc ) are excluded from the plot because the chalmers et al threshold did not classify any samples as tmb high . 
blue bars represent cancer types with a higher or equal number of tmb - high classications for the weill cornell medicine ( wcm ) threshold than the chalmers et al threshold . 
overall , the tmb - high and wcm - tmbhigh groups showed increased neoantigen counts compared with the tmb - low group . finally , mutational signatures were compared among tmb groups . 
distribution of predicted neoantigens for tmb high ( blue ) , tmb high weill cornell medicine ( wcm ; red ) , and tmb low ( teal ) for the cancer genome atlas ( tcga ) shown for ( a ) all cancers pooled together , ( b ) lung adenocarcinoma ( luad ) , and ( c ) bladder urothelial carcinoma ( blca )  . 
 ( a ) violin plots showing the distribution of the contribution of signature 10 ( defective polymerase - ) in tumor mutational burden ( tmb ) high , chalmers ( chm ) - tmb high , and tmb low . 
 ( b ) pca showing the signature contributions for uterine corpus endemetrial carcinoma ( ucec ) with tmb - high samples in blue , wcm - tmbhigh samples in red , and tmb - low samples in teal . 
only signatures 2 , 4 , 6 , 10 , 13 , 15 , 20 , 21 , and 26 were considered , to focus on signatures with the most relevant interpretations . 
for example , the chalmers et al cutoff of 20 mutations / mb is higher than the maximum tmb seen within tcga prostate cancer ( 14.13 mutations / mb ) ; however , using a lower threshold might be too lenient for tcga bladder cancer ( maximum tmb of 99.68 mutations / mb )  . 
the dramatic difference in tmb among cancer types led us to explore a tmb threshold applied in a cancer - specic manner . to a cancer - specic when comparing a pan - cancer threshold , we found we were able to maintain differences in survival trends between tmb high and tmb low , often while including more patients in the tmb - high group by using the cancer - specic threshold . 
using a tmb threshold that has a larger number of tmb - high classications while not having signicantly different survival trends than a more stringent threshold can potentially open treatment options to patients if they can be shown to respond well . 
hr , hazard ratio ; os , overall survival ; wcm , weill cornell medicine . seen in recent publications suggests that tmb alone is not a critical predictor of survival for nonimmunotherapy treatment response.6 , 37 , 38 differences in tmb distribution between the tcga and wcm advanced cohorts could be a result of biologic or technical differences . 
tcga contains primary tumor samples , and wcm advanced contains mostly metastatic tumor samples . when comparing different cohorts of patients , many technical differences can inuence tmb , making a static threshold potentially less accurate . 
after sequencing , the exact - 1 pipeline and tcga pipeline use different mutation callers and apply different quality lters . in addition , when comparing tmb among studies , it is important to understand the variants that were considered . 
 fernandez et al calculating tmb.6 , 38 , 39 targeted panels cover different genes and sizes of the genome and at a higher sequencing depth than wes , inuencing the estimated distribution of tmb . 
the panel must cover at least 2 mb of the genome to have high sensitivity and specicity when measuring tmb.8 a static threshold would not be able to adjust to the differing levels of tmb resulting from different panels . 
however , a dynamic threshold would be able to adjust for technical differences . the cancer - specic threshold adjusted for the variation in tmb ; however , the pan - cancer threshold classied more patients as tmb high for ucec . 
mutational signatures for these samples showed a cluster of patients with pole deciency , which led to a large number of mutations and a tmb distribution with a large iqr , 22 causing the cancerspecic threshold to be strict in this case . the survival analyses in this study were limited by a lack of patients undergoing immunotherapy treatment . 
in addition , the number of predicted neoantigens was compared per cancer , showing that a cancer - specic threshold identies patients with a higher number of neoantigens compared with tmb - low patients . moreover , tmb might depict only one factor in the success of immunotherapy . 
a recent study has shown that patients with melanoma and nonsmall - cell lung cancer with heterozygous hla class i exhibit improved survival compared with patients who are homozygous.40 because of the limitation of tcga data , we could not examine the role of hla class i in the current study . 
fernandez employment : celgene travel , accommodations , expenses : foundation medicine himisha beltran consulting or advisory role : janssen oncology , genzyme , glaxosmithkline , abbvie , astellas pharma , astrazeneca research funding : janssen ( inst ) , abbvie / stemcentrx ( inst ) , eli lilly ( inst ) travel , accommodations , expenses : janssen oncology bishoy m . 
faltas honoraria : digital science press publications research funding : eli lilly juan miguel mosquera research funding : personal genome diagnostics travel , accommodations , expenses : personal genome diagnostics david m . 
hoffmann la roche ag , novartis , astellas pharma research funding : eli lilly , janssen , millenium pharmaceuticals , sano patents , royalties , other intellectual property : us patent ( 7 , 767 , 393 and 7 , 229 , 774 ) , expression prole of prostate cancer , 2007 ; us patent ( 7 , 332 , 290 ) , detection of amacr cancer markers in urine , 2008 ; us patent ( 7 , 718 , 369 ) , recurrent gene fusions in prostate cancer , 2010 ; us patent ( 7 , 803 , 552 and 7 , 666 , 595 ) , biomarkers for predicting prostate cancer progression , 2010 ; us patent ( 7 , 981 , 609 b2 ) , methods for identifying and using snp panels , 2011 ; us patent ( 8 , 106 , 037 b2 ) , identication and treatment of estrogen responsive pca , 2012 ; us patent ( 9 , 090 , 899 b2 ) , methods of diagnosing and treating prostate cancer characterized by ndrg1 - erg fusion , 2015 ; us patent ( 9 , 458 , 213 b2 ) , compositions and methods for diagnosing prostate cancer based on detection of slc45a3 - elk4 fusion transcript , 2016 ; us patent ( 9 , 568 , 483 b2 ) , molecular subtyping , prognosis and treatment of prostate cancer , 2017 ; us patent ( 9 , 678 , 077 b2 ) , erg / tff3 / hmwck triple immunostain for detection of prostate cancer , 2017 ; us patent ( 61 , 408 , 341 ) , exploration of novel gene fusion in prostate cancer by rna - seq travel , accommodations , expenses : f . 
shah consulting or advisory role : astellas pharma , eli lilly japan research funding : gilead sciences ( inst ) , merck ( inst ) , boston biomedical ( inst ) , oncolys biopharma ( inst ) , bristol - myers squibb ( inst ) wei song employment : genentech ( i ) employment : cytokinetics ( i ) honoraria : foundation medicine , loxo consulting or advisory role : foundation medicine , loxo no other potential conicts of interest were reported . acknowledgment we thank juan sebastian andrade martinez for his previous work with the wcm advanced cohort and bhavneet bhinder for her advice on predicted neoantigens . references 2018 topalian sl , weiner gj , pardoll dm : cancer immunotherapy comes of age . 
j clin oncol 29 : 4828 - 4836 , 2011 topalian sl , hodi fs , brahmer jr , et al : safety , activity , and immune correlates of anti - pd - 1 antibody in cancer . 
n engl j med 366 : 2443 - 2454 , 2012 tray n , weber js , adams s : predictive biomarkers for checkpoint immunotherapy : current status and challenges for clinical application . 
cancer immunol res 6 : 1122 - 1128 , 2018 chalmers zr , connelly cf , fabrizio d , et al : analysis of 100 , 000 human cancer genomes reveals the landscape of tumor mutational burden . 
genome med 9 : 34 , 2017 zehir a , benayed r , shah rh , et al : mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10 , 000 patients . 
n engl j med 378 : 2093 - 2104 , rosenberg je , hoffman - censits j , powles t , et al : atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum - based chemotherapy : a single - arm , multicentre , phase 2 trial . 
lancet 387 : 1909 - 1920 , 2016 buchhalter i , rempel e , endris v , et al : size matters : dissecting key parameters for panel - based tumor mutational burden analysis . 
plimack er , dunbrack rl , brennan ta , et al : defects in dna repair genes predict response to neoadjuvant cisplatin - based chemotherapy in muscle - invasive bladder cancer . 
biometrics 56 : 337 - 344 , 2000 johnson db , frampton gm , rioth mj , et al : targeted next generation sequencing identies markers of response to pd - 1 blockade . 
rizvi h , sanchez - vega f , la k , et al : molecular determinants of response to anti - programmed cell death ( pd ) - 1 and anti - programmed death - ligand 1 ( pd - l1 ) blockade in patients with non - small - cell lung cancer proled with targeted next - generation sequencing . 
 no evidence of increased risk of breast cancer in women with lynch syndrome identied by multigene panel testing jessica stoll , ms , cgc1 ; eric rosenthal , scm , phd2 ; shelly cummings , ms , cgc2 ; jamie willmott , ms , cgc2 ; ryan bernhisel , mstat2 ; and sonia s . 
kupfer , md1 purpose prior estimates of breast cancer risk in women with lynch syndrome ( ls ) range from population risk to 18 - fold increased risk with reported differences by gene . 
standardized incidence ratios ( sirs ) and 95% cis of breast cancer were calculated compared with age - matched incidence in the general us female population and with women negative for pvs stratied by the test indication . results in total , 0.8% of women ( 30 , 362 of 441 , 966 women ) carried mmr gene pvs . 
women with pvs in pms2 and msh6 were tested more frequently for hboc , whereas those with pvs in mlh1 and msh2 / epcam were tested more frequently for ls . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction breast cancer risk in lynch syndrome ( ls ) has been debated , with published data indicating anywhere from no increased risk1 - 4 up to 18 - fold increased risk for women with a pathogenic variant ( pv ) in a mismatch repair ( mmr ) gene.5 - 9 a recent laboratorybased study reported a 2to 3 - fold increased incidence of breast cancer in women with pvs in msh6 and pms2 compared with the general us female population , but not in mlh1 or msh2 carriers.10 these authors used us female population data from the seer registry ; however , comparing cancer histories in a population heavily weighted toward women being assessed for hereditary breast and ovarian cancer ( hboc ) to cancer histories in the general population could result in ascertainment bias.11 - 13 specically , this approach likely increases the proportion of the sample with a personal diagnosis of breast cancer , resulting in elevated breast cancer rates regardless of mmr gene status . 
this ascertainment bias might be less evident for women with pvs in the higher penetrance mmr genes ( ie , mlh1 , msh2 , and epcam ) who are more likely to be ascertained for testing on the basis of personal or family history of early - onset colorectal or endometrial cancer or other ls - associated cancers . therefore , to properly estimate cancer risk , women with pvs should be compared with pv - negative women within the same testing population . quantifying breast cancer risk in women with ls has signicant implications for screening and risk reduction , especially for patients with ls caused by pvs in msh6 and pms2 , which are more prevalent than previously thought . 
 stoll et al context key objective using a large , laboratory - based database , we determined whether women with lynch syndrome ( ls ) are at increased risk of breast cancer compared with the general population and to a population undergoing genetic testing . knowledge generated women with ls are not at increased risk of breast cancer . 
these ndings are in line with results from the prospective ls database but contrast with other laboratory - based studies . relevance these ndings support average - risk breast cancer screening in women with ls . ascertained for suspicion of hboc and / or ls but were not found to have a pv by multigene panel testing . methods study population women tested with a single commercial laboratorys ( myriad genetic laboratories , salt lake city , ut ) multigene panel between september 2013 and november 2018 were included unless they resided in a state prohibiting use of deidentied genetic or clinical information for research . 
clinical and demographic data for participants were obtained from test request forms submitted by ordering health care providers . ls - associated cancers included colorectal , endometrial , ovarian , pancreatic , stomach , gastric , ureter , brain , small intestine , and biliary tract cancers . 
hboc - associated cancers included breast , ovarian , prostate , and pancreatic cancers . testing criteria individuals met providers indicated on the test requisition forms whether testing was ordered for suspicion of ls or hboc . 
for hboc , 2013 national comprehensive cancer network ( nccn ) guidelines were used , 14 but the contribution of prostate cancer was excluded because gleason score or metastatic disease information was not collected reliably . these exclusions were not expected to substantially inuence results but may have resulted in an underestimate of the number of women meeting hboc criteria . 
to directly evaluate rates against the recent publication that established a 2to 3fold elevated breast cancer incidence for msh6 and pms2 , 10 the us female population from the seer registry17 was used as one comparator group . 
to compensate for the ascertainment bias expected in a population selected for testing on the basis of suspicion of hereditary cancer risk , we also calculated the sirs using the following 3 additional comparator populations : women who tested negative for any pv who were ascertained for suspicion of hboc or ls ; women who were ascertained for hboc only ; and women who were ascertained for ls only . 
all analyses were performed using sas software ( sas institute , cary , nc )  . results demographic and clinical characteristics in total , 441 , 996 women met inclusion criteria ( appendix table a1 )  . 
among these , 3 , 362 women ( 0.8% ) were found to carry a single pv in an mmr gene ( mlh1 , msh2 , msh6 , pms2 , or epcam ) , and 438 , 634 women ( 99.2% ) were negative for pvs in any of the other genes included on the panel . 
epcam deletions were identied in only 11 women and therefore were included with msh2 for all analyses . demographic and clinical characteristics for women with mmr gene pv and all women tested are listed in table 1 . for women with an mmr gene pv , median age at testing was 47 years , and 56.8% of women reported white or caucasian ancestry . 
demographic and clinical characteristics of women with mmr gene pvs and all women undergoing multigene panel testing all women tested ( n = 441 , 996 ) women with mmr gene pv ( n = 3 , 362 ) characteristic age at testing , yearsa mean ( sd ) range mean ( sd ) range age at bc diagnosis , yearsb self - reported ancestry , no . 
similar differences in mutation spectrum were found for women meeting nccn testing criteria for ls versus hboc ( fig 1 )  . the majority of mlh1 and msh2 / epcam mutation carriers were ascertained for clinical suspicion of ls , whereas msh6 and pms2 mutation carriers were more commonly ascertained for suspicion of hboc ( fig 2 )  . phenotypic spectrum the majority of women with pvs in mlh1 and msh2 / epcam were affected by cancer , whereas most pms2 carriers were unaffected . 
for msh2 / epcam carriers , endometrial and colorectal cancers were the most common cancers reported ( n = 162 [ 24.8% ] and n = 150 [ 23.0% ] , respectively )  . 
carriers of mlh1 and msh2 / epcam pvs had lower rates of breast cancer , whereas there was no difference in sirs for msh6 or pms2 carriers compared with seer population breast cancer rates ( table 2 )  . table 3 lists sirs for women with mmr gene pvs compared with women with negative panel testing . 
when all women ascertained for hboc or ls were analyzed , breast cancer rates were lower in women with pvs in mmr genes . specically , breast cancer rates ranged from 22% to 50% lower than expected among the various mmr genes . 
similarly , when compared with women ascertained for suspicion of ls , breast cancer rates also did not differ between women with mmr gene pvs compared with pv - negative women . 
distribution of pathogenic variants ( pvs ) by mismatch repair gene on the basis of ascertainment for testing indicated by ordering provider and testing criteria met ( mlh1 , n = 462 ; msh2 / epcam , n = 653 ; msh6 , n = 985 ; pms2 , n = 1 , 262 )  . 
prevalence of mismatch repair pathogenic variants by ascertainment for lynch syndrome ( ls ) and hereditary breast and ovarian cancer syndrome ( hboc ) testing as indicated by ordering provider ( mlh1 , n = 462 ; msh2 / epcam , n = 653 ; msh6 , n = 985 ; pms2 , n = 1 , 262 )  . discussion data from multigene panel testing in ls have called into question the unresolved issue of whether breast cancer risk is increased in ls . 
although there is a wealth of existing literature on breast cancer in ls , data from these studies are conicting , with risk estimates ranging from no increased risk1 - 4 up to 18 - fold increased risk.5 - 9 recent studies report an increased breast cancer risk in women with pvs in msh6 and pms2 , but not in mlh1 and msh2 / epcam , 10 or an increased risk of breast cancer exclusive to msh6.19 in the present laboratory - based study , including a large sample of msh6 and pms2 pv carriers , previously published data showing an increased breast cancer risk in women with pvs in msh6 and / or pms2 were not replicated when the same methodology was used.10 moreover , the rate of breast cancer was found to be lower than expected among mlh1 and msh2 / epcam carriers . 
findings from the current study are in line with a recent report from the prospective ls database.20 the ndings in the current study likely differ from previous studies showing increased breast cancer risk in ls for several reasons . 
first , the current study includes the largest number of mmr gene pv carriers reported to date , including large numbers of msh6 and pms2 carriers , thereby increasing precision in breast cancer risk estimates compared with more moderately sized studies . 
moreover , this sample included a large proportion of women unaffected by cancer , which appropriately shifts the breast cancer estimates closer to those of the general us female population . colon endometrial breast ovarian other * unaffected mlh1 msh2 / epcam msh6 pms2 no mutation fig 3 . 
in these analyses , no difference in breast cancer incidence was found among women with or without mmr gene pvs whether they were ascertained for suspicion of hboc or ls . 
these results support current guidelines to follow average - risk breast cancer screening recommendations in all women with ls.22 consistent with ndings from other laboratory and population - based studies , these data demonstrate that pvs in msh6 and pms2 account for a much higher proportion of ls than previously estimated.10 , 23 also similar to previous studies , 23 , 24 this study found that the current ls testing criteria perform reasonably well for women with pvs in mlh1 and msh2 but miss close to half of women with pvs in msh6 and pms2 . 
 no increased breast cancer risk in lynch syndrome these results suggest that current clinical criteria are insufcient for identication of the full spectrum of patients with ls who would benet from appropriate surveillance.25 one limitation inherent to utilization of a laboratory - based testing population is that clinical information was limited to that which was included on the test requisition form as provided by ordering clinicians and therefore may not be wholly inclusive of individuals personal and family histories . 
however , we obtained essentially the same ndings through comparisons between different populations within this study , suggesting that underor overreporting of cancer history in any of these populations did not affect the ndings . 
although the study was designed specically to address ascertainment bias , the population included only women undergoing genetic testing for clinical suspicion of a hereditary cancer syndrome and may not be representative of the more general population with ls . however , this was at least in part mitigated by the fact that the comparator population was from the same testing population to ensure that any population differences between women with and without pvs were valid . in the largest study of mmr gene pv carriers identied by a multigene panel through a commercial laboratory , there was no evidence of increased breast cancer risk in women with pvs in mmr genes compared with the general us female population or with woman undergoing multigene panel testing . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . jessica stoll consulting or advisory role : invitae travel , accommodations , expenses : invitae eric rosenthal employment : myriad genetic laboratories stock and other ownership interests : myriad genetic laboratories shelly cummings employment : myriad genetics stock and other ownership interests : myriad genetics travel , accommodations , expenses : myriad genetics jamie willmott employment : myriad genetics stock and other ownership interests : myriad genetics research funding : myriad genetics travel , accommodations , expenses : myriad genetics ryan bernhisel employment : myriad genetics sonia s . 
kupfer honoraria : advance medical no other potential conicts of interest were reported . references baglietto l , lindor nm , dowty jg , et al : risks of lynch syndrome cancers for msh6 mutation carriers . 
j natl cancer inst 102 : 193 - 201 , 2010 pande m , wei c , chen j , et al : cancer spectrum in dna mismatch repair gene mutation carriers : results from a hospital based lynch syndrome registry . 
mller p , sepp al a tt , bernstein i , et al : cancer risk and survival in path_mmr carriers by gene and gender up to 75 years of age : a report from the prospective lynch syndrome database . 
gut 67 : 1306 - 1316 , 2018 therkildsen c , ladelund s , smith - hansen l , et al : towards geneand gender - based risk estimates in lynch syndrome ; age - specic incidences for 13 extracolorectal cancer types . 
 stoll et al oliveira ferreira f , napoli ferreira cc , rossi bm , et al : frequency of extra - colonic tumors in hereditary nonpolyposis colorectal cancer ( hnpcc ) and familial colorectal cancer ( fcc ) brazilian families : an analysis by a brazilian hereditary colorectal cancer institutional registry . 
fam cancer 3 : 41 - 47 , 2004 scott rj , mcphillips m , meldrum cj , et al : hereditary nonpolyposis colorectal cancer in 95 families : differences and similarities between mutation - positive and mutation - negative kindreds . 
win ak , young jp , lindor nm , et al : colorectal and other cancer risks for carriers and noncarriers from families with a dna mismatch repair gene mutation : a prospective cohort study . 
j clin oncol 30 : 958 - 964 , 2012 goldberg m , bell k , aronson m , et al : association between the lynch syndrome gene msh2 and breast cancer susceptibility in a canadian familial cancer registry . 
harkness ef , barrow e , newton k , et al : lynch syndrome caused by mlh1 mutations is associated with an increased risk of breast cancer : a cohort study . j med genet 52 : 553 - 556 , 2015 genet med 20 : 1167 - 1174 , 2018 10 . 
2018 : is breast cancer truly caused by msh6 and pms2 variants or is it simply due to a high prevalence of these variants in the population ? genet med 21 : 256 - 257 , 2019 12 . 
evans dg , woodward er , lalloo f , et al : are women with pathogenic variants in pms2 and msh6 really at high lifetime risk of breast cancer ? genet med , 2018 13 . 
dominguez - valentin m , sampson jr , sepp al a tt , et al : cancer risks by gene , age , and gender in 6350 carriers of pathogenic mismatch repair variants : findings from the prospective lynch syndrome database . 
giardiello fm , allen ji , axilbund je , et al : guidelines on genetic evaluation and management of lynch syndrome : a consensus statement by the us multisociety task force on colorectal cancer . 
klein , md2 purpose to assess the association between the oncotype dx genomic prostate score ( gps ) result and longterm oncological outcomes following radical prostatectomy ( rp )  . methods we evaluated the association of the gps result assayed from the index lesion from rp tissue with the risk of distant metastases ( dm ) and prostate cancerspecic mortality ( pcsm ) over the 20 years following rp in a stratied cohort sample of 428 patients from 2 , 641 treated between 1987 and 2004 . 
exploratory analysis using presurgical parameters and the gps test as prognostic variables was performed to assess the additional value of the gps test on 20 - year risk of dm and pcsm . 
model discrimination was measured using the area under the receiver operating characteristic curve . results the gps test appears to be independently associated with both 20 - year risk of dm and pcsm with a low false discovery rate . 
accuracy of models including clinical risk factors alone appeared to improve when including the gps test in assessing risk of both end points . conclusion the results suggest that the gps test provides information on the risk for the meaningful long - term outcomes of dm and pcsm . jco precis oncol 5 : 442 - 449 . 
2021 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction long - term cancer outcomes are an important consideration when deciding between active surveillance ( as ) and immediate treatment for newly diagnosed prostate cancer . 
multiple prospective as studies that predominantly include patients at the lowest risk of progression have demonstrated a low risk of distant metastasis ( dm ) and prostate cancerspecic mortality ( pcsm ) with extended follow - up.1 , 2 however , these excellent outcomes with as require strict selection criteria and stringent follow - up with frequent repeat prostate biopsies . 
based on these studies , the current use of as for newly diagnosed men is increasing and now includes expanded selection criteria , including younger men with longer life expectancy and those with biopsy - dened pathologic features that fall outside eligibility criteria of older studies.3 it is long - term as outunknown whether the excellent comes observed for low - risk disease can be maintained in this expanded group using only traditional clinical variables and existing surveillance strategies . for example , although both pivot and protect showed no statistically signicant difference in pcsm between observation and immediate treatment intermediate - risk patients with up to a 12.7 - year followup , 4 , 5 the condence intervals for the hazard ratio ( hr ) do not exclude substantial benet of radical prostatectomy ( rp )  . 
furthermore , the long - term results of scandinavian prostate cancer group trial 4 demonstrated an 11.7% absolute risk reduction in pcsm with rp , corresponding to a relative risk of 0.55 , compared with conservative management.6 with up to a 29 - year follow - up , the results of this study are more reective of the lifetime risk of cancer recurrence and death in men with intermediateor high - risk features . 
 gps assay associated with long - term prostate cancer outcomes context key objective localized prostate cancer has an extended natural history , making immediate treatment decisions difcult without the understanding of long - term cancer risks . 
we sought to evaluate the association between the genomic prostate score ( gps ) test and long - term prostate cancer outcomes : distant metastasis and prostate cancerspecic mortality following radical prostatectomy . knowledge generated the gps test appeared to be associated with both distant metastasis and prostate cancerspecic mortality at a 20 - year followup in both univariable and multivariable models , where model discrimination was improved when the gps results were included . relevance the use of the gps test can provide risk assessment of long - term prostate cancer outcomes , beyond clinical factors alone , for patients with localized prostate cancer . traditionally used clinical information in assessing risk of adverse pathology ( ap ) at rp.7 , 8 van den eeden et al9 established the association of the gps test with risk of dm and pcsm over 10 years . 
in this report , we sought to evaluate whether the gps test is also associated with 20 - year risk of dm and pcsm and whether its use improves risk assessment for these end points compared with clinical variables alone . methods using a stratied cohort sampling design , so that a weighted analysis of the study cohort is representative of all patients ( n = 2 , 641 ) who underwent rp between 1987 and 2004 at our institution , we selected 501 patients for inclusion . 
among the remaining 441 patients , the nal analysis cohort comprised the 428 patients with evaluable primary gleason pattern in prostatectomy tissue ( fig 1 )  . all patients were selected from our prospective institutional review board ( irb ) approved database that includes clinical staging , pathology from biopsy and rp , and followup information . 
follow - up and outcome data were obtained through subsequent clinic visits , telephone calls , and semiannual follow - up letters obtained through august 1 , 2019 , approximately 10 years after the previously reported cutoff.7 multiple data reviews and quality checks were performed to ensure delity of the data set . high grade at rp was dened as grade group 3 or above ( gleason score 4 + 3 or higher )  . 
the time to event in patients who died without an event or who were alive without an event at the end of follow - up was considered censored in these analyses . 
each patient was weighted using the inverse sampling fraction in his stratulin and weis robust variance estimate was used.12 in the multivariable analysis , the gps result , preoperative prostatespecic antigen ( psa ) , clinical stage , and biopsy grade were used as covariables . 
the hr for continuous gps results was reported per 20 - gps unit increase , which approximated the difference between the average gps value in the highest 25% and the average gps value in the lowest 25% of patients based on the gps value distribution from previous studies.7 , 13 preoperative psa value was included in the model as log2 ( psa value )  . 
characteristics of the patients in the cohort sample biopsy gleason grade , % brooks et al 61 ( 6 ) 62 ( 57 - 66 ) mean ( sd ) median ( iqr ) race , % white black / afro - caribbean asian / hispanic / others year of surgery , % surgical gleason grade results clinical tumor stage , % pathologic tumor stage % 3 + 4 3 + 5 4 + 3 4 + 5 5 , 5 + 4 low / very low intermediate high 1987 - 1989 1990 - 1994 1995 - 1999 2000 - 2004  . 
summary statistics and percentages are estimated using cohort sampling weights so they estimate the distribution of characteristics in the full cohort of 2 , 641 patients from which the cohort sample ( n = 428 ) was drawn . abbreviations : aua , american urological association ; psa , prostate - specic antigen . for overoptimism of effect estimates involving the gps test . therefore , all estimates using the gps test as a covariable were corrected for regression to the mean ( rm ) 14 and false discovery rates ( fdrs ) were reported rather than p values ( data supplement )  . 
applying the rm correction for gps test , the absolute risk estimates were computed as the weighted average of the absolute risk estimates over the study population patient age for each ap status , with cis derived using the delta method . 
receiver operating characteristic ( roc ) curve estimates were based on these absolute risk estimates , correcting for bias 444 2021 by american society of clinical oncology inherent in roc curves ( data supplement ) , with and without the gps results as a covariable . 
differences between models in the roc area under the curve ( auc ) were tested for signicance using the bootstrap . the overall cohort consisted of predominantly american urological association ( aua ) lowand intermediate - risk patients with prostate cancer , 55% and 35% , respectively ( table 1 )  . 
the distribution of gps results , accounting for the stratied cohort sampling weighting , in the overall population and by biopsy gleason grade is shown in figure 2 . the gps result appeared to be highly associated with both dm and pcsm at 20 years of follow - up . 
 gps assay associated with long - term prostate cancer outcomes total cohort 2 , 641 patients with prostatectomy 19872004 501 patients selected 127 with clinical recurrence 374 without clinical recurrence 441 patients evaluable for clinical / pathology / gene expression 428 patients with evaluable primary gleason pattern in prostatectomy tissue 51 excluded for insufficient tumor 7 did not meet inclusion criteria 2 outlier gene profile fig 1 . 
of 2 , 641 patients who underwent rp between 1987 and 2004 , a cohort of 501 patients was selected using a 1 : 3 sampling design , which included all 127 patients who did and 374 patients who did not have a clinical recurrence . 
rm - corrected estimates of the 20 - year absolute risk of dm and pcsm as a function of gps result among aua low - risk patients and favorable intermediaterisk patients have functional form similar to the estimates for the overall population , with a large increase in risk for the gps result  . 
the number of events in this subgroup was insufcient for multivariable analysis . discussion previous validation studies have clearly shown the gps test and other prostate biopsybased gene expression proles to predict the presence of ap ( grade group 3 or higher or extraprostatic disease ) in rp specimens or serial needle biopsies in men on as or post - rp.7 , 8 , 15 although ap on biopsy or rp is prognostic regarding risk of recurrence , dm , and pcsm , 16 its use as a surrogate end point for these outcomes in men considering starting or staying on surveillance has been questioned with no clear short - term alternative.17 in this study , we sought to assess whether the gps result , based on pathologic evaluation of the index lesion on rp , in the original discovery cohort of this biomarker appeared to be associated with the more clinically meaningful outcomes of dm and pcsm at long - term followup . 
absolute risks of these outcomes , considering death from causes other than prostate cancer as a competing risk , showed a lowslope relationship with the gps result from 1 to 29 , with an inection point evident at a score 30 , above which the risk of dm or pcsm increased substantially , reaching an absolute risk of 38% for dm and 13% for pcsm at a score of 60 ( fig 3 )  . 
furthermore , we found that including the gps results in models assessing risk of dm and pcsm at 20 years improved discrimination compared with using clinical variables alone ( tables 2 and 3 ; fig 4a and 4b )  . 
other commercially available gene expression proles have reported similar ndings , albeit at earlier time points.18 - 21 treatment because key drivers of biological progression in patients on as are yet to be elucidated , an open question is what aspect of tumor biology commercially available gene expression proles may be measuring . 
detailed pathological analysis of the canary - pass study , a large , well - characterized as cohort , identied the presence of cribriform or stromagenic histology as the strongest predictor of cancer recurrence after in men who progressed to requiring therapy.22 the presence of cribriform glands has been shown in other studies to be associated with higher rates of biochemical recurrence after rp or radiation , as well as both dm and pcsm , 23 , 24 likely driven by the observation that these glands exhibit genomic features characteristic of aggressive disease.25 in a recent study of 194 men with national comprehensive cancer network very low - , low - , or intermediate - risk disease considering as , we observed that only those with the gps results above 29 had cribriform histology present on biopsy ( falzarano et al , manuscript submitted for publication )  . 
gps , genomic prostate score . observed that a gps result 29 was associated with grade reclassication on subsequent biopsy in a cohort of lowand intermediate - risk men on as , whereas in an underpowered study using a cohort of lower - risk men whose mean gps results were 21 , no association with grade progression was observed.17 together , these table 2 . 
however , a number of published observations support a role for decision making in men with higher scores : ( 1 ) multiple biopsy - based studies have validated that higher gps scores are associated with traditional histologic measures of ap ( dened as grade group 3 or extraprostatic disease , both of which are associated with worse outcomes ) 7 - 9 ; ( 2 ) the association of higher gps scores with histologic variants ( cribriform [ falzarano et al , manuscript submitted for publication ] and stromagenic26 histology ) shown to be associated with higher rates of recurrence in an as cohort ; 22 and ( 3 ) long - term data from the current study that link higher gps scores to the risk of meaningful clinical end points ( dm and pcsm )  . 
rm - corrected estimates with 95% condence intervals of the 20 - year absolute risk of distant metastasis ( a ) and prostate cancerspecic mortality ( b ) as a function of the gps result . 
 a 100% brooks et al 100% gps grade stage spline ( psa ) grade stage spline ( psa ) reference gps grade stage spline ( psa ) grade stage spline ( psa ) reference 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% fig 4 . 
model - based rm - corrected , bias - corrected roc curve for dm ( a ) and pcsm ( b ) within 20 years of surgery with covariables : the gps result , high stage , high grade , and log2 psa . 
auc , area under the curve ; dm , distant metastases ; fpr , false positive rate ; gps , genomic prostate score ; pcsm , prostate cancerspecic mortality ; psa , prostate - specic antigen ; rm , regression to the mean ; roc , receiver operating characteristic ; tpr , true positive rate . strengths of this study include that the data were sourced from a prospectively maintained patient registry with locked baseline clinical , pathologic , and gene expression information ; centralized expert pathology review ; 20 - year followup , the longest reported in any similar study ; and independent verication of the statistical analysis . 
there are two limitations : ( 1 ) changes in standardized biopsy grading and stage migration since initial patient enrollment , although we note that all rp specimens were reviewed by an expert gu pathologist after the majority of substantive changes in prostate cancer grading occurred and ( 2 ) that this should be considered an exploratory analysis since the gps test was developed in this cohort . 
although the study by van den eeden et al9 largely replicates the results of this analysis , we believe that further validation in other cohorts is important to rmly establish our conclusions . in conclusion , with long - term follow - up , the gps test appears to be associated with both dm and pcsm and improves the accuracy of models containing clinical variables alone . 
these ndings suggest that genomic changes in the tumor tissue , quantied by the gps test , provide additional biological insight into the long - term risk of dm and pcsm . this information may be valuable to those considering as . prospective studies should be pursued to validate these results . affiliations 1scott department of urology , baylor college of medicine , houston , tx 2glickman urological and kidney institute , cleveland clinic , cleveland , 3department of anatomic pathology , university of alabama at birmingham , birmingham , al 4department of quantitative health sciences , cleveland clinic , cleveland , oh 5genomic health inc , an exact sciences corporation , redwood city , ca administrative support : tamer aboushwareb , eric a . 
 gps assay associated with long - term prostate cancer outcomes patents , royalties , other intellectual property : patent co - inventor on 17gene prostate assay with genomic health , inc conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . michael crager employment : exact sciences stock and other ownership interests : exact sciences travel , accommodations , expenses : genomic health ruixiao lu employment : genomic health , exact sciences stock and other ownership interests : genomic health , exact sciences consulting or advisory role : mission bio john abran employment : exact sciences stock and other ownership interests : exact sciences tamer aboushwareb employment : exact sciences stock and other ownership interests : exact sciences eric a . 
klein consulting or advisory role : grail no other potential conicts of interest were reported . references 2016 klotz l , vesprini d , sethukavalan p , et al : long - term follow - up of a large active surveillance cohort of patients with prostate cancer . 
j clin oncol 33 : 272 - 277 , 2015 tosoian jj , mamawala m , epstein ji , et al : intermediate and longer - term outcomes from a prospective active - surveillance program for favorable - risk prostate cancer . 
mahal ar , butler s , franco i , et al : conservative management of low - risk prostate cancer among young versus older men in the united states : trends and outcomes from a novel national database . 
n engl j med 377 : 132 - 142 , 2017 bill - axelson a , holmberg l , garmo h , et al : radical prostatectomy or watchful waiting in prostate cancer29 - year follow - up . 
n engl j med 379 : 2319 - 2329 , 2018 klein ea , cooperberg mr , magi - galluzzi c , et al : a 17 - gene assay to predict prostate cancer aggressiveness in the context of gleason grade heterogeneity , tumor multifocality , and biopsy undersampling . 
eur urol 66 : 550 - 560 , 2014 cullen j , rosner il , brand tc , et al : a biopsy - based 17 - gene genomic prostate score predicts recurrence after radical prostatectomy and adverse surgical pathology in a racially diverse population of men with clinically lowand intermediate - risk prostate cancer . 
eur urol 68 : 123 - 131 , 2015 van den eeden sk , lu r , zhang n , et al : a biopsy - based 17 - gene genomic prostate score as a predictor of metastases and prostate cancer death in surgically treated men with clinically localized disease . 
lin dw , zheng y , mckenney jk , et al : 17 - gene genomic prostate score test results in the canary prostate active surveillance study ( pass ) cohort . 
spratt de , youse k , deheshi s , et al : individual patient - level meta - analysis of the performance of the decipher genomic classier in high - risk men after prostatectomy to predict development of metastatic disease . 
klein ea , youse k , haddad z , et al : a genomic classier improves prediction of metastatic disease within 5 years after surgery in node - negative high - risk prostate cancer patients managed by radical prostatectomy without adjuvant therapy . 
cooperberg mr , davicioni e , crisan a , et al : combined value of validated clinical and genomic risk stratication tools for predicting prostate cancer mortality in a high - risk prostatectomy cohort . 
canter dj , freedland s , rajamani s , et al : analysis of the prognostic utility of the cell cycle progression ( ccp ) score generated from needle biopsy in men treated with denitive therapy . 
mckenney jk , wei w , hawley s , et al : histologic grading of prostatic adenocarcinoma can be further optimized : analysis of the relative prognostic strength of individual architectural patterns in 1275 patients from the canary retrospective cohort . 
kweldam cf , wildhagen mf , steyerberg ew , et al : cribriform growth is highly predictive for postoperative metastasis and disease - specic death in gleason score 7 prostate cancer . 
greenland ny , cowan je , chan e , et al : prostate biopsy histopathologic features correlate with a commercial gene expression assays reclassication of patient eur j cancer 66 : 26 - 33 , 2016 alterations . 
kornberg z , cooperberg mr , cowan je , et al : a 17 - gene genomic prostate score as a predictor of adverse pathology in men on active surveillance . 
 c clinical response to antiprogrammed death 1 after response and subsequent progression on antiprogrammed death ligand 1 therapy introduction immune checkpoint inhibitors are rapidly becoming a cornerstone in the treatment of nonsmall cell lung cancer ( nsclc )  . 
the programmed death 1 ( pd - 1 ) receptor and its two ligands , programmed death ligand 1 ( pd - l1 ; b7h1 ) and ligand 2 ( pd - l2 ; b7 - dc ) , negatively regulate t - cell activation , and expression of pd - l1 by tumor cells is an important mechanism of immune evasion.1 - 3 multiple agents have been developed to disrupt tumor - associated immune evasion by targeting either pd - 1 or pdl1 . 
nivolumab and pembrolizumab , both anti pd - 1 agents approved for advanced nsclc progressing after chemotherapy , confer an overall survival benefit and produce response rates in 16% to 23% of unselected patients with nsclc.4 - 6 food and drug administration approval was also recently extended to pembrolizumab in the first - line setting for patients with metastatic nsclc expressing pd - l1.7 atezolizumab , an antipd - l1 agent , has also demonstrated an overall survival benefit and has been approved for use in the second - line setting.8 the growing supply of treatment options has led to new questions of optimal sequencing and choice of therapy . 
in particular , the utility of serial treatment with immune checkpoint inhibitors has not been established . agents targeting pd - l1 block the interaction of pd - l1 expressed on tumor cells and tumorinfiltrating immune cells with pd - 1 and b7.1 expressed on t cells . 
studies of these agents , including atezolizumab , avelumab , and durvalumab , have demonstrated similar response rates and clinical benefit.9 - 14 although the effects of antipd - l1 are predicted to be similar to antipd - 1 , it has been speculated that the variation in mechanism may lead to distinct antitumor and toxicity profiles compared with the antipd - 1 agents.15 however , these agents have not been directly compared . 
he had been diagnosed approximately 2 years earlier with nsclc metastatic to bilateral cervical lymph nodes and bilateral adrenal glands . molecular testing showed that his tumor was egfr and kras wild type and alk translocation negative . 
before this evaluation , he received four lines of therapy , including platinum doublet chemotherapy , single - agent docetaxel , palliative chemoradiation with weekly carboplatin and paclitaxel , and erlotinib . he was enrolled in a phase i clinical trial of an antipd - l1 monoclonal antibody given in 21 - day cycles . 
he experienced only mild ( grade 1 ) rash and hypothyroidishe received a total of 16 cycles of antipd - l1 therapy before treatment was stopped per study protocol . the patient was followed with serial cross - sectional imaging and demonstrated continued pr on imaging for 16 months after therapy completion . 
after seven cycles of treatment , he was noted to have progression of disease in his right axilla and a new retropharyngeal ( rp ) mass . biopsy of the rp mass showed poorly differentiated carcinoma consistent with recurrent nsclc and similar to his initial biopsy . 
 chest , abdomen , and pelvis show a metastatic lesion within the right adrenal gland ( arrow ) before initiation of antiprogrammed death ligand 1 ( pd - l1 ) therapy ( panel a1 ) , which regressed after 16 cycles of treatment with antipd - l1 therapy ( panel a2 )  . 
repeat computed tomography imaging demonstrated progressive disease in the right adrenal gland before initiation of antipd - 1 therapy ( panel a3 ) , with subsequent improvement after 9 months of antipd1 therapy ( panel a4 )  . 
ccrt , concurrent radiotherapy ; lns , lymph nodes ; na , not available . palliative radiation therapy ( 30 gy in 10 fractions ) to both his right axilla and rp space . 
two weeks after radiation therapy , repeat imaging demonstrated stability of the rp mass , decrease in the size of the right axillary lymph node , and enlarging adrenal masses . 
imaging of the face after 6 weeks showed a pr in the rp space , and body imaging after 8 weeks of treatment showed a pr in both the right axilla and right adrenal gland ( fig 1a )  . 
he tolerated treatment well , with no immune - related adverse events for 24 cycles , after which he was noted to have progression limited to his right axilla , with a core biopsy confirming nsclc . 
unfortunately , he died several days after this procedure at an outside facility ; the cause of death was unclear . statement of informed consent all human investigations were performed after approval by a local human investigations committee and in accord with an assurance filed with and approved by the department of health and human services , and all data were anonymized to protect the identities of participants involved in the research . 
informed consent from the patient for such research was obtained . longitudinal assessment of pd - l1 and pd - l2 expression biopsy samples were obtained at the time of diagnosis ( biopsy 1 ) , before repeat treatment with antipd - l1 ( biopsy 2 ) , before treatment with anti - pd - 1 ( biopsy 3 ) , and after progression anti pd - 1 therapy ( biopsy 4 ; fig 1b )  . 
however , tissue for this first sample was extremely limited , and possible pd - l1 positivity could not be ruled out because of the size of the tissue specimen , particularly because subsequent specimens from the patient demonstrated a highly heterogeneous pattern of pd - l1 expression with rare positive stromal cells , whereas pd - l2 staining was robust throughout the tumor . 
after his initial disease progression while receiving antipd - l1 therapy , repeat biopsy demonstrated no tumor pd - l1 staining ( with limited stromal pd - l1 positivity ) , and pd - l2 remained strongly positive ( 70% ; fig 1c )  . 
similar results were demonstrated in biopsy 3 before starting pd - 1 therapy and biopsy 4 after progression on this final line of treatment , with strong pd - l2 expression in tumor , moderate pd - l1 staining in stroma , and absence of pd - l1 expression in tumor . 
representative immunohistochemistry samples from each time point are shown in figure 2 . targeted next - generation sequencing genomic testing using a hybrid capture - based next - generation sequencing platform assessing exons from 315 genes ( foundationone ) of the patients tumor at the time of progression while taking nivolumab ( biopsy 4 ) identified seven potentially functional alterations , including rictor amplification , arid1a t2030fs * 3 , arid2 q904 * , kdm5c e656 * , slit2 g498 * , tet2 e1851 * , and tp53 r280i . 
thirty - three variants of unknown significance were identified , and a total mutation burden of 53 mutations / megabase was estimated , placing this patient in the top 7% of all tumor specimens tested by this method.16 thus , the response observed in this high mutation - load patient was consistent with clinical observations.17 no alterations were identified in jak1 , jak2 , cd274 , pdcd1lg2 , pten , myc , or ctnnb1 in this patient , which were previously reported mechanisms of t - cell exclusion and / or resistance to antipd - 1 therapy.18 - 21 next - generation sequencing data were only available on the final biopsy specimen ; thus , changes in mutation burden over time cannot be assessed . gene expression analysis sufficient tissue for expression analysis ( nanostring pancancer immune profiling panel ) was available for two of the four biopsy time points : post - therapy a , which was sampled before beginning the second treatment course of antipd - l1 , and post - therapy b , sampled at disease progression on antipd - l1 , before radiation therapy followed by nivolumab . 
comparison of the expression of immune genes between these two samples demonstrated substantial downregulation of cd274 ( pd - l1 ) mrna and upregulation of several immunosuppressive genes , including il6 and ptgs2 ( cyclooxygenase - 2 ; fig 3a )  . 
it could be hypothesized that distinct immune checkpoints ( eg , pd - l1 v pd - l2 ) could divergently mediate therapeutic resistance to checkpoint inhibitors and support preclinical studies testing this hypothesis . this patients tumor was pd - l1 negative at diagnosis , with some positivity in the stroma , on the basis of our limited sample , but strongly pd - l2 positive . this finding suggests that the significance of pd - l2 positivity in response to pd - l1 or pd - 1 targeted therapies may be a useful subject of study . 
 ( a ) rna collected from archival although these studies used different clones for detection.27 , 28 using our own methods for detection of pd - l2 in a tissue microarray cohort of 43 patients with nsclc , we found that this patient was consistently among the top 5% of pd - l2 expressers . 
 tissue blocks were analyzed by nanostring pancancer immune profiling ( 730 immune - related genes )  . normalized data were compared between the post - therapy a ( preresistance ) and posttherapy b ( postresistance ) specimens for log2 fold change . 
 ( b ) genes involved in the interferon - gamma signaling reactome ( reactome.org ; accession : m965 ) were selected from the pancancer immune profiling gene set and plotted by fold change . this analysis demonstrated a general decrease in interferon - gamma signaling within the tumor microenvironment at disease progression on antiprogrammed death ligand 1 ( pd - l1 )  . 
pdl1 interacts with programmed death 1 ( pd1 ) and b7 - 1 ( cd80 ) to promote immune effector cell suppression , whereas pd - l2 can bind pd - 1 and rgmb , promoting effector suppression and respiratory tolerance in lung - resident macrophages , respectively.22 , 23 apcs , antigen - presenting cells ; cox2 , cyclooxygenase - 2 . be associated with earlier stage of disease ( stage i v all others ) , but not with gender or smoking status ( appendix fig a1 )  . during the second treatment with antipd - l1 , which was accompanied by short - term stable disease and subsequent progression , we observed maintenance of high pd - l2 expression and downregulation of pd - l1 mrna expression accompanied by simultaneous decrease of interferongamma response signatures . 
we also observed upregulation of immunosuppressive genes , such as cyclooxygenase - 2 ( recently shown to be important in mediating antipd - 1 responsiveness29 ) and interleukin - 6 , which was shown to decrease after initial treatment with pd - l1 targeted therapy.30 thus , the changes in gene expression within the tumor microenvironment in this patient were consistent with previous studies . importantly , there were multiple intervening therapies in this patient during the approximately 20month period after the initial pretreatment biopsy , limiting the inferences that can be made during the initial therapy with antipd - l1 . 
balko , leora horn manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors despite his prior treatments with palliative radiation ; however , he did receive an additional course of palliative radiation after his third biopsy and preceding his antipd - 1 therapy , and a role for radiation in sensitizing his tumor to anti - pd - 1 therapy cannot be ruled out . although anecdotal , this case report demonstrates an important clinical finding : patients who become resistant to antipd - l1 may still benefit from pd - 1 targeted therapies , possibly due to a switch from dependency on pd - l1 to pd - l2 for maintenance of immunosuppression . however , the presence of consistently high pd - l2 staining argues against a direct mechanism of pd - l2mediated de novo resistance to antipd - l1 therapy . 
moreover , pd - l2 mrna did not seem to be upregulated or changed during acquisition of resistance to antipd - l1 therapy . however , the ratio of pd - l2 to pd - l1 mrna increased substantially from 3.6 - fold to 14.6 - fold during this period and may be a useful metric to test experimentally in the future for association with resistance to antipd - l1 treatment . 
although more investigation is needed in the context of preclinical studies and clinical trials , this report supports investigations of sequencing antipd - l1 and antipd - 1 therapies to elucidate mechanisms of cross - resistance and derive maximal patient benefit from these agents . 
johnson consulting or advisory role : bristol - myers squibb , genoptix , merck research funding : incyte patents , royalties , other intellectual property : intellectual property and patents pending surrounding use of mhc - ii and response to immune therapy leora horn honoraria : biodesix consulting or advisory role : bristol - myers squibb , merck , bayer , xcovery , genentech , boehringer ingelheim , eli lilly , abbvie , astrazeneca research funding : astrazeneca ( inst ) other relationship : bristol - myers squibb justin m . 
balko research funding : incyte patents , royalties , other intellectual property : patent on use of hla - dr / mhc expression to predict response to immunotherapies references 677 - 704 , 2008 1 . 
mu cy , huang ja , chen y , et al : high expression of pd - l1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation . 
borghaei h , paz - ares l , horn l , et al : nivolumab versus docetaxel in advanced nonsquamous nonsmall - cell lung 2018 - 2028 , 2015 cancer . 
rittmeyer a , barlesi f , waterkamp d , et al : atezolizumab versus docetaxel in patients with previously treated nonsmall - cell lung cancer ( oak ) : a phase 3 , open - label , multicentre randomised controlled trial . 
brahmer jr , tykodi ss , chow lq , et al : safety and activity of anti - pd - l1 antibody in patients with advanced cancer . n engl j med 366 : 2455 - 2465 , 2012 10 . 
brahmer jr , rizvi na , luzky j , et al : clinical activity and biomarkers of medi4736 , an anti - pd - l1 antibody , in patients with nsclc . 
rizvi n , brahmer j , ignatius s , et al : safety and clinical activity of medi4736 , an anti - programmed cell death - ligand 1 ( pd - l1 ) antibody , in patients with nonsmall - cell lung cancer ( nsclc )  . 
spira ai , park k , mazi`eres j , et al : efficacy , safety and predictive biomarker results from a randomized phase ii study comparing mpdl3280a vs docetaxel in 2l / 3l nsclc ( poplar )  . 
spigel dr , chaft je , gettinger sn , et al : clinical activity and safety from a phase ii study ( fir ) of mpdl3280a ( antipdl1 ) in pd - l1selected patients with nonsmall - cell lung cancer ( nsclc )  . 
kelly k , patel mr , infante jr , et al : avelumab ( msb0010718c ) , an anti - pd - l1 antibody , in patients with metastatic or locally advanced solid tumors : assessment of safety and tolerability in a phase i , open - label expansion study . 
xiao y , yu s , zhu b , et al : rgmb is a novel binding partner for pd - l2 and its engagement with pd - l2 promotes respiratory tolerance . 
he j , hu y , hu m , et al : development of pd - 1 / pd - l1 pathway in tumor immune microenvironment and treatment for nonsmall - cell lung cancer . 
yearley j , gibson c , yu n , et al : pd - l2 expression in human tumors : relevance to anti - pd - 1 therapy in cancer . presented at european society for medical oncology annual meeting , vienna , austria , september 25 - 29 , 2015 27 . 
calles a , liao x , sholl lm , et al : expression of pd - 1 and its ligands , pd - l1 and pd - l2 , in smokers and never smokers with kras - mutant lung cancer . 
kim my , koh j , kim s , et al : clinicopathological analysis of pd - l1 and pd - l2 expression in pulmonary squamous cell carcinoma : comparison with tumor - infiltrating t cells and the status of oncogenic drivers . 
postow ma , callahan mk , barker ca , et al : immunologic correlates of the abscopal effect in a patient with melanoma . 2035 - 2036 , 2012 n engl j med 366 : 925 - 931 , 2012 34 . 
programmed death ligand 2 ( pd - l2 ) expression across 43 patients with nonsmall cell lung cancer ( nsclc )  . ( a ) a tissue microarray series of 43 patients with nsclc was stained for pd - l2 expression and analyzed by histoscore ( h - score ; % of cells staining positive 3 intensity [ 0 - 3 + ] )  . 
 c clinical observation of improved outcomes of patients with brca 1 / 2 breast cancer treated with everolimus prior to dna - damaging therapy introduction the mammalian target of rapamycin ( mtor ) pathway comprises mtor complexes 1 and 2 ( mtorc1 / 2 ) and is primarily responsible for maintaining cellular growth and proliferation . 
although it is known to act as a catalyst toward anabolic synthesis , mtorc1 also is responsible for mitigating cellular catabolic processes , namely autophagy.1 mtorc2 complements mtorc1 in anabolic processes.2 phosphoinositide 3 - kinase ( pi3k ) and phosphatase and tensin homolog ( pten ) genetic mutations frequently are found in many tumor types , including breast cancer.3 these genes are important in regulating the downstream activation of the mtor pathway . 
many drugs have been developed to target the mtor pathway , including the mtorc1 inhibitor ( mtori ) everolimus.2 , 4 poly ( adp - ribose ) polymerase ( parp ) is a family of enzymes that repairs damaged dna . 
treatment with a parp inhibitor ( parpi ) prevents dna repair in cancer cells during homologous recombination ( hr ) events in patients with brca1 / 2 mutations , which leads to cell death through synthetic lethality.5 brca1 / 2 germline mutations are found in approximately 5% of unselected breast cancers , with higher prevalence in high - risk cohorts.6 activation of the pi3k / mtor pathway is important in decreasing the effect of brca1 / 2 mutations by stabilizing hr.7 ibrahim et al8 showed that when pi3k is inhibited , hr is impaired , which results in sensitization of brca wild - type triple - negative breast cancer cells to parpis . 
the details of the cohort are listed in table 1 . our analysis revealed that six patients among the 14 treated in brocade had experienced exceptional responses to treatment ; these six had been treated with the mtori everolimus before commencing study therapy . 
the patient with the second - best response had a documented pten deletion and nearly the same pfs at 29.2 months but did not receive veliparib with her carboplatin doublet . discussion most of the preclinical data of the role of the pik3ca / mtor pathway in dna damage sensitivity was carried out with concurrent therapeutic combinations . 
 these adaptive changes can persist over time , which allows one to use a treatment to sensitize a patients tumors to a subsequent therapy.14 , 15 the observed difference in pfs between eve + and eve patients raises the possibility that prior mtori exposure may render brca1 / 2 mutated breast cancer more susceptible to subsequent dna - damaging therapy . the interaction between activating pik3ca mutations or pten deletions and this sensitizing effect by mtori cannot be fully evaluated in this limited case series . 
however , it seems that the presence of these genomic abnormalities did not preclude those patients from deriving a significant benefit from subsequent dna - damaging therapy . in addition to the usual limitations and biases of case series , our analysis was limited by the heterogeneity of patient characteristics , prior treatments received , and study treatment assigned . 
this precludes us from specifically concluding how much of the observed clinical benefit in the eve + group was a result of improving the activity of the cytotoxic chemotherapy or the veliparib ( if given )  . 
in addition , veliparib did not significantly increase the median pfs of the experimental arm compared with the control arm in the overall study , so the effect of the treatment assignment may be minor in explaining the difference between the eve + and eve arms . 
the pfs of these two patients was similar to the main study population , so including them in the analysis with the other estrogen receptor positive patients is unlikely to change the main analysis . 
future studies in patients with brca1 / 2 mutations also should examine sequential treatment strategies by administering pi3kcai / mtori agents for one to two cycles as a sensitizing therapy before switching to a parpi or dna - damaging cytotoxic chemotherapy . 
han research funding : abbvie ( inst ) , prescient therapeutics ( inst ) , tapimmune ( inst ) , seattle genetics ( inst ) , g1 therapeutics ( inst ) , bristol - myers squibb ( inst ) , pfizer ( inst ) , novartis ( inst ) hatem h . 
lee moffitt cancer center and research institute , tampa , fl . presented at the 2017 annual meeting of the american society of clinical oncology , chicago , il , june 2 - 6 , 2017 . prior presentation references 10 : 18 , 2018 2012 150 , 2009 2017 1 . 
ibrahim yh , garca - garca c , serra v , et al : pi3k inhibition impairs brca1 / 2 expression and sensitizes brca - proficient triple - negative breast cancer to parp inhibition . 
mo w , liu q , lin cc , et al : mtor inhibitors suppress homologous recombination repair and synergize with parp inhibitors via regulating suv39h1 in brca - proficient triple - negative breast cancer . 
juvekar a , burga ln , hu h , et al : combining a pi3k inhibitor with a parp inhibitor provides an effective therapy for brca1 - related breast cancer . 
beuvink i , boulay a , fumagalli s , et al : the mtor inhibitor rad001 sensitizes tumor cells to dna - damaged induced apoptosis through inhibition of p21 translation . 
oreilly t , mcsheehy pmj , wartmann m , et al : evaluation of the mtor inhibitor , everolimus , in combination with cytotoxic antitumor agents using human tumor models in vitro and in vivo . 
han hs , diras v , robson m , et al : veliparib with temozolomide or carboplatin / paclitaxel versus placebo with carboplatin / paclitaxel in patients with brca1 / 2 locally recurrent / metastatic breast cancer : randomized phase ii study . 
miller , md4 purpose next - generation sequencing ( ngs ) of tumor and germline dna is foundational for precision oncology , with rapidly expanding diagnostic , prognostic , and therapeutic implications . 
although few question the importance of ngs in modern oncology care , the process of gathering primary molecular data , integrating it into electronic health records , and optimally using it as part of a clinical workow remains far from seamless . numerous challenges persist around data standards and interoperability , and clinicians frequently face difculties in managing the growing amount of genomic knowledge required to care for patients and keep up to date . methods this review provides a descriptive analysis of genomic data workows for ngs data in clinical oncology and issues that arise from the inconsistent use of standards for sharing data across systems . 
questions about their provenance and timeliness of updating remapotentially useful standards for sharing genomic data , such as hl7 fhir and mcode , remain primarily in the research and / or development stage . 
2019 by american society of clinical oncology introduction next - generation sequencing ( ngs ) of tumor and inherited ( germline ) genomes has revolutionized and rened cancer treatment during the past two decades and is now vital for evaluating therapeutic opportunities in many solid and hematologic malignancies.1 currently , ngs panels including sets of genes are the most widespread method of rapidly identifying sequence variation in patients with cancer . 
ngs panels provide information for a variety of purposes , cluding diagnostics ( eg , determination of sarcoma subtype ) , hereditary risk assessment ( eg , lynch syndrome ) , prognosis ( eg , kras mutations in lung adenocarcinoma ) , and treatment selection ( eg , biomarkers for immunotherapy responsiveness , such as tumor mutational burden and microsatellite instability ; therapeutic selection for clinically actionable alterations , such as braf v600e in melanoma ; and biomarkers of resistance , such as loss of b2m for immunotherapy ) .2 , 3 as a metric of the signicance of ngs in oncology care , 29 of the 43 national comprehensive cancer network clinical practice guidelines denote specic sequence - based biomarkers important for clinical care ( table 1 )  . 
of these 29 guidelines for treatment of cancer by site , 12 include both somatic and germline biomarkers , 16 include somatic only , and one includes germline only . recently , there has been an increased focus on performing ngs on matched normal samples ( either adjacent tissue or blood ) to compare with tumor biopsies . 
 conway et al context key objective the amount of molecular knowledge required for patient care is exploding , but the minimal adoption of genomic data standards in electronic health records compounds the challenges facing oncologists . 
using examples from asco cancerlinq , this review covers the current status of next - generation sequencing , describes relevant data standards , highlights the pilot of the genomic smart - on - fhir application , and provides a call to action for laboratories and electronic health record vendors to ensure that the chain of data custody for structured genomic content remains unbroken and reaches the clinician . knowledge generated several genomic knowledge bases for variant interpretation are available to support clinicians . 
nonetheless , transmitting nextgeneration sequencing results via pdf hinders the use of decision support tools intended for clinical care , including trial selection , and blocks the downstream reuse of genomic data . relevance the consistent use of evolving standards ( eg , hl7 fhir and mcode ) should improve genomic data interoperability and care quality . 
however , the full integration of genotype and phenotype will require an innovative and socially driven commitment by all oncologic ecosystem participants , beginning with sequencing laboratories , to better leverage technologic solutions . also embedded in guidelines and reimbursement for certain treatments ( eg , poly [ adp - ribose ] polymerase inhibitors for ovarian cancer )  . 
multiple molecularly targeted agents have now been incorporated into standard patient management and reected in numerous guidelines . clinical quality measures the 2019 merit - based incentive payment system ( mips ) of the centers for medicare and medicaid services quality payment program contains 24 general oncology measures , two of which involve genetic testing ( kras and nras in patients with colorectal cancer ) .14 in addition , a number of clinical quality measures in the asco quality oncology practice initiative reference ngs testing , including nonsmall - cell lung cancer ( nsclc ) measures evaluating the use and turnaround time of molecular testing in patients with adenocarcinoma histology.15 as clinical quality measurement evolves from clinicianfocused process measures to more patient - centric outcome measures , it is reasonable to expect that more measures will incorporate results from ngs panels and potentially circulating tumor - free dna testing to foster appropriate diagnostic testing and therapy selection that affect patient outcomes . molecular testing has become essential to oncology care.12 survey data from 2017 showed that more than 75% of oncologists used ngs tests to guide cancer treatment . 
inclusion of genetic testing in nccn cancer site guidelines s , g , b , or n cancer subtype details cancer site acute lymphoblastic leukemia ( adult and aya ) acute lymphoblastic leukemia ( pediatric ) acute myeloid leukemia aids - related kaposi sarcoma anal carcinoma bladder cancer bladder cancer upper tract tumors urothelial carcinoma of the prostate primary carcinoma of the urethra ewing sarcoma family of tumors giant cell tumor of the bone bone chondrosarcoma chordoma osteosarcoma breast cancer cns cancer cervical cancer lymphoma chronic myeloid leukemia colon / rectal cancer colon cancer rectal cancer chronic lymphocytic leukemia / small lymphocytic esophageal and esophagogastric junction cancers gastric cancer gestational trophoblastic neoplasia hairy cell leukemia head and neck cancers hepatobiliary cancers hodgkin lymphoma kidney cancer malignant pleural mesothelioma melanoma cutaneous melanoma uveal melanoma multiple myeloma / other plasma cell neoplasms multiple myeloma systemic light chain amyloidosis waldenstr om macroglobulinemia / lymphoplasmacytic lymphoma myelodysplastic syndromes myeloproliferative neoplasms ( continued on following page ) efciently assess eligibility criteria . 
barriers include insufcient clinician knowledge , training , and condence regarding the use of ngs results.20 , 21 although many clinicians may take laboratorybased interpretations at face value , numerous knowledge bases have been created to aid oncologists and clinical researchers in the curation and interpretation of ngs data . these were developed largely because of the recognition that institutional ( local ) knowledge based on a limited sampling of the cancer population , including that possessed by testing laboratories , would not be sufcient to type , capture the genomic diversity of any cancer common or rare . 
perhaps a surprise to clinicians , many commonly tested genes , such as brca1 and brca2 , show considerable disagreement by interpreting laboratories about the pathogenicity of genetic variants.22 one of the earliest resources to address this issue was mycancergenome , 23 created at vanderbilt university in 2009 . 
since that time , there have been numerous entrants into an increasingly crowded eld , with the most well - known publicly available resources for variant interpretation being civic , 24 oncokb , 25and clinvar , 26 the latter of which is the rst fda - recognized public genetic variant database , housing clingen expert - curated data.27 each of these efforts was developed in relative isolation and with slightly different goals , leading to a divergence of data models , evidentiary models , and user experiences . 
the variant interpretation in cancer consortium , a driver project of the global alliance for genomics and health , 28 was founded with the mission statement to help unify and harmonize the disparate interpretation in cancer data sources . 
inclusion of genetic testing in nccn cancer site guidelines ( continued ) cancer site neuroendocrine and adrenal tumors s , g , b , or n cancer subtype details neuroendocrine tumors of the gi tract , lung , and thymus ( carcinoid tumors ) neuroendocrine tumors of the pancreas adrenal gland tumors pheochromocytoma / paraganglioma men1 men2 non - hodgkins lymphomas chronic lymphocytic leukemia / small lymphocytic lymphoma b - cell lymphomas primary cutaneous b - cell lymphomas t - cell lymphomas nonmelanoma skin cancers basal cell skin cancer dermatobrosarcoma protuberans merkel cell carcinoma squamous cell skin cancer nonsmall - cell lung cancer occult primary ( cancer of unknown primary ) ovarian cancer epithelial ovarian cancer ( including fallopian tube cancer and primary peritoneal cancer ) pancreatic adenocarcinoma penile cancer prostate cancer small - cell lung cancer soft tissue sarcoma systemic mastocytosis testicular cancer thyroid carcinoma uterine neoplasms endometrial carcinoma uterine sarcoma vulvar cancer thymomas and thymic carcinomas note . 
categorization of national comprehensive cancer network ( nccn ) guideline44 by cancer site showing whether the guideline recommends somatic testing ( s ) , germline testing ( g ) , both ( b ) , or neither ( n )  . 
genetic testing refers to short variations , structural sequence variations , cytogenetic analyses , and mismatch repair assays . abbreviations : aya , adolescent or young adult ; men1 , multiple endocrine neoplasia type 1 ; men2 , multiple endocrine neoplasia type 2 . several challenges associated with knowledge bases must be considered . 
although the aforementioned knowledge bases are free to users , they 4 2019 by american society of clinical oncology are generally not integrated into the clinical workow.30 the second is trustworthiness and culpability . 
can the assertions made by knowledge bases be trusted ? if advice is erroneous , the primary responsibility clearly lies with the managing clinician , but can knowledge bases also be held culpable ? furthermore , advice can become outdated ; this must be expected to some degree , given the exponential growth of the eld and the lag between the announcement of trial results , publication of ndings , fda labeling , and revision of variant interpretations ; however , poor recency of data in knowledge bases will diminish trust . 
many knowledge bases spend much effort on attractive so - called skins and search abilities , but the degree to which clinicians nd these interfaces useful has not been extensively studied . technological challenges of data integration : opportunities with fhir and mcode integration of ngs results into the clinical workow via tackle several ehr interfaces must technologic challenges . interpreted genomic results are in particular , generally reported in pdf format , which cannot support electronic search , clinical decision support ( cds ) , or secondary use.31 moreover , the lack of a common data model for genomic testing impedes all use cases by hampering data storage and interoperability between ehr systems and / or data warehouses , thereby hindering the use of cds tools intended to assist clinicians in caring for their patients . these issues have prompted the emergence of initiatives aimed at improving genomic data standards and facilitating genomic data interoperability . 
the top section shows total count of prognostic or predictive assertions related to this gene - variant combination , the breakdown by data source ( ve are shown ) , and the breakdown by assertion type per association for molecular pathology ( amp ) / asco / college of american pathologists ( cap ) guidelines.46 the middle section cross - references drugs and diseases with the gene of interest ( rst column ) , as well as potentially related genes ( adjacent columns ) , which are automatically determined ; heatmap colors indicate the quantity of evidence for a drug - gene or diseasegene correlation . 
the vicc meta - knowledgebase user guide is available online.47 auspices of sync for genes.36 these reports were generated from proprietary pipelines used for the fmi foundationone and foundationact ngs panels . 
although customers typically receive a pdf of the ndings and interpretations , fmi also sends xml les , based on a custom internal standard used to generate the pdfs , upon request . 
these xml les include more detailed information than that presented in the pdf , some of which is for research use only , and can be parsed to generate information relevant for fhir resources and elements ( eg , variant allele fraction , sequencing depth and functional effect for mutations , absolute copy number and copy - number ratio for copy - number events , number of supporting read pairs and identity of both genes involved for rearrangements , and genomic location for all alterations )  . 
briey , clinical elds such as patient , sample , and physician information could be mapped to patient , specimen , and physician fhir resources , respectively . genomic elds such as alterations ( eg , braf p.v600e , amplication , rearrangement ) , therapies , and clinical trials were mapped to elements of the observation genetics resource , with more detailed genetic information ( eg , chromosome , start position , end position , variant allele frequency , coverage , and variant type ) mapped to the molecular sequence resource . 
the entire fmi report was represented as a diagnostic report resource , which included references to the patient , physician , specimen , observation genetics , and molecular sequence resources . 
the modular design ( fig 2 ) was architected with four major goals : assist in clinical interpretation of variants within context , provide links to external resources , facilitate point - of - care physician - patient conversations , and demonstrate that smart - on - fhir technology can be used for a clinicogenomic use case . 
cancerlinq aggregates structured and unstructured data from ehrs and other sources via direct feeds and processes the data in a series of cloud - based databases where it normalized and deidentied for secondary reuse.40 database the cancerlinq database , now representing more than 1.5 million patients , contains vast amounts of detailed longitudinal clinical data , such as demographics , diagnoses , laboratory results , and medications . 
however , a signicant volume of the data is not computable , because many critical concepts ( cancer staging , biomarkers , disease progression , and adverse events ) reside primarily in text notes and other unstructured documents and not in discrete elds . 
smart ( sustainable medical apps , reusable technology ) precision cancer medicine ( pcm ) architecture diagrathe application is designed to be modular and extensible , particularly in the ability to add presentation modules and interfaces to external data sources . 
unique text strings are shown for two well - known egfr gene variations abstracted during curation of unstructured genomic reports that were obtained from electronic health records of practices participating in cancerlinq . 
although natural language processing technology itself has not yet demonstrated sufcient precision or recall to be used as a standalone solution , it has been successfully embedded in the curation workbench used by the data abstractors , where it functions to surface putative data elements from unstructured text that can then be conrmed or disregarded . curation of genomic data from ngs panels in cancerlinq as noted , ngs panels are typically brought into the ehr as pdfs or scanned faxes and therefore are not computable . however , since 2017 , cancerlinq has been extracting high - value genomic information via user interfaceassisted data abstraction . 
cancerlinq has also obtained and processed structured genomic reports in xml format and is evaluating automated processes to scan and extract data from reports with standardized formats . the magnitude of the genomic data gap in native ehrs is considerable and shows how poorly precision oncology is supported . 
similarly , for advanced nsclc , 85.3% of curated patient records had epidermal growth factor receptor tests , but structured epidermal growth factor receptor data were found in native ehrs for only 1.7% of all records for patients with advanced nsclc . it is worth noting that although cancerlinq has developed various technical solutions to structuring genomic data as it is housed in ehrs , all ngs data are originally generated as structured data by molecular diagnostics laboratories . 
the data capture of the various chemistries that underlie ngs sequencing , as well as the components of bioinformatic pipelines ( sequence aligners , variant callers ) and even clinical report production , comprises machine - readable , structured genomic data . 
the technical solutions cancerlinq has painstakingly developed would not be necessary at all if the originally structured data were provided . a stark example of how data quality is diminished when optical character recognition , needed to parse genomic reports , is applied , particularly to scanned images ( eg , faxed reports ) and tables , is listed in table 2 . 
 conway et al of national comprehensive cancer network guidelines as well as payment incentives ( eg , mips ) and quality programs ( eg , quality oncology practice initiative )  . 
clinical trial eligibility in oncology increasingly incorporates genomic biomarkers , and the fda has been modernizing its policies and review processes for cancer , resulting in an avalanche of new agents and approved indications for targeted therapies.41 there is no dearth of cds tools to help translate patient data into quality care , nor is there a lack of technical solutions to transmit , harmonize , curate , store , or otherwise manage data . 
these problems are not primarily technical , and solving them requires a will to action , with social incentives and disincentives . for example , ngs test results that are natively structured are sent to ordering physicians and stored in their ehrs , but in a manner that requires costly and data - lossy curation to restructure back into usable information . 
considering that the genomic results are reimbursed through federal and private insurers , but the reporting format impedes their utility to clinicians , we call for all molecular diagnostic laboratories to provide structured data as part of routine reporting at the request of ordering physicians and their practices . 
this would help laboratories meet the denition of interoperability by the 21st century cures act34 to facilitate health information exchange without special effort on the part of the user while avoiding the act 's prohibition of information blocking . it is notable that on the basis of curated records , structured brca1 or brca2 gene test results are extremely underrepresented among the 10 ehrs used by the more than 50 cancerlinq practices . 
it is possible to integrate genomic and clinical data from apis externally to native ehrs , but greater cooperation is needed from vendors to export clinical data for cds use and to provide a seamless experience for the clinician , whose attention is tethered to the ehr . adoption of data standards , including hl7 fhir and mcode , by all the entities that generate , transmit , and receive health information would also facilitate the implementation of precision oncology . 
the endless customizability of ehrs is at odds with health information technology interoperability and suboptimally supports automated quality measure reporting as required by mips.42 , 43 the dearth of coding for cancer staging , laboratory data , and medications within ehrs could be rectied by adopting widely used standard vocabularies , which would facilitate the computable representation of patients oncology records as envisioned by mcode . 
miller , md , cancerlinq , american society of clinical oncology , 2318 mill rd , suite 800 , alexandria , va 22314 ; e - mail : robert.miller@asco.org. support supported by award no . 
 ngs data challenges and solutions references freedman an , klabunde cn , wiant k , et al : use of next - generation sequencing tests to guide cancer treatment : results from a nationally representative survey of oncologists in the united states . 
mol oncol 8 : 859 - 873 , 2014 kamps r , brando rd , bosch bj , et al : next - generation sequencing in oncology : genetic diagnosis , risk prediction and cancer classication . 
int j mol sci 18 : e308 , 2017 jones s , anagnostou v , lytle k , et al : personalized genomic analyses for cancer mutation discovery and interpretation . 
sci transl med 7 : 283ra53 , 2015 cibulskis k , lawrence ms , carter sl , et al : sensitive detection of somatic point mutations in impure and heterogeneous cancer samples . 
nat biotechnol 31 : 213 - 219 , 2013 carter sl , cibulskis k , helman e , et al : absolute quantication of somatic dna alterations in human cancer . 
nat biotechnol 30 : 413 - 421 , 2012 robson me , bradbury ar , arun b , et al : american society of clinical oncology policy statement update : genetic and genomic testing for cancer susceptibility . j clin oncol 33 : 3660 - 3667 , 2015 karow j : tumor - only sequencing may misguide therapy but many labs omit matched control . 
may - misguide - therapy - many - labs - omit - matched - control damodaran s , berger mf , roychowdhury s : clinical tumor sequencing : opportunities and challenges for precision cancer medicine . 
saigi m , alburquerque - bejar jj , sanchez - cespedes m : determinants of immunological evasion and immunocheckpoint inhibition response in non - small cell patterns of carcinoma evolution . 
gao p , zhang r , li j : comprehensive elaboration of database resources utilized in next - generation sequencing - based tumor somatic mutation detection . biochim biophys acta rev cancer . 
j clin oncol 32 : 1317 - 1323 , 2014 johnson l - m , valdez jm , quinn ea , et al : integrating next - generation sequencing into pediatric oncology practice : an assessment of physician condence and understanding of clinical genomics . 
lawler m , siu ll , rehm hl , et al : all the worlds a stage : facilitating discovery science and improved cancer care through the global alliance for genomics and 29 . 
hughes ks , ambinder ep , hess gp , et al : identifying health information technology needs of oncologists to facilitate the adoption of genomic medicine : recommendations from the 2016 american society of clinical oncology omics and precision oncology workshop . 
li m , datto m , duncavage e : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
 during this period , because of a strong family history of breast cancer ( her mother and maternal aunt were diagnosed at age 50 years ) , the patient was referred for genetic counseling and germline testing . 
tumor next - generation sequencing of her resected brain metastasis detected the following genomic events : deleterious tp53 mutation , pi3k3ca exon 20 hotspot mutation , atm deletion mutation ( same as germ line dna sequencing ) and heterozygous atm loss , fgfr1 amplification ( validated by fluorescent in situ hybridization ; 10 gene copies ) , myc amplification , and ccnd1 amplification . 
the primary recommendation of the mtb was recruitment into the expansion cohort of a clinical trial with a poly ( adp - ribose ) polymerase ( parp ) inhibitor available on site . 
table 1 summarizes the existing knowledge in three large public databasesclinical interpretations of variants in cancer ( civic ) , 1 cancer genome interpreter ( cgi ) , 2 and oncokb3on driver genomic events considered predictive biomarkers for targeted therapies in cancer ( atm loss or mutation , pik3ca h1047r mutation , and fgfr1 amplification )  . 
the information is organized according to the level of evidence supporting each gene variantdrug - disease association , ranging from results of preclinical experiments to case reports and from early to late clinical trials . 
each database provided unique and relevant interpretations for single genomic events , and the collective information was helpful when making a therapeutic decision , such as with pik3ca mutations matched to pi3k pathway inhibitors and fgfr1 amplifications matched to fgfr inhibitors in breast cancer . 
of note , some biomarker - drug interactions were described only in different tumor types , including parp inhibitors in the setting of atm loss or mutation in prostate cancer . 
summary of existing knowledge in three public databases with genomic biomarkers for cancer drugs gene alteration atm loss or mutation database predictive association civic response drug evidence level disease pmid case study prostate cancer 26510020 conference ( abstract number ) sensitivity temozolomide preclinical melanoma , glioblastoma 23960094 response early trials prostate cancer 26510020 olaparib ( parp inhibitor ) olaparib ( parp inhibitor ) response vx - 970 ( atr inhibitor ) case report colorectal cancer ena 2015 ( a48 ) response cisplatin early trials bladder cancer 26238431 sensitivity temozolomide preclinical oncokb response olaparib ( parp inhibitor ) compelling biologic evidence pik3ca civic sensitivity preclinical breast cancer h1047r mutation melanoma , glioblastoma 23960094 prostate cancer 26510020 20453058 , 21558396 sensitivity preclinical her2 - positive breast cancer 22294718 resistance trastuzumab case study her2 - positive breast cancer 21594665 response early trials breast cancer 28331003 , 27672108 asco 2015 ( 2500 ) response oncokb response late trials her2 - positive breast cancer 27091708 compelling clinical evidence breast cancer 27672108 asco 2015 ( 2501 ) fgfr1 civic response amplification clinical , case study breast cancer 23658459 , 27179038 no response bgj398 ( fgfr inhibitor ) lucitanib ( multikinase fgfr inhibitor ) response early trials breast cancer 25193991 clinical breast cancer 27870574 oncokb response azd4547 ( fgfr inhibitor ) compelling clinical evidence lung cancer asco 2014 ( 8015 ) note . 
civic , cgi , and oncokb databases were accessed on august 10 , 2017 . abbreviations : cgi , cancer genome interpreter ; civic , clinical interpretations of variants in cancer ; ena , aacr - nci - eortc ; her2 , human epithelial growth factor receptor 2 ; parp , poly ( adp - ribose ) polymerase ; pmid , pubmed identification number . pictilisib ( pi3k inhibitor ) , ch5132799 ( pi3k inhibitor ) azd5363 ( akt inhibitor ) + trastuzumab / lapatinib taselisib ( pi3k inhibitor ) , copanlisib ( pi3k inhibitor ) , azd5363 ( akt inhibitor ) everolimus + trastuzumab + chemotherapy copanlisib ( pi3k inhibitor ) , mln1117 ( pi3k inhibitor ) , others dovitinib ( fgfr inhibitor ) , azd4547 ( fgfr inhibitor ) and consistent across databases . 
both cgi and oncokb present compelling clinical evidence that pik3ca mutations in breast cancer may be targetable with potent and selective pi3k alpha inhibitors , with response rates up to 30% . 
regarding fgfr1 amplifications , antitumor activity of fgfr inhibitors varied depending on the drug being investigated , and response rates were on average lower than those reported for selective pi3k alpha inhibitors in pik3ca - mutated breast cancer . 
 despite the limited preclinical and early clinical data on parp inhibitor activity in an atm loss or mutation setting , mtb members prioritized this therapy in a clinical trial setting because of the biologic evidence of complete atm deficiency ( germ line pathogenic atm mutation plus loss of wild - type atm allele in tumor dna sequencing ) coupled with the patients clinical history of longterm response to platinum - based chemotherapy . 
there are emerging retrospective data on increased efficacy of pd1 / pdl1 blockade in tumors with gene alterations in the dna damage repair pathway , 8 and prospective trials are being designed ( eg , nct03040791 and nct03236935 )  . 
mayer ia , abramson vg , formisano l , et al : a phase ib study of alpelisib ( byl719 ) , a pi3kspecific inhibitor , with letrozole in er + / her2metastatic breast cancer . 
goyal l , saha sk , liu ly , et al : polyclonal secondary fgfr2 mutations drive acquired resistance to fgfr inhibition in patients with fgfr2 fusion - positive cholangiocarcinoma . 
teo m , seier k , ostrovnaya i , et al : dna damage repair and response ( ddr ) gene alterations ( alt ) and response to pd1 / pdl1 blockade in platinum - treated metastatic urothelial carcinoma ( muc )  . 
 acquired ctnnb1 mutation drives immune checkpoint inhibitoracquired resistance in a microsatellite instabilityhigh gastroesophageal adenocarcinoma with brain metastases introduction the landscape of advanced gastric and esophageal adenocarcinomas continues to evolve , partly owing to improved molecular characterization identifying genomically defined subgroups that may benefit from matched therapies.1 - 3 the keynote - 059 and attraction - 2 trials have demonstrated efficacy for the antiprogrammed death - 1 ( pd - 1 ) antibodies pembrolizumab and nivolumab in gastroesophageal adenocarcinomas ( geas ) after two or more prior lines of therapy.3 , 4 the keynote - 059 study population included seven known patients with microsatellite instabilityhigh ( msi - h ) tumors , among whom the response rate was 57% . 
outside of msi - h tumors , changes in neoantigen landscape , acquired jak1 mutations , mhc - i loss , beta - 2microglobulin mutations affecting antigen presentation , and expression of alternate checkpoints have been shown to mediate acquired resistance to immune checkpoint blockade.9 - 12 with the tumor agnostic approval of pembrolizumab for msi - h tumors in 2017 , a better understanding of acquired resistance is increasingly important . 
 standard - of - care human epidermal growth factor receptor 2 immunohistochemical testing was negative , and he received first - line fluorouracil and oxaliplatin with palliative radiotherapy to a painful bone metastasis with a best response of stable disease . 
at the time of first - line progression , his liver biopsy was screened for microsatellite instability , found to be msi - h , and he was enrolled in a clinical trial of ipilimumab 1 mg / kg and nivolumab 3 mg / kg ( table 1 )  . 
after complete radiographic response of distant disease lasting 18 months , he received palliative radiotherapy ( 0.414 gy ) concurrent with nivolumab every 2 weeks for endoscopically improved , but residual , disease and remained in the trial . 
genomic alterations with preclinical and / or clinical evidence supporting resistance to immune checkpoint inhibitors are shown in bold . abbreviations : gej , gastroesophageal junction ; msi - h , microsatellite instabilityhigh ; mut , mutation ; tmb , tumor mutation burden ; wt , wild type . the resection specimen revealed persistence of an 8.5 - cm segment of poorly differentiated gej adenocarcinoma positive for lymphovascular invasion . all lymph nodes were negative , and evidence of significant treatment effect was seen ( tumor regression grade 2 ) , overall staged as ypt3ypn0 with negative margins . 
the surgical specimen was subjected to hybrid - capture comprehensive genomic profiling using a commercial assay ( foundationone , foundation medicine , cambridge , ma ) , which confirmed msi - h ( table 1 )  . 
during the treatment time encompassing stereotactic brain radiotherapy and nivolumab , washout biopsy samples were taken from a stable subcutaneous scalp metastasis , and an intramuscular rectus femoris lesion reappeared on restaging imaging ( table 1 )  . 
in an effort to explore putative resistance mechanisms after excellent durable response to ipilimumab and nivolumab , the samples were all subjected to the same hybrid - capture comprehensive genomic profiling assay ( table 1 ; fig 1 )  . 
after stereotactic brain radiotherapy , the patient went on to enroll in a third - line trial of paclitaxel in combination with the anti - dkk1 monoclonal antibody dkn - 01 ( clinicaltrials . gov identifier : nct02013154 ) and achieved stable disease lasting 4 months , ultimately , with progression in the brain and extracranial sites concurrently . 
clonal divergence and acquired ctnnb1 in a patient with microsatellite instabilityhigh ( msi - h ) gastroesophageal junction adenocarcinoma with disease progression while receiving ipilimumab in combination with nivolumab after a durable response . 
however , the sampled site of cns progression contained private alterations known or suspected to mediate immuno oncology resistance not seen at other disease sites ( fig 1 )  . 
the ctnnb1 t41a and d32y mutations are known ( t41a ) or predicted ( d32y ) stabilizing mutations that lead to t - cell exclusion and resistance to antiprogrammed death - ligand 1 therapies in preclinical models.17 although less well studied , the d32y mutation should increase stability owing to proximity to the cki and gsk3b phosphorylation sites and is a part of the recognition motif for b - trcp ( e3 ligase for b - catenin )  . 
the additional ctnnb1 n387k is not well characterized functionally , although there is some evidence for it leading to increased b - catenin activity in liver cancer.18 stabilizing or gain - of - function ctnnb1 alterations are recurrent in gea and other tumor types and may be associated with a worse outcome , although clinical immunotherapy - specific outcome data are lacking ( fig 3 ) .2 , 19 , 20 it is also possible that the blood - brain barrier may have reduced the efficacy of checkpoint blockade , allowing for the tissue - specific development of the ctnnb1 alterations we observed . 
however , because our methods are limited to dna - based assessment , we cannot exclude nongenomic mediators of acquired resistance that are known to exist.24 overall , our patient may add early support to the possibility of tissue - contextspecific acquired resistance under selective pressure of immune checkpoint inhibitors , although we cannot draw significant conclusions from a single patient . 
in an analogy to oncogene - driven gea , intertumoral heterogeneity may emerge as an acquired resistance mechanism in msi - driven gea.2 in fact , a recent correlative article from a phase ii trial of pembrolizumab in advanced gea identified intratumoral msi heterogeneity in a patient refractory to therapy.6 if further validated , our observation may have implications beyond gea and inform rational , and perhaps individualized , salvage strategies for msi - h tumors progressing while the patient is taking checkpoint inhibitor therapy . 
klempner stock and other ownership interests : tp therapeutics speakers ' bureau : foundation medicine research funding : leap therapeutics ( inst ) , astellas pharma ( inst ) travel , accommodations , expenses : eli lilly alexa b . 
ali employment : foundation medicine leadership : incysus stock and other ownership interests : exelixis , blueprint medicines , agios , genocea biosciences patents , royalties , other intellectual property : patents via foundation medicine , patents via seres health on microbiome stuff in non - neoplastic disease ( i ) charlotte d . 
kubicky consulting or advisory role : bristol - myers squibb , eisai , array biopharma , loxo , bayer , arqule , blueprint medicines , novartis speakers ' bureau : bristol - myers squibb , eisai consulting or advisory role : eli lilly , boston biomedical , astellas pharma matthew h . 
fuchs cs , doi t , jang rw , et al : safety and efficacy of pembrolizumab monotherapy in patients with previously treated advanced gastric and gastroesophageal junction cancer : phase 2 clinical keynote - 059 trial . 
kang yk , boku n , satoh t , et al : nivolumab in patients with advanced gastric or gastrooesophageal junction cancer refractory to , or intolerant of , at least two previous chemotherapy regimens ( ono - 4538 - 12 , attraction - 2 ) : a randomised , double - blind , placebo - controlled , phase 3 trial . 
janjigian yy , bendell j , calvo e , et al : checkmate - 032 study : efficacy and safety of nivolumab and nivolumab plus ipilimumab in patients with metastatic esophagogastric cancer . 
overman mj , lonardi s , wong kym , et al : durable clinical benefit with nivolumab plus instability - high metastatic in dna mismatch repair - deficient / microsatellite ipilimumab colorectal cancer . 
shitara k , zgrolu m , bang yj , et al : pembrolizumab versus paclitaxel for previously treated , advanced gastric or gastro - oesophageal junction cancer ( keynote - 061 ) : a randomised , openlabel , controlled , phase 3 trial . 
smyth ec , wotherspoon a , peckitt c , et al : mismatch repair deficiency , microsatellite instability , and survival : an exploratory analysis of the medical research council adjuvant gastric infusional chemotherapy ( magic ) trial . 
 o multi - institutional , prospective clinical utility study evaluating the impact of the 92 - gene assay ( cancertype id ) on final diagnosis and treatment planning in patients with metastatic cancer with an unknown or unclear diagnosis purpose metastatic cancers of unknown primary or with unclear diagnoses pose diagnostic and management challenges , often leading to poor outcomes . 
the current study assessed the clinical impact of the 92 - gene assay on diagnostic and treatment decisions for patients with unknown or uncertain diagnoses . methods patients in this prospective , multi - institutional , decision - impact study included those for whom the 92 - gene assay was ordered as part of routine care . 
the key study objective of clinical impact was calculated on the basis of changes in final diagnosis and treatment after testing . results data collection included 444 patients , 107 physicians ( 73 oncologists and 34 pathologists ) , and 28 sites . 
 physicians reported that the 92 - gene assay was used broadly for diagnostic dilemmas that ranged from single suspected tumor type ( 29% ) to a differential diagnosis of two or more suspected tumor types ( 30% ) or cancers of unknown primary ( 41% )  . 
integration of 92 - gene assay results led to a change in the recommended treatment in 47% of patients . conclusion findings from this clinical utility study demonstrate that the 92 - gene assay led to a change in treatment decisions in every other patient case . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction metastatic cancers that are initially characterized as unknown primary ( cup ) , or with other features that may lead to diagnostic uncertainty with respect to tumor type diagnosis , can prolong the clinicopathologic workup and result in delays in the initiation of treatment , additional costs , and relatively poor patient outcomes.1 - 3 sachdev p . 
schnabel fadi braiteh author affiliations and support information ( if applicable ) appear at the end of this article . licensed under the creative commons attribution 4.0 license corresponding author : sachdev p . 
numerous diagnostic and treatment strategies , such as enhanced pathologic techniques , molecular classification of tumor type and subtype , comprehensive mutational profiling , and novel combination regimens of cytotoxics plus biolo gics , have been proposed without universal consensus.4 - 10 investigational approaches to personalize the systemic treatment of metastatic cancer solely on the basis of the patients somatic genomic alterations , disregarding histologic context , have had mixed and disappointing results.11 - 14 results from several recent phase ii studies that evaluated targeted agents for specific genomic alterations across a variety of tumor types have provided evidence that mutations are not targetable in similar manners across tumor types.11 , 12 thus , the clinical utility of this approach is unclear . 
 whereas driver mutations matter , the integration of tumor type and subtype remains critical when considering the efficacy of a targeted therapy aimed at a putative driver mutation.15 given that empirical chemotherapy approaches are associated with poor prognosis in patients with cup or uncertain diagnoses , it might be more effective to refocus on methods with which to identify patient tumor type and subtype to guide therapy.1 , 16 gene expression profilingbased molecular classification of tumors might prove efficacious by helping to identify the primary tumor type and histologic subtype for patients with unknown or uncertain diagnoses . 
the primary objective was to assess clinical impact on the basis of changes in diagnostic and treatment decisions after the incorporation of 92 - gene assay results . methods study design observational in nature , the current study was prospectively defined to evaluate multidisciplinary clinical utility in patients for whom physicians ordered the 92 - gene assay as a clinical workup tool to help identify or narrow the tumor type and subtype diagnosis . 
prespecified objectives were to examine the diagnostic and clinical utility of the 92 - gene assay in oncology and pathology practice to characterize indications of use , and to evaluate its potential integration and impact on patient management by comparing changes in diagnosis and treatment selection after testing . data collection physicians were required to complete standard ized , discipline - specific questionnairespathology and oncologyafter receiving a 92 - gene assay test report . 
 the pathology ecrf consisted of 11 multiple choice questions and four questions that required written responses ( biopsy quality , number and types of immunohistochemistry [ ihc ] performed , and 92 - gene assay result )  . 
the medical oncologist ecrf consisted of 11 multiple choice questions and five questions that required written responses ( time between biopsy and treatment , clinical diagnosis for first - line treatment , name of first - line treatment , other diagnostic or clinical considerations for choosing first - line therapy , and 92 - gene assay result )  . 
 rna quality determined to be quantity not sufficient , in which a molecular prediction could not be determined , physicians completed quantity not sufficient ecrfs that consisted of four multiple choice questions and two questions that required written response ( biopsy quality and number of ihc performed )  . 
ecrf forms are included in the data supplement . physicians were instructed on the web portal and ecrf via a standardized training progra for the pathology questionnaire ( data supplement : pathology ecrf question 7 and oncology ecrf question 8 , respectively ) , physicians were instructed to align terminology with the 50 tumor types and subtypes classified by the 92 - gene assay . 
the testing laboratory was blinded to the working clinical diagnosis from the ecrf . 92 - gene assay the 92 - gene assay was performed as previously described.17 , 20 in brief , the assayreal - time rt pcrwas performed on isolated total rna from tumor cells that were enriched by either microdissection or laser microdissection . 
a prespecified computational algorithm generated probabilities for primary tumor type and subtype on the basis of the degree of similarity of the submitted sample to a reference database of gene expression information from more than 2 , 000 tumors of known tumor type . 
the test report provided a prediction of the main tumor type and subtype on the basis of the highest relative probability and any additional tumor types that cannot be ruled out . 
the report also provided a list of tumor types that could be excluded with 95% confidence . statistical analysis statistical considerations of study size were based on an anticipated treatment recommendation change rate of 35% , targeting a sample size of at least 156 patients to ensure a 95% two - sided ci width of 15% . 
the primary objective of the study was to measure the clinical impact of the 92 - gene assay results on the basis of changes in patient treatment , the narrowing of treatment options , or the elimination of a treatment option ( a , b , or c on question 15 of the oncology questionnaire in the data supplement )  . 
patients were considered eligible for a targeted agent if the physician responded a , " " b , " or c in question 15 and there was a us food and drug administrationapproved targeted therapy that was recommended by the national comprehensive cancer network for the molecular diagnosis provided by the 92 - gene assay . results patient enrollment began in february 2013 and closed october 2014 after enrolling 444 patients with 107 physicians73 medical oncologists and 34 pathologistsfrom 28 sites21 oncology sites and seven pathology sitesacross the united states . 
of the 444 patients enrolled , 397 had sufficient rna for analysis , which corresponded to an overall analytical success rate of 89% , taking into account samples that were determined to have insufficient tissue on pathology review before testing . 
a patient flow diagram is shown in figure 1 . biopsy and tumor characteristics the most common metastatic biopsy sites included the liver ( 23% ) , lymph nodes ( 17% ) , and lung ( 14% ; fig 2a )  . 
patient flow diagram showing the disposition of 92 - gene assay testing . patients submitted for testing ( n = 444 ) not reportable insufficient tissue or rna ( n = 47 ) reportable results ( n = 397 ) resulted to oncologist ( n = 271 ) resulted to pathologist ( n = 126 ) 92 - gene assay results ( n = 271 ) 92 - gene assay results ( n = 126 ) indeterminate ( n = 12 ) indeterminate ( n = 6 ) molecular diagnosis ( n = 259 ) molecular diagnosis ( n = 120 ) excluded did not receive anticancer therapy performance deaths patient choice other ( n = 56 ) ( n = 16 ) ( n = 8 ) ( n = 7 ) ( n = 25 ) patient received anticancer therapy ( n = 203 ) diagnostic testing and preassay diagnosis factors that contributed to an oncologists decision to order the 92 - gene assay were multidisciplinary and included the following : no primary site of origin after clinical review and imaging ( 42% ) , a pathology report that indicated a differential diagnosis ( 21% ) or that indicated an unknown primary site ( 20% ) , and distinguishing between new cancer versus recurrence ( 16% )  . 
 data that were collected to better characterize the sequence of diagnostic testing demonstrated that 72% of physicians responded that patients had pathology and ihc studies performed before the 92 - gene assay , 14% of samples were submitted for pathology and ihc evaluation and the 92 - gene assay concurrently , and approximately 14% of samples were submitted without an indication of the diagnostic sequence . 
the most common imaging tests were computed tomography scans ( 86% ) , fusion positron emission tomography / computed tomography scans ( 57% ) , magnetic resonance imaging ( 29% ) , ultrasound ( 28% ) , regular film radiographs ( 11% ) , or mammogram ( 11% ; data not shown )  . for pathologists , inconclusive ihc ( 50% ) was the most common reason for ordering the 92 - gene assay . 
the mean number of ihc stains performed before the molecular assay was ordered was 10 ( median , nine ; range , zero to 23 ; data not shown )  . 92 - gene assay results and impact on diagnosis of the 397 patients with sufficient tissue and rna for a reportable result , the 92 - gene assay provided a molecular - based tumor type and histologic subtype diagnosis in 379 patients ( 95.5% ) , whereas 4.5% had an indeterminate molecular diagnosis ( fig 1 )  . 
 ( * ) other indicates biopsy sites with fewer than three cases , encompassing uterus , gallbladder , gastroesophageal junction , spine , spleen , thyroid , and salivary gland . 
fna , fine - needle aspiration . liver lymph node lung peritoneal cavity or omentum soft tissue bone / marrow pleural cavity / pleura colon brain rectum skin breast bladder stomach pelvic mass retroperitoneum pericardial cavity adrenal gland pancreas mediastinum esophagus ovary duodenum other * core excision endoscopic other percentage percentage types . 
the most common diagnoses were pancreaticobiliary ( 21.9% ) , squamous cell carcinoma ( 10.1% ) , lung adenocarcinoma ( 9.3% ) , and intestinal ( 8.6% ) type tumors ( fig 3 )  . working diagnoses before the 92 - gene assay were assessed in the medical oncology subset ( n = 271 )  . 
preassay working diagnoses were reported as a single suspected site in 79 patients ( 29% ) , a differential diagnosis of two or more suspected sites in 80 patients ( 30% ) , and cup in 112 patients ( 41% ; fig 4a )  . 
comparison of the 92 - gene assay molecular diagnoses with preassay working diagnoses demonstrated that the assay provided a tumor type diagnosis that was not initially suspected in a large proportion of patients ( fig 4b )  . 
in patients with a single suspected primary site ( n = 79 ) , the 92 - gene assay confirmed the suspected diagnosis in 60% of patients but provided a tumor type result that was not initially suspected in 39% of patients . 
in patients with a differential diagnosis ( n = 80 ) , the 92 - gene assay narrowed the diagnosis in 66% of patients and provided a tumor type result that was not initially suspected in 27% of patients . 
in patients for whom the pathology report indicated cup site ( n = 112 ) , the assay provided a molecular tumor type prediction in 97% of patients . impact on treatment planning of the 271 oncologist - submitted cases , 203 patients ( 75% ) received anticancer treatment after the 92 - gene assay results were made available . 
the most common reasons for patients not receiving treatment were a rapid deterioration in the patients performance status ( 29% ) , patient death ( 14% ) , and a patient declination of treatment ( 13% ; fig 1 )  . 
molecular cancer classification predictions from submitted cases using the 92 - gene assay ( n = 397 )  . pancreaticobiliary squamous cell carcinoma lung adenocarcinoma intestine gastroesophageal adenocarcinoma urinary bladder neuroendocrine ovary breast adenocarcinoma liver hepatocellular carcinoma sarcoma head and neck salivary gland carcinoma prostate adenocarcinoma endometrial adenocarcinoma cervix adenocarcinoma melanoma kidney mesothelioma germ cell thyroid brain indeterminate skin basal cell carcinoma percentage ( fig 5a )  . 
the assay resulted in similar changes in treatment recommendations in all three scenarios of the original working diagnosis48% of cases with a single suspected primary ( n = 79 ) , 49% of differential diagnoses with two or more primaries ( n = 80 ) , and 42% of cases with an unknown primary site ( n = 112 )  . subset analysis within the most commonly predicted tumor type classesthose with 20 casesdemonstrated that physicians changed their recommended treatment in 58% of gi cancers ( n = 81 ) , 54% of gynecologic and breast cancers ( n = 28 ) , and 44% of lung cancers ( n = 27 ; fig 5b )  . 
mutational biomarker testing was most commonly ordered after the 92 - gene assay rendered a diagnosis of lung ( 81% ) or colorectal cancer ( 67% )  . discussion results from this large , multisite study have demonstrated that the 92 - gene assay was used across a spectrum of diagnostic uncertainty , beyond cups , and included cases with differential diagnoses and those with a suspected diagnosis for confirmatory testing . 
in addition , the assay provided a diagnosis that was not initially suspected in a substantial proportion of patients who had either a single suspected diagnosis or differential diagnosis before testing with the 92 - gene assay . 
 this clinical impact was more pronounced in gi and gynecologic and breast tumor types , which suggests that the 92 - gene assay may have additional utility in particular metastatic presentations in which standard approaches may be limited . 
 distribution of 92 - gene assay diagnostic results within the preassay working diagnosis classifications . differential diagnosis ( 30% ) unknown primary ( 41% ) single suspected site ( 29% ) indeterminate indeterminate indeterminate provided new diagnosis provided new diagnosis confirmed suspected diagnosis confirmed suspected diagnosis provided new diagnosis single suspected site differential diagnosis unknown primary approaches , what is both the current and future relevance of establishing the tumor type or cellular context of a metastatic cancer ? recent results from a number of basket trials have demonstrated that knowledge of tumor type and cellular context remains fundamental for the interpretation of potentially targetable dna mutations and the recommendation of treatment approaches in metastatic cancer . 
whereas molecularly targeted agents have been demonstrated to be effective in tumors that harbor a matching molecular alteration , a growing understanding of the importance of molecular heterogeneity and cellular context is emerging . 
for example , the efficacy of the targeted braf inhibitor , vemurafenib , has been shown to be mixed across a diverse set of nonmelanoma cancers with a braf v600 mutation11 and is well known to have poor efficacy in braf - mutated colorectal cancers.11 , 13 similarly , early results from the mypathway basket trial have demonstrated variable response rates in patients with identical mutations across different tumor types.14 finally , in a phase ii study in which patients with a specific molecular alteration were randomly assigned to receive treatment with a molecularly targeted agent or physicians choice of treatment , there was no difference in median progression - free survival between the two treatment groups.12 these data suggest that the effectiveness of targeting putative driver mutations with molecularly targeted agents may be dependent on the specific cellular context or tumor type . 
 no , treatment regimen was not changed ( 53% ) yes , treatment regimen was changed ( 47% ) gi cancers gynecological and breast cancers lung cancers treatment regimen was not changed ( 42% ) yes , treatment regimen was changed ( 58% ) treatment regimen was not changed ( 46% ) yes , treatment regimen was changed ( 54% ) treatment regimen was not changed ( 56% ) yes , treatment regimen was changed ( 44% ) fig 5 . 
 ( b ) impact of the 92 - gene assay on treatment decisions within tumor type subgroups that received therapy : gi cancers ( n = 81 ) , gynecologic and breast cancers ( n = 28 ) , and lung cancers ( n = 27 )  . breast adenocarcinoma , and lung adenocarcinoma.23 - 25 results presented here reinforce the continued relevance of tumor type diagnosis in optimizing treatment strategies that can potentially affect patient outcomes . 
with regard to pancreaticobiliary tumors , the 92 - gene assay also reports additional subtyping into gallbladder adenocarcinoma , pancreatic adenocarcinoma , or cholangiocarcinoma , which may inform treatment decisions for surgery type , neoadjuvant chemotherapy , or site - directed and targetable agents . 
in addition , a significant proportion of tumor types predicted by the 92 - gene assay , including lung adenocarcinoma , lung squamous , colorectal , gastroesophageal , urinary bladder , and neuroendocrine tumors , have not only specific chemotherapy approaches , but also us food and drug administrationapproved molecularly targeted therapies or immunotherapies . 
 decision making , with similar overall results , which supports the clinical utility of the assay.34 , 35 strengths of this study include the large number of patients and contributing physicians , which contributed to the generalizability of the study results . 
first , this was an observational study and no data on outcomes were collected ; however , previous studies have demonstrated that the use of the 92 - gene assay improved survival in patients with cup who were treated on the basis of assay results.16 , 34 although multiple aspects of the study design were prespecified and carefully planned , such as study physicians independently completing questionnaires , a blinded design such that the testing laboratory did not have knowledge of working diagnoses , and a preplanned statistical analysis , bias is an inherent feature of observational studies . 
this aspect of the cross - functional integration of molecular diagnostic results warrants investigation in future studies . this study demonstrated that the 92 - gene assay affected diagnosis and treatment selection in a significant proportion of patients , which supports the clinical utility of the assay as a standardized molecular approach to help streamline additional diagnostic testing in patients with metastatic cancer with unknown or uncertain diagnoses . 
schnabel data analysis and interpretation : all authors manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors lauren e . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . stock and other ownership interests : biotheranostics brock e . 
 fadi braiteh stock and other ownership interests : bristol - myers squibb , insys therapeutics , agios , clovis oncology , tesaro honoraria : genentech , insys therapeutics , bristol - myers squibb , amgen , boehringer ingelheim , astrazeneca , bayer , celgene , ipsen , incyte , taiho pharmaceutical , lexicon , ariad pharmaceuticals , eli lilly , abbott nutrition , heron consulting or advisory role : amgen , bristol - myers squibb , clovis oncology , boehringer ingelheim , ipsen , insys therapeutics , ambry genetics , genentech , eli lilly , incyte , bayer , sanofi , regeneron , astrazeneca , merck , celgene , lexicon , pfizer , merrimack pharmaceuticals speakers ' bureau : amgen , bristol - myers squibb , genentech , merck , pfizer , eli lilly , ipsen , insys therapeutics , incyte , boehringer ingelheim , astrazeneca , celgene , taiho pharmaceutical , merrimack pharmaceuticals , biotheranostics travel , accommodations , expenses : genentech , bristolmyers squibb , amgen , celgene , clovis oncology , tesaro , ipsen , insys therapeutics , incyte , heron , astrazeneca , medimmune , novartis , boehringer ingelheim , lexicon , taiho pharmaceutical , bayer , bayer , onyx pharmaceuticals , exelixis , regeneron , sanofi , merrimack pharmaceuticals , pfizer research funding : biotheranostics acknowledgment we thank andrea c . 
grschel s , bommer m , hutter b , et al : integration of genomics and histology revises diagnosis and enables effective therapy of refractory cancer of unknown primary with pdl1 amplification . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors based on molecular profiles : early results from mypathway , an open - label , phase iia umbrella basket study . 
hainsworth jd , rubin ms , spigel dr , et al : molecular gene expression profiling to predict the tissue of origin and direct site - specific therapy in patients with carcinoma of unknown primary site : a prospective trial of the sarah cannon research institute . 
weiss lm , chu p , schroeder be , et al : blinded comparator study of immunohistochemical analysis versus a 92 - gene cancer classifier in the diagnosis of the primary site in metastatic tumors . 
kim b , schroeder b , schnabel ca , et al : physician - reported clinical utility of the 92 - gene molecular classifier in tumors with uncertain diagnosis following standard clinicopathologic evaluation . 
ahmed ka , caudell jj , el - haddad g , et al : radiosensitivity differences between liver metastases based on primary histology suggest implications for clinical outcomes after stereotactic body radiation therapy  . 
spetzler d , xiao n , burnett k , et al : multi - platform molecular profiling of 1 , 180 patients increases median overall survival and influences treatment decision in 53% of cases . 
nystrom sj , hornberger jc , varadhachary gr , et al : clinical utility of gene - expression profiling for tumor - site origin in patients with metastatic or poorly differentiated cancer : impact on diagnosis , treatment , and survival . 
raghav k , mhadgut h , mcquade jl , et al : cancer of unknown primary in adolescents and young adults : clinicopathological features , prognostic factors and survival outcomes . 
hainsworth jd , schnabel ca , erlander mg , et al : a retrospective study of treatment outcomes in patients with carcinoma of unknown primary site and a colorectal cancer molecular profile . 
j nat cancer inst 105 : 782 - 790 , 2013 appendix we thank the following principal investigators of the clinical study sites who participated in the study : paul adams , md ( genesys hurley cancer institute ) , solhail akbani , md ( baptist hospitals of southeast texas ) , stephen anthony , do ( evergreen hematology - oncology ) , andrew j . 
brenner , md ( the university of texas health science center at san antonio ) , david campbell , md ( grass valley hematology - oncology medical group ) , arvind chaudhry , md , phd ( medical oncology associates ) , william gilles , md ( dignity health ) , h . 
oxnard , md1 noninvasive genotyping of plasma cell - free dna is being integrated into cancer care at an astonishing rate , as its potential to radically increase access to personalized cancer care is increasingly recognized.1 whereas next - generation sequencing ( ngs ) of tumor has been commoditized and democratized ( with many academic hospitals offering their own clinical laboratory improvement amendmentscertied tests ) , plasma ngs is primarily sent out for testing at commercial laboratories . 
however , although these tests are ordered and reported en masse , the causes of falsepositive or false - negative plasma ngs results have only gradually become apparent ( fig 1 )  . false - negative results have long been recognized as common with blood - based assays . 
we and others identied that assay sensitivity is closely related to clinical factors such as stage and metastatic spread , tumor dna in cases suggesting limited shed of missed by plasma ngs.2 , 3 false positives have also been described with many plasma genotyping assays and are routinely attributed to tumor heterogeneity.4 although heterogeneity is clearly a source of tumor / plasma discordance in cancers that have developed drug resistance , 5 , 6 many false positives can be attributed to dna shed from normal cells , including germline variants or noncancerous somatic variants such as clonal hematopoiesis ( ch ) .7 - 9 the latter is particularly challenging because ch can involve cancer - associated genes ( eg , tp53 , jak2 , kras )  . 
yet when such mutations are identied in plasma , they may not reect true tumor genotype . what is least understood is how technical factors related to assay performance contribute to false - positive and false - negative results . 
probe design and specics of library generation are often considered proprietary and are only minimally described in the few analytical validation studies that have been published . this is the context motivating the publication by stetson et al10 in jco precision oncology . 
the authors sent aliquots of plasma to four commercial clinical laboratory improvement amendmentscertied laboratories for plasma ngs and compared this to ngs performed on matched tumor and normal tissue at foundation medicine . 
the vendors were blinded to tumor ngs results but knew that their plasma ngs results would be compared with orthogonal tissue results , as well as with plasma results from other laboratories . 
following bioinformatic analysis and variant calling , binary alignment map les were provided by the vendors to the authors for independent , unblinded , post - hoc sequence analysis . with this design , the authors were not only able to address many of the pitfalls that plagued prior vendor comparison studies , 11 , 12 but they could also investigate interassay technical factors . 
more precisely , stetson et al10 were able to account for known germline variants , investigate mutation - calling biases , and examine whether false negatives were as the result of stochastic sample biases or thresholding nuances of the vendors bioinformatic lter . the results should give pause . 
positive predictive value ( ppv ) against tissue ranged from 36% to 80% . however , results improved when limited to mutations called at an allelic fraction ( af ) greater than 1% , with three vendors achieving a ppv of 100% for these higher af calls . 
note that variants detected at less than 1% af are routinely reported by each vendor , and such sensitivity is advertised as a unique strength of plasma ngs assays . false - positive variants tended to be novel with no reports in somatic variant databases , and these were often related to vendor - specic mutational biases . false negatives were attributed to bioinformatic ltering of suspected germline variants and limitations from high signal - to - noise ratio . 
taken together , these results point to recurrent contributions from technical differences in the bioinformatic pipelines of the assays , assay sensitivity , or plain error . the current study is far from perfect and leaves room for improvement . 
the study cohort consisted primarily of early - stage cancers ( 21 of 24 patients had stage i and ii cancers ) , which are not the intended population for plasma ngs . 
beyond these established biologic factors , technical factors may be an underappreciated source of erroneous plasma ngs . technical factors tett chnical factor tumor dna shed tumor heterogeneity ( particularly at resistance ) tett chnical factors technical factors white blood cell dna ( clonal hematopoiesis , germline variants ) finally , reference truth was extrapolated across four different assays . 
furthermore , this analysis does not broach the challenges of reporting plasma ngs results . one could imagine that a forward - thinking laboratory might recognize the limitations of plasma ngs in its reporting , noting the risk of false positives for low af or ch - associated variants , as well as the risk of false negatives when low tumor shed is apparent . as the use of plasma ngs becomes increasingly widespread in cancer care , there remains a clear need for concordance studies such as this one . 
 editorial policy , please refer to or ascopubs.org / po / authorcenter . patents , royalties , other intellectual property : patent pending on noninvasive blood - based monitoring of genomic alterations in cancer ( inst ) cloud p . 
paweletz honoraria : astrazeneca korea , bio - rad consulting or advisory role : dropworks research funding : guardant health , astrazeneca travel , accommodations , expenses : astrazeneca korea geoffrey r . 
oxnard honoraria : chugai pharma , bio - rad , sysmex , guardant health consulting or advisory role : astrazeneca , inivata , sysmex , takeda , loxo oncology , ignyta , dropworks , grail no other potential conicts of interest were reported . references aggarwal c , thompson jc , black ta , et al : clinical implications of plasma - based genotyping with the delivery of personalized therapy in metastatic non - small cell lung cancer . 
jama oncol 2 : 1014 - 1022 , 2016 bettegowda c , sausen m , leary rj , et al : detection of circulating tumor dna in earlyand late - stage human malignancies . 
sci transl med 6 : 224ra24 , 2014 forshew t , murtaza m , parkinson c , et al : noninvasive identication and monitoring of cancer mutations by targeted deep sequencing of plasma dna . 
sci transl med 4 : 136ra68 , 2012 oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced non - smallcell lung cancer . 
clin cancer res 24 : 4437 - 4443 , 2018 oxnard gr , hu y , mileham kf , et al : assessment of resistance mechanisms and clinical implications in patients with egfr t790m - positive lung cancer and acquired resistance to osimertinib . 
hu y , alden rs , odegaard ji , et al : discrimination of germline egfr t790m mutations in plasma cell - free dna allows study of prevalence across 31 , 414 cancer patients . 
clin cancer res 23 : 7351 - 7359 , 2017 slavin tp , banks kc , chudova d , et al : identication of incidental germline mutations in patients with advanced solid tumors who underwent cell - free circulating tumor dna sequencing . 
stetson d , ahmed a , nuttall brb , et al : orthogonal comparison of four plasma ngs tests with tumor suggests technical factors are a major source of assay discordance . 
paweletz cp , sacher ag , raymond ck , et al : bias - corrected targeted next - generation sequencing for rapid , multiplexed detection of actionable alterations in cell - free dna from advanced lung cancer patients . 
guibert n , hu y , feeney n , et al : amplicon - based next - generation sequencing of plasma cell - free dna for detection of driver and resistance mutations in advanced non - small cell lung cancer . 
 mutational landscape in resected periampullary adenocarcinoma : relationship with morphology and clinical outcome sebastian lundgren , md1 ; soe olsson hau , md1 ; jacob elebro , md , phd1 ; margareta heby , md , phd1 ; emelie karnevi , phd1 ; bj orn nodin , phd1 ; jakob eberhard , md , phd1 ; karolina holm , phd1 ; johan staaf , phd1 ; g oran b . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction periampullary adenocarcinoma is a collective term for tumors arising in the area surrounding the ampulla of vater , including the head of the pancreas , the duodenum , and the common bile duct . 
a morphologic classication into intestinal type ( i - type ) and pancreatobiliary type ( pb - type ) has been shown to provide better prognostic information than anatomic origin , with the former having a more favorable clinical outcome.1 - 3 although a plethora of mutations has been documented in pancreatic cancer , 4 - 7 the mutational landscape of other periampullary cancers has been less studied . 
no targeted therapies have proven to be efcient , and adjuvant chemotherapy therefore remains standard of care after resection of these tumors . hence , there is evident need for additional studies on the distribution and clinical signicance of the mutational landscape in the full range of periampullary including pancreatic cancer , adenocarcinomas , enable a better patient stratication and identify potential responders to targeted therapies . 
 lundgren et al context ( cid : 129 ) to what extent does morphology inuence the mutational landscape in periampullary adenocarcinomas , and can we nd novel prognostic and predictive biomarkers that differ by morphology , so as to better characterize this heterogeneous group of tumors in a clinical context ? ( cid : 129 ) the results presented here demonstrate that the distribution and prognostic impact of mutations in key genes indeed differ according to morphology and that emphasis on morphology rather than anatomy is of importance . 
approval for the study was obtained from the ethics committee of lund university ( reference number 445 / 07 ) , whereby the committee waived the need for consent other than the option to opt ; out . next - generation sequencing tissue cores of 1 - mm diameter were taken from tumor cellenriched formalin - xed parafn - embedded tissue . dna extraction was performed using the qiagen generead ( qiagen , hilden , germany ) kit for formalin - xed parafnembedded tissue according to the manufacturers instructions . 
forty - one ( 39.8% ) were classied as i - type and 62 ( 60.2% ) as pb - type . a panel of 70 cancer - associated genes was put together for this study and characterized using illumina truseq custom amplicon assay ( illumina , san diego , ca ) with a miseq instrument according to manufacturers instructions . 
detected mutations were screened against the cosmic and exac databases , to lter single - nucleotide polymorphisms commonly reported in different populations . immunohistochemical analysis of brahma - related gene 1 protein expression immunohistochemical analysis of brahma - related gene 1 ( brg1 ) protein expression , 4 - m tissue microarray sections were automatically pretreated in the pt - link system ( dako , glostrup , denmark ) and stained in an automated immunostainer ( autostainer plus , dako ) using the dako envision flex + detection system , peroxidase / dab , rabbit / mouse with the monoclonal anti - brg1 antibody clone g - 7 ( santa cruz biotechnology , dallas , tx ) , diluted 1 : 25 . brg1 was expressed in the tumor cell nuclei and present in the majority of tumor cells in positive cases . 
kaplanmeier analysis log - rank test was applied to illustrate any difference in 5 - year overall survival ( os ) , and cox regression proportional hazard models were used to estimate hazard ratios ( hrs ) for death within 5 years in both univariable and multivariable analysis . 
multivariable cox regression included adjustment for age ( continuous ) , t - stage ( t1 or t2 v t3 or t4 ) , n - stage ( negative v positive nodal status ) , grade ( well to moderate v poor ) , morphology ( i - type v pb - type ) in the entire cohort , adjuvant chemotherapy ( none v any ) , invasion into vascular and lymphatic structures , and perineural growth . 
the proportional hazard ( ph ) assumption was tested using cox regression with a timedependent covariate analysis , whereby the ph assumption was considered to be satised when the factor time interaction was nonsignicant . 
the ph assumption was also evaluated graphically using log - minus - log plots . in the entire cohort , three cases were excluded in the survival analyses : one patient with a pb - type tumor who was lost to follow - up because of emigration , and two patients with i - type tumors who died as a result of complications from the initial surgical treatment . 
to estimate the interaction treatment and selected bioeffect between adjuvant markers , the following interaction variable was constructed : any adjuvant treatment ( + / ) biomarker ( + / )  . 
all data generated or analyzed during this study are included in this published article ( data supplement )  . results distribution of mutations according to morphologic type there were no signicant differences in the distribution of clinicopathological characteristics between cases with known and unknown mutational status ( data supplement )  . the frequency of mutations according to morphologic type is shown in figure 1 and further outlined in the data supplement . 
the frequency of mutations according to anatomic origin is shown in the data supplement . prognostic impact of the most frequent mutations kaplan - meier analyses demonstrate a signicantly prolonged os for patients with i - type compared with patients with pb - type tumors ( data supplement )  . 
when stratifying for anatomic origin , the survival curves cluster into two groups concordant with morphology ( data supplement )  . of the nine most frequently mutated genes outlined above , only apc , erbb3 , kras , and smarca4 were shown to confer a prognostic value , depending on the context . 
apc mutation was not prognostic in i - type tumors ( data not shown ) , and erbb3 mutation was not prognostic in analyses stratied for morphology ( data not shown )  . kras mutation was the strongest predictor of survival , as visualized in figure 2 . 
when stratifying for ampullary origin , kras mutation remained a prognostic factor in i - type tumors ( p = 0.033 ; fig 2d ) but was not prognostic in pb - type tumors ( fig 2e )  . 
 intestinal type pancreatobiliary type tp53 kras rnf43 smad4 erbb3 smarca4 cdkn2a lundgren et al tp53 kras rnf43 smad4 smarca4 cdkn2a erbb3 genetic alteration no alterations truncating mutation in - frame mutation missense mutation fig 1 . 
the genetic alterations were classied as either truncating , missense , or in - frame mutations . mutation did not differ according to adjuvant chemotherapy in i - type tumors ( data not shown )  . 
in the entire cohort , high brg1 expression was an adverse prognostic factor in patients not receiving adjuvant chemotherapy but was not prognostic in patients receiving adjuvant chemotherapy ( data not shown )  . 
the mutational burden of the analyzed genes did not differ signicantly between i - type and pb - type tumors ( p = .577 ) and was not prognostic in the whole cohort or stratied for morphologic type . discussion morphology is emerging as an important prognostic factor in periampullary adenocarcinoma , 1 , 2 and to our knowledge , this is the rst study to comprehensively map common cancer - associated mutations in the full range of periampullary adenocarcinoma , with particular reference to morphology . 
these ndings are in line with a study on 112 cancers of ampullary origin , demonstrating that the mutational spectrum in i - type tumors resembles that of colorectal cancer , and the mutational spectrum in pb - type tumors resembles that of pancreatic cancer.12 in the current study , mutations in apc but not in cdkn2a were found to be prognostic . 
given that no pb - type tumors harbored apc mutations , it is plausible to assume that the link between these mutations and a favorable outcome in the entire cohort is mainly due to their association with i - type morphology . 
the same line of reasoning applies to the association between erbb3 mutations , being more prevalent in i - type tumors , and a prolonged survival in the entire cohort . 
adding to this , there was no signicant association between her3 expression and erbb3 mutational status , and the clinical utility of assessment of erbb3 mutation status for prognostication purposes needs additional validation . kras mutation was found to signify a signicantly shorter survival in patients with i - type tumors , also when adjusted for established clinical factors , whereas patients with kras wild - type i - type tumors could indeed be classied as long - term survivors . 
as of yet , epidermal growth factor receptor ( egfr ) inhibition has not shown clinical efcacy in pancreatic cancer , 15 but no study has investigated the efcacy of egfr inhibitors in periampullary adenocarcinoma in relation to morphology . although none of the patients in this study had received egfr - inhibiting treatment , the ndings indicate that some ras wild - type periampullary cancers of i - type may indeed benet from such treatment . 
 mutational spectrum and morphology in periampullary cancer kras wt kras mut p = .033 kras wt kras mut p = .018 time since diagnosis ( months ) time since diagnosis ( months ) 2 . 
kaplan - meier curves visualize differences in 5 - year overall survival for patients with kras - mutated ( mut ) and wild - type ( wt ) tumors in ( a ) the entire cohort , intestinal - type tumors , ( c ) pancreatobiliary - type tumors , ( d ) intestinal - type amtumors , and ( e ) pullary pancreatobiliary - type pullary tumors . kras wt kras mut p = .680 kras wt kras mut p = .033 time since diagnosis ( months ) time since diagnosis ( months ) no . 
 lundgren et al smarca4 wt smarca4 mut p = .050 smarca4 wt smarca4 mut p = .803 time since diagnosis ( months ) time since diagnosis ( months ) score 0 score 2 score 3 brg1 low brg1 hight p = .061 brg1 low brg1 high p = .037 time since diagnosis ( months ) time since diagnosis ( months ) no . 
relationship of smarca4 mutation status and brahma - related gene 1 ( brg1 ) protein expression with overall survival according to adjuvant chemotherapy in patients with pancreatobiliary ( pb ) - type tumors . 
 ( a , b ) kaplanmeier curves visualizing differences in 5 - year overall survival in patients with pb - type tumors according to smarca4 mutation status ( a ) without adjuvant chemotherapy , and ( b ) with adjuvant chemotherapy . ( c ) sample immunohistochemical images of brg - 1 expression . 
 ( d , e ) kaplan - meier analyses visualizing differences in 5 - year overall survival in patients with pb - type tumors according to low and high brg1 expression ( d ) without adjuvant chemotherapy , and ( e ) with adjuvant chemotherapy . 
 mutational spectrum and morphology in periampullary cancer despite the lack of a signicant interaction between smarca4 mutation and adjuvant treatment , this observation led us to further explore the expression and prognostic value of brg1 , the protein encoded by smarca4 , in tumors from the full study cohort . 
brg1 was found to be an adverse factor in patients who did not receive adjuvant chemotherapy and , in addition , there was a signicant interaction with adjuvant chemotherapy in pbtype tumors . 
one study on pancreatic cancer ( n = 68 ) failed to demonstrate an association between brg1 expression and gemcitabine response or survival , 17 but the nding of a potential predictive role for smarca4 / brg1 in the current study merits further validation in additional patient cohorts . to our knowledge , this is the rst study to map the prevalence and prognostic signicance of mutations in common cancer - associated genes in the full spectrum of periampullary adenocarcinoma . 
in particular , kras mutation status may be a suitable biomarker for prognostication in patients with i - type tumors , and smarca4 / brg1 expression may add value in the prediction of response to adjuvant chemotherapy treatment in patients with pbtype tumors . 
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diagn pathol 9 : 80 , 2014 elebro j , heby m , warfvinge cf , et al : expression and prognostic signicance of human epidermal growth factor receptors 1 , 2 and 3 in periampullary adenocarcinoma . 
philip pa , benedetti j , corless cl , et al : phase iii study comparing gemcitabine plus cetuximab versus gemcitabine in patients with advanced pancreatic adenocarcinoma : southwest oncology group - directed intergroup trial s0205 . 
 c signicant and durable clinical response to sorafenib and radiation therapy for a patient with stage iv hepatocellular carcinoma and lrrk2 mutation linlin yang , md1 ; patrick wald , md1 ; sameek roychowdhury , md , phd1 ; anne m . 
the level was elevated to alpha fetoprotein ( afp ) 57 , 400 ng / ml , and ct - guided biopsy of the large hepatic lesion revealed stage iv hcc . 
because of radiographic concern for disease progression and invasion into the transverse colon , sorafenib was discontinued and radiation therapy was in addition , started 1 week after sorafenib was discontinued . 
the two large masses were irradiated to a dose of 40 gy in 16 fractions using a conformal radiation plan ( fig 1d )  . repeat ct scans 1 month later showed a large decrease in the size of not only the two radiated extrahepatic lesions but also the unirradiated hepatic lesions , and it was decided to restart the patient on maintenance sorafenib ( 200 mg twice daily ) 6 weeks after radiation . 
on the basis of this impressive response , sorafenib doses were held , and the patient underwent laparoscopic partial left hepatectomy of segment 4b with removal of additional primary liver and extrahepatic omental masses . 
at abdominal masses showed no residual 3 weeks after surgery , sorafenib was restarted at 200 mg twice daily but was discontinued after 2 more months because of sorafenib - related pulmonary toxicity . 
after this exceptional response , we performed next - generation sequencing of whole blood lymphocytes and tumor dna from the diagnostic biopsy specimen ( table 1 ) , which revealed mutations in lrrk2 ( k1316n ) , lrp1b ( v4250a , y4256c ) , tp53 ( v157insa , a nonframeshift insertion of three base pairs : cgc ) , and notch3 ( g501s )  . 
 yang et al started sorafenib 400 mg twice a day sorefenib stopped and rt delivered started maintenance sorafenib 200 mg twice a day surgery sorafenib 200 mg twice a day sorafenib discontinued 9 10 11 12 14 47 52 time ( months ) 180000 160000 140000 120000 100000 80000 60000 40000 20000 fig 1 . 
 ( right ) disappearance of intrahepatic lesions noted in fig 1b . lrrk2 we turned our attention to lrrk2 , a gene that has been linked to the pathogenesis of parkinson disease with known roles in the antioxidant response.2 , 3 ionizing radiation generates reactive oxygen species ( ros ) , which are critical for the ability of radiation to promote tumor cell kill through dna damage . 
the degree of ros generation correlates with serum levels of advanced oxidation protein products and clinical effectiveness in sorafenib - treated patients with hcc.4 because ros are in the response to both radiation and sorafenib , critical through promotion of dna damage , we hypothesized that this mutation in lrrk2 could alter the response to both therapies . 
we observed adequate transfected wild - type ( wt ) and k1316n expression of leucine - rich repeat kinase 2 ( lrrk2 ) protein in multiple cell lines ( appendix fig a3 for rna and protein ; primer sequences intable a1 )  . 
we also tested genetic silencing of lrrk2 with small interfering rna ( sirna ) and found that lrrk2 depletion likewise sensitized hcc cells to sorafenib ( figs 2c and 2d )  . 
in addition , we found that expression of the lrrk2 k1316n mutation in both hela and hepg2 cells resulted in notable radiation sensitization in radiation clonogenic assays , whereas wt lrrk2 had no radiosensitizing effects ( figs 2e and 2f )  . because of the previously identied role of lrrk2 in ros , we assessed whether wt lrrk2 or the k1316n mutant altered levels of ros in cancer cells . 
 ( a , b ) overexpression of lrrk2 k1316n signicantly increases sorafenib sensitivity on hcc cells . ( c , d ) genetic silencing of lrrk2 with sirna sensitizes hcc cells to sorafenib treatment . 
sorafenib sensitivity on hcc cells was determined by alamarblue assay ( thermofisher , waltham , ma ) 72 hours after transfection , whereas radiation sensitivity was determined by clonogenic assay . 
similarly , we found that lrrk2 k1316n greatly increased hydrogen peroxide ( h2o2 ) levels , another known mediator of ros damage , and this increase was reversed by treatment of cells with ebselen , an h2o2 scavenger ( figs 3d , 3e , and 3f )  . 
finally , the addition of the superoxide scavenger tiron greatly attenuated the radiation sensitivity induced by lrrk2 k1316n in radiation clonogenic assays ( figs 4a and 4b )  . discussion in this case of a patient with an exceptional response to sorafenib and radiation , we identied a novel lrrk2 k1316n mutation that induced sorafenib and radiation sensitivity in various hcc cell lines , consistent with our clinical observations . 
 ( * ) signicant difference from corresponding tiron or ebselen treatment group ; ( ) signicant difference from empty vector ; ( ) signicant difference from lrrk2 wild type ( wt )  . 
hepg2 and hela cells transfected with wild - type ( wt ) lrrk2 and lrrk2 k1316n were preincubated with dmso or tiron ( 1 mm ) and then exposed to 4 gy of radiation . 
thus , we hypothesize that this lrrk2 mutation increases lrrk2 kinase activity and enhances sorafenib and radiation - induced apoptotic cell death signaling via increased p53 and p21 activity . our in vitro data suggest that this lrrk2 k1316n mutation like other canonical lrrk2 mutations increases ros , observed in parkinson disease . 
thus , another potential hypothesis to explain the radiosensitivity of disease in this patient is that increased lrrk2 kinase activity in lrrk2 mutants may render cells more susceptible to oxidative stress imparted by radiotherapy through creation of ros , such as oxygen free radicals.6 multiple publications show that lrrk2 mutations induce synergistic or additive cell killing in the presence of oxidative stress.7 , 8 taken together , the presence of increased oxidative stress imparted by an activating lrrk2 mutation could prime tumor cells to increased radiation - mediated cell death . the near - complete pathologic response and more than 4 - year disease - free interval since completion of all therapy are remarkable results from a low to moderate dose of radiation and a drug that rarely induces complete response ( 0% in the sharp trial )  . 
it is also interesting to note the dramatic radiographic responses seen in lesions that were not directly radiated , which suggests an abscopal effect from the combination of sorafenib and radiotherapy . 
certainly , we cannot overlook that the dramatic responses observed may have been due in part to the development of an antitumor immune response , which has been previously attributed to both sorafenib and radiation.9 , 10 indeed , the radiologic progression initially noted after sorafenib started may be related to pseudoprogression and development of an early immune response or apoptosis / necrosis of the tumor , despite the observation of the most dramatic clinical and radiographic response with postradiation sorafenib . in conclusion , these data support a role for a novel , previously unidentied lrrk2 mutation that promotes sorafenib and radiation sensitivity through an ros - dependent mechanism . in addition , the combination of radiation and sorafenib also may have induced an antitumor immune response . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . patrick wald employment : riverside radiation oncology sameek roychowdhury stock and other ownership interests : johnson & johnson ( i ) consulting or advisory role : incyte , abbvie , qed therapeutics research funding : takeda , ignyte anne m . 
noonan consulting or advisory role : helsinn healthcare no other potential conicts of interest were reported . references llovet jm , ricci s , mazzaferro v , et al : sorafenib in advanced hepatocellular carcinoma . 
n engl j med 359 : 378 - 390 , 2008 goldwurm s , di fonzo a , simons ej , et al : the g6055a ( g2019s ) mutation in lrrk2 is frequent in both early and late onset parkinsons disease and originates from a common ancestor . 
j med genet 42 : e65 , 2005 loefer da , klaver ac , coffey mp , et al : increased oxidative stress markers in cerebrospinal uid from healthy subjects with parkinsons diseaseassociated lrrk2 gene mutations . 
front aging neurosci 9 : 89 , 2017 coriat r , nicco c , ch ereau c , et al : sorafenib - induced hepatocellular carcinoma cell death depends on reactive oxygen species production in vitro and in vivo . mol cancer ther 11 : 2284 - 2293 , 2012 5 . 
ho dh , kim h , kim j , et al : leucine - rich repeat kinase 2 ( lrrk2 ) phosphorylates p53 and induces p21 ( waf1 / cip1 ) expression . 
gene 532 : 18 - 23 , 2013 yang d , li t , liu z , et al : lrrk2 kinase activity mediates toxic interactions between genetic mutation and oxidative stress in a drosophila model : suppression by curcumneurobiol dis 47 : 385 - 392 , 2012 8 . 
ho v , lim ts , lee j , et al : tlr3 agonist and sorafenib combinatorial therapy promotes immune activation and controls hepatocellular carcinoma progression . oncotarget 6 : 27252 - 27266 , 2015 10 . 
stable post - operative changes are seen , and there is no evidence of disease . 1 / 30 / 14 : afp decrease to 26 , 343 ng / ml but radiographic progression seen . sorafenib radiation surveillance 2 / 17 / 14 - 3 / 10 / 14 : received 40 gy in 16 fractions . 4 / 21 / 14 - 6 / 20 / 14 : sorafenib restarted at 200 mg twice daily . 
quantitative polymerase chain reaction ( qpcr ) analysis ( a ) and western blot ( b ) conrmed overexpression of wild - type ( wt ) lrrk2 and lrrk2 - k1316n ( mutant ) on four cell lines 72 hours after transfection . 
 telomerase is a prognostic marker of poor outcome and a therapeutic target in neuroblastoma andrea roderwieser , phd1 , 2 ; frederik sand , md1 ; esther walter , md1 ; janina fischer , md1 ; judith gecht , dmedsci1 ; christoph bartenhagen , phd1 , 2 ; sandra ackermann , phd1 , 2 ; felix otte1 ; anne welte1 , 2 ; yvonne kahlert1 , 2 ; daniela lieberz , phd1 ; falk hertwig , phd3 ; h . 
christian reinhardt , md2 , 4 ; thorsten simon , md1 ; martin peifer , phd2 ; monika ortmann , md2 ; reinhard b uttner , md2 ; barbara hero , md1 ; roderick j . 
osullivan , phd5 ; frank berthold , md1 ; and matthias fischer , md1 , 2 purpose telomere maintenance is a hallmark of high - risk neuroblastoma ; however , the contribution of telomerase and alternative lengthening of telomeres ( alt ) to clinical phenotypes has remained unclear . 
the presence of activated telomerase ( ie , tert rearrangements , mycn amplication , or high tert expression without these alterations ) was associated with poorest overall survival and was an independent prognostic marker in multivariable analyses . 
we also found that the telomerase - interacting compound 6 - thio - 2 ( cid : 1 ) - deoxyguanosine effectively inhibited viability and proliferation of neuroblastoma cells bearing activated telomerase . 
 roderwieser et al context key objective to determine the prognostic value of telomerase activation in primary neuroblastoma and to evaluate telomerase as a therapeutic target in preclinical neuroblastoma models . knowledge generated alterations associated with telomerase activation ( ie , tert rearrangements , mycn amplication , or elevated tert expression levels in the absence of these alterations ) occur in approximately one - third of primary neuroblastomas . 
the telomerase - interacting compound 6 - thio - 2 ( cid : 1 ) - deoxyguanosine efciently inhibits growth of telomerase - positive neuroblastoma cell lines in vitro and in vivo . relevance telomerase activation predicts adverse outcomes in primary neuroblastoma and may thus contribute to improving risk estimation for patients with neuroblastoma . 
preclinical studies establish telomerase as a promising therapeutic target in telomerase - positive neuroblastoma , suggesting that telomerase - interacting compounds should be evaluated in clinical trials . represent a therapeutic target in affected patients.9 in line with this notion , studies have reported on high levels of telomerase activity in unfavorable neuroblastoma phenotypes in a limited number of patients.10 - 12 by contrast , inactivating atrx mutations along with activation of the alternative lengthening of telomeres ( alt ) pathway and low tert mrna levels have been identied in a separate high - risk subgroup lacking mycn and tert alterations.6 , 13 in addition , we recently demonstrated that telomere maintenance mechanisms , in general , are a hallmark of high - risk neuroblastoma.14 it has remained unclear , however , whether telomerase and alt activation delineate distinct clinical subgroups and whether activated telomerase is a suitable therapeutic target in neuroblastoma . here , we set out to assess the prognostic value of biomarkers indicating telomerase or alt activation in patients with neuroblastoma and to examine the potential relevance of telomerase as a molecular target in this malignancy . 
to this end , we determined the genomic status of tert and mycn , characteristics of alt as well as tert expression levels in large cohorts of pretreatment neuroblastomas , and examined their association with prognostic markers and patient outcome . we also assessed growth characteristics of neuroblastoma cell lines on treatment with different telomeraseinteracting compounds in vitro and in vivo . 
we found that tert rearrangements occurred exclusively in patients age 18 months or older at diagnosis ( n = 46 of 254 [ 18.1% ] ; fig 1b ) , and they were not associated with being older than 5 years at diagnosis ( n = 11 of 66 [ 16.7% ] ; appendix fig a2a )  . 
tert rearrangements were predominantly detected in stage 4 disease and mycnnonamplied tumors ( n = 41 of 218 [ 18.8% ] and n = 41 of 361 [ 11.4% ] , respectively ; fig 1b ) , and were also associated with genomic losses at chromosome 1p and 11q ( appendix fig a2a )  . 
in addition , the presence of tert rearrangements was strongly associated with high - risk disease according to the international neuroblastoma risk stratication system17 ( n = 41 of 220 [ 18.6% ] ; fig 1b )  . outcome of patients whose tumors harbored tert rearrangements was poor in terms of event - free survival ( efs ) and overall survival ( os ) , and similar to that of patients whose tumors carried mycn amplication ( fig 1c )  . 
the prognostic relevance of tert rearrangements was substantiated by multivariable analyses , in which the presence of such alterations predicted adverse os independently of the established variables stage , age , and mycn and 1p status , which are currently used for patient risk assessment in germany18 ( p = .050 ; fig 1d , data supplement )  . 
we also validated that tert rearrangements and mycn amplication were strongly associated with elevated tert mrna levels in neuroblastoma in a large cohort of 379 neuroblastoma tumors14 ( fig 1e )  . alt denes an unfavorable patient subgroup that differs from patients with telomerase - positive neuroblastoma activation of the alt pathway has been suggested to be a marker of poor outcome in neuroblastoma13 ; however , its overall prevalence and its association with patient characteristics and courses have largely remained unclear . therefore , we assessed apbs , a hallmark of alt - positive tumors , in 273 of 457 neuroblastomas ( fig 2a ; table 1 ; appendix fig a1 ; data supplement )  . 
apbs were observed mainly in tumors of patients age 18 months or older at diagnosis ( n = 47 of 159 [ 29.6% ] ; fig 2b ) and occurred in approximately half of the patients who were older than 5 years at diagnosis ( n = 18 of 38 [ 47.4% ] ; appendix fig a2b )  . 
in addition , genomic alterations of atrx were identied in eight of 109 evaluable cases ( 7.3% ) , all of which were apb positive , whereas no atrx mutations were found in 12 additional apb - positive cases that ( 11.0% ; data supplement )  . 
as expected , 14 apbs were associated with low tert expression levels and increased telomere length ratios , which is in line with alt activation ( appendix fig a3a and a3b )  . telomerase and alt activation discriminate subgroups of patients with neuroblastoma with distinct outcome in our previous studies , we had observed that a small number of neuroblastomas harbored elevated tert expression levels and increased telomerase activity despite lacking genomic alterations of tert and mycn.14 therefore , we had dened a tert expression threshold that identied tert and mycn wild - type tumors whose tert mrna levels were comparable to those of tumors bearing tert or mycn alterations , referred to as tert hi14 . 
in the current study , we applied this threshold to the cohort for which information on genomic tert and mycn alterations and alt and tert expression was available ( n = 223 )  . 
the tert hi cohort consisted of 10 tumors ( 4.5% ; appendix fig a3a ) , which all were derived from patients age 18 months or older and mainly from stage 4 disease ( data supplement )  . 
outcome of these patients was as poor as that of patients with neuroblastomas bearing tert rearrangements and worse than that of patients with mycnamplied or alt - positive tumors ( appendix fig a4 )  . furthermore , we found three tumors positive for alt showing high tert expression levels ; it remains unclear , therefore , whether these tumors depend on alt or telomerase activation . together , our ndings suggest that telomerase activation , resulting from tert rearrangements , mycn amplication , or alternative events , may constitute a mechanistically dened clinical subgroup in neuroblastoma . 
telomerase activation was strongly associated with stage 4 , age older than 18 months at diagnosis , loss of 1p , and high - risk disease ( fig 3a )  . 
by contrast , absence of both telomerase activation and alt ( ie , absence of telomere maintenance mechanisms ) was predominantly associated with age younger than 18 months at diagnosis , stages 1 to 3 and 4s , and nonhigh - risk disease ( appendix fig a5 )  . 
tert mrna levels were slightly higher in tumors with complete response compared with tumors with partial response or stable disease ( appendix fig a6a )  . this nding was probably mainly due to better responses of mycn - amplied tumors ; however , the data might also suggest that alt - positive tumors are more indolent in response to induction therapy than tumors with telomerase activation ( appendix fig a6b )  . 
additional studies on larger patient cohorts are needed to address this question denitely . efs of patients whose tumors bore telomerase activation was similar to that of patients with alt - positive tumors and signicantly worse than that of patients whose tumors lacked telomere maintenance mechanisms ( fig 3b )  . 
by contrast , os of patients with telomerasepositive neuroblastomas was poorer than that of patients with alt - positive and telomere maintenancenegative tumors ( fig 3b )  . in multivariable analysis based on efs , both telomerase activation and alt were independent prognostic markers in addition to age , whereas only telomerase and age were independent prognostic markers in multivariable analysis based on fig 1 . 
 ( d ) multivariable cox regression analysis of patients with neuroblastoma ( n = 457 ) for os considering the following variables : age , stage , mycn status , 1p status , and tert status . 
 ( a ) detection of alt - associated pml nuclear bodies ( apbs ) indicating alt by combined telomere uorescent in situ hybridization and direct pml immunouorescence in tumor cells of sample nbc - 16 . 
 ( a ) frequencies of telomerase activation ( ie , mycn amplication , tert rearrangements , or tert hi ) in patient subgroups dened by stage ( blue , stage 4 ; red , stages 1 to 3 , 4s ) , age ( blue , 18 months or older ; red , younger than 18 months ) , risk group ( blue , hr ; red , nhr ) , 1p status and 11q status ( blue , deletion or imbalance [ del / im ] ; red , normal )  . 
 ( c ) multivariable cox regression analysis of neuroblastoma patients ( n = 223 ) for efs and os considering the following variables : age , stage , 1p status , telomerase activation , and alt . 
growth characteristics and dna damage signaling analysis of neuroblastoma cell lines with 6 - thio - 2 ( cid : 1 ) deoxyguanosine ( 6 - thio - dg ) treatment . 
 clinical relevance of telomerase activation in neuroblastoma telomerase is a therapeutic target in telomerase - activated neuroblastoma because telomerase activation denes a large group of high - risk neuroblastomas with poor outcome , we evaluated its potential value as therapeutic target in vitro and in vivo . 
to this end , the growth inhibitory effect of three different telomerase - interacting compounds ( 6 - thio - dg , 19 bibr - 1532 , 20 , 21 and costunolide22 ) was assessed in lines bearing tert rearhuman neuroblastoma cell rangements or mycn amplication , as well as altpositive cell lines and normal human bj broblasts as control . 
using two different assays , we found that cell viability upon treatment with 6 - thio - dg was impaired more effectively in telomerase - positive than in altlines and bj cells ( fig 4a - 4d ; appendix positive cell fig a7 )  . 
bibr - 1532 and costunolide treatment also reduced cell viability at lower concentrations in cell lines bearing telomerase activation than in alt - positive cell lines , using trypan blue exclusion assay ( appendix fig a8 ) ; however , this result was not conrmed for bibr1532 using a luminescent cell viability assay ( appendix fig a9 )  . 
moreover , growth inhibitory effects of these two compounds occurred in bj cells at similar or even lower concentrations in comparison with telomerase - positive cell lines , suggesting poor on - target specicity ( appendix fig a7 )  . 6 - thio - dg has been reported as a potent cytotoxic drug in telomerase - positive tumors that acts via telomere dysfunction leading to induction of apoptosis , reduction of proliferation , and senescence.19 , 23 - 25 therefore , we assessed the effect of 6 - thio - dg on apoptosis in telomeraselines . 
we obpositive and - negative neuroblastoma cell served a strong increase in apoptotic cells in cell line clb - ga , which harbors a tert rearrangement ( fig 4e and 4f ) , whereas the effect was less pronounced in the mycnamplied cell line imr - 32 ( fig 4g and 4h )  . 
by contrast , 6 - thio - dg treatment did not induce apoptosis in the altpositive cell line sk - n - fi ( fig 4i and 4j )  . 
this nding was supported by strong upregulation of cleaved caspase 7 in clb - ga , 25 whereas upregulation was weaker in imr - 32 cells and lacking in sk - n - fi ( fig 4k )  . 
we also examined levels of p21waf1 / cip1 as a marker of cell cycle arrest and found a strong and concentration - dependent p21waf1 / cip1 increase on 6 - thio - dg treatment in both telomerasepositive cell lines but not in alt - positive neuroblastoma cells ( fig 4k )  . line clb - ga , results from in vitro experiments suggested that 6thio - dg may represent a promising compound for the treatment of telomerase - positive neuroblastoma . 
to this end , the tert rearrangement - positive cell the two mycn - amplied cell lines be ( 2 ) c and imr - 32 , the tert hi cell line sh - sy5y , and the alt - positive cell line sk - n - fi were injected subcutaneously in immunodecient arthymic nude mice . 
growth of clb - ga and sh - sy5y cells was strongly impaired upon 6 - thio - dg treatment as compared with control animals , resulting in signicantly improved os of the treated mice ( fig 5a and 5b )  . 
in be ( 2 ) c xenografts , 6 - thio - dgtreated mice had signicantly better os , though the effect on tumor growth was moderate ( fig 5a and 5b )  . 
by contrast , 6 - thio - dg treatment had no effects on tumor growth or survival in imr - 32 and sk - n - fi xenograft models ( fig 5a and 5b )  . discussion in this study , we demonstrated that telomerase is activated in a large group of patients with high - risk neuroblastoma with poor outcome , who may thus potentially lines treated with fig 4 . 
because of limited compound effects , a growth inhibition curve could not be calculated for the alternative lengthening of telomeres ( alt ) - positive cell line chla - 90 . 
all experiments were performed in triplicate in at least two independent experiments . ( c ) cell viability determined by celltiter - glo assay ( promega , madison , wi ) in neuroblastoma cell lines treated with various concentrations of 6 - thio - dg . 
 ( k ) protein expression levels of p21waf1 / cip1 and cleaved caspase 7 after treatment with various concentrations of 6 - thio - dg for 48 hours , as determined by western blot analysis ; - actin was used as loading control . 
overall , approximately 10% and 20% of untreated tumors harbored tert rearrangements and mycn amplication , respectively , both of which have been shown to induce tert expression.6 , 26 in addition , tert mrna levels were elevated in approximately 4% of untreated tumors that lacked genomic aberrations of tert or mycn . 
all three alterations occurred mutually exclusively and were associated with similarly poor patient outcome . moreover , we have shown previously that enzymatic activity of telomerase is elevated in tumors of these subgroups.6 , 14 thus , neuroblastoma with tert or mycn alterations or high tert expression in the absence of these alterations may be considered as a single clinical subgroup that is dened by telomerase activation . 
telomerase activation was also a strong prognostic marker thus emphasizing its potential value for neuroblastoma risk assessment.10 in multivariable analyses , in addition , we found that alt occurs in approximately one - sixth of primary neuroblastomas . 
this nding is in line with those of a previous report suggesting that alt activation may be associated with a more indolent but eventually deadly course of the disease.13 in fact , we observed that os of patients with alt - positive tumors plateaued at 10 years , whereas that of patients with telomerase - positive tumors remained stable after 5 years ( fig 3b )  . 
together , our ndings indicate that telomerase and alt activation are strong and independent prognostic markers in neuroblastoma ( fig 3c ) and suggest these alterations may be considered for patient risk assessment formation on telomere maintenance with additional molecular data , such as copy number alterations , gene expression proles , or ras and p53 pathway gene mutations , 14 , 27 , 28 may rene clinico - genetic classication of neuroblastoma in future studies , ultimately allowing precise risk estimation and treatment in clinical practice . integration of stratication of patients solely on the basis of a dened set of biomarkers . in addition , we demonstrated that telomerase represents a potential therapeutic target in patients with telomerasepositive neuroblastoma , who have poor outcome with current treatment protocols . 
treatment of neuroblastoma cell lines with 6 - thio - dg impaired cell viability at signicantly lower concentrations in telomerase - activated than in altpositive cell lines and normal human broblasts , suggesting on - target specicity of this compound.19 , 23 - 25 , 29 , 30 the cytotoxic effect of 6 - thio - dg was obviously associated with induction of apoptosis and , potentially , with induction of cell cycle arrest . 
in neuroblastoma xenografts , 6 - thio - dg strongly impaired tumor growth of tert rearrangement - positive and tert hi neuroblastoma cells , whereas the inhibitory effect on mycn - amplied neuroblastoma was less pronounced . 
although the reason for these differences remains unclear , the broad effect of mycn on one may speculate that oncogenic pathways8 , 31 , 32 may counteract inhibition of single downstream molecules , such as telomerase . 
the distinct inhibitory effects may also be due to incorporation of a higher fraction of 6 - thio - dg into the genomic dna in mycn - amplied xenografts , related to different growth characteristics of these tumors , at least in the ultrafast growing sk - n - be ( 2 ) c xenografts . 
it has been demonstrated that 6 - thio - dg is incorporated into telomeres and genomic dna , whereas the toxic effects of 6 - thio - dg are elicited primarily by incorporation into telomeres.19 thus , higher doses of 6 - thio - dg might be required for efcient growth inhibition in very - fast - growing neuroblastomas . 
this suggestion is supported by the observation that 6 - thio - dg increased the probability of animal survival signicantly in mycn - amplied sk - n - be ( 2 ) c xenografts , whereas no growth inhibitory effects were observed in mycn - amplied imr - 32 tumors ( fig 5 )  . 
christian reinhardt , matthias fischer financial support : matthias fischer administrative support : reinhard b uttner , matthias fischer provision of study material or patients : janina fischer , h . 
osullivan , frank berthold , matthias fischer collection and assembly of data : andrea roderwieser , frederik sand , janina fischer , felix otte , anne welte , thorsten simon , monika ortmann , reinhard b uttner , barbara hero , frank berthold , matthias fischer data analysis and interpretation : andrea roderwieser , esther walter , janina fischer , judith gecht , christoph bartenhagen , sandra ackermann , yvonne kahlert , daniela lieberz , falk hertwig , martin peifer , reinhard b uttner , barbara hero , roderick j . 
christian reinhardt consulting or advisory role : abbvie , astrazeneca , vertex , merck research funding : gilead sciences thorsten simon consulting or advisory role : eusa pharma ( inst ) , merck ( inst ) reinhard b uttner stock and other ownership interests : targos molecular pathology , tamp honoraria : astrazeneca , abbvie , bayer , bristol - myers squibb , boehringer ingelheim , merck serono , msd , novartis , qiagen , pzer , roche research funding : roche ( inst ) frank berthold consulting or advisory role : ymabs pharmaceuticals , united therapeutics matthias fischer consulting or advisory role : novartis honoraria : janssen - cilag no other potential conicts of interest were reported . acknowledgment we thank the patients and their parents for making available the tumor specimens analyzed in this study . 
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 appendix roderwieser et al neuroblastoma cohort tert cohort ( n = 457 ) analysis of tert rearrangements alt cohort ( n = 273 ) telomerase cohort ( n = 223 ) analysis of tert rearrangements and apbs analysis of tert rearrangements , apbs , and gene expression fig a1 . 
a cohort of 457 patients with neuroblastoma was analyzed for the presence of tert rearrangements . for 273 of these 457 patients , activation of the alternative lengthening of telomeres ( alt ) pathway was examined . 
 ( a ) frequencies of tert rearrangements ( tert - ra ) in patient subgroups dened by age ( blue , 5 years or older ; red , younger than 5 years ) , and chromosome 1p and 11q copy number status ( blue , deletion or imbalance [ del / im ] ; red , normal )  . 
tumors were categorized according to the presence of tert rearrangements , mycn amplication , alternative lengthening of telomeres ( alt ) , elevated tert expression in the absence of tert or mycn alterations ( tert hi ) , or lack of telomere maintenance mechanisms ( others )  . patients whose tumors were both alt positive and tert hi were excluded from analysis . 
 ( b ) telomere length ratios of tumors and matched normal controls ( computed from whole genome sequencing and whole exome sequencing data14 ) in tumor subgroups dened by tert rearrangements , mycn amplication , alt , or lack of these alterations ( others )  . 
frequencies of absence of telomere maintenance mechanisms in patient subgroups dened by age ( blue , 18 months or older ; red , younger than 18 months ) , stage ( blue , stage 4 ; red , stages 1 to 3 , 4s ) , and risk group ( blue , hr ; red , nhr )  . 
 ( a ) association of tert expression levels as determined by microarrays in neuroblastomas ( n = 83 ) according to residual / stable disease or complete response of metastases at the end of induction therapy . 
 ( b ) frequencies of tert rearrangements ( blue , tert - ra ; red , tert - wt ) , mycn amplication ( blue , mycn amplied ; red , nma ) , and alt activation ( blue , alt pos ; red , alt neg ) in patients with residual / stable disease at the end of induction therapy . 
relative cell viability determined by celltiter - glo assay in human normal bj broblasts treated with various concentrations of 6 - thio - 2 ( cid : 1 ) deoxyguanosine ( 6 - thio - dg ) , bibr - 1532 , and costunolide . 
 mutational signature and transcriptomic classification analyses as the decisive diagnostic tools for a cancer of unknown primary roger olofsson bagge akif demir joakim karlsson babak alaei - mahabadi purpose cancer of unknown primary is a group of metastatic tumors in which the standard diagnostic workup fails to identify the site of origin of the tumor . 
the potential impact of precision oncology on this group of patients is large , because actionable driver mutations and a correct diagnosis could provide treatment options otherwise not available for patients with these fatal cancers . 
nilsson , phd , goteborgs universitet sahlgrenska akademin , sahlgrenska cancer center , medicinaregatan ( continued ) patients and methods here we describe a patient whose tumor was misdiagnosed at least three times . 
next - generation sequencing , a patient - derived xenograft mouse model , and bioinformatics were used to identify an actionable mutation , predict resistance development to the targeted therapy , and correctly diagnose the origin of the tumor . 
transcriptomic classification was benchmarked using the cancer genome atlas ( tcga )  . results despite the lack of a known primary tumor site and the absence of diagnostic immunohistochemical markers , the origin of the patients tumor was established using the novel bioinformatic workflow . 
2018 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license : introduction massively parallel sequencing of tumor dna and rna offers new possibilities to identify actionable mutations and enables precision medicine in the clinic.1 , 2 identification of a high mutational load ( eg , resulting from carcinogen exposure or dna repair defects3 ) can also provide information on increased responsiveness to immunotherapies , such as antiprogrammed death 1 antibodies.4 trials such as the nci - match ( national cancer institute molecular analysis for therapy choice ) trial , the tapur ( targeted agent and profiling utilization registry ) study , and the nct - master ( molecularly aided stratification for tumor eradication ) study5 , 6 include next - generation sequencing ( ngs ) to identify actionable mutations and will generate new data to inform future trial designs or best clinical practice . 
because their prognosis is poor , 7 patients may benefit significantly from extended genomic analysis not only for identification of targeted treatments but also for identification of the primary site of the tumor.8 , 9 targeted therapies directed against a particular driver mutation can act differently in different tumor types10 ; therefore , it may be beneficial to identify both the actionable mutation and the primary tumor site . here we describe an unusual case of a patient seeking a second opinion while receiving therapy with pembrolizumab for a presumed lung cancer . 
however , a subsequent fluorescent in situ hybridization analysis failed to confirm the alk positivity , and re - evaluation of the original specimen suggested instead a largecell neuroendocrine carcinoma of the lung . at the patients request , the cell block sections were re - examined in may 2016 at the sahlgrenska university hospital in sweden . 
cytologic and immunohistochemical ( ihc ) examination of the original specimen suggested a malignant neuroendocrine neoplasm ( nen ) , possibly a malignant paraganglioma ( pgl ) with differential diagnoses being gangliocytic neuroblastoma , malignant melanoma , merkel cell carcinoma , carcinoid , and small - cell lung cancer ( fig 1 )  . 
 analysis of an extirpated subcutaneous metastasis revealed that the tumor was positive for the neuroendocrine markers tyrosine hydroxylase ( th ) , chromogranin a , synaptophysin ( syn ) , neu - n , and cd99 , as well as for the neural crest markers sox10 and s100 . 
in contrast , the tumor was negative for cytokeratins as well as the melanoma markers tyrosinase , hmb45 ( pmel ) , and melan - a ( fig 1 ; appendix figs a1 to a3 )  . 
a patient - derived xenograft ( pdx ) mouse model was generated as previously described11 from the subcutaneous metastasis , and the xenograft was also positive for th , syn , sox10 , and s100 ( fig 1 )  . 
taken together , these analyses argue for a nen of unknown primary , which accounts for > 10% of these diagnoses.12 urinary dopamine levels were transiently elevated ( approximately three times the upper limit of normal ) , but repeated measurements of plasma metanephrines ( including metoxytyramine ) were negative , arguing against a primary pgl . 
ngs and multiplex ligation dependent probe amplification analyses of genes previously associated with hereditary pgl were all negative , 13 , 14 and there was no family history of pgl . 
array comparative genomic hybridization and whole - genome sequencing analyses of the metastasis revealed an aneuploid tumor with copy number alterations preferentially affecting whole chromosomes and chromosome arms , including gain of 1q , 2 , 7 , 8 , 9p24.3 - p23 , 9q , 13 , 15 , 16 , 20 , and 21 and loss of 1p , 9p23 - p13.2 ( homozygous loss of cdkn2a ) , 10 , and 18 ( fig 2a and 2b ; appendix table a1 ; data supplement )  . 
morphologic and immunohistochemical characteristics of the cell block from the ( a - f ) second opinion endobronchial ultrasound - guided aspiration , ( g - l ) subcutaneous metastatic lesion , and ( m - r ) patient - derived xenograft model . 
 [ 18f ] fluorodeoxyglucosepositron emission tomography / ct examination at sahlgrenska university hospital ( fig 3a ) showed progression , and the patient had clinical symptoms of stridor and shortness of breath . 
both were negative , ruling out the use of metaiodobenzylguanidineor peptide receptormediated radionuclide therapy . after a multidisciplinary conference in august 2016 , the patient started with the braf inhibitor ( brafi ) dabrafenib ( based on the v600e mutation )  . 
a rapid partial response was observed , with shrinkage of the subcutaneous metastases , disappearance of clinical symptoms including the stridor , and metabolic response on [ 18f ] fluorodeoxyglucosepositron emission tomography / ct after evaluation at 8 weeks ( fig 3a )  . 
unfortunately , he rapidly deteriorated ; a ct brain scan revealed massive progression of the brain metastases , and the patient died shortly thereafter . in the pdx model generated from the subcutaneous metastasis , treatment with the brafi dabrafenib alone or in combination with the meki trametinib was tested . 
notably , there was no effect of the brafi alone , whereas in two of three mice , the combination treatment led to markedly slower disease progression , which correlated with a reduction of phosphorylation of the mek target erk ( fig 3b )  . 
the lack of complete inhibition of erk phosphorylation could be explained by extended analyses of the genome sequencing of the subcutaneous metastasis , which revealed p124l and f53l mutations in map2k1 ( encoding mek1 ; fig 2d )  . 
these mutations have previously been associated with resistance to brafi in melanoma.17 comprehensive immunoprofiling to establish the correct diagnosis to diagnose this unusual case of presumed nen , additional morphologic and ihc analyses were performed ( appendix table a2 ; appendix figs a1 to a3 )  . 
analyses of all specimens , including the brain metastasis , showed that the tumor had partly epithelioid and partly elongated cells , with moderate nuclear pleomorphism but no prominent nucleoli , squamous differentiation , or gland formation . 
 ( a ) somatic copy number variants ( cnvs ) , single - nucleotide variants ( snvs ) , expressed fusion genes ( thick green lines ) , and somatic structural variants revealed by whole genome sequencing and ( b ) array comparative genomic hybridization . 
 ( c ) somatic variants detected with whole - genome and rna sequencing in braf ( v600e ) and ( d ) map2k1 ( p124l and f53l )  . carcinoma , malignant pgl , neuroblastic tumor , or malignant melanoma could be entertained . 
although such cells may be lost in malignant forms , and these may also have a more diffuse growth pattern , the extent of atypia and necrosis and the complete lack of nesting and sustentacular cell pattern would be unusual . 
a neuroblastic tumor would be unusual at the patients age and with this dissemination pattern , and the lack of ganglion cell differentiation and nb84 expression is not compatible with this diagnosis . 
lack of such differentiation may occur in undifferentiated neuroblastoma , but the patients tumor did not show the typical small blue round - cell tumor pattern characteristic of this entity . 
neural and neuroendocrine differentiation , as exemplified by focal reactivity for syn , chromogranin - a , and th , has been noted in melanoma18 but has always been accompanied by at least one of the melanoma markers hmb45 , tyrosinase , or melan - a . 
 ( a ) tumor load in patient visualized on [ 18f ] fluorodeoxyglucose ( [ 18f ] fdg ) positron emission tomography / computed tomography at baseline ( top panels ) and after 3 months of treatment with the braf inhibitor dabrafenib ( bottom panels )  . 
 ( b ) patient - derived xenograft ( pdx ) model in nonobese severe combined immune - deficient interleukin - 2 chain receptor knockout mice ( nog mice )  . 
mutational signatures have emerged as powerful tools to determine the somatic mutational processes underlying the development of tumors.19 , 20 by calculating the frequencies of base substitutions across the genome , we observed a dominance of c > t transitions that preferentially occurred in dipyrimidine trinucleotide contexts ( fig 4a ) and that were biased toward the untranscribed strand ( poisson test , false discovery rate < 0.05 ) in both the skin and brain metastases . 
this pattern closely matched a known mutational signature from the catalogue of somatic mutations in cancer ( signature 7 ) associated with ultraviolet radiation ( uv ) induced damage ( fig 4a ) , which is only found in cancers originating from sun - exposed areas.19 , 20 a decomposition of the mutational spectrum into known mutational signatures , using the mutationalpatterns r package , 21 yielded an estimated 80% contribution from signature 7 in both samples . 
these results strongly indicate that the primary tumor had originated from the skin . the classic melanoma markers tyr , pmel , and mlana and the merkel cell carcinoma marker cytokeratin 20 were all negative , both by ihc analyses and by rna sequencing ( rna - seq )  . 
therefore , we instead compared the transcriptome of the present tumor with that of 9 , 583 other tumors from 32 cancer types sequenced by the cancer genome atlas ( tcga ) consortiunotably , for both the skin and brain metastases , the strongest correlating samples were melanomas ( fig 4b )  . 
dct and sox10 were also elevated in xenografts from brafi - treated mice , which correlated with suppression of the neuroendocrine markers chgb , th , and eno2 ( appendix fig a4 ; data supplement )  . 
taken together , our findings of an uv - induced mutational signature , braf / map2k1 / cdkn2a mutations , the dissemination pattern , the expression of s100 and sox10 , and the correlation with the transcriptome profiles of tcga melanomas strongly indicated that the present tumors represented metastases from an unknown primary melanoma with neuroendocrine differentiation . pan - cancer transcriptome - based classification can predict the cellular origin of a tumor to investigate how transcriptome - based classification performance generalizes beyond the present case , we performed leave - one - out cross validation of k nearest neighbor classification on the pan - cancer data set , varying the number of nearest neighbors ( k ) considered , in terms of spearman correlation , from one to 50 ( appendix )  . 
however , certain cancers were more difficult to predict than others ; for example , rectal and colon adenocarcinomas were difficult to distinguish , as were squamous carcinoma of the lung , squamous carcinoma of the head and neck , and bladder cancer , which share molecular characteristics22 ( fig 5a )  . an additional evaluation was performed on an independent set of previously published data11 and unpublished rna - seq data of melanoma pdxs , uveal melanoma metastases , and one lung adenocarcinoma , yielding an overall accuracy of 0.84 ( appendix fig a5c )  . 
 ( a ) trinucleotide substitution frequencies observed in exonic regions of whole - genome sequencing data from the skin ( top ) and brain metastases ( middle ) , as well as the ultraviolet radiation ( uv ) - associated reference signature from catalogue of somatic mutations in cancer ( bottom )  . 
acc , adrenocortical carcinoma ; blca , bladder urothelial carcinoma ; brca , breast invasive carcinoma ; cesc , cervical squamous cell carcinoma and endocervical adenocarcinoma ; chol , cholangiocarcinoma ; coad , colon adenocarcinoma ; dlbc , lymphoid neoplasm diffuse large b - cell lymphoma ; esca , esophageal carcinoma ; gbm , glioblastoma multiforme ; hnsc , head and neck squamous cell carcinoma ; kich , kidney chromophobe ; kirc , kidney renal clear cell carcinoma ; kirp , kidney renal papillary cell carcinoma ; lgg , brain lower - grade glioma ; lihc , liver hepatocellular carcinoma ; luad , lung adenocarcinoma ; lusc , lung squamous cell carcinoma ; meso , mesothelioma ; ov , ovarian serous cystadenocarcinoma ; paad , pancreatic adenocarcinoma ; pcpg , pheochromocytoma and paraganglioma ; prad , prostate adenocarcinoma ; read , rectum adenocarcinoma ; sarc , sarcoma ; skcm , skin cutaneous melanoma ; stad , stomach adenocarcinoma ; tgct , testicular germ cell tumor ; thca , thyroid carcinoma ; thym , thymoma ; ucs , uterine carcinosarcoma ; ucec , uterine corpus endometrial carcinoma ; uvm , uveal melanoma . uveal melanomas were misclassified according to the organs where metastases were sampled , suggesting that normal tissue contamination could influence transcriptome - based predictions . 
 notably , however , both the skin and brain metastases of the present case were predicted to be melanoma by this classifier . ultraviolet radiation signature identifies incorrectly labeled tcga lung tumors to further study the utility of uv signature analysis in detecting misdiagnosed tumors , we investigated all squamous lung cancer samples in tcga . 
transcriptome - based analyses predicted one to be head and neck squamous cell carcinoma and the others to be lung squamous carcinomas ( fig 5a ; appendix fig a6a )  . 
melanoma could be ruled out by a lack of decisive melanoma markers and common driver mutations , as well as by high expression of the squamous / basal marker tp6325 ( appendix fig a6b )  . 
 although these samples still shared many catalogue of somatic mutations in cancer mutations with skin melanomas ( appendix fig a6c ) , this is likely explained by spuriously shared mutations with highly mutated tumors in general . 
 ( a ) t - distributed stochastic neighbor embedding ( tsne ) projection of the pan - cancer data set , together with the main case skin and brain metastasis samples , as well as the independent data set used for additional validation of the transcriptome - based classifier . 
notably , skin squamous and basal cell carcinomas are absent from tcga and therefore cannot be excluded as possible diagnoses for either of the three lung squamous cancers in tcga with an uv signature . 
the uv signature is a biomarker that can indicate an origin from a sun - exposed site . discussion here we demonstrate the application of mutational signature analysis and transcriptomic profiling as an adjunct in the diagnosis of a cup with neuroendocrine differentiation . 
although the tumor had a braf mutation , the morphology was ambiguous and atypical for malignant melanoma.27 at least three hospitals had misdiagnosed the tumor , because the following features did not support a melanoma diagnosis : the tumor in the thoracic region was believed to be the primary , and the patient had never had a skin lesion removed ; classic melanocyte markers were negative , whereas the tumor was positive for several neuroendocrine markers ; and s100 and sox10 were expressed , but these markers may also be expressed in nen . 
indeed , the possibility of a diagnosis of melanoma was confirmed by a specialist in nen pathology ( r.r.d.k. ) who was blinded to the genome analyses . we benchmarked the performance of transcriptome based cancer type classification according to our method using tcga data and found high accuracy in general . 
 although beyond the scope of our study to investigate , we expect that other signatures associated with site - specific carcinogen exposure , such as tobacco smoke , could also be found informative when deciphering the primary origin of tumors . current knowledge indicates that skin melanocytes may have two developmental origins : directly from the neural crest or via schwann cell progenitors ( scps ) migrating along nerves in the developing embryo.28 interestingly , scps also generate the chromaffin cells of the adrenal gland.29 it is therefore tempting to speculate that coexpression of the neuroendocrine markers th , chromogranin a , and syn in the present tumor was the result of a transformation event involving an scp - generated sox10 - positive / mitf - positive / dct - positive melanoblast . 
 ( d ) estimated contributions of the uv signature in samples from tcga head and neck squamous cell carcinoma ( hnsc ) cohort , with site of origin noted according to available clinical metadata . 
given the approval of targeted therapies and immunotherapies for melanoma and lung cancer , a change of diagnosis could have real clinical impact within a short timeframe . despite the incorrect diagnosis , we would argue that the treatments our patient received included those used in the clinical management of malignant melanoma , such as dabrafenib and pembrolizumab . 
one might argue that he should have received the dabrafenib and trametinib combination treatment upfront given the mapk1 mutation , 17 but at that time , this mutation was unknown to the clinician , and the diagnosis was pgl , meaning that dabrafenib was already off label . 
possibly , a recently postulated triple combination targeting braf , mek , and erk would have been beneficial in this case.30 in summary , this is to our knowledge the first case study where mutational signature analysis has been used as a decisive adjunct to routine histopathology to ascertain the diagnosis of metastatic deposits from a cup . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . roger olofsson bagge consulting or advisory role : amgen consulting or advisory role : bristol - myers squibb speakers bureau : roche joakim karlsson no relationship to disclose babak alaei - mahabadi no relationship to disclose berglind o . 
nilsson stock and other ownership interests : exocure ventures , oxcia travel , accommodations , expenses : merck sharp & dohme sweden acknowledgment this research would not have been possible without the cooperation of the patient and his close relatives , to whom we extend our sincere gratitude . 
sequence data generated for this study has been deposited at the european genome - phenome archive , which is hosted by the ebi and the crg under accession number egas00001003026 . 
wagle n , berger mf , davis mj , et al : high - throughput detection of actionable genomic alterations in clinical tumor samples by targeted , massively parallel sequencing . 
stadler zk , battaglin f , middha s , et al : reliable detection of mismatch repair deficiency in colorectal cancers using mutational load in next - generation sequencing panels . 
golfinopoulos v , pentheroudakis g , salanti g , et al : comparative survival with diverse chemotherapy regimens for cancer of unknown primary site : multiple - treatments meta - analysis . 
dasari a , shen c , halperin d , et al : trends in the incidence , prevalence , and survival outcomes in patients with neuroendocrine tumors in the united states . 
muth a , abel f , jansson s , et al : prevalence of germline mutations in patients with pheochromocytoma or abdominal paraganglioma and sporadic presentation : a population - based study in western sweden . 
hoadley ka , yau c , wolf dm , et al : multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origcell 158 : 929 - 944 , 2014 23 . 
adameyko i , lallemend f , aquino jb , et al : schwann cell precursors from nerve innervation are a cellular origin of melanocytes in skcell 139 : 366 - 379 , 2009 29 . 
 appendix patient the patient received oral and written information and signed the informed consent according to the ethical approval at the regional ethical review board ( #289 - 12 )  . 
the patient and his relatives have approved the publication of this case report . immunohistochemical staining of tumor tissues biopsies from patient tumor and patient - derived xenograft ( pdx ) tumor were fixed in neutral buffered formalin and embedded in paraffsections ( 3 to 5 microns ) were placed on positively charged glass slides and subjected to antigen retrieval using envision flex target retrieval solution ( high power of hydrogen ; agilent technologies , santa clara , ca )  . 
the following primary antibodies were used : anti - alk ( clone alk1 ; dako ) , antibeta - catenin ( bd610154 ; becton dickinson , franklin lakes , nj ) , anti - cd56 ( clone 123c3 ; dako ) , anti - cd99 ( clone epr3097y ; epitomics , burlingame , ca ) , antichromogranin a ( clone lk2h10 ; millipore , burlington , ma ) , anti - cdx2 ( ab76541 ; abcam , cambridge , united kingdom ) , anticytokeratin 8 / 18 ( ncl - 5d3 ; leica , wetzlar , germany ) , anticytokeratin 20 ( clone ks20.8 ; dako ) , antihigh molecular weight cytokeratin ( clone ae1 / 3 ; dako ) , anti - hmb45 ( m0634 ; dako ) , anti - ki67 ( clone mib - 1 ; dako ) , antimelan a ( clone a103 ; dako ) , anti - nb84 ( ncl - nb84 ; leica ) , anti neu - n ( mab377 ; millipore ) , anti - nfp ( m0762 ; dako ) , anti - pax8 ( 10336 - 1 - ap ; proteintech , chicago , il ) , anti - s100 ( catalog no ir504 ; dako ) , anti - sox10 ( clone ep268 ; epitomics ) , antisynaptophysin ( clone dak - synap ; dako ) , anti - ttf1 ( clone spt24 ; novocastra , wetzlar , germany ) , and antityrosine hydroxylase ( clone 1b5 ; novocastra )  . extraction of dna and rna from tumor snap - frozen tumor pieces were homogenized using bullet blender ( next advance , troy , ny )  . 
dna and rna were extracted using the allprep dna / rna kit ( qiagen , hilden , germany )  . dna sequencingbased variant calling raw dna sequencing reads were aligned to the hg19 reference genome with bwa ( version 0.7.12 ; options mem and - m ; li h , et al : bioinformatics 25 : 1754 - 1760 , 2009 )  . 
the resulting bam files were recalibrated and indels were jointly realigned for the tumor and normal samples with gatk ( version 3.3.0 ; mckenna a , et al : genome res 20 : 1297 - 1303 , 2010 )  . 
variant calling was performed with gatk haplotypecaller on the tumor and normal sample separately and with mutect ( version 1.1.5 ; cibulskis k , et al : nat biotechnol 31 : 213 - 219 , 2013 ) on both samples paired . rna sequencingbased variant calling raw rna sequencing reads were aligned to the hg19 reference genome with star ( verison 2.5.2b ; dobin a , et al : bioinformatics 29 : 15 - 21 , 2013 ) in two passes , with the parameter sjdboverhang 125 . 
variant calling was subsequently performed using gatk haplotypecaller with the options - dontusesoftclippedbases , - stand_call_conf 20.0 , and - stand_emit_conf 20.0. variant filtering to obtain a sensitive set of whole - genome sequencing variant calls that included indels , the union of the gatk tumor and mutect somatic calls was first constructed . 
before the set operations , the variant vcf files were left normalized using bcftools ( two steps : options norm - m - both in the first , and norm f human_g1k_v37.fasta in the second ; the reference fasta file was obtained from the gatk resource bundle )  . 
subsequently , the sets were annotated with annovar ( wang k , et al : nucleic acids res 38 : e164 , 2010 ) to identify and discard all variants present with 1% frequency in either the 1000 genomes database ( version august 2015 ; 1000g2015aug_all ) or esp6500 ( esp6500siv2_all ) or listed in a version of dbsnp 138 , from which flagged somatic and clinically associated variants had been removed ( snp138nonflagged )  . 
 mutational signature analysis variants detected by whole - genome sequencing , as described under dna sequencingbased variant calling , were annotated using annovar ( as described ) , after which all variants that were either nonexonic or present in the snp138nonflagged , esp6500siv2_all , or 1000g2015aug_all databases were removed . 
the principle behind the fitting algorithm is to find the nonnegative linear combination of the predefined mutational signatures that best explains all mutations in a given sample , which is done by solving a nonnegative least squares optimization problem.21 this allowed the estimation of the relative contributions of the known mutational signatures in a given sample . 
p values were adjusted using the benjamini - hochberg method . for mutational signature analysis of the cancer genoma atlas ( tcga ) tumors , annotated somatic mutation calls ( mutect2 ) in vcf format on the basis if whole - exome sequencing data were downloaded for 561 lung squamous cell carcinoma ( lusc ) and 528 head and neck squamous cell carcinoma tumors from the genomic data commons portal ( gov / ; accessed on november 24 , 2017 , and february 26 , 2018 , respectively )  . 
gene read counts were derived with htseq - count ( parameters : - m intersection - strict - s no ; anders s , et al : bioinformatics 31 : 166 - 169 , 2015 )  . 
reads per kilobase per million normalized values were calculated , taking into account the max mature transcript length of each gene and using robust size factors as previously described for the deseq method ( anders s , et al : genome biol 11 : r106 , 2010 )  . 
 implementation and cross - validation of transcriptome - based cancer type classification to classify a sample using the k - nearest neighbor approach , spearman correlation coefficients were first calculated between the sample of interest and all samples in the tcga pan - cancer data set . 
leave - one - out cross - validation was performed by in turn excluding each sample from the tcga data set and classifying it using correlations to the remaining samples . 
the final classifier was then subjected to additional validation on an independent data set composed of 30 skin melanoma pdxs , 13 uveal melanoma metastases ( four skin and nine liver ) , and one lung adenocarcinoma primary tumor sample , all preprocessed and normalized as described for the main case samples . 
only mutations in cancer gene census genes listed in the catalogue of somatic mutations in cancer and annotated as missense_mutation , nonsense_mutation , nonstop_mutation , in_frame_ins , in_frame_del , frame_ shift_ins , or frame_shift_del and found in any of the three lusc samples were included . 
for additional removal of germ line variants , we first used the somatic call function with the - f parameter where the discord files were generated from seven normal genomes ( unpublished data ) and then removed events in which both breakpoints overlapped with known population variants the database of genomic variants database ( macdonald jr , et al : nucleic acids res 42 : d986 - d992 , 2014 )  . array - based comparative genomic hybridization genomic dna was extracted from fresh - frozen tumor tissues of the subcutaneous and brain metastases as previously described ( persson f , et al : oncogene 27 : 3072 - 3080 , 2008 )  . 
array comparative genomic hybridization ( cgh ) analysis was performed using the human genome cgh microarray 244k oligonucleotide arrays ( g4411b ; agilent technologies ) as recommended by the manufacturer . 
slides were scanned using an agilent high - resolution c microarray scanner , followed by data extraction and normalization using feature extraction ( version 12.0.1.1 ; agilent technologies ) with linear normalization ( protocol cgh_1200_jun14 )  . 
 generation of a pdx model a 3 3 3 mm piece of a biopsy of a subcutaneous metastasis of the patient was transplanted into the flank of immunocompromised , nonobese severe combined immune - deficient interleukin - 2 chain receptor knockout mice ( nog mice ; taconic , copenhagen , denmark ) to form xenografts . 
when the first pdx tumor reached 150 mm3 , it was passaged to new mice ( passage 2 ) , which subsequently were passaged to 10 mice , where tumor finally grew in eight mice ( passage 3 )  . 
these mice were treated 5 days per week for a minimum of 3 weeks with either vehicle or trametinib 0.3 mg / kg twice per day ( selleck chem , houston , tx )  . 
all animal experiments were performed in accordance with eu directive 2010 / 63 ( regional animal ethics committee of gothenburg approval #36 - 2014 )  . rna sequencing analysis of drug - treated pdx models rna sequencing reads were aligned to a combined human ( hg19 ) and mouse ( mm10 ) reference genome using star , with default settings . 
read counts were then binned to genes using the htseq ( version 0.6.0 ; zhao m , et al : nucleic acids res 44 : d1023 - d1031 , 2016 ) command htseq - count ( parameters - s reverse and - m intersection - strict ) , as well as the human reference genome annotation gencode ( version 17 )  . 
differential expression analysis was carried out using deseq2 ( cancer genome atlas network : nature 517 : 576 - 582 , 2015 ) , with the parameter alpha = 0.05 , considering vehicle - treated and untreated mice both as controls relative to the dabrafeniband trametinib - treated samples . 
enrichment analyses were carried out using msiddb ( love mi , et al : genome biol 15 : 550 , 2014 ) , using gene ontology biological process gene sets ( anders s , et al : bioinformatics 31 : 166 - 169 , 2015 ) , considering genes with an absolute log2 fold change > 2 and adjusted p values < .01. 
differential expression analysis of ( a ) dabrafenib - treated mice compared with vehicle and untreated controls and ( b ) dabrafenib and trametinib combinationtreated mice compared with vehicle and untreated controls . 
 ( a ) classification error ( defined as 1 - accuracy ) per cancer type obtained during leave - one - out cross - validation of the k - nearest neighbor classification approach on the cancer genome atlas data set , with spearman correlation coefficients calculated using all coding genes as the measure of similarity between samples . 
 ( c ) performance with k = 6 on an independent validation set , using unrelated skin melanoma patient - derived xenograft models , uveal melanoma skin and liver metastasis samples , and one lung adenocarcinoma primary tumor . 
 ( a ) tcga cancer types ranked by mean spearman correlation of the top six most strongly correlating samples in each cohort : tcga - 21 - 1079 ( top ) , tcga - 90 - a4ed ( middle ) , and tcga - 18 - 3409 ( bottom )  . 
 ( b ) expression of selected marker genes relevant when considering a melanoma diagnosis : tcga - 21 - 1079 ( top ) , tcga - 90 - a4ed ( middle ) , and tcga - 18 - 3409 ( bottom )  . 
 ( c ) frequency of each driver gene mutation found in the ultraviolet radiationassociated lusc tumors , in each of the different tcga cancer types , relative to the total number of samples in each cohort . 
2 , 3 we used our recently reported in silico tool ( dpyd - varifier ) 7 to predict that p.t132a would be deleterious to function , which was subsequently validated using ex vivo and in vitro approaches . 
these methodologies permitted us to determine an activity score for the patient that was used to calculate a safe adjusted dose of fu for adjuvant therapy . this study was approved by the mayo clinic institutional review board . 
dpyd - varifier was used to predict the function of p.t132a.7 the effect of p.t132a on dpd activity was measured in vitro as previously described.5 - 7 total rna was isolated from the patients blood sample using the paxgene blood rna kit ( preanalytix , hombrechtikon , switzerland ) , and cdna was reverse transcribed using oligo ( dt ) 15 primers ( thermo fisher scientific , waltham , ma )  . 
the full - length dpyd open reading frame was amplified ( primers : 5 - gtttgtcactggcagactcg - 3 , 5 - ttcacagcaactgtttcacaaa - 3 ) using q5 high fidelity dna polymerase ( new england biolabs , ipswich , ma )  . 
the polymerase chain reaction ( pcr ) product was cloned into the pjet1.2 vector , and 20 cloned constructs were sequenced at the mayo clinic gene analysis shared resource to determine the cis or trans conformation of the variants . 
peripheral blood mononuclear cells ( pbmcs ) were isolated and assessed ex vivo for dpd activity as previously described.9 , 10 additional pcr and quantitative pcr reactions were performed with primers specific to canonically ( e13 / 14 : 5 - ctcttgataaggacattgtgacaaa - 3 , 5 - tttgcagctcttgcgatgc - 3 ) and alternatively spliced dpyd ( e13 / 15 : 5 - ctcttgataagattgtgattgctagc - 3 , 5 - tttgcagctcttgcgatgc - 3 )  . 
 patient presentation ( bowel function alteration and red blood cell per rectum ) colonoscopy pathology staging ct scan 1st cycle began toxicity symptoms dose reduction 2nd cycle radiotherapy only radiotherapy completed 1st cycle began ( reduced mfolfox6 ) 46 - hour infusion 2nd cycle 4th cycle ( dose increased ) 5th cycle ( initial dose ) 8th cycle 10th cycle in silico prediction , ex vivo , and in vitro dpd assay , clonal rna sequencing dpyd sequencing ( mayo medical lab ) blood sample collection surgical resection diagnostic tests neoadjuvant cape + radio variants testing adjuvant mfolfox6 30 34 543 635 no . 
a 32 - year - old male with a history of gastroesophageal reflux and hyperlipidemia presented with altered bowel function and bright red blood per rectu colonoscopy revealed a polyp in the cecum and a mass approximately 10 cm within the rectosigmoid colon . 
a staging computed tomography scan of the chest , abdomen , and pelvis showed no evidence of distal metastases , which suggested stage iii disease . the patient was treated shortly after diagnosis with neoadjuvant therapy that consisted of capecitabine ( 825 mg m2 for 5 days each week ) and radiotherapy ( 50.4 gy in 28 fractions )  . 
 during hospitalization , absolute neutrophil count decreased to 700 cells / l ( normal reference range , 1 , 500 to 8 , 000 cells / l ) , which prompted discontinuation of therapy late in the second week of treatment . 
one and a half months after surgery , the patient was scheduled to begin modified infusional fu , leucovorin , and oxaliplatin ( mfolfox6 ) adjuvant therapy . because of the severe toxicity during the initial neoadjuvant therapy , genetic testing was ordered . 
dpd activity in the patients pbmcs was reduced by 64% ( 92.4 pmol fu min1 mg1 ) compared with previously reported reference values obtained from individuals who do not carry a deleterious dpyd variant10 ( 254.0 pmol fu min1 mg1 ; fig 2a )  . 
to determine whether allelic imbalance of dpyd was occurring , allele - specific pcr primers e13 / 14 and e13 / 15 were used to amplify canonical and alternatively spliced dpyd ( fig 2b , top )  . 
 ( a ) dihydropyrimidine dehydrogenase ( dpd ) enzyme activity was measured in peripheral blood mononuclear cells isolated from the patient and was compared with reference values previously published by our laboratory for peripheral blood mononuclear cells from individuals who do not carry known deleterious variants in dpyd ( noncarriers ) and those heterozygous for * 2a ( * 2a / wild type [ wt ] ) .10 ( b ) polymerase chain reaction primers were designed as indicated ( arrows ) to amplify across the exon 13 / 14 and 13 / 15 junctions to detect the presence of alternative splicing in the patient and a heterozygous carrier of * 2a . 
this level of reduction is similar to that observed for the well - studied p.d949v variant5 , 7 ; thus , the dpd activity score would be expected to be similar . 
finally , the decision was made to administer two additional cycles of therapy that did not contain oxaliplatin because of accumulating grade 1 neuropathy ( fig 1 , gold box )  . 
the patient has completed the 10th cycle of adjuvant therapy and has responded well to the treatment with no severe toxicity and no physical evidence of cancer progression . discussion this report outlines the effective management of toxicity in a patient with rectal cancer treated with an fu - based regimen using a precision oncology approach . 
to date , genetic testing of dpyd variants is not mandated in the united states , and complete dpd deficiency is only listed as a contraindication to fu treatment.11 many studies have increasingly performed functional characterizations of dpyd variants , 3 , 5 - 7 , 9 , 10 , 12 , 13 and the clinical pharmacogenetics implementation consortium has published activity - based fu dosing guidelines for many of the reported dpyd variants.8 however , a paucity of information exists with respect to fu dose adjustments in compound heterozygous variant carriers . in this case report , dpyd genotype and variant function data were used to provide guidance for adjuvant fu dosing in a patient who carries a complex dpyd genotype that includes a novel variant of unknown significance . 
the 38% reduction in in vitro dpd function attributed to p.t132a also is consistent with the approximately 36% activity measured in the patients pbmcs because the p.t132a variant in the patients genome is present in a compound complex heterozygous state ( trans ) with dpyd * 2a . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . shikshya shrestha no relationship to disclose colbren ( scout ) trogstad - isaacson no relationship to disclose timothy j . 
lee am , shi q , pavey e , et al : dpyd variants as predictors of 5 - fluorouracil toxicity in adjuvant colon cancer treatment ( ncctg n0147 )  . 
meulendijks d , henricks lm , sonke gs , et al : clinical relevance of dpyd variants c.1679t > g , c.1236g > a / hapb3 , and c.1601g > a as predictors of severe fluoropyrimidine - associated toxicity : a systematic review and meta - analysis of individual patient data . 
offer sm , wegner nj , fossum c , et al : phenotypic profiling of dpyd variations relevant to 5 - fluorouracil sensitivity using real - time cellular analysis and in vitro measurement of enzyme activity . 
lee am , shi q , alberts sr , et al : association between dpyd c.1129 - 5923 c > g / hapb3 and severe toxicity to 5 - fluorouracil - based chemotherapy in stage iii colon cancer patients : ncctg n0147 ( alliance )  . 
 o crizotinib and surgery for long - term disease control in children and adolescents with alk - positive inammatory myobroblastic tumors toby trahair , mbbs , phd1 , 2 , 3 ; andrew j . 
we retrospectively analyzed the outcome of patients with ap - imt treated with crizotinib to document response , toxicity , survival , and features associated with relapse . methods the cohort comprised eight patients with ap - imt treated with crizotinib and surgery . 
both harbored ranbp2 - alk fusions and responded to alternative alk inhibitors ; one ultimately died as a result of progressive disease , whereas the other remains alive on treatment . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction inammatory myobroblastic tumors ( imts ) are rare and predominantly arise in the abdomen , pelvis , and chest . 
approximately 40% to 60% of imts express anaplastic lymphoma kinase ( alk ) and have associated alk gene rearrangements.1 - 4 multiple alk fusion partners have been identied , including , but limited to , cars , 5 tpm3 , tpm4 , 2 , 6 cltc , 7 , 8 ranbp2 , 9 sec31l1 , 10 ppfib1 , 11 fn1 , 12 igfbp5 , thbs1 , 13 dctn1 , rrbp1 , tfg , 6 and eml4.14 cryptic translocations that involve ros , etv6 , and ntrk also have been identied in alk - negative imt.15 - 18 currently , a poor understanding exists of how the alk fusion partner inuences the clinical behavior of imt , in cases of epithelioid inammatory myoexcept broblastic sarcoma ( eims ) , a clinically aggressive tumor associated with a high mortality before the introduction of alk inhibitors ( alkis ) .19 , 20 eims is characterized by perinuclear or nuclear membrane alk staining6 , 19 , 21 ; cd30 expression19 , 20 , 22 , 23 ; and characteristic alk fusion partners , namely ranbp2 , 19 , 20 , 23 rrbp1 , 6 and eml4.14 surgery remains central to imt therapy . 
surgical resection with clear margins results in excellent outcomes.24 , 25 although incomplete resection is associated with recurrence , favorable outcomes have been reported with adjuvant chemotherapy.24 - 26 alkis have been shown to be safe and effective in alkrearranged tumors , including imts , 27 - 30 but no randomized data have compared alki treatment and chemotherapy . 
some authors continue to advocate surgery and adjuvant chemotherapy.25 to better dene the utility of alki treatment in alk - positive imt ( ap - imt ) , we analyzed the outcome of a cohort of eight patients with ap - imts treated with crizotinib . 
 trahair et al context key objective to document the long - term outcome of a cohort of children and teenagers diagnosed with widespread , multifocal , alk - positive inammatory myobroblastic tumors ( ap - imts ) treated in a crizotinib compassionate use access program . knowledge generated crizotinib and surgery are effective for widespread ap - imts in most patients who are able to stop treatment safely . 
disease control in the aggressive imt variant ranbp2 - alk rearranged epithelioid inammatory myobroblastic sarcoma was suboptimal , with early , on - treatment relapses . relevance many patients with ap - imt have excellent outcomes with crizotinib and surgery , which conrms that crizotinib should be considered as rst - line therapy for patients with widespread or unresectable disease . 
the poorer outcomes in patients with ranbp2 - alkrearranged epithelioid inammatory myobroblastic sarcoma suggests that specic patient subgroups , dened by pathology and alk translocation partner , could benet from intensied or combination therapy . methods participants and treatment the cohort comprised eight patients diagnosed with apimt from 2009 onward and who were treated with crizotinib . 
patients were treated at four childrens cancer including sydney childrens hospital ( n = 5 ) , centers , queensland childrens hospital ( n = 1 ) , princess margaret hospital ( n = 1 ) , and starship childrens hospital ( n = 1 )  . the cohort includes all patients diagnosed with ap - imt from 2009 onward . 
alk locus rearrangement was detected using uorescence in situ hybridization ( fish ) with a dual - color break - apart probe ( vysis ; abbott laboratories , abbott park , il )  . crizotinib was provided by pzer ( new york , ny ) using a noncommercial supply management system for alkrearranged malignancies . 
eligibility criteria for patients the pzer with ap - imt were based on version 6.0 of named - patient early - access guidance dated november 1 , 2011 , and included a diagnosis of an ap - imt , adequate organ function , stable neurologic disease , and no clinical or laboratory evidence of signicant cardiorespiratory disease.31 for ap - imt , demonstration of alk positivity by ihc alone was sufcient . 
crizotinib was used at the pediatric maximum tolerated dose ( 280 mg / m2 / dose twice a day orally ) .28 , 29 a treatment cycle was dened as 28 days , and crizotinib was continued at physician discretion while there was evidence of clinical benet . 
guidelines for safety monitoring ; lifestyle recommendations , including contraception ; management of adverse events ( aes ) ; dose modication for toxicity and ae reporting ; grading ; and attribution were as recommended in the pzer crizotinib named - patient earlyaccess reference guide.32 briey , recommended clinical monitoring for safety included monthly blood tests , including a full blood count and liver function tests , for the rst 3 months then repeated as clinically indicated . 
it was recommended that aes be identied throughout the entire course of treatment with grading , causality , and reporting to the pzer drug safety unit within 24 hours of identifying each ae . 
disease staging and treatment response were determined locally by clinical examination and laboratory assessment combined with anatomic ( computed tomography [ ct ] , magnetic resonance imaging , and / or ultrasound ) and functional ( positron emission tomography [ pet ] / ct scan ) imaging . 
single diameter measurements were used to quantify treatment response on the basis of response evaluation criteria in solid tumors ( recist ) version 1.1.33 a complete response ( cr ) was dened as disappearance of all lesions , partial response ( pr ) as a 30% or greater reduction in the diameters of all lesions with no new lesions , progressive disease ( pd ) as a 20% or greater increase in the diameters of all lesions or the appearance of new sites of disease , and stable disease as no evidence of signicant change in all lesions to qualify as pd , pr , or cr . outcomes and toxicity were analyzed retrospectively . toxicity guidelines data were graded according to the common terminology criteria for adverse events ( version 4 )  . 
the primary outcomes of interest were progressionfree survival ( pfs ) and overall survival ( os ) since the commencement of crizotinib , the incidence of severe treatment - related toxicities ( dened as grade 3 ) , and features associated with treatment failure . 
pfs and os were determined by kaplan - meier method using medcalc version 18 ( medcalc , ostend , belgium ) statistical software . the study was approved by the sydney childrens hospital network human research ethics committee ( lnr / 14 / schn / 90 )  . 
 crizotinib and surgery for alk - positive imts the declaration of helsinki , and informed consent was obtained from the patients parents or legal guardians . results clinical and pathologic features molecular analyses tumor samples from the ve patients treated at sydney childrens hospital were subjected to rna extraction for rna capture sequencing to identify alk fusion partners.34 sequencing libraries were prepared from 1 g of patient rna using the kapa stranded rna - seq library preparation kit ( roche , basel , switzerland )  . capture was performed according to manufacturers instructions using custom biotinylated probes complementary to the coding regions of genes associated with chromosomal translocations in imt . 
captured libraries were sequenced to standard depth with 125 - base pair paired - end sequencing using the hiseq 2500 kit v4 ( illumina , san diego , ca )  . 
three patients with alk nuclear membrane staining were found to have ranbp2 - alk translocations , whereas two patients with diffuse cytoplasmic alk staining were found to have sec31a - alk and cltc - alk fusions ( fig 1 ; table 1 )  . outcome after treatment with crizotinib and surgery the median time from diagnosis to commencing crizotinib was 19 days ( range , 5 to 1 , 504 days ; table 2 )  . 
histopathology of the resected alk - positive epithelioid inammatory myobroblastic sarcoma tumor from patient 2 that demonstrates ( a ) epithelioid / rhabdoid tumor cells , ( b ) prominent perinuclear / nuclear membrane alk staining on immunohistochemistry , ( c ) cd30 + tumor cells on immunohistochemistry , and ( d ) conrmation of an alk translocation by uorescence in situ hybridization using a dual - color break - apart probe ( vysis ; abbott laboratories , abbott park , il )  . 
the photograph demonstrates the alk translocation ( the split red and green signals in the lower portion of the image ) and one normal alk locus signal ( red and green signals adjacent to each other in upper portion of the image ) in a patient tumor cell . 
 ( e ) schematic representation of sec31a - , cltc - , and ranbp2 - alk fusions identied in ve patients from sydney childrens hospital by rna capture sequencing . long - term naproxen before developing progressive hepatic dysfunction . 
three patients were critically unwell and commenced crizotinib treatment in the intensive care unit ( table 2 )  . all patients had a clinical response to crizotinib ( table 2 )  . in three patients , surgery was performed at the point of maximal tumor shrinkage to achieve a cr . 
analysis of resected tumors showed extensive treatment - induced changes characterized by brous , hyalinized nodules , a patchy inammatory inltrate with foamy macrophages , and scattered foci of dystrophic calcication . 
in patient 4 , plump spindle - shaped cells weakly staining for alk - 1 in a nuclear membrane / perinuclear pattern were identied in the resected residual tumor ( fig 2 )  . 
patient 1 , who had multifocal abdominal and hepatic disease , had no evidence of tumor shrinkage on ct scan but had a complete metabolic response to crizotinib on uorodeoxyglucose pet scan and normalization of abnormal liver function tests . the eight patients , both with ranbp2 - alk two of rearranged tumors , experienced disease relapse within the rst year of crizotinib treatment despite initial crs with crizotinib ( table 2 ; fig 3 )  . 
treatment and outcome of alk - positive inammatory myobroblastic tumor cohort best response duration of crizotinib therapy patient clinical status at commencement of crizotinib crizotinib alone after surgery , years pfs , years total , years additional therapy and current treatment duration off crizotinib , years outcome after crizotinib surgery crizotinib surgery off treatment relapse crizotinib ceritinib ongoing pleural and off treatment alive ceased ceased ceased ceased ceased ceased off treatment alive off treatment alive relapse ceritinib vinorelbine , methotrexate , nsaid off treatment alive ceased ongoing current status alive alive dead alive time from diagnosis to crizotinib , days 1 , 504 ascites , pleural effusions icu admission pericardial effusions with tamponade icu admission massive ascites , abdominal compartment syndrome , liver and renal impairment icu admission abbreviations : cr , complete response ; icu , intensive care unit ; na , not applicable ; ned , no evidence of disease ; nsaid , nonsteroidal anti - inammatory drug ; pd , progressive disease ; pfs , progression - free survival ; pr , partial response ; sd , stable disease . with intermittent granulocyte colony - stimulating factor ( 5 g / kg subcutaneous injection once or twice per week ) without crizotinib dose reduction . 
crizotinib adverse effects contributed to decisions to cease treatment in two patients . patient 3 , who had preexisting mild , chronic dermatitis , experienced multiple episodes of cellulitis , and patient 4 experienced multiple fractures ( fig 3 )  . 
both patients remain well , off crizotinib , and in continuing cr . clinical outcome after recurrence on crizotinib despite initial crs , two patients with ranbp2 - alk eims experienced on - treatment disease recurrence after 4 and 10 months of crizotinib . 
at recurrence , there were bilateral pleural effusions , largevolume ascites , multiple intra - abdominal peritoneal masses , and several lesions that indented the capsular surface of the liver . 
there was a signicant tumor response within 2 weeks of the dose increase , which was sustained at most disease sites and accompanied by resolution of pleural effusions , ascites , and peritoneal nodules . 
 ( d ) alk immunohistochemistry of resected residual tumor demonstrates frequent spindle - shaped cells with weak nuclear membrane / perinuclear alk staining . achieved a partial tumor response of 2 months.39 , 40 after progression on ceritinib , the patient was commenced on low - dose chemotherapy ( vinorelbine , methotrexate , and ketorolac ) , 41 which resulted in a complete metabolic response on uorodeoxyglucose pet scan , but with progressive calcication and no objective shrinkage on ct scan and magnetic resonance imaging . 
the tumor progressed after 6 months , and the patient died 23 months after the initial diagnosis . discussion imts are rare tumors in children , teenagers , and adults commonly characterized by alk gene rearrangement15 - 18 , 42 that are targetable with alkis.19 , 27 - 30 here , we present the clinical features , treatment , and outcome of a cohort of patients with ap - imts treated with crizotinib at the pediatric maximum tolerated dose.28 , 29 there are limitations to this study , being a retrospective analysis of outcome and toxicity in a compassionate access cohort . 
eligibility was based on a standardized guideline , which included demonstration of alk positivity on ihc , adequate organ function , and the absence of alternative treatment options as judged by the clinician . 
the access program provided guidance on safety monitoring and a requirement that clinicians identify , report , grade , and attribute causality for aes within 24 hours of their identication . 
 ( a ) kaplan - meier analysis of overall survival ( os ) and progression - free survival ( pfs ) since commencement of crizotinib treatment . ( b ) swimmer plot analysis of individual patient outcomes since diagnosis , including treatment received and episodes of disease progression , surgery , cellulitis , and fractures . 
although ranbp2 - alkrearranged eims has previously been identied as an aggressive ap - imt subtype , 19 , 22 we also observed life - threatening complications associated with a cltc - alkrearranged imt . 
early consideration of an alk rearrangement in the appropriate clinical context , particularly when patients present with extensive disease , is important for rapid diagnosis and timely initiation of alki treatment . 
this study demonstrates that crizotinib is tolerable and effective in patients with life - threatening complications , with three patients treated in the intensive care setting because of malignant effusions and one patient with associated hepatic impairment . 
despite this , oral crizotinib was well tolerated and resulted in rapid clinical responses . the phase i / ii crizotinib trial conducted by the us childrens oncology group ( cog ) demonstrated high response rates ( cr , 36% ; pr , 50% ) with a favorable toxicity prole in children and teenagers with ap - imts.28 , 29 a lower objective response rate ( 50% ) was observed in an adult crizotinib trial for ap - imt.30 our response data are concordant with the cog study , with all patients experiencing a clinical response . 
factors that underlie differences in responses between pediatric and adult patients are not clear ; however , the maximum tolerated dose in children ( 280 mg / m2 ) 28 is higher than the adult dose of 250 mg ( approximately equivalent to 140 mg / m2 ) .30 at steady state , the pediatric dose ( 280 mg / m2 ) results in a 2.5 - fold higher plasma area under the curve compared with the adult dose of 250 mg , 43 which suggests that the response of apimt to crizotinib may be partly due to plasma crizotinib levels . 
of note , ve ( 62.5% ) of the eight patients ceased crizotinib without experiencing relapse or pd , which indicates that possible to cease crizotinib in ap - imt without rapid disease recurrence as has been reported in alcl.44 the toxicity prole observed was in line with the cog clinical trial.28 , 29 the most notable ongoing toxicity during crizotinib treatment was moderate to severe neutropenia , which was managed by intermittent granulocyte colony - stimulating factor . 
recurrent toxicity contributed to the decision to cease crizotinib in two patients , one because of multiple fractures ( n = 3 ) and one because of multiple episodes of cellulitis ( n = 4 )  . 
cd30 - directed therapy using brentuximab vedotin is effective in relapsed hodgkin lymphoma and alcl , 62 , 63 in patients with cd30 + a strategy that may be useful ranbp2 - alk eims . in summary , with acknowledgment of the limitations of our retrospective cohort study , the outcomes indicate that crizotinib and surgery are effective in long - term disease control for the majority of patients with ap - imt . 
walker , james blackburn , draga barbaric data analysis and interpretation : toby trahair , chelsea mayoh , andrew c . wood , santosh valvi , james blackburn , erin e . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . toby trahair stock and other ownership interests : csl andrew c . 
wood patents , royalties , other intellectual property : pat055863 - us - pct , inventor assigned to genomics institute of the novartis research foundation involving combination inhibition of alk tim r . 
mercer speakers bureau : oxford nanopore patents , royalties , other intellectual property : inventor of two patents related to synthetic rna and dna controls for next - generation sequencing karen l . 
mackenzie research funding : komipharm ( inst ) no other potential conicts of interest were reported . acknowledgment we thank our surgical colleagues for the surgical care of these patients , including susan adams , bruce currie , and guy henry from sydney childrens hospital ; japinder khosa from perth childrens hospital ; and craig mcbride from lady cilento childrens hospital , brisbane . 
we acknowledge the pathologists martin weber and ella sugo , anatomical pathology , south eastern pathology service ; mary suter , cytogenetics laboratory , sydpath , st vincents hospital ; and the technical assistance of lisa morgan , emily mould , carol wadham , and angela xie at the childrens cancer institute . references grifn ca , hawkins al , dvorak c , et al : recurrent involvement of 2p23 in inammatory myobroblastic tumors . 
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j pathol 241 : 316 - 323 , 2017 bridge ja , kanamori m , ma z , et al : fusion of the alk gene to the clathrin heavy chain gene , cltc , in inammatory myobroblastic tumor . 
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takeuchi k , soda m , togashi y , et al : pulmonary inammatory myobroblastic tumor expressing a novel fusion , ppfibp1 - alk : reappraisal of anti - alk immunohistochemistry as a tool for novel alk fusion identication . 
am j surg pathol 41 : 773 - 780 , 2017 jiang q , tong hx , hou yy , et al : identication of eml4 - alk as an alternative fusion gene in epithelioid inammatory myobroblastic sarcoma . 
antonescu cr , suurmeijer aj , zhang l , et al : molecular characterization of inammatory myobroblastic tumors with frequent alk and ros1 gene fusions and rare novel ret rearrangement . 
mario - enrquez a , wang wl , roy a , et al : epithelioid inammatory myobroblastic sarcoma : an aggressive intra - abdominal variant of inammatory myobroblastic tumor with nuclear membrane or perinuclear alk . 
yu l , liu j , lao iw , et al : epithelioid inammatory myobroblastic sarcoma : a clinicopathological , immunohistochemical and molecular cytogenetic analysis of ve additional cases and review of the literature . 
kimbara s , takeda k , fukushima h , et al : a case report of epithelioid inammatory myobroblastic sarcoma with ranbp2 - alk fusion gene treated with the alk inhibitor , crizotinib . 
zhou j , jiang g , zhang d , et al : epithelioid inammatory myobroblastic sarcoma with recurrence after extensive resection : signicant clinicopathologic characteristics of a rare aggressive soft tissue neoplasint j clin exp pathol 8 : 5803 - 5807 , 2015 23 . 
moss e yp , lim ms , voss sd , et al : safety and activity of crizotinib for paediatric patients with refractory solid tumours or anaplastic large - cell lymphoma : a childrens oncology group phase 1 consortium study . 
moss e yp , voss sd , lim ms , et al : targeting alk with crizotinib in pediatric anaplastic large cell lymphoma and inammatory myobroblastic tumor : a childrens oncology group study . 
sch offski p , suiarsky j , gelderblom h , et al : crizotinib in patients with advanced , inoperable inammatory myobroblastic tumours with and without anaplastic lymphoma kinase gene alterations ( european organisation for research and treatment of cancer 90101 create ) : a multicentre , single - drug , prospective , non - randomised phase 2 trial . 
davis mp , van dongen s , abreu - goodger c , et al : kraken : a set of tools for quality control and analysis of high - throughput sequence data . 
nicorici d , satalan m , edgren h , et al : fusioncatcher a tool for nding somatic fusion genes in paired - end rna - sequencing data , 2014 . 
geoerger b , schulte j , zwaan cm , et al : phase i study of ceritinib in pediatric patients ( pts ) with malignancies harboring a genetic alteration in alk ( alk plus ) : safety , pharmacokinetic ( pk ) , and efcacy results . 
chang w , brohl as , patidar r , et al : multidimensional clinomics for precision therapy of children and adolescent young adults with relapsed and refractory cancer : a report from the center for cancer research . 
bertocchini a , lo zupone c , callea f , et al : unresectable multifocal omental and peritoneal inammatory myobroblastic tumor in a child : revisiting the role of adjuvant therapy . 
balis fm , thompson pa , mosse yp , et al : first - dose and steady - state pharmacokinetics of orally administered crizotinib in children with solid tumors : a report on advl0912 from the childrens oncology group phase 1 / pilot consortiucancer chemother pharmacol 79 : 181 - 187 , 2017 44 . 
thorne - nuzzo t , williams c , catallini a , et al : a sensitive alk immunohistochemistry companion diagnostic test identies patients eligible for treatment with j thorac oncol 9 : 295 - 306 , 2014 crizotinib . 
maesako y , izumi k , okamori s , et al : inv ( 2 ) ( p23q13 ) / ran - binding protein 2 ( ranbp2 ) - alk fusion gene in myeloid leukemia that developed in an elderly 49 . 
touriol c , greenland c , lamant l , et al : further demonstration of the diversity of chromosomal changes involving 2p23 in alk - positive lymphoma : 2 cases expressing alk kinase fused to cltcl ( clathrin chain polypeptide - like )  . 
tokuda k , eguchi - ishimae m , yagi c , et al : cltc - alk fusion as a primary event in congenital blastic plasmacytoid dendritic cell neoplasgenes chro53 . 
chikatsu n , kojima h , suzukawa k , et al : alk + , cd30 - , cd20large b - cell lymphoma containing anaplastic lymphoma kinase ( alk ) fused to clathrin heavy chain gene ( cltc )  . 
de paepe p , baens m , van krieken h , et al : alk activation by the cltc - alk fusion is a recurrent event in large b - cell lymphoma . 
mcmanus dt , catherwood ma , carey pd , et al : alk - positive diffuse large b - cell lymphoma of the stomach associated with a clathrin - alk rearrangement . 
cerchietti l , damm - welk c , vater i , et al : inhibition of anaplastic lymphoma kinase ( alk ) activity provides a therapeutic approach for cltc - alk - positive human diffuse large b cell lymphomas . 
van roosbroeck k , cools j , dierickx d , et al : alk - positive large b - cell lymphomas with cryptic sec31a - alk and npm1 - alk fusions . 
pro b , advani r , brice p , et al : brentuximab vedotin ( sgn - 35 ) in patients with relapsed or refractory systemic anaplastic large - cell lymphoma : results of a phase ii study . 
 heterogeneity and coexistence of t790m and t790 wild - type resistant subclones drive mixed response to third - generation epidermal growth factor receptor inhibitors in lung cancer zofia piotrowska mehlika hazarrethinam coleen rizzo brandon nadres emily e . 
contributed equally to this work . ( continued ) purpose third - generation epidermal growth factor receptor ( egfr ) inhibitors like nazartinib are active against egfr mutationpositive lung cancers with t790m mediated acquired resistance to initial anti - egfr treatment , but some patients have mixed responses . methods multiple serial tumor and liquid biopsies were obtained from two patients before , during , and after treatment with nazartinib . 
next - generation sequencing and droplet digital polymerase chain reaction were performed to assess heterogeneity and clonal dynamics . results we observed the simultaneous emergence of t790m - dependent and - independent clones in both patients . 
serial plasma droplet digital polymerase chain reaction illustrated shifts in relative clonal abundance in response to various systemic therapies , confirming a molecular basis for the clinical mixed radiographic responses observed . conclusion heterogeneous responses to treatment targeting a solitary resistance mechanism can be explained by coexistent tumor subclones harboring distinct genetic signatures . 
2018 by american society of clinical oncology introduction epidermal growth factor receptor ( egfr ) tyrosine kinase inhibitors ( tkis ) are effective therapies against advanced egfr - mutant lung cancer , but outcomes are limited by acquired resistance.1 egfr t790m accounts for 50% to 60% of resistance to erlotinib , gefitinib , and afatinib , whereas other mechanisms , including bypass pathway activation ( eg , met or errb2 amplification , mutations in braf and pik3ca ) and lineage shifts ( eg , small - cell transformation , epithelial - to - mesenchymal transition ) , are less commonly observed.2 , 3 third - generation egfr inhibitors yield responses in 40% to 60% of t790m - positive tumors , and osimertinib is us food and drug administration approved in this setting.4 other third - generation tkis , including nazartinib ( egf816 ; novartis , cambridge , ma ) , 5 are in development . 
however , tumors can harbor extensive molecular heterogeneity , potentially affecting response to subsequent therapies.6 we present two cases of egfr - mutant lung cancer treated with nazartinib after developing t790m . 
sequist , department of medicine , massachusetts general hospital , 32 fruit st , yawkey 7b , boston , ma 02114 ; e - mail : lvsequist@ partners.org. methods patients patients were treated with nazartinib in a phase i / ii trial ( clinicaltrials.gov identifier : nct02108964 ) at massachusetts general hospital ( mgh ; patient 1 : 200 mg daily , patient 2 : 150 mg daily )  . 
patients provided informed consent under an institutional review board approved protocol allowing next - generation sequencing ( ngs ) and exploratory research on their biopsies . clinical genotype assessments tissue biopsies were genotyped at mgh with three clinical laboratory improvement amendments ( clia ) - certified assays : snapshot version 4 , a multiplex polymerase chain reaction ( pcr ) allele - specific assay for somatic mutations in 23 cancer - related genes ( appendix table a1 ; snapshot ; applied biosystems7 ) ; a pcr sizing assay for in - frame egfr insertions or deletions ; and snapshot ngs , an anchored multiplex pcr assessing 39 ( v1 ) or 91 ( v2 ) genes ( appendix table a1 )  . 
foundation medicines foundationone ngs ( cambridge , ma ) assesses the entire coding sequence of 315 genes and select introns of 28 genes.8 tissue met amplification was assessed at mgh with a clia - certified dual - color fluorescence in situ hybridization ( fish ) assay using the 07q001b550 c - met probe ( chromosome 7q31 met , leica biosystems , wetzlar , germany ) and a copy number probe ( cep7 ; abbott - vysis , lake bluff , il )  . 
the remaining plasma samples were analyzed by droplet digital pcr ( ddpcr )  . plasma ctdna isolation and ddpcr ctdna was extracted from plasma using the qiaamp circulating nucleic acid kit ( qiagen , hilden , germany ) per manufacturers instructions . 
pcr reactions were performed using 10 l final volume containing 5 l lightcycler 480 sybr green i qpcr master mix , 2x ( roche , basel , switzerland ) and line - 1 ( 12.5 mol ) forward and reverse primers . 
droplets were analyzed with the qx200 droplet reader ( bio - rad ) for fluorescent measurement of fam and hex probes . quantasoft software ( bio - rad ) was used to obtain fractional abundance and copy number variations of mutant dna alleles in wild - type / normal background . 
 fractional abundance was calculated as ( nmut / [ nmut + nwt ] ) 100 , where nmut is number of mutant events and nwt is number of wt events per reaction . 
positive and negative droplet counts were used to calculate the concentration of target and reference dna sequences as previously described.10 normal control dna ( human genomic dna [ promega , madison , wi ] ) , and no dna template controls were included . 
 results patient 1 a 68 - year - old never - smoking woman presented with progressive neurologic symptoms and was found to have a lung mass with multiple brain , pulmonary , lymph node , and hepatic metastases . 
 a trans - bronchial biopsy confirmed lung adenocarcinoma and multiplexed , allele - specific pcr ( snapshot version 4 ) identified an egfr exon 19 deletion ( del19 )  . 
a symptomatic brain metastasis was resected , and erlotinib 150 mg daily was initiated with good response in all sites of disease . the cancer progressed after 9 months of erlotinib , and biopsy of a growing liver metastasis ( fig 1b ; day 35 ) was analyzed via targeted ngs ( snapshot ngs - v1 ) , revealing both the original egfr del19 and acquired egfr t790m . 
 hence , she began a clinical trial of nazartinib ( clinicaltrials.gov identifier : nct02108964 ; fig 1a , days 1 to 392 ) and achieved a partial response.11 after 11 months on nazartinib , she developed a solitary progressing liver metastasis in a location distinct from her prior lesion ( fig 1b , day 332 )  . 
in addition , a braf v600e mutation , not present on the preor post erlotinib biopsy samples , was detected as the likely nazartinib - resistant mechanism within this lesion . 
shortly after the ablation , further progression was noted within the thorax ( day 388 )  . ctdna analysis provides a noninvasive means of monitoring the genomic evolution of cancer during treatment . 
in this patient , multiple plasma samples were taken for ctdna analyses , including ngs ( guardant360 ; guardant health , redwood city , ca ) and ddpcr ( fig 1d )  . 
on nazartinib initiation , ddpcr revealed a sharp decline in both egfr del19 ( fig 1d , days 0 to 100 , gray line ) , and t790m ( red line ) , suggesting effective inhibition of the dominant egfr del19 / t790m subclone . 
by day 100 , the mutant allele fraction ( maf ) of del19 began to increase again , this time without rebound of t790m , suggesting outgrowth of a distinct t790wt subclone . 
the increase in egfr del19 was coincident with emergence of a previously undetected braf v600e mutation ( ddpcr ; blue line ) , consistent with the egfr del19 / t790wt / braf v600e subclone detected in the contemporaneous biopsy of the progressing liver metastasis . 
interestingly , ablation of the liver metastasis ( fig 1c , day 351 ) caused braf v600e levels to drop below detection in ctdna , corresponding with radiographic regression , and suggesting this subclone was likely unique to the ablated metastasis . meanwhile , egfr t790m ( fig 1d , red line ) reappeared around day 200 , accompanying a continued increase in total del19 , emergence of egfr c797s ( ddpcr ; gold line ) , and clinical progression of disease in the thorax . 
c797s drives resistance to third - generation egfr tkis by disrupting the key cysteine residue at the drug - binding site.12 - 15 although tissue biopsy was not obtained , we hypothesize based on increasing plasma levels of t790m and c797s that an egfr del19 / t790m / c797s clone may have been driving disease progression in the thorax . 
consistent with this , guardant360 ngs ctdna analysis on day 402 revealed egfr del19 ( maf 4.5% ) , t790m ( 1.1% ) , and c797s ( 0.6% ) in cis configuration without detectable braf v600e ( fig 1c , day 402 ; appendix figs a1a and a1b )  . although several novel treatment strategies for egfr c797s are being studied , 16 , 17 there are currently no targeted therapies against t790m / c797s available in the clinic . 
thus , the patient discontinued nazartinib and started carboplatin / pemetrexed chemotherapy ( fig 1a , days 400 to 547 ) with an initial brief response , followed by disease progression in the liver and thorax 5 months later ( fig 1b , day 547 )  . 
a repeat plasma sample ( guardant360 ngs ) demonstrated increasing maf of egfr del19 ( 44.8% ) and t790m ( 12.3% ) , whereas egfr c797s ( 0.5% ) remained stable ( fig 1c , day 555 ; appendix fig a1c )  . 
the pattern of ddpcr detected ctdna clones during carboplatin / pemetrexed chemotherapy ( fig 1d , days 400 to 547 ) suggests there are ( at least ) two subclones present . 
plasma g360 ngs day 555 ( maf ) egfr del19 egfr del19 egfr del19 ( 4.5% ) egfr del19 ( 44.8% ) egfr t790m egfr t790m ( 1.1% ) egfr t790m ( 12.3% ) egfr c797s ( 0.6% ) egfr c797s ( 0.5% ) braf v600e tp53 r333 * tp53 r333 * ddr2 r806 * ddr2 r806 * apc h1172h ( 0.4% ) , hnf1a t196t ( 0.2% ) rit1 e94k ( 0.4% ) , raf1 d457h ( 0.2% ) , tp53 c238y ( 0.2% ) , pik3ca i211v ( 0.1% ) nazartinib carboplatin + pemetrexed osi + doce * ablation of t790 wt , braf v600e liver lesion egfr del19 braf v600e egfr t790m egfr c797s time ( days ) fig 1 . 
 ( c ) results of clinical genotyping on days 35 , 351 ( tissue biopsies tested by snapshot next - generation sequencing [ ngs ] panel ) , and days 402 and 555 ( liquid biopsies , guardant360 )  . 
on day 548 , treatment was switched to docetaxel plus osimertinib ( fig 1a ) , which yielded a response in the thorax but progression in other sites , including the liver . 
interestingly , the high t790m levels quantified through ddpcr before reintroduction of a t790m specific inhib itor ( this time , osimertinib ) may provide a molecular predictor of the retreatment effect sometimes observed clinically , as in this patient.19 in summary , this case highlights the complexity and heterogeneity of resistance in egfr - mutant lung cancer and provides insight into clonal evolution via complementary longitudinal ctdna and tumor analyses . 
here , nazartinib resistance was attributed to two genetically distinct subclones , one braf v600e / egfr t790wt , the second egfr t790m / c797s , each of which followed a distinct trajectory in response to systemic and local therapies . patient 2 a 48 - year - old never - smoking woman presented with flank pain and was found to have a lung mass with metastases to mediastinal lymph nodes , ovary , liver , adrenal gland , and bones , and several small brain metastases . 
ngs ( foundationone ) showed egfr del19 , tp53 r273c , and the emergence of t790m ( fig 2c , day 29 , core 1 ) , prompting treatment with nazartinib . 
 interestingly , parallel ngs ( snapshot ngs - v1 ) of a second core from the same liver biopsy demonstrated egfr del19 without t790m , but met amplification was detected by fish ( abnormal signal clusters , unquantifiable ; fig 2c , day 29 , core 2 )  . after 2 months receiving nazartinib ( fig 2a , days 1 to 57 ) , a mixed response was observed , with progression of multiple liver metastases but reduction in mediastinal lymph nodes and lung mass ( fig 2b , day 54 )  . 
biopsy of the progressive hepatic metastasis on day 60 ( fig 2c ) analyzed by ngs ( snapshot ngs - v1 ) revealed egfr del19 without t790m , tp53 r273c , and high - level met amplification by fish ( abnormal signal clusters , unquantifiable )  . 
 on the basis of met amplification observed within the liver and ctdna , nazartinib was discontinued and combination therapy initiated ( fig 2a , days 70 to 100 ) with erlotinib ( 100 mg daily ) and crizotinib ( 250 mg daily ) , a regimen effective in another patient with egfr - mutant , met - amplified cancer.20 scans 1 month later showed reduction in the met - amplified liver metastasis that previously underwent biopsy but marked disease progression elsewhere ( fig 2b , day 100 )  . 
as expected , egfr del19 and t790m declined on nazartinib initiation ( fig 2d , days 1 to 25 , gray and red lines ) , but we observed a coincident increase in normalized met copy number ( ddpcr ; fig 2d , blue line )  . 
taken together with the biopsy and radiographic findings , the increasing met signal likely corresponds to a clone residing within the hepatic metastases , whereas the decline in t790m may arise from a nazartinib - sensitive clone within the thorax . 
humerus biopsy day 68 egfr del19 egfr del19 egfr del19 ( 2.4% ) egfr del19 egfr del19 egfr del19 egfr t790m egfr t790m ( 1.1% ) tp53 r273c tp53 r273c tp53 r273c ( 2.3% ) tp53 r273c tp53 r273c met amp met amp met amp tert p702p ( 1.4% ) , ccnd1 e122k ( 0.4% ) nazartinib erlotinib / crizotinib 1000 egfr del19 egfr t790m met copy # time ( days ) fig 2 . 
 ( c ) results of tissue genotyping with snapshot next - generation sequencing ( ngs ) panel on days 29 , 60 , 61 , and 68 and guardant360 plasma test ( mutant allele frequencies [ mafs ] in parentheses ) on day 57 . 
 discussion these two egfr - mutant lung cancer cases with serial tissue and plasma biopsies present a unique opportunity to study clonal evolution in depth during diverse molecularly targeted therapies . 
 our findings illustrate the critical role heterogeneity plays in resistance to targeted therapies and the clinical challenge posed by genetically distinct subpopulations that respond differentially to treatment . heterogeneity in the context of egfr resistance has been documented in both autopsy and ctdna - based studies.14 , 21 , 22 we previously described an afatinib - resistant pleural effusion , which was t790m positive on clinical testing , but single - cell cloning revealed both t790m mutant and t790wt subclones . 
in a cohort of such patients , either subpopulation could spawn the dominant driver of subsequent resistance to third - generation egfr tkis.6 recently , blakely et al23 demonstrated increasing heterogeneity of an egfr - mutant cancer across multiple lines of therapy through whole - exome sequencing of serial biopsies and autopsy specimens . 
the two cases reported here build on these observations and reveal that one can clinically document coexistent , genetically distinct subpopulations differentially responding as predicted by their genotype in real time to therapies . 
 indeed , chabon et al14 also demonstrated that coexistence of t790m and met amplification within ctdna predicted poorer outcomes than t790m alone on a third - generation tki.14 in contrast , patient 1 had only t790m detected on erlotinib progression and enjoyed a durable response to nazartinib . 
her ultimate nazartinib resistance was driven initially by a t790wt / braf - mutant clone localized to a solitary liver metastasis that was transiently eradicated from ctdna by directed ablation and , subsequently , by a t790m / c797s subclone . 
 although egfr c797s has previously been described in osimertinib , olmutinib , and rociletinib resistance , 12 - 15 to our knowledge this is the first report of c797s emergent after nazartinib . 
 broader efforts may be required moving forward to more fully characterize resistance . locally ablative therapies ( lat ) to treat sites of oligoprogression are often used to prolong disease control among patients with egfrand alk - positive disease.26 in patient 1 , microwave ablation of an oligoprogressive egfr del19 / braf v600e - mutant liver metastasis caused the corresponding ctdna signal to fall below detectable levels , suggesting local therapy can effectively eradicate a focally restricted resistant clone and lending biologic rationale to the use of lat . 
in this patient , ablation did not provide sufficient overall disease control , likely because of the egfr del19 / t790m / c797s subclone already detectable in plasma at the time of ablation , which emerged clinically shortly thereafter . 
sometimes , as in patient 1 , plasma identifies resistance mechanisms not detected in tissue ( here , egfr c797s ) , although its utility is limited in patients with low levels of ctdna . 
here , we used a broad ngs - based ctdna panel at key points of disease progression and ddpcr , which is generally more sensitive and cost - effective than serial ngs , to retrospectively quantify known genetic variants in plasma samples banked during treatment . 
although this strategy was well suited to our longitudinal analysis , in practice providers must select the most appropriate test on the basis of gene coverage , turnaround time , and cost . ultimately , our cases illustrate the critical role of cancer heterogeneity in targeted therapy acquired resistance and provide a glimpse of a future where real - time information gleaned from tracking resistant clones during treatment could be clinically meaningful . 
currently , t790m status is used to determine whether a patient should be treated with second - line osimertinib , 1 but our data suggest that looking beyond t790m might have relevance ; incorporating broader tissue and plasma panels into routine testing may identify other clones influencing the success of subsequent therapies . 
future clinical trials should incorporate longitudinal ctdnaand tissue - based assessments during therapy to develop a more complete understanding of dynamic evolution under the pressure of treatment and which clones survive and drive resistance . 
iafrate stock and other ownership interests : archer biosciences consulting or advisory role : debiopharm group , chugai pharmaceutical , roche research funding : blueprint medicines patents , royalties , other intellectual property : archerdx exclusive license to anchored multiplexpcr technology dora dias - santagata no relationship to disclose ignaty leshchiner no relationship to disclose nicholas a . 
engelman employment : novartis stock and other ownership interests : loxo oncology , agios pharmaceuticals , kura oncology , gatekeeper pharmaceuticals , novartis consulting or advisory role : novartis , agios pharmaceuticals , loxo oncology , clovis oncology , ventana medical systems , g1 therapeutics , sanofi , chugai pharmaceutical , warp drive bio , asana biosciences , emd serono , synta , allostery , genentech , aveo / biodesix , merck , cell signaling technology , endo pharmaceuticals , astrazeneca , glaxosmithkline , amgen , bristol - myers squibb , cancer progress research funding : novartis , jounce therapeutics , sanofi , araxes pharma , astrazeneca , amgen ( inst ) patents , royalties , other intellectual property : i am a co - inventor on a patent application that has been licensed to ventana / roche other relationship : third rock ventures aaron n . 
corcoran consulting or advisory role : avidity nanomedicines , taiho pharmaceutical , merrimack , genentech , n - of - one , astex pharmaceuticals , amgen , bristol - myers squibb , roche , shire , fog pharma , loxo oncology , roivant sciences , warp drive bio research funding : astrazeneca , sanofi stock and other ownership interests : guardant health lecia v . 
lanman employment : guardant health , veracyte leadership : guardant health , biolase stock and other ownership interests : guardant health , biolase research funding : guardant health consulting or advisory role : astrazeneca , genentech , bristol - myers squibb , pfizer research funding : boehringer ingelheim ( inst ) , clovis oncology ( inst ) , genentech ( inst ) , merrimack ( inst ) , novartis ( inst ) , astrazeneca ( inst ) , johnson & johnson ( inst ) , merck ( inst ) , pfizer ( inst ) , guardant health ( inst ) , incyte ( inst ) susan moody employment : novartis institutes for biomedical research stock and other ownership interests : novartis consulting or advisory role : dominion diagnostics ( i ) travel , accommodations , expenses : dominion diagnostics ( i ) other relationship : acadia healthcare ( i ) affiliations zofia piotrowska , mehlika hazar - rethinam , coleen rizzo , brandon nadres , emily e . 
 coyne memorial fund ( l.v.s. ) , uniting against lung cancer ( z.p. ) , the lungevity foundation ( z.p. ) , a damon runyon clinical investigator award ( r.b.c. ) , and national institutes of health / national cancer institute grants no . 
yu ha , arcila me , rekhtman n , et al : analysis of tumor specimens at the time of acquired resistance to egfr - tki therapy in 155 patients with egfr - mutant lung cancers . 
tan ds - w , yang jc - h , leighl nb , et al : updated results of a phase 1 study of egf816 , a thirdgeneration , mutant - selective egfr tyrosine kinase inhibitor ( tki ) , in advanced non - small cell lung cancer ( nsclc ) harboring t790m . 
piotrowska z , niederst mj , karlovich ca , et al : heterogeneity underlies the emergence of egfrt790 wild - type clones following treatment of t790m - positive cancers with a thirdgeneration egfr inhibitor . 
dias - santagata d , akhavanfard s , david ss , et al : rapid targeted mutational analysis of human tumours : a clinical platform to guide personalized cancer medicine . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
yu ha , tian sk , drilon ae , et al : acquired resistance of egfr - mutant lung cancer to a t790m - specific egfr inhibitor : emergence of a third mutation ( c797s ) in the egfr tyrosine kinase domajama oncol 1 : 982 - 984 , 2015 13 . 
thress ks , paweletz cp , felip e , et al : acquired egfr c797s mutation mediates resistance to azd9291 in non - small cell lung cancer harboring egfr t790m . 
song hn , jung ks , yoo kh , et al : acquired c797s mutation upon treatment with a t790mspecific third - generation egfr inhibitor ( hm61713 ) in non - small cell lung cancer . 
uchibori k , inase n , araki m , et al : brigatinib combined with anti - egfr antibody overcomes osimertinib resistance in egfr - mutated non - small - cell lung cancer . 
chang gc , tseng ch , hsu kh , et al : predictive factors for egfr - tyrosine kinase inhibitor retreatment in patients with egfr - mutated non - small - cell lung cancera multicenter retrospective sequence study . 
gainor jf , niederst mj , lennerz jk , et al : dramatic response to combination erlotinib and crizotinib in a patient with advanced , egfr - mutant lung cancer harboring de novo met amplification . 
suda k , murakami i , katayama t , et al : reciprocal and complementary role of met amplification and egfr t790m mutation in acquired resistance to kinase inhibitors in lung cancer . 
blakely cm , watkins tbk , wu w , et al : evolution and clinical impact of co - occurring genetic alterations in advanced - stage egfr - mutant lung cancers . 
ohashi k , sequist lv , arcila me , et al : lung cancers with acquired resistance to egfr inhibitors occasionally harbor braf gene mutations but lack mutations in kras , nras , or mek1 . 
weickhardt aj , scheier b , burke jm , et al : local ablative therapy of oligoprogressive disease prolongs disease control by tyrosine kinase inhibitors in oncogene - addicted non - small - cell lung cancer . 
 ( a ) results of next - generation sequencing of circulating tumor dna ( ctdna ) by guardant360 for patient 1 , day 402 ( progression on nazartinib )  . 
 ( c ) results of subsequent ctdna analysis on day 555 ( progression on carboplatin / pemetrexed ) with an overall increase in the mutant allele fraction of egfr del19 and t790 , whereas egfr c797s declined slightly . 
we sought to examine a large genomic data set to comprehensively characterize non - v600 braf alterations in lung cancer . patients and methods a total of 23 , 396 patients with lung cancer provided data to assay with comprehensive genomic profiling . 
data were reviewed for predicted pathogenic braf base substitutions , short insertions and deletions , copy number changes , and rearrangements . results adenocarcinomas represented 65% of the occurrences ; nsclc not otherwise specified ( nos ) , 15% ; squamous cell carcinoma , 12% ; and small - cell lung carcinoma , 5% . 
braf was altered in 4.5% ( 1 , 048 of 23 , 396 ) of all tumors ; 37.4% ( n = 397 ) were braf v600e , 38% were braf non - v600e activating mutations , and 18% were braf inactivating . 
2018 by american society of clinical oncology introduction the rapid development of genotype - directed management of metastatic nonsmall - cell lung carcinoma ( nsclc ) has emerged as the paradigm for precision oncology . 
ali , md , phd , 150 second st , foundation medicine , cambridge , ma 02139 ; e - mail : siraj@ foundationmedicine.com. within lung cancers , small series have described other oncogenic braf point mutations in exons 11 and 15.9 - 12 however , because of the small sample size of prior studies and the focused sequencing methodologies that can miss important classes of genomic alterations , such as rearrangements , a complete landscape of braf alterations in lung cancers is lacking.13 - 15 given the therapeutic action ability of diverse braf alterations , we hypothesized that analysis by comprehensive genomic profiling ( cgp ) would refine the braf landscape and identify additional subsets of patients who may be candidates for targeted therapy . 
the hybrid capture next - generation sequencingbased assay used identifies genomic alterations in 186 , 236 , or 315 genes : base substitutions , insertions / deletions , copy number alterations , and gene fusions via intron baiting for 14 , 19 , and 31 genes , as previously described.16 dna was extracted from 40 - micron scrolls of formalin - fixed paraffin - embedded tissue , and cgp was performed on hybridization captured , adaptor ligationbased libraries to a mean coverage depth of greater than 500 . 
before sequencing , all patient cases were reviewed by a board - certified pathologist to establish adequacy of submitted material but not to confirm or overturn the submitted histologic diagnosis . 
testing was performed in a clinical laboratory improvement amendmentscertified , college of american pathologistsaccredited reference laboratory ( foundation medicine , cambridge , ma )  . ordinal relationships were examined with the mann - whitney u test ; categoric relationships were examined with the pearson 2 test , and the yates continuity correction was applied when applicable . 
literature review defined kinase - activating base substitutions in braf as follows : g464a , g464e , g464v , g466a , f468c , g469a , g469r , g469s , g469v , v471f , n581s , e586k , f595l , l597q , l597r , l597s , l597v , and k601e . 
base substitutions that inactivate braf kinase activity were defined as follows : g466e , g466r , g466v , g469e , d594a , d594e , d594g , d594h , d594n , d594v , d594y , g596r , t599i . results the histologic breakdown and basic clinicopathologic features are listed in table 1 . 
 there were differences among histologic subtypes : 5.5% of adenocarcinomas and 1% ( 42 of 3 , 948 ) of squamous and small - cell tumors harbored braf alterations ( table 2 )  . 
of the 10 sclcs with braf alterations , nine were kinase activating , one was v600e , and two were g469v ( fig 1a )  . overall , non - v600e activating mutations accounted for 37.9% ( 402 of 1 , 061 ) of all braf alterations . 
within non - v600e mutations ( n = 402 ) , codon 469 ( g469 ) alterations represented 34% ( 135 of 402 ) , and g469a was the most common alteration ( 23% ; 94 of 402 ; table 2 )  . 
 frequency of point mutation within braf protein ( n = 1 , 048 ) ras - binding domain protein kinase domain v600 , 400 g469 , 171 g466 , 105 g464 , 38 k483 , 7 n581 , 67 d594 , 77 k601 , 63 g596 , 31 l597 , 16 amino acid residue tmb level ( mutations / mb ) low ( < 6 ) intermediate ( 6 and < 20 ) high ( 20 ) braf setd2 tp53 stk11 kras pik3ca keap1 smad4 rictor variant type point mutation / indel deletion fusion / rearrangement amp and fusion / rearrangement braf v600e amplification truncation amp and point mutation / indel other multiple number of samples : 1 , 047 fig 1 . 
intragenic distribution of braf point mutations and co - occurrence of braf alterations with alterations of other cancer - related genes within braf in a series of 23 , 396 patient cases with lung cancer . 
braf f595l and g596d were found once each and may have affected kinase activity.17 additional kinase - inactivating mutations included changes at position g466 in the p - loop in 54% ( 104 of 193 ) , as follows : g466v ( 35.2% ; 68 of 193 ) , g466e ( 5.7% ; 11 of 193 ) , and g466r ( 3.1% ; six of 193 )  . 
nearly half ( 44% ) of setd2 alterations in the braf v600e occurrences were loss of heterozygosity ( loh ) , which was more frequent than the occurrence of loh ( 22% ) across a large set of setd2 altered nsclc tumors ( data not shown )  . 
this assessment included any known / likely kras variant . small activating insertions and deletions of braf were rare ( 2% ; 23 of 1 , 061 tumors ) and were predominantly in adenocarcinoma ( 91% ; 21 of 23 tumors )  . 
such deletions were l485_ n486 > f , l485_n486 > y ( n = 2 ) , n486_p490del ( n = 3 ) , v487_p492 > a ( n = 2 ) , t488_p492del , and a489_q493del ( n = 3 ) , which are all adjacent or within the alpha c - helix , as previously described.14 , 15 other oncogenic braf deletions were g593_a598del , t599_v600 > m , v600_w604 > r , and v600_w604 > e ( n = 1 each )  . 
 oncogenic braf deletions and insertions were largely mutually exclusive from other national comprehensive cancer networkdesignated nsclc oncogenic alterations except for one tumor that harbored both t599_v600inst and erbb2 amplification , quantitatively estimated as seven copies ( table 2 )  . rearrangements that consisted of n - terminal deletions ( ntds ) , exonic deletions , kinase domain duplications ( kdds ) , and fusions of braf were identified at a frequency of 4.3% ( 46 of 1 , 061 braf alterations ; fig 2 )  . 
kdd events occurred in 0.76% ( eight of 1 , 048 tumors ) and appended exons that coded for the full kinase domain of braf to the 3 - prime end of the wildtype gene . 
one tumor had a breakpoint in intron 7 , appended to exons 7 through 18 ; two tumors had a breakpoint in intron 8 ; four tumors had breakpoints in intron 9 ; and one tumor had a breakpoint in intron 10 ( fig 2 )  . 
predicted fusions of braf were found in 2.9% ( 30 of 1 , 048 ) of tumors , which provided an overall frequency of 2.8% ( 30 of 1 , 061 ) of all braf alterations . 
 twenty - six tumors had identifiable fusion partners of braf , and the following recurrent fusions occurred twice each : armc10 , dock4 , and trim24 ; three tumors harbored snd1braf . 
fusions that involved agap3 , agk , ap3b1 , btfl34 , eps15 , eys , ghr , grm8 , lmo7 , mkrn1 , nup214 , parp12 , ptpn13 , stat3 , trim4 , trio , and zc3hav4 occurred once each ( fig 2 )  . paired samples were available for a small subset of tumors ( n = 16 )  . 
among seven braf v600e adenocarcinomas , five had new mutations in ras family members , including four mutations in kras , and one in nras ( table 3 )  . 
patients with chronologically separated specimens were assayed by comprehensive genomic profiling , and genomic alterations present in the latter specimen are highlighted in this table . abbreviation : tmb , tumor mutational burden . * original braf alteration for each was braf v600e . discussion here , we present , to our knowledge , the largest assessment of braf alterations and expand upon the understanding of activating genomic braf aberrations across lung cancers . 
pathologic activation of the ras / raf / mek / erk ( mapk ) pathway is observed across multiple tumor types , and braf alterations in lung cancer can be targeted by mek inhibitors or pan - raf inhibitors . 
preclinical data suggest that mapk pathway activation that results from braf activating ( including non - v600e ) alterations may be sensitive to targeting downstream signaling nodes mek and erk.18 our data suggest that non - v600e braf alterations are recurrent in nsclc and warrant additional clinical exploration . raf proteins ( including braf ) have similar structures , which contain three conserved regions ( cr1 , cr2 , and cr3 ) .19 cr1 contains ras - binding and cysteine - rich domains ( called rbd and crd , respectively ) , that bind ras . 
 cr2 is a serine - threoninerich domain , which functions as an inhibitory domain upon binding of the 14 - 3 - 3 regulatory prote cr3 encompasses the kinase domain , which includes sites for binding of atp ( the p - loop ) and braf substrates mek1 and mek2 . 
in this large patient subset , use of the multikinase inhibi tors sorafenib , and the closely structurally related compound regorafenib , which have activity against c - raf , an obligate physiologic heterodimerization partner for braf , may be a more rational approach . 
indeed , sorafenib activity in in two nsclc tumors with activating mutations , g469a and g469v , was demonstrated recently.17 , 20 orthogonal support from translational studies demonstrated decreased signaling activity , with a dimerization - impaired form of braf g469a ( r509h ) compared with wildtype braf g469a , in contrast with only a slight ( 7% ) reduction for the analogous dimerization impaired form of braf v600e ( r509h ) , which is consistent with the operation of the r509h form as a promoter.21 we hypothesize that response to single - agent braf inhibitors in g469 alterations would be limited by paradoxical activation of raf / mek / erk signaling caused by the current approved braf inhibitors ( vemurafenib , dabrafenib ) , and we expect that newer pan - raf inhibitors , such as plx8394 , may have broader utility against both v600 and non - v600 mutant forms of braf in nsclc.22 across cancers , the mutagenic processes most frequently observed in each anatomic tumor type exhibit some well - described variation.23 in this series , we identified recurrent braf mutations at g464 , g466 , and particularly g469 , which typically are not observed in melanoma.24 this observation hints at different underlying carcinogenic processes between melanoma ( ultraviolet lightinduced dna damage ) and lung cancer ( often smoking - induced damage )  . 
in preclinical studies of deletion in the alpha c - helix of braf , single to multiple amino acids deletions have been modeled with some gain in braf kinase activity less than that of the 486 through 490 deletion , but this insertion of f / y was not modeled . 
this change may mimic the poorly characterized l747p mutation in egfr or the conserved exon 19 insertion , which also results in l747p mutation.26 , 27 it remains to be understood how l485_n486 > f / y activates braf and any associated sensitivity to a pan - raf inhibitor . 
larger series with treatment data will be needed to address a possible role for how tissue and / or the genomic context of a given braf alteration would affect clinical responsiveness . 
for example , in this data set , 0.7% of lung carcinoma tumors harbored focal braf amplification , which by itself is not known to serve as an oncogenic driver , but more than half of these co - occurred with braf non - v600e point mutations without other oncogenic driver alterations ( data not shown )  . 
stk11 alterations in particular may be functionally related to braf alterations , as was shown in melanoma cells in which braf v600e suppressed lkb1 function , which allowed activation of ampk.28 , 29 such interaction has not been described for non - v600e braf mutants , but it is conceivable that stk11 mutations on one allele , coupled with inactivation of residual wild - type stk11 protein by a mutated braf protein , may abrogate stk11 function . we observed a diversity of braf rearrangements , including ntds , kdds , and braf fusions , in this series . 
previously , variably sized deletions of exons 2 through 9 or less in braf ( ntd - braf ) were described only in preclinical models of vemurafenib - resistant melanoma and lung cancer.30 , 31 to our knowledge , this is the first report of ntd - braf in lung cancer samples ( none , to our knowledge , with prior raf - directed therapy ) , and it suggests that mechanisms other than splicing at the rna level can underlie ntd . 
across the series , braf fusions lack the ras - binding auto inhibitory domain found in the n - terminal half of braf , akin to braf ntds , and the n - terminal fusion partner often harbors a con stitutive dimerization or oligomerization motif . 
 not needed for braf fusion activity , although is required.40 , 41 kinase some dimerization fusions may emerge as resistance mechanisms to targeted therapy , such as epidermal growth factor receptor inhibitors.42 , 43 limited clinical histories were available for patient cases in the series , but , for one tumor with egfr exon 19 deletion ( t790m negative ) , a trim24 - braf fusion was observed with erlotinib resistance , which suggests that the braf fusion may drive resistance . 
 resistance to small molecule inhibitors is universal , and the landscape of braf - mutant lung cancers treated with braf inhibition is not known.44 among a small subset of tumors with paired samples and clinical data , genomic alterations not present in the pretreatment specimen existed in the post - treatment specimens and may correlate with acquired resistance ( table 3 )  . 
we observed a braf fusion upon resistance to vemurafenib , which mimicked a recent description of a fusion - based resistance to vemurafenib in melanoma.45 in a second tumor , loss of map2k4 and biallelic inactivation of smarca4 was seen at progression . 
several tumors also had activating kras / nras mutations upon progression . overall , we report the largest series , to our knowledge , to examine all classes of braf alterations in lung cancers . 
although limited by disease heterogeneity , incomplete clinical annotation , and no independent confirmation of histology , the series identifies multiple non - v600 aberrations that tend to be mutually exclusive with other oncogenic drivers in lung cancer . 
 whether non - v600 identifies a good prognostic group , as in colorectal cancer , is of interest but is not answerable from our data.46 likewise , it is unclear whether non - v600e alterations are responsive to existing therapies ; clinical trials are needed . 
trabucco employment : foundation medicine research funding : foundation medicine travel , accommodations , expenses : foundation medicine jon chung employment : foundation medicine stock and other ownership interests : foundation medicine mark rosenzweig employment : foundation medicine stock and other ownership interests : foundation medicine kai wang no relationship to disclose vamsidhar velcheti honoraria : novartis , foundation medicine , merck , bristolmyers squibb , genentech , roche consulting or advisory role : clovis oncology , genentech , bristol - myers squibb , merck , celgene , foundation medicine , astrazeneca , medimmune , genoptix , amgen , takeda research funding : genentech ( inst ) , trovagene ( inst ) , eisai ( inst ) , oncoplex diagnostics ( inst ) , alkermes ( inst ) , nantomics ( inst ) , genoptix ( inst ) , altor bioscience ( inst ) , merck ( inst ) , bristol - myers squibb ( inst ) , atreca ( inst ) , heat biologics ( inst ) , leap therapeutics ( inst ) travel , accommodations , expenses : astrazeneca / medimmune , eisai , foundation medicine , merck garrett m . 
frampton employment : foundation medicine stock and other ownership interests : foundation medicine nir peled consulting or advisory role : astrazeneca , boehringer ingelheim , bristol - myers squibb , lilly , msd , novartis , pfizer , roche speakers ' bureau : astrazeneca , boehringer ingelheim , bristol - myers squibb , lilly , msd , novartis , pfizer , roche molly murray employment : foundation medicine young kwang chae consulting or advisory role : foundation medicine , boehringer ingelheim , biodesix , counsyl , astrazeneca , guardant health speakers ' bureau : merck , genentech , roche travel , accommodations , expenses : hanmi lee a . 
albacker employment : foundation medicine stock and other ownership interests : foundation medicine hatim husain consulting or advisory role : astrazeneca , abbvie , foundation medicine speakers ' bureau : astrazeneca , merck , bristol - myers squibb research funding : pfizer ( inst ) travel , accommodations , expenses : astrazeneca , merck , bristol - myers squibb , foundation medicine , abbvie james suh employment : foundation medicine stock and other ownership interests : foundation medicine sherri millis employment : foundation medicine venkataprasanth reddy employment : foundation medicine stock and other ownership interests : foundation medicine travel , accommodations , expenses : foundation medicine julia elvin employment : foundation medicine stock and other ownership interests : foundation medicine ryan j . 
 consulting or advisory role : revolution medicine , array biopharma , novartis , astrazeneca stock and other ownership interests : foundation medicine speakers ' bureau : novartis research funding : ignyta , revolution medicine vincent miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center shridar ganesan employment : merck ( i ) stock and other ownership interests : ibris , inspirata , merck ( i ) consulting or advisory role : inspirata , novartis patents , royalties , other intellectual property : i hold two patents for digital imaging that may be licensed to ibris and inspirata travel , accommodations , expenses : inspirata alexa schrock employment : foundation medicine sai - hong ou honoraria : pfizer , roche pharma ag , genentech , roche , ariad , takeda , novartis , astrazeneca , foundation medicine consulting or advisory role : pfizer , roche , genentech , novartis , astrazeneca , takeda , ariad , foundation medicine speakers ' bureau : genentech , astrazeneca , takeda , ariad research funding : pfizer ( inst ) , roche pharma ag ( inst ) , astrazeneca ( inst ) , medimmune ( inst ) , clovis oncology ( inst ) , ariad ( inst ) , ignyta ( inst ) , peregrine pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , astellas pharma ( inst ) , chugai pharma ( inst ) siraj ali employment : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine ; patents via seres health on microbiomes in non - neoplastic disease ( i ) affiliations yuri sheikine , vancouver general hospital , vancouver , british columbia , canada ; dean pavlick , sally e . 
 klempner , the angeles clinic and research institute and cedars - sinai medical center , los angeles ; hatim husain , university of california san diego , san diego ; trever g . 
rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as first - line treatment for european patients with advanced egfr mutationpositive nonsmall - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
planchard d , besse b , groen hjm , et al : dabrafenib plus trametinib in patients with previously treated braf ( v600e ) - mutant metastatic nonsmall - cell lung cancer : an open - label , multicentre phase 2 trial . 
planchard d , kim tm , mazieres j , et al : dabrafenib in patients with braf ( v600e ) - positive advanced non - small - cell lung cancer : a single - arm , multicentre , open - label , phase 2 trial . 
villaruz lc , socinski ma , abberbock s , et al : clinicopathologic features and outcomes of patients with lung adenocarcinomas harboring braf mutations in the lung cancer mutation consortiucancer 121 : 448 - 456 , 2015 13 . 
chen sh , zhang y , van horn rd , et al : oncogenic braf deletions that function as homodimers and are sensitive to inhibition by raf dimer inhibitor ly3009120 . 
kordes m , rring m , heining c , et al : cooperation of braf ( f595l ) and mutant hras in histiocytic sarcoma provides new insights into oncogenic braf signaling . 
rring m , herr r , fiala gj , et al : distinct requirement for an intact dimer interface in wildtype , v600e , and kinase - dead b - raf signaling . 
okimoto ra , lin l , olivas v , et al : preclinical efficacy of a raf inhibitor that evades paradoxical mapk pathway activation in protein kinase braf - mutant lung cancer . 
lee lh , gasilina a , roychoudhury j , et al : real - time genomic profiling of histiocytoses identifies early - kinase domain braf alterations while improving treatment outcomes . 
allen jm , schrock ab , erlich rl , et al : genomic profiling of circulating tumor dna in relapsed egfr - mutated lung adenocarcinoma reveals an acquired fgfr3 - tacc3 fusion . 
klempner sj , bazhenova la , braiteh fs , et al : emergence of ret rearrangement co - existing with activated egfr mutation in egfr - mutated nsclc patients who had progressed on first or second - generation egfr tki . 
kulkarni a , al - hraishawi h , simhadri s , et al : braf fusion as a novel mechanism of acquired resistance to vemurafenib in braf v600e mutant melanoma . 
carl barrett , phd1 ; and jeeyun lee , md3 purpose some gastric cancers harbor met gene amplications that can be targeted by selective met inhibitors to achieve tumor responses , but resistance eventually develops . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction gastric cancer is the fth most common cancer worldwide and the third leading cause of cancerrelated death.1 gastric cancer is a heterogenous disease , the drivers of which remain unclear , making it difcult to treat these patients . 
pembrolizumab was approved for treating gi malignancies with mismatch repair deciencies and for third - line programmed death ligand 1positive gastric cancer based on its recently observed promising antitumor activity.2 , 3 however , it remains urgent to identify a subset of patients with gastric cancer who may benet from molecularly targeted agents . 
one such potential target is met amplication , which is observed in approximately 5% of patients with gastric cancer.3 - 9 savolitinib ( azd6094 , hmpl - 504 , volitinib ) is a potent small - molecule met kinase inhibitor that inhibits met inhibitory concentration kinase at a half - maximal ( ic50 ) of 4 nm and met phosphorylation in tumor cells . savolitinib was found to inhibit cell growth in vitro against tumors with met gene amplication in the absence of hepatocyte growth factor stimulation , with ic50 generally , 10 nm . 
 met inhibitor in met - amplied gastric cancer context key objective we evaluated the mechanisms of acquired resistance to savolitinib in 3 patients with met - amplied gastric cancer who showed a clinical response and then cancer progression using tumor and circulating tumor dna ( ctdna ) sequencing . knowledge generated ctdna is a powerful tool for identifying potential genomic aberrations that emerge during therapy to confer resistance to targeted therapies . 
using a next - generation sequencing 100 - gene panel , we identied the target mechanisms of resistance met d1228v / n / h and y1230c mutations or high copy number met gene amplications that emerge when resistance to savolitinib develops in patients with met - amplied gastric cancer . relevance we demonstrated the utility of ctdna in gastric cancer and conrmed this approach using baseline tumor tissue or rebiopsy . we identied various met mutations previously unidentied in gastric cancer upon progression to savolitinib , as well as met amplications as drivers of savolitinib resistance during monotherapy treatment of patients with met - amplied gastric cancer . circulating tumor dna ( ctdna ) from baseline until progression every 2 cycles to evaluate the tumor biology during treatment with targeted agents . 
the human investigations were performed after approval by a local human investigations committee ( institutional review board ) and in accordance with an assurance led with and approved by the health authority in korea . 
this prospective open - label trial was designed as a single - arm phase ii study at an academic cancer center ( clinicaltrials.gov identier : nct02449551 )  . treatment was administered as follows : savolitinib 800 mg once a day until disease progression or unacceptable toxicity . 
tumor tissue and plasma ctdna samples from 3 patients enrolled in the viktory trial were obtained before treatment , during therapy , and at the time of progression . next - generation sequencing a targeted next - generation sequencing 100 - gene panel was used to analyze matched tumor dna at baseline and progression and longitudinal ctdna to determine the mechanisms of acquired resistance to savolitinib ( az translational genomics labs , boston , ma )  . 
genes included in this panel are provided in appendix figure a1a . sequencing libraries were prepared using the kapa biosystems hyperprep kit ( wilmington , ma ) and roche nimblegen hybridization capture reagents ( basel , switzerland )  . 
raw sequencing data were processed using the bcbio framework.12 the quality of sequencing data was assessed using the multiqc report.13 fastq les were aligned to the reference genome hg38 using bwa mem aligner , 14 yielding a median depth of coverage of 2 , 000 ( range , 1 , 300 - 6 , 600 ) in plasma samples and 1 , 200 ( range , 1 , 100 - 1 , 400 ) in tissue samples . 
variants were called using vardict15 and classied into the following 3 tiers from high to low based on the likelihood of the variant contributing to cancer - relevant processes : known , likely , and unknown.15 copy number analysis of sequencing data were performed using the copy number caller seq2c.16 all plasma samples were analyzed together as a plasma cohort , whereas tissue samples were processed together as a tissue cohort . 
mapping and sequencing statistics for formalin - xed parafn - embedded tumor tissue ( fig a1b ) and ctdna from frozen plasma ( fig a1c ) indicated that  . 90% and 96% of the 100 genes were sequenced with 500 coverage in tumor and ctdna , respectively , providing a quality data set for analysis . et immunohistochemistry and met fluorescent in situ hybridization met immunohistochemistry ( ihc ) was performed using the rabbit monoclonal primary antibody , confirm antitotal met ( sp44 ; ventana medical systems , tucson , az ) according to the manufacturers protocol . 
 frigault et al performed using dual - color dna - specic met / cep7 probes ( abnova , walnut , ca ) , as described previously.5 , 8 results we analyzed the rst 3 patients enrolled in the savolitinib trial , which enrolls patients with gastric cancer with met amplication in the salvage setting . 
patients were screened for the presence of met amplication by tumor sequencing as part of the viktory trial and are conrmed wild type for met.11 the viktory trial screened 715 patients with genomic sequencing , and the incidence of met amplication was 3.5% ( 25 of 715 patients )  . 
in this study , we tested the feasibility of using a 100 - gene panel in patients with met - amplied gastric cancer serially from baseline until disease progression using both matched biopsies and ctdna . 
the patient had microsatellite - stable and her2 - negative gastric cancer ( fig 1a ) and experienced rapid progression after 5 cycles of capecitabine plus oxaliplatin ( initially stable disease ) to extensive dissemination in the lymph nodes ( fig 1b )  . 
the patient responded dramatically to 2 cycles of savolitinib ( fig 1b , middle panel ) , which was concordant with the decrease in the allele frequency of tp53 p190l ( 7% ) , met 1.4 copy number , and myc 3.4 copy number in the ctdna . 
notably , although the patient maintained a radiologic and clinical partial response ( pr ) to savolitinib , low frequencies of met d1228h ( 5% ) , met d1228n ( 5% ) , met d1228v ( 35% ) , and met y1230c ( 3% ) were newly detected . 
met d1228n is a resistant mutation to crizotinib in lung cancer.17 in addition , met d1228v has been shown to induce resistance to type 1 met tyrosine kinase inhibitors in lung cancer.18 finally , met y1230c is associated with resistance to crizotinib in nonsmall - cell lung cancer.19 the high concordance for the copy number level between ctdna 100 - gene sequencing and tumor rebiopsy specimen sequencing supports the usefulness of copy number evaluation from longitudinal plasma ctdna ( appendix fig a2 )  . 
at disease progression , the patient had mostly experienced progression to bone and bone marrow metastases rather than previous lymph nodes , suggesting the emergence of an aggressive clone that spread to the bone and bone marrow at progression . 
our data suggest that , rather than met amplications , met mutations drove the rapid progression observed in this patient ( fig 1c )  . the second patient was a 74 - year - old woman who was diagnosed with gastric cancer with extensive liver metastases at presentation and enrolled in the viktory trial at diagnosis . 
after 4 cycles of savolitinib , with her pr conrmed radiologically , the met copy number was 2.7 and showed a slight increase from diploid , and a tp53 g245d ( 2% ) variant began to emerge as detected in the ctdna . 
after 6 months ( 7 cycles of savolitinib ) , the patient developed radiologic progression . interestingly , in contrast to the newly emerging mutations conferring resistance to savolitinib in patient 1 ( fig 1 ) , the second patient developed a high level of met amplication of 13 copies and cdk6 copy number of 3.9 at progression ( fig 2a )  . 
although both genes are proximal to one another on chromosome 7 , they are not part of the same amplicon because the trap and pik3cg gene loci are between met and cdk6 , and they remained at a normal copy number in both the ctdna and tumor samples at progression ( fig 2b , top panel )  . 
high concordance was observed between the ctdna and tumor tissue dna ( liver metastasis ) with a reincrease in met amplication and cdk6 amplication that was not detected at pr . 
in addition , the ctdna sample at progression indicated that the tp53 g245d substitution containing the clone had expanded , as suggested by an increase from a 2% allele fraction ( at the third follow - up ) to a 22% allele fraction ( at progression )  . 
this was conrmed in the progression rebiopsy , where the same tp53 g245d substitution that was not detected in normal dna had subsequently increased to an 82% allele fraction ( fig 2b , bottom panel )  . the third patient was a 31 - year - old woman who underwent total gastrectomy for gastric cancer in 2013 . 
pathology , clinical assessment , and circulating tumor dna ( ctdna ) monitoring for patient 1 ( b5 - 002 ) identies met d1228 and y1230 mutations as mechanism of resistance to savolitinib . 
 ( a ) hematoxylin and eosin ( he ) , met immunohistochemistry ( ihc ) , and met uorescence in situ hybridization ( fish ; left to right ) from baseline tumor tissue . 
 ( b ) tumor assessment by radiologic assessment at baseline before savolitinib treatment , after 2 cycles at time of partial response ( pr ) , and after 4 cycles at time of progressive disease ( pd )  . 
clinical assessment and circulating tumor dna ( ctdna ) monitoring for patient 2 ( b5 - 001 ) identify on - target high - level met amplication ( amp ) as mechanism of resistance to savolitinib . 
 ( a ) radiologic assessment of tumor at baseline before savolitinib treatment ( follow - up [ fu ] 1 ) ; after 2 cycles ( fu2 ) and 4 cycles ( fu3 ) , both at time of partial response ( pr ) ; and after 7 cycles at time of progressive disease ( pd )  . 
 ( b ) copy number prole of ctdna plasma ( top ) and tumor tissue or normal dna at progression ( bottom ) , where each sample is represented by a different color at baseline ( fu1 ) , after 2 cycles ( fu2 ) and 4 cycles ( fu3 ) , and after 7 cycles at time of progression . patient was administered postoperative chemoradiation therapy with a ts - 1 plus oxaliplatin regimen and developed recurrent metastases to both ovaries 18 months later . 
 ( a ) hematoxylin and eosin ( he ) , met immunohistochemistry ( ihc ) , and met uorescence in situ hybridization ( fish ; left to right ) from baseline tumor tissue . 
 ( b ) tumor assessment by radiologic assessment at baseline before savolitinib treatment , after 2 cycles at time of partial response ( pr ) , and after 8 cycles at time of progressive disease ( pd )  . 
 frigault et al discussion the aim of this exploratory study was to determine whether relapses on savolitinib in patients with gastric cancer after an initial pr resulted from the development of on - target or bypass mechanisms of resistance . 
furthermore , we established the utility of ctdna from plasma as a matrix for monitoring changes in dna alterations during the course of treatment of gastric cancer and demonstrated the concordance of genomic alterations in ctdna to tumor biopsy both before treatment with a targeted therapy and at the time of progression . 
the efcacy of the trial will be reported once the trial completes recruitment ; here , we focused on the utility of ctdna sequencing to identify potential resistance mechanisms in gastric cancer after met inhibition . 
we identied the following two potential mechanisms of acquired resistance at progression following a dramatic response to savolitinib in met - amplied gastric cancer : newly developed met mutations previously only described in lung cancer and an increase in the met copy number at resistance , which decreased during the clinical response but increased again at disease progression . in the rst patient , newly emerging met d1228h ( 31% ) , met d1228n ( 12% ) , met d1228v ( 1% ) , and met y1230c ( 1% ) mutations were detected with the sudden onset of disease progression to multiple bones and the bone marrow . 
this patient died of rapid progression immediately after developing resistance to savolitinib therapy . mutations in met , including at positions d1228 and y1230 , are among the reported mechanisms of acquired resistance in preclinical models21 , 22 and have been reported to confer clinical resistance to met inhibition in lung cancer.17 , 19 the cocrystal structure of the met kinase domain and met kinase inhibitor revealed an important binding interaction of the met inhibitor and y1230 and explains the abrogation of compound binding with the emergence of mutations at this residue . 
in addition , d1228 is engaged in interactions with other residues to maintain the activation loop in a conformation that enables the critical interaction between y1230 and the met inhibitor.21 these results demonstrate that the on - target resistance mutations emerge in met - amplied gastric cancer treated with a selective met kinase inhibitor . 
therefore , upregulation of met ( in a savolitinib - responsive tumor ) similar to her2 ( in a lapatinib - responsive tumor ) may confer resistance via epithelial to mesenchymal transition and by promoting a metastatic phenotype.26 , 27 an on - target met copy number gain as a mechanism of resistance has been reported in lung cancer , where one met exon 14skipping patient became a responder to a met inhibitor in the clinic ; however , at the time of progression , the exon 14 met allele was amplied , but not the wild - type allele of met , 28 suggesting that highlevel amplication of the met gene is a mechanism of resistance to met inhibitors across indications . in 2 of 3 patients studied , ctdna was detectable in the circulation , harboring somatic alterations in the met pathway that changed over time and were conrmed by rebiopsy at recurrence . 
our data agree with this hypothesis ; in the rst patient , tp53 p190l changed from a fraction of 44% at baseline to 7% during pr and then back to 13% at disease progression , which was further conrmed in tumor tissue with a 17% allele fraction harboring the same mutation in tp53 . 
 met inhibitor in met - amplied gastric cancer contained no detectable ctdna tp53 until after 4 cycles , at which point a g245d amino acid substitution was detectable at a 2% allele fraction and increased to 22% at progression . 
the same alteration was identied by rebiopsy at an 82% allele fraction . in 1 of 3 patients studied , no mutations in any of the 100 genes on the high - coverage deep - sequencing gene panel were detected , and therefore , this patients tumors may not be shedding ctdna into circulation . 
this patient demonstrates the limitations of the use of ctdna to the 57% - 87% of patients with gastric cancer who are shedding ctdna from their tumor into circulation.30 , 31 this patient highlights that tumor collection at the time of progression will still be important for identifying resistance mechanisms to targeted treatments in nonshedders and to understand tumor heterogeneity . in summary , ctdna is a powerful tool for identifying potential genomic aberrations that emerge during therapy to confer resistance to targeted therapies . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . aleksandra markovets employment : astrazeneca stock and other ownership interests : astrazeneca barrett nuttall employment : astrazeneca , cvs health ( i ) stock and other ownership interests : astrazeneca , cvs health ( i ) travel , accommodations , expenses : astrazeneca , cvs health ( i ) se hoon park honoraria : merck sharp & dohme , sano - aventis consulting or advisory role : eli lilly , janssen oncology research funding : kura oncology , ono pharmaceutical esha a . 
gangolli employment : clovis oncology stock and other ownership interests : astrazeneca , clovis oncology , novartis , merck , bristol - myers squibb , jounce therapeutics , unum therapeutics , incyte , alcon , guardant health , titan medical travel , accommodations , expenses : clovis oncology peter g.s. 
nat rev dis primers 3 : 17036 , 2017 fuchs c , doi t , jang r , et al : keynote - 059 cohort 1 : efcacy and safety of pembrolizumab ( pembro ) monotherapy in patients with previously treated advanced gastric cancer . 
n engl j med 372 : 2509 - 2520 , 2015 lee j , ou sh , lee jm , et al : gastrointestinal malignancies harbor actionable met exon 14 deletions . 
mod pathol 26 : 1632 - 1641 , 2013 lee j , kim km , kang wk , et al : innovative personalized medicine in gastric cancer : time to move forward . 
nature 513 : 202 - 209 , 2014 lee j , seo jw , jun hj , et al : impact of met amplication on gastric cancer : possible roles as a novel prognostic marker and a potential therapeutic target . 
oncol rep 25 : 1517 - 1524 , 2011 ahn s , brant r , sharpe a , et al : correlation between mek signature and ras gene alteration in advanced gastric cancer . 
ou si , young l , schrock ab , et al : emergence of preexisting met y1230c mutation as a resistance mechanism to crizotinib in nsclc with met exon 14 skipping . 
kim st , banks kc , pectasides e , et al : impact of genomic alterations on lapatinib treatment outcome and cell - free genomic landscape during her2 therapy in her2 + gastric cancer patients . 
tiedt r , degenkolbe e , furet p , et al : a drug resistance screen using a selective met inhibitor reveals a spectrum of mutations that partially overlap with activating mutations found in cancer patients . 
kim dc , park kr , jeong yj , et al : resistance to the c - met inhibitor krc - 108 induces the epithelial transition of gastric cancer cells . 
oliveras - ferraros c , corominas - faja b , cuf s , et al : epithelial - to - mesenchymal transition ( emt ) confers primary resistance to trastuzumab ( herceptin )  . 
shi j , li f , yao x , et al : the her4 - yap1 axis promotes trastuzumab resistance in her2 - positive gastric cancer by inducing epithelial and mesenchymal transition . 
awad mm , bahcall m , sholl lm , et al : mechanisms of acquired resistance to met tyrosine kinase inhibitors ( tkis ) in met exon 14 ( metex14 ) mutant non - small cell lung cancer ( nsclc )  . 
 ( b ) sequencing and mapping statistics of tissue samples and ( c ) plasma circulating tumor dna ( ctdna ) describing number of sequenced reads , percentage of aligned reads , percentage of duplicated reads , percentage of reads mapped on targets ( + 200 base pairs ) , percentage of usable reads , average depth of coverage , gc content ( percentage of nitrogenous bases that are either guanine or cytosine ) , average insert size , and percentage of targeted bases with given coverage . 
 next - generation sequencing reveals potentially actionable alterations in the majority of patients with lymphoid malignancies purpose next - generation sequencing ( ngs ) identifies potentially targetable alterations by us food and drug administration ( fda ) approved drugs and / or by available experimental agents that may not have otherwise been contemplated . 
many targeted drugs have been developed for diverse solid cancers ; a smaller number of genomically targeted drugs have been approved for lymphoid malignancies . materials and methods we analyzed ngs results from 60 patients with various lymphoid malignancies and found 224 alterations ( median per patient , three alterations )  . results forty - nine patients ( 82% ) had potentially actionable alterations with the use of fdaapproved drugs and / or experimental therapies ; only 11 patients ( 18% ) had no theoretically actionable alterations . 
only three patients ( 5% ) had an alteration for which an approved drug in the disease is available ( on label ) ; 45 patients ( 75% ) had an alteration for which an approved drug is available for another disease ( off label )  . 
of note , 19 ( 32% ) of 60 patients had intermediate to high tumor mutational burden , which may predict response to certain immunotherapy agents . conclusion ngs identifies alterations that may be pharmacologically tractable in most patients with lymphoid malignancies , albeit with drugs that have usually been developed in the context of solid tumors . 
goodman michael choi matthew wieduwilt carolyn mulroney caitlin costello garrett frampton vincent miller razelle kurzrock author affiliations and support information ( if applicable ) appear at the end of this article . corresponding author : aaron m . 
goodman , md , moores cancer center , university of california san diego , 3855 health science dr , la jolla , ca 92093 ; e - mail : a1goodman@ucsd.edu. introduction the lymphoid malignancies have diverse biologic and clinical behavior and typically are treated with multiagent chemotherapy . 
many therapeutic regimens for b - cell non - hodgkin lymphomas and leukemias also incorporate the anti - cd20 monoclonal antibody rituximab , which has improved patient outcomes.1 treatment of metastatic solid tumors , like lymphoid malignancies , has also relied heavily on the use of cytotoxic chemotherapy . however , over the past decade , the treatment paradigm for metastatic solid tumors has shifted away from chemotherapy toward matching oncogenic driver mutations with targeted therapy ( precision medicine ) .2 - 4 for example , in patients with braf - mutated metastatic melanoma , the braf inhibitor vemurafenib markedly increases overall survival compared with chemotherapy.5 numerous targeted therapies are now approved by the us food and drug administration ( fda ) for patients with metastatic solid tumors across a wide array of histologies ( data supplement )  . 
the first example of targeted therapeutic efficacy is the hematologic disorder chronic myelogenous leukemia , which has been transformed by the use of agents that affect bcr - abl kinase activity . despite the rapid success in development , approval , and use of small - moleculetargeted agents in patients with solid malignancies , a paucity of such therapies approved for use in lymphoid malignancies remains ( data supplement )  . 
 small - moleculetargeted therapeutics are not an available option . the rapid technological advances in nextgeneration sequencing ( ngs ) have allowed oncologists to sequence tumor exomes in a clinically meaningful period of time.6 - 8 some studies have shown that patients with solid malignancies treated with matched therapy have improved outcomes , 9 and meta - analyses in approximately 85 , 000 patients have supported this finding.10 - 12 the targeting of alterations such as braf can result in responses across a wide variety of cancers , including lymphoid malignancies ( eg , hairy cell leukemia ) .13 , 14 although not all malignancies that harbor braf mutations will respond equally well to braf inhibition , the strategy of crosscancer basket trials has been established as highly worthwhile . ngs accurately identifies substitutions , indels , copy number alterations , and gene fusions in hematologic malignancies.15 in this report , we use this technology to analyze the genomic alterations in a cohort of 60 patients with various lymphoid malignancies to estimate the frequency of theoretically actionable alterations . 
this study was performed and consents obtained in accordance with university of california san diego institutional review board guidelines . tumor samples from tissue ( table 1 ) or peripheral blood were collected from 60 patients and submitted for ngs to foundation medicine , a laboratory improvement amendments clinical certified laboratory for ngs . 
the foundationone heme panel was used , which is a hybrid capturebased ngs test.16 the methods used in this assay have been described in detail in previous reports.7 , 15 the foundationone heme assay simultaneously detects all genomic alterations , including base pair substitutions , indels , copy number alterations , and select gene rearrangements , in 405 cancer - related genes . 
for tumor mutational burden ( tmb ) , the number of somatic mutations detected on ngs are quantified , and that value was extrapolated to the whole exome by using a validated algorithm described in detail in earlier publications.17 , 18 alterations with known and likely effects on functional status are not counted . definition of actionable alteration an alteration was defined as potentially actionable if its protein product is a component of a molecularly defined pathway for which there is at least one available fda - approved drug or investigational drug that may affect the function of the protein product of the alteration or the immediate downstream effectors of the protein product or that differentially recognizes the protein in tumor versus normal cells . 
the protein products of genomic alterations were considered to be functional the genomic alterations have been previously identified as relevant to cancer in the cosmic database , 19 which catalogs recurrent somatic alterations in cancer . 
novel base substitution , indel , and rearrangement alterations that result in truncations and homozygous copy number deletions that occur in tumor suppressor genes were considered to have likely functional implications . 
novel genomic alterations that occur at the same position as known alterations as well as alterations with conflicting evidence with regard to implication for function were subject to review by an internal panel of subject matter experts to determine functional status of the relevant protein product on the basis of all available evidence , including , but not limited to , the exac , dbsnp , and clinvar databases.20 - 22 data and statistical analyses patient characteristics were obtained through electronic medical record review . 
descriptive statistics were used , including medians , ranges , and frequencies . results patient characteristics sixty patients ( 35 men [ 58% ] and 25 women [ 42% ] ) with lymphoid malignancies were identified ( table 1 )  . 
the most common malignancy in the cohort was chronic lymphocytic leukemia ( cll ; 32% ) , followed by acute lymphoblastic leukemia ( all ; 20% ) , multiple myeloma ( 18% ) , diffuse large b - cell lymphoma ( dlbcl ; 10% ) , follicular lymphoma ( 8% ) , and other lymphoid neoplasms ( 5% )  . 
however , the actual alteration in nras differed between the two patients ( nras g13d v nras q61r )  . the median number of alterations detected per patient was three ( range , zero to 14 )  . 
as demonstrated in figure 1b , seven patients ( 12% ) had no reportable alterations , 10 ( 17% ) had one alteration , and 43 ( 71% ) had two or more alterations . 
the maximum number of alterations identified was 14 , which was observed in two patients ( 3% ) , one with cll , the other with dlbcl . of note , all patients with dlbcl had five or more alterations . actionable alterations potentially actionable alterations were identified in all disease subtypes ( table 2 )  . 
all disease subtypes , except marginal zone lymphoma , had alterations targetable by fda - approved drugs . forty - nine patients ( 82% ) had potentially actionable alterations with fda - approved drugs and / or experimental therapies ( clinical trials ) , whereas 11 patients ( 18% ) had no theoretically actionable alterations . depicted in figure 2 is the number of patients with potentially fda actionable alterations per disease group . 
only three patients had an alteration for which an approved drug in the disease is available ( on label ) , whereas 45 patients ( 75% ) had an alteration for which an approved drug is available in another disease ( off label )  . 
eleven patients had no targetable alterations ; these patients included seven with no detected genomic alterations . two patients with b - cell all ( b - all ) and one with waldenstr om macroglobulinemia had alterations targetable with on - labelapproved drugs ( fig 3 )  . 
high tmb was seen in five patients ( 8% ) , intermediate in 14 ( 23% ) , and low in 40 ( 67% )  . tmb was not available for one patient ( 2% )  . intermediate to high tmb was noted across almost all histologies ( except for marginal zone lymphoma , natural killer / t - cell lymphoma , and cutaneous t - cell lymphoma )  . 
three patients with multiple myeloma had an intermediate level of tmb . discussion this study demonstrates that in the majority of patients ( 82% ) with lymphoid malignancies whose disease was interrogated by ngs , alterations were found that might be pharmacologically tractable , a percentage similar to the 77% reported by he et al15 for diverse hematologic malignancies . 
another study reported that 70% of patients with various solid tumors had an alteration that was theoretically actionable with an approved drug.24 this finding is similar for lymphoid malignancies , with 75% of the current study patients having an alteration potentially actionable by an approved drug . 
table 3 lists therapeutics with their corresponding targets that were identified in the patient cohort . one of the major obstacles to performing comprehensive genomic profiling of clinical specimens is the necessity for an adequate tumor sample . 
unlike solid malignancies where invasive biopsy is almost always required to obtain a tissue sample , comprehensive genomic profiling of lymphoid malignancies often can be performed with the use of peripheral blood and / or bone marrow . in this cohort , 54% of patients had specimens obtained from blood and / or bone marrow . tumors can acquire new mutations as they progress , which underscores the importance of obtaining new tissue when available for sequencing at the time of therapeutic decision making . 
 other not actionable , 2 mm not actionable , 2 other actionable , actionable , actionable , cll not actionable , fl not actionable , 2 actionable , 3 actionable , dlbcl actionable , fig 2 . 
in one study , 42% of patients did not have metastatic disease at the time of their biopsy.24 lymphoid malignancies are more amenable to repeat biopsy , with more than one half of the current cohort having tissue available from the blood and / or bone marrow . 
this accessibility facilitates repeat ngs to guide therapy . over the past decade , 26 orally administered targeted therapies have been fda approved for the treatment of 13 different solid malignancies ( data supplement )  . 
over the same period , only five orally administered targeted therapies have been approved for the treatment of lymphoid malignancies ( philadelphia chromosomepositive all , cll , follicular lymphoma , mantle cell lymphoma , and waldenstr om macroglobulinemia )  . the current data demonstrate that 45 patients ( 75% ) had an alteration that theoretically could have been targeted with an approved , albeit offlabel , drug , whereas only three patients ( 5% ) had an alteration for which an approved on - label drug was available . 
in a similar study in patients with solid malignancies , 20% had an alteration targeted by on - label agents , whereas 67% had an alteration targetable by off - label use.24 ( of note , the study used the same definitions for actionability as were used in this analysis . ) currently , a paucity of orally administered targeted therapy is available for onlabel use in lymphoid malignancies . 
however , as our findings suggest , the majority of cases likely will have potentially targetable alterations . the mutational landscape for numerous lymphomas has now been well characterized by several whole - exome sequencing studies.46 , 47 for example , mll2 , crebbp , and tp53 alterations in dlbcl , as described by pasqualucci et al , 46 were some of the most common recurrent mutations in the current cohort of patients with dlbcl . 
this alteration was also the most common ( 12% ) in a cohort of patients with cll sequenced by puente et al.47 furthermore , 82% of our patients had alterations that were pharmacologically tractable , similar to the 77% reported by he et al15 in diverse hematologic malignancies . 
these similar findings in other cohorts provide further support of the generalizability of our findings to other large cohorts of lymphoid malignancies . myd88 , an adapter protein used by toll - like receptors , was shown to be mutated ( myd88 l265p ) in  . 
90% of patients with waldenstr om macroglobulinemia ( lymphoplasmacytic lymphoma ) .48 this mutation not only is relatively specific for the disease but also predicts clinical presentation and survival.49 myd88 signaling is important in lymphomagenesis and signals through btk.50 in a phase ii trial , ibrutinib produced an overall response rate of 91% in a group of previously treated patients with waldenstr om macroglobulinemia , which led to its fda approval.37 furthermore , response rates to ibrutinib were significantly higher in patients with myd88 mutations versus wild - type myd88.51 in the current cohort , myd88 l265p mutation was found in the one patient with waldenstr om macroglobulinemia . 
however , myd88 alterations were also identified in one patient with dlbcl ( myd88 l265p ) , one with follicular lymphoma ( myd88 s219c ) , and one with primary cns lymphoma ( myd88 l265p )  . 
myd88 mutations have been discerned in marginal zone b - cell lymphoma , 52 cll , 53 dlbcl , 54 and primary cns lymphoma.55 preliminary data from a phase i trial of single - agent ibrutnib in four patients with cns lymphoma demonstrated responses in two of the three patients evaluated . 
in a phase i trial of 17 patients with relapsed mantle cell lymphoma treated with single - agent palbociclib , five ( 18% ) achieved progression - free survival of  . 
1 year , with one complete and two partial responses.58 the high percentage of cdkn2a / b alterations in the current study population gives further rationale for designing trials with cdk inhibitors in lymphoid malignancies . activating mutations in braf were found in three patients with cll ( two with braf g469a and one with braf v600e ) and one patient with multiple myeloma ( braf v600e )  . 
these mutations are potentially targetable by the braf inhibitors vemurafenib and dabrafenib and the mek inhibitors trametinib and cobimetinib.5 , 27 braf alterations have been identified as a driver mutation and as a biomarker for sensitivity to braf inhibition in both hairy cell leukemia and erdheim - chester disease.59 - 61 braf alterations have been found in approximately 3% of patients with cll.62 in the current study , two patients had mutations that led to an alanine substitution for a glycine at position 469 . 
ngs readily identified philadelphia chromosomelike b - all and can assist in selecting patients for clinical trials of targeted agents aimed at improving the poor outcome of these patients.15 higher neoantigen burden , which may be largely predicted by tmb , has been associated with higher objective response rates and progressionin patients treated with pd - 1 free survival blockade.67 , 68 melanoma , lung cancer , and renal cell carcinoma , all of which are highly responsive to pd - 1 blockade , have been shown to have a high degree of mutational burden from wholegenome and - exome sequencing studies.69 in the current cohort , tmb ranged from one or fewer to 140 ( data supplement )  . 
intermediate to high tmb was noted across almost all histologies ( except for marginal zone lymphoma , natural killer / t - cell lymphoma , and cutaneous t - cell lymphoma )  . 
the majority of the patients had cll , multiple myeloma , and all , whereas other nonhodgkin lymphomas were less well represented . although the majority of patients had actionable mutations theoretically targeted by fda - approved drugs , in practice , insurance approval for off - label drug use often is difficult to obtain.73 in addition , no standard definition exists for a targetable alteration , and the level of evidence needed to support this is a matter of debate . 
the number of actionable alterations discussed in this article may be overestimated , but even so , these patients should still be directed toward clinical trials that target these alterations so that the responsiveness or lack thereof can be determined . 
numerous additional clinical trials with a standardized definition of what constitutes a targetable alteration are needed to determine the extent to which patients respond when an alteration is theoretically druggable . 
currently , a number of such trials are ongoing ( clinicaltrials.gov identifiers nct02534675 , nct00851032 , and nct02465060 )  . in conclusion , we found that most patients with lymphoid malignancies have unique and complex molecular portfolios . 
goodman administrative support : caitlin costello , razelle kurzrock provision of study materials or patients : michael choi , matthew wieduwilt , carolyn mulroney , caitlin costello collection and assembly of data : aaron m . 
 novartis , merck , roche / genetech , genentech mypath , foundation medicine , pfizer , guardant health , merck serono patents , royalties , other intellectual property : four patents travel , accommodations , expenses : win , emd serono , gateway research advisory committee , icrp , win 2015 , caris centers of excellence , aacr , association of american cancer institutes , emd serono & quintiles , global biomarkers consortium , guardant health , global source ventures / novena therapeutics , ascpt , meyers consulting , fda - ocra , genentech , orbimed / global source ventures , sylvester cancer center , journal of precision medicine , curematch , lynx group , mayo clinic cancer center , kaiser permanente , pancan , cedars - sinai , medimmune / jk associates medical communications group affiliations aaron m . 
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 radiogenomics analysis of intratumor heterogeneity in a patient with high - grade serous ovarian cancer britta weigelt , phd1 ; hebert alberto vargas , md1 ; pier selenica1 ; felipe c . 
reis - filho , md , phd1 ; and evis sala , md , phd1 , 3 introduction the overall survival of patients with high - grade serous ovarian cancer ( hgsoc ) has not improved during the past 20 years.1 studies have revealed that genomic intratumor heterogeneity correlates with poor survival2 , 3 and specic patterns of malignant cell spread in hgsoc.4 the physical distribution of malignant clones across the peritoneal cavity may be nonrandom , because some sites harbor genetically diverse clones.5 these region - specic properties may invasion and expansion , modulate malignant cell thereby shaping evolutionary selection.3 , 6 our understanding of genomic heterogeneity in hgsoc is limited to single biopsies ; little is known about individual spatial and temporal variation across various tumor sites.2 - 5 hence , developing imaging methods for guiding tissue sampling to physiologically and metabolically distinct tumor habitats is desirable . 
such approaches will be vital for new clinical trials , especially those combining immunologic and genomically targeted therapies . here we use lesion - specic three - dimensional ( 3d ) molds for phenotypic image - guided tumor sampling to ensure spatial colocation of imaging , histology , and genomic data , critical for understanding tumor biology . phenotypic imaging maps of heterogeneity ( ie , imaging habitats ) of two hgsoc sites were obtained by combining perfusion , diffusion , and metabolic maps derived from multiparametric imaging . 
four days before primary cytoreductive surgery the patient underwent same - day multiparametric magnetic resonance imaging ( mpmri ; discovery mr750 3t mri system , ge healthcare , chicago , il ) of the abdomen and pelvis and 18f - uorodeoxyglucose ( fdg ) positron emission tomography ( pet ) / computed tomography ( ct ; discovery pet / ct system , ge healthcare )  . 
details of image acquisition , analysis , and mr - pet / ct coregistration are described in the appendix and in appendix table a1 . cluster - guided specimen sampling imaging habitats were identied using k - means clustering of the standardized uptake value ( suv ) , diffusion coefcient ( d ) , perfusion fraction ( f ) , and transfer constant ( dynamic contrast - enhanced parameter ktrans ) voxels , with the number of clusters ( k ) being xed to k = 3.7 custom - made 3d molds of the lesions were printed using a 3d printer ( makerbot replicator 2 ; makerbot , brooklyn , ny ) on the basis of manual segmentation of the right ovarian tumor and metastatic implant on the axial t2 - weighted mri ( appendix )  . 
tissue samples of each imaging cluster were obtained , cut in half , and formalin - xed and parafn - embedded for histopathologic analysis or snap - frozen for genomic analysis ( fig 1a )  . histologic review and immunohistochemistry the three right ovarian mass hgsoc imaging - based habitats ( labeled blue , yellow , or green ) and the omental implant ( blue ) were reviewed by two pathologists blinded to the habitat assignment . 
 ( a ) phenotypically distinct areas of a highgrade serous ovarian cancer ( hgsoc ) were identied by k - means clustering of imaging features derived from magnetic resonance imaging ( mri ) and positron emission tomography ( pet ) / computed tomography . 
the distinct imaging habitats ( labeled blue , yellow , and green ) were sampled from the surgically removed primary hgsoc and metastatic implant using a three - dimensional ( 3d ) mold . 
half of each imaging - based tissue area was formalin - xed parafnembedded for histopathologic review and immunohistochemical analysis , and the other half was ash - frozen for whole - exome sequencing analysis . 
 ( b ) micrographs of representative hematoxylin and eosin ( h and e ) stained sections of the imaging - based hgsoc areas ( top row ) , and immunohistochemical analysis of cd31 , ki - 67 , ca - ix , and hif - 1 . 
 ( c ) imaging features associated with the distinct color areas as dened by k - means clustering of standardized uptake values ( suv ) , diffusion coefcient ( d ) , dynamic contrast - enhanced dce parameter ( ktrans ) , and water volume fraction owing through microvessels ( f ) are plotted on top , and the histopathologic features of the distinct imaging - based tumor areas are shown at the bottocolor - coding according to the legend . dw , diffusion - weighted ; fdg , 18f - uorodeoxyglucose ; til , tumor - inltrating lymphocyte . nonsynonymous somatic mutations ( 74% ) not detected in all tumor areas by wes were independently validated using a custom hybrid - capture targeted massively parallel sequencing assay . 
the clusters were labeled blue ( lowest ktrans , highest suv , highest f ) , yellow ( highest d , lowest f ) , or green ( lowest d , highest ktrans , lowest suv )  . 
the right ovarian cancer contained all three distinct imagingbased clusters , whereas the omental metastatic implant was entirely composed of one cluster ( blue ; fig 1a ) , suggestive of phenotypic imaging heterogeneity in the primary hgsoc . tissue from the distinct imaging - based clusters for immunohistochemical and genetic analyses was obtained through a custom - made 3d mold ( fig 1a )  . imaging - based clusters are underpinned by distinct growth patterns and expression of hypoxia - related markers phenotypic imaging - based clusters were associated with distinct histopathologic growth patterns . 
 ( d ) unsupervised hierarchical clustering of the somatic mutations ( left ) and of the gene copy number alterations ( right ) identied not to be shared by all four imaging - based hgsoc areas using wards algorithm and euclidean distance . left , genes affected by mutations are shown in black ; right , amplication , gain , loss , and deletion are shown in dark red , red , blue , and dark blue , respectively . 
the values on the edges of the clustering are derived from pvclust analysis to assess the stability of the clusters ; both the approximatelyunbiased and bootstrap - probability p values were 100% . 
 weigelt et al signature 1 signature 3 other signatures mutant wild type somatic mutations copy number alterations amplification no change deletion gain loss ovary green ovary yellow ovary blue omentum blue ovary green ovary yellow ovary blue omentum blue 7q11 amp 2q11 amp ovary yellow ovary green tp53 pot1 16p12 amp 6p21 amp ovary blue fig 2 . 
the expression levels and patterns of the hypoxia - marker hif - 1 and its downstream target ca - ix correlated with the distinct areas dened by multiparametric imaging ( fig 1c )  . 
consistent with the imaging ndings , which demonstrated that the ovary blue and omentum blue areas displayed the highest f , an mri marker of tissue vascularity , cd31 immunohistochemical assessment revealed a higher density of tumor neovascularization ( 4 + ) in the ovary blue and omentum blue areas than in the ovary green and ovary yellow areas ( 2 + ; fig 1c )  . 
furthermore , distinct patterns and levels of ca - ix expression were observed among these areas , with ovary yellow and green having h - scores of 80 , compared with 230 and 195 in ovary blue and omentum blue , respectively . 
for hif - 1 , a reverse pattern was found , with ovary yellow and ovary green areas displaying high levels of hif - 1 expression ( h - scores , 115 ) compared with ovary blue and omentum blue ( h - scores , 75 and 60 , respectively ; figs 1b and 1c )  . imaging - based clusters are underpinned by distinct repertoires of genetic alterations high - depth wes of microdissected tumor and normal samples revealed 50 nonsynonymous somatic mutations , including a tp53 p.p47tfs * 5 frameshift mutation , which were shared among all four imaging - based tumor areas ( fig 2 )  . 
twenty - eight nonsynonymous somatic mutations were shared exclusively between ovary blue and omentum blue , and 46 between the ovary green and ovary yellow components ( fig 2a )  . 
although all imaging habitats displayed genomics features of homologous recombination dna repair deciency ( dominant mutational signature 3 , high large - scale transition scores ; fig 2c ) , hierarchical clustering of the 146 nonsynonymous somatic mutations or the 718 gene copy number alterations not shared among all four imagingbased areas revealed that both at the mutational and gene copy number levels , ovary blue and omentum blue clustered together and were distinct from ovary yellow and ovary green ( fig 2d )  . 
several somatic mutations found to be subclonal in ovary blue , including mutations affecting auts2 , eif2ak4 , and trim41 , became clonal in omentum blue ( fig 2a ) , and a subset of genes affected by copy number losses in ovary blue became homozygously deleted in omentum blue ( eg , chromosome 18q23 ; fig 2d )  . discussion several studies have demonstrated that hgsocs display spatial and temporal genetic heterogeneity , 2 - 6 , 22 with tumors composed of genetically distinct clones and exhibiting distinct evolutionary trajectories and mechanisms of therapy resistance.4 , 23 , 24 our observations support the contention that phenotypic and genetic analysis of single biopsy or single tumor samples may not provide a sufciently accurate representation of hgsoc heterogeneity.4 this proof - of - principle study suggests that multiparametric imaging assessment may provide a noninvasive surrogate for intratumor genetic heterogeneity and may guide precise tissue sampling . 
given the evidence supporting the impact of intratumor genetic heterogeneity on tumors metastatic ability and resistance to therapeutic interventions , 25 the results of this proof - of - principle observation provide the basis for studies to dene the type of sampling required for the assessment of preand posttreatment hgsocs . 
it should be noted , however , that multiparametric imaging for tumor heterogeneity and tumor evolution assessment requires not only manual segmentation of the imaging data but also standardization of the image acquisition protocols across different imaging platforms . targeted therapy and immunotherapy are being progressively applied earlier in the treatment of patients with cancer , and certain agents are anticipated to become part of front - line therapy . 
minimally invasive methods for disease monitoring in the form of circulating cell - free plasma dna hold great promise in the assessment of genetic heterogeneity within a patient.26 - 28 our imaging habitatbased method would constitute a noninvasive and complementary approach to such endeavors , given that it may allow for the sampling of selected image habitats without having to analyze every section of every tumor , an impossible task particularly in the metastatic and recurrent settings . 
for more information about asco 's conict of interest policy , please refer to or po.ascopubs.org / site / ifc . britta weigelt consulting or advisory role : genentech ( i ) , invicro ( i ) , ventana medical systems ( i ) , volitionrx ( i ) , paige.ai ( i ) , goldman sachs ( i ) felipe c . 
 weigelt et al niamh conlon employment : abbvie ( i ) stock and other ownership interests : merck ( i ) alexandra snyder employment : adaptive biotechnologies , merck stock and other ownership interests : merck consulting or advisory role : driver group research funding : bristol - myers squibb travel , accommodations , expenses : genentech , bristol - myers squibb robert a . 
assignee : the regents of the university of california august 18 , 2015 ( inst ) ; application : compositions and methods for the diagnosis and treatment of ovarian cancers that are associated with reduced smarca4 gene expression or protein function ( inst ) ; royalties from published books : cambridge university press and springer publishing dennis s . 
chi leadership : csurgeries stock and other ownership interests : bovie medical , verthermia , intuitive surgical , transenterix consulting or advisory role : bovie medical , verthemia ginger j . 
reis - filho consulting or advisory role : genentech , invicro , ventana medical systems , volitionrx , paige.ai , goldman sachs evis sala honoraria : siemens healthineers speakers ' bureau : siemens healthineers travel , accommodations , expenses : siemens healthineers no other potential conicts of interest were reported . references siegel rl , miller kd , jemal a : cancer statistics , 2018 . 
ca cancer j clin 68 : 7 - 30 , 2018 schwarz rf , ng ck , cooke sl , et al : spatial and temporal heterogeneity in high - grade serous ovarian cancer : a phylogenetic analysis . 
cell 173 : 1755 - 1769.e22 , 2018 bashashati a , ha g , tone a , et al : distinct evolutionary trajectories of primary high - grade serous ovarian cancers revealed through spatial mutational proling . j pathol 231 : 21 - 34 , 2013 5 . 
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dowsett m , nielsen to , ahern r , et al : assessment of ki67 in breast cancer : recommendations from the international ki67 in breast cancer working group . int j gynecol pathol 34 : 437 - 444 , 2015 working group 2014 . 
detre s , saclani jotti g , dowsett m : a quickscore method for immunohistochemical semiquantitation : validation for oestrogen receptor in breast carcinomas . j clin pathol 48 : 876 - 878 , 1995 110 : 1030 - 1034 , 2018 15 . 
schultheis am , ng ck , de filippo mr , et al : massively parallel sequencing - based clonality analysis of synchronous endometrioid endometrial and ovarian cancer res 72 : 5454 - 5462 , 2012 carcinomas . 
the examination was performed on a 3t magnetic resonance imaging ( mri ) whole - body scanner unit ( discovery mr750 ; ge healthcare , chicago , il ) using a dedicated multichannel torso phased - array coil as the receive coil . the standard protocol included the following sequences acquired in two - dimensional t2 - weighted fast spin - echo , t1an axial plane : weighted gradient - echo . 
single - shot incoherent motion intravoxel ( ivim ) based diffusion - weighted images were obtained using echoplanar imaging with a pair of motion - probing gradients along three orthogonal axes , using a chemical shift selective fat - saturation technique . 
dynamic contrast - enhanced ( dce ) images were acquired before and after the intravenous injection of gadopentetate dimeglumine ( magnevist ; berlex laboratories , montville , nj ; 0.1 mmol per kilogram of body weight at a rate of 2 ml / s ) using an automatic injector ( medrad , bayer , pittsburgh , pa )  . 
the patient was scanned on a dedicated positron emission tomography ( pet ) / computed tomography ( ct ) system ( discovery 690 ; ge healthcare , chicago , il )  . 
a standard - of - care acquisition protocol was applied with an intravenous injection of approximately 400 to 455 mbq 18f - uorodeoxyglucose after at least 6 hours of fasting and documentation of blood glucose less than 200 mg / dl followed by a 60 - minute uptake period . 
subsequently , a low - dose , attenuation - correction ct scan ( 120 to 140 kv , approximately 80 ma ) was acquired with the patient in the supine position , followed by acquisition of pet emission images of the whole body ( 3 minutes per bed position , ve to six bed positions )  . imaging analysis and mr - pet coregistration . 
using anatomic landmarks available on axial t2 - weighted mr images and on the axial pet and low - dose ct images , the tumor regions outlined on mri were coregistered with pet . 
this was achieved by calculating the mean location ( corresponding to the center of mass of the tumor ) on the mri image ( t2w ) and the corresponding mean location on the pet / ct image . 
rigid registration was selected to ensure that the geometry ( and the voxel count ) of the region of interest ( roi ) for each slice was maintained between mri and pet . 
the translational matrix was applied to the roi for each slice to determine the displacement of the roi . the ivim biexponential model proposed by le bihan et al ( le bihan d , et al : radiology 168 : 497 - 505 , 1988 ; le bihan d , et al : radiology 161 : 401 - 407 , 1986 ; le bihan d , et al : magn reson med 10 : 324 - 337 , 1989 ) was used to estimate the diffusion parameters for each lesion outlined , including diffusion coefcient ( d ) and the volume fraction of the water owing through the microvessels ( f )  . 
the segmented analysis procedure ( callot v , et al : magn reson med 50 : 531 - 540 , 2003 ; yao l , et al : acad radiol 7 : 27 - 32 , 2000 ) was used to estimate ivim parameters on a voxel - wise basis . 
pharmacokinetic analysis of the dce data were carried out using a one - compartment tofts model ( tofts ps : j magn reson imaging 7 : 91 - 101 , 1997 )  . 
for each outlined lesion , voxel - wise estimates of the volume transfer constant between the blood plasma and the extravascular extracellular space ( ktrans [ min1 ] ) was calculated . 
arterial input function was calculated using a biexponent model as previously described ( weinmann hj , et al : physiol chem phys med nmr 16 : 167 - 172 , 1984 ; priest an , et al : magn reson med 63 : 1044 - 1049 , 2010 )  . gaussian smoothing was applied to all raw mr images before processing with variance of half a voxel , to smooth the parameters . 
for voxels that had estimated parameters that exceeded the bounds set by the tting curve , the values were set to nan ( not - a - number )  . 
once all voxels were tted , the voxels with nan value were replaced by an average of the surrounding voxels so that each voxel had an estimated parameter for the purpose of clustering . the standardized uptake values ( suv ) of the voxels contained within each lesion on 18f - uorodeoxyglucose pet was calculated based on the standard expression given by kinahan et al ( kinahan pe , et al : transl oncol 2 : 223 - 230 , 2009 )  . three - dimensional printing methodology . 
custom made threedimensional ( 3d ) molds were printed based on manual segmentation of the ovarian primary tumor and metastatic implants on the preoperative axial t2 - weighted mr images . 
the 3d model was then compared with the mr images and manually adjusted to resolve any discrepancies using meshlab ( meshlab , visual computing labisti - cnr ) , an extensible mesh processing system , aimed at editing and rendering unstructured 3d triangular meshes . 
the nal 3d models of each lesion were imported into openscad ( openscad , the openscad developers ) , 3d cad modeling software , which was used to create an internal cavity that exactly shaped each lesion according to the mri shape and contour . 
the slits for slicing each lesion were designed into the molds at 5 - mm intervals , which corresponded to the slice thickness and locations of the axial t2w fast relaxation fast spin echo mr images . 
 graft - versus - host diseasefree antitumoral signature after allogeneic donor lymphocyte injection identied by proteomics and systems biology xiaowen liu , phd1 , 2 ; zongliang yue , ms3 ; yimou cao , ms1 ; lauren taylor , md2 ; qing zhang , ms4 ; sung w . 
chen , phd3 ; huanmei wu , phd1 ; and sophie paczesny , md , phd2 purpose as a tumor immunotherapy , allogeneic hematopoietic cell transplantation with subsequent donor lymphocyte injection ( dli ) aims to induce the graft - versus - tumor ( gvt ) effect but often also leads to acute graftversus - host disease ( gvhd )  . 
our novel six - step systems biology analysis involved removing common proteins and gvhd - specic proteins , creating a protein - protein interaction network , calculating relevance and penalty scores , and visualizing candidate biomarkers in gene networks . 
we then performed a second proteomics experiment in a validation set of patients who experienced gvt without acute gvhd after dli for comparison with the proteome of patients before dli . 
an independent experiment with single - cell proling in tumor antigenactivated t cells from a patient with posthematopoietic cell transplantation relapse was performed . results the approach provided a list of 46 proteins in the training set , and 30 proteins in the validation set were associated with gvt without gvhd . 
finally , the single - cell proling in activated t cells found 43 of the 61 genes . novel markers , such as rpl23 , ilf2 , cd58 , and crtam , were identied and could be extended to other antitumoral responses . conclusion our multiomic analysis provides , to our knowledge , the rst human plasma signature for gvt without gvhd . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction allogeneic hematopoietic cell transplantation ( hct ) is one of the most effective forms of tumor immunotherapy available to date . 
the lymphocytes in the donor graft recognize and eliminate residual tumoral cells through the graft - versus - tumor ( gvt ) effect , and thus , donor lymphocyte injection ( dli ) often is used at the time of relapse post - hct to induce gvt . 
despite the correlation of gvt indirect evidence for a gvt reaction and gvhd , separate from gvhd has been reported in large cohorts of hct patients or when dli is administered to induce remission in hct patients who have experienced a relapse.1 , 2 gvt activity can be increased by targeted therapy , as has been shown with sorafenib in flt3 internal tandem duplicationmutant leukemia cells.3 the gvt effect is mediated by minor histocompatibility antigens ( mihags ) on recipient leukemic cells that are recognized by donor cd4 + and cd8 + t cells.4 mihags can mediate gvt without inducing gvhd if they are only expressed by the recipient hematopoietic cells ( ie , minor h antigen [ ha ] - 1 , ha - 2 , bcl2a1 , and hb - 1 ) .5 , 6 nonhistocompatibility antigen proteins expressed by tumor cells , called tumor - associated antigens ( survivin , wilms tumor 1 , proteinase 3 ) also can mediate gvt activity.7 cytokines ( ie , interleukin - 15 [ il - 15 ] ) 3 ; checkpoint proteins , such as pd - 1 / pd - l18 ; and t - cell trafcking modulators9 are other possible mediators of gvt . our study aim was to develop a proteomic signature to identify gvt without gvhd after allogeneic dli . 
we attempted to do so through plasma proteomics and systems biology analyses of patients in relapse after hct who received donor lymphocytes as immunotherapy . knowledge generated our novel six - step systems biology analysis involved the removal of common proteins and gvhd - specic proteins , creation of a protein - protein interaction network , calculation of relevance and penalty scores , and visualization of candidate biomarkers in gene networks to dene a unique , biologically relevant 61 - protein signature of gvhd - free gvt . 
forty - three of the 61 genes also were found in an independent experiment using massive single - cell proling of tumor antigenactivated t cells from a patient who experienced post - hct relapse . relevance this multiomic analysis provides the rst human plasma signature for gvhd - free gvt to our knowledge . 
risk stratication on the basis of the gvt without gvhd protein signature would allow for customized treatment plans after hct . of 61 proteins that are signicantly expressed in the plasma of hct patients who received dli for tumor relapse . 
the methodology for this study is described in the data supplement . results plasma proteomics of gvhd - free gvt in a training set our initial approach to identifying gvt - specic biomarkers was to undertake a proteomics analysis . 
we reasoned that the best setting to observe a gvt effect without any effect of chemotherapy or preparative conditioning regimen would be after dli is given for relapse of malignant disease . 
our rst proteomics experiment ( ipas01 ) was designed to distinguish proteomes that predict gvhd - free gvt from those that predict gvt with gvhd after dli in a training set . 
we compared a pool of plasma samples taken approximately 30 days after dli from ve patients without relapse and without gvhd ( gvt - positive , gvhd - negative ) labeled with the heavy isotope and compared them with a pool of plasma samples at matched time points from 11 patients without relapse but with concomitant gvhd ( gvt - positive , gvhd - positive ) labeled with the light isotope ( fig 1 )  . 
no signicant differences between groups were observed for patient age , disease status ( all but one with morphologic relapse , and only one patient did not receive chemotherapy ) , dli donor type , and dli dose , and all patients were off immunosuppression . 
 gvhd - free antitumoral signature after donor lymphocyte injection gvt - positive , and gvhd - positive post - dli ( ipas - 01 ) gvt - positive , and gvhd - negative pre - dli ( ipas - 02 ) gvt - positive , and gvhd - negative post - dli ( ipas - 01 and - 02 ) isotopic labeling light isotopic labeling heavy immunodepletion six most abundant proteins acrylamide alkylation samples mixed fig 1 . 
the intact - protein analysis system ( ipas ) compared graft - versus ( gvt ) positive and graft - versus - host disease tumor ( gvhd ) negative postdonor lymphocyte injection ( dli ; heavy isotope ) with gvt - positive and gvhd - positive postdli ( light isotope ) samples in a training set ( ipas - 01 ) , and gvt - positive and gvhd - negative post - dli ( heavy isotope ) with gvt - positive and gvhd - negative pre - dli ( light isotope ) samples in a validation set ( ipas - 02 )  . anion - exchange chromatography ( n = 8 ) reversed - phase chromatography ( n = 60 ) liquid chromatography - ms / ms software tool ( data supplement )  . 
after this data processing , the ipas - 01 experiment provided a list of 46 proteins associated with a gvt signature without gvhd post - dli in the training set ( table 1 )  . 
although we started with ipas proteins with a heavy / light ratio greater than 1.2 , the systems biology process may have resulted in a nal score of less than 1 ; however , because all these proteins are gvt specic , they all are included in the nal gvhd - free gvt signature . 
the rst node is centered around cd8 ( cd8a ) and includes cd58 , il1a , ly75 , fas , and gpnmb and a secondary node centered around raf1 and containing guk1 , ephb4 , and cxcl12 . 
our data with the cd8a - centered node strongly suggest that gvt is majorly mediated by cd8 t cells and gvhd less so , which provides an opportunity to separate gvt and gvhd by manipulating these two t - cell subsets differently . 
as a proof of concept , we performed the experiment without applying these proteins to show that their nal scores have not been signicantly changed by the use of penalty scores ( table 1 ; data supplement )  . plasma proteomics of gvhd - free gvt in a validation set our second proteomics experiment ( ipas - 02 ) used a validation set that was designed to compare a pool of samples from ve gvt - positive , gvhd - negative ( labeled with the heavy isotope ) patients with a pool of samples from the same patients taken before dli when the patients were in relapse and thus gvt negative ( labeled with the light isotope ; fig 1 )  . 
a major cluster is constituted by centromere protein m ( cenpm ) , nup160 , wapal , dhx37 , stx7 , and il enhancer binding factor 2 ( ilf2 ) , which are all proteins implicated in the activation of cytotoxic t cells consistent with a gvt response ( table 2 )  . 
for example , cenpm ( also called pane1 ) is a known mihag expressed on b - lymphoid cells that is highly relevant to gvt - mediated post - dli.12 the protein encoded by ilf2 is a transcription factor needed for t - cell expression of il - 2.13 the second cluster is similar to the one found in ipas - 01 centered on map3k6 and dbnl . final 61 - protein gvhd - free gvt signature after dli the next step was to combine the proteins found in the training and validation sets . 
combined analysis of the two ipas experiments yielded 61 proteins : 49 with a nal combined score of greater than 1 and 12 with a nal combined score of less than 1 , all specic to gvt without gvhd ( table 1 )  . 
these include one similar to that seen in ipas - 01 centered on cd8a , one similar to that seen in ipas - 02 centered on cenpm - wapal and also containing dhx37 , and one found in both experiments and centered on map3k6 and dbnl . 
this additional cluster is centered around cse1l , which is a ras - related nuclear protein with a potential role in ras / raf / mapk signaling in t cells . 
some potential important novel markers , such as cytotoxic and regulatory t - cell molecule ( crtam ) , which has been shown to determine the cd4 + cytotoxic t - lymphocyte lineage , 14 are not part of a cluster . 
 ( a ) intact - protein analysis system ( ipas ) - 01 : geneterrain visualization shown in 2d for the graftversus - tumor ( gvt ) signature obtained by comparing gvt - positive , graft - versus - host disease ( gvhd ) negative with gvt - positive , gvhd - positive postdonor lymphocyte injection ( dli ) samples . 
a mean reads number of 381 , 079 per cell and median gene number of 1 , 091 per cell were analyzed for an average of 1 , 436 cells per condition . 
among the 61 genes identied in the proteomic gvhd - free gvt signature , 11 were more highly expressed in prame - specic t cells compared with cmvspecic t cells or nonreactive t cells . 
the expression prole of four representative gvt markers in the single - cell rna sequencing analysis of prame - specic t cells ( rpl23 , ilf2 , cd58 , and crtam ) is shown in figure 3 . 
rpl23 is a component of the 60s ribosomal subunit and has been shown to link the oncogenic ras signaling to p53 - mediated tumor suppression.15 t - cell responses to rpl23 also are increased in autoimmune diseases , 16 which suggests a role for the ras / raf / mapk pathway in t cells that is currently underexplored . 
the cd58 / cd2 axis is the primary costimulatory pathway for cd8 + t cells that lack cd28 , which suggests an alternate activation mechanism during gvt for exhausted t cells.17 crtam is upregulated in cd4 + and cd8 + t cells and encodes a type 1 transmembrane protein with v and c1 - like immunoglobulin domains.18 it has been shown to negatively regulate zeb1 ( zinc nger e - boxbinding homeobox 1 ) in t cells19 and to determine the cd4 + cytotoxic t - lymphocyte lineage , 14 and it might determine the cd8 + cytotoxic t - lymphocyte lineage as well . 
expression prole of four representative graft - versus - tumor ( gvt ) markers in single - cell rna sequencing analysis of prameand cytomegalovirus ( cmv ) pp65specic t cells . 
one systems biology novelty of our approach was the performance of a one - layer extension on ppi data using interactions one node away from the gene in the original list that are called outer genes . 
a strength of our approach is that we ltered out nonrelevant proteins using a penalty score , which led to a more - specic list of candidate proteins and avoided contaminants . 
this study favored a large - scale proteomics approach as opposed to a hypothesis - driven candidate approach.21 we have shown that for gvhd markers , this method is efcient in discovering new candidate markers.10 , 11 compared with our previous studies , we experimentally removed the gvhd proteins by assigning them the light isotope . 
of note , this approach showed that the proteins identied and their ratio have not been inuenced much by the implementation of penalty scores . in an independent experiment with single - cell proling of t cells from a patient with relapse after hct that were activated in vitro , 43 of the 61 proteomic genes also were found in activated t cells , which suggests that the proteins identied are biologically relevant in different antitumoral responses . the biology of the gvt markers not yet described is as follows . 
the function of trpc4ap ( transient receptor potential cation channel subfamily c member 4associated protein ) has been shown to be involved in the ubiquitination of e3 ligase skp222 and the activation of c - jun nh ( 2 ) terminal kinase and transcription factor ap - 1.23 guanylate kinase 1 ( guk1 ) is an enzyme that catalyzes the transfer of a phosphate group from atp to guanosine monophosphate ( gmp ) to form guanosine diphosphate ( gdp ) and is believed to be a good target for cancer chemotherapy.24 its expression on tumor - specic t cells was not previously reported . 
pin1 ( peptidylprolyl cis / trans isomerase , nimainteracting 1 ) catalyzes the cis / trans isomerization of peptidyl - prolyl peptide bonds and thus catalytically regulates the postphosphorylation conformation of its substrates and is involved in the regulation of t - cell biology . 
in particular , its implication has been shown in systemic lupus erythematosus and t - cell acute lymphoblastic leukemia progression.25 - 27 wapl cohesin release factor has been shown to restrict chromatin loop extension.28 of note , it was part of a microrna - mrna network in allogeneic t - cell responses.29 fermitin family member 3 ( fermt3 ) a member of the kindlins that mediates ppi involved in integrin activation . 
mutations in this gene cause the auadhesion deciency tosomal leukocyte syndrome - 3.30 its role in t cells has not been studied . cd160 is another surface protein tightly expressed on peripheral cytotoxic cd8 t lymphocytes and natural killer cells , 31 and soluble cd160 enhances cd8 + t cells , which results in increased interferon - , il - 2 , and tumor necrosis recessive factorsecretion as well as cytolysis against tumor cells in vitro and in vivo.32 not surprisingly , serglycin ( srgn ) , which serves as a mediator of granule - mediated apoptosis through the macromolecular complex of granzymes and perforin and determines the secretory granule repertoire of cytotoxic t lymphocytes , 33 was also upregulated in this study . 
the fas cell surface death receptor ( fas ) that plays a critical role in the activation of the death - inducing signaling complex with fas - associated death domain protein and triggers a downstream caspase cascade that leads to apoptosis34 also was upregulated . the chemokine cxcl12 has been proposed to be able to distinguish immune cells that induce gvt going to the bone marrow from immune cells that induce gvhd.35 it is one of the rare proteins for which the nal score was modied more than twice by the penalty score but is still included overall as a gvt proteseveral of the tripartite motif ( trim ) members encode for mihags.21 although trim42 , 22 , and 37 are located on chromosomes 3 , 11 , and 17 , respectively , trim39 is on chromosome 6 in the major histocompatability class i region and , in our study , showed global expression on activated t cells . 
raf1 is the cellular homolog of viral raf proto - oncogene ( v - raf ) and is also a map3k that functions downstream of the ras family of membrane - associated gtpases to which it binds directly . 
cse1l is a ras - related nuclear protein that binds strongly to nuclear localization signal - free importin - , and this complex is then released in the cytoplasm by the combined action of ranbp1 and rangap1 . 
one particular protein of nont - cell origin , il - 1 , was found to be elevated in the plasma of patients with gvt response and may play a signicant role in antitumor effects . 
of note , il - 1engineered tumor cells rarely develop into tumors , and if they do , the tumors are quickly destroyed through a mechanism that involves cd8 + t cells and natural killer cells , and il - 1 tumor immunoediting microenvironment.36 although our approach identied several proteins for gvhd - free gvt , there are limitations in this study . 
third , the systems biology pipeline relies on knowledge from the published domain , which makes the application of this method to diseases that have not been well studied , such as gvt , difcult . 
chen travel , accommodations , expenses : wuxi apptec sophie paczesny patents , royalties , other intellectual property : inventor on a patent entitled methods of detection of graft - versus - host disease ( us 20130115232a1 , wo 2013066369a3 ) no other potential conicts of interest were reported . acknowledgment the raw mass spectrometry data are publicly available at massive ( massive.ucsd.edu ) with the identication number msv000081057 . 
we thank gregory yanik , md , for providing the patient samples through a university of michigan - indiana university material transfer agreement . references ringd en o , labopin m , gorin nc , et al : is there a graft - versus - leukaemia effect in the absence of graft - versus - host disease in patients undergoing bone marrow transplantation for acute leukaemia ? br j haematol 111 : 1130 - 1137 , 2000 bar m , sandmaier bm , inamoto y , et al : donor lymphocyte infusion for relapsed hematological malignancies after allogeneic hematopoietic cell transplantation : prognostic relevance of the initial cd3 + t cell dose . 
mathew nr , baumgartner f , braun l , et al : sorafenib promotes graft - versus - leukemia activity in mice and humans through il - 15 production in flt3 - itdmutant leukemia cells . 
blood 91 : 2197 - 2207 , 1998 bleakley m , otterud be , richardt jl , et al : leukemia - associated minor histocompatibility antigen discovery using t - cell clones isolated by in vitro stimulation of naive cd8 + t cells . 
blood 115 : 4923 - 4933 , 2010 dossa rg , cunningham t , sommermeyer d , et al : development of t - cell immunotherapy for hematopoietic stem cell transplantation recipients at risk of leukemia relapse . 
leuk lymphoma 45 : 2229 - 2237 , 2004 koestner w , hapke m , herbst j , et al : pd - l1 blockade effectively restores strong graft - versus - leukemia effects without graft - versus - host disease after delayed adoptive transfer of t - cell receptor gene - engineered allogeneic cd8 + t cells . 
blood 117 : 1030 - 1041 , 2011 kim ym , sachs t , asavaroengchai w , et al : graft - versus - host disease can be separated from graft - versus - lymphoma effects by control of lymphocyte trafcking with fty720 . 
vander lugt mt , braun tm , hanash s , et al : st2 as a marker for risk of therapy - resistant graft - versus - host disease and death . 
brickner ag , evans am , mito jk , et al : the pane1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in b - lymphoid 13 . 
kao pn , chen l , brock g , et al : cloning and expression of cyclosporin aand fk506 - sensitive nuclear factor of activated t - cells : nf45 and nf90 . 
cancer res 76 : 5030 - 5039 , 2016 ito y , hashimoto m , hirota k , et al : detection of t cell responses to a ubiquitous cellular protein in autoimmune disease . 
leitner j , herndler - brandstetter d , zlabinger gj , et al : cd58 / cd2 is the primary costimulatory pathway in human cd28 - cd8 + t cells . 
yeh jh , sidhu ss , chan ac : regulation of a late phase of t cell polarity and effector functions by crtacell 132 : 846 - 859 , 2008 19 . 
bachireddy p , hainz u , rooney m , et al : reversal of in situ t - cell exhaustion during effective human antileukemia responses to donor lymphocyte infusion . blood 123 : 1412 - 1421 , 2014 21 . 
tissue antigens 81 : 183 - 193 , 2013 jamal a , swarnalatha m , sultana s , et al : the g1 phase e3 ubiquitin ligase truss that gets deregulated in human cancers is a novel substrate of the s - phase e3 ubiquitin ligase skp2 . 
soond sm , terry jl , riches dw : truss , a tumor necrosis factor receptor - 1 - interacting protein , activates c - jun nh ( 2 ) - terminal kinase and transcription factor ap - 1 . 
ruppert r , moser m , sperandio m , et al : kindlin - 3 - mediated integrin adhesion is dispensable for quiescent but essential for activated hematopoietic stem cells . j exp med 212 : 1415 - 1432 , 2015 31 . 
kotsiou e , davies jk : new ways to separate graft - versus - host disease and graft - versus - tumour effects after allogeneic haematopoietic stem cell transplantation . br j haematol 160 : 133 - 145 , 2013 36 . 
 o integrative molecular analysis of patients with advanced and metastatic cancer verena sailer , md1 ; kenneth wa eng , ms1 ; tuo zhang , phd1 ; rohan bareja , ms1 ; david j . 
pisapia , md1 ; alexandros sigaras , ms1 ; bhavneet bhinder , ms1 ; alessandro romanel , phd2 ; david wilkes , phd1 ; evan sticca , ms1 ; joanna cyrta , md1 ; rema rao , md1 ; sheena sahota , md1 ; chantal pauli , md1 ; shaham beg , md1 ; samaneh motanagh , md1 ; myriam kossai , md1 ; jacqueline fontugne , md1 ; loredana puca , phd1 ; hanna rennert , phd1 ; jenny zhaoying xiang , md1 ; noah greco , mba1 ; rob kim1 ; theresa y . 
in patients with more than one sequenced tumor sample ( n = 146 ) , 84.62% of actionable mutations were concordant . conclusion integrative analysis may uncover informative alterations for an advanced pan - cancer patient population . 
2019 by american society of clinical oncology introduction genomic proling is widely used in cancer care to identify actionable alterations for individual patients within the context of precision medicine ( pm ) .1 however , only 2% to 11% of those patients with sequencing performed receive a genomically matched therapy , which may be a result of the availability and accessibility of clinical trials , 2 - 6 patient factors and comorbidities , disease state or alternative options , or patient preference.5 despite these drawbacks , pm studies continue to provide insights into the molecular underpinnings of cancer . 
we and others have shown that performing whole - exome sequencing ( wes ) and rna sequencing is feasible in a clinical setting and may provide relevant information beyond targeted gene panels in certain settings.3 , 7 , 8 sequencing matched tumor and normal ( germline ) dna has the additional benet of uncovering unforeseen hereditary conditions , including cancer risk mutations . 
in particular , germline dna repair defects ( drds ) are more common than previously anticipated across adult advanced cancer populations.9 - 11 robinson et al12 published their met500 cohort with wes and rna sequencing data from 500 patients with metastatic disease of varied tumor primary and biopsy sites . 
our study , obtained from our own cohort of 759 samples from 515 patients with advanced and metastatic cancer , complements this data set as it adds wes data from approximately the same number of patients . 
 sailer et al context key objective we assessed whether developing a multidimensional precision oncology program is feasible and informative for patients with cancer with advanced disease . knowledge generated we established a comprehensive clinical genomics program for this patient group . 
the rate of clinically relevant alterations across 515 patients with advanced solid tumors was 39% by whole - exome sequencing , which was improved by 16% by adding rna sequencing . 
germline dna was obtained from blood samples ( circulating mononuclear cells ) , buccal swabs , or benign tissue as described previously.3 part of the data presented in this manuscript have already been published.3 , 13 , 15 wes was performed on each patients tumor / matched germline dna pair using previously described protocols.3 we used a clinical - grade wes testexome cancer test version 1approved by new york state department of health ( id# 43032 ) and described in detail in rennert et al.16 this approach allows for assessment of more than 21 , 000 genes through the development and implementation of computational approaches for tumor mutational burden and neoepitope analysis , as well as integration with other data , including rna sequencing , to improve the identication of clinically relevant and actionable alterations . 
to evaluate the concordance of tier 1 and tier 2 alterations between multiple samples from the same patient and to gain high - delity results , a cutoff of 20% for variant allele frequency was used . 
in addition , we developed patientderived organoids from fresh tissues using previously described protocols , 4 , 17 and we used cell lines to functionally validate outlier targetable alterations . wes alterations were categorized on the basis of on their actionability and clinical or biologic relevance.3 alterations in 49 actionable or clinically signicant genes were reported within category 1 , alterations in 508 known cancer - associated genes within category 2 , and somatic alterations of unknown signicance within category 3.3 we developed an open - access , dynamic , web - based pm knowledge base as an interactive online tool where variants are carefully interpreted in the context of tumor type.18 wes results were conveyed to the referring physician in the form of an exome cancer test version 1 report.3 selected cases are presented at a regular , continuing medical education accredited pm tumor board , which discusses sequencing results in the context of a patients history , available literature , and treatment options , including active clinical trials . pathogenic germline ndings were reported to the referring physician if they occurred in any of the genes deemed reportable by the american college of medical genetics and genomics ( acmg ) , 19 and these patients were referred for genetic counseling and results were conrmed by targeted testing in a clinical laboratory improvement amendments / clinical laboratory evaluation programcertied laboratory . study cohort demographic data were obtained through electronic health record search . 
 precision oncology for patients with advanced disease primary site ( 515 patients ) biopsy / resection site ( 759 samples ) breast ( 13 ) pancreas ( 18 ) kidney ( 46 ) soft tissue ( 8 ) brain ( 98 ) lung ( 16 ) hematologic malignancies ( 24 ) colon and / or rectum ( 41 ) bladder ( 57 ) prostate ( 105 ) primary recurrent metastatic brain ( 150 ) liver ( 64 ) bla d d er ( 69 ) b o n e ( 71 ) ly m p h n o d e ( 85 ) c olo n ( 18 ) l u n g ( 44 ) kid n ey ( 26 ) prostate ( 33 ) sample characteristic metastatic primary recurrent 34 - year - old female , er - positive breast carcinoma , mastectomy , brca1 / 2 germline negative chest wall recurrence , er / pr / her2 unknown , excision and radiotherapy no cancer detected metastatic disease seven systemic therapies precision medicine consent liver biopsy , akt1 mutation detected by wes cdk4 / 6 inhibitor ( palbociclib ) liver progress + 3 years + 20 years + 3 years + 1 year + 1 year + 1 year fig 1 . 
the most common somatic alterations were found in tp53 ( 33% ) , cdkn2a ( 11% ) , apc ( 10% ) , kmt2d ( 8% ) , pten ( 8% ) , and brca2 ( 7% ; fig 2 )  . multiple samples three hundred eighty - two spatially and temporally heterogeneous samples from 146 patients underwent wes . 
of these , 185 ( 48.4% ) were metastatic , 153 ( 40.1% ) were primary , and 44 ( 11.5% ) were recurrent tumor samples . most samples were paired primary and metastatic tumors , and concordance of alterations between primary and metastatic samples in clinically informative genes is shown in figure 3 . 
paired primarymetastatic tumor samples from 59 patients were the most frequent combination . spatially and / or temporally heterogeneous metastases and primary tumors from 35 and 29 patients , respectively , also underwent sequencing . 
 ( e ) different kras mutations in two morphologically similar lung cancer samples from the same patient , thus conrming two separate primary tumors . this cohort ( data supplement )  . 
the most frequently mutated genes were chek2 ( 11 patients ) , brca2 ( nine patients ) , brca1 ( nine patients ) , msh6 ( nine patients ) , and atm ( four patients ; fig 2 )  . 
in likely pathogenic variants in 38 addition , 44 additional patients in msh6 ( 14 cases ) ; apc ( seven cases ) ; chek2 ( ve cases ) ; pole , pms2 , msh2 brca1 , and atm ( three cases each ) ; and tp53 , cdh1 , and brip1 ( one case each ) were discovered in our cohort . the prevalence of drd in our cohort of patients with metastatic prostate cancer was 14.3% , with brca2 mutations being the most frequent variants ( data supplement )  . pathogenic germline drds were found in 9.2% of patients with primary brain tumors . 
we did not identify pathogenic mutations involving mismatch repair genes , which have been described in primary brain tumor patients , in particular as biallelic losses.25 chek2 was altered in four cases , including one medulloblastoma . 
 ( b ) high frequency of germline mutations in breast , prostate , and lung tumors is observed . germline drd defects , 12 had received platinum - based chemotherapy with follow - up available . 
an additional targetable ncoa4ret fusion , which has been described in papillary thyroid cancer , nonsmall - cell lung cancer , and colorectal cancer , was found in a brain metastasis of a patient with unknown primary.28 rna sequencing rna sequencing was performed when sufcient fresh frozen tumor tissue was available after wes . 
of these 89 patients , 50 did not harbor a targetable genomic alteration identied by wes , resulting in an increase in the rate of actionable alteration detection of approximately 15% . 
we conrmed the drug sensitivity of select outlier genes using cell line experiments compared with randomly selected drugs ( fig 5 )  . nine novel fusions in a variety of cancer types were detected . 
a novel rbm47 - cdk12 gene fusion was found in a prostate cancer bone metastasis , which was conrmed organoids part of the program was the development of patient - derived organoids from patient biopsies for high - throughput drug screening.4 altogether 60 organoids were developed from 98 patients with an overall success rate of 61% . 
a pathogenic msh6 mutation was detected in a 26 - year - old patient with metastatic breast carcinoma who had undergone previous outside testing for brca1 / 2 germline mutations with a negative result . 
other potentially signicant variants in drd genes , such as palb2 , would have been missed as well.30 this case underlines the importance of multigene panel or wes testing in patients not only with suspected hereditary cancer but also in those , especially young patients , with metastatic disease to detect underrecognized germline alterations.31 tumor evolution and clonality . 
a deleterious effect has been predicted for this variant.33 in a previously published analysis of 13 uterine carcinosarcomas analyzed by targeted sequencing , eight cases demonstrated 100% identical mutations in both the carcinoma and sarcoma part.34 in contrast , we observed an early divergence with only the one pten mutation of 54 nonsynonymous shared mutations . 
this case again demonstrates the importance of using next - generation sequencing to correctly identify synchronous primary tumors.39 another patient with metastatic neuroblastoma was conrmed to have an activating alk mutation ( r1275q ) in both primary tumor and bone marrow metastasis . 
the patient has had stable disease for 13 months on therapy with sequential alk inhibitors40 ( fig 3 )  . discussion we have established a pm program for patients with advanced cancer using tumor / normal wes and integrative molecular proling to detect genomic and other actionable alterations , improve clinical decision making , and study tumor evolution in a pan - cancer cohort . 
the patient population in our study is distinct from several reported studies in that our focus was on the evaluation of advanced tumors.41 , 42 the majority of patients ( 70.8% ) presented the time of with metastatic or recurrent disease at enrollment . whereas publicly accessible molecular data for several tumor types are available , both for primary and metastatic cancers , 43 , 44 these specimens are rarely matchedthat is , obtained from the same patient.45 our cohort of patients with locally advanced and metastatic cancer allowed for the collection of multiple matched , often primary and metastatic samples , resulting in a unique feature of this cohort : genomic data from spatially and / or temporally heterogeneous , matched tissue samples were available in 146 patients . 
of clinical importance is the nding that almost 85% of category 1 alterations were shared between multiple samples from the same patient . these results are in concordance with published data in specic cancer types . 
in one study , when comparing actionable mutations between presurgery biopsies and resected specimen in patients with nonsmall - cell lung cancer , the concordance rate was found to be 79%.46 similar ndings have been reported for primary and recurrent breast cancer , with 86.6% of mutations and 85.5% of scnas being concordant.47 our data conrm that it may reasonable to select the most accessible location or even archival material from the primary tumor for molecular analysis for certain cancer types.48 , 49 new research indicates that pathogenic germline mutations are more frequently found in patients with advanced cancer , 9 , 12 and our data conrm this . 
germline mutations that involve the dna repair pathway , found in 10.7% of our patients , are predictive of response to parp inhibitors and platinum - based therapy and have important familial plications for cancer screening.50 family history was indicative of a heritable component in only one half of these patients . 
this fusion might result in the loss of cdk12 activity , which has recently been described to delineate a distinct immunogenic subtype of metastatic castrationresistant prostate cancer.51 in our study , wes was prioritized over rna sequencing whenever tissue availability was of concern . 
biopsies in our study were usually performed for diagnostic purposes in the context of clinical care , hence the lower availability of fresh frozen tissue for additional rna sequencing . targeted sequencing of cancer - related genes offers several advantages over wes , including deeper coverage , quicker turnaround time , lower cost , and fewer requirements for an elaborate computational pipeline . 
here , we show that wes and rna sequencing may provide an additional and information in certain settings , complimentary layer of particularly for patients with advanced cancer who experience progression after standard therapies . 
wes also considers the rapidly expanding spectrum of actionable alterations , including alterations for which targeted treatment may not be available at the time of analysis , but for which clinical trials might be planned . 
in addition , whereas targeted nextgeneration sequencing testing of somatic dna are sometimes ordered as part of clinical care , using germline dna as normal control for wes necessitates securing informed consent . implementing - omic data into clinical care requires an interdisciplinary teagenomic data and its interpretation in the context of tumor type and primary site must be easily accessible to enable clinicians to integrate the data into everyday patient care . 
 precision oncology for patients with advanced disease one limitation of our study is the lack of uniformity of the cohort in terms of tumor type and therapy , which prevents us from making generalized statements about treatment response . 
this data set also reports on a large number of matched temporally and spatially heterogeneous samples , highlighting the concordance of actionable alterations in different samples . as other pm trials have shown , uncovering genomic alterations may not be sufcient in identifying actionable alterations . 
contributed equally to this work and share senior authorship . support supported by the englander institute for precision medicine of weill cornell medicine and new york presbyterian , the translational research program of the department of pathology and laboratory medicine at weill cornell medicine . 
macdonald , eleni andreopoulou , olivier elemento , himisha beltran provision of study materials or patients : noah greco , rob kim , bishoy m . faltas , eleni andreopoulou , linda t . 
pisapia , david wilkes , joanna cyrta , sheena sahota , chantal pauli , shaham beg , samaneh motanagh , myriam kossai , jacqueline fontugne , jenny zhaoying xiang , noah greco , rob kim , theresa y . 
pisapia , alexandros sigaras , bhavneet bhinder , alessandro romanel , david wilkes , evan sticca , rema rao , shaham beg , loredana puca , rob kim , mirjam blattner - johnson , eleni andreopoulou , linda t . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . alexandros sigaras employment : weill cornell medical college loredana puca employment : petra pharma corporation bishoy m . 
faltas honoraria : digital science press publications research funding : eli lilly david rickman research funding : janssen oncology eleni andreopoulou honoraria : astrazeneca , sirtex , svb leerink , abbvie , eisai , nanostring technologies consulting or advisory role : astrazeneca , sirtex , svb leerink , abbvie , eisai , nanostring technologies speakers bureau : r - pharm us travel , accommodations , expenses : astrazeneca , sirtex , svb leerink , abbvie , eisai , nanostring technologies kevin holcomb research funding : fujirebio diagnostics expert testimony : johnson & johnson linda t . 
vahdat honoraria : polyphor , r - pharm , athenex , seattle genetics , osmol therapeutics consulting or advisory role : berg pharma speakers bureau : eisai research funding : polyphor ( inst ) , immunomedics ( inst ) , seattle genetics ( inst ) patents , royalties , other intellectual property : patent pending on the application for bmd progenitor cells ( inst ) douglas s . 
 sailer et al koen van besien stock and other ownership interests : hemogenyx consulting or advisory role : hemogenyx , actinium pharmaceuticals , cellectis , tessa therapeutics research funding : miltenyi biotec , actinium pharmaceuticals , juno therapeutics , fate therapeutics christopher e . 
ocean consulting or advisory role : celgene , tyme speakers bureau : daiichi sankyo travel , accommodations , expenses : daiichi sankyo ana molina honoraria : american society of clinical oncology consulting or advisory role : eisai , exelixis , novartis , janssen oncology manish a . 
shah consulting or advisory role : astellas pharma , eli lilly research funding : gilead sciences ( inst ) , merck ( inst ) , boston biomedical ( inst ) , oncolys biopharma ( inst ) , bristol - myers squibb ( inst ) david m . 
nanus consulting or advisory role : genentech research funding : novartis ( inst ) , boehringer ingelheim ( inst ) , zenith epigenetics ( inst ) qiulu pan employment : caris life sciences stock and other ownership interests : caris life sciences francesca demichelis patents , royalties , other intellectual property : co - inventor on a patent led by the university of michigan and brigham and womens hospital covering the diagnostic and therapeutic elds for ets fusions in prostate cancer , diagnostic eld licensed to gen - probe scott t . 
tagawa consulting or advisory role : medivation , astellas pharma , dendreon , janssen oncology , bayer , genentech , endocyte , immunomedics , karyopharm therapeutics , abbvie , tolmar , qed , amgen , sano , pzer research funding : eli lilly ( inst ) , sano ( inst ) , janssen oncology ( inst ) , astellas pharma ( inst ) , progenics ( inst ) , millennium pharmaceuticals ( inst ) , amgen ( inst ) , bristol - myers squibb ( inst ) , dendreon ( inst ) , rexahn pharmaceuticals ( inst ) , bayer ( inst ) , genentech ( inst ) , newlink genetics ( inst ) , inovio pharmaceuticals ( inst ) , astrazeneca ( inst ) , immunomedics ( inst ) , novartis ( inst ) , aveo ( inst ) , boehringer ingelheim ( inst ) , merck ( inst ) , stem centrx ( inst ) , karyopharm therapeutics ( inst ) , abbvie ( inst ) , medivation ( inst ) , endocyte ( inst ) , exelixis ( inst ) , clovis oncology ( inst ) travel , accommodations , expenses : sano , immunomedics , amgen wei song employment : genentech ( i ) , cytokinetics ( i ) honoraria : foundation medicine , loxo consulting or advisory role : foundation medicine , loxo juan miguel mosquera research funding : personal genome diagnostics travel , accommodations , expenses : personal genome diagnostics mark a . 
nature 537 : s63 , 2016 beltran h , eng k , mosquera jm , et al : whole - exome sequencing of metastatic cancer and biomarkers of treatment response . 
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 impact of the spop mutant subtype on the interpretation of clinical parameters in prostate cancer purpose molecular characterization of prostate cancer , including the cancer genome atlas , has revealed distinct subtypes with underlying genomic alterations . 
one of these core subtypes , spop ( speckle - type poz protein ) mutant prostate cancer , has previously only been identifiable via dna sequencing , which has made the impact on prognosis and routinely used risk stratification parameters unclear . methods we have developed a novel gene expression signature , classifier ( subclass predictor based on transcriptional data ) , and decision tree to predict the spop mutant subclass from rna gene expression data and classify common prostate cancer molecular subtypes . 
we then validated and further interrogated the association of prostate cancer molecular subtypes with pathologic and clinical outcomes in retrospective and prospective cohorts of 8 , 158 patients . results the subclass predictor based on transcriptional data model showed high sensitivity and specificity in multiple cohorts across both rna sequencing and microarray gene expression platforms . 
we found that the spop mutant subclass was associated with lower frequency of positive margins , extraprostatic extension , and seminal vesicle invasion at prostatectomy ; however , spop mutant cancers were associated with higher pretreatment serum prostate - specific antigen ( psa )  . 
despite high pretreatment psa , the spop mutant subtype was associated with a favorable prognosis with improved metastasis - free survival , particularly in patients with high - risk preoperative psa levels . conclusion using a novel gene expression model and a decision tree algorithm to define prostate cancer molecular subclasses , we found that the spop mutant subclass is associated with higher preoperative psa , less adverse pathologic features , and favorable prognosis . 
2018 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license : introduction prostate cancer is a clinically and molecularly heterogeneous disease.1 - 4 current risk stratification guidelines , such as those from the national comprehensive cancer network , 5 the american urological association / american society for therapeutic radiology and oncology , 6 and the european association of urologyeuropean society for radiotherapyoncologyinternational society of geriatric oncology7 use clinical and pathologic parameters , including the level of prostate - specific antigen ( psa ) , to guide management decisions for clinically localized disease . 
psa is also used in a number of other clinical scenarios in prostate cancer , including initial diagnosis , monitoring for recurrence after primary therapy , and monitoring disease burden and treatment response for metastatic disease . the emerging next - generation dna and rna sequencing data point toward distinct molecular subclasses of prostate cancer , 2 - 4 , 8 , 9 but their clinical impact remains unclear . 
 genome atlas ( tcga ) study identified seven core prostate cancer subtypes , which were defined by underlying genomic alterations.4 one key molecular subclass , which represents approximately 10% of prostate cancers , harbors recurrent missense mutations in the e3 ubiquitin ligase component spop.2 - 4 , 10 - 13 to date , identification of spop mutations has required mutational analysis using genomic ( dna ) sequencing . 
in contrast , gene expression ( rna ) data are widely available from a variety of prostate cancer cohorts , often with the long follow - up necessary to define prognostic impact.14 - 21 here , we reported the first development and validation of the subclass predictor based on transcriptional data ( scapt ) model , which we used to define key prostate cancer molecular subclasses , including spop mutant cancer , using gene expression data . 
we used the scapt model to predict the molecular subclass from a retrospective cohort that included 1 , 626 patient samples and a prospective cohort that included 6 , 532 samples using genome - wide microarray gene expression data from a clinically available prognostic assay ( decipher ; genomedx biosciences , vancouver , bc , canada ) , and we explored the clinicopathologic and prognostic associations of key molecular subclasses of prostate cancer . methods prostate cancer tumor samples and microarray data we used a total of 8 , 559 radical prostatectomy ( rp ) tumor expression profiles for training , testing , and validation . 
for training and testing , we used rna sequencing expression and dna mutation data from tcga prostate cancer project ( n = 333 ) 4 and the weill cornell medicine ( wcm ) sequencing ( n = 68 ) cohort . 
for validation , expression profiles of retrospective ( n = 1 , 626 ) and prospective ( n = 6 , 532 ) cohorts were derived from the decipher genomics resource information database ( grid ) registry ( clinicaltrials.gov identifier : nct02609269 )  . 
 the retrospective grid cohort was pooled from seven published microarray studies : cleveland clinic , 22 erasmus mc , 23 johns hopkins , 15 memorial sloan kettering cancer center , 1 mayo clinic ( mayo i and mayo ii ) , 20 , 24 and thomas jefferson university.21 the prospective grid cohort was from clinical use of the decipher test . 
dna and rna from the tcga and wcm cohorts were extracted from fresh frozen rp tumor tissue , as previously described.4 rna from the grid cohorts was extracted from routine formalin - fixed , paraffin - embedded rp tumor tissues , amplified , and hybridized to human exon 1.0 st microarrays ( thermo fisher scientific , waltham , ma ) .24 , 25 spop mutant transcriptional signature we developed the spop mutant transcriptional signature , which includes 212 genes differentially expressed between spop mutant and wild - type samples from tcga prostate cancer rna sequencing data4 ( fig 1 )  . 
the overlap between two methods was significant ( p < 2.2 1016 ) , which confirmed similar results when applying these methods . scapt development on the basis of spop mutant transcriptional signature and the support vector machine model to predict tumors in the spop mutant subclass in the absence of dna sequencing datathat is , microarray data setswe developed the scapt model on the basis of the support vector machine ( svm ) model.28 - 30 given a set of training data marked with two categories , svm builds a model that assigns testing data into one category or the other , which makes it a nonprobabilistic binary linear classifier ( fig 2a )  . 
 ( a ) spop mutant transcriptional signature that included 212 differentially expressed genes between spop mutant and wild - type samples from the cancer genome atlas ( tcga ) non - ets fusion rna sequencing ( rna - seq ) data . 
 ( b ) significant enrichment of spop mutant case from 68 weill cornell medicine ( wcm ) prostate cancer samples with spop mutant transcriptional signature on the basis of hierarchical clustering . 
erg , erg - fusion position ; ets : other ets fusion positive ; fdr , false - discovery rate . z - scores of spop mutant signature from tcga cohort . 
 by performing 10 - fold cross - validation on the tcga training data set , we found costcost of constraints violationequal to 0.04 to yield the model with the highest sensitivity and specificity . 
next , using a decision tree and previously developed microarray - based classifiers for the erg - positive and ets - positive subtypes , 25 we classified the remaining cases in each cohort . 
high accuracy and confidence of spop mutant ( spopmut ) subclass prediction on weill cornell medicine ( wcm ) and gene expression omnibus ( geo ) data set by the scapt ( subclass predictor based on transcriptional data ) model . 
data from taylor et al , 1 erho et al , 24 and kelin et al.22 wt , wild type . spopwt spopmut support vectors large margin spop other taylor et al ( n = 150 ) erho et al ( n = 545 ) klein et al ( n = 182 ) prediction : spopmut sensitivity , 89% ; specificity , 95% spopwt spop wild type combined cohort to test the statistical association between spop mutant status and clinical variables , including age , race , preoperative psa , gleason score , lymph node invasion , surgical margin status , extracapsular extension , and seminal vesicle invasion . 
we evaluated the associations between spop mutant status and patient outcomes , including biochemical recurrence , metastasis , and prostate cancerspecific mortality , on the basis of kaplan - meier analysis . 
as spop mutations are mutually exclusive with erg and other ets rearrangements , 13 we excluded prostate cancer samples with ets fusions to define spop mutantspecific gene expression effects ( fig 1a )  . 
among those 212 genes , we found that upregulated genes from the spop mutant subgroup were enriched in transport vesicle membrane and oxidoreductase activity , but that there was no significant enriched function in downregulated genes from the spop mutant subgroup ( data supplement )  . 
in this independent cohort , we found significant enrichment ( p < 1.9103 ) of spop mutant tumors in one subcluster based on unsupervised clustering ( fig 1b and data supplement )  . 
we next used the spop mutant transcriptional signature in a locked svm modelthat is , with fixed parameters on the basis of the training step using the tcga cohort ( see methods ) to generate scores for each sample in the wcm cohort . 
the spop mutant prediction and its impacts on clinical and prognostic outcomes from retrospective ( n = 1 , 626 ) and prospective grid ( n = 6 , 532 ) cohorts . 
 ( a ) the pie chart of predicted molecular subclasses from the retrospective cohort with 1 , 626 samples , on the basis of the scapt ( subclass predictor based on transcriptional data ) model and decision tree . 
 ( c ) the pie chart of predicted molecular subclasses from the prospective grid cohort with 6 , 532 samples , on the basis of the scapt model and decision tree . 
erg , erg - fusion position ; ets , other ets fusion positive ; or , odds ratio ; psa , prostate - specific antigen . the grid microarray expression data , which do not have spop mutation annotation from dna analysis . 
between 9% and 15% of samples in the cohorts were predicted as spop mutant subclass , which is consistent with the known prevalence of spop mutations at the genomic level in previous prostate cancer studies3 , 4 , 10 , 12 ( fig 2b )  . 
overall , these results demonstrated that the scapt model predicted spop mutant subclass on the basis of the transcriptional data with high accuracy and confidence . molecular subtyping of 8 , 158 patients using the scapt and decision tree we applied the scapt model and decision tree to 8 , 158 patients from retrospective and prospective grid cohorts . 
among the retrospective cohort with 1 , 626 rp specimens , we predicted 9% ( range , 2% to 13% ) of samples to be spop mutant subclass ( data supplement )  . 
among the prospective cohort with 6 , 532 rp specimens , we predicted 8% of cases to be spop mutant subclass , 41% as erg positive , 12% as ets positive , 39% as other subtype , and 1% as conflict cases ( fig 3c )  . 
 the percentage of each molecular subclass was consistent with that reported in previous prostate cancer studies , 1 - 4 , 14 , 25 which supports the validity of our approach . spop mutant subclass associated with favorable pathology at rp we used binominal univariable analysis to compare clinical and pathologic characteristics between spop mutant and wild - type subclasses ( figs 3b and 3d )  . 
spop mutant subclass was less likely to harbor adverse pathologic features , such as positive surgical margins , extraprostatic extension , and seminal vesicle invasion , compared with wild - type subclass in both retrospective and prospective cohorts ( figs 3b and 3d )  . 
these data suggest that the spop mutant subclass was associated with more favorable pathologic outcomes at rp , but expresses higher levels of psa and was associated with tumors from older men , whereas the opposite was true in the erg - positive subclass of prostate cancer . consistent association between spop mutation and higher psa in multiple cohorts the inverse association of psa and prognosis in specific prostate cancer subtypes has potential clinical implications . 
to independently validate the association of spop mutant status and higher preoperative psa , we examined multiple distinct rp cohorts ( grid , tcga , taylor , and wcm )  . 
we observed a similar trend of higher preoperative psa in spop mutant cases , and the spop mutant subclass was more enriched in the higher psa subgroups ( psa > 10 ; fig 4a )  . 
on univariable analysis across cohorts , spop mutant status was significantly associated with higher preoperative psa ( fig 4b ) , whereas erg fusion status was significantly associated with lower preoperative psa ( fig 4c )  . spop mutation is associated with favorable clinical outcomes after rp on kaplan - meier analysis , the spop mutant subclass had the highest biochemical - free , metastasis - free , and lowest prostate cancer specific mortality compared with erg - positive , ets - positive , and other subtypes in the retrospective grid cohort ( fig 5b and data supplement )  . 
whereas long - term outcomes were not available for the prospective grid cohort , we evaluated the association with metastasis risk using the decipher score , a validated metric for prostate cancer metastatic potential.22 , 24 , 37 , 38 we found fewer spop mutant tumors in the decipher high - risk score ( > 0.6 ) group compared with the lowand average - risk groups ( data supplement ) , which again is consistent with a favorable prognosis subtype . 
together , these data support that spop mutant prostate cancer had favorable prognosis after rp , despite its association with higher preoperative psa . favorable prognosis in high - risk psa subgroup in the spop mutant subclass pretreatment psa level is a standard component of risk stratification for prostate cancer , with a psa level > 20 ng / ml considered to be high risk by a number of risk assessment methods.39 - 41 consistent with this , higher psa was associated with worse metastasis - free survival and prostate cancerspecific mortality in the retrospective cohorts ( fig 5a ) ; however , spop mutant status had a dramatic effect on prognosis within the subgroup of patients with high psa . 
 among patients with a psa level > 20 ng / ml , spop mutant tumors were associated with better clinical outcomes , which were comparable to the lowest psa subgroup ( psa < 10 ng / ml ; fig 5d )  . 
 ( a ) enrichment of spopmut cases among higher psa subgroups from prospective grid , the cancer genome atlas ( tcga ) , taylor , and weill cornell medicine ( wcm ) cohorts . 
 ( a ) clinical outcome difference between lower , average , and higher psa groups via kaplan - meier analyses for metastasis ( met ) and prostate cancerspecific mortality ( pcsm ) free survival rates . 
 society of geriatric oncology guidelines for clinically localized prostate cancer5 - 7 classify patients with a pretreatment psa level of > 20 as high risk , even in the absence of other adverse prognostic features . 
this results in increased treatment burden , and it is recommended that patients classified as high risk who opt for radiotherapy undergo 2 to 3 years of concurrent androgen - deprivation therapy compared with a duration of 4 to 6 months for patients with intermediate risk disease . 
knowledge of the molecular subtype of prostate cancer and its impact on psa level could therefore improve risk stratification , sparing unnecessary treatment burden and directing higher - intensity therapy to patients who are truly at higher risk ; however , whether molecular subtype will actually add clinical value to the current risk stratification toolsor if it adds additional information compared with such tests as decipherremains unclear . 
additional studies will be necessary to optimally deploy these strategies clinically , but it is clear that molecular subtyping should be considered in future clinical trial designs . recent advances in technology have increased our understanding of molecular subclasses of prostate cancer , but clinical and biologic differences among the key erg - positive , ets - positive , and spop mutant subclasses remain poorly understood . 
biologically , prostate cancer cells that harbor mutant spop may produce more psa on a per - cell basis as a result of enhanced androgen transcription , essentially leading to higher psa from fewer cancer cells , whereas the opposite may be true from erg - positive tumors . 
these hypotheses need to be rigorously tested in future functional and clinical studies . as a result of the retrospective nature of the cohorts and small sample size of case cohort studies , survival analysis was inevitably affected by baseline risk . 
although the favorable prognosis was consistent with improved clinical outcomes in the spop mutant subclass , these survival results need to be independently validated in additional clinical trials to be generalizable . in conclusion , we have developed the scapt model to predict spop mutant subclass purely on the basis of transcriptional data with high confidence and accuracy . 
we believe this work not only builds a prediction model for spop mutant prostate cancers and expands the data types usable for the interrogation of clinical outcomes , but it also reinforces the concept that molecular subtyping of prostate cancer can alter the interpretation of the current standard of care risk stratification methods . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . deli liu no relationship to disclose mandeep takhar employment : genomedx mohammed alshalalfa employment : genomedx nicholas erho employment : genomedx jonathan shoag no relationship to disclose travel , accommodations , expenses : genomedx robert b . 
rubin research funding : eli lilly , janssen pharmaceuticals bruce trock consulting or advisory role : genomedx consulting or advisory role : myriad genetics research funding : myriad genetics , mdxhealth eric a . 
den consulting or advisory role : genomedx speakers ' bureau : bayer research funding : medivation , astellas pharma , genomedx travel , accommodations , expenses : genomedx scott a . 
tomlins leadership : strata oncology stock and other ownership interests : strata oncology consulting or advisory role : abbvie , janssen pharmaceuticals , astellas pharma , medivation , strata oncology , sanofi , almac diagnostics research funding : astellas pharma ( inst ) , medivation ( inst ) , genomedx ( inst ) patents , royalties , other intellectual property : coauthor on a patent issued to the university of michigan on ets gene fusions in prostate cancer travel , accommodations , expenses : strata oncology daniel e . 
spratt no relationship to disclose elai davicioni employment : genomedx leadership : genomedx stock and other ownership interests : genomedx patents , royalties , other intellectual property : cancer diagnostics using biomarkers 20140066323 travel , accommodations , expenses : genomedx andrea sboner no relationship to disclose christopher e . 
barbieri patents , royalties , other intellectual property : coinventor on a patent application filed regarding spop mutations in prostate cancer by weill cornell medicine travel , accommodations , expenses : genomedx acknowledgment ashley e . 
ross stock and other ownership interests : genomedx honoraria : healthtronics consulting or advisory role : healthtronics research funding : metamark genetics we thank the patients with prostate cancer and families who contributed to this research . 
barbieri , newyork - presbyterian hospital , new york , ny ; mandeep takhar , mohammed alshalalfa , nicholas erho , and elai davicioni , genomedx bioscience , vancouver , british columbia , canada ; robert b . 
johnson mh , ross ae , alshalalfa m , et al : spink1 defines a molecular subtype of prostate cancer in men with more rapid progression in an at risk , natural history radical prostatectomy cohort . 
ross ae , johnson mh , yousefi k , et al : tissue - based genomics augments post - prostatectomy risk stratification in a natural history cohort of intermediateand high - risk men . 
nguyen pl , haddad z , ross ae , et al : ability of a genomic classifier to predict metastasis and prostate cancer - specific mortality after radiation or surgery based on needle biopsy specimens . 
spratt de , yousefi k , deheshi s , et al : individual patient - level meta - analysis of the performance of the decipher genomic classifier in high - risk men after prostatectomy to predict development of metastatic disease . 
karnes rj , bergstralh ej , davicioni e , et al : validation of a genomic classifier that predicts metastasis following radical prostatectomy in an at - risk patient population . 
klein ea , yousefi k , haddad z , et al : a genomic classifier improves prediction of metastatic disease within 5 years after surgery in node - negative high - risk prostate cancer patients managed by radical prostatectomy without adjuvant therapy . 
boormans jl , korsten h , ziel - van der made aj , et al : identification of tdrd1 as a direct target gene of erg in primary prostate cancer . 
bennett kp , campbell c : support vector machines : hype or hallelujah ? sigkdd explor 2 : 113 , 2000 technol 2 : 1 - 27 , 2011 30 . 
fine sw , gopalan a , leversha ma , et al : tmprss2 - erg gene fusion is associated with low gleason scores and not with high - grade morphological features . 
zhou ck , young d , yeboah ed , et al : tmprss2 : erg gene fusions in prostate cancer of west african men and a meta - analysis of racial differences . 
schaefer g , mosquera jm , ramoner r , et al : distinct erg rearrangement prevalence in prostate cancer : higher frequency in young age and in low psa prostate cancer . 
font - tello a , juanpere n , de muga s , et al : association of erg and tmprss2 - erg with grade , stage , and prognosis of prostate cancer is dependent on their expression levels . 
marrone m , potosky al , penson d , et al : a 22 gene - expression assay , decipher ( genomedx biosciences ) to predict five - year risk of metastatic prostate cancer in men treated with radical prostatectomy . 
cooperberg mr , pasta dj , elkin ep , et al : the university of california , san francisco cancer of the prostate risk assessment score : a straightforward and reliable preoperative predictor of disease recurrence after radical prostatectomy . 
damico av , whittington r , malkowicz sb , et al : biochemical outcome after radical prostatectomy , external beam radiation therapy , or interstitial radiation therapy for clinically localized prostate cancer . 
 c intratumoral cd3 + and cd8 + t - cell densities in patients with dna mismatch repairdecient metastatic colorectal cancer receiving programmed cell death - 1 blockade sakti chakrabarti , md1 ; lucas j . 
sinicrope , md1 introduction the programmed cell death - 1 ( pd - 1 ) pathway represses type 1 t - helper cell cytotoxic immune responses and is upregulated in many tumors and in their microenvironment . 
blockade of this pathway with antibodies to pd - 1 has led to robust clinical responses in patients with many tumor types , including a subset of metastatic colorectal cancers ( mcrcs ) with decient dna mismatch repair ( dmmr ) .1 the clinical benet of pd - 1 blockade by pembrolizumab in dmmr mcrc was rst reported in a phase ii study in which the objective response rate ( orr ) and immunerelated progression - free survival rate at 20 weeks were 40% ( four of 10 patients ) and 78% ( seven of nine patients ) , respectively.2 expansion of this cohort to 40 patients indicated an orr of 52%.1 among dmmr tumors , mechanisms underlying responsiveness or resistance to pd - 1 blockade remain unknown , 1 and a predictive biomarker has not been identied within this tumor subtype . colorectal cancers ( crcs ) with dmmr have high microsatellite instability ( msi - h ) and are associated with a high mutational burden , neoantigen load , and typically a dense lymphocytic inltrate.3 in a recent report , the densities of cd3 + and cd8 + t cells were found to be signicantly more heterogeneous in dmmr versus procient mismatch repair ( pmmr ) colon cancers and were shown to prognostically stratify patients with both dmmr and pmmr tumors.4 , 5 on the basis of these data , we hypothesized that the responsiveness of dmmr mcrcs to immunotherapy may be related to the extent of inltration . 
tumor response was assessed by radiographic studies using response evaluation criteria in solid tumors ( recist ) version 1.1.6 the study was approved by the institutional review board of mayo clinic . informed consent was obtained from all patients before collection of data and tumor specimens for analysis . tumors were analyzed for mmr protein ( mlh1 , msh2 , msh6 , and pms2 ) expression by immunohistochemistry ( ihc ) or using a polymerase chain reactionbased assay for msi.7 in archival primary colorectal tumor tissues , cd3 + and cd8 + t - lymphocyte staining was performed by ihc ( ventana medical systems , tucson , az ) , and t - cell densities were determined in tumor core ( ct ) and invasive margin ( im ) by automated image analysis ( appendix fig a1 )  . 
analyses of cd3 + and cd8 + density scores and programmed death ligand 1 ( pd - l1 ) expression were performed by investigators blinded to clinical characteristics and patient outcomes . 
density values for cd3 + and cd8 + t cells , calculated by dividing the cell count by the area ( square millimeters ) within ct or im compartments , were used to calculate a density score ( 0 to 100 ) for each marker by compartment ( cd3 + im , cd3 + ct , cd8 + im , and cd8 + ct ) , as previously described.5 pd - l1 protein expression in tumor cells and in tumor - inltrating immune cells was determined by ihc using ventana pd - l1 ( sp263 ) rabbit monoclonal antibody and was graded according to the percentage of tumor cells with membranous pdl1 expression or as the percentage of positive immune cells in the inammatory inltrate.8 patient and tumor characteristics , details of prior treatment , and pembrolizumab response data are listed in table 1 . 
box plots illustrating the distribution of cd3 + and cd8 + t - cell density ( score range , 0 to 100 ) at the tumor core ( ct ) and the invasive margin ( im ) among the responders and nonresponders . 
responders are dened as patients with complete responses ( crs ) and partial responses ( prs ) ; nonresponders are dened as those with stable disease ( sd ) or progressive disease ( pd )  . regimens received was one ( range , one to four regimens ; one regimen in seven patients , two regimens in three patients , and four regimens in two patients )  . 
median follow - up time of the study cohort since initiation of pembrolizumab was 19.5 months ( range , 9 to 41 months ) in all patients , 21 months ( range , 16 to 24 months ) in nonresponders , and 16 months ( range , 9 to 41 months ) in responders . patient radiographic response data were as follows : two complete responses ( crs ) , ve partial responses ( prs ) , four patients with stable disease , and one patient with progressive disease . 
among pembrolizumab - treated patients who had a cr or pr ( n = 7 ) , the median time to response was 12 weeks ( range , 6 to 40 weeks )  . to examine the relationship between t - cell density scores and treatment response , patients were divided into responders ( cr and pr , n = 7 ) and nonresponders ( stable disease and progressive disease , n = 5 ) to pembrolizumab . median values and ranges of cd3 + and cd8 + t - cell density scores at the ct ( cd3 + ct and cd8 + ct ) and im ( cd3 + im and cd8 + im ) in each response category are provided in figure 1 and table 2 . 
all median t - cell density scores were higher in responders than in nonresponders , with score for cd3 + ct ( 74 v 52 , differences that were greatest respectively ) and cd8 + ct ( 88 v 37 , respectively ) , as shown in figure 1 . 
all median t - cell density scores were higher in the patient group in whom disease control was achieved for more than 12 months ( n = 8 ) , with the greatest score difference seen for cd8 + ct ( 88 v 36 for patients with disease control for more than 12 months v less than 12 months , respectively )  . median tumor cell pd - l1 expression was 2% ( range , 1% to 40% ) among responders and 1% ( range , 0% to 60% ) among nonresponders . 
median pd - l1 expression in intratumoral immune cells was 5% for both responders ( range , 2% to 25% ) and nonresponders ( range , 1% to 10% )  . 
of the ve nonresponding patients , one patients tumor underwent testing for a jak2 mutation , which was not detected . discussion the ability to distinguish high versus low immunogenic tumors could enable selection of patients who are more likely to benet from immunotherapy . 
we used an automated analysis of cd3 + and cd8 + tumor - inltrating lymphocyte density quantitation that was initially reported in stage ii colon cancers and found to be signicantly prognostic independent of mmr status.9 more recently , we validated this platform in nearly 600 stage iii colon cancers from a clinical trial of folfox - based adjuvant chemotherapy where cd3 + and cd8 + t - cell densities were shown to prognostically stratify patients with both dmmr and pmmr colon cancers.5 we found that both cd3 + and cd8 + t - cell density scores in ct and im were higher in patients with dmmr tumors who beneted from pembrolizumab , both in terms of objective response rate and duration of disease control . despite variability observed in our small patient cohort , median density scores of t - cell markers were higher in responders versus nonresponders and in those in whom disease control was achieved for more than versus less than 12 months . 
conversely , these response data suggest that lower t - cell densities are associated with immunotherapy resistance in dmmr tumors and are consistent with the reported nding that lower t - cell densities are associated with worse survival in patients with dmmr stage iii colon cancers receiving cytotoxic chemotherapy.5 our data are consistent with the ndings that increased intratumoral cd8 + t cells can predict response to immunotherapy in patients with metastatic melanoma10 and in patients with head and neck cancers.11 median follow - up of 21 months among nonresponders exceeded the median follow - up time of 12.5 months that was reported in the largest published series of patients with dmmr mcrcs treated with pd - 1 blockade.1 in that study , the mean time to the best radiographic response was 7 months , 1 suggesting that our study follow - up time was of sufcient duration to detect response outcomes . studies have shown higher expression of tumor pd - l1 protein in dmmr versus pmmr crcs , 3 and our analysis of the cancer genome atlas database revealed higher pd - l1 mrna levels in dmmr versus pmmr primary colon cancers ( unpublished data )  . 
in the current study of patients with dmmr and msi - h crcs , we found similar median levels of pd - l1 expression on tumor cells and on inltrating immune cells in responders versus nonresponders . studies of crc , expression of pd - l1 has not been shown to be a predictive biomarker , in contrast to data in patients with nonsmall - cell lung cancer . 
furthermore , a phase ii study of patients with dmmr mcrcs treated with pembrolizumab found that the expression of pd - l1 was not signicantly associated with progression - free survival or overall survival.2 insight into responses to antipd - 1 blockade in dmmr colon cancers is suggested by functional analysis in a responding patient in which rapid in vivo expansion of neoantigen - specic t - cell clones that were reactive to mutant neopeptides were found in the tumor.1 immune inltration may reect the tumors clonal evolution during treatment . 
potential causes of immunotherapy resistance include 2 - microglobulin and jak1 / 2 mutations , 12 and in our study , only one of ve nonresponders was tested for a jak2 mutation , which was not detected . 
other potential biomarkers in dmmr tumors such as the elimination of neoepitopes and differences in t - cell clonal diversity and tumor microenvironment proles warrant additional study . it is important to recognize that dmmr and msi - h are predictive biomarkers for responsiveness to immune checkpoint inhibitors . 
tumor mutation burden ( tmb ) may be a promising biomarker and has been studied in relationship to a t - cellinamed gene expression prole across 22 tumor types.13 although tmb and t - cell gene expression signature were poorly correlated , their combination jointly predicted responders to pembrolizumab , suggesting that features of neoantigenicity and t - cell activation.13 using tmb as a biomarker for immunotherapy responsiveness has important limitations in that not all mutations generate neoantigens , the correlation of tmb with neoantigen load is variable , and an established cutoff point remains unresolved.14 they may capture distinct in summary , we observed that higher cd3 + and cd8 + t - cell densities were associated with higher objective response rates and duration of disease control in patients with dmmr mcrc treated with pembrolizumab . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . andrea muranyi employment : roche stock and other ownership interests : roche patents , royalties , other intellectual property : roche travel , accommodations , expenses : roche june clements employment : ventana medical systems stock and other ownership interests : ventana medical systems research funding : ventana medical systems shalini singh employment : ventana medical systems stock and other ownership interests : ventana medical systems patents , royalties , other intellectual property : method of identifying treatment - responsive nonsmall - cell lung cancer using alk as a marker ( inst ) joleen m . 
hubbard consulting or advisory role : bayer ( inst ) research funding : boston biomedical ( inst ) , senhwa biosciences ( inst ) , bayer ( inst ) , merck ( inst ) , taiho pharmaceutical ( inst ) , treosbio ( inst ) , effector therapeutics ( inst ) robert r . 
mcwilliams consulting or advisory role : merrimack ( inst ) , zeno pharmaceuticals , newlink genetics ( inst ) research funding : prism biolab ( inst ) , genentech ( inst ) , novartis ( inst ) , newlink genetics ( inst ) , eli lilly ( inst ) , aduro biotech ( inst ) , pzer ( inst ) , sano ( inst ) , merck ( inst ) , bristol - myers squibb ( inst ) kandavel shanmugam employment : ventana medical systems stock and other ownership interests : roche patents , royalties , other intellectual property : patents ( inst ) travel , accommodations , expenses : ventana medical systems frank a . 
sinicrope stock and other ownership interests : illumina honoraria : imedex , american society of clinical oncology consulting or advisory role : roche ( inst ) , bristol - myers squibb ( inst ) , ventana medical systems ( inst ) , haliodx ( inst ) research funding : ventana medical systems ( inst ) travel , accommodations , expenses : ventana medical systems no other potential conicts of interest were reported . references le dt , durham jn , smith kn , et al : mismatch repair deciency predicts response of solid tumors to pd - 1 blockade . 
science 357 : 409 - 413 , 2017 le dt , uram jn , wang h , et al : pd - 1 blockade in tumors with mismatch - repair deciency . 
n engl j med 372 : 2509 - 2520 , 2015 llosa nj , cruise m , tam a , et al : the vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter - inhibitory checkpoints . 
cancer discov 5 : 43 - 51 , 2015 pag `es f , mlecnik b , marliot f , et al : international validation of the consensus immunoscore for the classication of colon cancer : a prognostic and accuracy study . 
lancet 391 : 2128 - 2139 , 2018 yoon hh , shi q , heying en , et al : intertumoral heterogeneity of cd3 + and cd8 + t - cell densities in the microenvironment of dna mismatch - repair - decient colon cancers : implications for prognosis . 
eur j cancer 45 : 228 - 247 , 2009 sinicrope fa , mahoney mr , smyrk tc , et al : prognostic impact of decient dna mismatch repair in patients with stage iii colon cancer from a randomized trial of folfox - based adjuvant chemotherapy . 
j clin oncol 31 : 3664 - 3672 , 2013 el jabbour t , ross js , sheehan ce , et al : pd - l1 protein expression in tumour cells and immune cells in mismatch repair protein - decient and - procient colorectal cancer : the foundation study using the sp142 antibody and whole section immunohistochemistry . 
j clin pathol 71 : 46 - 51 , 2018 gibbs ph , hutchinson r , tran b , et al : immune prole and survival outcomes in stage 2 colon cancer . 
partlov a s , bou cek j , kloudov a k , et al : distinct patterns of intratumoral immune cell inltrates in patients with hpv - associated compared to non - virally induced head and neck squamous cell carcinoma . 
 ( a ) hematoxylin and eosin ( he ) stained whole - tissue sections were manually annotated to outline the entire tumor region ( red line ) and demarcate the invasive margin ( orange line )  . 
 ( b and c ) annotations were transferred onto adjacent cd3 + and cd8 + ihc images with an algorithm outlining the invasive margin ( im ; green line ) and core of the tumor ( ct ; red line )  . 
the cfdna next - generation sequencing ( ngs ) analysis was performed at guardant health ( guardant360 ; redwood city , ca ) , a clinical laboratory improvement amendmentscertified , college of american pathologistsaccredited , new york state department of healthapproved laboratory . 
the exons of 73 cancer genes were captured using biotinylated custom bait oligonucleotides ( agilent , santa clara , ca ) , resulting in a capture footprint of 148 , 000 base pairs ( 78 kb )  . 
he received adjuvant androgen deprivation therapy and radiation therapy ( 70 gy in 35 fractions ) and developed biochemical recurrence 1 year later , when he was treated with bicalutamide . 
germline testing confirmed a heterozygous brca2 c.5946delt mutation in the patient , which was inherited from his father , who had died as a result of colon cancer at age 81 years . after liver biopsy , the patient was treated with olaparib ( 400 mg twice per day ) , resulting in rapid reduction of psa from 821 to 300 ng / ml and improvement of lymphadenopathy and liver lesions . 
 in addition to multiple brca2 mutations , ctdna analyses revealed the following alterations : tp53 f113fs ; gata3 d336d ; arid1a s1755t ; myc p72a ; and amplification of myc , kras , ccnd2 , and braf . because the biallelic reversion of both germline and truncal somatic brca1 / 2 alterations contrasted the generally accepted model of monoallelic reversion of germline brca1 / 2 mutations , we attempted to estimate the relative prevalence of germline versus somatic brca1 / 2 reversion events in patients with mcrpc using a large genomic database including comprehensive ctdna results from more than 40 , 000 patients with a variety of solid tumors . 
a third case of reversion was identified , but the patient had no previous exposure to platinum - based chemotherapy or parp inhibitors . discussion we report a case of acquired resistance to olaparib in brca2 germlinepositive mcrpc resulting from multiple acquired brca2 reversion mutations of both the germline mutation and a second - hit somatic mutation on the opposite allele . 
this case is similar to one recently reported by goodall et al , 22 in which acquired reversion mutations restored the open reading frame of not only the primary germline mutation but also the secondary loss - of - function mutation . 
 although previous studies in ovarian cancer have established that reversion of the germline allele is necessary and sufficient to restore normal brca protein function , this case suggests functional comparability of the variants despite their origin ( ie , somatic or germline )  . 
this observation challenges the established model of brca1 / 2 reversion as restricted to germline mutations and suggests that the germline or somatic origin of the allele may not play a critical biologic role in this mechanism of resistance . furthermore , this case is a powerful illustration of convergent evolution of multiple brca2 reversion mutations arising in different clones of the metastatic lesion or within multiple metastases ( fig 2 ) , as has been described.23 other studies of acquired resistance have compared ctdna with tissue - based testing on biopsies from multiple metastatic lesions in the same patient . 
these studies have shown that a single tissue biopsy often does not capture the full spectrum of acquired resistance mutations , whereas ctdna may provide a more global summary of tumor heterogeneity , as seen in this case.24 , 25 ctdna analyses also enable monitoring and early detection of mutations driving treatment resistance to parp inhibitors , with meaningful clinical implications . once a brca1 / 2 mutation is detected , longitudinal monitoring with ctdna can be relevant for early detection of reversion brca1 / 2 mutations to predict resistance to parp inhibitors , as illustrated by the case presented here . 
in women with platinum - resistant ovarian cancer , presence of brca reversion mutations was a more accurate predictor of response to subsequent platinum or parp inhibitor therapy than duration of response to previous lines of platinum therapy.26 another study identified reversion of germline brca1 / 2 mutations in high - grade serous ovarian carcinoma using ctdna and was able to predict treatment responses.27 there are limited data on the prevalence of brca reversion mutations and rates of resistance to platinum or parp inhibitors in mcrpc . 
circulating tumor dna profiling of a patient experiencing disease progression during treatment with a poly ( adp - ribose ) polymerase inhibitor , showing a known germline frameshift mutation and somatic second - hit frameshift mutation , as well as 11 additional frameshift mutations . 
phe2000fs time platinum - resistant ovarian cancer range from 25% to 70% , but these are based on small series.18 , 26 analysis of genomic data from large databases may be one way to overcome this limitation . 
although genomic testing on tissue before parp inhibitor therapy was performed for the index case , this information was not available for the additional patient cases showing evidence of reversion mutation . 
all patients underwent cfdna analysis at the time of clinical progression , suggesting that they had developed platinum or parp inhibitor resistance , but the duration of their response during therapy or presence of reversion mutations before exposure is unknown . 
 with regard to the retrospective cohort analysis , our brca2 germline mutation rate was lower than that previously described in the literature.28 possible explanations for this include exclusion of putative germline missense and nonsense mutations in the analysis and overly restrictive germline maf thresholds resulting in exclusion of putative germline mutations in patients with more severe allele imbalance . 
lastly , one patient case with evidence of a reversion mutation had no prior exposure to parp inhibitors or platinu review of the patients treatment history revealed treatment with taxane - based chemotherapy , radium - 223 , and mitoxantrone . 
at the time of relapse , a bone marrow biopsy was performed , and a patient - derived cell line showed loss of the fanconi anemia phenotype because of monoallelic reversion of the brca2 mutation and restoration of wild - type brca2 function . 
the authors suggest that dna intercalating agents such as mitoxantrone may have the ability to induce reversion mutations and lead to resistance . compared with biopsy , cfdna analyses allow easier monitoring and potentially earlier detection of mutations that result in treatment resistance . 
cfdna analysis , which allows detection of both somatic and germline mutations in a single test , is well suited to distinguish whether a somatic brca mutation represents a second - hit loss of function or a reversion of the germline brca mutation . 
giles manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors sahithi pamarthy no relationship to disclose vinay sagar no relationship to disclose stephen fairclough employment : guardant health stock and other ownership interests : guardant health research funding : guardant health patents , royalties , other intellectual property : guardant health travel , accommodations , expenses : guardant health justin odegaard employment : guardant health stock and other ownership interests : guardant health richard b . 
 alice fan stock and other ownership interests : molecular decisions consulting or advisory role : verily research funding : calithera biosciences patents , royalties , other intellectual property : stanford patent has been licensed young kwang chae consulting or advisory role : foundation medicine , boehringer ingelheim , biodesix , counsyl , astrazeneca , guardant health , speakers bureau : merck , genentech / roche travel , accommodations , expenses : hanmi massimo cristofanilli honoraria : domp farmaceutici , pfizer consulting or advisory role : domp farmaceutici , newomics , vortex biosciences maha h . 
hussain honoraria : onclive , sanofi research funding : genentech ( inst ) , pfizer ( inst ) , pcctc ( inst ) , astrazeneca ( inst ) patents , royalties , other intellectual property : title : systems and methods for tissue imaging , 3676 our file , serial no . 
giles employment : actuate leadership : actuate honoraria : novartis consulting or advisory role : novartis travel , accommodations , expenses : medimmune , novartis , foundation medicine affiliations benedito a . 
cleton - jansen am , collins n , lakhani sr , et al : loss of heterozygosity in sporadic breast tumours at the brca2 locus on chromosome 13q12 - q13 . 
collins n , mcmanus r , wooster r , et al : consistent loss of the wild type allele in breast cancers from a family linked to the brca2 gene on chromosome 13q12 - 13 . 
tutt an , lord cj , mccabe n , et al : exploiting the dna repair defect in brca mutant cells in the design of new therapeutic strategies for cancer . 
bouwman p , jonkers j : molecular pathways : how can brca - mutated tumors become resistant to parp inhibitors ? clin cancer res 20 : 540 - 547 , 2014 13 . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
epstein ji , egevad l , amin mb , et al : the 2014 international society of urological pathology ( isup ) consensus conference on gleason grading of prostatic carcinoma : definition of grading patterns and proposal for a new grading systeam j surg pathol 40 : 244 - 252 , 2016 22 . 
quigley d , alumkal jj , wyatt aw , et al : analysis of circulating cell - free dna identifies multiclonal heterogeneity of brca2 reversion mutations associated with resistance to parp inhibitors . 
de mattos - arruda l , weigelt b , cortes j , et al : capturing intra - tumor genetic heterogeneity by de novo mutation profiling of circulating cell - free tumor dna : a proof - of - principle . 
christie el , fereday s , doig k , et al : reversion of brca1 / 2 germline mutations detected in circulating tumor dna from patients with high - grade serous ovarian cancer . 
ikeda h , matsushita m , waisfisz q , et al : genetic reversion in an acute myelogenous leukemia cell line from a fanconi anemia patient with biallelic mutations in brca2 . 
 long - term complete response of an androgen receptorpositive triple - negative metastatic breast cancer to abiraterone acetate introduction several gene expression array studies have identified a new breast cancer subtype characterized by the expression of the androgen receptor ( ar ) , absence of the estrogen receptor ( er ) , and paradoxical expression of many genes that are typically expressed in er - positive luminal tumors.1 - 3 two thirds of this tumor subtype are human epidermal growth factor receptor 2 ( her2 ) positive . 
these ar - positive and triple - negative tumors have been identified as a stable subtype in two series of tnbcs.4 , 5 of note , ar is also expressed in approximately 75% of er - positive / her2 - negative breast cancers.6 however , clinical and preclinical data suggest that ars act as a tumor suppressor gene in er - positive tumors and as an oncogene in er - negative tumors.7 in both retrospective series8 and prospective clinical trials , 9 - 11 the following immunohistochemistry ( ihc ) definition is classically used to identify this breast cancer subtype : ar positive and er , progesterone receptor ( pr ) negative , and her2 negative . 
there is no universal consensus on defining ar positivity to date , although the accepted definition of ar positivity is 10% . preclinical in vitro2 , 12 - 14 and in vivo14 , 15 data suggest that androgens are involved in the development and growth of the mda - mb - 453 cell line , which is used as an in vitro model for the ar - positive tnbc subtype.2 consequently , in three prospective clinical trials , either competitive antagonists of the ar ( bicalutamide or enzalutamide ) or androgen synthesis suppressor , cyp17 inhibitor ( abiraterone acetate , aa ) , were evaluated in ar - positive tnbcs . 
 she received neoadjuvant chemotherapy ( three cycles of fluorouracil , epirubicin , and cyclophosphamide followed by three cycles of docetaxel ) followed by mastectomy and axillary lymph node dissection ( level 1 and 2 )  . 
 the arrows show the index masses with changes with therapies . multiple sclerosis and administered interferon at a dose of 30 g per week . in november 2007 , the patient was diagnosed with an ipsilateral axillary lymph node relapse without distant metastasis . 
no further treatment was proposed . in july 2013 , she presented with a 4 - cm left axillary mass with skin involvement ( fig 1a ) and two supraclavicular 1 - cm lymph nodes ; a core biopsy of the axillary mass confirmed tnbc , with a proliferation index ( ki - 67 ) of 15% . 
on the computed tomography scan ( figs 1b and 1c ) , in addition to the axillary mass , several chest soft tissue masses were observed ; no visceral or bone metastasis was observed . 
the patient was included in the ucbg 2012 - 1 trial and received experimental treatment with 1 , 000 mg of aa and 10 mg of prednisone administered orally , daily . 
ten months after the treatment initiation , clinical and computed tomography scan evaluations showed that the patient achieved a cr ( on the basis of the response evaluation criteria in solid tumors criteria )  . 
after 4 years , the patient is still receiving the treatment with aa and low - dose prednisone , with a prolonged cr ( figs 1d - 1f )  . 
concomitantly , she has been receiving interferon treatment for multiple sclerosis . further ihc analyses later performed on the 2006 mastectomy and the 2007 axillary relapse showed that 100% of cells were ar positive . 
 furthermore , ihc analyses on additional markers in the 2013 axillary relapse confirmed a pronounced apocrine feature with marked forkhead box a1 and gross cystic disease fluid protein 15 expression . 
ongoing clinical trials on ar - positive triple negative breast cancer in metastatic setting single agent combination antiandrogen ar antagonist enzalutamide ( phase iii , clinicaltrials.gov identifier : nct02929576 , withdrawn ) bicalutamide v chemotherapy ( phase iii / first line , clinicaltrials . gov identifier : nct03055312 , recruiting ) darolutamide v chemotherapy ( randomized phase ii , first and second line , clinicaltrials.gov identifier : nct03383679 , recruiting ) bicalutamide plus cdk4 / 6 inhibitor ribociclib ( phase i / ii , clinicaltrials.gov identifier : nct03090165 , recruiting ) bicalutamide plus cdk4 / 6 inhibitor palbociclib ( phase i / ii , clinicaltrials.gov identifier : nct02605486 , recruiting ) enzalutamide plus pi3k inhibitor alpelisib ( phase i , clinicaltrials.gov identifier : nct03207529 , not yet recruiting ) or plus pi3k inhibitor taselisib ( phase i / ii , clinicaltrials.gov identifier : nct02457910 , recruiting ) cyp17a1 inhibitors orteronel ( tak - 700 ; phase ii , clinicaltrials.gov identifier : nct01990209 , recruiting ) seviteronel ( phase i / ii , clinicaltrials.gov identifier : nct02580448 , recruiting ) selective ar modulator cr1447 ( phase ii , clinicaltrials.gov identifier : nct02067741 , recruiting ) enobosarm plus pembrolizumab ( phase ii , clinicaltrials.gov identifier : nct02971761 , recruiting ) enobosarm ( phase ii , clinicaltrials.gov identifier : nct02368691 , terminated ) abbreviations : ar , androgen receptor ; cdk , cyclin - dependent kinase ; her2 , human epidermal growth factor receptor 2 ; pi3k , phosphatidylinositol 3 - kinase . from a clinicians perspective , the cbrs at 6 months ( 19% to 29% ) observed in this breast cancer subtype in the three prospective trials testing ar antagonist or androgen synthesis inhibitor are modest but clinically relevant . 
patients with ar - positive tnbcs seem to be older compared with the whole tnbc population.9 - 11 , 17 they also tend to relapse after a long interval after the primary tumor treatment . 
in our opinion , in ar - positive tnbcs , treatment choice between antiandrogens and chemotherapy could take into account different factors , including disease - free interval , tumor burden , and the need for rapid disease / symptom control , similar to luminal erand / or pr - positive tumors . 
of note , it is important to remember that progression - free survival for first - line chemotherapy is merely 4 to 6 months.18 , 19 from a researchers perspective , cbrs at 6 months with antiandrogens in this tumor subtype are much lower than those observed with anti - aromatase inhibitors used as single agents in luminal erand / or pr - positive cancers . 
in the latter group , the historical cbrs range from 49% to 59% for first - line and from 24% to 37% for second - line treatments , respectively.20 hence , there is a need to improve these results in ar - positive tnbcs , and we briefly outline four key areas that could be improved . first , ihc is probably not ideal to identify this breast cancer subtype . 
of note , in the enzalutamide trial , 10 patients with low ar ( 1% to 9% ) were included in an exploratory cohort , and some of these patients responded . 
in two large series of tnbcs analyzed by gene expression array , 11% and 17% of tumors were ar positive.4 , 5 in a tnbc series analyzed by ihc , 22% to 35% of patients were ar positive.8 it is difficult to explain this difference . 
hence , there is a need to better identify this subtype in clinical practice , for example , by using rna signatures with a selected number of transcripts.5 second , predictors of antiandrogen treatment sensitivity in this breast cancer subtype should be developed . 
new preclinical models are needed ; the reliability of the mda - mb - 453 cell line has been in question because of genomic alterations , such as ar mutation.22 , 23 this ar q865h mutation in its ligand - binding domain is reported to decrease sensitivity to dihydrotestosterone , limiting its use to test antiandrogen therapy.22 a new patient - derived xenograft model of molecular apocrine tumors , directly derived from patients , could be a solution.24 finally , we need better drugs and combinations . 
 however , both enzalutamide and abiraterone have shown to improve the sensitivity of tnbc cell lines toward immune - mediated lysis , providing potential rationale for combination of immunotherapy with antiandrogen therapy.25 in conclusion , we believe that this patients case is important for clinicians . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center  . thomas grellety consulting or advisory role : novartis herv bonnefoi consulting or advisory role : abbvie , astellas pharma , bayer healthcare pharmaceuticals , innocrin pharmaceuticals , puma biotechnology research funding : bayer ( inst ) travel , accommodations , expenses : roche , pfizer acknowledgment we thank ravi nookala , phd , of institut bergoni for the medical writing service . affiliations thomas grellety and herv bonnefoi , institut bergoni unicancer , universit bordeaux , institut national de la sant et de la recherche mdicale u1218 , institut national de la sant et de la recherche mdicale cic1401 ; and thomas grellety , gaetan macgrogan , camille chakiba , michele kind , and herv bonnefoi , institut bergoni , bordeaux , france . support supported by the fondation pour la lutte contre le cancer pour les recherches medico - biologiques , site de recherche intgre sur le cancer - bordeaux recherche intgre oncologie , and groupement interrgional de recherche clinique et dinnovation sud - ouest outre - mer grant no . 
vera - badillo fe , templeton aj , de gouveia p , et al : androgen receptor expression and outcomes in early breast cancer : a systematic review and meta - analysis . 
hickey te , robinson jl , carroll js , et al : minireview : the androgen receptor in breast tissues : growth inhibitor , tumor suppressor , oncogene ? mol endocrinol 26 : 1252 - 1267 , 2012 8 . 
gucalp a , tolaney s , isakoff sj , et al : phase ii trial of bicalutamide in patients with androgen receptor - positive , estrogen receptor - negative metastatic breast cancer . 
traina ta , miller k , yardley da , et al : results from a phase 2 study of enzalutamide ( enza ) , an androgen receptor ( ar ) inhibitor , in advanced ar + triple - negative breast cancer . 
bonnefoi h , grellety t , tredan o , et al : a phase ii trial of abiraterone acetate plus prednisone in patients with triple - negative androgen receptor positive locally advanced or metastatic breast cancer ( ucbg 12 - 1 )  . 
lehmann bd , bauer ja , schafer jm , et al : pik3ca mutations in androgen receptor - positive triple negative breast cancer confer sensitivity to the combination of pi3k and androgen receptor inhibitors . 
arce - salinas c , riesco - martinez mc , hanna w , et al : complete response of metastatic androgen receptor - positive breast cancer to bicalutamide : case report and review of the literature . 
oshaughnessy j , schwartzberg l , danso ma , et al : phase iii study of iniparib plus gemcitabine and carboplatin versus gemcitabine and carboplatin in patients with metastatic triple - negative breast cancer . 
miles dw , diras v , corts j , et al : first - line bevacizumab in combination with chemotherapy for her2 - negative metastatic breast cancer : pooled and subgroup analyses of data from 2447 patients . 
moore nl , buchanan g , harris jm , et al : an androgen receptor mutation in the mda - mb - 453 cell line model of molecular apocrine breast cancer compromises receptor activity . 
vranic s , gatalica z , wang zy : update on the molecular profile of the mda - mb - 453 cell line as a model for apocrine breast carcinoma studies . 
lumbar spine magnetic resonance imaging ( mri ) demonstrated multiple bone lesions , and computed tomography scans revealed a 4 - cm right - sided hilar lung mass , regional thoracic lymphadenopathy , multiple hepatic metastases , a left - sided adrenal metastasis , and several osseous lesions ( fig 1a )  . 
biopsies of the subcarinal lymph node and the left - side adrenal lesion were performed , which confirmed adenocarcinoma of lung origin ( figs 2a and 2b )  . the patient was treated with radiation therapy to the painful vertebral metastasis and stereotactic radiosurgery to two brain lesions . 
200 genes ( foundationone ; foundation medicine , cambridge , ma ) revealed an egfr exon 19 deletion ( del19 ) mutation , a tp53 v173l mutation , an egfr amplification , and an rb1 loss of exons 18 and 19 . oral erlotinib 150 mg daily was initiated in may 2015 , and uniform disease response was evident on restaging scans in july 2015 ( fig 1b )  . 
however , in october 2015 , repeat imaging showed significant growth in a single liver lesion and a new 2.6 - cm lesion in the spleen , with continued response elsewhere , including the brain ( fig 1c )  . repeat biopsy of the enlarging liver lesion in november 2015 revealed nests of highly atypical cells with finely dispersed chromatin , inconspicuous nucleoli , and abundant mitoses ( figs 2c to 2e )  . immunohistochemical stains were positive for synaptophysin and chromogranin and negative for thyroid transcription factor 1 and napsin a . 
the overall features were consistent with small - cell lung cancer ( sclc ) transformation.4 ngs that consisted of targeted hotspot evaluation in 39 genes ( snapshot ngs ; massachusetts general hospital , boston , ma ) confirmed the presence of the original egfr del19 and tp53 mutations and showed additional mutations in pik3ca ( g545l ) , pik3ca ( g726l ) , erbb3 ( g337a ) , and fbxw7 ( l8p )  . 
a patientderived xenograft generated from this biopsy specimen lacked rb protein expression but retained minimal egfr expression and demonstrated activation of the mitogen - activated protein kinase and akt pathways ( fig 3c ) , likely as a result of the presence of an activating pik3ca mutation . the patient was treated with carboplatin and etoposide chemotherapy and ongoing erlotinib . 
scans after four cycles of chemotherapy showed a mixed response with slight regression in the previously biopsied ( sclc - transformed ) liver metastasis , stable brain metastases , and multiple new distinct sites of hepatic progression ( fig 1d )  . 
 ( a to e ) selected radiographic images of the liver illustrate involvement with cancer . treatments and key biopsy ( bx ) results ( tissue or liquid ) are indicated underneath each image in chronologic order . 
of note , the guardant360 assay can detect rb1 inactivating mutations but not allelic losses . we interpreted the emergence of an egfr t790m positive clone as the most likely resistance mechanism within the growing liver nodules . 
the patient discontinued chemotherapy , and the t790m - specific tki osimertinib7 was administered in march 2016 . restaging in june 2016 ( performed with mri because of renal dysfunction ) revealed that the hepatic lesions that had most recently progressed on chemotherapy ( presumed t790m positive ) had stabilized , but the liver lesion biopsied in 2015 ( sclc histology at that time ) had again enlarged with no other sites of systemic or intracranial progression ( fig 1e )  . 
a repeat plasma guardant360 test in june 2016 confirmed that egfr t790m was now undetectable , but increases were found in the mafs of pik3ca e726k ( 50.9% ) , pik3ca e545k ( 54.3% ) , tp53 v173l ( 54.8% ) , and egfr del19 ( 45.5% ; fig 4 )  . 
 ( a ) hematoxylin and eosin ( h&e ) stain and ( b ) thyroid transcription factor 1 ( ttf - 1 ) immunostain of fine - needle aspiration of a subcarinal lymph node at diagnosis show adenocarcinoma with diffuse ttf - 1 expression consistent with a lung primary . 
 ( c ) h&e stain and ( d ) synaptophysin ( syn ) and ( e ) ttf - 1 immunostains of a liver core biopsy at the time of acquired resistance to erlotinib demonstrate small - cell lung cancer with solid nests of highly atypical epithelial cells with finely dispersed chromatin , inconspicuous nucleoli , and brisk mitotic activity . 
 ( a ) summary of all evaluable probes across all chromosomes ( red , genomic gains ; blue , genomic losses ) shows diffuse losses across chromosome 13 , including the rb1 gene locus . 
 ( b ) a magnified view of four specific genes on chromosome 13 shows that all examined loci of rb1 are lost , with only blue signals and complete absence of red signals . 
 ( c ) tissue obtained from the november 2015 liver biopsy ( which shows sclc ) wasusedtogenerateapatient - derivedxenograft ( pdx ) inannsgmouseandsubsequentlypassagedthroughadditionalnsgmice.thepdxtumordemonstrated sclc histologic features consistent with the patient biopsy sample ( not shown )  . 
a western blot demonstrates relative protein levels of egfr , p - akt ( s473 ) , p - erk , rb , p16 , and actin ( loading control ) among pdx tumors ( mgh1529 ; two tumors shown ) with control lung adenocarcinoma ( adenoca ) and sclc samples for comparison . genetic characteristics of the various cell lines ( rb1 , p16 , and egfr exon 19 deletion [ del19 ] mutations [ mut ] ) are shown above the western blot . 
however , one resistant clone with sclc morphology emerged clinically in october 2015 , and genotyping confirmed an additional private pik3ca e545l mutation , which often is seen in sclc - transformed egfr mutant clones.4 during chemotherapy , we believe that the sclc clone diminished , whereas another clone that harbored an acquired egfr t790m mutation and perhaps two other distinct pik3ca mutations ( e545k and e726k ) emerged as observed in plasma ctdna in march 2016 . although a tissue biopsy specimen could not be obtained at that time , on the basis of our prior observation that sclc and adenocarcinoma populations can oscillate in response to specific treatment4 and that t790m is rarely seen in sclc - transformed tissue biopsy specimens , we hypothesize that the t790m - positive clone detected in plasma maintained an adenocarcinoma phenotype . 
the t790m clone was no longer detectable by june 2016 after treatment with osimertinib , but one or more other clones became dominant with increasing mafs , and subsequently , the disease became refractory to therapy ( fig 4 )  . 
this elevation in ctdna and subsequent clinical decline mirror data that demonstrated the correlation of increased mafs and decreased overall survival.9 - 11 in addition , the patient had a tumor with baseline rb1 mutation that was expected to lead to loss of function and is believed to play an essential role in the histologic transformation to sclc among egfr mutant cancers . 
we previously demonstrated that rb1 is universally lost in sclc - transformed cancers , although not sufficiently for transformation.12 recent work has demonstrated that baseline rb1 loss among egfr mutant tumors is a strong predictor for subsequent sclc transformation.13 as the clinical use of ngs panels increases and baseline inactivating rb1 mutations are more frequently detected , more data to understand the implications will be required , including a better understanding of the critical steps that lead to the lineage shift , so that we can develop more - effective treatment strategies.14 finally , this case report illustrates the potential utility of longitudinal molecular profiling during targeted therapy . 
at present , two mutationspecific food and drug administrationapproved plasma tests may be used to select egfr tkis.15 , 16 clinical practice is rapidly evolving , but on the basis of current evidence , it is reasonable to offer plasma genotyping upon progression with frontline egfr tkis to evaluate for t790m . 
 testing is negative for t790m , reflexive tissue biopsy should be performed because approximately 30% of ctdna test results will be false negative.17 the exact role of liquid biopsies for serial monitoring requires more rigorous evaluation , but a key appeal is that tumor biopsy findings may underestimate the full spectrum of resistant clones present at the time of progression.6 , 18 , 19 in practice , we commonly see the type of mixed radiographic response as observed in this patient with regression of some sites but continued growth in others . 
heterogeneity among distinct cancer subclones may explain such disparate responses , and longitudinal plasma testing might be a valuable adjunct to tissue biopsies to understand the dynamic evolution of various clones . for example , although tissue biopsy may offer critical information about the histology and molecular alterations of a specific progressing lesion , it may lack information about other sites of disease . 
conversely , ctdna genotyping could paint a more - complete picture of the competing resistance clones within a patient , although precise determination of which molecular alterations coexist within one clone or in one anatomic site are not currently possible . 
indeed , other studies have demonstrated that multiple resistance mechanisms can be detected within plasma , 18 and we and others have observed that longitudinal ctdna analyses can track the rise and fall of distinct subclones.6 , 20 in summary , this case report illustrates that the relative magnitude of resistant subclones can fluctuate in response to therapy , that liquid biopsies hold great potential to detect and monitor distinct genetic subpopulations within a patient , and that the presence of a baseline rb1 mutation in an egfr mutant cancer and subsequent sclc transformation raises important questions about monitoring such patients . 
for those with egfr mutant lung cancers , both tissue and liquid biopsy specimens can yield critical information to elucidate dominant clones that drive cancer growth at various time points . 
mooradian no relationship to disclose zofia piotrowska consulting or advisory role : boehringer ingelheim , astrazeneca , ariad pharmaceuticals , takeda pharmaceuticals , superdimension research funding : guardant health ( inst ) benjamin j . 
drapkin no relationship to disclose dora dias - santagata no relationship to disclose nicolas marcoux honoraria : bristol - myers squibb konstantinos arnaoutakis honoraria : foundation medicine , precision advisors , ariad pharmaceuticals consulting or advisory role : foundation medicine , precision advisors , ariad pharmaceuticals rebecca j . 
nagy employment : guardant health stock and other ownership interests : guardant health richard lanman employment : guardant health , veracyte leadership : guardant health stock and other ownership interests : guardant health , veracyte research funding : guardant health anthony j . 
farago honoraria : foundation medicine consulting or advisory role : pharmamar , merrimack , takeda pharmaceuticals , abbvie , intervention insights research funding : pharmamar ( inst ) , abbvie ( inst ) , astrazeneca ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) , loxo ( inst ) , ignyta ( inst ) travel , accommodations , expenses : pharmamar , abbvie , stemcentrx mari mino - kenudson consulting or advisory role : merrimack , h3 biomedicine , acd pharmaceuticals , roche , genentech aaron n . 
sequist honoraria : astrazeneca consulting or advisory role : astrazeneca , genentech , roche , bristol - myers squibb , pfizer research funding : boehringer ingelheim ( inst ) , clovis oncology ( inst ) , genentech ( inst ) , merrimack ( inst ) , novartis ( inst ) , astrazeneca ( inst ) , johnson & johnson ( inst ) , pfizer ( inst ) affiliations meghan j . 
sequist lv , yang jc , yamamoto n , et al : phase iii study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with egfr mutations . 
rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as first - line treatment for european patients with advanced egfr mutation - positive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
yu ha , arcila me , rekhtman n , et al : analysis of tumor specimens at the time of acquired resistance to egfr - tki therapy in 155 patients with egfr - mutant lung cancers . 
piotrowska z , niederst mj , karlovich ca , et al : heterogeneity underlies the emergence of egfrt790 wild - type clones following treatment of t790m - positive cancers with a third - generation egfr inhibitor . 
cancer discov 5 : 713 - 722 , 2015 janne pa , yang jc , kim dw , et al : azd9291 in egfr inhibitor - resistant non - small - cell lung cancer . 
schwaederle mc , patel sp , husain h , et al : utility of genomic assessment of blood - derived circulating tumor dna ( ctdna ) in patients with advanced lung adenocarcinoma . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced nonsmall - cell lung cancer . 
suda k , murakami i , sakai k , et al : small cell lung cancer transformation and t790m mutation : complimentary roles in acquired resistance to kinase inhibitors in lung cancer . 
 systematic assessment of tumor purity and its clinical implications syed haider , phd1 , 2 ; svitlana tyekucheva , phd3 , 4 ; davide prandi , phd5 ; natalie s . 
laird , phd10 ; chris sander11 , 12 ; wenyi wang , phd13 ; francesca demichelis , phd5 , 14 ; massimo loda , md15 , 16 ; and paul c . 
as molecular proles are frequently generated using bulk tissue sections , they represent an admixture of multiple cell types ( including immune , stromal , and cancer cells ) interacting with each other . 
bulk mrna and microrna proles were subject to in silico deconvolution to estimate cancer cellspecic mrna and microrna proles . results we present a systematic comparison of 10 tumor purity estimation methods on a cohort of 333 prostate tumors . 
limited concordance between dnaand mrna - derived purity estimates remained a general pan - cancer phenomenon when tested in an additional 4 , 497 tumors spanning 12 cancer types . conclusion the choice of tumor purity estimation method may have a profound impact on the interpretation of genomic assays . 
taken together , these data highlight the need for improved assessment of tumor purity and quantitation of its inuences on the molecular hallmarks of cancers . jco precis oncol 4 : 995 - 1005 . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction the tumor microenvironment represents an admixture of multiple cell types and complex interactions between bona de cancer cells and surrounding stromal and immune cells.1 because a majority of highthroughput experiments are performed on bulk tissue samples , the resulting signal is usually confounded by nonmalignant tumor - adjacent cells ( tacs )  . variable tumor content and variable tac composition can impinge upon interpretations of molecular data and subsequent clinical decisions.2 - 4 to delineate true residual signal representing individual cell populations , it is crucial to accurately estimate tumor purity . 
tumor purity represents the fraction of cancer cells in a tumor and can be estimated either by expert pathologists reviewing tumor sections5 or in silico ( using epigenomic , genomic , or transcriptomic proles ) .6 pathologic estimates can be inconsistent5 and pragmatically may not always represent the region of tumor that is subject to molecular proling . 
although in silico estimates could circumvent these problems , it remains unclear to what extent these estimates vary across purity calling methods and with the underlying type of biomolecule ( eg , dna v rna )  . 
previous studies have quantied the pan - cancer purity landscape2 , 7 and compared a panel of tools for estimating tumor purity.6 however , systematic benchmarking of in silico tumor purity against matched pathologic estimates and its association with multimodal clinico - genomic proles remains to be elucidated . 
herein , we present systematic benchmarking of 10 purity estimation methods using dna , mrna , and microrna ( mirna ) proles in a 333patient clinically - coherent cohort8 with matched multiobserver pathologic estimates of purity . 
to determine the context specicity of these algorithms , we compared tumor purity estimates from multiobserver pathology to those from multiple algorithms working on different biomolecules ( eg , dna , rna )  . knowledge generated tumor purity estimates from in silico tools varied signicantly from pathology estimates . 
we recommend parameterizing genomic analyses with tumor purity estimated from the matched molecular analyte being analyzed . relevance tumor purity is a key criterion for sample inclusion in clinico - genomic studies and subsequent interpretation of molecular results . 
computational tools often require purity estimates ; we show that these are inuenced by the selected purity estimator . both molecularly driven clinical trials , as well as therapeutic and theranostic decisions , may be affected by these choices . quantify how molecular correlates of tumor purity can skew clinico - genomic interpretations as the result of variable estimates of cancer cell fraction . 
integer infers purity , ploidy , and subclonality from paired tumor and normal samples using the following principles : ( 1 ) models the relationship between the observed allelic frequencies and the underlying copy number changes , and the possible existences and impacts of multiple subclones that may often mislead inferences if not explicitly modeled ; ( 2 ) simultaneous statistic inference on the basis of both copy number changes and major allelic frequencies ; ( 3 ) restoration of information lost as a result of the guanine - cytosine content and actual sizes of each library insert and other specic biases of each genomic location ; ( 4 ) avoid making inferences when the signal - to - noise ratio is not ideal because of technical artifacts ; ( 5 ) an explicit modeling of whole - genome duplication events and whole - chromosome duplication events , which are common in cancer genomics and have huge impacts on the accurate inference of purity and ploidy ; the cancer genome atlas pan - cancer purity estimates were generated using processed rna - seq data ( for isopure ) downloaded from ( download version 2015 ) and snp6 array level - 1 data ( for ascat ) downloaded from gdc data portal . consensus pathology , dna , and mrna purity estimates multiobserver pathology reviews yielded purity ranges , 8 which were further collapsed into single - point estimates using the median value of purity range in deciles . 
dna ( absolute , ascat , clonet , integer , oncosnp ) and mrna ( demix and isopure - r ) based purity estimates were aggregated using median dna and mrna estimates , respectively . availability of data and materials all processed data are available either in the data supplement or uploaded to as speciﬁed in the data supplement . 
 haider et al ethics approval and consent to participate tissue contributing sites followed appropriate consent documentation and approved submission of cases to the cancer genome atlas , as detailed in the original publication.8 results prostate cancer presents complex intraand interpatient heterogeneity . 
it is an ideal model to study heterogeneity because of frequent surgical management via radical prostatectomy of the whole gland , allowing spatio - genomic studies.9 , 10 we collated pathologic , molecular , and clinical data sets from the cancer genome atlas ( tcga ) prostate marker study , which comprised 333 patients.8 purity estimates from multiple pathologists were consolidated , resulting in point estimates as previously described8 ( see methods )  . 
for a subset of cases , both top and bottom tissue block slides ( with sections acquired for molecular analysis in between these ) were assessed by multiple pathologists , demonstrating moderate correlation between pathologists ( top sections : pearsons r = 0.64 , p = 6.23 107 ; bottom sections : pearsons r = 0.53 , p = 8.93 103 ; data supplement fig 1a - b )  . 
a similar trend was observed between the pathology estimates of top and bottom sections ( pearsons r = 0.59 , p = 2.03 1012 ; data supplement fig 1c ) , highlighting potential inuence of spatial heterogeneity . 
in silico estimates of tumor purity were generated using nine methods11 - 18 that leverage dna ( methylation or copy number data ) , mrna , or mirna proles ( data supplement tables 1 and 2 ; methods )  . 
of note , leuc estimates on the basis of dna methylation data were right skewed , with a median purity of 0.9 ( leuc - other = 0.33 , p = 1.44 1095 , wilcoxon rank sum test )  . 
interestingly , all these methods were based on dna proles ( genomic or epigenomic ) , suggesting intrinsic limitations in estimating tumor purity from dna - based assays in this setting . 
these limitations could be explained by the dna prole itself , because samples with failed purity estimates exhibited quiet genomes with low numbers of somatic single nucleotide variants ( snvs ; data supplement fig 2a - b )  . 
pathology calls did not show clear evidence of low purity ; however , rnabased methods predicted a trend toward low purity for a subset of samples ( data supplement fig 2c )  . 
however , it is probable that some may also represent quiet cancer genomes , which are now increasingly recognized as a real phenomenon , particularly in prostate cancer.8 , 19 inspection of the complete sample set revealed no association with histologic heterogeneity ( rationalized as gleason score10 ; fig 1b , data supplement fig 3a - b )  . 
hence , we preclude spatial heterogeneity as the primary factor underlying this lack of concordance . these data highlight that variation and error proles among the intraplatform estimates are probably correlated and suffer from similar intrinsic limitations , independent of the specic algorithm used . 
therefore , we created consensus dna and mrna purity estimates using the median for each class of methods , hereafter referred to as dna and mrna estimates ( see methods )  . 
the differences between pathology estimates and either dna or mrna estimates were strongly correlated ( pearsons r = 0.81 , p = 4.68 1079 ; fig 2 ) , with 29.13% of cases demonstrating agreement ( within 15% purity of each other )  . 
 ( a ) distribution of tcga prostate tumor purity estimates ( n = 333 ) using in silico methods and consolidated multiobserver pathology reviews ; ( b ) patient - wise purity estimates grouped by gleason score . 
data from integer were available for 107 samples using the low - pass dna sequencing data ; ( c ) pearson correlation between purity estimates inferred using in silico methods and pathology reviews . 
of the dnaand mrna - based estimates , only two samples displayed discordant directions of effect relative to pathologic estimates ( purple and yellow dots in fig 2 ) , highlighting overall similarity in error proles of the underlying biomolecules . next , we assessed whether the key transcriptional and genomic biomarkers that underpin prostate cancer biology are dependent on tumor purity . 
to further delineate the relationship between tumor purity and somatic mutations , we stratied purity estimates by the mutation status of a panel of recurrently altered genes in prostate cancer.8 tumor purity determined by at least one prole was associated with six genes , including erg fusions and spop , foxa1 , and tp53 point mutations ( false discovery rate [ fdr ] adjusted p , .25 , wilcoxon rank sum test ; fig 3d , data supplement table 3 )  . 
for these six genes , tumor purity was moderately higher in mutant samples . to characterize this association between driver gene status and tumor purity , we evaluated the associations between tumor purity and the variant allele frequency ( vaf ) in samples carrying mutations ( fig 3e )  . 
however , pathology estimates of tumor purity were unable to accurately capture the vaf of these recurrently altered genes . next , we evaluated whether pathology , dna , mrna , and mirna purity estimates vary in their associations with individual genes or mirnas and to what extent these can be overcome by using in silico deconvolution.15 each of the four consensus purity estimators was individually correlated with ve molecular proles ( bulk / nave and deconvolved mrna abundance , bulk / nave and deconvolved mirna abundance , and bulk copy number data ; deconvolved proles were generated using isopure )  . 
nave mrna and mirna proles exhibited the greatest proportion of features correlated with tumor purity , which diminished after in silico deconvolution , highlighting potentially confounding tacs . with the exception of nave mirna proles , purity estimates were inversely correlated with molecular proles regardless of the underlying purity estimation prole ( data supplement fig 5a - f )  . 
these data suggest that the presence of genomic and transcriptomic correlates of tumor purity are likely to confound biologic and clinical interpretations . because dnaand mrna - based assays are most commonly used in cancer genomics , we asked if the purity estimates from these two analytes are comparable in other cancers . 
given the strong intra - analyte correlation ( fig 1c ) , we considered a representative dna - based method ( ascat ) and an mrna - based method ( isopure ) to estimate tumor purity for an additional 12 cancer types ( 4 , 497 tumor samples ) from tcga project ( fig 4b , prostate cancer data discussed above is shown for reference only )  . overall , all cancer types showed an average purity of at least 0.56. 
these data further underscore the importance of using analyte - matched purity estimates for bioinformatics analysis and subsequent interpretation . discussion herein , we provide evidence that tumor purity estimates manifest intrinsic properties of the underlying information used for purity estimation and exhibit only modest interprole concordance . 
however , interpathologist variation observed in our study , as well as previous studies , suggests that there are probably some inaccuracies in these estimates because of their subjectivity / qualitative nature.5 , 21 these discrepancies may also be a result of the lack of full spatial heterogeneity of the pathologic slide . 
genomic correlates of tumor purity as summarized using androgen receptor ( ar ) signature score ( a ) , percent genome altered ( [ pga ] , b ) , and mutation burden ( c )  . 
statistical tests were performed for genes with more than three mutant samples . therefore , idh1 , rb1 , akt1 , and chd1 ( displayed with x ) were deemed inappropriate for statistical testing . 
 ( a ) correlation between purity estimates derived using pathology , dna , mrna , and microrna ( mirna ) proles and molecular proles ( mrna.naive = bulk mrna abundance , mrna.isopure = deconvolved mrna abundance , mirna.naive = bulk mirna abundance , mirna.isopure = deconvolved mirna abundance , and cna = bulk copy number data ; deconvolved rna proles were generated using isopure )  . 
each feature ( genes for mrna and copy number aberration [ cna ] proles , mirnas for mirna proles ) was correlated with tumor purity estimators ( pathology , dna , rna , mirna ) separately . 
 ( b ) distribution of tumor purity estimates across 13 tcga tumor types ( 4 , 830 tumors ) using an in silico dna - based ( ascat ) and mrna - based ( isopure ) method . 
for clinico - genomic sequencing studies requiring a minimum purity threshold for inclusion in the study , an alternative to pathology estimates is to infer purity directly from the analyte by performing low - pass dna sequencing to lter low - purity samples.22 in addition to poor concordance between pathology and dna / rna - based tumor purity in prostate cancer , our pan - cancer data reported herein suggest that the purity estimates from dna and mrna proles also show limited concordance . 
the concordance between purity estimators also varies depending upon the tumor type and patterns of somatic changes it exhibits ( eg , dna - based methods rely on the presence of copy number aberrations )  . 
furthermore , previous studies have reported varying levels of concordance in purity estimates inferred from dnaand rna - based methods.2 , 23 for instance , aran et al2 show much stronger concordance between estimate24 ( rna - based purity estimator ) and absolute13 ( dna - based purity estimator ) compared with the rnaand dna - based methods in our study . 
because purity estimates vary across methods , consensus estimates on the basis of matched analyte type may further improve purity estimates and may also overcome missing values and normalize outlier estimates . 
after condent purity estimates have been created , one way to account for these is to adjust bioinformatics and statistical analyses for tumor purity , as stressed in previous studies.2 , 7 , 15 because bulk tumor proles are heterogeneous compositions of tumor cells and tacs featuring complex interplay , it is crucial to interpret the clinico - genomic proles in the context of the underlying heterogeneity.25 many in silico deconvolution techniques have been developed to estimate relative abundance of different cell types , 24 , 26 , 27 as well as techniques that transcriptomic11 , 12 , 15 , 18 , 23 and explicitly generate residual genomic14 proles of tumor - only and stromal - only cells . 
herein , we recommend researchers to consider deconvolution of bulk proles into individual component proles ( e.g. , cancer and stromal proles ) to improve sensitivity and specicity of downstream analyses.4 , 15 affiliations 1ontario institute for cancer research , toronto , ontario , canada 2the breast cancer now toby robins research centre , the institute of cancer research , london , united kingdom 3department of data sciences , dana - farber cancer institute , boston , 4department of biostatistics , harvard t.h. 
boutros provision of study material or patients : massimo loda collection and assembly of data : syed haider , svitlana tyekucheva , davide prandi , andrew wei xu , peter w . 
park honoraria : pzer consulting or advisory role : neuroinammation newco patents , royalties , other intellectual property : patent on mutational signature - based detection of homologous recombination deciency peter w . 
laird consulting or advisory role : progenity , anchordx patents , royalties , other intellectual property : received royalties annually through 2018 for inventions licensed to epigenomics ag by usc travel , accommodations , expenses : anchordx wenyi wang stock and other ownership interests : genomic health francesca demichelis patents , royalties , other intellectual property : co - inventor on a patent led by the university of michigan and the brigham and womens hospital covering the diagnostic and therapeutic elds for ets fusions in prostate cancer . 
boutros consulting or advisory role : biosymetrics patents , royalties , other intellectual property : holds patents on multiple biomarkers no other potential conicts of interest were reported . acknowledgment the results published herein are based , in part , upon data generated by tcga pilot project established by the national cancer institute and the national human genome research institute . 
nature 501 : 346 - 354 , 2013 fox ns , haider s , harris al , et al : landscape of transcriptomic interactions between breast cancer and its microenvironment . 
nat commun 10 : 3116 , 2019 smits aj , kummer ja , de bruin pc , et al : the estimation of tumor cell percentage for molecular testing by pathologists is not accurate . 
mod pathol 27 : 168 - 174 , 2014 yadav vk , de s : an assessment of computational methods for estimating purity and clonality using genomic data derived from heterogeneous tumor tissue samples . 
brief bioinform 16 : 232 - 241 , 2015 zheng x , zhang n , wu hj , et al : estimating and accounting for tumor purity in the analysis of dna methylation data from cancer studies . 
wang l , sebra rp , sfakianos jp , et al : a reference prole - free deconvolution method to infer cancer cell - intrinsic subtypes and tumor - type - specic stromal 24 . 
miller , md3 ; and razelle kurzrock , md2 , 5 purpose high - grade neuroendocrine cervical cancer ( hgnecc ) is an uncommon malignancy with limited therapeutic options ; treatment is patterned after the histologically similar small - cell lung cancer ( sclc )  . 
these results were subsequently compared with a cohort of 1 , 800 sclcs . results the median age of patients with hgnecc was 40.5 years ; 83 patients ( 85.6% ) harbored high - risk human papillomavirus ( hpv )  . 
overall , 294 genomic alterations ( gas ) were identied ( median , 2 gas / sample ; average , 3.0 gas / sample , range , 0 - 25 gas / sample ) in 109 distinct genes . 
seventy - one patients ( 73% ) had 1 alteration that was theoretically druggable . comparing hgnecc with sclc , signicant differences in tmb , microsatellite instability , hpv - positive status , and in pik3ca , myc , pten , tp53 , arid1a , and rb1 alteration rates were found . conclusion this large cohort of patients with hgnecc demonstrated a genomic landscape distinct from sclc , calling into question the biologic and therapeutic relevance of the histologic similarities between the entities . furthermore , 73% of hgnecc tumors had potentially actionable alterations , suggesting novel treatment strategies for this aggressive malignancy . jco precis oncol 4 : 972 - 987 . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction the treatment of solid malignancies has evolved and is perhaps best exemplied by the approach to non small - cell lung cancer , for which molecular characterization and use of targeted agents have emerged as standard therapeutic paradigms . 
recently , the cancer genome atlas ( tcga ) completed and published the integrated genomic and molecular characterization of cervical cancer.1 in addition to data previously released for both ovarian ( high - grade serous ) and endometrial ( endometrioid and serous ) cancers , this publication completed the molecular and genomic evaluation of the most common gynecologic malignancies.2 , 3 traditionally , cervical cancer clinical trials have excluded less common histologies such as high - grade neuroendocrine cervical carcinoma ( hgnecc )  . 
despite the low incidence of hgnecc ( , 2% of all cervical cancers ) the oncologic impact is signicant because these tumors exhibit more aggressive clinical characteristics.4 , 5 unfortunately , the 5 - year overall survival rate for patients with early - stage disease is only a 36% , and those with metastatic spread face an even more dismal prognosis . 
given these poor outcomes , patients with hgnecc represent an area of unmet clinical need . developing therapeutic options for patients with rare tumors is challenging , relying on international collaboration , as well as small case series or retrospective reports rather than prospective clinical trials . 
 genomic landscape of high - grade neuroendocrine cervix cancer context key objective to dene the molecular landscape of high - grade neuroendocrine cervical cancer in a large cohort of patients . knowledge generated high - grade neuroendocrine cervical cancer appears molecularly distinct from the histologically similar small - cell lung cancer . up to 73% of patients samples harbored potentially actionable alterations , informing novel treatment strategies . relevance continued understanding of the molecular underpinnings of high - grade neuroendocrine cervical carcinoma will be critical to driving drug discovery for this disease . approved by the food and drug administration ( fda ) specically for hgnecc.8 recently , 2 reports of exceptional responses to immune checkpoint inhibition in patients with recurrent hgnecc were published.9 , 10 to better understand these responses and to identify molecular aberrations underlying this uncommon malignancy , we examined the genomic landscape of hgnecc . 
the submitting physicians provided specication of a poorly differentiated , neuroendocrine tumor type of cervical origin , which was then independently reviewed by a gynecologic pathologist ( j.e. ) to conrm high - grade neuroendocrine pathologic features in the pathology report and / or the representative sample of tumor submitted for sequencing ( grade 3 cytomorphologic features , some component of small - cell or large - cell carcinoma histology , and / or positivity for neuroendocrine markers )  . 
ngs data were generated by foundationone ( foundation medicine ; cambridge , ma )  . the study was performed in accordance with university of california , san diego , institutional review board guidelines for a de - identied database . 
the test sequences the entire coding region of 182 or , more recently , 236 or 315 cancer - related genes plus up to 47 introns of up to 19 genes often rearranged or altered in cancer to an average depth of coverage of  . 
500.11 the pathologic diagnosis of each case was conrmed on routine hematoxylinand eosin - stained slides and all samples forwarded for dna extraction contained a minimum of 20% tumor nuclear area . 
microsatellite instability ( msi ) status was evaluable in 75 hgnecc and 1 , 573 sclc cases . the sequencing methods used for comprehensive genomic proling have been validated and reported previously ( appendix ) .12 , 13 the optimized loci used to evaluate msi status were selected from a total set of 1 , 897 that have adequate coverage on all versions of the assay . 
there was no need to extend beyond the rst principal component , because it explained approximately 50% of the total data variance , whereas none of the other principal components explained  . 
msi - low calls are not made because there was no gold - standard test set , but we presume such samples would signicantly overlap with the msiambiguous category reported here . 
the cutoff of 20 coding mutations / mb is approximately equal to 400 nonsynonymous mutations per exome.15 human papillomavirus detection in addition , the presence of high - risk human papillomavirus ( hpv ) was examined in submitted specimens , as previously reported.16 hybrid - capture reagents included baits designed to capture unique regions of select viral genomes including hpv - 16 and - 18 . 
a total hpv - 16 / 18 aligned read count of 5 reads per million was considered a positive hpv status , and , 5 reads per million was considered hpv not detected.16 end points and statistical methods descriptive statistics were used to summarize the baseline patient characteristics . 
all statistical tests were carried out using graphpad prism , version 6.0 ( graphpad software , san diego , ca )  . results characterization of gas in hgnecc the median age of the cohort was 40.5 years ( range , 25 - 77 years )  . 
the most frequently reported number of gas per sample was 2 , with a range of 0 - 25 ( average , 3.0 gas / sample ; fig 2 )  . 
5% of samples are depicted . variants of unknown signicance excluded . genomics in hpv - positive versus - negative patients when examining distribution of gas on the basis of hpv detection status , a signicant difference was identied in the frequency of several gas , including pik3ca , tp53 , pten , arid1a , and rb1 , all of which were more frequent in the hpv - negative subgroup ( table 1 )  . 
two of the 3 cases were both msi - h and tmb - h and harbored the highest numbers of identiable gas in the cohort.17 , 18 the third case , with a nonsense mutation near the 3 ( cid : 3 ) end of the coding sequence ( msh2 r929 * ) , was mss and tmb - l . the 2 msh - 2mutant msi - h cases were both of small - cell histology and accounted for all msi - h cases out of the 75 no . 
furthermore , a total of 40 patient samples ( 41.2% ) harbored mutations in the pi3k / akt / mtor pathway ; mutations in pik3ca were identied in 47.5% of these samples ( n = 19 ) , and pten mutations were reported in 35% ( n = 14 )  . 
the small - cell subset of hgnecc samples showed analogous gene mutation differences from sclc . discussion neuroendocrine carcinoma is an uncommon but aggressive variant accounting for approximately 1.5% of all newly diagnosed cervical cancers.20 the great majority of these lesions are high - grade largeor small - cell subtypes , with only rare reports of well - differentiated cervical carcinoid tumors.20 the treatment of patients with hgnecc remains table 2 . 
 eskander et al clinically challenging , with limited response rates to chemotherapy ; however , anecdotal reports of exceptional responders have been described.9 , 10 the paradigm for management of hgnecc has been informed by the treatment of the more commonly diagnosed ( and histologically similar ) sclc , which accounts for approximately 15% of all lung cancer cases . 
in prior studies , whole - genome sequencing of 110 sclc specimens identied essentially ubiquitous tp53 and rb1 inactivating mutations , with biallelic losses of each gene respectively in 100% and 93% of cases without chromothripsis.21 in an effort to better dene the molecular landscape of hgnecc , we evaluated the comprehensive genomic proling of 97 patient samples . 
at least 1 characterized alteration was identied in 88 patient samples ( 90.7% ) and of these , 72 had a potentially pharmacologically tractable alteration . interestingly , the frequency and distribution of gas identied in this cohort of patients are similar and distinct from mutational patterns described in the more common hpvrelated cervical cancer histologies.1 as detailed in tcgas integrated genomic characterization of cervical cancer ( ie , squamous , adenocarcinoma , and adenosquamous histologies ) , mutations in the pik3ca gene were the most frequently identied aberration , occurring in 26% of samples , approximating the nearly 20% rate in our cohort . in addition , signicantly mutated genes reported by the tcga , identied in similar proportions in this patient cohort , included arid1a ( 7% in tcga and 9.3% in our cohort ) and kras ( 6% in tcga and 8.2% in our cohort )  . 
these molecular differences may be reective of the varying histologies or , potentially , the differential highrisk hpv detection rates ( 85.6% in our cohort v 95% in the tcga ) .1 importantly , the high - risk hpv rate in our cohort should be interpreted with caution because the assay used has not undergone formal concordance study with gold standard tests such as hybrid capture and can detect only hpv 16 / 18 . our own , much larger cohort of sclc samples ( n = 1 , 800 ) recapitulates prior studies and had a strikingly different molecular portfolio when compared with hgnecc samples . 
msi - h status was also more common in the hgnecc cohort whereas tmb - i / tmb - h was more common in sclc ( despite the lack of msi - h status )  . 
the use of everolimus , or an alternate mtor or pik3ca inhibitor , may be considered in such circumstances , although the utility of a pik3ca mutation in predicting response to single - agent everolimus in the presence of multiple gas remains limited.26 , 27 although less frequently identied , alterations in the hrd pathway were detected in 9.3% of patient samples , potentially supporting use of a poly - adp ribose polymerase inhibitor . 
the identication of both tmb - h ( n = 2 ) and gas in mismatch repair genes ( n = 3 ) may also inform the use of immune checkpoint inhibition.28 in may 2017 , the fda approved pembrolizumab for the treatment of mismatch repairdecient or msi - h solid tumors that progressed after prior therapy . 
more recently , the fda accepted and granted priority review to a supplemental biologics license application for pembrolizumab for the treatment of adult and pediatric patients with unresectable or metastatic solid tumors with tissue tmb - h whose disease has progressed after prior treatment and who have no satisfactory alternative treatment options , supported by data from the phase ii keynote - 158 trial . 
notably , there are 2 published case reports of patients with recurrent , treatment - refractory hgnecc with exceptional and durable responses to checkpoint inhibition ; 1 of these tumors was from our current hgnecc cohort and had a mismatch repair defect and the other lacked correlative genomic testing.9 , 10 last , the identication of arid1a ( 9.3% ) and smarca4 ( 4.1% ) mutations may predict sensitivity to an alternate therapeutic strategy.29 homeostasis requires balanced arid1a and ezh2 activity , facilitated via chromatinmediated gene expression . 
 genomic landscape of high - grade neuroendocrine cervix cancer aberrations were identied in patients with msi - h lesions , possibly reecting that the smarca4 may be a passenger mutation resulting from the underlying msi . 
furthermore , of the 4 cases with smarca4 alterations , 1 was hpv - 18 positive and another was p16 positive by immunohistochemical assessment . despite the large sample size and robust genomic data , this study has limitations . 
95% of the identied high - risk hpv strains.30 this report highlights the potential therapeutic utility of genomic testing in patients with this uncommon disease.27 interest , despite the histologic similarity between hgnecc and sclc , which has led to the latter being used as a model for treating the former , the molecular portfolio of these 2 entities is strikingly different . 
therefore , plausible that patients with hgnecc may benet from alternative therapeutic strategies . it is not anticipated that traditional prospective trials will accrue sufcient patient numbers in this disease setting , and novel study designs , including umbrella , basket , and platform trials , should be considered given the presence of actionable targets . 
interestingly , the rst reported cohort of the dart trial ( clinicaltrials.gov identier : nct02834013 ) 31 was the neuroendocrine cohort , with a 44% overall response rate in those with high - grade disease . 
eskander consulting or advisory role : pzer , clovis oncology , astrazeneca / medimmune , tesaro , merck speakers bureau : clovis oncology , astrazeneca / medimmune , roche travel , accommodations , expenses : clovis oncology , astrazeneca / medimmune , roche , merck , pzer julia elvin employment : foundation medicine stock and other ownership interests : hoffman - laroche laurie gay employment : foundation medicine , invitae stock and other ownership interests : foundation medicine , naveris , invitae consulting or advisory role : invitae , naveris jeffrey s . 
ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine consulting or advisory role : celsius therapeutics research funding : foundation medicine vincent a . 
 eskander et al leadership : foundation medicine , revolution medicines stock and other ownership interests : foundation medicine , mirati therapeutics , revolution medicines patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center razelle kurzrock leadership : curematch , curemetrix stock and other ownership interests : curematch , idbydna , soluventis honoraria : roche , eusa pharma , neogenomics laboratories , biocom , neomed therapeutics , advanced therapeutics , lek , aacr , chugai pharma usa , wiley consulting or advisory role : actuate therapeutics , loxo , xbiotech , neomed , roche , gaido , soluventis , pzer , merck speakers bureau : roche , guardant health ( inst ) research funding : sequenom ( inst ) , merck serono ( inst ) , genentech ( inst ) , pzer ( inst ) , foundation medicine ( inst ) , incyte ( inst ) , konica minolta ( inst ) , grifols ( inst ) , omniseq ( inst ) , debiopharm group ( inst ) , boerhinger ingelheim ( inst ) travel , accommodations , expenses : roche , eusa pharma , neogenomics laboratories , biocom , neomed therapeutics , advanced therapeutics , lek , aacr , chugai pharma usa , wiley no other potential conicts of interest were reported . references cancer genome atlas research network : integrated genomic and molecular characterization of cervical cancer . 
nature 497 : 67 - 73 , 2013 [ erratum : nature 500 : 242 , 2013 ] cancer genome atlas research network : integrated genomic analyses of ovarian carcinoma . 
nature 474 : 609 - 615 , 2011 [ erratum : nature 490 : 298 , 2012 ] gardner gj , reidy - lagunes d , gehrig pa : neuroendocrine tumors of the gynecologic tract : a society of gynecologic oncology ( sgo ) clinical document . gynecol oncol 122 : 190 - 198 , 2011 satoh t , takei y , et al : gynecologic cancer intergroup ( gcig ) consensus review for small cell carcinoma of the cervix . 
int j gynecol cancer 24 : s102 - s108 , 2014 ( 9 suppl 3 ) fukuoka m , masuda n , furuse k , et al : a randomized trial in inoperable non - small - cell lung cancer : vindesine and cisplatin versus mitomycin , vindesine , and cisplatin versus etoposide and cisplatin alternating with vindesine and mitomycj clin oncol 9 : 606 - 613 , 1991 kubota k , hida t , ishikura s , et al : etoposide and cisplatin versus irinotecan and cisplatin in patients with limited - stage small - cell lung cancer treated with etoposide and cisplatin plus concurrent accelerated hyperfractionated thoracic radiotherapy ( jcog0202 ) : a randomised phase 3 study . 
lancet oncol 15 : 106 - 113 , 2014 ishikawa m , kasamatsu t , tsuda h , et al : prognostic factors and optimal therapy for stages i - ii neuroendocrine carcinomas of the uterine cervix : a multi - center retrospective study . 
gynecol oncol 148 : 139 - 146 , 2018 paraghamian se , longoria tc , eskander rn : metastatic small cell neuroendocrine carcinoma of the cervix treated with the pd - 1 inhibitor , nivolumab : a case report . 
sharabi a , kim ss , kato s , et al : exceptional response to nivolumab and stereotactic body radiation therapy ( sbrt ) in neuroendocrine cervical carcinoma with high tumor mutational burden : management considerations from the center for personalized cancer therapy at uc san diego moores cancer center . oncologist 22 : 631 - 637 , 2017 11 . 
ross js , fakih m , ali sm , et al : targeting her2 in colorectal cancer : the landscape of amplication and short variant mutations in erbb2 and erbb3 . 
genome med 9 : 34 , 2017 johnson db , frampton gm , rioth mj , et al : targeted next generation sequencing identies markers of response to pd - 1 blockade . 
chae yk , pan ap , davis aa , et al : path toward precision oncology : review of targeted therapy studies and tools to aid in dening actionability of a molecular lesion and patient management support . 
kato s , kurasaki k , ikeda s , et al : rare tumor clinic : the university of california san diego moores cancer center experience with a precision therapy 25 . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors on the basis of molecularp : results from mypathway , an openlabel , phase iia multiple basket study . 
j clin oncol 36 : 536 - 542 , 2018 janku f , hong ds , fu s , et al : assessing pik3ca and pten in early - phase trials with pi3k / akt / mtor inhibitors . 
alejo m , alemany l , clavero o , et al : contribution of human papillomavirus in neuroendocrine tumors from a series of 10 , 575 invasive cervical cancer cases . pract 4 : 17 , 2017 papillomavirus res 5 : 134 - 142 , 2018 31 . 
patel sp , othus m , chae yk , et al : a phase ii basket trial of dual anti - ctla - 4 and anti - pd - 1 blockade in rare tumors ( dart ) s1609 : the neuroendocrine cohort . presented at the american association for cancer research annual meeting , atlanta , ga , march 29 - april 3 , 2019 ( abstr ct039 )  . 
briey , after pathologic review to conrm sufcient tumor nuclei ( minimum , 20% ) and mitigate pathologic inconsistencies , at least 50 ng of dna was extracted from 40 tumor samples provided as formalin - xed , parafnmicrons of embedded tissue blocks . 
the samples were assayed using adaptorligation and hybrid - capture next - generation sequencing ( foundationone ) for all coding exons from 182 ( version 1 ) , 287 ( version 2 ) , or 315 ( version 3 ) cancer - related genes plus selected introns from 14 ( version 1 ) , 19 ( version 2 ) , or 28 ( version 3 ) genes frequently rearranged in cancer . sequencing of captured libraries was performed using hisequation 2500 / 4000 ( illumina , san diego , ca ) to a mean exon coverage depth of  . 
500 , and resultant sequences were analyzed using both an algorithmic pipeline and manual curation for base substitutions , small insertions or deletions ( indels ) , copy number alterations ( focal amplications and homozygous deletions ) , and selected gene fusions , as previously described.13 clinically relevant genomic alterations were dened as alterations targetable by anticancer drugs currently available on the market or in registered clinical trials . 
germline variants documented in the dbsnp database ( dbsnp142 ; nlm.nih.gov / snp / ) , with 2 counts in the exac database ( broadinstitute.org / ) , or recurrent variants of unknown signicance that were predicted by an internally developed algorithm to be germline were removed , with the exception of known driver germline events.12 known , conrmed somatic alterations deposited in the catalog of somatic mutations in cancer ( version 62 ) were highlighted as biologically signicant , as were inactivating events in tumor suppressor genes . 
 o majority of b2m - mutant and - decient colorectal carcinomas achieve clinical benet from immune checkpoint inhibitor therapy and are microsatellite instability - high sumit middha , phd1 ; rona yaeger , md1 ; jinru shia , md1 ; zsoa k . 
hechtman , md1 purpose microsatellite instability - high ( msi - h ) colorectal carcinomas ( crcs ) show high rates of response to immune checkpoint inhibitors ( ios )  . 
all b2m - mutant crcs were assessed for expression of b2m , major histocompatibility complex class i , and programmed death - 1 ligand ( pd - l1 ) via immunohistochemistry and average cd3 + and cd8 + tumor - inltrating lymphocyte counts against a control group of msi - h b2m wild - type crcs . results fifty - nine ( 3.4% ) of 1 , 751 patients with crc harbored b2m mutations , with 84% ( 77 of 92 ) of the mutations predicted to be truncating . 
b2m mutation status was not associated with major histocompatibility complex class i expression , kras or braf mutation , tumor - inltrating lymphocyte level , or pd - l1 expression after adjustment for msi status . 
of 13 patients with b2m - mutant crc who received programmed death - 1 or pd - l1 ios , 11 ( 85% ) achieved clinical benet , dened as stable disease or partial response using response evaluation criteria in solid tumors criteria . conclusion b2m mutations occur in approximately 24% of msi - h crcs and are usually associated with loss of b2m expression . 
 middha et al context do b2m - mutant and decient colorectal carcinomas ( crcs ) respond to immune checkpoint inhibitors ? we found that b2m mutations are enriched in microsatellite instability - high crc , that these mutations commonly occur at coding microsatellite loci , and that these b2m mutations are truncating and associated with loss of b2m expression . 
of 13 patients with b2m - mutant crc and available recist data , six achieved stable disease , ve achieved partial response , and one experienced pseudoprogression . b2m mutations are not predictive of primary resistance to immune checkpoint inhibition in crc . administrationcleared , next - generation sequencing assay that interrogates  . 
presence of loss of heterozygosity ( loh ) was assessed via allelespecic copy - number analysis using the fraction and allelespecic copy number estimates from tumor sequencing ( facets ) algorithm14 and , in cases of low tumor content , via comparison of b2m mutation variant allele frequency against median variant allele frequency . 
clinical parameters , clinical response to immune checkpoint inhibitor therapy all patients with crc with msk - impact data and b2m mutations who underwent therapy with immune checkinhibitors ( ios ; durvalumab , nivolumab , or pempoint brolizumab ) before july 2018 were assessed for b2m expression ( ihc ) , response , stable disease ( sd ) , and progressive disease ( pd )  . 
formal response evaluation criteria in solid tumors ( recist ) scores were assessed via radiologic data as follows : complete response ( cr ) , disappearance of all target lesions , conrmed at 4 weeks ; partial response ( pr ) , 30% decrease , conrmed at 4 weeks ; pd , 20% increase over smallest sum observed ; and sd , meeting none of the other criteria . patients were deemed to have experienced clinical benet from ios if recist results were sd , pr , or cr . ihc staining for b2m using a polyclonal antibody with concentration of 1 : 6 , 000 ( catalog #a0072 ; dako , santa clara , ca ) , mhc class i using a monoclonal antibody with concentration of 1 : 200 ( catalog #14 - 9958 ; e - bioscience , carlsbad , ca ) , cd3 using a monoclonal antibody with concentration of 1 : 200 ( catalog #ncl - l - cd3 - 565 ; leica , lincolnshire , il ) , cd8 using a monoclonal antibody with concentration of 1 : 100 ( catalog #m7103 ; dako ) , and pd - l1 using a monoclonal antibody with concentration of 1 : 100 ( catalog #13684 ; cell signaling , danvers , ma ) was performed on all crcs with b2m mutations with available tissue as well as a set of 26 randomly selected wild - type ( wt ) crcs with in - house resection specimens and mskimpact testing ( performed between january 1 , 2014 , and october 31 , 2017 ) that were matched to the b2m - mutant group for prevalence of msi status . 
complete loss of b2m on ihc ( 0% of tumor cells with b2m expression ) was interpreted as loss of b2m expression . statistical analyses associations were assessed using pearsons 2 test with simulated p value based on 2 , 000 replicates for low count data . 
a cox proportional hazards model was tted to the data to calculate survival using the covariates of b2m mutation status , age at diagnosis , pathologic stage , msi status , proximal versus distal status , and kras , nras , braf , and pik3ca mutation status . 
these were each assessed through both univariable and multivariable cox regressions . r survival and survminer software packages were used to perform this analysis ( r foundation , vienna , austria )  . results molecular findings we rst sought to determine the spectrum of b2m mutations in a cohort of patients with crc ( n = 1 , 751 ) with msk - impact data ( appendix fig a1 )  . 
next , we classied the samples on the basis of whether they were microsatellite stable ( mss ) or unstable ( msi - h ) on the basis of genomic data . 
a medullary carcinoma of the colon shows immune - cell expression of programmed death - 1 ligand at the tumor - stroma interface , loss of b2m expression in tumor cells , retention of strong diffuse mhc class i expression in tumor cells ,  . 
twelve samples showed either loh or copy - neutral loh along with a clonal mutation , suggesting biallelic loss . b2m expression , mhc class i expression , and til level to evaluate the functional outcome of b2m mutations , we examined protein expression in samples with available tissue ( figs 1b and 1c ; appendix table a2 )  . 
because pdl1 status has been used as a predictive marker of ios and linked to expression of mhc class 1 , we performed pd - l1 ihc in available b2m - mutant and wt patient cases . 
thirtytwo ( 73% ) of 44 b2m - mutant and 13 ( 50% ) of 26 b2m wt crcs were positive for pd - l1 expression ( immune cells in tumor - stroma interface )  . 
average median cd3 + count per hpf was 22.3 in b2m - mutant and 37.3 in b2m wt crcs , whereas average median cd8 + count per hpf was 15.1 and 49 for b2m - mutant and b2m wt crcs , respectively . 
in comparison with b2m wt crcs matched for msi status , b2m - mutant crcs tended to have lower average levels of cd3 and cd8 per patient case ( fig 2a ) , these differences did not reach statistical significance . 
age , stage , and kras , nras , and braf status were associated with os , whereas pik3ca mutation status , msi status , and proximal versus distal location were not associated with os ( fig 2b ; appendix table a2 )  . response to immunotherapy because b2m mutations were identied as a possible resistance mechanism for checkpoint inhibition , we identied 13 ( msi - h , n = 11 ; mss , n = 2 ) patients with crc with b2m mutations who subsequently received io therapy , and we evaluated their response to treatment . 
 ( a ) box plot graphs of average cd3 ( left ) and cd8 ( right ) counts in b2m - mutant vs wild - type ( wt ) colorectal carcinoma ( crc ) show that median average cd3 and cd8 trend toward higher values in b2m wt carcinoma . ( b ) multivariable model showing the clinical variables associated with os of patients with crc in our cohort . 
analysis of their tumor response by recist criteria demonstrated pr in ve patients , sd in six patients , and pd in one patient , likely pseudoprogression ( fig 3a ; appendix table a3 )  . 
this response rate is in line with recent publications of ios in msi - h crc.8 , 9 of the two patients with mss tumors , one had an ultramutated pole hotspot mutated tumor and experienced tumor growth with io treatment but was able to continue treatment for 1 year , likely to be pseudoprogbecause growth was thought ression , and the other patient had sd during treatment with a combination of io treatment and targeted therapy ( after progression with targeted therapy alone )  . 
the association of b2m mutations with complete loss of expression on whole sections of pole s459f best response pseudoprogression partial response stable disease treatment stopped ongoing 40% 80% msi status io therapy b2m expression fig 3 . 
immune checkinhibitor ( io ) repoint sponse in b2m - mutant colorectal cancer ( crc )  . waterfall plot of io response in b2m - mutant crc ( colored by microsatellite instability [ msi ] status , type of b2m mutation , and type of io received )  . 
indeed , in the 42 b2m - mutant crcs analyzed by facets , 60% had evidence of either two clonal b2m mutations or one clonal b2m mutation and loh ( appendix table a1 )  . 
it is possible that the remaining b2m - mutant crcs have epigenetic modications as a mechanism of b2m silencing . unlike previous studies , 3 we found that b2m protein loss is not correlated with loss of mhc class i expression . 
we show that b2m mutation and loss do not correlate with mhc class i loss of expression , although the effect of b2m loss on the functional competence of mhc class i is known to be deleterious.16 limitations to our study include the retrospective analysis and relatively small number of patients treated with ios , as well as limitations in molecular testing for epigenetic issues and allele specicity of b2m mutations . most importantly , our study shows that b2m mutation and loss in immunotherapy - naive crc do not predict primary resistance to ios . 
we saw that most patients had some degree of regression with treatment ; larger , prospective studies are needed to clarify if the response rate and duration of response vary by b2m mutation status . however , our data indicate that patients with crc whose tumors harbor b2m mutations should not be excluded from io treatment . 
the clinical benet demonstrated in patients with b2m - decient crc who received ios may have resulted from the fact that despite b2m loss , there was still evidence of functional neoantigen recognition , as indicated by the high number of tils ( median , 22.3 per hpf ) still present in b2m - decient msi - h crc . 
 middha et al leonard saltz consulting or advisory role : mcneil ( i ) research funding : taiho pharmaceutical neil segal consulting or advisory role : bristol - myers squibb , pzer , astrazeneca / medimmune , imugene , roche / genentech , pieris pharmaceuticals , synlogic , aduro biotech , kyn therapeutics , boehringer ingelheim , merck , puretech , horizon pharma , emd serono , gritstone oncology , chugai pharma , trm oncology , ifm therapeutics , psioxus therapeutics research funding : medimmune , bristol - myers squibb , pzer , roche / genentech , merck , incyte marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network / astrazeneca tagrisso rfp advisory committee , takeda pharmaceuticals , bristol - myers squibb , bayer healthcare pharmaceuticals , merck ( i ) research funding : loxo ( inst ) , helsinn therapeutics ahmet zehir no relationship to disclose jaclyn f . 
hechtman honoraria : medscape consulting or advisory role : navigant consulting , axiom biotechnologies research funding : bayer no other potential conicts of interest were reported . references roberts ad , ordway dj , orme im : listeria monocytogenes infection in beta 2 microglobulin - decient mice . 
infect immun 61 : 1113 - 1116 , 1993 schaible ue , collins hl , priem f , et al : correction of the iron overload defect in beta - 2 - microglobulin knockout mice by lactoferrin abolishes their increased susceptibility to tuberculosis . 
j exp med 196 : 1507 - 1513 , 2002 sade - feldman m , jiao yj , chen jh , et al : resistance to checkpoint blockade therapy through inactivation of antigen presentation . 
nat commun 8 : 1136 , 2017 janikovits j , m uller m , krzykalla j , et al : high numbers of pdcd1 ( pd - 1 ) - positive t cells and b2m mutations in microsatellite - unstable colorectal cancer . oncoimmunology 7 : e1390640 , 2017 grasso cs , giannakis m , wells dk , et al : genetic mechanisms of immune evasion in colorectal cancer . 
cancer discov 8 : 730 - 749 , 2018 le dt , uram jn , wang h , et al : pd - 1 blockade in tumors with mismatch - repair deciency . 
n engl j med 372 : 2509 - 2520 , 2015 riaz n , havel jj , makarov v , et al : tumor and microenvironment evolution during immunotherapy with nivolumab . 
cell 171 : 934 - 949.e16 , 2017 le dt , durham jn , smith kn , et al : mismatch repair deciency predicts response of solid tumors to pd - 1 blockade . 
science 357 : 409 - 413 , 2017 overman mj , mcdermott r , leach jl , et al : nivolumab in patients with metastatic dna mismatch repair - decient or microsatellite instability - high colorectal cancer ( checkmate 142 ) : an open - label , multicentre , phase 2 study . 
clendenning m , huang a , jayasekara h , et al : somatic mutations of the coding microsatellites within the beta - 2 - microglobulin gene in mismatch repairdecient colorectal cancers and adenomas . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
consort diagramutation and immunohistochemistry analyses included data from 1 , 751 patients with colorectal cancer ( crc ) with memorial sloan kettering integrated mutation proling of actionable cancer targets testing . 
 comparative rna - sequencing analysis benefits a pediatric patient with relapsed cancer clinical detection of sequence and structural variants in known cancer genes points to viable treatment options for a minority of children with cancer.1 to increase the number of children who benefit from genomic profiling , gene expression information must be considered alongside mutations.2 , 3 although high expression has been used to nominate drug targets for pediatric cancers , 4 , 5 its utility has not been evaluated in a systematic way.6 we describe a child with a rare sarcoma that was profiled with whole - genome and rna sequencing ( rna - seq ) techniques . 
although the tumor did not harbor dna mutations targetable by available therapies , incorporation of gene expression information derived from rna - seq analysis led to a therapy that produced a significant clinical response . 
histology revealed a mitotically active , epithelioid - to - spindled cell tumor in patternless sheets , interrupted by thick fibrous bands and foci of necrosis ( figs 1c to 1d )  . 
focal immunohistochemical positivity was observed for pan - cytokeratin ( ae1 / ae3 ) and synaptophys the tumor was negative for glial fibrillary acidic protein ( gfap ) , wilms tumor 1 ( wt1 ) , myo - d1 , myogenin , smooth muscle actin , nonphosphorylated and phosphorylated neurofilament protein , cd34 , cd31 , hmb - 45 , s - 100 , leukocyte common antigen , and baf47 / ini - 1 ( retained nuclear positivity )  . 
a diagnosis of desmoplastic small round cell tumor ( dsrct ) was favored initially.7 because ewsr1 breakapart fluorescence in situ hybridization confirmed an ewsr rearrangement but concomitant wt1 breakapart fluorescence in situ hybridization was negative , the molecular criterion for dsrct was not met , and a final diagnosis of poorly differentiated sarcoma , not otherwise specified , was rendered . 
the patient received six cycles of induction chemotherapyifosfamide , carboplatin , and etoposidefollowed by autologous stem - cell transplantation with a high - dose preparative regimen of carboplatin , thiotepa , and etoposide as well as 54 gy of focal radiation to the location of the original tumor . 
we compared the rna - seqderived tumor gene expression profile of patient 1 with similarly derived profiles of 10 , 668 samples that represented 38 pediatric and adult tumor types studied by the the cancer genome atlas ( tcga ) and therapeutically actionable research to generate effective treatments ( target ) .11 , 12 rna - seq reads from different laboratories were reanalyzed with a single computational pipeline to reduce batch effects.13 we searched for tumors in this homogeneously processed compendium in which expression profiles were similar to those of patient 1 by using tumormap.14 the tumor gene expression profile for patient 1 resembled lung cancers ( fig 2a ) , the site of the metastasis . 
lung adenocarcinoma ( luad ) samples formed four groups in the tumormap ( fig 2b ) and the sarcoma of patient 1 clustered with the 354 luad tumors of the terminal respiratory unit and proximal - inflammatory molecular subtype ( fig 2c ) , associated with the activation of receptor tyrosine kinases ( rtk ) .15 to define the transcriptional programs that drove placement of the patients tumor with the lung cancers , we conducted gene set enrichment analysis16 with genes differentially expressed between the luad cluster that contained the tumor of patient 1 ( n = 354 ) and the remaining samples in the compendium ( n = 10 , 314 ) ; we also repeated this analysis and compared the cluster for patient 1 with the remaining luad samples ( n = 529 )  . 
both analyses revealed the overexpression of members of the il6 / jak / stat3 signaling pathway ( appendix fig a2 ) , which suggests that the activation of shared signaling programs likely contributed to the tumor transcriptional phenotype of patient 1 in addition to the site of the metastatic sample . 
we next searched for genes that were significantly overexpressed in the patients tumor compared with the whole compendium and compared with only the sarcomas by using outlier statistics3 , 17 ( data supplement )  . 
consolidating the fusion - based and the rtk - based mechanisms of il6 / jak / stat3 activation , we reconstructed a candidate pathway driving patient 1s cancer ( fig 3 , appendix fig a4 )  . 
a decision was made to use ruxolitinib given ( 1 ) the over - expression of atf1 target genes , ( 2 ) the over - expression of jak1 , and ( 3 ) the available pediatric dosing information.20 in addition , ruxolitinib was favored over crizotinib because it targets downstream of both ewsr1 - atf1 and the over expressed rtks , whereas crizotinib only targets the rtks . 
t2 - weighted coronal sequence revealed a large , primarily hyperintense tumor that arises from the ventral aspect of the left tentorium , invaginating into the superior aspect of the cerebellum and causing diffuse edema there ( b ) preoperative mri . 
 higher magnification depicts epithelioid tumor cells ( eg , long arrow ) and a mitotic figure ( short arrow ) ; many of the tumor cells exhibit somewhat vacuolated cytoplasm ( h&e ; 400 magnification )  . 
diffuse membranous immunostaining is appreciated for ( g ) epithelial membrane antigen ( ema ) , and ( h ) cd99 ( all photomicrographs taken at 200 magnification )  . lethargy , was mostly bed - ridden , and had a lansky play - performance score21 of 60 ( fig 4 )  . 
the patient tolerated this therapy without significant toxicity and had stabilization of the previously rapidly growing lung nodules by recist , 22 and his lansky score improved to 90 to 100 for 5 months . 
 a patient 1 's tumor rna - seq prole in the context of 38 tumor types lung adenocarcinoma lung adenocarcinoma molecular subtypes : proximal proliferative proximal - inammatory terminal respiratory unit patient 1 patient 1 cluster patient 1 lung adenocarcinoma lung squamous cell carcinoma acute lymphoblastic leukemia acute myeloid leukemia adrenocortical cancer bladder urothelial carcinoma brain lower grade glioma breast invasive carcinoma cholangiocarcinoma cervical & endocervical cancer colon adenocarcinoma diffuse large b - cell lymphoma esophageal carcinoma pathology test data , multiple tumor types glioblastoma multiforme head & neck squamous cell carcinoma kidney chromophobe kindney clear cell carcinoma kidney papillary cell carcinoma liver hepatocellular carcinoma lung adenocarcinoma lung squamous cell carcinoma mesothelioma neuroblastoma ovarian serous cystadenocarcinoma pancreatic adenocarcinoma pheochromocytoma & paraganglioma prostate adenocarcinoma rhabdoid tumor rectum adenocarcinoma sarcoma skin cutaneous melanoma stomach adenocarcinoma testicular germ cell tumor thymoma thyroid carcinoma uterine carcinosarcoma uterine corpus endometrioid carcinoma uveal melanoma wilms tumor fig 2 . 
 ( c ) a zoomed - in view of the cluster for patient 1 and the surrounding area that contains luad tumors , now colored according to luad molecular subtypes ( proximal proliferative , blue ; proximal inflammatory , green ; terminal repiratory unit , red )  . 
the family requested that ruxolitinib be restarted for quality of life , and the patient again showed dramatic improvement in clinical status and an unexpected prolonged period of stable disease until dose reduction because of myelosuppression was required . 
 ( a ) candidate pathway that drove tumorigenesis in patient 1 was reconstructed on the basis of outlier analysis , differential expression analysis compared with normal tissues , copy number information , and literature mining ( appendix methods )  . 
all gene expression outliers depicted in the figure ( gene names written in red font ) were significant in all three comparisons : patient 1 versus all cancers , patient 1 versus lung adenocarcinomas , and patient 1 versus sarcomas . 
 ( b ) the tumor in patient 1 expresses jak1 at a strikingly higher level than those seen in all 10 , 668 tumors , which are represented by 38 tumor types studied by the tcga11 and target ( denoted pancan ) , 12 including lung adenocarcinomas ( luads ) and sarcomas ( sarcs )  . 
 ( c ) jak1 is an attractive molecular target for patient 1s tumor because it is downstream of the ewsr1 - atf1 fusion and the activate receptor tyrosine kinases ( rtks )  . 
previous functional studies implicated stat3 as an oncogene in sarcomas25 ; the current case report builds on this work and prompts investigation into the potential clinical utility of targeting this pathway . 
of note was the patients marked and rapid clinical response to treatment , which suggests that response may have been related to the modulation of cytokine expression by the medication . 
ultimately , a randomized clinical trial is necessary to assess the benefit of molecular approaches compared with the standard of care . the case also highlights tumor heterogeneity : although the majority of the metastases remained stable , one lesion rapidly became resistant . 
during treatment , the patients status improved significantly according to both body weight and lansky play performance score , which reached normal levels ; thus , the ruxi dose was increased to 60 mg twice per day at day 29 . 
 ruxi was restarted at day 348 , and a response again was noted according to both body weight and lansky performance score . ruxi at 60 mg twice daily ruxi at 40 mg twice daily ruxi stopped ; progression lansky weight ruxi stopped ruxi at 40 mg twice daily time ( days ) a serial molecular analysis of the heterogeneous lesions could inform the mechanisms of resistance ; however , it was not pursued because of the familys wishes . 
rod rassekh no relationship to disclose yaoqing shen no relationship to disclose martin jones no relationship to disclose chris dunham no relationship to disclose stephen yip no relationship to disclose sreeja leelakumari no relationship to disclose jing zhu no relationship to disclose duncan mccoll no relationship to disclose teresa swatloski no relationship to disclose sofie r . 
mungall no relationship to disclose david haussler no relationship to disclose janessa laskin honoraria : boehringer ingelheim , roche canada , astrazeneca , pfizer research funding : astrazeneca ( inst ) , roche canada ( inst ) steven j.m. 
stuart stock and other ownership interests : five3 genomics patents , royalties , other intellectual property : pending patent on creb1 inhibition for treatment of metastatic prostate cancer colleen jantzen no relationship to disclose we thank the patients family and all of our patient families for their support . 
we thank the bc cancer foundation , alex 's lemonade stand foundation for childhood cancer research , the team finn foundation , the california initiative to advance precision medicine , unravel pediatric cancer , team g childhood cancer foundation , and the st baldricks foundation . 
we thank poul sorensen , md , phd , for his assistance with the fluorescence in situ studies and charles vaske , phd , for manuscript review . affiliations yulia newton , jingchun zhu , duncan mccoll , teresa swatloski , sofie salama , david haussler , joshua m . 
stuart , and olena morozova , university of california santa cruz genomics institute ; sofie salama and david haussler , howard hughes medical institute , university of california , santa cruz , ca ; s . 
lee , and colleen jantzen , british columbia childrens hospital and british columbia children 's hospital research institute ; stephen yip , tony ng , and janessa laskin , british columbia cancer ; and yaoqing shen , martin jones , sreeja leelakumari , yussanne ma , richard moore , andrew j . 
marra , canada 's michael smith genome sciences centre , british columbia cancer , vancouver , british columbia , canada . supported by the bc cancer foundation , an alex 's lemonade stand foundation for childhood cancer innovation grant , the team finn foundation , the california initiative to advance precision medicine , unravel pediatric cancer , the team g . 
is supported by the canada research chairs program and the cihr ( fdn - 143288 )  . presented at the american association for cancer research advances in pediatric cancer research conference , november 9 - 12 , 2015 , fort lauderdale , fl . support prior presentation references 1 . 
laskin j , jones s , aparicio s , et al : lessons learned from the application of whole - genome analysis to the treatment of patients with advanced cancers . 
le rhun e , chamberlain mc , zairi f , et al : patterns of response to crizotinib in recurrent glioblastoma according to alk and met molecular profile in two patients . 
xu j , gong b , wu l , et al : comprehensive assessments of rna - seq by the seqc consortium : fda - led efforts advance precision medicine . 
neder l , scheithauer bw , turel ke , et al : desmoplastic small round cell tumor of the central nervous system : report of two cases and review of the literature . 
loh ml , tasian sk , rabin kr , et al : a phase 1 dosing study of ruxolitinib in children with relapsed or refractory solid tumors , leukemias , or myeloproliferative neoplasms : a childrens oncology group phase 1 consortium study ( advl1011 )  . 
 appendix online methods reference compendium we obtained the cancer genome atlas ( tcga ) 11 and therapeutically applicable research to generate effective treatments ( target ) 12 rna sequencing fragments per kilobase of transcript per million mapped reads ( fpkm12 ; trapnell c , et al : natl biotechnol 28 : 511 - 515 , 2010 ) gene expression data from the public data hub in the university of california santa cruz xena browser ( vivian j , et al : nat biotechnol 35 : 314 - 316 , 2017 ; goldman m , et al : nucleic acids res 43 : d812 - d817 , 2015 )  . 
the tcga and target data sets were processed with the same rna - seq pipeline ( star2 [ dobin a , et al : bioinformatics 29 : 15 - 21 , 2013 ] and rsem [ li b , et al : bmc bioinformatics 12 : 323 , 2011 ] ) by the university of california santa cruz genomics institute.13 we extracted tumor samples from these data sets and combined them into a single cohort that contained multiple adult and pediatric tumor types ( n = 10 , 668 )  . 
the expression of 18 , 357 protein - coding genes was measured ( data supplement )  . gene expression outlier analysis gene - level reads per kilobase of transcript per million mapped reads ( rpkm ) expression measurements for patient 1 's tumor were generated according to the previously published method.3 we used these data to compute fpkms by dividing each value by two . 
we proceeded to quantile normalize the expression values , using theoretical exponential distribution ( rate parameter = 1 ) as a background distribution ( bolstad bm , et al : bioinformatics 19 : 185 - 193 , 2003 )  . 
we then performed gene expression outlier analysis3 , 17 to identify transcripts significantly enriched in the patient 's tumor compared with 10 , 668 cancer samples in the reference compendium ( pan - cancer outlier analysis )  . 
we also performed the same analysis using only the 529 lung adenocarcinoma ( luad ) tumors as a reference ( luad - only outlier analysis ) and using 262 sarcoma tumors as a reference ( sarcoma - only outlier analysis )  . 
we analyzed the outlier genes for enrichment of specific pathways and signaling networks that could be targeted by available therapies using msigdb ( liberzon a et al : bioinformatics 27 : 1739 - 1740 , 2011 )  . the pan - cancer outlier analysis of the tumor in patient 1 compared with the reference compendium ( n = 10 , 668 ) revealed 906 genes significantly overexpressed in the patients tumor . 
the luad - only outlier analysis3 , 17 ( n = 529 ) revealed 1 , 176 up - outlier genes , and the same analysis against sarcomas ( n = 262 ) revealed 1 , 196 genes . 
we used the drug gene interaction database ( dgidb ; wagner ah , et al : nucleic acids res 44 : d1036 - d1044 , 2016 ) to identify genes whose protein products could be targeted by clinically available inhibitors . 
samples that cluster together exhibit similar gene expression profiles . we first computed pairwise spearman correlations ( brown gw : arch pediatr adolesc med 146 : 682 , 1992 ) between rnaseqderived gene expression profiles of all tumor pairs in our reference cohort ( n = 10 , 668 ) , including the tumor in patient 1 ( n = 10 , 669 )  . 
this produced a square correlation matrix with 10 , 669 columns and 10 , 669 rows . the tumormap method seeks to project high - dimensional genomic observations onto a two - dimensional plane while preserving original sample - to - sample distances . 
openord treats the similarities as spring constants and searches for a configuration among the samples that produces an arrangement to minimize the spring tension of the system as much as possible . 
 furthermore , to avoid overlapping and crowding samples in the dense graph components , openord ( x , y ) coordinates are snapped to their nearest hexagon to arrange all of the samples on a tiling of regular hexagons . 
the api provides the ability to interactively navigate , zoom , and explore various annotations of locations on the map , analogous to google maps and google earth applications . we applied the tumormap method to the reference cohort of 10 , 668 tumors together with the tumor of patient 1 by using transcriptional profiles of 18 , 357 genes ( data supplement )  . 
of note , the reference compendium contained 262 heterogeneous sarcomas from the tcga11 cohort . statistical robustness of tumormap placement the tumormap method belongs to the family of nearest neighbor classification methods . 
specifically , we wanted to assess whether the local neighborhood of patient 1 remains stable when only subsets of genes are used to compute pairwise similarities between samples . we subsampled , without replacement , gene expression features at 80% of the original gene features . 
we computed a local neighborhood specificity ( lnspecificity ) score as follows : lnspecificity = 1 __ * i = 1 n = 1000 s true s i _______ , | s i | where s is a set of nearest neighbors for either true or subsampled computation and |s| is the nearest neighbor set cardinality . this score represents average overlap , as a fraction , of the true local neighborhood and the perturbed local neighborhoods across all subsampling iterations . 
the sp score of patient 1 was 0.885 , which indicated that 88.5% of the top neighbors were consistently the same across 1 , 000 perturbations to the gene expression profiles , from which the tumormap visualization was computed . identification of genes associated with the patient 1 cluster in tumormap we identified genes differentially expressed between the luad cluster containing patient 1 and the 10 , 668 tumor samples in the compendium by using the linear model for microarray analysis method ( ritchie me , et al : mucleic acids res 43 : e47 , 2015 )  . 
the gene set enrichment analysis ( gsea ) 16 of the resulting list of genes revealed that il6 / jak / stat3 signaling pathway annotation was significantly enriched among these genes ( false discovery rate q value , 1.787e4 with 39 genes in the leading edge )  . we also identified differentially expressed genes between the luad cluster containing patient 1 and the remaining luad tumors in the compendium ( data supplement )  . 
the gsea analysis16 of this gene list revealed that members of the il6 / jak / stat3 signaling pathway were still driving the creation of the luad cluster containing patient 1 's tumor ( false discovery rate q value , 0 with 36 genes in the leading edge )  . identification of druggable targets we used the dgidb ( liberzon a et al : bioinformatics 27 : 1739 - 1740 , 2011 ; wagner ah , et al : nucleic acids res 44 : d1036 - d1044 , 2016 ) resource to search for drug targets among the genes that were upregulated in patient 1 by gene expression outlier analysis . 
for each sample in the cohort as well as for patient 1 , we ranked genes relative to the expression of all genes within that sample ( ie , the highest expressed gene was ranked as 1 )  . 
the percentiles are displayed in figure 3 for 12 genes that can be targeted by known cancer drugs , according to dgidb analysis ( liberzon a et al : bioinformatics 27 : 1739 - 1740 , 2011 ; wagner ah , et al : nucleic acids res 44 : d1036 - d1044 , 2016 )  . differential expression analysis relative to normal tissues for all of the genes in the reconstructed candidate driver pathway that are targetable by cancer drugs ( fig 3 ) we computed the fold change between the expression in patient 1 's tumor and in normal cells . 
for each gene , we mean - aggregated normal expression into a single value and computed log change of the expression of that gene in patient 1 compared with the aggregated normal value . histologic techniques and immunohistochemistry the tumor tissue was routinely fixed in 10% buffered formalin and then was processed in an automated fashion . 
all primary antibodies were either diluted or received as a ready to use prediluted solution from the relevant vendor . fluorescence in situ hybridization fluorescence in situ hybridization analysis was performed on formalin - fixed paraffin - embedded tumor tissue . 
antibodies and vendors primary antibody ki67 vimentin epithelial membrane antigen pan - cytokeratin ( ae1 / ae3 ) neuron specific enolase desmin synaptophysin cd99 myo - d1 myogenin s - 100 cd34 cd31 hmb - 45 leukocyte common antigen baf - 47 / ini - 1 abbreviation : rtu , ready to use . 
two forms of the fusion were identified ; the ewsr1 - atf1 form had higher expression than the reciprocal atf1ewsr1 forbzip , basic leucine zipper domain , which mediates sequence specific dna binding properties and the leucine zipper that is required to hold together ( dimerize ) two dna binding regions ; pkid , phosphorylated kinase - inducible - domain ; rrm , rna recognition motif ; zf , zn - finger in ran binding protein and others . 
all gene expression outliers depicted in this figure were significant in all three comparisons : patient 1 versus all cancers , patient 1 versus lung adenocarcinomas , and patient 1 versus sarcomas . 
romero - cordoba et al we appreciate the commentary and analysis by romero - cordoba et al1 in their response to our recent article in jco precision oncology.2 our research groups pursue the common goal of elucidating immunologic differences between breast cancers that could inform future immunotherapy strategies . 
all immune metrics , including counts of tumor inltrating lymphocytes and other immune cells , as well as immune gene signatures , represent a continuum and display substantial variations across samples . nevertheless , it is possible to dene more versus less immune - rich cancers by any of these metrics , and for some types of analyses , such dichotomization is necessary , even using an arbitrary threshold . our study focused on the question of whether immune - rich estrogen receptor ( er ) positive and triple - negative ( tn ) breast cancers ( bcs ) have similar or different immune microenvironments . 
to perform this comparison across multiple types of molecular data , we had to dene a shared denition of immunerichness ; we used the 75th percentile of a total tumor inltrating lymphocyte gene expression score , which encompasses the expression of 11 immune cell populations , calculated across all samples as the threshold . 
we were delighted to see that this admittedly arbitrary denition of immune - richness corresponded reasonably well with the tnbc immune classication developed by romero - cordoba et al3 : 62% of our immune - rich cancers were classied as ima ( t cell - inamed ) and 21% as imc ( immune warm )  . 
several tnbc classications have been proposed using this method.3 - 6 despite the high and mostly positive correlation in immune gene expression in breast cancer tissues , the different classication schemas yield discordant results in a substantial minority of cases , partly because of different design principles and partly because of methodology . 
however , which classication schema or molecular marker is most useful in the clinic is yet to be determined in clinical trials . the clear majority of immune biomarker studies in breast cancer focus on tnbc because of the higher prevalence of tumor inltrating lymphocytes in this subtype . 
we hope that our article draws attention to immunotherapy opportunities in er - positive cancers , because in absolute numbers , there are more immune - rich er - positive cancers than immune - rich tnbcs . 
omeara t , marczyk m , qing t , et al : immunological differences between immune - rich estrogen receptor - positive and immune - rich triple - negative breast cancers . 
prado - v azquez g , g amez - pozo a , trilla - fuertes l , et al : a novel approach to triple - negative breast cancer molecular classication reveals a luminal immune - positive subgroup with good prognoses . 
j ez equel p , loussouarn d , gu erin - charbonnel c , et al : gene - expression molecular subtyping of triple - negative breast cancer tumours : importance of immune response . 
sohal , md , mph1 a 53 - year - old woman with no signicant medical history presented to her primary care physician with fatigue and anorexia in may 2016 . 
she subsequently underwent a liver biopsy in september 2016 , and the pathology was consistent with adenocarcinoma ( strongly and diffusely positive for ck7 , with weak , patchy expression for ck20 ; programmed death ligand 1 , 2% )  . 
she was diagnosed with cancer of unknown primary , despite extensive workup revealing caudal - type homeobox 2 , thyroid transcription factor 1 , napsin a , gata - 3 , arginase , receptor 2 , and human epidermal growth factor mammaglobin negativity on immunohistochemical staining and no lesions on esophagogastroduodenoscopy or repeat colonoscopy ; however , the pathology was thought to be suggestive of a biliary origin . she received three doses of uorouracil and oxaliplathowever , she deteriorated clinically . 
genomic sequencing performed on her initial biopsied tissue ( foundationone ; foundation medicine , cambridge , ma ) was notable for mutations in braf v600e , apc , and cdkn2a ( table 1 )  . 
her case was reviewed at our genomics tumor board in december 2016 , and the recommendation was made to trial ( nci - match [ national pursue either a clinical cancer institute molecular analysis for therapy choice ; clinicaltrials.gov identier : nct02465060 ] or mypathway [ clinicaltrials.gov identier : nct02091141 ] ; each trial , the vemurafenib arm was open and available at the time ) or off - label treatment with dabrafenib and trametinib based on the asco 2016 update.1 at our institution , if two outside experts agree with the scientic data presented , off - label therapy can be approved on a case - by - case basis . given that she was not a clinical trial candidate because of the high bilirubin , off - label treatment with dabrafenib and trametinib was started in december 2016 . 
it is important to note that hepatic dysfunction , although closely monitored , is not a contraindication to initiating treatment with dabrafenib and trametinib . although serum alt elevations can occur in approximately 35% to 42% of patients receiving combination therapy , elevations greater than ve times the upper limit of normal occur in only 4% of such patients . 
in addition , there have been no instances of clinically apparent acute liver injury or hepatic failure in prelicensure studies , and there have been no published reports of clinically apparent hepatotoxicity with either drug.2 our patient was noted to experience clear clinical benet , with her total bilirubin and ast normalizing within just 2.5 months of treatment initiation and the remainder of her liver function enzymes continuing to downtrend . 
she had remarkable disease response , with radiographic ( fig 1 ) and clinical improvement until may 2018 ( a duration of almost 18 months ) , at which point there was concern about radiographic progression . 
cisplatin and gemcitabine were considered but not agreed to by the patient and family because of prior potential failure of oxaliplatin . her other option was to pursue a phase i clinical trial at an outside institution . 
her case was again discussed at the genomics tumor board in july 2018 , at which time there was deemed to be no targetable therapeutic option beyond the braf alteration , for which she had already been treated . 
interestingly , her second biopsy did identify some additional , albeit nontherapeutic , alterations not seen on the rst : rictor amplication and mtap loss of exons 6 to 8 . 
in a study analyzing these alterations in melanoma cell lines , it was found that v600e - mutant braf inhibits mammalian target of rapamycin complex 2induced phosphorylation of akt ser473 in the presence of pten . 
therapeutic implications of genomic sequencing , november 2016 sadaps and sohal fda - approved therapies genomic alterations detected * patients tumor type another tumor type potential clinical trials braf v600e none cobimetinib dabrafenib regorafenib nct01827384 nct01531361 ( phase i , targeting braf ) nct02034110 ( phase ii , targeting braf , mek ) trametinib nct02465060 ( phase ii , nci - match ) vemurafenib nct02693535 ( phase ii , tapur study ) apc r856c cdkn2a / b : cdkn2a loss exon 1 and cdkn2b loss none none none none none none abbreviations : fda , us food and drug administration ; nci - match , national cancer institute molecular analysis for therapy choice ; tapur , targeted agent and proling utilization registry . * variants of unknown signicance , 10 ; microsatellite stable ; low tumor mutational burden , 4 . discovered rictor amplication contributed to the development of braf inhibitor resistance . cleveland clinic to allow publication of this case have been obtained . per the request of the patient and her family and preliminary data extrapolated from the keynote 022 phase ii trial ( clinicaltrials.gov identier : nct02130466 ) , pembrolizumab ( via compassionate use ) was added to her regimen of dabrafenib plus trametinib starting in august 2018 . 
although high tumor mutational burden ( tmb ) has been demonstrated to be a useful biomarker in predicting immunotherapy responsiveness , 4 , 5 most studies ( eg , mypathway ) have used much higher tmb cutoffs , generally of at least 16 or more mutations per megabase , which far exceeds the tmb score obtained for our patient in either of her next - generation sequencing reports . 
she died in january 2019 , secondary to septic shock with multiorgan failure . because the patient died , consent could not be obtained ; however , no identifying personal health information has been disclosed . 
patients who receive standard - of - care gemcitabine and cisplatin chemotherapy have an overall survival ( os ) benet of 3.6 months when compared with gemcitabine monotherapy.6 even then , most patients develop treatment resistance within a few months , and median os remains less than 1 year . 
outcomes for second - line therapies have not been promising , with median progression - free survival of only a few months.7 - 13 recent advances in precision medicine have allowed for the identication of potentially targetable mutations in the effort to improve therapeutic outcomes in this population . promising targets identied in cholangiocarcinoma thus far include idh 1 / 2 , fgfr , her2 , and , the focus of our patient case , braf mutations.14 braf encodes protein kinases that function downstream of ras as part of the mitogenactivated protein kinase signaling cascade , which promotes cell proliferation , survival , and transformation . 
therapeutic implications of repeat genomic sequencing , june 2018 fda - approved therapies genomic alterations detected * patients tumor type another tumor type potential clinical trials braf v600e none cobimetinib dabrafenib regorafenib trametinib vemurafenib nct02091141 ( phase ii , mypathway ) nct02070549 ( phase i , targeting mek ) nct01989585 ( phase i / ii ) nct02428712 ( phase i / ii , targeting braf / craf ) nct02693535 ( phase ii , tapur study ) nct02034110 ( phase ii , targeting braf , mek ) apc r856c cdkn2a / b : cdkn2a loss exon 1 and cdkn2b loss riktor amplication none none none none none none none none nct02795156 ( phase ii ) nct02466802 ( phase i ) nct02097225 ( phase i ) nct01531361 ( phase i ) nct02576444 ( phase ii ) nct02142803 ( phase i ) nct03065062 ( phase i ) nct02159989 ( phase i ) nct02719691 ( phase i ) nct02583542 ( phase i / ii ) mtap loss of exons 6 to 8 none none none abbreviations : fda , us food and drug administration ; tapur , targeted agent and proling utilization registry . * variants of unknown signicance , 10 ; microsatellite stable ; low tumor mutational burden , 1 . and hyperactivate its downstream signaling.15 braf mutations are reported in up to 22% of biliary tract cancers , among which it is most commonly seen in intrahepatic cholangiocarcinoma . 
braf mutations have typically been associated with poor prognosis in patients with cholangiocarcinoma , including worse os , higher tumor grade , and chemotherapy resistance.16 there have been a few case reports on the use of dabrafenib and trametinib in cholangiocarcinoma , describing remarkable clinical and radiographic responses in patients who experienced progression during standard - of - care chemotherapy and who continued to do well 5 to 9 months out.17 , 18 most recently , a phase ii international clinical trial of dabrafenib plus trametinib in braf - mutated rare gi cancers was reported as a late - breaking presentation at the 30th european organisation for research and treatment of cancernational cancer instituteamerican association for cancer research symposium on molecular targets and cancer therapeutics . 
sohal honoraria : foundation medicine consulting or advisory role : perthera research funding : novartis ( inst ) , celgene ( inst ) , oncomed ( inst ) , bayer healthcare pharmaceuticals ( inst ) , genentech ( inst ) , bristol - myers squibb ( inst ) , agios ( inst ) , incyte ( inst ) , loxo ( inst ) travel , accommodations , expenses : foundation medicine no other potential conicts of interest were reported . references flaherty k , davies ma , grob jj , et al : genomic analysis and 3 - y efcacy and safety update of combi - d : a phase iii study of dabrafenib ( d ) + trametinib ( t ) vs d monotherapy in patients with unresectable or metastatic braf v600e / k - mutant cutaneous melanoma . 
plos one 7 : e42598 , 2012 chan ta , yarchoan m , jaffee e , et al : development of tumor mutation burden as an immunotherapy biomarker : utility for the oncology clinic . 
ann oncol 30 : 44 - 56 , 2019 legrand f , gandara d , mariathasan s , et al : association of high tissue tmb and atezolizumab efcacy across multiple tumor types . 
j clin oncol 36 , 2018 ( suppl ; abstr 12000 ) valle j , wasan h , palmer dh , et al : cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer . 
croitoru a , gramaticu i , dinu i , et al : fluoropyrimidines plus cisplatin versus gemcitabine / gemcitabine plus cisplatin in locally advanced and metastatic biliary tract carcinomaa retrospective study . 
fornaro l , vivaldi c , cereda s , et al : second - line chemotherapy in advanced biliary cancer progressed to rst - line platinum - gemcitabine combination : a multicenter survey and pooled analysis with published data . 
brugnara s , sicher m , bonandini em , et al : treatment with combined dabrafenib and trametinib in brafv600e - mutated metastatic malignant melanoma : a case of long - term complete response after treatment cessation . 
loaiza - bonilla a , clayton e , furth e , et al : dramatic response to dabrafenib and trametinib combination in a braf v600e - mutated cholangiocarcinoma : implementation of a molecular tumour board and next - generation sequencing for personalized medicine . 
presented at the 30th eortc - nci - aacr symposium on molecular targets and cancer therapeutics , dublin , ireland , november 13 - 16 , 2018 ( abstr 2 ) 20 . 
 c profound immunotherapy response in mismatch repair - deficient breast cancer introduction because most patients with cancer do not benefit from immunotherapies such as anti - pd1 , the identification of biomarkers to select patients for immunotherapy is crucial . 
mismatch repair deficiency ( dmmr ) is strongly associated with responsiveness to anti - pd11 in gi and endometrial cancers and can be readily detected using immunohistochemistry ( mlh1 , msh2 , mhs6 , and pms2 ) and / or polymerase chain reaction ( pcr ) for microsatellite instability ( msi )  . 
the response rate to anti - pd1 in dmmr colorectal cancers was no less than 40% , and was even 70% in other gi and endometrial dmmr cancers , whereas it was 0% in mmr - proficient colorectal cancer.1 although not routinely tested , dmmr can be found in other cancer types outside gi and endometrial cancers , in a frequency of approximately 2% to 3%.2 breast cancer is an example of a tumor type in which dmmr is rare ( frequency is 0% to 1% ) .3 responses to checkpoint blockade in breast cancer are relatively infrequent ( 5% to 19% ) 4 but can be durable , illustrating the urgent need for predictive biomarkers to select those few patients who could benefit from immunotherapy . 
three years before , she received treatment for a triple negative breast carcinoma ( tnbc )  . morphology , immunohistochemistry , and cancer gene - panel sequencing of the lung and stomach and the archived breast biopsy findings confirmed the diagnosis of lung and stomach metastases from the former tnbc ( appendix )  . immunohistochemistry findings showed loss of mlh1 and pms2 , and somatic hypermethylation of the mlh1 promotor was found . 
however , focal pd - l1 expression was observed on the tils . short - term response was seen with palliative carboplatin , yet , after six cycles , progression was observed . 
in addition , a partial response of all other lesions was observed as determined by response evaluation criteria in solid tumors ( recist ) 1.1.5 after 12 cycles , this response is still ongoing . in this case , extensive revision of three seemingly separate lesions led to the identification of this rare dmmr breast cancer . 
we would like to bring this case to the attention of colleagues to make a case for msi testing in patients with metastatic cancer beyond the typical lynch - associated cancers . 
linn jolanda van dieren all authors : netherlands cancer institute , amsterdam , netherlands . the dutch cancer society provided funding for the setup and data management of the clinical trial in which the patient in this report is participating , as well as translational fellowships to m.k. 
 ( a ) a deep 5 - cm ulcerative tumor located at the lesser curvature of the stomach . ( b ) disappearance of the tumor after three cycles of nivolumab . 
horlings , jolanda van dieren collection and assembly of data : marleen kok , hugo m . horlings , petur snaebjornsson , myriam chalabi , sabine c . linn , jolanda van dieren data analysis and interpretation : marleen kok , hugo m . horlings , petur snaebjornsson , ton n . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . marleen kok research funding : bristol - myers squibb ( inst ) travel , accommodations , expenses : roche hugo m . 
horlings no relationship to disclose petur snaebjornsson no relationship to disclose references 2509 - 2520 , 2015 myriam chalabi research funding : bristol - myers squibb ( inst ) travel , accommodations , expenses : roche / genentech ton n . 
schumacher employment : kite pharma stock and other ownership interests : kite pharma , neon therapeutics , aimm therapeutics honoraria : neon therapeutics , aimm therapeutics research funding : merck , bristol - myers squibb christian u . 
blank consulting or advisory role : genentech ( inst ) , msd oncology ( inst ) , bristol - myers squibb ( inst ) , novartis ( inst ) , glaxosmithkline ( inst ) , pfizer ( inst ) , astrazeneca ( inst ) , eli lilly ( inst ) research funding : bristol - myers squibb ( inst ) , novartis ( inst ) travel , accommodations , expenses : pfizer , roche sabine c . 
linn consulting or advisory role : astrazeneca ( inst ) , ibm ( inst ) , bayer ( inst ) research funding : genentech ( inst ) , astrazeneca ( inst ) , bristol - myers squibb ( inst ) patents , royalties , other intellectual property : named inventor on a patent for a brca1ness gene expression classifier in breast cancer ( inst ) travel , accommodations , expenses : roche jolanda van dieren no relationship to disclose 1 . 
hall mj , gowen k , sanford em , et al : evaluation of microsatellite instability ( msi ) status in 11 , 573 diverse solid tumors using comprehensive genomic profiling ( cgp )  . 
subsequently , microsatellite instability ( msi ) was assessed by a pentaplex polymerase chain reaction - based assay using fluorescently labeled primers of five mononucleotide repeat targets ( bat25 , bat26 , nr24 , nr21 , nr27 ) , followed by fragment analysis . 
promoter methylation status of mmr genes was assessed by multiplex ligation - dependent probe amplification ( mlpa , me011 - b2 kit ; mrc holland , amsterdam , netherlands )  . 
in brief , paraffin sections were cut at 4 mm , heated at 75c for 28 minutes , and deparaffinized in the instrument with ez prep solution ( roche )  . 
two pathologists scored pd - l1 staining based on the percentage of positive tumor and / or immune cells . assessment of tumor - infiltrating lymphocytes tumor - infiltrating lymphocytes ( tils ) were evaluated according to the guidelines of the international tils working group for invasive breast carcinoma ( luen sj , et al : lancet oncol 18 : 52 - 62 , 2017 ; salgado r , et al : ann oncol 26 : 259 - 271 , 2015 )  . 
dmmr / microsatellite high ( msi - h ) breast cancers were among the responding tumors that led to the recent us food and drug administration accelerated approval of pembrolizumab for any patient with a dmmr / msi - h metastatic solid tumor.2 the authors conclude that they would like to make a case for msi testing in patients with metastatic cancer beyond typical lynch - associated cancers.1 i also propose the study of whether patients now identified with dmmr breast cancers might harbor germline mismatch repair ( mmr ) - mutated genes and , therefore , would be molecularly defined as lynch syndrome family members . the us food and drug administration accelerated approval and suggestion by kok et al represent an opportunity to clarify whether breast cancer should be considered a lynch - associated cancer . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . steven sorscher honoraria : celgene , pfizer , puma biotechnology speakers bureau : celgene , pfizer , puma biotechnology travel , accommodations , expenses : celgene , pfizer , puma biotechnology 1 . 
 e beyond the androgen receptor : targeting actionable drivers of prostate cancer see accompanying article doi : more than 70 years ago , huggins et al1 exploited the exquisite and unique dependence of prostate cancer ( pc ) on androgenic hormones for survival and growth using a treatment strategy that foreshadowed the clinical benefits underlying the application of precision oncology . 
today , variations of androgen receptor ( ar ) pathway targeting remain the cornerstone of the initial management of men with metastatic pc , usually with gratifying responsesalthough unfortunately few cures . 
however , the advent and application of technologies capable of deep assessments of the full spectrum of molecular aberrations that underlie the development and progression of malignant neoplasms now forecast the near - term potential of new therapeutic approaches poised to change the near - exclusive reliance on ardirected interventions as initial treatment for advanced pc . studies using whole - exome sequencing and comprehensive rna sequencing , supported through the cancer genome atlas2 and stand up to cancer / prostate cancer foundation3 initiatives , have profiled hundreds of localized and castration - resistant metastatic pcs ( crpcs ) , resulting in the identification of numerous recurrent driver aberrations that comprise signaling programs and pathways for which there are pharmacologic inhibitors . 
lacking in these previous studies was an assessment of untreated metastatic tumors . in the article accompanying this editorial , abida et al4 have taken the first step in filling this knowledge gap . using a predefined gene panel , abida et al4 evaluated mutations and copy losses in key oncogenes and tumor suppressors across a spectrum of localized pc , untreated metastatic disease , and crpc . 
the tumor assays were conducted in a clinical laboratory improvement amendments environment using real - world clinical samples , which demonstrates the feasibility of molecular profiling to guide therapeutics , even in challenging situations such as evaluating bone metastases . overall , a high frequency of potentially actionable alterations were identified that included components of the phosphatidylinositol 3 - kinase ( pi3k ) pathway ( 24% ) , mitogen - activated protein kinase pathway ( 5% ) , and b - catenin program ( 15% )  . this study also confirmed previous reports that aberrations in genes involved in dna repair are common , particularly those involved in the process of homologous recombination repair.5 overall , 22% of tumors had somatic aberrations in a dna repair gene , and 19% were found to have a putative deleterious germ line mutation . 
this pc subtype is notable for therapeutics such as poly ( adp - ribose ) polymerase inhibitors and platinum - based chemotherapy that could exploit a dna repair vulnerability.6 , 7 however , it is important to recognize that it is currently unclear whether each gene comprising the dna repair machinery will exhibit enhanced responses to these agents , nor is it clear whether each type of aberration within a particular dna repair gene will confer congruent outcomes with respect to treatment response . 
preclinical studies using functional readouts of dna repair proficiency and clinical trials coupled with careful assessments of each dna damage response gene aberration will be required to confirm predictive associations . overall , there was substantial heterogeneity in the composition of putative cancer drivers between individuals , a finding that supports avoiding a onesize - fits - all approach to therapy . 
 number of matched tumors from the same patient demonstrated consistent truncal aberrations in tumor suppressors such as tp53 and brca2 and amplification of ar across each tumor evaluated . these results indicate that sampling a single metastatic site provides a reasonable assessment of the major molecular alterations that may influence treatment decisions . 
however , analyses of tumors across intervals of time and treatment indicated that molecular evolution does occur , with higher mutation counts in tumors sampled at later time points . consequently , it is not clear whether sampling a primary tumor or a metastatic tumor early in the course of disease will accurately reflect driver or resistance mechanisms that are operating later in the course of cancer progression . a distinguishing feature of the abida et al4 study involves the acquisition and assessment of metastatic tumors that had not been exposed to ar - directed therapy or other systemic treatment . comparing the features of these metastases with those of localized tumors serves to identify drivers of the metastatic process , and comparing their molecular composition with that of tumors withstanding ar - directed therapy serves to identify mechanisms contributing to treatment resistance . in the first comparison , it is notable that aberrations in ar did not occur , emphasizing that ar mutation and copy - number gain , frequent events in crpc , occur in response to pressures exerted by ar pathway inhibition and rarely or never arise as de novo drivers of pc . 
in addition to ar , the frequency of aberrations in tp53 , rb1 , pten , and atm was significantly increased in crpc compared with metastatic noncastrate disease , indicating potential roles in mediating castration resistance . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . interactions with the ar or ar program or if they promote pc growth through mechanisms that bypass ar requirements . 
although enriched in crpc , a substantial number of treatment - nave tumors did exhibit mutations or structural alterations in these genes , suggesting that cotargeting the aberrant pathways concurrently with the ar may serve to circumvent predestined pathways of resistance . that drug combinations current therapy for metastatic pc generally follows a paradigm of sequentially deploying a given therapeutic such as androgen deprivation to the point of resistance , switching to another agent such as abiraterone or enzalutamide , and treating again to resistance , until options are exhausted . 
nelson consulting or advisory role : janssen oncology , astellas pharma , genentech acknowledgment supported by national institutes of health grants no . p30ca15704 and p50ca097186 , us department of defense grant no . 
cancer genome atlas research network : the molecular taxonomy of primary prostate cancer . arch surg 43 : 209 - 223 , 1941 cell 163 : 1011 - 1025 , 2015 3 . 
mateo j , carreira s , sandhu s , et al : dna - repair defects and olaparib in metastatic prostate cancer . n engl j med 373 : 1697 - 1708 , 2015 7 . 
schweizer mt , zhou xc , wang h , et al : the influence of prior abiraterone treatment on the clinical activity of docetaxel in men with metastatic castration - resistant prostate cancer . 
smith mr , saad f , rathkopf de , et al : clinical outcomes from androgen signaling - directed therapy after treatment with abiraterone acetate and prednisone in patients with metastatic castration - resistant prostate cancer : post hoc analysis of cou - aa - 302 . 
30 years ago with the identication of the reciprocal translocation , t ( 11 ; 22 ) ( q24 ; q12 ) , otherwise known as ews - fl1.1 , 2 in the time since , multiple other fusion partners with ews have been identied that t a similar ewing sarcoma phenotype.3 , 4 when ews fusions are not identied , tumors with histologic features of ewing sarcoma have been labeled as primitive neuroectodermal tumors . 
in 2012 , pierron et al5 identied a subset of ewing - like tumors harboring paracentric inversion on the short arm of chromosome x , resulting in the fusion of the bcor and ccnb3 genes.5 since that discovery , several small case series have further elucidated the clinical , morphologic , and genomic differences that make this diagnosis distinct from other round cell sarcomas , most notably ewing sarcoma.6 - 8 though distinct from ewing sarcoma , most bcorccnb3fused sarcomas ( bcs ) are treated with upfront compressed chemotherapy with vincristine , doxorubicin , cyclophosphamide , ifosfamide , and etoposide plus local control with surgery and / or radiation . bcs shares similar event - free and overall survival rates with the standard ews - fli1fused ewing sarcoma using this treatment strategy.6 - 8 despite the growing knowledge base related to bcs , little is known about potential drug targets related to this disease entity , especially with regard to treatment of disease recurrence . 
we highlight the treatment of a young patient who had multiply - relapsed disease with the us food and drug administrationapproved cyclindependent kinase 4 / 6 ( cdk4 / 6 ) inhibitor palbociclib ; the tumor harbored a bcor - ccnb3 fusion and a germline variant in cdkn2b , and treatment resulted in a complete response and no evidence of disease 25 months into therapy . case history our male patient initially presented in 2010 at 1 year of age with a xed mass on his back . 
a core needle biopsy was performed , which revealed a malignant , small , round , blue cell tumor along with small amounts of benign brofatty tissue and skeletal muscle . 
immunohistochemical stains were positive for cd99 , fli1 , and vimentin and were negative for nse , synaptophysin , myf4 , gaf , cd45rb , and tdt consistent with a primitive neuroectodermal tumor . 
the patient started chemotherapy per childrens oncology group protocol aews0031 , regimen b2 , with ifosfamide , etoposide , vincristine , doxorubicin , and cyclophosphamide . gross total resection was not feasible at the time per neurosurgery , and the patient received 57.6 gy of proton beam radiation in october 2010 . 
2 years but then developed multiple local recurrences without metastases from 2013 to 2017 and underwent numerous surgeries , along with multiple different early - phase childrens oncology group therapeutic studies , as outlined in the timeline in figure 1a . 
after the most recent recurrence in october 2016 , the patient was referred to our pediatric cancer precision genomics prograbecause of the ndings outlined here in the results , we chose to start palbociclib in february 2017 . 
 diagnosis radiation debulking surgeries ( n = 4 ) case report molecular tumor analysis debulking surgery debulking surgery april 2010 2010 july 2013 2014 2015 2016 june 2016 2016 2016 2017 march 2019 initial therapy aews0031 arm b : vincristine , doxorubicin , cyclophosphamide , ifosfamide , etoposide cog phase i advl1212 : crizotinib , cyclophosphamide , topotecan phase ii advl1322 : pazopanib phase i advl1315 : axitinib metronomic therapy molecular - guided therapy palbociclib initial diagnosis before palbociclib 2 cycles of palbociclib 3 cycles of palbociclib 2 years of palbociclib fig 1 . 
there was concern for progression after 2 cycles of palbociclib , but there was a 2 - month lag between the before palbociclib scan and actually starting drug , so interval progression likely occurred in this timeframe . 
ned , no evidence of disease . results whole - genome sequencing , rna sequencing ( rna - seq ) analysis , germline exome sequencing , and protein evaluation were performed at the clinical laboratory improvement amendment ( clia ) approved laboratory , nantomics ( culver city , ca )  . 
the mutational burden of the tumor was relatively low at 75 , 046 somatic mutations , with only 88 somatic mutations mapping to protein coding regions ( circos plot in fig 2a )  . 
additionally , an undescribed somatic mutation in the smo gene ( smo n476s ) was identied in the tumor , and germline sequencing revealed a cdkn2b n41d missense variant , which was heterozygous in both the germline and tumor genomes of this patient . 
rna - seq was also performed by nantomics , and mrna transcripts were ranked by abundance , which could be associated with increased pathway activity and sensitivity to a targeted drug . 
with the exception of the destruction box in ccnb3 , all functional domains from each encoded protein remain intact in the bcor - ccnb3 fusion prote ( c ) the cdk4 / 6 pathway is a gene regulatory program controlled by multiple tiers of protein kinases and transcriptional regulators . increases in cyclin - d or cdk4 or 6 protein can lead to phosphorylation of the rb1 - e2f tumor suppressor complex . 
upon phosphorylation of rb1 , the e2f1 - 3 transcription factors are released from the complex and are able to bind to the promoters of target genes , driving activation of transcription . 
in the several case series describing bcs , the median age of diagnosis is in the teenage years , with the youngest patient recorded at age 2.5 - 8 , 11 again , because of the age of presentation , one could be concerned about an inherited cancer syndrome . 
this congenital tumor also harbored a smarcb1 / ini1 gene deletion common to malignant rhabdoid tumor , epithelioid sarcomas , and epithelioid malignant peripheral nerve sheath tumor that also , table 1 . 
overexpression of relevant tumor - promoting pathways gene status gene function ccnd1 ccnd2 cdk4 e2f1 e2f2 e2f3 cdc25a cdc25c cdc25b top2a bub1 bub1b overexpressed activating cyclin for cdk4 and cdk6 overexpressed activating cyclin for cdk4 and cdk6 overexpressed rb1 protein kinase overexpressed rb1 - regulated transcription factor overexpressed rb1 - regulated transcription factor overexpressed rb1 - regulated transcription factor overexpressed e2f - regulated gene overexpressed e2f - regulated gene overexpressed e2f - regulated gene overexpressed e2f - regulated gene overexpressed e2f - regulated gene overexpressed e2f - regulated gene note . 
the expression status for a gene was classied as overexpressed if its tpm exceeded the genes upper 5th percentile.30 abbreviations : cdk , cyclin - dependent kinase ; e2f , e2 transcription factor ; rb1 , retinoblastoma gene ; tpms , transcripts per million . when found germline , is known to cause rhabdoid tumor predisposition syndrome.12 - 16 in the case reported by alfaro - cervello et al , 12 ini1 germline analysis was not performed . 
our patient also harbored a germline heterozygous missense variant , cdkn2b n41d . is unclear what role this germline cdkn2b n41d variant could play in sarcomagenesis , as cancer risks associated with cdkn2a / b gene variants include melanoma , pancreatic cancer , and astrocytomas.17 , 18 there is a recent short report from jouenne et al19 that found an increased risk of soft tissue sarcoma development with germline loss of cdkn2a , though no data exist conrming this risk with cdkn2b variants . 
sunita et al20 showed that the specic cdkn2b n41d variant , which encodes p15 ( ink4b ) , is unable to bind to the cdk6 protein , leading to loss of function of cdkn2b , which could lead to dysregulated control of s - phase entry . 
though this variants contribution to tumorigenesis is intriguing , ckdn2b was normally expressed in our patients tumor , and there are no data suggesting that this impaired binding to cdk6 leads to mrna overexpression along multiple levels of the cdk4 / 6 pathway . despite discovering alterations of several key regulators of the cdk4 / 6 pathway in this tumor , none have been proven to serve as clinical biomarker for sensitivity to cdk4 / 6 inhibitors.21 in a preclinical ewing sarcoma orthotopic xenograft model with cdkn2a deletion , palbociclib was able to greatly suppress growth despite doxorubicin resistance of this model.22 in other sarcoma subtypes , palbociclib reduced tumor burden in murine preclinical models.23 - 25 clinically , there is phase ii evidence of palbociclibs efcacy in adults with liposarcoma26 , 27 and leiomyosarcoma.28 despite growing evidence in these sarcomas , there are no published data testing cdk4 / 6 inhibitors in bcs . 
for our patient with bcs , the expression status for a gene was classied as overexpressed if its tpm exceeded the genes upper 5th percentile of per - gene rna - seq by expectation - maximization ( rsem ) transcript - per - million ( tpm ) values for a collection of rna sequencing ( rna - seq ) datasets from the cancer genome atlas normal samples . 
in the study by pierron et al , 5 10 bcs tumors were analyzed for certain mrna expression ; overexpression was determined as described in pierron manuscript methods . abbreviations : bcs , bcor - ccnb3fused sarcomas ; nd , not disclosed . pathway , and ( 3 ) the presence of a germline cdkn2b variant . 
ferguson , md , ms , 705 riley hospital dr , ri 4340 , indianapolis , in 46202 ; twitter : @rileychildrens ; e - mail : micjferg@iu.edu. support supported by grant no . 
cancer genet cytogenet 21 : 185 - 208 , 1986 turc - carel c , aurias a , mugneret f , et al : chromosomes in ewings sarcoma : i . 
an evaluation of 85 cases of remarkable consistency of t ( 11 ; 22 ) ( q24 ; q12 )  . cancer genet cytogenet 32 : 229 - 238 , 1988 ginsberg jp , de alava e , ladanyi m , et al : ews - fli1 and ews - erg gene fusions are associated with similar clinical phenotypes in ewings sarcoma . 
j clin oncol 17 : 1809 - 1814 , 1999 shing dc , mcmullan dj , roberts p , et al : fus / erg gene fusions in ewings tumors . 
cancer res 63 : 4568 - 4576 , 2003 pierron g , tirode f , lucchesi c , et al : a new subtype of bone sarcoma dened by bcor - ccnb3 gene fusion . 
nat genet 44 : 461 - 466 , 2012 peters tl , kumar v , polikepahad s , et al : bcor - ccnb3 fusions are frequent in undifferentiated sarcomas of male children . 
mod pathol 28 : 575 - 586 , 2015 puls f , niblett a , marland g , et al : bcor - ccnb3 ( ewing - like ) sarcoma : a clinicopathologic analysis of 10 cases , in comparison with conventional ewing sarcoma . 
am j surg pathol 38 : 1307 - 1318 , 2014 kao , yc , owosho aa , sung ys , et al : bcor - ccnb3 fusionpositive sarcomas : a clinicopathologic and molecular analysis of 36 cases with comparison to morphologic spectrum and clinical behavior of other round cell sarcomas . 
riggi n , suv `a ml , suv `a d , et al : ews - fli - 1 expression triggers a ewings sarcoma initiation program in primary human mesenchymal stem cells . 
matsuyama a , shiba e , umekita y , et al : clinicopathologic diversity of undifferentiated sarcoma with bcor - ccnb3 fusion : analysis of 11 cases with a reappraisal of the utility of immunohistochemistry for bcor and ccnb3 . 
chan ak , han sj , choy w , et al : familial melanoma - astrocytoma syndrome : synchronous diffuse astrocytoma and pleomorphic xanthoastrocytoma in a patient with germline cdkn2a / b deletion and a signicant family history . 
campa d , pastore m , gentiluomo m , et al : functional single nucleotide polymorphisms within the cyclin - dependent kinase inhibitor 2a / 2b region affect pancreatic cancer risk . 
oncotarget 7 : 57011 - 57020 , 2016 jouenne f , chauvot de beauchene i , bollaert e , et al : germline cdkn2a / p16ink4a mutations contribute to genetic determinism of sarcoma . 
agarwal sk , mateo cm , marx sj : rare germline mutations in cyclin - dependent kinase inhibitor genes in multiple endocrine neoplasia type 1 and related states . j clin endocrinol metab 94 : 1826 - 1834 , 2009 21 . 
murakami t , singh as , kiyuna t , et al : effective molecular targeting of cdk4 / 6 and igf - 1r in a rare fus - erg fusion cdkn2a - deletion doxorubicin - resistant ewings sarcoma patient - derived orthotopic xenograft ( pdox ) nude - mouse model . 
perez m , muoz - galv an s , jim enez - garca mp , et al : efcacy of cdk4 inhibition against sarcomas depends on their levels of cdk4 and p16ink4 mrna . oncotarget 6 : 40557 - 40574 , 2015 24 . 
vlenterie m , hillebrandt - roeffen mh , schaars ew , et al : targeting cyclin - dependent kinases in synovial sarcoma : palbociclib as a potential treatment for synovial sarcoma patients . 
b ohm mj , marienfeld r , j ager d , et al : analysis of the cdk4 / 6 cell cycle pathway in leiomyosarcomas as a potential target for inhibition by palbociclib . 
dickson ma , tap wd , keohan ml , et al : phase ii trial of the cdk4 inhibitor pd0332991 in patients with advanced cdk4 - amplied well - differentiated or dedifferentiated liposarcoma . 
dickson ma , schwartz gk , keohan ml , et al : progression - free survival among patients with well - differentiated or dedifferentiated liposarcoma treated with cdk4 inhibitor palbociclib : a phase 2 clinical trial . 
elvin ja , gay lm , ort r , et al : clinical benet in response to palbociclib treatment in refractory uterine leiomyosarcomas with a common cdkn2a alteration . oncologist 22 : 416 - 421 , 2017 1072 - 1074 , 2015 29 . 
we developed and validated a genome - wide dna methylationbased classifier to differentiate between osteosarcoma , ewing sarcoma , and synovial sarcoma . methods dna methylation status of 482 , 421 cpg sites in 10 ewing sarcoma , 11 synovial sarcoma , and 15 osteosarcoma samples were determined using the illumina infinium humanmethylation450 array . 
this classifier was validated on data drawn from the cancer genome atlas synovial sarcoma , target - osteosarcoma , and a recently published series of ewing sarcoma . results methylation profiling revealed three distinct patterns , each enriched with a single sarcoma subtype . 
within the validation cohorts , all samples from the cancer genome atlas were accurately classified as synovial sarcoma ( 10 of 10 ; sensitivity and specificity , 100% ) , all but one sample from target - osteosarcoma were classified as osteosarcoma ( 85 of 86 ; sensitivity , 98% ; specificity , 100% ) , and 14 of 15 ewing sarcoma samples were classified correctly ( sensitivity , 93% ; specificity , 100% )  . 
two additional clinical samples that were difficult to classify by morphology and molecular methods were classified as osteosarcoma ; one had been suspected of being a synovial sarcoma and the other of being ewing sarcoma on initial diagnosis . conclusion osteosarcoma , synovial sarcoma , and ewing sarcoma have distinct epigenetic profiles . 
ewing sarcoma ( ews ) , synovial sarcoma ( ss ) , and osteosarcoma ( os ) are among the most common malignant solid tumors in children.1 although these tumors can occur in similar anatomic locations , optimal management and treatment strategies differ substantially depending on the tumor type.2 , 3 accurate diagnosis is thus paramount for clinical management , but can be challenging . histologically , ews is mainly composed of small round blue cells4 but occasionally consists of larger , more pleomorphic cells , making the diagnosis of ews based solely on histopathological analysis unreliable.5 the discovery of the ewsr1 - fli1 fusion detected by fluorescent in situ hybridization ( fish ) has significantly improved diagnostic accuracy , but the fusion is only present in approximately 85% of samples that are histologically consistent with ews.6 in the remainder of cases , most tumors harbor a fusion of the ewsr1 gene with a different member of the e - 26 transformation specific family of transcription factors.7 , 8 , 9 , 10 , 11 ss typically has a biphasic appearance consisting of epithelioid and fibroblast - like spindle cell components ; however , a monophasic spindle cell s . 
morphologic variants include spindle - cell os ( resembling fibrosarcoma or monophasic synovial sarcoma ) , high - grade pleomorphic os ( resembling undifferentiated pleomorphic sarcoma ) , or small round blue cell os ( resembling classic ews ) .12 the presence of osteoid deposition is helpful in the histologic diagnosis , but this deposition may not be present in very poorly differentiated specimens or small biopsy specimens . 
a wide range of copy number changes , most frequently including chromosomes 6p , 8q , 13q , and 17p , have been observed.15 - 18 additionally , the presence of the ewsr1 - fli1 translocation in a rare subset of small - cell os further complicates diagnostics.19 - 22 previous work has demonstrated the feasibility of classifying small round cell tumors using machinelearning techniques and artificial neural networks trained on gene expression data , 23 or support vector machinebased classifiers to distinguish between sarcoma subtypes with variable sensitivity and specificity ( range , 50% to 100% ) .24 a new approach to aid the diagnosis of solid tumors is based on the molecular signatures of genomewide dna methylation profiles . 
this technique has been pioneered as a powerful diagnostic tool in pediatric brain tumors and has been shown to be superior for risk stratification compared with standard histopathology.25 - 27 for the purposes of classification alone , the advantage of methylation is resolution . 
there is also an advantage of decreased noise , because methylation is more invariant to formalin fixation , time to fixation , cold ischemia time , temperature out of the body , immune status of the host , and several other factors that affect gene expression . 
integrative dna - methylation analysis has been examined as a tool to classify high - grade soft - tissue sarcoma , including ss , but not os or ews.28 the aim of the current study was to determine if methylation profiling can be used to accurately distinguish among ss , os , and ews . methods tissue collection and dna extraction eighty tissue samples from patients newly diagnosed with os , ews , and ss were obtained from the archives of the departments of pathology at the new york university langone medical center ( nyulmc ) , memorial sloan kettering cancer center , and montefiore medical center . 
oss were diagnosed on the basis of histologic features and , where available , absence of fusions characteristic of ews and ss . to develop the classifier , we selected histopathologically classic ( os , ss , ews ) and molecularly confirmed ( ss , ews ) reference samples . 
for the ith probe , the m value was calculated as a log2 transform of a ratio of the b value . mi log2 = 1 2 b a one - way analysis of variance via empirical bayes method ( r package limma ) was conducted to select for the most differentially methylated probes among the different sarcoma subtypes . 
more than 8 , 556 probes were found to be significantly differentially methylated ; however , many were very highly correlated ( data supplement ) ; thus , we selected the top 400 probes with the greatest median absolute deviation in b values ( data supplement )  . the random forest algorithm was used for classification . 
the number of trees was set at 400 with a number of variables tried at each node of 305 ( tuned to reduce out - of - bag error ) and minimum node size of 1 . validation samples we validated our classifier using the cancer genome atlas ( tcga ) , target - osteosarcoma ( target - os ; from childrens oncology group and the hospital for sick children , toronto , canada ) , and ews samples from a recently published series . 
raw signal intensity data ( idat ) files from tcga sss ( n = 10 ) were downloaded from the legacy archives of the genomic data commons.32 similarly , idat files and normalized gene quantification from mrna sequencing belonging to the discovery cohort of target - os ( n = 86 ) were accessed through the target data matrix ( matrix )  . 
idat files from huertas - martinez et al ( institut dinvestigacio biomedica de bellvitge [ idibell ] ) were obtained from the corresponding author.33 genomic pathway analysis information probes in the classifier were annotated using the ( version 1.2 ; humanmethylation450 manifest illumina )  . 
genomic including dna sequence and coordinates of gene coding regions were obtained from the university of california santa cruz genome browser database.34 we accessed 113 probes corresponding to enhancers and gene coding regions , using the molecular signatures database , a gene - set based pathway analysis . 
in total , we interrogated the overlap of classifier genes with 4 , 729 curated gene sets , which include known chemical and genetic perturbations ( n = 3 , 500 ) , gene sets derived from the kegg ( n = 186 ) , biocarta ( n = 217 ) , and reactome ( n = 674 ) pathways , and canonical pathways curated by domain experts ( n = 1 , 329 ) .35 data access idat files of the training set have been deposited in gene expression omnibus , accession number gse97529 . 
sample location included the appendicular ( 11 of 36 ; 31% ) and axial skeleton ( 23 of 36 ; 64% ) ; biopsy sites of two samples ( 5% ) were unknown . sarcomas showed distinct patterns of methylation unsupervised hierarchical clustering using the 400 most differentially methylated probes among the training set demonstrated three distinct molecular phenotypes corresponding to the pathologic diagnoses ( fig 1a )  . 
patient characteristics of the training set ( n = 36 ) sample histology primary tumor location translocation or fusion lateral side of the right knee mediastinal lymph node orbit ( secondary os ) lymph node left talus tibia back femur epidural tibia femur mandible femur femur right tibia right knee neck left knee right neck left foot thigh chest wall foot shoulder abdomen thigh foot right distal femur left tibia right femur right shoulder left pelvis left humerus left shoulder wall ewsr1 - fli1 ewsr1 - fli1 ewsr1 - fli1 ewsr1 - fli1 ewsr1 - fli1 ewsr1 - fli1 ewsr1 - fli1 ewsr1 - fli1 ewsr1 - fli1 x : 18 x : 18 x : 18 syt rearrangement syt rearrangement syt rearrangement abbreviations : ews , ewing sarcoma ; n / a , not applicable ; os , osteosarcoma ; ss , synovial sarcoma . with known genetic probes of interest . 
the closest probe to fli1 is 5 mb away ( cg13153466 ) , corresponding to the promoter region of asam , a coxsackieand adenovirus - receptorlike membrane protein . no probes lie near the cytogenetic band 18q11.2 belonging to the syt translocation partner of synovial sarcoma ; the closest four probes in chromosome 18 lie in a 0.1 - mb strip at 18q23 , notably hypomethylated in ss . 
the gray arrow indicates the sample from the targetosteosarcoma ( targetos ) dataset ( target40 - pasefs ) that demonstrated hypermethylation in several cpg islands uncharacteristic of other osteosarcomas . accurate methylation - based classification of bone sarcomas when the classifier was applied to the training cohort , all samples were accurately classified with a minimum margin score of 0.2 , thus confirming internal validity ( table 3 )  . 
within the validation cohorts , all samples ( 10 of 10 ) from tcga were accurately classified as ss , 14 of 15 ews samples were accurately classified as ews , and all tumors from target - os ( 85 of 86 ) were classified as os except one sample , which was classified as ews ( table 4 )  . 
a single case from the ews validation set that classified as a ss did not have rna data available for confirmatory studies . the discrepant os sample , target - 40pasefs ( highlighted by the arrow in fig 1a and displayed in fig 1b ) , was submitted from an 18 - year - old white man who was enrolled and treated in childrens oncology group clinical trial aost0331 . 
 ( b ) multidimensional scaling plot of random forest classifier samples demonstrates this target - os sample ( red triangle ) was classified as ewing sarcoma ( ews )  . 
 ( c ) rna sequence analysis of the sample target - os sample identified as ews by methylation classifier . the target - 40pasefs sample shows overexpression of ewsr1 and etv1 . 
rna expression data are consistent with the methylation sarcoma classifier diagnosis of ews . have been misclassified as ews at the time of original diagnosis , we investigated ewsr1 , fli1 , erg , and etv1 mrna transcript counts , available in the target - os dataset , to examine the common gene products of oncogenic ews fusions . 
although the expression of fli1 was similar to other samples in the cohort , we noted a striking overexpression of etv1 ( n = 70 transcripts per kilobase million ) and ewsr1 ( n = 200 transcripts per kilobase million ) compared with the other os samples in the dataset ( fig 1c )  . 
we hypothesized that the tumor harbored a ewsr1 / etv1 rearrangement consistent with ews ; however , after manually curating the transcript sequence reads , there was no evidence of an etv1 fusion , suggesting an alternative mechanism leading to etv1 upregulation . clinical application to illustrate the clinical utility of our classifier , we present two challenging clinical cases . 
the first is that of a 16 - year - old girl with a past medical history of rhabdomyosarcoma of the right orbit initially diagnosed in 2007 and treated with chemotherapy and radiation therapy . 
immunohistochemical stains were positive for vimentin , cd56 , and desmin , but tumor cells were negative for myogenin , myo - d1 , s - 100 , cam 5.2 , ae 1 / 3 , bcl - 2 , cd99 , cd34 , and ema . 
fish testing using a dual - color breakapart probe showed a rearrangement involving ss18 ( syt ; 88.0% of cells ; data not shown ) , raising the concern that the tumor may represent ss . 
however , satb2 was positive by immunohistochemistry , suggesting osteoblastic differentiation.36 ssx1 and ssx2 , the usual fusion partners for syt in synovial sarcoma , had no abnormalities . we performed methylation profiling on the 2016 recurrence sample . 
moreover , it indicates robustness of a methylation - based sarcoma classifier not only in de novo but also in radiation - induced os . the second difficult case was a 2 - year - old girl who had a mass involving t7 - 9 of the thoracic spine with radiographic evidence of pulmonary metastases . 
she received intensive multiagent chemotherapy and involved field radiation therapy to the spine . three years later at the age of 5 , she presented with recurrent disease , including a left - sided pleural mass and hilar adenopathy . 
pathology revealed viable disease with similar morphologic findings as her initial disease . at the age of 15 , the patient presented with headache , pupillary asymmetry , and slurred speech . 
targeted dna ( msk - impact37 ) and rna ( archer fusionplex ; archerdx , boulder , co ) were nondiagnostic but revealed a genomically unstable tumor with numerous mutations , which would be unusual for translocation - driven sarcomas such as ews . quantitative predictive probabilities derived from the random forest model were used to classify this sample , with a predicted probability of 48% os , 32% ss , and 20% ews . 
in the scenario in which os and ews compose the differential diagnosis , os was the most likely and ews the least likely grouping ( fig 2f )  . discussion in sarcomas lacking pathognomonic gene fusions , diagnostic differentiation can be extremely challenging , even with the help of modern diagnostic tools including immunohistochemistry and cytogenetics.38 - 41 additionally , tumor heterogeneity and sampling errors can significantly confound the diagnosis . 
ancillary cytogenetic studies such as fish can assist in differentiating between morphologically similar tumor specimens45 ; however , these tests frequently fail to identify a pathognomonic genetic abnormality and can occasionally produce false - positive results . the humanmethylation450 array is a rapid and cost - effective method for genome - wide quantitative profiling of the methylation of cpg loci.29 to explore the utility of dna - based methylation profiling in patients with sarcoma , we used a similar approach that has been shown to be highly accurate and reproducible in subclassifying other histologically similar tumors ( ie pediatric brain tumors ) .46 using random forest modeling , we developed a classifier that we successfully validated using ss and os samples from two publicly available datasets and an ews validation set obtained from a recently published series.33 in our training set , our classifier correctly identified all 36 samples as the tumor type determined by a combination of clinical , histologic , and table 3 . 
when applied to our validation cohorts ( tcga and target - os ) , all 10 ss samples and 85 of 86 of the os samples ( 99% ) were correctly classified . 
our analysis shows that the case target - 40pasefs may represent a tumor that is biologically related to ews , 47 as indicated by overexpression of ewsr1 and etv1 , illustrating the power and usefulness of methylation - based profiling for discovery . our study highlights the epigenetic heterogeneity present in ews despite a single recurrent oncogenic fusion driver . 
contingency table for validation set * pathology diagnosis random forest classification , validation set data ( n = 110 ) , no . abbreviations : ews , ewing sarcoma ; os , osteosarcoma ; ss , synovial sarcoma . * within the validation set , all patients were correctly classified within the the cancer genome atlas ( ss ) , huertas - martinez et al ( ews ) , and target - osteosarcoma ( target - os ) cohorts , with the exception of one sample from target - os ( target - 40 - pasefs ) that is classified as a es and one es sample from the huertas - martinez et al cohort that is classified as an ss . single test , thereby expediting the diagnosis . 
on the basis of positive fluorescent in situ hybridization analysis , the tumor was initially diagnosed as a synovial sarcoma ; however , multidimensional scaling plot of the classifier including the patients tumor ( red triangle ) ( c ) shows that the tumor classifies with osteosarcoma . 
 study comparing the performance and costeffectiveness of our methylation classifier and traditional immunohistochemical and fish testing could represent a fruitful avenue of research . in summary , we developed and validated a dna methylationbased classifier that accurately differentiated three of the most common subtypes of bone sarcomas . 
meyers stock and other ownership interests : amgen , bayer , e.i. dupont , henry schein , jazz pharmaceuticals , mednax , novartis , procter & gamble , sigma - aldrich honoraria : france foundation ( i ) consulting or advisory role : boehringer ingelheim ( i ) speakers bureau : france foundation ( i ) travel , accommodations , expenses : takeda , intermune ( i ) david j . 
pisapia no relationship to disclose richard gorlick stock and other ownership interests : oncolytics consulting or advisory role : oncolytics marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network / boehringer ingelheim afatinib targeted therapy advisory committee , national comprehensive cancer network / astrazeneca tagrisso request for proposal advisory committee research funding : loxo ( inst ) kristen thomas no relationship to disclose matija snuderl stock and other ownership interests : arca biopharma , bluebird bio , cellceutix , epizyme , geron , isis pharmaceuticals , sevion therapeutics , acceleron pharma , catalyst pharmaceutical travel , accommodations , expenses : silicon biosystems matthias a . 
pisapia , weill cornell medical college , new york ; and richard gorlick , albert einstein college of medicine , bronx , ny . supported in part by kids of new york university langone and the friedberg charitable foundation and through the nih / nci cancer center support grant no . 
werier j , yao x , caudrelier jm , et al : a systematic review of optimal treatment strategies for localized ewings sarcoma of bone after neo - adjuvant chemotherapy . 
surg oncol 25 : 16 - 23 , 2016 isakoff ms , bielack ss , meltzer p , et al : osteosarcoma : current treatment and a collaborative pathway to success . j clin oncol 33 : 3029 - 3035 , 2015 4 . 
sorensen ph , lessnick sl , lopez - terrada d , et al : a second ewings sarcoma translocation , t ( 21 ; 22 ) , fuses the ews gene to another ets - family transcription factor , erg . 
nat genet 6 : 146 - 151 , 1994 jeon is , davis jn , braun bs , et al : a variant ewings sarcoma translocation ( 7 ; 22 ) fuses the ews gene to the ets gene etv1 . 
kaneko y , yoshida k , handa m , et al : fusion of an ets - family gene , eiaf , to ews by t ( 17 ; 22 ) ( q12 ; q12 ) chromosome translocation in an undifferentiated sarcoma of infancy . 
peter m , couturier j , pacquement h , et al : a new member of the ets family fused to ews in ewing tumors . oncogene 14 : 1159 - 1164 , 1997 12 . 
goldblum jr , folpe al , weiss sharon w : general considerations , in goldblum jr , folpe al , weiss sharon w , eds : enzinger and weisss soft tissue tumors . 
yen cc , chen wm , chen th , et al : identification of chromosomal aberrations associated with disease progression and a novel 3q13.31 deletion involving lsamp gene in osteosarcoma . 
kresse sh , ohnstad ho , paulsen eb , et al : lsamp , a novel candidate tumor suppressor gene in human osteosarcomas , identified by array comparative genomic hybridization . 
dragoescu e , jackson - cook c , domson g , et al : small cell osteosarcoma with ewing sarcoma breakpoint region 1 gene rearrangement detected by interphase fluorescence in situ hybridization . 
hill da , osullivan mj , zhu x , et al : practical application of molecular genetic testing as an aid to the surgical pathologic diagnosis of sarcomas : a prospective study . 
sturm d , orr ba , toprak uh , et al : new brain tumor entities emerge from molecular classification of cns - pnets . cell 164 : 1060 - 1072 , 2016 26 . 
hovestadt v , remke m , kool m , et al : robust molecular subgrouping and copy - number profiling of medulloblastoma from small amounts of archival tumour material using high - density dna methylation arrays . 
huertas - martnez j , court f , rello - varona s , et al : dna methylation profiling identifies ptrf / cavin - 1 as a novel tumor suppressor in ewing sarcoma when co - expressed with caveolin - 1 . 
raney bj , dreszer tr , barber gp , et al : track data hubs enable visualization of user - defined genome - wide annotations on the ucsc genome browser . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
kilpatrick se , ward wg , chauvenet ar , et al : the role of fine - needle aspiration biopsy in the initial diagnosis of pediatric bone and soft tissue tumors : an institutional experience . 
bridge rs , rajaram v , dehner lp , et al : molecular diagnosis of ewing sarcoma / primitive neuroectodermal tumor in routinely processed tissue : a comparison of two fish strategies and rt - pcr in malignant round cell tumors . 
 ntrk alterations in pediatric highrisk malignancies identied through european clinical sequencing programs constitute promising drug targets over the last 2 years , two neurotrophic - tropomyosin receptor tyrosine kinase ( ntrk ) inhibitors have been approved for a tissue - agnostic indication in adults and children , namely , larotrectinib and entrectinib both in the united states and europe . 
although ntrkfusionpositive malignancies are rare , patients with tumors harboring this alteration may greatly benet from those innovative targeted therapies . molecular characterization platforms have been set up in europe over the last 5 years to identify targetable alterations in tumors of individual pediatric patients and to guide therapeutic prioritization . 
mappyacts , coordinated in france ( molecular proling for pediatric and young adult cancer treatment stratication ; clinicaltrials.gov identier : nct02613962 ) , and in ( individualized form , coordinated in germany therapy for relapsed malignancies in childhood ; eudract number : 2018 - 000127 - 14 ) , are the largest european efforts , offering real - time clinical sequencing to relapsed or refractory or very high - risk pediatric patients from 13 european countries.1 - 3 they are part of the innovative therapies for children with cancer ( itcc ) precision medicine and new drug development program , matching patients tumor molecular proles to new drugs in early clinical trials . alterations of the ntrk 1 - 3 were recently investigated in approximately 3 , 500 pediatric tumor samples across tumor types , with fusions involving these genes being detected in 0.34% of samples and amplication , mutation , and mrna overexpression occurring less frequently.4 although ntrk fusions are druggable by selective ( eg , larotrectinib and entrectinib ) and less specic small molecules , 4 - 11 reports on response of nonfusion ntrk alterations to ntrk inhibitors ( ntrki ) are lacking to date.4 since its initiation in january 2016 until july 2020 , approximately 690 patients with relapsed , refractory , or very high - risk pediatric cancer have been screened through mappyacts , whereas approximately 1 , 100 patients were screened through the inform registry from february 2015 until july 2020 . 
apart in mappyacts patients inform who were included because of being very high risk at rst diagnosis , all other tumor samples were obtained at relapse or progression ( table 1 )  . ntrk fusions were the most frequent alterations ( n = 25 ) , with ntrk1 , 2 , 3 being involved in 9 / 25 , 8 / 25 , and 8 / 25 patients , respectively . 
even for previously not described fusions , the similar structure to known ntrk fusions , with conservation of the tyrosine kinase domain , supports the hypothesis of an activation of the tyrosine kinase domain of ntrk . single nucleotide variants ( snvs ) were detected in 14 tumors ( 3 ntrk1 , 7 ntrk2 , and 4 ntrk3 ) , with six snvs being located in the kinase domain of the respective protein , one snv each in the immunoglobulin i - set domain and the leucine - rich domain , respectively , and the remaining six snvs falling outside of annotated functional domains . two cases harbored a focal amplication of ntrk1 genomic locus . a total of 17 patients with tumors harboring an ntrk fusion were treated with the specic ntrki larotrectinib . 
of these , four patients from germany , four patients from sweden , four patients from france , and one patient from poland were enrolled in an early phase clinical trial . 
complete resection of the tumor , stabilization with conventional therapy , missing availability of the drug ( before the initiation of pediatric trials ) , and loss to follow - up were stated as reasons not to treat with ntrki . 
one patient with ntrk1 amplication as nonfusion ntrk alteration received larotrectinib in a clinical trial setting . through this pan - european initiative , we demonstrate the feasibility and value of next - generation sequencing to identify children and adolescents with an ntrk tumor alteration - harboring malignancy ( across all types ) who may be eligible for ntrki therapy . addition , it allows the capture of all potential ntrk fusion partners and novel snvs in functionally relevant domains of ntrk . 
the current development of additional pediatric sequencing programs ( ie , smpaeds in the united kingdom , ither in the netherlands , and the danish program ) as part of the itcc strategy will provide an expanded access to targeted agents for children and adolescents across europe . elke pfaff , md hopp childrens cancer center heidelberg ( kitz ) , heidelberg , germany ; pediatric glioma research group , german cancer research center ( dkfz ) , heidelberg , germany ; department of pediatric oncology , hematology and immunology , heidelberg university hospital , heidelberg , germany tiphaine adam de beaumais , md clinical research department , gustave roussy cancer center , villejuif , france antonin marchais , phd bioinformatics and inserm u1015 , gustave roussy cancer center , university paris - saclay , villejuif , france cornelis m . 
van tilburg , md hopp childrens cancer center heidelberg ( kitz ) , heidelberg , germany ; department of pediatric oncology , hematology and immunology , heidelberg university hospital , heidelberg , germany ; clinical cooperation unit pediatric oncology , german cancer research center ( dkfz ) , heidelberg , germany german cancer consortium ( dktk ) , germany ; kitz clinical trial unit , hopp childrens cancer center heidelberg ( kitz ) , german cancer research center ( dkfz ) and heidelberg university hospital , heidelberg , germany mirjam blattner - johnson , phd hopp childrens cancer center heidelberg ( kitz ) , heidelberg , germany ; pediatric glioma research group , german cancer research center ( dkfz ) , heidelberg , germany uta dirksen , md german cancer consortium ( dktk ) , partner essen , germany ; university hospital essen , pediatrics iii , hematology and oncology , west german cancer centre , essen , germany ingrid ra , md , phd department of pediatric oncology and hematology , karolinska university hospital , solna , sweden ; skane university hospital , lund , sweden birgit geoerger , md , phd department of pediatric and adolescent oncology , gustave roussy cancer center , inserm u1015 , university parissaclay , villejuif , france gudrun schleiermacher , md equipe siric rtop recherche translationelle en oncologie p ediatrique , institut curie , paris , france ; siredo : care , innovation and research for children , adolescents and young adults with cancer , institut curie , paris , france stefan m . 
 correspondence hematology and immunology , heidelberg university hospital , heidelberg , germany ; division of pediatric neurooncology , german cancer consortium ( dktk ) and german cancer research center ( dkfz ) , heidelberg , germany data analysis and interpretation : elke pfaff , antonin marchais , ingrid ra , birgit geoerger , gudrun schleiermacher , stefan m . 
the inform project is nancially supported by the german consortium for translational cancer research ( dktk ) , german cancer aid , the german childhood cancer foundation , the german cancer research center ( dkfz ) , bild e.v. 
pster provision of study materials or patients : elke pfaff , uta dirksen , ingrid ra , birgit geoerger , olaf witt collection and assembly of data : elke pfaff , tiphaine adam de beaumais , antonin marchais , cornelis m . 
van tilburg consulting or advisory role : novartis , bayer uta dirksen consulting or advisory role : lilly , ipsen ingrid ra consulting or advisory role : bayer birgit geoerger consulting or advisory role : roche or genentech , merck kgaa , boehringer ingelheim , bayer , azd , novartis gudrun schleiermacher honoraria : bristol - myers squibb research funding : bristol - myers squibb , pzer , msdavenir , roche travel , accommodations , expenses : roche stefan m . 
pster research funding : lilly , bayer , roche , pharmamar , pzer patents , royalties , other intellectual property : patent on utilizing dna methylation proling for tumor classication olaf witt consulting or advisory role : novartis , astrazeneca , janssen research & development , bristol - myers squibb , roche , bayer , dayonetx research funding : biomed valley discoveries david t.w. 
worst bc , van tilburg cm , balasubramanian gp , et al : next - generation personalised medicine for high - risk paediatric cancer patients : the inform pilot study . 
harttrampf ac , lacroix l , deloger m , et al : molecular screening for cancer treatment optimization ( moscato - 01 ) in pediatric patients : a singleinstitutional prospective molecular stratication trial . 
 correspondence berlanga p , pierron g , lacroix l , et al : can pediatric and adolescent patients with recurrent tumors benet from a precision medicine program ? the european mappyacts experience . 
j clin oncol 37 , 2019 ( suppl ; abstr 10018 ) okamura r , boichard a , kato s , et al : analysis of ntrk alterations in pan - cancer adult and pediatric malignancies : implications for ntrk - targeted therapeutics . 
cancer discov 7 : 400 - 409 , 2017 laetsch tw , dubois sg , mascarenhas l , et al : larotrectinib for paediatric solid tumours harbouring ntrk gene fusions : phase 1 results from a multicentre , open - label , phase 1 / 2 study . 
lancet oncol 19 : 705 - 714 , 2018 konicek bw , capen ar , credille km , et al : merestinib ( ly2801653 ) inhibits neurotrophic receptor kinase ( ntrk ) and suppresses growth of ntrk fusion bearing tumors . 
oncotarget 9 : 13796 - 13806 , 2018 van tilburg cm , dubois sg , albert cm , et al : larotrectinib efcacy and safety in pediatric trk fusion cancer patients . 
j clin oncol 37 , 2019 ( suppl ; abstr 10010 ) robinson gw , gajjar aj , gauvain km , et al : phase 1 / 1b trial to assess the activity of entrectinib in children and adolescents with recurrent or refractory solid tumors including central nervous system ( cns ) tumors . 
hong ds , dubois sg , kummar s , et al : larotrectinib in patients with trkfusionpositive solid tumours : a pooled analysis of three phase 1 / 2 clinical trials . 
robson , md10 purpose screening and prevention decisions for women at increased risk of developing breast cancer depend on genetic and clinical factors to estimate risk and select appropriate interventions . 
a combined risk score ( crs ) of an 86single - nucleotide polymorphism polygenic risk score and the tyrer - cuzick v7.02 clinical risk estimator was generated with attenuation for confounding by family history . 
calibration and discriminatory accuracy of the crs were evaluated in two independent validation cohorts of women of european ancestry ( n = 1 , 615 and n = 518 )  . 
risk stratication with a 20% risk threshold was compared between crs and tyrer - cuzick in an independent clinical cohort ( n = 32 , 576 )  . results simulation studies conrmed that the fixed - stratied method produced accurate risk estimation across patients with different family history . 
in an analysis with both crs and tyrer - cuzick as predictors of breast cancer , crs added signicant discrimination independent of that captured by tyrer - cuzick ( p , 1011 in validation 1 ; p , 107 in validation 2 )  . 
in an independent cohort , 18% of women shifted breast cancer risk categories from their tyrer - cuzickbased risk compared with risk estimates by crs . conclusion integrating clinical and polygenic factors into a risk model offers more effective risk stratication and supports a personalized genomic approach to breast cancer screening and prevention . jco precis oncol 5 : 307 - 316 . 
2021 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction mammography and adjuvant treatment of early - stage disease are widely used for the mitigation of morbidity and mortality in invasive breast cancer.1 effective prevention requires the identication of higher - risk individuals , which has typically involved family history evaluation , assessment of clinical and lifestyle factors , and testing for the presence of pathogenic variants ( pvs ) in a limited number of highor moderate - risk breast cancer genes.2 although guidelines for pv carriers have long recommended enhanced screening and discussion of surgical prevention measures , they do not adequately address the increased risk for unaffected pv - negative women with a strong family history of breast cancer . 
we examined confounding and interaction between an 86 - snp polygenic risk score and each clinical factor in the tyrer - cuzick model to inform the development of a combined clinical and polygenic risk score . 
20% lifetime risk of developing breast cancer . improved risk stratication from the combined risk score can help target the use of more intense screening strategies for unaffected women with truly elevated risk based on clinical and genetic factors . here , we describe the development and validation of a strategy for integrating a snp score with a validated clinical risk model , with attenuation for correlated effects . 
clinical testing cohort data were subdivided according to successive time intervals to ensure the independence of development and validation activities ( fig 1 ) ; this included two development sets ( development 1 and 2 ) , one validation set ( validation 1 ) , and one clinical performance population . 
genetic testing for all patients was performed at a clinical laboratory improvement amendments - approved and college of american pathology - approved laboratory ( myriad genetics , inc . , salt lake city , ut ) by next - generation sequencing.34 hybridization probes for the 86 snps in the prs were included in the sequencing panel.33 women were eligible if they were of age 18 - 84 years , of european ancestry ( ashkenazi and non - ashkenazi ; selfreported ) , and negative for likely pvs or pvs in 11 breast cancerrelated genes ( brca1 , brca2 , tp53 , pten , stk11 , cdh1 , palb2 , chek2 , atm , nbn , and bard1 )  . patients were excluded as unaffected controls if they reported a history of ductal carcinoma in situ , lobular carcinoma in situ , hyperplasia , or unspecied breast disease . women were excluded from the clinical testing cohort if they were submitted from states that disallow the research use of samples after completion of genetic testing . 
all studies were conducted with institutional review board ( irb ) oversight ( quorum review irb #31713 , 32556 , 32608 )  . development 1 included women from the clinical testing cohort unaffected by cancer of any type accrued between june 1 , 2017 and august 11 , 2017 ( n = 5 , 489 )  . 
development 2 ( n = 141 , 160 ) included 112 , 232 women unaffected by breast cancer and 28 , 928 breast cancer cases from the clinical testing cohort accrued between august 11 , 2017 and january 11 , 2019 . 
clinical characteristics of this set were previously reported.33 validation 1 consisted of women in the clinical testing cohort accrued between june 1 , 2017 and august 10 , 2017 ( n = 1 , 615 )  . 
controls included unaffected women submitted for genetic testing because of possible hereditary nonpolyposis colorectal cancer syndrome as these patients have a breast cancer family history consistent with that of general population controls . for validation 2 , a consecutive series of women who presented to four breast cancer screening centers ( elizabeth wende breast care , the breast center of nwa , bethesda health , and cuda women 's health center / cape cod healthcare ) were prospectively recruited between february 6 , 2017 and november 11 , 2017 ( n = 518 )  . 
 integrating clinical and polygenic factors to predict cancer risk cohort description noisulcni noisulcxe accrual findings development n = 5 , 489 clinical testing cohort : 18 - 84 years old , european ancestry , pv - negative , from states that allow research use of data or samples post - testing history of breast cancer , dcis , lcis , hyperplasia , or unspecified breast disease ; eligible for validation set 1 development n = 141 , 160 clinical testing cohort : same inclusion criteria as development set 1 controls could not have a history of dcis , lcis , hyperplasia , or unspecified breast disease validation 1 clinical testing cohort : same basic inclusion or exclusion criteria as development set 2 cases : breast cancer diagnosis , submitted for testing due to suspicion of hboc within 1 year of diagnosis controls : unaffected women submitted for testing due to suspicion of hnpcc prospectively acquired case - control study enrolled from imaging centers cases : pathologically confirmed first diagnosis of invasive breast cancer 12 mos . preenrollment or incident breast cancer 6 mos . 
for each factor , we conducted a simple linear regression with prs as the dependent variable and the clinical factor as the independent variable . from these models , we examined regression coefcients , p values based on f - statistics , and pearson correlation coefcients . design of the combined risk score . 
to calculate absolute risk incorporating tyrer - cuzick and prs , we developed a fixed - stratied method ( data supplement ) to attenuate prs after xing the effects of confounded factors from the tyrer - cuzick model and to constrain risk separately within strata of the confounders . 
absolute risk for a woman in strata k was calculated as follows : ( cid : 3 ) exp { prs + ck } , ( cid : 1 ) 1 tc where represents the per - unit log ( or ) of the prs from a multivariable logistic regression model with the effect of breast cancer family history xed . 
the combined risk score ( crs ) was validated in two independent studies ( table 1 ) : a clinical testing set ( validation 1 ; n = 1 , 615 ) and the prospective case - control cohort ( validation 2 ; n = 518 )  . 
exploratory analyses tested calibration of the crs by comparing average absolute risk estimates with those from the tyrer - cuzick model ; with proper calibration , we expected to observe the same average risk for tyrer - cuzick as for the crs among unaffected controls . 
these analyses were conducted separately for rlr and 5 - year risk of developing breast cancer . weighted logistic regression was used with weights for unaffected controls calculated such that average tyrercuzick rlr matched general population rates ( data supplement )  . performance of the 86 - snp prs . 
in contrast , for women with family history , the fixed - stratied method matched risks from multivariable co - estimation , whereas the unadjusted model overestimated risk ( data supplement )  . clinical validations validation 1 included 988 ( 61% ) breast cancer cases and 627 ( 39% ) unaffected controls ( table 1 )  . 
overall , 362 ( 22% ) patients reported breast cancer in 1 rst - degree relative and 620 ( 38% ) in 1 second - degree relative . the prospectively collected case - control validation 2 included 256 ( 49% ) breast cancer cases and 262 ( 51% ) unaffected controls ( table 1 )  . 
nearly one third ( 29% ) of patients reported breast cancer in a rst - degree relative and 204 ( 39% ) patients in 1 second - degree relative . in both validations , rlr and 5 - year risk estimates of the crs and tyrer - cuzick were signicantly associated with breast cancer ( table 2 and fig 2 )  . 
in a model with both risk predictors , the crs added signicant discrimination independent of that captured by tyrer - cuzick for both rlr ( p , 1011 in validation 1 ; p , 107 in validation 2 ) and 5year risk ( p , 1011 in validation 1 ; p , 107 in validation 2 ; data supplement )  . although all patients in validation 2 provided complete tyrer - cuzick risk factor questionnaires , data on tyrercuzick variables were incomplete for some patients in validation 1 . 
analyses were repeated in the subset of patients with complete information for all tyrer - cuzick risk factors with results similar to those from the full data set ( data supplement )  . calibration of the crs was examined by comparing average risk estimates with those from tyrer - cuzick in the control samples from either validation . 
 hughes et al average remaining lifetime risk in validation cohort 1 ( n = 627 ) average 5 - year risk in validation cohort 1 ( n = 627 ) crs mean ( 95% ci ) tyrercuzick mean ( 95% ci ) fig 3 . 
estimates for average remaining lifetime ( a and c ) and 5 - year risk ( b and d ) for unaffected controls in validation 1 ( a - b ) and validation 2 ( c - d )  . patients were grouped into 5 - year age bins , and average risks were evaluated according to crs and tyrer - cuzick . 95% cis are also reported . 
addition of the 86 - snp score signicantly improved discrimination relative to the tyrer - cuzick model for predicting risk of breast cancer , and the crs model showed excellent calibration across age groups . 
20% - 25% lifetime risk of breast cancer to improve early - stage cancer detection.36 in a clinical testing population , we showed that for 33% of pv - negative women , the crs - estimated risk was  . 
distribution of crs risk estimates in unaffected women . remaining lifetime risk ( rlr ) in a population of unaffected women ( clinical performance population , n = 32 , 576 ; excluded women with ductal carcinoma in situ , lobular carcinoma in situ , hyperplasia , or unspecied breast disease ) according to crs with thresholds at 20% ( increased ) and 50% ( high ) rlr . 
blue squares indicate patients with discordance between the scores ( eg , the tyrer - cuzick model produced a score that indicated a patient had low rlr , but the same patient was determined to have increased rlr by the crs model )  . 
crs , combined risk score . performance integrating clinical and polygenic factors to predict cancer risk increased risk ( crs > 20% ) 33.1% high risk ( crs > 50% ) 0.6% 3000 2500 2000 1500 1000 rlr according to crs ( % ) remaining lifetime risk according to tyrer - cuzick ( 20% ) increased ( > 20% ) crs total 56.5% n = 18 , 398 10.5% n = 3 , 407 67.0% n = 21 , 805 8.0% n = 2 , 619 25.0% n = 8 , 152 33.0% n = 10 , 771 64.5% n = 21 , 017 35.5% n = 11 , 559 100% n = 32 , 576 tyrer - cuzick or crs - based risk discordant increased risk ( > 20% ) high risk ( > 50% ) tyrer - cuzick remaining lifetime risk ( % ) women ( 8% ) who would have been categorized as , 20% using tyrer - cuzickestimated risk alone . combinations of prss with the tyrer - cuzick model have been presented previously , generally without attenuation for confounding . 
an 88 - snp score was added to tyrercuzick under the assumption of independence in a nested case - control study.25 although the snp score added disit had poor crimination to the tyrer - cuzick prediction , calibration . 
first , previous examinations of confounding often evaluated the correlations between a prs and a model rather than individual clinical factors.21 , 25 , 26 this approach might have obscured associations between prs and clinical factors with a stronger genetic component . 
second , genetic overlap is more likely with a larger prs since casting a wider for cancer - associated snps is more likely to also the genetic component of capture a larger fraction of clinical risk . 
1 rst - degree relative or those with second - degree relatives . limitations of the present study include a potential ascertainment bias in the clinical testing population cohorts . qualication for genetic testing is often based on family history . 
it has been previously shown that this potential bias can be avoided by accounting for family history in a multivariable model.38 , 39 consequently , all analyses presented here were conducted by multivariable co - estimation . 
in support of this approach , results from validation 1 ( clinical testing samples ) were similar to results obtained in validation 2 , for which participants were prospectively collected and were unaffected by potential bias due to selection for hereditary cancer testing . 
evaluation of the crs in a prospectively collected , unselected populationbased sample is being pursued.40 future work to expand assessment tools such as tyrer - cuzick and the prs for non - european ancestries will be required to apply these tools to other ancestry populations . 
however , subanalysis of validation 1 in women with complete tyrer - cuzick information showed similar results as the whole data set , indicating that missing information did not substantially affect the analysis . this crs model is suitable for reporting age - specic risk of developing breast cancer for unaffected women of european descent with or without signicant family history . 
it is currently the only tyrer - cuzickbased model fully adjusted for the shared risk between snps and family history and is therefore less likely to overestimate risk in women with a family history of breast cancer . 
lanchbury data analysis and interpretation : elisha hughes , placede tshiaba , susanne wagner , eric rosenthal , benjamin roa , shannon gallagher , stephanie meek , wade hedegard , susan m . 
 integrating clinical and polygenic factors to predict cancer risk benjamin roa employment : myriad genetics leadership : myriad genetics stock and other ownership interests : myriad genetics research funding : myriad genetics patents , royalties , other intellectual property : intellectual property held by employer myriad genetics travel , accommodations , expenses : myriad genetics shannon gallagher employment : myriad genetics stock and other ownership interests : myriad genetics stephanie meek employment : myriad genetics stock and other ownership interests : myriad genetics kathryn dalton speakers bureau : myriad genetics danna f . 
domchek honoraria : astrazeneca , clovis oncology , bristol - myers squibb research funding : astrazeneca , clovis oncology judy garber consulting or advisory role : novartis , gtx , helix biopharma , konica minolta , aleta biotherapeutics , h3 biomedicine , kronos bio research funding : novartis , ambry genetics , invitae , myriad genetics other relationship : susan g . 
lanchbury employment : myriad genetics leadership : myriad genetics stock and other ownership interests : myriad genetics patents , royalties , other intellectual property : i am an inventor on multiple patents led by myriad genetics travel , accommodations , expenses : myriad genetics alexander gutin employment : myriad genetics stock and other ownership interests : myriad genetics , gilead sciences mark e . 
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jco precis oncol 4 : 585 - 592 , 2020 judkins t , leclair b , bowles k , et al : development and analytical validation of a 25 - gene next generation sequencing panel that includes the brca1 and brca2 genes to assess hereditary cancer risk . 
newberg , phd3 ; meagan montesion , phd3 ; and peter mu - hsin chang , md , phd1 , 2 introduction salivary gland tumors comprise a rare group of neoplasms that arise from salivary ducts , most of which are benign.1 the parotid gland is the most frequently involved site and comprises more than 80% of salivary tumors.2 of the parotid tumors , approximately 75% are benign , whereas the remaining 25% are malignant.3 the most frequent subtype of malignant salivary gland cancer is mucoepidermoid carcinoma , followed by adenoid cystic carcinoma.4 salivary gland tumors have been linked to exposure to high ultraviolet radiation.5 salivary gland cancer has a poor prognosis ( 5 - year overall survival , 50% ) and a high propensity for recurrence.6 the mainstay of treatment of salivary gland cancers is surgery in patients with early and locally advanced disease.7 in metastatic or recurrent disease , consensus is lacking on the optimal regimen for systemic chemotherapy.8 the low prevalence of this disease precludes large - scale randomized trials , and current clinical data are mostly derived from small clinical studies . 
current data , although dated , support cisplatin , anthracyclines , uorouracil , and cyclophosphamide as active agents against salivary gland cancer.9 - 11 in general , response to chemotherapy is limited ( a review of response to chemotherapy was published by laurie and licitra , 12 with response rates mostly ranging from 10% to 30% ) , and chemotherapy is mostly for palliative control.8 , 12 experts have urged a coordinated effort for clinical trials to further elucidate the role of chemotherapy in this patient group , 12 but this proves to be difcult , owing to the rarity of this cancer . the success of epidermal growth factor receptor ( egfr ) inhibitors in head and neck cancers13 and lung cancers14 has sparked hope for the possibility of use in salivary gland tumors because egfr is sometimes upregulated in this disease group.15 however , attempts to use egfr inhibitors , such as getinib and cetuximab , have largely been disappointing , with limited response.16 , 17 multiple mechanisms have been proposed for the lack of response to egfr inhibition in salivary gland cancers.18 to gain additional insight into the egfr expression pattern in salivary gland cancers , the dna and protein status of more than 800 tumor samples were analyzed for egfr gene alteration and protein levels in a retrospective study.19 the study revealed a high frequency of egfr low prevalence of egfr gene protein expression , mutation , and , more interestingly , no occurrence of egfr gene amplication . this is signicantly different from studies of head and neck squamous cell carcinomas , in which the prevalence of egfr amplication has been reported to be 17%20 and was linked to worse clinical outcome . 
interestingly , studies in colorectal cancer have shown that egfr gene amplication is linked to better outcomes and response to cetuximab.21 , 22 conversely , egfr amplication is proposed as an intrinsic resistance mechanism in lung cancer , 23 although other studies report conicting results , demonstrating egfr amplication associated with better outcomes with tyrosine kinase inhibitors ( tkis ) in patients with advanced nonsmall - cell lung cancer.24 therefore , the exact role of egfr amplication in response to egfr inhibition therapy remains to be claried . in this case report , we describe an unusual patient with a salivary gland cancer , refractory to initial chemotherapy agents but demonstrating signicant response to afatinib , an egfr and human epidermal growth factor receptor 2 tki . 
 ( c ) a computed tomography scan was performed 3 months after starting afatinib , showing marked decrease in size of the tumor ( right )  . pathology studies conrmed the diagnosis of high - grade salivary ductal carcinoma with inltration to the nontumor portion of the parotid gland . 
copy number ( cn ) plot ( top ) and minor allele frequency plot ( bottom ) showing marked increase in copy number of epidermal growth factor receptor . after discussion with the patient , the decision was made to initiate a therapeutic regimen of the egfr inhibitor afatinib ( 40 mg , every other day )  . 
written informed consent for publication of this case report was obtained from the patient . discussion afatinib is a tki that irreversibly inhibits the human egfr and human epidermal growth factor receptor 2 kinases.26 it has proven clinical activity and is approved for treatment of nonsmall - cell lung cancer with mutant egfr27 ( as shown in the lux - lung 3 and lux - lung 6 trials )  . 
interestingly , afatinib showed signicant activity against tumors harboring the deletion of exon 19 in egfr , while lacking activity against the common l858r point mutation , 27 , 28 highlighting the differences in biology between different egfr mutation types.29 afatinib was shown to be effective in advanced head and neck cancers , with benet progression - free survival30 , 31 ; however , little data exist for afatinib in salivary gland cancers . although some subtypes of salivary gland tumors exhibit egfr overexpression , 15 it is generally established that egfr inhibitors such as cetuximab are ineffective against salivary gland tumors . 
mechanistically , egfr amplication has been linked to higher spatial density of the receptor and thus linked to heightened dimerization and signaling.32 interestingly , as described in a recent case report , a patient with triple - negative breast cancer with egfr amplication was also successfully treated with cetuximab.33 a recent study retrospectively investigated a subgroup of esophageal cancers with egfr amplication.34 although previous studies have failed to demonstrate efcacy of egfr inhibition in esophageal cancers , 35 this study demonstrated a high response rate ( objective response rate , 58% ; disease control rate , 100% ) in this patient population.34 the ndings in our case report are in line with the aforementioned studies , highlighting the prognostic value of egfr amplication with treatment with egfr inhibitors . 
our patient is unique in that egfr amplication is extremely rare in salivary gland tumors , 19 compared with higher prevalence in triple - negative breast cancer ( up to 92% ) 36 or in esophageal cancer ( 7% ) .37 these reports that egfr amplication may indicate a favorsuggest able response to egfr inhibitors , regardless of the tumor site . 
this highlights the importance of genetic proling of individual tumors to determine personalized treatment of patients . in conclusion , we report a patient with a high copy number of egfr who demonstrated good response to egfr inhibition therapy . 
newberg employment : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : im on a patent stemming from research in my graduate school days related to identifying location biomarkers using imaging techniques . 
head neck surg 8 : 177 - 184 , 1986 liu y , li h , qin l , et al : prognostic factors in malignant sublingual salivary gland tumors . 
j oral maxillofac surg 75 : 1542 - 1548 , 2017 spitz mr , sider jg , newell gr , et al : incidence of salivary gland cancer in the united states relative to ultraviolet radiation exposure . 
head neck surg 10 : 305 - 308 , 1988 johnston ml , huang sh , waldron jn , et al : salivary duct carcinoma : treatment , outcomes , and patterns of failure . 
head neck 38 : e820 - e826 , 2016 ( suppl 1 ) lewis ag , tong t , maghami e : diagnosis and management of malignant salivary gland tumors of the parotid gland . 
otolaryngol clin north am 49 : 343 - 380 , 2016 aleri s , granata r , bergamini c , et al : systemic therapy in metastatic salivary gland carcinomas : a pathology - driven paradigm ? oral oncol 66 : 58 - 63 , 2017 licitra l , cavina r , grandi c , et al : cisplatin , doxorubicin and cyclophosphamide in advanced salivary gland carcinoma . 
proc natl sci counc repub china b 25 : 90 - 96 , 2001 jakob ja , kies ms , glisson bs , et al : phase ii study of getinib in patients with advanced salivary gland cancers . 
chen z , chen j , gu y , et al : aberrantly activated areg - egfr signaling is required for the growth and survival of crtc1 - maml2 fusion - positive mucoepidermoid carcinoma cells . 
temam s , kawaguchi h , el - naggar ak , et al : epidermal growth factor receptor copy number alterations correlate with poor clinical outcome in patients with head and neck squamous cancer . 
hirsch fr , herbst rs , olsen c , et al : increased egfr gene copy number detected by uorescent in situ hybridization predicts outcome in non - small - cell lung cancer patients treated with cetuximab and chemotherapy . 
nukaga s , yasuda h , tsuchihara k , et al : amplication of egfr wild - type alleles in non - small cell lung cancer cells confers acquired resistance to mutationselective egfr tyrosine kinase inhibitors . 
cappuzzo f , hirsch fr , rossi e , et al : epidermal growth factor receptor gene and protein and getinib sensitivity in non - small - cell lung cancer . 
sk alov a a , vanecek t , sima r , et al : mammary analogue secretory carcinoma of salivary glands , containing the etv6 - ntrk3 fusion gene : a hitherto undescribed salivary gland tumor entity . 
solca f , dahl g , zoephel a , et al : target binding properties and cellular activity of afatinib ( bibw 2992 ) , an irreversible erbb family blocker . 
yang jc , wu yl , schuler m , et al : afatinib versus cisplatin - based chemotherapy for egfr mutation - positive lung adenocarcinoma ( lux - lung 3 and lux - lung 6 ) : analysis of overall survival data from two randomised , phase 3 trials . 
kato t , yoshioka h , okamoto i , et al : afatinib versus cisplatin plus pemetrexed in japanese patients with advanced non - small cell lung cancer harboring activating egfr mutations : subgroup analysis of lux - lung 3 . 
kaneda t , yoshioka h , tamiya m , et al : differential efcacy of cisplatin plus pemetrexed between l858r and del - 19 in advanced egfr - mutant non - squamous non - small cell lung cancer . 
machiels jp , haddad ri , fayette j , et al : afatinib versus methotrexate as second - line treatment in patients with recurrent or metastatic squamous - cell carcinoma of the head and neck progressing on or after platinum - based therapy ( lux - head & neck 1 ) : an open - label , randomised phase 3 trial . 
clement pm , gauler t , machiels jp , et al : afatinib versus methotrexate in older patients with second - line recurrent and / or metastatic head and neck squamous cell carcinoma : subgroup analysis of the lux - head & neck 1 trial . 
suntharalingam m , winter k , ilson d , et al : effect of the addition of cetuximab to paclitaxel , cisplatin , and radiation therapy for patients with esophageal cancer : the nrg oncology rtog 0436 phase 3 randomized clinical trial . 
 c dramatic response to sequential braf inhibition in braf v600emutant metastatic lung adenocarcinoma introduction lung [ aq1 ] [ aq2 ] cancer is the leading cause of cancer related death.1 targeted therapy directed toward molecular pathways that drive nonsmall - cell lung cancer ( nsclc ) has dramatically improved treatment options . 
some of the oncogenic drivers identified in lung adenocarcinoma involve the ras - raf - mitogen - activated protein kinase ( mapk ) kinase ( mek ) - extracellular regulated kinase ( erk ) pathway ( mapk - erk pathway ) .2 braf is a key protein in mapk - erk signaling . 
 mutations in braf [ aq3 ] are present in 1% to 3% of nsclc and lead to constitutive activation of the mapk - erk pathway , promoting cancer cell proliferation and survival . 
several other mutations of braf have been identified and are grouped as non - v600e mutations.3 patients with nsclc harboring braf v600e mutations are sensitive to braf inhibitors ( brafis ) alone or in combination with mek inhibitors ( mekis ) .4 - 6 after initial response , patients typically experience relapse as a result of various resistance mechanisms . 
imaging revealed a left - side hilar lung mass , enlarged mediastinal lymph nodes , vertebral body sclerotic lesions , small bilateral pleural effusions , and a pericardial effusion consistent with metastatic disease . 
the repeat biopsy specimen was sent for massively parallel dna sequencing using the illumina hiseq2000 platform ( foundation medicine , cambridge , ma ) to characterize base substitutions , short insertions and deletions , copy number alterations , and selected fusions across 287 cancer - related genes . 
several tumor mutations , including braf v600e , mapk1 ( r306h ) , smad4 ( r361c ) , and setd2 ( splice site 65_71 + 13del20 ) , were identified . 
 a through lymphangitic spread ( fig 2 ) that required oxygen supplementation at 5 l / min and showed malignant involvement of the right - side iliopsoas muscle , which caused significant pa after palliative radiation to the right - side pelvic mass for pain control , he began a combination of dabrafenib 150 mg twice daily and trametinib 2 mg daily . 
at the same time , the patient became oxygen independent , and functional status improved , which allowed him to fulfill his desire to travel to his home country to visit family . more than 5 months later , after dose reduction for grade 3 fatigue , his lung mass again increased in size . 
by that point , he had survived 3.5 years since diagnosis of nsclc with a response duration of 12 months on braf targeted therapy . discussion in this era of personalized medicine , targeted therapies against braf v600e and mek have changed the standard of care in the treatment of braf - mutated metastatic melanoma . 
two brafis , dabrafenib and vemurafenib , with or without mekis , trametinib and cobimetinib , are approved for braf v600emutant metastatic melanoma.7 , 8 such targeted medications also have clinical activity in braf v600emutant nsclc.4 - 6 , 9 , 10 in a retrospective analysis of the european euraf cohort , 35 patients with braf - mutant nsclc treated with brafis were identified . 
after first - line therapy with brafis , overall survival ( os ) was 25.3 months in patients with braf v600emutant nsclc ( n = 29 ) and 11.8 months in patients with nonv600e braf mutations ( n = 6 ) .9 response to chest before and after initiation of vemurafenib , at 2 weeks , and at 3 months demonstrated a significant decrease in the size of the rightside upper - lobe mass [ aq4 ] and improvement of the air space disease , with an overall decrease in tumor volume of greater than 90% ( fig 1 )  . 
the dosage of vemurafenib was reduced twice for grade 3 fatigue , recurrent grade 2 skin rash , and transaminitis . after maintaining response for 7 months on vemurafenib , the patients tumor burden escalated fig 1 . 
computed tomography scans of chest demonstrating response to combination dabrafenib and trametinib treatment brafis also was documented in a phase ii basket trial of vemurafenib in braf v600mutant positive nonmelanoma cancers with an overall response rate ( orr ) of 42% in 20 patients with nsclc.10 in a prospective multicenter phase ii clinical trial , planchard et al5 evaluated the safety and efficacy of dabrafenib trametinib in nsclc . 
previously treated patients ( n = 78 ) and treatment - nave patients ( n = 6 ) responded to dabrafenib alone with a median progression - free survival ( pfs ) of 5.5 months , a median os of 12.7 months , and an orr of 33% . 
testing for braf is recommended as part of the initial molecular analysis in patients with lung cancer of adenocarcinoma histology and is considered in some patients with squamous subtype.11 all patients with braf v600emutant nsclc eventually progress after brafis with or without mekis . 
resistance mechanisms are not as well characterized in nsclc compared with braf v600emutant melanoma.12 secondary resistance mechanisms in melanoma include ras mutations , braf splice variants , braf v600 amplifications , mek1 / 2 mutations , and non - mapk pathway alterations.13 in nsclc , lin et al14 described two classes of resistance patterns to vemurafenib using a preclinical model of hcc364 braf v600emutant lung cancer cells . 
in contrast , expression of c - junmediated egfr ligands that promote continued egfr signaling represents another class.14 plx9394 , a novel brafi , evades resistance caused by alternatively spliced truncated braf v600e , but subsequent resistance occurs through egfr - mediated ras - mammalian target of rapamycin signaling.15 in the clinical setting , kras , tp53 , and cdkn2a mutations were acquired in one patient after progression on dabrafenib.16 currently , there is no standard therapy for patients with braf v600emutant nsclc after progression on brafis . 
in a phase i / ii study , the combination of brafi and meki was evaluated in patients with braf v600mutant metastatic melanoma who had disease progression on a single - agent brafi . 
one of these patients had a partial response to dabrafenib monotherapy after prior treatment with vemurafenib.9 another case report showed a similar response ; a patient who progressed on vemurafenib achieved a 3 - month clinical benefit with single - agent dabrafenib.18 our patient attained clinical response for an additional 5 months with an alternative brafi , dabrafenib , combined with the meki trametinib after progression on vemurafenib . 
two prior case studies described the benefit of combination treatment after progression on initial brafi monotherapy.19 , 20 similar to our patient , schmid et al19 reported on a patient who responded to combination dabrafenib and trametinib for 6 months after progression on vemurafenib . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . sushma jonna no relationship to disclose affiliations all authors : georgetown university , washington , dc . clinical trials or have already progressed on other lines of therapy . 
chen d , zhang lq , huang jf , et al : braf mutations in patients with non - small cell lung cancer : a systematic review and meta - analysis . 
planchard d , besse b , groen hjm , et al : dabrafenib plus trametinib in patients with previously treated braf ( v600e ) - mutant metastatic non - small cell lung cancer : an open - label , multicentre phase 2 trial . 
planchard d , kim tm , mazieres j , et al : dabrafenib in patients with braf ( v600e ) - positive advanced non - small - cell lung cancer : a single - arm , multicentre , open - label , phase 2 trial . 
planchard d , smit ef , groen hjm , et al : dabrafenib plus trametinib in patients with previously untreated brafv600e - mutant metastatic non - small - cell lung cancer : an open - label , phase 2 trial . 
lindeman ni , cagle pt , aisner dl , et al : updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors : guideline from the college of american pathologists , the international association for the study of lung cancer , and the association for molecular pathology . 
johnson db , menzies am , zimmer l , et al : acquired braf inhibitor resistance : a multicenter meta - analysis of the spectrum and frequencies , clinical behaviour , and phenotypic associations of resistance mechanisms . 
okimoto ra , lin l , olivas v , et al : preclinical efficacy of a raf inhibitor that evades paradoxical mapk pathway activation in protein kinase braf - mutant lung cancer . 
rudin cm , hong k , streit m : molecular characterization of acquired resistance to the braf inhibitor dabrafenib in a patient with braf - mutant non - small - cell lung cancer . 
johnson db , flaherty kt , weber js , et al : combined braf ( dabrafenib ) and mek inhibition ( trametinib ) in patients with brafv600 - mutant melanoma experiencing progression with single - agent braf inhibitor . 
schmid s , siano m , joerger m , et al : response to dabrafenib after progression on vemurafenib in a patient with advanced braf v600e - mutant bronchial adenocarcinoma . 
 outcomes of chemotherapy for microsatellite instablehigh metastatic colorectal cancers purpose microsatellite instable - high ( msi - h ) colorectal cancers ( crcs ) are known to carry better survival in the local disease stage even without treatment . 
the influence of types of treatment on survival of msi - h metastatic crcs ( mcrcs ) is still unclear and is evaluated in this study . materials and methods patients with msi - h mcrc treated with first - line chemotherapy , with or without bevacizumab , identified in the israeli population - based molecular epidemiology of colorectal cancer ( mecc ) study , were diagnosed between 1998 and 2013 and followed up until may 2017 ; msi status was determined by comparing 10 markers in tumor and normal tissue . 
dates of metastases and death and treatment details were extracted from oncology records . results among 590 patients treated for mcrc , 106 ( 18% ) had msi - h tumors . 
although treatment advances with modern protocols including antivascular endothelial growth factor and antiepidermal growth factor receptor ( egfr ) monoclonal antibodies1 - 3 prolonged median overall survival ( os ) of patients with crc to > 2 years , mcrc , unfortunately , is still an incurable disease in most cases . k - ras proto - oncogene ( kras ) or n - ras proto oncogene ( nras ) mutation ; the presence of a braf v600e mutation has a detrimental effect on overall survival ; the presence of human epidermal growth factor receptor 2 ( her2 ) in mcrc tumors suggests the possibility of benefit from anti - her2 biologic treatment ; and , more recently , microsatellite instable ( msi ) high tumors have been identified as candidates for immunotherapy.4 - 7 molecular heterogeneity in metastatic crcs influences treatment and outcome . 
anti - egfr agents are less effective in the presence of a loss of genomic stability is identified as an early step in colon cancer tumorigenesis and has pathologic , phenotypic , and clinical consequences.8 - 11 stephen b . 
 molecular analysis enables better understanding and separation of colorectal tumors now identified as carrying differences in clinical outcome.12 , 13 under this classification , tumors defined as consensus molecular subtype - 1 ( cms1 ) are characterized as microsatellite instable ( msi - h ) and hypermutated tumors.14 the clinical importance and exclusiveness of msi - h crcs have formerly been established.15 - 18 msi - h tumors have significantly better prognosis at early local stages of disease and rarely metastasize . 
cases identified in the mecc study were approached for risk - factors interview and contributed a venous blood sample after signing a consent form approved by carmel medical center in israel and the university of southern california in the united states . 
dna was extracted from both the peripheral blood and the tissue sample and was routinely studied for msi status , kras and braf mutations , as well as known founder mutations in mmr genes , mostly in ashkenazi jews . 
clinical follow - up included identification of treatment modalities used during the whole follow - up period and identification of changes in disease status , including identification of new metastases . 
details of treatments , as well as date of identification of metastases ( at diagnosis or during disease progression ) , were extracted from the medical files of the patients , which were extracted by experienced senior physicians . for this study , mecc cases that presented with metastatic disease at diagnosis or developed metastases during follow - up were sought . 
in addition to cases from the mecc population - based series , we recruited into the study other mcrc cases that were evaluated in our laboratory on the basis of their clinical presentation or suspicion of lynch syndrome to increase the size of the case series . 
detailed treatment options were grouped into three categories : fu or capecitabine only , combination chemotherapy ( oxaliplatin - based or irinotecan - based in combination with fluoropyrimidine ) , and combination chemotherapy with bevacizumab . 
 metastases with information on treatment details , and have available tissue and germline dna to analyze msi and braf status . laboratory assay dna was extracted using a commercial dna extraction kit . 
five loci of mononucleotide repeats ( bat25 , bat26 , bat40 , - catenin , and tgfbrii ) and five of dinucleotide repeats ( d2s123 , d5s346 , d10s197 , d17s250 , and d18s58 ) were amplified and tested . 
those include the five original bethesda panel markers and five additional monoand dinucleotide markers.33 results for each marker were registered as stable , unstable , equivocal , or suspected as loss of heterozygosity . 
most msi - h cases were also validated by immunohistochemistry tests of the mmr genes . mutations in codon 600 of braf were identified by direct sequencing of exon 15 of the braf gene . 
similarly , kras mutations in exon 12 , 13 , and 61 were identified using a taqman - based single - nucleotide polymorphism genotyping assay on the abi prism 7900ht sequence detection system ( applied biosystems ) in a 96 - well format . until the recorded date of death ( of any cause ) or the last date of available follow - up . 
overall survival was estimated with the use of kaplanmeier method and presented using medians and 2 - year and 5 - year survival probabilities . differences in os were summarized using hazard ratios ( hrs ) , estimated using cox proportional hazard modeling . 
the predictive effect of msi status on treatment effects was assessed using cox proportional hazard models with terms for treatment , msi status , and their interaction results patient population the study population consisted of 106 msi - h mcrc ( met / msi - h ) cases for which first - line chemotherapy - based treatment was identified . 
the metastatic msi - h group had mean age at treatment of 63 15 years , 54% female , 86% with jewish ethnicity , and 48% presenting at stage iv at diagnosis . 
generally , the met / mss group had comparable sex and ethnicity ( jewish / non - jewish ) distribution , as well as frequency of presenting at stage iv at diagnosis . 
this distribution reflects that fact that the patients with met / msi - h were younger at diagnosis ( proportion of patients younger than 50 years : 25% in met / msi - h group and only 10% in met / mss group )  . 
overall survival was calculated from the start date of first treatment of the first identified metastasis ( either at time of crc diagnosis or at time of disease progression ) and treatments for metastatic disease common first - line treatment chemotherapy for the metastatic disease in the msi - h group included fu + leucovorin ( lcv ) or capecitabine only in 21% ( n = 22 ) , combination chemotherapy protocols ( irinotecan based or oxaliplatin based ) in 33% ( n = 35 ) , and combination chemotherapy ( irinotecan based or oxaliplatin based ) and bevacizumab in 46% ( n = 49 ) of cases . 
demographic and disease characteristics of study participants with metastatic colorectal cancer by microsatellite instability status ( n = 590 ) characteristic metastatic msi - h ( n = 106 ) metastatic mss ( n = 484 ) age younger than 50 years at diagnosis female sex jewish ethnicity braf mutation positive stage iv at diagnosis right colon primary tumor location 26 ( 25 ) 57 ( 54 ) 88 * ( 86 ) 19 ( 18 ) 45 ( 48 ) 39 ( 38 ) note . 
 figure 1 presents frequency of the three classes across period years . second - line treatment was given to 55% of the patients treated before 2003 , 63% of those treated in 2003 to 2006 , and 68% of patients treated in 2007 and after . 
no difference in number of treatment lines was noticed between cases with msi - h and cases with mss tumors , probably reflecting the fact that in those years the treating oncologists were unaware of the msi status at time of treatment . in addition , in the msi - h population , the baseline characteristics of patients in the different treatment regimen were comparable ( data not shown )  . 
figure 4 presents os by treatment group , separately for patients with met / mss ( fig 4a ) and patients with met / msi - h ( fig 4b ) , in the homogenous population of braf - wt cases . 
 a sensitivity analysis adjusting for study period revealed similar results . prognostic effect of msi status in the braf - wt population the prognostic role of msi status in the metastatic disease setting was assessed in the population of patients who were indicated to receive an fu - only regimen as having the smallest or even no treatment effect , according to the literature . 
overall survival of patients with microsatellite instable , brafwild type metastatic colorectal cancer by treatment groups in the in the molecular epidemiology of colorectal cancer study , northern israel ( n = 87 )  . 
the mecc study reported here has recruited and collected materials over a time span of 17 years from close to 6 , 000 cases of crc and served as the source for the 590 cases who were diagnosed with , or later developed , metastases and for whom detailed treatment information was available . 
all cases participating in the final analysis were evaluated for their msi status , which was performed in only approximately two thirds of all cases recruited into mecc , with enrichment for suggestive phenotypes . in accordance with other published literature , cases with msi - high mcrc in our study were younger at diagnosis and had a higher proportion of braf mutations , 33 , 34 with a similar proportion of right - sided tumors . 
although msi - h tumors are described as having a much better prognosis than mss tumors for early - stage disease , the proportion of tumors diagnosed with metastases at the time of diagnosis was similar between msi - h cases and mss cases . 
the leading randomized controlled trials evaluating the effect of various chemotherapy regimens in mcrc were not randomized by msi status , and most have not provided subgroup analyses of the results according to their msi status . our study has three major findings . 
we verified that the survival of patients with mcrc indeed improved with the introduction of new chemotherapies but showed that the improvement in survival was limited to the met / mss , braf - wt cases and was not evident in the met / msi - h , braf - wt group of patients . 
we were unable to have a comparison with an observation - only group , and no future studies are likely to include such an arour observational data found no difference in os among metastatic msi - h cases between cases treated with a basic fu + lcv regimen only , compared with cases treated with any ( oxaliplatin or irinotecan ) combination chemotherapy with or without bevacizumab , whereas a significant os improvement was found with advanced chemotherapy in met / mss cases . 
similarly , we did not have any cases that were treated with immune checkpoint inhibitors at the time interval of the current analysis . few reports evaluated the effect of chemotherapy in met / msi - h cases . 
although early studies reported a positive effect of high - dose fu + lcv in patients with met / msi - h in comparison with patients with met / mss , 35 , 36 more recent studies of fu , in combination with irinotecan or oxaliplatin , did not show benefit.16 , 31 , 34 , 37 one study31 showed a trend toward benefit of oxaliplatin - based treatment in cases of msi - h mcrc . 
our data do not have enough power to evaluate specific therapeutic regimens . we chose to exclude from our study all metastatic cases that were not treated at all , because of the inability to assess the causes for no treatment and biases that could stem from it . 
comparison of os results in met / mss and met / msi - h groups supports biologic differences , with a baseline survival advantage for the msi - h group . 
effect of combination treatment on overall survival of patients with metastatic , brafwild type colorectal cancer by microsatellite instability status in the molecular epidemiology of colorectal cancer study , northern israel . 
 published data on the effect of braf mutation on prognosis in patients with met / msi - h were published on a group of crc stages ii to iv , 38 potentially underrepresenting the few cases with stage iv disease . 
extremely poor outcomes in this patient group support the use of the folfoxiri ( oxaliplatin , irinotecan , fluorouracil , leucovorin ) protocol in the unselected group of braf - mutated cases . 
given the much higher prevalence of braf mutations in the msi - h cases , 11 , 26 , 39 it is important to study the outcomes of folfoxiri in this subgroup of patients with met / msi - h . our study is limited by the relatively modest sample size of patients with msi - h mcrc . 
 advantages of our population - based sampling of incident cases with long - term follow - up include generalizability to community practice in a large and well - defined population . 
the representative , observational nature of the data from a large health system , in combination with molecular evaluation of all tumor tissues , offers a framework for understanding clinical practice over nearly two decades in a universally insured group of patients in a single country . msi status is becoming a cornerstone of individualized decision making with regard to a variety of potential oncological interventions , and this trend is likely to continue with the introduction of immunotherapy.40 , 41 routine mutational profiling of mcrc and specifically msi testing and other assays that designate deficient mismatch repair have already been recommended.39 , 42 , 43 the failure of conventional advanced treatments to improve survival in the meaningful subset of met / msi - h cases emphasizes the need to evaluate the role of the newly introduced immunotherapy as a first - line treatment in these cases with msi - h / braf - wt mcrc . in conclusion , therapeutic approaches to mcrc have changed dramatically during the past two decades , with increasing reliance on the molecular features of tumors to inform treatment decisions . 
gruber stock and other ownership interests : teva pharmaceuticals provision of study material or patients : flavio lejbkowicz collection and assembly of data : all authors data analysis and interpretation : katerina shulman , ofra barnett - griness , joel k . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . katerina shulman no relationship to disclose vered friedman no relationship to disclose joel k . 
gruber employment : brogent technologies leadership : brogent technologies stock and other ownership interests : brogent technologies , fulgent therapeutics consulting or advisory role : myriad genetics , fulgent therapeutics research funding : myriad genetics ( inst ) flavio lejbkowicz no relationship to disclose gad rennert no relationship to disclose affiliations katerina shulman , hillel yaffe medical center , hadera ; katerina shulman , ofra barnett - griness , vered friedman , flavio lejbkowicz , and gad rennert , carmel medical center , and technion , haifa , israel ; joel k . 
zhou sw , huang yy , wei y , et al : no survival benefit from adding cetuximab or panitumumab to oxaliplatin - based chemotherapy in the first - line treatment of metastatic colorectal cancer in kras wild type patients : a meta - analysis . 
chan dl , pavlakis n , shapiro j , et al : does the chemotherapy backbone impact on the efficacy of targeted agents in metastatic colorectal cancer ? a systematic review and meta - analysis of the literature . 
siena s , sartore - bianchi a , di nicolantonio f , et al : biomarkers predicting clinical outcome of epidermal growth factor receptor - targeted therapy in metastatic colorectal cancer . 
tol j , dijkstra jr , klomp m , et al : markers for egfr pathway activation as predictor of outcome in metastatic colorectal cancer patients treated with or without cetuximab . 
mller ci , schulmann k , reinacher - schick a , et al : predictive and prognostic value of microsatellite instability in patients with advanced colorectal cancer treated with a fluoropyrimidine and oxaliplatin containing first - line chemotherapy . 
sargent dj , marsoni s , monges g , et al : defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil - based adjuvant therapy in colon cancer . 
weitzel jn , blazer kr , macdonald dj , et al : genetics , genomics , and cancer risk assessment : state of the art and future directions in the era of personalized medicine . 
webber em , kauffman tl , oconnor e , et al : systematic review of the predictive effect of msi status in colorectal cancer patients undergoing 5fu - based chemotherapy . 
des guetz g , schischmanoff o , nicolas p , et al : does microsatellite instability predict the efficacy of adjuvant chemotherapy in colorectal cancer ? a systematic review with meta - analysis . 
ribic cm , sargent dj , moore mj , et al : tumor microsatellite - instability status as a predictor of benefit from fluorouracil - based adjuvant chemotherapy for colon cancer . 
cremolini c , loupakis f , masi g , et al : folfoxiri or folfoxiri plus bevacizumab as first - line treatment of metastatic colorectal cancer : a propensity score - adjusted analysis from two randomized clinical trials . 
van cutsem e , khne c - h , lng i , et al : cetuximab plus irinotecan , fluorouracil , and leucovorin as first - line treatment for metastatic colorectal cancer : updated analysis of overall survival according to tumor kras and braf mutation status . 
peeters m , price tj , cervantes a , et al : randomized phase iii study of panitumumab with fluorouracil , leucovorin , and irinotecan ( folfiri ) compared with folfiri alone as secondline treatment in patients with metastatic colorectal cancer . 
tran b , kopetz s , tie j , et al : impact of braf mutation and microsatellite instability on the pattern of metastatic spread and prognosis in metastatic colorectal cancer . 
goldstein j , tran b , ensor j , et al : multicenter retrospective analysis of metastatic colorectal cancer ( crc ) with high - level microsatellite instability ( msi - h )  . 
liang j - t , huang k - c , lai h - s , et al : high - frequency microsatellite instability predicts better chemosensitivity to high - dose 5 - fluorouracil plus leucovorin chemotherapy for stage iv sporadic colorectal cancer after palliative bowel resection . 
brueckl wm , moesch c , brabletz t , et al : relationship between microsatellite instability , response and survival in palliative patients with colorectal cancer undergoing first - line chemotherapy . 
kim je , hong ys , ryu m - h , et al : association between deficient mismatch repair system and efficacy to irinotecan - containing chemotherapy in metastatic colon cancer . 
overman mj , mcdermott r , leach jl , et al : nivolumab in patients with metastatic dna mismatch repair - deficient or microsatellite instability - high colorectal cancer ( checkmate 142 ) : an open - label , multicentre , phase 2 study . 
dunne pd , oreilly pg , coleman hg , et al : stratified analysis reveals chemokine - like factor ( cklf ) as a potential prognostic marker in the msi - immune consensus molecular subtype cms1 of colorectal cancer . 
the aim of our study was to investigate whether ctdna can be used for response monitoring in neuroblastoma . methods one hundred forty - nine plasma samples from 56 patients were analyzed by quantitative polymerase chain reaction ( qpcr ) for total cell free dna ( cfdna ; albumin and - actin ) and ctdna ( hypermethylated rassf1a )  . 
ctdna results were compared with mrna - based minimal residual disease ( qpcr ) in bone marrow ( bm ) and blood and clinical patient characteristics . results ctdna was detected at diagnosis in all patients with high - risk and stage m neuroblastoma and in 3 of 7 patients with localized disease . 
the discrepancies indicated either low - level bm inltration ( ctdna negative / mrna positive ) or primary tumor / soft tissue lesions with no bm involvement ( ctdna positive / mrna negative )  . conclusion ctdna can be used for monitoring disease in patients with neuroblastoma . 
it is likely that ctdna can originate from both primary tumor and metastases and may be of special interest for disease monitoring in patients who experience relapse in other organs than bm . jco precis oncol 4 : 291 - 306 . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction neuroblastoma is the most common extracranial solid tumor of childhood . in approximately 50% , patients present with high - risk ( hr ) disease and are treated with intensive multimodality treatment protocols that encompass induction therapy , primary tumor surgery , myeloablative chemotherapy with autologous stem - cell rescue , local irradiation , and anti - gd2based immunotherapy1 ( appendix fig a1 )  . 
despite this intensive therapy , in approximately one half of hr patients , the tumor will relapse and result in a fatal outcome.2 assessment of treatment response is based on the international neuroblastoma response criteria . 
meta - iodobenzylguanidine ( mibg ) scintigraphy , imaging ( magnetic resonance imaging / positron emission tomography scans ) , and bone marrow ( bm ) examinations by histology or ( immuno ) cytology are combined to assess the extent of disease.3 because the median age at diagnosis is 18.8 months , 4 response evaluation that is based on imaging and bm testing often must be performed under general anesthesia . 
 van zogchel et al context key objective can hypermethylated rassf1a be used as a circulating tumor dna ( ctdna ) marker for minimal residual disease detection in neuroblastoma ? knowledge generated when testing cell free dna , we were able to detect tumor - derived hypermethylated rassf1a in all patients with stage m disease at diagnosis . 
comparison between ctdna and bloodor bone marrow ( bm ) derived mrna revealed that discrepancies were found when bm inltration was low or when there were primary tumor lesions without bm involvement . relevance ctdna is an interesting source for monitoring disease in patients with neuroblastoma . 
our data indicate that testing of both ctdna and mrna increases the sensitivity of molecular disease monitoring . several studies have shown the feasibility of detecting mutations or tumor - specic translocations in the ctdna by high - depth targeted sequencing or mutation - specic pcr to monitor disease in various types of adult cancer.13 - 19 however , neuroblastoma tumors , like many pediatric tumors , lack recurrent mutations and translocations . 
broader analysis , such as whole - exome sequencing ( wes ) and shallow whole - genome sequencing ( swgs ) , to detect tumor - specic mutations or copy number alterations have been performed successfully using cfdna of patients with neuroblastoma at diagnosis and relapse.20 - 26 nevertheless , these techniques are only informative when the ctdna content is approximately 10%27 and are , therefore , less suited for the detection of mrd , when the tumor burden is low . in contrast to the copy number alterations and tumorspecic mutations , a methylation - specic qpcr assay could potentially be a more general and sensitive ctdna marker . 
hypermethylated rassf1a ( rassf1am ) can be detected in bm with a similar sensitivity as mrna and has shown added value in mrna - negative bm.28 in addition , rassf1am already has been described as a prognostic ctdna marker at diagnosis.29 the aim of this study was to investigate the feasibility of using ctdna ( rassf1am in plasma ) to monitor treatment response in patients with hr / stage m neuroblastoma . 
we retrospectively performed qpcr for rassf1am on cfdna from stored remains of previously collected plasma samples of patients with localized or metastatic neuroblastoma at diagnosis and for patients with hr / stage m neuroblastoma during treatment and at relapse . 
to test the additional value of ctdna monitoring , we compared it with other techniques for disease monitoring : mibg scans , urinary catecholamines , immunocytology , and rt - qpcr rna - based mrd detection in bm and peripheral blood ( pb )  . methods between 2013 and 2016 , from all consecutively diagnosed patients who were included in this study ( n = 56 ) , 149 pb samples for mrna and cfdna and 105 bm samples for mrna were tested . 
pediatric pb control samples were collected from the amsterdam umc ( appendix table a1 )  . sample collection , dna isolation , bisulte conversion , and real - time qpcr methods for sample collection , dna isolation , bisulte conversion , and real - time qpcr for rassf1am28 and mrna markers31 can be found in the appendix and appendix table a2 . data analysis total cfdna was quantied by qpcr for albumin ( alb ) or - actin ( actb )  . 
all statistical analyses were performed with spss version 23 ( ibm corporation , chicago , il ) or graphpad prism 8 ( graphpad software , la jolla , ca ) software . results patients and samples from 48 patients with hr and / or stage m and 8 patients with non - hr neuroblastoma , 149 samples were tested in this study ( fig 1 )  . 
six of the 149 patient samples and 12 of 73 healthy control samples were not included for rassf1am qpcr because too much dna had been lost during bisulte conversion ( fig 1 )  . 
flowchart of samples tested for total cell free dna ( cfdna ) , number of samples excluded after too much dna had been lost after bisulte conversion , and number of samples tested for circulating tumor dna ( ctdna ) by hypermethylated rassf1a ( rassf1am )  . 
the percentage of ctdna of total cfdna , calculated with the equation [ rassf1am / ( rassf1am + unmethylated rassf1a ) 100 ] , was 94% ( range , 82% - 98% ) in the 14 diagnostic samples from patients with stage m disease . 
compared with the individual mrna markers , rassf1am was more often positive , but the combined mrna markers identied the same positive samples as rassf1am ( appendix table a4 )  . in 93 matched bm mrna and ctdna ( pb ) samples , double - negative or double - positive results were found in 77% ( fig 3b )  . 
in contrast to pb , the bm mrna panel identied more positive samples than ctdna , and the individual markers phox2b and th correlated best with rassf1am ( appendix table a4 )  . discrepant findings between ctdna and pb or bm mrna mrd discrepant results between ctdna and mrna were detected in 27 pb and 21 bm samples , respectively , and listed in table 3 and appendix table a4 . 
from 3 of 5 bm mrna - negative / ctdnapositive samples , cryopreserved bm cells were available and tested all negative for rassf1ain some patients ( n850 , n865 , n732 ) , the high levels of ctdna probably correlated with the large primary or local relapse tumors , and these patients had no or very little bm inltration . in the ctdna - negative samples , in general , the cfdna levels were lower , with a median of 6.1 ng / ml for the bm mrna - positive / ctdna - negative and 1.52 ng / ml for the pb mrna - positive / ctdna - negative samples . 
from 15 of negative samples , cryopreserved cells were available and tested for rassf1am ; of the 5 positive samples , 4 were not in the quantitative range , which indicated low levels of bm inltration . in the samples from patients n777 , n798 , n2011 , n2014 , and n802 , no ctdna was detected . 
begin induction indicates until 2 courses of induction therapy ; mid - induction indicates after 3 - 5 courses of induction chemotherapy , unless additional courses were given after 6 courses , and samples before last course were also included at this time point ; end induction indicates at the end of induction therapy ; surveillance indicates during follow - up or at relapse suspicion ; and event indicates relapse or progression . 
of samples diagnosis samples localized and stage ms stage m follow - up samples beginning of induction therapy mid - induction therapy end of induction therapy postconsolidation surveillance progression relapse / refractory disease relapse therapy note . 
positive indicates that rassf1am circulating tumor dna was detected and quantied , and negative indicates that no rassf1am circulating tumor dna was detected . abbreviations : pnq , positive not quantiable ; rafssf1am , hypermethylated rafssf1a . complete remission at that time . 
patient n802 was treated for an isolated cns relapse . in the samples from patients n2012 , n2013 , n2016 , n2024 , n2029 , and n2031 , no ctdna was detected , while low amounts of mrna were detected in the bm . in the case of restricted , minimal bm disease , mrna detection was more sensitive than ctdna ( appendix table a4 )  . 
however , in some patients , a primary tumor was still present ( median , 50 mm ) while ctdna was negative ( table 3 )  . discussion ctdna in plasma is a powerful source for the detection of tumor - derived aberrations in a minimally invasive setting . many ctdna studies in adults for the detection of mrd are based on detection of tumor - specic mutations by targeted sequencing or digital droplet pcr ( ddpcr ) .12 , 16 , 17 because recurrent mutations are not common in neuroblastoma , 32 tumor - specic aberrations need to be characterized before they can be used as an mrd marker . 
however , temporospatial heterogeneity has been reported in neuroblastoma by several studies , 20 , 26 which raises the question of whether we should only use the small part of the tumor that is derived from the biopsy to design tumor - specic mrd markers . 
first , it is a sensitive marker , with a sensitivity of 1 tumor cell in 105 mononuclear cells.28 second , rassf1am qpcr can be used in all patients with stage m neuroblastoma because it has been shown that rassf1a is hypermethylated in all previously tested stage m neuroblastoma tumors.28 third , detection of rassf1am is less costly compared with wes and even swgs ( approximately 40and 10 - fold less expensive , respectively )  . 
hypermethylation of rassf1a has been described in several types of cancer and in physiologic circumstances in placental cells.33 rassf1a is not methylated in normal hematologic cells.28 , 33 , 34 however , in 2 of 61 samples from healthy individuals , we detected very low , nonquantiable levels of rassf1ain diagnosis or relapse other diagnosis or relapse other n = 11 n = 12 n = 23 n = 16 n = 12 n = 124 positive negative positive negative n = 66 n = 12 n = 4 n = 36 n = 4 n = 1 negative ctdna ( rassf1am ) positive negative ctdna ( rassf1am ) positive fig 3 . 
 van zogchel et al addition , in other studies , infrequent detection of rassf1am has been observed in plasma samples from healthy control participants.35 , 36 therefore , when detecting verylow levels of rassf1am in patients with neuroblastoma ( indicated as pnq range ) , results should be analyzed with caution . it has been shown that neuroblastoma tumors shed high amounts of ctdna in the plasma.25 , 26 , 37 in the current study , we found a median cfdna concentration of 73.1 ng / ml at diagnosis for patients with stage m disease . 
however , the levels we found are lower compared with previously published studies.25 , 26 , 37 this inconsistency may be due to differences in isolation of cfdna because we did not use a circulating nucleic acid kit or to differences in quantication methods . 
we found the majority of the cfdna ( 94% at diagnosis ) to be tumor derived in patients with stage m or hr disease , which is also supported by previous research.26 , 37 we tested 143 samples from 54 patients with neuroblastoma and detected ctdna in 57 samples . 
ctdna was detected at diagnosis in all 14 patients with stage m and 4 of 8 patients with localized and stage ms neuroblastoma . misawa et al29 described detection of rassf1am at diagnosis in the serum of 17 of 68 patients ( all stages ) and in 11 of 18 patients with stage m disease . 
there are two likely causes for the increased ctdna detection in our study . first , we used plasma , whereas misawa et al tested serum , which is known to be more contaminated by genomic dna originated from leukocytes during ex vivo clotting.38 second , misawa et al used conventional pcr , which is less sensitive than qpcr . 
our group has previously described that hypermethylation of rassf1a is variable in tumors of patients with stage ms ( median , 65% ) and localized ( median , 30% ) disease28 ; therefore , the level of ctdna can be slightly underestimated in these patients when using rassf1am as marker . we compared the performance of ctdna with pb and bm mrna in 128 and 93 samples , respectively . 
most patients in whom we detected relatively high levels of ctdna compared with pb or bm mrnas still had considerable tumor volumes or negative or low mibg scores ( data not shown ) ; therefore , it is likely that the ctdna in these patients originated from the primary tumor . 
two of these patients were in complete remission but in the other 15 patients , considerable tumor volumes were detected on imaging or urine catecholamines were still positive , which indicate the need to optimize pre - analytic sample handling and prospective study of cfdna kinetics in well - characterized patient cohorts with available paired ( nuclear ) imaging and bm assessment . while the detection of ctdna is very promising for future mrd studies , the current study has some limitations . stored remains were used , which resulted in missing samples and paired clinical data . 
for detection and quantication of low levels of ctdna in the plasma , dna extraction methods can be optimized with an isolation method specic for cfdna , and ddpcr39 may be a more suited technique compared with qpcr.40 moreover , large amounts of cfdna ( up to 96% ) could be destroyed during bisulte conversion41 ; therefore , we are investigating alternative methylation - specic ddpcr methods . 
previous studies showed that rassf1a was the most frequent hypermethylated tumor suppressor gene in neuroblastoma as well as identied other hypermethylated tumor suppressor genes , and inclusion of these genes as mrd markers might increase the sensitivity.42 , 43 in this study , we used rassf1am as a ctdna marker . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . no potential conicts of interest were reported . references pinto nr , applebaum ma , volchenboum sl , et al : advances in risk classication and treatment strategies for neuroblastoma . 
j clin oncol 33 : 3008 - 3017 , 2015 park jr , kreissman sg , london wb , et al : effect of tandem autologous stem cell transplant vs single transplant on event - free survival in patients with high - risk neuroblastoma : a randomized clinical trial . 
jama 322 : 746 - 755 , 2019 park jr , bagatell r , cohn sl , et al : revisions to the international neuroblastoma response criteria : a consensus statement from the national cancer institute clinical trials planning meeting . 
j clin oncol 35 : 2580 - 2587 , 2017 london wb , castleberry rp , matthay kk , et al : evidence for an age cutoff greater than 365 days for neuroblastoma risk group stratication in the childrens oncology group . 
j clin oncol 23 : 6459 - 6465 , 2005 viprey vf , gregory wm , corrias mv , et al : neuroblastoma mrnas predict outcome in children with stage 4 neuroblastoma : a european hr - nbl1 / siopen study . 
j clin oncol 32 : 1074 - 1083 , 2014 cheung nk , ostrovnaya i , kuk d , et al : bone marrow minimal residual disease was an early response marker and a consistent independent predictor of survival after anti - gd2 immunotherapy . 
j clin oncol 33 : 755 - 763 , 2015 kreissman sg , seeger rc , matthay kk , et al : purged versus non - purged peripheral blood stem - cell transplantation for high - risk neuroblastoma ( cog a3973 ) : a randomised phase 3 trial . 
lancet oncol 14 : 999 - 1008 , 2013 burchill sa , beiske k , shimada h , et al : recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the international neuroblastoma response criteria bone marrow working group . 
cancer 123 : 1095 - 1105 , 2017 stutterheim j , zappeij - kannegieter l , versteeg r , et al : the prognostic value of fast molecular response of marrow disease in patients aged over 1 year with stage 4 neuroblastoma . 
gormally e , caboux e , vineis p , et al : circulating free dna in plasma or serum as biomarker of carcinogenesis : practical aspects and biological signicance . mutat res 635 : 105 - 117 , 2007 2017 12 . 
mcbride dj , orpana ak , sotiriou c , et al : use of cancer - specic genomic rearrangements to quantify disease burden in plasma from patients with solid tumors . genes chromosomes cancer 49 : 1062 - 1069 , 2010 16 . 
tsao sc , weiss j , hudson c , et al : monitoring response to therapy in melanoma by quantifying circulating tumour dna with droplet digital pcr for braf and 18 . 
van roy n , van der linden m , menten b , et al : shallow whole genome sequencing on circulating cell - free dna allows reliable noninvasive copy - number proling in neuroblastoma patients . 
chicard m , colmet - daage l , clement n , et al : whole - exome sequencing of cell - free dna reveals temporo - spatial heterogeneity and identies treatmentresistant clones in neuroblastoma . 
yanik ga , parisi mt , shulkin bl , et al : semiquantitative mibg scoring as a prognostic indicator in patients with stage 4 neuroblastoma : a report from the 31 . 
stutterheim j , gerritsen a , zappeij - kannegieter l , et al : detecting minimal residual disease in neuroblastoma : the superiority of a panel of real - time quantitative 32 . 
chan kc , ding c , gerovassili a , et al : hypermethylated rassf1a in maternal plasma : a universal fetal dna marker that improves the reliability of noninvasive prenatal diagnosis . 
scheffer pg , de haas m , van der schoot ce : the controversy about controls for fetal blood group genotyping by cell - free fetal dna in maternal plasma . 
white he , dent cl , hall vj , et al : evaluation of a novel assay for detection of the fetal marker rassf1a : facilitating improved diagnostic reliability of noninvasive opin hematol 18 : 467 - 473 , 2011 prenatal diagnosis . 
wong fc , sun k , jiang p , et al : cell - free dna in maternal plasma and serum : a comparison of quantity , quality and tissue origin using genomic and epigenomic approaches . 
 circulating tumor dna in neuroblastoma appendix sample collection clinical samples were collected in edta tubes and processed within 24 hours , and 2 ml peripheral blood ( pb ) or 0.5 ml bone marrow ( bm ) was transferred to paxgene blood rna tubes ( qiagen , venlo , the netherlands ) and stored at 20c . 
mononuclear cells were isolated from the remaining bm sample by ficoll density centrifugation and cryopreserved in 10% dimethyl sulfoxide . dna isolation and bisulte conversion dependent on the available plasma volume , dna was extracted by using the qiaamp dna mini blood kit ( qiagen ) for 200 l plasma , or the magna pure 96 isolation robot ( roche , basel , switzerland ) for 500 - 1 , 000 l plasma and eluted in h2o . 
converted dna samples were used directly or stored at 20c . real - time quantitative polymerase chain reaction real - time quantitative polymerase chain reaction ( qpcr ) was performed as previously described ( van wezel em , et al : j mol diagn 17 : 43 - 52 , 2015 )  . 
primer and probe sequences have been described previously28 ( scheffer pg , et al : bjog 118 : 1340 - 1348 , 2011 ) and are listed in appendix table a2 . 
qpcr for alb , actb , and unmethylated rassf1a was performed in duplicate ; hypermethylated rassf1a was tested in triplicate . pb and bm mrna reverse transcriptase qpcr total rna from whole blood and bm samples was extracted using the paxgene blood rna kit ( qiagen ) according to the manufacturers instructions . 
cdna was synthesized and reverse transcriptase ( rt ) qpcr , with a maximum of 50 cycles was performed for - glucuronidase ( gusb ) , paired ctdna in neuroblastoma like homeobox 2b ( phox2b ) , tyrosine hydroxylase ( th ) , dopa decarboxylase ( ddc ) , growth - associated protein 43 ( gap43 ) , cholinergic receptor nicotinic - 3 ( chrna3 ) , and dopamine - hydroxylase ( dbh ) , as has been described previously.31 expression was normalized to gusb expression using the following equation : [ normalized threshold cycle ( ct ) = ( ctgusb ctmarker ) ]  . 
samples were scored for positivity according to previously published thresholds9 , 31 ( stutterheim j , et al : j clin oncol 26 : 5443 - 5449 , 2008 )  . 
oncogenic met activation results from a hijacking of various aspects of the canonical met pathway otherwise normally active in development and wound healing.2 the canonical pathway consists of hgf ligand - induced dimerization , tyrosine kinase domain activation , and the subsequent activation of a downstream signaling cascade resulting in the met - driven oncophenotype . 
shouldnt a targeted therapy be directed toward a susceptible cancer cell that harbors the target ? but what was the biomarker to reliably signify that a tumor was truly met dependent and would derive substantial benet from anti - met therapy ? the leading biomarker candidate was , and still is , met amplication , which has severalfold higher protein expression as observed by mass spectrometry compared with nonamplied tumors.8 although rare , amounting to only approximately 5% of gea ( the highest incidence of any cancer ) , met amplication has been the most consistent and reliable biomarker both of overall poor prognosis and predictive benet from agents targeting the receptor.3 , 4 , 12 - 14 however , other potential predictive biomarkers have also been proposed , including met tyrosine kinase phosphorylation ( difcult to measure ) , 15 met activating mutation or fusion ( rare ) , met exon 14 skipping or deletion ( mostly lung cancer ) , 16 high hgf tumor and / or serum levels ( not consistently demonstrated ) , and met overexpression ( in the absence of met amplication ) .17 focus on the latter overexpression ensued , likely because this was generally than the more stringent genomic more prevalent denition of amplication , making this more expedient and lucrative to study . 
what exactly is the denition of met overexpression ? met expression by immunohistochemistry ( ihc ) has many descriptions , including cellular distribution ( membranous , cytoplasmic , and / or nuclear staining ) , extensity ( the percentage of tumor cells actually staining ) , and intensity ( the semiquantied staining strength from 0 to 3 + )  . 
 catenacci death ligand 1 ( pd - l1 ) , various alterations in the scoring criteria can change a tumor from met negative to met positive , or vice versa , and dramatically alter positivity incidence with slight scoring modications . 
benet in the met - high population ( 64% incidence in that study ) hinged on a small retrospective subgroup of 47 and 21 patients receiving rilotumumab and placebo , respectively.18 unfortunately , the competition to be rst to market and , therefore , the rush to initiate phase iii studies with rilotumumab in rilomet - 1 ( international phase iii multicenter , randomized , double - blind , placebocontrolled trial of rilotumumab plus epirubicin , cisplatin , and capecitabine as first - line therapy in patients with advanced met - positive gastric or gastroesophageal junction adenocarcinoma ) and onartuzumab in metgastric ( fluorouracil , leucovorin , and oxaliplatin with or without onartuzumab in her2 - negative , met - positive gastroesophageal adenocarcinoma ) , without more attention to these issues , resulted in a fundamental lack of biomarker understanding . 
because the questions were unasked , the answers were unknown . importantly , thus , the market - based engines steamed on ahead with the following two bits of data : one patient who responded to an lbab monotherapy5 and one retrospective small subgroup analysis of a phase ii randomized study.18 unfortunately , ultimately both lbab phase iii studies were negative.10 , 11 regrettably , neither study successfully enriched for a population who would benet from met lbabs . 
notably , for the rilotumumab studies , the initial antibody prototype assay and , the actual scoring system used in the phase ii study were changed for the prospective phase iii study to ofcially codevelop a companion diagnostic assay . the assay in rilomet - 1 likely inadequately selected any optimal patient population for treatment , with most patients ( 81% ) screened considered positive for met expression using the lax criteria of greater than or equal to 1 + intensity at 25% or greater extensity staining ( hardly selecting ) and most patients having low - level expression.11 prospectively testing formalin - xed parafn - embedded ( ffpe ) freshly cut samples from recently diagnosed patients in rilomet - 1 compared with a retrospective batched archival ffpe analysis in the phase ii study may also have contributed to the incidence discrepancy between the studies , because ihc sensitivity is decreased with older ffpe samples and , when still positive in light of this , would represent those indeed , true benet randomized phase ii samples likely having the highest levels of expression to begin with . 
conversely , the metgastric study had only the slightly more stringent criteria of positivity for eligibility of greater than or equal to 1 + intensity at 50% or greater extensity staining ( ie , more cells needed to be expressing at least low - level amounts of protein )  . 
notably , in this latter underpowered subgroup analysis , the hr for overall survival was 0.64 in favor of onartuzumab ( p = .062 ) , and the median survival increased from 9.7 months to 11 months with onartuzumab.10 could this have been statistically signicant with the originally intended number of patients ? because the question was unasked ( study terminated early ) , the answer is unknown . 
in the end , the optimal met biomarker assay , scoring , and positivity criteria of expression for lbabs , if any , remain undened . nevertheless , even if we could rewind and do things differently with this hindsight ( could we perform prospective , adequately accrued , randomized phase ii and / or iii studies with only the highest ihc criteria amounting to approximately 20% to 30% of all patients with gea ? ) , the marginal absolute benets that could be realistically achieved with lbab in an optimal expressing subgroup , as exemplied by the metgastric subgroup analysis hr of 0.64 , lend pause to question whether this would really provide signicant clinical improvement . 
yet , frustratingly , met amplication , now serving as a driver oncogene in this scenario , is the biomarker subset to likely derive the most benet from targeted met receptor inhibitors . 
in comparison , pd - l1 checkpoint blockade monotherapies , despite the hysteria , ultimately demonstrated an approximate 10% response rate in the third - line or higher setting in gea all - comers , with a median progression - free survival time of 2 months , and only a 13.3% response rate ( 19 of 143 patients ) in the subset of pd - l1positive , microsatellite stable ( mss ) selected patients , with a median dor of less than 8 months ( only ve patients , or ve [ 28% ] of 19 responders or ve [ 3% ] of 143 pd - l1positive / mss patients , had dor greater than 12 months ) .25 yet on the basis of these results , pembrolizumab was recently successfully pursued for accelerated approval for the entire pd - l1positive / mss subset by the us food and drug administration.25 , 26 similar clinical outcomes are consistently reported in patients with met - amplied gea ( with comparable incidence of responses lasting greater than 12 months )  . 
in the face of being an uncommon genomic event , this discussion still matters for patients with gea with tumors harboring met amplication . this amg337 example , along with other met inhibitors , has well - elucidated reasons for perceived failure . 
these include underappreciating the general poor prognosis of met - amplied tumors in single - arm , late - line studies , intrapatient heterogeneity with regions harboring and not harboring met amplication , and the presence of concurrent genomic aberrations in some met - amplied cells . all of these factors could lead to relatively quick progression on monotherapy either outright or shortly after an initial response in most , but importantly not all , responding patients . 
however , the ideal randomized and combination therapy studies have been elusive to date because of the infeasibility of classic clinical study designs and the requirement of extraordinarily high numbers of patients screened to identify the met - amplied subset , with the consequent abandonment of additional investigation by pharma because of this apparently high - risk and low - yield scenario . 
with met , supportive evidence abounds that there are patients who derive benet from met inhibitors , particularly those with met - amplied tumors , yet to date , no met - specic inhibitor is approved by the us food and drug administration in any met - specic indication for any tumor . 
lack of progress can be attributed to a number of issues including inadequate validation of biomarker cut - offs , along with the inherent hurdles to developing drugs in lowincidence biomarker populations , coupled with complex intrapatient heterogeneous biology rendering monotherapy in late - line therapy less likely to impress , 34 and certainly so for accelerated approval paths . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . daniel catenacci honoraria : genentech , eli lilly , amgen , oncoplex diagnostics , foundation medicine , taiho pharmaceutical , genmab , nantomics , guardant health , merck , bristol - myers squibb , gritstone oncology , five prime therapeutics , astellas pharma consulting or advisory role : genentech , amgen , merck , eli lilly , taiho pharmaceutical , bristol - myers squibb , astellas pharma speakers ' bureau : guardant health , foundation medicine , genentech , eli lilly , merck references giordano s , di renzo mf , ferracini r , et al : p145 , a protein with associated tyrosine kinase activity in a human gastric carcinoma cell line . 
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clin cancer res 20 : 1666 - 1675 , 2014 catenacci dv , liao wl , thyparambil s , et al : absolute quantitation of met using mass spectrometry for clinical application : assay precision , stability , and correlation with met gene amplication in ffpe tumor tissue . 
plos one 9 : e100586 , 2014 shah ma , wainberg za , catenacci dv , et al : phase ii study evaluating 2 dosing schedules of oral foretinib ( gsk1363089 ) , cmet / vegfr2 inhibitor , in patients with metastatic gastric cancer . 
shah ma , bang yj , lordick f , et al : effect of uorouracil , leucovorin , and oxaliplatin with or without onartuzumab in her2 - negative , met - positive gastroesophageal adenocarcinoma : the metgastric randomized clinical trial . 
catenacci dvt , tebbutt nc , davidenko i , et al : rilotumumab plus epirubicin , cisplatin , and capecitabine as rst - line therapy in advanced met - positive gastric or gastro - oesophageal junction cancer ( rilomet - 1 ) : a randomised , double - blind , placebo - controlled , phase 3 trial . 
smolen ga , sordella r , muir b , et al : amplication of met may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor pha665752 . 
j clin oncol 29 : 4803 - 4810 , 2011 jardim dlf , tang c , gagliato ddm , et al : analysis of 1 , 115 patients tested for met amplication and therapy response in the md anderson phase i clinic . 
awad mm , oxnard gr , jackman dm , et al : met exon 14 mutations in non - small - cell lung cancer are associated with advanced age and stage - dependent met genomic amplication and c - met overexpression . 
neoplasia 7 : 75 - 84 , 2005 iveson t , donehower rc , davidenko i , et al : rilotumumab in combination with epirubicin , cisplatin , and capecitabine as rst - line treatment for gastric or oesophagogastric junction adenocarcinoma : an open - label , dose de - escalation phase 1b study and a double - blind , randomised phase 2 study . 
shah ma , cho jy , tan ib , et al : a randomized phase ii study of folfox with or without the met inhibitor onartuzumab in advanced adenocarcinoma of the stomach and gastroesophageal junction . 
wilke h , muro k , van cutsem e , et al : ramucirumab plus paclitaxel versus placebo plus paclitaxel in patients with previously treated advanced gastric or gastrooesophageal junction adenocarcinoma ( rainbow ) : a double - blind , randomised phase 3 trial . 
kwak el , lorusso p , hamid o , et al : clinical activity of amg 337 , an oral met kinase inhibitor , in adult patients ( pts ) with met - amplied gastroesophageal junction ( gej ) , gastric ( g ) , or esophageal ( e ) cancer . 
hong ds , lorusso pm , hamid o , et al : phase 1 study of amg 337 , a highly selective small - molecule met inhibitor , in patients with advanced solid tumors . 
van cutsem e , karaszewska b , kang yk , et al : a multicenter phase 2 study of amg 337 in patients with met - amplied gastric / gastroesophageal junction / esophageal adenocarcinoma and other solid tumors . 
fuchs cs , doi t , jang rw , et al : safety and efcacy of pembrolizumab monotherapy in patients with previously treated advanced gastric and gastroesophageal junction cancer : phase 2 clinical keynote - 059 trial . 
catenacci dvt , park h , uronis he , et al : margetuximab ( m ) plus pembrolizumab ( p ) in erbb2 - amplied pd - l1 + gastroesophageal adenocarcinoma ( gea ) post trastuzumab ( t )  . 
lancet oncol 16 : 1276 - 1278 , 2015 joshi ss , maron sb , lomnicki s , et al : personalized antibodies for gastroesophageal adenocarcinoma ( pangea ) : a phase ii precision medicine trial ( nct02213289 )  . 
j natl cancer inst 97 : 249 - 250 , 2005 jardim dl , groves es , breitfeld pp , et al : factors associated with failure of oncology drugs in late - stage clinical development : a systematic review . 
hamid o , gajewski tf , frankel ae , et al : epacadostat plus pembrolizumab in patients with advanced melanoma : phase 1 and 2 efcacy and safety results from echo - 202 / keynote - 037 . 
long gv , dummer r , hamid o , et al : epacadostat ( e ) plus pembrolizumab ( p ) versus pembrolizumab alone in patients ( pts ) with unresectable or metastatic melanoma : results of the phase 3 echo - 301 / keynote - 252 study . 
gene amplication , or increased gene dosage , leads to increased messenger rna expression , which leads to exponential increase in the amount of met protein receptor ( green ) expression at the membrane . 
 c sustained exceptional response to poly ( adp - ribose ) polymerase inhibition plus temozolomide in metastatic melanoma with dna repair deficiency introduction the median overall survival ( os ) of patients with melanoma with brain metastases was 4.7 months in a retrospective series from 1986 to 2004.1 in historical series of such patients , median os was 7.5 months for those diagnosed between 2000 and 2008 , 8.5 months for those diagnosed between 2009 and 2010 , and 22.5 months for those diagnosed from 2011 on.1 - 5 the availability of newer systemic therapies , such as braf inhibitors and immunotherapy , in patients with melanoma and brain metastases are directly attributable to the increased length in survival.2 - 5 better understanding of the biology of advanced melanoma could help researchers to identify targetable aberrations in the relapsed refractory setting , and integrative clinical sequencing may allow us to exploit additional therapeutic modalities . 
the tumor was ulcerated with an involved marg he had a wide local excision and left - side axillary dissection that showed residual melanoma to a depth of 12 mm , clear margins , and zero of 14 lymph nodes positive for melanoma . 
after work - up , he was found to be eligible and was treated with high - dose interleukin - 2 and received a first cycle ( 11 of a planned 14 doses ) and a second cycle ( eight of a planned 14 doses )  . 
the patient was then treated in a double - blind randomized controlled clinical trial with temozolomide 200 mg / m2 plus placebo or combined with either 20 or 40 mg abt - 888 ( veliparib ) , a parp inhibitor . 
the patient remained on treatment , continuing with temozolomide 150 mg / m2 once per day for days 1 to 5 of a 28 - day cycle and veliparib 40 mg twice per day on days 1 to 7 . 
his metastases did not progress , and he continues to live without treatment , with follow - up ct and mri scans revealing no progression ( fig 1 )  . given the exceptional response , the patient was followed through the indiana university health precision genomics biobank , and his archived brain tissue underwent whole - exome sequencing ( nantomics , culver city , ca )  . 
information at the gene level ( oncogene or tumor suppressor , driver of tumorigenesis , etc ) and specific to the mutation ( activating or disruptive , conservation score , location within recurrently mutated hotspot , etc ) is used . 
gene type is obtained using data from the catalog of somatic mutations in cancer cancer gene census.8 driver status is obtained from a pan - cancer publication across 15 and more the cancer genome atlas cancer types , and clusters of mutations are discovered using oncodriveclust.9 best - fit parameters for tumor ploidy are found by gradient descent . 
in the joint log likelihood calculation , a set of common allelic states are used to determine the expected relative coverage and majority allele fraction values for each state , given tumor ploidy . on the basis of this analysis , somatic tumor changes revealed a single copy number loss of brca2 , atm , chek1 , atr , fancm , and rad21 , which indicates dna repair deficiencies ( fig 2 )  . 
pathway analysis was performed using ingenuity pathway analysis.15 bard1 , brca1 - associated ring domain 1 ; fanc , fanconi anemia complementation group ; nhej1 , nonhomologous end - joining factor 1 ; plk1 , polo - like kinase 1 ; rb , retinoblastoma ; rx , treatment ; xrcc1 , x - ray repair cross - complementing group 1 . 
in addition , a germline recq1 i46r dna mutation was observed and is predicted to be possibly damaging through polyphen - 2.11 recql ( also known as recq1 ) helicases are a family of dna unwinding enzymes involved in the maintenance of chromosome stability and play a role in cellular response to oxidative dna damage.12 , 13 recent data have suggested that recql deficiency results in decreased homologous recombination and hyperactivation of parp.13 viziteu et al14 demonstrated that recql deficiency in myeloma cell lines displays sensitivity to parp inhibitors . 
there was an increase in the presence of other pathogenic alterations , which included braf g469r , cdh1 p429s , apc q1090 * , nf1 q803 * , and tp53 r248w . 
moving inward from the outside : intrachromosomal structural variants ( lines ) , overall copy number ( grey ) , majority and minority allele fractions ( light red and blue , respectively ) , single nucleotide variants and small indels ( colored dots ) , and interchromosomal structural variants . 
 ( b ) a total of 8 , 040 somatic variants were identified in this patient sample and categorized according to each variants annotation and supporting data as described in the case report . 
additional findings are presented in the circular genome and tumor mutation burden plots shown in figure 3 , and mutations are included in the data supplement . discussion our patient was treated in a randomized , controlled , double - blind , phase ii study of temozolomide combined with placebo or veliparib ( clinicaltrials.gov identifier : nct00804908 )  . 
 the randomization was 1 : 1 : 1 , and results were published by middleton et al.16 this report is the first to our knowledge of a patient treated successfully with chemotherapy and a parp inhibitor who had observed somatic losses in homologous recombination pathways with a recql germline mutation and who had an loh of 42.4%. 
 recently , the ariel2 trial was completed with the parp inhibitor rucaparib ; participants had relapsed , platinum - sensitive , high - grade ovarian carcinoma with brca mutations and an loh with a cutoff of 14%.18 the investigators observed a higher progression - free survival benefit in patients with high loh treated with parp inhibition . 
the investigators also suggested that patients with ovarian cancer who are brca wild type but with a high loh may derive benefit from parp inhibitor therapy as a future research hypothesis . in addition to the potential implications from high loh , evaluation of hrd or dna repair deficiency in melanoma registered with cbioportal reveals an overall incidence of dna repair mutations in 41% of samples , including 5% brca1 , 10% brca2 , 10% atm , 2.9% recql , and less than 10% for other fig 4 . 
although we cannot pinpoint the exact aberrations associated with sensitivity to therapy , we believe that the single - loss combinations in dna repair pathways and loh play the most important role to parp sensitivity . reported here is an extraordinary case of response to parp inhibition that may have been kismet on the basis of the plethora of aberrations in dna repair , germline recql , and loh of 42.4%. 
hypothesis - generating points from this report include the evaluation of the role of monoallelic and biallelic dna repair dysfunction , loh , or novel biomarkers or algorithms associated with hrd because this patient would not have been expected to derive such a continued benefit . 
logan consulting or advisory role : prometheus laboratories research funding : abbott laboratories ( inst ) , abraxis bioscience ( inst ) , acceleron pharma ( inst ) , amgen ( inst ) , argos therapeutics ( inst ) , astrazeneca ( inst ) , aveo pharmaceuticals ( inst ) , biovex ( inst ) , bristol - myers squibb ( inst ) , eisai ( inst ) , eli lilly ( inst ) , glaxosmithkline ( inst ) , roche ( inst ) , immatics biotechnologies ( inst ) , merck ( inst ) , novartis ( inst ) , pfizer ( inst ) , synta pharmaceuticals ( inst ) , threshold pharmaceuticals ( inst ) , millennium pharmaceuticals ( inst ) , tracon pharmaceuticals ( inst ) , cerulean pharma ( inst ) , emd serono ( inst ) , prometheus laboratories ( inst ) , macrogenics ( inst ) , peloton therapeutics ( inst ) , iovance biotherapeutics ( inst ) , medimmune ( inst ) , dynavax technologies ( inst ) affiliations all authors : indiana university school of medicine and iu simon cancer center , indianapolis , in . support supported by indiana university health with assistance from the indiana clinical and translational sciences institute and funded in part by national center for advancing translational sciences , clinical and translational sciences award ul1tr001108 . 
sloot s , chen ya , zhao x , et al : improved survival of patients with melanoma brain metastases in the era of targeted braf and immune checkpoint therapies . 
long gv , trefzer u , davies ma , et al : dabrafenib in patients with val600glu or val600lys braf - mutant melanoma metastatic to the brain ( break - mb ) : a multicentre , open - label , phase 2 trial . 
goldberg sb , gettinger sn , mahajan a , et al : pembrolizumab for patients with melanoma or non - small - cell lung cancer and untreated brain metastases : early analysis of a non - randomised , open - label , phase 2 trial . 
telli ml , hellyer j , audeh w , et al : homologous recombination deficiency ( hrd ) status predicts response to standard neoadjuvant chemotherapy in patients with triple - negative or brca1 / 2 mutation - associated breast cancer . 
hu h , huff cd , moore b , et al : vaast 2.0 : improved variant classification and disease - gene identification using a conservation - controlled amino acid substitution matrix . 
middleton mr , friedlander p , hamid o , et al : randomized phase ii study evaluating veliparib ( abt - 888 ) with temozolomide in patients with metastatic melanoma . 
middleton mr , grob jj , aaronson n , et al : randomized phase iii study of temozolomide versus dacarbazine in the treatment of patients with advanced metastatic malignant melanoma . 
swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
 molecular and clinical characterization of a claudin - low subtype of gastric cancer purpose claudin - low molecular subtypes have been identified in breast and bladder cancers and are characterized by low expression of claudins , enrichment for epithelial - to - mesenchymal transition ( emt ) , and tumor - initiating cell ( tic ) features . 
we evaluated whether the claudin - low subtype also exists in gastric cancer . materials and methods four hundred fifteen tumors from the cancer genome atlas ( tcga ) gastric cancer mrna data set were clustered on the claudin , emt , and tic gene sets to identify claudin - low tumors . 
compared with gs subtype , claudin - low subtype had significant activation in rho family of gtpases signaling , which appears to play a key role in its emt and tic properties . 
in the acrg data set , 28 of 300 samples were classified as claudinlow tumors by the 24 - gene predictor and were phenotypically similar to the initially derived claudinlow tumors . 
2017 by american society of clinical oncology introduction claudin - low tumors have been identified as a molecular subtype originally in breast cancer and more recently in bladder cancer on the basis of gene expression profiling.1 , 2 these distinct tumors are characterized by the low expression of tight - junction claudins , enrichment for epithelial - to - mesenchymal transition ( emt ) markers , and tumor - initiating cell ( tic ) features.1 , 2 clinically , claudin - low tumors , which lack luminal differentiation marker expression , are associated with poor prognosis compared with luminal tumors.1 , 3 similar to breast , bladder , and other types of cancer , gastric cancer is a heterogeneous disease and arises from multiple genetic and epigenetic aberrations . 
to better understand the biology that drives gastric cancer , molecular evaluation of gastric cancer has been performed and has led to the identification of key dysregulated pathways and the development of molecular classifications.4 gene expressionbased classifiers were reported by the asian cancer research group ( acrg ) 5 and by lei et al.6 the acrg divided gastric cancer tumors into four subtypes ( microsatellite unstable [ msi ] , emt , and tumor protein 53 active and inactive ) , and lei et al classified gastric cancer tumors into three subtypes ( proliferative , metabolic , and mesenchymal )  . on the basis of an integrative analysis of molecular profiling data , the cancer genome atlas ( tcga ) proposed the following four molecular subtypes of gastric cancer tumors : epstein - barr virus ( ebv ) positive , msi , chromosomally unstable ( cin ) , and genomically stable ( gs ) .7 this classification system first categorizes tumors by ascopubs.org / journal / po jco precision oncology tomohiro f . 
vincent author affiliations appear at the end of this article . supported by the university cancer research fund of the lineberger comprehensive cancer center at the university of north carolina at chapel hill and the university of north carolina at chapel hill oncology clinical translational research training program ( 5k12ca120780 )  . presented at the asco 2017 gastrointestinal cancers symposium , san francisco , ca , january 1921 , 2017 . corresponding author : benjamin g . 
however , gs subtype was found to have some notable features , including enrichment of lauren diffuse histologic type and alterations in the genes associated with cell adhesion and motility , such as cdh1 and rhoa mutations and cldn18 - arhgap fusions . 
missing values were imputed using the k - nearest neighbor imputation method , and gene expression values were median centered across each gene . results gene expression signatures tight - junction claudin , emt , and tic gene signatures were used in the classification of a claudin - low subtype . 
the set of claudins used was identified by herschkowitz et al.9 the bidirectional pan - cancer emt signature was derived by tan et al.10 the gastric cancerspecific tic signature was derived from the publicly available gene expression omnibus gene expression data set ( gse53276 )  . 
proliferation and immune gene signatures were derived by hippo et al11 and iglesia et al , 12 respectively . identification of a claudin - low subtype data were clustered on the claudin , emt , and tic gene sets using average linkage clustering with a centered correlation similarity metric on the cluster 3.0 platform ( stanford university , stanford , ca )  . 
sigclust2 r software ( statistical significance of clustering 2 ) was run on the node to perform a gaussian distribution analysis , which expands to the entire gene set for each increasing node . 
the data supplement contains additional details on methods . identification of a claudin - low subtype in gastric cancer from adjacent clusters of we performed unsupervised hierarchical clustering on 415 gastric cancer samples from tcga data set using gene signatures representative of biologic characteristics known to define the claudin - low subtype of breast and bladder cancers.1 , 2 this unsupervised hierarchical clustering with these gene signatures revealed a distinct cluster that had characteristics of claudin - low tumors ( fig 1a , highlighted in red )  . 
to ensure that the set of tumors within the presumed claudin - low cluster were homogeneous and distinct tumors , we performed a gaussian distribution analysis ( data supplement )  . 
this method identified a conserved node of 46 tumors ( 11.1% ) that had consensus enrichment for claudin - low features , and these tumors , therefore , were defined as claudin - low subtype . 
classification of the nonclaudin - low tumors ( n = 369 ) was as follows : ebv , 31 tumors ; msi , 79 tumors ; cin , 218 tumors ; and gs , 41 tumors . 
because the claudin - low tumors were identified originally in breast cancer , we applied the previously defined breast cancerspecific claudin - low classifier to tcga gastric cancer samples and found a significant enrichment ( p , .001 , fishers exact test ) of the breast cancerdefined claudin - low tumors within the gastric claudin - low cluster ( data supplement )  . 
this finding further supports the notion that claudin - low tumors exhibit features of previously defined claudin - low tumors . gene expression signature analysis relative to tic and stem - cell features , breast cancer claudin - low tumors express low levels of proliferation genes and are likely slowercycling tumors.1 , 3 we examined proliferation gene expression of gastric cancer by subtype using a previously reported gastric cancerspecific proliferation signature.11 expression of the proliferation - associated genes in the claudinlow subtype was lowest among all the subtypes ( figs 2a and 2b )  . 
despite the apparent similarity to gs subtype , the claudin - low subtype expressed significantly lower levels of proliferation genes than gs subtype ( p , .001 , bonferronicorrected pairwise t test )  . 
 another notable feature of the claudin - low subtype of breast and bladder cancers is high expression of immune cell genes , including t and b cells.1 , 2 to characterize the immune response in gastric claudin - low tumors , we evaluated gene signatures associated with immune cells by molecular subtype using previously defined signatures that correspond to tumor - infiltrating immune cells.12 heat maps of immune signature expression across the 415 samples in order by subtype showed generally high expression in ebv , claudin - low , and gs ( fig 3a )  . 
on the basis of the z scores of the gene signature , expression of all immune signatures other than the b - cell signature was highest in the ebv subtype ( data supplement )  . to assess the level of active immunosuppression , we examined the expression of a previously defined panel of immune checkpoint molecules ( immunosuppression signature ) 2 and found that it was also highest in the ebv subtype ( fig 3b ; data supplement )  . 
this observation is in line with the previous report by strong et al.13 wherein ebvpositive gastric carcinoma samples expressed high levels of the cytotoxic t - cell and natural killer cell inhibitor indoleamine - 2 , 3 - dioxygenase 1 , which leads to the induction of immune tolerance to tumors despite the elevated immune cell infiltration . 
similar to the previous analysis of bladder cancer , a clear correlation existed between the immune signatures and the immunosuppression signature across all gastric cancer subtypes2 ( data supplement )  . the prognostic impact of tumor - immune infiltrates , especially tumor - infiltrating lymphocytes ( tils ) , has been reported in multiple tumor types.14 , 15 for instance , the presence of cd8 + tils was associated with favorable survival outcomes.16 - 18 some studies evaluated the prognostic relevance of tils in gastric cancer , but the results were not consistent across them.19 - 21 we performed cox proportional hazards regression modeling for each immune gene signature , including immunosuppression signature , across all tumors and within each subtype . 
none of the signatures were prognostic in tcga gastric cancer samples ( data not shown )  . we explored potential mechanisms of immune response by examining predicted neoantigen burden and levels of cytokines and chemokines among subtypes . 
 ( a ) heat map of supervised clustering of gastric cancer subtypes across previously identified proliferationassociated genes ( n = 415 )  . ( b ) box plot of proliferation gene signature z scores across gastric cancer subtypes ( n = 415 )  . significance was determined by one - way analysis of variance ( anova )  . 
 ( b ) box plot of immune suppression gene signature z scores across gastric cancer subtypes ( n = 415 )  . significance was determined by one - way analysis of variance ( anova )  . 
 ( c ) and ( d ) box plots show the number of predicted neoantigens with a half - maximal inhibitory concentration , 150 nm by tumor molecular subtype . significance was determined by kruskalwallis exact test ( n = 357 )  . ( e ) box plot of cytokine and chemokine signature z scores across gastric cancer subtypes ( n = 415 )  . significance was determined by one - way anova . 
cin , chromosomally unstable ; ebv , epstein - barr virus ; gs , genomically stable ; igg , immunoglobulin g ; mac th1 , macrophageassociated t helper 1 ; msi , microsatellite unstable . difference in immune infiltration among subtypes because the predicted neoantigen burdens of ebv , claudin - low , and gs subtypes were lower than that of cin subtype and relatively similar to one another ( fig 3d )  . we next examined the relative expression of a previously defined panel of cytokines and chemokines by subtype2 and observed a strong correlation between this expression signature and multiple immune signatures , including immunosuppression signature ( data supplement )  . 
overall , claudin - low tumors appeared to have a high level of immune infiltration but lacked active immunosuppression within the tumor microenvironment . pathway analysis given that claudin - low tumors were primarily found in gs subtype , we performed ingenuity pathway analysis ( ipa ) and gene set enrichment analysis to understand the gene expression patterns that differentiate claudin - low tumors from nonclaudin - low gs tumors . 
ipa revealed that claudin - low subtype had significant activation in rho family of gtpases signaling and significant inactivation in rho guanine nucleotide dissociation inhibitor ( gdi ) signaling compared with gs subtype ( fig 4 )  . 
rhoa ( a member of the rho family of gtpases ) signaling has been reported to promote cancer stem - celllike phenotype in diffuse - type gastric cancer.22 in addition , claudinlow subtype had higher levels of actin cytoskeleton and integrin signaling relative to signaling levels in gs subtype . 
these observations are in keeping with the tic and emt phenotypes , which are defining characteristics of claudin - low tumors . mutation and copy number alteration analysis we next examined somatic mutations and copy number alterations using tcga data set to compare genomic events of claudin - low subtype with those of nonclaudin - low gs subtype ( data supplement )  . 
tcga network reported rhoa and cdh1 mutations , and cldn18 - arhgap fusions were enriched in gs subtype.7 we did not observe significant differences in the frequency of these genomic events between the two subtypes ( data supplement )  . 
nonclaudin - low tumors ( n = 272 ) were classified into the following subtypes : ebv , 17 tumors ; msi , 63 tumors ; cin , 162 tumors ; and gs , 30 tumors . 
we found that claudin - low subtype in the acrg data set was phenotypically similar to the initially derived claudin - low subtype in the discovery tcga data set as measured by expression of the claudin , emt , and tic gene signatures ( data supplement )  . 
bar graph of the most significantly activated ( gold ) or inactivated ( blue ) signaling pathways in claudin - low subtype ( n = 46 ) compared with gs subtype ( n = 41 )  . significance was determined using ipa software . 
to further compare prognosis for claudin - low subtype with gs subtype , with adjustment for potential confounders ( age and pathologic stage ) , we performed cox proportional hazards regression modeling by setting gs type as a reference group ( table 1 )  . 
in addition , we assessed a prognostic value of claudin - low subtype in patients with stage iii cancer because it was the largest subgroup with the most equal distribution of subtypes . 
similar hrs from the two independent data sets suggest that claudin - low subtype of gastric cancer has a poor prognosis as a result of its unique biologic features . tcga overall survival by subtype acrg overall survival by subtype claudin fig 5 . 
prognosis of claudin - low tumors in ( a ) the cancer genome atlas ( tcga ) and ( b ) the asian cancer research group ( acrg ) data sets . unadjusted kaplan - meier plots show overall survival of gastric cancer by molecular subtype . significance was determined by log - rank test ( n = 415 in tcga cohort ; n = 300 in the acrg cohort )  . 
thus , we especially focused on comparing molecular and clinical features of claudin - low tumors with those of nonclaudin - low gs tumors . claudin - low gastric cancer was defined by low expression levels of tight - junction claudins , high levels of emt , and enrichment for tic signatures . 
in addition , compared with gs subtype , claudin - low subtype had the lower expression of proliferation signature , significant activation in rho family of gtpases signaling , and enrichment of cell motility and adhesion - associated pathways . furthermore , the claudin - low subtype conferred significantly worse prognosis than the gs subtype , which had comparable survival to the cin and ebv subtypes . 
these observations support the claim that claudin - low subtype is distinct from gs subtype and warrants additional validation in prospective studies . we report a 24 - gene classifier that accurately classifies gastric cancer into claudin - low and non claudin - low subtypes . 
we applied this classifier to the acrg data set and identified 28 claudin - low tumors that were phenotypically similar to the initially derived claudin - low subtype in the discovery data set . 
adjusted for age and pathologic stage ( n = 415 in tcga cohort ; n = 300 in the acrg cohort )  . abbreviations : acrg , asian cancer research group ; cin , chromosomally unstable ; ebv , epsteinbarr virus ; gs , genomically stable ; msi , microsatellite unstable ; tcga , the cancer genome atlas . are also characterized by low expression of claudins and enrichment for tic signatures . 
the combined expression of emt and cancer stem - cell makers was reported to be associated with aggressive clinical features , such as vascular invasion and metastasis , and prognostic of poorer disease - free and overall survival in a study of 276 patients with gastric cancer.25 moreover , emt and / or stem - celllike biologic processes have been linked to resistance to chemotherapy and radiation.26 - 28 the mechanisms of therapeutic resistance include relative quiescence , expression of atp - binding cassette transporters , an active dna - repair capacity , and a resistance to apoptosis.29 recent work by yoon et al22 demonstrated a role of rhoa , a member of the rho family of gtpases , in gastric cancer stem - like cells ( cscs ) for the maintenance of emt phenotypes and chemotherapy resistance . 
furthermore , rhoa pathway inhibition reversed chemoresistance in diffuse gastric cscs resistant to fluorouracil and cisplatof note , our ipa revealed that , compared with gs subtype , the most significantly activated and inactivated pathway in claudin - low subtype were rho family of gtpases signaling and rhogdi signaling , respectively . 
gdis are regulators of rho gtpases and prevent activation of gtpases by acting as molecular chaperones.30 all things considered , dysregulated rho gtpase signaling appears to play a key role in the biology of gastric claudinlow tumors and contributes to the poor prognosis . 
sanoff research funding : bayer ag ( inst ) , novartis ( inst ) , merck ( inst ) , precision biologics ( inst ) , immunomedics ( inst ) references 1 . 
sabatier r , finetti p , guille a , et al : claudin - low breast cancers : clinical , pathological , molecular and prognostic jci insight 1 : e85902 , 2016 characterization . 
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ryu hs , park dj , kim hh , et al : combination of epithelial - mesenchymal transition and cancer stem cell - like phenotypes has independent prognostic value in gastric cancer . 
takaishi s , okumura t , tu s , et al : identification of gastric cancer stem cells using the cell surface marker cd44 . stem cells 27 : 1006 - 1020 , 2009 27 . 
miller , md3 ; and razelle kurzrock , md2 , 5 purpose high - grade neuroendocrine cervical cancer ( hgnecc ) is an uncommon malignancy with limited therapeutic options ; treatment is patterned after the histologically similar small - cell lung cancer ( sclc )  . 
these results were subsequently compared with a cohort of 1 , 800 sclcs . results the median age of patients with hgnecc was 40.5 years ; 83 patients ( 85.6% ) harbored high - risk human papillomavirus ( hpv )  . 
overall , 294 genomic alterations ( gas ) were identied ( median , 2 gas / sample ; average , 3.0 gas / sample , range , 0 - 25 gas / sample ) in 109 distinct genes . 
seventy - one patients ( 73% ) had 1 alteration that was theoretically druggable . comparing hgnecc with sclc , signicant differences in tmb , microsatellite instability , hpv - positive status , and in pik3ca , myc , pten , tp53 , arid1a , and rb1 alteration rates were found . conclusion this large cohort of patients with hgnecc demonstrated a genomic landscape distinct from sclc , calling into question the biologic and therapeutic relevance of the histologic similarities between the entities . furthermore , 73% of hgnecc tumors had potentially actionable alterations , suggesting novel treatment strategies for this aggressive malignancy . jco precis oncol 4 : 972 - 987 . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction the treatment of solid malignancies has evolved and is perhaps best exemplied by the approach to non small - cell lung cancer , for which molecular characterization and use of targeted agents have emerged as standard therapeutic paradigms . 
recently , the cancer genome atlas ( tcga ) completed and published the integrated genomic and molecular characterization of cervical cancer.1 in addition to data previously released for both ovarian ( high - grade serous ) and endometrial ( endometrioid and serous ) cancers , this publication completed the molecular and genomic evaluation of the most common gynecologic malignancies.2 , 3 traditionally , cervical cancer clinical trials have excluded less common histologies such as high - grade neuroendocrine cervical carcinoma ( hgnecc )  . 
despite the low incidence of hgnecc ( , 2% of all cervical cancers ) the oncologic impact is signicant because these tumors exhibit more aggressive clinical characteristics.4 , 5 unfortunately , the 5 - year overall survival rate for patients with early - stage disease is only a 36% , and those with metastatic spread face an even more dismal prognosis . 
given these poor outcomes , patients with hgnecc represent an area of unmet clinical need . developing therapeutic options for patients with rare tumors is challenging , relying on international collaboration , as well as small case series or retrospective reports rather than prospective clinical trials . 
 genomic landscape of high - grade neuroendocrine cervix cancer context key objective to dene the molecular landscape of high - grade neuroendocrine cervical cancer in a large cohort of patients . knowledge generated high - grade neuroendocrine cervical cancer appears molecularly distinct from the histologically similar small - cell lung cancer . up to 73% of patients samples harbored potentially actionable alterations , informing novel treatment strategies . relevance continued understanding of the molecular underpinnings of high - grade neuroendocrine cervical carcinoma will be critical to driving drug discovery for this disease . approved by the food and drug administration ( fda ) specically for hgnecc.8 recently , 2 reports of exceptional responses to immune checkpoint inhibition in patients with recurrent hgnecc were published.9 , 10 to better understand these responses and to identify molecular aberrations underlying this uncommon malignancy , we examined the genomic landscape of hgnecc . 
the submitting physicians provided specication of a poorly differentiated , neuroendocrine tumor type of cervical origin , which was then independently reviewed by a gynecologic pathologist ( j.e. ) to conrm high - grade neuroendocrine pathologic features in the pathology report and / or the representative sample of tumor submitted for sequencing ( grade 3 cytomorphologic features , some component of small - cell or large - cell carcinoma histology , and / or positivity for neuroendocrine markers )  . 
ngs data were generated by foundationone ( foundation medicine ; cambridge , ma )  . the study was performed in accordance with university of california , san diego , institutional review board guidelines for a de - identied database . 
the test sequences the entire coding region of 182 or , more recently , 236 or 315 cancer - related genes plus up to 47 introns of up to 19 genes often rearranged or altered in cancer to an average depth of coverage of  . 
500.11 the pathologic diagnosis of each case was conrmed on routine hematoxylinand eosin - stained slides and all samples forwarded for dna extraction contained a minimum of 20% tumor nuclear area . 
microsatellite instability ( msi ) status was evaluable in 75 hgnecc and 1 , 573 sclc cases . the sequencing methods used for comprehensive genomic proling have been validated and reported previously ( appendix ) .12 , 13 the optimized loci used to evaluate msi status were selected from a total set of 1 , 897 that have adequate coverage on all versions of the assay . 
there was no need to extend beyond the rst principal component , because it explained approximately 50% of the total data variance , whereas none of the other principal components explained  . 
msi - low calls are not made because there was no gold - standard test set , but we presume such samples would signicantly overlap with the msiambiguous category reported here . 
the cutoff of 20 coding mutations / mb is approximately equal to 400 nonsynonymous mutations per exome.15 human papillomavirus detection in addition , the presence of high - risk human papillomavirus ( hpv ) was examined in submitted specimens , as previously reported.16 hybrid - capture reagents included baits designed to capture unique regions of select viral genomes including hpv - 16 and - 18 . 
a total hpv - 16 / 18 aligned read count of 5 reads per million was considered a positive hpv status , and , 5 reads per million was considered hpv not detected.16 end points and statistical methods descriptive statistics were used to summarize the baseline patient characteristics . 
all statistical tests were carried out using graphpad prism , version 6.0 ( graphpad software , san diego , ca )  . results characterization of gas in hgnecc the median age of the cohort was 40.5 years ( range , 25 - 77 years )  . 
the most frequently reported number of gas per sample was 2 , with a range of 0 - 25 ( average , 3.0 gas / sample ; fig 2 )  . 
5% of samples are depicted . variants of unknown signicance excluded . genomics in hpv - positive versus - negative patients when examining distribution of gas on the basis of hpv detection status , a signicant difference was identied in the frequency of several gas , including pik3ca , tp53 , pten , arid1a , and rb1 , all of which were more frequent in the hpv - negative subgroup ( table 1 )  . 
two of the 3 cases were both msi - h and tmb - h and harbored the highest numbers of identiable gas in the cohort.17 , 18 the third case , with a nonsense mutation near the 3 ( cid : 3 ) end of the coding sequence ( msh2 r929 * ) , was mss and tmb - l . the 2 msh - 2mutant msi - h cases were both of small - cell histology and accounted for all msi - h cases out of the 75 no . 
furthermore , a total of 40 patient samples ( 41.2% ) harbored mutations in the pi3k / akt / mtor pathway ; mutations in pik3ca were identied in 47.5% of these samples ( n = 19 ) , and pten mutations were reported in 35% ( n = 14 )  . 
the small - cell subset of hgnecc samples showed analogous gene mutation differences from sclc . discussion neuroendocrine carcinoma is an uncommon but aggressive variant accounting for approximately 1.5% of all newly diagnosed cervical cancers.20 the great majority of these lesions are high - grade largeor small - cell subtypes , with only rare reports of well - differentiated cervical carcinoid tumors.20 the treatment of patients with hgnecc remains table 2 . 
 eskander et al clinically challenging , with limited response rates to chemotherapy ; however , anecdotal reports of exceptional responders have been described.9 , 10 the paradigm for management of hgnecc has been informed by the treatment of the more commonly diagnosed ( and histologically similar ) sclc , which accounts for approximately 15% of all lung cancer cases . 
in prior studies , whole - genome sequencing of 110 sclc specimens identied essentially ubiquitous tp53 and rb1 inactivating mutations , with biallelic losses of each gene respectively in 100% and 93% of cases without chromothripsis.21 in an effort to better dene the molecular landscape of hgnecc , we evaluated the comprehensive genomic proling of 97 patient samples . 
at least 1 characterized alteration was identied in 88 patient samples ( 90.7% ) and of these , 72 had a potentially pharmacologically tractable alteration . interestingly , the frequency and distribution of gas identied in this cohort of patients are similar and distinct from mutational patterns described in the more common hpvrelated cervical cancer histologies.1 as detailed in tcgas integrated genomic characterization of cervical cancer ( ie , squamous , adenocarcinoma , and adenosquamous histologies ) , mutations in the pik3ca gene were the most frequently identied aberration , occurring in 26% of samples , approximating the nearly 20% rate in our cohort . in addition , signicantly mutated genes reported by the tcga , identied in similar proportions in this patient cohort , included arid1a ( 7% in tcga and 9.3% in our cohort ) and kras ( 6% in tcga and 8.2% in our cohort )  . 
these molecular differences may be reective of the varying histologies or , potentially , the differential highrisk hpv detection rates ( 85.6% in our cohort v 95% in the tcga ) .1 importantly , the high - risk hpv rate in our cohort should be interpreted with caution because the assay used has not undergone formal concordance study with gold standard tests such as hybrid capture and can detect only hpv 16 / 18 . our own , much larger cohort of sclc samples ( n = 1 , 800 ) recapitulates prior studies and had a strikingly different molecular portfolio when compared with hgnecc samples . 
msi - h status was also more common in the hgnecc cohort whereas tmb - i / tmb - h was more common in sclc ( despite the lack of msi - h status )  . 
the use of everolimus , or an alternate mtor or pik3ca inhibitor , may be considered in such circumstances , although the utility of a pik3ca mutation in predicting response to single - agent everolimus in the presence of multiple gas remains limited.26 , 27 although less frequently identied , alterations in the hrd pathway were detected in 9.3% of patient samples , potentially supporting use of a poly - adp ribose polymerase inhibitor . 
the identication of both tmb - h ( n = 2 ) and gas in mismatch repair genes ( n = 3 ) may also inform the use of immune checkpoint inhibition.28 in may 2017 , the fda approved pembrolizumab for the treatment of mismatch repairdecient or msi - h solid tumors that progressed after prior therapy . 
more recently , the fda accepted and granted priority review to a supplemental biologics license application for pembrolizumab for the treatment of adult and pediatric patients with unresectable or metastatic solid tumors with tissue tmb - h whose disease has progressed after prior treatment and who have no satisfactory alternative treatment options , supported by data from the phase ii keynote - 158 trial . 
notably , there are 2 published case reports of patients with recurrent , treatment - refractory hgnecc with exceptional and durable responses to checkpoint inhibition ; 1 of these tumors was from our current hgnecc cohort and had a mismatch repair defect and the other lacked correlative genomic testing.9 , 10 last , the identication of arid1a ( 9.3% ) and smarca4 ( 4.1% ) mutations may predict sensitivity to an alternate therapeutic strategy.29 homeostasis requires balanced arid1a and ezh2 activity , facilitated via chromatinmediated gene expression . 
 genomic landscape of high - grade neuroendocrine cervix cancer aberrations were identied in patients with msi - h lesions , possibly reecting that the smarca4 may be a passenger mutation resulting from the underlying msi . 
furthermore , of the 4 cases with smarca4 alterations , 1 was hpv - 18 positive and another was p16 positive by immunohistochemical assessment . despite the large sample size and robust genomic data , this study has limitations . 
95% of the identied high - risk hpv strains.30 this report highlights the potential therapeutic utility of genomic testing in patients with this uncommon disease.27 interest , despite the histologic similarity between hgnecc and sclc , which has led to the latter being used as a model for treating the former , the molecular portfolio of these 2 entities is strikingly different . 
therefore , plausible that patients with hgnecc may benet from alternative therapeutic strategies . it is not anticipated that traditional prospective trials will accrue sufcient patient numbers in this disease setting , and novel study designs , including umbrella , basket , and platform trials , should be considered given the presence of actionable targets . 
interestingly , the rst reported cohort of the dart trial ( clinicaltrials.gov identier : nct02834013 ) 31 was the neuroendocrine cohort , with a 44% overall response rate in those with high - grade disease . 
eskander consulting or advisory role : pzer , clovis oncology , astrazeneca / medimmune , tesaro , merck speakers bureau : clovis oncology , astrazeneca / medimmune , roche travel , accommodations , expenses : clovis oncology , astrazeneca / medimmune , roche , merck , pzer julia elvin employment : foundation medicine stock and other ownership interests : hoffman - laroche laurie gay employment : foundation medicine , invitae stock and other ownership interests : foundation medicine , naveris , invitae consulting or advisory role : invitae , naveris jeffrey s . 
ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine consulting or advisory role : celsius therapeutics research funding : foundation medicine vincent a . 
 eskander et al leadership : foundation medicine , revolution medicines stock and other ownership interests : foundation medicine , mirati therapeutics , revolution medicines patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center razelle kurzrock leadership : curematch , curemetrix stock and other ownership interests : curematch , idbydna , soluventis honoraria : roche , eusa pharma , neogenomics laboratories , biocom , neomed therapeutics , advanced therapeutics , lek , aacr , chugai pharma usa , wiley consulting or advisory role : actuate therapeutics , loxo , xbiotech , neomed , roche , gaido , soluventis , pzer , merck speakers bureau : roche , guardant health ( inst ) research funding : sequenom ( inst ) , merck serono ( inst ) , genentech ( inst ) , pzer ( inst ) , foundation medicine ( inst ) , incyte ( inst ) , konica minolta ( inst ) , grifols ( inst ) , omniseq ( inst ) , debiopharm group ( inst ) , boerhinger ingelheim ( inst ) travel , accommodations , expenses : roche , eusa pharma , neogenomics laboratories , biocom , neomed therapeutics , advanced therapeutics , lek , aacr , chugai pharma usa , wiley no other potential conicts of interest were reported . references cancer genome atlas research network : integrated genomic and molecular characterization of cervical cancer . 
nature 497 : 67 - 73 , 2013 [ erratum : nature 500 : 242 , 2013 ] cancer genome atlas research network : integrated genomic analyses of ovarian carcinoma . 
nature 474 : 609 - 615 , 2011 [ erratum : nature 490 : 298 , 2012 ] gardner gj , reidy - lagunes d , gehrig pa : neuroendocrine tumors of the gynecologic tract : a society of gynecologic oncology ( sgo ) clinical document . gynecol oncol 122 : 190 - 198 , 2011 satoh t , takei y , et al : gynecologic cancer intergroup ( gcig ) consensus review for small cell carcinoma of the cervix . 
int j gynecol cancer 24 : s102 - s108 , 2014 ( 9 suppl 3 ) fukuoka m , masuda n , furuse k , et al : a randomized trial in inoperable non - small - cell lung cancer : vindesine and cisplatin versus mitomycin , vindesine , and cisplatin versus etoposide and cisplatin alternating with vindesine and mitomycj clin oncol 9 : 606 - 613 , 1991 kubota k , hida t , ishikura s , et al : etoposide and cisplatin versus irinotecan and cisplatin in patients with limited - stage small - cell lung cancer treated with etoposide and cisplatin plus concurrent accelerated hyperfractionated thoracic radiotherapy ( jcog0202 ) : a randomised phase 3 study . 
lancet oncol 15 : 106 - 113 , 2014 ishikawa m , kasamatsu t , tsuda h , et al : prognostic factors and optimal therapy for stages i - ii neuroendocrine carcinomas of the uterine cervix : a multi - center retrospective study . 
gynecol oncol 148 : 139 - 146 , 2018 paraghamian se , longoria tc , eskander rn : metastatic small cell neuroendocrine carcinoma of the cervix treated with the pd - 1 inhibitor , nivolumab : a case report . 
sharabi a , kim ss , kato s , et al : exceptional response to nivolumab and stereotactic body radiation therapy ( sbrt ) in neuroendocrine cervical carcinoma with high tumor mutational burden : management considerations from the center for personalized cancer therapy at uc san diego moores cancer center . oncologist 22 : 631 - 637 , 2017 11 . 
ross js , fakih m , ali sm , et al : targeting her2 in colorectal cancer : the landscape of amplication and short variant mutations in erbb2 and erbb3 . 
genome med 9 : 34 , 2017 johnson db , frampton gm , rioth mj , et al : targeted next generation sequencing identies markers of response to pd - 1 blockade . 
chae yk , pan ap , davis aa , et al : path toward precision oncology : review of targeted therapy studies and tools to aid in dening actionability of a molecular lesion and patient management support . 
kato s , kurasaki k , ikeda s , et al : rare tumor clinic : the university of california san diego moores cancer center experience with a precision therapy 25 . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors on the basis of molecularp : results from mypathway , an openlabel , phase iia multiple basket study . 
j clin oncol 36 : 536 - 542 , 2018 janku f , hong ds , fu s , et al : assessing pik3ca and pten in early - phase trials with pi3k / akt / mtor inhibitors . 
alejo m , alemany l , clavero o , et al : contribution of human papillomavirus in neuroendocrine tumors from a series of 10 , 575 invasive cervical cancer cases . pract 4 : 17 , 2017 papillomavirus res 5 : 134 - 142 , 2018 31 . 
patel sp , othus m , chae yk , et al : a phase ii basket trial of dual anti - ctla - 4 and anti - pd - 1 blockade in rare tumors ( dart ) s1609 : the neuroendocrine cohort . presented at the american association for cancer research annual meeting , atlanta , ga , march 29 - april 3 , 2019 ( abstr ct039 )  . 
briey , after pathologic review to conrm sufcient tumor nuclei ( minimum , 20% ) and mitigate pathologic inconsistencies , at least 50 ng of dna was extracted from 40 tumor samples provided as formalin - xed , parafnmicrons of embedded tissue blocks . 
the samples were assayed using adaptorligation and hybrid - capture next - generation sequencing ( foundationone ) for all coding exons from 182 ( version 1 ) , 287 ( version 2 ) , or 315 ( version 3 ) cancer - related genes plus selected introns from 14 ( version 1 ) , 19 ( version 2 ) , or 28 ( version 3 ) genes frequently rearranged in cancer . sequencing of captured libraries was performed using hisequation 2500 / 4000 ( illumina , san diego , ca ) to a mean exon coverage depth of  . 
500 , and resultant sequences were analyzed using both an algorithmic pipeline and manual curation for base substitutions , small insertions or deletions ( indels ) , copy number alterations ( focal amplications and homozygous deletions ) , and selected gene fusions , as previously described.13 clinically relevant genomic alterations were dened as alterations targetable by anticancer drugs currently available on the market or in registered clinical trials . 
germline variants documented in the dbsnp database ( dbsnp142 ; nlm.nih.gov / snp / ) , with 2 counts in the exac database ( broadinstitute.org / ) , or recurrent variants of unknown signicance that were predicted by an internally developed algorithm to be germline were removed , with the exception of known driver germline events.12 known , conrmed somatic alterations deposited in the catalog of somatic mutations in cancer ( version 62 ) were highlighted as biologically signicant , as were inactivating events in tumor suppressor genes . 
all four patients received selpercatinib ( capsule or liquid formulation ) orally in continuous 28 - day cycles at a starting dose of 90 mg / m2 twice per day . this dose was intended to deliver exposure equivalent to the recommended adult phase ii dose of 160 mg twice per day . 
one patient was enrolled to the 80 - mg cohort of trial ( clinicaltrials.gov identier : nct03157128 ) and underwent intrapatient dose escalation to 160 mg per protocol . the ongoing selpercatinib phase i / ii pharmacokinetic analysis serial blood samples were collected for pharmacokinetic analyses . 
molecular analysis showed that the tumor harbored an ret exon 6 deletion ( d378_ g685.e ) , and the patient was treated sequentially with four multitargeted kinase inhibitors : vandetanib , sunitinib , cabozantinib , and lenvatinib , which were each discontinued because of either adverse events or lack of efcacy . 
a 7 - year - old boy with neonatal hypotonia , abdominal pain , hollow feet , and unexplained laryngeal spasms was referred for diagnostic exome sequencing of genomic dna ( sureselect xt clinical research exome , agilent , santa clara , ca )  . 
this revealed a constitutional de novo ret m918t mutation , a pathogenic variant associated with men2b that confers the highest risk of early onset mtc.16 the patient was subsequently diagnosed with an mtc and underwent thyroidectomy with tracheostomy followed by vandetanib therapy , which was discontinued because of the onset of grade 3 colitis . selpercatinib was subsequently initiated at 90 mg twice per day . 
magnetic resonance imaging demonstrated new lung nodules indicative of metastatic disease . an rna - based next - generation sequencing ( ngs ) fusion assay ( solid fusion assay v2 ; archerdx , boulder , co ) indicated that the tumor harbored an myh10 - ret gene fusion . 
the patient was started on vandetanib and achieved a partial tumor response by response evaluation criteria in solid tumors ( recist ) 1.1.22 however , after 4 months of treatment , she developed disease progression at the primary site . 
repeat imaging after 6 months of treatment showed complete resolution of the paraspinal mass and a 50% decrease in the size of the retroperitoneal , pelvic , and lumbosacral mass ( fig 2a - d )  . 
during repair of congenital bilateral inguinal hernias , a 2 - month - old girl was found to have a right renal mass , and she underwent a right nephrectomy . 
patient 1 : magnetic resonance imaging scans of ( a ) right lateral and ( b ) left anterior liver metastatic lesions at baseline and after 22 months of treatment with selpercatinib of ( c ) right lateral and ( d ) left anterior in a heavily pretreated patient with ret - mutated medullary thyroid cancer . 
patient 3 : ( a , c ) computed tomography ( ct ) scans of the abdomen at baseline and ( b , d ) after 6 months of treatment with selpercatinib , revealing multiple paraspinal retroperitoneal and pelvic lesions in a patient with infantile myobroma / hemangiopericytoma harboring an myh10 - ret fusion . 
a partial response was observed after one cycle of selpercatinib ; after six cycles , the paraspinal lesion had completely resolved , and the patient regained lower extremity neurologic function . 
patient 4 : ct scans at baseline of ( e ) the lungs and ( g ) brain and after 8 months of treatment with selpercatinib of ( f ) the lungs and ( h ) brain in a patient with an specc1l - ret fusionpositive congenital mesoblastic nephroma and infantile brosarcoma . 
patient 5 : ct scans ( i , k ) at baseline and ( j , l ) after 2 months of treatment with selpercatinib of the left foot in a patient with an ncoa4 - ret fusionpositive lipobromatosis . 
abdominal ultrasound and chest x - ray identied a mass in the left kidney and a large left lower lung mass ; computed tomography imaging conrmed these lesions and identied additional , multiple small lung lesions . 
biopsy of a lung mass revealed high - grade spindle cell sarcoma consistent with an infantile brosarcoma ; pathology review of the original right nephrectomy specimen conrmed the initial diagnosis of mesoblastic nephroma . 
the patient received additional chemotherapy with cyclophosphamide and topotecan , but treatment was complicated by febrile neutropenia and enterococcus faecalis ventriculitis . dna - based ngs ( using memorial sloan kettering integrated mutation proling of actionable cancer targets [ msk - impact ] ) of the tumor from the renal and lung masses identied the same specc1l - ret gene fusion , and the patient initiated treatment with selpercatinib . after two cycles , a partial response was observed with a 41% tumor reduction , ( fig 2e - h )  . 
a selpercatinib concentration of 4.5 ng / ml was achieved in cerebrospinal uid at a dose level of 48 mg twice per day ( 90 mg / m2 per dose )  . upon detection and resection of an isolated metastasis at the right posterior temporal occipital junction , the dose of selpercatinib was increased to 94 mg ( 180 mg / m2 ) twice per day , raising the concentration of the drug to 16 ng / ml in the cerebrospinal uid . 
 selpercatinib in pediatric ret - altered cancer libretto - 001 ( 160 mg , day 8 ) patient 3 patient 4 patient 5 6 , 000 4 , 000 2 , 000 time ( hours ) fig 3 . 
plasma samples from three patients revealed that adequate plasma concentrations of selpercatinib were achieved ( greater than ret wild - type concentration that inhibits 90% [ ic90 ] ) , which were within the range seen in adult patients treated with the recommended dose of 160 mg in the libretto - 001 trial . 
the gray area represents the 95% cis for the median plasma concentrations observed in patients treated in the libretto - 001 trial ( red circles )  . fusion , which was conrmed by whole genome and transcriptome analysis . 
selpercatinib was initiated , and imaging after 2 months revealed a partial response by recist 1.1 , with a 59% reduction in tumor volume and resolution of tumor inltration of the metatarsals ( fig 2i - l )  . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . sandya govinda raju employment : loxo oncology dahlia henry employment : loxo oncology stock and other ownership interests : loxo oncology , allergan , axovant sciences , palatin technologies steve smith consulting or advisory role : various , loxo oncology patents , royalties , other intellectual property : various patents and applications travel , accommodations , expenses : various s . 
cox employment : bayer , loxo oncology , merck kgaa , amgen , day one biopharmaceuticals stock and other ownership interests : loxo oncology , bayer , merck kgaa , amgen , day one biopharmaceuticals patents , royalties , other intellectual property : us patent 62 / 318 , 041 issued to loxo oncology ( inst ) julia glade bender consulting or advisory role : abbvie ( inst ) research funding : celgene ( inst ) , merck ( inst ) , pzer ( inst ) , amgen ( inst ) , ignyta ( inst ) , bristol - myers squibb ( inst ) , eisai ( inst ) , novartis ( inst ) , eli lilly ( inst ) , loxo oncology ( inst ) , genentech ( inst ) travel , accommodations , expenses : novartis , amgen , merck , bayer , genentech uncompensated relationships : springworks therapeutics , bristol - myers squibb a . 
lindsay frazier stock and other ownership interests : decibel therapeutics consulting or advisory role : decibel therapeutics peter anderson stock and other ownership interests : healios consulting or advisory role : enlivity patents , royalties , other intellectual property : patent for glutamine and trehalose compositions ; priority date 13 september 2013 ; app 14 / 470 , 545 , led august 27 , 2014 ; patent 61 / 878 , 084 issued december 26 , 2017 other relationship : enlivity alberto s . 
pappo honoraria : bayer , roche consulting or advisory role : merck , loxo oncology / bayer , eusa pharma no other potential conicts of interest were reported . acknowledgments we thank the patients and their families and contributing clinical staff across all sites . 
medical writing services were provided by jim heighway of cancer communications and consultancy , knutsford , united kingdom , and were funded by loxo oncology , stamford , ct . references kato s , subbiah v , marchlik e , et al : ret aberrations in diverse cancers : next - generation sequencing of 4 , 871 patients . 
clin cancer res 23 : 1988 - 1997 , 2017 ceccherini i , pasini b , pacini f , et al : somatic in frame deletions not involving juxtamembranous cysteine residues strongly activate the ret proto - oncogene . oncogene 14 : 2609 - 2612 , 1997 prescott jd , zeiger ma : the ret oncogene in papillary thyroid carcinoma . 
nat commun 9 : 4821 , 2018 ferrara r , auger n , auclin e , et al : clinical and translational implications of ret rearrangements in non - small cell lung cancer . 
j thorac oncol 13 : 27 - 45 , 2018 cordioli mi , moraes l , bastos au , et al : fusion oncogenes are the main genetic events found in sporadic papillary thyroid carcinomas from children . 
thyroid 27 : 182 - 188 , 2017 fenton cl , lukes y , nicholson d , et al : the ret / ptc mutations are common in sporadic papillary thyroid carcinoma of children and young adults . 
j clin endocrinol metab 85 : 1170 - 1175 , 2000 prasad ml , vyas m , horne mj , et al : ntrk fusion oncogenes in pediatric papillary thyroid carcinoma in northeast united states . 
cancer 122 : 1097 - 1107 , 2016 vanden borre p , schrock ab , anderson pm , et al : pediatric , adolescent , and young adult thyroid carcinoma harbors frequent and diverse targetable genomic alterations , including kinase fusions . 
carvalho d , mackay a , bjerke l , et al : the prognostic role of intragenic copy number breakpoints and identication of novel fusion genes in paediatric high 11 . 
al - ibraheemi a , folpe al , perez - atayde ar , et al : aberrant receptor tyrosine kinase signaling in lipobromatosis : a clinicopathological and molecular genetic grade glioma . 
antonescu cr , suurmeijer aj , zhang l , et al : molecular characterization of inammatory myobroblastic tumors with frequent alk and ros1 gene fusions and rare novel ret rearrangement . 
elisei r , cosci b , romei c , et al : prognostic signicance of somatic ret oncogene mutations in sporadic medullary thyroid cancer : a 10 - year follow - up study . j clin endocrinol metab 93 : 682 - 687 , 2008 16 . 
latteyer s , klein - hitpass l , khandanpour c , et al : a 6 - base pair in frame germline deletion in exon 7 of ret leads to increased ret phosphorylation , erk activation , and men2a . 
wirth lj , kohno t , udagawa h , et al : emergence and targeting of acquired and hereditary resistance to multikinase ret inhibition in patients with ret - altered 20 . 
drilon ae , subbiah v , oxnard gr , et al : a phase 1 study of loxo - 292 , a potent and highly selective ret inhibitor , in patients with ret - altered cancers . 
antonescu cr , dickson bc , swanson d , et al : spindle cell tumors with ret gene fusions exhibit a morphologic spectrum akin to tumors with ntrk gene 26 . 
wells sa jr , robinson bg , gagel rf , et al : vandetanib in patients with locally advanced or metastatic medullary thyroid cancer : a randomized , double - blind 28 . 
schlumberger m , jarzab b , cabanillas me , et al : a phase ii trial of the multitargeted tyrosine kinase inhibitor lenvatinib ( e7080 ) in advanced medullary thyroid 30 . 
gautschi o , milia j , filleron t , et al : targeting ret in patients with ret - rearranged lung cancers : results from the global , multicenter ret registry . 
yoh k , seto t , satouchi m , et al : vandetanib in patients with previously treated ret - rearranged advanced non - small - cell lung cancer ( luret ) : an open - label , multicentre phase 2 trial . 
kasi , mbbs , md , ms1 ; judong yang , md , ms2 ; phani keerthi surapaneni , mbbs1 ; tanios bekaii - saab , md3 ; daniel h . 
borad , md3 purpose recent advances in molecular diagnostic technologies have allowed for the evaluation of solid tumor malignancies via noninvasive blood sampling , including circulating tumor dna ( ctdna ) proling . 
after excluding variants of unknown signicance , therapeutically relevant alterations were observed in 76 patients ( 55% ) , including braf mutations , erbb2 amplications , fgfr2 fusions , fgfr2 mutations , and idh1 mutations seen in 21% of patients . 
a different spectrum of alterations was observed in patients with early - onset btc ( younger than age 50 years ) compared with older patients ( older than age 50 years )  . conclusion data on ctdna in btc is currently limited . 
intact circulating tumor cells and cell - free dna ( cfdna ) from both leukocytes and tumors ( circulating tumor dna [ ctdna ] ) can now be isolated and analyzed using advanced sequencing methods . 
ctdna has been shown to carry tumor - specic genetic or epigenetic alterations , such as point mutations , copy number variations , chromosomal rearrangements , and dna methylation patterns.1 - 4 the testing approaches in this eld are two dominant polymerase chain reaction ( pcr ) based ( digital pcr ) and next - generation sequencing ( ngs )  . 
ngs approaches have the ability to investigate a larger these number of genes simultaneously ; however , techniques have historically been limited by the need for high sensitivity and specicity and the cost associated with sequencing . 
this approach allows for agnostic analysis of large portions of the genome and can identify multiple mutations with increased sensitivity.5 evaluation of ctdna is particularly attractive as it enables the assessment of patient - specic tumoral genetic / epigenetic alterations while allowing for serial monitoring of tumor genomes in a noninvasive , convenient , and accurate manner . 
 mody et al biliary tract cancers ( btcs ) are an uncommon malignancy arising from epithelial cells that line the biliary tree and have a rapidly fatal course , with 5 - year survival rates of less than 10%.1 , 2 although relatively rare in western countries , btcs are more prevalent in southeast asia.3 in the united states , btcs account for approximately 2% of all new cancer diagnoses and have been rising in incidence during the past few decades.4 - 10 patients with cholangiocarcinoma stand to gain immensely from the use of ctdna given that diagnosis currently is more often made at advanced stages of disease ; recurrences are common , despite the pursuit of potentially curable treatments , such as surgery in a subset of patients ; biopsies are not always obtained or often yield suboptimal quantities of tumor cells for tissue - based genomic proling ; and multiple genomic alterations , which are potential therapeutic targets , are known to occur in btcs . 
genetic alterations that have been identied include activating kras mutations , tp53 mutations / deletions , idh1 and idh2 mutations , fgfr2 gene fusions , cdkn2a / b mutations / deletions , and , less commonly , ntrk gene fusions and alterations in erbb2 ( her2 ) , met , braf , nras , and pik3ca.6 , 7 therefore , the purpose of the current study was to examine the results of ctdna testing from a large cohort of patients with btc at a national cancer institute comprehensive cancer center , acquired in the course of clinical practice , and to characterize the mutational landscape of btcs using this technology . 
data analysis from this patient cohort was reviewed and approved by the mayo clinic institutional review board . demographic information and the date of blood collection were available for all patients . 
additional patient information was collected from the mayo clinic electronic medical record and included conrmation of btc diagnosis ; risk factors , including history of hepatitis a , b , or c , nonviral chronic liver disease , diabetes , obesity , or primary sclerosing cholangitis ; and pertinent clinical information , such as treatment status at the time of ctdna testing and history of liver - directed therapy . comprehensive genomic testing in plasma cfdna was extracted from whole blood collected in 10 - ml streck tubes . 
samples were shipped to a clinical laboratory improvement actcertied , college of american pathologistsaccredited laboratory ( guardant health )  . after double ultracentrifugation , 5 to 30 ng cfdna was isolated for digital sequencing . cfdna fragments , both leukocyte and tumor derived , were simultaneously sequenced . 
analytical sensitivity allowed for the detection of one to two mutant fragments in a 10 - ml blood sample ( 0.1% limit of detection ) with analytic specicity of more than 99.9999%. twelve copy number alterations were reported as the absolute gene copy number in plasma . 
as most cfdna is leukocyte derived , the gene copy number is generally 2.0. tumor - derived dna shed into the bloodstream raises this value , but , because of the relative proportions of tumorderived versus leukocyte - derived cfdna , is typically a minor contributor . 
in the 68 - gene panel , eight genes were retired from the single - nucleotide variation gene set , whereas coverage of gene amplications expanded from three to 16 genes , and the addition of detection of fusions in four genes and insertions or deletion of bases ( indels ) in one gene was made . 
the 70 - gene panel included all national comprehensive cancer network somatic genomic targets , cluding complete or critical exon coverage in 30 and 40 genes , respectively , amplications in 14 genes , fusions in six genes , and indels in three genes . 
the majority of samples in this study ( n = 122 ) were tested using the 73 - gene panel . to determine if an alteration was therapeutically relevant , we dened therapeutically relevant as any gene alteration with an oncokb level of evidence of 1 , 2a , 2b , 3a , 3b , or r1.8 oncokb is a precision oncology knowledge base that contains information about the effects and treatment implications of specic cancer gene alterations.8 oncokb contains detailed information about specic alterations in 477 cancer genes compiled from various sources , cluding guidelines from the us food and drug administration ( fda ) , national comprehensive cancer network , asco , clinicaltrials.gov , and the scientic literature . 
level 4 includes those variants that are considered predictive of response to targeted agents on the basis of compelling biologic , nonclinical evidence . by these standards , a gene is considered therapeutically relevant if there is supporting evidence that the gene is a driver of tumorigenesis , and wherein actionability of the gene can refer to either sensitivity and / or resistance to a drug ( s )  . 
in addition , there must be a clinically available agent , including clinical trials , that targets the gene / protein for a specic agent , and there must be at least preclinical evidence that supports its role in targeting the specic gene / protein . on the basis of the aforementioned criteria , the following genes were considered actionable : akt1 , alk , araf , atm , braf , brca1 , brca1 , ccnd1 , ccnd2 , cdk4 , cdk6 , cdkn2a , ctnnb1 , egfr , erbb2 , esr1 , fgfr1 , fgfr2 , gnas , idh1 , idh2 , jak2 , kras , met , map2k1 , mlh1 , mtor , myc , ntrk1 , ntrk3 , pdgfra , pik3ca , raf1 , ptpn11 , stk11 , tsc1 , and vhl . 
finally , variants found in actionable genes that did not alter the amino acid sequencethat is , synonymous mutationsand / or those that lacked any supporting functional evidence of pathogenicity were excluded . statistical analysis the distribution of each continuous variable is summarized by its mean , standard deviation , and range . 
the relationship between gene types with regard to mutation and amplication , as well as synonymous and targetable status , was evaluated using spearmans rank correlation and was displayed in the correlation matrix in which nonsignicant correlations are marked with a blank in the graph . 
a small subset of patients had underlying chronic liver disease , such as primary sclerosing cholangitis ( 8% ) or other chronic liver disease ( 6% )  . landscape of alterations among a total of 138 ctdna samples , 105 ( 76% ) of these included one or more alterations when variants of unknown signicance were excluded . 
tp53 and kras were the two most common alterations in all subtypes of disease , followed by fgfr2 in 7% ( intrahepatic subtype ) , arid1a ( extrahepatic subtype ) and cdk6 , apc , and smad4 ( gallbladder subtype )  . 
in the more prevalent of the subtypes ( intrahepatic ) in our sampleaside from fgfr2pik3ca and idh1 were fairly commonly altered . a different spectrum of alterations was observed in patients with early - onset btc ( younger than age 50 years ) compared with older patients ( older than age 50 years ) , although overall the differences were not statistically signicant ( p = .55 ; fig 3 )  . 
other alterations which were more common in younger patients included pik3ca , met , and braf . therapeutically relevant alterations after excluding variants of unknown signicance , therapeutically relevant alterations were seen in 55% ( n = 76 ) of the total cohort of samples . 
several risk factors for btc have been described , with most etiologies resulting in long - standing inammation.9 , 12 the only standard systemic therapy for the disease is gemcitabine and cisplatthere is no standard second - line therapy that has been shown to unequivocally improve survival for patients who experience disease progression on gemcitabine / cisplatin.13 btcs are traditionally classied by location as intrahepatic , extrahepatic , and gallbladder on the basis of their presumed site of origin within the biliary duct systethis 4 2019 by american society of clinical oncology mody et al 65 ( 28 - 87 ) 85 ( 69 ) 22 ( 18 ) 16 ( 13 ) 60 ( 48 ) 63 ( 52 ) 42 ( 35 ) 38 ( 30 ) 43 ( 35 ) 21 ( 17 ) 14 ( 12 ) 10 ( 8 ) 2 ( 1 ) 6 ( 5 ) 15 ( 11 ) 30 ( 22 ) 26 ( 19 ) 19 ( 14 ) 48 ( 35 ) anatomic classication has been effective in differentiating btcs with regard to epidemiology , etiology , clinical presentation , and treatment.9 early diagnosis is ideal given that surgical resection or liver transplantationfor perihilar tumorsoffers the best chance at cure ; however , majority of patients are diagnosed with advanced - stage disease precluding surgical management . 
of particular importance , pathologic tissue - based conrmation of btc can be challenging given that clinical yield is often suboptimal and insufcient , especially for dna extraction to enable genomic proling . 
this is primarily a result of the more desmoplastic nature of many btc tumors . with the availability of next - generation genomic proling in recent years , a number of genomic subtypes of btc have been described , many of which are dened by therapeutically targetable genomic alterations.7 , 14 - 18 for example , fgfr fusion rearrangements and idh1 and idh2 mutations have emerged as signicant driver mutation genomic subtypes . 
at present , several targeted drugs are in clinical development , with encouraging results thus far . for the above reasons , the potential for use of ctdna in the management of btc is of particular interest . 
investigations of the use of ctdna in cca have previously been hampered by the rarity of the disease , the relatively incomplete understanding of the genetics of this cancer , and the lack of a validated and widely used ctdna assay . 
this has now changed with recent increased focus on the disease ; recent characterization of the btc genome by several studies , including that from the cancer genome atlas ; and also with the availability of a number of commercially available ctdna assays in clinical practice.14 , 15 , 19 thus far , data on ctdna in cholangiocarcinoma have been sparse . 
andersen and jakobsen20 have demonstrated the early feasibility of a ctdna assay in btc , using a multiplex digital pcr assay to screen for 31 mutations in kras , nras , braf , and pik3ca . 
the authors then performed the assay on serum from ve patients with btc with known tumor mutations and six patients who were known to be wild type for the assayed mutations . 
the assay correctly identied the ve known mutations , whereas none of the six wild - type samples had mutations identied in cfdna . goyal and colleagues21 demonstrated the usefulness of ctdna in serial monitoring of disease and for the detection of resistance mechanisms in the setting of targeted therapy . among 32 patients enrolled in a phase ii study of the fgfr inhibitor bgj398 , nine patients ( 28% ) had fgfr2 fusions detected and four patients were enrolled in the trial . 
pivotal trials exploring inhibitors of fgfr and idh in the management of btc specically are ongoing ( clinicaltrials.gov identiers : nct02924376 , nct03230318 , nct02052778 , and nct02989857 )  . other data have highlighted the application of erbb2 and braf targeted therapies in the disease.22 - 27 for these reasons , it is critically important to identify therapeutically relevant alterations in patients with cholangiocarcinoma . we demonstrate , for the rst time to our knowledge , the ability to detect a broad array of therapeutically relevant alterations using ctdna testing at a signicant rate ( 55% ) in patients with btc . 
whereas the oncokb system is an extensive , comprehensive , and curated precision oncology knowledge base that is capable of supporting precision oncology treatment decisions , should be noted that universally accepted denitions of actionable alterations are not currently available.8 more practically , a total of 21% of patients in our cohort were cohort ; fig 3 . 
 circulating tumor dna in biliary tract cancer identied as having actionable alterations in braf , erbb2 , fgfr2 , and idh1 , for which there are dedicated clinical trials available for patients with btc or for which currently available data exist on the treatment of patients with btc with existing targeted therapies . limitations , our study does have other including the moderate sample size , its retrospective nature , and the fact that patients are derived from a single institution . 
we also have limited clinical data as a result of the retrospective nature of the study . ctdna in btc has been an underexplored area , but our study for the rst time to our knowledge demonstrates the true feasibility of ctdna testing in this disease . 
we believe there is a rationale for ctdna to be incorporated in cholangiocarcinoma trials going forward so as to further explore the technology as a means for identifying patients with actionable genomic alterations , as a biomarker of response , and for serial monitoring of disease and detection of resistance mechanisms , as well as for prognostic purposes . 
borad manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors policy , please refer to or ascopubs.org / po / authorcenter . kabir mody consulting or advisory role : celgene , genentech , merrimack pharmaceuticals , eisai , astrazeneca , vicus therapeutics research funding : fibrogen , senhwa biosciences , ariad pharmaceuticals , tracon pharma , medimmune , agios , arqule , taiho pharmaceutical pashtoon m . 
kasi consulting or advisory role : taiho pharmaceutical ( inst ) , ipsen ( inst ) , bristol - myers squibb ( inst ) , advanced accelerator applications ( inst ) , array biopharma ( inst ) , celgene ( inst ) tanios bekaii - saab consulting or advisory role : amgen ( inst ) , glenmark , ipsen ( inst ) , eli lilly ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) , bayer ( inst ) , celgene ( inst ) , genentech ( inst ) , abbvie , incyte ( inst ) , imugene , immuneering other relationship : exelixis , merck , sillajen , armo biosciences daniel h . 
ahn consulting or advisory role : celltrion ( i ) , merrimack pharmaceuticals , astellas pharma , eisai , cardinal health , paradigm , lexicon amit mahipal research funding : taiho pharmaceutical jason s . 
nat med 20 : 548 - 554 , 2014 sia d , hoshida y , villanueva a , et al : integrative molecular analysis of intrahepatic cholangiocarcinoma reveals 2 classes that have different outcomes . gastroenterology 144 : 829 - 840 , 2013 valle jw , lamarca a , goyal l , et al : new horizons for precision medicine in biliary tract cancers . 
shaib yh , davila ja , mcglynn k , et al : rising incidence of intrahepatic cholangiocarcinoma in the united states : a true increase ? j hepatol 40 : 472 - 477 , 2004 11 . 
chan - on w , kuwahara k , kobayashi n , et al : cholangiocarcinomas associated with long - term inammation express the activation - induced cytidine deaminase and germinal center - associated nuclear protein involved in immunoglobulin v - region diversication . 
j gastrointest oncol 7 : 797 - 803 , 2016 javle m , bekaii - saab t , jain a , et al : biliary cancer : utility of next - generation sequencing for clinical management . 
goyal l , saha sk , liu ly , et al : polyclonal secondary fgfr2 mutations drive acquired resistance to fgfr inhibition in patients with fgfr2 fusion - positive cholangiocarcinoma . 
j clin oncol 33 , 2017 ( suppl ; abstr e15137 ) javle m , churi c , kang hc , et al : her2 / neu - directed therapy for biliary tract cancer . 
kocsis j , aroksz all asi a , andr as c , et al : combined dabrafenib and trametinib treatment in a case of chemotherapy - refractory extrahepatic braf v600e mutant cholangiocarcinoma : dramatic clinical and radiological response with a confusing synchronic new liver lesion . 
loaiza - bonilla a , clayton e , furth e , et al : dramatic response to dabrafenib and trametinib combination in a braf v600e - mutated cholangiocarcinoma : implementation of a molecular tumour board and next - generation sequencing for personalized medicine . 
golan t , raitses - gurevich m , kelley rk , et al : overall survival and clinical characteristics of brca - associated cholangiocarcinoma : a multicenter retrospective a brief review . 
 circulating tumor dna predicts the response and prognosis in patients with early breast cancer receiving neoadjuvant chemotherapy shunying li , phd1 , 2 ; hongna lai , phd1 , 2 ; jieqiong liu , md1 , 2 ; yujie liu , phd1 , 2 ; liang jin , md1 , 2 ; yudong li , phd1 , 2 ; fengtao liu , md1 , 2 ; yuhua gong , ms3 ; yanfang guan , phd3 ; xin yi , phd3 ; qianfeng shi , md1 , 2 ; zijie cai , md1 , 2 ; qian li , md1 , 2 ; ying li , md1 ; mengdi zhu , md1 , 2 ; jingru wang , md1 , 2 ; yaping yang , md1 , 2 ; wei wei , md4 ; dong yin , phd1 , 2 ; erwei song , md , phd1 , 2 ; and qiang liu , md , phd1 , 2 purpose many patients with breast cancer still relapse after curative treatment . 
serial plasma samples before , during , and after nac and paired tumor biopsies were harvested and subjected to deep targeted sequencing using a large next - generation sequencing panel that covers 1 , 021 cancerrelated genes . results positive baseline ctdna was detected in 21 of 44 patients before nac . 
more importantly , positive baseline ctdna is signicantly associated with worse disease - free survival ( p = .011 ) and overall survival ( p = .004 ) in patients with early breast cancer , especially in estrogen receptornegative patients . conclusion our study demonstrated that ctdna can be used to predict tumor response to nac and prognosis in early breast cancer , providing information to tailor an individuals therapeutic regimen . jco precis oncol 4 : 244 - 257 . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women worldwide.1 although early breast cancer is a curable disease , up to 40% of such patients still relapse after surgery.2 it is believed that breast cancer is a systemic disease and that clinically undetectable micrometastases often happened before the diagnosis.3 , 4 how to distinguish patients with early breast cancer with high relapse risk from those with low risk remains a critical and challenging clinical question . neoadjuvant chemotherapy ( nac ) is often used in patients with locally advanced or triple - negative / her2positive early breast cancer before surgery to decrease tumor size and enable breast - conserving surgery . 
the current consensus to monitor response to nac is clinical examination backed up by radiologic and sonographic measures.5 however , these measures are quite subjective and have poor interand intraobserver reproducibility.6 moreover , given the heterogeneity of cancer , a reliable method to examine the overall response from local , regional , and micrometastatic lesions in patients with early breast cancer is still lacking . circulating tumor dnas ( ctdnas ) are mutated gene fragments that are exclusively shed by cancer cells into blood , 7 which can be detected by digital polymerase chain reaction ( dpcr ) 8 or sequencing . 
 ctdna predicts prognosis in breast cancer context key objective can the presence and dynamic change of circulating tumor dna ( ctdna ) predict the tumor response and prognosis in patients with breast cancer receiving neoadjuvant chemotherapy ( nac ) ? knowledge generated using a next - generation sequencing panel of 1 , 021 genes , positive baseline ctdna was identied in 21 of 44 patients with early breast cancer before nac . 
positive ctdna before nac was associated with signicantly worse disease - free survival and overall survival , especially in estrogen receptor ( er ) negative patients . relevance our study demonstrates that ctdna has signicant value to complement imaging to monitor tumor response and predict prognosis in patients with early breast cancer , providing information to tailor an individuals therapeutic regimen . 
the high relapse rate in er - negative patients with positive ctdna indicates that it may be necessary to escalate treatment in these high - risk patients . patients with early breast cancer after surgery , ctdna by dpcr predicted metastasis at 7.9 - 11 months earlier than clinical relapse.11 , 12 a few studies also explored the role of ctdna in patients with breast cancer who received nac . 
it was reported that ctdna change during nac was correlated with tumor response13 - 15 or relapse , 16 , 17 although two of them only used dpcr to detect a single gene ( metrassf1a14 or tp5316 ) , and the other study only correlated the ctdna disappearance after the rst cycle of nac with two patients who achieved pathologic complete response.13 advances in next - generation sequencing ( ngs ) 18 technology have enabled the rapid identication and broad coverage of tumor - specic genomic alterations in cell - free dna ( cfdna ) of individual patients.19 - 21 the ngs assay provides an opportunity to screen more genes at a single time point and avoid missing mutations absent in biopsy because of intratumor heterogenicity . 
for patient cohort , sample collection and processing , and ngs sequencing and data analysis , please see the data supplement . statistical analysis the primary end point of this study was to evaluate the clinical value of the presence and dynamic change of ctdna to predict the tumor response and prognosis in patients with breast cancer treated with neoadjuvant chemotherapy . 
all statistical analyses were performed with graphpad prism version 6.0 or r version 3.4.1. results clinicopathological features of patients a total of 52 patients with early breast cancer who received nac were prospectively enrolled in the study since 2013 . eight patients were excluded from further study because of insufcient cfdna in plasma before nac ( fig 1 )  . the clinicopathological features of the 44 patients in the study are listed in table 1 and the data supplement . 
after surgery , the patients were followed up every 6 months . during the follow - up ( median , 46 months ; range , 11 - 68 months ) , 11 patients ( 25% ) had distant metastasis , and 9 of them died of metastatic breast cancer . baseline mutated dnas in plasma and tumor all baseline plasma samples , matched blood cells , and 32 paired tumor biopsy samples underwent parallel targeted ngs to screen for point mutations and structural variants , using the ngs panel of 1 , 021 cancer - related genes that covers a region of 1.1 mb ( data supplement ) .24 after deduplication , the median depths of unique coverage were 984 for primary tumor and 1 , 225 for plasma dna . 
the analysts were blinded to clinical and follow - up information during the analysis of sequencing data . among the 44 patients , 21 patients were found to have positive baseline ctdna . 
a total of 72 mutations in 46 genes were found in these 21 samples , with 1 - 8 ( median , 3 ) mutations per sample ( data supplement )  . 
tp53 ( 20 / 32 ) , pi3kca ( 13 / 32 ) , nf1 ( 5 / 32 ) , and gata3 ( 4 / 32 ) were the most frequently mutated genes in tumor ( fig 2c )  . in the 32 patients with both tumor and plasma sequencing data , 14 had concordant / partially concordant mutations in plasma and tumor , 16 patients had mutations only in tumor with negative ctdna , and the other two patients ( p034 and p036 ) had completely different mutations in tumor and plasma ( fig 2b )  . 
in the 16 patients with positive ctdna , 14 ( 87.5% ) of them had one or more mutations conrmed independently in tumor sequencing data , indicating that most positive ctdna represents tumor - specic mutations in the primary tumors . 
among the 33 concordant mutations found in both tumor and plasma of the 14 patients , tp53 ( 12 / 14 ) and pik3ca ( 4 / 14 ) mutations were the most frequent ( fig 2d )  . changes of ctdna and tumor response during nac the current gold standard to monitor tumor response during nac is ultrasound or magnetic resonance imaging , although it is subjective and sometimes falls behind the histologic changes . 
to explore whether ctdna surpasses imaging in monitoring or predicting overall tumor response , all plasma samples collected during nac in the 20 patients ( 1 patient was excluded because of insufcient cfdna in subsequent plasma samples ) with positive baseline ctdna were sequenced and compared with baseline data to track the dynamic changes of ctdna during nac ( data supplement )  . in this study , tumor response was dened as cr / pr / sd / pd using recist1.1 criteria when the imaging data of local / regional tumor before surgery was compared with that before nac . 
of the 20 assessable patients , 11 did not respond to nac ( including a patient with pd ) , and 9 responded to nac ( response rate , 45% )  . however , patient 038 ( p038 ) , who was dened as pr according to imaging , had an increasing ctdna amount ( clonal variant allele frequency [ vaf ] ) during all cycles of nac , 25 and a persisting high ctdna even after surgery , although she did not have any clinical metastasis before surgery . 
follow - up showed that she had multiple distant metastases ( liver , lung , and bone ) at 21 months and died at 46 months after surgery ( appendix fig a2 )  . 
the increase of ctdna in this case indicated the early micrometastases undetected by imaging did not respond to nac well , although the primary tumor did , suggesting that ctdna is better than imaging of local tumor to monitor the overall response to nac . next we did a longitudinal analysis of ctdna changes during nac and correlated it with local tumor response . p038 was excluded from this analysis because of discrepant tumor and ctdna data . 
the ctdna amount ( clonal vaf ) in baseline plasma was set as 100% , and those in subsequent plasma from the same patient were normalized it was found that patients who to the baseline value . responded to nac had more decreasing ctdna than patients who did not respond to nac ( fig 3a )  . 
the difference was signicant after the second cycle ( p = .03 ) and all cycles of nac ( p = .02 ) but was not statistically signicant after the rst cycle of nac ( p = .290 ; figs 3b - 3d )  . next , we constructed roc22 curves to compare the efcacy of ctdna amount ( clonal vaf ) and ultrasound to predict the response to nac ( fig 4 )  . 
it was found that clonal vaf after 2 cycles , clonal vaf before surgery , and ultrasound after 2 cycles were all predictive of the response to nac before surgery . 
the optimal criteria of clonal vaf in predicting the response to nac , as determined by the roc plots , are 40% drop in clonal vaf after 2 cycles and 60% drop in clonal vaf before surgery , respectively . 
the optimal criterion of ultrasound after 2 cycles was 20% decrease in the longest diameter of tumor in our study . concordant mutations between tumor and plasma were also identied in two patients with smaller tumor and negative lymph nodes ( t2n0m0 )  . 
the er - negative patients with negative baseline ctdna had 100% dfs and os , whereas those with positive baseline ctdna had a dfs / os of only approximately 36% . among the 20 patients with positive baseline ctdna ( one patient ruled out due to lack of subsequent samples ) , 6 of them turned negative and 14 remained positive for the ctdna before surgery . 
six out of 8 ( 75% ) er - negative patients with persistent positive ctdna relapsed , whereas only 1 out of 3 ( 33% ) such patients with negative ctdna before surgery relapsed , although the difference was not statistically signicant because of small sample size ( appendix figure a4 )  . discussion in this study , we used targeted deep sequencing of cfdna with a panel of 1 , 021 cancer - related genes to screen for mutated ctdna in patients with early breast cancer and found that 21 out of 44 patients had positive ctdna . 
more importantly , positive ctdna before nac is signicantly associated with worse prognosis in patients with early breast cancer , with the signicance mainly derived from er - negative patients . many patients with early breast cancer still relapse after surgery because of clinically undetectable micrometastases . 
current imaging methods are not sensitive enough to detect micrometastatic lesions ; even the most sensitive positron emission tomographycomputed tomography has the resolution limit at 4 mm.26 recently , ctdna has been suggested to be an ideal tumor biomarker because of its specicity , stability , and sensitivity . 
it is shown to be better than traditional biomarkers and imaging in predicting the relapse or progression in both early and metastatic cancer.9 , 11 , 12 , 27 - 29 however , the best method to measure ctdna is uncertadpcr is cheaper and easier , but it only measures one or few known mutations . 
to our knowledge , this study used the largest ngs panel so far to look for ctdna in patients with early breast cancer . using the large 1 , 021 - gene panel , somatic mutations were found in all the tumor biopsies . 
more importantly , positive ctdna was found in 47% of patients with early breast cancer and 73% of er - negative patients , which was consistent with previous reports.12 , 16 , 17 , 30 , 31 the majority of the ctdna was conrmed in tumor mutation data , suggesting that ctdna can be used to detect tumor - specic fig 2 . 
 ( c , d ) clonal vaf in no - response patients was signicantly higher than that in response patient after ( c ) 2 , and ( d ) all cycles of nac . 
in the 21 patients with positive baseline ctdna , 16 patients had paired plasma and tumor biopsy samples for ngs analysis , and 14 / 16 had at least one mutation conrmed in primary tumors . 
with the high specicity , at 98.72% , the discrepancy between the mutations in plasma and the mutations in tumor may be explained by the small tumor biopsy specimen not representing the whole primary tumor or micrometastasis . 
but this study also identied 24% ( 5 / 21 ) of patients with positive ctdna who had mutations other than tp53 and pi3kca . interestingly , our data showed that dynamic change of ctdna can be used to monitor and predict the response to nac in early breast cancer . 
kaplan - meier curve compares the disease - free survival ( dfs ) and overall survival ( os ) in patients with breast cancer with positive or negative baseline circulating tumor dna ( ctdna )  . 
nbc , negative baseline ctdna ; pbc , positive baseline ctdna . the change of ctdna level after 2 cycles of nac predicted local tumor response to nac better than ultrasound , although this was not statistically signicant . 
interestingly , none of the four er - negative patients with negative baseline ctdna relapsed during the follow - up , which was strikingly different from the high relapse rate in er - negative patients with positive baseline ctdna . 
other limitations of this study include limited sample size and suboptimal nac cycles , which hinder further subgroup analysis . in conclusion , our study demonstrates the feasibility and validity of ctdna in patients with early breast cancer receiving nac . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . yuhua gong employment : geneplus - beijing yanfang guan employment : geneplus - beijing xin yi employment : geneplus - beijing qiang liu consulting or advisory role : novartis , astrazeneca speakers bureau : pzer , roche , novartis , astrazeneca , eisai no other potential conicts of interest were reported . references bray f , ferlay j , soerjomataram i , et al : global cancer statistics 2018 : globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries . 
ca cancer j clin 68 : 394 - 424 , 2018 early breast cancer trialists collaborative group ( ebctcg ) , davies c , godwin j , et al : relevance of breast cancer hormone receptors and other factors to the efcacy of adjuvant tamoxifen : patient - level meta - analysis of randomised trials . 
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cancer res 72 : 5145 - 5149 , 2012 ; discussion 5150 schwarzenbach h , hoon ds , pantel k : cell - free nucleic acids as biomarkers in cancer patients . 
nat rev cancer 11 : 426 - 437 , 2011 bernard - tessier a , jeannot e , guenat d , et al : clinical validity of hpv circulating tumor dna in advanced anal carcinoma : an ancillary study to the epitopeshpv02 trial . 
clin cancer res25 : 2109 - 2115 , 2019 dawson sj , tsui dw , murtaza m , et al : analysis of circulating tumor dna to monitor metastatic breast cancer . 
schiavon g , hrebien s , garcia - murillas i , et al : analysis of esr1 mutation in circulating tumor dna demonstrates evolution during therapy for metastatic breast 11 . 
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robin x , turck n , hainard a , et al : proc : an open - source package for r and s + to analyze and compare roc curves . 
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 li et al et * 4 2 weeks after surgery no liver metastasis after surgery liver metastasis after 1 cycle b aselin e after 2 cycles b efore surg ery after 3 cycles after surg ery cycles 24 months fig a2 . 
et * 4 , 4 cycles of epirubicin and docetaxel . p031 ( t2n0m0 ) p041 ( t2n0m0 ) tuba3c tp53 raf1 pik3ca notch2 tsc2 tp53 gab2 dnmt3a brd3 fig a3 . 
although targeted therapies , including vandetanib and cabozantinib , are used to treat mtc , resistance ( both intrinsic and acquired ) to these therapeutic approaches warrants a need to better understand the broad - spectrum anomalies that govern this phenomenon , especially in patients with aggressive disease . 
using whole - exome sequencing ( wes ) and shallow whole - genome sequencing ( swgs ) of the primary tumor , adjacent normal bone marrow tissues , and blood , we identified three germline single nucleotide polymorphisms ( snps ) within the ret proto - oncogene that remained undetected using routine hospital genetic testing procedures . 
supported by findings from both wes and swgs , we report on the occurrence of a chromothripsis - like pattern in thyroid cancer , which involved shattering of chromosome 4 leading to complete abrogation of normal chromosomal function , along with dramatic widespread copy - number aberrations across both primary tumor and bone marrow samples . a 36 - year - old man with a body mass index ( bmi ) of 45 presented with a 4 - week history of chest pain , dyspnea , and 22 kg of unintentional weight loss ( overview of the patients clinical course is presented in the data supplement )  . 
the working diagnosis was carcinoma of unknown primary . a bone marrow biopsy was performed , and immunohistochemical ( ihc ) staining revealed round and polygonal malignant cells arranged in nests and cord - like patterns with round to oval nuclei containing coarse granular chromatin patterns adjacent to areas of extensive tumor necrosis ( fig 1a1 )  . focal staining was positive for calcitonin ( fig 1a2 ) , chromogranin ( data supplement ) , thyroid transcription factor 1 ( data supplement ) , and synaptophysin ( data supplement ) , but it was negative for cd45 ( data supplement )  . 
a diagnosis of mtc was established . these results led to a thyroid ultrasound , which showed a 4 - cm hypoechoic nodule at the lower pole of the left lobe and a 1.4 - cm hypervascular echogenic focus inferomedially to it ( fig 1b )  . 
ihc of the primary tumor revealed pleomorphic polygonal and ascopubs.org / journal / po jco precision oncology sudipto das deirdre kelly bruce moran kathleen han niall mulligan ciara barrett patrick g . 
radiologic detection of primary tumor and immunohistochemical analysis of primary thyroid and bone marrow sample . ( a ) bone marrow biopsy sample stained with ( 1 ) hematoxylin and eosin staining showed infiltration of the marrow space by a proliferation of pleomorphic round to oval cells with scant eosinophilic cytoplasm and inconspicuous nucleoli . 
 ( 2 ) positive staining of the bone marrow biopsy observed for calciton ( b ) thyroid ultrasound shows a 1.4 - cm hyperechoic nodule at the lower pole of the left lobe of thyroid ( as indicated by the red arrow ) , suggestive of a primary thyroid malignancy . 
 ( d ) primary tumor sample stained with ( 1 ) hematoxylin and eosin shows that the primary tumor consisted primarily of pleomorphic polygonal and spindle cells arranged in nests separated by fibrous strand . 
serum calcitonin was 5 , 000 ng / l ( normal range , , 10 ng / l ) , and carcinoembryonic antigen was 200 mg / l ( normal range , , 5 mg / l )  . routine germline ret mutation testing in exons 10 , 11 , 13 , 14 , and 16 was negative . 
the patient died on day 31 from multiorgan failure . postmortem examination showed metastatic mtc , with complete replacement of the left lobe of the thyroid involving para - esophageal tracheal lymph nodes and the pleural surface of both lungs extending into the parenchyma . 
after receiving written informed consent from the patients family for research activities to be carried out postmortem , dna extracted from the primary tumor , adjacent normal bone marrow tissue , and blood was used to carry out wes and swgs ( data supplement )  . results chromothripsis - like pattern in genomic data . we revealed a dramatic landscape , concordant between wes and swgs ( fig 2a ) , of wideranging copy - number aberrations ( cnas ) across the primary tumor , two distinct regions of bone marrow , which showed clear tumor cell infiltration ( bm - mets1 , bm - mets2 ) , and adjacent normal tissue . 
by using the blood sample as a baseline , losses ranging from regions of several megabases to entire chromosomes were evident across all tumor samples ( fig 2a ; data supplement ) with a single gain occurring on the q arm of chromosome 1 . 
genome - wide copy - number aberrations and identification of chromothripsis - like pattern across adjacent normal tissue , blood , primary tumor , and bone marrow samples showing somatic mutations from whole - exome sequencing ( wes )  . 
 ( a ) absolute copy - number aberrations ( y - axis : 1 , loss ; 3 , gain ) across chromosomes 1 to 21 ( x - axis , denoted by blue lines , centromere denoted by dashed pink line ) compared by using data from wes and shallow wholegenome sequencing across all sample types . 
 ( b ) the copy - number aberration profile for chromosome 4 across the all samples demonstrating the chromothripsis - like pattern event generated for a 30 - kb bin using the wes data . 
ctlp is a catastrophic event involving tens to hundreds of chromosomal rearrangements up to the whole - chromosome level , which can contribute to development of a highly unstable and aggressive disease.1 - 7 the ctlp observed in this patient led to generation of approximately 30 to 40 segments with a typical oscillation of cna between two states ( loss and normal copy number ) .7 although we did carry out genomic analysis of the other metastatic lesions , including the para - esophageal , para - tracheal , and lung metastases , they were not included in the detailed genomic assessment described in this study because of unacceptable sequencing data quality . germline mutations in ret and somatic mutations in disease - associated genes . 
germline mutation analysis using wes from blood and adjacent normal tissue did not reveal any ret variants that are currently used diagnostically ( confirming the posthumous germline ret mutation profiling )  . 
however , we identified three annotated snps within ret ( rs1800861 , rs2742241 , and rs2742243 ) that were fixed with a mutant allele frequency of 1 across all samples including blood and normal tissue . we were able to validate these orthogonally across all samples by using sanger sequencing ( data supplement )  . 
the rs1800861 ( l769l , exon 13 leuctt / leuctg ) variant has previously been reported as a predisposing factor for development of aggressive mtc.8 , 9 of the other two mutations , rs2742243 ( c / t variant ) has been associated with papillary thyroid carcinoma10 and rs2742241 ( g / a variant ) has not been shown to be associated with thyroid cancer . furthermore , our somatic mutational analysis identified 109 somatic mutations , 70% of which occurred in only one of the samples ( fig 2c ; data supplement )  . 
among the nonsynonymous variants of note in disease - associated are missense mutations genes , including gse1 , cldn12 , and trim28 , which have been previously implicated as playing a role in disease processes such as metastasis , migration , and proliferation across various cancer types11 - 15 and may have had a role as potential drivers of disease progression in this patient . 
the differences in mutational patterns between the two bone marrow samples suggest marked genetic heterogeneity between tumor deposits in the same tissue . discussion the patients high bmi made palpation of a neck mass impossible , and the lack of an abnormality on routine imaging in addition to the development of dic and deteriorating respiratory status made this an extremely difficult clinical and diagnostic case . 
remarkably , it was the analysis of the bone marrow that led to the diagnosis of mtc , which further exemplified the severity of the disease in this patient showing complete infiltration of the bone marrow , potentially contributing to a total bone marrow collapse and resulting in development of dic.16 , 17 posthumously , routine clinical germline ret mutation testing did not identify any mutations within this gene . we sought to determine why this young man developed such an aggressive mtc and if it could be explained by the interplay of genomic events . our results support the hypothesis of a catastrophic event such as the evident ctlp , along with widespread copy - number alterations that may be regarded as contributing factors toward the observed phenotype.4 , 5 , 18 although ctlp has been detected in 2% to 3% of all cancers , 1 , 3 , 7 , 19 - 21 to the best of our knowledge , this is the first report to demonstrate ctlp in any form of thyroid cancer . in addition to de novo events that have an impact on cancer phenotype , known genetic alterations can often predispose patients to aggressive disease development . 
variants in the ret protooncogene are commonly associated with mtc and are used as a primary diagnostic test to identify familial and sporadic cases.22 - 25 these tests encompass well - established hot spots within ret and have largely been proven effective in detecting patients with conventional mtc . 
however , unusual patients such as this one require detailed analysis of such genes with the intent of identifying rare mutations that might either explain the atypical phenotype or allow it to be molecularly diagnosed . 
the germline ret l769l variant , previously associated with increased risk of aggressive mtc , 8 , 10 , 22 as well as the other two mutations discovered here , which have not previously been reported in mtc , could have an additive effect on increased risk of development of an aggressive phenotype . 
 ret snps were shown to have increased risk for lymph node and distant metastases at diagnosis ( odds ratio , 5.84 ) and also for developing metastases earlier.8 importantly , our results exemplify how the inclusion of more variants in clinical assays may be of use , especially in diagnostically challenging patients . although identification of mutations in individual disease - associated loci , as observed through the somatic mutation analysis , allows us to generate an appropriate hypothesis surrounding the events that could have led to the development of metastasis , the genetic heterogeneity observed across all resection samples of this patient underpin why , in the clinical setting , targeting specific mutational events therapeutically is rarely effective . 
however , it is also important to note that the study reported here describes a single patient with a rare and atypical mtc . multifactorial genomic events , including germline and somatic mutations in functionally important genes together with widespread cna ( including ctlp ) supports the need for pragmatic treatment approaches using combinations of targeted agents and possibly even immunotherapeutic approaches.26 - 28 within the context of this study and in patients who have disease of a similar nature , it is tempting to speculate that the presence of ctlp would likely lead to generation of multiple neoantigens , suggesting a potential benefit of immunotherapy.26 however , it is noteworthy that such immunotherapeutic strategies to date have not been used for patients with mtc . 
although mutational load may be inferred from routine panel - based genetic testing when only a few hot spot mutations are screened , such as in mtc , it is unlikely that candidates for immunotherapy will be identified , suggesting that a deeper examination of the genome may provide better diagnostic and therapeutic information . in conclusion , our results provide a critical insight into potential drivers of an aggressive phenotype of mtc and , in parallel , present a vital opportunity to identify effective therapeutic strategies based on the evident abnormalities in a given patient . 
kelly collection and assembly of data : sudipto das , deirdre kelly , bruce moran , kathleen han , niall mulligan , ciara barrett , peter mcmahon , hendrik f . 
relationships are self - held unless noted . deirdre kelly no relationship to disclose bruce moran no relationship to disclose kathleen han no relationship to disclose niall mulligan no relationship to disclose ciara barrett no relationship to disclose patrick g . 
van essen no relationship to disclose kate connor no relationship to disclose diether lambrechts no relationship to disclose bauke ylstra no relationship to disclose consulting or advisory role : carrick therapeutics research funding : carrick therapeutics travel , accommodations , expenses : oncomark darran p . 
gallagher employment : oncomark leadership : oncomark stock and other ownership interests : oncomark acknowledgment we thank lisa dwane , brian mooney , and camille hurley for their technical assistance on the study and the family of the patient for consenting to the study . affiliations sudipto das , deirdre kelly , bruce moran , kathleen han , niall mulligan , ciara barrett , peter mcmahon , john mccaffrey , kate connor , william m . 
oconnor , royal college of surgeons in ireland ; deirdre kelly , kathleen han , niall mulligan , ciara barrett , peter mcmahon , john mccaffrey , and catherine m . 
van essen and bauke ylstra , vrije universiteit medical center , amsterdam , the netherlands ; and diether lambrechts , vesailus research center , vlaams instituut voor biotechnologie , katholieke universiteit , leuven , belgium . supported by funding from the mater foundation , by postdoctoral fellowship crf13das from the irish cancer society ( s.d. ) , by grant no . 
cai h , kumar n , bagheri hc , et al : chromothripsis - like patterns are recurring but heterogeneously distributed features in a survey of 22 , 347 cancer genome screens . 
sromek m , czetwerty nska m , skasko e , et al : the frequency of selected polymorphic variants of the ret gene in patients with medullary thyroid carcinoma and in the general population of central poland . 
liao s , song w , liu y , et al : familial multinodular goiter syndrome with papillary thyroid carcinomas : mutational analysis of the associated genes in 5 cases from 1 chinese family . 
wei c , cheng j , zhou b , et al : tripartite motif containing 28 ( trim28 ) promotes breast cancer metastasis by stabilizing twist1 protesci rep 6 : 29822 , 2016 15 . 
yang y , cheon s , jung mk , et al : interleukin - 18 enhances breast cancer cell migration via down - regulation of claudin - 12 and induction of the p38 mapk pathway . 
forment jv , kaidi a , jackson sp : chromothripsis and cancer : causes and consequences of chromosome shattering . j gen intern med 21 : c6 - c8 , 2006 study . 
ye l , santarpia l , cote gj , et al : high resolution array - comparative genomic hybridization profiling reveals deoxyribonucleic acid copy number alterations associated with medullary thyroid carcinoma . 
 parental perspectives on whole - exome sequencing in pediatric cancer : a typology of perceived utility purpose to explore how parents of pediatric patients with cancer perceived the utility of clinical tumor and germline whole - exome sequencing ( wes ) results . patients and methods we conducted longitudinal interviews with parents of a diverse pediatric cancer population before disclosure of wes results ( n = 64 ) , then 1 to 8 months ( n = 33 ) after disclosure . 
2017 by american society of clinical oncology introduction genomic sequencing ( gs ) technologies including whole - exome sequencing ( wes ) and wholegenome sequencing are increasingly being used in cancer clinical research and care.1 - 5 these technologies are typically valued for their potential to allow evidence - based changes to patients treatment . 
this is particularly true in the context of pediatric practice , in which ethical reservations about the impact of gs information have limited its use in generating direct clinical benefits.6 however , recent policy and position statements have criticized these narrow definitions of utility and argue that testing and disclosure provide value for families and society in addition to individual patients and may be appropriate in a wider set of cases.7 - 10 many have argued for a more comprehensive and empirically informed understanding of the utility of gs and have urged that perspectives of individuals receiving sequencing results should be incorporated into the development of clinical practice and coverage guidelines.11 - 14 the hypothetical interest of parents in receiving gs results for their children with conditions other than cancer has been reported.15 - 17 two studies have described the perceptions of parents of children with rare diseases18 and autism spectrum disorders19 regarding gs ; these parents found the information useful for reproductive and financial planning19 as well as for finding a conclusive diagnosis and helping with the coping process.18 recent studies have found that adults undergoing sequencing reported finding value in increased self - awareness , 20 , 21 satisfaction of curiosity , 20 legitimation of their condition , 21 and support from others.21 to the best of our knowledge , no research has evaluated how parents of pediatric patients with cancer who have received their childs gs results conceptualize the utility of the information . 
the basic3 study enrolled 287 pediatric patients ; the consent process and reporting components of the trial as well as the initial germline and tumor exome results have been described.22 , 23 the study includes providing clinical wes of blood samples for all patients and tumor samples when available ( 80% of patients enrolled ) ; results were reported in the medical record . 
the study was approved by the institutional review board of baylor college of medicine . tumor and germline wes results were disclosed to parents by the patients primary oncologist and a study genetic counselor . 
it was explicitly stated in both the informed consent document and consent session that it was unlikely that wes information would provide immediate clinical benefit to patients in terms of current treatment and that the tumor wes results in particular might be more clinically useful if the tumor recurred . 
parents were given a copy of their childs wes report ( s ) in addition to a counseling letter summarizing findings from the germline wes report in lay language . we conducted semistructured interviews with one parent of basic3 study patients in either english or spanish as preferred by the parent . 
to ensure that we would have sufficient participation throughout the longitudinal project , we oversampled for predisclosure interviews ( n = 64 )  . these were conducted with parents after consent and enrollment in the study but before disclosure of wes results ( predisclosure )  . 
interviews lasted an average of 50 minutes and were conducted at a time and location convenient for the parent , most frequently at the time of one of their childs clinical appointments . 
all potentially identifying information was removed , and parents were referred to only by their study identification number , noted in brackets after each quotation . we calculated descriptive statistics for parents demographic characteristics , evaluating differences between interview participants and overall study participants sex and race / ethnicity by using x2 tests . 
all p values were two - sided and with statistical significance set at p , .05. results our study enrolled a highly diverse set of families . demographic information from all parents who completed predisclosure interviews are provided in table 1 . 
 families enrolled in basic3 study ( n = 287 ) not interviewed for predisclosure enrolled after predisclosure goal met scheduling conflicts , unable to be contacted , and / or disclosed before predisclosure interview ineligible due to tumor unavailability declined predisclosure interview withdrawal ( n = 223 ) ( n = 102 ) ( n = 80 ) ( n = 35 ) ( n = 1 ) ( n = 5 ) completed predisclosure interview ( n = 64 ) not disclosed scheduling conflicts and / or unable to be contacted ( n = 3 ) ( n = 3 ) received wes results disclosure ( n = 61 ) not interviewed for postdisclosure scheduling conflicts and / or unable to be contacted patient death before postdisclosure interview declined postdisclosure interview ( n = 28 ) ( n = 19 ) ( n = 8 ) ( n = 1 ) completed postdisclosure interview ( n = 33 ) fig 1 . 
representative quotes illustrating each dimension are included in table 3 . although the focus of this article is the range of ways in which parents found participation in the basic3 study valuable , it is worth noting that parents described few concerns about having gs information . clinical utility parents were informed as part of the consent process that participation in the study was highly unlikely to change their childs treatment plan . even so , in predisclosure interviews some parents expressed hope that their childrens wes results might provide clinical benefits for their children either in the short term or in the future , including tailored treatments and information about the need for additional genetic testing or surveillance . as expected , only a minority ( 33% of those interviewed postdisclosure ) of tumor reports included somatic mutations categorized by the laboratory as being of proven or potential clinical relevance.23 even when such a mutation was found , the information was not used immediately because the child was undergoing standard treatment . 
however , some parents , including both those whose child was found to have a tumor mutation and those who received a negative report , believed that it could be used to personalize treatment in the future if the tumor returned . before and after disclosure , parents were clear that germline genomic information was valuable not only for their child with cancer . 
they hoped that the tumor or germline wes reports would offer an answer to a question bothering them . of the 33 families interviewed postdisclosure , only 11 had received information identifying a potentially clinically relevant mutation in the childs tumor , and one had received information about a cancer - related germline mutation . that most parents did not get information explaining their childs cancer caused disappointment among some participants whereas others were grateful for any findings that were returned . some parents looking to explain their childs cancer articulated in predisclosure interviews that they hoped the results would offer exculpatory information . 
they felt that a better understanding of the cancers source would remove the guilt associated with worrying that they had passed the disease on or that they had done something to cause it . 
relief from guilt associated with the possibility of passing on a cancer gene was also identified by parents as a source of utility after receiving genomic information about tumor mutations . there were parents who stated that the information offered them peace of mind after they had received a negative result . 
all demographics were self - reported at the time of enrollment in the larger basic3 study . abbreviation : na , not applicable . * non - hispanic or latino , other category includes asian ( 4 ) , black or african american ( 8 ) , two or more races ( 2 ) , declined or na ( 9 )  . participation in the basic3 study . 
interestingly , many parents discussed the psychological impact of genomic information on themselves rather than on the patient or other family members . one of the strongest themes emerging from our analysis was that parents felt the need to know why table 2 . 
parents descriptions of utility of tumor and germline wes before ( pre ) or after ( post ) disclosure findings patient id post post medically actionable finding post tumor mutation finding description clinical utility tailored treatment of patient thats what my hope is , is that of course any genetic information that they find would be helpful to do that , just to find what would be the most effective therapy for him . but , if for some reason , ( knocks on table ) [ the tumor ] came back , they could use that basic3 study and figure out from the genetics of his tumor what to do , and he would essentially have his own regimen and treatment plan or whatever . additional testing for patient it will just help us in the future with her , and itll help me in the future if i decide to have another baby . 
so we know what to look for , we know the signs , we know what to do with her to help prevent things returning or prevent more disease and illness . testing or surveillance for others it showed us some of the mutations that we really need to look at their possible representation and thats what we did . 
we dont know when its going to show so we will keep informing next generations and making sure that they check this . i really wanted to know if it was genetic , just for the fact if i needed to get my daughter [ the patients sibling ] testedbut when they said that it wasnt , then that made me reallythat i didnt need to get her a whole body scanned for anything . ( laughs ) so that was my real reason for doing it is to make sure she didnt need to be checked . psychological utility understanding cause of cancer they possibly could figure out why this happens too . 
parents descriptions of utility of tumor and germline wes ( continued ) description before ( pre ) or after ( post ) disclosure post findings patient id of course , the study doesnt point out what caused it exactly or why that was . 
yeah , that was the only disappointment , but otherwise i guess whatever the information we have so far is not bad . curiosity i want to know everything there is to know about [ child ] and us . 
it really justits just really interesting to find out these things relief from guilt how did it develop ? i dont know like , is it that something that we carried ? is it something i did while i was pregnant ? what caused it ? mainly thats what we want to know . as long as theres nothing that we did that caused itor nothing that we couldve possibly ever have done to cause iti suppose that [ would be ] a relief . you know it was a relief to know that its just something that his body just grew on its own and didnti didnt pass downso justi dont know , just a reliefsomething off your shoulders . peace of mind i guess peace of mind at some level for her thatyou knowtheres not an inherited mutation . 
parents descriptions of utility of tumor and germline wes ( continued ) before ( pre ) or after ( post ) disclosure findings patient id post post post description pragmatic utility reproductive considerations she also has a gene that if her future partner has the same kind ofif they carry the same kind of gene together , their kids mightwell , well tell her when she grow up . 
she might go to the doctors to find out whats going on . so now its like , if we ever do plan on having more children , its kind of like , we have to go get our genetics testing done first before we even think about having a baby , and then while imwhenever i do get pregnantthen we have to get the genetic testing while the babys still inside of me . 
so its likeits a lot to think about . future planning butfor me , the benefits are just like mainly her future stuff and knowing that we could have another child that has cancer...id rather know than not know and it just pop up and surprise us like it did with her . 
if i know its coming , you cani can better handle myself like , okay , thats fine , well be prepared , well handle it just like we handled this one , except now we know what to expect . abbreviations : id , identification ( number ) ; nf , no medically actionable , cancer - related , or tumor mutation findings ; wes , whole - exome sequencing . finally , several parents identified the value of being able to plan for the future and the possibility of additional health problems . 
although few of the parents in our postdisclosure cohort ( three [ 9% ] of 33 ) received results indicating that their family was at increased genetic risk for other cancers or diseases , many reported being grateful for any information that could prevent them from being surprised by a similar occurrence again , consistent with results in the predisclosure interviews.25 discussion the results of this qualitative analysis demonstrate that parents perception of the value of wes information in the context of childhood cancer extends well beyond a narrow definition of clinical utility focused on diagnosis , prognosis , and therapeutic management . 
our diverse cohort of parents described other dimensions of utility , including the psychological and pragmatic value of having this information . existing literature typically classifies any utility that is not grounded in a change of medical management as personal utility . 
 clinical decision making at the time of the disclosure was found in our analysis and in other work.21 with respect to psychological utility , our results mirror those suggesting that parents and patients place enormous value on establishing a causal explanation for a disease or condition.19 , 21 similarly , our data support a finding that parents of children with serious diseases seek confirmation that the disease was not the fault of the parents.18 as other studies have shown , 19 , 20 our participants reported that having genomic information may be valuable in and of itself , even if it does not lead to any significant changes in their lives or experiences . 
the possibility that this information could have an impact on reproductive decision making was identified as important for many parents in other contexts as well.19 available literature on pediatric gs has not described the extent to which utility for individuals other than the patient is considered by those making decisions about wes , although this is clearly a theme in adult contexts . 
finally , our study has described parents perceptions in the unique context of decision making about wes in pediatric cancer . the experiences and perspectives of the parents interviewed offer evidence to buttress emerging critiques of a narrow view of utility . 
our data support the view of botkin et al13 that , softer outcomes , such as psychological impacts , behavior changes , and social impacts , are particularly important in the assessment of many genetic tests and illustrate the importance of recognizing a wide range of utility . 
the finding that parents of pediatric patients with cancer perceive the value of genomic information so broadly has several significant ethical implications for whether and how wes is integrated into clinical practice . first , a few of the responses given by parents suggest that their perceived value of wes information may be disproportionate to its clinical implications . 
despite being told that it was unlikely that the wes results would change or improve their childs medical care in the short term , many parents still cited hopes of tailored treatment . 
in addition , the peace of mind that parents described having after getting a negative germline report may reflect an overestimation of the familys risk reduction estimate , even though the disclosure and counseling letter notes that wes does not identify all cancer susceptibility findings . although it is not novel to note concerns about the accuracy of decision makers understanding of complex genomic data , the question of how to ethically account for parents possibly exaggerated perceptions of the utility of a negative report ( there are no reliable data on how exaggerated this might be ) seems particularly salient in the context of pediatric cancer . second , the data demonstrate that parents put significant weight on the perceived benefits of wes information for themselves , their other children , and other family members . 
the nature of genetic and genomic technologies is that they have implications for individuals beyond the index patient , an aspect that parents seemed acutely aware of and that shaped their experience with wes . this finding raises questions about the degree to which utility for nonpatients should have an impact on sequencing recommendations , health care coverage , and disclosures . third , it is uncertain which of these varied types of utility should be used to justify a recommendation for genomic sequencing of a patient and his or her family . 
although it is clear that clinical utility could justify the use of this technology by thirdparty payers , it has not been generally accepted that the possibility of finding psychological or pragmatic utility offer the same grounding for a testing recommendation . 
consideration should be given to whether these types of utility are sufficient justification for wes or whether they ought to be given second - tier status . finally , the insights offered by this study raise questions about the nature of the informed consent process in the clinical and research settings for gs that is used with parents of children with serious diseases . 
current practice is to identify and clearly communicate possible psychological and social risks ( such as anxiety or loss of insurance ) , but corresponding possible benefits ( such as peace of mind or reproductive planning ) are not typically discussed ; instead , the focus is usually on narrow definitions of potential medical benefits . 
our results suggest that this differential treatment of the different types of risks and benefit may not be coherent and that both may be of great importance to decision makers . 
 experience , which suggests that additional work is required to ensure that all of the appropriate risks and benefits are discussed with parents who are consenting to genomic trials or testing . our study had several limitations . 
first , this interview - based analysis used open - ended questions and so was not designed to assess the prevalenceofvariousperspectiveson genomic information . accurate characterization of the frequency of various responses would require a methodology that included greater numbers of participants and more discrete response options . 
second , because parents understanding of their childs wes results was not directly evaluated , we were not able to characterize parents comprehension of the information they were given at disclosure or the degree to which their understanding of utility was shaped by various elements of the study ( ie , the consent process , education and counseling , and the information contained in the tumor and germline reports )  . finally , because our study was conducted with parents of pediatric patients with cancer , our findings might be limited to this population and may not be generalizable to other pediatric patient populations . longitudinal interviews with a diverse cohort of parents of pediatric patients with cancer identified a broad range of anticipated and experienced utility through the integration of wes into their childrens medical care . 
parents cited possible clinical utility for the patient , themselves , and for their other family members , and also described psychological and pragmatic benefits for these parties as well . 
mcguire no relationship to disclose acknowledgments the authors thank robin raesz - martinez , study coordinator , sarah scollon and katie bergstrom , study genetic counselors , and uma ramamurthy and her team at the baylor college of medicine institute for clinical and translational research and especially thank the oncologists and parents who participated in this study . janet malek , jill o . 
worthey ea , mayer an , syverson gd , et al : making a definitive diagnosis : successful clinical application of whole exome sequencing in a child with intractable inflammatory bowel disease . 
yang y , muzny dm , reid jg , et al : clinical whole - exome sequencing for the diagnosis of mendelian disorders . n engl j med 369 : 1502 - 1511 , 2013 4 . 
american college of medical genetics and genomics ( acmg ) board of directors : clinical utility of genetic and genomic services : a position statement of the american college of medical genetics and genomics . 
botkin jr , belmont jw , berg js , et al : points to consider : ethical , legal , and psychosocial implications of genetic testing in children and adolescents . 
waisbren se , back dk , liu c , et al : parents are interested in newborn genomic testing during the early postpartum genomics 18 : 151 - 159 , 2015 period . 
mccullough lb , slashinski mj , mcguire al , et al : is whole - exome sequencing an ethically disruptive technology ? perspectives of pediatric oncologists and parents of pediatric patients with solid tumors . 
to inform targeted treatment strategies , 3 , 476 clinically advanced prostate tumors were analyzed by cgp for genomic alterations ( gas ) and signatures of genomic instability . methods prostate cancer samples ( 1 , 660 primary site and 1 , 816 metastatic site tumors from unmatched patients ) were prospectively analyzed by cgp ( foundationone assay ; foundation medicine , cambridge , ma ) for gas and genomic signatures ( genome - wide loss of heterozygosity [ gloh ] , microsatellite instability [ msi ] status , tumor mutational burden [ tmb ] )  . results frequently altered genes were tp53 ( 44% ) , pten ( 32% ) , tmprss2 - erg ( 31% ) , and ar ( 23% )  . 
potentially targetable gas were frequently identied in dna repair , phosphatidylinositol 3 - kinase , and ras / raf / mek pathways . dna repair pathway gas included homologous recombination repair ( 23% ) , fanconi anemia ( 5% ) , cdk12 ( 6% ) , and mismatch repair ( 4% ) gas . 
metastatic site tumors were enriched for the 11q13 amplicon ( ccnd1 / fgf19 / fgf4 / fgf3 ) and gas in ar , lyn , myc , ncor1 , pik3cb , and rb1 compared with primary tumors . conclusion routine clinical cgp in the real - world setting identied gas that are investigational biomarkers for targeted therapies in 57% of cases . 
2019 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction genomic alterations ( gas ) that drive prostate cancer have been elucidated by proling primary tumors.1 , 2 comprehensive genomic proling ( cgp ) is increasingly used for routine clinical management of patients with prostate cancer , 3 with accumulating evidence associating gas with responses to therapy . 
 chung et al context key objective in this study , we use real - world data from routine prospective clinical genomic proling to evaluate genomic alterations ( gas ) and genomic signatures in primary and metastatic prostate cancer . knowledge generated we evaluated the landscape of gas in prostate cancer and identied those that are enriched in metastatic site tumors . approximately 3% of cases had high microsatellite instability and / or high tumor mutational burden status . 
genomic signatures , including microsatellite instability , tumor mutational burden , and genome - wide loss of heterozygosity , may further rene biomarker development for poly ( adp - ribose ) polymerase inhibitors and immunotherapies . samples using a validated assay16 ( foundationone ; foundation medicine , cambridge , ma ; appendix table a1 )  . 
the pathologic diagnosis of each case was conrmed on routine hematoxylin and eosinstained slides . results were analyzed for gas and gene signatures ( tmb , msi , genome - wide loss of heterozygosity [ gloh ] )  . 
germline / somatic mutation calls were predicted without a matched normal ; in validation testing of 480 tumor - only sequencing calls against matched normal samples , accuracy was 95% for somatic and 99% for germline calls.17 enrichment was dened as the difference in ga frequency between metastatic and primary sites . 
activating braf or raf1 fusions / rearrangements were observed in 1.2% of cases ( 35 braf and seven raf1 ; appendix fig a2 )  . the pi3k / akt / mammalian target of rapamycin ( mtor ) pathway was frequently altered ( 40.8% ; figs 1b and 1c )  . as expected , pten gas were frequent , and we observed in pik3ca , activating mutations and amplications pik3cb , akt1 / 2 / 3 , mtor , and rictor and loss - offunction alterations in pik3r1 / 2 and tsc1 / 2 . 
ras / raf / mek pathway alterations ( 5.7% ; figs 1b and 1f ) included oncogenic braf mutations ( appendix fig a2 ) , braf / raf1 rearrangements , activating mutations in raf1 / araf , kras / nras / hras and map2k1 / 2 , and nf1 loss - of - function alterations . 
 chung et al cdk12 ga ( p , .001 ) , and braf rearrangements / mutations ( p , .001 ; fig 1b )  . to compare primary and metastatic site tumors , we assessed relative enrichment in gas ( fig 1g ; appendix table a3 )  . 
predicted germline mutations were identied in 57.8% of brca2 - , 25.0% of brca1 - , 35.8% of atm - , 80.0% of chek2 - , 52.2% of fanca - , 42.3% of msh2 - , 20.0% of msh6 - , 25.0% of mlh1 - , and 44.4% of pms2 - mutated cases ; only 9.2% of cdk12 - mutated cases harbored a predicted germline mutation ( fig 2c )  . genomic signatures : gloh in addition to gas in individual dna repair genes , genomic signatures represent the phenotypic readout of deleterious dna repair and are potential predictive biomarkers . 
we evaluated the association between gloh and dna repair gas ( fig 2d )  . consistent with the established relationship between brca1 / 2 and hrd , 21 brca1 / 2 - altered cases ( both predicted germline and somatic mutations ) were more frequently gloh - h compared with the overall data set ( fig 2d )  . to evaluate the potential relevance of non - brca1 / 2 dna repair genes as biomarkers for parp inhibition , we assessed their association with gloh . 
gloh - h frequency was comparable between the overall data set and cases with non - brca1 / 2 dna repair gas , although cases with homozygous deletions in non - brca1 / 2 hrr pathway or fa / icl pathway genes were more frequently gloh - h ( appendix fig a4b )  . 
 ( b ) frequency of major pathway alterations , including ets fusions , braf rearrangements / mutations , spop / cul3 mutations , cdk12 gas , idh1 / 2 mutations , ar gas , phosphatidylinositol 3 - kinase ( pi3k ) pathway gas , homologous recombination gas , g1 / s - cell cycle gas , wnt pathway gas , fanconi anemia / interstrand crosslink repair ( fa / icl ) repair pathway gas , ras / raf / mek pathway gas ( other than braf ) , mismatch repair gas , and pole mutations ( see figs 1c to 1f for details )  . 
 ( c to f ) gas identied in each pathway , including the ( c ) pi3k / akt / mammalian target of rapamycin ( mtor ) pathway , ( d ) g1 / s - cell cycle pathway , ( e ) wnt pathway , and ( f ) ras / raf / mek pathway ( nf1 mutation , rearrangement , or copy number loss ; braf or raf1 mutations or rearrangements ; araf , k / n / h - ras , or map2k1 / 2 mutations were included ) ; percent of altered cases is indicated . 
genes that were enriched ( difference between primary site v metastatic site frequency ) by at least 2% are indicated ( all p , .05 by fishers exact test [ two - tailed ] ) , with genes enriched at least twofold indicated in red ( all p , .001 by fishers exact test [ two - tailed ] )  . 
msi status was assessable for 3 , 326 cases , and overall , 87 of 3 , 326 of cases ( 2.6% ) were msi - h ( 2.0% primary site , 3.1% metastatic site tumors )  . 
for the remaining cases , tmb - h phenotype was not attributed to msi - h or pole . landscape of ets fusions and ar gas in total , 1 , 236 ets fusions were detected ( one sample harbored two ets fusions ; fig 1b )  . 
tmprss2 - erg comprised the majority ( 87.7% ) of ets fusions ( fig 3a ) , with the remaining consisting of etv1 ( 8.5% ) , etv4 ( 2.6% ) , and etv5 ( 1.2% ) with diverse fusion partners , including several not previously described ( fig 3b )  . 
we also identied breakpoints that juxtaposed tmprss2 with the intergenic region upstream of erg ( 0.1 to 75 kb upstream ; fig 3c ) ; similar upstream intergenic breakpoints were observed for tmprss2 fusions with etv4 ( 10 of 32 cases ) and etv5 ( two of 15 cases )  . ar gas are associated with castration - resistant disease and were most enriched in metastatic site tumors ( 39.7% of metastatic site and 3.7% of primary site tumors ; fig 1g )  . 
five hundred fty - ve of 557 cnas were amplications ( median copy number , 24 ; range , six to 366 ) ; two homozygous deletions that encompass exons 5 to 7 or exons 5 to 8 are likely activating.27 ar missense mutations were observed in 6.9% of cases ( 1.3% primary site and 11.9% metastatic site ) , and most were ligandbinding domain antiandrogen resistance mutations ( fig 4b ) .28 finally , ar rearrangements were observed in 1.3% of cases ( 0.4% primary site and 2.2% metastatic site ) ; however , because the sequencing strategy was not explicitly designed to detect ar rearrangements and does not fully cover ar intronic regions , the true frequency of ar rearrangements is difcult to establish . 
gas that co - occur with targetable gas ( fig 1b ) and their impact on response to therapy warrant consideration in biomarker - driven trials . msi - h and tmb - h have been associated with immunotherapy benet , 10 , 24 , 25 and gloh - h has been associated with parp inhibitor benet21 ; therefore , assessment of signatures of genomic instability potentially expands the population of patients addressable with immunotherapy or targeted therapy . we identied a subset of tmprss2 rearrangements fused to upstream intergenic regions of erg , etv4 , or etv5 as well as novel ets fusion partners . 
 cgp of primary and metastatic prostate tumors etv4 ( 32 ; 2.6% ) etv5 ( 15 ; 1.2% ) etv1 ( 105 ; 8.5% ) tmprss2 - erg ( 1 , 084 ; 87.7% ) etv1 etv4 etv5 intron 4 ( n = 38 ) erg gene ( nm_004449 chr21 : 40033704 - 39751950 ) intron 3 ( n = 940 ) intron 1 ( n = 58 ) upstream ( 108 bp - 75 kbp ) ( n = 39 ) intron 2 ( n = 9 ) erg exon 1 genomic coordinates : chr21 : 40020000 40040000 40060000 40080000 40100000 40120000 fig 3 . 
consistent with distinct molecular subsets of prostate cancer , 1 , 8 ets fusions were mutually exclusive with spop / cul3 , cdk12 mutations , or braf rearrangements / mutations that may potentially comprise a clinically relevant genomic subset.30 , 31 diverse ar gas can mediate androgen axis inhibitor resistance28 , 29 and were strongly enriched in metastatic site tumors . 
ar rearrangements that disrupted the ligand - binding domain can mediate resistance to current ar inhibitors.29 development of novel ar inhibitors that target the diverse spectrum of ar alterations is needed . 
 ( a ) ar genomic alterations ( gas ) were identied in 721 cases , including amplication ( red ) , mutation ( green ) , and rearrangements ( purple )  . 
of note , 11q13 amplication is associated with endocrine resistance in breast cancer and potentially targetable by broblast growth factor receptor inhibitors32 - 34 and , therefore , warrants investigation in prostate cancer . 
second , cdkn2a gas were enriched in metastatic site tumors ( 1.9 - fold ) , and although not previously described in primary versus metastatic studies , 1 - 3 acquired cdkn2a alterations were described in enzalutamide - resistant tumors.15 metastatic enrichment of cell cycle gas suggests that cdk4 / 6 inhibitors could be explored . 
 cgp of primary and metastatic prostate tumors certain hrr genes , mmr genes , or msi status ; identication of such dna repair ga by tumor sequencing warrants follow - up germline testing and genetic counseling . 
however , compared with a recent study , 3 we identify similar relative proportions of germline / somatic mutations for frequently mutated hrr genes ( brca2 , atm , chek2 )  . 
furthermore , another study of prostate cancer similarly identied germline mutations for many of the dna repair genes evaluated here.37 we also describe potential cdk12 germline mutations ( four of 12 were rs138292741 )  . 
although not identied in one recent study of cdk12 in prostate cancer , 8 germline truncating cdk12 mutations were described in other studies , cluding prostate cancer , 38 - 40 and in germline databases.40 although preclinical studies have identied non - brca1 / 2 hrr genes , their association with hrd phenotype in clinical samples is unclear . 
identication of gloh - h cases lacking dna repair gas ( appendix fig a4a ) may have clinical value , but further investigation in parp inhibitor trials is required to assess the clinical utility of gloh as a biomarker in prostate cancer . similarly , dna repair gas were associated with msi - h or tmb - h genomic signatures that are associated with benet from immunotherapy ( fig 2 ; appendix fig a5 )  . 
a subset of msi / tmb - h cases do not harbor corresponding dna repair gas potentially because gas may occur in genes involved in dna repair that have not yet been discovered , complex rearrangements with intronic breakpoints may be challenging to detect , or genomic instability can occur through mechanisms such as gene silencing or extrinsic dna damage . limitations of this study should be acknowledged . 
furthermore , because samples were collected for routine clinical testing , primary samples in this cohort may be biased toward patients who have received prior treatment or developed metastatic disease that resulted in a distinct genomic landscape from untreated primary tumors . 
chung employment : foundation medicine ninad dewal employment : foundation medicine stock and other ownership interests : foundation medicine ethan sokol employment : foundation medicine paul mathew honoraria : exelixis patents , royalties , other intellectual property : patent pending on new therapeutic invention travel , accommodations , expenses : exelixis robert whitehead employment : unitedhealthcare , cancer treatment centers of america sherri z . 
pal honoraria : novartis , medivation , astellas pharma consulting or advisory role : pzer , novartis , aveo , myriad pharmaceuticals , genentech , exelixis , bristol - myers squibb , astellas pharma , ipsen , eisai research funding : medivation richard j . 
lee consulting or advisory role : janssen pharmaceuticals , exelixis research funding : janssen pharmaceuticals andrea necchi employment : bayer ag ( i ) stock and other ownership interests : bayer ag ( i ) honoraria : roche , merck , astrazeneca , janssen pharmaceuticals , foundation medicine consulting or advisory role : merck sharp & dohme , roche , bayer ag , astrazeneca , clovis oncology , janssen pharmaceuticals , incyte , seattle genetics , astellas pharma , bristol - myers squibb , rainier therapeutics research funding : merck sharp & dohme ( inst ) , astrazeneca ( inst ) travel , accommodations , expenses : roche , merck sharp & dohme , astrazeneca , janssen pharmaceuticals other relationship : bayer ag ( i ) jeffrey p . 
gregg consulting or advisory role : astrazeneca , bristol - myers squibb , roche , foundation medicine speakers bureau : astrazeneca , foundation medicine , bristol - myers squibb primo lara jr honoraria : pzer consulting or advisory role : exelixis , pzer , astrazeneca , bayer ag , genentech , roche , janssen pharmaceuticals , bristol - myers squibb , abbvie , turnstone bio , foundation medicine , merck , cellmax life , nektar research funding : millennium pharmaceuticals ( inst ) , polaris ( inst ) , glaxosmithkline ( inst ) , genentech ( inst ) , aragon pharmaceuticals ( inst ) , janssen pharmaceuticals ( inst ) , heat biologics ( inst ) , tracon pharma ( inst ) , merck ( inst ) , pharmacyclics ( inst ) , incyte ( inst ) emmanuel s . 
antonarakis honoraria : sano , dendreon , medivation , janssen pharmaceuticals , essa , astellas pharma , merck , astrazeneca , clovis oncology consulting or advisory role : sano , dendreon , medivation , janssen pharmaceuticals , essa , astellas pharma , merck , astrazeneca , clovis oncology research funding : janssen pharmaceuticals ( inst ) , johnson & johnson ( inst ) , sano ( inst ) , dendreon ( inst ) , aragon pharmaceuticals ( inst ) , exelixis ( inst ) , millennium pharmaceuticals ( inst ) , genentech ( inst ) , novartis ( inst ) , astellas pharma ( inst ) , tokai pharmaceuticals ( inst ) , merck ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , constellation pharmaceuticals ( inst ) patents , royalties , other intellectual property : co - inventor of a biomarker technology that has been licensed to qiagen travel , accommodations , expenses : sano , dendreon , medivation vincent a . 
miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine consulting or advisory role : revolution medicines patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center jeffrey s . 
 cgp of primary and metastatic prostate tumors research funding : bayer ag ( inst ) , bristol - myers squibb ( inst ) , glaxosmithkline ( inst ) , medivation ( inst ) , takeda pharmaceuticals ( inst ) , novartis ( inst ) , pzer ( inst ) , bn immunotherapeutics ( inst ) , tracon pharma ( inst ) , rexahn pharmaceuticals ( inst ) , amgen ( inst ) , astrazeneca ( inst ) , active biotech ( inst ) , bavarian nordic ( inst ) , calithera biosciences ( inst ) , celldex ( inst ) , eisai ( inst ) , genentech ( inst ) , immunomedics ( inst ) , janssen pharmaceuticals ( inst ) , merck ( inst ) , newlink genetics ( inst ) , prometheus ( inst ) , sano ( inst ) no other potential conicts of interest were reported . references abeshouse a , ahn j , akbani r , et al : the molecular taxonomy of primary prostate cancer . 
nat genet 50 : 645 - 651 , 2018 abida w , armenia j , gopalan a , et al : prospective genomic proling of prostate cancer across disease states reveals germline and somatic alterations that may affect clinical decision making . 
n engl j med 373 : 1697 - 1708 , 2015 de bono js , de giorgi u , massard c , et al : pten loss as a predictive biomarker for the akt inhibitor ipatasertib combined with abiraterone acetate in patients with metastatic castration - resistant prostate cancer ( mcrpc )  . 
mateo j , ganji g , lemech c , et al : a rst - time - in - human study of gsk2636771 , a phosphoinositide 3 kinase beta - selective inhibitor , in patients with advanced 8 . 
clin cancer res 23 : 5981 - 5992 , 2017 2018 lee l , ali s , genega e , et al : aggressive - variant microsatellite - stable pole mutant prostate cancer with high mutation burden and durable response to immune checkpoint inhibitor therapy . 
cell 161 : 1215 - 1228 , 2015 [ erratum : cell 162 : 454 , 2015 ] joseph jd , lu n , qian j , et al : a clinically relevant androgen receptor mutation confers resistance to second - generation antiandrogens enzalutamide and arn509 . 
han gc , hwang j , wankowicz sam , et al : genomic resistance patterns to second - generation androgen blockade in paired tumor biopsies of metastatic castration - resistant prostate cancer . 
mateo j , chakravarty d , dienstmann r , et al : a framework to rank genomic alterations as targets for cancer precision medicine : the esmo scale for clinical actionability of molecular targets ( escat )  . 
bajrami i , frankum jr , konde a , et al : genome - wide proling of genetic synthetic lethality identies cdk12 as a novel determinant of parp1 / 2 inhibitor sensitivity . 
blazek d , kohoutek j , bartholomeeusen k , et al : the cyclin k / cdk12 complex maintains genomic stability via regulation of expression of dna damage response 21 . 
swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
coleman rl , oza am , lorusso d , et al : rucaparib maintenance treatment for recurrent ovarian carcinoma after response to platinum therapy ( ariel3 ) : a randomised , double - blind , placebo - controlled , phase 3 trial . 
carbone dp , reck m , paz - ares l , et al : first - line nivolumab in stage iv or recurrent non - small - cell lung cancer . 
giltnane jm , hutchinson ke , stricker tp , et al : genomic proling of er + breast cancers after short - term estrogen suppression reveals alterations associated with endocrine resistance . 
luen sj , asher r , lee ck , et al : association of somatic driver alterations with prognosis in postmenopausal , hormone receptor - positive , her2 - negative early breast cancer : a secondary analysis of the big 1 - 98 randomized clinical trial . 
soria j - c , debraud f , bahleda r , et al : phase i / iia study evaluating the safety , efcacy , pharmacokinetics , and pharmacodynamics of lucitanib in advanced solid tumors . 
brovkina oi , shigapova l , chudakova da , et al : the ethnic - specic spectrum of germline nucleotide variants in dna damage response and repair genes in hereditary breast and ovarian cancer patients of tatar descent . 
popova t , mani e e , boeva v , et al : ovarian cancers harboring inactivating mutations in cdk12 display a distinct genomic instability pattern characterized by large tandem duplications . 
abida w , bryce ah , vogelzang nj , et al : preliminary results from triton2 : a phase ii study of rucaparib in patients ( pts ) with metastatic castration - resistant prostate cancer ( mcrpc ) associated with homologous recombination repair ( hrr ) gene alterations . 
clarke n , wiechno p , alekseev b , et al : olaparib combined with abiraterone in patients with metastatic castration - resistant prostate cancer : a randomised , double - blind , placebo - controlled , phase 2 trial . 
 cgp of primary and metastatic prostate tumors appendix methods approval for this study , including a waiver of informed consent and health insurance portability and accountability act waiver of authorization , was obtained from the western institutional review board ( protocol 20152817 )  . 
comprehensive genomic proling ( cgp ) results were reported clinically by prospective sequencing of tissue samples from 3 , 476 unique patients with prostate cancer ( august 2014 to february 2018 ) using a validated hybrid capture - based cgp assay ( foundationone [ baitset version t7 was used during this period ] ) 16 in a clinical laboratory improvement amendmentscertied , college of american pathologistsaccredited , new york stateapproved laboratory ( foundation medicine , cambridge , ma )  . 
for patients with multiple submitted samples , only a single sample was included , and the sample with the highest sequencing quality metrics was included . age and site of specimen collection were abstracted from the accompanying pathology reports , clinical notes , and requisition forms submitted by the treating physician . 
specimens included 1 , 660 primary site tumors and 1 , 816 metastatic site tumors ( appendix fig a1 )  . the pathologic diagnosis of each case was conrmed on routine hematoxylin and eosinstained slides , and all samples forwarded for dna extraction contained a minimum of 20% tumor nuclei . 
cgp was performed on hybridization - captured , adaptor ligation - based libraries to a median coverage depth of 743 for 395 cancer - related genes plus select introns from 31 genes frequently rearranged in cancer ( appendix table a1 )  . 
for ets fusions , targeted regions were tmprss2 ( introns 1 to 3 ) , erg ( all exons ) , etv1 ( introns 3 to 4 ) , etv4 ( intron 8 ) , and etv5 ( introns 6 to 7 )  . insertions / deresults were analyzed for base substitutions , short letions , rearrangements , and copy number alterations ( amplication and homozygous deletion )  . 
genomic alterations ( gas ) were called as reportable if the specic variant was present in the catalog of somatic mutations in cancer database ( forbes et al : nucleic acids res 45 : d777 - d783 , 2017 ) , if the variant has been characterized as pathogenic , or if the variant had likely functional status ( disruptive alterations in tumor suppressor genes ) ; all other variants were classied as variants of unknown signicance . to compare relative enrichments in primary site and metastatic site tumors , genes altered at a 2% or greater frequency were assessed for enrichment . 
enrichment was dened as the difference in frequency of gene alteration between metastatic site and primary site samples . to determine microsatellite instability status , 114 intronic homopolymer repeat loci on the foundationone panel were analyzed for length variability and compiled into an overall microsatellite instability score through principal components analysis ( chalmers et al : genome med 9 : 34 , 2017 )  . 
tumor mutational burden was calculated as the number of somatic base substitutions or insertions / deletions per megabase of the coding region target territory of the test ( 1.1 mb ) after ltering to remove known somatic and deleterious mutations and extrapolating that value to the exome or genome as a whole ( chalmers et al : genome med 9 : 34 , 2017 )  . 
tumor mutational burden was categorized as low ( fewer than six mutations / mb ) , intermediate ( six to 20 mutations / mb ) , or high ( 20 mutations / mb or more ; chalmers et al : genome med 9 : 34 , 2017 )  . 
germline , somatic , and zygosity statuses for mutations were determined without matched normal tissue as previously described17 ; in validation testing of 480 germline / somatic calls from tumor - only sequencing with matched normal reference samples , accuracy was 95% for somatic calls and 99% for germline calls . 
biallelic inactivation was dened as mutations under loss of heterozygosity ( loh ) as determined by zygosity status.17 percent genome - wide loh ( gloh ) was used as a marker of homologous recombination deciency and calculated as described , and a gloh score of 14% or greater was dened as gloh - high.21 potentially targetable gas were dened as those that have been associated with response to targeted therapy in prostate cancer , homologous recombination repair gas that have been associated with responses to poly ( adp - ribose ) polymerase inhibitors , or gas associated with response to targeted therapy in multiple other tumor types and were ranked according to modied european society for medical oncology scale for clinical actionability of molecular targets criteria.18 ln ( unk ) ln ( d ) ln ( l ) liver bone lung other prostate ( n = 1 , 660 ) metastasis ( n = 1 , 816 ) neuroendocrine ( 4% ) undifferentiated ( 3% ) acinar ( 93% ) fig a1 . 
for fusion , exons ( e ) are annotated with the last exon included in the 5 ( cid : 3 ) partner and the rst exon included in the 3 ( cid : 3 ) partner . 
in addition , braf rearrangements at intron 9 ( n = 6 ) and intron 7 ( n = 1 ) and raf1 rearrangement at intron 7 with no clear fusion partner were identied ( data not shown )  . 
 ( a ) genome - wide loss of heterozygosity ( gloh ) score was assessable for 2 , 624 cases , and cases with 14% or more gloh were designated loh - high ( loh - h )  . 
loh - h cases were assessed for the presence ( green ) or absence ( yellow ) of a genomic alteration ( ga ) in the homologous recombination repair ( hrr ) or fanconi anemia / interstrand crosslink repair ( fa / icl ) pathway . 
 ( b ) genes were grouped together into hrr pathway ( excluding non - brca1 / 2 ) or fa / icl pathway ( fanc ) ; genes were categorized as in figure 2a . 
 ( a ) microsatellite instability ( msi ) status was assessable for 3 , 326 cases , and each case was designated as msi - high ( msi - h ) , msi - low ( msi - l ) , or microsatellite stable ( mss )  . 
the percent tmb - high ( tmb - h ) or tmb of 10 or more mutations ( mut ) / mb cases in primary site and metastatic site tumors is indicated . 
 m is there room for personalized medicine in small - cell lung cancer ( sclc ) ? remarkable activity of pazopanib in refractory fgfr1 - amplied ed - sclc alessandro russo , md1 , 2 ; david arias ron , md3 ; marika rasschaert , md3 ; hans prenen , md , phd3 ; ranee mehra , md1 ; katherine scilla , md1 ; patrick pauwels , phd3 ; and christian rolfo , md , phd , mba , drhc1 , 3 introduction small - cell lung cancer ( sclc ) is a malignant neuroendocrine tumor that represents almost 15% of total lung cancers , characterized by distinct clinicopathologic , biologic , and therapeutic features.1 in contrast to the impressive progress made in non - sclc ( nsclc ) , 2 , 3 treatment of sclc remained relatively unchanged for more than 30 years . 
only recently the advent of immune checkpoint inhibitors introduced novel options in the therapeutic armamentarium of sclc.4 - 7 despite accelerated approval of these agents , several relevant questions remain unanswered . 
unfortunately , for the vast majority of patients survival rates are still quite disappointing . during the last several years , different studies have shed light on the genetic background of sclc , and a new model of sclc subtypes has been proposed.8 sclcs have a mean mutation rate of 7.4 nonsynonymous mutations per million base pairs , and alterations in tumor - suppressor genes are the most frequent ndings , 9 with mutations in rb1 ( 91% ) and tp53 ( 98% ) being nearly ubiquitous.10 alterations in the notch family genes are detected in 25% of sclcs10 and have a crucial role in sclc tumorigenesis , disease progression , and chemoresistance.11 recently , delta - like protein 3 ( dll3 ) , a notch inhibitory ligand , has emerged as a potential target in sclc , and the dll3 - targeted antibody - drug conjugate rovalpituzumab tesirine is under active clinical development.11 unfortunately , most of the genome alterations found in sclc are not easily therapeutically tractable . interestingly , broblast growth factor receptor 1 ( fgfr1 ) amplications have been reported in a small subgroup of patients ( 5%7% ) 10 , 12 - 14 and might represent a potentially exploitable therapeutic target . preclinical data have shown in vitro and in vivo activity of the fgfr inhibitor pd173074 on sclc growth.15 multiple fgfr inhibitors have been developed , and different nonselective agents have been evaluated in unselected patients with sclc with relatively modest activity.16 , 17 to date , the role of these agents has not been evaluated in fgfr - amplied sclc . the development of new diagnostic methods , cluding liquid biopsy and next - generation sequencing ( ngs ) , offers novel opportunities to identify potentially exploitable genetic alterations in this tumor type . 
to our knowledge , we present for the rst time a case of a heavily pretreated patient with advanced sclc with an fgfr1 amplication who experienced a durable and remarkable response to third - line pazopanib . informed consent for use of the radiology images was obtained by the patient presented . case report a 49 - year - old woman without relevant medical antecedents was diagnosed in august 2015 with ct3 n3 m0 sclc . 
at that time , she presented with nodules in the same lung lobe ( stage iiib according to tnm , 8th edition ) , and with symptomatic superior vena cava ( svc ) syndrome , including dyspnea , cyanosis , fatigue , and weight loss . 
at that time , additional local treatment with radiation was not possible , and there was a decline in clinical condition ( eastern cooperative oncology group performance status 2 )  . treatment with carboplatin - etoposide was initiated but did not result in a clinical response after two cycles , as the computed tomography ( ct ) scan in october 2016 showed additional progression of disease . 
hence , she started second - line topotecan , but after 10 weeks new lung progression was found in imaging studies . in january 2017 , the patient was referred to the phase iearly clinical trials unit of the university of antwerp . a liquid biopsy with a 73 - gene ngs plasma test ( guardant360 ; guardanthealth , redwood city , ca ) was performed to determine the existence of somatic alterations and targetable mutations . 
on the basis of these results and the absence of clinically approved drugs at that time , off - label pazopanib 800 mg / d was started on february 20 , 2017 after notication of the local ethical committee . pazopanib is a tyrosine kinase inhibitor with highly selective activity against vascular endothelial growth factor receptors ( vegfr - 1 , - 2 , and - 3 ; ic50 , 10 - 47 nm ) , platelet - derived growth factor receptors ( pdgfrand - ; ic50 , 84 nm ) , c - kit ( ic50 , 74 nm ) , and fgfr - 1 , - 2 , and - 319 ( ic50 , 140 nm ) , and it is currently approved for the treatment of metastatic renal cell cancer and soft - tissue sarcoma . after 2 months of treatment , in april 2017 a ct scan evaluation showed a partial response according to recist to perform real - time monitoring of the disease , in may a second liquid biopsy was carried out , demonstrating a molecular response to the treatment with a spectacular decrease of molecular tumor burden , with fgfr1 amplication undetectable in plasma cfdna ( fig 2 ) , as well as myc and raf1 amplications . in june 2017 , the patient developed dyspnea and chest pain , with right - sided crackles on auscultation . 
a chest x - ray showed a possible growth in the right upper quadrant with right - sided bronchus obliteration and lateral expansion of lung mass , in addition to enlargement of a right apical nodule . 
cdbca - vp16 , carboplatin - etoposide ; cddpvp16 , cisplatin - etoposide ; cfdna , cell - free dna ; ecog ps2 , eastern cooperative oncology group performance status 2 ; ld - sclc , limited disease smallcell lung cancer ; nd , not detectable ; ngs , next - generation sequencing ; pd , progressive disease ; rt , radiotherapy ; svcs , superior vena cava syndrome . nonetheless , we performed a uorodeoxyglucosepositron emission tomography ( fdg - pet ) scan in july 2017 for a better interpretation of previous results and noted a consolidation in the right hemithorax compatible with a necrotic process and not with tumor progression ( fig 3a ) , further conrming the decision of maintaining treatment with pazopanib at the same dose . in september 2017 , a new liquid biopsy analysis was repeated , showing an increase of amount of cfdna , with a total of eight somatic alterations detected and reappearance of fgfr1 amplication . 
an fdg - pet scan ( fig 3b ) and a ct scan documented progression of disease in both the thorax and abdomen , with enlargement of the primary tumor , mediastinal adenopathy , mesenteric lesions , and a left adrenal gland nodule . 
 ( a ) fdgpet scan in july 2017 shows an increase in size of the consolidation process in the right thorax half , with current conuent intense ring - shaped tracer stacking ( presumably component central necrosis / cysteous transformation )  . 
 ( b ) fdg - pet scan in february 2018 documenting progression of disease in both the thorax and the abdomen . inhibitor either in clinical trial or as compassionate use . unfortunately , treatment was poorly tolerated , and in april 2018 radiotherapy to the mediastinum was performed because of re - emergence of svc syndrome . 
the patient ultimately died in july 2018 as a result of clinical deterioration and further disease progression . discussion the fgfr family comprises four transmembrane tyrosine kinase members ( fgfr 1 - 4 ) and a fth member without tyrosine kinase domain ( fgfr - 5 ) that has been recently identied . 
the main pathways include ras / mapk , dagpkc , and pi3k / akt / mtor , which result in nuclear activation of genomic compounds related to cell growth , differentiation , proliferation , motility , angiogenesis , and apoptosis20 ( appendix fig a1 )  . in clinical practice , interest in fgfr as a potential targetable pathway in several tumor types is based on evidence of uncontrolled oncogenesis , tumor progression , and development of resistance as a result of aberrant fgfr - fgf signaling axis . 
genetic aberrations of fgfr members have been reported in multiple solid tumors.21 - 23 interestingly , fgfr1 amplications ( 8q12 locus ) have been described in different cancers , 24 , 25 including 5% - 7% of sclc , 10 , 12 - 14 and fgfr blockade has demonstrated preclinical in vivo and in vitro activity in sclc , 15 with increased sensitivity in cell lines with higher copies of fgfr1.14 moreover , fgfr1 amplication has been correlated with poorer outcomes after platinum - etoposide chemotherapy.26 these data suggest that fgfr1 could represent a valuable target for sclc . 
multiple selective and nonselective fgfr inhibitors have been developed to date ( appendix fig a1 ) , and in april 2019 the us food and drug administration granted accelerated approval of erdatinib for the treatment of bladder cancer with fgfr3 or fgfr2 alterations . 
 remarkable activity of pazopanib in fgfr - amplied sclc reported a sustained and remarkable response to pazopanib in a heavily pretreated patient with ed - sclc relines and with svc fractory to two previous treatment syndrome . 
interestingly , plasma ngs results predicted radiographic disease progression a few months in advance , further conrming the utility of serial liquid biopsies in oncogene - driven tumors treated with targeted inhibitors . these results are quite impressive and suggest that an appropriate selection strategy might improve the outcome of patients with ed - sclc , who have been traditionally considered to have low responses to targeted therapy strategies . 
ctcs are prognostic factors for survival in both limited29 and ed - sclc , 30 , 31 and their decline during treatment has been correlated with response to chemotherapy31 , 32 and also pazopanib.33 some studies have also shown the utility of molecular analysis of ctcs to depict the genomic proles and evolutionary history of sclc.34 therapeutic decision - making process and might pave the way for the development of targeted therapies through rationally designed clinical trials . 
furthermore , liquid biopsy can also allow real - time monitoring of patients treated with targeted therapies , predicting disease progression in advance compared with conventional radiographic methods , 35 , 36 as reported in our case . 
no data were reported in fgfr - amplied tumors . emergence of cdk6 amplication and raf1 amplication at progression suggests the involvement of these abnormalities in the development of resistance to pazopanib . interestingly , activation of erk1 / 2 has been associated with lines , 37 pazopanib resistance in synovial sarcoma cell suggesting that activation of alternative downstream signaling pathways such as ras / raf / erk / mek might represent an escape strategy to fgfr blockage in sclc . 
furthermore , multiple cdk4 / 6 inhibitors are under active evaluation in sclc in combination with chemotherapy , and alterations of cell cycle regulation genes can represent a potential target . in the present case , the use of a commercially available 73 - gene plasma ngs in our case demonstrates the utility of this diagnostic tool also in patients with sclc to select the best therapeutic strategy and might provide a useful minimally invasive source for tumor genotyping in a disease that is classically associated with small histologic tumor samples . 
the biomolecular information derived by these tests can guide the in conclusion , we reported here for the rst time a dramatic and sustained response to a nonselective fgfr inhibitor in a refractory , heavily pretreated ed - sclc harboring an fgfr1 amplication through a plasma ngs test . 
fgfr1 amplication can represent a novel therapeutic target in sclc , and clinical evaluation of novel selective fgfr inhibitors in this selected population is awaited . affiliations 1university of maryland medical center greenebaum comprehensive cancer center , baltimore , md 2a.o. 
 russo et al ranee mehra stock and other ownership interests : glaxosmithkline ( i ) consulting or advisory role : genentech , bayer research funding : astra zeneca patrick pauwels consulting or advisory role : astra zeneca , boehringer ingelheim , bristolmyers squibb belgium ( inst ) , msd oncology ( inst ) , roche molecular diagnostics , pzer ( inst ) , bayer ( inst ) research funding : pzer ( inst ) , roche molecular diagnostics ( inst ) christian rolfo consulting or advisory role : mylan speakers bureau : novartis , msd , guardanthealth travel , accommodations , expenses : ialsc , astrazeneca research funding : novartis , sano , grant from antwerp university hospital , belgium no other potential conicts of interest were reported . acknowledgment we thank amelie lyssens for her assistance in the material supply . references gazdar af , bunn pa , minna jd : small - cell lung cancer : what we know , what we need to know and the path forward . 
nat rev cancer 17 : 725 - 737 , 2017 [ erratum : nat rev cancer 17 : 765 , 2017 ] corrales l , scilla k , caglevic c , et al : immunotherapy in lung cancer : a new age in cancer treatment , in naing a and hajjar j ( eds ) : immunotherapy . 
cham , switzerland , springer international publishing , 2018 , pp 65 - 95 russo a , franchina t , ricciardi grr , et al : a decade of egfr inhibition in egfr - mutated non small cell lung cancer ( nsclc ) : old successes and future perspectives . 
n engl j med 379 : 2220 - 2229 , 2018 antonia sj , l opez - martin ja , bendell j , et al : nivolumab alone and nivolumab plus ipilimumab in recurrent small - cell lung cancer ( checkmate 032 ) : a multicentre , open - label , phase 1 / 2 trial . 
cancer cell 33 : 853 - 861.e4 , 2018 [ erratum : cancer cell 35 : 329 , 2019 ] chung hc , lopez - martin ja , kao sc - h , et al : phase 2 study of pembrolizumab in advanced small - cell lung cancer ( sclc ) : keynote - 158 . 
j clin oncol 36 , 2018 ( suppl 15 ; abstr 8506 ) rudin cm , poirier jt , byers la , et al : molecular subtypes of small cell lung cancer : a synthesis of human and mouse model data . 
nat rev cancer 19 : 289 - 297 , 2019 [ erratum : nat rev cancer 19 : 415 , 2019 ] sabari jk , lok bh , laird jh , et al : unravelling the biology of sclc : implications for therapy . 
schultheis am , bos m , schmitz k , et al : fibroblast growth factor receptor 1 ( fgfr1 ) amplication is a potential therapeutic target in small - cell lung cancer . 
pardo oe , latigo j , jeffery re , et al : the broblast growth factor receptor inhibitor pd173074 blocks small cell lung cancer growth in vitro and in vivo . 
koinis f , agelaki s , karavassilis v , et al : second - line pazopanib in patients with relapsed and refractory small - cell lung cancer : a multicentre phase ii study of the hellenic oncology research group . 
nat rev cancer 17 : 318 - 332 , 2017 jiang t , gao g , fan g , et al : fgfr1 amplication in lung squamous cell carcinoma : a systematic review with meta - analysis . 
park js , lee j - s , kim ey , et al : the frequency and impact of fgfr1 amplication on clinical outcomes in korean patients with small cell lung cancer . 
sun j - m , lee kh , kim b - s , et al : pazopanib maintenance after rst - line etoposide and platinum chemotherapy in patients with extensive disease small - cell lung cancer : a multicentre , randomised , placebo - controlled phase ii study ( kcsg - lu12 - 07 )  . 
rolfo c , mack pc , scagliotti gv , et al : liquid biopsy for advanced non - small cell lung cancer ( nsclc ) : a statement paper from the iaslc . 
tay ry , fern andez - guti errez f , foy v , et al : prognostic value of circulating tumour cells in limited - stage small - cell lung cancer : analysis of the concurrent oncedaily versus twice - daily radiotherapy ( convert ) randomised controlled trial . 
hou j - m , krebs mg , lancashire l , et al : clinical signicance and molecular characteristics of circulating tumor cells and circulating tumor microemboli in patients with small - cell lung cancer . 
hou j - m , greystoke a , lancashire l , et al : evaluation of circulating tumor cells and serological cell death biomarkers in small cell lung cancer patients undergoing chemotherapy . 
messaritakis i , politaki e , plataki m , et al : heterogeneity of circulating tumor cells ( ctcs ) in patients with recurrent small cell lung cancer ( sclc ) treated with 34 . 
su z , wang z , ni x , et al : inferring the evolution and progression of small - cell lung cancer by single - cell sequencing of circulating tumor cells . 
oxnard gr , paweletz cp , kuang y , et al : noninvasive detection of response and resistance in egfr - mutant lung cancer using quantitative next - generation genotyping of cell - free plasma dna . 
clin cancer res 20 : 1698 - 1705 , 2014 iwama e , sakai k , azuma k , et al : monitoring of somatic mutations in circulating cell - free dna by digital pcr and next - generation sequencing during afatinib treatment in patients with lung adenocarcinoma positive for egfr activating mutations . 
each one of the four members of the fgfr family presents an intracellular region , which involves a juxtamembrane domain that acts as a binding side for phosphotyrosinebinding domains , a transmembrane domain that allows ligand - dependent activation as a result of the stabilization of receptor conformation , and an extracellular ligand binding domain including three immunoglobulin domains that act as binding points for fgfr ligands ( olsen et al : proc natl acad sci usa 101 : 935 - 940 , 2004 )  . 
the fgf family is composed of almost 22 ligands that have been identied in humans , and each one is recognized by a specic fgfr member ( beenken et al : nat rev drug discov 8 : 235 - 253 , 2009 )  . 
although from their analysis , her2 exon 20 mutations emerge as the most clearly actionable driver in nsclc , the authors themselves pinpoint ambiguous functional roles of individual her2 aberrations ( ie , activating mutations , amplication , and overexpression ) , as well as their reciprocal associations . therefore , her2 biology may be different in lung cancer compared with breast or gastric cancers , possibly because of specic microenvironmental and tumor - host interactions . as pointed out by the authors , a substantial heterogeneity exists in therapeutic approaches and clinical results of her2 - targeting strategies in this context . this is well exemplied by two clinical nsclc cases harboring different her2 alterations ( her2 mutation and all three her2 aberrationsmutation , amplication , and protein overexpression , respectively ) we discuss herein . case 1 a 44 - year - old woman , never - smoker , presented with stage ivb lung adenocarcinoma ( egfr - wt , alk and ros1 nonrearranged , pd - l1 1% )  . 
she was then treated with two courses of atezolizumab , resulting in clinico - radiologic hyperprogression.2 next - generation sequencing ( ngs ) based molecular proling revealed a her2 exon 20 insertion ( p.a775_ g776insyvma ) ; neither gene amplication nor protein expression was detected . 
she then received whole - brain radiotherapy ( rt ) for an isolated brain progression and was subsequently enrolled in a clinical trial of poziotinib ; treatment was poorly tolerated , and diffuse extracranial progression rapidly ensued . 
rechallenge with trastuzumab and vinorelbine obtained no clinical benet , and she went on to receive gemcitabine single agent and subsequently best supportive care on further clinicoradiologic disease progression . case 2 a 48 - year - old woman , never - smoker , presented with stage iib lung adenocarcinoma in 2011 , when she underwent left inferior lobectomy and medilymphadenectomy followed by adjuvant astinal cisplatin / vinorelbine . 
in 2018 , disease recurred in multiple metastatic sites ; right supraclavicular lymph node rebiopsy showed egfr - wt , alk and ros1 nonrearranged , and pd - l1 1% , and she received rstline platinum - based chemotherapy with partial response , followed by stereotactic body rt on residual tumor sites ; multisite disease progression was observed 2 months after treatment completion . 
she was then started on trastuzumab emtansine ( tdm - 1 ) in march 2020 , experiencing dramatic clinical benet with disappearance of all disease - related symptoms , in particular dyspnea , and performance status improvement ( from 2 to 0 , according to eastern cooperative oncology group ) ; treatment is currently ongoing . the two cases presented highlight the inherent complexity of her2 biology in nsclc and the difculties in reliably predicting the clinical outcome of specic her2 - targeted interventions . 
in case 1 , a clinically meaningful response to trastuzumab - based treatment was observed , in line with other reports , 3 - 5 even in the presence of an isolated exon 20 mutation not accompanied by gene amplication and in the absence of protein expression by immunohistochemistry . 
intrigued by the relatively unexpected response , occurring after hyperprogression to pd - l1 blockade , we analyzed fcriiia polymorphisms and found that the patient carried a valine / valine ( v / v ) variant , which has been correlated to better response to trastuzumab - based treatment in breast cancer.6 in line with the presence of a polymorphism potentially fostering antibody - dependent cell - mediated cythe patients peripheral blood totoxicity ( adcc ) , mononuclear cells displayed a marked and specic trastuzumab - dependent cytotoxic activity toward her2 - overexpressing targets in vitro ( fig 1 )  . 
on the other hand , such a mechanism did not prove relevant in case 2 , in which no response to trastuzumab - based treatment was observed , despite gene amplication and protein overexpression accompanying exon 20 insertion . 
 ( a ) computed tomographic imaging of primary lesion at baseline ( showing atezolizumab progression , left ) and at rst assessment during trastuzumab - based therapy ( right )  . 
 ( b ) mcf7 target cells , expressing ( mcf7 - her2 ) or not expressing ( mcf7 - evector ) her2 antigen , were rst stained with carboxyuorescein succinimidyl ester ( cfse ) 5 mm and celltrace 5 mm , respectively . 
mixed targets ( ratio 1 : 1 ) were incubated for 18 hours at different ratios ( 2 : 1 , 4 : 1 , 8 : 1 , 12 : 1 ) with peripheral blood mononuclear cells ( pbmcs ) isolated from peripheral blood of the patient in the presence of rituximab ( 10 mg / ml ) or trastuzumab ( 10 mg / ml )  . 
tumor cellspecic lysis by pbmcs was analyzed by ow cytometry ( representative plot of 8 : 1 ratio on the left ) and quantied as percentage of lysis ( right ) using the formula : ( 1 ( % mcf7 - her2 or mcf7 - evector + trastuzumab / tot target ) / ( %mcf7 - her2 or mcf7 - evector + rituximab / tot target ) 100 )  . 
fitc , uorescein isothiocyanate ; v450 - ct , v450 cell trace . overall , her2 aberrations in nsclc convey important prognostic10 and potentially predictive information ; however , prospective studies should be designed not only according to patients stratication for specic her2 aberration ( s ) or combinations but also it is mandatory to evaluate the patients immunologic features , especially for antibody - based treatments . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . zhao j , xia y : targeting her2 alterations in nonsmall - cell lung cancer : a comprehensive review . 
jco precis oncol 4 : 411 - 425 , 2020 kato s , goodman a , walavalkar v , et al : hyperprogressors after immunotherapy : analysis of genomic alterations associated with accelerated growth rate . 
clin cancer res 23 : 4242 - 4250 , 2017 chuang jc , stehr h , liang y , et al : erbb2 - mutated metastatic non - small cell lung cancer : response and resistance to targeted therapies . 
mazi `eres j , barlesi f , filleron t , et al : lung cancer patients with her2 mutations treated with chemotherapy and her2 - targeted drugs : results from the european euher2 cohort . 
mazi `eres j , peters s , lepage b , et al : lung cancer that harbors an her2 mutation : epidemiologic characteristics and therapeutic perspectives . j clin oncol 31 : 1997 - 2003 , 2013 6 . 
musolino a , naldi n , bortesi b , et al : immunoglobulin g fragment c receptor polymorphisms and clinical efcacy of trastuzumab - based therapy in patients with her - 2 / neu - positive metastatic breast cancer . 
j clin oncol 26 : 1789 - 1796 , 2008 li b , ofn m , hembrough t , et al : p1.13 - 43 molecular and imaging predictors of response to ado - trastuzumab emtansine in patients with her2 mutant lung cancers : an exploratory phase 2 trial . 
j thorac oncol 13 : s599 , 2018 li bt , shen r , buonocore d , et al : ado - trastuzumab emtansine for patients with her2 - mutant lung cancers : results from a phase ii basket trial . 
j clin oncol 36 : 2532 - 2537 , 2018 peters s , stahel r , bubendorf l , et al : trastuzumab emtansine ( t - dm1 ) in patients with previously treated her2 - overexpressing metastatic non - small cell lung cancer : efcacy , safety , and biomarkers . 
milella m , nuzzo c , bria e , et al : egfr molecular proling in advanced nsclc : a prospective phase ii study in molecularly / clinically selected patients pretreated with chemotherapy . 
 cd33_pgx6_score predicts gemtuzumab ozogamicin response in childhood acute myeloid leukemia : a report from the childrens oncology group lata chauhan , phd1 ; miyoung shin , phd1 ; yi - cheng wang , ms2 ; michael loken , phd3 ; jessica pollard , md4 , 5 ; richard aplenc , md , msce6 ; betsy a . 
lamba , phd1 purpose the us food and drug administration recently announced reapproval of gemtuzumab ozogamicin ( go ) for treatment of cd33 - positive acute myeloid leukemia ( aml ) , thus opening up opportunities to develop strategies for effective use of go . 
we further developed a composite cd33 pharmacogenetics ( pgx ) score using six cd33 snps ( cd33_pgx6_score ) for association with clinical outcome . results four cd33 snps were associated with cell surface cd33 levels and clinical response in the go versus no - go arms . 
 chauhan et al context key objective the key objective of this study was to identify and establish the clinical impact of cd33 genetics on treatment response to gemtuzumab , a recently approved cd33 - directed immunotherapeutic agent in patients with acute myeloid leukemia . knowledge generated our results identied multiple cd33 single nucleotide polymorphisms ( snps ) of prognostic signicance that were associated with gemtuzumab clinical response . 
patients with a cd33_pgx6_score of 0 or higher had higher cd33 expression levels compared with patients with a score of less than 0 and demonstrated improved disease - free survival and reduced risk of relapse when given gemtuzumab . 
no improvement from gemtuzumab was observed in patients with a cd33_pgx6_ score of less than 0 . relevance our results hold promise for developing strategies to personalize the use of cd33 - directed agents such as gemtuzumab using cd33 genetics . 
in addition , cd33 expression levels have been correlated with disease characteristics such as presence of flt3 internal tandem duplications ( itds ) , npm1 mutations , and high - risk group features.20 , 21 recently , we reported a splicing single nucleotide polymorphism ( snp ) in cd33 rs12459419 ( c.t ) , resulting in a shorter isoform of cd33 that lacks exon 2 ( cd33 - d2 ) .22 lack of exon 2 results in loss of the igv domain within the cd33 protethis is clinically relevant , because the igv domain is recognized by go and other cd33 antibodies used in clinical immunophenotyping of aml specimens . 
our results showed signicant association of rs12459419 with diagnostic leukemic cell surface cd33 intensity ( determined using igv targeting p67.6 antibody ) , as well as differential response in go versus no - go treatment arms.22 specically , patients with at least one copy of the variant t allele ( ct / tt genotypes ) derived no benet from addition of go . 
in contrast , patients with homozygous cc genotype showed signicantly better survival ( event - free survival [ efs ] and disease - free survival [ dfs ] ) as well as lower risk of relapse with the addition of go to standard chemotherapy.22 in contrast to these results , an investigation in the united kingdom mrc adult aml cohorts showed consistent association of the splicing snp with cd33 cell surface intensity but no association with clinical outcome.23 this was most likely a result of the inclusion of multiple go random assignments and varying go doses in these studies23 or the presence of other cd33 snps . 
these observations and an anticipated increase in the use of go as a result of its recent approval prompted us to evaluate the prognostic role of other snps within cd33 in the context of the go - based randomized cog aaml0531 trial . 
details of the study design , treatment regimen , and clinical outcome have been reported previously.17 overall , 1 , 022 patients with de novo aml ( age , 0 to 29 years ) were randomly assigned to either standard ve - course chemotherapy ( no - go arm ; n = 511 ) or the same chemotherapy plus two doses of go 3 mg / m2 ( go arm ; n = 511 ) during induction 1 and intensication 2 . as described previously , low - risk features included t ( 8 ; 21 ) , inv ( 16 ) , or t ( 16 ; 16 ) ; high - risk features included monosomy 7 , monosomy 5 / 5q deletion , or persistent disease at end of induction 1 . 
patients not classied as low or high risk were categorized as intermediate risk and received hematopoietic stem - cell transplantation if suitable donor was available.17 , 21 specimens from patients who consented for biology studies were used in this study . 
the institutional review boards of all participating institutions approved the clinical protocol , and the cog myeloid disease biology committee approved this research study . genotyping of cd33 snps on the basis of our previous ndings , 24 , 25 we selected cd33 snps with potential functional or clinical relevance that occurred with the minimum allele frequency of greater than 0.10 in either white or black patients . 
genotype data on rs12459419 ( ala14 val ) splicing were already available in this cohort.22 all cd33 snps had a call rate of greater than 0.98 , and all but rs1803254 were in accordance with the hardy - weinberg equilibriuin addition , we genotyped a novel 4base pair ( bp ) ccgg deletion variation ( rs201074739 ) that creates a premature termination codon and thus loss of cd33 using a polymerase chain reactionrestriction fragment length polymorphismbased recent data suggest method ( data supplement )  . 
for cd33 snps rs12459419 , rs1803254 , rs35112940 , rs6136475 , and rs201074739 , with variant alleles associated with lower leukemic cell surface cd33 intensity and inferior response by treatment arms , the genotype score was 0 for wild - type ( wt ) / wt genotype , 1 for wt / variant genotype , and 2 for variant / variant genotype ( for rs201074739 , none of the patients were the variant allele )  . 
for cd33 snp homozygous for rs2455069 , with variant allele associated with high cd33 cell surface intensity and superior response by treatment arms , the genotype score was 0 for wt / wt genotype , 1 for wt / variant genotype , and 2 for variant / variant genotype . composite cd33_pgx6_score = ( individual genotype scores of six snps [ rs12459419 , rs1803254 , rs35112940 , rs201074739 , rs61736475 , and rs2455069 ] )  . 
 chauhan et al statistical analyses genotype and clinical data were available for 938 patients . clinical outcome data for patients enrolled in cog aaml0531 were current as of september 30 , 2014 . 
the median follow - up time for eligible patients with de novo aml who were alive at last contact and included in our analysis was 1 , 856 days ( range , 4 to 2 , 829 days )  . 
the kaplan - meier method was used to estimate overall survival ( os ; dened as time from study entry to death ) , efs ( dened as time from study entry until failure to achieve cr during induction , relapse , or death ) , and dfs ( dened as time from end of course 1 for patients in cr until relapse or death ) .28 relapse risk ( rr ) was calculated by cumulative incidence methods dened as time from the end of induction 1 for patients in cr to relapse or death , where deaths without a relapse were considered competing events . 
cox proportional hazards models were used to estimate the hazard ratios ( hrs ) for os and efs.29 the 2 test was used to test the signicance of observed differences in proportions , and fishers exact test was used when data were sparse . 
in contrast , for missense snp rs2455069 , the presence of the variant g allele was associated with higher cd33 levels ( p , .001 ; table 1 and data supplement )  . 
no difference in distribution within arms , in risk groups , or by flt3 - itd status was observed ( data supplement ) for these snps , indicating the effect of these snps on cd33 cell surface intensity might not be associated with risk group characteristics . 
rs201074739 and rs61736475 occurred with a low frequency , and thus analysis within each group was limited by small sample size within genotype groups . in a subset of patients ( n = 496 ) , we had mrna levels of total cd33 available from the rna sequencing data . 
as shown in the data supplement , variant alleles for 3 ( cid : 1 ) utr rs1803254 ( p = .010 ) and the rs201074739 - ccgg deletion that results in a premature termination codon ( p = .003 ) were associated with low cd33 mrna expression . association of cd33 snps with clinical outcome by treatment arm ( no - go v go ) survival estimates and rr indicated differences in treatment arm by cd33 snps ( table 2 )  . 
although limited by numbers , we observed that presence of variant alleles for rs1803254 ( c allele ) , rs35112940 ( a allele ) , rs201074739 ( ccgg deletion ) , and rs61736475 ( c allele ) within the 12459419 cc genotype resulted in no signicant outcome benet from adding go to treatment ( data supplement )  . 
presence of the wt / reference genotype for these snps in conjunction with the wt / reference cc genotype for rs12459419 resulted in significant improvement in outcome with the addition of go . 
 cd33_pgx and gemtuzumab response in aml across all genotype groups , which was expected given that the variant allele is associated with higher cd33 expression . development of composite cd33_pgx6_score and its association with cd33 expression and benet from go on the basis of the results for individual cd33 snps as well as our previous ndings regarding rs12459419 splicing snp , 22 we developed a composite cd33_pgx6_score for each patient by taking into account the directional genotype scores of six snps ( rs12459419 , rs1803254 , rs2455069 , rs35112940 , rs61736475 , and rs201074739 ) , as described in patients and methods . 
a higher snp score was associated with higher cd33 cell surface expression , and as the score decreased , it was predictive of lower cd33 levels ( p , .001 ; fig 3a )  . 
signicant benet of adding go , compared with the standard no - go arm , was observed only for the score categories of 0 and greater than 1 ( p , .05 ) , and no difference in outcome was observed for the 3 to 4 and 2 to 1 categories . 
given this observation and that a score of 0 indicated reference allele for each genotype , we dichotomized patients into those with a cd33_pgx6_score of 0 or higher and those with scores less than 0 ( presence of least one detrimental allele )  . 
the cd33_pgx6_score was created for each patient using genotype information for the following six single nucleotide polymorphisms ( snps ) : rs12459419 , rs1803254 , rs35112940 , rs2455069 , rs61736475 , and rs201074739 . 
genotype scores are as follows : wild type ( wt ) / wt = 0 , wt / variant ( var ) = 1 , and var / var = 2 for rs12459419 , rs1803254 , rs35112940 , rs61736475 , and rs201074739 and wt / var = 1 and var / var = 2 for rs2455069 . 
association of dichotomized cd33 - pgx6_score ( rs12459419 , rs35112940 , rs1803254 , rs2455069 , rs61736475 , and rs201074739 ) with clinical outcome by go versus no - go arms in all patients and by risk group no - go no . 
association of dichotomized cd33 - pgx6_score ( rs12459419 , rs35112940 , rs1803254 , rs2455069 , rs61736475 , and rs201074739 ) with clinical outcome by go versus no - go arms in all patients and by risk group ( continued ) no - go no . 
success of go has resulted in development of multiple new cd33 - directed therapeutics that are currently at different stages of development and investigation , such as fc - engineered unconjugated antibodies ( bi 836858 [ mab 33.1 ] ) , antibody - drug conjugates ( sgncd33a [ vadastuximab talirine ] , imgn779 , and ave9633 ) , radioimmunoconjugates ( actinium - 225 lintuzumab ) , biand trispecic antibodies ( amg330 , amg673 , amv564 , and 161533 trike fusion protein ) , and chimeric antigen receptormodied immune effector cells.30 thus , timely to dene pharmacogenetic determinants of go response . we recently reported that the cd33 splicing polymorphism rs12459419 c.t regulates production of an alternatively spliced isoform of cd33 lacking exon 2 and , hence , the igv domain of cd33.22 our previous results demonstrated that rs12459419 was a signicant predictor of cell surface cd33 intensities as well as clinical response . 
association of dichotomized cd33_pgx6_score ( rs12459419 , rs35112940 , rs1803254 , rs2455069 , rs61736475 , and rs201074739 ) with clinical outcome by go versus no - go arms in patients with aml patients by race no - go no . 
our results show that the rs1803254 , rs2455069 , rs201074739 , and rs35112940 snps in cd33 are signicantly associated with cell surface cd33 expression as well as outcome by treatment arm ( go v no - go ) within the context of the cog in the 3 ( cid : 1 ) utr aaml0531 trial . 
the novel variant rs201074739 is a 4 - bp deletion in exon 3 that changes the reading frame , creating a premature regulates cd33 transcript termination codon and thus nonfunctional truncated cd33 protethe transcript with premature termination is degraded by the cellular nonsense - mediated decay pathway , 26 consistent with the observed association of this variant with cd33 mrna levels . rs35112940arg304gly and rs61736475 - ser305pro occur close to the itim motif of cd33 and thus might inuence cd33 function . 
our results demonstrate the clinical impact of these snps , warranting additional investigation in independent clinical cohorts as well as in - depth molecular these snps for impact on cd33 characterization of mrna , protein stability , cd33 trafcking and signaling , and efcacy of other cd33 - directed therapies . given that a patient with aml can inherit different genotype combinations for snps ( as shown in cd33 onco - print ; data supplement ) , it is important to evaluate the association of genotype combinations for the most inuential snps in relation to cd33 cell surface levels and go clinical response . 
a cd33_pgx6_score of 0 or higher was associated with signicantly higher cd33 expression , better efs and dfs , and lower rr in the go arm compared with the nogo arin patients with a score of less than 0 , cd33 expression was low , and the addition of go did not provide any clinical benet . 
in addition , as shown in figure 3b , there is overlap in the cd33 expression levels between the scores , indicating that the cd33 expression may not be the sole determinant of response . 
in fact , our previous data show that patients heterozygous ( ct ) for the splicing snp rs12459419 , despite having intermediate levels of cd33 , did not benet from the addition of go , implying co - occurrence of the short isoform with the full - length isoform compromising the efcacy . 
furthermore , as indicated earlier , the presence of the coding or splicing variations can affect cd33 intracellular trafcking , localization , internalization rates of the go - cd33 complex , recycling , and signaling as the mechanisms underlying the observed impact on response . 
pollard et al21 showed that patients classied as low cd33 expressers ( quartile 1 ) on the basis of cd33 cell surface levels do not derive benet from go , whereas patients classied as high cd33 expressers ( quartiles 2 to 4 ) show signicant improvement with go . 
we investigated the interaction of snps within high cd33 expressers ( quartiles 2 to 4 , as per pollard et al21 ) and observed that , despite being in a high cd33 expression category , the presence of a detrimental variant allele for cd33 snp compromises benet from adding go to chemotherapy ( data not shown )  . 
our results show that in black patients with a cd33_pgx6_score of less than 0 , 54.5% of patients have the cc genotype , compared with only 6% of white patients with the cc genotype for the rs12459419 splicing group . 
given that the cd33_pgx6_score considers multiple cd33 snps in addition to the splicing snp , it is more likely to identify patients who are less likely to benet from go or other cd33 - directed therapies across different ethnic groups . 
the novel snp rs201074739 , a 4 - bp ccgg deletion that results in truncated protein , never occurs in patients in the rs12459419 tt genotype group ( data supplement ) but has been observed in patients with the cc and ct genotypes for rs12459419 , indicating its contribution to lower cd33 levels and perhaps response within the rs12459419 cc and ct genotype groups . given the anticipated increase in go use with its recent approval and several new cd33 - directed therapies in the pipeline , development of the cd33_pgx6_score is timely and critical to establish its ability and clinical utility to personalize go treatment in aml . 
our future work is targeted toward validating the cd33 score in other clinical cohorts ( united kingdom trials ) and an upcoming study from cog that plans to incorporate go - containing regithe cd33_pgx6_score holds mens . 
lamba provision of study material or patients : richard aplenc collection and assembly of data : lata chauhan , yi - cheng wang , michael loken , jessica pollard , richard aplenc , betsy a . 
alonzo , soheil meshinchi , jatinder k . lamba manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors michael loken employment : hematologics , hematologics ( i ) leadership : hematologics , hematologics ( i ) stock and other ownership interests : hematologics , hematologics ( i ) consulting or advisory role : newlink genetics richard aplenc honoraria : sigma - tau expert testimony : wiggin and dana travel , accommodations , expenses : sigma - tau irwin d . 
16 / 091 , 720 entitled biomarkers of anti - leukemic therapy no other potential conicts of interest were reported . acknowledgment we thank the childrens oncology group acute myeloid leukemia reference laboratory for providing specimens for the study . 
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leukemia 27 : 843 - 851 , 2013 burnett ak , hills rk , hunter ae , et al : the addition of gemtuzumab ozogamicin to low - dose ara - c improves remission rate but does not signicantly prolong survival in older patients with acute myeloid leukaemia : results from the lrf aml14 and ncri aml16 pick - a - winner comparison . 
burnett ak , hills rk , milligan d , et al : identication of patients with acute myeloblastic leukemia who benet from the addition of gemtuzumab ozogamicin : results of the mrc aml15 trial . 
castaigne s , pautas c , terr e c , et al : effect of gemtuzumab ozogamicin on survival of adult patients with de - novo acute myeloid leukaemia ( alfa - 0701 ) : a randomised , open - label , phase 3 study . 
delaunay j , recher c , pigneux a , et al : addition of gemtuzumab ozogamycin to chemotherapy improves event - free survival but not overall survival of aml patients with intermediate cytogenetics not eligible for allogeneic transplantation : results of the goelams aml . 
hills rk , castaigne s , appelbaum fr , et al : addition of gemtuzumab ozogamicin to induction chemotherapy in adult patients with acute myeloid leukaemia : a meta - analysis of individual patient data from randomised controlled trials . 
cooper tm , franklin j , gerbing rb , et al : aaml03p1 , a pilot study of the safety of gemtuzumab ozogamicin in combination with chemotherapy for newly diagnosed childhood acute myeloid leukemia : a report from the childrens oncology group . 
gamis as , alonzo ta , meshinchi s , et al : gemtuzumab ozogamicin in children and adolescents with de novo acute myeloid leukemia improves event - free survival by reducing relapse risk : results from the randomized phase iii childrens oncology group trial aaml0531 . 
 cd33_pgx and gemtuzumab response in aml jen ey , ko cw , lee je , et al : fda approval : gemtuzumab ozogamicin for the treatment of adults with newly diagnosed cd33 - positive acute myeloid leukemia . clin cancer res 24 : 3242 - 3246 , 2018 19 . 
norsworthy kj , ko cw , lee je , et al : fda approval summary : mylotarg for treatment of patients with relapsed or refractory cd33 - positive acute myeloid leukemia . 
pollard ja , alonzo ta , loken m , et al : correlation of cd33 expression level with disease characteristics and response to gemtuzumab ozogamicin containing chemotherapy in childhood aml . 
pollard ja , loken m , gerbing rb , et al : cd33 expression and its association with gemtuzumab ozogamicin response : results from the randomized phase iii childrens oncology group trial aaml0531 . 
lamba jk , chauhan l , shin m , et al : cd33 splicing polymorphism determines gemtuzumab ozogamicin response in de novo acute myeloid leukemia : report from randomized phase iii childrens oncology group trial aaml0531 . 
mortland l , alonzo ta , walter rb , et al : clinical signicance of cd33 nonsynonymous single - nucleotide polymorphisms in pediatric patients with acute myeloid leukemia treated with gemtuzumab - ozogamicin - containing chemotherapy . 
papageorgiou i , loken mr , brodersen le , et al : ccgg deletion ( rs201074739 ) in cd33 results in premature termination codon and complete loss of cd33 expression : another key variant with potential impact on response to cd33 - directed agents . 
 eleven month progressionfree survival on vemurafenib monotherapy in a patient with recurrent and metastatic braf v600emutated glioblastoma who grade 4 introduction glioblastoma multiforme ( gbm ) accounts for 3% to 4% of all cancer - related deaths , despite having an average incidence rate of 3.19 / 100 , 000 population.1 , 2 prognosis for gbm remains poor , with standard - of - care therapy providing only a 16to 20 - month median overall survival.3 , 4 braf kinase inhibitors have transformed melanoma treatment and have successfully been used in other tumor types containing the braf v600e mutation.5 - 8 there are several reports of improved survival with the use of braf - directed therapy in braf - positive gbm , although the efficacy of braf inhibitors in nonmelanoma cancers has not been systematically explored . 
a basket study of 122 patients with braf v600e mutations in various nonmelanoma cancer types found three out of six patients with gbm experienced tumor regression with vemurafenib.8 a pediatric case series presented three pediatric patients with braf v600e - mutant high - grade gliomas treated with vemurafenib , including a patient with clinical and neurologic improvement 20 months after starting treatment.9 robinson et al10 reported a case of complete clinical regression of a braf v600emutant pediatric gbm treated with braf inhibitor therapy . 
the patient underwent a craniotomy with resection , and pathology demonstrated glioblastoma , who grade 4 , isocitrate dehydrogenase ( idh - 1 ; wild - type immunohistochemistry and by sequencing )  . 
further analysis showed a tert mutation and wildtype for both atrx and egfr . immediate postoperative imaging showed residual enhancement superior to the resection cavity , treated with gamma knife radiosurgery . 
subsequent treatment consisted of fractionated intensitymodulated radiation therapy of 60 gy at 2 gy per fraction with concurrent daily temozolomide 75 mg / m2 , followed by monthly cycles of maintenance temozolomide . 
her treatment was then switched to bevacizumab monotherapy . eleven months postsurgery , or 4 months after switching to bevacizumab monotherapy , she was found to have a right hemispheric focus of duralbased enhancement concerning for local recurrence and underwent repeat gamma knife radiosurgery . 
within 2 months of starting vemurafenib , she was weaned off all opioid medications , dexamethasone , baclofen , and gabapentshe reported a significant improvement in her quality of life and was once again able to work with physical therapy to improve her chronic left - sided weakness . 
five months after starting vemurafenib , her brain and spine mri demonstrated significant improvement in intracranial leptomeningeal disease and in the nodular leptomeningeal enhancement along the conus medullaris and multiple nerve roots of the cauda equina . 
eleven months after starting vemurafenib monotherapy , the patient reported feeling well overall and had achieved stable disease on brain and spine mri ( fig 3 )  . she denied any headaches , visual changes , back pain or neuropathy , or gi or genitourinary symptoms . 
she was rated 80% on the karnofsky performance status scale . discussion this case report is consistent with several others in showing significantly improved survival with the use of braf kinase inhibitors in braf v600e positive gbm . 
no epithelioid or rhabdoid morphology was identified . spine , which demonstrated drop metastases throughout the thoracic and lumbosacral spine , specifically on the posterior cord from t10 - 12 , anterior to the conus medullaris , and throughout the cauda equina . 
an mri of the spine 1 month later showed a significant increase in the number and size of the abnormal cord enhancement lesions . one month after being diagnosed with metastatic recurrent glioblastoma , the patient was referred to our clinic , and we recommended addition of carboplatin area under the curve 4 intravenously monthly to her regimen of bevacizumab 10 mg / kg intravenously every 2 weeks . 
at the time of vemurafenib approval 2 months later and before starting the braf inhibitor , neuraxis imaging demonstrated development of leptomeningeal enhancement around the lobe resection cavity and initial right parietal considerable progression of metastatic spinal cord disease . 
 plasma concentration ratio for dabrafenib was 10 - fold higher compared with vemurafenib.17 in the dabrafenib plus trametinib versus vemurafenib alone in unresectable or metastatic braf v600e / k cutaneous melanoma ( combi - v ) trial , patients with braf - mutated metastatic melanomas who were treated with the combination of dabrafenib and mek inhibitor trametinib had significantly longer pfs than those treated with vemurafenib monotherapy.18 furthermore , dabrafenib appears to be less nephrotoxic than vemurafenib.19 nonmelanoma tumorspecific studies of braf inhibitors are difficult to conduct because of the relative rarity of these tumors and the low frequencies of braf mutations . 
these limitations also extend to basket trials of braf inhibitors , such as the national cancer institutemolecular analysis for therapy choice ( nci - match ; eay131 - h protocol ) evaluating dabrafenib and trametinib in patients with braf v600e or v600k mutations . 
despite these limitations , case reports of exceptional responders to targeted therapies such as ours support the value of genetics - guided therapy for activating braf mutations and driver mutations in general in gbm and other rare cancers , underscoring the need to systematically investigate these targeted drugs . the case reports can also be of value in supporting current off - label therapy of these rare mutations in tumor types not listed in the us food and drug administrationapproved indications . 
this microscopic image shows an immunohistochemical study for braf v600e . the tumor cells and their processes are positive for the mutated form of the protein . although there is a therapeutic potential of braf inhibitors in gbm , the experience with these drugs in melanoma treatment suggests that resistance will surely emerge in tumors responsive to this therapy . several mechanisms mediating resistance to braf inhibitors through mitogen - activated protein kinase ( mapk ) reactivation have been described , and coadministration of braf inhibitors with selective mapk inhibitors has led to significant improvement in progression - free survival in melanoma.15 coadministration with alternative survival pathway inhibitors , such as vascular endothelial growth factor and phosphatidylinositol 3 - kinase inhibitors , may also need to be evaluated.16 it could be further argued that dabrafenib might be the preferred braf kinase inhibitor . 
tran honoraria : novocure , monteris medical consulting or advisory role : novocure research funding : novocure , merck , novartis , tocagen , northwest biotherapeutics , vbl therapeutics , stemline therapeutics patents , royalties , other intellectual property : a molecular cell diary system , chemotherapeutic methods to target differentiated tumor cells , generep and nscore : method and apparatus for improved determination of node influence in a network , core master regulators of glioblastoma stem cells , conversion of glioblastoma multiforme cells into regulatory immune cells , novel targets that prevent cancer cells from evading the immune system and synergize with current immunotherapies travel , accommodations , expenses : novocure , monteris medical , novartis , tocagen , vbl therapeutics ashley p . 
 ( b ) the same regions are denoted by arrows on follow - up imaging performed 8 months later , showing improvement in metastatic disease and near - complete resolution of some lesions . references med 6 : 1359 - 1370 , 2014 1 . 
stupp r , mason wp , van den bent mj , et al : radiotherapy plus concomitant and adjuvant temozolomide for epidemiol biomarkers prev 23 : 1985 - 1996 , 2014 glioblastoma . 
stupp r , taillibert s , kanner aa , et al : maintenance therapy with tumor - treating fields plus temozolomide vs temozolomide alone for glioblastoma : a randomized clinical trial . 
myall nj , neal jw , cho - phan cd , et al : long - term survival of a patient with nonsmall - cell lung cancer harboring a v600e mutation in the braf oncogene . 
hyman dm , puzanov i , subbiah v , et al : vemurafenib in multiple nonmelanoma cancers with braf v600 mutations . n engl j med 373 : 726 - 736 , 2015 9 . 
schindler g , capper d , meyer j , et al : analysis of braf v600e mutation in 1 , 320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma , ganglioglioma and extra - cerebellar pilocytic astrocytoma . acta neuropathol 121 : 397 - 405 , 2011 14 . 
jang s , atkins mb : which drug , and when , for patients with braf - mutant melanoma ? lancet oncol 14 : e60 - e69 , 2013 17 . 
kieran mw , cohen kj , doz f , et al : complete radiographic responses in pediatric patients with brafv600 - positive tumors including high - grade gliomas : preliminary results of an ongoing phase 1 / 2a safety and pharmacokinetics ( pk ) study of dabrafenib . 
grob jj , amonkar mm , karaszewska b , et al : comparison of dabrafenib and trametinib combination therapy with vemurafenib monotherapy on health - related quality of life in patients with unresectable or metastatic cutaneous braf val600 - mutation - positive melanoma ( combi - v ) : results of a phase 3 , open - label , randomised trial . 
 o evaluation of the her / pi3k / akt family signaling network as a predictive biomarker of pathologic complete response for patients with breast cancer treated with neratinib in the i - spy 2 trial purpose in the i - spy 2 trial ( investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2 ) , the panerythroblastic oncogene b inhibitor neratinib was available to all hormone receptor ( hr ) / human epidermal growth factor receptor 2 ( her2 ) subtypes and graduated in the hr - negative / her2 - positive signature . 
we hypothesized that neratinib response may be predicted by baseline her2 epidermal growth factor receptor ( egfr ) signaling activation / phosphorylation levels independent of total levels of her2 or egfr proteins . materials and methods complete experimental and response data were available for between 130 and 193 patients . 
in qualifying analyses , which used logistic regression and treatment interaction analysis , 18 protein / phosphoprotein , 10 mrna , and 12 dna biomarkers that related to her family signaling were evaluated . 
 exploratory analysis of her family signaling in patients with triple - negative ( tn ) disease found that activation of egfr y1173 ( p = .005 ) and her2 y1248 ( pher2 ) ( p = .019 ) were positively associated with pathologic complete response . 
exploratory analysis in this pegfr / pher2activated tn subgroup identified elevated levels of estrogen receptor ( p < .006 ) in these patients . conclusion activation of her family phosphoproteins associates with response to neratinib , but only egfr y1173 and stmn1 appear to add value to the graduating signature . 
 introduction the i - spy 2 trial ( investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2 ; clinicaltrials.gov identifier : nct01042379 ) is a phase ii , adaptive neoadjuvant therapy trial in which the primary goal was to determine the predictive probabilities of phase iii trial success for various targeted therapeutics . 
patients with locally advanced , high - risk breast cancer had their diseases assigned to one of eight subtypes on the basis of hormone receptor ( hr ) , human epidermal growth factor receptor 2 ( her2 ) , and mammaprint - based high1 / ( ultra ) high2 risk statuses.1 - 3 neratinib , a pan - erythroblastic oncogene b ( erbb ) inhibitor , was available to patients with all tumor subtypes in the i - spy 2 trial , and the agent availability was graduated in the hr negative / her2 - positive signature.4 neratinib has shown activity against her2 - positive metastatic breast cancer , and there is also evidence for activity against her2 - negative tumor cells in vitro.5 , 6 because neratinib was available to all patients in the trial , this study provided an opportunity to test the efficacy of the drug in patients with her2 - negative tumors . cell linebased preclinical studies have implicated alterations in her / akt / mtor family genes on the dna , mrna , or protein level as predictive of neratinib response.7 - 11 however , recent her2 therapybased clinical trials in which her family biomarkers at the total protein and / or mrna level were analyzed for response prediction found that none provided predictive value compared with the conventional , us food and drug administrationapproved immunohistochemistry ( ihc ) / fluorescence in situ hybridizationbased her2 / estrogen receptor ( er ) testing methods.12 - 14 previous work with the i - spy 1 trial cohort revealed that her2 phosphorylation was highly concordant with her2 expression.15 that study identified a subpopulation of patients who had her2 negative disease by standard testing yet had her2 phosphorylation levels similar to patients with her2 - positive disease . 
the her2 activation seen in these patients in the her2 - negative subtype appeared coincident with epidermal growth factor receptor ( egfr ) activation . on the basis of the mechanism of action of neratinib as a potent her family kinase inhibitor combined with observations of i - spy 1 trial results , we postulated that her2 and egfr activation / phosphorylation may be predictive of neratinib response independent of total her2 status in the i - spy 2 trial ; this hypothesis was motivation for an exploratory analysis within the her2 - negative and triple - negative ( tn ) subpopulation . 
our analysis of her / phosphoinositide - 3 - kinase ( pi3k ) / akt signaling family components in pretreatment samples from the neratinib and concurrent control arms of the i - spy 2 trial was performed at a unique multiomic level . 
we report results from the following : ( 1 ) assessment of selected mutation / copy number alterations by exome sequencing , ( 2 ) mrna expression levels by expression microarrays , and ( 3 ) analysis of protein / phosphoprotein levels by reverse phase protein arrays ( rppas ) as specific biomarkers of neratinib response . 
rppa analysis assessed the ability of 18 protein / phosphoproteins that comprise the known drug targets of neratinib ( egfr , her2 ) and downstream effector molecules , such as shc transforming protein 1 ( shc ) and mammalian target of rapamycin ( mtor ) / ak thyoma ( akt ) signaling components to predict complete pathologic response ( pcr ) to neratinib . 
 patients randomly assigned to the experimental arm were treated with neratinib in addition to standard chemotherapy ( neratinib + t > ac ) .4 in patients with her2 - positive disease , neratinib was administered in place of trastuzumab ( appendix fig a1 )  . 
consort diagram that outlines the number of patients included in the neratinib and control arms of the i - spy 2 trial ( investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2 ) and those included in the subsequent analyses . 
cnv , copy number variation ; rppa , reverse phase protein array . primary efficacy analysis cohort neratinib concurrent control pretreatment expression data available neratinib concurrent control ( n = 193 ) ( n = 115 ) ( n = 78 ) ( n = 193 ) ( n = 115 ) ( n = 78 ) withdrew consent for use of tissue ( n = 2 ) pretreatment samples available for additional biomarker assessment ( n = 191 ) neratinib concurrent control ( n = 115 ) ( n = 76 ) insufficient material ( n = 23 ) insufficient material ( n = 61 ) rppa data available ( n = 168 ) neratinib ( n = 106 ) concurrent control ( n = 62 ) somatic mutation and cnv data available neratinib concurrent control ( n = 168 ) ( n = 78 ) ( n = 52 ) expression by using agilent 44k expression arrays ( agendia , irvine , ca ) , signaling protein activation by rppa , and dna sequencing ( approximately 2 , 000 - gene mini - cancer genome ; utrecht , the netherlands )  . 
details of sample preparation and data processing are provided in the data supplement . in our prespecified analysis plan , 16 logistic regression was used to assess association with pcr in the control and neratinib treated populations individually . 
relative biomarker performance between arms ( biomarker - by - treatment interaction ) was assessed with a logistic model ( pcr approximately equaled treatment + biomarker + [ treatment biomarker ] )  . 
 if a continuous biomarker did not follow a normal distribution , we applied a nonparametric method that discretized the score by using a series of cut points , and we applied logistic modeling for the dichotomized biomarker at each cut point ; significance was assessed by permutation testing . 
for significant biomarkers , bayesian logistic regression analysis was performed , as previously described , with the following model : pcr hr + her2 + biomarker + treatment + ( treatment hr ) + ( treatment her2 ) + ( treatment biomarker ) .16 in additional analyses , parametric t tests ( for normally distributed data ) or nonparametric wilcoxon rank sum tests ( for non - normally distributed data ) were used to assess association of protein end points with pcr in the control and neratinib - treated populations individually . 
hr , hormone receptor ; tn , triple negative . curves were generated for end points associated with pcr in the neratinib - treated arm of the trial to identify potential cut points for biomarker positivity rates within selected patient subtypes . 16 patients with mutated pik3ca achieved pcr in the neratinib arm compared with 24 ( 39% ) of 62 patients with wild - type pik3ca . 
however , pik3ca mutation status did not show a significant biomarker - by - treatment interaction . results evaluation of her family gene point mutations and dna amplification / loss as predictors of neratinib sensitivity we evaluated somatic mutations and copy number variations ( cnvs ) in 12 her family linked signaling pathway genesegf , egfr , her2 , nrg1 , igf1r , pik3ca , akt1 , pten , stmn1 , and mtorby targeting exome sequencing across all evaluable patients . 
 consistent with publications that link pik3ca mutation with resistance to neratinib and other her2 - targeted agents , 14 , 17 - 19 only two ( 13% ) of association of her family gene expression with response to neratinib we evaluated 10 predefined her family signaling genes as expression biomarkers of neratinib response in all evaluable patients ( appendix table a1 )  . 
her2 expression was significantly associated with sensitivity to neratinib combination therapy ( and in the her2 - positive subpopulation ) , and igf1r , to resistance ; neither gene was associated with response in the control arm of the trial ( appendix tables a1 and a2 )  . 
within the her2 negative subset , stmn1 was associated with response to neratinib ( p = .0023 ) and not control , and this result showed a significant biomarker - by - treatment interaction ( p = .0036 ; fig 3a ; appendix table a3 )  . 
 2 neratinib arm : her2 control arm : her2 hr + her2 no pcr no pcr her2 subset stmn1 - low stmn1 - high unselected her2 her2 / stmn1 - high pcr probability pcr probability fig 3 . 
 ( d ) bayesian estimated pcr probability distributions in ( left ) the unselected her2 - negative subset and ( right ) the her2 - negative / stmn1 - high subset in the control ( blue ) and neratinib ( gold ) arms . of patients with her2 - negative disease ( 29 of 106 patients ) had stmn1 - high status ( fig 3b - c )  . 
by using bayesian modeling , the estimated pcr rate of patients with her2 negative / stmn1 - high status in the treatment arm was 57% compared with 17% in the control arm ( fig 3d ) ; the predictive probability of phase iii success in this subset was 97% . her family protein signaling activation as predictors of neratinib sensitivity we evaluated 18 her family signaling proteins / phosphoproteins as biomarkers of neratinib response by using rppa data from pretreatment , laser capture microdissection ( lcm ) purified tumor epithelia across all evaluable patients in the neratinib and concurrent control arms . 
six of the 18 her family biomarkers tested ( egfr y1068 , egfr y1173 , egfr y992 , her2 total , her2 y1248 , and shc y317 ) were associated with pcr in neratinib - treated patients but not in concurrent control - arm patients in the trial ( table 1 )  . 
we dichotomized patients by their egfr y1173 intensity values into high and low groups ( optimal cut point of 4 , 501 , which maximized the significance of the biomarker - by - treatment interaction term ) , and we evaluated the distribution of pcr rates ( table 2 )  . 
bold indicates p < 0.05. the predictive probability of phase iii success ( appendix table a4 )  . comparison of her family gene expression , protein , phosphoprotein , and mutation data unsupervised clustering revealed that total protein levels of her - family genes clustered with its gene expression level , whereas phosphoprotein levels were more highly correlated to one another than to their respective gene or total protein expression levels ( fig 4 )  . 
pik3ca mutations ( 28 of 130 patients ) were not associated with altered levels of pik3ca expression of protein / phosphoprotein ; however , they were associated with lower levels of stmn1 and egfr gene expression and higher levels of the phosphoproteins pten s380 and akt s473 ( data not shown )  . exploratory analysis of her family signaling in patients with tn disease although neratinib graduated in the hr negative / her2 - positive signature , this signature was characterized by ihc / f luorescence in situ hybridizationbased analysis of total her2 expression and did not measure her2 or egfr phosphorylation . 
pcr rates for egfr y1173 and her2 y1248 biomarker high / low groups by hr / her2 subtype in neratinib - treated and control groups subtype neratinib ( n = 106 ) control ( n = 62 ) egfr y1173 ( riu > 4501 cutoff ) egfr y1173 high egfr y1173 low egfr y1173 high egfr y1173 low no . 
two - way plots of egfr y1173 and her2 y1248 for neratinib treated and concurrent controls demonstrated that nine ( 82% ) of 11 patients with tn disease who exhibited elevated phospho - egfr ( pegfr ) and phospho - her2 ( pher2 ) levels experienced a pcr in response to neratinib treatment compared with four ( 36% ) of 11 with tn disease among the concurrent controls ( table 2 ; fig 5a - b )  . 
bayesian evaluation of egfr y1173 and her2 y1248 as biomarkers for neratinib response in the tn population revealed a comparable probability of success in a phase iii trial to that of the graduated hr negative / her2 - positive population ( fig 5c ; appendix tables a4 and a5 )  . 
in this pegfr / pher2 - high group of tn tumors , 16 ( 76% ) of 21 patients had erlevels greater than the median value for the tn population compared with 11 ( 37% ) of 30 patients in the rest of the tn population ( fig 5d )  . 
in the neratinib - treated population alone , 11 ( 35% ) of 31 patients had pegfr / pher2 - high status , and nine ( 82% ) of 11 patients had total er levels greater than the tn population median . discussion as precision cancer medicine evolves into concomitant processes of discovery , validation , and development of biomarkers predictive of therapeutic response alongside the clinical assessment of drug efficacy , there is increasing emphasis on identification of predictive biomarker candidates as early as possible . 
the subset of biomarkers represented include phosphoproteins associated with response to neratinib , and their associated mrna and total protein levels ( scaled to a median of 0 and standard deviation of 1 )  . 
 egfr , epidermal growth factor receptor ; erbb , erythroblastic oncogene b ; her2 , human epidermal growth factor receptor 2 ; hr , hormone receptor ; na , not available ; shc , shc transforming protein ; tn , triple negative . neratinib arm control arm treatment receptor subtype pten loss pik3ca mut egf mut her2 mut egfr total egfr mrna stmn1 mrna erbb3 total erbb3 mrna egfr y1068 egfr y1173 her2 y1248 shc y317 egfr y992 erbb3 y1289 her2 total her2 mrna no pcr no pcr treatment receptor subtype mutation status heatmap color key neratinib control hr / her2 + hr + / her2 + hr + / her2 mutated wild type 3 2 1 0 1 2 3 value proteomic / phosphoproteomic biomarkers were measured in all pretreatment biopsy samples . 
given that her2 total protein levels do not strictly correlate with her2 phosphorylation in the her2 - negative setting , 15 and given that the phosphorylation status of her2 protein ( compared with total her2 ) provides significant information on breast cancer survival , 23 - 27 we postulated that phosphorylation levels of the neratinib drug targets her2 and egfr in pretreatment biopsy specimens would correlate with pcr in both her2 - positive and her2 negative tumors . our data revealed that coactivation of her2 and egfr correlated with pcr in both her2 positive and tn tumors . 
the pcr rate in the pegfr / pher2 - high tn population , according to an optimal cut point for both markers , was 82% ( nine of 11 patients ) in the treatment arm , compared with 36% ( four of 11 patients ) with pcr in the control ar although these data are exploratory , this pcr rate was higher than the 55% of patients who had a complete response observed in the graduated hr negative / her2 - positive arm for neratinib.4 in keeping with these results , in which phosphorylated egfr levels rather than total egfr were predictive for pcr , phosphorylated egfr has been shown to be a potential predictive marker for anti - egfr therapies in other tumors , such as nonsmall - cell lung cancer.28 , 29 also , our expression analysis produced only one predictive biomarker for neratinib sensitivity , stmn1 , a gene implicated in her2 pathway signaling and chemotherapy sensitivity in non - her2 amplified cell lines.30 - 33 although our data indicate that phosphorylation levels of her2 and egfr correlate with pcr in neratinib - treated patients with breast cancer , a number of caveats in the study limit generalizability . 
the adaptive design of the trial allowed for efficient and quick graduation ( or termination ) of agent / marker combinations on the basis of their estimated likelihood of phase iii success . 
analysis of epidermal growth factor receptor ( egfr ) y1173 and human epidermal growth factor receptor 2 ( her2 ) y1248 as biomarkers for neratinib response in patients with triple - negative ( tn ) disease enrolled in the i - spy 2 trial ( investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2 )  . 
 ( a ) and ( b ) two - way plots of egfr y1173 and her2 y1248 intensities for tn ( a ) control group and ( b ) neratinib - treated patients . 
other studies with her family inhibitors have shown that neratinib , in combination with insulin - like growth factor receptor ( igfr ) inhibitors had antiproliferative effects in her2 non - overexpressing breast cancer cell lines , and pilot clinical studies of lapatinib treatment in various metastatic cancers showed a correlation of pher2 with lapatinib response.37 , 38 what is the basis for her2 and egfr activation in the tn subpopulation ? the frequency of her2 and egfr mutations in the neratinib cohort is too small ( n = 4 and 3 , respectively ) to be a contributing factor . 
in the absence of genomic alterations related to her family signaling when her activation is seen , a logical postulate is that the process is mediated by ligand - driven events . 
recently , it has been shown that neuregulin 1 ( nrg1 or heregulin ) and her2 phosphorylation coincidentally occurred in a subset of her2 - negative tumors and that inhibition of egfr or her2 or both receptors reduced breast cancer stem cell survival and self - renewal.39 although we evaluated nrg1 in our study and did not find any correlation with pcr or her2 activation status ( table 1 ) , we did explore the possibilities of other ligand - driven events underpinning the her2 and egfr activation observed in tn tumors , namely estrogen signaling . 
estrogen can exert nongenomic activity called membrane - initiated steroid signaling through binding with er at low concentrations40 ; er may exist in a signalsome complex with a variety of receptor tyrosine kinases , such as igfr , 41 egfr , 42 , 43 or her2 , 44 , 45 and lead to activation of egfr , her2 , and igfr121 in the absence of gene transcription . 
 we found that erlevels in the tn cohort were higher in pegfr / pher2 - high tumors than in tumors that were not her2 / egfr activated ( fig 5 )  . 
this result could provide a potential explanation for the paradoxical finding of her2 / egfr activation in tn cancers in the absence of her2 genomic alterations and must be confirmed in independent study sets . the lcm - rppa workflow used in this study provides a powerful and unique approach to quantitatively measure the activated signaling architecture of a large number of cancer - related pathways , including the her family , from microscopic quantities of tissue . 
this technology and workflow is especially well suited for clinical sample assessment.46 , 47 past studies have revealed the need for lcm to accurately assess phosphorylated and total protein levels , and to facilitate biomarker evaluation in the context of high and low tumor cell content.48 moreover , unlike ihc - based approaches that can be adversely effected by choice of antigen retrieval method , the rppa technique utilizes fully denatured protein in which phospho - epitopes are fully linearized and recognized by the cognate primary antibody . patients with tn breast cancer have a paucity of targeted therapeutic options available . 
given the pcr rate observed and biomarker positive prevalence we found in the tn setting , we believe these data provide a strong molecular rationale to consider prospective validation of the findings in patients with tn disease who received neratinib . 
to our knowledge , this study is the first of its kind to quantitatively assess activated her family signaling in the context of clinical response to her - directed therapies in a tn population . 
 the biomarker findings , although prespecified , ultimately are based on small numbers of patients and must be confirmed with larger patient populations in independent clinical studies to validate fig 5 . 
wulfkuhle honoraria : dava oncology patents , royalties and other intellectual property : coinventor on filed george mason universityassigned patents related to phosphorylated her2 and egfr response predictors for her family directed therapeutics . 
gallagher no relationship to disclose lamorna brown - swigart no relationship to disclose gillian hirst no relationship to disclose laura esserman consulting or advisory role : blue cross blue shield association research funding : merck travel , accommodations , expenses : blue cross blue shield association donald berry employment : berry consultants leadership : berry consultants stock and other ownership interests : berry consultants consulting or advisory role : berry consultants travel , accommodations , expenses : berry consultants minetta liu research funding : eisai ( inst ) , seattle genetics ( inst ) , novartis ( inst ) , roche ( inst ) , genentech ( inst ) , grail ( inst ) , merck ( inst ) , janssen diagnostics ( inst ) travel , accommodations , expenses : grail , mreck , celgene , agena bioscience , menarini silicon biosystems , cynvenio biosystems john w . 
park stock and other ownership interests : merrimack consulting or advisory role : genentech ( inst ) speakers ' bureau : genentech , pfizer , agendia ( i ) lodewyk f.a. 
petricoin iii leadership : perthera , ceres nanosciences stock and other ownership interests : perthera , ceres nanosciences , avant diagnostics consulting or advisory role : perthera , ceres nanosciences , azgen , avant diagnostics research funding : ceres nanosciences ( inst ) , glaxosmithkline ( inst ) , abbvie ( inst ) , symphony evolution ( inst ) patents , royalties , other intellectual property : national institutes of health patents licensing fee distribution / royalty . 
6 - 10 , 2016 , and the annual meeting of the american society for clinical oncology , chicago , il , may 29 - june 2 , 2015 . support prior presentation references 1 . 
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baselga j , corts j , im sa , et al : biomarker analyses in cleopatra : a phase iii , placebocontrolled study of pertuzumab in human epidermal growth factor receptor 2 - positive , first - line metastatic breast cancer . 
wolf dm , yau c , sanil a , et al : dna repair deficiency biomarkers and the 70 - gene ultra - highrisk signature as predictors of veliparib / carboplatin response in the i - spy 2 breast cancer trial . 
loibl s , majewski i , guarneri v , et al : pik3ca mutations are associated with reduced pathological complete response rates in primary her2 - positive breast cancer : pooled analysis of 967 patients from five prospective trials investigating lapatinib and trastuzumab . 
lopez s , cocco e , black j , et al : dual her2 / pik3ca targeting overcomes single - agent acquired resistance in her2 - amplified uterine serous carcinoma cell lines in vitro and in vivo . 
lee av , guler bl , sun x , et al : oestrogen receptor is a critical component required for insulinlike growth factor ( igf ) - mediated signalling and growth in mcf - 7 cells . 
lee av , cui x , oesterreich s : cross - talk among estrogen receptor , epidermal growth factor , and insulin - like growth factor signaling in breast cancer . 
thor ad , liu s , edgerton s , et al : activation ( tyrosine phosphorylation ) of erbb - 2 ( her - 2 / neu ) : a study of incidence and correlation with outcome in breast cancer . 
cicenas j , urban p , kng w , et al : phosphorylation of tyrosine 1248 - erbb2 measured by chemiluminescence - linked immunoassay is an independent predictor of poor prognosis in primary breast cancer patients . 
frogne t , laenkholm av , lyng mb , et al : determination of her2 phosphorylation at tyrosine 1221 / 1222 improves prediction of poor survival for breast cancer patients with hormone receptorpositive tumors . 
digiovanna mp , stern df , edgerton sm , et al : relationship of epidermal growth factor receptor expression to erbb - 2 signaling activity and prognosis in breast cancer patients . 
hudelist g , kstler wj , czerwenka k , et al : her - 2 / neu and egfr tyrosine kinase activation predict the efficacy of trastuzumab - based therapy in patients with metastatic breast cancer . 
wang f , wang s , wang z , et al : phosphorylated egfr expression may predict outcome of egfr - tkis therapy for the advanced nsclc patients with wild - type egfr . 
saal lh , johansson p , holm k , et al : poor prognosis in carcinoma is associated with a gene expression signature of aberrant pten tumor suppressor pathway activity . 
drury sc , detre s , leary a , et al : changes in breast cancer biomarkers in the igf1r / pi3k pathway in recurrent breast cancer after tamoxifen treatment . 
zhu hw , jiang d , xie zy , et al : effects of stathmin 1 silencing by sirna on sensitivity of esophageal cancer cells eca - 109 to paclitaxel . 
chakraborty a , hatzis c , digiovanna mp : co - targeting the her and igf / insulin receptor axis in breast cancer , with triple targeting with endocrine therapy for hormone - sensitive disease . 
lee cy , lin y , bratman sv , et al : neuregulin autocrine signaling promotes self - renewal of breast tumor - initiating cells by triggering her2 / her3 activation . 
arpino g , wiechmann l , osborne ck , et al : crosstalk between the estrogen receptor and the her tyrosine kinase receptor family : molecular mechanism and clinical implications for endocrine therapy resistance . 
song rx , barnes cj , zhang z , et al : the role of shc and insulin - like growth factor 1 receptor in mediating the translocation of estrogen receptor to the plasma membrane . 
pietras rj , arboleda j , reese dm , et al : her - 2 tyrosine kinase pathway targets estrogen receptor and promotes hormone - independent growth in human breast cancer cells . 
akbani r , becker kf , carragher n , et al : realizing the promise of reverse phase protein arrays for clinical , translational , and basic research : a workshop report : the rppa ( reverse phase protein array ) society . 
i - spy2 trial ( investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2 ) schema for patients in the control and experimental therapy arms . 
 o undetectable ras - mutant clones in plasma : possible implication for anti - egfr therapy and prognosis in patients with ras - mutant metastatic colorectal cancer mohamed bouchahda , md1 - 4 ; raphael saffroy , phd5 ; abdoulaye karabou e , md2 , 6 ; jocelyne hamelin , phd5 ; pasquale innominato , md , phd2 , 7 , 8 ; faouzi saliba , md , phd9 ; francis l evi , md , phd1 , 2 , 8 ; nelly bosselut , md5 ; and antoinette lemoine , pharmd , phd5 purpose combining cetuximab with chemotherapy provides clinical benet to 60% of the patients with ras wild - type ( ras - wt ) metastatic colorectal cancer ( mcrc )  . 
the patients with raswt ctdna received cetuximab + uorouracil , leucovorin , and irinotecan ( folfiri ) , whereas those with ras - mt ctdna were treated with the oncologists choice of therapy . results of 16 registered patients , 11 were male and ve female . 
they were age 48 to 81 years , and they had unresectable metastatic adenocarcinoma from the colon ( n = 11 ) or rectum ( n = 5 ) , with a median of two metastatic sites . 
in the patients with wt ctdna , objective tumor response rate was 50.0% , including one complete response and four partial responses after a median number of 6 courses of cetuximab + folfiri ( range , 1 to 16 courses )  . 
this was particularly the case for the combination of cetuximab with uorouracil , leucovorin , and irinotecan ( folfiri ) or uorouracil , leucovorin , and oxaliplatin ( folfox ) .1 , 2 however , several studies have shown that the survival of those patients with tumor ras mutations ( ras - mt ) was shorter than for those with ras - wild - type ( ras - wt ) tumors.3 , 4 since 2014 , it has been recommended that only patients with ras - wt mcrc should receive an anti - egfr targeted agent.5 ras mutations have been found in 30% to 50% of patients with mcrc , 6 - 9 which makes these patients ineligible for egfr - targeted therapies . recent studies have demonstrated that the analysis of circulating tumor dna ( ctdna ) in blood samples , through its ability to recapitulate tumor heterogeneity , is a remarkable surrogate of tumor biopsy for detecting mutations.10 - 12 this technique has the advantage of being less invasive than a tissue biopsy and can be easily repeated over time . 
 anti - egfr therapy for ctdna ras - wt despite ras - mt in tumor tissue context key objective to determine whether the lack of ras mutation in a liquid biopsy supports the administration of an antiepidermal growth factor receptor ( egfr ) antibody , despite earlier documentation of a pathogenic ras mutation in the primary colorectal cancer ( crc )  . knowledge generated nearly half the patients in this pilot study received cetuximab - based chemotherapy , because no ras mutation was detected in the liquid biopsy , despite such mutations having been found in the primary tumor . 
the progression - free survival and the overall survival of these heavily pretreated patients largely exceeded those in patients whose liquid biopsy revealed ras mutation and who received chemotherapy only . relevance tracking the gain or loss of ras - mutated cancer clones through liquid biopsies along the course of crc disease may have a profound impact on its therapeutic management . 
this is achieved through enabling the administration of anti - egfr that was initially rejected on the basis of previous molecular testing of tissue . in the characterization of the dynamics of acquired resistance to anti - egfr therapies.13 to date , studies with liquid biopsy have been selectively focused on the early detection of the appearance of ras - mt clones in tumor deposits by analyzing ctdna in blood samples from patients with ras - wt primary crc13 as biomarkers of an increasing resistance to anti - egfr agents . 
indeed , no specication in the european marketing authorisation for cetuximab mentions that tumor ras testing should be determined on solid tumor tissue in order to allow for anti - egfr administration . 
this is also the case for the recommendations by the french high health authority regarding cetuximab use . all consecutive patients treated between august 2017 and february 2019 at one of three participating centers in france were screened for inclusion in this pilot study . 
inclusion required histologic or cytologic proof of colorectal adenocarcinoma , with a kirsten rat sarcoma viral oncogene homolog ( kras ) or neuroblastoma rat sarcoma viral oncogene homolog ( nras ) mutation ( nras - mt ) from a tissue biopsy . 
the massarray ultraseek procedure involves a 3 - step process consisting of the initial polyinactivation of unincorporated merase chain reaction , nucleotides by shrimp alkaline phosphatase , and a singlebase primer extension according to the manufacturers protocol . 
the products were then nano - dispensed onto a matrix - loaded silicon chip ( spectrochipii , agena bioscience ) , and the mutations were detected by matrixassisted laser desorption - ionizationtime of ight mass spectrometry . 
for our study , the sensitivity for the detection of clinically relevant ras gene mutations in ctdna was 88% for patients with crc liver metastases , in good agreement with bettegowda et al.16 we currently use these highly sensitive mass spectrometry ctdna analyses for monitoring treatment of patients with nonsmall - cell lung cancer or breast cancer in routine oncology practice . study treatment all patients had ras - mt tissue biopsies . 
the study population was divided in two groups according to the results of the ctdna mutational analysis : group 1 included the patients with ras - mt also found in plasma ; group 2 included the patients with ras - wt ctdna . 
the sum of the largest diameters of the target lesions was computed on the inclusion imaging and used as baseline for the quantication of tumor downsizing and response categorization according to recist . 
response was classied as complete response , partial response , stable , or progressive disease.15 statistical consideration this pilot exploratory study included consecutive patients , and no sample size was dened a priori . 
the durations of progression - free survival ( pfs ) and overall survival ( os ) were measured from inclusion until the date of progression or death , respectively , or that of last follow up , with the database locked on may 25 , 2019 . 
all analyses were performed with intentto - treat using spss v18.0 software ( spss , chicago , il )  . results patients sixteen patients with unresectable mcrc and ras - mt in a cancer tissue were registered at one of three centers in initial ras mutational status was dethis pilot study . termined in the resected primary tumor for 11 patients ( 69% ) or in a tumor biopsy for 5 patients ( 31% )  . 
 anti - egfr therapy for ctdna ras - wt despite ras - mt in tumor tissue two or more chemotherapy regimens for metastatic disease . two groups of patients were identied according to the results of ctdna mutational analysis at inclusion . 
group 1 received the investigators choice of chemotherapy regimens , which included bevacizumab + folfox for three patients , aibercept + folfiri for three patients , and regorafenib for one patient . 
baseline characteristics were similar in the 2 groups ( table 1 )  . safety data for the nine patients with ctdna ras - wt ( group 2 ) overall , the cetuximab + folfiri regimen was well tolerated . 
the main grade 1 or 2 clinical toxicities were fatigue , diarrhea , nausea or vomiting , mucositis , acneiform rash , and alopecia ( 6 [ 67% ] of 9 for all )  . 
this fourth - line protocol was well tolerated , with the most severe toxicities being grade 2 leukopenia , neutropenia , fatigue , and acneiform rash . discussion the patients in this study had advanced and chemotherapy - resistant metastatic disease , without any imbalance in apparent overall tumor burden that could reasonably inuence the liquid biopsy results . 
thus , no evidence supports the possibility that the differences in detection of ctdna ras mutation would be related to between - patient variations in tumor burden . the identication of a mutation in kras or nras from a cancer tissue biopsy precludes the use of anti - egfr treatment in association with chemotherapy against mcrc , based on consistent evidence.5 our group rst reported the occurrence of acquired kras mutations along the progression of colorectal metastases in patients treated with cetuximab . 
we then hypothesized that the late acquisition of kras mutations could represent a possible mechanism of secondary resistance to anti - egfr antibodies.18 however , the assumption of persisting ras - mt genotype for patients who received chemotherapy has not been challenged before now because of the expected evolutionary advantage of ras - mt clones.19 yet in this pilot study , we found that nearly half the patients with mcrc displayed no detectable ras - mt in ctdna , although their cancer tissue genotyping had demonstrated ras - mt . 
this nding raised several questions . first , is there a concordance between the ras mutational status of tumor tissues and that of ctdna ? several studies have highlighted discrepant results in ras mutational status for 10% - 15% of the patients tested , depending on the method used.11 , 14 , 20 vidal et al explained such plasmaversus - tissue ras discrepancies with spiral and temporal heterogeneity in ras - mt tumor clones within the tumor tissue.11 according to thierry et al , 20 such discrepancies could relate to the use of biopsies . 
other discrepancies that seemed to affect concordance were long intervals between assessments of the ras status in tumor tissue and that in the blood sample , resection of the tumor at the time of blood draw , tissue analyzed . grasselli et al14 ascribed the discrepancies in ras status to differences in technical sensitivity of the methods used for analysis or to heterogeneity . tumor site , and type of second , is there a change in ras status over time and / or during treatment ? this question does not yet have a clear answer . 
we were the rst to report 1 case of acquired kras mutation in metastases after progression under combined anti - egfr and doublet chemotherapy in 12 patients with kras - wt mcrc.18 other authors have highlighted the concurrent detection of sensitive and resistant clones to anti - egfr antibodies within tumor deposits from different locations in the same patient.21 - 24 this multiclonality could explain the dissociated antitumor responses that are frequently encountered in clinical practice . 
such tumor heterogeneity assumes the existence of wt clones sensitive to anti - egfr treatment alongside resistant mutant clones . interestingly , the team of raimondi et al25 recently reported the disappearance of ras - mt clones in ctdna after tumor progression while receiving bevacizumab and chemotherapy in 4 patients with ras - mt mcrc . 
 anti - egfr therapy for ctdna ras - wt despite ras - mt in tumor tissue feb 19 , 2019 before cetux + folfiri may 29 , 2019 after 6 courses sept 19 , 2019 after 12 courses target lesion target lesion target lesion fig 4 . 
case report illustrating exceptional efcacy of cetuximab + uorouracil , leucovorin , and irinotecan ( cetux + folfiri ) as fourth - line chemotherapy received by a 72 - year - old patient with kras mutation in tumor tissue but kras wild - type in liquid biopsy , upon inclusion in the study . 
indeed , the observed median pfs and os in these patients were similar to those reported for patients with ras - wt tumor tissue who received this combination as rst - line treatment for mcrc . 
our internal steering committee proposed to stop the pilot study , once the information was adequate for designing a randomized trial for testing the hypothesis further on the basis of an apparent three - fold increase in the median pfs of the experimental treatment compared with cetuximab - free chemotherapy . the intriguing and encouraging results of this exploratory trial need to be conrmed in randomized clinical trials . such validation steps are particularly relevant because all our patients with ras - wt ctdna received cetuximab ; thus , we cannot differentiate between a prognostic and a predictive role of ras - wt ctdna for outcomes of patients receiving cetuximab - based chemotherapy . 
 bouchahda et al than prognostic of an aggressive tumor biology.34 such consideration would further support the hypothesis of an added benet from cetuximab in this subgroup of patients . yet recent evidence suggests that the proportion of ras - mt in ctdna was a prognostic indicator for both os and pfs in patients with mcrc.35 this nding raised the question of a potential divide on the clinical signicance between tumor and ctdna genotyping , which this study cannot respond to . 
the fact that orrs exceeded 40% and were similar in both treatment groups suggested that all the patients included in the study were not completely resistant to chemotherapy and that those with ras - mt in the liquid biopsy could have a worse prognosis , as proposed by elz et al.35 however , single - agent cetuximab achieved only a 10% orr in patients with chemotherapy refractory mcrc , 36 despite prolonging pfs by 4 months . 
taken together , the literature results support a much greater value for pfs prolongation compared with orr as an efcacy end point , which supports the hypothesis that the liquid biopsy results have a predictive value . the lack of a prespecied sample size and the low number of patients in each group constitute the main limitations of our pilot salvage study . 
the three - fold increase in median pfs in the absence of any apparent bias does suggest that antiegfrbased chemotherapy could represent a promising option for nearly half the patients with initially documented ras - mt on tumor tissue at a later stage of their disease . our study was in line with the chronos study ( clinicaltrials.gov identier : nct03227926 ) , 37 whose design is based on the concept that crc genome adapts dynamically to intermittent anti - egfr drug schedules . 
a follow - up on our study would involve a randomized clinical trial in which eligible patients with ras - mt on solid tumor tissue undergo ras mutational status assessment in ctdna upon progression on chemotherapy . 
 anti - egfr therapy for ctdna ras - wt despite ras - mt in tumor tissue references venook ap , niedzwiecki d , lenz hj , et al : effect of rst - line chemotherapy combined with cetuximab or bevacizumab on overall survival in patients with kras wild - type advanced or metastatic colorectal cancer : a randomized clinical trial . 
jama 317 : 2392 - 2401 , 2017 tejpar s , stintzing s , ciardiello f , et al : prognostic and predictive relevance of primary tumor location in patients with ras wild - type metastatic colorectal cancer : retrospective analyses of the crystal and fire - 3 trials . 
modest dp , ricard i , heinemann v , et al : outcome according to kras - , nrasand braf - mutation as well as kras mutation variants : pooled analysis of ve randomized trials in metastatic colorectal cancer by the aio colorectal cancer study group . 
ann oncol 27 : 1746 - 1753 , 2016 de roock w , claes b , bernasconi d , et al : effects of kras , braf , nras , and pik3ca mutations on the efcacy of cetuximab plus chemotherapy in chemotherapy - refractory metastatic colorectal cancer : a retrospective consortium analysis . 
lancet oncol 11 : 753 - 762 , 2010 van cutsem e , cervantes a , nordlinger b , et al : metastatic colorectal cancer : esmo clinical practice guidelines for diagnosis , treatment and follow - up . 
ann oncol 25 : iii1 - iii9 , 2014 douillard jy , oliner ks , siena s , et al : panitumumab - folfox4 treatment and ras mutations in colorectal cancer . 
heinemann v , von weikersthal lf , decker t , et al : folfiri plus cetuximab versus folfiri plus bevacizumab as rst - line treatment for patients with metastatic colorectal cancer ( fire - 3 ) : a randomised , open - label , phase 3 trial . 
lancet oncol 15 : 1065 - 1075 , 2014 van cutsem e , lenz hj , k ohne ch , et al : fluorouracil , leucovorin , and irinotecan plus cetuximab treatment and ras mutations in colorectal cancer . 
j clin oncol 33 : 692 - 700 , 2015 peeters m , kafatos g , taylor a , et al : prevalence of ras mutations and individual variation patterns among patients with metastatic colorectal cancer : a pooled analysis of randomised controlled trials . 
vidal j , muinelo l , dalmases a , et al : plasma ctdna ras mutation analysis for the diagnosis and treatment monitoring of metastatic colorectal cancer patients . ann oncol 28 : 1325 - 1332 , 2017 12 . 
grasselli j , elez e , carat `u g , et al : concordance of bloodand tumor - based detection of ras mutations to guide anti - egfr therapy in metastatic colorectal cancer . 
bouchahda m , macarulla t , liedo g , et al : feasibility of cetuximab given with a simplied schedule every 2 weeks in advanced colorectal cancer : a multicenter , retrospective analysis . 
lacina l , coma m , dvor ankov a b , et al : evolution of cancer progression in the context of darwinisanticancer res 39 : 1 - 16 , 2019 20 . 
thierry ar , el messaoudi s , mollevi c , et al : clinical utility of circulating dna analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti - egfr treatment . 
raimondi c , nicolazzo c , belardinilli f , et al : transient disappearance of ras mutant clones in plasma : a counterintuitive clinical use of egfr inhibitors in ras mutant metastatic colorectal cancer . 
zhou m , yu p , hou k , et al : effect of ras status on anti - egfr monoclonal antibodies + 5 - fu infusion - based chemotherapy in rst - line treatment of metastatic colorectal cancer : a meta - analysis . 
giantonio bj , catalano pj , meropol nj , et al : bevacizumab in combination with oxaliplatin , uorouracil , and leucovorin ( folfox4 ) for previously treated metastatic colorectal cancer : results from the eastern cooperative oncology group study e3200 . 
van cutsem e , tabernero j , lakomy r , et al : addition of aibercept to uorouracil , leucovorin , and irinotecan improves survival in a phase iii randomized trial in patients with metastatic colorectal cancer previously treated with an oxaliplatin - based regimen . 
tabernero j , yoshino t , cohn al , et al : ramucirumab versus placebo in combination with second - line folfiri in patients with metastatic colorectal carcinoma that progressed during or after rst - line therapy with bevacizumab , oxaliplatin , and a uoropyrimidine ( raise ) : a randomised , double - blind , multicentre , phase 3 study . 
sobrero af , maurel j , fehrenbacher l , et al : epic : phase iii trial of cetuximab plus irinotecan after uoropyrimidine and oxaliplatin failure in patients with metastatic colorectal cancer . 
peeters m , oliner ks , price tj , et al : analysis of kras / nras mutations in a phase iii study of panitumumab with folfiri compared with folfiri alone as second - line treatment for metastatic colorectal cancer . 
de stefano a , rosanova m , malapelle u , et al : discordance in ras mutations between primary colon tumor and metastases : a real event or a matter of methodology ? int j biol markers 32 : e474 - e477 , 2017 34 . 
elez e , chianese c , sanz - garca e , et al : impact of circulating tumor dna mutant allele fraction on prognosis in ras - mutant metastatic colorectal cancer . 
siena s , bardelli a , sartore - bianchi a , et al : exploiting clonal evolution and liquid biopsy to overcome resistance to anti - egfr treatment in metastatic colorectal cancer : the chronos trial . 
lolkema1 introduction the vhl gene is a tumor suppressor gene located at chromosome 3p25 , 1 and its protein has multiple functions linked to multiple effector proteins.2 aberrations in the vhl gene are associated with sporadic clear cell renal cell carcinomas ( ccrccs ) , sporadic hemangioblastomas , pheochromocytomas , pancreatic islet cell tumors , endolymphatic sac tumors , and benign cysts affecting various organs.3 germline inthe vhl gene causes the autosomal activation of dominant von hippel - lindau ( vhl ) syndrome . 
vhl is rarely mutated outside of the context of rcc or vhl syndromeassociated malignancies.4 biallelic vhl inactivation caused by genetic and epigenetic alterations ( including dna methylation , histone modications , and coincidental loss of genes localized adjacent to the vhl chromosome locus ) has been described.2 in both hereditary and sporadic tumors , vhl mutations are heterogeneous.5 the encoded vhl protein ( pvhl ) plays an important role in ubiquitination and proteosomal degradation of hypoxia inducible factor - 1 ( hif - 1 )  . 
our aim was to investigate the ( epi ) genetic background of this patients disease and to determine whether the identied vhl mutation could be a driving mutation in this patients fdcs . methods case report in 2013 , a 29 - year - old female patient presented with a large abdominally located mass and multiple hepatic lesions . 
a histologic biopsy from a liver lesion showed an epithelioid and spindle cell malignant neoplasm with scattered lymphocytes . immunohistochemistry ( ihc ) results matched the pattern of fdcs with exfollicular dendritic cell markers cd21 , pression of cd23 , and cd359 ( fig 1a - c )  . 
on the basis of radiologic diagnosis , the pattern did not t an rcc . this was further supported by a negative paired box gene 8 ( pax8 ) ihc result ( fig 1d )  . the clinical course of our patients disease is outlined in figure 2 . 
the second - line treatment was pazopanib , which resulted in unexpected stable disease for 22 months . rendering additional the patient participated in the dutch national center for personalized cancer treatment ( cpct ) - 02 program , 10 tumor sampling for whole - genome sequencing ( wgs ) analysis before and after treatment with pazopanib . 
wgs was performed on the illumina hiseq x platform with 100 ng dna as input using standard protocols ( pairedend 2 150 base pairs ; illumina , san diego , ca )  . within the framework of cpct - 02 , all germline variants are ltered , which guarantees that only somatic variants are reported . 
hif - 1 ihc showed positive nuclear staining ( e ) , and glucose transporter 1 ( glut1 ) ihc showed positive staining ( f )  . results genomic landscape the identied c.119delc vhl frameshift mutation led to a truncated pvhl ( p.pro40fs ; fig 4 )  . 
both biopsies showed genome - wide aberrations , with large chromosomal copy number alterations , structural rearrangements , and mutations in a broad spectrum of loci ( fig 5 )  . 
the patient received eight cycles of chop ( cyclophosphamide , doxorubicin , vincristine , prednisone ) , which resulted 6 months later ( t6 ) in metabolic complete remission ( cr )  . 
after that ( t36 ) , the patient showed pd and participated in a phase 1 trial with immunotherapy ( cd40 agonistic monoclonal antibody ) in combination with vanucizumab ( anti - angiopoietin - 2 and anti - vegf [ vascular endothelial growth factor ] ) for 11 months . 
both results provide circumstantial evidence for functional loss of pvhl.7 , 10 discussion our analysis revealed a unique patient with fdcs harboring a somatic functional vhl aberration , which is the rst description of a vhl mutation in sarcoma.14 because fdcs is a rare mesenchymal neoplasm with a largely unknown and rather complex genetic landscape15 and is unknown for harboring vhl aberrations , we veried specic ihcbased markers to conrm the diagnosis and to reject metastatic rcc ( mrcc ) as an alternative diagnosis . 
the positive hif - 1 ihc result may indicate a functional loss of pvhl ; in this patient , it was a consequence of mutation and potential methylation - derived changes leading to biallelic vhl gene inactivation . 
although most evidence in fdcs has been found for the involvement of the ras / raf signaling pathway , 16 , 17 we did not identify mitogen - activated protein kinase ( mapk ) alterations in our patient . 
a mutant allele - specic imbalance as a consequence of allele - specic amplication has been described.18 , 19 however , this seems to be a more common aspect with activating mutations . 
recurrent vhl locus amplications have not been described in ccrcc ; therefore , it does not seem to be a common aberration . however , these reports did not specically investigate post - tyrosine kinase samples.2 , 20 , 21 numerous nonspecic chromosomal translocations were present in the pretreatment biopsy , with a remarkable decline in the number of structural variants ( svs ) following treatment with pazopanib . 
treatment of cancer may have an inuence on involved processes and subsequently on patterns and frequency of svs.22 alterations in the number of svs between time points in a patients malignancy may also be a consequence of tumor evolution and selective survival of subclonal populations as a result of the selective pressure of treatment , similar to variable somatic alterations that cause the emergence of drug resistance.23 the changes in the genomic landscape between the two biopsies obtained in our patient could be associated with the observed disease response during treatment with pazopanib.24 moreover , the fact that the mutated vhl gene has been amplied in our patients disease over time emphasizes its importance and potential as a driver mutation . upregulated vegf is associated with the hif pathway . 
pazopanib , a multikinase inhibitor with activity against the vegf receptor and platelet - derived growth factor and receptors , is effective in the treatment of mrcc25 and metastatic sarcoma.26 during treatment with pazopanib , our patient had stable disease for 22 months . 
in march 2016 , progressive disease was revealed on a conventional ct scan ( a ) and on a positron emission tomography scan ( b ) before the start of treatment with pazopanib . 
untranslated regions ( utr ) and exonic regions are depicted by rectangular boxes ( colored dark blue and dark gold , respectively ) , and intronic regions are depicted by black lines . 
 ( c ) protein sequences of wild - type ( wt ) pvhl and mutant pvhl , colored by protein domains ; ( gxeex ) 8 is shown in light blue , - domain in light gold , and - domain in red . 
 ( the vhl gene model is on the basis of ensembl transcript nm_000551.2 and the protein model is on the basis of the uniprotkb p40337 entry . ) red asterisk indicates stop codon . 
moreover , saygin et al27 performed a pooled analysis of data from 462 patients with fdcs , showing a median survival for patients with metastatic disease of 9 months ( range , 0.25 to 72 months ) and a 2 - year survival rate of 15.8% ; these results suggest that the long pfs is not the result of better prognostic features of fdcs . 
the relatively long pfs during our patients last treatment , which consisted of the combination of a cd40 agonistic monoclonal antibody and an antiangiopoietin - 2 and anti - vegf bispecic monoclonal antibody , could possibly be explained by the effect of the latter drug ( vanucizumab )  . 
these data imply that the vhl mutation in our patient may predict a biologic behavior more similar to mrcc than to a metastatic sarcoma in response to treatment with pazopanib . 
these ndings are not sufcient to make an argument for vegf - targeted therapies in fdcs , because this is the rst case of a vhl - mutated fdcs as far as we know . indirect evidence for pvhl functional although hif stabilization and glut1 accumulation are , at the most , loss , a limitation of this case report is the absence of methylation analysis looking further into epigenetic silencing of the vhl gene . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . florence atra travel , accommodations , expenses : abbvie , astellas pharma paul j . 
van diest consulting or advisory role : panterhei ( inst ) patents , royalties , other intellectual property : ddx3 as a biomarker for cancer and methods related thereto ( inst ) geert j.l.h. 
we thank hartwig medical foundation for providing wholegenome sequencing data and for performing analysis of these data . moreover , this patient was entered into the center for personalized cancer treatment ( cpct ) - 02 study . 
medicine ( baltimore ) 76 : 381 - 391 , 1997 lawrence ms , stojanov p , mermel ch , et al : discovery and saturation analysis of cancer genes across 21 tumour types . 
annu rev pathol 10 : 263 - 289 , 2015 greijer ae , van der groep p , kemming d , et al : up - regulation of gene expression by hypoxia is mediated predominantly by hypoxia - inducible factor 1 ( hif - 1 )  . j pathol 206 : 291 - 304 , 2005 lymphoproliferative disorders associated with immune deciencies and histiocytic and dendritic cell neoplasms , in wahed a and dasgupta a : hematology and coagulation : a comprehensive review for board preparation , certication and clinical practice . 
center for personalized cancer treatment : development of a platform for next - generation dna sequencing based personalized treatment for cancer patients : protocol to obtain biopsies from patients with locally advanced or metastatic cancer ( cpct 02 biopsy protocol )  . 
chen x , schulz - trieglaff o , shaw r , et al : manta : rapid detection of structural variants and indels for germline and cancer sequencing applications . bioinformatics 32 : 1220 - 1222 , 2016 iliopoulos o , levy ap , jiang c , et al : negative regulation of hypoxia - inducible genes by the von hippel - lindau proteproc natl acad sci usa 93 : 10595 - 10599 , 1996 14 . 
soh j , okumura n , lockwood ww , et al : oncogene mutations , copy number gains and mutant allele specic imbalance ( masi ) frequently occur together in tumor cells . 
beroukhim r , brunet jp , di napoli a , et al : patterns of gene expression and copy - number alterations in von - hippel lindau disease - associated and sporadic clear cell carcinoma of the kidney . 
van allen em , wagle n , stojanov p , et al : whole - exome sequencing and clinical interpretation of formalin - xed , parafn - embedded tumor samples to guide amsterdam , netherlands , elsevier , 2013 , pp 39 - 71 precision cancer medicine . 
borad mj , champion md , egan jb , et al : integrated genomic characterization reveals novel , therapeutically relevant drug targets in fgfr and egfr pathways in sporadic intrahepatic cholangiocarcinoma . 
j pathol 221 : 125 - 138 , 2010 van der graaf wt , blay jy , chawla sp , et al : pazopanib for metastatic soft - tissue sarcoma ( palette ) : a randomised , double - blind , placebo - controlled phase 3 trial . 
morgan , md1 , 2 purpose using nonenrichment - based , potentially more sensitive epic sciences circulating tumor cell ( ctc ) platform , we sought to detect and characterize ctcs in untreated , high - risk localized prostate cancer and to evaluate their clinical implication . methods between 2012 and 2015 , blood samples were prospectively collected from patients with national comprehensive cancer network high - risk localized prostate cancer undergoing either radiotherapy ( xrt ) plus androgen deprivation therapy or radical prostatectomy ( rp ) with curative intent . 
samples were analyzed with the epic sciences platform with 4 ( cid : 1 ) , 6 - diamidino - 2 - phenylindole , cd45 , cytokeratin ( ck ) , and androgen receptor ( ar ) n - terminal staining . 
bcr occurred more frequently in the rp group than xrt ( 15 of 26 v one of 19 ) , with most patients in the xrt group continuing to receive androgen deprivation therapy . 
2019 by american society of clinical oncology introduction the existence of disseminated tumor cells detectable in the blood or bone marrow of men with clinically localized prostate cancer has long been known.1 - 4 yet , molecular characterization of these cells in the localized setting has remained elusive because of the extremely low ratio of tumor to normal cells in circulation . 
with recent advances in molecular imaging , we now know that many men with clinically localized disease have positron emission tomographyvisible metastatic disease , 5 - 9 yet these modalities are expensive and may only detect lesions consisting of at least several million tumor cells . 
additional methods of detecting otherwise occult , disseminated disease are still needed , because these may inform primary treatment decisions such as the use of systemic therapy . circulating tumor cells ( ctcs ) may provide an approach for identifying men with micrometastatic disease at the time of diagnosis . 
 salami et al context key objective to determine if circulating tumor cells ( ctcs ) can predict treatment response in high - risk localized prostate cancer . knowledge generated phenotypic and genomic features of ctcs may predict biochemical recurrence in high - risk localized prostate cancer . 
individual ctcs can be sequenced in this setting , conrming their tumor origin . relevance detection and characterization of ctcs may facilitate identication of patients with high - risk prostate cancer who could benet from multimodal therapy . in the advanced prostate cancer setting , the identication and characterization of ctcs is now poised to help guide appropriate treatment selection , because detection of the ar - v7 splice variant in ctcs predicts resistance to second - generation antiandrogens in men with metastatic castration - resistant prostate cancer ( mcrpc ) .10 - 12 previous efforts to reliably detect and analyze ctcs in localized prostate cancer , however , have not led to clinically useful assays because of the low clinical sensitivity and challenges associated with characterizing the individual detected ctcs.13 - 23 in men with mcrpc , a high - throughput digital imaging approach has been shown to increase the sensitivity of ctc detection while also providing additional molecular and morphologic granularity at the single - cell level.24 - 26 this same approach may offer one avenue to improving the sensitivity and clinical implications of ctc detection in the localized disease setting . 
adt , androgen deprivation therapy ; ctc , circulating tumor cell ; rp , radical prostatectomy ; xrt , radiation therapy . methods cohort description after institutional review board approval , we conducted a prospective study of 49 patients ( fig 1 ) with untreated national comprehensive cancer network ( nccn ) dened high - risk prostate cancer.27 these men had at least one of the following : grade group 4 or 5 disease , prostate - specic antigen ( psa ) greater than 20 , and / or clinical stage t2c or greater . 
all patients completed bone scans and computerized tomography ( ct ) scans of the abdomen and pelvis demonstrating no evidence of metastatic disease before undergoing local therapy ( either radiotherapy [ xrt ] plus androgen deprivation therapy [ adt ] or radical prostatectomy [ rp ] ) with curative intent . 
the samples were analyzed with the epic sciences ctc platform ( epic sciences , san diego , ca ) , which uses an enrichment - free approach to ctc detection , with all nucleated cells analyzed on a glass slide using digital pathology . 
 ( % ) unless otherwise noted . abbreviations : bcr , biochemical recurrence , bmi , body mass index ; n / a , not applicable ; psa , prostate - specic antigen ; rp , radical prostatectomy . subtypes inclusive of ck - negative ctcs , ctc clusters , and apoptotic ctcs ( fragmented nuclei ) .28 additional parameters included cell size , shape , nuclear area , and presence of macronucleoli , along with ar expression , uniformity , and cellular localization . 
ctc phenotypic heterogeneity was quantied using the shannon index , as previously described.29 samples from four patients failed quality control testing , leaving a nal analyzable cohort size of 45 patients ( fig 1 )  . single - cell whole - genome sequencing and copy number variation analysis single ctc copy number variation ( cnv ) analysis based on low - pass whole - genome sequencing was performed as previously described.30 , 31 briey , individual ctcs were isolated from the slides , dropped into eppendorf tubes , and whole - genome amplied using seqplex enhanced dna amplication kit from sigma ( sigma aldrich , st louis , mo )  . 
amplied dna was used for library construction using nebnext ultra dna library prep kit for illumina ( new england biolabs , ipswich , ma ) and 2 150 pairedend sequencing was performed using illumina nextsequation 500 sequencer . 
dna cnvs were determined using an existing , standardized single - cell cnv analysis pipeline.30 data collection and statistical analyses we tracked relevant demographic , clinical , and pathologic data for each patient and entered all data into a secure electronic health insurance portability and accountability actcompliant database . 
a diversity of ctc subtypes was detected , including arpositive cells , ck - positive cells , ck - negative ctcs , 28 and clusters of ctcs ( fig 2a )  . 
ctcs with detectable ar expression were observed in 24.4% ( 11 of 45 ) of the cohort . compared with patients who underwent xrt , those who underwent rp had a higher rate of bcr ( 15 of 26 v one of 19 ; p , .001 ) , although most patients in the xrt group continued receiving adt . 
 ( b ) comparison of total ctc / ml of blood , presence of androgen receptor ( ar ) positive ctcs , ctc phenotypic heterogeneity or shannon index ( si ) , metastases , and bcr for each patient . 
 ( c ) association of bcr and development of radical metastases after prostatectomy to total ctc / ml , ar - positive ctc / ml , and ctc phenotypic heterogeneity . 
all of the patients who underwent rp and xrt who experienced metastasis ( to bone , regional lymph nodes , and abdominal viscera ) during follow - up were preceded by bcr . 
patients experiencing bcr had signicantly greater numbers of arpositive ctcs , and patients developing metastases had signicantly more total ctcs and ar - positive ctcs ( figs 2b and 2c )  . single - cell sequencing and morphology analysis of all detected ctcs was performed on patient 252 , who had the highest ctc count in the rp group ( 4.7 ctcs / ml of blood )  . 
single - cell sequencing results identied genomic aberrations consistent with malignant origin in six ( 46% ) of the 13 ctcs sequenced ( fig 3b )  . moreover , heterogeneous genomic proles were observed in these six ctcs . 
pten loss was detected in this ctc akin to those ctcs found in patients with mcrpc previously analyzed with similar methodology.24 , 29 two ctcs had 12p loss and 22q loss ( prole b ) ; both of these alterations have been reported in this study , we detected ctcs in more than two - thirds of patients , demonstrating the feasibility of detecting and characterizing ctc morphology and genomics in men with localized prostate cancer . 
oncologic end points were not evaluable in this time frame for patients who underwent radiotherapy , because most of these patients continued to receive adt . various liquid biopsy approaches have been interrogated in the past for risk stratifying patients with apparent clinically localized prostate cancer with varying success . 
for example , reverse transcriptase polymerase chain reaction assay for psa , 34 - 36 an indirect approach to detect psaexpressing cells in circulation , demonstrated no signicant advantage in preoperative staging of prostate cancer . similarly , recent approaches to identifying prostate cancer ctcs have widely varied in their capacity to detect ctcs in men with localized disease.13 - 23 , 37 , 38 thus , unlike in metastatic disease , where ctc number is associated with survival , the clinical or prognostic relevance of ctc detection in localized prostate cancer remains unclear . 
this stands in contrast to other tumor types , where the detection of ctcs in localized disease is considered prognostic.39 , 40 there are a number of existing , tissue - based molecular markers for risk stratication in prostate cancer ; therefore , a key question is whether the molecular information obtained through analysis of ctcs could have any clinical relevance beyond tissue - based approaches.41 - 44 yet , prostate cancer is characterized by tumor multifocality as well as intraand interfocal genomic and transcriptomic heterogeneity , which may affect the clinical performance of tests assessing a single tumor focus.45 - 49 similarly , metastatic prostate cancer , ctcs are diverse in their phenotypic appearance and genomic makeup.29 a liquid biopsy approach may provide one avenue for addressing tumor heterogeneity and biopsy under - sampling by allowing a broader sample of potentially clinically signicant tumor cells . in line with this hypothesis , we observed signicant phenotypic diversity within the same patient and from patient to patient in our cohort of men with untreated high - risk localized prostate cancer . 
this molecular information could allow for clinical utility related to risk stratication and prediction of treatment response . the role for molecular diagnostics in localized prostate cancer continues to evolve and now also includes advanced molecular imaging techniques.5 - 9 the ultimate goal is the prediction and / or identication of early metastatic disease to guide improved treatment and long - term oncologic outcomes . 
compared with men who underwent radical prostatectomy , we observed a relatively lower rate of bcr in men who received radiation therapy , likely due to the effect of adt in this subgroup . 
furthermore , the phenotypic and genomic make - up of ctcs may provide additional prognostic and predictive information . to our knowledge , the current study is the rst to characterize ctcs at the combined protein and whole - genome single - cell level in localized prostate cancer . 
before routine clinical implementation , larger studies with longer follow - up are needed to validate our ndings and further investigate the association between ctc proles and the likelihood of subsequent metastasis . 
third , we did not measure ctcs intraor postoperatively to account for the impact of surgery on ctc count or evaluate the clearance of ctcs after surgery . in this study , we demonstrate that ctcs are not only detectable but also can be further proled in men with localized high - risk prostate cancer . 
the association of ctc subtypes , including those with high ar protein expression , with bcr and metastases suggests that characterization of ctcs may provide useful information for risk stratication and the need for multimodal therapy . 
palapattu stock and other ownership interests : nantkwest consulting or advisory role : janssen scientic affairs research funding : minomic patents , royalties , other intellectual property : implantable nanotechnology for long - term testosterone delivery functionalized ducial marker for drug delivery brent k . 
 salami et al ryon graf employment : epic sciences research funding : epic sciences travel , accommodations , expenses : epic sciences jessica louw employment : epic sciences , epic sciences ( i ) , navican genomics stock and other ownership interests : navican genomics travel , accommodations , expenses : navican genomics adam jendrisak employment : epic sciences stock and other ownership interests : epic sciences research funding : epic sciences lyndsey dugan employment : navican genomics , human longevity stock and other ownership interests : navican genomics consulting or advisory role : human longevity ( inst ) , chromacode yipeng wang employment : epic sciences stock and other ownership interests : epic sciences scott a . 
tomlins employment : strata oncology leadership : strata oncology stock and other ownership interests : strata oncology consulting or advisory role : abbvie , janssen , astellas medivation , strata oncology , sano , almac diagnostics research funding : astellas medivation ( inst ) , genomedx ( inst ) patents , royalties , other intellectual property : i am a coauthor on a patent issued to the university of michigan on ets gene fusions in prostate cancer . 
the diagnostic eld of use has been licensed to hologic / genprobe , who has sublicensed some rights to ventana medical systems / roche . travel , accommodations , expenses : strata oncology ryan dittamore employment : epic sciences leadership : epic sciences stock and other ownership interests : epic sciences patents , royalties , other intellectual property : patents pending felix y . 
feng leadership : pfs genomics stock and other ownership interests : pfs genomics consulting or advisory role : dendreon , emd serono , janssen oncology , ferring pharmaceuticals , sano , bayer , blue earth diagnostics , celgene , medivation / astellas research funding : zenith epigenetics patents , royalties , other intellectual property : i helped develop a molecular signature to predict radiation resistance in breast cancer , and this signature was patented by the university of michigan , my employer . 
morgan consulting or advisory role : myriad genetics , terumobct research funding : myriad genetics ( inst ) , mdxhealth ( inst ) , genomedx ( inst ) no other potential conicts of interest were reported . references ch ery l , lam hm , coleman i , et al : characterization of single disseminated prostate cancer cells reveals tumor cell heterogeneity and identies dormancy associated pathways . 
oncotarget 5 : 9939 - 9951 , 2014 todenh ofer t , hennenlotter j , faber f , et al : signicance of apoptotic and non - apoptotic disseminated tumor cells in the bone marrow of patients with clinically localized prostate cancer . 
prostate 75 : 637 - 645 , 2015 lilleby w , stensvold a , mills ig , et al : disseminated tumor cells and their prognostic signicance in nonmetastatic prostate cancer patients . 
morgan tm , lange ph , porter mp , et al : disseminated tumor cells in prostate cancer patients after radical prostatectomy and without evidence of disease predicts biochemical recurrence . 
oncotarget 8 : 84180 - 84192 , 2017 selns km , kr uger - stokke b , elschot m , et al : 18f - fluciclovine pet / mri for preoperative lymph node staging in high - risk prostate cancer patients . 
eur radiol 28 : 3151 - 3159 , 2018 bauman g , martin p , thiessen jd , et al : [ 18f ] - dcfpyl positron emission tomography / magnetic resonance imaging for localization of dominant intraprostatic foci : first experience . 
eur urol focus 4 : 702 - 706 , 2016 schmuck s , von klot ca , henkenberens c , et al : initial experience with volumetric 68ga - psma i&t pet / ct for assessment of whole - body tumor burden as a quantitative imaging biomarker in patients with prostate cancer . 
scher hi , graf rp , schreiber na , et al : nuclear - specic ar - v7 protein localization is necessary to guide treatment selection in metastatic castration - resistant prostate cancer . 
scher hi , graf rp , schreiber na , et al : assessment of the validity of nuclear - localized androgen receptor splice variant 7 in circulating tumor cells as a predictive biomarker for castration - resistant prostate cancer . 
scher hi , lu d , schreiber na , et al : association of ar - v7 on circulating tumor cells as a treatment - specic biomarker with outcomes and survival in castration13 . 
meyer cp , pantel k , tennstedt p , et al : limited prognostic value of preoperative circulating tumor cells for early biochemical recurrence in patients with localized resistant prostate cancer . 
shao c , liao cp , hu p , et al : detection of live circulating tumor cells by a class of near - infrared heptamethine carbocyanine dyes in patients with localized and metastatic prostate cancer . 
davis jw , nakanishi h , kumar vs , et al : circulating tumor cells in peripheral blood samples from patients with increased serum prostate specic antigen : initial results in early prostate cancer . 
kauffman ec , lee mj , alarcon sv , et al : lack of impact of robotic assisted laparoscopic radical prostatectomy on intraoperative levels of prostate cancer 100 : 608 - 610 , 2009 circulating tumor cells . 
punnoose ea , ferraldeschi r , szafer - glusman e , et al : pten loss in circulating tumour cells correlates with pten loss in fresh tumour tissue from castrationresistant prostate cancer patients . 
werner sl , graf rp , landers m , et al : analytical validation and capabilities of the epic ctc platform : enrichment - free circulating tumour cell detection and characterization . 
scher hi , graf rp , schreiber na , et al : phenotypic heterogeneity of circulating tumor cells informs clinical decisions between ar signaling inhibitors and taxanes in metastatic prostate cancer . 
anantharaman a , friedlander t , lu d , et al : programmed death - ligand 1 ( pd - l1 ) characterization of circulating tumor cells ( ctcs ) in muscle invasive and variation analysis . 
urology 43 : 765 - 775 , 1994 ignatoff jm , oefelein mg , watkin w , et al : prostate specic antigen reverse transcriptase - polymerase chain reaction assay in preoperative staging of prostate cancer . 
shariat sf , gottenger e , nguyen c , et al : preoperative blood reverse transcriptase - pcr assays for prostate - specic antigen and human glandular kallikrein for prediction of prostate cancer progression after radical prostatectomy . 
helo p , cronin am , danila dc , et al : circulating prostate tumor cells detected by reverse transcription - pcr in men with localized or castration - refractory prostate cancer : concordance with cellsearch assay and association with bone metastases and with survival . 
erho n , crisan a , vergara ia , et al : discovery and validation of a prostate cancer genomic classier that predicts early metastasis following radical prostatectomy . dju066 , 2014 118 : 3072 - 3077 , 2006 plos one 8 : e66855 , 2013 42 . 
cuzick j , swanson gp , fisher g , et al : prognostic value of an rna expression signature derived from cell cycle proliferation genes in patients with prostate cancer : a retrospective study . 
cuzick j , berney dm , fisher g , et al : prognostic value of a cell cycle progression signature for prostate cancer death in a conservatively managed needle biopsy cohort . 
klein ea , cooperberg mr , magi - galluzzi c , et al : a 17 - gene assay to predict prostate cancer aggressiveness in the context of gleason grade heterogeneity , tumor multifocality , and biopsy undersampling . 
 bayesian adaptive design for finding the maximum tolerated sequence of doses in multicycle dose - finding clinical trials purpose statistical designs for traditional phase i dose - finding trials consider dose limiting toxicity in the first cycle of treatment . 
 therefore , it is desirable to identify the maximum tolerated sequence of three doses across three cycles of treatment . methods motivated by a three - cycle dose - finding clinical trial for a rare cancer with a jak inhibitor , we proposed and implemented a simple bayesian adaptive dose - cycle finding ( basyc ) design that allows intercycle and intrapatient dose modification . 
because of the patient - specific dosing strategy over cycles , the basyc design is suited as a method in precision oncology . results basyc is simple and transparent because its algorithm can be summarized as two tabulated decision rules before the trial starts , allowing physicians to visually examine these rules . 
2018 by american society of clinical oncology introduction phase i oncology clinical trials are typically first - in - human studies characterized by a small number of patients with a goal of determining the maximum tolerated dose of an experimental drug . 
numerous dose - finding designs have been developed , such as the 3 + 3 design , 1 the continual reassessment method , 2 and the modified toxicity probability interval ( mtpi ; mtpi - 2 ) methods , 3 - 5 as recently reviewed by sverdlov et al.6 most of the dose - finding trials consider dlt within the first cycle of treatment as the primary outcome and do not allow multicycle intrapatient dose modification . 
 for example , a new developmental drug may be considered as an additional treatment in chemotherapy that includes other standard drugs over multiple cycles . our work was motivated by and applied to such a trial at the university of chicago for a rare form of leukemia . 
in this trial , a janus kinase inhibitor , ruxolitinib , was added to a three cycle us food and drug administration ( fda ) approved chemotherapy backbone that includes cycle - specific drugs ( table 1 )  . 
scheme of the dose - cycle finding clinical trial induction ( course 1 ) four - drug bfmbased regimen consolidation ( course 2 ) cyclophosphamide vincristine dexamethasome pegylated asparaginase cytarabine mercaptopurine methotrexate ( it ) interim maintenance ( course 3 ) delayed intensification ( course 4 ) methotrexate ( iv ) vincristine pegylated asparaginase methotrexate ( it ) doxorubicin cyclophosphamide vincristine dexamethasome pegylated asparaginase cytarabine thioguanine methotrexate ( it ) maintenance ( course 5 ) vincristine dexamethasone mercaptopurine methotrexate ( po ) methotrexate ( it ) screening ruxolitinib ruxolitinib ruxolitinib 4 weeks 4 - week treatment , 4 - week washout 4 - week treatment , 4 - week washout 4 - week treatment , 4 - week washout monitoring 2 - 3 years note . 
the chemotherapy backbone is based on the c10403 regimen.7 ruxolitinib is added to courses 2 to 4 as a three - cycle treatment , with potentially varying doses across cycles . abbreviations : bfm , berlin - frankfurt - munster ; it , intrathecally ; iv , intravenously ; po , orally . dose - finding study , which aimed to identify the maximum tolerated sequence ( mts ) of ruxolitinib dose levels across three cycles . 
the mts is defined as the highest sequence of three doses at which the probability of toxicity per cycle ( tpc ) is no more than or close to a prespecified target response rate pt , given that the probability of toxicity at each cycle is also no more than or close to pt . 
three doses of ruxolitinib were considered for each of the three cycles ; therefore , there were a total of 27 possible dose - cycle combinations for three cycles of treatment . the complication of the trial was the intrapatient dose modifications over cycles . 
such approaches usually work well when the sample size of the trial is sufficiently large , as seen in the dynamic treatment regimen methods , 8 reinforcement learning , 9 and sequential multiple assignment randomized trials.10 these approaches aim to better reflect the intrinsically multistage , adaptive structure of medical decisions , in both trial design and analysis of the trial data . 
the first is lee et al , 17 who proposed an innovative phase i / ii design to adaptively and dynamically optimize a patients dose in each of two cycles of therapy based on the joint binary cycle - specific efficacy and toxicity outcomes . 
 begin treatment end of treatment cycle 1 cycle 2 cycle 3 patient enrollment ith cohort trial end mts selection first cohort dar - 1 select dose level ( cid : 143 ) treat cohort at di and collect data stop ? treat at stop ? exit trial dar - 2b treat at stop ? nth cohort exit trial patients without dlts dar - 2a patients with dlts patients without dlts patients with dlts patients without dlts patients with dlts dar - 2a dar - 2b dar - 2a dar - 2b treat at treat at treat at treat at exit trial fig 1 . 
basyc consists of decisions to determine the dose in each cycle for the enrolled patients and to select the maximum tolerated sequence ( mts ) when the trial is completed . 
in each cycle , the dose allocation rule ( dar ) selects the doses for treating patients , in which dar - 1 selects the dose for cycle 1 and dar - 2 selects the dose for subsequent cycles . 
dar - 2 is composed of two subcases : dar - 2a and dar - 2b , which are applied to the two subcohortspatients without dose - limiting toxicities ( dlts ) and patients with dlts , respectively . 
for example , a patient might exit the trial after one cycle of treatment ( potentially because of toxicity [ indicated by stop ] ) based on the dcr applied in cycle 1 . 
figure 1 illustrates the general scheme of the basyc design for the motivating trial with three doses and three cycles of treatment . dcr and dar the basic concept of the dcr is that if the probability of toxicity of the lowest dose in the next cycle is considered lower than the target rate pt , the cohort can move to the next cycle for additional treatment ( ie , the decision is a go )  . 
technical details of the dcr are given in the appendix . if the dcr is a go for a cohort , the cohort will enter the next cycle for additional treatment . 
first , the dar always assigns the first cohort patients at the lowest dose level in all the cycles of treatment , which is a default rule to ensure the safety of the lowest dose in all three cycles . 
in this case , dar - 1 follows the same decision rule as in the mtpi - 2 design.5 specifically , the dose level is decided based on the observed toxicity data from previously enrolled patients treated at the most recent dose in the first cycle . 
this ad hoc rule is needed to protect patient safety . the two subcohorts undergo the dcr and dar separately hereafter until all three cycles of treatment are completed or until they exit the trial . 
motivated by the mtpi - 2 design , 5 dose allocation is presented in the form of dose escalation , stay , or de - escalation to give the candidate dose level at the next cycle relevant to the dose level of the cycle we currently consider . 
 this table is applied to the dose at which the current cohort is treated and the dose at which the previous cohort is most recently treated in the next cycle . 
the two tables can be generated and predetermined before the trial starts for examination by physicians . in the dar , we used the mtpi - 2 design as the backbone for decision making . 
the default values of 1 and 2 are 0.05. dose - finding algorithm to begin a trial , the basyc design treats the first cohort of patients at the lowest dose level in all three cycles . 
then , after the toxicity outcomes from the first cycle are observed , the dcr decides whether the cohort will go to the next cycle , and the dar decides which dose level to use if the dcr decision is go . 
this process is repeated until all cohorts finish all cycles of treatment unless the trial is terminated early because of excessive toxicity ( safety rules are given in the appendix )  . 
as a default rule , the cohort at a given cycle must wait for the cohort at the next cycle to complete follow - up before it can enter the next cycle . we demonstrate in table 2 how the basyc design guides the motivating dose - cycle trial with three doses and three cycles . 
specifically , once the current cohort completes the first cycle of treatment with observed outcome data , the next cohort can be enrolled and assigned a dose level in the first cycle . 
for example , in table 2 , in week 24 , after cohort 3 completed the first - cycle treatment at dose level 2 , the next cohort ( cohort 4 ) was enrolled and treated at dose level 1 as its first - cycle therapy . 
each column number represents the number of patients treated , and each row number represents the number of patients experiencing toxicity , at a given dose where the decision is applied . 
 ( a ) go ( g ) / no - go ( ng ) decisions according to the dose continuous rule ( dcr ) , in which the letters in blue and red represent g and ng decisions , respectively . 
 ( b ) dose allocation decisions according to the dose allocation rule ( dar ) , in which the letters in blue , gold , green , and red represent dose escalation ( e ) , staying at the current dose ( s ) , dose de - escalation ( d ) , and dose de - escalation and exclusion ( du ) because of toxicity , respectively . 
this table is applied to the dose at which the current cohort is treated and the dose at which the previous cohort is treated in the next cycle . mts selection at the end of the trial , among all the candidate sequences of dose - cycle combinations , a sequence of three doses for three cycles would be selected as the mts . 
we provided the true toxicity probabilities of the three dose levels in the first cycle and assumed that the toxicity probability would increase by 10% per cycle at the same dose level . 
for each scenario , 10 , 000 simulated trials are conducted on computer . operating characteristics the simulation results for scenarios 1 to 6 are summarized in table 3 , with four sections per scenario . 
recall that the mts is defined as the highest sequence of three safe doses with the true probability of tpc ( calculated as the average of the three true toxicity probabilities corresponding to the three dose levels ) closest to pt and falling in the ei ( pt 1 , pt + 2 )  . 
if a scenario has no dose sequence falling in the ei , the highest sequence of three safe doses with the true probability of tpc less than pt is considered the true mts . 
no practical methods are available , and the two existing designs17 , 18 in the literature are either highly complex or tailored for specific trials . perhaps the most attractive feature of the basyc design is its simplicity and transparency , as shown in figure 2 . 
we can see that escalation and de - escalation will never happen if the true toxicity probability of a dose is located in the higher interval ( pt + 2 , 1 ) and the lower interval ( 0 , pt + 1 ) , respectively . in this report , we implemented basyc for a realworld trial with three dose levels for three cycles of treatment . 
for example , a total of 53 = 125 sequences are available for trials with five dose levels and three cycles . basyc uses independent beta / binomial models as the working model to estimate the toxicity probability of each dose in each cycle based on all patients data at that dose / cycle . 
 alternatively , one could consider a dependent model , such as the markov model in fernandes et al , 18 to estimate the toxicity probabilities . in basyc , we focused on the dose - cycle trials with binary outcomes . 
with larger sample sizes , one can adopt dose - insertion methods23 , 24 to allow insertion of new doses when necessary . an mts with fixed dose levels at the end of the trial . 
for example , one may not allow a higher dose level than that in mts for any cycle of the treatment in the policy . lastly , a formal personalized dosing policy would follow the spirit of lee et al17 and use model - based inference on multiple cycles . 
stock w , luger sm , advani as , et al : favorable outcomes for older adolescents and young adults ( aya ) with acute lymphoblastic leukemia ( all ) : early results of us intergroup trial c10403 . 
doussau a , asselain b , le deley mc , et al : dose - finding designs in pediatric phase i clinical trials : comparison by simulations in a realistic timeline framework . 
doussau a , thibaut r , paoletti x : dose - finding design using mixed - effect proportional odds model for longitudinal graded toxicity data in phase i oncology clinical trials . 
guo w , ni y , ji y : teams : toxicityand efficacy - based dose insertion design with adaptive model selection for phase i / ii dose - escalation trials in oncology . 
 appendix notation suppose under consideration is a total of i candidate dose levels and j treatment cycles , indexed by i = 1 , 2 , , i ; j = 1 , 2 , , j . 
let k index the cohort , k = 1 , 2 , , k , where k is the maximum number of cohorts that will be recruited to the trial . during dose - cycle finding ( not for maximum tolerated sequence [ mts ] selection ) , we use simple and independent beta priors across doses and cycles and an independent binomial likelihood as a working model for the estimation of pij . 
the application in bayesian adaptive dose - cycle finding ( basyc ) is detailed here . dose continuation rule for cohort k that has been treated at dose i in cycle j , the dose continuation rule ( dcr ) for cohort continuation ( whether the cohort can continue treatment at cycle ( j + 1 ) ) is described as follows : if cohort k has finished the last cycle of therapy ( ie , j = j ) , cohort k exits the trial . if cohort k has not finished all cycles of therapy ( ie , j < j ) , and if pr { p1 , j + 1 > pt|data } > , where is a prespecified cutoff probability and often set to be close to 1 ( eg , = 0.95 ) , stop cohort k for any additional treatment , and patients in cohort k exit the trial . 
 otherwise , continue this cohort k to the next cycle ( j + 1 )  . this algorithm states that when the probability that toxicity of the lowest dose level ( dose level 1 ) in cycle ( j + 1 ) is higher than the target pt does not exceed a large value , cohort continuation is allowed . 
let x1 , j + 1 and n1 , j + 1 be the cumulative number of patients ( across all the previous and current cohorts ) with dose - limiting toxicity ( dlt ) events and treated at dose level 1 at cycle ( j + 1 ) , respectively . 
this beta distribution is used to calculate the posterior probability . dose allocation rule if cohort k in cycle j can proceed to the next cycle , the dose assigned to the next cycle ( j + 1 ) is dependent on the cumulative data from both the current cycle j and the next cycle ( j + 1 )  . 
such data accumulate all the toxicity outcomes from previously treated cohorts at this dose . then , consider a cohort of patients that has just completed treatment in the first cycle at a dose level . 
 here , the previous cohort refers to the cohort of patients enrolled just before the current cohort , and the toxicity data or the data ( also hereinafter ) refer to the data from all enrolled patients treated at the corresponding dose . 
specifically , given data1 and dose1 , the mtpi - 2 design will output a candidate dose level i1 , and given data2 and dose2 , mtpi - 2 will output another candidate dose i2 . 
specifically , given the dose ( dose1 ) in cycle 1 and the candidate dose i2 that mtpi - 2 gives based on data2 and dose2 from cycle 2 , dar - 2b selects the dose i = min { max { dose1 1 , 1 } , i2 } , the minimum of the dose 1 level lower than the current dose in cycle 1 , and i2 as the final dose for treating the cohort in cycle 2 . note that because of the cohort split , the previously enrolled subcohorts may have been treated in cycle 2 at two dose levels , say dose21 and dose22 . 
the outcome data from both subcohorts , denoted as data21 and data22 , will be used to inform the cycle 2 dose for the subsequent cohort that will start from the first cycle . 
in particular , for each of the data21 and data22 , the mtpi - 2 design will give two candidate doses i21 and i22 as the potential dose for the subsequent cohort in cycle 2 . 
then , the minimum of i21 and i22 will be provided as the candidate dose , denoted as i2 = min { i21 , i22 } , for the subsequent cohort as the cycle 2 candidate dose . 
the same principle is applied to decide the dose level in cycle 3 if needed . walkthrough of the hypothetic trial table 2 summarizes how the basyc design guides the motivating dose - cycle trial with three doses and three cycles . 
at this point , cohort 2 is enrolled and assigned to the next higher dose , dose 2 ( d2 ) , according to the dar decision , because cohort 1 has not exhibited any toxicity . 
at week 16 , cohort 1 completes treatment at d1 in cycle 2 with 0 toxicity outcome , and cohort 2 completes treatment at d2 in cycle 1 with 1 dlt outcome . 
the single patient with dlt , patient 4 , is de - escalated to d1 , and the remaining two patients without dlts , patients 5 and 6 , stay at d2 . 
this process continues for subsequent cohorts and cycles at weeks 24 , 32 , and so on , until all 15 patients ( or five cohorts ) have completed treatment at week 56 . safety rule applied in basyc for safety , the basyc design applies two additional safety rules throughout the entire trial : rule 1 ( early termination ) : for any cycle , if the toxicity probability of the lowest dose is higher than the target pt with a large probabilitythat is , if j * { 1 , 2 , , j } such that pr { p1j * > pt|data } > , where is close to 1 ( say = = 0.95 ) terminate the trial early . rule 2 ( dose exclusion ) : if the toxicity probability of dose i in cycle j is higher than the target pt with a large probability , pr { pij > pt|data } > , exclude doses i and the higher doses from cycle j and higher cyclesthat is , doses { i , i + 1 , , i } will never be used in the trial in cycles { j , j + 1 , , j } again . the posterior probabilities in both rules are calculated based on the simple beta / binomial calculation proposed in the mtpi design.3 details are provided in appendix under dose continuation rule . mts definition and selection after the enrollment and dose - cycle finding are completed , in the mts selection , we use a frequentist and nonparametric probability model to make inference . 
specifically , we essentially want to estimate the toxicity per cycle ( tpc ) qs , which is the average of the three toxicities of the doses in a sequence s ( described under definition of mts )  . 
we then construct an unbiased estimate of tpc qs ( described under estimation of qs ) and order - transform them based on isotonic regression ( described under mts selection )  . 
for each sequence s , we define the cumulative dose level as the sum of the dose levels across three cycles ( ie , d * s = ds1 + ds2 + ds3 )  . 
for each sequence s , denote pdsj , j the probability of toxicity for the dose in cycle j of sequence s , j = 1 , 2 , 3 . 
a sequence s is unacceptable if the toxicity probability of each dose in each cycle is higher than pt with a large probability , defined as j = 1 , 2 , 3 , pr { pdsj , j > pt|data } > , where dsj denotes dose levels { 1 , 2 , 3 } and is close to 1 , say = 0.95. 
the posterior probability pr { pdsj , j > pt|data } is based on a simple beta / binomial hierarchic model , similar to the calculation under dose continuation rule . 
 among all the safe sequences that satisfy the condition above , we want to select the highest sequence with overall qs close to pt , subject to the toxicity probability of the same dose level nondecreasing across cycles . 
however , the problem with that approach is that because of random chance , a sequence with a higher order ( and therefore higher toxicity ) might have a smaller qs than a lower - ordered sequence . 
for example , it is possible that the toxicity data for sequence 1 , 1 , 1 include only one toxicity event in all three cycles , whereas the data for sequence 2 , 1 , 1 include no toxicity event . 
however , sequence 2 , 1 , 1 is in theory a more toxic sequence , because it uses dose level 2 in cycle 2 instead of dose level 1 . 
therefore , selecting the sequence based on the distance of |qs pt| is not valid , because we would select 1 , 1 , 1 as the mts , not 2 , 1 , 1 in this case . to address this issue , we propose an isotonic transformation and calculate transformed qs values using the pool adjacent violator algorithm ( pava ) 20 just like in the mtpi design3 , 4 and mtpi - 2 design.5 here , we still use the sequence - specific data for the isotonic transformation . 
when there is a tie between m sequences , there are m ! possible simple orderings among thethen , assuming with probability 1 / m ! that one of the orderings , say ordering w , is true , we apply pava to the following ordering w of qs , w = 1 , 2 , , m !  . 
then , finally , the sequence dl * * that has nonincreasing dose levels across three cycles and the largest frequency f l * is selected as the estimated mts . 
therefore , the dose levels of the mts cannot be increasing , or else the lower dose in the early cycle can always be replaced by the higher dose in the later cycle . 
these results show that basyc is strong in protecting patient safety . scenario 2 presents a similar pattern of dose - toxicity relationship to scenario 1 , except that the toxicity probability of dose level 2 is now below pt = 30% . 
this suggests that the basyc design is nimble and can reach high dose - cycle combinations even with small sample sizes . scenario 4 presents an opposite case to scenario 3 , because all the doses are overly toxic with toxicity probabilities above pt . 
this scenario demonstrates the ability of the basyc design to stop the trial quickly in case of excessive toxicity in all dose sequences . scenarios 5 and 6 reflect situations where there is a leap in toxicity probabilities across the three doses . 
oxnard , md1 noninvasive genotyping of plasma cell - free dna is being integrated into cancer care at an astonishing rate , as its potential to radically increase access to personalized cancer care is increasingly recognized.1 whereas next - generation sequencing ( ngs ) of tumor has been commoditized and democratized ( with many academic hospitals offering their own clinical laboratory improvement amendmentscertied tests ) , plasma ngs is primarily sent out for testing at commercial laboratories . 
however , although these tests are ordered and reported en masse , the causes of falsepositive or false - negative plasma ngs results have only gradually become apparent ( fig 1 )  . false - negative results have long been recognized as common with blood - based assays . 
we and others identied that assay sensitivity is closely related to clinical factors such as stage and metastatic spread , tumor dna in cases suggesting limited shed of missed by plasma ngs.2 , 3 false positives have also been described with many plasma genotyping assays and are routinely attributed to tumor heterogeneity.4 although heterogeneity is clearly a source of tumor / plasma discordance in cancers that have developed drug resistance , 5 , 6 many false positives can be attributed to dna shed from normal cells , including germline variants or noncancerous somatic variants such as clonal hematopoiesis ( ch ) .7 - 9 the latter is particularly challenging because ch can involve cancer - associated genes ( eg , tp53 , jak2 , kras )  . 
yet when such mutations are identied in plasma , they may not reect true tumor genotype . what is least understood is how technical factors related to assay performance contribute to false - positive and false - negative results . 
probe design and specics of library generation are often considered proprietary and are only minimally described in the few analytical validation studies that have been published . this is the context motivating the publication by stetson et al10 in jco precision oncology . 
the authors sent aliquots of plasma to four commercial clinical laboratory improvement amendmentscertied laboratories for plasma ngs and compared this to ngs performed on matched tumor and normal tissue at foundation medicine . 
the vendors were blinded to tumor ngs results but knew that their plasma ngs results would be compared with orthogonal tissue results , as well as with plasma results from other laboratories . 
following bioinformatic analysis and variant calling , binary alignment map les were provided by the vendors to the authors for independent , unblinded , post - hoc sequence analysis . with this design , the authors were not only able to address many of the pitfalls that plagued prior vendor comparison studies , 11 , 12 but they could also investigate interassay technical factors . 
more precisely , stetson et al10 were able to account for known germline variants , investigate mutation - calling biases , and examine whether false negatives were as the result of stochastic sample biases or thresholding nuances of the vendors bioinformatic lter . the results should give pause . 
positive predictive value ( ppv ) against tissue ranged from 36% to 80% . however , results improved when limited to mutations called at an allelic fraction ( af ) greater than 1% , with three vendors achieving a ppv of 100% for these higher af calls . 
note that variants detected at less than 1% af are routinely reported by each vendor , and such sensitivity is advertised as a unique strength of plasma ngs assays . false - positive variants tended to be novel with no reports in somatic variant databases , and these were often related to vendor - specic mutational biases . false negatives were attributed to bioinformatic ltering of suspected germline variants and limitations from high signal - to - noise ratio . 
taken together , these results point to recurrent contributions from technical differences in the bioinformatic pipelines of the assays , assay sensitivity , or plain error . the current study is far from perfect and leaves room for improvement . 
the study cohort consisted primarily of early - stage cancers ( 21 of 24 patients had stage i and ii cancers ) , which are not the intended population for plasma ngs . 
beyond these established biologic factors , technical factors may be an underappreciated source of erroneous plasma ngs . technical factors tett chnical factor tumor dna shed tumor heterogeneity ( particularly at resistance ) tett chnical factors technical factors white blood cell dna ( clonal hematopoiesis , germline variants ) finally , reference truth was extrapolated across four different assays . 
furthermore , this analysis does not broach the challenges of reporting plasma ngs results . one could imagine that a forward - thinking laboratory might recognize the limitations of plasma ngs in its reporting , noting the risk of false positives for low af or ch - associated variants , as well as the risk of false negatives when low tumor shed is apparent . as the use of plasma ngs becomes increasingly widespread in cancer care , there remains a clear need for concordance studies such as this one . 
 editorial policy , please refer to or ascopubs.org / po / authorcenter . patents , royalties , other intellectual property : patent pending on noninvasive blood - based monitoring of genomic alterations in cancer ( inst ) cloud p . 
paweletz honoraria : astrazeneca korea , bio - rad consulting or advisory role : dropworks research funding : guardant health , astrazeneca travel , accommodations , expenses : astrazeneca korea geoffrey r . 
oxnard honoraria : chugai pharma , bio - rad , sysmex , guardant health consulting or advisory role : astrazeneca , inivata , sysmex , takeda , loxo oncology , ignyta , dropworks , grail no other potential conicts of interest were reported . references aggarwal c , thompson jc , black ta , et al : clinical implications of plasma - based genotyping with the delivery of personalized therapy in metastatic non - small cell lung cancer . 
jama oncol 2 : 1014 - 1022 , 2016 bettegowda c , sausen m , leary rj , et al : detection of circulating tumor dna in earlyand late - stage human malignancies . 
sci transl med 6 : 224ra24 , 2014 forshew t , murtaza m , parkinson c , et al : noninvasive identication and monitoring of cancer mutations by targeted deep sequencing of plasma dna . 
sci transl med 4 : 136ra68 , 2012 oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced non - smallcell lung cancer . 
clin cancer res 24 : 4437 - 4443 , 2018 oxnard gr , hu y , mileham kf , et al : assessment of resistance mechanisms and clinical implications in patients with egfr t790m - positive lung cancer and acquired resistance to osimertinib . 
hu y , alden rs , odegaard ji , et al : discrimination of germline egfr t790m mutations in plasma cell - free dna allows study of prevalence across 31 , 414 cancer patients . 
clin cancer res 23 : 7351 - 7359 , 2017 slavin tp , banks kc , chudova d , et al : identication of incidental germline mutations in patients with advanced solid tumors who underwent cell - free circulating tumor dna sequencing . 
stetson d , ahmed a , nuttall brb , et al : orthogonal comparison of four plasma ngs tests with tumor suggests technical factors are a major source of assay discordance . 
paweletz cp , sacher ag , raymond ck , et al : bias - corrected targeted next - generation sequencing for rapid , multiplexed detection of actionable alterations in cell - free dna from advanced lung cancer patients . 
guibert n , hu y , feeney n , et al : amplicon - based next - generation sequencing of plasma cell - free dna for detection of driver and resistance mutations in advanced non - small cell lung cancer . 
rna - seq allows the use of a method called the percent splicing index to quantify allelic expression by mapping rna - seq reads from alternate or aberrant transcripts relative to reference ( normal ) transcripts as described by schafer et al.7 we have recently published data demonstrating that the variant brca2 c.426 - 12_426 - 8delgtttt not only results in partial skipping of exon 5 ( brca2 r.426_475del ) , but this splicing event was also seen in 20 of 345 control patients.8 laboratories should proceed with caution when weighting functional evidence in variant curation , because interpretation of these results can be complex . 
in this specic case , the rna - seq results and the new clinical data prompted reassessment and resulted in reclassication of this brca2 variant . the article by nix et al6 highlights the need for laboratories to increase collaboration , particularly when siloed clinical information can shed light onto discrepant classications . 
submission of a classication and its justication to dynamic databases such as clinvar is a critical step in this process , 9 because data sharing among clinical diagnostic laboratories improves the resolution of variant interpretation differences.10 in summary , we believe that innovation , such as introduction of rna - seq to hereditary cancer clinical genetic testing as well as participation in multi - institutional efforts designed to increase transparency between laboratories , is of utmost importance for advancing variant classication that will ultimately benet patients . rachid karam , md , phd ; holly laduca , ms ; marcy e . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . rachid karam employment : ambry genetics research funding : ambry genetics travel , accommodations , expenses : ambry genetics holly laduca employment : ambry genetics stock and other ownership interests : ambry genetics marcy e . 
richardson employment : ambry genetics tina pesaran employment : ambry genetics stock and other ownership interests : ambry genetics travel , accommodations , expenses : ambry genetics elizabeth chao employment : ambry genetics no other potential conicts of interest were reported . references cummings bb , marshall jl , tukiainen t , et al : improving genetic diagnosis in mendelian disease with transcriptome sequencing . 
sci transl med 9 : eaal5209 , 2017 fr esard l , smail c , ferraro nm , et al : identication of rare - disease genes using blood transcriptome sequencing and large control cohorts . 
nat med 25 : 911 - 919 , 2019 gonorazky hd , naumenko s , ramani ak , et al : expanding the boundaries of rna sequencing as a diagnostic tool for rare mendelian disease . 
am j hum genet 104 : 466 - 483 , 2019 lee h , huang ay , wang l - k , et al : diagnostic utility of transcriptome sequencing for rare mendelian diseases . 
genet med 22 : 490 - 499 , 2020 karam r , conner b , laduca h , et al : assessment of diagnostic outcomes of rna genetic testing for hereditary cancer . 
nix p , mundt e , manley s , et al : functional rna studies are a useful tool in variant classication but must be used with caution : a case study of one brca2 variant . 
jco precis oncol 4 : 730 - 735 , 2020 schafer s , miao k , benson cc , et al : alternative splicing signatures in rnaseq data : percent spliced in ( psi )  . 
curr protoc hum genet 87 : 11.16.111.16.14 , 2015 landrith t , li b , cass aa , et al : splicing prole by capture rna - seq identies pathogenic germline variants in tumor suppressor genes . 
considering that the codon of asparagine is aac or aat , we wondered if the adenine base editor ( abe ) , which induces at to gc conversion at specific site , could be used to reduce pd - 1 suppression by changing the glycosylated residue in car - t cells . 
our study suggested that the single base editors can be used to augment car - t cell therapy . keywords : glycosylation , single base editing , car - t cell , pd - 1 to the editor , chimeric antigen receptor t ( car - t ) cells are not satisfying in treating solid tumors [ 1 ]  . 
crispr / cas9 can downregulate pd - 1 [ 2 ] but also potentially leads to carcinogenesis , because success of such tools relies on suppressing dna damage response [ 3 ]  . 
as n - linked glycosylation is restricted on asparagine coded by aac or aat , adenine base editors ( abe ) can convert at to gc base pair [ 5 ] , and may be used to diminish such glycosylation . 
the delivery of grna using lentivirus is efficient [ 6 ] , so we constructed lentiviral vectors simultaneously expressing mesothelin - directed car and grna targeting non - specific sites ( scramble ) or n74 of pdcd1 ( grna ) , under 2 independent promoters ( additional file 1 : figure s1a )  . 
2h ( cid : 150 ) ( additional files 3 and 4 )  . single base editing can modulate the stability and function of target protein by changing a single residue [ 8 ]  . 
compared with crispr / cas9 , abe has narrower editing window and much less frequent off - target events [ 9 ] , representing a safer and more precise approach for gene editing . 
abe - mediated point mutation can downregulate the inhibitory pd - 1 , therefore providing an alternative approach to augment t cell immunotherapy . authors contributions yz and fl devised the project and supervised the experiments . 
all authors read and approved the final manuscript . funding this work received funds from the national key research and development program ( 2018yfc1313400 , 2016yfc1303501 ) ; national natural science foundation of china ( 81771781 , u1804281 )  . availability of data and materials all supporting data are included in the manuscript and supplemental files . additional data are available upon reasonable request to corresponding author . ethics approval and consent to participate all experiment procedures were followed the protocol approved by institute committee on ethics . 
and all animal experiments were performed in accordance with the ethics approval obtained from the first affiliated hospital of zhengzhou university animal ethics committee . consent for publication not applicable competing interests the authors declare that they have no competing interests . author details 1biotherapy center , the first affiliated hospital of zhengzhou university , zhengzhou 450052 , henan , china . 
3school of life sciences , zhengzhou university , zhengzhou 450052 , henan , china . 4henan key laboratory for tumor immunology and biotherapy , zhengzhou 450052 , henan , china . received : 3 november 2019 accepted : 13 november 2019 supplementary information supplementary information accompanies this paper at 1186 / s13045 - 019 - 0831 - 5 . next - generation sequencing directs therapy and delineates a clonal relationship in mast cell sarcoma and acute myeloid leukemia timothy j . 
collins jr , md1 a 61 - year - old man was diagnosed with acute myeloid leukemia ( aml ) associated with trisomy 8 cytogenetic abnormality and flt3 itd mutation . 
post - transplantation , he showed no morphologic or ow cytometric evidence of disease and his bone marrow chimerism was more than 95% donor cells at several timepoints post - transplantation , including 24 months after the transplant . 
he received tacrolimus post - transplantation and developed mild chronic graft - versus - host disease that was limited to the skthe patient remained on tacrolimus . twenty - nine months after transplantation , he presented with abdominal pacomputed tomography scan of the abdomen and pelvis revealed a known , unchanged ventral abdominal hernia , but also showed a new lytic lesion on the inferior pubic ramus that was concerning for a new malignancy ( fig 1a )  . 
flow cytometry of the lesion demonstrated a predominance of cd117bright positive , cd34 - negative myeloid cells suspected to be mast cells that did not aberrantly express cd2 or cd25 ( fig 3 )  . 
these cells showed atypical expression of fcri ( cd64 ) , which is seen upon mast cell activation . immunohistochemical analysis demonstrated that the cells were positive for cd117 and weakly positive for mast cell tryptase and negative for cd2 , cd25 , and cd34 ( fig 4 )  . 
altogether , supportive of a diagnosis of mast cell sarcoma ( mcs )  . the tumor next - generation sequencing ( foundationone heme panel ; foundation medicine , cambridge , ma ) on the biopsy was signicant for kit y503_f504insay , idh2 r140q , and srsf2 p95h mutations , but was negative for flt3 mutations . 
the particular kit mutation detected has been reported to be responsive to imatinib.1 - 4 the idh2 mutation has not previously been identied in mast cell neoplasms.5 the patient was treated with imatinib 400 mg per day in addition to radiation to the sacrum and pubic ramus4 , 500 cgy in 25 fractions . 
he remains without evidence of aml recurrence 71 months after hematopoietic sct . the original diagnosis of aml and subsequent bone marrow evaluations were reviewed in light of the development of mcs . 
cytogenetic studies revealed 47xy , + 8 [ 13 ] / 46 , xy [ 7 ] and uorescence in situ hybridization was positive for an extra signal for runx1t1 ( 8q22 ) in 59 of 200 interphase cells . 
sanger sequencing on dna from a saliva sample demonstrated wild - type germline idh2 and srsf2 . identical idh2 and srsf2 mutations implied a clonal relationship between the mcs and aml ( fig 5 )  . 
 brown et al context and mast cell sarcoma ? can tools , such as next - generation sequencing ( ngs ) , delineate a clonal relationship in a patient with acute myeloid leukemia we identied a probable clonal relatedness between the two neoplasms , with both sharing identical idh2 and srsf2 mutations . 
this report provides anecdotal evidence of a clonal relationship between acute myeloid leukemia and mast cell sarcoma that is developmentally feasible and describes a unique scenario in which ngs was effective at directing therapy . 
clinicians should consider performing ngs in patients with two neoplasms , particularly if a progenitor cell is feasible , as well as in patients with mast cell disorders and a neoplasm . the clonal progenitor that harbored the idh2 and srsf2 mutations persisted . 
shared mutations between aml and mcs support the existence of a preleukemic clonal progenitor that developed into aml initially and later into mcs . mcs was rst described in 1986 and is one of many diseases arising from the spectrum of mastocytosis.11 - 13 it typically presents as a solitary mass that may arise anywhere in the body and has intensely avid uorodeoxyglucose uptake on petcomputed tomography scan.9 - 11 , 14 - 20 diagnosis depends on the synthesis of pathologic and immunophenotypic that are features in a patient with an aggressive , initially sarcoma - like tumor.16 , 17 although initially localized , these tumors typically metastasize fig 1 . 
 ( a ) computed tomography ( ct ) scan of the abdomen and pelvis obtained for evaluation of abdominal pain incidentally revealed a destructive lesion of the left inferior pubic ramus that measured approximately 3 cm 2 cm , worrisome for malignancy . 
survival ranges from 2 months to 8 years , with a median survival of 6 months.10 , 12 , 15 , 16 , 18 - 21 cells are usually more atypical than those observed in systemic mastocytosis , with an epithelioid appearance with abundant granular cytoplasm and bilobed or multilobed nucleoli.15 , 22 mcs can be misdiagnosed on initial pathologic review given its rarity and resemblance to other tumor types , including lymphomas . 
these cells often lose markers that are characteristic for mast cells , which further complicates the diagnosis.20 , 23 evaluation of markers that are helpful to establish the diagnosis include cd2 , cd25 , cd30 , cd33 , cd34 , cd45 , cd117 , and mast cell tryptase.12 , 18 absence of cd117 positivity should call into question the diagnosis of a mast cell disorder . 
mast cell tryptase is highly specic for mast cell lineage , but may only show variable positivity in mcs.12 , 23 other immunohistochemical markers that overlap with melanoma , lymphomas , or other histiocytic processes have been reported.12 in the absence of another concurrent myeloid neoplasm , serum tryptase elevation strongly supports the diagnosis of a mast cell disorder.12 , 23 mcs have possessed kit mutations in eight of 15 cases that have reported on kit mutation testing , including the present case.10 , 11 , 19 , 20 cells with kit mutations fig 2 . 
flow cytometry of pubic ramus lesion detected a population of medium - sized to large cells , which accounted for approximately 60% of the total events , with high orthogonal light scatter indicative of high granularity / internal complexity ( red , abnormal cells ; blue , lymphocytes )  . 
abnormal cells were cd15 negative , cd117 bright positive , cd7 equivocal , cd13 + , cd2 , cd25 , cd36 , and cd64 + ( variable intensity )  . 
by immunohistochemistry , tumor cells were positive for cd117 and mast cell tryptase ( weakly ; top ) but negative for cd2 and cd25 ( bottom )  . cd25 remain cd117 bright on immunohistochemistry and are unaffected by changes induced by these mutations.24 kit mutations do not affect the ow cytometric detection of cd117 . therapy of mcs is poorly dened but typically involves surgery and radiation to localized lesions ; chemotherapy is usually ineffective.19 , 20 we would suggest that all patients with suspected mcs have kit sequenced . 
results of next - generation sequencing on both mcs and aml mutated allele genomic alteration frequency , % detected fda - approved therapy for patients tumor kit ( y503_ f504insay ) idh2 ( r140q ) srsf2 ( p95h ) flt3 ( s451f ) idh2 ( r140q ) ptpn11 d61h - subclonal n308dsubclonal srsf2 ( p95h ) imatinib none * none none none none enasidenib ( 2017 ) common myeloid progenitor cell idh2 srsf2 idh2 srsf2 flt3 ptpn11 mast cell idh2 srsf2 note . 
enasidenib was approved by the fda in 2017 , several years after the patient had aml . abbreviations : aml , acute myeloid leukemia ; fda , us food and drug administration ; mcs , mast cell sarcoma . * although enasidenib is not fda approved for mcs , it is conceivable that the drug , which targets mutant idh2 , may have activity in this patients case . and thus remains sensitive to imatinib.28 mcs with d816v mutations may be sensitive to other tyrosine kinase inhibitors , including avapritinib , which selectively targets the d816v mutation and is well tolerated.29 , 30 of interest , the idh2 mutation in our patients case is shared between aml and mcs , which suggests that it was present in a lessdifferentiated common progenitor cell . 
collins jr employment : university of texas southwestern medical centersimmons cancer center honoraria : optumhealth research funding : agios , arog , bristol - myers squibb , celgene no other potential conicts of interest were reported . references 2011 guo t , hajdu m , agaram np , et al : mechanisms of sunitinib resistance in gastrointestinal stromal tumors harboring kitay502 - 3ins mutation : an in vitro mutagenesis screen for drug resistance . 
br j haematol 173 : 323 - 326 , 2016 cancer genome atlas research network ; ley tj , miller c , et al : genomic and epigenomic landscapes of adult de novo acute myeloid leukemia . 
cell 168 : 1041 - 1052.e18 , 2017 yamashita a , saito t , akaike k , et al : mast cell sarcoma of the sternum , clonally related to an antecedent germ cell tumor with a novel d579del kit mutation . virchows arch 470 : 583 - 588 , 2017 10 . 
georgin - lavialle s , aguilar c , guieze r , et al : mast cell sarcoma : a rare and aggressive entityreport of two cases and review of the literature . 
krauth mt , f odinger m , rebuzzi l , et al : aggressive systemic mastocytosis with sarcoma - like growth in the skeleton , leukemic progression , and partial loss of mast cell differentiation antigens . 
verstovsek s , tefferi a , cortes j , et al : phase ii study of dasatinib in philadelphia chromosome - negative acute and chronic myeloid diseases , including systemic j cancer 42 : 1093 - 1103 , 2006 mastocytosis . 
brunner am , neuberg ds , wander sa , et al : isocitrate dehydrogenase 1 and 2 mutations , 2 - hydroxyglutarate levels , and response to standard chemotherapy for patients with newly diagnosed acute myeloid leukemia . 
schuurhuis gj , heuser m , freeman s , et al : minimal / measurable residual disease in aml : a consensus document from the european leukemianet mrd working party . 
leach , ms , lgc1 ; jessica marquard , ms , lgc1 ; manmeet ahluwalia , md1 ; hetty carraway , md , mba1 ; petros grivas , md , phd2 ; davendra p.s. 
recent data sets indicate that the incidence of hereditary cancer may be as high as 17.5% in patients with cancer , and a notable subset is missed if screening is solely by family history and current syndrome - based testing guidelines . identication of germline variants has implications for both patients and their families . 
we aimed to summarize genes linked to hereditary cancer and the somatic and germline panels that include such genes . methods germline predisposition genes were chosen if commercially available for testing . 
penetrance was dened as low , moderate , or high according to whether the gene conferred a 0% to 20% , 20% to 50% , or 50% to 100% lifetime risk of developing the cancer or , when percentages were not available , was estimated on the basis of existing literature descriptions . results we identied a total of 89 genes linked to hereditary cancer predisposition , and we summarized these genes alphabetically and by organ systewe considered four germline and six somatic commercially available panel tests and quantied the coverage of germline genes across thecomparison between the number of genes that had germline importance and the number of genes included in somatic testing showed that many but not all germline genes are tested by frequently used somatic panels . conclusion the inclusion of cancer - predisposing genes in somatic variant testing panels makes incidental germline ndings likely . 
2019 by american society of clinical oncology introduction cancer is a genetically driven disease triggered by the accumulation of variants in genes responsible for regulation of cell growth , invasion , survival , and migration . 
most cancers are sporadic and result from acquired genetic changes known as somatic variants , which are caused by stochastic events that accumulate over time as well as by specic environmental factors , such as smoking and ultraviolet radiation , among others.1 precision oncology currently consists of an effort to understand the acquired genetic variants that underlie each patients cancer , which thus allows the opportunity to tailor therapy to a patients individual tumor biology.2 although tumor testing previously involved sequencing of individual genes or short hotspot fragments of dna , recent advances in next - generation sequencing ( ngs ) technology have enabled much more frequent and precise identication of pathogenic variants.2 , 3 such sequencing platforms are rapidly becoming faster and cheaper , which makes them as available and as popular as many other clinical testing tools.4 , 5 germline variants occur in germ cells , which are responsible for reproduction . 
although somatic variants occur in a single cell and are passed down only to that cells offspring , germline variants exist in all somatic cells and can inuence cancer susceptibility by affecting multiple cellular processes ( eg , cancer clone growth , somatic variant acquisition rates , and carcinogen metabolism ) .1 discovery and understanding of germline variants are of major clinical importance to the patient diagnosed with a hereditary cancer ; the identication of an inherited genetic variant often has therapeutic and prognostic value for a patient undergoing therapy . 
 akras et al showing promise in the context of both somatic and germline variants in brca and other homologous repair genes and represent a breakthrough niche in precision oncology.7 therapeutic consequences of other germline ndings are also expanding ; this is best highlighted by the recent nding that lynch syndromerelated cancers are exquisitely sensitive to programmed cell death - 1 inhibition.8 finally , a germline nding is clinically important in that members of the patients broader family who also carry inherited pathogenic variants will be at an increased risk of cancer as well.9 targeted surveillance is important for all carriers of germline pathogenic variants , whether they are cancer survivors or asymptomatic relatives ; the vast majority of inherited cancer predisposition syndromes increase the risk of multiple cancers , and targeted surveillance for the discrete subset of cancers associated with that particular gene should be undertaken . 
in addition , data support that gene - specic , directed cancer surveillance programs can be life - saving for carriers.10 , 11 thus , identication of pathogenic variants in the germline is critical for the improvement of health outcomes in cancer treatment and prevention . previously , it had been estimated that a small minority of cancers ( 5% to 10% ) are a result of inherited germline pathogenic variants.9 emerging data sets in the post - ngs era indicate that the incidence of hereditary cancer may be as high as 17.5% in patients who carry a diagnosis of cancer and even higher in specic histologic types , such as ovarian cancer and urothelial carcinoma.12 - 14 multiple studies have shown that somatic tumor dna sequencing may lead to incidental ndings of hereditary risk.15 , 16 this realization magnies the potential impact of somatic testing and prompts the need for a re - evaluation of companion germline diagnostic methodology . 
in this review , we provide a summary of genes related to hereditary cancer and assess the comprehensiveness of somatic and germline panels that test them . genes with germline implications table 1 catalogs cancer predisposition genes organized by tumor type ; each row lists all associated germline genes for a given tumor type . 
as an alternate reference , table 2 lists cancer predisposition genes in alphabetical order ; each row lists associated cancers and the estimated penetrance of these cancers for that particular gene . 
the dual table organization allows an oncologist to use either the clinical presentation or the results of somatic genetic testing as a screening tool to understand if a patients family history or somatic genetic testing result , respectively , may represent a germline variant . reviewed genes were chosen on the basis of their identication as cancer - susceptibility genes for which commercial germline testing is available from the four major germline testingfocused companies in the united states . 
for each cancer predisposition gene in table 2 , associated cancers are broadly categorized by the estimated penetrance categories ( high , medium , and low penetrance ) for that gene . 
penetrance was dened as low , moderate , or high , according to a 0% to 20% , 20% to 50% , or 50% to 100% lifetime chance of developing that particular cancer or , when percentages were unavailable , an estimate based on descriptions in the existing literature.17 it is important to note that different studies may declare varying penetrance rates for a gene , so we aimed to reect the ndings of the literature at large . as seen in table 2 , cancer - associated genes have greatly varying penetrance rates for different phenotypes ( ie , different types of cancer )  . 
for example , pathogenic variants in lynch syndrome genes ( ie , epcam , mlh1 , msh2 , msh6 , pms2 ) are not highly penetrant for pancreatic cancer but are for colorectal cancer . 
these differences in penetrance also underscore the importance of gene - based , rather than syndromic or histology - based , interpretations , given the unique physiology that underlies specic variants . in addition , several germline variants confer an increased risk only if they are biallelic ( ie , both alleles of the gene carry pathogenic variants )  . 
if genes are not marked as such , they can be assumed to confer risk when monoallelic ( ie , only one allele is altered )  . in the appropriate scenario , these tables can be used to guide genetic testing and counseling ; however , they should the current clinical be used with an understanding of limitations of such data . 
cancer subtypes and associated hereditary genes that confer risk ( continued ) cancer type associated germline genes skin ( scc ) small bowel ( ac ) thyroid acd , blm , cdkn2a , fancc , recql4 , terc , tert , tp53 apc , bmpr1a , epcam , mlh1 , msh2 , msh6 , mutyh , pms2 , smad4 , stk11 apc ( ptc ) , hras , mutyh , pten ( ftc , ptc ) , ret ( mtc ) , wrn ( ftc , ptc ) abbreviations : ac , adenocarcinoma ; acc , adrenocorticocarcinoma ; all , acute lympocytic leukemia ; aml , acute myeloid leukemia ; bcc , basal cell carcinoma ; cel , chronic eosinophilic leukemia ; cll , chronic lymphocytic leukemia ; cmml , chronic myelomonocytic leukemia ; eoc , epithelial ovarian cancer ; erms , embryonal rhabdomyosarcoma ; ftc , follicular thyroid cancer ; gbm , glioblastoma multiforme ; gist , gi stromal tumor ; hb , hepatoblastoma ; hcc , hepatocellular carcinoma ; lam , lymphangioleiomyomatosis ; mds , myelodysplastic syndrome ; mel , melanoma ; mtc , medullary thyroid cancer ; nb , nephroblastoma ; nsclc , nonsmall - cell lung cancer ; pnet , pancreatic neuroendocrine tumors ; ppb , pleuropulmonary blastoma ; ptc , papillary thyroid cancer ; rcc , renal cell carcinoma ; scc , squamous cell carcinoma ; sccoht , small - cell carcinoma of the ovary , hypercalcemic type ; scst , sex cord stromal tumors ; sctat , sex cord tumors with annular tubules ; seb , sebaceous gland carcinoma ; sega , supependymal giant cell astrocytoma ; tcc , transitional cell carcinoma ; t - pll , t - cell prolymphocytic leukemia . clinical benet to the patient . 
testing relatives can help identify those at an increased risk of developing similar cancers ; however , a challenge to counseling still exists , because these variants lack the cohesive screening guidelines of their high - penetrance counterparts , and they may require a more individualized plan . 
differences between the number of genes currently tested in widely available germline panels , even compared with the number of genes linked to cancer predisposition as listed in table 2 , underscore the rapidly shifting understanding of clinically pertinent germline genes and the wide differences in opinion about what constitutes a clinically useful gene list . variants noted as rare ( labeled - r in table 2 ) are included for a more comprehensive guide to known germline - related malignancies . 
given the young age at which cancers develop in these patients or the other profound organ dysfunction associated with the respective syndromes , germline variants in such genes are unlikely to be carried through generations . furthermore , these syndromes often have clearly associated features that readily identify them clinically , and genetic testing only conrms a suspected diagnosis . 
one such example is wrn , the gene implicated in werner syndrome ; individuals with this disease display severe growth retardation , lipodystrophy , and progeria that would be obvious on examination . 
although familial inheritance of these genes has on occasion been described , such cases should be regarded as the rare exception rather than the rule . currently available somatic variant panels somatic variant panels test many more genes than their germline counterparts do . 
some prominent somatic tests are caris molecular intelligence ( caris life sciences , irving , tx ) , foundationone cdx ( foundation medicine , cambridge , ma ) , genpath onkosight ( genpath oncology , elmwood park , nj ) , msk - impact ( memorial sloan kettering cancer center , new york city , ny ) , omniseq comprehensive ( omniseq corporation , buffalo , ny ) , and tempus xt ( tempus labs , chicago , il )  . 
table 4 lists the number of genes in these somatic panels and the number of genes in each panel with germline implications ( according to table 2 ) at the time of publication . 
in general , a notable number of genes with germline implications are sequenced within common somatic panels . importantly , however , no somatic panel covers all known genes of germline importance , and sizeable numbers of cancer - susceptibility genes are excluded . currently available germline variant panels discussion several companies offer panels that test genes linked to hereditary cancer for various body systems . 
well - established companies with longer commercial track records include ambry genetics ( aliso viejo , ca ) , genedx ( gaithersburg , md ) , invitae ( san francisco , ca ) , and myriad genetics ( salt lake city , ut )  . 
criteria for inclusion of a company in this summary were a primary focus in commercial clinical laboratory improvement amendmentscertied germline testing and exclusion of direct - to - consumer testing or testing several cancer - related genes hold germline implications , and these genes are not uniformly included in currently available germline panel tests ( table 3 )  . 
given the state of current germline testing guidelines , which are syndrome based , and available commercial germline panels , which increasingly are organ - system based , a degree of disconnect between what is clinically relevant and what is offered with germline panels is to be expected . 
our group found that tumor sequencing had a notable number of potential germline variants and that referrals based on somatic variant sequencing could reveal otherwise unknown germline variants.22 within the 315 - gene panel used for the initial 222 - patient cohort , 41 genes ( 13% ) were classied as genes known to cause cancer susceptibility when mutated in the germline . 
coverage of genes within comprehensive or body systemspecic panel offerings from each major germline testing company ambry cancer type genedx invitae myriad * breast / gynecologic colorectal melanoma pancreatic prostate renal frequently somatically mutated in tumors , including tp53 , apc , and cdkn2a , were excluded , 91 patients ( 41% ) had a potential germline variant . 
twenty - three patients ( 10% ) were evaluated by genetics professionals ; three genetics referrals resulted directly from somatic testing results , which ultimately yielded one previously unknown germline variant . 
with formal integration of medical genetics into somatic testing review by the genomics tumor board at our institution , the overall percentage of referrals to genetics increased from 15% to 33% , and 41% of those referrals were the direct result of a somatic test review.23 in another study , speare et al24 reviewed 74 germline tests that followed identication of somatic variants in the tumor specimens of 54 patients and found that 10 variants ( 13.5% ) were conrmed in the germline . 
last , schrader et al25 surveyed germline variants in 187 genes that overlapped with a concurrent 341 - gene tumor somatic panel and identied pathogenic germline variants in 246 ( 15.7% ) of 1 , 566 patients . 
moreover , of the 198 individuals with variants in genes specically associated with increased cancer risk , only 81 of these germline changes ( 40.9% ) were concordant with the tumor type . 
that is , more than half of the patients who had pathogenic germline variants likely would not have had a recommendation for germline testing of that particular gene on the basis of their cancer type alone . 
in our own data , a high proportion of patients who were discovered by somatic tumor testing alone to carry a germline pathogenic variant ( ve of seven patients , or 71.4% ) did not meet any guidelinebased genetic testing criteria.23 together , these data demonstrate that somatic variant sequencing can inform germline testing and act as a screening tool for inherited germline carrier status , which suggests an additional clinical purpose for somatic variant testing separate from the intended goal of screening for possible targeted therapies . * myriad myrisk simultaneously tests 28 genes linked to hereditary cancers in various body systems . 
although some of these tests ask for blood or saliva samples and can test germline variants , they are primarily advertised as panels used to test tumor samples for somatic variants . abbreviation : mskcc , memorial sloan kettering cancer center . in a 1 , 000 - patient cohort with tumor and normal dna sequenced with a 202 - gene panel , meric - bernstam et al20 reported 43 patients ( 4.3% ) with potentially germline variants . 
more than half of these germline variantpositive individuals ( 101 patients ) would not have met guideline - based criteria for clinical germline testing . these studies add credence to the idea that the identication of germline variants from somatic testing can benet patients who would not otherwise have been identied by means of striking personal or family histories and would likely identify inherited cancer susceptibility in additional patients that targeted germline panel or syndrome testing alone may miss . although somatic variant testing can unearth germline variants that traditional means of screening have not , it is important to point out that some common types of germline variants are not picked up with the current technology used for somatic sequencing . 
large rearrangements , inversions , promoter variants , and pseudogene differentiation are not consistently addressed with current somatic sequencing . thus , a germline variant that involves a large rearrangement of brca or is within pms2 , which is notoriously difcult to distinguish from its pseudogene , may be missed these genes or misinterpreted despite the inclusion of within the somatic test panel.26 , 27 despite technical differences between germline and somatic testing , somatic sequencing still has promise as a clinically useful , but not independently adequate , tool for the screening of inherited cancer predisposition . 
somatic tumor sequencing may be considered additive to a proper family history and of particular utility in screening the subset of patients whose family or tumor histology would not otherwise meet traditional germline genetic testing criteria . tables 3 and 4 demonstrate the complexity of current offerings in germline and somatic variant testing . 
even within the category of commercial germline testing , the composition of clinically relevant genes within panels designed for the same organ ( eg , breast ) varies widely from company to company . 
these tables underscore the difculty of interpreting exactly which genes are clinically relevant , whether in a germline or somatic setting , and in some ways reect the issues of offering tests in a eld with a rapidly expanding knowledge base . 
correspondingly , one of the largest challenges in the writing of this review was the expansive and rapidly changing nature of both genes and tumors , which require multidisciplinary expertise and multiple revisions to interpret emerging data . although these tables are designed as a foundational primer specically for the oncologist and should serve well as a quick reference in clinic , the involvement of genetic counselors whenever available in the interpretation of germline implications of somatic testing and the orders for follow - up germline testing is recommended . in conclusion , although family history remains the most reliable screening tool for inherited cancer predisposition syndromes , several studies suggest that a clinically notable subset of germline carriers would be missed by family history alone . because limited family history or an incomplete gene penetrance can result in ambiguity , family historybased germline testing alone may be inadequate to completely assess a patients , and the familys , hereditary cancer risk . 
the plethora of cancer susceptibility genes tested in widespread somatic testing tumor panels makes incidental germline ndings likely , and numerous studies have conrmed that somatic testing can be a useful , but not fully adequate , screen for germline variants . 
sohal , pauline funchain collection and assembly of data : zade akras , brandon bungo , brandie h . leach , jessica marquard , manmeet ahluwalia , hetty carraway , davendra p.s. 
leach consulting or advisory role : invitae speakers ' bureau : myriad genetics references manmeet ahluwalia stock and other ownership interests : mimivax honoraria : prime oncology , elsevier , itamar medical ( i ) consulting or advisory role : monteris medical , astrazeneca , bristol - myers squibb , abbvie , cbt pharmaceuticals , kadmon , vbi vaccines research funding : novartis , tracon pharma , novocure , spectrum pharmaceuticals , lilly , imclone , boehringer ingelheim , astrazeneca hetty carraway consulting or advisory role : amgen , agios , myriad genetics , celgene speakers ' bureau : baxalta , celgene , novartis , jazz pharmaceuticals research funding : celgene travel , accommodations , expenses : celgene , agios , jazz pharmaceuticals , novartis , baxalta petros grivas consulting or advisory role : genentech , merck , bristol - myers squibb , astrazeneca , biocept , clovis oncology , emd serono , seattle genetics , foundation medicine , driver , pzer , qed therapeutics , heron speakers ' bureau : genentech , bristol - myers squibb research funding : mirati therapeutics ( inst ) , genentech ( inst ) , roche ( inst ) , merck ( inst ) , oncogenex ( inst ) , bayer ( inst ) , pzer ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) davendra p.s. 
sohal honoraria : foundation medicine consulting or advisory role : perthera research funding : novartis ( inst ) , celgene ( inst ) , oncomed ( inst ) , bayer ( inst ) , genentech ( inst ) , bristol - myers squibb ( inst ) , agios ( inst ) , incyte ( inst ) travel , accommodations , expenses : foundation medicine pauline funchain consulting or advisory role : eisai no other potential conicts of interest were reported stratton mr : exploring the genomes of cancer cells : progress and promise . 
science 331 : 1553 - 1558 , 2011 friedman aa , letai a , fisher de , et al : precision medicine for cancer with next - generation functional diagnostics . 
nat rev cancer 15 : 747 - 756 , 2015 kamps r , brando rd , bosch bj , et al : next - generation sequencing in oncology : genetic diagnosis , risk prediction and cancer classication . 
nature 470 : 204 - 213 , 2011 biesecker lg : opportunities and challenges for the integration of massively parallel genomic sequencing into clinical practice : lessons from the clinseq project . genet med 14 : 393 - 398 , 2012 castro e , goh c , leongamornlert d , et al : effect of brca mutations on metastatic relapse and cause - specic survival after radical treatment for localised prostate cancer . 
n engl j med 373 : 1697 - 1708 , 2015 le dt , durham jn , smith kn , et al : mismatch repair deciency predicts response of solid tumors to pd - 1 blockade . 
science 357 : 409 - 413 , 2017 lu kh , wood me , daniels m , et al : american society of clinical oncology expert statement : collection and use of a cancer family history for oncology providers . j clin oncol 32 : 833 - 840 , 2014 10 . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
carlo mi , zhang l , mandelker d , et al : cancer predisposing germline mutations in patients ( pts ) with urothelial cancer ( uc ) of the renal pelvis ( r - p ) , ureter ( u ) and bladder ( b )  . 
catenacci dvt , amico al , nielsen sm , et al : tumor genome analysis includes germline genome : are we ready for surprises ? int j cancer 136 : 1559 - 1567 , 2015 16 . 
cheon jy , mozersky j , cook - deegan r : variants of uncertain signicance in brca : a harbinger of ethical and policy issues to come ? genome med 6 : 121 , 2014 19 . 
meric - bernstam f , brusco l , daniels m , et al : incidental germline variants in 1 , 000 advanced cancers on a prospective somatic genomic proling protocol . 
pearlman r , frankel wl , swanson b , et al : prevalence and spectrum of germline cancer susceptibility gene mutations among patients with early - onset oncol 27 : 795 - 800 , 2016 colorectal cancer . 
klek s , heald b , milinovich a , et al : genetic counseling ( gc ) and germline ( gl ) testing rates after adoption of an integrated clinical cancer genetics ( ccg ) approach to genomics tumor board ( gtb )  . 
this study aimed to describe the hr pathway mutations and hrd status and determine their association with treatment response and outcome in patients with pdac . patients and methods we performed a retrospective analysis of tumor samples from patients treated at indiana university for locally advanced or metastatic pdac . 
patients were included if they received gemcitabine plus nanoparticle albumin - bound paclitaxel ( control ) or fluorouracil , oxaliplatin , leucovorin , and irinotecan ( folfirinox ) and had adequate follow - up to assess survival and response to therapy . 
tumor analysis generated a three - biomarker hrd score and mutation data for 44 genes . results ninety - one samples met inclusion criteria , and 78 samples ( formalin - fixed paraffinembedded , n = 15 ; fine - needle aspiration , n = 63 ) generated mutation data . 
hrd analysis was successful for 57 samples ( hrd score : median , 18 ; range , 5 to 61 ) ; the primary cause of failure was low tumor cellularity . 
2018 by american society of clinical oncology introduction pancreatic ductal adenocarcinoma ( pdac ) is the fourth leading cause of cancer - related mortality in the united states.1 most patients present with advanced disease where palliative chemotherapy is minimally effective with high rates of toxicity , and the lack of meaningful benefit associated with targeted therapy continues to be a challenge.2 gemcitabine plus nanoparticle albumin - bound ( nab ) paclitaxel ( g + nab - p ) and fluorouracil , oxaliplatin , leucovorin , and irinotecan ( folfirinox ) are commonly used first - line treatment regimens for advanced disease , with objective response rates of 23% to 31% and median overall survival ( os ) times of , 1 year.3 , 4 kras and tp53 mutations remain the most common molecular abnormalities in pdac , 5 in the absence of effective therapy targeting these mutations . 
genomic analysis has revealed the presence of low - frequency , potentially actionable mutations in patients with pdac6 ; 15% of patients with pdac may carry a mutation in the safi shahda kirsten m . 
therefore , a score of 42 was not chosen as a cutoff in this study ; instead , we decided to evaluate the range of scores and correlate outcome based on hrd scores greater than and less than the median , without intending to establish the median as the cutoff . data collection data were retrieved from a retrospective database ( under a separate irb - approved protocol ) of patients who were diagnosed with pdac and who were partially or completely observed at our institution . 
the data were housed in an oncore ( forte research systems , madison , wi ) database per institutional guidelines . determination of radiographic response and outcome the principal investigator reviewed all images of eligible patients and analyzed response to therapy per response evaluation criteria in solid tumors ( recist ) v1.1. 
median follow - up time for the 16 patients without progression was 9 months ( 11 months for the six patients without progression who were alive at end of study )  . 
pfs and os were calculated from the time of initiating second - line therapy . tissue samples the direct smears prepared from the pancreatic fine - needle aspirates ( fna ) were screened by a cytopathologist ( a.a.i. ) to assure adequacy for molecular testing . 
 formalin - fixed paraffin - embedded ( ffpe ) cell blocks were prepared in a subset of the fnas . the hematoxylin and eosinstained sections cut from those blocks were also examined by a cytopathologist ( a.a.i. ) , and the block was considered acceptable for molecular testing if the estimated tumor cellularity was at least 20% . 
either the entire paraffin cell block or sections cut from the block ( one 4to 5 - mm hematoxylin and eosin slide followed by five consecutive 10 - mm unstained slides ) were submitted for analysis . tissue processing and dna extraction ffpe blocks and fna smears from pretreatment samples were retrieved . 
mutations identified were only included in the analysis if they were classified as deleterious or suspected deleterious on the basis of previously described criteria.21 to calculate the hrd score for samples analyzed by custom hybridization sequencing assay , reads covering snp positions were used to generate allelic imbalance profiles as described previously.14 hrd score was defined as the unweighted sum of loh , tai , and lst scores ( hrd = loh + tai + lst )  . statistical analysis clinical characteristics were compared between treatment groups using nonparametric methods ( wilcoxon rank sum or fishers exact test )  . 
response was coded as a binary variable ( complete response [ cr ] + partial response [ pr ] v stable disease [ sd ] + progressive disease [ pd ] or cr + pr v pd , unless otherwise noted ) , and logistic regression analysis was used to test association with clinical and molecular variables . 
hrd score was dichotomized at the median of 18 ( score , or > 18 ) , and median survival times were estimated using kaplan - meier curves compared by log - rank tests . 
all p values are two - sided , with significance set at p = .05. study approval and ethical considerations this was a retrospective analysis , and at the time of analysis , most patients had died as a result of the advanced nature of their illness . 
patients received care at the discretion of their treating oncologist , with genetic counseling and testing as deemed appropriate . results study population ninety - one patients met inclusion criteria , of whom seven had inadequate tissue for analysis after a second screening process by a pathologist from myriad genetics , 14 had ffpe tissue , and 70 had fna ( fig 1 )  . patients characteristics and treatment patient characteristics are listed in table 1 . 
summary of sample screening and number of samples used in the final analysis . hrd , homologous recombination deficiency . tumor samples ( n = 91 ) final clinical data merged with molecular results ( n = 84 ) inadequate tissue for molecular analysis ( n = 7 ) mutation screen and hrd score failed ( n = 6 ) mutation screen passed ( n = 78 ) brca1 / 2 mutation ( n = 6 ) brca1 / 2 wild type ( n = 72 ) hrd score failed ( n = 1 ) hrd score passed ( n = 5 ) hrd score failed ( n = 20 ) hrd score passed ( n = 52 ) mutant with no hrd score ( n = 1 ) mutants with hrd score ( n = 5 ) wild type with no hrd score ( n = 20 ) wild type with hrd score ( n = 52 ) 5 to 61 , with a median score of 18 . 
six brca1 / 2 mutants were detected in this cohort , and hrd score analysis was successful in five of six patients , with scores ranging from 13 to 61 and a median score of 44 . 
loss of the second allele of the mutant locus via either loh or a second deleterious mutation was observed in three of the six mutant samples , with hrd scores of 43 , 52 , and 53 , respectively . 
in comparison , the median hrd score observed in brca1 / 2 mutation carriers with breast or ovarian tumors was 65 , suggesting that the distribution of hrd scores may differ between pdac and these other tumor types , consistent with our preclinical observations . description of brca mutation functionality and pathogenicity six patients harbored brca mutations , four brca1 mutations and two brca2 mutations . individual mutations and hrd scores associated with those mutations are listed in table 2 . association of hrd with response to folfirinox for the purpose of this study we explored the following two scenarios , which were disease response defined as cr + pr versus sd + pd or cr + pr versus pd ( fig 2 )  . 
the composite variables that were assessed were any pathogenic mutation regardless of the status of the second allele and any brca1 / 2 mutation regardless of the status of the second allele against response , pfs , and os . 
in a subgroup analysis of metastatic status , with or without adjusting for treatment , hrd score was not significant for any of the outcomes ; however , the sample size was not large enough to test whether hrd behaves differently in these two groups . discussion this is the first study to our knowledge to evaluate the relationship between hr gene mutations , hrd score , and response to chemotherapy in patients with pdac . 
higher hrd score was correlated with brca1 / 2 mutations but was not associated with the presence of other hr brca1 mutation brca2 mutation brca1 / 2 wild type brca1 mutation brca2 mutation brca1 / 2 wild type fig 2 . 
 ( a ) complete response ( cr ) or partial response ( pr ) versus stable disease or progressive disease ( pd ) , and ( b ) cr or pr versus pd . 
distribution of homologous recombination deficiency ( hrd ) scores across samples of locally advanced ( la ) and metastatic pancreatic ductal adenocarcinoma ( pdac )  . max , maximum ; min , minimum ; qu , quartile ; sd , standard deviation . time from start of metastatic therapy ( months ) pathway mutations . 
one possible explanation for the absence of an elevated hrd score despite the presence of an hr pathway mutation is the retained functional allele in the affected genes . although conducted retrospectively , our study demonstrates the feasibility of performing genomic analysis on fna samples available from patients with pdac . 
on the contrary , however , this study also highlights the challenge of conducting correlative studies in patients with pdac ; most patients with pancreatic malignancies are diagnosed based on endoscopic ultrasound - guided fna only.22 in addition , high nontumor content , likely as a result of abundant desmoplastic stroma , often led to low dna content and thus a high assay failure rate . interestingly , we experienced a higher failure rate associated with core needle biopsy samples as opposed to fna samples , as a result of higher levels of nontumor dna content , even after macrodissection . 
 despite the low incidence of dna repair deficiency in pdac , it continues to represent an attractive target for drug development.23 several clinical trials using single agents or drug combinations are ongoing . 
in addition , patients with pdac tend to have a higher symptom burden and experience rapid progression after first - line therapy , frequently prohibiting second - line therapy.3 , 4 therefore , the optimal timing for testing such methods to select patients for subsequent therapies will likely be early in the disease process to inform secondline therapy.24 although our study does not address the presence of heterogeneity between the primary and metastatic lesions , some literature exists regarding hrd heterogeneity in other tumor types . for example , a study of patients with triplenegative breast cancer compared hr score in three different areas from the same primary tumor.25 overall , there was no significant difference among these samples , and the authors concluded that heterogeneity for hrd metrics was small and did not influence hrd score or status from three samples in the same tumor . whether hrd is more common in metastatic sites remains unknown in pdac ; however , a higher rate of germline mutations associated with dna repair genes ( without examining hrd score ) was found in metastatic prostate cancer when compared with localized disease.26 this study has limitations . 
it is a single - institution , nonrandomized , retrospective study comparing two commonly used chemotherapy regimens and including patients who had locally advanced and metastatic disease , which may carry different biology and represent a challenge in assessing response to therapy in locally advanced pdac . 
we had a relatively high assay failure rate in both fna and ffpe , although it was higher in the ffpe samples . in conclusion , this study did not demonstrate a correlation between hrd score and radiographic response to platinum - based therapy in patients with pdac . 
although hr gene mutations or hrd score may represent targets for drug development in pdac , larger prospective studies may be needed to evaluate the utility of these markers and their correlation with response to dna - damaging agents . 
for more information about ascos conflict of interest policy , please refer to or ascopubs.org / po / author - center . safi shahda consulting or advisory role : merrimack , bayer research funding : myriad genetics kirsten m . 
timms employment : myriad genetics stock and other ownership interests : myriad genetics patents , royalties , other intellectual property : austria ( e840080 , 3012329 ) , china pr ( zl 201280070358.0 ) , denmark , european patent convention , finland , france , great britain , ireland , the netherlands , spain , sweden , and switzerland ( 2582847 , 3012329 ) , germany ( 602011031723.7 , 3012329 ) , italy ( 502016000131029 , 3012329 ) , japan ( 6117194 ) , new zealand ( 625468 ) , united states ( 9 , 574 , 229 ; 9 , 279 , 156 ; 9 , 388 , 427 ) travel , accommodations , expenses : myriad genetics ashley a . 
 milan radovich no relationship to disclose sulfikar ibrahim no relationship to disclose brian allen employment : myriad genetics , grail stock and other ownership interests : myriad genetics , grail bert h . 
oneil , indiana university school of medicine , indianapolis ; ashley a . ibrahim and sulfikar ibrahim , indiana university health ball memorial hospital , muncie , in ; and kirsten m . 
moore mj , goldstein d , hamm j , et al : erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer : a phase iii trial of the national cancer institute of canada clinical trials group . j clin oncol 25 : 1960 - 1966 , 2007 3 . 
von hoff dd , ervin t , arena fp , et al : increased survival in pancreatic cancer with nab - paclitaxel plus gemcitabine . med 364 : 1817 - 1825 , 2011 n engl j med 369 : 1691 - 1703 , 2013 jones s , zhang x , parsons dw , et al : core signaling pathways in human pancreatic cancers revealed by global genomic analyses . 
risch ha , mclaughlin jr , cole de , et al : population brca1 and brca2 mutation frequencies and cancer penetrances : a kin - cohort study in ontario , canada . 
yang d , khan s , sun y , et al : association of brca1 and brca2 mutations with survival , chemotherapy sensitivity , and gene mutator phenotype in patients with ovarian cancer . 
mccabe n , turner nc , lord cj , et al : deficiency in the repair of dna damage by homologous recombination and sensitivity to poly ( adp - ribose ) polymerase inhibition . 
timms km , abkevich v , hughes e , et al : association of brca1 / 2 defects with genomic scores predictive of dna damage repair deficiency among breast cancer subtypes . 
telli ml , timms km , reid j , et al : homologous recombination deficiency score predicts response to platinumcontaining neoadjuvant chemotherapy in patients with triple - negative breast cancer . 
mills gb , timms km , reid je , et al : homologous recombination deficiency score shows superior association with outcome compared with its individual score components in platinum - treated serous ovarian cancer . 
 alk testing trends and patterns among community practices in the united states purpose targeted therapy of alk in patients with metastatic nonsquamous nonsmall - cell lung cancer ( nsclc ) and alk rearrangements improves outcomes compared with chemotherapy . 
logistic regression was used to examine the association between demographic and clinical characteristics and testing for alk rearrangements . results of 31 , 483 patients analyzed from the database , 16 , 726 patients ( 53.1% ) were tested for alk rearrangements . 
median ( interquartile range ) time from laboratory receipt of sample to first alk test result was 7 ( 7 ) days ; median time from advanced diagnosis to first alk test result was 25 ( 29 ) days . 
patients who were older , male , had a history of smoking , lived in non - western us regions , and who had recurrent disease or squamous histology were less likely to be tested for alk . 
2018 by american society of clinical oncology introduction recent advances in molecular genetics have increased the opportunity for precision medicine in metastatic nonsquamous nonsmall - cell lung cancer ( nsclc )  . 
for instance , translocation of the anaplastic lymphoma kinase ( alk ) gene is found in approximately 2% to 8% of all patients with nsclc and occurs primarily in patients who are never - smokers or light ex - smokers with adenocarcinoma histology.1 - 4 in addition to representing a distinct molecular nsclc subtype , alk rearrangements are indicative of tumor cell susceptibility to alk inhibitors.5 targeted inhibition of alk in patients with alk rearrangements can decrease tumor burden and significantly increase progression - free survival compared with chemotherapy.3 , 6 the first alk inhibitor , crizotinib , along with the vysis alk break apart fish probe kit , was given accelerated approval by the us food and drug administration ( fda ) in 2011 . 
since then , other firstand second - generation alk inhibitors have been approved , including ceritinib in 2014 and alectinib in 2015 . the college of american pathologists , international association for the study of lung cancer , and association for molecular pathology ( cap / iaslc / amp ) molecular testing and national comprehensive cancer network ( nccn ) peter b . 
 nsclc treatment guidelines recommend testing for alk , egfr , and ros1 in lung adenocarcinomas and in nsclc where an adenocarcinoma component cannot be excluded , regardless of clinical characteristics.1 , 7 , 8 fluorescent in situ hybridization ( fish ) or immunohistochemistry ( ihc ) are assays recommended by cap / iaslc / amp guidelines for the detection of alk rearrangements before selection of targeted therapy.8 in addition to fish , the alk ( d5f3 ) cdx assay ( ventana medical systems , tucson , az ) and foundationone cdx ( foundation medicine , cambridge , ma ) are ihc and next - generation sequencing ( ngs ) assays , respectively , that are fda approved to identify patients eligible for alk inhibitor treatment . 
alk rearrangements could also be detected using polymerase chain reaction , but no fda - approved assay is available to date , and this method is not currently recommended in guidelines.8 numerous challenges associated with molecular testing of malignancies may affect adoption of molecular testing guidelines and performance of alk testing in clinical practice . 
a major challenge in molecular testing is inadequate tissue samples.9 , 10 for example , in a study that examined the workflow of molecular testing for 157 lung cancer specimens , successful testing of all molecular targets could not be completed for 19% of specimens.11 additional challenges may include tumor heterogeneity , complex results that require multidisciplinary input , education and training of laboratory and staff , availability of assays , and patient affordability.9 , 10 in a retrospective analysis of 814 patients in new jersey and maryland with nonsquamous nsclc who were identified using the cota database ( cota , new york , ny ) , which extracts and organizes data from electronic health records , only 65% underwent alk testing , indicating limited integration of molecular testing recommendations into clinical practice.12 given the clinical benefit of alk inhibitor therapy in patients with nsclc with alk rearrangements , evaluation of alk testing using large , real - world , geographically diverse data sources is warranted . 
the objective of this study was to assess real - world alk testing patterns among community practices in the united states in patients with advanced nsclc from january 2011 through may 2017 . methods study overview and database description this was a retrospective analysis using the flatiron health electronic health record ( ehr ) derived database , a longitudinal , demographically and geographically diverse database.13 the database includes > 265 cancer clinics ( approximately 800 sites of care ) representing more than 2 million active us patients with cancer available for analysis . 
the current analysis was limited to data from community practices and included 34 , 911 patients diagnosed with advanced nsclc ( stage iiib or iv by american joint committee on cancer 7th edition ) from january 2011 through may 2017 . 
insurance information was categorized into commercial ( including those with dual medicare or medicaid coverage ) , medicare ( including dual medicaid coverage ) , medicaid , and other / unknown . 
 ( % ) unless otherwise noted . abbreviations : alk , anaplastic lymphoma kinase ; iqr , interquartile range , q3 - q1 ; nos , not otherwise specified ; nsclc , nonsmall - cell lung cancer . date specimens were received by the laboratory to the test report date . data analysis statistical analyses were primarily descriptive , with frequencies calculated for categorical data and means , medians , interquartile ranges ( iqrs ) , and standard deviations ( sds ) calculated for continuous data . 
odds ratios ( ors ) and 95% cis were calculated by logistic regression to examine the association between demographic and clinical characteristics and testing for alk rearrangements . results patient characteristics overall , 34 , 911 patients with advanced nsclc were identified from the flatiron health ehr - derived database from january 2011 through may 2017 ; 31 , 483 met the selection criteria and were included in the analysis . 
most of the samples used for alk testing were tissue ( 93.7% ; 15 , 664 of 16 , 726 ) ; 2.6% ( 439 of 16 , 726 ) were blood , and the rest were unknown . 
of the total 21 , 639 patients with nonsquamous histology , 66.9% ( 14 , 478 of 21 , 639 ) were tested for alk , and of the 7 , 923 patients with squamous histology , 18.5% ( 1 , 466 of 7 , 923 ) were tested for alk . 
 among the patients who had nonsquamous histology and who were not tested for alk , 87.2% ( 6 , 025 of 6 , 913 ) had a history of smoking , and among those with a squamous histology and who were either tested or not tested for alk , 91.5% ( 1 , 320 of 1 , 443 ) and 95.6% ( 5 , 992 of 6 , 268 ) , respectively , had a history of smoking . 
overall , 16 , 726 of 31 , 483 ( 53.1% ) patients were tested for alk , with 633 of 16 , 726 ( 3.8% ) patients positive for alk ( 581 of 14 , 478 [ 4.0% ] nonsquamous histology , 24 of 1 , 466 [ 1.6% ] squamous histology , and 28 of 782 [ 3.6% ] not otherwise specified histology )  . alk testing patterns alk testing rates by year of advanced diagnosis steadily increased over time from an average of 32.4% ( 1 , 093 of 3 , 371 ) in 2011 to 62.1% ( 3 , 526 of 5 , 680 ) in 2016 for all patients with advanced nsclc , from 41.1% ( 935 of 2 , 273 ) in 2011 to 75.1% ( 2 , 947 of 3 , 924 ) in 2016 for patients with nonsquamous histology , and from 12.1% ( 101 of 832 ) in 2011 to 26.5% ( 372 of 1 , 403 ) in 2016 for patients with squamous histology ( fig 1a )  . 
with test 2012 2014 year 178 201 245 368 395 471 483 446 525 595 593 625 665 765 717 746 728 801 891 820 860 805 787 702 631 2015 2013 2016 2017 squamous , no . 
 age , years 55 - 64 v < 55 65 - 74 v < 55 75 - 84 v < 55 85 + v < 55 female v male race asian v white black or african american v white other race v white missing v white hispanic v non - hispanic ethnicity histology nos v non squamous squamous v non squamous disease type recurrent v de novo unknown v de novo smoking status history of smoking v no history unknown history v no history insurance medicaid v commercial medicare v commercial other v commercial region midwest v west northeast v west south v west missing v west fig 2 . 
the overall median ( iqr ) turnaround time from the date samples were received by the laboratory to the test report date was 7 ( 7 ) days and ranged from 6 days for fish to 12 days for ngs ( table 2 )  . 
the overall median ( iqr ) time from advanced diagnosis date to first alk test result date was 25 ( 29 ) days ( table 2 ) and differed by type of test and insurance type . 
 median ( iqr ) times from advanced diagnosis to first alk test result for fish were 20 ( 19 ) , 22 ( 31 ) , and 23 ( 27 ) days for commercial insurance , medicaid , and medicare , respectively , and for ngs were 37 ( 34 ) , 58 ( 159 ) , and 41 ( 105 ) days , respectively ( table 2 )  . 
on the basis of tests conducted by foundation medicine ( cambridge , ma ) laboratory , the overall agreement between ngs and fish was 97.6% ( 360 of 369 )  . 
the percentage of tests positive by ngs that were initially negative by fish was 1.9% ( 7 of 369 )  . treatment before alk test results among patients tested for alk who had valid tissue collection and test result dates , 21.5% ( 3 , 290 of 15 , 320 ) initiated therapy before receiving their first alk test results . 
percentage of patients initiating treatment before first anaplastic lymphoma kinase ( alk ) test result . discussion this retrospective analysis of patients with advanced nsclc treated at community practices between january 2011 and may 2017 found that 53.1% of all patients , 66.9% of patients with nonsquamous histology , and 18.5% of patients with squamous histology were tested for alk rearrangements . 
cap / iaslc / amp and nccn guidelines recommend alk testing be conducted for patients with nsclc that has an adenocarcinoma component.7 , 8 however , alk testing may also be considered for patients with nsclc who have mixed histology , who are of young age , or who are without a history of tobacco exposure.7 , 8 the current analysis confirms and extends the findings of a retrospective analysis of 814 patients with nonsquamous nsclc treated in community centers in the united states , which found an overall 65% alk testing rate for the years 2013 to 2015.12 alk testing rates increased over time , reaching 75% in 2016 in patients with nonsquamous histology , indicating improvement in adherence to alk testing recommendations as well as physician recognition of the clinical benefits of alk targeted therapy . 
 the reasons for lack of testing were not captured in the flatiron health database , although possible reasons may include evolution of best practices and guidelines across the study period , patient disease severity , and insufficient tissue ( as was observed in 16% to 21% of patients in other nsclc studies ) .4 , 14 , 15 blood - based assays could address the issue of insufficient tissue , and research in this field is emerging.16 patients with squamous histology were less likely to be alk tested , which is consistent with guidelines that state there is no evidence to recommend alk testing in patients with typical squamous histology who do not have other clinical features to prompt alk testing.7 , 8 in the current analysis , testing rates increased over time in patients with squamous histology , and , overall , 1.6% had an alk rearrangement . 
the increased testing rate for patients with squamous histology may be due to a general increase in the perceived benefit of alk inhibitors for alk - positive nsclc by physicians or to the release of cap / iaslc / amp and nccn guidelines that suggest selectively testing patients with squamous histology , such as patients who are never - smokers or former light smokers.1 , 17 the likelihood of alk testing decreased as age increased . 
patients with alk - positive disease in clinical studies tended to be younger , nonsmokers , and have adenocarcinoma , perhaps increasing the likelihood that these patients would have been tested for alk positivity despite guidelines recommending that all patients with nonsquamous disease be tested . 
in addition , the current analysis suggests there may also be socioeconomic barriers to alk testing ; medicaid patients were 40% less likely to be tested than patients with commercial insurance . 
collectively , these results suggest that additional education on alk testing in the community oncology setting is warranted . in our study , fish was the most commonly ordered genomic testing method , which was not unexpected , given that fish was the only recommended and fda - approved assay during the majority of the time period for our study . 
 updated 2018 cap / iaslc / amp and nccn guidelines now recommend fish or ihc for alk testing.7 , 8 with the rapidly changing landscape of fda - approved assays , such as the 2017 approval of the foundationone cdx ngs assay , the distribution of assays used will likely continue to evolve . 
indeed , in our study we observed a trend of declining use of fish and an increase in the use of ngs and , to a lesser extent , ihc . 
there is a trend in clinical practice to use ngs as the primary testing method to identify all targetable alterations.18 , 19 turnaround times for laboratory receipt of sample to test results were consistently less than 2 weeks across different test types , including ngs . 
the reasons for this variation are unknown ; however , potential reasons for longer times for some patients may include the ordering process for biomarker testing , sample shipment to the laboratory , and sample processing . 
for example , a previous study in metastatic colon cancer found that testing performed at a nonlocal or noncancer centeraffiliated laboratory was associated with greater likelihood of a testing delay.20 the current study was conducted in the community setting , and the majority of biomarker testing was done in commercial laboratories , which may be one of the possible explanations for the observed variance in times . 
in addition , the 14 - day rule may also have contributed to the variation in times for some medicare patients , because testing may have been delayed to avoid reimbursement complexities . numerically , ngs had a longer time from advanced diagnosis to alk test result for both medicare and medicaid patients . 
given that turnaround times for ngs were similar across insurance types and only accounted for a minor portion of the time from advanced diagnosis to alk test result , this may imply that delays in ordering ngs , which was not an approved test during the study period , may have existed . 
additional research is needed to determine if this trend will continue in the future , particularly given the 2017 simultaneous fda approval of ngs tests for tumor profiling and centers for medicare & medicaid services approval of coverage for fda - approved ngs tests , which may substantially improve the ability to provide more timely access to ngs testing.21 , 22 the lack of alk testing or delay in results may have serious clinical implications , given the efficacy of alk inhibitors in patients with alk rearrangements.3 , 6 , 23 , 24 in a head - to - head comparison , a second - generation alk inhibitor ( alectinib ) exhibited a median progression - free survival of 34.8 months , compared with 10.9 months with a first - generation alk inhibitor ( crizotinib ) .24 , 25 delays in alk testing may potentially lead to delayed treatment , 15 and , as is seen in other cancers , delays in treatment may lead to poorer outcomes.26 , 27 although additional research is needed to assess the impact of delays on outcomes , specifically in alk - positive nsclc , alk testing is needed to help facilitate timely treatment with an efficacious alk inhibitor . day - level granularity of biomarker testing ordered date is not always available in ehr data , which has limited our ability to interpret and determine reasons for observed delays in testing . 
 last , this study focused on community practices in the united states , and hence the results of this study may not be generalizable to all clinical practices . the results of the current analysis indicate that the frequency of alk testing in patients with advanced nsclc treated in community centers in the united states has increased over time . 
 however , certain subgroups of patients , such as patients receiving medicaid , were less likely to be tested , suggesting that additional education on genomic testing and investigation into socioeconomic barriers is warranted . 
 laura chu employment : genentech stock and other ownership interests : roche ravindra gupta employment : genentech katja schulze employment : genentech stock and other ownership interests : genentech matthew a . 
gubens consulting or advisory role : bristol - myers squibb , ariad pharmaceuticals , genentech , astrazeneca , abbvie , novartis , mersana therapeutics research funding : celgene ( inst ) , merck ( inst ) , novartis ( inst ) , genentech ( inst ) , oncomed ( inst ) acknowledgment medical writing and editorial assistance was provided by erin p . 
illei , johns hopkins university school of medicine , baltimore , md ; william wong , laura chu , ravindra gupta , and katja schulze , genentech , south san francisco ; matthew a . 
gubens , university of california , san francisco , ca ; and ning wu , pro unlimited , boca raton , fl . support supported by genentech , south san francisco , ca . prior presentation presented in part at the international association for the study of lung cancer 18th world conference on lung cancer , yokohama , japan , october 15 - 18 , 2017 . 
lindeman ni , cagle pt , beasley mb , et al : molecular testing guideline for selection of lung cancer patients for egfr and alk tyrosine kinase inhibitors : guideline from the college of american pathologists , international association for the study of lung cancer , and association for molecular pathology . 
korpanty gj , graham dm , vincent md , et al : biomarkers that currently affect clinical practice in lung cancer : egfr , alk , met , ros - 1 , and kras . 
lindeman ni , cagle pt , aisner dl , et al : updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors : guideline from the college of american pathologists , the international association for the study of lung cancer , and the association for molecular pathology . 
distasio m , chen y , rangachari d , et al : molecular testing turnaround time for non - small cell lung cancer in routine clinicalpractice confirms feasibility of cap / iaslc / amp guideline recommendations : a single - center analysis . 
mayo - de - las - casas c , garzn ibez m , jordana - ariza n , et al : an update on liquid biopsy analysis for diagnostic and monitoring applications in non - small cell lung cancer . 
suh jh , johnson a , albacker l , et al : comprehensive genomic profiling facilitates implementation of the national comprehensive cancer network guidelines for lung cancer biomarker testing and identifies patients who may benefit from enrollment in mechanism - driven clinical trials . 
jensen ts , chin j , baldwin j , et al : proposed decision memo for next generation sequencing ( ngs ) for medicare beneficiaries with advanced cancer ( cag - 00450n )  . 
soria jc , tan dsw , chiari r , et al : first - line ceritinib versus platinum - based chemotherapy in advanced alk - rearranged non - small - cell lung cancer ( ascend - 4 ) : a randomised , open - label , phase 3 study . 
garcia , md3 ; amir momeni - boroujeni , md1 ; qi gao , mls1 ; jeeyeon baik , ba1 ; dory londono , bs1 ; ryma benayed , phd1 ; allison sigler , ma1 ; michael haddadin , md2 ; alexander v . 
goldberg , md , phd2 ; yanming zhang , md1 ; wenbin xiao , md , phd1 ; and caleb ho , md1 introduction ighcytokine receptorlike factor 2 ( crlf2 ) rearrangement is the most common mechanism of overexpression of crlf2 in precursor b - cell acute lymphoblastic leukemia ( b - all ) ; however , p2ry8 - crlf2 rearrangement or alternative alterations leading to crlf2 overexpression have also been observed , which are associated with increased relapse rate and progression.1 although many myeloid neoplasms , particularly acute myeloid leukemia ( aml ) , are associated with various recurrent genetic rearrangements and fusions , 2 alterations leading to crlf2 overexpression are exceedingly rare in aml . 
one patient with myelodysplastic syndrome with excess blasts - 1 ( mds - eb1 ) with crlf2 overexpression via multiple copies of an isodicentric y chromosome , without evidence of crlf2 rearrangement , has been reported.3 to our knowledge , we report the rst patient with aml arising from an mds , with a p2ry8 - crlf2 fusion , which responded to a myeloid regimen in a clinical trial of hypomethylating agent and immune checkpoint blockade combination therapy . 
at mskcc , karyotype analysis of the aml bone marrow aspirate specimen showed a normal male karyotype , and fish analysis was negative for the presence of kmt2a ( mll ) , evi1 , and cbfb translocations , and tp53 deletion . 
a targeted nextgeneration sequencing ( nsg ) panel ( msk - impact heme panel ; mskcc , new york , ny ) was performed on the bone marrow specimen obtained at the time of the aml diagnoses , which showed the alterations including mutations in asxl1 , listed in table 1 , kmt2d , runx1 , sf3b1 , tet2 , phf6 , rad21 , and xbp1 . 
evaluation of this bone marrow sample using an anchored multiplex polymerase chain reaction based , targeted rna sequencing assay for detection of fusions ( archer fusionplex , archerdx , boulder , co ) revealed the presence of a fusion between exon 1 of p2ry8 and exon 1 of crlf2 ( fig 1c )  . 
cell sorting , molecular characterization , and immunostaining characterized the myeloid origin of this fusion and subsequent signaling effects . relevance this patient was treated with the novel combination of decitabine and ipilimumab with an outstanding response . 
immune cell inltrates in the bone marrow with treatment suggests the response was related to the immune checkpoint inhibition . additional study of p2ry8 - crlf2 fusions and response to immunotherapy is warranted and could have implications for fusion discovery in novel disease contexts . sorting to isolate the leukemic blasts , myelomonocytic cells , lymphocytes . 
fish using the crlf2 breakand normal apart probe revealed the crlf2 rearrangement in a subset of the myeloid blasts ( 27.5% ) and myelomonocytic cells ( 19.5% ) but not in b cells ( fig 2 )  . fish evaluation of the prior bone marrow biopsy with mdseb1 was also performed , which showed the presence of the p2ry8 - crlf2 rearrangement in 10% of the cells ( appendix fig a1c )  . 
oncoscan array ( thermofisher , waltham , ma ) of the mds - eb1 biopsy did not reveal any unbalanced genomic changes or copy - neutral loss of heterozygosity ( data not shown ) , consistent with the normal fish study observed by the outside institution a year prior . 
targeted ngs sequencing ( msk - impact heme panel ) of the mds - eb1 bone marrow specimen also showed overlapping mutations with the aml bone marrow sample ( table 1 ) , but with acquisition of new mutations ( phf6 , rad21 , xbp1 ) in the aml sample , suggesting a myeloid primed hematopoietic stem cell / progenitor origin and clonal evolution with expansion of the clone with the p2ry8 - crlf2 rearrangement , likely due to acquisition of a loss - of - function mutation in phf6 , which led to downregulation of genes involved in normal b - cell development.4 total rna sequencing of the aml bone marrow specimen further conrmed the presence of the p2ry8 - crlf2 fusion along with overexpression of crlf2 ( fig 3a )  . 
principal component analysis comparing our patient with cohorts of aml or b - all from the target study5 showed that the patients neoplasm clustered with other patients with aml , conrming the myeloid differentiation of the blasts ( fig 3b )  . gene ontology analysis showed transcriptome - wide increases in cytokine - related signaling pathway genes potentially related to the p2ry8 - crlf2 fusion , as well as genes involved in regulating hematopoietic progenitor cell differentiation and the spliceosome ( fig 3c )  . because it is known that stat5 is activated by p2ry8crlf2 fusion in b - all , we performed immunohistochemistry ( ihc ) using phosphorylation - specic antibodies that showed increased stat5 phosphorylation and partial erk phosphorylation but not stat3 phosphorylation ( fig 4a )  . 
gene expression and ihc were largely correlated ; however , the results are interpreted in the context of the importance of signaling in leukemia overall , and these are not necessarily specic for p2ry8 - crlf2 fusion . cell sorting myeloid blasts monocytes 27.5% ( 55 / 200 ) 19.5% ( 22 / 113 ) bone marrow aspirate fluorescence in - situ hybridization 0% ( 0 / 174 ) b lymphocytes crlf2 fusion positive fig 2 . 
 ( b ) principal component analysis plot showing 2 strongest principal components ( pc1 on x - axis and pc2 on y - axis ) from publicly available data from aml ( green dots ) and b - all ( red dots ) patient samples compared with our patient with p2ry8 - crlf2 fusion ( blue dot )  . 
he was consented and enrolled in a study regimen consisting of standard - dose decitabine plus investigational administration of the cytotoxic t - lymphocyte associated protein - 4 ( ctla - 4 ) antagonist , ipilimumab ( clinicaltrials.gov identier : nct02890329 )  . 
after 2 cycles of combination therapy , he achieved complete remission with incomplete count recovery and shortly thereafter a full complete remission ( table 2 ) with negative measurable residual disease ( mrd ) status by a ow cytometric assay with a detection limit of 1 in 2 , 500 cells . 
this was accompanied by a robust molecular response with undetectable levels of the previously detected phf6 , runx1 , tet2 , and rad21 mutations , and a marked reduction in the variant allele frequencies for the mutations asxl1 ( 0.4% ) and sf3b1 ( 1.4% ) , likely representing mrd below the detection limit of ow cytometry . 
ihc was performed on the checkpoint molecules including programmed death ( pd ) - 1 , pd - ligand 1 ( pd - l1 ) and pd - l2 ( fig 4b )  . 
 ( c ) using multiplex immunouorescence microscopy , with digital image analysis , we found that the patients bone marrow was highly enriched with cd8 plus granzyme b ( gzmb ) plus t cells . 
we further identied a population of cd3 + cd4 + t cells at baseline . discussion crlf2 is the protein product of a synonymous gene located in the par1 on the short arm of the x chromosome and the y chromosome . 
it forms a heterodimer with interleukin 7 receptor alpha and is involved in lymphoid signaling pathways . activation of this receptor leads to activation of janus family tyrosine kinases and stat5 . 
deregulated expression of this gene has been described as the characteristic alteration in philadelphia chromosomelike b - all in adolescent and adult patients.7 , 8 russell et al9 showed that this subgroup of b - all has either a translocation juxtaposing the crlf2 to the igh gene on chromosome 14 ( t ( x ; 14 ) ( p22 ; q32 ) or t ( y ; 14 ) ( p11 ; q32 ) ) or a deletion of par1 region , resulting in overexpression of the crlf2 gene . 
this has been observed in down syndromerelated all as well as nondown syndrome all , supporting the cooperation of crlf2 events with other genetic alterations in progression of disease.11 however , irrespective of the clone size , presence of this rearrangement is associated with a higher relapse rate.12 there have been suggestions that the increased relapse rate may be due to other concurrent alterations , such as ikzf1 deletion.13 however , other studies have shown the rearrangement as well as the crlf2 overexpression to be independent predictors of worse outcome in b - all.14 , 15 until now , only one patient with myeloid neoplasm with crlf2 overexpression has been reported . 
however , this patient was associated with multiple copies of an isodicentric y chromosome and not associated with p2ry8crlf2 rearrangement.3 to our knowledge , our patient represents the rst p2ry8 - crlf2 rearrangement myeloid neoplasms , which in our patient was an aml arising from the underlying mds . 
the ow - sorted fish the p2ry8 - crlf2 was not analysis has shown that present in the b lymphocytes of this patient but only in the myeloid - lineage cells ( myeloid blasts and myelomonocytic cells )  . 
in addition , based on the results from total rna sequencing , our patients neoplasm clustered with other patients with aml , providing additional evidence for the myeloid differentiation of the blasts . 
furthermore , the alterations observed in this patient , including the p2ry8affiliations 1department of pathology , memorial sloan kettering cancer center , new york , ny 2leukemia service , department of medicine , memorial sloan kettering cancer center , new york , ny 3department of medical oncology , dana - farber cancer institute , boston , 4department of pathology , brigham and womens hospital , boston , ma crlf2 rearrangement , were present at the mds stage , suggesting a stem cell origin with selective pressure leading to development of aml . phf6 mutations are rare in de novo aml but relatively common in aml - mrc and exceedingly rare in b - all.16 , 17 phf6 and dnmt3a are the most common mutations in mixed phenotype acute leukemias ( mpal ) with t - lineage differentiation.18 phf6 is a critical component in lineage determination of precursor cells , especially between t and b lineages , and it is believed that a functioning phf6 is required for b - cell differentiation leading to selective abundance of this mutation in t - all and mpal with t - lineage differentiation.18 , 19 this notion is further supported by the observation that phf6 suppression leads to impaired tumor progression in b - all.4 it has also been shown that pax5 - decient pro - b cells can undergo myeloid - lineage differentiation in the presence of transcription factors , such as gata or cebpa.20 in our patient , the immunophenotype and rna expression data unequivocally point toward myeloid differentiation . 
although it is difcult to ascertain the functional consequences of the p2ry8 - crlf2 rearrangement and the acquired phf6 mutation at the time of aml transformation , we hypothesize that the two may interact in a way that led to the development of aml instead of b - all . remarkably , this patient with aml - mrc and phf6 , runx1 , tet2 , and rad21 mutations had an mrdnegative complete response by ow cytometry after just 4 cycles of combination decitabine with ipilimumab . 
although the complete results from clinical trials of these agents are needed to evaluate the efcacy and safety of this this patients exceptional response could combination , suggest the unique molecular characteristics of his tumor the p2ry8 - crlf2 may have been important , namely , fusion . 
recent evidence in head and neck cancers with oncogenic chromosomal rearrangements suggests that gene fusions may be a source of immunogenic neoantigens and stimulate t - cell responses that could be unleashed after immune checkpoint blockade.21 this patients t cells were positive for pd - 1 , suggesting a relation to the response to ctla - 4 blockade . 
garcia , wenbin xiao , caleb ho administrative support : jeeyeon baik , allison sigler collection and assembly of data : umut aypar , justin taylor , jacqueline s . garcia , amir momeni - boroujeni , qi gao , dory londono , ryma benayed , michael haddadin , alexander v . 
goldberg , yanming zhang , wenbin xiao , caleb ho data analysis and interpretation : umut aypar , justin taylor , jacqueline s . garcia , amir momeni - boroujeni , alexander v . 
garcia consulting or advisory role : abbvie research funding : abbvie ( inst ) , pzer ( inst ) , genentech ( inst ) , eli lilly ( inst ) maria e . 
arcila honoraria : invivoscribe , biocartis references mikhail roshal honoraria : celgene consulting or advisory role : agios , auron research funding : agios , roche , boehringer ingelheim ana lako employment : bristol - myers squibb scott j . 
rodig honoraria : perkin elmer , bristol - myers squibb consulting or advisory role : bristol - myers squibb research funding : bristol - myers squibb , merck , afmed therapeutics , kite pharma patents , royalties , other intellectual property : patent pending for use of antigalectin - 1 antibodies for diagnostic use travel , accommodations , expenses : roche , bristol - myers squibb aaron d . 
goldberg honoraria : dava oncology consulting or advisory role : abbvie , celgene , daiichi sankyo research funding : abbvie ( inst ) , adc therapeutics ( inst ) , pzer ( inst ) , arog ( inst ) travel , accommodations , expenses : arog open payments link : 1195852 / summary wenbin xiao research funding : stemline therapeutics ( inst ) caleb ho honoraria : invivoscribe travel , accommodations , expenses : invivoscribe no other potential conicts of interest were reported . acknowledgment we acknowledge the members of the memorial sloan kettering cancer center diagnostic molecular laboratory , cytogenetics laboratory , ow cytometry laboratory , and immunohistochemical stain laboratory for their support in performing the relevant assays . schm ah j , fedders b , panzer - gr umayer r , et al : molecular characterization of acute lymphoblastic leukemia with high crlf2 gene expression in childhood . pediatr blood cancer 64 : e26539 , 2017 arber da , orazi a , hasserjian r , et al : the 2016 revision to the world health organization classication of myeloid neoplasms and acute leukemia . 
meacham ce , lawton ln , soto - feliciano ym , et al : a genome - scale in vivo loss - of - function screen identies phf6 as a lineage - specic regulator of leukemia cell therapeutic target . 
nature 555 : 371 - 376 , 2018 patel ss , weirather jl , lipschitz m , et al : the microenvironmental niche in classic hodgkin lymphoma is enriched for ctla - 4 - positive t cells that are pd - 1negative . 
curr opin pharmacol 8 : 249 - 254 , 2008 vesely c , frech c , eckert c , et al : genomic and transcriptional landscape of p2ry8 - crlf2 - positive childhood acute lymphoblastic leukemia . 
leukemia 31 : 1491 - 1501 , 2017 russell lj , capasso m , vater i , et al : deregulated expression of cytokine receptor gene , crlf2 , is involved in lymphoid transformation in b - cell precursor acute lymphoblastic leukemia . 
potter n , jones l , blair h , et al : single - cell analysis identies crlf2 rearrangements as both early and late events in down syndrome and non - down syndrome leukemia . 
morak m , attarbaschi a , fischer s , et al : small sizes and indolent evolutionary dynamics challenge the potential role of p2ry8 - crlf2 - harboring clones as main relapse - driving force in childhood all . 
dou h , chen x , huang y , et al : prognostic signicance of p2ry8 - crlf2 and crlf2 overexpression may vary across risk subgroups of childhood b - cell acute lymphoblastic leukemia . 
fang q , zhao x , li q , et al : ikzf1 alterations and expression of crlf2 predict prognosis in adult chinese patients with b - cell precursor acute lymphoblastic leukemia . 
xiao w , bharadwaj m , levine m , et al : phf6 and dnmt3a mutations are enriched in distinct subgroups of mixed phenotype acute leukemia with t - lineage differentiation . 
presence of the p2ry8 - crlf2 fusion in earlier bone marrow biopsy with myelodysplastic syndrome ( mds )  . ( a ) photomicrographs of the bone marrow biopsy showing mds with excess blast - 1 ( mds - eb1 )  . 
 molecular characterization and clinical outcomes of primary gleason pattern 5 prostate cancer after radical prostatectomy pedro isaacsson velho , md1 ; david lim , ms1 ; hao wang , phd1 ; jong chul park , md1 ; harsimar b . 
lotan , md1 purpose very high - risk prostate cancer ( pc ) is associated with poor response to local and systemic treatments ; however , few cases have been molecularly proled . 
clinicopathologic and genomic parameters were correlated with biochemical relapse , metastasis - free survival , time to castration resistance , and overall survival using cox proportional hazards models . results of patients with somatic sequencing data and clinical follow - up , 34% had dna repair gene mutations , including 22% ( 11 of 49 ) with homologous recombination and 12% ( six of 49 ) with mismatch repair gene alterations . 
these data are retrospective and hypothesis generating . conclusion potentially actionable homologous recombination and mismatch repair alterations are observed in a signicant proportion of patients with very high - risk pc at the time of radical prostatectomy . 
2019 by american society of clinical oncology introduction the national comprehensive cancer network very high - risk prostate cancer ( pc ) subset includes patients with t3b to t4 disease or more than four biopsy cores with gleason score 8 to 10 ( grade group 4 / 5 ) or those presenting with gleason score 5 + 5 = 10 or 5 + 4 = 9 ( primary gleason pattern 5 [ pg5 ] ; grade group 5 ) .1 these patients have the poorest oncologic outcomes , with higher relapse rates , frequent development of distant metastasis , poor response to systemic treatments , and short survival.2 - 4 previous studies have suggested that the proportion of gleason pattern 5 tumor on biopsy or surgical specimen denes a subset with independent prognostic implications and distinct outcomes after local and systemic treatments.5 - 8 although we have a wealth of data substantiating the poor prognosis of patients with predominant or pg5 , the molecular we know surprisingly little about alterations in these poorly differentiated tumors . among the 333 primary prostate tumors proled for the cancer genome atlas ( tcga ) , only eight patient cases ( 2% ) had pg5.9 because the prevalence of targetable alterations is relatively increased in other aggressive histologic subtypes of pc , 10 - 12 it is critically important to ll in these gaps in our molecular characterization of this disease . evolving data on metastatic castration - resistant pc ( mcrpc ) have demonstrated that druggable genomic alterations are not uncommon . 
 isaacsson velho et al context key objective we performed an integrated clinical and genomic analysis of prostate tumors with primary gleason pattern 5 at radical prostatectomy to identify potentially actionable alterations . knowledge generated in this very high - risk patient subset , adverse pathologic ndings and common molecular alterations , such as tp53 mutation , pten loss , and erg expression , were associated with poor outcomes . 
importantly , one third of patients had pathogenic mutations in dna repair genes , including in the homologous repair and mismatch repair pathways . relevance these data support the notion that aggressive subsets of primary prostate cancer are enriched for actionable genomic alterations and could inform the design of prospective clinical trials in this population . ( pd - 1 ) inhibitors.18 yet how these results in mcrpc might translate into treatment for clinically localized disease remains unclear . 
several clinical trials evaluating perioperative therapies in patients with high - risk localized including androgen - deprivation therapy ( adt ) , 19 antiandrogens , 20 and docetaxel , 21 have failed to demonstrate relevant benet . 
however , the utility of neoadjuvant and / or adjuvant parp and pd - 1 inhibitors has not yet been studied , in large part because of the relative rarity of these alterations in primary pc cohorts . here , we assembled a consecutive cohort of clinically localized prostate tumors with pg5 and long - term clinical follow - up after radical prostatectomy ( rp )  . 
to maximize duration of oncologic follow - up with the most recent pathologic specimens possible to preserve dna quality , we queried the pathology database for consecutive rps with pg5 from 2005 to 2015 and identied 60 patient cases with available tissue and clinical follow - up ( appendix fig a1 )  . immunohistochemistry tissue microarrays were constructed as previously reported.11 immunostaining for erg , pten , and p53 status was performed using previously reported and genetically validated rabbit monoclonal antibodies and staining and scoring protocols.22 - 26 punches from regions with the highest percentage of gleason pattern 5 tumor . 
dna was extracted from formalin - xed parafn - embedded material as previously described.11 next - generation tumor sequencing targeted next - generation deep sequencing to evaluate 262 cancer - related genes at a 500 average depth was performed using uw - oncoplex ( university of washington , seattle , wa ) as previously described.27 reported alterations were limited to those deemed pathogenic or likely pathogenic in the clinvar database . 
germline mutations were inferred by expert molecular pathologist review through cross - referencing against the clinvar database and by variant allele fraction in the context of tumor content , ploidy , and loss of heterozygosity status.28 , 29 two patients ( two of 49 ) did not have tumor dna available for uwoncoplex but had prior somatic sequencing performed using a clinical platform for clinical care purposes ( pgdx ; foundation medicine , cambridge , ma , and baltimore , md ) and were included in the analysis . 
patient cases in which tumor purity and / or dna quality was not high enough to exclude false - negative results are listed in the data supplement . germline sequencing altogether , 55% patient cases ( 27 of 49 ) underwent germline sequencing ( data supplement )  . 
 ( % ) a conrmatory test , 31 and metastasis - free survival ( mfs ) , dened as the time from rp to the rst detection of distant metastasis on imaging or death resulting from any cause , whichever occurred rst . 
time to castration resistance ( tcr ) , dened as the time from adt to two consecutive elevations in psa at least 4 weeks apart , and overall survival ( os ) , dened as the time from rp to death , were also assessed . 
univariable and multivariable cox proportional hazards models were used to estimate hazard ratios ( hrs ) and corresponding 95% cis and test for the association of the variables with clinical outcomes . 
because this study was hypothesis generating , we did not perform corrections for multiple comparisons . results baseline characteristics demographic , clinical , and pathologic characteristics of the pg5 cohort ( n = 60 ) at rp are listed in table 1 . 
at surgery , most patients had pathologic evidence of locally advanced disease , including nonorgan - conned ( pt3 / t4 ) tumors ( 54 [ 90% ] of 60 ) , seminal vesicle invasion ( svi ) ( 35 [ 58% ] of 60 ) , positive lymph nodes ( 16 [ 27% ] of 60 ) , and positive surgical margins ( 28 [ 47% ] of 60 )  . 
of the 37 patients who received either salvage or adjuvant radiation therapy , 27 ( 73% ) also received adjuvant adt . also , of the 16 patients who had positive lymph nodes ( pn + ) , 11 ( 69% ) received adjuvant adt . prevalence of gene mutations overall , 81.7% ( 49 of 60 ) of patients had available sequencing data on somatic genomic alterations ( data supplement ) , and 35% ( 17 of 49 ) of these had at least one pathogenic mutation in a gene involved in dna repair ( table 2 ; data supplement )  . 
overall , 55% ( 27 of 49 ) had germline sequencing results available , including six patients with presumed germline homologous recombination alterations on the basis of somatic sequencing , all of which were conrmed on germline sequencing ( brca2 , n = 3 ; brca1 , chek2 , and palb2 , n = 1 each )  . 
in addition to these alterations in homologous recombination genes , mmr deciency was found in 12% ( six of 49 ) of patients ( all in msh2 and included in our previous immunohistochemistry [ ihc ] study11 )  . other mutations common in pc were also identied ( data supplement )  . 
of 57 patients who had tumors evaluated by ihc , 51% ( 29 of 57 ) had pten protein loss . foxa1 mutations were seen in 12% ( six of 49 ) of patients , and spop mutations were seen in 8% ( four of 49 ) of patients . 
several pathologic characteristics were associated with increased risk of bcr on univariable analysis , including svi ( appendix table a1 ; appendix fig a2a ) and presence of ductal or intraductal histology ( appendix table a1 ; appendix fig a2b )  . 
multivariable models were t separately for each genomic alteration , adjusting for ductal or intraductal histology and svi ; however , none of the genomic alterations remained signicantly associated with bcr ( appendix table a2 )  . overall , 43% ( 26 of 60 ) of the cohort developed metastasis , with a median mfs of 86.4 months ( 95% ci , 40.7 months to not reached )  . 
visceral disease was seen in 31% of patients who developed metastasis , with liver ( 23% ) and lung ( 19% ) the most common sites . among clinicopathologic factors on univariable analysis , presence of svi ( table 3 ; fig 1a ) and ductal or intraductal histology ( table 3 ; fig 1b ) , along with patient age , psa , and nodal status , were both associated with increased risk of metastasis ( fig 1b )  . 
kaplan - meier analysis of metastasis - free survival in primary gleason pattern 5 cohort by ( a ) seminal vesicle invasion ( svi ) status , ( b ) ductal or intraductal ( doi ) histology status , ( c ) tp53 mutation status , and ( d ) pten protein status using immunohistochemistry . 
we chose a subset of patients with established unfavorable clinical outcomes who were diagnosed before evidence of metastatic disease and received the same initial local treatment to minimize potential biases associated with extent of disease . 
almost all patients had nonorganconned disease , more than half had svi , and a quarter had node - positive disease , demonstrating the aggressive behavior of these poorly differentiated tumors . the prevalence of genomic abnormalities in the dna repair pathway in clinically localized pg5 seems to be comparable to or perhaps even higher than that in patients with mcrpc . pritchard et al13 showed that 12% of patients with mcrpc table 5 . 
abnormalities in homologous recombination and mmr genes could have potential therapeutic implications for the treatment of all stages of pc , including the design of adjuvant and neoadjuvant trials in the pg5 population . 
in addition , our ndings have important implications for the families of patients with very high - risk disease , given that 82% of patients with homologous recombination mutations in this study had apparent underlying germline mutations . 
these ndings clearly support the recent national comprehensive cancer network guidelines , which recommend genetic testing in all patients with high - risk or very high - risk pc . our results show that there are pathologic and molecular features that are useful for risk stratication after rp . 
patients with svi or ductal or intraductal histology have more than four times greater risk of metastasis , and tp53 mutation , pten loss , and erg expression were associated with increased risk of biochemical failure and metastasis . 
notably , our study validates current clinicopathologic variables used for risk stratication and suggests that pathologic ndings after rp may be more signicant for risk prediction than molecular alterations in multivariable models . 
however , where full pathologic data are not available ( eg , in patients who do not undergo rp ) , molecular alterations could be of added value . future studies should evaluate prospectively the utility of tp53 mutation and pten loss in well - designed biomarkerdriven clinical trials cohorts , particularly in the biopsy setting . in addition , although we believe that our data are unique and informative , these results should be viewed as preliminary because of relatively the small numbers and multiplicity of clinical , pathologic , and molecular data points , which limit the power of the observations with regard to outcomes . 
it is likely that a prospective evaluation with additional numbers and follow - up time will allow for a more precise study of the prognostic signicance of clinicopathologic and molecular parameters in this group of patients . the pivotal role of adt in pc with gleason scores 9 to 10 has been called into question in recent years.8 a recent large retrospective study of patients undergoing external - beam radiation therapy in combination with adt compared outcomes of patients with gleason score 8 versus 9 to 10 , suggesting that those in the higher gleason group may derive less benet than patients with gleason score 8.8 recent studies have shown that the addition of either docetaxel21 , 32 or abiraterone33 , 34 improves os in the locally advanced or metastatic hormone - sensitive setting , with higher benet among high - risk patients , including those with high disease burden , 32 t3 to t4 disease , 21 , 34 psa greater than 40 ng / ml , 21 , 34 and gleason score 8 to 10.21 , 33 , 34 our data suggest that compared with lower - grade tumors , pg5 tumors may be less dependent on androgens and potentially more dependent on alternative oncogenic drivers , including defects in dna repair pathways . limitations of the study include the fact that it was retrospective , and clinical follow - up and dna sequencing were available only for a subset of patients , although the clinicopathologic parameters of patients with and without follow - up and sequencing were not signicantly different ( appendix fig a1 )  . 
also , the quality of the archival dna and tumor purity were not uniformly high , and false negatives at sequencing could not be excluded in all patient cases , suggesting that our results may actually underestimate the prevalence of dna repair alterations among pg5 tumors . 
 isaacsson velho et al in conclusion , we performed the rst integrated clinical and genomic analysis to our knowledge of pg5 prostate tumors at rp , a subset that has not been well represented in other sequencing efforts . 
clinical outcomes and response to conventional treatment are worse compared with lower - risk groups , and adverse pathologic ndings and common molecular alterations such as tp53 mutation , pten loss , and erg expression are associated with poor outcomes . 
the relatively high incidence of hrd and mmr deciency observed herein could inform the design of multimodal clinical trials employing parp inhibitors or pd - 1 / pd - 1 ligand monoclonal antibodies in very high - risk patients with clinically localized disease . 
contributed equally to this work . support supported by the patrick walsh research fund ; by national cancer institute ( nci ) , national institutes of health , prostate specialized program of research excellence grant no . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . travel , accommodations , expenses : astrazeneca , astellas pharma , pzer , merck serono , merck michael a . 
carducci consulting or advisory role : astellas pharma , abbvie , roche / genentech , pzer , foundation medicine research funding : bristol - myers squibb ( inst ) , pzer ( inst ) , astrazeneca ( inst ) , gilead sciences ( inst ) , emd serono ( inst ) , effector therapeutics ( inst ) samuel r . 
antonarakis honoraria : sano , dendreon , medivation , janssen biotech , essa , astellas pharma , merck , astrazeneca , clovis oncology consulting or advisory role : sano , dendreon , medivation , janssen biotech , essa , astellas pharma , merck , astrazeneca , clovis oncology research funding : janssen biotech ( inst ) , johnson & johnson ( inst ) , sano ( inst ) , dendreon ( inst ) , aragon pharmaceuticals ( inst ) , exelixis ( inst ) , millennium pharmaceuticals ( inst ) , genentech ( inst ) , novartis ( inst ) , astellas pharma ( inst ) , tokai pharmaceuticals ( inst ) , merck ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , constellation pharmaceuticals ( inst ) patents , royalties , other intellectual property : coinventor of biomarker technology licensed to qiagen travel , accommodations , expenses : sano , dendreon , medivation mario a . 
eisenberger leadership : veru stock and other ownership interests : veru honoraria : sano , pzer consulting or advisory role : astellas pharma , ipsen , bayer healthcare pharmaceuticals , sano , pzer research funding : sano , tokai pharmaceuticals , genentech travel , accommodations , expenses : bayer healthcare pharmaceuticals , astellas pharma , sano , pzer , veru pedro isaacsson velho honoraria : bayer healthcare pharmaceuticals speakers bureau : astrazeneca , pzer , bristol - myers squibb research funding : bristol - myers squibb , pzer ( inst ) expert testimony : bayer healthcare pharmaceuticals tamara l . 
prostate cancer 2018 : 151 epstein ji , zelefsky mj , sjoberg dd , et al : a contemporary prostate cancer grading system : a validated alternative to the gleason score . 
eur urol 69 : 428 - 435 , 2016 sundi d , tosoian jj , nyame ya , et al : outcomes of very high - risk prostate cancer after radical prostatectomy : validation study from 3 centers . 
cancer 125 : 391397 , 2019 sundi d , wang vm , pierorazio pm , et al : very - high - risk localized prostate cancer : denition and outcomes . 
prostate cancer prostatic dis 17 : 57 - 63 , 2014 ellis cl , partin aw , han m , et al : adenocarcinoma of the prostate with gleason score 9 - 10 on core biopsy : correlation with ndings at radical prostatectomy and prognosis . 
j urol 190 : 2068 - 2073 , 2013 cheng l , davidson dd , lin h , et al : percentage of gleason pattern 4 and 5 predicts survival after radical prostatectomy . 
cancer 110 : 1967 - 1972 , 2007 tilki d , chen mh , wu j , et al : surgery vs radiotherapy in the management of biopsy gleason score 9 - 10 prostate cancer and the risk of mortality . 
marshall ch , fu w , wang h , et al : prevalence of dna repair gene mutations in localized prostate cancer according to clinical and pathologic features : association of gleason score and tumor stage . 
clin cancer res 23 : 6863 - 6874 , 2017 isaacsson velho p , silberstein jl , markowski mc , et al : intraductal / ductal histology and lymphovascular invasion are associated with germline dna - repair gene mutations in prostate cancer . 
powell ij , tangen cm , miller gj , et al : neoadjuvant therapy before radical prostatectomy for clinical t3 / t4 carcinoma of the prostate : 5 - year followup , phase ii southwest oncology group study 9109 . 
vale cl , burdett s , rydzewska lhm , et al : addition of docetaxel or bisphosphonates to standard of care in men with localised or metastatic , hormone - sensitive prostate cancer : a systematic review and meta - analyses of aggregate data . 
tosoian jj , almutairi f , morais cl , et al : prevalence and prognostic signicance of pten loss in african - american and european - american men undergoing 23 . 
lotan tl , wei w , ludkovski o , et al : analytic validation of a clinical - grade pten immunohistochemistry assay in prostate cancer by comparison with pten radical prostatectomy . 
maughan bl , guedes lb , boucher k , et al : p53 status in the primary tumor predicts efcacy of subsequent abiraterone and enzalutamide in castration - resistant prostate cancer . 
pritchard cc , salipante sj , koehler k , et al : validation and implementation of targeted capture and sequencing for the detection of actionable mutation , copy number variation , and gene rearrangement in clinical cancer specimens . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
na r , zheng sl , han m , et al : germline mutations in atm and brca1 / 2 distinguish risk for lethal and indolent prostate cancer and are associated with early age 31 . 
cookson ms , aus g , burnett al , et al : variation in the denition of biochemical recurrence in patients treated for localized prostate cancer : the american urological association prostate guidelines for localized prostate cancer update panel report and recommendations for a standard in the reporting of surgical outcomes . 
n engl j med 377 : 352 - 360 , 2017 james nd , de bono js , spears mr , et al : abiraterone for prostate cancer not previously treated with hormone therapy . 
pathology database query for consecutive radical prostatectomies ( rps ) with primary gleason score 5 ( pg5 ) from 2005 to 2015 , where 60 patient cases were identied with available tissue and clinical follow - up . 
 c significant tumor response to the poly ( adp - ribose ) polymerase inhibitor olaparib in heavily pretreated patient with ovarian carcinosarcoma harboring a germline rad51d mutation introduction brca1 and brca2 are the best - known examples of proteins that contribute to repair of double - strand dna breaks via the homologous recombination repair ( hrr ) pathway . the rad51 paralogs rad51b , rad51c , and rad51d are proteins that are also involved in the hrr pathway . 
parp inhibitors are rationally designed drugs that inhibit parp1 by trapping the inactivated enzyme onto the single - strand break , generating a potential block for cellular dna replication.5 - 7 trapped parp - dna complexes can lead to the stalling and / or collapse of replication forks , resulting in the generation of more deleterious double - strand breaks.8 in cells unable to carry out hrr because of brca mutation , the added genotoxic stress of inhibition of single - strand repair is cytotoxic and referred to as synthetic lethality.5 , 7 in vitro and mice studies support the use of parp inhibition , not only in the presence of brca mutation but also in malignancy with other genetic defects that affect hrr , such as rad51 deletion.9 given the rarity of rad51 mutations , there is limited clinical evidence in this context . we describe a heavily pretreated patient with ovarian carcinosarcoma , with known germline rad51d mutation , that exhibited a remarkable and durable response to parp inhibitor therapy . 
subsequently the patient experienced remissions of varying degrees and durations , with additional lines of therapy including paclitaxel , cisplatin , ifosfamide , and doxorubictwo years before presentation of this case report , the patient was enrolled in a phase i clinical trial using a novel pd - 1 inhibitor . 
 a writing , the patient continues to take olaparib , with no evidence of disease regrowth . discussion synthetic lethality is a term used to describe the combination of two defects that do not affect cell viability when each occurs individually but are cytotoxic when they occur simultaneously in a single cell.7 , 10 parp inhibitors have been used to exploit synthetic lethality in brca1and brca2 - mutant ovarian , breast , prostate , and other cancers . 
 however , hrr deficiency extends beyond cancers harboring brca1 / 2 mutations , and the term brcaness has been coined to describe the clinical and biologic features of such tumors . 
 this not only includes similar histomorphological features such as basal - like phenotype in breast cancers or high - grade serous morphology in ovarian cancers , but also similar immunophenotypic profile ( eg , triple - negative breast cancers ) , drug sensitivity ( eg , to platinum agents and parp inhibitors ) , as well as prognosis.11 - 13 the brcaness phenotype is seen in tumors with loss - of - function mutations involving other hrr pathway genes , including atm , atr , bard1 , brip1 , mre11a , palb2 , rad50 , rad51d , rad54 , nbs1 , chek1 , and chek2 , as well as components of the fanconi anemia repair pathway.13 , 14 in vitro and mice studies have demonstrated efficacy of parp inhibitors against cancer cells with defects in rad51 . 
separate studies by shah et al , cruz et al , and wang et al found that breast or ovarian cancer cells with absent or low levels of rad51 were sensitive to parp inhibitors.15 - 17 another study observed that parp inhibitor sensitivity was increased by 1 , 000 - fold in cells that are rad51 knockdown.14 cruz et al and wang et al additionally demonstrated that the presence of rad51 conferred resistance to parp inhibitors in these cells.16 , 17 in their experiments on triple - negative breast cancer cells , wang et al postulated that it may be possible to expand the sensitivity of triple - negative breast cancer to parp inhibitors by targeting rad51.17 other studies found increased sensitivity to parp inhibition when rad51 inhibitors were coadministered.10 , 18 interestingly , falchi et al found the addition of a brca2 - rad51 disruptor to parp inhibition to be synergistic , rather than merely additive , which is probably a consequence of fig 1 . 
 ( a ) baseline computed tomography ( ct ) scan of pelvis , demonstrating heterogeneous mass ( arrowhead ) , ( b ) ct scan after 8 weeks on olaparib , showing excellent partial response in pelvic mass ( arrowhead ) , and ( c ) ct scan after 16 weeks on olaparib , showing additional disease response ( arrowhead )  . disease response for 16 months , followed by rapid and significant tumor regrowth ( fig 1a )  . the patient had previously been determined to have a germline mutation in rad51d , evidenced by a heterozygous variant c.263 + 1g > a . 
 this mutation has previously been described in ovarian cancer.4 with the possibility of defective hrr in the patients tumor as the result of loss of the remaining functional rad51d allele , a therapeutic trial of olaparib was initiated . 
we are not aware of other case reports that have demonstrated disease response to parp inhibitors in tumors with an underlying rad51d mutation . our clinical experience is consistent with preclinical models and adds to the current limited clinical evidence of benefit of parp inhibition in the context of rad51 mutations . 
chandran no relationship to disclose ian kennedy no relationship to disclose the simultaneous inhibition of two dna - repair mechanisms.10 a 2016 phase ii study ( ariel2 ; a study of rucaparib in patients with platinum - sensitive , relapsed , high - grade epithelial ovarian , fallopian tube , or primary peritoneal cancer ) used the parp inhibitor rucaparib in recurrent , platinum - sensitive , high - grade ovarian carcinoma . 
 of 206 patients , there were seven with rad51 mutations : one patient with rad51b mutation with stable disease , who was receiving rucaparib ; four patients with rad51c mutations ( three partial responses and one patient with stable disease ) ; and two patients with rad51d mutation , both with stable disease.19 using baseline and postprogression biopsy samples from the same study , kondrashova et al reported that in five of six patients with rad51c and rad51d truncation mutations that subsequently developed resistance to rucaparib , there were one or more secondary mutations restoring the open reading frame . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
song h , dicks e , ramus sj , et al : contribution of germline mutations in the rad51b , rad51c , and rad51d genes to ovarian cancer in the population . 
tan dsp , rothermundt c , thomas k , et al : brcaness syndrome in ovarian cancer : a casecontrol study describing the clinical features and outcome of patients with epithelial ovarian cancer associated with brca1 and brca2 mutations . 
mccabe n , turner nc , lord cj , et al : deficiency in the repair of dna damage by homologous recombination and sensitivity to poly ( adp - ribose ) polymerase inhibition . 
shah mm , dobbin zc , nowsheen s , et al : an ex vivo assay of xrt - induced rad51 foci formation predicts response to parp - inhibition in ovarian cancer . 
cruz c , castroviejo - bermejo m , gutirrez - enrquez s , et al : rad51 foci as a functional biomarker of homologous recombination repair and parp inhibitor resistance in germline brca - mutated breast cancer . 
swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
kondrashova o , nguyen m , shield - artin k , et al : secondary somatic mutations restoring rad51c and rad51d associated with acquired resistance to the parp inhibitor rucaparib in high - grade ovarian carcinoma . 
tan author affiliations and support information ( if applicable ) appear at the end of this article . creative commons attribution non commercial no derivatives 4.0 license corresponding author : david s.p. 
indeed , hrd - related genetic aberrations may confer a similar clinical phenotype to gboc , 7 , 8 including improved responses and longer progression - free survival ( pfs ) in patients receiving maintenance parpi treatment.9 - 11 few effective treatment options exist for patients with recurrent hgsoc ( rhgsoc ) , especially in the context of malignant bowel obstruction , where the use of bevacizumab is associated with a higher risk of bowel perforation.12 we present the case of a patient with a germline pathogenic rad51c mutation who received salvage treatment for terminal malignant bowel obstruction with sixth - line carboplatin and pegylated liposomal doxorubicin ( pld ) , followed by maintenance olaparib . a 57 - year - old chinese woman was referred to our institution after hgsoc relapse . 
she first received the hgsoc diagnosis at 53 years of age , with no family history of breast or ovarian cancer , and had received initial treatment elsewhere ( table 1 )  . 
at first relapse of hgsoc ( after a 28 - month pfi ) , she received carboplatin plus pld every 4 weeks for six cycles , followed by secondary debulking . 
she received four cycles of carboplatin every 3 weeks with dose - dense paclitaxel once per week , followed by two cycles of cisplatin substituted for carboplatin , owing to grade 2 carboplatin hypersensitivity . 
complete response was achieved with a nadir ca - 125 concentration of 90 u / ml . three months later , however , the patients ca - 125 level rose to 571 u / ml , at which point she transferred care to our institution . 
clinical presentation and treatment summary treatment received at another institution presentation and treatment date may 2010 july 2010 july - nov 2010 mar 2013 apr - june 2013 july 2013 sept - nov 2013 june 2014 july - nov 2014 jan 2015 feb 2015 apr - june 2015 june 2015 june - nov 2015 feb - june 2016 june 2016 july - oct 2016 nov 2014 ca - 125 level nadir of 90 u / ml . complete response determined from repeated ct scan . transfer of care to our institution genetic testing performed using invitae panel . hgsoc diagnosed at another institution . 
 switched to cisplatin ( 75 mg / m2 ) and paclitaxel ( 175 mg / m2 ) every 3 weeks for two more cycles in view of grade 2 hypersensitivity to carboplatin . biochemical recurrence ; ttp : 3 months ; ca - 125 level : 571 u / ml . 
no measurable disease seen on ct scan . genetic testing showed pathogenic grad51c mutation . symptomatic hgsoc relapse ; pfi now 5 months ; ca - 125 level : 3 , 145 u / ml . 
intestinal obstruction resolved with conservative management . switched to fourth - line carboplatin ( auc 5 ) with desensitization protocol and gemcitabine ( 1 , 000 mg / m2 , days 1 and 8 ) every 3 weeks . carboplatin ( auc 5 ) treatment continued with desensitization protocol and gemcitabine ( 1 , 000 mg / m2 , days 1 and 8 ) every 3 weeks ; total of eight cycles with partial response . 
ct scan shows residual 3.6 - cm pelvic implant . ct scans showed new ascites and increasing size of peritoneal implant . patient enrolled in phase i study of low - dose whole abdominal radiation therapy ( 60 cgy fractions , 2 d / wk , twice on each of those days ) , in combination with weekly paclitaxel ( 80 mg / m2 )  . 
pfs while receiving olaparib treatment : 7 months . underwent surgical exploration and defunctioning jejunostomy . july 2017 multiple levels of malignant small - bowel obstruction developed . patient referred to hospice care . 
she joined a phase i trial of kpt330 ( clinicaltrials.gov identifier : nct02078349 ) , but her disease progressed after 2 months , and she had an intestinal obstruction from worsening peritoneal metastases . 
after eight cycles , sustained partial response ( pr ) was achieved according to gynecologic cancer intergroup criteria.14 the ca - 125 concentration improved from 7 , 550 u / ml to 368 u / ml . 
the patient was given a chemotherapy holiday . unfortunately , after another short ttp of 2 months , there was symptomatic disease progression , and the patient was enrolled in a phase ii clinical trial of low - dose , whole - abdomen radiation combined with weekly paclitaxel treatment . 
this approach was based on the knowledge that genomic hrd could enable prediction of response to dna - damaging agents such as carboplatin and topoisomerase iia inhibitors.15 the patient chose carboplatin plus pld every 4 weeks , and her disease responded according to gynecologic cancer intergroup criteria . 
the patient was experiencing grade 2 fatigue and , in view of emerging data at that time on the efficacy of parpis in tumors with hrd , 16 a complete break from treatment , versus off - label maintenance therapy with olaparib capsules , was discussed . 
 she chose the latter option . because of financial constraints , the patient was prescribed olaparib capsules 200 mg twice daily ( rather than the 400 - mg twice - daily standard dosing ) , but her disease continued to respond , with a reduction in ca - 125 level from 1 , 405 u / ml to a nadir of 44.7 u / ml ( fig 2 )  . 
after 6 months of olaparib treatment , scans confirmed pr by response evaluation criteria in solid tumors ( recist ) , version 1.1 , with improved ascites and resolution of a perihepatic peritoneal deposit ( fig 1 )  . unfortunately , the patients disease relapsed a month later with small - bowel obstruction and required parenteral nutrition . 
 ( a and b ) june 2016 ( preplatinum rechallenge ) : left lung lesion ( curved arrow ) , ascites , and peritoneal thickening ( double arrow )  . 
 ( e and f ) april 2017 resolution of ascites and of the deposit at the hepatic hilum after 6 months of olaparib treatment ( originally seen in ( d ) , arrow )  . many to be a preterminal event.17 retrospective data show limited responses from chemotherapy.17 yet , this patient responded remarkably to sixthline carboplatin plus pld and , per the recent us food and drug administration approval for maintenance parpis , derived significant benefit from olaparib , even at an off - label dose of 200 mg twice daily . 
notably , biologically effective inhibition of parp by olaparib capsules has been demonstrated even at 100 mg twice daily.18 to our knowledge , this report is the first to describe the clinical effect of this specific grad51c variant on treatment selection and patient outcome in rhgsoc . the cancer genome atlas project showed approximately 30% of hgsoc cases harbor nonbrca1 / 2 mutation - related hrd . 
 more recently , olaparib , 7 - 9 niraparib , 10 and rucaparib16 were approved for use in patients who have received at least two prior treatments of platinum - based chemotherapy and are platinum responsive . 
of note , all patients eligible for the landmark phase iii trials , 9 - 11 leading to approval of parpi for this indication , had experienced a ttp after penultimate platinum exposure of 6 months , which was not the case in our patient . efficacy data on resensitization to platinum based chemotherapy and thresholds constituting an effective pfi are ambiguous . 
dramatic reduction of ca - 125 level from 17 , 256 u / ml to 867.7 u / ml was noted after the patient commenced receiving carboplatin plus pld treatment . 
analyses revealed a frameshift - truncating mutation of tp53bp1 , which is associated with olaparib resistance.33 reversion mutations in rad51c have also been described in acquired rucaparib resistance.34 unfortunately , at the time of disease progression , surgeons were unable to obtain an adequate tumor sample for analysis of resistance mechanisms in the patient in the current report . in conclusion , patients with ovarian cancer with grad51c mutations may benefit from similar treatment paradigms as brca1 / 2 - mut disease . 
 darwin tay no relationship to disclose valerie heong consulting or advisory role : roche ; astrazeneca patents , royalties , other intellectual property : royalties received for drug venetoclax travel , accommodations , expenses : astrazeneca soo chin lee honoraria : roche ; astrazeneca ; pfizer ; novartis consulting or advisory role : pfizer ; novartis ; astrazeneca ; roche ; eisai research funding : eisai ; taiho pharmaceutical ; bayer ; aslan pharmaceuticals expert testimony : novartis travel , accommodations , expenses : roche ; novartis ; pfizer ; act genomics yee liang thian no relationship to disclose pei yi ong no relationship to disclose samuel g.w. 
jeyasekharan honoraria : msd oncology yi wan lim no relationship to disclose siew eng lim no relationship to disclose research funding : astrazeneca ; perkin elmer travel , accommodations , expenses : perkin elmer joseph ng no relationship to disclose jeffrey j.h. 
tan honoraria : astrazeneca ; roche consulting or advisory role : astrazeneca ; roche research funding : astrazeneca ( inst ) ; karyopharm therapeutics ( inst ) ; bayer ( inst ) travel , accommodations , expenses : merck sharp & dohme ; merck serono ; astrazeneca ; bayer ; roche affiliations natalie y.l. 
alsop k , fereday s , meldrum c , et al : brca mutation frequency and patterns of treatment response in brca mutation - positive women with ovarian cancer : a report from the australian ovarian cancer study group . 
song h , dicks e , ramus sj , et al : contribution of germline mutations in the rad51b , rad51c , and rad51d genes to ovarian cancer in the population . 
swisher em , harrell mi , lin k , et al : brca1 and rad51c promoter hypermethylation confer sensitivity to the parp inhibitor rucaparib in patients with relapsed , platinum - sensitive ovarian carcinoma in ariel2 part 1 . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
pujade - lauraine e , ledermann ja , selle f , et al : olaparib tablets as maintenance therapy in patients with platinum - sensitive , relapsed ovarian cancer and a brca1 / 2 mutation ( solo2 / engot - ov21 ) : a double - blind , randomised , placebo - controlled , phase 3 trial . 
coleman rl , oza am , lorusso d , et al : rucaparib maintenance treatment for recurrent ovarian carcinoma after response to platinum therapy ( ariel3 ) : a randomised , double - blind , placebocontrolled , phase 3 trial . 
rustin gj , vergote i , eisenhauer e , et al : definitions for response and progression in ovarian cancer clinical trials incorporating recist 1.1 and ca 125 agreed by the gynecological cancer intergroup ( gcig )  . 
tan ds , kaye sb : chemotherapy for patients with brca1 and brca2 - mutated ovarian cancer : same or different ? am soc clin oncol educ book 35 : 114 - 121 , 2015 16 . 
swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
kaye sb , lubinski j , matulonis u , et al : phase ii , open - label , randomized , multicenter study comparing the efficacy and safety of olaparib , a poly ( adp - ribose ) polymerase inhibitor , and pegylated liposomal doxorubicin in patients with brca1 or brca2 mutations and recurrent ovarian cancer . 
gayarre j , martn - gimeno p , osorio a , et al : characterisation of the novel deleterious rad51c p.arg312trp variant and prioritisation criteria for functional analysis of rad51c missense changes . 
hatanaka a , yamazoe m , sale je , et al : similar effects of brca2 truncation and rad51 paralog deficiency on immunoglobulin v gene diversification in dt40 cells support an early role for rad51 paralogs in homologous recombination . 
yokoyama h , sarai n , kagawa w , et al : preferential binding to branched dna strands and strand - annealing activity of the human rad51b , rad51c , rad51d and xrcc2 protein complex . 
somyajit k , mishra a , jameei a , et al : enhanced non - homologous end joining contributes toward synthetic lethality of pathological rad51c mutants with poly ( adp - ribose ) polymerase . 
ghiringhelli f , richard c , chevrier s , et al : efficiency of olaparib in colorectal cancer patients with an alteration of the homologous repair proteworld j gastroenterol 22 : 10680 - 10686 , 2016 33 . 
kondrashova o , nguyen m , shield - artin k , et al : secondary somatic mutations restoring rad51c and rad51d associated with acquired resistance to the parp inhibitor rucaparib in high - grade ovarian carcinoma . 
others have an intermediate phenotype and are classified as glomus tumors of uncertain malignant potential ( gt - ump ) .3 herein , we present a case of an 18 - year - old patient with two glomus tumors involving the lateral and posterior cords of the right brachial plexus . 
physical examination demonstrated no neurologic deficit . imaging mr imaging of the right upper extremity demonstrated two distinct brachial plexus lesions , one emanating from the distal aspect of the lateral cord and the other from the posterior cord ( fig 1 )  . 
after extensive discussion with the neurosurgeon , the patient opted for surgical exploration with open biopsy and possible removal of the infraclavicular tumors to provide a histologic diagnosis and guidance on further management . intraoperative findings open exploration of the right infraclavicular brachial plexus was performed . 
these were gently mobilized but did not stimulate and were divided , allowing the lesion to be removed en bloc and sent for histopathological analysis . in the posterior cord region , the axillary nerve was clearly enlarged with tumor . 
 ( a ) on coronal fluid - sensitive magnetic resonance sequence through the chest , the right brachial plexus demonstrates asymmetric hyperintensity and thickening of the c5 , c6 , c7 , and t1 nerve roots and upper trunk ( not shown ) in addition to two discrete masses arising from the distal brachial plexus involving the posterior ( short arrow ) and lateral ( long arrow ) cords . 
 ( c ) on diffusion weighted imaging with apparent diffusion coefficient mapping , there is qualitative and quantitative restricted diffusion ( apparent diffusion coefficient [ adc ] values range from 0.6 to 1 103 mm2 / s in the larger mass and 0.9 to 1 103 mm2 / s in the smaller mass )  . 
on frozen section , this tumor appeared relatively benign , but given its widespread involvement with the axillary nerve , further aggressive dissection of the remaining tumor was deferred to avoid morbid neurologic deficits . histopathology and molecular analysis histopathologic analysis revealed a well circumscribed proliferation of relatively monomorphic cells with round nuclei and abundant eosinophilic cytoplasm ( figs 2a and 2b )  . 
mart1 was faint , caldesmon was focal , cd34 highlighted blood vessels , ini - 1 expression was intact , and ki - 67 proliferation index was mild to moderate . 
these findings together led to the diagnosis of malignant glomus tumor per current criteria.3 molecular genetic analysis using polymerase chain reactionbased sequencing of braf exon 15 revealed the braf v600e mutation at a mutant allele frequency of 40% . 
 to support this finding , we performed immunohistochemical analysis using antibodies directed against phosphoextracellular signal - regulated kinase ( erk ) ; this revealed variable immunostaining , with both cytoplasmic and nuclear immunoreactivity . 
in addition to isolated braf testing , a conventional cytogenetic analysis of the tumor revealed a normal 46 xx karyotype in all cells examined . therapeutic management management of this patients malignant tumor was discussed at the johns hopkins hospital multidisciplinary sarcoma tumor board . 
 ( a ) low - power view of glomus tumor associated with peripheral nerve fascicles , hematoxylin and eosin sta ( b ) tumor cells had ample eosinophilic cytoplasm and evident mitotic activity . 
mr imaging and 18f - fluorodeoxy - d - glucose positron emission tomographycomputed tomography obtained 2 weeks after the addition of trametinib demonstrated interval decrease in tumor size ( table 1 )  . 
 mr imaging at 3 and 6 months after initiating raf inhibitor therapy showed continued decrease in tumor size ( figs 3 and 4 ) and at 9 months showed no further change in size or imaging characteristics relative to most recent prior imaging . 
she experiences no pain , and the extremity function is normal . discussion the ras - raf - mek - erk signaling pathway is a frequent site of oncogenic mutations , with approximately 30% of human cancer harboring at least one mutation that results in its dysregulation . 
activating braf mutations are found in approximately 7% of cancers , roughly half of malignant melanomas , 5 and in other tumors , including colorectal , ovarian , papillary thyroid , and nonsmall - cell lung cancers , gliomas , and histiocytic disorders.6 - 12 the most common mutation , v600e , results in constitutive activation of the erk pathway . small molecule inhibitors of raf kinase were first studied in clinical trials of patients with melanoma . 
to determine whether the benefit of targeted raf inhibition would extend to nonmelanoma tumors with braf v600e , a phase ii basket trial explored the use of vemurafenib in patients selected for this mutation . 
this study demonstrated modest antitumor activity , although not all tumors with braf mutation responded , thus enforcing that a single driver oncogene may be associated with a nonuniform response to targeted therapy.16 braf mutations also occur rarely in a wide spectrum of cancers , including 1.4% of soft tissue sarcomas.17 a single patient with braf - mutated clear cell sarcoma was treated in the vemurafenib basket trial and sustained a partial response.16 the role of raf inhibitors in other sarcomas , however , has not been explored . glomus tumors are mesenchymal neoplasms that are rare and generally benign.18 they can occur as solitary or multiple soft tissue tumors and are most commonly located on the digits , supporting early hypotheses about their origin in the temperature - sensing glomus body . 
in a targeted sequencing analysis , three of 28 glomus tumors were found to harbor braf v600e mutations and a single tumor mutation in kras.19 a single case of a glomus tumor of the median nerve was found by next - generation sequencing to harbor a braf v600e mutation.20 this patient , and those in the retrospective analysis of 28 cases , was treated surgically without consideration for systemic therapy . 
finally , in a collection of 102 glomus tumors ( 56% benign , 15% gt - ump , and 29% malignant ) braf v600e mutation was detected in six tumors ( 6% ) , all of which were classified as either malignant glomus tumor or gt - ump ( and 0 of 57 benign glomus tumors harbored braf mutations ) .21 these data , although reflective of an overall small sample size , are suggestive of braf mutation as a predictor of more aggressive clinical behavior . 
 interestingly , glomus tumor has previously been associated with neurofibromatosis type 1 , 22 suggesting a shared terminal event in activation of erk signaling in these tumors . our patients response to targeted braf inhibition is an extraordinary one and highlights promise for future use in sarcomas with this mutation . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center  . andrea cuviello no relationship to disclose aviad zick research funding : merck ( inst ) , eli lilly ( inst ) shivani ahlawat no relationship to disclose fausto j . 
folpe al , fanburg - smith jc , miettinen m , et al : atypical and malignant glomus tumors : analysis of 52 cases , with a proposal for the reclassification of glomus tumors . 
kimura et , nikiforova mn , zhu z , et al : high prevalence of braf mutations in thyroid cancer : genetic evidence for constitutive activation of the ret / ptc - ras - braf signaling pathway in papillary thyroid carcinoma . 
mcarthur ga , chapman pb , robert c , et al : safety and efficacy of vemurafenib in braf ( v600e ) and braf ( v600k ) mutation - positive melanoma ( brim - 3 ) : extended follow - up of a phase 3 , randomised , open - label study . 
hauschild a , grob jj , demidov lv , et al : dabrafenib in braf - mutated metastatic melanoma : a multicentre , open - label , phase 3 randomised controlled trial . 
dahlin lb , scherman p , besjakov j , et al : intraneural glomus tumor of uncertain malignant potential and with braf mutation in the median nerve an unusual case . 
karamzadeh dashti n , bahrami a , lee sj , et al : braf v600e mutations occur in a subset of glomus tumors , and are associated with malignant histologic characteristics . 
dahlin lb , mller g , anagnostaki l , et al : glomus tumours in the long finger and in the thumb of a young patient with neurofibromatosis - 1 ( nf - 1 )  . 
voss ( continued ) purpose with prospective clinical sequencing of tumors emerging as a mainstay in cancer care , an urgent need exists for a clinical support tool that distills the clinical implications associated with specific mutation events into a standardized and easily interpretable format . 
to this end , we developed oncokb , an expert - guided precision oncology knowledge base . methods oncokb annotates the biologic and oncogenic effects and prognostic and predictive significance of somatic molecular alterations . 
potential treatment implications are stratified by the level of evidence that a specific molecular alteration is predictive of drug response on the basis of us food and drug administration labeling , national comprehensive cancer network guidelines , disease - focused expert group recommendations , and scientific literature . results to date , > 3 , 000 unique mutations , fusions , and copy number alterations in 418 cancerassociated genes have been annotated . 
with the shift from single analyte tests and small hotspot panels to larger gene panels and whole - exome and - genome platforms , interpretation of the clinical significance of the increasing number of genomic alterations identified in individual tumors has become a challenge . 
a smaller subset are known or suspected functionally significant mutations with no clear therapeutic implications , and the smallest subset consists of known driver mutations that are clinically actionable . the information that discriminates whether an alteration is clinically actionable can reside in various silos , including us food and drug administration ( fda ) labeling , national comprehensive cancer network ( nccn ) guidelines , conference proceedings , disease - focused expert group recommendations , and the scientific literature . 
therefore , an urgent need exists for a clinical support tool that distills this information into a standardized and easily interpretable format that democratizes its access to clinicians of all knowledge levels and at all centers . 
to address these limitations , we describe oncokb , a comprehensive precision oncology knowledge base that offers oncologists detailed , evidence - based information about individual somatic mutations and structural alterations present in patient tumors to support optimal treatment decisions . 
oncokb content is supervised by a dedicated panel of physicians and cancer biologists who review and edit biomarker - associated investigational therapeutic strategies ( data supplement )  . through continual dialogue with the scientific and medical community , oncokb integrates clinical best practices as defined by institutionwide , multidisciplinary disease management teams ( fig 1 )  . 
oncokb communicates information on the biomarker - guided use of fdaapproved therapies and investigational agents under evaluation in clinical trials and highlights negative clinical results to discourage off - label use of expensive targeted therapies shown to be ineffective in specific mutational contexts . methods oncokb includes biologic , clinical , and therapeutic information curated from multiple unstructured information resources , including guidelines and recommendations derived from fda labeling , nccn guidelines , other diseasespecific expert and advocacy group recommendations , and the medical literature ( fig 1 )  . with recognition that clinical implications vary substantially on the basis of specific alterations within a gene and tumor context , information in oncokb is hierarchically organized by gene , alteration , tumor type , and clinical implication ( fig 1 )  . 
oncokb information is publicly available through an interactive web site19 and incorporated into the cbioportal for cancer genomics , 20 - 22 where patient genomic alterations are annotated with information from oncokb and their biologic effects and clinical implications are summarized , which facilitates cancer researcher and clinician interpretation of complex genomic data ( fig 1 )  . 
3 , 000 alterations in 418 cancer - associated genes ( table 1 )  . levels of evidence to communicate the clinical utility of individual mutant alleles consistently , a level of evidence classification system was developed ( fig 2 ) that takes into account the site of tumor origin by recognizing that the effects of targeted inhibitors vary by tumor lineage , even in cancers that share the same mutant allele ( fig 3 )  . 
potentially actionable alterations in a specific cancer type are assigned to one of four levels that are based on the strength of evidence that the mutation is a predictive biomarker of drug sensitivity to fdaapproved or investigational agents for a specific indication . 
level 1 includes genes for which specific alterations have been recognized by the fda as predictive of response to an fda - approved drug in a particular disease context ( data supplement )  . examples include braf v600e and either vemurafenib or dabrafenib as monotherapy or in combination with the mek inhibitors cobimetinib or trametinib in melanoma ; egfr l858r and erlotinib , afatinib , or gefitinib in nonsmall - cell lung cancer ( nsclc ) ; and mutations in exons 9 and 11 of kit and imatinib , sunitinib , and regorafenib in gi stromal tumors . 
in total , 82 alterations in 12 genes are considered level 1 ( data supplement )  . in recognition that some alterations in what would be considered a level 1 gene are intrinsically resistant to currently available fda - approved drugs or fall outside explicit fda approval , oncokb assigns individual alterations to a level of evidence as opposed to the entire gene . 
 variant databases gene alteration tumor type clinical implications incorporation of feedback onc kb oncokb.org web site statistical recurrence treatment guidelines akt1 scientific literature data sources breast cancer ovarian cancer lung cancer e17k e40k l52r q79k amplification prevalence prognosis nccn guidelines standard therapy clinical genomics annotation committee oncokb api cbioportal msk clinical reports investigational therapy curation review variant curation oncokb access fig 1 . 
data sources : alterations are identified by their recurrence18 from public variant databases ( cbioportal , cosmic [ catalogue of somatic mutations in cancer ] , the memorial sloan kettering [ msk ] impact internal clinical sequencing cohort ) , and by prior knowledge available in the literature . biologic and clinical therapeutic implications of alterations are curated from several public resources , including disease - specific treatment guidelines ; abstracts from major conference proceedings , such as asco , european society of medical oncology , and american association for cancer research ; and the scientific literature through pubmed . 
level 2a includes alterations that are not fda - recognized biomarkers but are considered standard care predictive biomarkers of response to an fda - approved therapy in specific cancer types . 
these alterations are highlighted in expert panel guidelines , such as the nccn compendium and asco clinical practice guidelines . several level 2a associations involve rare cancer types ( eg , braf v600e mutant histiocytosis ) or small subpopulations of common cancers ( eg , braf v600e mutant lung cancer ) , and therefore , accrual to a prospective randomized phase iii clinical study may not be feasible . 
because the total number of patients affected often is small , an application for an fda indication may never be filed , but disease experts consider the biomarker - drug association as a standard off - label use . 
in total , 11 genes with 85 alterations are currently considered level 2a ( data supplement )  . as an example , met exon 14 alterations are present in approximately 3% of nsclc27 and represent a distinct , molecularly defined subpopulation of lung adenocarcinomas that are mutually exclusive with tumors that harbor activating mutations in egfr and kras and fusions of alk , ros1 , and ret.28 - 30 an adequately powered randomized trial that compares the met inhibitor crizotinib with other standard approaches , such as chemotherapy and immunotherapy , has not yet been performed partly because of the rarity of these alterations . 
however , durable complete or partial responses to crizotinib and cabozantinib in patients with met exon 14altered lung cancers have been reported.27 , 31 although widely available as a result of its fda approval for use in alk fusionpositive nsclc , crizotinib is not explicitly fda approved in the setting of met exon 14altered nsclc . 
because the nccn guidelines consider off - label prescription of crizotinib a standard treatment approach for patients with lung cancer with met exon 14 alterations , 32 they are classified as level 2a ( fig 2 )  . 
although vemurafenib is not fda approved for use in patients with these specific braf v600e mutant indications , the off - label use of vemurafenib is a well - supported treatment option included in the nccn guidelines on the basis of compelling clinical data33 - 35 ( fig 3 )  . level 2b includes alterations that are standard predictive biomarkers of drug sensitivity in other tumor types but for which data in the tumor in question are either lacking or negative to date . 
for example , braf v600e mutations have been identified in several cancer types , including urothelial carcinomas and germ cell tumors , 36 , 37 for which no clinical response data are reported in the literature . 
in these tumors , the use of raf inhibitors in patients with braf mutant tumors remains investigational , and braf v600e is therefore classified as level 2b36 ( fig 3 )  . 
in patients with braf v600e mutant colorectal cancer , braf inhibitors , such as vemurafenib , have been tested with disappointing results.38 such negative data are referenced in oncokb and argue against the use of raf inhibitor monotherapy in patients with braf v600e colorectal cancers . 
level 3a includes mutations that are candidate predictive biomarkers of drug response on the basis of off - label use of fdaapproved drugs or investigational agents not yet fda approved for any indication . 
for the former , the evidence that supports the predictive value of the alteration is not considered sufficient to warrant a change in standard clinical practice , and disease experts would consider the use of the fda - approved drug in this context to be investigational . 
 into oncokb in real time . oncokb access : oncokb data are available for public use through an interactive web site19 and the cbioportal for cancer genomics22 and are used internally to annotate msk clinical reports . 
api , application program interface ; nccn , national comprehensive cancer network . activity has been reported , and the mutation - drug association is classified as level 3b in all other tumor types . 
fifty - five alterations in 25 genes are considered level 3 ( data supplement )  . a representative example of a level 3 alteration is akt1 e17k ( fig 3 )  . 
promising clinical activity consistent with preclinical studies of this compound has been reported with the investigational panakt inhibitor azd5363 in patients with akt1 e17k mutant breast , lung , cervical squamous , and endometrial cancers.40 - 42 on the basis of these emerging clinical data , akt1 e17k is classified as a level 3a mutation in breast , cervical , endometrial , ovarian , and lung cancers . 
a more complex example of level 3 alterations are erbb2 missense mutations that are present in a minority of a broad range of human cancers43 and that often arise in patients without erbb2 amplification or human epidermal growth factor receptor 2 ( her2 ) protein overexpression.44 these erbb2 mutants demonstrate varying degrees of sensitivity to her2 - selective kinase inhibitors , such as lapatinib and neratinib44 ( fig 3 )  . 
level 4 alterations are candidate predictive biomarkers of response to either fdaapproved or investigational agents on the basis of compelling laboratory data and an absence of substantiating compelling clinical data . 
although anecdotal responses to targeted agents may have been demonstrated in individual patients whose tumor harbored a level 4 alteration , the data are not sufficiently robust to indicate that the presence of the mutation is associated with significantly greater activity than tumors that lack the alteration . 
for example , although studies in mouseand patient - derived xenograft models have suggested that mammalian target of rapamycin or akt - targeted inhibitors may be effective in pten - null tumors , 46 the clinical data that support pten loss as a predictive biomarker of response to phosphatidylinositol 3 - kinase , akt , or mammalian target of rapamycin inhibitors in patients are limited and conflicting.47 - 52 therefore , classification of loss - of - function pten alterations as a level 4 alteration indicates that patients with pten - deficient tumors would be rational candidates for a clinical trial of investigational phosphatidylinositol 3 - kinase pathway inhibitors alone or in combination with other agents , but that the use of such agents outside the context of a clinical trial is not yet supported by the sum of the clinical data . 
additional examples of level 4 alterations include nf1 inactivating alterations , which may be predictive of response to mek1 / 2 inhibitors , 53 and egfr exon 20 insertions in lung adenocarcinomas that respond poorly to erlotinib54 , 55 but which may be sensitive to ap32788 , an investigational inhibitor of egfr and her256 ( fig 3 )  . 
because oncokb levels are dynamic , mutations currently classified as level 4 may be reclassified as level 3 or higher if additional compelling clinical data emerge . levels of resistance ( levels r1 to r3 )  . 
oncokb classifies mutations that have been shown to confer resistance to specific targeted therapies into one of three levels on the basis of the strength of the evidence that the mutation is predictive of treatment resistance ( fig 2 )  . 
level r1 includes mutational events for which there is sufficient evidence to recommend routine testing for the mutation to identify , with a high likelihood , patients who will not respond to a standard therapy . identification of r1 mutations , therefore , would lead to a recommendation that the associated therapy be withheld in patients whose tumors harbor the mutation . 
by definition , level r1 mutations predict for resistance to fda - approved drugs , and testing for such mutations is typically recommended by expert guidelines , such as those published by the nccn . 
level r1 alterations include activating ras mutations in colorectal cancer , which predict for resistance to the egfr - targeted monoclonal antibodies cetuximab and panitumumab57 , 57a ; egfr t790m mutations in nsclc , which predict for intrinsic and acquired resistance to the egfr tyrosine kinase inhibitors ( tkis ) erlotinib , afatinib , and gefitinib58 ; and pdgfra d842v , which predicts for oncokb annotation metric no . 
 level level level level level level level level level fda - recognized biomarker predictive of response to an fdaapproved drug in this indication standard care biomarker predictive of response to an fdaapproved drug in this indication * standard care biomarker predictive of response to an fdaapproved drug in another indication but not standard care for this indication compelling clinical evidence supports the biomarker as being predictive of response to a drug in this indication , but neither biomarker nor drug is standard care compelling clinical evidence supports the biomarker as being predictive of response to a drug in another indication , but neither biomarker nor drug is standard care compelling biologic evidence supports the biomarker as being predictive of response to a drug , but neither biomarker nor drug is standard care standard care biomarker predictive of resistance to an fda - approved drug in this indication compelling clinical evidence supports the biomarker as being predictive of resistance to a drug , but neither biomarker nor drug is standard care compelling biologic evidence supports the biomarker as being predictive of resistance to a drug , but neither biomarker nor drug is standard care standard therapeutic implications * includes biomarkers that are recommended as standard care by the nccn or other expert panels but not necessarily fda recognized for a particular indication investigational therapeutic implications possibly directed to clinical trials hypothetical therapeutic implications on the basis of preliminary , nonclinical data standard therapeutic implications hypothetical therapeutic implications on the basis of preliminary , nonclinical data fig 2 . 
standard therapeutic implications include food and drug administration ( fda ) recognized biomarkers that are predictive of response to an fda - approved drug in a specific indication ( level 1 ) and standard care biomarkers that are predictive of response to an fda - approved drug in a specific indication ( level 2a )  . 
investigational therapeutic implications include fda - approved biomarkers predictive of response to an fdaapproved drug detected in an off - label indication ( level 2b ) , fdaor nonfda - recognized biomarkers that are predictive of response to novel targeted agents that have shown promising results in clinical trials ( level 3a ) , and nonfdarecognized biomarkers that are predictive of response to novel targeted agents on the basis of compelling biologic data ( level 4 )  . 
nccn , national comprehensive cancer network . resistance to imatinib in patients with gi stromal tumors.59 alterations classified as levels r2 and r3 have hypothetical therapeutic implications and include alterations that are predictive of drug resistance on the basis of clinical and biologic data , respectively , but their use in guiding treatment decisions is considered investigational ( fig 2 )  . 
for example , although multiple strategies to reverse the oncogenic effects of tp53 loss have been explored in laboratory studies and early - phase clinical trials , 61 , 62 the agents tested do not directly target tp53 , their activity is typically not restricted to tp53 mutant models , and clinical trials that test these agents either have been aborted because of lack of efficacy or do not use tp53 status as a selection criterion.63 thus , although genomic alterations in tp53 are typically oncogenic , oncokb does not consider them therapeutically actionable . 
90% of alterations in oncokb have curated biologic effects and are classified as oncogenic but are not associated with actionability . actionable alterations across cancer types although targeted inhibitors have been shown to improve clinical outcomes in melanoma and lung cancer among others , 64 the broader clinical utility of large panel or whole - exome testing remains undefined . 
implicit in the designation of a level of evidence for each branch is whether the biomarker is food and drug administration ( fda ) recognized standard care or investigational and whether it is predictive of response to a drug that is fda approved or currently being tested in clinical trials . 
90% of samples harbored at least one known oncogenic mutation , only 41% had one or more alterations for which compelling clinical data currently exist to justify the use of a standard or an investigational agent ( levels 1 to 3b ) ( fig 4a )  . 
level 1 and 2a alterations were most common in melanoma ( 44% ) , ovarian cancer ( 21% , which includes 65 of 312 samples that have either germline or somatic brca1or brca2 - inactivating mutations ) , soft tissue sarcomas ( 19% on the basis of cdk4 amplifications , which predict response to palbociclib in welldifferentiated and dedifferentiated liposarcomas but not in other soft tissue sarcomas ) , nsclc ( 14% ) , esophagogastric cancer ( 13% ) , and breast cancer ( 12% )  . 
however , whereas 44% of melanomas had mutations that predict for clinical benefit with standard therapies in patients with melanoma , the majority of actionable alterations in lgg were associated with only investigational implications , with the most common mutation being idh1 r132c ( 77% of lgg samples ) , a level 3b alteration that is based on promising clinical data with the idh1 inhibitor ag - 120 in patients with acute myeloid leukemia ( figs 4a and 4b )  . 
in total , just over 10% of all samples had a level 3a mutation as their highest actionable alteration , a cohort of patients for which enrollment in a clinical trial would represent a compelling treatment option after standard treatments . 
in addition , approximately 15% of samples had level 3b alterations as their highest actionable event , ie , alterations for which promising clinical data have been observed in an investigational setting in another cancer type . on average , there were approximately three oncogenic mutations per sample , and the number of known oncogenic mutations per sample in tumor types was independent of overall mutation burden ( fig 4c )  . 
for example , although renal cell cancers had , on average , one oncogenic mutation per sample , these mutations were typically inactivating in tumor suppressors , such as vhl and pbrm1 , which are not clinically actionable at this time . 
in contrast , although thyroid cancers also on average had approximately one oncogenic mutation per sample ( fig 4c ) , these alterations were typically actionable , such as ret fusions and braf and nras mutations . 
frequencies of level of evidence 1 to 3 assignments in the cancer genome atlas cohorts . patient samples from 19 cancer types ( the cancer genome atlas ) are classified by the alteration that carries the highest level of evidence . 
 ( a ) inset pie chart : fraction of samples across all cancer types that carry a mutation considered actionable according to the levels of evidence , oncogenic but not actionable , or variants of unknown significance ( vus )  . 
cell color as shown in the key for the inset pie chart ( a )  . columns indicate sample tumor type , rows indicate gene alteration present in sample , and numbers indicate the percentage of samples per tumor type that harbor an alteration in each gene . 
 ( c ) each patient sample was classified by the number of oncogenic alterations or the number of actionable alterations . shown is the mean number of actionable ( black ) , oncogenic ( dark gray ) , or total ( gray ) mutations per sample per tumor type . 
 ( d ) each tumor type was evaluated for the percentage of samples that carry zero , one , two , three , or four or more actionable mutations per sample ( indicated in shades of blue )  . 
 mutations ( 31% , 25% , and 22% , respectively ) , which is consistent with data that demonstrate that these tumor types are driven by multiple oncogenic mutations in nonredundant pathways65 - 68 ( fig 4d ) and may explain why targeted monotherapies have shown disappointing results to date in some of these cancer types.69 , 70 discussion since the introduction of imatinib for chronic myeloid leukemia more than a decade ago , 71 , 72 a growing number of drugs that target specific genetic alterations required for tumor initiation and progression have been shown to significantly improve outcomes in molecularly defined populations of patients with cancer.64 , 73 - 74a although tumor genetic testing is now part of routine patient care in an increasing number of tumor types , interpretation of variants remains an important challenge , and in major academic cancer centers , a significant proportion of physicians report low confidence in their ability to make optimal treatment recommendations on the basis of genomic information.75 although multiple classification systems exist for the annotation of germline variants , 76 , 77 efforts to define the clinical utility of somatic alterations have been limited to established biomarkers , 78 - 80 and prior efforts often have classified actionability as a binary variable that results in the grouping of biomarkers that are fda recognized with those that are nonactionable but oncogenic . 
to this end , we assigned each mutation to one of four levels that are based on available clinical and laboratory data that support the use of the mutation as a predictive biomarker . 
standard therapeutic implications are classified as either level 1 or 2a to recognize that not all mutation - drug associations used in standard practice have been recognized by the fda . 
moreover , there are cases where off - label use of an fda - approved drug is explicitly not warranted because of existing data that argue against the use of a targeted agent in a specific cancer type . 
for example , although the braf inhibitor vemurafenib is a standard treatment option for patients with braf v600e mutant melanoma or nsclc , robust clinical reports from multiple independent centers demonstrated that patients with braf v600e mutant colorectal cancer do not respond to raf inhibitors , at least as monotherapy.38 , 84 similarly , although erbb2 amplification predicts for the clinical utility of her2 - targeted therapies in breast and esophagogastric cancers , the clinical activity of trastuzumab in erbb2 - amplified lung cancers has been disappointing.85 braf v600e and erbb2 amplification are , therefore , level 2b when detected in colorectal cancers . 
 individual mutant alleles within a single gene may be functionally distinct , with different predictive value and , therefore , individual therapeutic implications , which complicates the development of clinical decision support tools , particularly in the context of often - vague fda labeling and expert guidelines , such as those provided by the nccn , that may not define at a granular level whether specific mutations within a gene are predictive of drug response . 
to address this complexity , oncokb groups mutations in level 1 genes , such as egfr and kit , according to whether they are biologically active , whether there are preclinical data to suggest that the allele is sensitive or resistant to the matched targeted agents , and whether there are clinical data to suggest clinical sensitivity or intrinsic resistance to the approved targeted therapy . 
the challenge of rare drug - sensitive variants in level 1 genes also highlights the need for consortia efforts , such as the american association for cancer research project genie ( genomics evidence neoplasia information exchange ) , which should allow for the collection of clinical response data for rare alleles to help to guide the treatment of patients with these less common mutations . although new laboratory and clinical data are continually generated , fda labels and professional guidelines are updated at irregular intervals . 
for example , although explicit fda approval of crizotinib in ros1 - rearranged nsclc did not occur until march 2016 , offlabel use of crizotinib in patients with ros1 fusion - positive lung cancers has been considered standard of care by several expert groups for some time.86 - 88 as another example , whereas kras was initially considered a level 3a alteration in nsclc on the basis of promising data from the randomized phase ii study that supplemented standard chemotherapy with a mek inhibitor , 89 the subsequent phase iii trial showed no survival benefit with this combination , 90 which is consistent with negative data associated with mek inhibitor use in kras mutant pancreatic and colorectal cancers.91 , 92 nonetheless , anecdotal clinical and compelling preclinical data support the use of kras as a predictive biomarker of sensitivity to novel mek and erk inhibitors alone or in combination with other agents.93 - 97 oncokb thus recognizes and reassigns the level of evidence of mutational events , if appropriate , on the basis of newer , moredefinitive , and negative randomized clinical data , which take precedence over prior preliminary clinical findings . to incorporate new clinical and research findings , we have made the oncokb annotation available publically through an interactive web site19 and through the cbioportal for cancer genomics.22 both systems include a comment feature to facilitate crowdsourcing curation of this knowledge base . 
user suggestions are evaluated by the scientific team and incorporated into oncokb through periodic updates . oncokb is also an active member in efforts to promote harmonization of variant annotation across existent knowledge bases and is participating in both clingen and the global alliance for genomic health through the variant interpretation cancer consortium . in the future , we will curate information about mutational signatures , such as overall mutation burden and the possible link to immunotherapy , mutational clonality , and the impact on drug sensitivity of co - occurrence of specific oncogenic and actionable mutations . 
rudolph , ederlinda paraiso collection and assembly of data : debyani chakravarty , jianjiong gao , sarah phillips , ritika kundra , hongxin zhang , tara soumerai , moriah h . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . debyani chakravarty no relationship to disclose jianjiong gao no relationship to disclose sarah phillips no relationship to disclose ritika kundra no relationship to disclose hongxin zhang no relationship to disclose jiaojiao wang no relationship to disclose julia e . 
chang no relationship to disclose rona yaeger consulting or advisory role : glaxosmithkline , advaxis sarat chandarlapaty honoraria : chugai pharmaceutical , foresite capital , astrazeneca , sermonix pharmaceuticals , macrogenics , agendia research funding : eli lilly ( inst ) , novartis ( inst ) travel , accommodations , expenses : chugai pharmaceutical , macrogenics , astrazeneca tiffany a . 
traina consulting or advisory role : genentech , roche , eisai , mundipharma , medivation , pfizer , astrazeneca , bayer ag , immunomedics , merck research funding : medivation , eisai , pfizer , novartis , myriad genetics , innocrin pharmaceuticals , astrazeneca paul k . 
paik honoraria : celgene , bristol - myers squibb , eli lilly , ariad pharmaceuticals consulting or advisory role : celgene , eli lilly , ariad pharmaceuticals , bristol - myers squibb , celgene research funding : celgene , emd serono travel , accommodations , expenses : emd serono alan l . 
ho consulting or advisory role : oncology consortium , bristol - myers squibb , eisai , genzyme , merck , novartis , sun pharmaceutical industries , kura oncology speakers bureau : medscape , omniprex america , eli lilly , genentech , roche , astrazeneca , bayer ag , kura oncology , kolltan pharmaceuticals , eisai , astrazeneca travel , accommodations , expenses : janssen pharmaceuticals , merck , kura oncology feras m . 
hantash employment : quest diagnostics stock and other ownership interests : quest diagnostics patents , royalties , other intellectual property : patents on molecular methods , but not related to manuscript andrew grupe employment : quest diagnostics stock and other ownership interests : quest diagnostics patents , royalties , other intellectual property : patents or patent applications travel , accommodations , expenses : quest diagnostics shrujal s . 
danila honoraria : astellas pharma , angle , bayer ag , janssen pharmaceuticals consulting or advisory role : angle , bayer ag research funding : prostate cancer foundation , genentech , janssen pharmaceuticals ( inst ) patents , royalties , other intellectual property : gene expression profile associated with prostate cancer travel , accommodations , expenses : cambridge healthtech institute , prostate cancer foundation , angle , bayer ag , american austrian foundation open medical institute , global technology community , janssen pharmaceuticals , oncology education mrinal gounder honoraria : amgen , daiichi sankyo , karyopharm therapeutics , tracon pharmaceuticals , amgen consulting or advisory role : daiichi sankyo , karyopharm therapeutics , epizyme speakers bureau : amgen travel , accommodations , expenses : amgen travel , accommodations , expenses : daiichi sankyo , karyopharm therapeutics james j . 
harding no relationship to disclose dana rathkopf consulting or advisory role : janssen pharmaceuticals research funding : janssen pharmaceuticals ( inst ) , medivation ( inst ) , celgene ( inst ) , takeda pharmaceuticals ( inst ) , millennium pharmaceuticals ( inst ) , ferring pharmaceuticals ( inst ) , novartis ( inst ) , taiho pharmaceutical ( inst ) , astrazeneca ( inst ) , genentech ( inst ) , roche ( inst ) , tracon pharmaceuticals ( inst ) alexander n . 
shoushtari consulting or advisory role : vaccinex , castle biosciences , immunocore research funding : bristol - myers squibb , immunocore travel , accommodations , expenses : bristol - myers squibb neerav shukla no relationship to disclose martin h . 
voss honoraria : novartis consulting or advisory role : novartis , calithera biosciences , natera , glaxosmithkline , exelixis , pfizer , alexion pharmaceuticals research funding : pfizer , bristol - myers squibb , genentech , roche travel , accommodations , expenses : novartis , takeda pharmaceuticals matthew d . 
hellmann consulting or advisory role : bristol - myers squibb , merck , genentech , astrazeneca , medimmune , novartis , janssen pharmaceuticals research funding : bristol - myers squibb , genentech , roche ederlinda paraiso no relationship to disclose ahmet zehir no relationship to disclose gopa iyer no relationship to disclose yelena y . 
levine stock and other ownership interests : critical outcome technologies consulting or advisory role : clovis oncology , mtrap , biodesix , tesaro maeve lowery consulting or advisory role : agios , celgene antonio omuro consulting or advisory role : stemline therapeutics , juno therapeutics , bristol - myers squibb , oxigene , alexion pharmaceuticals , astrazeneca , inovio pharmaceuticals , merck michael a . 
postow honoraria : bristol - myers squibb , merck consulting or advisory role : amgen , bristol - myers squibb , novartis research funding : bristol - myers squibb ( inst ) , novartis ( inst ) , array biopharma ( inst ) , infinity pharmaceuticals ( inst ) travel , accommodations , expenses : bristol - myers squibb leonard b . 
riely consulting or advisory role : novartis , genentech research funding : novartis ( inst ) , roche ( inst ) , genentech ( inst ) , millennium pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , pfizer ( inst ) , infinity pharmaceuticals ( inst ) , ariad pharmaceuticals ( inst ) patents , royalties , other intellectual property : patent application submitted that covers pulsatile use of erlotinib to treat or prevent brain metastases ( inst ) travel , accommodations , expenses : novartis marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network , boehringer ingelheim , astrazeneca research funding : loxo oncology ( inst ) david m . 
 jos e baselga leadership : infinity pharmaceuticals , varian medical systems , grail , foghorn stock or other ownership : pmv pharma , juno therapeutics , infinity pharmaceuticals , grail , varian medical systems , tango , foghorn , aura biomedical , apogen , northern biologics honoraria : pmv pharma , juno therapeutics , infinity pharmaceuticals , grail , northern biologics consulting or advisory role : eli lilly , novartis , grail patents , royalties , other intellectual property : combination therapy using pdk1 and pi3k inhibitors . 
may 16 travel , accommodations , expenses : roche / genentech paul sabbatini research funding : bristol - myers squibb ( inst ) , ludwig institute for cancer research ( inst ) david b . 
solit honoraria : loxo oncology , pfizer consulting or advisory role : pfizer , loxo oncology nikolaus schultz no relationship to disclose acknowledgment we thank the following individuals for serving as oncokb curators : andrew intlekofer , eric smith , piro lito , jaclyn hechtman , dmitriy zamarin , wassim abida , mythili koneru , weiyi toy , pedram razavi , philip iaquinta , byron lee , martin dalin , matthew jones , elizabeth adams , karuna ganesh , olga guryanova , carolyn jackson , william terry , yu chen , ping chi , eduard reznik , aphrothiti hanrahan , sevin turcan , philip watson , neeman mohibullah , elena goldberg , aaron viny , emily foley , samuel kaffenberger , andrew winer , connie batlevi , helen won , lindsay saunders , kinisha gala , philip jonsson , fiona brown , eneda toska , i~nigo landalopez , and tripti shrestha - bhattarai . affiliations debyani chakravarty , jianjiong gao , sarah phillips , ritika kundra , hongxin zhang , jiaojiao wang , julia e . 
j am med inform assoc 10.1093 / jamia / ocw148 [ epub ahead of print on october 27 , 2016 ] institute of precision medicine : welcome to the precision medicine knowledgebase . 
kim kb , kefford r , pavlick ac , et al : phase ii study of the mek1 / mek2 inhibitor trametinib in patients with metastatic braf - mutant cutaneous melanoma previously treated with or without a braf inhibitor . 
paik pk , drilon a , fan pd , et al : response to met inhibitors in patients with stage iv lung adenocarcinomas harboring met mutations causing exon 14 skipping . 
ma pc , jagadeeswaran r , jagadeesh s , et al : functional expression and mutations of c - met and its therapeutic inhibition with su11274 and small interfering rna in non - small cell lung cancer . 
ma pc , kijima t , maulik g , et al : c - met mutational analysis in small cell lung cancer : novel juxtamembrane domain mutations regulating cytoskeletal functions . 
frampton gm , ali sm , rosenzweig m , et al : activation of met via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to met inhibitors . 
planchard d , besse b , groen hj , et al : dabrafenib plus trametinib in patients with previously treated braf ( v600e ) mutant metastatic non - small cell lung cancer : an open - label , multicentre phase 2 trial . 
davies br , greenwood h , dudley p , et al : preclinical pharmacology of azd5363 , an inhibitor of akt : pharmacodynamics , antitumor activity , and correlation of monotherapy activity with genetic background . 
hyman dm , smyth l , bedard pl , et al : abstract b109 : azd5363 , a catalytic pan - akt inhibitor , in akt1 e17k mutation positive advanced solid tumors . 
hyman d , piha - paul s , rod on j , et al : abstract pd5 - 05 : neratinib for erbb2 mutant , her2 non - amplified , metastatic breast cancer : preliminary analysis from a multicenter , open - label , multi - histology phase ii basket trial . cancer res 76 : pd5 - 05 , 2016 46 . 
lin j , sampath d , nannini ma , et al : targeting activated akt with gdc - 0068 , a novel selective akt inhibitor that is efficacious in multiple tumor models . 
clin cancer res 19 : 1760 - 1772 , 2013 de bono js , de giorgi u , massard c , et al : pten loss as a predictive biomarker for the akt inhibitor ipatasertib combined with abiraterone acetate in patients with metastatic castration - resistant prostate cancer ( mcrpc )  . 
ma cx , luo j , naughton m , et al : a phase i trial of bkm120 ( buparlisib ) in combination with fulvestrant in postmenopausal women with estrogen receptor - positive metastatic breast cancer . 
rodon j , bra~na i , siu ll , et al : phase i dose - escalation and - expansion study of buparlisib ( bkm120 ) , an oral panclass i pi3k inhibitor , in patients with advanced solid tumors . 
yang l , clarke mj , carlson bl , et al : pten loss does not predict for response to rad001 ( everolimus ) in a glioblastoma orthotopic xenograft test panel . 
naidoo j , sima cs , rodriguez k , et al : epidermal growth factor receptor exon 20 insertions in advanced lung adenocarcinomas : clinical outcomes and response to erlotinib . 
yasuda h , park e , yun ch , et al : structural , biochemical , and clinical characterization of epidermal growth factor receptor ( egfr ) exon 20 insertion mutations in lung cancer . 
gonzalvez f , zhu x , huang w - s , et al : abstract 2644 : ap32788 , a potent , selective inhibitor of egfr and her2 oncogenic mutants , including exon 20 insertions , in preclinical models . 
druker bj , sawyers cl , kantarjian h , et al : activity of a specific inhibitor of the bcr - abl tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the philadelphia chromosome . n engl j med 344 : 1038 - 1042 , 2001 72 . 
druker bj , talpaz m , resta dj , et al : efficacy and safety of a specific inhibitor of the bcr - abl tyrosine kinase in chronic myeloid leukemia . 
tsimberidou am , wen s , hong ds , et al : personalized medicine for patients with advanced cancer in the phase i program at md anderson : validation and landmark analyses . 
macarthur dg , manolio ta , dimmock dp , et al : guidelines for investigating causality of sequence variants in oncol 32 : 1317 - 1323 , 2014 human disease . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
simon rm , paik s , hayes df : use of archived specimens in evaluation of prognostic and predictive biomarkers . j natl cancer inst 101 : 1446 - 1452 , 2009 81 . 
conti rm , bernstein ac , villaflor vm , et al : prevalence of off - label use and spending in 2010 among patentprotected chemotherapies in a population - based cohort of medical oncologists . 
langer cj , stephenson p , thor a , et al : trastuzumab in the treatment of advanced non - small - cell lung cancer : is there a role ? focus on eastern cooperative oncology group study 2598 . 
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cell 131 : 1190 - 1203 , 2007 janne pa , shaw at , pereira jr , et al : selumetinib plus docetaxel for kras - mutant advanced non - small - cell lung cancer : a randomised , multicentre , placebo - controlled , phase 2 study . 
clin lung cancer 17 : e1 - e4 , 2016 infante jr , fecher la , falchook gs , et al : safety , pharmacokinetic , pharmacodynamic , and efficacy data for the oral mek inhibitor trametinib : a phase 1 dose - escalation trial . 
rinehart j , adjei aa , lorusso pm , et al : multicenter phase ii study of the oral mek inhibitor , ci - 1040 , in patients with advanced non - small - cell lung , breast , colon , and pancreatic cancer . 
hu - lieskovan s , mok s , homet moreno b , et al : improved antitumor activity of immunotherapy with braf and mek inhibitors in braf ( v600e ) melanoma . 
kakavand h , wilmott js , menzies am , et al : pd - l1 expression and tumor - infiltrating lymphocytes define different subsets of mapk inhibitor - treated melanoma patients . 
loi s , dushyanthen s , beavis pa , et al : ras / mapk activation is associated with reduced tumor - infiltrating lymphocytes in triple - negative breast cancer : therapeutic cooperation between mek and pd - 1 / pd - l1 immune checkpoint inhibitors . 
yoon yk , kim hp , han sw , et al : kras mutant lung cancer cells are differentially responsive to mek inhibitor due to akt or stat3 activation : implication for combinatorial approach . 
 precision oncology and the universal health coverage system in japan hiromichi ebi , md , phd1 , 2 , 3 and hideaki bando , md , phd4 although precision oncology is transforming clinical management of patients with cancer , many hospitals face challenges to effectively implement precision oncology . 
the most promising development is that tests to prole the genomes of select cancers are now fully covered by the national health insurance systein may 2019 , two gene panels were approved with reimbursement : foundationone cdx cancer genomic prole and oncoguide ncc oncopanel system , the latter of which was developed in japan . 
to make better use of scarce resources , the reimbursement is restricted to patients with solid tumors that have progressed on standard chemotherapy , rare tumors , or tumors of unknown primary . 
in addition , japans national cancer center launched a center for cancer genomics and advanced therapeutics ( c - cat ) that collects genomic information and clinical characteristics of patients who received genomic proling tests . 
the centralized system under the national health insurance system could be a double - edged sword . although tight regulation may make it hard to keep up with the rapid development of precision oncology , a federated ecosystem for sharing clinical and genomic data will be a precious asset and allow for shared access to data . 
high - cost medical expense benet also denes maximum out - of - pocket payment month according to household income of the patient . for example , the maximum is roughly $800 for a household income of approximately $34 , 000$70 , 000 . 
although for - prot insurance companies have voluntary health insurance programs , holding this type of insurance does not exempt an individual from mandatory enrollment in the universal health coverage scheme . 
the role of voluntary health insurance is supplemental and complements social health insurance benet packages . prices for all drugs and devices reimbursed by the universal health coverage system are the ofcial price set by the government ( japanese ministry of health , labor , and welfare [ mhlw ] )  . 
if a physician uses an unapproved device or off - label drug , any costs related to the patient , including but not limited to blood tests and physician fees , are not reimbursed . 
therefore , any cancer genomic proling tests must be examined and approved by the pharmaceuticals and medical devices agency ( pmda ) and mhlw before insurance reimbursement is set by the government , unless all the cost is to be paid by the patients . 
 ebi et al context key objective to summarize the framework of precision oncology recently arranged in japan . knowledge generated the japanese government designated core , hub , and liaison hospitals for cancer genomic medicine . 
also , reimbursement applies only to patients who nished standard chemotherapy , as a way to restrict potentially unnecessary investigations . relevance each country must design a precision oncology initiative under their distinct social care systejapan is an example of a centralized precision oncology under a universal health care coverage system . for clinical trials , even when gene proling testing identies actionable alterations . the japanese mhlw has a structured system to promote the development of drugs and devices under governmental regulations . 
although system a is designed for intervention with approved drugs / devices or minimally invasive intervention with unapproved drugs / devices , system b is designed for unapproved drugs or medical technologies . 
the patientrequested therapy system is also expected to be used as the japanese version of what the united states has hailed as the compassionate use prograto start off - label drug treatment using patient - requested therapy systems , it is necessary to pass several review processes equivalent to clinical trials , and the cost of the unapproved medical care should be fully paid by the patients . 
second , similar to the breakthrough therapy designation by the us food and drug administration , mhlw has the sakigake program , an accelerated inspection scheme for rapid authorization of unapproved drugs and devices . 
beginning around 2010 , pan - cancer genome screening started in japan by using researchuse only next - generation sequencing ( ngs ) panels.2 to promote genome - based clinical trials , the nationwide genome screening consortium for lung cancer was launched in february 2013 . 
the group , lc - scrum - japan , originally aimed to identify patients harboring ros1 and ret fusions originally discovered in japan.3 in february 2014 , gi - screen - japan multicenter screening project also started for gi cancer . 
the project began to screen for braf , nras , and pik3ca mutations in patients with metastatic colorectal cancer , using multiplex polymerase chain reaction ( pcr ) and luminex ( xmap ) technology.4 in february 2015 , these two groups merged into scrumjapan and started to use oncomine comprehensive assays as a screening platform.5 with the advent of plasma - based genome screening , scrum - japan subsequently started plasma - based ngs using guardant 360 . 
the screening and associated clinical trials have been research based , funded by government agencies through grant mechanisms ( the japan agency for medical research and development or the national cancer center research and development fund ) as well as by pharmaceutical companies . 
therefore , patients were not required to pay screening costs for any of these studies . from february 2013 to december 2018 , 6 , 860 and 6 , 391 patients were enrolled in lc - scrum - japan and gi - screenjapan , respectively . 
on the basis of this screening platform , 28 umbrella and 20 basket - type genome - based studies have been conducted.6 one of the notable accomplishments was a clinical trial of vandetanib , for patients with ret - rearranged nonsmall - cell lung cancer ( nsclc ) identied by the screening . 
 precision oncology in japan results of clinical trials conducted by lc - scrum japan led to the approval of crizotinib for the treatment of ros1translocated nsclc and the combination of trametinib and dabrafenib for use in patients with braf v600e - mutant nsclc . 
scrum - japan also contributed to the approval of in vitro diagnostic testing by pmda including simultaneous detection of ras and braf mutations in colorectal cancer , 8 oncomine dx target test cdx ngs panel for egfr and braf mutations and ros1 and alk translocations in nsclc , pcr - based microsatellite instability testing for solid tumors , 9 and plasma - based ras mutation testing for colorectal cancer.10 another notable genome screening is the top - gear ( trial of oncopanel for gene proling to estimate both adverse events and response ) project , which is a prospective cohort study conducted by japans national cancer center ( ncc ) to investigate the feasibility and utility of an ngsbased panel customized for japanese patients with cancer ( ncc oncopanel ) .11 this work was carried out within the context of the sakigake program and clinical utility was investigated within the advanced medical care b system . during the second stage of the top - gear project , 187 patients with advanced solid tumors obtained the gene proling data , and 25 ( 13.3% ) of them have received molecular - targeted therapy on the basis of their genome alterations.12 the achievements of this project led to governmental approval for the oncoguide ncc oncopanel system . some other medical university hospitals are also developing their own genome screening systems . 
these tests are performed under the advanced medical care b systekyoto university hospital ( oncoprime ) 13 , 14 and keio university hospital ( plessision - rapid ) offer their screening service outside of the national health care systerecently , keio university hospital started a whole - exon sequencing service ( plessision - exome )  . 
importantly , the report also concluded that cancer genome medicine should be accomplished under a universal health coverage systeafter the report , the japanese government designated 11 hospitals throughout japan to serve as core hospitals for cancer genomic medicine . 
the eligibility to be designed as a hub hospital is based on their ability to organize their resources and infrastructure akin to the core hospitals , which have their own molecular tumor board ( mtb ) and organic capabilities to run clinical trials . in addition , core hospitals have more responsibilities to train fellows and clinical coordinators and to be involved in translational research . 
liaison hospitals , on the other hand , are dependent on core and hub hospitals for their sequencing , reports , and mtb ( fig 1 )  . from the academia side , three major japanese cancerrelated societies ( the japanese society of medical oncology [ jsmo ] , the japanese society of clinical oncology [ jsco ] , and the japanese cancer association [ jca ] ) issued consensus clinical practice guidelines for ngs - based cancer tests in october 2017.18 the guideline dened the evidence level of each genomic alteration suitable for the japanese medical care system in harmony with classications of evidence levels set by regulators in the united states and european union ( eu ) .19 in june 2018 , japans national cancer center founded the center for cancer genomics and advanced therapeutics ( c - cat ) to coordinate an integrated network of core , hub , and liaison hospitals . 
c - cat is expected to aggregate and deploy cancer genomic medicine information to advance the quality of health care offered under the universal health coverage system and to devise new modalities of health care ( fig 2 )  . 
first , the center has been constructing a cancer knowledge database ( ckdb ) optimized for japan to assist in decision making by mtbs . c - cat will collect and share updated information on clinical trials , promoting and improving matching between patients genomic data and clinical trials . 
oncoguide ncc oncopanel system is a 114 - gene ngs panel for tumor and germline analysis developed by japans ncc and health instrument maker sysmex corp , and foundationone cdx cancer genomic prole ( foundation medicine ) sequences 324 genes and also can detect microsatellite instability . oncoguide ncc oncopanel system was approved as a device combining the oncoguide ncc oncopanel kit and the oncoguide ncc oncopanel analyzing systein contrast , although foundation medicine had to be reviewed 11 core hospitals 34 hub hospitals 122 liaison hospitals their quality for sample analysis by pmda for the approval , pmda could not perform assessment as an in vitro diagnostic because the analysis was done in the united states . 
first , physicians submit tumor samples for foundationone cdx analysis to foundation medicine , and foundation medicine performs sequencing and reports the variant calls in an xml le , uploaded to a portal site created by chugai , a subsidiary of roche . 
because this step is not subject to reimbursement , and to set up a procedure suitable for reimbursement , physicians need to download the xml le from the portal site and then send it back to foundation medicine . 
in addition , the japanese act on the protection information denes genomic sequence as of personal personal information that was amended in response to the general data protection regulation enacted by the eu . 
on comply with the law , written consent consent , samples are sent to third parties outside of japan to analyze personal information and sequence data . after approval by pmda , the mhlw granted the use of these two gene panel analyses and set the ofcial price of reimbursement at the end of may 2019 . 
the second reimbursement is 480 , 000 , applied after the physician explains the results to patients following train personnel for precision medicine play central role in running clinical trials based on molecular profiling translational research run molecular tumor board run clinical trials based on molecular profiling provide patients with genomic testing interpretation is supported by core and hub hospitals refer patients to core and hub hospitals for clinical trial fig 1 . 
the test is approved for patients with solid tumors that have progressed on standard chemotherapy , with rare tumors , or with cancers of unknown primary . foundationone cdx is also approved for the use of companion diagnostic tests , such as those for the detection of egfr , ras , and braf mutations during standard care . for these companion dihowever , agnostic tests is much cheaper . 
furthermore , the results obtained outside of companion diagnostics will not be taken the reimbursement into consideration for decision making about treatment even when the test analyzes a number of genes . 
if a physician uses a test result during standard care , the difference for companion between the amount of reimbursement diagnostics and the actual cost paid to foundation medicine will be a decit for the hospital . 
if the physician wants to use the test results when the patient experiences progression while receiving standard therapy , additional reimbursement can be submitted as a fee for mtb ( 480 , 000 )  . 
 precision oncology in japan patient dies during standard care or fails to follow - up , it is unlikely that genome proling would be used during standard therapy in japan under current reimbursement rules . although only a few hospitals run these genome panels inhouse , most hospitals outsource these genomic proling tests to a clinical testing company . 
laboratories in core and hub hospitals are required to be certied by independent organizations such as iso15189 to handle patient samples . clinical testing companies also have clinical laboratory improvement amendments certication . 
formalin - xed and parafn - embedded samples ( and also blood samples in case of oncoguide ncc oncopanel system ) are prepared in each hospital and sent to the company . 
turnaround time in general is 16 to 22 days . to fulll its function as a data repository and to facilitate access to clinical data , a requirement of test granted by the government is that physicians need to submit detailed clinical data from patients with cancer to c - cat , including diagnostics , treatment , and outcomes information , as well as the raw bam or fastq and vcf or xml les ( fig 2 ; in japan , each hospital has an appendix table a1 )  . electronic platform that contains individual health records for patients . 
test results are usually reported in pdf les that are incorporated into electronic medical records , making it difcult for physicians to match patients with ongoing clinical trials or to identify patients who are eligible for new drugs when they become available . thus , a central data repository at c - cat will make it easier to identify candidates for clinical trials in a timely manner . using the submitted data , c - cat also issues their own report . 
ckdb1 is further divided into four databases : marker database listing genomic abnormalities such as egfr mutation or brca1 germline mutation ; drug database listing approved drugs or drugs under clinical trials in and outside of japan and their targets ; evidence database curated from biologic , clinical , and therapeutic information in multiple public information resources , including civic ( clinical interpretation of variants in cancer ) , brca exchange , clinvar , and cosmic ( catalogue of somatic mutations in cancer ) ; and a clinical trial database generated from clinicaltrials.gov as well as several japanese clinical trial registries . 
for instance , a patient with a druggable mutation needs to be treated by the appropriate drug , even as an off - label indication in the absence of a clinical trial or current approval . 
patient - requested therapy system , a japanese compassionate use program , has to be initiated by the request from a patient to the government with required documentation from physician . because the preparation of documents is a heavy burden for physicians , the system has not been widely adopted . also , as mentioned above , the cost of the drug has to be fully paid by the patient . 
to enable large basket / umbrella trials , a core facility supporting the protocol creation , drug distribution , monitoring , and audit will be needed , similar to the cancer therapy evaluation program in the united states . the other big challenge facing japanese precision oncology is the timing of reimbursements for genome proling tests . 
in addition , it was difcult to grant the use of cancer genomic proling during standard care because clear evidence for the benet of testing did not exist when consensus clinical practice guidelines for ngs - based cancer testing were published . 
for example , french clinical trials suggested the use of molecularly targeted agents outside established indications do not improve progression - free survival for heavily pretreated patients with cancer when compared with oncologists preferred treatment regimen.20 however , patients who experienced progression while receiving standard chemotherapy tend to have poor performance status and may not have enough time to wait for the results of genomic testing ; even in the very best case scenarios , their tumors will continue to grow and their health worsen each day that they are required to wait . 
currently , uncovered genome screening services are the only option for patients without rare tumors and before disease progression with standard therapy . quality and sustainability of mtbs are also challenging . 
however , a problem that arises is that it is challenging to demand this level of participation of physicians , given their limited availibility.21 overall , the cost for precision oncology is a heavy burden for hospitals . 
to put this into global perspective , memorial sloan kettering cancer center has already achieved clinical sequencing of 10 , 000 patients in the united states.22 furthermore , the united kingdom nished sequencing for  . 
in september 2019 , the mhlv unveiled a project with the goal of sequencing whole genomes from 100 , 000 patients with cancer over 3 years , a number chosen by referring to the united kingdoms sequencing achievements . 
although details of the project have not emerged yet , it is expected to result in the analyses of fresh frozen samples in collaboration with biobanks at core and hub hospitals . 
as the primary aim is to accelerate the development of new diagnostics and treatments , the project should be research based and funded by the japanese government and private sources . 
although the primary purpose of this initiative in precision oncology is to harness articial telligence ( ai ) for improved genomic analyses and drug target identication , the use of ai could connect genomic data with other prioritized areas in this initiative , such as pathology and radiology . 
the relatively homogenous genetic background of the japanese population and the detailed clinical outcomes collected by c - cat will be an advantage when harnessing the power of genomic data to develop new therapies . 
also , the genomic and clinical data would be integrated with other japanese databases , such as the medical genomics japan variant database and the japanese multi omics reference panel.23 conclusion in conclusion , precision oncology covered by the health insurance system has just begun in japan . 
on the other hand , although the japanese health care system has so far achieved excellent health outcomes with a relatively low cost , 24 the centralized structure under the national health insurance system with its inherent tight regulation may cause difculty in keeping up with the rapid development of precision oncology . 
in the united kingdom , genomics england was founded as a subsidiary limited company , as it was the most effective way to ensure the project running as quickly as possible . 
in the united states , for instance , protocols and treatment decisions surrounding personalized medicine are largely decided by the institutions treating the patients , with governing bodies like the national cancer institute playing ancillary roles and the ability to pay for patient care largely dictated by individuals private insurance policies . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . hiromichi ebi honoraria : taiho pharmaceutical , astrazeneca consulting or advisory role : merck serono , eli lilly , astellas pharma hideaki bando honoraria : taiho pharmaceutical , lilly japan , takeda , chugai pharma , sano , yakult honsha research funding : astrazeneca , sysmex no other potential conicts of interest were reported . acknowledgment we thank anthony faber , md ( vcu massey cancer center ) , for critical reading of the manuscript . references 48 : 559 - 564 , 2018 sakamoto h , rahman m , nomura s , et al : japan health system review . 
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li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular proling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
nucleic acids res 46 : d551 - d557 , 2018 ikegami n , yoo bk , hashimoto h , et al : japanese universal health coverage : evolution , achievements , and challenges . 
anthony blau rebecca boddicker ron bose seruga bostjan theresa boyle priscilla brastianos james brigarolas thomas brown mark burkard john burn jaume capdevila richard carvajal daniel catenacci jamie chaft marjan champine chang chan james chen mark chiang elena gabriela chiorean david chism atish choudhury james cleary eric collisson howard colman ryan corcoran brian crompton daniel crona william dahut adil daud michael davies kurtis davies francesca demichelis jennifer diamond adam dicker max diehn robert doebele alexander drilon steven dubois suhendan ekmekcioglu aymen elfiky leif ellisen anna farago phillip febbo sara federico michael feldman emmanouil fokas james ford william foulkes terence friedlander shirish gadgeel vijayakrishna gadi judy garber michael gatza thomas george giuseppe giaccone marios giannakis hannah gilmore bert glader ramaswamy govindan lipika goyal stacy gray joshua gruber matthew gubens michael hall heather hampel sigurdis haraldsdottir andrew harris derrick haslem eric haura florette hazard yijing he andrew hendifar daniel hertz cinta hierro kim hirshfield david hsu franklin huang willy hugo hatim husain a . 
 steven isakoff gopa iyer katherine janeway yelena janjigian filip janku randy jensen rinath jeselsohn neil johnson hossein khiabanian samuel klempner todd knepper wendy kohlmann takashi kohno scott kopetz carl koschmann vessela kristensen ian krop shivaani kummar marc ladanyi albert lai aly - khan lalani josh lauring alexander lazar dung le christophe le tourneau sarah leary sooyoun lee benjamin levy mark lewis jessica lin geoffrey lindeman jeffrey lipton stephen liu roger lo tamara lotan christine lovly ying lu david lum patrick ma subha madhavan luke maese roberta maestro david mangum juan martin - liberal renato martins clinton mason joaquin mateo david mcconkey bruno medeiros alexander menzies rodney miles c . 
chung , phd1 purpose brca1 or brca2 loss of function results in homologous recombination deciency ( hrd ) , which is targetable by poly ( adp - ribose ) polymerase ( parp ) inhibitors and other dna - damaging agents . 
in cancers associated with germline brca1 / 2 alterations ( brca1 / 2 - associated cancers : breast , ovarian , pancreatic , prostate ) , brca1 / 2 alterations result in hrd and are biomarkers for parp inhibitor use . 
across cancer types , biallelic brca1 / 2 alteration was associated with increased gloh versus monoallelic or wild - type brca1 / 2 ; predicted germline or somatic mutations were both associated with elevated gloh . conclusion biallelic brca1 / 2 alterations were associated with elevated gloh in diverse cancer types , including those not traditionally associated with brca1 / 2 cancer syndromes . 
biomarker development for parp inhibitors should integrate methods to distinguish biallelic from monoallelic brca1 / 2 status , and biallelic brca1 / 2 alteration should be broadly evaluated across cancer types as a biomarker for underlying hrd and parp inhibitor sensitivity . jco precis oncol 4 : 442 - 465 . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction brca1 and brca2 encode critical components of the homologous recombination ( hr ) dna repair pathway that maintains genomic stability.1 germline brca1 / 2 ( gbrca1 / 2 ) alterations are associated with elevated risk for breast , ovarian , pancreatic , and prostate cancer ( brca1 / 2 - associated cancers ) , 2 , 3 and tumors that arise in brca1 / 2 mutation carriers have often lost the wild - type allele.4 synthetic lethal interactions between brca1 / 2 loss of function and poly ( adp - ribose ) polymerase inhibitors ( parpi ) underlie development and regulatory approval of parpi targeted therapy for ovarian , breast , and pancreatic cancer.1 , 5 companion diagnostic testing for gbrca1 / 2 and somatic brca1 / 2 ( sbrca1 / 2 ) alteration1 can guide parpi therapy selection . brca1 or brca2 loss - of - function results in hr deciency ( hrd ) and accumulation of chromosomal rearrangements and copy number alterations . 
 pan - cancer analysis of biallelic brca1 / 2 context key objective brca1 / 2 loss - of - function alterations result in homologous recombination deciency ( hrd ) and are biomarkers for poly ( adpribose ) polymerase ( parp ) inhibitor sensitivity in breast , ovarian , prostate , and pancreatic cancer . 
to determine the relevance of brca1 / 2 alterations across cancer types , we evaluated the pan - cancer landscape of brca1 / 2 alterations and their association with the genome - wide loss - of - heterozygosity ( gloh ) marker of hrd . knowledge generated the fraction of brca1 / 2 alterations that were biallelic differed by cancer type and predicted germline / somatic status . 
brca1 / 2 alterations were most frequently biallelic in breast , ovarian , prostate , and pancreatic cancer ; in other cancer types , 44% of brca1 / 2 alterations were biallelic . 
percent gloh was calculated as a signature of hrd as previously described.9 , 10 see the appendix for full methods . results to assess the prevalence of brca1 / 2 genomic alterations across cancer types , we examined cgp results from 234 , 154 tumors sequenced as part of routine clinical care . overall , brca1 / 2 alterations were observed in 4.7% of cases ( brca1 , 2.1% ; brca2 , 2.7% ; fig 1a ; appendix fig a1 )  . 
brca1 and brca2 alterations were most frequent in ovarian ( 10.5% ) and prostate cancer ( 9.6% ) , respectively ; unlike brca2 , brca1 alterations were infrequent in prostate cancer . brca1 / 2 homozygous deletions were infrequent except in prostate cancer , where brca2 deletions were observed at a 2.6% frequency and accounted for 25% of brca1 / 2altered cases . 
brca1 / 2 mutations were distributed throughout the length of each gene , and most were truncating events ( appendix fig a2 )  . germline / somatic status for brca1 / 2 short variant mutations was predicted using validated computational methods.18 we also evaluated performance of germline / somatic predictions in this study . 
fraction ( % ) of ( b ) brca1 or ( c ) brca2 mutations predicted to be germline v somatic was determined for each cancer type . see the data supplement for detailed data . computational methods correctly identied 21 ( 91% ) as predicted germline variants . 
second , because cell - free dna ( cfdna ) sequencing can often distinguish germline from somatic variants , 19 we evaluated 52 brca1 / 2 germline / somatic predictions from tissue samples and evaluated patientmatched cfdna ngs results . 
as expected , the majority of mutations were predicted to be germline in brca1 / 2 - associated cancers ( brca1 , 58.1% ; brca2 , 56.8% ) , but predicted sbrca1 / 2 mutations comprised an appreciable proportion of brca1 / 2 mutations . 
 ( a ) fraction of brca1 / 2 - altered cases with biallelic or monoallelic alteration was determined . brca1 / 2 - altered cases were evaluated for class of alteration identied and classied as biallelic ( multiple brca1 or multiple brca2 alterations in the same sample , homozygous deletion , biallelic short variant mutation [ loss of heterozygosity of the wild - type allele ] ) , monoallelic ( heterozygous mutation ) , or unknown ( zygosity status could not be determined )  . 
a lower - bound estimate was established by assessing biallelic cases as a fraction of all brca1 / 2 - altered cases , including those with unknown biallelic / monoallelic status ( see data supplement )  . 
for cases where biallelic / monoallelic status could be determined , we estimated the fraction of brca1 / 2 - altered cases with biallelic alteration ( biallelic fraction )  . 
although biallelic fraction was lower in nonbrca1 / 2 - associated cancers ( p , .0001 , fishers exact test ) , biallelic alterations nonetheless comprised 43.6% of brca1 / 2 - altered cases : biallelic fraction was  . 
1% frequency in at least 13 cancer types . in prostate cancer , predicted gbrca1 / 2 and sbrca1 / 2 mutations were separately assessed for biallelic fraction ( fig 3 ; appendix fig a5 )  . 
for brca1 / 2associated cancers , both predicted gbrca1 / 2 ( 90.8% ) and sbrca1 / 2 ( 81.2% ) mutations were frequently biallelic , whereas in nonbrca1 / 2 - associated cancers , fewer predicted gbrca1 / 2 ( 46.4% ) and sbrca1 / 2 ( 25.4% ) mutations were biallelic . 
in ovarian and breast cancer , the majority of brca1 and brca2 mutations were biallelic , both for predicted germline and the majority of somatic mutations . brca2 ( gbrca2 , 87.6% ; sbrca2 , 75.0% ) mutations were biallelic , but brca1 ( gbrca1 , 40.0% ; sbrca1 , 22.2% ) mutations were less frequently biallelic . 
cases with 16% gloh were classied as gloh - high on the basis of the cutoff established in the ariel3 trial of rucaparib in ovarian cancer.10 biallelic brca1 / 2 alterations were associated with gloh - high across every cancer type examined . 
the fraction of cases that were gloh - high was signicantly increased for biallelic brca1 / 2 - altered compared with wild - type cases ( fig 4a , appendix fig a6a ) , whereas the fraction of monoallelic brca1 / 2 cases that were gloh - high was similar to wild type . 
signicant association of biallelic but not monoallelic alterations with gloh - high was observed both for brca1 and for brca2 when assessed individually ( figs 4b and 4c )  . 
in some cancer types , significant but modest elevation in tumor mutational burden ( tmb ) was observed for biallelic brca1 / 2 - mutated cases ( predicted germline or somatic ) versus wild type ; however , the association with tmb was not consistent across cancer types ( appendix fig a7 )  . 
monoallelic sbrca1 / 2 - mutated ( but not gbrca1 / 2 ) cases were commonly associated with elevated tmb versus wild type , and such mutations may be a consequence of increased mutation rate . although biallelic brca1 / 2 status was consistently associated with gloh - high , the magnitude was variable for each cancer type , with the greatest association observed in pancreatic ( or , 22.5 ) , biliary ( or , 21.5 ) , endometrial ( or , 17.2 ) , unknown primary ( or , 16.1 ) , and ovarian ( or , 14.9 ) cancer ( fig 4a )  . 
more than 75% of cases with biallelic brca1 / 2 alterations were gloh - high for ovarian , breast , pancreatic , unknown primary , and endometrial cancer , whereas fewer than half were gloh - high for prostate and colorectal cancer . 
25% of brca1 / 2 wildtype cases were gloh - high for ovarian , breast , lung , and gastric / esophageal cancer . the 16% gloh - high cutoff was clinically validated in ovarian cancer9 , 10 and requires optimization for other cancer types . 
consistent with the ndings using a gloh cutoff - based approach , gloh scores were higher in brca1 / 2 biallelic versus wild - type cases across all cancer types evaluated ; increased gloh score was also observed when biallelic brca1 and brca2 were evaluated independently . hrd signatures ( including gloh - high ) may identify additional brca1 / 2 wild - type tumors potentially suitable for parpi.9 , 10 , 20 overall , 19.3% of cases were gloh - high compared with 3.2% of cases with biallelic brca1 / 2 alteration , and for most cancer types , the frequency of gloh - high was greater than biallelic brca1 / 2 alterations ( data supplement ) ; distribution of gloh scores varied between cancer types . 
to inform rational cancer typespecic gloh - high cutoffs , we assessed the performance of different gloh - high thresholds to classify biallelic brca1 / 2 compared with wild - type cases . 
plotting sensitivity to detect cases with biallelic brca1 / 2 alteration and specicity ( percentage of brca1 / 2 wild - type cases negative for the glohhigh biomarker ) , we identied a cutoff that maximized the combined sensitivity and specicity score ( fig 6 )  . 
the identied cutoff for ovarian cancer was 15.1% , which was consistent with the 14% and 16% cutoffs identied in clinical trials.9 , 10 discussion the development of parpi for brca1 / 2 altered ovarian , breast , and pancreatic cancer1 , 5 is predicated on synthetic lethality interactions between brca1 / 2 loss of function and parpi / trapping . 
emerging clinical trial data suggest that brca1 / 2 alteration may also be predictive of parpi response in prostate cancer.1 , 11 - 13 although responses to parpi have been documented in other cancer types , 14 , 21 - 23 the relevance of brca1 / 2 alterations in nonbrca1 / 2associated cancers remains unclear.4 to understand the landscape and phenotypic consequence of brca1 / 2 alterations , we assessed our data set of 234 , 154 cancer specimens sequenced using a clinicalgrade cgp assay . 
brca1 / 2 alterations were frequent in brca1 / 2 - associated cancers but also observed in a signicant fraction ( 3% ) of nonbrca1 / 2 - associated cancers . 
predicted germline mutations comprised the majority of brca1 / 2 mutations in brca1 / 2 - associated cancers ; however , it is notable that 43% were predicted somatic ( of which 81% were biallelic ) given data that support sbrca1 / 2 alteration as a biomarker for parpi in ovarian and prostate cancer.1 , 11 - 13 brca1 / 2 alterations were assessed for biallelic versus monoallelic status to distinguish likely loss of function from biologically neutral alterations . 
of note , although monoallelic alteration in brca1 / 2 mutation carriers may have a subtle haploinsufcient phenotype , 24 it does not lead to severe hrd or sensitivity to platinum - based chemotherapy.25 consistent with the established role of brca1 / 2 in the pathogenesis of brca1 / 2 - associated cancers , 90% of brca1 / 2 - altered cases were biallelic with high biallelic fraction both for predicted gbrca1 / 2 and sbrca1 / 2 . 
differences in brca1 and brca2 biallelic fraction were observed : brca2 was more frequently biallelic in prostate and small - cell lung cancer , and brca1 was more frequently biallelic in endometrial cancer , esophageal scc , colorectal cancer , and melanoma . 
the frequency of ( a ) brca1 / 2 , ( b ) brca1 , or ( c ) brca2 biallelic , monoallelic , and wild - type cases that were gloh - high was compared across cancer types . 
50 biallelic brca1 / 2 - altered samples were assessed individually , and all other cancer types were grouped together and analyzed as a single group ( other )  . 
association between brca1 / 2 biallelic alteration and genome - wide loss of heterozygosity ( gloh ) score as a continuous variable . gloh score was assessed in ( a ) brca1 / 2 , ( b ) brca1 , and ( c ) brca2 biallelic v wild - type cases across cancer types . 
boxes span the rst and third quartiles , and the median is denoted by the horizontal line in the box ; whiskers indicate maximum and minimum values within 1.5 the interquartile range ; black dots indicate outlier events . 
 100 sokol et al ovarian prostate breast endometrial pancreatic bladder / uc gastric / esophageal adenocarcinoma biliary tract colorectal nsclc % gloh sensitivity specificity % gloh % gloh fig 6 . 
 pan - cancer analysis of biallelic brca1 / 2 differences suggest that brca1 and brca2 may have tissue specicities outside breast and ovarian different cancer . to determine whether brca1 / 2 alterations lead to hrd in both brca1 / 2 - associated and nonbrca1 / 2 - associated cancers , we evaluated gloh in brca1 / 2 - altered versus type evaluated , wild - type cases . 
monoallelic alterations may be found in sporadic cancers from gbrca1 / 2 carriers or as somatic passenger mutations . distinguishing biallelic from monoallelic status may be an important consideration for rening brca1 / 2 alteration as a predictive biomarker for parpi . 
in brca1 / 2 - associated cancers , brca1 / 2 alteration status alone has proven sufcient as a predictive biomarker , 1 likely explained by the majority of brca1 / 2 alterations in this context being biallelic . 
nevertheless , parpi trials in prostate cancer have incorporated brca1 / 2 biallelic status into biomarker development , 13 and our nding of signicantly lower biallelic fraction for brca1 ( v brca2 ) in prostate cancer suggests that integrating biallelic status could rene predictive biomarkers in this setting . the lack of parpi clinical activity in nonbrca1 / 2 - associated cancers reported previously could be explained by grouped analysis of biallelic and monoallelic brca1 / 2 alterations.4 because of lower rates of biallelic alteration in nonbrca1 / 2 - associated cancers , parpi clinical trials will likely require patient selection strategies that incorporate methods to discriminate biallelic from monoallelic brca1 / 2 alterations . our ndings are consistent with a recent study that demonstrated an elevated hrd composite score in biallelic but not heterozygous brca1 / 2 cases relative to wild type in an aggregate set of nonbrca1 / 2 - associated cancers.4 a strength of the current study was that the large data set size enabled analysis of biallelic brca1 / 2 separately from monoallelic alterations , independent assessment of brca1 and brca2 , and evaluation of nonbrca1 / 2 - associated cancers as individual cancer types rather than in aggregate , which may have enabled identication of associations between biallelic brca1 / 2 alterations and an hrd signature across cancer types not previously described.4 if brca1 / 2 biallelic alterations functionally result in hrd , they should represent a targetable vulnerability to parpi and platinum - based chemotherapy , irrespective of whether they are drivers of disease pathogenesis or bystander passenger alterations.26 our data demonstrate that biallelic brca1 / 2 alteration in nonbrca1 / 2 - associated cancers are associated with the gloh - high signature of hrd and , therefore , warrant investigation in parpi trials . 
although biallelic brca1 / 2 alterations occur at low prevalence in nonbrca1 / 2 - associated cancers , basket trials that led to the tumor - agnostic approvals of ntrk inhibitors for ntrk fusion - positive tumors and pembrolizumab for microsatellite instabilityhigh tumors27 demonstrate feasibility of therapeutic development for rare pan - cancer biomarkers . 
clinical trials such as the tapur study ( clinicaltrials.gov identier : nct02693535 ) that treatment arm for brca1 / 2 - altered includes a parpi cancers will inform parpi development in nonbrca1 / 2associated cancers , and analyses may benet from consideration of monoallelic versus biallelic status . although gloh - high is associated with clinical benet from rucaparib in ovarian cancer , 9 , 10 understanding of the gloh biomarker in other cancer types is required . 
evaluation of sensitivity and specicity of varying gloh thresholds to distinguish biallelic brca1 / 2 - altered and wild - type cases may inform development of disease - specic gloh - high cutoffs . 
in future studies , analysis of brca1 / 2 wild - type , gloh - high cases may be a discovery tool for characterizing brca1 / 2 vuss and for prioritizing candidate non - brca1 / 2 hr pathway gene biomarkers.9 although detection of gloh - high in brca1 / 2 wild - type cases potentially expands the patient population addressable by parpi , the utility of gloh - high requires validation in prospective trials for nonovarian cancers . limitations of this study should be acknowledged . 
first , we focus on gloh as a biomarker for hrd ; other hrd markers were not evaluated , including telomeric allelic imbalance ( tai ) , large - scale transition ( lst ) , mychoice hrd ( combination loh / tai / lst score ; myriad genetics , salt lake city , ut ) , signature 3 , and hrdetect.7 , 8 , 28 - 33 in parpi trials , hrd biomarkers have focused on approaches that are compatible with targeted ngs assays ( gloh , mychoice hrd ) , which are used in routine clinical practice.1 , 9 , 10 in contrast , signature 3 and hrdetect signatures have been evaluated in the research setting using whole - exome or whole - genome sequencing ; novel methods may enable future clinical assessment of signature 3 with targeted assays.34 second , germline / somatic status predictions using tumor - only sequencing and computational methods are less denitive compared with matched normal sequencing used in other studies.4 , 18 third , using brca1 / 2 biallelic versus wild - type status to rene gloh - high cutoffs is confounded by some brca1 / 2 wild - type gloh - high cases that are hrd because of brca1 / 2 alteration independent mechanisms , such as brca1 / 2 methylation or alteration in other hr genes . 
 sokol et al in the future ; however , we focused on brca1 / 2 because other hr pathway genes have not been consistently predictive of clinical response to parpi , 11 - 13 , 36 and robust clinical evidence supporting predictive biomarker genes beyond brca1 / 2 is lacking . 
finally , clinical outcomes data that associate parpi response with biallelic brca1 / 2 alteration and gloh were not available and require evaluation in clinical trials . we demonstrate that biallelic brca1 / 2 alterations are associated with elevated gloh across all cancer types evaluated and may therefore represent a therapeutic vulnerability targetable by parpi . 
ali , shridar ganesan , jon h . chung financial support : brian alexander administrative support : brian alexander provision of study material or patients : hossein khiabanian , primo n . 
gregg consulting or advisory role : astrazeneca , bristol - myers squibb , roche , foundation medicine , luminex speakers bureau : astrazeneca , foundation medicine , bristol - myers squibb primo n . 
 pan - cancer analysis of biallelic brca1 / 2 research funding : merck sharp & dohme ( inst ) , astrazeneca ( inst ) , ipsen ( inst ) travel , accommodations , expenses : roche , merck sharp & dohme , astrazeneca , janssen pharmaceuticals , rainier therapeutics other relationship : bayer ag ( i ) vincent a . 
miller employment : foundation medicine leadership : foundation medicine , revolution medicines stock and other ownership interests : foundation medicine , mirati therapeutics , revolution medicines patents , royalties , other intellectual property : periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center brian alexander employment : foundation medicine leadership : foundation medicine stock and other ownership interests : roche research funding : eli lilly ( inst ) , puma ( inst ) , celgene ( inst ) open payments link : 854258 / summary siraj m . 
ali employment : foundation medicine , eqrx , eqrx ( i ) leadership : incysus stock and other ownership interests : exelixis , blueprint medicines , agios , genocea biosciences consulting or advisory role : revolution medicines , azitra ( i ) , princepx tx ( i ) patents , royalties , other intellectual property : patents through foundation medicine , patents through seres health on microbiome stuff in nonneoplastic disease ( i ) shridar ganesan employment : merck ( i ) stock and other ownership interests : ibris , inspirata , merck ( i ) consulting or advisory role : inspirata , novartis , roche , foghorn therapeutics , foundation medicine , merck sharp & dohme patents , royalties , other intellectual property : two patents for digital imaging that may be licensed to ibris and inspirata travel , accommodations , expenses : inspirata other relationship : national cancer institute / national institutes of health jon h . 
chung employment : foundation medicine stock and other ownership interests : foundation medicine no other potential conicts of interest were reported . references 105 : 812 - 822 , 2013 1 . 
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j natl cancer inst 110 : 704 - 713 , 2018 swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
coleman rl , oza am , lorusso d , et al : rucaparib maintenance treatment for recurrent ovarian carcinoma after response to platinum therapy ( ariel3 ) : a randomised , double - blind , placebo - controlled , phase 3 trial . 
abida w , bryce ah , vogelzang nj , et al : preliminary results from triton2 : a phase ii study of rucaparib in patients ( pts ) with metastatic castration - resistant prostate cancer ( mcrpc ) associated with homologous recombination repair ( hrr ) gene alterations . 
smith mr , sandhu sk , kelly wk , et al : phase ii study of niraparib in patients with metastatic castration - resistant prostate cancer ( mcrpc ) and biallelic dnarepair gene defects ( drd ) : preliminary results of galahad . 
sandhu sk , schelman wr , wilding g , et al : the poly ( adp - ribose ) polymerase inhibitor niraparib ( mk4827 ) in brca mutation carriers and patients with sporadic cancer : a phase 1 dose - escalation trial . 
slavin tp , banks kc , chudova d , et al : identication of incidental germline mutations in patients with advanced solid tumors who underwent cell - free circulating tumor dna sequencing . 
moore kn , secord aa , geller ma , et al : niraparib monotherapy for late - line treatment of ovarian cancer ( quadra ) : a multicentre , open - label , single - arm , 21 . 
necchi a , raggi d , giannatempo p , et al : exceptional response to olaparib in brca2 - altered urothelial carcinoma after pd - l1 inhibitor and chemotherapy 22 . 
hierro c , matos i , martin - liberal j , et al : agnostic - histology approval of new drugs in oncology : are we already there ? clin cancer res 25 : 3210 - 3219 , 2019 28 . 
timms km , abkevich v , hughes e , et al : association of brca1 / 2 defects with genomic scores predictive of dna damage repair deciency among breast cancer 31 . 
telli ml , timms km , reid j , et al : homologous recombination deciency ( hrd ) score predicts response to platinum - containing neoadjuvant chemotherapy in patients with triple - negative breast cancer . 
marshall ch , sokolova ao , mcnatty al , et al : differential response to olaparib treatment among men with metastatic castration - resistant prostate cancer harboring brca1 or brca2 versus atm mutations . 
 pan - cancer analysis of biallelic brca1 / 2 appendix supplementary methods approval for this study , including a waiver of informed consent and health insurance portability and accountability act waiver of authorization , was obtained from the western institutional review board ( irb ; protocol #20152817 )  . 
cgp results included in this study were from tumor tissue specimens ( n = 234 , 154 ) submitted as part of routine clinical care ( december 2013 - march 2019 ) ; for patient characteristics , see the data supplement . 
in validation testing of 480 tumor - only sequencing calls against matched normal specimens , accuracy was 95% for somatic and 99% for germline calls ( sun jx , et al : plos comput biol 14 : e1005965 , 2018 ) ; in assessment of zygosity calls , signicant enrichment in mutations with loss of heterozygosity ( loh ) was observed for tumor suppressor genes ( sun jx , et al : plos comput biol 14 : e1005965 , 2018 )  . 
to evaluate performance of germline / somatic computational predictions in this series , we compared predictions against a subset of cases with available results from patient - matched germline testing or cell - free dna ( cfdna ) ngs that were performed as previously described ( clark ta , et al : j mol diagnostics 20 : 686 - 702 , 2018 ; khiabanian h , et al : jco precis oncol 2 : 1 - 15 , 2018 ) ; brca1 / 2 genetic testing on a subset of patients at the rutgers cancer institute of new jersey were analyzed under the auspices of an irb - approved protocol . 
germline - like af was dened as mutations observed in cfdna at 40% - 60% af ( except for cfdna samples with high circulating tumor dna fraction [  . 20% ] that were excluded from the analysis as ambiguous )  . 
brca1 / 2 alterations were categorized as biallelic , monoallelic , or unknown . biallelic alterations were mutations with loh of the wild - type allele , as determined by zygosity status ( sun jx , et al : plos comput biol 14 : e1005965 , 2018 ) ; homozygous deletion ; or 2 brca1 or 2 brca2 alterations in a sample . 
percent genome - wide loh ( gloh ) was calculated as a signature of hrd as previously described ( coleman rl , et al : lancet 390 : 1949 - 1961 , 2017 ; swisher em , et al : lancet oncol 18 : 75 - 87 , 2017 )  . 
this prole was segmented and interpreted using afs of sequenced snps to estimate copy number ( ci ) and minor allele count ( mi ) at each segment ( i )  . 
two types of loh segments were excluded from the calculation of percent gloh : loh segments that spanned 90% of a whole chromosome or chromosome arm because these loh events usually arise through non - hrd mechanisms ( eg , mitotic nondisjunction ) and regions in which loh inference was ambiguous . 
for each tumor , the percent gloh was computed as 100 the total length of nonexcluded loh regions ( xi ) divided by the total length of nonexcluded regions of the genome . 
 sokol et al brca1 / 2 short variant homozygous deletion rearrangement multiple brca1 brca2 short variant homozygous deletion rearrangement multiple brca1 brca2 breast cancer ovarian cancer breast cancer ovarian cancer fig a1 . 
growth factor receptor 2 ( her2 ) status was determined on the basis of the presence or absence of a copy number amplication ; triple - negative breast cancer ( tnbc ) was dened as er - negative , her2 - negative samples . 
 somatic af germline af cfdna germline af cfdna somatic af 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 brca1 / 2 variant allele frequency ( % ) predicted germline ( n = 22 ) predicted somatic ( n = 30 ) cfdna gbrca sbrca predicted germline predicted somatic sokol et al predicted germline predicted somatic breast cancer ovarian cancer fig a3 . 
 ( a and b ) comparison of tissue - based germline / somatic predictions to brca1 / 2 allele frequencies ( afs ) in patient - matched cell - free dna ( cfdna )  . 
mutations with germline af were dened as those identied in cfdna at 40% - 60% af , except for cases with high circulating tumor dna fraction ( 20% ) that were excluded from the analysis as ambiguous af . 
 ( c - e ) predicted germline / somatic status calls were made for each brca1 or brca2 short variant mutation . for mutations yielding a successful call , frequency of predicted germline v somatic mutation was determined for ( c ) brca1 / 2 across cancer types , ( d ) brca1 / 2 ( grouped and individually ) overall ( n = 5 , 845 ) for brca1 / 2 - associated cancers ( breast , ovarian , pancreatic , prostate ; n = 3 , 061 ) and nonbrca1 / 2 - associated cancers ( n = 2 , 784 ) , and ( e ) brca1 / 2 for the subset of ovarian and breast cancer cases where molecular / histologic subtype information was available . 
relative fraction of brca1 / 2 - altered cases with biallelic or monoallelic alteration was determined for ( a ) brca1 across cancer types and ( b ) brca2 across cancer types . 
 ( c ) brca1 / 2 ; ( d ) brca1 ; ( e ) brca2 for overall , brca1 / 2 - associated cancers , and nonbrca1 / 2 - associated cancers ; and ( f ) brca1 / 2 for the subset of ovarian and breast cancer cases where molecular / histologic subtype information was available . 
 ( a ) brca1 / 2 mutations ( grouped ) with a germline ( gbrca1 / 2 ) or somatic ( sbrca1 / 2 ) prediction were evaluated for biallelic / monoallelic status for all cancers , brca1 / 2 - associated cancers ( as a group and as individual cancer types ) , and nonbrca1 / 2 - associated cancers ( see data supplement )  . 
cases with somatic brca1 / 2 ( sbrca1 / 2 ) or germline brca1 / 2 ( gbrca1 / 2 ) biallelic mutation , monoallelic mutation , or wild - type ( wt ) brca1 / 2 were plotted against tumor mutational burden ( tmb in mutations / mb ; log10 score )  . 
box and whisker plot where the box spans the rst and third quartiles , the median is denoted by the horizontal line in the box , and whiskers indicate maximum and minimum values within 1.5 the interquartile range ( see data supplement )  . 
 ( a ) the frequency of brca1 / 2 biallelic , monoallelic , and wild - type cases that were high genome - wide loss of heterozygosity ( gloh - high ) was compared in the subset of ovarian and breast cancer cases where molecular / histologic subtype information was available ( see data supplement )  . 
 ( b ) frequency of predicted germline brca1 / 2 ( gbrca1 / 2 ) biallelic , monoallelic , and wild - type cases that were gloh - high ( see data supplement )  . 
 ( c ) frequency of predicted somatic brca1 / 2 ( sbrca1 / 2 ) biallelic , monoallelic , and wild - type cases that were gloh - high ( see data supplement )  . 
egfr tyrosine kinase inhibitors ( tkis ) markedly increase progression - free survival in advanced disease.17 , 18 this discovery opened a new era of precision medicine leading to accelerated development and subsequent approval of drugs for several actionable genomic alterations in nsclc . 
while the molecular proling of nsclc is considered a standard of care in the advanced setting , the utility of molecular testing and targeted therapies in the adjuvant setting in early - stage nsclc has yet to be established . 
the br19 trial compared getinib versus placebo in a similar population.21 the trial closed prematurely after results of another trial showed detrimental effect of getinib after chemoradiation in stage iii nsclc.23 however , egfrmutant nsclc accounted for only 4% ( n = 15 ) of the patients . 
patients with resected stage ib - iiia ( american joint committee on cancer [ ajcc ] 7th edition ) adenocarcinoma with sensitizing egfr mutation were randomly assigned 1 : 1 to adjuvant osimertinib at the standard dose of 80 mg / d or matching placebo . 
random assignment was stratied by stage ( ib v ii v iiia ) , type of egfr mutation ( exon 19 del v l858r ) , and race ( asian v non - asian ) , but not based on receipt of adjuvant chemotherapy . 
patients received assigned treatment for a planned 3 - year duration or until disease recurrence , completion they met predetermined criteria for discontinuation . treatment , or if six - hundred eighty - two patients were randomly assigned to osimertinib ( n = 339 ) or placebo ( n = 343 )  . 
the published data are an unplanned interim analysis with the dfs data being only 33% mature ( 11% in osimertinib and 55% in placebo ) at the data cutoff date of january 17 , 2020 . 
similarly , dfs in the overall population ( including stage ib ) was also signicantly longer with a consistent benet seen across all predened subgroups ( age , smoking , race , stage , and type of egfr mutation )  . 
first , positron emission tomography - computed tomography ( pet - ct ) scan and brain mri were not mandated at screening in the study protocol.27 in the united states , pet - ct scan and brain mri are considered the standard of care for the complete staging of early - stage nsclc.28 data on proportion of patients with complete staging information ( pet - ct and brain mri ) in either arm are not available . 
in the adura trial , the median dfs and 2 - year dfs rate in the placebo arm are signicantly lower than those reported in historical trials , suggesting that suboptimal staging at baseline could have contributed to higher proportion of patients being understaged.29 , 30 compared to control arm , osimertinib showed an 82% reduction in risk for cns recurrence or death . 
however , this should be interpreted with caution , given limited follow - up and patients with occult intracranial metastatic disease missed on brain ct scans who get osimertinib are at an advantage because of potent cns activity . 
data regarding the recurrence rates adjusted by country or region of enrollment may be important as petct and brain mri are not readily available worldwide in resource - limited settings . second , about 40% of the patients did not get adjuvant chemotherapy . 
to accurately assess the benet of adjuvant osimertinib , adjuvant chemotherapy should have been offered to all patients in the control arm and not just left to the discretion of the attending physician . 
 commentary enrollment , port was considered a valid treatment option for resected stage iiia n2 disease . in retrospect , not allowing port and not mandating standard of care adjuvant chemotherapy with proven os benet among participants in the placebo arm may be more of an ethical consideration for patients and investigators rather than a confounding variable . 
in the adjuvant setting , in an asymptomatic context , daily treatment with osimertinib for up to 3 years can lead to considerable toxicity , especially if we consider that some patients have been already cured with surgery with or without adjuvant chemotherapy . in addition to some of the limitations mentioned above , several important aspects related to study outcomes remain to be dened . 
in the adjuvant setting , it is not known whether the 2 - year dfs superiority with egfr directed therapy will translate into an os benet with osimertinib . data from adjuvant / ctong 1104 trial suggest that the substantial dfs benet did not translate into os , a goldstandard measure of patient impact in the adjuvant setting . although pfs may be a reasonable surrogate for os in the metastatic setting , higher level of evidence is needed before advocating for dfs as a proxy for os benet in patients receiving adjuvant cancer therapies . 
unlike adaura , the alchemist trial is testing adjuvant egfrand alkdirected therapy using os as the primary end point . furthermore , it has been suggested that targeted therapies as cytostatic agents suppress micrometastatic disease progression , thereby delaying recurrence but not eliminating the disease completely . 
despite multiple studies across solid tumors , to our knowledge , imatinib is the only tki that has shown improved os when given in the adjuvant setting of high - risk gi stromal malignancies , 31 a disease with completely different molecular background compared with lung adenocarcinomas . 
of note , in the osimertinib arm of the adaura trial , about 15% of the patients who stopped the drug at 3 years experienced dfs event in the 6 - 9 months after drug discontinuation . 
this might suggest that recurrence - free survival is directly related to the length of drug exposure , and therefore adjuvant osimertinib might only delay recurrence and not cure the disease . 
in adaura , at the data cutoff , approximately 46% of patients in the placebo arm experienced disease reto know the post - protocol currence . treatment patterns , specically the proportion of patients in the placebo arm who received osimertinib at progression . if this percentage is lower than expected , then the dfs benet seen in adaura might only reect the suboptimal treatment of control arm upon progression . 
if similar it will be critical os can be achieved in the placebo arm with osimertinib treatment on recurrence , one can speculate that osimertinib has minimal effect on the natural history of the disease and is only delaying the inevitable relapsed disease . 
one can anticipate that having a nonngsbased molecular testing at the time of initial detection of the egfr mutation will make it hard to tease out the acquired , potentially actionable mechanisms of resistance at the dfs event . 
furthermore , development of acquired resistance mechanisms at relapse on osimertinib in the adjuvant setting could result in these patients missing the window of opportunity to receive osimertinib in the metastatic setting . robust biomarkers to predict durable clinical benet to adjuvant therapy are lacking . 
thus , it might not be as cost - effective to justify the use of osimertinib in the absence of an os benet , an issue that is contentious but is an important consideration from a societal healthcare perspective . 
furthermore , a proportion of patients who are probably cured of lung cancer with surgery alone may have unnecessary exposure to osimertinib and its toxicities with added hospitalizations . lowand middle - income countries might be affected the most because of lack of access to testing and drugs , as well as prohibitive costs . 
assessment of the quality of life ( qol ) while on therapy and beyond will also be a crucial parameter to consider the overall benet of treatment . in conclusion , os is the most important and clinically meaningful end point in the adjuvant setting . 
given the lack of evidence to demonstrate dfs as a validated surrogate end point with targeted therapy in the adjuvant setting , mature os data are recommended before a denitive statement on adjuvant osimertinib in egfr - mutant nsclc . we also worry that wide adoption of adura in the community might lead to lower use of adjuvant chemotherapy in favor of osimertinib . 
with the idmc decision to unblind and terminate the study early based on the perceived dfs benet , we might never know whether adjuvant osimertinib leads to a superior os versus treatment with osimertinib upon rst evidence of relapse . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . sonam puri consulting or advisory role : astrazeneca rodrigo dienstmann consulting or advisory role : roche , boehringer ingelheim speakers bureau : roche , ipsen , sano , msd oncology , servier , amgen research funding : merck no other potential conicts of interest were reported . references siegel rl , miller kd , jemal a : cancer statistics , 2020 . 
ca cancer j clin 70 : 7 - 30 , 2020 besse b , le chevalier t : developments in the treatment of early nsclc : when to use chemotherapy . 
ann thorac surg 83 : 409 - 418 , 2007 pignon jp , tribodet h , scagliotti gv , et al : lung adjuvant cisplatin evaluation : a pooled analysis by the lace collaborative group . 
j clin oncol 26 : 3552 - 3559 , 2008 kris mg , gaspar le , chaft je , et al : adjuvant systemic therapy and adjuvant radiation therapy for stage i to iiia completely resected non - small - cell lung cancers : american society of clinical oncology / cancer care ontario clinical practice guideline update . 
j clin oncol 35 : 2960 - 2974 , 2017 arriagada r , auperin a , burdett s , et al : adjuvant chemotherapy , with or without postoperative radiotherapy , in operable non - small - cell lung cancer : two metaanalyses of individual patient data . 
lancet 375 : 1267 - 1277 , 2010 strauss gm , herndon je , maddaus ma , et al : adjuvant paclitaxel plus carboplatin compared with observation in stage ib non - small - cell lung cancer : calgb 9633 with the cancer and leukemia group b , radiation therapy oncology group , and north central cancer treatment group study groups . 
j clin oncol 26 : 5043 - 5051 , 2008 burdett s , rydzewska l , tierney jf , et al : a closer look at the effects of postoperative radiotherapy by stage and nodal status : updated results of an individual participant data meta - analysis in non - small - cell lung cancer . 
lung cancer 80 : 350 - 352 , 2013 burdett s , stewart l : postoperative radiotherapy in non - small - cell lung cancer : update of an individual patient data meta - analysis . 
robinson cg , patel ap , bradley jd , et al : postoperative radiotherapy for pathologic n2 non - small - cell lung cancer treated with adjuvant chemotherapy : a review of the national cancer data base . 
mikell jl , gillespie tw , hall wa , et al : postoperative radiotherapy is associated with better survival in non - small cell lung cancer with involved n2 lymph nodes : results of an analysis of the national cancer data base . 
corso cd , rutter ce , wilson ld , et al : re - evaluation of the role of postoperative radiotherapy and the impact of radiation dose for non - small - cell lung cancer using the national cancer database . 
fukuoka m , wu yl , thongprasert s , et al : biomarker analyses and nal overall survival results from a phase iii , randomized , open - label , rst - line study of getinib versus carboplatin / paclitaxel in clinically selected patients with advanced non - small - cell lung cancer in asia ( ipass ) , j clin oncol 29 : 2866 - 2874 , 2011 inoue a , kobayashi k , maemondo m , et al : updated overall survival results from a randomized phase iii trial comparing getinib with carboplatin - paclitaxel for chemo - nave non - small cell lung cancer with sensitive egfr gene mutations ( nej002 )  . 
zhong wz , wang q , mao wm , et al : getinib versus vinorelbine plus cisplatin as adjuvant treatment for stage ii - iiia ( n1 - n2 ) egfr - mutant nsclc ( adjuvant / ctong1104 ) : a randomised , open - label , phase 3 study . 
kelly k , altorki nk , eberhardt wee , et al : adjuvant erlotinib versus placebo in patients with stage ib - iiia nonsmall - cell lung cancer ( radiant ) : a randomized , double - blind , phase iii trial . 
wu yl , zhong w , wang q , et al : ctong1104 : adjuvant getinib versus chemotherapy for resected n1 - n2 nsclc with egfr mutationnal overall survival analysis of the randomized phase iii trial 1 analysis of the randomized phase iii trial . 
douillard jy , rosell r , de lena m , et al : adjuvant vinorelbine plus cisplatin versus observation in patients with completely resected stage ib - iiia non - small - cell lung cancer ( adjuvant navelbine international trialist association [ anita ] ) : a randomised controlled trial . 
arriagada r , de radiomedic - ina i , santiago c , et al : cisplatin - based adjuvant chemotherapy in patients with completely resected nonsmall - cell lung cancer . n engl j med 350 : 351 - 360 , 2004 joensuu h , eriksson m , sundby hall k , et al : survival outcomes associated with 3 years vs 1 year of adjuvant imatinib for patients with high - risk gastrointestinal stromal tumors : an analysis of a randomized clinical trial after 10 - year follow - up . 
abbosh c , frankell a , garnett a , et al : phylogenetic tracking and minimal residual disease detection using ctdna in early - stage nsclc : a lung tracerx study . cancer res 80 , 2020 ( suppl ; abstr ct023 ) 36 . 
li n , ou w , ye x , et al : pemetrexed - carboplatin adjuvant chemotherapy with or without getinib in resected stage iiia - n2 non - small cell lung cancer harbouring egfr mutations : a randomized , phase ii study . 
zhong wz , chen kn , chen c , et al : erlotinib versus gemcitabine plus cisplatin as neoadjuvant treatment of stage iiia - n2 egfr - mutant non - small - cell lung cancer ( emerging - ctong 1103 ) : a randomized phase ii study . 
pennell na , neal jw , chaft je , et al : select : a phase ii trial of adjuvant erlotinib in patients with resected epidermal growth factor receptor - mutant non - smallcell lung cancer . 
precursors and carcinomas were immunohistochemically compared and analyzed for mutations , gene amplications , microsatellite instability , and tumor mutational burden using an next - generation sequencing panel containing 523 cancer - related genes . 
in addition , presence of fusion genes was analyzed using a panel of 55 genes . results sixty percent of neuroendocrine cases ( 6 / 10 ) presented with a precursor lesion , either intracholecystic papillary neoplasm ( n = 3 ) or biliary intraepithelial neoplasia ( n = 3 )  . 
moreover , nearly half of the ne gbcs carried at least one potentially actionable molecular alteration , highlighting the importance of molecular testing in this highly lethal cancer . jco precis oncol 5 : 473 - 484 . 
2021 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction neuroendocrine neoplasms ( nens ) can arise from almost any anatomical site including , but not limited to , the lung , the prostate , and throughout the gi and hepatobiliary tract . 
two major categories of nens have been described : well - differentiated neuroendocrine tumors ( nets ) and poorly differentiated neuroendocrine carcinomas ( necs ) , the latter being high grade by denition.1 morphologically , necs are classied as either pure small - cell or large - cell nec , and they might also be present together with an adenocarcinoma ( ac ) component . 
they nonneuroendocrine neoplasms ( minens ) when both the neuroendocrine component ( generally nec ) and the non - neuroendocrine component ( generally ac ) comprise at least 30% of the neoplasm.2 regardless of the anatomical site , necs and minens are highly aggressive tumors and carry a dismal prognosis . neuroendocrine as mixed classied necs can either arise de novo or during progression of an epithelial neoplasm , for example , because of a therapy resistance mechanism.3 the molecular features of necs have been best described in lung , pancreas , and prostate , but the cell of origin of necs has been a matter of controversy , specically in tumors with a mixed phenotype ( ie , minens )  . 
this study was approved by the radboud university medical center medical ethics committee ( 2018 - 4126 )  . histopathologic review formalin - xed parafn - embedded ( ffpe ) blocks were selected on the basis of pathology reports and histopathologic review . 
histologic typing and grading , including description of a precursor lesion ( either bilin or icpn ) , was performed using the who histologic classication of tumors of the gallbladder ( fth edition ) .2 t classication was performed using the american joint committee on cancer tumor - node - metastasis classication system ( eighth edition ) .12 additionally , presence of vascular , lymphatic , and perineural invasion was assessed . immunohistochemistry immunohistochemistry ( ihc ) was performed on 4 - mm whole - slide ffpe sections semiautomated using the lab vision autostainer ( immunologic , duiven , the netherlands ) for muc2 , muc5ac , muc6 , and ttf - 1 essentially as described before13 or fully automated using tissue - tek genie ( sakura finetek , torrance , ca ) for chromogranin a , cd56 , ck7 , ck20 , ki - 67 , ema ( muc1 ) , p53 , and synaptophysin with ready - to - use reagents . 
discrepancies in scoring were reevaluated until agreement . nucleic acid extraction for molecular analyses , dna and rna were isolated from ffpe - derived tumor tissue and the precancerous lesion if available . 
dna was isolated , quantied , and precipitated manually as described before.14 after precipitation , the nal concentration was determined using the qubit high sensitivity kit ( thermo fisher scientic , waltham , ma )  . rna was isolated using the reliaprep ffpe total rna miniprep system ( promega , madison , wi ) according to the manufacturers protocol , omitting the dnase treatment step . 
 histologic and molecular features of ne gallbladder cancer subsequently , 60 ng of either dna or rna was used as input for the library preparation . library preparation dna and rna library preparations were performed separately using the hybrid capture - based trusight oncology ( tso ) 500 dna and rna library preparation kits , respectively ( illumina , san diego , ca ) according to the manufacturers protocol essentially as described before.15 the tso500 dna assay targets 523 cancer - related genes for assessment of singleand multiple - nucleotide variants , gene amplications , tumor mutational burden ( tmb ) , and microsatellite instability ( msi ) , whereas the rna kit targets 55 genes for fusion gene analyses ( data supplement )  . 
in short , dna samples were fragmented , and rna samples were used for rstand second - strand cdna synthesis . next , samples underwent end - repair and a - tailing , followed by unique molecular identier ligation and barcoding . two target capture and purication steps , allowing for maximal target enrichment , were followed by polymerase chain reaction amplication and purication . 
libraries were quantied and normalized for uniform library representation . sequencing and data analysis sequencing was performed on a nextseq 500 system ( illumina ) , with 10 dna libraries on a nextseq high - output cassette or 16 rna libraries on a nextseq mid - output cassette . 
the number of gene copies in the tumor cells was estimated on the basis of the relative coverage corrected for the percentage of neoplastic cells in the sample . variant annotation was performed as described before via an in - housedeveloped pipeline.17 variants were ltered by exclusion of ( 1 ) variants outside exons and splice site regions ( 8 / + 8 ) except those in the tert promoter region , ( 2 ) synonymous variants , unless present in a splice site region , ( 3 ) variants present with a frequency of  . 
0.1% in the exac ( version 0.2 ) database , ( 4 ) variants with a variant allele frequency ( vaf ) of , 5% , and ( 5 ) variants with , 5 variant reads . 
remaining variants were manually inspected and curated and classied into ve classes on the basis of their level of evidence for pathogenicity , largely on the basis of american college of medical genetics / association for molecular pathology guidelines18 , 19 : class 1 , not pathogenic ; class 2 , unlikely pathogenic ; class 3 , possibly pathogenic ; class 4 , likely pathogenic ; and class 5 , pathogenic . 
potential effects on splicing were evaluated on the basis of the majority vote of splicesitefinder - like , maxentscan , nnsplice , and genesplicer ( all available from alamut visual version 2.13 [ sophia genetics , lausanne , switzerland ] )  . 
details on classes and their interpretation are provided in figure a1 , online only . variant - specic level 1 , 2 , 3 , or 4 evidence for actionability across all tumor types was derived from oncokb.21 level 1 and 2 biomarkers are dened as fda - recognized or standard care biomarkers , respectively , that are predictive of response to fda - approved drugs in specic tumor types . for level 3a biomarkers , there is compelling clinical evidence of drug response in a specic indication , and level 3b alterations are predictive of response to an fdaapproved or investigational drug in another indication . level 4 alterations comprise alterations for which there is compelling biological evidence that predicts response to a drug . 
seven cases ( 70% ) were pure high - grade net or nec , whereas three tumors ( 30% ) showed both ac and nec features and were classied as minens . 
data on type of systemic therapy are not available . tumors ( 50% ) were large - cell nec , four cases ( 40% ) were small - cell nec , and one case ( 10% ) was a net grade 3 , hereafter collectively referred to as ne gbc . 
in 90% of cases , at least one tumor component ( either nec or , if present , ac ) showed angioinvasion ; lymphatic invasion and perineural invasion were observed in 80% and 30% of cases , respectively . neuroendocrine expression with synaptophysin , chromogranin a , and cd56 conrmed neuroendocrine histology and was absent in the precancerous lesions ( table 2 and fig 1 )  . 
tumor characteristics case diagnosis ptnm ( m location ) icpn ( mixed type ) with nec ( sc ) t2anx bilin with nec ( sc ) t3nx nec ( sc ) t3n2m1 ( liver ) icpn ( intestinal type ) with nec ( lc ) t3n1 icpn ( biliary type ) with minen ( lc ) t2an1 bilin with minen t3nx ( lc ) nec ( lc ) nec ( lc ) t2nx t3nxm1 ( peritoneum ) 476 2021 by american society of clinical oncology abbreviations : + ,  . 
neuroendocrine marker expression with synaptophysin was positive in the nec and negative in the icpn and ac ( chromogranin a and cd56 were comparable ; table 2 ) , whereas epithelial marker expression ( ck 7 ) was positive in the icpn and ac and negative in the nec ( ck20 - negative in all three ; table 2 )  . 
since tp53 is frequently altered in necs of other anatomical origin , case 5 served as an exemplary case for which p53 ihc was performed next to a molecular screening , which was done for all cases . 
both the ac and nec components showed aberrant p53 expression , whereas the icpn component showed predominantly a normal p53 expression with only a small region with overexpression ( fig 1 )  . 
 de bitter et al molecular characteristics discussion for each case , dna and rna were isolated from the precursor lesion , the nec component and , if available , the ac component . 
quality control parameters of dna and rna libraries including reference values are listed in the data supplement . for case 1 , both the nec and icpn components did not meet quality control for msi , tmb , and cnv assessment . all likely pathogenic and pathogenic singleand multiplenucleotide variants ( classes 4 and 5 ) were considered potentially clinically relevant and are listed in figure 2 and the data supplement . 
a variety of other genes harbored potentially clinically relevant mutations in individual cases . mutations in tp53 were shared among the different precursor , ac , and nec components in all but one case ( case 8 ) , where two different mutations were observed in the ac versus the nec . 
interestingly , the icpn of case 5 did share the same tp53 mutation with the invasive tumor components , albeit with a low vaf ( 8.7% ) , reecting the limited region with p53 overexpression ( fig 1 )  . 
three cases ( 30% ) harbored potentially actionable variants ( level 1 ) in atm , brca2 , and rad54l , albeit with a low vaf for the atm variant of case 4 . in six cases , gene amplications were observed in a variety of genes including potentially actionable amplications of erbb2 ( level 1 ) and mdm2 ( level 3a ) , the latter being in a case without a tp53 mutation ( fig 2 )  . 
whereas the majority of mutations were shared among different precancerous , ac , and nec components , for gene amplications this was for a cnne1 amplication that not observed , except was shared between the ac and nec , but not the icpn , of case 5 . a fusion involving fgfr3 and tacc3 genes was observed in all three components of case 5 ( fig 2 )  . 
despite these phenotypical differences , the shared tp53 mutation in the different components in ve of six cases strongly suggests a common origin for neuroendocrine and epithelial components of ne gbc . 
in addition , 50% of the cases also had cholelithiasis , which is a well - known risk factor for pure gallbladder ac , 26 and may further reinforce the concept of a common origother studies of digestive system necs also support the lineage transformation hypothesis with molecular analyses.5 , 27 , 28 nearly all cases had multiple pathogenic or likely pathogenic mutations in a variety of genes with tp53 , ctnnb1 , rb1 , and atm being the most frequently involved . 
our ndings are largely in line with observations in lung , prostate , and pancreatic necs , where tp53 and rb1 are altered in roughly 80% of cases.3 interestingly , also pure gallbladder ac is molecularly highly heterogeneous with tp53 as the most frequently mutated gene ( 47.1% - 59% of cases ) .29 however , large - scale molecular data remain scarce and are mainly derived from endemic regions , which could hamper the extrapolation of ndings to nonendemic regions . gene amplications ( n = 6 cases ) were observed in a subset of cases and were in all but one case not shared between the different components . 
this suggests that , in contrast to mutations that are shared between the precursor and invasive components , oncogenic amplications typically occur as a later event in carcinogenesis , which has been observed before in esophageal ac.30 in case 5 , a ccne1 amplication was shared between the ac and nec , but not the icpn . 
potentially clinically relevant mutations ( class 4 and class 5 ) , gene amplications , fusion genes , microsatellite status , and total tumor mutational burden , identied by tso500 . 
another limitation of this study is the limited information on used therapeutic regimens , such as the type and outcome of adjuvant systemic therapy received by three of the patients , because of the retrospective anonymized nature of this study . in conclusion , we provide a comprehensive histopathologic and molecular overview of a very rare and understudied tumor type . 
ligtenberg employment : synthon ( i ) honoraria : astrazeneca ( inst ) , msd ( inst ) consulting or advisory role : astrazeneca ( inst ) , bayer ( inst ) , bristol - myers squibb ( inst ) , janssen ( inst ) , merck sharp and dohme ( inst ) , novartis ( inst ) , roche ( inst ) , lilly ( inst ) research funding : astrazeneca ( inst ) , bristol - myers squibb ( inst ) no other potential conicts of interest were reported . acknowledgment we would like to thank mandy hermsen , lisa plettenburg , and chinook ophelders for technical assistance ; palga and the ncr for providing data ; and pathology laboratories ( pathan , gelre hospitals , olvg , meander medical center , and martini hospital ) for providing tumor tissue . references rindi g , klimstra ds , abedi - ardekani b , et al : a common classication framework for neuroendocrine neoplasms : an international agency for research on cancer ( iarc ) and world health organization ( who ) expert consensus proposal . 
mod pathol 31 : 1770 - 1786 , 2018 klimstra ds , lam ak , paradis v , et al : tumours of the gallbladder and extrahepatic bile ducts , in who classication of tumours , 5th edition : digestive system tumours . 
am j surg pathol 11 : 71 - 86 , 1987 ( suppl 1 ) furlan d , cerutti r , genasetti a , et al : microallelotyping denes the monoclonal or the polyclonal origin of mixed and collision endocrine - exocrine tumors of the gut . 
world j surg 29 : 92 - 101 , 2005 yao jc , hassan m , phan a , et al : one hundred years after carcinoid : epidemiology of and prognostic factors for neuroendocrine tumors in 35 , 825 cases in the united states . 
j clin oncol 26 : 3063 - 3072 , 2008 sciarra a , missiaglia e , trimech m , et al : gallbladder mixed neuroendocrine - non - neuroendocrine neoplasm ( minen ) arising in intracholecystic papillary neoplasm : clinicopathologic and molecular analysis of a case and review of the literature . 
meguro y , fukushima n , koizumi m , et al : a case of mixed adenoneuroendocrine carcinoma of the gallbladder arising from an intracystic papillary neoplasm associated with pancreaticobiliary maljunction . 
acosta am , hamedani fs , kajdacsy - balla a , et al : primary mixed adenoneuroendocrine carcinoma of the gallbladder in a 55 - year - old female patient : a case report and review of the literature . 
de bitter tjj , van der linden rla , van vliet s , et al : colorectal metastasis to the gallbladder mimicking a primary gallbladder malignancy : histopathological and molecular characteristics . 
eijkelenboom a , kamping ej , kastner - van raaij aw , et al : reliable next - generation sequencing of formalin - xed , parafn - embedded tissue using single 15 . 
kroeze li , de voer rm , kamping ej , et al : evaluation of a hybrid capture - based pan - cancer panel for analysis of treatment stratifying oncogenic aberrations and molecule tags . 
eijkelenboom a , tops bbj , van den berg a , et al : recommendations for the clinical interpretation and reporting of copy number gains using gene panel ngs analysis in routine diagnostics . 
neveling k , feenstra i , gilissen c , et al : a post - hoc comparison of the utility of sanger sequencing and exome sequencing for the diagnosis of heterogeneous diseases . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
scardoni m , vittoria e , volante m , et al : mixed adenoneuroendocrine carcinomas of the gastrointestinal tract : targeted next - generation sequencing suggests a monoclonal origin of the two components . 
voss mh , hierro c , heist rs , et al : a phase i , open - label , multicenter , dose - escalation study of the oral selective fgfr inhibitor debio 1347 in patients with advanced solid tumors harboring fgfr gene alterations . 
chung , phd1 purpose brca1 or brca2 loss of function results in homologous recombination deciency ( hrd ) , which is targetable by poly ( adp - ribose ) polymerase ( parp ) inhibitors and other dna - damaging agents . 
in cancers associated with germline brca1 / 2 alterations ( brca1 / 2 - associated cancers : breast , ovarian , pancreatic , prostate ) , brca1 / 2 alterations result in hrd and are biomarkers for parp inhibitor use . 
across cancer types , biallelic brca1 / 2 alteration was associated with increased gloh versus monoallelic or wild - type brca1 / 2 ; predicted germline or somatic mutations were both associated with elevated gloh . conclusion biallelic brca1 / 2 alterations were associated with elevated gloh in diverse cancer types , including those not traditionally associated with brca1 / 2 cancer syndromes . 
biomarker development for parp inhibitors should integrate methods to distinguish biallelic from monoallelic brca1 / 2 status , and biallelic brca1 / 2 alteration should be broadly evaluated across cancer types as a biomarker for underlying hrd and parp inhibitor sensitivity . jco precis oncol 4 : 442 - 465 . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction brca1 and brca2 encode critical components of the homologous recombination ( hr ) dna repair pathway that maintains genomic stability.1 germline brca1 / 2 ( gbrca1 / 2 ) alterations are associated with elevated risk for breast , ovarian , pancreatic , and prostate cancer ( brca1 / 2 - associated cancers ) , 2 , 3 and tumors that arise in brca1 / 2 mutation carriers have often lost the wild - type allele.4 synthetic lethal interactions between brca1 / 2 loss of function and poly ( adp - ribose ) polymerase inhibitors ( parpi ) underlie development and regulatory approval of parpi targeted therapy for ovarian , breast , and pancreatic cancer.1 , 5 companion diagnostic testing for gbrca1 / 2 and somatic brca1 / 2 ( sbrca1 / 2 ) alteration1 can guide parpi therapy selection . brca1 or brca2 loss - of - function results in hr deciency ( hrd ) and accumulation of chromosomal rearrangements and copy number alterations . 
 pan - cancer analysis of biallelic brca1 / 2 context key objective brca1 / 2 loss - of - function alterations result in homologous recombination deciency ( hrd ) and are biomarkers for poly ( adpribose ) polymerase ( parp ) inhibitor sensitivity in breast , ovarian , prostate , and pancreatic cancer . 
to determine the relevance of brca1 / 2 alterations across cancer types , we evaluated the pan - cancer landscape of brca1 / 2 alterations and their association with the genome - wide loss - of - heterozygosity ( gloh ) marker of hrd . knowledge generated the fraction of brca1 / 2 alterations that were biallelic differed by cancer type and predicted germline / somatic status . 
brca1 / 2 alterations were most frequently biallelic in breast , ovarian , prostate , and pancreatic cancer ; in other cancer types , 44% of brca1 / 2 alterations were biallelic . 
percent gloh was calculated as a signature of hrd as previously described.9 , 10 see the appendix for full methods . results to assess the prevalence of brca1 / 2 genomic alterations across cancer types , we examined cgp results from 234 , 154 tumors sequenced as part of routine clinical care . overall , brca1 / 2 alterations were observed in 4.7% of cases ( brca1 , 2.1% ; brca2 , 2.7% ; fig 1a ; appendix fig a1 )  . 
brca1 and brca2 alterations were most frequent in ovarian ( 10.5% ) and prostate cancer ( 9.6% ) , respectively ; unlike brca2 , brca1 alterations were infrequent in prostate cancer . brca1 / 2 homozygous deletions were infrequent except in prostate cancer , where brca2 deletions were observed at a 2.6% frequency and accounted for 25% of brca1 / 2altered cases . 
brca1 / 2 mutations were distributed throughout the length of each gene , and most were truncating events ( appendix fig a2 )  . germline / somatic status for brca1 / 2 short variant mutations was predicted using validated computational methods.18 we also evaluated performance of germline / somatic predictions in this study . 
fraction ( % ) of ( b ) brca1 or ( c ) brca2 mutations predicted to be germline v somatic was determined for each cancer type . see the data supplement for detailed data . computational methods correctly identied 21 ( 91% ) as predicted germline variants . 
second , because cell - free dna ( cfdna ) sequencing can often distinguish germline from somatic variants , 19 we evaluated 52 brca1 / 2 germline / somatic predictions from tissue samples and evaluated patientmatched cfdna ngs results . 
as expected , the majority of mutations were predicted to be germline in brca1 / 2 - associated cancers ( brca1 , 58.1% ; brca2 , 56.8% ) , but predicted sbrca1 / 2 mutations comprised an appreciable proportion of brca1 / 2 mutations . 
 ( a ) fraction of brca1 / 2 - altered cases with biallelic or monoallelic alteration was determined . brca1 / 2 - altered cases were evaluated for class of alteration identied and classied as biallelic ( multiple brca1 or multiple brca2 alterations in the same sample , homozygous deletion , biallelic short variant mutation [ loss of heterozygosity of the wild - type allele ] ) , monoallelic ( heterozygous mutation ) , or unknown ( zygosity status could not be determined )  . 
a lower - bound estimate was established by assessing biallelic cases as a fraction of all brca1 / 2 - altered cases , including those with unknown biallelic / monoallelic status ( see data supplement )  . 
for cases where biallelic / monoallelic status could be determined , we estimated the fraction of brca1 / 2 - altered cases with biallelic alteration ( biallelic fraction )  . 
although biallelic fraction was lower in nonbrca1 / 2 - associated cancers ( p , .0001 , fishers exact test ) , biallelic alterations nonetheless comprised 43.6% of brca1 / 2 - altered cases : biallelic fraction was  . 
1% frequency in at least 13 cancer types . in prostate cancer , predicted gbrca1 / 2 and sbrca1 / 2 mutations were separately assessed for biallelic fraction ( fig 3 ; appendix fig a5 )  . 
for brca1 / 2associated cancers , both predicted gbrca1 / 2 ( 90.8% ) and sbrca1 / 2 ( 81.2% ) mutations were frequently biallelic , whereas in nonbrca1 / 2 - associated cancers , fewer predicted gbrca1 / 2 ( 46.4% ) and sbrca1 / 2 ( 25.4% ) mutations were biallelic . 
in ovarian and breast cancer , the majority of brca1 and brca2 mutations were biallelic , both for predicted germline and the majority of somatic mutations . brca2 ( gbrca2 , 87.6% ; sbrca2 , 75.0% ) mutations were biallelic , but brca1 ( gbrca1 , 40.0% ; sbrca1 , 22.2% ) mutations were less frequently biallelic . 
cases with 16% gloh were classied as gloh - high on the basis of the cutoff established in the ariel3 trial of rucaparib in ovarian cancer.10 biallelic brca1 / 2 alterations were associated with gloh - high across every cancer type examined . 
the fraction of cases that were gloh - high was signicantly increased for biallelic brca1 / 2 - altered compared with wild - type cases ( fig 4a , appendix fig a6a ) , whereas the fraction of monoallelic brca1 / 2 cases that were gloh - high was similar to wild type . 
signicant association of biallelic but not monoallelic alterations with gloh - high was observed both for brca1 and for brca2 when assessed individually ( figs 4b and 4c )  . 
in some cancer types , significant but modest elevation in tumor mutational burden ( tmb ) was observed for biallelic brca1 / 2 - mutated cases ( predicted germline or somatic ) versus wild type ; however , the association with tmb was not consistent across cancer types ( appendix fig a7 )  . 
monoallelic sbrca1 / 2 - mutated ( but not gbrca1 / 2 ) cases were commonly associated with elevated tmb versus wild type , and such mutations may be a consequence of increased mutation rate . although biallelic brca1 / 2 status was consistently associated with gloh - high , the magnitude was variable for each cancer type , with the greatest association observed in pancreatic ( or , 22.5 ) , biliary ( or , 21.5 ) , endometrial ( or , 17.2 ) , unknown primary ( or , 16.1 ) , and ovarian ( or , 14.9 ) cancer ( fig 4a )  . 
more than 75% of cases with biallelic brca1 / 2 alterations were gloh - high for ovarian , breast , pancreatic , unknown primary , and endometrial cancer , whereas fewer than half were gloh - high for prostate and colorectal cancer . 
25% of brca1 / 2 wildtype cases were gloh - high for ovarian , breast , lung , and gastric / esophageal cancer . the 16% gloh - high cutoff was clinically validated in ovarian cancer9 , 10 and requires optimization for other cancer types . 
consistent with the ndings using a gloh cutoff - based approach , gloh scores were higher in brca1 / 2 biallelic versus wild - type cases across all cancer types evaluated ; increased gloh score was also observed when biallelic brca1 and brca2 were evaluated independently . hrd signatures ( including gloh - high ) may identify additional brca1 / 2 wild - type tumors potentially suitable for parpi.9 , 10 , 20 overall , 19.3% of cases were gloh - high compared with 3.2% of cases with biallelic brca1 / 2 alteration , and for most cancer types , the frequency of gloh - high was greater than biallelic brca1 / 2 alterations ( data supplement ) ; distribution of gloh scores varied between cancer types . 
to inform rational cancer typespecic gloh - high cutoffs , we assessed the performance of different gloh - high thresholds to classify biallelic brca1 / 2 compared with wild - type cases . 
plotting sensitivity to detect cases with biallelic brca1 / 2 alteration and specicity ( percentage of brca1 / 2 wild - type cases negative for the glohhigh biomarker ) , we identied a cutoff that maximized the combined sensitivity and specicity score ( fig 6 )  . 
the identied cutoff for ovarian cancer was 15.1% , which was consistent with the 14% and 16% cutoffs identied in clinical trials.9 , 10 discussion the development of parpi for brca1 / 2 altered ovarian , breast , and pancreatic cancer1 , 5 is predicated on synthetic lethality interactions between brca1 / 2 loss of function and parpi / trapping . 
emerging clinical trial data suggest that brca1 / 2 alteration may also be predictive of parpi response in prostate cancer.1 , 11 - 13 although responses to parpi have been documented in other cancer types , 14 , 21 - 23 the relevance of brca1 / 2 alterations in nonbrca1 / 2associated cancers remains unclear.4 to understand the landscape and phenotypic consequence of brca1 / 2 alterations , we assessed our data set of 234 , 154 cancer specimens sequenced using a clinicalgrade cgp assay . 
brca1 / 2 alterations were frequent in brca1 / 2 - associated cancers but also observed in a signicant fraction ( 3% ) of nonbrca1 / 2 - associated cancers . 
predicted germline mutations comprised the majority of brca1 / 2 mutations in brca1 / 2 - associated cancers ; however , it is notable that 43% were predicted somatic ( of which 81% were biallelic ) given data that support sbrca1 / 2 alteration as a biomarker for parpi in ovarian and prostate cancer.1 , 11 - 13 brca1 / 2 alterations were assessed for biallelic versus monoallelic status to distinguish likely loss of function from biologically neutral alterations . 
of note , although monoallelic alteration in brca1 / 2 mutation carriers may have a subtle haploinsufcient phenotype , 24 it does not lead to severe hrd or sensitivity to platinum - based chemotherapy.25 consistent with the established role of brca1 / 2 in the pathogenesis of brca1 / 2 - associated cancers , 90% of brca1 / 2 - altered cases were biallelic with high biallelic fraction both for predicted gbrca1 / 2 and sbrca1 / 2 . 
differences in brca1 and brca2 biallelic fraction were observed : brca2 was more frequently biallelic in prostate and small - cell lung cancer , and brca1 was more frequently biallelic in endometrial cancer , esophageal scc , colorectal cancer , and melanoma . 
the frequency of ( a ) brca1 / 2 , ( b ) brca1 , or ( c ) brca2 biallelic , monoallelic , and wild - type cases that were gloh - high was compared across cancer types . 
50 biallelic brca1 / 2 - altered samples were assessed individually , and all other cancer types were grouped together and analyzed as a single group ( other )  . 
association between brca1 / 2 biallelic alteration and genome - wide loss of heterozygosity ( gloh ) score as a continuous variable . gloh score was assessed in ( a ) brca1 / 2 , ( b ) brca1 , and ( c ) brca2 biallelic v wild - type cases across cancer types . 
boxes span the rst and third quartiles , and the median is denoted by the horizontal line in the box ; whiskers indicate maximum and minimum values within 1.5 the interquartile range ; black dots indicate outlier events . 
 100 sokol et al ovarian prostate breast endometrial pancreatic bladder / uc gastric / esophageal adenocarcinoma biliary tract colorectal nsclc % gloh sensitivity specificity % gloh % gloh fig 6 . 
 pan - cancer analysis of biallelic brca1 / 2 differences suggest that brca1 and brca2 may have tissue specicities outside breast and ovarian different cancer . to determine whether brca1 / 2 alterations lead to hrd in both brca1 / 2 - associated and nonbrca1 / 2 - associated cancers , we evaluated gloh in brca1 / 2 - altered versus type evaluated , wild - type cases . 
monoallelic alterations may be found in sporadic cancers from gbrca1 / 2 carriers or as somatic passenger mutations . distinguishing biallelic from monoallelic status may be an important consideration for rening brca1 / 2 alteration as a predictive biomarker for parpi . 
in brca1 / 2 - associated cancers , brca1 / 2 alteration status alone has proven sufcient as a predictive biomarker , 1 likely explained by the majority of brca1 / 2 alterations in this context being biallelic . 
nevertheless , parpi trials in prostate cancer have incorporated brca1 / 2 biallelic status into biomarker development , 13 and our nding of signicantly lower biallelic fraction for brca1 ( v brca2 ) in prostate cancer suggests that integrating biallelic status could rene predictive biomarkers in this setting . the lack of parpi clinical activity in nonbrca1 / 2 - associated cancers reported previously could be explained by grouped analysis of biallelic and monoallelic brca1 / 2 alterations.4 because of lower rates of biallelic alteration in nonbrca1 / 2 - associated cancers , parpi clinical trials will likely require patient selection strategies that incorporate methods to discriminate biallelic from monoallelic brca1 / 2 alterations . our ndings are consistent with a recent study that demonstrated an elevated hrd composite score in biallelic but not heterozygous brca1 / 2 cases relative to wild type in an aggregate set of nonbrca1 / 2 - associated cancers.4 a strength of the current study was that the large data set size enabled analysis of biallelic brca1 / 2 separately from monoallelic alterations , independent assessment of brca1 and brca2 , and evaluation of nonbrca1 / 2 - associated cancers as individual cancer types rather than in aggregate , which may have enabled identication of associations between biallelic brca1 / 2 alterations and an hrd signature across cancer types not previously described.4 if brca1 / 2 biallelic alterations functionally result in hrd , they should represent a targetable vulnerability to parpi and platinum - based chemotherapy , irrespective of whether they are drivers of disease pathogenesis or bystander passenger alterations.26 our data demonstrate that biallelic brca1 / 2 alteration in nonbrca1 / 2 - associated cancers are associated with the gloh - high signature of hrd and , therefore , warrant investigation in parpi trials . 
although biallelic brca1 / 2 alterations occur at low prevalence in nonbrca1 / 2 - associated cancers , basket trials that led to the tumor - agnostic approvals of ntrk inhibitors for ntrk fusion - positive tumors and pembrolizumab for microsatellite instabilityhigh tumors27 demonstrate feasibility of therapeutic development for rare pan - cancer biomarkers . 
clinical trials such as the tapur study ( clinicaltrials.gov identier : nct02693535 ) that treatment arm for brca1 / 2 - altered includes a parpi cancers will inform parpi development in nonbrca1 / 2associated cancers , and analyses may benet from consideration of monoallelic versus biallelic status . although gloh - high is associated with clinical benet from rucaparib in ovarian cancer , 9 , 10 understanding of the gloh biomarker in other cancer types is required . 
evaluation of sensitivity and specicity of varying gloh thresholds to distinguish biallelic brca1 / 2 - altered and wild - type cases may inform development of disease - specic gloh - high cutoffs . 
in future studies , analysis of brca1 / 2 wild - type , gloh - high cases may be a discovery tool for characterizing brca1 / 2 vuss and for prioritizing candidate non - brca1 / 2 hr pathway gene biomarkers.9 although detection of gloh - high in brca1 / 2 wild - type cases potentially expands the patient population addressable by parpi , the utility of gloh - high requires validation in prospective trials for nonovarian cancers . limitations of this study should be acknowledged . 
first , we focus on gloh as a biomarker for hrd ; other hrd markers were not evaluated , including telomeric allelic imbalance ( tai ) , large - scale transition ( lst ) , mychoice hrd ( combination loh / tai / lst score ; myriad genetics , salt lake city , ut ) , signature 3 , and hrdetect.7 , 8 , 28 - 33 in parpi trials , hrd biomarkers have focused on approaches that are compatible with targeted ngs assays ( gloh , mychoice hrd ) , which are used in routine clinical practice.1 , 9 , 10 in contrast , signature 3 and hrdetect signatures have been evaluated in the research setting using whole - exome or whole - genome sequencing ; novel methods may enable future clinical assessment of signature 3 with targeted assays.34 second , germline / somatic status predictions using tumor - only sequencing and computational methods are less denitive compared with matched normal sequencing used in other studies.4 , 18 third , using brca1 / 2 biallelic versus wild - type status to rene gloh - high cutoffs is confounded by some brca1 / 2 wild - type gloh - high cases that are hrd because of brca1 / 2 alteration independent mechanisms , such as brca1 / 2 methylation or alteration in other hr genes . 
 sokol et al in the future ; however , we focused on brca1 / 2 because other hr pathway genes have not been consistently predictive of clinical response to parpi , 11 - 13 , 36 and robust clinical evidence supporting predictive biomarker genes beyond brca1 / 2 is lacking . 
finally , clinical outcomes data that associate parpi response with biallelic brca1 / 2 alteration and gloh were not available and require evaluation in clinical trials . we demonstrate that biallelic brca1 / 2 alterations are associated with elevated gloh across all cancer types evaluated and may therefore represent a therapeutic vulnerability targetable by parpi . 
ali , shridar ganesan , jon h . chung financial support : brian alexander administrative support : brian alexander provision of study material or patients : hossein khiabanian , primo n . 
gregg consulting or advisory role : astrazeneca , bristol - myers squibb , roche , foundation medicine , luminex speakers bureau : astrazeneca , foundation medicine , bristol - myers squibb primo n . 
 pan - cancer analysis of biallelic brca1 / 2 research funding : merck sharp & dohme ( inst ) , astrazeneca ( inst ) , ipsen ( inst ) travel , accommodations , expenses : roche , merck sharp & dohme , astrazeneca , janssen pharmaceuticals , rainier therapeutics other relationship : bayer ag ( i ) vincent a . 
miller employment : foundation medicine leadership : foundation medicine , revolution medicines stock and other ownership interests : foundation medicine , mirati therapeutics , revolution medicines patents , royalties , other intellectual property : periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center brian alexander employment : foundation medicine leadership : foundation medicine stock and other ownership interests : roche research funding : eli lilly ( inst ) , puma ( inst ) , celgene ( inst ) open payments link : 854258 / summary siraj m . 
ali employment : foundation medicine , eqrx , eqrx ( i ) leadership : incysus stock and other ownership interests : exelixis , blueprint medicines , agios , genocea biosciences consulting or advisory role : revolution medicines , azitra ( i ) , princepx tx ( i ) patents , royalties , other intellectual property : patents through foundation medicine , patents through seres health on microbiome stuff in nonneoplastic disease ( i ) shridar ganesan employment : merck ( i ) stock and other ownership interests : ibris , inspirata , merck ( i ) consulting or advisory role : inspirata , novartis , roche , foghorn therapeutics , foundation medicine , merck sharp & dohme patents , royalties , other intellectual property : two patents for digital imaging that may be licensed to ibris and inspirata travel , accommodations , expenses : inspirata other relationship : national cancer institute / national institutes of health jon h . 
chung employment : foundation medicine stock and other ownership interests : foundation medicine no other potential conicts of interest were reported . references 105 : 812 - 822 , 2013 1 . 
nature 571 : 576 - 579 , 2019 [ erratum : nature 577 : e1 , 2020 ] golan t , hammel p , reni m , et al : maintenance olaparib for germline brca - mutated metastatic pancreatic cancer . 
nature 534 : 47 - 54 , 2016 [ erratum : nature 566 : e1 , 2019 ] davies h , glodzik d , morganella s , et al : hrdetect is a predictor of brca1 and brca2 deciency based on mutational signatures . 
j natl cancer inst 110 : 704 - 713 , 2018 swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
coleman rl , oza am , lorusso d , et al : rucaparib maintenance treatment for recurrent ovarian carcinoma after response to platinum therapy ( ariel3 ) : a randomised , double - blind , placebo - controlled , phase 3 trial . 
abida w , bryce ah , vogelzang nj , et al : preliminary results from triton2 : a phase ii study of rucaparib in patients ( pts ) with metastatic castration - resistant prostate cancer ( mcrpc ) associated with homologous recombination repair ( hrr ) gene alterations . 
smith mr , sandhu sk , kelly wk , et al : phase ii study of niraparib in patients with metastatic castration - resistant prostate cancer ( mcrpc ) and biallelic dnarepair gene defects ( drd ) : preliminary results of galahad . 
sandhu sk , schelman wr , wilding g , et al : the poly ( adp - ribose ) polymerase inhibitor niraparib ( mk4827 ) in brca mutation carriers and patients with sporadic cancer : a phase 1 dose - escalation trial . 
slavin tp , banks kc , chudova d , et al : identication of incidental germline mutations in patients with advanced solid tumors who underwent cell - free circulating tumor dna sequencing . 
moore kn , secord aa , geller ma , et al : niraparib monotherapy for late - line treatment of ovarian cancer ( quadra ) : a multicentre , open - label , single - arm , 21 . 
necchi a , raggi d , giannatempo p , et al : exceptional response to olaparib in brca2 - altered urothelial carcinoma after pd - l1 inhibitor and chemotherapy 22 . 
hierro c , matos i , martin - liberal j , et al : agnostic - histology approval of new drugs in oncology : are we already there ? clin cancer res 25 : 3210 - 3219 , 2019 28 . 
timms km , abkevich v , hughes e , et al : association of brca1 / 2 defects with genomic scores predictive of dna damage repair deciency among breast cancer 31 . 
telli ml , timms km , reid j , et al : homologous recombination deciency ( hrd ) score predicts response to platinum - containing neoadjuvant chemotherapy in patients with triple - negative breast cancer . 
marshall ch , sokolova ao , mcnatty al , et al : differential response to olaparib treatment among men with metastatic castration - resistant prostate cancer harboring brca1 or brca2 versus atm mutations . 
 pan - cancer analysis of biallelic brca1 / 2 appendix supplementary methods approval for this study , including a waiver of informed consent and health insurance portability and accountability act waiver of authorization , was obtained from the western institutional review board ( irb ; protocol #20152817 )  . 
cgp results included in this study were from tumor tissue specimens ( n = 234 , 154 ) submitted as part of routine clinical care ( december 2013 - march 2019 ) ; for patient characteristics , see the data supplement . 
in validation testing of 480 tumor - only sequencing calls against matched normal specimens , accuracy was 95% for somatic and 99% for germline calls ( sun jx , et al : plos comput biol 14 : e1005965 , 2018 ) ; in assessment of zygosity calls , signicant enrichment in mutations with loss of heterozygosity ( loh ) was observed for tumor suppressor genes ( sun jx , et al : plos comput biol 14 : e1005965 , 2018 )  . 
to evaluate performance of germline / somatic computational predictions in this series , we compared predictions against a subset of cases with available results from patient - matched germline testing or cell - free dna ( cfdna ) ngs that were performed as previously described ( clark ta , et al : j mol diagnostics 20 : 686 - 702 , 2018 ; khiabanian h , et al : jco precis oncol 2 : 1 - 15 , 2018 ) ; brca1 / 2 genetic testing on a subset of patients at the rutgers cancer institute of new jersey were analyzed under the auspices of an irb - approved protocol . 
germline - like af was dened as mutations observed in cfdna at 40% - 60% af ( except for cfdna samples with high circulating tumor dna fraction [  . 20% ] that were excluded from the analysis as ambiguous )  . 
brca1 / 2 alterations were categorized as biallelic , monoallelic , or unknown . biallelic alterations were mutations with loh of the wild - type allele , as determined by zygosity status ( sun jx , et al : plos comput biol 14 : e1005965 , 2018 ) ; homozygous deletion ; or 2 brca1 or 2 brca2 alterations in a sample . 
percent genome - wide loh ( gloh ) was calculated as a signature of hrd as previously described ( coleman rl , et al : lancet 390 : 1949 - 1961 , 2017 ; swisher em , et al : lancet oncol 18 : 75 - 87 , 2017 )  . 
this prole was segmented and interpreted using afs of sequenced snps to estimate copy number ( ci ) and minor allele count ( mi ) at each segment ( i )  . 
two types of loh segments were excluded from the calculation of percent gloh : loh segments that spanned 90% of a whole chromosome or chromosome arm because these loh events usually arise through non - hrd mechanisms ( eg , mitotic nondisjunction ) and regions in which loh inference was ambiguous . 
for each tumor , the percent gloh was computed as 100 the total length of nonexcluded loh regions ( xi ) divided by the total length of nonexcluded regions of the genome . 
 sokol et al brca1 / 2 short variant homozygous deletion rearrangement multiple brca1 brca2 short variant homozygous deletion rearrangement multiple brca1 brca2 breast cancer ovarian cancer breast cancer ovarian cancer fig a1 . 
growth factor receptor 2 ( her2 ) status was determined on the basis of the presence or absence of a copy number amplication ; triple - negative breast cancer ( tnbc ) was dened as er - negative , her2 - negative samples . 
 somatic af germline af cfdna germline af cfdna somatic af 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 brca1 / 2 variant allele frequency ( % ) predicted germline ( n = 22 ) predicted somatic ( n = 30 ) cfdna gbrca sbrca predicted germline predicted somatic sokol et al predicted germline predicted somatic breast cancer ovarian cancer fig a3 . 
 ( a and b ) comparison of tissue - based germline / somatic predictions to brca1 / 2 allele frequencies ( afs ) in patient - matched cell - free dna ( cfdna )  . 
mutations with germline af were dened as those identied in cfdna at 40% - 60% af , except for cases with high circulating tumor dna fraction ( 20% ) that were excluded from the analysis as ambiguous af . 
 ( c - e ) predicted germline / somatic status calls were made for each brca1 or brca2 short variant mutation . for mutations yielding a successful call , frequency of predicted germline v somatic mutation was determined for ( c ) brca1 / 2 across cancer types , ( d ) brca1 / 2 ( grouped and individually ) overall ( n = 5 , 845 ) for brca1 / 2 - associated cancers ( breast , ovarian , pancreatic , prostate ; n = 3 , 061 ) and nonbrca1 / 2 - associated cancers ( n = 2 , 784 ) , and ( e ) brca1 / 2 for the subset of ovarian and breast cancer cases where molecular / histologic subtype information was available . 
relative fraction of brca1 / 2 - altered cases with biallelic or monoallelic alteration was determined for ( a ) brca1 across cancer types and ( b ) brca2 across cancer types . 
 ( c ) brca1 / 2 ; ( d ) brca1 ; ( e ) brca2 for overall , brca1 / 2 - associated cancers , and nonbrca1 / 2 - associated cancers ; and ( f ) brca1 / 2 for the subset of ovarian and breast cancer cases where molecular / histologic subtype information was available . 
 ( a ) brca1 / 2 mutations ( grouped ) with a germline ( gbrca1 / 2 ) or somatic ( sbrca1 / 2 ) prediction were evaluated for biallelic / monoallelic status for all cancers , brca1 / 2 - associated cancers ( as a group and as individual cancer types ) , and nonbrca1 / 2 - associated cancers ( see data supplement )  . 
cases with somatic brca1 / 2 ( sbrca1 / 2 ) or germline brca1 / 2 ( gbrca1 / 2 ) biallelic mutation , monoallelic mutation , or wild - type ( wt ) brca1 / 2 were plotted against tumor mutational burden ( tmb in mutations / mb ; log10 score )  . 
box and whisker plot where the box spans the rst and third quartiles , the median is denoted by the horizontal line in the box , and whiskers indicate maximum and minimum values within 1.5 the interquartile range ( see data supplement )  . 
 ( a ) the frequency of brca1 / 2 biallelic , monoallelic , and wild - type cases that were high genome - wide loss of heterozygosity ( gloh - high ) was compared in the subset of ovarian and breast cancer cases where molecular / histologic subtype information was available ( see data supplement )  . 
 ( b ) frequency of predicted germline brca1 / 2 ( gbrca1 / 2 ) biallelic , monoallelic , and wild - type cases that were gloh - high ( see data supplement )  . 
 ( c ) frequency of predicted somatic brca1 / 2 ( sbrca1 / 2 ) biallelic , monoallelic , and wild - type cases that were gloh - high ( see data supplement )  . 
our real - time tumor sequencing program , which makes precision treatment decisions for patients with cancer , produces matched germline information , providing a unique opportunity to efficiently implement pharmacogenetics and benefit patients . methods the germline genetic database from the michigan oncology sequencing ( mi - oncoseq ) program was searched for 21 clinically actionable polymorphisms in five cancer - relevant genes : tpmt , dpyd , cyp2c19 , cyp3a5 , and ugt1a1 . 
the medical records of mi - oncoseq patients with actionable phenotypes were searched for receipt of relevant drugs and to determine whether having genetic information at the time of treatment would have led to a treatment recommendation . results all nine variants in tpmt , dpyd , and cyp2c19 that were detected in mi - oncoseq were confirmed by external genotyping . 
on the basis of retrospective assessment of 115 adult and pediatric patient records , 4.3% ( n = 5 ) had a potentially clinically actionable phenotype for tpmt , dpyd , or cyp2c19 and received a relevant medication . 
 after accounting for differences in adult and pediatric recommendations , three of these patients could have received a treatment recommendation at the time of prescribing . conclusion germline genotype determinations for tpmt , dpyd , and cyp2c19 can be used to make evidence - based treatment recommendations in mi - oncoseq patients . 
 although the proportion of patients for whom recommendations can be made is small , this added value to mi - oncoseq and patient care comes at no additional genotyping cost . 
2018 by american society of clinical oncology introduction the genomes of patients ( germline ) and their tumors ( somatic ) each provide useful information to guide precision medicine in oncology . 
vali dated pharmacogenetic associations exist for several germline polymorphisms with com monly used cancer treatments , including thiopu rines ( tpmt ) , 1 fluorouracil ( fu ) / capecitabine ( dpyd ) , 2 irinotecan ( ugt1a1 ) , 3 and tacrolimus ( cyp3a5 ) .4 additionally , there are several known associations with supportive care agents com monly used in patients during treatment , such fungal prophylaxis with voriconazole ( cyp2c19 ) 5 and antiemetic treatment with ondansetron ( cyp2d6 ) .6 despite their established clinical validity , few of these associations have been implemented into clinical practice . 
st jude childrens research hospital has led the way , implementing pre emptive pharmacogenetic testing to guide per sonalized treatment of several genedrug pairs.7 the experiences of early adopters has identified formidable challenges to implementing phar macogenetics into clinical practice , 8 , 9 which require substantial investment and expertise.10 , 11 daniel l . 
mody arul chinnaiyan author affiliations and support information ( if applicable ) appear at the end of this article . the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health . corresponding author : daniel l . 
although there is debate about the necessity and feasibility of demon strating clinical utility for pharmacogenetic implementation , 12 , 13 the lack of uptake indicates that health systems are not willing to incur the costs of pharmacogenetic implementation for unproven clinical benefit . 
however , available germline genetic information should be con sidered when making treatment decisions.14 in anticipation of a time when genetic informa tion is available for many patients , perhaps as a result of the proliferation of directtoconsumer genotyping , 15 the pharmacogenetic community has developed evidencebased pharmacogenetic treatment guidelines.3 , 16 in oncology , there has been a tremendous expan sion in the availability of genetic information as a result of the proliferation of tumor sequenc ing programs that use somatic genetic informa tion to personalize selection of targeted cancer treatments.17 although some programs analyze only the somatic genome , others have found that matched germline analysis improves quality control18 and enables simultaneous assessment of familial predisposition to cancer.19 this cre ates a unique situation in which germline genetic information for clinically relevant pharmaco genes is freely available.20 however , the oppor tunity to use these data in clinical practice has not yet been capitalized upon . the michigan oncology sequencing ( mi oncoseq ) program at the university of michi gan comprehensive cancer center ( umccc ) performs targeted sequencing of somatic and matched germline dna , in addition to somatic wholetranscriptome analysis . 
21 the targeted sequencing panel includes approximately 100 pharmacogenes that could be used to provide evidencebased pharmacogenetic treatment rec ommendations , if the accuracy of the germline genetic determinations are verified . 
detailed study information including inclusion and exclusion criteria , data analysis and processing , and return of results has been pre viously published.2123 briefly , patients treated at umccc with refractory tumors are invited to participate in a research protocol in which they provide tumor and blood samples for matched genetic sequencing , among other somatic anal yses . 
 since the initiation of mioncoseq , the dna sequencing platform has transitioned from wholeexome sequencing to a targeted exon sequencing panel , and sequencing methods have been previously described in detail.21 , 23 this tar geted panel sequences primarily exonic regions of approximately 1 , 700 genes , including approx imately 100 pharmacogenes selected based on curation within pharmgkb ( appendix table a1 )  . 
these five genes were selected based on their relevance to cancer treatment or sup portive care , likelihood of clinician interest in prospective implementation of the genedrug pairs , and existence of evidencebased treatment guidelines from the clinical pharmacogenet ics implementation consortium ( cpic ) 16 or the dutch pharmacogenetics working group ( dpwg ) .3 the polymorphisms in each gene were selected based on the validated variant lists within each guideline . 
note that ugt1a1 * 28 ( rs8175347 ) and * 80 ( rs887829 ) were both included to represent the ugt1a1 * 28 geno type , because they are highly linked , and * 80 is often substituted for * 28 as a result of its relative ease of genotyping.24 the mioncoseq germ line genetic database was screened to identify all variant calls at any of these polymorphisms for all patients sequenced on onco1700_v4 , and variant calls were compiled in a single data set for further analysis . confirmatory genotyping genotype determinations from onco1700 were manually screened for potentially unreliable calls by assessing standard sequencing quality con trol parameters , including read depth . 
in the second stage , polymor phisms with > five but < 30 occurrences were selected , by prioritizing patients who also car ried a variant at one of the common snp posi tions . 
finally , as many samples as necessary were selected that carried these common variants so that each variant was represented in five samples . selected samples were sent for college of american pathologistsaccredited , clinical lab oratory improvement amendmentsapproved genotyping at genelex laboratories ( seat tle , wa )  . 
genotypes were obtained using a laboratorydeveloped , multiplex polymerase chain reactionbased test followed by single base primer extension for variant detection by mass spectrometry ( massarray analyzer 4 system ; agena bioscience , san diego , ca )  . 
percent concordance for each snp was calculated as the number of concordant genotypes divided by the total number of samples compared . retrospective analysis of clinically actionable phenotypes patient genotypes were translated to activity phenotypes based on the appropriate cpic26 or dpwg3 guidelines ( appendix table a2 )  . 
genotype data for ugt1a1 * 28 ( * 80 ) and cyp3a5 * 3 was only available for the 25 samples sent for confirma tory genotyping ; therefore , these patients and genotypes were included in assessments of clin ical usefulness . 
medical record screening was performed using an automated screening tool ( emerse27 ) that searches the text of notes in michart , the version of epic used at mich igan medicine . 
text used to screen the medical records included all generic and brand drug names and commonly used acronyms ( eg , fu for fluourouracil )  . a pharmacy student , in consultation with a phar macist with oncology pharmacogenomic expertise , manually reviewed the electronic medical record for each patient who had an actionable pheno type and received the relevant drug to determine whether a treatment modification would have been recommended had this genetic information been available to the clinician before treatment initia tion . 
these patients represent the diversity of the mioncoseq cohort , including adult ( n = 82 ; 71% ) and pediatric patients ( n = 33 ; 29% ) who were evenly divided between male ( n = 58 ; 50% ) and female sex ( n = 57 ; 50% ) , were primarily white ( n = 97 ; 84% ) , and had a variety of solid ( n = 80 ; 70% ) and liquid tumor types ( n = 35 ; 30% )  . no genotype determinations for cyp3a5 * 3 , ugt1a1 * 28 , or ugt1a1 * 80 were made by onco1700 because of low read depth ( cyp3a5 * 3 and ugt1a1 * 80 ) or sequence repeat misalign ment ( ugt1a1 * 28 ) ; therefore , these polymor phisms were excluded from genotype concordance analyses . 
across the 18 remaining snps , a total of 139 variant calls were made in these 115 samples , and nine unique snps were detected in at least one patient ( table 1 )  . 
the frequency of actionable phenotypes was highest for cyp2c19 ( 43.5% ) , followed by cyp3a5 ( 20% ) , ugt1a1 ( 20% ) , and tpmt ( 13% ) , and lowest for dpyd ( 3.5% ) , as expected . the electronic medical record for each patient carrying an actionable phenotype was screened for relevant drugs . 
one patient with cyp2c19 rapid metabolizer phenotype never received voriconazole ; two patients with cyp2c19 rapid metabolizer phenotype received voriconazole treatment , but they were pediatric patients , so no dose adjustment is recommended per cpic guidelines5 ; and one patient with ugt1a1 poor metabolizer phenotype treated with irinotecan received a standard pediatric dose ( 49 mg / m2 ) , which is below the recommended threshold for dose adjustment based on dpwg guidelines.3 three patients with actionable phenotypes who received the relevant drug , in whom a treatment recommendation could have been made , were identified . 
one patients dose was held after 5 days because of neutro penia ( absolute neutrophil count [ anc ] , 200 ) , and the second patients dose was reduced to 70% of the standard dose after 7 days because of neutropenia ( anc , 500 )  . 
 are many challenges to pharmacogenetic imple mentation , 10 but perhaps the primary challenge is the current lack of evidence of clinical util ity to justify the upfront cost of establishing a pharmacogenetic service.31 , 32 in anticipation of a future in which genomic information is more readily available , cpic and other groups have published evidencebased treatment recommen dations for patients with known genotypes.3 , 16 there is a unique opportunity to integrate evidencebased pharmacogenetic treatment into tumor sequencing programs that analyze germline genetic information , 20 such as the mioncoseq program at umccc.21 the objective of this analysis was to confirm the accuracy of germ line genotype determinations produced during mioncoseq sequencing and then to retrospec tively assess the clinical usefulness of integrating pharmacogenetics into mioncoseq . attempted confirmatory genotyping of 21 clin ically actionable snps in five cancerrelevant pharmacogenes confirmed genotyping accu racy for common and uncommon variants in three genes ( tpmt , dpyd , and cyp2c19 ) but revealed an inability to genotype common vari ants in cyp3a5 and ugt1a1 . 
this finding is easily explained by the targeted exonic coverage of onco1700 and the location of these polymor phisms at a splice site ( cyyp3a5 * 3 , rs776746 ) and in the promoter region ( ugt1a1 * 28 , rs8175347 )  . 
other variants that were not detected are extremely rare , and several have not been found in white patients . prior studies have estimated that > 90% of the population has an actionable phenotype of at least one candidate gene33 , 34 ; however , this is only relevant if the patient is treated with the drug of interest and the guidelines apply to the patient . 
 in our cohort , 4% ( five of 115 ) of patients had a potentially actionable phenotype in cyp2c19 , tpmt , or dpyd and received the relevant drug , and in 2.6% ( three of 115 ) of patients , a guideline based treatment recommendation could have been made . manual review of these three patients identified several interesting findings . 
despite tpmt gen otype information available at the time of treat ment , two patients with heterozygous genotypes initiated treatment at standard mercaptopurine doses , per the childrens oncology group protocols on which they were enrolled . 
cpic recommends a preemptive 30% to 70% dose decrease with enhanced monitoring and titration based on tolerability.1 although germline tpmt determination from mioncoseq would not have changed these patients treatment in any way , it would have prevented external genetic testing , resulting in cost savings to the health systethe third patient carried a dpyd geno type that confers risk of severe toxicity with fu , which was not known at the time of treatment . 
 cpic guidelines recommend a preemptive dose reduction of 50% with monitoring and titra tion.2 although it is impossible to attribute tox icity to any single factor , it is interesting that two patients experienced toxicity requiring a reactive dose reduction , which may have been prevented if care had been based on cpic guidelines . the 2% to 4% absolute increase in the propor tion of patients with clinically actionable findings from germline pharmacogenetics represents a minimal estimate , because the emerse screen ing tool does not automatically screen prescrib ing data , and there is some chance that a patient with an actionable phenotype received a relevant medication that was never mentioned in a clini cal note . 
regardless , this represents a meaning ful increase in the clinically actionable findings from our tumor sequencing program.35 in addi tion to its usefulness for quality control , 18 , 36 matched germline analysis identifies validated cancer predisposition variants in an estimated 15% of patients.19 several tumor sequencing programs , including mioncoseq , 21 use their matched germline dna for this purpose.37 , 38 our results represent a critical first step toward integration of germline pharmacogenetics into mioncoseq . although several programs have reported that pharmacogenetics is considered in their deci sion making , 39 , 40 we are not aware of any detailed reports of the integration of pharmacogenetics into these programs . 
 used to inform irinotecan treatment decisions3 or how the incidental finding of gilberts syn drome would be conveyed to the patient.42 , 43 generalization of our estimate of the proportion of patients who could benefit from pharmacog enetic implementation in other tumor sequenc ing programs is challenging for several reasons . 
 first , there are differences in the frequencies of clinically actionable alleles or phenotypes among racial cohorts.44 additionally , institutional differ ences in the distribution of tumors that are treated and sent for sequencing could dramatically affect this estimate . 
 pharmacogenetic integration would be most ben eficial in programs that sequence tumors early in treatment , particularly at institutions that treat many pediatric patients with all . the proportion of patients who would benefit from pharmacogenetic implementation could be increased in several ways . 
mody , arul chinnaiyan substantial upfront investment to build and maintain clinical decision support within the electronic health record , hire or train individu als with pharmacogenetic expertise , and provide clinician and patient education . 
pharmacoge netic implementation within tumor sequencing programs that analyze matched germline dna is particularly efficient because there is no geno typing cost and the bioinformatic workflow to detect actionable germline phenotypes can be integrated into the existing infrastructure . 
additional work is necessary to develop infrastructure to embed active clini cal decision support into the electronic health record so that actionable phenotypes can be stored and used indefinitely.7 , 11 in conclusion , onco1700 produces reliable germline pharmacogenetic information for three clinically relevant pharmacogenes ( tpmt , dpyd , and cyp2c19 )  . 
elliott ls , henderson jc , neradilek mb , et al : clinical impact of pharmacogenetic profiling with a clinical decision support tool in polypharmacy home health patients : a prospective pilot randomized controlled trial . 
caudle ke , dunnenberger hm , freimuth rr , et al : standardizing terms for clinical pharmacogenetic test results : consensus terms from the clinical pharmacogenetics implementation consortium ( cpic )  . 
hanauer da , mei q , law j , et al : supporting information retrieval from electronic health records : a report of university of michigans nineyear experience in developing and using the electronic medical record search engine ( emerse )  . 
luzum ja , pakyz re , elsey ar , et al : the pharmacogenomics research network translational pharmacogenetics program : outcomes and metrics of pharmacogenetic implementations across diverse healthcare systems . 
ji y , skierka jm , blommel jh , et al : preemptive pharmacogenomic testing for precision medicine : a comprehensive analysis of five actionable pharmacogenomic genes using nextgeneration dna sequencing and a customized cyp2d6 genotyping cascade . 
stockley tl , oza am , berman hk , et al : molecular profiling of advanced solid tumors and patient outcomes with genotypematched clinical trials : the princess margaret impact / compact trial . 
crews kr , gaedigk a , dunnenberger hm , et al : clinical pharmacogenetics implementation consortium ( cpic ) guidelines for codeine therapy in the context of cytochrome p450 2d6 ( cyp2d6 ) genotype . 
hicks jk , bishop jr , sangkuhl k , et al : clinical pharmacogenetics implementation consortium ( cpic ) guideline for cyp2d6 and cyp2c19 genotypes and dosing of selective serotonin reuptake inhibitors . 
 retrospective survival analysis of patients with resected pancreatic ductal adenocarcinoma and a germline brca or palb2 mutation shun yu , md1 , 2 ; parul agarwal , md1 ; ronac mamtani , md1 , 2 ; heather symecko , mph2 ; kelsey spielman , ms2 ; mark ohara , md1 , 2 ; peter j . 
reiss , md1 , 2 purpose germline mutations in the homologous recombination genes brca1 , brca2 , and palb2 confer an increased risk for pancreatic ductal adenocarcinoma ( pdac )  . 
2019 by american society of clinical oncology introduction ( pdac ) adenocarcinoma pancreatic ductal a highly lethal cancer with a 5 - year overall survival rate of 9% in unselected patients , despite multimodality treatment that includes surgery , chemotherapy , and radiation.1 for patients who have undergone curative intent surgery for early - stage the perioperative therapeutic landscape pdac , is shifting ; several recent studies demonstrated improved overall survival ( os ) using specic combinations of chemotherapy and radiation in the neoadjuvant and adjuvant settings.2 - 4 however , treatment of resected pancreatic cancer has yet to be tailored to subsets of patients according to the underlying mechanism of tumor development . 
pdac that develops in the setting of a germline mutation in brca1 , brca2 , or palb2 , for example , may represent a biologically unique group that would benet from tailored perioperative management . tumors that develop in the setting of pathogenic germline mutations in the dna - repair genes brca1 , brca2 , or palb2 are well understood to have heightened sensitivity to therapies that induce doublestranded dna breaks.5 - 7 as a result of ineffective homologous recombination , the tumor cells are unable to repair the dna damage caused by such treatments , which include platinum chemotherapies , 8 and cell death results . we and others have shown with retrospective data that patients with advanced pdac and a germline mutation in brca1 , brca2 , or palb2 have improved os when exposed to platinum - based therapies , such as oxaliplatin and cisplatin.9 , 10 most recently , yurgelun et al11 published an analysis that included seven germline brca mutation carriers who had undergone resection for pdac . 
 yu et al context key objective to examine whether platinum - based chemotherapy improved outcomes for patients with surgically resected pancreatic ductal adenocarcinoma and pathogenic germline mutations in brca1 , brca2 , or palb2 . knowledge generated platinum - based chemotherapy may be associated with increased survival for patients with early - stage pancreatic ductal adenocarcinoma who have pathogenic germline brca1 , brca2 , or palb2 mutations . 
this was especially true for patients who received platinum therapy in the perioperative setting . relevance these results suggest that platinum - based perioperative chemotherapy may be preferable for patients with pancreatic cancer who have germline mutations in brca1 , brca2 , or palb2 . 
therefore , awareness of such a mutation in the early - stage disease setting may be of value . was noted that this group of patients had signicantly longer os than noncarriers overall , and this difference seemed to be enhanced when oxaliplatin therapy was delivered in the recurrent disease setting.11 collectively , these ndings are consistent with literature about other brca mutation associated cancers , such as ovarian and breast cancer.12 - 16 data to describe the effect of perioperative platinum on early - stage pdac caused by a mutation in brca or palb2 are sparse . 
patients were identied one of two ways : enrollment in the university of pennsylvania basser center for brca registry or identication via the penn medicine patient electronic medical record ( emr )  . 
the emr was mined using pennseek ( university of pennsylvania , philadelphia , pa ) , which allows for targeted keyword searches of unstructured or semistructured medical documents . notably , patients who were in the mutation - positive group were identied in both the basser registry and the emr , whereas those in the mutation - negative group were identied solely from the emr . 
data were collected and extracted using the emr , paper records , and the social security death index . patients diagnosed with nonmetastatic pdac who had resection between undergone curative intent surgical january 1 , 1995 , and march 31 , 2018 , were included . those in the mutation - positive group were dened as patients with a known pathogenic germline mutation in brca1 , brca2 , or palb2 . 
collected data included demographics ; clinical history ; personal history of prior malignancies ; family history of cancers ; pathology results at time of resection ; germline and somatic sequencing results ; and treatments related to pdac , including surgical interventions , radiation , and systemic chemotherapy . 
each patient in the mutation - positive group was matched with two patients in the mutation - negative group by stage at diagnosis , age at diagnosis , year of diagnosis , and sex . 
the institutional review board at the university of pennsylvania approved this study . os was dened as the time from the date of diagnosis of pdac to the date of death for any cause . 
the os for patients exposed to platinumbased treatments ( including cisplatin , oxaliplatin , or carboplatin ) during the course of the disease was compared with the os for patients who had not been exposed to such treatment . 
whenever possible , specic regimen details were recorded . time to progression time to progression ( ttp ) was dened as the time from the date of surgical resection to the documented date of recurrence or death . 
 survival of patients with pdac and brca or palb2 mutation platinum - based treatments in the perioperative period was compared with the ttp for patients who had not been exposed to such treatment . statistical analysis univariable analysis was performed using fishers exact test for dichotomous variables and the t test and wilcoxon rank sum test for normal and nonnormal continuous variables , respectively . 
survival curves by mutation carrier status were estimated using the kaplan - meier method and were compared using the log - rank test . in subgroup analyses , separate cox regression models examined the following a priori subgroups : patients who received no platinum therapy , patients who received platinum therapy in any setting ( adjuvant , neoadjuvant , palliative ) , and patients who received platinum only in the perioperative setting ( adjuvant or neoadjuvant )  . each patient in the mutation - positive group was matched to two patients in the mutation - negative group using a greedymatch algorithm18 without replacement on the basis of four patient factors : pathologic stage at resection , if available , or clinical stage if the former was unavailable ; age at diagnosis ; year of diagnosis ; and sex . 
during matching , stage at diagnosis was given the most weight , followed by age at diagnosis and year of diagnosis , and then followed by sex . each patient in the mutation - positive group was successfully matched to two eligible patients in the mutationnegative group . all analyzed variables were 100% complete with the exceptions of family history ( missing in 11% ) , node status ( missing in 7% ) , margin status ( missing in 11% ) , timing of germline testing ( missing in 8% ) , time to progression ( missing in 6% ) , and use of platinum therapy ( missing in 7% )  . 
all statistical analyses were performed using sas 9.4 ( sas institute , cary nc )  . results patient characteristics overall , 32 patients with pdac were identied for the mutation - positive group ( table 1 ) , and the mutational breakdown was as follows : brca1 ( n = 6 ) , brca2 ( n = 23 ) , brca1 and brca2 ( n = 1 ) , and palb2 ( n = 2 )  . 
the mean age of patients in the mutationnegative group was 60 years ( range , 44 to 85 years )  . data points were missing in both groups of patients . 
to mitigate possible bias as a result of missing data , we performed a sensitivity analysis to compare those who had missing data with those who had no missing data . 
we found no statistically signicant differences in patient age , sex , stage at diagnosis , receipt of platinum therapy , receipt of perioperative platinum therapy , ttp , or os between these groups . personal and family history of cancer fourteen patients ( 44% ) in the mutation - positive group had a personal history of a prior malignancy . 
details of these results can be found in appendix table a1 . clinical and treatment characteristics of patients all patients underwent curative - intent surgical resection ( table 2 )  . 
of these , three patients in the mutation - positive group and 10 in the mutation - negative group received perioperative infusional 5 - uorouracil , irinotecan and oxaliplatin ( folfirinox ) , whereas the remaining patients received other platinum - containing regimens . 
 ( % ) unless otherwise indicated . abbreviations : dcis , ductal carcinoma in situ ; iqr , interquartile range ; r0 , negative margin ; r1 , microscopically positive margins ; r2 , macroscopically positive margins ; sd , standard deviation . group received palliative platinum chemotherapy upon recurrence . node and margin status nodal disease was detected in 18 patients ( 72% ) in the mutation - positive group and in 50 patients ( 78% ) in the mutation - negative group ( p = .58 ; table 1 )  . 
several subgroup analyses were performed . those in the mutation - positive group had signicantly longer median os ( mos ) than those in the mutation - negative group first , patients in both groups who received platinum therapy at any time during treatment course were evaluated . 
of patients mutation negative mutation positive time ( months ) no platinum perioperative platinum + censored log - rank p = .3722 + censored log - rank p = .0193 mutation negative mutation positive mutation negative mutation positive time ( months ) no . 
 full cohort no platinum + censored log - rank p = .2557 + censored log - rank p = .5427 mutation negative mutation positive mutation negative mutation positive time ( months ) time ( months ) no . 
of patients mutation negative mutation positive platinum in any setting perioperative platinum + censored log - rank p = .0685 mutation negative mutation positive + censored log - rank p = .2134 mutation negative mutation positive yu et al no . 
time to progression of patients by mutation - positive and mutation - negative statuses : ( a ) full cohort ; ( b ) patients who received any platinum therapy ( neoadjuvant , adjuvant , or palliative ) ; ( c ) patients who received no platinum therapy ; and ( d ) patients who received platinum in the neoadjuvant or adjuvant setting . nding in the advanced cancer setting . 
however , we also observed an improvement in mos when both groups receive perioperative ( neoadjuvant , adjuvant , or both ) platinum therapya nding that , to our knowledge , has not previously been described . 
after analysis was adjusted for margin and nodal status , the mos was not reached for patients in the mutation - positive group who received perioperative platinum ( median follow - up , 22.8 months ) , whereas patients in the mutation - negative group had an mos of 23.1 months in the same setting . 
furthermore , we observed prolonged ttp for patients in the mutationpositive group who received perioperative platinum therapy compared with those who did not ; again , the median ttp was not reached in patients who received perioperative platinum therapy . chemotherapy in the curative - intent setting . 
prospective studies are required to conrm this observation . one of the key strengths of this study is that the cohort of patients with brca1 , brca2 , or palb2 mutations was matched to a cohort without these mutations according to age at diagnosis , year of diagnosis , sex , and disease stage . in addition , the multivariable model accounted for several additional variables that might have inuenced patient outcomes : nodal status at resection , margin positivity , and resectability at diagnosis . 
the resectability variable allowed control of the analysis for patients who had locally advanced disease at the time of diagnosis and who might , therefore , have inherently different outcomes compared with those whose disease was resectable at presentation . when patients in both groups did not receive perioperative platinum - based therapy , there was no signicant difference in mos . 
although the number of patients enrolled in the study was small , the results suggest that patients who are positive for mutations benet preferentially from platinum - based this study has several important limitations that should be highlighted . 
because of the small number of patients in the mutation - positive group enrolled in the study , we included all patients for whom we had survival data . third , there is the risk of a potential survival bias as a result of the timing of germline testing . 
of the 24 patients in the mutation - positive group for whom we had dates of germline testing , six had testing before the diagnosis of pdac , and the remaining 19 patients had testing at a median of 10.5 months after diagnosis ; 10 of these patients were tested in the perioperative period , and nine were tested after disease recurrence . 
we are unable to account for this bias in this retrospective study because of the small number of tested patients in the mutationnegative group , which highlights the need for prospective studies . finally , the mutation - negative group may have inadvertently contained patients who were brca or palb2 carriers . 
however , this would have plausibly biased results toward the null hypothesis . these ndings are hypothesis generating only , but they may have several potential implications within the current neoadjuvant and adjuvant therapy treatment landscape . we observed a trend toward improved ttp and a signicantly longer mos in patients in the mutation - positive group who were treated with platinum chemotherapy in the perioperative setting . 
importantly , recent data from hu et al19 demonstrate that a substantial portion of patients who are positive for mutations do not have the classic clinical features associated with genetic syndromes , such as a positive family history or personal history of cancer . therefore , using an all - comer approach of early testing for patients with pdac seems rational and may improve outcomes for those who carry a pathogenic homologous recombination mutation . second , recent data from conroy et al3 have shown a substantial os benet in patients with resected pdac when adjuvant modied folfirinox , compared with gemcitabine therapy , is used.3 however , because of its substantial toxicities , folfirinox must be reserved for patients who have a robust performance status . 
at this time , patients who have undergone curative - intent surgery but who are not well enough to receive folfirinox do not have a dened course , and many are likely to receive nonplatinum treatments , such as gemcitabine plus capecitabine2 or gemcitabine plus nab - paclitaxel . 
given the nding that platinum - based perioperative therapy may preferentially benet patients who have a positive mutation status , chemotherapy combinations , such as cisplatin plus gemcitabine or infusional uorouracil , leucovorin , and oxaliplatin , might be preferable for these patients if folfirinox cannot be used . in conclusion , we present a small cohort study in which we observed an os benet when patients with resected pdac and a brca or palb2 mutation received platinum therapy in the perioperative setting . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . ronac mamtani consulting or advisory role : genentech , roche mark o ' hara consulting or advisory role : karyopharm therapeutics travel , accommodations , expenses : astrazeneca / medimmune peter j . 
o ' dwyer consulting or advisory role : genentech , bristol - myers squibb , boehringer ingelheim research funding : bristol - myers squibb , pzer , novartis , genentech , mirati therapeutics , celgene , glaxosmithkline , bbi healthcare , merck , pharmacyclics , bayer , five prime therapeutics , forty seven , amgen susan m . 
domchek honoraria : astrazeneca , clovis oncology , bristol - myers squibb research funding : astrazeneca ( inst ) , clovis oncology ( inst ) , pharmamar ( inst ) kim a . 
reiss research funding : lilly ( inst ) , clovis oncology ( inst ) , bristol - myers squibb ( inst ) , tesaro ( inst ) no other potential conicts of interest were reported . references siegel rl , miller kd , jemal a : cancer statistics , 2018 . 
neoptolemos jp , palmer dh , ghaneh p , et al : comparison of adjuvant gemcitabine and capecitabine with gemcitabine monotherapy in patients with resected pancreatic cancer ( espac - 4 ) : a multicentre , open - label , randomised , phase 3 trial . 
j clin oncol 36 , 2018 ( suppl ; abstr lba4001 ) van tienhoven g , versteijne e , suker m , et al : preoperative chemoradiotherapy versus immediate surgery for resectable and borderline resectable pancreatic cancer ( preopanc - 1 ) : a randomized , controlled , multicenter phase iii trial . 
genet med 17 : 569 - 577 , 2015 dann rb , deloia ja , timms km , et al : brca1 / 2 mutations and expression : response to platinum chemotherapy in patients with advanced stage epithelial ovarian cancer . 
mol cell 7 : 263 - 272 , 2001 golan t , kanji zs , epelbaum r , et al : overall survival and clinical characteristics of pancreatic cancer in brca mutation carriers . 
yurgelun mb , chittenden ab , morales - oyarvide v , et al : germline cancer susceptibility gene variants , somatic second hits , and survival outcomes in patients with resected pancreatic cancer . 
genet med 21 : 213 - 223 , 2018 isakoff sj , mayer el , he l , et al : tbcrc009 : a multicenter phase ii clinical trial of platinum monotherapy with biomarker assessment in metastatic triplenegative breast cancer . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
 ( % ) variable timing of germline testing before diagnosis during perioperative period at recurrence somatic brca testing ( controls only ) germline testing results ( controls only ) negative variant of uncertain signicance non - brca pathogenic mutation note . 
one is the growing recognition that complex diseases such as cancer are multifactorial and may be attributed to harmful changes on multiple - omics levels and on the pathway level . 
when individual genes in an important pathway have relatively weak signals , it can be challenging to detect them on their own , but the aggregated signal in the pathway can be considerably stronger and hence easier to detect with the same sample size . 
to address these challenges , there is a growing body of literature on knowledge - guided statistical learning methods for analysis of high - dimensional - omics data that can incorporate biological knowledge such as functional genomics and functional proteomics . 
2019 by american society of clinical oncology introduction rapid advances in technologies have led to generation of high - dimensional - omics data , such as genomics , transcriptomics , and metabolomics data , in many biomedical studies . 
they have been used to build prediction models for disease risk or progression and for adaptive response to treatment , uncover molecular signatures associated with a disease that provide insights about disease mechanism , and identify potential therapeutic targets . 
in particular , they achieve simultaneous variable selection and model estimation and can be used to analyze data where the sample size is less than the number of - omics features . 
for example , when expression levels of individual genes in an important pathway associated with cancer risk have relatively weak signals , it can be challenging to detect them on their own , but the aggregated signal in the pathway can be considerably stronger and hence easier to detect with the same sample size . 
the vast majority of existing statistical learning methods are entirely data driven and fail to incorporate biological knowledge such as functional genomics and functional proteomics that can be represented by a graph ( table 1 )  . extensive research in the past has yielded everfunctions of deepening knowledge of biological genes and gene products , which have been shown to function through pathways and networks . 
 zhao , chang , and long context key objective to review advances in knowledge - guided statistical learning methods for analysis of - omics data and spur wider and more frequent applications of such methods in basic , translational , and clinical research toward the goal of advancing precision oncology . knowledge generated the knowledge - guided analysis strategy offers a number of advantages . 
the strategy can also facilitate the integration of multimodal - omics data through the incorporation of biological knowledge about the functional relationship between different modalities . relevance knowledge - guided statistical learning methods can be used to construct accurate models for predicting cancer risk , prognosis , and response ; uncover molecular signatures that are predictive of cancer risk , disease progression , or patient response to treatment , which can inform novel targets for therapeutic development ; and identify cancer subtypes related to molecular differences , which offers insights about optimizing treatment strategy for each subtype , an important step toward precision oncology . associated with disease risk or progression . 
in addition , a two - step approach , 22 conducting clustering analysis followed by annotating clusters by a gene set enrichment analysis , has been shown to improve power in clustering analysis of gene expression data . 
similar biological knowledge is also available for other types of - omics data , and table 1 lists representative databases for such biological knowledge . recent methodologic research23 - 25 has also provided strong evidence about the advantages of knowledgeguided statistical learning methods that can incorporate the aforementioned biological knowledge , compared with statistical learning methods that do not use such biological knowledge . 
knowledge - guided statistical learning methods enable selection of important pathways instead of individual - omics features and improve power in uncovering important features , particularly those with weak signals . 
in addition , such a strategy can also facilitate the integration of multimodal - omics data through the incorporation of biological knowledge about the functional relationship between different modalities ( eg , expression quantitative trait loci and metabolomic quantitative trait loci )  . 
as such , this knowledge - guided data - driven approach is particularly powerful and useful for analysis of - omics data in complex diseases such as cancer . knowledge - guided statistical learning methods have wide applications in precision medicine , including precision oncology . 
the knowledge - guided supervised learning methods can be used to construct prediction models for disease risk and prognosis , which can then be used to identify higher risk groups more accurately and tailor interventions to individual patients.26 they can also be used to uncover molecular signatures that are predictive of disease risk , disease progression , or patient response to treatment , which can inform novel targets for therapeutic development . 
the knowledge - guided unsupervised learning methods , such as biclustering , can be used to identify disease subgroups and important pathways associated with each subgroup.27 identication of subgroups related to molecular differences offers insights about optimizing treatment strategy for each subgroup and is an important step toward developing a precision medicine approach for complex diseases such as cancer . in this review , we survey advances in knowledge - guided statistical learning methods for analysis of highdimensional - omics data in precision medicine , many of which have been applied to analysis of cancer data . 
representative databases for various types of biological knowledge database full name knowledge kyoto encyclopedia of genes and genomes2 metabolic pathways reactome pathway database3 metabolic and signaling pathways mummichog mummichog4 metacyc metabolic pathways from all domains of life6 invitrogen ipath invitrogen ipath7 metabolomic pathway metabolic pathways metabolic pathways ingenuity pathways knowledge base8 gene regulatory and signaling pathways biocyc pathway / genome database collection9 metabolic pathways transpath transpath10 gene regulatory and signaling pathways cell signaling technology pathway11 signaling pathways targetscan12 mirbase : the microrna database13 gene - microrna regulatory network gene - microrna regulatory network probabilistic identication of combinations of target sites14 gene - microrna regulatory network mirdb15 microrna data integration portal16 gene - microrna regulatory network gene - microrna regulatory network biological general repository for interaction datasets17 protein and genetic interactions kegg reactome ipkb biocyc targetscan mirbase pictar mirdb mirdip biogrid consensuspathdb consensuspathdb18 integrative database for molecular interactions and certain clinical outcomes . 
depending on the type of clinical outcomes , methods have been proposed to handle binary data ( eg , the disease status ) , continuous data ( eg , time to death ) , categorical data ( eg , cancer subtypes ) , and censored data ( eg , time to cancer recurrence )  . 
through this learning procedure , we can identify important genomic features that are highly associated with clinical end points , build a prediction rule that will be crucial for future clinical practice , or even achieve both simultaneously . 
correspondingly , prior biological or structure knowledge is supposed to be incorporated within feature selection or the prediction process to rene the model for a more accurate and interpretable result . x : - omics predictors yi = xi + ei biological knowledge g = < v , e > penalty for ( frequentist ) prior for ( bayesian ) fig 1 . 
knowledge - guided linear regression model where y is the clinical outcome of interest and x is the set of high - dimensional - omics features or predictors , and g = , v , e . 
is the graph containing the biological knowledge about - omics predictors with v denoting the set of nodes ( ie , - omics features ) and e denoting the set of edges . 
to incorporate g = , v , e . , a penalty for is used in a frequentist framework , and a prior distribution for is used in a bayesian framework . for ease of illustration , we introduce a few notations without loss of generality . 
suppose a study recruits n patients with an outcome of interest ( eg , disease status ) and p predictors ( ie , confounders and genomics features ) collected for each patient . 
the existing biological knowledge ( eg , pathway information , gene - gene network ) among predictors is represented as a direct or indirect graph among genomics features , denoted as g . 
figure 1 provides an example of a knowledge - guided linear regression model in both the frequentist and bayesian frameworks . the main distinction between the frequentist and bayesian approaches is their view on probability . short , frequentist approaches try to solve the exact value of probability on the basis of the event they observed , whereas bayesian methods do not assume there is xed probability of the event but treat this probability itself also as randoin terms of model tting , frequentist models do not assign prior distribution for the unknown parameters and eventually end up with a point estimate for them , whereas bayesian models need such a prespecied prior and produce a posterior distribution for each parameter . 
 zhao , chang , and long results in their differences in how to incorporate biological knowledge , computation , and interpretation of analysis results . frequentist approach the majority of knowledge - guided frequentist methods for analysis of high - dimensional - omics data are based on a regression framework to study the association between - omics features x and an outcome of interest y [ ie , y f ( x , ) ] , with being the set of p unknown regression coefcients to be estimated . 
to induce the regression coefcients , sparsity or shrinkage for a penalty function , p ( ) , is often introduced , leading to the so - called penalized regression . in the case where biological knowledge among predictors x is available and can be represented by a graph g , we can also use p ( ) to incorporate such biological knowledge ( fig 1 )  . 
specically , when there is an edge between features i and j in g , a common strategy is to induce similar shrinkage effects between their corresponding parameters i and j . the work of li and li23 represents one of the earliest attempts to incorporate gene network information into feature selection for cancer genomics application . 
to incorporate the pathway information in kegg , they proposed a network - constrained penalty on the the following two assumptions : rst , genes basis of connected by an edge have similar functions and , therefore , are expected to have smoothed regression parameters ; and second , the connected genes have higher probability to be selected or not selected simultaneously . 
the penalty itself consists of an l1 - norm to achieve global sparsity as the usual penalization procedure does , as well as a quadratic form on the network laplacian matrix to induce smoothness across the biological graph . 
using this method , they identied gene subnetworks that were highly correlated with survival time of patients with glioblastoma ( see fig 2 in the article by li and li23 ) , and some of these subnetworks were not identied by other existing learning methods that did not use pathway information . 
although some of the subnetworks were supported by previously published work , some have not been reported in the literature , and these novel gene signatures can inform the molecular underpinnings of glioblastoma prognosis or even novel targets for therapeutic development . after li and li , 23 a number of extensions have been proposed . 
to handle binary outcome , zhang et al28 adopted the network - constrained penalty under a logistic regression model that was used to predict a specic breast cancer subtype on the basis of gene expression data from the cancer genome atlas ( tcga ) consortium while incorporating protein - protein interactions . 
their method was applied to a breast cancer study to identify genes and subnetworks that are predictive of survival while incorporating biologic network from kegg . besides extensions to handle different outcome types , pan et al24 proposed a new penalty on the basis of lrnorit reduces the computational costs with a smaller number of tuning parameters and allows two connected genes to both upand downregulate one anothers expression while maintaining the grouping effect . 
a few subsequent extensions include those by kim et al30 to modify the penalty by removing the constraint of a similar magnitude of the connected biomarkers , which is biologically more meaningful , and tian et al31 to use a multinomial logit model for cancer subtype prediction . overall , these works focus on incorporating individuallevel relationships between - omics biomarkers under the assumption that connected biomarkers are more likely to affect clinical outcome in a coordinated way . more recently , for analysis of gene and microrna biomarkers , zhao et al26 proposed a hierarchical group penalty , which incorporates pathway membership , gene network , and microrna - gene regulatory network information into a semiparametric accelerated failure time model to predict prostate cancer recurrence after surgery . 
compared with previous works , hierarchical group penalty further allows a group - level sparsity ( ie , genes in the same pathway are also more likely to be associated or not associated with clinical outcome at the same time )  . 
zhao et al26 were among the rst to include both group - level membership and within - group connectivity and induce sparsity at both the pathway level and gene level . 
the article by zhao et al26 treats the unknown component of the biological graph as missing data and proposes a multiple - imputation approach for handling missing edges in the graph . 
 knowledge - guided statistical learning methods for - omics data for both methods , similar to the regression setting , biological knowledge represented by graph is incorporated into the model using penalty functions , where the sparsity and grouping effects are also expected . 
the knowledgeguided svm has been used to predict clinical outcome of patients with glioblastoma using genomic biomarkers.32 bayesian approach although frequentist models are traditionally considered canonical , bayesian approaches have attracted increasing interest in recent years as a result of their ease in incorporating prior information and quantifying uncertainty . as such , they have played an important role in the development of knowledge - guided supervised learning methods . when analyzing high - dimensional - omics data , prior distributions that lead to variable selection or shrinkage are used in a bayesian model to improve prediction and identify important features . 
there is an extensive body of literature on bayesian variable selection.34 different from variable selection achieved through a penalty in a frequentist paradigm , bayesian variable selection can be achieved in two ways , namely , selection on the basis of a point - mass mixture prior or regularization through a shrinkage prior . the former35 directly includes or excludes a predictor in the model by introducing a binary selection indicator . 
the downside with a shrinkage prior is that it does not directly provide results on feature selection ; in addition , a subsequent truncation step , often ad hoc , is required to obtain the nal set of selected predictors . similar to existing frequentist methods , the majority of knowledge - guided bayesian supervised learning methods are developed for regression models . 
as an extension of the work by li and zhang , 37 stingo et al39 proposed a bayesian hierarchical variable selection regression model incorporating both pathway membership and gene network information with application to breast cancer microarray data . 
zhe et al40 tackled the following limitation of the method from stingo et al39 : all the pathways that a selected gene belongs to are also selected , which is overly restrictive and may not always be meaningful . 
to remove this restriction as well as reduce the computation , they proposed a bayesian joint pathway and gene selection model that uses a graph laplacian matrix to encode biological knowledge and a variational bayesian algorithm for model estimation . 
the analyses of a gene expression data set using their method yielded a more accurate prediction model for survival time in palymphoma than several tients with diffuse large b - cell existing learning methods that did not use biological information . beyond genomics , zhang et al41 analyzed molecular inversion probe data to identify genes and probes that are associated with clinically relevant subtypes of breast cancer . 
their method uses information on biological grouping of gene and probe - within - gene levels to dene a hierarchical selection procedure through a point - mass mixture prior for gene selection and a shrinkage prior for probe selection . 
as the dimension of - omics data increases , there is a growing interest in using bayesian shrinkage priors for knowledge - guided variable selection and prediction as a result of potential computational savings . 
rockova and lesaffre42 developed a bayesian model for hierarchical feature selection at the pathway level and gene level on the basis of bayesian lasso.36 subsequently , chang et al25 developed a novel adaptive structured shrinkage prior to incorporate biological knowledge in a bayesian regression model . 
in general , with the computational advantage of the bayesian shrinkage prior , this research direction is expected to become even more active for analysis of large - scale - omics data . besides regression , knowledge - guided bayesian methods have also been developed for discriminant analysis44 and svm45 for analysis of high - dimensional genomics data . 
unsupervised learning methods are typically used as a data mining approach to help explore and visualize large - scale data ; see chapter 14 in hastie et al.1 the existing knowledge - guided unsupervised learning methods have been focused in two areas , namely , clustering analysis and dimension reduction , for which either a frequentist or a bayesian approach can be used . clustering analysis the goal of clustering analysis is to group patients on the basis of their similarities in , for example , genomic features . in terms of application to cancer - omics , clustering analysis has been widely used to uncover cancer subtypes , which is essential to understand tumor heterogeneity and optimize prevention and treatment strategy accordingly . 
they mainly used the number of edges connected with each gene to dene weights in the clustering procedure . recently , more advanced integrative clustering methods have been proposed to jointly analyze multimodality cancer - omics data while incorporating biological information . 
their method is able to jointly handle different data types ( continuous and discrete ) , which is well suited to analyze multiomics data sets in cancer , including gene expression , copy number , rna sequencing , and single nucleotide polymorphisto incorporate biological knowledge , a bayesian adaptive structured shrinkage prior is placed on the factor loading matrix , which encourages the - omics features connected in a graph to have zero or nonzero loading simultaneously in the same factor . 
to reduce heavy computation , they developed an efcient variational expectation - maximization algorithm for estimation , making their method scalable to the analysis of high - dimensional - omics data . dimension reduction dimension reduction methods can be used to project highdimensional - omics features into a lower dimension space either to better understand or visualize the data structure or to facilitate assessing dependence between two sets of ( cid : 129 ) ( cid : 129 ) ( cid : 129 ) subtypes identified in wz prior for z : ( zli ) exp ( |zli| ) prior for w : ( wjl ) exp ( |wjl| ) prior for : l l ~ ln ( , g ) biological knowledge : g = < v , e > insights on subtypes and molecular signatures prediction ( cid : 129 ) ( cid : 129 ) ( cid : 129 ) ( cid : 129 ) ( cid : 129 ) ( cid : 129 ) fig 2 . 
this method was used to conduct biclustering analysis of gene expression data , dna methylation data , and dna copy number data from a glioblastoma data set from the cancer genome atlas . 
li et al27 incorporated biological knowledge into the sparse principal component analysis , a popular dimension reduction tool for high - dimensional data , to identify genes associated with glioblastoma cancer subtypes . 
they extended and investigated two networkbased penalties , the grouped penalty by pan et al24 and the fused lasso penalty by tibshirani et al.50 the resulting sparse principal component analysis algorithm is able to incorporate biological network information in the principal component loadings and achieve more accurate and biologically meaningful dimension reduction . 
similarly , biologic knowledge has also been incorporated into the canonical correlation analysis51 and the coinertia analysis ( cia ) 52 for assessing dependence between two - omics data sets . 
applied to analysis of gene expression data and protein abundance data in the nci - 60 cell line data set , the knowledge - guided cia52 method was able to project the high - dimensional - omics feature to a lower dimensional space in which the 10 different types of cancers were clearly separated ( see fig 1 in article by min et al52 )  . 
of note , both canonical correlation analysis and cia are popular multivariate statistical methods for integrative analysis and have become popular in analysis of multimodality - omics data in cancer studies . in addition , liu et al53 developed a knowledge - guided approach to use expression data and coexpression network information to improve de novo discovery of driver pathways in cancer on the basis of mutation data . 
analyses of three cancer data sets using their method revealed new driver pathways that were not uncovered by other methods including , particularly , driver genes with less frequent mutations that are much more difcult to detect . 
these new driver pathways may offer insights about cancer biology and inform novel targets for screening and for therapeutic development . discussion statistical learning methods have been proven powerful for analysis of high - dimensional - omics data in modern biomedical research but have some important limitations . 
to address these limitations , the knowledge - guided strategy , as reviewed here , has drawn increasing interest in recent years and has been shown to yield biologically more interpretable and meaningful results . 
software tools for many of the methods reviewed earlier have been made publicly available ( table 2 )  . knowledge - guided statistical although substantial progress has been made in the delearning velopment methods , there is still much room for additional methodologic developments and improvements . 
one area for future research is to assess robustness of knowledgeguided methods to mis - specication of biological knowledge , because in practice , biological knowledge represented by a graph g is known to be incomplete and include table 2 . 
existing work27 has demonstrated that some knowledge - guided supervised learning methods are fairly robust to mis - specied biological knowledge g , but more research is needed for other types of statistical learning methods . 
to further enhance robustness , it is also of signicant interest to combine knowledge - guided methods with learning biological knowledge graphs from observed data , for which there is little research besides that by zhao et al.26 in addition , most of the existing knowledge - guided bayesian methods may not be scalable to analysis of big - omics data that can have hundreds of thousands or even millions of features , and more research on efcient computation algorithms is needed . although knowledge - guided statistical learning methods have drawn growing interest in methodologic communities , this review will they have not been widely used by cancer researchers , and the ndings from the methods publications reviewed in this article largely remain to be validated in subsequent studies . 
our hope is that raise the awareness and spur wider and more frequent applications of knowledge - guided methods in basic , transto advance lational , and clinical precision medicine , particularly for complex diseases such as cancer . 
nucleic acids res 28 : 27 - 30 , 2000 croft d , okelly g , wu g , et al : reactome : a database of reactions , pathways and biological processes . 
subramanian a , tamayo p , mootha vk , et al : gene set enrichment analysis : a knowledge - based approach for interpreting genome - wide expression proles . proc natl acad sci usa 102 : 15545 - 15550 , 2005 23 . 
zhao y , chung m , johnson ba , et al : hierarchical feature selection incorporating known and novel biological information : identifying genomic features related to prostate cancer recurrence . 
stingo fc , chen ya , tadesse mg , et al : incorporating biological information into linear models : a bayesian approach to the selection of pathways and genes . ann appl stat 5 : 1978 - 2002 , 2011 40 . 
polley , phd1 and ying kuen cheung , phd2 applications in early - phase cancer trials have motivated the development of many statistical designs since the late 1980s , including dose - nding methods , futility screening , treatment selection , and early stopping rules . these methods are often proposed to address the conventional cytotoxic therapeutics for neoplastic diseases and cancer . 
recent advances in precision medicine have motivated novel trial designs , most notably the idea of master protocol ( eg , platform trial , basket trial , umbrella trial , n - of - 1 trial ) , for the evaluation of molecularly targeted cancer therapies . 
in this article , we review the concepts and methodology of early - phase cancer trial designs with a focus on dose nding and treatment screening and put these methods in the context of platform trials of molecularly targeted cancer therapies . 
tumors once thought to be of the same histologic type may now be considered heterogeneous and differentiated on the basis of genomic biomarkers and may be treated differently according to the biomarker expression prole . 
because the subject subgroups dened by specic genetic abnormalities of the tumors may only represent a small fraction of the disease population , the concept of precision oncology trials for targeted therapies has created enormous challenges in recruiting patients with rare genomic subtypes . 
this recruitment challenge , coupled with the need to test multiple targeted therapies in relatively small molecularly dened patient subgroups in an efcient manner , has led to the rethinking of cancer drug development paradigto meet these challenges , the concept of master protocols has been introduced to increase the speed of oncologic therapy development and evaluation . 
the main goal of constructing a master protocol is to enable sharing of operational infrastructure and key design elements across substudies so as to achieve better coordination and efciency than can be achieved in single trials designed and conducted independently in biomarker - dened subpopulations.1 - 5 a platform trial is a randomized trial in a single histology that involves multiple treatments and multiple biomarkers under a master protocol . 
instead , rather than presuming we know which therapy is appropriate for which biomarker stratum , randomization is used for treatment assignment within each biomarker stratutable 1 gives a schema of biomarker - based inclusion criteria in a hypothetical platform trial . 
 polley and cheung context key objective this article provides a critical review of early - phase cancer trial statistical methods and discusses their practicality under the novel platform trial paradigm for evaluating molecularly targeted therapies . knowledge generated although many trial design principles for cytotoxic agents still hold relevance for molecularly targeted therapies , new design and analysis issues , including delayed toxicities and efcacy - tolerability trade - offs , should be considered when designing studies involving these agents . 
early - phase platform trials facilitate efciency gains by allowing within - trial information sharing of safety and efcacy data . relevance this article provides a practical guide for early - phase platform trial designs in the era of precision medicine . 
novel statistical methods continue to be needed to harness the information garnered in these new - generation trial designs to help expedite oncologic drug development and approval . for neoadjuvant therapeutic response with imaging and molecular treatment of analysis 2 ( i - spy2 ) trial women with locally advanced breast cancer.8 , 9 methodologic innovations in a platform trial may include midtrial outcome - adaptive randomization to favor treatments with higher response rates within the respective biomarkerdened stratum.10 , 11 the treatment effects of various experimental therapies are often estimated according to a bayesian hierarchical model by borrowing outcome information across molecularly dened biomarker subgroups.10 , 11 at the same time , bayesian decision rules may be incorporated to determine when and whether therapies with low probabilities of success should be discontinued and therapies with high probabilities of future success should advance to subsequent conrmatory studies ; in this way , novel therapies may be added or dropped in a perpetual manner.10 , 12 , 13 because of the need for midtrial adaptations , platform trials often use short - term end points , such as absence of disease progression at 8 weeks ( as in battle ) or pathologically conrmed complete response ( as in i - spy2 )  . 
although the bayesian inferential structure provides a convenient and efcient framework for analysis , it does not protect the type i error rate against multiple comparisons in the conventional sense . 
as such , phase ii platform trials are generally regarded as exploratory , and positive ndings from these trials will require independent conrmation for the promising drug - biomarker stratum in subsequent phase iii trials . cancer trial designs have been well established for cytotoxic agents . 
specically , phase i trials are dose - nding studies that examine safety of the drug and estimate the maximum - tolerated dose ( mtd ) , 14 and phase ii trials are designed to screen new drugs on the basis of pilot efcacy such as clinical response.15 , 16 the general objective of early - phase cancer trials is to identify and recommend a promising dose regimen of a new agent for conrmatory investigation in multicenter phase iii randomized clinical trials . 
the rationale for using toxicity as a surrogate for efcacy can be traced back to the suggestion of using nitrogen mustards in the treatment of neoplastic diseases in the 1960s.18 the most commonly used statistical design for a phase i oncology dose - nding trial is the 3 + 3 design . 
this is a rulebased design in which the number of dose levels is xed in advance and the dose escalation or de - escalation decision is based on the number of dlts that patients experience at a given dose . 
the 3 + 3 design has received extensive examinations and has been demonstrated to produce poor statistical properties , including treating many patients at doses that are below the biologically active level and imprecise estimation of dlt probability because of the relatively small number ( three to six ) of patients treated at each dose cohort.19 - 21 to address the deciencies of the 3 + 3 design , many dosending designs have been proposed since the 1990s that purport to improve statistical accuracy and trial efciency in the context of chemotherapy trials.22 the most prominent work among these is an adaptive design known as the continual reassessment method ( crm ) .23 , 24 the crm uses a mathematical model to relate the dose levels to the probability of dlt . 
after the rst patient has been treated at the dose determined by the initial dose - toxicity curve , the toxicity information is combined statistically with the initial curve , and an updated dose - toxicity curve is estimated using a bayesian methodology . 
the crm has been shown to improve the precision in estimating the mtd and to increase the number of patients assigned to the selected mtd when compared with the standard 3 + 3 method.25 - 27 the latter property has important ethical implications because traditional methods will likely treat many patients at low and , hence , inefcacious the crm fullls the therapeutic doses . purpose of phase i cancer trials , which are often the last resort of the patients who have exhausted other alternative treatment options . 
during its early inception , the crm drew some criticisms particularly with regard to the possibility of exposing patients to doses that are likely to cause dlt.28 , 29 to address these concerns , several trials used a hybrid approach that combined the model - based crm with a rulebased initial dose - escalation plan ( clinicaltrial.gov identiers : nct03141203 , nct03733990 , and nct03028766 ) .28 - 31 in this regard , once an mtd is identied , a new chemotherapy will be studied in phase ii trials , which are proof - of - concept studies looking for early indication of antitumor activity . 
this objective is often achieved with a single - arm trial design using clinical response as an end point , with the possibility of stopping a trial early as a result of futility.16 , 23 , 32 , 33 for instance , to have 80% statistical power to reject a 25% response rate in favor of a 45% response rate of a new drug at 5% signicance , a single - arm , xed - sample size trial will need to enroll 36 patients and seek to observe 14 responses in the enrolled patients . 
alternatively , a simon two - stage design16 will rst enroll 17 patients and then enroll an additional 24 patients ( ie , a total of 41 patients ) only if there are at least six responses in the rst 17 patients . 
because of the provision for futility stopping , the simon design will , on average , treat fewer patients with inefcacious drugs , thus allocating resources to the more promising ones . 
however , to demonstrate promise of a new drug , an adaptive design with futility stopping will always require a larger maximum sample size than a xed trial ( as in the example just provided )  . 
a participant with a given biomarker is eligible for random assignment to a drug and dose among those marked by a checkmark , which indicates that the drug is a candidate for the biomarker and that the dose does not exceed the maximum - tolerated dose . 
these noncytotoxic agents tend to induce long - lasting mild toxicities , and hence , the assumption of acute toxicities may not apply.35 for some agents , prolonged administration may be required to achieve desirable therapeutic benet , thus increasing the likelihood of late - onset or cumulative toxicities that may manifest many months after the initiation of treatment . 
therefore , for phase i trials of targeted therapies , it is critical to identify the mtd with respect to a longer observation window , so as to ensure future investigations are limited to a tolerated dose range . the 3 + 3 design and crm - type designs dictate that accrual of new patients be suspended until all enrolled patients have been fully evaluated for dlt . 
when delayed toxicities are anticipated , the trial length may be prohibitively long because of the need for frequent accrual suspension and the lengthy dlt observation window.36 , 37 a dose - nding design that does not require suspension of patient accrual while waiting for dlt information on previously treated patients to mature is the time - to - event continual reassessment method ( titecrm ) .38 the dose allocation scheme of the tite - crm follows closely the original crm paradigm but additionally leverages the time - to - toxicity and partial follow - up information in all enrolled patients when estimating the mtd . 
since its introduction , the tite - crm design has been successfully implemented in trials at major academic cancer centers , 39 - 48 as well as us national cancer institutesponsored cooperative groups trials such as radiation therapy oncology group 0813 trial ( clinicaltrials.gov identier : nct00750269 )  . in situations where patients become available to a trial at a rapid rate , the tite - crm could accrue many patients at a given dose level before anyone has been observed for a meaningfully long period . 
therefore , it may be useful to impose a waiting window between cohorts of patients , so as to allow adequate follow - up before adaptation is made.49 - 51 tolerability and efcacy trade - off for molecularly targeted agents and immunotherapies , it may not be adequate to only consider toxicity and tolerability in a dose - nding trial . 
second , a higher dose of a new compound may be less efcacious than a lower dose52 or may see limited benets on the basis of considerations of pharmacokinetic properties.53 hence , the mtd may not be an ideal recommended phase ii dose , although it is still useful to dene the upper safety limit by the mtd . 
third , dlts may not be observed with these agents , and mtd may not be determined at the conclusion of dose escalation.54 , 55 these are the scenarios where both toxicity and activity end points should be used to guide dose recommendations.54 , 55 yan et al56 proposed an adaptive bayesian phase i / ii efftox design that guides dose selection on the basis of the tradeoffs between the probabilities of treatment toxicity and efcacy . 
paoletti and postel - vinay55 discussed the ideal setting for the efftox design and contended that such design is best suited when a homogeneous disease population is well - dened for drug sensitivity and an early biomarker for efcacy is available . 
houede et al57 model clinical utilities on the basis of both toxicity and clinical response in a combination therapy trial , which aims to choose the optimal dose pair of a chemotherapy and a biologic agent . finally , li et al58 proposed an approach to identify the best drug at an efcacious and safe dose for a given biomarker subtype in an early - phase platform trial . 
as cancer trials increasingly focus on combination therapies , it is critical to leverage efcacy information as well as toxicity in dosing decisions . treatment screening in a randomized platform trial in a single - arm , proof - of - concept trial , the interpretation of the results and conclusion of the trial depend on the specication of what would constitute a poor ( null ) response rate on the basis of historical data or a priori clinical experience . 
although response dened by tumor shrinkage is an indicator of activity for cytotoxic drugs , progression - free survival ( pfs ) may be more appropriate for early indication of efcacy of a targeted therapy whose mechanism does not necessarily shrink tumors . 
pfs is a less specic end point than tumor shrinkage because untreated patients may survive without progression for a period of time , whereas patients not treated with chemotherapy are unlikely to experience response . 
furthermore , as precision medicine challenges the fundamental assumption of tumor homogeneity , the appropriate null response rate may vary with biomarker expression . in a platform trial where several drugs are considered for a biomarker subtype ( table 1 ) , drug screening can be based on randomized comparison and ranking , thereby eliminating some subjectivity in choosing a null response rate . 
others have proposed adaptive screening strategies such as a two - stage design , 60 , 61 whereby the rst screening stage aims to select a promising drug among several for additional evaluation , and continuous monitoring using the sequential probability ratio test.62 these innovative adaptive designs have been shown to increase the nal dose selection accuracy while treating more patients with better drugs during the trial and can accommodate situations where there is a concurrent standard therapy . 
indeed , the infrastructure developed under a master protocol , in conjunction with the use of these innovative adaptive designs , facilitates unbiased randomized comparisons of potential drug candidates in a platform trial in a timely and efcient manner . pooling information across subtypes in a platform trial in an early - phase platform trial , where a drug may be given to patients across biomarker subtypes , the safety prole of the drug can be evaluated using pooled information across subgroups . 
specically , it is often reasonable to assume there is a common mtd for all biomarker subtypes when the biomarker is agnostic to adverse reactions to the drug.58 the concept of combining toxicity information from different diseases is not new , because phase i cancer trials of chemotherapy are often conducted in patients with heterogeneous malignancies ( eg , solid tumors )  . 
however , implementing this concept in the context of a platform trial provides much needed relevant safety information of the drug in patients with rare biomarker subtypes . when assessing a drugs efcacy in a given biomarker subtype , a statistically unbiased approach is to consider the observed response or pfs rate using only data of patients with that subtype . 
this analytical approach may make it difcult to understand the drugs effect in rare biomarker subtypes . is that a potential advantage of running a platform trial a targeted therapys efcacy for a given biomarker can potentially be analyzed based on its efcacy in other subtypes of the same histology , when the drug is hypothesized to work through a common molecular mechanism in all subtypes . 
 polley and cheung this analysis adds another level in the hierarchy of bayesian analysis , it is called hierarchical bayesian modeling ( hbm )  . several authors have examined and proposed the use of hbm in platform trials.58 , 63 , 64 it has been noted that borrowing information using hbm can be counterproductive when the drugs effects vary across biomarker subtypes.63 this is conceivable when a drug has nonspecic anticancer activity that is not specic to the molecule . 
although ongoing work has been suggested to model multiple sources of exchangeable drug effects in conjunction with hbm , 64 it is critical to evaluate the clinical situations as to whether there is a strong reason for expecting a common drug effect across subgroups . discussion there are many potential efciency gains associated with a platform trial.2 , 5 first , the use of a common genomic screening platform to identify patients eligible for the trial often results in shorter recruitment time and lower screen failure rate . 
second , the use of centralized governance bodies under a master protocol ( eg , the scientic review committee , institutional review board , or data and safety monitoring committee ) can ensure streamlined and efcient oversight for all substudies . 
for example , the trial innovation network represents a collaborative national network that aims to enhance operational efciency by leveraging the resources and expertise of the clinical and translational science award.65 third , the speed of drug development and evaluation is improved by the common infrastructure that allows opening or closure of substudies of new agents and biomarkers more expeditiously . other related trial designs for biomarker - based cancer drug development under the broad denition of a master protocol include basket trials and umbrella trials.1 , 3 , 4 a basket trial evaluates a targeted therapy on multiple disease subtypes ; specically , each basket evaluates the therapeutic effect of a targeted therapy for several cancer types that have a common molecular biomarker or genetic alteration . 
basket trials are typically nonrandomized ; on the basis of a prespecied genetic alteration , patients are assigned to a regimen that is expected to be active for their tumor . 
these biomarker - dening strata may be single - arm phase ii or phase ii / iii trials that randomly assign patients to either the matched targeted therapy or some standard therapy ( or placebo )  . 
an example of an umbrella trial is the adjuvant lung cancer enrichment marker identication and sequencing trial ( alchemist ) for early - stage nonsmall - cell lung cancer.66 although these novel trials provide a platform to answer clinical questions that traditional clinical trial design may not afford , it is important to appreciate that these gains are accompanied by increased logistical challenges and coordination efforts.67 for example , a platform trial requires tremendous resources and time commitment to establish the common trial infrastructure . 
often , a platform trial would involve multiple agents developed by different industry partners ; the contract negotiation with multiple stakeholders in terms of data sharing or coordination across substudies will likely pose additional communication the assay platform used to screen challenges . 
finally , patients for eligibility and assignment to substudies will also need to be cleared with an investigational device exemption by the us food and drug administration , further adding regulatory complexities . despite these challenges , the opportunities afforded by the new - generation platform trials are enormous . 
from a study design perspective , as we have illustrated , a single platform trial under a master protocol can facilitate information sharing of safety and efcacy data across disease subtypes in the statistical analysis.58 to date , most existing statistical methods have been proposed that deal with safety and efcacy questions independently ( eg , crm for safety dose nding , hbm for efcacy evaluation )  . 
 early - phase platform trials manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors inst = my institution . 
semin oncol 42 : 724 - 730 , 2015 renfro la , sargent dj : statistical controversies in clinical research : basket trials , umbrella trials , and other master protocolsa review and examples . 
n engl j med 377 : 62 - 70 , 2017 kim es , herbst rs , wistuba ii , et al : the battle trial : personalizing therapy for lung cancer . 
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cancer prev res ( phila ) 5 : 1144 - 1154 , 2012 jakubowiak aj , grifth ka , reece de , et al : lenalidomide , bortezomib , pegylated liposomal doxorubicin , and dexamethasone in newly diagnosed multiple myeloma : a phase 1 / 2 multiple myeloma research consortium trial . 
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polley my : practical modications to the time - to - event continual reassessment method for phase i cancer trials with fast patient accrual and late - onset toxicities . stat med 30 : 2130 - 2143 , 2011 52 . 
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houede n , thall pf , nguyen h , et al : utility - based optimization of combination therapy using ordinal toxicity and efcacy in phase i / ii trials . 
 harnessing clinical sequencing data for survival stratication of patients with metastatic lung adenocarcinomas ronglai shen , phd1 ; axel martin , ms1 ; ai ni , phd1 ; matthew hellmann , md1 ; kathryn c . 
we sought to extend the use of broad - panel sequencing results to survival stratication and clinical outcome prediction . methods by using sequencing results from a cohort of 1 , 054 patients with advanced lung adenocarcinomas , we developed oncocast , a machine learning tool for survival risk stratication and biomarker identication . results with oncocast , we stratied this patient cohort into four risk groups on the basis of tumor genomic prole . 
2019 by american society of clinical oncology introduction with the growth of precision medicine programs driven by genomic testing as well as recent us food and drug administration clearance of next - generation sequencing ( ngs ) platforms for clinical use , there has been rapid growth in the availability of broad - panel sequencing data for patients with cancer . 
zehir et al1 delineated the molecular landscape of 10 , 000 metastatic cancers in a pretreated real - world cohort sequenced by the memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) platform , a hybridization capture - based ngs panel that can detect mutations and copy number alterations in 341 or more cancer - associated genes.2 this study showed that 37% of patients harbored at least one therapeutically actionable alteration , and 11% were matched to genome - directed clinical trials . in patients with lung adenocarcinoma , tumor genotyping is now an essential step in routine clinical decision making . 
to determine treatment , patients with lung adenocarcinomas are currently categorized on the basis of the presence of mutated driver oncogenes ( eg , egfr , alk , ros1 , and braf )  . 
a multiinstitutional study characterized genetic aberrations across 10 genes in 733 tumor samples and identied an oncogenic driver in 64% of the patients.3 by using broad - panel sequencing , jordan et al4 reported on a single - institution experience of 860 patients with metastatic lung adenocarcinoma . 
more than 37% of patients received a matched therapy , and the use of matched therapy was strongly inuenced by the level of pre - existent clinical evidence that the mutation identied predicts the drug response . although the focus of tumor genotyping has been on ascertaining mutations that identify therapeutic targets , there is considerable unexplained variability in clinical outcomes , even within specic molecular subsets of patients with metastatic cancer . 
 shen et al death - ligand 1 ( pd - 1 / pd - l1 ) inhibitors.5 however , additional markers are needed to predict durable benet and long - term survival among these patients . 
some studies have explored the effects of single co - occurring alterations on outcome in patients with egfr - mutant and kras - mutant lung adenocarcinoma , and they observed that the presence or absence of pairs of co - occurring events could be used to identify those patients with a poor prognosis most in need of novel therapeutic approaches.3 , 6 - 8 however , a systematic approach is needed to additionally improve our understanding of survival and treatment outcome of patients . thus , we developed oncocast , a computational tool for survival stratication , and applied it to a large clinical series of patients with metastatic lung adenocarcinomas to improve the understanding of heterogeneity in clinical outcome for these patients and the mutation and comutational patterns that underlie such heterogeneity . 
such real - world evidence can supplement information from randomized clinical trials in that the results are more generalizable to patients treated outside randomized clinical trials , and the larger sample sizes of real - world data sets allow subset analysis that clinical trials are not powered for . 
the open - source computational pipeline that we have developed can facilitate the application of statistical and machine learning approaches for clinico - genomics analysis of precision medicine data sets . methods the oncocast method is described in the data supplement . 
electronic medical records were used to identify patient clinical factors as well as survival outcomes as previously described.4 overall survival ( os ) was dened as the time from date of diagnosis of advanced disease ( stage iv or recurrent cancer ) until date of death or last follow - up . 
however , a fraction ( 21% ) of the tumors were sampled and sequenced more than 6 months from the date of metastatic disease , with 16% sequenced at more than 1 year and 8% sequenced at more than 2 years , which represents older samples used for sequencing analysis . 
those patients with older samples which had been taken at initial diagnosis of from their initial advanced lung cancer were immortal sampling time to the time of referral for msk - impact sequencing . 
this interval can be long for a small fraction of patients ( 8% with a delayed interval of more than 2 years , as mentioned earlier ) , which introduces survival bias . 
details of lefttruncation analysis are described in the data supplement . the most frequently mutated genes were tp53 ( 55.1% ) , kras ( 30% ) , egfr ( 29.4% ) , stk11 ( 17.7% ) , and keap1 ( 17.7% ; data supplement )  . 
among the frequently comutated gene pairs , stk11 and keap1 were comutated in 10% of the tumors , and kras and stk11 were comutated in 9% of the tumors ( data supplement )  . prognostic relevance and clonality of cancer genes to dene the prognostic signicance of msk - impact panel genes that were sequenced , we developed oncocast , a machine learning tool for survival risk stratication and biomarker identication by implementing a lassopenalized proportional hazards regression for deriving prediction rules for os and feature selection ( data supplement )  . 
oncocast uses an ensemble learning strategy by repeatedly splitting the cohort into training and test sets that generate an ensemble of classiers with varying selection of genes and gene combinations ( data supplement )  . the median number of prognostic genes selected was 19 ( range , 4 - 67 genes ) , with a total of 169 cancer genes selected at least once by the ensemble learner . 
the relative prognostic importance of each gene was measured by how often it was selected ( fraction of the models containing the gene ) and its average regression coefcients , which determined the corresponding weights for individual genes in the scoring rule . 
circle size is proportional to mutation frequency . targeted and immunotherapies than subclonal events.9 - 11 we thus used the high depth of coverage afforded by our sequencing data ( mean coverage , 758 ) to determine the clonality of the gene alterations found to be associated with prognosis in the analysis we outlined earlier . 
 shen et al oncocast : an integrated prognostic scoring system based on ngs tumor proling in addition , improvement . oncocast aggregates prognostic effects across the sequenced cancer genes to derive a genomic risk score for each patient ( scaled from 0 to 10 ) , with a higher score indicating a greater likelihood of shorter survival . 
there was a difference of more than 6 units in average risk score between the highand low - risk groups . the oncocast classication substantially outperformed all of the individual genes as a predictor of os ( fig 3b )  . 
oncocast risk score remained a highly signicant predictor after adjusting for clinical variables and treatment types as potential confounding factors in a multivariable cox regression model ( data supplement )  . to conrm the validity of the oncocast survival stratication , we applied it to a separate data set of patients with mostly early - stage nonsmall - cell lung cancer obtained from the cancer genome atlas lung adenocarcinoma analysis . 
stk11 is a tumor suppressor gene that encodes for the serine / threonine kinase lkb1 that functions as a negative regulator of mammalian target of rapamycin ( mtor ) signaling . 
to examine the status of the other allele in such cases , we performed allele - specic cna using the fraction and allele - specic copy number estimates from tumor sequencing ( facets ) algorithm.15 strikingly , more than 90% of stk11 mutant tumors had evidence of biallelic inactivation through loss of heterogeneity ( loh ) of chromosome 19p ( data supplement )  . 
the majority of keap1 mutations were missense , and the mutant copy was frequently duplicated ( present as copy - neutral loh and uniparental gains ) as reected in the average total copy number . we also explored whether other tumor characteristics such as tumor mutational signature or intratumor heterogeneity were prognostic and associated with oncocast risk score . previously dened smoking - associated and apolipoprotein b mrna editing enzyme , catalytic polypeptide - like ( apobec ) signatures were the most prevalent mutational signatures in this cohort . 
however , mutation burden did not provide additional prognostic value beyond oncocast risk score in a multivariable cox model ( data supplement )  . clonal diversity was calculated for each tumor by summarizing the cancer cell fraction for all somatic mutations using the shannon index . 
 ( a ) kaplan - meier plot of survival curves for the four risk subgroups ( low , intermediate - low [ int - low ] , intermediate - high [ int - high ] , and high )  . 
the average risk score ( avg rs ) , median overall survival ( os ) , and 1 - year and 3 - year survival probabilities are reported for each group . 
 ( b ) forest plot of hazard ratios ( hrs ) for age , sex , smoking status , and individual driver gene alterations compared with the oncocast integrated scoring approach . genotypes associated with risk groups to better understand the association between targetable cancer driver mutations and oncocast risk score , we explored the distribution of genotypes within four major risk categories ( fig 5 )  . 
the dening characteristic for the patients in the low - risk group without egfr , alk , ret , and ros1 alterations was an absence of any of the poor prognostic gene alterations . 
this suggests that stk11 and keap1 comutation denes a cohort of patients with resected lung adenocarcinoma at higher risk of disease progression and cancerspecic mortality . survival stratication in specic treatment subsets we then sought to demonstrate the ability of the oncocast technique to explore tumor genomic predictor outcomes in last to death or the subset of patients with mutated driver oncogenes treated with kinase inhibitors . 
the model , oncocast - tr ( targeted therapies model ) , was derived using the genomic proles and survival outcomes from the start of therapy for the 387 patients who received targeted therapies ( egfr , alk , ros )  . 
tp53 is strongly associated with worse survival outcome ( fig 6c ) , with 96% of patients in the tr2 group harboring concurrent tp53 mutations and 0% concurrent tp53 mutations in the tr1 group . 
arid1a also showed association with poor survival with 11 ( 78% ) of 14 arid1a mutations in tr2 . interactive tool for visualizing and exploring mutation pattern and survival to facilitate clinical translation and research use of the data , we created an interactive web application ( data that allows for visualization of mutation supplement ) ros1 fusion , 6% ret fusion , low - intermediate tp53 , egfr , smarca4 , 2% tp53 , ret fusion , 1% tp53 , alk fusion , 1% tp53 , ros1 fusion , 2% tp53 , smarca4 , 3% egfr , 40% tp53 , kras , tp53 , egfr , tp53 , egfr , alk fusion , other , 30% kras , 17% tp53 , 24% high - intermediate keap1 , 2% stk11 , 5% tp53 , kras , stk11 , 6% tp53 , kras , keap1 , 6% tp53 , keap1 , kras , keap1 , kras , stk11 , stk11 , keap1 , smarca4 , 7% tp53 , kras , stk11 , keap1 , 8% stk11 , keap1 , 8% high tp53 , stk11 , keap1 , smarca4 , 6% kras , stk11 , keap1 , 28% tp53 , stk11 , keap1 , 25% tp53 , stk11 , 10% kras , stk11 , keap1 , smarca4 , 16% fig 5 . 
 ( c ) gene importance plot for the oncocast - tr model . patterns and individualized prediction of os to be generated on the basis of a user - dened mutational prole and clinical characteristics . 
along with a patients clinical prole , the application outputs the predicted survival probabilities and 95% cis . although the tool was built using the largest metastatic lung adenocarcinoma cohort to date , it was designed to be dynamically updated as new data are incorporated into the model . discussion tumors from patients with lung adenocarcinoma have a high frequency of actionable oncogenic drivers.16 , 17 the introduction of targeted therapy has transformed the clinical care of patients with lung adenocarcinoma by incorporating tumor genotyping into therapeutic decision making . 
by using an ensemble learning approach , we demonstrated the prognostic utility of data from a large panel ngs assay interrogating 341 cancer - associated genes in 1 , 054 patients with metastatic lung adenocarcinoma treated with currently available therapies . 
we show that it is a practical approach to molecular stratication in metastatic lung adenocarcinoma and provides the ability to identify biomarkers that predict survival outcome . overlaying the oncocast risk score with the tumor genomic landscape revealed novel biologic and clinical insights . 
the major prognostic genes associated with poor survival included stk11 , keap1 , tp53 , kras , and smarca4 . remarkably , comutations of both stk11 and keap1 dened an exceptionally high - risk prole with a short median os of 7.3 months and an increase of more than 6 units in risk score compared with a low - risk group , corresponding to an hr of 4.6. 
some of the favorable prognostic genes may reect the availability of effective targeted therapies in the case of egfr , alk , and ros1 . however , in patients with these targetable oncogenes , there is heterogeneity in the additional mutations their tumors harbor . 
for example , our model revealed that mutations in tp53 and arid1a dene a high - risk subgroup with shorter survival in the tyrosine kinase inhibitortreated patient cohort . is novel in that conversely , some of the unfavorable prognostic genes may reect negative associations with treatment responses ( eg , the recent observation that stk11 inactivation is associated with poor responses to immunotherapy18 )  . 
mutations in keap1 are associated with chemotherapy resistance and poor survival . some studies have suggested that targeting nrf2 may enhance chemotherapy sensitization.19 , 20 mtor is a kinase downstream of lkb1 ( stk11 ) , and mtor inhibitors have been proposed as a potential therapeutic approach in stk11 - mutant tumors.21 smarca4 was also identied as a major driver of a poor prognosis factor in metastatic lung adenocarcinoma in our study . 
 shen et al complexes that regulate genomic instability and dna repair . smarca4 was mutated in 9.4% of lung adenocarcinomas in the msk - impact cohort , with 42% of the mutations being truncating and residing in regions of loh . 
with increasing sample sizes , we will be poised to identify outcome associations with rare alterations , and we will be better able to explain the heterogeneity in clinical outcomes for patients with advanced lung adenocarcinoma . in addition , the oncocast risk score described here provides a valuable tool for more accurately determining the prognosis of patients enrolled in clinical trials or included in real - world data sets . 
although conventional clinical factors such as age , sex , and performance status have long been used , our data indicate that we can signicantly improve the description of patient populations by incorporating the genomic risk scores in our understanding to allow better comparisons of groups of patients enrolled in clinical trials or included in real - world data sets . with the growth of precision medicine programs driven by genomic testing as well as recent us food and drug administration clearance of ngs platforms for clinical use , there will be a rapid growth in the availability of broad sequencing data for patients with a variety of cancers and growing integration with clinical data through institutional databases and electronic health records . 
riely manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors matthew hellmann stock and other ownership interests : shattuck labs honoraria : astrazeneca , bristol - myers squibb consulting or advisory role : bristol - myers squibb , merck , genentech , astrazeneca / medimmune , novartis , janssen , nektar therapeutics , syndax pharmaceuticals , mirati therapeutics , shattuck labs research funding : bristol - myers squibb ( inst ) patents , royalties , other intellectual property : a patent has been led by memorial sloan kettering ( pct / us2015 / 062208 ) for the use of tumor mutation burden for prediction of immunotherapy efcacy , which is licensed to personal genome diagnostics ( inst ) travel , accommodations , expenses : astrazeneca , bristol - myers squibb kathryn c . 
rudin consulting or advisory role : bristol - myers squibb , abbvie , seattle genetics , harpoon therapeutics , genentech , astrazeneca , ascentage pharma , bicycle therapeutics , celgene , daiichi sankyo , ipsen , loxo oncology , pharmamar , elucida oncology research funding : abbvie / stemcentrx ( inst ) , viralytics ( inst ) , merck ( inst ) , daiichi sankyo ( inst ) michael f . 
solit stock and other ownership interests : loxo oncology consulting or advisory role : pzer , loxo oncology , illumina , intezyne technologies , vivideon therapeutics travel , accommodations , expenses : merck maria arcila consulting or advisory role : astrazeneca travel , accommodations , expenses : astrazeneca , invivoscribe technologies , raindance technologies marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network / astrazeneca tagrisso request for proposal advisory committee , takeda pharmaceuticals , bristol - myers squibb , bayer ag , merck ( i ) research funding : loxo oncology ( inst ) , helsinn therapeutics gregory j . 
riely research funding : novartis ( inst ) , genentech ( inst ) , millennium pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , pzer ( inst ) , innity pharmaceuticals ( inst ) , ariad pharmaceuticals ( inst ) patents , royalties , other intellectual property : patent application submitted covering pulsatile use of erlotinib to treat or prevent brain metastases ( inst ) travel , accommodations , expenses : merck sharp & dohme no other potential conicts of interest were reported . references zehir a , benayed r , shah rh , et al : mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10 , 000 patients . 
nat med 23 : 703 - 713 , 2017 cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
j mol diagn 17 : 251 - 264 , 2015 kris mg , johnson be , berry ld , et al : using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs . 
jama 311 : 1998 - 2006 , 2014 jordan ej , kim hr , arcila me , et al : prospective comprehensive molecular characterization of lung adenocarcinomas for efcient patient matching to approved and emerging therapies . 
hellmann md , nathanson t , rizvi h , et al : genomic features of response to combination immunotherapy in patients with advanced non - small - cell lung cancer . cancer cell 33 : 843 - 852.e4 , 2018 arbour kc , jordan e , kim hr , et al : effects of co - occurring genomic alterations on outcomes in patients with kras - mutant non - small cell lung cancer . 
clin cancer res 24 : 334 - 340 , 2018 yu ha , suzawa k , jordan e , et al : concurrent alterations in egfr - mutant lung cancers associated with resistance to egfr kinase inhibitors and characterization of mtor as a mediator of resistance . 
clin cancer res 24 : 3108 - 3118 , 2018 yu ha , sima cs , shen r , et al : prognostic impact of kras mutation subtypes in 677 patients with metastatic lung adenocarcinomas . 
j thorac oncol 10 : 431 - 437 , 2015 de bruin ec , mcgranahan n , mitter r , et al : spatial and temporal diversity in genomic instability processes denes lung cancer evolution . 
rizvi h , sanchez - vega f , la k , et al : molecular determinants of response to anti - programmed cell death ( pd ) - 1 and anti - programmed death - ligand 1 ( pd - l1 ) blockade in patients with non - small - cell lung cancer proled with targeted next - generation sequencing . 
azzoli cg , temin s , giaccone g : 2011 focused update of 2009 american society of clinical oncology clinical practice guideline update on chemotherapy for stage iv non - small - cell lung cancer . 
directors challenge consortium for the molecular classication of lung adenocarcinoma , shedden k , taylor jm , et al : gene expression - based survival prediction in lung adenocarcinoma : a multi - site , blinded validation study . 
ren d , villeneuve nf , jiang t , et al : brusatol enhances the efcacy of chemotherapy by inhibiting the nrf2 - mediated defense mechanisproc natl acad sci u 21 . 
tagal v , wei s , zhang w , et al : smarca4 - inactivating mutations increase sensitivity to aurora kinase a inhibitor vx - 680 in non - small cell lung cancers . 
 impact of genetic ancestry on outcomes in ecog - acrin - 5103 purpose racial disparity in breast cancer outcomes exists between african american and white women in the united states . 
we have evaluated the impact of genetically determined ancestry on disparity in efficacy and therapy - induced toxicity for patients with breast cancer in the context of a randomized , phase iii adjuvant trial . methods this study compared outcomes between 386 patients of african ancestry ( aa ) and 2 , 473 patients of european ancestry ( ea ) in a randomized , phase iii breast cancer trial , ecogacrin - 5103 . 
the primary efficacy end point , invasive diseasefree survival ( dfs ) , and clinically significant toxicities were compared , including anthracycline - induced congestive heart failure , taxane - induced peripheral neuropathy ( tipn ) , and bevacizumab - induced hypertension . results overall , aas had significantly inferior dfs ( p = .002 ; hazard ratio , 1.5 ) compared with eas . 
2017 by american society of clinical oncology introduction african american patients with breast cancer have inferior efficacy outcomes compared with other races.1 , 2 the reason for this imbalance is multifactorial and includes higher stage , higher grade , more triple - negative breast cancer ( tnbc ) , and poorer responsiveness to chemotherapy.3 , 4 these clinical imbalances have previously been attributed to both socioeconomic factors and a different underlying biology of the tumor.2 , 5 patients of african ancestry ( aa ) also experience more adverse drug reactions , including an increase in clinically important toxicities for chemotherapeutic agents . 
although less well characterized , these toxicity disparities are also likely multifactorial and include inherited genetic variation and comorbidities , among other factors.6 - 10 much of the prior work has been based on selfreported race . 
 node - negative breast cancer to intravenous doxorubicin and cyclophosphamide ( ac ) every 2 or 3 weeks ( at discretion of treating physician ) for four cycles followed by 12 weeks of paclitaxel ( 80 mg / m2 once per week ) alone ( arm a ) or to the same chemotherapy with either concurrent bevacizumab ( arm b ) or concurrent plus sequential bevacizumab ( arm c ) ; figure 1a . 
patients with estrogen receptor ( er ) positive disease were ln positive , had a tumor > 5 cm or a tumor measuring 1 to 5 cm , with a recurrence score > 11 . genome - wide association study germline dna from whole blood and companion clinical outcome data were available in 3 , 126 patients . 
if arm c significantly improved dfs relative to arm a , then in the second step , a comparison of arm b with arm a was performed.14 in this correlative study , dfs and overall survival ( os ) were evaluated using kaplan - meier methodology . 
the differences in outcomes between aas and eas were compared with the application of cox proportionalhazards models using either univariate or multivariate analysis , which were corrected with significant covariates . 
to identify the best regression model , a forward and a backward stepwise selection procedure were performed separately to evaluate variable associations , and the akaike information criterion was used to determine the inclusion of potential confounders such as race , menopausal status , age , weight , height , side of cancer involvement , er status , histologic grade , nuclear grade , ln status , type of surgery , types of ac schedule , and dose reductions in ac or paclitaxel . both procedures returned the same model with six predictors , which are listed in the data supplement . dfs was the primary end point of the parent trial ; it was defined as invasive dfs and calculated from the date of random assignment to the date of first treatment failure ( invasive ipsilateral , local / regional invasive , distant recurrence , invasive contralateral breast cancer , invasive nonbreast second primary , or death from any cause [ whichever occurred first ] )  . cases with incomplete follow - up , without a documented invasive dfs event ( including those who developed squamous or basal cell skin cancers or in situ carcinomas of any site as their only event ) were censored at the date of last disease evaluation . 
we previously published biomarker data on each of these toxicities.15 , 17 , 18 in the present study , we sought to compare the frequency of all toxicities between aas and eas and to assess the impact of race on dose modifications . 
statistical analysis was performed using r ( version 3.3.0 ) and the x2 test . odds ratio ( or ) was used to evaluate the significance and magnitude of the differences in the toxicity frequency between aas and eas . chf cases for this study included individuals who had centrally reviewed , cardiologist - adjudicated chf . 
to be selected for inclusion in ecogacrin - 5103 , patients must have had no history of clinically significant cardiovascular disease . cardiovascular health was monitored at the start and during the trial by multigated angiograms or echocardiograms . 
cardiac events included chf , decrease in left ventricle ejection fraction , acute coronary syndrome , supraventricular tachycardia , and myocardial dysfunction diagnosed by a cardiologist . tipn cases for this study were defined as those experiencing either grades 2 to 4 or grades 3 to 4 tipn as assessed by ctcae . 
to serve as a tipn case , the patient must have received at least one dose of paclitaxel , and the neuropathy event must have occurred during treatment or within 3 months of the last dose of therapy . hypertension cases for this study were defined as those experiencing a systolic blood pressure ( sbp )  . 
blood pressure values were collected as part of standard clinical assessment before administration of therapy throughout the conduct of the trial . results genotyped group from ecog - acrin5103 and genetic ancestry a total of 3 , 394 germline dna samples from patients were collected as part of the planned correlative protocol within ecog - acrin5103 ( fig 1b )  . 
the outcomes of the entire genotyped cohort ( all ancestries combined ) used in this correlative study were almost identical to the parent trial4 ( data supplement )  . demographic data and disease comparisons table 1 summarizes the important demographic comparisons between aas and eas . 
we assessed for the differences in the parent trial stratification factors as well as other known outcome predictors , including age , hormone receptor status ( er positive v tnbc ) , ln status , height , weight , type of surgery , and tumor grade . 
there were no significant differences overall in ln status . genetic ancestry as a predictor of efficacy in ecog - acrin - 5103 dfs was the primary efficacy end point of the parent trial . 
an imbalance in associated comorbidities or environmental factors cannot be excluded as a contributing cause , and unfortunately , those data were not collected in ecog - acrin - 5103 . 
dose reductions in paclitaxel also negatively affected dfs for all ancestries ( p = .02 ; data supplement ) ; however , having a dose modification in paclitaxel did not affect dfs for eas , but it did significantly cause an inferior dfs for aas ( fig 4 )  . the difference may have been explained by more severe dose reductions in aas . 
the outcomes for the subgroup genotyped , however , were similar to the parent population , thus minimizing the concern for subgroup bias . we also compared the results of the genetic ancestry with self - reported race , and there was no significant difference in the conclusion . 
work in other disease phenotypes , however , has demonstrated that use of self - reported race as a surrogate for genetic ancestry was not perfect.25 - 27 prior work has demonstrated that  . 
9% of patients cannot supply , or choose not to supply , ancestry information.28 thus , the real impact of accurately self - reported race is probably larger than the 9% that was observed , as the result of misclassification alone . 
although genotype may provide more insight toward underlying biologic diversity , selfreported race still likely represents a reasonable surrogate for genotypic variation in the routine clinical setting for consideration of metabolism and drug toxicity . we previously evaluated genetic markers to predict some of the most clinically important toxicities in ecog - acrin - 5103 : bevacizumabinduced hypertension ( using various definitions ) , tipn , and cardiologist - adjudicated anthracyclineinduced chf.15 , 17 , 18 in this study we compared the likelihood of each of these clinically relevant toxicities between aas and eas . 
these data support prior work from a single - institution retrospective analysis as well as data in pediatric populations that revealed higher likelihood of anthracycline - induced chf in aas . 
similarly , a prior single - institution retrospective analysis demonstrated higher risk of tipn for aas.8 - 10 because aas experience greater toxicity , it is highly unlikely that the imbalance in efficacy is a result of exposure as the result of pharmacokinetic considerations , as the end organs are clearly being affected . 
as expected , all ancestries that had dose reductions in the ac portion of the chemotherapy had inferior dfs ; this difference , however , was present for both eas and aas . 
the significant difference in dfs as the result of paclitaxel dose modifications for aas may have been because of the markedly more severe dose reductions ; this was evidencedby alower meannormalizeddosein thosewho had dose reductions ( p = .03 ) , in large part because table 2 . 
this study did not evaluate socioeconomic factors , which are known to be important variables in outcomes.5 however , in the context of a randomized phase iii trial , many of these inequities are minimized . in conclusion , this study highlights the need to better understand the biologic differences in normal breast biology , tumor biology , and the inherited genetic differences between women of aa and ea . 
it also highlights the need to better personalize counseling when discussing the risk - to - benefit ratio for aas , for whom the disease - specific outcomes are inferior and drug - specific toxicities are higher . 
ecog - 1199 previously demonstrated that the dosing of docetaxel every 3 weeks was as effective but had fewer dose reductions for aas in a similar clinical setting.20 future trials should investigate whether another taxane with less risk of tipn , such as docetaxel , might be more effective for aas in the adjuvant breast cancer setting . 
sparano stock and other ownership interests : metastat consulting or advisory role : genentech , novartis , astrazeneca , celgene , eli lilly , celldex , pfizer , prescient therapeutics , juno therapeutics , merrimack research funding : prescient therapeutics ( inst ) , deciphera ( inst ) , genentech ( inst ) , merck ( inst ) , novartis ( inst ) , merrimack ( inst ) george w . 
sledge jr leadership : syndax stock and other ownership interests : syndax honoraria : symphogen consulting or advisory role : symphogen , coherus biosciences , radius health , peregrine pharmaceuticals , taiho pharmaceutical research funding : genentech ( inst ) travel , accommodations , expenses : nektar , radius health , taiho pharmaceutical kathy d . 
miller consulting or advisory role : nektar , tesaro , merck research funding : genentech ( inst ) , merrimack ( inst ) , entremed ( inst ) , taiho pharmaceutical ( inst ) , macrogenics ( inst ) , medivation ( inst ) , novartis ( inst ) , seattle genetics ( inst ) affiliations support bryan p . 
schneider , fei shen , guanglong jiang , milan radovich , lang li , laura gardner , dongbing lai , tatiana foroud , and kathy d . miller , indiana university school of medicine , indianapolis , in ; anne oneill , dana farber cancer instituteecog - acrin biostatistics center , boston , ma ; joseph a . 
keenan t , moy b , mroz ea , et al : comparison of the genomic landscape between primary breast cancer in african american versus white women and the association of racial differences with tumor recurrence . 
desantis c , jemal a , ward e : disparities in breast cancer prognostic factors by race , insurance status , and education . jama 295 : 2492 - 2502 , 2006 cancer causes control 21 : 1445 - 1450 , 2010 americans . 
kidd rs , curry tb , gallagher s , et al : identification of a null allele of cyp2c9 in an african - american exhibiting toxicity to phenytopharmacogenetics 11 : 803 - 808 , 2001 8 . 
bhatnagar b , gilmore s , goloubeva o , et al : chemotherapy dose reduction due to chemotherapy induced peripheral neuropathy in breast cancer patients receiving chemotherapy in the neoadjuvant or adjuvant settings : a single - center experience . 
cobb rj , thomas cs , laster pirtle wn , et al : self - identified race , socially assigned skin tone , and adult physiological dysregulation : assessing multiple dimensions of race in health disparities research . 
miller k , oneill am , dang ct , et al : bevacizumab ( bv ) in the adjuvant treatment of her2 - negative breast cancer : final results from eastern cooperative oncology group e5103 . 
schneider bp , li l , radovich m , et al : genome - wide association studies for taxane - induced peripheral neuropathy in ecog - 5103 and ecog - 1199 . 
killelea bk , yang vq , wang sy , et al : racial differences in the use and outcome of neoadjuvant chemotherapy for breast cancer : results from the national cancer data base . 
halder i , shriver m , thomas m , et al : a panel of ancestry informative markers for estimating individual biogeographical ancestry and admixture from four continents : utility and applications . 
louwers yv , lao o , fauser bc , et al : the impact of self - reported ethnicity versus genetic ancestry on phenotypic characteristics of polycystic ovary syndrome ( pcos )  . 
 c distinct dicer1 hotspot mutations identify bilateral tumors as separate events introduction dicer1 syndrome1 predisposes to a variety of cancers , including pleuropulmonary blastoma ( ppb ) , 2 ovarian sertoli - leydig cell tumor ( slct ) , 3 embryonal rhabdomyosarcoma , 4 and kidney tumors.5 - 8 in dicer1 syndrome , most patients bear a germline null mutation in dicer1 , and the tumors uniformly bear a second - hit missense substitution at one of five hotspot positions ( 1705e , 1709d , 1809g , 1810d , and 1813e )  . a wide range of clinical phenotypes can be seen in dicer1 syndrome5 , 9 ; some patients are asymptomatic , whereas others develop multiple tumors . 
in the classic cases , patients with a germline null mutation develop different somatic hotspot mutations in each tumor.7 , 10 - 16 however , in some patients , multiple tumors arise from germline mosaicism of the hotspot mutation , with subsequent somatic loss of the wild - type allele.17 - 19 here we report a patient with dicer1 syndrome who developed four tumor types at six anatomic sites over the course of 12 years . 
because we found no other mutations to explain her particularly severe clinical course , we speculate that her subsequent tumors were a product of the intense chemotherapy and radiation regimens she received . methods informed consent was obtained from the patient and her guardian before collection of tumor specimens . 
when needed , pcr products were subcloned into pcr2.1topo ( thermo - fisher scientific , waltham , ma ) before sequencing . for tp53 sequencing , coding regions were amplified using the accel - amplicon comprehensive tp53 panel ( swift biosciences , ann arbor , mi )  . 
reads were trimmed using trimmomatic21 in the galaxy project and were aligned to human genome grch38 using bwa - mem22 ; 88% mapped to the tp53 locus , producing a mean depth of 13 , 367x in targeted regions . 
variants were called using the freebayes algorithm v0.9.20 using frequency - based pooled calling instead of simple diploid calling , which allowed the algorithm to detect subclonal mutations.23 results clinical presentation the patient ( cmcw11 ) presented originally at 5 years of age with a left kidney mass . 
she was treated with 10 months of combination chemotherapy ( vincristine , doxorubicin , cyclophosphamide , irinotecan , and etoposide ) targeted at both anaplastic wilms tumor and sarcoma , together with 2 , 100 cgy whole - abdomen irradiation given in 14 fractions over 19 days . when the patient was 10 years old , a new mass arose in her remaining kidney , with a histology similar to her prior tumor , and she was diagnosed with relapsed wilms tumor ( fig 1b )  . 
 on completion , she underwent right radical nephrectomy and started hemodialysis . this patients kidney tumors were diagnosed initially as wilms tumor , but in retrospect their unique histologic features are more consistent with anaplastic sarcoma of the kidney . 
this entity was first described in 2007 , after our patients kidney tumor first arose.24 biallelic dicer1 mutations have been seen in both wilms tumor and anaplastic sarcoma of the kidney , which may represent neoplastic degeneration from cystic nephroma.6 - 8 at 12 years of age , the patient developed multiple nodules throughout her thyroid . 
thyroidectomy revealed follicular adenomas ( fig 1c )  . at 13 years of age , a mass arising from her bladder was diagnosed as embryonal rhabdomyosarcoma with focal cartilaginous differentiation ( fig 1d )  . 
sanger sequencing chromatograms demonstrating the patients ( a ) dicer1 germline frameshift mutation and ( b ) rnase iiib hotspot mutations in her ovarian sertoli - leydig cell tumors ( slcts ) ; ( c ) left ( l ) kidney tumor ; and ( d ) right ( r ) kidney tumor . 
since that time , she has been observed for > 2 years , with no further disease . dicer1 mutations we performed whole - exome sequencing on the patients first kidney tumor and germline dna as table 1 . 
her tumor bore an additional somatic hotspot mutation ( c.5425g > a ; p.g1809r ; fig 2 and table 1 )  . we then sequenced the rnase iii domains of dicer1 in archival specimens from her other tumors . 
to sequence strands separately , we subcloned the pcr amplicon spanning this mutation and found that both single - nucleotide variants occurred on the same strand , making the tumor heterozygous for a single somatic hotspot mutation ( appendix fig a1 )  . 
contrary to our clinical suspicion at the time , her contralateral kidney tumor was , in fact , a second primary . we also sequenced dicer1 from her thyroid follicular adenoma and bladder rhabdomyosarcoma . 
bladder rhabdomyosarcomas have been associated previously with dicer1 syndrome , although specific discussion of their histologic features was not reported.4 her two ovarian tumors were detected at the same time , and it was unclear whether they represented distinct primary tumors or metastatic spread . 
her initial renal tumor bore a p.v274l variant , as we described previously.20 however , her remaining tumors had no additional somatic tp53 variants and did not undergo loss of heterozygosity at tp53 . discussion clinically , the discovery of a second tumor with similar histology can raise the question of whether it is a recurrence or a new primary tumor . 
had we known that her second kidney tumor was , in fact , metachronous , she might have avoided the increased toxicity of chemotherapy regimens designed for relapsed or refractory tumors . 
in fact , the cartilaginous elements of her bladder tumor led us to question whether it could instead represent a distant recurrence of her renal sarcoma , although its distinct dicer1 mutation proved its independence . thus , this case highlights a powerful but often overlooked application of clinical tumor sequencing : better understanding of the biology behind tumor formation . 
as sequencing costs fall , clinicians should be cognizant that tumor sequencing can help distinguish between new primary tumors and recurrences . in this case , sequencing at initial diagnosis would have also had the added benefit of alerting clinicians to her potential dicer1 status . 
in the case of dicer1 syndrome , because second hit mutations occur at such stereotyped positions , sequencing separate tumors could even occur by simple sanger sequencing rather than by next - generation sequencing . 
in patients with dicer1 syndrome , dedicated tests may be developed for identifying these stereotyped mutations , perhaps including circulating tumor dna assays as a noninvasive screening method . this is one of the most severe cases in the literature of multiple distinct somatic dicer1 mutations arising in a patient . 
shukla no relationship to disclose jason wang no relationship to disclose veena rajaram no relationship to disclose gordan vujanic no relationship to disclose tamra slone no relationship to disclose dinesh rakheja no relationship to disclose james f . 
slade i , bacchelli c , davies h , et al : dicer1 syndrome : clarifying the diagnosis , clinical features and management implications of a pleiotropic tumour predisposition syndrome . 
schultz ka , pacheco mc , yang j , et al : ovarian sex cord - stromal tumors , pleuropulmonary blastoma and dicer1 mutations : a report from the international pleuropulmonary blastoma registry . 
schultz ka , harris a , messinger y , et al : ovarian tumors related to intronic mutations in dicer1 : a report from the international ovarian and testicular stromal tumor registry . 
durieux e , descotes f , mauduit c , et al : the co - occurrence of an ovarian sertoli - leydig cell tumor with a thyroid carcinoma is highly suggestive of a dicer1 syndrome . 
klein s , lee h , ghahremani s , et al : expanding the phenotype of mutations in dicer1 : mosaic missense mutations in the rnase iiib domain of dicer1 cause glow syndrome . 
brenneman m , field a , yang j , et al : temporal order of rnase iiib and loss - of - function mutations during development determines phenotype in dicer1 syndrome : a unique variant of the two - hit tumor suppression model . 
vujani gm , kelsey a , perlman ej , et al : anaplastic sarcoma of the kidney : a clinicopathologic study of 20 cases of a new entity with polyphenotypic features . 
pugh tj , yu w , yang j , et al : exome sequencing of pleuropulmonary blastoma reveals frequent biallelic loss of tp53 and two hits in dicer1 resulting in retention of 5p - derived mirna hairpin loop sequences . 
 o plasma androgen receptor copy number status at emergence of metastatic castration - resistant prostate cancer : a pooled multicohort analysis anuradha jayaram , md1 ; anna wingate , msc1 ; daniel wetterskog , phd1 ; vincenza conteduca , md , phd2 ; daniel khalaf , md3 ; mansour taghavi azar sharabiani , phd4 ; fabio calabr `o , md5 ; lorraine barwell , md6 ; susan feyerabend , md7 ; enrique grande , md8 ; alberto martinez - carrasco , msc9 ; albert font , md , phd10 ; alfredo berruti , md11 ; cora n . 
sternberg , md12 ; rob jones , ma , md , phd6 ; florence lefresne , md13 ; marjolein lahaye , msc13 ; shibu thomas , phd14 ; shilpy joshi , phd15 ; dong shen , md , phd14 ; deborah ricci , phd14 ; michael gormley , phd14 ; axel s . 
wyatt , md , phd3 ; and gerhardt attard , md , phd1 purpose increases in androgen receptor ( ar ) copy number ( cn ) can be detected in plasma dna when patients develop metastatic castration - resistant prostate cancer . 
2019 by american society of clinical oncology introduction standard of care treatment at development of metastatic castration - resistant prostate cancer ( mcrpc ) is inhibition of androgen receptor ( ar ) signaling with either abiraterone acetate administered with prednisone ( aap ; 5 mg twice a day ) or enzalutamide . 
 jayaram et al context key objective several studies have assessed plasma dna , including the androgen receptor ( ar ) , as a biomarker for patient stratication . variable plasma ar copy number ( cn ) parameters have been used , making it challenging to interpret differences in associations with outcome observed across multiple analyses . 
the pcr2023 cohort ( clinicaltrials.gov identier : nct01867710 ) was an international , multi - institutional , open - label , parallel - arm , phase ii study of abiraterone acetate in asymptomatic or minimally symptomatic , chemotherapy - nave patients with mcrpc.13 patients were randomly assigned 1 : 1 : 1 : 1 to abiraterone acetate and one of four different glucocorticoid regimens ( prednisone 5 mg twice a day or once a day or 2.5 mg twice a day or dexamethasone 0.5 mg once a day ) to evaluate tolerability . 
the nal cohort , the british columbia ( bc ) cohort , was from a randomized phase ii trial of aap versus enzalutamide ( clinicaltrials.gov identier : nct02125357 ) that was sponsored and conducted by the bc cancer agency.12 for the biomarker analyses from all four studies , only patients with histologically conrmed prostate adenocarcinoma and no neuroendocrine differentiation ; progressive disease despite castrate levels of serum testosterone ( , 50 ng / dl ) ; ongoing medical or prior surgical castration ; and no prior treatment with chemotherapy , aap , or enzalutamide were included . 
all four studies obtained institutional review board and ethics committee approval and were conducted in accordance with the declaration of helsinki and the good clinical practice guidelines of the international conference on harmonization . 
psa levels in cohort a and the premiere and bc cohorts were measured as previously described.10 disease was evaluated radiographically using computed tomography scans of the chest , pcr2023 cohort a pcr2023 patients randomly assigned ( n = 164 ) baseline plasma samples ( n = 151 ) samples without long dna fragments ( n = 98 ) samples with long dna fragments ( n = 53 ) samples optimal for final analysis ( n = 135 ) samples without long dna fragments after size selection ( n = 37 ) samples with long dna fragments after size selection ( n = 16 ) patients on enzalutamide excluded ( n = 2 ) ar copy number ar copy number premiere british columbia crossover study ar copy number ar copy number samples optimal for final analysis ( n = 133 ) ar copy number density maximum log - likelihood density copy number cut point of 1.92 relative hr cumulative relative hr 1.92 cutoff based on maximum log - likelihood ar copy number ar copy number fig 1 . 
 ( b ) ar copy number ( cn ) distribution across the four clinical cohorts ( pcr2023 , cohort a , premiere , and british colombia crossover study ) included in the pooled analysis ( n = 501 )  . 
serum lactate dehydrogenase ( ldh ) and alkaline phosphatase ( alp ) were measured at screening or on day 1 of cycle 1 . outcomes plasma samples were collected prospectively in all four studies with the primary aim of studying associations between plasma dna aberrations and clinical outcome as secondary exploratory objectives . 
for full denitions of outcome measures , see the data supplement . for exploratory analysis of the association between ar status at development of mcrpc and time on adt , the outcome measure of time from start of uninterrupted adt to start of aap , abiraterone acetate plus dexamethasone ( aad ) , or enzalutamide was used . 
the association of clinically relevant baseline factors ( previously shown to be associated with prognosis15 - 17 for os and progression - free survival [ pfs ] ) was examined using univariable and multivariable cox regression models that were performed with stepwise variable selection to identify the prognostic factors for os and pfs . 
all tests were two sided , and an error of 5% was considered signicant . results clinical cohorts and plasma ar status between june 2013 and october 2014 , 164 patients were recruited onto the pcr2023 study at 22 centers in ve countries . 
visual inspection showed that the cut point region overlapped with the turning point of decreasing local hr identied via the maximum log - likelihood statistics approach.14 at increasing cns , the decreasing trend became so prominent that it reached a plateau close to an hr of 1 ( fig 1d )  . 
the clinical prognostic factors selected have been previously demonstrated to be strong independent predictors of os and pfs for ar - targeted therapies.15 , 17 - 20 using stepwise backward elimination , plasma ar gain ; high alp , ldh , and psa ; and the presence of bone and visceral metastases were all dependently associated with poorer os ( table 2 )  . 
for cohort a and the premiere cohort , 69.2% ( 36 of 52 patients ) and 60% ( 56 of 94 patients ) of patients were stage m0 at diagnosis , respectively , whereas in the bc cohort , only men who had metastatic disease detected on computed tomography of the chest , abdomen , and pelvis or whole - body bone scan at start of continuous adt were included . 
the median times from start of adt to second - generation hormone treatment of cohort a and the premiere and bc cohorts were 91 , 165 , and 64 weeks , respectively . 
 plasma ar in mcrpc pcr2023 cohort a time receiving adt ( weeks ) 4 , 096 time receiving adt ( weeks ) 4 , 096 premiere british columbia crossover study time receiving adt ( weeks ) 4 , 096 time receiving adt ( weeks ) 4 , 096 fig 3 . 
in addition , to account for differences in frequency of psa and imaging measurements , end points , follow - up times , and treatment , the mfp was stratied by trial . 
 jayaram et al second - generation ar - targeting agents when the response to adt was shorter than 12 months.23 - 25 rapidly expands after treatment resistance . initiation , leading to to appropriately interrogate our main aim , we focused our genomic analysis solely on ar . 
in an exploratory analysis in two similar but nonrandomized cohorts , we reported that the outcome of patients with mcrpc at a similar stage as patients in our study population but who were treated with taxane chemotherapy was worse in patients with higher circulating dna but not those with plasma ar gain.30 randomized studies are required to investigate whether ar gain can be used for treatment selection . 
 plasma ar in mcrpc daniel khalaf consulting or advisory role : bayer fabio calabr `o consulting or advisory role : pzer susan feyerabend consulting or advisory role : janssen - cilag a grant from astellas to support the conduct of the trial . 
wyatt , gerhardt attard provision of study materials or patients : vincenza conteduca , lorraine barwell , susan feyerabend , enrique grande , albert font , alfredo berruti , cora n . 
wyatt , gerhardt attard collection and assembly of data : anuradha jayaram , anna wingate , daniel wetterskog , vincenza conteduca , lorraine barwell , susan feyerabend , alberto martinez - carrasco , albert font , alfredo berruti , cora n . sternberg , rob jones , dong shen , deborah ricci , michael gormley , axel s . 
wyatt , gerhardt attard data analysis and interpretation : anuradha jayaram , anna wingate , daniel wetterskog , daniel khalaf , mansour taghavi azar sharabiani , fabio calabr `o , susan feyerabend , enrique grande , cora n . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . anna wingate research funding : janssen ( inst ) travel , accommodations , expenses : janssen vincenza conteduca consulting or advisory role : bayer , astellas pharma , janssen - cilag , sano travel , accommodations , expenses : bayer , astellas pharma , janssencilag , sano enrique grande honoraria : pzer , bristol - myers squibb , ipsen , roche , eisai , eusa pharma , msd , genzyme , adacap , novartis , pierre fabre , lexicon , celgene , janssen - cilag , astellas pharma consulting or advisory role : msd , pzer , ipsen research funding : mtem / threshold ( inst ) , roche , pzer ( inst ) , astrazeneca ( inst ) , ipsen ( inst ) travel , accommodations , expenses : bristol - myers squibb , genentech , pzer , janssen - cilag alfredo berruti consulting or advisory role : janssen - cilag , astellas pharma speakers bureau : janssen - cilag research funding : astellas pharma ( inst ) , janssen - cilag ( inst ) travel , accommodations , expenses : janssen - cilag , sano cora n . 
 jayaram et al shibu thomas employment : janssen research & development leadership : janssen research & development stock and other ownership interests : janssen research & development research funding : janssen oncology patents , royalties , other intellectual property : four patents ( inst ) travel , accommodations , expenses : janssen research & development kim n . 
chi honoraria : sano , janssen , astellas pharma , bayer consulting or advisory role : essa , astellas pharma , janssen , sano , amgen , bayer , astrazeneca , roche research funding : janssen ( inst ) , astellas pharma ( inst ) , bayer ( inst ) , sano ( inst ) , tokai pharmaceuticals ( inst ) , eli lilly / imclone ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) , roche ( inst ) dong shen employment : janssen research & development stock and other ownership interests : janssen research & development deborah ricci employment : janssen stock and other ownership interests : janssen patents , royalties , other intellectual property : janssen ( inst ) travel , accommodations , expenses : janssen michael gormley employment : janssen research & development stock and other ownership interests : johnson & johnson patents , royalties , other intellectual property : i have several patents pending where i am listed as the inventor of things that have been developed in the course of my work at janssen research and development ( inst ) travel , accommodations , expenses : janssen research & development axel s . 
merseburger honoraria : janssen - cilag , astellas pharma , ipsen , roche , bristol - myers squibb , eisai , takeda , pzer , novartis consulting or advisory role : msd oncology , bristol - myers squibb , janssen - cilag , astellas pharma , ipsen speakers bureau : ipsen research funding : novartis ( inst ) , astrazeneca ( inst ) , janssen - cilag ( inst ) , bristol - myers squibb ( inst ) , clovis oncology ( inst ) travel , accommodations , expenses : janssen - cilag , astellas pharma , ipsen bertrand tombal honoraria : amgen , astellas pharma , bayer , ferring , sano , janssen , pzer , myovant sciences consulting or advisory role : astellas pharma , bayer , ferring , janssen , takeda , steba biotech , sano speakers bureau : amgen , janssen research funding : ferring ( inst ) travel , accommodations , expenses : amgen , astellas pharma , bayer , ferring , janssen , sano ugo de giorgi consulting or advisory role : pzer , janssen , astellas pharma , sano , bristol - myers squibb , bayer , ipsen , merck research funding : sano ( inst ) , astrazeneca ( inst ) , roche ( inst ) travel , accommodations , expenses : bristol - myers squibb , ipsen , janssen , pzer enrique gonzalez - billalabeitia travel , accommodations , expenses : bristol - myers squibb , pzer , bristolmyers squibb , janssen - cilag , astellas pharma , sano , roche , pzer alexander w . 
wyatt consulting or advisory role : genzyme speakers bureau : janssen research funding : janssen gerhardt attard honoraria : janssen , astellas pharma , janssen ( i ) consulting or advisory role : janssen - cilag , veridex , ventana medical systems , astellas pharma , medivation , novartis , millennium , abbott laboratories , essa , bayer , pzer speakers bureau : janssen , astellas pharma , takeda , sano , ventana medical systems research funding : janssen ( inst ) , arno therapeutics ( inst ) , innocrin pharma ( inst ) patents , royalties , other intellectual property : i am on the institute of cancer research rewards to inventors list of abiraterone acetate travel , accommodations , expenses : janssen , astellas pharma , medivation , ventana medical systems , abbott laboratories , bayer , essa , janssen ( i ) , astellas pharma ( i ) , pzer other relationship : institute of cancer research no other potential conicts of interest were reported . acknowledgment we thank the study participants and study centers . 
we acknowledge all the staff at the spanish oncology genitourinary group for their support in running the premiere trial and apoyo a la investigacion clinica en espana for data management . references beer tm , armstrong aj , rathkopf de , et al : enzalutamide in metastatic prostate cancer before chemotherapy . 
n engl j med 371 : 424 - 433 , 2014 ryan cj , smith mr , de bono js , et al : abiraterone in metastatic prostate cancer without previous chemotherapy . 
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nat genet 50 : 645 - 651 , 2018 kumar a , coleman i , morrissey c , et al : substantial interindividual and limited intraindividual genomic diversity among tumors from men with metastatic prostate cancer . 
nat med 22 : 369 - 378 , 2016 kallio hml , hieta r , latonen l , et al : constitutively active androgen receptor splice variants ar - v3 , ar - v7 and ar - v9 are co - expressed in castration - resistant prostate cancer metastases . 
conteduca v , wetterskog d , sharabiani mta , et al : androgen receptor gene status in plasma dna associates with worse outcome on enzalutamide or abiraterone for castration - resistant prostate cancer : a multi - institution correlative biomarker study . 
annala m , vandekerkhove g , khalaf d , et al : circulating tumor dna genomics correlate with resistance to abiraterone and enzalutamide in prostate cancer . cancer discov 8 : 444 - 457 , 2018 13 . 
attard g , merseburger as , arlt w , et al : assessment of the safety of glucocorticoid regimens in combination with abiraterone acetate for metastatic castrationresistant prostate cancer : a randomized , open - label phase 2 study . 
halabi s , lin cy , kelly wk , et al : updated prognostic model for predicting overall survival in rst - line chemotherapy for patients with metastatic castrationresistant prostate cancer . 
chi kn , kheoh t , ryan cj , et al : a prognostic index model for predicting overall survival in patients with metastatic castration - resistant prostate cancer treated with abiraterone acetate after docetaxel . 
rathkopf de , smith mr , de bono js , et al : updated interim efcacy analysis and long - term safety of abiraterone acetate in metastatic castration - resistant prostate cancer patients without prior chemotherapy ( cou - aa - 302 )  . 
ryan cj , smith mr , fizazi k , et al : abiraterone acetate plus prednisone versus placebo plus prednisone in chemotherapy - naive men with metastatic castrationresistant prostate cancer ( cou - aa - 302 ) : final overall survival analysis of a randomised , double - blind , placebo - controlled phase 3 study . 
armstrong aj , lin p , higano cs , et al : development and validation of a prognostic model for overall survival in chemotherapy - nave men with metastatic castration - resistant prostate cancer . 
de laere b , oeyen s , mayrhofer m , et al : tp53 outperforms other androgen receptor biomarkers to predict abiraterone or enzalutamide outcome in metastatic castration - resistant prostate cancer . 
de laere b , van dam pj , whitington t , et al : comprehensive proling of the androgen receptor in liquid biopsies from castration - resistant prostate cancer reveals novel intra - ar structural variation and splice variant expression patterns . 
loriot y , eymard jc , patrikidou a , et al : prior long response to androgen deprivation predicts response to next - generation androgen receptor axis targeted drugs in castration resistant prostate cancer . 
bellmunt j , kheoh t , yu mk , et al : prior endocrine therapy impact on abiraterone acetate clinical efcacy in metastatic castration - resistant prostate cancer : post - hoc analysis of randomised phase 3 studies . 
hung j , taylor ar , divine gw , et al : the effect of time to castration resistance on outcomes with abiraterone and enzalutamide in metastatic prostate cancer . clin genitourin cancer 14 : 381 - 388 , 2016 26 . 
wang l , dehm sm , hillman dw , et al : a prospective genome - wide study of prostate cancer metastases reveals association of wnt pathway activation and increased cell cycle proliferation with primary resistance to abiraterone acetate - prednisone . 
antonarakis es , lu c , luber b , et al : clinical signicance of androgen receptor splice variant - 7 mrna detection in circulating tumor cells of men with metastatic castration - resistant prostate cancer treated with rstand second - line abiraterone and enzalutamide . 
scher hi , graf rp , schreiber na , et al : assessment of the validity of nuclear - localized androgen receptor splice variant 7 in circulating tumor cells as a predictive biomarker for castration - resistant prostate cancer . 
torquato s , pallavajjala a , goldstein a , et al : genetic alterations detected in cell - free dna are associated with enzalutamide and abiraterone resistance in castration - resistant prostate cancer . 
baseline characteristics of pcr2023 cohort by ar status abiraterone chemotherapy naive ( n = 133 ) ar normal ( n = 111 , 83.5% ) ar gain ( n = 22 , 16.5% ) characteristic median age , years ( range ) median pretreatment psa , mg / l ( range ) median pretreatment ldh , uln ( range ) median pretreatment alp , uln ( range ) site of metastases , no . 
malignant cells have a high proliferative fraction and typically express b - cell lymphoma 2 ( bcl2 ) , interferon regulatory factor 4 ( irf4 ) , and b - cell lymphoma 6 ( bcl6 ) on immunohistochemistry . gene expression proling is associated with constitutive nuclear factor ( nf ) - b pathway activation and up - regulation of apoptosis - inhibiting genes , similar to ndings in abc dlbcl . 
mutations of myd88 , resulting in constitutive nf - b activation , are reported in pbdlbcl - lt in up to 69% of cases6 , 7 and are associated with a worse clinical outcome.7 apoptosis gene expression proling in pcdlbcl - lt has shown not only high expression levels of antiapoptotic genes but also concomitant downstream inhibition of the intrinsic apoptosis pathway , as reported in chemotherapy - refractory nodal abc dlbcl.8 , 9 treatment of relapsed / refractory pcdlbcl - lt remains a signicant challenge , with many patients succumbing to uncontrolled disease.3 current therapeutic options , including access to clinical trials , are limited because of both the rarity of disease and the often frail , comorbid patient population . 
small case series of patients with myd88 - mutated pcdlbcl - lt have demonstrated responses to inhibition of b cell receptor ( bcr ) signaling via bruton 's tyrosine kinase ( btk ) inhibition , although responses are of short duration ( 4 to 40 weeks ) and associated with rapid emergence of resistance - conferring mutations.10 , 11 the efcacy of the specic bcl2 inhibitor venetoclax in pcdlbcl - lt remains unexplored . 
this study was approved by the university hospitals of leicester nhs trust ( uk ; 06 / q2501 / 122 )  . research and development written informed consent was obtained for publication purposes . expression of cd20 , cd79a , paired box 5 ( pax5 ) , bcl2 , bcl6 , irf4 , and cd10 , with a ki67 fraction of 90% ( figs 1a - 1d )  . 
 ( a ) hematoxylin and eosin staining , ( b ) interferon regulatory factor 4 ( irf4 ) , ( c ) b - cell lymphoma 2 ( bcl2 ) , and ( d ) ki67 expression . 
in 2010 , he relapsed again with left leg cutaneous involvement only , adjacent to the previously treated sites , and received additional treatment with four cycles of r - chop and radiotherapy ( 40 gy in 15 fractions ) , again attaining clinical response . 
two additional relapses in the left leg with progressively shorter disease - free intervals were treated with radiotherapy alone in 2011 and 2012 ( 4 gy , single fraction )  . 
subsequently , in 2016 , an additional relapse , treated with rituximab , gemcitabine , cyclophosphamide , vincristine , and prednisolone , was refractory to chemotherapy . because of high expression of bcl2 and the development of chemorefractory disease , in january 2017 we obtained venetoclax through the uk abbvie compassionate access scheme . 
repeat staging by ct and 18f - labeled uorodeoxyglucosepositron emission tomography / ct scans conrmed that disease remained localized to the left lower leg , as shown in figure 1e . 
the dose of venetoclax was escalated to 1 , 200 mg because of published data in dlbcl and follicular lymphoma conrming safety and tolerability of this dose.12 however , 1 , 200 mg was not tolerated because of persistent grade 2 diarrhea and grade 2 fatigue , and thus 800 mg was adopted as a maintenance dose . 
biopsy after 2 weeks of treatment showed a complete histologic and immunohistochemical response , although ighv polymerase chain reaction showed persistence of clonal b cells ( data not shown )  . 
treatment with venetoclax was stopped 1 month after demonstration of metabolic remission . however , within 4 weeks of stopping venetoclax , disease recurred , once again conned to the left lower leg , but at different anatomic sites . 
an additional biopsy was performed , which conrmed relapsed disease and showed persistent high - level bcl2 expression ( data not shown )  . venetoclax was recommenced at a dose of 400 / 800 mg on alternate days , with only mild gastrointestinal ( gi ) toxicity ( grade 1 common terminology criteria for adverse events )  . eighteen months later , with continued daily venetoclax , his disease remains in complete remission . whole - exome dna sequencing was performed only on the last relapse sample because of a lack of suitable material from prior biopsies . 
call thresholds were a minimum of 10 variant reads , depth 25 reads , variant allele frequency 10% , scale - invariant feature transform score less than 0.35 , polyphen greater than 0.7. 
potential genetic drivers of this malignancy included previously described arid1a and notch2 mutations ( table 1 ) , as well as biallelic deletion of rb1 and cdkn2a and monoallelic loss of tp53 ( fig 2 )  . 
 ( a , b ) somatic copy number analysis of the tumor biopsy was carried out using varscan and sequenza programs against patient germline dna . frequency of copy number ( log2 ratio ) obtained by pairwise comparison of read depths along the sequenced exome . 
dyer honoraria : roche pharma ag , abbvie , sandoz speakers ' bureau : roche research funding : roche ( inst ) , gilead sciences ( inst ) , astex pharmaceuticals ( inst ) , bioinvent ( inst ) travel , accommodations , expenses : abbvie no other potential conicts of interest were reported . references sokol l , naghashpour m , glass lf : primary cutaneous b - cell lymphomas : recent advances in diagnosis and management . 
blood 105 : 3768 - 3785 , 2005 grange f , beylot - barry m , courville p , et al : primary cutaneous diffuse large b - cell lymphoma , leg type : clinicopathologic features and prognostic analysis in 60 cases . 
arch dermatol 143 : 1144 - 1150 , 2007 vermeer mh , geelen fa , van haselen cw , et al : primary cutaneous large b - cell lymphomas of the legs . 
arch dermatol 132 : 1304 - 1308 , 1996 senff nj , noordijk em , kim yh , et al : european organization for research and treatment of cancer and international society for cutaneous lymphoma consensus recommendations for the management of cutaneous b - cell lymphomas . 
 successful treatment with single - agent venetoclax pham - ledard a , cappellen d , martinez f , et al : myd88 somatic mutation is a genetic feature of primary cutaneous diffuse large b - cell lymphoma , leg type . j invest dermatol 132 : 2118 - 2120 , 2012 pham - ledard a , beylot - barry m , barbe c , et al : high frequency and clinical prognostic value of myd88 l265p mutation in primary cutaneous diffuse large b - cell lymphoma , leg - type . 
jama dermatol 150 : 1173 - 1179 , 2014 cillessen sa , hess cj , hooijberg e , et al : inhibition of the intrinsic apoptosis pathway downstream of caspase - 9 activation causes chemotherapy resistance in diffuse large b - cell lymphoma . 
clin cancer res 13 : 7012 - 7021 , 2007 van galen jc , hoefnagel jj , vermeer mh , et al : proling of apoptosis genes identies distinct types of primary cutaneous large b cell lymphoma . 
fox lc , yannakou ck , ryland g , et al : molecular mechanisms of disease progression in primary cutaneous diffuse large b - cell lymphoma , leg type during 11 . 
davids ms , roberts aw , seymour jf , et al : phase i rst - in - human study of venetoclax in patients with relapsed or refractory non - hodgkin lymphoma . 
li l , pongtornpipat p , tiutan t , et al : synergistic induction of apoptosis in high - risk dlbcl by bcl2 inhibition with abt - 199 combined with pharmacologic loss 14 . 
paulli m , lucioni m , maf a , et al : primary cutaneous diffuse large b - cell lymphoma ( pcdlbcl ) , leg - type and other : an update on morphology and treatment . 
 prospective longitudinal ctdna workow reveals clinically actionable alterations in ovarian cancer jaana oikkonen , phd1 ; kaiyang zhang , msc1 ; liina salminen , md2 ; ingrid schulman1 ; kari lavikka1 ; noora andersson , phd1 ; erika ojanper a1 ; sakari hietanen , md , phd2 ; seija gr enman , md , phd2 ; rainer lehtonen , phd1 ; kaisa huhtinen , phd3 ; olli carp en , md , phd1 , 3 , 4 ; johanna hynninen , md , phd2 ; anniina f arkkil a , md , phd1 , 4 , 5 ; and sampsa hautaniemi , d tech1 purpose circulating tumor dna ( ctdna ) detection is a minimally invasive technique that offers dynamic molecular snapshots of genomic alterations in cancer . 
although ctdna markers can be used for early detection of cancers or for monitoring treatment efcacy , the value of ctdna in guiding treatment decisions in solid cancers is controversial . 
we applied the workow to a prospective cohort consisting of 78 ctdna samples from 12 patients with high - grade serous ovarian cancer before , during , and after treatment . these longitudinal data sets were analyzed using our open - access ctdna - tailored bioinformatics analysis pipeline and in - house translational oncology knowledgebase to detect clinically actionable genomic alterations . 
the alterations were ranked according to the european society for medical oncology scale for clinical actionability of molecular targets . results our results show good concordance of mutations and copy number alterations in ctdna and tumor samples , and alterations associated with clinically available drugs were detected in seven patients ( 58% )  . treatment of one chemoresistant patient was changed on the basis of detection of erbb2 amplication , and this ctdna - guided decision was followed by signicant tumor shrinkage and complete normalization of the cancer antigen 125 tumor marker . conclusion our results demonstrate a proof of concept for using ctdna to guide clinical decisions . 
analysis of circulating tumor dna ( ctdna ) is an approach with the potential of overcoming all three obstacles.1 indeed , ctdna sampling is a clinically attractive , minimally invasive technique that is based on the observation that tumor cells leak dna to the bloodstream , where it can be captured by genomic assays . 
 oikkonen et al context key objective the aim of the study was to identify the clinical usefulness of circulating tumor dna ( ctdna ) in the treatment of high - grade serous ovarian cancer ( hgsoc ) and to provide a clinical workow for reliable detection of ctdna alterations . knowledge generated we show that longitudinal ctdna sampling can be used to detect response to platinum - taxane primary therapy after the rst cycles of chemotherapy and to identify clinically applicable mutations and copy number alterations . 
in one patient with chemoresistant hgsoc , ctdna - guided treatment including trastuzumab was administered during tumor progression and yielded tumor shrinkage . relevance the early detection of poor - responding patients allows the design of alternative treatment strategies in hgsoc . 
especially for platinum - resistant patients who have limited treatment options , rapid discovery of resistant cell populations can provide an early opportunity to interfere with the development of recurrence . 
this is a substantial improvement in the management of recurrent solid cancers , where tumors are not usually sampled either because of the risk of potential intervention complications or simply because the sample could be insufcient or not representative of the disease . ctdna analysistailored bioinformatics pipelines , and a translational oncology knowledgebase to identify clinically actionable mutations and copy number alterations ( cnas )  . 
we demonstrate the usefulness of this approach in high - grade serous ovarian cancer ( hgsoc ) , which is the most common and aggressive form of epithelial ovarian cancer , with a 5 - year survival rate of 43%.7 the current standard - of - care therapy for advanced hgsoc consists of cytoreductive surgery and platinum - paclitaxel chemotherapy.8 although the initial response to standard - of - care therapy is usually good , nearly all patients eventually relapse and have limited treatment options . 
the samples were stored at 80c as 1 to 2 ml aliquots frozen less than 2 hours after collection to avoid lysis of the white blood cells.1 cell - free dna ( cfdna ) was extracted from plasma samples and subjected to 1000 targeted illumina hi - seq sequencing at bgi ( bgi europe a / s , copenhagen , denmark ) using the oseq solid cancer panel with more than 500 clinically actionable genes ( data supplement )  . dna was isolated from 21 liquid ln2 snap - frozen tumor tissue and ascites samples ( data supplement ) , as well as from whole blood ( germline ) buffy coat samples , and was sequenced using the oseq panel with coverage of 200 . mutation and copy number detection mutation detection was performed in the computational ecosystem anduril10 using the ctdna data analysis pipeline , which is based on best practices for sequencing data analysis ( data supplement )  . 
filtering steps included detection of likely spurious mutations by identifying clusters of variants shared between patients but not known in cancer , 12 and functionally irrelevant variants by a combined annotation dependent depletion13 score of less than 15 . 
to estimate the change in mutational proles , we identied the largest proportion of signicantly decreased variants during treatment compared with the pretreatment sample ( p , .01 , fishers exact test )  . 
because of varying numbers of mutations in the pretreatment samples , the proportion of signicantly decreased variants was corrected for mutation counts . pathway analysis was performed for genes with a nondecreasing mutation frequency ( p , .01 , fishers exact test ; data supplement ) using the consensuspathdb15 algoriththe consensuspathdb integrates data from 32 databases and allows comprehensive over - representation analysis for mutations . immunohistochemistry and in situ hybridization potentially clinically actionable alterations were validated through immunohistochemistry ( ihc ) and in situ hybridization for alterations classied as most prominent ( escat , the european society for medical oncology scale for clinical actionability of molecular targets ) 16 and shown to exist in patients tumor tissue ( data supplement )  . results ctdna panel reliably captures mutations and cnas we collected 78 longitudinal ctdna samples and 21 tumor tissue samples from 12 unselected patients with hgsoc ( data supplement )  . 
we applied a sequencing panel of more than 500 genes , followed by variant and cna calling , ltering , and prioritization of clinically relevant aberrations as shown in fig 1a and the data supplement . 
altogether , 265 mutations in 185 genes ( data supplement ) and cna aberrations in 113 genes ( data supplement ) passed our calling and ltering criteria . the median concordance of mutations detected from plasma to tumor tissue samples from the same patient was 79% ( fig 1b ; data supplement )  . 
 oikkonen et al 12 patients with hgsoc variant and cna calling targeted sequencing mutation discovery filtering plasma , sampled longitudinally cancer gene panel cna detection germline dna tumor tissue validation clinical application interpretation prioritization early response monitoring vaf dynamics drug relevance translational oncology knowledgebase tumor burden and mutational composition during therapy plasma only shared tissue only patient 12 months 12 months fig 1 . 
pfi , platinum - free interval . ctdna mutation proles reect long - term treatment response and reveal targetable pathways in patients with aggressive hgsoc ctdna allows several ways to monitor treatment response . for hgsoc , parkinson et al4 suggested the use of tp53 measured from ctdna in disease monitoring . 
we observed that , consistent with their results , tp53 vaf was below 0.5% in all except one patient during primary chemotherapy and was increased in all patients at disease progression ( fig 2 )  . 
these results suggest that the changes in vafs detected from ctdna samples can be used for the early identication of patients with poor response to chemotherapy . we hypothesized that genes whose mutation vafs remained stable or increased during treatment in poor responders could reveal pathways crucial for chemoresistance . 
pathways enriched in the poor - responding patients in comparison with the good - responding patients included transcription , p53 , and chromatin regulatory pathways and dna double - strand break repair pathways ( data supplement )  . 
these alterations are predicted to cause sensitivity to cdk2 / 425 and / or cdk4 / 6 inhibitors.26 regardless of the presence of hr - deciencyconferring alterations , this patient , who had no residual tumor after surgery , progressed 8.9 months after the last platinum cycle . ctdna identies actionable mutations in patients with hgsoc we prioritized and interpreted all 265 mutations and 113 cnas using the translational oncology knowledgebase , which integrates information from 11 databases and 14 scientic articles ( data supplement )  . 
the knowledgebaseprioritized mutations and cnas revealed four major targetable processes : mammalian target of rapamycin ( mtor ; patients eoc429 , eoc49 ) , dna repair ( patients eoc429 , eoc677 , and eoc1067 ) , epidermal growth factor receptor ( egfr ; patients eoc587 , eoc736 , and eoc568 ) , and cyclin dependent kinases ( cdks ; patient eoc1067 )  . the highest allele frequency mutations matching tumor content were detected in pten ( patient eoc49 ) and nf1 ( patient eoc429 ) , which cause overactivation of the mtor pathway.20 , 22 patients with the activated mtor pathway have been shown to be responsive to phosphatidylinositol 3 - kinase / mtor inhibitors.22 we validated overactivity of the mtor pathway in the two patients with ihc from tumor tissue samples ( fig 3 ; data supplement )  . 
in patient eoc49 , pten mutation vaf in omentum ( 8% and 6% for pretreatment and interval samples , respectively ) and plasma matched the pattern of tp53 mutation , indicating that tumor cells and presents an early event in tumor evolution ( data supplement )  . 
pten was detected in a follow - up sample at 8 months after primary treatment with vaf of 0.1% , conrming its persistence during therapy . the pten mutation exists in all we observed mutations and cnas in genes associated with dna repair in three patients . 
in patient eoc677 , we detected mutations in brca2 and stag2 , which potentially confer homologous recombination ( hr ) dna repair deciency and sensitivity to poly - adp ribose polymerase ( parp ) inhibitors . however , the frequencies of these mutations were low in the tumor tissues ( 0.2% for brca2 and 3.1% for stag2 ) , suggesting subclonal events . 
in patient eoc429 , we detected a germline deletion in rad51c from plasma , which has been shown to confer hr deciency and parp inhibitor sensitivity , 21 and , consistent with this , the patient had a prolonged response to platinum - based chemotherapy . 
we also detected a somatic deletion of fanca in patient three patients had mutations or cnas associated with sensitivity to drugs targeting the epidermal growth factor receptor ( egfr ) pathway . 
patient eoc568 had an erbb4 mutation , which is predicted to confer sensitivity to egfr inhibitors.27 patient eoc587 had a mapk mutation and a simultaneous mapk1 amplication , which are associated with resistance to the egfr targeting therapies.28 , 29 we detected a high copycount erbb2 amplication in the ctdna in patient eoc736 . the erbb2 locus is amplied in 3% to 11% of hgsocs30 , 31 and is a predictive biomarker for trastuzumab . 
the erbb2 amplication was validated in primary omentum tissue using snp genotypebased cna detection and in the tumor tissue sample from interval debulking surgery with in situ hybridization and ihc ( figs 4a and 4b )  . patient eoc736 was treated with nact , and she participated in a clinical trial after primary treatment , receiving maintenance bevacizumab and olaparib or placebo ( fig 4c )  . 
although she had a clinical and radiologic complete response to primary therapy , tp53 vaf remained detectable throughout the treatment , which , on the basis of our ndings , indicates a poor response to platinum taxane ( data supplement )  . 
this patient also suffered from severe paclitaxel - induced neuropathy after primary treatment , and therefore , pegylated liposomal doxorubicin ( pld ) was started at the rst relapse instead of dose - dense paclitaxel therapy . 
pld was discontinued after four cycles because of constantly rising ca125 ( from 193 to 769 ) and progression in lymph nodes as evidenced by a computed tomography scan . the erbb2 amplication detected in on the basis of ctdna , treatment of patient eoc736 was changed to trastuzumab 600 mg subcutaneously once every 3 weeks , combined with reduced doses of carboplatin ( auc4 ) and dose - dense paclitaxel ( 80 mg / m2 on days 1 and 8 )  . 
the patient responded to this combination well , and a biochemical complete response ( ca - 125 value reduced from 840 iu / ml to 19 iu / ml ) was achieved after only three treatment cycles ( fig 4c )  . 
pathogenic mutation in nf1 was detected in circulating tumor dna ( ctdna ) , leading to functionally overactive mammalian target of rapamycin ( mtor ) signaling in the tumor tissue . 
 ( a ) mutational frequencies in a good - responding patient ( eoc429 ) during primary treatment and follow - up . variant allele frequency ( vaf ) values are normalized for the frequency level to show relative changes in the levels . 
 ( b ) mtor pathway activation was detected in primary tumor tissue with higher staining for phosphorylated mtor ( pmtor ) and e4 - bp1 compared with normal ovarian tissue . 
scale bar , 100 m ( c ) in addition to having a complete response on the basis of recist 1.1 , the response to primary therapy was good , as indicated by cancer antigen 125 ( ca - 125 ) values that stayed low during treatment and follow - up . 
cadd , combined annotation dependent depletion ; hgsoc , high - grade serous ovarian cancer ; pte , primary treatment end ; 4e - bp , 4e - binding protein . sequencing of more than 500 genes from liquid biopsies , a ctdna data analysis pipeline , and a translational oncology knowledgebase to enable longitudinal analysis of ctdna during therapy and guide personalized cancer treatment decisions . 
the ctdna analysis pipeline and translational oncology knowledgebase are open - source and available with documentation ( data supplement )  . we detected high ctdnatumor congruence , which , together with the absence of alterations from the blood cells used as germline samples and comprehensive ltering of mutations through clustering , maximizes the likelihood that the reported alterations originated from tumor and not from other sources , such as clonal hematopoiesis.1 indeed , our ltering approach using longitudinal samples removed false - positive alterations in all patients . 
rara was within the amplied region in plasma but not in primary omentu ( b ) erbb2 protein was overexpressed in cancer tissue ( arrowheads ) in immunohistochemistry ( ihc ) and the erbb2 gene was amplied in dna in situ hybridization ( ish ) analysis ( scale bars , 20 mm )  . 
the most informative comparisons for predicting treatment outcome are samples at the preoperative stage , after two cycles of chemotherapy , and after debulking surgery , because they provide information on treatment response and on persisting tumor subclones we identied potentially clinically applicable mutations or cnas in more than one half of the patients with hgsoc . these included two patients with mtor pathwayactivating mutations that were validated . 
both patients had a good response to the platinum - based rst - line therapy , but in the case of relapse they could most likely benet from mtor inhibitors alone or in combination with chemotherapy.22 in patient eoc429 , we identied alterations conferring both mtor pathway activation and hr deciency , which suggests potential benet from combination treatment with , for example , mtor inhibition and parp inhibition . 
in addition , two patients had more than one concurrent , potentially targetable alteration . patient eoc1067 progressed after 9 months of platinum - based therapy despite the fanca deletion conferring hr deciency , potentially because of the contribution of the cdk alterations on cell - cycle regulation and tumor progression.32 on the basis of the concurrent ckdn1b and ckdn2b deletions , this patient could potentially benet from a combination targeting both pathways , for instance , parp inhibitor and cdk4 / 6 inhibitor . these results indicate that longitudinal ctdna sampling can detect and monitor a relapse at an early stage and identify potentially effective drug combinations . we identied a high - condence erbb2 amplication in the ctdna of a patient with platinum - resistant hgsoc and used this information to change the patients treatment to include trastuzumab with reduced doses of platinum and paclitaxel . 
thus , rapid response to the ctdna - guided treatment , detected by complete normalization of ca - 125 values within 2 months after starting the combination treatment , was surprising . 
earlier studies that have combined erbb2 inhibitors with platinum and paclitaxel report similar encouraging responses for relapsed patients with advanced hgsoc.31 although the number of patients in these studies and our study remains small because of the low number of patients with erbb2 - amplied hgsoc , these results warrant testing erbb2 amplication from relapsed hgsoc patients with advanced disease . ctdna sampling and sequencing with a large panel offers several benets to physicians . 
for a detailed description of the disclosure categories , or for more information about ascos conict of interest policy , please refer to or po.ascopubs.org / site / ifc sakari hietanen consulting or advisory role : tesaro , astrazeneca travel , accommodations , expenses : roche pharma ag olli carp en stock and other ownership interests : orion corporation honoraria : merck kgaa , roche pharma ag sampsa hautaniemi stock and other ownership interests : nanostring technologies no other potential conicts of interest were reported . acknowledgment we thank the patients who participated in this study , dr tuulia vallius and dr zoltan maliga for critical review of the manuscript , ma tiia pelkonen for proof reading , and the csc - it center for science ltd . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american pathologists joint review . 
j clin oncol 36 : 1631 - 1641 , 2018 khan kh , cunningham d , werner b , et al : longitudinal liquid biopsy and mathematical modeling of clonal evolution forecast time to treatment failure in the prospect - c phase ii colorectal cancer clinical trial . 
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science359 : 926 - 930 , 2018 parkinson ca , gale d , piskorz am , et al : exploratory analysis of tp53 mutations in circulating tumour dna as biomarkers of treatment response for patients with relapsed high - grade serous ovarian carcinoma : a retrospective study . 
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wright aa , bohlke k , armstrong dk , et al : neoadjuvant chemotherapy for newly diagnosed , advanced ovarian cancer : society of gynecologic oncology and american society of clinical oncology clinical practice guideline . 
mateo j , chakravarty d , dienstmann r , et al : a framework to rank genomic alterations as targets for cancer precision medicine : the esmo scale for clinical actionability of molecular targets ( escat )  . 
swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
cen l , carlson bl , schroeder ma , et al : p16 - cdk4 - rb axis controls sensitivity to a cyclin - dependent kinase inhibitor pd0332991 in glioblastoma xenograft cells . biol 19 : 1371 - 1378 , 2017 2013 2015 27 . 
 precision medicine across the cancer continuum : implementation and implications for cancer disparities advances in health care , including genomic technologies and reduction in the cost of dna sequencing , create new opportunities and implications for individualized health care regarding the potential for specific treatments based on a persons genetic makeup . 
precision oncology discoveries and implementation can be extended across the cancer continuum , from individualized screening recommendations to targeted treatments based on a persons risk profile encompassing genetic , environmental , and / or behavioral factors.4 although the concept of precision medicine may not be entirely new , its visibility has increased considerably with initiatives such as the all of us precision medicine research program5 supported by the us governments investment of $70 million . summarized by collins and varmus , 6 the goal of this initiative is to lay the groundwork for ultimately developing treatment strategies based on the specific characteristics of an individuals tumor , with eventual application to other chronic diseases . 
importantly , the all of us research program includes a focus on patient engagement , which will be critical to its success in reaching goals such as the recruitment of 1 million americans to provide the level and types of data necessary to understand the clinical impact of precision medicine . with the implementation of precision medicine comes a concomitant need for ethically responsible and effective communication strategies to clearly convey this benefit to patients . 
a recent study examined communication between genetic counselors and patients and reported a mismatch ( eg , too much information , not the right kind of information ) between the information provided by the counselors and the understanding and preferences of the audience.7 there is confusion among clinicians about how to use genomic data at the point of care8 ; thus , with greater implementation of precision medicine approaches will come a need for clinician training about how best to apply these approaches and communicate their potential benefits ( and risks ) to patients.4 at present , it is not clear how well the clinical and public health communities are prepared to communicate about precision medicine to patients , particularly those with limited resources , education , or health literacy . 
holt , university of maryland , college park ; arif hussain , robert wachbroit , and jessica scott , university of maryland , baltimore , md . corresponding author : cheryl l . 
 recent studies3 , 9 have suggested an important role for implementation science , and chambers et al9 extended this to include the role of the learning health care system , a concept advocated by the institute of medicine , in successfully implementing precision medicine . 
implementation science can provide theories , methods , and guidance for examining issues such as the role of context ( eg , diverse array of health care systems ) in implementing precision medicine , identification of influences on evidence - based practice usage , and implementation strategies . 
implementation issues will likely arise around the recommendation of wearable devices and ecological momentary assessment , 10 which , again , will benefit from community engagement and a multidisciplinary approach to implementation . a 2015 commentary by riley et al10 recommends involvement of behavioral and social sciences because of the potential impact of behavioral and environmental factors in determining responsiveness to treatment . 
it is likely that as precision medicine is implemented on a population scale , disciplines such as economics , health services research , behavioral medicine , communication , public health , and psychology will become increasingly relevant . we believe it is critical to take a patient - centered perspective on precision medicine in particular , because it involves different treatments for different people , which , for minorities and the poor , could amplify feelings of mistrust in the medical system and could result in adverse medical outcomes . 
similar to the confusion that was created when the us preventive service task force made changes in recommendations to cancer screening guidelines , 11 the complexity of precision medicine has the potential to significantly magnify medical mistrust . 
continuous patient engagement involves actively listening to and incorporating patient perspectives through all phases of research , including selecting and prioritizing research topics and questions , identifying appropriate outcomes , weighing in on data collection methods , and helping interpret and disseminate results.12 in the context of precision oncology and disparities , patient engagement may involve the inclusion of individuals from under - represented groups on research advisory boards so that they can help prioritize cancers they think impact their communities most , provide input on acceptable ways to communicate precision oncology - based treatment strategies , and ensure patient representation and recruitment in clinical trials while minimizing risk and maximizing benefit . advances in precision medicine are likely to benefit the medically underserved last and could increase existing health disparities.3 , 4 this may be particularly true in circumstances where precision medicine relies on clinical samples and / or data from electronic medical records , because people with no regular source of care could be excluded.13 there are several pathways by which the implementation of precision oncology could widen cancer disparities . 
first , to the extent that discoveries in precision oncology are based on a majority population ( eg , white people ) , findings may not generalize to minorities ( eg , african americans , hispanic / latino , and asian americans ) ; thus , these groups may not reap as much benefit . 
second , there are likely to be geographic ( eg , urban , rural ) , demographic ( eg , race , ethnicity , age , sex ) , and socioeconomic ( eg , income , education , neighborhood context ) differences in the trajectory at which precision oncology approaches become available to patients . 
this is illustrated in a report of a sample of rural and appalachian physicians in which 66% indicated that accessing a genetic counselor was somewhat or extremely difficult.14 it was reported that most patients living in a rural area would have to travel to an academic medical center to access genetic counseling services , creating a financial and psychological burden.15 third , there could be a gradient among patients most likely to be served by or willing to participate in precision oncology , starting with early adopters , the most affluent and insured , followed by the poor and people without health insurance at the other end of the spectrum . paradoxically , the poor suffer the greatest burden of cancer disparities and may be most in need of precision approaches to cancer screening and treatment , but this group is more likely to be left behind . 
among the several factors that are particularly pertinent to the poor in terms of increasing their cancer risk are an unhealthy diet beginning in childhood , 16 living in areas considered food deserts or areas with greater environmental exposures to carcinogens resulting in chronic stress and allostatic load , 16 and greater tobacco use.17 the poor are more likely to be negatively affected by factors such as housing segregation , 18 and health care access has recently become uncertain with possible changes to the affordable care act . 
given these challenges , it is apparent that the poor and those in rural areas would be among the last to have access to and reap the potential benefits of precision oncology . responsibilities of patients , insofar as they have a responsibility for their own care , by obligating them to undergo genetic tests and to inform health professionals of their results when appropriate . we have several suggestions for the field of precision oncology to move forward in consideration of cancer disparities . 
as an initial step , we need to gain a better understanding of what precision medicine and precision oncology mean to different stakeholders such as clinicians and patients . through stakeholder engagement , including a patient - centered approach , diverse voices should be considered at both the provider and the recipient levels . 
knowledge and acceptability should be determined as precursors to implementation . we currently know almost nothing about what people in the community know , particularly medically underserved people or those low in health literacy , nor do we know how they feel about precision medicine and how they will respond to its ever - growing application in clinical settings.4 similarly , we know little about the perspective of cancer clinicians and cancer researchers on precision oncology and how this may influence educating patients and guiding treatments , particularly in underserved patient populations . precision medicine promises to identify therapies and drugs that are tailored to the individual patients genetic makeup or their tumor and other patient - specific factors . 
because much of this promise relies on information obtained from the genetic testing or sequencing of the individual patient , many of the ethical issues raised by genetic testing ( eg , concerns about misuse of genetic information ) carry over to the implementation of precision medicine . 
does the advent of precision medicine mean that no physician should treat a patient with certain drugs ( eg , clopidogrel ) without first a genetic analysis indicating whether the patient is a good metabolizer of that drug ? precision medicine may involve a different configuration of physician and patient responsibilities . 
for example , precision medicine may shape the responsibilities of clinicians toward their patients by obligating them to know the results from genetic tests before treatment and to work closely with genetic counselors to inform patients of their vulnerabilities and treatment options . 
indeed , there could be some things that certain patients may not want to know about ( eg , genetic risk for cancer ; predisposition to disease )  . this could also include nonmedical information such as a discovery of nonpaternity . 
although there might be a predisposition among physicians not to disclose information that the patient does not want to know , this can challenge the professionals responsibilities and obligations , especially if the information is clinically actionable . in summary , we recommend that those in the field of precision oncology make a concerted effort to consider cancer disparities , not only by including diverse participant / patient groups but also by pursuing research questions that consider diversity . 
for instance , such questions might include the examination of targeted therapies for triple negative breast cancer in black women and advancing research on biologic explanations for why black men are more likely to die of prostate cancer than white men . 
in the face of large epidemiologic trials suggesting little benefit from prostate cancer screening , there remains a challenging question about what black men can do about prostate cancer mortality , especially if they have a family history . culturally appropriate strategies and messaging will need to be developed and evaluated to communicate about precision medicine with patients , and providers will need training and increased knowledge to be able to effectively integrate precision medicine into their practices . 
we recommend community and patient engagement as part of future precision oncology research in these areas to gain important perspectives relevant to the diverse patient populations that otherwise could be missed . 
holt no relationship to disclose arif hussain consulting or advisory role : novartis , astrazeneca , bayer , bristol - myers squibb research funding : sotio , merck , bayer , agensys , clovis oncology , cerulean pharma robert wachbroit no relationship to disclose jessica scott no relationship to disclose references 1 . 
n engl j med 372 : 793 - 795 , 2015 joseph g , pasick rj , schillinger d , et al : information mismatch : cancer risk counseling with diverse underserved patients . 
gray sw , park er , najita j , et al : oncologists and cancer patients views on whole - exome sequencing and incidental findings : results from the canseq study . 
chambers da , feero wg , khoury mj : convergence of implementation science , precision medicine , and the learning health care system : a new model for biomedical research . 
riley wt , nilsen wj , manolio ta , et al : news from the nih : potential contributions of the behavioral and social sciences to the precision medicine initiative . 
gehlert s : precision behavioral medicine in cancer , personalization across the cancer care continuu37th annual meeting and scientific sessions , pre - conference workshop , washington , dc , 2016 14 . 
 immunological differences between immune - rich estrogen receptorpositive and immune - rich triple - negative breast cancers tess omeara , ba1 ; michal marczyk , phd1 , 2 ; tao qing , phd1 ; vesal yaghoobi , md3 ; kim blenman , ms , phd1 ; kimberly cole , md3 ; vasiliki pelekanou , md , phd3 , 4 ; david l . 
we compared expression of immune cell markers , immune function metagenes , and immuno - oncology therapeutic targets among immune - rich subtypes . results relative fractions of resting mast cells ( tcga padj = .009 ; metabric padj = 4.09e - 15 ) , cd8 + t cells ( tcga padj = .015 ; metabric padj = 0.390 ) , and m2 - like macrophages ( tcga padj = 4.68e - 05 ; metabric padj = .435 ) were higher in immune - rich er - positive bcs , but m0 - like macrophages ( tcga padj = 0.015 ; metabric padj = .004 ) and m1 - like macrophages ( tcga padj = 9.39e - 08 ; metabric padj = 6.24e - 11 ) were higher in immune - rich tnbcs . 
ninety - one immune - related genes ( eg , cxcl14 , csf3r , tgf - b3 , lrrc32 / garp , tgfb - r2 ) and a transforming growth factor ( tgf - ) response metagene were signicantly overexpressed in immune - rich er - positive bcs , whereas 41 immune - related genes ( eg , ifng , pd - l1 , ctla4 , magea4 ) were overexpressed in immune - rich tnbcs in both discovery and validation data sets . 
tgfpathway member genes correlated negatively with expression of immune activation markers ( ifng , granzyme - b , perforin ) and positively with m2 - like macrophages ( il4 , il10 , and mmp9 ) and regulatory t - cell ( foxp3 ) markers in both subtypes . conclusion different immunotherapy strategies may be optimal in immune - rich er - positive bc and tnbc . drugs targeting the tgfpathway and m2 - like macrophages are promising strategies in immune - rich erpositive bcs to augment antitumor immunity . jco precis oncol 4 : 767 - 779 . 
 omeara et al context key objective does the immune microenvironment of immune - rich estrogen receptorpositive breast cancer ( er - positive bc ) differ from the immune microenvironment of immune - rich triple - negative bc ( tnbc ) ? knowledge generated relative fractions of mast cells and m2 - like macrophages are higher in the immune microenvironment of immune - rich erpositive bc , whereas fractions of m0 - like and m1 - like macrophages are higher in immune - rich tnbc . 
her2 - positive patients were excluded , and only er - positive / her2 - negative ( herein designated er - positive ; tcga , n = 627 ; metabric , n = 1 , 186 ) and er - negative / her2 - negative ( herein designated tnbc ; tcga , n = 191 ; metabric , n = 279 ) patients were included in this analysis . 
we obtained formalin - xed parafn - embedded tissues from 63 tnbcs and 53 erpositive , treatment - nave , stage i to iii bcs from yale pathology tissue archives , matched by distribution of histologic grade , for quantitative immunouorescence ( qif ) and automated quantitative analysis ( aqua )  . assessment of immune cell content and selection of immune - rich patients a gene expression signature score developed by danaher et al19 was used to score each tcga and metabric patient for total tils . 
the total til gene signature score is the average of scores representing 11 immune cell subpopulations ( cd45 , macrophages , cd8 + t cells , t cells , cytotoxic cells , exhausted cd8 + cells , th1 cells , b cells , neutrophils , natural killer [ nk ] cells , cd56dim nk cells )  . log2 + 1 - transformed expression was used to calculate total til gene signature scores from tcga ( rna - seq ) and metabric ( microarray ) data . 
the til metagene score distribution was plotted for the pooled er - positive bc and tnbc patients , and patients with scores in the top 25th percentile of the distribution were dened as immune - rich . for 156 basal - like cancers in tcga ( er - positive bc , n = 21 ; tnbc , n = 135 ) , histologic til counts were also available.20 , 21 for these patients , we assessed the correlation between histologic til count and til gene expression score . 
we also assessed correlations between til gene signature score and pam50 subtype , tumor mutation burden ( tmb ) , and ki - 67 expression level in the tcga data when this information was available ( er - positive bc , n = 191 ; tnbc , n = 697 )  . 
pam50 subtypes and tmb categories were obtained from a previous publication , and ki - 67 categories were dened as greater than or less than the median log2 + 1 - transformed ki - 67 expression across tcga patients.22 survival analysis progression - free survival ( pfs ) and overall survival ( os ) times were available for 807 tcga patients ( er - positive bcs , n = 189 ; tnbcs , n = 618 ) .23 pfs and os were compared by er subtype within immune - rich patients ( erpositive bcs , n = 114 ; tnbcs , n = 84 ) and within nonimmune - rich patients ( er - positive bcs , n = 504 ; tnbcs , n = 105 ) using the survival and survminer r packages.24 assessment of immune cell subpopulations cibersort , an analytical tool that uses support vector regression , was used to estimate the relative proportions of 9 aggregated immune cell populations and 22 subpopulations in immune - rich er - positive bcs and immunerich tnbcs.25 , 26 the average relative fraction and standard error of the mean of each immune cell population were compared by subtype . 
 immunological differences between immune - rich er - positive bcs and tnbcs calculated gene expression signature scores of overall macrophages , m1 - like macrophages , m2 - like macrophages , and the transforming growth factor ( tgf - ) signaling response were obtained from previous publications and were compared between patients with erpositive bcs and those with tnbcs.27 - 29 slopes of linear regression curves between total tils and metagene expression signatures were compared by using a t test based on standard error in the linear regression models.30 formalinxed parafn - embedded tissue microarrays were stained for the nuclear marker 4 ( cid : 1 ) , 6 - diamidino - 2 - phenylindole and the pan - macrophage marker cd68 , and multiplexed qif and aqua were used to quantify expression levels in yale er - positive bc and tnbc patients as previously described.31 differential gene expression analysis the deseq2 ( tcga , rna - seq data ) and limma ( metabric , microarray data ) r packages were used for differential gene expression analysis of 779 immunerelated genes ( nanostring pancancer io 360 panel , nanostring technologies , seattle , wa ) and 137 currently available immuno - oncology drug target genes between immune - rich er - positive bcs and immune - rich tnbcs.32 - 34 availability of rna expression data for each member gene in tcga and metabric databases is summarized in the data supplement . 
genes with false discovery rate padj , .05 were considered signicantly differentially expressed for a broad investigation of differentially regulated immune pathways . statistical analysis all statistical analyses were performed in r v 3.5.0. 
in tcga , the 75th percentile til metagene score was 5.92 , and it categorized 119 er - positive bcs ( 19% ) and 89 tnbcs ( 45% ) as being immune rich . 
the total til gene signature scores correlated moderately but signicantly with histologic til scores in tcga ( r = 0.44 ; p = 1.18e - 08 ; fig 2a ) when these data were available . 
in tcga , there was no statistically signicant difference between the distribution of pam50 subtypes or ki - 67 expression levels between immune - rich and nonimmune - rich er - positive bcs . 
patients with immune - rich er - positive bcs had signicantly higher tmb than patients with nonimmune - rich er - positive bcs ; this difference was not seen among patients with tnbcs . 
for a subset of tcga patients with available survival formation ( n = 804 ) , pfs and os were compared by subtype ( data supplement )  . immune - rich patients had similar pfs and os regardless of er status . 
in contrast , among the nonimmune - rich cancers , os was signicantly longer in patients with er - positive bcs than in patients with tnbcs ( p = .043 ) , as expected . immune cell subpopulations in the tumor microenvironment of immune - rich er - positive bcs and immune - rich tnbcs by using cibersort , we found higher relative fractions of t cells ( padj = .009 ) and mast cells ( padj = .0005 ) in patients with immune - rich er - positive bcs compared with those who had immune - rich tnbcs ( fig 3a ) in tcga . in patients with immune - rich tnbcs , the relative fractions of macrophages ( padj = .026 ) and nk cells ( padj = .0007 ) were higher relative to those in patients with immune - rich er - positive bcs . 
the higher relative fractions of t cells ( padj = .005 ) and mast cells ( padj = 1.73e - 20 ) in immunerich er - positive bcs and the higher relative fraction of macrophages ( padj = 1.22e - 06 ) in immune - rich tnbcs were also observed in metabric ( fig 3b )  . 
qif also showed higher overall expression of the pan - macrophage marker cd68 in tnbc tissues relative to tissue from erpositive bcs from yale pathology archives ( p = .011 ; fig 3c )  . next , we examined differences among 22 immune cell subpopulations . in immune - rich er - positive tumors in tcga , the relative fractions of cd8 + t cells ( padj = .015 ) and resting mast cells ( tcga padj = .009 ) were higher relative to those in immune - rich tnbc tumors in both data sets ( fig 3d )  . 
in contrast , the relative fractions of m0 - like macrophages ( padj = .015 ) and m1 - like macrophages ( padj = 9.39e - 08 ) were signicantly higher in patients with immune - rich tnbcs compared with those with immunerich er - positive bcs . 
 ( a - b ) gene signature scores were calculated for 11 immune cell subpopulations for patients with er - positive bcs and tnbcs in ( a ) the cancer genome atlas ( tcga ) and ( b ) molecular taxonomy of breast cancer international consortium ( metabric ) databases , as described by danaher et al.19 the x - axis indicates immune cell subpopulations plus the total tumor - inltrating lymphocyte ( til ) population . 
immune - rich erpositive bcs had higher m2 - like macrophage ( p = .012 ) and tgfresponse ( p = 0.035 ) metagene expression scores ( fig 4a )  . 
the subtype - specic differences in m1 - like macrophage ( p = 1.85e - 04 ) and tgfresponse ( p = .029 ) metagene scores signicantly increased with higher total til gene signature scores , whereas the differences in overall macrophage ( p = .786 ) and m2 - like macrophage ( p = .097 ) scores between cancer subtypes were not specic to immune - rich patients ( fig 4b )  . 
 ( a ) for a subset of basal - like patients from the cancer genome atlas ( tcga ) with previously published histologic til scores ( n = 156 ) according to danaher et al , 19 the total til gene signature scores ( x - axis ) were correlated with histologic stromal til counts ( y - axis )  . 
 ( b - d ) for all patients with er - positive bcs ( n = 697 ) and tnbcs ( n = 191 ) in tcga , patients were classied as immune cell high ( danaher et l19 total til gene signature score in the top 25th percentile ) or low ( danaher total til gene signature scores in the bottom 75th percentile ) , represented on the x - axis . 
distributions of ( b ) pam50 subtypes , ( c ) ki - 67 expression categories and ( d ) tumor mutation burden ( tmb ) categories were compared between patients with immune - rich high and low er - positive bcs and tnbcs separately . 
pam50 and tmb categories were obtained from previous publications , and ki - 67 categories were dened as greater than ( high ) or less than ( low ) the median ki - 67 expression level across all patients . differential expression of immune - related genes and immuno - oncology drug target genes between immune - rich er - positive bcs and immune - rich tnbcs at the individual gene level , 132 of 754 immune - related genes were signicantly overexpressed in tcga immunerich er - positive bcs . 
ninety - one of these immune - related genes , including mast cell genes ms4a2 and cpa3 and therapeutic targets cxcl14 , cx3cr1 , and potential csf3r , were also upregulated in metabric patients with immune - rich er - positive bcs ( data supplement )  . immune - rich tnbcs , 303 immune - related genes were overexpressed compared with immune - rich er - positive bcs in tcga , and 218 of these were validated in metabric . 
sixteen of 136 available therapeutic target genes were overexpressed in tcga patients with immune - rich er - positive bcs , and 12 of these , including 3 members of the tgfsignaling pathway ( tgf - 3 , lrrc32 / garp , and tgf - r2 ) , were also overexpressed in metabric patients with immune - rich er - positive bcs ( table 1 ; data supplement )  . 
for patients from ( a ) the cancer genome atlas ( tcga ) and ( b ) molecular taxonomy of breast cancer international consortium ( metabric ) databases , 9 aggregate immune cell populations are listed on the x - axis , and the average fraction of each immune cell population for a certain subtype is quantied on the y - axis . 
 ( c ) quantitative immunouorescence ( qif ) signal was compared between estrogen receptorpositive breast cancer ( er - positive bc ; , n = 53 ) and triple - negative bc ( tnbc ; n = 63 ) formalin - xed parafn - embedded tumor microarray tissues . 
subtype is shown on the x - axis , and cd68 qif signal in the total 4 ( cid : 1 ) , 6 - diamidino - 2 - phenylindole ( dapi ) compartment is quantied on the y - axis . 
for ( d ) tcga and ( e ) metabric patients , 22 immune cell populations derived by cibersort are listed on the x - axis , and the average fraction of each immune cell population for a certain subtype is quantied on the y - axis . 
false discovery rate padj values are provided for tcga ( er - positive bcs , n = 119 ; tnbcs , n = 86 ) and metabric ( er - positive bcs , n = 225 ; tnbcs , n = 140 ) patients . 
comparison of macrophage population and transforming growth factor ( tgf - ) response metagene expression scores between patients with immune - rich estrogen receptorpositive breast cancers ( er - positive bcs ) and those with triple - negative bcs ( tnbcs ) in the cancer genome atlas ( tcga )  . 
 ( a ) metagene expression scores for overall macrophages , m1 - like macrophages , m2 - like macrophages , and tgfresponse were compared between immune - rich er - positive bc ( red , n = 119 ) and immune - rich tnbc ( blue , n = 86 ) patients in tcga . 
 ( b ) for all patients with er - positive bcs ( n = 697 ) and tnbcs ( n = 191 ) in tcga , gene expression signature scores for overall macrophages , m1 - like macrophages , m2 - like macrophages , and tgfresponse ( y - axis ) were plotted against total til gene signature scores ( x - axis )  . 
linear regression lines for each subtype with 95% cis are shown in red ( er - positive bc ) or blue ( tnbc )  . and the cancer - testis antigen , magea4 , were also overexpressed in immune - rich tnbcs in metabric . correlation between tgfpathway expression and other immune - related genes tgfis a pleotropic cytokine that can stimulate regulatory induce m2 - like macrophage polarization , and t cells , suppress effector t cells , nk cells , and dendritic cells . furthermore , tgfsignaling has been linked to an immune - excluded phenotype in cancer . 
we therefore examined correlations between the differentially expressed tgfpathway members ( tgf - 3 , lrrc32 , and tgfr2 ) and the expression levels of the 53 immuno - oncology drug target genes that were differentially expressed in both data sets ( 12 upregulated in immune - rich er - positive bcs , 41 upregulated in immune - rich tnbcs ) across immunerich tnbcs and er - positive bcs . in both tcga and metabric , expression of the tgfpathway members correlated positively with each other and negatively with most immune activation markers ( fig 5a - b ; data supplement )  . 
similar correlation patterns were seen between tgfpathway members and immune activation marker expression when only immune - rich er - positive bcs were analyzed ( fig 5c - d )  . 
we also noted a positive correlation between expression of tgf - 1 and the m2 - like macrophage markers il4 ( r = 0.24 ) , il10 ( r = 0.38 ) , and mmp9 ( r = 0.43 ) as well as the regulatory t - cell marker foxp3 ( r = 0.42 ) in the tcga data . discussion in this report , we used a gene expression signature of total tils to identify patients with immune - rich er - positive bcs and tnbcs in 2 publicly available databases to compare the tumor immune microenvironment of these subtypes . the patients with immune - rich er - positive bcs had signicantly higher tmb than patients with nonimmune - rich er - positive bcs , but no difference in tmb was seen between immune - rich and nonimmune - rich tnbcs . 
for ( a ) the cancer genome atlas ( tcga ) and ( b ) molecular taxonomy of breast cancer international consortium ( metabric ) patients , correlation matrices display the pearson correlation coefcients between expression levels of 53 immuno - oncology drug targets across patients with immune - rich estrogen receptorpositive breast cancers ( er - positive bcs ) and triple - negative bcs ( tnbcs )  . 
for tcga , the number of patients with immune - rich er - positive bcs was 119 , and for tnbcs , it was 86 ; for metabric , the number of patients with immune - rich er - positive bcs was 225 , and for tnbcs , it was 140 . 
for ( c ) tcga and ( d ) metabric patients , correlation matrices display the pearson correlation coefcients between expression levels of 53 immuno - oncology drug targets in patients with immune - rich er - positive bc alone . 
the number of patients with immune - rich er - positive bcs in tcga was 119 , and in metabric , it was 225 . results are consistent with previous reports.11 , 36 approximately 70% of the immune - rich er - positive bcs we identied were luminal a molecular subtype , and the pam50 subtype distribution and ki - 67 expression were similar between immune - rich and nonimmune - rich er - positive bcs . an os difference was detected between patients with nonimmune - rich er - positive bcs relative to nonimmunerich tnbcs , but no similar statistically signicant difference was seen between patients with immune - rich er - positive bcs and immune - rich tnbcs ( both groups had excellent long - term survival )  . 
by using deconvolution methods , we observed differences in the relative fractions of immune cell subpopulations between immune - rich er - positive bcs and immune - rich tnbcs . immune - rich er - positive bcs had higher relative fractions of m2 - like macrophages , cd8 + t cells , and resting mast cell populations , whereas immunerich tnbcs had overall higher macrophage content and higher m0 - like and m1 - like macrophage populations . 
higher overall cd68 protein expression in tnbcs was also conrmed by immunohistochemistry . interestingly , immune - rich er - positive bcs had higher expression of a tgfsignaling metagene and showed coordinated overexpression of tgf - 3 , tgf - r2 , and lrrc32 compared with immune - rich tnbcs . 
the tgfpathway is activated by ligands tgf - 1 , tgf - 2 , and tgf3 that bind to membrane - bound serine / threonine protein receptor kinases composed of subunits tgf - r1 and tgfr2 . 
although the tgfligand isoforms are more than 75% similar in amino acid structure , tgf - r2 has increased afnity for tgf - 1 and tgf - 3 relative to tgf2.37 lrrc32 encodes the garp protein that tethers tgfligands to the cell membrane to prime its activation.38 the simultaneous upregulation of the ligand , receptor , and signaling components suggests a functional role for the tgfpathway in immune - rich er - positive bcs . the immune - attenuating function of tgfsignaling in the tumor microenvironment has been extensively studied in vitro and in vivo . 
in early in tumorigenesis , tgfacts as a tumor suppressor , inducing apoptosis in premalignant cells and inhibiting proliferation.39 after tumor formation , tgfpromotes cancer progression through suppressing the host antitumor immune response.40 among other roles , tgfinhibits cd8 + effector t - cell function , inhibits the th1 helper t - cell phenotype , activates foxp3 - positive regulatory t cells , drives m2 - like macrophage polarization , and excludes immune cells from the tumor compartment.41 - 45 the distinct roles of the different tgfisoforms in cancer remain undened . 
among the 3 isoforms , tgf - 3 is the most potent stimulator of neovascularization in models of wound healing and the most potent inhibitor of granulocyte - macrophage colonystimulating factormediated hematopoiesis in the bone marrow . 
tgf - 1 and tgf - 3 are equally potent stimulators of extracellular matrix collagen deposition in models of pulmonary brosis.46 overall , high tgfsignaling is also associated with lesser response to immune checkpoint inhibitors.41 , 42 on an expression level , we found higher relative fractions of cd8 + t cells in immune - rich er - positive bcs as well as higher tgfsignaling ; these cd8 + t cells in immune - rich er - positive patients may therefore lack robust effector cytotoxic function or be excluded from the tumor compartment by augmented tgfsignaling . tgfcan be released by cancer cells as well as by m2 - like macrophages in a positive feedback loop to attenuate antitumor immunity.47 in several studies , the presence of m2 - like macrophages in bc was associated with poor prognosis , and reprogramming of macrophages toward the m1 - like phenotype has been shown to increase responsiveness to immune checkpoint therapy in experimental models.48 , 49 a recent study found that a macrophage - dependent autochthonous mouse model of luminal btype bc , inhibition of class iia histone deacetylase ( hdac ) by tmp195 induces the recruitment and differentiation of antitumor - type macrophages , reduces tumor burden and metastases , and enhances the durability of tumor reduction when combined with chemotherapy regimens or t - cell checkpoint blockade.50 this observation in an animal model of bc is consistent with our ndings in human tissues . 
our analyses also showed that expression of tgfpathway members correlated negatively with the expression of many immune activation markers including interferon ( ifn - ) , granzymes , and perforin , but correlated positively with m2 - like macrophage markers il4 , il10 , mmp9 , and regulatory t - cell marker foxp3 . 
although m2 - like macrophages in the tumor microenvironment may be the source of tgfsignaling , high levels of apolipoprotein b mrna editing enzyme , catalytic polypeptide - likeassociated mutational signatures that have been previously identied in patients with immunerich er - positive bcs may also contribute to upregulation of the tgfpathway in tumor cells and downstream recruitment of m2 - like macrophages.36 deconvolution of the leukocyte compartment also revealed a higher relative fraction of mast cells in immune - rich erpositive bcs ; this was corroborated by overexpression of the mast cell markers cpa3 and ms4a2 in patients with immune - rich er - positive bcs compared with those who have immune - rich tnbcs . 
how these cells inuence antitumor immunity is unknown , but high mast cell inltration in bc has been associated with er expression and favorable prognosis in earlier studies.51 , 52 in summary , our analysis of gene expression data is consistent with a more attenuated immune microenvironment mediated by increased tgfsignaling and m - 2 macrophage presence in immune - rich er - positive bcs . 
in this subset of er - positive bcs , we observed high relative fractions of cd8 + t cells , mast cells , and m2 - like macrophages with lower expression of granzyme - b , perforin , and ifn -  . 
in addition , practical assays that can distinguish patients with immune - rich and tgf - - high from patients with tgf - - low er - positive bcs are needed before these results can be applied in the clinic . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . vasiliki pelekanou employment : sano david l . 
rimm stock and other ownership interests : pixel gear honoraria : amgen , bristol myers squibb , ventana medical systems consulting or advisory role : perkin elmer , bristol myers squibb , astrazeneca , agendia , ultivue , merck , daiichi sankyo , glaxosmithkline , konica minolta , nanostring technologies , nextcure , cell signaling technology , roche , paige , cepheid , sano research funding : cepheid ( inst ) , perkin elmer ( inst ) , astrazeneca / medimmune ( inst ) , nextcure ( inst ) , eli lilly ( inst ) , nanostring technologies ( inst ) , navigate biopharma ( inst ) , ultivue ( inst ) , konica minolta ( inst ) , amgen ( inst ) patents , royalties , other intellectual property : rarecyte circulating tumor cells ; quantitative immunouorescence ( aqua ) ( inst ) travel , accommodations , expenses : ventana medical systems , nextcure , bristol - myers squibb lajos pusztai consulting or advisory role : h3 biomedicine , merck , novartis , seattle genetics , syndax pharmaceuticals , athenex , astrazeneca , genentech , bristol myers squibb , clovis oncology , immunomedics , eisai , almac diagnostics research funding : merck ( inst ) , genentech ( inst ) , seattle genetics ( inst ) , astrazeneca ( inst ) travel , accommodations , expenses : astrazeneca uncompensated relationships : nanostring technologies open payments link : 110878 / summary no other potential conicts of interest were reported . references bianchini g , qi y , alvarez rh , et al : molecular anatomy of breast cancer stroma and its prognostic value in estrogen receptor - positive and - negative cancers . j clin oncol 28 : 4316 - 4323 , 2010 karn t , pusztai l , holtrich u , et al : homogeneous datasets of triple negative breast cancers enable the identication of novel prognostic and predictive signatures . 
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 use of a targeted exome next - generation sequencing panel offers therapeutic opportunity and clinical benet in a subset of patients with advanced cancers scott kopetz , md , phd1 ; kenna r . 
jack lee , md1 ; jiexin zhang , ms1 ; beate litzenburger , phd1 ; vijaykumar holla , phd1 ; walter kinyua , ms1 ; emily broaddus1 ; molly s . 
broaddus , md , phd1 purpose smaller hotspot - based next - generation sequencing ( ngs ) panels have emerged to support standard of care therapy for patients with cancer . 
survival and the impact of matched therapy use were determined by kaplan - meier estimate , log - rank test , and cox proportional hazards regression . results the larger ngs panel identied at least one alteration in an actionable gene not previously identied in the smaller sequencing panel in 214 ( 41% ) of 521 of enrolled patients . 
with current clinical molecular diagnostics , sequencing is faster , uses less tumor dna , and costs less when genes are multiplexed into small next - generation sequencing ( ngs ) panels . 
historically , the focus was often on the identication of mutations found in hotspotsareas of the genome where known drivers are frequently mutated , many of which can be mapped to therapeutic intervention . 
these panels typically emphasize genes that are recommended by cancer treatment guidelines . an emerging utility of ngs is the screening of other types of cancers for mutations in clinically actionable genes that can be targeted therapeutically . 
such treatment approaches are associated with improved in retrospective analyses screening , 1 prosurvival spective trials , 2 - 4 and meta - analyses , 5 - 7 but this approach is not universally embraced.8 , 9 an important issue in these studies is the low number of patients who benet from this approach . 
 kopetz et al context key objective can additional actionable information be identied by expanding sequencing coverage to the full exome of hundreds of genes beyond hotspot analysis and , secondarily , does that additional information affect patient outcomes ? knowledge generated only a small number of genes have alterations that are specically listed in a us food and drug administrationapproved indication that would match a patient with cancer to a specic actionable agentfor example , braf v600e to vemurafenib . 
limited access to targeted therapy trials is an issue , as some trials enroll only certain tumor types or are only active in a few geographic sites . in patients with advanced cancer that is refractory to standard treatment , any benet gained from the detection of mutations in clinically actionable genes beyond those detected using focused hotspot sequencing panels remains unknown . 
hotspot - focused ngs panels fail to robustly identify copy number alterations or mutations that are found outside commonly altered loci , which would represent an advantage of larger ngs initiative was to panels . 
enrollment criteria included the following : adult patient with pathologic documentation of a single solid tumor malignancy ; completed frontline treatment and any standard treatments that extended life by at least 3 months ; eastern cooperative oncology group performance status of 0 or 1 ; no active brain metastases ; and completed tumor testing using a smaller sequencing panel and either disease progression on a matched therapy that targeted a previously identied actionable nding or no clinically actionable mutations detected . molecular assays and decision support that consisted of hotspot mutation testing was primarily performed on archival formalin - xed , parafn - embedded primary and metastatic cancers acquired during the course of routine clinical care using a 46or 50 - gene ngs panel described previously10 ( actionable genes summarized in the data supplement )  . 
subsequent testing used a larger ngs the entire coding regions of panel 409 cancer - related genes with methodologies previously detailed11 ( actionable genes and details of larger ngs panel provided in the data supplement )  . 
the protocol did not provide for image - guided biopsies of the most recent metastasis for testing ; median time since tissue acquisition to ngs was 467 days ( appendix fig a1 )  . relevant germline ndings were reported to the referring oncologist.12 for comparisons with protocol patients , the cancer genome atlas ( tcga ) datagene mutations and gene amplicationsfor the same 409 genes from 316 ovarian high - grade serous carcinomas , 212 colorectal adenocarcinomas , and 230 lung adenocarcinomas were extracted using cbioportal.org. 
for patients with colorectal cancer , mismatch repair was assessed as part of routine clinical care.13 somatic mutations and amplications that were reported for each patient were reviewed by the precision oncology decision support team.14 , 15 this involved annotation for function and potential clinical trial matching for actionability . 
characteristics for patients enrolled and tested in the prospective protocol characteristic female male performance status missing race asian black white hispanic other unknown tumor type bladder breast colorectal endometrial hawaiian / pacic islander gastric / gastroesophageal junction germ cell tumor head and neck lung neuroendocrine others ovarian prostate renal sarcoma thyroid age , years 20 20 - 30 30 - 40 40 - 50 50 - 60 60 - 70  . 
for the smaller 46and 50 - gene ngs panel , 34 and 38 genes , respectively , were considered actionable , whereas for the larger 409 - gene ngs panel , 96 genes were actionable ( data supplement )  . statistical analysis study sample size was dictated by the capacity of the clinical laboratory improvement amendmentscertied clinical molecular diagnostics laboratory that performed the 409 - gene ngs panel , estimated to be 25 cases per week . with a target of 600 patients , it was determined a priori that the rate of identication of alterations in actionable genes could be estimated with a standard error of no more than 0.02. 
we used fishers exact test to assess whether the gene mutation frequency was signicantly different between protocol patients with colorectal adenocarcinoma , lung adenocarcinoma , or ovarian high - grade serous carcinoma and corresponding tcga data sets . 
the probability of overall survival was determined by kaplan - meier estimate , and we used a log - rank test to assess the statistical signicance of the difference between patient groups . 
to evaluate the effect of clinical variables on overall survival , we performed multivariable survival analysis using a cox proportional hazards regression model that included variables that were signicant ( p , .05 ) in a univariable analysis . results characteristics of the patient population enrolled in the protocol a total of 675 patients were enrolled , with large - panel testing completed in 569 patients . 
five hundred twenty - one patients who received at least 6 months of follow - up or who died within 6 months were included in the analysis ( appendix fig a2 )  . colorectal cancer , sarcoma , head and neck cancer , ovarian cancer , and breast cancer were the most frequently represented cancers in the cohort ( table 1 ) , but the protocol enrolled patients with a broad spectrum of cancer types . sequencing results once material was received by the molecular diagnostics laboratory , a median of 13 days passed before the molecular reports were nalized and entered into the electronic medical record ( appendix fig a3 )  . 
of the 521 patients who were tested with the 409 - gene ngs assay , 214 ( 41% ) had at least one tumor alteration in an actionable gene that was not identied in a prior assay using a smaller ngs panel ( figs 1a and 1b )  . full 409 - gene ngs assay results are summarized for all patients in the data supplement . 
not counting the sequencing results from the smaller panel , the mean number of alterations detected in actionable genes in the 409 - gene panel was 1.0 ( range , 0 to 66 ; fig 1c )  . 
after the application of decision support , 201 ( 41% ) of 495 alterations in actionable genes were considered to affect the function of the gene and were deemed actionable ( mean per patient , 0.4 ; range , 0 to 5 ; fig 1d )  . 
the rate of detection of an alteration in an actionable gene with additional testing was dependent on tumor type , with low rates observed in thyroid and ovarian cancers ( fig 2a )  . 
alterations in a variety of actionable genes from the 409 - gene panel were identied , with the most common mutations in genes nf1 , mdm2 , atm , kdr , notch2 , erbb2 , mtor , and pten ( fig 2b )  . matching tested patients to targeted therapy of the 214 patients who had alterations in actionable genes in the large ngs platform that were not detected in the previous small - panel ngs testing , 40 ( 19% ) were matched gender race male female unknown other hispanic hawaiian / pacific islander white black asian disease site others thyroid sarcoma renal ovarian lung colorectal breast age , years > q3 : 65 - 85 q2 - q3 : 56 - 65 q1 - q2 : 46 - 56 < q1 : 18 - 46 fig 1 . 
on average , only one alteration in an actionable gene per tumor was detected . ( d ) on average , only 0.4 actionable alterationsmutation or amplication in an actionable gene that is known or has inferred biologic functionwere detected per tumor . 
 ( a ) stacked bar chart shows the percent of cases for each broad class of tumor type listed with 0 , 1 , 2 , or 3 or more alterations in actionable genes not detected in the smaller testing panel . data are shown for tumor types in which at least 10 patient tumors were sequenced . 
these patients had mutations in actionable genes that were classied as actionable , potentially actionable , or a variant of unknown signicance but the oncologist chose a therapy that was not a match to the gene alterations treating with matched therapy found . 
reasons for not protocols were similar to those from a previously reported patient population18 and included declining performance status , a desire to seek treatment closer to home , or an oncologist who did not consider the gene mutation clinically actionable or the targeted therapy sufciently active in the given tumor type ( data supplement )  . decision support decision support was critical as alterations were detected in a number of actionable genes that were less familiar to oncologists , and the availability of matching clinical trials varied during the study period . 
given the hypermutated nature of this patients tumor , it was felt that none of the mutations would act as a driver or represented a good target for matched therapy ; thus , they were not annotated ( fig 3a )  . 
not counting the sequencing results from the smaller panel , nearly 70% of tumor alterations were identied in patients with tumors that were found to be in the top quartile of overall tumor mutation burden . 
 kopetz et al ( n = 201 ; 41% ) yes : inferred ( n = 35 ; 17% ) potentially ( n = 81 ; 40% ) yes : literature based ( n = 85 ; 42% ) ( n = 66 ; 13% ) ( n = 11 ; 2% ) unknown ( n = 21 ; 44% ) all gene alterations ( n = 2 , 673 ) bottom quartile 3rd quartile 2nd quartile top quartile alterations in actionable genes ( n = 495 ) actionable alterations ( n = 201 ) bottom quartile 3rd quartile bottom quartile 2nd quartile top quartile 3rd quartile top quartile 2nd quartile fig 3 . 
forty - four percent of the alterations found in actionable genes were variants of unknown signicance ( unknown ) , and 13% were not annotated because they were all from one hypermutated patient ( na )  . 
of the alterations with known function , 2% are known to be benign or not actionable ( no ) , with the remainder having some annotated function on the basis of the existing literature . 
presence of clinically actionable alterations was not related to high overall tumor mutation burden . application of decision support , the number of actionable alterations was poorly correlated with mutation burden , as less than 50% of actionable alterations in actionable genes are in the top quartile of mutation load ( fig 3b )  . 
thus , tumor mutation burden was not a good predictor of the presence of a clinically actionable alteration . patient outcomes neither the number of overall gene alterations in the tumor , nor the presence of alterations in actionable genes signicantly inuenced patient survival ( figs 4a and 4b )  . 
survival was assessed by manual curation of the medical record along with the date when a patient was last known to be alive as documented by a physician note or date of death provided by obituary , tumor registry , or reported by a family member . 
this study is limited by the lack of randomization ; however , comparison of matched and unmatched patients helps to minimize concerns related to comorbidities or declining performance status as an explanation for the differences in outcomes observed in figures 4c and 4d , as all patients in this analysis received another line of therapy , including participation in clinical trials . two patients were found to have deleterious germline mutations that were not previously known to the patient or oncologistpatient management was not altered in either case . 
 ( a - d ) kaplan - meier plots for overall survival by ( a ) total number of alterations ( actionable and nonactionable ) detected , ( b ) whether the patient had a tumor with an alteration in an actionable gene , ( c ) type of therapy and absence of alterations in actionable genes , and ( d ) whether the patient had a tumor with an alteration in an actionable gene on the 409 - genes next - generation sequencing panel received matched treatment . 
only treatment with matched targeted therapy ( c and d ) was associated with signicantly improved survival ( p = .017 compared with patients treated with nonmatched therapy )  . 
tcga did not select for patients with advanced cancers ; therefore , we compared mutation frequencies in our cohort with those of the tcga for colorectal adenocarcinoma , lung adenocarcinoma , and ovarian high - grade serous carcinoma.19 - 21 these three cancers were examined because of their relatively homogeneous histology and sufcient representation in the study . 
for colorectal cancer , kras mutation frequency was higher in our patient population , which was consistent with their resistance to epidermal growth factor receptordirected therapy22 ( data supplement )  . 
protocol patients also had a signicantly higher incidence of tp53 mutations ( 72% v 52% ; p = .004 ; data not shown )  . only 3% of protocol colorectal cancers were microsatellite instabilityhigh compared with 13% in tcga . 
at risk : wild type 507 439 315 197 112 508 440 318 198 113 508 439 319 199 114 time ( months ) time ( months ) time ( months ) mutation mutation mutation no . 
 ( a - j ) kaplan - meier plots for overall survival for nine actionable genes commonly altered in clinical protocol patients ( a - i ) and for the most common cancer types represented in the protocol ( j )  . 
only two clinically actionable genescdkn2a and cdkn2bhad lower frequencies of alteration in protocol patients with lung adenocarcinoma ( data supplement )  . discussion survival for the overall cohort was poor in this institutionwide prospective protocol that enrolled patients with advanced solid tumor malignancies refractory to treatment . for 41% of these patients , we identied an alteration in a clinically actionable gene that was not detected by previous small - panel testing . 
the overall utility of singleas opposed to sequential smallpanel , then large - panel testinglarge - panel ngs testing combined with decision support can be estimated by combining the 11% enrollment in clinical trials from our prior 46and 50 - gene panel study18 with the 8% derived from the incremental addition of the large - panel testing in this study . 
as metastases can accumulate mutations over time , this represents another potential reason for limited patient benet . with increasing gene targets and matched drugs , it is becoming increasingly difcult for an individual physician to reasonably interpret the molecular ndings from larger ngs panels . 
the importance of decision support a comprehensive precision medicine strategy is highlighted by the fact that less than one half of actionable alterations in actionable genes had been reported previously in the literature . 
over time , at least some of these variants of undetermined signicance may be found to be activating mutations after they are examined in functional assays . examples of variants that have been reclassied as activating mutations in a functional genomics platform26 include pdgfra k385m , pik3ca e110del , and ret d627n . 
the reliability of quality decision support is crucial as patients with advanced cancers have a nite lifespan . patients with advanced cancers that are refractory to standard therapy may have mutational frequencies of individual genes that differ from published frequencies derived from unselected patients . 
this was apparent in patients with colorectal cancer and ovarian high - grade serous carcinoma in whom frequencies likely reected a bias toward more clinically aggressive or treatment - resistant tumor biology . frontline treatment of ovarian cancer is for example , platinum - based chemotherapy ; is possible that , by selecting for patients with platinum - resistance , the protocol also selected for patients with ovarian cancers with therapydriven alterations in the molecular landscape . 
the phenomenon is supported by the comparison of survival populations from stage - matched patients in tcga and clinical trials , where patient survival from clinical trials was substantially lower.27 patients with advanced cancers may have higher incidences of mutations associated with other forms of treatment resistance , such as egfr t790m and esr1 mutations.28 the potentially unique molecular features in patients with advanced , chemotherapy - resistant cancers may represent one factor that contributes to the the matched targeted therapy low success rate of approach . treatment directed against actionable genes remains suboptimal . 
pathways that were previously thought to be universally targetable have proven to be difcult to treat , such as the phosphatidylinositol 3 - kinase / akt pathway , 29 or subject to variation in results by tumor type , such as braf30 or her2 / 3 mutations.31 , 32 in protocol patients , we were unable to ascertain any strong individual signals of activity for particular alterations in specic tumor types in part because of insufcient numbers of patients . in conclusion , these results demonstrate the utility of largepanel ngs testing when combined with decision support . the derived benet is realized in only a small subset of patients . 
future efforts should emphasize high - quality and timely decision support and minimize barriers to patient enrollment , with the ultimate goal of broadly delivering precision medicine to patients with solid tumor malignancies . 
broaddus , md , phd , department of pathology , unit 85 , university of texas md anderson cancer center , 1515 holcombe blvd , houston , tx 77030 ; twitter : @mdandersonnews ; e - mail : rbroaddus@ mdanderson.org. support supported , in part , by undesignated donor funds from the md anderson annual funds program provided by the provosts ofce , khalifa bin zayed al nahyan foundation , cancer prevention research institute of texas grant no . 
for more information about asco 's conict of interest policy , please refer to or po.ascopubs.org / site / ifc . scott kopetz stock and other ownership interests : molecularmatch , navire consulting or advisory role : roche , genentech , emd serono , merck , karyopharm therapeutics , amal therapeutics , navire pharma , symphogen , holy stone , biocartis , amgen , novartis research funding : amgen ( inst ) , sano ( inst ) , biocartis ( inst ) , guardant health ( inst ) , array biopharma ( inst ) , genentech ( inst ) , emd serono ( inst ) , medimmune ( inst ) , novartis ( inst ) j . 
jack lee consulting or advisory role : abbvie kopetz et al beate litzenburger employment : qiagen funda meric - bernstam honoraria : sumitomo group , dialectica consulting or advisory role : genentech , inection biosciences , pieris pharmaceuticals , clearlight diagnostics , darwin health , samsung bioepis , spectrum pharmaceuticals , aduro biotech , origimed , xencor , debiopharm group research funding : novartis , astrazeneca , taiho pharmaceutical , genentech , calithera biosciences , debiopharm group , bayer , aileron therapeutics , puma biotechnology , cytomx therapeutics , jounce therapeutics , zymeworks , curis , pzer , effector therapeutics , abbvie , boehringer ingelheim ( i ) , guardant health ( inst ) no other potential conicts of interest were reported . references tsimberidou am , iskander ng , hong ds , et al : personalized medicine in a phase i clinical trials program : the md anderson cancer center initiative . 
massard c , michiels s , fert e c , et al : high - throughput genomics and clinical outcome in hard - to - treat advanced cancers : results of the moscato 01 trial . 
cancer discov 7 : 586 - 595 , 2017 radovich m , kiel pj , nance sm , et al : clinical benet of a precision medicine - based approach for guiding treatment of refractory cancers . 
cancer res 76 : 3690 - 3701 , 2016 jardim dl , schwaederle m , wei c , et al : impact of a biomarker - based strategy on oncology drug development : a meta - analysis of clinical trials leading to fda approval . 
j natl cancer inst 107 : djv253 , 2015 schwaederle m , zhao m , lee jj , et al : impact of precision medicine in diverse cancers : a meta - analysis of phase ii clinical trials . 
j clin oncol 33 : 3817 - 3825 , 2015 schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
singh rr , patel kp , routbort mj , et al : clinical validation of a next - generation sequencing screen for mutational hotspots in 46 cancer - related genes . 
singh rr , patel kp , routbort mj , et al : clinical massively parallel next - generation sequencing analysis of 409 cancer - related genes for mutations and copy number variations in solid tumours . 
bartley an , luthra r , saraiya ds , et al : identication of cancer patients with lynch syndrome : clinically signicant discordances and problems in tissue - based mismatch repair testing . 
cancer prev res ( phila ) 5 : 320 - 327 , 2012 johnson a , zeng j , bailey am , et al : the right drugs at the right time for the right patient : the md anderson precision oncology decision support platfordrug discov today 20 : 1433 - 1438 , 2015 15 . 
liang h , cheung lw , li j , et al : whole - exome sequencing combined with functional genomics reveals novel candidate driver cancer genes in endometrial cancer . genome res 22 : 2120 - 2129 , 2012 33 : 2753 - 2762 , 2015 18 . 
andr e f , bachelot t , commo f , et al : comparative genomic hybridisation array and dna sequencing to direct treatment of metastatic breast cancer : a multicentre , prospective trial ( safir01 / unicancer )  . 
hyman dm , piha - paul sa , rodon j , et al : neratinib for erbb2 mutant , her2 non - amplied , metastatic breast cancer : preliminary analysis from a multicenter , open - label , multi - histology phase ii basket trial . 
 t clinical benet of larger ngs panels assessed for eligibility ( n = 675 ) excluded ( n = 101 ) died before testing declined to return to the institution for treatment insufficient tissue for testing ngs testing ( n = 574 ) final analysis ( n = 521 ) excluded ( n = 53 ) no death within 6 months follow - up < 6 months cms46 / 50 testing ( n = 507 ) without cms46 / 50 ( n = 14 ) no actionable gene found ( n = 371 ) actionable gene found , then progression ( n = 78 ) actionable gene found , no progression ( n = 58 ) cms400 testing ( n = 507 ) no actionable genes in cms400 that are not identified in cms46 / 50 ( n = 301 ) no actionable gene identified in cms400 ( n = 6 ) cms400 testing ( n = 14 ) 1 actionable gene in cms400 not identified in cms46 / 50 ( n = 206 ) no additional actionable gene identified in cms400 ( n = 307 ) 1 actionable gene identified in cms400 ( n = 8 ) 1 actionable gene in cms400 not in previous panel ( n = 214 ) matched therapy ( n = 40 ) nonmatched therapy ( n = 108 ) no therapy ( n = 66 ) fig a2 . 
summary of the turnaround time ( in days ) for large panel next - generation sequencing testing to be nalized once tissue was received by the clinical molecular diagnostics lab . 
polley , phd1 and ying kuen cheung , phd2 applications in early - phase cancer trials have motivated the development of many statistical designs since the late 1980s , including dose - nding methods , futility screening , treatment selection , and early stopping rules . these methods are often proposed to address the conventional cytotoxic therapeutics for neoplastic diseases and cancer . 
recent advances in precision medicine have motivated novel trial designs , most notably the idea of master protocol ( eg , platform trial , basket trial , umbrella trial , n - of - 1 trial ) , for the evaluation of molecularly targeted cancer therapies . 
in this article , we review the concepts and methodology of early - phase cancer trial designs with a focus on dose nding and treatment screening and put these methods in the context of platform trials of molecularly targeted cancer therapies . 
tumors once thought to be of the same histologic type may now be considered heterogeneous and differentiated on the basis of genomic biomarkers and may be treated differently according to the biomarker expression prole . 
because the subject subgroups dened by specic genetic abnormalities of the tumors may only represent a small fraction of the disease population , the concept of precision oncology trials for targeted therapies has created enormous challenges in recruiting patients with rare genomic subtypes . 
this recruitment challenge , coupled with the need to test multiple targeted therapies in relatively small molecularly dened patient subgroups in an efcient manner , has led to the rethinking of cancer drug development paradigto meet these challenges , the concept of master protocols has been introduced to increase the speed of oncologic therapy development and evaluation . 
the main goal of constructing a master protocol is to enable sharing of operational infrastructure and key design elements across substudies so as to achieve better coordination and efciency than can be achieved in single trials designed and conducted independently in biomarker - dened subpopulations.1 - 5 a platform trial is a randomized trial in a single histology that involves multiple treatments and multiple biomarkers under a master protocol . 
instead , rather than presuming we know which therapy is appropriate for which biomarker stratum , randomization is used for treatment assignment within each biomarker stratutable 1 gives a schema of biomarker - based inclusion criteria in a hypothetical platform trial . 
 polley and cheung context key objective this article provides a critical review of early - phase cancer trial statistical methods and discusses their practicality under the novel platform trial paradigm for evaluating molecularly targeted therapies . knowledge generated although many trial design principles for cytotoxic agents still hold relevance for molecularly targeted therapies , new design and analysis issues , including delayed toxicities and efcacy - tolerability trade - offs , should be considered when designing studies involving these agents . 
early - phase platform trials facilitate efciency gains by allowing within - trial information sharing of safety and efcacy data . relevance this article provides a practical guide for early - phase platform trial designs in the era of precision medicine . 
novel statistical methods continue to be needed to harness the information garnered in these new - generation trial designs to help expedite oncologic drug development and approval . for neoadjuvant therapeutic response with imaging and molecular treatment of analysis 2 ( i - spy2 ) trial women with locally advanced breast cancer.8 , 9 methodologic innovations in a platform trial may include midtrial outcome - adaptive randomization to favor treatments with higher response rates within the respective biomarkerdened stratum.10 , 11 the treatment effects of various experimental therapies are often estimated according to a bayesian hierarchical model by borrowing outcome information across molecularly dened biomarker subgroups.10 , 11 at the same time , bayesian decision rules may be incorporated to determine when and whether therapies with low probabilities of success should be discontinued and therapies with high probabilities of future success should advance to subsequent conrmatory studies ; in this way , novel therapies may be added or dropped in a perpetual manner.10 , 12 , 13 because of the need for midtrial adaptations , platform trials often use short - term end points , such as absence of disease progression at 8 weeks ( as in battle ) or pathologically conrmed complete response ( as in i - spy2 )  . 
although the bayesian inferential structure provides a convenient and efcient framework for analysis , it does not protect the type i error rate against multiple comparisons in the conventional sense . 
as such , phase ii platform trials are generally regarded as exploratory , and positive ndings from these trials will require independent conrmation for the promising drug - biomarker stratum in subsequent phase iii trials . cancer trial designs have been well established for cytotoxic agents . 
specically , phase i trials are dose - nding studies that examine safety of the drug and estimate the maximum - tolerated dose ( mtd ) , 14 and phase ii trials are designed to screen new drugs on the basis of pilot efcacy such as clinical response.15 , 16 the general objective of early - phase cancer trials is to identify and recommend a promising dose regimen of a new agent for conrmatory investigation in multicenter phase iii randomized clinical trials . 
the rationale for using toxicity as a surrogate for efcacy can be traced back to the suggestion of using nitrogen mustards in the treatment of neoplastic diseases in the 1960s.18 the most commonly used statistical design for a phase i oncology dose - nding trial is the 3 + 3 design . 
this is a rulebased design in which the number of dose levels is xed in advance and the dose escalation or de - escalation decision is based on the number of dlts that patients experience at a given dose . 
the 3 + 3 design has received extensive examinations and has been demonstrated to produce poor statistical properties , including treating many patients at doses that are below the biologically active level and imprecise estimation of dlt probability because of the relatively small number ( three to six ) of patients treated at each dose cohort.19 - 21 to address the deciencies of the 3 + 3 design , many dosending designs have been proposed since the 1990s that purport to improve statistical accuracy and trial efciency in the context of chemotherapy trials.22 the most prominent work among these is an adaptive design known as the continual reassessment method ( crm ) .23 , 24 the crm uses a mathematical model to relate the dose levels to the probability of dlt . 
after the rst patient has been treated at the dose determined by the initial dose - toxicity curve , the toxicity information is combined statistically with the initial curve , and an updated dose - toxicity curve is estimated using a bayesian methodology . 
the crm has been shown to improve the precision in estimating the mtd and to increase the number of patients assigned to the selected mtd when compared with the standard 3 + 3 method.25 - 27 the latter property has important ethical implications because traditional methods will likely treat many patients at low and , hence , inefcacious the crm fullls the therapeutic doses . purpose of phase i cancer trials , which are often the last resort of the patients who have exhausted other alternative treatment options . 
during its early inception , the crm drew some criticisms particularly with regard to the possibility of exposing patients to doses that are likely to cause dlt.28 , 29 to address these concerns , several trials used a hybrid approach that combined the model - based crm with a rulebased initial dose - escalation plan ( clinicaltrial.gov identiers : nct03141203 , nct03733990 , and nct03028766 ) .28 - 31 in this regard , once an mtd is identied , a new chemotherapy will be studied in phase ii trials , which are proof - of - concept studies looking for early indication of antitumor activity . 
this objective is often achieved with a single - arm trial design using clinical response as an end point , with the possibility of stopping a trial early as a result of futility.16 , 23 , 32 , 33 for instance , to have 80% statistical power to reject a 25% response rate in favor of a 45% response rate of a new drug at 5% signicance , a single - arm , xed - sample size trial will need to enroll 36 patients and seek to observe 14 responses in the enrolled patients . 
alternatively , a simon two - stage design16 will rst enroll 17 patients and then enroll an additional 24 patients ( ie , a total of 41 patients ) only if there are at least six responses in the rst 17 patients . 
because of the provision for futility stopping , the simon design will , on average , treat fewer patients with inefcacious drugs , thus allocating resources to the more promising ones . 
however , to demonstrate promise of a new drug , an adaptive design with futility stopping will always require a larger maximum sample size than a xed trial ( as in the example just provided )  . 
a participant with a given biomarker is eligible for random assignment to a drug and dose among those marked by a checkmark , which indicates that the drug is a candidate for the biomarker and that the dose does not exceed the maximum - tolerated dose . 
these noncytotoxic agents tend to induce long - lasting mild toxicities , and hence , the assumption of acute toxicities may not apply.35 for some agents , prolonged administration may be required to achieve desirable therapeutic benet , thus increasing the likelihood of late - onset or cumulative toxicities that may manifest many months after the initiation of treatment . 
therefore , for phase i trials of targeted therapies , it is critical to identify the mtd with respect to a longer observation window , so as to ensure future investigations are limited to a tolerated dose range . the 3 + 3 design and crm - type designs dictate that accrual of new patients be suspended until all enrolled patients have been fully evaluated for dlt . 
when delayed toxicities are anticipated , the trial length may be prohibitively long because of the need for frequent accrual suspension and the lengthy dlt observation window.36 , 37 a dose - nding design that does not require suspension of patient accrual while waiting for dlt information on previously treated patients to mature is the time - to - event continual reassessment method ( titecrm ) .38 the dose allocation scheme of the tite - crm follows closely the original crm paradigm but additionally leverages the time - to - toxicity and partial follow - up information in all enrolled patients when estimating the mtd . 
since its introduction , the tite - crm design has been successfully implemented in trials at major academic cancer centers , 39 - 48 as well as us national cancer institutesponsored cooperative groups trials such as radiation therapy oncology group 0813 trial ( clinicaltrials.gov identier : nct00750269 )  . in situations where patients become available to a trial at a rapid rate , the tite - crm could accrue many patients at a given dose level before anyone has been observed for a meaningfully long period . 
therefore , it may be useful to impose a waiting window between cohorts of patients , so as to allow adequate follow - up before adaptation is made.49 - 51 tolerability and efcacy trade - off for molecularly targeted agents and immunotherapies , it may not be adequate to only consider toxicity and tolerability in a dose - nding trial . 
second , a higher dose of a new compound may be less efcacious than a lower dose52 or may see limited benets on the basis of considerations of pharmacokinetic properties.53 hence , the mtd may not be an ideal recommended phase ii dose , although it is still useful to dene the upper safety limit by the mtd . 
third , dlts may not be observed with these agents , and mtd may not be determined at the conclusion of dose escalation.54 , 55 these are the scenarios where both toxicity and activity end points should be used to guide dose recommendations.54 , 55 yan et al56 proposed an adaptive bayesian phase i / ii efftox design that guides dose selection on the basis of the tradeoffs between the probabilities of treatment toxicity and efcacy . 
paoletti and postel - vinay55 discussed the ideal setting for the efftox design and contended that such design is best suited when a homogeneous disease population is well - dened for drug sensitivity and an early biomarker for efcacy is available . 
houede et al57 model clinical utilities on the basis of both toxicity and clinical response in a combination therapy trial , which aims to choose the optimal dose pair of a chemotherapy and a biologic agent . finally , li et al58 proposed an approach to identify the best drug at an efcacious and safe dose for a given biomarker subtype in an early - phase platform trial . 
as cancer trials increasingly focus on combination therapies , it is critical to leverage efcacy information as well as toxicity in dosing decisions . treatment screening in a randomized platform trial in a single - arm , proof - of - concept trial , the interpretation of the results and conclusion of the trial depend on the specication of what would constitute a poor ( null ) response rate on the basis of historical data or a priori clinical experience . 
although response dened by tumor shrinkage is an indicator of activity for cytotoxic drugs , progression - free survival ( pfs ) may be more appropriate for early indication of efcacy of a targeted therapy whose mechanism does not necessarily shrink tumors . 
pfs is a less specic end point than tumor shrinkage because untreated patients may survive without progression for a period of time , whereas patients not treated with chemotherapy are unlikely to experience response . 
furthermore , as precision medicine challenges the fundamental assumption of tumor homogeneity , the appropriate null response rate may vary with biomarker expression . in a platform trial where several drugs are considered for a biomarker subtype ( table 1 ) , drug screening can be based on randomized comparison and ranking , thereby eliminating some subjectivity in choosing a null response rate . 
others have proposed adaptive screening strategies such as a two - stage design , 60 , 61 whereby the rst screening stage aims to select a promising drug among several for additional evaluation , and continuous monitoring using the sequential probability ratio test.62 these innovative adaptive designs have been shown to increase the nal dose selection accuracy while treating more patients with better drugs during the trial and can accommodate situations where there is a concurrent standard therapy . 
indeed , the infrastructure developed under a master protocol , in conjunction with the use of these innovative adaptive designs , facilitates unbiased randomized comparisons of potential drug candidates in a platform trial in a timely and efcient manner . pooling information across subtypes in a platform trial in an early - phase platform trial , where a drug may be given to patients across biomarker subtypes , the safety prole of the drug can be evaluated using pooled information across subgroups . 
specically , it is often reasonable to assume there is a common mtd for all biomarker subtypes when the biomarker is agnostic to adverse reactions to the drug.58 the concept of combining toxicity information from different diseases is not new , because phase i cancer trials of chemotherapy are often conducted in patients with heterogeneous malignancies ( eg , solid tumors )  . 
however , implementing this concept in the context of a platform trial provides much needed relevant safety information of the drug in patients with rare biomarker subtypes . when assessing a drugs efcacy in a given biomarker subtype , a statistically unbiased approach is to consider the observed response or pfs rate using only data of patients with that subtype . 
this analytical approach may make it difcult to understand the drugs effect in rare biomarker subtypes . is that a potential advantage of running a platform trial a targeted therapys efcacy for a given biomarker can potentially be analyzed based on its efcacy in other subtypes of the same histology , when the drug is hypothesized to work through a common molecular mechanism in all subtypes . 
 polley and cheung this analysis adds another level in the hierarchy of bayesian analysis , it is called hierarchical bayesian modeling ( hbm )  . several authors have examined and proposed the use of hbm in platform trials.58 , 63 , 64 it has been noted that borrowing information using hbm can be counterproductive when the drugs effects vary across biomarker subtypes.63 this is conceivable when a drug has nonspecic anticancer activity that is not specic to the molecule . 
although ongoing work has been suggested to model multiple sources of exchangeable drug effects in conjunction with hbm , 64 it is critical to evaluate the clinical situations as to whether there is a strong reason for expecting a common drug effect across subgroups . discussion there are many potential efciency gains associated with a platform trial.2 , 5 first , the use of a common genomic screening platform to identify patients eligible for the trial often results in shorter recruitment time and lower screen failure rate . 
second , the use of centralized governance bodies under a master protocol ( eg , the scientic review committee , institutional review board , or data and safety monitoring committee ) can ensure streamlined and efcient oversight for all substudies . 
for example , the trial innovation network represents a collaborative national network that aims to enhance operational efciency by leveraging the resources and expertise of the clinical and translational science award.65 third , the speed of drug development and evaluation is improved by the common infrastructure that allows opening or closure of substudies of new agents and biomarkers more expeditiously . other related trial designs for biomarker - based cancer drug development under the broad denition of a master protocol include basket trials and umbrella trials.1 , 3 , 4 a basket trial evaluates a targeted therapy on multiple disease subtypes ; specically , each basket evaluates the therapeutic effect of a targeted therapy for several cancer types that have a common molecular biomarker or genetic alteration . 
basket trials are typically nonrandomized ; on the basis of a prespecied genetic alteration , patients are assigned to a regimen that is expected to be active for their tumor . 
these biomarker - dening strata may be single - arm phase ii or phase ii / iii trials that randomly assign patients to either the matched targeted therapy or some standard therapy ( or placebo )  . 
an example of an umbrella trial is the adjuvant lung cancer enrichment marker identication and sequencing trial ( alchemist ) for early - stage nonsmall - cell lung cancer.66 although these novel trials provide a platform to answer clinical questions that traditional clinical trial design may not afford , it is important to appreciate that these gains are accompanied by increased logistical challenges and coordination efforts.67 for example , a platform trial requires tremendous resources and time commitment to establish the common trial infrastructure . 
often , a platform trial would involve multiple agents developed by different industry partners ; the contract negotiation with multiple stakeholders in terms of data sharing or coordination across substudies will likely pose additional communication the assay platform used to screen challenges . 
finally , patients for eligibility and assignment to substudies will also need to be cleared with an investigational device exemption by the us food and drug administration , further adding regulatory complexities . despite these challenges , the opportunities afforded by the new - generation platform trials are enormous . 
from a study design perspective , as we have illustrated , a single platform trial under a master protocol can facilitate information sharing of safety and efcacy data across disease subtypes in the statistical analysis.58 to date , most existing statistical methods have been proposed that deal with safety and efcacy questions independently ( eg , crm for safety dose nding , hbm for efcacy evaluation )  . 
 early - phase platform trials manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors inst = my institution . 
semin oncol 42 : 724 - 730 , 2015 renfro la , sargent dj : statistical controversies in clinical research : basket trials , umbrella trials , and other master protocolsa review and examples . 
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triplenegative breast cancer ( tnbc ) is dened by the lack of expression of estrogen receptor ( er ) , progesterone receptor , and human epidermal growth factor receptor 2 ( her2 ) .1 , 2 this subtype represents 15% to 20% of all breast cancers and is associated with the worst outcome of all subtypes , with greater tendency to distant recurrence in general and visceral metastasis in particular , including brain metastasis.3 , 4 to date , chemotherapy remains the standard of care for tnbc.5 molecular stratication of tnbc will have treatment implications.6 for example , approximately 40% of tnbc patients have expression of programmed death - ligand 1 ( pd - l1 ) protein in immune cells , and this biomarker predicts survival benet from anti - pd - l1 therapy in combination with chemotherapy in the rst - line metastatic setting.7 in addition , approximately 10% of patients with tnbc harbor a germline brca1 / 2 mutation , which confers sensitivity to platinum and / or poly ( adpribose ) polymerase ( parp ) inhibitors.2 , 8 parp is involved in the repair of dna single - strand breaks via the base excision pathway . 
parp inhibitors such as olaparib or talazoparib lead to an accumulation of double - strand dna breaks , resulting in the activation of homologous recombination repair , which can compensate for the lack of activity of the base excision pathway and repair the dna damage.9 however , patients with defects in the homologous recombination dna repair pathway cannot repair dna damages caused by parp inhibitors , and the tumor cell eventually dies ( a term known as synthetic lethality )  . the patient is a 46 - year - old woman diagnosed in december 2015 with stage iib ( ct3cn1 ) moderately differentiated invasive papillary carcinoma with marked tumoral inltrating lymphocytes10 ( 60% ) and the presence of vascular invasion . 
she had a mastectomy and lymphadenectomy in june 2016 . analysis of the surgical specimen revealed extensive invasive residual disease ( ypt2ypn1 ) with a tnbc images of vascular inphenotype and abundant vasion . 
she then underwent adjuvant radiation to the breast and started adjuvant endocrine therapy with tamoxifen ( clinical decision based on baseline er positivity )  . in august 2017 , a positron emission tomography scan revealed multiple pathologic deposits in the bone , lung , and mediastinum and a prepectoral lesion . 
biopsy of the lesion conrmed recurrence of the disease ( gata3 positivity ) and a tnbc phenotype with a ki - 67 of 70% and androgen receptornegative and tumor inltrating lymphocytes around 10% ( fig 1 )  . 
no germline mutation was detected . here , we describe a heavily pretreated patient with tnbc brain metastasis and a brca1 somatic mutation with a remarkable and durable response to parp inhibitor therapy . 
to our knowledge , this is the rst case report to demonstrate disease response to parp inhibition in a tnbc without a germline brca1 or brca2 mutation . the patient was treated with carboplatin and gemcitabine for six cycles , and she achieved a partial response ( fig 2 )  . 
 ( a ) the tumor ( hematoxylin and eosin stain ) was composed of neoplastic inltrative nests with scattered stromal tumor - inltrating lymphocytes with many images of ( b ) lymphovascular invasion ( cd31 )  . 
 ( g ) the tumor had a high proliferation index ( ki - 67 ) , and ( h ) programmed death - ligand 1 ( pd - l1 ) was negative in both the neoplasm and the stromal cells . emergency department with progressively worsening headache . 
a brain magnetic resonance imaging scan showed multiple metastases , the largest being found in the right frontal lobe with surrounding edema ( fig 3a )  . the patient consented to participate in a research project in which tumor proling at the dna and rna level was performed with results discussed at a molecular tumor board . the prepectoral lesion was used for all the molecular analyses . 
the results revealed somatic mutations in brca1 ( s1253fs * 10 9435_9436delgt ) and tp53 ( s37fs * 6 * ) , a low tumor mutational burden ( four mutations per megabase ) , and a stable microsatellite status . 
at the rna level , an ncounter - based breast cancer 360 panel was performed.11 this assay includes 752 breast cancerrelated genes and 23 signatures , the tumor inammation signature , 12 the pam50 subtype predictor , 13 and the tnbctype classications14 , 15 ( fig 4 )  . 
results revealed a pam50 basal - like subtype with the following features : high expression of brca - ness and dna scar signatures , high expression of proliferationrelated genes , low expression of androgen receptorand estrogen - regulated genes , low expression of cd8 t cells and pd - l1 , high expression of immunosuppressive genes or signatures such as transforming growth factorand regulatory t - cell signatures , and a tnbctype mesenchymal subtype . on the basis of these results , off - label use of olaparib 300 mg twice per day was indicated . 
consideration was given to introducing corticosteroids or delivering whole - brain radiotherapy ( wbrt ) , but after discussion with the patient , a clinical decision was made to start olaparib under close observation and without the addition of either radiotherapy or corticosteroids . 
after 2 weeks of treatment , neurologic symptoms improved , and a restaging magnetic resonance imaging scan at week 8 demonstrated a signicant reduction in the size of the brain lesions and disappearance of associated cerebral edema ( fig 3b )  . 
brca proteins play a critical role in the homologous recombination dna repair pathway.16 in the presence of a brca germline mutation , one allele is affected , and the occurrence of a genetic alteration in the other allele ( eg , through methylation or loss of heterozygosity ) leads to a nonfunctional brca and the appearance of breast cancer , among other cancers.9 to date , two phase iii clinical trials have shown that parp inhibition with olaparib17 or talazoparib18 is superior to standard chemotherapy in terms of progression - free survival in her2 - negative advanced breast cancer harboring a brca germline mutation . 
 short title neoadjuvant doxorubicin + cyclophosphamide followed by paclitaxel radiation completed sep 2016 ; adjuvant tamoxifen carboplatin + gemcitabine with clinical response ; treatment stopped for toxicities palliative radiotherapy for bone metastasis ; progression on capecitabine + vinorelbine olaparib initiated dec 2015 jul 2016 aug 2017 feb 2018 may 2018 diagnosed with stage iib ( ct3n1 ) ; er , 50% ; pr , 0% ; her2 - ki - 67 , 80% right mastectomy and alnd ; ypt2n1 ; tnbc ; ki - 67 , 40% metastatic disease , bone , lymph node , subcutaneous tissue ; tnbc reappearance of pain and progression of bone metastases brain metastases fig 2 . 
alnd , axillary lymph node dissection ; er , estrogen receptor ; her2 , human epidermal growth factor receptor 2 ; pr , progesterone receptor ; tnbc , triple - negative breast cancer ; yp , pathologic staging after neoadjuvant therapy . patients with germline brca1 / 2 - mutated advanced breast cancer . 
brain magnetic resonance imaging before and after olaparib monotherapy . images before olaparib therapy show a cortical enhancing lesion on gadolinum - enhanced t1 - weighted imaging in the inferior frontal gyrus with perilesional edema visualized on uid - attenuated inversion recovery ( flair ) sequencing , as well as diffuse dural enhancement in the right hemisphere . 
selected signature scores are shown with the tumor inammation signature ( tis ) and pam50 signatures at the core ; the other signature scores are shown as bars around the riscores range from 0 to 1 mapped to quantiles of the population with invasive breast carcinoma in the cancer genome atlas ( tcga )  . 
apm , antigen processing machinery ; ar , androgen receptor ; brca - ness , brcaness signature ; cldl - ness , claudin - low subtype signature ; diffrn , differentiation ; dna scar , dna scar signature ; er , estrogen receptor ; her2 - e , her2 - enriched ; inm chmkn , inammatory chemokines ; pd - 1 , programmed cell death protein 1 ; pdl1 , programmed death - ligand 1 ; pgr , progesterone receptor ; tigit , t cell immunoreceptor and ig and itims domains ; treg , regulatory t cell . somatic mutations may also arise in genes involved in homologous recombination . 
early - phase clinical trials of parp inhibitors in metastatic tnbc and germline brca1 / 2 wild - type her2 - negative breast cancer with specic somatic genomic alterations such as brca1 / 2 mutations are underway ( eg , nct02401347 ; phase ii talazoparib in brca1 + brca2 wild - type & triple - neg / her2 - negative breast cancer / solid tumors and nct03330847 ; to assess safety and efcacy of agents targeting dna damage repair with olaparib versus olaparib monotherapy )  . the long - lasting response of breast cancer brain metastasis to olaparib in the absence of any other treatment is worth discussing . 
first , this suggests that olaparib , which had not previously been thought to cross the blood - brain barrier , 28 is able to get to the site of the tumor . 
concordant with this , other case reports with olaparib monotherapy have described regression of brain metastasis.29 , 30 second , olaparib , and other highly effective targeted systemic therapies , allow the delay of wbrt.31 - 35 this is important because wbrt can have a negative impact on quality of life and long - term neurocognitive functioning.36 thus , strategies to avoid , delay , or abrogate the effects of wbrt using systemic targeted therapies should be prioritized . in summary , comprehensive genomic alteration testing may provide novel clinical strategies for personalized therapy in advanced tnbc with improvement in overall survival and quality of life . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / author - center . tom as pascual consulting or advisory role : genentech research funding : genentech ( inst ) , msd oncology ( inst ) consulting or advisory role : bristol - myers squibb , diaceutics research funding : novartis travel , accommodations , expenses : pzer , boehringer ingelheim , menarini , msd , takeda pharmaceuticals , thermo fisher scientic maria vidal consulting or advisory role : novartis / pzer speakers bureau : novartis / pzer , genentech , eisai travel , accommodations , expenses : pzer montserrat muoz consulting or advisory role : eli lilly speakers bureau : roche travel , accommodations , expenses : roche , eli lilly aleix prat employment : novartis ( i ) honoraria : pzer , novartis , roche , msd oncology , eli lilly , daiichi sankyo consulting or advisory role : nanostring technologies ( inst ) , amgen , roche , novartis , pzer , bristol - myers squibb research funding : roche ( inst ) , novartis ( inst ) patents , royalties , other intellectual property : pct / ep2016 / 080056 : her2 as a predictor of response to dual her2 blockade in the absence of cytotoxic therapy travel , accommodations , expenses : daiichi sankyo other relationship : oncolytics biotech no other potential conicts of interest were reported . cristina teixid o honoraria : msd , pzer references bauer kr , brown m , cress rd , et al : descriptive analysis of estrogen receptor ( er ) - negative , progesterone receptor ( pr ) - negative , and her2 - negative invasive breast cancer , the so - called triple - negative phenotype : a population - based study from the california cancer registry . 
nature 490 : 61 - 70 , 2012 carey la , perou cm , livasy ca , et al : race , breast cancer subtypes , and survival in the carolina breast cancer study . 
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clin cancer res 13 : 4429 - 4434 , 2007 cardoso f , senkus e , costa a , et al : 4th eso - esmo international consensus guidelines for advanced breast cancer ( abc 4 )  . 
ann oncol 29 : 1634 - 1657 , 2018 prat a , adamo b , cheang mc , et al : molecular characterization of basal - like and non - basal - like triple - negative breast cancer . 
oncologist 18 : 123 - 133 , 2013 schmid p , adams s , rugo hs , et al : atezolizumab and nab - paclitaxel in advanced triple - negative breast cancer . 
n engl j med 379 : 2108 - 2121 , 2018 ahn sg , kim sj , kim c , et al : molecular classication of triple - negative breast cancer . 
j breast cancer 19 : 223 - 230 , 2016 ashworth a : a synthetic lethal therapeutic approach : poly ( adp ) ribose polymerase inhibitors for the treatment of cancers decient in dna double - strand break repair . 
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lehmann bd , jovanovi c b , chen x , et al : renement of triple - negative breast cancer molecular subtypes : implications for neoadjuvant chemotherapy j clin invest 121 : 2750 - 2767 , 2011 selection . 
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 o prospective genomic profiling of prostate cancer across disease states reveals germline and somatic alterations that may affect clinical decision making see accompanying editorial doi : purpose a long natural history and a predominant osseous pattern of metastatic spread are impediments to the adoption of precision medicine in patients with prostate cancer . 
to establish the feasibility of clinical genomic profiling in this disease , we performed targeted deep sequencing of tumor and normal dna from patients with locoregional , metastatic noncastrate , and metastatic castration - resistant prostate cancer . patients and methods patients consented to genomic analysis of their tumor and germline dna . a hybridization capture - based clinical assay was used to identify single - nucleotide variations , small insertions and deletions , copy number alterations , and structural rearrangements in more than 300 cancer - related genes in tumors and matched normal blood . results we successfully sequenced 504 tumors from 451 patients with prostate cancer . 
twenty - seven percent of patients harbored a germline or a somatic alteration in a dna damage repair gene that may predict for response to poly ( adp - ribose ) polymerase inhibition . 
in contrast , comparative analysis across disease states revealed that apc alterations were enriched in metastatic tumors , whereas atm alterations were specifically enriched in castration - resistant prostate cancer . conclusion through genomic profiling of prostate tumors that represent the disease clinical spectrum , we identified a high frequency of potentially actionable alterations and possible drivers of disease initiation , metastasis , and castration resistance . 
2017 by american society of clinical oncology wassim abida joshua armenia anuradha gopalan ryan brennan michael walsh david barron daniel danila dana rathkopf michael morris susan slovin brigit mclaughlin kristen curtis david m . 
although molecular profiling is not yet considered a standard - of - care for patients with this disease , new evidence points to enhanced treatment response in specific molecular contexts , paving the way for therapy selection on the basis of tumor molecular characteristics . 
all tumors were reviewed by pathologists who specialize in genitourinary oncology for confirmation of malignant histology of prostatic origin . sequencing and analysis we used the msk - impact assay as previously described10 , 11 ( fig 1a )  . 
the assay was performed in a clinical laboratory improvement amendments certified laboratory and designed to robustly identify single - nucleotide variations , small insertions and deletions ( indels ) , somatic copy number alterations , and structural rearrangements in more than 300 cancerrelated genes in formalin - fixed and paraffinembedded tumors and matched normal blood . somatic variant analysis was performed as described , 10 , 11 with germline variants identified in matched blood samples filtered out in the somatic analysis process . 
somatic findings were reported in the electronic medical record and anonymized and uploaded to cbioportal for visualization and analysis.12 - 14 clonality of mutations was estimated as cancer cell fraction15 and implemented in the facets algorithm.16 beginning in may 2015 , patients were given the option to consent to analysis of germline variants that were identified via sequencing of normal blood samples . 
germline analysis of 76 known cancer - predisposing genes was performed as previously described.16a additional methods are provided in the data supplement . results targeted dna sequencing of tumornormal pairs from patients with prostate cancer using the msk - impact assay , we successfully profiled 504 tumors from 451 patients with prostate cancer who presented to the clinic ( fig 1a and data supplement )  . 
in total , 348 patients ( 77% ) had metastatic prostate cancer , 53 ( 12% ) had biochemical recurrence after definitive therapy , and 50 ( 11% ) had locoregional disease ( fig 1b )  . 
metastatic tumors that were successfully profiled were obtained from lymph node ( 45% ) , bone ( 22% ) , liver ( 14% ) , lung ( 5% ) , and other soft tissue sites ( 14% ; fig 1c )  . 
unlike the tcga and su2c - pcf studies , tumors that were profiled represented all three prostate cancer clinical states : locoregional , metastatic noncastrate , and metastatic castration resistant . we began with 746 biopsy / surgical samples to successfully sequence the 504 tumors reported above , with an overall success rate of 68% ( appendix fig a1 )  . 
 a tumor pathology review matched normal extraction library construction target gene capture somatic analysis ( snvs , indels , cnvs , fusions ) germline analysis ( with specific consent ) tumor report in emr genetics report in emr metastatic tumor site other soft tissue lung liver bone patient disease state ( highest known ; n = 451 patients ) locoregional biochemically recurrent metastatic sample type prostate metastasis disease state at time of tumor tissue collection ( n = 504 tumors ) locoregional locoregional locoregional metastatic noncastrate metastatic castration resistant fig 1 . 
their disease state at the time of tissue collection for the 504 tumors that were profiled is represented at bottotumors that were profiled represented all three prostate cancer clinical disease states : locoregional , metastatic noncastrate , and metastatic castration resistant . 
cnv , copy number variation ; emr , electronic medical record , msk - impact , memorial sloan kettering - integrated mutation profiling of actionable cancer targets ; snv , single - nucleotide variation . states and are shown in fig 2 , grouped in pathways that are potentially clinically actionable . prospectively acquired primary tcga tumors ( appendix fig a3 )  . overall , the frequency of alterations in genes of interest in prostate cancer was similar for mcrpc tumors that were profiled in the msk - impact and su2c - pcf mcrpc data sets4 ( appendix fig a2 ) but demonstrated now in a clinical practice setting . 
however , notable differences were observed when comparing primary localized tumors in the msk - impact data set with those that were profiled in the prospective tcga study , including a higher frequency of alterations in tp53 and foxa1 in msk - impact tumors ( appendix fig a3 )  . 
this is most likely a result of the more aggressive nature of the primary localized tumors in the msk - impact cohort , which were predominantly obtained from patients who subsequently developed biochemically recurrent and metastatic disease ( fig 1b ) relative to the in total , 24% of patients carried somatic alterations in the pi3k / akt pathway , including in pten , pik3ca , pik3cb , pik3r1 , akt1 , and akt3 ( appendix fig a4 )  . 
alterations in commonly affected genes in prostate cancer ( eg , ar , pten , tp53 , foxa1 ) are shown in addition to genes in potentially actionable or biologically relevant pathways . 
mapk , mitogen - activated protein kinase ; msk - impact , memorial sloan ketteringintegrated mutation profiling of actionable cancer targets ; pi3k , phosphatidylinositol - 3kinase . homologous recombination , including brca2 , brca1 , atm , fanca , rad50 , palb2 , and cdk1217 - 21 ( fig 3a and appendix fig a7 )  . a recent multi - institutional study identified a high frequency of ddr gene alterations in the germline of patients with advanced prostate cancer.22 of patients in our data set , 221 underwent formal germline analysis , the first 124 of whom were included in the previously reported study.22 of these 221 patients , 42 ( 19% of total ) had a known or likely pathogenic germline mutation in brca2 ( 9% of total ) , chek2 ( 4% ) , atm ( 2% ) , brca1 ( 1% ) , fh ( 1% ) , and pms2 , nbn , palb2 , and brip1 ( , 1% each ; fig 3b and appendix table a1 )  . 
whereas germline ddr gene alterations may predict for response to parp inhibition or platinum agents , somatic - only alterations in these genes without a germline event may still predict for drug response.9 of the 221 patients who underwent germline analysis , 27% demonstrated alterations in brca2 , brca1 , atm , or chek2 , either in the germline or somatically ( fig 3c )  . 
of note , germline analysis alone accounted for approximately one half of these patients only , which suggests that both germline and somatic analysis should be performed to identify patients with ddr gene deficiency . in total , 3% of patients had tumors with somatic alterations in mismatch repair ( mmr ) genes msh2 , mlh1 , pms2 , or msh6 . 
these tumors accounted for the majority of samples with the highest mutation counts on msk - impact profiling ( fig 3d ) , which were confirmed to be enriched for previously described mmr and microsatellite instability signatures23 , 24 ( appendix fig a8 )  . 
identification of mmr - deficient prostate cancers may have immediate clinical applicability , given recent data that suggest sensitivity of such tumors to immune checkpoint blockade in colorectal cancer and other malignancies.25 - 27 overall , 36% of patients were found to have a potentially actionable alteration by using the oncokb annotation platform27a ( appendix fig a9 )  . this platform does not include non - brca / atm germline alterations , missense alterations of unknown significance , and genes whose clinical significance is less clear , including cdk12 and fanca . 
as genomic alterations undergofurther characterization and new trials and drug targets emerge , the frequency of alterations that are defined as actionable may increase . comparative analysis of somatic alterations across disease states a unique aspect of this data set is that it includes genetic profiles of tumors that represent all three clinical states : locoregional , metastatic noncastrate , and metastatic castration resistant . 
consistent with previous studies , 3 , 4 we identified recurrent areas of copy number loss that involved chromosomes 6q , 8p , 13q , and 16q , and areas of copy number gain that involved chromosomes 1q , 3q , 7 , 8q , and x ( fig 4 )  . 
 displayed the highest burden of copy number alterations , whereas those that represented locoregional disease displayed the lowest ( fig 4 and appendix fig a10 )  . aiming to identify possible genomic drivers of disease progression , we performed a selective enrichment analysis to identify genes that were more frequently altered in mcrpc compared with locoregional disease ( fig 5a )  . 
ar amplification / mutation was the most enriched alteration in mcrpc , as shown in previous studies.3 , 4 other genes that were more commonly altered in mcrpc included tp53 , rb1 , pten , apc , atm , fanca , and cdk12 . we performed a similar analysis that compared alterations in mcrpc with metastatic noncastrate prostate cancer ( fig 5b )  . 
the high enrichment of alterations in ar in mcrpc relative to both locoregional and metastatic non - castrate disease serves as a positive control , consistent with the known role of ar as a driver of castration resistance.28 - 31 beyond frequent amplification of the gene , ar antiandrogen resistance mutations were identified in tumors from patients with mcrpc ( appendix fig a11 ) , including an f877l enzalutamide / arn509 resistance mutation32 , 33 that was found in the tumor of a patient who experienced progression after 4 years of treatment on arn509 . 
four locoregional tumors were found to have mutations in ar , including an h875y mutation that is known to confer resistance to flutamide29 , 34 in a prostatectomy 16 17 18 19 20 21 22 disease state : locoregional metastatic noncastrate metastatic castration resistant sample type : prostate metastasis scna fig 4 . 
represented here are regions of amplification ( red ) or deletion ( blue ) with chromosomes listed horizontally ( top ) in the msk - impact , su2c - pcf ( metastatic castration - resistant prostate cancer [ crpc ] ) , and tcga ( primary localized prostate cancer ) data sets . 
msk - impact tumors are sorted by disease state at time of tissue acquisition , from locoregional ( bottom , gray ) to metastatic crpc ( top , blue )  . 
the level of enrichment is represented as difference in frequency between the two indicated classes ( x - axis ) and its significance ( p value , y - axis )  . 
amp , amplification ; homdel , homozygous deletion ; mut , mutation . sample from a patient who was treatment - nave but who had received dutasteride for benign prostate enlargement . in addition to ar , we again identified enrichment of alterations in tp53 , rb1 , pten , and atm in mcrpc compared with metastatic noncastrate disease . 
enrichment of these genes in mcrpc relative to both earlier disease states implicates them in the development of castration resistance , a finding that is of particular interest for atm , a gene that is involved in ddr . 
fanca and cdk12 , two other dna repair genes , did not show statistically significant mcrpc compared with metastatic noncastrate disease as they did versus locoregional disease , enrichment although there is a trend that suggests a role in castration resistance as well ( fig 5d )  . 
when analysis was limited to metastatic tumors or lymph nodes only , similar trends for enrichment were observed , although statistical significance was not always reached because of smaller sample size ( appendix fig a12 )  . only two genes were enriched in metastatic noncastrate versus locoregional disease : apc and , to a lesser extent , arid5a ( fig 5c )  . 
these findings will require functional validation in the laboratory . matched samples identify clonal alterations in prostate cancer in total , 44 patients had more than one tumor site profiled by msk - impact , including 16 with a matched primary localized tumor and a subsequent metastatic tumor . 
tumors from the same patient that were acquired at a later time point typically had a higher mutation count ( fig 6a )  . given the high frequency of tp53 alterations that were observed in primary localized tumors in this data set and prior reports of aggressive behavior of prostate tumors that harbor tp53 alterations , 36 - 37a we sought to determine whether tp53 alterations were present in tumors early in their evolution or whether they were acquired later at disease progression . 
tp53 mutations were clonal , including in cases in which both a primary localized tumor and a later metastasis were available ( cancer cell fraction > 0.9 in the later metastasis )  . 
likewise , we found that somatic alterations in brca2 were present in matched tumors , which again suggested that somatic brca2 loss of function alterations occur early in tumorigenesis for affected patients . conversely , alterations in ar did not occur early in matched samples ( fig 6b , patients p - 0003597 and p - 0004910 ) , which is consistent with treatment - related changes that promote castration resistance . 
of note , other potentially actionable alterations may arise later in disease evolution , as was the case for patient p - 0002149 ( fig 6b ) , who acquired an activating pik3ca e545k mutation ( appendix fig a4b ) in a recurrent tumor nearly 3 years after his radical prostatectomy . to confirm the above findings , we performed phylogenetic analysis on cases in which several matched tumors were available from the same patient ( fig 6c )  . 
for patient p - 0003511 , ar amplification was truncal castration - resistant tumors , which is consistent with its well - characterized role in castration resistance.28 as the number of patients with prostate cancer who are profiled longitudinally throughout their clinical care increases , such findings may provide insight into clonal driver events that promote disease progression and site - specific metastasis . discussion unlike previous prostate cancer genomic studies , we profiled tumors that represent the clinical spectrum of the disease , from locoregional to metastatic noncastrate and metastatic castrationresistant prostate cancer , which enabled comparisons of genomic landscape across disease states using a single assay . 
whereas locoregional tumors in this data set typically represented more aggressive disease than tcga , patients with such tumors are those in greatest need of new treatment approaches and profiling of their tumors may have particular clinical relevance . an increase in copy number alterations and mutation frequency was evident in mcrpc compared with earlier disease states , as was an increased frequency of alterations in ar , tp53 , rb1 , and pten . 
spop mutations , however , were more frequent in the earlier disease states , which suggested better outcomes for patients with spop - mutant tumors , possibly through increased sensitivity to adt . 
in this analysis , apc and atm emerged as candidate genes of interest that may independently contribute to metastasis and castration resistance , respectively , pending functional validation in the laboratory . 
these genes , like atm , are involved in ddr , alluding to a possible role for dna repair defects in the development of castration resistance . we also found that tp53 alterations are early clonal events in matched tumor samples from individual patients . 
a notable exception ( * ) involves a bladder metastasis and a bone metastasis acquired 5 months later , where the patient had received salvage radiation to the pelvis , possibly explaining the higher mutation count in the earlier bladder tumor . 
 ( b ) somatic alterations in tp53 ( red ) , brca2 ( blue ) , ar ( gold ) , and pik3ca ( green ) in matched tumors in the data set , including localized primaries and later metastases ( green box ) and other matched tumors from the same patients . 
crpc , castration - resistant prostate cancer . available , it will be possible to validate this finding , guiding more aggressive treatment approaches early on for these patients . overall , we identified potentially actionable alterations , including hotspot activating alterations in genes that are known drug targets , consistent with findings of the su2c mcrpc study , but this time in a prospective clinical practice setting . 
these findings have immediate therapeutic relevance , given the recently reported sensitivity of these tumors to parp inhibition9 or immune checkpoint blockade.25 the higher frequency of dna repair alterations that were identified via integrative germline and somatic analyses strongly argues for performing both germline and somatic genomic analyses in all patients with advanced prostate cancer who will require systemic treatment , irrespective of screening on the basis of family history . in summary , this study shows that a large genomic data set that represents the clinical spectrum of prostate cancer can provide mechanistic insight into possible genomic drivers of disease initiation , metastasis , and drug resistance . 
our ability to profile metastatic tumors allowed us to detect the evolution of potential driver alterations in matched tumors from individual patients , identifying alterations in tp53 and brca2 as early events that may confer a more aggressive phenotype . 
scher provision of study materials or patients : wassim abida , joshua armenia , daniel danila , michael morris , susan slovin , marc ladanyi , liying zhang , victor e . 
scher collection and assembly of data : wassim abida , joshua armenia , ryan brennan , michael walsh , david barron , daniel danila , michael morris , susan slovin , brigit mclaughlin , kristen curtis , david m . 
scher data analysis and interpretation : wassim abida , joshua armenia , anuradha gopalan , michael walsh , daniel danila , dana rathkopf , michael morris , susan slovin , jeremy c . 
robson honoraria : astrazeneca consulting or advisory role : mckesson , astrazeneca research funding : astrazeneca ( inst ) , abbvie ( inst ) , myriad genetics ( inst ) , biomarin ( inst ) , medivation ( inst ) , tesaro ( inst ) travel , accommodations , expenses : astrazeneca vijai joseph stock and other ownership interests : juno therapeutics daniel danila honoraria : astellas scientific , medical affairs , angle , bayer , xian - janssen pharmaceutical consulting or advisory role : angle , bayer research funding : prostate cancer foundation , genentech , janssen research & development ( inst ) patents , royalties , other intellectual property : gene expression profile associated with prostate cancer travel , accommodations , expenses : cambridge healthtech institute , prostate cancer foundation , angle , bayer , american austrian open medical institute , global technology community , xian - janssen pharmaceutical , oncology education dana rathkopf consulting or advisory role : janssen oncology research funding : janssen oncology ( inst ) , medivation ( inst ) , celgene ( inst ) , takeda ( inst ) , millennium pharmaceuticals ( inst ) , ferring ( inst ) , novartis ( inst ) , taiho pharmaceutical ( inst ) , astrazeneca ( inst ) , genentech ( inst ) , tracon pharma ( inst ) michael morris consulting or advisory role : astellas pharma , bayer , endocyte research funding : bayer ( inst ) , sanofi ( inst ) , endocyte ( inst ) , progenics ( inst ) travel , accommodations , expenses : bayer , endocyte susan slovin consulting or advisory role : bayer brigit mclaughlin no relationship to disclose kristen curtis no relationship to disclose david m . 
durack stock and other ownership interests : adient medical consulting or advisory role : adient medical patents , royalties , other intellectual property : patent disclosures , provisionals submitted for instrument to analyze biopsy specimens ( unrelated to the small renal masses topic )  . patent holder for device related to x - ray imaging data collection ( unrelated to small renal masses topic ) , july 2008 . other relationship : society of interventional radiology foundation stephen b . 
solomon leadership : devicor medical products stock and other ownership interests : johnson & johnson , progenics , aspire bariatrics consulting or advisory role : ge healthcare , angiodynamics , medtronic research funding : ge healthcare ( inst ) , angiodynamics ( inst ) patents , royalties , other intellectual property : ge healthcare - patents pending ( inst ) , aspire bariatrics ahmet zehir no relationship to disclose aijazuddin syed no relationship to disclose jianjiong gao no relationship to disclose debyani chakravarty no relationship to disclose hebert alberto vargas no relationship to disclose kenneth offit no relationship to disclose mark t.a. 
kantoff stock and other ownership interests : bellicum pharmaceuticals , metamark genetics , placon , druggablity technologies , tarveda consulting or advisory role : bavarian nordic , janssen , millennium pharmaceuticals , morphosys , pfizer , astellas pharma , bellicum pharmaceuticals , bind biosciences , endocyte , metamark genetics , medivation , merck , mtg therapeutics , oncocellmdx , oncogenex , sotio , sanofi , tokai pharmaceuticals , bayer , genentech , cristal therapeutics , ipsen , omnitura , gtx , tarveda , druggablity technologies , progenity research funding : medivation ( inst ) , sanofi ( inst ) , oncogenex ( inst ) , aragon pharmaceuticals ( inst ) , amgen ( inst ) , astellas pharma ( inst ) , bayer ( inst ) , bavarian nordic ( inst ) , dendreon ( inst ) , exelixis ( inst ) , janssen ( inst ) patents , royalties , other intellectual property : method for predicting the risk of prostate cancer morbidity and mortality , predicting and treating prostate cancer , methods for predicting likelihood of responding to treatment , chromosome copy number gain as a biomarker of urothelial carcinoma lethality , drug combinations to treat cancer , somatic ercc2 mutations correlate with cisplatin sensitivity in muscle - invasive urothelial carcinoma ( patent ) , up - to - date royalties , wolters - kluwer royalties expert testimony : sanofi , janssen travel , accommodations , expenses : sanofi , janssen , bind biosciences , bavarian nordic , millennium pharmaceuticals david b . 
sawyers leadership : novartis stock and other ownership interests : novartis , agios , blueprint medicines , beigene , oric pharmaceuticals consulting or advisory role : novartis , blueprint medicines , agios , beigene , oric pharmaceuticals patents , royalties , other intellectual property : xtandi nikolaus schultz no relationship to disclose howard i . 
grasso cs , wu ym , robinson dr , et al : the mutational landscape of lethal castration - resistant prostate cancer . 11 - 22 , 2010 nature 487 : 239 - 243 , 2012 7 . 
hyman dm , solit db , arcila me , et al : precision medicine at memorial sloan kettering cancer center : clinical next - generation sequencing enabling next - generation targeted therapy trials . 
cheng dt , mitchell t , zehir a , et al : msk - impact : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
joshi pm , sutor sl , huntoon cj , et al : ovarian cancer - associated mutations disable catalytic activity of cdk12 , a kinase that promotes homologous recombination repair and resistance to cisplatin and poly ( adp - ribose ) polymerase inhibitors . 
bajrami i , frankum jr , konde a , et al : genome - wide profiling of genetic synthetic lethality identifies cdk12 as a novel determinant of parp1 / 2 inhibitor sensitivity . 
mccabe n , turner nc , lord cj , et al : deficiency in the repair of dna damage by homologous recombination and sensitivity to poly ( adp - ribose ) polymerase inhibition . 
mskimpact versus su2cpcf dataset comparison . frequencies of alterations in select genes in metastatic crpc tumors from the msk - impact dataset ( orange ) versus the su2cpcf dataset ( red )  . 
mmr / msi mutation signatures in hypermutated tumors . k - means clustering of mutations / mb ( mut / mb ) across all 504 tumors identified two distinct clusters : cluster1 with mutations / mb10 . 
actionable alterations are ranked by level of evidence ( level 2b : standard of care biomarker predictive of response to an fda - approved drug in another indication , but not standard of care for this indication ; level 3a : compelling clinical evidence supports the biomarker as being predictive of response to a drug in this indication , but neither biomarker nor drug are standard of care ; level 3b : compelling clinical evidence supports the biomarker as being predictive of response to a drug in another indication , but neither biomarker nor drug are standard of care ; level 4 : compelling biological evidence supports the biomarker as being predictive of response to a drug , but neither biomarker nor drug are standard of care )  . 
variants were considered pathogenic or likely pathogenic per american college of medical genetics and genomics guidelines . somatic second hit is reported in column two for patients with germline dna damage repair gene mutations . 
of interest , several of the patients who harbored a germline brca2 c.5946delt ashkenazi founder mutation did not have a detectable somatic second hit in this gene , suggesting a nonclassical pathogenic mechanism for this mutation . 
in this article , we cover topics related to the analysis of prognostic factors , centering on factors that are both relevant at the time of diagnosis or initial treatment and during treatment . 
we then review the state - of - the art methods for dynamic prediction and compare the strengths and limitations of these methods . although static models will continue to play an important role in oncology , developing and validating dynamic models of clinical outcomes need to take a higher priority . 
2019 by american society of clinical oncology introduction the identication of prognostic factors and building of risk assessment prognostic models will continue to play a major role in 21st century medicine in patient management and decision making.1 prognostic factors in oncology associate host and tumor variables treatment.2 with clinical outcomes independent of gospodarowicz et al3 classied factors as either tumor related , host , or environmental . 
tumor - related factors are variables related to the tumor and reveal the tumor biology and pathology , such as size of tumor , lymph node involvement , presence of metastasis , and molecular markers ( overexpression of pten gene , presence of androgen receptor variant ar - v7 )  . 
finally , environmental factors are external to the patient , such as access to health care.3 prognostic models are increasingly used in the design , conduct , and analysis of clinical trials . for example , trials of prostate cancer , randomization was stratied by the predicted survival probability determined by the prognostic model of overall survival.4 - 7 in the tailorx trial , oncotype dx , a 21 - gene score that predicts the likelihood of in several recurrence , is used to classify women with breast cancer by their risk group of recurrence.8 prognostic factors also have been used for enriching the patient population in trials with targeted therapies . 
for example , in the toga trial , patients with human epireceptor 2positive gastric dermal growth factor cancer were randomly assigned to either trastuzumab plus chemotherapy or chemotherapy alone.9 in this article , we cover topics that are related to the analysis of prognostic factors , centering on factors that are relevant both at the time of diagnosis or initial treatment and during treatment or follow - up . 
we make a distinction between static and dynamic predictive models and provide a review of the state - of - the - art methods for dynamic predictive models to promote them for future use . knowledge generated to date , most risk assessment models in oncology have been based on static prognostic models . 
in patients with advanced cancer , the disease has substantially evolved race , age , performance status , body mass index , host factors tumor factors psa , ldh , alkaline phosphatase , p53 , etc clinical outcomes overall survival progression - free survival objective response rate prediction window all available information up to t timeline study baseline landmark time horizon time fig 1 . 
 developing and validating risk assessment models and has a heterogeneous presentation within the patient.10 the interand intrapatient variability should be taken into account in statistical modeling.11 , 12 dynamic prediction incorporates time - dependent covariates so that risk prediction would be continually updated with new observations to reect the patients prognosis . we dene terminology that is typically used in dynamic risk prediction . 
standard variable selection approaches , such as forward selection , backward selection , and so forth , with logistic regression for binary end points , 13 and proportional hazards regression for timeto - event end points , 14 have been applied . 
criticism for the stepwise variable selection has been well documented.15 , 16 of note , classication trees , such as recursive partitioning for both binary and time - to - event end points , 17 - 20 frequently have been used.2 , 21 - 26 we concentrate on penalized methods that t and shrink p predictors and in doing so , reduce the variance of the coefcient estimates.27 thus , these methods would improve the accuracy of the model.28 the least absolute shrinkage and selection operator ( lasso ) and adaptive lasso ( alasso ) have been widely used to develop prognostic models of clinical outcomes.29 , 30 we will briey describe ridge regression and penalized methods . 
ridge regression minimizes the residual sum of squares function , but a caveat is that it does not reduce all the coefcients exactly to 0.28 let yi be the response , xij the jth covariate value ( j = 1 , 2 , , p ) corresponding to the ith individual , j the regression coefcient jth covariate , and a tuning parameter . 
similar to ridge regression , the rst term in lasso is the residual sum of squares , and lasso minimizes this function subject to the l1 penalty ( eq 1 ) : ( cid : 1 ) i ( cid : 1 ) 1 yi ( cid : 1 ) j ( cid : 1 ) 1 j xij ( cid : 2 ) + ( cid : 1 ) ( cid : 2 ) j ( cid : 2 ) ( cid : 2 )  . j ( cid : 1 ) 1 a large tuning parameter causes the coefcient estimates to be equal to 0 , thus the lasso will have the sparsity property.28 lasso is an improvement over ridge regression , although it has the main limitation of tending to select too many unimportant variables , and it performs poorly in situations when p  . 
the alasso enjoys the oracle property.30 , 34 elastic net regression uses a combination of l1 penalty and ridge l2 penalty and is a compromise between lasso and ridge regression . 
n is that it retains more than n variables in the model.35 hastie et al28 provided a thorough comparison of these shrinkage techniques . we applied lasso and alasso from calgb 90401 , a phase iii clinical trial in advanced prostate cancer , with the overall goal of building a model of overall survival.5 we had 22 variables that were common between calgb 90401 and the enthuse trial ( external data set ) .36 because of missing data in the covariates , we used methods to impute them as described by white and royston.37 the regressions estimates from the cox proportional hazards model , lasso , and alasso , are listed in table 1 . 
we considered lasso and alasso and applied both the akaike information criterion and the bayesian information criterion to choose the optimal model of overall survival . lasso and alasso selected eight and nine variables , respectively ( table 1 )  . 
figure 2 shows the solution lactate depath for alasso , and we observe that hydrogenase ( ldh ) greater than the upper limit of normal and eastern cooperative oncology group ( ecog ) performance status were selected early in the l1 path compared with the other variables . 
this is followed by visceral disease , alkaline phosphatase , albumin , hemoglobin , pain , bone metastases , and then psa ( the bayesian information criterion stopped at psa )  . 
the nal model selected the following prognostic factors : ldh greater than the upper limit of normal , ecog performance status , metastatic site ( presence of visceral disease , presence of bone metastases ) , psa , alkaline phosphatase , albumin , hemoglobin , and analgesic opioid use . we have focused on variable selection when the number of predictors is small relative to the sample size . 
in recent years , a few statistical studies extended the penalized method for the joint modeling of longitudinal data and survival outcomes.42 , 43 the general idea is to postulate the joint likelihood linking the two submodels through latent random variables and to add shrinkage operators to select xed and random effects . 
while these methods have not been implemented in oncology , the 4 2019 by american society of clinical oncology heterogeneity of treatment effect ( hte ) is another important area to consider when building prognostic models . hte is the nonrandom , explainable variability in the direction and magnitude of treatment effects for individuals within a population.44 there are different sources for hte , and hte may arise from an underlying causal mechanism , artifacts , measurements , or methods.45 , 46 one main goal of hte analyses is to predict whether a patient might benet from a treatment . 
when we turned to our prognostic model of overall survival in prostate cancer , we computed a risk score from the estimated regression coefcients and the predicted survival at 24 months using the baseline cumulative hazard . 
we observe that patient 2 had a worse predicted survival probability at 24 months than patient 1 . another important task in static predictive modeling is to construct prognostic risk groups , which can be formed on the basis of their quantiles from the estimated linear predictor . 
t , we can update the risk prediction by calculating i * ( ut ( cid : 5 ) )  . there are two general dynamic risk prediction frameworks : joint modeling and landmark analysis . 
joint modeling comprises two linked submodels , one for the longitudinal process and one for the time - to - event data , and both depend on a common set of latent random variables.54 , 55 in particular , the longitudinal data usually are modeled by a linear mixed - effects model . 
the cox regression coefcient quanties the association between the latent longitudinal process and the hazard rate at time t . the longitudinal process and the event time process are assumed to be independent given the latent random effects , and the joint likelihood can be derived . 
the model can be estimated either using a frequentist approach that attains maximum likelihood through an expectationmaximization algorithm54 , 56 - 58 or a bayesian approach that uses markov chain monte carlo to obtain posterior means.59 - 61 while assuming that the parameters are readily estimated from the observed data , the conditional probability i * ( ut ) can be computed . 
a monte carlo estimate of i * ( ut ) can be obtained by sampling the random effects and the parameters from the corresponding distributions.62 on the other hand , landmarking63 - 66 consists of a series of related cox regression models , where each one is dened at a distinct landmark time t.63 - 66 for each pair of { u , t } , a separate model is tted to the individuals who remain in the study and have not yet experienced the event of interest . 
joint modeling models the dual distribution of the longitudinal process and the failure times and hence satises the consistency conditions for dynamic prediction.68 moreover , it exploits the full information of collected data and takes into account the measurement error of the longitudinal data . 
joint modeling also takes a considerable amount of effort to estimate the parameters , and the computational cost is high because it involves complicated joint distribution and numerical integration . landmarking circumvents the aforementioned model assumptions and computational burden , but it is not a comprehensive probability model of in contrast , the longitudinal process and failure times and , as such , does not satisfy the consistency conditions . 
another major shortcoming is that landmarking only considers the patients at risk at the landmark time and does not fully explore the information . several articles have focused on the comparison of joint modeling and landmarking . 
ferrer et al71 compared the two approaches in the case of model misspecication , and they aimed for predicting competing risks of prostate cancer from psa history . dynamic risk prediction ( joint modeling ) relies on model assumptions , and hence , its performance suffers from model misspecication . 
we have previously explored whether psa decline at different landmark times is prognostic for overall survival in patients with advanced prostate cancer.79 fontein et al80 developed a dynamic model for predicting overall survival in patients with breast cancer . 
other applications of dynamic models have been implemented to prostate cancer82 - 85 and colorectal cancer.86 we demonstrated the application of dynamic risk prediction using the datatop study , 87 a clinical trial that examined the benets of deprenyl and - tocopherol in slowing the progression of parkinson disease ( pd ) .88 multiple longitudinal biomarkers were collected in the datatop study , including unied pd rating scale total score , modied hoehn and yahr scale , and schwab and england activities of daily living . 
 developing and validating risk assessment models trajectories ( fig 3a ) and risk of functional disability ( fig 3b )  . patient 169 , who had more severe disease with earlier development of functional disability , and patient 718 , who had less severe disease , were selected to illustrate the patient - specic predictions at clinically relevant future time points conditional on their available longitudinal measurements . 
 ( a ) predicted unied parkinson disease rating scale ( updrs ) trajectories and ( b ) predicted conditional failure probabilities for patient 169 ( top rows ) and patient 718 ( bottom rows )  . 
 halabi , li , and luo joint modeling , our major interest was to predict probability of functional disability after visits at time t given the patients longitudinal proles and event - free status up to time t . 
overtting occurs when a high predictive accuracy is estimated from a model that has been applied to the training set but has low accuracy when assessed in an independent data set.90 a good example of overtting is provided in halabi and owzar.2 the validation of a prognostic model is considered a critical step after a risk assessment model has been built . 
there are two types of validation : external and internal.16 , 89 , 90 external validation , where the frozen model from the training data is applied to an independent data set , is the most rigorous approach . 
of note , other types of resampling methods , such as cross - validation , bootstrapping , and bootstrapping using 0.632 + , are considered appropriate approaches to model validation.32 , 53 , 91 , 92 investigators plot assessment of the models performance usually is conducted by examining the calibration and discriminative ability of the model . 
using data from two phase iii clinical trials , we evaluated the overall survival model for calibration at different landmarks.36 , 93 figure 4a shows that the predicted survival probabilities at 18 months in the enthuse 33 trial were close to the proportion of patients who survived 18 months . 
the rst two points ( circles ) show that the model overpredicted the proportion of patients who survived 12 months , whereas the third and fth data points show that the model underpredicted the proportion of patients who survived 12 months . discrimination describes the ability of a prognostic model to distinguish between patients with and without the outcome of interest.16 several metrics are used to report the performance of a model . 
these models are anticipated to be implemented in both the design and the conduct of future trials . although static models will continue to play an important role in oncology , developing and validating dynamic models of clinical outcomes need to take a higher priority . 
one of the limitations is that modelers may be constrained by the lack of access to the longitudinal biomarker data ; therefore , the next generation of risk assessments are highly recommended to take into consideration the longitudinal biomarker data and outcomes so that predictions are updated . in summary , risk assessment will remain an important research task in precision oncology . 
calibration of the overall survival model for observed and predicted survival probability at ( a ) 18 months and ( b ) 12 months . pertinent to patient outcomes , dene the primary end point a priori , justify the sample size , and describe the appropriate methods for variable selection and model assessment . 
lastly , they should be validated using external data sets if available . an understanding of the longitudinal relationship between host and tumor - related factors and their impact on clinical outcomes is critical . 
we expect to see an upsurge in dynamic risk assessments in oncology , and as such , the american joint committee on cancer and the transparent reporting of a multivariable prediction model for individual prognosis or diagnosis guidelines should be extended to dynamic predictive modeling . 
regardless of whether static or dynamic modeling is the primary objective , we envision that this review will bridge gaps in knowledge and motivate investigators to take risk assessment as a discipline by itself . 
 halabi , li , and luo references osullivan b , brierley j , gospodarowicz m : prognosis and classication of cancer , in osullivan b , brierley jd , dcruz ak , et al : uicc manual of clinical oncology ( ed 9 )  . 
halabi s , lin cy , kelly wk , et al : updated prognostic model for predicting overall survival in rst - line chemotherapy for patients with metastatic castrationresistant prostate cancer . 
j clin oncol 32 : 671 - 677 , 2014 kelly wk , halabi s , carducci m , et al : randomized , double - blind , placebo - controlled phase iii trial comparing docetaxel and prednisone with or without bevacizumab in men with metastatic castration - resistant prostate cancer : calgb 90401 . 
morris mj , heller g , bryce ah , et al : alliance a031201 : a phase iii trial of enzalutamide ( enz ) versus enzalutamide , abiraterone , and prednisone ( enz / aap ) for metastatic castration resistant prostate cancer ( mcrpc )  . 
j clin oncol 37 , 2019 ( suppl ; abstr 5008 ) sparano ja , gray rj , makower df , et al : adjuvant chemotherapy guided by a 21 - gene expression assay in breast cancer . 
n engl j med 379 : 111 - 121 , 2018 bang yj , van cutsem e , feyereislova a , et al : trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of her2 - positive advanced gastric or gastro - oesophageal junction cancer ( toga ) : a phase 3 , open - label , randomised controlled trial . 
miller kd , oneill a , gradishar w , et al : double - blind phase iii trial of adjuvant chemotherapy with and without bevacizumab in patients with lymph nodepositive and high - risk lymph node - negative breast cancer ( e5103 )  . 
rudloff u , jacks lm , goldberg ji , et al : nomogram for predicting the risk of local recurrence after breast - conserving surgery for ductal carcinoma in situ . 
fizazi k , higano cs , nelson jb , et al : phase iii , randomized , placebo - controlled study of docetaxel in combination with zibotentan in patients with metastatic castration - resistant prostate cancer . 
halabi sp , pi l : statistical considerations for developing and validating prognostic models of clinical outcomes , in kelly do , kevin w , halabi s ( eds ) : oncology clinical trials : successful design , conduct , and analysis ( ed 2 )  . 
gabler nb , duan n , liao d , et al : dealing with heterogeneity of treatment effects : is the literature up to the challenge ? trials 10 : 43 , 2009 46 . 
dane a , spencer a , rosenkranz g , et al : subgroup analysis and interpretation for phase 3 conrmatory trials : white paper of the efspi / psi working group on 51 . 
kattan mw , hess kr , amin mb , et al : american joint committee on cancer acceptance criteria for inclusion of risk models for individualized prognosis in the practice of precision medicine . 
rizopoulos d , hateld la , carlin bp , et al : combining dynamic predictions from joint models for longitudinal and time - to - event data using bayesian model averaging . 
yao f , m uller hg , clifford aj , et al : shrinkage estimation for functional principal component scores with application to the population kinetics of plasma folate . biometrics 59 : 676 - 685 , 2003 74 . 
yan f , lin x , li r , et al : functional principal components analysis on moving time windows of longitudinal data : dynamic prediction of times to event . 
halabi s , conaway mr , small ej , et al : serum prostate specic antigen as a predictor of survival in prostate cancer patients treated with second - line hormonal 80 . 
proust - lima c , taylor jm : development and validation of a dynamic prognostic tool for prostate cancer recurrence using repeated measures of posttreatment psa : a joint modeling approach . 
s `ene m , taylor jm , dignam jj , et al : individualized dynamic prediction of prostate cancer recurrence with and without the initiation of a second treatment : development and validation . 
kr ol a , ferrer l , pignon jp , et al : joint model for left - censored longitudinal data , recurrent events and terminal event : predictive abilities of tumor burden for cancer evolution with application to the ffcd 2000 - 05 trial . 
moons kg , altman dg , reitsma jb , et al : transparent reporting of a multivariable prediction model for individual prognosis or diagnosis ( tripod ) : explanation checklist . 
petrylak dp , vogelzang nj , budnik n , et al : docetaxel and prednisone with or without lenalidomide in chemotherapy - naive patients with metastatic castrationresistant prostate cancer ( mainsail ) : a randomised , double - blind , placebo - controlled phase 3 trial . 
halabi s , pi l , lin c - y : developing and validating prognostic models of clinical outcomes , in halabi s , michiels s ( eds ) : textbook of clinical oncology : a statistical perspective . 
giannetta , md1 ; gianluca tomasello , md1 ; stefano stabile , phd1 ; valentina motta , phd1 ; spyridon alexiadis , md1 ; francesco scaglione , md , phd1 , 2 ; angelo vanzulli , md1 , 2 ; massimo torre , md1 ; emanuela bonoldi , md1 ; silvio m . 
veronese , phd1 ; salvatore siena , md1 , 2 ; and andrea sartore - bianchi , md1 , 2 introduction epidermal growth factor receptor ( egfr ) gene mutations are strong oncogenic drivers in a subset of nonsmall - cell lung cancer ( nsclc )  . 
their inhibition with specic tyrosine kinase inhibitors ( tkis ) represents a successful example of precision medicine.1 nevertheless , disease progression almost invariably occurs after 9 - 14 months of treatment with either getinib , erlotinib , or afatinib ( rstand second - generation tkis ) 2 - 4 and after 19 months with osimertinib ( a thirdgeneration tki ) 5 because of the development of acquired therapeutic resistance . several mechanisms of tki resistance have been reported , with different mechanisms for rst - , second - , 6 and third - generation tkis.7 the acquisition of t790m mutation in exon 20 of egfr represents the most frequent mechanism ( more than 50% of patients ) of disease progression with getinib , erlotinib , or afatinib , which can be overcome by osimertinib and other thirdgeneration inhibitors . 
however , a frequent cause of third - generation tki resistance is the development of mutations at the egfr c797 codon , because they prevent binding to the egfr active site.8 , 9 in these patients , preclinical studies have shown that c797s mutation can be found in the same ( in cis ) or different ( in trans ) t790m - mutated alleles , or in other patients the t790m mutation can be lost.10 in clinical practice , resistant mechanisms to different tkis can be detected on disease progression through tissue rebiopsy . 
the isolation and analysis of circulating tumor dna ( ctdna ) through liquid biopsy is also recommended and widely used for identication of acquired resistance mutations of egfr.11 however , not only target - dependent resistance mechanisms can occur . 
after 9 months , the patient experienced disease progression in the liver ( data supplement ) , and a liquid biopsy revealed the acquisition of egfr t790m mutation ( fig 1b ; table 1 )  . 
almost 12 months later , dyspnea worsened , with evidence of increased right pleural effusion and disease progression in the lung , lymph nodes , and liver ( data supplement )  . a new liquid biopsy identied persistence of t790m plus the acquisition of c797s in trans conformation ( table 1 ; fig 1 - c )  . 
synopsis of therapeutic history and histologic and molecular analysis of the present patient showing tumor evolution toward small - cell lung cancer ( sclc ) transformation and accumulation of epidermal growth factor receptor ( egfr ) genetic abnormalities . 
in the case of sclc transformation , a favorable response to etoposide and platinum can be expected25 ; therefore , a rebiopsy should be performed when a histologic transformation is suspected ( eg , aggressive progression )  . in this case report , which describes a representative patient , different subclones harboring distinct resistance mechanisms coexisted . 
the initial ratio of t790m / activating mutation was below the threshold of 0.05 and , according to the ndings of chabon et al , 24 the response to osimertinib was limited to a stable disease by recist criteria . 
this ratio deeply decreased ( table 1 ) at progression after 12 months of therapy with osimertinib when c797s appeared concomitantly with a small - cell transformation , after a total of 20 months of treatment with tkis . 
at this time point , we observed a reduction of t790m / del19 ratio in ctdna , which was similar to the c797s / del19 ratio ( table 1 )  . 
 pizzutilo et al in conclusion , our case report underlines the importance of monitoring the evolution of tissue rebiopsy for identifying heterogeneous resistance mechanisms , especially in patients with aggressive progression , the disease and of to better dene potential treatment modications . 
pizzutilo , calogero lauricella , giulio cerea , massimo torre , salvatore siena , andrea sartore - bianchi financial support : salvatore siena provision of study materials or patients : elio g . 
giannetta , stefano stabile , valentina motta , spyridon alexiadis , angelo vanzulli , emanuela bonoldi , silvio m . veronese , salvatore siena , andrea sartore - bianchi data analysis and interpretation : elio g . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . angelo vanzulli travel , accommodations , expenses : bracco diagnostics , ge healthcare emanuela bonoldi consulting or advisory role : bayer silvio m . 
mitsudomi t , morita s , yatabe y , et al : getinib versus cisplatin plus docetaxel in patients with non - small - cell lung cancer harbouring mutations of the epidermal growth factor receptor ( wjtog3405 ) : an open label , randomised phase 3 trial . 
lancet oncol 11 : 121 - 128 , 2010 rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as rst - line treatment for european patients with advanced egfr mutationpositive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
lancet oncol 13 : 239 - 246 , 2012 sequist lv , yang jc - h , yamamoto n , et al : phase iii study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with egfr mutations . 
j clin oncol 31 : 3327 - 3334 , 2013 soria j - c , ohe y , vansteenkiste j , et al : osimertinib in untreated egfr - mutated advanced non - small - cell lung cancer . 
 case report thress ks , paweletz cp , felip e , et al : acquired egfr c797s mutation mediates resistance to azd9291 in non - small cell lung cancer harboring egfr t790m . nat med 21 : 560 - 562 , 2015 10 . 
niederst mj , hu h , mulvey he , et al : the allelic context of the c797s mutation acquired upon treatment with third - generation egfr inhibitors impacts sensitivity to subsequent treatment strategies . 
rolfo c , mack pc , scagliotti gv , et al : liquid biopsy for advanced non - small cell lung cancer ( nsclc ) : a statement paper from the iaslc . 
tan dsw , yom ss , tsao ms , et al : the international association for the study of lung cancer consensus statement on optimizing management of egfr mutation - positive non - small cell lung cancer : status in 2016 . 
j thorac oncol 11 : 946 - 963 , 2016 jukna a , montanari g , mengoli mc , et al : squamous cell carcinoma transformation concurrent with secondary t790m mutation in resistant egfr - mutated adenocarcinomas . 
yu ha , arcila me , rekhtman n , et al : analysis of tumor specimens at the time of acquired resistance to egfr - tki therapy in 155 patients with egfr - mutant 75ra26 , 2011 lung cancers . 
oxnard gr , hu y , mileham kf , et al : assessment of resistance mechanisms and clinical implications in patients with egfr t790m - positive lung cancer and acquired resistance to osimertinib . 
yang z , yang n , ou q , et al : investigating novel resistance mechanisms to third - generation egfr tyrosine kinase inhibitor osimertinib in non - small cell lung cancer patients . 
leone a : c797s and t790m egfr mutations in non - small cell lung cancer : in trans or in separate clones ? j thorac oncol 13 : e21 - e22 , 2018 19 . 
wang z , yang j - j , huang j , et al : lung adenocarcinoma harboring egfr t790m and in trans c797s responds to combination therapy of rstand thirdgeneration egfr tkis and shifts allelic conguration at resistance . 
zhou z , zhao y , shen s et al : durable clinical response of lung adenocarcinoma harboring egfr 19del / t790m / in trans - c797s to combination therapy of rstand third - generation egfr tkis . 
minari r , bordi p , del re m , et al : primary resistance to osimertinib due to sclc transformation : issue of t790m determination on liquid re - biopsy . 
piotrowska z , niederst mj , karlovich ca , et al : heterogeneity underlies the emergence of egfrt790 wild - type clones following treatment of t790m - positive cancers with a third - generation egfr inhibitor . 
marcoux n , gettinger sn , okane g , et al : egfr - mutant adenocarcinomas that transform to small - cell lung cancer and other neuroendocrine carcinomas : clinical outcomes . 
they demonstrated that one or a number of molecular alterations could commonly be identied in the circulating dna of these patients . their key nding was that 55% of these patients demonstrated therapeutically relevant alterations in their ctdna.1 because these alterations would be unexpected in patients without an underlying malignancy , their comprehensive work suggests another tool to aid in the often difcult task of diagnosing btc . the authors comment that of particular importance , pathologic tissue - based conrmation of btc can be challenging given that clinical yield is often suboptimal and insufcient1 in fact , despite using endoscopic retrograde cholangiopancreatography with brushings ( including forceps biopsies ) , endoscopic retrograde cholangiopancreatography with endoscopy , percutaneous cholangioscopy , and cholangioscopy with spyglass , diagnosing btc in patients suspected to have btc remains particularly challenging . 
for example , in 2018 , a worldwide study revealed that 8% to 22% of patents turned out to have benign disease on microscopic examination of resected specimens.2 mody and cleary3 recently reviewed the potential usefulness of evaluating circulating dna in diagnosing hepatocellular carcinoma and other cancers . 
rizvi et al4 and wasenang et al5 reviewed the evidence and presented data that differentially methylated circulating cellfree dna regions might aid in distinguishing malignant from nonmalignant causes of signs and symptoms in patients where btc is included as part of the differential diagnosis . 
a case of molecularly diagnosed cholangiocarcinoma on the basis of clinical suspicion and after multiple failed attempts at a tissue diagnosis has also been described.7 if btc is suspected , the lack of ctdna consistent with btc could result from the patients having only a small tumor volume and shedding of insufcient mutated dna to be identied . 
however , if the circulating dna included one or more of the molecular alterations described in the study by mody et al1 and in reports by others , it is almost certainly reasonable to conclude that there is an underlying btc in a clinical setting consistent with the patient harboring a btc and no other apparent malignancy . 
aside from being derived from a different tumor of origin , only a couple of the alterations they described would be potentially unrelated to a nonmalignant source , although , for example , identication of circulating brcamutated dna might imply a germline mutation without an underlying related malignancy , and circulating mutated tp53 might be related to clonal hematopoietic cells of indeterminate potential.8 an expert panel of the american society of clinical oncology and the college of american pathologists concluded that , in general , the evidence is currently lacking to support the use of cell - free dna evaluation for early - stage cancer diagnosis.9 however , btc in particular is often difcult to diagnose , even at a locally advanced stage . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / author - center . steven sorscher honoraria : celgene , pzer , puma speakers bureau : celgene , pzer , puma travel , accommodations , expenses : celgene , pzer , puma references 1 . 
wasenang w , chaiyarit p , proungvitaya s , et al : serum cell - free dna methylation of opcml and hoxd9 as a biomarker that may aid in differential diagnosis between cholangiocarcinoma and other biliary diseases . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american pathologists joint review . 
j clin oncol 36 : 1631 - 1641 , 2018 jaiswal s , fontanillas p , flannick j , et al : age - related clonal hematopoiesis associated with adverse outcomes . 
the other two articles are case reports of breast and gastroesophageal cancer with mismatch repair deficiency ( dmmr ) where immunotherapy resulted in remarkable responses . these articles are highly relevant because the us food and drug administration ( fda ) approved pembrolizumab in may 2017 for the treatment of all unresectable or metastatic cancer with msi / dmmr irrespective of tissue origit was the first time that any therapy had been approved for cancers that share a specific genetic feature irrespective of site of origfurthermore , in late july , nivolumab was approved for msi / dmmr metastatic colorectal cancer . 
the fda approvals are based on studies showing disease control rates of 70% to 90% in patients with colorectal cancer and noncolorectal msi / dmmr cancers , most of whom with disease that was refractory to standard chemotherapy upon entering these trials.1 - 3 these trials had not reached median progression - free survival1 , 2 or overall survival1 - 3 at the time of publication , indicating that the responses are durable . msi is defined as a difference in length of microsatellites ( dna nucleotide repeats ) when comparing tumor dna with normal dna from the same individual . 
deficiency in the mmr system , a dna repair system in charge of correcting errors during dna replication , causes a distinct accumulation of errors in microsatellites , as well as a hypermutated phenotype . 
the mmr system is most commonly inactivated in tumors by either somatic inactivation via methylation of the mlh1 gene , 4 germline mutations in the mmr genes ( mlh1 , msh2 , msh6 , and pms2 [ lynch syndrome ] ) , 5 or , more rarely , somatic mmr mutations.6 , 7 dmmr can be detected by two widely implemented methods : immunohistochemistry ( tumor is stained for the four mmr proteins ) and msi testing via polymerase chain reaction ( pcr )  . 
these two methods have a high concordance.8 traditionally , msi testing has been done via pcr focusing on five predefined microsatellites ( monoor dinucleotide microsatellites ) , which were initially chosen during a meeting in bethesda in 1997.9 , 10 with the advent of ngs with targeted gene sequencing or whole exome / genome sequencing , myriad microsatellites can now be investigated simultaneously with computational tools . 
at least four different computational tools have been developed : msings , 11 msisensor , 12 mantis , 13 and mosaic.14 so how common is msi / dmmr in cancer ? it is well known that the highest msi / dmmr incidences are observed in endometrial cancer ( 20% to 30% ) , 15 - 17 colorectal cancer ( 12% to 15% ) , 18 , 19 and gastric cancer ( 15% to 20% ) , 20 although rates may be lower when focusing on metastatic disease ( 4% to 5% in colorectal cancer in populationbased studies ) .21 , 22 bonneville et al23 examined msi with mantis software in 11 , 139 tumors ( 39 cancer types ) , mostly derived from the cancer genome atlas , using whole exome sequencing data ( including 2 , 530 microsatellites )  . 
the study by middha et al24 describes lower rates of msi compared with bonneville et al , 23 but it is probably as a result of the more advanced stages examined in the study by middha et al . 
both studies looked at mutational burden and found that msi tumors have a significantly higher mutational burden compared with microsatellite stable ( mss ) tumors . do msi cases found via msi - ngs always signify tumors with dmmr ? or could they have other etiologies causing msi ? mantis had previously been validated against samples with msi by pcr in colorectal cancer , endometrial cancer , gastric cancer , esophageal cancer , and prostate cancer , as well as uterine carcinomasarcoma.13 in the study by bonneville et al , 23 the investigators examined ngs data of the mmr genes and found mmr mutations in some cases , but do not have data on mlh1 hypermethylation , which is the most common reason for msi . 
in the study by middha et al , 24 the msi status was validated in 138 colorectal and endometrial cancer cases by performing mmr immunohistochemistry and conventional msi by pcr . 
they found a high concordance between the methods ( 99.4% ) , and msi by ngs seems slightly more sensitive than msi by pcr because they discovered three msi cases by msisensor that scored as msi - low or mss on pcr but were dmmr on immunohistochemistry . 
furthermore , they investigated 456 other cancer cases that scored low ( 3 to 10 ) on msisensor and found that only 3.5% were discordant ( msi by pcr ) , reflecting the fact that most cases with low msi are not truly dmmr.10 ideally , validation would be done for every tumor type to ensure that msi is detected across a range of different cancers and truly represents dmmr . 
in the study by middha et al , 24 one case with a pole mutation was msh6 deficient and msi ; all other pole - mutated cases were mss by msisensor and had normal mmr immunohistochemistry , suggesting that msi in pole - mutated cases is secondary to mutations in mmr genes . other studies have described similar findings.6 , 25 the case reports are great examples of how tumors that are not typically associated with msi status can respond dramatically to immunotherapy . 
in the study by kok et al , 26 a 69 - year - old patient with metastatic breast cancer responded to nivolumab , with complete resolution of a malignant gastric ulcer after three cycles and an ongoing response after 12 cycles of nivolumab . 
in the study by klempner et al , 27 an 85 - yearold patient with stage iii gastroesophageal adenocarcinoma , deemed not a surgical candidate , had a complete response to pembrolizumab , with an ongoing response for 8 months . 
because mlh1 - hypermethylated tumors are more commonly observed in older patients , 16 , 20 , 28 a patients age should not deter physicians from investigating msi / dmmr . with the fdas recent approval of immunotherapy in msi / dmmr advanced cancers , and the finding of this molecular signature across a broad range of tumor types , screening all metastatic cancer cases for msi should become standard practice . 
companies and / or institutions who offer multiple gene sequencing panels should strongly consider integrating msi analysis into their existing ngs protocols and include this as part of their reporting . 
some panels are already reporting this , such as the foundation one platform . it is unclear how many of the msi cases found in the studies by bonneville et al23 and middha et al24 were caused by lynch syndrome . 
in prior studies , 10% to 15% of msi / dmmr colorectal cancer cases and 10% of msi / dmmr endometrial cancer cases were caused by lynch syndrome.21 , 22 , 29 lynch syndrome can increase the risk of many of the malignancies found to be msi in the studies presented in jco precision oncology ; thus it is important for physicians to refer patients with msi / dmmr cancers to the genetics clinic ( the exception being mlh1 / pms2 - absent cancers that test positive for mlh1 hypermethylation , because most of these cases are somatic )  . 
overman mj , mcdermott r , leach jl , et al : nivolumab in patients with metastatic dna mismatch repair - deficient or microsatellite instability - high colorectal cancer ( checkmate 142 ) : an open - label , multicentre , phase 2 study . 
kane mf , loda m , gaida gm , et al : methylation of the hmlh1 promoter correlates with lack of expression of hmlh1 in sporadic colon tumors and mismatch repair - defective human tumor cell lines . 
haraldsdottir s , hampel h , tomsic j , et al : colon and endometrial cancers with mismatch repair deficiency can arise from somatic , rather than germline , mutations . 
bartley an , luthra r , saraiya ds , et al : identification of cancer patients with lynch syndrome : clinically significant discordances and problems in tissue - based mismatch repair testing . 
boland cr , thibodeau sn , hamilton sr , et al : a national cancer institute workshop on microsatellite instability for cancer detection and familial predisposition : development of international criteria for the determination of microsatellite instability in colorectal cancer . 
billingsley cc , cohn de , mutch dg , et al : polymerase e ( pole ) mutations in endometrial cancer : clinical outcomes and implications for lynch syndrome testing . 
 sunitinib in the treatment of thymoma and associated autoimmune neutropenia introduction autoimmune disorders such as myasthenia gravis or pure red cell aplasia ( prca ) are frequent in patients with thymoma . 
thymoma - associated neutropenias , however , are rare ; to the best of our knowledge , only 17 patients have been described.1 - 16 for these patients , the diagnosis of neutropenia occurred simultaneously with that of thymoma or , less often , during the disease course , 2 , 4 - 8 , 10 - 16 and was frequently associated with myasthenia gravis , hypogammaglobulinemia , or anemia.2 - 12 , 14 - 16 granulopoiesis has been described as being completely or almost completely absent or as being arrested at the promyelocyte stage.2 - 7 , 9 , 10 , 12 - 16 bone marrow ( bm ) lymphocytosis is frequent and predominantly the result of t - cell infiltration.2 , 6 , 7 , 10 , 13 in one patient , a monoclonal rearrangement of the t - cell receptor ( tcr ) beta chain was found.8 thymoma - associated neutropenia is likely of autoimmune etiology ( ie , autoimmune neutropenia [ ain ] )  . 
antinuclear antibodies were present4 , 10 - 12 , 14 or not6 , 12 , 15 ; antineutrophil cytoplasmic antibodies were positive in one article , 11 with a decrease in titer upon neutrophil recovery . 
antigranulocyte antibodies were not detected5 , 9 , 12 or present10 ; in the latter case , the antibodies did not react with common human neutrophil antigens.10 studies consistently showed decreased granulocyte - macrophage colony formation in the bm of patients or controls in the presence of patients serum , 5 - 7 , 9 , 10 , 12 whereas formation was not affected by control sera or patients lymphocytes or thymoma cells.5 , 7 , 10 the inhibitory activity of the patients serum was present in the immunoglobulin g fraction.5 , 9 , 10 the treatment of thymoma - associated ain resembles that of prca17 and comprises the use of immunosuppressants ( eg , corticosteroids , 5 , 7 - 13 cyclosporine , 13 - 15 azathioprine , 9 cyclophosphamide , 5 , 10 , 12 mycophenolate , 14 or alemtuzumab14 ) , intravenous immunoglobulins , granulocyte colony - stimulating factor , plasmapheresis , and / or thymectomy.5 , 7 , 9 - 15 neutrophil response varied widely among the patients described . the tyrosine kinase inhibitor sunitinib has recently been shown to be effective against thymic carcinoma and , less frequently , type b thymoma in a phase ii trial and in retrospective analyses.18 , 19 sunitinib inhibits the receptors for the plateletderived growth factor receptor , vascular endothelial growth factor receptor ( vegfr ) , and stem cell factor ( kit ) , as well as other kinases . 
the patient provided written informed consent for the research use of the clinical data and biomaterial in accordance with the declaration of helsinki , as approved by the local ethics committee ( university of freiburg )  . the patient , a 33 - year - old man , was diagnosed with thymoma b2 in masaoka stage iva ( table 1 )  . he received three courses of cisplatin , doxorubicin , and cyclophosphamide ( pac ) chemotherapy followed by total thymectomy and resection of adjacent structures , leaving only microscopic tumor residues . 
shortly thereafter , computed tomography scans revealed new lesions in the right cardiophrenic angle and mediastinuthe patient then agreed to receive radiotherapy . radiotherapy was interrupted after 20 gy because of neutropenia and anemia . 
the neutrophils spontaneously rapidly recovered , but the anemia persisted until corticosteroid therapy was started . after recovery of the blood counts and stopping corticosteroids , and in the light of new lesions , radiotherapy was reinitiated but again stopped heiko becker konrad auman rainer claus nikolas von bubnoff ulrich j . 
diagnosed with thymoma b2 in masaoka stage iva . biopsy negative for variants ( non - synonymous and allele frequency > 10% ) in braf exon 15 ; egfr exons 18 , 19 , 20 , 21 ; kras exons 2 , 3 , 4 ; kit exons 9 , 11 , 13 , 17 ; nras exons 2 , 3 ; pdgfra exon 18 ; pi3kca exons 9 , 20 . 
bold font indicates days being on sunitinib treatment ( v being off treatment )  . abbreviations : gat , granulocyte agglutination test ; gift , granulocyte immunofluorescence test ; nd , not determined . * the interval between the end of cycle 2 and the start of cycle 3 was prolonged by 4 weeks because of patients personal reasons ; during this interval , granulocyte colony - stimulating factor was prophylactically applied once per week . computed tomography scan : progressive disease ; discontinuation of sunitinib treatment after 20 gy because of neutropenia , this time not coinciding with anemia . during the following treatment break , the blood counts completely recovered , but the thymoma lesions slowly progressed , which was associated with increased pain and deterioration of the patients general condition . 
at the same time , the patient developed a severe neutropenia ; the lowest wbc count at presentation was 1.290 3 109 / l with bands accounting for 8% , segmented cells 12% , basophils 2% , monocytes 15% , and lymphocytes 62% . 
immunoglobulin g was decreased ( 420 mg / dl ) ; c - reactive protein and c3d levels were increased , and procalcitonin was repeatedly normal . the patient had not received any thymoma - specific systemic therapy or radiotherapy for approximately fig 1 . 
the patient had taken valproic acid for epilepsy since adolescence . bm examination showed hypoplasia and a marked left shift of the granulopoiesis with almost complete absence of mature granulocytes ( fig 1 ) , which was associated with hyperplasia of the erythropoiesis and megakaryopoiesis , and an interstitial t - cell infiltration ( cd1a , predominantly terminal deoxynucleotidyl transferasenegative )  . in the blood , the cd4 : cd8 and tcr alpha / beta : gamma / delta cell ratios were slightly decreased ( table 2 )  . 
although a low cd4 : cd8 ratio as a result of the relative increase of cd8 + cells has been inconsistently described in patients with thymoma , 24 , 25 our patient had normal cd8 + cell counts . 
no clonal tcr gamma rearrangement was detected . in the search for granulocyte autoantibodies , we performed granulocyte agglutination ( gat ) and granulocyte immunofluorescence tests ( gifts ; table 1 )  . 
in enzyme immunoassays , no reactions were observed against hla class i antigens or human neutrophil antigens cd16b , cd11a , cd11b / cd18 , and cd177 . the neutropenia repeatedly displayed prompt but short - lasting responses to granulocyte colony - stimulating factor application . 
this decision was based on the efficacy of sunitinib against thymoma , 18 , 19 our reluctance to apply chemotherapy during neutropenia , and consideration of the immunomodulatory effect of sunitinib.21 - 23 we decided to use sunitinib at a dose of 25 mg / day because adverse effects were common at higher doses in patients with thymoma18 and in 6 - week cycles ( 4 weeks of treatment followed by 2 weeks without treatment )  . sunitinib treatment was associated with a rapid increase in neutrophil counts and decrease in inflammation parameters and granulocyte antibodies ( table 1 )  . 
moreover , the computed tomography scan showed stable disease . because adverse effects had been minimal during cycle 1 , we increased the dose to 37.5 mg / day in cycle 2 , which resulted in mucositis , sinus arrhythmia , and decreased left ventricular contractility , all of which improved during treatment break . 
from cycle 3 on , we kept the sunitinib dose at 25 mg / day and used an alternative application schedule ( ie , 3 - week cycles consisting of 2 weeks of treatment and 1 week without treatment , in accordance with a current trial [ nct01621568 ; sunitinib for advanced thymus cancer following earlier treatment ] )  . 
subsequent treatments included paclitaxel and gemcitabine . at the last follow - up , approximately 4 months after sunitinib was stopped , the patient remained without neutropenia and no granulocyte antibodies were detected . the rationale for the initial trials of sunitinib in patients with thymoma was the expression and mutation profile of distinct signaling molecules . in retrospective analyses of the thymoma biopsy from the diagnosis , immunohistochemistry showed weak expression of vegfr1 and vegfr2 and no expression of kit . 
in miseq ( illumina , san diego , ca ) - based sequencing of thymoma cells microdissected on the basis of their morphology , no variants were identified in the genes analyzed ( table 1 )  . sunitinib alters the ctla4 expression on cd8 + t cells and pd1 expression on regulatory t cells in patients with thymoma , and low ctla4 and decrease of pd1 are associated with shorter survival among patients with thymoma who are treated with sunitinib.18 although we have no data on pd1 or ctla4 , immunohistochemistry showed moderate to high pd - l1 expression on the thymoma cells . discussion the patient was diagnosed with thymoma b2 in masaoka stage iva ; he developed prca under radiotherapy , which resolved with corticosteroid treatment , and ain under progression of the thymoma , which resolved with sunitinib treatment . although we cannot exclude with certainty another cause for the ain besides the thymoma , the causative relationship is supported by the simultaneous occurrence of the ain and the progression of the thymoma and by the previous reports describing the development of ain in patients with thymoma.1 - 16 considering that the etiology of thymomaassociated ain is a matter of debate , we here demonstrate a clear association between neutrophil counts and the detection of autoantibodies against granulocytes that were present at diagnosis and disappeared under treatment . one may speculate that the neutropenic episodes under radiotherapy that were once associated with prca may have been temporary occurrences of the ain . 
however , bm findings ( ie , hypoplastic erythropoiesis , maturing granulopoiesis ) and the rapid and stable wbc recovery without immunosuppressive therapy suggest that these neutropenic episodes were indeed related to radiotoxicity . to the best of our knowledge , this is the first description of a thymoma - associated autoimmune disorder that resolved under sunitinib . 
in the study by thomas et al , 18 two patients developed prca or hypogammaglobulinemia while receiving sunitinib ; three patients had autoimmune disorders before receiving sunitinib and not during treatment , but it was not specified whether these disorders were present when sunitinib therapy was started ( as opposed to earlier ) and then improved under therapy . because our report is based on clinical observations , we cannot exclude that the neutropenia only coincidentally improved during sunitinib treatment . 
sachs no relationship to disclose konrad aumann research funding : novartis ( inst ) travel , accommodations , expenses : novartis rainer claus honoraria : janssen oncology , roche , novartis consulting or advisory role : roche , janssen oncology speakers bureau : janssen oncology travel , accommodations , expenses : janssen oncology , abbvie , roche nikolas von bubnoff honoraria : novartis , bristol - myers squibb consulting or advisory role : novartis research funding : novartis travel , accommodations , expenses : novartis cornelius f . 
waller consulting or advisory role : mylan , teva , boehringer ingelheim , bristol - myers squibb , astrazeneca , msd , roche travel , accommodations , expenses : bristol - myers squibb acknowledgment we thank ingrid bartsch for flow cytometry analyses and the molecular tumor board ( medical center , university of freiburg , germany ) for the thorough and elaborate discussion of the case . 
schweiz med wochenschr 97 : 1606 - 1611 , 1967 jacobson bm , castleman b : case 1 - 1971anemia , granulocytopenia and terminal sepsis in a 70 - year - old woman . n engl j med 284 : 39 - 47 , 1971 4 . 
nagashima s , yamato h , saito k , et al : cd8 + agranular lymphocyte proliferative disorder with t - cell receptor betachain gene rearrangement associated with thymoma and neutropenia [ in japanese ]  . 
mathieson pw , oneill jh , durrant st , et al : antibody - mediated pure neutrophil aplasia , recurrent myasthenia gravis and previous thymoma : case report and literature review . 
kobayashi m , hasegawa t , iwabuchi s , et al : the effect of thymectomy on myasthenia gravis , thrombocytopenia , and granulocytopenia associated with thymoma : report of a case . 
hirokawa m , sawada k , fujishima n , et al : long - term response and outcome following immunosuppressive therapy in thymoma - associated pure red cell aplasia : a nationwide cohort study in japan by the prca collaborative study group . 
thomas a , rajan a , berman a , et al : sunitinib in patients with chemotherapy - refractory thymoma and thymic carcinoma : an open - label phase 2 trial . 
finke jh , rini b , ireland j , et al : sunitinib reverses type - 1 immune suppression and decreases t - regulatory cells in renal cell carcinoma patients . 
ozao - choy j , ma g , kao j , et al : the novel role of tyrosine kinase inhibitor in the reversal of immune suppression and modulation of tumor microenvironment for immune - based cancer therapies . 
gu y , zhao w , meng f , et al : sunitinib impairs the proliferation and function of human peripheral t cell and prevents t - cell - mediated immune response in mice . 
hoffacker v , schultz a , tiesinga jj , et al : thymomas alter the t - cell subset composition in the blood : a potential mechanism for thymoma - associated autoimmune disease . 
faden , md1 , 2 ; roberto gomez - casal , ms3 ; diego alvarado , phd4 ; and umamaheswar duvvuri , md , phd3 introduction the epidermal growth factor receptor / human epidermal growth factor receptor ( her ) family plays a critical role in the growth of numerous human cancers . multiple therapeutics targeted to members of the her family have been developed , including the epidermal growth factor receptortargeting monoclonal antibody cetuximab , which is fda approved for treatment of head and neck squamous cell carcinoma ( hnscc )  . her3 / erbb3 is expressed in most solid tumors , including hnscc , and has been shown to correlate with a poor prognosis.1 , 2 phosphorylation of erbb3 ( perbb3 ) affects numerous pathways , including activation of the pi3k / akt pathway , driving cell survival and growth . 
for this reason , blocking erbb3 activity could be an effective strategy to decrease tumor growth and resistance to other tyrosine kinase inhibitors.3 - 5 cdx - 3379 is a human immunoglobulin g1 lambda monoclonal antibody that binds erbb3 and inhibits its activity by multiple mechanisms . 
after pathologic conrmation by biopsy , informed consent was obtained , the patient was enrolled in nct02473731 and given two 1 , 000 mg intravenous doses of cdx - 3379 . 
a signiresponse was noted within the rst cant clinical 48 hours after administration of the rst dose , with concomitant reduction in pain from 8 of 10 to 2 of 10 . on examination before surgical extirpation , the primary tumor was found to have a nearly complete response , demonstrating a 92% reduction in greatest dimension . 
tumor and germline dna underwent whole exome sequencing ( wes ) library construction with the illumina truseq kit ( san diego , ca ) , with exonic regions captured using agilent sureselect v4 ( santa clara , ca )  . 
rna underwent library preparation with the illumina truseq rna access kit followed by pairedend ( 2 76 ) sequencing on an illumina nextseq 500 , with average coverage of 45 million reads per sample . primary processing of sequence data was performed using illumina casava software v1.8. 
somatic mutations and copy number variants ( cnvs ) were identied by variantdx as previously described.10 all nonsynonymous coding variants , splice site variants , indels , and cnvs above or below 2.0 were considered potentially deleterious . 
after adapter trimming with cutadapt v1.5 , reads were mapped to the human reference genome grch37 / 38 using hisat2 v2.0.4 , and gene expression was quantitated using htseq v0.9. 
differential gene expression was performed using edger v3.20.9. quantitative real - time polymerase chain reaction total rna was isolated from human hnscc cell lines ( te6 , kyse510 , te9 , fadu , pecapj34 , cal33 , scc4 , bhy , osc19 , scc25 ) using the qiagen rneasy kit . 
 faden et al real - time polymerase chain reaction ( pcr ) was performed using the bio - rad iq sybr green supermix and applied biosystems 7900ht fast real - time pcr systethe following primers were used to detect fzd3 mrna : 5 ( cid : 2 ) - gcttccacagtgacacaagg - 3 ( cid : 2 ) and 5 ( cid : 2 ) - accatacactgccagccata - 3 ( cid : 2 )  . to detect erbb3 mrna , the following primers were used : 5 ( cid : 2 ) - actctccatatcccttcctctc - 3 ( cid : 2 ) and 5 ( cid : 2 ) - gtttcctccctttcctctct - 3 ( cid : 2 )  . pcr amplications were run in triplicate for each cell line . comparative quantication of gene expression was performed using the ct method , and values were referenced to gapdh mrna expression . erbb3 phosphorylation erbb3 phosphorylation was measured by veratag assay ( san francisco , ca ) on ffpe tissue.6 colony - formation assays five thousand cells per well were seeded in six - well plates and allowed to attach overnight . 
the area covered by stained cells was measured using imagej . statistical analysis was performed using graphpad prism 7 . perbb3 was assessed by veratag assay to examine changes in activated erbb3 pretreatment to post - treatment , revealing a 69% decrease from 0.45 to less than 0.14 ( lower limit of quantitation of the assay ) , demonstrating successful blockage of erbb3 activation by cdx - 3379 . 
we hypothesized that loss of somatic alterations from pretreatment to post - treatment , in combination with a signicant reduction in clinical tumor volume , could represent selective killing of clones more sensitive to cdx - 3379 and thus give insight terrogating the list of alterations that were present in the tumor pretreatment but not post - treatment ( on the basis of known mechanisms of action of each gene ) identied amplication of fzd3 ( frizzled class receptor 3 ) , a receptor in the wnt signaling pathway , as a possible candidate conferring sensitivity to cdx - 3379 ( table 1 )  . 
rna - seq demonstrated a 2.5 - foldhigher expression of fzd3 pretreatment versus post - treatment ( p , 4.5e - 13 ) , consistent with the 3.0 amplication identied on wes in the pre ( fig 1a )  . 
six percent ( 28 of 496 ) of hnsccs in tcga possess an fzd3 alteration that could result in an increase in downstream wnt signaling , consistent with the case reported here ( one amplication and 27 mrna upregulations )  . 
patients with hnsccs from tcga that possess an fzd3 alteration have poorer overall survival , suggesting that alterations in this gene may be important in the biology of a subset of hnsccs ( fig 1b )  . tumor to explore the possibility that fzd3 plays a role in sensitivity to erbb3 inhibition , we examined fzd3 mrna expression lines , by quantitative real - time pcr in 10 hnscc cell nding high variability among cell lines ( fig 1c )  . 
to test the hypothesis that increased fzd3 expression could render cells more sensitive to cdx - 3379 , akin to the pretreatment tumor , we performed colony - formation assays with table 1 . 
the role of wnt signaling pathways in cancer is well established.11 fzd upregulation has been identied in numerous cancers , correlates with poor outcomes , and is an active area of investigation for targeted therapeutics.11 , 12 fzd3 , in particular , is believed to play a role in both the canonical and noncanonical wnt pathways and has been found to be upregulated in lung , esophageal , and colon cancers.13 - 15 here , we report fzd3 gene amplication and overexpression in pretreatment but not post - treatment tumor from an exceptional responder to cdx - 3379 . 
review of fzd3 in hnsccs from tcga supports the potential biologic importance of alterations in this gene . we further demonstrate that fzd3 expression levels correlate with sensitivity to cdx - 3379 and that activation of fzd3 by its ligand wnt3a further potentiates the effects of cdx - 3379 . 
the mechanism by which fzd3 confers sensitivity to erbb3 inhibition is unclear ; however , the wnt and erbb pathways have been reported to closely interact and collude in tumorigenesis.16 , 17 it remains to be seen whether this is by convergence on a common downstream molecule , such as - catenin , 18 or via effects on different target genes . 
in breast cancer , wnt overexpression activates signaling via erbb.19 , 20 interestingly , we identied a tight correlation between erbb3 and fzd3 mrna expression levels in hnscc cell lines , suggesting a similar relationship and highlighting the interconnected nature of these singling pathways . 
images of a representative colony formation ( top ) with quantication of stained area represented in bar graph ( middle ) and culture additives ( bottom ) p , .001 for all comparisons by one - way analysis of variance . 
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a major barrier is that many conventional designs are inadequate for modern drug development , yet most novel adaptive designs are difcult to understand , require complicated statistical modeling , demand complex computation , and need expensive infrastructure for implementation . 
the objective of this article is to introduce and review a class of novel adaptive designs , known as model - assisted designs , to remove this barrier and increase the use of novel adaptive designs . 
model - assisted designs enjoy superior performance comparable to more complicated , model - based adaptive designs , but their decision rule can be pretabulated and included in the protocolthus implemented as simply as the conventional designs . 
we review state - of - the - art model - assisted designs for phase i clinical trials for single - agent , drug - combination and late - onset toxicity scenarios . 
as a result , conventional designs ( eg , the 3 + 3 design ) are still dominantly used despite relatively poor performance . there is an urgent need to increase the adoption of novel adaptive designs . the objective of this article is to introduce and review a class of novel phase i and ii designs , known as model - assisted designs , 10 - 12 to overcome this quandary of simplicity versus performance . 
model - assisted designs yield superior performance compared with the conventional algorithm - based designs and are comparable to more complicated ( model - based ) designs . with the model - assisted designs , the decision rule can be pretabulated and included in the protocol and , thus , implemented in as simple a way as the conventional designs . 
 yuan et al context key objective is there any novel adaptive design that is simple to implement ? knowledge generated this article introduces and reviews a class of novel phase i and ii designs , known as model - assisted designs , to provide a stateof - the - art approach to overcome the quandary of simplicity versus performance that hinders the adoption of novel adaptive designs . 
model - assisted designs yield superior performance compared with more complicated model - based designs , but the decision rule can be pretabulated and included in the protocol and , thus , implemented in as simple a way as the conventional designs . relevance model - assisted designs are easy to implement and have great potential to improve the efciency and success rate of earlyphase trials . estimate the dlt rate , and has a greater tendency to underdose patients ( ie , it treats patients at the doses lower than the mtd )  . 
despite decades of advocacy by statisticians , the use of the crm , however , is still limited because of statistical and computational complexity of the design.8 , 9 for appropriate use , the crm requires specialized expertise to choose and calibrate the dose - toxicity model and to re - estimate the model at each decision of dose escalation / de - escalation . 
examples of model - assisted designs include the modied toxicity probability interval ( mtpi ) design23 and its variation , mtpi - 224 ; bayesian optimal interval ( boin ) design25 , 26 ; and keyboard design22 ( fig 1 )  . 
given the data observed at current dose , mtpi makes the decision of dose escalation and de - escalation on the basis of the unit probability mass ( upm ) of the three intervals . 
the upm of an interval is dened as the posterior probability that p is within the interval divided by the length of the interval , calculated according to a statistical model known as the beta - binomial model . 
comparison of design characteristics among algorithm - based , model - based , and model - assisted phase i designs algorithm based model assisted model based design characteristic transparency and simplicity in the protocol dose escalation / de - escalation rule can be predetermined and included avoids computation - intensive , repeated estimation of the dose - toxicity curve model to make interim decisions flexibility size targets any prespecied dlt rate allows decision making when the cohort size deviates from the planned no . 
6 sample size can be calibrated to ensure good operating characteristics performance identies the mtd accurately allocates a high percentage of patients to the mtd provides good overdose control abbreviations : dlt , dose - limiting toxicity ; mtd , maximum - tolerated dose . the dose stays at the current dose . 
one deciency of mtpi is that it overly downweighs the overdosing probability , because the overdosing interval is typically wider than the proper dosing interval , which leads to a high risk of overdose of patients ( ie , treatment of a high percentage of patients at doses greater than the mtd ) .22 the keyboard design addresses the overdosing issue of mtpi by dening a series of equal - width dosing intervals ( or keys ) to represent the potential locations of p.22 the proper dosing interval is called the target key . 
the design makes the decision of dose escalation and de - escalation by examining the relative position between the target key and the strongest key , in which the strongest key is dened as the interval that the true dlt is most likely located . 
if the strongest key is on the left ( or right ) side of the target key , the observed data suggest that the current dose is most likely underdosing ( or overdosing ) ; thus , the design escalates ( or de - escalate ) the dose ; otherwise , the dose stays at the current dose . 
the keyboard design outperforms the mtpi with substantially lower risk of overdosing patients and better accuracy to identify the mtd.11 , 22 the variation of the mtpi ( ie , mtpi - 224 ) adopts the same dose escalation / de - escalation rule as the keyboard design but is less transparent . 
the mtpi - 2 relies on complicated procedures , such as occams razor and model selection . compared with the mtpi / mtpi - 2 and keyboard designs , the boin design is more straightforward and transparent ( fig 1 )  . 
let p denote the observed dlt rate at the current dose , dened as the number of patients experiencing dlt at the current dose divided by the total number of dltevaluable patients treated at the current dose . the boin design makes dose escalation / de - escalation recommendations simply by comparing p with prespecied dose escalation ( e ) and de - escalation ( d ) boundaries , as illustrated in figure 2 and described as follows : 1 . 
repeat step 2 until the prespecied maximum sample size is reached or the number of patients treated on a single level reaches a certain number ( eg , 12 )  . 
at that point , select the mtd as the dose at which the dlt estimate is closest to the target.25 , 26 figure 2 provides the default , optimal dose escalation and deescalation boundaries ( e , d ) for commonly used target dlt rates ,  . 
for patient safety , boin imposes an overdose control rule : if the observed data indicate that there is more than a 95% chance that the current dose is higher than and at least three patients have been treated , the current and higher doses are eliminated from the trial . 
to determine the next dose , the modied toxicity probability interval design calculates and compares the values of the three unit probability masses ( upms ) , whereas the keyboard design compares the location of the strongest key with respect to the target key . 
bayesian optimal interval compares the observed dlt rate at the current dose with the optimized dose escalation boundary , e , and de - escalation boundary , d . the simple and intuitive structure of boin gives it several unique advantages . 
because boin guarantees deescalation of the dose when the observed toxicity rate p is higher than the de - escalation boundary d , it is particularly easy for clinicians and regulatory agents to assess the safety of a trial with boin . 
for example , given a target dlt rate of = 0.25 , we know a priori that a phase i trial using boin guarantees de - escalation of the dose if the observed dlt rate is higher than 0.298. 
suppose that , for a phase i trial with a new compound , the regulatory agency mandates that the dose must be de - escalated if the observed toxicity rate is higher than 0.25. 
we can easily fulll that requirement by setting the target dlt rate at = 0.21 , such that boin guarantees de - escalation of the dose if the p 0.25. 
such exibility and observed toxicity rate of transparency are the important advantages of boin compared with the other model - assisted designs , such as mtpi / mtpi - 2 and keyboard designs . because model - assisted designs are built upon rigorous statistical theory , like model - based designs , they also enjoy the same exibility as the model - based designs . 
in addition , unlike the 3 + 3 design , for which the dose escalation and de - escalation decisions can be made only when we have three or six evaluable patients , boin allows decision making with incomplete cohorts in the face of dropouts as a result of dlt inevaluability . 
boin design has been used for various treatment agents , including chemotherapy ( clinicaltrials . gov identier : nct02942264 ) , radiotherapy ( clinicaltrials.gov identier : nct03114462 ) , checkpoint inhibitor ( clinicaltrials . gov identier : nct03600155 ) , monoclonal antibody ( clinicaltrials.gov identier : nct03577704 ) , oncolytic virus ( clinicaltrials.gov identier : nct02705196 ) , and t - cell immunotherapy ( clinicaltrials.gov identier : nct03318900 )  . boin also has also been used in nononcology trials , such as stem cell therapy for stroke patients with stroke.35 we used an ongoing national cancer institute phase i to ii trial ( clinicaltrials.gov identier : nct02942264 ) to illustrate the design . 
one of the trials objectives of was to identify the mtd of tg02 , a pyrimidine - based multikinase inhibitor combined with temozolomide in adult patients with recurrent anaplastic astrocytoma or glioblastoma . because of lack of effective treatments for this patient population , the target dlt rate was set at a relatively high value of 0.35. 
according to the toxicity prole of tg02 in other patients with cancer , the principal vestigator chose 200 mg of tg02 as the starting dose . according to the boin design , the dose will be escalated if the observed dlt rate at the current dose is lower than e = 0.276 , and it will be deescalated if the observed dlt rate is greater than d = 0.419 ( fig 2 )  . 
for example , if the dlt takes up to 8 weeks to evaluate and the accrual rate is one patient per week , on average , then ve new patients could be accrued while investigators wait to evaluate the previous three patients outcomes . 
the question is this : how can new patients receive timely treatment when the previous patients outcomes are pending ? a few model - based designs have been proposed to address this logistic issue , including the time - to - event crm ( titecrm ) 38 and data - argumentation crm ( da - crm ) .39 these designs support continuous accrual and yield superior performance in nding the mtd.39 , 40 however , like the crm , they are statistically and computationally complex and require repeated model tting after each cohort , which limits their use . model - assisted designs , including time - to - event boin ( tite - boin ) 29 and time - to - event keyboard ( tite - keyboard ) designs , 41 provide a well - performing and yet easy - toimplement approach to address late - onset toxicity . 
table 2 shows the tite - boin decision rule with a cohort size of three patients , which can be generated easily using the software described in the software section . 
during the trial , at the current dose , we counted the number of patients , the number of patients who experienced dlt , and the number of pending patients and their standard total follow - up times ( stft ) ; we then used the table to make the dose escalation / de - escalation decision . 
to illustrate the use of the tite - boin decision table , suppose that three patients have been treated at the current dose : one had a dlt , one had no dlt , and one has dlt data pending . 
according to table 2 , if the stft of the pending patient is greater than 0.88 , we treat the next cohort at the same dose ; otherwise , we de - escalate the dose . 
suppose that the next cohort of three patients is treated at the same dose and that , among the six treated patients , one patient had a dlt , two had no dlt , and three have dlt data pending . 
unlike single - agent trials with a string of ordered doses , combinations in the dose matrix are only partially ordered in toxicity , and multiple mtds ( ie , the mtd contour ) may exist in the dose matrix ( appendix fig a1 )  . 
numerous designs have been proposed to nd an mtd or the mtd contour for combination trials.28 , 42 - 52 almost all are model - based designs using a strategy similar to that of a crm : devise a model to describe the dose - toxicity surface and then , on the basis of accumulating data , continuously update the model estimate to select a dose for the new patient . 
because of their statistical and computational complexity , despite good performance , these model - based designs are rarely used for conducting trials . model - assisted designs provide a simple and robust approach to phase i combination trials.43 one example is the boin combination design , 28 which makes the decision of dose escalation / de - escalation according to the same rule as the single - agent boin design described in the modelassisted designs section , and thereby inherits the singleagent designs simplicity and good performance . 
the only difference is that , in combination trials , when we decide to escalate or de - escalate the dose , there is more than one neighboring dose to which we can move . 
dose escalation and de - escalation rule for tite - boin with a target dlt rate of 0.3 and a cohort size of three patients , with up to nine patients treated at a dose no . 
for example , simons optimal ( or minimax ) two - stage design53 could be classied as a model - assisted design in the sense that it is derived from a statistical model ( ie , a binomial model for response ) , but its go / no - go decision rule can be predetermined when the design parameters are specied . 
simons optimal two - stage designs are appropriate for the simple setting in which the end point is binary and quickly ascertainable , and these designs allow only one interim look . phase ii trials sometimes are more complicated and have more than one end point . 
table 3 provides four trial examples with different types of end points ( eg , ordinal end point and coprimary end points ) .54 - 56 in addition , multiple interim looks are useful to improve the exibility and efcacy of the trial , especially in basket and platform trials.57 - 59 the bayesian optimal phase ii ( bop2 ) design60 provides a simple , exible , and efcient model - assisted design to allow multiple interims and handle different types of phase ii trials under a unied framework . the key feature of bop2 is that , although the end points in the four examples are clinically different , they all can be represented using a variable y with k distinct categories , statistically known as a multinomial random variable . 
as an example , consider a treatment that is deemed ineffective if the objective response rate ( orr ) is , in which is a threshold prespecied by clinicians ( eg , = 20% )  . 
at each interim , the go / no - go decision is made according to the following bayesian stopping criteria : stop the trial if pr ( orr | data )  . 
given the response rate deemed ineffective ( ie , the null hypothesis ) and the response rate deemed desirable ( ie , the alternative hypothesis ) , the value of cn is chosen such that the type i error rate is controlled at a prespecied level and the statistical power is maximized . 
 ( see zhou et al60 for details . ) similar bayesian stopping criteria can be applied to other types of end points to determine whether the treatment promising.60 in the equation , as a model - assisted design , one important advantage of the bop2 design is that its stopping boundary can be enumerated and included in the trial protocol before the onset of the trial . 
when they conduct the trial , clinicians simply count the number of relevant events and make the go / no - go decision according to whether that count exceeds the boundary or not . 
if the end point requires a long time to be scored , clinicians may have to suspend the accrual and wait for the interim data mature to make interim decisions . 
use the design decision table to conduct the trial and make adaptive decisions ( eg , dose escalation / stay / deescalation or go / no - go )  . discussion one major barrier for the adaptation of novel adaptive designs is that these designs often are complicated to implement . 
model - assisted designs offer the superior performance compared with the more complicated , model - based adaptive designs but , once designed , can be implemented in as simple a way as the conventional designs . 
the approach establishes a new kiss principle : keep it simple and smart ! as in all trial designs , the design parameters for modelassisted designs must be carefully chosen to reect the clinical setting and the study objective . 
for the boin design , the target dlt rate can vary with the type of phase i studies . the maximum sample size must be realistic and attainable . for the bop2 design , the choice of using a binary end point or coprimary end point , and their null and target rates , depends on which disease type is studied and how effective the standard of care is . 
the number of interim analyses should account for both design efciency and the logistic complexity . the choice of designs parameters should be validated and carefully calibrated through extensive computer simulation to ensure that the design has desirable operating characteristics under a variety of scenarios . 
jack lee consulting or advisory role : abbvie no other potential conicts of interest were reported . references kessel m : the problems with todays pharmaceutical business : an outsiders view . 
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 o bayesian adaptive design for finding the maximum tolerated sequence of doses in multicycle dose - finding clinical trials purpose statistical designs for traditional phase i dose - finding trials consider dose limiting toxicity in the first cycle of treatment . 
 therefore , it is desirable to identify the maximum tolerated sequence of three doses across three cycles of treatment . methods motivated by a three - cycle dose - finding clinical trial for a rare cancer with a jak inhibitor , we proposed and implemented a simple bayesian adaptive dose - cycle finding ( basyc ) design that allows intercycle and intrapatient dose modification . 
because of the patient - specific dosing strategy over cycles , the basyc design is suited as a method in precision oncology . results basyc is simple and transparent because its algorithm can be summarized as two tabulated decision rules before the trial starts , allowing physicians to visually examine these rules . 
2018 by american society of clinical oncology introduction phase i oncology clinical trials are typically first - in - human studies characterized by a small number of patients with a goal of determining the maximum tolerated dose of an experimental drug . 
numerous dose - finding designs have been developed , such as the 3 + 3 design , 1 the continual reassessment method , 2 and the modified toxicity probability interval ( mtpi ; mtpi - 2 ) methods , 3 - 5 as recently reviewed by sverdlov et al.6 most of the dose - finding trials consider dlt within the first cycle of treatment as the primary outcome and do not allow multicycle intrapatient dose modification . 
 for example , a new developmental drug may be considered as an additional treatment in chemotherapy that includes other standard drugs over multiple cycles . our work was motivated by and applied to such a trial at the university of chicago for a rare form of leukemia . 
in this trial , a janus kinase inhibitor , ruxolitinib , was added to a three cycle us food and drug administration ( fda ) approved chemotherapy backbone that includes cycle - specific drugs ( table 1 )  . 
scheme of the dose - cycle finding clinical trial induction ( course 1 ) four - drug bfmbased regimen consolidation ( course 2 ) cyclophosphamide vincristine dexamethasome pegylated asparaginase cytarabine mercaptopurine methotrexate ( it ) interim maintenance ( course 3 ) delayed intensification ( course 4 ) methotrexate ( iv ) vincristine pegylated asparaginase methotrexate ( it ) doxorubicin cyclophosphamide vincristine dexamethasome pegylated asparaginase cytarabine thioguanine methotrexate ( it ) maintenance ( course 5 ) vincristine dexamethasone mercaptopurine methotrexate ( po ) methotrexate ( it ) screening ruxolitinib ruxolitinib ruxolitinib 4 weeks 4 - week treatment , 4 - week washout 4 - week treatment , 4 - week washout 4 - week treatment , 4 - week washout monitoring 2 - 3 years note . 
the chemotherapy backbone is based on the c10403 regimen.7 ruxolitinib is added to courses 2 to 4 as a three - cycle treatment , with potentially varying doses across cycles . abbreviations : bfm , berlin - frankfurt - munster ; it , intrathecally ; iv , intravenously ; po , orally . dose - finding study , which aimed to identify the maximum tolerated sequence ( mts ) of ruxolitinib dose levels across three cycles . 
the mts is defined as the highest sequence of three doses at which the probability of toxicity per cycle ( tpc ) is no more than or close to a prespecified target response rate pt , given that the probability of toxicity at each cycle is also no more than or close to pt . 
three doses of ruxolitinib were considered for each of the three cycles ; therefore , there were a total of 27 possible dose - cycle combinations for three cycles of treatment . the complication of the trial was the intrapatient dose modifications over cycles . 
such approaches usually work well when the sample size of the trial is sufficiently large , as seen in the dynamic treatment regimen methods , 8 reinforcement learning , 9 and sequential multiple assignment randomized trials.10 these approaches aim to better reflect the intrinsically multistage , adaptive structure of medical decisions , in both trial design and analysis of the trial data . 
the first is lee et al , 17 who proposed an innovative phase i / ii design to adaptively and dynamically optimize a patients dose in each of two cycles of therapy based on the joint binary cycle - specific efficacy and toxicity outcomes . 
 begin treatment end of treatment cycle 1 cycle 2 cycle 3 patient enrollment ith cohort trial end mts selection first cohort dar - 1 select dose level ( cid : 143 ) treat cohort at di and collect data stop ? treat at stop ? exit trial dar - 2b treat at stop ? nth cohort exit trial patients without dlts dar - 2a patients with dlts patients without dlts patients with dlts patients without dlts patients with dlts dar - 2a dar - 2b dar - 2a dar - 2b treat at treat at treat at treat at exit trial fig 1 . 
basyc consists of decisions to determine the dose in each cycle for the enrolled patients and to select the maximum tolerated sequence ( mts ) when the trial is completed . 
in each cycle , the dose allocation rule ( dar ) selects the doses for treating patients , in which dar - 1 selects the dose for cycle 1 and dar - 2 selects the dose for subsequent cycles . 
dar - 2 is composed of two subcases : dar - 2a and dar - 2b , which are applied to the two subcohortspatients without dose - limiting toxicities ( dlts ) and patients with dlts , respectively . 
for example , a patient might exit the trial after one cycle of treatment ( potentially because of toxicity [ indicated by stop ] ) based on the dcr applied in cycle 1 . 
figure 1 illustrates the general scheme of the basyc design for the motivating trial with three doses and three cycles of treatment . dcr and dar the basic concept of the dcr is that if the probability of toxicity of the lowest dose in the next cycle is considered lower than the target rate pt , the cohort can move to the next cycle for additional treatment ( ie , the decision is a go )  . 
technical details of the dcr are given in the appendix . if the dcr is a go for a cohort , the cohort will enter the next cycle for additional treatment . 
first , the dar always assigns the first cohort patients at the lowest dose level in all the cycles of treatment , which is a default rule to ensure the safety of the lowest dose in all three cycles . 
in this case , dar - 1 follows the same decision rule as in the mtpi - 2 design.5 specifically , the dose level is decided based on the observed toxicity data from previously enrolled patients treated at the most recent dose in the first cycle . 
this ad hoc rule is needed to protect patient safety . the two subcohorts undergo the dcr and dar separately hereafter until all three cycles of treatment are completed or until they exit the trial . 
motivated by the mtpi - 2 design , 5 dose allocation is presented in the form of dose escalation , stay , or de - escalation to give the candidate dose level at the next cycle relevant to the dose level of the cycle we currently consider . 
 this table is applied to the dose at which the current cohort is treated and the dose at which the previous cohort is most recently treated in the next cycle . 
the two tables can be generated and predetermined before the trial starts for examination by physicians . in the dar , we used the mtpi - 2 design as the backbone for decision making . 
the default values of 1 and 2 are 0.05. dose - finding algorithm to begin a trial , the basyc design treats the first cohort of patients at the lowest dose level in all three cycles . 
then , after the toxicity outcomes from the first cycle are observed , the dcr decides whether the cohort will go to the next cycle , and the dar decides which dose level to use if the dcr decision is go . 
this process is repeated until all cohorts finish all cycles of treatment unless the trial is terminated early because of excessive toxicity ( safety rules are given in the appendix )  . 
as a default rule , the cohort at a given cycle must wait for the cohort at the next cycle to complete follow - up before it can enter the next cycle . we demonstrate in table 2 how the basyc design guides the motivating dose - cycle trial with three doses and three cycles . 
specifically , once the current cohort completes the first cycle of treatment with observed outcome data , the next cohort can be enrolled and assigned a dose level in the first cycle . 
for example , in table 2 , in week 24 , after cohort 3 completed the first - cycle treatment at dose level 2 , the next cohort ( cohort 4 ) was enrolled and treated at dose level 1 as its first - cycle therapy . 
each column number represents the number of patients treated , and each row number represents the number of patients experiencing toxicity , at a given dose where the decision is applied . 
 ( a ) go ( g ) / no - go ( ng ) decisions according to the dose continuous rule ( dcr ) , in which the letters in blue and red represent g and ng decisions , respectively . 
 ( b ) dose allocation decisions according to the dose allocation rule ( dar ) , in which the letters in blue , gold , green , and red represent dose escalation ( e ) , staying at the current dose ( s ) , dose de - escalation ( d ) , and dose de - escalation and exclusion ( du ) because of toxicity , respectively . 
this table is applied to the dose at which the current cohort is treated and the dose at which the previous cohort is treated in the next cycle . mts selection at the end of the trial , among all the candidate sequences of dose - cycle combinations , a sequence of three doses for three cycles would be selected as the mts . 
we provided the true toxicity probabilities of the three dose levels in the first cycle and assumed that the toxicity probability would increase by 10% per cycle at the same dose level . 
for each scenario , 10 , 000 simulated trials are conducted on computer . operating characteristics the simulation results for scenarios 1 to 6 are summarized in table 3 , with four sections per scenario . 
recall that the mts is defined as the highest sequence of three safe doses with the true probability of tpc ( calculated as the average of the three true toxicity probabilities corresponding to the three dose levels ) closest to pt and falling in the ei ( pt 1 , pt + 2 )  . 
if a scenario has no dose sequence falling in the ei , the highest sequence of three safe doses with the true probability of tpc less than pt is considered the true mts . 
no practical methods are available , and the two existing designs17 , 18 in the literature are either highly complex or tailored for specific trials . perhaps the most attractive feature of the basyc design is its simplicity and transparency , as shown in figure 2 . 
we can see that escalation and de - escalation will never happen if the true toxicity probability of a dose is located in the higher interval ( pt + 2 , 1 ) and the lower interval ( 0 , pt + 1 ) , respectively . in this report , we implemented basyc for a realworld trial with three dose levels for three cycles of treatment . 
for example , a total of 53 = 125 sequences are available for trials with five dose levels and three cycles . basyc uses independent beta / binomial models as the working model to estimate the toxicity probability of each dose in each cycle based on all patients data at that dose / cycle . 
 alternatively , one could consider a dependent model , such as the markov model in fernandes et al , 18 to estimate the toxicity probabilities . in basyc , we focused on the dose - cycle trials with binary outcomes . 
with larger sample sizes , one can adopt dose - insertion methods23 , 24 to allow insertion of new doses when necessary . an mts with fixed dose levels at the end of the trial . 
for example , one may not allow a higher dose level than that in mts for any cycle of the treatment in the policy . lastly , a formal personalized dosing policy would follow the spirit of lee et al17 and use model - based inference on multiple cycles . 
stock w , luger sm , advani as , et al : favorable outcomes for older adolescents and young adults ( aya ) with acute lymphoblastic leukemia ( all ) : early results of us intergroup trial c10403 . 
doussau a , asselain b , le deley mc , et al : dose - finding designs in pediatric phase i clinical trials : comparison by simulations in a realistic timeline framework . 
doussau a , thibaut r , paoletti x : dose - finding design using mixed - effect proportional odds model for longitudinal graded toxicity data in phase i oncology clinical trials . 
guo w , ni y , ji y : teams : toxicityand efficacy - based dose insertion design with adaptive model selection for phase i / ii dose - escalation trials in oncology . 
 appendix notation suppose under consideration is a total of i candidate dose levels and j treatment cycles , indexed by i = 1 , 2 , , i ; j = 1 , 2 , , j . 
let k index the cohort , k = 1 , 2 , , k , where k is the maximum number of cohorts that will be recruited to the trial . during dose - cycle finding ( not for maximum tolerated sequence [ mts ] selection ) , we use simple and independent beta priors across doses and cycles and an independent binomial likelihood as a working model for the estimation of pij . 
the application in bayesian adaptive dose - cycle finding ( basyc ) is detailed here . dose continuation rule for cohort k that has been treated at dose i in cycle j , the dose continuation rule ( dcr ) for cohort continuation ( whether the cohort can continue treatment at cycle ( j + 1 ) ) is described as follows : if cohort k has finished the last cycle of therapy ( ie , j = j ) , cohort k exits the trial . if cohort k has not finished all cycles of therapy ( ie , j < j ) , and if pr { p1 , j + 1 > pt|data } > , where is a prespecified cutoff probability and often set to be close to 1 ( eg , = 0.95 ) , stop cohort k for any additional treatment , and patients in cohort k exit the trial . 
 otherwise , continue this cohort k to the next cycle ( j + 1 )  . this algorithm states that when the probability that toxicity of the lowest dose level ( dose level 1 ) in cycle ( j + 1 ) is higher than the target pt does not exceed a large value , cohort continuation is allowed . 
let x1 , j + 1 and n1 , j + 1 be the cumulative number of patients ( across all the previous and current cohorts ) with dose - limiting toxicity ( dlt ) events and treated at dose level 1 at cycle ( j + 1 ) , respectively . 
this beta distribution is used to calculate the posterior probability . dose allocation rule if cohort k in cycle j can proceed to the next cycle , the dose assigned to the next cycle ( j + 1 ) is dependent on the cumulative data from both the current cycle j and the next cycle ( j + 1 )  . 
such data accumulate all the toxicity outcomes from previously treated cohorts at this dose . then , consider a cohort of patients that has just completed treatment in the first cycle at a dose level . 
 here , the previous cohort refers to the cohort of patients enrolled just before the current cohort , and the toxicity data or the data ( also hereinafter ) refer to the data from all enrolled patients treated at the corresponding dose . 
specifically , given data1 and dose1 , the mtpi - 2 design will output a candidate dose level i1 , and given data2 and dose2 , mtpi - 2 will output another candidate dose i2 . 
specifically , given the dose ( dose1 ) in cycle 1 and the candidate dose i2 that mtpi - 2 gives based on data2 and dose2 from cycle 2 , dar - 2b selects the dose i = min { max { dose1 1 , 1 } , i2 } , the minimum of the dose 1 level lower than the current dose in cycle 1 , and i2 as the final dose for treating the cohort in cycle 2 . note that because of the cohort split , the previously enrolled subcohorts may have been treated in cycle 2 at two dose levels , say dose21 and dose22 . 
the outcome data from both subcohorts , denoted as data21 and data22 , will be used to inform the cycle 2 dose for the subsequent cohort that will start from the first cycle . 
in particular , for each of the data21 and data22 , the mtpi - 2 design will give two candidate doses i21 and i22 as the potential dose for the subsequent cohort in cycle 2 . 
then , the minimum of i21 and i22 will be provided as the candidate dose , denoted as i2 = min { i21 , i22 } , for the subsequent cohort as the cycle 2 candidate dose . 
the same principle is applied to decide the dose level in cycle 3 if needed . walkthrough of the hypothetic trial table 2 summarizes how the basyc design guides the motivating dose - cycle trial with three doses and three cycles . 
at this point , cohort 2 is enrolled and assigned to the next higher dose , dose 2 ( d2 ) , according to the dar decision , because cohort 1 has not exhibited any toxicity . 
at week 16 , cohort 1 completes treatment at d1 in cycle 2 with 0 toxicity outcome , and cohort 2 completes treatment at d2 in cycle 1 with 1 dlt outcome . 
the single patient with dlt , patient 4 , is de - escalated to d1 , and the remaining two patients without dlts , patients 5 and 6 , stay at d2 . 
this process continues for subsequent cohorts and cycles at weeks 24 , 32 , and so on , until all 15 patients ( or five cohorts ) have completed treatment at week 56 . safety rule applied in basyc for safety , the basyc design applies two additional safety rules throughout the entire trial : rule 1 ( early termination ) : for any cycle , if the toxicity probability of the lowest dose is higher than the target pt with a large probabilitythat is , if j * { 1 , 2 , , j } such that pr { p1j * > pt|data } > , where is close to 1 ( say = = 0.95 ) terminate the trial early . rule 2 ( dose exclusion ) : if the toxicity probability of dose i in cycle j is higher than the target pt with a large probability , pr { pij > pt|data } > , exclude doses i and the higher doses from cycle j and higher cyclesthat is , doses { i , i + 1 , , i } will never be used in the trial in cycles { j , j + 1 , , j } again . the posterior probabilities in both rules are calculated based on the simple beta / binomial calculation proposed in the mtpi design.3 details are provided in appendix under dose continuation rule . mts definition and selection after the enrollment and dose - cycle finding are completed , in the mts selection , we use a frequentist and nonparametric probability model to make inference . 
specifically , we essentially want to estimate the toxicity per cycle ( tpc ) qs , which is the average of the three toxicities of the doses in a sequence s ( described under definition of mts )  . 
we then construct an unbiased estimate of tpc qs ( described under estimation of qs ) and order - transform them based on isotonic regression ( described under mts selection )  . 
for each sequence s , we define the cumulative dose level as the sum of the dose levels across three cycles ( ie , d * s = ds1 + ds2 + ds3 )  . 
for each sequence s , denote pdsj , j the probability of toxicity for the dose in cycle j of sequence s , j = 1 , 2 , 3 . 
a sequence s is unacceptable if the toxicity probability of each dose in each cycle is higher than pt with a large probability , defined as j = 1 , 2 , 3 , pr { pdsj , j > pt|data } > , where dsj denotes dose levels { 1 , 2 , 3 } and is close to 1 , say = 0.95. 
the posterior probability pr { pdsj , j > pt|data } is based on a simple beta / binomial hierarchic model , similar to the calculation under dose continuation rule . 
 among all the safe sequences that satisfy the condition above , we want to select the highest sequence with overall qs close to pt , subject to the toxicity probability of the same dose level nondecreasing across cycles . 
however , the problem with that approach is that because of random chance , a sequence with a higher order ( and therefore higher toxicity ) might have a smaller qs than a lower - ordered sequence . 
for example , it is possible that the toxicity data for sequence 1 , 1 , 1 include only one toxicity event in all three cycles , whereas the data for sequence 2 , 1 , 1 include no toxicity event . 
however , sequence 2 , 1 , 1 is in theory a more toxic sequence , because it uses dose level 2 in cycle 2 instead of dose level 1 . 
therefore , selecting the sequence based on the distance of |qs pt| is not valid , because we would select 1 , 1 , 1 as the mts , not 2 , 1 , 1 in this case . to address this issue , we propose an isotonic transformation and calculate transformed qs values using the pool adjacent violator algorithm ( pava ) 20 just like in the mtpi design3 , 4 and mtpi - 2 design.5 here , we still use the sequence - specific data for the isotonic transformation . 
when there is a tie between m sequences , there are m ! possible simple orderings among thethen , assuming with probability 1 / m ! that one of the orderings , say ordering w , is true , we apply pava to the following ordering w of qs , w = 1 , 2 , , m !  . 
then , finally , the sequence dl * * that has nonincreasing dose levels across three cycles and the largest frequency f l * is selected as the estimated mts . 
therefore , the dose levels of the mts cannot be increasing , or else the lower dose in the early cycle can always be replaced by the higher dose in the later cycle . 
these results show that basyc is strong in protecting patient safety . scenario 2 presents a similar pattern of dose - toxicity relationship to scenario 1 , except that the toxicity probability of dose level 2 is now below pt = 30% . 
this suggests that the basyc design is nimble and can reach high dose - cycle combinations even with small sample sizes . scenario 4 presents an opposite case to scenario 3 , because all the doses are overly toxic with toxicity probabilities above pt . 
this scenario demonstrates the ability of the basyc design to stop the trial quickly in case of excessive toxicity in all dose sequences . scenarios 5 and 6 reflect situations where there is a leap in toxicity probabilities across the three doses . 
 acquired met exon 14 alteration drives secondary resistance to epidermal growth factor receptor tyrosine kinase inhibitor in egfr - mutated lung cancer ken suzawa , md , phd1 ; michael ofn , md1 ; adam j . 
in this study , we used targeted ngs with memorial sloan kettering integrated mutation proling of actionable cancer targets ( msk - impact ) , 12 immunohistochemistry , cell - free dna testing , and uorescence in situ hybridization to evaluate acquired resistance mediated by metex14 . 
furthermore , we used in vitro functional studies to establish metex14 as a novel mechanism of acquired resistance to egfr tkis . we used msk - impact , a large - panel ngs assay , to detect mutations , copy - number alterations , and select gene fusions involving up to 468 cancer - associated genes.12 metex14 was introduced into pc9 and h1975 cells as follows . 
briey , full - length metex14 was polymerase chain reaction amplied and subcloned into plenti - cmv - blast lentiviral vector ( plasmid 17451 ; addgene , cambridge , ma )  . 
the patient restarted erlotinib ( 100 mg daily ) with clinical and radiologic response for 12.5 months , at which time computed tomography revealed an increase in the dominant right upper lobe mass . 
 ( b - d ) representative images showing ( b ) baseline scan ( at time of progression during osimertinib monotherapy ) , ( c ) response to crizotinib monotherapy , and ( d ) response to combined crizotinib and osimertinib therapy . 
 ( * ) the patient initially received 1.4 months of combination osimertinib and savolitinib in a clinical trial , but treatment was changed to monotherapy with osimertinib because of intolerable toxicity . 
 ( ) as of 10 months of ongoing treatment with osimertinib and crizotinib . l858r mutation , no evidence of egfr t790m , and a new metex14 ( c.2899g.a ) alteration and met amplication ( fold change , 2.5 ; fig 1a ; appendix table a1 )  . 
therapy was changed to osimertinib with savolitinib daily ( clinicaltrials . gov identier : nct02143466 ) for 1.4 months , after which savolitinib was stopped because of toxicity and single - agent osimertinib 80 mg daily was continued . 
the patient continued to receive combination therapy with durable clinical and radiographic benet for more than 9 months . to dene the role of metex14 in mediating resistance to egfr tkis , we generated two isogenic egfr - mutant nonsmall - cell lung cancer ( nsclc ) cell models using pc9 ( exon 19 deletion ) and h1975 cells ( l858r and t790m ) by transduction with lentiviral vectors driving expression of metex14 ( fig 2a )  . 
western blot analysis showed that phosphorylation of egfr and its downstream effectors akt and erk was inhibited by osimertinib in pc9 cells transduced with empty plasmids ( pc9 empty ) , but phosphorylation of egfr , metex14 , and downstream effectors remained unaffected by osimertinib treatment in pc9 metex14 cells ( fig 2b )  . 
each condition was assayed in triplicate determinations ; data were normalized for cell number by measuring cell viability and shown relative to the control group ( mean 6 standard deviation )  . 
combination treatment with epidermal growth factor receptor ( egfr ) and met inhibitors is effective against met exon 14 skipping alteration ( metex14 ) induced drug resistance in egfr - mutant nonsmall - cell lung cancer cells . 
 ( c ) cells were treated with osimertinib ( 1 mm ) , crizotinib ( 1 mm ) , or a combination of the two agents for 3 hours , and lysates were subjected to immunoblotting . 
 ( e ) caspase 3 / 7 activity was analyzed in pc9 metex14 cells that were treated with osimertinib ( 1 mm ) , crizotinib ( 200 nm ) , or a combination of osimertinib ( 1 mm ) and crizotinib ( 200 nm ) for 48 hours . 
each condition was assayed in triplicate determinations , and data were normalized for cell number by measuring cell viability and are shown relative to the control group ( mean 6 standard deviation )  . 
pc9 metex14 cells showed a reduction in osimertinib - induced caspase 3 / 7 activation compared with pc9 - empty cells ( p , .001 ; fig 2d )  . 
together , these results indicate that metex14 induces resistance to osimertinib in egfr - mutant nsclc cells . investigated whether metex14 - mediated rewe next sistance to egfr tkis could be overcome by combination therapy with egfr and met inhibitors . 
as expected , crizotinib inhibited metex14 phosphorylation in pc9 metex14 cells ; however , phosphorylation of egfr , akt , and erk remained largely unchanged ( fig 3a ) , suggesting that egfr is still signaling effectively in pc9 metex14 cells . similarly , crizotinib was ineffective at modulating growth of egfr - mutated cell lines , with or without metex14 expression ( fig 3b )  . 
in agreement with these results , inhibition of egfr and met caused signicantly dual higher activation of caspase 3 / 7 ( fig 3e )  . discussion our study highlights the importance of serial and diverse molecular analyses , including ngs , to evaluate acquired alterations in the post - tki setting . 
although the patient did not respond to met - targeted therapy alone , the patient continued to have a durable clinical response to combination osimertinib and crizotinib , with stable disease by recist criteria and without notable toxicity . 
crizotinib restored sensitivity to egfr tkis ; however , crizotinib alone was not enough to suppress growth . two previous reports have demonstrated co - occurrence of egfr and metex14 mutations . 
in the rst report , three ( 0.2% ) of 1 , 590 patients with nsclc harbored concomitant egfr and metex14 mutations , one of whom received combination treatment with met ( volitinib ) and egfrtargeted therapies ( getinib ) , yielding a partial response.16 the second report noted one patient case of sarcomatoid carcinoma with egfr and metex14 alterations.17 in our msk - impact testing experience of 866 patient cases of egfr - mutant lung adenocarcinomas ( as of october 15 , 2018 ) , only two patients showed this combination of alterations : the patient described here , with acquired resistance , and a patient separate primaries at diagnosis . in whom the alterations were present the clinical benet of the combination of metand egfrtargeted therapies in patients with nsclc with acquired met amplicationmediated resistance to egfr tkis has trials , with varying tolerability been explored in clinical dependent upon the agents being combined.18 our ndings provide a rationale for future clinical evaluation of this combination approach , given its tolerability and efcacy in this case , for patients with egfr and metex14 mutations . furthermore , given the recent reports of secondary - site mutations in the met kinase domain , such as d1228n / v and y1230c , as mechanisms of acquired resistance to crizotinib in patients with metex14 , 19 it is plausible that these secondary met mutations will also emerge as mechanisms of resistance to the combination of osimertinib and crizotinib . we found that expression of metex14 upregulated phosphorylation of egfr itself , presumably via crossphosphorylation , because met is known to interact with egfr and drive the activity of egfr , 20 which resulted in blunting of the inhibition of egfr phosphorylation by egfr tkis . 
yu , romel somwar , marc ladanyi data analysis and interpretation : ken suzawa , michael ofn , adam j . schoenfeld , igor odintsov , daniel lu , alexander drilon , helena a . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter michael ofn consulting or advisory role : pharmamar travel , accommodations , expenses : bristol - myers squibb , merck sharp & dohme maria e . 
rudin consulting or advisory role : bristol - myers squibb , abbvie , seattle genetics , harpoon therapeutics , genentech / roche , astrazeneca , ascentage pharma , bicycle therapeutics , celgene , daiichi sankyo , ipsen , loxo , pharmamar , elucida oncology , bridge medicines research funding : abbvie / stemcentrx ( inst ) , viralytics ( inst ) , merck ( inst ) , daiichi sankyo ( inst ) alexander drilon honoraria : medscape , onclive , peervoice , physicians education resources , targeted oncology , more health , research to practice , foundation medicine , peerview consulting or advisory role : ignyta , loxo , tp therapeutics , astrazeneca , pzer , blueprint medicines , genentech / roche , helsinn therapeutics , beigene , hengrui therapeutics , exelixis , bayer healthcare pharmaceuticals , tyra biosciences , verastem , takeda / ariad / millennium pharmaceuticals , bergenbio , more health patents , royalties , other intellectual property : wolters kluwer ( royalties for pocket oncology ) other relationship : merck , glaxosmithkline , teva pharmaceuticals industries , taiho pharmaceutical helena a . 
yu consulting or advisory role : astrazeneca , eli lilly research funding : clovis oncology ( inst ) , astrazeneca ( inst ) , astellas pharma ( inst ) , eli lilly ( inst ) , novartis ( inst ) , pzer ( inst ) , daiichi sankyo ( inst ) travel , accommodations , expenses : eli lilly other relationship : astellas pharma gregory j . 
 acquired met exon 14 alteration and resistance to egfr tkis references yu ha , suzawa k , jordan e , et al : concurrent alterations in egfr - mutant lung cancers associated with resistance to egfr kinase inhibitors and characterization of mtor as a mediator of resistance . 
clin cancer res 24 : 3108 - 3118 , 2018 ofn m , somwar r , rekhtman n , et al : acquired alk and ret gene fusions as mechanisms of resistance to osimertinib in egfr - mutant lung cancers . 
jama oncol 4 : 1527 - 1534 , 2018 bean j , brennan c , shih jy , et al : met amplication occurs with or without t790m mutations in egfr mutant lung tumors with acquired resistance to getinib or erlotinib . 
proc natl acad sci usa 104 : 20932 - 20937 , 2007 onozato r , kosaka t , kuwano h , et al : activation of met by gene amplication or by splice mutations deleting the juxtamembrane domain in primary resected lung cancers . 
j thorac oncol 4 : 5 - 11 , 2009 kong - beltran m , seshagiri s , zha j , et al : somatic mutations lead to an oncogenic deletion of met in lung cancer . 
cancer res 66 : 283 - 289 , 2006 paik pk , drilon a , fan pd , et al : response to met inhibitors in patients with stage iv lung adenocarcinomas harboring met mutations causing exon 14 skipping . 
cancer discov 5 : 842 - 849 , 2015 drilon ae , camidge dr , ou s - hi , et al : efcacy and safety of crizotinib in patients ( pts ) with advanced met exon 14 - altered non - small cell lung cancer ( nsclc )  . 34 , 2016 ( suppl ; abstr 108 ) drilon a , ou s - h , clark j , et al : antitumor activity and safety of crizotinib in patients with met exon 14 - altered advanced non - small cell lung cancer . 
felip e , horn l , patel jd , et al : tepotinib in patients with advanced non - small cell lung cancer ( nsclc ) harboring met exon 14 - skipping mutations : phase ii trial . 
wolf j , han j , nishio m , et al : geometry mono - 1 : phase ii , multicenter study of met inhibitor capmatinib ( inc280 ) in egfr wt , met - dysregulated advanced nsclc . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
pennell na , neal jw , chaft je , et al : select : a phase ii trial of adjuvant erlotinib in patients with resected epidermal growth factor receptormutant nonsmallcharacteristics . 
borsu l , intrieri j , thampi l , et al : clinical application of picodroplet digital pcr technology for rapid detection of egfr t790m in next - generation sequencing libraries and dna from limited tumor samples . 
ou si , govindan r , eaton kd , et al : phase i results from a study of crizotinib in combination with erlotinib in patients with advanced nonsquamous non - small cell lung cancer . 
in cohort b , the orr and disease control rate were 75% and 75% . conclusion capmatinib in combination with erlotinib demonstrated safety proles consistent with prior studies . we observed efcacy in specic patient populations . 
early studies evaluated onartuzumab ( an anti - met monoclonal antibody ) in combination the phase iii with erlotinib in recurrent nsclc.16 , 17 in the phase ii study , patients with met 2 + or 3 + by immunohistochemistry ( ihc ) demonstrated a signicant improvement in survival end points compared with low met expression . 
despite this , trial was negative.18 tivantinib , a non - atp competitive small molecule met inhibitor , was evaluated in combination with erlotinib in a phase ii trial of unselected pretreated patients . 
the combination did not meet its overall efcacy goal ; however , a planned subset analysis showed a trend toward prolonged progression - free survival ( pfs ) in patients with met amplication.19 this lack of efcacy may be due to the lack of specicity of tivantinib for the met pathway compared with other met inhibitors.20 capmatinib ( inc280 ) is a highly potent and selective oral met inhibitor that has recently been approved for the treatment of tumors with met exon 14 mutations.21 in the phase i study , antitumor activity was observed in pretreated patients with egfr wild - type ( wt ) tumors with met dysregulation . 
because of overlapping signaling pathways , we sought to determine whether the combination of capmatinib with erlotinib would be safe and demonstrate an efcacy signal in patients with egfror metaltered tumors . knowledge generated we demonstrated that the combination of capmatinib and erlotinib is safe . 
however , it restored sensitivity to erlotinib and promoted apoptosis in nsclc models rendered erlotinib resistant by hgf.23 on the basis of the data available during trial development , we propose that the combination of capmatinib plus erlotinib would be safe and show efcacy in patients with egfr - mutated tumors experiencing disease progression on erlotinib due to met activating bypass pathways . 
the initial phase of the study was a dose escalation to determine the maximum tolerated dose ( mtd ) of capmatinib plus erlotinib ; there was no intrapatient dose escalation . 
patients in cohort a must have an egfr activating mutation and a biopsy at the time of progression that shows evidence of met positivity . treatment capmatinib capsules were administered orally every 12 hours of a 28 - day cycle at 100 - 600 mg . 
a dose - limiting toxicity ( dlt ) was dened as per the study protocol ( data supplement )  . pk blood samples were collected for patients in the doseescalation cohort on cycle 1 , day 15 ; cycle 2 , days 1 and 15 ; and cycles 3 and 4 , day 1 . 
analysis of hgf levels was conducted in duplicate by enzyme - linked immunosorbent assay ( r&d systems , minneapolis , mn ; cat dhg00 ) as per the manufacturers instructions . statistical methods all patients who had at least one dose of study therapy were included in the analyses . 
twelve and ve patients were enrolled in cohorts a and b , respectively ( fig 1 )  . patient demographics and disease characteristics patient demographics and disease characteristics are listed in table 1 . 
in the doseescalation cohort and cohort a , 67% ( patients 1 and 313 ) and 92% ( all except patient 29 ) had prior treatment with at least one egfr tki . 
swimmers plot demonstrating the cohort , met , and epidermal growth factor receptor ( egfr ) status of each patient and corresponding response status and progression - free survival of each patient . 
patient 30 was found to have an egfr t790m mutation on a biopsy sample taken after egfr tyrosine kinase inhibitor ( tki ) treatment ; however , on immediate pretreatment biopsy this mutation was not detected . 
a waterfall plot of best response for all evaluable patients is shown in figure 3 . the aes that were possibly , probably , or denitely attributed to the study drugs are shown in table 2 for all dose levels . 
the most common aes of any grade were acneiform rash ( 62.9% ) , fatigue ( 51% ) , nausea ( 45.7% ) , diarrhea , lower extremity edema , vomiting , and hypoalbuminemia ( 37% each )  . 
in patients who received the recommended phase ii dose ( rp2d ) , the most common aes were acneiform rash ( 65% ) , fatigue and nausea ( both 60% ) , vomiting ( 55% ) , and hypoalbuminemia and edema ( both 50% ) ; additional aes are detailed in appendix table a1 . there were no grade 5 toxicities related to the drug combination . dlt and mtd in dose level 5 , one patient developed grade 3 neutropenia possibly related to treatment . 
dose level 6 served as the expansion of dose level 5 , given that 600 - mg capsules were previously shown to be pharmacokinetically equivalent to 400 - mg tablets . 
six patients received dose modications of capmatinib due to nausea ( 2 ) , alt abnormalities ( 1 ) , edema ( 1 ) , low neutrophil count ( 1 ) , and elevated amylase ( 1 )  . 
five patients received dose modications of erlotinib due to paronychia ( 2 ) , acneiform rash ( 1 ) , creatinine increase ( 1 ) , and diarrhea ( 1 )  . 
four patients received dose modications of both drugs for lipase elevation ( 1 ) , creatinine increase ( 1 ) , lymphopenia ( 1 ) , and lung inammation / pneumonitis ( 1 )  . the pk properties of erlotinib and capmatinib were examined during dose escalation on cycle 1 , day 15 at multiple time points after drug administration . 
the same 400mg dose of capmatinib in the tablet formulation demonstrates a higher drug exposure with greater variation ( appendix table a2 ) compared with the capsule formulation , 182 2021 by american society of clinical oncology although it was not statistically signicant . in addition , erlotinib showed a dose - dependent change of systemic drug exposure ( 150 mg v 100 mg ; appendix fig a1b and appendix table a2 ) ; coadministration with capmatinib did not have signicant impact on erlotinib pk ( appendix table a2 )  . 
overall , the range of erlotinib exposures during dose escalation was similar to previously published results.28 efcacy across all evaluable patients , the orr was 31% ( 8 / 26 ; fig 3 )  . 
among patients with pr or cr , the responses were prolonged , from patient 24 , who discontinued study after apart 3 months because of pneumonia / pneumonitis ; vestigators could not rule out possible drug - related pneumonitis , and thus the patient was discontinued from study . two responders were treatment nave ( 20 and 24 ) ; the remainder had at least one prior regimen ( table 1 )  . dose - escalation cohort fifteen of the 18 patients in the dose - escalation cohort were evaluable . 
two patients ( patients 21 and 23 ) withdrew from the study by individual choice , one patient ( patient 33 ) developed new brain metastasis and withdrew , and one patient ( patient 30 ) had a prolonged hospitalization for a cerebrovascular event deemed unrelated to study drugs . 
efcacy data across dose - escalation cohort dose level median cycles ( range ) 2 ( 2 - 4 ) 5 ( 4 - 16 ) 2 ( 1 - 7 ) 2 ( 2 - 8 ) 4 ( 1 - 10 ) 4 ( 4 - 29 ) inc280 , mg twice a day erlotinib , mg once daily evaluable patients complete response partial response stable disease progressive disease overall response rate abbreviation : inc280 , capmatinib . 1 ( 33% ) 3 ( 100% ) disease control rate 2 ( 67% ) 2 ( 67% ) 1 ( 33% ) 2 ( 67% ) 1 ( 33% ) five egfr - wt patients were enrolled in cohort b ; one patient ( patient 25 ) was not evaluable because of death during cycle 1 deemed unrelated to the study drugs . 
efcacy data across dose - expansion cohorts dosing and response groups cohort a median cycles ( range ) 2 ( 1 - 33 ) 3 ( 1 - 45 ) inc280 , mg twice daily erlotinib , mg once daily evaluable patients complete response partial response stable disease progressive disease overall response rate , % disease control rate , % cohort b abbreviations : inc280 , capmatinib ; ne , not evaluable . at the recommended phase ii dosing , responses were seen in patients with met amplication by ngs and / or 3 + ihc expression and those with met exon 14 mutations , but not in patients with 2 + ihc expression . 
a study in chinese patients with getinib plus capmatinib also reported that 2 + ihc expression was not predictive of response unless it was accompanied by a gene copy number of 5.29 the met / cen7 ratios were not fully reported in this study , but a ratio  . 
serial blood hgf levels were measured to evaluate baseline levels and treatment - related changes in patients over time ; however , no overt correlations with baseline hgf levels and treatment outcomes were observed ( data not shown )  . the patient population with the greatest potential to benet from the combination treatment was cohort a , which enrolled egfr - positive patients who had experienced progression on egfr tkis . 
additional evaluation of an egfr - tki plus a met - tki to overcome this common resistance mechanism is warranted . affiliations 1helen diller family comprehensive cancer center , university of california , san francisco , ca 2university of california davis comprehensive cancer center , sacramento , ca 3mount sinai tisch cancer institute , new york , ny subject matter of this manuscript . 
mccoach honoraria : novartis , genentech , guardant health consulting or advisory role : astrazeneca speakers bureau : novartis research funding : novartis , revolution medicine travel , accommodations , expenses : loxo , eli lilly , takeda pharmaceuticals aiming yu stock and other ownership interests : johnson & johnson patents , royalties , other intellectual property : patents : ( 1 ) yu am , wang wp , chen qx , li mm . 
gandara honoraria : astrazeneca consulting or advisory role : astrazeneca ( i ) , guardant health ( i ) , oncocyte ( i ) , amgen , io biotech ( i ) , merck , roche / genentech , boehringer ingelheim , inivata , novartis research funding : roche / genentech ( i ) , merck ( i ) , amgen ( i ) travel , accommodations , expenses : boehringer ingelheim jonathan w . 
lara consulting or advisory role : janssen research funding : aragon pharmaceuticals ( i ) , janssen biotech ( i ) , tracon pharma ( i ) , merck ( i ) , pharmacyclics ( i ) , incyte ( i ) , taiho pharmaceutical ( i ) matthew gubens consulting or advisory role : bristol myers squibb , roche / genentech , astrazeneca , heron , boehringer ingelheim , takeda pharmaceuticals , beyondspring pharmaceuticals , inivata research funding : celgene ( i ) , merck ( i ) , novartis ( i ) , roche / genentech ( i ) , oncomed ( i ) philip c . 
mack honoraria : guardant health consulting or advisory role : astrazeneca , guardant health , amgen research funding : boehringer ingelheim consulting or advisory role : roche / genentech , regeneron , abbvie , emd serono , merck , pzer , astrazeneca , novartis , symphogen , inivata , bristol myers squibb , takeda pharmaceuticals , eli lilly , amgen , genmab , targeted oncology , genentech research funding : emd serono ( i ) , genentech ( i ) , abbvie ( i ) , five prime therapeutics ( i ) , regeneron ( i ) , astellas pharma ( i ) , tizona therapeutics , ( i ) eli lilly ( i ) , novartis ( i ) , amgen ( i ) patents , royalties , other intellectual property : author royalties for uptodate , an evidence - based , peer - reviewed information resource , available via the web , desktop , and pda travel , accommodations , expenses : astrazeneca , roche / genentech , merck , regeneron , eli lilly , abbvie , emd serono no potential conicts of interest were reported . acknowledgment we thank patients and their families for participation in this study . karen kelly honoraria : merck references finocchiaro g , toschi l , gianoncelli l , et al : prognostic and predictive value of met deregulation in non - small cell lung cancer . 
ann transl med 3 : 83 , 2015 go h , jeon yk , park hj , et al : high met gene copy number leads to shorter survival in patients with non - small cell lung cancer . 
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science 316 : 1039 - 1043 , 2007 bean j , brennan c , shih jy , et al : met amplication occurs with or without t790m mutations in egfr mutant lung tumors with acquired resistance to getinib or erlotinib . 
proc natl acad sci usa 104 : 20932 - 20937 , 2007 cappuzzo f , j anne pa , skokan m , et al : met increased gene copy number and primary resistance to getinib therapy in non - small - cell lung cancer patients . ann oncol 20 : 298 - 304 , 2009 piotrowska z , thress ks , mooradian m , et al : met amplication ( amp ) as a resistance mechanism to osimertinib . 
le x , puri s , negrao mv , et al : landscape of egfr - dependent and - independent resistance mechanisms to osimertinib and continuation therapy beyond progression in egfr - mutant nsclc . 
drilon a , cappuzzo f , ou si , et al : targeting met in lung cancer : will expectations nally be met ? j thorac oncol 12 : 15 - 26 , 2017 15 . 
spigel dr , ervin tj , ramlau ra , et al : randomized phase ii trial of onartuzumab in combination with erlotinib in patients with advanced non - small - cell lung 18 . 
spigel dr , edelman mj , obyrne k , et al : results from the phase iii randomized trial of onartuzumab plus erlotinib versus erlotinib in previously treated stage iiib or iv non - small - cell lung cancer : metlung . 
sequist lv , von pawel j , garmey eg , et al : randomized phase ii study of erlotinib plus tivantinib versus erlotinib plus placebo in previously treated non - smallcell lung cancer . 
calles a , kwiatkowski n , cammarata bk , et al : tivantinib ( arq 197 ) efcacy is independent of met inhibition in non - small - cell lung cancer cell lines . 
liu x , wang q , yang g , et al : a novel kinase inhibitor , incb28060 , blocks c - met - dependent signaling , neoplastic activities , and cross - talk with egfr and her22 . 
bang y - j , su w - c , nam d - h , et al : phase i study of the safety and efcacy of inc280 in patients with advanced met - dependent solid tumors . 
lara ms , holland ws , chinn d , et al : preclinical evaluation of met inhibitor inc - 280 with or without the epidermal growth factor receptor inhibitor erlotinib in non - small - cell lung cancer . 
paik pk , drilon a , fan pd , et al : correction : response to met inhibitors in patients with stage iv lung adenocarcinomas harboring met mutations causing exon 14 skipping . 
paik pk , drilon a , fan pd , et al : response to met inhibitors in patients with stage iv lung adenocarcinomas harboring met mutations causing exon 14 skipping . 
cancer discov 5 : 842 - 849 , 2015 jorge se , schulman s , freed ja , et al : responses to the multitargeted met / alk / ros1 inhibitor crizotinib and co - occurring mutations in lung adenocarcinomas with met amplication or met exon 14 skipping mutation . 
fukudo m , ikemi y , togashi y , et al : population pharmacokinetics / pharmacodynamics of erlotinib and pharmacogenomic analysis of plasma and cerebrospinal uid drug concentrations in japanese patients with non - small cell lung cancer . 
wu yl , zhang l , kim dw , et al : phase ib / ii study of capmatinib ( inc280 ) plus getinib after failure of epidermal growth factor receptor ( egfr ) inhibitor therapy in patients with egfr - mutated , met factor - dysregulated non - small - cell lung cancer . 
wolf j , overbeck tr , han j , et al : capmatinib in patients with high - level met - amplied advanced nonsmall cell lung cancer ( nsclc ) : results from the phase 2 geometry mono - 1 study . 
camidge dr , otterson ga , clark jw , et al : crizotinib in patients ( pts ) with met - amplied non - small cell lung cancer ( nsclc ) : updated safety and efcacy ndings from a phase 1 trial . 
caparica r , yen ct , coudry r , et al : responses to crizotinib can occur in high - level met - amplied non - small cell lung cancer independent of met exon 14 33 . 
wolf j , seto t , han j , et al : results of the geometry mono - 1 phase ii study for evaluation of the met inhibitor capmatinib ( inc280 ) in patients with metex14 mutated advanced non - small cell lung cancer . 
yu h , ahn m , kim s , et al : ct032 tatton phase ib expansion cohort : osimertinib plus savolitinib for patients ( pts ) with egfr - mutant , met - amplied nsclc after progression on prior rst / second - generation epidermal growth factor receptor ( egfr ) tyrosine kinase inhibitor ( tki )  . 
sequist l , lee js , han j , et al : ct033 tatton phase ib expansion cohort : osimertinib plus savolitinib for patients ( pts ) with egfr - mutant , met - amplied nsclc after progression on prior third - generation epidermal growth factor receptor ( egfr ) tyrosine kinase inhibitor ( tki )  . 
treatment - related adverse events by cohort and dose level ( continued ) dose level 1 dose level 2 dose level 3 dose level 4 dose level 5 dose level 6 expansion adverse event gr 1 / 2 gr 3 gr 1 / 2 gr 3 gr 1 / 2 gr 3 gr 1 / 2 gr 3 gr 1 / 2 gr 3 gr 1 / 2 gr 3 gr 1 / 2 gr 3 total gastroesophageal reux disease oral mucositis hyponatremia stroke proteinuria back pain peripheral sensory neuropathy abbreviations : gr , grade ; inr , international normalized ratio ; sae , signicant adverse event . 
 response of a metastatic breast carcinoma with a previously uncharacterized erbb2 g776v mutation to human epidermal growth factor receptor 2targeted therapy introduction we present a case of a patient with advanced estrogen receptor / progesterone receptorpositive , her2 - negative ( by fluorescent in situ hybridization ) breast cancer , refractory to estrogen receptortargeted therapy . 
in vitro experiments showed that erbb2 g776v is an oncogenic driver , and further largescale analysis of genomic profiles of breast carcinomas suggested the existence of a distinct subtype of her2 - negative breast cancer driven by a combination of activating erbb2 kinase domain mutations and inactivating map3k1 alterations . 
these observations suggest that patients with breast cancer who have relapsed / refractory disease can benefit from cancer genomic profiling to identify unanticipated opportunities for targeted therapy , including the use of combinatorial therapies with anti - her2 agents for those cases with erbb2 mutations . management of estrogen receptor ( er ) / progesterone receptor ( pr ) - positive breast cancer refractory to endocrine therapy presents a clinical challenge , although recent studies demonstrating the acquisition of esr1 mutations1 - 5 or alterations in the pi3k / mtor pathway6 - 8 have identified mechanisms of such resistance . 
in addition , unlike the breast cancer described in this case report , where the tumor was her2 negative , a small subset of er - positive breast cancers are also her2 positive and have been dubbed triple - positive cases ; in the absence of her2 - targeted therapy , these have a poor prognosis and worse response to endocrine therapy relative to er - positive breast cancers in general , 9 - 11 but they have been reported to respond to anti - her2 agents.11 , 12 recently , several lines of investigation have demonstrated that mutations in the her2 gene ( erbb2 ) are oncogenic drivers in breast carcinoma , as evidenced by patient responses to her2 - targeted therapy . 
erbb2 mutations such as s310f , l755s , v777l , and l869q have been shown to be strong drivers associated with clinical sensitivity to her2 - targeted agents.13 - 16 in addition , erbb2 mutations have been reported to be prevalent in invasive lobular breast carcinoma , which is typically er / pr positive , her2 negative , and cdh1 mutant.17 - 19 we report on a patient with advanced breast cancer harboring a previously uncharacterized erbb2 g776v variant , who derived clinical benefit from treatment with trastuzumab plus pertuzumab ( tp ) regimens . 
furthermore , breast carcinomas with erbb2 kinase domain ( kd ) mutations are marked by cosegregation with map3k1 alterations , and the majority of breast cancer cases with the g776v mutation are er positive , which are not general features of her2 - positive or erbb2 - mutant breast cancer and , thus , may denote a distinct class of breast carcinoma driven by erbb2 kd alterations . methods comprehensive genomic profiling dna was extracted from 40 microns of formalinfixed paraffin - embedded sections , and comprehensive yakov chudnovsky runjun d . 
 genomic profiling was performed in a clinical laboratory improvement amendments certified , college of american pathologistsaccredited reference laboratory ( foundation medicine ) on hybridization - captured , adaptor ligationbased libraries to a mean coverage depth of  . 
the construct was then shuttled into the pcfg5 retroviral vector ( which includes a zeocyn resistance marker and ires - gfp sequence ) using the in - fusion hd cloning system kit ( clonetech laboratories , mountain view , ca ) , and verified using sanger sequencing . retroviral particles were produced using fnx amphotrophic packaging cells . 
cells were selected under 10 mg / ml zeocin for 3 weeks . agar colony - formation assays , wells were seeded with 7 , 000 cells and grown for 2 weeks before imaging . 
codons 23652 were defined as the extracellular domain ( ecd ) , and codons 720987 were defined as the kd.22 the p value was calculated using logistic regression , and p values were corrected for multiple testing using the falsediscovery rate23 method to generate q values . the odds ratio ( or ) for each variant type is calculated from the logistic regression coefficient ; an or  . 
1.0 indicates more frequent co - occurrence than expected by chance , whereas an pr , 1.0 indicates less frequent co - occurrence than expected by chance . results case description for western blots , cells were serum starved for 6 hours before lysate harvesting . 
lysates were probed using the following antibodies : her2 ( monoclonal , ab - 17 ) from thermo fisher scientific ( waltham , ma ) , phospho - her2 ( py1248 ) from emd millipore ( billerica , ma ) , p44 / 42 mapk and phospho p44 / 42 mapk from cell signaling technologies ( danvers , ma )  . 
in july 2003 , the patient presented with a t2 n0 m0 , er - positive , pr - positive , her2negative ( nonamplified by fluorescent in situ hybridization , 2 + by immunohistochemistry ) invasive ductal carcinoma of the left breast . 
in december 2005 , she developed metastasis to the sternum and lung , and was initially treated with ovarian suppression and zoledronate . she received radiation therapy to the sternal lesions in december 2008 and again in may 2012 . 
additional antiestrogen therapies used included letrozole ( january 2009 to january 2011 and again from february 2012 to may 2013 ) and fulvestrant ( february 2011 to january 2012 )  . in early 2013 , positron emission tomographycomputed tomography ( pet - ct ) scans showed increasing activity in the lung lesions , and the patient underwent a video - assisted thoracoscopic surgery procedure in may 2013 . 
the ca15 - 3 tumor marker level decreased with capecitabine ( down from 391 to 85 u / ml , but then rising to 213 u / ml ) and also with estradiol ( decreasing further to 74 u / ml , but then rising to 132 u / ml ; fig 1 )  . 
trastuzumab , pertuzumab , and exemestane were started in october to november 2014 , and the patient responded well to this , with marked improvement on pet - ct scan and decrease in ca15 - 3 level to 43 u / ml . 
however , by february 2015 , the ca15 - 3 level rose to 58 u / ml and the patients treatment was changed to nabpaclitaxel , trastuzumab , and pertuzumab . 
the ca15 - 3 level decreased to a nadir of 31 u / ml ( normal range for ca15 - 3 is 0 to 31 u / ml ) , the pet - ct scan showed no areas of increased uptake , and the patient remained on this regimen for a total of seven cycles . 
in december 2015 , pet - ct showed new uptake in her lung nodules , and the patient enrolled in an unrelated clinical trial . preclinical characterization of her2 ( erbb2 ) g776v the her2 g776v mutant was stably overexpressed in nih 3t3 cells by retroviral transduction . capecitabine estradiol trastuzumab pertuzumab exemestane nabpaclitaxel trastuzumab pertuzumab maintanence trastuzumab date ascopubs.org / journal / po jco precision oncology 3 fig 1 . 
immunoblotting showed that these cell lines expressed elevated levels of active phosphorylated mapk ( similar to cells expressing her2 v777l ) , but nonelevated levels of phosphorylated her2 , which may be an artifact of the overexpression system ( fig 2a )  . 
we also assayed these cell lines for colony formation in soft agar . we found that cells expressing her2 g776v formed colonies at a rate similar to that of the her2 v777l mutant ( figs 2b and 2c )  . 
together , these results suggest that g776v is an activating mutation , causing increased signaling through the her2 / mapk pathway and allowing for anchorage - independent growth of nih 3t3 cells . genomic co - occurrence of map3k1 alterations with erbb2 alterations among the 10 , 790 breast carcinoma cases that have undergone cgp in our laboratory , 1 , 329 ( 12.3% ) have been found to harbor alterations in erbb2 , including 1 , 041 cases ( 9.6% ) with erbb2 amplification , 229 cases ( 2.1% ) with erbb2 short variants ( substitutions or inframe insertions or deletions ) , and 59 ( 0.5% ) with both ( table 1 )  . 
to identify additional genomic variants that may cooperate with erbb2 mutations in the pathogenesis of her2 - negative breast cancer , we calculated the co - occurrence of alterations in each of 345 genes profiled at foundation medicine with various alterations in erbb2 ( data supplement )  . 
as discussed , cdh1 alterations have been reported to co - occur with erbb2 mutations in the context of invasive lobular breast carcinoma.17 - 19 tbx3 has also been reported to be enriched for mutations in lobular breast carcinoma , 25 as well in neuroendocrine breast cancer , 26 but its role in breast cancer is not entirely clear : it has been suggested to be a tumor suppressor27 , 28 and an oncogene29 , 30 in different studies . 
we chose to further examine the tumor suppressor gene map3k1 , which was mutated in the patient described in this case report . alterations in map3k1 co - occur with erbb2 short variants significantly more frequently than expected by chance in breast cancer cases profiled in our laboratory , but erbb2 alterations , in general , are not significantly enriched for cooccurrence with map3k1 variants ( table 1 )  . furthermore , although activating mutations in erbb2 are found in both the ecd and the kd , 21 only kd mutations , and not ecd mutations , are significantly enriched for co - occurrence with map3k1 alterations ( table 1 )  . 
map3k1 alterations have been identified in 513 ( 4.8% ) of the 10 , 790 breast carcinoma cases profiled at foundation medicine and at a similar frequency range of 0% to 8% of breast cancers in several large - scale sequencing studies.27 , 31 - 33 map3k1 inactivation via mutation or copy number loss has been reported to be particularly prevalent in the luminal a subtype of breast cancer , which is distinct from the subtype that is characterized by erbb2 amplification.33 , 34 consistent with this , among the breast carcinoma cases profiled using our hybrid capture based assay , erbb2 copy number alterations co - occur with map3k1 variants significantly less frequently than expected by chance ( table 1 )  . 
notably , although subtype data were not available for the breast cancer cases profiled in our laboratory , logistic regression g776va v777l her2 wt parental ascopubs.org / journal / po jco precision oncology fig 2 . 
 ( a ) western blot showing expression of active phosphorylated and total her2 and mapk in untransduced ( parental ) nih 3t3 cells and those transduced with wt human her2 or the indicated mutants . 
g776va and g776vb are duplicate cell lines that were transduced independently . ( b ) representative images and ( c ) quantification of soft - agar colony formation ( anchorage - independent growth ) by untransduced ( parental ) nih 3t3 cells and those transduced with wt human her2 or the indicated mutants . 
 analysis to control for alteration status of the genes reported to be most frequently mutated in luminal a breast cancer ( ie , tp53 , pik3ca , gata3 , and cdh1 ) 33 retained significant enrichment of cooccurrence of map3k1 alterations with all erbb2 short variants and with erbb2 kd short variants ( table 2 )  . 
this indicates that the co - occurrence of erbb2 and map3k1 variants in a subset of breast carcinoma cases is independent of the status of luminal aassociated genes and cannot be attributed solely to these cases belonging to the luminal a subtype . 
these findings suggest that there may be a particular subtype of her2 - negative , predominantly er - positive breast cancer driven by a combination of activating erbb2 kinase domain mutations and inactivating map3k1 alterations . discussion this is the first demonstration of clinical response to tp regimens ( tp plus exemestane followed by tp plus nab - paclitaxel ) in a patient with a her2 kinase domain mutation ( erbb2 g776v ) and expands our knowledge of treatment options for patients with metastatic breast cancer containing erbb2 mutations . 
although it cannot be excluded that the clinical benefit was due to the exemestane or nab - paclitaxel , these findings are consistent with four previous studies that have reported responses of patients with erbb2 - mutant ( s310f , l755s , v777l , l869q ) breast cancer to antiher2 agents including antibodies and tyrosine kinase inhibitors , with a cytotoxic chemotherapy backbone in three of the four cases.13 - 16 in addition , studies in cultured cells and animal xenograft models have shown that multiple erbb2 mutations associated with erbb2 amplification - negative breast cancer are activating , oncogenic , and sensitive to the irreversible pan - her inhibitor neratinib and , in some cases , to the reversible pan - her inhibitor lapatinib and the anti - her2 antibody trastuzumab.21 the effects of the g776v mutation on her2 functions had not previously been characterized in published studies , although one study reported that this alteration is untargetable.35 however , including the substitution similar alterations , g776s36 and the compound substitution and insertion g776vinsc , 37 - 39 have been shown to be activating . 
 her2 - targeted agents have shown high rates of clinical benefit in her2 - positive breast cancers and are approved for both er - negative and erpositive patients.11 , 12 the clinical data for her2negative breast carcinoma cases driven by erbb2 mutations are considerably more limited . 
a recent large - scale study of nonamplification genomic alterations in erbb2 in breast cancer18 reported three patients who responded to anti - her2 therapy , two with er - positive tumors and one with triple - negative cancer.13 - 15 , 18 consistent with this , the patient we report here had er - positive cancer , and additional analysis showed that 80% ( four of five ) of breast cancers profiled at foundation medicine and found to carry the erbb2 g776v mutation were er positive . 
the previous study reported that several genes are frequently coaltered with erbb2 in erbb2 - nonamplified breast cancers , particularly the tumor suppressor tp53 and the oncogene pik3ca.18 here , we show that erbb2 - nonamplified breast carcinomas driven by erbb2 kinase domain mutations specifically are significantly enriched for co - alteration with the tumor suppressor map3k1 . 
although frequent alteration of map3k1 in the luminal / erpositive breast cancer subtype has been reported , 33 the current study provides evidence of association between erbb2 and map3k1 mutations independent of subtype and suggests that activating mutations in the erbb2 kinase domain may cooperate with inactivation of map3k1 to promote cancer progression . 
additional work will also be needed to elucidate the effects of er status on the clinical efficacy of her2 - targeted agents in erbb2 - mutated breast cancer . in this patients case , the clinical decision to use tp - based regimens was based on prior reports of similar erbb2 mutations in breast cancer or nsclc.36 - 42 , 46 , 48 the preclinical experiments reported here confirmed that erbb2 g776v is an activating mutation , and the patient had a good clinical outcome on tp - based therapy . 
this study adds to a body of evidence supporting the use of her2 - targeted therapy in patients with metastatic breast cancer with erbb2 mutations , although given that the tp - based regimens in this case included exemestane or nab - paclitaxel that may have significantly contributed to the clinical benefit , caution must be exercised in applying these findings to the treatment of other patients . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . yakov chudnovsky employment : foundation medicine stock and other ownership interests : foundation medicine travel , accommodations , expenses : foundation medicine runjun d . 
schrock employment : foundation medicine stock and other ownership interests : foundation medicine caitlin connelly employment : foundation medicine kyle gowen employment : foundation medicine stock and other ownership interests : foundation medicine travel , accommodations , expenses : foundation medicine garrett m . 
miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan - kettering cancer center jeffrey s . 
ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine honoraria : pfizer , emd merck serono research funding : foundation medicine siraj m . 
ali employment : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine , patents via seres health related to microbiome in non - neoplastic disease ( inst ) ron bose honoraria : genentech , novartis consulting or advisory role : genentech affiliations yakov chudnovsky , alexa b . 
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schiavon g , hrebien s , garcia - murillas i , et al : analysis of esr1 mutation in circulating tumor dna demonstrates evolution during therapy for metastatic breast cancer . 
tokunaga e , kimura y , oki e , et al : akt is frequently activated in her2 / neu - positive breast cancers and associated with poor prognosis among hormone - treated patients . 
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ali sm , alpaugh rk , downing sr , et al : response of an erbb2 - mutated inflammatory breast carcinoma to human epidermal growth factor receptor 2 - targeted therapy . 
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chumsri s , weidler j , ali s , et al : prolonged response to trastuzumab in a patient with her2 - nonamplified breast cancer with elevated her2 dimerization harboring an erbb2 s310f mutation . 
grellety t , soubeyran i , robert j , et al : a clinical case of invasive lobular breast carcinoma with erbb2 and cdh1 mutations presenting a dramatic response to anti - her2 - directed therapy . 
ross js , wang k , sheehan ce , et al : relapsed classic e - cadherin ( cdh1 ) - mutated invasive lobular breast cancer shows a high frequency of her2 ( erbb2 ) gene mutations . 
ross js , gay lm , wang k , et al : nonamplification erbb2 genomic alterations in 5605 cases of recurrent and metastatic breast cancer : an emerging opportunity for anti - her2 targeted therapies . 
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minami y , shimamura t , shah k , et al : the major lung cancer - derived mutants of erbb2 are oncogenic and are associated with sensitivity to the irreversible egfr / erbb2 inhibitor hki - 272 . 
shimamura t , ji h , minami y , et al : non - small - cell lung cancer and ba / f3 transformed cells harboring the erbb2 g776insv_g / c mutation are sensitive to the dual - specific epidermal growth factor receptor and erbb2 inhibitor hki - 272 . 
suzuki t , fujii a , ohya j , et al : antitumor activity of a dual epidermal growth factor receptor and erbb2 kinase inhibitor mp - 412 ( av - 412 ) in mouse xenograft models . 
wang se , narasanna a , perez - torres m , et al : her2 kinase domain mutation results in constitutive phosphorylation and activation of her2 and egfr and resistance to egfr tyrosine kinase inhibitors . 
buttitta f , barassi f , fresu g , et al : mutational analysis of the her2 gene in lung tumors from caucasian patients : mutations are mainly present in adenocarcinomas with bronchioloalveolar features . 
arcila me , chaft je , nafa k , et al : prevalence , clinicopathologic associations , and molecular spectrum of erbb2 ( her2 ) tyrosine kinase mutations in lung adenocarcinomas . 
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de gr`eve j , moran t , graas mp , et al : phase ii study of afatinib , an irreversible erbb family blocker , in demographically and genotypically defined lung adenocarcinoma . 
kris mg , camidge dr , giaccone g , et al : targeting her2 aberrations as actionable drivers in lung cancers : phase ii trial of the pan - her tyrosine kinase inhibitor dacomitinib in patients with her2 - mutant or amplified tumors . 
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bryce purpose genomic testing has increased the quantity of information available to oncologists . unfortunately , many identified sequence alterations are variants of unknown significance ( vuss ) , which thus limit the clinicians ability to use these findings to inform treatment . 
we applied a combination of in silico prediction and molecular modeling tools and laboratory techniques to rapidly define actionable vuss . materials and methods exome sequencing was conducted on 308 tumors from various origins . most single nucleotide alterations within gene coding regions were vuss . 
a subset of receptor tyrosine kinase vuss was characterized by laboratory comparison of each vus versus its wild - type counterpart in terms of expression and signaling activity . results the study identified 4 , 327 point mutations of which 3 , 833 were vuss . 
three vuss ( fgfr2 f276c , fgfr4 r78h , and kdr g539r ) showed increased basal or ligand - stimulated erk phosphorylation compared with their wild - type counterparts , which suggests that they support transformation . 
for example , braf v600e is well accepted as a therapeutic target in metastatic melanoma.7 however , a current challenge in genomic oncology care is the evaluation of the potential therapeutic significance of large numbers of uncharacterized , nonsynonymous sequence alterations referred to as variants of unknown significance ( vuss ) in potentially oncogenic proteins . when novel vuss are identified , the clinical team must try to draw conclusions about whether the variant is a driver mutation or has no significance for cancer pathogenesis . 
although some recurrent mutations within oncogenic proteins have been characterized as having an effect on protein function and thus , the promotion of transformation , novel vuss identified through clinical genomic testing often are lacking functional information . the definition of the therapeutic value of vuss is a current unmet need.8 we demonstrate how in silico analysis and experimental laboratory studies can rapidly determine the potential therapeutic value of a vus . 
by using bioinformatic analysis of exome sequencing results , a large number of potentially deleterious vuss that are therapeutically targetable were identified with a high frequency of occurrence in kinases . 
the mayo clinic irb approved the in vitro functional studies of somatic mutations identified in tumors of patients enrolled in the cim oncology service ( irb 15 - 003386 )  . 
redcap ( research electronic data capture ) hosted at the mayo clinic was used to collect and store clinical follow - up data.10 results vuss are the most common findings in tumor exome sequencing analysis targeted and exome sequencing were performed on heterogeneous solid ( 57% ) and hematologic ( 43% ) malignancies ( data supplement )  . 
vuss , which constitute mutations that are functionally uncharacterized or previously unreported in the cosmic ( catalogue of somatic mutations in cancer ) database , 11 comprised the majority ( 89% ) of the point mutations observed in this cohort ( fig 1a )  . 
obstacles to clinical use of these data resulted from large numbers of vuss identified in each patient , uncertainty whether identified vuss are potential drivers of transformation , and whether encoded proteins were therapeutically targetable . to begin to address these difficulties , vuss were filtered into a subgroup of 905 in genes that encode proteins therapeutically targetable by food and drug administrationapproved drugs or by investigational agents available through clinical trials . 
protein class analysis demonstrated that rtks and serine / threonine kinases represent the largest therapeutically targetable functional classes , with mutations in both hematologic and solid tumors ( fig 1b )  . these 905 therapeutically targetable vuss were then evaluated in silico , and those determined to be benign by two prediction tools were removed , which left 522 potentially deleterious , therapeutically targetable vuss in 226 patients ( data supplement ) or 12% of the total variants detected ( fig 1a )  . 
the distribution of protein classes represented in this deleterious , therapeutically targetable subgroup ( fig 1c ) was similar to that of the therapeutically targetable vus group ( fig 1b )  . 
 a significant findings , 11% targetable , deleterious , vuss , 12% other vuss , receptor tyrosine kinase serine / threonine kinase notch receptor dna endonuclease endocytosis nonreceptor tyrosine kinase tumor suppressor pi3k family ubiquitin ligase apoptotic regulator growth inhibition receptor tyrosine kinase serine / threonine kinase notch receptor endocytosis nonreceptor tyrosine kinase dna endonuclease pi3k family histone deacetylase tumor suppressor hormone receptor fgf receptor ubiquitin ligase growth inhibition hematologic solid no . 
 ( a ) frequency of significant findings and uncharacterized / unreported variants of unknown significance ( vuss ) in 4 , 327 point mutations reported in 308 patient tumors , including solid and hematologic malignancies . 
 ( b ) frequency of the most commonly observed protein classes in 905 therapeutically targetable vuss . with pymol software ( schrodinger , new york , ny ; data supplement ) , which predicted potentially altered function on the basis of their location within the proteins kinase domain ( data supplement )  . in silico and functional characterization of rtk vuss identified a subset of new targetable mutations ten rtk vuss from varied solid and hematologic malignancies ( data supplement ) and in reinterest were selected for gions of functional additional evaluation by in vitro testing . 
in addition , sequence alignment across species demonstrated that the wt residue that corresponded to each of the 10 vuss was completely conserved among diverse species , including mouse , rat , bovine , and human proteins . 
biochemical studies of r78h demonstrated no significant differences from wt fgfr4 in overall expression levels when expressed in kmch - 1 cholangiocarcinoma cells ( figs 2b and 2c )  . 
cellular localization of wt and r78h fgfr4 was also similar and exhibited mainly a plasma membrane distribution with intracellular labeling of probable golgi membranes ( fig 2e )  . however , r78h fgfr4 exhibited a small , but significantly elevated fgf2 - stimulated phosphoerk ( perk ) level compared with wt ( figs 2b and 2d )  . two kdr vuss , g55e and g539r , are located in the extracellular domain in proximity to amino acids that form disulfide bonds ( c53 and c530 ) ; thus , these amino acid substitutions may potentially affect neighboring disulfide bonds . 
 ( c ) frequency of the most commonly observed protein classes in 522 therapeutically targetable , potentially deleterious vuss . cells that expressed g55e kdr ( figs 3a and 3c )  . kdr variants and wt exhibited a similar punctate distribution by immunofluorescence ( fig 3d )  . vuss g251e , v484m , and t643m of pdgfra and variants v258l and v316m of pdgfrb were investigated . 
all variants fall in the extracellular domain of their respective receptor except t643m , which occurs within the pdgfrb kinase domav258l was predicted to be functionally benign but was of interest for in vitro study because of its ig3 location . 
pdgfr vuss were generally similar to their wt counterparts in terms of expression , distribution of polypeptide species on western blots , pdgf - stimulated perk levels , and cellular localization ( data supplement ) , except that pdgfra v484m was significantly lower in expression and pdgfstimulated perk levels than wt ( data supplement ) and pdgfrb v316m was significantly lower in expression than wt ( data supplement )  . f276c mutant is a new , constitutively active form of fgfr2 although predicted in silico to be benign , fgfr2 k41e , identified in an acute myeloid leukemia , was selected for additional study because of its location in the extracellular ig1 of fgfr2 and its unknown effect on ligand binding in nearby ig2 and ig3 ( data supplement )  . 
the fgfr2 f276c mutation identified here from a cholangiocarcinoma was also in a single cholangiocarcinoma in the cosmic database but has not been characterized.11 , 12 f276c is located in an extracellular , ig - like c2 - type 3 domain13 where ligand binding occurs.14 a different amino acid substitution at the same residue , f276v , has been reported in crouzon syndrome.15 modeling of wt and f276c fgfr2 showed that the extracellular receptor of fgfr2 contains an intrinsic disulfide bond between c278 and c342 in ig3 ( fig 4a , shown in gold )  . 
these data suggest that the f276c variant is functionally altered relative to the wt form . when fgfr2 proteins were expressed in kmch - 1 cells , immunofluorescence microscopy showed that the wt and k41e fgfr2 proteins were localized mainly to the cell surface and occurred on intracellular structures , likely endosomes and the golgi apparatus ( fig 4c )  . 
in contrast , the f276c variant exhibited an endoplasmic reticulumlike appearance and bright golgi - like intracellular structures in most cells , with only a minority of cells showing an obvious plasma membrane distribution ( fig 4c )  . 
each protein was expressed as an approximately 130 - kda polypeptide as detected by western blot ( fig 4d )  . however , the f276c variant was expressed at a higher level and the k41e variant at a lower level compared with wt ( fig 4d ) , although cells were transfected with equal amounts of dna for the different fgfr2 constructs . functional characteristics of fgfr2 proteins were assessed by studying perk signaling . 
because the f276c fgfr2 variant is expressed at higher levels than wt when equal amounts of dna were used , a lower ratio of f276c construct dna was used for transfection in the following experiments so that resulting levels of wt and f276c fgfr2 proteins were comparable ( figs 4e and 4f ) : kmch - 1 cells transfected with fgfr2 constructs were treated for 16 hours in serum - free media with fgf2 , lysed , and analyzed by western blot . 
the expression of wt or k41e fgfr2 in the absence of fgf2 increased perk levels beyond control levels , and treatment with fgf2 led to approximately fivefold increases in perk compared with control for both wt and k41e . 
however , expression of f276c fgfr2 significantly increased the perk level in the absence of fgf2 compared with wt fgfr2 , with little increase upon treatment with fgf2 ( figs 4e and 4g )  . 
in summary , f276c fgfr2 has high expression , altered cellular distribution , and increased constitutive activity compared with wt . finally , the sensitivity of wt and f276c fgfr2 activities to treatment with the fgfr inhibitor bgj398 were compared . 
at concentrations between 0 and 100 nm , erk phosphorylation was similarly partially inhibited by bgj398 in cells that expressed wt or f276c , and both fgfr2 forms were completely inhibited by bgj398 at 200 nm ( figs 5a and 5b )  . 
right panel : after 1 day , the cells were incubated with and without 20 ng / ml fgf2 in 0.1% bovine serum albumin / dmem for 16 hours at 37c . 
values are mean 6 se normalized to wt ( + fgf2 ) levels . bracket indicates groups within treatment types ( 2fgf or + fgf ) among fgfr4 - transfected samples that were significantly different ( p , .05 ) from each other in twotailed t tests . 
 ( e ) hucct - 1 cells were transfected with flag - tagged wt and r78h fgfr4 for 2 days and then processed for immunofluorescence by using an anti - flag antibody . 
he was originally treated in a clinical trial with gemcitabine , cisplatin , and silmitasertib , which resulted in a partial response . he remained on this treatment of 10 months , at which time his disease progressed and he was switched to capecitabine and oxaliplattwo months later , the disease became refractory to that regimen . 
after 1 day , the cells were serum starved in 0.1% bovine serum albumin / dmem overnight and then incubated with and without 25 ng / ml vascular endothelial growth factor ( vegf ) for 10 min at 37c . 
cell samples were then lysed and subjected to western blot analysis for flag - kdr , phospho - erk ( perk ) , total erk , and vinculequal amounts of total protein were loaded per lane . 
g55e expression of full - length receptor ( approximately 200 kda ) was decreased compared with wt , with the appearance of approximately 70to 80 - kda fragments , which suggested decreased stability / increased degradation of the g55e forperk was barely detectable in g55e samples and not increased by vegf treatment . 
brackets indicate groups within treatment types ( 2vegf or + vegf ) among kdr - expressing samples that were significantly different ( p , .05 ) from one another in two - tailed t tests . 
 ( d ) hucct - 1 cells were transfected with flag - tagged wt and variants of unknown significance kdr for 2 days and then processed for immunofluorescence with an anti - flag antibody . 
on the basis of this finding , the patient started on bgj398 ( clinicaltrials.gov identifier : nct02150967 ) , achieved a partial response to therapy after 2 months ( figs 5c and 5d ) , and maintained the response for an additional 4 months , at which time new lesions developed that led to discontinuation of bgj398 . 
as a result of our in vitro studies of f276c , a mechanism of action has now been correlated with this observed clinical response of the tumor to bgj398 . discussion the volume of new data from individual and group sequencing efforts ( eg , the cancer genome atlas ) has rapidly expanded the understanding of the incidence and frequency of mutations in disparate cancers . 
few mutations have extensive preclinical and clinical evidence that support the effectiveness of targeted therapies , and genomic testing often reveals that tumors have numerous vuss , including variant sequences for which no functional data are available . mutations that have not been functionally characterized present a significant and growing challenge to the treating physician . 
the extracellular receptor of fgfr2 contains an intrinsic disulfide bond between c278 and c342 in the immunoglobulin - like domain 3 ( ig3 ; shown in gold )  . residue f276 is highlighted in gray and is proximal to the disulfide bridge . 
the fgfr2 f276c variant ( highlighted in red ) may lead to the introduction of aberrant disulfide bonds that could alter the activation state of the prote ( b ) sequence alignment shows that residue f276 is highly conserved among the fgfr2 family from zebrafish to humans ( sequence alignment performed by using clustal omega [ embl - ebi , wellcome genome campus , uk ] ; uniprotkb entry numbers are shown )  . 
 ( c ) hucct - 1 cholangiocarcinoma cells transfected with the wt , k41e , and f276c fgfr2 forms for 2 days were fixed , permeabilized , and processed for immunofluorescence by using the flag antibody . 
kmch - 1 cells were transfected with fgfr2 forms by using a 3.5 / 5 ratio of f276c / wt and k41e dna to adjust expression of the f276c protein to similar levels as the wt forafter 1 day , cells were switched to serum - free medium with or without 20 ng / ml fgf2 and incubated an additional 16 hours at 37c before lysis . 
lysates were analyzed by western blot for expression of fgfr2 - flag , phospho - erk ( perk ) , total erk , and vincul ( f ) quantitation of fgfr2 levels in western blots as in ( e )  . 
brackets indicate groups within treatment types ( 2fgf or + fgf ) among fgfr2 - expressing samples that were significantly different ( p , .05 ) from one another in two - tailed t tests . 
 ( a ) kmch - 1 cells were transfected with f276c and wild type ( wt ) by using a 3.5 / 5 dna ratio , respectively , to normalize expression levels . 
values are mean 6 se and expressed as percent inhibition of perk signal compared with cells with no bgj398 . response of fgfr2 f276ccontaining tumor to bgj398 in ( c ) september 2015 ( pretreatment , with magnetic resonance imaging showing a 30.1mm tumor diameter [ red line ] ) and ( d ) october 2015 ( postinitiation of treatment , with pan - fgfr inhibitor bgj398 magnetic resonance imaging showing tumor shrinkage to an 18.2 - mm diameter [ red line ] )  . fgfr2flag perk fgfr2flag perk f276c bgj398 ( nm ) 50 100 200 f276c bgj398 ( nm ) becomes how to quickly assess the potentially deleterious effect of vuss that occur in therapeutically targetable genes . 
with multiple uncharacterized / unreported mutations returned for each patient , a method of prioritizing which variants to study in depth is necessary . we addressed this problem by developing an approach that uses an in silico filtering process to prioritize mutations of the highest biologic and clinical interest . 
of these 10 variants , seven were found to be altered in expression or activity relative to the wt protein , and three of these ( fgfr2 f276c , fgfr4 r78h , and kdr g539r ) demonstrated greater activity than their wt counterparts ( table 1 ) , which suggests that these mutations play a role in promoting oncogenesis . 
in contrast , four vuss ( fgfr4 k41e , kdr g55e , pdgfra v484m , and pdgfrb v316m ) exhibited reduced expression compared with their wt counterparts , and thus were unlikely to be involved in oncogenesis in the tumors where they occurred . 
these findings not only support the strength of our in silico analysis in predicting whether vuss are functionally altered but also point to the inability to distinguish among activating , deactivating , and destabilizing mutations . optimization of appropriate cellular models is important in methods development for functional evaluation of vuss . 
for more understanding of the functional significance of vuss , these studies should be followed by experiments that use cell types that match the vus tumor of origin and evaluate end points such as cell growth and viability . fgfr2 f276c was identified as a vus of potential interest because of the presence of several factors , including prediction of a deleterious effect by two algorithms , 3d modeling that suggests an increase in activity on the basis of its location in the ligand - binding ig3 , and proximity to a key disulfide bond . 
dashes ( ) indicate no change versus wild - type protein . abbreviation : vus , variant of unknown significance . * increases or decreases in activity ( phospho - erk levels ) indicate changes seen versus wild type upon stimulation with ligand , except for fgfr2 f276c , which exhibited increased constitutive activity . constitutive receptor dimerization and activation , which lead to a variety of skeletal and craniosynostosis disorders ( eg , crouzon and pfeiffer syndromes ) .16 - 18 our molecular modeling combined with the demonstration that f276c fgfr2 is more highly expressed and constitutively active than the wt receptor suggests that this mutation alters disulfide bonds , which alters receptor dimerization and activity similarly to the fgfr2 mutations seen in craniosynostosis syndromes . 
by using in vitro studies , we show that the f276c fgfr2 variant is sensitive to bgj398 , a panfgfr inhibitor , which was also reflected clinically in the response of a patients tumor when treated with bgj398 as part of a clinical trial ( fig 5 )  . 
however , additional studies , such as the testing of bgj398 effectiveness in impeding growth of organoids or xenografts that express wt versus f276c , are needed to confirm that fgfr2 f276c is actionable . the ability to identify potentially actionable vuss from numerous vuss for each patient tumor would simplify therapeutic choices . 
in silico analysis requires only hours to conduct and as demonstrated here , can yield a subset of vuss that encode therapeutically targetable proteins likely to be altered in function by their variant sequence . in vitro functional studies are more time consuming and may not fit practically within a patients progression timeline , but they may yield moredefinitive findings . 
gawryletz no relationship to disclose sikander ailawadhi consulting or advisory role : amgen , takeda pharmaceuticals , novartis research funding : pharmacyclics ( inst ) travel , accommodations , expenses : amgen , takeda pharmaceuticals , novartis stephen m . 
ansell honoraria : webmd , research to practice research funding : bristol - myers squibb ( inst ) , celldex therapeutics ( inst ) , seattle genetics ( inst ) , merck ( inst ) , affimed ( inst ) , trillium therapeutics ( inst ) kelly k . 
halfdanarson consulting or advisory role : lexicon ( inst ) , ipsen ( inst ) , merrimack ( inst ) research funding : esanex ( inst ) , ipsen ( inst ) , boston biomedical ( inst ) thai h . 
 research funding : merck , bristol - meyers squibb , roche ( inst ) , genentech ( inst ) , x4p pharmaceuticals ( inst ) , amgen ( inst ) prashant kapoor consulting or advisory role : sanofi ( inst ) research funding : amgen ( inst ) , takeda pharmaceuticals ( inst ) aaron s . 
mansfield consulting or advisory role : trovagene ( inst ) , genentech ( inst ) , bristol - myers squibb ( inst ) travel , accommodations , expenses : bristol - myers squibb nathalie meurice no relationship to disclose amulya a . 
nowakowski consulting or advisory role : celgene ( inst ) , morphosys ( inst ) , genentech ( inst ) research funding : celgene ( inst ) animesh pardanani no relationship to disclose sameer a . 
feldman consulting or advisory role : infinity pharmaceuticals patents , royalties , other intellectual property : inventor of technology for which the mayo clinic holds an unlicensed patent or has submitted a patent application ( inst ) ramesh k . 
ramanathan honoraria : taiho pharmaceutical , cerulean pharma , pharmacyclics , insys therapeutics , novocure consulting or advisory role : cerulean pharma , novocure , insys therapeutics , pharmacyclics research funding : abbvie ( inst ) , celgene ( inst ) , merck ( inst ) , schering plough ( inst ) , merrimack ( inst ) , boston biomedical ( inst ) , berg ( inst ) , superlab far east ( inst ) steven i . 
robinson travel , accommodations , expenses : tricon pharmaceuticals raoul tibes research funding : novartis ( inst ) , merck ( inst ) , seattle genetics ( inst ) , boehringer ingelheim ( inst ) , astellas pharma ( inst ) , astex pharmaceuticals ( inst ) heidi d . 
mcwilliams consulting or advisory role : merrimack ( inst ) research funding : prism biolab ( inst ) , genentech ( inst ) , novartis ( inst ) , newlink genetics ( inst ) , eli lilly ( inst ) , aduro biotech ( inst ) , pfizer ( inst ) , sanofi ( inst ) travel , accommodations , expenses : astrazeneca mrinal m . 
wieben patents , royalties , other intellectual property : champions oncologylicensed ip - targeted therapy with abiraterone acetate ( $0 received ) , wuxi appteclicensed ip - breast cancer xenografts ( $12 , 737 to mayo clinic ; inst ) , affymetrix - licensed ip on several variants in drug processing enzymes ( $0 in past 2 years ) , life technologieslicensed ip on several variants in drug processing enzymes ( $0 in past 2 years ) tammy m . 
kennedy patents , royalties , other intellectual property : patent on unrelated work ( us patent 9 , 458 , 189 ) for ligation of stapled polypeptides issued october 4 , 2016 eric w . 
borad stock and other ownership interests : glaxosmithkline , gilead sciences consulting or advisory role : g1 therapeutics , td2 , fujifilm ( inst ) , agios ( inst ) , insys therapeutics ( inst ) , novartis ( inst ) , arqule ( inst ) , celegene ( inst ) , inspyr therapeutics , halozyme therapeutics ( inst ) research funding : boston biomedical ( inst ) , mirna therapeutics ( inst ) , senhwa biosciences ( inst ) , medimmune ( inst ) , bioline ( inst ) , agios ( inst ) , halozyme therapeutics ( inst ) , celgene ( inst ) , threshold pharmaceuticals ( inst ) , toray ( inst ) , dicerna pharmaceuticals ( inst ) , sillajen ( inst ) , eisai ( inst ) , taiho pharmaceuticals ( inst ) , emd serono ( inst ) , isis pharmaceuticals ( inst ) , incyte ( inst ) , sun biopharma ( inst ) , ariad pharmaceuticals ( inst ) , imclone systems ( inst ) travel , accommodations , expenses : arqule , celgene , astrazeneca aminah jatoi research funding : entera health , boston biologics martin e . 
kennedy , university of georgia , athens , ga . supported by the mayo clinic center for individualized medicine . presented at the science of team science 2016 conference , phoenix , az , may 16 - 19 , 2016 . support prior presentation references 35 : 49 - 62 , 2016 1 . 
horak p , fr ohling s , glimm h : integrating next - generation sequencing into clinical oncology : strategies , promises onco targets ther 9 : 7355 - 7365 , 2016 and pitfalls . 
harris pa , taylor r , thielke r , et al : research electronic data capture ( redcap ) a metadata - driven methodology and workflow process for providing translational research informatics support . 
steinberger d , vriend g , mulliken jb , et al : the mutations in fgfr2 - associated craniosynostoses are clustered in five structural elements of immunoglobulin - like domain iii of the receptor . 
lajeunie e , heuertz s , el ghouzzi v , et al : mutation screening in patients with syndromic craniosynostoses indicates that a limited number of recurrent fgfr2 mutations accounts for severe forms of pfeiffer syndrome . 
ratisoontorn c , fan gf , mcentee k , et al : activating ( p253r , c278f ) and dominant negative mutations of fgfr2 : differential effects on calvarial bone cell proliferation , differentiation , and mineralization . 
connor claire bascuana jamil asselah nathaniel bouganim vronique poulin jacques jolivet petro vafiadis philippe le guillaume martel frdric lemay annie beaudoin khashayar rafatzand prosanto chaudhury jeffrey barkun peter metrakos victoria marcus atilla omeroglu george chong mohammad r . 
foulkes steven gallinger george zogopoulos ( continued ) purpose we investigated the translational value of reflex testing for germline mutations in four homology - directed dna repair predisposition genes ( brca1 , brca2 , palb2 , and atm ) in consecutive patients with pancreatic adenocarcinoma . methods one hundred fifty patients with french - canadian ( fc ) ancestry were evaluated for founder mutations , and 114 patients were subsequently assessed by full gene sequencing and multiplex ligation - dependent probe amplification for nonfounder mutations . 
2018 by american society of clinical oncology introduction pancreatic adenocarcinoma ( pac ) remains a lethal malignancy.1 - 3 although novel combinations of systemic therapies have been evaluated in pac over the past decade , 4 , 5 these efforts have not resulted in marked improvements in patient survival . 
these clinical failures , together with the projection that pac will be the second leading cause of cancer death by 2030 , 6 highlight the urgent need for both early detection and precision therapy strategies for pac . genomic subtypes of pac have been described and may inform therapies . 
in this large , two - center study , we assessed whether reflex genetic testing of brca1 , brca2 , palb2 , and atm should be offered to all or select patients with incident pac . methods study participants two case series were evaluated . 
the fc study participants included consecutively enrolled patients with a clinical diagnosis of pac and self - reported fc ancestry ( one or more grandparents ) enrolled in the quebec pancreas cancer study ( qpcs ) 30 between april 2012 and june 2017 . 
 the montreal - toronto ( mt ) study participants included patients , unselected for ancestry or family history , with a clinical diagnosis of pac who were consecutively enrolled in the qpcs or ontario pancreas cancer study31 between august 2014 and december 2015 . 
dna from circulating lymphocytes , saliva , and / or tissue specimens was obtained from participants and their relatives for germline genetic testing , segregation , and / or somatic studies ( data supplement )  . 
ethics approval was obtained for both studies . clinical variables and family history data were extracted from the qpcs and ontario pancreas cancer study registries.30 , 31 eligibility for clinical genetic testing was assessed using the national comprehensive cancer network ( nccn ) brca - related breast and / or ovarian cancer syndrome testing criteria v2.2016.32 for the nccn criteria , a second primary cancer in the proband was considered equivalent to a cancer in a close blood relative , and all prostate cancers were assumed to have a gleason score < 7 , because these were unavailable . fc founder mutation screening screening for the 20 fc founder mutations ( 11 brca1 , eight brca2 , and one palb2 ; listed with historical names in data supplement ) 21 - 26 was performed on a rolling basis . 
screening was performed by polymerase chain reaction and bead - based fluorescent detection on the luminex 200 platform26 , 33 or by sanger sequencing ( primers listed in data supplement )  . full gene sequencing in the fc study , full gene sequencing of brca1 , brca2 , palb2 , and atm was performed after founder mutation testing ( data supplement )  . 
pathogenic mutation prevalence french - canadian study ( n = 150 ) montreal - toronto study ( n = 236 ) founder panel testing ( n = 150 ) full gene testing ( n = 114 * ) ashkenazi jewish ( n = 30 ) nonfounder ( n = 206 ) no . 
these vus were assessed for pathogenicity using in silico prediction algorithms ( data supplement )  . multiplex ligation - dependent probe amplification patients with pac with fc ancestry have not previously been characterized for mutations in these four genes . 
therefore , fc study participants were evaluated for large insertions / deletions or rearrangements in brca1 , brca2 , palb2 , and atm by multiplex ligation - dependent probe amplification ( mlpa ; data supplement )  . statistical analysis categorical and continuous patient variables were compared using fishers exact test or t test , respectively . 
for the os analyses , atm carriers were included in the noncarrier group , because it remains uncertain whether these patients exhibit similar genomic and therapeutic profiles as brca1 , brca2 , and palb2 carriers . 
fc patients who underwent founder testing only were not considered in statistical analyses , because nonfounder mutations could not be excluded . results mutation carriers if samples were available , pathogenic variants and vus predicted pathogenic were assessed for segregation in relatives affected with pac or associated cancers and for loss - of - heterozygosity ( loh ) or somatic silencing of the wild - type allele in the tumors ( data supplement )  . one hundred seventy - four patients underwent reflex fc founder mutation testing during the study period . 
notably , an fc patient who was excluded from our analyses because of a final pathologic diagnosis of ampullary carcinoma was found to carry an atm mutation ( table 2 )  . 
considering these results , mlpa was performed only for brca1 and brca2 for the subsequent 24 samples , because large alterations are more common in these genes , 39 - 42 with no mutations identified . two - hundred ninety - one patients were enrolled during the mt study period . 
 table 2 shows mutation details as well as clinical and family history variables for the individual carriers . the results from segregation and somatic silencing analyses of mutation and vus carriers are described in the data supplement . 
the pedigrees of the palb2 mutation carriers demonstrate cosegregation of the germline mutation with pac in family fc - 2 ( fig 1a ) and a pac phenocopy in family fc - 58 ( fig 1b )  . 
the pedigrees for the remaining fc study carriers are shown in the data supplement . clinicopathologic characteristics , including responses to chemotherapy of all carriers , are shown in table 2 , with additional mutation details in the data supplement . 
a family history ( one or more firstor second - degree relatives ) of pac ( p = .03 ) , breast cancer ( p = .02 ) , or prostate cancer ( p = .009 ) were each significantly associated with carrier status . 
a previous study of patients with incident pac reported a brca1 and brca2 mutation prevalence of 4.6%.13 this observation was driven by a 12.1% founder mutation prevalence among aj patients , whereas the prevalence among non - aj patients was 3.7%.13 hu et al14 reported a similar prevalence of 3.1% among 96 unselected patients with pac without aj ancestry . 
moreover , one of the two rare nonfounder mutations identified did not originate from the fc branch of the patients pedigree , whereas the second nonfounder mutation was identified in an adopted patient where paternal ancestry could not be confirmed . 
although we did not identify any brca1 mutations , pathogenic mutations in brca1 are less common than in brca2 in pac , and our study is not sufficiently powered to exclude a contribution of brca1 mutations.13 , 47 , 48 considering the low cost of targeted mutation screening , we recommend founder mutation testing include all 20 fc founder mutations . in the fc study case series , two carriers had the palb2 c.2323c > t ( p.gln775 * ) fc founder mutation . 
the observation of cosegregation with disease , as well as loh of the wild - type allele in the tumors of affected carriers in family fc - 2 , provides further support of a causal role for this palb2 mutation in pac . among patients with pac with fc ancestry , a single pathogenic atm mutation was identified ( 0.9% ; 95% ci , 0% to 5.3% ) , which has only been previously reported as a somatic variant.50 an additional germline atm mutation , with loh in the tumor , was identified in an fc patient with a final diagnosis of ampullary carcinoma . 
a recent genomic study of ampullary carcinomas described mutations in atm ; however , it was not indicated whether these mutations originated in the germline.51 no atm mutations were identified among the patients reporting aj ancestry . 
consistent with these findings are reports showing absence of large genomic changes in brca1 , brca2 , and / or palb2 in families with breast / ovarian cancer with fc ancestry , 22 , 42 , 52 , 53 aj ancestry , 54 and ethnically diverse families.13 , 41 , 55 , 56 large genomic changes in atm are also rare.57 collectively , these studies suggest that mlpa analyses for these four genes in patients with incident pac should not be necessary . in recent years , numerous studies have demonstrated that brca1and brca2 - deficient pac tumors are sensitive to platinum - based chemotherapies or parpis.7 , 9 - 11 although less well established , similar findings in palb2 - deficient tumors treated with dna damaging agents have been reported.58 , 59 here , we report two patients with germline palb2 mutations treated with platinum - based chemotherapy . 
much less is known about the clinical behavior of patients with pac with atm mutations , and the genomes of atm - deficient pac tumors infrequently exhibit the genomic hallmarks associated with hdr deficiency.7 , 8 although there is some evidence that atm - deficient breast and prostate cancers have sensitivity to olaparib ( a parpi ) , 60 , 61 the chemotherapeutic sensitivities to platinum - based agents and parpis in atm deficient pac remains unknown . 
although this was not an intent - to - treat study , the carrier status of participants may have influenced chemotherapy choices as well as decisions to include surgical and ablative approaches to manage advanced disease . 
because these treatment decisions were not uniform , and in the absence of intent - to - treat clinical trial data , it is not possible to fully assess the benefit of platinum therapy in carriers . increased numbers of mutation - associated neoantigens8 and favorable responses to immunotherapy.62 although genetic studies have reported the prevalence of germline mutations in these genes among patients with incident pac to be low ( 0% to 2% ) , 14 , 16 these studies have not considered large genomic changes ( eg , exon deletions ) , which are prevalent in patients with lynch syndrome.63 considering the promise of immunotherapy , genetic studies to fully assess the prevalence of mismatch repair germline mutations in pac are merited . although our study was focused on hdr genes , additional genetic subtypes of pac may be amenable to precision oncology strategies . 
the genomes of pac tumors with deficiency in one or more of the dna mismatch repair genes ( mlh1 , msh2 , msh6 , and pms2 ) exhibit finally , although age at diagnosis and family history have not consistently been shown to be predictors of brca1 or brca2 carrier status in pac , 13 , 64 significant associations were observed in our study . 
nearly one in six patients with pac diagnosed at age 50 years or younger were found carriers noncarriers p = .068 carriers noncarriers p = .049 * 1 , 000 2 , 000 2 , 500 3 , 000 1 , 500 2 , 000 1 , 500 time ( days ) 1 , 000 time ( days ) no . 
kaplan - meier survival curves for ( a ) all - stages ( 0 - iv ) carriers versus noncarriers , ( b ) late - stage ( iii / iv ) carriers versus noncarriers , and ( c ) late - stage ( iii / iv ) carriers treated with platinum versus noncarriers . 
we found that patients with pac meeting family history criteria of two or more firstor second - degree relatives with pancreas , breast , ovarian , or prostate cancer on the same side of the family , or one such relative and a second primary of one of these cancer types , identified carriers with similar sensitivity and specificity to the nccn guidelines . 
in the interim , in view of existing health care systems and considering our observed founder mutation frequencies , we recommend reflex founder mutation testing of patients with incident pac with fc or aj ancestry and reflex full - gene sequencing for these four hdr genes in patients without founder ancestry or who test negative for founder mutations who are diagnosed at age 50 years or younger or who meet our described family history criteria . 
 foulkes provision of study material or patients : adeline cuggia , ayelet borgida , spring holter , claire bascuana , jamil asselah , nathaniel bouganim , vronique poulin , jacques jolivet , petro vafiadis , philippe le , guillaume martel , frdric lemay , annie beaudoin , khashayar rafatzand , prosanto chaudhury , jeffrey barkun , peter metrakos , william d . 
connor , claire bascuana , jamil asselah , nathaniel bouganim , vronique poulin , jacques jolivet , petro vafiadis , philippe le , guillaume martel , annie beaudoin , prosanto chaudhury , jeffrey barkun , peter metrakos , george chong , mohammad r . 
smith no relationship to disclose cavin wong no relationship to disclose adeline cuggia no relationship to disclose ayelet borgida no relationship to disclose spring holter no relationship to disclose anita hall no relationship to disclose ashton a . 
 jamil asselah consulting or advisory role : roche canada research funding : celgene nathaniel bouganim no relationship to disclose vronique poulin honoraria : amgen jacques jolivet no relationship to disclose petro vafiadis no relationship to disclose philippe le no relationship to disclose guillaume martel stock and other ownership interests : abbott laboratories honoraria : sanofi canada travel , accommodations , expenses : baxter frdric lemay honoraria : esperas pharma consulting or advisory role : esperas pharma prosanto chaudhury honoraria : novartis , ipsen , astellas pharma consulting or advisory role : ipsen travel , accommodations , expenses : novartis jeffrey barkun travel , accommodations , expenses : baxter canada peter metrakos honoraria : novartis , ipsen , astellas pharma consulting or advisory role : ipsen , novartis speakers ' bureau : amgen travel , accommodations , expenses : novartis , ipsen victoria marcus no relationship to disclose atilla omeroglu no relationship to disclose george chong no relationship to disclose mohammad r . 
foulkes no relationship to disclose steven gallinger no relationship to disclose annie beaudoin honoraria : amgen , novartis , roche canada consulting or advisory role : amgen , novartis travel , accommodations , expenses : novartis , roche canada khashayar rafatzand no relationship to disclose george zogopoulos consulting or advisory role : astellas pharma , baxalta , ipsen research funding : diazon pharmaceuticals patents , royalties , other intellectual property : tc - ptp inhibitors as apc activators for immunotherapy travel , accommodations , expenses : baxalta affiliations alyssa l . 
foulkes , and george zogopoulos , research institute of the mcgill university health centre ; jamil asselah , nathaniel bouganim , khashayar rafatzand , prosanto chaudhury , jeffrey barkun , peter metrakos , victoria marcus , atilla omeroglu , william d . 
rahib l , smith bd , aizenberg r , et al : projecting cancer incidence and deaths to 2030 : the unexpected burden of thyroid , liver , and pancreas cancers in the united states . 
andrei a - z , hall a , smith al , et al : increased in vitro and in vivo sensitivity of brca2 - associated pancreatic cancer to the poly ( adp - ribose ) polymerase - 1 / 2 inhibitor bmn 673 . 
lowery ma , kelsen dp , stadler zk , et al : an emerging entity : pancreatic adenocarcinoma associated with a known brca mutation : clinical descriptors , treatment implications , and future directions . 
cavallone l , arcand sl , maugard cm , et al : comprehensive brca1 and brca2 mutation analyses and review of french canadian families with at least three cases of breast cancer . 
cote s , arcand sl , royer r , et al : the brca2 c.9004g > a ( e2002k ) [ corrected ] variant is likely pathogenic and recurs in breast and / or ovarian cancer families of french canadian descent . 
tischkowitz m , sabbaghian n , hamel n , et al : contribution of the palb2 c.2323c > t [ p.q775x ] founder mutation in well - defined breast and / or ovarian cancer families and unselected ovarian cancer cases of french canadian descent . 
ghadirian p , robidoux a , nassif e , et al : screening for brca1 and brca2 mutations among french - canadian breast cancer cases attending an outpatient clinic in montreal . 
belanger mh , dolman l , arcand sl , et al : a targeted analysis identifies a high frequency of brca1 and brca2 mutation carriers in women with ovarian cancer from a founder population . 
oros kk , ghadirian p , greenwood cmt , et al : significant proportion of breast and / or ovarian cancer families of french canadian descent harbor 1 of 5 brca1 and brca2 mutations . 
borgida ae , ashamalla s , al - sukhni w , et al : management of pancreatic adenocarcinoma in ontario , canada : a population - based study using novel case ascertainment . 
higgs je , harkness ef , bowers nl , et al : the brca2 polymorphic stop codon : stuff or nonsense ? j med genet 52 : 642 - 645 , 2015 35 . 
cavalieri s , funaro a , pappi p , et al : large genomic mutations within the atm gene detected by mlpa , including a duplication of 41 kb from exon 4 to 20 . 
ameziane n , van den ouweland amw , adank ma , et al : lack of large genomic deletions in brip1 , palb2 , and fancd2 genes in brca1 / 2 negative familial breast cancer . 
foulkes wd , ghadirian p , akbari mr , et al : identification of a novel truncating palb2 mutation and analysis of its contribution to early - onset breast cancer in french - canadian women . 
kwong a , ng eko , wong clp , et al : identification of brca1 / 2 founder mutations in southern chinese breast cancer patients using gene sequencing and high resolution dna melting analysis . 
martin dc , mark bl , triggs - raine bl , et al : evaluation of the risk for tay - sachs disease in individuals of french canadian ancestry living in new england . 
moisan a - m , fortin j , dumont m , et al : no evidence of brca1 / 2 genomic rearrangements in high - risk french - canadian breast / ovarian cancer families . 
gunard f , pedneault cs - l , ouellette g , et al : evaluation of the contribution of the three breast cancer susceptibility genes chek2 , stk11 , and palb2 in non - brca1 / 2 french canadian families with high risk of breast cancer . 
pylks k , erkko h , nikkil j , et al : analysis of large deletions in brca1 , brca2 and palb2 genes in finnish breast and ovarian cancer families . 
rouleau e , jesson b , briaux a , et al : rare germline large rearrangements in the brca1 / 2 genes and eight candidate genes in 472 patients with breast cancer predisposition . 
villarroel mc , rajeshkumar nv , garrido - laguna i , et al : personalizing cancer treatment in the age of global genomic analyses : palb2 gene mutations and the response to dna damaging agents in pancreatic cancer . 
 circulating human papillomavirus dna as a biomarker of response in patients with locally advanced cervical cancer treated with definitive chemoradiation purpose to determine whether plasma human papillomavirus ( hpv ) dna predates clinical recurrence and compare its accuracy with 3 - month fluorodeoxyglucose positron emission tomography ( fdg - pet ) in locally advanced cervical cancer . methods this prospective multicenter study accrued 23 women with stage ib to iva cervical cancer planned for definitive chemoradiation therapy ( crt )  . 
all patients underwent routine staging investigations , including thoracic , abdominal , and pelvic computed tomography scan and pelvic magnetic resonance imaging , and were treated with definitive standard external beam radiotherapy ( 45 in 1.8 - gy daily fractions ) to the pelvis with or without inclusion of para aortic lymph nodes , weekly cisplatin chemotherapy ( 40 mg / m2 ) , and brachytherapy . 
all patients were examined routinely every 3 months during the first 2 years after crt and every 6 months thereafter . hpv genotyping was performed by qpcr ( sacace biotechnologies , como , italy ) using genomic dna isolated from baseline cervical swabs . 
at each time point , 20 ml of edta blood was collected at each center and then promptly transported on ice to the central laboratory where plasma was immediately separated and stored at 80c . 
after purification of cfdna from 5 to 10 ml of plasma ( circulating nucleic acid kit ; qiagen , hilden , germany ) , total cfdna concentration was determined using qubit fluorometric quantitation ( thermo fisher scientific , waltham , ma )  . 
absolute quantification of hpv genotype - specific dna was performed on 10 ng of cfdna by dpcr using the qx200 droplet digital pcr system ( bio - rad laboratories , hercules , ca )  . 
each 20 - l reaction contained a final concentration of 900 nm of each primer , 250 nm of each taqman probe , and 1 droplet dpcr master mix ( ddpcr supermix for probes ; bio - rad )  . 
multiplexed taqman assays ( primers and probes from bio - rad ) targeted the e6 and e7 open reading frames from the hpv genotype detected from the baseline swab for each patient . 
 before their use on cfdna , each dpcr assay was evaluated for technical performance using negative controls ( fragmented human genomic dna supplied in the sacace biotechnologies hpv genotyping kit ) and positive controls ( fragmented genomic dna from genotype - matched cervical cancer tissue )  . 
flow of patients by plasma human papillomavirus ( hpv ) and fluorodeoxyglucose positron emission tomography ( pet ) results at the end of or 3 months after chemoradiation therapy ( crt )  . 
given that the e6 and e7 values were highly correlated ( pearsons correlation coefficient , 0.997 ) , their average value was used for all analyses . tumor response on the 3 - month fdg - pet was defined per previous studies into three categories : complete metabolic response , partial metabolic response , and progressive disease.5 consistent with the conventional definitions used to categorize 3 - month fdg - pet in patients with cervical cancer who underwent definitive radiotherapy , 5 the latter two categories were considered a positive test . 
pfs was measured from the date of diagnosis and defined as the time to disease progression / relapse ( biopsy proven relapse or progression of disease on serial imaging ) 5 , 6 or death . 
as of the cutoff date for this analysis ( january 16 , 2018 ) , median follow - up from the date of diagnosis for patients without relapse was 24 months ( range , 18 to 31 months )  . 
currently , at least six active phase iii studies are evaluating this question worldwide ( clinicaltrials.gov identifiers : nct02853604 , nct01414608 , nct00980954 , nct02036164 , nct03468010 , and nct02703961 )  . 
thus , many patients will be subjected needlessly to additional toxicities of these investigational agents , and real benefits may be missed as a result of the inclusion of lower - risk patients . 
representative example of a patient with stage iib cervical cancer who had a false - positive fluorodeoxyglucose positron emission tomography scan ( maximum standardized uptake value , 4.6 ) at approximately 3 months after chemoradiation therapy ( crt ) , with no definite residual disease on subsequent imaging or clinical examination to date ( 25 months after crt )  . 
although the specificity of fdg - pet might have improved with repeated or later ( ie , > 3 months post - treatment ) assessment , the added cost and delay in diagnosing recurrent disease would be major drawbacks of this approach . 
we anticipate that this approach would result in more - successful adjuvant trials , thus enabling personalized and adaptive treatment regimens . strengths of the current study include the prospective design and serial analysis of plasma hpv dna . 
this study may not be applicable to modern cohorts because it was conducted before concurrent cisplatin chemotherapy became standard of care and included patients with small - cell carcinoma ( rare histology with more - aggressive behavior )  . 
this may be explained by more - modern radiation techniques that are leading to improved locoregional control.21 , 22 our follow - up time was limited , although the majority of recurrences in cervical cancer occur within 2 years . 
 bratman administrative support : kathy han provision of materials or patients : kathy han , eric leung , lisa barbera , jennifer croke , anthony fyles , robert wolfson , scott v . 
bratman data analysis and interpretation : kathy han , eric leung , lisa barbera , anthony fyles , ur metser , michael milosevic , melania pintilie , zhen zhao , scott v . 
di grappa no relationship to disclose anthony fyles no relationship to disclose ur metser no relationship to disclose michael milosevic no relationship to disclose melania pintilie no relationship to disclose robert wolfson no relationship to disclose zhen zhao no relationship to disclose kathy han no relationship to disclose eric leung no relationship to disclose scott v . 
 affiliations kathy han , eric leung , lisa barbera , elizabeth barnes , jennifer croke , anthony fyles , ur metser , michael milosevic , robert wolfson , and scott v . 
bratman , princess margaret cancer centre , university health network ; and eric leung , lisa barbera , elizabeth barnes , and robert wolfson , odette cancer centre , sunnybrook health sciences centre , toronto , ontario , canada . supported by the university of toronto department of radiation oncology collaborative seed grant . 
is supported by the princess margaret cancer foundation 's gattuso - slaight personalized cancer medicine fund . presented at the american society of radiation oncology annual meeting , san diego , ca , september 24 - 27 , 2017 , and the canadian association of radiation oncology annual meeting , toronto , ontario , canada , september 13 - 16 , 2017 . support prior presentation references 1 . 
chemoradiotherapy for cervical cancer meta - analysis collaboration : reducing uncertainties about the effects of chemoradiotherapy for cervical cancer : a systematic review and meta - analysis of individual patient data from 18 randomized trials . 
rose pg , java j , whitney cw , et al : nomograms predicting progression - free survival , overall survival , and pelvic recurrence in locally advanced cervical cancer developed from an analysis of identifiable prognostic factors in patients from nrg oncology / gynecologic oncology group randomized trials of chemoradiotherapy . 
schwarz jk , siegel ba , dehdashti f , et al : metabolic response on post - therapy fdg - pet predicts patterns of failure after radiotherapy for cervical cancer . 
ho cm , yang ss , chien ty , et al : detection and quantitation of human papillomavirus type 16 , 18 and 52 dna in the peripheral blood of cervical cancer patients . 
wang wy , twu cw , lin wy , et al : plasma epstein - barr virus dna screening followed by 18f - fluoro - 2 - deoxy - d - glucose positron emission tomography in detecting posttreatment failures of nasopharyngeal carcinoma . 
jeannot e , becette v , campitelli m , et al : circulating human papillomavirus dna detected using droplet digital pcr in the serum of patients diagnosed with early stage human papillomavirusassociated invasive carcinoma . 
sturdza a , ptter r , fokdal lu , et al : image guided brachytherapy in locally advanced cervical cancer : improved pelvic control and survival in retroembrace , a multicenter cohort study . 
charra - brunaud c , harter v , delannes m , et al : impact of 3d image - based pdr brachytherapy on outcome of patients treated for cervix carcinoma in france : results of the french stic prospective study . 
 functional rna studies are a useful tool in variant classication but must be used with caution : a case study of one brca2 variant paola nix , phd1 ; erin mundt , ms1 ; susan manley , ms / cgc , mba1 ; bradford coffee , phd1 ; and benjamin roa , phd1 the use of genetic testing to evaluate hereditary cancer risk is increasing , because it can inform clinical management and cancer treatment decisions . 
a germline pathogenic variant in one of these genes is associated with an increased risk of cancer and therefore merits altered including increased surveilmedical management , lance and the option of risk - reducing surgeries.1 in addition , the identication of a pathogenic brca1 or brca2 variant may affect treatment decisions and clinician recommendations for women with breast and / or ovarian cancer.1 - 4 given these clinical implications , laboratories accurately that classify variants as part of hereditary cancer genetic testing . is essential although established guidelines from the american college of medical genetics ( acmg ) and association for molecular pathology ( amp ) provide a framework to help with variant classication , 5 there may be challenges interpreting the available evidence for some variants.6 , 7 this includes some sequence variants that have the potential to affect mrna splicing . 
a notable fraction of disease - causing variants in cancer predisposition genes affect splicing.8 - 10 many of these occur at the 5 ( cid : 1 ) and 3 ( cid : 1 ) exon - intron boundaries , and the potential impact on splicing is well established , based on the expected sequence at the canonical splice donor and acceptor ( 6 1 , 2 splice sites ) .5 however , sequence variants located beyond these canonical splice sites may also alter mrna processing . 
pathogenicity for these variants is more difcult to assess , and additional functional evidence is often required to determine the actual impact of the variants on splicing . clinical laboratories commonly use functional rna studies to aid in the classication of variants that may affect splicing . 
although these studies can provide useful evidence about the pathogenicity of such variants , there are important caveats to consider , and care must be taken in both how the studies are performed and how the data are interpreted.11 , 12 one important consideration when using rna analysis to classify variants that may affect splicing is that the observation of a splice defect alone may not be sufcient evidence for classication . 
aberrant splicing may result either in no normal transcript produced by the variant allele ( complete splice defect ) or in some residual normal transcript produced by the variant allele.13 these partial , or leaky , splice defects may result in enough functional transcript being produced to support normal protein function . 
therefore , it is essential to quantify any aberrant splice product and determine whether the variant allele produces any normal transcript when using rna analysis in variant classication . functional rna studies often involve reverse transcription polymerase chain reaction ( rt - pcr ) analysis of rna extracted from a patient blood sample or lymphoblastoid cell line . 
one method to distinguish variant - specic transcripts is to use informative single nucleotide in the same pcr polymorphisms ( snps ) present amplicon as the putative spliceogenic variant being tested ( ie , tag snps )  . 
in the case of a heterozygous variant carrier , these tag snps can differentiate the transcripts produced by the normal allele versus the variant allele , especially when the variant allele is located in the intron and not present in the processed mrna . 
rt - pcr products representing these transcripts can be cloned into a vector system or used directly as templates for sequencing analysis . laboratories that do not require rna analysis to include allele - specic transcript quantication for variant classication , either as part of their own studies or from published studies , may misclassify splice - related variants and potentially report false - positive results . 
in addition , care must be taken in assigning pathogenicity if a variant allele has been shown to produce functional transcript , because it may be unclear whether the amount transcript could support normal protein function . 
 commentary splice variants causing brca2 exon 3 skipping , a variant allele may express up to 20% aberrant product yet not be associated with even a moderate risk of cancer . case study of brca2 c.426 - 12_426 - 8del one example of a splice variant with a complex interpretation is brca2 c.426 - 12_426 - 8delan intronic variant resulting in the deletion of ve nucleotides from the dna sequence adjacent to the splice acceptor site of exon 5 . 
published functional studies support this prediction , with studies by zhang et al in 200916 and sanz et al in 201017 demonstrating that this variant causes skipping of exon 5 , which is out of frame . 
however , both studies also noted that the variant allele produces full - length transcript , indicating a partial splice defect . the variant recently published rna analyses from one clinical testing laboratory showed that brca2 c.426 - 12_426 - 8del caused aberrant splicing , resulting in the deletion of exon 5.18 , 19 there was no allele - specic quantication of however , normal transcripts to determine how much , if any , was produced from the variant allele . 
still , the authors concluded that this variant could be classied as likely pathogenic in accordance with acmg / amp guidelines5 on the basis of rna studies demonstrating abnormal splicing from ( evidence category ps3 ) , absence of population controls ( evidence category pm2 ) , and in silico splicing models predicting a weakening of the native site ( evidence category pp3 ) .18 conversely , our laboratory initially classied brca2 c.42612_426 - 8del as a vus on the basis of the evidence available at the time ( fig 1 )  . 
this evidence included the 2 previously published functional studies showing that the variant allele produced some normal transcript.16 , 17 these functional data alone are not sufcient to determine whether the variant is associated with increased cancer risk , because there is uncertainty in how much normal transcript must be expressed by the variant allele to support normal brca2 function . 
although population databases and in silico modeling can provide useful evidence for classication , these tools also must be considered carefully . benign variants also can be rare ( eg , absent from population controls ) , and in silico splicing models cannot discriminate between a full or partial splice defect . 
as a result , this body of evidence was not sufcient for our laboratory to classify brca2 c.42612_426 - 8del as likely pathogenic . our laboratory was later able to reclassify brca2 c.42612_426 - 8del from vus to benign using additional clinical evidence . 
this included evidence from a validated history weighting algorithm ( hwa ) that scores a variant on the basis of the personal and family histories of multiple individuals who carry the specic variant of interest.20 the variant - specic score is compared with matched pathogenic controls ( individuals with known pathogenic variants in brca2 ) and matched benign controls ( individuals with only benign variants in brca2 and other breast and ovarian cancer - risk genes ; appendix fig a1 )  . 
in this case , the hwa called brca2 c.426 - 12_426 - 8del benign , indicating that carriers did not have a personal or family in brca2 history consistent with a pathogenic variant ( appendix fig a1 )  . in addition , co - occurrence of a vus with known pathogenic variants in the same gene can provide evidence that a variant is benign on the basis of associated clinical phenotypes . 
this provides additional evidence that time of brca2 c.426 - 12_426 - 8del hwa and co - occurrence data are consistent with a benign classication and suggest the variant allele does produce enough functional transcript to support normal brca2 function . 
the recent uptake in laboratories performing their own functional rna studies for variant classication can lead to denitive , actionable ndings for more patients ; however , it is critical that rna analysis be held to a high standard to ensure that variant classications also retain a high level of accuracy . to this end , there are several important considerations in the classication of variants in cancer predisposition genes that may affect splicing . 
snp , single - nucleotide polymorphism . some functional transcript , additional clinical evidence may be needed to determine whether the variant is pathogenic . the importance of this additional clinical evidence is exemplied by brca2 c.426 - 12_426 - 8del . 
karam et al18 came to the conclusion that the variant was likely pathogenic on the basis of rna analysis that identied aberrant splicing , in combination with population frequency and in silico modeling data ; however , the rna analysis performed in that study did not elucidate whether the transcript . 
the variant allele produced any functional previously published rna analyses were considered insufcient for a pathogenic classication by our laboratory because of evidence that the variant allele does produce some normal transcript . 
previous studies have highlighted the complex situation that discordant classications create for patients and providers regarding patient management and the potential for discordant test results for family members tested at different laboratories.6 , 7 second , classications made without careful consideration of rna analysis performed by the laboratory or from published studies may result in a false - positive test result . 
professional societies provide recommendations for the medical management of patients with a pathogenic variant in brca2 , including consideration of risk - reducing surgical interventions and eligibility for cancer drug therapies.1 - 4 the example of brca2 c.426 - 12_426 - 8del highlights the need for caution when interpreting functional evidence from rna analysis for variants that may affect splicing to avoid potential mismanagement and inappropriate medical intervention . in this case , variant classication made without full the partial splice defect consideration of resulted in a classication of likely pathogenic for a variant that our laboratory classied as benign when assessed with additional clinical evidence . acmg / amp guidelines provide useful guidance for variant classication5 but do not inform on every caveat or complexity that may be encountered during variant classication . given the severity of the potential clinical consequences of incorrectly classifying a benign variant as pathogenic , additional guidance for interpreting rna sequencing data may be necessary to enhance accurate classication of splicerelated variants . 
this could involve a stepwise process until sufcient data ( from functional rna studies and other sources ) are obtained to accurately perform variant classication ( fig 1 )  . 
for example , the rna transcripts observed in a patient who carries a sequence variant should be compared with the range of full - length and alternative transcripts that naturally occur in normal controls . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . erin mundt employment : myriad genetics stock and other ownership interests : myriad genetics susan manley employment : myriad genetics stock and other ownership interests : myriad genetics bradford coffee employment : myriad genetic laboratories stock and other ownership interests : myriad genetics benjamin roa employment : myriad genetics leadership : myriad genetics stock and other ownership interests : myriad genetics research funding : myriad genetics patents , royalties , other intellectual property : intellectual property held by employer myriad genetics ( inst ) travel , accommodations , expenses : myriad genetics no other potential conicts of interest were reported . acknowledgment we thank krystal brown , phd , and stephanie meek , phd , for their assistance in preparing the manuscript . references daly mb , pilarski r , berry mp et al : nccn clinical practice guidelines in oncology : genetic / familial high - risk assessment : breast , ovarian , and pancreatic ( version 1.2020 ) , nccn clinical practice guidelines in oncology , 2020 . 
 nix et al armstrong d , plaxe s , alvarex r , et al : nccn clinical practice guidelines in oncology : ovarian cancer including fallopian tube cancer and primary peritoneal cancer , version 4.2017. 
curr oncol 25 : s151 - s160 , 2018 richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
genet med 17 : 405 - 424 , 2015 gradishar w , johnson k , brown k , et al : clinical variant classication : a comparison of public databases and a commercial testing laboratory . 
oncologist 22 : 797 - 803 , 2017 balmaa j , digiovanni l , gaddam p , et al : conicting interpretation of genetic variants and cancer risk by commercial laboratories as assessed by the prospective registry of multiplex testing . 
j clin oncol 34 : 4071 - 4078 , 2016 rhine cl , cygan kj , soemedi r , et al : hereditary cancer genes are highly susceptible to splicing mutations . 
whiley pj , de la hoya m , thomassen m , et al : comparison of mrna splicing assay protocols across multiple laboratories : recommendations for best practice in 12 . 
brnich se , abou tayoun an , couch fj , et al : recommendations for application of the functional evidence ps3 / bs3 criterion using the acmg / amp sequence standardized clinical testing . 
gelli e , colombo m , pinto am , et al : usefulness and limitations of comprehensive characterization of mrna splicing proles in the denition of the clinical relevance of brca1 / 2 variants of uncertain signicance . 
sanz dj , acedo a , infante m , et al : a high proportion of dna variants ofbrca1 and brca2 is associated with aberrant splicing in breast / ovarian cancer patients . 
pruss d , morris b , hughes e , et al : development and validation of a new algorithm for the reclassication of genetic variants identied in the brca1 and brca2 genes . 
the new era of precision medicine complicates communication even further as a result of our increasing reliance on genomic data and the varying psychological responses to genomic - based treatments and their expected outcomes . 
however , many of the communication issues are actually similar to perennial long - standing communication issues in oncology , which center on providing hope when breaking bad news and ensuring that adequate informed consent to treatments is obtained . 
we highlight these new communication issues that focus on clinical and ethical questions ( ie , informed consent , shared decision making , patient autonomy , and uncertainty in oncologic treatments ) and provide guidance on working with each scenario . 
in this article , we address common reactions of patients to genomic information and provide thoughtful communication suggestions using a shared decision making framework to help patients cope with the inherent distress - provoking uncertainties in oncology practice . introduction the practice of oncology depends on adequate communication between patients and clinicians . although the science of cancer medicine is advancing exponentially , information technology advances are seriously impeding the critical component of face - to - face communication . 
the key communication issues in oncology are always twofold : the delivery of emotionally charged bad news while sustaining hope in the presence of a lifethreatening prognosis and ensuring that patients understand the information that is provided.1 the advent of precision medicine now complicates the discussion of these issues in oncology in significant ways.2 , 3 although there is a growing emphasis on providing humanistic care in oncology ( eg , palliative care initiatives ) , the increasingly complicated science and public hype , coupled with limited time to talk with patients in busy oncology clinics , significantly threaten this goal . 
science and technology have yet again raced ahead of the human element , which depends on communication to foster those critical elements of mutual respect and trust . the application of genomic medicine is now accessible for the majority of oncology patients because the cost of genomic sequencing has decreased 1 millionfold from a decade ago.4 - 6 the development of molecular diagnostic , prognostic , and predictive biomarkers continues to change the landscape of clinical oncology.7 furthermore , technologic advances facilitate the use of largescale databases that store genomic data , such as ascos cancerlinq and ibm watson.8 - 11 also , research trial design in precision oncology is evolving from the randomized controlled trial to designs that harness these large data - collection initiatives and evaluate responses to drugs across multiple cancer subtypes ( ie , basket trial design ) .12 , 13 at the same time that genomic science and technology have created new research and clinical treatment paradigms , social media and drug direct - to - consumer marketing in the united states has brought information to the public with remarkable speed and fervor.14 , 15 although there are advantages to patients being informed consumers , handling this complex information without any interpretation of its meaning is not helpful.16 this breadth and rapidity of information adds an additional strain to the already complicated communication that occurs between clinicians and their patients.17 direct - to - consumer marketing is new and often amplifies drug benefits beyond reality . 
mcfarland , memorial sloan kettering cancer center , west harrison ; and elizabeth blackler , smita banerjee , and jimmie holland , memorial sloan kettering cancer center , new york , corresponding author : daniel c . 
 understand the implications for the overall cancer experience , eg , their satisfaction or dissatisfaction with their oncology care , or how this information influences patients treatment choices.15 its implications may be greatest when considering the delicate end - of - life conversations that are critical to quality oncology care.18 therefore , it is critical that we understand patients experiences in this new technologic age of genomic medicine . 
the shared decision making ( sdm ) model of communication promotes patient activation and engagement in health care and is an optimal model to guide communication tasks in the precision medicine era.19 , 20 the sdm model describes how clinician and patient jointly participate in making a health decision , having discussed the options and their benefits and harms , as well as having considered the patients values , preferences , and circumstances . 
it respects patient autonomy and leads to improved patient satisfaction when patients are faced with clinical options for which there is not a clearly correct choice , as is frequently encountered in the context of precision oncology.21 - 24 in this article , we highlight the psychological , social , and ethical issues that are part of our new treatments , and we provide communication suggestions on the basis of sdm . 
patients emotional reactions should be considered before , during , and after sdm to ensure effective communication . i won the lottery : the lucky biology club member jane was a 58 - year - old teacher who was diagnosed with stage iv adenocarcinoma of the lung that harbored an epidermal growth factor receptor mutation . her cancer responded well to erlotinib . 
after 15 months , her cancer began to grow as it became increasingly resistant to erlotinib , and chemotherapy was required to treat it . jane became extremely angry and frustrated that her cancer had progressed and found it hard to appreciate the benefit that the drug provided in light of its failure . she remained in shock and disbelief and had a difficult time accepting the need to adjust her treatment to the new situation . the lucky biology club members have driver actionable mutations such as epidermal growth factor receptor , anaplastic lymphoma kinase , ros1 , and brafv600e . 
subsequently , discussions about health care proxies , financial planning , and wills are often delayed . in terms of discussion , it is important to acknowledge the implications of disease biology and to corroborate and correct the information that they have from other sources . 
using sdm principles , the clinician could initiate a discussion of options by saying , there are some specific treatment options for you , on the basis of your cancers genomic make - up . 
however , the clinician must also discuss the framework for treatment and prognosis : although the cancer is still not curable , the mutation indicates that we have some additional treatment options . 
the clinician may say , given this news , it seems like a good time to talk about what to do next or we are in a different place now . 
is it okay if we talk more about the other options and next steps ? addressing the underlying emotions and acknowledging fear are important tools that should be dealt with before focusing on sdm . 
sdm can help provide clinical decision transparency and ameliorate disappointments with limited treatment effectiveness . i failed the test : the unlucky biology club member sarah was a 73 - year - old patient with newly diagnosed nonsmall - cell lung cancer who had no actionable mutation . 
at her first visit , she was clutching an article from the new york times and yelling , i want the serial killer ! she had come to memorial sloan kettering cancer center in hopes of enrolling in a clinical trial with first - line immunotherapy . when she did not meet trial entry criteria because of her pd - l1 status , she became enraged at being denied the opportunity that she had heard and read about . 
the article she held on to described how immunotherapy unleashes a so - called serial killer that ravages all cancer cells in the body , which is understandably what she wanted.29 sarah had delayed seeking cancer treatment in hopes of having the right mutation to enroll in a clinical trial . 
the unlucky biology club members feel that their tumor biology has conspired against them ; they are angered by the treatment options that are available for others but not for thethey feel that it is not fair that other patients with the same cancer are treated with more desirable targeted therapies . 
this may resonate with failures in their life before having cancer . it is crucial to validate their emotions of anger , fear , frustration , and worry by saying , i cant imagine what it has been like for you to hear this news ; i can see this is very upsetting . 
is it okay if we talk a bit more about what this means ? in this scenario , it is important to educate and reorient the patient who has been stuck on one idea as a result of their limited information . 
as part of sdm , the clinician should provide information on benefit and risk , such as , this information helps us plan the very best treatment for you . clinicians should be careful about comparing patients and especially about providing unsolicited information . this is an opportunity to re - establish / realign with the patients goals . 
given what you know about your illness , whats most important to you ? or , as you think about the future , are there situations or things that you want to make sure you accomplish or avoid ? explain that treatment options can be re - evaluated and tailored to the patients wishes . 
it also sends a positive message that although one opportunity for treatment did not work out , the team provides realistic hope and direction , despite a disappointment . a new drug has just come out : a novel treatment michael was a 47 - year - old patient with sinonasal undifferentiated carcinoma , a rare form of head and neck cancer . 
although the drug had never been tested with his specific head and neck cancer , sinonasal undifferentiated carcinoma , his clinicians believed that he had to be informed about the new treatment because he was young and otherwise healthy and , if it worked , would possibly provide a durable response . 
he did not want to complicate or disrupt the decision to accept hospice care , yet he felt ethically bound to offer a treatment for which michael had become eligible . this case highlights an ethical dilemma associated with the interface of advancing science and clinical care . 
it illustrates how the emergence of rapidly changing oncology treatments begets new levels of uncertainty in the cancer experience ( eg , no specific data for a cancer subtype , even though pembrolizumab had been approved for head and neck cancers )  . 
clinicians are taught to provide answers , which can be frustrating when new data become available and raise new questions and more uncertainty ( eg , does the drug work in this cancer subtype ? )  . 
medical training teaches clinicians how to handle uncertainty , but it does not teach them how to share its presence in a constructive way for patients.30 a full discussion was undertaken with the patient about the absence of data , the ethical reason for informing him of the new agent , and the inherent complications of considering another new treatment . 
he decided to stay with his decision to begin hospice care . it is important to discuss patients emotions about clinical uncertainty and the various treatment options , framed in the context of their current clinical situation : i realize this is all very complicated ; let me try to explain these options in the setting of your illness . 
also , it is important to frequently determine their level of understanding ( facilitates deliberation ) : can you tell me , in your words , what you understand ? also , it is helpful to review the patients clinical information together as a background to discussing the uncertainty . in situations of greatest clinical uncertainty , it is important to assure the patient that you are available for further discussion of their concerns and questions . 
a partnering statement , conveying the sense of were in this together and i will continue to work with you , is helpful to demonstrate a personal commitment to providing the best care possible despite the clinical uncertainty . 
patients who feel that they are in a partnership with their clinicians are better able to trust them and tolerate clinical uncertainty.31 i know i can beat it : the denier don was a 68 - year - old patient with progressive metastatic colon cancer who had received all conventional lines of cancer treatment . 
don said that he was the exception , frequently finding ways to explain how the negative clinical facts did not apply to hihe made many references to newly available targeted therapies that he believed could be used to treat his cancer . hope is a critical component of coping , especially in a time of great need . 
these offers are often made in the face of overwhelming disease biology . for most advanced cancers , precision oncology finds driver mutations in only a minority of cases , and the goal of most early trial design is still safety and not necessarily efficacy . 
these are difficult concepts to explain to patients who will probably only hear that there is hope . typically , educating the denier will require revisiting the topic several times to try to arrive at a shared understanding of the actual situation : you may be eligible for a trial if your cancer has a certain mutation ; however , taking the drug in this basket trial may not extend your life . 
when the patients condition worsens , the education may have to become more pointed : scans dont always reflect what is going on in terms of tumor growth and a change in functional status is an indication that the cancer is progressing . 
i hear you saying that what is most important to you is and i understand that you want to make sure to avoid the following this demonstrates that they have been heard , ensures that the goal of hope is not being taken away , and helps the patient to evaluate options . 
it is also important to keep in mind that the denier fluctuates in their level of denial and may partially accept the reality of the situation . patients often maintain two levels of awareness and acknowledgment of the situation . 
one is cognitive : i know how ill i athe other is emotional : i simply cant believe i could die . both exist simultaneously in many patients , which accounts for these day - to - day attitudinal changes . an ongoing positive clinician - patient relationship allows these feelings to emerge , and a more realistic discussion can occur about end - of - life planning , for example . 
often , a patients underlying fear will ameliorate in the context of working closely together . i have done my research : the overly or misinformed patient barbara was a 49 - year - old well - educated professional with nonsmall - cell lung cancer metastatic to brain and bone . 
barbara had strong opinions about the new treatment choices for lung cancer and was unwilling to consider therapies that were more appropriate for her type of disease . this case illustrates the complexity of precision oncology and the level of sophisticated genomic knowledge that is required to have a fully informed conversation with a patient . 
she explained the difference between inherited dna mutations , for which olaparib could be used in the presence of an inherited brca2 mutation in ovarian cancer , and the more common somatic mutations present in noninherited cancers such as her brca2 - mutated nonsmall - cell lung cancer . 
in this situation , correcting misinformation and misunderstanding is essential : there are many exciting cancer treatments , but whats important is that we find the right one for you . some patients want all available information and want to actively participate in their treatment decision . 
also , uncovering each patients major concerns is critical : what information would be most helpful for me to know about you ? then , it is important to acknowledge the patients concerns : this must be very upsetting for you . 
this acknowledges the importance of alliance.32 a request for a specific cancer treatment may be driven by an attempt to reach a goal , which may be modifiable in certain cases . 
it is crucial to demonstrate that you clearly understand their request : i hear you saying that whats most important is to continue treating your cancer to ensure you can reach your goal . case continued : a rebiopsy after progression revealed a her2 mutation that made her eligible for a basket trial . the oncologist invited another discussion of her tumor dna mutations and informed her that a newly found her2 mutation would qualify her for a basket trial . 
the previous genomics discussion on inherited and somatic mutations helped her understand that she would be treated with a group of patients with different cancers , who would all have the same somatic tumor dna mutation , to assess safety and perhaps efficacy in accordance with the trial objectives . she understood alternative treatment options and was offered a decision delay to facilitate deliberation and decision making . 
so far , the literature remains limited.7 , 33 , 34 patients acknowledge the promise of precision medicine but also consistently raise concerns about the discovery of incidental findings , information overload , complications from additional biopsies with delay in definitive cancer treatment , and the lack of a clear benefit.35 the risk of ineffective communication is that patients misunderstand the nature and seriousness of their diseases , which may lead to more aggressive and futile cancer treatments at the end of life or to increased fear and anxiety , while increasing stress and burnout in members of the medical team.36 on the other hand , effective communication enhances shared decision making , decreases patient distress , and increases patient satisfaction and trust in the medical team.25 shared decision making is the communication style that is preferred by most patients.37 patients make choices that are guided by the clinicians information and recommendations . 
patients may also have preferences on a spectrum of communication styles that can range from paternalistic ( ie , the patient is a passive recipient of their care ) to the clinician who is a nondirective information provider or educator ( ie , the patient is given choices and decides on management according to his / her preferences ) .30 it can be helpful to simply ask how the patient and family would like to make decisions , and the clinician can discuss this spectrum to gain clarification . 
discussing therapeutic uncertainty is often a difficult task for clinicians and leads to clinician behaviors of not disclosing all of the information , not talking about it , or oversimplifying the information.30 understandably , this can leave patients with a distorted account of their clinical situation . 
they may make sense of the situation by prescribing to alternative introducing so - called explanations or hopes , extramedical values , or seeking other remedies ( eg , alternative treatments ) when presented with clinical uncertainty.30 the idea of effective disclosure has been proposed as a potential goal in discussing uncertainty with patients . 
this idea supposes that patients can tolerate a certain amount of uncertainty and that extra vigilance is required by the clinician to ensure that they are given the tools and information they need to engage in decision making.30 effectively , extra care is taken in those situations of greater uncertainty to provide information and to ensure that the patient understands . 
this protects autonomy and increases patient trust in the long run . also , addressing underlying emotions , hopes , and fears is an effective way to ensure good communication and informed consent . 
a conversation analysis found that patients who are more assertive draw more empathy from their oncologists , whereas passive patients elicited much less emotional reciprocation from the oncologists.39 clinicians may use open - ended questions , such as , id like to switch gears and check in to see how you are feeling about all of this technical information ? or more closed - ended questions , such as , many patients may feel overwhelmed by all of the scientific information weve discussed ; im wondering if that is true for you ? in addition , checking understanding and emotional reactions enhances the clinician - patient relationship and is key in the setting of uncertainty . directly focusing on the therapeutic aspects of the relationshipby assessing communication preference , checking understanding , and searching for underlying emotional issuesdevelops rapport and may bolster patients resilience in the face of clinical uncertainty . 
the clinician may say something such as : im wondering if there is anything that might be difficult for you to discuss with me ? is there anything that i might be able to do to make this better for you ? or i have a sense that you are someone who likes x , y , and z ; please help me understand if i am correct . 
realigning goals may be accomplished by saying something such as : it is my goal to provide you with the best information for us to make a decision that works for you ; please let me know if that is also your goal or if youd like to focus on additional issues . this human connection dimension of medical care is needed to withstand the uncertainty . 
clinicians frequently underestimate the therapeutic power of their relationships with patients.40 a strong therapeutic bond is fundamental , not simply useful.41 patients have varying abilities to trust , hope , and have faith in their clinicians . 
the relationship is strengthened by focusing on agreed - upon common goals and tasks.32 it is always appropriate to discuss the nuts and bolts of the therapeutic relationship , especially when the clinician perceives that it may not be firmly established or upon entering areas of clinical uncertainty that are inherent in the precision medicine era . summary precision medicine presents a new era of communication in oncology . 
this article has presented some of the common patient reactions to precision medicine and basic communication issues in oncology . communication can be used to enhance rapport , trust , and support . 
zachariae r , pedersen cg , jensen ab , et al : association of perceived physician communication style with patient satisfaction , distress , cancer - related self - efficacy , and perceived control over the disease . 
blanchette ps , spreafico a , miller fa , et al : genomic testing in cancer : patient knowledge , attitudes , and expectations . cancer 120 : 3066 - 3073 , 2014 35 . 
politi mc , studts jl , hayslip jw : shared decision making in oncology practice : what do oncologists need to know ? oncologist 17 : 91 - 100 , 2012 38 . 
kenny da , veldhuijzen w , weijden tv , et al : interpersonal perception in the context of doctor - patient relationships : a dyadic analysis of doctor - patient communication . 
differences in the relative fractions of immune cell subpopulations between immune - rich er - positive and tnbcs3 suggested disparate immunotherapy strategies in the two immune - rich subsets . concerning the study of tnbc immune microenvironment , we recently observed the existence of at least three immune - clusters ( im ) in tnbcs by computing transcriptional - based deconvolution algorithms.4 ima , considered hot or t cell - inamed , shows greater enrichment of cytotoxic t cells , inferred by deconvolutions tools or by histologic til counts , and accounts for approximately 30% of the tnbcs analyzed . 
finally , imc was described as warm tumors , because intrinsic immune activities are effectively mounted , but tumors are able to escape and suppress such responses , as indicated by the greater abundance of tregs and the lower expression of pdl - 1 and pd - 1 compared with ima . 
on the basis of our ndings , we would like to comment that besides the heterogeneity of tnbcs in terms of immune - cell inltration , the immune - rich subtype represents immune - related transcriptional heterogeneity , with signicant implications for potential immunotherapeutic strategies . consistent with the genomic characterization of immune - cell inltration obtained in our tnbc cohort , the incidence of immune - enriched ima in tcga and metabric data sets does not exceed 30% ( fig 1a ) , supporting the reproducibility of our immune clusters , especially for hot tumors . 
 ( a ) proportion of immuno - clusters ( im ) previously reported in romero et al4 with the ital - mex , molecular taxonomy of breast cancer international consortium ( metabric ) , and the cancer genome atlas ( tcga ) datasets . 
 ( b ) donut plot of the percentage of immuno - clusters among tumors classied as immune - rich or non - immune - rich from the tcga and metbric cohorts . 
by applying cluster analysis of our immune clusters to tcga and metabric data sets , we found that immune - rich tnbcs ( high til metagene described by omeara et al1 ) are composed mainly of ima ( n = 160 , 62% ) , followed by imc ( n = 54 , 21% ; fig 1b ) , with cases categorized as imb ( n = 44 , 17% ) also present . 
when considering the distribution of high til metagenes ( top 25th percentile ) applied by omeara et al1 in our cohort ( ital - mex , n = 158 ) and classied according to immune - related clusters , we conrmed that immune - rich cases ( ima ) are almost exclusively integrated by high til metagene tumors ( n = 41 , 94% )  . however , we also observed that a relevant percentage of high til cases are also distributed among imc ( n = 17 , 32% ) and less represented in the cold imb immune cluster ( n = 11 , 18% ; fig 1c )  . 
omeara t , marczyk m , qing t , et al : immunological differences between immune - rich estrogen receptor - positive and immune - rich triple - negative breast cancers . 
 a rationale and design of the targeted agent and profiling utilization registry study purpose case reports and small prospective trials suggest that administering targeted therapies to patients with advanced cancer and an identified genomic target may be associated with clinical benefit . 
the targeted agent and profiling utilization registry ( tapur ) study , a phase ii prospective , nonrandomized , multibasket pragmatic clinical trial , aims to identify signals of drug activity when us food and drug administration approved drugs are matched to prespecified genomic targets in patients with advanced cancer , outside of approved indications . methods patients eligible to participate in tapur are age 12 years and have advanced measurable or evaluable solid tumors , multiple myeloma , or b - cell non - hodgkin lymphoma . 
secondary end points include safety , progression - free survival , and overall survival . results more than 1 , 000 participants have thus far been registered , and more than 800 have been treated with a tapur study drug . 
two study cohorts have permanently closed to enrollment because of lack of antitumor activity , and 12 have expanded to the second stage of enrollment after promising preliminary activity . conclusion the tapur study will describe the efficacy and toxicity of the targeted drugs used outside of their approved indications when matched to a somatic genomic variant . evidence is building through reports of clinical trials , case reports , and clinical anecdotes to suggest that patient outcomes may be improved when a targeted agent is matched to a genomic alteration present in a patients tumor.1 - 6 clinical reports to date suggest that 30% to 80% of advanced solid tumors harbor potentially actionable genomic variants.7 - 10 in a meta - analysis of 570 phase ii studies of new anticancer agents , schwaederle et al11 examined response rate ( rr ) , progression - free survival ( pfs ) , and overall survival ( os ) for 32 , 149 patients who received a personalized treatment strategy versus those who did not . 
 of the md anderson cancer center experience of genomic profiling of patients with solid tumors with advanced disease , the impact ( initiative for molecular profiling and advanced cancer therapy ) study . 
the moscato - 01 ( molecular screening for cancer treatment optimization ; clinicaltrials.gov identifier : nct01566019 ) trial enrolled patients with treatment - resistant progressive metastatic cancers with lesions accessible to biopsy to perform genomic profiling.14 this study compared pfs using therapy based on genomic assessment with pfs for the most recent therapy during which the patient had experienced disease progression . 
le tourneau et al17 published the results of a randomized phase ii trial comparing therapy on the basis of tumor molecular profiling versus physician choice of therapy in patients with refractory cancer . 
this article will describe the rationale and design of the targeted agent and profiling utilization registry ( tapur ) study , a large pragmatic precision medicine basket trial with the overarching goal of describing the efficacy and toxicity of targeted anticancer drugs used outside of their approved indications for treatment of patients with advanced cancer based on a tumor genomic profile . methods objectives the primary objective of tapur is to evaluate the antitumor activity of commercially available targeted anticancer drugs used outside of their fda - approved indications for treatment of patients with advanced solid tumors , multiple myeloma , or b - cell non - hodgkin lymphoma with a genomic alteration known to be a drug target . 
secondary objectives include determination of pfs , os , and safety . design the tapur study is a phase ii prospective , nonrandomized , open - label clinical trial that aims to define signals of drug activity . 
list of available tapur study treatments for adults and pediatric patients study drug population to which available adults and children age 12 - 17 years adults only adults and children age 12 - 17 years adults and children age 16 - 17 years adults and children age 13 - 17 years adults and children age 12 - 17 years adults and children age 16 - 17 years adults only adults and children age 12 - 17 years adults and children age 12 - 17 years adults and children age 12 - 17 years nivolumab + ipilimumab adults only pembrolizumab adults and children age 12 - 17 years axitinib bosutinib cetuximab crizotinib dasatinib erlotinib olaparib palbociclib regorafenib sunitinib temsirolimus trastuzumab + pertuzumab adults only vemurafenib + cobimetinib adults only vismodegib adults and children age 12 - 17 years note . 
the study includes participants as young as 12 years of age with a tumor harboring a genomic alteration known to be a target of or to predict sensitivity to at least one of the 16 therapeutic options available in the study ( table 1 )  . 
eligible participants must have a tumor genomic variant identified on a test performed in a laboratory that has certification under the clinical laboratory improvement amendments and accreditation by the college of american pathologists . 
patients are matched to at least one of the available study treatments through a set of protocol - defined genomic matching rules or after review of a participant case by the tapur molecular tumor board ( mtb )  . 
the study uses simons optimal two - stage design for cohort analysis . tapur was designed independently by asco staff and volunteer leaders , with input from patient advocates , community - based investigators , the initial collaborating pharmaceutical companies , and the asco cancer research committee . 
the study was registered on clinicaltrials.gov ( nct02693535 ) before study launch . a data and safety monitoring board ( dsmb ) , consisting of a group of independent experts not involved in the conduct of tapur , meets biannually to monitor the study data and outcomes . 
 their primary functions include assessing the safety and efficacy of study treatments , safeguarding the interests and safety of trial participants , and ensuring that study results are both credible and reported to the medical community in a timely manner . 
for expanded cohorts , it will review all final cohort analyses and make recommendations regarding release of the cohort data . study population and recruitment the pragmatic approach of tapur allows for broad eligibility criteria , including eastern cooperative oncology group performance status of 0 to 2 , age 12 years , prior malignancies or positive hiv status , and previously treated but clinically stable brain metastases . 
tapur inclusion and exclusion criteria comport fully with recently published recommendations by asco and friends of cancer research for broadening eligibility criteria for cancer clinical trials.23 participating sites include both academic and community centers . study treatment decision once a potential participant is identified , the clinical site obtains informed consent and registers the participant in the tapur electronic data capture ( edc ) platform as shown in figure 1 . 
if a study drug is identified and the drug - specific inclusion and exclusion criteria are met , the clinical site completes the drug specific informed consent process and enrolls the participant in the study . 
 a screening consent general eligibility criteria met registration treatment 1 treatment 2 , continued tapur matching to study drug ( s ) based on genomic criteria ( or approved by mtb ) treatment decision by treating physician target variant 1 target variant 2 screening continued drug - specific eligibility criteria met tumor type 1 tumor type 2 tumor type 1 tumor type 2 enrolled and placed into cohorts enrollment cohort 1 cohort 2 cohort 3 cohort 4 fig 1 . 
the mtb , composed of clinical oncologists , molecular pathologists , and patient advocates , reviews the genomic test results , pathology reports , and clinical histories of submitted patient cases and identifies potential treatment matches among the study treatments . 
of the patient cases reviewed by the mtb , approximately 65% resulted in identification of a study drug option . permitted at the discretion of the treating physician in accordance with the recommended dose modifications contained in the approved prescribing information . 
management of aes is performed according to institutional standards of care , informed by the package insert . tumor measurements and radiologic evaluations and evaluation of clinical disease status are performed every 8 weeks after initiation of treatment for the first 16 weeks , and then every 12 weeks if the treatment continues , and at the end of study treatment , if possible . 
study treatment is continued until progressive disease is documented . after progression of disease during any study treatment , participants may be reassessed to determine eligibility for treatment with another study drug after a minimum of 30 days has elapsed from the last dose of the previous drug and any aes have resolved to grade 2 or stabilized . 
all general and drug - specific eligibility criteria must be reconfirmed , and the participant must sign a new drug - specific consent form . treatment schedule and interval of evaluations ae and sae criteria study treatments are administered according to the recommended starting dose and schedule described in the package insert of each drug . 
events not resolved at the end of study treatment require follow - up every 30 days until the event resolves to ctcae grade 2 . tapur requires reporting of ctcae grade 3 to 4 aes that are possibly , probably , or definitely related to the study treatment , whether expected or not . 
all saes , regardless of grade , relatedness to study drug , or expectedness , must also be reported , including all deaths . end points the primary end point is objective response at 8 weeks or sd documented at 16 weeks . 
tapur study milestones date milestone march 14 , 2016 study launch at 37 clinical site locations with 14 study drugs first participant registered ( consented ) first participant enrolled ; two additional drugs added for total of 16 study drugs additional drug added for total of 17 study drugs 100th participant registered november 2016 100th participant enrolled march 2016 april 2016 may 2016 august 2016 april 2017 june 2017 august 2017 september 2017 november 2017 december 2017 february 2018 march 2018 33 clinical sites added for total of 70 locations 500th participant registered additional therapy added for 19 study drugs first cohort reaches stage one 500th participant enrolled 1 , 000th participant registered 10th cohort reaches stage one 43 clinical sites added for total of 113 locations abbreviation : tapur , targeted agent and profiling utilization registry . the basis of the presence , absence , or unequivocal progression of the lesions . 
participants who have no tumor evaluation beyond baseline and are alive with no signs of progressive disease at the time of leaving the study will be replaced in their cohort with another participant . secondary end points include grade 3 to 5 aes and saes , pfs , response duration , and os . 
the null hypothesis is that the probability of objective response or sd of at least 16 weeks duration is 15% , versus the alternative hypothesis that it is at least 35% . 
after 28 participants in the cohort have been observed for the primary outcome , if seven or more participants achieve objective response or sd of at least 16 weeks duration , the null hypothesis will be rejected , and we will declare that the study drug is active in the cohort of participants defined by tumor type and genomic alteration . 
this design has a 54% chance of early stopping if the null hypothesis is true . estimates of the rr and 95% ci will be presented for each cohort upon closure ( at either stage one or stage two ) .24 the kaplan - meier method will be used to estimate the duration of response , pfs , and os distributions . results table 3 summarizes key study milestones . 
figure 2 also shows the distribution of genomic targets by tumor type ( fig 2b ) and study drug ( fig 2c ) , highlighting the heterogeneity of genomic targets and tumor types , resulting in 336 unique cohorts at the time of this writing . 
targeted agent and profiling utilization registry ( tapur ) study registration , enrollment , and cohort creation by ( a ) month , ( continued on next page ) many cancers that are driven by specific molecular alterations that can be targeted with drugs that inhibit aberrant signaling pathways in tumor cells . 
thus , the targeting of bcr - abl alterations in chronic myeloid leukemia , flt3 alterations in acute myeloid leukemia , egfr mutations and alk translocations in nonsmallcell lung cancer , braf mutations in melanoma and nonsmall - cell lung cancer , and human epidermal growth factor receptor 2 overexpression in breast and gastric cancers , among others , represent examples of the successful application of precision medicine approaches in clinical oncology that have extended the lives of many patients.1 , 25 - 30 interest in precision medicine has been further fueled by reported responses ranging from modest to exceptional when targeted treatments are selected based on large - scale genomic tumor profiling.5 the widespread availability of commercial next generation sequencing tests presents opportunities for patients who have exhausted standard treatment options to pursue treatments with investigational agents or approved drugs prescribed outside of their labeled indications . the tapur study stemmed from the recognition that the rapid dissemination of genomic profiling provides an opportunity to learn from the application of precision cancer medicine in practice while at the same time providing a framework for clinical decision support for clinical oncologists who are struggling to interpret the complex genomic data they now confront in practice . 
 b vegfr3 ( flt4 ) mutation , amplification , or overexpression vegfr1 ( flt1 ) mutation , amplification , or overexpression smo mutation lck mutation prkdc mutation pold1 mutation msh2 mutation vegfr3 ( flt4 ) mutation or amplification ewsr1nrya3 fusion cdk12 mutation ccnd3 bcr - abl mutation alk fusion or mutation msi high status egfr mutation rictor amplification vegfr1 ( flt1 ) mutation or amplification csf1r mutation or amplification vegfr2 ( kdr ) mutation , amplification , or overexpression vhl mutation or amplification ptch1 deletion or inactivating mutation mlh1 mutation tsc2 mutation pdgfra mutation or amplification palb2 mutation stk11 mutation vegfr2 ( kdr ) mutation or amplification ret mutation or amplification pdgfr ? mutation or amplification kit mutation atm mutation tsc1 mutation pole mutation fgfr3 mutation or amplification cdk6 amplification raf1 mutation or amplification pten mutation kit mutation or amplification braf mutation or amplification akt1 mutation flt3 mutation or amplification brca1 / brca2 mutation met amplification or mutation pik3ca mutation mtor mutation fgfr2 mutation or amplification ccnd1 amplification fgfr1 mutation or amplification cdk4 amplification atm mutation or deletion braf_v600e / d / k / r mutation erbb2 / erbb3 mutation , amplification , or overexpression kras , nras , and braf wild type ( all must be wild type ) germline or somatic brca1 / brca2 inactivating mutations high tumor mutational burden cdkn2a loss or mutation total fig 2 . 
 ( a ) april 2018 registration , enrollment , and cohort creation in progress at the time of this report ; ( b , c ) the number in each box indicates the number of patients . 
 c vegfr3 ( flt4 ) mutation , amplification , or overexpression vegfr1 ( flt1 ) mutation , amplification , or overexpression smo mutation lck mutation prkdc mutation pold1 mutation msh2 mutation vegfr3 ( flt4 ) mutation or amplification ewsr1nrya3 fusion cdk12 mutation ccnd3 bcr - abl mutation alk fusion or mutation msi high status egfr mutation rictor amplification vegfr1 ( flt1 ) mutation or amplification csf1r mutation or amplification vegfr2 ( kdr ) mutation , amplification , or overexpression vhl mutation or amplification ptch1 deletion or inactivating mutation mlh1 mutation tsc2 mutation pdgfra mutation or amplification palb2 mutation stk11 mutation vegfr2 ( kdr ) mutation or amplification ret mutation or amplification pdgfr ? mutation or amplification kit mutation atm mutation tsc1 mutation pole mutation fgfr3 mutation or amplification cdk6 amplification raf1 mutation or amplification pten mutation kit mutation or amplification braf mutation or amplification akt1 mutation flt3 mutation or amplification brca1 / brca2 mutation met amplification or mutation pik3ca mutation mtor mutation fgfr2 mutation or amplification ccnd1 amplification fgfr1 mutation or amplification cdk4 amplification atm mutation or deletion braf_v600e / d / k / r mutation erbb2 / erbb3 mutation , amplification , or overexpression kras , nras and braf wild type ( all must be wild type ) germline or somatic brca1 / brca2 inactivating mutations high tumor mutational burden cdkn2a loss or mutation total fig 2 . 
 ( continued ) ( c ) ibrance ( pfizer , new york , ny ) , lynparza ( astrazeneca , wilmington , de ) , sutent ( pfizer ) , keytruda ( merck , kenilworth , nj ) , torisel ( pfizer ) , erbitux ( eli lilly , indianapolis , in ) , perjeta ( genentech , south san francisco , ca ) , herceptin ( genentech ) , cotellic ( genentech ) , zelboraf ( genentech ) , opdivo ( bristol - myers squibb , new york , ny ) , yervoy ( bristol - myers squibb ) , stivarga ( bayer , whippany , nj ) , xalkori ( pfizer ) , sprycel ( bristol - myers squibb ) , erivedge ( genentech ) , inlyta ( pfizer ) , bosulif ( pfizer ) , and tarceva ( genentech )  . 
tapur study initial cohort expansions and closures study drug tumor type variant ovarian cancer kras , nras , and braf wild type colorectal cancer braf_v600e / d / k / r mutation breast cancer colorectal cancer germline or somatic brca1 / brca2 inactivating mutations atm mutation or deletion head and neck cancer soft tissue sarcoma malignant neoplasm of bronchus and lung cdkn2a loss or mutation cdk4 amplification cdkn2a loss or mutation breast cancer colorectal cancer uterine cancer high tumor mutational burden high tumor mutational burden high tumor mutational burden colorectal cancer erbb2 / erbb3 mutation , amplification , or overexpression expansion to stage two cetuximab cobimetinib + vemurafenib olaparib palbociclib pembrolizumab pertuzumab + trastuzumab sunitinib closed at stage one palbociclib breast cancer fgfr1 mutation or amplification pancreatic cancer malignant neoplasm of gallbladder and bile ducts cdkn2a loss or mutation abbreviation : tapur , targeted agent and profiling utilization registry . data collection , and discretion left to the treating physician regarding choice of which tumor biospecimen to interrogate and which genomic profiling test to use . 
however , tapur uses standard treatment response and toxicity criteria for assessment of efficacy and toxicity outcomes , structured data collection , protocol - specified evaluation times , and a standard simons twostage statistical design to assess the primary end point as well as an independent dsmb to provide recommendations on cohort expansion and closure . 
importantly , the study also provides educational opportunities and clinical decision support tools to treating physicians in the form of protocol - specified genomic matching rules and access to an mtb to assist in interpretation of genomic test reports . although study results are not yet available , preliminary information about the status of certain cohorts suggests that the study can provide meaningful information . 
 enrollment rates when the frequency of genomic targets was not well defined for many tumor types , identifying clinical launch sites that routinely used genomic profiling , arranging for drug distribution , and building an experienced clinical trial management team to operate the study . 
mangat no relationship to disclose susan halabi consulting or advisory role : tokai pharmaceuticals consulting or advisory role : eisai , bayer healthcare pharmaceuticals travel , accommodations , expenses : bayer healthcare pharmaceuticals suanna s . 
bruinooge no relationship to disclose elizabeth garrett - mayer stock and other ownership interests : abbott laboratories , abbvie consulting or advisory role : tactical therapeutics , okava pharmaceuticals ajjai alva consulting or advisory role : eisai , astrazeneca , genentech / roche speakers bureau : astrazeneca research funding : genentech ( inst ) , novartis ( inst ) , bristol - myers squibb ( inst ) , bind biosciences ( inst ) , acerta pharma ( inst ) , merck sharp & dohme ( inst ) , prometheus laboratories ( inst ) , covance ( inst ) , mirati therapeutics ( inst ) , united biosource ( inst ) , ariad pharmaceuticals ( inst ) , astrazeneca ( inst ) , pfizer ( inst ) , genentech / roche ( inst ) , hoosier cancer research network ( inst ) , bayer healthcare pharmaceuticals ( inst ) katherine a . 
stella no relationship to disclose emile voest consulting or advisory role : interna , biogeneration ventures research funding : novartis ( inst ) , glaxosmithkline ( inst ) , genentech / roche ( inst ) , pfizer ( inst ) , astrazeneca ( inst ) , eisai ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) kathleen j . 
kim honoraria : celgene , eli lilly , astrazeneca , boehringer ingelheim consulting or advisory role : eli lilly , celgene , astrazeneca , boehringer ingelheim travel , accommodations , expenses : eli lilly , celgene , astrazeneca , boehringer ingelheim richard l . 
kim , carolinas healthcare systems levine cancer institute , charlotte , nc ; ajjai alva , university of michigan ; jane perlmutter , gemini group , ann arbor ; philip j . 
janeway , data - farber / boston childrens cancer and blood disorders center , boston , ma ; emile voest , netherlands cancer institute , amsterdam , the netherlands ; and navin pinto , seattle childrens hospital , seattle , wa . 
wagle n , grabiner bc , van allen em , et al : activating mtor mutations in a patient with an extraordinary response on a phase i trial of everolimus and pazopanib . 
cobain ef , robinson dr , wu y - m , et al : clinical impact of high - throughput sequencing in patients with advanced cancer : lessons learned from the michigan oncology sequencing center . 
jardim dl , schwaederle m , wei c , et al : impact of a biomarker - based strategy on oncology drug development : a meta - analysis of clinical trials leading to fda approval . 
tsimberidou am , wen s , hong ds , et al : personalized medicine for patients with advanced cancer in the phase i program at md anderson : validation and landmark analyses . 
massard c , michiels s , fert c , et al : high - throughput genomics and clinical outcome in hardto - treat advanced cancers : results of the moscato 01 trial . 
haslem ds , van norman sb , fulde g , et al : a retrospective analysis of precision medicine outcomes in patients with advanced cancer reveals improved progression - free survival without increased health care costs . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , openlabel , proof - of - concept , randomised , controlled phase 2 trial . 
cheson bd , fisher ri , barrington sf , et al : recommendations for initial evaluation , staging , and response assessment of hodgkin and non - hodgkin lymphoma : the lugano classification . 
barrington sf , mikhaeel ng , kostakoglu l , et al : role of imaging in the staging and response assessment of lymphoma : consensus of the international conference on malignant lymphomas imaging working group . 
kim es , bruinooge ss , roberts s , et al : broadening eligibility criteria to make clinical trials more representative : american society of clinical oncology and friends of cancer research joint research statement . 
druker bj , talpaz m , resta dj , et al : efficacy and safety of a specific inhibitor of the bcr - abl tyrosine kinase in chronic myeloid leukemia . 
slamon dj , leyland - jones b , shak s , et al : use of chemotherapy plus a monoclonal antibody against her2 for metastatic breast cancer that overexpresses her2 . 
bang yj , van cutsem e , feyereislova a , et al : trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of her2 - positive advanced gastric or gastrooesophageal junction cancer ( toga ) : a phase 3 , open - label , randomised controlled trial . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors on the basis of molecular profiles : results from mypathway , an open - label , phase iia multiple basket study . 
butler , mph ; kuo guo , ms ; molly holoubek , ccrn ; samiha islam ; linda miller , msn , rn , ocn ; shamika ranasinghe , ms ; brittany m . 
 drug - related pneumonitis in the era of precision cancer therapy drug - related pneumonitis as a result of novel cancer therapy provides new challenges for providers of cancer care in the era of precision medicine . 
here , we provide a detailed review of drug - related pneumonitis that develops during precision cancer therapies using immune - checkpoint inhibitors and molecular targeting agents , and we summarize the emerging data that have been obtained by recent investigations to provide a state - of - the - art overview for clinicians involved in cancer care . we focus on immune - checkpoint inhibitorrelated pneumonitis , which is an immune - related adverse event of growing interest and increasing clinical significance in current oncology practice that has rapidly expanding access to these agents . 
clinical characteristics , radiographic spectrum , and risk factors and outcome of pneumonitis are described for each class of agents , and current treatment guidelines and monitoring recommendations are discussed . 
with recent advances in precision cancer therapy using molecular targeting agents and immune - checkpoint inhibitors ( icis ) , pneumonitis in patients who are treated with novel anticancer agents is increasingly recognized as a significant clinical challenge . 
in addition , rapidly expanding access to icis , such as programmed death 1 ( pd - 1 ) and programmed death - ligand 1 ( pd - l1 ) inhibitors has brought new and emerging challenges . 
recent studies of pneumonitis in patients who are treated with these novel agents have provided important new knowledge and insight that are relevant to cancer care providers across disciplines . this review focuses on drug - related pneumonitis during precision cancer therapies , including icis and molecular targeting agents , and provides an overview for clinicians who are involved in the care of patients with cancer . 
the spectrum of radiographic manifestations of pneumonitis is presented in the context of patient management . we also provide up - to - date guidelines and recommendations for the treatment and monitoring of pneumonitis during these therapies . 
 been made to characterize the radiographic patterns of pneumonitis caused by various therapeutic agents.1 , 2 several recent reports have indicated that the radiographic patterns of drug - related pneumonitis may be categorized according to patterns that correspond to american thoracic society / european respiratory society ( ats / ers ) classifications of idiopathic interstitial pneumonias and related disorders , 3 which are based on ct findings and their extent and distributions on imaging.2 , 4 - 8 in these studies of pneumonitis that developed during treatment with molecular targeting agents and icis , the commonly noted radiographic patterns of pneumonitis included cryptogenic organizing pneumonia ( cop ) pattern , nonspecific interstitial pneumonia ( nsip ) pattern , hypersensitivity pneumonitis ( hp ) pattern , and acute interstitial pneumonia ( aip ) / acute respiratory distress syndrome ( ards ) pattern2 , 4 - 10 ( table 1 )  . 
radiographic patterns of pneumonitis have been shown to correlate with the toxicity grades assessed by the common terminology criteria for adverse events.5 recent advances in precision cancer therapy have brought the new and emerging challenges of pneumonitis as a result of treatment with novel agents . 
regulatory approvals for these agents have recently been granted , including nivolumab for melanoma , nonsmall - cell lung cancer ( nsclc ) , renal cell carcinoma ( rcc ) , and hodgkin lymphoma ; pembrolizumab for melanoma , nsclc , hodgkin lymphoma , and squamous cell head and neck cancer ; and atezolizumab for urothelial carcinoma and nsclc . 
combination therapy of nivolumab and ipilimumab , a cytotoxic t - lymphocyte - associated protein 4 inhibitor , has also been approved for treatment of advanced melanoma and is actively investigated in nsclc and other malignancies . as a result , these agents are rapidly expanding their treatment roles in clinical oncology practice , and more agents are in the drug development and testing pipeline . immune - checkpoint inhibition of these agents is associated with a unique set of toxicities that are known as immune - related adverseevents ( iraes ) .11 - 14 among a variety of iraes that can affect organs from head to toe , ici - related pneumonitis is a clinically significant and potentially life - threatening irae and is therefore recognized as an event of special interest . phase i trials of pd - 1 inhibitors have resulted in pneumonitis - related deaths in patients with advanced solid tumors , including nsclc , melanoma , and colorectal cancer.15 - 18 in a report of long - term safety in a nsclc cohort that was treated in a phase i trial , pneumonitis occurred in 7% ( 9 of 129 patients ) , with three pneumonitisrelated deaths.15 in a phase ii trial of nivolumab in squamous nsclc , pneumonitis was one of the most common iraes , occurring in 5% of patients ( 6 of 117 patients ) , including four patients with grade 3 pneumonitis.19 more recent data suggest that pneumonitis can be an even more significant issue in patients who are treated with combination therapies . 
radiographic pattern was associated with clinical severity of pneumonitis represented by toxicity grades , where aip / ards pattern had the highest grade , followed by cop pattern , whereas nsip pattern and hp pattern had lower grade ( median grade : 3 , 2 , 1 , 1 ) .5 results indicate that the radiographic patternbased approach , in addition to early and accurate diagnosis , may help guide patient management and follow - up in the setting of pneumonitis . 
another recent study of pd - 1 / pd - l1 inhibitor - related pneumonitis has also reported the diverse radiographic and pathologic features of the entity , 25 further indicating the importance of a standardized common language to describe the findings of ici - related pneumonitis.10 treatment , clinical course , and management guidelines treatment of ici - related pneumonitis consists mainly of holding back on the use icis and administering corticosteroids ; however , some cases may be refractory to corticosteroids and require additional treatment . 
 ( d ) aip / ards pattern is characterized by diffuse or multifocal ggos or consolidations , along with lung volume loss and traction bronchiectasis . ( n = 12 ) or as intravenous therapy ( n = 5 )  . 
although the cause of death was solely attributed to pneumonitis in one patient alone , the serious outcome in 12% ( 5 of 43 ) of patients again emphasizes the clinical significance of the entity.25 twelve patients were retreated with icis after complete clinical resolution of initial pneumonitis , and three patients experienced recurrent pneumonitis , which again resolved by drug holding ( n = 1 ) or oral corticosteroids ( n = 2 ) as in the course of their initial pneumonitis treatment.25 a recently published guideline of pneumonitis management is in agreement with the observations in these reports.31 the guideline recommends oral corticosteroid treatment , including prednisone 1 to 2 mg / kg / d or methylprednisolone 0.5 to 1 mg / kg / d in mild to moderate cases . 
in mild to moderate cases , bronchoscopy is recommended to exclude infectious etiologies before starting immunosuppression.31 in severe cases , hospitalization is necessary , with treatment to include high - dose corticosteroids , such as methylprednisolone 2 to 4 mg / kg / d , and additional immunosuppression , including mycophenolate mofetil , cyclophosphamide ; infliximab can be administered if necessary.31 although limited to a small percentage of patients , an additional unique phenomenon of pneumonitis flare has been reported where ici - related pneumonitis recurs after the termination of corticosteroid taper and in the absence of ici retreatment after the initial episode of pneumonitis has been successfully treated ( fig 2 )  . 
this was first reported in a patient with nsclc who was treated with commercial nivolumab and who experienced pneumonitis.6 the patient was successfully treated with corticosteroids for the initial pneumonitis ; however , the patient experienced another episode of pneumonitis after completing corticosteroid taper without resuming pd - 1 inhibitor therapy or starting any other therapy , indicating a pneumonitis flare.6 although similar to the initial presentation both clinically and radiographically , flare pneumonitis tends to be more severe and extensive than the initial episode . 
such a phenomenon has not been described in the setting of pneumonitis related to other anticancer agents and this that indicates the clinical course of icirelated pneumonitis may be more complex than other drug - related pneumonitis in some patients . in a series of 20 patients with ici - related pneumonitis , one patient with lymphoma experienced two episodes of pneumonitis flare in which pneumonitis came back with similar but more extensive radiographic presentations compared with the initial episode after completion of corticosteroid taper without pd - 1 retreatment5 ( fig 2 )  . 
this observation further confirms the phenomenon , which is likely unique to iraes and may involve autoimmune mechanisms . unmet clinical needs that require additional investigations although several recent reports provide important knowledge and observations of ici - related pneumonitis , significant knowledge gaps still exist for this emerging entity . 
because early and accurate diagnosis is key for this mostly treatable conditionwith good response to corticosteroids in the majority of the casesadditional studies are needed to identify risk factors and early markers for pneumonitis to improve diagnostic accuracy . treatment and clinical management guidelines need to be further optimized as the immuneoncology community accumulates the experience of this entity . 
pneumonitis with a cryptogenic organizing pneumonia ( cop ) pattern in a 33 - year - old female with hodgkin lymphoma who was treated with nivolumab and ipilimumab combination therapy , with a recurrence during retreatment ( 2a ; ah ) and two episodes of pneumonitis flare after completion of corticosteroid taper ( 2b ; ah )  . 
reprinted with permission from nishino et al.5 2a : ( a and b ) computed tomography ( ct ) scan of the chest at 1.4 months of therapy demonstrated ground - glass and reticular opacities and consolidations with multifocal distribution , which are indicative of a cop pattern of pneumonitis ( arrowheads )  . 
 ( 2b : a and b ) the patient completed 2 months of corticosteroid taper , and after 1 month , experienced another episode of pneumonitis with a similar radiographic pattern , without nivolumab retreatment or other systemic therapy , which indicated a pneumonitis flare . 
 ( 2b : e and f ) the 2.7 - month course of corticosteroid taper was completed , and after 2 weeks , the patient again developed a pneumonitis flare with a similar radiographic pattern as prior episodes . 
findings included lymphocyte - predominant interstitial pneumonitis ( arrowhead , 2b [ g ] , hematoxylin and eosin stain , 3200 ) with rare eosinophils ( arrow , 2b [ g ] ) , and areas of organizing pneumonia with fibroblast plugs and foamy macrophages filling the airspaces ( asterisks , 2b [ h ] , hematoxylin and eosin stain , 3200 )  . 
the patient started another course of prednisone taper with subsequent clinical improvement and is schedule for a follow - up ct scan . in addition , awareness of pneumonitis related to icis other than pd - 1 / pd - l1 inhibitors is becoming increasingly important . 
everolimus is approved for the treatment of several cancers , including advanced rcc , advanced pancreatic neuroendocrine tumors ( nets ) , subependymal giant cell astrocytomas , and advanced hormone receptorpositive , human epireceptor 2negative dermal growth factor breast cancer ( in combination with exemestane )  . 
temsirolimus is approved for the treatment of advanced rcc . drug - related pneumonitis is a recognized class effect toxicity of mtor inhibitors and has been described in detail in the context of advanced rcc treatment . 
in a study by maroto et al , 34 among 178 patients with rcc who were treated with singleagent temsirolimus , 52 ( 29% ) had radiographically identified drug - related pneumonitis . 
of these , 16 patients ( 31% ) had respiratory symptoms at the time of the onset of radiographically detected pneumonitis , whereas others ( 36 of 52 ; 69% ) were asymptomatic and thus may not be clinically diagnosed for pneumonitis . 
the estimated cumulative probability of radiologically identified drug - related pneumonitis was 21% ( 95% ci , 15% to 29% ) at 8 weeks , 31% ( 95% ci , 24% to 40% ) at 16 weeks , and 45% ( 95% ci , 36% to 57% ) at 13 months.34 another study of 46 patients with rcc who were treated with temsirolimus or everolimus reported that 14 patients ( 30% ) developed drug - related pneumonitis.35 median time of the onset of radiologic manifestations of pneumonitis was 56 days ( range , 31 to 214 days )  . 
these 14 patients more frequently achieved stable disease than did others , which indicates a possibility of pneumonitis as a treatment benefit marker.35 reports of mtor inhibitorrelated pneumonitis in tumors other than rcc indicate a somewhat different incidence of pneumonitis according to tumor types . 
radiographic patterns of mtor inhibitorrelated pneumonitis have been characterized in patients with wm and in those with advanced net.7 , 8 in both cohorts , the most common findings were bilateral ground - glass opacities ( ggos ) and reticular opacities , sometimes with consolidations , in a peripheral and lower distribution . 
cop pattern was the leading pattern observed in the majority of cases ( 70% in wm and 57% in net ) , followed by nsip pattern ( 30% in wm and 36% in net ) , whereas one patient in the net cohort demonstrated the hp pattern.7 , 8 similar imaging manifestations are noted in other cohorts with mtor inhibitor related pneumonitis.34 , 35 recognition of the characteristic radiographic patterns of pneumonitis during mtor inhibitor therapy helps with the early detection and accurate diagnosis of this entity . clinical management of mtor inhibitorrelated pneumonitis mostly depends on the severity of clinical symptoms . 
egfr - tki therapy should be discontinued , and systemic corticosteroids are usually administered.49 although egfr - tkirelated pneumonitis has mostly been a specific concern among the japanese population and has often had a low clinical impact in other populations , a recent observation from a trial of a novel ici and third - generation egfr - tki , osimertinib , raises a new concern in the setting of combination therapy . 
 agents may limit , in part , the destructive remodeling of the lung and impair the lungs ability to respond to injury.37 , 49 , 52 these possible mechanisms are also likely modified by various host and environmental factors , which further complicates the issue.52 likewise , risk factors for pneumonitis need to be defined , especially for ici - related pneumonitis , to allow for optimal patient selection with regard to treatment safety . 
the risk of recurrent pneumonitis after reintroduction of therapy is another important debate for these agents , as they may be the only available or effective therapy for patients with advanced cancers.5 , 37 the impact of radiotherapy on pneumonitis is a major topic , given a number of ongoing combination therapy trials that use ici and radiotherapy.53 in some studies of patients with advanced nsclc who were treated with egfr - tkis , prior thoracic irradiation and concomitant radiotherapy have been noted as risk factors for pneumonitis.49 , 54 although the exact effect of radiotherapy on ici - related pneumonitis awaits additional data , prior chest radiation has influenced the radiographic appearance of pd - 1 pneumonitis in patients with lymphoma , which indicates a need for caution in treatment monitoring.30 prior reports have often focused on radiation to the chest ; however , radiotherapy in the extrathoracic sites may require more attention given the concept of abscopal effects , which are increasingly discussed in the context of ici and radiation treatment.53 in the clinical setting , drug - related pneumonitis is ultimately a diagnosis of clinical correlation and exclusion , and there is no specific test for the entity.55 among various lung conditions that can affect patients with advanced cancer who undergo systemic therapy , infection is an important differential diagnosis that should be excluded before starting corticosteroid therapy . 
fiberoptic bronchoscopy is helpful for this purpose as it allows for targeted and deep sampling of specimens for microbiology , cytology , and histology via bronchoalveolar lavage and transbronchial biopsy.55 a minority of patients may present with respiratory distress in the absence of radiographic confirmation , thereby leading to delays in appropriate management . 
of note , some of the findings of pneumonitis , such as ggos , are not adequately evaluated on chest radiographs , and thus diagnostic chest ct scans are needed to sensitively detect and fully characterize the lung abnormalities in the setting of suspected pneumonitis . in conclusion , knowledge of drug - related pneumonitis has increasingly emerged in recent investigations , providing important clues for accurate diagnosis , patient management , and monitoring . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . mizuki nishino honoraria : bayer yakuhin consulting or advisory role : bristol - myers squibb , worldcare clinical , toshiba medical systems research funding : merck ( inst ) drug - related pneumonitis in the era of precision cancer therapy the following represents disclosure information provided by authors of this manuscript . 
stephen hodi employment : dana - farber cancer institute consulting or advisory role : merck sharp & dohme , novartis , genentech , amgen , emd serono research funding : bristol - myers squibb ( inst ) , merck sharp & dohme ( inst ) , genentech ( inst ) , novartis ( inst ) patents , royalties , other intellectual property : patent pending as per institutional policy , patent pending royalties received on mica - related disorders application to institution per institutional policy travel , accommodations , expenses : novartis , bristol - myers squibb other relationship : bristol - myers squibb , genentech nikhil h . 
travis wd , costabel u , hansell dm , et al : an official american thoracic society / european respiratory society statement : update of the international multidisciplinary classification of the idiopathic interstitial pneumonias . 
nishino m , chambers es , chong cr , et al : anti - pd - 1 inhibitor - related pneumonitis in nonsmall - cell lung cancer . cancer immunol res 4 : 289 - 293 , 2016 7 . 
nishino m , brais lk , brooks nv , et al : drug - related pneumonitis during mammalian target of rapamycin inhibitor therapy in patients with neuroendocrine tumors : a radiographic pattern - based approach . 
nishino m , boswell en , hatabu h , et al : drug - related pneumonitis during mammalian target of rapamycin inhibitor therapy : radiographic pattern - based approach in waldenstr om macroglobulinemia as a paradigoncologist 20 : 1077 - 1083 , 2015 9 . 
nishino m , tirumani sh , ramaiya nh , et al : cancer immunotherapy and immune - related response assessment : the role of radiologists in the new arena of cancer treatment . 
weber js , dummer r , de pril v , et al : patterns of onset and resolution of immune - related adverse events of special interest with ipilimumab : detailed safety analysis from a phase 3 trial in patients with advanced melanoma . 
gettinger sn , horn l , gandhi l , et al : overall survival and long - term safety of nivolumab ( anti - programmed death 1 antibody , bms - 936558 , ono - 4538 ) in patients with previously treated advanced nonsmall - cell lung cancer . 
rizvi na , mazi`eres j , planchard d , et al : activity and safety of nivolumab , an anti - pd - 1 immune checkpoint inhibitor , for patients with advanced , refractory squamous nonsmall - cell lung cancer ( checkmate 063 ) : a phase 2 , single - arm trial . 
rizvi na , hellmann md , brahmer jr , et al : nivolumab in combination with platinum - based doublet chemotherapy for first - line treatment of advanced nonsmall - cell lung cancer . 
nishino m , giobbie - hurder a , hatabu h , et al : incidence of programmed cell death 1 inhibitor - related pneumonitis in patients with advanced cancer : a systematic review and meta - analysis . 
fehrenbacher l , spira a , ballinger m , et al : atezolizumab versus docetaxel for patients with previously treated nonsmall - cell lung cancer ( poplar ) : a multicentre , open - label , phase 2 randomised controlled trial . 
rittmeyer a , barlesi f , waterkamp d , et al : atezolizumab versus docetaxel in patients with previously treated nonsmall - cell lung cancer ( oak ) : a phase 3 , open - label , multicentre randomised controlled trial . 
antonia s , goldberg sb , balmanoukian a , et al : safety and antitumour activity of durvalumab plus tremelimumab in nonsmall cell lung cancer : a multicentre , phase 1b study . 
ghobrial im , gertz m , laplant b , et al : phase ii trial of the oral mammalian target of rapamycin inhibitor everolimus in relapsed or refractory waldenstr om macroglobulinemia . 
dabydeen da , jagannathan jp , ramaiya n , et al : pneumonitis associated with mtor inhibitors therapy in patients with metastatic renal cell carcinoma : incidence , radiographic findings and correlation with clinical outcome . 
soria jc , shepherd fa , douillard jy , et al : efficacy of everolimus ( rad001 ) in patients with advanced nsclc previously treated with chemotherapy alone or with chemotherapy and egfr inhibitors . 
janne pa , gurubhagavatula s , yeap by , et al : outcomes of patients with advanced nonsmall cell lung cancer treated with gefitinib ( zd1839 , iressa ) on an expanded access study . 
lynch tj , bell dw , sordella r , et al : activating mutations in the epidermal growth factor receptor underlying responsiveness of nonsmall - cell lung cancer to gefitinib . 
burotto m , manasanch ee , wilkerson j , et al : gefitinib and erlotinib in metastatic nonsmall cell lung cancer : a metaanalysis of toxicity and efficacy of randomized clinical trials . 
kudoh s , kato h , nishiwaki y , et al : interstitial lung disease in japanese patients with lung cancer : a cohort and nested case - control study . 
gemma a , kudoh s , ando m , et al : final safety and efficacy of erlotinib in the phase 4 polarstar surveillance study of 10 708 japanese patients with nonsmall - cell lung cancer . 
ding pn , lord sj , gebski v , et al : risk of treatment - related toxicities from egfr tyrosine kinase inhibitors : a metaanalysis of clinical trials of gefitinib , erlotinib , and afatinib in advanced egfr - mutated nonsmall cell lung cancer . j thorac oncol 12 : 633 - 643 , 2017 49 . 
min jh , lee hy , lim h , et al : drug - induced interstitial lung disease in tyrosine kinase inhibitor therapy for nonsmall cell lung cancer : a review on current insight . 
ahn m , yang j , yu h , et al : osimertinib combined with durvalumab in egfr - mutant nonsmall cell lung cancer : results from the tatton phase ib trial . 
hotta k , kiura k , tabata m , et al : interstitial lung disease in japanese patients with nonsmall cell lung cancer receiving gefitinib : an analysis of risk factors and treatment outcomes in okayama lung cancer study group . 
we completely agree with the authors that better ways of selecting patients with melanoma for snb are required , because approximately 80% of patients who currently have an snb are found to be sn negative . 
improved methods of identifying those at low risk of being sn positive are needed so that they are not unnecessarily subjected to the procedure , while those at higher risk are not excluded . bellomo et al1 propose that using their gene expression prole ( gep ) test will achieve greater accuracy , but we are not convinced that this is correct . 
first , we have several concerns about the statistical methodology used in their study to support the claim that a combination of two clinicopathologic ( cp ) features and an eight - gene test ( a cp - gep model ) improves the accuracy of selecting patients for snb . and second , we have evidence that prognostic estimates of sn status equivalent to or better than those provided by the cp - gep model of bellomo et al1 can be achieved using readily available cp parameters only . one of our statistical concerns is that the study had a relatively small sample size of patients who underwent snb ( n = 754 ) and only 128 were sn positive . the small sample size used for model development may explain why there was no apparent improvement in performance when more complex cp models were used compared with the use of breslow thickness and age only . another statistical concern is that the model reported by bellomo et al1 was assessed only by internal crossvalidation , and external validation was not performed . this is important , because there is a high probability that external validation using an unrelated data set will demonstrate a reduced discrimination ( c statistic ) compared with the development set because models tend to overt the data set on which they were developed.2 the apparent gain in discrimination achieved by the cp - gep model relative to the memorial sloan kettering cancer center ( mskcc ) nomogram ( from 77% to 82% ) is therefore likely to be is externally validated . reduced when the model furthermore , the c statistics had considerable overlap , and there is no evidence that there was a statistically signicant improvement . the 95% cis of in addition , the reported snb reduction rate reported by bellomo et al1 is based upon a negative predictive value ( npv ) threshold of 95% . 
however , the npv of any test decreases as the prevalence of the condition increases ; thus , in practice , it cannot be xed.3 therefore , it would be more informative for both clinicians and patients to indicate the rate reductions of snb with individual risk thresholds rather than the npv . the above statistical concerns , regardless of question of whether the gep test improved prognostic accuracy remains . 
it should be noted that the c statistic obtained by using the mskcc nomogram was considerably higher in the report by bellomo et al1 than in both our study and the original mskcc study , 5 which probably reects selection bias in their study sample that excluded thick tumors . 
the 5% absolute improvement in the c statistic reported in their study combining cp parameters ( two only ) and molecular risk factors ( an eight - gene test ) is less than the 6.2% achieved by our new model , which used cp parameters only . for all of the above reasons , we are not convinced that the addition of gep to cp parameters improves the reliability of sn - positivity estimates . 
scolyer , phd melanoma institute australia and faculty of medicine and health , the university of sydney , sydney , and department of tissue oncology and diagnostic pathology , royal prince alfred hospital and nsw health pathology , camperdown , nsw , australia john f . 
scolyer employment : royal prince alfred hospital consulting or advisory role : bristol myers squibb , glaxosmithkline , merck sharp & dohme , neracare , novartis amgen , myriad genetics , qbiotics research funding : the ainsworth foundation ( inst ) , national health and medical research council travel , accommodations , expenses : bristol myers squibb , novartis john f . 
bellomo d , arias - mejias sm , ramana c , et al : model combining tumor molecular and clinicopathologic risk factors predicts sentinel lymph node metastasis in primary cutaneous melanoma . 
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wong sl , kattan mw , mcmasters km , et al : a nomogram that predicts the presence of sentinel node metastasis in melanoma with better discrimination than the american joint committee on cancer staging systeann surg oncol 12 : 282 - 288 , 2005 6 . 
scolyer ra , judge mj , evans a , et al : data set for pathology reporting of cutaneous invasive melanoma : recommendations from the international collaboration on cancer reporting ( iccr )  . 
 impact of the spop mutant subtype on the interpretation of clinical parameters in prostate cancer purpose molecular characterization of prostate cancer , including the cancer genome atlas , has revealed distinct subtypes with underlying genomic alterations . 
one of these core subtypes , spop ( speckle - type poz protein ) mutant prostate cancer , has previously only been identifiable via dna sequencing , which has made the impact on prognosis and routinely used risk stratification parameters unclear . methods we have developed a novel gene expression signature , classifier ( subclass predictor based on transcriptional data ) , and decision tree to predict the spop mutant subclass from rna gene expression data and classify common prostate cancer molecular subtypes . 
we then validated and further interrogated the association of prostate cancer molecular subtypes with pathologic and clinical outcomes in retrospective and prospective cohorts of 8 , 158 patients . results the subclass predictor based on transcriptional data model showed high sensitivity and specificity in multiple cohorts across both rna sequencing and microarray gene expression platforms . 
we found that the spop mutant subclass was associated with lower frequency of positive margins , extraprostatic extension , and seminal vesicle invasion at prostatectomy ; however , spop mutant cancers were associated with higher pretreatment serum prostate - specific antigen ( psa )  . 
despite high pretreatment psa , the spop mutant subtype was associated with a favorable prognosis with improved metastasis - free survival , particularly in patients with high - risk preoperative psa levels . conclusion using a novel gene expression model and a decision tree algorithm to define prostate cancer molecular subclasses , we found that the spop mutant subclass is associated with higher preoperative psa , less adverse pathologic features , and favorable prognosis . 
2018 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license : introduction prostate cancer is a clinically and molecularly heterogeneous disease.1 - 4 current risk stratification guidelines , such as those from the national comprehensive cancer network , 5 the american urological association / american society for therapeutic radiology and oncology , 6 and the european association of urologyeuropean society for radiotherapyoncologyinternational society of geriatric oncology7 use clinical and pathologic parameters , including the level of prostate - specific antigen ( psa ) , to guide management decisions for clinically localized disease . 
psa is also used in a number of other clinical scenarios in prostate cancer , including initial diagnosis , monitoring for recurrence after primary therapy , and monitoring disease burden and treatment response for metastatic disease . the emerging next - generation dna and rna sequencing data point toward distinct molecular subclasses of prostate cancer , 2 - 4 , 8 , 9 but their clinical impact remains unclear . 
 genome atlas ( tcga ) study identified seven core prostate cancer subtypes , which were defined by underlying genomic alterations.4 one key molecular subclass , which represents approximately 10% of prostate cancers , harbors recurrent missense mutations in the e3 ubiquitin ligase component spop.2 - 4 , 10 - 13 to date , identification of spop mutations has required mutational analysis using genomic ( dna ) sequencing . 
in contrast , gene expression ( rna ) data are widely available from a variety of prostate cancer cohorts , often with the long follow - up necessary to define prognostic impact.14 - 21 here , we reported the first development and validation of the subclass predictor based on transcriptional data ( scapt ) model , which we used to define key prostate cancer molecular subclasses , including spop mutant cancer , using gene expression data . 
we used the scapt model to predict the molecular subclass from a retrospective cohort that included 1 , 626 patient samples and a prospective cohort that included 6 , 532 samples using genome - wide microarray gene expression data from a clinically available prognostic assay ( decipher ; genomedx biosciences , vancouver , bc , canada ) , and we explored the clinicopathologic and prognostic associations of key molecular subclasses of prostate cancer . methods prostate cancer tumor samples and microarray data we used a total of 8 , 559 radical prostatectomy ( rp ) tumor expression profiles for training , testing , and validation . 
for training and testing , we used rna sequencing expression and dna mutation data from tcga prostate cancer project ( n = 333 ) 4 and the weill cornell medicine ( wcm ) sequencing ( n = 68 ) cohort . 
for validation , expression profiles of retrospective ( n = 1 , 626 ) and prospective ( n = 6 , 532 ) cohorts were derived from the decipher genomics resource information database ( grid ) registry ( clinicaltrials.gov identifier : nct02609269 )  . 
 the retrospective grid cohort was pooled from seven published microarray studies : cleveland clinic , 22 erasmus mc , 23 johns hopkins , 15 memorial sloan kettering cancer center , 1 mayo clinic ( mayo i and mayo ii ) , 20 , 24 and thomas jefferson university.21 the prospective grid cohort was from clinical use of the decipher test . 
dna and rna from the tcga and wcm cohorts were extracted from fresh frozen rp tumor tissue , as previously described.4 rna from the grid cohorts was extracted from routine formalin - fixed , paraffin - embedded rp tumor tissues , amplified , and hybridized to human exon 1.0 st microarrays ( thermo fisher scientific , waltham , ma ) .24 , 25 spop mutant transcriptional signature we developed the spop mutant transcriptional signature , which includes 212 genes differentially expressed between spop mutant and wild - type samples from tcga prostate cancer rna sequencing data4 ( fig 1 )  . 
the overlap between two methods was significant ( p < 2.2 1016 ) , which confirmed similar results when applying these methods . scapt development on the basis of spop mutant transcriptional signature and the support vector machine model to predict tumors in the spop mutant subclass in the absence of dna sequencing datathat is , microarray data setswe developed the scapt model on the basis of the support vector machine ( svm ) model.28 - 30 given a set of training data marked with two categories , svm builds a model that assigns testing data into one category or the other , which makes it a nonprobabilistic binary linear classifier ( fig 2a )  . 
 ( a ) spop mutant transcriptional signature that included 212 differentially expressed genes between spop mutant and wild - type samples from the cancer genome atlas ( tcga ) non - ets fusion rna sequencing ( rna - seq ) data . 
 ( b ) significant enrichment of spop mutant case from 68 weill cornell medicine ( wcm ) prostate cancer samples with spop mutant transcriptional signature on the basis of hierarchical clustering . 
erg , erg - fusion position ; ets : other ets fusion positive ; fdr , false - discovery rate . z - scores of spop mutant signature from tcga cohort . 
 by performing 10 - fold cross - validation on the tcga training data set , we found costcost of constraints violationequal to 0.04 to yield the model with the highest sensitivity and specificity . 
next , using a decision tree and previously developed microarray - based classifiers for the erg - positive and ets - positive subtypes , 25 we classified the remaining cases in each cohort . 
high accuracy and confidence of spop mutant ( spopmut ) subclass prediction on weill cornell medicine ( wcm ) and gene expression omnibus ( geo ) data set by the scapt ( subclass predictor based on transcriptional data ) model . 
data from taylor et al , 1 erho et al , 24 and kelin et al.22 wt , wild type . spopwt spopmut support vectors large margin spop other taylor et al ( n = 150 ) erho et al ( n = 545 ) klein et al ( n = 182 ) prediction : spopmut sensitivity , 89% ; specificity , 95% spopwt spop wild type combined cohort to test the statistical association between spop mutant status and clinical variables , including age , race , preoperative psa , gleason score , lymph node invasion , surgical margin status , extracapsular extension , and seminal vesicle invasion . 
we evaluated the associations between spop mutant status and patient outcomes , including biochemical recurrence , metastasis , and prostate cancerspecific mortality , on the basis of kaplan - meier analysis . 
as spop mutations are mutually exclusive with erg and other ets rearrangements , 13 we excluded prostate cancer samples with ets fusions to define spop mutantspecific gene expression effects ( fig 1a )  . 
among those 212 genes , we found that upregulated genes from the spop mutant subgroup were enriched in transport vesicle membrane and oxidoreductase activity , but that there was no significant enriched function in downregulated genes from the spop mutant subgroup ( data supplement )  . 
in this independent cohort , we found significant enrichment ( p < 1.9103 ) of spop mutant tumors in one subcluster based on unsupervised clustering ( fig 1b and data supplement )  . 
we next used the spop mutant transcriptional signature in a locked svm modelthat is , with fixed parameters on the basis of the training step using the tcga cohort ( see methods ) to generate scores for each sample in the wcm cohort . 
the spop mutant prediction and its impacts on clinical and prognostic outcomes from retrospective ( n = 1 , 626 ) and prospective grid ( n = 6 , 532 ) cohorts . 
 ( a ) the pie chart of predicted molecular subclasses from the retrospective cohort with 1 , 626 samples , on the basis of the scapt ( subclass predictor based on transcriptional data ) model and decision tree . 
 ( c ) the pie chart of predicted molecular subclasses from the prospective grid cohort with 6 , 532 samples , on the basis of the scapt model and decision tree . 
erg , erg - fusion position ; ets , other ets fusion positive ; or , odds ratio ; psa , prostate - specific antigen . the grid microarray expression data , which do not have spop mutation annotation from dna analysis . 
between 9% and 15% of samples in the cohorts were predicted as spop mutant subclass , which is consistent with the known prevalence of spop mutations at the genomic level in previous prostate cancer studies3 , 4 , 10 , 12 ( fig 2b )  . 
overall , these results demonstrated that the scapt model predicted spop mutant subclass on the basis of the transcriptional data with high accuracy and confidence . molecular subtyping of 8 , 158 patients using the scapt and decision tree we applied the scapt model and decision tree to 8 , 158 patients from retrospective and prospective grid cohorts . 
among the retrospective cohort with 1 , 626 rp specimens , we predicted 9% ( range , 2% to 13% ) of samples to be spop mutant subclass ( data supplement )  . 
among the prospective cohort with 6 , 532 rp specimens , we predicted 8% of cases to be spop mutant subclass , 41% as erg positive , 12% as ets positive , 39% as other subtype , and 1% as conflict cases ( fig 3c )  . 
 the percentage of each molecular subclass was consistent with that reported in previous prostate cancer studies , 1 - 4 , 14 , 25 which supports the validity of our approach . spop mutant subclass associated with favorable pathology at rp we used binominal univariable analysis to compare clinical and pathologic characteristics between spop mutant and wild - type subclasses ( figs 3b and 3d )  . 
spop mutant subclass was less likely to harbor adverse pathologic features , such as positive surgical margins , extraprostatic extension , and seminal vesicle invasion , compared with wild - type subclass in both retrospective and prospective cohorts ( figs 3b and 3d )  . 
these data suggest that the spop mutant subclass was associated with more favorable pathologic outcomes at rp , but expresses higher levels of psa and was associated with tumors from older men , whereas the opposite was true in the erg - positive subclass of prostate cancer . consistent association between spop mutation and higher psa in multiple cohorts the inverse association of psa and prognosis in specific prostate cancer subtypes has potential clinical implications . 
to independently validate the association of spop mutant status and higher preoperative psa , we examined multiple distinct rp cohorts ( grid , tcga , taylor , and wcm )  . 
we observed a similar trend of higher preoperative psa in spop mutant cases , and the spop mutant subclass was more enriched in the higher psa subgroups ( psa > 10 ; fig 4a )  . 
on univariable analysis across cohorts , spop mutant status was significantly associated with higher preoperative psa ( fig 4b ) , whereas erg fusion status was significantly associated with lower preoperative psa ( fig 4c )  . spop mutation is associated with favorable clinical outcomes after rp on kaplan - meier analysis , the spop mutant subclass had the highest biochemical - free , metastasis - free , and lowest prostate cancer specific mortality compared with erg - positive , ets - positive , and other subtypes in the retrospective grid cohort ( fig 5b and data supplement )  . 
whereas long - term outcomes were not available for the prospective grid cohort , we evaluated the association with metastasis risk using the decipher score , a validated metric for prostate cancer metastatic potential.22 , 24 , 37 , 38 we found fewer spop mutant tumors in the decipher high - risk score ( > 0.6 ) group compared with the lowand average - risk groups ( data supplement ) , which again is consistent with a favorable prognosis subtype . 
together , these data support that spop mutant prostate cancer had favorable prognosis after rp , despite its association with higher preoperative psa . favorable prognosis in high - risk psa subgroup in the spop mutant subclass pretreatment psa level is a standard component of risk stratification for prostate cancer , with a psa level > 20 ng / ml considered to be high risk by a number of risk assessment methods.39 - 41 consistent with this , higher psa was associated with worse metastasis - free survival and prostate cancerspecific mortality in the retrospective cohorts ( fig 5a ) ; however , spop mutant status had a dramatic effect on prognosis within the subgroup of patients with high psa . 
 among patients with a psa level > 20 ng / ml , spop mutant tumors were associated with better clinical outcomes , which were comparable to the lowest psa subgroup ( psa < 10 ng / ml ; fig 5d )  . 
 ( a ) enrichment of spopmut cases among higher psa subgroups from prospective grid , the cancer genome atlas ( tcga ) , taylor , and weill cornell medicine ( wcm ) cohorts . 
 ( a ) clinical outcome difference between lower , average , and higher psa groups via kaplan - meier analyses for metastasis ( met ) and prostate cancerspecific mortality ( pcsm ) free survival rates . 
 society of geriatric oncology guidelines for clinically localized prostate cancer5 - 7 classify patients with a pretreatment psa level of > 20 as high risk , even in the absence of other adverse prognostic features . 
this results in increased treatment burden , and it is recommended that patients classified as high risk who opt for radiotherapy undergo 2 to 3 years of concurrent androgen - deprivation therapy compared with a duration of 4 to 6 months for patients with intermediate risk disease . 
knowledge of the molecular subtype of prostate cancer and its impact on psa level could therefore improve risk stratification , sparing unnecessary treatment burden and directing higher - intensity therapy to patients who are truly at higher risk ; however , whether molecular subtype will actually add clinical value to the current risk stratification toolsor if it adds additional information compared with such tests as decipherremains unclear . 
additional studies will be necessary to optimally deploy these strategies clinically , but it is clear that molecular subtyping should be considered in future clinical trial designs . recent advances in technology have increased our understanding of molecular subclasses of prostate cancer , but clinical and biologic differences among the key erg - positive , ets - positive , and spop mutant subclasses remain poorly understood . 
biologically , prostate cancer cells that harbor mutant spop may produce more psa on a per - cell basis as a result of enhanced androgen transcription , essentially leading to higher psa from fewer cancer cells , whereas the opposite may be true from erg - positive tumors . 
these hypotheses need to be rigorously tested in future functional and clinical studies . as a result of the retrospective nature of the cohorts and small sample size of case cohort studies , survival analysis was inevitably affected by baseline risk . 
although the favorable prognosis was consistent with improved clinical outcomes in the spop mutant subclass , these survival results need to be independently validated in additional clinical trials to be generalizable . in conclusion , we have developed the scapt model to predict spop mutant subclass purely on the basis of transcriptional data with high confidence and accuracy . 
we believe this work not only builds a prediction model for spop mutant prostate cancers and expands the data types usable for the interrogation of clinical outcomes , but it also reinforces the concept that molecular subtyping of prostate cancer can alter the interpretation of the current standard of care risk stratification methods . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . deli liu no relationship to disclose mandeep takhar employment : genomedx mohammed alshalalfa employment : genomedx nicholas erho employment : genomedx jonathan shoag no relationship to disclose travel , accommodations , expenses : genomedx robert b . 
rubin research funding : eli lilly , janssen pharmaceuticals bruce trock consulting or advisory role : genomedx consulting or advisory role : myriad genetics research funding : myriad genetics , mdxhealth eric a . 
den consulting or advisory role : genomedx speakers ' bureau : bayer research funding : medivation , astellas pharma , genomedx travel , accommodations , expenses : genomedx scott a . 
tomlins leadership : strata oncology stock and other ownership interests : strata oncology consulting or advisory role : abbvie , janssen pharmaceuticals , astellas pharma , medivation , strata oncology , sanofi , almac diagnostics research funding : astellas pharma ( inst ) , medivation ( inst ) , genomedx ( inst ) patents , royalties , other intellectual property : coauthor on a patent issued to the university of michigan on ets gene fusions in prostate cancer travel , accommodations , expenses : strata oncology daniel e . 
spratt no relationship to disclose elai davicioni employment : genomedx leadership : genomedx stock and other ownership interests : genomedx patents , royalties , other intellectual property : cancer diagnostics using biomarkers 20140066323 travel , accommodations , expenses : genomedx andrea sboner no relationship to disclose christopher e . 
barbieri patents , royalties , other intellectual property : coinventor on a patent application filed regarding spop mutations in prostate cancer by weill cornell medicine travel , accommodations , expenses : genomedx acknowledgment ashley e . 
ross stock and other ownership interests : genomedx honoraria : healthtronics consulting or advisory role : healthtronics research funding : metamark genetics we thank the patients with prostate cancer and families who contributed to this research . 
barbieri , newyork - presbyterian hospital , new york , ny ; mandeep takhar , mohammed alshalalfa , nicholas erho , and elai davicioni , genomedx bioscience , vancouver , british columbia , canada ; robert b . 
johnson mh , ross ae , alshalalfa m , et al : spink1 defines a molecular subtype of prostate cancer in men with more rapid progression in an at risk , natural history radical prostatectomy cohort . 
ross ae , johnson mh , yousefi k , et al : tissue - based genomics augments post - prostatectomy risk stratification in a natural history cohort of intermediateand high - risk men . 
nguyen pl , haddad z , ross ae , et al : ability of a genomic classifier to predict metastasis and prostate cancer - specific mortality after radiation or surgery based on needle biopsy specimens . 
spratt de , yousefi k , deheshi s , et al : individual patient - level meta - analysis of the performance of the decipher genomic classifier in high - risk men after prostatectomy to predict development of metastatic disease . 
karnes rj , bergstralh ej , davicioni e , et al : validation of a genomic classifier that predicts metastasis following radical prostatectomy in an at - risk patient population . 
klein ea , yousefi k , haddad z , et al : a genomic classifier improves prediction of metastatic disease within 5 years after surgery in node - negative high - risk prostate cancer patients managed by radical prostatectomy without adjuvant therapy . 
boormans jl , korsten h , ziel - van der made aj , et al : identification of tdrd1 as a direct target gene of erg in primary prostate cancer . 
bennett kp , campbell c : support vector machines : hype or hallelujah ? sigkdd explor 2 : 113 , 2000 technol 2 : 1 - 27 , 2011 30 . 
fine sw , gopalan a , leversha ma , et al : tmprss2 - erg gene fusion is associated with low gleason scores and not with high - grade morphological features . 
zhou ck , young d , yeboah ed , et al : tmprss2 : erg gene fusions in prostate cancer of west african men and a meta - analysis of racial differences . 
schaefer g , mosquera jm , ramoner r , et al : distinct erg rearrangement prevalence in prostate cancer : higher frequency in young age and in low psa prostate cancer . 
font - tello a , juanpere n , de muga s , et al : association of erg and tmprss2 - erg with grade , stage , and prognosis of prostate cancer is dependent on their expression levels . 
marrone m , potosky al , penson d , et al : a 22 gene - expression assay , decipher ( genomedx biosciences ) to predict five - year risk of metastatic prostate cancer in men treated with radical prostatectomy . 
cooperberg mr , pasta dj , elkin ep , et al : the university of california , san francisco cancer of the prostate risk assessment score : a straightforward and reliable preoperative predictor of disease recurrence after radical prostatectomy . 
damico av , whittington r , malkowicz sb , et al : biochemical outcome after radical prostatectomy , external beam radiation therapy , or interstitial radiation therapy for clinically localized prostate cancer . 
 high - content biopsies facilitate molecular analyses and do not increase complication rates in patients with advanced solid tumors purpose precision oncology relies on frequent pathologic , molecular , and genomic assessments of tumor tissue to guide treatment selection , evaluate pharmacodynamic effects of novel agents , and determine drug resistance mechanisms . 
it remains unknown how the need for serial biopsies with large numbers of tumor cores relates to tissue yields and biopsy complication rates . materials and methods in this study , we performed a retrospective analysis of 199 focal liver biopsies performed in 143 patients in the setting of oncologic research protocols ( research biopsy group ) over a 4 - year period at a single - intervention oncology service . 
practice patterns and complication rates were compared with those related to 1 , 522 consecutive biopsies performed in 1 , 154 patients in whom two cores were obtained for standard clinical management of patients ( standard biopsy )  . results in the research biopsy group , 1 , 100 tissue cores ( average , 5.5 cores per procedure ) were harvested and distributed to trial sponsors , internal research laboratories , and pathology services . 
in the standard biopsy control group , major complications were observed in 1.4% of procedures ( 22 of 1 , 522 ) and minor complications in 0.2% ( three of 1 , 522 )  . 
2017 by american society of clinical oncology introduction precision oncology aims to adapt treatment decisions based on the molecular characteristics of an individual tumor , thereby increasing the chance for a successful clinical outcome.1 tumors are increasingly being classified into subsets based on molecular profile with corresponding tailored treatment . 
there are substantial data demonstrating metastatic tissue can harbor unique genomic alterations compared with the primary tumor , and heterogeneity can also be seen among different metastatic sites in the same patient.2 , 3 it is generally accepted that the use of genomics and biomarkers substantially increases the success rate of clinical trials while lowering their cost.4 despite the rapid emergence of liquid biopsies ( eg , circulating tumor dna , 5 exosomes , circulating tumor cells ) , image - guided , minimally invasive biopsies remain the gold standard in procuring sufficient malignant tissue for molecular analysis . there are several reasons for sampling increasing amounts of tissue more frequently : the realization that tumors can adapt rapidly to therapeutic pressures and that molecular analysis of progression site biopsy , rather than primary specimen , is crucial to identify mechanisms of resistance ; the nathan e . 
 increasing use of immunotherapy trials where treatment response assessment can be difficult by imaging ( eg , pseudoprogression ) ; the emergence of more sophisticated analytic models , including patient - derived tumor cell lines and xenografts , requiring additional tissue ; the fact that a single biopsy core ( 10 mm3 ) maximally contains 30 million cells but often much fewer because of stroma and other factors ; and as a precautionary method to ensure that genomic analysis is indeed possible , because results can help with potential selection of matched targeted therapy . 
for example , in a large recent federal trial of personalized cancer medicine , 13% of tumor biopsy samples could not be used for genomic analysis.6 a recent trend in image - guided biopsies has therefore been to collect more cores ( four to 10 cores ) than necessary for traditional pathology workup ( eg , one to two cores )  . additional cores can be harvested with the use of coaxial systems that allow repeat 18 - gauge samples to be obtained through a hollow needle placed under image guidance . 
with such an approach , consecutive tissue fragments can then be used for immunohistochemistry , fluorescence in situ hybridization analysis , genomic analysis , protein and phosphoprotein biomarker testing , and establishment of patient - derived models , including cell lines , organoids , and mouse xenografts.7 these multicore biopsies , which we herein term highcontent biopsies ( hcbx ) , are thus attractive in initiating and evaluating genotype - centric drug trials and may be critically important for comprehensive genomic and proteomic analyses . 
with increased core sampling , a critical question remains : is there an increased complication rate associated with more extensive tissue harvesting ? data on the safety of research biopsies are limited , 8 , 9 in part because such data are typically not reported in clinical trials , and there is no standard consensus for the reporting of adverse events.10 the technical success rates of obtaining adequate cores after repeat sampling are largely unpublished , although anecdotal reports describe potential reduction in sample quality after repeat sampling.11 furthermore , performing biopsies within research protocols requires more complex logistics , with dedicated personnel and workflows . the main goal of this study was to evaluate if the increased number of cores obtained for research biopsies was associated with increased complication rates or decreased sample quality . 
to address these questions , we performed a retrospective analysis of focal tumor biopsies in the liver performed in the setting of specific oncologic research protocols at a single institution over a 4 - year period . 
practice patterns and complication rates were compared with those of 1 , 522 consecutive biopsies performed in 1 , 154 patients for standard clinical management during the same time period . 
of these , 199 biopsies were performed in 143 patients in the setting of oncologic research protocols ( research biopsy group ) ; 1 , 522 were performed in 1 , 154 patients for standard clinical management ( standard biopsy group )  . in the standard biopsy group , patients were referred from their oncologists directly to the interventional oncology service . 
these cores were placed in formalin and sent to pathology for standard processing : hematoxylin and eosin staining , immunohistochemistry as needed , and , more recently , molecular testing ( snapshot or foundation medicine [ fm ] , as discussed in pathology analysis ) when requested by referring oncologists . for research biopsies , however , a dedicated staff research coordinator was responsible for coordinating the entire process : arranging for the biopsy scheduling , determining and discussing the number of cores needed with the interventional radiologist , and collecting the samples and distributing them to their appropriate destinations . 
this was performed according to a standard operating procedure ( sop ) associated with each research study in which these patients were enrolled and was prospectively logged in an institutional database . 
for research biopsies , a dedicated staff member ( biopsy coordinator ) is responsible for coordinating the entire process : arranging for the scheduling , discussing the number of cores , and collecting the samples and distributing them to their appropriate destinations . the most common workflow involves obtaining five cores : in formalin for pathology , including mutational analyses ; fixed in formalin for 6 to 24 hours and subsequently transferred to ethanol , to be sent to trial sponsor ; in fetal bovine serum ( fbs ) , for development of patientderived models and drug testing ; in formalin for intrainstitutional research laboratories ; and snap frozen for tissue bank . prior imaging coaxial biopsy research / clinical trial biopsy > 5 cores formalin formalin formalin snap frozen patient 1 - 2 cores analysis of : size location growth approach routine pathology tissue type ihc mutations analysis of : tissue type pathway analysis mutations drug response oncologist interventional radiologist biopsy coordinator the fourth in formalin for intra - institutional research laboratories ; and the fifth , which was snap frozen ( fig 1 )  . 
patients in the research group were consented for additional risk of bleeding and other complications . image - guided biopsy procedures all procedures were performed using 18 - gauge coaxial , semiautomatic needle biopsy cutting needles ( bard mission ; bard biopsy systems , tempe , az ) under imaging guidance . 
50 , 000 / ml and an international normalized ratio of prothrombin time , 1.5 for all patients undergoing liver biopsy . in the research biopsies , 54.3% of the procedures were performed using ultrasound guidance ( supersonic imagine , aix - en - provence , france ; hitachi aloka medical , tokyo , japan ) , and 45.7% were performed using computed tomography guidance ( general electrics , fairfield , ct )  . 
a total of 193 procedures were performed using moderate sedation ( intravenous use of fentanyl and midazolam ) , three biopsies were performed with general anesthesia or monitored anesthesia care , and three procedures were performed with local anesthetic only ( lidocaine )  . complication rates were determined by review of medical notes and imaging studies obtained up to 30 days after the procedure ( research biopsies ) and of a prospectively maintained departmental database ( research and standard biopsies ; hi - iq ; conexsys , lincoln , ri )  . 
complications were classified as major ( including all patients who required more therapy other than clinical support , required hospitalization , or experienced an unplanned increase in the level of care , permanent adverse sequelae , or death ) or minor ( others ) .13 we did not record intraprocedural pain scores , because a majority of patients received conscious sedation in a patient - focused manner and by different care providers . 
all patient cases with negative results were reviewed , and clinical and imaging follow - up was used to determine whether they were true or false negatives . pathology analysis a negative biopsy was defined as 0% tumor cells . when rare tumor cells were present , they were noted , and the diagnosis was positive for malignancy . 
this was performed in house with polymerase chain reaction ( pcr ) based assay ( pcr - snapshot14 ) or targeted nextgeneration sequencing ( ngs ) tumor genotyping assay ( ngs - snapshot ) .15 selected samples were sent to fm for a different comprehensive genomic profiling using ngs.16 statistical analysis descriptive statistics were used to summarize results . 
table 1 summarizes patient demographics . because of heterogeneity among the clinical trial protocols , the mean number of cores obtained in the research cohort varied ( fig 3a )  . 
for a majority of procedures ( n = 186 of 199 ; hcbx ) , a larger number of cores ( range , four to 10 ; mean , 5.7 cores ) was obtained . 
in a smaller characteristic age , years mean range male female cancer type breast lung colorectal other primary stage * complication minor major 57.9 28 - 84 60 ( 30.2 ) 139 ( 69.8 ) 68 ( 34.2 ) 55 ( 27.6 ) 26 ( 13.1 ) 50 ( 25.1 ) 1 ( 0.5 ) 5 ( 2.5 ) 3 ( 1.5 ) 190 ( 95.5 ) 2 ( 1.0 ) 1 ( 0.5 ) * patient cases of stage i to iii disease correspond to cases of intrahepatic cholangiocarcinoma or hepatocellular carcinoma ; patient cases of stage iv disease represent all other neoplasms . number of procedures ( n = 13 of 199 ) , fewer cores ( range , two to three ; mean , 2.8 cores ) were sufficient to satisfy trial requirements and / or research needs . sample processing and analysis the 1 , 100 cores were distributed to their appropriate destination by the research biopsy coordinator according to the sop of each study ( fig 3b ) ; 229 cores ( from 127 procedures ) were sent to trial sponsors , 121 cores ( from 110 procedures ) were sent for development of patient - derived cell lines , 466 cores ( from 192 procedures ) were sent to other in - house research laboratories for various studies , and 284 cores ( from 174 procedures ) were sent to standard pathology . 
among the procedures from which samples were sent to standard pathology ( n = 174 ) , molecular analysis was attempted on 73 : multiplex pcr - based snapshot in 13 , ngs - based snapshot in 39 , and foundationone in 19 ; two samples had insufficient tissue for molecular analysis . the success rates of different analyses for research biopsies are summarized in figure 3c . 
 ( a ) distribution of number of cores per biopsy ( median , five ; mean , 5.5 6 standard deviation , 1.5 ; range , two to 10 cores )  . 
of these , four were shown to be true negatives ( ie , no evidence of metastasis or persistent local response after treatment on followup ) and were excluded from the analyses of success for the different tests . 
of the evaluated parameters ( table 2 ) , none were clearly associated with either satisfactory or nondiagnostic samples . the complication rates in the standard biopsy cohort were similar , with major complications observed in 1.4% of procedures ( 22 of 1 , 522 )  . the major complications were hematomas requiring transfusion or intervention ( n = 15 ) , pneumothorax requiring chest tube ( n = 1 ) , hemobilia requiring endoscopic retrograde cholangiopancreatogram ( n = 1 ) , infection requiring admittance ( n = 1 ) and / or percutaneous drainage ( n = 1 ) , and death ( n = 3 ; all secondary to bleeding )  . 
the minor complication rate was 1.0% ( n = 2 ; both were hematomas managed conservatively : one patient with metastatic breast cancer [ seven cores ] and one with colorectal cancer [ five cores ] )  . image - guided biopsies have long played an important role in the management of cancer . 
these findings are not entirely unexpected , because we used a coaxial biopsy technique that allows for multiple cores to be harvested through a single coaxial needle , therefore minimizing the number of capsular punctures probably a factor for decreasing the risk of complications in biopsies.24 one evident finding of our study is the progressive increase of research biopsies over the study period . this is consistent with the increasing popularity of molecularly targeted trials , as well as the realization that hcbx are technically feasible and safe . 
it has already been reported that genetic analysis is contingent on sufficient material.6 accordingly , different methods of molecular analysis in our data had results that ranged from 79% ( fm ) to 100% ( pcr - based snapshot )  . although ngs can more efficiently detect a wider range of molecular aberrations , more material is needed compared with older pcrbased techniques . to streamline research biopsies and accommodate larger numbers of procedures , we implemented a specific workflow with dedicated staff for efficient communication among clinicians , researchers , trial sponsors , and interventional radiologists , as well as collection and distribution of samples . 
although we did not measure the exact length of time required for the procedures in this study , we had previously determined that additional core sampling only required a few minutes longer , unless the coaxial needle had to be repositioned because of poor sample material ( eg , necrotic core , cystic tumor , fragmented specimen )  . 
interestingly , in our experience , the order in which cores were harvested was not associated with lower success rates for pathology , contrary to prior published reports.11 the samples sent for cell - line creation had a lower success rate . 
although this may have been related to the sample volume , it may also have resulted from other variables beyond our control ( amount of tumoral cells required for successful cell - line creation , timing , distance , or personnel , among others )  . 
interestingly , however , within the samples that were used for cell - line creation , the number of cores was not associated with cellline creation success rate . our study was designed to answer simple questions : how successful are hcbx , and what is their complication rate ? this is of timely interest ; data on the safety of research biopsies are limited , because they are often not reported in clinical trials.10 as such , our study focused on so - called all comers in a large oncologic interventional care setting rather than on specific subsets of patients , biopsy sites , or operators . 
 3% pi3k - akt signaling pathway fgfr1 / 2 / 3 / 4 and fgf familiy mapk / ras signaling pathway cyclin - dependent kinase related tp53 egfr esr1 other negative fig 4 . 
the genomic abnormalities detected in our study were similar to those found in several large studies17 and reflect the study population involving breast , lung , and colorectal cancer and cholangiocarcinoma trials at our institution . 
mapk , mitogen - activated protein kinase ; pi3k , phosphatidylinositol 3 - kinase . thoracic lesions , and similar studies may be warranted for those cohorts . our study has several limitations . 
medical records were reviewed for complications exclusively in the research biopsy group , although this would , if anything , overestimate the relative risk of complications compared with that of standard biopsies . 
only data on liver biopsies were reviewed , and therefore , additional studies are required to verify if the conclusions apply to other organs frequently biopsied , such as lungs , lymph nodes , and bones . 
vadvala , aditya bardia , jeffrey engelman , peter r . mueller , dejan juric , ralph weissleder financial support : dejan juric , ralph weissleder administrative support : dejan juric , ralph weissleder provision of study material or patients : laura spring , dejan juric collection and assembly of data : nathan e . 
frenk , laura spring , alona muzikansky , mari mino - kenudson , amy ly , aditya bardia , dianne finkelstein , dejan juric , ralph weissleder manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors the field of blood - based biomarkers in oncology for disease monitoring and molecular analysis is rapidly evolving . 
liquid biopsies can be used to detect circulating extracellular vesicles25 or tumor cells within a blood specimen , providing a tool to monitor cancer noninvasively.26 similarly , a number of technologies have also been developed to identify and track tumor - specific mutations via circulating free dna in the blood and urine.5 as these technologies are further developed and validated , they might replace some of the tissue biopsies in certain research and clinical settings . however , tissue biopsies allow for sampling of specific lesions and evaluation of tissue architecture and the tumor microenvironment , including proteins , phosphoproteins , and varied cytokines . image - guided biopsies will therefore continue to remain important for phenotyping tumor responses , identifying tumor heterogeneity , identifying therapeutic resistance , and characterizing host responses in the era of immunotherapy and targeted therapies . 
additional advances in image - guided tumor sampling , including lower computed tomography doses , shorter image acquisition times , and systems for acquiring , processing , and analyzing smaller samples may further expand the use of hcbx . 
mueller consulting or advisory role : cook medical patents , royalties , other intellectual property : royalties from cook ( percutaneous catheters ) dejan juric consulting or advisory role : novartis , emd serono , eisai ralph weissleder consulting or advisory role : moderna therapeutics , alivio therapeutics , tarveda therapeutics , lumicell , t2 biosystems , bioanalytix acknowledgment we thank all members of the division of interventional and abdominal radiology who contributed to the review of cases and / or performing of biopsies , in particular steven dawson , md , ashraf thabet , md , ronald arellano , md , suvranu ganguli , md , avinash kambadakone , md , michael zalis , md , and omar zurkiya , md , phd . 
j clin oncol 31 : 1904 - 1911 , 2013 juric d , castel p , griffith m , et al : convergent loss of pten leads to clinical resistance to a pi ( 3 ) ka inhibitor . 
friedman aa , letai a , fisher de , et al : precision medicine for cancer with next - generation functional diagnostics . nat rev cancer 15 : 747 - 756 , 2015 8 . 
el - osta h , hong d , wheler j , et al : outcomes of research biopsies in phase i clinical trials : the md anderson cancer center experience . 
hopper kd , grenko rt , tenhave tr , et al : percutaneous biopsy of the liver and kidney by using coaxial technique : adequacy of the specimen obtained with three different needles in vitro . 
dias - santagata d , akhavanfard s , david ss , et al : rapid targeted mutational analysis of human tumours : a clinical platform to guide personalized cancer medicine . 
giorgio a , tarantino l , de stefano g , et al : complications after interventional sonography of focal liver lesions : a 22 - year single - center experience . 
babaei jandaghi a , lebady m , zamani aa , et al : a randomised clinical trial to compare coaxial and noncoaxial techniques in percutaneous core needle biopsy of renal parenchyma . 
im h , shao h , park yi , et al : label - free detection and molecular profiling of exosomes with a nano - plasmonic sensor . nat biotechnol 32 : 490 - 495 , 2014 26 . 
mody , md , ms2 introduction somatic mutations in b - cell lymphoma 6 ( bcl6 ) corepressor ( bcor ) are reported in solid and hematologic malignancies in adults and children . 
bcor mediates transcriptional repression through epigenetic modication believed to cause oncogenesis through both overexpression or loss of tumor suppressor function.1 it is found to affect multiple diverse downstream gene targets and plays a role in development and function across many tissue lineages , especially the cns.2 , 3 in pediatric malignancies , bcor - ccnb3 fusion is prominent in undifferentiated round cell sarcoma ( uds ) , which , until recently , was treated similarly to ewing sarcoma ( ews )  . 
now considered its own genetic entity , bcor - ccnb3 fusions are typically identied in uds using transcriptome sequencing ( rna - seq ) and are present in up to 14% of uds cases.4 , 5 internal tandem duplications in bcor are found in pediatric cns neuroepithelial tumors and clear cell sarcoma of the kidney , 6 , 7 and mutations are reported in rhabdomyosarcoma and diffuse intrinsic pontine glioma ( dipg ) .8 , 9 however , uncertainty remains regarding the genomic landscape and signicance of bcor alterations in pediatric malignancies . in this article , we describe bcor alterations identied in our integrative clinical sequencing ( ics ) cohort in patients with sarcoma and high - grade glioma ( hgg ) these alterations in and explore the landscape of publicly available databases . 
furthermore , we examine their histopathologic features and co - occurring genomic aberrations and propose prospective targeted treatment strategies . methods pediatric and young adult patients ( , 26 years ) described were consented and enrolled in a prospective , institutional review boardapproved ( hum# : ics trial pediatricmichigan oncology 00056496 ) sequencing ( peds - mioncoseq ) at c.s. 
details of the sequencing procedures and bioinformatics analyses have been previously described.10 briey , somatic mutations , copy number variants / alterations , insertions and deletions , gene fusions , and gene expression are analyzed.11 , 12 investigation of clinical relevance of somatic variants uses an integrated approach incorporating technical considerations , variant and patient - specic information , and published data . st judes pediatric cancer data portal ( pecan ) was used ( accessed january 2019 ) to explore bcor alterations in a larger cohort of pediatric patients . 
the pecan database is a pediatric cancer genome repository containing whole - genome sequencing , wholeexome sequencing , and rna - seq data on more than 4 , 300 patients . 
 mankuzhy et al of the 86 sarcoma cases sequenced at the time of this report , 18 osteosarcomas and ve malignant peripheral nerve sheath tumors did not contain bcor alterations . twenty - seven of the remaining 63 cases contained clinically relevant diagnostic fusions , one ( 3.7% ) of which ( pax3 - foxo1positive alveolar rhabdomyosarcoma ) also had a bcor alteration . 
of the 15 patients with dipg sequenced , three ( 20% ) harbored mutations in bcor . pathologic evaluation of the bcor - mutant sarcoma cases showed both round - cell ( ewing - like ) morphology and the recently recognized variant with spindle - cell sarcoma morphology.17 figure 1 shows noncohesive sheets of malignant round to ovoid cells , with primitive - appearing chromatin , high mitotic activity , and necrosis . 
there were interlacing short fascicles with whorled pattern and foci of somewhat epithelioid congurations around larger vessels , mimicking synovial sarcoma and malignant peripheral nerve sheath tumor . mitotic gures were also identied . 
vimentin immunostain was diffusely strongly positive , and cd99 was negative . review of the pecan data set revealed 16 patients with tumors harboring nonsynonymous bcor alterations , ranging in age from 2 to 21 years ( table 2 )  . 
of the nine hggs , six contained concurrent gfr alterations , including two egfr and four platelet - derived growth factor receptor a mutations . the frequency of bcor mutations of primary adult cancers in tcga was 15 of 393 ( 3.8% ) in sarcoma and two of 273 ( 0.7% ) in hgg , particularly gbm . 
in the mi - oncoseq data set with advanced or refractory adult patients , sarcoma had a bcor mutation frequency of three of 199 ( 1.5% ) and gbm one of 49 ( 2% )  . discussion through integrative analysis of a cohort of high - risk pediatric malignancies and review of other large genomic databases , our study further describes the frequency and distribution of bcor alterations in pediatric sarcoma and high - grade glial malignancies . 
we recognized a higher overall prevalence of bcor alterations in these pediatric malignancy subtypes compared with their adult counterparts , both in primary cases ( tcga ) and refractory / advanced cases ( mi - oncoseq )  . 
this could be explained by bcors role in embryonic stem - cell differentiation , hematopoiesis , and regulation of vertebrate laterality and germline bcor mutations causing lenz microphthalmia and oculofaciocardiodental syndromes , which display fig 1 . 
 ( d ) cd99 immunostain ( 20 ) is negative . phenotypically overlapping skeletal , cns , and ocular effects , supporting its pleotropic developmental function.1 , 3 , 18 our study shows relatively high prevalence of bcor alterations in pediatric hgg ( 17% ) and in pediatric sarcomas , which are negative for fusions and other pathognomonic molecular alterations ( 11% ) , emphasizing the need for ics analysis to better dene molecular subtypes of these cases . 
our study also conrms earlier observations of bcor alterations in fusion - positive and - negative alveolar rhabdomyosarcoma.19 , 20 however , our ics cohort did not detect any bcor alterations in classic ewsr1 - rearranged ews or osteosarcoma , as seen in pecan . previous research has suggested that the prognostic signicance of bcor alterations could be tissue and diagnosis specic , with bcor - mutant leukemia being associated with a worse prognosis and no survival difference detected when comparing bcor - ccnb3 and ews.4 , 21 , 22 however , because of our cohorts small sample size and a lack of availability of outcomes data in larger data sets , additional investigation is needed to truly dene prognostic signicance of bcor alterations , specically in hgg . a novel nding in our cohort is the high frequency ( 57% ) of concurrent bcor and gfr alterations . 
gfrs are typically expressed on cell surfaces and play important roles in cell growth , survival , angiogenesis , and metastasis.23 the concurrent gfr mutations seen in our cohort are all known to be gain - of - function mutations resulting in constitutive activation of the gfr pathway and increased cell proliferation.24 - 27 bcl6 depletion results in downregulation of kinases in the receptor tyrosine kinase pathway , suggesting a potential link between bcl6 and related proteins such as bcor with receptor tyrosine kinase signaling that could be further explored to determine the signicance of these mutations.28 the bcl6 / bcor previous research has suggested that complex could serve as a potential therapeutic target . reported downstream targets of bcor activity include bcl6 targets , hoxa cluster genes , notch homolog 1 targets , and sonic hedgehog effectors such as gli1 and gli2.2 , 29 - 31 bcor can interact with specic histone deacetylase of both class i and ii , which increases the repression of transcriptional activity.32 the effect of histone deacetylase inhibitors on tumors with bcor mutations warrants additional investigation to assess the biologic relevance of these interactions , particularly in patients with dipg who also harbor histone mutations . 
 bcor alterations in pediatric solid tumors molecular and genomic characterization of tumors can complement histology - based classications of human cancers.35 recognizing which alterations are clinically meaningful and developing effective targeted treatment strategies can further contribute to dening this new cancer routine genomic proling can taxonomy . identify potential drivers of oncogenesis and possible targetable aberrations that can stimulate development of novel investigations of therapeutic approaches and additional in addition , molecular mechanisms of cancer . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . chandan kumar patents , royalties , other intellectual property : royalty for um agr #3199.101 recurrent gene fusions in prostate cancer licensed to gen - probe , inc . no other potential conicts of interest were reported . references hum mol genet 16 : 1773 - 1782 , 2007 1 . 
hilton en , manson fdc , urquhart je , et al : left - sided embryonic expression of the bcl - 6 corepressor , bcor , is required for vertebrate laterality determination . gearhart md , corcoran cm , wamstad ja , et al : polycomb group and scf ubiquitin ligases are found in a novel bcor complex that is recruited to bcl6 targets . mol cell biol 26 : 6880 - 6889 , 2006 3 . 
plos one 3 : e2814 , 2008 kao yc , owosho aa , sung ys , et al : bcor - ccnb3 fusion positive sarcomas : a clinicopathologic and molecular analysis of 36 cases with comparison to morphologic spectrum and clinical behavior of other round cell sarcomas . 
am j surg pathol 42 : 604 - 615 , 2018 peters tl , kumar v , polikepahad s , et al : bcor - ccnb3 fusions are frequent in undifferentiated sarcomas of male children . 
mod pathol 28 : 575 - 586 , 2015 argani p , kao y - c , zhang l , et al : primary renal sarcomas with bcor - ccnb3 gene fusion : a report of 2 cases showing histologic overlap with clear cell sarcoma of kidney , suggesting further link between bcor - related sarcomas of the kidney and soft tissues . 
am j surg pathol 41 : 1702 - 1712 , 2017 sturm d , orr ba , toprak uh , et al : new brain tumor entities emerge from molecular classication of cns - pnets . 
cell 164 : 1060 - 1072 , 2016 cramer sl , miller al , pressey jg , et al : pediatric anaplastic embryonal rhabdomyosarcoma : targeted therapy guided by genetic analysis and a patient - derived xenograft study . 
mackay a , burford a , carvalho d , et al : integrated molecular meta - analysis of 1 , 000 pediatric high - grade and diffuse intrinsic pontine glioma . 
tirode f , surdez d , ma x , et al : genomic landscape of ewing sarcoma denes an aggressive subtype with co - association of stag2 and tp53 mutations . 
 mankuzhy et al juckett lt , lin di , madison r , et al : a pan - cancer landscape analysis reveals a subset of endometrial stromal and pediatric tumors dened by internal tandem duplications of bcor . 
li w - s , liao i - c , wen m - c , et al : bcor - ccnb3 - positive soft tissue sarcoma with round - cell and spindle - cell histology : a series of four cases highlighting the pitfall of mimicking poorly differentiated synovial sarcoma . 
shern jf , chen l , chmielecki j , et al : comprehensive genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic axis in fusion - positive and fusion - negative tumors . 
puls f , niblett a , marland g , et al : bcor - ccnb3 ( ewing - like ) sarcoma : a clinicopathologic analysis of 10 cases , in comparison with conventional ewing sarcoma . 
bellus ga , spector eb , speiser pw , et al : distinct missense mutations of the fgfr3 lys650 codon modulate receptor kinase activation and the severity of the skeletal dysplasia phenotype . 
de gunst mm , gallegos - ruiz mi , giaccone g , et al : functional analysis of cancer - associated egfr mutants using a cellular assay with yfp - tagged egfr 28 . 
proc natl acad sci usa 114 : 3981 - 3986 , 2017 [ erratum : proc intracellular domamol cancer 6 : 56 , 2007 natl acad sci usa 114 : e4317 , 2017 ] 29 . 
tiberi l , bonnefont j , van den ameele j , et al : a bcl6 / bcor / sirt1 complex triggers neurogenesis and suppresses medulloblastoma by repressing sonic hedgehog signaling . 
schnidar h , eberl m , klingler s , et al : epidermal growth factor receptor signaling synergizes with hedgehog / gli in oncogenic transformation via activation of the mek / erk / jun pathway . 
wei , md1 cdk12 is a kinase that regulates several key cellular processes , including transcription and maintenance of genomic stability.1 the prevalence of cdk12 alterations is higher in metastatic castration - resistant prostate cancer ( mcrpc ; 7% ) compared with primary prostate cancer ( 1% ) , which indicates an association with an aggressive phenotype.2 in the articles accompanying this editorial in jco precision oncology , schweizer et al3 and antonarakis et al4 report on the clinical features and outcomes of patients with cdk12 - altered prostate cancer in two independent retrospective multicenter series . 
in both studies , the source of tumor dna included primary prostate , metastatic sites , and circulating tumor dna sequenced for cdk12 alteration status through a variety of commercial and inhouse sequencing platforms . schweizer et al3 identied 52 patients who harbored cdk12 mutations , and antonarakis et al4 identied 60 . in both studies , there was a roughly even split incidence of monoallelic or biallelic cdk12 inactivation . because of limitations in the diverse sequencing platforms used , neither series required biallelic mutations . 
in the schweizer series , median time from initiation of androgen deprivation therapy ( adt ) to development of crpc was 1.4 years , and in the antonarakis series , median progression - free survival ( pfs ) after initiation of adt was 12.3 months , which indicates short responses to primary adt . 
median overall survival ( os ) from time of metastasis was 3.9 years in the schweizer series , and median os from initiation of adt was 40.8 months in the antonarakis data set , which suggests poor prognosis . 
in the schweizer cohort , the unconrmed 50% prostate - specic antigen decline ( psa50 ) response rate to rst - line abiraterone and enzalutamide for crpc was approximately 70% ; however , median pfs was short at approximately 9 months . 
first , antonarakis et al required a conrmatory psa at least 4 weeks after the initial psa50 response , which was not required in the schweizer series and likely contributed to the lower psa response rate in the antonarakis study . 
de novo metastatic presentation is a poor prognostic factor that likely contributed to the shorter pfs with rst - line abiraterone and enzalutamide in the antonarakis study . these data are consistent with another recently published retrospective series by reimers et al5 that included 46 patients with advanced prostate cancer with cdk12 mutations . 
of note , the patients with cdk12 loss - of - function alterations had worse clinical outcomes compared with those with other mutations , including atm , brca1 / 2 , and tp53 . 
taken together , all three studies have consistently shown that cdk12 alterations portend aggressive clinical features and relative resistance to second - generation androgen signaling inhibitors . while the antonarakis and schweizer series represent the largest cdk12 - altered cohorts to date , they are both limited by their retrospective nature . 
clinical assessments were variable and not standardized , and there were no comparisons of cdk12 alteration with other genomically dened cohorts ( as in the reimers et al5 study )  . 
regardless , the authors should be commended for assembling these large data sets that help to inform on the prognostic implications of cdk12 - mutated prostate cancer . furthermore , these series include the largest cohort to date of outcomes in cdk12 - mutated prostate cancer after immune checkpoint blockade , shedding light on its predictive potential . 
two phase iii , randomized , placebocontrolled trials of ipilimumab in the postdocetaxel and predocetaxel mcrpc settings failed to meet their primary end point of os.6 , 7 pembrolizumab alone in mcrpc leads to low rates of either psa or radiographic response.8 while the prevalence of mismatch repair ( mmr ) deciency in crpc is low ( , 5% ) , 9 deciencies in mmr proteins are associated with higher psa and objective responses to pd - 1 / pd - l1 inhibitors.10 the response rate to pd - 1 / pd - l1 inhibitors in crpc is higher than the prevalence of mmr deciency , and some patients achieve responses without harboring mmr - decient tumors , which suggests that there are other currently undetermined biomarkers of clinical benet . cdk12 biallelic inactivating mutations lead to the formation of ftd , which results in increased prevalence of gene fusions , elevated antigen load , and inltration of t cells into the tumor microenvironment.2 these ndings suggest that tumors that harbor cdk12 loss may be immunogenic and susceptible to immunotherapy . in a small cohort of 4 pretreated patients with cdk12 - mutant mcrpc treated with an anti - pd - 1 immune checkpoint inhibitor , 2 experienced a marked psa decline.2 this suggests the potential of cdk12 as a predictive biomarker for immunotherapy , a hypothesis that can be scrutinized further with new data from the schweizer and antonarakis series . in the antonarakis series , 9 heavily pretreated men with mcrpc received a pd - 1 inhibitor , of whom 3 ( 33% ) achieved a conrmed psa50 response . 
in the schweizer series , 19 patients received diverse immunotherapy regimens , including pembrolizumab , atezolizumab , nivolumab and ipilimumab , durvalumab and tremelimumab , and atezolizumab and radium - 223 . 
objective responses were not assessed in this series . heterogeneity in patient population , prior therapies , and treatment regimens likely account for the differences in clinical outcomes to immune checkpoint inhibitors observed in these two studies . 
furthermore , the rate of objective responses and duration of disease control remain unclear . ultimately , prospective data are needed to further dene the role of cdk12 as a potential biomarker to predict response and benet to immune checkpoint inhibitors in advanced prostate cancer . 
toward this end , multiple prospective clinical trials are currently under way to explore outcomes to immune checkpoint inhibition in patients with prostate cancer who harbor cdk12 alterations or other dna damage repair defects ( table 1 )  . 
of note , these studies are prospectively enriching for genomic alterations of interest and cover a spectrum of disease states , including biochemically recurrent prostate cancer , metastatic hormone - sensitive prostate cancer , and mcrpc . 
for example , cdk12 loss is associated with amplication of cell cycle genes ccn1 and cdk4 , which suggests that cdk12 - mutated tumors may be susceptible to cdk4 / 6 inhibition.11 the combination of atezolizumab and abemaciclib is being studied in cdk12 - altered mcrpc ( clinicaltrials.gov identier : nct04272645 )  . optimal patient selection using clinical and genomic / molecular characteristics along with effective combination strategies are likely to move the needle for immunotherapy strategies in prostate cancer . 
despite the limitations of retrospective analysis , these data with rich clinical annotation suggest that patients with prostate cancer with cdk12 mutations have a poor prognosis and that novel therapies are needed . 
wei honoraria : onclive research funding : bristol - myers squibb travel , accommodations , expenses : corvus pharmaceuticals no other potential conicts of interest were reported . references blazek d , kohoutek j , bartholomeeusen k , et al : the cyclin k / cdk12 complex maintains genomic stability via regulation of expression of dna damage response genes . 
cell 173 : 1770 - 1782.e14 , 2018 schweizer mt , ha g , gulati r , et al : cdk12 - mutated prostate cancer : clinical outcomes with standard therapies and immune checkpoint blockade . 
eur urol 77 : 333 - 341 , 2020 kwon ed , drake cg , scher hi , et al : ipilimumab versus placebo after radiotherapy in patients with metastatic castration - resistant prostate cancer that had progressed after docetaxel chemotherapy ( ca184 - 043 ) : a multicentre , randomised , double - blind , phase 3 trial . 
lancet oncol 15 : 700 - 712 , 2014 beer tm , kwon ed , drake cg , et al : randomized , double - blind , phase iii trial of ipilimumab versus placebo in asymptomatic or minimally symptomatic patients with metastatic chemotherapy - naive castration - resistant prostate cancer . 
j clin oncol 38 : 395 - 405 , 2020 abida w , cheng ml , armenia j , et al : analysis of the prevalence of microsatellite instability in prostate cancer and response to immune checkpoint blockade . jama oncol 5 : 471 - 478 , 2019 10 . 
agarwal n , loriot y , mcgregor ba , et al : cabozantinib ( c ) in combination with atezolizumab ( a ) in patients ( pts ) with metastatic castration - resistant prostate cancer ( mcrpc ) : results of cohort 6 of the cosmic - 021 study . 
 acquired alk and ret gene fusions as mechanisms of resistance to osimertinib in egfr - mutant lung cancers introduction approximately 20% of patients with metastatic lung adenocarcinoma have somatic activating mutations in the epidermal growth factor receptor ( egfr ) gene , egfr . 
1 patients with egfr - mutant lung adenocarcinomas have a 70% response rate to first - line egfrtyrosine kinase inhibitor ( tki ) therapy ( ie , erlotinib , gefitinib , or afatinib ) .2 egfr t790m is the dominant resistance mechanism to earlier - generation egfr - tkis.3 osimertinib is approved by the us food and drug administration for the treatment of egfr - mutant lung cancers that have an acquired egfr t790m after failure of a previous egfr - tki4 and now is approved in the first - line setting as well.5 response to osimertinib eventually is followed by progression with known resistance mechanisms , including small - cell transformation3 , 6 ; acquired egfr mutations , including g796 / c797 , l792 and l718 / g7197 ; and non - egfr mediated resistance , including alterations / amplification in met , her2 , braf , mek , kras , and pik3ca . 
ngs with archerdx and msk - impact confirmed an acquired eml4 - alk fusion ( eml4 exons 1 through 6 fused with alk exons 20 through 29 ; c.667 + 516 : eml4_c.3173 - 415 : alkinv ) in addition to the egfr exon19del and t790m mutations . 
the patient started combination treatment with osimertinib 80 mg daily and crizotinib 200 mg twice daily , remained on treatment with stable disease ( by recist version 1.1 ) , and continued to receive clinical benefit from treatment , with no report of toxicity . 
the preosimertinib biopsy was a fine - needle aspiration ( fna ) and was partially fragmented on staining , whereas the acquired resistance ( ar ) sample was a core - needle biopsy . 
the pretreatment egfr exon19del staining was performed with epidermal growth factor receptor ( egfr ) e746 ; the post - treatment ihc stained more diffusely for egfr exon19del , likely related to the egfr amplification ( fold change : 3.0 ) noted on next - generation sequencing with msk - impact . 
 the ihc data suggest the presence of the alk rearrangement and egfr mutation within the same cell ( however , this cannot be confirmed by targeted next - generation sequencing because of technical limitations )  . 
 case 2 a 68 - year - old woman , who was never a smoker , presented with a 9 - cm right apical lung mass , right hilar lymphadenopathy , and a left temporal lobe mass ( staging t4n1m1b )  . 
 ( of note , no alk rearrangement was detected on ngs or fish . ) the patient started treatment with osimertinib and had an initial disease response , but oligoprogression occurred in the lung after 6 months . 
 ( a ) osimertinib and crizotinib in patient case 1 after one cycle ( 28 days ) of treatment ; image shows overall stable disease ( 0% best response )  . 
 ( b ) osimertinib and alectinib in patient case 2 after one cycle ( 28 days ) of treatment ; image shows regression of the dominant right upper lobe mass ( 25% reduction )  . precombination targeted therapy postcombination targeted therapy precombination targeted therapy postcombination targeted therapy l858r and t790m mutations as well as the new eml4 - alk rearrangement ( eml4 exons 1 through 2 fused to alk exons 20 through 29 ; c.208 + 4890 : eml4_c.3173 - 373 : alkinv ; fig 3b ; appendix table a2 )  . 
the patient started treatment with alectinib 300 mg twice daily and osimertinib 80 mg daily ; the first interval scan showed a decrease in the dominant right lung mass ( 25% reduction by recist version 1.1 ) as well as ongoing clinical benefit and no report of toxicity at the last follow - up . 
a right lung biopsy showed the known egfr l747s and l858r mutations as well as a new ncoa4 - ret fusion on msk - impact ; the fusion was confirmed by archerdx . 
 ( * ) treatment ongoing as of april 1 , 2018 . which showed that the fusion partner did not influence ret activity in cell lines.19 , 20 each condition was assayed in triplicate in at least two independent experiments . 
the pc9 cells were used to assess the effect of ret fusions on sensitivity of growth and apoptosis ( caspase 3 / 7 activity ) in the presence of osimertinib and cabozantinib . 
however , erk1 / 2 phosphorylation was not sensitive to osimertinib treatment in pc9 - pcx4.1ccdc6 - ret cells but was diminished with cabozantinib treatment ( fig 4b )  . 
these results suggest that ret rearrangements can cause bypass activation of growth - promoting pathways in cells that express oncogenic egfr . growth of pc9 cells that stably expressed ccdc6 - ret or kif5b - ret rearrangements was at least 10 - fold less sensitive to osimertinib than pc9 - pcx4.1empty cells were ( 50% inhibitory concentrations described in fig 4e )  . 
expression of ret fusions in pc9 cells prevented osimertinib - induced activation of caspase 3 / 7 , similar to the effect on growth rate ( fig 4f [ left panel ] )  . 
treatment of control - group pc9 cells or pc9 - ret cells with cabozantinib did not lead to activation of caspase 3 / 7 ( fig 4f [ left panel ] )  . 
these results suggest that ret rearrangements can induce resistance to osimertinib in cells that have egfr mutations and that response to osimertinib is restored when it is used in combination with cabozantinib . in conclusion , osimertinib is now a first - line treatment for metastatic egfr - mutant lung cancers.5 although potential resistance mechanisms to osimertinib have been identified , these have primarily been observed after later - line osimertinib in the setting of egfr t790m mutations and have focused on ctdna.9 - 11 many alterations identified as potential resistance mechanisms are seen concurrently with egfr in pretreatment samples , including amplifications of met and her2 as well as pik3ca mutations , which makes the comparison with pretreatment tissue critical to identify truly acquired alterations.13 as ngs becomes standard of care , it is feasible and fruitful to molecularly profile tumors broadly before treatment and at progression to identify acquired alterations that mediate resistance . it remains unclear whether mechanisms of resistance to first - line osimertinib will differ from mechanisms of resistance to later - line osimertinib or earlier - generation egfr - tkis . 
osimertinib is a potent mutant - egfr inhibitor that inhibits egfr t790m , so there is a potential to see novel on - target21 , 22 and off - target23 - 25 resistance mechanisms . 
this is corroborated by the low frequency ( 15% to 25% ) of egfr c797s and other acquired egfr alterations after osimertinib use compared with 60% frequency of acquired egfr t790m after use of earlier generation egfr - tkis.9 , 13 , 22 we may see new acquired alterations not previously seen or seen rarely with earlier - generation egfr - tkis . 
 moreover , although cabozantinib can inhibit growth of egfrand ret - mutant / rearranged cell lines , a combination of osimertinib and cabozantinib induced apoptosis . at our institution , among 174 patients with egfr - mutant lung cancer in whom ngs was performed on tumor tissue after progression developed during treatment with erlotinib or afatinib , we found no alk or ret fusions . 
it is unknown whether this potential enrichment of acquired fusions is related to the more potent egfr inhibition of osimertinib or to the later - line setting after multiple lines of egfr inhibition . 
pc9 cells were infected with lentivirus that harbored an empty plasmid ( pcx4.1 - empty ) or pcx4.1 with either ccdc6 - ret or kif5b - ret complementary dna ( cdna ) , and cells that expressed the plasmids were selected to generate stable cell lines . 
 ( a ) cell extracts were immunoblotted ( indicated by ib ) for ( left panel ) total ret ( left panel ) or ( right panel ) phosphorylated ret . 
 ( b ) cell lines were treated with either osimertinib 0.05 m or cabozantinib or 0.25 m for 1 h ; cell extracts were prepared and then immunoblotted for the indicated proteins . 
 ( c ) cells were treated with increasing concentrations ( as indicated on the figure ) of ( left panel ) osimertinib or ( right panel ) cabozantinib for 96 h , and then growth was determined . 
 ( f ) cells were treated with the indicated concentrations of ( left panel ) osimertinib or ( right panel ) cabozantinib for 48 hours , and then caspase 3 / 7 enzymatic activity determined . 
 ( g ) pc9 cells that expressed either pcx4.1 - empty vector or pcx4.1ccdc6 - ret were treated with the indicated concentrations of cabozantinib in the presence of osimertinib 0.1 m for 48 h , and then caspase 3 / 7 enzymatic activity was determined . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . michael offin no relationship to disclose romel somwar research funding : helsinn healthcare travel , accommodations , expenses : helsinn healthcare natasha rekhtman no relationship to disclose ryma benayed no relationship to disclose jason c . 
li consulting or advisory role : roche , biosceptre international , thermofisher scientific , mersana , guardant health research funding : roche ( inst ) , genentech ( inst ) , illumina ( inst ) , biomed valley discoveries ( inst ) , astrazeneca ( inst ) , grail ( inst ) gregory j . 
riely research funding : novartis ( inst ) , roche ( inst ) , genentech ( inst ) , millenium ( inst ) , glaxosmithkline ( inst ) , pfizer ( inst ) , infinity pharmaceuticals ( inst ) , ariad ( inst ) patents , royalties , other intellectual property : patent application submitted covering pulsatile use of erlotinib to treat or prevent brain metastases ( inst ) travel , accommodations , expenses : merck sharp & dohme charles m . 
kris consulting or advisory role : astrazeneca , regeneron william travis speakers ' bureau : genentech alexander drilon consulting or advisory role : ignyta , loxo , tp therapeutics , astrazeneca , pfizer , blueprint medicines , genentech , roche , takeda , helsinn therapeutics , beigene maria e . 
arcila consulting or advisory role : astrazeneca travel , accommodations , expenses : astrazeneca , invivoscribe , raindance technologies marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network , boehringer ingelheim , astrazeneca ( tagrisso rfp advisory committee ) research funding : loxo ( inst ) helena a . 
jui , the chinese university of hong kong , sha tin new town , hong kong special administrative region , china . supported in part by the national cancer institute of the national institutes of health grants no . 
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national cancer institute : osimertinib and necitumumab in treating patients with egfr - mutant stage iv or recurrent nonsmall - cell lung cancer who have progressed on a previous egfr tyrosine kinase inhibitor ( nct02496663 )  . 
okamoto k , kodama k , takase k , et al : antitumor activities of the targeted multi - tyrosine kinase inhibitor lenvatinib ( e7080 ) against ret gene fusiondriven tumor models . 
arai s , kita k , tanimoto a , et al : in vitro and in vivo anti - tumor activity of alectinib in tumor cells with ncoa4 - ret . 
klempner sj , mehta p , schrock ab , et al : cis - oriented solvent - front egfr g796s mutation in tissue and ctdna in a patient progressing on osimertinib : a case report and review of the literature . 
ou si , horn l , cruz m , et al : emergence of fgfr3 - tacc3 fusions as a potential bypass resistance mechanism to egfr tyrosine kinase inhibitors in egfr - mutated nsclc patients . 
detailed molecular and immunohistochemical characterization of patient case 1 presented timing of molecular test diagnosis type of molecular test result tissue pcra impactb 15base pair egfr exon 19 deletion detected egfr exon19 p.e746_a750del ; tp53 exon10 p.r337l ; atr exon41 p.d2331y detected erlotinib progression cfdnac egfr t790m detected ( 0.79% ) d tissue pcrc egfr t790m detected ( 15.15% ) d alk : negative ; egfr exon 19 deletion : positive osimertinib + necitumumab ihce alk : positive ; egfr exon 19 deletion : positive progression impactb ihce impactb archerdxf fishg egfr exon19 p.e746_a750del ; egfr exon20 p.t790m ; kmt2c exon38 p.d2690n ; rad51b exon7 p.l209v detected no alk fusion detected no evidence of alk rearrangement . 
the single base extension product is detected by tandem mass - spectrometry on a sequenom massarray spectrometer ( sequenom , san diego , ca )  . bmsk - impact ( integrated mutation profiling of actionable cancer targets ) was used to identify specific mutations in 468 genes . cdigital pcr of cfdna and / or tissue amplification of part of egfr exon 20 in the presence of fluorescent probes specific to the wild - type and mutant alleles . dratio of mutant allele / ( mutant + wild - type allele )  . eimmunohistochemistry ( ihc ) for pertinent proteins performed with the following clones : egfr - exon19del ( egfr - e746 ) : clone 6b6 ; alk : clone d5f3 . farcher fusionplex custom solid panel ( archer fusionplex , boulder , co ) uses the anchored multiplex pcr used to detect gene fusions in tumor samples consisting of 62 cancer - related genes previously reported to be involved in chromosomal rearrangements . 
the single base extension product is detected by tandem mass - spectrometry on a sequenom massarray spectrometer . carcher fusionplex custom solid panel uses the anchored multiplex pcr used to detect gene fusions in tumor samples consisting of 62 cancer - related genes previously reported to be involved in chromosomal rearrangements . 
 lynch syndrome and breast cancer risk : weighing the data wendy kohlmann , ms , cgc1 as we continue to expand our understanding of the genotype - phenotype correlations in lynch syndrome ( ls ) , we must be cautious in the design of studies and interpretation of ndings to responsibly serve our patients with ls with as accurate information as possible . 
although pathogenic variants ( pvs ) in the mismatch repair ( mmr ) genes mlh1 , msh2 / epcam , msh6 , and pms2 are all associated with ls , each gene is associated with a unique risk prole . 
mlh1 and msh2 have been associated with the highest risk for ls - associated cancers , whereas risk for colorectal cancer is signicantly lower for msh6 and pms2 pv carriers.1 in studies of high - risk colorectal cancer cohorts , msh6 and pms2 pvs are less commonly identied than mlh1 and msh2 , but use of clinical multigene panel testing is expanding the identication of carriers of pvs in these genes , often in individuals and families lacking the classic pattern of cancers associated with ls.2 , 3 the large data sets being generated by genetic testing laboratories can be assets for research and can allow for gene - specic risk analysis . however , analytical caution needs to be taken to account for the biases that arise from patient selection for clinical genetic testing , and family history and clinical data provided to commercial laboratories may be incorrect or inaccurate . in the article accompanying this editorial , stoll et al2 add data from a large cohort of mmr carriers and demonstrate a successful strategy for using commercial laboratory data to assess associations between pvs and cancer risk . 
however , to account for the study population having a more signicant cancer history than the general population , pv carriers were also compared with other subpopulations within the laboratory cohort , including women who were negative for a pv in any cancer predisposition gene when testing was performed for evaluation for hereditary breast or ovarian cancer ( hboc ) and / or ls , evaluation for hboc only , and evaluation for ls only . no excess in breast cancer risk was noted when compared with seer or any laboratory comparison group for any of the mmr genes . in the cohort in the study by stoll et al , 2 pvs in msh6 and pms2 were more common in women undergoing genetic testing for the indication of hboc than in women being tested as a result of suspicion of ls . other studies of clinical testing populations have also found a predominance of msh6 and pms2 pvs in individuals meeting hboc guidelines rather than ls.4 however , this pattern does not conrm that these genetic variants increased the risk for breast cancer . this distribution of pvs in laboratory cohorts likely reects that , other than msh6 - associated endometrial cancer risk , pvs in msh6 and pms2 confer only a modest risk for colorectal and other lsassociated cancers . 
stoll et al2 have provided a systematic examination of a large laboratory cohort using the proper comparison groups to put the frequency of msh6 and pms2 pvs by testing criteria into context and have provided strong evidence against the association of breast cancer with any of the mmr genes . before widespread panel testing , most analyses of breast cancer risk and ls had come from small studies.5 data from studies with larger samples are now becoming available . 
signicant breast cancer history may be a result of other factors , and genetic testing of other affected family members may be warranted to determine whether there is an additional pv segregating in the family and contributing to that cancer risk . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . wendy kohlmann employment : biofire diagnostics ( i ) no other potential conicts of interest were reported . references dominguez - valentin m , sampson jr , sepp al a tt , et al : cancer risks by gene , age , and gender in 6350 carriers of pathogenic mismatch repair variants : findings from the prospective lynch syndrome database . 
cancer epidemiol biomarkers prev 26 : 404 - 412 , 2017 espenschied cr , laduca h , li s , et al : multigene panel testing provides a new prospective on lynch syndrome . 
mller p , sepp al a tt , bernstein i , et al : cancer risk and survival in path_mmr carriers by gene and gender up to 75 years of age : a report from the prospective lynch syndrome database . 
gut 67 : 1306 - 1316 , 2018 ten broeke sw , van der klift hm , tops cmj , et al : cancer risks for pms2 - associated lynch syndrome . 
j clin oncol 36 : 2961 - 2968 , 2018 slavin tp , maxwell kn , lilyquist j , et al : the contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk . 
npj breast cancer 3 : 22 , 2017 therkildsen c , ladelund s , smith - hansen l , et al : towards geneand gender - based risk estimates in lynch syndrome ; age - specic incidences for 13 extracolorectal cancer types . 
evans dg , woodward er , lalloo f , et al : are women with pathogenic variants in pms2 and msh6 really at high lifetime risk of breast cancer ? genet med 13 . 
2018 : is breast cancer truly caused by msh6 and pms2 variants or is it simply due to a high prevalence of these variants in the population ? genet med 21 : 256 - 257 , 2019 14 . 
 c treatment of patients with lobular breast cancer harboring human epidermal growth factor receptor 2 mutation with her2 - directed therapy introduction human epidermal growth factor receptor 2 ( her2 ) is a tyrosine kinase encoded by the erbb2 gene . 
mutations in cdh1 have been reported in 62% to 100% of cases of invasive lobular carcinoma , and many investigators consider a mutation in cdh1 to represent a genomic alteration characteristic of lobular carcinoma.5 mutations in erbb2 have been reported in six of 22 ( 27% ) cdh1 - mutated lobular carcinomas.5 the presence of activating somatic mutations in erbb2 could provide a rationale for her2 targeted therapy , 4 , 6 and there have now been several case reports of successful treatment of patients with such mutations with her2 targeted therapy.7 - 10 we initially identified a patient with progressive metastatic lobular carcinoma resistant to chemotherapy and hormonal regimens , whose liver metastasis harbored both cdh1 and erbb2 mutations . 
on the basis of the presence of the erbb2 mutation , we elected to treat the patient with her2 - targeted therapy , and she has achieved significant and durable responses to three consecutive her2 - targeted therapies during a 2.5year period of time , despite the lack of erbb2 amplification . after our experience with this patient , we then reviewed all of our patients with metastatic lobular carcinoma of the breast for whom next generation sequencing had been obtained . 
on the basis of reports that erbb2 exon 20 insertion has been shown to confer sensitivity to trastuzumab and tyrosine kinase inhibitors , we elected to treat the patient with dual her2 blockade with the combination of trastuzumab and pertuzumab.2 , 3 treatment was continued for 8 months , with major subjective clinical improvement , which included better appetite and resolution of abdominal pa clinical response was confirmed by ct scan on april 4 , 2016 showing a significant decrease in hepatic lesions , which has been maintained ( fig 2 )  . 
by july 2017 , the carcinoembryonic antigen , ca19 - 9 , and alkaline phosphatase increased aga ado - trastuzumab emtansine was discontinued , and she was started on third - line anti - her2directed therapy with lapatinib and capecitabine in july 2017 . she once again demonstrated significant response in all four tumor markers as well as her liver enzymes ( figs 3 and 4 )  . 
 only one of the five was also her2 amplified and had received prior her2 - directed therapy . all five patients have received her2 - targeted therapy for their metastatic disease , and four have achieved clinical responses as defined by response evaluation criteria in solid tumors ( recist ; tables 1 and 2 )  . 
liver biopsy showing infiltrating cords and nests of tumor cells ( july 2011 )  . in december 2010 , tumor markers were noted to be elevated , and a magnetic resonance imaging scan of the abdomen demonstrated hepatic metastases . 
 subsequent positron emission tomography / computed tomography ( ct ) scan showed progression of her liver lesions , and an ultrasound guided biopsy of the liver in july 2011 confirmed metastatic lobular carcinoma ( fig 1 )  . 
as with the primary tumor , the tumor specimen from liver biopsy was estrogen receptor and progesterone receptor positive and her2 negative by ihc . during the next 2 years she was treated with several chemotherapy regimens , including carboplatin and gemcitabine , capecitabine , and eribul she experienced progression on these regimens after initial responses of approximately 6 months , and progression of liver metastases was seen in august of 2014 . eribulin was then discontinued and she was started on tamoxifen , followed by exemestane and everolimus . 
her disease did not respond to these treatments , and she presented to cancer treatment centers of america at midwestern regional medical center for a second opinion in march of 2015 with extensive hepatic and bone metastases . 
comprehensive metabolic panel : alkaline phosphatase , ast , and alt . has received only two cycles of treatment , and it is too early to evaluate for radiologic response , although her tumor markers have fallen significantly . discussion we have identified erbb2 mutations in five of 16 patients ( 31% ) with cdh1 - mutated metastatic lobular carcinoma , an incidence similar to that previously reported.5 our experience suggests that such patients may well respond favorably to her2 - targeted therapy despite the absence trastuzumab and pertuzumab of her2 amplification . 
if the prevalence of erbb2 mutations is confirmed in larger studies , it would indicate that a significant number of patients with metastatic lobular carcinoma are likely to benefit from her2 - targeted therapy . 
this , together with the fact that one patient ( patient 3 ) had received no systemic treatment before biopsy , suggests that erbb2 mutation may be an intrinsic characteristic of lobular carcinoma of the breast and does not necessarily arise from prior systemic therapy . there is currently considerable interest in the possibility that patients with her2 - mutated breast cancer can be successfully treated with drugs already approved by the us food and drug administration for her2 - amplified disease . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . speakers ' bureau : astrazeneca , pfizer , r - pharm us , agendia bradford a . 
ali employment : foundation medicine leadership : incyte stock and other ownership interests : exelixis , blueprint medicines , agios , genocea biosciences patents , royalties , other intellectual property : patents via foundation medicine , patents via seres health on microbiome in non - neoplastic disease ( i ) ankur r . 
parikh consulting or advisory role : foundation medicine maurie markman employment : cancer treatment centers of america consulting or advisory role : amgen , celgene , crititech , pfizer , myriad genetics , merck , clovis oncology dennis l . 
 expert testimony : actavis , acorn research , apotex , emcure pharmaceuticals , dr reddy 's laboratories , fresenius kabi , hikma pharmaceuticals , hospira , pharma international , sagent pharmaceuticals , strides arcolab , sun pharma , wockhardt jeffrey s . 
ross employment : foundation medicine leadership : foundation medicine references stock and other ownership interests : foundation medicine research funding : foundation medicine arun kadamkulam syriac no relationship to disclose sara j . 
wolff ac , hammond me , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
ross js , slodkowska ea , symmans wf , et al : the her - 2 receptor and breast cancer : ten years of targeted anti - her - 2 therapy and personalized medicine . 
ross js , wang k , sheehan ce , et al : relapsed classic e - cadherin ( cdh1 ) - mutated invasive lobular breast cancer shows a high frequency of her2 ( erbb2 ) gene mutations . 
ali sm , alpaugh rk , downing sr , et al : response of an erbb2 - mutated inflammatory breast carcinoma to human epidermal growth factor receptor 2 - targeted therapy . 
chumsri s , weidler j , ali s , et al : prolonged response to trastuzumab in a patient with her2nonamplified breast cancer with elevated her2 dimerization harboring an erbb2 s310f mutation . 
ben - baruch ne , bose r , kavuri sm , et al : her2 - mutated breast cancer responds to treatment with single - agent neratinib , a second - generation her2 / egfr tyrosine kinase inhibitor . 
shih j , bashir b , gustafson ks , et al : cancer signature investigation : erbb2 ( her2 ) - activating mutation and amplification - positive breast carcinoma mimicking lung primary . 
hyman dm , piha - paul sa , rodon j , et al : neratinib in her2 or her3 mutant solid tumors : summit , a global , multi - histology , open - label , phase 2 basket study . 
 use of cabozantinib in a patient with egfr - mutated nonsmall - cell lung cancer harboring acquired ccdc6 - ret fusion young kwang chae , md , mph , mba1 ; pedro viveiros , md1 ; caio t . 
nine months after initiation of therapy , however , the patient displayed growth of the known right lung lesion as well as a new subcentimeter nodule in the right lower lobe . 
genomic proling with ctdna next - generation sequencing ( ngs ; guardant health , redwood city , ca ) identied an egfr t790m mutation with a variant allele frequency of 0.2% ( fig 1b ) the patient was then started on osimertinib . 
ct scans after 7 months showed a new right - sided pleural effusion , with an associated dense consolidation that was considered to represent either atelectasis or therapy - related pneumonitis . 
repeat ctdna ngs no longer detected the t790m mutation but presented new mutations in pik3ca ( g118d , 0.7% ) and tp53 ( h193l , 0.4% ) as well as a ccdc6 - ret rearrangement with a frequency of 0.1% ( fig 1c )  . 
instead , she opted to continue osimertinib with the addition of bevacizumab . fourteen months from the start of osimertinib , the patient had recurrent malignant effusions with possible pleural - based metastases concerning for disease progression . 
the tumor response mapping illustrates the mutant allele percentage ( percent cell - free dna [ cfdna ] ) of observed somatic variants at each sample submission time point . the somatic alteration burden value refers to the maximum percentage cfdna detected at each time point and has been used to illustrate how tumor progeny is responding to the selective pressure of treatment . 
three months after cabozantinib initiation , ct imaging still could not visualize her primary tumor but demonstrated decreased burden of bilateral pulmonary nodules as well as a discrete increase in pre - existent liver metastases . 
the patient reported marked improvement of dyspnea and pain as well as increased activity tolerance . discussion we report emergence of ccdc6 - ret rearrangement in the setting of osimertinib use in a patient with egfr t790mmutated nsclc . 
the c797s egfr mutation is detected in ctdna in nearly 40% of these patients and has been implicated in osimertinib resistance.9 - 12 other less commonly reported mutations include met , erbb2 ( human epidermal growth factor receptor ) , braf , egfr ( l718q ) , kras , and g12s.6 , 13 - 16 many of these occurred with the loss of egfr t790m mutation , suggesting that t790m - positive clones were suppressed , but t790m wild - type cells with other driver mutations were able to mediate resistance.6 epithelial - mesenchymal transition transformation have also been changes and small - cell reported in some cases of resistance . 
nonetheless , approximately 15% to 20% of the cases , the mechanism of acquired resistance to egfr tkis is yet to be discovered . in this patient , we identied an uncommon acquired ccdc6 - ret rearrangement that is also found in approximately 1% to 2% of nsclcs at primary diagnosis.17 this rearrangement raises interest as a result of its possible role in resistance , emerging under the selective pressure of treatment . 
fusion of the ret tyrosine kinase domain with an upstream coiled - coil domain has been shown to promote self - dimerization , resulting in constitutive signaling via prosurvival and proliferative pathways.18 ccdc6 - ret rearrangements accounted for 23% of genetic alterations in a recent international registry of ret - rearranged lung cancers.19 at the initial diagnosis , de novo ret rearrangements have been considered to be mutually exclusive with other common driver mutations , such as those in egfr , braf , kras , and alk.17 trials assessing clinical and pathologic characteristics of these de novo ret rearrangement suggest increased frequency in nonsmokers and patients with poorly differentiated adenocarcinomas , solid subtype , younger age , asian origin , or small tumors ( 3 cm or smaller ) with n2 disease.17 , 20 , 21 germline gain - of - function mutations in ret predispose carriers to multiple endocrine neoplasia type 2 , whereas somatic gain - of - function ret mutations have been commonly reported in sporadic medullary thyroid cancer and papillary thyroid cancer.22 , 23 several commercially available multikinase inhibitors , such as vandetanib , cabozantinib , sorafenib , sunitinib , lenvatinib , and ponatinib , have shown activity against ret kinase ( table 1 )  . 
one example is loxo - 292 , which had a good toxicity prole and showed activity against retmutated or including those with previous resistance to multikinase inhibitors ( table 2 )  . translocated tumors , cabozantinib is recommended by the national comprehensive cancer network for treatment of medullary thyroid and clear cell renal cancers . 
its use in ret - mutated tumors is approved by the us food and drug administration and has been reported in a phase ii clinical trial of patients with nsclc.24 in this trial , an overall response rate of 28% was reported with a favorable safety prole . 
this dose has comparable plasma exposure ( area under the plasma concentration - time curve ) as the us food and drug administrationapproved dose of 140 mg per day used for the treatment of patients with metastatic medullary thyroid carcinoma.19 , 24 , 38 after 3 months of cabozantinib use in our patient , there was a reduction in ctdna level of ccdc6 - ret rearrangement and signicant improvement in reported symptoms . 
ctdna has been useful in demonstrating molecular response without the need for tissue biopsies and is a convenient way to assess for driver mutations and molecular response.39 absolute levels of ctdna have also been signicantly correlated with tumor volume measured by ct and positron emission tomographyct imaging.40 , 41 however , it has so far been used as an exploratory measure of response . 
clinical and imaging data remain sovereign . it is still unclear whether the presence of a rare yet targetable mutation represents a putative resistance mechanisfor this patient , the ret rearrangement was possibly not the only mechanism of acquired resistance . loss of t790m coincided with the emergence of several mutations in oncogenic drivers , although none have been proven to be associated with osimertinib resistance . 
furthermore , retrospective data sets can complement clinical trial results , as exemplied in a recently published global registry of retdirected treatment outcomes.19 in conclusion , we report a case of acquired ccdc6 - ret rearrangement in a patient with egfr - mutated nsclc . treatment with the ret inhibitor cabozantinib led to signicant clinical improvement and associated reduction in levels of ccdc6 - ret detected from ctdna . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . young kwang chae consulting or advisory role : foundation medicine , boehringer ingelheim , biodesix , counsyl , astrazeneca , guardant health , takeda , genentech , immuneoncia , hanmi speakers ' bureau : genentech , merck , astrazeneca , eli lilly research funding : abbvie , bristol - myers squibb , lexent bio , freenome , biodesix travel , accommodations , expenses : hanmi wade t . 
iams travel , accommodations , expenses : emd serono no other potential conicts of interest were reported . references diaz la jr , bardelli a : liquid biopsies : genotyping circulating tumor dna . 
j clin oncol 32 : 579 - 586 , 2014 pao w , chmielecki j : rational , biologically based treatment of egfr - mutant non - small - cell lung cancer . 
nat rev cancer 10 : 760 - 774 , 2010 kosaka t , yatabe y , endoh h , et al : analysis of epidermal growth factor receptor gene mutation in patients with non - small cell lung cancer and acquired resistance to getinib . 
clin cancer res 12 : 5764 - 5769 , 2006 j anne pa , yang jc , kim dw , et al : azd9291 in egfr inhibitor - resistant non - small - cell lung cancer . 
n engl j med 372 : 1689 - 1699 , 2015 soria jc , ohe y , vansteenkiste j , et al : osimertinib in untreated egfr - mutated advanced non - small - cell lung cancer . 
n engl j med 378 : 113 - 125 , 2018 planchard d , loriot y , andr e f , et al : egfr - independent mechanisms of acquired resistance to azd9291 in egfr t790m - positive nsclc patients . 
j clin oncol 31 : 3639 - 3646 , 2013 choueiri tk , escudier b , powles t , et al : cabozantinib versus everolimus in advanced renal - cell carcinoma . 
 cabozantinib in patient with nsclc with acquired ccdc6 - ret fusion 82 : 294 - 298 , 2013 e121 - e123 , 2016 2012 35 : 1403 - 1410 , 2017 2014 10 . 
yu ha , tian sk , drilon ae , et al : acquired resistance of egfr - mutant lung cancer to a t790m - specic egfr inhibitor : emergence of a third mutation ( c797s ) in the egfr tyrosine kinase domajama oncol 1 : 982 - 984 , 2015 12 . 
niederst mj , hu h , mulvey he , et al : the allelic context of the c797s mutation acquired upon treatment with third - generation egfr inhibitors impacts sensitivity to subsequent treatment strategies . 
klempner sj , bazhenova la , braiteh fs , et al : emergence of ret rearrangement co - existing with activated egfr mutation in egfr - mutated nsclc patients who had progressed on rstor second - generation egfr tki . 
sun j - m , ahn m - j , choi y - l , et al : clinical implications of t790m mutation in patients with acquired resistance to egfr tyrosine kinase inhibitors . 
gautschi o , milia j , filleron t , et al : targeting ret in patients with ret - rearranged lung cancers : results from the global , multicenter ret registry . 
lin c , wang s , xie w , et al : the ret fusion gene and its correlation with demographic and clinicopathological features of non - small cell lung cancer : a metaanalysis . 
yeganeh mz , sheikholeslami s , dehbashi behbahani g , et al : skewed mutational spectrum of ret proto - oncogene exon 10 in iranian patients with medullary thyroid carcinoma . 
yakes fm , chen j , tan j , et al : cabozantinib ( xl184 ) , a novel met and vegfr2 inhibitor , simultaneously suppresses metastasis , angiogenesis , and tumor growth . 
wells sa jr , robinson bg , gagel rf , et al : vandetanib in patients with locally advanced or metastatic medullary thyroid cancer : a randomized , double - blind phase iii trial . 
motzer rj , hutson te , glen h , et al : lenvatinib , everolimus , and the combination in patients with metastatic renal cell carcinoma : a randomised , phase 2 , open - label , multicentre trial . 
cortes je , kim dw , pinilla - ibarz j , et al : ponatinib efcacy and safety in philadelphia chromosome - positive leukemia : final 5 - year results of the phase 2 pace trial . 
drilon ae , subbiah v , oxnard gr , et al : a phase 1 study of loxo - 292 , a potent and highly selective ret inhibitor , in patients with ret - altered cancers . 
drilon a , rekhtman n , arcila m , et al : cabozantinib in patients with advanced ret - rearranged non - small - cell lung cancer : an open - label , single - centre , phase oncol 36 , 2018 ( suppl 15 ; abstr 102 ) 2 , single - arm trial . 
thompson jc , yee ss , troxel ab , et al : detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next - generation sequencing of cell - free circulating tumor dna . 
nat med 20 : 548 - 554 , 2014 imamura f , uchida j , kukita y , et al : monitoring of treatment responses and clonal evolution of tumor cells by circulating tumor dna of heterogeneous mutant egfr genes in lung cancer . 
this article describes medical oncologists condence with genomic testing and the association between genomic condence and test use . methods we used data from the 2017 national survey of precision medicine in cancer treatment to characterize oncologists condence with genomic testing . 
genomic condence was examined separately by type of test user : next - generation sequencing ( ngs ) only , gene expression ( ge ) only , both ngs and ge , or nonuser . predictors of genomic condence were examined with multinomial logistic regression . 
the association between genomic condence and test use was examined with multivariable linear regression . results more than 75% of genomic test users were either moderately or very condent about using results from multimarker tumor panel tests to guide patient care . 
condence with using multimarker tumor panel tests was highest among both ngs and ge test users , with 60.1% very condent in using test results , and lowest among ngs - only test users , with 38.2% very condent in using test results . 
oncologists were most condent in using single - gene tests and least condent in using whole - genome or - exome sequencing to guide patient care . genomic condence was positively associated with self - reported test use . 
in adjusted models , training in genomics , larger patient volume , and treating patients with solid tumors predicted higher genomic condence . onsite pathology services and receipt of electronic medical record alerts for genomic testing predicted lower genomic condence . conclusion oncologists condence varies by testing platform , patient volume , genomic training , and practice infrastructure . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction the landscape of cancer care has changed dramatically over the past decade as therapies are increasingly personalized to the molecular characteristics of a patients tumor.1 whereas genomic testing in oncology started with testing for single mutations in specic genes , advances in molecular proling has spurred the development and now widespread use of multimarker tumor panels.2 , 3 these panels assess different types of genomic alterations commonly detected through nextgeneration sequencing ( ngs ) and gene expression ( ge ) proles . 
such testing can identify therapeutic targets that can be treated with particular anticancer agents and determine when individuals are at higher risk for recurrence and may need therapy.3 , 4 although not standard practice as of yet , whole - genome or whole - exome sequencing is a newer technology that shows promise in informing patient care.4 the rapid proliferation of genomic testing has outpaced the development of practice guidelines on their appropriate use.5 , 6 multimarker tumor panel tests generate vast data about the biology of a patients tumor . 
in cases where the tumor can be successfully sequenced , an actionable target is identied for 39%90% of patients , a wide range that varies by patient population and the size of the panel test.7 - 10 although testing reports typically highlight multimarker panel potentially benecial therapies and relevant clinical trials , individual providers must navigate the process of communicating results to their patients and selecting an appropriate targeted therapy . 
furthermore , multimarker panel testing frequently identies variants of unknown signicance or germline variants , which can further complicate interpretation and care management.11 - 13 very few studies have examined providers attitudes about genomic testing . 
 oncologist condence in genomic testing context key objective this article provides unique data about oncologists in the united states and their condence in using results from commercially available multimarker tumor panel tests to guide decisions about patient treatment and management . knowledge generated oncologists who used both next - generation sequencing ( ngs ) and gene expression tests reported the highest condence in using multimarker tumor panel results to guide patient care ; oncologists who only used ngs tests had the lowest condence . 
oncologists with higher condence were more likely to report using results from multimarker tumor panels to inform patient care . relevance these ndings underscore the importance of genomic training and suggest other providerand practice - level factors that can be leveraged to build oncologists condence in using genomic testing . providers lack condence in using new genomic tests , which could limit the extent to which new tests are appropriately applied in practice.5 , 6 , 14 , 15 for example , providers report less condence in using multimarker tumor panels compared with single - gene tests , which are well established for certain patient populations.6 in addition , providers vary in their understanding of how to interpret and act upon results from whole - genome sequencing , which is not available outside of clinical trials.16 given the potential of precision oncology , there is a need to better understand oncologists attitudes and experiences with genomic testing and to identify factors that foster genomic testing condence . this article builds upon previous studies by describing oncologists condence with genomic testing and the association between condence and genomic test use over the past 12 months . 
oncologists condence in using multimarker tumor panels was rst examined in relation to their condence in using single - gene somatic tests and whole - genome or - exome sequencing . 
oncologists condence was then further characterized by exploring the individualand practice - level characteristics associated with condence and assessing whether their condence was associated with their use of multimarker tumor panels in practice . methods data and study population this study uses data from the national survey of precision medicine in cancer treatment , a nationally representative study of how oncologists use genomic testing in practice . eligible providers were identied from the american medical association physician masterle and selected using multistage probability sampling . 
additional information about the study design and methods have been published elsewhere.17 the survey is available upon request . measures the national survey of precision medicine in cancer treatment collected detailed data about oncologists condence in using genomic testing ; their genomic test use ; and demographics , specialty , and practice characteristics . 
condence was assessed for genomic tests for individual genes or chromosomal alterations ( referred to as single - gene tests ) , commercially available multimarker tumor panel tests , and whole - genome or - exome sequencing . 
response options were very condent , moderately condent , a little condent , or not at all condent . type of genomic test user was determined on the basis of oncologists reported use of 18 specic multimarker tumor panels in the past 12 months . 
the multimarker tumor panel tests included commercially available ge tests ( eg , oncotype dx breast ; genomic health , redwood city , ca ) , commercially available ngs tests ( eg , foundationone ; foundation medicine , cambridge , ma ) , and noncommercial panels performed at an academic medical center . 
training in genomics was also captured and dened as any formal training ( eg , instruction during residency / fellowship , professional lectures or seminars , symposiums , conferences , continuing medical education ) in the use of genomic testing . practice setting information was assessed with questions about the number of unique patients with cancer treated per month . 
responses were categorized as academic medical center or medical school and nonacademic setting . infrastructure to support precision medicine was assessed with a series of questions about whether the respondents primary practice had the following genomic testing services : onsite pathology , internal policies or protocols for use of genomic and biomarker testing , an electronic medical record ( emr ) that alerts when a genomic test is recommended , or a genomic / molecular tumor board . 
response options were categorized as yes and no or do not know . analysis weighted percentages were calculated to describe the sample and provider condence in using single - gene tests , multimarker tumor panel tests , and whole - genome or - exome sequencing . 
two sets of models were examined : one that adjusted for type of genomic test user and a second that simultaneously adjusted for type of genomic test user and the other physician and practice characteristics in the model . 
results are presented as odds ratios and adjusted percentages , also referred to as predicted margins.19 multivariable linear regression was then used to assess whether condence was associated with the percentage of patients for whom multimarker panel test results guided patient care in the past 12 months . 
multivariable models adjusted for years since graduation , sex , race , census region , specialty , training in genomics , practice setting , patient volume , and infrastructure for precision medicine . 
results are presented as adjusted percentages.19 all analyses were conducted using sudaan release 11.0 statistical software ( rti international , research triangle park , nc ) and weighted to account for the complex sampling approach and survey nonresponse . results sample characteristics the sample comprised 1 , 281 oncologists ( table 1 )  . 
participants reported a range of institutional resources to support precision medicine , with onsite pathology and internal policies or protocols for use of genomic and biomarker testing reported most frequently . genomic test use was reported by 95.4% of the sample , with 58.6% using both ngs and ge tests , 26.4% using only ngs tests , and 10.3% using only ge tests . condence using genomic tests in practice condence using multimarker tumor panel tests to guide decisions about patient treatment and management was highest among ngs and ge users , with 60.1% very condent and 35.6% moderately condent in using test results . 
even among oncologists who did not use multimarker tumor panel tests in the past 12 months , 27.1% were very condent and 37.8% were moderately condent in using test results ( fig 1b )  . in contrast to multimarker tumor panel tests , oncologists were more condent in using somatic single - gene tests and less condent in using whole - genome or - exome sequencing ( figs 1a and 1c )  . 
30 female male race white asian other region midwest northeast south west specialty solid and hematologic malignancies hematologic malignancies only solid tumors only training in genomics practice setting practice afliation academic medical center or medical school other practice settingb no . 
percentages may not sum to 100 because of missing data . bother settings include a medical center not afliated with a medical school , community hospital , ofce - based practice , health maintenance organization or integrated health care system , and other . guide decisions about patient treatment and management . in the fully adjusted models ( table 2 ) and in models that only adjusted for the type of genomic test user ( appendix table a1 ) , oncologists with training in genomics had higher condence than those who did not . 
likewise , oncologists who treated 100 patients per month had higher condence than those who treated , 49 patients per month . compared with oncologists who treated both solid and hematologic malignancies , those who only treated solid tumors were more condent , whereas those who treated only hematologic malignancies were less condent in using results of multimarker panel tests to guide patient care . oncologists who practice in institutions with onsite pathology or emr alerts for recommended genomic tests had lower condence than those who practice in institutions without onsite pathology or emr alerts . 
for example , ngs and ge users who were very condent used multimarker panel test results to inform care for 30.97% of their patients ( who had received testing ) compared with 18.44% among ngs and ge users who were not at all or a little condent . 
oncologists condence in using results from ( a ) single - gene tests , ( b ) commercially available multimarker somatic panels , and ( c ) whole - genome or - exome sequencing to guide decisions about patient treatment and management ( n = 1 , 281 )  . 
on the basis of 2 tests for independence , overall differences in condence among 4 types of genomic test users were statistically signicant at p , .001 for single - gene tests , multimarker tumor panels , and whole - genome or - exome sequencing . discussion the current study adds to the literature on provider attitudes about genomic testing . 
most ge panel tests are used to inform the treatment of patients with breast cancer , and there are clear guidelines for test results.20 , 21 for users of other multimarker tumor panel tests , the application of test results to patient management is guideline endorsed for a limited set of clinical indications . testing and the interpretation of tumor panel tests to inform care is more complicated than using single - gene tests . 
at the time the survey was conducted , there were only consensus recommendations for multimarker tumor panel testing in treating nonsmall - cell lung cancer.23 despite this , providers condence using multimarker tumor panel tests was relatively high , with  . 75% of genomic test users reporting that they were either moderately or very condent in using results to guide patient care . 
these ndings are consistent with other studies of provider condence.15 , 24 - 26 oncologists condence also varied by test type , which is consistent with previous research.6 single - gene tests have been used for several decades as companion diagnostics to targeted therapies ; thus , clinical appropriateness and utility are well prescribed.22 even among genomic test nonusers , more than half reported being very condent in using single - gene tests to guide patient care . 
presented are adjusted percentages from multivariable linear regression models that were adjusted for years since graduation , sex , race , census region , specialty , training in genomics , practice setting , and infrastructure for precision medicine . 
historically , providers who treat patients with a hematologic malignancy have technologies ( eg , ow cytometry for relied on different immunophenotyping ) or single - gene somatic tests to identify relevant targeted therapies.27 in contrast , use of multimarker tumor panel tests has been more common in the treatment of solid tumors.23 the importance of experience was also highlighted in our ndings that providers who treat  . 
100 patients per month had higher condence than providers who treat , 50 . providers in high - volume clinics have more opportunity to encounter patients for whom multimarker tumor panel tests are warranted . 
indeed , patient volume has been associated with better treatment outcomes in other settings.28 providers in lower - volume clinics may benet from additional resources , including clinical decision support , to strengthen evidence - based use of genomic testing in treatment planning.29 our ndings underscore the importance of genomic training to increase provider condence in using multimarker panel tests to guide patient care . 
training was dened broadly and included any instruction during residency or fellowship , professional lectures , conferences , or continuing medical education in the use of genomic testing . additional research should identify the specic competencies needed by different types of oncologists and be tailored to support providers who demonstrate relatively low condence in using genomic testing in practice . 
several categories of training could be pursued , including revision of the curricula of medical and nursing schools to provide training in genomics ; advanced training of physicians and other practitioners in genomics ; and continuing medical education to help practicing clinicians to stay up to date on advances in the eld.30 , 31 strategies such as case - based learning , clinical decision - support tools , administrative support for prior authorization , and clinical trial matching may be helpful in strengthening provider condence around genomic testing at the point of care and supporting successful implementation of precision medicine programs.29 genomic testing services available in an oncologists primary practice were either not associated with provider condence or , paradoxically , associated with lower condence . 
oncologists who reported that their practice had onsite pathology spent a lower percentage of time in delivering patient care ( data not shown ) , which could have inuenced their condence in using genomic test results to guide treatment decisions . 
oncologists in settings with onsite pathology could also have been more likely to have a research focus outside of genomics , although that is not something we can explore with our data . in addition , it is possible that other features of the practice setting or availability of appropriate training were more important drivers of provider condence . 
this work should address both the knowledge and the skills to use genomic testing appropriately and to act on test results as well as system - level resources to support precision oncology . our study results should be considered in light of a few limitations . 
first , providers were asked generally about their condence in using genomic test results to guide patient care ; we do not know how condence with a particular testing approach may vary on the basis of specic patient subgroups or clinical scenarios . 
however , participants are representative of practicing oncologists in the united states in terms of age , sex , and geographic location , and all analyses were weighted to adjust estimates for survey nonresponse . 
de moor , phd , mph , healthcare delivery research program , division of cancer control and population sciences , national cancer institute , 9609 medical center dr , room 3e438 , rockville , md 20850 ; e - mail : demoorjs@mail.nih.gov. support supported by the national cancer institute ( nci ) , national human genome research institute , and american cancer society through nci contract no . 
gray stock and other ownership interests : magenta therapeutics ( i ) consulting or advisory role : grail industries , magenta therapeutics ( i ) expert testimony : riley and associates no other potential conicts of interest were reported . references kalia m : biomarkers for personalized oncology : recent advances and future challenges . 
morganti s , tarantino p , ferraro e , et al : complexity of genome sequencing and reporting : next generation sequencing ( ngs ) technologies and implementation of precision medicine in real life . 
crit rev oncol hematol 133 : 171 - 182 , 2019 el - deiry ws , goldberg rm , lenz hj , et al : the current state of molecular testing in the treatment of patients with solid tumors , 2019 . 
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genet test mol biomarkers 19 : 657 - 665 , 2015 tan o , shrestha r , cunich m , et al : application of next - generation sequencing to improve cancer management : a review of the clinical effectiveness and costeffectiveness . 
j clin oncol serv res 9 : 131 , 2009 33 : 2753 - 2762 , 2015 johnson db , dahlman kh , knol j , et al : enabling a genetically informed approach to cancer medicine : a retrospective evaluation of the impact of comprehensive tumor proling using a targeted next - generation sequencing panel . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
freedman an , klabunde cn , wiant k , et al : use of next - generation sequencing tests to guide cancer treatment : results from a nationally representative survey of oncologists in the united states . 
duffy mj , harbeck n , nap m , et al : clinical use of biomarkers in breast cancer : updated guidelines from the european group on tumor markers ( egtm )  . 
godin g , b elanger - gravel a , eccles m , et al : healthcare professionals intentions and behaviours : a systematic review of studies based on social cognitive theories . 
implement sci 3 : 36 , 2008 johnson lm , valdez jm , quinn ea , et al : integrating next - generation sequencing into pediatric oncology practice : an assessment of physician condence and understanding of clinical genomics . 
allegretti m , fabi a , buglioni s , et al : tearing down the walls : fda approves next generation sequencing ( ngs ) assays for actionable cancer genomic 33 . 
pennell na , mutebi a , zhou z - y , et al : economic impact of next - generation sequencing versus single - gene testing to detect genomic alterations in metastatic nonsmall - cell lung cancer using a decision analytic model . 
unadjusted models of provider and practice characteristics associated with condence in using genomic test results to guide patient management not at all or a little condent moderately condent very condent moderately v not at all or a little condent very condent v not at all or a little condent total wtd % 95% ci wtd % 95% ci wtd % 95% ci 95% ci 95% ci overall p characteristic provider years since graduation 10 11 - 20 21 - 30  . 
unadjusted multinomial logistic regression models controlled for type of genomic test use , which was dened according to the multimarker somatic panel tests each oncologist used in the past 12 months . 
lum , md1 ; shirley zhu , md , mph1 ; sujay vennam1 ; erna forg o , md1 ; sushama varma , ms1 ; kristen ganjoo , md1 ; trevor hastie , phd3 ; rafck bowen , phd1 ; maria debiec - rychter , md , phd2 ; and matt van de rijn , md , phd1 purpose the preoperative distinction between uterine leiomyoma ( lm ) and leiomyosarcoma ( lms ) is difcult , which may result in dissemination of an unexpected malignancy during surgery for a presumed benign lesion . an assay based on circulating tumor dna ( ctdna ) could help in the preoperative distinction between lm and lms . 
we also proled 36 lm tumor specimens by exome sequencing to develop a panel for targeted detection of point mutations in ctdna of patients with lm . results we identied tumor - derived cnas in the plasma dna of 50% ( six of 12 ) of patients with lm . 
we identied only two recurrently mutated genes in lm tumors ( med12 and acly )  . conclusion our results show that lms do shed dna into the circulation , which provides an opportunity for the development of ctdna - based testing to distinguish lm from lms . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction uterine leiomyomas ( lms ) are common benign smooth muscle tumors that may present with symptoms similar to those associated with uterine leiomyosarcoma ( lms ) , a rare malignant tumor with a poor prognosis.1 with the exception of endometrial lesions , most uterine masses are not biopsied before surgery , and the preoperative distinction between benign and malignant uterine smooth muscle tumors relies primarily on clinical evaluation and imaging . 
as a result , patients may undergo surgery without a denitive distinction between the two entities . it is estimated that one in nine women in the united states will undergo a hysterectomy for benign gynein their lifetime.2 cologic indications , such as lm , power morcellation used to be performed in many in 2014 the us food and drug such cases , until administration discouraged the use of power morcellation for the removal of the uterus or uterine masses , after reporting that this procedure may lead to inadvertent retroperitoneal spread of an unsuspected malignancy in one in 305 patients.3 however , intraabdominal manual morcellation is still performed in patients with large masses diagnosed as lm , and although this is less aggressive than power morcellation , it still carries a risk for dissemination of an unexpected lms . 
risk factors that may favor the diagnosis of lms over lm include postmenopausal status , tamoxifen use , history of retinoblastoma , pelvic irradiation , hereditary leiomyomatosis , and renal cell cancer syndrome , 4 but these factors do not always correlate with a diagnosis of lms . also , the new emerging magnetic resonance imaging techniques present unsatisfactory positive predictive values for the distinction between lm and lms.4 therefore , there is a high need for improved methods for preoperative discrimination between benign lm and malignant tumors . 
in an attempt to improve the distinction between lm and lms , nagai et al5 developed a revised preoperative sarcoma score ( rpress ) based on patients age , serum lactate dehydrogenase ( ldh ) levels , and endometrial cytology ndings . 
 przybyl et al context key objective we sought to explore the feasibility of distinguishing between uterine leiomyoma and leiomyosarcoma based on the analysis of tumor - specic aberrations in circulating tumor dna ( ctdna )  . 
for this purpose , in the current study we aimed to determine whether uterine leiomyomas shed dna into the circulation . knowledge generated we demonstrate that it is feasible to detect ctdna derived from leiomyomas . 
in the limited number of cases analyzed to date , we show that these benign tumors do not carry alterations in tumor suppressor genes that can be detected in the ctdna of most patients with leiomyosarcoma . relevance our ndings are a rst and necessary step toward developing a blood test that , together with clinical and imaging information , ultimately could help clinicians to better evaluate the risk of leiomyosarcoma in a patient presenting with a uterine mass . was developed in a group of 63 patients with lm and lms but has not yet been validated in an independent study . we recently demonstrated that tumor - associated genetic aberrations can be detected in the circulating tumor dna ( ctdna ) of patients with lms.6 in that study , we used two technologies to detect different classes of genomic aberrations in plasma dna : cancer personalized proling by deep sequencing ( capp - seq ) for the analysis of point mutations , 7 , 8 and a genome - wide interrogation of copy number alterations ( cnas ) by shallow whole - genome sequencing.9 , 10 using these two approaches , we were able to detect ctdna in six of seven patients with lms with  . 
the study was approved by the stanford university institutional review board ( irb - 31067 )  . proling plasma cell - free dna cell - free dna was extracted from plasma using qiaamp circulating nucleic acid kit ( qiagen )  . 
sequencing libraries were prepared with the truseq chip preparation kit ( illumina , foster city , ca ) and sequenced using hiseq 2500 instrument ( illumina ) ( data supplement )  . plasma ldh measurement plasma ldh levels were measured on a roche cobas 8000 ( roche , indianapolis , in ) automated platform ( data supplement )  . proling tumor specimens genomic dna extracted from 12 archival lm tumor specimens and germline dna extracted from patient - matched blood leukocytes were used for genome - wide copy number proling with the oncoscan ffpe assay ( thermo fisher scientic / affymetrix , santa clara , ca )  . thirty - six archival lm tumor specimens and normal patientmatched counterpart tissues or peripheral blood leukocytes were used for whole - exome sequencing using the seqcap ez human exome v3.0 library ( roche ) on hiseq 4000 instrument ( illumina ) ( data supplement )  . results tumor - derived copy number aberrations can be detected in plasma dna of patients with lm to explore the possibility of developing a blood - based test for the distinction between lm and lms , we rst determined whether lm can shed dna in the blood . 
we calculated genome - wide segmented z - scores in plasma cell - free dna , and we intersected each genomic region of cnas identied in the eight lm tumor specimens with the segments identied in plasma dna of the matching patients . 
of the 33 cnas detected in the eight tumor specimens , 30 cnas had the same type of copy number alteration in tumor and plasma dna ( ie , matching dna copy number gain or loss ; data supplement )  . 
1.5 in cell - free dna , we found 11 tumor - derived cnas in the plasma dna of six patients with lm ( correlation between log2 ratio in the tumor and z - score in the plasma determined by linear regression was r2 = 0.74 ; p value = .0007 ; table 3 ; appendix fig a1 )  . 
selected large dna cnas identied in lm tumor specimens were not detected in plasma specimens , which is most likely the result of the limit of detection of our method and / or the effect of stochastic sampling of blood specimens . 
we have previously demonstrated that it is possible to detect tumorderived cnas in cell - free dna among patients with lms using the same technology as applied to the patients with lm in the current study.6 we sought to verify whether the aberrations found in ctdna of patients with lm in the present report are specic to these benign tumors or whether the same aberrations could be found also in lms tumor specimens . 
for this purpose , we reanalyzed the previously generated genomic proles of 22 tumors from seven patients with lms using exactly the same criteria as were used for the lm tumor specimens . 
our results show that 10 of 11 genomic aberrations found in the ctdna of patients with lm were also present in the tumor dna of patients with lms analyzed in our previous study , 6 with a median of four aberrations per lms tumor sample ( range , 1 - 8 )  . 
these results demonstrate that the majority of cnas found in lm tumors are not specic for this entity ; therefore , proling these cnas in plasma dna may not be useful to distinguish between lm and lms . 
on the other hand , cnas found in lm specimens did not affect any of the frequently deleted tumor suppressor genes in lms ( eg , tp53 , rb1 , atrx , pten , atm , or cdkn2a )  . 
therefore , the detection of cnas in these tumor suppressor genes in ctdna may favor a diagnosis of lms . patient age and levels of ldh do not allow for distinction between lm and lms previous studies have proposed that ldh level measurements and patient age may be helpful in the distinction between lm and lms.5 , 11 we evaluated the ldh levels in plasma specimens of nine of 12 patients with lm proled for the presence of ctdna and eight untreated patients with uterine lms . 
in the rpress algorithm , the cutoff of serum ldh levels indicative of lms was set at 279 u / l.5 in our group of patients , the sensitivity and specicity values of this test were found to be only 50% and 89% , respectively . 
the mean age of the patients with lm and lms was 48 and 54 years , respectively ( t test two - tailed p value = .19 ; fig 2b )  . 
this performance was poorer than the ndings reported in the rpress study , where the sensitivity , specicity , positive predictive value , and negative predictive value were 93% , 65% , 45% , and 97% , respectively.5 low mutation burden in lm previous genomic studies of lm have identied med12 as the most frequently mutated gene in these lesions , but these studies did not report on other recurrent somatic mutations in these tumors.12 - 14 however , med12 mutations have been reported also in a subset of lms tumors , and therefore these mutations cannot be used for a molecular distinction between lm and lms.15 to investigate whether lm tumors might have recurrently mutated genes other than med12 , we performed whole - exome sequencing on 36 pairs of matched tumor and normal dna specimens from patients with lm . 
mutations in exon 2 of med12 were identied in 39% ( 14 of 36 ) of lm tumor specimens , and the acly gene was mutated in 6% ( two of 36 ) of lm tumor specimens ( data supplement )  . 
mutations in these two genes were present in 15 of 36 patients , with a median of one mutation per tumor ( range , 0 - 2 across all 36 tumors )  . 
all med12 mutations identied in our study were previously reported in the cosmic database ( cosmic v84 ; accessed on november 12 , 2018 ) .16 our results show that recurrent mutations other than in the med12 gene rarely occur in lm . 
importantly , we did not detect in any lm case any of the mutations in tumor suppressor genes that are frequently mutated in lms , such as tp53 , rb1 , pten , atrx , atm , and arid1a.6 this indicates that mutations in these driver genes may be highly specic to malignant tumors such as lms . 
because lms do not carry mutations in these tumor suppressor genes , we propose that detection of these mutations in plasma dna of patients with uncertain diagnosis , using the capp - seq lms - specic panel developed in our previous study , may favor the diagnosis of lms . discussion the clinical utility of ctdna proling has been widely demonstrated in malignant tumors that harbor highly recurrent mutations.7 , 8 , 17 , 18 currently , the two most frequently used sequencing - based approaches for ctdna monitoring include deep targeted sequencing for ultrasensitive quantitative analysis of point mutations , such as capp - seq , and shallow whole - genome sequencing for the detection of cnas . 
histologic appearance of representative leiomyoma ( lm ) cases with ( a ) no detectable dna copy number alterations , ( b ) 20 dna copy number alterations , and ( c ) six dna copy number alterations . 
however , it is not practical to design an lm - specic capture panel for detection of point mutations by deep targeted sequencing of ctdna , because we identied only two recurrently mutated genes in a low percentage of 36 lm tumors . we assume that an approximately two - fold increase of coverage may result in improving the reliability of the calls , by reducing the number of false - positive calls and increasing the number of true - positive calls.22 moreover , most of the patients included in our study had multifocal disease , and we expect that proling all nodules from these patients would reveal a wider spectrum of cnas and thus allow for the detection of overlapping aberrations between plasma and tumor dna in a higher percentage of patients with lm . although ctdna released from malignant tumors has been well characterized in multiple types of cancer , ctdna derived from benign lesions has not been extensively studied . an incidental nding of lm - derived dna in the circulatory system has been reported in pregnant women undergoing noninvasive prenatal testing ( nipt )  . 
dharajiya et al19 reported unexpectedly abnormal nipt proles in 55 of 450 , 000 pregnant women tested for fetal aneuploidy ; 20 of these 55 women were known to have had lm at the time of nipt . 
but the genomic prole of a matching tumor was examined only in a single patient from this group to conrm levels of cell - free dna were indeed that the abnormal derived from this lm.19 although these incidental ndings indicated that lm can shed dna into the circulatory system , the overall sensitivity of shallow whole - genome sequencing for the detection of lm - derived ctdna has not been investigated . 
in the current study , by prospective analysis of plasma and tumor samples of 12 patients with lm , we found ctdna in 50% of these patients , which is a comparable detection rate to the current sensitivity of ctdna detection in many stage i cancers.20 , 21 importantly , the nipt assays used for detecting fetal abnormalities usually focus solely on screening for monosomy or trisomy of chromosomes 13 , 18 , and / or 21 . 
in the current study , instead of focusing only on selected chromosomes , we performed a genome - wide analysis in which we aimed to detect not only chromosome - wide aberrations but also small focal aberrations . 
we performed shallow whole - genome sequencing at approximately 0.1 genomewide coverage ; however , we expect that deeper sequencing would allow for the detection of ctdna in a higher percentage of patients with lm . 
on the basis of previously published studies , it must be noted that the development of a reliable assay for the distinction between lm and lms is challenged by the great difference in prevalence of these two entities . 
 circulating tumor dna in leiomyoma predictive value for any positive result indicating lms would be only 2% , while the negative predictive value of a negative result would be 99.99%. 
on the basis of the genomic proles of lm and lms , we propose that it may be more practical to apply an assay that would rule out the diagnosis of lm . 
this could be possible by applying capp - seq for detection of mutations in tumor suppressor genes that are frequently mutated in lms but have never been reported in lm . 
regardless , given the statistical considerations on the prevalence of lm and lms , validation of such assay in the clinical setting may be challenging and would require a very large prospective study . in summary , we demonstrate in the current study that a substantial portion of lms do shed dna into the circulatory systethese ndings provide an opportunity to develop a noninvasive test for distinction between lm and lms on the basis of ctdna in plasma . 
lyon , iarc , 2014 , in kurman rj , carcangiu ml , herrington cs , et al ( eds ) : who classication of tumours of female reproductive organs . 
nagai t , takai y , akahori t , et al : highly improved accuracy of the revised preoperative sarcoma score ( rpress ) in the decision of performing surgery for patients presenting with a uterine mass . 
nat biotechnol 34 : 547 - 555 , 2016 1 : 814 - 819 , 2015 334 : 252 - 255 , 2011 bayindir b , dehaspe l , brison n , et al : noninvasive prenatal testing using a novel analysis pipeline to screen for all autosomal fetal aneuploidies improves pregnancy management . 
goto a , takeuchi s , sugimura k , et al : usefulness of gd - dtpa contrast - enhanced dynamic mri and serum determination of ldh and its isozymes in the differential diagnosis of leiomyosarcoma from degenerated leiomyoma of the uterus . 
m akinen n , mehine m , tolvanen j , et al : med12 , the mediator complex subunit 12 gene , is mutated at high frequency in uterine leiomyomas . 
wan r , wang z , lee jj , et al : comprehensive analysis of the discordance of egfr mutation status between tumor tissues and matched circulating tumor dna in advanced non - small cell lung cancer . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced nonsmall - cell lung cancer . 
sci transl med van beek dm : detection of copy number variation using shallow whole genome sequencing data to replace array - comparative genomic hybridization analysis [ masters thesis ]  . 
heitzer e , ulz p , belic j , et al : tumor - associated copy number changes in the circulation of patients with prostate cancer identied through whole - genome 27 . 
karczewski kj , francioli lc , tiao g , et al : variation across 141 , 456 human exomes and genomes reveals the spectrum of loss - of - function intolerance across 37 . 
correlation between log2 ratio in tumor dna and z - score in cell - free dna for 11 tumor - derived copy number alterations detected in plasma of leiomyoma patients . 
although from their analysis , her2 exon 20 mutations emerge as the most clearly actionable driver in nsclc , the authors themselves pinpoint ambiguous functional roles of individual her2 aberrations ( ie , activating mutations , amplication , and overexpression ) , as well as their reciprocal associations . therefore , her2 biology may be different in lung cancer compared with breast or gastric cancers , possibly because of specic microenvironmental and tumor - host interactions . as pointed out by the authors , a substantial heterogeneity exists in therapeutic approaches and clinical results of her2 - targeting strategies in this context . this is well exemplied by two clinical nsclc cases harboring different her2 alterations ( her2 mutation and all three her2 aberrationsmutation , amplication , and protein overexpression , respectively ) we discuss herein . case 1 a 44 - year - old woman , never - smoker , presented with stage ivb lung adenocarcinoma ( egfr - wt , alk and ros1 nonrearranged , pd - l1 1% )  . 
she was then treated with two courses of atezolizumab , resulting in clinico - radiologic hyperprogression.2 next - generation sequencing ( ngs ) based molecular proling revealed a her2 exon 20 insertion ( p.a775_ g776insyvma ) ; neither gene amplication nor protein expression was detected . 
she then received whole - brain radiotherapy ( rt ) for an isolated brain progression and was subsequently enrolled in a clinical trial of poziotinib ; treatment was poorly tolerated , and diffuse extracranial progression rapidly ensued . 
rechallenge with trastuzumab and vinorelbine obtained no clinical benet , and she went on to receive gemcitabine single agent and subsequently best supportive care on further clinicoradiologic disease progression . case 2 a 48 - year - old woman , never - smoker , presented with stage iib lung adenocarcinoma in 2011 , when she underwent left inferior lobectomy and medilymphadenectomy followed by adjuvant astinal cisplatin / vinorelbine . 
in 2018 , disease recurred in multiple metastatic sites ; right supraclavicular lymph node rebiopsy showed egfr - wt , alk and ros1 nonrearranged , and pd - l1 1% , and she received rstline platinum - based chemotherapy with partial response , followed by stereotactic body rt on residual tumor sites ; multisite disease progression was observed 2 months after treatment completion . 
she was then started on trastuzumab emtansine ( tdm - 1 ) in march 2020 , experiencing dramatic clinical benet with disappearance of all disease - related symptoms , in particular dyspnea , and performance status improvement ( from 2 to 0 , according to eastern cooperative oncology group ) ; treatment is currently ongoing . the two cases presented highlight the inherent complexity of her2 biology in nsclc and the difculties in reliably predicting the clinical outcome of specic her2 - targeted interventions . 
in case 1 , a clinically meaningful response to trastuzumab - based treatment was observed , in line with other reports , 3 - 5 even in the presence of an isolated exon 20 mutation not accompanied by gene amplication and in the absence of protein expression by immunohistochemistry . 
intrigued by the relatively unexpected response , occurring after hyperprogression to pd - l1 blockade , we analyzed fcriiia polymorphisms and found that the patient carried a valine / valine ( v / v ) variant , which has been correlated to better response to trastuzumab - based treatment in breast cancer.6 in line with the presence of a polymorphism potentially fostering antibody - dependent cell - mediated cythe patients peripheral blood totoxicity ( adcc ) , mononuclear cells displayed a marked and specic trastuzumab - dependent cytotoxic activity toward her2 - overexpressing targets in vitro ( fig 1 )  . 
on the other hand , such a mechanism did not prove relevant in case 2 , in which no response to trastuzumab - based treatment was observed , despite gene amplication and protein overexpression accompanying exon 20 insertion . 
 ( a ) computed tomographic imaging of primary lesion at baseline ( showing atezolizumab progression , left ) and at rst assessment during trastuzumab - based therapy ( right )  . 
 ( b ) mcf7 target cells , expressing ( mcf7 - her2 ) or not expressing ( mcf7 - evector ) her2 antigen , were rst stained with carboxyuorescein succinimidyl ester ( cfse ) 5 mm and celltrace 5 mm , respectively . 
mixed targets ( ratio 1 : 1 ) were incubated for 18 hours at different ratios ( 2 : 1 , 4 : 1 , 8 : 1 , 12 : 1 ) with peripheral blood mononuclear cells ( pbmcs ) isolated from peripheral blood of the patient in the presence of rituximab ( 10 mg / ml ) or trastuzumab ( 10 mg / ml )  . 
tumor cellspecic lysis by pbmcs was analyzed by ow cytometry ( representative plot of 8 : 1 ratio on the left ) and quantied as percentage of lysis ( right ) using the formula : ( 1 ( % mcf7 - her2 or mcf7 - evector + trastuzumab / tot target ) / ( %mcf7 - her2 or mcf7 - evector + rituximab / tot target ) 100 )  . 
fitc , uorescein isothiocyanate ; v450 - ct , v450 cell trace . overall , her2 aberrations in nsclc convey important prognostic10 and potentially predictive information ; however , prospective studies should be designed not only according to patients stratication for specic her2 aberration ( s ) or combinations but also it is mandatory to evaluate the patients immunologic features , especially for antibody - based treatments . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . zhao j , xia y : targeting her2 alterations in nonsmall - cell lung cancer : a comprehensive review . 
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mazi `eres j , peters s , lepage b , et al : lung cancer that harbors an her2 mutation : epidemiologic characteristics and therapeutic perspectives . j clin oncol 31 : 1997 - 2003 , 2013 6 . 
musolino a , naldi n , bortesi b , et al : immunoglobulin g fragment c receptor polymorphisms and clinical efcacy of trastuzumab - based therapy in patients with her - 2 / neu - positive metastatic breast cancer . 
j clin oncol 26 : 1789 - 1796 , 2008 li b , ofn m , hembrough t , et al : p1.13 - 43 molecular and imaging predictors of response to ado - trastuzumab emtansine in patients with her2 mutant lung cancers : an exploratory phase 2 trial . 
j thorac oncol 13 : s599 , 2018 li bt , shen r , buonocore d , et al : ado - trastuzumab emtansine for patients with her2 - mutant lung cancers : results from a phase ii basket trial . 
j clin oncol 36 : 2532 - 2537 , 2018 peters s , stahel r , bubendorf l , et al : trastuzumab emtansine ( t - dm1 ) in patients with previously treated her2 - overexpressing metastatic non - small cell lung cancer : efcacy , safety , and biomarkers . 
milella m , nuzzo c , bria e , et al : egfr molecular proling in advanced nsclc : a prospective phase ii study in molecularly / clinically selected patients pretreated with chemotherapy . 
responses to avelumab were observed regardless of pd - l1 expression , the presence or absence of tumor - inltrating therapy , and lymphocytes , multiple prior lines of somatic or germline origin of mmrd , which suggests that baseline clinical and pathologic characteristics could not predict response . 
ten of the 12 tumors were determined to be mmrd by oncopanel on the basis of mutational signature analysis using two independent algorithms , 4 , 5 which was consistent with the ihc determination . 
the remaining 2 tumors were determined to be mmrp by oncopanel and microsatellite stable using polymerase chain reactionthat is , both oncopanel and polymerase chain reaction were discordant with ihcand none of them responded to avelumab . 
 ( b ) frequency of common jak1 and b2m mutations found in our data set are compared with the frequency in the mismatch repairdecient ( mmrd ) endometrial cancers ( ecs ) from the cancer genome atlas ( tcga )  . 
fs , frameshift ; fsdel , frameshift deletion ; fsins , frameshift insertion ; nr , nonresponder ; or , objective response ; r , responder ; recur , recurrent . 
therefore , these 2 tumors were more likely to be mmrp and were excluded from the analysis . of the remaining 10 patients ( table 1 ) with tumors determined to be mmrd using both ihc and oncopanel , 3 exhibited an objective response to avelumab ( responders ) , whereas 7 did not ( nonresponders )  . 
all 7 nonresponders harbored either jak1 ( 6 tumors ) or b2m mutations ( 1 tumor ) , while only 1 of the 3 responders harbored a jak1 mutation ( fisher exact test , two - sided p = .067 ; fig 1a )  . 
in addition , of the 7 nonresponders , 4 harbored two mutations of jak1 ( 3 tumors ) or b2m ( 1 tumor ) , possibly reecting biallelic inactivation of these genes . 
all jak1 mutations were frameshift ( deletions or insertions ) , with the exception of two missense mutations that occurred together with frameshift mutations : a missense mutation q750r on the pseudokinase domain ( exon 16 ) and a missense mutation l1071p toward the end of the kinase domain ( exon 23 )  . 
frameshift jak1 mutations involved the hotspot position k860 / p861 ( deletions in 5 tumors and insertion in 1 tumor ) and the hotspot position p430 / l431 ( insertions in 2 tumors )  . 
 ( a ) number of indels , deletions , insertions , and tumor mutational burden , dened as the number of nonsynonymous mutations per mb for responders ( r ) and nonresponders ( nr ) in our data set . 
 ( b ) same as panel a comparing patients with endometrial cancer ( ec ) in the cancer genome atlas ( tcga ) data set with and without frameshift jak1 mutations . 
furthermore , in another data set ( msk - imp ) , which included 29 patients with recurrent mmrd ecs ( dened by ihc ) , 6 the overall frameshift jak1 mutations was 51.7% cidence of signicantly higher than that in the tcga data set , but comparable with that in our data set ( fig 1c )  . 
nonresponders had a signicantly higher number of total deletion mutations compared with responders ( p = .03 ) , but the number of total insertion mutations was not different ( fig 2a )  . 
 ( a ) exposure ( left ) and fraction ( right ) , dened as the exposure divided by the total number of single - nucleotide variants of signatures 20 . 
bottom : blue and red boxes and points indicate cases with and without frameshift ( fs ) jak1 mutations in whole - exome sequencing data from the cancer genome atlas ( tcga )  . 
to calculate the fraction , the exposure of each mmrd signature is divided by the sum of exposures of all mmrd signatures ( the contribution of the non - mmrd signatures is not shown as they did not vary signicantly across data sets )  . 
konstantinopoulos , md , phd , dana - farber cancer institute , 450 brookline ave , boston , ma 02115 ; e - mail : panagiotis_ konstantinopoulos@dfci.harvard.edu. support supported by the ludwig center at harvard and by a fund in memory of bina sareen . 
gulhan patents , royalties , other intellectual property : a provisional patent application is being drafted for an algorithm developed by the author for which a coversheet provisional has been led on september 24 , 2018 , titled computational method to identify mutational signatures from sequencing data ( inst ) joyce f . 
liu consulting or advisory role : tesaro , mersana , clovis oncology , genentech , glaxosmithkline research funding : genentech ( inst ) , astrazeneca ( inst ) , boston biomedical ( inst ) , atara biotherapeutics ( inst ) , acetylon ( inst ) , bristolmyers squibb ( inst ) , agenus ( inst ) , cytomx therapeutics ( inst ) , regeneron ( inst ) , tesaro ( inst ) , clovis oncology ( inst ) , surface oncology ( inst ) , 2x oncology ( inst ) , vigeo therapeutics ( inst ) , aravive ( inst ) , arch oncology ( inst ) travel , accommodations , expenses : astrazeneca , merck uncompensated relationships : merck , astrazeneca ursula a . 
konstantinopoulos consulting or advisory role : merck , vertex , astrazeneca , pzer , emd serono , tesaro , bayer research funding : pzer ( inst ) , eli lilly ( inst ) , tesaro ( inst ) , merck serono ( inst ) , astrazeneca ( inst ) , merck ( inst ) , bayer ( inst ) no other potential conicts of interest were reported . references aghajanian c , sill mw , darcy km , et al : phase ii trial of bevacizumab in recurrent or persistent endometrial cancer : a gynecologic oncology group study . 
j clin oncol 29 : 2259 - 2265 , 2011 alvarez ea , brady we , walker jl , et al : phase ii trial of combination bevacizumab and temsirolimus in the treatment of recurrent or persistent endometrial carcinoma : a gynecologic oncology group study . 
gynecol oncol 129 : 22 - 27 , 2013 konstantinopoulos pa , luo w , liu jf , et al : phase ii study of avelumab in patients with mismatch repair decient and mismatch repair procient recurrent / persistent endometrial cancer . 
j mol diagn 19 : 84 - 91 , 2017 gulhan dc , lee jj , melloni gem , et al : detecting the mutational signature of homologous recombination deciency in clinical samples . 
nat genet 51 : 912 - 919 , 2019 soumerai te , donoghue mta , bandlamudi c , et al : clinical utility of prospective molecular characterization in advanced endometrial cancer . 
clin cancer res 24 : 5939 - 5947 , 2018 alexandrov lb , nik - zainal s , wedge dc , et al : signatures of mutational processes in human cancer . 
gao j , shi lz , zhao h , et al : loss of ifn - gamma pathway genes in tumor cells as a mechanism of resistance to anti - ctla - 4 therapy . 
albacker la , wu j , smith p , et al : loss of function jak1 mutations occur at high frequency in cancers with microsatellite instability and are suggestive of 15 . 
howitt be , shukla sa , sholl lm , et al : association of polymerase e - mutated and microsatellite - instable endometrial cancers with neoantigen load , number of tumor - inltrating lymphocytes , and expression of pd - 1 and pd - l1 . 
 returning individual genetic research results to research participants : uptake and outcomes among patients with breast cancer purpose understanding the outcomes of returning individual genetic research results to participants is critical because some genetic variants are found to be associated with health outcomes and have become available for clinical testing . materials and methods brca1 / 2 - negative women with early - onset breast cancer , multiple primary cancers , or a family history of breast cancer who participated in a gene discovery cancer registry were offered the opportunity to learn their individual genetic research results of 24 breast cancer susceptibility genes with a genetic counselor after predisclosure genetic counseling . 
outcomes included uptake of research results , knowledge , informed choice , psychosocial adjustment , uncertainty , satisfaction , and uptake of clinical confirmation testing . results four hundred two potential participants were contacted . 
sixty - four percent of participants had clinical confirmation testing when recommended , including all participants with a mutation in a high - penetrance gene . conclusion uptake of genetic research results may be lower than anticipated by hypothetical reports and small select studies . 
2018 by american society of clinical oncology introduction as large studies with banked dna are increasingly used in genetic sequencing studies , there has been increasing debate over the obligation to share individual research results with research participants.1 - 7 over time , genetic variants identified in research may be found to be associated with health outcomes and may become available in clinical practice.8 , 9 arguments against returning research results include a blurring of the distinction between research and clinical care , misunderstanding , the right not to know , and costs.3 , 7 , 10 - 17 others argue that results should be returned based on the principles of beneficence , autonomy , reciprocity , and respect for persons.6 , 18 - 25 there remains no consensus on how and which results should be returned.1 - 7 , 26 - 28 numerous studies have reported high interest in receiving research results , even if the patients knew there was nothing they could do about the results.29 - 35 although some argue that only actionable results from a laboratory certified by angela r . 
 the clinical laboratory improvement amendments ( clia ) should be returned , 3 , 36 , 37 others argue that research results should be offered to patients more broadly , allowing patients to make decisions about which results they would like to receive , with recommendations for clia confirmation for results that could change clinical care.6 , 20 , 21 , 38 - 41 in the few studies reporting uptake of research results ( as opposed to hypothetical interest ) , uptake varied considerably ( 17% to > 80% ) in cohorts with cancer.42 - 48 most studies returned results with recommendations to have confirmatory clia testing , but none report on uptake of confirmatory testing , 42 - 44 , 48 , 49 and the majority of these studies were small , with select populations and limited outcomes.42 - 45 , 50 - 52 in the returning genetic research panel results for breast cancer susceptibility ( respect ) study , we sought to evaluate uptake of research results in patients with breast cancer who participated in the research registry , factors associated with uptake of results , and participant cognitive and affective outcomes . 
five hundred two samples from brca1 / 2 - negative women with breast cancer and with a diagnosis at < 40 years of age , multiple primary cancers , or at least three firstor second - degree relatives with breast cancer were selected for sequencing of 24 breast cancer susceptibility genes ( cdh1 , cdkn2a , mlh2 , msh2 , epcam , msh6 , mutyh homozygous , pms2 , pten , stk11 , tp53 , atm , bap1 , bard1 , bmpr1a , brip1 , chek2 , mre11a , mutyh heterozygous , nbn , palb2 , rad50 , rad51c , and rad51d ) for related research.53 sequencing was by either targeted or whole - exome hybrid capture methodologies , as described.53 - 55 all potential participants were checked against a death index and were confirmed to have permission for recontact . 
the letter explained that everyone whose research sample had been tested was being invited to participate in the study ( ie , that the invitation was not an indication of a positive result )  . 
this was the only method provided to participants to receive their research results . predisclosure and result disclosure genetic counseling predisclosure genetic counseling sessions using the tiered - binned model were conducted by genetic counselors ( n = 5 ) via phone or in person.56 , 57 genetic counselors were blinded to the results at predisclosure and completed counseling checklists for all sessions . 
genetic counselors shared the need for clinical confirmation when applicable and any clinical genetic testing consistent with current clinical practice . confirmatory testing in a clia - certified laboratory participants with a deleterious or likely deleterious research result in any gene and a variant of uncertain significance ( vus ) in a high penetrance gene were recommended to have clinical confirmation of the research finding . 
 assessed for eligibility ( n = 582 ) contacted by letter for ror ( n = 402 ) excluded ineligible deceased no permission to recontact ( n = 180 ) ( n = 35 ) * ( n = 64 ) ( n = 81 ) ineligible no response mailing returned called but not reached discussed ror but never enrolled declined ( n = 16 ) ( n = 194 ) ( n = 14 ) ( n = 180 ) ( n = 62 ) ( n = 23 ) interested in ror and consented completed t0 survey ( n = 107 ) ( n = 93 ) received predisclosure genetic counseling ( n = 91 by phone ; n = 1 in person ) completed t1 survey ( n = 86 ) declined receipt of results ( n = 9 ) || received genetic research result ( n = 81 by phone ; n = 2 in person ) deleterious / likely deleterious results vus results no findings ( n = 13 ) # ( n = 16 ) ( n = 54 ) completed t2 survey ( after disclosure ; n = 70 ) fig 1 . 
study flow diagra ( * ) seven patients were men , 12 had clinical multigene panel testing ( mgpt ) , five had return of results ( ror ) before returning genetic research panel results for breast cancer susceptibility ( respect ) study , and research charts were not available for 11 . 
 ( ) reasons for declining included concerns about research or time burdens , not being interested in genetic information , only wanting actionable results , preferring clinical testing , and concerns about uncertainty or distress . 
 ( ) three participants reported concerns about distress , three reported concerns about uncertainty , three reported a preference for clinical testing , three reported that they were not ready , and one was not ready at the time because of relative in hospice . 
we adjusted for individuals within the same family using cluster - corrected standard errors.73 in regression models , we excluded 26 of 372 participants who had missing data for continuous variables , race , or proband status . we used paired t tests for psychosocial responses between the first and second survey rounds . 
because only seven pairs of individuals within a same family had longitudinal data ( 7 2 = 14 individuals ) , we did not account for familial correlation in longitudinal analyses . 
we assumed exchangeable correlation matrices for generalized estimating equations . we did not find significant differences when comparing baseline response means between those who completed the first two surveys and those who completed all three surveys ( p > .28 in all cases )  . 
 similarly , we did not find significant differences in the second set of survey responses when comparing the same two groups ( p > .075 in all cases )  . 
a positive attitude about genetic testing was defined by a sum score at or greater than the midpoint of the scale.71 , 72 a more informed choice is defined by adequate knowledge and a testing decision concordant with attitudes about genetic testing . 
all other categories are considered less informed . results participant characteristics all of the 402 potential participants contacted had a history of breast cancer ; the mean age at diagnosis was 40.2 years old ( table 1 )  . 
 in a multiple logistic regression analysis , nonresponders were more likely to be older at the time of their first cancer diagnosis , to not have been seen in the clinical program ( eg , participated in research only ) , and to be nonwhite ( table 2 )  . 
variables included in the models were as follows : race ( nonwhite v white ) , married ( yes , no , or missing ) , children ( yes , no , or missing ) , seen in clinical cancer risk program ( yes , no , or missing ) , age at time of survey , number of fdrs or sdrs with breast cancer , number of fdrs or sdrs with any cancer , age at first diagnosis , and time since sample received . 
three hundred forty - six potential participants had complete data . abbreviations : fdr , first - degree relative ; or , odds ratio ; respect , returning genetic research panel results for breast cancer susceptibility ; sdr , second - degree relative . declined participation ( eg , communicated that they did not want to participate ) or passively declined participation ( eg , never responded after multiple contacts ) after learning about the study were more likely to have been younger age at cancer diagnosis and to have had a greater number of relatives with cancer ( table 2 )  . 
in a multiple logistic regression analysis , those who consented were more likely to be younger at the time of contact , married , and white ( table 2 )  . 
nine participants who had predisclosure counseling chose not to receive their results . informed decision making eighty - nine percent of participants ( 74 of 83 participants ) who had predisclosure counseling and completed relevant measures were classified as making an informed choice as defined by the mmic ( table 3 )  . 
making an informed choice was not associated with any demographic characteristics but was associated with a 2.0 - point change - score increase in research knowledge ( t test , p = .048 ) and marginally statistically significant 3.0 - point change score in total knowledge ( t test , p = .095 ) compared with participants who did not make an informed choice . patient - reported outcomes with receipt of genetic research results knowledge increased significantly after predisclosure counseling and receipt of results ( table 4 )  . 
frequency of making an informed choice using the tiered - binned predisclosure genetic counseling model adequate knowledge of genetic testing positive attitudes toward testing proceeded with receipt of research results all participants ( n = 83 ) , no . 
 ( % ) 74 ( 89 ) category informed choices category 1 category 2 category 3 category 4 category 5 category 6 category 7 category 8 less informed choices 9 ( 11 ) note . 
 anxiety declined significantly for participants with a positive and negative result , but cancer worry only declined significantly for those with a negative result . clinical confirmation testing and changes in medical management sixty - four percent of participants ( 14 of 22 participants ) had the recommended clinical confirmation testing ( fig 2 ) , and all recommended confirmation testing was covered by insurance , although one patient had a small out - of - pocket cost . 
clinical testing ( 14 samples with variants where confirmation testing was recommended and three samples where participants elected to have clinical testing to confirm a vus in a moderate - penetrance gene ) identified 16 of 17 variants identified by the research laboratory ( 94% ; appendix table a2 )  . 
for two samples , the research classification was vus and the clinical result by the commercial laboratory was benign , although other clinical laboratories also classify the variant as vus ( thus , discordant when compared with the call by the specific clinical laboratory used but not discordant with results in clinvar ) .75 for two samples , the research classification was likely deleterious , and the clinical result was reported as a vus . 
finally , clinical testing identified three additional variants that were not reported on research testing because they were considered likely benign by the research laboratory . discussion to our knowledge , this is the largest study offering a range of research genetic cancer susceptibility results ( eg , actionable mutations as well as no identified mutations , genes of variable clinical utility ) to research participants and collecting comprehensive outcomes as well as uptake of confirmatory testing in a commercial clia laboratory . 
although hypothetical query and some published studies have suggested high interest in receiving genetic research results , 29 , 30 , 48 , 76 in this study , we contacted > 400 research participants and only a small subset elected to receive research results . 
nonetheless , patient - reported outcomes for those who elected to receive results were favorable , with no significant increase in distress or uncertainty , even among subgroups receiving a positive or vus result . 
we found increases in understanding of the limitations of research testing and that the majority of participants made informed decisions about receiving individual genetic research results . of note , in our study , we were not able to reach 50% of research participants . 
although some studies have reported higher contact rates , 43 , 48 our rate is consistent with that in the kathleen cuningham foundation consortium for research into familial breast cancer cohort , where 62% of participants did not acknowledge receipt of an invitation to receive results.46 these data highlight that it may be more difficult than anticipated to reach research participants in some cohorts . 
 disparities in uptake and highlighting the need to better understand how to clearly communicate the opportunity to receive research results among diverse populations . in addition , 22% of research participants contacted actively or passively declined . 
these data are consistent with other studies reporting that some participants are not interested in receiving research results.49 , 76 , 79 , 80 until further data are available , researchers and institutional review boards considering return of results should anticipate that there will be a subset of participants who will not be interested in receiving results . although uptake was lower than anticipated in our study , the majority of participants who spoke with a genetic counselor elected to receive their research results , and most met criteria for making an informed choice with favorable patient - reported outcomes . 
our findings are consistent with the favorable patient - reported outcomes reported in the colon cancer family registry , 47 , 48 and our study included additional knowledge measures , patients with a wide range of results , and greater sociodemographic diversity . 
many studies have suggested that research participants and the lay public report interest in receiving genetic research results even when there is unclear utility or uncertainty.29 - 32 our findings support the premise that providing participants with a choice to receive a wide range of results may not be associated with negative psychosocial outcomes . 
outcomes could differ with different approaches , and providing results with a genetic provider for all research all ( 14 of 22 participants ) high - penetrance d / ld research result ( 3 of 3 participants ) moderate - penetrance d / ld research result ( 6 of 10 participants ) high - penetrance vus ( 5 of 9 participants ) participants will be challenge given the limited number of genetic counselors.81 importantly , all participants with a deleterious or likely deleterious finding in a high - penetrance gene proceeded with confirmation testing , and for the most part , this testing was covered by insurance . 
 however , returning only results obtained in a clia - certified research laboratory would deny some individuals critical medically actionable information that could be relevant to their health if subsequently confirmed in a clinical laboratory . 
 for a small number of participants , the return of results and confirmation in a clinical laboratory did change their medical management and , even more frequently , could have medical implications for their relatives . 
equally important , all 92 participants who had predisclosure counseling were candidates for clinical testing ( eg , they were all reasonable candidates for clinical multigene panel testing ) , but only 32% proceeded with some clinical testing . 
longitudinal data and further data will be needed to understand whether declining available clinical testing is based on informed preferences or potential misunderstandings or reliance on research results . although this study is , to our knowledge , the largest report of patient - reported outcomes in a sociodemographically diverse population receiving a wide range of genetic panel research results , we acknowledge several limitations . 
in addition , participants who did not respond could have had higher baseline distress , and these results may not reflect outcomes for all individuals who are contacted to receive research results . 
maxwell no relationship to disclose laura digiovanni employment : carevive research funding : carevive ( inst ) amanda brandt no relationship to disclose danielle mckenna no relationship to disclose jessica long employment : depuy synthes companies ( i ) stock and other ownership interests : depuy synthes companies ( i ) travel , accommodations , expenses : depuy synthes companies ( i ) jacquelyn powers employment : carevive systems honoraria : cureconnect consulting or advisory role : carevive systems travel , accommodations , expenses : hospital of the university of pennsylvania jill e . 
domchek honoraria : astrazeneca , clovis oncology research funding : astrazeneca ( inst ) , clovis oncology ( inst ) , abbvie ( inst ) , pharmamar ( inst ) the pennsylvania department of health specifically disclaims responsibility for any analyses , interpretations , or conclusions . 
wallace se , kent a : population biobanks and returning individual research results : mission impossible or new directions ? hum genet 130 : 393 - 401 , 2011 5 . 
jarvik gp , amendola lm , berg js , et al : return of genomic results to research participants : the floor , the ceiling , and the choices in between . 
daly mb , pilarski r , berry m , et al : nccn guidelines insights : genetic / familial high - risk assessment : breast and ovarian , version 2.2017. 
berg js , amendola lm , eng c , et al : processes and preliminary outputs for identification of actionable genes as incidental findings in genomic sequence data in the clinical sequencing exploratory research consortiugenet med 15 : 860 - 867 , 2013 12 . 
fernandez cv , santor d , weijer c , et al : the return of research results to participants : pilot questionnaire of adolescents and parents of children with cancer . 
bollinger jm , scott j , dvoskin r , et al : public preferences regarding the return of individual genetic research results : findings from a qualitative focus group study . 
fullerton sm , wolf wa , brothers kb , et al : return of individual research results from genome - wide association studies : experience of the electronic medical records and genomics ( emerge ) network . 
wakefield ce , thorne h , kirk j , et al : improving mutation notification when new genetic information is identified in research : a trial of two strategies in familial breast cancer . 
keogh la , fisher d , sheinfeld gorin s , et al : how do researchers manage genetic results in practice ? the experience of the multinational colon cancer family registry . 
cacioppo cn , chandler ae , towne mc , et al : expectation versus reality : the impact of utility on emotional outcomes after returning individualized genetic research results in pediatric rare disease research , a qualitative interview study . 
maxwell kn , hart sn , vijai j , et al : evaluation of acmg - guideline - based variant classification of cancer susceptibility and non - cancer - associated genes in families affected by breast cancer . 
maxwell kn , wubbenhorst b , d ' andrea k , et al : prevalence of mutations in a panel of breast cancer susceptibility genes in brca1 / 2 - negative patients with early - onset breast cancer . 
bradbury ar , patrick - miller l , long j , et al : development of a tiered and binned genetic counseling model for informed consent in the era of multiplex testing for cancer susceptibility . 
lerman c , narod s , schulman k , et al : brca1 testing in families with hereditary breast - ovarian cancer : a prospective study of patient decision making and outcomes . 
kelly k , leventhal h , marvin m , et al : cancer genetics knowledge and beliefs and receipt of results in ashkenazi jewish individuals receiving counseling for brca1 / 2 mutations . 
pieterse ah , van dulmen am , beemer fa , et al : cancer genetic counseling : communication and counselees post - visit satisfaction , cognitions , anxiety , and needs fulfillment . 
cella d , hughes c , peterman a , et al : a brief assessment of concerns associated with genetic testing for cancer : the multidimensional impact of cancer risk assessment ( micra ) questionnaire . 
o ' neill sc , white db , sanderson sc , et al : the feasibility of online genetic testing for lung cancer susceptibility : uptake of a web - based protocol and decision outcomes . 
fiallos k , applegate c , mathews dj , et al : choices for return of primary and secondary genomic research results of 790 members of families with mendelian disease . 
wakefield ce , ratnayake p , meiser b , et al : for all my familys sake , i should go and find out : an australian report on genetic counseling and testing uptake in individuals at high risk of breast and / or ovarian cancer . 
crook a , plunkett l , forrest le , et al : connecting patients , researchers and clinical genetics services : the experiences of participants in the australian ovarian cancer study ( aocs )  . 
keogh la , southey mc , maskiell j , et al : uptake of offer to receive genetic information about brca1 and brca2 mutations in an australian population - based study . 
ultrasound imaging identied a hypoechoic nodule in the right breast ; right axillary lymph node ne - needle aspiration biopsy performed at previous hospital on day 13 revealed invasive carcinoma with a focal micropapillary pattern ( fig 1 )  . immunostaining of estrogen receptor ( er ) and progesterone receptor ( pgr ) was evaluated by using the allred score and the allred scores were positive ( proportion score [ ps ] 1 ( , 1% ) , intensity score [ is ] 2 ) and negative ( ps 0 , is 0 ) , respectively.12 , 13 the tumor was human epidermal growth factor 2 ( her2 ) negative ( immunohistochemistry [ ihc ] 0 )  . 
given these results , she was diagnosed with stage iv breast cancer . targeted next - generation sequencing ( ngs ) analysis using the foundationone companion diagnostic panel ( foundation medicine , cambridge , ma ) was performed on the right axillary lymph node specimen . 
the result of ngs was reported on day 58 , and the ngs identied a ccdc6 - ret fusion ( c1 ; r12 ) with no other reported genomic alterations known to contribute to human breast tumorigenesis , including none in brca1 or brca2 . 
consistent with local standard - of - care guidelines , she received treatment with tamoxifen plus goserelin from day 14 to day 91 , but these were discontinued due to progression in the right breast and new lesions detected in the left lower lung . 
rebiopsy of the right breast tumor revealed the following results : er allred score 2 ( ps 1 [ , 1% ] , is 1 ) , pgr allred score 2 ( ps 1 , is 1 ) , her2 ihc 2 + , her2 uorescence in situ hybridization negative ( her2 / her2 / cep ( centromere ) 17 = 0.9 ) , and programmed death ligand 1 ( sp142 ) expression on tumor - inltrating immune cells of 1% - 4% . 
on day 126 , she was started on treatment with selpercatinib at the recommended phase 2 dose of 160 mg twice daily in the libretto - 001 study after providing written informed consent from the patient to publish information and images . 
 case report sorafenib and vandetanib , multikinase inhibitors with preclinical inhibitory activity against ret , have been used to treat unselected patients with breast cancer , but minimal clinical activity was observed.14 , 15 a patient with ncoa4 - retpositive breast cancer experienced a partial response to the multikinase inhibitor cabozantinib in combination with trastuzumab and exemestane although the cabozantinib dose was reduced for toxicity , the total time on treatment was short , and the relative contribution of each agent to the overall antitumor activity was not known.8 in addition , although cabozantinib has preclinical inhibitory activity against ret , its much stronger inhibition of other kinases ( eg , vegfr2 ) likely accounts for its clinical activity.16 , 17 in contrast , in the current case , the highly selective and potent ret inhibitor selpercatinib demonstrated a durable singleagent response in a patient with ret fusionpositive breast cancer . to our knowledge , this is the rst report of a breast cancer patient with a complete and sustained response to selective , ret - targeted therapy and adds to the diversity of ret fusionpositive tumor types that may benet from selective ret inhibition . 
rothenberg , kazuhiko nakagawa financial support : elizabeth olek administrative support : elizabeth olek provision of study materials or patients : elizabeth olek collection and assembly of data : satomi watanabe , tomoyuki otani , takeshi yoshida , kazuko sakai , elizabeth olek , s . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . masayuki takeda honoraria : astrazeneca kk , chugai pharma , bristol - myers squibb , novartis , ono pharmaceutical kazuko sakai honoraria : roche diagnostics , bio - rad , astrazeneca , chugai pharma elizabeth olek employment : loxo oncology inc stock and other ownership interests : loxo oncology , inc travel , accommodations , expenses : loxo oncology , inc s . 
michael rothenberg employment : loxo , pzer stock and other ownership interests : loxo , pzer jennifer kherani employment : loxo oncology at lilly , ajax health , aisling capital , summus global leadership : ajax health and aisling capital stock and other ownership interests : ajax health and aisling capital consulting or advisory role : ajax health and aisling capital travel , accommodations , expenses : ajax health and aisling capital , loxo pearl p . 
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allison kh , hammond meh , dowsett m , et al : estrogen and progesterone receptor testing in breast cancer : american society of clinical oncology / college of american pathologists guideline update . 
miller kd , trigo jm , wheeler c , et al : a multicenter phase ii trial of zd6474 , a vascular endothelial growth factor receptor - 2 and epidermal growth factor receptor tyrosine kinase inhibitor , in patients with previously treated metastatic breast cancer . 
drilon a , rekhtman n , arcila m , et al : cabozantinib in patients with advanced ret - rearranged non - small - cell lung cancer : an open - label , single - centre , phase 17 . 
schlumberger m , elisei r , m uller s , et al : overall survival analysis of exam , a phase iii trial of cabozantinib in patients with radiographically progressive 2 , single - arm trial . 
konat e , phd1 ; biswajit das , phd3 ; chris karlovich , phd3 ; chih - jian lih , phd3 ; eric polley , phd9 ; richard simon , dsc1 ; ming - chung li , phd1 ; richard piekarz , md , phd1 ; and james h . 
although patients and physicians were blinded to the sequencing and random assignment results , a higher pretreatment dropout rate was observed in the control arm ( 22% ) compared with the experimental arm ( 6% ; p = .038 ) , suggesting that some patients may have had prior tumor mutation proling performed that led to a lack of participation in the control arm . conclusion further investigation , better annotation of predictive biomarkers , and the development of more effective agents are necessary to inform treatment decisions in an era of precision cancer medicine . 
increasing prevalence of tumor mutation proling and preference for targeted therapy make it difcult to use a randomized phase ii design to evaluate targeted therapy efcacy in an advanced disease setting . jco precis oncol 5 : 133 - 144 . 
to explore this , we conducted a randomized , histology - agnostic clinical to examine whether patients with advanced , refractory cancer who had a tumor mutation in a gene in one of three signaling pathways ( dna repair , pi3k , or ras / raf / mek ) were more likely to derive clinical benet if treated with regimens targeting that pathway ( the experimental arm ) than if they were treated with regimens that did not ( the control arm )  . 
 chen et al context key objective a molecular aberration in a patients tumor is expected to render the tumor susceptible to a drug targeting that aberration , but randomized , controlled , blinded phase ii trials conrming the efcacy of this precision medicine approach are both sparse and challenging to design . knowledge generated efcacy in patients with study - dened actionable mutations in the dna repair , ras / raf / mek , or akt / pi3k / mtor pathways either did not achieve the target objective response rate ( trametinib or adavosertib with carboplatin ) or indicated futility despite accrual challenges ( everolimus or veliparib with temozolomide )  . 
patients randomly assigned to the nontargeted control arm had a higher pretreatment dropout rate than the experimental arm , suggesting a preference for targeted therapy based on prestudy genetic proling . relevance the prevalence of genetic data makes it challenging to randomly assign patients to a nontargeted control arbetter geneand variant - specic biomarkers that predict response to drugs are needed for patients with cancer . patients were required to have measurable disease , be willing to undergo tumor biopsy to establish presence of a study - dened actionable mutation of interest ( amoi ) , and have tumor amenable to interventional radiologyguided percutaneous biopsy with a 16to 18 - gauge neeindicated and dle ; excisional biopsy was allowed if evaluable . 
patients who had prior treatment with any of the investigational agents were eligible to participate but were not assigned that same agent . agent - specic eligibility criteria are included in the data supplement . this trial was conducted under a national cancer institute ( nci ) - sponsored investigational new drug application with institutional review board approval . 
protocol design and conduct followed all applicable regulations , guidances , and local policies ( clinicaltrials.gov identier : nct01827384 )  . the investigators obtained informed consent from each participant . trial design this was a multihistology , multicenter , randomized phase ii study of four investigational drug regimens demonstrated to inhibit the dna repair pathway , ras / raf / mek pathway , or akt / pi3k / mtor pathway . 
eligible patients with an amoi detected were randomly assigned 2 : 1 to receive the recommended phase ii dose of either ( 1 ) a predened targeted regimen based on mutation status ( experimental arm ) or ( 2 ) a regimen , chosen from the four study regimens , that did not target their amois ( control arm )  . 
the specic study amois ( data supplement ) were selected on the basis of published functional evidence or implications for protein translation and pathway function ; they were detected in patient samples via a clinical laboratory improvement amendments ( clia ) sequencing assay developed and validated by the molecular characterization laboratory at the frederick national laboratory for cancer research , as previously described.5 patient eligibility determination and randomized treatment assignment were performed with the genemed informatics system as described.6 if more than one amoi was detected for a patient randomly assigned to the experimental arm , the targeted treatment was based on the selection of higher allele frequency ; if allele frequencies were within 15% , the patient was assigned to the targeted treatment cohort with the fewest patients enrolled ( with the option to receive the other treatment regimen upon disease progression )  . 
if a patient randomly assigned to the control arm had multiple nontargeted treatment options , their regimen was chosen based on the proportions of treatment assignments on the experimental arm , so as to balance the two arms with respect to proportions receiving each of the four regimens . for the purpose of assessing the primary end point of this trial , only response to the rst regimen was used . 
adverse events ( aes ) were graded according to nci common toxicity criteria version 4.0 until march 31 , 2018 , when version 5.0 was implemented ; all aes were mapped to version 5.0 before the nal analysis . 
 random assignment and clinical outcome on the nci - mpact trial study agents veliparib ( abt - 888 ; nsc 737664 ) , adavosertib ( azd1775 ; nsc 751084 ) , trametinib ( nsc 763093 ) , and everolimus ( nsc 733504 ) were supplied by the division of cancer treatment and diagnosis , nci , under collaborative research and development agreements with abbvie ( north chicago , il ) , astrazeneca ( cambridge , united kingdom ) , glaxosmithkline ( brentford , united kingdom ) , and novartis ( basel , switzerland ) , respectively . 
study drugs were administered at the recommended phase ii doses and schedules ( data supplement )  . statistical study design the accrual ceiling for each regimen cohort of the experimental arm was set at 30 patients to discriminate between tumor response rates of 20% versus 5% . 
the design included n = 198 enrolled n = 5 did not have biopsy n = 193 had biopsy collected n = 13 insufficient tumor or dna n = 1 declined to participate detceted ioma on dah 17 = n detceted ioma na dah 801 = n n = 7 had no targeted regimen n = 5 had no control regimen but were offered treatment n = 64 randomly assigned to targeted arm n = 32 randomly assigned to control arm n = 10 assigned everolimus n = 25 assigned trametinib n = 3 assigned veliparib plus tmz n = 10 assigned everolimus n = 10 assigned trametinib n = 2 assigned veliparib plus tmz n = 26 assigned adavosertib plus carboplatin n = 10 assigned adavosertib plus carboplatin n = 7 clinically excluded n = 4 died n = 4 declined to participate n = 7 clinically excluded n = 1 died n = 7 declined to participate n = 6 received adavosertib plus carboplatin n = 8 received everolimus n = 20 received trametinib n = 3 received veliparib plus tmz n = 3 received everolimus n = 6 received trametinib n = 2 received veliparib plus tmz n = 8 off treatment : n = 2 death ( pd ) n = 4 pd n = 1 toxicity n = 1 withdrew n = 20 off treatment : n = 19 pd n = 1 withdrew n = 3 off treatment : n = 3 pd n = 3 off treatment : n = 3 pd n = 6 off treatment : n = 5 pd n = 1 withdrew n = 2 off treatment : n = 1 pd n = 1 withdrew n = 6 off treatment : n = 5 pd n = 1 toxicity n = 18 received adavosertib plus carboplatin n = 18 off treatment : n = 13 pd n = 1 intercurrent illness n = 3 toxicity n = 1 withdrew fig 1 . 
seven patients had an amoi but could not be randomly assigned because they were ineligible for the targeted treatment ( six patients : pancreatic cancer with an ras mutation ; one patient : unknown reason )  . 
 chen et al an interim futility analysis ; if no objective responses were observed among the initial 12 patients in each experimental cohort , the cohort was to be terminated early ( with 54% likelihood under the null hypothesis ) , with 93% condence that the response rate would be lower than the target 20% rate . 
this design yields at least 84% power to detect a true objective response rate of at least 20% and at least 0.94 probability of a negative result if the true objective response rate was no more than 5% . 
four - month progression - free survival ( pfs ) , dened as the time from random assignment to progression or death from any cause ( whichever comes rst ) , was evaluated as a secondary end point using kaplan - meier estimates and cis calculated using greenwoods formula . 
twelve or more instances of 4 - month pfs ( at least 40% ) among the 30 patients in an experimental cohort was to be considered promising ; this would occur with 90% likelihood if the true 4 - month pfs rate is 50% ( median pfs of 4 months ) and with 5% likelihood if the true 4 - month pfs rate is 25% ( median pfs of 2 months )  . results enrollment and treatment assignment one hundred ninety - eight patients who met study eligibility criteria were enrolled from january 2014 to april 2018 ( fig 1 , table 1 , data supplement ) , 108 ( 55% ) of whom underwent a tumor biopsy procedure and ultimately had a study - actionable mutation detected ( fig 2a )  . 
patient characteristics characteristic patients enrolled median age , years ( range ) sex ( female / male ) median number of prior lines of therapy ( range ) a patients with karnofsky performance status ( % ) dna repair pi3k ras / raf / mek patients with an amoi in a targeted pathwayb number 60 ( 23 - 83 ) 109 / 89 4 ( 1 - 12 ) abbreviations : amoi , actionable mutation of interest . areported for patients who received at least one dose of study agent on nci - mpact . breported for all patients with an amoi detected on nci - mpact . 
patients with an amoi in more than one pathway are counted here for each affected pathway . 136 2021 by american society of clinical oncology the genes with highest frequency of study amois were tp53 and kras ( 56% and 46% of patients with an amoi , respectively ) ( fig 2c , data supplement )  . 
all patients assigned to the two dna repair pathway cohorts of the experimental arm had a tp53 mutation and 75% of patients assigned to the trametinib experimental cohort had a kras mutation . 
the majority of amois were nonsynonymous single - nucleotide variants ( data supplement )  . toxicity thirty ( 45% ) of the 66 patients who received at least one dose of study agent ( s ) experienced an ae of grade 3 or greater that was considered at least possibly related to study treatment ( data supplement )  . 
seven patients died within 30 days of their last dose of study drugs but none of the on - study deaths were considered related or likely related to the study treatments . treatment compliance interim analysis revealed unanticipated differences in compliance for patients who were assigned to targeted versus nontargeted therapy ( fig 3a )  . 
the overall percentage of patients assigned to the control arm who never initiated treatment was 47% ( 15 / 32 ) , signicantly higher than the rate for patients assigned to the experimental arm ( 23% ; 15 / 64 ; p = .034 , two - sided , by fishers exact test )  . 
evaluation of the reasons why patients came off study , as documented in the clinical database , reveals that a signicantly higher percentage of the patients randomly assigned to the control arm chose not to start treatment ( 22% [ 7 / 32 ] ) compared with the percentage of patients in the experimental arm who chose not to start treatment ( 6% [ 4 / 64 ] ; p = .038 , two - sided , by fishers exact test )  . interim futility analysis accrual to the veliparib plus tmz and everolimus cohorts was slow such that accrual did not reach the 12 - patient threshold for interim analysis of the primary end point , objective response rate ; no responses were measured on either regimen ( fig 3b )  . 
slow accrual to the veliparib plus tmz arm was because of the fact that every dna repair pathway amoi detected on study was within tp53 ; on the basis of preclinical evidence that loss - of - function mutations in tp53 affect regulation of cell cycle progression , patients who were randomly assigned to the experimental arm with these mutations were assigned adavosertib plus carboplatin as their rst - line study treatment if eligible , not veliparib plus tmz . 
pfs events were experienced by 12 / 17 randomly assigned and treated patients in the control arm ; among the patients randomly assigned to the experimental cohorts , pfs events were documented in 6 of 8 treated with everolimus , 20 of 20 treated with trametinib , 13 of 18 treated with adavosertib plus carboplatin , and 3 of 3 treated with veliparib plus tmz . 
caution must be used in comparing the individual experimental cohorts to the control arm as the restriction of an experimental cohort to a particular target may be prognostic of better ( or worse ) pfs . 
 ( a ) nci - mpact treatment compliance rates : the percent of randomly assigned patients who initiated their assigned study treatment or did not start treatment because of death , clinical exclusion that developed after enrollment , or patient choice . 
 ( b ) the proportions of randomly assigned and treated patients who experienced objective response ( complete response or conrmed partial response ) are presented by arm or targeted treatment cohort . 
none of the three patients in the experimental veliparib plus tmz cohort were progressionfree at 4 months ( ci not provided because of small sample size )  . two patients experienced sustained stable disease ( sd ) for a noteworthy 24 cycles of targeted treatment ( fig 5 )  . one patient with endometrial cancer and pik3ca h1047l and pten r130 * amois received everolimus and experienced sd for 24 cycles before progressing . 
the other patient , a 52 - year - old woman with low - grade ovarian cancer with an nras q61r amoi , experienced sd for 27 cycles of trametinib treatment before her disease progressed . 
in the experimental adavosertib plus carboplatin cohort , one patient with endometrial carcinoma and tp53 r213 * and pik3ca c420r amois had an unconrmed pr before coming off treatment because of toxicity . 
none of the patients who crossed over at disease progression from the control arm to a targeted treatment or from one targeted treatment to another experienced clinical benet on the crossover regimen ( data supplement )  . discussion highly effective , tailored therapy targeting specic genetic aberrations is a primary goal of precision medicine.1 , 2 reports of exceptional responders , retrospective studies , and several nonrandomized trials indicate clinical benet for genome - driven treatments , prompting optimism and discussion about the appropriate evidence framework for precision oncology.3 , 8 - 24 the results of our study , which was important considerdesigned in 2012 , highlight several ations for randomized precision oncology studies in the advanced disease setting that have evolved since then.25 - 28 with the greater prevalence of genetic tests and emphasis on precision medicine , it may be very challenging to randomly assign patients to a nontargeted control arour data suggest that some patients and physicians may have had prior tumor mutation prole knowledge and , when randomly assigned to the control arm , appeared to show a bias favoring the presumed precision medicine approach , declining to participate in the study . 
the low dropout rate on the similarly designed shiva trial may be explained by having the patients randomly assigned to a physicians choice control arm ; results from the shiva trial also did not demonstrate the superiority of molecularly targeted agents over the control treatment.25 the results of the winther and coppo trials , two nonrandomized studies , describe clinical benet for a small number of patients with personalized treatment but the pfs - based end point was not met in either study.29 , 30 our results are similar in that the two statistically evaluable experimental cohorts indicate that neither trametinib nor adavosertib plus carboplatin was more effective than standard therapy at achieving objective response when assigned to target nci - mpact - dened amois in ras / raf / mek or dna repair pathways ( eg , kras gain of function or tp53 loss of function mutations , respectively )  . 
 random assignment and clinical outcome on the nci - mpact trial experimental arm control arm 120 - day rate median 120 - day rate median 100 150 200 250 300 350 100 150 200 250 300 350 days since random assignment days since random assignment number at risk number at risk everolimus experimental cohort trametinib experimental cohort 120 - day rate median 120 - day rate median 100 150 200 250 300 350 days since random assignment number at risk 100 150 200 250 300 350 days since random assignmentt number at risk ( b ) control arm , fig 4 . 
 chen et al x age at dx prior disease category cycles best response werdhtiw yticixot withdrew toxicity illness toxicity werdhtiw female genitourinary genitourinary lower gi breast lower gi female genitourinary gender female male female male female female female male female female male male female female female female male female male male female female male male female female female female female male female male male female female female female female male male male female female male female female female male male male female male female male male male male female female female female elamef genitourinary head and neck genitourinary sarcoma genitourinary lower gi lower gi lower gi lung upper gi genitourinary neuroendocrine lower gi genitourinary lower gi lower gi lower gi lower gi lower gi breast sarcoma lower gi genitourinary gi upper head and neck breast lower gi melanoma lower gi breast sarcoma lower gi lower gi genitourinary genitourinary lower gi lung lower gi lower gi lower gi lower gi lower gi breast lower gi lower gi lung lower gi lower gi lower gi lower gi lower gi lower gi female genitourinary lower gi melanoma melanoma lung genitourinary female genitourinary fig 5 . 
each patients detected nci - mpact amois are listed and color - coded to indicate the level of evidence that the mutation is susceptible to the assigned nci - mpact treatment ( based on the information in the oncokb and civic precision oncology knowledge bases at the time of writing )  . 
where available , the results of whole exome sequencing are presented as the number of genetic alterations detected that are annotated in oncokb as either oncogenic ( # oncogenic mutations ) or as oncogenic and actionable with available therapeutic agents ( # oncokb mutations )  . 
 random assignment and clinical outcome on the nci - mpact trial medicine approach , the lack of signicant response on the experimental arm argues for need of better therapies and further validation . 
extensive genetic sequencing and clinical trials evaluating broader aspects of pathway alterations may reveal additional information about actionable sensitivity markers . the targeted treatment assignments in nci - mpact were made based on molecular aberrations that were expected to confer therapeutic sensitivity at the level of a signaling pathway . 
since the trials initiation , the cancer research community has been engaged in an ongoing effort identify geneand variant - specic biomarkers that predict response to drugs.31 - 37 retrospective review of two precision oncology knowledge bases , oncokb and civic , suggests that there is little curated evidence to support many of the amoi - drug associations targeted in the ncimpact trial , which is consistent with the lack of clinical activity . 
for example , tp53 mutations , which were detected in every nci - mpact patient randomly assigned to a targeted dna repair inhibitor cohort , are understood to be oncogenic but have remained largely undruggable.38 the experimental trametinib cohort is the exception in that for each ras / mek / erk pathway amoi that was detected , there is evidence curated in oncokb or civic suggesting effective inhibition by trametinib . 
notably , all three patients who were treated with trametinib to target an nras q61r mutation experienced clinical benet , in line with published clinical evidence of mek inhibitor activity in patients carrying an nras q61 mutation.39 most of the patients treated in the experimental trametinib arm carried an amoi in kras . 
although there is preclinical support for treating kras amois with an mek inhibitor , the results of that approach were modest in this study , perhaps because of reported mechanisms of kras mutant resistance to atpnoncompetitive mek inhibitors.40 - 42 the presence of a therapy - targeted amoi furthermore , in a patient tumor does not indicate whether the amoi is a driver or passenger mutation.43 , 44 in nci - mpact , the most frequently detected amoisthose in tp53 and kraswere detected in all four treatment cohorts and might have contributed to disease progression in cases where treatment was assigned to target an amoi in a different pathway ( eg , akt / pi3k / mtor pathway )  . 
treatment assignment in such cases was made based on the amoi with the highest allele frequency but there is precedent for subclonal molecular alterations driving disease progression.45 - 48 two of the three patients who tolerated everolimus treatment and had amois conned to only the pi3k pathway experienced prolonged sd ( 10 cycles )  . it is important to note that we were able to achieve a successful biopsy collection rate of  . 
90%.49 even with a turnaround time of 10 days for sequencing results , 5 19 biopsied patients ( 10% ) progressed to the point of ineligibility or death before treatment initiation , reecting the advanced disease status of this patient population . 
biopsy sampling could not have identied spatially or temporally isolated tumor subclones that may have been driving tumor growth or conferring resistance in these patients , 43 , 44 , 50 , 51 challenges that can be explored further in preclinical studies as can the presence of putative driver mutations in other signaling pathways . we are completing the analysis of a preclinical study performed in parallel with the nci - mpact trial using xenograft models derived from the tumors of cancer patients to overcome the hurdles inherent to clinical investigations of molecularly targeted treatment response ( manuscript in preparation )  . 
our preliminary data suggest that functional in vivo evidence of activity in molecularly dened models should be the basis on which to evaluate clinical benet of targeted agents in specied patient subgroups . 
chen , md , national cancer institute , 31 center drive , room 3a44 , bethesda , md 20814 ; twitter : @ncitreatment ; e - mail : chenali@ mail.nih.gov. support this project has been funded in whole or in part with federal funds from the national cancer institute , national institutes of health , under contract no . 
mickey williams , richard piekarz , james h . doroshow provision of study materials or patients : shivaani kummar , geraldine osullivan coyne , funda meric - bernstam , stephen leong , chris karlovich collection and assembly of data : alice p . 
konat e , biswajit das , chris karlovich , chih - jian lih , eric polley , richard simon , ming - chung li , richard piekarz , james h . 
mickey williams research funding : illumina patents , royalties , other intellectual property : i was a co - inventor of the dlbcl cell of origin patent recently led by the nih kanwal p . 
trends mol med 23 : 874 - 898 , 2017 tate jg , bamford s , jubb hc , et al : cosmic : the catalogue of somatic mutations in cancer . 
nucleic acids res 47 : d941 - d947 , 2019 lih c - j , sims dj , harrington rd , et al : analytical validation and application of a targeted next - generation sequencing mutation - detection assay for use in treatment assignment in the nci - mpact trial . 
j mol diagn 18 : 51 - 67 , 2016 zhao y , polley ec , li m - c , et al : genemed : an informatics hub for the coordination of next - generation sequencing studies that support precision oncology clinical trials . 
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blood 100 : 1965 - 1971 , 2002 sawyers cl , hochhaus a , feldman e , et al : imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis : results of a phase ii study . 
obrien sg , guilhot f , larson ra , et al : imatinib compared with interferon and low - dose cytarabine for newly diagnosed chronic - phase chronic myeloid leukemia . 
fukuoka m , wu yl , thongprasert s , et al : biomarker analyses and nal overall survival results from a phase iii , randomized , open - label , rst - line study of getinib versus carboplatin / paclitaxel in clinically selected patients with advanced non - small - cell lung cancer in asia ( ipass )  . 
rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as rst - line treatment for european patients with advanced egfr mutationpositive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
hauschild a , grob jj , demidov lv , et al : dabrafenib in braf - mutated metastatic melanoma : a multicentre , open - label , phase 3 randomised controlled trial . lancet 380 : 358 - 365 , 2012 16 . 
haslem ds , van norman sb , fulde g , et al : a retrospective analysis of precision medicine outcomes in patients with advanced cancer reveals improved progression - free survival without increased health care costs . 
massard c , michiels s , ferte c , et al : high - throughput genomics and clinical outcome in hard - to - treat advanced cancers : results of the moscato 01 trial . cancer discov 7 : 586 - 595 , 2017 19 . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors on the basis of molecular proles : results from mypathway , an open - label , phase iia multiple basket study . 
freidlin b , allegra cj , korn el : moving molecular proling to routine clinical practice : a way forward ? j natl cancer inst 112 : 773 - 778 , 2019 25 . 
le tourneau c , delord j - p , gonalves a , et al : molecularly targeted therapy based on tumour molecular proling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
herbst rs , gandara dr , hirsch fr , et al : lung master protocol ( lung - map ) - a biomarker - driven protocol for accelerating development of therapies for squamous cell lung cancer : swog s1400 . 
goncalves a , bachelot t , lusque a , et al : high - throughput genome analysis and therapeutic decision for patients with her2 - negative metastatic breast cancer : first feasibility and molecular results of the randomized phase ii study safir02 breast ( ucbg - 0105 / 1304 )  . 
tuxen iv , rohrberg ks , oestrup o , et al : copenhagen prospective personalized oncology ( coppo ) clinical utility of using molecular proling to select patients to phase i trials . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
dummer r , schadendorf d , ascierto pa , et al : binimetinib versus dacarbazine in patients with advanced nras - mutant melanoma ( nemo ) : a multicentre , open - label , randomised , phase 3 trial . 
ferry - galow kv , datta v , makhlouf hr , et al : what can be done to improve research biopsy quality in oncology clinical trials ? j oncol pract 14 : e722 - e728 , 2018 50 . 
 c deep response to anti - pd - 1 therapy of metastatic neurobromatosis type 1 - associated malignant peripheral nerve sheath tumor with cd274 / pd - l1 amplication berna c . 
ozdemir , md , phd1 , 2 ; pierre bohanes , md3 ; bettina bisig , md , phd1 ; edoardo missiaglia , phd1 ; petros tsantoulis , md , phd4 ; george coukos , md , phd1 , 5 , 6 ; michael montemurro , md1 ; krisztian homicsko , md , phd1 , 5 , 6 ; and olivier michielin , md , phd1 , 5 , 6 introduction malignant peripheral nerve sheath tumors ( mpnsts ) , also known as neurobrosarcomas , are a rare type of highly aggressive soft tissue sarcomas originating from peripheral nerve branches or sheaths . 
more than 50% of these tumors are found in individuals with neurobromatosis type 1 , evolve from benign neurobromas , and are associated with worse outcome than are sporadic tumors.1 patients with metastatic mpnst have a 3 - year survival of only 14%.2 surgery in combination with adjuvant chemotherapy and radiotherapy is the current standard of care.3 although complete resection is potentially curative , local or distant relapse frequently occurs.4 mpnsts are resistant to chemotherapy through different mechanisms , including the upregulation of drug efux transporters.5 radiotherapy can improve local control but does not increase patient survival.3 overall , treatment options are limited for recurrent or metastatic disease . molecular proling of mpnsts identied frequent mutations in the mapk pathway ( nf1 , rassf9 ) ; loss tumor suppressor genes , such as tp53 and cdkn2a ; and alterations in core components of the polycomb repressive complex 2 ( prc2 ) : eed and suz12.6 , 7 still , with some notable exceptions , targeted therapies are generally not successful in the treatment of mpnst.8 , 9 typically , mpnsts present with low programmed death ligand - 1 ( pd - l1 ) expression9 but with relatively high numbers of inltrating cytotoxic t cells and low abundance of immunosuppressive regulatory t cells.10 this immune phenotype would be suggestive of possible benet from immune checkpoint inhibition . 
after multidisciplinary evaluation , a second revision surgery with re - excision of the stump of the left peroneal nerve was realized by the end of october 2015 and showed negative surgical margins . in march 2016 , 5 months after the re - excision , computed tomography ( ct ) showed multiple pulmonary metastases , and a positron emission tomography - ct scan conrmed the absence of extrapulmonary disease . 
after three cycles of doxorubicin chemotherapy ( 75 mg / m2 ) , a partial response was achieved with signicant decrease of the voluminous paramediastinal pulmonary metastasis and stability of the other pulmonary nodules , per response evaluation criteria in solid tumors ( recist ) 1.1. 
we performed a comprehensive molecular analysis of a pulmonary metastasis specimen ( estimated to contain 50% tumor cells ; fig 1a ) and matched constitutional dna , using an inhouse , 400 - gene , next - generation sequencing panel . this is a capture - based panel developed at our institution , aimed at detecting clinically actionable somatic genomic alterations in 394 common cancer - associated genes ( data supplement )  . 
tumor mutation burden , dened as the number of nonsynonymous somatic mutations ( excluding splice variants ) divided by the size of the coding sequence covered by the panel , ( ie , 1.48 megabase ) , was 3.4 per megabase , within the range of previous descriptions.6 besides the known germline mutation of the nf1 gene , we observed a second somatic hit represented by the frameshift variant arg1136hisfs * 2 , interpreted as a loss - offunction mutation , as expected in mpnsts developed in the setting of neurobromatosis type 1 . 
among other alterations , a tp53 loss - of - function variant and a cdkn2a deletion were also identied , in line with published genomic data.6 furthermore , we detected an amplication of the including the cd274 / chromosomal region 9p23 - p24 , pd - l1 locus , in the context of a near - triploid genome . cd274 / pd - l1 locus amplication was conrmed by uorescence in situ hybridization analysis ( fig 1b ) and appeared to be associated with pd - l1 protein overexpression in almost 100% of tumor cells ( fig 1c )  . 
we could not detect any ny - eso - 1 expression ( fig 1g )  . on the basis of these ndings , the molecular tumor board recommended anti - pd - 1 therapy . 
therefore , it was decided to deliver treatments concomitantly.12 the patient began treatment with nivolumab 3 mg / kg intravenously every 2 weeks and received radiotherapy to the bilateral anterior pleural metastases at a dose of 39 gy ( 13 sessions at 3 gy ) at the beginning of july 2017 ( fig 2 )  . two months after starting nivolumab , a ct scan demonstrated a decrease in the size of all pleural and pulmonary lesions without any new lesions . 
 ( a ) microscopic examination of a pulmonary metastasis showed a spindle cell proliferation with marked pleomorphism , consistent with a high - grade mpnst ( hematoxylin - and - eosin stain ; original magnication , 200 )  . 
this was a laboratory - developed break - apart fish probe exploring rearrangements and copy number gains of cd274 / pd - l1 locus at 9p24.1 ; an average of nine red - green fusion signals per nucleus were observed , representing nonrearranged cd274 / pd - l1 loci . 
in contrast , a mean of only three blue signals per nucleus were present , corresponding to a centromeric control probe on chromosome 9 ( original magnication , 630 )  . 
 ( c ) by immunohistochemistry , almost 100% of tumor cells strongly expressed pd - l1 at their cell membrane ( pd - l1 , clone sp263 ; original magnication , 100 ) and ( d ) numerous tumor - inltrating t cells were present ( cd3 , clone 2gv6 ; original magnication , 100 )  . 
 ( e ) the vast majority of these were cd8 positive ( cd8 , clone c8 / 144b ; original magnication , 100 ) , whereas ( f ) foxp3 - expressing regulatory t cells represented less than 10% ( foxp3 , clone 236a / e7 ; original magnication , 100 )  . 
all upstream or downstream nonsense variants are also reported to be pathogenic or likely pathogenic . this variant has not been observed in the general population or in the consulted databases . 53% decrease of the target lesions according to recist 1.1 at 9 months remained stable at 12 and 15 months of nivolumab therapy ( fig 2 ) an increase of a pulmonary micronodule from 5 to 7 mm and the appearance of three small , aspecic micronodules were noted after 12 months of therapy . 
importantly , the radiologic improvement translated into improved general health status , with an increase in appetite and weight , a reduction of fatigue , and a decrease in opioid use for pleural pathe patients general condition improved rapidly , from eastern cooperative oncology group performance status 3 to 1 , 12 months from the start of the immunotherapy . 
a case of complete response to pembrolizumab in combination with procarbazine was reported in a patient with mpnst harboring 90% pd - l1 expression.13 , 14 to our knowledge , this is the rst report of a deep and prolonged response to nivolumab and radiotherapy in mpnst with cd274 / pd - l1 amplication . 
most notably , cd274 / pd - l1 amplication is found in 97% of patients with classic hodgkin lymphoma and is characterized by exceptionally high response rates of 70% to anti - pd - 1 therapy.14 , 15 however , cd274 / pd - l1 amplication has been detected across more than 100 different solid tumor types at varying frequencies , 16 , 17 including in triple - negative breast cancer ( 29% ) , 17 squamous cell carcinomas of the oral cavity ( 19% ) , 18 nonsmall - cell lung cancer ( 5% ) , 19 and colon cancer ( 3% ) .17 in a recent analysis of the cancer genome atlas sarcoma data set , cd274 / pd - l1 copy number gains were detected in 11% of the mpnst samples . 
in agreement with other studies , pd - l1 expression correlated with copy number gains of the corresponding genomic locus.12 cd274 / pd - l1 amplication in hodgkin lymphoma and nonsmall - cell lung cancer has been associated with poor prognosis15 , 20 and predicts response to anti - pd - 1 therapy.21 , 22 as in the patient in this case report , amplication of table 2 . 
13 jak2 , cd274 , ptprd cdkn2a , cdkn2b , fancg all genes in the region all genes in the region hnf1a , ncor2 , pole all genes in the region all genes in the region all genes in the region amplication deletion amplication amplication amplication deletion amplication deletion * only copy number gains of ve or more copies and deletions are reported in this table . 
computed tomography scans show the bilateral pleural metastases in june 2017 , before starting nivolumab therapy or radiotherapy and after 5 , 9 , 12 , and 15 months of nivolumab therapy , respectively . 
the irradiated pleural metastases are indicated by red arrows ; the nonirradiated lesions are indicated by blue arrows . the 9p24 locus typically involves both cd274 / pd - l1 and jak2 , with the 9p24 locus amplication situated 300 kb distally of cd274 / pd - l1 . 
jak2 activation causes increased pd - l1 transcription via the increased signal transducer and activator of transcription ( stat ) protein secretion22 and is associated with response to anti - pd - 1 therapies . 
in line with this , preclinical models have shown that loss of function mutations in the jak / stat pathway are correlated with impaired response to interferon receptor 1 and 2 signaling and could represent a mechanism of resistance to anti - pd - 1 therapies.23 - 25 it is difcult to estimate the contribution of radiotherapy to the exceptional response of the patient in this report . 
radiotherapy might potentiate the effect of immune checkpoint inhibitors through induction of immunogenic cell death with an increase in the number or diversity of tumorassociated antigens and improved inltration of immune cells , and by consequence , could partially overcome the immune suppressive tumor microenvironment , a phenomenon known as the abscopal effect.25 although we cannot exclude the presence of an abscopal effect , we currently have no direct supporting evidence . 
clinical trials testing different sequences and types of immunotherapy in combination with radiotherapy are ongoing and will help determine the extent of the abscopal effect.26 a major issue in immuno - oncology is the lack of predictive biomarkers . 
our report suggests that pd - 1 targeting alone or in combination with radiotherapy could be an effective strategy in mpnsts harboring amplication of cd274 / pd - l1 and could be an option for patients whose disease progresses after standard therapies . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . pierre bohanes honoraria : merck serono , eli lilly , abbvie , sano travel , accommodations , expenses : amgen , boehringer ingelheim , bristol - myers squibb , eli lilly petros tsantoulis honoraria : roche , pzer travel , accommodations , expenses : amgen george coukos consulting or advisory role : roche , astrazeneca , bristol - myers squibb speakers ' bureau : roche research funding : bristol - myers squibb ( inst ) , celgene ( inst ) , boehringer ingelheim ( inst ) , roche ( inst ) , iovance kite pharma ( inst ) , nextcure ( inst ) , geneostx ( inst ) , sano / aventis ( inst ) patents , royalties , other intellectual property : patents in the domain of antibodies and vaccines targeting the tumor vasculature as well as technologies related to t - cell expansion and engineering for t - cell therapy . 
receives royalties from the university of pennsylvania . krisztian homicsko consulting or advisory role : amgen , bristol - myers squibb , roche travel , accommodations , expenses : msd oncology olivier michielin consulting or advisory role : bristol - myers squibb , msd , novartis , roche , amgen research funding : msd , bristol - myers squibb expert testimony : bristol - myers squibb travel , accommodations , expenses : bristol - myers squibb , msd no other potential conicts of interest were reported . acknowledgment the patient has provided informed consent for the publication of this case report . we thank dr abderrahim zouhair , clinique de la source , lausanne , for providing the images of the irradiation elds . 
we also thank the fasma and philanthropia foundations that are supporting the romand network of oncology and our institutional molecular tumor board . references kolberg m , hland m , agesen th , et al : survival meta - analyses for .1800 malignant peripheral nerve sheath tumor patients with and without neurobromatosis type 1 . 
j neurosurg 126 : 319 - 329 , 2017 kahn j , gillespie a , tsokos m , et al : radiation therapy in management of sporadic and neurobromatosis type 1 - associated malignant peripheral nerve sheath tumors . 
front oncol 4 : 324 , 2014 zehou o , fabre e , zelek l , et al : chemotherapy for the treatment of malignant peripheral nerve sheath tumors in neurobromatosis 1 : a 10 - year institutional review . 
orphanet j rare dis 8 : 127 , 2013 peacock j.d. , cherba d , kampfschulte k , et al : molecular - guided therapy predictions reveal drug resistance phenotypes and treatment alternatives in malignant peripheral nerve sheath tumors . 
j transl med 11 : 213 , 2013 brohl a.s. , kahen e , yoder sj , et al : the genomic landscape of malignant peripheral nerve sheath tumors : diverse drivers of ras pathway activation . 
sci rep 7 : 14992 , 2017 lee w , teckie s , wiesner t , et al : prc2 is recurrently inactivated through eed or suz12 loss in malignant peripheral nerve sheath tumors . 
curr treat options oncol 16 : 328 , 2015 shurell e , singh as , crompton jg , et al : characterizing the immune microenvironment of malignant peripheral nerve sheath tumor by pd - l1 expression and presence of cd8 + tumor inltrating lymphocytes . 
budczies j , mechtersheimer g , denkert c , et al : pd - l1 ( cd274 ) copy number gain , expression , and immune cell inltration as candidate predictors for response to immune checkpoint inhibitors in soft - tissue sarcoma . 
payandeh , ms , sadeghi , m , sadeghi , e : complete response to pembrolizumab in a patient with malignant peripheral nerve sheath tumor : the rst case 14 . 
armand p , engert a , younes a , et al : nivolumab for relapsed / refractory classic hodgkin lymphoma after failure of autologous hematopoietic cell transplantation : extended follow - up of the multicohort single - arm phase ii checkmate 205 trial . 
straub m , drecoll e , pfarr n , et al : cd274 / pd - l1 gene amplication and pd - l1 protein expression are common events in squamous cell carcinoma of the oral oncol 4 : 1237 - 1244 , 2018 cancer . 
n engl j med 372 : 311 - 319 , 2015 ikeda s , goodman am , cohen pr , et al : metastatic basal cell carcinoma with amplication of pd - l1 : exceptional response to anti - pd1 therapy . 
luo n , formisano l , gonzalez - ericsson pi , et al : melanoma response to anti - pd - l1 immunotherapy requires jak1 signaling , but not jak2 . 
 first report of clinical response to venetoclax in early t - cell precursor acute lymphoblastic leukemia introduction early t - cell precursor acute lymphoblastic leukemia ( etp - all ) is a recently recognized , highrisk subgroup of all characterized by early differentiation arrest and distinct genetic and transcriptional features . 
etp - all represents 10% to 30% of t - cell all ( t - all ) cases in adults and 10% to 15% of cases in children , exhibits inferior response rates to chemotherapy , and is characterized by high relapse rates with dismal outcomes in the relapsed / refractory setting.1 recent insights into the biology of etpall using bh3 profiling have revealed bcl - 2 dependence2 with exquisite in vitro sensitivity to the bcl - 2selective antagonist venetoclax.3 herein , we report a patient with refractory etp - all and another with t - all and some features suggestive of the etp subtype who had excellent responses to venetoclax given in combination with cytotoxic chemotherapy . 
both patients received venetoclax off - label and provided written informed consent after receiving counseling about the unapproved nature of this treatment , the rationale for its use , and possible adverse effects from the medication . patient 1 a 71 - year - old woman was diagnosed with etpall in april 2017 with 80% bone marrow blasts . 
she received two cycles of hyperfractionated cyclophosphamide , vincristine , doxorubicin and dexamethasone ( hyper - cvad ) alternating with high - dose methotrexate and cytarabine and was refractory to treatment . 
 negative markers included cd1a , cd2 , surface cd3 , cd8 , cd14 , cd15 , cd19 , cd22 , cd25 , cd36 , cd41 , myeloperoxidase , and terminal deoxynucleotidyl transferase ( tdt )  . 
 a third cycle of mini - cvd was then administered , but venetoclax was held on day 31 of cycle yazan numan mansour alfayez abhishek maiti yesid alvarado elias j . 
at the time of writing , she remains in morphologic remission with progressively declining levels of mrd in the bone marrow ( 0.02% on cycle 3 day 44 ; 0.01% on cycle 3 day 78 )  . 
filgrastim was initiated on cycle 3 day 84 enabling resumption of venetoclax ( without chemotherapy ) on cycle 3 day 93 . patient 2 a 75 - year - old man was referred to our center with an outside diagnosis of refractory secondary acute myeloid leukemia ( aml )  . 
the patient had initially been diagnosed with myelodysplastic syndrome ( mds ) in june 2016 and treated with azacitidine for six cycles before it was discontinued , reportedly because of pancytopenia . 
initial bone marrow examination at our center in october 2017 showed 85% blasts with a phenotype consistent with t - all ( positive for cytoplasmic cd3 , cd5 , cd7 , cd10 ( partial ) , cd13 , cd34 , cd38 ( dim ) , cd56 ( dim ) , cd117 ( dim ) , and hla - dr and negative for cd1a , cd2 , surface cd3 , cd4 , cd8 , cd14 , cd15 , cd19 , cd22 , cd25 , cd33 , cd36 , cd41 , cd64 , cd123 , myeloperoxidase , and tdt )  . 
the blasts lacked cd1a and cd8 expression , while co expressing the myeloid markers cd13 , cd117 ( dim ) , and cd34 , consistent with etp - all ; however , cd5 expression was strong , which argued against etp - all . 
we reviewed his previous bone marrow slides performed at the outside facility ( august 2017 ) , which showed an acute leukemia ( 70% blasts ) without good evidence for a myeloid immunophenotype and dysplastic granulocytes . 
 flow cytometry performed at the outside facility had shown expression of cd5 ( moderate ) , cd7 ( bright ) , cd10 ( partial ) , cd11b ( partial ) , cd13 ( variable ) , cd33 ( variable ) , cd34 ( moderate ) , cd38 ( moderate ) , cd45 ( dim ) , cd56 ( partial ) , cd117 ( variable ) , cd123 ( partial ) , and hla - dr ( partial ) , and karyotyping had revealed monosomy 7 , trisomy 1 , and der ( 1 ; 22 ) ( q10 ; q10 )  . 
the reported immunophenotype of the blasts ( 7% ) was positive for cd34 , cd117 , hla - dr , cd13 , cd33 ( partial ) , cd38 , cd7 , myeloperoxidase , and cytoplasmic cd3 ( subset ) , and negative for cd2 , cd3 , cd4 , cd10 , cd11b , cd14 , cd15 , cd16 , cd19 , cd56 , cd64 , cd123 , and tdt . 
routinely stained sections of bone marrow core biopsy specimens obtained from patients with ( a ) t - cell acute lymphoblastic leukemia and ( c ) early t - cell precursor acute lymphoblastic leukemia demonstrate sheets of medium - sized blasts . 
immunohistochemical studies performed with anti - bcl - 2 antibody ( clone 100 / d5 , leica / novocastra , buffalo grove , il ) on formalin fixed , paraffin - embedded tissue of bone marrow core biopsy specimen of ( b ) the patient with t - cell acute lymphoblastic leukemia demonstrate that blasts show variable bcl - 2 expression . 
mutational analysis disclosed the known srsf2 mutation and tet2 variants , and a new mpl variant of uncertain orig the response , however , was transient , with a repeat bone marrow examination 4 weeks later showing persistent t - all with 10% blasts , background mds , and del ( 20q ) on karyotyping . 
the patient then received a third cycle of mini - cvd with venetoclax , and a follow - up bone marrow examination 4 weeks later was markedly hypocellular with no morphologic evidence of acute leukemia , but flow cytometry showed persistent mrd ( t - all ) at 1.2%. 
unfortunately , approximately 3 weeks afterward , he was found to have relapsed with 62% bone marrow blasts ; flow cytometry revealed 18.8% aberrant t lymphoblasts with no appreciable change in immunophenotype , whereas karyotyping and fluorescent in situ hybridization again showed del ( 20q ) and monosomy 7 . 
serial ngs was not performed . clonal selection and survival advantage mediated by antiapoptotic bcl - 2 family proteins have a central role in lymphoblast biology.5 , 6 abt - 263 ( navitoclax ) , the clinical equivalent of the prototypical bh3 - mimetic abt - 737 , 7 targeted both bcl - xl and bcl - 2 ; however , severe thrombocytopenia as a result of bcl - xl inhibition in megakaryocytes limited its application in the clinic.8 - 10 venetoclax ( formerly abt - 199 ) is a bcl - 2selective bh3 - mimetic that spares platelets while inducing apoptosis of tumor cells.6 by using bh3 profiling in patient - derived tumor xenograft models , chonghaile et al3 demonstrated that t - all cells are , in general , dependent on bcl - xl for survival . 
other investigators have reported similar findings and shown synergism between venetoclax and cytotoxic chemotherapy against t - all cell lines and primary samples.11 , 12 to our knowledge , we report the first clinical response of etp - all ( case 1 ) , which has been durable thus far , to venetoclax administered in combination with low - intensity chemotherapy . 
trajectory of circulating leukocyte count ( 103 / l ) and blast percentage from start of treatment for patient 1 with corresponding serum phosphate ( mg / dl ) and plasma uric acid levels ( mg / dl ) , and absolute blast count ( 103 / l )  . 
 this is in line with the preclinical data showing particular susceptibility of the etp subtype to bcl - 2 inhibition , with greater dependence on bcl - xl exhibited by other , more mature subtypes of t - all . 
given these preclinical data and such anecdotal clinical responses , as well as preclinical evidence of efficacy of venetoclax against mixed lineage leukemiarearranged b - cell all ( b - all ) , 13 a phase ib clinical trial ( clinicaltrials.gov identifier : nct03319901 ) testing the combination of venetoclax and minicvd in previously untreated adults with all ( both band t - all , including etp - all ) has been initiated at dana - farber cancer institute and md anderson cancer center . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . yazan numan no relationship to disclose mansour alfayez no relationship to disclose abhishek maiti research funding : celgene ( inst ) yesid alvarado no relationship to disclose elias j . 
jain n , lamb av , obrien s , et al : early t - cell precursor acute lymphoblastic leukemia / lymphoma ( etp - all / lbl ) in adolescents and adults : a high - risk subtype . 
chonghaile tn , roderick je , glenfield c , et al : maturation stage of t - cell acute lymphoblastic leukemia determines bcl - 2 versus bcl - xl dependence and sensitivity to abt - 199 . 
kantarjian h , ravandi f , short nj , et al : inotuzumab ozogamicin in combination with lowintensity chemotherapy for older patients with philadelphia chromosome - negative acute lymphoblastic leukaemia : a single - arm , phase 2 study . 
souers aj , leverson jd , boghaert er , et al : abt - 199 , a potent and selective bcl - 2 inhibitor , achieves antitumor activity while sparing platelets . 
roberts aw , advani rh , kahl bs , et al : phase 1 study of the safety , pharmacokinetics , and antitumour activity of the bcl2 inhibitor navitoclax in combination with rituximab in patients with relapsed or refractory cd20 + lymphoid malignancies . 
roberts aw , seymour jf , brown jr , et al : substantial susceptibility of chronic lymphocytic leukemia to bcl2 inhibition : results of a phase i study of navitoclax in patients with relapsed or refractory disease . 
wilson wh , oconnor oa , czuczman ms , et al : navitoclax , a targeted high - affinity inhibitor of bcl - 2 , in lymphoid malignancies : a phase 1 dose - escalation study of safety , pharmacokinetics , pharmacodynamics , and antitumour activity . 
anderson nm , harrold i , mansour mr , et al : bcl2 - specific inhibitor abt - 199 synergizes strongly with cytarabine against the early immature loucy cell line but not more - differentiated t - all cell lines . 
peirs s , matthijssens f , goossens s , et al : abt - 199 mediated inhibition of bcl - 2 as a novel therapeutic strategy in t - cell acute lymphoblastic leukemia . 
 economic impact of next - generation sequencing versus single - gene testing to detect genomic alterations in metastatic nonsmall - cell lung cancer using a decision analytic model nathan a . 
pennell , md , phd1 ; alex mutebi , phd2 ; zheng - yi zhou , phd3 ; marie louise ricculli , msc3 ; wenxi tang , ms3 ; helen wang3 ; annie guerin , msc4 ; tom arnhart , pharmd , ms2 ; anand dalal , phd2 ; medha sasane , phd2 ; kevin y . 
model outcomes for each strategy were time - to - test results , the proportion of patients identied harboring alterations with or without us food and drug administrationapproved therapies , and total testing costs . 
a budget impact analysis assessed the economic effects of increasing the proportion of ngs - tested patients . results in a hypothetical 1 , 000 , 000 - member health plan , 2 , 066 medicare - insured patients and 156 commercially insured patients were estimated to have mnsclc and to be eligible for testing . 
2019 by american society of clinical oncology lung cancer ( nsclc ) , introduction nonsmall - cell the most common type of lung cancer , 1 can be classied on the basis of the presence of genomic alterations in driver oncogenes , 2 , 3 such as egfr , alk , ros1 , kras , braf , met , her2 , ret , and ntrk1.2 - 4 in the united states , several theratarget egfr , alk , ros1 , and braf pies that v600e alterations5 - 7 have been approved by the us food and drug administration ( fda ) therapy for patients with metaacross lines of static nsclc ( mnsclc )  . 
other targeted therapies are in clinical including those trial phase , against met , her2 , ntrk1 , and ret alterations identier : nct01639508 ; ( clinicaltrials.gov nct02414139 ; nct02716116 ; nct01336634 ; nct02097810 )  . improve treatment targeted therapies have been demonstrated to signicantly response and progression - free survival7 - 9 ; therefore , it is important to identify patients with specic genomic alterations to individualize treatment and optimize outcomes.2 , 9 currently , a number of testing strategies are available with which to identify genomic alterations in nsclc . 
 pennell et al context key objective to determine the least costly strategy for testing for all recommended molecular abnormalities in patients with nonsmall - cell lung cancer ( nsclc ) at diagnosis . knowledge generated upfront next - generation sequencing ( ngs ) testing for all relevant molecular targets in nsclc is less costly than performing single - gene testing , no matter what testing strategy is used . 
upfront ngs testing also results in the largest number of patients with targetable genetic alterations being identied in the shortest period of time . relevance as more clinically relevant genetic targets in nsclc emerge that require routine testing at diagnosis , it is vital to identify the molecular testing strategy that is timeliest , spares the most tissue , and is most cost efcient , as this must be done in every new patient . 
our model illustrates that moving from sequential single - gene tests or even panels of tests to broader ngs testing for patients with advanced nsclc is already the best strategy in these three areas and will only become more relevant as the list of tests grows . 
stakeholders should consider moving to ngs as the preferred method for biomarker testing . single - gene tests for less common alterations.10 sequences of single - gene tests can also be used without hotspot panel , typically testing for the most common alterations rst and less common alterations last.10 in either case , running a sequence of single - gene tests can be time consuming and may require a relatively large tissue sample , which is not always available as nsclc is often detected at an advanced stage and only small biopsy samples are usually attainable.11 - 13 as a result , one or more rebiopsies may be needed to complete the sequence . 
in recent years , next - generation sequencing ( ngs ) has emerged as a reliable strategy to simultaneously test for multiple alterations using a single tissue sample14 ; however , only a small percentage of patients with nsclc currently receive ngs , 15 as a number of practical barriers hinder its wider adoption , including limited patient access , a lack of awareness and communication within medical care teams about the benets of ngs , limited coverage , and low reimbursement rates.16 , 17 in addition , given the relatively recent development of ngs , there is a need to better understand the economic implications of using ngs versus other testing strategies in real - world clinical practice . therefore , a decision analytic model was developed to estimate the difference in testing costs and time - to - test results between ngs and commonly used single - gene testing strategies among patients with mnsclc from the perspective of the centers for medicare & medicaid services ( cms ) and a us commercial payer . 
in addition , the budget impact of increasing ngs testing was assessed from the perspective of both cms and a us commercial payer . methods model overview a decision analytic model was developed in microsoft excel 2016 from the perspective of cms and a us commercial payer for patients with newly diagnosed mnsclc who were eligible for genomic testing to determine mnsclc alteration status and inform treatment selection . 
alterations included in the model involved the programmed cell death protein 1 and its ligand programmed death protein 1 ligand ( pd - l1 ) , as well as genes egfr , alk , ros1 , braf , kras , met , her2 , ret , and ntrk1 . 
four genomic testing strategies were considered . upfront ngs : at mnsclc diagnosis , patients received a panel that tested simultaneously for all alterations with and without fda - approved therapies ( egfr , alk , ros1 , braf , met , her2 , ret , and ntrk1 )  . sequential testing : patients received a sequence of singlegene tests for alterations with fda - approved therapies ( egfr , alk , ros1 , and braf )  . 
negative test results for all four alterations were followed by either a sequence of single - gene tests or ngs to test for alterations without fda - approved therapies ( met , her2 , ret , and ntrk1 )  . 
single - gene tests were assumed to be ordered in sequence , one at a time , after receiving negative results for each previous test . exclusionary testing : patients rst received a test for kras because kras is the most common mutation in lung adenocarcinoma , and as the genetic alterations of interest are mutually exclusive , a positive test would prevent the need for additional testing . 
negative results were followed by either a sequence of single - gene tests or ngs to test for alterations without fda - approved therapies ( met , her2 , ret , and ntrk1 )  . all four testing strategies included pd - l1 and routine immunohistochemistry testing , which were assumed not to time because they were conducted sitake additional multaneously with the rst test in sequential and exclusionary testing and at the same time as the hotspot panel and ngs . 
appropriate therapyeither targeted therapy or nontargeted therapy ( eg , chemotherapy and immunotherapy ) was selected on the basis of the test results , that is , a conrmed alteration with or without fdaapproved therapies or no known alteration . 
patients who received rst - line nontargeted therapy and who tested negative for alterations with fda - approved therapies were assumed to continue testing with ngs or single - gene tests for alterations without fda - approved therapies , provided that enough tissue sample remained . 
a proportion of patients with alterations without fda - approved therapies were assumed to receive a benet from participating in clinical trials of agents under development . model inputs epidemiology and population inputs . 
for cms , the diagnosis of metastatic nsclc ( biopsy performed ) sequential exclusionary hotspot panel positive for tested alteration negative for tested alteration positive for tested alteration negative for tested mutation negative for tested mutation negative for tested mutation positive for tested alteration exclusionary mutation ( kras positive ) appropriate therapy appropriate therapy appropriate therapy appropriate therapy appropriate therapy enough tissue / no need for rebiospy insufficient tissue / need rebiopsy negative for tested mutation positive for tested alteration successful rebiopsy no rebiopsy / failed rebiopsy continue testing until appropriate treatment appropriate treatment appropriate treatment continue testing until appropriate treatment legend : continue testing for next alteration until the decision is made for appropriate treatment . is there sufficient v insufficient tissue and need for rebiopsy to continue testing ? fig 1 . 
sequential testing : sequence of single - gene tests for egfr , alk , ros1 , and braf , followed by either a sequence of single - gene tests or ngs for met , her2 , ret , and ntrk1 . 
hotspot panel : panel testing simultaneously for egfr , alk , ros1 , and braf , followed by either a sequence of single - gene tests or ngs for met , her2 , ret , and ntrk1 . 
epidemiology , population , and testing cost inputs variable commercial payer source patient population in the health care plan , no . 1 , 000 , 000 1 , 000 , 000 assumption adults in the population of interest , % * cms : kaiser family foundation18 ; commercial : us census bureau19 no . 
unit costs for all genomic alteration testing were based on the 2017 clinical lab fee schedule23 for the medicare - insured population and from dalal et al15 for the commercially insured population using two us administrative claims databases ( table 1 )  . 
single - gene tests for kras , egfr , her2 , and braf were assumed to be performed using real - time polymerase chain reaction . pd - l1 tests were assumed to be performed using immunohistochemistry or uorescence in situ hybridization . for each testing strategy , the per - patient cost of sequential , exclusionary , and hotspot panel testing was considered to be the sum of the cost of each individual test for pd - l1 and alterations with or without fdaapproved therapiesegfr , alk , ros1 , braf , met , her2 , ret , ntrk1 , and krasmultiplied by the proportion of patients who received each test , which was determined by the results for the preceding testing and rebiopsy failure rate ( table 2 )  . 
the difference between the plan - total testing costs of a certain strategy and those of ngs was considered as the cost savings associated with ngs versus the strategy in question . 
as a result of insufcient tissue sample , 8% of patients were assumed to require rebiopsy to continue testing.22 of these patients , only 30% were assumed to be actually rebiopsied , with 15% experiencing failure with rebiopsy.28 on the basis of on handorf et al , 28 rebiopsy cost was $415.30 for medicare - insured patients and $1 , 399.80 for commercially insured patients . 
one half of patients who tested negative for alterations with fda - approved therapies and who received nontargeted therapy were assumed to continue testing for alterations without fda - approved therapies . 
of these patients , 75% were assumed to continue testing with ngs and 25% with one of the other testing strategies . model outputs model results for ngs were compared with those for sequential , exclusionary , and hotspot panel testing in terms of differences in the estimated total cost associated with testing , calculated separately for cms and the commercial payer ; time - to - test results that accounted for the time associated with test turnaround and rebiopsy , assumed to be the same for cms and the commercial payer ; and the proportion of patients identied by each testing strategy as harboring alterations with or without fda - approved therapies . budget impact analysis we conducted a budget impact analysis to assess the effects of increasing upfront ngs testing in cms and commercial plans . 
total cost and cost difference versus ngs medicare - insured patients ( n = 2 , 066 ) commercially insured patients ( n = 156 ) testing strategy sequential exclusionary hotspot panel total cost 2 , 190 , 499 3 , 721 , 368 3 , 584 , 177 4 , 331 , 295 note . 
costs are given in 2017 us dollars . abbreviation : ngs , next - generation sequencing . cost difference v ngs total cost cost difference v ngs 1 , 530 , 869 1 , 393 , 678 2 , 140 , 795 620 , 369 747 , 771 624 , 178 871 , 211 127 , 402 3 , 809 250 , 842 the other parameters at their base - case values . 
on the basis of commercial payer for the 156 commercially insured patients , ngs rates , remained the least expensive testing option , with cost savings of $3 , 809 versus exclusionary testing , $127 , 402 versus sequential testing , and $250 , 842 versus hotspot panel testing . time to receive test results the cumulative time to receiving full test results and initiating treatment , incorporating both testing time and time for rebiopsy , is a critical element in molecular testing . 
proportion of patients identied with alterations with or without fda - approved therapies patients identied with alterations with fda - approved therapies ( medicare insured , n = 446 ; commercially insured , n = 34 ) patients identied , % difference v ngs , % difference in no . 
the only exception was ngs versus exclusionary testing for commercial payersranging from $19 , 943 to $27 , 562likely because of the relatively small base - case cost savings results ( $3 , 809 )  . 
results were most sensitive to scenarios in which test unit costs and the proportion of patients tested for alterations without fdaapproved therapies were varied . discussion several testing strategies for the detection of genomic alterations in nsclc exist in clinical practice ; however , scant evidence is available on the comparative economic implications of using these strategies in patients with mnsclc . 
accordingly , an economic model was developed to estimate the time - to - test results ; the proportion of patients identied as harboring alterations with or without fda - approved therapies ; and testing costs associated with upfront ngs , sequential , exclusionary , and hotspot panel testing among patients with mnsclc . model results demonstrate that ngs was associated with the same ( as hotspot panel ) or shorter ( v exclusionary and sequential testing ) time - to - test results and lower testing costs than sequential , exclusionary , and hotspot panel testing . both ngs and hotspot panel testing had the shortest time - totest results ( 2 weeks ) , indicating that these two testing strategies may yield the shortest time to initiation of appropriate therapy among the four strategies considered here . of note , although the unit cost of ngs was higher than that of individual single - gene tests , the overall cost , which included testing and rebiopsy cost , to test for a broad range of alterations was the lowest for ngs . 
although no treatment is yet approved for these alterations , it is important to identify these patients to give them the opportunity to participate in clinical trials of investigational therapies . although prior studies have discussed the cost effectiveness and benets of various testing strategies in patients with nsclc , 31 - 34 the current study is , to our knowledge , the rst to quantify the economic impact of using ngs versus other testing strategies to detect a comprehensive list of alterationsegfr , alk , ros1 , braf , met , her2 , ret , ntrk1 , and krasin patients with mnsclc in the united states . 
from a clinical standpoint , the timely identication of genomic alterations in patients with mnsclc is key to initiating the most appropriate treatment course on the basis of either targeted therapy , if available , or nontargeted therapy . 
as the results of the current study indicate , upfront ngs seems to be the cheapest broad molecular testing strategy for genomic alterations in terms of both time - to - test results and testing costs . 
furthermore , unlike other testing strategies , ngs allows for the simultaneous screening of both common and less common alterations , which is important because less common alterations are less likely to be tested when using sequential testing strategies . 
in a retrospective review of genomic testing patterns in patients with nonsquamous mnsclc , only 63 of 814 patients were reported to have undergone testing for all seven alterations recommended by national comprehensive cancer network for patients with mnsclc , namely egfr , alk , ros1 , braf , met , ret , and her2.17 using ngs may increase the number of patients tested for including both common and less common alterations , those recommended by the national comprehensive cancer network , thereby providing more information to physicians and patients with which to make informed and timely treatment decisions . 
future studies should also assess the clinical implications of our nding that , as a result of longer time - totest results , patients using sequential and / or exclusionary testing are able to initiate targeted therapy nearly 3 weeks later than patients using ngs or hotspot panel testing . 
it is worth noting that , whereas this study suggests that ngs is a more efcient testing strategy than sequential , exclusionary , and hotspot panel testing , several barriers to wider adoption of ngs in clinical practice remain , including limited coverage and reimbursement.17 however , this may change over time given that the lower total testing costs and shorter initiationof ngs , together with a reduced need for rebiopsy , may reduce medical costs substantially and improve outcomes , beneting patients and payers alike . resultsand thus treatment time - to - test this study has some limitations . 
first , as the study is based on a modeling approach using a decision tree and budget impact inputs and assumptionssome of framework , the model which were based on expert opinionmay contain uncertainty or may have limited generalizability . 
last , the model included only testing costs and not therapy costs and benets , the inclusion of which would allow for an estimate of a wider spectrum of costs . in conclusion , for both cms and commercial payers , upfront ngs testing was associated with the same ( as ( v exclusionary and hotspot panel testing ) or shorter sequential testing ) time to the initiation of appropriate treatment , as well as substantial cost savings compared with all other testing strategies in patients with newly diagnosed mnsclc . 
pennell , alex mutebi , zheng - yi zhou , marie louise ricculli , wenxi tang , helen wang , annie guerin , tom arnhart , anand dalal , kevin y . 
pennell , alex mutebi , zheng - yi zhou , marie louise ricculli , wenxi tang , helen wang , annie guerin , tom arnhart , anand dalal , medha sasane , kevin y . 
pennell consulting or advisory role : astrazeneca , eli lilly , regeneron , cota healthcare , merck research funding : genentech ( inst ) , celgene ( inst ) , astrazeneca ( inst ) , pzer ( inst ) , merck ( inst ) , loxo ( inst ) , altor bioscience ( inst ) , spectrum pharmaceuticals ( inst ) , bristol - myers squibb ( inst ) tom arnhart employment : novartis , alexion pharmaceuticals stock and other ownership interests : alexion pharmaceuticals anand dalal employment : novartis stock and other ownership interests : novartis medha sasane employment : sano , novartis ( i ) stock and other ownership interests : sano , novartis ( i ) kevin y . 
wu employment : novartis , astrazeneca stock and other ownership interests : novartis , astrazeneca , gilead sciences , intercept pharmaceuticals , merck , organovo , takeda , seattle genetics , tg therapeutics kenneth w . 
transl respir med 1 : 6 , 2013 kohno t , nakaoku t , tsuta k , et al : beyond alk - ret , ros1 and other oncogene fusions in lung cancer . 
expert rev mol diagn 16 : 737 - 749 , 2016 barlesi f , mazieres j , merlio jp , et al : routine molecular proling of patients with advanced non - small - cell lung cancer : results of a 1 - year nationwide programme of the french cooperative thoracic intergroup ( ifct )  . 
lancet 387 : 1415 - 1426 , 2016 kris mg , johnson be , berry ld , et al : using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs . 
dalal aa , guerin a , mutebi a , et al : economic analysis of braf gene mutation testing in real world practice using claims data : costs of single gene versus panel tests in patients with lung cancer . 
levy bp , chioda md , herndon d , et al : molecular testing for treatment of metastatic non - small cell lung cancer : how to implement evidence - based recommendations . 
yang p , allen ms , aubry mc , et al : clinical features of 5 , 628 primary lung cancer patients : experience at mayo clinic from 1997 to 2003 . 
reck m , rodrguez - abreu d , robinson ag , et al : pembrolizumab versus chemotherapy for pd - l1 - positive non - small - cell lung cancer . 
chen d , zhang lq , huang jf , et al : braf mutations in patients with non - small cell lung cancer : a systematic review and meta - analysis . 
meng d , yuan m , li x , et al : prognostic value of k - ras mutations in patients with non - small cell lung cancer : a systematic review with meta - analysis . 
handorf ea , mcelligott s , vachani a , et al : cost effectiveness of personalized therapy for rst - line treatment of stage iv and recurrent incurable adenocarcinoma 29 . 
vanderlaan pa , yamaguchi n , folch e , et al : success and failure rates of tumor genotyping techniques in routine pathological samples with non - small - cell lung 5 : 293 - 305 , 2011 22 : 2616 - 2624 , 2011 cancer 18 : 651 - 659 , 2017 snapshot / 2005 prevalence / canques.html 375 : 1823 - 1833 , 2016 2014 cancer 81 : 1 - 10 , 2013 of the lung . 
as noted in a recent article by black et al2 in jco precision oncology , many laboratories , including ours , have included analysis of the upstream promoter region in routine pten analysis as a result of early literature that identified variants in this region in individuals with phts component cancers , with some variants reported to have a possible impact on translation.3 , 4 our laboratory , however , does not currently classify any pten promoter variant as pathogenic or likely pathogenic . 
thus , we also question the clinical utility of including this region in clinical diagnostic testing and offer our laboratorys experience in support of the proposal that analysis of the full pten promoter region may not be beneficial as a component of hereditary cancer panel testing . 
 this study was approved by the western institutional review board , puyallup , washington ( wirb 20162523 )  . to review briefly , black et al2 assessed cancer risks in individuals who underwent multigene hereditary cancer panel testing at their clinical diagnostic laboratory and identified no increased risk for cancer overall or for the major phts component cancers ( breast , colon , endometrial , thyroid , and renal ) in those with pten promoter variants of uncertain significance ( vus ) compared with those with wild - type pten . 
no statistically significant difference was observed in the frequency of pten promoter vus among those with breast , endometrial , thyroid , renal , or colorectal cancer compared with individuals with no personal history of cancer ( table 1 )  . 
within this cohort , individuals with vus in the minimum promoter region did not seem to have increased prevalence of phts component cancers compared with those with promoter vus outside this region ( table 2 )  . 
of note , variants in the minimum promoter region were found at a lower frequency in those with cancers of interest compared with individuals without cancer ; however , the power of these analyses was limited by sample size . as noted by black et al , 2 additional functional and clinical data are needed to better understand the impact of ultra - rare variants . 
the three promoter variants reported by teresi et al4 as leading to decreased promoter activity ( c . - 862g > t , c . - 854c > g , and c . - 765g > a ) by using the current reference sequence were either absent or present in only one of approximately 30 , 000 alleles in the genome aggregation database.6 furthermore , none of these variants were observed in our cancer panel cohort . 
pten promoter variant prevalence comparisons for core pten - associated cancers individuals with cancer individuals without cancer carrier * negative carrier * negative cancer type female breast endometrial thyroid colorectal renal breast and endometrial breast and thyroid or endometrial 37 , 648 1 , 308 3 , 082 breast and / or thyroid and / 44 , 352 note . 
mester manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors immediate family member , inst = my institution . 
zhou x - p , waite ka , pilarski r , et al : germline pten promoter mutations and deletions in cowden / bannayan - riley - ruvalcaba syndrome result in aberrant pten protein and dysregulation of the phosphoinositol - 3 - kinase / akt pathway . 
bekelman , md2 , 4 , 6 , 7 the senseless killings of george floyd , ahmaud arbery , breonna taylor , and countless others have led to a national reckoning on institutional racism . medicine , race has long been used as a heuristic , or mental shortcut , used by clinicians to process information and make decisions within the time and cognitive constraints of our healthcare system.1 however , using race as a heuristic in medical decision making has the potential to accentuate cognitive biases and lead to deviations from rational decision making , even among the most well - intentioned of clinicians.2 furthermore , it is well recognized that race is a social , not a biological , construct ; as a result , the broad race categories used in medicine are heterogeneously dened and may be incongruent with genetic ancestry , particularly among minority patients.3 , 4 because cancer is inherently a disease of genetic aberration , using these race categories to make decisions in oncology may lead to the delivery of ineffective , or even detrimental , patient care . the rapid advancement of precision oncology , the use of molecular and genetic testing to tailor cancer therapies to individual patients , has uniquely positioned the eld of oncology to move beyond racebased medicine . 
since the discovery of the rst targeted therapy , imatinib , in the 1990s , over 80 targeted agents have been approved by the us food and drug administration for the treatment of numerous solid organ and hematologic malignancies.5 , 6 some of these precision oncology applications correlate with patient race . 
for instance , epidermal growth factor receptor ( egfr ) mutations in nonsmall - cell lung cancer are found in over 60% of patients of asian descent and strongly predict sensitivity to egfr tyrosine kinase inhibitors.7 , 8 similarly , several founder mutations in brca1 / 2 have been observed in multiple ethnic groups , including patients with ashkenazi jewish ancestry , and can be used to predict sensitivity to poly ( adp - ribose ) polymerase inhibitors in patients with breast , ovarian , prostate , and pancreatic cancers.9 - 14 fortunately , the tools of precision oncology enable the administration of agents such as egfr and poly ( adpribose ) polymerase inhibitors to be guided by genetic testing rather than race or ancestry . however , differential use of precision oncology has the potential to widen the very racial disparities that the eld seeks to eliminate.15 indeed , multiple studies have demonstrated decreased rates of genetic counseling , germline and somatic genetic testing , targeted therapy administration , genomic database participation , and clinical trial enrollment among minorities compared with white patients.16 - 22 there are many barriers to achieving the promise of precision oncology , including lack of access to and payment for testing , targeted treatment , and counseling . 
additionally , one major challenge is simply a lack of awareness among minority patients and clinicians.23 the rapid advancement of precision oncology has made it difcult for many clinicians to have the time and expertise to select appropriate genetic testing , interpret and apply test results , and educate patients on their implications.24 this challenge may be especially pertinent among minority - serving physicians , who have been shown to be less likely to refer to genetics and order germline testing for diseases such as breast and colorectal cancer.25 the resultant use of race as a heuristic in the setting of these time and cognitive constraints may lead to decisions that are inuenced by implicit bias , unconscious stereotypes , or attitudes toward people that inuence ones actions and behaviors . 
much like the general population , physicians have been found to have implicit bias with a preference for white over black patients and to associate race with their assessments of patient intelligence , risk - taking behavior , and adherence to medical advice.26 - 29 in turn , these perceptions have been linked to poorer communication , biased treatment recommendations , and unequal access to quality healthcare for minority patients.30 , 31 in this paper , we propose three strategies to advance health equity in precision oncology ( table 1 )  . 
ehr - based strategies to advance health equity in precision oncology ehr strategy key features default orders preselected ehr orders that are automatically applied unless a clinician actively modies them behavioral economics principle nudge decision making by taking advantage of individual status quo bias , or the tendency to favor the current state of affairs automated patient dashboards programs that harness the automation and computing power of the ehr to identify candidate patients for a given diagnostic or therapeutic intervention minimize choice overload by searching through a clinicians full patient panel to identify the subset that is eligible for precision oncology care clinician - directed clinical integration of standardized clinical pathways into the ehr for minimize choice overload by simplifying and facilitating use at the point of care complex clinical decision making use of electronic patient portals to send educational materials prime patients to initiate conversations with their clinicians about precision oncology and communicate normative feedback on its uptake among other patients about precision oncology note . 
we propose that behavioral economic principles should be leveraged to promote health equity in precision oncology as well . to steer the ehr offers a scalable , cost - effective approach to harnessing behavioral economic principles to advance health equity in precision oncology . 
defaultspreselected choices that are applied unless an individual actively changes themare powerful tools that take advantage of the status quo bias to nudge decision making while minimizing cognitive effort and preserving choice . 
this approach has been shown to optimize cancer care delivery by increasing high - value medication prescribing and reducing unnecessary daily imaging during palliative radiotherapy.36 , 37 in the precision oncology space , default orders in the ehr can be used to make direct referrals to genetics for patients who meet guideline criteria on the basis of factors such as tumor type and age , clinical characteristics that can be easily identied in the ehr . 
 advancing health equity in precision oncology decision altogether.47 - 49 the ehr is well equipped to solve the problem of choice overload through automated patient dashboards , which can efciently search through all the patients cared for by a given clinician or at a particular practice to identify a more limited pool of individuals who are eligible for a given intervention . 
this approach has been leveraged by multiple clinical trial matching programs to match patient and tumor characteristics from the ehr to study eligibility criteria , thereby enabling oncologists to identify study opportunities for individual patients . 
these programs have been effective in facilitating increased clinical trial participation not only within academic institutions but also at community oncology practices , sites that are more likely to serve minority and underserved populations.50 , 51 similar automated patient dashboards should be leveraged to identify candidate patients for other precision oncology opportunities that remain underutilized by minorities , such as genetics referrals , germline and somatic testing , targeted therapy administration , and genomic database participation . 
because this strategy matches patients to precision oncology care using the automation and computing power of the ehr rather than the heuristics of human decision making , it is likely to be more effective than individual clinicians in advancing health equity . clinical decision support systems decision support also mitigates choice overload by guiding individuals toward more desirable decisions while preserving their freedom of choice . 
in precision oncology , clinical pathways have emerged as a decision support strategy for clinicians to handle the elds ever - expanding scope and complexity.52 however , these pathways are ineffective unless they are seamlessly integrated into routine clinical practice to maximize their usefulness . 
embedding clinical decision support systems into the ehr has been shown to facilitate decision making in other areas of cancer diagnosis , treatment , and supportive care , 53 and it has been associated with decreased racial disparities in the management such as hypertension.39 this approach should also be leveraged to standardize precision oncology care for all patients , regardless of race . of nononcologic conditions perhaps an even more compelling clinician decision aid is a patients own request for a given medical intervention . 
priming , the use of subtle stimuli to activate thoughts and ideas , 54 is a powerful tool that can be directly used by patients to support clinicians in overcoming the use of race as a heuristic . 
first , we recognize that our proposed strategies do not fully combat the lack of access , nancial language barriers , and challenges , health literacy or longstanding history of mistrust in the healthcare system that have also impeded the adoption of precision oncology among minority patients . 
to be successful , ehr - based strategies informed by the principles of behavioral economics must be implemented as part of a broader , multifaceted approach that addresses all these challenges . second , the application of behavioral economics in medicine is still in its nascency , so there is a paucity of data on the impact of choice architecture redesign on subgroups within the population . 
future studies should incorporate the emerging concept of precision nudging to tailor behavioral economics interventions to specic patient populations as a strategy to ensure equal effectiveness for all . finally , the implementation of ehr - based strategies may require initial investment of nancial and human resources that is not feasible at all institutions . 
as implementation efforts get underway , we must monitor for and address any signs of differential uptake so that our proposed strategies do not inadvertently widen existing inequities in care . in conclusion , in this article , we present three ehr - based strategies that leverage the principles of behavioral economics to nudge oncologists away from the use of race as a heuristic in their decision making . 
 lau - min et al 4abramson cancer center , perelman school of medicine , university of pennsylvania , philadelphia , pa 5division of translational medicine and human genetics , department of medicine , perelman school of medicine , university of pennsylvania , philadelphia , pa 6department of radiation oncology , perelman school of medicine , university of pennsylvania , philadelphia , pa 7department of medical ethics and health policy , perelman school of medicine , university of pennsylvania , philadelphia , pa otherwise noted . 
guerra leadership : freenome , guardant health , genentech stock and other ownership interests : crispr therapeutics , beam therapeutics , intellia therapeutics , editas medicine honoraria : lundbeck ( i ) consulting or advisory role : bristol - myers squibb ( i ) speakers bureau : janssen , pzer ( i ) research funding : bristol - myers squibb ( inst ) travel , accommodations , expenses : lundbeck , janssen ( i ) uncompensated relationships : tapestry networks justin e . 
bekelman honoraria : unitedhealthcare , national comprehensive cancer network , cvs health , optum , american journal of managed care research funding : pzer , united health group , north carolina blue cross blue shield , embedded healthcare no other potential conicts of interest were reported . acknowledgment we thank mitesh patel , md , mba , and callie scott , msc , both at the university of pennsylvania , for their helpful comments during the writing of this manuscript . 
new york , farrar , straus and giroux , 2011 zhang f , finkelstein j : inconsistency in race and ethnic classication in pharmacogenetics studies and its potential clinical implications . 
therapies - for - cancer / shi y , au js , thongprasert s , et al : a prospective , molecular epidemiology study of egfr mutations in asian patients with advanced non - small - cell lung cancer of adenocarcinoma histology ( pioneer )  . 
j thorac oncol 9 : 154 - 162 , 2014 soria j - c , ohe y , vansteenkiste j , et al : osimertinib in untreated egfr - mutated advanced nonsmall - cell lung cancer . 
n engl j med 378 : 113 - 125 , 2018 robson m , im s - a , senkus e , et al : olaparib for metastatic breast cancer in patients with a germline brca mutation . 
binder kar , mick r , ohara m , et al : abstract ct234 : a phase ii , single arm study of maintenance rucaparib in patients with platinum - sensitive advanced pancreatic cancer and a pathogenic germline or somatic mutation in brca1 , brca2 or palb2 . 
presley cj , soulos pr , chiang ac , et al : disparities in next generation sequencing in a population - based community cohort of patients with advanced non - small cell lung cancer . 
kwiatkowski k , coe k , bailar jc , et al : inclusion of minorities and women in cancer clinical trials , a decade later : have we improved ? cancer 119 : 2956 - 2963 , 22 . 
j health care poor underserved 20 : 896 - 913 , 2009 van ryn m , burke j : the effect of patient race and socio - economic status on physicians perceptions of patients . 
cooper la , roter dl , carson ka , et al : the associations of clinicians implicit attitudes about race with medical visit communication and patient ratings of interpersonal care . 
sharma s , guttmann d , small ds , et al : effect of introducing a default order in the electronic medical record on unnecessary daily imaging during palliative radiotherapy for adults with cancer . 
grebe ta , khushf g , chen m , et al : the interface of genomic information with the electronic health record : a points to consider statement of the american college of medical genetics and genomics ( acmg )  . 
lau - min ks , asher sb , chen j , et al : real - world integration of genomic data into the electronic health record : the pennchart genomics initiative . 
cheema pk , menjak ib , winterton - perks z , et al : impact of reex egfr / alk testing on time to treatment of patients with advanced nonsquamous nonsmallmayo clinic proc 95 : 476 - 487 , 2020 cell lung cancer . 
new york , ecco , 2004 iyengar ss , lepper mr : when choice is demotivating : can one desire too much of a good thing ? j personal soc psychol 79 : 995 - 1006 , 2000 jacoby j : perspectives on information overload . 
 balancing raf , mek , and egfr inhibitor doses to achieve clinical responses and modulate toxicity in braf v600e colorectal cancer introduction recent years have seen dramatic clinical advances in targeting the erk pathway with the us food and drug administration approval of several selective inhibitors of raf and mek.1 - 5 clinical trials of these agents indicate that near - complete inhibition of pathway signaling is necessary to effectively inhibit tumor growth.6 targeted approaches have been most successful in braf v600e tumors because of the relatively wide therapeutic index of raf inhibitors ( vemurafenib and dabrafenib ) , which inhibit signaling in cells with braf v600 mutants but cause paradoxical activation of erk signaling in normal cells that are wild type for raf kinases . 
combining these agents ( with raf and mek inhibitors for braf v600e melanoma or lung cancer and with raf , mek , and epidermal growth factor receptor [ egfr ] inhibitors for braf v600e colorectal cancer ) to profoundly inhibit erk signaling has led to improved antitumor activity.7 combination therapy has also been shown to offset the toxicities caused by raf inhibitors , such as the development of keratoacanthoma and squamous cell carcinoma , resulting from paradoxical erk activation with these agents . the role of raf inhibitors in offsetting mek inhibitor toxicity and the need for dose intensity to modulate opposing toxicities is less clear . 
in the phase iii trial of trametinib in melanoma , grade 3 or 4 acneiform dermatitis occurred in 8% of trametinib - treated patients , whereas in the phase iii trial of the combination of dabrafenib and trametinib , no patient had grade 3 or 4 acneiform dermatitis.8 , 9 combinations of raf and egfr inhibitors have also had a lower incidence of acneiform rash than seen with egfr inhibitors alone.7 , 10 , 11 this is also likely because of the opposite effects of raf and egfr inhibitors on mek activation in normal cells . 
we now report the course of a patient with braf v600e colorectal cancer treated with dabrafenib , trametinib , and panitumumab in a phase ii clinical trial and characterize the effect on toxicities of different dose levels of these agents in this patient . 
she received 6 months of adjuvant folfox ( folinic acid , fluorouracil , and oxaliplatin ) chemotherapy , and imaging 3 months after completion of adjuvant therapy showed recurrent disease with peritoneal carcinomatosis ( primarily omental caking ) and ascites . 
she then provided written informed consent to participate in a clinical trial for patients with braf v600e colorectal cancer and was started on the combination of dabrafenib ( 150 mg orally twice daily ) , trametinib ( 2 mg orally once daily ) , and panitumumab ( 6 mg / kg intravenously every 2 weeks ) , all at the full us food and drug administration approved single - agent doses ( fig 1a ) , with prophylactic doxycycline ( 28 - day cycles )  . 
during the first cycle , she developed grade 2 neutropenia that was attributed to dabrafenib ; therefore , the dose of this drug was decreased one dose level to 100 mg orally twice daily . 
despite these modifications , during the fourth cycle , the rash became grade 3 ; skin cultures were positive for methicillin - sensitive staphylococcus aureus , and she was treated with sulfamethoxazole and trimethopri given the lack of response of the rash to medical treatment , with sponsor approval , the dose of dabrafenib was escalated to 100 mg orally twice daily during the fifth cycle , which was followed by clinical improvement of skin toxicity to grade 1 . 
in terms of clinical efficacy , her disease was difficult to measure on imaging , but response evaluation criteria in solid tumors review was consistent with stable disease on the computed tomography evaluations at the 6 - , 12 - , and 18 - week assessments . 
the patient died as a result of disease progression 1 month after stopping the study treatment . discussion this case shows the opposing effects of raf inhibitors and egfr and mek inhibitors on skin and the importance of raf inhibitor dose - intensity for clinical efficacy and for offsetting toxicity in combination regimens with other erk pathway inhibitors . 
as the dose of dabrafenib was reduced , skin toxicity resulting from the egfr and mek inhibitors became more pronounced , and when the dabrafenib dose was raised , the toxicity improved , confirming that dabrafenib was modulating skin toxicity resulting from the egfr and mek inhibitors . 
differences in raf signaling underlie the differing effects of raf inhibitors seen in normal and tumor tissues ; in normal tissues , raf signals as homoand heterodimers activated by ras , whereas braf v600 mutants , which lead to high erk activation and feedback suppression of upstream signaling and ras , signal uniquely as constitutively activated monomers . 
in braf v600mutant tumors , this leads to suppression of activated monomers , and in tissues with wildtype raf kinases ( functioning as dimers ) , this leads to transactivation of raf dimers.12 , 13 thus , clinical efficacy and the modulation of toxicity in combination regimens are linked ; inhibition of braf v600e monomers and paradoxical activation of erk occur at the same doses . 
the absence of clinically appreciable opposing effects to egfr or mek inhibitors in normal tissues , as occurred in this case , thus suggests inadequate occupation of the first site of raf dimers in normal tissues as well as of braf v600e monomers.14 this case shows the fine balance of the raf inhibitor dose in the clinical range for treatment efficacy and adverse effects . 
when the raf inhibitor was reduced to a dose level of 50% of the recommended dose , toxicities from unopposed erk inhibition resulting from the mek and egfr inhibitors became problematic , and the tumor began progressing through treatment . 
also shown is inferred phosphorylated erk ( p - erk ) level in skin with inflection point from mild to severe erk inhibition occurring at the dabrafenib dose of 75 mg orally twice daily . 
 ( b ) grade 2 acneiform rash , paronychia , and splinter hemorrhages . raf inhibitor dose can rapidly improve toxicity but may not overcome resistance that has developed to treatment . 
although tumor progression in this case was likely multifactorial , incomplete inhibition of braf v600e at a lower dose of dabrafenib and the likely higher threshold to reestablish erk suppression after incomplete pathway inhibition may have contributed to the short duration of clinical benefit of approximately 4 months . 
 mario lacouture consulting or advisory role : novartis , novocure , legacy healthcare services , janssen research & development , adgero biopharmaceuticals , galderma , amryt pharmaceuticals , lindi , debiopharm group , merck , legacy healthcare services , helsinn healthcare , celldex , menlo therapeutics , johnson & johnson , roche research funding : veloce , us biotest jonathan hersch no relationship to disclose rona yaeger research funding : array biopharma , glaxosmithkline , novartis travel , accommodations , expenses : array biopharma affiliation all authors : memorial sloan - kettering cancer center , new york , ny . support references supported by the byrne fund ( r.y. ) and national institutes of health memorial sloan kettering cancer center core grant no . 
long gv , stroyakovskiy d , gogas h , et al : dabrafenib and trametinib versus dabrafenib and placebo for val600 braf - mutant melanoma : a multicentre , double - blind , phase 3 randomised controlled trial . 
planchard d , smit ef , groen hjm , et al : dabrafenib plus trametinib in patients with previously untreated brafv600e - mutant metastatic non - small - cell lung cancer : an open - label , phase 2 trial . 
van geel rmjm , tabernero j , elez e , et al : a phase ib dose - escalation study of encorafenib and cetuximab with or without alpelisib in metastatic braf - mutant colorectal cancer . 
yaeger r , yao z , hyman dm , et al : mechanisms of acquired resistance to braf v600e inhibition in colon cancers converge on raf dimerization and are sensitive to its inhibition . 
 dramatic response to crizotinib in a patient with lung cancer positive for an hla - drb1 - met gene fusion introduction nonsmall - cell lung cancer ( nsclc ) is a disease in which tumor growth is commonly driven by alterations along the receptor tyrosine kinase ras - rafmitogen - activated protein kinase pathway . 
the patient declined adjuvant chemotherapy . eighteen months after diagnosis , surveillance imaging with a fluorodeoxyglucose positron emission tomography / computed tomography ( ct ) scan demonstrated new lesions within the right and left lower lung lobe , as well as a pericardiac nodule and an enlarged precarinal lymph node . 
this sample was negative for alk and ros1 rearrangements . there was insufficient sample for additional molecular testing . treatment with carboplatin and pemetrexed was commenced for four cycles , followed by continuation maintenance pemetrexed with stable disease as best response . 
at her second opinion appointment , tissue from the patients second lung resection was subjected to additional molecular analysis . methods anchored multiplex polymerase chain reaction total nucleic acid was extracted from formalinfixed , paraffin - embedded processed material from the resection sample by using the agencourt kurtis d . 
 ( a ) schematic of the detected fusion with breakpoints between exons indicated ( transcript fusion breakpoints map to the following genomic coordinates [ hg19 ] : hla - drb1 , chromosome 6 : 32548024 ; met , chromosome 7 : 116414935 ; top )  . 
this rnabased assay allows for fusion gene detection without a priori knowledge of the fusion partner.18 the assay detected a previously undescribed fusion of hla - drb1 exon 5 ( nm_002124 ) to met exon 15 ( nm_000245 ) , which was predicted to be in - frame and to preserve the met kinase domain ( fig 1a )  . this finding was confirmed on a separate extraction and repeat analysis of the resection sample . 
whereas the 59 fusion partner is novel , previously described fusions to met , including fusions to c8orf34 , baiap2l1 , tfg , and clip2 , have also been reported to occur at exon 15.13 , 14 a reverse transcriptase polymerase chain reaction assay , followed by sanger sequencing of the product confirmed the existence of this fusion transcript ( fig 1a )  . expression of any fusion gene relies on the activity of the promoter of the 59 partner . 
although this does not directly demonstrate activity of the hladrb1 promoteras other b - subunit genes can also form the heterodimer in this protein complexit suggests the general expression of the gene class , which tends to be coordinately expressed . 
the observation of high hla class ii ( dr ) expression is in accordance with a previous study that demonstrated robust major histocompatibility class ii expression specifically in lung adenocarcinoma.19 to demonstrate the activation of hgfr - mediated signaling in the sample , we performed a pla.20 pla measures the formation of protein complexes , in this case , between hgfr and the adaptor gab1 , which occurs upon hgfr activation . 
this assay demonstrated hgfr - gab1 complex formation at levels similar to samples that were positive for met exon 14 skipping , which suggests that hgfr is activated ( fig 2 )  . of importance , the sample was found to be negative for other known oncogenic drivers of lung cancer , including alk and ros1 fusions , as well as met exon 14 skipping . 
any of these three aberrations would be expected to confer sensitivity to crizotinib ; however , if present in this sample , they would have been detected by the amp - based assay . 
lack of alk rearrangement was confirmed by immunohistochemistry . met amplification may also promote sensitivity to crizotinib ; however , met fluorescent in situ hybridization did not reveal an increase in met copy number . 
an ngs - based mutational assay ( illumina trusight tumor 26 ) that covers commonly mutated regions in 26 genes , including egfr , kras , and braf , revealed no nonsynonymous coding mutations . the patient began treatment with off - label oral crizotinib 250 mg twice daily . 
after 6 weeks of therapy , repeat ct showed complete resolution of the previously observed left upper lobe perihilar and right lower lobe lung nodules with no new lesions ( fig 3 )  . 
the patient experienced grade 1 fatigue , grade 1 dysgeusia , grade 2 anorexia , grade 1 visual disturbance , grade 1 transaminitis , grade 1 creatinine increase , grade 1 hyponatremia , and grade 3 hypokalemia ( grade 1 hypokalemia at baseline )  . 
this robust response remains at 8 months on therapy . discussion in this study , we described the discovery of a novel fusion gene that involves met in a tumor sample from a patient with nsclc who was negative for fig 2 . 
 ( a ) met - gab1 proximity ligation assay ( pla ) analysis of formalinfixed , paraffin - embedded samples from the hladrb1 - met patient , two different met exon 14 ( ex14 ) skippingpositive patients , and an alk rearrangementpositive patient ( negative control )  . scale bar is 50 microns . ( b ) quantification of pla analysis displayed as the average number of dots per nucleus . 
the hla - drb1 - met sample displayed a significantly higher pla score than did the alk sample , as determined by analysis of variance followed by bonferronis post hoc analysis . 
contrastenhanced computed tomography scans of the chest of the patient ( a ) before and ( b ) approximately 6 weeks and ( c ) 3 months after starting crizotinib 250 mg twice daily . 
arrows highlight lung lesions before and after therapy . shown are four of 14 lesions ; the remaining 10 lesions had regressed by 6 weeks , with an ongoing response at 3 months . other known oncogenic drivers , and , in particular , other known targets of crizotinib . 
the patient demonstrated a dramatic response to off - label treatment with crizotinib , a us food and drug administrationapproved small - molecule tyrosine kinase inhibitor with activity against hgfr . this finding suggests that the met fusion gene product serves as an oncogenic driver in the patients tumor . 
future studies are needed to determine the specific mechanism of action of this novel fusion gene , although overexpression of the met kinase domain may be sufficient to activate hgfr - mediated signaling . 
it is also tempting to speculate that , as the fusion occurred to met exon 15 , the lack of the ubiquitination motif that is found in exon 14 results in impaired degradationsimilar to the met exon 14 skipping isoform.21 for clinical purposes , it is now imperative to know the driver oncogene status of patients with nsclc . 
until recently , test a small number of genes to guide clinical decision making , and this could be achieved by using single - gene assays.22 however , the recent it was sufficient rapid expansion in the number of characterized driver oncogenes , the increase in the number of approved therapies , and the large number of clinical studies now available that are testing drugs that target these novel oncogenes necessitates the evaluation of multiple genes simultaneously . ngs - based assays are well suited to meet this requirement because of the ability to sequence in massively parallel fashion . 
the ability of this assay to detect fusions without prior knowledge of the fusion partner was critical in this case , as fusion of met to hla - drb1 has not been reported previously . in conclusion , we provide evidence that met fusions may be another class of actionable alteration in nsclc . 
doebele honoraria : pfizer , astrazeneca , ariad pharmaceuticals , guardant health consulting or advisory role : pfizer , oncomed , trovagene , ignyta , green peptide , astrazeneca research funding : mirati therapeutics ( inst ) , abbott molecular , loxo oncology ( inst ) , ignyta ( inst ) patents , royalties , other intellectual property : licensing fees from abbott molecular for patent pct / us2013 / 057495 , licensing fees for biologic material from ariad pharmaceuticals ( inst ) , licensing fees for biologic materials from gvkbio , licensing fees for biologic materials from loxo oncology ( inst ) travel , accommodations , expenses : ignyta , ariad pharmaceuticals dara l . 
drilon a , rekhtman n , arcila m , et al : cabozantinib in patients with advanced ret - rearranged non - small - cell lung cancer : an open - label , single - centre , phase 2 , single - arm trial . 
tabernero j , bahleda r , dienstmann r , et al : phase i dose - escalation study of jnj - 42756493 , an oral pan - fibroblast growth factor receptor inhibitor , in patients with advanced solid tumors . 
zou hy , li q , lee jh , et al : an orally available small - molecule inhibitor of c - met , pf - 2341066 , exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms . 
ou sh , kwak el , siwak - tapp c , et al : activity of crizotinib ( pf02341066 ) , a dual mesenchymal - epithelial transition ( met ) and anaplastic lymphoma kinase ( alk ) inhibitor , in a non - small cell lung cancer patient with de novo met amplification . 
frampton gm , ali sm , rosenzweig m , et al : activation of met via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to met inhibitors . 
paik pk , drilon a , fan pd , et al : response to met inhibitors in patients with stage iv lung adenocarcinomas harboring met mutations causing exon 14 skipping . 
awad mm : impaired c - met receptor degradation mediated by met exon 14 mutations in non - small - cell lung cancer . j clin oncol 34 : 879 - 881 , 2016 22 . 
lindeman ni , cagle pt , beasley mb , et al : molecular testing guideline for selection of lung cancer patients for egfr and alk tyrosine kinase inhibitors : guideline from the college of american pathologists , international association for the study of lung cancer , and association for molecular pathology . 
 immunological differences between immune - rich estrogen receptorpositive and immune - rich triple - negative breast cancers tess omeara , ba1 ; michal marczyk , phd1 , 2 ; tao qing , phd1 ; vesal yaghoobi , md3 ; kim blenman , ms , phd1 ; kimberly cole , md3 ; vasiliki pelekanou , md , phd3 , 4 ; david l . 
we compared expression of immune cell markers , immune function metagenes , and immuno - oncology therapeutic targets among immune - rich subtypes . results relative fractions of resting mast cells ( tcga padj = .009 ; metabric padj = 4.09e - 15 ) , cd8 + t cells ( tcga padj = .015 ; metabric padj = 0.390 ) , and m2 - like macrophages ( tcga padj = 4.68e - 05 ; metabric padj = .435 ) were higher in immune - rich er - positive bcs , but m0 - like macrophages ( tcga padj = 0.015 ; metabric padj = .004 ) and m1 - like macrophages ( tcga padj = 9.39e - 08 ; metabric padj = 6.24e - 11 ) were higher in immune - rich tnbcs . 
ninety - one immune - related genes ( eg , cxcl14 , csf3r , tgf - b3 , lrrc32 / garp , tgfb - r2 ) and a transforming growth factor ( tgf - ) response metagene were signicantly overexpressed in immune - rich er - positive bcs , whereas 41 immune - related genes ( eg , ifng , pd - l1 , ctla4 , magea4 ) were overexpressed in immune - rich tnbcs in both discovery and validation data sets . 
tgfpathway member genes correlated negatively with expression of immune activation markers ( ifng , granzyme - b , perforin ) and positively with m2 - like macrophages ( il4 , il10 , and mmp9 ) and regulatory t - cell ( foxp3 ) markers in both subtypes . conclusion different immunotherapy strategies may be optimal in immune - rich er - positive bc and tnbc . drugs targeting the tgfpathway and m2 - like macrophages are promising strategies in immune - rich erpositive bcs to augment antitumor immunity . jco precis oncol 4 : 767 - 779 . 
 omeara et al context key objective does the immune microenvironment of immune - rich estrogen receptorpositive breast cancer ( er - positive bc ) differ from the immune microenvironment of immune - rich triple - negative bc ( tnbc ) ? knowledge generated relative fractions of mast cells and m2 - like macrophages are higher in the immune microenvironment of immune - rich erpositive bc , whereas fractions of m0 - like and m1 - like macrophages are higher in immune - rich tnbc . 
her2 - positive patients were excluded , and only er - positive / her2 - negative ( herein designated er - positive ; tcga , n = 627 ; metabric , n = 1 , 186 ) and er - negative / her2 - negative ( herein designated tnbc ; tcga , n = 191 ; metabric , n = 279 ) patients were included in this analysis . 
we obtained formalin - xed parafn - embedded tissues from 63 tnbcs and 53 erpositive , treatment - nave , stage i to iii bcs from yale pathology tissue archives , matched by distribution of histologic grade , for quantitative immunouorescence ( qif ) and automated quantitative analysis ( aqua )  . assessment of immune cell content and selection of immune - rich patients a gene expression signature score developed by danaher et al19 was used to score each tcga and metabric patient for total tils . 
the total til gene signature score is the average of scores representing 11 immune cell subpopulations ( cd45 , macrophages , cd8 + t cells , t cells , cytotoxic cells , exhausted cd8 + cells , th1 cells , b cells , neutrophils , natural killer [ nk ] cells , cd56dim nk cells )  . log2 + 1 - transformed expression was used to calculate total til gene signature scores from tcga ( rna - seq ) and metabric ( microarray ) data . 
the til metagene score distribution was plotted for the pooled er - positive bc and tnbc patients , and patients with scores in the top 25th percentile of the distribution were dened as immune - rich . for 156 basal - like cancers in tcga ( er - positive bc , n = 21 ; tnbc , n = 135 ) , histologic til counts were also available.20 , 21 for these patients , we assessed the correlation between histologic til count and til gene expression score . 
we also assessed correlations between til gene signature score and pam50 subtype , tumor mutation burden ( tmb ) , and ki - 67 expression level in the tcga data when this information was available ( er - positive bc , n = 191 ; tnbc , n = 697 )  . 
pam50 subtypes and tmb categories were obtained from a previous publication , and ki - 67 categories were dened as greater than or less than the median log2 + 1 - transformed ki - 67 expression across tcga patients.22 survival analysis progression - free survival ( pfs ) and overall survival ( os ) times were available for 807 tcga patients ( er - positive bcs , n = 189 ; tnbcs , n = 618 ) .23 pfs and os were compared by er subtype within immune - rich patients ( erpositive bcs , n = 114 ; tnbcs , n = 84 ) and within nonimmune - rich patients ( er - positive bcs , n = 504 ; tnbcs , n = 105 ) using the survival and survminer r packages.24 assessment of immune cell subpopulations cibersort , an analytical tool that uses support vector regression , was used to estimate the relative proportions of 9 aggregated immune cell populations and 22 subpopulations in immune - rich er - positive bcs and immunerich tnbcs.25 , 26 the average relative fraction and standard error of the mean of each immune cell population were compared by subtype . 
 immunological differences between immune - rich er - positive bcs and tnbcs calculated gene expression signature scores of overall macrophages , m1 - like macrophages , m2 - like macrophages , and the transforming growth factor ( tgf - ) signaling response were obtained from previous publications and were compared between patients with erpositive bcs and those with tnbcs.27 - 29 slopes of linear regression curves between total tils and metagene expression signatures were compared by using a t test based on standard error in the linear regression models.30 formalinxed parafn - embedded tissue microarrays were stained for the nuclear marker 4 ( cid : 1 ) , 6 - diamidino - 2 - phenylindole and the pan - macrophage marker cd68 , and multiplexed qif and aqua were used to quantify expression levels in yale er - positive bc and tnbc patients as previously described.31 differential gene expression analysis the deseq2 ( tcga , rna - seq data ) and limma ( metabric , microarray data ) r packages were used for differential gene expression analysis of 779 immunerelated genes ( nanostring pancancer io 360 panel , nanostring technologies , seattle , wa ) and 137 currently available immuno - oncology drug target genes between immune - rich er - positive bcs and immune - rich tnbcs.32 - 34 availability of rna expression data for each member gene in tcga and metabric databases is summarized in the data supplement . 
genes with false discovery rate padj , .05 were considered signicantly differentially expressed for a broad investigation of differentially regulated immune pathways . statistical analysis all statistical analyses were performed in r v 3.5.0. 
in tcga , the 75th percentile til metagene score was 5.92 , and it categorized 119 er - positive bcs ( 19% ) and 89 tnbcs ( 45% ) as being immune rich . 
the total til gene signature scores correlated moderately but signicantly with histologic til scores in tcga ( r = 0.44 ; p = 1.18e - 08 ; fig 2a ) when these data were available . 
in tcga , there was no statistically signicant difference between the distribution of pam50 subtypes or ki - 67 expression levels between immune - rich and nonimmune - rich er - positive bcs . 
patients with immune - rich er - positive bcs had signicantly higher tmb than patients with nonimmune - rich er - positive bcs ; this difference was not seen among patients with tnbcs . 
for a subset of tcga patients with available survival formation ( n = 804 ) , pfs and os were compared by subtype ( data supplement )  . immune - rich patients had similar pfs and os regardless of er status . 
in contrast , among the nonimmune - rich cancers , os was signicantly longer in patients with er - positive bcs than in patients with tnbcs ( p = .043 ) , as expected . immune cell subpopulations in the tumor microenvironment of immune - rich er - positive bcs and immune - rich tnbcs by using cibersort , we found higher relative fractions of t cells ( padj = .009 ) and mast cells ( padj = .0005 ) in patients with immune - rich er - positive bcs compared with those who had immune - rich tnbcs ( fig 3a ) in tcga . in patients with immune - rich tnbcs , the relative fractions of macrophages ( padj = .026 ) and nk cells ( padj = .0007 ) were higher relative to those in patients with immune - rich er - positive bcs . 
the higher relative fractions of t cells ( padj = .005 ) and mast cells ( padj = 1.73e - 20 ) in immunerich er - positive bcs and the higher relative fraction of macrophages ( padj = 1.22e - 06 ) in immune - rich tnbcs were also observed in metabric ( fig 3b )  . 
qif also showed higher overall expression of the pan - macrophage marker cd68 in tnbc tissues relative to tissue from erpositive bcs from yale pathology archives ( p = .011 ; fig 3c )  . next , we examined differences among 22 immune cell subpopulations . in immune - rich er - positive tumors in tcga , the relative fractions of cd8 + t cells ( padj = .015 ) and resting mast cells ( tcga padj = .009 ) were higher relative to those in immune - rich tnbc tumors in both data sets ( fig 3d )  . 
in contrast , the relative fractions of m0 - like macrophages ( padj = .015 ) and m1 - like macrophages ( padj = 9.39e - 08 ) were signicantly higher in patients with immune - rich tnbcs compared with those with immunerich er - positive bcs . 
 ( a - b ) gene signature scores were calculated for 11 immune cell subpopulations for patients with er - positive bcs and tnbcs in ( a ) the cancer genome atlas ( tcga ) and ( b ) molecular taxonomy of breast cancer international consortium ( metabric ) databases , as described by danaher et al.19 the x - axis indicates immune cell subpopulations plus the total tumor - inltrating lymphocyte ( til ) population . 
immune - rich erpositive bcs had higher m2 - like macrophage ( p = .012 ) and tgfresponse ( p = 0.035 ) metagene expression scores ( fig 4a )  . 
the subtype - specic differences in m1 - like macrophage ( p = 1.85e - 04 ) and tgfresponse ( p = .029 ) metagene scores signicantly increased with higher total til gene signature scores , whereas the differences in overall macrophage ( p = .786 ) and m2 - like macrophage ( p = .097 ) scores between cancer subtypes were not specic to immune - rich patients ( fig 4b )  . 
 ( a ) for a subset of basal - like patients from the cancer genome atlas ( tcga ) with previously published histologic til scores ( n = 156 ) according to danaher et al , 19 the total til gene signature scores ( x - axis ) were correlated with histologic stromal til counts ( y - axis )  . 
 ( b - d ) for all patients with er - positive bcs ( n = 697 ) and tnbcs ( n = 191 ) in tcga , patients were classied as immune cell high ( danaher et l19 total til gene signature score in the top 25th percentile ) or low ( danaher total til gene signature scores in the bottom 75th percentile ) , represented on the x - axis . 
distributions of ( b ) pam50 subtypes , ( c ) ki - 67 expression categories and ( d ) tumor mutation burden ( tmb ) categories were compared between patients with immune - rich high and low er - positive bcs and tnbcs separately . 
pam50 and tmb categories were obtained from previous publications , and ki - 67 categories were dened as greater than ( high ) or less than ( low ) the median ki - 67 expression level across all patients . differential expression of immune - related genes and immuno - oncology drug target genes between immune - rich er - positive bcs and immune - rich tnbcs at the individual gene level , 132 of 754 immune - related genes were signicantly overexpressed in tcga immunerich er - positive bcs . 
ninety - one of these immune - related genes , including mast cell genes ms4a2 and cpa3 and therapeutic targets cxcl14 , cx3cr1 , and potential csf3r , were also upregulated in metabric patients with immune - rich er - positive bcs ( data supplement )  . immune - rich tnbcs , 303 immune - related genes were overexpressed compared with immune - rich er - positive bcs in tcga , and 218 of these were validated in metabric . 
sixteen of 136 available therapeutic target genes were overexpressed in tcga patients with immune - rich er - positive bcs , and 12 of these , including 3 members of the tgfsignaling pathway ( tgf - 3 , lrrc32 / garp , and tgf - r2 ) , were also overexpressed in metabric patients with immune - rich er - positive bcs ( table 1 ; data supplement )  . 
for patients from ( a ) the cancer genome atlas ( tcga ) and ( b ) molecular taxonomy of breast cancer international consortium ( metabric ) databases , 9 aggregate immune cell populations are listed on the x - axis , and the average fraction of each immune cell population for a certain subtype is quantied on the y - axis . 
 ( c ) quantitative immunouorescence ( qif ) signal was compared between estrogen receptorpositive breast cancer ( er - positive bc ; , n = 53 ) and triple - negative bc ( tnbc ; n = 63 ) formalin - xed parafn - embedded tumor microarray tissues . 
subtype is shown on the x - axis , and cd68 qif signal in the total 4 ( cid : 1 ) , 6 - diamidino - 2 - phenylindole ( dapi ) compartment is quantied on the y - axis . 
for ( d ) tcga and ( e ) metabric patients , 22 immune cell populations derived by cibersort are listed on the x - axis , and the average fraction of each immune cell population for a certain subtype is quantied on the y - axis . 
false discovery rate padj values are provided for tcga ( er - positive bcs , n = 119 ; tnbcs , n = 86 ) and metabric ( er - positive bcs , n = 225 ; tnbcs , n = 140 ) patients . 
comparison of macrophage population and transforming growth factor ( tgf - ) response metagene expression scores between patients with immune - rich estrogen receptorpositive breast cancers ( er - positive bcs ) and those with triple - negative bcs ( tnbcs ) in the cancer genome atlas ( tcga )  . 
 ( a ) metagene expression scores for overall macrophages , m1 - like macrophages , m2 - like macrophages , and tgfresponse were compared between immune - rich er - positive bc ( red , n = 119 ) and immune - rich tnbc ( blue , n = 86 ) patients in tcga . 
 ( b ) for all patients with er - positive bcs ( n = 697 ) and tnbcs ( n = 191 ) in tcga , gene expression signature scores for overall macrophages , m1 - like macrophages , m2 - like macrophages , and tgfresponse ( y - axis ) were plotted against total til gene signature scores ( x - axis )  . 
linear regression lines for each subtype with 95% cis are shown in red ( er - positive bc ) or blue ( tnbc )  . and the cancer - testis antigen , magea4 , were also overexpressed in immune - rich tnbcs in metabric . correlation between tgfpathway expression and other immune - related genes tgfis a pleotropic cytokine that can stimulate regulatory induce m2 - like macrophage polarization , and t cells , suppress effector t cells , nk cells , and dendritic cells . furthermore , tgfsignaling has been linked to an immune - excluded phenotype in cancer . 
we therefore examined correlations between the differentially expressed tgfpathway members ( tgf - 3 , lrrc32 , and tgfr2 ) and the expression levels of the 53 immuno - oncology drug target genes that were differentially expressed in both data sets ( 12 upregulated in immune - rich er - positive bcs , 41 upregulated in immune - rich tnbcs ) across immunerich tnbcs and er - positive bcs . in both tcga and metabric , expression of the tgfpathway members correlated positively with each other and negatively with most immune activation markers ( fig 5a - b ; data supplement )  . 
similar correlation patterns were seen between tgfpathway members and immune activation marker expression when only immune - rich er - positive bcs were analyzed ( fig 5c - d )  . 
we also noted a positive correlation between expression of tgf - 1 and the m2 - like macrophage markers il4 ( r = 0.24 ) , il10 ( r = 0.38 ) , and mmp9 ( r = 0.43 ) as well as the regulatory t - cell marker foxp3 ( r = 0.42 ) in the tcga data . discussion in this report , we used a gene expression signature of total tils to identify patients with immune - rich er - positive bcs and tnbcs in 2 publicly available databases to compare the tumor immune microenvironment of these subtypes . the patients with immune - rich er - positive bcs had signicantly higher tmb than patients with nonimmune - rich er - positive bcs , but no difference in tmb was seen between immune - rich and nonimmune - rich tnbcs . 
for ( a ) the cancer genome atlas ( tcga ) and ( b ) molecular taxonomy of breast cancer international consortium ( metabric ) patients , correlation matrices display the pearson correlation coefcients between expression levels of 53 immuno - oncology drug targets across patients with immune - rich estrogen receptorpositive breast cancers ( er - positive bcs ) and triple - negative bcs ( tnbcs )  . 
for tcga , the number of patients with immune - rich er - positive bcs was 119 , and for tnbcs , it was 86 ; for metabric , the number of patients with immune - rich er - positive bcs was 225 , and for tnbcs , it was 140 . 
for ( c ) tcga and ( d ) metabric patients , correlation matrices display the pearson correlation coefcients between expression levels of 53 immuno - oncology drug targets in patients with immune - rich er - positive bc alone . 
the number of patients with immune - rich er - positive bcs in tcga was 119 , and in metabric , it was 225 . results are consistent with previous reports.11 , 36 approximately 70% of the immune - rich er - positive bcs we identied were luminal a molecular subtype , and the pam50 subtype distribution and ki - 67 expression were similar between immune - rich and nonimmune - rich er - positive bcs . an os difference was detected between patients with nonimmune - rich er - positive bcs relative to nonimmunerich tnbcs , but no similar statistically signicant difference was seen between patients with immune - rich er - positive bcs and immune - rich tnbcs ( both groups had excellent long - term survival )  . 
by using deconvolution methods , we observed differences in the relative fractions of immune cell subpopulations between immune - rich er - positive bcs and immune - rich tnbcs . immune - rich er - positive bcs had higher relative fractions of m2 - like macrophages , cd8 + t cells , and resting mast cell populations , whereas immunerich tnbcs had overall higher macrophage content and higher m0 - like and m1 - like macrophage populations . 
higher overall cd68 protein expression in tnbcs was also conrmed by immunohistochemistry . interestingly , immune - rich er - positive bcs had higher expression of a tgfsignaling metagene and showed coordinated overexpression of tgf - 3 , tgf - r2 , and lrrc32 compared with immune - rich tnbcs . 
the tgfpathway is activated by ligands tgf - 1 , tgf - 2 , and tgf3 that bind to membrane - bound serine / threonine protein receptor kinases composed of subunits tgf - r1 and tgfr2 . 
although the tgfligand isoforms are more than 75% similar in amino acid structure , tgf - r2 has increased afnity for tgf - 1 and tgf - 3 relative to tgf2.37 lrrc32 encodes the garp protein that tethers tgfligands to the cell membrane to prime its activation.38 the simultaneous upregulation of the ligand , receptor , and signaling components suggests a functional role for the tgfpathway in immune - rich er - positive bcs . the immune - attenuating function of tgfsignaling in the tumor microenvironment has been extensively studied in vitro and in vivo . 
in early in tumorigenesis , tgfacts as a tumor suppressor , inducing apoptosis in premalignant cells and inhibiting proliferation.39 after tumor formation , tgfpromotes cancer progression through suppressing the host antitumor immune response.40 among other roles , tgfinhibits cd8 + effector t - cell function , inhibits the th1 helper t - cell phenotype , activates foxp3 - positive regulatory t cells , drives m2 - like macrophage polarization , and excludes immune cells from the tumor compartment.41 - 45 the distinct roles of the different tgfisoforms in cancer remain undened . 
among the 3 isoforms , tgf - 3 is the most potent stimulator of neovascularization in models of wound healing and the most potent inhibitor of granulocyte - macrophage colonystimulating factormediated hematopoiesis in the bone marrow . 
tgf - 1 and tgf - 3 are equally potent stimulators of extracellular matrix collagen deposition in models of pulmonary brosis.46 overall , high tgfsignaling is also associated with lesser response to immune checkpoint inhibitors.41 , 42 on an expression level , we found higher relative fractions of cd8 + t cells in immune - rich er - positive bcs as well as higher tgfsignaling ; these cd8 + t cells in immune - rich er - positive patients may therefore lack robust effector cytotoxic function or be excluded from the tumor compartment by augmented tgfsignaling . tgfcan be released by cancer cells as well as by m2 - like macrophages in a positive feedback loop to attenuate antitumor immunity.47 in several studies , the presence of m2 - like macrophages in bc was associated with poor prognosis , and reprogramming of macrophages toward the m1 - like phenotype has been shown to increase responsiveness to immune checkpoint therapy in experimental models.48 , 49 a recent study found that a macrophage - dependent autochthonous mouse model of luminal btype bc , inhibition of class iia histone deacetylase ( hdac ) by tmp195 induces the recruitment and differentiation of antitumor - type macrophages , reduces tumor burden and metastases , and enhances the durability of tumor reduction when combined with chemotherapy regimens or t - cell checkpoint blockade.50 this observation in an animal model of bc is consistent with our ndings in human tissues . 
our analyses also showed that expression of tgfpathway members correlated negatively with the expression of many immune activation markers including interferon ( ifn - ) , granzymes , and perforin , but correlated positively with m2 - like macrophage markers il4 , il10 , mmp9 , and regulatory t - cell marker foxp3 . 
although m2 - like macrophages in the tumor microenvironment may be the source of tgfsignaling , high levels of apolipoprotein b mrna editing enzyme , catalytic polypeptide - likeassociated mutational signatures that have been previously identied in patients with immunerich er - positive bcs may also contribute to upregulation of the tgfpathway in tumor cells and downstream recruitment of m2 - like macrophages.36 deconvolution of the leukocyte compartment also revealed a higher relative fraction of mast cells in immune - rich erpositive bcs ; this was corroborated by overexpression of the mast cell markers cpa3 and ms4a2 in patients with immune - rich er - positive bcs compared with those who have immune - rich tnbcs . 
how these cells inuence antitumor immunity is unknown , but high mast cell inltration in bc has been associated with er expression and favorable prognosis in earlier studies.51 , 52 in summary , our analysis of gene expression data is consistent with a more attenuated immune microenvironment mediated by increased tgfsignaling and m - 2 macrophage presence in immune - rich er - positive bcs . 
in this subset of er - positive bcs , we observed high relative fractions of cd8 + t cells , mast cells , and m2 - like macrophages with lower expression of granzyme - b , perforin , and ifn -  . 
in addition , practical assays that can distinguish patients with immune - rich and tgf - - high from patients with tgf - - low er - positive bcs are needed before these results can be applied in the clinic . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . vasiliki pelekanou employment : sano david l . 
rimm stock and other ownership interests : pixel gear honoraria : amgen , bristol myers squibb , ventana medical systems consulting or advisory role : perkin elmer , bristol myers squibb , astrazeneca , agendia , ultivue , merck , daiichi sankyo , glaxosmithkline , konica minolta , nanostring technologies , nextcure , cell signaling technology , roche , paige , cepheid , sano research funding : cepheid ( inst ) , perkin elmer ( inst ) , astrazeneca / medimmune ( inst ) , nextcure ( inst ) , eli lilly ( inst ) , nanostring technologies ( inst ) , navigate biopharma ( inst ) , ultivue ( inst ) , konica minolta ( inst ) , amgen ( inst ) patents , royalties , other intellectual property : rarecyte circulating tumor cells ; quantitative immunouorescence ( aqua ) ( inst ) travel , accommodations , expenses : ventana medical systems , nextcure , bristol - myers squibb lajos pusztai consulting or advisory role : h3 biomedicine , merck , novartis , seattle genetics , syndax pharmaceuticals , athenex , astrazeneca , genentech , bristol myers squibb , clovis oncology , immunomedics , eisai , almac diagnostics research funding : merck ( inst ) , genentech ( inst ) , seattle genetics ( inst ) , astrazeneca ( inst ) travel , accommodations , expenses : astrazeneca uncompensated relationships : nanostring technologies open payments link : 110878 / summary no other potential conicts of interest were reported . references bianchini g , qi y , alvarez rh , et al : molecular anatomy of breast cancer stroma and its prognostic value in estrogen receptor - positive and - negative cancers . j clin oncol 28 : 4316 - 4323 , 2010 karn t , pusztai l , holtrich u , et al : homogeneous datasets of triple negative breast cancers enable the identication of novel prognostic and predictive signatures . 
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cancer immunol res 3 : 326 - 332 , 2015 loi s , giobbe - hurder a , gombos a , et al : phase ib / ii study evaluating safety and efcacy of pembrolizumab and trastuzumab in patients with trastuzumabresistant her2 - positive metastatic breast cancer : results from the panacea ( ibcsg 45 - 13 / big 4 - 13 / keynote - 014 ) study . 
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tolaney sm , barroso - sousa r , keenan t , et al : randomized phase ii study of eribulin mesylate ( e ) with or without pembrolizumab ( p ) for hormone receptorpositive ( hr + ) metastatic breast cancer ( mbc )  . 
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statistics / breast - cancer - facts - and - gures / breast - cancer - facts - and - gures - 2017 - 2018.pdf van maaren mc , de munck l , strobbe lja , et al : ten - year recurrence rates for breast cancer subtypes in the netherlands : a large population - based study . 
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 novel alk fusion , ppfibp1 - alk , in pancreatic ductal adenocarcinoma responsive to alectinib and lorlatinib arjan gower , md , ms1 ; barry golestany , md2 ; jun gong , md3 ; aatur d . 
singhi , md , phd4 ; and andrew eugene hendifar , md , mph3 introduction pancreatic cancer is the seventh leading cause of cancer death among men and women worldwide , with  . 
430 , 000 deaths in 2018.1 despite advances in surgical techniques , radiation , and systemic treatment in the past few decades , overall survival for pancreatic cancer is extremely poor , with a 5 - year survival rate of 9%.2 current systemic treatment regimens for metastatic pancreatic cancer include folfirinox ( uorouracil , folic acid , oxaliplatin , and irinotecan ) , gemcitabine plus nab - paclitaxel , and liposomal irinotecan with uorouracil.3 - 6 approximately 88% - 95% of pancreatic adenocarcinomas harbor kras driver mutations . 
in row 1 , the circled hepatic lesion in the right lobe of the liver at baseline ( a ) before alectinib decreased in size ( b ) 2 months after alectinib . 
the patient experienced progression during alectinib treatment after 5 months and experienced progression during uorouracil , folic acid , oxaliplatin , and irinotecan treatment shortly thereafter . fluorescence in situ hybridization ( fish ) was negative . 
it has been shown that fish may have different sensitivities of detecting alk compared with next - generation sequencing or immunohistochemistry.27 of note , molecular proling was negative for kras , microsatellite instability , nf1 and p53 inactivating mutations , and cdkn2a / b loss . proling was positive for brca2 c.8007a.g ( silent ) and cdkn1b 407a.g , both of unknown clinical signicance . patient was started on folfirinox with radiographic response . 
however , because of the adverse effects of chemotherapy , including nausea and neuropathy , the patient was switched to alectinib 600 mg twice daily , given her alk fusion status . 
subsequent cell - free plasma ( guardant360 ; guardant health , redwood city , ca ) showed newly acquired alk mutations g1202r and v1180l in addition to the ppfibp1 - alk translocation . 
the patients disease has been stable on lorlatinib on 2 - month follow - up imaging , and she continues to be treated with lorlatinib ( figs 2 and 3 )  . discussion currently , there are no fda - approved targeted therapies for pancreatic cancer , and standard chemotherapy ( folfirinox ) for metastatic pancreatic cancer is quite toxic , with a median overall survival of approximately 11 months.3 to our knowledge , we present the seventh case of alk - positive metastatic pancreatic cancer with a novel ppfibp1 - alk fusion gene and the rst patient to be treated with alectinib as rst - line targeted therapy . 
the patient ultimately acquired alectinib - resistant alk mutations g1202r and v1180l ; however , the disease has been stable on the third - generation alk inhibitor lorlatinib . alk translocation in pancreatic cancer was rst described in 2017 , and there have been only 6 documented cases of alk - positive pancreatic adenocarcinoma through literature review ( table 1 )  . 
although the prevalence of the alk fusion gene is rare , at 0.16% , the prevalence increases to 1.3%.among patients , 50 years old12 the majority of alk fusion partners seen in nsclc includes eml4 , but other partners include strn , kcnq , klc1 , kif5b , ppm1b , and tgf genes.28 ppfibp1 - alk gene fusion was rst described in 2011 in a patient with pulmonary inammatory myobroblastic tumor29 and subsequently was described in epithelioid brous histiocytoma , 30 but it has never been described in nsclc or pancreatic cancer . 
of the 6 patients with alk - positive pancreatic cancer , 4 patients had eml4alk translocation , 1 patient had strn - alk , and 1 patient had a dctn1 - alk translocation ( table 1 )  . 
 case report 3 / 19 4 / 19 5 / 19 7 / 19 9 / 19 12 / 19 1 / 20 3 / 20 3w pain + bloating ; us , pet / ct : liver lesions folfirinox #2 - 6 ; dose reduction due to adverse effects ct imaging : stable disease biopsy with acquired resistance mutations ; progression on folfirinox #7 - 8 ; start lorlatinib biopsy : pancreatic adenocarcinoma ; folfirinox #1 ct imaging : stable disease ; start alectinib ct imaging : progressive disease ct imaging : stable disease inferior r hepatic lobe posterior r hepatic lobe folfirinox alectinib lorlatinib folfirinox 3 / 1 / 19 5 / 1 / 19 7 / 1 / 19 9 / 1 / 19 11 / 1 / 19 1 / 1 / 20 3 / 1 / 20 date fig 2 . 
# , dose number ; 3w , 3 - week ; ct , computed tomography ; folfirinox , uorouracil , folic acid , oxaliplatin , and irinotecan ; pet , positron emission tomography ; r , right ; us , ultrasound . and proliferative capabilities , ultimately leading to activation of different signaling pathways.28 in nsclc , alk fusion is an oncogenic driver that is generally mutually exclusive of kras mutation16 ; however , there are reports of concomitant kras and alk fusion double alteration that may confer worse prognosis.31 all 7 patients with alk - positive pancreatic cancer were kras wild type , suggesting , albeit in a small sample size , that alk translocation drives oncogenesis and that these 2 oncogenic drivers are mutually exclusive . 
this case demonstrates the rst time , to our knowledge , that alectinib has been used as the rst targeted therapy , and the development of known alectinib - resistant alk mutations suggests that alk - positive pancreatic cancer acquires resistance in a fashion similar to that of nsclc . there is a clear unmet medical need for novel therapeutic approaches in pancreatic cancer . 
a recent study in metastatic pancreatic cancer with germ - line brca mutations ( 7% prevalence ) regardless of kras mutation status showed increased pfs with the poly adp ribose polymerase inhibitor olaparib after rst - line platinum - based chemotherapy.4 in a recent phase ii basket study , 2 of 9 patients who had pancreatic cancer with her2 amplication / overexpression experienced responses to her2 - targeted therapies trastuzumab plus pertuzumab . 
additionally , a patient who had pancreatic cancer and braf gene fusion ( cux1 - braf ) had a partial response to the braf - targeted therapy vemurafenib.32 last , 3 patients who had pancreatic cancer with ntrk1 and ros1 gene fusions experienced responses to entrectinib , a targeted inhibitor of these genes.33 these studies are additional proof of concept that targeted therapies can be used successfully when paired with the right genomic alteration and that alk inhibitors may be of substantial clinical benet in the right patient . 
in row 1 , the circled hepatic lesion after disease progression on uorouracil , folic acid , oxaliplatin , and irinotecan ( a ) before lorlatinib was stable in size ( b ) 2 months after lorlatinib treatment . 
the patient currently continues to be treated with lorlatinib . 38% of kras wild - type tumors have genomic alterations that could activate the mapk signaling cascade.9 identication of molecular alterations in patients with kras wild - type status that may drive oncogenesis will help guide therapeutic intervention in a small but clinically relevant population . alk translocations are rare in pancreatic cancer . 
this is , to the rst report of ppfibp1 - alk rearour knowledge , rangement in pancreatic or nsclc , the rst reported patient with pancreatic cancer to be treated with alectinib as the rst targeted therapy , and the rst patient to be treated with lorlatinib after acquired resistance to alectinib with stabilization of disease . 
all 7 occurrences of alk - positive pancreatic cancer have been kras wild type ; given that 6 of the 7 patients were , age 50 years , there may be clinical benet to screen young patients with pancreatic cancer who are kras negative for the alk gene fusion , as they may benet from alk inhibitors , which may lead to improved overall survival . 
clinical characteristics of patients with alk - positive pancreatic cancer patient case age ( years ) alk rearrangement exon 13 eml4 - exon 20 alk ( 1 ) crizotinib ; ( 2 ) ceritinib ; ( 3 ) alectinib alk inhibitor ( in order of treatment ) ( 1 ) crizotinib ( 1 ) crizotinib unknown none ( 1 ) crizotinib ; ( 2 ) alectinib duration of survival ( months ) unknown unknown ppfibp1 - alk ( 1 ) alectinib ; ( 2 ) lorlatinib exon 6 eml4 - exon 20 alk exon 3 strn - exon 20 alk exon 6 eml4 - exon 20 alk exon 6 eml4 - exon 20 alk dctn1 - alk note . 
singhi , andrew eugene hendifar collection and assembly of data : arjan gower , barry golestany , andrew eugene hendifar data analysis and interpretation : arjan gower , jun gong , aatur d . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / authors / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . jun gong honoraria : amgen , astellas pharma , clinical congress consultants , qed therapeutics , exelixis , elsevier consulting or advisory role : amgen , astellas pharma , clinical congress consultants , qed therapeutics , exelixis , elsevier aatur d . 
singhi honoraria : foundation medicine andrew eugene hendifar consulting or advisory role : novartis , ipsen , perthera , celgene , abbvie research funding : ipsen travel , accommodations , expenses : halozyme no other potential conicts of interest were reported . references bray f , ferlay j , soerjomataram i , et al : global cancer statistics 2018 : globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries . 
ca cancer j clin 69 : 7 - 34 , 2019 conroy t , desseigne f , ychou m , et al : folfirinox versus gemcitabine for metastatic pancreatic cancer . 
n engl j med 364 : 1817 - 1825 , 2011 golan t , hammel p , reni m , et al : maintenance olaparib for germline brca - mutated metastatic pancreatic cancer . 
n engl j med 381 : 317 - 327 , 2019 von hoff dd , ervin t , arena fp , et al : increased survival in pancreatic cancer with nab - paclitaxel plus gemcitabine . 
wang - gillam a , li cp , bodoky g , et al : nanoliposomal irinotecan with uorouracil and folinic acid in metastatic pancreatic cancer after previous gemcitabinebased therapy ( napoli - 1 ) : a global , randomised , open - label , phase 3 trial . 
lancet 387 : 545 - 557 , 2016 biankin av , waddell n , kassahn ks , et al : pancreatic cancer genomes reveal aberrations in axon guidance pathway genes . 
nature 518 : 495 - 501 , 2015 singhi ad , george b , greenbowe jr , et al : real - time targeted genome prole analysis of pancreatic ductal adenocarcinomas identies genetic alterations that might be targeted with existing drugs or used as biomarkers . 
lin e , li l , guan y , et al : exon array proling detects eml4 - alk fusion in breast , colorectal , and nonsmall - cell lung cancers . 
kelly lm , barila g , liu p , et al : identication of the transforming strn - alk fusion as a potential therapeutic target in the aggressive forms of thyroid cancer . 
no e j , lovejoy a , ignatius ou , s - h , et al : alk mutation status before and after alectinib treatment in locally advanced or metastatic alk - positive nsclc : pooled analysis of two prospective trials . 
solomon bj , besse b , bauer tm , et al : lorlatinib in patients with alk - positive nonsmall - cell lung cancer : results from a global phase 2 study . 
zou hy , friboulet l , kodack dp , et al : pf - 06463922 , an alk / ros1 inhibitor , overcomes resistance to rst and second generation alk inhibitors in preclinical 27 . 
lin c , shi x , yang s , et al : comparison of alk detection by fish , ihc , and ngs to predict benet from crizotinib in advanced nonsmall - cell lung cancer . 
takeuchi k , soda m , togashi y , et al : pulmonary inammatory myobroblastic tumor expressing a novel fusion , ppfibp1 - alk : reappraisal of anti - alk immunohistochemistry as a tool for novel alk fusion identication . 
ulivi p , chiadini e , dazzi c , et al : nonsquamous , nonsmall - cell lung cancer patients who carry a double mutation of egfr , eml4 - alk or kras : frequency , clinical - pathological characteristics , and response to therapy . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors on the basis of molecular proles : results from mypathway , an open - label , phase iia multiple basket study . 
 c clinical response to antiprogrammed death 1 after response and subsequent progression on antiprogrammed death ligand 1 therapy introduction immune checkpoint inhibitors are rapidly becoming a cornerstone in the treatment of nonsmall cell lung cancer ( nsclc )  . 
the programmed death 1 ( pd - 1 ) receptor and its two ligands , programmed death ligand 1 ( pd - l1 ; b7h1 ) and ligand 2 ( pd - l2 ; b7 - dc ) , negatively regulate t - cell activation , and expression of pd - l1 by tumor cells is an important mechanism of immune evasion.1 - 3 multiple agents have been developed to disrupt tumor - associated immune evasion by targeting either pd - 1 or pdl1 . 
nivolumab and pembrolizumab , both anti pd - 1 agents approved for advanced nsclc progressing after chemotherapy , confer an overall survival benefit and produce response rates in 16% to 23% of unselected patients with nsclc.4 - 6 food and drug administration approval was also recently extended to pembrolizumab in the first - line setting for patients with metastatic nsclc expressing pd - l1.7 atezolizumab , an antipd - l1 agent , has also demonstrated an overall survival benefit and has been approved for use in the second - line setting.8 the growing supply of treatment options has led to new questions of optimal sequencing and choice of therapy . 
in particular , the utility of serial treatment with immune checkpoint inhibitors has not been established . agents targeting pd - l1 block the interaction of pd - l1 expressed on tumor cells and tumorinfiltrating immune cells with pd - 1 and b7.1 expressed on t cells . 
studies of these agents , including atezolizumab , avelumab , and durvalumab , have demonstrated similar response rates and clinical benefit.9 - 14 although the effects of antipd - l1 are predicted to be similar to antipd - 1 , it has been speculated that the variation in mechanism may lead to distinct antitumor and toxicity profiles compared with the antipd - 1 agents.15 however , these agents have not been directly compared . 
he had been diagnosed approximately 2 years earlier with nsclc metastatic to bilateral cervical lymph nodes and bilateral adrenal glands . molecular testing showed that his tumor was egfr and kras wild type and alk translocation negative . 
before this evaluation , he received four lines of therapy , including platinum doublet chemotherapy , single - agent docetaxel , palliative chemoradiation with weekly carboplatin and paclitaxel , and erlotinib . he was enrolled in a phase i clinical trial of an antipd - l1 monoclonal antibody given in 21 - day cycles . 
he experienced only mild ( grade 1 ) rash and hypothyroidishe received a total of 16 cycles of antipd - l1 therapy before treatment was stopped per study protocol . the patient was followed with serial cross - sectional imaging and demonstrated continued pr on imaging for 16 months after therapy completion . 
after seven cycles of treatment , he was noted to have progression of disease in his right axilla and a new retropharyngeal ( rp ) mass . biopsy of the rp mass showed poorly differentiated carcinoma consistent with recurrent nsclc and similar to his initial biopsy . 
 chest , abdomen , and pelvis show a metastatic lesion within the right adrenal gland ( arrow ) before initiation of antiprogrammed death ligand 1 ( pd - l1 ) therapy ( panel a1 ) , which regressed after 16 cycles of treatment with antipd - l1 therapy ( panel a2 )  . 
repeat computed tomography imaging demonstrated progressive disease in the right adrenal gland before initiation of antipd - 1 therapy ( panel a3 ) , with subsequent improvement after 9 months of antipd1 therapy ( panel a4 )  . 
ccrt , concurrent radiotherapy ; lns , lymph nodes ; na , not available . palliative radiation therapy ( 30 gy in 10 fractions ) to both his right axilla and rp space . 
two weeks after radiation therapy , repeat imaging demonstrated stability of the rp mass , decrease in the size of the right axillary lymph node , and enlarging adrenal masses . 
imaging of the face after 6 weeks showed a pr in the rp space , and body imaging after 8 weeks of treatment showed a pr in both the right axilla and right adrenal gland ( fig 1a )  . 
he tolerated treatment well , with no immune - related adverse events for 24 cycles , after which he was noted to have progression limited to his right axilla , with a core biopsy confirming nsclc . 
unfortunately , he died several days after this procedure at an outside facility ; the cause of death was unclear . statement of informed consent all human investigations were performed after approval by a local human investigations committee and in accord with an assurance filed with and approved by the department of health and human services , and all data were anonymized to protect the identities of participants involved in the research . 
informed consent from the patient for such research was obtained . longitudinal assessment of pd - l1 and pd - l2 expression biopsy samples were obtained at the time of diagnosis ( biopsy 1 ) , before repeat treatment with antipd - l1 ( biopsy 2 ) , before treatment with anti - pd - 1 ( biopsy 3 ) , and after progression anti pd - 1 therapy ( biopsy 4 ; fig 1b )  . 
however , tissue for this first sample was extremely limited , and possible pd - l1 positivity could not be ruled out because of the size of the tissue specimen , particularly because subsequent specimens from the patient demonstrated a highly heterogeneous pattern of pd - l1 expression with rare positive stromal cells , whereas pd - l2 staining was robust throughout the tumor . 
after his initial disease progression while receiving antipd - l1 therapy , repeat biopsy demonstrated no tumor pd - l1 staining ( with limited stromal pd - l1 positivity ) , and pd - l2 remained strongly positive ( 70% ; fig 1c )  . 
similar results were demonstrated in biopsy 3 before starting pd - 1 therapy and biopsy 4 after progression on this final line of treatment , with strong pd - l2 expression in tumor , moderate pd - l1 staining in stroma , and absence of pd - l1 expression in tumor . 
representative immunohistochemistry samples from each time point are shown in figure 2 . targeted next - generation sequencing genomic testing using a hybrid capture - based next - generation sequencing platform assessing exons from 315 genes ( foundationone ) of the patients tumor at the time of progression while taking nivolumab ( biopsy 4 ) identified seven potentially functional alterations , including rictor amplification , arid1a t2030fs * 3 , arid2 q904 * , kdm5c e656 * , slit2 g498 * , tet2 e1851 * , and tp53 r280i . 
thirty - three variants of unknown significance were identified , and a total mutation burden of 53 mutations / megabase was estimated , placing this patient in the top 7% of all tumor specimens tested by this method.16 thus , the response observed in this high mutation - load patient was consistent with clinical observations.17 no alterations were identified in jak1 , jak2 , cd274 , pdcd1lg2 , pten , myc , or ctnnb1 in this patient , which were previously reported mechanisms of t - cell exclusion and / or resistance to antipd - 1 therapy.18 - 21 next - generation sequencing data were only available on the final biopsy specimen ; thus , changes in mutation burden over time cannot be assessed . gene expression analysis sufficient tissue for expression analysis ( nanostring pancancer immune profiling panel ) was available for two of the four biopsy time points : post - therapy a , which was sampled before beginning the second treatment course of antipd - l1 , and post - therapy b , sampled at disease progression on antipd - l1 , before radiation therapy followed by nivolumab . 
comparison of the expression of immune genes between these two samples demonstrated substantial downregulation of cd274 ( pd - l1 ) mrna and upregulation of several immunosuppressive genes , including il6 and ptgs2 ( cyclooxygenase - 2 ; fig 3a )  . 
it could be hypothesized that distinct immune checkpoints ( eg , pd - l1 v pd - l2 ) could divergently mediate therapeutic resistance to checkpoint inhibitors and support preclinical studies testing this hypothesis . this patients tumor was pd - l1 negative at diagnosis , with some positivity in the stroma , on the basis of our limited sample , but strongly pd - l2 positive . this finding suggests that the significance of pd - l2 positivity in response to pd - l1 or pd - 1 targeted therapies may be a useful subject of study . 
 ( a ) rna collected from archival although these studies used different clones for detection.27 , 28 using our own methods for detection of pd - l2 in a tissue microarray cohort of 43 patients with nsclc , we found that this patient was consistently among the top 5% of pd - l2 expressers . 
 tissue blocks were analyzed by nanostring pancancer immune profiling ( 730 immune - related genes )  . normalized data were compared between the post - therapy a ( preresistance ) and posttherapy b ( postresistance ) specimens for log2 fold change . 
 ( b ) genes involved in the interferon - gamma signaling reactome ( reactome.org ; accession : m965 ) were selected from the pancancer immune profiling gene set and plotted by fold change . this analysis demonstrated a general decrease in interferon - gamma signaling within the tumor microenvironment at disease progression on antiprogrammed death ligand 1 ( pd - l1 )  . 
pdl1 interacts with programmed death 1 ( pd1 ) and b7 - 1 ( cd80 ) to promote immune effector cell suppression , whereas pd - l2 can bind pd - 1 and rgmb , promoting effector suppression and respiratory tolerance in lung - resident macrophages , respectively.22 , 23 apcs , antigen - presenting cells ; cox2 , cyclooxygenase - 2 . be associated with earlier stage of disease ( stage i v all others ) , but not with gender or smoking status ( appendix fig a1 )  . during the second treatment with antipd - l1 , which was accompanied by short - term stable disease and subsequent progression , we observed maintenance of high pd - l2 expression and downregulation of pd - l1 mrna expression accompanied by simultaneous decrease of interferongamma response signatures . 
we also observed upregulation of immunosuppressive genes , such as cyclooxygenase - 2 ( recently shown to be important in mediating antipd - 1 responsiveness29 ) and interleukin - 6 , which was shown to decrease after initial treatment with pd - l1 targeted therapy.30 thus , the changes in gene expression within the tumor microenvironment in this patient were consistent with previous studies . importantly , there were multiple intervening therapies in this patient during the approximately 20month period after the initial pretreatment biopsy , limiting the inferences that can be made during the initial therapy with antipd - l1 . 
balko , leora horn manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors despite his prior treatments with palliative radiation ; however , he did receive an additional course of palliative radiation after his third biopsy and preceding his antipd - 1 therapy , and a role for radiation in sensitizing his tumor to anti - pd - 1 therapy cannot be ruled out . although anecdotal , this case report demonstrates an important clinical finding : patients who become resistant to antipd - l1 may still benefit from pd - 1 targeted therapies , possibly due to a switch from dependency on pd - l1 to pd - l2 for maintenance of immunosuppression . however , the presence of consistently high pd - l2 staining argues against a direct mechanism of pd - l2mediated de novo resistance to antipd - l1 therapy . 
moreover , pd - l2 mrna did not seem to be upregulated or changed during acquisition of resistance to antipd - l1 therapy . however , the ratio of pd - l2 to pd - l1 mrna increased substantially from 3.6 - fold to 14.6 - fold during this period and may be a useful metric to test experimentally in the future for association with resistance to antipd - l1 treatment . 
although more investigation is needed in the context of preclinical studies and clinical trials , this report supports investigations of sequencing antipd - l1 and antipd - 1 therapies to elucidate mechanisms of cross - resistance and derive maximal patient benefit from these agents . 
johnson consulting or advisory role : bristol - myers squibb , genoptix , merck research funding : incyte patents , royalties , other intellectual property : intellectual property and patents pending surrounding use of mhc - ii and response to immune therapy leora horn honoraria : biodesix consulting or advisory role : bristol - myers squibb , merck , bayer , xcovery , genentech , boehringer ingelheim , eli lilly , abbvie , astrazeneca research funding : astrazeneca ( inst ) other relationship : bristol - myers squibb justin m . 
balko research funding : incyte patents , royalties , other intellectual property : patent on use of hla - dr / mhc expression to predict response to immunotherapies references 677 - 704 , 2008 1 . 
mu cy , huang ja , chen y , et al : high expression of pd - l1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation . 
borghaei h , paz - ares l , horn l , et al : nivolumab versus docetaxel in advanced nonsquamous nonsmall - cell lung 2018 - 2028 , 2015 cancer . 
rittmeyer a , barlesi f , waterkamp d , et al : atezolizumab versus docetaxel in patients with previously treated nonsmall - cell lung cancer ( oak ) : a phase 3 , open - label , multicentre randomised controlled trial . 
brahmer jr , tykodi ss , chow lq , et al : safety and activity of anti - pd - l1 antibody in patients with advanced cancer . n engl j med 366 : 2455 - 2465 , 2012 10 . 
brahmer jr , rizvi na , luzky j , et al : clinical activity and biomarkers of medi4736 , an anti - pd - l1 antibody , in patients with nsclc . 
rizvi n , brahmer j , ignatius s , et al : safety and clinical activity of medi4736 , an anti - programmed cell death - ligand 1 ( pd - l1 ) antibody , in patients with nonsmall - cell lung cancer ( nsclc )  . 
spira ai , park k , mazi`eres j , et al : efficacy , safety and predictive biomarker results from a randomized phase ii study comparing mpdl3280a vs docetaxel in 2l / 3l nsclc ( poplar )  . 
spigel dr , chaft je , gettinger sn , et al : clinical activity and safety from a phase ii study ( fir ) of mpdl3280a ( antipdl1 ) in pd - l1selected patients with nonsmall - cell lung cancer ( nsclc )  . 
kelly k , patel mr , infante jr , et al : avelumab ( msb0010718c ) , an anti - pd - l1 antibody , in patients with metastatic or locally advanced solid tumors : assessment of safety and tolerability in a phase i , open - label expansion study . 
xiao y , yu s , zhu b , et al : rgmb is a novel binding partner for pd - l2 and its engagement with pd - l2 promotes respiratory tolerance . 
he j , hu y , hu m , et al : development of pd - 1 / pd - l1 pathway in tumor immune microenvironment and treatment for nonsmall - cell lung cancer . 
yearley j , gibson c , yu n , et al : pd - l2 expression in human tumors : relevance to anti - pd - 1 therapy in cancer . presented at european society for medical oncology annual meeting , vienna , austria , september 25 - 29 , 2015 27 . 
calles a , liao x , sholl lm , et al : expression of pd - 1 and its ligands , pd - l1 and pd - l2 , in smokers and never smokers with kras - mutant lung cancer . 
kim my , koh j , kim s , et al : clinicopathological analysis of pd - l1 and pd - l2 expression in pulmonary squamous cell carcinoma : comparison with tumor - infiltrating t cells and the status of oncogenic drivers . 
postow ma , callahan mk , barker ca , et al : immunologic correlates of the abscopal effect in a patient with melanoma . 2035 - 2036 , 2012 n engl j med 366 : 925 - 931 , 2012 34 . 
programmed death ligand 2 ( pd - l2 ) expression across 43 patients with nonsmall cell lung cancer ( nsclc )  . ( a ) a tissue microarray series of 43 patients with nsclc was stained for pd - l2 expression and analyzed by histoscore ( h - score ; % of cells staining positive 3 intensity [ 0 - 3 + ] )  . 
laetsch author affiliations and support information ( if applicable ) appear at the end of this article . creative commons attribution non commercial no derivatives 4.0 license clinical trial information : nct02637687 . corresponding author : theodore w . 
in normal development , these genes regulate the growth , differentiation , and survival of neurons.14 , 15 gene fusions that involve one of these genes ( trk fusions ) have been described in a range of pediatric and adult cancers.16 - 25 in these fusions , the 3 region of the ntrk gene , which includes the kinase domain , is joined downstream of the promoter and 5 region of an unrelated gene . 
most reported fusions in extra cranial solid tumors to date have involved either ntrk1 or ntrk3 , with ntrk2 fusions more common in primary cns tumors.26 larotrectinib is the first highly selective inhibitor of all three trk kinases to enter clinical development . 
after two cycles of chemotherapy , disease progression continued , and an aortic pseudo aneurysm developed within the mass , which led to displacement of the inferior vena cava and ureteral impingement ( fig 1b )  . 
 ( e ) after 14 months of larotrectinib treatment . the patient was subsequently referred to our institution , where surgical intervention to debulk the tumor and repair the pseudo - aneurysm was offered . 
histologically , the tumor consisted of sheets of epithelioid cells with large eccentrically placed nucleus , occasional nucleoli , and abundant eosinophilic cytoplasm , mimicking rhabdoid cells ( fig 3 )  . 
immunohistochemically , the cells showed retained ini - 1 nuclear positivity and scattered cells positive for s - 100 protein and cd34 . postoperatively , gross residual tumor remained in the vertebral bodies . 
next generation sequencing ( ngs ) of her tumor was performed using foundationoneheme ( foundation medicine , cambridge , ma ) , a combined dna and rna sequencing hybrid capture - based assay that has been validated for clinical use without requirement for an orthogonal confirmatory assay.28 sequencing revealed a novel strn - ntrk2 fusion in which the kinase domain of ntrk2 is joined in frame to strn ( figs 3e and 3f )  . 
copy number loss of cdkn2b also was identified . two months after surgery , after informed consent , the patient was enrolled in the pediatric phase i trial of larotrectinib.29 she was treated with dose level 1 ( 100 - mg adult equivalent dose by simcyp modeling [ simcyp , sheffield , uk ] ) and received larotrectinib 75 mg twice a day continuously on the basis of her age and body surface area . 
pharmacokinetic assessment after the first dose of larotrectinib revealed an estimated 24 - hour area under the plasma concentration time curve ( auc0 - 24 ) of 2 , 980 ng h / ml , which was less than the protocol - specified threshold of 3 , 500 ng h / ml that would allow intrapatient dose escalation . 
pharmacokinetics at this dose demonstrated an estimated auc0 - 24 of 3 , 830 ng h / ml . at the time of initiation of larotrectinib , the patients lansky play - performance score ( lps ) was 60 . 
by 14 days after initiation of larotrectinib , the patient was noticeably less fatigued ( lps of 70 ) , the ascites had resolved by physical examination , and her back pain had resolved ( fig 4 )  . 
 ( d ) excised tumor specimen , including the aortic bifurcation . ureter position of vertebral bodies 2 , demonstrated resolution of pet avidity of all tumors , resolution of ascites , and significant reduction in the size of the residual pulmonary metastases and retroperitoneal tumors consistent with a partial response by response evaluation criteria in solid tumors ( recist ) version 1.1 ( figs 1d and 1e )  . 
five months after starting larotrectinib , the patient underwent uncomplicated closure of her colostomy and removal of her denver shunt . despite heavy pretreatment , the patient has tolerated larotrectinib well , with the only drug related adverse events consisting of mild ( common terminology criteria for adverse events [ version 4 ] grade 1 ) fatigue and intermittent grade 1 to 2 cytopenias . 
her response continues to deepen with complete resolution of her pulmonary metastasis and only residual pet - negative tissue at the site of tumor invasion of the vertebral bodies after 22 months of larotrectinib therapy as of february 2018 . 
 currently , the patients lps is 90 , and she has returned to school . discussion we describe a patient in whom tumor sequencing led to the identification of a novel ntrk2 fusion in a chemotherapy - refractory , metastatic , unresectable , undifferentiated sarcoma and successful treatment with larotrectinib . 
the identification of an ntrk2 fusion is consistent with other reports of oncogenic fusions in this histology.13 , 30 - 39 a wide range of tumors that harbor trk fusions has been reported to respond to larotrectinib.27 the activity of larotrectinib is striking in children , with a 93% confirmed objective response rate and all patients with trk fusions experiencing tumor regression.29 this patients tumor harbored a fusion of ntrk2 , and she was the sole patient in the initial 55 - patient data set to have a fusion of this gene.27 other rare ntrk2 fusions have been described in gliomas , specifically with vcl , agbl4 , qki , and nacc2 , 20 , 40 but to our knowledge , this report is the first of an ntrk2 fusion in a pediatric extracranial solid tumor . 
 ( b ) hematoxylin and eosin stain that shows epithelioid / rhabdoid cells with eccentric , pleomorphic nuclei ; occasional nucleoli and abundant eosinophilic cytoplasm ; and a single mitosis . 
 ( f ) rna sequencing reads that support the strn - ntrk2 fusion . ntrk3 suggests that these sarcomas should be evaluated for fusions of all three ntrk genes.41 strn on chromosome 2p22.2 encodes striatin , a ubiquitously expressed calmodulin - binding protein thyroid cancer , it is a common 5 fusion partner for alk also located on 2p.42 however , strn has not previously been reported as an ntrk fusion partner , which is consistent with the broad diversity of 5 fusion partners seen in trk fusion - positive cancers27 and is an important consideration when designing testing strategies for trk fusions . 
tests such as reverse transcriptase polymerase chain reaction only detect known fusion partners and are likely to miss tumors with uncommon or unique ntrk fusion genes , which can respond to trk inhibition . 
testing that uses hybrid capturebased ngs has the potential to detect a diversity of ntrk fusion partners , but careful attention must be paid to the initial substrate ( dna v rna ) , the target enrichment strategy ( hybrid capture v amplicon v anchored multiplex polymerase chain reaction ) , and the detailed probe design . the profound clinical response seen in this strn - ntrk2 fusion patient to larotrectinib establishes that the trk fusion is the key driver for this patients tumor . 
in the latter , despite high initial response rates , nearly all patients treated with vemurafenib or dabrafenib experience disease progression within 12 to 18 months of starting therapy.43 , 44 durable responses also have been observed in patients with fusions that involve ntrk1 and ntrk3 treated with larotrectinib.27 in conclusion , ngs of the undifferentiated sarcoma in our patient was critical in identifying a highly actionable kinase fusion ( strn - ntrk2 ) , which resulted in a profound and durable clinical response on matched treatment . 
laetsch manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors veena rajaram no relationship to disclose dean c . 
miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center lee a . 
cox employment : bayer ag , loxo oncology , merck , amgen stock and other ownership interests : loxo oncology , bayer ag , merck , amgen patents , royalties , other intellectual property : us patent 62 / 318 , 041 issued to loxo oncology ( inst ) theodore w . 
laetsch , the university of texas southwestern medical center ; tara pavlock , alison patterson , anne post , caitlyn ambrose , veena rajaram , andrew martin , steve megison , and theodore w . 
ferrari a , sultan i , huang tt , et al : soft tissue sarcoma across the age spectrum : a populationbased study from the surveillance epidemiology and end results database . 
pappo as , devidas m , jenkins j , et al : phase ii trial of neoadjuvant vincristine , ifosfamide , and doxorubicin with granulocyte colony - stimulating factor support in children and adolescents with advanced - stage nonrhabdomyosarcomatous soft tissue sarcomas : a pediatric oncology group study . 
antonescu cr , suurmeijer aj , zhang l , et al : molecular characterization of inflammatory myofibroblastic tumors with frequent alk and ros1 gene fusions and rare novel ret rearrangement . 
simon mp , pedeutour f , sirvent n , et al : deregulation of the platelet - derived growth factor b - chain gene via fusion with collagen gene col1a1 in dermatofibrosarcoma protuberans and giant - cell fibroblastoma . 
moss yp , voss sd , lim ms , et al : targeting alk with crizotinib in pediatric anaplastic large cell lymphoma and inflammatory myofibroblastic tumor : a childrens oncology group study . 
laetsch tw , roy a , xu l , et al : undifferentiated sarcomas in children harbor clinically - relevant oncogenic fusions and gene copy - number alterations : a report from the childrens oncology group . 
bourgeois jm , knezevich sr , mathers ja , et al : molecular detection of the etv6 - ntrk3 gene fusion differentiates congenital fibrosarcoma from other childhood spindle cell tumors . 
knezevich sr , garnett mj , pysher tj , et al : etv6 - ntrk3 gene fusions and trisomy 11 establish a histogenetic link between mesoblastic nephroma and congenital fibrosarcoma . 
el demellawy d , cundiff ca , nasr a , et al : congenital mesoblastic nephroma : a study of 19 cases using immunohistochemistry and etv6 - ntrk3 fusion gene rearrangement . 
laetsch tw , dubois sg , mascarenhas l , et al : larotrectinib for paediatric solid tumours harbouring ntrk gene fusions : phase 1 results from a multicentre , open - label , phase 1 / 2 study . 
italiano a , sung ys , zhang l , et al : high prevalence of cic fusion with double - homeobox ( dux4 ) transcription factors in ewsr1 - negative undifferentiated small blue round cell sarcomas . 
yamada y , kuda m , kohashi k , et al : histological and immunohistochemical characteristics of undifferentiated small round cell sarcomas associated with cic - dux4 and bcor - ccnb3 fusion genes . 
li ws , liao ic , wen mc , et al : bcor - ccnb3 - positive soft tissue sarcoma with round - cell and spindle - cell histology : a series of four cases highlighting the pitfall of mimicking poorly differentiated synovial sarcoma . 
omeara e , stack d , phelan s , et al : identification of an mll4 - gps2 fusion as an oncogenic driver of undifferentiated spindle cell sarcoma in a child . 
kao yc , sung ys , zhang l , et al : recurrent bcor internal tandem duplication and ywhaenutm2b fusions in soft tissue undifferentiated round cell sarcoma of infancy : overlapping genetic features with clear cell sarcoma of kidney . 
sugita s , arai y , aoyama t , et al : nutm2a - cic fusion small round cell sarcoma : a genetically distinct variant of cic - rearranged sarcoma . 
kelly lm , barila g , liu p , et al : identification of the transforming strn - alk fusion as a potential therapeutic target in the aggressive forms of thyroid cancer . 
hauschild a , grob jj , demidov lv , et al : dabrafenib in braf - mutated metastatic melanoma : a multicentre , open - label , phase 3 randomised controlled trial . 
 o tmprss2 - erg fusions unexpectedly identified in men initially diagnosed with nonprostatic malignancies purpose tmprss2 - erg gene fusions are frequently found in prostate cancer and are pathognomonic for prostatic origin a series of cancer cases assayed with comprehensive genomic profiling ( cgp ) in the course of clinical care , we reviewed the frequency of tmprss2 - erg fusions in patient tumors of various histologic subtypes . methods frequency of tmprss2 - erg fusions was determined in cgps from 64 , 263 cancer cases submitted to foundation medicine to assess genomic alterations suggesting benefit from targeted therapy . 
genomic results are presented from an index case of prostate cancer that underwent evolution from adenocarcinoma to pure squamous cell carcinoma . results tmprss2 - erg fusions were identified for 0.86% of male patients ( 250 of 29 , 030 ) and not found for female patients ( none of 35 , 233 )  . 
the index case is a patient with a large , left retrovesical mass diagnosed as squamous carcinoma by morphologic examination and a history of gleason score 9 prostate cancer with prior prostatectomy and salvage radiation therapy . 
a phylogenetic tree demonstrating clonal and histopathologic evolution of prostate cancer in the index patient was constructed . conclusion in this large cgp dataset , tmprss2 - erg fusion was seen in approximately 30% of prostate cancers regardless of histologic type ; on occasion , the fusion was detected in advanced cancers not initially carrying a diagnosis of prostate carcinoma . 
2017 by american society of clinical oncology introduction chromosomal translocations involving tmprss2 , an androgen - regulated gene encoding a transmembrane serine protease , and members of the ets transcription factor family including erg are frequently observed in prostate cancer.1 specifically , tmprss2 - erg fusions occur in approximately half of all prostate cancers and are believed to be a critical player in prostate carcinogenesis.2 tmprss2 - erg is involved in the promotion of cell migration , invasion , and tumor progression.1 , 3 the prognostic and predictive value of tmprss2erg fusions is still under active investigation ; however , some studies have suggested an association of the fusion gene with higher recurrence risk and possibly even increased risk of prostate cancerrelated death.4 - 6 clinically , tmprss2 - erg fusions can be detected in formalin - fixed paraffin - embedded tumor specimens through comprehensive genomic profiling ( cgp ) assays that use next - generation sequencing technology . 
the wide availability of this technology enables practicing clinicians to readily ascertain the presence of gene fusion events , such as those between tmprss2 and erg , in patient tumors . 
however , the presence of this fusion in a case where prostate cancer was not the diagnosis may dramatically alter the therapeutic approach . here , we review a large database of patients with advanced cancers who underwent cgp in the course of clinical care to ascertain the frequency of tmprss2 - erg in both prostate cancer and in patients with diagnoses of nonprostate cancer . 
an index case was identified to highlight the clinical impact of this approach . methods the methods used for this analysis have been previously described.7 in brief , formalin - fixed , paraffin - embedded slides or blocks from tumor samples were subjected to cgp in a clinical laboratory improvement amendmentscertified , college of american pathologistsaccredited , new york stateregulated reference laboratory ( foundation medicine )  . 
60 , 000 cancer cases submitted to foundation medicine by december 31 , 2015 , were used to determine the frequency of tmprss2 - erg fusions across histologic subtypes . genomic profiles for an index case of prostate cancer with histologic evolution from prostatic adenocarcinoma to pure squamous cell carcinoma were identified and described . 
an evolutionary tree was inferred using the neighbor - joining method , with the branch lengths being proportional to the number of genomic alterations acquired on the corresponding branch.8 the evolutionary analysis was conducted in mega7 ( net / home ) .9 approval for this study , including a waiver of informed consent and a health insurance portability and accountability act waiver of authorization , was obtained from the western institutional review board ( protocol no . 20152817 ) results the tmprss2 - erg fusion gene was detected exclusively in tumor samples obtained from male patients . 
other tumor types , for which tmprss2 - erg was not found , accounted for most of the samples ( n = 59 , 114 ) in the overall dataset ( table 2 )  . 
in the 2 , 139 patients with carcinoma of unknown primary , 28 ( 1.3% ) had tmprss2erg identified in their tumors and six of those were of the squamous cell subtype . 
he was noted to have a stage pt2 n0 mx tumor ; final histopathologic analysis identified his tumor to be of higher grade ( gleason score 9 [ 4 + 5 ] )  . 
by november 2009 , the patient was found to have a persistently detectable prostate - specific antigen ( psa ) level of 0.2 ng / ml , which increased to 0.3 ng / ml by january 2010 . 
the patient subsequently underwent salvage external beam radiation tomotherapy ( 64.8 gy 3 36 fractions ) to the prostatic fossa . despite radiation , his psa level continued to slowly rise over the next few years . in june 2011 , the patient underwent a liver biopsy for a slowly enlarging liver nodule that had been observed over the past 6 years . 
to palliate urinary and rectal obstruction from the enlarging pelvic mass , the patient underwent radical cystectomy with ileal conduit urinary diversion , low anterior resection with diverting colostomy , and small - bowel resection in september 2015 . 
tumor types for samples in the dataset ( n = 59 , 114 ) percentage of all samples tumor type lung adenocarcinoma colon adenocarcinoma breast invasive ductal carcinoma breast carcinoma ( nos ) unknown primary adenocarcinoma brain glioblastoma lung non - small cell lung carcinoma ( nos ) pancreas ductal adenocarcinoma ovary serous carcinoma lung squamous cell carcinoma abbreviation : nos , not otherwise specified . pelvic mass was confirmed to be pure squamous cell cancer . 
all three carcinoma specimens shared the tmprss2 - erg fusion , loss of pten exons 5 through 9 , the tp53 missense mutation v147g , and variants of unknown significance in the genes pdgfrb ( r177c ) and prkdc ( s1660y )  . 
in addition , the primary prostatic adenocarcinoma tumor ( june 2009 ) carried high - level amplifications of the genes foxp1 and tbx3 , whereas the squamous cell carcinoma specimen ( july 2015 ) and the prostatic adenocarcinoma metastasis ( september 2015 ) , which were both from the bladder , had homozygous loss of the adjacent genes cdkn2a and cdkn2b in common . 
the adenocarcinoma metastasis also harbored a subclonal nfe2l2 mutation ( w8c ) , and the squamous cell carcinoma carried a distinct variant of unknown significance ( cic g133c )  . 
no reportable genomic alterations were identified in the benign carcinoid specimen ; all its variants were of unknown significance , shared with the other specimens , and had variant allele frequencies consistent with germline status . 
in summary , comprehensive genomic analysis identified truncal prostatic adenocarcinoma drivers in the squamous cell carcinoma , including the unexpected tmprss2 - erg fusion event , and thus indicated that the squamous cell carcinoma had arisen from a prostatic adenocarcinoma . discussion prostate cancer is a highly heterogeneous malignancy on the genomic level . 
nevertheless , morphologic evaluation of prostate cancer overwhelmingly identifies this tumor type to be adenocarcinoma , albeit of varying grades within the same tumor ( intrapatient ) or across different patients tumors ( interpatient )  . 
here , we have reported an index case of prostate cancer with variant histology , specifically squamous cell carcinoma that was realized by the identification of tmprss2erg fusion in both the squamous ( divergent histology ) and in the primary prostatic adenocarcinoma specimen . 
histopathologic sections of the index patients tumors . ( a ) micrographs of hematoxylinand - eosin stained typical carcinoid biopsy specimen and the corresponding synaptophysin stain . ( b ) micrographs of representative sections of the patients metachronous squamous cell cancer in a metastatic pelvic mass . ( c ) metastatic prostatic adenocarcinoma obtained from a regional lymph node . 
 ( d ) metastatic prostatic adenocarcinoma adherent to small bowel . we observed tmprss2 - erg fusions in approximately 30% of prostate cancers , which were predominantly adenocarcinoma , but the frequency of fusion - positive cases held true across histologic prostate tumor types . 
tmprss2 - erg fusion was also detected in patients with advanced cancer who had a diagnosis of nonprostate tumors as defined by the treating teain addition to the index case of squamous carcinoma presented in this report , five additional patients who had squamous cell cancer were found to harbor tmprss2 - erg , a finding with likely therapeutic implications . 
in the absence of genomic information , a histologic phenotype not known to be associated with prostate cancer , such as squamous cell carcinoma , would trigger a different treatment algorithm , such as cytotoxic platinum - based chemotherapy . 
 squamous cell carcinoma ( pelvic mass ) cic g133c nfe2l1 w8c cdkn2a / b loss adenocarcinoma ( metastatic deposit ) adenocarcinoma , prostate ( primary tumor ) foxp1 amplification tbx3 amplification tmprss2 - erg fusion pten loss exons 5 - 9 tp53 v147g prkdc s1660y pdgfrb r177c carcinoid , liver ( benign specimen ) fig 2 . 
the evolutionary tree was inferred using the neighbor - joining method , 8 with the branch lengths being proportional to the number of genomic alterations acquired on the corresponding branch . evolutionary analyses were conducted in mega7.9 xenograft mouse models of tmprss - erg expression nonadenocarcinoma . primary squamous cell carcinoma of the prostate has been reported in the literature , but it is exceedingly rare.11 such cases may represent transformation of a prostatic adenocarcinoma primary , as in this index case . 
lara jr honoraria : pfizer consulting or advisory role : exelixis , pfizer , novartis , astrazeneca , bayer , roche , celgene , janssen , bristol - myers squibb , abbvie , turnstone bio research funding : millennium ( inst ) , polaris ( inst ) , glaxosmithkline ( inst ) , genentech ( inst ) , aragon established the correct clinical diagnosis and later revealed the pattern of clonal evolution and selection . most importantly , the finding of tmprss2 - erg in the squamous cell tumor provided support for the use of androgen deprivation therapy for the patient . there are important limitations to this analysis . our report focuses solely on tmprss2 - erg fusions . 
although this event represents the vast majority of ets rearrangements , tmprss2 can be rearranged with other biologically relevant partner genes that were not specifically studied here and that could be similarly pathognomonic for a prostatic origin of advanced cancer in men . 
elvin employment : foundation medicine stock and other ownership interests : foundation medicine mamta parikh no relationship to disclose ralph de vere white stock and other ownership interests : pfizer regina gandour - edwards no relationship to disclose christopher p . 
miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan - kettering cancer center siraj m . 
ali employment : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine ; patents via seres health on microbiome in non - neoplastic disease ( i ) primo n . 
p30 ca093373 to the university of california davis comprehensive cancer center . presented in part at the asco annual meeting , chicago , il , june 3 - 7 , 2016 . affiliations support prior presentation references 1 . 
wirth , md1 ; takashi kohno , phd2 ; hibiki udagawa , md , phd3 ; shingo matsumoto , md , phd3 ; genichiro ishii , md , phd3 ; kevin ebata , phd4 ; brian b . 
michael rothenberg , md , phd4 ; and koichi goto , md , phd3 introduction the ret receptor tyrosine kinase is oncogenically activated by ret gene fusions in 1% to 2% of nonsmall - cell lung cancers ( nsclc ) and by ret gene mutations in most medullary thyroid cancers ( mtc ) .1 although multikinase inhibitors ( mkis ) with nonspecic anti - ret activity are approved for the treatment of mtc irrespective of ret mutation status , their investigational use for patients with ret fusion - positive nsclc has been limited by substantial off - target adverse effects that lead to dose reductions and inadequate ret - specic inhibition.2 - 7 as a result , tumor responses to mkis have been infrequent and short lived , and a comprehensive molecular understanding of mki response and resistance is lacking . in contrast to the mkis , loxo - 292 is a highly selective , small molecule ret tyrosine kinase inhibitor ( tki ) with nanomolar potency against diverse ret alterations , favorable pharmacokinetic ( pk ) properties , and signicant cns penetration.8 in an ongoing , phase i / ii identier : study of loxo - 292 ( clinicaltrials.gov nct03157128 ) , conrmed partial responses ( prs ) were achieved in most ret - altered patients across different tumor histologies and diverse ret gene fusions and mutations . 
a 42 - yearold woman with nsclc had received nine prior systemic therapy regimens , including approved chemotherapy , immunotherapy , and investigational tkis , before the identication of a kif5b - ret fusion in a sample from a recurrent pleural fusion ( fig 1a , data supplement )  . 
she received alectinib and additional chemotherapy and then vandetanib ( fig 1a ) .6 she experienced a conrmed pr by response evaluation criteria in solid tumors ( recist ) 1.1 with vandetanib treatment , with improvement in multiple lung and pleural - based lesions . 
targeted ret gene sequencing of a biopsy specimen from a progressing right lung lesion identied a ret valine - to - leucine ( v804l ) gatekeeper mutation absent from a pretreatment tumor sample ( fig 1a , data supplement )  . v804l allele frequency in the progression sample was 15% by next - generation sequencing ( 120 of 799 reads ) compared with 0% in the pretreatment sample ( 0 of 847 reads ; data supplement )  . the patient received additional systemic chemotherapy with pemetrexed and amrubicin but experienced continued disease progression . 
after providing written informed consent , she started treatment with loxo292 in the phase i portion of the phase i / ii clinical trial at the recommended phase ii dose of 160 mg twice a day . 
 ( a ) various treatments the patient received for metastatic , kif5b - ret fusion - positive nonsmall - cell lung cancer are shown , together with the levels of carcinoembryonic antigen ( cea ; red diamonds ) and the sum of target lesions by response evaluation criteria in solid tumors ( recist ) 1.1 ( blue squares ) over time during treatment . 
the estimated levels of ret target inhibition at the 50% inhibitory concentration ( ic50 ) and 90% inhibitory concentration ( ic90 ) for kif5b - ret and kif5b - ret - v804l were modeled using actual patient human pharmacokinetic parameters ( data supplement )  . 
 ( c ) computed tomographic images of the patients metastatic lung and liver disease before and at the indicated times after initiating treatment with vandetanib ( left three pairs of panels ) or loxo - 292 ( right two pairs of panels )  . 
consistent with this , she experienced a rapid clinical and biochemical response to loxo - 292 , with decreased shortness of breath and reduction in serum carcinoembryonic antigen ( fig 1a )  . repeat imaging after 8 weeks of treatment demonstrated a 48% decrease in measurable tumor burden by recist 1.1 that was maintained after 16 weeks , indicating a conrmed pr ( figs 1a and 1c )  . 
the patient has received loxo - 292 for more than 11 months , is still receiving treatment , and is tolerating therapy well , without dose interruption , and with all treatment - emergent adverse events grade 1 or 2 . case 2 : hereditary ret v804m mtc . 
a 50 - year - old man with hereditary ret v804m multiple endocrine neoplasia 2a and mtc developed primary disease progression despite previous treatment with three anti - ret mkis ( fig 2a )  . before treatment with loxo - 292 , he was highly symptomatic , with abdominal pain from tumor inltration of the liver and severe tumor - related diarrhea . 
consistent with this , serum carcinoembryonic antigen and calcitonin levels decreased rapidly , together with resolution of diarrhea , abdominal distension , and abdominal parepeat imaging after 8 weeks of treatment indicated a complete response by recist 1.1 , which was conrmed after 12 weeks of treatment , with complete resolution of target lymph node lesions and nontarget liver memediastinal tastases and normalization of serum tumor markers ( figs 2c and 2d )  . 
the patients dose was increased to 160 mg twice a day as allowed by the protocol , with doseproportional increases in exposure and continuous more than ic90 ret v804m target coverage ( fig 2b ) , continued complete response , and continued normalization of tumor markers ( fig 2c )  . 
the patient experienced three adverse events : transient grade 3 altered mental status ( sedation ) , judged by the investigator to be related to concomitant treatment with anxiolytic medications and unrelated to loxo - 292 ; grade 1 constipation ; and grade 2 nausea . 
he remains in complete response and is still receiving treatment at more than 20 months after the start of loxo - 292 . loxo - 292 but not anti - ret mkis maintain inhibitory activity against ret gatekeeper mutations preclinically . 
in contrast to mkis , loxo - 292 is predicted to better accommodate the bulky leucine and methionine side chains in the gatekeeper residues without any steric interactions ( fig 3b )  . to determine the impact of ret kinase domain resistance mutations on inhibitor activity , loxo - 292 , vandetanib , and cabozantinib were tested in vitro against the puried wildtype and mutant ret kinase domains ( fig 3c )  . 
at physiologic ( 1 mm ) atp concentration , loxo - 292 had potent inhibitory activity against wild - type ret , ret v804l , and ret v804m kinases . the anti - ret mkis , in contrast , vandetanib and cabozantinib , displayed signicantly reduced activity against ret v804l / m compared with wildtype ret . 
these data demonstrate that loxo - 292 , but not the mkis vandetanib or cabozantinib , maintain potent inhibitory activity against ret gatekeeper mutations . discussion selective tkis targeting actionable kinase alterations have transformed the treatment landscape for several human cancers . 
although acquired resistance to tkis is universal , a detailed molecular understanding of resistance has led to the development of next - generation tkis that have extended disease control and clinical benet in resistant patients.16 , 17 in several instances , initial treatment with the next - generation inhibitor has led to more durable treatment responses than treatment with the rstgeneration tki and has become standard upfront systemic therapy.18 , 19 loxo - 292 combines features of both rstand nextgeneration tkis used to treat other single kinase - driven cancers . 
it is highly potent against and selective for diverse founder activating ret alterations in human cancers , and in preclinical experiments , it overcomes ret v804 gatekeeper mutations , which have been identied in rare patients with acquired resistance to anti - ret mkis . 
for both patients , inhibiintegration of preclinical ret target tory activity with the actual exposures safely achieved at the recommended phase ii dose of 160 mg twice a day allowed more than ic90 calculated target coverage of both wild - type and gatekeeper - mutated ret in patients ( figs 1b and 2b )  . 
 ( b ) pharmacokinetic analysis at steady state after 8 days of treatment with loxo - 292 at a dose of 80 mg , 120 mg , and 160 mg twice a day . 
the estimated levels of ret target inhibition at the 50% inhibitory concentration ( ic50 ) and 90% inhibitory concentration ( ic90 ) for wild - type ( wt ) ret and ret v804m were modeled as in fig 1b . 
the red arrows indicate diffuse inltration of the liver by tumor . lenvat , lenvatinib ; rt , radiation treatment ; vande , vandetanib . treatment with loxo - 292 after the prior mkis has signicantly extended the period of durable clinical and biochemical response and disease control . 
 wirth et al hibiki udagawa honoraria : astrazeneca , chugai pharmaceutical , bristol - myers squibb , ono pharmaceutical , msd , taiho pharmaceutical , amco , abbvie , boehringer ingelheim , daiichi sankyo consulting or advisory role : abbvie , boehringer ingelheim research funding : msd , abbvie , daiichi sankyo , amgen shingo matsumoto honoraria : pzer , novartis , chugai pharma , astrazeneca research funding : chugai pharma ( inst ) , novartis ( inst ) , lilly ( inst ) , merck serono ( inst ) , merck sharp & dohme ( inst ) kevin ebata employment : loxo oncology stock and other ownership interests : loxo oncology travel , accommodations , expenses : loxo oncology brian b . 
tuch employment : loxo oncology stock and other ownership interests : loxo oncology consulting or advisory role : kezar life sciences patents , royalties , other intellectual property : biomarkers for ntrk inhibition ; biomarkers for proteasome inhibition edward y . 
zhu employment : loxo oncology stock and other ownership interests : loxo oncology travel , accommodations , expenses : loxo oncology michele nguyen employment : loxo oncology stock and other ownership interests : loxo oncology travel , accommodations , expenses : loxo oncology steve smith consulting or advisory role : loxo oncology , various patents , royalties , other intellectual property : various patents and applications travel , accommodations , expenses : various lauren m . 
burkard employment : array biopharma , university of colorado hospital ( i ) stock and other ownership interests : array biopharma , pzer , pzer ( i ) james f . 
blake employment : array biopharma stock and other ownership interests : array biopharma patents , royalties , other intellectual property : i am listed as a co - author on approximately 46 patents ( inst ) kevin r . 
brandhuber employment : loxo oncology stock and other ownership interests : loxo oncology , array biopharma travel , accommodations , expenses : loxo oncology steve andrews employment : loxo oncology leadership : loxo oncology stock and other ownership interests : loxo oncology travel , accommodations , expenses : loxo oncology s . 
michael rothenberg employment : loxo oncology stock and other ownership interests : loxo oncology travel , accommodations , expenses : loxo oncology koichi goto honoraria : bristol - myers squibb , astrazeneca , pzer , chugai pharmaceutical , taiho pharmaceutical , ono pharmaceutical , novartis , eli lilly , boehringer ingelheim , quintiles , merck serono , life technologies , msd , abbvie , riken genesis , nippon kayaku , takeda pharmaceuticals , otsuka pharmaceutical , srl diagnostics consulting or advisory role : otsuka pharmaceutical research funding : msd , astrazeneca , taiho pharmaceutical , chugai pharmaceutical , boehringer ingelheim , ono pharmaceutical , sumitomo dainippon , takeda pharmaceuticals , novartis , daiichi sankyo , kyowa hakko kirin , astellas pharma , eisai , eli lilly , pzer , riken genesis , bristol - myers squibb , merck serono , ignyta , life technologies , research triangle institute d / b / a rti health solutions , janssen , xcoo , loxo oncology no other potential conicts of interest were reported . references ji jh , oh yl , hong m , et al : identication of driving alk fusion genes and genomic landscape of medullary thyroid cancer . 
wells sa jr , robinson bg , gagel rf , et al : vandetanib in patients with locally advanced or metastatic medullary thyroid cancer : a randomized , double - blind phase iii trial . 
j clin oncol 30 : 134 - 141 , 2012 drilon a , rekhtman n , arcila m , et al : cabozantinib in patients with advanced ret - rearranged non - small - cell lung cancer : an open - label , single - centre , phase 2 , single - arm trial . 
lancet oncol 17 : 1653 - 1660 , 2016 lin jj , kennedy e , sequist lv , et al : clinical activity of alectinib in advanced ret - rearranged non - small cell lung cancer . 
j thorac oncol 11 : 2027 - 2032 , 2016 yoh k , seto t , satouchi m , et al : vandetanib in patients with previously treated ret - rearranged advanced non - small - cell lung cancer ( luret ) : an open - label , multicentre phase 2 trial . 
lancet respir med , 5 : 42 - 50 , 2017 lee sh , lee jk , ahn mj , et al : vandetanib in pretreated patients with advanced non - small cell lung cancer - harboring ret rearrangement : a phase ii clinical trial . 
ann oncol 28 : 292 - 297 , 2017 brandhuber bb , haas j , tuch bb , et al : ena - 0490 the development of loxo - 292 , a potent , kdr / vegfr2 - sparing ret kinase inhibitor for treating patients with ret - dependent cancers . 
aacr - nci - eortc international conference on molecular targets and cancer therapeutics , munich , germany , november 29 - december 2 , 2016 oxnard gr , subbiah v , park k , et al : clinical activity of loxo - 292 , a highly selective ret inhibitor , in patients with ret fusion + non - small cell lung cancer : an update from asco 2018 . 
wirth lj , cabanillas me , sherman ej , et al : clinical activity of loxo - 292 , a highly selective ret inhibitor , in patients with ret - altered thyroid cancers : an update from asco 2018 . 
drilon ae , subbiah v , oxnard gr , et al : a phase 1 study of loxo - 292 , a potent and highly selective ret inhibitor , in patients with ret - altered cancers . 
yang , m , cai j , guo s , et al : rapid conversion to resistance , of a colon pdw with ret - fusion , by ponatinib treatment could potentially be attributed to the introduction of the gate keeper mutation , v804m . 
dagogo - jack i , stevens se , lin jj , et al : emergence of a ret v804m gatekeeper mutation during treatment with vandetanib in ret - rearranged nsclc . j thorac oncol 13 : e226 - e227 , 2018 15 . 
nat commun 9 : 625 , 2018 j anne pa , yang jc , kim dw , et al : azd9291 in egfr inhibitor - resistant non - small - cell lung cancer . 
ou sh , ahn js , de petris l , et al : alectinib in crizotinib - refractory alk - rearranged non - small - cell lung cancer : a phase ii gobal study . 
pao w , miller va , politi ka , et al : acquired resistance of lung adenocarcinomas to getinib or erlotinib is associated with a second mutation in the egfr kinase 2016 293 : 876 - 880 , 2001 domaplos med 2 : e73 , 2005 22 . 
katayama r , khan tm , benes c , et al : therapeutic strategies to overcome crizotinib resistance in non - small cell lung cancers harboring the fusion oncogene eml4 - alk . 
tumor biopsies taken prior to neoadjuvant treatment were analyzed by reverse - phase protein arrays ( rppa ) for expression levels of 210 bc - relevant ( phospho - ) proteins . 
similarly , the mrna virp score was validated ( p , .001 ) in an independent phase ii clinical trial ( promix )  . conclusion our virp score , integrating the expression of nine proteins and validated on mrna data both internally and in an independent clinical trial , may be used to increase the likelihood of benet from treatment with bevacizumab combined with chemotherapy in patients with her2 - negative bc . jco precis oncol 5 : 286 - 306 . 
2021 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction treatment of solid tumors using antiangiogenic therapy has been explored for several decades.1 , 2 discovery of vascular endothelial growth factor a ( vegf - a ) as a major culprit in tumor angiogenesis led to the development of bevacizumab , a recombinant humanized monoclonal antibody targeting vegf - a.3 addition of bevacizumab to various chemotherapy regimens has proven highly benecial in patients with several types of advanced solid tumors resulting in a signicant improvement in overall survival ( os ) and / or progression - free survival ( pfs )  . despite good response to bevacizumab therapy in many individual patients with breast cancer ( bc ) , randomly selected treatment populations do not demonstrate improved os.4 , 5 thus , the clinical use of bevacizumab in bc is limited and currently only approved in europe . 
regardless of this , activity is observed in subsets of patients pointing to the urgent need for novel biomarkers to select subpopulations in which adequate clinical benet can be achieved.6 , 7 one of the most obvious and biological plausible biomarkers was the plasma vegf - a level , and although promising in some studies , evidence from the latter meridian trial did not support its use for identifying patients with benet from added bevacizumab.8 other biomarkers at various molecular levels have been investigated such as soluble carbonic anhydrase ix , 9 brca1 / 2 mutations , 10 and dna methylation signatures.11 however , tissue protein expression and protein signatures have not previously been explored , although data at the proteomic level in general have proven highly valuable in drug response prediction models.12 in this study , we sought to establish protein expression features predicting the response to treatment with bevacizumab in combination with chemotherapy . 
 protein signature predicts response to chemotherapy plus bevacizumab context key objective the use of antivascular endothelial growth factor ( vegf ) targeted therapies in combination with chemotherapy in breast cancer ( bc ) is limited because of the lack of a predictive biomarker . 
in this study , protein expression in pretreatment tumor biopsies was used to develop a predictor for patients with large bcs . knowledge generated a nine - protein response predictor was established by lasso regression trained by a continuous response evaluation . 
the protein signature was validated using corresponding mrna expression in the same patient cohort as well as in an independent patient cohort . relevance our study supports using this protein signature as a predictive marker for vegf inhibition in combination with chemotherapy , and represents a novel opportunity for rational use of this therapy in patients with bc . samples collected from patients with bc prior to neotreatment ( nat ) with chemotherapy ( ctx ) or adjuvant chemotherapy in combination with bevacizumab ( bev plus ctx ) , and analyzed expression against treatment effect on tumor size and clinical outcomes . 
patients with human epidermal growth factor receptor 2 ( her2 ) negative , previously untreated , breast carcinomas with size 2.5 cm were included , and 67 and 71 patients randomly assigned to treatment with ctx or bev plus ctx , respectively . 
one hundred nine samples collected prior to treatment were available for protein analysis on rppa in the ctx treatment arm ( n = 55 ) and in the bev plus ctx treatment arm ( n = 54 ) ( appendix fig a1 )  . 
clinicopathologic characteristics of patients , including adverse events , have previously been described.13 in brief , the primary end point of the clinical study , pcr , was dened as pathologic stage ypt0 and ypn0 after end of therapy . 
in the subset of patients with available protein proles ( n = 109 ) , pcr rates were overall higher in the bev plus ctx ( 26 % ) compared with the ctx ( 13 % ) treatment arm , although not signicantly ( p = .094 ) ( appendix fig a2 )  . 
relative tumor size in percent after 24 weeks of nat was calculated as tumor size at the time of surgery ( longest diameter on histopathologic specimen ) relative to tumor size at week 0 ( mri if available or ultrasound or mammography )  . 
tumor cell content in samples was assessed by use of ascat15 as previously reported.16 the study was approved by the institutional protocol review board , the regional ethics committee , the norwegian medicines agency , performed in accordance with the declaration of helsinki , and informed consent was obtained . 
we further used an external cohort with similar treatment characteristics ( promix trial , clinicaltrials.gov identier : nct00957125 ) , with mrna expression data available , 17 for validation of the virp score . reverse phase protein arrays ( rppa ) proling of 210 cancer - relevant proteins of which 54 were in a phosphorylated state ( appendix table a1 ) were performed by the rppa18 core facility at md anderson cancer center ( houston , tx )  . 
tumor protein lysates were serially diluted two - fold for 5 dilutions ( from undiluted to 1 : 16 dilution ) , probed with antibodies , and visualized by dab colorimetric reaction . 
for development of the virp score , the continuous response parameter relative tumor size was used as outcome , whereas the other clinically used response parameters pcr and rcb were used for evaluation of predictive performance . 
relative importance of each model variable was assessed using the r - package relaimpo23 with metrics lmg . virp scores based on mrna data in the neoava and promix study were computed using the intercept and beta - coefcients determined from the protein data in the neoava study . 
the corresponding surrogate mrna scores were determined using quantile - normalized and probeaveraged mrna expressions from the genes corresponding to the proteins in the original protein signature , including the phosphoproteins . results protein prole of tumors before treatment using unsupervised hierarchical clustering on the expression of the 210 proteins in all 109 tumors collected prior to treatment , they cluster into groups signicantly related to tumor shrinkage and to pam50 subtypes , with most estrogen receptor ( er ) - negative patients ( 17 of 21 ) localizing in a separate subcluster ( appendix fig a4 )  . univariate spearman correlation analysis of protein expressions related to relative tumor size visualized by the nonvariable - dependent rst principal component demonstrated a positive yet limited relationship between the two treatment arms , with expression of almost half of proteins ( n = 103 ) being inversely correlated with response . 
the number of proteins signicantly ( p , .05 unadjusted ) related to response was higher in the ctx treatment arm ( n = 54 ) than in the bev plus ctx arm ( n = 38 ) , with only ten in common and of which two had rho - values with opposite signs ( appendix fig a5 and appendix table a2 )  . development of a protein signature score predicting response to treatment with bev plus ctx addition of bevacizumab to standard treatment with chemotherapy demonstrated benet compared with chemotherapy alone , although not signicant ( appendix fig a2 )  . to develop a signature predicting response to treatment with bev plus ctx , protein expression in biopsies taken prior to treatment were used from patients with available relative tumor size after nat ( n = 54 )  . 
low - variance proteins are likely to have low predictive value , thus a certain level of variance in expression should be present in order for clinical markers to be applicable . 
we thus reduced the original panel of 210 proteins by plotting the variance in expression and demonstrated a mixed distribution ( appendix fig a3 ) where only members belonging mainly to the proteins with higher variance ( n = 114 ; appendix table a3 ) were considered for use in the adaptive lasso regression model . 
a second iterative round of lasso on this subset of ten proteins gave the nal intercept and beta coefcients , of which one was shrunk to zero giving a nal signature of nine proteins with corresponding beta coefcients . 
 protein signature predicts response to chemotherapy plus bevacizumab the virp score for each patient was calculated as the sum of the intercept and beta coefcient weighted expression of the nine proteins . 
in univariate analysis , only the virp score , both as a continuous value and dichotomized ( low v high ) , was signicantly associated with pcr ( table 2 )  . 
no signicant association was found for tumor stage , pam50 subtypes , age , pgr , tp53 mutation , and nodal or er status . we next sought to establish and evaluate the corresponding mrna expression score as a surrogate to the virp protein score . 
by taking the corresponding genes from the virp ( including the parent protein for the phosphoprotein antibodies ) and using the predened lasso intercept and beta coefcients , the mrna score for the neoava patients ( mrna virp scores ) was calculated . 
this demonstrated a signicant increase , approximately doubling , in the percentage of both pcr responders ( p = .009 ) and low rcb ( p = .022 ) in the virp score selected patient population . 
use of the virp score for selecting patients to bev plus ctx demonstrated a clear benet with an increased response both by pcr ( p , .001 ) and rcb ( p , .001 ) compared with standard ctx treatment ( fig 3 )  . discussion we have dened a protein signature score named virp for use in large her2 - negative primary bcs that identies good responders to nat with chemotherapy plus bevacizumab . 
use of the virp score for selection to bev plus ctx therapy signicantly enriches for patients responding 290 2021 by american society of clinical oncology to treatment ( pcr or rcb - low )  . 
importantly , the robustness of the virp score was conrmed through validation in an independent clinical cohort ( promix )  . the lasso regression method was used to select proteins for the virp score . 
however , in silico evaluation the virp proteins under stimulus of bevacizumab treatment links them to response on relevant biological processes ( appendix fig a7 )  . ranking the relative importance of each protein in the virp signature is interesting with respect to , for example , the potential to use each or selected proteins as biomarkers separately . 
it appears that the syk protein is of high importance ( appendix fig a8 ) , which also coincides with being the only protein in the signature differently expressed ( p = .002 ) in pcr versus non - pcr patients . 
the syk protein is enriched in immune cells , but also expressed in breast epithelium and endothelial cells where it plays a role in angiogenesis.26 when passing from protein to mrna expression , functional dimension of activating or deactivating phosphorylations is lost . 
this potentially inuences the performance of the mrna virp score when used as surrogate for the original protein score , and could partly explain the observed drop in auc for the roc analysis between the protein and mrna data sets in the neoava trial ( fig 2a and appendix fig a6a )  . 
spearman correlation between expression of all 210 assessed proteins and mrnas showed that of the nine proteins in the signature , the four with the lowest correlation were phosphorylated or extracellularly localized proteins . 
however , the signature members had a signicantly ( p = .039 ) higher correlation compared with all proteins in the original data set ( appendix fig a9 )  . 
although the neoava protein data set is too small to draw conclusions , a previous observation of nearly 10 , 000 proteins in bc showed that established prognostic mrna - centered signatures ( ie , pam50 - ror , oncotype dx , and mammaprint ) in general had higher protein - mrna correlation than average for the human genome.27 we propose that because of the closer relation proteins have to phenotype , biomarker signatures being developed to identify features with clinical , and thus phenotypical impact , will represent genes with high expressional and translational penetrance . in the neoava clinical trial , patient subgroups were divided based on er expression ( using 1% positivity as cutoff ) , and better response ( as assessed by pcr and rcb ) was in general observed for the er - negative group , whereas only the er - positive group had signicant benet of added in univariate analysis , er bevacizumab . 
this is also consistent with the discrepancy seen in large studies regarding the benet of added bevacizumab in the er - positive or er - negative subpopulations of patients.28 - 30 the relative higher number of proteins signicantly correlated with tumor size after treatment in the ctx arm ( n = 54 ) suggested that a similar protein signature predicting response to chemotherapy alone might be developed . however , use of adaptive lasso regression only returned a signature of two proteins with low predictive performance , and not represented in the bev plus ctx virp signature . use of the virp score developed for bev plus ctx treatment applied on the ctx patients did demonstrate a signicantly lower score in the limited number of patients with pcr ( n = 7 ) , but not when assessed for the low - rcb patients ( n = 14 )  . 
this does not exclude the possibility that the proteins in the virp signature also reect sensitivity to the baseline chemotherapy when combined with bevacizumab . bulk tumor samples may have variable tumor cell content , which might inuence the readout of mrna or proteinbased molecular signatures.31 inuence of tumor cell content ( median of 42% ) on the virp signature , or on its individual proteins , did not demonstrate any signicant associations in the bev plus ctx - treated patients ( appendix fig a11 )  . 
furthermore , the virp score should be related to the mechanism of action of the anti - vegf therapy , which involves normal tissue constituents and in particular endothelial cells and vessels.32 thus , a composite tissue is likely required for the score to function . 
 ( a ) fraction of patients obtaining pcr when treated with standard ctx in all neoava patients ( n = 55 ) or bev plus ctx in all neoava patients ( n = 54 ) and selected based on virp score ( n = 22 )  . 
 ( b ) fraction of patients obtaining low rcb ( 0 and i ) when treated with standard ctx in all neoava patients ( n = 55 ) or bev plus ctx in all neoava patients ( n = 54 ) and selected based on virp score ( n = 22 )  . 
pcr , pathologic complete response ; rcb , residual cancer burden ; virp , vegf inhibition response predictor . 100% 100% random population ctx treatment ( low = 14 high = 41 ) random population bev plus ctx treatment ( low = 20 high = 34 ) virp selected population bev plus ctx treatment ( low = 15 high = 7 ) from treatment with bevacizumab in combination with chemotherapy.34 the virp score is predicting responses , but further studies are warranted to assess for impact on survival . although the discovered virp signature was established for patients treated with bev plus ctx in a neoadjuvant setting , it recapitulates biology related to treatment response and could have the potential to also predict response to bev plus ctx therapy in the metastatic setting . 
additionally , studies are initiated to investigate whether the virp signature recapitulates common response biology shared by other solid cancer types for which bevacizumab in combination with chemotherapy is commonly used.35 affiliations 1department of tumor biology , institute for cancer research , division of cancer medicine , oslo university hospital , the norwegian radium hospital , oslo , norway 2department of genetics , institute for cancer research , division of cancer medicine , oslo university hospital , the norwegian radium hospital , oslo , norway 3department of informaticsbiomedical informatics , university of oslo , oslo , norway 4k.g. 
mills , olav engebraaten provision of study materials or patients : ingrid hedenfalk , elin borgen , niklas loman , thomas hatschek , anne - lise brresen - dale , bjrn naume , gordon b . 
mills , olav engebraaten manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors thomas hatschek consulting or advisory role : roche , pzer , pierre fabre research funding : roche , pzer travel , accommodations , expenses : roche anne - lise brresen - dale employment : arctic pharma as , pubgene stock and other ownership interests : arctic pharma as gordon b . 
mills stock and other ownership interests : catena , signalchem , tarveda therapeutics , immunomet honoraria : nuevolution : astrazeneca , tarveda therapeutics , tesaro , symphogen , pdx pharmacy , immunomet , lilly consulting or advisory role : astrazeneca , signalchem , tarveda therapeutics , symphogen , takeda / millennium , pdx pharmacy , immunomet , lilly , turbine , ion pharma , zentalis research funding : adelson medical research foundation , astrazeneca , nanostring technologies , breast cancer research foundation , karus therapeutics , pzer , prospect creek foundation , tarveda therapeutics , ions pharmaceuticals , immunomet patents , royalties , other intellectual property : hrd assay to myriad genetics , dsp technology patent with nanostring travel , accommodations , expenses : astrazeneca , pzer , symphogen , chrysalis biomedical advisors , immunomet , michigan primary care consortium gunhild m . 
mlandsmo patents , royalties , other intellectual property : patent application submitted for a nine - protein / gene panel predicting response to anti vegf therapies in combination with chemotherapy olav engebraaten patents , royalties , other intellectual property : patent application pending for a biomarker for antibody drug conjugates , patent application submitted for a nine - protein / gene panel predicting response to anti vegf therapies in combination with chemotherapy no other potential conicts of interest were reported . references folkman j : anti - angiogenesis : new concept for therapy of solid tumors . 
 haugen et al roviello g , bachelot t , hudis ca , et al : the role of bevacizumab in solid tumours : a literature based meta - analysis of randomised trials . 
maru d , venook ap , ellis lm : predictive biomarkers for bevacizumab : are we there yet ? clin cancer res 19 : 2824 - 2827 , 2013 gampenrieder sp , westphal t , greil r : antiangiogenic therapy in breast cancer . 
miles d , cameron d , bondarenko i , et al : bevacizumab plus paclitaxel versus placebo plus paclitaxel as rst - line therapy for her2 - negative metastatic breast cancer ( meridian ) : a double - blind placebo - controlled randomised phase iii trial with prospective biomarker evaluation . 
eur j cancer 70 : 146 - 155 , 2017 janning m , m uller v , vettorazzi e , et al : evaluation of soluble carbonic anhydrase ix as predictive marker for efcacy of bevacizumab : a biomarker analysis from the geparquinto phase iii neoadjuvant breast cancer trial . 
fasching pa , loibl s , hu c , et al : brca1 / 2 mutations and bevacizumab in the neoadjuvant treatment of breast cancer : response and prognosis results in patients with triple - negative breast cancer from the geparquinto study . 
ali m , khan sa , wennerberg k , et al : global proteomics proling improves drug sensitivity prediction : results from a multi - omics , pan - cancer modeling 13 . 
silwal - pandit l , nord s , von der lippe gythfeldt h , et al : the longitudinal transcriptional response to neoadjuvant chemotherapy with and without bevacizumab 8 : 2278 - 2288 , 2018 approach . 
h oglander ek , nord s , wedge dc , et al : time series analysis of neoadjuvant chemotherapy and bevacizumab - treated breast carcinomas reveals a systemic shift in genomic aberrations . 
lu y , ling s , hegde am , et al : using reverse - phase protein arrays as pharmacodynamic assays for functional proteomics , biomarker discovery , and drug development in cancer . 
robin x , turck n , hainard a , et al : proc : an open - source package for r and s + to analyze and compare roc curves . 
loibl s , denkert c : how much information do we really need after neoadjuvant therapy for breast cancer ? j clin oncol 35 : 1029 - 1030 , 2017 25 . 
kazerounian s , duquette m , reyes ma , et al : priming of the vascular endothelial growth factor signaling pathway by thrombospondin - 1 , cd36 , and spleen subtype . 
nat commun 10 : 1600 , 2019 von minckwitz g , eidtmann h , rezai m , et al : neoadjuvant chemotherapy and bevacizumab for her2 - negative breast cancer . 
earl hm , hiller l , dunn ja , et al : efcacy of neoadjuvant bevacizumab added to docetaxel followed by uorouracil , epirubicin , and cyclophosphamide , for women with her2 - negative early breast cancer ( artemis ) : an open - label , randomised , phase 3 trial . 
garcia j , hurwitz hi , sandler ab , et al : bevacizumab ( avastin ( r ) ) in cancer treatment : a review of 15 years of clinical experience and future outlook . 
robert nj , di eras v , glaspy j , et al : ribbon - 1 : randomized , double - blind , placebo - controlled , phase iii trial of chemotherapy with or without bevacizumab for rst - line treatment of human epidermal growth factor receptor 2 - negative , locally recurrent or metastatic breast cancer . 
delaloge s , p erol d , courtinard c , et al : paclitaxel plus bevacizumab or paclitaxel as rst - line treatment for her2 - negative metastatic breast cancer in a multicenter national observational study . 
 protein signature predicts response to chemotherapy plus bevacizumab appendix patients assigned to treatment with chemotherapy with or without bevacizumab ( n = 138 ) patients randomly assigned to chemotherapy ( ctx ) ( n = 67 ) patients randomly assigned to chemotherapy and bevacizumab ( bev plus ctx ) ( n = 71 ) ineligible ( n = 1 ) ineligible ( n = 2 ) discontinued treatment ( n = 2 ) patients included in primary endpoint analysis ( n = 66 ) patients included in primary endpoint analysis ( n = 66 ) start of treatment ( n = 55 ) start of treatment ( n = 54 ) fig a1 . 
mixed distribution of protein expression variances ( solid green and blue lines ) ; only proteins with variance above intersection ( red line ) were selected for input in lasso regression to determine the virp ( appendix table a3 )  . 
 protein signature predicts response to chemotherapy plus bevacizumab relative tumor size ( p = .003 ) bevacizumab ( p = .815 ) pam50 ( p < .001 ) bevacizumab relative tumor size pam50 basal her2 luma lumb normal 150% fig a4 . 
rho - values derived from the spearman correlation of protein expression to relative tumor size for patients receiving the bev plus ctx combination ( x - axis ) and ctx treatment only ( y - axis ) with rst principal component of relation between the treatment arms ( stitched red line )  . 
in silico simulation with ingenuity pathway analysis ( version 47547484 , qiagen ) was used to predict biologic effects ( lower row ) affected by the virp member proteins ( second bottom row ) when inuenced by bevacizumab treatment ( top row ) through a set of intermediate proteins ( second top row )  . 
 ( c ) virp scores in rcb low ( 0 and i ) and high ( ii and iii ) patients . ( d ) fraction of patients obtaining pcr when treated with standard ctx in all neoava patients ( n = 55 ) or selected based on virp score ( n = 17 )  . 
 ( e ) fraction of patients obtaining low rcb ( 0 and i ) when treated with standard ctx in all neoava patients ( n = 55 ) or selected based on virp score ( n = 17 )  . 
 tissue / site - agnostic study of ribociclib for tumors with cyclin dcdk4 / 6 pathway genomic alterations : a phase ii , open - label , single - arm basket study julio peguero , md1 ; davendra p . 
grilley - olson , md6 ; vivek subbiah , md7 ; lincoln nadauld , md , phd8 ; das purkayastha , phd9 ; erica stealey , ms9 ; alejandro d . 
kang , phd9 ; and joseph paul eder , md10 purpose as part of the novartis signature program , this study evaluated the efcacy of ribociclib ( selective cyclin - dependent kinase 4 / 6 [ cdk4 / 6 ] inhibitor ) in patients with cyclin dcdk4 / 6 pathwayaberrant tumors . methods this was a phase ii , single - arm , signal - seeking study in patients with advanced malignancies that had progressed on or after standard treatment . 
clinical benet ( dened as the proportion of patients with response or stable disease at 16 weeks ) was the primary end point . results from 61 centers in the united states , 106 patients ( median age , 62.5 years ) were enrolled across multiple malignancies . 
in patients with solid tumors , the clinical benet rate was 18.1% ( n = 19 of 105 ) and the overall response rate was 2.9% ( n = 3 of 105 ) ; three partial responses occurred in patients with adenocarcinoma ( unknown primary ) , soft tissue sarcoma , and urothelial carcinoma . 
2019 by american society of clinical oncology introduction cyclin - dependent kinases 4 and 6 ( cdk4 / 6 ) , in conjunction with their protein regulator cyclin d1 ( encoded by ccnd1 ) , phosphorylate the retinoblastoma ( rb ) protein and regulate g1 cell - cycle progression . 
the p16 ( cdkn2a ) and p15 ( cdkn2b ) tumor suppressors and negaproteins are critical tive regulators of this pathway , mediating cellular senescence.1 , 2 arf ( p19 ) plays an important role in the p53 / mouse double minute 2 homolog ( mdm2 ) - dependent checkpoint ; arf expression leads to activation and stabilization of p53 by promoting mdm2 degradation.3 , 4 pathway alteration can result including from multiple mechanisms , rb mutation or loss , increased signaling through cdk4 and cdk6 amplication , cyclin d1 overexpression , and loss of inhibitors.1 cdk pathway alterations contribute to tumor proliferation and genomic instability , and have been linked to poorer clinical outcomes in multiple cancers . 
cdk4 / 6 and cdkn2a / b abnormalities were reported in 22.8% of patients with various cancers who underwent molecular proling by targeted next - generation sequencing.2 ribociclib is a selective cdk4 / 6 inhibitor that induces dose - dependent rb dephosphorylation , reducing cell proliferation via g1 arrest . 
ribociclib is approved in combination with an aromatase inhibitor as initial endocrine - based therapy for postmenopausal women with hormone receptorpositive / human epidermal growth factor receptor 2 ( her2 ) - negative advanced or metastatic breast cancer . 
 peguero et al context key objective to determine if tumor cyclin dcyclin - dependent kinase 4 / 6 ( cdk4 / 6 ) pathway alterations are an effective signal of efcacy for treatment with ribociclib , a selective cdk4 / 6 inhibitor . knowledge generated in this phase ii , signal - seeking study , 105 patients with heavily pretreated cyclin dcdk4 / 6 pathwayaberrant solid tumors received ribociclib treatment . 
although the primary end point of the study was not met , the clinical benet rate was 18.1% , with partial responses observed in three patients with single gene amplications in the cyclin dcdk4 / 6 pathway . relevance a deeper understanding of the molecular context of tumors is required to accurately match patients to ribociclib , either as a monotherapy or in combination with agents that target interconnected signaling pathways . we present ndings from a signal - seeking study that assessed ribociclib as part of the novartis signature program ( clinicaltrials.gov identier : nct02187783 ) , comprising a series of signal - nding basket protocols in which different single - agent targeted therapies were investigated in a tissueor site - agnostic indication , mutation - specic manner.6 , 7 in this study , patients preidentied as having cdk4 / 6 pathwayaberrant tumors were matched to ribociclib monotherapy . methods study design this was a phase ii , open - label , singletreatment arm , basket study in patients with solid tumors or hematologic malignancies with cdk4 / 6 pathway alterations and whose disease had progressed on or after standard treatment . patients received ribociclib 600 mg , once daily , on a 3week - on / 1 - week - off schedule until disease progression ( by investigator assessment ) , unacceptable toxicity , death , or discontinuation from study treatment ( eg , consent withdrawal , starting a new antineoplastic therapy , investigator decision )  . 
ribociclib dose was reduced to 400 mg , and then to 200 mg if an additional dose reduction was required ; ribociclib dose could not be reescalated . the primary end point was the clinical benet rate assoinvestigator asciated with ribociclib treatment by local sessment , dened as the proportion of patients achieving a complete response ( cr ) , partial response ( pr ) , or stable disease ( sd ) at 16 weeks or later by response evaluation criteria in solid tumors 1.1. 
patients who discontinued treatment before week 16 for reasons other than progressive disease ( pd ) were considered nonevaluable ; patients who had pd before week 16 were considered to have no clinical benet . 
overall response rate ( orr ; ie , cr or pr ) by local investigator assessment was the key secondary end point . patient eligibility criteria patients at least 18 years old with solid tumors or hematologic malignancies with cdk4 / 6 mutations or amplications , cyclin - d1 / d3 gene amplications , or p16 gene mutations or loss were enrolled . 
patients had at least one prior treatment of their recurrent , metastatic , and / or locally advanced disease and had no remaining standard therapy options expected to result in durable response . 
because of overlapping disease - oriented studies , the following tumor types were excluded : breast cancer ( except triple negative ) , liposarcoma , teratoma , castrate - resistant prostate cancer , melanoma , and mantle cell lymphoma . 
all patients gave written informed consent before treatment , according to federal and local stitutional guidelines . genomic proling gene aberrations were assessed in a local clinical laboratory improvement amendmentscertied laboratory before patient consent . 
once a patient was identied , vestigators contacted novartis study enrollment consideration.8 post hoc broad molecular proling using a next - generation dna sequencing panel of more than 280 cancerassociated genes9 was performed centrally on pretreatment tumor biopsy specimens from all patients . 
 mutation - specic , tissue / site - agnostic study of ribociclib statistical analysis tumor cohorts were formed when four or more patients with a particular tumor type were enrolled . 
the number of patients enrolled per tumor type was based on a patientsparing , adaptive design , allowing early closure of nonresponding groups and / or groups with early success . clinical benet was evaluated using a bayesian adaptive statistical design that allowed dynamic borrowing of information across cohorts on the basis of their degree of similarity.8 the statistical design used a hierarchical model that allowed dynamic borrowing of information between groups . 
this design allowed analyses with small sample sizes for each cohort . at least 10 patients were required to evaluate early futility , and at least 15 to evaluate early success and make a go or no - go decision for each tumor type cohort . 
if there was an 80% or greater probability that the response rate exceeded the historical rate , the group was considered a success . orr , pr , or cr on the basis of local investigator assessment were summarized , and 95% exact cis using the clopperpearson method were also reported.8 correlation between a specic genomic ( somatic ) dna prole , based laboratory analysis , and clinical benet was on central explored . 
in addition , ribociclib safety and tolerability were reported . results patients in total , 106 patients from 61 centers in the united states were enrolled across multiple malignancies between august 25 , 2014 , and may 4 , 2015 . 
of the 105 patients with solid tumors , 19 ( 18.1% ) achieved clinical benet ( primary end point ) ; the orr ( key secondary end point ) was 2.9% ( three prs ; table 2 )  . 
the rst patient , a 59 - year - old man with poorly differentiated adenocarcinoma of unknown primary with cdk6 amplication , achieved a pr ( 100% reduction in target lesions ) after the fourth cycle of ribociclib and was continuing therapy at last data cutoff with a duration of response of + 796 days . 
the second patient , a 25year - old man with poorly differentiated , round blue cell soft tissue sarcoma not otherwise specied with cdk4 amplication , had a pr ( 100% reduction ) after two cycles of ribociclib , with a duration of response of 330 days . 
the third patient , a 53 - year - old man with urothelial carcinoma of the bladder with ccnd1 amplication , also experienced a pr ( 37.5% reduction ) after two cycles of ribociclib , with a duration of response of 254 days . 
his bladder cancer had not responded to either rst - line cisplatin plus gemcitabine combination or apatorsen ( ogx - 427 ) an experimental antisense inhibitor targeting heat shock protein 27 . 
one patient with papillary serous ovarian carcinoma with cdk6 mutation ( not conrmed by central review ) experienced a tumor burden reduction of at least 30% , without subsequent conrmation , and had sd for 239 days . 
 ( % ) unless otherwise indicated . abbreviations : ecog , eastern cooperative oncology group ; ps , performance status . * gastroesophageal junction ( n = 4 ) , head and neck nonsquamous cell carcinoma ( n = 4 ) , unknown primary site ( n = 4 ) , uterus ( n = 4 ) , esophagus ( n = 3 ) , ovarian ( n = 3 ) , skin nonmelanoma ( n = 3 ) , cholangiocarcinoma ( n = 2 ) , colorectal ( n = 2 ) , renal cell carcinoma ( n = 2 ) , liver ( n = 2 ) , neuroendocrine ( n = 2 )  . 
other histologies ( n = 10 ) only included one each . based on local molecular proling . a patient can be counted in multiple categories ; therefore , the overall count can be  . 
100. 4 2019 by american society of clinical oncology there was no association between any specic genomic ( somatic ) dna prole and clinical benet per central analysis ( fig 3 )  . 
however , 18 of 19 patients ( 94.7% ) with clinical benet had tumors with a single hit in the cyclin d - cdk4 / 6 pathway and one patient with clinical benet had multiple mutational events ; patients with a pr had tumors with cdk4 / 6 or ccnd1 amplication . 
in total , 86 patients had a single cyclin dcdk4 / 6 pathway mutation and 10 patients had multiple pathway mutations ; central mutation analysis was not available in nine patients . 
the most common aes suspected to be related to the study drug included fatigue ( 31.1% ) , neutropenia ( 30.2% ; decreased neutrophils , 15.1% ) , and nausea ( 29.2% ; table 3 )  . 
of these , fatigue and nausea were typically mild . the most common grade 3 / 4 ae suspected to be related to the study drug was neutropenia ( 18.9% ; n = 20 ; decreased neutrophils , 7.5% , n = 8 ) ; only one incident of febrile neutropenia ( grade 3 ) occurred , but it was effectively managed with supportive care and antibiotics . 
 mutation - specic , tissue / site - agnostic study of ribociclib screened ( n = 176 ) enrolled ( n = 106 ) excluded * unacceptable laboratory value unacceptable test result unacceptable medical history intercurrent medical event did not meet diagnostic / severity criteria use of excluded medications patient withdrew consent other ( n = 70 ) ( n = 22 ) ( n = 15 ) ( n = 6 ) ( n = 6 ) ( n = 7 ) ( n = 8 ) ( n = 2 ) ( n = 10 ) still receiving study drug ( n = 1 ) discontinued disease progression patient / guardian decision adverse event death protocol deviation physician decision ( n = 105 ) ( n = 80 ) ( n = 9 ) ( n = 8 ) ( n = 3 ) ( n = 3 ) ( n = 2 ) consented for survival follow - up ( n = 89 ) death lost to follow - up ( n = 58 ) ( n = 31 ) fig 1 . 
as such , more patients have been identied with potentially actionable pathway activation , raising the hope of personalized medicine through targeted drug treatment.10 , 11 the design of basket trials enables molecularly driven enrollment and treatment assignment , with simultaneous testing of a single targeted agent in tumors of different histologies that harbor genetic alterations in the targeted pathway.12 - 14 the signature program was built on this approach and included eight agentspecic , signal - seeking phase ii basket trials of various targeted therapies in a histology - agnostic , mutation - specic manner , matching patients with potentially actionable mutations to the corresponding agent . 
the aim of the signature program was to support or refute the hypothesis that driver mutations determine therapeutic response to treatment in a histology - agnostic manner . this tissueor site - agnostic approach was recently evaluated in patients with solid tumors and led to the us food and drug administration granting accelerated approval to pembrolizumab for the rst tissueor site - agnostic indication.15 indeed , the prospects for the tissueor siteagnostic approach in oncology have never looked brighter , as the successful stories of larotrectinib and entrectinib continue to unfold.16 moreover , the food and drug administration has also recently granted marketing approval to several diagnostics based on next - generation sequencing : the praxis extended ras panel ( illumina , san diego , ca ) , the oncomine dx target test ( thermo fisher scientic , waltham , ma ) , and the foundationone cdx ( foundation medicine , cambridge , ma ) .17 - 19 these tests , and others under clinical investigation , could identify which patients with pathway - aberrant tumors may benet from targeted treatment options . 
thus , the aim for the present ribociclib signal - seeking study was to match patients with cdk4 / 6 pathway - aberrant tumors with the selective cdk4 / 6 inhibitor ribociclib . the primary end point of the study was the rate of clinical benet associated with ribociclib monotherapy , based on local investigator assessment . 
prs were observed in three patients , and 16 patients had sd for 16 weeks or longer . two patients with pr had the maximum percentage reduction from baseline in target lesions ( 100% )  . 
unlike the mutations affecting rb activation , cdkn2a mutation or loss concomitantly decreases the critical function of the p53 - stabilizing effect of the arf protein on the ubiquitin ligase mdm2 , mimicking many mutations in tp53 . 
other selective cdk4 / 6 inhibitors , palbociclib and abemaciclib , have shown low - rate monotherapy activity outside of breast cancer.20 - 23 similarly , a few extraordinary long - term responses have been observed with these agents in tumors with ccnd1cdk4 / 6 amplications . 
these observations may be partly due to the molecular context of the tumor . cyclin dcdk4 / 6 pathway activity is regulated by a number of mitogenic signals , including epidermal growth factors , signaling through the her2 and estrogen receptors , and the phosphoinositide 3 - kinase ( pi3k ) akt pathway.24 studies have suggested that response to targeted therapy may be affected by the presence of tumor mutations in related signaling pathways . 
 mutation - specic , tissue / site - agnostic study of ribociclib chromosome head and neck nonsquamous - cell carinoma head and neck squamous - cell carinoma adrenals bladder breast , triple negative cervix cholangiocarcinoma chordoma colorectal esophagus gallbladder ducts gastroesophageal junction gist kidneys liver lung , nonsmall - cell nonadenocarcinoma lung , nonsmall - cell adenocarcinoma lung , nonsmall - cell squamous lymphoma mesothelioma neuroendocrine ovarian pancreas penile prostate salivary gland sarcoma skin , nonmelanoma thyroid unknown primary uterus shrinkage mutation amplication loss rearrangement short variant fig 3 . 
the most common grade 3 / 4 aes suspected to be related to ribociclib was neutropenia ( 18.9% ) and decreased neutrophils ( 7.5% ) ; only one incident of febrile neutropenia ( grade 3 ) was reported , which was effectively managed with supportive care and antibiotics . 
no deaths due to aes were suspected to be related to study drug per the investigator . one study limitation was the restriction of cohorts by tumor type rather than molecular subtype . 
a second limitation was the exclusion of the most obvious indications for single - agent cdk4 / 6 inhibition : hormone - sensitive breast cancer , liposarcoma , teratoma , castrate - resistant prostate cancer , melanoma , and mantle cell lymphoma . 
this exclusion was due to overlapping disease - oriented studies in these indications , which already were in place by the time this tissueor site - agnostic study started . in conclusion , although no tumor - type cohort graduation was observed , preliminary activity was seen in tumors with single - hit ccnd1cdk4 / 6 amplications . 
these ndings investigation and suggest that in the warrant additional absence of ccnd1cdk4 / 6 amplications or in patients with multiple mutational events , cdk4 / 6 inhibition may require combination with other agents that provide additive or synergistic effects . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . julio peguero employment : oncology consultants leadership : oncology consultants davendra p . 
sohal honoraria : foundation medicine consulting or advisory role : perthera , ability pharma research funding : novartis ( inst ) , celgene ( inst ) , oncomed ( inst ) , bayer ( inst ) , genentech ( inst ) , bristol - myers squibb ( inst ) , agios ( inst ) , incyte ( inst ) , loxo ( inst ) bert h . 
oneil employment : eli lilly consulting or advisory role : bristol - myers squibb , merck karen kelly honoraria : merck consulting or advisory role : astrazeneca , roche , eli lilly , regeneron , abbvie , janssen , emd serono , merck , pzer , astrazeneca research funding : emd serono ( inst ) , genentech ( inst ) , abbvie ( inst ) , five prime therapeutics ( inst ) , regeneron ( inst ) patents , royalties , other intellectual property : author royalties for uptodate travel , accommodations , expenses : astrazeneca , roche , merck , regeneron , eli lilly , abbvie , emd serono , merck juneko e . 
grilley - olson consulting or advisory role : bayer , chimerix research funding : nanocarrier ( inst ) , genentech ( inst ) , seattle genetics ( inst ) , medimmune ( inst ) , pzer ( inst ) vivek subbiah consulting or advisory role : medimmune research funding : novartis ( inst ) , glaxosmithkline ( inst ) , nanocarrier ( inst ) , northwest biotherapeutics ( inst ) , roche ( inst ) , berg pharma ( inst ) , bayer ( inst ) , incyte ( inst ) , fujilm ( inst ) , pharmamar ( inst ) , d3 oncology solutions ( inst ) , pzer ( inst ) , amgen ( inst ) , abbvie ( inst ) , multivir ( inst ) , blueprint medicines ( inst ) , loxo ( inst ) , vegenics ( inst ) , takeda ( inst ) , alfasigma ( inst ) , agensys ( inst ) , idera ( inst ) , boston biomedical ( inst ) , inhibrx ( inst ) , exelixis ( inst ) travel , accommodations , expenses : pharmamar , bayer lincoln nadauld stock and other ownership interests : toma biosciences , citizen corporation consulting or advisory role : roche speakers bureau : genentech patents , royalties , other intellectual property : navican travel , accommodations , expenses : roche , chugai pharma das purkayastha employment : novartis pharmaceuticals erica stealey employment : novartis stock and other ownership interests : novartis alejandro d . 
clin cancer res 21 : 2905 - 2910 , 2015 kato s , schwaederle m , daniels ga , et al : cyclin - dependent kinase pathway aberrations in diverse malignancies : clinical and molecular characteristics . 
cell cycle 14 : 1252 - 1259 , 2015 zhang y , xiong y , yarbrough wg : arf promotes mdm2 degradation and stabilizes p53 : arf - ink4a locus deletion impairs both the rb and p53 tumor suppression pathways . 
cell 92 : 725 - 734 , 1998 ozenne p , eymin b , brambilla e , et al : the arf tumor suppressor : structure , functions and status in cancer . 
n engl j med 375 : 1738 - 1748 , 2016 kang bp , slosberg e , snodgrass s , et al : the signature program : bringing the protocol to the patient . 
n engl j med 377 : 62 - 70 , 2017 slosberg ed , kang bp , peguero j , et al : signature program : a platform of basket trials . 
oncotarget 9 : 21383 - 21395 , 2018 frampton gm , fichtenholtz a , otto ga , et al : development and validation of a clinical cancer genomic proling test based on massively parallel dna sequencing . 
lopez - chavez a , thomas a , rajan a , et al : molecular proling and targeted therapy for advanced thoracic malignancies : a biomarker - derived , multiarm , multihistology phase ii basket trial . 
patnaik a , rosen ls , tolaney sm , et al : efcacy and safety of abemaciclib , an inhibitor of cdk4 and cdk6 , for patients with breast cancer , non - small cell lung cancer , and other solid tumors . 
goldman jw , shi p , reck m , et al : treatment rationale and study design for the juniper study : a randomized phase iii study of abemaciclib with best supportive care versus erlotinib with best supportive care in patients with stage iv non - small - cell lung cancer with a detectable kras mutation whose disease has progressed after platinum - based chemotherapy . 
dickson ma , schwartz gk , keohan ml , et al : progression - free survival among patients with well - differentiated or dedifferentiated liposarcoma treated with cdk4 inhibitor palbociclib : a phase 2 clinical trial . 
edelman mj , redman mw , albain ks , et al : a phase ii study of palbociclib ( p ) for previously treated cell cycle gene alteration positive patients ( pts ) with stage iv squamous cell lung cancer ( scc ) : lung - map sub - study swog s1400c . 
ther adv med oncol 10 : 1 - 15 , 2018 janku f , hong ds , fu s , et al : assessing pik3ca and pten in early - phase trials with pi3k / akt / mtor inhibitors . 
kim s , tiedt r , loo a , et al : the potent and selective cyclin - dependent kinases 4 and 6 inhibitor ribociclib ( lee011 ) is a versatile combination partner in preclinical cancer models . 
 circulating cell - free tumor dna in the management of double primary tumors introduction synchronous lung and colorectal cancer ( crc ) occurrence in one individual is a rare event.1 - 3 the genomics of both malignancies have been extensively studied and found to be distinct . 
 broad molecular profiling in advanced non small - cell lung cancer is widely accepted and routinely used in selecting targeted therapy , because response rates ( rrs ) to single targeted agents are twoto three - fold higher than those to cytotoxic chemotherapy.4 - 11 in advanced crc , potentially targeted alterations are mostly detected in the braf gene ; however , the rr in crc is typically worse than in nonsmall - cell lung cancer ( rr range in crc , 10% to 15% )  . 
 in addition , in crc , expanded testing of the kras and nras genes is used as a negative predictor of antiepidermal growth factor receptor ( egfr ) antibody treatment.12 circulating cell - free tumor dna ( ctdna ) sequencing is a blood - based test , also known as liquid biopsy , which is currently widely used for tumor genomic profiling.13 in contrast to tissue biopsy , ctdna sequencing is a noninvasive approach and thus especially valuable where the tumor tissue obtained is insufficient for molecular testing . 
most importantly , liquid biopsy may provide a global summary of intraand intertumor heterogeneity.14 guardant 360 ( guardant health , redwood city , ca ) is a liquid biopsy test based on digital sequencing technology . 
 the clinical sensitivity and specificity of this test are comparable to those of tissue - based next - generation sequencing ( ngs ; analytic specificity of > 99.9999% at the per - nucleotide level ) .15 absolute variant allele fractions , including egfr and braf single - nucleotide variants , have been shown to tightly correlate with a well - validated orthogonal digital droplet polymerase chain reactionbased plasma analysis.16 we present a case of a patient with synchronous lung and colorectal cancers driven by two different oncogenic drivers ( egfr l858r and braf v600e for the lung and colorectal tumors , respectively )  . 
whereas the incidence of the abovementioned molecular aberrations in these tumor types ranges from 10% to 20% , 17 - 19 it is rare to discover both in the same individual . 
biopsy of the colonic mass confirmed a cdx2 - positive adenocarcinoma compatible with primary colonic orig positron emission tomographycomputed tomography scan showed the abovementioned tumor in the right colon and also demonstrated metastatic disease in the bones and a speculated right upper lobe ( rul ) lesion with an atelectatic component compatible with a second primary lung tumor ( fig 1a )  . 
a biopsy of both the rul lesion and the t9 lytic mass confirmed the diagnosis of metastatic lung adenocarcinoma in both tumor specimens ; tumor cells were positive for ttf1 and ck7 and negative for cdx2 and ck20 . because the rul lesion and the vertebral metastasis had insufficient tumor tissue for elizabeth dudnik tal twito iris faull addie dvir lior soussan - gutman ofer purim richard b . 
 ( a ) positron emission tomographycomputed tomography scan demonstrating metastatic disease in the spiculated right upper lobe ( rul ) lesion with an atelectatic component compatible with a second primary lung tumor . 
targeted ngs of the ctdna identified two driver mutations : egfr l858r at 2.4% mutant allele fraction ( maf ) and braf v600e mutation at 0.9% maf ( fig 2a )  . 
after the ctdna analysis , the tumor specimen obtained from the bone lesion was reviewed , and polymerase chain reaction analysis for egfr ( amoydx egfr 29 mutations detection ce - ivd kit ; xiamen , peoples republic of china ) was performed , confirming the presence of egfr l858r mutation in the bone metastasis . 
the origin of the braf v600e clone at that point remained unclear . when the patient was about to start treatment with anti - egfr therapy , she was diagnosed with acute large - bowel obstruction and underwent extended right hemicolectomy , which revealed moderately differentiated colonic adenocarcinoma infiltrating the pericolic fat with three involved regional lymph nodes and positive surgical margins ( pt3n2r1 )  . 
soon thereafter , the patient developed a large pericardial effusion , and pericardial window was performed , revealing no viable tumor in the pericardial tissue specimen . erlotinib was initiated , resulting in a significant shrinkage of the rul mass ; however , new liver metastases developed ( fig 1b )  . 
this time , braf testing as well as kras and nras testing was performed ( amoydx ce - ivd kit ) , revealing that the tumor was kras wild type and nras wild type and harbored a braf v600e mutation . after the diagnosis of metastatic crc , in march 2016 , capecitabine was added to erlotinib . 
although not known as driver mutations , these mutations are more common in crc than in lung cancer , and their mafs increased from the initial test to that performed after the patient received egfr inhibitors ( fig 2b )  . after the second ctdna testing , the treatment was switched to a combination of vemurafenib and erlotinib.20 - 22 however , the treatment was poorly tolerated ( severe diarrhea ) and was therefore discontinued after 1 month . 
previous genetic studies of crc have identified somatic mutations in four main candidate pathways : tgf - , wnt , p53 , and rtk - ras , all of which were identified as important contributors to crc development.29 among them , mutations in the smad4 , apc , fbxw7 , and braf genes were identified as recurrent mutations in patients with crc.29 - 31 in contrast , apc was suggested to be a gatekeeper , which regulates epithelial cells to develop into adenoma and even carcinoma cells.32 notably , not only did liquid biopsy detect both tumor driver mutations at the time of initial diagnosis , it also reflected the progression of the second primary colon tumor , early in the course of the disease , even before its growth was shown in imaging . 
a rise in the maf of braf v600e , a driver mutation , as well as other crc - associated mutations ( smad4 , apc , fbxw7 ) , alongside a drop in the maf of the egfr l858r mutation , suggested that the new retroperitoneal lesions were related to the crc . 
this finding is consistent with other reports showing how ctdna allele fractions serve as early indicators of response versus progression in earlyand late - stage crc.33 , 34 later on , liquid biopsy allowed simultaneous response monitoring of both tumors . 
however , it should be emphasized that for clinical application of monitoring with serial testing , the assay used must be validated not only for detecting mutations but also for accurate quantitation of variant allele fractions . 
our patient did not benefit from the braf inhibitor , possibly because single - agent braf inhibitor yields poor results in patients with crc with braf mutations.12 therefore , inhibition of multiple pathways and immunotherapy should have been considered.35 although tumor mutational burden was not assessed here , analysis of blood - derived ctdna demonstrated high mutation in november 2015 as well as in june 2016 ( fig 2 )  . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . elizabeth dudnik consulting or advisory role : boehringer ingelheim speakers bureau : boehringer ingelheim , roche , astrazeneca , pfizer , merck sharp & dohme , bristol - myers squibb , novartis research funding : boehringer ingelheim ofer purim no relationship to disclose richard b . 
lanman employment : guardant health , veracyte leadership : guardant health , biolase stock and other ownership interests : guardant health , biolase research funding : guardant health affiliations elizabeth dudnik and ofer purim , davidoff cancer center , rabin medical center , petach tikva ; tal twito , addie dvir , and lior soussan - gutman , teva pharmaceuticals industries , shoam , israel ; and iris faull and richard b . 
yun hr , yi lj , cho yk , et al : double primary malignancy in colorectal cancer patients : msi is the useful marker for predicting double primary tumors . 
chiang jm , yeh cy , changehien cr , et al : clinical features of second other - site primary cancers among sporadic colorectal cancer patients : a hospital - based study of 3 , 722 cases . 
zhou c , wu yl , chen g , et al : erlotinib versus chemotherapy as first - line treatment for patients with advanced egfr mutation - positive non - small - cell lung cancer ( optimal , ctong - 0802 ) : a multicentre , open - label , randomised , phase 3 study . 
sequist lv , yang jc , yamamoto n , et al : phase iii study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with egfr mutations . 
mazires j , barlesi f , filleron t , et al : lung cancer patients with her2 mutations treated with chemotherapy and her2 - targeted drugs : results from the european euher2 cohort . 
paik pk , drilon a , fan pd , et al : response to met inhibitors in patients with stage iv lung adenocarcinomas harboring met mutations causing exon 14 skipping . 
sarfaty m , moore a , neiman v , et al : ret fusion lung carcinoma : response to therapy and clinical features in a case series of 14 patients . 
prez - callejo d , romero a , provencio m , et al : liquid biopsy based biomarkers in non - small cell lung cancer for diagnosis and treatment monitoring . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
ulrich bc , nagy rj , odegaard ji , et al : cross - platform detection and quantification of actionable mutations in cell - free dna shows high concordance and correlation between next - generation sequencing and droplet digital pcr . 
pao w , miller va : epidermal growth factor receptor mutations , small - molecule kinase inhibitors , and non - small - cell lung cancer : current knowledge and future directions . 
sanz - garcia e , martinez am , elez e , et al : survival determinants with matched targeted therapies in braf mutant metastatic colorectal cancer ( mcrc )  . 
barlesi f , mazieres j , merlio jp , et al : routine molecular profiling of patients with advanced nonsmall - cell lung cancer : results of a 1 - year nationwide programme of the french cooperative thoracic intergroup ( ifct )  . 
ohashi k , sequist lv , arcila me , et al : lung cancers with acquired resistance to egfr inhibitors occasionally harbor braf gene mutations but lack mutations in kras , nras , or mek1 . 
blakely cm , watkins tbk , wu w , et al : evolution and clinical impact of co - occurring genetic alterations in advanced - stage egfr - mutant lung cancers . 
kim st , lee ws , lanman rb , et al : prospective blinded study of somatic mutation detection in cell - free dna utilizing a targeted 54 - gene next generation sequencing panel in metastatic solid tumor patients . 
tie j , wang y , tomasetti c , et al : circulating tumor dna analysis detects minimal residual disease and predicts recurrence in patients with stage ii colon cancer . 
we hypothesized that patients with pdac with germline brca1 , brca2 , or palb2 mutations may benefit preferentially from platinum - based chemotherapy . materials and methods twenty - nine individuals with deleterious germline mutations in brca1 , brca2 , or palb2 and a diagnosis of advanced pdac ( mut - positive ) were matched 2 : 1 to patients who were noncarrier or untested ( control ) by age at diagnosis , year of diagnosis , stage , and sex . 
patients were identified via one of two available databases at the university of pennsylvania : the basser center for brca registry or the university of pennsylvania electronic medical patient record . 
2018 by american society of clinical oncology introduction pancreatic ductal adenocarcinoma ( pdac ) is an increasingly prevalent and often rapidly fatal malignancy.1 - 3 even with the relatively recent introduction of gemcitabine plus paclitaxel4 and folinic acid , fluorouracil , irinotecan , and oxaliplatin ( folfirinox ) 5 chemotherapy regimens , the median overall survival for patients with inoperable disease remains abysmal at , 1 year . 
 clinically , breast and ovarian cancers associated with brca1 and brca2 mutations have heightened sensitivity to platinum - based chemotherapies and to parp inhibitors.16 - 22 these observations suggest that patients with pdac due to brca1 , brca2 , or palb2 germline mutations with resulting hrd might also benefit from similar therapeutic strategies . early data have suggested that patients with advanced pdac and a germline brca1 / 2 mutation who received platinum treatment had higher tumor response rates and improved overall survival compared with those who did not receive platinum.23 however , because of their high toxicity , platinum - based therapies such as folfirinox are often reserved for the younger , healthier patients . 
therefore , without an age - matched control group , it is unclear whether the inherent bias to select platinum - based treatment in healthier patients might have affected these results . we performed a retrospective cohort study of patients with unresectable pdac . 
these were matched 2 : 1 with patients who either had not been tested or were known not to have a germline deleterious mutation ( referred to collectively as control ) by age at diagnosis , year of diagnosis , stage at diagnosis , and sex . 
patients were identified one of two ways : enrollment in the university of pennsylvania basser center for brca registry or identification via the university of pennsylvania patient electronic medical record ( emr )  . 
the emr was mined using pennseek , which allows for targeted keyword searches of unstructured or semistructured medical documents . notably , patients who were mut - positive were identified in both the basser registry and the emr . 
data were collected and extracted using the emr , paper records , and patient and family reports , and online using the social security death index . patients diagnosed with either locally advanced or metastatic pdac between january 1 , 1995 , and december 31 , 2016 , were included . 
cases were defined as patients with a known pathogenic germline brca1 , brca2 , or palb2 mutation . controls were either confirmed mutation noncarriers or those who had not been tested . 
collected data included demographics , clinical history , personal history of prior malignancies , family history of cancers , and treatments related to pdac , including surgical interventions , radiation , and systemic chemotherapy . 
the institutional review board at the university of pennsylvania approved this study . overall survival overall survival ( os ) was defined as the time from the date of diagnosis of pdac to the date of death from any cause . 
the os for patients exposed to platinum - based treatments ( including cisplatin , oxaliplatin , or carboplatin and with at least one confirmed dose ) during the course of their disease were compared with the os for patients who had not been exposed to such treatment . 
whenever possible , specific regimen details were recorded . statistical analysis univariate analysis was performed using fishers exact test for dichotomous variables and students t test or wilcoxon rank sum test for normal and nonnormal continuous variables , respectively . hazard ratios ( hrs ) and 95% cis were calculated using the cox proportional hazards model . 
survival curves were compared using the log - rank test . each patient who was mut - positive was matched to two control patients using a greedy - match algorithm24 without replacement on the basis of four patient factors : stage at diagnosis , age at diagnosis , year of diagnosis , and sex . 
all statistical analyses were performed using sas 9.4 ( sas institute , cary , nc )  . results patients and tumor characteristics overall , 29 patients who were mut - positive were identified with the following mutational breakdown : brca1 ( n = 12 ) , brca2 ( n = 15 ) , and palb2 ( n = 2 )  . 
the median age of control patients was 63.5 years ( range , 38 to 82 years )  . demographics are summarized in table 1 . clinical and treatment characteristics of patients of the 87 patients , four mut - positive patients ( 13.8% ) and 12 control patients ( 20.7% ) received no systemic treatment of any kind . 
for patients who were mut - positive and who did receive systemic therapy , 18 of 25 cases ( 72.0% ) received platinum - based therapy during their treatment course : 12 ( 48.0% ) received oxaliplatin , three ( 12.0% ) received cisplatin , two ( 8.0% ) received both oxaliplatin and cisplatin , and the exact regimen was unknown for one patient . among control patients , 28 of 46 ( 60.8% ) received platinum - based therapy : 27 ( 96.4% ) received oxaliplatin , one received cisplatin ( 3.5% ) , and the regimen was unknown for one patient . 
among patients who were mut - positive and for whom this information was known , six ( 33% ) received a parp inhibitor and 12 ( 67% ) did not . 
the median os for patients who were mut - positive and had received platinumbased treatment was undefined ( at a median follow - up of 20.1 months ) , and the median os for control patients was 15.5 months ( fig 1 ; table 2 )  . 
 ( % ) unless otherwise noted . abbreviations ; fhx , family history ; folfirinox , folinic acid , fluorouracil , irinotecan , oxaliplatin ; folfox , folinic acid , fluorouracil , oxaliplatin ; iqr , interquartile range . mutations ( control ) when both receive platinumbased treatment . 
in mut - positive patients , the median os was not yet reached at median follow - up of 20.1 months , and the 1 - year os was 94% . however , when patients who are mut - positive and control patients who did not receive platinum therapy were compared , there was no os difference between groups . 
 ( a - c ) kaplanmeier survival curves demonstrating the overall survival for ( a ) the full cohort , ( b ) patients treated with platinum , and ( c ) patients treated without platinu ( d ) breakdown of hazard ratios for the full cohort of patients . this may suggest that mutational status is predictive of benefit to platinum - based therapies . in mut - positive patients treated with platinum therapy , the regimens were split nearly evenly between folfirinox ( folinic acid , fluorouracil , irinotecan and oxaliplatin ) , folfox ( folinic acid , fluorouracil andoxaliplatin ) , andcisplatin / gemcitabine.subgroup analysis comparing oxaliplatin to cisplatin did not demonstrate a difference between regimens , with curves clearly overlapping . 
it is possible that receiving a platinum agent , regardless of which one , is beneficial to patients with pdac who carry a germline brca1 , brca2 , or palb2 mutation . 
however , because cisplatin and oxaliplatin differ in their mechanisms of action and therefore trigger different dna repair pathways , it is also reasonable to hypothesize that there is a therapeutic difference between these agents in patients with an hrd . 
whether an oxaliplatin - based treatment or a cisplatin - based regimen is superior for this population is an area of active research . one of the key strengths of our study is the fact that we were able to fully match our cohort of patients with and without brca1 , brca2 , or palb2 mutations on age at diagnosis , year of diagnosis , sex , and stage . 
in our subgroup analysis of those who received platinum - based therapy , there continued to be no significant differences between mut - positive and control patients regarding these variables . 
golan et al23 reported an os benefit in patients with pdac with brca mutations who had received platinum , but because this study did not include a comparison with patients who did not carry mutations , it was unclear if the observed benefit was due to patient selection bias ( such as age ) or carrier status . 
our data suggest that patients with brca1 , brca2 , or palb2 mutations and pdac may be more likely to respond to platinum , similar to other cancer types , such as breast and ovarian . our study has several important limitations . 
patients in the registry were less likely to have received all their treatment at the university of pennsylvania , meaning that control patients were more uniformly treated at our single - institution academic center . 
however , we were able to collect treatment data for the vast majority of patients . second , because testing for germline brca1 / 2 and palb2 mutations in patients with pdac has only recently been offered as a standard part of clinical practice , our control group may have inadvertently contained patients who were carriers table 3 . 
comparison of baseline variables for patients receiving and not receiving platinum therapy subgroup controls cases year of diagnosis , median ( iqr ) 2014 ( 2010 - 2015 ) 2014 ( 2011 - 2015 ) patients who received platinum age at diagnosis , mean ( sd ) stage at diagnosis stage iii stage iv patients who received no platinum age at diagnosis , mean ( sd ) males males stage at diagnosis stage iii stage iv 57 ( 10 ) 17 ( 61 ) 7 ( 25 ) 21 ( 75 ) 67 ( 9 ) 15 ( 50 ) 3 ( 10 ) 27 ( 90 ) note . 
 ( % ) unless otherwise noted . abbreviations : iqr , interquartile range ; sd , standard deviation . 61 ( 9 ) 8 ( 44 ) 2 ( 11 ) 16 ( 89 ) 65 ( 14 ) 7 ( 64 ) 3 ( 27 ) 8 ( 73 ) year of diagnosis , median ( iqr ) 2013 ( 2009 - 2015 ) 2006 ( 2002 - 2014 ) .053 of brca or other homologous recombination deficiencies . 
however , if patients with germline brca1 , brca2 , or palb2 mutations had unintentionally been included in our control group , this would have plausibly biased toward the null hypothesis . a third limitation is potential survival bias due to the timing of brca or palb2 testing . 
it is possible that patients who survive their pdac diagnosis longer are more likely to undergo genetic testing . prospective cohorts are needed to overcome potential survival bias . our findings have several implications . 
given that patients who are mut - positive may have a substantial survival benefit when treated with platinumbased therapy , it might be beneficial for such patients to be selected for this treatment strategy up front when presenting with inoperable pdac . second , on the basis of our data , efforts should be made to maximize the identification of brca1 , brca2 , and palb2 mutation carriers either by expanding up - front germline testing or by confirming germline status when somatic sequencing identifies a mutation . 
although clinical clues , such as ashkenazi ancestry , a family history of brcaassociated malignancies , or a personal history of other cancers , can be helpful and in themselves may predict for response to platinum agents , 25 not all of the patients in our small cohort had these features . perhaps most importantly , our findings provide further evidence that pdac in patients with germline brca1 , brca2 , or palb2 mutations may represent a fundamentally biologically different group compared with sporadic pdac , suggesting that clinical trial and drug development strategies for this cohort should perhaps be different than for the general population of patients with pdac . indeed , clinical trials using parp inhibitors for patients with pdac and a brca1 , brca2 , or palb2 mutation are currently underway ( clinicaltrials.gov identifiers : nct03140670 and nct02184195 )  . it is unknown whether our observation would extend to patients with somatic alterations in brca1 , brca2 , or palb2 and whether allelespecific loss of heterozygosity would be required to observe the same biologic effect . 
at risk case control 17 time ( months ) in conclusion , we present a small cohort study in which we observe a substantial survival benefit when patients with advanced pdac and a brca or palb2 mutation are treated with platinumbased therapy . 
rahib l , smith bd , aizenberg r , et al : projecting cancer incidence and deaths to 2030 : the unexpected burden of thyroid , liver , and pancreas cancers in the united states . 
murphy km , brune ka , griffin c , et al : evaluation of candidate genes map2k4 , madh4 , acvr1b , and brca2 in familial pancreatic cancer : deleterious brca2 mutations in 17% . 
blanco a , de la hoya m , osorio a , et al : analysis of palb2 gene in brca1 / brca2 negative spanish hereditary breast / ovarian cancer families with pancreatic cancer cases . 
dann rb , deloia ja , timms km , et al : brca1 / 2 mutations and expression : response to platinum chemotherapy in patients with advanced stage epithelial ovarian cancer . 
isakoff sj , mayer el , he l , et al : tbcrc009 : a multicenter phase ii clinical trial of platinum monotherapy with biomarker assessment in metastatic triple - negative breast cancer . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
robson m , im sa , senkus e , et al : olaparib for metastatic breast cancer in patients with a germline brca mutation . oncol 28 : 1145 - 1153 , 2010 n engl j med 377 : 523 - 533 , 2017 23 . 
 contribution of inherited dna - repair gene mutations to hormone - sensitive and castrate - resistant metastatic prostate cancer and implications for clinical outcome siddhartha yadav , md1 ; steven n . 
couch , phd1 ; and manish kohli , md1 purpose to compare the prevalence of germline mutations in metastatic hormone - sensitive prostate cancer ( mhspc ) and metastatic castrate - resistant prostate cancer ( mcrpc ) and assess the impact of mutations on progression to castration resistance and overall survival . methods targeted sequencing of germline dna from 704 men ( 221 at the time of mhspc and 483 at the time of mcrpc ) enrolled in two advanced prostate cancer registries at mayo clinic between 2003 and 2013 was performed for 21 predisposition genes . 
mutations in atm , brca2 , chek2 , fancm , and tp53 were signicantly enriched ( odds ratio greater than 2.0 ) in the metastatic cohorts compared with reference controls . 
2019 by american society of clinical oncology introduction prostate cancer is a leading cause of cancer in men in the united states and worldwide.1 , 2 approximately 30% of patients experience a relapse with advanced disease after curative - intent local therapy for prostate cancer , 3 , 4 and between 5% and 7% of patients are diagnosed initially with advanced stage . 
however , the majority of patients with metastatic hormonesensitive prostate cancer ( mhspc ) eventually stop responding to adt and progress to metastatic castrate - resistant prostate cancer ( mcrpc )  . 
several factors have been implicated in the progression from mhspc to mcrpc , 5 - 8 but the role of mutations in cancer predisposition genes has not been adequately explored . inherited mutations in cancer predisposition genes , such as brca1 / 2 and other dna repair genes , are detected in approximately 4% of patients with localized prostate cancer and less than 1% of the general population.9 in comparison , 7% to 17% of men with mcrpc carry deleterious germline mutations in cancer predisposition genes , 9 - 13 which suggests a contributory role of these mutations in the progression from localized prostate cancer to mcrpc . 
however , the frequency of germline mutations in patients with mhspc is not known , and whether mutation carrier status can predict rapid progression from hormonal sensitivity to castration resistance is unclear . novel drug combinations with adt in patients with mhspc already have demonstrated efcacy in slowing progression to mcrpc and death , 14 - 16 but currently , interventions are not based on the presence of specic genomic aberrations or mutations . 
 yadav et al context key objective to compare the prevalence of germline mutations in metastatic hormone - sensitive prostate cancer ( mhspc ) and metastatic castrate - resistant prostate cancer ( mcrpc ) and assess the impact of mutations on progression to castration resistance and overall survival . knowledge generated in this analysis of 704 patients with metastatic prostate cancer enrolled in a prospective registry , germline mutations were observed in 9.4% of men , 11.8% of men with mhspc , and 8.3% of men with mcrpc . 
no signicant difference was found in time to progression from the hormonesensitive state to the castrate - resistant state or in overall survival between mutation carriers and noncarriers . relevance these ndings have signicant implications for informing prognosis and designing future clinical trials with targeted agents in patients with mhspc and mcrpc . mcrpc state , the targeting of mutations in dna repair genes with poly ( adp - ribose ) polymerase ( parp ) inhibitors , such as olaparib or platinum - based chemotherapy , 17 - 19 has demonstrated clinical benet . 
because the inuence of dna repair gene defects in mhspc on disease progression has not been determined , the use of parp inhibitors or carboplatin in this subcohort has not been explored . 
in this study , we undertook a comprehensive assessment to study the contributions of dna repair gene defects in mhspc and mcrpc and the implications for clinical outcomes . methods patient selection patients with advanced prostate cancer were derived from two prospective registries at mayo clinic . 
a second registry enrolled patients with mhspc and mcrpc between september 2009 and january 2013 . details of both cohorts have been published previously.20 - 23 a total of 826 patients with either mhspc or mcrpc enrolled in either registry were evaluated for inclusion in this study . 
patients without a germline dna sample or available clinical data were excluded from germline sequencing . patients were stratied into mhspc or mcrpc enrollment cohorts on the basis of hormone sensitivity or castration resistance at the time of enrollment . 
mhspc was dened as the development of metastasis as seen on bone imaging scans or computed tomography scans of the abdomen and pelvis , with or without a rising prostate - specic antigen ( psa ) , and no previous adt in the past 2 years . 
mcrpc was dened as the occurrence of either two serial psa rises during continuous adt measured at least 1 week apart with an absolute psa level greater than 2 ng / ml or detection of imaging - based progressive metastasis , whichever came rst . 
high volume of metastasis was dened as the presence of visceral metastases or four or more bone lesions with one or more beyond the vertebral bodies and pelvis per the e3805 study.14 data on initial diagnosis of localized cancer , including date of diagnosis , t stage , n stage , and gleason score , were collected by review of medical records the time of enrollment . 
for the mcrpc cohort , formation on date of mhspc diagnosis , psa and alkaline phosphatase ( alp ) levels , and volume of disease at mhspc diagnosis were collected by medical record review at the time of enrollment . 
this study was approved by the institutional review board at mayo clinic . sequencing and bioinformatics analysis germline dna from peripheral blood mononuclear cells was enriched for the coding regions and consensus splice sites of 21 cancer predisposition genes ( atm , bard1 , brca1 , brca2 , brip1 , chek2 , fancc , fancm , mre11a , mlh1 , msh2 , msh6 , nbn , palb2 , pten , rad50 , rad51c , rad51d , stk11 , tp53 , and xrcc2 ) using a custom ampliconbased qiaseq panel ( qiagen , hilden , germany )  . pooled libraries ( n = 728 ) were subjected to paired - end 150 - base pair sequencing in a single lane of a hiseq 4000 ( illumina , san diego , ca ) , which corresponds to a median coverage of 200 . 
 germline mutations and outcomes in metastatic prostate cancer detected with patterncnv version 1.1.3.27 annotations were provided through the bior toolkit , 28 which leverages dbnsfp version 3.0 , 29 clinvar , 30 clinical annotation of variants , 31 and population frequencies from genome aggregation database ( gnomad ) 32 and exome aggregation consortium33 nonthe cancer genome atlas controls . 
mutations were viewed with vcf - miner.34 interpretation of variants the pathogenicity of germline variants was determined using a framework consistent with established american college of medical genetics and genomics and association for molecular pathology guidelines.35 only patients with likely pathogenic or pathogenic mutations were classied as mutation carriers . 
in case - control association analysis , mutation frequencies by gene were compared with mutation frequencies for non - finnish whites in gnomad.32 large genomic rearrangements in all genes and gnomad lter non - pass variants were excluded from case - control association studies . 
odds ratios ( ors ) and 95% cis for the strength of association with prostate cancer were estimated by fishers exact test . gene - specic mutation enrichment in the mhspc enrollment cohorts relative to the mcrpc cohort were assessed by logistic regression adjusted for age at mhspc diagnosis and reported as ors with 95% cis . 
multivariable analysis for time to progression to mcrpc and overall survival was performed using a cox proportional hazards regression model that included age , initial presentation , gleason score , t and n stage at initial diagnosis , psa and alp level at mhspc diagnosis , volume of metastasis , and mutation status as covariates . 
seventeen patients who did not receive adt despite a diagnosis of metastatic prostate cancer were excluded from analysis of progression from hormone - sensitive stage to castration resistance but were included in the overall survival analysis . all tests were two - sided , and p , .05 was considered to indicate statistical signicance . local versus advanced disease at results of 728 samples subjected to germline sequencing , 17 failed coverage , and six were identied as duplicates ; one patient was excluded because of a diagnosis of neuroendocrine prostate cancer ( data supplement )  . 
the median duration of follow - up from mhspc diagnosis was 71 months for the entire cohort , 53 months for the mhspc cohort , and 77 months for the mcrpc cohort . 
atm , brca2 , chek2 , fancm , and tp53 mutations were signicantly enriched in patients with prostate cancer relative to controls , with ors ranging from 2.8 for chek2 to 15.9 for tp53 ( table 2 )  . 
for the entire cohort , the median time to progression from mhspc to mcrpc was 23.1 months for mutation carriers and 32.5 months for noncarriers ( p = .96 ; fig 1a )  . 
when the mhspc and mcrpc cohorts were analyzed separately , a signicant difference in the rate of progression from mhspc to mcrpc was not observed between mutation carriers and noncarriers in either cohort ( figs 1b and 1c )  . in univariable cox proportional hazards regression analysis , older age at diagnosis of mhspc , metastatic disease at initial presentation , gleason score greater than 7 , higher psa level , elevated alp level , and high volume of metastatic disease were signicant predictors of mhspc to mcrpc progression ( table 3 )  . 
in multivariable cox proportional hazards regression analysis , older age at diagnosis , gleason score greater than 7 , elevated alp level , and high volume of metastasis remained signicant predictors of progression to castration resistance ( table 3 )  . 
on further analysis by enrollment cohort , mutation status was not associated with progression to castration resistance in multivariable models of the mhspc or mcrpc cohorts ( data supplement )  . impact of germline mutations on overall survival ( 87.0% ) from the mcrpc enrollment cohort . 
no difference in overall survival was found from diagnosis of mhspc between patients with and without mutations ( median , 66 v 84 months ; p = .14 ; fig 2a )  . 
on further subset analysis by enrollment cohort , mutation status was not associated with overall survival in multivariable analysis of the mhspc or mcrpc cohorts ( data supplement )  . discussion this study showed that more than 10% of patients in the mhspc state of advanced prostate cancer harbor a germline mutation in one of the predisposition genes associated with the cellular response to dna damage and that the frequency of germline mutations is not signicantly different between patients with mhspc and mcrpc . 
comparison of the rate of progression from metastatic hormone - sensitive prostate cancer ( mhspc ) to metastatic castrate - resistant prostate cancer ( mcrpc ) between mutation carriers and noncarriers . 
this variant also has been noted to be more frequent in patients with lethal prostate cancer compared with low - risk prostate cancer.38 whether other variants in chek2 confer similar prostate cancer risk is unclear . 
for instance , in the study by pritchard et al , more than 50% of patients had a rst - degree relative with cancer , whereas in the study by castro et al , those with brca2 mutations had the greatest association with a family history of cancer . 
although to our knowledge the current study is the largest in terms of sample size among all reported studies , we did not evaluate family history of cancer , which is a limitation when comparing participating populations across different studies . 
 yadav et al with the general population in this study . in a similar comparison , pritchard et al9 found that six genes ( atm , brca1 , brca2 , chek2 , gen1 , and rad51d ) were signicantly enriched in mcrpc . 
fancm is considered a member of the fanconi anemia ( fa ) family of dna repair genes ( online mendelian inheritance in man 609644 ) , a component of the upstream fa pathway that mediates the assembly of the fa core complex , and a regulator of the s - phase checkpoint upon interstrand crosslink - induced dna damage.41 fancm has been associated with an increased risk of breast cancer.42 - 45 in the current study , three of the seven mutations noted in fancm were due to the deletion of exon 8 , which has not been reported previously to be associated with prostate cancer risk , and hence , needs to be validated in future studies . 
the association between germline tp53 mutations and prostate cancer risk is also a novel nding of this study but needs to be interpreted with caution because of the small number of mutation carriers and the potential for ageand / or therapy - related clonal hematopoiesis events , which can be seen in up to one quarter of tp53 mutation carriers.46 we did not attempt to conrm the mendelian inheritance of these tp53 mutations through analysis of dna from secondary tissue sources , 46 which is a limitation of this and other germline tp53 studies . 
based on the results of our study , additional studies should investigate associations between germline tp53 mutations and risk of prostate cancer while also accounting for clonal hematopoiesis events . germline mutation carriers with localized prostate cancer have been reported to have a higher rate of progression to metastatic disease and an overall poorer prognosis.47 - 52 this is also supported by signicant enrichment of mutation carriers among patients with metastatic prostate cancer compared with patients with localized prostate cancer.9 although mutation carriers are more likely to progress from localized prostate cancer to metastatic prostate cancer , our results suggest the rate of progression to castration resistance or death is not affected by mutation carrier status . 
the biologic mechanisms of metastasis and androgen resistance are distinct from each other.53 , 54 genomic analysis of metastatic prostate tumors has demonstrated that mutation or amplication of the androgen receptor gene is the most enriched alteration in mcrpc compared with mhspc tumors , and mutations in dna repair genes are less common.55 that in recent years , several clinical trials evaluated the addition therapeutic agents to adt in early mhspc of other stage , 14 , 15 , 56 - 58 with the goal of slowing down progression to mcrpc and improving overall survival . 
at present , targeted therapies for patients with dna repair defects , such as parp inhibitors , have been studied only in the mcrpc stage.17 our study suggests that future clinical trials with early addition of parp inhibitors or similar drugs to adt in the mhspc stage may be feasible . 
these somatic alterations in dna repair genes can arise during different stages of disease progression as a result of acquired resistance to therapy or as part of tumor evolution59 even in the absence of germline mutations . 
our study did not evaluate somatic mutations in dna repair genes in corresponding tumor material , but such patients are known to respond to parp inhibitors.17 in addition , parp inhibitors may potentially replace adt in a select group of patients with mhspc because of superior adverse effect proles , as is being evaluated in an ongoing phase ii clinical trial of parp inhibitors instead of adt in patients with mhspc.60 because this study was performed using prospective registries from a single institution , it has several strengths compared with similar prior studies . 
in addition , we did not account for treatment effect , although the majority of patients were enrolled before the publication of studies that established preferential use of docetaxel14 or abiraterone in the mhspc setting15 , 58 and were unlikely to receive these treatments outside a clinical trial . 
however , patients may have received these treatments at progression to castration resistance , which can potentially affect the overall survival analysis . finally , we did not take into account somatic mutations in dna repair genes , which can independently affect outcomes . this study demonstrated that approximately 10% of patients with metastatic prostate cancer harbor germline mutations in cancer predisposition genes , and it identied mutations in ve genes ( atm , brca2 , chek2 , fancm , and tp53 ) that are enriched in patients with advanced prostate cancer . 
couch consulting or advisory role : astrazeneca research funding : grail travel , accommodations , expenses : grail , qiagen other relationship : ambry genetics no other potential conicts of interest were reported . references siegel rl , miller kd , jemal a : cancer statistics , 2018 . 
ca cancer j clin 68 : 7 - 30 , 2018 ferlay j , soerjomataram i , dikshit r , et al : cancer incidence and mortality worldwide : sources , methods and major patterns in globocan 2012 . 
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prostate 74 : 820 - 828 , 2014 karantanos t , corn pg , thompson tc : prostate cancer progression after androgen deprivation therapy : mechanisms of castrate resistance and novel therapeutic approaches . 
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prostate 74 : 225 - 234 , 2014 keto cj , aronson wj , terris mk , et al : obesity is associated with castration - resistant disease and metastasis in men treated with androgen deprivation therapy after radical prostatectomy : results from the search database . 
bju int 110 : 492 - 498 , 2012 pritchard cc , mateo j , walsh mf , et al : inherited dna - repair gene mutations in men with metastatic prostate cancer . 
antonarakis es , lu c , luber b , et al : germline dna - repair gene mutations and outcomes in men with metastatic castration - resistant prostate cancer receiving rst - line abiraterone and enzalutamide . 
pomerantz mm , spis ak s , jia l , et al : the association between germline brca2 variants and sensitivity to platinum - based chemotherapy among men with metastatic prostate cancer . 
liu x , wu c , li c , et al : dbnsfp v3.0 : a one - stop database of functional predictions and annotations for human nonsynonymous and splice - site snvs . 
m unz m , ruark e , renwick a , et al : csn and cava : variant annotation tools for rapid , robust next - generation sequencing analysis in the clinical setting . genome med 7 : 76 , 2015 32 . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
castro e , romero - laorden n , del pozo a , et al : prorepair - b : a prospective cohort study of the impact of germline dna repair mutations on the outcomes of patients with metastatic castration - resistant prostate cancer . 
n aslund - koch c , nordestgaard bg , bojesen se : increased risk for other cancers in addition to breast cancer for chek2 * 1100delc heterozygotes estimated from the copenhagen general population study . 
peterlongo p , catucci i , colombo m , et al : fancm c.5791c.t nonsense mutation ( rs144567652 ) induces exon skipping , affects dna repair activity and is a familial breast cancer risk factor . 
neidhardt g , hauke j , ramser j , et al : association between loss - of - function mutations within the fancm gene and early - onset familial breast cancer . 
kiiski ji , tervasm aki a , pelttari lm , et al : fancm mutation c.5791c.t is a risk factor for triple - negative breast cancer in the finnish population . 
na r , zheng sl , han m , et al : germline mutations in atm and brca1 / 2 distinguish risk for lethal and indolent prostate cancer and are associated with early age at death . 
castro e , goh c , olmos d , et al : germline brca mutations are associated with higher risk of nodal involvement , distant metastasis , and poor survival outcomes 51 . 
castro e , goh c , leongamornlert d , et al : effect of brca mutations on metastatic relapse and cause - specic survival after radical treatment for localised in prostate cancer . 
abida w , armenia j , gopalan a , et al : prospective genomic proling of prostate cancer across disease states reveals germline and somatic alterations that may affect clinical decision making . 
kyriakopoulos ce , chen yh , carducci ma , et al : chemohormonal therapy in metastatic hormone - sensitive prostate cancer : long - term survival analysis of the randomized phase iii e3805 chaarted trial . 
tombal b , borre m , rathenborg p , et al : enzalutamide monotherapy in hormone - naive prostate cancer : primary analysis of an open - label , single - arm , phase 2 study . 
lancet oncol 15 : 592 - 600 , 2014 james nd , de bono js , spears mr , et al : abiraterone for prostate cancer not previously treated with hormone therapy . 
markowski mc , wang h , sullivan r , et al : phase ii trial of rucaparib ( without adt ) in patients with metastatic hormone - sensitive prostate cancer harboring germline dna repair gene mutations ( triumph )  . 
 integrative molecular analysis of patients with advanced and metastatic cancer verena sailer , md1 ; kenneth wa eng , ms1 ; tuo zhang , phd1 ; rohan bareja , ms1 ; david j . 
pisapia , md1 ; alexandros sigaras , ms1 ; bhavneet bhinder , ms1 ; alessandro romanel , phd2 ; david wilkes , phd1 ; evan sticca , ms1 ; joanna cyrta , md1 ; rema rao , md1 ; sheena sahota , md1 ; chantal pauli , md1 ; shaham beg , md1 ; samaneh motanagh , md1 ; myriam kossai , md1 ; jacqueline fontugne , md1 ; loredana puca , phd1 ; hanna rennert , phd1 ; jenny zhaoying xiang , md1 ; noah greco , mba1 ; rob kim1 ; theresa y . 
in patients with more than one sequenced tumor sample ( n = 146 ) , 84.62% of actionable mutations were concordant . conclusion integrative analysis may uncover informative alterations for an advanced pan - cancer patient population . 
2019 by american society of clinical oncology introduction genomic proling is widely used in cancer care to identify actionable alterations for individual patients within the context of precision medicine ( pm ) .1 however , only 2% to 11% of those patients with sequencing performed receive a genomically matched therapy , which may be a result of the availability and accessibility of clinical trials , 2 - 6 patient factors and comorbidities , disease state or alternative options , or patient preference.5 despite these drawbacks , pm studies continue to provide insights into the molecular underpinnings of cancer . 
we and others have shown that performing whole - exome sequencing ( wes ) and rna sequencing is feasible in a clinical setting and may provide relevant information beyond targeted gene panels in certain settings.3 , 7 , 8 sequencing matched tumor and normal ( germline ) dna has the additional benet of uncovering unforeseen hereditary conditions , including cancer risk mutations . 
in particular , germline dna repair defects ( drds ) are more common than previously anticipated across adult advanced cancer populations.9 - 11 robinson et al12 published their met500 cohort with wes and rna sequencing data from 500 patients with metastatic disease of varied tumor primary and biopsy sites . 
our study , obtained from our own cohort of 759 samples from 515 patients with advanced and metastatic cancer , complements this data set as it adds wes data from approximately the same number of patients . 
 sailer et al context key objective we assessed whether developing a multidimensional precision oncology program is feasible and informative for patients with cancer with advanced disease . knowledge generated we established a comprehensive clinical genomics program for this patient group . 
the rate of clinically relevant alterations across 515 patients with advanced solid tumors was 39% by whole - exome sequencing , which was improved by 16% by adding rna sequencing . 
germline dna was obtained from blood samples ( circulating mononuclear cells ) , buccal swabs , or benign tissue as described previously.3 part of the data presented in this manuscript have already been published.3 , 13 , 15 wes was performed on each patients tumor / matched germline dna pair using previously described protocols.3 we used a clinical - grade wes testexome cancer test version 1approved by new york state department of health ( id# 43032 ) and described in detail in rennert et al.16 this approach allows for assessment of more than 21 , 000 genes through the development and implementation of computational approaches for tumor mutational burden and neoepitope analysis , as well as integration with other data , including rna sequencing , to improve the identication of clinically relevant and actionable alterations . 
to evaluate the concordance of tier 1 and tier 2 alterations between multiple samples from the same patient and to gain high - delity results , a cutoff of 20% for variant allele frequency was used . 
in addition , we developed patientderived organoids from fresh tissues using previously described protocols , 4 , 17 and we used cell lines to functionally validate outlier targetable alterations . wes alterations were categorized on the basis of on their actionability and clinical or biologic relevance.3 alterations in 49 actionable or clinically signicant genes were reported within category 1 , alterations in 508 known cancer - associated genes within category 2 , and somatic alterations of unknown signicance within category 3.3 we developed an open - access , dynamic , web - based pm knowledge base as an interactive online tool where variants are carefully interpreted in the context of tumor type.18 wes results were conveyed to the referring physician in the form of an exome cancer test version 1 report.3 selected cases are presented at a regular , continuing medical education accredited pm tumor board , which discusses sequencing results in the context of a patients history , available literature , and treatment options , including active clinical trials . pathogenic germline ndings were reported to the referring physician if they occurred in any of the genes deemed reportable by the american college of medical genetics and genomics ( acmg ) , 19 and these patients were referred for genetic counseling and results were conrmed by targeted testing in a clinical laboratory improvement amendments / clinical laboratory evaluation programcertied laboratory . study cohort demographic data were obtained through electronic health record search . 
 precision oncology for patients with advanced disease primary site ( 515 patients ) biopsy / resection site ( 759 samples ) breast ( 13 ) pancreas ( 18 ) kidney ( 46 ) soft tissue ( 8 ) brain ( 98 ) lung ( 16 ) hematologic malignancies ( 24 ) colon and / or rectum ( 41 ) bladder ( 57 ) prostate ( 105 ) primary recurrent metastatic brain ( 150 ) liver ( 64 ) bla d d er ( 69 ) b o n e ( 71 ) ly m p h n o d e ( 85 ) c olo n ( 18 ) l u n g ( 44 ) kid n ey ( 26 ) prostate ( 33 ) sample characteristic metastatic primary recurrent 34 - year - old female , er - positive breast carcinoma , mastectomy , brca1 / 2 germline negative chest wall recurrence , er / pr / her2 unknown , excision and radiotherapy no cancer detected metastatic disease seven systemic therapies precision medicine consent liver biopsy , akt1 mutation detected by wes cdk4 / 6 inhibitor ( palbociclib ) liver progress + 3 years + 20 years + 3 years + 1 year + 1 year + 1 year fig 1 . 
the most common somatic alterations were found in tp53 ( 33% ) , cdkn2a ( 11% ) , apc ( 10% ) , kmt2d ( 8% ) , pten ( 8% ) , and brca2 ( 7% ; fig 2 )  . multiple samples three hundred eighty - two spatially and temporally heterogeneous samples from 146 patients underwent wes . 
of these , 185 ( 48.4% ) were metastatic , 153 ( 40.1% ) were primary , and 44 ( 11.5% ) were recurrent tumor samples . most samples were paired primary and metastatic tumors , and concordance of alterations between primary and metastatic samples in clinically informative genes is shown in figure 3 . 
paired primarymetastatic tumor samples from 59 patients were the most frequent combination . spatially and / or temporally heterogeneous metastases and primary tumors from 35 and 29 patients , respectively , also underwent sequencing . 
 ( e ) different kras mutations in two morphologically similar lung cancer samples from the same patient , thus conrming two separate primary tumors . this cohort ( data supplement )  . 
the most frequently mutated genes were chek2 ( 11 patients ) , brca2 ( nine patients ) , brca1 ( nine patients ) , msh6 ( nine patients ) , and atm ( four patients ; fig 2 )  . 
in likely pathogenic variants in 38 addition , 44 additional patients in msh6 ( 14 cases ) ; apc ( seven cases ) ; chek2 ( ve cases ) ; pole , pms2 , msh2 brca1 , and atm ( three cases each ) ; and tp53 , cdh1 , and brip1 ( one case each ) were discovered in our cohort . the prevalence of drd in our cohort of patients with metastatic prostate cancer was 14.3% , with brca2 mutations being the most frequent variants ( data supplement )  . pathogenic germline drds were found in 9.2% of patients with primary brain tumors . 
we did not identify pathogenic mutations involving mismatch repair genes , which have been described in primary brain tumor patients , in particular as biallelic losses.25 chek2 was altered in four cases , including one medulloblastoma . 
 ( b ) high frequency of germline mutations in breast , prostate , and lung tumors is observed . germline drd defects , 12 had received platinum - based chemotherapy with follow - up available . 
an additional targetable ncoa4ret fusion , which has been described in papillary thyroid cancer , nonsmall - cell lung cancer , and colorectal cancer , was found in a brain metastasis of a patient with unknown primary.28 rna sequencing rna sequencing was performed when sufcient fresh frozen tumor tissue was available after wes . 
of these 89 patients , 50 did not harbor a targetable genomic alteration identied by wes , resulting in an increase in the rate of actionable alteration detection of approximately 15% . 
we conrmed the drug sensitivity of select outlier genes using cell line experiments compared with randomly selected drugs ( fig 5 )  . nine novel fusions in a variety of cancer types were detected . 
a novel rbm47 - cdk12 gene fusion was found in a prostate cancer bone metastasis , which was conrmed organoids part of the program was the development of patient - derived organoids from patient biopsies for high - throughput drug screening.4 altogether 60 organoids were developed from 98 patients with an overall success rate of 61% . 
a pathogenic msh6 mutation was detected in a 26 - year - old patient with metastatic breast carcinoma who had undergone previous outside testing for brca1 / 2 germline mutations with a negative result . 
other potentially signicant variants in drd genes , such as palb2 , would have been missed as well.30 this case underlines the importance of multigene panel or wes testing in patients not only with suspected hereditary cancer but also in those , especially young patients , with metastatic disease to detect underrecognized germline alterations.31 tumor evolution and clonality . 
a deleterious effect has been predicted for this variant.33 in a previously published analysis of 13 uterine carcinosarcomas analyzed by targeted sequencing , eight cases demonstrated 100% identical mutations in both the carcinoma and sarcoma part.34 in contrast , we observed an early divergence with only the one pten mutation of 54 nonsynonymous shared mutations . 
this case again demonstrates the importance of using next - generation sequencing to correctly identify synchronous primary tumors.39 another patient with metastatic neuroblastoma was conrmed to have an activating alk mutation ( r1275q ) in both primary tumor and bone marrow metastasis . 
the patient has had stable disease for 13 months on therapy with sequential alk inhibitors40 ( fig 3 )  . discussion we have established a pm program for patients with advanced cancer using tumor / normal wes and integrative molecular proling to detect genomic and other actionable alterations , improve clinical decision making , and study tumor evolution in a pan - cancer cohort . 
the patient population in our study is distinct from several reported studies in that our focus was on the evaluation of advanced tumors.41 , 42 the majority of patients ( 70.8% ) presented the time of with metastatic or recurrent disease at enrollment . whereas publicly accessible molecular data for several tumor types are available , both for primary and metastatic cancers , 43 , 44 these specimens are rarely matchedthat is , obtained from the same patient.45 our cohort of patients with locally advanced and metastatic cancer allowed for the collection of multiple matched , often primary and metastatic samples , resulting in a unique feature of this cohort : genomic data from spatially and / or temporally heterogeneous , matched tissue samples were available in 146 patients . 
of clinical importance is the nding that almost 85% of category 1 alterations were shared between multiple samples from the same patient . these results are in concordance with published data in specic cancer types . 
in one study , when comparing actionable mutations between presurgery biopsies and resected specimen in patients with nonsmall - cell lung cancer , the concordance rate was found to be 79%.46 similar ndings have been reported for primary and recurrent breast cancer , with 86.6% of mutations and 85.5% of scnas being concordant.47 our data conrm that it may reasonable to select the most accessible location or even archival material from the primary tumor for molecular analysis for certain cancer types.48 , 49 new research indicates that pathogenic germline mutations are more frequently found in patients with advanced cancer , 9 , 12 and our data conrm this . 
germline mutations that involve the dna repair pathway , found in 10.7% of our patients , are predictive of response to parp inhibitors and platinum - based therapy and have important familial plications for cancer screening.50 family history was indicative of a heritable component in only one half of these patients . 
this fusion might result in the loss of cdk12 activity , which has recently been described to delineate a distinct immunogenic subtype of metastatic castrationresistant prostate cancer.51 in our study , wes was prioritized over rna sequencing whenever tissue availability was of concern . 
biopsies in our study were usually performed for diagnostic purposes in the context of clinical care , hence the lower availability of fresh frozen tissue for additional rna sequencing . targeted sequencing of cancer - related genes offers several advantages over wes , including deeper coverage , quicker turnaround time , lower cost , and fewer requirements for an elaborate computational pipeline . 
here , we show that wes and rna sequencing may provide an additional and information in certain settings , complimentary layer of particularly for patients with advanced cancer who experience progression after standard therapies . 
wes also considers the rapidly expanding spectrum of actionable alterations , including alterations for which targeted treatment may not be available at the time of analysis , but for which clinical trials might be planned . 
in addition , whereas targeted nextgeneration sequencing testing of somatic dna are sometimes ordered as part of clinical care , using germline dna as normal control for wes necessitates securing informed consent . implementing - omic data into clinical care requires an interdisciplinary teagenomic data and its interpretation in the context of tumor type and primary site must be easily accessible to enable clinicians to integrate the data into everyday patient care . 
 precision oncology for patients with advanced disease one limitation of our study is the lack of uniformity of the cohort in terms of tumor type and therapy , which prevents us from making generalized statements about treatment response . 
this data set also reports on a large number of matched temporally and spatially heterogeneous samples , highlighting the concordance of actionable alterations in different samples . as other pm trials have shown , uncovering genomic alterations may not be sufcient in identifying actionable alterations . 
contributed equally to this work and share senior authorship . support supported by the englander institute for precision medicine of weill cornell medicine and new york presbyterian , the translational research program of the department of pathology and laboratory medicine at weill cornell medicine . 
macdonald , eleni andreopoulou , olivier elemento , himisha beltran provision of study materials or patients : noah greco , rob kim , bishoy m . faltas , eleni andreopoulou , linda t . 
pisapia , david wilkes , joanna cyrta , sheena sahota , chantal pauli , shaham beg , samaneh motanagh , myriam kossai , jacqueline fontugne , jenny zhaoying xiang , noah greco , rob kim , theresa y . 
pisapia , alexandros sigaras , bhavneet bhinder , alessandro romanel , david wilkes , evan sticca , rema rao , shaham beg , loredana puca , rob kim , mirjam blattner - johnson , eleni andreopoulou , linda t . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . alexandros sigaras employment : weill cornell medical college loredana puca employment : petra pharma corporation bishoy m . 
faltas honoraria : digital science press publications research funding : eli lilly david rickman research funding : janssen oncology eleni andreopoulou honoraria : astrazeneca , sirtex , svb leerink , abbvie , eisai , nanostring technologies consulting or advisory role : astrazeneca , sirtex , svb leerink , abbvie , eisai , nanostring technologies speakers bureau : r - pharm us travel , accommodations , expenses : astrazeneca , sirtex , svb leerink , abbvie , eisai , nanostring technologies kevin holcomb research funding : fujirebio diagnostics expert testimony : johnson & johnson linda t . 
vahdat honoraria : polyphor , r - pharm , athenex , seattle genetics , osmol therapeutics consulting or advisory role : berg pharma speakers bureau : eisai research funding : polyphor ( inst ) , immunomedics ( inst ) , seattle genetics ( inst ) patents , royalties , other intellectual property : patent pending on the application for bmd progenitor cells ( inst ) douglas s . 
 sailer et al koen van besien stock and other ownership interests : hemogenyx consulting or advisory role : hemogenyx , actinium pharmaceuticals , cellectis , tessa therapeutics research funding : miltenyi biotec , actinium pharmaceuticals , juno therapeutics , fate therapeutics christopher e . 
ocean consulting or advisory role : celgene , tyme speakers bureau : daiichi sankyo travel , accommodations , expenses : daiichi sankyo ana molina honoraria : american society of clinical oncology consulting or advisory role : eisai , exelixis , novartis , janssen oncology manish a . 
shah consulting or advisory role : astellas pharma , eli lilly research funding : gilead sciences ( inst ) , merck ( inst ) , boston biomedical ( inst ) , oncolys biopharma ( inst ) , bristol - myers squibb ( inst ) david m . 
nanus consulting or advisory role : genentech research funding : novartis ( inst ) , boehringer ingelheim ( inst ) , zenith epigenetics ( inst ) qiulu pan employment : caris life sciences stock and other ownership interests : caris life sciences francesca demichelis patents , royalties , other intellectual property : co - inventor on a patent led by the university of michigan and brigham and womens hospital covering the diagnostic and therapeutic elds for ets fusions in prostate cancer , diagnostic eld licensed to gen - probe scott t . 
tagawa consulting or advisory role : medivation , astellas pharma , dendreon , janssen oncology , bayer , genentech , endocyte , immunomedics , karyopharm therapeutics , abbvie , tolmar , qed , amgen , sano , pzer research funding : eli lilly ( inst ) , sano ( inst ) , janssen oncology ( inst ) , astellas pharma ( inst ) , progenics ( inst ) , millennium pharmaceuticals ( inst ) , amgen ( inst ) , bristol - myers squibb ( inst ) , dendreon ( inst ) , rexahn pharmaceuticals ( inst ) , bayer ( inst ) , genentech ( inst ) , newlink genetics ( inst ) , inovio pharmaceuticals ( inst ) , astrazeneca ( inst ) , immunomedics ( inst ) , novartis ( inst ) , aveo ( inst ) , boehringer ingelheim ( inst ) , merck ( inst ) , stem centrx ( inst ) , karyopharm therapeutics ( inst ) , abbvie ( inst ) , medivation ( inst ) , endocyte ( inst ) , exelixis ( inst ) , clovis oncology ( inst ) travel , accommodations , expenses : sano , immunomedics , amgen wei song employment : genentech ( i ) , cytokinetics ( i ) honoraria : foundation medicine , loxo consulting or advisory role : foundation medicine , loxo juan miguel mosquera research funding : personal genome diagnostics travel , accommodations , expenses : personal genome diagnostics mark a . 
nature 537 : s63 , 2016 beltran h , eng k , mosquera jm , et al : whole - exome sequencing of metastatic cancer and biomarkers of treatment response . 
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cell 163 : 1011 - 1025 , 2015 isaka m , serizawa m , kenmotsu h , et al : comparison of clinically relevant mutation proles between preoperative biopsy and corresponding surgically resected specimens in japanese patients with non - small - cell lung cancer by amplicon - based massively parallel sequencing . 
kang hj , hwangbo b , lee js , et al : comparison of epidermal growth factor receptor mutations between metastatic lymph node diagnosed by ebus - tbna and primary tumor in non - small cell lung cancer . 
 response to selective ret inhibition with loxo - 292 in a patient with ret fusion - positive lung cancer with leptomeningeal metastases robin guo , md1 ; mark schreyer , ms1 ; jason c . 
hyman , md1 , 4 ; and alexander drilon1 , 4 introduction the development of leptomeningeal metastases is a poor prognostic factor in patients with advanced cancers.1 - 3 in nonsmall - cell lung cancers ( nsclcs ) , median overall survival of patients from the diagnosis of leptomeningeal disease is 1 to 2 months without treatment and up to 8 months with systemic therapy.4 - 6 furthermore , patients with leptomeningeal disease have historically had limited access to novel therapies in clinical trials . 
recent efforts from many groups , including the european society for medical oncology and the us food and drug administration ( fda ) , have encouraged the inclusion of patients with leptomeningeal metastases in clinical trials , in addition to promoting standardization of intracranial response assessments.7 - 9 these efforts are crucial given that many investigational agents have substantial cns activity and may improve outcomes in driver - positive cancers with leptomeningeal involvement.5 , 10 ret fusions are actionable oncogenic drivers that are identied in 1% to 2% of nsclcs.11 , 12 to date , chemotherapy and / or immunotherapy remain the only approved systemic therapies for these cancers . 
although these agents were found to be active in these patients , outcomes are modest a subset of compared with targeted therapies in other driverpositive lung cancers , and intracranial activity is poor.13 , 14 selective ret inhibitors currently in development , such as loxo - 292 and blu - 667 , have improved outcomes for patients with ret fusion - positive cancers because of increased potency and less offtarget toxicity.15 , 16 in september of 2018 , loxo - 292 received breakthrough therapy designation from the fda for treatment of patients with metastatic ret fusion - positive nsclcs ( as well as ret fusion - positive thyroid cancers and ret - mutant medullary thyroid cancer )  . 
in addition , conrmed intracranial responses and durable disease control have been achieved in patients with brain metastases in an ongoing phase i / ii trial of loxo - 292 for patients with ret fusion - positive cancers.15 its activity in leptomeningeal disease , however , has not previously been characterized . 
computed and positron - emission tomography imaging revealed a hypermetabolic 4.8lobe mass , mediastinal and hilar cm right adenopathy , and osseous metastases involving l1 , the sacrum , and the left anterolateral sixth rib . 
magnetic resonance imaging ( mri ) of the brain showed three subcentimeter enhancing foci in the right precentral lobe . gyrus , right parietal endobronchial biopsy of an r4 lymph node revealed adenocarcinoma with signet ring cell features ( fig 1a )  . tumor cells were positive for ttf - 1 and negative for p40 by immunohistochemistry . 
broad , hybrid capture based next - generation sequencing using the memorial sloan kettering integrated mutation proling of actionable cancer targetsmsk - impactand illumina hiseq 2500 ( illumina , san diego , ca ) 17 identied an eml4 - ret fusion ( fig 1b ) in addition to a tp53 p.p142tfs * 5 frameshift mutation . 
this eml4 - ret fusion was conrmed using a targeted rna - based anchored multiplex polymerase chain reaction archer fusion assay ( archer , boulder , co )  . with identication of the ret fusion , the patient was treated with the investigational anti - ret multikinase inhibitor rxdx - 105.18 , 19 although a conrmed partial response was initially achieved ( a near - complete response in her brain metastases ) , her course was marked by isolated asymptomatic intracranial progression requiring multiple radiation treatments . 
 guo et al eml4 ( nm_019063.3 ) ret ( nm_020630.4 ) 8 9 10 1112 13 14 15 16 1718 1912 13 1415 1617 18 ence eml4 ( + ) nm_019063.3|exon : 19 ret ( + ) nm_020630.4|exon : 12 fig 1 . 
 ( a ) a hematoxylin and eosinstained section from a cell block of a ne - needle aspiration specimen from a lower paratracheal lymph node conrmed a diagnosis of lung adenocarcinoma . 
clusters of malignant epithelial cells with signet - ring cell morphology ( eccentrically placed nuclei , focally prominent nucleoli , and abundant amount of cytoplasm containing grayish - blue mucin ) are shown . 
 ( b ) an in - frame ret fusion containing the ret tyrosine kinase domain was identied in extracted dna from this sample by broad , hybrid capturebased next - generation sequencing using the memorial sloan kettering integrated mutation proling of actionable cancer targetsmsk - impact and illumina hiseq 2500 ( illumina , san diego , ca )  . exon 19 of the 5 ( cid : 1 ) upstream gene partner eml4 was fused to exon 12 of 3 ( cid : 1 ) ret . 
this eml4 - ret fusion was conrmed using an rna - based anchored multiplex polymerase chain reaction ( archer , illumina miseq [ archer , boulder , co ] )  . four months later , the patient developed symptomatic progression of brain metastases and new leptomeningeal disease . 
she presented with left facial , tongue , and upper extremity tingling and worsening neck pathese symptoms were deemed to be secondary to leptomeningeal disease that was identied radiologically in the right hemisphere , predominantly in the right parietal lobe ( fig 2a ; top panel ) , recognizing that nonradiologically apparent disease was likely present in other areas.8 multiple brain metastases had also increased ( largest measuring 2.7 cm in the right frontal lobe ; fig 2a ; bottom panel )  . 
a cant lumbar puncture was recommended , but the patient declined ; a brain biopsy to potentially determine the mechanism of resistance to rxdx - 105 was not deemed safe . extracranial imaging again showed no evidence of disease . given that the patient was highly symptomatic with progressive symptoms , a single - patient use protocol of loxo - 292 was approved by the fda and institutional review board . 
magnetic resonance imaging axial brain images are shown ( a ) at baseline , ( b ) 5 weeks , and ( c ) 21 weeks after the initiation of loxo - 292 therapy in a patient with an eml4ret fusion - positive lung cancer . 
a representative right superior medial and right lower frontal intraparenchymal metastasis ( green arrows ) regressed with loxo - 292 therapy , along with several other metastases followed on serial intracranial response by recist ( response evaluation criteria in solid tumors ) v1.1 and a complete response in leptomeningeal disease by response assessment in neuro - oncology were achieved . 
a conrmed partial patient provided written informed consent before enrollment , and loxo - 292 was administered orally at 160 mg twice daily . this dose was selected based on preliminary safety and efcacy results from an ongoing phase i / ii trial of the drug ( clinicaltrials.gov identier : nct03157128 )  . imaging assessments ( mri of the brain and computed tomography of the chest , abdomen , and pelvis ) were performed every 8 weeks . response was assessed by recist ( response evaluation criteria in solid tumors ) version 1.1.20 response of leptomeningeal metastases was assessed in accordance with response assessment in neuro - oncology criteria.8 additional volumetric three - dimensional imaging was performed on subsequent scans ( sloan kettering advanced imaging lab , sail ; figs 3b and 3c )  . a clinical response to therapy was achieved within the rst week of therapy , with improvement and subsequent resolution of the patients neurologic symptoms . 
in addition , loxo - 292 therapy achieved complete resolution of leptomeningeal in neuroenhancement , with a response assessment oncology leptomeningeal score dropping from 1 at baseline to 0 at 8 weeks . 
volumetric assessment revealed a continued decrease in the total volume of signicant intracranial disease ( leptomeningeal and parenchymal ) , with a maximal shrinkage of 65% ( from 20.1 cm3 at baseline to 7 cm3 ) at 5 months ( fig 3c )  . the patient continues to receive therapy with loxo - 292 at 10.8 months , with ongoing radiologic disease control and no neurologic symptoms . 
volumetric three - dimensional magnetic resonance imaging analyses were performed on serial imaging performed at ( a ) baseline , ( b ) 5 weeks , and ( c ) 21 weeks . 
maximal volumetric disease regression of 65% was achieved at 21 weeks in the graph of total volume over time on loxo - 292 therapy as shown in the bottom panel . 
in nsclcs with leptomeningeal metastases , there is no consensus on the use of wholebrain radiotherapy , because it has not been shown to consistently improve survival.1 , 3 although systemic chemotherapy with more contemporary regimens ( including pemetrexed and / or bevacizumab ) and intrathecal chemotherapy have been shown to improve outcomes in select series , the development of new agents with higher response rates and more durable disease control continues to represent an unmet need for many patients.4 , 6 this report represents the rst description , to our knowledge , of leptomeningeal metastases responding to any systemic therapy in a patient with a ret fusion - positive cancer . 
a brisk and durable ongoing response to loxo - 292 was achieved in a patient with a ret fusion - positive lung cancer who had notable disease progression while 4 2019 by american society of clinical oncology receiving a prior multikinase inhibitor and multiple prior stereotactic radiosurgery treatments . 
in preliminary data from trial ( clinicaltrials.gov identier : an ongoing phase i / ii nct03157128 ) , all four patients with untreated measurable parenchymal metastases had conrmed intracranial responses to therapy accompanied by overall disease control.15 durable intracranial and extracranial disease control was likewise established in several other patients with untreated nonmeasurable brain metastases before loxo - 292 therapy . the activity of loxo - 292 in the cns can be attributed to several factors . 
the drug is active preclinically , with oral dosing in an orthotopic mouse model of a ret fusionpositive patient - derived tumor implanted into the brain.21 , 22 its potency and selectivity for ret are also likely contributory . 
using a highly active agent in the cns is crucial in ret fusion - positive lung cancers , because close to 25% of patients present with intracranial disease at baseline , whereas the lifetime prevalence of brain metastases approaches 50%.14 in addition , loxo - 292 was designed to target potential resistance mechanisms that can emerge from prior multikinase inhibitor use , such as ret v804m / l gatekeeper substitutions . 
 response to loxo - 292 in ret - rearranged leptomeningeal disease fusions are also identied in papillary thyroid , anaplastic thyroid , colorectal , pancreatic , and breast cancers ; spitzoid neoplasms ; and chronic myeloproliferative neoplasms.11 somatic and germline - activating ret mutations are likewise actionable drivers of oncogenesis that are identied in medullary thyroid cancers and potentially other malignancies.33 although the frequency at which intracranial metastases present is lower for many of these other cancers compared with nsclcs , metastatic disease in the cns can occur in some cases.34 in conclusion , selective ret inhibition with loxo - 292 achieved a clinically meaningful and conrmed response in a patient with a ret fusion - positive lung cancer with leptomeningeal disease and heavily pretreated brain metastases . 
although additional conrmation of this activity will help elucidate overall intracranial disease outcomes , this report underscores the potential of selective ret inhibition as a means of treating and preventing the occurrence of disease in the cns in patients with ret - dependent cancers of any histology . affiliations 1memorial sloan kettering cancer center , new york , ny 2loxo oncology , stamford , ct 3new york university langone medical center , new york , ny 4weill cornell medical college , new york , ny s . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . dahlia henry employment : loxo stock and other ownership interests : loxo , allergan , axovant sciences , palatin technologies mark g . 
kris consulting or advisory role : astrazeneca , regeneron , pzer travel , accommodations , expenses : astrazeneca , genentech other relationship : memorial sloan kettering cancer center robert j . 
young stock and other ownership interests : agios , alexion pharmaceuticals , biogen , celgene , gilead sciences , karyopharm therapeutics , spark therapeutics , regeneron , stemline therapeutics , vertex consulting or advisory role : agios , puma biotechnology , nordicneurolab , icon clinical research research funding : agios ( inst ) david m . 
future oncol 14 : 391 - 407 , 2018 omuro am , kris mg , miller va , et al : high incidence of disease recurrence in the brain and leptomeninges in patients with nonsmall cell lung carcinoma after response to getinib . 
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j thorac oncol 11 : 1962 - 1969 , 2016 park jh , kim yj , lee jo , et al : clinical outcomes of leptomeningeal metastasis in patients with non - small cell lung cancer in the modern chemotherapy era . lung cancer 76 : 387 - 392 , 2012 kim es , bruinooge ss , roberts s , et al : broadening eligibility criteria to make clinical trials more representative : american society of clinical oncology and friends of cancer research joint research statement . 
neuro - oncol 19 : 484 - 492 , 2017 le rhun e , weller m , brandsma d , et al : eano executive board and esmo guidelines committee : eano - esmo clinical practice guidelines for diagnosis , treatment and follow - up of patients with leptomeningeal metastasis from solid tumours . 
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drilon a , rekhtman n , arcila m , et al : cabozantinib in patients with advanced ret - rearranged non - small - cell lung cancer : an open - label , single - centre , phase 14 . 
oxnard gr , subbiah v , park k , et al : clinical activity of loxo - 292 , a highly selective ret inhibitor , in patients with ret fusion + non - small cell lung cancer : an update from asco 2018 . 
subbiah v , taylor m , lin j , et al : highly potent and selective ret inhibitor , blu - 667 , achieves proof of concept in a phase i study of advanced , ret - altered solid tumors . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
drilon ae , subbiah v , oxnard gr , et al : a phase 1 study of loxo - 292 , a potent and highly selective ret inhibitor , in patients with ret - altered cancers . 
yang jc - h , cho bc , kim d - w , et al : osimertinib for patients ( pts ) with leptomeningeal metastases ( lm ) from egfr - mutant nonsmall cell lung cancer ( nsclc ) : updated results from the bloom study . 
reungwetwattana t , nakagawa k , cho bc , et al : cns response to osimertinib versus standard epidermal growth factor receptor tyrosine kinase inhibitors in patients with untreated egfr - mutated advanced nonsmall - cell lung cancer . 
wu yl , ahn mj , garassino mc , et al : cns efcacy of osimertinib in patients with t790m - positive advanced nonsmall - cell lung cancer : data from a randomized 28 . 
costa db , shaw at , ou sh , et al : clinical experience with crizotinib in patients with advanced alk - rearranged nonsmall - cell lung cancer and brain phase iii trial ( aura3 )  . 
gadgeel s , peters s , mok t , et al : alectinib versus crizotinib in treatment - naive anaplastic lymphoma kinase - positive ( alk + ) non - small - cell lung cancer : cns efcacy results from the alex study . 
shaw at , kim tm , crin `o l , et al : ceritinib versus chemotherapy in patients with alk - rearranged non - small - cell lung cancer previously given chemotherapy and crizotinib ( ascend - 5 ) : a randomised , controlled , open - label , phase 3 trial . 
camidge dr , kim dw , tiseo m , et al : exploratory analysis of brigatinib activity in patients with anaplastic lymphoma kinase - positive nonsmall - cell lung cancer and brain metastases in two clinical trials . 
solomon bj , besse b , bauer tm , et al : lorlatinib in patients with alk - positive non - small - cell lung cancer : results from a global phase 2 study . 
 total number of alterations in liquid biopsies is an independent predictor of survival in patients with advanced cancers peter vu , md1 ; yulian khagi , md1 ; paul riviere , bs1 ; aaron goodman , md2 ; and razelle kurzrock , md1 purpose studies have demonstrated an association between quantity of circulating tumor dna ( ctdna ) and poorer survival . 
our ndings suggest that the total number of alterations in plasma may be an indicator of more aggressive tumor biology and therefore poorer survival . jco precis oncol 4 : 192 - 201 . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction five - year survival rates are incredibly variable among cancer types , ranging from more than 90% in prostate cancer to less than 8% in pancreas cancer , and depend heavily on clinical and pathologic stage.1 although repeat tissue biopsies during the course of treatment or at the time of progression may provide clinically important information , such biopsies are not routinely performed because they can be technically difcult , time consuming , medically invasive , and lead to complications . 
however , liquid biopsies , or cell - free dna ( cfdna ) obtained from blood plasma that contains fragments of circulating tumor dna ( ctdna ) shed from tumor cells into the bloodstream , can identify new actionable alterations and be performed repeatedly with minimal procedural risk.2 - 5 ctdna can then be analyzed using technologies such as digital polymerase chain reaction to detect specic known somatic variants ( eg , egfr t790m ) or next - generation sequencing ( ngs ) that uses massive parallel sequencing to detect up to thousands of somatic and germline alterations in a single run.6 in addition , genomic alterations found on liquid biopsies are often concordant with alterations found on tissue biopsy when obtained within close proximity to one another.7 - 9 a number of studies have demonstrated that there is an association between higher amounts of cfdna or ctdna and poorer survival , perhaps because percent ctdna ( %ctdna ) correlates with tumor burden.10 , 11 for the most part , these reports dichotomized the level of cfdna or ctdna at a cut - point ( often but not always at approximately 5% or 10% ctdna ) .10 , 12 - 18 in the case of surgical candidates , the cut - points may be lower . 
eligible patients included those who had solid tumor malignancies , never received immunotherapy treatment , and were evaluable for clinical correlations , including overall survival ( os ) from ctdna collection date . 
immunotherapytreated patients were omitted because a correlation with blood or tissue tumor mutational burden has been associated with better immunotherapy response and might therefore alter the survival curve.19 , 20 patients with amplications only in ctdna were omitted because the %ctdna for amplications could not be determined . 
in addition to os evaluation , patients data were also collected and analyzed for %ctdna ; the alteration with the highest allele fraction was calculated from all alterations , including variants of unknown signicance ( vuss ) , total number of vuss , and total number of alterations ( which included vuss )  . 
all values for the total number of ctdna alterations and the number of vuss were corrected for the length ( kilobase pairs [ kbp ] ) of dna sequenced based on the date sequencing was performed and multiplied by 100 ( appendix table a1 )  . 
all data were analyzed from the time of ctdna collection from plasma ( two 10 - ml blood tubes )  . this ctdna assay has a sensitivity and specicity of  . 
hazard ratios ( hrs ) for survival were calculated by comparing os above and below cutoffs and performed from the time of ctdna collection ; dichotomization for each variable ( ie , total number of alterations , total number of vuss , %ctdna ) was performed at the median . 
multivariate analyses were conducted using the wald 2 test from a cox proportional hazards model that included all variables with p .05 in univariate analyses ( ie , sex , age , total number of alterations , %ctdna ) , with the exception of vuss because these alterations are already encompassed within the total number of alterations variable . 
associations between %ctdna and total number of alterations were determined using spearmans rank - order correlation . bootstrapping using random sampling with replacement ( n = 1 , 000 bootstrap samples ) and multiple logistic regression analysis were performed , permitting the data of the sample study to be used as a surrogate for a larger population to validate the model . 
intermediate to high %ctdna was dichotomized at 5% because it had been found to be signicant in prior studies.10 the %ctdna for each patient was calculated using the alteration with the highest allele fraction , including variants of unknown signicance validation cohort , as was the case in our study.22 statistical analyses were carried out using prism version 7.0 ( graphpad , san diego , ca ) and r version 3.5 ( r foundation for statistical computing , vienna , austria )  . results patient characteristics this study included 418 patients who had ngs performed on plasma - derived ctdna and did not receive immunotherapy treatment . 
after correcting for the kbp length of dna sequenced for each sample , the median total number of ctdna alterations ( including vuss ) per patient was 1.46 ( range , 0 - 78.8 ) ; the median total number of vus alterations per patient was 0.66 ( range , 0 - 64.2 ) ; and the median %ctdna was 0.4% ( range , 0% - 80.3% ; table 1 )  . 
 total number of alterations in liquid biopsies is prognostic regression model showed that age , sex , and the total number of alterations were independently prognostic for survival ( table 1 )  . 
among these characteristics , only total number of alterations was signicantly associated with survival ( p , .0001 ; table 1 )  . discussion liquid biopsies have been incorporated into clinical practice as a means to obtain noninvasive molecular proling to identify specic oncogenic driver mutations or other alterations that can guide treatment selection . 
in this study , we evaluated the relationship between the total number of alterations and the %ctdna detected by liquid biopsy and survival outcomes in 418 patients with advanced cancers . 
in addition , we intentionally excluded patients who subsequently received immunotherapy treatment , because several studies have suggested that the use of immune checkpoint inhibitors may alter the survival curve in patients with increased tumor mutational burden.19 , 20 , 23 we demonstrate that both the total number of alterations and the %ctdna have prognostic value and correlate with one another , but only an intermediate to high ( 1.46 ) total number of alterations / kbp ( and not high %ctdna ) was independently associated with worse survival outcomes in multivariate analysis in patients with gi tumors ( table 2 ) , as well as in patients with a diverse group of advanced cancers ( table 1 )  . 
it is also plausible that the higher number of alterations and accompanying aggressive biology results in a higher tumor burden that yields a higher %ctdna ( rather than vice versa )  . a strength of our study is that we used sequencing technology that allows for the detection of %ctdna at a low level with high sensitivity and high specicity.21 , 24 in comparison , some prior studies have used low - depth sequencing , which is less capable of detecting ctdna . 
as a result , these studies were only able to conclude that the presence of ctdna was associated with worse outcomes compared with the absence of detectable ctdna.14 , 18 , 25 , 26 indeed , yang et al27 proposed that the presence or absence of ctdna should be added to the tnm staging classication of tumors because it has diagnostic , therapeutic , and prognostic value . 
when greater depth of ctdna sequencing was used , prior studies have reported that %ctdna is correlated with worse survival and also with increased tumor volume.10 , 11 , 14 we also demonstrated that %ctdna correlates with survival measured from the time of blood draw ( fig 1 ) , which suggests that the association between %ctdna and outcomes may be more reective of tumor burden . limitations to our ndings , given the there are several retrospective nature of the analysis . 
although our study used a relatively large sample of 418 patients , we included a diverse group of advanced cancers and , therefore , our ndings may not be applicable to certain tumor types . despite this , the variety of tumor types in our study may make our ndings more generalizable across advanced total no . 
in addition , 112 patients in this study had no detectable %ctdna , which may be due to low disease burden or due to limitations of the ctdna sequencing technique . 
it should also be noted that there was a different number of subgroups in the analysis of %ctdna and number of ctdna alterations ; hence , the conclusion that the total number of alterations and %ctdna have prognostic value and correlate with one another but that only the total number of alterations was independently associated with survival outcomes will need to be further examined and validated . 
also , we do not know whether this patient population is comparable with those who were not analyzed for ctdna because physicians chose not to perform the analysis or with patients who were lost to follow - up early and hence were inevaluable . 
finally , patients had a diverse array of prior treatments , some of which could have confounded the results ; patients treated with immunotherapy were excluded because cancers with higher mutational burden / number appear to do better on this modality . in conclusion , to our knowledge , this is the rst demonstration that the total number of alterations and %ctdna are highly correlated and have prognostic value . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . support supported in part by the joan and irwin jacobs fund and by national cancer institute grant no . 
 total number of alterations in liquid biopsies is prognostic aaron goodman consulting or advisory role : mitsubishi tanabe pharma , gerson lehrman group , jazz pharmaceuticals , speakers bureau : seattle genetics razelle kurzrock leadership : curematch stock and other ownership interests : curematch , idbydna , soluventis , actuate therapeutics , loxo , xbiotech , neo - med , roche , gaido , soluventis , pzer , merck speakers bureau : roche research funding : guardant health ( inst ) , sequenom ( inst ) , merck serono ( inst ) , genentech ( inst ) , pzer ( inst ) , foundation medicine ( inst ) , incyte ( inst ) , konica minolta ( inst ) , grifols ( inst ) , omniseq ( inst ) , debiopharm group ( inst ) , boerhinger ingelheim ( inst ) no other potential conicts of interest were reported . references siegel rl , miller kd , jemal a : cancer statistics , 2018 . 
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ai b , liu h , huang y , et al : circulating cell - free dna as a prognostic and predictive biomarker in non - small cell lung cancer . 
baumgartner jm , raymond vm , lanman rb , et al : preoperative circulating tumor dna in patients with peritoneal carcinomatosis is an independent predictor of progression - free survival . 
mehrotra m , singh rr , loghavi s , et al : detection of somatic mutations in cell - free dna in plasma and correlation with overall survival in patients with solid 17 . 
stover dg , parsons ha , ha g , et al : association of cell - free dna tumor fraction and somatic copy number alterations with survival in metastatic triple - negative 19 . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free 22 . 
j clin oncol 36 : 543 - 553 , 2018 ther 16 : 2598 - 2608 , 2017 cancer res 23 : 5729 - 5736 , 2017 circulating tumor dna . 
lecomte t , berger a , zinzindohou e f , et al : detection of free - circulating tumor - associated dna in plasma of colorectal cancer patients and its association with 27 . 
yang m , forbes me , bitting rl , et al : incorporating blood - based liquid biopsy information into cancer staging : time for a tnmb system ? ann oncol prognosis . 
intermediate to high %ctdna was dichotomized at 5% because it had been found to be signicant in prior studies.10 the %ctdna for each patient was calculated using the alteration with the highest allele fraction , including variants of unknown signicance . time ( months ) fig a2 . 
 clinical application of circulating tumor cells and circulating tumor dna in uveal melanoma purpose to evaluate the feasibility of using circulating tumor cells ( ctcs ) and circulating tumor dna ( ctdna ) for the management of uveal melanoma ( um )  . patients and methods low - coverage whole - genome sequencing was used to determine somatic chromosomal copy number alterations ( scnas ) in primary um tumors , ctdna , and whole - genome amplified ctcs . 
ctcs were immunocaptured using an antimelanoma associated chondroitin sulfate antibody conjugated to magnetic beads and immunostained for melanoma antigen recognised by t cells 1 ( mart1 ) / glycoprotein 100 ( gp100 ) / s100 calcium - binding protein ( s100 )  . 
ctdna was quantified using droplet digital polymerase chain reaction assay for mutations in the gnaq , gna11 , plc4 , and cysltr2 genes . results scna analysis of ctcs and ctdna isolated from a patient with metastatic um showed good concordance with the enucleated primary tumor . 
in a cohort of 30 patients with primary um , ctcs were detected in 58% of patients ( one to 37 ctcs per 8 ml of blood ) , whereas only 26% of patients had detectable ctdna ( 1.6 to 29 copies / ml )  . 
however , the frequent detection of ctcs in patients with early - stage um supports a model in which ctcs can be used to derive tumor - specific scna relevant for prognosis . 
monitoring of ctdna after treatment of the primary tumor allowed detection of metastatic disease earlier than 18f - labeled fluorodeoxyglucose positron emission tomography in two patients . conclusion the presence of ctcs in localized um can be used to ascertain prognostic scna , whereas ctdna can be used to monitor patients for early signs of metastatic disease . 
2018 by american society of clinical oncology introduction uveal melanoma ( um ) is the most common intraocular malignancy.1 despite successful control of the primary tumor within the eye , metastatic disease ultimately develops in up to 50% of patients , predominantly in the liver . 
currently , there are limited therapeutic options for metastatic um , and as a result there is a high mortality rate.2 extensive analysis of primary ums has defined molecular features of the tumor cells that predict , with a high degree of accuracy , a patients risk for development of metastases . 
biomarkers of poor prognosis include histopathological features of the tumor ; somatic copy number alterations ( scnas ) , such as loss of chromosome 3 , 6q , and 8q3 , 4 ; bap1 mutations5 , 6 ; and the differential expression of marker genes that include well - characterized cancer - associated factors.7 , 8 other features , such as gain in 6p and mutations in eif1ax and sf3b1 , are associated with better prognosis.4 , 6 given that metastasis in um arises from hematogenous dissemination , investigation of circulating tumor cells ( ctcs ) and circulating tumor dna ( ctdna ) could provide a unique opportunity for genetic analysis of the patients tumor through a simple and safe blood test . 
previous research has indicated that ctcs harbor genetic profiles representative of the primary tumor9 and can be used to detect tumor - specific mutations in other cancers.9 - 14 in um , amplification aaron beasley timothy isaacs muhammad a . 
gray , phd , school of medical and health sciences , edith cowan university , 270 joondalup dr , joondalup , wa 6027 , australia ; e - mail : e.gray@ecu. 
 of dna from a single ctc has been reported , with comparative genomic hybridization array showing copy number abnormalities associated with poor prognosis.15 a recent study described the detection of loss in chromosome 3 in ctcs using a modified fluorescence in situ hybridization technique as a method to aid prognostication of um patients likely to metastasize.16 an additional blood - based marker , commonly used to evaluate tumor burden and tumor - specific genetic features , is ctdna.17 , 18 the high proportion of recurrent hot spot mutations in um enables the opportune detection of ctdna using droplet digital polymerase chain reaction ( pcr )  . 
ums carry mutually exclusive activating mutations in gnaq , gna11 , plc4 , and cysltr2 , encompassing more than 90% of um patients.4 , 19 - 22 bidard et al18 detected ctdna in 84% of patients with um with metastatic disease and found ctdna levels to be an independent prognostic factor for both progression - free survival and overall survival.18 however , the presence and prognostic significance of ctdna in patients with primary um without detectable metastatic disease has yet to be evaluated . given that um harbors distinct scnas that correlate with poor prognosis ( l1p , l3 , l6q , g8q ) and good prognosis ( g6p ) , 4 their detection in ctcs may offer a minimally invasive method for prognostication . 
furthermore , because ctdna is highly correlated with tumor volume , 18 it may offer a minimally invasive method for detection of metastatic disease . here , we evaluated the viability of ctc and ctdna as suitable biomarkers to derive prognostic information in um . 
the blood of patients with primary um was analyzed for both the number of ctcs immunomagnetically captured using melanoma - associated chondroitin sulfate proteoglycan ( mcsp ) and the level of plasma ctdna . 
finally , we showed that ctdna monitoring allowed early detection of metastatic disease in two patients with um . western australia , were enrolled in the study between march 2014 and november 2016 . 
um was diagnosed by clinical and ultrasound examination performed by a specialist ophthalmologist to evaluate the size and location of the intraocular tumor , including the presence of ciliary body involvement . 
for ctc quantification , blood was collected in vacutainer k2 edta tubes ( bd biosciences , franklin lakes , nj ) , stored at 4c , and processed within 24 hours . 
 plasma was isolated by double centrifugation at 1 , 600 g for 10 minutes and stored at 80c . ctc capture , quantification , and lowpass whole - genome sequencing ctc isolation was adapted from a previously described protocol , 23 detailed in the data supplement . 
one hundred nanograms of whole - genome amplified ( wga ) dna ( data supplement ) was used to construct 200 - bp sequencing libraries using an neb next ultra fragment library kit ( new england biosciences , ipswich , ma ) and barcoded using the ion xpress barcode adapters 1 - 96 kit ( life technologies , carlsbad , ca )  . 
somatic mutations and scnas were analyzed using ion reporter 4.6 ( life technologies )  . ctdna quantification cell - free dna ( cfdna ) was extracted from 1 to 5 ml of plasma using a qiaamp circulating nucleic acid kit ( qiagen , hilden , germany ) according to the manufacturers instructions and stored at 80c . 
comparison between the genetic profile of the primary tumor , cell - free dna ( cfdna ) , two circulating tumor cells ( ctcs ) , and a single peripheral blood mononuclear cell ( pbmc ) in a patient with metastatic uveal melanoma . 
cells were stained with a combination of antibodies against the melanoma markers melanoma antigen recognised by t cells 1 ( mart1 ) / glycoprotein 100 ( gp100 ) / s100 calcium - binding protein ( s100 ; green ) , cd45 ( red ) , and 4 ' , 6 diamidino - 2 - phenylindole ( dapi ; blue ) , taken at 200 magnification . 
 ( c ) whole - genome sequencing somatic chromosomal copy number alteration profile of primary formalin - fixed paraffin - embedded tumor , cfdna , two ctcs , and a single pbmc . 
 tumor volume was calculated using a formula previously described.24 the numbers of captured ctcs in patients with and without monosomy of chromosome 3 were compared using a nonparametric mann - whitney u test . 
statistical analyses were performed using graphpad prism version 5.0 ( graphpad software , la jolla , ca )  . results analysis of somatic copy number alterations in ctcs from a patient with um with metastatic disease ctcs were isolated from peripheral venous blood of a patient with metastatic um ( patient 640 )  . 
in march 2017 , the patient was diagnosed with metastatic um , with bone marrow activity in the spine , pelvis , ribs , and both femora ( fig 1a )  . 
the accuracy and reliability of this wga method for the analysis of single um for assessment of scna were first demonstrated using individual cells from three um cell lines ( data supplement )  . two ctcs and a pbmc provided suitable wgadna material for whole - genome sequencing . 
multiplex ligand dependent probe amplification ( mlpa ) analysis of the primary tumor confirmed the chromosomal gains of 6p and 8p / 8q and the lack of evidence for scnas of 1p and 3p / 3q ( data supplement )  . 
 the two isolated ctcs also showed overlapping chromosomal gains and losses in comparison with the primary tumor , despite the primary tumor being removed 2 years earlier ( fig 1c )  . 
additional alterations found in ctcs comprised a gain in 6q in both cells , a gain of chromosome 22 in ctc1 , and a loss of chromosome 10 in ctc2 . 
the pbmc analyzed did not harbor any scnas ( fig 1c ) , same as multiple pbmcs used as negative controls in the validation experiments ( data supplement )  . 
the gna11 q209l mutation was detectable in dna derived from the primary tumor and in wga ctcs . sequencing of cfdna from the same blood sample from which ctcs were isolated also showed similar chromosome gains , with trends toward gains in some chromosomal alterations found in the primary tissue ; however , these did not reach the threshold to be called a true gain ( fig 1c )  . 
 droplet digital pcr targeting the gna11 q209l mutation in the cfdna indicated the presence of 15 , 460 copies / ml of mutant dna in plasma , with a 20.2% frequency abundance relative to normal dna . 
thus , despite the significant abundance of ctdna , this compartment is not as sensitive as ctcs for the analysis of scnas . these results illustrate that tumor - associated scna can be ascertained through genetic analysis of ctcs , providing important prognostic information . 
ctcs were identified by positive staining for melanoma antigen recognised by t cells 1 ( mart1 ) / glycoprotein 100 ( gp100 ) / s100 calcium - binding protein ( s100 ) and negative staining for cd45 ( fig 2a )  . 
a total of 15 of 26 ( 58% ) individuals with assessable results had at least one ctc in 8 ml of blood , with a range of one to 37 ctcs detected , and 14 ( 54% ) patients had two or more detectable ctcs . 
screening for all of these mutations was necessary in our study , because for most patients fine - needle aspirate biopsies were either not performed or provided limited amounts of dna that was used for mlpa testing and , therefore , we were unable to determine the tumors driver mutations before testing for ctdna . 
instead , all patient blood samples were tested for these mutations , because they have been reported to occur in > 90% of ums.19 , 20 , 22 we detected ctdna in eight of the 30 patients tested ( 23% ; range , 1.6 to 29 copies ) ; two patients had a gnaq q209l mutation , two had a gna11 q209l mutation , one had a gna11 q209p mutation , one had a gna11 r183c mutation , one had a plc4 d630y mutation , and one had a cysltr2 l129q mutation . 
green fluorescence ( af488 , mel ) indicates staining with a combination of antibodies against the melanoma markers mart1 / gp100 / s100 ; red fluorescence ( phycoerythrin [ pe ] ) indicates cd45 positivity ; and blue fluorescence ( 4 ' , 6 - diamidino - 2 - phenylindole , dapi ) indicates the presence of a nucleus . 
no correlation was found between ctc , ctdna , and tumor size . detectable mutations , ctdna levels were correlated with tumor size ( largest basal / apical diameter / volume ; figs 2g - 2i )  . ctdna for detection of metastatic um we also analyzed ctdna in eight patients with metastatic um . 
 retrospective analysis of longitudinal samples collected from two patients indicated that detection of ctdna preceded radiologic recognition of liver metastases . patient 656 had an enucleation 3 months before enrollment and a history of low - grade lymphoproliferative disorder and pulmonary embolis pathology of the enucleated tumor confirmed a choroidal melanoma , with callender classification mixed and no angiolymphatic invasion . 
a positron emission tomography ( pet ) scan 4 weeks before enrollment showed mildly fluorodeoxyglucose ( fdg ) - avid bilateral pelvic and inguinofemoral lymphadenopathy consistent with her history of a low - grade lymphoproliferative disorder . 
characteristics of patients with confirmed metastatic disease patient baseline ctdna ( copies / ml ) ctdna mutation primary um location ( eye ) gna11 q209l 5 , 745 3 , 300 15 , 160 gnaq q209l gna11 q209l gna11 q209l gna11 q209l gna11 q209l note . 
 ( c ) plasma ctdna levels in longitudinally collected samples from patient 433 with um , before and after the development of overt metastatic disease as shown by fluorodeoxyglucose positron emission tomography imaging of the liver . 
however , ctdna levels significantly increased to 1 , 380 copies / ml by week 96 , with the concurrent imaging indicating new extensive metastases to the left lobe of the liver and new bone metastases . 
these results illustrate that for um , ctdna can be used to track disease burden and has the potential to provide a complementary measure in addition to imaging . discussion our results provide the proof of concept for using blood - based biomarkers for prognostication and routine monitoring of patients with um . 
these preliminary findings warrant additional clinical studies to validate the use of these two biomarkers for the management of um . previously , scna profiles of the primary tumor have been shown to accurately identify patients with um at risk for developing metastatic disease.4 , 25 although fine - needle aspirate biopsies are performed worldwide , genetic testing of um primary tumors can be hampered by patients declining biopsy because of the perceived invasiveness of the procedure and the preferred use of sight - conserving therapies . 
nevertheless , up to 50% of patients with um will develop metastatic tumors after either short or long latency periods.26 thus , development of pre - emptive adjuvant therapies may be an important strategy for improving survival . 
in this context , the provision of a blood test for the identification of prognostic scna from ctcs would allow identification of patients at risk , aiding triaging of patients for clinical trials and more frequent systemic surveillance for metastatic disease . most ctc studies in um have been limited to ctc quantification . 
however , they have failed to find significant association between the levels of ctcs and disease prognosis.27 , 28 similarly , we found that most patients have detectable ctcs irrespective of the predicted propensity of their tumor to metastasize , indicating that the presence of these ctcs may not be associated with metastatic disease risk . 
however , our ctc detection was restricted to the detection of mcsp - expressing cells , and thus we cannot exclude that ctcs expressing other cell surface markers may also provide prognostic information , as shown in some studies.28 , 29 nevertheless , the opportunity to examine the genomic features in ctcs may offer a more accurate indication of patient metastatic risk , in comparison with simple quantification of ctcs . 
on the basis of the results presented here , methodologies to enhance ctc capture16 , 28 are needed for the optimal implementation of ctcs as a viable alternate source of tumor genetic material from which prognosis can be derived for most patients . sequencing of the matching cfdna revealed several large chromosomal gains , but because of the high abundance of normal cfdna , we could only detect trends toward chromosomal amplifications found in the ctcs and primary tumor . 
 patient 640 ( fig 1 ) had a high disease volume , with 15 , 460 copies / ml of gna11 q209l ctdna with a fractional abundance of 20% , whereas by contrast the highest ctdna level in our primary um study was found to be 29 copies / ml , with a fractional abundance of < 1% ; therefore , sequencing of patients with localized disease for scna profiles may prove ineffective . 
 previous studies investigating scna in cfdna similarly required the presence of a large fraction of ctdna present to obtain results similar to the tissue of origin.30 thus , although ctdna is much easier to isolate , analysis of scna in ctcs should prove to be a more effective means of analyzing prognostic scna . we also showed that ctdna is not commonly detectable in blood of patients with localized um . 
our findings in this study indicate that the low levels of detectable ctdna in patients with primary disease are not suitable for screening of patients at a high risk of developing metastasis . 
however , given the high proportion of hotspot mutations in um , 4 ctdna analysis may be a feasible minimally invasive method of monitoring metastatic disease burden and disease progression , as we have exemplified here . in conclusion , our study underscores the potential clinical use of liquid biopsy for um . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . aaron beasley no relationship to disclose muhammad a . 
khattak honoraria : msd oncology , novartis , merck serono consulting or advisory role : bristol - myers squibb , merck serono speakers ' bureau : merck serono , msd oncology , novartis research funding : msd oncology travel , accommodations , expenses : msd oncology , amgen , merck serono fred k . 
pereira no relationship to disclose kyle yau no relationship to disclose jacqueline bentel no relationship to disclose tersia vermeulen no relationship to disclose leslie calapre no relationship to disclose michael millward consulting or advisory role : roche , bristol - myers squibb , astrazeneca , merck sharp & dohme , novartis , boehringer ingelheim travel , accommodations , expenses : roche , merck sharp & dohme , bristol - myers squibb , astrazeneca elin s . 
gray research funding : merck sharp & dohme patents , royalties , other intellectual property : provisional patent on a blood test to detect melanoma based on auto - antibody detection travel , accommodations , expenses : bio - rad laboratories timothy isaacs travel , accommodations , expenses : pfizer melanie r . 
we also thank pauline zaenker , ashleigh mcevoy , and danielle bartlett for their phlebotomy services , william cunningham , md , and rian vijoen , md , for assisting with patient recruitment at royal perth hospital , and karen shakespeare for gathering the clinical data . affiliations aaron beasley , muhammad a . 
khattak , jaqueline bentel , and tersia vermeulen fiona stanley hospital , murdoch , western australia , australia . support supported by an edith cowan university postgraduate scholarship and a cancer council western australia honours scholarship ( a.b. ) ; a fellowship from the cancer research trust ( e.s.g. ) ; raine medical research foundation priming grant , ophthalmic research institute of australia grant , and western australia cancer council grant no . 
heitzer e , auer m , gasch c , et al : complex tumor genomes inferred from single circulating tumor cells by array - cgh and next - generation sequencing . 
gasch c , bauernhofer t , pichler m , et al : heterogeneity of epidermal growth factor receptor status and mutations of kras / pik3ca in circulating tumor cells of patients with colorectal cancer . 
carter l , rothwell dg , mesquita b , et al : molecular analysis of circulating tumor cells identifies distinct copy - number profiles in patients with chemosensitive and chemorefractory small - cell lung cancer . 
dago ae , stepansky a , carlsson a , et al : rapid phenotypic and genomic change in response to therapeutic pressure in prostate cancer inferred by high content analysis of single circulating tumor cells . 
suesskind d , ulmer a , schiebel u , et al : circulating melanoma cells in peripheral blood of patients with uveal melanoma before and after different therapies and association with prognostic parameters : a pilot study . 
 addition of chemotherapy is associated with decreased survival in early - stage ( t1 - 2n0m0 ) glottic squamous cell carcinoma treated with definitive radiotherapy purpose the role of chemoradiation ( crt ) in treating patients with early - stage glottic squamous cell carcinoma ( scc ) , especially for t2n0m0 glottic scc with impaired vocal cord mobility , remains unexplored . 
we sought to evaluate the impact of crt on survival in early - stage glottic scc by using the seer database . patients and methods we included patients with localized ( t1 - 4n0m0 ) glottic scc ( n = 4 , 743 ) diagnosed between 2004 and 2014 and treated with definitive radiotherapy ( rt ) alone , crt , or laryngectomy alone in the seer database . 
disease - specific mortality ( dsm ) was evaluated via multivariable regression using a competing risk model that accounts for other - cause mortality as a competing risk event for dsm . 
2019 by american society of clinical oncology introduction definitive treatment with radiotherapy ( rt ) for t1 - 2n0m0 squamous cell carcinomas ( scc ) of the glottis remains a standard of care in the united states and can help to preserve voice quality . 
several studies have shown lc rates for t1 and t2 glottic cancer treated with transoral laser microsurgery to range from 77% to 92% and 66% to 88% , respectively , 1 - 5 whereas open surgical procedures for glottic scc demonstrated a lc rate of 86% to 98%.2 , 6 - 10 the lc rate for early - stage glottic cancer treated with definitive rt depends significantly on t stage , with studies showing lc rates of 82% to 94% for t1 disease11 - 17 and 61% to 80% for t2 disease.11 - 13 , 15 - 19 the decreased lc for t2 glottic cancers treated with rt alone was thought to be attributed to a subset of patients with impaired vocal cord chenyang wang amar u . 
 the radiation therapy oncology group 9111 trial established concurrent crt as the standard treatment of t3 glottic cancer.28 the lc benefit associated with crt in the setting of t3 glottic cancer has raised the question of whether the subset of patients with t2 glottic cancer with impaired vocal cord mobility should be treated with crt as well , given the suboptimal outcomes with rt alone . 
seer routinely collects data on patient demographics , primary tumor site , morphology , stage at diagnosis , first course of treatment , and follow up for vital status . for this study , we selected patients diagnosed with n0m0 scc of the glottic larynx diagnosed from january 1 , 2004 , to december 31 , 2014 , who underwent definitive rt , crt , or subtotal or total laryngectomy . 
we excluded patients who had non - scc histology , unknown tumor grade , unknown race , unknown surgical status , unknown t stage , more than one primary malignancy , no active follow - up , or age younger than 18 years . 
this study was reviewed and approved by the institutional review board of the university of california , los angeles . statistical analysis extracted variables included age at diagnosis , sex , race , year of diagnosis , t stage , and tumor grade . 
analysis of variance , kruskal - wallis rank sum tests , and 2 tests were used to characterize differences in variables of interest between different treatment cohorts for continuous , ordinal , and categorical variables , respectively . 
 follow - up time was calculated via the reverse kaplan - meier method described by schemper and smith , 30 in which alive at last follow - up was designated the event of interest while death from any cause was censored . 
seer determines death and cause of death via algorithms to process death certificates , taking into account causes of deaths in conjunction with tumor sequence , site of original cancer diagnosis , and comorbidities . 
 to account for other - cause mortality ( ocm ) as a competing risk for dsm , multivariable regression analyses on the basis of the competing risk model as described by fine and gray31 were applied to the survival analysis while adjusting for age , year of diagnosis , sex , race , t stage , definitive treatment modality , and tumor grade . 
for t2n0 disease , an additional subset analysis was carried out on the basis of vocal cord impairment status to investigate whether patients with t2n0 disease with vocal cord impairment selectively benefit from crt . 
the validity of proportional hazards assumption was evaluated via schoenfeld residuals . to adjust for covariables and determine whether the addition of chemotherapy to rt affected dsm in patients with t1 - 4n0m0 glottic scc , we adopted propensity score matching ( psm ) via a nearest neighbor approach to balance patient characteristics including age , year of diagnosis , sex , race , and tumor grade between the rt and crt cohorts for each t stage . 
propensity score , first defined by rosenbaum and rubin32 as the probability of treatment assignment on the basis of a set of observed baseline patient characteristics , provides the means to retrospectively balance distribution of patient characteristics and thereby minimize effects of unmeasured confounders on treatment outcomes . 
the cumulative incidences of dsm and ocm after psm were plotted to illustrate the difference between rt and crt cohorts for each t stage , and their distributions were evaluated via a k - sample test described by gray.33 all statistical analyses were carried out using r version 3.4.1 ( r foundation for statistical computing , vienna , austria ) with two - sided testing and a statistical significance threshold of p = .05. results patient characteristics patient characteristics , stratified by definite treatment modality consisting of rt , crt , and subtotal or total laryngectomy , are listed in table 1 . 
patients who underwent rt alone tended to be older , with a median age of 66 years , compared with patients who underwent crt and surgery , who had median ages of 62 and 63 years , respectively . 
these patients were also more likely to present with higher tumor grade . multivariable regression for dsm and the results of the multivariable regression for dsm according to the competing risk model , accounting for ocm as a competing risk , are listed in table 2 . 
 plots of schoenfeld residuals versus failure times are shown in appendix figure a2 . subgroup multivariable regression for dsm using a competing risk model stratified by t stage the results of subgroup multivariable regression for dsm stratified by t stage are listed in table 3 . 
more recent year of diagnosis was not associated with any significant difference in dsm . cumulative incidence of dsm and ocm for rt and crt after psm patient characteristics stratified by definitive treatment modality before and after psm are listed in appendix tables a1 and a2 , respectively . 
a recent phase ii trial of concurrent crt with a fluoropyrimidine agent for t2n0m0 scc of the larynx ( 70% ) , oropharynx ( 16% ) , and hypopharynx ( 14% ) demonstrated a 3 - year lc rate of 89.0% and a 3 - year overall survival rate of 97.2%.29 another study by bhateja et al25 demonstrated superior lc in patients receiving crt for t2b - 3n0 - 2 glottic cancers compared with patients receiving rt alone for t2n0m0 glottic cancer with impaired vocal cord mobility ( t2bn0 ) , suggesting a potential therapeutic benefit of crt in this setting . 
in this study , 51.6% of the t2bn0 patients treated with rt alone received altered fractionation ( > 2.1 gy per fraction or twice - a - day fractionation ) compared with 38.5% of patients in the crt cohort . 
of note , the crt cohort contained only one patient with t2bn0 and one patient with t2bn1 ; therefore , it is difficult to assess the impact of crt on patients with t2bn0 disease . 
in accordance with the results from bhateja et al , 25 our study also demonstrated that patients with t3n0 glottic scc had decreased dsm with crt compared with rt alone . 
cumulative incidence plots of disease specific mortality ( dsm ) and other - cause mortality ( ocm ) for glottic squamous cell carcinoma at each t stage after propensity score matching . 
to our knowledge , our data from this study are the first to question the utility of chemotherapy in addition to rt for t2bn0 glottic scc . one possible hypothesis for the increase in dsm for patients with t1 - 2n0 glottic scc treated with crt may be treatment prolongation as a result of increased toxicity . 
rt treatment time prolongation past 40 days has been shown to correlate with decreased lc from 95% to 100% to 79% to 84%.13 , 34 additional factors that can contribute to the observed survival detriment in crt may be lower total rt dose and dose per fraction as a result of the higher combined toxicity with chemotherapy , because it has been established that total rt dose less than 65 gy results in decreased lc.2 , 10 , 13 , 14 , 35 furthermore , a randomized prospective study has shown that 2.25 gy per fraction , as compared with 2 gy per fraction , results in significantly improved lc.36 meanwhile , the survival benefit associated with chemotherapy in locally advanced glottic scc may be attributed to improved lc secondary to chemotherapy sensitization and / or additional regional and distant control , which could compensate for the decrease in lc associated with potential treatment prolongation . this study had several important limitations inherent to the seer database , including lack of information on radiation dose , treating physician experience , the type of treatment institution , and sequence of chemotherapy relative to rt , which prevented us from distinguishing between concurrent versus induction chemotherapy . 
however , given the relatively rare use of induction chemotherapy in the modern era for t1 - 2n0 glottic scc , it is reasonable to assume that the patients in the crt cohort of this study predominantly received concurrent crt . 
steinberg honoraria : viewray consulting or advisory role : viewray research funding : viewray , accuray travel , accommodations , expenses : viewray robert chin travel , accommodations , expenses : varian medical systems , brainlab affiliations chenyang wang , amar u . 
spector jg , sessions dg , chao ksc , et al : stage i ( t1 n0 m0 ) squamous cell carcinoma of the laryngeal glottis : therapeutic results and voice preservation . 
laccourreye o , muscatello l , laccourreye l , et al : supracricoid partial laryngectomy with cricohyoidoepiglottopexy for early glottic carcinoma classified as t1 - t2n0 invading the anterior commissure . 
marshak g , brenner b , shvero j , et al : prognostic factors for local control of early glottic cancer : the rabin medical center retrospective study on 207 patients . 
le q - tx , fu kk , kroll s , et al : influence of fraction size , total dose , and overall time on local control of t1t2 glottic carcinoma . 
cellai e , frata p , magrini sm , et al : radical radiotherapy for early glottic cancer : results in a series of 1087 patients from two italian radiation oncology centersi . 
warde p , osullivan b , bristow rg , et al : t1 / t2 glottic cancer managed by external beam radiotherapy : the influence of pretreatment hemoglobin on local control . 
mendenhall wm , parsons jt , million rr , et al : t1t2 squamous cell carcinoma of the glottic larynx treated with radiation therapy : relationship of dose - fractionation factors to local control and complications . 
frata p , cellai e , magrini sm , et al : radical radiotherapy for early glottic cancer : results in a series of 1087 patients from two italian radiation oncology centersii . 
garden as , forster k , wong p - f , et al : results of radiotherapy for t2n0 glottic carcinoma : does the 2 stand for twice - daily treatment ? int j radiat oncol biol phys 55 : 322 - 328 , 2003 20 . 
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hoang , md1 , 2 ; rui yang , md1 , 2 ; daniel weiser , md2 ; jonathan morris , md3 ; richard gorlick , md4 ; jonathan b . 
we have identied a single patient who was initially diagnosed with a benign osteoblastoma and subsequently presented years later with a high - grade osteosarcoma in the same anatomic region . 
a biopsy sample of the mass demonstrated conventional - type osteoblastoma with focal epithelioid features for which the patient underwent intralesional curettage ( fig 2 )  . subsequent imaging demonstrated a persistent abnormal signal in the sacrum , and a plan of continued surveillance was agreed upon . 
over the ensuing 4 - year imaging failed to demonstrate appreciable period , radiographic change , and the patients clinical course remained stable . five years later , the patient returned with increasing paimaging demonstrated an increase in the size of the mass , and an urgent open sacral biopsy was performed ( fig 3 )  . 
staging studies performed at that time were negative for distant disease . neoadjuvant chemotherapy was started in accordance with childrens oncology group protocol aost0331 ; however , because of severe pain , patient required semi - urgent surgical intervention and did not complete planned neoadjuvant treatment . 
the decision was made to manage conservatively , and as of july 2019 , surveillance demonstrated no evidence of disease . methods including the osteoblastoma , patient consent and institutional review board approval were obtained before study initiation . 
sequencing and data analysis were performed by novogene technology ( beijing , china ) to evaluate for structural variants , copy number variation , single nucleotide variants , and insertions / deletions . 
germline - matched comparison from each tumor was undertaken to evaluate for somatic changes . to evaluate for pathogenic variants , somatic analysis on small variants focused on changes that would elicit a coding or splice site change and were not common polymorphisms ( allele frequency , 0.01 in 1 , 000 genomes and exac databases )  . 
furthermore , we evaluated whether any variants were found in cancer mutational hotspots ( recurrence of more than ve in cosmic [ catalog of somatic mutations in cancer ] ) or were a truncating variant in a known tumor suppressor gene ( cancer gene census )  . 
 geller et al context key objective does genomic analysis of a patient who was initially diagnosed with a benign osteoblastoma and subsequently presented years later with a high - grade osteosarcoma in the same anatomic region demonstrate evidence of malignant transformation ? knowledge generated there was near - zero overlap in the somatic small variants , somatic copy number variation pattern , and predicted structural variants in the osteoblastoma compared with the osteosarcoma . 
findings from this study argue against malignant transformation as an evolving or stepwise process and conversely support two distinct neoplasms with dissimilar genetic makeups . relevance this study performed an in - depth genetic characterization of two distinct tumors that historically have been believed to be along the same spectrum of disease . 
in addition , a germline mutation in brip1 was discovered , which lends itself to additional investigation . pathogenic or likely pathogenic for a cancer predisposition syndrome . results whole - genome sequencing resulted in high - quality data , which generated an average 63 million reads per tumor , with more than 98% usable on the basis of quality metrics . 
after ltering and duplicate removal , mapping to hg19 resulted in an average sequencing depth of 29 and 32 for the benign and malignant tumors , respectively , and at least 4 coverage in more than 99% of the genome . somatic variant analysis revealed approximately 98 , 000 and 141 , 000 high - condence small variants in the benign and malignant tumor , respectively , which corresponded to somatic mutational burdens of 33 and 47 mutations / megabase ( mb )  . 
in the osteosarcoma , a single variant recurrent in cosmic of unclear oncologic signicance , znf429 p.n426k , was found as well as truncating mutations in two tumor suppressor genes , daxx and ncor1 . structural and copy number variation analysis revealed signicant alterations in both tumors . 
a striking number of somatic inversion events were found in both tumors ( approximately 1 , 750 per tumor ) , particularly compared with other structural changes , such as translocations , deletions , or tandem duplications ( each approximately 10 to 30 per tumor )  . 
 ( a ) a 2015 hematoxylin and eosinstained low - power ( 10 ) micrograph demonstrates ndings consistent with high - grade osteosarcoma , including high cellularity , multiple foci of tumor necrosis , and osteoid formation . 
 ( b ) a 2015 hematoxylin and eosinstained high - power ( 40 ) micrograph that demonstrates pleiomorphic and bizarre cellular features . overlap in the predicted structural variants in the osteoblastoma compared with the osteosarcoma . germline analysis using the methods described identied a single pathogenic germline mutation , brip1 r798x , identied with high condence in all three samples . 
a summary of genomic ndings is listed in table 1 . discussion the notion that osteoblastoma can undergo malignant transformation to osteosarcoma dates back to an original report in 1967.1 since then , 24 cases have appeared in the literature , none of which investigated the transformation event on a genomic level1 - 20 ( table 2 )  . 
given the lack of objective genetic data and short time to transformation in some cases , there is a distinct possibility that some of these reports represent coincidence or diagnostic error . a few prior reports have attempted to document malignant transformation through a limited analysis of sequential biopsies samples.4 , 18 , 21 however , these tumors were characterized only in terms of ploidy , thereby limiting understanding of the transformation process.4 , 18 , 21 although these reports suggested that transformation is associated with an increase in dna content , only one patients tumor showed an observable change in dna content over time . moreover , in the current study , we have demonstrated the benign and malignant tumors to have distinct genomic proles with almost no overlap in somatic changes . 
whether these tumors are driven in part by an unrecognized anatomic predisposition remains speculative . we also identied a germline mutation in the brip1 tumor suppressor gene , a member of the recq helicase family associated with brca1.22 , 23 brip1 mutations are associated with a predisposition to breast cancer , ovarian cancer , and fanconi anemia , 22 , 24 , 25 and brip1 is known to play a critical role in dna repair , with knockdown leading to chromosomal instability.26 of note in our patient , both the benign and the malignant tumors exhibited a higher - thantypical somatic mutation rate compared with most pediatric malignancies27 as well as a high number of structural variants with a preponderance of inversion events , which suggests a functional role of this germline nding . 
we have speculated that these changes may have accumulated because of brip1 partial dysfunction that led to chromoinstability and impaired dna crosslink repair.26 somal admittedly , denitive conclusions cannot be drawn on the basis of a single patient , and additional investigation seems warranted . although osteoblastoma infrequently has been associated with mutations involving rb , tp53 , fos , fosb , d - jun , and mdm2 , the genetic landscape of this tumor is still largely unknown.28 - 31 the osteoblastoma in this report is similar to previous reports in that we observed many structural table 1 . 
many osteosarcomas demonstrate chromosomal abnormalities that commonly involve tumor suppressor genes or dna helicases.32 , 33 however , osteosarcoma generally is recognized as exhibiting tremendous genetic complexity with no clearly identiable genetic driver . this heterogeneity may stem from chromothripsis , 34 - 36 markers of which have been described in 2% to 3% of all cancers and up to 33% of osteosarcomas . 
although the current case did not exhibit evidence of chromothripsis , it demonstrates an otherwise typical genomic prole for osteosarcoma with a high degree of chromosomal instability , increased ploidy , and truncating point mutations in several tumor suppressor genes . in some cases , in conclusion , although malignant transformation of osteoblastoma to osteosarcoma historically has been accepted , review of the literature has revealed a paucity of convincing evidence , and controversy has persisted . 
to our knowledge , this study is the rst to report an in - depth genetic characterization of two distinct tumors within the same patient and the same anatomic location . 
findings from this study argue against malignant transformation as an evolving or stepwise process and conversely supports two distinct neoplasms with dissimilar genetic makeups . this report also has uncovered a germline truncating mutation in brip1 in this patient , which raises the question of whether this serves as an underlying predisposition for both tumors . 
geller , md , monteore greene medical arts pavilion , 3400 bainbridge ave , bronx , ny 10467 ; e - mail : dgeller@monteore.org. support supported by clinical and translational science awards catalytic seed grant ul1 tr001073 ( d.s.g. ) from the national center for advancing translational sciences , a component of the national institutes of health . the content is solely the responsibility of the authors and does not necessarily represent the ofcial views of the national institutes of health . 
j bone joint surg am 57 : 424 - 426 , 1975 stutch r : osteoblastomaa benign entity ? orthopaed rev 4 : 27 , 1975 grace j , mccarthy s , stankovic r , et al : malignant transformation of osteoblastoma : study using image analysis microdensitometry . 
 genomic analysis does not support malignant transformation kunze e , enderle a , radig k , et al : aggressive osteoblastoma with focal malignant transformation and development of pulmonary metastases . 
sun x , brieo - enrquez ma , cornelius a , et al : fancj ( brip1 ) loss - of - function allele results in spermatogonial cell depletion during embryogenesis and altered processing of crossover sites during meiotic prophase i in mice . 
weber - lassalle n , hauke j , ramser j , et al : brip1 loss - of - function mutations confer high risk for familial ovarian cancer , but not familial breast cancer . 
radig k , schneider - stock r , mittler u , et al : genetic instability in osteoblastic tumors of the skeletal systepathol res pract 194 : 669 - 677 , 1998 31 . 
lorenz s , bary t , sun j , et al : unscrambling the genomic chaos of osteosarcoma reveals extensive transcript fusion , recurrent rearrangements and frequent novel tp53 aberrations . 
however , the clinical application of these targeted drugs and diagnostics often remains unclear . however , this approach is not newdoctors have been trying to provide personalized care with high precision for centuries . 
for example , during the last 150 years , virchow developed microscopic pathology , roentgen introduced radiographic imaging , and bloom and richardson proposed a tumor grading scale ; all represented major leaps forward in personalized medicine that we still apply.1a - 1e more recent examples include tests for estrogen receptor in breast cancer to guide endocrine therapy and evaluation of the cd20 antigen on b - cell lymphomas to guide the use of anti - cd20 monoclonal antibodies . so , whats new ? we have new and exciting tools that rival those giants on whose shoulders we stand . 
however , to deliver personalized medicine as precisely as possible , we need high levels of evidence that use of these new therapies and diagnostic tests does , in fact , improve patient care . we agree with the skeptics in oncology who question the benefit of precision oncology , but we maintain that the answer is not to declare it all hype and walk away but to continue to learn how best to use it . 
rather than hope for luck , clinicians should continue to use the scientific method to make evidence - based decisions . in may 2013 , journal of clinical oncology published a special series on precision oncology . the success of this special series led asco to recognize both the plethora of research that is occurring in this field and the need to disseminate this information effectively to the oncology community . 
the special issue of jco featured articles that outlined the current and emerging technologies for tumor genomic profiling , 1f ways to build personalized medicine infrastructure at major cancer centers , 2 and various applications of genomic profiling for different cancer diagnoses . 
it will also provide researchers and clinicians with a reliable source of high - quality precision oncology research , reviews , and commentary essential to real - world cancer care . 
in addition to original clinical research , the journal will publish articles about key care delivery issues , such as value , quality , and cost - effectiveness , and about ethical , legal , and social considerations to inform clinical practice and shape this rapidly evolving field . we are thrilled to have james m . 
ford is a professor of medicine ( oncology ) , pediatrics ( medical genetics ) , and genetics and is the director of the stanford cancer genetics clinic and the cancer genomics program at the stanford cancer institute . 
hayes , university of michigan comprehensive cancer center , ann arbor , mi ; and julie vose , university of nebraska medical center , omaha , ne corresponding author : daniel f . 
 genetic targets for personalized targeted therapies in breast and gastrointestinal cancer as well as in examination of the role of dna repair in the treatment and prevention of cancer in families and populations . 
with the commencement of the targeted agent and profiling utilization registry ( tapur ) study , asco aims to describe both the safety and the efficacy of targeted anticancer drugs to catalog genomic profiling tests and to learn about the utility of registry data . 
in addition , with the launch by asco of cancerlinq , oncologists will have access to a massive database of treatment options and results of those treatments from millions of patient records . 
vose consulting or advisory role : bio connections research funding : celgene ( inst ) , genentech ( inst ) , incyte ( inst ) , janssen biotech ( inst ) , acerta pharma ( inst ) , kite pharma ( inst ) , seattle genetics ( inst ) , novartis ( inst ) , amgen ( inst ) , bristol - myers squibb ( inst ) , allos therapeutics ( inst ) references 1a . 
hammond me , hayes df , dowsett m , et al : american society of clinical oncology / college of american pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer . 
wolff ac , hammond me , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
tbt reimbursement within the united states in the current regulatory environment is not tied to premarket evidence of clinical utility , resulting in a vicious cycle wherein low - level evidence of utility leads to poor reimbursement , thereby impeding investment in developing new , clinically valuable tbts supported by high - level evidence . 
precise one - to - one mapping of reimbursement to cost savings or cost effectiveness is precluded by an absence of formal cost - effectiveness analyses for many emerging tbts , and for more established tbts , it has become clear that such analyses may yield wildly variable , subjective estimates . 
to address these challenges , we propose a system of tiered reimbursement that rewards development of high - quality tbts within specic use contexts , supported by strong evidence of analytic validity and clinical utility . 
we propose three use contexts of tbts , each dened by its inuence on treatment decisions relative to the current standard of careopt - out , opt - in , and the use of appropriate , alternative , effective therapies ( opt - alt )  . 
the true clinical utility of a tbt is often difcult to determine , initiating a vicious cycle in which tbts are often undervalued and poorly reimbursed because of a lack of high - level evidence of clinical utility.1 an inconsistent regulatory and reimbursement environment results in an unfavorable return on investment in the research and development ( r&d ) needed to generate the high levels of evidence ( loes ) necessary to demonstrate the clinical utility of tbts.1 indeed , despite increasing recognition of the benets of personalized medicine , medicare coverage of many emerging tbts is being signicantly reduced or denied , 2 resulting in decreased investment in tbt r&d.3 reimbursement of tbts to date has been driven primarily by a combination of commodity and labor pricing ( ie , reagent and technical costs ) , market forces , and perceived clinical value . 
this is not an effective strategy to support long - term investment in high - quality tbts . we propose that objective and transparent tiers of tbt value be created based on high loes of analytic validity ( ie , accurate and reproducible measurement of a specic analyte ) and clinical utility ( ie , improves patient outcomes ) that meet minimal specied thresholds and address unmet medical needs.4 , 5 our proposal would potentially result in redistribution of health care dollars currently spent on needless or ineffective therapies while ensuring that patients who require and are likely to benet from treatment strategies receive theit will create pathways by which tbt manufacturers could receive guaranteed minimum rates of reimbursement ( fig 1 ) and thereby increase the likelihood that tbt developers obtain a return on r&d investment . 
we propose an alternative , value - based reimbursement which clinical utility can be assessed within specic use contexts , undergo formal review , and then provide a new value - based , tiered reimbursement rate . 
note that the use of appropriate , alternative , effective therapies ( opt - alt ) context includes both the initial treatment and the monitoring paradigms . fda , us food and drug administration ; icers , cost - effectiveness ratios ; qaly , quality - adjusted life - year . dinan et al proposed valuation status quo / conventional pricing holder of tbt seeks higher , value - based reimbursement value - based application to set new reimbursement cost - saving tbts opt - out evidence to reduce treatment % treatment reduction treatment cost pricing : function of costs saved cost - effective tbts evidence of beneficial treatment treatment outcomes ( qalys ) treatment costs pricing : function of icers opt - in opt - alt approved : new value - based reimbursement set formal review by fda or other regulatory body loes of robustness in the assay itself and that its use improves patient outcomes compared with not using it , respectively.4 - 6 we propose three major contexts of use for which a tbt might have clinical utility , and therefore value , designated as optout , opt - in , and the use of appropriate , alternative , effective therapies ( opt - alt ; table 1 )  . 
each of these terms refers to the clinical decision that might be made based on the results of the tbt compared with the decision made as standard of care ( soc ) without tbt results . 
these categories can be applied to the various use contexts considered for the tbt , including assessment of cancer risk / susceptibility , screening , differential diagnosis , prognosis , prediction , and monitoring.7 similar to therapeutics , the performance and therefore value of a tbt will vary within certain subpopulations of patients , underlying the need to specify the target population when assessing value within a specic use context . 
opt - out tbts have value and the potential to be directly cost saving by reducing the use of unnecessary or ineffective yet toxic and costly therapies , either by indicating that the patient does not need additional therapy ( prognosis ) or that the therapy under consideration is unlikely to work ( prediction ; table 1 )  . perhaps the most well - known example of opt - out tbts with high analytic validity and clinical utility are genomic tests used to determine whether adjuvant chemotherapy should be given to patients with estrogen receptorpositive , human epidermal growth factor receptor 2negative , and least ve node - negative breast cancer . 
there are at commercially available tbts in this category.8 , 9 the 21 - gene recurrence score ( rs ; oncotype dx ; genomic health , redwood city , ca ) is the most well studied and provides an illuminating case study . 
before introduction of the 21 - gene rs , adjuvant chemotherapy was recommended for 50% or more of patients with estrogen receptorpositive , human epidermal growth factor 2negative , node - negative breast cancer.10 the 21 - gene rs , which has high analytic validity , 11 identies a large percentage of this group of patients for whom adjuvant chemotherapy has little or no chance of improving cancer - related outcomes , and its use is now recommended for all women in this category.8 , 12 cost - effectiveness analyses of the 21 - gene rs have varied widely and involve complex and unavoidably subjective modeling that depends on assumptions made about management strategy , impact of treatment , time horizon , inputs.13 accepted patient population , and other model estimates of the impact of the 21 - gene rs assay suggest overall absolute reductions in chemotherapy of 7% to 14% in the roughly 50 , 000 women who are newly diagnosed every year in whom testing would be recommended ( table 2 )  . 
assuming an average total adjuvant chemotherapy cost of $80 , 000 ( including modern drugs and supportive care treatments ) , 14 annual chemotherapy cost savings for women in this category would be between $280 million to $560 million per year , signicantly outpacing the roughly $175 million spent on the test for these same women ( assuming $3 , 500 per test )  . 
in this case , the value of the tbt is generated by turning a minimally effective or is indicated , and unacceptable therapy into one that therefore acceptable , because of an altered risk - benet that justies or renes treatment . 
an opt - in tbt will typically increase costs , but still has value because it may result in improved long - term clinical outcomes . an example of an opt - in tbt is provided by analysis of germline susceptibility genes , such as brca1 or brca2 , or associated genes ( table 2 )  . 
in this case , the presence of a deleterious inherited germline mutation in the brca1 or brca2 ( or related ) gene substantially increases the odds of developing and dying as a result of breast and / or ovarian cancer . 
however , the risk of developing these cancers and the odds of mortality are reduced by as much as 90% and 35% to 75% with prophylactic mastectomy and salpingooophorectomy , respectively.15 , 16 in this case , these otherwise unacceptable interventions become appropriate because of the increased chances of long - term survival as a result of the intervention in a high - risk population ( table 2 )  . 
therefore , although such tests may be relatively expensive , their value in terms of lives saved , decreased long - term medical costs , and increased economic productivity and quality of life often outweigh the immediate costs of the tests and associated downstream medical expenditures . 
this tradeoff between added costs and increased quality of life is often measured through economic metrics , such as incremental costeffectiveness ratios , which are expressed in units of cost ( dollars ) per 1 year of full quality of life , or quality - adjusted life - years ( qalys )  . 
for example , the brca test listed in table 2 , when applied to the target population , saves on average 1 qaly for every $4 , 300 spent ( $4 , 300 / qaly ) .17 opt - alt tbt the opt - alt tbt paradigm is a subcategory of opt - in . 
in this case , the value of the tbt is to help select a strategy from two or more available choices that is most likely to benet the patient , thus enhancing the precision of his or her management . 
opt - alt tbts improve outcomes via the use of appropriate , alternative , effective therapies . an example of an opt - alt tbt is illustrated by somatic mutation testing in metastatic lung adenocarcinoma to drive the use of therapies directed against genomic abnormalities ( table 2 ; opt - alt : initial rx )  . 
these abnormalities occur in various genes , including egfr , kras , alk , ros1 , erbb2 , braf , ret , and ntrk1 , which are found collectively in more than 20% of patients with metastatic nonsmall - cell lung cancer using tbts on the basis of nextthese abnormalities is generation sequencing . 
each of linked to a specic therapy that targets the genomic abnormality ( eg , erlotinib and other tyrosine kinase inhibitors ) , 18 and approximately one quarter of these patients will benet from these agents . 
in contrast , chemotherapy , which has roughly the same likelihood of benet but is associated with substantially higher toxicities , remains an soc for many of the remaining patients.19 although the clinical value in terms of quality and quantity of life is established , it is a difcult and somewhat subjective exercise to determine the true economic value of these tests , which depend heavily on the relative benets of each of the drugs in each of the many settings in which they may be used across different populations . opt - out / monitoring or opt - in / monitoring tbt in an opt - in / monitoring scenario , a patient a variation on the opt - out , opt - in , or opt - alt schema is the monitoring paradigm ( table 1 )  . 
value arises from avoiding the toxicity and cost of continuing an ineffective therapy . clinically free of apparent disease , but the tbt results indicate an impending future cancer - related event which early intervention may be more effective than waiting until the event becomes clinically evident . 
an additional variant of the opt - alt / monitoring paradigm occurs if serial tbts are used as surrogates of more expensive or inconvenient , but strongly indicated , monitoring strategies . 
in this case , the patients cancer - related outcome may not be improved by the tbt per se , but the tbt helps the patient avoid other diagnostic studies , such as radiographic imaging . 
one should note that a given tbt can fall into one or more of these use context categories , depending on the disease to which the tbt is applied and the clinical scenario . as noted , the opt / monitoring paradigm has three possibilities : opt - out / monitoring , opt - in / monitoring , or opt - alt / monitoring . 
for solid malignancies , liquid biopsies include tumor - associated proteins ( eg , carcinoembryonic antigen [ cea ] / colorectal cancer ; ca125 / ovarian cancer ; prostate - specic antigen [ psa ] / prostate cancer , and muc1 , identied by ca15 - 3 or ca27.29 / breast cancer ) , circulating tumor cells , and cell - free tumor dna . opt - out / monitoring and opt - alt / monitoring tbts might be used in many settings , and their economic value may differ in each . 
introducing earlier treatment of such patients may result in higher overall survival rates , as with patients with isolated colorectal metastases detected by an increasing cea level , 22 or may not , as in patients with ovarian and breast cancers monitored with ca125 or ca15 - 3 , respectively.23 , 24 we consider serial cea for patients with a history of colorectal cancer to have high clinical utility , and we would place this tbt for this indication in tier 1 , suggesting that reimbursement should be based on more than just commodity pricing . 
in contrast , monitoring the latter two tbts has been shown in prospective randomized clinical trials to not result in longer overall survival . we would place these markers in tier 3 , or tier x , because their clinical and economic value is not clear ( table 3 )  . 
in contrast , results of a prospective , randomized clinical trial have demonstrated that identication and treatment of hepatic oligometastases in patients with stage ii to iii colorectal cancer results in a small but denite chance of cure22 and would therefore be placed in tier 1 ( table 2 )  . 
however , the development of high - level evidence of tbts in high - risk subsets might expand their indications in the future , thus offering an incentive for biotech companies to invest in generating high - level evidence demonstrating the clinically utility of tbts . in the metastatic setting , an increasing circulating tbt either directly indicates failure of the current therapy or suggests the need for more invasive imaging to determine whether the disease is progressing . 
in this case , such tbts provide both opt - out and opt - alt information because the soc is usually to change from the apparently inactive treatment to a second or later - line treatment ( opt - alt ) or to discontinue treatment altogether ( opt - out )  . 
although few , if any , prospective randomized clinical trials exist to demonstrate the clinical utility of these markers in this use context , in general , the opt - alt paradigm is considered to be sufcient to place these tbts ( at least the circulating proteins ) in tier 2 or 3 , and they are used routinely.25 our model would suggest that reimbursement for these tbts should be commensurate with their value in managing patient care . having dened the tbt use contexts and their relative value possibilities , we propose a semiquantitative , hierarchal , tiered - based approach to help determine a reimbursement structure for tbts ( table 3 )  . 
tiers dene tbts ranging from high analytic validity and clinical utility ( tier 1 ) to less accurate analytic validity or lower evidence of clinical utility ( tiers 2 and 3 ) and tbts without conrmed clinical value ( tier x )  . 
in each case , assessment of a tbt is dependent on its specic use context as an optout , opt - in , opt - alt , or opt / monitoring tbt ( table 1 )  . 
these savings could be redistributed within the health care systeone option would be to simply use the savings , especially those generated by opt - out tbts , to reduce the cost of care . 
we maintain that this approach is unattractive , because it is likely to stie innovation for new tbts , reduce the quality of data regarding a tbts clinical utility , and further enhance the existing vicious cycle.1 rather , we argue that the value of generating high - quality tbts should instigate a redistribution of health care spending directly ( in the case of opt - out tbts ) from therapeutics to diagnostics or indirectly ( in the case of opt - in tbts ) , on the basis of the case that immediate costs should be borne by society to obtain long - term cost effectiveness . 
such redistribution of nancial resources would then stimulate greater investment into diagnostics to support r&d and reward innovation and entrepreneurship . our tiered approach provides a clear and achievable opportunity for companies to develop high - quality tbts with a guarantee for a commensurate return on investment . 
rather , tbt proposes a strategy to redistribute third - party reimbursement from unnecessary or ineffective therapies to support tbts with high clinical utility to provide a baseline assurance of reimbursement and return on investment for entities investing in tbt development . 
importantly , tbts in tiers with poor evidence ( tier 3 ) or those that are purely informative with unclear implications for management ( tier x ) cannot be expected to recoup health care resources , and we argue that such tbts should receive little , if any , third - party reimbursement . although our system does not directly reward a tbt for having slightly better performance than another for a similar indication , it does so indirectly . 
therefore , a static determination of cost effectiveness for a tbt is unrealistic . in the example of the 21 - gene rs , the cost of adjuvant chemotherapy has increased dramatically in breast cancer , in part as a result of routine hematopoietic growth factor for dose - dense treatment.14 another issue is support whether tbts should be entered into clinical practice before high loes are available , with reimbursement on the basis of a coverage - with - evidence - development approach , which has been piloted by the centers for medicare and medicaid and its afliates.27 in this case , rather than insistence on either prospective clinical trials or prospectiveretrospective4 , 5 studies , the third party payer is willing to cover the use of a tbt , with the proviso that the user or manufacturer generates evidence supporting at least clinical validity if not utility for the assay . 
this strategy represents an intriguing but special circumstance outside of our model . a key limitation of our proposed system is that there will be justiable differences in opinions regarding the relative analytic validity , meaningfulness of different clinical end points , magnitude of outcomes , and strategies to generate loes for clinical utility . 
nonetheless , this formulation is a rst conceptual and quantitative step toward what would eventually be a rened , objective , and quantitative method to assign value to emerging tbts using a practical tiered reimbursement scheme , helping to break the vicious cycle of tumor biomarker value . finally , a consideration of the proposed system would not be complete without acknowledging the critical importance of practical barriers to implementation . 
although an indepth discussion of all barriers is beyond the scope of this article , it is worth noting that efforts to implement this proposed system will require input and approval from the involved stakeholders , 1 which include the life sciences industry , private third - party payers , government and regulatory agencies , health care systems , providers and larger medical associations , and patients and patient advocacy groups . 
however , the ultimate success of this system will require large - scale adoption to provide incentives sufcient to power the proposed virtuous cycle of investment in high - quality tbt development . in conclusion , we have proposed an objective , systematic , and semiquantitative approach to guide researchers , funders , test manufacturers , clinicians , patients , and health care systems to invest health care dollars into tbt r&d . 
by providing a tiered categorical solution with clear minimum reimbursement levels , we hope to provide assurance to investors of a solid potential for return on investment via r&d into high - quality clinically useful tbts . 
by increasing entry into the market for high - quality tbts , we argue that market forces will drive real - time selection of the most clinically local useful tbt available for a given clinical context , treatment practices , and population characteristics . 
lyman consulting or advisory role : g1 therapeutics , halozyme , partners healthcare , amgen , pzer , agendia , helsinn therapeutics , celldex ( i ) , janssen ( i ) , genomic health , mylan , samsung bioepis , spectrum pharmaceuticals research funding : amgen richard l . 
schilsky research funding : astrazeneca ( inst ) , bayer ( inst ) , bristol - myers squibb ( inst ) , genentech ( inst ) , eli lilly ( inst ) , merck ( inst ) , pzer ( inst ) , boehringer ingelheim ( inst ) travel , accommodations , expenses : varian daniel f . 
hayes stock and other ownership interests : oncimmune , inbiomotion consulting or advisory role : cepheid , freenome , cellworks , cvs caremark breast cancer expert panel , agendia research funding : astrazeneca ( inst ) , puma biotechnology ( inst ) , pzer ( inst ) , eli lilly ( inst ) , merrimack ( inst ) , menarini silicon biosystems ( inst ) patents , royalties , other intellectual property : royalties from licensed technology ; diagnosis and treatment of breast cancer . 
hayes is designated as inventor / co - inventor ; title : a method for predicting progression - free and overall survival at each follow - up time point during therapy of patients with metastatic breast cancer using circulating tumor cells . 
cancer - tests / #530d572f5955 teutsch sm , bradley la , palomaki ge , et al : the evaluation of genomic applications in practice and prevention ( egapp ) initiative : methods of the egapp working group . 
harris ln , ismaila n , mcshane lm , et al : use of biomarkers to guide decisions on adjuvant systemic therapy for women with early - stage invasive breast cancer : american society of clinical oncology clinical practice guideline . 
j clin oncol 34 : 1134 - 1150 , 2016 krop i , ismaila n , andre f , et al : use of biomarkers to guide decisions on adjuvant systemic therapy for women with early - stage invasive breast cancer : american society of clinical oncology clinical practice guideline focused update . 
cronin m , sangli c , liu ml , et al : analytical validation of the oncotype dx genomic diagnostic test for recurrence prognosis and therapeutic response prediction in node - negative , estrogen receptor - positive breast cancer . 
wang sy , dang w , richman i , et al : cost - effectiveness analyses of the 21 - gene assay in breast cancer : systematic review and critical appraisal . 
balmaa j , sanz j , bonll x , et al : genetic counseling program in familial breast cancer : analysis of its effectiveness , cost and cost - effectiveness ratio . 
lindeman ni , cagle pt , beasley mb , et al : molecular testing guideline for selection of lung cancer patients for egfr and alk tyrosine kinase inhibitors : guideline from the college of american pathologists , international association for the study of lung cancer , and association for molecular pathology . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american pathologists joint review . 
rustin gj , van der burg me : a randomized trial in ovarian cancer ( oc ) of early treatment of relapse based on ca125 level alone versus delayed treatment based on conventional clinical indicators ( mrc ov05 / eortc 55955 trials )  . 
van poznak c , somereld mr , bast rc , et al : use of biomarkers to guide decisions on systemic therapy for women with metastatic breast cancer : american society of clinical oncology clinical practice guideline . 
reed sd , dinan ma , schulman ka , et al : cost - effectiveness of the 21 - gene recurrence score assay in the context of multifactorial decision making to guide chemotherapy for early - stage breast cancer . 
eiermann w , rezai m , k ummel s , et al : the 21 - gene recurrence score assay impacts adjuvant therapy recommendations for er - positive , node - negative and node - positive early breast cancer resulting in a risk - adapted change in chemotherapy use . 
pilon d , queener m , lefebvre p , et al : cost per median overall survival month associated with abiraterone acetate and enzalutamide for treatment of patients with metastatic castration - resistant prostate cancer . 
schremser k , rogowski wh , adler - reichel s , et al : cost - effectiveness of an individualized rst - line treatment strategy offering erlotinib based on egfr mutation testing in advanced lung adenocarcinoma patients in germany . 
 plasma cell - free dna testing of patients with egfr mutant nonsmall - cell lung cancer : droplet digital pcr versus next - generation sequencing compared with tissue - based results christi m.j. 
aerts , md , phd1 ; and hendrikus jan dubbink , phd , 1 on behalf of erasmus mc cancer institute purpose to compare the results of plasma cell - free dna ( cfdna ) droplet digital pcr ( ddpcr ) and nextgeneration sequencing ( ngs ) on detection of epidermal growth factor receptor ( egfr ) primary activating mutations and p.t790m with results of tissue analysis in patients with egfr mutated nonsmall - cell lung cancer . methods all patients with egfr mutated nonsmall cell lung cancer for which a pathology and a plasma specimen were available upon progression between november 2016 and july 2018 were selected . 
for the detection of p.t790m , this was 75% ( = 0.49 ) for ddpcr as well as for ngs . conclusion mutual agreement is high between ngs and ddpcr in cfdna on the level of a specic mutation , with comparable ratio results . 
plasma testing of egfr primary activating mutations and p.t790m shows high concordance with pathology results , for ngs as well as for ddpcr , depending on the extent of the panel used . 
2019 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction among cancer deaths worldwide , nonsmall - cell lung cancer ( nsclc ) is the leading cause.1 the survival of metastasized disease is poor as illustrated by a 1 - year survival rate of 23% in the netherlands between 2010 and 2015.2 the choice of palliative systemic treatment currently depends on histologic subtype , programmed cell death ligand 1 ( pd - l1 ) expression , and the presence of specic genetic aberrations ( also known as driver mutations ) for which specic targeted therapies are available.3 today , is common practice to perform molecular analysis on a tissue biopsy specimen at the time of diagnosis.3 for nonsquamous nsclc , targeted therapies have been developed and registered for treating nsclc on the basis of the presence of genetic alterations in an expanding number of genes . 
 steendam et al context key objective to compare plasma cell - free dna mutation testing by droplet digital polymerase chain reaction ( ddpcr ) and next - generation sequencing ( ngs ) with their concordance with tissue testing . knowledge generated ddpcr and ngs yield comparable results , with similar sensitivity for the mutations that can be detected by both methods , and the concordance with tissue - based results is high . 
discordant cases tend to show intrathoracic and / or cns progression . the extent of mutations that can be discovered by ngs in one step is larger . relevance when searching for a resistance mechanism , ngs analysis of cell - free dna in plasma offers a more comprehensive view than ddpcr , with comparable precision at a single mutation level . 
when no mutations are detected in plasma , tissue - based investigation remains desirable . average to rst - generation tyrosine kinase inhibitors ( tkis ) .6 all patients , however , ultimately develop resistance to treatment with tkis and show progression of disease at some point . 
there are two main mechanisms of acquired resistance : pharmacologic ( eg , problems with compliance , dose reductions , reduced absorption or increased metainadequate cns penetration ) and biologic ( eg , bolism , altered drug target , bypass tracks , phenotypic change , downstream signaling pathways ) .7 the gatekeeper mutation p.t790m in egfr exon 20 is the most common resistance mechanism to rstand secondgeneration tkis ( erlotinib , getinib , and afatinib ) nsclc with an activating egfr mutation and occurs in more than 50% of patients.7 because the availability of osimertinib , a drug that overcomes the p.t790m resistance mechanism that shows high response rates and a substantial median pfs of 8 months , the detection of this gatekeeper mutation has been of utmost importance.8 , 9 in addition , there are other known resistance mechanisms for which targeted therapies are available in research or off - label settings . 
real - time pcr slightly im ( eg , cobas [ roche , basel , proves the detection limit switzerland ] , therascreen [ qiagen , valencia , ca ] ) , but a major improvement in sensitivity was achieved by the development of digital platforms that target specic mutations like droplet digital pcr ( ddpcr ) and beads , emulsion , amplication , and magnetics digital pcr.13 this requires a modest amount of time and cfdna to obtain reliable results . 
a lot of effort was invested in optimizing ngs for use on cfdna , with adjusted amplicon sizes for amplication of smaller dna fragments and application of molecular barcodes to recognize the needle in the haystack in low concentrations of ctdna in the total amount of cfdna.14 for optimal results , it is advised to use as much cfdna in the panel as possible . 
depending on the platform used , the lead time requires several working days , which is comparable to ngs on tissue specimens . this study compares the results of ddpcr ( bio - rad laboratories , hercules , ca ) and ngs ( ion torrent , thermo fisher scientic , waltham , ma ) for detection of primary activating and resistance p.t790m egfr mutations in plasma - derived cfdna . 
outcomes are compared with ngs results of conventional tissue biopsy or cytology . methods we included all patients with egfr mutated nsclc with progression on current therapy for which a tissue specimen ( histology / cytology ) was available in the same time frame and line of treatment as plasma analysis . 
we prospectively collected all data on requested plasma analyses . cfdna isolation blood was collected in 10 - ml cellsave preservative tubes ( cellsearch , menarini silicon biosystems , castel maggiore , italy ) and centrifuged for 20 minutes at 1 , 600 g . 
the dna was eluted in 50 l of buffer . ddpcr analysis the actual analysis of egfr activating ( exon 19 deletions and p.l858r ) and resistance ( p.t790m ) mutations was performed using ddpcr mutation assays ( bio - rad laboratories ) as previously described.15 ngs on pathology specimens and cfdna dna was isolated from formalin - xed parafn - embedded tissues enriched for neoplastic cells by manual microdissection as previously described.16 ngs analysis was performed by semiconductor sequencing with the ion s5 system ( thermo fisher scientic ) with the suppliers materials and protocols . 
library preparation was performed with 1 to 10 ng of tissue dna and 4 to 50 ng of cfdna , depending on the amount of tissue or cfdna available . libraries of tissue dna were prepared with a custom - made primer panel that encompassed , among others , egfr exons 18 to 21 , kras exons 2 to 4 , erbb2 exons 19 to 21 , braf exons 11 and 15 , and the entire coding region of tp53 using the ampliseq library kit 2.0 - 384 lv ( thermo fisher scientic ) ; cfdna library preparation was performed using the oncomine lung cfdna assay ( thermo fisher scientic )  . 
templates were prepared using the ion 520 & ion 530 kit - chef and sequenced with the ion s5 sequencing kit on an ion 530 chip ( thermo fisher scientic )  . sequence data were analyzed with variant caller version 5.6.0.4 ( thermo fisher scientic )  . 
results are reported as allele ratios ( mutated alleles / [ mutated + wild - type alleles ] 100% ) in the case of at least three positive droplets ( ddpcr ) or three unique molecules ( ngs )  . statistical analysis ratios of ddpcr and ngs for p.t790m as well as for the primary activating mutation when applicable . results between november 2016 and july 2018 , 162 patients underwent cfdna analysis on plasma collected in 10 - ml cellsave preservative tubes . 
we selected all 36 patients with egfr mutated nsclc with progression on current treatment of which a histology or cytology specimen was available in the same time frame and line of treatment . the population are listed in baseline characteristics of table 1 . results of plasma analyses agreement between ddpcr and ngs was 94% ( = 0.89 ) for egfr p.t790m detection in plasma and 86% ( = 0.63 ) for detection of the primary activating egfr mutation . 
table 2 lists results of plasma and tissue analyses for all patients . in six patients ( 16.7% ) , the primary activating mutation was a less common variant that was not present in the current ddpcr panel . 
at the level of p.t790m detection , better agreement was shown , with only one patient in whom p.t790m was detected by ngs but not by ddpcr . comparison with tissue results for ngs , concordance for the primary activating egfr mutation with tissue specimens was 83% ( 30 of 36 conrmed results )  . 
cohens was calculated to evaluate the agreement between ddpcr or ngs and tissue - based results for p.t790m detection and between ddpcr and ngs on cfdna for the primary activating mutation as well as for p.t790m. intraclass correlation coefcients were calculated for the in six patients ( 16.7% ) , all with intrathoracic progression , no mutations were detected in cfdna ( mutation - negative plasma ) , whereas the tissue analysis showed the presence of egfr p.t790m in three ( 50% ) of these patients . 
 ( % ) 66 ( 45 - 85 ) activating egfr mutation exon 21 p.l858r exon 21 other lines of therapy mean before pa specimen ( range ) smoking status male female never former current unknown pack - years 1 - 15 15 - 30  . 
one patient showed stable disease as best response to osimertinib , another died before subsequent therapy could be initiated , and the clinical course of the third patient remained unknown because he was treated elsewhere . discussion this study compared two accurate and promising techniques for detection of targetable genetic aberrations in nsclc in the same plasma samples and conrms the high concordance of cfdna with tumor tissue analysis as published earlier.13 , 17 we did not calculate negative predictive value or positive predictive value because we did not consider the tissue analysis as the gold standard ; it is tumor heterogeneity might a known phenomenon that result in a mutation - negative biopsy specimen while a mutation can be present in another ( metastasized ) region . our ndings support this because three patients had p.t790m detected in plasma but not in tissue . 
we do not believe that they had false - positive ndings but , rather , that the biopsy specimen did not represent the whole spectrum of genetic aberrations of the disease . ddpcr is considered a highly sensitive pcr platform that , next to high concordance with tissue - based results , also offers high sensitivity and specicity compared with earlier pcr assays ( taqman pcr with peptide nucleic acid ; therascreen ; cobas ; and beads , emulsion , amplication , and magnetics digital pcr ) .12 , 18 ngs has proven its qualities in molecular analysis of tissue and has promising evolving capabilities for mutation analysis of cfdna.19 - 21 both techniques yield results as a percent of the total ( ratio ) , which is considered a benet over methods that merely indicate positive or negative . the moderate agreement between cfdna and tissuebased results on p.t790m detection is partly the result of mutation - negative plasma samples . 
correlation between the ratio of mutant versus total cell - free dna of next - generation sequencing ( ngs ) and droplet digital polymerase chain reaction ( ddpcr ) results . 
the mutation - negative plasma subset has a substantial negative effect on the concordance between cfdna and tissue biopsy results . on the other hand , the three patients ( 8.3% ) in whom p.t790m was detected in cfdna but not in tumor tissue also contribute to the limitation of agreement . 
these three patients might represent the concept of tumor heterogeneity , where the location of the biopsy does not represent the full spectrum of genetic aberrations of the disease.24 , 26 the p.t790m - positive cells might represent a subclone of limited extent , or another ( not yet detected ) alternative resistance mechanism might be of greater inuence ( illustrated by the patient with a pik3ca mutation in the pathology specimen )  . 
the patients who showed p.t790m in tissue analysis but not in plasma all had intrathoracic and / or cns progression , which again supports the theory that those sites are associated with a lesser rate of shedding of tumor dna into blood.25 the concordance between ngs and ddpcr in cfdna of a specic genetic aberration that is targetable for ddpcr , like p.t790m , is higher than for the broader and heterogeneous group of activating egfr mutations because not all activating egfr mutations were present in the current ddpcr panel and , thus , will be missed . 
in such cases , it is unclear whether plasma is false negative for p.t790m because of limited sensitivity of ddpcr or true negative because of absence of ctdna , as the primary activating egfr mutation also could not be detected . by the food and drug administration and european medicines agency is a fact , the expectation is that in the near future , most patients will be treated with osimertinib upfront , and p.t790m detection will be of lesser importance . 
however , mechanisms of acquired resistance on rst - line osimertinib presented at the european society for medical oncology 2018 congress showed a shift toward more mesenchymal - epithelial transition factor amplications ( 14% ) ; some secondary egfr mutations , like p.c797s ( 7% ) ; and human epidermal growth factor receptor 2 amplications ( 2% ) .28 in this light , a broader approach to investigate the resistance mechanism upon progression seems desirable . in practice , frequently , only a limited amount of material for dna investigation is available , and this is also the case for blood samples . 
an advantage is the fast lead time because ddpcr can generate a quick answer for the clinician ( ie , within 1 working day when needed )  . ngs can explore a broad spectrum of genetic aberrations in a single run , and the possibilities to detect translocations and amplications are expanding quickly . 
thus , with the expanding knowledge of resistance mechanisms and possible targeted treatments ( in development ) for these , the detection of a broad set of genetic aberrations seems desirable . 
for example , a braf v600e mutation can appear next to the primary activating egfr mutation for which a dabrafenib and trametinib combination can be added to the current treatment . 
on the other hand , ngs is more time consuming and still much more expensive than ddpcr . both plasma - based approaches are limited by the fact that some resistance mechanisms need a tissue - based diagnosis ( eg , transformation to small - cell lung cancer )  . because osimertinib showed improved pfs when used in rst - line treatment in the azd9291 versus getinib or erlotinib in patients with locally advanced or metastatic nsclc ( flaura ) trial27 and registration for this indication in conclusion , our study demonstrates that results of egfr mutation detection in cfdna by ngs and ddpcr are comparable , with a high agreement when the ratio of egfr mutant alleles to wild - type alleles is compared . 
steendam research funding : astrazeneca ( inst ) travel , accommodations , expenses : roche , boehringer ingelheim , eli lilly cor van der leest honoraria : roche , boehringer ingelheim , bristol - myers squibb , abbvie consulting or advisory role : roche , abbvie , boehringer ingelheim , bristol - myers squibb jan h . 
aerts stock and other ownership interests : amphera consulting or advisory role : eli lilly , boehringer ingelheim , msd oncology , roche , bristol - myers squibb , amphera , astrazeneca , takeda pharmaceuticals patents , royalties , other intellectual property : dendritic cellbased immunotherapy ( inst ) , proteomics in nsclc ( inst ) hendrikus jan dubbink consulting or advisory role : pzer , personal genome diagnostics , eli lilly , astrazeneca , pzer , janssen pharmaceuticals research funding : astrazeneca no other potential conicts of interest were reported . acknowledgment we thank nadine van donk for her extensive efforts and support in obtaining the ddpcr results at the department of clinical chemistry , erasmus mc rotterdam . references bray f , ferlay j , soerjomataram i , et al : global cancer statistics 2018 : globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries . 
novello s , barlesi f , califano r , et al : metastatic non - small - cell lung cancer : esmo clinical practice guidelines for diagnosis , treatment and follow - up . 
ann oncol 27 : v1 - v27 , 2016 barlesi f , mazieres j , merlio jp , et al : routine molecular proling of patients with advanced non - small - cell lung cancer : results of a 1 - year nationwide programme of the french cooperative thoracic intergroup ( ifct )  . 
midha a , dearden s , mccormack r : egfr mutation incidence in non - small - cell lung cancer of adenocarcinoma histology : a systematic review and global map by ethnicity ( mutmapii )  . 
ann oncol 29 : i3 - i9 , 2018 camidge dr , pao w , sequist lv : acquired resistance to tkis in solid tumours : learning from lung cancer . 
nat rev clin oncol 11 : 473 - 481 , 2014 yang jc , ahn mj , kim dw , et al : osimertinib in pretreated t790m - positive advanced non - small - cell lung cancer : aura study phase ii extension component . j clin oncol 35 : 1288 - 1296 , 2017 9 . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american 11 . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced non - smallpathologists joint review . 
rolfo c , mack pc , scagliotti gv , et al : liquid biopsy for advanced non - small cell lung cancer ( nsclc ) : a statement paper from the iaslc . 
oncotarget 8 : 13611 - 13619 , 2017 van lier mg , wagner a , van leerdam me , et al : a review on the molecular diagnostics of lynch syndrome : a central role for the pathology laboratory . 
mok t , wu yl , lee js , et al : detection and dynamic changes of egfr mutations from circulating tumor dna as a predictor of survival outcomes in nsclc patients treated with rst - line intercalated erlotinib and chemotherapy . 
sacher ag , paweletz c , dahlberg se , et al : prospective validation of rapid plasma genotyping for the detection of egfr and kras mutations in advanced lung cancer . 
hou h , yang x , zhang j , et al : discovery of targetable genetic alterations in advanced non - small cell lung cancer using a next - generation sequencing - based circulating tumor dna assay . 
veldore vh , choughule a , routhu t , et al : validation of liquid biopsy : plasma cell - free dna testing in clinical management of advanced non - small cell lung cancer . 
karachaliou n , mayo - de las casas c , queralt c , et al : association of egfr l858r mutation in circulating free dna with survival in the eurtac trial . 
seki y , fujiwara y , kohno t , et al : circulating cell - free plasma tumour dna shows a higher incidence of egfr mutations in patients with extrathoracic disease progression . 
 clinically integrated sequencing alters therapy in children and young adults with high - risk glial brain tumors carl koschmann yi - mi wu chandan kumarsinha robert lonigro pankaj vats katayoon kasaian marcin cieslik xuhong cao bailey anderson kevin frank lili zhao john r . 
the goal of this study was to evaluate the feasibility , utility , and clinical impact of integrative clinical sequencing and genetic counseling in children and young adults with high - risk brain tumors . patients and methods fifty - two children and young adults with brain tumors designated by the treating neuro - oncologist to be high risk ( > 25% chance for treatment failure ; mean age , 10.2 years ; range , 0 to 39 years ) were enrolled in a prospective , observational , consecutive case series , in which participants underwent integrative clinical exome ( tumor and germline dna ) and transcriptome ( tumor rna ) sequencing and genetic counseling . 
results were discussed in a multi - institutional brain tumor precision medicine teleconference . results sequencing revealed a potentially actionable germline or tumor alteration in 25 ( 63% ) of 40 tumors with adequate tissue , of which 21 ( 53% ) resulted in an impact on treatment or change of diagnosis . 
seventeen of 20 patients ( 85% ) with glial tumors were found to have a potentially actionable result , which resulted in change of therapy in 14 ( 70% ) patients . 
 none of the sponsors played a role in the design ; data collection , management , analysis , and interpretation of the data ; preparation , review , or approval of the manuscript ; and decision to submit the manuscript for publication . corresponding author : rajen mody , md , ms , michigan medicine , d4207 medical professional building , po box 5718 , ann arbor mi , 48109 ; e - mail : rmody@ umich.edu. novel effective therapies that are based , ideally , on the unique biology of each tumor . in contrast to adult tumors , recent surveys of pediatric tumors have shown that most are driven by relatively few mutational events , many of which may be targetable with personalized clinically available agents.2 , 10 although precision medicine is an exciting potential therapeutic avenue for younger patients with brain tumors , this approach requires substantial optimization to successfully improve outcomes for children with these aggressive tumors . 
our group has successfully shown the feasibility and potential clinical utility of integrative clinical sequencing in the practice of precision oncology in pediatric and adult patients with relapsed and refractory disease , 11 , 12 and other pediatric groups have recently reported similar findings.13 , 14 two recent studies have reported targeted dna sequencing in pediatric patients with brain tumors.15 , 16 however , to our knowledge , no studies have been primarily designed to study the role of dna and rna sequencing of children and adolescents with high - risk brain tumors , with a focus on their treatment and follow - up . we therefore launched a clinical sequencing study to better understand the role of personalized genomic characterization and precision oncology in the management of these patients . 
 we performed clinically integrated tumor ( dna / rna ) and germline ( dna ) sequencing and genetic counseling for a prospective cohort of children and young adults with high - risk brain tumors . 
the pediatric mioncoseq study was approved by the institutional review board of the university of michigan medical school , and all patients or their parents or legal guardians provided informed consent ( written assent if > 10 years )  . 
 clinically integrated sequencing was performed according to previous published methodology ( data supplement ) .11 , 12 , 17 our study began using a whole - exome gene panel , as previously described . 
partway through the study of our cohort , the mioncoseq sequencing laboratory ( michigan center for translational pathology ) adjusted their panel from whole exome to a panel of 1 , 700 genes ( oncoseq1700 ) that covered all cancer consensus genes and fusion partners seen in their initially sequenced cohort ( > 1 , 000 tumors )  . 
the michigan center for translational pathology prospectively performed paired whole exome and 1 , 700 gene panel on a prospective cohort of tumors and confirmed no notable difference in reporting of cancer consensus gene alterations to clinicians . as previously described , 11 potentially actionable findings were defined as any genomic finding that could lead to a change in patient management by providing a targetable tumor molecular aberration , a change in diagnosis or risk stratification ( leading to an immediate potential therapeutic application ) , or a change in patient or family counseling by identifying cancer - related germline findings . 
for medulloblastoma , sonic hedgehog ( shh ) was the only subgroup designation considered actionable by itself . brain tumor precision medicine conference tumor sequencing results were reviewed in a multi - institutional brain tumor precision medicine teleconference held at the university of michigan and attended by seattle childrens hospital , university of california , san francisco , baylor , colorado childrens hospital , lurie childrens , childrens national , and childrens hospital of michigan . 
 opinion and to generate discussion of clinical trial availability . for discussion of targeted agents to be considered for patients , special attention was paid to likelihood of their penetration of bbb . 
for all agents ( available on or off trial ) , the university of michigan cns targeted agent prediction ( cns - tap ) rating system was developed , with seven criteria to score utility of the agent , including targeted pathway / relevance to sequencing results , preclinical data , pediatric phase i data , clinical data in cns tumors , active clinical trials for which patient is eligible , cns / bbb penetration , and relevant formulation ( pill v suspension ; see data supplement for sample checkbox evaluation of everolimus for a pik3ca mutation in a patient with diffuse intrinsic pontine glioma [ dipg ] )  . 
for patients with multiple targetable lesions , preference was given to lesions with higher variant allele fraction in tumor , when known . results feasibility of clinically integrated sequencing for children and young adults with brain tumors we screened 52 patients and enrolled 50 patients with high - risk primary brain tumors ( mean age , 10.2 years ; median age , 9.0 years ; range , 1 to 39 years ) between january 2014 and march 2017 ( fig 1 )  . 
patients were referred with a wide variety of brain tumor diagnoses , as long as their treating neuro - oncologist denoted high - risk status ( > 25% risk of progression or treatment failure )  . eighteen patients ( 36% ) had relapsed or refractory tumors ( of which we sequenced the original tumor in 12 cases ) , and 13 patients ( 26% ) had metastatic tumors at the time of enrollment ( table 1 )  . 
thirty - two patients were enrolled at the time of their original diagnoses , when they presented with either a high - risk neoplasm or a possible diagnosis that met criteria for highrisk neoplasm after pathology review . 
for all patients , tissue was obtained by standard - of - care diagnostic or therapeutic surgical procedures done either at the time of enrollment or at the time of an earlier procedure . 
of the 50 enrolled patients , 40 ( 80% ) had adequate tumor specimen for dna / rna sequencing , including 20 ( 50% ) glial tumors , 15 ( 38% ) embryonal tumors , and five ( 13% ) other tumors . 
patients with frozen tumor samples had a median time to results of 49 days . twenty - six tumors ( 65% ) underwent dna sequencing with a panel of 1 , 700 cancer - related genes ( oncoseq1700 ) , rather than whole - exome sequencing . 
rates of actionable findings were similar between both methodologies . clinical utility of integrated sequencing for children and young adults with brain tumors sequencing revealed a potentially actionable germline or tumor alteration in 25 ( 63% ) tumors , including five germline and 22 somatic tumor alterations ( two patients with both a distinct germline and tumor alteration ; table 1 )  . 
 this included 18 patients ( 45% ) who underwent a change in therapy on the basis of sequencing results and three patients ( 8% ) who received new genetic counseling for germline finding and future cancer risk . 
during the course of this study , none of these patients were found to have additional malignancies , although three of five of these patients ( and the sibling of one of these patients ) remain on annual surveillance programs for additional associated malignancies ( data supplement )  . we graded the actionable potential of the somatic mutations into category i to iv ( table a1 ) as described previously.13 category i included somatic mutations of established clinical utility ; category ii : mutations of potential utility ; category iii : other consensus cancer gene mutations ; and category iv : other genes . 
 total patients screened ( n = 52 ) enrolled patients ( n = 50 ) agreed to receive incidental genetic findings ( n = 48 ) patients with inadequate tissue , no further analysis done ( n = 8 ) patients with adequate quality and quantity of tissue ( n = 40 ) patients with incidental genetic findings returned ( n = 38 ) glial tumors ( n = 20 ) embryonal tumors ( n = 15 ) other cns tumor ( n = 5 ) ( n = 8 ) medulloblastoma ( n = 10 ) clival chordoma ( n = 1 ) anaplastic oligodendroglioma ( n = 2 ) rhabdomyosarcoma ( n = 1 ) choroid plexus tumor ( n = 2 ) pilocytic astrocytoma ( n = 2 ) pineoblastoma ( n = 1 ) meningioma ( n = 2 ) ependymoma ( n = 2 ) dipg ( n = 4 ) other glial ( n = 2 ) at / rt ( n = 2 ) etmr ( n = 1 ) fig 1 . 
children and their families were offered enrollment if they had a diagnosis of a primary brain tumor believed to have at least a 25% chance of treatment failure by their treating oncologist . 
at / rt , atypical teratoid / rhabdoid tumor ; dipg , diffuse intrinsic pontine glioma ; etmr , embryonal tumor with multilayered rosettes ; gbm , glioblastoma multiforme . actionable mutations per tumor ( range , 0 to 8 ; median , 2 )  . 
glial tumors showed more grade i to iii mutations per tumor ( average , 3.0 ; range , 0 to 8 ; median , 3 ) than embryonal tumors ( average , 1.6 ; range , 0 to 5 ; median , 1 ; data supplement )  . most targeted therapeutic interventions included use of tyrosine kinase inhibitors , histone deactylase inhibitors , or mechanistic target of rapamycin inhibitors . 
fifteen patients ( 38% ) underwent precision medicinebased therapy with agents selected through the cns - tap program , including 10 patients who also underwent radiation and one patient who underwent surgery and radiation ( data supplement )  . 
our group was heterogeneous in terms of tumor type , time of enrollment ( diagnosis or relapse ) , and whether targeted therapy was given in isolation or was followed by radiation therapy , thus limiting our ability to analyze whether targeted treatments improved treatment outcomes . 
 ( % ) 40 mean age , years median age , years histology glial tumors embyronal other metastatic local at diagnosis clinical characteristics at relapse or progression tissue used for sequencing original diagnostic material sample obtained at relapse ffpe frozen tissue feasibility adequate tissue ffpe adequate no . 
 germline finding 40 of 50 ( 80 ) therapy / diagnostic finding 26 of 36 ( 72 ) any actionable finding frozen tissue adequate 14 of 15 ( 96 ) action taken note : data presented as no . 
the most frequent reasons cited by clinicians or families of the five patients ( of 22 ) for not pursuing treatment on the basis of potentially actionable findings included selecting a nontargeted salvage chemotherapy regimen ( n = 3 ) or pursuing no therapy in the absence of clear clinical or radiologic progression ( n = 1 )  . 
 for one patient with a recurrent medulloblastoma and shh pathway upregulation , consideration of therapy with a targeted shh inhibitor , such as vismodegib , was considered , but no clinical trial was available , and the provider and family decided not to pursue off - trial use of the agent . actionable findings sequencing was of highest utility in glial tumors . 
he was treated with adjuvant temozolomide and lomustine ( ccnu ) , but magnetic resonance imaging obtained before third cycle ( including magnetic resonance spectroscopy ) was consistent with tumor progression before third cycle . 
sequencing of his tumor revealed h3f3a k27m mutation , tp53 mutation and copy loss , and cdk4 amplification and outlier expression ( all category iii ; figs 2a to 2c )  . 
 ( d ) magnetic resonance imaging of the brain ( t1 with contrast ) showed increase in size in enhancing mass after three cycles of adjuvant lomustine ( ccnu ) / temozolomide . 
tp53 , tumor protein 53 ; xrt , radiation . exception of mild hyperbilirubinemia and count suppression . as another example , a 1 - year old girl presented with a recurrent posterior fossa glioblastoma multiforme . 
she originally underwent a gross total resection at 8 months of age , multiagent chemotherapy according to children 's cancer group protocol 99703 , 20 and then focal radiation therapy with concomitant temozolomide ( after initial relapse )  . 
sequencing of her initial tumor revealed significant focal amplification of pdgfb ( 19 copies ; data supplement ) , outlier expression of pdgfb and pdgfra ( data supplement ) , and somatic mutations in setd2 , with the loss - of - function mutation in setd2 comprising 42% of the sequenced tumor fraction . 
 unfortunately , she developed additional progression and changed to a phase i trial . embryonal tumors were found to have potentially actionable results in five of 15 patients ( 33% )  . 
only two of these patients ( 13% ) underwent a change in their therapy on the basis of sequencing results ( table a1 ) both cases involved clarification or revision of the original diagnosis . thirty - eight ( 95% ) participating patients received germline sequencing results . 
 dna damage cell cycle epigenetic transcription signaling kinase cell cycle transcription tumor suppressor genes oncogenes signaling medulloblastoma subgroups glial ( n = 21 ) embryonal ( n = 15 ) gbm ( n = 8 ) dipg ( n = 4 ) other ( n = 9 ) medulloblastoma ( n = 10 ) at / rt ( n = 2 ) other ( n = 3 ) other cns tumors ( n = 5 ) fig 3 . 
 activating dna lesions ( mutation , amplification , or fusion ) were seen in 19 tumors ( 48% ) , most frequently in the mitogen - activated protein kinase signaling and mechanistic target of rapamycin / phosphoinositide 3 - kinase pathways . 
tumor suppressor mutations or deletions ( germline and / or somatic ) were seen in 32 tumors ( 80% ) , most frequently in dna damage or apoptotic signaling pathways . 
at / rt , atypical teratoid / rhabdoid tumor ; dipg , diffuse intrinsic pontine glioma ; gbm , glioblastoma multiforme ; snv , somatic nucleotide variant . deleterious germline snv somatic mutation deletion activating overexpression somatic mutation amplification germline and somatic snv amplification and overexpression other gene fusion present subtype present deletion and germline snv deletion and somatic snv are listed in table a1 . 
all patients with a new actionable pathogenic germline variant were seen in a cancer genetics clinic for counseling and additional testing in family members . molecular landscape of brain tumors in cohort tumor dna and rna sequencing allowed us to explore the molecular landscape of a diverse group of high - risk brain tumors from children and young adults . 
activating alterations ( mutation , amplification , or fusion ) in platelet - derived growth factor receptor ( pdgfr ) or fibroblast growth factor receptor ( fgfr ) pathways was seen in nine ( 45% ) of 20 glial tumors ( fig 3 ) , which is higher than previously published in any of these tumor subtypes.21 - 23 in addition , we saw potential tumor - driving rna fusions in eight ( 20% ) tumors , including novel fusions in three tumors ( lrp6 - etv6 fusion in a pediatric glioblastoma [ po - 3089 ] , an fgfr3 - phgdh fusion in a thalamic anaplastic oligodendroglioma [ po3124 ] , and an elf4 - smarca fusion in a bithalamic pediatric glioblastoma [ po - 3195 ] )  . 
the location of the fgfr3 breakpoint in this fusion is similar to that of reported fgfr3 - tacc3 fusions in adult glioblastoma multiforme.24 of note , overexpression of phosphoglycerate dehydrogenase has been shown to be a negative prognostic funding in adult glioma , possibly related to increase in glioma invasion and proliferation.25 sequencing allowed for medulloblastoma molecular group analysis in nine of 10 medulloblastomas with tissue suitable for rna library formation , by comparison with established groups of highly expressed genes in each group.26 our population of medulloblastomas was from all four groups but was enriched for group 4 tumors ( table a1 ; data supplement )  . 
one tumor ( po - 3063 ) had expression and copy number attributes of three subgroups ( wnt , shh , and group 3 )  . discussion younger patients with brain tumor diagnoses die as a result of their disease more commonly than those with any other tumor type.1 in an effort to tackle this problem , we sought to study a comprehensive integration of precision medicine into the management of this patient population . 
 a few recent studies have assessed the utility of sequencing pediatric cancers , 11 , 13 , 14 including targeted dna sequencing at diagnosis in pediatric patients with brain tumors.15 to our knowledge , our study is the first to report on the feasibility of integrating dna and rna sequencing into the clinical management of children and young adults with high - risk brain tumors at both diagnosis and relapse , along with their treatment and response . 
sequencing revealed a potentially actionable germline or somatic alteration in two thirds of brain tumors , of which one third resulted in an impact on treatment or change of diagnosis . our results demonstrate the utility of combining tumor exome dna and rna sequencing with germline dna sequencing in pediatric brain tumors . 
the brain tumors in our cohort harbored driving tumor mutations not previously known for their tumor type or seen only in a small number ( < 5% ) of tumors . 
in addition , this series supports the use of rna - seq , which identified fusions in 20% of tumors , allowed us study gene expression levels , and also helped us with identification of medulloblastoma subgroups . 
germline sequencing revealed germline cancer predisposition alterations in 13% of tumors , which is consistent with previous studies of pediatric malignancies that have included germline sequencing.11 , 13 our median turnaround time was suboptimal . 
future efforts for molecular profiling will be clinically more relevant if available within 1 to 2 weeks , and novel platforms have been able to bring the time down to days.27 the brain tumors in our younger patient cohort were found to have relatively few somatic mutational events , as compared with mutational surveys of adult brain tumors , which are more heavily mutated.28 , 29 as seen in previous surveys of pediatric cancer , 2 , 12 the somatic mutations that were present in our tumors , especially in our gliomas , were frequently targetable with personalized clinically available agents . 
of note , 14 of 17 ( 83% ) of our patients who underwent therapy changes on the basis of sequencing had a high - grade glial tumor , whereas only one of 23 ( 4% ) of our patients who did not undergo therapy change had a high - grade glial tumor . 
 although no formal survey was performed on this topic , multiple families expressed comfort with pursuing a targeted therapy without a formal second opinion because of the attendance and input from clinicians from multiple other childrens hospitals around the country . pediatric and young adult patients with gbm and dipg highlight the unique opportunities and drawbacks of precision medicine in this patient population . 
however , all of these tumors harbored multiple consensus cancer gene alterations ( category i to iii ) , with an average of 3.8 per tumor , and one with eight consensus cancer gene alterations . 
the selected targeted therapy may have led to the evolution of resistant subclonal tumor cell populations in tumors that progressed.30 recent studies have shown a variable degree of heterogeneity within pediatric and adult high - grade gliomas , which could account for treatment failure with the use of a single agent.31 , 32 current and future clinical trials will approach some of these issues by starting with multiple agents targeting nonredundant pathways.30 the timing of the biopsy is also likely important , because gliomas have also been shown to develop numerous mutations in response to cytotoxic chemotherapy and radiation.9 as a limitation of our study , a minority of our patients with progressive tumors underwent biopsy or resection at progression . 
nevertheless , targeted therapies chosen for some of these patients in our study contributed to potential clinical benefit ( although response attribution is uncertain because of previous irradiation in many cases )  . 
none of the patients receiving targeted therapy had grade 3 or 4 adverse events , cytopenia requiring transfusion , or admission for fever and neutropenia . the cns - tap tool provided a useful framework for selection of targeted agents on the basis of previously published data and the likelihood of bbb penetration . 
we are also developing a prospective trial , which will compare cns - tapselected therapy to clinician choice , as well as measuring outcomes for a more homogenous cohort prospectively compared with historical controls . 
these studies will be crucial to our understanding of the true value of targeted therapy for children with brain tumors . in summary , selection of personalized agents on the basis of integrative clinical sequencing of high - risk brain tumors of children and young adults is feasible and frequently results in change of management . 
maher , karin muraszko , patricia robertson collection and assembly of data : carl koschmann , yi - mi wu , marcin cieslik , xuhong cao , bailey anderson , kevin frank , andrew h . 
chinnaiyan , rajen mody data analysis and interpretation : carl koschmann , chandan kumar - sinha , robert lonigro , pankaj vats , katayoon kasaian , bailey anderson , kevin frank , lili zhao , john r . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . carl koschmann no relationship to disclose yi - mi wu no relationship to disclose chandan kumar - sinha no relationship to disclose robert lonigro no relationship to disclose pankaj vats no relationship to disclose katayoon kasaian no relationship to disclose marcin cieslik no relationship to disclose xuhong cao no relationship to disclose bailey anderson no relationship to disclose kevin frank no relationship to disclose lili zhao no relationship to disclose john r . 
 bernard marini speakers ' bureau : genentech / abbvie , shire jessica everett no relationship to disclose brendan mullan no relationship to disclose sandra camelo - piragua no relationship to disclose sriram venneti no relationship to disclose paul mckeever no relationship to disclose kathryn mcfadden no relationship to disclose andrew p . 
maher no relationship to disclose hugh garton no relationship to disclose karin muraszko no relationship to disclose patricia robertson no relationship to disclose dan robinson no relationship to disclose arul m . 
chinnaiyan consulting or advisory role : tempus rajen mody no relationship to disclose acknowledgment we thank the patients and families who participated in this study for their generosity and kindness . 
we also thank steven pipe , valerie opipari , maria castro , pedro lowenstein , becky sigler , coral grothe , lilly pritula , justin flees , javed siddiqui , christine brennan , erica rabban , terrence barrette , christine betts , karen giles , pallavi mohapatra , and xiaoxuan dong for their administrative or academic guidance ; none of them received compensation beyond their salaries at the university of michigan . 
we also thank participants and presenters in the university of michigan brain tumor precision medicine conference . affiliations carl koschmann , yi - mi wu , chandan kumar - sinha , robert lonigro , pankaj vats , katayoon kasaian , marcin cieslik , xuhong cao , kevin frank , lili zhao , andrew h . 
zureick , jessica everett , bailey anderson , brendan mullan , bernard marini , sandra camelo - piragua , sriram venneti , paul mckeever , kathryn mcfadden , andrew p . 
harris mh , dubois sg , glade bender jl , et al : multicenter feasibility study of tumor molecular profiling to inform therapeutic decisions in advanced pediatric solid tumors : the individualized cancer therapy ( icat ) study . 
ramkissoon sh , bandopadhayay p , hwang j , et al : clinical targeted exome - based sequencing in combination with genome - wide copy number profiling : precision medicine analysis of 203 pediatric brain tumors . 
kline cn , joseph nm , grenert jp , et al : targeted next - generation sequencing of pediatric neuro - oncology patients improves diagnosis , identifies pathogenic germline mutations , and directs targeted therapy . 
thorarinsdottir hk , rood b , kamani n , et al : outcome for children < 4 years of age with malignant central nervous system tumors treated with high - dose chemotherapy and autologous stem cell rescue . 
to our knowledge , there are no previous reports of basal cell carcinoma of the prostate occurring in association with a germline brca2 mutation , as in the patient reported here ; however , brca2 mutations , which are a recognized risk factor for prostate adenocarcinoma , are increasingly recognized as a driver for more aggressive disease5 and as a potential therapeutic target . 
olaparib , a poly ( adp ribose ) polymerase ( parp ) inhibitor , has shown high response rates in patients with prostate cancer and defects in homologous repair.6 unfortunately , many patients receiving treatment with parp inhibitors may develop reversion mutations that restore brca function and reduce sensitivity to parp inhibition or confer resistance . 
to our knowledge , there are no reports of the successful treatment of patients with metastatic prostate cancer after brca reversion mutations . in this article , we present a patient with a germline brca2 mutation who developed de novo metastatic basal cell carcinoma of the prostate and experienced a complete clinical response with olaparib . 
 after a mixed response , the patient underwent metastasectomy and subsequently had minimal to no evidence of disease while continuing to receive olaparib maintenance . in january 2016 , a 77 - year - old man with a normal psa presented with urinary obstruction . 
a computed tomography ( ct ) scan confirmed dozens of soft tissue nodules in all lobes of both lungs , suggestive of metastatic disease , as well as an enlarged prostate with a hypodense lesion in the left lobe causing mass effect on the urethra , suggestive of malignancy . 
tumor cells stained diffusely positive for bcl2 and ttf1 ; focally positive for ck903 , ck20 , p63 , and ar ; and negative for ck7 , napsin a , chromogranin , synaptophysin , psa , erg , nkx3.1 , uroplakin , gata3 , and s100 . 
overall , the immunophenotype and morphology were compatible with basal cell carcinoma of the prostate , basaloid variant ( fig 1 )  . the patients lung metastases continued to grow rapidly , he was no longer ambulatory , and he joseph e . 
bhatt author affiliations and support information ( if applicable ) appear at the end of this article . the content is solely the responsibility of the authors and does not necessarily represent the official views of harvard catalyst , harvard university , and its affiliated academic health care centers or the national institutes of health . corresponding author : joseph e . 
 ( a ) x100 , ( b ) x400 , ( c ) x200 , and ( d ) x400 ; prostate biopsies demonstrated a high - grade carcinoma with basaloid morphology : monotonous cells with high nuclear to - cytoplasmic ratio , arranged in solid nests , with peripheral palisading , frequent comedonecrosis . 
we met him for an initial visit at this time , and targeted sequencing of his tumor was performed by foundationone ( foundation medicine , cambridge , ma ) , revealing a mutation in brca2 : s1982fs * 22 , also known as 6174delt , present at 70% allele frequency . 
after 2 months of treatment , he developed progressive macrocytic anemia , which is a known adverse effect of olaparib , and his dose was reduced to 300 mg twice daily . 
three months after initiating treatment , a ct scan of the torso revealed a dramatic response , with the innumerable pulmonary nodules and masses decreased in size and quantity throughout the lungs ( fig 2c ) and shrinkage of the hypodense lesion in the prostate . eight months after beginning treatment , the patient continued receiving olaparib , which was further dose reduced to 200 mg twice daily for anemia . 
the second mutation restored the open reading frame presumably restoring brca2 function , thereby mediating resistance to olaparib . because of frequent complicated urinary tract infections , we wished to avoid cytotoxic chemotherapy , and we continued giving olaparib while adding atezolizumab and androgen deprivation therapy ( adt ) with leuprolide acetate . 
we did not treat with adt initially because of what we thought would be a low likelihood of response for this type of tumor on the basis of the limited case series available , 8 but we added it at this point because of limited low - toxicity options . 
imaging again showed a mixed response : the four new lung nodules seen on the previous scan had disappeared , other nodules were stable to smaller , but the largest right subpleural nodule and one additional nodule continued to grow and cavitate . 
 the two growing lung nodules were resected , and the patient continues to receive olaparib monotherapy , now 28 months after diagnosis , with his most recent scan showing only a single 1.4 - cm nodule that continues to shrink . discussion we present a patient who was successfully treated for a rare disease that has no standard of care . 
the patient is notable for rationally selected targeted therapy with parp inhibition in a new tumor subtype and for further success in treatment after the development of a brca2 reversion mutation that seemed to confer resistance . 
parp inhibition is showing increasing promise across tumor types that harbor defects in homologous recombination , and mechanisms and patterns of resistance are being elucidated.9 in prostate cancer , the first case series6 reporting the successful treatment of tumors harboring defects in homologous repair with olaparib showed a response in 88% of patients with dna repair defects . 
long - term data are still evolving and have not yet been reported in this setting , but a recent abstract10 suggests that , at least in combination with adt , olaparib results in a median pfs of approximately 13 months in patients with metastatic prostate cancer . 
at the time of progression , various mechanisms of resistance have been reported , including brca2 reversion mutations , as in this patient , 11 which may be polyclonal , suggesting that these reversion mutations may be relatively common under the evolutionary pressure of parp inhibition . in the patient presented here , we elected to add atezolizumab in the hope that there could be a synergistic effect with parp inhibition . 
 preclinical rationale for this approach is based on cell culture and xenograft models where parp inhibitors upregulate pd - l1 expression , thereby attenuating anticancer immunity , and pd - l1 inhibitors resensitize tumors to t - cell killing , increasing therapeutic efficacy.12 clinical trials investigating these combinations are ongoing.13 , 14 after adding atezolizumab , this patient experienced a mixed response , and with only two lung nodules remaining , we elected to proceed with a metastasectomy . 
if reversion mutations are indeed common under the evolutionary pressure of parp inhibition , we hypothesize that the patient remains disease free , at least in part , as a result of continued efficacy of immune checkpoint blockade . 
in conclusion , to our knowledge , this is the first report of the successful treatment of basal cell prostate cancer with a parp inhibitor , and more importantly , the first report of the successful treatment of a brca2 - mutated tumor after a reversion mutation . 
 bhatt collection and assembly of data : all authors data analysis and interpretation : all authors manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors relationships may not relate to the subject matter of this manuscript . 
simper nb , jones cl , maclennan gt , et al : basal cell carcinoma of the prostate is an aggressive tumor with frequent loss of pten expression and overexpression of egfr . 
chang k , dai b , kong y , et al : basal cell carcinoma of the prostate : clinicopathologic analysis of three cases and a review of the literature . 
banda k , swisher em , wu d , et al : somatic reversion of germline brca2 mutation confers resistance to poly ( adp - ribose ) polymerase inhibitor therapy . 
clarke n , wiechno pj , alekseev b , et al : olaparib combined with abiraterone in patients ( pts ) with metastatic castration - resistant prostate cancer ( mcrpc ) : a randomized phase ii trial . 
quigley d , alumkal jj , wyatt aw , et al : analysis of circulating cell - free dna identifies multiclonal heterogeneity of brca2 reversion mutations associated with resistance to parp inhibitors . 
karzai f , madan ra , owens h , et al : a phase 2 study of olaparib and durvalumab in metastatic castrate - resistant prostate cancer ( mcrpc ) in an unselected population . 
friedlander m , meniawy t , markman b , et al : a phase 1b study of the anti - pd - 1 monoclonal antibody bgb - a317 ( a317 ) in combination with the parp inhibitor bgb - 290 ( 290 ) in advanced solid tumors . 
suarez , md1 introduction genomic sequencing of colorectal cancers reveals that 16% have a very high tumor mutation burden ( tmb ) .1 three quarters of such tumors display microsatellite instability ( msi ) associated with silencing or somatic mutation of mismatch repair ( mmr ) genes . 
among advanced colorectal cancers , prevalence of pole mutations remains unknown , but only 3.5% are defective in mmr.2 pole proofreading and mmr act in concert to correct replication errors . because mmr - decient tumors often respond to therapy , 3 - 5 pole - mutated tuimmune checkpoint mors might also respond . 
pathology revealed moderately differentiated adenocarcinoma with two involved lymph nodes , classied as stage iiic disease ( pt4bn1b according to the american joint committee on cancer staging system , eighth edition )  . 
after adjuvant chemotherapy with uorouracil and oxaliplatin , computerized tomography ( ct ) scan showed no evidence of disease . three years later , biopsy of an enlarging left supraclavicular lymph node revealed a kras - mutated mss adenocarcinoma with abundant extracellular mucin and a lack of programmed death ligand 1 ( pd - l1 ) tumor cell expression by both e1l3n and sp263 antibody clones . 
the tumor - inltrating lymphocytes ( tils ) could not be assessed , because this was a lymph node metastasis and the primary tumor was unavailable . the patient received uorouracil and irinotecan plus bevacizumab . 
treatment was complicated by a small bowel stula , which required discontinuation of bevacizumab , partial small bowel resection , takedown of an enterorectal stula , and placement of a permanent rectal tube . after a 3 - month recovery from surgical complications , imaging showed new pulmonary metastases . 
the enlarging left supraclavicular nodal mass led to horner syndrome with ptosis and near syncope , which required palliative radiation . the left supraclavicular lymph node specimen obtained 3 years after surgery displayed approximately 20% tumor purity by histology . 
genomic proling with the stanford solid tumor actionable mutation panel , a hybrid capturebased next - generation sequencing assay , revealed an ultra - high tmb relative to colorectal carcinomas analyzed on the same panel ( fig 1b )  . additional testing demonstrated intact expression of mmr proteins as well as mss by polymerase chain reaction . 
the boxplot shows the median , interquartile range , and standard deviation for the tmb in 82 microsatellite - stable ( mss ) colorectal cancers ( crcs ) detected by the stanford solid tumor actionable mutation panel at the time of patient testing . 
the gray dots indicate tmb for those pole - mutated tumors reported to have responded to programmed death 1 ( pd - 1 ) blockade : two crcs and two endometrial cancers ( ecs )  . 
the box plots show the tmb for a survey of 859 tumors with likely driver mutations in mismatch repair genes that would produce microsatellite instability ( msi ) and 102 tumors with known or likely functional pole mutations ( adapted from chalmers et al16 )  . 
on the basis of the ultra - high tmb , estimated at 200 mutations / mb ( fig 1c ) , our molecular tumor board recommended immunotherapy with an immune checkpoint inhibitor . pembrolizumab was obtained for compassionate use . treatment led to a transient increase in the carcinoembryonic antigen ( cea ) from 2 , 742 ng / ml to a peak of 3 , 727 ng / ml followed by a decline to a plateau of 57 to 83 ng / ml which was maintained through 25 months of treatment and an additional 3 months of follow - up ( fig 2a )  . this was associated with resolution of pain and normalization of performance status from eastern cooperative oncology group status of 2 to 0 . 
bars above the graph indicate periods of treatment with uorouracil and irinotecan ( folfiri ) , uorouracil and oxaliplatin ( folfox ) , regorafenib , triuridine ( tfd ) and tipiracil ( tpi ) , radiation therapy ( xrt ) to the left supraclavicular mass , and the programmed death 1 ( pd - 1 ) inhibitor pembrolizumab . 
the delayed response was consistent with slow clearance of mucin after tumor cell death . the patient experienced a brief episode of localized herpes zoster and later a brief episode of asymptomatic grade 1 transaminitis . 
each episode was addressed by withholding one cycle of pembrolizumab , and each quickly resolved without sequelae . discussion in recent years , immune checkpoint blockade has emerged as a safe and effective treatment of many solid tumors . 
in particular , tumors with high level of msi and mismatch repair deciency have shown dramatic responses to treatment with inhibitors.4 , 5 similarly , accumulating immune checkpoint evidence suggests that tmb alone , independent of mmr status , correlates with response to immune checkpoint blockade for some tumor types.3 , 6 - 9 hence , mss tumors with an ultra - mutated phenotype as a result of mutations in pole or pold110 , 11 represent an intriguing subset of tumors that may also respond to immune checkpoint inhibitors . the case presented here adds to the few reports of polemutated tumors that responded to pd - 1 checkpoint blockade . 
by contrast , the responsive tumors were inconsistent in pd - l1 expression ( fig 1d )  . unlike patients in the other cases , the patient in this report began treatment with the greatest extent of disease and enjoyed the longest sustained response ( which continued beyond 28 months )  . 
radiologic density of tumors failed to change signicantly for 8 months before nally disappearing at 28 months . the dramatic response in this patient shows the potential benet of evaluating mss tumors for pole and possibly pold1 mutations . 
however , it is important to realize that some pole or pold1 mutations , particularly previously uncharacterized mutations , may prove to be passenger alterations with no effect on tmb.10 a prospective analysis of 80 , 853 patients with advanced solid tumors revealed known genomic alterations in pole in only 259 patients ( 0.3% ) , with a median tmb of 31 mutations / mb.17 the most common mutation was p.r446q ( n = 77 ) , which is uncharacterized , associated with low tmb ( less than ve mutations / mb ) , and predominantly germline . 
the two nextmost - common mutations , p.p286r ( n = 41 ) and p.v411l ( n = 29 ) , are both functional , associated with high tmb ( greater than 20 mutations / mb ) , predominantly somatic , and enriched in colorectal cancer and endometrial carcinoma . 
these were the mutations present in the four polemutant tumors that have responded to pd - 1 checkpoint blockade in this and other published reports ( fig 1d )  . however , not all pole - mutated tumors respond to checkpoint blockade . 
two colorectal cancers with the p.p286r mutation showed progressive and stable disease after 1 and more than 10 months of follow - up.15 both cases showed low levels of cd8 + tils . 
lo employment : genentech , roche stock and other ownership interests : genentech , roche gilbert chu patents , royalties , other intellectual property : rapid small volume detection of blood ammonia , patent no . 
nature 487 : 330 - 337 , 2012 koopman m , kortman ga , mekenkamp l , et al : decient mismatch repair system in patients with sporadic advanced colorectal cancer . 
br j cancer 100 : 266 - 273 , 2009 rizvi na , hellmann md , snyder a , et al : mutational landscape determines sensitivity to pd - 1 blockade in nonsmall - cell lung cancer . 
science 348 : 124 - 128 , 2015 le dt , durham jn , smith kn , et al : mismatch repair deciency predicts response of solid tumors to pd - 1 blockade . 
science 357 : 409 - 413 , 2017 le dt , uram jn , wang h , et al : pd - 1 blockade in tumors with mismatch - repair deciency . 
n engl j med 372 : 2509 - 2520 , 2015 snyder a , makarov v , merghoub t , et al : genetic basis for clinical response to ctla - 4 blockade in melanoma . 
n engl j med 371 : 2189 - 2199 , 2014 goodman am , kato s , bazhenova l , et al : tumor mutational burden as an independent predictor of response to immunotherapy in diverse cancers . 
mol cancer ther 16 : 2598 - 2608 , 2017 . kowanetz m , zou w , shames d , et al : oa20.01 : tumor mutation burden ( tmb ) is associated with improved efcacy of atezolizumab in 1l and 2l + nsclc patients . 
santin ad , bellone s , buza n , et al : regression of chemotherapy - resistant polymerase ( pole ) ultra - mutated and msh6 hyper - mutated endometrial tumors with nivolumab . 
wang c , gong j , tu ty , et al : immune proling of microsatellite instability - high and polymerase ( pole ) mutated metastatic colorectal tumors identies predictors of response to antipd - 1 therapy . 
schrock ab , fabrizio d , he y , et al : analysis of pole mutation and tumor mutational burden ( tmb ) across 80 , 853 tumors : implications for immune checkpoint inhibitors ( icpis )  . 
gaba author affiliations and support information ( if applicable ) appear at the end of this article . authors disclosures of potential conflicts of interest and contributions are found at the end of this article . corresponding author : steven f . 
powell , sanford cancer center , 1309 w 17th st , suite 101 , sioux falls , sd 57104 ; e - mail : steven.powell@sanfordhealth.org. introduction precision oncology ( po ) is a growing treatment approach in the era of next - generation sequencing ( ngs ) and matched therapies . 
our program developed a virtual molecular tumor board ( mtb ) strategy to help guide po care . materials and methods over 18 months , eligible adult patients with advanced , incurable solid tumor malignancies were enrolled in a molecular profiling ( mp ) study using the foundation medicine ngs panel . 
results were reviewed through a weekly , videoconferenced mtb conducted across our largely rural integrated health systerecommendations from the mtb were used to identify actionable alterations ( aas )  . 
secondary outcomes included the frequency of aas , genomic matched treatments , genomic matched clinical trial enrollment , and clinical outcomes . results a total of 120 participants with a variety of advanced tumor types were enrolled . 
2018 by american society of clinical oncology introduction the emergence of next - generation sequencing ( ngs ) and genomic matched treatments ( gmts ) has spawned the field of precision oncology ( po ) .1 now that ngs has moved from academic centers to routine clinical practice , 2 po is rapidly becoming a part of standard cancer care . 
 variability in clinician understanding , tumor tissue requirements , cost concerns , limited availability of gmts , and lack of infrastructure to support testing are barriers to ngs testing in clinical practice.6 effective cancer care delivery mechanisms are needed to understand the impact of po . community oncology centers represent a major venue for the growth of po . 
nearly 85% of patients with cancer are diagnosed and treated at community - based cancer centers.7 although ngs testing is now available to providers in these settings , assistance in interpretation and access to therapies can vary . 
 although collaborative academic - community mtbs are emerging , 10 this practice has not been widely used in the community , a setting that offers many challenges for po care delivery . access to therapies is perhaps the most significant challenge in po . 
 academic centers with large clinical trial portfolios demonstrate genomic - matched clinical trial ( gmct ) enrollment rates for 3% to 11% of participants with an actionable alteration ( aa ) .11 , 12 gmcts often enroll rare patient cohorts , and implementation at a community site can be challenging . 
highlighting this access issue is the fact that only approximately 2% to 5% of patients with cancer enroll in trials as part of standard cancer care.13 furthermore , for off label drug use , relevant evidence - based guidelines are limited and variability in insurance coverage exists , which can limit drug access for food and drug administration ( fda ) approved agents . 
 although newer clinical trial models , such as the national cancer institutemolecular analysis for therapy choice ( nci - match ) trial14 and asco targeted agent and profiling utilization registry ( tapur ) 15 study , seek to improve access , po therapy is often limited to major cancer centers.16 , 17 as commercial cancer ngs panels are becoming widely available to cancer providers , our cancer program sought to determine the best approach to deliver po care . 
we developed a centralized mtb with video conferencing to all oncology sites in our largely rural community cancer progra as part of this program , we developed a prospective observational study to determine the impact of this approach in this setting . 
 eligibility criteria included patients with incurable , solid tumor malignancies who were 18 years of age ; experienced disease progression while receiving one or more prior lines of therapy or had no standard first - line treatment options available ; had a recent tissue biopsy ( 14 weeks ) available for molecular testing ; had an eastern cooperative oncology group performance status of 1 ; and had adequate organ and bone marrow function for active cancer therapy . 
patients were excluded if they had primary brain cancer , had a concurrent uncontrolled malignancy , had symptomatic or untreated cns metastases , had uncontrolled concurrent illness , were pregnant or breastfeeding , or were of childbearing potential not using adequate contraception . participants who were interested in molecular profiling ( mp ) were approached for consent ( prescreening phase )  . 
if a participant did not have tumor tissue available for testing , a new biopsy was allowed if this was felt to be clinically appropriate by the enrolling investigator . study objectives the primary objective of the study was to determine the feasibility of mp in patients with advanced cancer in the community setting . 
secondary objectives included evaluation of patient and tumor characteristics , frequency of molecular alterations , influence of mp on treatment decisions , and patient outcomes . molecular profiling mp was performed in a clinical laboratory improvement amendmentscertified , new york stateaccredited , and college of american pathologistsaccredited laboratory ( foundation medicine [ fm ] , cambridge ma )  . 
technical descriptions and validation of genomic profiling assays have been extensively described in previous publications.18 - 20 in brief , at least 50 ng dna were extracted from 40 m formalin - fixed , paraffin - embedded tissue sections with at least 20% tumor cells . 
adaptor - ligated dna underwent hybrid capture for all coding exons of 236 , 315 , or 405 cancer - related genes plus select introns from 19 , 28 , or 31 genes frequently rearranged in cancer.18 - 20 captured libraries were sequenced to a median exon coverage depth of 528 ( hiseq ; illumina , san diego , ca )  . 
resultant sequences were analyzed for short variants ( base substitutions , insertions / deletions ) , copy number alterations ( focal amplifications and homozygous deletions ) , rearrangements , and select gene fusions . 
for the purposes of this study , treatment was recommended for an aa only if the gene variant had been previously reported as actionable in the peer - reviewed literature , either from clinical or preclinical data . 
variants of undetermined significance were included in treatment decisions , but first reviewed by genetics professionals to determine whether there was strong evidence of functionality in the peer reviewed literature . 
participants who did not have an aa and / or those who were treated with non - gmts were classified as being treated as standard of care ( soc )  . 
any participant with a variant with potential germline implications was offered a referral to a genetic counselor . clinical outcomes assessment all participants enrolled in the study were followed for clinical outcomes on the basis of their mp . 
 for participants treated with an fda - approved on - label or off - label drug , new baseline tumor assessments were required within 30 days before starting recommended treatment . 
after the initial 16 weeks , tumor assessments were performed per routine clinical practice and continued until disease progression or discontinuation because of toxicity . statistical analysis sample size was not calculated but set at 120 participants with testing performed . 
patients considered evaluable for response included those who received at least two treatment cycles ( or a minimum of 6 weeks of therapy ) and underwent at least one in - study disease assessment or experienced clinical disease progression . 
kaplan - meier curves were created in r software with the survival package . results patient and tumor characteristics a total of 144 participants were screened from june 2014 to december 2015 , with 120 eligible for enrollment . 
race distribution predominantly consisted of white ( n = 114 ; 95% ) participants ; however , two latino , two asian , one native american , and one african american participant were also included . 
 case presentation provider or designee review of mp results moderator mtb discussion medical oncology genetics specialists oncology subspecialists clinical research ancillary staff treatment recommendation on - label drug off - label drug clinical trials standard of care documentation direct to provider electronic medical record fig 1 . 
the sanford mtb encompasses a large , rural geographic area ( covering > 200 , 000 square miles ) , with cancer centers in fargo , nd ; sioux falls , sd ; bemidji , mn ; and bismarck , nd . 
in addition to the physician moderator and presenting provider , the mtb discussion included practicing medical oncologists , board - certified genetics specialists ( clinical genetics , molecular genetics , cytogenetics , and genetic counselors ) , oncology subspecialists ( radiology , pathology , interventional radiology , and surgery ) , clinical research , and ancillary staff ( nursing , pharmacy , and patient advocates )  . 
colorectal ( n = 20 ; 16.7% ) , nonsmall - cell lung ( n = 20 ; 16.7% ) , and breast ( n = 15 ; 12.5% ) cancers represented the most common disease types enrolled . 
rare tumors , representing one patient each , included bladder , germ cell , duodenal , neuroblastoma , salivary , and cutaneous squamous cell cancers . molecular pathway alterations of the 120 participants who had mp performed , 109 completed testing . 
genomic matched treatment categories treatment categories fda - approved on - label drug ( companion biomarker approved ) treatment is fda approved for the identified aa in the same tumor type and is a validated companion biomarker example : erlotinib for egfr mutated nonsmall - cell lung cancer fda - approved on - label drug ( biomarker relevant , but not approved ) treatment is fda approved for the tumor type and directed at an aa predictive of response , but not validated as a companion biomarker example : everolimus for i3k - activated breast cancer fda - approved off - label treatment is directed at an aa and is fda approved for use for another indication example : vemurafenib for braf v600e mutated nonsmall - cell lung cancer genomic matched clinical trial treatment is matched to an aa that is a required for enrollment in a clinical trial example : bgj398 ( fgfr inhibitor ) for fgfr1 mutated adenoid cystic carcinoma abbreviations : aa , actionable alteration ; egfr , endothelial growth factor receptor ; fda , food and drug administration . there was an average of 4.7 genomic alterations per patient , ranging from one to 15 genomic alterations each . 
on the basis of mtb review , an aa was identified , and treatment was recommended for 63 of the 109 of patients ( 58% ) who completed testing with the fm panel . influence on treatment decisions the consort diagram in figure 2 outlines the pathway from mp to treatment assignment . 
for those who did not pursue treatment , enrollment in hospice ( n = 10 ) because of declining clinical status or standard chemotherapy ( n = 7 ) because of patient or provider preference were the most commonly chosen treatment options . 
only six participants who sought targeted therapy were denied access either by clinical trial screen failure or lack of off - label access . treatment outcomes of the 39 participants treated with gmt , the overall response rate was 12.8% ( n = 5 ) , all of which were partial responses ( prs )  . 
a total of 14 patients ( 35.9% ) had a best response of stable disease ( sd ) , and 13 ( 33.3% ) had progressive disease ( pd )  . 
 figure 3 demonstrates the kaplan - meier curves with pfs and os data from each of these two groups . discussion implementation of an easily accessible , multidisciplinary team approach to ngs testing and review resulted in a high rate of gmt in our patient population . 
 ( * ) 31% of trials ( n = 5 ) identified as an exact genomic matched clinical trial ( gmct ) match on the foundation medicine ( fm ) report . 
because of rapid changes in genomic biomarkers , relevant data , and emerging treatment options , access to these clinical trials is critical in determining the efficacy of this approach . outlined in figure 2 , our mtb was able to identify additional participants who might have benefited from gmcts beyond what was listed as an exact molecular match on the fm report . 
by using this care delivery model , we were able to enroll 14.7% of those with aas on gmcts , the majority of whom were at our centers . until recently , access to gmcts has been largely limited to major academic centers . 
md anderson has the largest series of patients in this setting , showing gmct match rates of 11% for those with an aas.12 these major academic centers often have large phase i programs and a number of relevant clinical trials . 
however , in the community , these trials are often not as easily accessible because of physician , organizational , and patient factors.22 these challenges can be magnified further with po trials because they seek to enroll unique cohorts that require specialized screening through ngs tests . 
treatment options were available through novel umbrella / basket clinical trials ( novartis signature program , 23 - 28 genentech mypathway trial29 ) and with a limited number of off - label drug options . 
improving patient access to gmcts in the community has the potential to rapidly propel this field forward . as part of this study , our group not only learned how to implement this process in our multicenter health system , but also how to adapt the program to emerging trends in po . 
since the end of our study , the pd - 1 antibody pembrolizumab was subsequently approved by the fda for any solid tumor with msi - h status.31 through our genomic tumor board review of genomic results and emerging efficacy data in msi - h tumors , we were able to identify patients who may benefit from pd - 1 inhibitors.32 two patients in this study with msi - h tumors on the basis of msh6 and mlh1 loss were identified and had exceptional , durable responses to therapy . 
this scenario highlights the fact that a multidisciplinary approach , such as described here , can provide continuous education to clinicians on rapidly emerging data on genomic biomarkers and targeted therapies . 
as such , we have now developed a subsequent program to integrate additional biomarker tests , such as programmed death ligand - 1 expression , into our mtb review process . one limitation of our study was the heterogenous patient population and the heavily pretreated , advanced stage of diseases we included . 
although several rare tumors were allowed to proceed with testing and treatment with no prior therapy , the majority of participants were being evaluated for third - line or greater therapy . 
gemma , genetic exploration of the molecular basis of malignancy in adults . treatment gmct treatment gmct time ( days ) time ( days ) previous analyses of po - guided care have suggested improved pfs data in those treated with gmt compared with soc.34 although we did not directly compare outcomes with soc , we did find similar outcomes for those treated with gmt , regardless of gmct enrollment . 
despite this , our group strongly favors enrollment in gmcts to increase the knowledge base of relevant aas and outcomes of gmt . finally , it is important to consider the number of participants ( 16% ) who had treatment recommended but declined and enrolled in hospice before they could pursue therapy . 
as the cost of ngs testing decreases and use becomes more widespread , it is important to consider its role early in diagnosis to help define future therapeutic strategies for patients . 
as we learn more effective ways to deliver po care , we can develop evidence - based guidelines around the timing of testing , appropriate disease types , and treatment selection . overall , this study demonstrates the feasibility of a po care delivery model in a largely rural community oncology syste by developing a systematic process to provide ngs testing and review results , we achieved a high rate of gmt . 
powell research funding : merck sharp & dohme ( inst ) , novartis ( inst ) , genentech / roche ( inst ) , incyte ( inst ) , bristol - myers squibb ( inst ) , pfizer ( inst ) , vyriad ( inst ) elie g . 
chung employment : foundation medicine stock and other ownership interests : foundation medicine rachel anhorn employment : foundation medicine , pfizer stock and other ownership interests : foundation medicine anu g . 
tafe lj , gorlov ip , de abreu fb , et al : implementation of a molecular tumor board : the impact on treatment decisions for 35 patients evaluated at dartmouth - hitchcock medical center . 
lynam eb , leaw j , wiener mb : a patient focused solution for enrolling clinical trials in rare and selective cancer indications : a landscape of haystacks and needles . 
haslem ds , van norman sb , fulde g , et al : a retrospective analysis of precision medicine outcomes in patients with advanced cancer reveals improved progression - free survival without increased health care costs . 
 economic impact of next - generation sequencing versus single - gene testing to detect genomic alterations in metastatic nonsmall - cell lung cancer using a decision analytic model nathan a . 
pennell , md , phd1 ; alex mutebi , phd2 ; zheng - yi zhou , phd3 ; marie louise ricculli , msc3 ; wenxi tang , ms3 ; helen wang3 ; annie guerin , msc4 ; tom arnhart , pharmd , ms2 ; anand dalal , phd2 ; medha sasane , phd2 ; kevin y . 
model outcomes for each strategy were time - to - test results , the proportion of patients identied harboring alterations with or without us food and drug administrationapproved therapies , and total testing costs . 
a budget impact analysis assessed the economic effects of increasing the proportion of ngs - tested patients . results in a hypothetical 1 , 000 , 000 - member health plan , 2 , 066 medicare - insured patients and 156 commercially insured patients were estimated to have mnsclc and to be eligible for testing . 
2019 by american society of clinical oncology lung cancer ( nsclc ) , introduction nonsmall - cell the most common type of lung cancer , 1 can be classied on the basis of the presence of genomic alterations in driver oncogenes , 2 , 3 such as egfr , alk , ros1 , kras , braf , met , her2 , ret , and ntrk1.2 - 4 in the united states , several theratarget egfr , alk , ros1 , and braf pies that v600e alterations5 - 7 have been approved by the us food and drug administration ( fda ) therapy for patients with metaacross lines of static nsclc ( mnsclc )  . 
other targeted therapies are in clinical including those trial phase , against met , her2 , ntrk1 , and ret alterations identier : nct01639508 ; ( clinicaltrials.gov nct02414139 ; nct02716116 ; nct01336634 ; nct02097810 )  . improve treatment targeted therapies have been demonstrated to signicantly response and progression - free survival7 - 9 ; therefore , it is important to identify patients with specic genomic alterations to individualize treatment and optimize outcomes.2 , 9 currently , a number of testing strategies are available with which to identify genomic alterations in nsclc . 
 pennell et al context key objective to determine the least costly strategy for testing for all recommended molecular abnormalities in patients with nonsmall - cell lung cancer ( nsclc ) at diagnosis . knowledge generated upfront next - generation sequencing ( ngs ) testing for all relevant molecular targets in nsclc is less costly than performing single - gene testing , no matter what testing strategy is used . 
upfront ngs testing also results in the largest number of patients with targetable genetic alterations being identied in the shortest period of time . relevance as more clinically relevant genetic targets in nsclc emerge that require routine testing at diagnosis , it is vital to identify the molecular testing strategy that is timeliest , spares the most tissue , and is most cost efcient , as this must be done in every new patient . 
our model illustrates that moving from sequential single - gene tests or even panels of tests to broader ngs testing for patients with advanced nsclc is already the best strategy in these three areas and will only become more relevant as the list of tests grows . 
stakeholders should consider moving to ngs as the preferred method for biomarker testing . single - gene tests for less common alterations.10 sequences of single - gene tests can also be used without hotspot panel , typically testing for the most common alterations rst and less common alterations last.10 in either case , running a sequence of single - gene tests can be time consuming and may require a relatively large tissue sample , which is not always available as nsclc is often detected at an advanced stage and only small biopsy samples are usually attainable.11 - 13 as a result , one or more rebiopsies may be needed to complete the sequence . 
in recent years , next - generation sequencing ( ngs ) has emerged as a reliable strategy to simultaneously test for multiple alterations using a single tissue sample14 ; however , only a small percentage of patients with nsclc currently receive ngs , 15 as a number of practical barriers hinder its wider adoption , including limited patient access , a lack of awareness and communication within medical care teams about the benets of ngs , limited coverage , and low reimbursement rates.16 , 17 in addition , given the relatively recent development of ngs , there is a need to better understand the economic implications of using ngs versus other testing strategies in real - world clinical practice . therefore , a decision analytic model was developed to estimate the difference in testing costs and time - to - test results between ngs and commonly used single - gene testing strategies among patients with mnsclc from the perspective of the centers for medicare & medicaid services ( cms ) and a us commercial payer . 
in addition , the budget impact of increasing ngs testing was assessed from the perspective of both cms and a us commercial payer . methods model overview a decision analytic model was developed in microsoft excel 2016 from the perspective of cms and a us commercial payer for patients with newly diagnosed mnsclc who were eligible for genomic testing to determine mnsclc alteration status and inform treatment selection . 
alterations included in the model involved the programmed cell death protein 1 and its ligand programmed death protein 1 ligand ( pd - l1 ) , as well as genes egfr , alk , ros1 , braf , kras , met , her2 , ret , and ntrk1 . 
four genomic testing strategies were considered . upfront ngs : at mnsclc diagnosis , patients received a panel that tested simultaneously for all alterations with and without fda - approved therapies ( egfr , alk , ros1 , braf , met , her2 , ret , and ntrk1 )  . sequential testing : patients received a sequence of singlegene tests for alterations with fda - approved therapies ( egfr , alk , ros1 , and braf )  . 
negative test results for all four alterations were followed by either a sequence of single - gene tests or ngs to test for alterations without fda - approved therapies ( met , her2 , ret , and ntrk1 )  . 
single - gene tests were assumed to be ordered in sequence , one at a time , after receiving negative results for each previous test . exclusionary testing : patients rst received a test for kras because kras is the most common mutation in lung adenocarcinoma , and as the genetic alterations of interest are mutually exclusive , a positive test would prevent the need for additional testing . 
negative results were followed by either a sequence of single - gene tests or ngs to test for alterations without fda - approved therapies ( met , her2 , ret , and ntrk1 )  . all four testing strategies included pd - l1 and routine immunohistochemistry testing , which were assumed not to time because they were conducted sitake additional multaneously with the rst test in sequential and exclusionary testing and at the same time as the hotspot panel and ngs . 
appropriate therapyeither targeted therapy or nontargeted therapy ( eg , chemotherapy and immunotherapy ) was selected on the basis of the test results , that is , a conrmed alteration with or without fdaapproved therapies or no known alteration . 
patients who received rst - line nontargeted therapy and who tested negative for alterations with fda - approved therapies were assumed to continue testing with ngs or single - gene tests for alterations without fda - approved therapies , provided that enough tissue sample remained . 
a proportion of patients with alterations without fda - approved therapies were assumed to receive a benet from participating in clinical trials of agents under development . model inputs epidemiology and population inputs . 
for cms , the diagnosis of metastatic nsclc ( biopsy performed ) sequential exclusionary hotspot panel positive for tested alteration negative for tested alteration positive for tested alteration negative for tested mutation negative for tested mutation negative for tested mutation positive for tested alteration exclusionary mutation ( kras positive ) appropriate therapy appropriate therapy appropriate therapy appropriate therapy appropriate therapy enough tissue / no need for rebiospy insufficient tissue / need rebiopsy negative for tested mutation positive for tested alteration successful rebiopsy no rebiopsy / failed rebiopsy continue testing until appropriate treatment appropriate treatment appropriate treatment continue testing until appropriate treatment legend : continue testing for next alteration until the decision is made for appropriate treatment . is there sufficient v insufficient tissue and need for rebiopsy to continue testing ? fig 1 . 
sequential testing : sequence of single - gene tests for egfr , alk , ros1 , and braf , followed by either a sequence of single - gene tests or ngs for met , her2 , ret , and ntrk1 . 
hotspot panel : panel testing simultaneously for egfr , alk , ros1 , and braf , followed by either a sequence of single - gene tests or ngs for met , her2 , ret , and ntrk1 . 
epidemiology , population , and testing cost inputs variable commercial payer source patient population in the health care plan , no . 1 , 000 , 000 1 , 000 , 000 assumption adults in the population of interest , % * cms : kaiser family foundation18 ; commercial : us census bureau19 no . 
unit costs for all genomic alteration testing were based on the 2017 clinical lab fee schedule23 for the medicare - insured population and from dalal et al15 for the commercially insured population using two us administrative claims databases ( table 1 )  . 
single - gene tests for kras , egfr , her2 , and braf were assumed to be performed using real - time polymerase chain reaction . pd - l1 tests were assumed to be performed using immunohistochemistry or uorescence in situ hybridization . for each testing strategy , the per - patient cost of sequential , exclusionary , and hotspot panel testing was considered to be the sum of the cost of each individual test for pd - l1 and alterations with or without fdaapproved therapiesegfr , alk , ros1 , braf , met , her2 , ret , ntrk1 , and krasmultiplied by the proportion of patients who received each test , which was determined by the results for the preceding testing and rebiopsy failure rate ( table 2 )  . 
the difference between the plan - total testing costs of a certain strategy and those of ngs was considered as the cost savings associated with ngs versus the strategy in question . 
as a result of insufcient tissue sample , 8% of patients were assumed to require rebiopsy to continue testing.22 of these patients , only 30% were assumed to be actually rebiopsied , with 15% experiencing failure with rebiopsy.28 on the basis of on handorf et al , 28 rebiopsy cost was $415.30 for medicare - insured patients and $1 , 399.80 for commercially insured patients . 
one half of patients who tested negative for alterations with fda - approved therapies and who received nontargeted therapy were assumed to continue testing for alterations without fda - approved therapies . 
of these patients , 75% were assumed to continue testing with ngs and 25% with one of the other testing strategies . model outputs model results for ngs were compared with those for sequential , exclusionary , and hotspot panel testing in terms of differences in the estimated total cost associated with testing , calculated separately for cms and the commercial payer ; time - to - test results that accounted for the time associated with test turnaround and rebiopsy , assumed to be the same for cms and the commercial payer ; and the proportion of patients identied by each testing strategy as harboring alterations with or without fda - approved therapies . budget impact analysis we conducted a budget impact analysis to assess the effects of increasing upfront ngs testing in cms and commercial plans . 
total cost and cost difference versus ngs medicare - insured patients ( n = 2 , 066 ) commercially insured patients ( n = 156 ) testing strategy sequential exclusionary hotspot panel total cost 2 , 190 , 499 3 , 721 , 368 3 , 584 , 177 4 , 331 , 295 note . 
costs are given in 2017 us dollars . abbreviation : ngs , next - generation sequencing . cost difference v ngs total cost cost difference v ngs 1 , 530 , 869 1 , 393 , 678 2 , 140 , 795 620 , 369 747 , 771 624 , 178 871 , 211 127 , 402 3 , 809 250 , 842 the other parameters at their base - case values . 
on the basis of commercial payer for the 156 commercially insured patients , ngs rates , remained the least expensive testing option , with cost savings of $3 , 809 versus exclusionary testing , $127 , 402 versus sequential testing , and $250 , 842 versus hotspot panel testing . time to receive test results the cumulative time to receiving full test results and initiating treatment , incorporating both testing time and time for rebiopsy , is a critical element in molecular testing . 
proportion of patients identied with alterations with or without fda - approved therapies patients identied with alterations with fda - approved therapies ( medicare insured , n = 446 ; commercially insured , n = 34 ) patients identied , % difference v ngs , % difference in no . 
the only exception was ngs versus exclusionary testing for commercial payersranging from $19 , 943 to $27 , 562likely because of the relatively small base - case cost savings results ( $3 , 809 )  . 
results were most sensitive to scenarios in which test unit costs and the proportion of patients tested for alterations without fdaapproved therapies were varied . discussion several testing strategies for the detection of genomic alterations in nsclc exist in clinical practice ; however , scant evidence is available on the comparative economic implications of using these strategies in patients with mnsclc . 
accordingly , an economic model was developed to estimate the time - to - test results ; the proportion of patients identied as harboring alterations with or without fda - approved therapies ; and testing costs associated with upfront ngs , sequential , exclusionary , and hotspot panel testing among patients with mnsclc . model results demonstrate that ngs was associated with the same ( as hotspot panel ) or shorter ( v exclusionary and sequential testing ) time - to - test results and lower testing costs than sequential , exclusionary , and hotspot panel testing . both ngs and hotspot panel testing had the shortest time - totest results ( 2 weeks ) , indicating that these two testing strategies may yield the shortest time to initiation of appropriate therapy among the four strategies considered here . of note , although the unit cost of ngs was higher than that of individual single - gene tests , the overall cost , which included testing and rebiopsy cost , to test for a broad range of alterations was the lowest for ngs . 
although no treatment is yet approved for these alterations , it is important to identify these patients to give them the opportunity to participate in clinical trials of investigational therapies . although prior studies have discussed the cost effectiveness and benets of various testing strategies in patients with nsclc , 31 - 34 the current study is , to our knowledge , the rst to quantify the economic impact of using ngs versus other testing strategies to detect a comprehensive list of alterationsegfr , alk , ros1 , braf , met , her2 , ret , ntrk1 , and krasin patients with mnsclc in the united states . 
from a clinical standpoint , the timely identication of genomic alterations in patients with mnsclc is key to initiating the most appropriate treatment course on the basis of either targeted therapy , if available , or nontargeted therapy . 
as the results of the current study indicate , upfront ngs seems to be the cheapest broad molecular testing strategy for genomic alterations in terms of both time - to - test results and testing costs . 
furthermore , unlike other testing strategies , ngs allows for the simultaneous screening of both common and less common alterations , which is important because less common alterations are less likely to be tested when using sequential testing strategies . 
in a retrospective review of genomic testing patterns in patients with nonsquamous mnsclc , only 63 of 814 patients were reported to have undergone testing for all seven alterations recommended by national comprehensive cancer network for patients with mnsclc , namely egfr , alk , ros1 , braf , met , ret , and her2.17 using ngs may increase the number of patients tested for including both common and less common alterations , those recommended by the national comprehensive cancer network , thereby providing more information to physicians and patients with which to make informed and timely treatment decisions . 
future studies should also assess the clinical implications of our nding that , as a result of longer time - totest results , patients using sequential and / or exclusionary testing are able to initiate targeted therapy nearly 3 weeks later than patients using ngs or hotspot panel testing . 
it is worth noting that , whereas this study suggests that ngs is a more efcient testing strategy than sequential , exclusionary , and hotspot panel testing , several barriers to wider adoption of ngs in clinical practice remain , including limited coverage and reimbursement.17 however , this may change over time given that the lower total testing costs and shorter initiationof ngs , together with a reduced need for rebiopsy , may reduce medical costs substantially and improve outcomes , beneting patients and payers alike . resultsand thus treatment time - to - test this study has some limitations . 
first , as the study is based on a modeling approach using a decision tree and budget impact inputs and assumptionssome of framework , the model which were based on expert opinionmay contain uncertainty or may have limited generalizability . 
last , the model included only testing costs and not therapy costs and benets , the inclusion of which would allow for an estimate of a wider spectrum of costs . in conclusion , for both cms and commercial payers , upfront ngs testing was associated with the same ( as ( v exclusionary and hotspot panel testing ) or shorter sequential testing ) time to the initiation of appropriate treatment , as well as substantial cost savings compared with all other testing strategies in patients with newly diagnosed mnsclc . 
pennell , alex mutebi , zheng - yi zhou , marie louise ricculli , wenxi tang , helen wang , annie guerin , tom arnhart , anand dalal , kevin y . 
pennell , alex mutebi , zheng - yi zhou , marie louise ricculli , wenxi tang , helen wang , annie guerin , tom arnhart , anand dalal , medha sasane , kevin y . 
pennell consulting or advisory role : astrazeneca , eli lilly , regeneron , cota healthcare , merck research funding : genentech ( inst ) , celgene ( inst ) , astrazeneca ( inst ) , pzer ( inst ) , merck ( inst ) , loxo ( inst ) , altor bioscience ( inst ) , spectrum pharmaceuticals ( inst ) , bristol - myers squibb ( inst ) tom arnhart employment : novartis , alexion pharmaceuticals stock and other ownership interests : alexion pharmaceuticals anand dalal employment : novartis stock and other ownership interests : novartis medha sasane employment : sano , novartis ( i ) stock and other ownership interests : sano , novartis ( i ) kevin y . 
wu employment : novartis , astrazeneca stock and other ownership interests : novartis , astrazeneca , gilead sciences , intercept pharmaceuticals , merck , organovo , takeda , seattle genetics , tg therapeutics kenneth w . 
transl respir med 1 : 6 , 2013 kohno t , nakaoku t , tsuta k , et al : beyond alk - ret , ros1 and other oncogene fusions in lung cancer . 
expert rev mol diagn 16 : 737 - 749 , 2016 barlesi f , mazieres j , merlio jp , et al : routine molecular proling of patients with advanced non - small - cell lung cancer : results of a 1 - year nationwide programme of the french cooperative thoracic intergroup ( ifct )  . 
lancet 387 : 1415 - 1426 , 2016 kris mg , johnson be , berry ld , et al : using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs . 
dalal aa , guerin a , mutebi a , et al : economic analysis of braf gene mutation testing in real world practice using claims data : costs of single gene versus panel tests in patients with lung cancer . 
levy bp , chioda md , herndon d , et al : molecular testing for treatment of metastatic non - small cell lung cancer : how to implement evidence - based recommendations . 
yang p , allen ms , aubry mc , et al : clinical features of 5 , 628 primary lung cancer patients : experience at mayo clinic from 1997 to 2003 . 
reck m , rodrguez - abreu d , robinson ag , et al : pembrolizumab versus chemotherapy for pd - l1 - positive non - small - cell lung cancer . 
chen d , zhang lq , huang jf , et al : braf mutations in patients with non - small cell lung cancer : a systematic review and meta - analysis . 
meng d , yuan m , li x , et al : prognostic value of k - ras mutations in patients with non - small cell lung cancer : a systematic review with meta - analysis . 
handorf ea , mcelligott s , vachani a , et al : cost effectiveness of personalized therapy for rst - line treatment of stage iv and recurrent incurable adenocarcinoma 29 . 
vanderlaan pa , yamaguchi n , folch e , et al : success and failure rates of tumor genotyping techniques in routine pathological samples with non - small - cell lung 5 : 293 - 305 , 2011 22 : 2616 - 2624 , 2011 cancer 18 : 651 - 659 , 2017 snapshot / 2005 prevalence / canques.html 375 : 1823 - 1833 , 2016 2014 cancer 81 : 1 - 10 , 2013 of the lung . 
 clinical utility of gene expression classifiers in men with newly diagnosed prostate cancer purpose tissue - based gene expression classifiers ( gecs ) may assist with management decisions in patients with newly diagnosed prostate cancer . 
we sought to assess the current use of gec tests and determine how the test results are associated with primary disease management . methods in this observational study , patients diagnosed with localized prostate cancer were tracked through the michigan urological surgery improvement collaborative registry . 
practice patterns , predictors of gec use , and effect of gec results on disease management were investigated . results of 3 , 966 newly diagnosed patients , 747 ( 18.8% ) underwent gec testing . 
this results in an estimate that , for every nine men with favorable risk of cancer who undergo gec testing , one additional patient may have their disease initially managed with as . 
although active surveillance ( as ) is now recognized as a preferred treatment option for patients with low - risk prostate cancer , more than one third of patients will experience disease progression that requires treatment.1 - 3 men with favorable intermediate risk prostate cancer are rarely entered onto as , yet growing data suggest that some may be safely treated with as to avoid immediate radical therapy.4 multiple new tissue biomarkers have surfaced to aid in risk stratification of newly diagnosed patients , with a goal of improving prognostic accuracy compared with routine clinicopathologic variables . 
on the basis of multiple retrospective development and validation studies , there are now three widely available tissue - based gene expression classifiers ( gecs ) for use in this setting : prolaris ( myriad genetics , salt lake city , ut ) , oncotype dx prostate ( genomic health , redwood city , ca ) , and decipher prostate biopsy ( genomedx , vancouver , canada ) .5 - 12 these tools are now included in the national comprehensive cancer network ( nccn ) jonathan c . 
 guidelines as options to aid in decision making for men with newly diagnosed localized prostate cancer , although other national guidelines caution against their routine use.13 , 14 each of these tests are rna - based prognostic biomarkers that analyze a distinct multigene panel that predicts oncologic end points , which range from the likelihood of harboring unrecognized high - grade disease to death as a result of prostate cancer at 10 years . 
 although published data have demonstrated the prognostic utility of these tests compared with clinicopathologic data alone , 7 , 9 , 11 , 12 there is limited understanding of how these tools influence treatment decisions in the contemporary setting . one unique aspect of these tests is the continuous scale used to report risk , whether for near - term adverse pathology end points or more distant survival end points . 
this is in contrast to breast cancer , for which analogous tests have become incorporated into guidelines as a result of multiple prospective trials that validate discrete cut points for management decisions.15 - 17 because the emergence of multiple gec tests each with various measurement scales , thresholds , outcomes , and reporting toolsis recent , it is unclear how these tests affect real - world management decisions . to address this complex issue in localized prostate cancer , we sought to perform a prospective clinical utility study by tracking the use of gecs in patients with newly diagnosed prostate cancer and comparing treatment decisions according to gec result . 
to capture gec use and subsequent management across a broad cohort of patients , this study was performed through the michigan urological surgery improvement collaborative ( music ) , a consortium of more than 90% of urology practices in the state of michigan . 
we hypothesized that gec use would be highly variable and that results would correlate closely with subsequent treatment decisions , specifically that rates of as would be higher in gec - defined low - risk patients . methods study population within music . 
in each participating practice , trained abstractors prospectively enter a standardized set of demographic and pathologic data for every patient with a new prostate cancer diagnosis into an electronic clinical registry . 
abstractors also enter data related to treatmentradical prostatectomy , radiotherapy , or other ( primary androgen deprivation therapy , high - intensity focused ultrasound , cryotherapy , or other ablative technologies ) and follow - up data . 
each site obtains regulatory exemption from local institutional review boards to participate in music , and funding is provided by blue cross blue shield of michigan . in this analysis , we included all men who were newly diagnosed with clinically localized prostate cancer between january and september 2017 . 
subsequent treatment decisions ( as , prostatectomy , or radiotherapy ) were tracked until the analytic data set was locked on april 1 , 2018 . clinical and pathologic variable definitions data elements collected included age , ethnicity , prostate - specific antigen ( psa ) level , number of positive cores from biopsy , primary and secondary gleason patterns , prostate volume , clinical t stage , body mass index ( bmi ) , and comorbidity scores . 
prostate magnetic resonance imaging ( mri ) was categorized according to maximum prostate imaging and reporting data system ( pirads ) version 2 score as reassuring ( pirads 1 to 3 ) or nonreassuring ( pirads 4 to 5 )  . 
using standard clinicopathologic parameters , patients were categorized according to the favorable - risk prostate cancer stratification that has been widely disseminated in the music consortium as a way to identify patients who may be most appropriate for as.18 favorable - risk prostate cancer was defined by a biopsy gleason score of 6 or low - volume , gleason 3 + 4 = 7 disease ( defined as three or fewer cores positive and no core with > 50% cancer volume )  . 
 accordance with published data and test reports for each of these assays.19 a summary of the three assays and cut points used to define gec low - risk disease are listed below : decipher biopsy ( genomedx biosciences ) : microarray platform , with score composed of 22 rna biomarkers reported on a scale of 0 to 1 . 
threshold for low versus high risk is 0.45. oncotype dx prostate ( genomic health ) : quantitative real - time polymerase chain reaction platform , with score composed of 17 genes and reported on a scale of 0 to 100 . 
 threshold for low versus high risk is the probability of adverse surgical pathology greater than 20% . prolaris ( myriad genetics ) : quantitative real - time polymerase chain reaction platform , with score composed of 46 genes and reported on a scale of 1 to 10 . 
threshold for low versus high risk is 10 - year mortality risk of 3% or greater . for patients who had more than one test performed ( n = 13 ) , only the initial test was included in the analysis . end points the primary study outcome was gec testing status ( did not undergo gec testing v underwent gec testing with result below threshold v underwent gec testing with result above threshold )  . 
patients whose data were lost to follow - up , deceased , or switched practices for follow - up care were excluded from the evaluation of treatment because music no longer collected treatment information . 
patients who were still missing treatment data at the time of analysis because of a lag in data entry were also excluded . statistical analyses patient characteristics were compared between those who did and did not undergo gec testing using 2 tests for the overall cohort as well as for patients with favorable - risk prostate cancer . 
the proportion of patients with a test result above the threshold was then summarized by the type of the test used ( prolaris v oncotype dx v decipher )  . 
the primary management strategy was then compared across three groups defined by the performance and result of the gec test : no test , gec test below the threshold , and gec test above the threshold . 
the model included the performance and result of the gec test ( above or below the threshold ) as the primary predictor ( with no test as the reference group )  . 
the model also adjusted for patient and practice - level characteristics , including patient age , ethnicity , psa level , biopsy gleason score , clinical t stage , comorbidity , bmi , practice size , and practice type ( academic v private )  . 
among this subgroup , patients who underwent a gec test were more likely to be african american and to have a higher biopsy gleason score ( 3 + 4 v 6 ) , fewer positive cores , and nccn intermediaterather than low - risk cancer ( p < .05 for each ; appendix table a1 )  . 
seven practices ordered a gec test on more than 50% of patients with newly diagnosed prostate cancer . among the 747 patients who underwent gec testing , 439 ( 59% ) were tested with prolaris , 81 ( 11% ) with oncotypedx , and 227 ( 30% ) with decipher . 
gec testing was performed in 367 ( 17.6% ) of 2 , 080 patients with unfavorable risk ; 61 of these men ( 16.6% ) had a low - risk result , and 17 ( 27.9% ) elected as for initial management . of the 1 , 487 patients with favorable - risk disease but with treatment data , 320 ( 22% ) underwent gec testing . 
only 30 patients with favorable - risk disease underwent both mri and gec testing ; this represented 16% of patients who underwent mri and 9% of patients who underwent gec testing ( appendix table a2 )  . 
gec testing and use of as appeared to be independent of each other at a provider level , which indicated the absence of any provider - specific effect ( correlation coefficient r = 0.16 ; appendix fig a2 )  . with 63% of patients with favorable - risk prostate cancer harboring gec low - risk disease in this cohort ( 203 of 320 who underwent gec testing ) , these data suggest that , for every nine patients with favorable - risk disease who undergo testing , one patient will receive as who would have otherwise undergone primary therapy . 
 conversely , 117 ( 37% ) of 320 gec - tested patients with favorable - risk prostate cancer were considered molecularly high risk , and 46 ( 39% ) of these 117 patients selected initial definitive management compared with 337 ( 29% ) of 1 , 167 untested patients . 
patient and practice characteristics stratified by gec testing ( continued ) characteristic all patients ( n = 3 , 966 ) gec test performed ( n = 747 ) gec test not performed ( n = 3 , 219 ) practice size , no . 
more granular clinical tools to quantify risk , including the cancer of the prostate risk assessment25 score and memorial sloan kettering cancer center26 nomograms , can be affected greatly by variability among pathologists and are still somewhat limited in their prognostic accuracy.27 , 28 emerging tools , such as multiparametric mri ( mpmri ) , may improve the initial detection of more aggressive tumors , but variability in the performance and interpretation of mpmri may limit its generalizability for now.29 gec testing provides an additional objective tool for risk stratification . 
prospective data suggest that up to 67% of patients may be reclassified into different risk categories when genomic risk is considered in addition to conventional clinicopathologic parameters of risk.11 despite this , limited data describe the use of gec testing in this setting , and the effect of these tools on clinical decision making has not been well established . 87 112 152 243 133 165 practice ascopubs.org / journal / po jco precision oncology fig 1 . 
practice - level rate of genomic test after diagnosis : rate of gene expression classifier ( gec ) testing in newly diagnosed men with prostate cancer at each practice ( practices are numbered 1 through 44 )  . 
 the overall rates and rates within patient subgroups are presented separately . overall ( n = 747 ) patients with favorable risk ( n = 336 ) patients with unfavorable risk ( n = 411 ) patients with gleason score 3 + 4 = 7 ( n = 296 ) patients with gleason score 3 + 3 = 6 ( n = 215 ) a number of unique findings are presented in this study , because this is the first study , to our knowledge , to report on a prospective observational cohort of patients with and without gec testing . 
approximately 20% of patients diagnosed during the study period underwent a gec test , and there was wide variation in testing patterns across different urology practices in the state of michigan . 
fitting with the current approval details for each test , which are primarily for men with favorable - risk disease , these tests were more likely to be obtained in the setting of clinically less aggressive disease . 
patients were more likely to undergo testing if seen at a larger urology practice ( with more than 10 providers ) , which may relate to incorporation of gec testing into clinical care pathways at some of these practices . the analysis of the primary end point , clinical utility of gec testing in favorable - risk prostate cancer , provides key information about how these tests affect treatment decisions . 
this is one of the key measures used by the molecular diagnostic services program developed by palmetoto gba to determine medicare coverage , and prospective data that compare tested and untested patients . 
multivariable logistic regression model to assess variables associated with active surveillance among patients with favorable - risk prostate cancer variable gec ( ref : no test ) below threshold above threshold race ( ref : white ) african american biopsy gleason grade ( ref : 6 ) clinical t stage ( ref : ct1 ) ct2 or higher charlson comorbidity index ( ref : 0 ) practice size , no . 
the model also included random intercepts for practices . abbreviations : bmi , body mass index ; gec , gene expression classifier ; or , odds ratio ; psa , prostate - specific antigen ; ref , reference . underwent as instead of surgery or radiation for every nine patients tested and that one additional patient underwent primary therapy rather than as for every 26 patients tested . 
in the unfavorable - risk group , utility was less apparent : 17% of tested patients had gec low risk disease , and only 28% of these men received as . finally , it is important to note that meaningful differences existed among the three tests . 
 although we did not have sufficient data to compare test results for patients with more than one gec assay performed , we found substantial variability in the likelihood of gec highversus gec low - risk disease , depending on the test performed . 
for example , in the subgroup with a gleason score of 3 + 3 = 6 , 13.7% of patients tested with prolaris had results that were considered above the threshold , whereas 58.3% were above the threshold as reported by decipher . 
 importantly , although most of these test - specific thresholds have not been validated , they are included in test reports provided to patients and providers.30 this issue highlights the challenges of applying thresholds to tests developed and validated for assessment along a continuum of risk and also emphasizes the need for research to compare the predictive accuracy of each of these tests . 
although that was not the case in this cohort , additional study about appropriate and consistent thresholds for these tests is paramount . it is important note key limitations to this study . 
 some patients could have undergone gec testing outside of a music urology practice , such as by a radiation oncologist , and this would only be captured in the registry if the test results were provided to the primary urologist . 
an example of such a trial is the ongoing genomics in michigan impacting observation or radiation ( g - minor ) trial in the postprostatectomy setting.31 however , in the absence of a similar trial in patients with newly diagnosed disease , prospective observational data can provide critical insights into how these tests are guiding clinical decisions . these limitations notwithstanding , strengths of this study include prospective data collection on nearly 4 , 000 consecutively enrolled patients with newly diagnosed prostate cancer . 
although other studies have sought to evaluate treatment decisions related to gec testing , none included concurrently diagnosed patients without testing , which is vital to understand the clinical utility of these tests.32 - 35 in conclusion , these data indicate that gec testing has clinical utility in the favorable - risk setting , and testing of patients with favorable - risk disease is more common in men with higher psa levels and higher - grade disease who may not typically be considered as candidates . 
gec testing also is being performed in patients with intermediate - risk and high - risk unfavorable disease , for whom the clinical implications are less apparent , and there is marked variability in how gec tests are being used . 
spratt no relationship to disclose speakers ' bureau : myriad genetics , genomic health , genomedx , mdxhealth , strand diagnostics research funding : myriad genetics ( inst ) , genomic health ( inst ) , genomedx ( inst ) , mdxhealth ( inst ) , neogenomics laboratories ( inst ) frank n . 
morgan consulting or advisory role : myriad genetics , terumobct research funding : myriad genetics ( inst ) , mdxhealth ( inst ) , genomedx ( inst ) affiliations jonathan c . 
morgan , university of michigan , ann arbor ; and kirk wojno , comprehensive urology , royal oak , mi . supported by blue cross blue shield of michigan ; the department of defense physician research training award no . 
bokhorst lp , valdagni r , rannikko a , et al : a decade of active surveillance in the prias study : an update and evaluation of the criteria used to recommend a switch to active treatment . 
newcomb lf , thompson im , jr , boyer hd , et al : outcomes of active surveillance for clinically localized prostate cancer in the prospective , multi - institutional canary pass cohort . 
tosoian jj , mamawala m , epstein ji , et al : intermediate and longer - term outcomes from a prospective active - surveillance program for favorable - risk prostate cancer . 
cuzick j , stone s , fisher g , et al : validation of an rna cell cycle progression score for predicting death from prostate cancer in a conservatively managed needle biopsy cohort . 
cuzick j , swanson gp , fisher g , et al : prognostic value of an rna expression signature derived from cell cycle proliferation genes in patients with prostate cancer : a retrospective study . 
van den eeden sk , lu r , zhang n , et al : a biopsy - based 17 - gene genomic prostate score as a predictor of metastases and prostate cancer death in surgically treated men with clinically localized disease . 
klein ea , cooperberg mr , magi - galluzzi c , et al : a 17 - gene assay to predict prostate cancer aggressiveness in the context of gleason grade heterogeneity , tumor multifocality , and biopsy undersampling . 
spratt de , zhang j , santiago - jimnez m , et al : development and validation of a novel integrated clinical - genomic risk group classification for localized prostate cancer . 
spratt de , yousefi k , deheshi s , et al : individual patient - level meta - analysis of the performance of the decipher genomic classifier in high - risk men after prostatectomy to predict development of metastatic disease . 
barcenas ch , sinha ak , raghavendra as , et al : outcomes after chemotherapy in early - stage breast cancer ( ebc ) patients who underwent a 21 - gene expression assay . 
reese ac , pierorazio pm , han m , et al : contemporary evaluation of the national comprehensive cancer network prostate cancer risk classification systeurology 80 : 10751079 , 2012 25 . 
cooperberg mr , pasta dj , elkin ep , et al : the university of california , san francisco , cancer of the prostate risk assessment score : a straightforward and reliable preoperative predictor of disease recurrence after radical prostatectomy . 
morgan tm , miller dc , dunn r , et al : prospective randomized trial of genomic classifier impact on treatment decisions in patients at high risk of recurrence following radical prostatectomy ( g - minor )  . 
dallera ma , maddala t , polychronopoulos l , et al : utility of the oncotype dx prostate cancer assay in clinical practice for treatment selection in men newly diagnosed with prostate cancer : a retrospective chart review analysis . 
eure g , germany r , given r , et al : use of a 17 - gene prognostic assay in contemporary urologic practice : results of an interim analysis in an observational cohort . 
rev urol 18 : 123 - 132 , 2016 appendix for number needed to test calculations , we assumed that the distribution of test results in untested patients would reflect the distribution of test results in tested patients . 
we first asked the following : of the 1 , 167 patients with favorable risk of cancer without a gene expression classifier ( gec ) test , how many patients with a gec test result below threshold would likely be reallocated to active surveillance ( as ) if they had undergone gec testing ? we calculated this as ( 203 / 320 ) 1 , 167 = 740 , which means that approximately 740 patients with low - risk gec exist of the 1 , 167 untested men . 
similarly , we assumed that , of the 667 untested patients with favorable risk of cancer who initially received as in this cohort , ( 203 / 320 ) 667 , or 429 of these men , are truly gec low risk . 
thus , use of a gec test should shift 562 429 , or 133 , patients with low - risk gec to as and result in a number needed to test of 1 , 167 / 133 , or 8.8 patients with favorable risk of cancer to shift one patient to as . conversely , of the 1 , 167 patients with favorable risk of cancer without a gec test , we asked how many patients with a gec test result above the threshold would likely be reallocated to definitive treatment if they had undergone gec testing . 
we assumed that ( 117 / 320 ) 1 , 167 , or 427 , would be gec high risk , using the same approach as for low risk , and that 427 39.3% , or 168 patients , would undergo primary therapy . 
summary of primary management in patients with favorable - risk disease according to the performance and result of prostate mri and gec testing test result total active surveillance definitive treatment no treatment no mri no gec test gec test < threshold gec test > threshold reassuring mri no gec test gec test < threshold gec test > threshold nonreassuring mri no gec test gec test < threshold gec test > threshold 564 ( 56.6 ) 144 ( 77 ) 46 ( 45.1 ) 77 ( 78.6 ) 9 ( 69.2 ) 5 ( 71.4 ) 34 ( 55.7 ) 0 ( 0 ) 3 ( 37.5 ) 299 ( 30 ) 27 ( 14.4 ) 41 ( 40.2 ) 14 ( 14.3 ) 2 ( 15.4 ) 2 ( 28.6 ) 17 ( 27.9 ) 2 ( 100 ) 3 ( 37.5 ) 134 ( 13.4 ) 16 ( 8.6 ) 15 ( 14.7 ) 7 ( 7.1 ) 2 ( 15.4 ) 0 ( 0 ) 10 ( 16.4 ) 0 ( 0 ) 2 ( 25 ) note . 
by law , we are the custodians of diagnostic tissues , and as such , we must comply with legal regulations covering the preservation and use of these tissues.1 as representatives of our hospitals and specialty , we want to be helpful collaborators in providing the diagnostic tissues necessary for the care of oncology patients . 
and as patient advocates , because of our legal role as custodians of these tissues , many of us harbor serious concerns about the current informed consent processes and standard practices for tissue handling during clinical trials and translational research . 
the vast majority of tissue - based assays used in the clinic ( including dna sequencing , rna and fusion protein identication by in situ hybridization , and immunohistochemistry to detect protein expression ) can be performed on formalinxed , parafn - embedded ( ffpe ) tissues . 
simultaneously , there is less diagnostic tissue available on each patient because of smaller biopsies acquired as part of lessinvasive sampling techniques , including small - caliber core biopsies or ne - needle aspiration ( fna ) cell blocks , earlier identication of small tumors , and successful neoadjuvant lead to marked tumor response . treatments that conservative estimates from our clinical practice suggest that up to 50% of tissue in diagnostic biopsies is used during routine standard - of - care testing , with more being used for challenging cases ( unpublished data )  . 
typical clinical scenarios from our practices illustrate the potential tensions between the availability of tissue after standard - of - care testing ( the supply ) and subsequent demands for tissues for research . 
their eligibility for these trials inevitably will lead to a request by the investigators for tissue submission ( usually 10 to 15 but sometimes 25 unstained slides or the entire parafn block ) , often as a condition of enrollment . 
pathologists are the caretakers of all tissues removed during clinical care , 1 and there are federal , state , and sometimes local laws governing how pathologists must treat those tissues . 
 mccall and dry of american pathologists ( cap ) require that pathologists store archival parafn tissues in a retrievable manner for a minimal time , which includes rules prohibiting exhaustion of the tissue during storage . 
these laws were intended to protect a patients ability to access the tissue for subsequent diagnostic testing , transfer of care , or medicolegal inquiries . as noted , many patients have only limited amounts of tissue available after standard - of - care testing . 
it is not uncommon for a pathologist to examine a block of tissue and realize that submitting the material desired by one clinical trial will completely exhaust the remaining biopsy sample . 
if they refuse to fulll the request , the patient will not qualify for the study , which will upset the patient and oncologist and will deny the patient a therapy with the potential to extend their survival and / or improve their quality of life . local trial enrollment , pathologists , when confronted with a request for limited biopsy tissue as a condition of stitutional review boards , and hospital legal counsel in the united states struggle with a catch - 22 situation : national institutes of healthsponsored clinical trials require submission of research tissue , whereas the centers for medicare and medicaid services ( another federal agency ) reimburse testing only in clia - certied laboratories ( that must adhere to tissue retention guidelines )  . 
clia requires that parafn blocks be retained for 2 years , and cap ( which has clia - deemed status and is the accrediting body for many us pathology laboratories ) requires that pathology laboratories retain parafn blocks for 10 years.4 , 5 state laws may have different retention requirements , and laboratories must comply with the most stringent applicable requirement . 
if a patient were appropriately informed , would that patient elect to provide two slides for biomarker research or trial eligibility and not provide 15 slides for downstream , unnamed correlative science ? as caretakers of the parafn archival tissues , pathologists take special care to avoid exhausting diagnostic biopsy samples in any retrospective translational research project . should correlative science performed as part of a clinical trial be different ? are clinical trial sponsors using ( for research or clinical purposes ) all the tissue they are collecting on enrolled patients ? and what about the submitted tissues from patients who are found to be ineligible for the study ? recent cancer clinical trials have reported accrual rates of 14% to 46%.7 , 8 patients are risking their existing tissues for an uncertain or even low chance of participation , without fully understanding the risks of tissue exhaustion or the trial needs for eligibility testing versus correlative science ( appendix )  . should the pathologist make executive decisions in these cases regarding the most judicious use of the remaining tissue ? as seasoned pathologists , we recall a time in which these individual patient scenarios would be discussed directly with oncologists ; however , the ever - increasing volume of patients , the remarkable number of available clinical trials , the practice of patients seeking second ( and third and fourth ) opinions far from home , and other pressures on general medical practice are making these conversations increasingly difcult to have . there are ethical and practical concerns here . 
patients may believe that the available clinical trial provides their only hope of benet from additional therapy . however , from prior experience , oncologists and pathologists know that the patient may not respond to the rst clinical trial agent and may want to enroll in a second clinical trial . 
 precision pathology in precision medicine mimicked metastatic disease or had developed a second cancer that had metastasized ( or that presents multifocally , such as multiple myeloma ) whereas the original cancer was controlled . 
without diagnostic conrmation of the tissues procured for research , these patients have a small but real risk of receiving the wrong treatment , which could shorten in misleading data and untheir life span and result necessary expense for the study sponsor . recommendations fortunately , we believe that there are multiple approaches available to address these concerns . 
working together , regulators , pathology departments , investigators , and clinical trial sponsors can improve how we prioritize limited tissue samples and how we inform patients of the risks and benets of using remnant clinical diagnostic samples for research . although we do not recommend major changes to clia or cap tissue retention regulations , we do feel a minor change is needed , namely explicit authorization to exhaust clinical diagnostic tissues if required for clinical trial enrollment ; we do not recommend similar authorization for unnamed correlative science as part of a clinical trial . 
we believe the minor modications we propose would continue to protect patients terests , while clarifying and permitting tissue exhaustion in exceptional circumstances . future diagnostic and medicolegal we also recommend changes to informed consent clinical trials . 
patients need to understand what materials are required for study enrollment , what materials are requested for possible future correlative science , benets and risks of providing materials for future correlative science , and their risks if their diagnostic tissues are exhausted in the process of getting tissues for study enrollment or future correlative science . 
such truly informed consent would fulll the intention of the common rule for protecting human research subjects9 and could make it easier for pathologists , institutional review boards , and legal counsel to approve exceptions to laws and regulations governing conservation of diagnostic tissues . for sponsors who receive more limited correlative science tissues , scientic review committees could prioritize the use of the material . 
other sources of cancer biologic materials , such as cell - free dna ( cfdna ) in peripheral blood could be considered , because cfdna may be most representative of the mutational prole of aggressive , metastatic tumor clones . 
in fact , the fda approved cfdna assays to direct treatment in egfr - mutated nonsmall - cell lung cancer.10 when possible , clinical trial sponsors should design studies so that research - only biopsies can be taken at the same time as routine clinical diagnostic biopsies . 
research - only biopsies performed under specic study consent do not fall under state , clia , or cap custodianship requirements , allowing entire parafn blocks to be submitted directly to the sponsor . finally , we urge sponsors to obtain diagnostic conrmation before enrolling patients into clinical trials . 
the incidence of such errors is unknown and is worthy of study . new approaches in pathology pathology departments also can make important changes . they can and should make it easier for clinical trial sponsors to obtain additional tissue sections to facilitate correlative science . 
this would encourage sponsors to reduce their upfront minimum acceptable requirements . this also could be accomplished by using consent language that gives the pathology department permission to send additional unstained slides or ( in case the patient dies ) a representative block or blocks for additional correlative science to the sponsor . 
sponsors could leverage virtual biobank technology to keep track of the samples potentially available to them for correlative science . pathology departments should review and update their protocols for processing small biopsy and cytology samples to maximize their potential downstream utility . 
one clear best practice emerging from recent studies is the separation of multiple biopsy cores into individual parafn blocks.11 - 14 this saves tissue for research and facilitates retention of representative diagnostic material per cap and clia regulations . 
although this prevents wasting tissue , it does require workows that may not be compatible with busy clinical histology laboratories and may be more suitable for research laboratories and biorepositories . 
 mccall and dry laboratories are reimbursed for interrupting clinical workows for high - priority assessments ( intraoperative consultations , cytologic adequacies ) , and research pathologists and laboratories should also be appropriately reimbursed for these activities . for downstream molecular during the biopsy procedure , cytopathologists or cytotechnologists can perform an immediate adequacy assessment in the procedural suite ( so - called rapid on - site evaluation [ rose ] )  . 
because feedback is immediate , rose increases the likelihood that research - only procedures will be successful in obtaining diagnostic tissue sufcient testing.15 during clinical and diagnostic biopsy procedures , rose is generally performed with simple designations such as diagnostic or nondiagnostic . 
in the era of precision pathology , varying degrees of diagnosis may be desired : enough for denitive diagnosis , enough for denitive diagnosis plus standard - of - care ancillary testing , or enough for denitive diagnosis plus standard - of - care ancillary testing and submission of tissue for clinical trial research . 
upfront planning and budgeting by sponsors of rose for research - only biopsies will ensure the availability of specialized cytotechnologists or cytopathologists and will increase the probability that the sponsor obtains sufcient malignant tissues for study . 
if the cytopathologist cannot be on site , telecytopathology has been successful and may decrease costs.16 , 17 in summary , pathologists want to support research and want to help their patients enroll in clinical trials . 
but we are concerned that current practices could result in morbidity for our patients in the form of additional biopsy procedures , may not meet with patient expectations of tissue availability after the clinical trial , and may explicitly violate federal regulations , state laws , and the standards of laboratory accrediting bodies . 
mccall other relationship : college of american pathologists no other potential conicts of interest were reported . references dry s , grody ww , papagni p : stuck between a scalpel and a rock , or molecular pathology and legal - ethical issues in use of tissues for clinical care and research : what must a pathologist know ? am j clin pathol 137 : 346 - 355 , 2012 sepulveda ar , hamilton sr , allegra cj , et al : molecular biomarkers for the evaluation of colorectal cancer : guideline from the american society for clinical pathology , college of american pathologists , association for molecular pathology , and the american society of clinical oncology . 
j clin oncol 35 : 1453 - 1486 , 2017 lindeman ni , cagle pt , aisner dl , et al : updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors : guideline from the college of american pathologists , the international association for the study of lung cancer , and the association for molecular pathology . 
amaria rn , prieto pa , tetzlaff mt , et al : neoadjuvant plus adjuvant dabrafenib and trametinib versus standard of care in patients with high - risk , surgically resectable melanoma : a single - centre , open - label , randomised , phase 2 trial . 
lozano md , echeveste ji , abengozar m , et al : cytology smears in the era of molecular biomarkers in non - small cell lung cancer : doing more with less . 
aisner dl , rumery md , merrick dt , et al : do more with less : tips and techniques for maximizing small biopsy and cytology specimens for molecular and ancillary testingthe university of colorado experience . 
harada s , agosto - arroyo e , levesque ja , et al : poor cell block adequacy rate for molecular testing improved with the addition of diff - quik - stained smears : need for better cell block processing . 
khurana kk , graber b , wang d , et al : telecytopathology for on - site adequacy evaluation decreases the nondiagnostic rate in endoscopic ultrasound - guided ne - needle aspiration of pancreatic lesions . 
identifying sufciently expanded short nucleotide repeat sequences in the tumor compared with non - neoplastic tissue is diagnostic for microsatellite instability ( whether it is the result of sporadic or inherited causes )  . however , if a clinical trial sponsor is already aware of a tumor mutation that has a hereditary cancer association , submitting non - neoplastic tissue to the sponsor could conrm a sporadic versus a hereditary origit should be noted that some commercial providers of highthroughput next - generation sequencing assays have elected to neither simultaneously test non - neoplastic germ line tissue nor provide interpretation of a possible inherited mutation within their reported results on the basis of increased mutant allele fraction . 
 window - of - opportunity study of valproic acid in breast cancer testing a gene expression biomarker purpose the anticancer activity of valproic acid ( vpa ) is attributed to the inhibition of histone deacetylase . 
we previously published the genomically derived sensitivity signature for vpa ( gdssvpa ) , a gene expression biomarker that predicts breast cancer sensitivity to vpa in vitro and in vivo . we conducted a window - of - opportunity study that examined the tolerability of vpa and the ability of the gdss - vpa to predict biologic changes in breast tumors after treatment with vpa . patients and methods eligible women had untreated breast cancer with breast tumors larger than 1.5 cafter a biopsy , women were given vpa for 7 to 12 days , increasing from 30 mg / kg / d orally divided into two doses per day to a maximum of 50 mg / kg / d . 
fifty percent of women ( three of six ) with triple - negative breast cancer had a ki - 67 reduction of at least 10% compared with 17% of other women . 
treatment individualization has been accomplished to a limited extent with the use of hormone receptor testing to determine a patients eligibility for antiestrogen therapy and human epidermal growth factor receptor 2 ( her2 ) testing to determine eligibility for anti - her2 therapies . however , with the ability to analyze tumors on the basis of their genomic profiles , further individualization may be possible for additional cancer therapies . inhibitors have histone deacetylase ( hdac ) shown promise in breast cancer in vitro , although this promise has not yet translated to clinical benefit . 
 effects , including increasing the expression of tumor suppression genes , 2 increasing the expression of cell cycle regulators , 3 increasing the expression of mediators of apoptosis , 4 - 7 decreasing proteasomemediated degradation of tumor suppressor genes , 8 decreasing oncoprotein levels via loss of hsp90mediated protection , 9 - 11 decreasing mitotic stability , 12 and decreasing angiogenesis.13 , 14 hdac inhibitors potentiate the apoptotic effect of anthracyclines on breast cancer cell lines.15 valproic acid ( vpa ) is an antiepileptic discovered in 1882 , which has been used clinically since 1962 . vpa inhibits class i and class iia hdacs.16 , 17 in vitro and in vivo studies show that vpa at clinically relevant concentrations inhibits the proliferation of breast cancer cell lines and xenografts.18 - 21 vpa has been used in breast cancer in combination with chemotherapy in three small trials22 - 24 in which response rates ranged from 33% to 70% , but the relative contribution of vpa and other drugs could not be determined . 
we previously published a gene expression signature that predicted the sensitivity of breast cancer to vpa in vitro and in vivo.21 we refer to this signature as the genomically derived sensitivity signature for vpa ( gdss - vpa )  . before committing to phase i and ii studies , it is important to establish the biologic effect of a drug and get preliminary data on potential biomarkers . in window - of - opportunity studies , women with newly diagnosed breast cancer receive a drug between the diagnostic breast biopsy and planned surgical resection or the start of neoadjuvant therapy . 
the primary objectives were to determine safety and tolerability of vpa , whether vpa levels correlate with histone acetylation in peripheral blood mononuclear cells ( pbmcs ) during treatment , and whether either vpa levels or histone acetylation in pbmcs predicts histone acetylation in tumor samples after treatment . 
key secondary objectives were to assess the sensitivity and specificity of the gdss - vpa to predict histologically measured antitumor activity of vpa in breast cancer , to assess the sensitivity and specificity of the gdss - vpa to predict antitumor activity of vpa in breast cancer as measured by dce - mri scanning , and to determine whether women have dose - limiting toxicities during or after treatment with vpa over 7 to 12 days . eligibility women with any stage of breast cancer who had received no prior treatment were potentially eligible for the trial . 
adult women were eligible if they had eastern cooperative oncology group performance status of 0 to 2 , a biopsy - proven invasive adenocarcinoma of the breast 1.5 cm or larger by clinical examination or imaging , were able to provide consent , and either were scheduled for breast surgery or were willing to have an endof - study biopsy . 
exclusion criteria included pregnancy ; need for immediate chemotherapy ; hypersensitivity to vpa or its components ; peanut allergy ( because some vpa formulations contain peanut oil ) ; inadequate bone marrow , kidney , or liver function ( greater than grade 1 by common terminology criteria for adverse events v3 ) ; were immunocompromised as a result of medications or hiv ; used other antiepileptics or medications that interact with vpa ; had inborn errors of metabolism ; had a history of pancreatitis ; were on a ketogenic diet ; were unable to have an mri scan ; or had a tumor that was unlikely to yield adequate tissue for genomic studies . conduct of the study women were enrolled between the biopsy that diagnosed their breast cancer and the start of therapy for the breast cancer , and all provided informed consent . 
estrogen receptor ( er ) , progesterone receptor ( pr ) , and her2 were assessed by using standard diagnostic methods.29 , 30 the percent of invasive cancer cells that expressed ki - 67 in samples before and after treatment with vpa was determined manually by a certified expert pathologist ( r.e.f. ) , who used an eyeball estimate . 
if no intolerable grade 2 or higher adverse effects ( aes ) were present , the dose was increased every 3 days by 10 mg / kg / d to a maximum of 50 mg / kg / d . 
dce - mri , dynamic contrast - enhanced magnetic resonance imaging ; pbmc , peripheral blood mononuclear cell ; vpa , valproic acid . screened ( n = 39 ) enrolled ( n = 31 ) completed vpa ( n = 29 ) screen failed no tumor on biopsy unable to isolate rna infection unwilling to do rebiopsy could not have mri withdrew consent ( n = 8 ) ( n = 1 ) ( n = 2 ) ( n = 1 ) ( n = 1 ) ( n = 2 ) ( n = 1 ) withdrawn change in diagnosis withdrew consent ( n = 2 ) ( n = 1 ) ( n = 1 ) missing some data post treatment mri : ( n = 4 ; 3 could not complete , and 1 technical problem ) pbmc histone acetylation : ( n = 4 ; insufficient pbmcs collected ) evaluable for ( n = 31 ) tolerability ( n = 29 ) ki - 67 pbmc histone acetylation ( n = 25 ) ( n = 25 ) dce - mri ( 7 days ) of vpa had been given or after 24 doses ( 12 days ) of vpa had been given . once the patient had received the highest tolerated dose of vpa treatment for at least four doses , a dce - mri scan was performed followed by either surgical excision of the tumor per standard of care or a repeat core biopsy . 
pbmcs were frozen , and histone acetylation was assessed by using western blot ( acetyl - h3 antibody #9649s and beta - actin antibody #3700s ; cell signaling technology , danvers , ma )  . the gdss - vpa was calculated by using rna from the pretreatment and post - treatment specimens as previously described.21 parameters for the gene expression signature were locked before the trial . 
cognitive effects were assessed by using the short portable mental status questionnaire ( spmsq ) .31 statistical analysis a sample size of 33 patients was planned to give a power of 80% to detect a correlation of 0.5 between the vpa level on the day of surgery and change in tumor or peripheral blood histone acetylation from day 0 to the day of surgery at a twosided a = .025. 
accuracy of predictions was determined by using area under the receiver operating characteristic ( roc ) curves calculated by using the proc r package.32 cis for roc curves were calculated by using the method of delong.33 thirty - three patients would result in a precision for estimating the specificity of the gdss - vpa of 6 14% if the specificity were 0.8 and 6 17% if the specificity were 0.6. 
 results patient characteristics between june 2010 and august 2014 , 39 patients were screened , 31 were enrolled , and 29 were treated with vpa ( fig 1 )  . 
all patients completed the study . safety and tolerability of the 31 women who started vpa treatment , 16 ( 51.6% ) completed the 50 mg / kg / d dose . 
characteristics of participants in vast characteristic median age , years ( range ) tumor size 54 ( 31 - 73 ) tumor grade er - positive / her2 - negative er - positive / her2 - positive er - negative / her2 - positive triple - negative study . 
all aes resolved quickly upon stopping vpa . pharmacodynamics most women ( 68% ) had increased pbmc histone 3 ( h3 ) acetylation after treatment with vpa ( fig 2a )  . 
there seemed to be a threshold effect at 100 mg / ml of vpa , because no women with vpa levels below this level had a doubling of pbmc h3 acetylation . 
however , change in histone acetylation was inconsistent ; three of eight women with serum vpa levels above 200 mg / ml had less than a doubling of h3 acetylation . 
h3 acetylation in tumors could not be assessed because of technical issues . we assessed the change in gene expression from before to after vpa treatment in breast tumors by using the gdss - vpa , which was designed by comparing gene expression patterns in breast cancer cells before and after in vitro treatment with vpa.21 the gene expression pattern in the posttreatment samples was significantly more like that seen in cells treated in vitro with vpa ( mean difference , 0.2063 on a scale of 0 to 1 , with 0 being the most like untreated cells and 1 being the most like vpa - treated cells ; p , .001 by paired t test )  . 
thirty - one percent of the women with er - positive / her2 - negative breast cancer had a decrease in ki - 67 of at least 10% , and 50% of women with triple - negative breast cancer ( tnbc ) had a decrease in ki - 67 of 10% or greater with vpa treatment . 
there was no detectable difference in age , grade , tumor size , baseline ki - 67 , final vpa level , or tumor subtype between the four responding women and the rest of the group , with the exception of the lack of her2 - positve tumors among the responders . 
in particular , of the four women with tumors whose ki - 67 decreased by 20% or more , two tumors were er - positive / pr - positive and two were triplenegative , two were grade 3 and two were grade 2 . in addition to using gdss - vpa as an indicator of exposure to vpa , we tested its ability as a biomarker on the pretreatment tumor rna to predict sensitivity to vpa . 
with the optimal cutoff chosen post hoc on the basis of the roc curves , the gdss - vpa had a sensitivity of 75% and a specificity of 64% for predicting a decrease in ki - 67 of at least 20% . 
 ( a ) scatter plot comparing serum valproic acid ( vpa ) level on the last day of treatment and the change in peripheral blood mononuclear cell histone 3 acetylation . 
the change in histone 3 acetylation is calculated as the ratio of the normalized expression of acetylated histone 3 by western blot on the final day of treatment to that before treatment . 
one tumor , which had a decrease from 60% to 40% in ki - 67 , was grade 3 on the pretreatment biopsy and grade 2 on the posttreatment lumpectomy , and one tumor was grade 2 on the pretreatment biopsy and grade 3 on the post - treatment lumpectomy . 
window - ofopportunity studies in breast cancer have been completed in the united kingdom , sweden , and canada.25 , 26 , 37 - 41 in the united states , windowof - opportunity studies have been conducted but have been more challenging.42 - 46 despite the lack of evidence of harm from short delays in definitive therapy , women in the united states are often reluctant to wait more than 2 weeks between seeing a surgeon and having surgery.41 the vast trial enrolled 94% of the goal population over 4 years . 
without treatment , the absolute change in ki - 67 between biopsy and surgery averages 2% to 4%.47 by contrast , large changes in ki - 67 were seen in some women in our trial , particularly women with tnbc . 
this contrasts with in vitro and in silico data from our group and from others.21 , 48 the reason for this lack of sensitivity may be related to the differences in expression of the various hdacs among breast cancer subtypes.49 , 50 our results complement another recent windowof - opportunity study of hdac inhibitors in breast cancer . 
other ongoing studies are combining hdac inhibitors with chemotherapy ( nct02632071 , nct02393794 ) , endocrine therapy ( nct02820961 , nct02115282 , nct02395627 ) , or immunotherapy ( nct02708680 , nct02453620 , nct02395627 )  . 
 ( a ) hematoxylin and eosin stain from biopsies before ( left ) and after ( right ) treatment with valproic acid ( vpa ) showing fibrosis typical of a post - treatment effect . 
like any biomarker , gdss - vpa was not perfect . discrepant response to drug and biomarker prediction could be the result of tumor heterogeneity , subtherapeutic vpa levels , deficiencies in the signature , or insufficient time of treatment ; our sample size was too small for a multivariable analysis . vpa faces significant challenges as an anti breast cancer agent . 
although by traditional measures it is well tolerated with no grade 4 toxicities , many of the grade 2 toxicities , including dizziness , sleepiness , and cognitive slowing , are intolerable over long periods of time and are difficult to ameliorate with standard supportive care . 
 ( a ) scatter plot comparing genomically derived sensitivity signature for valproic acid ( gdss - vpa ) prediction to the change in ki - 67 from the pretreatment tumor to the post - treatment tumor . the prediction ranges from 0 ( predicted most resistant ) to 1 ( predicted most sensitive )  . 
 ( b ) receiver operating characteristic curve for the gdss - vpa predictor for predicting a decrease in ki - 67 of 20% . finally , the nonlinear relationship between vpa serum levels and pbmc histone acetylation changes suggests pharmacogenetic effects that may make vpa ineffective in some people . therefore , other hdac inhibitors may be more appropriate for future studies . weaknesses of our study include the small sample size and the inability to assess histone acetylation changes in tumors . 
conclusions about the relationship between tumor subtype and sensitivity to vpa are hypothesis generating only . finally , the short time period of treatment may not have been long enough to see the full effect of treatment . we were able to complete a window - ofopportunity trial of vpa in breast cancer and to test a gene expression biomarker of sensitivity to vpa . 
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kim hr , kim ej , yang sh , et al : trichostatin a induces apoptosis in lung cancer cells via simultaneous activation of the death receptor - mediated and mitochondrial pathway ? exp mol med 38 : 616 - 624 , 2006 6 . 
zhang xd , gillespie sk , borrow jm , et al : the histone deacetylase inhibitor suberic bishydroxamate regulates the expression of multiple apoptotic mediators and induces mitochondria - dependent apoptosis of melanoma cells . 
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zhou q , agoston at , atadja p , et al : inhibition of histone deacetylases promotes ubiquitin - dependent proteasomal degradation of dna methyltransferase 1 in human breast cancer cells . 
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bali p , pranpat m , bradner j , et al : inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90 : a novel basis for antileukemia activity of histone deacetylase inhibitors . 
phiel cj , zhang f , huang ey , et al : histone deacetylase is a direct target of valproic acid , a potent anticonvulsant , mood stabilizer , and teratogen . 
hodges - gallagher l , valentine cd , bader se , et al : inhibition of histone deacetylase enhances the anti - proliferative action of antiestrogens on breast cancer cells and blocks tamoxifen - induced proliferation of uterine cells . 
candelaria m , gallardo - rinc on d , arce c , et al : a phase ii study of epigenetic therapy with hydralazine and magnesium valproate to overcome chemotherapy resistance in refractory solid tumors . 
arce c , perez - plasencia c , gonzalez - fierro a , et al : a proof - of - principle study of epigenetic therapy added to neoadjuvant doxorubicin cyclophosphamide for locally advanced breast cancer . 
m unster p , marchion d , bicaku e , et al : phase i trial of histone deacetylase inhibition by valproic acid followed by the topoisomerase ii inhibitor epirubicin in advanced solid tumors : a clinical and translational study . 
hadad sm , coates p , jordan lb , et al : evidence for biological effects of metformin in operable breast cancer : biomarker analysis in a pre - operative window of opportunity randomized trial . 
klintman m , dowsett m : early surrogate markers of treatment activity : where are we now ? j natl cancer inst monogr 2015 ( 51 ) : 24 - 28 , 2015 28 . 
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wolff ac , hammond me , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
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mehta s , hughes np , buffa fm , et al : assessing early therapeutic response to bevacizumab in primary breast cancer using magnetic resonance imaging and gene expression profiles . 
singletary se , atkinson en , hoque a , et al : phase ii clinical trial of n ( 4 - hydroxyphenyl ) retinamide and tamoxifen administration before definitive surgery for breast neoplasia . 
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curigliano g , g omez pardo p , meric - bernstam f , et al : ribociclib plus letrozole in early breast cancer : a presurgical , window - of - opportunity study . 
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gandini s , guerrieri - gonzaga a , pruneri g , et al : association of molecular subtypes with ki - 67 changes in untreated breast cancer patients undergoing pre - surgical trials . 
mawatari t , ninomiya i , inokuchi m , et al : valproic acid inhibits proliferation of her2 - expressing breast cancer cells by inducing cell cycle arrest and apoptosis through hsp70 acetylation . 
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m uller bm , jana l , kasajima a , et al : differential expression of histone deacetylases hdac1 , 2 and 3 in human breast cancer : overexpression of hdac2 and hdac3 is associated with clinicopathological indicators of disease progression . 
 decision - making preferences about secondary germline findings that arise from tumor genomic profiling among patients with advanced cancers purpose in patients with advanced cancers , tumor genomic profiling ( tgp ) can reveal secondary germline findings ( sgfs ) with regard to inherited disease risks . 
this study examined the process by which patients with advanced cancers would decide about whether to learn these sgfs and their preferences about specific challenging decision scenarios , including whether patients should be required to receive sgfs and whether sgfs should be returned to the family after a patients death . patients and methods we conducted qualitative semistructured interviews with 40 patients with advanced breast , bladder , colorectal , or lung cancer who had undergone tgp . 
data were collected on participants perspectives about the hypothetical decision to learn their sgfs , including their anticipated approach to the decision - making process , and their preferences about challenging decision scenarios . 
data were evaluated by thematic content analysis . results we identified themes with regard to participants preferred degree of decisional autonomy , perceived vital role of doctors , information needs , and anticipated process of deliberation . 
most participants stated that patients should be able to make a choice about receiving actionable sgfs , and a majority stated that sgfs should be available to family after a patients death . conclusion these results provide insight into sgf decision - making processes of patients with advanced cancers , which can allow clinicians to provide patients with optimal decision support in this context . 
these germline variants are considered secondary findings when actively sought by researchers or clinicians ( or incidental findings when not ) because they arise outside the original purpose of tgp.1 , 2 secondary germline findings ( sgfs ) that indicate risks for various health conditions are likely to be detected in a sizable minority of patients who receive tgp ; for example , presumed pathogenic germline variants have been observed jada g . 
this decision is likely to be challenging , particularly for patients with advanced cancers who are currently the primary users of tgp ( because of its utility for identifying eligibility for clinical trials of novel therapeutics5 , 6 )  . 
these individuals must choose whether to learn information about their future disease risks and potential shared familial risks while facing a poor prognosis and the psychosocial challenges of a terminal diagnosis.7 although patients with varying stages of cancer have reported interest in receiving such information from tgp in real8 and hypothetical9 - 11 settings , how patients decide whether to learn sgfs is unclear . 
understanding the decision - making processes of patients with advanced cancers would allow clinicians to anticipate patient informational and decision support needs in this context . the current study describes processes by which patients with advanced cancers decide whether to learn sgfs that arise from tgp . 
we analyzed qualitative data collected through an investigation of attitudes about sgfs among patients who received tgp at our institution.12 these patients were informed about the possible incidental discovery of germline variants during tgp consent conducted by their primary medical oncologists ; however , because our institution did not routinely conduct secondary analyses at the time of this study , none of the patients had made a definitive decision about learning their sgfs . 
 we also assessed preferences with regard to specific challenging decision scenarios , including whether patients should be required to receive sgfs and whether sgfs should be returned to the family after a patients death . study methods are described in detail elsewhere.12 in brief , we recruited 40 adults with advanced breast , bladder , colorectal , or lung cancer who had undergone tgp with an institutional somatic sequencing panel ( msk - impact [ memorial sloan kettering - integrated mutation profiling of actionable targets ] 13 , 14 )  . 
 the memorial sloan kettering cancer center institutional review board approved this study . individual semistructured interviews15 - 18 were conducted with participants in person or by telephone on the basis of participant preference . 
we categorized participant responses into four key themes and relevant subthemes ( indicated by italicized text ) ; illustrative quotes appear in table 2 . theme 1 : degree of decisional autonomy as participants considered how they would decide whether to learn sgfs , a spectrum emerged with regard to participants preferred degree of decisional control and autonomy from close others . 
influential factors for this perspective included a view that the decision was my choice because it involved highly personal information fundamentally related to my body and a desire to avoid burdening others , particularly family , with potentially distressing information . a second group of participants preferred that close others play a consultative role in the decision making process . 
these participants anticipated communicating with close others about the option to learn sgfs and would consider their advice and opinions but would ultimately make the final decision on their own . 
some in this group noted that their familys views were highly valued but would not be determinants in their decision making . finally , a smaller group preferred that close others serve as active partners in decision making . 
others explained that their family members should be actively involved in this decision because sgfs may have direct health - related implications for them . participants who anticipated the involvement of others in their decision making also described their process of selecting close others for communication about the option of learning sgfs . 
decision - making process and preferences regarding secondary germline findings and illustrative participant quotes participant quote theme theme 1 : degree of decisional autonomy patient as an autonomous decision maker well , i think that would be up to me to decide , so i wouldnt be asking my family what they think about doing that . 
 ( f / cc ) close others play a well , id like their input , but ultimately , i make the decisions for what...kind of consultative role in the decision - making process treatment....i would make the final decision . 
 ( f / brc ) close others serve as active partners in decision making process of selecting close others for communication i would expect my wife to be involved....i trust her knowledge and judgment in these matters . 
i dont think my husband could deal with it , so i dont want him burdened with it , and i dont think my step - kids have enough...skin in the game , so to speak , that they should actually be involved in making the decision and i guess i feel similarly about sisters and brother that thats too distant . 
 ( f / cc ) theme 2 : vital role of doctors nature and quality of the doctor - patient relationship primary source of relevant and valuable information theme 3 : information needs clinical benefit meaning tainty testing procedure believe me , its been a rough road , and so like i said , my oncologist and i , we have a good understanding . 
he was the one that put me in the tumor profiling and also on this new research , and anything he decides with me , im okay with it because we have that doctor - patient trust . 
i mean , you know , if they tell me to go get this test , i go get that test....i do it because i think its in the interest of my health.you know , you would have to make a strong argument for that case , but if he was insistent , i would do it . 
 ( m / blc ) i think from a personal standpoint i would ask...how realistic do you think , or how probable do you think , something that came up as high risk is likely to happen , or is there anything i can do to prevent it ? i mean , its more so in the latter that i would care about more if theres anything i can do to prevent it , to minimize the risk . 
is it certain mutations , is it only certain diseases that were talking about , or...is it kind of open ended ? ...i guess i would want to learn more and hear more about the science of what the mutations might mean . 
 ( m / cc ) degree of scientific uncerwhat im trying to have connection with is that if this testing were predictive of something , they would be more interested than if [ with ] this testing , nobody understood or knew how to interpret the results . 
so i guess it would be depending [ on ] how far along the continuum we are in being able to use this information [ that ] would make a difference . 
 ( m / lc ) yes , and if its nothing invasive and they wont...poke me anymore and they wont do anything to me , its fine with me....i would like to know . 
decision - making process and preferences regarding secondary germline findings and illustrative participant quotes ( continued ) theme participant quote who will have access who would have that information , would health care providers...have to have access to that or insurance providers have to have access to that information ? ( f / brc ) yes , what are the possible ramifications , like everything ? like the question that i have now , like what havent i thought of that could be a possible ramification of knowing ? yeah . 
 ( f / brc ) negative implications or harms theme 4 : process of deliberation ing process deliberative decision - makthe only potential benefit i see is if it discovers something that could be dealt with and prevent serious illness or genetic problems in the future . 
 ( m / cc ) take time to decide i think i would want to think about it and talk about it a little more before i made that quick decision , yeah . 
but i dont think its something that we would shy away fro ( m / lc ) well , i think i would research mutations first and find out a little about it before i answered him , but my nature is to go ahead and find out as much information as i can . 
 ( f / blc ) no need to engage in i would say , great , where do i sign ? when i first got diagnosed , i offered to extensive deliberation have my dna sequenced , and the doctor said , why would you bother ? theres only 30 markers , and weve already looked at theso yeah , to me , it was like a no - brainer and required no thought . 
so for me , its really once i understand what exactly ill be getting out of the study or what benefit it can provide , thats enough of what i need to make a decision on . 
i wouldnt want to procrastinate...it would be my decision....id discuss with family members...my husband and my sister , but...it would be my decision ultimately , and id really want to make it quickly . 
the interviewer described secondary germline findings as follows : i mentioned that with tumor genomic profiling , sometimes the lab will also look for mutations in the genes in your normal cells . 
 abbreviations : blc , bladder cancer ; brc , breast cancer ; cc , colorectal cancer ; f , female ; lc , lung cancer ; m , male . theme 2 : vital role of doctors participants perceived their doctors ( ie , oncologists ) as a vital influence on their decision making . 
for example , several participants reported great trust in their doctors on the basis of a foundation of past experiences and certainty that their doctors will act in their best interests . 
several participants anticipated that their doctors would possess expertise with regard to a range of issues relevant to sgfs and thus could help them to acquire all essential information . theme 3 : information needs participants described a typology of information that they would require to make an educated decision about learning sgfs , including an explanation of whether sgfs would provide a clinical benefit to the patient , his or her family , or other cancer patients and whether these benefits would outweigh possible harms ; assistance in interpreting the meaning of sgfs , such as the degree of certainty of the results and meaning of specific mutations ; degree of scientific uncertainty of sgfs and confidence in their applicability to health decisions ; description of the testing procedure in terms of the invasiveness of sample acquisition ; information about who will have access to the findings ( eg , insurers , health care providers ) ; and negative implications or harms of learning sgfs for the patient and family , including unanticipated consequences . 
many participants stated that they would ask questions about these issues to feel adequately informed , yet a minority doubted that they would have any specific questions if presented with this decision primarily because of placing a high innate value on sgfs . theme 4 : process of deliberation two preferences emerged among participants with regard to the necessity to engage in an extensive decision - making process . 
 ( a detailed description of these perceived benefits and harms is provided elsewhere.12 ) participants described procedural aspects of their deliberation and expressed a preference to take time to decide , during which they would consider the option on their own and seek out information about the value of sgfs . 
furthermore , a few participants expressed a preference to conduct independent research to learn more about receiving sgfs and the meaning of potential mutations . a minority of the sample articulated no need to engage in an extensive deliberation to determine their interest in sgfs . 
 finally , some described a sense of urgency about learning sgfs and stated the necessity to gain and act upon this information quickly to benefit their current health directly . preferences with regard to decision scenarios during the interview , participants were presented with challenging scenarios and asked to describe their preferences for how clinicians should handle these situations . 
first , in response to the debate about the disclosure of sgfs , 26 - 29 we asked participants whether findings about diseases that have effective medical interventions or medication adverse effects ( ie , actionable sgfs ) should always be returned to patients . 
most participants ( 28 [ 70% ] of 40 ) stated that patients should be able to choose whether to receive this information , whereas a minority ( 12 [ 30% ] of 40 ) stated that such information should always be disclosed to patients . participants also were asked to decide whether they believed that if actionable sgfs were detected after a patients death , then these findings should be made available to a patients family or significant other . 
a subset of participants ( 16 [ 69.5% ] of 23 ) also expressed a belief that patients should be required to provide consent for this disclosure before their death , such as at the time of agreeing to tgp , whereas fewer ( seven [ 30.5% ] of 23 ) deemed patient consent unnecessary . 
preferences with regard to specific decision scenarios that involve secondary germline findings and illustrative participant quotes participant quote question should actionable secondary germline findings * always be returned to patients ? yes ( 30% ) yes , i agree with that because they may not want to know , but theyre still going to be affected by it . 
because at least theyd have the opportunity to know...whats going on with thethey may not want to know , and it may be painful , but i think that they should be told . 
i think of a very close friend that was diagnosed with cancer , and he was in his 20s , and he survived , but when i learned that i had cancer , i reached out to him , and he said that at his lowest point he begged , i dont want to know any more information . 
and that was part of the healing for him , so i always think about that because that was a poignant point that he made , and i think its so personal . 
 ( f / brc ) should actionable secondary germline findings be made available to a patients family or significant other if a patient has died ? yes ( 90% ) unsure ( 5% ) no ( 5% ) should nonactionable secondary germline findings be made available to a patients family or significant other if a patient has died ? yes . 
well , if it in any way could...impact the timing of treatment or care for someone else in the family , they should....i would want them to know about it....i guess at the end of the day that you should get consent from the patient...as to what youre going to do with anything...you take from the ( m / lc ) you got a coin ; you wanna flip a coin ? because the problem that comes to me is that my family is very tight , and it wouldnt be a problem with my family , but you always have a family that [ is ] ...on the outs , so to speak , and if you tell one , you got to tell all . 
 ( m / lc ) thats a good question because im thinking that if the spouse , for example , were told after the person passed away that we had discovered this , i guess the first reaction would be , how come we didnt discover it earlier while the person was still alive and there may have been time for some kind of treatment ? so it might cause some kind of anger . 
preferences with regard to specific decision scenarios that involve secondary germline findings and illustrative participant quotes ( continued ) question unsure ( 2.5% ) no ( 15% ) participant quote i can project how i might think in the future , but its hard for me to say at this time in a practical way how i would feel about , you know , releasing . 
if they want the information they should go and get it...and i think that if they get it just because it was available for me , like as part of my estate , heres her genetic testing , and again , ill use my brother because he has the kids . 
if...he sees in black and white that theres an indicator that we have a genei have a gene so that becomes a family gene - - so hes now gotten a worry he didnt ask for in his life . 
 abbreviations : blc , bladder cancer ; brc , breast cancer ; cc , colorectal cancer ; f , female ; lc , lung cancer ; m , male . * in the interview , actionable secondary germline findings were described as follows : there are different ways to think about the many kinds of mutations or disease risks that you could theoretically learn about . 
when doctors know that someone has one of these mutations , they can recommend ways to help prevent a disease from developing or help find it earlier when it is more likely to be treatable . 
the doctors may also change the kinds of medications that they prescribe . in the interview , nonactionable secondary germline findings were described as follows : it is also possible that the lab will find mutations for conditions that do not have recommended or effective medical interventions . 
 but when two carriers of the same recessive mutation have a child , then the child could have the disease . unsure about whether actionable sgfs should be available to a patients family after death ( two [ 5% ] of 40 ) or stated that such information should not be made available ( two [ 5% ] of 40 )  . 
 preferences against disclosure were due to concerns about negative emotional implications of such information for families . participants were similarly asked to decide whether they believed that sgfs about diseases without effective medical interventions or that indicate one is a healthy carrier for recessive diseases ( ie , nonactionable sgfs ) should be made available to a patients family after a patients death . 
most participants who provided an opinion regarding consent reported that patients should be required to consent to the disclosure of this information to their families ( 11 [ 92% ] of 12 )  . 
fewer ( six [ 15% ] of 40 ) stated that nonactionable sgfs should not be made available to family after a patients death because of concerns about negative emotional reactions and the limited ability to intervene with such diseases . 
 finally , when comparing the preferences of participants with regard to the return of actionable versus nonactionable sgfs to family after a patients death , 22.5% ( nine of 40 ) were discordant in their preferences across these scenarios . discussion this study clarifies the decision - making processes of patients with advanced cancer with regard to sgfs from tgp . 
however , consistent with other medical decision contexts , 30 - 34 variability existed in participants preferences for involving others , including spouses / partners , children , and siblings , in their decision making . 
consequently , when presenting the option of learning sgfs , clinicians must allow patients to solicit input from close others and help to navigate challenges inherent in decision making with multiple individuals.35 additional research should investigate how such interpersonal influences shape , hinder , or support patients sgfs decision making . participants anticipated that their doctors ( ie , oncologists ) would be the primary source of guidance for this decision . 
participants also anticipated that they would have extensive questions about the benefits , harms , interpretation , and process of obtaining sgfs and would expect their oncologists to provide answers . 
however , research has demonstrated that this may not be feasible because many oncologists have limited experience with germline testing and express concerns about their ability to address challenges presented by sgfs.8 several approaches may help to bridge this gap between patient expectations and oncologist preparedness , including oncologist - targeted communication training , novel patient education materials , and referral to genetic counselors to address patient questions . 
future research should evaluate which of these approaches are most effective at achieving the delicate balance between meeting patients information needs and practical challenges of cancer care delivery ( eg , time demands , workforce limitations )  . 
research should also examine how various models of patient education ( eg , oncologist led , genetics professional led ) influence patient sgfs decisions and how patients weigh the opinions of various care providers in this context . many participants anticipated a preference to undergo a thoughtful deliberation about the prospect of learning sgfs . 
research suggests that the adoption of a more intuitive decision - making approach can yield similar outcomes to deliberative decision making , 36 although both approaches have benefits and drawbacks.37 of note , some participants preferences for a quick decision were motivated by beliefs that sgfs would provide clinical utility or necessitate urgent action for them to reap health benefits . 
accordingly , clinicians must ensure that all patients , including those immediately enthusiastic or accepting of sgfs , accurately understand the limitations of this risk information . these results also provide insight into preferences of patients with advanced cancers with regard to challenging scenarios that involve the return of sgfs . 
consistent with american college of medical genetics and genomics recommendations4 and expert opinion , 38 most participants stated that patients should choose whether they want to receive actionable sgfs from tgp . 
participants generally were more supportive of the return of actionable sgfs to family after a patients death than nonactionable sgfs ; although , in both instances , a majority supported the sharing of this information with family largely because of perceived family health benefits . 
the qualitative design enabled an in - depth analysis of the decision - making preferences of a sample of patients with advanced cancers diverse in diagnosis , sex , and health status . 
however , the majority was well - educated ( 85% reporting at least some college ) ; decision - making preferences and processes of these individuals may differ from those with less formal education . 
additional limitations are that this sample was racially and ethnically homogenous , recruited from one institution , and assessed at a time when the decision about learning sgfs was hypothetical in nature ; thus , the findings may not be generalizable to the broader population of patients with advanced cancers treated in other care settings who are navigating this decision in real time . 
recommendations for developing precision oncology programs with regard to how to manage and support patient decision making about secondary germline findings from tumor genomic profiling develop educational materials about tumor genomic profiling ( tgp ) and secondary germline findings ( sgfs ) that can be easily disseminated to and understood by close others ( eg , siblings , children , spouses / partners ) who may play a role in a patients decision making . ensure that individuals who lead education and consent discussions about the return of sgfs are prepared to help patients with varying preferences for decisional autonomy from their close others . patients attribute high trust and expertise to their oncologists ; therefore , prepare oncologists to serve as a primary resource who can provide balanced advice to patients about sgf decisions . create patient educational materials that provide clear information about the potential benefits and harms of sgfs . 
 distinguish between potential outcomes of sgfs for patients ( with a consideration of their cancer stage and prognosis ) and their families . ensure that patients understand that the decision to undergo tgp is separate from the decision about return of sgfs and that varying potential benefits and harms of each choice exist . structure education and consent discussions about tgp and the return of sgfs to be temporally flexible and , therefore , capable of accommodating patients preferences to take time to deliberate , seek additional input from close others , and conduct independent research . give patients a choice about the return of actionable sgfs . 
either opt - in or opt - out models of germline variant management could allow such patient choice , but each has unique implications for resources to support informed patient decision making and subsequent uptake of sgfs . require patients to make decisions about the management of actionable and nonactionable sgfs in the event of their death at the time of consenting to tgp and the return of sgfs . in conclusion , this study provides important insight into how patients with advanced cancers approach the decision to learn sgfs and informs how developing precision oncology programs can manage the reporting of germline variants from tgp ( table 4 )  . 
patient preferences for involvement in this decision can be accommodated in both opt - in and opt - out models , although these models likely will differ in the resources necessary to support patient deliberation and in the number of patients who select to receive sgfs.42 , 43 although most patients likely will want to retain decisional control in this context , some will desire time and space to include family and other influential figures in their decision making . 
whereas the tgp decision may be time sensitive because of treatment implications , patients may benefit from efforts to ensure that the decision to receive sgfs can be pursued on a different temporal schedule that aligns with their preferences for information seeking and deliberation . 
thus , educational and communication interventions targeted to patients , their families , and oncologists are needed to provide clear information that contextualizes the meaning of sgfs in the advanced cancer setting , assist the weighing of benefits and harms , and allow patients to explore and express their preferences about specific categories of sgfs and management of this information in the event of their death . 
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kaphingst ka , ivanovich j , biesecker bb , et al : preferences for return of incidental findings from genome sequencing among women diagnosed with breast cancer at a young age . 
 o predicting expected absolute chemotherapy treatment benet in women with early - stage breast cancer using endopredict , an integrated 12 - gene clinicomolecular assay hatem soliman , md1 ; darl d . 
flake ii , phd2 ; anthony magliocco , md1 ; mark robson , md3 ; lee schwartzberg , md4 ; priyanka sharma , md5 ; krystal brown , phd2 ; saskia wehnelt2 ; ralf kronenwett , md , phd6 ; alexander gutin , phd2 ; johnathan lancaster , md , phd2 ; jack cuzick , phd7 ; and william gradishar , md8 purpose previous studies have shown endopredict ( epclin ) , a test that integrates 12 - gene expression data with nodal status and tumor size , to be predictive for risk of distant recurrence in women with estrogen receptorpositive , human epidermal growth factor receptor 2negative early - stage breast cancer . 
here , we modeled expected absolute chemotherapy benet on the basis of epclin test results . methods the effect of chemotherapy was modeled using previously validated 10 - year risk of distant recurrence as a function of epclin score for patients treated without chemotherapy . 
the early breast cancer trialists collaborative group performed a meta - analysis of more than 100 clinical trials in more than 100 , 000 women to evaluate the benet of adjuvant chemotherapy.6 although the in individual benet trials varied considerably , 3 this meta - analysis demonstrated an average 30% relative reduction in distant recurrence among women who received chemotherapy compared with those who did not.6 for many patients with er - positive disease , the risk of distant recurrence is sufciently low that et alone is adequate . 
in these patients , the adverse effects associated with chemotherapy may outweigh any benet provided by a reduced risk of disease recurrence . historically , identication of these low - risk patients has relied on clinicopathologic parameters , such as nodal status , tumor size , and tumor grade . 
 soliman et al context key objective can an integrated , 12 - gene , clinicomolecular assay predict absolute benet from chemotherapy for women with estrogen receptor ( er ) positive , human epidermal growth factor receptor 2 ( her2 ) negative breast cancer ? knowledge generated in this model , patients with low 12 - gene scores showed no difference in recurrence - free survival with or without chemotherapy , whereas those with high 12 - gene scores showed a marked increase in recurrence - free survival with the addition of chemotherapy . 
overall , this study suggests that the 12 - gene clinicomolecular score is able to predict absolute benet of chemotherapy for women with er - positive , her2 - negative breast cancer . relevance accurate risk assessment is critical for determining appropriate treatment of women with breast cancer . 
breast cancer prognostic assays like the 12 - gene clinicomolecular score have been shown to provide more information about risk than standard clinical information alone in predicting recurrence - free survival in the absence of treatment . 
these new data support the ability of the 12 - gene clinicomolecular score to also predict absolute benet from chemotherapy to help to guide chemotherapy decisions for women with er - positive , her2 - negative breast cancer . are inadequate to guide adjuvant chemotherapy decisions reliably.7 thus , when risk is dened using clinicopathologic features alone , a subset of truly low - risk patients receives unnecessary chemotherapy , whereas others at high risk forgo chemotherapy and experience avoidable disease recurrence . to address this clinical dilemma , multigene expression prognostic assays have been developed for patients with er - positive , human epidermal growth factor receptor 2 ( her2 ) negative early - stage breast cancer . 
several such assays are clinically available , with variable performance according to nodal status , and early ( 0 to 5 years ) versus late ( 5 or more years ) recurrence.8 - 11 these assays better quantify residual risk of recurrence after surgery and et than clinicopathologic features alone , which enables physicians to tailor the use of adjuvant therapies ( et alone v et + c ) more accurately . 
to this end , evidence - based practice guidelines now indicate that prognostic assays are appropriate tools to help guide decision making.12 - 14 one such test is endopredict ( myriad genetics , salt lake city , ut ) , which integrates a 12 - gene molecular score with nodal status and tumor size into a combined clinicomolecular score ( epclin )  . 
previous studies have validated that epclin accurately predicts the risk of distant metastases in patients with er - positive , her2 - negative breast cancers.11 , 15 - 18 although this information can reliably identify patients at sufciently low risk so that chemotherapy may be avoided safely , the ability of epclin to predict benet from the addition of chemotherapy has yet to be fully dened . 
ideal approaches to evaluate this are limited because archival samples from previous randomized trials of et 6 c in appropriate patients largely have been exhausted.3 , 5 a prospective trial of epclin that includes an arm randomized to receive no chemotherapy would now be unethical , given the established benet of chemotherapy for patients with high - risk disease . 
alternative study designs , therefore , are required to explore the scope of epclins ability to predict adjuvant chemotherapy benet for patients with er - positive , her2 - negative earlystage breast cancer . so far , classic factors that inuence breast cancer prognosis have not shown an interaction between the relative effect of adjuvant chemotherapy on absolute risk of recurrence.19 furthermore , the absolute benet of chemotherapy depends on the patients baseline risk of developing recurrent disease.19 as such , as we evaluate a biomarker , it is helpful to understand the principal drivers of its chemopredictive ability . 
one possibility is that chemopredictive ability is driven by high prognostic accuracy , that is , the ability of the biomarker to differentiate those patients at highest versus lowest absolute risk of recurrence and , thus , those latter patients for whom chemotherapy cannot yield any clinically meaningful benet . 
alternatively , it is possible that a biomarkers chemopredictive ability is driven by differences in relative chemotherapy benet between higher versus lower biomarker - dened patient groups ( ie , interaction between the biomarker score and relative chemotherapy benet )  . in the current study , we describe a modeling study that uses epclin score , absolute risk of recurrence , and reduction of this risk associated with the addition of chemotherapy to estimate the differences in absolute risk for distant recurrence across epclin scores . 
we demonstrate that for a molecular marker such as epclin , patients with the highest baseline absolute risk for recurrence experience the greatest absolute benet from chemotherapy . furthermore , we establish that the key factor in any biomarkers chemopredictive capacity is the ability to predict accurately the baseline absolute risk of recurrence . 
through this modeling , we estimate the differences in absolute risk of distant recurrence across epclin scores and examine the impact of different amounts of statistical interaction between epclin and chemotherapy on these estimates . methods gene expression assay the 12 - gene mrna expression assay has been previously described in detail.20 - 22 in brief , the expression of three proliferation - related target genes ( birc5 , dhcr7 , ube2c ) , ve hormone receptorrelated target genes ( azgp1 , il6st , mgp , rbbp8 , stc2 ) , and three normalization genes ( calm2 , oaz1 , rpl37a ) were measured by reversetranscription quantitative polymerase chain reaction . 
a 12gene molecular score was calculated as the linear combination of the normalized target gene expression.11 , 22 the clinicomolecular score epclin was calculated by combining the 12 - gene molecular score with tumor size and the number of positive lymph nodes and was reported as a numerical score from 1 to 6.11 , 22 tumors with an epclin score of less than 3.3 are considered low risk for distant recurrence and tumors with scores of 3.3 or greater are considered high risk for distant recurrence.11 , 17 , 18 statistical methods the ability of epclin to provide an estimate of expected chemotherapy benet was modeled , as shown in figure 1 . the primary component was the prognostic value of epclin to predict the rate of distant recurrence in the rst 10 years of follow - up . 
 soliman et al 10 - year distant recurrence in patients treated with et + c ( treated ) was estimated by introducing a relative chemotherapy benet for a particular epclin score , hrtreatment , to the untreated risk according to a proportional hazards model ( fig 1b ) , as in equation 1 : rtreated ( cid : 1 ) 1 1 runtreated ( cid : 1 ) ( cid : 3 ) hrtreatment mathematically , the dependence of the relative chemotherapy benet on the risk of distant recurrence was modeled using a main effect for chemotherapy and an interaction between treatment status and the prognostic signature.23 interactions were modeled as a linear dependence of the logarithm of hr for treatment on epclin score according to equation 2 : ( cid : 3 ) ( cid : 3 ) ( cid : 3 ) ( cid : 1 ) hrtreatment ( cid : 1 ) ( cid : 1 ) ln hroverall ( cid : 1 ) epclin epclinavg in this equation , represents the interaction strength and denes how strongly the relative chemotherapy benet for a patient depends on epclin score , epclinavg is the mean epclin score in the population , and hroverall is the hazard ratio for the chemotherapy benet in the population . 
with the maximum value of determined for an interaction giving no chemotherapy benet at epclinmin , weaker interactions were modeled using a percentage of the maximum value of ( fig 1b )  . finally , the absolute benet ab from chemotherapy for treated patients was determined according to equation 3 ( fig 1c ) : december 8 , 2017 , who received valid test results . formalin - xed parafn - embedded breast resections of treatment - nave er - positive , her2 - negative breast tissue were tested . 
these scores also were used to calculate epclinavg . results we examined the impact of different magnitudes of treatment effect and interaction strengths on the estimated 10 - year risk of distant recurrence ( table 1 )  . 
maximal interaction strength ( max = 0.159 ) was rst determined by assuming that a patients relative chemotherapy benet is 0 at the lowest possible epclin score and maintaining the hroverall of treatment at 0.7. 
the corresponding hrtreatment as a function of epclin score is shown in figure 2 . hrtreatment is 1 for a patient with an epclin score of 1 and 0.45 for a patient with the maximum observed epclin score of 6 . 
the risk of distant recurrence in the treated arm also was evaluated in a conservative scenario that assumed no interaction between chemotherapy benet and epclin score ( ie , 0% interaction strength , constant hrtreatment )  . 
in this scenario , the relative chemotherapy benet was equal to the overall chemotherapy benet ( = 0 ; hrtreatment = 0.7 ; fig 2 )  . after max was determined , risk of distant recurrence was calculated for scenarios with a variety of interaction strengths between epclin and chemotherapy . 
figure 3a shows the relationship between epclin score and predicted risk of distant recurrence by 10 years for untreated and treated patients under interaction strength ( 0% , 50% , and 100% )  . 
absolute chemotherapy benet for patients with low and high epclin scores assuming different overall relative chemotherapy benet ( 20% , 30% , or 40% ) for different relative interaction strengths ( 0% , 50% , or 100% ) absolute chemotherapy benet ( % ) 20% relative chemotherapy benet low epclin high epclin 0% relative interaction ( no interaction ) 50% relative interaction ab ( cid : 1 ) runtreated rtreated 100% relative interaction ( maximal ) 30% relative chemotherapy benet low epclin high epclin absolute chemotherapy benet for the low - risk category was calculated as a mean absolute chemotherapy benet for all low - risk patients , and the same went for the high - risk category . 
 predicting chemotherapy benet in early - stage breast cancer ln ( hrtreatment ) = ln ( hroverall ) ( epclin epclinavg ) 0% interaction strength ( = 0 ) 100% interaction strength ( = 0.159 ) epclin score fig 2 . 
patient benet from chemotherapy ( hrtreatment ) according to epclin score at no interaction ( 0% ) and maximal interaction ( 100% )  . recurrence is low for low epclin scores , which results in small absolute chemotherapy benet . 
for example , the expected absolute benet of chemotherapy treatment for a patient with low epclin scores ranged from 0.3% ( epclin = 1 ) to 2.9% ( epclin = 3.3 ) for 0% interaction strength and from 0% to 3.0% for 100% interaction strength . 
similar results were obtained when overall relative chemotherapy benet was modeled to be lower ( 20% ; fig 3b ) or higher ( 40% ; table 1 ; fig 3c )  . the various risk estimates across all scenarios of interaction strength were applied to a clinical cohort of patients with er - positive , her2 - negative breast resections and representative epclin scores ( n = 2 , 185 )  . 
the full epclin distribution is shown in figure 4 . overall , 1 , 275 samples ( 58% ) were low risk , and 910 ( 42% ) were high risk . 
risk of distant recurrence as a function of epclin score for different interaction strengths under different assumptions about overall relative chemotherapy benet : ( a ) 30% , ( b ) 20% , and ( c ) 40% . 
epclin scores in pretreatment estrogen receptorpositive , human epidermal growth factor receptor 2negative breast resection tissue ( n = 2 , 185 )  . the absolute chemotherapy benet in lowand high - risk patients was calculated as a function of interaction strength ( fig 5 )  . 
indeed , even with the strongest interaction , the absolute chemotherapy benet for a 30% relative benet in high - risk patients is 7.3% , which is not dramatically different from the 5.3% benet modeled in the patients with high ( 3.3 ) epclin score patients with low ( < 3.3 ) epclin score 40% overall benefit 30% overall benefit 20% overall benefit case of no interaction ( table 1 )  . 
similarly , in low - risk patients , the absolute benet is 1.5% when we assume the strongest interaction , which is close to a 1.8% benet in the case of no interaction . discussion previous studies have demonstrated that epclin accurately identies patients at low risk for distant recurrence 10 years after surgery , with improved prognostic performance compared with clinical features alone or other molecular tests.11 , 15 - 18 for these low - risk patients , it is clear that the addition of adjuvant chemotherapy likely will not yield any clinically meaningful risk reduction . 
here , we evaluated the capacity of epclin to identify patients with er - positive , her2 - negative earlystage breast cancer who may benet most from adding adjuvant chemotherapy to et . 
thus , we evaluated the impact by modeling across a range of potential chemotherapy effects sizes and interaction strengths with epclin . to minimize potential error associated with individual trial values , the absolute benet of chemotherapy in women with various epclin scores was modeled using wellestablished estimates of the average relative benet of adjuvant chemotherapy . 
this risk is particularly noteworthy in interventions in early - stage breast studies of adjuvant cancer because event rates may be low and distant recurrence may manifest many years or even decades after the initial diagnosis and treatment . 
as a result , the absolute predicted chemotherapy benet in low - risk patients was small and largely unaffected by average chemotherapy effect or interaction strength , whereas the toxicity of therapy remains the same . 
this association is irrespective of interaction strength between epclin and predicted chemotherapy benet , and the impact of any interaction on absolute benet is much smaller than the ability to accurately estimate absolute risk . as is typical of modeling studies , this study is not without limitations . 
of note , the approximately 30% reduction in distant recurrence was selected on the basis of its association with chemotherapy regimens that are more consistent with modern patterns of care in contrast to more historic regimens . other aspects of the statistical model are also limitations of the study . 
it is assumed that the relative benet of chemotherapy linearly depends on epclin score ( eq 2 ) , which is the simplest mathematical implementation of dependence . linear dependence also was used to demonstrate interaction between chemotherapy and a prognostic score.8 in statistical analysis , the most common implementation of interaction between variables has the same functional forin addition , only positive interaction between chemotherapy benet and epclin score was included in this analysis because it is expected that high expression of the proliferation - related genes in epclin is associated with higher rather than lower relative chemotherapy benet . 
conversely , high expression of the hormone receptorrelated genes is expected to be associated with smaller relative chemotherapy benet because er - negative tumors have a much stronger response to chemotherapy than er - positive tumors . 
this again corresponds to a positive interaction because the hormone receptorrelated genes contribute negatively to the epclin score . in summary , this modeling analysis demonstrates that epclin is able to predict which patients will gain maximum absolute benet from chemotherapy . 
in addition , these data indicate that the predicted absolute impact of chemotherapy on distant recurrence depends much more on the prognostic ability of the biomarker to predict an individual womans risk of recurrence accurately . 
endopredict has been shown in multiple studies to predict risk of distant recurrence accurately in women with er - positive , her2 - negative early - stage breast cancer who receive 5 years of et , with an accuracy similar to the other best breast prognostic tests and substantially better than the 21 - gene recurrence score ( oncotype dx ; genomic health , redwood city , ca ) .18 , 24 this modeling indicates identied a substantial proportion of that endopredict women whose risk of breast cancer recurrence was so low that the addition of adjuvant chemotherapy would not produce a clinically meaningful benet on recurrence risk . conversely , patients with high epclin scores were predicted to experience the greatest benet from chemotherapy regardless of the simulated interaction strength between epclin and predicted chemotherapy benet . 
flake , anthony magliocco , mark robson , lee schwartzberg , priyanka sharma , ralf kronenwett , alexander gutin , johnathan lancaster , jack cuzick , william gradishar collection and assembly of data : darl d . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . hatem soliman consulting or advisory role : celgene , astrazeneca , eli lilly , novartis , pzer , puma biotechnology research funding : amgen ( inst ) travel , accommodations , expenses : astrazeneca , eli lilly darl d . 
flake employment : myriad genetics stock and other ownership interests : myriad genetics patents , royalties , other intellectual property : multiple patents related to the development of tests at myriad genetics anthony magliocco stock and other ownership interests : protean biodiagnostics , the genomic cancer institute , proscia honoraria : bristol - myers squibb , merck , illumina , leica consulting or advisory role : bristol - myers squibb , merck , proscia , roche , illumina speakers bureau : bristol - myers squibb research funding : biotheranostics , roche , genentech patents , royalties , other intellectual property : various patents pending as co - inventor through moftt cancer center travel , accommodations , expenses : menarini silicon biosystems , illumina , bristol - myers squibb , merck , ventana medical systems mark robson honoraria : astrazeneca consulting or advisory role : mckesson , astrazeneca , merck , pzer , daiichi sankyo research funding : astrazeneca ( inst ) , myriad genetics ( inst ) , invitae ( inst ) , abbvie ( inst ) , tesaro ( inst ) , medivation ( inst ) travel , accommodations , expenses : astrazeneca , pzer lee schwartzberg stock and other ownership interests : vector oncology honoraria : pzer , amgen , nanostring technologies , novartis , napo , taiho pharmaceutical , genentech , e.r. 
squibb and sons , helsinn therapeutics , genomic health , myriad genetics , astrazeneca consulting or advisory role : bristol - myers squibb , pzer , helsinn therapeutics , caris life sciences , spectrum pharmaceuticals , tesaro , genomic health , astrazeneca , e.r. 
squibb and sons , amgen , f . hoffmann - la roche , genentech , eli lilly , novartis , merck , biocept , abbvie , biotheranostics , athenex , coherus biosciences speakers bureau : coherus biosciences , puma biotechnology research funding : amgen ( inst ) , glaxosmithkline ( inst ) , spectrum pharmaceuticals ( inst ) , medivation ( inst ) , bayer ag ( inst ) , genentech ( inst ) , pzer ( inst ) , tesaro ( inst ) , sano ( inst ) , e.r. 
n engl j med 319 : 1681 - 1692 , 1988 early breast cancer trialists collaborative group : tamoxifen for early breast cancer : an overview of the randomised trials . 
lancet 351 : 1451 - 1467 , 1998 early breast cancer trialists collaborative group ( ebctcg ) : effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15 - year survival : an overview of the randomised trials . 
lancet 365 : 1687 - 1717 , 2005 peto r , davies c , godwin j , et al : comparisons between different polychemotherapy regimens for early breast cancer : meta - analyses of long - term outcome among 100 , 000 women in 123 randomised trials . 
curr treat options oncol 16 : 23 , 2015 paik s , tang g , shak s , et al : gene expression and benet of chemotherapy in women with node - negative , estrogen receptor - positive breast cancer . 
j clin oncol 24 : 3726 - 3734 , 2006 van de vijver mj , he yd , vant veer lj , et al : a gene - expression signature as a predictor of survival in breast cancer . 
gnant m , filipits m , greil r , et al : predicting distant recurrence in receptor - positive breast cancer patients with limited clinicopathological risk : using the pam50 risk of recurrence score in 1478 postmenopausal patients of the abcsg - 8 trial treated with adjuvant endocrine therapy alone . 
filipits m , rudas m , jakesz r , et al : a new molecular predictor of distant recurrence in er - positive , her2 - negative breast cancer adds independent information to conventional clinical risk factors . 
coates as , winer ep , goldhirsch a , et al : tailoring therapies - - improving the management of early breast cancer : st gallen international expert consensus on the primary therapy of early breast cancer 2015 . 
harris ln , ismaila n , mcshane lm , et al : use of biomarkers to guide decisions on adjuvant systemic therapy for women with early - stage invasive breast cancer : american society of clinical oncology clinical practice guideline . 
dubsky p , filipits m , jakesz r , et al : endopredict improves the prognostic classication derived from common clinical guidelines in er - positive , her2 - negative 17 . 
martin m , brase jc , calvo l , et al : clinical validation of the endopredict test in node - positive , chemotherapy - treated er + / her2breast cancer patients : results early breast cancer . 
buus r , sestak i , kronenwett r , et al : comparison of endopredict and epclin with oncotype dx recurrence score for prediction of risk of distant recurrence after endocrine therapy . 
warf mb , rajamani s , krappmann k , et al : analytical validation of a 12 - gene molecular test for the prediction of distant recurrence in breast cancer . 
sestak i , buus r , cuzick j , et al : comparison of the performance of 6 prognostic signatures for estrogen receptor - positive breast cancer : a secondary analysis of a randomized clinical trial . 
 applying precision oncology principles in radiation oncology radiation therapy is a critical component in the curative management of many solid tumor types , and advances in radiation delivery techniques during the past decade have led to improved disease control and quality of life for patients . 
during the same period , remarkable advances have also been made in understanding the genomic landscape of tumors ; however , treatment decisions in radiation oncology continue to depend primarily on clinical and histopathologic characteristics rather than on the genetic features of the tumor or the patient . 
with the development of novel genomic techniques and their increasing use in clinical practice , radiation oncology is uniquely positioned to leverage these advances to identify novel biomarkers that could inform radiation dose , field , and the use of concurrent systemic agents . 
2018 by american society of clinical oncology introduction radiation plays a central role in cancer management , and it is estimated that more than half of all patients with cancer will receive radiation therapy during their treatment course.1 radiation is used in a variety of clinical contexts , including in the definitive management of several solid tumor types as well as in palliation of symptoms associated with advanced disease.2 many of the changes in radiation oncology in recent decades have been driven by advances in imaging and dosimetry that have resulted in the ability to deliver higher radiation doses to tumor while minimizing the dose to surrounding normal tissue.3 in contrast , advances in understanding tumor biology and genetics have affected radiation oncology practice less to date , particularly when compared with other oncology specialties.4 , 5 currently , genomic biomarkers are rarely used to inform the use of radiation therapy . 
instead , clinical - pathologic factors , such as tumor size , histology , lymph node involvement , and surgical margin status , continue to drive radiation oncology standards of practice . 
thus , although radiation is a precision treatment modality in a spatial and anatomic sense , the potential to incorporate tumor genomic features as a precision tool in radiation oncology has not yet been realized . 
efforts by radiobiologists to understand and model these differences have driven current clinical strategies , such as dose fractionation ( ie , delivering a fractional dose of radiation each day over several weeks ) , that exploit differences in the radiation sensitivity of tumor and normal cells . 
the development of massively parallel sequencing and other high - throughput techniques has led to an explosion in available tumor genomic data , which provide a unique opportunity to map the landscape of radiation response across tumor types . 
nevertheless , defining the underlying genomic determinants of differential radiation response remains challenging for several reasons . historically , the tumoricidal effects of radiation were believed to be mediated primarily through dna damage , but accumulating evidence suggests that radiation has numerous effects on the sophia c . 
 tumor and microenvironment that vary on the basis of anatomic site , tumor histology , radiation dose and fractionation , and the use of concurrent therapies.6 , 7 therefore , the molecular underpinnings of radiation response may vary within and among tumor types and may be strongly dependent on clinical and treatment factors . when delivered in the neoadjuvant or definitive settings , radiation is often combined with cytotoxic chemotherapy , and separating the effects of each agent on tumor response is difficult . 
conversely , when radiation is used in the adjuvant setting , no measurable tumor is present , and response is defined by lack of tumor recurrence over months or years , which can be affected by factors beyond tumor cell radiosensitivity . finally , although comprehensive genomic profiling of thousands of tumors has been performed through efforts such as the cancer genome atlas , these studies often pool cases that represent diverse clinical settings and disease states , and detailed treatment and response data are often not available . 
furthermore , even when an association between a specific genomic event and treatment response is observed , rigorous experimental work is required to validate the association and establish causality . experimental systems to study radiation sensitivity many of the tenets of radiobiology were developed and validated using radiosensitivity assays , including in vitro approaches such as clonogenic cell survival and in vivo approaches using transplantable tumor systems.8 although these assays have been invaluable in establishing the mechanisms of radiation - mediated cell killing and the properties of dose fractionation , the assays are often time consuming , technically challenging , and difficult to scale . 
therefore , one of the most important challenges currently facing the field is the development of efficient and reliable techniques that faithfully recapitulate the effects of radiation to yield insights at both the cellular and tissue levels . in an attempt to comprehensively characterize associations between genomic features and radiation sensitivity , yard et al9 profiled radiation sensitivity across 533 genomically annotated cell lines representing 26 tumor types using a validated high - throughput assay . 
perhaps one of the most surprising findings from this study was the large variation in radiation sensitivity observed within and among lineages : many tumor types exhibited a greater than five - fold difference in survival between the mostand least - sensitive cell lines . 
conversely , mutations in several dna repair genes , such as brca1 , mlh1 , and atr , were associated with high point mutation frequency , low levels of aneuploidy , and radiation sensitivity . 
 whole - transcriptome gene expression profiling revealed that upregulation of pathways involved in dna damage response , cell cycle , and chromatin organization were associated with radiation sensitivity , whereas increased expression of genes in pathways involved in cellular signaling ( janus kinase - signal transducers and activators of transcription [ jak / stat ] and phosphatidylinositol 3 - kinase [ pi3k ] pathways ) , stem - cell state , and inflammation were associated with radiation resistance . 
this study is one of the largest efforts to date to investigate the association of tumor genomic features with radiation sensitivity and highlights the potential to leverage large data sets to study cellular radiation response . many other groups have studied gene expression patterns in an attempt to define transcriptional signatures of radiation response . 
several studies have identified an association between increased clonogenic survival of primary tumor cells irradiated ex vivo and worse clinical outcomes after radiation - based treatment.17 , 18 in addition , immunohistochemical ( ihc ) readouts of dna repair functionsuch as formation of functional dna repair foci after ex vivo tumor irradiationhave been described as a potential biomarker for predicting radiosensitivity.19 genetic changes that are unique to the experimental system.21 given the expanding role of numerous genomic and experimental platforms for tumor profiling , an important challenge will be developing tools for integrating multi - omic tumor data to derive clinically relevant insights . 
it is likely that the most powerful clinical insights will be achieved by combining analyses of large genomic data sets with functional data derived from experimental systems ( fig 1 )  . candidate clinical radiation biomarkers finally , recent efforts have focused on developing platforms for establishing patient - derived organoid and xenograft models that can serve as a living biobank to test drugs or other therapy combinations.20 the goal of these approaches is to identify personalized treatment approaches in a time frame that can be useful for individual patients . 
organoid and xenograft systems likely reflect more elements of relevant tumor biology than two - dimensional cell culture systems ; however , these systems are often more expensive and time consuming to develop and may select for dna repair pathway alterations have been studied extensively as potential biomarkers of radiation sensitivity , and several clinically relevant associations between dna repair deficiency and tumor biology have been described ( fig 2 )  . 
5 - fu , 5 - fluorouracil ; ai , aromatase inhibitor ; ar , androgen receptor ; bet , bromodomain and extra - terminal domain ; dna - pkcs , dna - dependent protein kinase catalytic subunit ; dnmt , dna methytransferase ; egfr , epidermal growth factor receptor ; er / pr , estrogen receptor / progesterone receptor ; fgfr , fibroblast growth factor receptor ; hdac , histone deacetylase ; her2 , human epidermal growth factor receptor 2 ; hif1 , hypoxia - inducible factor 1 ; lhrh , luteinizing hormonereleasing hormone ; msi , microsatellite instability ; parp , poly ( adp ) ribose polymerase ; pd - 1 , programmed cell death1 ; pd - l1 , programmed cell deathligand 1 ; serd , selective estrogen receptor degrader ; serm , selective estrogen receptor modulator ; tmb , tumor mutational burden ; vegf , vascular endothelial growth factor . properties and predicts response to conventional chemotherapy and immune checkpoint blockade25 , 26 ; however , msi is not currently a biomarker that directly affects radiation decision making . 
somatic mutations in the nucleotide excision repair gene ercc2 are associated with improved response to cisplatin - based chemotherapy and chemoradiotherapy in muscle invasive bladder cancer.27 , 28 deleterious germline and somatic mutations in homologous recombination ( hr ) genes such as brca1 , brca2 , palb2 , and atm occur across tumor sites . 
although the rates are highest in breast and ovarian cancers , a subset of other tumors also have hr gene alterations or have mutational patterns suggestive of functional hr loss.29 because hr is a primary repair mechanism for radiation - induced dna double strand breaks ( dsbs ) , it has been hypothesized that hr - deficient tumors may have heightened radiation sensitivity . 
however , defining the clinical role of hr gene alterations as a radiation biomarker has been challenging for several reasons.30 radiation is not part of the standard treatment of ovarian cancer and is typically used in the adjuvant setting in breast cancer , making it more challenging to assess treatment response than in settings where radiation is used to treat gross disease . 
 carriers treated with lumpectomy and adjuvant radiation for localized breast cancer have reported higher rates of ipsilateral breast event rates ( tumor recurrence and / or second primary tumors ) among brca1 / 2 carriers , whereas others have found no difference.32 - 34 the rate of contralateral breast cancer is significantly higher in brca1 / 2 carriers than in noncarriers , 35 and the role of germline brca1 / 2 status in driving clinical management ( including prophylactic mastectomy and oophorectomy ) is an active area of investigation with important medical and socioeconomic implications . homologous recombination deficiency was also recently identified as a relatively common feature of metastatic prostate tumors , 36 and germline hr alterations are surprisingly frequent among men with metastatic disease.37 hr - deficient prostate cancers have aggressive clinical behavior and respond to poly ( adp ribose ) polymerase ( parp ) inhibitors38 , 39 ; however , it is not known if hr alterations predict improved response to definitive radiation for men with localized prostate cancer . 
similarly , the prognostic and predictive implications of hr pathway alterations in other tumor types in which radiation is used in the first - line settingsuch as gastric and pancreatic cancershave not yet been defined . 
 in pancreatic cancer , patients with a family history of breast , ovarian , or pancreatic cancer had significantly improved survival after platinum - based chemotherapy compared with patients with sporadic disease , suggesting that germline brca1 / 2 - mediated hr deficiency may contribute to treatment response.40 - 42 somatic mutations in the dsb checkpoint kinase ataxia telangiesctasia mutated ( atm ) have been associated with durable responses to radiation for recurrent or metastatic lesions , 43and increased ihc staining of the dsb nuclease mre11 is associated with improved outcomes after radiotherapy in muscle - invasive bladder cancer.44 , 45 in addition to dna repair pathway alterations , numerous other tumor genomic features have been correlated with clinical radiation response ( fig 2 )  . 
activating kras mutations are associated with resistance to radiation as well as to numerous conventional and targeted systemic agents.46 , 47 oncogene activation can result in increased oxidative stress in tumor cells , and mutations in keap1 , or the keap1 - binding region of nfe2l2 ( which encodes nuclear factor erythroid 2 - related factor 2 [ nrf2 ] ) , can result in stabilization of nrf2 and transcriptional upregulation of oxidative stress response genes . 
keap1 mutations are associated with radiation resistance in cell lines and have been shown to drive tumor formation and radiation resistance in a mouse model of lung squamous cell cancer.48 , 49 keap1 mutations are enriched in patients with lung cancer who develop local recurrence after radiation , consistent with a role in mediating radiation resistance.48 the advent of next - generation sequencing techniques has allowed genomic analyses to be performed from small amounts of formalin - fixed paraffin - embedded tumor tissue.50 in addition to characterizing features of primary ( pretreatment ) tumors , these techniques can also be applied to post - treatment residual or recurrent tumors to provide a window into mechanisms of radiation - induced tumor evolution . 
 sequencing of preand post - chemoradiotherapy rectal tumors suggest enrichment of tp53 or co - occurring kras / tp53 mutations in poorly responding tumors , 51 , 52 and enrichment of kras / tp53 mutations were also noted in liver metastases that recurred after proton - based stereotactic radiotherapy.53 analysis of preand post - treatment anal cancer showed enrichment or emergence of mutations in known cancer genes , such as tp53 , pik3ca , and fbxw7.54 interestingly , in both the rectal and anal tumor cohorts , numerous subclonal mutations were gained or lost through treatment , but there was no change in overall mutational burden . 
 finally , there is immense interest in the use of minimally invasive techniques , such as circulating tumor dna ( ctdna ) and circulating tumor cell ( ctc ) analyses , to monitor disease status and treatment response . 
it is already clear that many of the common genomic features of tumors have important prognostic implicationsthat is , the genomic event is associated with better ( or worse ) clinical outcomes independent of the type of treatment . 
however , to advance precision radiation oncology , it will also be critical to identify genomic features that , although perhaps not strongly prognostic , are predictive of favorable response to radiation . 
however , despite the lack of mutation - based biomarkers , several gene expression signatures have been validated as prognostic and predictive tools in a variety of radiation settings . one of first gene expression signatures to be widely used in clinical practice was the oncotypedx test , which measures expression of a panel of genes involved in estrogen receptor signaling and cell proliferation.61 although the assay was initially validated to predict benefit of adjuvant chemotherapy in estrogen receptor positive breast cancer , it has now been studied in other contexts and can be used to estimate risk of locoregional recurrence after radiation for invasive cancer62 or ductal carcinoma in situ.63 similar approaches have also been used in the post - mastectomy setting.64 clinical trials are ongoing to investigate if transcriptional signatures can be used to prospectively identify women at low risk of locoregional recurrence for whom post - lumpectomy radiation can be omitted ( clinicaltrials.gov identifier : nct02653755 ) .65 gene expressionbased assays are also being used to inform management of prostate cancer . 
lin et al77 showed that higher pretreatment plasma ebv titers ( > 1 , 500 copies / ml ) were associated with worse outcomes after chemotherapy and radiation in patients with stage iii and iv npc . 
historically , patients with medulloblastoma were defined as standard risk or high risk on the basis of clinical features , and chemotherapy and radiation decisions were driven by clinical risk group . 
however , elegant work by several groups has identified four consensus molecular subtypes of medulloblastoma with distinct biology and clinical behavior.78 patients with tumors harboring mutations in the wingless - type mmtv1 integration site ( wnt ) pathway ( such as ctnnb1 ) have extremely good prognosis , patients with group 3 tumors have extremely poor prognosis , and patients with group 4 tumors or tumors with alterations in the sonic hedgehog ( shh ) pathway ( such as ptch1 , gli3 , mycn ) have intermediate prognoses . 
for example , the childrens oncology group is examining the role of reduced - dose craniospinal irradiation ( csi ) and chemotherapy for wntdriven medulloblastoma ( clinicaltrials.gov identifier : nct02724579 )  . 
similarly , st judes hospital is evaluating different radiation and drug combinations across molecular subtypes ( clinicaltrials.gov identifier : nct01878617 ) : patients with low - risk wnt pathway mutations will receive dose - reduced csi , and patients with shh alterations will receive an shh inhibitor ( vismodegib ) after four cycles of standard chemotherapy . 
if successful , these studies have the potential to maximize cure rates while minimizing the known acute and late toxicities of chemotherapy and csi in pediatric patients . in current practice , the prescribed radiation dose is defined by tumor histology , with doses reduced when necessary to avoid exceeding normal ( nontumor ) tissue tolerance . 
instead , normal ( nontumor ) radiation dose limits are typically selected to limit the rate and severity of toxicity to an acceptably low level ( often 5% )  . 
therefore , prospectively identifying the patients at highest risk for normal tissue toxicity could inform mitigation strategies such as dose de - escalation or aggressive supportive care in the most sensitive patients and , conversely , could allow for dose escalation in the majority of the population who are at lower risk for normal tissue toxicity.86 many preclinical and retrospective clinical studies have attempted to identify germline factors that affect radiation sensitivity.87 , 88 candidate gene approaches focusing on genes involved in dna damage signaling and repair have identified associations between specific germline alleles in dna repair genes and increased normal tissue sensitivity to radiation , but many of these associations have failed to validate in larger cohorts.89 genome - wide association studies have also identified associations between specific single nucleotide polymorphisms and radiation sensitivity . 
although some of these alleles occur in genes known to play a role in cancer biology or tissue injury , many exist in noncoding regions of the genome and / or have no obvious role in damage - related cell signaling . 
therefore , additional work will be needed to validate these associations and uncover the relevant biology . in addition to defining predictive biomarkers of radiation toxicity , efforts have also focused on developing functional assays to profile and predict normal tissue toxicity . 
for example , increased radiation - induced apoptosis of circulating cd8 + t lymphocytes from patients undergoing breast radiotherapy was associated with increased risk of late breast fibrosis.90 ideally , genetic and functional data reflecting the sensitivity of both the tumor and normal tissue to radiation would be used to personalize radiation dose for individual patients . one of the most feared and life - threatening late toxicities of radiation is radiation - induced second malignancy ( smn )  . 
smns typically develop in normal tissues adjacent to the treated tumor and have unique genomic features and aggressive clinical behavior.91 - 93 the risk of developing an smn depends on multiple factors , including age at radiation , anatomic site , and radiation dose . 
 germline mutations in known cancer genes such as tp53 and rb1 can increase the risk of smn after radiation.94 however , accurately predicting smn risk is challenging in part because radiation - induced tumors often take years to develop ; therefore , patients who present with an smn were often treated using techniques that no longer reflect the standard of care . 
radiosurgery ( rs ) - based treatments are now commonly used in many disease settings and are capable of precisely delivering high doses of radiation in just a single fraction or over a small number of treatments.95 rs - based approaches minimize toxicity by using advanced delivery techniques to reduce normal tissue radiation exposure and can also increase patient convenience by decreasing the number of treatment visits . currently , the decision to pursue rs - based treatment is dependent on clinical factors such as tumor size and location . 
for example , the classic radiobiology tenets of sublethal damage repair and cell cycle redistribution between fractionated radiation treatments are likely less relevant in rs - based therapy.96 rs doses may increase the extent of dna damage or create dna lesions that are more difficult to repair than those created by conventional doses . 
 however , the contribution of each of these factors has not been firmly established , and much of the observed efficacy of rs treatments may be due simply to the ability to deliver a higher biologically relevant dose.99 , 100 tumor genomic features that affect the dose dependent radiation response have the potential to serve as biomarkers to inform selection of the dose and fractionation scheme . 
specifically , rs - based dose escalation may be particularly beneficial in specific populations of cells , such as hypoxic cells or cancer stem cells , that are traditionally considered to be radioresistant . 
in addition , the potential to induce systemic immune responses using rs is of substantial interest in the setting of combined immune checkpoint blockade . radiotherapy with charged particles ( such as protons or carbon ions ) is becoming increasingly common in some developed countries . 
charged particles have distinct physical properties that make them attractive for treatment of specific tumor types.101 in addition to their unique physical properties that can be exploited to minimize dose to normal tissues , charged particles may induce tumor cell death via distinct mechanisms . 
for example , charged particles may be less dependent on oxidative intermediates to create lethal dna damage and may therefore be more effective for hypoxic tumors.102 in addition , emerging evidence suggests that charged particles may elicit a host immune response that is distinct from the immune response elicited by conventional ( photon - based ) radiation , 103 and studies reporting associations between specific genomic features and response to particle therapy are now emerging.53 radiomics and molecular imaging imaging - based analyses are a particularly promising tool for precision radiotherapy because they are noninvasive and can be used longitudinally to assess tumor response during and after treatment . 
radiomics is an emerging field of study that aims to characterize and quantify imaging properties of tumors and correlate these features with available genomic or clinical data to define new image - based biomarkers.104 quantifying novel anatomic and functional features of tumors can provide relevant clinical insights , and examples of the potential utility of radiomics based analyses are now being reported . 
in one large study , computed tomographybased imaging features such as tumor shape , contrast , and texture had independent prognostic power in several cohorts of lung and head and neck cancers.105 radiomics features also correlate with genomic properties ; for example , arterial phase imaging traits were associated with a doxorubicin resistance transcriptional program in hepatocellular carcinoma.106 functional imaging studies also have the potential to be rich sources of radiomics data . 
positron emission tomography ( pet ) tracer uptake ( as measured by standard uptake value ) has been studied as a potential prognostic and predictive biomarker in several tumor types , 107 and more complex metrics that incorporate both anatomic and functional outputs are now being explored . advanced imaging techniques allow improved visualization of both tumor and normal tissue , and many of these imaging modalities are now routinely integrated into radiation oncology practice . 
daily imaging with either two - dimensional ( x - rays ) or three - dimensional ( computed tomography ) techniques is used to ensure set - up reproducibility and minimize the effect of interfraction changes in anatomy ( such as bowel or bladder filling )  . 
intrafraction motion can also be managed using techniques that account for positional uncertainty in the planning phase or directly track the position of an implantable beacon and adjust the radiation field in real time . in addition to techniques for ensuring reproducible patient position and accounting for slight variations in anatomy , advanced imaging modalities ( such as magnetic resonance imaging or pet ) can also be used to measure tumor response during the treatment course . 
 adaptive planningupdating the radiation plan during the treatment course on the basis of tumor responsehas the potential to maximize dose delivery to areas of persistent tumor while sparing normal tissue in areas where tumor has regressed.108 - 110 for example , an attractive property of particle therapy is the potential to use pet techniques to monitor in vivo dosimetry . 
 pet imaging can be used to detect positron emissions created from inelastic nuclear collisions of protons with elements in tissues , thereby creating a radiation dose distribution from proton111 , 112 or carbon ion113 radiation . 
 for adaptive radiation planning on the basis of delivered dose to the target and adjacent normal tissue.114 prospective studies are being conducted in multiple disease settings to study the impact of adaptive on - treatment planning on outcome and toxicity . 
early results from single - arm studies suggest that dose escalation using adaptive radiation techniques can be performed safely and are associated with promising local control rates115 - 117 ; however , results from randomized trials ( such as clinicaltrials.gov identifier : nct01507428 ) will be required to confirm that shrinking the radiation fields does not compromise disease control by sparing regions that appear normal on imaging but harbor residual microscopic disease . identifying and targeting tumor hypoxia many tumors contain hypoxic regions , and hypoxic conditions are known to induce tumor cell radioresistance.118 a direct measure of tumor hypoxia can be performed using an oxygenation probe , but this method is invasive and cannot readily distinguish between viable hypoxic regions of the tumor and dead / necrotic tumor . 
 noninvasive functional imagingbased methods are also available , such as 18f - fluoromisonidazole pet imaging , which uses a hypoxia - sensitive chemical probe . measures of hypoxia can also be estimated from harvested tumor specimens . 
for example , increased ihc levels of hypoxia - inducible factor 1 ( hif1 ) have been associated with worse outcomes after radiotherapy in several clinical contexts , 119 and in a randomized trial for muscle invasive bladder cancer , only tumors with high hif1 staining benefitted from the addition of the antihypoxia agents carbogen and nicotinamide to radiation.120 gene expression signatures have also been used to identify tumor hypoxia . 
the rationale for combining radiation with chemotherapy was outlined by steel124 and others in the 1970s , and despite preceding the advent of modern molecular cancer biology , this contextual framework remains relevant to understanding the interplay of radiation with chemotherapy and targeted agents . systemic agents have the potential to interact with radiation to produce additive , synergistic , or antagonistic effects , and the net biologic response is dependent on both tumor and host genomic features ( fig 2 )  . 
despite the widespread use of concurrent chemoradiotherapy regimens , the molecular features that drive response and / or toxicity with these combinations remain poorly understood , and treatment decisions continue to be driven primarily by clinical factors , such as patient age , performance status , and tumor histology , rather than by tumor genomic features . 
 however , given the proven efficacy of combined chemoradiotherapy approaches , future efforts should focus not only on discovering novel targets and therapies but also on defining reliable genomic biomarkers that predict response to existing cytotoxic regimens . molecularly targeted therapies have transformed the management of diseases such as chronic myelogenous leukemia and nonsmall - cell lung cancer . 
as with conventional chemotherapy , there are several potential therapeutic advantages to combining targeted agents with radiation ( table 1 ) .125 , 126 in the primary tumor , targeted agents may radiosensitize tumor cells , resulting in improved local control and / or allowing for radiation dose de - escalation . 
the sequence of therapies is an important factor that may affect the interactions between radiation and targeted agents at the cellular and systemic levels , and the best outcomes will be achieved when the correct combination is provided in the correct genomic context with the correct timing ( fig 2 )  . given that dna damage is a major mechanism of radiation - induced cell killing , there has been significant interest in combining targeted inhibitors of dna repair with radiation.127 , 128 as large genomic studies continue to define the landscape of dna repair pathway deficiencies across tumor types , it will be critical to match dna repair inhibitors with the tumor genomic contexts in which maximum benefit can be achieved when combined with radiation . 
inhibitors of parp are the most clinically advanced dna repair directed agents and are now approved for the treatment of hr - deficient tumors , such as those with brca1 / 2 mutations.129 preclinical evidence suggests that parp inhibition may synergize with radiation to kill tumor cells , and clinical trials combining parp inhibitors with radiation are now open . 
it is unknown whether adding parp inhibition to radiation will primarily benefit hr - deficient tumorsthe setting in which the largest parp inhibitor monotherapy effect has been observedor whether parp inhibition might best be used to sensitize hr - proficient tumors to radiation - induced dna damage . 
in addition to parp inhibitors , combination trials of radiation plus inhibitors of numerous different kinases that are involved in orchestrating the cellular response to dna damage ( including atm , atr , chek1 / 2 , wee1 , and prkdc [ dna - pkcs ] ) are also underway ( table 1 )  . inhibition of receptor tyrosine kinaseswith either antibodies or small moleculesrepresents one of the most well - developed and successful targeted therapeutic strategies to date , and inhibitors of several families of receptor tyrosine kinases are us food and drug administration approved . 
 repair genes such as prkdc ( encoding dnapkcs ) to promote radioresistance , 145 - 147 and this ar - mediated radioresistance can be countered with adt . the androgen receptor is also found in a subset of breast tumors , and increased ar transcript levels are associated with radioresistance and increased risk of local recurrence.148 , 149 in preclinical systems , blocking ar signaling with enzalutamide led to increased levels of dna damage and cell death after radiation , and adt and radiation cooperate to decrease cell growth in orthotopic breast cancer models.9 interestingly , although the interactions between radiation and estrogen signaling in breast cancer have been extensively studied , there is no standard for sequencing of antiestrogen therapy and radiation in the breast - conserving or post - mastectomy setting.150 radiation is also being investigated in combination with modulators of intracellular signaling pathways . 
for example , activating pik3ca mutations are common in head and neck squamous cell cancers , and increased activity of the pik3 / akt serine / threonine kinase ( akt ) / mammalian target of rapamycin ( mtor ) signaling pathway drives cellular growth , proliferation , and a radioresistant phenotype . 
 accordingly , depletion or pharmacologic inhibition of pi3k impairs dna repair and confers radiosensitivity in cell lines with activating pik3ca mutations.151 , 152 numerous drugs targeting epigenetic processes are also being tested in combination with radiation . 
 for example , the histone deacetylase inhibitor vorinostat has shown radiosensitizing effects in preclinical studies.153 , 154 a clinical trial combining vorinostat with temozolomide and radiation in newly diagnosed glioblastoma multiforme showed the combination to be safe ; however , the primary efficacy end point was not met.155 , 156 as the portfolio of targeted therapies continues to expand , and as targeted agents become approved for first - line use in more settings , significant work will be needed to understand the interactions of these agents with radiation . 
the identification of reliable predictive biomarkers for both the targeted agent and radiation will be critical to ensuring that combinations are applied in the most rational way to maximize benefit . combining radiation with immunotherapy inhibitors the discovery and clinical development of therapies that modulate the host immune response has transformed oncology practice , and immune checkpoint ( anticytotoxic t - cell lymphocyte - 4 [ ctla - 4 ] , anti - programmed cell death 1 [ pd1 ] , and antiprogrammed death ligand 1 [ pd - l1 ] ) are now approved in numerous clinical settings . 
given the widespread incorporation of these agents , there is intense interest in combining immunotherapies with radiation , and the topic has been addressed comprehensively in several recent reviews.157 - 159 the interaction between the immune system and radiation has been appreciated for decades160 , 161 ; however , the tumor and host genomic features governing the interplay between immune directed therapies and radiation are incompletely understood . 
remarkable case reports of an abscopal effect , in which radiation drives regression of lesions outside the radiation field , have become more common in the era of immune checkpoint inhibitors , 162 , 163 but the mechanisms remain unclear , and it is currently impossible to predict which patients may experience a systemic response to localized radiation . 
it is likely that tumor and host genomic features as well as clinical factors such as radiation field size , dose , and fractionation pattern play a role . in preclinical models , radiation has been shown to have both immune - stimulating and immune suppressive effects . 
advances in imaging and dosimetry have allowed radiation to become remarkably precise in its physical delivery ; however , recent advances in cancer biology and genomics must now be translated into clinical radiation oncology practice to further maximize the therapeutic potential of radiation . many oncology centers are now performing routine genomic analysis of primary tumor specimens from patients . 
although these assays often identify genomic features that are known to be prognostic , translating these findings into actionable treatment decisions at the point of care remains a significant challenge ( fig 3 ) .169 the recent advent of noninvasive biomarkers such as circulating tumor dna and circulating tumor cellswill likely become integral techniques in monitoring disease response and identifying resistance mechanisms . incorporating genomic biomarkers to shift radiation oncology practice represents a unique challenge . 
radiation has complex , dose - dependent cellular effects and is often combined with systemic agents ; therefore , defining reliable signatures of radiation response will require unraveling intricate interactions among signals and effectors . 
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rieckmann t , tribius s , grob tj , et al : hnscc cell lines positive for hpv and p16 possess higher cellular radiosensitivity due to an impaired dsb repair capacity . 
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 genomic characterization of erbb2 - driven biliary cancer and a case of response to ado - trastuzumab emtansine sebastian mondaca , md1 ; pedram razavi , md , phd1 , 2 ; chongrui xu , md , phd1 ; michael ofn , md1 ; mackenzie myers , ms1 ; maurizio scaltriti , phd1 ; jaclyn f . 
abou - alfa , md , mba1 , 2 purpose biliary tract cancers ( btcs ) , which include intrahepatic cholangiocarcinoma ( icc ) , extrahepatic cholangiocarcinoma ( ehc ) , and gallbladder cancer ( gbc ) , have limited treatment options . 
twenty - eight patients ( 5.4% ) had erbb2 alterations , including 2.7% with erbb2 gene amplication , 2.3% with erbb2 mutation , and 0.4% with concurrent amplication and mutation . 
frequent co - altered genes in this cohort were tp53 ( 54% ) , pik3ca ( 21% ) , and cdkn2a ( 18% ) ; kras amplication / mutation was found in 7% of patients . 
2019 by american society of clinical oncology introduction biliary tract cancer ( btc ) comprises three clinically and molecularly distinct entities , intrahepatic cholangiocarcinoma ( icc ) , extrahepatic cholangiocarcinoma ( ehc ) , and gallbladder cancer ( gbc )  . 
genomic proling of btcs with next - generation sequencing ( ngs ) platforms has shown that the three clinical subtypes have signicant molecular differences.5 whereas all three have a high prevalence of tp53 and cdkn2a mutations , genomic alterations in fgfr2 and idh1 are mostly limited to icc ( approximately 20% each )  . 
 mondaca et al context key objective to describe the landscape of erbb2 genomic alterations in biliary tract cancer and illustrate a case of a patient who responded to antihuman epidermal growth factor receptor 2 ( her2 ) targeted therapy . knowledge generated erbb2 amplication or mutations were identied in 28 ( 5.4% ) of 517 patients with biliary tract cancer . 
erbb2 - driven biliary tract tumors had higher concurrent alterations of tp53 and pik3ca , while kras alterations seemed to be less common . one patient with erbb2 - amplied extrahepatic cholangiocarcinoma had a partial response to the her2 - targeted antibodydrug conjugate ado - trastuzumab emtansine . relevance potentially targetable erbb2 amplication and mutation are present in a signicant group of biliary tract tumors , particularly in gallbladder cancer . 
this study supports prospective testing for erbb2 alterations to develop novel anti - her2 strategies in this population . rst - line chemotherapy compared with patients with erbb2 wild type . on the basis of demonstrated improved os in pivotal phase iii trials , drugs that target her2 are standard treatment in her2 - overexpressed breast and gastric cancers.9 , 10 given the low incidence of btcs , the clinical characterization of relevant molecular subgroups , such as erbb2 - amplied or erbb2 - mutant btcs , has been limited , and the clinical experience with anti - her2 agents in this group has been largely anecdotal . 
a recent basket trial showed objective response in four ( 36% ) of 11 patients with erbb2amplied or erbb2 - mutant btc treated with the combination of the anti - her2 antibodies trastuzumab and pertuzumab.11 the aim of the current study was to comprehensively describe the clinical and molecular characteristics of erbb2 - amplied or erbb2 - mutant btcs identied within the context of a prospective tumor proling initiative at memorial sloan kettering ( msk ) cancer center . we also report a durable response to the her2 - targeted antibody - drug conjugate ado - trastuzumab emtansine ( t - dm1 ) in a patient with erbb2 - amplied btc enrolled in a basket trial ( clinicaltrials.gov identier : nct02675829 )  . methods patients demographic data and treatment histories were collected for all patients with erbb2 - altered btc who consented for prospective tumor genomic proling using the us food and drug administrationauthorized msk - impact assay between february 2014 and june 2018 . 
informed consent for tumor proling and clinical data collection was obtained under the genomic proling in cancer patients protocol ( clinicaltrials.gov identier : nct01775072 ) , which was approved by the msk institutional review board . data collection clinical data were extracted from electronic medical records . variables collected were demographic characteristics ( age , sex , race ) , eastern cooperative oncology group performance status , tumor type ( icc , ehc , gbc ) , metastatic sites , systemic treatments received , and clinical outcomes . 
all tumors were prospectively reviewed to conrm histology and to estimate tumor purity . molecular proling and genomic analysis memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) was performed as previously described in a clinical laboratory improvement amendmentscertied laboratory.12 , 13 msk - impact sequences at high coverage all exons and selected intronic and noncoding regions of up to 468 cancer - associated genes . 
baseline characteristics characteristic illustrative case one patient with metastatic erbb2 - amplied ehc was treated with t - dm1 in a basket trial at msk for patients with her2 - amplied or her2 - mutant cancers in the cohort of other solid tumors . 
methods and partial results of this trial have been presented previously , 15 , 16 but to our knowledge , this would be the rst publication about the patient with btc from the basket trial . 
this study was approved by the msk institutional review board , and all patients signed informed consent in accordance with the precepts of the declaration of helsinki . results patients and molecular prole during the period considered in this study , 517 patients with btc underwent msk - impact testing . 
in the subset of patients with btc with erbb2 - amplied or erbb2 - mutant tumors , the median age at diagnosis was 64 years ( range , 27 - 82 years ) , and the most frequent stage of presentation was stage iv ( 50% )  . 
the most frequently mutated domain of her2 was the extracellular domain ( 32% ) , and all mutations in this region arose at the s310 codon ( fig 1 )  . missense mutations represented the most common alteration in the mutated group ( 94% )  . 
of patients age , years median ( range ) male female race white asian black unknown primary tumor stage at diagnosis genomic alteration erbb2 amplication erbb2 mutation erbb2 amplication / mutation first sites of metastasis liver lung bone peritoneum other btc , no . 
the patient then received adjuvant gemcitabine , but within 3 months of initiation of systemic chemotherapy , imaging showed progression of disease in the posterior mediastinum , liver , and peritoneua biopsy specimen of the table 3 . 
aa , amino acid ; furin - like , furin - like cysteine - rich region ; gf_recept - iv , growth factor receptor domain iv ; pkinase_tyr , protein tyrosine kinase domain ; recep_l , receptor l domain . mediastinal metastasis conrmed recurrence ; immunohistochemistry revealed 3 + her2 protein overexpression . the patient was then treated with t - dm1 3.6 mg / kg once every 21 days in a basket trial and achieved a conrmed partial response after 12 weeks of treatment ( fig 3 )  . 
despite ongoing treatment with t - dm1 , the patients disease progressed after 8.6 months with the development of a new 2.2 - cm ( short - axis ) retrocaval lymph node . 
cancer antigen 19 - 9 was 10 , 700 u / ml before starting t - dm1 and decreased to 73 u / ml after achieving a conrmed partial response , with cancer antigen 19 - 9 remaining stable at the time of disease progression ( 62 u / ml )  . 
however , with the development of a new site of disease , t - dm1 was discontinued . discussion this study reports that potentially targetable erbb2 amplication or mutation was present in 5.4% of a cohort of 517 patients with btc who underwent prospective tumor molecular characterization at our institution . 
a similar gradient was described in a previous study that reported 16% , 11% , and 3% of erbb2 amplication in gbc , ehc , and icc , respectively.5 we also found that msi - high tumors were more frequent in the erbb2 - altered cohort compared with tumors without these alterations . 
similar ndings have been described in erbb2 - mutant colon cancer17 ; however , it is not clear whether this represents a biologically meaningful association or passenger erbb2 mutations are more frequent in tumors with an increased tumor mutational burden . 
erbb2activating mutations have been previously described to be present in 1% to 8% of btcs ; the potential therapeutic signicance of these mutations is an active area of research.5 as an example , 20 patients with her2 - mutant btc were enrolled in a basket trial of the pan - her tyrosine kinase inhibitor neratinib ( clinicaltrials.gov identier : nct01953926 ) , with two ( 10% ) achieving a partial response.18 this trial the clinical activity of found that neratinib varied as a function of tumor site , with the most promising clinical activity observed in her2 - mutant breast cancer ( 32% ) but no recist responses in several other cancer types , including bladder and colon cancers.19 the likelihood of clinical response to neratinib also varied as a function of mutation type . 
in another basket study , the response rate of patients with her2 - mutant nonsmall - cell lung cancer to t - dm1 was 44%.15 it remains unknown whether her2targeted antibody drug conjugates would have similar activity in her2 - mutant btc . we report here a patient with erbb2 - amplied btc who achieved a partial response to the her2 - targeted antibodydrug conjugate t - dm1 . 
 mutation count mutation spectrum msi type cancer type sample type erbb2 egfr erbb3 erbb4 fgfr2 pik3ca akt1 tsc2 tp53 mdm2 smad4 arid1a arid1b arid2 kmt2c kras cdkn2a idh1 bap1 mondaca et al stable indeterminate instable primary metastatic female male 100% genomic alterations missense mutation ( putative driver ) missense mutation ( unknown significance ) truncating mutation in - frame mutation fusion amplification deletion no alterations fig 2 . 
ehc , extrahepatic cholangiocarcinoma ; gbc , gallbladder cancer ; icc , intrahepatic cholangiocarcinoma ; msi , microsatellite instability . basket trials , conduct of dedicated trials that include an erbb2 - altered btc cohort has been more challenging . currently , a phase ii trial in china is evaluating the combination of gemcitabine plus oxaliplatin and trastuzumab in erbb2 - amplied btc ( clinicaltrials.gov identier : nct02836847 )  . 
her2 overexpression by immunohistochemistry and uorescence in situ hybridization has been pivotal for the approval of her2 - targeted therapies in breast and gastric cancers.9 , 10 however , tissuebased ngs analyses can identify both mutation and amplication along with other potential actionable targets . given the good correlation for amplication among these methods14 and the increasing clinical use of ngs , we believe that this approach represents an appropriate inclusion criterion in clinical trials . 
first , the cohort represents a selected population treated at a single academic cancer center that favors clinical trial enrollment ; hence , the patients with btc studied may have had different characteristics compared with those being treated in a community setting . 
 erbb2 amplication and mutation in biliary tract cancer baseline 3 months 8.6 months 30% present 80% present new retrocaval ln target lesions ( liver ) nontarget lesions new lesions 10 , 000 8 , 000 6 , 000 4 , 000 2 , 000 months fig 3 . 
ca , cancer antigen ; ln , lymph node . reported treatment is greater than 1.3 times the pfs on the prior line of therapy , the reported treatment can be considered benecial.26 in the case presented here , the patient progressed within 3 months after starting adjuvant chemotherapy and had a pfs of 8.6 months on rst - line t - dm1 , which supports a clinically meaningful benet . 
abou - alfa , md , memorial sloan kettering cancer center , 300 e 66th st , new york , ny 10065 ; twitter : @gaboualfa , @sloan_ kettering ; e - mail : abou - alg@mskcc.org. support supported by national institutes of health memorial sloan kettering cancer center core grant no . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . sebastian mondaca consulting or advisory role : foundation medicine pedram razavi consulting or advisory role : novartis research funding : grail ( inst ) , illumina ( inst ) chongrui xu honoraria : astrazeneca , boehringer ingelheim , eli lilly , msd , novartis , pzer , roche consulting or advisory role : boehringer ingelheim , pzer travel , accommodations , expenses : astrazeneca , boehringer ingelheim , eli lilly , novartis , pzer , roche , bristol - myers squibb , msd michael ofn consulting or advisory role : pharmamar , novartis , targeted oncology travel , accommodations , expenses : bristol - myers squibb , merck sharp & dohme maurizio scaltriti stock and other ownership interests : loxo oncology honoraria : menarini , adc therapeutics consulting or advisory role : menarini research funding : menarini ( inst ) , immunomedics ( inst ) , targimmune therapeutics ( inst ) , puma biotechnology ( inst ) , daiichi sankyo ( inst ) patents , royalties , other intellectual property : pi3k inhibitors and uses thereof , us provisional application no . 
62 / 742 , 163 , inventors : maurizio scaltriti , dan heller , jos e baselga , yosef shamay , carles monterrubio , amaia arruabarrena ( inst ) ; methods for detecting her2 dimerization in patients with her2 mutant lung cancers and predicting responsiveness to ado - trastuzumab emtansine therapy , sk2018038 - 01 , inventors : maurizio scaltriti , bob t . 
solit stock and other ownership interests : loxo oncology consulting or advisory role : pzer , loxo oncology , illumina , intezyne technologies , vivideon therapeutics , eli lilly travel , accommodations , expenses : merck kgaa bob t . 
li consulting or advisory role : roche , biosceptre international , thermo fisher scientic , mersana , guardant health , hengrui therapeutics research funding : roche ( inst ) , genentech ( inst ) , illumina ( inst ) , biomed valley discoveries ( inst ) , astrazeneca ( inst ) , grail ( inst ) , daiichi sankyo ( inst ) , hengrui therapeutics ( inst ) , guardant health ( inst ) , amgen ( inst ) ghassan k . 
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identication and monitoring the molecular mechanisms of acquired resistance to cetuximab or panitumumab in crc is important to anticipate the failure of anti - egfr antibodies and design strategies for additional treatments . several mechanisms of acquired resistance to antiegfr have been described in metastatic crc ( mcrc )  . 
 maurel et al context key objective the key objective was to evaluate the predictive and prognostic value of ras and braf mutation dynamics in plasma from patients with metastatic colorectal cancer treated with antiepidermal growth factor receptor ( anti - egfr ) therapy within two clinical trials ( posiba and pulse )  . knowledge generated baseline mutant ras / braf circulating tumor ( ct ) dna was a negative predictive marker of response to anti - egfr therapy , as well as a poor prognostic factor . 
the emergence of ctdna ras / braf mutations had limited relevance to the time to progression to anti - egfr therapy but conferred poor prognosis . relevance this work conrms the clinical utility of ctdna for ras / braf molecular testing to guide treatment decisions in patients with metastatic colorectal cancer ; however , clinical impact of ras / braf dynamics deserves further characterization . of ras , braf , or egfr mutations that drive acquired resistance in approximately 50% of patients.5 , 6 the use of circulating tumor ( ct ) dna for genotyping the tumor ( liquid biopsy ) offers clear advantages compared with repeated tumor tissue biopsies , including the minimally invasive method and no need for a tumor lesion accessible to biopsy . 
our objective was to determine the role of ctdna ras and braf mutations at the time of progression to anti - egfr therapy . methods study design and patient population the pulse and posiba studies were both prospective biomarker - design trials . 
patients were recruited into the pulse trial from november 2010 to april 2013 in 24 spanish centers and treated with infusional uorouracil , leucovorin , and oxaliplatin ( folfox ) - 6 plus panitumumab ( 6 mg / kg )  . 
patients were recruited into the posiba trial from july 2011 to may 2015 in 28 spanish centers and treated with folfox6 or uorouracil , leucovorin , and irinotecan ( folfiri ; at the investigators choice ) plus biweekly cetuximab ( 500 mg / m2 )  . in both trials , cytotoxic drugs were administered for 6 months , and anti - egfr monotherapy was continued until pd or unacceptable toxicity occurred . 
extended ras mutational analysis ( including kras / nras exons 2 , 3 , and 4 ) started in october 2013 in the pulse trial and in october 2015 in the posiba trial after protocol amendments . 
competitive allele - specic taqman pcr ( castpcr ; thermo fisher scientic , waltham , ma ) and an ultrasensitive sequencing technique ( digital polymerase chain reaction [ pcr ] ) were used to reanalyze tissue ras determination in patients with discrepancies between basal liquid biopsy ( mutation ) and formalin - xed parafn - embedded ( ffpe ) specimens ( wt )  . ctdna ras and braf mutational analysis this was an exploratory translational study with a primary objective of investigating measurement of ras and braf v600 - mutant ctdna in patients treated in the posiba and pulse trials using the idylla ctras and ctbraf mutation assay ( biocartis , jersey city , nj )  . 
plasma samples were collected basally , and serum samples were collected every 2 months in the pulse trial and every 3 months in the posiba trial , until pd was detected according to response evaluation criteria in solid tumors ( recist ) v1.1. 
two blood samples were analyzed for each patient for this study : a baseline plasma sample and a serum sample collected at the time of the last available determination . integrated ctdna purication and allele - specic quantitative pcr followed by automated data analysis was performed using the idylla ctkras and ctnras mutation assays to detect ras exon 2 , 3 , and 4 mutations and braf v600 mutations using 0.5 to 1 ml of plasma or serum per assay . 
the investigators who performed plasma genotyping were blinded to the clinical patient information . statistical analysis imaging was performed every 8 weeks in the pulse trial and every 12 weeks in the posiba trials and response was based on recist v1.1 as assessed by the investigators . progression - free survival ( pfs ) was dened as time from enrollment to disease progression ( recist criteria ) , death , or end of follow - up , whichever came rst . 
concordance was assessed using the kappa statistic and the characteristics of the diagnostic test using the sensitivity , specicity , and positive and negative predictive values by collapsing ras plus braf versus wt . 
statistical analysis was performed with spss statistics 22 ( spss , chicago , il ) and sas 9.4 software ( sas institute , cary , nc )  . results patient characteristics and concordance of baseline ras and braf mutation determination in plasma versus tissue the study ow chart is presented in figure 1 . 
kaplan - meier estimates of ( a ) progression - free survival and ( b ) overall survival , on the basis of ( c ) solid and ( d ) liquid biopsies . 
blood extraction was performed in group 1 ( between blood extraction and pd ) with a median ( p25 to p75 ) of + 7 days ( 1 to + 23 days ) and in group 2 with a median of 157 days ( 76 to 330 days ) , with the date of progression as the index date . the emergence of ras / braf mutations in ctdna was observed in 15 patients after initiation of therapy ( 11% ; appendix table a3 , online only )  . 
however , one of 11 patients ( 9% ) with acquired mutations in ctdna received anti - egfr compounds after disease progression compared with 22 of 79 ( 27.8% ) of those who remained free of mutations , potentially contributing to improved os . discussion our study indicates that ras and braf testing in baseline if not better , pfs and os ctdna results in a similar , discrimination compared with tissue testing in patients treated with rst - line chemotherapy plus anti - egfr therapy . 
 maurel et al 20116 20601 10019 10129 10140 10039 10027 10149 23209 10090 10153 20804 10131 10082 20401 10004 10159 20602 10006 10182 20306 10124 10115 21119 10122 22008 10167 22901 20611 10174 10126 10189 10147 22001 mutational status ( os bar ) : negativitzation no change overall response rate ( pfs bar ) : metastasectomy end of treatment liquid biopsy 1 , 000 1 , 200 1 , 400 time ( days ) fig 4 . 
progression - free survival ( pfs ) and overall survival ( os ) in patients with mutant ras and braf on basal liquid biopsy ( n = 34 )  . 
in a recent study with 43 patients , oncobeam demonstrated 72.1% concordance with tissue results , whereas idylla demonstrated 46.5% concordance with kras tissue results.15 finally , we observed that the subgroup of patients who had ras or braf mutations on tissue and wt on ctdna was enriched with patients with low tumor burden ( ldh , 1 uln )  . 
our results analyzing plasma samples at baseline and serum samples at progression differ with the analysis from the aspecct third - line phase iii panitumumab study , which showed that patients with emergent ras mutations at progression had similar pfs and os versus patients whose status remained wt at progression.19 in our study , patients with ras and braf with acquired mutations that were initially 2 wt showed signicantly poorer survival than those patients who remained wt at progression ; also , patients with disappearance of mutations showed better os . 
we have to note that patients in our study had been treated in rst - line therapy ( compared with third line in the aspecct trial ) and , therefore , second - line egfr therapies in truly sensitive patients ( those with liquid biopsies that remain wt ) would affect survival . 
moreover , we could not rule out that at pd , in patients with a poor prognosis ( identied by a high level of ldh at disease progression ) , we would have more chance to detect acquired mutations in ctdna . 
our study addresses most of the potential limitations from previous analyses , such as low number of patients , treatment heterogeneity ( including type of treatment and mixed rst , second , and third lines ) , and lack of a control group ( analysis of ras and braf in samples of patients without progression or before pd )  . 
 clinical impact of circulating tumor ras and braf mutations with rst - line therapy with currently accepted schedules of chemotherapy ( folfox and folfiri ) and approved antiegfr compounds ( cetuximab and panitumumab ) to establish differences between both groups ( 14% and 8% , respectively ; p = .47 ) , and global mutation acquisition was observed in only 11% of patients . 
this suggests that other mechanisms beyond ras and braf acquired mutations will probably play an important role , not only in the subset of patients without acquired ras and braf mutations , but also in the group of patients with acquired ras and braf mutations . 
n engl j med 360 : 1408 - 1417 , 2009 van cutsem e , lenz hj , k ohne ch , et al : fluorouracil , leucovorin , and irinotecan plus cetuximab treatment and ras mutations in colorectal cancer . 
j clin oncol 33 : 692 - 700 , 2015 douillard jy , siena s , cassidy j , et al : randomized , phase iii trial of panitumumab with infusional uorouracil , leucovorin , and oxaliplatin ( folfox4 ) versus folfox4 alone as rst - line treatment in patients with previously untreated metastatic colorectal cancer : the prime study . 
j clin oncol 28 : 4697 - 4705 , 2010 douillard jy , oliner ks , siena s , et al : panitumumab - folfox4 treatment and ras mutations in colorectal cancer . 
montagut c , dalmases a , bellosillo b , et al : identication of a mutation in the extracellular domain of the epidermal growth factor receptor conferring cetuximab resistance in colorectal cancer . 
montagut c , argil es g , ciardiello f , et al : efcacy of sym004 in patients with metastatic colorectal cancer with acquired resistance to anti - egfr therapy and molecularly selected by circulating tumor dna analyses : a phase 2 randomized clinical trial . 
jama oncol 4 : e175245 , 2018 pietrantonio f , vernieri c , siravegna g , et al : heterogeneity of acquired resistance to anti - egfr monoclonal antibodies in patients with metastatic colorectal cancer . 
clin cancer res 23 : 2414 - 2422 , 2017 diaz la jr , williams rt , wu j , et al : the molecular evolution of acquired resistance to targeted egfr blockade in colorectal cancers . 
siena s , sartore - bianchi a , garcia - carbonero r , et al : dynamic molecular analysis and clinical correlates of tumor evolution within a phase ii trial of panitumumab - based therapy in metastatic colorectal cancer . 
normanno n , esposito abate r , lambiase m , et al : ras testing of liquid biopsy correlates with the outcome of metastatic colorectal cancer patients treated with rst - line folfiri plus cetuximab in the capri - goim trial . 
grasselli j , elez e , carat `u g , et al : concordance of bloodand tumor - based detection of ras mutations to guide anti - egfr therapy in metastatic colorectal cancer . 
ann oncol 29 : 1211 - 1219 , 2018 jacobs b , claes b , pomella v , et al : analytical and clinical validation of the idylla ( cid : 1 ) ctkras and ctnras - braf liquid biopsy tests identies mcrc patient groups with high and low ctdna shedding . 
ras and braf mutational status in baseline plasma sample and secondary extraction serum sample : characteristics of patients with ctdna ras / braf mutation in baseline and second liquid biopsy plasma liquid biopsy ( basal ) serum liquid biopsy ( secondary ) , no . ras mutated braf mutated patient primary side basal liquid biopsy site of metastasis response surgery of second liquid metastasis biopsy maurel et al mutated braf mutated 2 wt total rectum kras ( a146p / t / v ) plus nras ( g13r / v ) liver left colon nras ( g13r / v ) liver , lung sigmoid kras ( g12d ) liver , lung sigmoid nras ( g13r / v ) liver , peritoneum sigmoid kras ( g12v ) node right side kras ( a146p / t / v ) local relapse sigmoid kras ( k117n ) liver , lung left colon kras ( g12v ) peritoneum rectum kras ( g12r ) liver , lung rectum kras ( k117n ) liver , lung right side braf ( v600e / d ) liver , peritoneum rectum nras ( q61h ) rectum braf ( v600e / d ) right side braf ( v600e / d ) kras ( g12d ) liver liver liver liver rectum kras ( a146p / t / v ) liver , node , suprarenal  . 
ras and braf mutational status in baseline plasma sample and secondary extraction serum sample : characteristics of patients with ctdna ras / braf mutation in baseline and second liquid biopsy ( continued ) serum liquid biopsy ( secondary ) , no . 22008 10122 10115 10006 20602 10004 10082 10153 22001 10189 10174 21119 10131 10090 rectum nras ( g61k ) liver , lung sigmoid nras ( g61r ) liver , lung right side kras ( a59t / e / g ) liver rectum kras ( a146p / t / v ) liver , lung left colon nras ( g61h ) liver , node , peritoneum  . 
 o palbociclib in patients with pancreatic and biliary cancer with cdkn2a alterations : results from the targeted agent and proling utilization registry study tareq al baghdadi , md1 ; susan halabi , phd2 ; elizabeth garrett - mayer , phd3 ; pam k . 
schilsky , md3 purpose the targeted agent and proling utilization registry ( tapur ) study identies signals of antitumor activity of commercially available targeted agents in patients with advanced cancers that harbor genomic alterations known as drug targets . 
in this article , data from two cohorts of patients with pancreatic and biliary cancers with cdkn2a loss or mutation treated with palbociclib are reported . methods eligible patients age 12 years and older with advanced measurable or evaluable solid tumors are provided treatment according to protocol - specied genomic matching rules . 
secondary end points include safety , progression - free survival ( pfs ) , and overall survival ( os )  . results between july 2016 and november 2017 , 12 and 10 patients with pancreatic and biliary cancer , respectively , with cdkn2a loss or mutation were treated with palbociclib . 
despite recent advances , outcomes remain poor with a median overall survival ( os ) of less than 12 months in stage iv biliary and pancreatic cancers , 1 - 4 and new therapies are urgently needed . cyclin - dependent kinase ( cdk ) inhibitor 2a ( cdkn2a ) encodes p16ink4a , which plays an important role in cell - cycle regulation through inhibition of cdk4 / 6 . cdkn2a loss or mutation is found in a wide array of malignancies and may lead to increased cdk activity.5 in a report of the mutational landscape of advanced pancreatic cancer , 46.5% of tumors harbored alterations in cdkn2a.6 palbociclib is an orally available selective cdk inhibitor approved for the treatment of hormone receptorpositive , human epidermal growth factor receptor 2 ( her2 ) negative breast cancer in combination with endocrine therapy . the targeted agent and proling utilization registry ( tapur ) study is a prospective , phase ii , pragmatic basket trial designed to identify signals of antitumor activity of commercially available targeted agents in patients with advanced cancers that harbor genomic alterations known to be drug targets . 
this study examined whether patients with advanced biliary and pancreatic cancer with cdkn2a loss or mutation would be responsive to palbociclib , an oral cyclin - dependent kinase inhibitor . knowledge generated the results demonstrated that palbociclib monotherapy has no meaningful clinical activity in patients with cdkn2a mutated or deleted advanced pancreatic or biliary adenocarcinoma . relevance off - label prescribing of palbociclib for the treatment of patients with pancreatic and biliary tract cancers with cdkn2a loss or mutation is not recommended . 
these patients should be offered participation in clinical trials of new treatment approaches . methods trial design the rationale and study design have been reported previously in detail.7 patients with advanced cancer and no standard treatment options are eligible if they have a tumor that harbors a genomic alteration known to be a target of or to predict sensitivity to one of the available study drugs . the primary study end point is antitumor activity , dened as objective response ( or ) at 8 weeks or later or stable disease ( sd ) at 16 weeks or later from the time of enrollment . secondary end points include progression - free survival ( pfs ) , os , and toxicity . 
patients are matched to one of according to prespecied protocol matching rules on the basis of genomic testing performed by laboratories selected by clinical sites that have certication under the clinical laboratory improvement amendments and accreditation from the college of american pathologists . 
treating physicians may choose to consult with the tapur molecular tumor board for appropriate targeted treatment options where none or multiple treatment matches surface within the tapur matching rules or if the treating physician would like to propose a treatment match outside the matching rules . 
radiographic assessment and clinical evaluation for response evaluation are performed at 8 and 16 weeks after initiation of treatment ; then every 12 weeks while the patient remains in the study ; and then at the end of study treatment , where possible . 
patients are placed in multiple parallel cohorts dened by study drug , genomic alteration , and tumor type . each cohort has the same optimal simon two - stage design , with a total possible cohort size of 28 patients . 
the design requires 10 patients to be enrolled in a cohort in stage i and if fewer than two patients have or or sd of at least 16 weeks duration , the cohort is permanently closed . 
if at least seven patients in the total cohort have a response of at least 16 weeks duration , the null hypothesis is rejected , and it is concluded that a signal of activity has been identied . patients eligible patients are required to meet both protocoland drugspecic inclusion and exclusion criteria . 
protocol - specic eligibility criteria include advanced measurable or evaluable solid tumors per recist version 1.1 ; eastern cooperative oncology group ( ecog ) performance status ( ps ) of 0 , 1 , or 2 ; and a protocol - specied genomic target identied by a test performed in a laboratory that has clinical laboratory improvement amendments certication and college of american pathologists accreditation . 
 palbociclib in pancreatic / biliary cancer with cdkn2a alterations addition , to match to palbociclib , patients must have a cancer type other than breast and not have received food or drugs within 7 days before the start of study treatment that are known to be cyp3a4 inhibitors or inducers . 
detailed inclusion and exclusion criteria for tapur have been previously reported.7 patients treated with palbociclib received standard doses ( 125 mg orally daily for 21 days followed by 7 days off treatment to complete a 28 - day cycle ) until disease progression . 
all patients were tested for alterations using next - generation sequencing ( ngs ) platforms , with more than half the patients determined to have cdkn2a loss ( table 1 )  . patients in both cohorts continued treatment with palbociclib until disease progression , and no patients withdrew from the study . trial oversight the tapur study protocol was reviewed and approved by a central institutional review board and , in some cases , by the local institutional review board at participating sites . patients provide written consent before any screening activities or data collection . 
the tapur study was designed by asco staff with input from asco volunteer members and the participating pharmaceutical companies . the tapur data and safety monitoring board ( dsmb ) is an independent board appointed by asco and meets biannually to monitor the study and review the safety and efcacy ndings . 
in the case of the cohorts reported herein , the dsmb reviewed the safety and efcacy data and determined that both cohorts should permanently close at the end of stage i enrollment and that the ndings be released . study end points the primary end point is or , dened as complete or partial response at or before 16 weeks , or sd at 16 weeks or later as reported per recist version 1.1. 
 al baghdadi et al results patients with pancreatic cancer twelve patients with pancreatic cancer and cdkn2a loss or mutation treated with palbociclib were enrolled in the study across seven sites from july 25 , 2016 , to april 28 , 2017 . 
the median age was 62 years ( range , 52 to 70 years ) ; 67% of patients were male ; and 10 patients were white , one black , and one other . 
other common genomic alterations noted in this cohort were mutations in braf , fgfr1 , frs2 , kras , and tp53 , with kras mutations being the most commonly reported in 10 of 12 patients . two patients who were enrolled and received treatment were subsequently found to be ineligible because they had not met the eligibility requirement for a hemoglobin level of 9.0 g / dl or greater at the time of enrollment . 
of the 10 eligible patients , nine experienced progression at or before 8 weeks ; the remaining patient had tumor progression at 16 weeks . all patients were included in the analysis for safety , pfs , and os . 
no other grade 3 or higher adverse event or serious adverse event was observed as at least possibly related to palbociclib . patients with biliary cancer ten patients with biliary cancer with cdkn2a loss or mutation treated with palbociclib were enrolled in the study across six sites from august 9 , 2016 , to november 11 , 2017 . 
other genomic alterations noted in this cohort included arid1a , atm , braf , fgfr2 , fh , kras , nras , pik3ca , and vhl . all 10 patients experienced progression at or before 10 weeks . 
four patients experienced grade 3 or 4 adverse events of thrombocytopenia at least possibly related to palbociclib . discussion this phase ii study in patients with advanced pancreatic or biliary cancers with cdkn2a loss or mutation treated with single - agent palbociclib demonstrated no clinical activity . the toxicity is similar to previous reports of monotherapy with palbociclib . cdkn2a is a tumor suppressor gene frequently altered by mutations , deletions , and epigenetic silencing . 
of patients loss mutation loss cdkn2a / 2b homozygous loss p16ink4a a4_p11del , p16ink4a l16fs * 9 , loss exons 2 - 3 splice site 151 - 2a.g p16ink4a r58 * and p14arf p72l p16ink4a a36fs * 17 , p16ink4a n71fs * 49 and p14arf q85fs * 50 + splice site 151 - 1g.c truncation exon 2 foundationone mi - oncoseq foundationone foundationone foundationone foundationone foundationone foundationone abbreviations : mi - oncoseq , michigan oncology sequencing project ; ngs , next - generation sequencing . cdkn2a mutations / deletions occur frequently in pancreatic adenocarcinoma.11 moreover , alterations that abrogate the rb / p16 tumor suppressor pathway are seen in virtually all pancreatic carcinomas.12 cdkn2a mutations occur in a minority of cholangiocarcinomas and are associated with a poor prognosis.13 , 14 prior studies of multiple rb - positive tumors have shown that most are sensitive to some degree to cdk4 / 6 inhibition.15 - 18 preclinical studies assessing the impact of cdk4 / 6 inhibition on pancreatic cancer cell lines and xenograft models showed conicting data . 
for example , one study showed that palbociclib inhibited the growth of pancreatic cancer cell lines but resulted in upregulation of the expression of genes that promote invasion and metastasis.19 another showed that established pancreatic cell lines displayed a relatively weak response to palbociclib , but palbociclib had potent activity in patient - derived xenografts.20 palbociclib is approved for the treatment of patients with advanced hormone receptorpositive , her2 - negative breast cancer in combination with an aromatase inhibitor or fulvestrant.21 , 22 palbociclib showed promising activity in patients with liposarcoma23 and cdkn2a - mutated nonsmallcell lung cancer.24 however , palbociclib was not effective in lung cancer25 or unselected patients with squamous cell urothelial cancer.26 prior studies have suggested several mechanisms of resistance to cdk4 / 6 inhibitors in human cancer cell lines and xenograft models , including cdk4 amplication , rb loss , and cyclin e1 amplication.27 - 31 the potential for prior exposure to systemic chemotherapy to induce resistance to palbociclib through these or other mechanisms has not been explored but might have contributed to the lack of efcacy of palbociclib in this trial . 
it is conceivable , therefore , that the use of palbociclib alone or in combination earlier in the treatment course could be benecial . the association of cdkn2a mutations / deletions with clinical activity of cdk4 / 6 inhibitors is undetermined . 
in patients with hormone receptorpositive , her2 - negative breast cancer , cdkn2a mutations do not predict activity of either palbociclib or ribociclib.32 , 33 a report revealed that cdkn2a mutant cancers show only intermediate sensitivity to cdk4 / 6 inhibition with abemaciclib , whereas genetic events that activate d - type cyclins were associated with high sensitivity.30 the effects of combining other targeted agents with palbociclib in pancreatic adenocarcinoma cell lines have been explored . 
the addition of a mek inhibitor31 and a mammalian target of rapamycin inhibitor30 , 34 was synergistic . the latter may relate to metabolic reprogramming of pancreatic cancer cells by cdk4 / 6 inhibition , which results in increased oxidative phosphorylation , increased consumption of glucose and glutamine , and activation of mammalian target of rapamycin pathway.34 these and other novel combinations hopefully will allow successful treatment of pancreatic adenocarcinoma and other malignancies on the basis of a sound understanding of mechanisms that underlie cancer cell resistance . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . tareq al baghdadi stock and other ownership interests : array biopharma , astrazeneca , biogin , bristol - myers squibb , merck , portola pharmaceuticals , spectrum pharmaceuticals , tracon pharma , celgene , seattle genetics , sunesis pharmaceuticals honoraria : cardinal health , medscape consulting or advisory role : celgene , bristol - myers squibb , heron therapeutics travel , accommodations , expenses : cardinal health , celgene , bristolmyers squibb , heron therapeutics susan halabi consulting or advisory role : eisai , ferring pharmaceuticals vaibhav sahai consulting or advisory role : celgene , halozyme , newlink genetics , ipsen , incyte research funding : celgene ( inst ) , bristol - myers squibb ( inst ) , agios ( inst ) , incyte ( inst ) , clovis oncology ( inst ) , debiopharm group ( inst ) , fibrogen ( inst ) , halozyme ( inst ) , medimmune ( inst ) , rafael pharmaceuticals ( inst ) , ipsen ( inst ) ricardo h . 
alvarez employment : cancer treatment centers of america leadership : cancer treatment centers of america consulting or advisory role : eisai , puma biotechnology , r - pharma , pzer speakers bureau : eisai , pzer other relationship : eisai , puma biotechnology , pzer edward s . 
kim honoraria : astrazeneca , boehringer ingelheim , pzer , merck , takeda pharmaceuticals , roche , genentech consulting or advisory role : astrazeneca , boehringer ingelheim , pzer , merck , takeda pharmaceuticals , roche , genentech research funding : boehringer ingelheim , merck , ignyta , genentech , roche travel , accommodations , expenses : astrazeneca , boehringer ingelheim , takeda pharmaceuticals , genentech , roche , pzer , merck kathleen j . 
schilsky research funding : astrazeneca ( inst ) , bayer ag ( inst ) , bristol - myers squibb ( inst ) , genentech ( inst ) , roche ( inst ) , eli lilly ( inst ) , merck ( inst ) , pzer ( inst ) , boehringer ingelheim ( inst ) , varian ( inst ) no other potential conicts of interest were reported . references conroy t , desseigne f , ychou m , et al : folfirinox versus gemcitabine for metastatic pancreatic cancer . 
n engl j med 364 : 1817 - 1825 , 2011 von hoff d.d. , ervin t , arena fp , et al : increased survival in pancreatic cancer with nab - paclitaxel plus gemcitabine . 
n engl j med 369 : 1691 - 1703 , 2013 valle j , wasan h , palmer dh , et al : cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer . 
n engl j med 362 : 1273 - 1281 , 2010 sahai v , catalano pj , zalupski mm , et al : nab - paclitaxel and gemcitabine as rst - line treatment of advanced or metastatic cholangiocarcinoma : a phase 2 clinical trial . 
witkiewicz ak , knudsen ke , dicker ap , et al : the meaning of p16 ( ink4a ) expression in tumors : functional signicance , clinical associations and future developments . 
cell cycle 10 : 2497 - 2503 , 2011 aguirre aj , nowak ja , camarda nd , et al : real - time genomic characterization of advanced pancreatic cancer to enable precision medicine . 
fry dw , harvey pj , keller pr , et al : specic inhibition of cyclin - dependent kinase 4 / 6 by pd 0332991 and associated antitumor activity in human tumor 16 . 
finn rs , dering j , conklin d , et al : pd 0332991 , a selective cyclin d kinase 4 / 6 inhibitor , preferentially inhibits proliferation of luminal estrogen receptor - positive human breast cancer cell lines in vitro . 
baughn lb , di liberto m , wu k , et al : a novel orally active small molecule potently induces g1 arrest in primary myeloma cells and prevents tumor growth by specic inhibition of cyclin - dependent kinase 4 / 6 . 
young rj , waldeck k , martin c , et al : loss of cdkn2a expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the cdk4 / 6 inhibitor pd0332991 in melanoma cell lines . 
cristofanilli m , turner nc , bondarenko i , et al : fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone - receptor - positive , her2negative metastatic breast cancer that progressed on previous endocrine therapy ( paloma - 3 ) : final analysis of the multicentre , double - blind , phase 3 randomised controlled trial . 
dickson ma , schwartz gk , keohan ml , et al : progression - free survival among patients with well - differentiated or dedifferentiated liposarcoma treated with cdk4 inhibitor palbociclib : a phase 2 clinical trial . 
gopalan pk , pinder mc , chiappori a , et al : a phase ii clinical trial of the cdk 4 / 6 inhibitor palbociclib ( pd 0332991 ) in previously treated , advanced non - small cell lung cancer ( nsclc ) patients with inactivated cdkn2a . 
edelman mj , redman mw , albain ks , et al : a phase ii study of palbociclib ( p ) for previously treated cell cycle gene alteration positive patients ( pts ) with stage iv squamous cell lung cancer ( scc ) : lung - map sub - study swog s1400c . 
rose tl , chism dd , alva as , et al : phase ii trial of palbociclib in patients with metastatic urothelial cancer after failure of rst - line chemotherapy . 
herrera abreu mt , asghar u , elliot r , et al : pi3 kinase / mtor inhibition increases sensitivity of er positive breast cancers to cdk4 / 6 inhibition by blocking cell cycle re - entry driven by cyclind1 and inducing apoptosis . 
gong x , litcheld lm , webster y , et al : genomic aberrations that activate d - type cyclins are associated with enhanced sensitivity to the cdk4 and cdk6 31 . 
finn rs , crown jp , lang i , et al : the cyclin - dependent kinase 4 / 6 inhibitor palbociclib in combination with letrozole versus letrozole alone as rst - line treatment of oestrogen receptor - positive , her2 - negative , advanced breast cancer ( paloma - 1 / trio - 18 ) : a randomised phase 2 study . 
andre f , stemmer sm , campone m , et al : ribociclib + letrozole for rst - line treatment of hormone receptor - positive ( hr + ) , human epidermal growth factor receptor 2 - negative ( her2 - ) advanced breast cancer ( abc ) : efcacy by baseline tumor markers . 
 genomic and proteomic alterations in desmoplastic small round bluecell tumors purpose desmoplastic small round blue - cell tumors ( dsrcts ) are sarcomas that contain the t ( 11 ; 22 ) ( p13 ; q12 ) translocation ews - wt1 fusion protebecause this is a rare tumor type , prospective clinical trials in dsrct are challenging . 
patients are treated in a manner similar to those with ewing sarcoma ; however , differences in prognosis and clinical presentation suggest fundamental differences in biology and potentially different therapeutic implications . 
 methods thirty - five dsrct tumors were assessed using next - generation sequencing , protein expression ( immunohistochemistry ) , and gene amplification ( chromogenic in situ hybridization or fluorescence in situ hybridization )  . 
independent analysis by rna sequencing confirmed higher ar expression from an independent data set of ews - wt1 fusionpositive dsrcts compared with ewing sarcoma and a pan - cancer analysis . 
dsrcts had somatic mutations that were identified in tp53 and foxo3 , averaged five mutations per megabase , and no programmed death - ligand 1 expression was detected in any dsrct samples . 
2018 by american society of clinical oncology ajaz bulbul john paul shen joanne xiu pablo tamayo hatim husain author affiliations and support information ( if applicable ) appear at the end of this article . corresponding author : hatim husain , md , university of california san diego , 3855 health sciences #3011 , la jolla , ca 92093 ; e - mail : hhusain@ucsd.edu. introduction desmoplastic small round blue - cell tumors ( dsrcts ) originate from a cell with multilineage potential and are defined by a molecular hallmark with ews - wt1 reciprocal translocation.1 dsrcts are rare , highly aggressive mesenchymal tumors , with only 200 to 450 affected patients with the disease described to date.1 , 2 ewing sarcoma and dsrcts are treated in a similar manner in the clinic ; however , dsrcts have been represented in limited numbers in sarcoma studies . 
the clinical presentations of these diseases are different , with important comparisons being the site of disease presentation and poor prognosis in dsrct groups . despite aggressive therapy , median survival ranges from 17 to 25 months , 2 , 3 and the 5 - year survival rate remains around 15%3 with higher survival reported among those who underwent removal of at least 90% of the tumor with an absence of extraperitoneal metastasis.4 almost all of these tumors contain the t ( 11 ; 22 ) ( p13 ; q12 ) translocation that fuses ews with wt1 leading to production of a chimeric protein with transcriptional regulatory activity . 
 and pelvic cavity of young adult men in 88% to 97% of cases , 2 , 5 whereas ewing sarcoma commonly occurs in the axial skeleton.7 despite similar histologic morphology , differences in disease presentation and prognosis likely reflect the impact of different biologic factors , including differences in the transcriptional effects of the ews - wt1 fusion relative to the ews - fli1 fusion observed in ewing sarcoma . although there is no standard clinical management strategy for dsrct , treatment usually includes neoadjuvant high - dose cyclophosphamide , doxorubicin , and vincristine , alternating with ifosfamide and etoposide chemotherapy combined with aggressively attempted r0 resection.4 , 8 , 9 recent accelerated us food and drug administration ( fda ) approval in october 2016 of the compound olaratumab , a platelet - derived growth factor receptor a inhibitor , was based on a highly significant improvement in overall survival of 26.5 months in soft tissue sarcomas of histologic subtypes for which an anthracycline containing regimen may be appropriate . 
however , only one patient in the phase ii olaratumab study had round blue - cell sarcoma , and that patient was negative for the ews translocation.10 patients with dsrcts are poorly represented in clinical trials as a result of the rare nature of the disease . 
a list of ongoing clinical trials in this disease is provided in the data supplement . the aim of the current study was to understand the molecular characteristics of dsrcts by analyzing 35 patients with molecularly and immunohistochemically profiled tumors and to compare these with ewing sarcoma to explore therapeutic options and potential oncogenic vulnerabilities for this extremely rare and aggressive cancer type . 
dna from formalin fixed , paraffin - embedded samples was sequenced using the nextseq ( 592 genes selected on the basis of the cosmic database ; agilent sure select xt ; illumina , san diego , ca ) and miseq ( 47 genes ; truseq ) , to evaluate mutation and gene amplification . 
the full list of markers surveyed is available.11 twenty - four tumors were sequenced with the 45 - gene panel , and 11 tumors were sequenced with the 592 - gene panel . 
additional data for ar and epidermal growth factor receptor ( egfr ) ihc in other tumors , including prostate cancer , lung cancer , breast cancer , kidney cancer , endometrial cancer , and glioblastoma , were obtained from a larger cohort of 127 , 000 pan - cancer tumor samples analyzed . we used 2 tests for comparison , and statistical significance was determined as p < .05. 
 ( a ) enrichment scores ( single - sample gene set enrichment analysis ) of selected transcriptional signatures across alveolar rhabdomyosarcoma , desmoplastic small round blue cell tumors , ewing sarcoma , and synovial sarcoma . 
desmoplastic small round blue - cell tumor ( dsrct ) sarcomas are clearly overenriched in androgen receptor ( ar ) , epidermal growth factor receptor ( egfr ) , and ews - wt1 ( kts positive / negative ) signatures compared with those of other sarcomas . 
arms , alveolar rhabdomyosarcoma ; es , ewing sarcoma ; ss , synovial sarcoma . the degree to which the genes in a particular gene set are coordinately upregulated or downregulated within each sample . 
in this manner , ssgsea projects a single sample gene expression profile from the space of single genes onto the space of gene sets . the signature for ar is the hallmark_ androgen_response gene set from the molecular signatures database hallmark subcollection.13 the egfr signature corresponds to the egfr_up.v1_up / dn gene sets from the molecular signatures database subcollection c6.14 the ews - wt1 ( kts positive / negative ) signatures were generated from the geo data set gse53301.15 the ews - fli1 signature was made from the 20 genes listed in figure 1 from mendiola et al.16 transcriptional data sets the multiple sarcoma data set shown in figure 1a is a subset of the data set from filion et al.17 sarcoma samples in figure 1b are the dsrct subset from filion et al.17 prostate samples , shown in figure 1b , consisted of 22 primary prostate cancer samples from the geo data set gse32269.18 endometrial samples are 30 samples from the endometrial cancer data set from raeder et al.19 glioblastoma multiforme samples consisted of a subset of 30 samples from the glioblastoma multiforme data set from verhaak et al.20 results demographic data from the 35 patients whose tumors underwent clinical molecular testing are provided in the data supplement . 
 ( a ) thirty - five desmoplastic small round blue - cell tumors ( dsrcts ) went through molecular profiling using a commercial , multiplatform profiling service ( caris life sciences )  . 
specific tests performed included sequencing ( next - generation sequencing [ seq ] ) , protein expression ( immunohistochemistry [ ihc ] ) , and gene amplification ( chromogenic in situ hybridization or fluorescence in situ hybridization [ ish ] )  . 
 patients with ewing sarcoma had lung and bone involvement , which is consistent with the known distribution pattern of these tumors.21 in the 35 dsrcts , increased expression of top2a was observed in 63% of cases , topo1 in 63% , pten in 62% , ar in 59% , and egfr in 42% of tumors . 
increased expression of tubb3 was observed in 56% of tumors , mgmt in 55% , ts in 52% , rrm1 in 43% , and ercc1 in 24% ( fig 2a )  . 
comparing dsrcts with ewing sarcoma tumors , we found that both ar ( 59% v 3% ; p = 1.7e10 ) and tubb3 ( 56% v 29% ; p = .03 ) were expressed in significantly more dsrcts ( fig 2b )  . 
a full list of all of the patients with ihc staining is provided in the data supplement , which includes a description of staining thresholds . given that both the ar and egfr pathways can be effectively targeted with fda - approved drugs , we sought to further evaluate the activity of these pathways in dsrcts . 
we obtained gene expression profiles for 28 dsrcts as well as profiles for ewing sarcoma , alveolar rhabdomyosarcomas , and synovial sarcoma for comparison with other primitive sarcomas.17 performing ssgsea , we found that the majority of dsrcts was enriched for the ar signature , which indicated that these tumors coordinately upregulated genes that are known to be highly expressed when the ar pathway is activated ( fig 1a )  . 
as expected , ewing sarcoma tumors were enriched for the ews - fli1 signature , and dsrcts were enriched for the ews - wt1 signature , which is consistent with the translocation event pathognomonic for each tumor . to evaluate the significance of ar pathway activation in dsrcts , we made comparisons with tumors that are known to be driven by the ar pathway . 
compared with 59% ar expression in dsrct , ihc results indicated that ar expression was most frequent in prostate cancer ( 94% [ n = 1 , 340 tumors ] ) ; however , dsrct was greater than any other tumor type , including breast cancer ( 50% [ n = 1 , 962 tumors ] ) , kidney cancer ( 30% [ n = 40 tumors ] ) , and the pan - cancer analysis , which demonstrated < 30% expression ( n = 127 , 000 )  . 
a pan - cancer analysis of egfr expression demonstrated a percentage of expression ( 53% [ n > 20 , 000 ] ) that was similar to the 42% observed in dsrct . 
furthermore , tubb3 expression ( 51% [ n = 66 , 000 ] ) and top2a ( 69% [ n = 77 , 000 ] ) was similar to that observed in dsrct . pd - l1 expression ( sp142 ) was not detected in tumors tested ( zero of four ) , whereas programmed death 1 ( pd - 1 ) staining on tumor infiltrating lymphocytes ( tils ) was found in 25% of tumors tested ( two of eight )  . 
pd - l1 expression on tumor cells in ewing sarcoma was absent ( zero of 10 ) , and pd - 1 expression on tils was observed in 32% ( six of 19 ) of ewing sarcoma tumors ( fig 2 )  . 
tumor mutational load was calculated in 11 ewing sarcoma tumors , and mean tumor mutational load was five mutations per megabase ( range , three to eight mutations )  . next - generation sequencing revealed a synonymous tp53 g245g mutation and a foxo3 mutation ( l382fs ) in dscrt , whereas six tp53 mutations ( 13% ) , two apc mutations ( l1129s and i1307k ) , one brca1 ( c.301 + 1g > a ) , and one ctnnb1 ( t41a ) mutation were identified in ewing sarcoma . 
there was no copy number amplification identified in dsrct samples for cmet , her2 ( human epidermal growth factor receptor 2 ) , and egfr with in situ hybridization ( data supplement )  . discussion our understanding of the biology of dsrcts and their distinction from other sarcomas is evolving . 
 apart from the canonical ews - wt1 fusion , some major differences between dsrct and ewing sarcoma reflect those between ar and egfr expression . significantly higher ar expression in dsrcts compared with ewing sarcoma tumors suggest the possibility of a mechanistic link between ews - wt1 activation and androgen receptor expression . 
various ews - wt1 proteins regulate the promoter activity of egr - 1 , a zinc finger transcription factor that contributes to ar pathway activation.25 , 26 egr - 1 knockdown by small interfering rna inhibited activity of the ar signaling pathway.27 evidence for ar activation in dsrcts was observed by ihc as well as the coordinate regulation of the ar - responsive transcriptome . 
the degree of ar enrichment in dsrcts was higher than that of many other tumors , and additional work is needed to understand the clinical sensitivity of these tumors to ar inhibition . fda - approved novel ar inhibitors , including enzalutamide , and cyp17a1 inhibitors , including abiraterone , will need to be explored and provide a potential opportunity for continued drug expansion in this space . 
the three patients who had benefit from complete androgen blockade ( cab ) had normal testosterone levels before cab , whereas the three who did not experience a response had baseline castrate levels of testosterone.28 in vitro studies that evaluated the functional status of ar using western blot analysis and the stimulation of growth by dihydrotestosterone in mtt calorimetric assays in 27 patient tumors suggested that ar in dsrcts is functional , and inhibition of basal and testosterone - stimulated growth by flutamide could be achieved.28 a phase ib study in prostate cancer has combined treatment with enzalutamide and docetaxel chemotherapy and has resulted in a 50% decline from baseline in prostate - specific antigen in 19 of 20 patients , and a phase ii study is ongoing in prostate cancer ( clinicaltrials.gov identifier : nct02685267 ) .29 given the poor outcomes found with conventional therapy , the role of combinatorial therapy with ar inhibitors alone or in combination with chemotherapy in dsrtc deserves additional clinical investigation . tumor mutational load may affect response rates to immunotherapy in melanoma and nonsmallcell lung cancers.30 unfortunately , single - agent antipd - 1 antibodies , to date , have had limited efficacy across sarcomas , and an ongoing phase ii study ( sarc028 ) is evaluating the role of pembrolizumab across various sarcoma histologies ( clinicaltrials.gov identifier : nct02301039 ) .31 none of the patients in our cohort had identifiable tumoral pd - l1 expression by sp142 antibody testing , and the significance of pd - 1 positive tils is unclear at this time . in conclusion , there are distinguishing molecular characteristics between dsrcts and ewing sarcoma . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . ajaz bulbul consulting or advisory role : pfizer , astrazeneca travel , accommodations , expenses : pfizer , astrazeneca joanne xiu employment : caris life sciences pablo tamayo no relationship to disclose hatim husain consulting or advisory role : astrazeneca , abbvie , foundation medicine speakers ' bureau : astrazeneca , merck , bristol - myers squibb research funding : pfizer ( inst ) travel , accommodations , expenses : astrazeneca , merck , bristol - myers squibb , foundation medicine , abbvie affiliations ajaz bulbul , john paul shen , pablo tamayo , and hatim husain , university of california san diego , la jolla , ca ; and joanne xiu , caris life sciences , phoenix , az . funded by university of california san diego moores cancer center institutional funds and national institutes of health grants no . 
honor c , amroun k , vilcot l , et al : abdominal desmoplastic small round cell tumor : multimodal treatment combining chemotherapy , surgery , and radiotherapy is the best option . 
tap wd , jones rl , van tine ba , et al : olaratumab and doxorubicin versus doxorubicin alone for treatment of soft - tissue sarcoma : an open - label phase 1b and randomised phase 2 trial . 
creighton cj , hilger am , murthy s , et al : activation of mitogen - activated protein kinase in estrogen receptor alpha - positive breast cancer cells in vitro induces an in vivo molecular phenotype of estrogen receptor alpha - negative human breast tumors . 
mendiola m , carrillo j , garca e , et al : the orphan nuclear receptor dax1 is up - regulated by the ews / fli1 oncoprotein and is highly expressed in ewing tumors . 
raeder mb , birkeland e , trovik j , et al : integrated genomic analysis of the 8q24 amplification in endometrial cancers identifies atad2 as essential to myc - dependent cancers . 
verhaak rg , hoadley ka , purdom e , et al : integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in pdgfra , idh1 , egfr , and nf1 . 
gerald wl , rosai j , ladanyi m : characterization of the genomic breakpoint and chimeric transcripts in the ews - wt1 gene fusion of desmoplastic small round cell tumor . 
sukhatme vp , cao xm , chang lc , et al : a zinc finger - encoding gene coregulated with c - fos during growth and differentiation , and after cellular depolarization . 
fine rl , shah ss , moulton ta , et al : androgen and c - kit receptors in desmoplastic small round cell tumors resistant to chemotherapy : novel targets for therapy . 
morris mj , rathkopf de , novotny w , et al : phase ib study of enzalutamide in combination with docetaxel in men with metastatic castration - resistant prostate cancer . 
burgess ma , crowley j , reinke dk , et al : sarc 028 : a phase ii study of the anti - pd1 antibody pembrolizumab ( p ) in patients ( pts ) with advanced sarcomas . 
 eleven month progressionfree survival on vemurafenib monotherapy in a patient with recurrent and metastatic braf v600emutated glioblastoma who grade 4 introduction glioblastoma multiforme ( gbm ) accounts for 3% to 4% of all cancer - related deaths , despite having an average incidence rate of 3.19 / 100 , 000 population.1 , 2 prognosis for gbm remains poor , with standard - of - care therapy providing only a 16to 20 - month median overall survival.3 , 4 braf kinase inhibitors have transformed melanoma treatment and have successfully been used in other tumor types containing the braf v600e mutation.5 - 8 there are several reports of improved survival with the use of braf - directed therapy in braf - positive gbm , although the efficacy of braf inhibitors in nonmelanoma cancers has not been systematically explored . 
a basket study of 122 patients with braf v600e mutations in various nonmelanoma cancer types found three out of six patients with gbm experienced tumor regression with vemurafenib.8 a pediatric case series presented three pediatric patients with braf v600e - mutant high - grade gliomas treated with vemurafenib , including a patient with clinical and neurologic improvement 20 months after starting treatment.9 robinson et al10 reported a case of complete clinical regression of a braf v600emutant pediatric gbm treated with braf inhibitor therapy . 
the patient underwent a craniotomy with resection , and pathology demonstrated glioblastoma , who grade 4 , isocitrate dehydrogenase ( idh - 1 ; wild - type immunohistochemistry and by sequencing )  . 
further analysis showed a tert mutation and wildtype for both atrx and egfr . immediate postoperative imaging showed residual enhancement superior to the resection cavity , treated with gamma knife radiosurgery . 
subsequent treatment consisted of fractionated intensitymodulated radiation therapy of 60 gy at 2 gy per fraction with concurrent daily temozolomide 75 mg / m2 , followed by monthly cycles of maintenance temozolomide . 
her treatment was then switched to bevacizumab monotherapy . eleven months postsurgery , or 4 months after switching to bevacizumab monotherapy , she was found to have a right hemispheric focus of duralbased enhancement concerning for local recurrence and underwent repeat gamma knife radiosurgery . 
within 2 months of starting vemurafenib , she was weaned off all opioid medications , dexamethasone , baclofen , and gabapentshe reported a significant improvement in her quality of life and was once again able to work with physical therapy to improve her chronic left - sided weakness . 
five months after starting vemurafenib , her brain and spine mri demonstrated significant improvement in intracranial leptomeningeal disease and in the nodular leptomeningeal enhancement along the conus medullaris and multiple nerve roots of the cauda equina . 
eleven months after starting vemurafenib monotherapy , the patient reported feeling well overall and had achieved stable disease on brain and spine mri ( fig 3 )  . she denied any headaches , visual changes , back pain or neuropathy , or gi or genitourinary symptoms . 
she was rated 80% on the karnofsky performance status scale . discussion this case report is consistent with several others in showing significantly improved survival with the use of braf kinase inhibitors in braf v600e positive gbm . 
no epithelioid or rhabdoid morphology was identified . spine , which demonstrated drop metastases throughout the thoracic and lumbosacral spine , specifically on the posterior cord from t10 - 12 , anterior to the conus medullaris , and throughout the cauda equina . 
an mri of the spine 1 month later showed a significant increase in the number and size of the abnormal cord enhancement lesions . one month after being diagnosed with metastatic recurrent glioblastoma , the patient was referred to our clinic , and we recommended addition of carboplatin area under the curve 4 intravenously monthly to her regimen of bevacizumab 10 mg / kg intravenously every 2 weeks . 
at the time of vemurafenib approval 2 months later and before starting the braf inhibitor , neuraxis imaging demonstrated development of leptomeningeal enhancement around the lobe resection cavity and initial right parietal considerable progression of metastatic spinal cord disease . 
 plasma concentration ratio for dabrafenib was 10 - fold higher compared with vemurafenib.17 in the dabrafenib plus trametinib versus vemurafenib alone in unresectable or metastatic braf v600e / k cutaneous melanoma ( combi - v ) trial , patients with braf - mutated metastatic melanomas who were treated with the combination of dabrafenib and mek inhibitor trametinib had significantly longer pfs than those treated with vemurafenib monotherapy.18 furthermore , dabrafenib appears to be less nephrotoxic than vemurafenib.19 nonmelanoma tumorspecific studies of braf inhibitors are difficult to conduct because of the relative rarity of these tumors and the low frequencies of braf mutations . 
these limitations also extend to basket trials of braf inhibitors , such as the national cancer institutemolecular analysis for therapy choice ( nci - match ; eay131 - h protocol ) evaluating dabrafenib and trametinib in patients with braf v600e or v600k mutations . 
despite these limitations , case reports of exceptional responders to targeted therapies such as ours support the value of genetics - guided therapy for activating braf mutations and driver mutations in general in gbm and other rare cancers , underscoring the need to systematically investigate these targeted drugs . the case reports can also be of value in supporting current off - label therapy of these rare mutations in tumor types not listed in the us food and drug administrationapproved indications . 
this microscopic image shows an immunohistochemical study for braf v600e . the tumor cells and their processes are positive for the mutated form of the protein . although there is a therapeutic potential of braf inhibitors in gbm , the experience with these drugs in melanoma treatment suggests that resistance will surely emerge in tumors responsive to this therapy . several mechanisms mediating resistance to braf inhibitors through mitogen - activated protein kinase ( mapk ) reactivation have been described , and coadministration of braf inhibitors with selective mapk inhibitors has led to significant improvement in progression - free survival in melanoma.15 coadministration with alternative survival pathway inhibitors , such as vascular endothelial growth factor and phosphatidylinositol 3 - kinase inhibitors , may also need to be evaluated.16 it could be further argued that dabrafenib might be the preferred braf kinase inhibitor . 
tran honoraria : novocure , monteris medical consulting or advisory role : novocure research funding : novocure , merck , novartis , tocagen , northwest biotherapeutics , vbl therapeutics , stemline therapeutics patents , royalties , other intellectual property : a molecular cell diary system , chemotherapeutic methods to target differentiated tumor cells , generep and nscore : method and apparatus for improved determination of node influence in a network , core master regulators of glioblastoma stem cells , conversion of glioblastoma multiforme cells into regulatory immune cells , novel targets that prevent cancer cells from evading the immune system and synergize with current immunotherapies travel , accommodations , expenses : novocure , monteris medical , novartis , tocagen , vbl therapeutics ashley p . 
 ( b ) the same regions are denoted by arrows on follow - up imaging performed 8 months later , showing improvement in metastatic disease and near - complete resolution of some lesions . references med 6 : 1359 - 1370 , 2014 1 . 
stupp r , mason wp , van den bent mj , et al : radiotherapy plus concomitant and adjuvant temozolomide for epidemiol biomarkers prev 23 : 1985 - 1996 , 2014 glioblastoma . 
stupp r , taillibert s , kanner aa , et al : maintenance therapy with tumor - treating fields plus temozolomide vs temozolomide alone for glioblastoma : a randomized clinical trial . 
myall nj , neal jw , cho - phan cd , et al : long - term survival of a patient with nonsmall - cell lung cancer harboring a v600e mutation in the braf oncogene . 
hyman dm , puzanov i , subbiah v , et al : vemurafenib in multiple nonmelanoma cancers with braf v600 mutations . n engl j med 373 : 726 - 736 , 2015 9 . 
schindler g , capper d , meyer j , et al : analysis of braf v600e mutation in 1 , 320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma , ganglioglioma and extra - cerebellar pilocytic astrocytoma . acta neuropathol 121 : 397 - 405 , 2011 14 . 
jang s , atkins mb : which drug , and when , for patients with braf - mutant melanoma ? lancet oncol 14 : e60 - e69 , 2013 17 . 
kieran mw , cohen kj , doz f , et al : complete radiographic responses in pediatric patients with brafv600 - positive tumors including high - grade gliomas : preliminary results of an ongoing phase 1 / 2a safety and pharmacokinetics ( pk ) study of dabrafenib . 
grob jj , amonkar mm , karaszewska b , et al : comparison of dabrafenib and trametinib combination therapy with vemurafenib monotherapy on health - related quality of life in patients with unresectable or metastatic cutaneous braf val600 - mutation - positive melanoma ( combi - v ) : results of a phase 3 , open - label , randomised trial . 
morphology and ow cytometry phenotype of the bone marrow and peripheral blood were consistent with a diagnosis of t - cell acute lymphoblastic leukemia ( t - all )  . 
targeted next - generation sequencing ( ngs ) based mutational proling ( foundation medicine , cambridge , ma ) of the bone marrow specimen demonstrated the following pathogenic molecular events : mtorc1483y , mtort1977k , nrasg12d , ptenr234 * ( 29base pair frameshift , subclonal ) , loss of p16ink4a , loss of exons 2 and 3 from p14arf , and phf6r116 * ( asterisk denotes a nonsense mutation )  . 
written informed consent for the study described herein was obtained from the patient . induction chemotherapy using a pediatric - type approach was initiated per the cancer and leukemia group b 10403 protocol.1 the patient achieved complete cytomorphologic remission as demonstrated on the day 28 bone marrow assessment . 
bone marrow biopsy performed on day 32 demonstrated continued morphologic and cytogenetic remission with full donor chimerism . at a clinic visit 11 days after discharge , the patient complained of progressive dyspnea and cough . 
there was no morphologic evidence of t - all in the biopsy sample . fluorescence in situ hybridization studies performed on tissue touch imprints with representative neoplastic hs cells demonstrated a male karyotype and were positive for the tcr , myc , and tcr rearrangements detected at t - all diagnosis ( figs 1e and 1f )  . molecular studies performed on hs parafn tissue blocks were also positive for tcr gene rearrangements , as described earlier . diagnostic t - all and hs lung biopsy specimens were submitted for ngs to foundation medicine , a clinical laboratory improvement amendmentscertied laboratory service . 
foundationone heme ( foundation medicine ) is a combination dnaand rna - sequencing assay using a hybrid - capture ngs technology with the capability to detect base substitutions , insertions and deletions , copy number variants , and select fusions from a panel of more than 400 protein - coding genes , 250 rna transcripts , and 30 selected introns thought to play a role in hematologic malignancies . 
positive immunohistochemistry for ( c ) cd163 and ( d ) cd68 conrms histiocytic lineage . ( e ) fluorescence in situ hybridization studies performed on tissue touch imprints show ghost outline for the large neoplastic histiocytes . 
 ( f ) breakapart probes ( cytocell , tarrytown , ny ) identied recurrent rearrangements of the myc locus at 8q24 and the tcr locus at 7q34 in 112 of 200 cells analyzed , identical to those present at the time of t - cell actute lymphoblastic leukemia diagnosis . 
tmb is reported to be low if a level of 5 mutations per megabase ( muts / mb ) or lower is detected , intermediate if a level of 6 to 19 muts / mb is detected , and high if a level of 20 muts / mb or greater is detected . 
the biopsy sample tested negative for brafv600e mutation.2 the patient was treated with a chemotherapy regimen of ifosfamide , carboplatin , and etoposide.3 bone marrow assessment before therapy initiation showed full donor chimerism and was negative for t - all and hs . 
cycle 2 was started on day 20 , with the positron emission tomographyct scan at this time demonstrating intensively positron emission tomographyavid lesions throughout both lungs ( fig 2a )  . 
therefore , we chose to treat the patient with ipilimumab because he presented with predominantly extramedullary relapsed disease and did not respond to a standard chemotherapeutic regimen aimed at hs . 
in a 2016 series of 28 patients with relapsed hematologic malignancies after allogeneic transplantation , ipilimumab was particularly active in patients with acute leukemia with extramedullary relapse.4 in addition , recent analysis of tmb across the spectrum of histiocytic neoplasms by goyal et al5 found that hs harbors a higher tmb than the other tumor types examined and that patients with hs would likely benet from further investigation into treatment with checkpoint inhibitor therapies . 
repeat imaging after one dose of ipilimumab showed substantial reduction of disease burden ( fig 2b ) , but shortly thereafter , the patient presented with a total - body rash that was ultimately proven to be grade 4 cutaneous graft - versus - host disease ( gvhd )  . although ngs - based mutational proling of the hs specimen had not revealed immediately targetable molecular lesions , mutations in nras may portend sensitivity to map kinase pathway inhibitors through activation of downstream signaling.6 a previous report of a patient with atypical chronic myeloid leukemia containing nrasg12d exhibited a durable response to trametinib7 ( a mek inhibitor )  . 
gounder et al8 have also reported on the successful use of trametinib in a patient presenting with hs ( and previous follicular lymphoma ) harboring an activating mutation in map2k1 . 
a recent analysis of the us national cancer institute seer database uncovered 159 reported patients with hs in the period between 2000 and 2014.9 although hs can arise sporadically , a considerable proportion of hss occur in patients with previous or synchronous hematopoietic malignancies and have been found to harbor identical bor t - cell receptor rearrangements as those observed in the associated tumors . 
 ( a ) maximumintensity coronal and fused axial projections from positron emission tomography ( pet ) and computed tomography ( ct ) after one cycle of ifosfamide , carboplatin , and etoposide chemotherapy show progressive disease burden with several pet - avid foci and malignant rightsided pleural effusion . 
in the case of this patient , such explanations would include dedifferentiation of t - all to an uncommitted stem cell that subsequently differentiates in a myeloid - biased fashion ; residual neoplastic cells responding to intrinsic or extrinsic inuences with changes in transcription factor levels , cytokine signaling , or epigenetic modication that mediate transdifferentiation to hs ; and the presence of a shared clonal precursor giving rise to both tumors ( appendix fig a2 )  . because we conducted longitudinal ngs - based analysis , the patients clinical course can be understood by deconstructing the molecular evidence available . 
two of the seven molecular lesions identied in our patients diagnostic t - all bone marrow specimen , specically the loss of p16ink4a and loss of exons 2 and 3 from p14arf , were not present in the post - transplantation hs specimen , which was resequenced by ngs using the same assay . 
consistent with a previous report by brunner et al12 of hs arising in the context of this makes hypotheses based on transdifferentiation or dedifferentiation or redifferentiation mechanisms improbable , because such genomic losses are considered irreversible . follicular lymphoma , the cdkn2a gene encodes for tumor suppressor proteins p16ink4a and p14arf through use of different open reading frames and alternative splice isoforms . 
specically , using a mouse model , they demonstrated that normal ink4a / arf and pten tumor suppressor function is required to preserve homeostasis in hematopoietic compartments and constrain emergence of hs . 
mutational proling of our patients hs specimen did not reveal genetic deletions in the cdkn2a locus , which had been detected in the diagnostic t - all sample , as might be suspected by the ndings of carrasco et al.13 however , their analysis of human hss revealed that alternative mechanisms , such as hypermethylation of the p16ink4a and p14arf promoters , might also develop , mediating loss of tumor suppressor function and subsequent development of hs in cooperation with pten inactivation . ngs - based mutational proling from the t - all diagnostic specimen and the specimen of the hs that presented after transplantation was also notable for the presence of phf6116 *  . 
recent analysis has uncovered that phf6 mutations are early events in t - all leukemogenesis and that phf6 loss propagates transcriptional networks associated with leukemia stem - cell activity.14 this allows for the construction of a disease progression model whereby enhanced expression of phf6116 * promoted the divergence of t - all from a bipotential neoplastic progenitor population that also gave rise to hs . 
given the acquisition of additional genetic events in the t - all genome , it is possible that our patients t - all branched from the malignant precursor later than the hs but clinically presented rst.15 because establishing the diagnosis of hs is difcult , a history of lymphoid neoplasm should warrant suspicion on the part of the clinician . 
although much of the literature on the disease is limited to case reports , hs is an exceedingly rare neoplasm with a rapid clinical course and no standard treatment options . 
grifths , md , department of medicine , roswell park comprehensive cancer center , elm and carlton sts , buffalo , ny ; twitter : @roswellpark ; e - mail : elizabeth.grifths@roswellpark.org. sheila sait speakers bureau : celgene relationships are self - held unless noted . 
grifths honoraria : novartis , abbvie consulting or advisory role : celgene , boston scientic , persimmune , new link genetics , otsuka , alexion pharmaceuticals , partner therapeutics research funding : genentech ( inst ) , celgene ( inst ) , astex pharmaceuticals ( inst ) , apellis pharmaceuticals ( inst ) , astex pharmaceuticals ( inst ) , celldex therapeutics ( inst ) no other potential conicts of interest were reported . acknowledgment we thank eunice wang , md ; james thompson , md ; evelena ontiveros , md , phd ; philip mccarthy , md ; and maureen ross , md , phd , for providing clinical care and wish to dedicate this article to the memory of the patient described herein . references advani as , sanford b , luger s , et al : frontline - treatment of acute lymphoblastic leukemia ( all ) in older adolescents and young adults ( aya ) using a pediatric regimen is feasible : toxicity results of the prospective us intergroup trial c10403 ( alliance )  . 
moskowitz ch , bertino jr , glassman jr , et al : ifosfamide , carboplatin , and etoposide : a highly effective cytoreduction and peripheral - blood progenitor - cell mobilization regimen for transplant - eligible patients with non - hodgkins lymphoma . 
j clin oncol 17 : 3776 - 3785 , 1999 davids ms , kim ht , bachireddy p , et al : ipilimumab for patients with relapse after allogeneic transplantation . 
hematol oncol clin north am 31 : 705 - 719 , 2017 khanna v , pierce st , dao kh , et al : durable disease control with mek inhibition in a patient with nras - mutated atypical chronic myeloid leukemia . 
n engl j med 378 : 1945 - 1947 , 2018 kommalapati a , tella sh , durkin m , et al : histiocytic sarcoma : a population - based analysis of incidence , demographic disparities , and long - term outcomes . blood 131 : 265 - 268 , 2018 10 . 
feldman al , arber da , pittaluga s , et al : clonally related follicular lymphomas and histiocytic / dendritic cell sarcomas : evidence for transdifferentiation of the follicular lymphoma clone . 
huang w , qiu t , zeng l , et al : high frequency of clonal ig and t - cell receptor gene rearrangements in histiocytic and dendritic cell neoplasms . 
carrasco dr , fenton t , sukhdeo k , et al : the pten and ink4a / arf tumor suppressors maintain myelolymphoid homeostasis and cooperate to constrain histiocytic sarcoma development in humans . 
wendorff aa , quinn sa , rashkovan m , et al : phf6 loss enhances hsc self - renewal driving tumor initiation and leukemia stem cell activity in t - all . 
computed tomography ( ct ) scan at the time of presentation to oncology care demonstrates ( a ) a 20 15 cm mass within the anterior mediastinum and ( b ) splenomegaly . 
 roloff et al common progenitor leukemogenesis hs oncogenesis nras g12d pten r234fs phf6 r116 * mtor c1483y , t1977k cdkn2a ( p16 ink4a loss ) p14 arf ( loss of exons 2 - 3 ) tcell acute lymphoblastic leukemia transdifferentiation mtor c1483y , t1977k cdkn2a ( p16 ink4a loss ) p14arf ( loss of exons 2 - 3 ) nras g12d pten r234fs phf6 r116 * histiocytic sarcoma fig a2 . 
the schematic depicts potential routes for stepwise oncogenesis of t - cell acute lymphoblastic leukemia ( t - all ) and histiocytic sarcoma ( hs ) from a common malignant precursor cell . 
shared mutational composition in nras , pten , and phf6 likely arose in the progenitor cell , whereas the acquisition of mutations in mtor and cdkn2a ( p16ink4a / p14arf losses ) occurred privately in t - all . 
possible transdifferentiation events showing the development of hs from t - all and vice versa are also depicted but are unlikely given the irreversibility of genomic losses in p16ink4a and p14arf ( dashed line )  . 
 prospective genomic profiling of prostate cancer across disease states reveals germline and somatic alterations that may affect clinical decision making see accompanying editorial doi : purpose a long natural history and a predominant osseous pattern of metastatic spread are impediments to the adoption of precision medicine in patients with prostate cancer . 
to establish the feasibility of clinical genomic profiling in this disease , we performed targeted deep sequencing of tumor and normal dna from patients with locoregional , metastatic noncastrate , and metastatic castration - resistant prostate cancer . patients and methods patients consented to genomic analysis of their tumor and germline dna . a hybridization capture - based clinical assay was used to identify single - nucleotide variations , small insertions and deletions , copy number alterations , and structural rearrangements in more than 300 cancer - related genes in tumors and matched normal blood . results we successfully sequenced 504 tumors from 451 patients with prostate cancer . 
twenty - seven percent of patients harbored a germline or a somatic alteration in a dna damage repair gene that may predict for response to poly ( adp - ribose ) polymerase inhibition . 
in contrast , comparative analysis across disease states revealed that apc alterations were enriched in metastatic tumors , whereas atm alterations were specifically enriched in castration - resistant prostate cancer . conclusion through genomic profiling of prostate tumors that represent the disease clinical spectrum , we identified a high frequency of potentially actionable alterations and possible drivers of disease initiation , metastasis , and castration resistance . 
2017 by american society of clinical oncology wassim abida joshua armenia anuradha gopalan ryan brennan michael walsh david barron daniel danila dana rathkopf michael morris susan slovin brigit mclaughlin kristen curtis david m . 
although molecular profiling is not yet considered a standard - of - care for patients with this disease , new evidence points to enhanced treatment response in specific molecular contexts , paving the way for therapy selection on the basis of tumor molecular characteristics . 
all tumors were reviewed by pathologists who specialize in genitourinary oncology for confirmation of malignant histology of prostatic origin . sequencing and analysis we used the msk - impact assay as previously described10 , 11 ( fig 1a )  . 
the assay was performed in a clinical laboratory improvement amendments certified laboratory and designed to robustly identify single - nucleotide variations , small insertions and deletions ( indels ) , somatic copy number alterations , and structural rearrangements in more than 300 cancerrelated genes in formalin - fixed and paraffinembedded tumors and matched normal blood . somatic variant analysis was performed as described , 10 , 11 with germline variants identified in matched blood samples filtered out in the somatic analysis process . 
somatic findings were reported in the electronic medical record and anonymized and uploaded to cbioportal for visualization and analysis.12 - 14 clonality of mutations was estimated as cancer cell fraction15 and implemented in the facets algorithm.16 beginning in may 2015 , patients were given the option to consent to analysis of germline variants that were identified via sequencing of normal blood samples . 
germline analysis of 76 known cancer - predisposing genes was performed as previously described.16a additional methods are provided in the data supplement . results targeted dna sequencing of tumornormal pairs from patients with prostate cancer using the msk - impact assay , we successfully profiled 504 tumors from 451 patients with prostate cancer who presented to the clinic ( fig 1a and data supplement )  . 
in total , 348 patients ( 77% ) had metastatic prostate cancer , 53 ( 12% ) had biochemical recurrence after definitive therapy , and 50 ( 11% ) had locoregional disease ( fig 1b )  . 
metastatic tumors that were successfully profiled were obtained from lymph node ( 45% ) , bone ( 22% ) , liver ( 14% ) , lung ( 5% ) , and other soft tissue sites ( 14% ; fig 1c )  . 
unlike the tcga and su2c - pcf studies , tumors that were profiled represented all three prostate cancer clinical states : locoregional , metastatic noncastrate , and metastatic castration resistant . we began with 746 biopsy / surgical samples to successfully sequence the 504 tumors reported above , with an overall success rate of 68% ( appendix fig a1 )  . 
 a tumor pathology review matched normal extraction library construction target gene capture somatic analysis ( snvs , indels , cnvs , fusions ) germline analysis ( with specific consent ) tumor report in emr genetics report in emr metastatic tumor site other soft tissue lung liver bone patient disease state ( highest known ; n = 451 patients ) locoregional biochemically recurrent metastatic sample type prostate metastasis disease state at time of tumor tissue collection ( n = 504 tumors ) locoregional locoregional locoregional metastatic noncastrate metastatic castration resistant fig 1 . 
their disease state at the time of tissue collection for the 504 tumors that were profiled is represented at bottotumors that were profiled represented all three prostate cancer clinical disease states : locoregional , metastatic noncastrate , and metastatic castration resistant . 
cnv , copy number variation ; emr , electronic medical record , msk - impact , memorial sloan kettering - integrated mutation profiling of actionable cancer targets ; snv , single - nucleotide variation . states and are shown in fig 2 , grouped in pathways that are potentially clinically actionable . prospectively acquired primary tcga tumors ( appendix fig a3 )  . overall , the frequency of alterations in genes of interest in prostate cancer was similar for mcrpc tumors that were profiled in the msk - impact and su2c - pcf mcrpc data sets4 ( appendix fig a2 ) but demonstrated now in a clinical practice setting . 
however , notable differences were observed when comparing primary localized tumors in the msk - impact data set with those that were profiled in the prospective tcga study , including a higher frequency of alterations in tp53 and foxa1 in msk - impact tumors ( appendix fig a3 )  . 
this is most likely a result of the more aggressive nature of the primary localized tumors in the msk - impact cohort , which were predominantly obtained from patients who subsequently developed biochemically recurrent and metastatic disease ( fig 1b ) relative to the in total , 24% of patients carried somatic alterations in the pi3k / akt pathway , including in pten , pik3ca , pik3cb , pik3r1 , akt1 , and akt3 ( appendix fig a4 )  . 
alterations in commonly affected genes in prostate cancer ( eg , ar , pten , tp53 , foxa1 ) are shown in addition to genes in potentially actionable or biologically relevant pathways . 
mapk , mitogen - activated protein kinase ; msk - impact , memorial sloan ketteringintegrated mutation profiling of actionable cancer targets ; pi3k , phosphatidylinositol - 3kinase . homologous recombination , including brca2 , brca1 , atm , fanca , rad50 , palb2 , and cdk1217 - 21 ( fig 3a and appendix fig a7 )  . a recent multi - institutional study identified a high frequency of ddr gene alterations in the germline of patients with advanced prostate cancer.22 of patients in our data set , 221 underwent formal germline analysis , the first 124 of whom were included in the previously reported study.22 of these 221 patients , 42 ( 19% of total ) had a known or likely pathogenic germline mutation in brca2 ( 9% of total ) , chek2 ( 4% ) , atm ( 2% ) , brca1 ( 1% ) , fh ( 1% ) , and pms2 , nbn , palb2 , and brip1 ( , 1% each ; fig 3b and appendix table a1 )  . 
whereas germline ddr gene alterations may predict for response to parp inhibition or platinum agents , somatic - only alterations in these genes without a germline event may still predict for drug response.9 of the 221 patients who underwent germline analysis , 27% demonstrated alterations in brca2 , brca1 , atm , or chek2 , either in the germline or somatically ( fig 3c )  . 
of note , germline analysis alone accounted for approximately one half of these patients only , which suggests that both germline and somatic analysis should be performed to identify patients with ddr gene deficiency . in total , 3% of patients had tumors with somatic alterations in mismatch repair ( mmr ) genes msh2 , mlh1 , pms2 , or msh6 . 
these tumors accounted for the majority of samples with the highest mutation counts on msk - impact profiling ( fig 3d ) , which were confirmed to be enriched for previously described mmr and microsatellite instability signatures23 , 24 ( appendix fig a8 )  . 
identification of mmr - deficient prostate cancers may have immediate clinical applicability , given recent data that suggest sensitivity of such tumors to immune checkpoint blockade in colorectal cancer and other malignancies.25 - 27 overall , 36% of patients were found to have a potentially actionable alteration by using the oncokb annotation platform27a ( appendix fig a9 )  . this platform does not include non - brca / atm germline alterations , missense alterations of unknown significance , and genes whose clinical significance is less clear , including cdk12 and fanca . 
as genomic alterations undergofurther characterization and new trials and drug targets emerge , the frequency of alterations that are defined as actionable may increase . comparative analysis of somatic alterations across disease states a unique aspect of this data set is that it includes genetic profiles of tumors that represent all three clinical states : locoregional , metastatic noncastrate , and metastatic castration resistant . 
consistent with previous studies , 3 , 4 we identified recurrent areas of copy number loss that involved chromosomes 6q , 8p , 13q , and 16q , and areas of copy number gain that involved chromosomes 1q , 3q , 7 , 8q , and x ( fig 4 )  . 
 displayed the highest burden of copy number alterations , whereas those that represented locoregional disease displayed the lowest ( fig 4 and appendix fig a10 )  . aiming to identify possible genomic drivers of disease progression , we performed a selective enrichment analysis to identify genes that were more frequently altered in mcrpc compared with locoregional disease ( fig 5a )  . 
ar amplification / mutation was the most enriched alteration in mcrpc , as shown in previous studies.3 , 4 other genes that were more commonly altered in mcrpc included tp53 , rb1 , pten , apc , atm , fanca , and cdk12 . we performed a similar analysis that compared alterations in mcrpc with metastatic noncastrate prostate cancer ( fig 5b )  . 
the high enrichment of alterations in ar in mcrpc relative to both locoregional and metastatic non - castrate disease serves as a positive control , consistent with the known role of ar as a driver of castration resistance.28 - 31 beyond frequent amplification of the gene , ar antiandrogen resistance mutations were identified in tumors from patients with mcrpc ( appendix fig a11 ) , including an f877l enzalutamide / arn509 resistance mutation32 , 33 that was found in the tumor of a patient who experienced progression after 4 years of treatment on arn509 . 
four locoregional tumors were found to have mutations in ar , including an h875y mutation that is known to confer resistance to flutamide29 , 34 in a prostatectomy 16 17 18 19 20 21 22 disease state : locoregional metastatic noncastrate metastatic castration resistant sample type : prostate metastasis scna fig 4 . 
represented here are regions of amplification ( red ) or deletion ( blue ) with chromosomes listed horizontally ( top ) in the msk - impact , su2c - pcf ( metastatic castration - resistant prostate cancer [ crpc ] ) , and tcga ( primary localized prostate cancer ) data sets . 
msk - impact tumors are sorted by disease state at time of tissue acquisition , from locoregional ( bottom , gray ) to metastatic crpc ( top , blue )  . 
the level of enrichment is represented as difference in frequency between the two indicated classes ( x - axis ) and its significance ( p value , y - axis )  . 
amp , amplification ; homdel , homozygous deletion ; mut , mutation . sample from a patient who was treatment - nave but who had received dutasteride for benign prostate enlargement . in addition to ar , we again identified enrichment of alterations in tp53 , rb1 , pten , and atm in mcrpc compared with metastatic noncastrate disease . 
enrichment of these genes in mcrpc relative to both earlier disease states implicates them in the development of castration resistance , a finding that is of particular interest for atm , a gene that is involved in ddr . 
fanca and cdk12 , two other dna repair genes , did not show statistically significant mcrpc compared with metastatic noncastrate disease as they did versus locoregional disease , enrichment although there is a trend that suggests a role in castration resistance as well ( fig 5d )  . 
when analysis was limited to metastatic tumors or lymph nodes only , similar trends for enrichment were observed , although statistical significance was not always reached because of smaller sample size ( appendix fig a12 )  . only two genes were enriched in metastatic noncastrate versus locoregional disease : apc and , to a lesser extent , arid5a ( fig 5c )  . 
these findings will require functional validation in the laboratory . matched samples identify clonal alterations in prostate cancer in total , 44 patients had more than one tumor site profiled by msk - impact , including 16 with a matched primary localized tumor and a subsequent metastatic tumor . 
tumors from the same patient that were acquired at a later time point typically had a higher mutation count ( fig 6a )  . given the high frequency of tp53 alterations that were observed in primary localized tumors in this data set and prior reports of aggressive behavior of prostate tumors that harbor tp53 alterations , 36 - 37a we sought to determine whether tp53 alterations were present in tumors early in their evolution or whether they were acquired later at disease progression . 
tp53 mutations were clonal , including in cases in which both a primary localized tumor and a later metastasis were available ( cancer cell fraction > 0.9 in the later metastasis )  . 
likewise , we found that somatic alterations in brca2 were present in matched tumors , which again suggested that somatic brca2 loss of function alterations occur early in tumorigenesis for affected patients . conversely , alterations in ar did not occur early in matched samples ( fig 6b , patients p - 0003597 and p - 0004910 ) , which is consistent with treatment - related changes that promote castration resistance . 
of note , other potentially actionable alterations may arise later in disease evolution , as was the case for patient p - 0002149 ( fig 6b ) , who acquired an activating pik3ca e545k mutation ( appendix fig a4b ) in a recurrent tumor nearly 3 years after his radical prostatectomy . to confirm the above findings , we performed phylogenetic analysis on cases in which several matched tumors were available from the same patient ( fig 6c )  . 
for patient p - 0003511 , ar amplification was truncal castration - resistant tumors , which is consistent with its well - characterized role in castration resistance.28 as the number of patients with prostate cancer who are profiled longitudinally throughout their clinical care increases , such findings may provide insight into clonal driver events that promote disease progression and site - specific metastasis . discussion unlike previous prostate cancer genomic studies , we profiled tumors that represent the clinical spectrum of the disease , from locoregional to metastatic noncastrate and metastatic castrationresistant prostate cancer , which enabled comparisons of genomic landscape across disease states using a single assay . 
whereas locoregional tumors in this data set typically represented more aggressive disease than tcga , patients with such tumors are those in greatest need of new treatment approaches and profiling of their tumors may have particular clinical relevance . an increase in copy number alterations and mutation frequency was evident in mcrpc compared with earlier disease states , as was an increased frequency of alterations in ar , tp53 , rb1 , and pten . 
spop mutations , however , were more frequent in the earlier disease states , which suggested better outcomes for patients with spop - mutant tumors , possibly through increased sensitivity to adt . 
in this analysis , apc and atm emerged as candidate genes of interest that may independently contribute to metastasis and castration resistance , respectively , pending functional validation in the laboratory . 
these genes , like atm , are involved in ddr , alluding to a possible role for dna repair defects in the development of castration resistance . we also found that tp53 alterations are early clonal events in matched tumor samples from individual patients . 
a notable exception ( * ) involves a bladder metastasis and a bone metastasis acquired 5 months later , where the patient had received salvage radiation to the pelvis , possibly explaining the higher mutation count in the earlier bladder tumor . 
 ( b ) somatic alterations in tp53 ( red ) , brca2 ( blue ) , ar ( gold ) , and pik3ca ( green ) in matched tumors in the data set , including localized primaries and later metastases ( green box ) and other matched tumors from the same patients . 
crpc , castration - resistant prostate cancer . available , it will be possible to validate this finding , guiding more aggressive treatment approaches early on for these patients . overall , we identified potentially actionable alterations , including hotspot activating alterations in genes that are known drug targets , consistent with findings of the su2c mcrpc study , but this time in a prospective clinical practice setting . 
these findings have immediate therapeutic relevance , given the recently reported sensitivity of these tumors to parp inhibition9 or immune checkpoint blockade.25 the higher frequency of dna repair alterations that were identified via integrative germline and somatic analyses strongly argues for performing both germline and somatic genomic analyses in all patients with advanced prostate cancer who will require systemic treatment , irrespective of screening on the basis of family history . in summary , this study shows that a large genomic data set that represents the clinical spectrum of prostate cancer can provide mechanistic insight into possible genomic drivers of disease initiation , metastasis , and drug resistance . 
our ability to profile metastatic tumors allowed us to detect the evolution of potential driver alterations in matched tumors from individual patients , identifying alterations in tp53 and brca2 as early events that may confer a more aggressive phenotype . 
scher provision of study materials or patients : wassim abida , joshua armenia , daniel danila , michael morris , susan slovin , marc ladanyi , liying zhang , victor e . 
scher collection and assembly of data : wassim abida , joshua armenia , ryan brennan , michael walsh , david barron , daniel danila , michael morris , susan slovin , brigit mclaughlin , kristen curtis , david m . 
scher data analysis and interpretation : wassim abida , joshua armenia , anuradha gopalan , michael walsh , daniel danila , dana rathkopf , michael morris , susan slovin , jeremy c . 
robson honoraria : astrazeneca consulting or advisory role : mckesson , astrazeneca research funding : astrazeneca ( inst ) , abbvie ( inst ) , myriad genetics ( inst ) , biomarin ( inst ) , medivation ( inst ) , tesaro ( inst ) travel , accommodations , expenses : astrazeneca vijai joseph stock and other ownership interests : juno therapeutics daniel danila honoraria : astellas scientific , medical affairs , angle , bayer , xian - janssen pharmaceutical consulting or advisory role : angle , bayer research funding : prostate cancer foundation , genentech , janssen research & development ( inst ) patents , royalties , other intellectual property : gene expression profile associated with prostate cancer travel , accommodations , expenses : cambridge healthtech institute , prostate cancer foundation , angle , bayer , american austrian open medical institute , global technology community , xian - janssen pharmaceutical , oncology education dana rathkopf consulting or advisory role : janssen oncology research funding : janssen oncology ( inst ) , medivation ( inst ) , celgene ( inst ) , takeda ( inst ) , millennium pharmaceuticals ( inst ) , ferring ( inst ) , novartis ( inst ) , taiho pharmaceutical ( inst ) , astrazeneca ( inst ) , genentech ( inst ) , tracon pharma ( inst ) michael morris consulting or advisory role : astellas pharma , bayer , endocyte research funding : bayer ( inst ) , sanofi ( inst ) , endocyte ( inst ) , progenics ( inst ) travel , accommodations , expenses : bayer , endocyte susan slovin consulting or advisory role : bayer brigit mclaughlin no relationship to disclose kristen curtis no relationship to disclose david m . 
durack stock and other ownership interests : adient medical consulting or advisory role : adient medical patents , royalties , other intellectual property : patent disclosures , provisionals submitted for instrument to analyze biopsy specimens ( unrelated to the small renal masses topic )  . patent holder for device related to x - ray imaging data collection ( unrelated to small renal masses topic ) , july 2008 . other relationship : society of interventional radiology foundation stephen b . 
solomon leadership : devicor medical products stock and other ownership interests : johnson & johnson , progenics , aspire bariatrics consulting or advisory role : ge healthcare , angiodynamics , medtronic research funding : ge healthcare ( inst ) , angiodynamics ( inst ) patents , royalties , other intellectual property : ge healthcare - patents pending ( inst ) , aspire bariatrics ahmet zehir no relationship to disclose aijazuddin syed no relationship to disclose jianjiong gao no relationship to disclose debyani chakravarty no relationship to disclose hebert alberto vargas no relationship to disclose kenneth offit no relationship to disclose mark t.a. 
kantoff stock and other ownership interests : bellicum pharmaceuticals , metamark genetics , placon , druggablity technologies , tarveda consulting or advisory role : bavarian nordic , janssen , millennium pharmaceuticals , morphosys , pfizer , astellas pharma , bellicum pharmaceuticals , bind biosciences , endocyte , metamark genetics , medivation , merck , mtg therapeutics , oncocellmdx , oncogenex , sotio , sanofi , tokai pharmaceuticals , bayer , genentech , cristal therapeutics , ipsen , omnitura , gtx , tarveda , druggablity technologies , progenity research funding : medivation ( inst ) , sanofi ( inst ) , oncogenex ( inst ) , aragon pharmaceuticals ( inst ) , amgen ( inst ) , astellas pharma ( inst ) , bayer ( inst ) , bavarian nordic ( inst ) , dendreon ( inst ) , exelixis ( inst ) , janssen ( inst ) patents , royalties , other intellectual property : method for predicting the risk of prostate cancer morbidity and mortality , predicting and treating prostate cancer , methods for predicting likelihood of responding to treatment , chromosome copy number gain as a biomarker of urothelial carcinoma lethality , drug combinations to treat cancer , somatic ercc2 mutations correlate with cisplatin sensitivity in muscle - invasive urothelial carcinoma ( patent ) , up - to - date royalties , wolters - kluwer royalties expert testimony : sanofi , janssen travel , accommodations , expenses : sanofi , janssen , bind biosciences , bavarian nordic , millennium pharmaceuticals david b . 
sawyers leadership : novartis stock and other ownership interests : novartis , agios , blueprint medicines , beigene , oric pharmaceuticals consulting or advisory role : novartis , blueprint medicines , agios , beigene , oric pharmaceuticals patents , royalties , other intellectual property : xtandi nikolaus schultz no relationship to disclose howard i . 
grasso cs , wu ym , robinson dr , et al : the mutational landscape of lethal castration - resistant prostate cancer . 11 - 22 , 2010 nature 487 : 239 - 243 , 2012 7 . 
hyman dm , solit db , arcila me , et al : precision medicine at memorial sloan kettering cancer center : clinical next - generation sequencing enabling next - generation targeted therapy trials . 
cheng dt , mitchell t , zehir a , et al : msk - impact : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
joshi pm , sutor sl , huntoon cj , et al : ovarian cancer - associated mutations disable catalytic activity of cdk12 , a kinase that promotes homologous recombination repair and resistance to cisplatin and poly ( adp - ribose ) polymerase inhibitors . 
bajrami i , frankum jr , konde a , et al : genome - wide profiling of genetic synthetic lethality identifies cdk12 as a novel determinant of parp1 / 2 inhibitor sensitivity . 
mccabe n , turner nc , lord cj , et al : deficiency in the repair of dna damage by homologous recombination and sensitivity to poly ( adp - ribose ) polymerase inhibition . 
mskimpact versus su2cpcf dataset comparison . frequencies of alterations in select genes in metastatic crpc tumors from the msk - impact dataset ( orange ) versus the su2cpcf dataset ( red )  . 
mmr / msi mutation signatures in hypermutated tumors . k - means clustering of mutations / mb ( mut / mb ) across all 504 tumors identified two distinct clusters : cluster1 with mutations / mb10 . 
actionable alterations are ranked by level of evidence ( level 2b : standard of care biomarker predictive of response to an fda - approved drug in another indication , but not standard of care for this indication ; level 3a : compelling clinical evidence supports the biomarker as being predictive of response to a drug in this indication , but neither biomarker nor drug are standard of care ; level 3b : compelling clinical evidence supports the biomarker as being predictive of response to a drug in another indication , but neither biomarker nor drug are standard of care ; level 4 : compelling biological evidence supports the biomarker as being predictive of response to a drug , but neither biomarker nor drug are standard of care )  . 
variants were considered pathogenic or likely pathogenic per american college of medical genetics and genomics guidelines . somatic second hit is reported in column two for patients with germline dna damage repair gene mutations . 
of interest , several of the patients who harbored a germline brca2 c.5946delt ashkenazi founder mutation did not have a detectable somatic second hit in this gene , suggesting a nonclassical pathogenic mechanism for this mutation . 
 c acral lentiginous melanoma harboring a ros1 gene fusion with clinical response to entrectinib purpose ros1 gene fusions demonstrate oncogenic activity , and patients with nonsmall - cell lung cancer ( nsclc ) harboring a ros1 fusion benefit from the use of a ros1 inhibitor ; however , clinical response to ros1 inhibitors remains largely uncharacterized outside of nsclc . 
ros1 fusions have been identified in multiple tumor types but have not been reported in cutaneous melanoma . patients and methods tumors from 22 patients with acral lentiginous melanoma ( alm ) were analyzed with targeted rna sequencing to detect fusions in ros1 , ntrk1 , ntrk2 , ntrk3 , and alk genes . 
the patient underwent a dramatic and durable response to entrectinib , with a recist ( version 1.1 ) partial response of 238% at 3 months and 255% at 11 months . 
ohara ii seed grant program , an american cancer society institutional research grant to university of colorado cancer center , the amy davis foundation , and the moore family foundation . the university of colorado molecular tumor board ( at which patient case was discussed ) is supported in part by the colorado center for personalized medicine . clinical trial information : nct02097810 . corresponding author : kasey l . 
cdna was synthesized using the verso cdna kit ( thermo fisher scientific , waltham , ma ) , followed by pcr amplification using gotaq ( promega , madison , wi ) and gopcros1specific primers ( sense , 59 - agagcttgaggcaaacaa - 39 ; antisense , 59 - ccactgctgttccttcataca - 39 )  . 
purified pcr products were sequenced using the bigdye terminator cycle sequencing ready reaction kit ( version 3.1 ; applied biosystems , foster city , ca )  . clinical trial startrk - 1 ( studies of tumor alterations responsive to targeting receptor kinases ) is an ongoing multicenter phase i / iia open - label study of oral entrectinib ( rxdx - 101 ) in patients with locally advanced or metastatic cancer who have a confirmed ros1 , ntrk1 , ntrk2 , ntrk3 , or alk fusion . 
a fusion containing exons 1 to 4 of the gopc gene and exons 36 to 42 of the ros1 gene was detected in one patient ( table 1 ; fig 1a )  . 
expression of the gopc - ros1 fusion transcript was confirmed using reverse - transcriptase pcr followed by sanger sequencing ( fig 1b ) , and ihc of the tumor sample confirmed ros1 protein expression ( fig 1c )  . the patient with the gopc - ros1 fusion is a 46 - year - old man who was initially diagnosed with alm in 2010 . 
the patient then underwent amputation of the fifth toe with left groin sentinel lymph node biopsy followed by amputation of the fourth left toe and resection of a satellite nodule . 
because of high - risk disease , the patient underwent complete left inguinal lymph node dissection and received adjuvant biochemotherapy with cisplatin , vinblastine , dacarbazine , aldesleukin , and interferon alfa - 2b . approximately 1 year later , a recurrence was detected in the surgical scar , and this was reresected , followed later by recurrent in - transit metastases . mutation testing showed the tumor was wild type for braf and kit genes . 
 table 1 demographic and clinical characteristics of patients with aml in rna sequencing screen patient case no . age ( years ) stage location fusion detected * tissue left thumbnail bed sole , left foot plantar surface , right foot plantar surface , left foot right heel left heel left thumbnail right heel left thumb sole , left foot right heel right heel subcutaneous subcutaneous lymph node lymph node brain brain brain brain brain brain gopc - ros1 abbreviation : aml , acral lentiginous melanoma . * indicates no fusions were detected . patient with ros1 fusion responds to treatment with entrectinib given the ros1 fusion and the potential for response to targeted treatment , treatment options included off - label crizotinib , which is effective in ros1 - positive nsclc tumors , or a phase i clinical trial of the cns - penetrant ros1 / trk / alk inhibitor entrectinib . 
the patient was found eligible for this trial and provided written informed consent . at baseline , the patient had several palpable masses in the left leg , ranging from approximately 1 to 4 cm ( figs 2a to 2c ) , as well as a 12 - mm nodal mass in the paratracheal lymph node and a small right subpleural pulmonary nodule ( data not shown )  . the patient initiated entrectinib 600 mg orally once per day , and after 2 weeks , four of the five main lesions on the left leg were no longer palpable . 
staining was scored on a scale from 0 to 2 + , and all samples scored a 0 , except for two samples from one patient , which were scored 1 + and corresponded to the study patient described here ( table 2 )  . 
 ( b ) dna sequencing chromatogram of real - time polymerase chain reaction product confirms a gopc - ros1 fusion and validates the gopc exon 4 and ros1 exon 36 breakpoints . 
 ( c ) immunohistochemistry images ( magnification , 103 ) show tumor - specific staining for ros1 ( left ) in the patient tumor sample . he , hematoxylin and eosin . fig 2 target lesion responses to entrectinib at baseline ( column one ) , 3 months ( column two ) , and 11 months ( column three )  . ( a ) anterior proximal shin subcutaneous nodule ( yellow arrow )  . 
i = immediate family member , inst = my institution . for other cancer types where ros1 fusions have been detected , such as nsclc , but lower than frequencies reported for spitzoid neoplasms and spitzoid melanomas.8 , 17 we did not find any ros1 expressionpositive cases in 30 superficial spreading or nodular melanoma cases , consistent with the previous study conducted on 374 cutaneous melanomas.10 we also did not find any ros1 expressionpositive cases in the 18 mucosal melanomas ; however , the number of patients needed to estimate the true frequency likely needs to be much larger . 
although we found the overall ros1 fusion frequency to be 1.3% across all melanoma subtypes , the dramatic treatment response described in this report should prompt additional screening and studies of ros1 fusions in patients with melanoma . although entrectinib has shown activity against ros1 fusion tumors in nsclc , 18 this is the first report to our knowledge of entrectinib response against a ros1 fusion outside of lung cancer . entrectinib has previously demonstrated antitumor response in patients with tumors that are positive for alk or ntrk1 - 3 gene fusions in indications outside of nsclc , such as colorectal cancer , 19 , 20 astrocytoma , 18 and mammary analog secretory carcinoma.21 altogether , the tumor response observed in this patient with ros1 - positive melanoma is adding toagrowingbodyofliteraturesuggestinggenefusions in ros1 , ntrk1 - 3 , and alk may be generally responsive to their respective tyrosine kinase inhibitors , irrespective of the tumor histology context.18 , 22 - 24 published online on po.ascopubs.org on march 2 , 2017 . relationships may not relate to the subject matter of this manuscript . 
charest a , lane k , mcmahon k , et al : fusion of fig to the receptor tyrosine kinase ros in a glioblastoma with an interstitial del ( 6 ) ( q21q21 )  . 
birch ah , arcand sl , oros kk , et al : chromosome 3 anomalies investigated by genome wide snp analysis of benign , low malignant potential and low grade ovarian serous tumours . 
murphy da , ely ha , shoemaker r , et al : detecting gene rearrangements in patient populations through a 2 - step diagnostic test comprised of rapid ihc enrichment followed by sensitive next - generation sequencing . 
drilon a , de braud fg , siena s , et al : entrectinib , an oral pan - trk , ros1 , and alk inhibitor in tki - nave patients with advanced solid tumors harboring gene rearrangements : updated phase i results . 
sartore - bianchi a , ardini e , bosotti r , et al : sensitivity to entrectinib associated with a novel lmna - ntrk1 gene fusion in metastatic colorectal cancer . 
drilon a , li g , dogan s , et al : what hides behind the masc : clinical response and acquired resistance to entrectinib after etv6 - ntrk3 identification in a mammary analogue secretory carcinoma ( masc )  . 
hong ds , farago af , brose ms , et al : clinical safety and activity from a phase i study of loxo - 101 , a selective trka / b / c inhibitor , in solid - tumor patients with ntrk gene fusions . 
vassal g , faivre l , geoerger b , et al : crizotinib in children and adolescents with advanced ros1 , met , or alk - rearranged cancer : results of the acse phase ii trial . 
approximately 20% to 25% metastasize to distant sites and are generally associated with poor response to treatment.1 outcomes with systemic chemotherapy have historically been poor.2 with improved understanding of molecular biology of these tumors , we are beginning to identify promising potential therapeutic targets . in particular , the human epidermal growth factor receptor 2 / neu ( her2 / neu ) oncogene , one of the tyrosine kinase receptors , is overexpressed in salivary duct carcinomas ( 44% ) , salivary gland adenocarcinomas ( 21% ) , and adenoid cystic carcinomas ( 2% ) , among other subtypes.3 trastuzumab , a monoclonal antibody that binds to the extracellular domain of her2 / neu , inhibits the proliferation of tumor cells that overexpress her2 / neu.4 patients with her2 / neu - overexpressing sgns are often treated with trastuzumab - containing regimens with encouraging results , but disease progression is inevitable , even with a favorable initial response . 
the antibody - drug conjugate adotrastuzumab emtansine ( t - dm1 ) is currently approved for treating her2 - positive metastatic breast cancer that has progressed on therapy with trastuzumab . this antibody - drug conjugate selectively directs the cytotoxic drug emtansine to tumor cells with her2 / neu expression , thereby minimizing systemic toxicities.5 results favorable given the seen with adotrastuzumab emtansine in patients with breast cancer , it is logical to consider administering it to patients with sgns that overexpress her2 / neu after they have progressed on trastuzumab with or without chemotherapy . 
sequential her2 / neu - directed therapy has been successfully used with sustained clinical benet , albeit in a single patient.6 here we report on the clinical outcomes of seven patients with metastatic sgns who were treated at our institution with adotrastuzumab emtansine ( table 1 ) , and we summarize the clinical course and response to therapy of one patient . a 45 - year - old man presented with a painless right neck mass in late 2013 . 
fine - needle aspiration revealed a high - grade carcinoma and positron emission tomography / ct ( pet / ct ) showed multiple uorodeoxyglucose ( fdg ) - avid lymph nodes in the right cervical region . 
surgical pathology revealed a 3.9 - cm high - grade salivary duct carcinoma that was positive for androgen receptor ( ar ) and her2 / neu ( 3 + ) by immunohistochemistry ( ihc ) staining . 
because of the high - risk features of extracapsular nodal extension and close margins , he received adjuvant chemotherapy with cisplatin and radiation therapy once per week in october 2014 . 
 ( c ) restaging scan in june 2018 after ado - trastuzumab emtansine shows a complete metabolic response of previously noted metastatic lymphadenopathy . currently a void in the therapeutic guidelines for managing this disease . 
standard treatment of sgns involves surgical resection with or without adjuvant radiation therapy . locoregional recurrence and metastases are common , at which time palliative chemotherapy is considered , but response rates are generally poor , and the duration of response is brief without a clear favorable impact on survival . 
given the aggressive nature of these tumors and the limited treatment options upon relapse and metastasis , there has been an increased interest in developing targeted therapies . two such targets , the ar and her2 / neu receptor tyrosine kinase , are currently used in clinical practice with variable results.7 a relatively large retrospective case series has reported favorable outcomes using androgen deprivation therapy in ar - positive sgns with good tolerability and an overall survival benet.8 the success of her2 / neu - directed therapy in breast cancer has resulted in multiple case reports that show benet from using trastuzumab - based regimens in treating sgns.4 , 9 despite initial success with targeted therapy , however , progression is inevitable . mechanisms of resistance have not been extensively explored . 
therefore , it seems reasonable to consider the use of ado - trastuzumab emtansine upon progression on trastuzumab , drawing on its demonstrated activity and clinical benet in the analogous clinical scenario in patients with advanced her2 / neu - positive breast cancer.10 by contrast to the previous reports , we describe one of the largest single - institution experiences with her2 / neutargeted therapy in patients with metastatic sgns . 
most of our patients ( n = 6 of 7 ) experienced progression after trastuzumab , which highlights the role of sequential her2 / neu therapy described by almquist et al.6 all of our patients tumors demonstrated her2 / neu overexpression , and most had a high degree of her2 / neu receptor positivity . in our retrospective case series , there was variability in the methods for her2 / neu testing that reect the natural evolution of institutional testing practices . 
nevertheless , clinical benet was seen regardless of the method of testing diagnosed with high - grade right parotid salivary carcinoma solitary liver metastasis retroperitoneal lymph node metastases progression of retroperitoneal lymph node metastases surgical resection with modied radical neck dissection left hepatic lobectomy cisplatin and rt bicalutamide and leuprolide carboplatin and paclitaxel ado - trastuzumab emtansine trastuzumab and pertuzumab maintenance time ( months ) fig 2 . 
 swed , cohen , and aggarwal ( ihc , uorescence in situ hybridization , or next - generation sequencing ) , conrming the role of a her2 / neu - targeted approach . 
for example , the patient discussed here initially experienced a complete metabolic and radiographic response and derived a sustained clinical benet with a progression - free survival of 29 months that is ongoing . progression - free survival in all of our patients ranged from 6 to 29 months , and overall survival ranged from 9 to 33 months . 
all of the patients experienced varying degrees of neuropathy , which proved to be dose - limiting in patient 2 . thrombocytopenia was another adverse effect of therapy , which led to interruption of therapy in one patient . 
cohen honoraria : bristol - myers squibb consulting or advisory role : heat biologics , takeda pharmaceuticals , cerulean pharma , kolltan pharmaceuticals , zymeworks , pzer research funding : heat biologics ( inst ) , macrogenics ( inst ) , merck ( inst ) , takeda pharmaceuticals ( inst ) , cleave biosciences ( inst ) , celldex ( inst ) travel , accommodations , expenses : heat biologics takeda pharmaceuticals , kolltan pharmaceuticals , cerulean pharma , zymeworks , bristol - myers squibb , pzer charu aggarwal consulting or advisory role : genentech , bristol - myers squibb , eli lilly , celgene , medimmune research funding : genentech ( inst ) , incyte ( inst ) , macrogenics ( inst ) , merck sharp & dohme ( inst ) , astrazeneca / medimmune ( inst ) no other potential conicts of interest were reported . references bradley pj : distant metastases from salivary glands cancer . 
otolaryngol head neck surg 154 : 1041 - 1046 , 2016 alotaibi am , alqarni ma , alnobi a , et al : human epidermal growth factor receptor 2 ( her2 / neu ) in salivary gland carcinomas : a review of literature . 
j clin diagn res 9 : ze04 - ze08 , 2015 limaye sa , posner mr , krane jf , et al : trastuzumab for the treatment of salivary duct carcinoma . 
oncologist 18 : 294 - 300 , 2013 girish s , gupta m , wang b , et al : clinical pharmacology of trastuzumab emtansine ( t - dm1 ) : an antibody - drug conjugate in development for the treatment of her2 - positive cancer . 
cancer chemother pharmacol 69 : 1229 - 1240 , 2012 almquist d , umakanthan jm , ganti ak : sequential her2 - targeted therapy in salivary ductal carcinoma with her2 / neu overexpression and a concomitant erbb2 mutation . 
head neck pathol 12 : 95 - 104 , 2018 boon e , van boxtel w , buter j , et al : androgen deprivation therapy for androgen receptor - positive advanced salivary duct carcinoma : a nationwide case series of 35 patients in the netherlands . 
head neck 40 : 605 - 613 , 2018 van boxtel w , boon e , weijs wlj , et al : combination of docetaxel , trastuzumab and pertuzumab or treatment with trastuzumab - emtansine for metastatic salivary duct carcinoma . 
baron jm , boster bl , barnett cm : ado - trastuzumab emtansine ( t - dm1 ) : a novel antibody - drug conjugate for the treatment of her2 - positive metastatic breast cancer . 
vitanza , md1 , 3 ; and bonnie cole , md1 , 4 introduction xeroderma pigmentosum ( xp ) is a rare autosomal recessive disorder caused by a nucleotide excision repair decit , resulting in sensitivity to uv radiation.1 individuals with xp are 10 , 000 times more likely to develop cutaneous squamous cell carcinoma ( scc ) , with initial presentation at a median age of 9 years.2 metastatic skin cancer is the leading cause of death ( 34% ) , with a median survival of 32 years.2 few options for effective systemic therapy of advanced cutaneous scc are available , 3 , 4 and there is no standard therapy for patients with xp who have developed metastatic skin malignancies.5 there is a critical need for effective therapy for advanced skin cancers , and patients with xp represent a unique need because they have the potential to suffer catastrophic adverse effects from standard chemotherapeutic agents.6 immune checkpoint blockade has demonstrated efcacy in tumors characterized by high mutational burincluding scc.7 , 8 therefore , cutaneous scc is den , a good candidate for additional investigation of response to checkpoint inhibitors.9 - 13 a recent phase i / ii clinical trial demonstrated partial response in 41% of patients receiving the programmed cell death 1 ( pd - 1 ) inhibitor cemiplimab.14 case reports in patients with xp and advanced scc describe response with pd - 1 inhibitors , including pembrolizumab.5 , 15 - 17 however , these reports do not provide a molecular mechanistic rationale for the use of pd - 1 inhibitors specic to this patient population , nor do they provide support for the use of these agents for long - term disease management . 
we report stable disease for 2 years in response to pembrolizumab in a patient with xp and metastatic cutaneous scc , along with molecular ndings to support the use of pd - 1 inhibitors in patients with xp . 
extensive perineural spread along the right inferior alveolar nerve and right third through sixth in addition to lepcranial nerves was observed , tomeningeal spread into the right aspect of the posterior fossa ( figs 1a to 1d )  . biopsies of sites concerning for malignant skin lesions were performed , including separate left lower eyelid , right conjunctiva and cornea , and right preauricular masses ; all were diagnostic of scc . 
perineural spread of squamous cell carcinoma ( a to d ) at time of diagnosis and ( e to h ) after therapy , shown by contrast - enhanced t1 - weighted magnetic resonance imaging . 
 ( a ) axial image through face at level of parotid gland shows asymmetric nodular enhancement throughout right parotid gland , as well as more anterior nodular supercial enhancing tissue ( arrow )  . 
 ( b ) axial , ( c ) coronal , and ( d ) sagittal images through head at level of cavernous sinus and right pterygopalatine fossa show extensive abnormal enhancing tissue within cavernous sinus , extending anteriorly into the right orbital apex , as well as posteriorly toward posterior fossa ( arrowheads )  . 
 ( e and f ) five months after initiation of therapy , imaging shows dramatic reduction in tumor burden , both ( e , arrow ) in supercial tissues of face and ( f , arrowheads ) within cavernous sinus and orbital apex . 
 ( g and h ) thirteen months after initiation of therapy , disease burden had diminished further , both ( g , arrow ) in supercial tissues of the face and ( h , arrowheads ) within the cavernous sinus and orbital apex . were also tested and did not show any unexpected mutations ( data supplement )  . 
both tumors showed a signicant strand bias ( p , .001 ) , concordant with the transcription - coupled repair function of the nucleotide excision repair complex ( fig 2b )  . 
on the basis of encouraging reported results in msi - high malignancies to immune checkpoint blockade , pembrolizumab ( 2 mg / kg intravenously every 3 weeks ) was initiated 1 month after diagnosis , using the recent us food and drug administration indication for msi - high malignancies to support insurance approval . after ve cycles of pembrolizumab , follow - up imaging demonstrated a considerable decrease in tumor bulk and resolution of leptomeningeal disease along the right pons and cerebellum ( figs 1e to 1h )  . 
the patient experienced resolution of the chronic cough and reported substantial improvement in the facial pain ( initially the pain required scheduled opioid medications , but they have now been discontinued without pain )  . 
mutation prole analysis of the corneal and conjunctival primary tumors , along with their parotid lymph node metastases ( met ) , was performed on identied somatic single nucleotide variants.19 ( a ) all four tumors show mutation patterns consistent with base excision repair deciency ( catalogue of somatic mutations in cancer mutation signature 7 )  . 
 ( b ) analysis of mutation signatures based on the transcribed gene strand shows a strong bias of c.t mutations on the untranscribed strand ( 1 , 683 v 348 mutations on the transcribed strand ) , consistent with the transcription - coupled nature of the nucleotide excision repair pathway . 
 ( c ) comparison and set overlap of somatic variants identied in four sequenced samples reveals two unrelated primary tumors ( corneal and conjunctival ) , each with unique metastases to the parotid lymph nodes . 
 steineck et al on clinical examutational analyses demonstrated that the ocular lesions had different mutations but a similar mutational burden compared with her metastatic tumor ( data supplement )  . 
mutations implicated in pd - 1 resistance , including wnt , were not identied in analysis of the patients tumors.21 - 23 topical uorouracil , which is efcacious in cutaneous scc associated with xp , was initiated in the affected eye , and no additional tumor progression was noted after 1 month of local treatment . discussion we report the case of a pediatric patient with xp who demonstrated long - term partial response of advanced cutaneous scc to pembrolizumab with development of no clinically signicant adverse effects . 
acknowledges the potential limitation of pd - 1 inhibitors for the treatment of scc involving immune - privileged sites , such as the cornea . xp represents a decit in dna nucleotide excision repair , resulting in sensitivity to uv radiation . 
loss of xpc ( or xpe ) is consistent with decient global genomic repair ( ggr ) but relatively preserved transcription - coupled repair ( tcr ) ; this contrasts with loss of xpa , xpb , or xpd , which results in a ggr - negative / tcr - negative phenotype.24 , 25 the tcrnegative / ggr - positive mutational signature of xpc is demonstrated in this patient with a greater burden of c.t mutations on the untranscribed strand ( fig 2b )  . 
the novel report of our patients mutational signature enriches our understanding of xp and further supports the potential efcacy of immune checkpoint blockade with pd - 1 inhibitors in the treatment of advanced scc among patients with xp . 
in addition , we were able to use molecular methods to demonstrate the coincidental development of two independent primary lesions with two paired metastatic lesions to better understand the origin of this patients metastatic disease . this report describes long - term sustained stable disease , including response of signicant burden of disease in the cns . 
of the existing published reports using pd - 1 inhibitors in patients with xp , the longest reported time to follow - up was 18 months , 16 with other studies describing therapy for no more than 1 year.5 , 15 , 17 our patients sustained response supports the use of pd - 1 inhibitors for long - term disease management in this setting . 
moreover , pd - 1 inhibitors have the potential to do this with minimal adverse effects , providing a quality - oflife benet , which is an important consideration for individuals with advanced cancer . our patients right corneal lesion demonstrated discordant poor response . 
the cornea is immune privileged26 and does not have t - cell immunosurveillance , potentially limiting the ability of immune checkpoint blockade to treat malignancies in the cornea and other immune - privileged sites , such as the testis . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . niklas krumm stock and other ownership interests : reference genomics andrew w . 
stacey honoraria : immunocore travel , accommodations , expenses : immunocore no other potential conicts of interest were reported . references kraemer kh , digiovanna jj : xeroderma pigmentosum , in adam mp , ardinger hh , pagon ra , et al ( eds ) : genereviews . 
seattle , wa , university of washington , 2003 bradford pt , goldstein am , tamura d , et al : cancer and neurologic degeneration in xeroderma pigmentosum : long term follow - up characterises the role of dna repair . 
maubec e , petrow p , scheer - senyarich i , et al : phase ii study of cetuximab as rst - line single - drug therapy in patients with unresectable squamous cell carcinoma of the skj clin oncol 29 : 3419 - 3426 , 2011 deinlein t , lax sf , schwarz t , et al : rapid response of metastatic cutaneous squamous cell carcinoma to pembrolizumab in a patient with xeroderma pigmentosum : case report and review of the literature . 
eur j cancer 83 : 99 - 102 , 2017 sumiyoshi m , soda h , sadanaga n , et al : alert regarding cisplatin - induced severe adverse events in cancer patients with xeroderma pigmentosuintern med 56 : 979 - 982 , 2017 7 . 
science 351 : 1463 - 1469 , 2016 rizvi na , hellmann md , snyder a , et al : cancer immunology : mutational landscape determines sensitivity to pd - 1 blockade in non - small cell lung cancer . science 348 : 124 - 128 , 2015 bauml j , seiwert ty , pster dg , et al : pembrolizumab for platinumand cetuximab - refractory head and neck cancer : results from a single - arm , phase ii study . j clin oncol 35 : 1542 - 1549 , 2017 10 . 
chang al , kim j , luciano r , et al : a case report of unresectable cutaneous squamous cell carcinoma responsive to pembrolizumab , a programmed cell death protein 1 inhibitor . 
stevenson ml , wang cq , abikhair m , et al : expression of programmed cell death ligand in cutaneous squamous cell carcinoma and treatment of locally advanced disease with pembrolizumab . 
salomon g , maza a , boulinguez s , et al : efcacy of anti - programmed cell death - 1 immunotherapy for skin carcinomas and melanoma metastases in a patient with xeroderma pigmentosubr j dermatol 178 : 1199 - 1203 , 2018 17 . 
hauschild a , eichstaedt j , m obus l , et al : regression of melanoma metastases and multiple non - melanoma skin cancers in xeroderma pigmentosum by the pd1 - antibody pembrolizumab . 
pritchard cc , salipante sj , koehler k , et al : validation and implementation of targeted capture and sequencing for the detection of actionable mutation , copy number variation , and gene rearrangement in clinical cancer specimens . 
kruizinga r , scherer - rath m , schilderman jb , et al : the life in sight application study ( lisa ) : design of a randomized controlled trial to assess the role of an assisted structured reection on life events and ultimate life goals to improve quality of life of cancer patients . 
adra n , einhorn lh , althouse sk , et al : phase ii trial of pembrolizumab in patients with platinum refractory germ - cell tumors : a hoosier cancer research network study gu14 - 206 . 
 steineck et al appendix molecular methods tumor and germline samples were processed , sequenced , and analyzed using the university of washington laboratory medicine uwoncoplex cancer gene panel ( university of washington , seattle , wa ) .18 single nucleotide variants and indels in targeted regions were called from aligned bam les using the genome analysis toolkit ( broad institute , cambridge , ma ) and the variant detection in massively parallel sequencing datavarscan . 
in this study , metex14 ( c.2899g.a ) was detected by targeted next - generation sequencing with memorial sloan kettering integrated mutation proling of actionable cancer targets ( msk - impact ) in a case of advanced lung adenocarcinoma driven by egfr l858r mutation , after 12.5 months of frontline egfrtyrosine kinase inhibitor ( tki ) treatment . 
interestingly , metex14 co - occurred with met amplication and egfr t790m at the time of progression , as revealed by digital polymerase chain reaction on cell - free dna ( but not conrmed on tumor tissue )  . 
earlier , we described a patient whose primary resection sample tested positive for both egfr l858r and metex14 ( c3028 + 1g.t ) , but not for met amplication , as assessed by next - generation sequencing.2 in the current study , although the patient did not respond to met - targeted therapy alone , a combination of crizotinib ( met - tki ) and osimertinib ( third - generation egfr - tki ) led to durable disease control.1 however , we believe that additional key points should be discussed in more detail . 
targeted sequencing did not assess unknown potential mechanisms of resistance . it would have been interesting to learn which other pathways were abnormally activated in this tumor , such as fas or components of the nf - b pathway that specically enhanced cell death induced by rstgeneration egfr - tki erlotinib and caused acquired resistance to third - generation egfr - tki rociletinib in egfr - mutant lung cancer cells.3 , 4 met amplication should be further investigated . 
two recent studies reported a response to a combination of osimertinib and crizotinib in patients with nonsmallcell lung cancer ( nsclc ) harboring egfr l858r , while a rebiopsy showed met amplication after disease progression on erlotinib.5 , 6 similarly , a dramatic response was achieved using crizotinib and erlotinib in a case of met amplication emerging in the setting of egfr l858r mutation , suggesting the potential role of erlotinib and crizotinib combination , 5 which was not tested in vitro . 
in this model , crizotinib monotherapy was unable to modulate cell growth and switch off the egfr downstream signaling . although the drugs were used at a low concentration and no analysis was performed to assess pharmacointeractions , 7 crizotinib restored sensitivity to logical osimertinib in both pc9 and h1975 metex14 models . mechanistically , it was shown that the combination inhibited activation of egfr , met , as well as downstream akt and erk.1 these ndings underline the effectiveness of different tki combinations in inhibition of multiple signaling cascades to overcome cancer cell resistance.8 in particular , met signaling plays a crucial role in driving egfr - tki resistance in oncogene - addicted nsclc , and met inhibition can be a successful strategy to recover sensitivity in cases of met aberrations . 
despite the striking clinical activity of the combination in the current study , there is often a large gap between preclinical evaluations of tki combinations and clinical outcome.9 thus , the identication of predictive biomarkers for response could be useful for proper selection of patients who would likely benet from the therapeutic combination . 
we recently identied p - her3 and p - pras40 as potential candidates to assess the synergy between erlotinib and crizotinib in vitro.10 erlotinib and crizotinib showed remarkable synergy in the ludlu squamous - cell lung cancer ( scc ) cell line , which does not harbor egfrsensitizing mutations , met amplication , or metex14 . interestingly , other investigated scc cell lines ( h1703 and skmes - 1 ) , which were also wild - type for egfr and met , reacted differently ( but still additive ) , suggesting a role for the differential expression of p - her3 and p - pras40 expression in this synergy . 
because we observed a synergistic effect of the combination treatment of erlotinib and crizotinib only in scc cells that showed concordant expression of p - her3 and p - pras40 , we assume that p - her3 and p - pras40 may be candidate biomarkers for effective egfr + met blockade in patients with scc.10 taking into account our ndings , other components of the egfr and met pathways , including p - her3 and p - pras40 , should be investigated in tissues obtained from patients receiving a combination of egfr - tki and met - tki . in conclusion , we are indebted to suzawa et al1 for their elegant study . 
 correspondence alessandro leonetti , md amsterdam umc , vu university medical center , cancer center amsterdam , amsterdam , the netherlands , and university hospital of parma , parma , italy marcello tiseo , md , phd university hospital of parma and university of parma , parma , italy christian rolfo , md , phd , mba university of maryland greenebaum comprehensive cancer center , baltimore , md nele van der steen , phd amsterdam umc , vu university medical center , cancer center amsterdam , amsterdam , the netherlands , and center for oncological research ( core ) , university of antwerp , wilrijk , belgium godefridus j . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . marcello tiseo consulting or advisory role : astrazeneca , bristol - myers squibb , msd , boehringer ingelheim , takeda research funding : astrazeneca christian rolfo consulting or advisory role : mylan speakers bureau : novartis , msd , boehringer ingelheim , guardant health travel , accommodations , expenses : astrazeneca godefridus j . 
peters consulting or advisory role : taiho pharmaceutical , clear creek bio , ellipses pharma research funding : rexahn pharmaceuticals ( inst ) travel , accommodations , expenses : rexahn pharmaceuticals , taiho pharmaceutical no other potential conicts of interest were reported . references suzawa k , ofn m , schoenfeld aj , et al : acquired met exon 14 alteration drives secondary resistance to epidermal growth factor receptor tyrosine kinase inhibitor in egfr - mutated lung cancer . 
clin lung cancer 19 : 35 - 41 , 2018 bivona tg , hieronymus h , parker j , et al : fas and nf - b signalling modulate dependence of lung cancers on mutant egfr . 
nature 471 : 523 - 526 , 2011 galvani e , sun j , leon lg , et al : nf - b drives acquired resistance to a novel mutant - selective egfr inhibitor . 
oncotarget 6 : 42717 - 42732 , 2015 giroux - leprieur e , dumenil c , chinet t : combination of crizotinib and osimertinib or erlotinib might overcome met - mediated resistance to egfr tyrosine kinase inhibitor in egfr - mutated adenocarcinoma . 
j thorac oncol 13 : e232 - e234 , 2018 zhu vw , schrock ab , ali sm , et al : differential response to a combination of full - dose osimertinib and crizotinib in a patient with egfr - mutant nonlung cancer and emergent met amplication . 
lung cancer small cell ( auckl ) 10 : 21 - 26 , 2019 bijnsdorp iv , giovannetti e , peters gj : analysis of drug interactions . methods mol biol 731 : 421 - 434 , 2011 tong cws , wu wkk , loong hhf , et al : drug combination approach to overcome resistance to egfr tyrosine kinase inhibitors in lung cancer . cancer lett 405 : 100 - 110 , 2017 yang z , tam ky : combination strategies using egfr - tki in nsclc therapy : learning from the gap between pre - clinical results and clinical outcomes . 
van der steen n , leonetti a , keller k , et al : decrease in phospho - pras40 plays a role in the synergy between erlotinib and crizotinib in an egfr and cmet wild - type squamous non - small cell lung cancer cell line . 
de moor author affiliations and support information ( if applicable ) appear at the end of this article . creative commons attribution non commercial no derivatives 4.0 license corresponding author : andrew n . 
freedman , phd , epidemiology and genomics research program , division of ( continued ) purpose there are no nationally representative data on oncologists use of next - generation sequencing ( ngs ) testing in practice . 
the purpose of this study was to investigate how oncologists in the united states use ngs tests to evaluate patients with cancer and to inform treatment recommendations . methods the study used data from the national survey of precision medicine in cancer treatment , which was mailed to a nationally representative sample of oncologists in 2017 ( n = 1 , 281 ; cooperation rate = 38% )  . 
of these oncologists , 34.0% used them often to guide treatment decisions for patients with advanced refractory disease , 29.1% to determine eligibility for clinical trials , and 17.5% to decide on off - label use of food and drug administrationapproved drugs . 
oncologists younger than 50 years of age , holding a faculty appointment , having genomics training , seeing more than 50 unique patients per month , and having access to a molecular tumor board were more likely to use ngs tests . conclusion in 2017 , most oncologists in the united states were using ngs tests to guide treatment decisions for their patients . 
multimarker panels also include dna and rna analysis through next - generation sequencing ( ngs ) technologies , including custom panels that profile multiple actionable driver genes and tumor characteristics that may guide the selection of targeted therapies . despite advances in precision oncology , comparatively little empirical research is available that assesses the expectations and experiences of oncologists in the united states who may use these genomic tools to care for patients with cancer . 
to date , most studies have evaluated physicians experiences with specific genomic tests for individual gene mutations.3 - 5 studies that have attempted to assess providers experiences with custom ngs tumor panels6 - 9 have focused on intended rather than actual use . 
furthermore , previous studies of physicians use of ngs tests were on the basis of small samples and were conducted in tertiary referral centers ; as a result , their findings may not represent how oncology is practiced in the united states . 
currently , there are no nationally representative data describing oncologists awareness , knowledge , and use of ngs testing to inform patient care , especially in community practice settings . with the increasing application of precision oncology , understanding how oncologists use genomic tests in their practice and the factors that affect their use is essential to ensure that patients who can benefit receive appropriate testing and follow - up . 
the purpose of this study was to investigate how oncologists in the united states use ngs tests to evaluate patients with cancer and inform treatment recommendations . methods data source this study used data from the national survey of precision medicine in cancer treatment , a nationally representative survey of hematologists , hematologists / oncologists , and oncologists sponsored by the national cancer institute ( nci ) , the national human genomic research institute , and the american cancer society . 
 of the remaining 3 , 378 oncologists with verified contact information , 1 , 281 completed the survey ( cooperation rate = 38%10 )  . a survey packet containing a personalized invitation letter from the nci , an endorsement letter from asco , a pen with the studys name printed on it , a self - administered paper questionnaire , and a business reply envelope was mailed to eligible oncologists . 
participants received a $50 honorarium for completing the survey . the questionnaire ( data supplement ) took 20 minutes to complete on average , and data were collected from february through may of 2017 . 
information was ascertained about oncologists demographics , training , academic affiliation , specialty , and patient volume , and their practice characteristics , including practice setting , structure , and resources to support genomic testing . 
 information about oncologists use of commercially available multimarker tumor panels and noncommercial tumor panels performed at academic medical centers was collected , as were data on the clinical scenarios in which ngs test were used . 
because of differences in how survey questions were worded ( eg , some explicitly stated the exclusion of oncotype dx ) , sensitivity analyses were conducted to examine whether oncologists who reported using other gene expression tests , including the breast cancer index , mammaprint , prosigna , and / or myplan lung cancer , differed in their use of ngs tests from those who did not use these gene expression tests . 
the survey weights were applied in all analyses of the data and provide statistical adjustment so that respondents are representative of the population of practicing oncologists in the united states . each oncologist was classified as a user of ngs tests if he or she reported using at least one specific multimarker panel with ngs technologies profiling multiple actionable driver genes for guiding treatment decisions in the past 12 months . 
multivariable models were estimated to examine the independent association between each physician demographic and practice characteristic and the likelihood of ngs testing , when adjusting for the other characteristics in the model . 
all analyses were conducted using sudaan release 11.0.1 ( rti international , research triangle park , nc )  . results characteristics of survey respondents characteristics of the 1 , 281 oncologists who completed the survey are listed in table 1 . 
 more than one half of respondents ( 56.9% ) spent at least 90% of their time in patient care and almost one half ( 49.3% ) spent less than 10% of their time teaching ( data not shown )  . prevalence of ngs test use and predictors of test use to guide treatment decisions overall , 75.6% of oncologists reported using ngs tests in the past 12 months to guide treatment decisions ; use differed according to the physicians demographic and practice characteristics ( table 2 )  . 
oncologists who treated only patients with hematologic malignancies were less likely to use ngs tests than were oncologists who treated both solid and hematologic malignancies and those who only treated patients with solid tumors . 
oncologists who held a faculty appointment , had some training in genomics , or were part of a practice that had a molecular tumor board were more likely to use ngs tests than were those who did not have these characteristics . 
in addition , those who treated fewer than 50 patients with cancer per month were more likely to use ngs tests than were oncologists who treated more than 50 patients with cancer per month . 
the likelihood of ngs use was not statistically significantly associated with other provider and practice characteristics assessed . in this survey , oncologists in the united states were asked how often they used 11 commercially available ngs tests or a noncommercial ngs test during the past 12 months ( fig 1 )  . 
moreover , among oncologists who ordered any of these ngs tests ( n = 959 ) , 28.2% ordered one test , 31.7% ordered two tests , 23.3% ordered three tests , and 16.7% ordered four or more tests in the past 12 months . ngs test use practice patterns oncologists use of ngs test results varied by clinical presentation ( fig 2 )  . 
characteristics of oncologist respondents in the united states to the 2017 national survey of precision medicine in cancer treatment ( continued ) total ( n = 1 , 281 ) unweighted percentage ( % ) * weighted percentage ( % ) * characteristic no . 
we found that oncologists who used the breast cancer index , mammaprint , prosigna , and / or myplan lung cancer did not differ in the way they answered these questions in nine out of 10 comparisons . 
when analyses were restricted to oncologists who treated a high volume of patients with breast cancer , there were no differences in test use between oncologists who used the breast cancer index , mammaprint , and prosigna and those who did not ( data not shown )  . respondents were explicitly directed to exclude oncotype dx when answering questions about ngs testing for different clinical purposes . 
use of ngs testing during the past 12 months among oncologists in the united states , by physician demographics and practice characteristics ( continued ) total ( n = 1 , 281 ) ngs user ( unadjusted % ) ngs user ( adjusted % ) 95% ci adjusted or ( 95% ci ) characteristic sees patients at academic center or medical school no . 
twenty - five percent ( n = 319 ) indicated that they referred their patients to another location or provider for ngs testing ; among these oncologists , 84.4% ( n = 257 ) referred to an academic medical center , 82.1% ( n = 240 ) to a clinical trial , and 18.2% ( n = 51 ) to an oncologist outside of their practice ( data not shown )  . discussion in this nationally representative study of 1 , 281 oncologists , we examined how ngs tests are currently used in clinical practice and whether use is influenced by physician and practice characteristics . 
 initial diagnosis rare cancers advanced refractory disease unknown origin never rarely sometimes often reported using ngs test results to guide patient care , and the use of ngs tests differed by oncologists demographic and practice characteristics . 
 for example , younger oncologists were more likely to be ngs test users , suggesting that they may have had more recent training in genomic testing or that they are more receptive to the incorporation of ngs testing in their practice . 
 having a faculty appointment and access to a molecular tumor board were also associated with ngs test use , perhaps reflecting greater access to in - house ngs tests in academic settings or more involvement in clinical research . 
 not surprisingly , oncologists who treated only patients with hematologic malignancies were less likely to use ngs tests than were oncologists who treated both solid and hematologic malignancies or those who treated only patients with solid tumors , suggesting that ngs tests may be ordered reflexively by pathologists rather than by oncologists . 
use of next generation sequencing tests by clinical purpose during the past 12 months among oncologists in the united states . molecular testing for patients with hematologic malignancies . oncologists reported using ngs most often for patients with advanced refractory disease , but also used these tests often for patients diagnosed with a rare cancer or cancers of unknown origin and / or for patients with an initial diagnosis of cancer . 
these results may reflect oncologists use of ngs testing to inform treatment strategies when established therapies have failed or when there is uncertainty about the usefulness of existing treatment guidelines for less common clinical situations . 
with the recent approval of multiple therapeutic agents targeting specific driver mutations , oncologists may send a patients tumors for sequencing with the hope of identifying treatments that are potentially efficacious for their patients particular molecular tumor subtype . 
in our survey , more than one third of oncologists reported using ngs test results often to guide the use of an fda approved therapy . advances in precision oncology pose challenges for oncologists . 
at the time of the survey in may 2017 , nonsmall - cell lung cancer was the only cancer type for which there was a consensus recommendation for ngs use.13 furthermore , no professional group other than the national comprehensive cancer network has yet issued evidence - based guidelines recommending the use of ngs testing . 
 however , as the number of single - gene or focused - indication ( eg , microsatellite instability ) tests that are components of the standardized evaluation of common malignancies has grown , oncologists are increasingly faced with decisions about whether to order several targeted tests or a single ngs panel . there has also been an effort by pharmaceutical companies to develop tissue - agnostic cancer drugs that target a specific genetic marker independent of tumor type.14 given the prospect of tissue - agnostic labeling , the use of ngs will only grow , and in the absence of clinical guidelines informing ngs use , it is likely that many providers will experience uncertainty about how to clearly integrate this information into clinical care decisions . 
 treatment recommendations , suggesting that oncologists are already confronted with a large volume of genomic information that they must interpret . that ngs test results are frequently ambiguous presents another challenge for oncologists . 
in addition , 25% indicated that they referred patients to other providers for ngs testing , possibly suggesting a lack of expertise or resources for ordering and interpreting ngs tests . 
several academic and commercial groups have responded to this need , developing online decision support resources for genomic medicine , such as personalized cancer therapy , my cancer genome , and oncokb , with the goal of providing an accessible platform to oncologists.15 however , more work is needed to ensure that patients who can benefit from these new technologies receive appropriate testing and follow - up . a third challenge for oncologists is the lack of clinical usefulness data for many of the available ngs tests . 
two major , ongoing clinical trials , ( national cancer institute nci - match molecular analysis for therapy choice ) , 16 and pediatric match ( national cancer institute childrens oncology group pediatric match trial ) , will generate evidence to establish clinical usefulness to fill some of these gaps . 
furthermore , ascos targeted agent and profiling utilization registry ( tapur ) study17 and cureone ( formerly called med - c ) are building registries of ngs testing data to enhance informed treatment recommendations difficulty in interpreting results never / rarely sometimes often understanding of how testing is being used and its impact on patient outcomes.18 insurers are also recognizing the cost efficiencies of ngs panel testing as opposed to multiple single - gene testing for specific cancer - site indications but are grappling with a lack of clinical usefulness data for testing cancers broadly . 
 effective march 16 , 2018 , the centers for medicare & medicaid services has allowed non fda - approved assays run in clinical laboratory improvement amendmentscertified laboratories to be reimbursed , dependent on decisions made by local medicare administrative contractors.19 it is unclear whether and under what circumstances commercial private payers will decide to reimburse for ngs testing broadly for cancer . 
our findings offer an important baseline that could be used to evaluate the impact of these coverage decisions on ngs test use because our data were collected before implementation of these key changes in coverage . our study has several limitations . 
first , the cooperation rate was lower than that of previous surveys of physicians on the topic of genomic and genetic test use , 7 , 20 and responders may have differed from nonresponders in terms of their genomic testing practices and other characteristics such as academic affiliation . 
however , respondents are representative of the population of practicing oncologists in the united states in terms of age , sex , and geographic location on the basis of statistical adjustment for nonresponse using data available on the survey 's sample frame . another limitation is that we were unable to assess how oncologists use the tests in specific clinical situations and for which cancer types they are ordering these tests . 
however , these limitations are offset by several strengths of the study , including the nationally representative sample of practicing oncologists in a wide variety of practice settings and the large sample size , which allowed us to analyze multiple factors associated with ngs use and to examine a variety of practice patterns . the majority of oncologists in the united states use ngs tests to guide patient care , even in the absence of evidence - based practice guidelines . 
schilsky research funding : astrazeneca ( inst ) , bayer ag ( inst ) , bristol - myers squibb ( inst ) , genentech / roche ( inst ) , eli lilly ( inst ) , merck ( inst ) , pfizer ( inst ) deborah schrag consulting or advisory role : journal of the american medical association research funding : pfizer other relationship : journal of the american medical association naoko i . 
simonds stock and other ownership interests : cvs health ( i ) , walgreens boots alliance , anthem foundation , anthem foundation ( i ) , celgene ( i ) , cvs health , johnson & johnson , johnson & johnson ( i ) , walgreens boots alliance ( i ) , labcorp , labcorp ( i ) , valeant pharmaceuticals international , valeant pharmaceuticals international ( i ) , endo pharmaceuticals ( i ) , aetna ( i ) helmneh m . 
miller fa , hayeems rz , bytautas jp , et al : testing personalized medicine : patient and physician expectations of next - generation genomic sequencing in late - stage cancer care . 
gray sw , park er , najita j , et al : oncologists and cancer patients views on whole - exome sequencing and incidental findings : results from the canseq study . 
johnson lm , valdez jm , quinn ea , et al : integrating next - generation sequencing into pediatric oncology practice : an assessment of physician confidence and understanding of clinical genomics . 
 appendix physician characteristics included age , sex , race or ethnicity , faculty appointment , training in genomic testing ( eg , instruction throughout residency and / or fellowship , professional lectures or seminars , symposiums , conferences , or continuing medical education )  . 
practice characteristics , such as region , urbanicity , type of practice ( solo , single specialty group , multispecialty group , other ) , academic affiliation , and number of unique patients with cancer seen per month were also collected . 
in addition , questions were asked about whether the oncologists practice setting has a genomic or molecular tumor board , policies for genomic testing use , and / or an electronic medical record that provides alerts when a genomic test is recommended . 
an oncologists specialty was defined using information on type ( s ) of patients with cancer seen : only patients with solid tumors , only those with hematologic malignancies , or both . 
age and demographic characteristics other than race or ethnicity were obtained from the american medical association masterfile . an oncologists was classified as a user of next - generation sequencing ( ngs ) tests if he or she reported use , in the past 12 months , of specific multimarker panels with ngs technologies profiling multiple actionable driver genes used to guide treatment decisions . 
the following tests were included in this definition : cancerselect or cancer complete , caris molecular intelligence or target now , cgi complete , foundationone , foundationoneheme , foundationact , gps cancer , guardant360 , omniseq comprehensive , onkosight tumor panels , solid tumor mutation panel ( arup laboratories ) , and noncommercial tumor panels . 
multimarker tests that use gene - expression profiling ( eg , oncotype dx for breast cancer ) , primarily used to predict prognosis and / or the likelihood of recurrence , were not classified as ngs tests . 
neil hayes , md , lineberger comprehensive cancer center , university of north carolina at chapel hill , cb #7295 , 125 mason farm road , chapel hill , nc 27599 ; e - mail : hayes@ med.unc.edu. purpose a 73 - year - old woman with metastatic colon cancer experienced a complete response to chemotherapy with dose - intensified irinotecan that has been durable for 5 years . 
we sequenced her tumor and germ line dna and looked for similar patterns in publicly available genomic data from patients with colorectal cancer . patients and methods tumor dna was obtained from a biopsy before therapy , and germ line dna was obtained from blood . 
the somatic mutation and clinical annotation data files from the colon ( n = 461 ) and rectal ( n = 171 ) adenocarcinoma data sets were downloaded from the cancer genome atlas data portal and analyzed for patterns of mutations and clinical outcomes in patients with poleand / or pold1mutated tumors . results the pattern of alterations included apc biallelic inactivation and microsatellite instability high ( msi - h ) phenotype , with somatic inactivation of mlh1 and hypermutation ( estimated mutation rate > 200 per megabase )  . 
colonoscopy showed a cecal mass , and biopsy showed invasive adenocarcinoma . she had blood transfusions , with an appropriate increase in her hemoglobher medical history was significant for back surgery , depression , diverticulitis , hyperlipidemia , hypothyroidism , and osteoporosis . 
the objective of this study was to define the maximum - tolerated dose of irinotecan in folfiri plus bevacizumab as firstline therapy in metastatic colorectal cancer , with dosing of irinotecan based on ugt1a1 genotype.1 her ugt1a1 genotype was * 1 / * 28 , and she was assigned to receive irinotecan at a dose of 310 mg / m2 intravenously ( iv ) , 72% higher than the standard dose of 180 mg / m2 . 
she received standard doses of the other drugs : fu 400 mg / m2 iv bolus , then 2 , 400 mg / m2 iv over 46 hours ; leucovorin 200 mg / m2 iv ; and bevacizumab 5 mg / kg iv . 
dna from tumor and normal samples were processed through the usual lccc1108 production pipeline of targeted capture sequencing as described previously , with a modification for small - volume input of less than 1 mg of input tumor dna.3 briefly , tumor and normal samples were sequenced using commercial protocols ( agilent sureselect custom targeted panel of approximately 250 genes ; santa clara , ca ) and the illumina hiseq platform ( san diego , ca ) in either singleor paired - end format of 75 and 100 bases per end . average depth of coverage for target sequences was 665 reads in the tumor sample and 293 reads in the germline sample . 
whole - exome sequencing was performed , and the identical informatics pipeline was applied . focal validation of the pold1 mutation was performed on the whole - genomeamplified dna using sanger sequencing . 
primers were designed to flank exons 18 and 26 of pold1 and exon 6 of pole : pold1 exon 18 : 59 - gcatgattctctccccgacag - 39 ( forward ) and 59 - ctgccctgggcccatctca - 39 ( reverse ) ; pold1 exon 26 : 59 - ccgggagtctgagctgtatc - 39 ( forward ) and 59 - cagggtcgggacatggca - 39 ( reverse ) ; and pole exon 6 : 59 - ctcttgaaccaatgagcgtga - 39 ( forward ) and 59 - gtcgttctgaaccgctgatg - 39 ( reverse )  . 
polymerase chain reaction amplification and sanger sequencing were performed using a previously validated protocol within the unc hospitals molecular genetics laboratory ( chapel hill , nc )  . the somatic mutation and clinical annotation data files from the colon ( n = 461 ) and rectal ( n = 171 ) adenocarcinoma data sets were downloaded from the cancer genoma atlas ( tcga ) data portal.4 of these 632 patient cases , 430 ( colon , n = 330 ; rectum , n = 99 ; unknown , n = 1 ) contained both somatic mutation and clinical annotation data points . 
select protein - coding mutations in genes previously associated with colon cancer are listed in table 1 . review of the mutant allele fractions of apc and pik3ca was potentially informative as to the nature of the tumor genome . 
protein - coding mutations selected from 1 , 192 somatic mutations identified ; organized top to bottom by maf . abbreviations : cosmic , catalogue of somatic mutations in cancer ; maf , mean allelic frequency ; na , not applicable ; q , 210 3 log10 ( probability of sequencing error )  . * q score not defined for this deletion event . mutation in 1489 followed by genome doubling , resulting in two mutant alleles and two wild - type alleles . 
this event was followed by independent separate mutations , resulting in 25% mutant allele fractions and complete loss of functional apc . the mutation in pik3ca , as well as most of the more than 1 , 000 mutations , occurred after genome doubling , because the most common mutant allele fractions were approximately 25% . tumor evolution in this manner suggests a mechanism by which a cell with so many mutations might remain viable by offering four alleles to retain at least one functional form of the gene . despite the large number of mutations , the cancer genome remained structurally intact ( fig 3 )  . 
genomes without such copy - number changes are uncommon , with nearly every colon cancer demonstrating amplifications or deletions.7 the only evidence of genome instability in this patient case involved low - level gains of chromosomes 17 and 19 and perhaps loss of a portion of chromosome 13 . 
if the supposition of a tetraploid genome is correct , these events might best be explained by loss or gain of an additional chromosome from an average copy of four alleles . 
interestingly , it has been suggested that tumors that retain wild - type tp53 and / or are of the msi - h phenotype are more likely to have a stable genome configuration , such as we saw in our patient case.8 , 9 in our patient case , despite the large number of mutations , no evidence for tp53 mutation was detected . 
this is also consistent with the observation that the frequency of tp53 mutations is lower in msi - h tumors.10 although the msi - h phenotype with hypermutation in association with mlh1 alteration is an established entity , 10 the overall number of mutations in this patient case was particularly striking , leading us to ask whether any additional alterations might play a role . 
specifically , we queried whether previously reported mutations in pole might be present.8 because pole was not included in the original hybrid capture set , the remaining dna was submitted for wholeexome sequencing , and no pole mutation was detected . 
we then looked for associations of pole and pold1 mutations with both overall mutation rate and the pattern of hyper - indel hypermutation seen in our patient case ( fig 5a )  . 
by contrast , cancers with pold1 mutations were not associated with ultra - high mutation rates , although the median number of nonsilent mutations was much higher ( 893 mutations ) than the median for the entire data set ( 134 mutations )  . 
cancers with pole and / or pold1 mutations generally were hypermutated , with only seven patient cases ( 1.6% ) having fewer than 200 nonsilent somatic mutations . we considered the possibility that , because the set of samples being considered included those with the highest mutation rates , mutations of pold1 might simply occur by random chance . 
further arguing against chance alone , we noted that pold1 mutations were not observed in any of the patient cases of pole ultramutated tumors but rather uniquely in patients with msi - h phenotype with high numbers of indels . 
lollipop plots showing mutations in ( a ) pole and ( b ) pold1 from the cancer genome atlas ( tcga ) , as well as ( b ) the mutation in pold1 from our patient . 
this gene model includes important functional domains of the gene : dna_pol_b_exo1 , dna polymerase beta exonuclease 1 domain ; dna_pol_b , dna polymerase , family b domain ; duf1744 , domain of unknown function found on epsilon catalytic subunit of dna polymerase ; zf - , zf - c4pol ( c4 - type zinc - finger of dna polymerase delta )  . 
all domains were annotated from the pfam database . msi - h , microsatellite instability high . dna_pol_b_exo1 dna_pol_b duf1744 1200 1600 2000 2286 aa dna_pol_b_exo1 dna_pol_b zf - .. reported mutation 1000 1107 aa missense mutation nonsense mutation nonhypermutated > 2 , 000 mutations hyper indels msi - h because pold1 mutations were associated with msi - h phenotype , we considered the possibility that these mutations were a passenger event confined to the biology of msi - h cancers . 
comparison of indel rate in msi - h cancers on the basis of pold1 mutation status revealed that msi - h cancers with pold1 mutations exhibited approximately 60% more indels than msi - h cancers without pold1 mutations ( p , .03 ; fig 5b )  . 
however , polehypermutated cancers exhibited approximately 50% fewer indels than hypermutated cancers without pole mutations ( p , .001 ; fig 5c )  . discussion crs to chemotherapy have been described previously in patients with colorectal cancer . 
a recent meta - analysis of chemotherapy plus bevacizumab reported a cr rate of 2.4% based on aggregate data from seven randomized controlled trials.13 because these responses are infrequent , and many of them occurred in the era before genomic profiling of tumors , the links between tumor genomics and exceptional unclear . 
having observed a cr to folfiri and bevacizumab that has been durable for 5 years in the patient case presented here , sequencing of tumor and germline dna was undertaken to search for alterations associated with this exceptional response . 
msi - h tumors typically had hypermethylation and mlh1 silencing , whereas hypermutated tumors without msi - h typically had somatic mutations in one or more mismatchrepair genes and / or pole . 
right - sided colon cancers were recently reported to be associated with worse prognosis compared with left - sided colon cancers in a systematic review and metaanalysis , 14 but it is not clear whether the same would be true for hypermutated right - sided colon cancers . dna polymerases e ( pole ) and d ( pold1 ) are the two main enzymes involved in human dna replication . 
germline and somatic mutations in the exonuclease ( proofreading ) domains of pole and pold1 are associated with ultramutated colorectal and endometrial cancers.12 , 15 whole - genome sequencing in our patient case showed a hypermutated ( but not ultramutated ) phenotype in this right - sided tumor with many indels including a codon deletion of mlh1 , so the decision was made to pursue whole - exome sequencing to search for additional mutations in pole or pold1 . 
tumors with somatic mutations in pole ( blue ) , pold1 ( gold ) , or both pole and pold1 ( gray ) are distinguished from tumors without pole or pold1 mutations ( red )  . ( b ) indels in pold1mutant versus wild - type ( wt ) tumors with microsatellite instability high ( msi - h ) phenotype from tcga . 
number of indel was calculated for the 67 msi - h tumors from the colorectal tcga data set . msi - h tumors that also had pold1 somatic mutations exhibited more indels as compared with msi - h tumors without pold1 somatic mutations ( mean pold1 - mutant indels , n = 230 ; mean pold1 wt indels , n = 144 )  . 
 ( c ) indels among hypermutated ( n = 50 ) , nonhypermutated ( n = 356 ) , and polehypermutated ( n = 24 ) tumors from tcga ( total , n = 430 ; mean hypermutated indels , n = 176 ; mean nonhypermutated indels , n = 6 ; mean polehypermutated indels , n = 91 )  . 
of indels pold1 mutant 7 , 000 6 , 000 5 , 000 4 , 000 3 , 000 2 , 000 1 , 000 * p < .001 analysis of tcga confirmed that the observed phenotype was similar to other tumors with pold1 mutations . 
to our knowledge , this is a pattern that has not previously been reported in the literature . two previous studies have attempted to relate pole mutations to colorectal cancer outcomes . in a population - based cohort with microsatellitestable stage i to iv colorectal cancer , stenzinger et al16 reported a trend toward shorter survival for patients with pole mutations compared with their counterparts without pole mutations . along the same lines , there was a statistically significantly increased mortality for patients with stage iii to iv colorectal cancer receiving chemotherapy with pole mutations compared with their counterparts without pole mutations.16 in contrast , domingo et al17 recently reported that pole mutations were associated with a lower risk of recurrence compared with microsatellitestable stage ii to iii colorectal cancers , with correlative data suggesting that these tumors were more immunogenic . 
rizvi et al18 showed that higher nonsynonymous mutation burden was associated with markers of immunogenicity and improved outcomes with programmed death - 1 blockade in nonsmall - cell lung cancer . 
in our analyses of data from tcga , there were no statistically significant differences in survival based on overall mutation status , pole mutation status , pold1 mutation status , or combined pole and pold1 mutation status ( appendix fig a1 )  . an important limitation of this study is that it was impossible to isolate the genomic alterations most important in this patients exceptional response . 
 mutation burden and immunogenicity , 18 it is also possible that chemotherapy induced an endogenous immune response to a variety of neoantigens in this patients cancer . in conclusion , an exceptional response in a patient with metastatic colorectal cancer to folfiri plus bevacizumab led us to identify a somatic mutation in pold1 . 
grilley - olson consulting or advisory role : sanofi research funding : bayer , novartis ( inst ) , peregrine pharmaceuticals ( inst ) , nanocarrier ( inst ) , genentech ( inst ) , seattle genetics ( inst ) , medimmune ( inst ) , glaxosmithkline ( inst ) , pfizer ( inst ) xiaobei zhao no relationship to disclose stergios j . 
moschos consulting or advisory role : merck sharp & dohme , amgen , paometheus , castle biosciences research funding : merck sharp & dohme , pharmacyclics , amgen , genentech / roche travel , accommodations , expenses : bristol - myers squibb , merck sharp & dohme , novartis blase n . 
neil hayes leadership : genecentric stock and other ownership interests : genecentric consulting or advisory role : genecentric patents , royalties , other intellectual property : i hold several diagnostic patents or pending patents in the area of solid tumor diagnostics federico innocenti patents , royalties , other intellectual property : royalties from the mayo foundation on the ugt1a1 testing acknowledgment we thank the patient for her enthusiasm in participating in the clinical trial and subsequent correlative studies . 
neil hayes , and federico innocenti , university of north carolina at chapel hill , chapel hill , nc ; and elena marangon , bianca posocco , and guiseppe toffoli , centro di riferimento oncologico , aviano , italy . supported by national cancer institute award no . 
therasse p , arbuck sg , eisenhauer ea , et al : new guidelines to evaluate the response to treatment in solid tumors : european organisation for research and treatment of cancer , national cancer institute of the united states , national cancer institute of canada . 
zhao x , wang a , walter v , et al : combined targeted dna sequencing in non - small cell lung cancer ( nsclc ) using uncseq and ngscopy , and rna sequencing using uncqer for the detection of genetic aberrations in nsclc . plos one 10 : e0129280 , 2015 4 . 
westra jl , boven lg , van der vlies p , et al : a substantial proportion of microsatellite - unstable colon tumors carry tp53 mutations while not showing chromosomal instability . 
mertz tm , sharma s , chabes a , et al : colon cancer - associated mutator dna polymerase d variant causes expansion of dntp pools increasing its own infidelity . 
zhang g , zhou x , lin c : efficacy of chemotherapy plus bevacizumab as first - line therapy in patients with metastatic colorectal cancer : a meta - analysis and up - date . 
petrelli f , tomasello g , borgonovo k , et al : prognostic survival associated with left - sided vs right - sided colon cancer : a systematic review and meta - analysis . 
domingo e , freeman - mills l , rayner e , et al : somatic pole proofreading domain mutation , immune response , and prognosis in colorectal cancer : a retrospective , pooled biomarker study . 
rizvi na , hellmann md , snyder a , et al : cancer immunology : mutational landscape determines sensitivity to pd - 1 blockade in non - small cell lung cancer . 
bertagnolli mm , redston m , compton cc , et al : microsatellite instability and loss of heterozygosity at chromosomal location 18q : prospective evaluation of biomarkers for stages ii and iii colon cancera study of calgb 9581 and 89803 . 
rmer mu , jensen nf , nielsen sl , et al : top1 gene copy numbers in colorectal cancer samples and cell lines and their association to in vitro drug sensitivity . 
 genomic assessment of bloodderived circulating tumor dna in patients with colorectal cancers : correlation with tissue sequencing , therapeutic response , and survival purpose genomic alterations in blood - derived circulating tumor dna ( ctdna ) from patients with colorectal cancers were correlated with clinical outcomes . patients and methods next - generation sequencing of ctdna ( 54to 73 - gene panel ) was performed in 94 patients with colorectal cancer . results most patients ( 96% ) had metastatic or recurrent disease at the time of blood draw . 
altogether , 74 patients ( 79% ) had one or more nonsynonymous alterations , 69 ( 73% ) had one or more potentially actionable alterations , and 61 ( 65% ) had an alteration actionable by a drug approved by the us food and drug administration ( on or off label )  . 
ctdna of 5% or more from blood tests as well as egfr and erbb2 ( her2 ) nonsynonymous alterations correlated with worse survival ( but only erbb2 remained significant in multivariable analysis )  . 
globally , there were 1.4 million new cases and 693 , 900 deaths in 2012 , with an increase in incidence and mortality rates in developing countries.1 , 2 at diagnosis , approximately 20% of patients have distant metastatic disease.3 for decades , systemic therapy used fluorouracil as the main active agent . 
addition of irinotecan and oxaliplatin , as well as the recently developed inhibitors that target vegf ( bevacizumab , aflibercept , and regorafenib ) and egfr ( cetuximab and panitumumab ) , have markedly improved the outcome of patients with metastatic colorectal cancer . 
however , prognosis remains poor ( median progression - free survival [ pfs ] , 10 months ; median overall survival [ os ] , 19 to 28 months ; 5 - year survival , 10%4 , 5 )  . 
 there is an unmet need to better understand the clinically relevant biology of colorectal cancer . the molecular characteristics of colorectal cancer are better understood because of advances in next - generation sequencing ( ngs ) technology.6 categorizing patients on the basis of their underlying molecular features has been proposed ( ie , consensus molecular subtypes ) , 7 and several genomic markers are now routinely used in the clinic to guide treatment . 
examples of genomically guided us food and drug administration ( fda ) approved therapies include anti - egfr agents ( cetuximab and panitumumab ) among patients with wild - type ras8 - 10 and pembrolizumab ( anti - programmed cell death protein 1 antibody ) for microsatellite instability - high or mismatch repairdeficient colorectal cancer.11 in additon , adding vemurafenib ( braf inhibitor ) to irinotecan and cetuximab demonstrated better clinical outcome among patients with brafv600mutated colorectal cancer.12 however , despite the recent progress in targeted therapy approaches , more than 50% of patients do not respond to the aforementioned regimens , and an increased understanding of the molecular underpinnings of the disease is needed . 
the study followed the guidelines of the university of california , san diego , internal review board , the declaration of helsinki for the predict study ( nct02478931 ; profile related evidence determining individualized cancer therapy ) , and any investigational therapy for which the patients gave consent . sequencing , concordance rate , matched therapy , and actionability ctdna sequencing was performed by guardant health and has been previously described ( data supplement ) .16 , 23 , 24 overall , 76 ( 81% ) of 94 patients had ngs performed on tumor tissue using the foundationone assay . 
the methods have been previously described ( data supplement ) .25 tissue ngs and plasma ctdna tests were compared by using the kappa statistic ( data supplement ) .26 we retrospectively analyzed the treatments given after ctdna testing and compared the clinical outcomes among patients who received matched and unmatched therapies ( data supplement ) .27 statistical analysis statistical analysis was performed by m.c.s. 
pfs and os were analyzed by using the kaplan - meier method28 and the logrank test ( univariable analysis ) ; a cox regression model ( multivariable analysis ) was used to compare variables . 
among the 74 patients with one or more nonsynonymous ctdna alterations , 59 ( 79.7% ) had distinct gene alteration portfolios ; 15 patients ( 20.3% ) had similar genomic alteration portfolios ( when alterations were considered at the gene level only , irrespective of the specific variant )  . 
this was the case only when patients had a small number of alterations ( three or fewer ) and included mostly the three genes with the most frequent alterations : tp53 , kras , and apc ( fig 1b )  . 
the other altered genes in our population ( fewer than five patients had the alteration ) were abl1 , akt1 , alk , araf , atm , brca1 , ccnd1 , ccnd2 , ccne1 , cdh1 , cdk6 , cdkn2a , ctnnb1 , esr1 , fgfr3 , gnaq , gnas , hras , jak3 , kit , map2k1 , mlh1 , mtor , notch1 , nras , pten , raf1 , rb1 , ret , rhoa , rit1 , ros1 , sdk6 , smo , stk11 , vhl . 
alterations of unknown significance ( variant of unknown significance ; vuss ) versus characterized mutations ( indels , amplifications , fusions , and single nucleotide variant ( snv ) point mutations ) were considered at the variant level . 
we did not observe a difference in the concordance rate when the time interval between the blood draw and tissue biopsy were taken into consideration ( a cutoff of 6 months was used because it was the median time interval )  . 
although apc alterations seemed to be detected more frequently in the tissue than in the plasma ( 22 patients were positive in both tests , 27 patients were positive in tissue only , and one patient was positive in plasma only ) , braf alterations seemed to be detected more frequently in the ctdna test ( eight patients were positive in both tests , two were positive in tissue only , and nine were positive in plasma only )  . 
in contrast , braf amplification was seen only with ctdna testing ( n = 8 )  . os analysis first , we analyzed the impact of several clinical variables on os calculated from the time of diagnosis ( table 2 )  . 
after the multivariable analysis , patients in whom an alteration was detected with ctdna of 5% or more still had statistically significant survival ( fig 2a )  . among 76 patients who had tissue ngs analysis , patients with braf alterations had significantly worse os , and patients with lung metastases had better os ( statistically significant after the multivariable analysis )  . 
 however , only erbb2 alterations remained significant in the multivariable model ( fig 2b )  . analysis of outcomes for patients with matched therapy versus patients with unmatched therapy outcomes of the patients who were treated with matched therapy after their ctdna testing ( n = 17 ) versus those who were given unmatched therapy ( n = 18 ) were evaluated ( figure 3a ; data supplement )  . 
altogether , 65% of patients in the matched therapy group attained sd for 6 months or more , pr , or cr versus 31% of patients in the unmatched therapy group ( p = .060 in univariable analysis ; p = .045 in multivariable analysis [ multivariable analysis was performed using line of therapy as a confounding variable ] )  . 
in addition , patients in the matched therapy group had a median pfs of 6.1 months compared with 2.3 months for patients in the unmatched therapy group ( p = .143 for univariable analysis ; p = .079 for multivariable analysis )  . 
 finally , patients who received matched therapy survived longer than unmatched therapy group , with a median os ( calculated from the treatment start date ) not reached ( at 11.1 months ) fig 1 . 
only patients with at least one alteration in one of these genestp53 , kras , apc , braf , pik3ca , egfr , myc , or erbb2are displayed ( n = 71 patients ; the other 23 patients had no alterations in the represented genes and their corresponding columns would have been empty or white )  . 
 ( d ) pie charts representing the potential actionability of the detected alterations in the overall population ( n = 94 ; left ) and in patients with at least one nonsynonymous alteration ( n = 74 ; right )  . 
only variables that were significant in the univariable models ( log - rank test ) were included in the multivariable analysis ( cox regression model ) , with the final model containing only significant covariables in the multivariable analyses ( forward stepwise selection model )  . 
the wald statistic tests the unique contribution of each variable ; the higher the wald statistic , the higher the association or contribution in the model . abbreviations : ctdna , circulating tumor dna ; nr , not reached . * if only alterations are included in the multivariable analysis , braf ( p = .034 ) and erbb2 ( p = .004 ) remain significantly associated with shorter survival . 
kaplan - meier curves for overall survival analysis from ( a ) the time of diagnosis and ( b ) the time of blood draw used for the ctdna testing . 
 ( b ) kaplan - meier curves comparing the progression - free survival ( pfs ) for the patients with matched treatment ( n = 17 ) v patients with unmatched treatment ( n = 18 )  . 
 ctdna alterations at baseline : ctdna alterations at progression : arid1a k1808k ( 1.5% ) mtor e162v ( 6.6% ) apc e422 * ( 2.2% ) tp53 s127f ( 1.9% ) apc i1307fs ( 15.9% ) apc e422 * ( 33.8% ) tp53 s127f ( 39% ) egfr amplification time from initiation of therapy with irinotecan plus cetuximab ( months ) imaging obtained pretreatment 4 months post - treatment 9 months post - treatment fig 4 . 
 a 49 - year - old - man with metastatic adenocarcinoma of the rectum had a history of previous treatment with ( 1 ) capecitabine plus oxaliplatin and ( 2 ) fluorouracil plus irinotecan plus bevacizumab ; ( 3 ) treatment on a clinical trial with anti - cd73 included fourthline therapy with irinotecan plus cetuximab . 
 ( b ) the patient showed initial tumor regression , but at 9 months , the tumor progressed with new lung metastases and lymphangitic spread ( red arrow )  . 
ctdna among previously observed alterations increased approximately 20 - fold ( 33.8% for apc e422 * ; 39% for tp53 s127f ) along with emerging alterations , including mtor e162v , apc i1307fs , and egfr amplification . 
 ( ) only levels of ctdna mutations were quantified using %ctdna and represented ; egfr amplification was detected at progression but not quantified . overall , 79% of patients ( 74 of 94 ) had one or more nonsynonymous ctdna alterations ( table 1 )  . 
the most frequently characterized alterations were in tp53 ( 51% of patients ) followed by kras ( 34% ) , apc ( 27% ) , braf ( 16% ) , pik3ca ( 16% ) , and egfr ( 15% ) genes ( fig 1 )  . 
hong et al29 previously reported that the ctdna ngs had 100% sensitivity for tissue - detected as well as digital droplet polymerase chain reactionbased plasma - detected brafv600e mutation among patients with colorectal cancer , which is consistent with our data . 
for instance , sensitivity of ctdna for detection of patients with tissue apc positivity was 44.9% ; sensitivity of ctdna for detection of those with tissue braf positivity was 80% ( this analysis was not restricted to brafv600e )  . 
the apparent low capacity for detecting apc alterations could have implications for plasma monitoring of this alteration . we did not observe differences in the concordance rate of driver alterations when we compared patients with a time interval between the blood draw and tissue biopsy of 6 months or less versus more than 6 months ( data supplement )  . 
 this observation differs from previous reports , which showed that the longer the time interval between the two tests , the lower the rate of concordance.20 , 30 the small number of patients in each of our subgroups may have confounded our ability to discern such differences . importantly , certain clinical and ctdna factors were associated with survival outcome . 
 when survival was evaluated from the time of blood draw to the last follow - up , the presence of apc , braf , pik3ca , egfr , myc , and erbb2 nonsynonymous alterations ( including single nucleotide variations and amplifications ) were all associated with poor overall survival ( all p < .05 by univariable analysis ) , and erbb2 alterations remained significant after the multivariable analysis ( p < .001 ) ( table 2 ; fig 2 )  . 
many of these poor prognostic alterations are potentially targetable with either fda - approved drugs ( onor off - label use ) or with investigational agents currently in clinical development . 
for instance , patients often received concomitant chemotherapy , and the putative interaction between tp53 and bevacizumab could have inflated the results.37 - 39 these considerations are important because some matched therapies have not proved to be effective in colorectal cancers.40 , 41 although the precision medicine approach of matching patients with genomically or immunotargeted treatments is potentially promising , there are challenges for implementing this strategy ( eg , tumor heterogeneity and genomic complexity among patients )  . 
in this study , we observed a median of three alterations per patient ( range , zero to 30 alterations ) , and among 74 patients with one or more alterations , there were no two patients with identical molecular profiles . 
along with this notion , we have also presented a patient with metastatic rectal adenocarcinoma whose ctdna level increased by 20 - fold when his tumor showed progression of lung metastases and lymphangitic spread while he was receiving treatment with irinotecan and cetuximab ( fig 4 )  . 
emergence of alterations similar to those in ctdna and other anomalies such as the appearance of kras alterations have been implicated in the evolution of resistance in patients with colorectal cancer who were treated with egfr inhibitors.46 there were several limitations to our study . 
third , it would be interesting to determine the impact of microsatellite stability status on the ctdna alteration profile ; this analysis could not be performed in this study because only 26 of our patients had microsatellite testing and 25 of them were microsatellite stable . 
despite these limitations , our study provided an in - depth investigation of the clinical utility of ctdna testing among patients with colorectal cancer . in conclusion , we have interrogated tumors from 94 patients with mostly advanced - stage colorectal cancer who had clinical - grade ngs performed on blood - derived ctdna . 
 although the number of patients receiving targeted therapy in our study was modest , this is the first study , to our knowledge , to demonstrate objective evidence of the clinical utility of ctdna ngs in metastatic colorectal cancer across multiple biomarkers beyond brafv600e . 
 stock and other ownership interests : merck ( i ) travel , accommodations , expenses : merck ( i ) amirali talasaz employment : guardant health leadership : guardant health shumei kato no relationship to disclose maria c . 
lanman employment : guardant health , veracyte leadership : guardant health , biolase stock and other ownership interests : guardant health , biolase research funding : guardant health victoria m . 
raymond employment : trovagene , guardant health stock and other ownership interests : trovagene , guardant health stock and other ownership interests : guardant health research funding : guardant health patents , royalties , other intellectual property : guardant health travel , accommodations , expenses : guardant health razelle kurzrock leadership : curematch stock and other ownership interests : curematch , idbydna consulting or advisory role : actuate therapeutics , loxo oncology , xbiotech , neo - med , roche speakers bureau : roche research funding : guardant health ( inst ) , sequenom ( inst ) , merck serono ( inst ) , genentech ( inst ) , pfizer ( inst ) , foundation medicine ( inst ) , incyte ( inst ) , konica minolta ( inst ) support references 2015 affiliations shumei kato , maria c . 
van der geest lg , lam - boer j , koopman m , et al : nationwide trends in incidence , treatment and survival of colorectal cancer patients with synchronous metastases . 
cassidy j , clarke s , daz - rubio e , et al : xelox vs folfox - 4 as first - line therapy for metastatic colorectal cancer : no16966 updated results . 
heinemann v , von weikersthal lf , decker t , et al : folfiri plus cetuximab versus folfiri plus bevacizumab as first - line treatment for patients with metastatic colorectal cancer ( fire - 3 ) : a randomised , open - label , phase 3 trial . 
kopetz s , mcdonough sl , morris vk , et al : randomized trial of irinotecan and cetuximab with or without vemurafenib in braf - mutant metastatic colorectal cancer ( swog 1406 )  . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
schwaederl mc , patel sp , husain h , et al : utility of genomic assessment of blood - derived circulating tumor dna ( ctdna ) in patients with advanced lung adenocarcinoma . 
thierry ar , el messaoudi s , mollevi c , et al : clinical utility of circulating dna analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti - egfr treatment . 
hong ds , morris vk , el osta b , et al : phase ib study of vemurafenib in combination with irinotecan and cetuximab in patients with metastatic colorectal cancer with brafv600e mutation . 
sartore - bianchi a , trusolino l , martino c , et al : dual - targeted therapy with trastuzumab and lapatinib in treatment - refractory , kras codon 12 / 13 wild - type , her2 - positive metastatic colorectal cancer ( heracles ) : a proof - of - concept , multicentre , open - label , phase 2 trial . 
jardim dl , schwaederle m , wei c , et al : impact of a biomarker - based strategy on oncology drug development : a meta - analysis of clinical trials leading to fda approval . 
schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
said r , hong ds , warneke cl , et al : p53 mutations in advanced cancers : clinical characteristics , outcomes , and correlation between progression - free survival and bevacizumab - containing therapy . 
schwaederl m , lazar v , validire p , et al : vegf - a expression correlates with tp53 mutations in nonsmall cell lung cancer : implications for antiangiogenesis therapy . 
hochster hs , uboha n , messersmith w , et al : phase ii study of selumetinib ( azd6244 , arry142886 ) plus irinotecan as second - line therapy in patients with k - ras mutated colorectal cancer . 
kim st , lee j , park sh , et al : prospective phase ii trial of everolimus in pik3ca amplification / mutation and / or pten loss patients with advanced solid tumors refractory to standard therapy . 
janku f , huang hj , claes b , et al : braf mutation testing in cell - free dna from the plasma of patients with advanced cancers using a rapid , automated molecular diagnostics systemol cancer ther 15 : 1397 - 1404 , 2016 44 . 
tie j , wang y , tomasetti c , et al : circulating tumor dna analysis detects minimal residual disease and predicts recurrence in patients with stage ii colon cancer . 
the analytical and clinical validation of guardant360 was conducted in conformance with evidentiary standards established by the standards for reporting of diagnostic accuracy , reporting of tumor marker studies , evaluation of genomic applications in practice and prevention , recent next - generation sequencing ( ngs ) , and standardization of clinical testing biomarker guidelines.16 the sequencing uses molecular barcoding and hybrid capture followed by ngs of the critical exons in a panel of 54 to 73 genes and reports all four major types of genomic alterations ( point mutations , insertions - deletions [ indels ] , fusions , and copy number amplifications ) 16 , 23 see the data supplement for detailed gene panels . 
the analytic sensitivity reaches detection levels of one to two single mutant fragments from a 10 - ml blood sample ( 0.1% limit of detection ) , and analytic specificity is greater than 99.9999%. 
the fractional concentration or mutation allele frequency ( maf ) for a given somatic mutation is calculated as the fraction of ctdna harboring that mutation in a background of wild - type ctdna fragments at the same nucleotide position . 
when characterized alterations are referred to , vuss were excluded in the analysis . overall , 76 ( 81% ) of 94 patients who had ctdna results also had ngs performed by agencies accredited by clinical laboratory improvement amendment and the college of american pathologists on tumor tissue using the foundationone assay ( hybrid capture–based ngs ; 182 , 236 , or 315 genes , depending on the time period )  . 
 kappa values range from 1 ( perfect agreement ) to 0 ( no agreement other than what would be expected by chance ) .26 matched therapy and actionability we retrospectively analyzed the treatments given after ctdna testing and compared the clinical outcomes among patients who received matched and unmatched therapies . 
patients were evaluable if therapy was administered for more than 10 days . actionability implies that the protein product of a genomic abnormality can be affected by a specific targeted drug.27 a potentially actionable alteration was defined as an alteration that was either the direct target ( such as an egfr inhibitor targeting an egfr mutation ) or a pathway component ( such as an mtor inhibitor for a pik3ca mutation [ because mtor is downstream of pik3ca ] ) that could be targeted by at least one us food and drug ( fda ) approved or investigational drug in a clinical trial . 
actionability was considered at the variant level ; vuss ( functional consequences and clinical significance of these gene variants are not established , as opposed to characterized alterations ) were considered nonactionable . statistical analysis medians and 95% cis or ranges were reported . 
progression - free survival and overall survival were analyzed by the kaplan - meier method , 28 and the log - rank test ( univariable analysis ) or cox regression model ( multivariable analysis ) was used to compare variables . 
 sequential her2 - targeted therapy in salivary ductal carcinoma with her2 / neu overexpression and a concomitant erbb2 mutation introduction salivary duct carcinoma ( sdc ) is a rare malignancy arising from the major and minor salivary glands.1 , 2 though curative intent surgery followed by radiation is the treatment of choice , locoregional recurrences and distant metastases are frequently reported.1 , 3 cytotoxic chemotherapeutic agents such as platinum analogs , anthracyclines , and taxanes have been effective in treating sdc but there is no established standard of care.4 overexpression of her2 / neu is frequently seen ; thus , trastuzumab can be an effective treatment option.3 however , the role of other her2 - targeting agents after trastuzumab failure is unclear . 
this involved a multiplex polymerase chain reaction using the ion ampliseq cancer hotspot primer pool and the ion ampliseq library kit ( both from thermo fisher scientific , waltham , ma )  . 
 data files were analyzed using the ion torrent variant caller software and integrated genomic viewer ( thermo fisher scientific ) using hg19 reference sequence and nextgene software ( softgenetics , state college , pa )  . 
in addition , three possible germline polymorphisms ( met n375s , tp53 p72r , and flt c.1310 3t > c ) were also detected in the tumor specimen . the patient was treated postoperatively with concurrent radiation to the parotid bed and right neck ( 6 , 600 cgy in 33 fractions over 48 days ) , with high - dose cisplatin ( 100 mg / m2 every 3 weeks for two cycles ) as radiation sensitizer . 
 he underwent a left modified radical neck dissection followed by concurrent radiation ( 6 , 000 cgy in 30 fractions over 42 days to the left neck and 6 , 600 cgy in 33 fractions over 48 days to the axilla ) with weekly carboplatin ( area under the curve , 2 ) because of marginal renal function . 
he had a possible anaphylactic reaction to pertuzumab and hence received trastuzumab monotherapy for seven additional cycles and had a partial response . the patient was disease free for 6 months and then a biopsy - proven recurrence was found on his sk lapatinib treatment was restarted and continued for 9 months ; there was a complete clinical response in his sk the patient is currently on a drug holiday , with no obvious recurrence . 
biopsy - proven skin recurrence of salivary duct carcinoma with complete resolution with lapatinib treatment . two years after the initial recurrence , the patient underwent a wedge resection of a solitary metastasis in the right lower lobe of the lung . nine months later , disease progression was detected in the right axillary and mediastinal lymph nodes . 
 however , because of the response of the sdc to her2 - targeted therapies.3 her2 overexpression is associated with higher recurrence risk , shorter survival , and aggressive clinical behavior with early regional and distant metastasis.2 taxanes and trastuzumab have been used anecdotally in metastatic sdc with her2 / neu overexpression with a good response , 7 but the role of targeted therapy remains unclear . in this case report , we have demonstrated the successful sequential use of her2 - targeted therapy in her2 - overexpressing metastatic sdc . 
after an initial response to trastuzumab docetaxel combination therapy , her2 - targeted agents , with differing mechanisms of action , were used sequentially with good response and prolonged disease control . 
the patient received , sequentially , trastuzumab ( which binds to the extracellular segment of the her2 receptor , neutralizing it and also producing antibody dependent cellular cytotoxicity ) , lapatinib ( which binds to the atp - binding pocket of the her2 protein kinase domain and inhibits tyrosine kinase activity ) , and ado - trastuzumab emtansine ( an antibody - drug conjugate in which trastuzumab inhibits the her2 receptor and the cytotoxic maytansinoid portion of the conjugate is delivered intracellularly and produces cell death )  . 
also , to the best of our knowledge , this is only the second report of ado trastuzumab emtansine use in sdc8 and the first describing sequential her2 targeting in this disease . sequential her2 - directed therapy is well established in metastatic her2 - positive breast cancer , with frontline chemotherapy along with dual her2 - targeted therapy with trastuzumab and pertuzumab followed by other her2 targeted agents.9 a higher absolute erbb2 gene copy number results in shorter time to metastasis in trastuzumab - untreated early breast cancer but longer progression - free survival upon initiation of trastuzumab - based treatment.10 though her2 - positive tumors are highly sensitive to the receptor blockade , resistance has been shown to develop through three main mechanisms : ( 1 ) incomplete blockade as a result of redundant ligands and receptors that enable alternative dimerization patterns ; ( 2 ) reactivation of pathway signaling at or downstream of the receptor layer , such as activating mutations in the pik3ca pathway ; and ( 3 ) use of pre existing or acquired alternate pathways , such as hormone - receptor pathways.11 interestingly , the patient in this case report had an erbb2 v777l mutation in a metastatic specimen before exposure to her2 - targeted therapy . 
the incidence of erbb2 mutations in sdc is uncommon , with a recent series reporting a mutation in four of 37 patients studied.12 all these mutations were in exons 17 through 19 ( namely , i767m , d769y , g776v , and v842i ) , but this series did not identify the v777l mutation . 
the coexistence of her2 amplification and her2 mutation is extremely uncommon because most of the her mutations reported in breast cancer have been seen in patients with her2 neu amplification - negative disease.13 there are limited data on the role of this mutation in breast cancer . 
v777l is present in the proximity of the kinase domain dfg motif of the erbb2 gene ( exon 20 ) and functions as an activating mutation.13 the effects of the erbb2 v777l mutation on response to her2 - targeted therapy are less clear . 
some in vitro studies suggest that this mutation did not affect the sensitivity to her2 - directed therapies in proliferation assays13 ; however , a recent report suggests that the presence of this mutation confers resistance to trastuzumab.14 because the coexistence of her2 amplification and erbb2 mutation is extremely rare , the effect of this combination on response to her2 - targeted therapy is unknown . 
 however , it is possible that the combination of these two events may have led to the excellent response seen in the patient in this report . though this is a single report , we believe that testing for the presence of an erbb2 mutation may be indicated in patients with sdc who have her2 amplification . 
sequential her2 targeting , similar to the breast cancer paradigm , may be a feasible approach in her2 - overexpressing sdcs , especially in those with a concomitant activating erbb2 mutation . 
locati ld , perrone f , cortelazzi b , et al : clinical activity of androgen deprivation therapy in patients with metastatic / relapsed androgen receptor - positive salivary gland cancers . 
van boxtel w , boon e , weijs wlj , et al : combination of docetaxel , trastuzumab and pertuzumab or treatment with trastuzumab - emtansine for metastatic salivary duct carcinoma . 
borley a , mercer t , morgan m , et al : impact of her2 copy number in ihc2 + / fish - amplified breast cancer on outcome of adjuvant trastuzumab treatment in a large uk cancer network . 
 personalized clinical decision making through implementation of a molecular tumor board : a german single - center experience rouven hoefflin anna - lena geiler ralph fritsch rainer claus julius wehrle patrick metzger meike reiser leman mehmed lisa fauth dieter henrik heiland thalia erbes friedrich stock agnes csanadi cornelius miething britta weddeling frank meiss dagmar von bubnoff christine dierks isabell ge volker brass steffen heeg henning schfer martin boeker justyna rawluk gian kayser simone hettmer hauke busch christoph peters martin werner justus duyster tilman brummer melanie boerries ( continued ) purpose dramatic advances in our understanding of the molecular pathophysiology of cancer , along with a rapidly expanding portfolio of molecular targeted drugs , have led to a paradigm shift toward personalized , biomarker - driven cancer treatment . 
here , we report the 2 - year experience of the comprehensive cancer center freiburg molecular tumor board ( mtb ) , one of the first interdisciplinary molecular tumor conferences established in europe . 
the role of the mtb is to recommend personalized therapy for patients with cancer beyond standard - of - care treatment . methods this retrospective case series includes 198 patients discussed from march 2015 through february 2017 . 
the mtb guided individual molecular diagnostics , assessed evidence of actionability of molecular alterations , and provided therapy recommendations , including approved and off - label treatments as well as available matched clinical trials . results the majority of patients had metastatic solid tumors ( 73.7% ) , mostly progressive ( 77.3% ) after a mean of 2.0 lines of standard treatment . 
 treatment recommendations were implemented in 33 of 104 patients ( 31.7% ) , of whom 19 ( 57.6% ) showed stable disease or partial response , including 14 patients ( 7.1% of the entire population ) receiving off - label treatments . conclusion personalized extended molecular - guided patient care is effective for a small but clinically meaningful proportion of patients in challenging clinical situations . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license personalized cancer medicine uses molecular biomarkers for standard - of - care treatment stratification , such as activating braf mutations for the treatment of melanoma with braf inhibitors.1 in parallel , it has become evident that therapeutic strategies with targeted drugs are no longer specific for the treatment of distinct entities but rather for particular molecular profiles across different cancers.2 - 4 thus , testing for single - drug targets can provide therapeutic information , but its predictive value may vary between entities . 
although an activating braf v600e mutation will predict response to braf inhibitors in melanoma , 1 it may not do so in colorectal cancers because of epidermal growth factor receptor ( egfr ) feedback activation with requirement of additional egfr targeting.5 , 6 moreover , non - v600 braf mutations might not be responsive to braf inhibition at all.7 thus , one - mutationone - drug approaches may be ineffective , especially in heavily pretreated patients with cancer . 
examples include the selection of ras mutant clones in colorectal cancer treated with egfr antibodies , such as cetuximab or panitumumab , 11 or the acquisition of a secondary egfr t790m kinase domain mutation mediating resistance to egfr kinase inhibitors , such as gefitinib or erlotinib in non small - cell lung cancer.12 , 13 this increasing amount of complexity requires tools to translate individual information into personalized treatment concepts . 
the initial discussion includes a clinical case presentation , review of the pathology data and the tumor - specific genetic landscape , known molecular predictive or prognostic markers , active clinical trials , and potential inand off - label molecular targeted treatments . 
 diagnostic results are presented to the mtb by the molecular pathology and / or the computational biologist teaafter discussion , treatment recommendations are given and are supported by levels of evidence ( data supplement )  . 
these are based on published molecular biomarker recommendations.14 patients and patient informed consent all patients discussed ( n = 198 ) were included in this retrospective single - center case series . 
patients with individual or family history indicative of germline disease - causing mutations were referred to the institute of human genetics for counseling and possibly germline genetic analyses . diagnostic molecular pathology appropriate tissues were subjected to molecular analyses as recommended by the mtb ( fig 1 )  . 
 a molecular diagnostic tests 36 16 immunohistochemistry per case 15 6 ddseq ( exon / hotspot ) hpv testing tngs rna - seq ( total = 867 ) ( total = 492 ) 7 - 8 antibodies ( total = 139 ) type of tngs 48 - gene panel 8 - gene panel brca1 / brca2 54 - gene myeloid panel 1 antibody 2 antibodies 3 antibodies 4 antibodies 5 antibodies 6 antibodies not annotated cosmic ( no hotspot ) hotspot ( not actionable ) actionable ( sensitizing ) actionable ( resistence ) tumor entities brain colorectal melanoma thyroid other gastrointestinal fig 1 . 
 ( a ) the panels depict the type of molecular diagnostic testing performed ( left panel ) and specify the number of immunohistochemical stains ( one to eight antibodies ) per case ( middle panel ) as well as the type of targeted next - generation sequencing ( tngs ) library sequenced ( right panel )  . 
tngs was performed either by a custom panel ( eight - gene panel ) , a 48 - gene panel ( truseq amplicon cancer panel , illumina , san diego , ca ) , a 54 - gene myeloid panel ( trusight myeloid sequencing panel , illumina ) or a custom brca1 / 2 consortium panel . 
differentially expressed genes were identified using the limma voom package from r / bioconductor.34 , 35 results from march 2015 through february 2017 , 49 mtb meetings were attended by a median of 16 physicians and scientists , ensuring continuous interdisciplinary data interpretation and discussions with diagnostic and therapeutic decision making . 
the workflow of the mtb included a case and literature review , molecular diagnostic recommendations , and follow - up discussions of the molecular diagnostic results , including treatment recommendations ( appendix fig a1 )  . 
these included 305 diagnostic and 104 treatment recommendations . patient characteristics the average patient age at the time of the initial mtb presentation was 58 years ( range , 1 to 85 years )  . 
the majority of patients ( n = 146 ; 73.7% ) suffered from metastatic disease , and 77.3% ( n = 153 ) showed disease progression while receiving the standard treatment ( table 2 )  . 
of all diagnostic recommendations , 234 ( 76.7% ) were implemented , resulting in 867 single diagnostic tests ( mean , five per patient ) , including 815 routine molecular tests and 52 extended genetic analyses ( fig 1a , left panel )  . routine molecular diagnostics included immunohistochemical ( ihc ) staining for biomarkers ( n = 492 ; fig 1a , middle panel ) , such as programmed death - ligand 1 ( pd - l1 ) and mismatch repair proteins , in situ hybridizations ( ish ) for gene copy number analyses ( n = 92 ) , and testing for microsatellite instability and / or gene hotspot variations ( n = 89 ) and tngs ( n = 139 ; fig 1a )  . 
the most frequent cosmic annotated sequence variants detected by tngs occurred in tp53 , brca1 , kdr , kit , kras , pik3ca , brca2 , and braf ( fig 1b ; data supplement )  . 
therapeutically relevant mutations in hotspot regions were identified in 41 of 139 patients ( 29.5% ) , including drug - sensitizing variants in braf , pik3ca , idh1 , egfr , and kit , as well as drug resistance variants in kras and nras . extended genetic analyses including exome and transcriptome assays were performed for 36 patients ( 18.2% ; wes and rna - seq : n = 35 ; rna - seq only : n = 1 )  . 
patient characteristics characteristic total , no . female male ( range ) tumor type soft tissue unknown primary site colorectal urogenital thyroid breast lung skin hepatobiliary upper gi tract hematologic neuroendocrine pediatric head and neck others complete remission localized disease metastatic disease 2 to 3 rare tumor others solid tumors ( n = 189 ) : stage at presentation no . 
ninety of 104 treatment recommendations ( 86.5% ) were either off - label therapies ( n = 77 ) or trial inclusions ( n = 13 )  . the implementation rate of treatment recommendations was 31.7% ( 33 of 104 )  . 
in - label recommendations were pursued in nine of 14 cases ( 64.3% ) , whereas off - label recommendations and trial inclusions were implemented in only 28.6% ( 22 of 77 ) and 15.4% ( two of 13 ) of the cases , respectively . 
intended trial inclusion in 11 patients failed because of poor performance status or patient death ( n = 5 ) , closed trial arm ( n = 4 ) , or patient will ( n = 2 )  . 
main reasons for nonimplementation of treatment recommendations included loss to follow - up ( 22.5% ) , recommendation in the future ( 19.7% ) , patient death ( 16.9% ) , patient will ( 14.1% ) , and medical reasons ( 14.1% ; data supplement )  . 
of note , evidence level of individual off - label recommendations did not affect implementation rates ( data not shown )  . clinical outcome dgi and target databases , respectively ( data supplement )  . 
 the disease impact of non - hotspot mutations is more difficult to evaluate ; however , it can lead to in 33 patients with implemented treatment recommendations , partial remissions ( pr ) and stable diseases ( sd ) were seen in 11 ( 33.3% ) and eight patients ( 24.2% ; table 1 ) , respectively . 
of 14 responders receiving off - label therapies , eight ( 57.1% ) showed a progression - free survival ( pfs ) ratio ( pfs2 / pfs1 ; pfsr ) > 1.3 , supporting the impact of the recommended therapies.36 three patients had a pfsr < 1.3 with ongoing responses , meaning that their pfsr is still increasing . 
to assess whether implementation of treatment recommendations affected overall survival from first mtb discussion , we analyzed all patients with stage iv malignancies according to three subgroups ( n = 148 ; fig 3 )  . 
the median survival was not reached for patients with implemented treatment recommendations ( n = 33 recommendations pursued ; 95% ci , 9 months to not reached ) , 8 months for patients for whom treatment recommendations were not implemented ( n = 43 recommendations not pursued ; 95% ci , 3 to 10 months ) , and 10 months for patients who did not receive a treatment recommendation ( n = 72 no recommendations ; 95% ci , 7 to 17 months )  . 
predictive biomarkers for individualized immunotherapies are emerging and changing rapidly , with strong differences between entities.44 here , we used ihc for programmed cell death protein 1 ( pd - 1 ) / pd - l1 , tumor - infiltrating lymphocytes , microsatellite instability testing , and mutational burden assessment as predictive biomarkers . 
in the near future , identifying individual cancer neoantigens might allow a more precise prediction of responses to immunotherapies.45 this highlights the importance of an interdisciplinary mtb team that analyzes and interprets biomarkers to identify patients who might benefit from off label immuno - oncology treatments . in an mtb workflow , the portfolio of molecular diagnostic tests , as well as criteria to match and prioritize targeted therapies to molecular biomarkers , affects the probability to identify patients with actionable targets . 
our approach shares similarities with memorial sloan kettering integrated mutation profiling of actionable cancer targets ( msk - impact ) , focusing on therapeutically targetable biomarkers for fast clinical decision making and referral of patients to available clinical trials.51 targeted drug combinations might offer better dcr over single - agent therapies.52 - 55 in part , this is due to crosstalk between signaling pathways as well as spatial and temporal clonal heterogeneity , especially in patients with advanced cancer who failed standard - of - care treatment.56 , 57 most current programs for precision oncology use prespecified , genetically matched , single - agent treatments ( nci - match , clinicaltrials.gov identifier : nct02465060 ; or targeted agent and profiling utilization registry [ tapur ] , clinicaltrials.gov identifier : nct02693535 )  . 
these patients did not suffer from grade 3 to 4 adverse effects , although treatment combinations may bear a higher risk of toxicity.58 earlier referral to an mtb ( eg , after failure of first - line treatment ) might prevent the institution of ineffective treatments , improve the implementation rate , and increase the likelihood of success of molecular biomarkermatched treatments . 
 because of the low sample size and the heterogeneous composition of patients in the cohorts , the validity of this survival analysis is limited . access to molecular biomarkermatched , off label agents for cancer treatment is limited . 
in a recent single - center study , only 5% of molecular biomarkermatched treatment recommendations were implemented , mainly because of limited access to clinical trials or to restricted use of drugs outside their marketed label.59 thus , it is crucial to build up platforms for patients and treating physicians to link individual molecular information of the tumor to appropriate nonapproved drugs and available clinical trials . 
in rare entities , and especially in the setting of treatment - refractory cancers , precision oncology networks should allow hypothesis - driven in vitro studies and validation in small sets of individuals . 
thus , within the concept of patient - centric , biomarker - driven trial designs , 60 an mtb might constitute a critical tool to identify informative patients for clinical trials of targeted therapies in rare molecular subgroups . in summary , this mtb experience illustrates that patient management , on the basis of individual molecular biomarker profiling and analysis , is feasible in patients beyond standard - of - care treatment . 
 hauke busch no relationship to disclose christoph peters leadership : university medical center freiburg honoraria : university medical center freiburg travel , accommodations , expenses : novartis martin werner honoraria : diakovere consulting or advisory role : roche , novartis , johnson & johnson research funding : agilent ( inst ) , novartis ( inst ) , roche ( inst ) justus duyster honoraria : novartis , genentech , pfizer consulting or advisory role : novartis , roche travel , accommodations , expenses : novartis tilman brummer no relationship to disclose melanie boerries no relationship to disclose silke lassmann honoraria : novartis , astrazeneca travel , accommodations , expenses : novartis , astrazeneca nikolas von bubnoff honoraria : astrazeneca , amgen , bristol - myers squibb consulting or advisory role : novartis research funding : novartis travel , accommodations , expenses : novartis acknowledgment we thank the team of the genomics and proteomics core facility , german cancer research center / dkfz , heidelberg , germany , for their sequencing service . affiliations all authors : university of freiburg , freiburg ; ralph fritsch , julius wehrle , cornelius miething , christoph peters , martin werner , justus duyster , tilman brummer , melanie boerries , silke lassmann , and nikolas von bubnoff , german cancer consortium , partner site freiburg , and german cancer research center , heidelberg ; rainer claus , augsburg medical center , augsburg ; and hauke busch , university of lbeck , lbeck , germany . supported by comprehensive cancer center freiburg and by deutsches konsortium fr translationale krebsforschung grant no . 
hoadley ka , yau c , wolf dm , et al : multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origcell 158 : 929 - 944 , 2014 3 . 
hong ds , morris vk , el osta b , et al : phase ib study of vemurafenib in combination with irinotecan and cetuximab in patients with metastatic colorectal cancer with brafv600e mutation . 
goss g , tsai cm , shepherd fa , et al : osimertinib for pretreated egfr thr790met - positive advanced non - small - cell lung cancer ( aura2 ) : a multicentre , open - label , single - arm , phase 2 study . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
kovaleva v , geissler al , lutz l , et al : spatio - temporal mutation profiles of case - matched colorectal carcinomas and their metastases reveal unique de novo mutations in metachronous lung metastases by targeted next generation sequencing . 
kumar p , henikoff s , ng pc : predicting the effects of coding non - synonymous variants on protein function using the sift algorithnat protoc 4 : 1073 - 1081 , 2009 32 . 
von hoff dd , stephenson jj jr , rosen p , et al : pilot study using molecular profiling of patients tumors to find potential targets and select treatments for their refractory cancers . 
johnson db , dahlman kh , knol j , et al : enabling a genetically informed approach to cancer medicine : a retrospective evaluation of the impact of comprehensive tumor profiling using a targeted next - generation sequencing panel . 
massard c , michiels s , fert c , et al : high - throughput genomics and clinical outcome in hardto - treat advanced cancers : results of the moscato 01 trial . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
tos , tumorboard online system . online registration ( tos ) > 5 days prior to mtb treating physician analysis : adherence to recommendation entity expert review literature check trial availability molecular pathology review pathology review biomarkers treatment decision patient analysis : outcome 1 . 
 o impact of variable rna - sequencing depth on gene expression signatures and target compound robustness : case study examining brain tumor ( glioma ) disease progression alexey stupnikov paul g . 
mcart , phd , msc , bioinformatics group , health sciences ( continued ) purpose gene expression profiling can uncover biologic mechanisms underlying disease and is important in drug development . 
the impact of sequencing depth alteration on rna - seqbased downstream analyses such as gene expression connectivity mapping is not known , where this method is used to identify potential therapeutic compounds for repurposing . methods in this study , published rna - seq profiles from patients with brain tumor ( glioma ) were assembled into two disease progression gene signature contrasts for astrocytoma . 
gene signatures were subsampled to simulate sequencing alterations and analyzed in connectivity mapping to investigate target compound robustness . results data loss to gene signatures led to the loss , gain , and consistent identification of significant connections . 
 building , centre for cancer research and cell biology , queens university belfast , 97 lisburn rd , belfast , bt9 7bl , united kingdom ; twitter : @drdmcart ; e - mail : d.mcart@qub.ac.uk. measurement is rna sequencing ( rna - seq ) of cdna transcripts in a high - throughput manner . 
rna - seq has wider analytical capabilities , including single nucleotide variants , insertion - deletions , gene splice variants , post - transcriptional modifications , and gene fusion detection , but remains costly.6 , 7 experimental techniques developed to minimize sequencing costs include sample multiplexing . 
 multiplexing involves labeling each sample library with a barcode identifier , allowing multiple libraries to be pooled and sequenced simultaneously , reducing costs.7 - 10 smaller volumes of rna are analyzed for multiplexed samples ; thus , the downside to multiplexing is reduced sequencing depth for this library type . accurate assessment of transcripts depends on length , abundance , and mappability to the reference and sufficient sequencing depth , particularly for genes with low transcript abundance.11 , 12 sequencing depth alterations can affect the detection of differentially expressed genes ( degs ) and potentially the accuracy of rna - seqbased downstream analysis . 
few studies have assessed the impact of sequencing depth alterations on rna - seq downstream applications.13 more studies are required , particularly to assess applications that rely on precise gene signatures , informative in classifying cancer subtypes and improved prognostic and predictive outcomes.14 , 15 a gene signature is summarized by degs that collectively represent the most prominent features of a cancer subtype or disease progression phenotype . 
this could be particularly problematic for connectivity mapping that examines a gene expression signature contrast with the aim of predicting potentially therapeutic us food and drugapproved target compounds for repurposing.16 there is urgent need for new targeted therapies for gliomas , which are the most common form of primary brain tumor . 
gliomas can be classified from grade i to iv on the basis of histologic and molecular information.17 depending on the cell of origin , each neoplasm is classified as an astrocytoma , oligodendroglioma , or ependymoma . 
herein , reference gene signatures were compiled from publically available sequenced tumors for astrocytoma disease progression.2 subsampling was applied to simulate sequencing depth alterations of gene signatures , and the performance of connectivity mapping was assessed . 
results reveal that information loss to gene signatures significantly affects target compound robustness . methods published whole transcriptome sequencing data of brain tumor biopsy specimens from adults ( accession : gse48865 ; bao et al2 ) was downloaded from the sequence read archive.25 on average , samples had 50 million reads each . 
reads were quality controlled using trimmomatic software26 and aligned using bowtie2 , 27 allowing one mismatch against the human genome version hg38.28 aligned reads were mapped to genes from the grch38.81 annotation29 using samexplorer software.30 , 31 to benchmark a diverse range in performance of the rna - seq analysis , mapped reads were subsampled to simulate samples with a range of lower cdna library sequencing depths using a bioinformatics pipeline32 ( appendix fig a1 ; data supplement )  . 
the impact of information loss to gene signatures for degs , gene ontology ( go ) terms , and target compound detection was assessed using differential expression , go , and gene expression connectivity mapping analysis , respectively , with deseq2 , goseq , and the qub accelerated drug and transcriptomic connectivity ( quadratic ) software33 - 35 ( data supplement )  . 
the reproducibility of significant connections to the library of integrated cellular signatures identified for all cell lines and neuronal specific cell lines ( data supplement ) by quadratic was investigated16 , 36 - 38 ( data supplement )  . 
results and associated false discovery rates ( fdrs ) were visualized using the r packages venndiagram and ggplot2.39 , 40 results assessment of the l - h and h - h gene expression signatures l - h ( dataset_i ) and h - h ( dataset_ii ) gene signature contrasts comprised 47 and 33 patients , respectively ( data supplement )  . 
 when data input was reduced , the fdr for the number of degs detected increased linearly and by approximately the same amount for both data sets ( appendix fig a2 )  . 
dataset_i gene signature therefore demonstrated better resilience to data loss for deg detection compared with dataset_ii . for the full data set ( f = 1.0 ) , > 200 go terms described the functions of the degs identified for dataset_i ( appendix fig a3a )  . 
given this low number , which reduced to zero on f = 0.5 , go results for subsampled dataset_ii are not depicted . impact of information loss to gene signatures used in gene expression connectivity mapping for the full data set , a greater number of significant reverse ( rev ) and progress ( prog ) connections were identified for dataset_i compared with dataset_ii ( fig 2a )  . 
effect of decreased cdna library sequencing depth on the number of differentially expressed genes ( degs ) detected from ( a ) dataset_i , and ( b ) dataset_ii gene signatures ( data supplement )  . 
visualization of the global stratification ability of ( c ) dataset_i , low to high ( l - h ) , and ( d ) dataset_ii , high to high ( h - h ) gene signatures . 
heatmap was generated using unsupervised hierarchical clustering with the full rna - seq data ( f = 1 ) and depicts the gene expressional patterns of the top 100 differentially expressed genes identified between the gene signature contrast groups . 
the who disease grades of samples as determined by bao et al2 are overlaid . target compound identification , dataset_i was therefore more resilient to alterations in cdna sequencing depth compared with dataset_ii . the impact of data loss to gene signatures and the reproducibility of connectivity mapping is presented in figures 3 - 5 . 
for example , 3 , 135 rev connections were identified for dataset_i using f = 1.0 ; this increased to approximately 5 , 000 when subsampled to f = 0.01 , but approximately 60% of compounds were infrequently detected ( figs 3b and 3c )  . 
for dataset_i , when 50% of reads were removed , approximately 62.5% rev ( approximately 2 , 500 of 4 , 000 ) and approximately 50% prog significant connections ( approximately 1 , 500 of 3 , 000 ) were identified with every iteration ( fig 3 )  . 
for dataset_ii , when 50% of reads were removed , just approximately 13% rev ( approximately 400 of 3 , 000 ) and 9% prog significant connections ( approximately 180 of 2 , 000 ) were identified with every iteration ( fig 4 )  . 
 significant connections that potentially could progress ( prog ) or reverse ( rev ) the disease phenotype and fdrs are plotted against the data fraction included in the analysis ( f )  . 
when affected by data loss , connectivity mapping results were more robust for dataset_i compared with the dataset_ii gene signature . reducing data to the gene signature led to the loss , gain , and consistent identification of significant connections to target compounds ( fig 5 )  . 
for dataset_i , a large proportion of the significant connections across all cell lines ( 69% ; 2 , 195 of 3 , 135 ) and neuronal - specific cell lines ( 70% ; 144 of 205 ) were detected with all data fractions . 
similarly for dataset_ii , a proportion of the significant connections across all cell lines ( 7% ; 100 of 1 , 339 ) and neuronal specific cell lines ( 5% ; nine of 172 ) were detected with all data fractions . 
for example , 350 and 105 compounds were detected across all cell lines for dataset_i , f = 0.01 , that were not identified by the full data set ( fig 5a )  . 
when 50% of the reads were removed , nine and 27 target compounds identified for neuronal specific cell lines were lost , respectively , for dataset_i and ii ( figs 5c and 5d ; table 1 )  . 
thus , for dataset_ii , more target compounds identified by the full gene signature were lost , and some of these included compounds of potential clinical utility for glioma , such as suramin and dasatinib ( table 1 )  . 
a comparison of the rate of impact of data loss on go terms and significant connections detection for dataset_i can be seen by comparing figures a3 and 2a . discussion understanding molecular pathways and regulatory networks driving cancer is essential for the development of new therapies . 
gene expression profiling using rna - seq has led to the development of clinically relevant gene signatures that are informative for cancer subtypes.14 , 15 rnaseq experimental approaches such as sample multiplexing reduce cdna sequencing depth and potentially affect gene signature accuracy . 
significant connections to target compounds identified from the neuronal derived cell lines using ( c ) dataset_i and ( d ) dataset_ii across gene signatures are also compared . more patient gene expression profiles matched their who grade classifications . 
results support the subjective nature of tumor classification , which has interobserver variability.41 gene signatures provided a framework to assess connectivity mapping output for a well - performing accurate versus a poorer - performing less accurate contrast . characterization of the disease progression gene signatures revealed they differed in biologic complexity . 
thus , the l - h gene signature was more resilient to data loss for deg detection and had greater test sensitivity compared with the h - h gene signature . 
overall , the number of significant connections detected for the l - h gene signature was greater , most likely explained by the heterogeneous biologic mechanisms involved in lowto high - grade astrocytoma transition . 
with data loss , this fdr threshold was reached by the l - h and h - h gene signatures , respectively , when 70% and just 1% of reads were removed . 
thus , the l - h gene signature was more resilient to data loss for the detection of significant connections to target compounds using connectivity mapping . subsampling of gene signatures for connectivity mapping revealed that the suite of significant connections to target compounds became modified with data loss . 
compounds lost by the h - h gene signature ( who grade iii to iv ) included suramin , lopinavir , dasatinib , and vincristine , which have already been considered as glioma treatments . 
suramin , an anticancer agent , inhibits the binding of growth factors understood to play a role in glioma progression , angiogenesis , and radioresistance and has been used to treat newly diagnosed gbms.42 , 43 lopinavir , a protease inhibitor , has reached phase ii clinical trials for the treatment of high - grade glioma.44 dasatinib , a kinase inhibitor that acts on members of the src family of kinases , is well studied in glioma and has shown preclinical promise.45 vincristine , a spindle poison , is used in combination with procarbazine and lomustine to treat high - grade glioma and has also been successful in a phase iii trial for the treatment of low - grade gliomas.22 , 46 , 47 reductions in transcript abundance probably led to the loss of low - abundance genes from the full gene signature and altered the degs detected , leading to the loss of these connectivity mapping connections . 
fewer significant connections identified by the full data sets were lost by the l - h gene signature compared with the h - h gene signature , suggesting it was more resilient to data loss . 
similarly , in another subsampling rna - seq study of healthy organisms from multiple taxa , highly expressed genes regulating metabolism and pathogenesis of disease were consistently identified even when downsampling rna - seq reads to only 1 million reads , 13 thereby corroborating our findings from diseased tumors . results highlight the need for determining the optimal cdna sequencing depth for accurately identifying degs when compiling gene signatures . 
in the future , rna standard and spike - in controls may be useful to inform rna - seq best practices.48 the accuracy of a gene signature was particularly important when carrying out additional downstream analyses , such as connectivity mapping . 
given the instability of gene expression , perhaps using ontology types or ontotypes49 to characterize contrast phenotypes may be a more reliable approach compared with gene lists in connectivity mapping . 
other target compounds sensitive to data loss are being tested for their biologic efficacy against glioma stem cells using clonogenic cell survival assays and western blot analyses in ongoing studies by this research group . 
roddy no relationship to disclose manuel salto - tellez consulting or advisory role : philips healthcare , visiopharm , bristol - myers squibb , merck patents , royalties , other intellectual property : co - inventor of qupath ( open - source system for digital pathology analysis ) darragh g . 
mcart , queens university belfast ; tom flannery , estelle healy , and manuel salto - tellez , belfast health and social care trust , belfast , united kingdom ; alexey stupnikov , johns hopkins university , baltimore , md ; hayley p . 
 kurian , brain tumour research centre , university of bristol , bristol , united kingdom ; and frank emmert - streib , tampere university of technology , tampere , finland . supported by funding from the brainwaves northern ireland charity ( registered charity number : nic103464 )  . 
bao zs , chen hm , yang my , et al : rna - seq of 272 gliomas revealed a novel , recurrent ptprz1 - met fusion transcript in secondary glioblastomas . 
verhaak rg , hoadley ka , purdom e , et al : integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in pdgfra , idh1 , egfr , and nf1 . 
wang l , si y , dedow lk , et al : a low - cost library construction protocol and data analysis pipeline for illumina - based strand - specific multiplex rna - seq . 
liu y , zhou j , white kp : rna - seq differential expression studies : more sequence or more replication ? bioinformatics 30 : 301 - 304 , 2014 13 . 
turkington rc , hill la , mcmanus d , et al : association of a dna damage response deficiency ( ddrd ) assay and prognosis in early - stage esophageal adenocarcinoma . 
lamb j , crawford ed , peck d , et al : the connectivity map : using gene - expression signatures to connect small molecules , genes , and disease . 
okamoto y , di patre pl , burkhard c , et al : population - based study on incidence , survival rates , and genetic alterations of low - grade diffuse astrocytomas and oligodendrogliomas . 
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stupp r , hegi me , mason wp , et al : effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase iii study : 5 - year analysis of the eortc - ncic trial . 
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effect of decreased cdna library sequencing depth on the number of gene ontology ( go ) terms identified for ( a ) dataset_i and ( b ) the false discovery rate ( fdr ) for the number of go terms identified . 
 multiplex gene profiling of cellfree dna in patients with metastatic melanoma for monitoring disease purpose hotspot blood cell - free dna ( cfdna ) biomarker assays have limited utility in profiling tumor heterogeneity and burden and in capturing regional metastasis with low disease burden in patients with melanoma . 
we investigated the utility of a sensitive 54cancer gene digital next - generation sequencing approach targeting blood cfdna single nucleotide variants ( snvs ) and copy number amplification for monitoring disease in patients with melanoma with regional or distant organ metastasis ( dom )  . patients and methods a total of 142 blood samples were evaluated by digital next - generation sequencing across two patient cohorts . 
cohort 2 consisted of 12 overlapping patients who were longitudinally monitored after complete lymph node dissection to dom . results in cohort 1 , cfdna snvs were detected in 75% of patients . 
in cohort 2 , 98 longitudinal blood samples along with matched regional and distant metastases from 12 stage iii patients were analyzed before complete lymph node dissection and throughout disease progression . 
2018 by american society of clinical oncology introduction after surgical resection of regional metastatic melanoma , longitudinal molecular profiling would greatly aid in monitoring for recurrence , progression , and therapeutic efficacy.1 molecular profiling of melanoma biopsies or tumors becomes more challenging when patients experience progression to distant organ metastasis ( dom ) as a result of procedure - related morbidity risks and limited sampling efficiency in capturing tumor heterogeneity.2 blood cell - free dna ( cfdna ) biomarkers are minimally invasive and potentially allow routine monitoring of molecular changes in patients cancer over the course of therapy and follow - up.3 this would enable monitoring of treatment efficacy , 4 recurrence , and / or subclonal mutation ( s ) tracking as tumors evolve or relapse.5 unfortunately , no blood - based melanoma biomarker is available for early detection of recurrence , particularly in patients with stage iii melanoma rendered clinically disease free upon surgery , except the problematic surrogate biomarker , serum lactate dehydrogenase ( ldh ) .6 to address this need , we pioneered the investigation of selena y . 
 cfdna utility7 - 9 and explored different types of cfdna biomarkers for monitoring patients with melanoma.4 , 9 - 12 a comprehensive profile of cfdna mutations given the recent genomic classification of cutaneous melanoma ( braf , nras , nf1 , and triple wild - type ) 13 , 14 would provide real - time monitoring of postoperative residual disease to capture progression and enable earlier detection of recurrence compared with clinical or radiologic detection . 
hotspot target blood assays ( braf mutations ) may not be suitable to comprehensively profile metastatic melanomas given the high intratumor heterogeneity.15 - 17 blood cfdna profiling using a cancer panel can address this heterogeneity issue if highly sensitive for metastatic disease . 
overall , melanoma cfdna profiling could provide a clinically informative approach for monitoring disease progression , heterogeneity , and earlier detection of dom . in this study , we systemically assessed the utility of profiling melanoma blood cfdna using a sensitive digital next - generation sequencing ( ngs ) assay that includes a panel of 54 cancer genes in our clinically well - annotated patient cohorts with melanoma , with follow - up during a clinically disease - free period . 
specifically , we focused on longitudinal cfdna follow - up analysis in patients before curative surgery of american joint committee on cancer ( ajcc ) stage iii regional metastatic disease and throughout disease progression until the development of dom . 
the second cohort ( cohort 2 ) included patients overlapping with cohort 1 ( table 1 ) who were enrolled in a us food and drug administrationregistered phase iii clinical trial for ajcc stage iii melanoma ( clinicaltrials.gov identifier : nct00052130 ; appendix fig a1 ) .19 briefly , after complete lymph node dissection ( clnd ) to render patients clinically disease free , patients were randomly assigned to one of the following two treatment arms : bacillus calmette - gurin plus canvaxin melanoma vaccine ( cancervax , carlsbad , ca ) or bacillus calmette - gurin plus placebo , with all patient belonging to the treatment ar no statistically significant clinical difference between the two randomized treatment arms was reported.20 cohort 2 patients were selected on the basis of available blood samples during follow - up that were in accordance with the clinical trials protocol . 
ninety - eight blood samples were collected at baseline ( before clnd or before study ) and during follow - up with available paired formalin fixed paraffin - embedded ( ffpe ) tumors . 
specifically , the blood was collected before clnd ( ajcc stage iii disease ) and during follow - up every 2 to 4 months before dom ( ajcc stage iv disease )  . 
the follow - up time points were based on defined patient visits at 2 , 4 , and 12 months in year 1 and every 6 months in years 2 and 3 . 
standard clinical follow - up consisted of a patient visit with serum ldh blood testing , x - ray imaging every 3 months , and annual ct of the chest , abdomen , and pelvis along with brain ct or magnetic resonance imaging . 
this study was performed in accordance with reporting recommendations for tumor marker prognostic studies.21 sample collection and dna purification peripheral blood was collected , and serum was isolated , centrifuged , and filtered before cryopreservation in aliquots at 80c , as previously described7 , 11 under good laboratory practice conditions . 
the panel covers all known frequent melanoma driver mutations14 and includes full exon coverage of 18 genes , critical exons for the 36 remaining genes ( ie , having somatic mutations reported in the catalogue of somatic mutations in cancer23 ) , and three copy number variations ( cnvs )  . 
 the variant allele fraction ( vaf ) was calculated as the number of cfdna molecules with variants at a given nucleotide position divided by the total number of unique cfdna molecules at that position . 
cfdna snvs were categorized as somatic variants through referencing the catalogue of somatic mutations in cancer database23 or as variants of uncertain significance upon additional reference to the database of short genetic variation . 
snv load was calculated on the basis of the number of unique snvs per patient excluding cnvs . validation of digital ngs to validate cfdna snvs identified by digital ngs , dna from the resected matched tumor ( regional or distant metastasis ) was subjected to custom targeted sequencing using a truseq custom amplicon panel ( illumina , san diego , ca ) as performed by the john wayne cancer institute sequencing center . 
libraries were prepared with the truseq custom amplicon low input library prep kit ( illumina ) , following the manufacturers protocol , and sequenced on the illumina miseq with 150 - base pair single - end reads . 
raw sequencing reads were trimmed using trimmomatic ( version 0.33 ) , 24 mapped to the human genome ( 1000 genomes ( b37 ) build ) using bwa ( version 0.7.12 ) 25 at default settings , and processed using gatk ( version 3.4 ) 26 base quality score recalibration and indel realignment in accordance with the gatk best practices recommendations . 
the number of reads mapping to each locus of interest was counted using the mpileup function in samtools.27 concordance analysis snv concordance in paired tumor tissue and blood samples was determined as positive when the mutation was found in both tumor and cfdna or negative when cfdna snvs were not detected in the paired tumor . 
comparison of cfdna analysis versus radiologic imaging or ldh for detection of dom was assessed using fishers exact test , where an ldh cutoff value of 190 u / l was used.6 fishers exact and f tests were performed for categorical and continuous variables , respectively . 
cfdna status cutoff values were evaluated using the cutp ( ) function in statistical r package , survmisc.28 the gompertz survival regression29 - 31 was used to evaluate the disease - free survival ( dfs ) differences between cfdna snv groups . 
a single blood sample obtained from patients in cohort 1 was analyzed for known os outcome based on ( a ) snv load and ( b ) snv burden . regression was used for adjusting clinical factors in multivariable analyses . 
all statistical analyses were performed with r studio ( r studio , boston , ma )  . results cfdna snvs and association with outcomes the digital ngs assay was used to analyze 54 clinically relevant cancer genes covering all major melanoma driver genes ( appendix table a2 ) in blood cfdna of patients with ajcc stage ii , iii , or iv melanoma ( cohort 1 ; appendix table a1 ) collected before dom relapse or elective surgery . 
 the most frequently mutated genes , braf , tp53 , and nras ( appendix fig a2 ) , align with those previously reported in our studies in the melanoma tissue mutational landscape.13 , 14 to confirm that cfdna somatic snvs ( appendix table a3 ) were tumor derived , custom targeted amplicon sequencing was performed in matched tumor dna ( cohort 1 )  . 
multivariable analysis showed that higher snv load and burden were independent prognostic factors for worse os and dfs after adjusting for age , sex , and m category ( appendix tables a6 and a7 )  . 
 a pre - op < 0.1 clinically disease free disease progression curative sg ( clnd ) 2 - 4 mos 2 - 4 mos 2 - 4 mos 2 - 4 mos 2 - 6 mos cfdna snvs / cnvs detected recurrence lead time pre - op clinical recurrence 0 - 13 mos follow - up after recurrence pre - op sb12 disease free distant mets pre - op disease free distant mets pik3ca ( i663s ) met ( r1166q ) braf ( v600k ) ezh2 ( y646f ) myc ( l97f ) tp53 ( l111q ) apc ( t1689t ) egfr ( p589p ) ldh ( nd ) < 0.1 time after clnd ( mos ) time after clnd ( mos ) fig 2 . 
clnd , complete lymph node dissection ; cnv , copy number variation ; mets , metastases ; mos , months ; ldh ( nd ) , lactate dehydrogenase not done longitudinally ; pre - op , preoperative ; sg , surgery ; snv , single nucleotide variant ; vaf , variant allele fraction . cfdna profiling after clnd digital ngs was performed to longitudinally profile cfdna snvs in 98 blood samples collected from 12 patients with melanoma ( cohort 2 )  . 
the cfdna analysis of all serially collected blood samples is summarized per patient over longitudinal follow - up for somatic snvs ( appendix table a8 ) and variants of uncertain significance ( appendix table a9 )  . 
the most frequent snvs detected were in tp53 ( 75% ) and braf ( 58% ; fig 3a ) , reflecting frequently reported metastatic melanoma dna mutations.14 lack of preclnd cfdna snv detection in three patients was unlikely to be a result of low tumor burden , because all three patients had stage iiic disease with positive lymph nodes ( sb3 , n = 9 ; sb7 , n = 1 ; and sb10 , n = 1 )  . 
increasing vaf ( p = .019 ) and snv burden ( p = .039 ) after relapse was strongly associated with disease progression in this patient cohort ( cohort 2 ; fig 3b )  . earlier detection of dom by longitudinal cfdna analysis we evaluated whether longitudinal cfdna profiles can detect residual or progressive disease after clnd to provide earlier detection of dom compared with clinical or radiologic imaging . 
 given their utility in ajcc staging32 and emerging prognostic utility in immunotherapy , ldh levels were also evaluated for recurrence monitoring.33 , 34 ldh values were longitudinally assessed in eight patients ( fig 4 )  . 
 ( b ) significant correlation of increasing variant allele fraction ( vaf ; left ) and total number of cfdna variants ( right ) with tumor burden in 11 patients . 
 in patient sb11 , the presence of new somatic snvs ( brafv600e and akt1e17k ) during follow - up and upon dom suggests the value of cfdna monitoring for tumor heterogeneity after surgery . 
altogether , the longitudinal cfdna snv profiles suggest the possibility of monitoring disease through detecting the dynamic cfdna snv levels during disease - free follow - up . met and egfr cfdna amplification the recent association of cnv detection in melanoma tumors with clinical outcome and treatment response35 , 36 has yet to be clearly demonstrated in blood . 
furthermore , two of four patients with preoperative egfr / met cfdna amplification had detectable egfr / met cfdna amplification postoperatively , reflecting residual disease presence . cfdna monitoring captures tumor evolution given high tumor heterogeneity , cfdna monitoring was evaluated to determine whether dynamic or evolving tumor snv profiles can be captured , precluding the need for repetitive invasive tissue biopsies . 
 the tumor - cfdna concordance , defined as the presence of cfdna snvs in any serially collected blood sample to any matched tumors sequenced , ranged from 66% to 100% , with an average concordance of 81.5% ( appendix table a11 )  . 
 cfdna profiling in patient sb4 ( fig 5a ) captured heterogeneous tumor clones 2 to 7 months before distant metastasis biopsy , as revealed by detection of cdkn2a and braf somatic snvs in blood . 
 we focused on cfdna profiling during longitudinal follow - up at clinically relevant times , namely before curative elective surgery , during disease - free follow - up , and at relapse . 
this is critical for understanding how to use long - term longitudinal cfdna analysis to best guide management of patients with melanoma . the study explores the application of cfdna analysis in melanoma for earlier recurrence detection compared with standard radiologic imaging after surgery , and the results support previous oncologic blood cfdna studies.38 - 41 however , this study provides a novel view of cfdna snv dynamics . 
the assay containing a cancer panel that includes known melanoma driver genes minimizes the need for tumor dna sequencing to identify baseline snvs and proved advantageous in profiling dynamic cfdna snv levels , particularly for snvs not detectable at the time of surgery . 
longitudinal follow - up captured dynamic cfdna snv levels , reflecting tumor heterogeneity , and the potential increase of subclonal cfdna mutation levels upon relapse , potentially indicative for alternative treatment regimens.49 , 50 the 54cancer gene panel also permitted evaluation of cnvs and snv load and burden in melanoma blood cfdna . 
recently , the association of melanoma tumor cnvs with therapeutic outcomes has suggested their potential use for monitoring disease.36 , 37 cfdna cnv utility was evident because egfr , erbb2 , and met cfdna amplifications were detected during follow - up , suggesting that cfdna amplification has the potential to monitor for occult micrometastatic residual disease after surgery . 
this study demonstrates the feasibility of detecting cfdna amplification of metastasis driver genes in melanoma , supporting the reported associations of met / egfr amplification with tumor progression and poor outcome.51 , 52 cfdna snv load and burden were independent prognostic factors for os in stage iv patients when age , sex , and m category were not significantly different between the dichotomized groups . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . snv load and burden factors were also analyzed as continuous variables ( data not shown ) and demonstrated the same finding . 
the association of cfdna snv load or burden status with worse os supports recent findings of a high cfdna snv load ( > three snvs ) correlating with immunotherapy response.53 altogether , this highlights a cfdna snv load and burden trend that needs further validation in a larger patient cohort in a multicenter study . 
in addition , future studies are needed to determine the impact of longitudinal follow - up cfdna monitoring in patients with early - stage regional melanoma . to our knowledge , this is the first study to report blood cfdna multiplex gene analysis in patients with regional ajcc stage iii melanoma metastasis over longitudinal follow - up through dom . 
 amirali talasaz employment : guardant health leadership : guardant health stock and other ownership interests : guardant health research funding : guardant health patents , royalties , other intellectual property : guardant health travel , accommodations , expenses : guardant health dave s.b. 
hoon consulting or advisory role : guardant health acknowledgment we acknowledge dr ian hutchinson , phd , editorial services , for his support in proofreading and editing of this article , and dr yuuki iida , md , for technical assistance in preparation of the article . 
lanman and amirali talasaz , guardant health , redwood city , ca ; and shu - ching chang , medical data research center at providence saint josephs health , portland , or . supported by the dr miriam and sheldon g . 
nicholl mb , elashoff d , takeuchi h , et al : molecular upstaging based on paraffin - embedded sentinel lymph nodes : ten - year follow - up confirms prognostic utility in melanoma patients . 
wittner c , park s , rifai n , et al : circulating tumor cells and circulating tumor dna as a real time liquid biopsy approach , in rifai n ( ed ) : tietz textbook clinical chemistry and molecular diagnostics ( ed 6 )  . 
zhang j , yao yh , li bg , et al : prognostic value of pretreatment serum lactate dehydrogenase level in patients with solid tumors : a systematic review and meta - analysis . 
hoshimoto s , kuo ct , chong kk , et al : aim1 and line - 1 epigenetic aberrations in tumor and serum relate to melanoma progression and disease outcome . 
harbst k , lauss m , cirenajwis h , et al : multiregion whole - exome sequencing uncovers the genetic evolution and mutational heterogeneity of early - stage metastatic melanoma . 
hoshimoto s , shingai t , morton dl , et al : association between circulating tumor cells and prognosis in patients with stage iii melanoma with sentinel lymph node metastasis in a phase iii international multicenter trial . 
morton d , mozzillo n , thompson j , et al : an international , randomized , phase iii trial of bacillus calmette - guerin ( bcg ) plus allogeneic melanoma vaccine ( mcv ) or placebo after complete resection of melanoma metastatic to regional or distant sites . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
li h , durbin r : fast and accurate short read alignment with burrows - wheeler transfor bioinformatics 30 : 2114 - 2120 , 2014 bioinformatics 25 : 1754 - 1760 , 2009 26 . 
martens a , wistuba - hamprecht k , geukes foppen m , et al : baseline peripheral blood biomarkers associated with clinical outcome of advanced melanoma patients treated with ipilimumab . 
wilson ma , zhao f , khare s , et al : copy number changes are associated with response to treatment with carboplatin , paclitaxel , and sorafenib in melanoma . 
almodovar k , iams wt , meador cb , et al : longitudinal cell - free dna analysis in patients with small cell lung cancer reveals dynamic insights into treatment efficacy and disease relapse . 
chang ga , tadepalli js , shao y , et al : sensitivity of plasma brafmutant and nrasmutant cell - free dna assays to detect metastatic melanoma in patients with low recist scores and non - recist disease progression . 
sanmamed mf , fernndez - landzuri s , rodrguez c , et al : quantitative cell - free circulating brafv600e mutation analysis by use of droplet digital pcr in the follow - up of patients with melanoma being treated with braf inhibitors . 
tsao sc - h , weiss j , hudson c , et al : monitoring response to therapy in melanoma by quantifying circulating tumour dna with droplet digital pcr for braf and nras mutations . 
disease - free survival ( dfs ) for cellfree dna ( cfdna ) single nucleotide variant ( snv ) load and burden in patients with stage iv melanoma ( cohort 1 )  . 
the kaplan - meier curves for ( a ) two cfdna snv load groups and for ( c ) two cfdna snv burden groups with the fitted ( dashed lines ) gompertz curves . 
lennerz author affiliations and support information ( if applicable ) appear at the end of this article . creative commons attribution non commercial no derivatives 4.0 license ( continued ) purpose targeted therapy is the cornerstone of treatment of advanced egfr - mutant nonsmall - cell lung cancer ( nsclc )  . 
here , we assessed workflows designed to rapidly identify patients with actionable egfr mutations and reduce time to initiation ( tti ) of epidermal growth factor receptor ( egfr ) directed therapy . patients and methods we performed rapid testing for egfr l858r mutations and exon 19 deletions on paraffin - embedded or frozen section biopsy specimens from newly diagnosed patients with metastatic nsclc by using an egfr - specific assay ( rapid test )  . 
to determine clinical utility , we assessed concordance with ngs results , turnaround time , and tti of egfr therapy , and we evaluated reimbursement data . results between january 2015 and september 2017 , we performed 243 rapid egfr tests and identified egfr mutations in 43 patients ( 18% )  . 
escalation of the initiative into an interdisciplinary ultra - rapid next - day frozen - section workflow for highly symptomatic patients ( n = 8 ) resulted in a reduction in the median ( standard deviation ) turnaround time to 1 0.4 days and allowed several patients to initiate therapy within 1 week of biopsy . 
2018 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license : introduction nonsmall - cell lung cancer ( nsclc ) is a heterogeneous disease composed of unique molecular subsets with distinct clinical outcomes.1 , 2 multiple randomized studies have established the superiority of molecularly targeted therapies versus chemotherapy for the treatment of egfr - mutant and alk - positive nsclc.3 - 6 in other molecular subsets , single - arm studies confirm that treatment with targeted therapies can induce durable responses.7 , 8 as drugs that target these molecular drivers are approved for first - line treatment , genotyping in newly diagnosed nsclc is considered the standard of care.9 because of the need to interrogate a growing number of genes , next - generation sequencing ( ngs ) has largely replaced traditional single - gene assays.10 guidelines endorsed by oncology and pathology societies recommend that molecular testing turnaround times not exceed 10 working days.9 genotyping by ngs requires complex bioinformatics that can create treatment delays . 
we selected egfr mutant nsclc on the basis of its relatively high prevalence ( 10% to 15% ) among patients with nsclc , 12 the lack of overlap between egfr mutations and other clinically relevant molecular alterations , 13 and the fact that egfr inhibitors were readily available for hospitalized patients . 
in the first phase ( january 2015 to may 2017 ) , rapid egfr testing was performed on tissue specimens from consecutive patients diagnosed with metastatic nsclc who were never smokers / minimal smokers ( 10 pack years ) or who were hospitalized at the time of diagnosis ( data supplement )  . 
we also identified a comparator population composed of 121 patients with egfr - mutant nsclc who were diagnosed before implementation of the rapid testing ( ie , historical cohort ; data supplement )  . 
isolated nucleic acids from tumor specimens were analyzed with our ngs assay that uses anchored multiplex pcr to detect single - nucleotide variants and insertions / deletions in a target set of cancer - related genes.15 in addition , we examined fusion transcripts and met exon 14 skipping by using an rna - based ngs solid - fusion assay.15 statistical and economic analysis for contingency analyses , we used t tests , fishers exact tests , and 2 statistics . 
we defined turnaround - time from the date of accessioning to the date of reporting , and we defined tti from the date of the diagnostic biopsy to the date of ingestion of an egfr tyrosine kinase inhibitor ( tki )  . 
 for economic assessment , we reviewed line items of reimbursement data ( january 3 , 2017 , to february 8 , 2018 ) and extracted ( by payor ) the number of encounters and claim adjustment codes , and defined reimbursed as a payment greater than 0 . 
note that there is a ( variable ) delay from reporting to treatment decision and initiation of therapy because of cost coverage determination , preauthorization requirements , etc.the ultra - rapid egfr testing pathway ( pathway c ) is a multidisciplinary workflow designed to improve turnaround time using fresh tissue ( frozen sections ) to extract nucleic acids . 
among the remaining 234 patients , 23 ( 10% ) did not have ngs results because of insufficient tissue or technical failure of ngs ( data supplement )  . diagnostic utility of the rapid egfr assay improvement in turnaround time . 
first , we compared the turnaround times of the rapid and ngs assays by reviewing all specimens submitted for genotyping between july 2014 and january 2017 ( fig 2b )  . 
through ngs , we identified one additional low - level ( allelic fraction , approximately 4% ) p.l858r case , which was missed by rapid egfr testing ( fig 3 )  . 
the mutation spectrum of the tumors that underwent rapid testing is depicted in figure 3 , and the data supplement shows probabilities of therapeutically actionable variants . clinical utility of rapid egfr testing tti of tki therapy . 
p values result from t test ( age ) , fishers exact test ( sex , histology ) , or 2 test ( ethnicity , smoking )  . * includes the patient with a false - negative egfr - positive result . tki as the primary measure of clinical utility of the rapid egfr test . 
we reviewed the medical records of the 39 patients with egfr - mutant disease who underwent both rapid testing and ngs to determine whether tki therapy was initiated before ngs results . 
 validation implementation of rapid egfr testing ngs panel rapid wt rapid mut rapid egfr assay rapid egfr assay exon 19 exon 20 exon 21 del / ins t790m l858r variant fig 2 . 
scatter plots portray turnaround times of all 243 rapid egfr samples ( jan 2015 to may 2017 ; black , egfr wild type [ wt ] ; red , egfr mutation [ mut ] detected ) and all specimens that underwent ngs ( gray dots ) during this period . 
a case report of a patient who benefitted from this effort and a summary of the experience to date are described in the next section . therapeutic utility of ultra - rapid testing . 
osimertinib was selected on the basis of its activity against leptomeningeal disease.19 within days , the cough resolved , and the patient experienced marked improvement in his swallowing and regained the ability to walk . 
figure 4d illustrates the difference in turnaround time for the rapid and ultra - rapid tests and the time to initiation of tki therapy for patients tested through each workflow ( fig 4d inset )  . 
the sites of disease and presenting symptoms of these patients are detailed in the data supplement , which also summarizes our ultra - rapid workflow . rapid testing is operationally and financially sustainable . 
the heatmap portrays clinicopathologic features ( top three rows ) , rapid egfr results , and key molecular drivers along with the results of next generation sequencing ( ngs ) based fusion detection , fluorescence in - situ hybridization ( fish ) , and ngs panel results . 
key findings include ( 1 ) an isolated false - negative rapid egfr result ( arrow ) , ( 2 ) the inability of the rapid egfr test to detect egfr mutations at other residues , ( 3 ) identification of at least one underlying driver mutation in more than 50% of all tested cases by using the integrated molecular diagnostic approach , and ( 4 ) the association between clinicopathologic features and certain key drivers ( eg , never - smoking women with adenocarcinoma and egfr v > 10 pack - year smoking history and kras )  . 
 review of 214 clinical encounters with 1 , 475 line items of reimbursement data showed large variability between payors ( range , 0% to 100% ) and that , overall , 63% of encounters received reimbursement ( fig 4f )  . 
these data indicate that rapid egfr testing in our practice is operationally and financially sustainable . discussion here , we report the results of a quality improvement initiative to explore parallel testing with a rapid egfr assay and ngs in newly diagnosed nsclc . 
our findings emphasize the diagnostic , clinical , and therapeutic utility of rapid egfr testing . metastatic nsclc is a devastating and incurable disease for which the prognosis is highly dependent on identification of actionable molecular drivers.2 the established efficacy of us food and drug administrationapproved targeted therapies in specific nsclc subsets and the increasing number of investigational compounds underscore the necessity of identification of molecular alterations in a timely fashion . 
 ( a ) event curve that shows rapid egfr test reporting times and a comparison of the tyrosine kinase inhibitor ( tki ) initiation times ( relative to date of diagnosis ) for patients in the rapid and historical cohorts . 
the second block shows the 49% of patients ( n = 17 of 35 patients ) with egfr - mutant disease who started a tki before next - generation sequencing ( ngs ) results were available . 
 ( c ) response to the egfr inhibitor osimertinib in a patient with nonsmall - cell lung cancer who underwent ultra - rapid egfr testing : ( left ) pretreatment image and ( right ) response after 3 weeks ; arrow indicates primary tumor . 
 ( d ) comparison of rapid test times ( average ) and the eight patients tested with the ultra - rapid protocol ( fig 1c ) ; inset shows event curve comparison of time to initiation of tki between the rapid and ultra - rapid subsets . 
 ( e ) turnaround times for rapid ( gray ) and ultra - rapid ( blue ) workflows in a 9 - month extended standardof - care evaluation phase ; red , cases with an egfr mutation . 
several studies suggest that genotyping with multiple single - gene assays may reduce reporting time20 , 21 ; however , selective interrogation of the ever - expanding set of targets is impractical and may be limited by tissue availability.22 at the time our initiative began , consensus guidelines supported testing newly diagnosed patients with nsclc for egfr mutations and rearrangements in alk and ros1.9 these mutually exclusive genetic alterations are predominantly found in neveror light - smokers.13 , 23 testing these alterations in two stages ( ie , restriction of testing for rearrangements to patients whose disease is wild type for egfr mutations ) may lead to unnecessary delays between diagnosis and initiation of treatment for a significant proportion of patients and potentially steer symptomatic patients away from molecularly targeted treatments . 
through co - testing , we detected alk and ros1 rearrangements in 11 and six patients , respectively , and identified molecular alterations with promising investigational therapies in more than 40 additional patients ( fig 3 )  . 
for example , in a recent retrospective analysis that involved 15 community oncology centers , the median turnaround time for egfr testing and ngs was 23 days and 30 days , respectively.24 although this practice is likely more common in the community setting , where in - house testing is infrequent , one study demonstrated that 19% of patients with egfr mutations or alk rearrangements at an academic medical center in canada initiated first - line chemotherapy before testing results became available.11 the sequence of therapies may be particularly important for those patients initially treated with immunotherapy , because recent studies demonstrate that immunotherapy is seldom effective for egfr - mutant nsclc and that treatment with immunotherapy before targeted therapy increases the likelihood of the development of significant toxicities.25 - 27 given these data , we believe that rapid testing should be performed in parallel with full ngs genotyping . with a parallel - testing approach , we successfully performed rapid testing and ngs in 90% of patients in our initiative with material from a single biopsy . 
because half of the patients with tissue / dna that was insufficient for the full ngs were effectively tested for both alk and ros1 rearrangements using either fluorescence in - situ hybridization or the fusion assay , 95% of the patients in the rapid cohort met the minimum molecular testing requirement proposed by current asco guidelines . 
although pcr - based plasma genotyping is an expedient alternative to ngsbased tissue genotyping , the sensitivity of liquid biopsy for detection of egfr mutations is lower than what we report here with the rapid assay.29 , 30 despite the high sensitivity and specificity of the rapid assay , half of the patients in our study did not start tki therapy before ngs results were available ( fig 4b )  . 
 because tti was assessed retrospectively for the historical cohort , the median of 37 days between diagnosis and tki therapy might be an overestimate ; however , a similar delay was observed in another study.31 it also is conceivable that the difference in tti might be explained by process improvements over time that have shortened the interval between requests for and receipt of a drug from specialty pharmacies . 
because tki use predated ngs results in approximately 50% of patients , the reduction in time to tki initiation in the rapid group relative to the historical cohort might also reflect faster ngs reporting times . 
the scale of ngs panels currently precludes significant reduction , and the median and mean turnaround times for ngs results in the ultrarapid cohort were 9 and 10 days , respectively . 
 as a result of the short duration of follow - up of our rapid cohort and imbalances between the rapid and historical groups ( including enrollment of patients in the rapid cohort into a consolidation radiation protocol ) , we were unable to evaluate differences in progression - free survival on egfr - directed therapy . 
lennerz the potential to defer brain radiation for these patients underscore the importance of rapid identification of egfr mutations.41 , 42 the small number of patients with egfr - mutant disease in our study also limits the potential impact of our findings . 
however , many laboratories are proficient in performing targeted egfr genotyping , 43 and our extended standard of care evaluation ( fig 4e ) and reimbursement analysis ( fig 4f ) indicate operational and financial sustainability . in summary , we demonstrate that expedited egfr genotyping enables early intervention with targeted therapies and allows symptomatic patients to access effective treatments . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . ibiayi dagogo - jack honoraria : foundation medicine consulting or advisory role : boehringer ingelheim administrative support : christopher g . 
farago honoraria : foundation medicine vania nose no relationship to disclose consulting or advisory role : pharmamar , takeda , abbvie , loxo , stemcentrx research funding : pharmamar ( inst ) , abbvie ( inst ) , astrazeneca ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) , loxo ( inst ) , ignyta ( inst ) miguel rivera consulting or advisory role : loxom asubio ( i ) speakers ' bureau : pfizer ( i ) research funding : advanced cell diagnostics , affymetrix travel , accommodations , expenses : pharmamar , abbvie , stemcentrx patents , royalties , other intellectual property : patents with affymetrix justin f . 
gainor honoraria : merck , incyte , ariad , novartis , pfizer consulting or advisory role : genentech , bristol - myers squibb , theravance , loxo , takeda , array biopharma , amgen research funding : merck ( inst ) , novartis ( inst ) , genentech , bristol - myers squibb ( inst ) , adaptimmune ( inst ) , astrazeneca ( inst ) , ariad , jounce therapeutics ( inst ) , blueprint medicines ( inst ) , moderna therapeutics ( inst ) , tesaro ( inst ) travel , accommodations , expenses : affymetrix ginger jiang employment : novartis ( i ) zofia piotrowska consulting or advisory role : boehringer ingelheim , astrazeneca , ariad , takeda , superdimension , guardant health , novartis , abbvie research funding : novartis ( inst ) , ariad ( inst ) , takeda ( inst ) , guardant health ( inst ) rebecca s . 
heist consulting or advisory role : boehringer ingelheim research funding : abbvie ( inst ) , novartis ( inst ) , roche ( inst ) , incyte ( inst ) , celgene ( inst ) , mirati therapeutics ( inst ) , peregrine pharmaceuticals ( inst ) , exelixis ( inst ) , millenium ( inst ) , debiopharm group ( inst ) , corvus pharmaceuticals ( inst ) valentina nardi stock and other ownership interests : ksq therapeutics ( i ) , navicor ( i ) consulting or advisory role : thermo fisher scientific ( i ) , cell signaling technology ( i ) , raze ( i ) , caloric tests ( i ) dora dias - santagata no relationship to disclose long p . 
le stock and other ownership interests : archer biosciences consulting or advisory role : archer biosciences patents , royalties , other intellectual property : i am a co - inventor of the anchored multiplex pcr technology which is licensed to archerdx . 
sequist honoraria : astrazeneca consulting or advisory role : astrazeneca , genentech , roche , bristol - myers squibb , pfizer research funding : boehringer ingelheim ( inst ) , clovis oncology ( inst ) , genentech ( inst ) , merrimack ( inst ) , novartis ( inst ) , astrazeneca ( inst ) , johnson & johnson ( inst ) , merck ( inst ) , pfizer ( inst ) , guardant health ( inst ) , incyte ( inst ) martha pitman consulting or advisory role : medtronic inga t . 
shaw honoraria : pfizer , novartis , roche , genentech , foundation medicine consulting or advisory role : pfizer , novartis , genentech , roche , ariad , ignyta , blueprint medicines , daiichi sankyo , emd serono , taiho pharmaceutical , kso therapeutics , natera , loxo , takeda research funding : pfizer , novartis , roche , genentech , a . 
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leighl nb , rekhtman n , biermann wa , et al : molecular testing for selection of patients with lung cancer for epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors : american society of clinical oncology endorsement of the college of american pathologists / international association for the study of lung cancer / association for molecular pathology guideline . 
gainor jf , varghese am , ou sh , et al : alk rearrangements are mutually exclusive with mutations in egfr or kras : an analysis of 1 , 683 patients with nonsmall - cell lung cancer . 
dias - santagata d , akhavanfard s , david ss , et al : rapid targeted mutational analysis of human tumours : a clinical platform to guide personalized cancer medicine . 
gainor jf , niederst mj , lennerz jk , et al : dramatic response to combination erlotinib and crizotinib in a patient with advanced , egfr - mutant lung cancer harboring de novo met amplification . 
yang jc - h , cho bc , kim d - w , et al : osimertinib for patients ( pts ) with leptomeningeal metastases ( lm ) from egfr - mutant nonsmall - cell lung cancer ( nsclc ) : updated results from the bloom study . 
barlesi f , mazieres j , merlio jp , et al : routine molecular profiling of patients with advanced non small - cell lung cancer : results of a 1 - year nationwide programme of the french cooperative thoracic intergroup ( ifct )  . 
distasio m , chen y , rangachari d , et al : molecular testing turnaround time for nonsmallcell lung cancer in routine clinicalpractice confirms feasibility of cap / iaslc / amp guideline recommendations : a single - center analysis . 
drilon a , wang l , arcila me , et al : broad , hybrid capture - based next - generation sequencing identifies actionable genomic alterations in lung adenocarcinomas otherwise negative for such alterations by other genomic testing approaches . 
garassino mc , cho bc , kim jh , et al : durvalumab as third - line or later treatment for advanced nonsmall - cell lung cancer ( atlantic ) : an open - label , single - arm , phase 2 study . 
lee ck , man j , lord s , et al : clinical and molecular characteristics associated with survival among patients treated with checkpoint inhibitors for advanced nonsmall - cell lung carcinoma : a systematic review and meta - analysis . 
sholl lm , aisner dl , varella - garcia m , et al : multi - institutional oncogenic driver mutation analysis in lung adenocarcinoma : the lung cancer mutation consortium experience . 
sacher ag , paweletz c , dahlberg se , et al : prospective validation of rapid plasma genotyping for the detection of egfr and kras mutations in advanced lung cancer . 
lee ck , davies l , wu yl , et al : gefitinib or erlotinib vs chemotherapy for egfr mutation positive lung cancer : individual patient data meta - analysis of overall survival . 
yang jc , wu yl , schuler m , et al : afatinib versus cisplatin - based chemotherapy for egfr mutationpositive lung adenocarcinoma ( lux - lung 3 and lux - lung 6 ) : analysis of overall survival data from two randomised , phase 3 trials . 
vergnenegre a , massuti b , de marinis f , et al : economic analysis of first - line treatment with erlotinib in an egfr - mutated population with advanced nsclc . 
ting j , tien ho p , xiang p , et al : cost - effectiveness and value of information of erlotinib , afatinib , and cisplatin - pemetrexed for first - line treatment of advanced egfr mutationpositive nonsmall - cell lung cancer in the united states . 
lu s , ye m , ding l , et al : cost - effectiveness of gefitinib , icotinib , and pemetrexed - based chemotherapy as first - line treatments for advanced nonsmall - cell lung cancer in china . 
handorf ea , mcelligott s , vachani a , et al : cost effectiveness of personalized therapy for firstline treatment of stage iv and recurrent incurable adenocarcinoma of the lung . 
isla d , de castro j , juan o , et al : costs of adverse events associated with erlotinib or afatinib in first - line treatment of advanced egfr - positive nonsmall - cell lung cancer . 
park k , tan eh , obyrne k , et al : afatinib versus gefitinib as first - line treatment of patients with egfr mutationpositive nonsmall - cell lung cancer ( lux - lung 7 ) : a phase 2b , open - label , randomised controlled trial . 
schuler m , wu yl , hirsh v , et al : first - line afatinib versus chemotherapy in patients with non small - cell lung cancer and common epidermal growth factor receptor gene mutations and brain metastases . 
goss g , tsai cm , shepherd fa , et al : cns response to osimertinib in patients with t790mpositive advanced nsclc : pooled data from two phase ii trials . 
 o real - time genomic characterization of metastatic pancreatic neuroendocrine tumors has prognostic implications and identifies potential germline actionability purpose we assessed the usefulness of real - time molecular profiling through next - generation sequencing ( ngs ) in predicting the tumor biology of advanced pancreatic neuroendocrine tumors ( pannets ) and in characterizing genomic evolution . methods patients with metastatic pannets were recruited in the routine clinical practice setting ( between may 2014 and march 2017 ) for prospective ngs of their tumors as well as for germline analysis using the memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) sequencing platfor when possible , ngs was performed at multiple time points . results ngs was performed in 96 tumor samples from 80 patients . 
sequencing of preand post - treatment samples revealed tumor - grade progression ; clonal evolution patterns were also seen ( molecular resistance mechanisms and chemotherapy - associated mutagenesis )  . 
germline genetic analysis identified clinically actionable pathogenic or likely pathogenic variants in 14 of 88 patients ( 16% ) , including mutations in high - penetrance cancer susceptibility genes ( men1 , tsc2 , and vhl )  . conclusion a clinical ngs platform reveals pertubations of biologic pathways in metastatic pannets that may inform prognosis and direct therapies . 
2018 by american society of clinical oncology introduction pancreatic neuroendocrine tumors ( pannets ) represent 1% to 2% of all pancreatic neoplasms , but they have a rising incidence and prevalence.1 - 4 whole - exome and whole - genome sequencing , largely of resected pannets , has defined genomic events and improved our understanding of disease pathogenesis.5 , 6 these efforts have identified changes in chromatin remodeling , telomere maintenance , and mammalian target of nitya raj ronak shah zsofia stadler semanti mukherjee joanne chou brian untch janet li virginia kelly muyinat osoba leonard b . 
in addition to the well - characterized hereditary syndromes associated with pannet development ( multiple endocrine neoplasia 1 , von hippel - lindau syndrome , neurofibromatosis type 1 ) , recent data suggest the presence of germline alterations in dna damage repair genes in some patients.6 , 7 treatments for unresectable pannets include targeted and cytotoxic therapies , as well as liver directed approaches.8 - 13 no data exist to guide therapy for pannets with defined genomic alterations . 
in addition , disease heterogeneity poses a significant challenge in defining the selection and sequencing of therapy . to that end , using an institutional matched tumor - normal platform , we prospectively performed next - generation sequencing ( ngs ) of tumors in patients with metastatic well differentiated ( wd ) pannets . 
 we also hypothesized that pannets evolve over time with a more aggressive behavior that could be characterized biologically through ngs of repeat biopsies after treatment . methods study population written informed consent was obtained from patients with a diagnosis of wd pannet using a prospective , institutional review board approved protocol ( clinicaltrials.gov identifier : nct01775072 )  . 
pathologic features of the tumor samples ( differentiation and grade ) were determined according to the 2017 who classification , which recognizes the existence of a grade 3 ( g3 ) category within wd pannets ; historical values for differentiation and grade were collected through review of the electronic medical records . 
electronic medical records were also reviewed for patient data on demographics , treatments , and survival . sequencing of tumor samples ngs was performed using memorial sloan ketteringintegrated mutation profiling of actionable cancer targets ( msk - impact ) , a matched tumor - normal sequencing platform approved for clinical use by the new york state department of health , to detect base substitutions , small indels , copy number changes , and selected gene rearrangements.14 - 17 updated assay versions were released between 2014 and 2017 , and ngs of tumor tissue consisted of deep sequencing of all exons and selected introns of 275 ( preclinical pilot test ) , 341 ( version 1 ) , 410 ( version 2 ) , or 468 ( version 3 ) cancer - related genes . 
genotyping was performed using the pilot test for two of 96 samples ( 2% ) , version 2 for 16 of 96 samples ( 17% ) , version 3 for 57 of 96 samples ( 59% ) , and version 4 for 21 of 96 samples ( 22% )  . 
actionable genetic alterations were defined as changes that could guide therapy , including standard of care and investigational treatments ( annotated according to oncokb ) .18 germline sequencing germline variants from the .bam file of the normal dna sequence were called using the mutect and gatk haplotype caller as described previously ; all variants with < 1% population frequency in the exome aggregation consortium database were interpreted.19 , 20 germline analysis included 76 genes known to be associated with hereditary cancer predisposition , including all cancer predisposition genes identified in the american college of medical genetics and genomics guidelines.19 , 21 , 22 variants were interpreted on the basis of american college of medical genetics and genomics criteria . loss of heterozygosity loss of heterozygosity ( loh ) was determined through analysis of total , allele - specific , and integer dna copy number genome - wide using facets.23 statistical analysis descriptive statistics were used to characterize the study population and the somatic and germline genetic results . 
overall survival ( os ) , defined as the time from the date of first tissue acquisition ( from which ngs was completed ) until death or last known follow - up , was estimated by kaplan - meier methods . 
all p values were two sided , and p < .05 was considered to indicate statistical significance . results clinical characteristics as of march 2017 , ngs had been performed on 96 tumor samples from 80 patients ( eight patients with two samples analyzed ; four patients with three samples analyzed )  . 
in 29 patients , the primary tumor was sequenced , and in 58 patients , a metastatic tumor was sequenced ; in seven patients , both primary and metastases were sequenced ( in one instance , sequencing took place synchronously )  . 
recurrent somatic alterations occurred in key pathways and functional groups : chromatin remodeling factors ( crfs ) , histone methyltransferases ( hmts ) , and mtor pathway genes . somatic alterations in crfs ( daxx , atrx , pbrm1 , smarcb1 , arid5b , arid2 , arid1b , and arid1a ) were observed in 52 of 80 patients ( 65% )  . 
baseline patient characteristics ( continued ) wd pannets with msk - impact results systemic therapy somatostatin analogs ( octreotide long - acting release or lanreotide ) everolimus sunitinib platinum drug chemotherapy alkylating drug chemotherapy peptide receptor radionuclide therapy hepatic arterial embolization ( bland , radiotherapy , or chemotherapy ) 58 ( 73 ) 29 ( 36 ) 8 ( 10 ) 18 ( 23 ) 46 ( 58 ) 12 ( 15 ) 35 ( 44 ) note . 
 the clinical behavior of these tumors , including the single g1 tumor , was more aggressive . ten tumor samples ( collected from six patients ) harbored braf alterations : two of 10 g1 ( 20% ) , three of 10 g2 ( 30% ) , and five of 10 g3 ( 50% )  . 
 in addition to two tumors with v600e mutations , several non - v600 braf alterations were seen ( k601e , t599k , and t310i ) , and one tumor expressed both braf g596d and e451k mutations . survival with a median follow - up for survivors of 27.6 months , we observed 14 deaths ; median os was 118.9 months ( 95% ci , 99.6% - not achieved )  . 
tp53 alterations were observed in 10 of 80 patients ( 13% ) ; all were characterized as likely oncogenic . somatic loh in 50% of the genome was highly prevalent ; it was identified in 55 of 95 tested samples ( 58% )  . 
loh was observed recurrently in chromosomes 1 , 2 , 3 , 6 , 8 , 10 , 11 , 15 , 16 , 21 , and 22 ( appendix fig a1 )  . ngs results for the first sequenced sample in all patients are summarized in figure 1 . 
in the recurrently altered genes , there were no statistical differences in genetic spectrum on the basis of g1 versus g2 tumors , but a trend was noted of g2 and g3 tumors harboring tp53 , setd2 , and braf alterations . novel recurrently altered genes in this metastatic cohort , we observed a much higher frequency of setd2 alterations ( 14 of 80 patients [ 18% ] ) in comparison with previous investigations , and a novel finding of braf molecular mechanisms of resistance . 
genomic landscape of pancreatic neuroendocrine tumors , including the first sequenced sample for each patient ( n = 80 ) , as identified by the memorial sloan kettering - integrated mutation profiling of actionable cancer targets . 
 loh , loss of heterozygosity ; mb , megabyte ; mtor , mammalian target of rapamycin . post - treatment samples were available for three patients receiving the mtor inhibitor everolimus and for one patient receiving the braf v600e inhibitor vemurafenib . in all three patients receiving everolimus , mtor pathway alterations were identified at progression . 
on disease progression , a biopsy specimen revealed an oncogenic pten mutation ( q298 * ) , and the sampled areas of tumor progressed from g1 to g3 ( appendix figure a2 )  . 
this pten mutation was seen previously at a low allele frequency when the original pancreas tumor was sequenced , reflecting the possible emergence of this clone through targeted therapy . patient 2 received everolimus for 8 weeks , at which point treatment was held for infectious complications . 
a liver lesion increased in size and a biopsy was performed ; ngs identified a hotspot mutation in rheb ( y35s ) .25 subsequent sampling and ngs of this same liver lesion after 16 months of observation demonstrated the rheb alteration at a higher allele frequency . 
the msk - impact version used to sequence the pretreatment sample did not interrogate rheb , so the allele fraction of the rheb mutation is unknown in this sample ; however , it increased notably after treatment with andprogression on everolimus . patient 3 received everolimus for > 9 months before disease progression . 
at disease progression , ngs of a growing liver lesion demonstrated loss of this igf2 alteration , acquisition of a tsc2 splice site mutation ( x161_ splice ) , and an akt2 mutation ( g16d ; appendix figure a4 )  . 
surprisingly , no shared mutations were noted in sequencing of the two samples ; clinically , however , the disease was consistent with a single primary , suggesting the emergence of a previously unrecognized clone through treatment , although the possibility of a second primary cannot be excluded . patient 4 , whose tumor harbored a braf v600e mutation , enrolled in a basket clinical trial with vemurafenib and developed an nras hotspot mutation ( q61r ; appendix figure a5 ) .26 chemotherapy - associated mutagenesis . 
two patients received alkylating agents , and the third received oxaliplat a review of the tumors exposed to alkylating drugs showed that the variants shared by both samples were observed at a higher allele fraction compared with those unique to the individual samples , and the evolutionary pattern demonstrated a predominant c > t signature consistent with alkylating - agent associated mutagenesis ; in addition , consistent with previous observations , acquired alterations were observed in the mismatch repair genes for both tumors ( fig 3 ) .27 - 29 the sampled tumor in both patients increased from g1 to g3 . germline genetic testing germline testing for 76 cancer - associated genes was performed in 88 patients , 78 in the original prospective cohort and an additional 10 with surgically resected pannets . 
fourteen likely pathogenic germline alterations were identified in known cancer susceptibility genes ( 16% ) : three in established high - penetrance genes ( men1 , vhl , and tsc2 [ 3% ] ) , four in moderate - penetrance genes ( chek2 in four patients [ 5% ] ) , and seven in low - penetrance genes ( monoallelic mutyh in four patients and apc i1307k in three patients [ 8% ] )  . 
notably , we identified a germline tsc2 mutation in a 32 - year - old patient who did not exhibit the classic clinical features of tuberous sclerosis complex ( tsc ) and did not meet the criteria for tsc genetic testing ; magnetic resonance imaging of the brain identified tubers and radial bands , typical of tsc ( appendix figure a6 )  . 
among these patients , as best therapy response , two of 12 ( 17% ) had tumor regression , six of 12 ( 50% ) had stable disease , and one of 12 ( 8% ) had progressive disease ; three of 12 ( 25% ) are still receiving treatment . 
in the remaining 46 patients without somatic mtor pathway alterations , 17 of 46 ( 37% ) received everolimus ; five of 17 ( 29% ) had tumor regression , seven of 17 ( 41% ) had stable disease , four of 17 ( 24% ) had progressive disease , and there was an unknown response in one patient ( lost to follow - up )  . 
results from ngs led to the enrollment of three patients into clinical trials , but with little response noted . to our knowledge , this is the first study to evaluate the real - time clinical usefulness of ngs for patients with advanced , metastatic pannets . 
setd2 alterations have been implicated in the pathogenesis of many cancers ( breast , lung , acute lymphoblastic leukemia , renal cell carcinoma , and gliomas ) , supporting a role for setd2 as a tumor suppressor across many diseases.34 - 41 given our findings , one might consider closer monitoring and / or cytotoxic therapies in patients with setd2or table 3 . 
levels 1 and 2a include food and drug administration ( fda ) - recognized and standardof - care biomarkers predictive of response to an fda - approved drug for pancreatic neuroendocrine tumors . 
level 2b includes fda - approved biomarkers predictive of drug response in another cancer , but not fda or national comprehensive cancer network - compendium listed for pancreatic neuroendocrine tumors . 
level 3a includes alterations for which clinical evidence links the biomarker to drug response for pancreatic neuroendocrine tumors , but neither the biomarker nor the drug are standard of care in any cancer type . 
level 3b includes alterations for which clinical evidence links the biomarker to drug response for other cancers , but neither the biomarker nor the drug are standard of care in any cancer type . 
level 4 includes alterations in which preclinical evidence potentially links the biomarker to drug response , but neither the biomarker nor the drug are standard of care in any cancer type . abbreviation : msk - impact , memorial sloan kettering - integrated mutation profiling of actionable cancer targets . high penetrance high penetrance high penetrance moderate penetrance moderate penetrance moderate penetrance low penetrance low penetrance low penetrance recessive allele recessive allele braf - altered tumors and a higher burden of disease earlier in the treatment course . our study identifies pertubations of biologic pathways in metastatic pannets that were found in previous whole - exome and whole genome studies evaluating mostly localized pannets.5 , 6 we observed alterations largely along three pathways : crfs , hmts , and mtor . 
our study clarifies pannet genetics exclusively in the metastatic cohort , the group which gains immediate clinical value from realtime sequencing as we make steps toward precision medicine . in addition , we found that 16% of patients harbored a germline pathogenic or likely pathogenic alteration in high - , moderate - , or low - penetrance cancer predisposition genes . 
in looking at those patients found to have mutations in low or moderate - penetrance genes , it is notable that both chek2 and monoallaleic mutyh were noted in previous germline assessments of patients with pannets.6 notably , the patient in our series with a tsc2 germline mutation did not meet the clinical criteria for tsc genetic testing ; the identification of this mutation was incidental . 
 because mutations in men1 and tsc2 are two of the most common somatic findings in pannets , our finding of germline mutations within these two high - penetrance genes demonstrates the importance of these genes in pannet pathogenesis . 
although the contribution of the other germline alterations to pannet susceptibility has yet to be elucidated , our data suggest that there is likely a larger - than - anticipated germline contribution in this disease . 
given the lack of predictable phenotypic findings in most highand moderate - risk mutation carriers , combined with the relative rarity of this tumor subtype , consideration of genetic risk assessment for all patients with pannets is not unreasonable . 
such germline findings would have a profound impact on long - term cancer surveillance and cascade genetic testing of at - risk family members . in a small number of patients , somatic mutational signatures guided therapy . 
we identified somatic mtor alterations in a much greater proportion of patients ( 43% ) than described previously , but the presence or absence of an mtor pathway mutation was not predictive of response or resistance to treatment.8 our numbers are small , however , so additional work is needed to clarify the role of mtor pathway alterations in predicting response and / or resistance to targeted mtor pathway inhibitors . our series included only patients with wd tumors , and consistent with previous findings , rb1 alterations were essentially absent and were observed in only one post - treatment sample exposed to chemotherapy.43 , 44 wd pannets are often thought of as cancer in slower motion , and we have used our sequencing efforts to help clarify pannet evolutionary patterns over time and through therapy . 
in the 12 patients who underwent ngs of tumor tissue at multiple time points , we noted increased tumor grade in eight patients ( 67% ) , associated with marked genetic evolution after treatment . 
to our knowledge , we are the first to observe this evolutionary pattern in pannets.27 - 29 in conclusion , comprehensive molecular profiling of pannet samples using archival tissue is feasible . 
in a disease in which the natural history is thought to be indolent , we have demonstrated that the cancer transforms with grade and genetic progression over time and as therapy progresses . 
the practical usefulness of this analysis remains limited at this time ; our data suggest a higher - than - expected germline mutation rate , and consideration of genetic risk assessment for all patients with pannets is reasonable . 
in addition , given the heterogeneity of the disease , rapid , readily available , and clinically feasible ngs should be considered in clinical trials to help stratify these patients . 
specifically , our numbers are too small to clarify the genetic predictors of response or resistance to commonly used therapies in pannets , the prognostic effects of the identified genetic alterations , or the patterns of genetic evolution ( because only 12 patients underwent sequencing at multiple time points )  . 
klimstra , diane reidy - lagunes financial support : nitya raj , diane reidy - lagunes administrative support : nitya raj , joanne chou , virginia kelly , diane reidy - lagunes provision of study material or patients : nitya raj , leonard b . 
 data analysis and interpretation : nitya raj , ronak shah , zsofia stadler , semanti mukherjee , joanne chou , brian untch , janet li , muyinat osoba , leonard b . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . nitya raj research funding : novartis ( inst ) ronak shah no relationship to disclose leonard b . 
saltz consulting or advisory role : mcneil ppc ( i ) research funding : taiho pharmaceutical diana mandelker no relationship to disclose marc ladanyi honoraria : merck ( i ) consulting or advisory role : nccn / boehringer ingelheim afatinib targeted therapy advisory committee , national comprehensive cancer network / astrazeneca tagrisso request for proposal advisory committee research funding : loxo ( inst ) michael f . 
berger research funding : illumina zsofia stadler consulting or advisory role : allergan ( i ) ; genentech ( i ) , regeneron pharmaceuticals ( i ) , optos ( i ) , adverum ( i ) david s . 
klimstra stock and other ownership interests : paige.ai consulting or advisory role : wren laboratories , ipsen semanti mukherjee employment : regeneron pharmaceuticals stock and other ownership interests : regeneron pharmaceuticals research funding : regeneron pharmaceuticals diane reidy - lagunes honoraria : novartis consulting or advisory role : ipsen , pfizer , novartis research funding : novartis acknowledgment the authors thank the patients who kindly agreed to participate in this research effort . joanne chou no relationship to disclose brian untch no relationship to disclose support prior presentation affiliations nitya raj , ronak shah , zsofia stadler , semanti mukherjee , joanne chou , brian untch , janet li , virginia kelly , muyinat osoba , leonard b . 
presented , in part , as poster presentations at the 2015 gastrointestinal cancers symposium ( san francisco , ca , january 15 - 17 , 2015 ) and 2016 gastrointestinal cancers symposium ( san francisco , ca , january 21 - 23 , 2016 )  . 
dasari a , shen c , halperin d , et al : trends in the incidence , prevalence , and survival outcomes in patients with neuroendocrine tumors in the united states . 
yao jc , hassan m , phan a , et al : one hundred years after carcinoid : epidemiology of and prognostic factors for neuroendocrine tumors in 35 , 825 cases in the united states . 
yao jc , phan at , chang dz , et al : efficacy of rad001 ( everolimus ) and octreotide lar in advanced lowto intermediate - grade neuroendocrine tumors : results of a phase ii study . 
yao jc , lombard - bohas c , baudin e , et al : daily oral everolimus activity in patients with metastatic pancreatic neuroendocrine tumors after failure of cytotoxic chemotherapy : a phase ii trial . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
wagle n , berger mf , davis mj , et al : high - throughput detection of actionable genomic alterations in clinical tumor samples by targeted , massively parallel sequencing . 
cheng dt , prasad m , chekaluk y , et al : comprehensive detection of germline variants by mskimpact , a clinical diagnostic platform for solid tumor molecular oncology and concurrent cancer predisposition testing . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
kim jy , brosnan - cashman ja , an s , et al : alternative lengthening of telomeres in primary pancreatic neuroendocrine tumors is associated with aggressive clinical behavior and poor survival . 
singhi ad , liu tc , roncaioli jl , et al : alternative lengthening of telomeres and loss of daxx / atrx expression predicts metastatic disease and poor survival in patients with pancreatic neuroendocrine tumors . 
marinoni i , kurrer as , vassella e , et al : loss of daxx and atrx are associated with chromosome instability and reduced survival of patients with pancreatic neuroendocrine tumors . 
piva f , santoni m , matrana mr , et al : bap1 , pbrm1 and setd2 in clear - cell renal cell carcinoma : molecular diagnostics and possible targets for personalized therapies . 
liu l , guo r , zhang x , et al : loss of setd2 , but not h3k36me3 , correlates with aggressive clinicopathological features of clear cell renal cell carcinoma patients . 
walter dm , venancio os , buza el , et al : systematic in vivo inactivation of chromatin - regulating enzymes identifies setd2 as a potent tumor suppressor in lung adenocarcinoma . 
tang lh , basturk o , sue jj , et al : a practical approach to the classification of who grade 3 ( g3 ) well - differentiated neuroendocrine tumor ( wd - net ) and poorly differentiated neuroendocrine carcinoma ( pd - nec ) of the pancreas . 
yachida s , vakiani e , white cm , et al : small cell and large cell neuroendocrine carcinomas of the pancreas are genetically similar and distinct from well - differentiated pancreatic neuroendocrine tumors . 
tbt reimbursement within the united states in the current regulatory environment is not tied to premarket evidence of clinical utility , resulting in a vicious cycle wherein low - level evidence of utility leads to poor reimbursement , thereby impeding investment in developing new , clinically valuable tbts supported by high - level evidence . 
precise one - to - one mapping of reimbursement to cost savings or cost effectiveness is precluded by an absence of formal cost - effectiveness analyses for many emerging tbts , and for more established tbts , it has become clear that such analyses may yield wildly variable , subjective estimates . 
to address these challenges , we propose a system of tiered reimbursement that rewards development of high - quality tbts within specic use contexts , supported by strong evidence of analytic validity and clinical utility . 
we propose three use contexts of tbts , each dened by its inuence on treatment decisions relative to the current standard of careopt - out , opt - in , and the use of appropriate , alternative , effective therapies ( opt - alt )  . 
the true clinical utility of a tbt is often difcult to determine , initiating a vicious cycle in which tbts are often undervalued and poorly reimbursed because of a lack of high - level evidence of clinical utility.1 an inconsistent regulatory and reimbursement environment results in an unfavorable return on investment in the research and development ( r&d ) needed to generate the high levels of evidence ( loes ) necessary to demonstrate the clinical utility of tbts.1 indeed , despite increasing recognition of the benets of personalized medicine , medicare coverage of many emerging tbts is being signicantly reduced or denied , 2 resulting in decreased investment in tbt r&d.3 reimbursement of tbts to date has been driven primarily by a combination of commodity and labor pricing ( ie , reagent and technical costs ) , market forces , and perceived clinical value . 
this is not an effective strategy to support long - term investment in high - quality tbts . we propose that objective and transparent tiers of tbt value be created based on high loes of analytic validity ( ie , accurate and reproducible measurement of a specic analyte ) and clinical utility ( ie , improves patient outcomes ) that meet minimal specied thresholds and address unmet medical needs.4 , 5 our proposal would potentially result in redistribution of health care dollars currently spent on needless or ineffective therapies while ensuring that patients who require and are likely to benet from treatment strategies receive theit will create pathways by which tbt manufacturers could receive guaranteed minimum rates of reimbursement ( fig 1 ) and thereby increase the likelihood that tbt developers obtain a return on r&d investment . 
we propose an alternative , value - based reimbursement which clinical utility can be assessed within specic use contexts , undergo formal review , and then provide a new value - based , tiered reimbursement rate . 
note that the use of appropriate , alternative , effective therapies ( opt - alt ) context includes both the initial treatment and the monitoring paradigms . fda , us food and drug administration ; icers , cost - effectiveness ratios ; qaly , quality - adjusted life - year . dinan et al proposed valuation status quo / conventional pricing holder of tbt seeks higher , value - based reimbursement value - based application to set new reimbursement cost - saving tbts opt - out evidence to reduce treatment % treatment reduction treatment cost pricing : function of costs saved cost - effective tbts evidence of beneficial treatment treatment outcomes ( qalys ) treatment costs pricing : function of icers opt - in opt - alt approved : new value - based reimbursement set formal review by fda or other regulatory body loes of robustness in the assay itself and that its use improves patient outcomes compared with not using it , respectively.4 - 6 we propose three major contexts of use for which a tbt might have clinical utility , and therefore value , designated as optout , opt - in , and the use of appropriate , alternative , effective therapies ( opt - alt ; table 1 )  . 
each of these terms refers to the clinical decision that might be made based on the results of the tbt compared with the decision made as standard of care ( soc ) without tbt results . 
these categories can be applied to the various use contexts considered for the tbt , including assessment of cancer risk / susceptibility , screening , differential diagnosis , prognosis , prediction , and monitoring.7 similar to therapeutics , the performance and therefore value of a tbt will vary within certain subpopulations of patients , underlying the need to specify the target population when assessing value within a specic use context . 
opt - out tbts have value and the potential to be directly cost saving by reducing the use of unnecessary or ineffective yet toxic and costly therapies , either by indicating that the patient does not need additional therapy ( prognosis ) or that the therapy under consideration is unlikely to work ( prediction ; table 1 )  . perhaps the most well - known example of opt - out tbts with high analytic validity and clinical utility are genomic tests used to determine whether adjuvant chemotherapy should be given to patients with estrogen receptorpositive , human epidermal growth factor receptor 2negative , and least ve node - negative breast cancer . 
there are at commercially available tbts in this category.8 , 9 the 21 - gene recurrence score ( rs ; oncotype dx ; genomic health , redwood city , ca ) is the most well studied and provides an illuminating case study . 
before introduction of the 21 - gene rs , adjuvant chemotherapy was recommended for 50% or more of patients with estrogen receptorpositive , human epidermal growth factor 2negative , node - negative breast cancer.10 the 21 - gene rs , which has high analytic validity , 11 identies a large percentage of this group of patients for whom adjuvant chemotherapy has little or no chance of improving cancer - related outcomes , and its use is now recommended for all women in this category.8 , 12 cost - effectiveness analyses of the 21 - gene rs have varied widely and involve complex and unavoidably subjective modeling that depends on assumptions made about management strategy , impact of treatment , time horizon , inputs.13 accepted patient population , and other model estimates of the impact of the 21 - gene rs assay suggest overall absolute reductions in chemotherapy of 7% to 14% in the roughly 50 , 000 women who are newly diagnosed every year in whom testing would be recommended ( table 2 )  . 
assuming an average total adjuvant chemotherapy cost of $80 , 000 ( including modern drugs and supportive care treatments ) , 14 annual chemotherapy cost savings for women in this category would be between $280 million to $560 million per year , signicantly outpacing the roughly $175 million spent on the test for these same women ( assuming $3 , 500 per test )  . 
in this case , the value of the tbt is generated by turning a minimally effective or is indicated , and unacceptable therapy into one that therefore acceptable , because of an altered risk - benet that justies or renes treatment . 
an opt - in tbt will typically increase costs , but still has value because it may result in improved long - term clinical outcomes . an example of an opt - in tbt is provided by analysis of germline susceptibility genes , such as brca1 or brca2 , or associated genes ( table 2 )  . 
in this case , the presence of a deleterious inherited germline mutation in the brca1 or brca2 ( or related ) gene substantially increases the odds of developing and dying as a result of breast and / or ovarian cancer . 
however , the risk of developing these cancers and the odds of mortality are reduced by as much as 90% and 35% to 75% with prophylactic mastectomy and salpingooophorectomy , respectively.15 , 16 in this case , these otherwise unacceptable interventions become appropriate because of the increased chances of long - term survival as a result of the intervention in a high - risk population ( table 2 )  . 
therefore , although such tests may be relatively expensive , their value in terms of lives saved , decreased long - term medical costs , and increased economic productivity and quality of life often outweigh the immediate costs of the tests and associated downstream medical expenditures . 
this tradeoff between added costs and increased quality of life is often measured through economic metrics , such as incremental costeffectiveness ratios , which are expressed in units of cost ( dollars ) per 1 year of full quality of life , or quality - adjusted life - years ( qalys )  . 
for example , the brca test listed in table 2 , when applied to the target population , saves on average 1 qaly for every $4 , 300 spent ( $4 , 300 / qaly ) .17 opt - alt tbt the opt - alt tbt paradigm is a subcategory of opt - in . 
in this case , the value of the tbt is to help select a strategy from two or more available choices that is most likely to benet the patient , thus enhancing the precision of his or her management . 
opt - alt tbts improve outcomes via the use of appropriate , alternative , effective therapies . an example of an opt - alt tbt is illustrated by somatic mutation testing in metastatic lung adenocarcinoma to drive the use of therapies directed against genomic abnormalities ( table 2 ; opt - alt : initial rx )  . 
these abnormalities occur in various genes , including egfr , kras , alk , ros1 , erbb2 , braf , ret , and ntrk1 , which are found collectively in more than 20% of patients with metastatic nonsmall - cell lung cancer using tbts on the basis of nextthese abnormalities is generation sequencing . 
each of linked to a specic therapy that targets the genomic abnormality ( eg , erlotinib and other tyrosine kinase inhibitors ) , 18 and approximately one quarter of these patients will benet from these agents . 
in contrast , chemotherapy , which has roughly the same likelihood of benet but is associated with substantially higher toxicities , remains an soc for many of the remaining patients.19 although the clinical value in terms of quality and quantity of life is established , it is a difcult and somewhat subjective exercise to determine the true economic value of these tests , which depend heavily on the relative benets of each of the drugs in each of the many settings in which they may be used across different populations . opt - out / monitoring or opt - in / monitoring tbt in an opt - in / monitoring scenario , a patient a variation on the opt - out , opt - in , or opt - alt schema is the monitoring paradigm ( table 1 )  . 
value arises from avoiding the toxicity and cost of continuing an ineffective therapy . clinically free of apparent disease , but the tbt results indicate an impending future cancer - related event which early intervention may be more effective than waiting until the event becomes clinically evident . 
an additional variant of the opt - alt / monitoring paradigm occurs if serial tbts are used as surrogates of more expensive or inconvenient , but strongly indicated , monitoring strategies . 
in this case , the patients cancer - related outcome may not be improved by the tbt per se , but the tbt helps the patient avoid other diagnostic studies , such as radiographic imaging . 
one should note that a given tbt can fall into one or more of these use context categories , depending on the disease to which the tbt is applied and the clinical scenario . as noted , the opt / monitoring paradigm has three possibilities : opt - out / monitoring , opt - in / monitoring , or opt - alt / monitoring . 
for solid malignancies , liquid biopsies include tumor - associated proteins ( eg , carcinoembryonic antigen [ cea ] / colorectal cancer ; ca125 / ovarian cancer ; prostate - specic antigen [ psa ] / prostate cancer , and muc1 , identied by ca15 - 3 or ca27.29 / breast cancer ) , circulating tumor cells , and cell - free tumor dna . opt - out / monitoring and opt - alt / monitoring tbts might be used in many settings , and their economic value may differ in each . 
introducing earlier treatment of such patients may result in higher overall survival rates , as with patients with isolated colorectal metastases detected by an increasing cea level , 22 or may not , as in patients with ovarian and breast cancers monitored with ca125 or ca15 - 3 , respectively.23 , 24 we consider serial cea for patients with a history of colorectal cancer to have high clinical utility , and we would place this tbt for this indication in tier 1 , suggesting that reimbursement should be based on more than just commodity pricing . 
in contrast , monitoring the latter two tbts has been shown in prospective randomized clinical trials to not result in longer overall survival . we would place these markers in tier 3 , or tier x , because their clinical and economic value is not clear ( table 3 )  . 
in contrast , results of a prospective , randomized clinical trial have demonstrated that identication and treatment of hepatic oligometastases in patients with stage ii to iii colorectal cancer results in a small but denite chance of cure22 and would therefore be placed in tier 1 ( table 2 )  . 
however , the development of high - level evidence of tbts in high - risk subsets might expand their indications in the future , thus offering an incentive for biotech companies to invest in generating high - level evidence demonstrating the clinically utility of tbts . in the metastatic setting , an increasing circulating tbt either directly indicates failure of the current therapy or suggests the need for more invasive imaging to determine whether the disease is progressing . 
in this case , such tbts provide both opt - out and opt - alt information because the soc is usually to change from the apparently inactive treatment to a second or later - line treatment ( opt - alt ) or to discontinue treatment altogether ( opt - out )  . 
although few , if any , prospective randomized clinical trials exist to demonstrate the clinical utility of these markers in this use context , in general , the opt - alt paradigm is considered to be sufcient to place these tbts ( at least the circulating proteins ) in tier 2 or 3 , and they are used routinely.25 our model would suggest that reimbursement for these tbts should be commensurate with their value in managing patient care . having dened the tbt use contexts and their relative value possibilities , we propose a semiquantitative , hierarchal , tiered - based approach to help determine a reimbursement structure for tbts ( table 3 )  . 
tiers dene tbts ranging from high analytic validity and clinical utility ( tier 1 ) to less accurate analytic validity or lower evidence of clinical utility ( tiers 2 and 3 ) and tbts without conrmed clinical value ( tier x )  . 
in each case , assessment of a tbt is dependent on its specic use context as an optout , opt - in , opt - alt , or opt / monitoring tbt ( table 1 )  . 
these savings could be redistributed within the health care systeone option would be to simply use the savings , especially those generated by opt - out tbts , to reduce the cost of care . 
we maintain that this approach is unattractive , because it is likely to stie innovation for new tbts , reduce the quality of data regarding a tbts clinical utility , and further enhance the existing vicious cycle.1 rather , we argue that the value of generating high - quality tbts should instigate a redistribution of health care spending directly ( in the case of opt - out tbts ) from therapeutics to diagnostics or indirectly ( in the case of opt - in tbts ) , on the basis of the case that immediate costs should be borne by society to obtain long - term cost effectiveness . 
such redistribution of nancial resources would then stimulate greater investment into diagnostics to support r&d and reward innovation and entrepreneurship . our tiered approach provides a clear and achievable opportunity for companies to develop high - quality tbts with a guarantee for a commensurate return on investment . 
rather , tbt proposes a strategy to redistribute third - party reimbursement from unnecessary or ineffective therapies to support tbts with high clinical utility to provide a baseline assurance of reimbursement and return on investment for entities investing in tbt development . 
importantly , tbts in tiers with poor evidence ( tier 3 ) or those that are purely informative with unclear implications for management ( tier x ) cannot be expected to recoup health care resources , and we argue that such tbts should receive little , if any , third - party reimbursement . although our system does not directly reward a tbt for having slightly better performance than another for a similar indication , it does so indirectly . 
therefore , a static determination of cost effectiveness for a tbt is unrealistic . in the example of the 21 - gene rs , the cost of adjuvant chemotherapy has increased dramatically in breast cancer , in part as a result of routine hematopoietic growth factor for dose - dense treatment.14 another issue is support whether tbts should be entered into clinical practice before high loes are available , with reimbursement on the basis of a coverage - with - evidence - development approach , which has been piloted by the centers for medicare and medicaid and its afliates.27 in this case , rather than insistence on either prospective clinical trials or prospectiveretrospective4 , 5 studies , the third party payer is willing to cover the use of a tbt , with the proviso that the user or manufacturer generates evidence supporting at least clinical validity if not utility for the assay . 
this strategy represents an intriguing but special circumstance outside of our model . a key limitation of our proposed system is that there will be justiable differences in opinions regarding the relative analytic validity , meaningfulness of different clinical end points , magnitude of outcomes , and strategies to generate loes for clinical utility . 
nonetheless , this formulation is a rst conceptual and quantitative step toward what would eventually be a rened , objective , and quantitative method to assign value to emerging tbts using a practical tiered reimbursement scheme , helping to break the vicious cycle of tumor biomarker value . finally , a consideration of the proposed system would not be complete without acknowledging the critical importance of practical barriers to implementation . 
although an indepth discussion of all barriers is beyond the scope of this article , it is worth noting that efforts to implement this proposed system will require input and approval from the involved stakeholders , 1 which include the life sciences industry , private third - party payers , government and regulatory agencies , health care systems , providers and larger medical associations , and patients and patient advocacy groups . 
however , the ultimate success of this system will require large - scale adoption to provide incentives sufcient to power the proposed virtuous cycle of investment in high - quality tbt development . in conclusion , we have proposed an objective , systematic , and semiquantitative approach to guide researchers , funders , test manufacturers , clinicians , patients , and health care systems to invest health care dollars into tbt r&d . 
by providing a tiered categorical solution with clear minimum reimbursement levels , we hope to provide assurance to investors of a solid potential for return on investment via r&d into high - quality clinically useful tbts . 
by increasing entry into the market for high - quality tbts , we argue that market forces will drive real - time selection of the most clinically local useful tbt available for a given clinical context , treatment practices , and population characteristics . 
lyman consulting or advisory role : g1 therapeutics , halozyme , partners healthcare , amgen , pzer , agendia , helsinn therapeutics , celldex ( i ) , janssen ( i ) , genomic health , mylan , samsung bioepis , spectrum pharmaceuticals research funding : amgen richard l . 
schilsky research funding : astrazeneca ( inst ) , bayer ( inst ) , bristol - myers squibb ( inst ) , genentech ( inst ) , eli lilly ( inst ) , merck ( inst ) , pzer ( inst ) , boehringer ingelheim ( inst ) travel , accommodations , expenses : varian daniel f . 
hayes stock and other ownership interests : oncimmune , inbiomotion consulting or advisory role : cepheid , freenome , cellworks , cvs caremark breast cancer expert panel , agendia research funding : astrazeneca ( inst ) , puma biotechnology ( inst ) , pzer ( inst ) , eli lilly ( inst ) , merrimack ( inst ) , menarini silicon biosystems ( inst ) patents , royalties , other intellectual property : royalties from licensed technology ; diagnosis and treatment of breast cancer . 
hayes is designated as inventor / co - inventor ; title : a method for predicting progression - free and overall survival at each follow - up time point during therapy of patients with metastatic breast cancer using circulating tumor cells . 
cancer - tests / #530d572f5955 teutsch sm , bradley la , palomaki ge , et al : the evaluation of genomic applications in practice and prevention ( egapp ) initiative : methods of the egapp working group . 
harris ln , ismaila n , mcshane lm , et al : use of biomarkers to guide decisions on adjuvant systemic therapy for women with early - stage invasive breast cancer : american society of clinical oncology clinical practice guideline . 
j clin oncol 34 : 1134 - 1150 , 2016 krop i , ismaila n , andre f , et al : use of biomarkers to guide decisions on adjuvant systemic therapy for women with early - stage invasive breast cancer : american society of clinical oncology clinical practice guideline focused update . 
cronin m , sangli c , liu ml , et al : analytical validation of the oncotype dx genomic diagnostic test for recurrence prognosis and therapeutic response prediction in node - negative , estrogen receptor - positive breast cancer . 
wang sy , dang w , richman i , et al : cost - effectiveness analyses of the 21 - gene assay in breast cancer : systematic review and critical appraisal . 
balmaa j , sanz j , bonll x , et al : genetic counseling program in familial breast cancer : analysis of its effectiveness , cost and cost - effectiveness ratio . 
lindeman ni , cagle pt , beasley mb , et al : molecular testing guideline for selection of lung cancer patients for egfr and alk tyrosine kinase inhibitors : guideline from the college of american pathologists , international association for the study of lung cancer , and association for molecular pathology . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american pathologists joint review . 
rustin gj , van der burg me : a randomized trial in ovarian cancer ( oc ) of early treatment of relapse based on ca125 level alone versus delayed treatment based on conventional clinical indicators ( mrc ov05 / eortc 55955 trials )  . 
van poznak c , somereld mr , bast rc , et al : use of biomarkers to guide decisions on systemic therapy for women with metastatic breast cancer : american society of clinical oncology clinical practice guideline . 
reed sd , dinan ma , schulman ka , et al : cost - effectiveness of the 21 - gene recurrence score assay in the context of multifactorial decision making to guide chemotherapy for early - stage breast cancer . 
eiermann w , rezai m , k ummel s , et al : the 21 - gene recurrence score assay impacts adjuvant therapy recommendations for er - positive , node - negative and node - positive early breast cancer resulting in a risk - adapted change in chemotherapy use . 
pilon d , queener m , lefebvre p , et al : cost per median overall survival month associated with abiraterone acetate and enzalutamide for treatment of patients with metastatic castration - resistant prostate cancer . 
schremser k , rogowski wh , adler - reichel s , et al : cost - effectiveness of an individualized rst - line treatment strategy offering erlotinib based on egfr mutation testing in advanced lung adenocarcinoma patients in germany . 
 o survival outcomes by tp53 mutation status in metastatic breast cancer purpose to determine the significant genomic alterations in patients with metastatic breast cancer ( mbc ) and survival outcomes in common genotypes . patients and methods high - depth next - generation sequencing was performed for 202 genes in tumor and normal dna from 257 patients with mbc , including 165 with estrogen receptor / progesterone receptorpositive and human epidermal growth factor receptor 2 ( her2 [ hormone receptor positive ( hr + ) ] ) positive , 32 with her2 - positive , and 60 with triple - negative ( estrogen receptor / progesterone receptornegative and her2 - negative ) disease . 
kaplan - meier survival analysis was performed in the discovery set , in patients with breast cancer analyzed in the cancer genome atlas , and in a separate cohort of 98 patients with mbc who underwent clinical genomic testing . results significantly mutated genes ( smgs ) varied by histology and tumor subtype , but tp53 was an smg in all three subtypes . 
emerging data have shown that the targeting of some of these alterations , such as akt and her2 mutations , may have antitumor efficacy.1 , 2 most proof - of - principle genomically selected trials are conducted in the metastatic setting , whereas many molecular characterization efforts , such as the cancer genome atlas ( tcga ) , are performed in operable breast cancer.3 to design and interpret genotype selected trials effectively , the determination of the genomic profile of patients with metastatic breast cancer ( mbc ) , the frequency of genomic alterations and co - alterations , and the effect of common alterations on prognosis is critical . we determined the genomic profile of patients with mbc in a prospective study and report the significantly altered genes in various breast cancer subtypes . 
 an additional cohort of 98 patients with hr + mbc who underwent clinical genomic testing funda mericbernstam xiaofeng zheng maryam shariati senthil damodaran chetna wathoo lauren brusco mehmet esat demirhan coya tapia agda karina eterovic reva k . 
 were identified as a validation cohort ; patients had undergone testing with foundationone ( foundation medicine , cambridge , ma ) , ion ampliseq comprehensive cancer panel ( thermo fisher scientific , waltham , ma ) , or oncomine comprehensive assay ( thermo fisher scientific )  . 
ngs of 202 genes ( t200 platform ; data supplement ) was performed on tumor and normal dna samples as previously described.4 assays were performed while blinded to the clinical outcomes . 
 reporting was done consistent with reporting recommendations for tumor marker prognostic studies guidelines.5 molecular inversion probe ( mip ) arrays were performed as previously described.6 , 7 esr1 mutation status was tested using the qx200 droplet digital pcr system ( biorad laboratories , hercules , ca ) with primers to assess the following four esr1 mutations : y537c ( 1980a > g ) , y537n ( 1979t > a ) , y537s ( 1980a > c ) , and d538g ( 1983a > g ; data supplement )  . 
quantitative analysis was performed using quantasoft software ( bio - rad laboratories )  . bioinformatics analysis comprehensive methods for bioinformatics analysis has been previously published.4 for copy number calls , high amplification and high deletion was defined as an estimated copy number of five and 0.6 on ngs analysis and five and one on mip analysis . 
discrete independence statistic controlling for observations with varying event rates ( discover ) , a statistical test for detecting co - occurrence and mutual exclusivity in cancer genomics data , was used.8 unlike traditional approaches such as fishers exact test , discover is based on a null model that takes into account the overall tumor - specific alteration rates when deciding whether alterations co - occur more or less often than expected by chance . 
multiple testing was adjusted using false discovery rate ( fdr )  . recurrence - free survival ( rfs ) was calculated from the date of initial breast cancer diagnosis to the date of first local or distant relapse , death , or last follow - up . 
overall survival ( os ) was calculated from the date of mbc diagnosis . results somatic alterations two hundred sixty - eight samples from 257 patients were sequenced ( table 1 )  . 
distribution by tumor subtype was as follows : 165 patients ( 64.2% ) with estrogen receptor / progesterone receptorpositive ( er / pr + ) and human epidermal growth factor 2negative ( her2 ) hr + breast cancer ; 60 with triple - negative breast cancer ( tnbc ) ; and 32 with her2 + breast cancer ( 24 with er / pr + and her2 + and eight with er / pr and her2 + )  . 
forty - eight patients ( 18.7% ) had stage iv disease at presentation . a heat map of the top 50 mutated genes and 50 copy numberaltered ( cna ) genes are shown in the data supplement . 
with rfs 12 months postneoadjuvant chemotherapy postneoadjuvant endocrine therapy locoregional recurrence distant metastases soft tissue bone liver lung other patients with both primary and recurrence / metastasis ( n = 11 ) primary and recurrence primary and metastasis no . 
in her2 hr + breast cancer , a significant gain was seen of fgfr1 , gnas , smarca4 , cpamd8 , crebbp , fgfr3 , hnf1a , lrp1 , and nfkb2 and a loss of csmd1 . 
of the 36 amplifications detected by ngs , 22 were confirmed by mip arrays ( data supplement ) , including all four patients with fgfr1 amplification , all five with gnas amplification , three of four with smarca4 amplification , and all four with notch2 amplifications . 
of 11 deletions detected by ngs , nine were confirmed by mip arrays , including all three patients with nf1 and all three with pten deletions . alterations in actionable genes overall , 244 patients ( 94.9% ) had an alteration in at least one potentially actionable gene.9 of note , mutations differ in their functional consequences ; thus , not all mutations may be actionable.9 , 10 furthermore , a genomic profile may not be considered actionable because of co alterations or other patient variables . 
actionable alterations included well - recognized alterations , such as pik3ca mutations ( 24% ) and fgfr amplifications ( 10% ) , as well as less - frequent but clinically compelling alterations , such as akt1 mutations ( 3% ) and her2 mutations ( 3% )  . 
in addition , there were potentially actionable rarer alterations , such as an inactivating mutation in ptch1 , an activating mutation in idh1 , and high - level amplification of egfr . 
of note , 117 patients ( 72% ) with alterations in an actionable gene had a co - alteration in another potentially actionable gene . we previously reported that most brca1 / 2 alterations in breast cancer are germline.11 , 12 however , we observed potentially deleterious somatic alterations in dna damage repair genes brca1 / 2 , palb2 , atm , and rad51 . 
 no differences were found among these cohorts . genomic alterations in 191 primary versus 77 recurrent / metastatic tumors were compared ( data supplement ) ; no significant differences were found by fishers exact test . 
of the hr + primary tumors , 78 were chemotherapy - nave , 39 were postneoadjuvant we compared the alterations seen in this study with that in the tcga breast cohort ( data supplement )  . 
the mbc cohort was enriched for some alterations , such as tp53 mutations , compared with the tcga series . by ngs , 251 of 257 patients had esr1 sequencing ; however , only 151 patients had adequate coverage of esr1 . 
 two other patients with hr + breast cancer whose primary tumors did not show an esr1 mutation subsequently had ngs of a new distant metastatic lesion not included in this analysis , and this testing uncovered esr1 mutations . because a 4% esr1 mutation rate in mbc is lower than what we and others have reported , 13 , 14 we also used droplet digital polymerase chain reaction for four hot spot mutations ( esr1 y537s , y537c , y537n , and d538g ) in 49 patients with dna from metastatic tumor samples.13 , 14 thirty - eight patients had endocrine therapy before the biopsy of the metastasis ( 31 in the adjuvant setting , seven for recurrent disease )  . 
two of these patients also had t200 sequencing , and one was found to have the same esr1 mutation ( d538g ) , whereas the other , although the same dna was used , did not have the mutation detected , which suggests that droplet digital polymerase chain reaction may be more sensitive for detection . 
one patient had four lines of endocrine therapy in the metastatic setting , whereas the other had adjuvant tamoxifen . genomic profile and prognosis in hr + breast cancer a heat map and bar plot of the 50 most commonly altered genes in hr + breast cancer are shown in fig 2 . 
we tested for the co - occurrence between the top 50 altered genes in hr + breast cancer using the discover algoriththe p and q values for all the gene pairs are listed in the data supplement . 
with an fdr of 0.1 , we found co - alterations in fgfr3 and cripak , which are colocalized on chromosome 4 ; co - alterations in cpamd8 , smarca4 , and notch3 , which are colocalized on chromosome 19 ; and co alterations in crebbp and tsc2 , which are colocalized on chromosome 16 . 
with an fdr of 0.1 , gata3 alterations were mutually exclusive with tp53 alterations . in precision oncology trials , treatment often is given to patients with selected alterations ; thus , we assessed the effect of common genomic alterations on pfs in patients with hr + breast cancer . 
patients with missense tp53 mutations had a longer rfs than those with other types of tp53 mutations , but this difference was not significant ( p = .055 ; fig 3b )  . tp53 mutations were associated with a shorter os in patients with hr + breast cancer ( p = .003 ; fig 3c )  . 
tp53 mutation type ( missense v other ) was not associated with os or pfs on first - line endocrine therapy . when patients with pik3ca mutation / amplification , akt1 mutation / amplification , pten mutation / deletion , fgfr1 / 3 amplifications , gata3 mutations , or map3k1 / map2k4 mutation / deletion were compared with patients who lack these alterations , no significant difference was found in pfs in the first - line metastatic setting on any therapy ( data supplement ) as well as in patients treated with endocrine therapy ( data supplement )  . 
 alteration type del / mut amp / mut pik3ca tp53 gata3 notch1 fgfr1 cdh1 notch3 gnas csmd1 lrp1 cripak map3k1 mll3 fgfr3 ryr2 tsc2 crebbp lrp2 hnf1a akt1 hydin smarca4 spen hmcn1 mll2 cpamd8 znf536 csmd3 arid1a nfkb2 pten ercc5 igf1r daxx ush2a men1 bap1 pkhd1 notch2 il6r erbb2 dnmt3a top1 tgfbr2 fgfr4 pkhd1l1 spta1 tbc1d4 fig 2 . 
the samples are presented in order , with the most common alterations on the leamp , amplification ; del , deletion ; mut , mutation . discussion tp53 was an smg in all breast cancer subtypes , but mutations were more frequent in hr tumors . 
tp53 mutations were seen in 41% of patients with mbc in our sample compared with 30% in the tcga , which represents patients with earlier - stage disease ( fig 1 ) and higher rates of tp53 mutations in hr + breast cancer ( 29% v 18% )  . 
 of note , when ellis et al20 compared aromatase inhibitorsensitive versus aromatase inhibitor resistant tumors in the neoadjuvant setting ( clinicaltrials.gov identifier : nct00265759 ) , the tp53 signaling was enriched in resistant tumors ( 38% of the aromatase inhibitorresistant and 17% in the sensitive group )  . 
 however , emerging therapeutics have been targeting mutant p53.21 , 22 an urgent need exists for novel therapies for tp53 mutant tumors . in the current study , patients had a variety of genomic alterations . 
the frequency of alterations in this pathway may differ on the basis of patient population ( tumor subtype and histology and other variables ) as well as the assay and bioinformatics pipeline . 
in most breast cancer series , this potentially actionable pathway is the most frequently altered ; thus , these alterations are being actively pursued in trials with pi3k / akt / mtor inhibitors.2 , 23 , 24 cdh1 mutations , as expected , were almost exclusively found in invasive lobular carcinoma . 
mutations in map3k1 and map2k4 already have been reported in hr + breast cancer.20 ellis et al20 reported a frequency of 15.5% for map3k1 and map2k4 in er + breast cancer . 
the prognostic and predictive value of gata3 needs to be evaluated further . although our ngs platform primarily analyzes commonly mutated genes in cancer , it can provide copy number information.4 indeed , we identified common cnas , such as gain in fgfr1 and her2 . 
we recently reported that when ngs demonstrates high - level amplification , we are able to validate cnas on an orthogonal platform , such as fluorescent in situ hybridization.25 ngs - based detection of cnas is limited to high - level losses or gains ; thus , we may have underestimated the frequency of copy number changes . 
however , several of these cnas , such as notch alterations and nf1 loss , have therapeutic implications and need additional study . we had too few matched primary and recurrence samples to systematically study genomic evolution in this series . 
in another study , we reported that in 33 matched primary and recurrent tumors , 97 ( 87% ) of 112 somatic mutations were concordant.26 more recently , lefebvre et al27 reported the genomic profiling results of patients who underwent a biopsy of mbc in the context of the safir - 01 , safir - 02 , shiva ( tumor molecular profiling versus conventional therapy in patients with refractory cancer ) , or moscato ( molecular screening for cancer treatment optimization ) prospective trials . 
however , in lefebvre et al , eight genes ( esr1 , fsip2 , fras1 , osbpl3 , edc4 , palb2 , igfn1 , and agrn ) were more frequently mutated in mbc than in early breast cancer profiles in tcga , which suggests that systematic assessment of metastatic tissue in mbc may lead to identification of additional genomic alterations . the current study had some additional limitations . 
we may not have captured molecular profiles of patients who rapidly progressed while on therapy and succumbed to their disease , or alternately , those who responded well were not perceived as needing molecular characterization . 
our panel also did not include mdm2 and mdm4 , two genes that could be amplified to negatively regulate the tp53 axis in patients with wild - type tp53 . in conclusion , genomic profiling has identified multiple potentially actionable alterations . 
mills shaw provision of study materials or patients : funda mericbernstam , maryam shariati , chetna wathoo , lauren brusco , mehmet esat demirhan , filip janku , kenna r . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . funda meric - bernstam honoraria : dialecta , sumitomo group consulting or advisory role : genentech , inflection biosciences , pieris pharmaceuticals , clearlight diagnostics , darwin health , grail research funding : novartis , astrazeneca , taiho pharmaceutical , genentech , calithera biosciences , debiopharm group , bayer ag , aileron therapeutics , puma biotechnology , cytomx therapeutics , jounce therapeutics , zymeworks , curis , pfizer , effector therapeutics xiaofeng zheng no relationship to disclose maryam shariati no relationship to disclose senthil damodaran no relationship to disclose chetna wathoo no relationship to disclose lauren brusco employment : celgene mehmet esat demirhan no relationship to disclose coya tapia research funding : armo biosciences travel , accommodations , expenses : incyte agda karina eterovic no relationship to disclose reva k . 
basho employment : davita ( i ) , castlewood treatment center ( i ) honoraria : insight strategy advisors , navigant consulting consulting or advisory role : heron therapeutics , puma biotechnology naoto t . 
ueno no relationship to disclose filip janku stock and other ownership interests : trovagene consulting or advisory role : deciphera , trovagene , novartis , sequenom , foundation medicine , guardant health , immunome research funding : novartis , biomed valley discoveries , roche , agios pharmaceutical , astellas pharma , deciphera , symphogen , plexxikon , piqur therapeutics , fujifilm other relationship : bio - rad laboratories aysegul sahin no relationship to disclose jordi rodon consulting or advisory role : novartis , eli lilly , imclone systems , servier , orion , peptomyc research funding : novartis , bayer ag russell broaddus no relationship to disclose tae - beom kim no relationship to disclose john mendelsohn leadership : merrimack stock and other ownership interests : merrimack patents , royalties , other intellectual property : royalty payments from university of california , san diego travel , accommodations , expenses : merck kenna r . 
mills shaw no relationship to disclose debu tripathy consulting or advisory role : novartis , nektar , pfizer , puma biotechnology research funding : genentech ( inst ) , roche ( inst ) , novartis ( inst ) travel , accommodations , expenses : novartis gordon b . 
 patents , royalties , other intellectual property : licensed technology hrd assay to myriad genetics acknowledgment travel , accommodations , expenses : astrazeneca , symphogen , ionis pharmaceuticals , medimmune , eli lilly , novartis , immunome , pfizer we thank kristin hargraves and the khalifa institute for personalized cancer therapy clinical research team for assistance with data curation and kurt evans for technical assistance . ken chen no relationship to disclose affiliations all authors : the university of texas md anderson cancer center , houston , tx . support supported by the sheikh khalifa al nahyan ben zayed institute for personalized cancer therapy , an astrazeneca foundation grant , national cancer institute grant no . 
connally breast cancer research endowment , cancer prevention research institute of texas precision oncology decision support core rp150535 , the bosarge foundation , and md anderson cancer center support grant no . 
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although targeted therapies , including vandetanib and cabozantinib , are used to treat mtc , resistance ( both intrinsic and acquired ) to these therapeutic approaches warrants a need to better understand the broad - spectrum anomalies that govern this phenomenon , especially in patients with aggressive disease . 
using whole - exome sequencing ( wes ) and shallow whole - genome sequencing ( swgs ) of the primary tumor , adjacent normal bone marrow tissues , and blood , we identified three germline single nucleotide polymorphisms ( snps ) within the ret proto - oncogene that remained undetected using routine hospital genetic testing procedures . 
supported by findings from both wes and swgs , we report on the occurrence of a chromothripsis - like pattern in thyroid cancer , which involved shattering of chromosome 4 leading to complete abrogation of normal chromosomal function , along with dramatic widespread copy - number aberrations across both primary tumor and bone marrow samples . a 36 - year - old man with a body mass index ( bmi ) of 45 presented with a 4 - week history of chest pain , dyspnea , and 22 kg of unintentional weight loss ( overview of the patients clinical course is presented in the data supplement )  . 
the working diagnosis was carcinoma of unknown primary . a bone marrow biopsy was performed , and immunohistochemical ( ihc ) staining revealed round and polygonal malignant cells arranged in nests and cord - like patterns with round to oval nuclei containing coarse granular chromatin patterns adjacent to areas of extensive tumor necrosis ( fig 1a1 )  . focal staining was positive for calcitonin ( fig 1a2 ) , chromogranin ( data supplement ) , thyroid transcription factor 1 ( data supplement ) , and synaptophysin ( data supplement ) , but it was negative for cd45 ( data supplement )  . 
a diagnosis of mtc was established . these results led to a thyroid ultrasound , which showed a 4 - cm hypoechoic nodule at the lower pole of the left lobe and a 1.4 - cm hypervascular echogenic focus inferomedially to it ( fig 1b )  . 
ihc of the primary tumor revealed pleomorphic polygonal and ascopubs.org / journal / po jco precision oncology sudipto das deirdre kelly bruce moran kathleen han niall mulligan ciara barrett patrick g . 
radiologic detection of primary tumor and immunohistochemical analysis of primary thyroid and bone marrow sample . ( a ) bone marrow biopsy sample stained with ( 1 ) hematoxylin and eosin staining showed infiltration of the marrow space by a proliferation of pleomorphic round to oval cells with scant eosinophilic cytoplasm and inconspicuous nucleoli . 
 ( 2 ) positive staining of the bone marrow biopsy observed for calciton ( b ) thyroid ultrasound shows a 1.4 - cm hyperechoic nodule at the lower pole of the left lobe of thyroid ( as indicated by the red arrow ) , suggestive of a primary thyroid malignancy . 
 ( d ) primary tumor sample stained with ( 1 ) hematoxylin and eosin shows that the primary tumor consisted primarily of pleomorphic polygonal and spindle cells arranged in nests separated by fibrous strand . 
serum calcitonin was 5 , 000 ng / l ( normal range , , 10 ng / l ) , and carcinoembryonic antigen was 200 mg / l ( normal range , , 5 mg / l )  . routine germline ret mutation testing in exons 10 , 11 , 13 , 14 , and 16 was negative . 
the patient died on day 31 from multiorgan failure . postmortem examination showed metastatic mtc , with complete replacement of the left lobe of the thyroid involving para - esophageal tracheal lymph nodes and the pleural surface of both lungs extending into the parenchyma . 
after receiving written informed consent from the patients family for research activities to be carried out postmortem , dna extracted from the primary tumor , adjacent normal bone marrow tissue , and blood was used to carry out wes and swgs ( data supplement )  . results chromothripsis - like pattern in genomic data . we revealed a dramatic landscape , concordant between wes and swgs ( fig 2a ) , of wideranging copy - number aberrations ( cnas ) across the primary tumor , two distinct regions of bone marrow , which showed clear tumor cell infiltration ( bm - mets1 , bm - mets2 ) , and adjacent normal tissue . 
by using the blood sample as a baseline , losses ranging from regions of several megabases to entire chromosomes were evident across all tumor samples ( fig 2a ; data supplement ) with a single gain occurring on the q arm of chromosome 1 . 
genome - wide copy - number aberrations and identification of chromothripsis - like pattern across adjacent normal tissue , blood , primary tumor , and bone marrow samples showing somatic mutations from whole - exome sequencing ( wes )  . 
 ( a ) absolute copy - number aberrations ( y - axis : 1 , loss ; 3 , gain ) across chromosomes 1 to 21 ( x - axis , denoted by blue lines , centromere denoted by dashed pink line ) compared by using data from wes and shallow wholegenome sequencing across all sample types . 
 ( b ) the copy - number aberration profile for chromosome 4 across the all samples demonstrating the chromothripsis - like pattern event generated for a 30 - kb bin using the wes data . 
ctlp is a catastrophic event involving tens to hundreds of chromosomal rearrangements up to the whole - chromosome level , which can contribute to development of a highly unstable and aggressive disease.1 - 7 the ctlp observed in this patient led to generation of approximately 30 to 40 segments with a typical oscillation of cna between two states ( loss and normal copy number ) .7 although we did carry out genomic analysis of the other metastatic lesions , including the para - esophageal , para - tracheal , and lung metastases , they were not included in the detailed genomic assessment described in this study because of unacceptable sequencing data quality . germline mutations in ret and somatic mutations in disease - associated genes . 
germline mutation analysis using wes from blood and adjacent normal tissue did not reveal any ret variants that are currently used diagnostically ( confirming the posthumous germline ret mutation profiling )  . 
however , we identified three annotated snps within ret ( rs1800861 , rs2742241 , and rs2742243 ) that were fixed with a mutant allele frequency of 1 across all samples including blood and normal tissue . we were able to validate these orthogonally across all samples by using sanger sequencing ( data supplement )  . 
the rs1800861 ( l769l , exon 13 leuctt / leuctg ) variant has previously been reported as a predisposing factor for development of aggressive mtc.8 , 9 of the other two mutations , rs2742243 ( c / t variant ) has been associated with papillary thyroid carcinoma10 and rs2742241 ( g / a variant ) has not been shown to be associated with thyroid cancer . furthermore , our somatic mutational analysis identified 109 somatic mutations , 70% of which occurred in only one of the samples ( fig 2c ; data supplement )  . 
among the nonsynonymous variants of note in disease - associated are missense mutations genes , including gse1 , cldn12 , and trim28 , which have been previously implicated as playing a role in disease processes such as metastasis , migration , and proliferation across various cancer types11 - 15 and may have had a role as potential drivers of disease progression in this patient . 
the differences in mutational patterns between the two bone marrow samples suggest marked genetic heterogeneity between tumor deposits in the same tissue . discussion the patients high bmi made palpation of a neck mass impossible , and the lack of an abnormality on routine imaging in addition to the development of dic and deteriorating respiratory status made this an extremely difficult clinical and diagnostic case . 
remarkably , it was the analysis of the bone marrow that led to the diagnosis of mtc , which further exemplified the severity of the disease in this patient showing complete infiltration of the bone marrow , potentially contributing to a total bone marrow collapse and resulting in development of dic.16 , 17 posthumously , routine clinical germline ret mutation testing did not identify any mutations within this gene . we sought to determine why this young man developed such an aggressive mtc and if it could be explained by the interplay of genomic events . our results support the hypothesis of a catastrophic event such as the evident ctlp , along with widespread copy - number alterations that may be regarded as contributing factors toward the observed phenotype.4 , 5 , 18 although ctlp has been detected in 2% to 3% of all cancers , 1 , 3 , 7 , 19 - 21 to the best of our knowledge , this is the first report to demonstrate ctlp in any form of thyroid cancer . in addition to de novo events that have an impact on cancer phenotype , known genetic alterations can often predispose patients to aggressive disease development . 
variants in the ret protooncogene are commonly associated with mtc and are used as a primary diagnostic test to identify familial and sporadic cases.22 - 25 these tests encompass well - established hot spots within ret and have largely been proven effective in detecting patients with conventional mtc . 
however , unusual patients such as this one require detailed analysis of such genes with the intent of identifying rare mutations that might either explain the atypical phenotype or allow it to be molecularly diagnosed . 
the germline ret l769l variant , previously associated with increased risk of aggressive mtc , 8 , 10 , 22 as well as the other two mutations discovered here , which have not previously been reported in mtc , could have an additive effect on increased risk of development of an aggressive phenotype . 
 ret snps were shown to have increased risk for lymph node and distant metastases at diagnosis ( odds ratio , 5.84 ) and also for developing metastases earlier.8 importantly , our results exemplify how the inclusion of more variants in clinical assays may be of use , especially in diagnostically challenging patients . although identification of mutations in individual disease - associated loci , as observed through the somatic mutation analysis , allows us to generate an appropriate hypothesis surrounding the events that could have led to the development of metastasis , the genetic heterogeneity observed across all resection samples of this patient underpin why , in the clinical setting , targeting specific mutational events therapeutically is rarely effective . 
however , it is also important to note that the study reported here describes a single patient with a rare and atypical mtc . multifactorial genomic events , including germline and somatic mutations in functionally important genes together with widespread cna ( including ctlp ) supports the need for pragmatic treatment approaches using combinations of targeted agents and possibly even immunotherapeutic approaches.26 - 28 within the context of this study and in patients who have disease of a similar nature , it is tempting to speculate that the presence of ctlp would likely lead to generation of multiple neoantigens , suggesting a potential benefit of immunotherapy.26 however , it is noteworthy that such immunotherapeutic strategies to date have not been used for patients with mtc . 
although mutational load may be inferred from routine panel - based genetic testing when only a few hot spot mutations are screened , such as in mtc , it is unlikely that candidates for immunotherapy will be identified , suggesting that a deeper examination of the genome may provide better diagnostic and therapeutic information . in conclusion , our results provide a critical insight into potential drivers of an aggressive phenotype of mtc and , in parallel , present a vital opportunity to identify effective therapeutic strategies based on the evident abnormalities in a given patient . 
kelly collection and assembly of data : sudipto das , deirdre kelly , bruce moran , kathleen han , niall mulligan , ciara barrett , peter mcmahon , hendrik f . 
relationships are self - held unless noted . deirdre kelly no relationship to disclose bruce moran no relationship to disclose kathleen han no relationship to disclose niall mulligan no relationship to disclose ciara barrett no relationship to disclose patrick g . 
van essen no relationship to disclose kate connor no relationship to disclose diether lambrechts no relationship to disclose bauke ylstra no relationship to disclose consulting or advisory role : carrick therapeutics research funding : carrick therapeutics travel , accommodations , expenses : oncomark darran p . 
gallagher employment : oncomark leadership : oncomark stock and other ownership interests : oncomark acknowledgment we thank lisa dwane , brian mooney , and camille hurley for their technical assistance on the study and the family of the patient for consenting to the study . affiliations sudipto das , deirdre kelly , bruce moran , kathleen han , niall mulligan , ciara barrett , peter mcmahon , john mccaffrey , kate connor , william m . 
oconnor , royal college of surgeons in ireland ; deirdre kelly , kathleen han , niall mulligan , ciara barrett , peter mcmahon , john mccaffrey , and catherine m . 
van essen and bauke ylstra , vrije universiteit medical center , amsterdam , the netherlands ; and diether lambrechts , vesailus research center , vlaams instituut voor biotechnologie , katholieke universiteit , leuven , belgium . supported by funding from the mater foundation , by postdoctoral fellowship crf13das from the irish cancer society ( s.d. ) , by grant no . 
cai h , kumar n , bagheri hc , et al : chromothripsis - like patterns are recurring but heterogeneously distributed features in a survey of 22 , 347 cancer genome screens . 
sromek m , czetwerty nska m , skasko e , et al : the frequency of selected polymorphic variants of the ret gene in patients with medullary thyroid carcinoma and in the general population of central poland . 
liao s , song w , liu y , et al : familial multinodular goiter syndrome with papillary thyroid carcinomas : mutational analysis of the associated genes in 5 cases from 1 chinese family . 
wei c , cheng j , zhou b , et al : tripartite motif containing 28 ( trim28 ) promotes breast cancer metastasis by stabilizing twist1 protesci rep 6 : 29822 , 2016 15 . 
yang y , cheon s , jung mk , et al : interleukin - 18 enhances breast cancer cell migration via down - regulation of claudin - 12 and induction of the p38 mapk pathway . 
forment jv , kaidi a , jackson sp : chromothripsis and cancer : causes and consequences of chromosome shattering . j gen intern med 21 : c6 - c8 , 2006 study . 
ye l , santarpia l , cote gj , et al : high resolution array - comparative genomic hybridization profiling reveals deoxyribonucleic acid copy number alterations associated with medullary thyroid carcinoma . 
 o application of circulating cell - free tumor dna proles for therapeutic monitoring and outcome prediction in genetically heterogeneous metastatic melanoma ren ata v araljai , msc1 ; kilian wistuba - hamprecht , phd2 ; teola seremet , md phd3 ; joey mark s . 
becker , md phd1 ; julia newton - bishop , md4 ; andreas stang , md mph1 ; bart neyns , md phd3 ; benjamin weide , md2 ; dirk schadendorf , md1 ; and alexander roesch , md1 purpose circulating cell - free tumor dna ( ctdna ) reects the heterogeneous spectrum of tumor - specic mutations , especially in systemic disease . 
2019 by american society of clinical oncology introduction circulating cell - free tumor dna ( ctdna ) has emerged as a potential noninvasive blood biomarker for monitoring tumor load and detecting clinically actionable driver and resistance mutations in patients with melanoma who are receiving therapy.115 most studies published on ctdna in melanoma have been , in essence , proof - of - principle reports investigating technical feasibility or focusing on ctdna courses of individual patients ( mostly brafv600e - positive patients ) based on retrospective sample and data collections . 
however , this does not account for the divergence of mutational states in blood versus tissue , because of intraand intertumor heterogeneity or the different detection sensitivities of bloodversus tissue - based assays . 
 v araljai et al melanoma monitored for the most frequently occurring mutations in braf , nras , and the promoter region of the tert geneestimated to cover 80% of patients.1 we established ctdna thresholds for disease monitoring and examined the relationship between ctdna responses to therapies , including mitogen - activated protein kinase ( mapk ) and immune checkpoint inhibition , and correlated the dynamic changes of ctdna with clinical benet . 
plasma samples were collected as part of standard care or within clinical trials . patients were treated with mapk signaling - targeted drugs ( n = 58 ) , immune checkpoint inhibitors ( n = 33 ) , or with signaling targeted therapy in combination with chemotherapy ( n = 5 ; data supplement )  . 
patients were eligible for plasma monitoring on the basis of histologically conrmed stage iii or iv melanoma and positive detection status for brafv600e , nrasq61 , tertc228t , or tertc250t mutations in a previous tumor biopsy specimen . 
patients were included in ctdna monitoring when they had available baseline and a minimum of three consecutive plasma samples during systemic therapy collected at approximately 4to 6 - week follow - up intervals . 
overview of ( a ) the training cohort ( n = 96 patients with stage iii and iv disease ) and ( b ) the validation cohort ( n = 35 patients with stage iii and iv disease )  . 
correlation of baseline circulating cell - free tumor dna ( ctdna ) levels with metastatic stage and routine serum markers in the training cohort . correlation of baseline brafv600e ctdna levels with increasing metastatic tumor load in ( a ) lymph nodes ( n1 , n = 3 ; n2 , n = 3 ; and n3 , n = 14 ) and ( b ) organs ( m1a , n = 5 ; m1b , n = 5 ; m1c , n = 41 )  . 
 v araljai et al results tomography ( ct ) , magnetic resonance imaging ( mri ) , or ultrasound imaging ( us ) was carried out at baseline and at every 6 to 12 weeks ( 6 2 weeks of plasma sampling time points )  . 
technical assay details and statistical data processing are described in the data supplement . patient validation cohort in total , 104 plasma samples from 35 patients with advanced stage iii or iv disease were prospectively collected either within pharmaceutical clinical trials , early access programs , or biobanking protocols at the department of dermatology of the t ubingen university medical center and the department of medical oncology of the universitair ziekenhuis in brussels ( fig 1b ; data supplement )  . 
informed consent procedures were followed ( approval nos . 316 / 2018bo2 and bun 143201421920 , respectively )  . patients from t ubingen were treated with pembrolizumab ( n = 17 ) or nivolumab plus ipilimumab ( n = 4 ) at the department of dermatology of the t ubingen university medical center . 
the eligibility criteria were the same as for the training cohort . patients with available baseline and a minimum of two consecutive plasma samples during systemic therapy were included in ctdna monitoring . 
radiologic tumor response evaluation of the validation cohort was performed according to immune - related response criteria ( brussels ) or response evaluation criteria in solid tumors ( recist ) 1.1 ( t ubingen )  . ctdna level correlated with melanoma tumor burden and was a risk factor for disease progression at baseline previous reports suggested a correlation between ctdna levels and tumor burden in patients with melanoma.2 - 7 for this study , candidate mutations for ctdna monitoring were selected on the basis of the most frequently occurring genomic changes in melanoma ( estimated to cover 80% of patients ; data supplement )  . 
correlation of mutant tertprom ctdna levels with metastatic progression from locoregional ( stage iiib , n = 2 ; or iiic , n = 6 ) to systemic disease ( stage iv ; n = 14 ) in patients with tertprom - mutated melanomas . 
 ( e ) correlation of baseline serum lactate dehydrogenase ( ldh ) and s100 levels with metastatic progression in brafv600e - positive patients ( gray line , upper limit of normal )  . 
 ( f - k ) correlation of serum ldh and s100 levels with brafv600e ( n = 274 and n = 216 , respectively ) , nrasq61 ( n = 97 and n = 67 , respectively ) , and mutant tertprom ( n = 152 and n = 124 , respectively ) ctdna levels in the training cohort . 
changes in the brafv600e circulating cell - free tumor dna ( ctdna ) levels correlate with therapy response ( pfs )  . and progression - free survival changes in mean brafv600e ctdna levels after therapy initiation relative to baseline ( bl ) in patients who received ( a ) immune checkpoint inhibition therapy ( n = 18 patients ) and ( b ) signaling targeted therapy ( n = 33 )  . 
 ( * ) p , .01 , ( ) p , .001 , and ( ) p , .001 from unpaired t test . the data represent mean 6 sem . kaplan - meier plots are for pfs of the same patients with melanoma assessed by routine radiologic scans . 
 ( c ) immune checkpoint inhibition ( patients with ctdna decrease [ n = 6 ] v increase [ n = 12 ] ) and ( d ) signaling targeted therapy ( patients with ctdna decrease [ n = 12 ] v increase [ n = 21 ] )  . 
 ( e , f ) scatter dot plots of brafv600e ctdna levels of responders versus nonresponders grouped according to ( e ) radiologic response 10 weeks after receiving any therapy or ( f ) radiologic pfs at 6 months of therapy ( mann - whitney u test )  . 
furthermore , the roc analysis allowed determination of ctdna thresholds that we could use subsequently to evaluate the predictive impact of ctdnas during therapy ( data supplement )  . to assess ctdna levels under targeted and immune inhibition , 540 plasma samples from 76 checkpoint patients were analyzed by ddpcr and compared with radiologic staging . 
targeted therapies seemed to decrease ctdna levels faster than immune checkpoint inhibitors ( mean decrease of 31% v 15% at week 4 ; p , .001 ; combined analysis of data from figs 3a and 3b , and figs 4a , 4b , 4c , and 4d , differences remained signicant : unpaired t test p , .001 for all time points )  . 
changes in mean nrasq61 and tertprom ctdna levels after therapy initiation relative to baseline ( bl ) in patients who received ( a , c ) immune checkpoint inhibition therapy , ( n = 10 and n = 12 , respectively ) and ( b , d ) signaling targeted therapy , ( n = 7 and n = 10 , respectively )  . 
scatter dot plots of nrasq61 and ( f ) tertprom ctdna levels of patients assessed at week 10 after receiving any therapy , grouped according to radiologic pfs at 6 months ( mann - whitney u test )  . points represent individual patients ; median with interquartile range is indicated for each plot . 
accordingly , the median was 494.5 copies / ml in patients with a pfs shorter than 6 months ( ie , signicantly higher than in patients with a pfs longer than 6 months [ mann - whitney u test p , .001 ] ; fig 3f )  . 
 ( a ) changes in mean ctdna levels after therapy initiation relative to baseline ( bl ) from patients who received immune checkpoint inhibition therapy ( n = 35 patients )  . 
 ( b ) kaplan - meier plot for progressionfree survival ( pfs ) of the same patients with melanoma who received immune checkpoint inhibition therapy who were assessed by routine radiologic scans ( patients with ctdna decrease [ n = 15 ] v increase [ n = 20 ] )  . 
regarding absolute copy numbers of ctdna across all patients treated , the median ctdna value of nonresponders was 144 copies / ml at 12 weeks of treatment , which was signicantly higher than in responders ( 12 copies / ml ; mann - whitney u test p , .001 ; fig 5c ) and above the threshold dened by roc analysis in the training cohort ( data supplement )  . 
the median was 127 copies / ml in patients with a pfs shorter than 6 months and , thus , signicantly higher than in patients with a pfs longer than 6 months ( 14 copies / ml ; mann - whitney u test p = .003 ; fig 5d )  . 
 ( c - d ) scatter dot plots of brafv600e , nrasq61 , and tertprom ctdna levels of responders versus nonresponders , grouped according to ( c ) radiologic response 12 weeks after receiving any therapy or ( d ) radiologic pfs at 6 months of therapy ( mann - whitney u test )  . 
at risk : samples tissue ngs tissue ddpcr plasma ddpcr tissue ngs tissue ddpcr plasma ddpcr tissue ngs tissue ddpcr plasma ddpcr tissue ngs tissue ddpcr plasma ddpcr tissue ngs detected not detected tissue ddpcr brafv600e plasma ddpcr brafv600e nrasq61 hotspot tert c228t tert c250t not detected nrasq61 hotspot tert c228t tert c250t not detected fig 6 . 
kaplan - meier plots for ( a ) radiologic progression - free survival ( pfs ) and ( b ) overall survival ( os ) of patients with brafv600e - positive tumors who received mapk signaling targeted therapy ( n = 21 with positive nrasq61 detection in bl plasma v n = 12 without detection )  . 
 ( c ) overview of 51 brafv600e - , nrasq61 - , and tertprom - matched tumor tissue and plasma samples of patients who had available tumor biopsy specimen at therapy bl . 
plasma was sampled at bl ( ie , before systemic therapy initiation ) and analyzed for ctdnas by droplet digital polymerase chain reaction ( ddpcr )  . to test if potential differences in the assay sensitivities of next - generation sequencing ( ngs ) versus ddpcr accounted for the observed discrepancies of the patients nrasq61 mutational states , we obtained genomic dna isolated from 51 melanoma tissue samples that previously underwent amplicon - targeted ngs and reanalyzed them on the ddpcr platforindeed , of 12 patients with brafv600e mutation whose plasma samples were positive for nrasq61 ctdna at baseline , but with negative tumor tissue according to ngs , eight were also positive in tissue samples when tested with the ddpcr assay ( fig 6c ; data supplement )  . however , in another four cases , only plasma ddpcr could detect the positive mutational nrasq61 state , whereas the tumor sample remained negative by ddpcr . 
 v araljai et al discussion to make precision oncology a reality , highly predictive , noninvasive monitoring technologies must be identied . the application of ctdna to metastatic melanoma has been reported to be a promising tool in terms of detecting the relevant tumor - derived mutant dna over time.2 - 6 , 10 - 19 here , we also considered the clinical signicance of background mutations in the ctdna of healthy individuals for statistical normalization at baseline and then later under therapy . 
this consideration is critical because mutant ctdna , derived from aging cells or from undiscovered cancer cells , also can occur throughout the lifetime of individuals without a known diagnosis of cancer.20 regarding differences between healthy plasma donors and patients with melanoma with radiologically conrmed active tumor mass , we intended to establish a molecular cutoff value with true clinical relevance using roc analysis . 
we conclude that concrete ctdna thresholds are prognostic for outcome and represent a reliable practical tool for routine diagnostics even in the context of background mutant ctdna . another goal of this study was to statistically validate a therapeutic predictive threshold on the basis of roc analyses of longitudinally assessed plasma samples from patients with melanoma with timely matching ct / mri scans . 
accordingly , patients whose ctdna levels were above a threshold of 35.85 copies / ml brafv600e , 51.5 copies / ml nrasq61 , and 32.83 copies / ml tertprom ctdna over 6 to 8 weeks despite therapy had signicantly shorter pfs as compared with patients whose ctdna levels remained below these thresholds . 
all the ctdna assays applied here were signicantly better indicators of measurable disease than were ldh or s100 levels . despite the practicability of molecular thresholds , our study indicates that the mean change in the ctdna level over consecutive time points compared with the baseline represents an additional signicant indicator of treatment effectiveness . 
our study also showed that ctdna changes relative to the previous sampling time point or even the rate of ctdna increase per week ( data supplement ) can serve as an early hint of progression in patients receiving therapy . tumor tissuebased mutational testing ( eg , by ngs with a sensitivity of approximately 1%21 ) is currently used as clinical state - of - the - art guidance for therapy selection for patients with metastatic melanoma . 
it does not account for intertumor and intrapatient heterogeneity . the issue of instead , liquid biopsies should better assess the full genetic heterogeneity of solid cancers at the systemic disease level . here , we analyzed baseline plasma samples with highly sensitive ddpcr and compared the results with existing ngs - based tissue proles . 
this may indicate that the amplicon - targeted ngs protocol , which was routinely applied to assess the mutational tumor status in our cohorts , may not be sensitive enough to detect low - frequency nras mutations within bulk tumors ( eg , when originating only from minor cell subpopulations )  . 
to test this assumption , we obtained genomic dna isolated from 51 melanoma tissue samples that previously underwent amplicon - targeted ngs and reanalyzed them on the ddpcr platforindeed , of 12 patients with brafv600e mutation whose plasma samples were positive for nrasq61 ctdna at baseline , while having a negative ngs status for nrasq61 , tumor tissue of eight also was positive when tested with the ddpcr assay . 
in contrast , enhancement of the ngs coverage from 103 to 177 and reduction of the mutant discovery threshold from 5% to 1% ( mutant allele frequency ) did not increase the nrasq61 detection rate in a control experiment with selected tissue samples ( data supplement )  . 
previous data suggest that nrasq61 ctdna emerges upon mapki therapy.3 , 16 accordingly , we observed increasing levels of nrasq61 during mapki the absolute nrasq61 copy numbers remained below the brafv600e copy level.3 , 16 therapy ; however , in summary , our study ndings support the importance of ctdna analysis as a valid clinical tool for the provision of real - time information on current treatment response and prediction of future response probability . 
in addition , the comparison of plasma - based ctdna analysis with tumor tissuebased , amplicon - targeted ngs indicated considerable differences in diagnostic sensitivity , particularly with regard to intrapatient tumor heterogeneity . 
vogel , susanne horn , dirk schadendorf , alexander roesch financial support : julia newton - bishop , dirk schadendorf administrative support : klaus griewank , dirk schadendorf , alexander roesch provision of study materials or patients : teola seremet , peter a . 
horn , bart neyns , dirk schadendorf , alexander roesch collection and assembly of data : ren ata v araljai , kilian wistubahamprecht , teola seremet , antje sucker , jan - malte placke , peter a . horn , nils von neuhoff , susanne horn , j urgen c . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . teola seremet employment : janssen ( i ) , glaxosmithkline ( i ) honoraria : novartis , janssen consulting or advisory role : novartis speakers ' bureau : novartis travel , accommodations , expenses : novartis , msd oncology , janssen , leo pharma klaus griewank patents , royalties , other intellectual property : exon 4 mutations in gnaq and gna11 , a rare gene mutation in melanoma . 
horn stock and other ownership interests : share certicates ( aktien ) of different companies patents , royalties , other intellectual property : patent applications support supported by the european union 's horizon 2020 research and innovation program under marie sklodowska - curie grant no . 
 v araljai et al lipson ej , velculescu ve , pritchard ts , et al : circulating tumor dna analysis as a real - time method for monitoring tumor burden in melanoma patients undergoing treatment with immune checkpoint blockade . 
j immunother cancer 2 : 42 , 2014 sanmamed mf , fern andez - land azuri s , rodrguez c , et al : quantitative cell - free circulating brafv600e mutation analysis by use of droplet digital pcr in the follow - up of patients with melanoma being treated with braf inhibitors . 
clin chem 61 : 297 - 304 , 2015 lee jh , long gv , boyd s , et al : circulating tumour dna predicts response to anti - pd1 antibodies in metastatic melanoma . 
ann oncol 28 : 1130 - 1136 , 2017 gangadhar tc , savitch sl , yee ss , et al : feasibility of monitoring advanced melanoma patients using cell - free dna from plasma . 
nazarian r , shi h , wang q , et al : melanomas acquire resistance to b - raf ( v600e ) inhibition by rtk or n - ras upregulation . 
nature 468 : 973 - 977 , 2010 shi h , hugo w , kong x , et al : acquired resistance and clonal evolution in melanoma during braf inhibitor therapy . 
haselmann v , gebhardt c , brechtel i , et al : liquid proling of circulating tumor dna in plasma of melanoma patients for companion diagnostics and monitoring of braf inhibitor therapy . 
santiago - walker a , gagnon r , mazumdar j , et al : correlation of braf mutation status in circulating - free dna and tumor and association with clinical outcome across four brafi and meki clinical trials . 
gonzalez - cao m , mayo - de - las - casas c , molina - vila ma , et al : braf mutation analysis in circulating free tumor dna of melanoma patients treated with braf 13 . 
chang ga , tadepalli js , shao y , et al : sensitivity of plasma brafmutant and nrasmutant cell - free dna assays to detect metastatic melanoma in patients with low recist scores and non - recist disease progression . 
schreuer m , meersseman g , van den herrewegen s , et al : quantitative assessment of braf v600 mutant circulating cell - free tumor dna as a tool for therapeutic monitoring in metastatic melanoma patients treated with braf / mek inhibitors . 
lee jh , long gv , menzies am , et al : association between circulating tumor dna and pseudoprogression in patients with metastatic melanoma treated with antiprogrammed cell death 1 antibodies . 
xi l , pham th - t , payabyab ec , et al : circulating tumor dna as an early indicator of response to t - cell transfer immunotherapy in metastatic melanoma . 
blumenthal , md1 ; and reena philip , phd1 purpose next - generation sequencing ( ngs ) oncology panels are becoming integral in hospital and academic settings to guide patient treatment and enrollment in clinical trials . 
although ngs technologies have revolutionized decision - making for cancer therapeutics , physicians may face many challenges in parsing and prioritizing ngs - based test results to determine the best course of treatment for individual patients . 
here , we discuss the presentations and discussion highlights across the four sessions of the workshop . methods the goal of the public workshop was to engage stakeholders and solicit input from experts in precision oncology to discuss the integration of complex ngs data into patient management and regulatory innovation within the precision oncology community . 
the us food and drug administration gathered representatives from academia , industry , patient advocacy , government , and professional organizations for a series of presentations followed by panel discussions . 
after the workshop , the transcript and speaker presentation slides were reviewed and summarized for manuscript preparation . results speakers and panelists provided diverse perspectives on the integration of ngs technology into patient care for oncology and on the complexities that surround data interpretation and sharing . 
2019 by american society of clinical oncology introduction the goal of precision oncology is to use the genetic informationgermline or somaticfrom a patient with cancer to help determine which drug ( s ) might be most effective in treating his or her disease . 
next - generation sequencing ( ngs ) is increasingly used in precision oncology because of its ability to identify many mutations simultaneously , thereby maximizing the amount of information obtained from a single test.1 the global endeavor to identify somatic mutations within a patients tumor genome has led to an unprecedented amount of genomic information , with varying degrees of associated clinical evidence . 
ngs can report a vast number of mutations which may lead to clinician uncertainty in the interpretation and prioritization of mutations with respect to the clinical signicance and optimal course of treatment.2 in january 2017 , the association for molecular pathology ( amp ) , asco , and the college of american pathologists ( cap ) published a joint consensus recommendation for standards and guidelines for the interpretation and reporting of sequence variants in cancer3 ; however , stakeholders have not consistently implemented these recommendations . 
 horne et al context key objective to provide an overview of a public workshop hosted by the us food and drug administration that discussed key concepts and considerations for the classication and interpretation of genetic variants in precision oncology . knowledge generated key highlights and ndings from the discussion include the conclusion that as the use of next - generation sequencing oncopanels in the clinical setting increases , it is critical to develop a framework that ensures accurate and consistent interpretation of genetic variants . 
in addition , transparency in data sourcing and reporting methodologies , as well as frequent updating of variant interpretations , are necessary to advance science and appropriately care for patients . relevance as precision medicine advances , clear communication among stakeholders in oncology will be key to creating an environment that facilitates generating and sharing data that have value to patients . treatement options , in addition to providing tumor proling data.6 , 7 with fda approval and authorization of these two ngs - based pan - tumor oncology panels ( oncopanels ) , the agency was in a unique position to foster a dialogue among key stakeholders about this innovative technology . input on january 29 , 2018 , the fda held a public workshop to engage stakeholders and solicit from experts in precision oncology and patient representatives to discuss how genetic sequencing data could be best implemented in patient management . 
to set the stage for the workshop discussion , dr philip outlined the fdas new regulatory pathway for ngs tumor - proling tests , as summarized in the fdas center for devices and radiologic health fact sheet published online on november 15 , 2017.8 as described by dr philip , the agencys three - tiered approach for reporting biomarkers in tumor - proling ngs tests includes the following : level 1 , companion diagnostic claims ; level 2 , cancer mutations with evidence of clinical signicance ; and level 3 , cancer mutations with potential clinical signicance . 
tumor - proling ngs tests may include companion diagnostic claims ( level 1 ) that are prescriptive for a specic therapy and are supported by the clinical validity of the test for each biomarker reported . 
bowles , phd , myriad genetic laboratories best practices for the use of clinical databases for variant classication and facmg interpretation in clinical oncology session 4 : future directions for data sharing , standardization , and establishing consistency in precision oncology dane dickson , md cureone us food and drug administration public workshop : variant classication dennis dean , phd seven bridges genomics creating a community view : standards , analyses , and data to advance robert grossman , phd university of chicago the important role of data commons for sharing variant classication and consistency in precision oncology interpretation data in precision oncology len lichtenfeld , md american cancer society cancer action panelist network * afliations noted in the table are based on speaker afliations at the time of the public workshop and may not reect the current afliation . validity has not been demonstrated either in professional guidelines or with the test , but is suggestive on the basis of clinical / biologic evidence . session 1 : overview of the state of science for sequence variant classication in oncology and its practical use in treating patients use of an individuals mutation prole for clinical decision making is driving the ever - expanding number of biomarkerbased clinical trials and clinical regimens in precision oncology . 
 horne et al vincent miller , md , indicated that fmi lters out germline mutations but further noted that when reporting germline mutations , laboratories need to have an operation in place , or at least have an understanding with the clinicians , so that the information can be followed up with genetic counseling . comprehensive variant annotation is necessary to inform treatment selection and match patients to clinical trials . 
the number of patients who are matched to either clinical trials or personalized treatments on the basis of their genomic data were reported by panelists to be between 10% and 37% . 
to compile and interpret the evidence , scientists and pathologists continually gather new information from guidelines , published literature , public databases , clinical trials , fdaapproved therapeutic labeling , and other sources . 
this comprehensive information is then used to sort biomarkers , either manually or in combination with bioinformatics software algorithms , on the basis of levels of evidence . panelists concluded that a key to the success of somatic genotyping will be developing statistical tools and bioinformatics infrastructure to identify clinically meaningful mutations . 
dr berger noted that as more sequencing data accumulates , integrated analyses of big data will allow for the identication of novel hotspot mutations occurring at frequencies greater than would be expected by chance . this approach would allow for these novel mutations to be directly tested in targeted clinical trials to assess drug sensitivity . 
panelists noted that the eld will continue to evolve and become more challenging with the increase in tissue - agnostic trials in which patients are enrolled on the basis of biomarker status , irrespective of the tumor type , which could , in turn , lead to an increase in tissue - agnostic drug indications . 
john deeken , md , also highlighted some of the unique challenges faced when running an in - house 50 - gene oncopanel at a community - based hospital system . these challenges included the cost of developing and supporting an oncopanel test and the lack of reimbursement for the majority of ngs testing performed . 
donna roscoe , phd , noted that the fda is striving to ensure that there are validated tests on the market that will allow for dynamic use within clinical settings to enable clinicians to optimize patient decisions in oncology and other therapeutic areas . session 2 : levels of evidence required for reporting variants and guiding patient treatment widespread implementation of ngs data in clinical settings has been accompanied by challenges in standardization , interpretation , and reporting of genetic tests . 
shashikant kulkarni , phd , provided an overview of the proposed guidelines published by a multidisciplinary working group convened by amp , asco , and cap for variant interpretation and reporting.3 the four - tiered system to categorize somatic sequence variations on the basis of their levels of evidence are as follows : tier i , variants with strong clinical signicance ; tier ii , variants with potential clinical signicance ; tier iii , variants of unknown clinical signicance ; and tier iv , variants deemed benign or likely benign.3 guidelines were developed to provide a general framework , on the basis of an evidence - based variant classication approach , for the standardized interpretation and reporting of somatic variants in cancer . 
for example , variants with strong clinical evidence include those with level a evidence ( fda - approved therapies or included in professional guidelines ) or level b evidence ( well - powered studies with consensus from experts in the eld )  . 
variants with potential clinical evidence are those with level c evidence ( fda - approved therapies for different tumor types or investigational therapies of multiple small published studies with some consensus ) or level d evidence ( preclinical trials or found in a few case reports without consensus )  . in general , it is widely accepted that tests should be optimized to ensure coverage across targets and high sensitivity for detecting low - frequency mutations ; however , as noted in the consensus manuscript , 3 the eld is split on the use of variant allele frequency ( vaf ) in reporting and clinical practice . 
one perspective , provided from a workshop attendee , was that if frequencies are too low , the detected mutations could represent subclonal events and may not necessarily be appropriate therapeutic targets . 
on the contrary , apostolia - maria tsimberidou , md , phd , noted that , as a clinical investigator , there is utility in having allele fraction present in the patient report as the data could be used to interpret clinical outcomes , particularly because patients have multiple molecular alterations . 
a consensus was not reached by workshop panelists or attendees regarding the clinical use or validity of reporting vaf in precision oncology . standardizing the annotation and reporting of somatic mutations from ngs data remains a challenge . 
john pfeifer , md , phd , pointed out that there is a gray zone between where the technical portion of the ngs test ends and the practice of medicine begins . 
although many of the speakers and panelists used the amp / asco / cap guidelines , several groups used a slightly modied tiering system . panelists generally agreed that treatment options for tier ii and below , as noted in the amp / asco / cap guidelines , should be considered only once tier i treatment options have been exhausted . 
moreover , panelists expressed the need to create a databasethat is , a knowledge basethat can assist in resolving discordances in variant interpretation and allow for the generation of community standards . session 3 : best practices for use of public / private databases for variant classication and interpretation in oncology to positively affect patient care , mutations detected using ngs technology must be appropriately interpreted and classied . 
the panel unanimously agreed that a known drawback to using publicly available databases for variant classication is that interpretation is not always consistent . heidi rehm , phd , noted that the quality of the data rests in the hands of those who deposit the information into the database and that amassing sufcient information to provide high - quality classication data requires global sharing . kenna shaw , phd , afrmed that standards and rules are needed to not only interpret but reinterpret variant classication , and she summarized the efforts being conducted at her institution to amass a personalized cancer therapy knowledge base . 
dr shaw provided an overview of aacr project genie ( genomics evidence neoplasia information exchange ) , which presently includes 17 institutions across the world that are working together to develop a regulatorygrade registry that aggregates and links cancer genomic data with clinical outcomes from tens of thousands of cancer patients . 
project genie will allow for sharing of the accumulated data with the global research community through cbioportal and the synapse platform with the noted caveat that the underlying functional evidence int the knowledge base will continue to change over time . panelists agreed that databases should be transparent about data sources , methods , and rules used for reporting mutation - associated clinical claims . 
karla bowles , phd , noted that subjective tools , such as literature reviews , population data , and structural analysis , should be used with caution , as interpretations from these data are often sources of discrepancies . 
the number of session 4 : future directions for data sharing , standardization , and establishing consistency in precision oncology many mutations identied by genomic tests for oncology are novel , rare , or otherwise lack conclusive evidence to support a clinical these mutations will only increase as additional genes are added to oncopanels and the costs of sequencing decrease . understanding the clinical utility of these mutations requires addressing barriers that prevent accurate classication . 
dane dickson , md , cited improved data sharing as a critical need and described the utility of high - quality databases that include both diagnostic results and clinical outcomes . dennis dean , phd , described the need for interoperability of complex systems and tools to enable analysis of large data sets . interoperability requires standards and languages to support reproducibility . 
dean discussed rabix , a suite of open - source development tools for creating and running computation workows , as a development environment that uses common workow language to promote the analysis of combined large data sets . 
robert grossman , phd , emphasized the need for data commonsthat is , a unied data system that promotes sharingsuch as the national cancer institute that use data models and genomic data commons , metadata services to harmonize and share cancer genomic datasets . 
grossman discussed the concept of leveraging funders , journal editors , and payers to incentivize the data sharing required to build a data commons . as a patient advocate , len lichtenfeld , md , discussed the challenges of translating the information generated by large - scale projects to communities that lack the resources and infrastructure to access the latest information necessary to improve care . 
 horne et al in conclusion , precision oncology is a rapidly evolving eld . with more oncopanels being developed , and as more genomic and patient outcome data accumulate , physicians are faced with new challenges in interpreting ngs - based test results to determine the best treatment course for each patient . 
in addition , there are multiple public and private databases available that aid in variant classication and interpretation ; however , methods used to classify variants and how these databases align remain unclear . 
to that end , the fda recently nalized a guidance document entitled , use of public human genetic variant databases to support clinical validity for genetic and genomic - based in vitro diagnostics . 
david litwack employment : prevail therapeutics travel , accommodations , expenses : prevail therapeutics no other potential conicts of interest were reported . acknowledgment the authors thank all event staff at the us food and drug administration for helping to execute a seamless public workshop that was benecial to both in - person and online attendees . 
the authors thank the workshop presenters who graciously provided their permission to publish details about their titles and summaries in the manuscript table and text . references strom sp : current practices and guidelines for clinical next - generation sequencing oncology testing . 
n engl j med 375 : 711 - 713 , 2016 li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
us food and drug administration : use of public human genetic variant databases to support clinical validity for genetic and genomic - based in vitro diagnostics : guidance for stakholders and food and drug administration staff . 
us food and drug administration : public workshop - - weighing the evidence : variant classication and interpretation in precision oncology , january 29 , 2018 , workshops and conferences ( medical devices )  . 
 biomarkers intratumoral transcriptome heterogeneity is associated with patient prognosis and sidedness in patients with colorectal cancer treated with anti - egfr therapy from the co.20 trial elisa fontana , md , phd1 , 2 ; gift nyamundanda , phd1 , 2 ; david cunningham , md3 ; dongsheng tu , phd4 ; maggie c.u. 
siu , md8 ; francesco sclafani , md3 , 9 ; katherine eason , phd1 ; chanthirika ragulan , msc1 , 2 ; maria antonietta bali , md , phd10 , 11 ; sanna hulkki - wilson , bsc1 ; jonathan m . 
waring , mbbs , phd13 ; mirella giordano , phd14 ; patrick lawrence , ms1 , 2 ; daniel nava rodrigues , md3 ; ruwaida begum , bsc3 ; jeremy d . 
price , md16 ; chiara cremolini , md17 , 18 ; naureen starling , mbbs , mrcp , md ( res ) 3 ; filippo pietrantonio , md19 , 20 ; livio trusolino , md , phd21 , 22 ; christopher j . 
ocallaghan , dvm , msc , phd4 ; and anguraj sadanandam , phd1 , 2 purpose metastatic colorectal cancers ( mcrcs ) assigned to the transit - amplifying ( ta ) crcassigner subtype are more sensitive to antiepidermal growth factor receptor ( egfr ) therapy . 
progression - free survival ( pfs ) , overall survival ( os ) , and disease control rate ( dcr ) according to ta - ness classication were assessed by univariate and multivariate analyses . results the ta - ness was measured in 772 samples from 712 patients . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction epidermal growth factor receptor ( egfr ) - targeting antibodies cetuximab and panitumumab are available treatment options for approximately 40% of patients with metastatic colorectal cancer ( mcrc ) .1 patient selection based on ras and braf wild - type status and sidedness has improved overall response rates and survival outcomes . 
nevertheless , 30% - 60% of eligible patients do not benet from these expensive drugs.2 - 4 as a shift from the traditional paradigm of negative molecular selection , we previously demonstrated that the transit - amplifying ( ta ) crcassigner ( crca ) subtype was enriched for cetuximab - responsive tumors , 5 a nding independently validated in a clinical study , 6 in a panel of crc xenografts5 and cell lines.5 , 7 however , responses were also seen in other groups , such as the poorly differentiated stem - like subtype , 5 , 7 albeit at a lower frequency . 
 intratumoral transcriptome heterogeneity and anti - egfr therapy context key objective to evaluate whether the presence of the transit - amplifying ( ta ) subtype gene signature ( dubbed as ta - ness classication ) representing the intratumoral transcriptome heterogeneity is associated with antiepidermal growth factor receptor ( egfr ) therapy outcomes . knowledge generated the ta - ness classication is an easily detectable biomarker of intratumoral transcriptome heterogeneity , which was retrospectively evaluated in 712 patient samples , including those from a clinical ( co.20 ) trial , which showed prognostic signicance in patients treated with anti - egfr therapy . 
this biomarker provides additional biologic insights for the association between ras / braf wild - type left - sided tumors ( enriched for ta - high ) and anti - egfr therapy benet . relevance with further validation , ta - ness may represent a positive selection biomarker for patients with ras / braf wild - type left - sided metastatic colorectal cancer who are most likely to benet from anti - egfr therapy . ta subtype tumors are characterized by gene signatures similar to normal ta cells of the colonic crypt , that is , those in transit between stem cells in the crypt base and differentiated cells at the top of the crypt.5 after asymmetric division , stem cells generate rapidly proliferating ta cells characterized by increased egfr expression that eventually differentiate into goblet cells and enterocytes.8 , 9 we evaluated a hypothesis that tumors with increased ta gene signature expression ( irrespective of ta or other subtypes ) may be associated with anti - egfr therapy outcomes . 
the discovery cohort included chemorefractory patients ( n = 84 ) who had received anti - egfr therapy as a single agent or in combination with chemotherapy after progression while receiving irinotecan ( during or within 3 months from the end of treatment ) as part of standard treatment at the royal marsden hospital ( rmh ; n = 59 ; united kingdom , ethics committee : 10 / h0308 / 28 ; and clinicaltrials.gov identier : nct02112357 ) or within the context of a case - control study in italian institutions ( pressing , n = 25 ; ethics committee area vasta nord ovest number 1333 / 173 )  . 
nineteen and 12 patients from the rmh cohort were treated before the implementation of kras testing ( august 2009 ) and extended ras testing ( december 2011 ) , respectively.11 , 12 all patient samples from the pressing study had extended ras / braf wildtype tumors . one of the clinical validation cohorts included 121 patients with kras exon 2 wild - type tumors who had received single - agent cetuximab within the control arm of the co.20 phase iii randomized clinical trial ( clinicaltrials.gov identier : nct00640471 ) .13 this correlative analysis was approved by the joint canadian cancer trial group and australasian gastrointestinal trial group ( cctg / agitg ) correlative sciences and tumor biology committee . two additional public gene expression datasets ( n = 397 ; not treated with anti - egfr therapy ) of primary crc samples ( gse39582 ; n = 328 ) and liver mcrc lesions ( gse73255 ; n = 69 ) were evaluated.14 , 15 only samples with known kras wild - type status were selected . nucleic acids extraction formalin - xed parafn - embedded ( ffpe ) tissues were least evaluated by a trained pathologist ; areas with at 30% of tumor content were marked on hematoxylin and eosin slides and macrodissected in unstained slides ( 7to 10 - m thickness )  . 
after deparafnization , total rna and dna were simultaneously isolated using the ambion recoverall kit ( discovery ) or qiaamp nucleic acid ffpe tissue kit ( validation ) and quantied with nanodrop 2000 spectrophotometer ( thermo fisher , waltham , ma ) according to the manufacturers instructions . 
 a fontana et al discovery cohort retrospective tissue collections validation cohort 1 prospectively enrolled in the co.20 study outcome cohort ta - ness no . median pfs ( months ) univariate analysis hazard ratio ( 95% ci ) log - rank multivariate analysis hazard ratio ( 95% ci ) cox regression rmh cohort ( n = 90 ) italian cohort ( n = 42 ) patients ( out of 374 in cetuximab arm ) ( n = 163 ) patients with quality rna ( n = 65 ) patients with quality rna ( n = 25 ) patients with quality rna ( n = 146 ) excluded because of low tumor content in the block or low rna conc . 
 after extraction ( n = 25 ) patients excluded because of qc flag after ncounter analysis ( n = 6 ) patients excluded for anti - egfr in refractory setting as re - challenge ( n = 17 ) patients excluded because of qc flag after ncounter analysis ( n = 0 ) patients for final analysis ( n = 59 ) patients for final analysis ( n = 25 ) patients for final analysis ( n = 121 ) patients excluded because of low tumor content in the block or low rna conc . 
covariates included in the validation cohort : eastern cooperative oncology group performance status , sex , age , baseline lactate dehydrogenase level , baseline alkaline phosphatase , baseline hemoglobin , number of disease sites , number of previous chemotherapy drug classes , prior vegfr target therapy , and presence of liver metastases . 
conc. , concentration ; egfr , epidermal growth factor receptor ; pdx , patient - derived xenograft ; qc , quality control ; rmh , royal marsden hospital . ve published crca - 38 centroids ( expression summary of each gene in each subtype ) 16 and gene expression , each sample was assigned either to ta - high ( increased expression of ta signature genes ) or ta - low ( reduced expression ) ta - ness classes . 
when gene expression proles were compared with the ve crca - 38 centroids , ve correlation coefcients ( one for each subtype - centroid ) were calculated for each sample . 
the coefcients were then ranked from highest to lowest ; ta - high samples were those with a correlation coefcient value for the ta centroid ranking within the rst three highest values ; ta - low samples were those with a correlation coefcient for the ta centroid , which is second to last or the lowest . 
therefore , the ta - ness classication represents a measure of transcriptome - based intratumoral heterogeneity in mcrc , based on the idea that each sample can contain more than one subtype . 
this best cut - off for ta - ness classication was established based on the highest accuracy in dening disease control , measured as area under the curve ( auc ) of a receiver operating characteristic ( roc ) curve ( appendix fig a1 )  . statistical analysis progression - free survival ( pfs ) was the primary endpoint . overall survival ( os ) , disease control rate ( dcr ) , and response rate were secondary endpoints . 
fishers exact test was used to compare categorical variables , and wilcoxon signed rank test with p , .05 was used to assess the association between ta - ness classes and percentage of tumor shrinkage ( using recist ) criteria in a subgroup of the discovery cohort . 
although the statistical analysis of discovery cohort was performed by the institute of cancer research statistician , the validation cohort was independently analyzed by cctg / agitg investigators blinded to the biomarker cut - off analysis . 
additional methods are available in the data supplement . results retrospective antiegfr - treated tumor samples from 205 patients were identied from the discovery and validation ( co.20 ) cohorts after clinical review and quality control of the tumor blocks and tumor - derived rna ( fig 1a )  . 
 intratumoral transcriptome heterogeneity and anti - egfr therapy cohort from 30 patients along with 30 patient - derived xenografts ( pdxs ; derived from the patient tumors ) that were treated with anti - egfr therapy or vehicle ( control ) were subjected to the same crca gene expression assay . 
in addition , publicly available gene expression microarray data for 80 patients with mcrc ( treated with anti - egfr therapy ) was included as an additional clinical validation cohort.17 this publicly available cohort also served to validate ta - ness classication using a different platform ( microarrays ; fig 1a )  . patient characteristics for discovery and validation co.20 cohorts are shown in the data supplement . 
with the exception of sex , there were no signicant differences in patients characteristics between the co.20 subgroup included in this analysis and the overall co.20 clinical trial cohort ( data supplement )  . using conventional subtyping , 15 of 84 samples belonged to the ta subtype in the discovery cohort . 
the association of ta - ness classication with both pfs and dcr remained signicant after adjusting for multiple variables in both the discovery and validation cohorts ( fig 1b )  . 
conversely , after adjusting for multiple variables , signicant association of ta - ness with os was only borderline ( or not signicant with p = .1 in the discovery cohort and p = .06 in the validation cohort ; data supplement ) ; postprogression treatment information was not available . in the discovery cohort , ta - high tumors ( 62% ; n = 52 ) were predominantly ras / braf wild - type ( 69% ; n = 36 ) and were found in the left side of the colon ( 79% ; n = 41 )  . 
this result was mirrored in the experimental cohort , 18 , 19 in which 30 ras / braf wild - type liver metastases were classied into ta - high ( n = 16 ) and ta - low ( n = 14 ) classes . 
the percentage of cetuximab - induced tumor volume change in the pdx - based mouse - propagated patient metastatic tumors was signicantly associated ( p , .042 ) with the ta - ness signature ( fig 3b )  . in the discovery cohort , the ta - ness classication was assessed using samples from primary tumors in 76% of patients and samples from metastatic sites in 24% of patients . 
to further conrm that the association was independent of the diagnostic sample of origin and to further validate the results , we examined the khambata - ford publicly available ( microarray ) dataset17 of mcrc samples from patients treated with cetuximab . 
to further conrm that the ta - ness can be assessed in both primary tumors and metastatic lesions , kras wild - type samples from two publicly available datasets14 , 15 were selected ; 328 primary tumors and 69 liver metastases were classied into ta - high and ta - low . 
similar distribution of the two classes was demonstrated ( fig 3c )  . beyond ras / braf mutational status , sidedness is a recognized selection factor for anti - egfr therapy benet : patients with left - sided tumors benet more than patients with right - sided tumors.4 however , the biology behind this association remains unclear . 
first , we further conrmed signicant association ( p , .001 ) between ta - ness classication and sidedness in kras wild - type primary tumors ( gse39582 ; fig 3d )  . 
then , we sought to discover whether the ta - ness classication could further rene the selection of patients in addition to ras / braf status and sidedness . within discovery and validation cohorts ( n = 205 ) , highsensitivity next - generation sequencing ras / braf mutational analysis was available for 118 patients : 71 were classied as ras / braf wild - type , of which 53 were assigned to ta - high ( 75% ) class . 
 ( a ) a waterfall plot showing a subgroup of patients within the discovery cohort ( n = 35 ) showing disease response ( treated with anti epidermal growth factor receptor [ egfr ] drug ) according to recist criteria and ta - ness classication . 
 ( b ) a waterfall plot showing change in tumor ( percent ) volume in anti - egfrtreated mouse - propagated patient tumor samples ( n = 30 ) compared with matched control treated ( baseline ; n = 30 ) mouse - propagated patient tumors . 
the bars in the graph show ta - ness classication for the matched patient metastatic liver samples ( n = 30 ) , and the bars below the graph show the same classication for matched mousepropagated patient tumors ( treated v control )  . 
this classication has the advantage of providing a qualitative assessment in all the samples , including the non - ta subtypes , overcoming the limitations posed by intratumoral heterogeneity when using the conventional molecular subtyping classication as a potential tool to assess benet from anti - egfr therapy . 
kaplan - meier survival curves of patients with transit - amplifying ( ta ) - high versus ta - low tumors treated with antiepidermal growth factor receptor ( egfr ) therapy . 
 fontana et al ( and potentially clinical benet ) in patients treated with anti - egfrbased therapy in our discovery cohort ; this was validated in a kras exon 2 wild - type trial cohort of cetuximab - onlytreated patients , 13 which has the advantage of properly assessing prognostic value in a homogeneously treated population and in the absence of confounding effect of chemotherapy . 
moreover , ta - low assignment was enriched for ras / braf - mutant tumors , providing a potential alternative method to estimate prognosis and may be a treatment benet from anti - egfr therapy when the mutational status is missing . 
this signature and its association with anti - egfr treatment outcomes were also conrmed in the publicly available samples from patients with mcrc17 and the preclinical pdx models treated with cetuximab.18 , 19 finally , the taness classication retained prognostic signicance when assessed in either archival primary tumors or metastatic samples in multiple cohorts . 
this is highly clinically relethe classication can be vant , because it means that assessed in metastatic lesions when the primary tumor sample is not available or of poor quality ; however , intrapatient concordance was not assessed ; therefore , additional validation is required . several studies have now evaluated the association between single genes or micrornas ( ereg / areg , her2 , her3 , epha2 , or mir - 31 - 3p ) and responses to anti - egfr therapy.20 in contrast , we evaluated a rened form of our previously published gene expression signature ( with multiple genes ) to identify biologically different crc subtypes with distinct cellular phenotypes.5 , 16 the subtypes summarize a complex network of pathways potentially associated with therapeutic responses , simplifying multiple levels of information derived from heterogeneous samples . hence , the deployment of subtypes and their signatures , instead of single genes , has the advantage of reducing the dimension of complexity without losing biologic information . 
although the crca and consensus molecular subtype ( cms ) classications are highly concordant , 16 , 21 cms classication was not assessed here because it is technically challenging to dichotomize samples into two groups based on the current cms classier ( with multiple centroids )  . this study has some limitations . 
the identication of such patients in the context of clinical trials is challenging ; in fact , the negative predictive value of ras / braf mutations was retrospectively demonstrated in multiple clinical trials , and to our knowledge , none of them was designed with an up - front prospective inclusion of extended ras / braf wild - type tumors . 
last , this was a proof - of - concept study and was retrospectively designed on preexisting tissue collections in the absence of a control group , limiting the assessment of a ta - ness biomarker as prognostic rather than predictive . 
shapiro , chiara cremolini , filippo pietrantonio , livio trusolino , anguraj sadanandam data analysis and interpretation : elisa fontana , gift nyamundanda , david cunningham , dongsheng tu , maggie c . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . elisa fontana consulting or advisory role : astellas pharma ( i ) , celgene ( i ) , servier ( i ) , bristol myers squibb ( i ) patents , royalties , other intellectual property : patent no : 1716712.3 pending ( i ) travel , accommodations , expenses : bristol myers squibb ( i ) , servier ( i ) gift nyamundanda patents , royalties , other intellectual property : prognostic and treatment response prediction in gastric cancer priority patent csc / bp7295892 , patient classication and prognostic method patent application number pct / ep2019 / 053845 , patent application number 2011213.2 david cunningham stock and other ownership interests : ovibio consulting or advisory role : ovibio research funding : astrazeneca ( inst ) , amgen ( inst ) , sano ( inst ) , merrimack ( inst ) , celgene ( inst ) , medimmune ( inst ) , bayer ( inst ) , 4sc ( inst ) , clovis oncology ( inst ) , eli lilly ( inst ) , janssen ( inst ) , merck ( inst ) maggie c . 
siu leadership : treadwell therapeutics ( i ) stock and other ownership interests : agios ( i ) consulting or advisory role : merck , astrazeneca / medimmune , morphosys , roche , loxo , voronoi , oncorus , symphogen , mirati therapeutics , gsk , seattle genetics , treadwell therapeutics , arvinas , navire research funding : bristol myers squibb ( inst ) , genentech ( inst ) , glaxosmithkline ( inst ) , merck ( inst ) , novartis ( inst ) , pzer ( inst ) , medimmune ( inst ) , astrazeneca ( inst ) , boehringer ingelheim ( inst ) , bayer ( inst ) , amgen ( inst ) , astellas pharma ( inst ) , shattuck labs ( inst ) , symphogen ( inst ) , avid ( inst ) , mirati therapeutics ( inst ) , intensity therapeutics ( inst ) , karyopharm therapeutics ( inst ) francesco sclafani consulting or advisory role : bayer research funding : bayer ( inst ) , astrazeneca ( inst ) , roche ( inst ) , bristol myers squibb ( inst ) travel , accommodations , expenses : bayer , eli lilly jonathan m . 
waring employment : astrazeneca leadership : pillar biosciences , xing technologies , ori healthcare stock and other ownership interests : genentech , roche ( i ) , pillar biosciences , xing technologies consulting or advisory role : pillar biosciences , xing technologies , ori healthcare travel , accommodations , expenses : pillar biosciences , xing technologies timothy j . 
 fontana et al travel , accommodations , expenses : roche , servier naureen starling honoraria : eli lilly , msd oncology , merck serono , pierre fabre , servier consulting or advisory role : servier , astra zeneca , pzer research funding : astra zeneca ( inst ) , pzer / emd serono ( inst ) , bms ( inst ) travel , accommodations , expenses : msd oncology filippo pietrantonio consulting or advisory role : amgen , merck serono , bayer , eli lilly , sano , roche , servier research funding : bristol myers squibb livio trusolino honoraria : eli lilly , astrazeneca , merck research funding : symphogen ( inst ) , merus ( inst ) , pzer ( inst ) , servier ( inst ) , menarini ( inst ) anguraj sadanandam consulting or advisory role : ploughshare innovations research funding : merck , pierre fabre , bristol myers squibb patents , royalties , other intellectual property : patent colorectal cancer classication with differential prognosis and personalized therapeutic responses ( patent number pct / ib2013 / 060416 ) , prognostic and treatment response prediction in gastric cancer priority patent csc / bp7295892 , patent patient classication and prognostic method ( gep - net ) priority patent ep18425009.0 , patent molecular predictors of therapeutic response to specic anti - cancer agents ( patent number us9506926b2 ) no other potential conicts of interest were reported . acknowledgment we thank all patients and families participating in the studies included in this article . 
tebbutt , hagen kennecke , gabriella fontanini , eugenia zanella , and andrea bertotti for their contributions in the studies included in this article . references van cutsem e , cervantes a , adam r , et al : esmo consensus guidelines for the management of patients with metastatic colorectal cancer . 
ann oncol 27 : 1386 - 1422 , 2016 de roock w , claes b , bernasconi d , et al : effects of kras , braf , nras , and pik3ca mutations on the efcacy of cetuximab plus chemotherapy in chemotherapy - refractory metastatic colorectal cancer : a retrospective consortium analysis . 
lancet oncol 11 : 753 - 762 , 2010 cremolini c , morano f , moretto r , et al : negative hyper - selection of metastatic colorectal cancer patients for anti - egfr monoclonal antibodies : the pressing case - control study . 
ann oncol 28 : 3009 - 3014 , 2017 tejpar s , stintzing s , ciardiello f , et al : prognostic and predictive relevance of primary tumor location in patients with ras wild - type metastatic colorectal cancer : retrospective analyses of the crystal and fire - 3 trials . 
jama oncol 3 : 194 - 201 , 2017 [ erratum : jama oncol 3 : 1742 , 2017 ] sadanandam a , lyssiotis ca , homicsko k , et al : a colorectal cancer classication system that associates cellular phenotype and responses to therapy . 
woolston a , khan k , spain g , et al : genomic and transcriptomic determinants of therapy resistance and immune landscape evolution during anti - egfr treatment in colorectal cancer . 
stem cells int 2017 : 7602951 , 2017 yang yp , ma h , starchenko a , et al : a chimeric egfr protein reporter mouse reveals egfr localization and trafcking in vivo . 
national institute for health and care excellence : cetuximab for the rst - line treatment of metastatic colorectal cancer : technical appraisal guidance [ ta176 ]  . 30 : 520 - 527 , 2019 12 . 
national institute for health and care excellence : panitumumab in combination with chemotherapy for the treatment of metastatic colorectal cancer ( terminated appraisal ) : technical appraisal guidance [ ta240 ]  . 
marisa l , de reyni `es a , duval a , et al : gene expression classication of colon cancer into molecular subtypes : characterization , validation , and prognostic value . 
plos med 10 : e1001453 , 2013 isella c , brundu f , bellomo se , et al : selective analysis of cancer - cell intrinsic transcriptional traits denes novel clinically relevant subtypes of colorectal cancer . 
khambata - ford s , garrett cr , meropol nj , et al : expression of epiregulin and amphiregulin and k - ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab . 
bertotti a , migliardi g , galimi f , et al : a molecularly annotated platform of patient - derived xenografts ( xenopatients ) identies her2 as an effective therapeutic target in cetuximab - resistant colorectal cancer . 
zanella er , galimi f , sassi f , et al : igf2 is an actionable target that identies a distinct subpopulation of colorectal cancer patients with marginal response to 20 . 
receiver operating characteristic ( roc ) curve to determine the best cut - off to dene disease control rate in the discovery cohort . 1 represents samples classied into transit - amplifying ( ta ) tumor with highest ( rank ) correlation with crcassigner ( crca ) - 38 centroids . 
 ( a ) kaplan - meier survival curve of patients with transit - amplifying ( ta ) tumor versus non - ta tumor in the discovery cohort subtyped using conventional subtyping approach . 
 molecular and clinical characterization of a claudin - low subtype of gastric cancer purpose claudin - low molecular subtypes have been identified in breast and bladder cancers and are characterized by low expression of claudins , enrichment for epithelial - to - mesenchymal transition ( emt ) , and tumor - initiating cell ( tic ) features . 
we evaluated whether the claudin - low subtype also exists in gastric cancer . materials and methods four hundred fifteen tumors from the cancer genome atlas ( tcga ) gastric cancer mrna data set were clustered on the claudin , emt , and tic gene sets to identify claudin - low tumors . 
compared with gs subtype , claudin - low subtype had significant activation in rho family of gtpases signaling , which appears to play a key role in its emt and tic properties . 
in the acrg data set , 28 of 300 samples were classified as claudinlow tumors by the 24 - gene predictor and were phenotypically similar to the initially derived claudinlow tumors . 
2017 by american society of clinical oncology introduction claudin - low tumors have been identified as a molecular subtype originally in breast cancer and more recently in bladder cancer on the basis of gene expression profiling.1 , 2 these distinct tumors are characterized by the low expression of tight - junction claudins , enrichment for epithelial - to - mesenchymal transition ( emt ) markers , and tumor - initiating cell ( tic ) features.1 , 2 clinically , claudin - low tumors , which lack luminal differentiation marker expression , are associated with poor prognosis compared with luminal tumors.1 , 3 similar to breast , bladder , and other types of cancer , gastric cancer is a heterogeneous disease and arises from multiple genetic and epigenetic aberrations . 
to better understand the biology that drives gastric cancer , molecular evaluation of gastric cancer has been performed and has led to the identification of key dysregulated pathways and the development of molecular classifications.4 gene expressionbased classifiers were reported by the asian cancer research group ( acrg ) 5 and by lei et al.6 the acrg divided gastric cancer tumors into four subtypes ( microsatellite unstable [ msi ] , emt , and tumor protein 53 active and inactive ) , and lei et al classified gastric cancer tumors into three subtypes ( proliferative , metabolic , and mesenchymal )  . on the basis of an integrative analysis of molecular profiling data , the cancer genome atlas ( tcga ) proposed the following four molecular subtypes of gastric cancer tumors : epstein - barr virus ( ebv ) positive , msi , chromosomally unstable ( cin ) , and genomically stable ( gs ) .7 this classification system first categorizes tumors by ascopubs.org / journal / po jco precision oncology tomohiro f . 
vincent author affiliations appear at the end of this article . supported by the university cancer research fund of the lineberger comprehensive cancer center at the university of north carolina at chapel hill and the university of north carolina at chapel hill oncology clinical translational research training program ( 5k12ca120780 )  . presented at the asco 2017 gastrointestinal cancers symposium , san francisco , ca , january 1921 , 2017 . corresponding author : benjamin g . 
however , gs subtype was found to have some notable features , including enrichment of lauren diffuse histologic type and alterations in the genes associated with cell adhesion and motility , such as cdh1 and rhoa mutations and cldn18 - arhgap fusions . 
missing values were imputed using the k - nearest neighbor imputation method , and gene expression values were median centered across each gene . results gene expression signatures tight - junction claudin , emt , and tic gene signatures were used in the classification of a claudin - low subtype . 
the set of claudins used was identified by herschkowitz et al.9 the bidirectional pan - cancer emt signature was derived by tan et al.10 the gastric cancerspecific tic signature was derived from the publicly available gene expression omnibus gene expression data set ( gse53276 )  . 
proliferation and immune gene signatures were derived by hippo et al11 and iglesia et al , 12 respectively . identification of a claudin - low subtype data were clustered on the claudin , emt , and tic gene sets using average linkage clustering with a centered correlation similarity metric on the cluster 3.0 platform ( stanford university , stanford , ca )  . 
sigclust2 r software ( statistical significance of clustering 2 ) was run on the node to perform a gaussian distribution analysis , which expands to the entire gene set for each increasing node . 
the data supplement contains additional details on methods . identification of a claudin - low subtype in gastric cancer from adjacent clusters of we performed unsupervised hierarchical clustering on 415 gastric cancer samples from tcga data set using gene signatures representative of biologic characteristics known to define the claudin - low subtype of breast and bladder cancers.1 , 2 this unsupervised hierarchical clustering with these gene signatures revealed a distinct cluster that had characteristics of claudin - low tumors ( fig 1a , highlighted in red )  . 
to ensure that the set of tumors within the presumed claudin - low cluster were homogeneous and distinct tumors , we performed a gaussian distribution analysis ( data supplement )  . 
this method identified a conserved node of 46 tumors ( 11.1% ) that had consensus enrichment for claudin - low features , and these tumors , therefore , were defined as claudin - low subtype . 
classification of the nonclaudin - low tumors ( n = 369 ) was as follows : ebv , 31 tumors ; msi , 79 tumors ; cin , 218 tumors ; and gs , 41 tumors . 
because the claudin - low tumors were identified originally in breast cancer , we applied the previously defined breast cancerspecific claudin - low classifier to tcga gastric cancer samples and found a significant enrichment ( p , .001 , fishers exact test ) of the breast cancerdefined claudin - low tumors within the gastric claudin - low cluster ( data supplement )  . 
this finding further supports the notion that claudin - low tumors exhibit features of previously defined claudin - low tumors . gene expression signature analysis relative to tic and stem - cell features , breast cancer claudin - low tumors express low levels of proliferation genes and are likely slowercycling tumors.1 , 3 we examined proliferation gene expression of gastric cancer by subtype using a previously reported gastric cancerspecific proliferation signature.11 expression of the proliferation - associated genes in the claudinlow subtype was lowest among all the subtypes ( figs 2a and 2b )  . 
despite the apparent similarity to gs subtype , the claudin - low subtype expressed significantly lower levels of proliferation genes than gs subtype ( p , .001 , bonferronicorrected pairwise t test )  . 
 another notable feature of the claudin - low subtype of breast and bladder cancers is high expression of immune cell genes , including t and b cells.1 , 2 to characterize the immune response in gastric claudin - low tumors , we evaluated gene signatures associated with immune cells by molecular subtype using previously defined signatures that correspond to tumor - infiltrating immune cells.12 heat maps of immune signature expression across the 415 samples in order by subtype showed generally high expression in ebv , claudin - low , and gs ( fig 3a )  . 
on the basis of the z scores of the gene signature , expression of all immune signatures other than the b - cell signature was highest in the ebv subtype ( data supplement )  . to assess the level of active immunosuppression , we examined the expression of a previously defined panel of immune checkpoint molecules ( immunosuppression signature ) 2 and found that it was also highest in the ebv subtype ( fig 3b ; data supplement )  . 
this observation is in line with the previous report by strong et al.13 wherein ebvpositive gastric carcinoma samples expressed high levels of the cytotoxic t - cell and natural killer cell inhibitor indoleamine - 2 , 3 - dioxygenase 1 , which leads to the induction of immune tolerance to tumors despite the elevated immune cell infiltration . 
similar to the previous analysis of bladder cancer , a clear correlation existed between the immune signatures and the immunosuppression signature across all gastric cancer subtypes2 ( data supplement )  . the prognostic impact of tumor - immune infiltrates , especially tumor - infiltrating lymphocytes ( tils ) , has been reported in multiple tumor types.14 , 15 for instance , the presence of cd8 + tils was associated with favorable survival outcomes.16 - 18 some studies evaluated the prognostic relevance of tils in gastric cancer , but the results were not consistent across them.19 - 21 we performed cox proportional hazards regression modeling for each immune gene signature , including immunosuppression signature , across all tumors and within each subtype . 
none of the signatures were prognostic in tcga gastric cancer samples ( data not shown )  . we explored potential mechanisms of immune response by examining predicted neoantigen burden and levels of cytokines and chemokines among subtypes . 
 ( a ) heat map of supervised clustering of gastric cancer subtypes across previously identified proliferationassociated genes ( n = 415 )  . ( b ) box plot of proliferation gene signature z scores across gastric cancer subtypes ( n = 415 )  . significance was determined by one - way analysis of variance ( anova )  . 
 ( b ) box plot of immune suppression gene signature z scores across gastric cancer subtypes ( n = 415 )  . significance was determined by one - way analysis of variance ( anova )  . 
 ( c ) and ( d ) box plots show the number of predicted neoantigens with a half - maximal inhibitory concentration , 150 nm by tumor molecular subtype . significance was determined by kruskalwallis exact test ( n = 357 )  . ( e ) box plot of cytokine and chemokine signature z scores across gastric cancer subtypes ( n = 415 )  . significance was determined by one - way anova . 
cin , chromosomally unstable ; ebv , epstein - barr virus ; gs , genomically stable ; igg , immunoglobulin g ; mac th1 , macrophageassociated t helper 1 ; msi , microsatellite unstable . difference in immune infiltration among subtypes because the predicted neoantigen burdens of ebv , claudin - low , and gs subtypes were lower than that of cin subtype and relatively similar to one another ( fig 3d )  . we next examined the relative expression of a previously defined panel of cytokines and chemokines by subtype2 and observed a strong correlation between this expression signature and multiple immune signatures , including immunosuppression signature ( data supplement )  . 
overall , claudin - low tumors appeared to have a high level of immune infiltration but lacked active immunosuppression within the tumor microenvironment . pathway analysis given that claudin - low tumors were primarily found in gs subtype , we performed ingenuity pathway analysis ( ipa ) and gene set enrichment analysis to understand the gene expression patterns that differentiate claudin - low tumors from nonclaudin - low gs tumors . 
ipa revealed that claudin - low subtype had significant activation in rho family of gtpases signaling and significant inactivation in rho guanine nucleotide dissociation inhibitor ( gdi ) signaling compared with gs subtype ( fig 4 )  . 
rhoa ( a member of the rho family of gtpases ) signaling has been reported to promote cancer stem - celllike phenotype in diffuse - type gastric cancer.22 in addition , claudinlow subtype had higher levels of actin cytoskeleton and integrin signaling relative to signaling levels in gs subtype . 
these observations are in keeping with the tic and emt phenotypes , which are defining characteristics of claudin - low tumors . mutation and copy number alteration analysis we next examined somatic mutations and copy number alterations using tcga data set to compare genomic events of claudin - low subtype with those of nonclaudin - low gs subtype ( data supplement )  . 
tcga network reported rhoa and cdh1 mutations , and cldn18 - arhgap fusions were enriched in gs subtype.7 we did not observe significant differences in the frequency of these genomic events between the two subtypes ( data supplement )  . 
nonclaudin - low tumors ( n = 272 ) were classified into the following subtypes : ebv , 17 tumors ; msi , 63 tumors ; cin , 162 tumors ; and gs , 30 tumors . 
we found that claudin - low subtype in the acrg data set was phenotypically similar to the initially derived claudin - low subtype in the discovery tcga data set as measured by expression of the claudin , emt , and tic gene signatures ( data supplement )  . 
bar graph of the most significantly activated ( gold ) or inactivated ( blue ) signaling pathways in claudin - low subtype ( n = 46 ) compared with gs subtype ( n = 41 )  . significance was determined using ipa software . 
to further compare prognosis for claudin - low subtype with gs subtype , with adjustment for potential confounders ( age and pathologic stage ) , we performed cox proportional hazards regression modeling by setting gs type as a reference group ( table 1 )  . 
in addition , we assessed a prognostic value of claudin - low subtype in patients with stage iii cancer because it was the largest subgroup with the most equal distribution of subtypes . 
similar hrs from the two independent data sets suggest that claudin - low subtype of gastric cancer has a poor prognosis as a result of its unique biologic features . tcga overall survival by subtype acrg overall survival by subtype claudin fig 5 . 
prognosis of claudin - low tumors in ( a ) the cancer genome atlas ( tcga ) and ( b ) the asian cancer research group ( acrg ) data sets . unadjusted kaplan - meier plots show overall survival of gastric cancer by molecular subtype . significance was determined by log - rank test ( n = 415 in tcga cohort ; n = 300 in the acrg cohort )  . 
thus , we especially focused on comparing molecular and clinical features of claudin - low tumors with those of nonclaudin - low gs tumors . claudin - low gastric cancer was defined by low expression levels of tight - junction claudins , high levels of emt , and enrichment for tic signatures . 
in addition , compared with gs subtype , claudin - low subtype had the lower expression of proliferation signature , significant activation in rho family of gtpases signaling , and enrichment of cell motility and adhesion - associated pathways . furthermore , the claudin - low subtype conferred significantly worse prognosis than the gs subtype , which had comparable survival to the cin and ebv subtypes . 
these observations support the claim that claudin - low subtype is distinct from gs subtype and warrants additional validation in prospective studies . we report a 24 - gene classifier that accurately classifies gastric cancer into claudin - low and non claudin - low subtypes . 
we applied this classifier to the acrg data set and identified 28 claudin - low tumors that were phenotypically similar to the initially derived claudin - low subtype in the discovery data set . 
adjusted for age and pathologic stage ( n = 415 in tcga cohort ; n = 300 in the acrg cohort )  . abbreviations : acrg , asian cancer research group ; cin , chromosomally unstable ; ebv , epsteinbarr virus ; gs , genomically stable ; msi , microsatellite unstable ; tcga , the cancer genome atlas . are also characterized by low expression of claudins and enrichment for tic signatures . 
the combined expression of emt and cancer stem - cell makers was reported to be associated with aggressive clinical features , such as vascular invasion and metastasis , and prognostic of poorer disease - free and overall survival in a study of 276 patients with gastric cancer.25 moreover , emt and / or stem - celllike biologic processes have been linked to resistance to chemotherapy and radiation.26 - 28 the mechanisms of therapeutic resistance include relative quiescence , expression of atp - binding cassette transporters , an active dna - repair capacity , and a resistance to apoptosis.29 recent work by yoon et al22 demonstrated a role of rhoa , a member of the rho family of gtpases , in gastric cancer stem - like cells ( cscs ) for the maintenance of emt phenotypes and chemotherapy resistance . 
furthermore , rhoa pathway inhibition reversed chemoresistance in diffuse gastric cscs resistant to fluorouracil and cisplatof note , our ipa revealed that , compared with gs subtype , the most significantly activated and inactivated pathway in claudin - low subtype were rho family of gtpases signaling and rhogdi signaling , respectively . 
gdis are regulators of rho gtpases and prevent activation of gtpases by acting as molecular chaperones.30 all things considered , dysregulated rho gtpase signaling appears to play a key role in the biology of gastric claudinlow tumors and contributes to the poor prognosis . 
sanoff research funding : bayer ag ( inst ) , novartis ( inst ) , merck ( inst ) , precision biologics ( inst ) , immunomedics ( inst ) references 1 . 
sabatier r , finetti p , guille a , et al : claudin - low breast cancers : clinical , pathological , molecular and prognostic jci insight 1 : e85902 , 2016 characterization . 
lei z , tan ib , das k , et al : identification of molecular subtypes of gastric cancer with different responses to pi3 - kinase inhibitors and 5 - fluorouracil . 
tan tz , miow qh , miki y , et al : epithelial - mesenchymal transition spectrum quantification and its efficacy in deciphering survival and drug responses of cancer patients . 
strong mj , xu g , coco j , et al : differences in gastric carcinoma microenvironment stratify according to ebv infection intensity : implications for possible immune adjuvant therapy . 
gooden mj , de bock gh , leffers n , et al : the prognostic influence of tumour - infiltrating lymphocytes in cancer : a systematic review with meta - analysis . 
shen z , zhou s , wang y , et al : higher intratumoral infiltrated foxp3 + treg numbers and foxp3 + / cd8 + ratio are associated with adverse prognosis in resectable gastric cancer . 
yoon c , cho sj , aksoy ba , et al : chemotherapy resistance in diffuse - type gastric adenocarcinoma is mediated by rhoa activation in cancer stem - like cells . 
ryu hs , park dj , kim hh , et al : combination of epithelial - mesenchymal transition and cancer stem cell - like phenotypes has independent prognostic value in gastric cancer . 
takaishi s , okumura t , tu s , et al : identification of gastric cancer stem cells using the cell surface marker cd44 . stem cells 27 : 1006 - 1020 , 2009 27 . 
martin , md1 introduction we read with interest two recently published articles in jco precision oncology that provide valuable insight regarding stk11 , an emerging biomarker in nonsmall - cell lung cancer ( nsclc ) .1 , 2 initially , shen et al1 detailed their ndings with the statistical learning tool oncocast . 
they identied stk11 as one of several prognostic genes afliated with poor survival from 1 , 054 patients with advanced lung adenocarcinoma proled with msk - impact next - generation sequencing ( ngs )  . 
by dividing the cohort into genomic risk - stratication groups , the authors highlighted the complexity of stk11 co - mutation patterns with other high - risk genes , including keap1 , kras , tp53 , and smarca4 . 
in the article by bange et al , 2 the authors retrospectively demonstrated heterogeneity of outcomes among patients with stk11 - mutated , advanced nsclc on the basis of co - mutation status . they reported that kras co - mutated cases had worse progression - free and overall survival outcomes after rst - line chemotherapy compared with tp53 . taken together , these ndings and others3 - 5 indicate that further work is warranted to characterize stk11 as an nsclc biomarker and to identify unique therapeutic approaches for patients with high - risk stk11mutated nsclc . 
a lumpectomy was performed in april 2014 , and adjuvant treatment included 12 doses of weekly paclitaxel , 1 year of trastuzumab , a partial breast radiation , and continuation on letrozole . 
a biopsy showed lung adenocarcinoma , and positron emission tomography ( pet ) / ct showed no additional disease . she received a robotic lobectomy , and pathologic staging revealed iiia disease . 
postoperative pet / ct in august showed a mildly hypermetabolic 1 - cm focus in the right middle lobe of the lung without other disease ; magnetic resonance imaging of the brain was negative . after radiologic review , it was initially believed that this focus represented postsurgical hypermetabolism , and close follow - up was recommended . 
she received adjuvant cisplatin and pemetrexed on day 1 of 21 - day cycles ; ct scans in october , after two of four cycles , showed no new disease . 
pet / ct revealed hypermetabolic disease limited to the 4 - cm adrenal mass . the adrenal and right middle - lobe lung lesions were biopsied , and both conrmed lung adenocarcinoma . she was referred for surgical evaluation but was not a candidate . 
testing included immunohistochemistry ( ihc ) , tumor mutational burden ( tmb ) , and 592 - gene ngs including egfr , braf , met , and her2 , as well as the previously mentioned high - risk genes stk11 , keap1 , kras , and tp53 ( nextseq ; illumina [ san diego , ca ] ) .6 ros1 and alk fusions were detected using the archerdx fusion assay ( archer fusionplex solid tumor kit )  . 
proling showed no targetable mutations , programmed death - ligand 1 ( pd - l1 ) ihc expression 0% ( 22c3 ; dako [ santa clara , ca ] ) , and tmb 15 mutations / megabase . 
no keap1 or tp53 mutations were identied . intravenous nivolumab 3 mg every 2 weeks and 1 mg / kg ipilimumab every 6 weeks were initiated in june 2018 . both treatments were withheld in july when the patient developed symptomatic autoimmune hyperthyroidism that necessitated treatment with methimazole and atenolol . 
nivolumab was resumed in august with symptom resolution , when ct scans showed a small increase in size of her adrenal metastasis with reduction of the right middle - lobe lesion . 
 nivolumab / ipilimumab in stk11 - mutated nonsmall - cell lung cancer by bange et al.2 although characterized by caris life sciences as a pathogenic mutation , we recognize that explicit pathogenicity of r39fs cannot be conrmed by commercial ngs alone . 
however , it should be noted that several studies have shown that frameshift variants ( including those of exon 1 ) generally express little stk11 protein by ihc , which serves as a reliable marker for loss of stk11.15 , 16 we recognize the complexity of dening high - risk stk11 molecular subsets from commercial ngs . 
martin consulting or advisory role : seattle genetics no other potential conicts of interest were reported . references shen r , martin a , ni a , et al : harnessing clinical sequencing data for survival stratication of patients with metastatic lung adenocarcinomas . 
cancer discov 8 : 822 - 835 , 2018 arbour kc , jordan e , kim hr , et al : effects of co - occurring genomic alterations on outcomes in patients with kras - mutant non - small cell lung cancer . 
clin cancer res 24 : 334 - 340 , 2018 skoulidis f , arbour kc , hellmann md , et al : association of stk11 / lkb1 genomic alterations with lack of benet from the addition of pembrolizumab to platinum doublet chemotherapy in non - squamous non - small cell lung cancer . 
vanderwalde a , spetzler d , xiao n , et al : microsatellite instability status determined by next - generation sequencing and compared with pd - l1 and tumor mutational burden in 11 , 348 patients . 
cancer med 7 : 746 - 756 , 2018 rizvi h , sanchez - vega f , la k , et al : molecular determinants of response to anti - programmed cell death ( pd ) - 1 and anti - programmed death - ligand 1 ( pd - l1 ) blockade in patients with non - small - cell lung cancer proled with targeted next - generation sequencing . 
nat rev drug discov 18 : 197 - 218 , 2019 ready n , hellmann md , awad mm , et al : first - line nivolumab plus ipilimumab in advanced non - small - cell lung cancer ( checkmate 568 ) : outcomes by programmed death ligand 1 and tumor mutational burden as biomarkers . 
frank r , schefer m , merkelbach - bruse s , et al : clinical and pathological characteristics of keap1and nfe2l2 - mutated non - small cell lung carcinoma 13 . 
p ecuchet n , laurent - puig p , mansuet - lupo a , et al : different prognostic impact of stk11 mutations in non - squamous non - small - cell lung cancer . 
 o orthogonal comparison of four plasma ngs tests with tumor suggests technical factors are a major source of assay discordance daniel stetson , ms1 ; ambar ahmed , ms1 ; xing xu , phd2 ; barrett r.b. 
2019 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction increased reliance on liquid biopsy specimens for oncologic clinical decisions emphasizes the critical need for sensitive , specic multigene tests to detect genetic aberrations . 
plasma - based genotyping tests are approved for clinical use by the us food and drug administration but are limited to recurrent hot spot mutations or specic genes such as egfr and kras.1 next - generation sequencing ( ngs ) gene panel assays , in contrast , analyze thousands to millions of contiguous base pairstens to hundreds of genes for somatic alterations at low variant allele frequency ( vaf )  . 
the food and drug administration has approved the oncomine ( thermo fisher scientic , waltham , ma ) , 2 memorial sloan ketteringintegrated mutation proling of actionable cancer targets , 3 and foundationone ( foundation medicine [ fmi ] , cambridge , ma ) 4 gene panels . 
the need for noninvasive testing of a patients current mutational status , 5 coupled with the increased throughput6 and sensitivity7 of ngs , has led to the commercialization of circulating tumor dna ( ctdna ) ngs services . studies have examined the correlation between tumor and plasma mutational proling . 
in the context of egfr testing , thress et al8 used orthogonal genotyping methods to show that acquired resistance variants detected in plasma , but not in tumor , were reproduced by different plasma genotyping plattumor heterogeneity alone forms , suggesting that may explain tissue - plasma discordance for these hotspot assays . 
for broader gene - panel mutation detection in plasma by ngs , kuderer et al9 compared actionable variants found with plasma testing versus tumor testing and found only 22% variant concordance ( 10 of 45 variants )  . 
 stetson et al to address questions regarding sources of ctdna assay discordance , baseline plasma samples from 24 patients were sent to four ctdna sequencing vendors , and reported variants were compared with independently tested , timematched , tumor - normal tissue pairs . 
we supplied challenging samples with limited ctdna from early - stage cancers , which tested the limits of sensitivity and positive predictive value ( ppv ) but may have resulted in higher than expected failure and discrepancy rates . 
the aim of this study was to improve performance of ctdna ngs assays and inform physicians of clinical use . their potential limitations methods samples twenty - four matched , tumor - normal pairs with matched plasma from lung , breast , ovary , and prostate cancers ( table 1 ) were obtained from three commercial biobanking companies . 
plasma was collected 1 to 4 hours before patient surgery or drug treatment via double - spun edta tubes . tumor ngs assays matched tumor - normal tissue samples were sent to fmi as formalin - xed parafn - embedded blocks or genomic dna puried from frozen tissues ( 400 ng )  . 
variant calls and raw data for the matched tumor - normal pairs were returned to astrazeneca and , after matched normal single - nucleotide polymorphisms ( snps ) from tumor calls were subtracted , were used as a presumed somatic truth set for comparison with plasma results . plasma ngs assays plasma samples for each patient ( 8 ml ) were thawed , mixed , pooled , and dispensed into four 2 - ml aliquots . 
general results were discussed with each vendor to better understand laboratory methodologies and reporting criteria . sample identity verication to conrm that all returned sample data traced back to the correct patient , sample identity was veried by germline snp clustering analysis from tumor , normal , and plasma samples . 
a comprehensive set of snps was dened by intersecting all vendor bed les , then the corresponding snps were used to generate phylogenetic clusters between tissue and plasma for each vendor . 
these analyses , using the open source clearup algorithm13 ( astrazeneca - ngs / clearup ) , conrmed that the plasma samples were properly handled and matched by all vendors , except for eight samples incorrectly identied by vendor a . 
the identity matching and the pattern of the four discordant samples ( ie , samples were transposed ) suggest that vendor a mislabeled the samples , possibly during the preanalytical laboratory process , because the mismatch was found in both the gender quality - control check and subsequent sequencing . two blocks of the rst comparative tumor and plasma variant analyses the four variant reports were used for all orthogonal comparisons among assays without additional modications . 
non - shedders were patients with no evidence of tumor - plasma or plasma - plasma concordant variants . was not reported by vendor c despite its presence in that vendors raw data , possibly because of a bioinformatic ltering of suspected germline variants . 
tp53 v143m ( fig 2c ) was missed owing to elevated background noise in the vendor a assay , but it was clearly present and reported by vendor c . results variant - level concordance all reported plasma variants were plotted according to mutant vaf ( fig 1 ) and vendor ( data supplement )  . 
higher - frequency plasma variants ( approximately 50% vaf ) were veried as germline by comparison with the tumor - normal sequencing data . germline snps were reported as variants by all except vendor c , but these snps were excluded from any concordance calculations . 
overall , vendor c had the highest sensitivity ( 89% ) and vendor b had the highest ppv ( 80% )  . vendor d had a ppv of 36% owing to the high level of fps ( table 2 )  . 
above a vaf of 1% , vendors a , b , and c had a ppv of 100% ; below 1% vaf , vendor a had the lowest values for sensitivity ( 10% ) and ppv ( 17% )  . analysis of fps fps were identied by examining the raw alignment data from the replicate plasmas relative to tumor and normal samples and resulted from nonspecic variant calling and sequencing noise at nonreference bases . 
examples of nonspecic variant calls include fp brca2 l1103f ( fig 3a ) and erbb2 splice acceptor ( fig 3b ) calls not present in vendor cs data . fp signatures the 40 fp calls identied in this study ( table 2 ) were enriched for vendor - specic mutational biases ( data supplement )  . 
most fp calls were novel variants not found in somatic variant databases ( data supplement ) ; 100% of fp variants reported by vendor a , 100% by vendor c , and 95% by vendor d were novel . analysis of fns patient - level concordance to understand the nature of fn variant calls , raw aligned data were examined for tumor , normal , and plasma samples . 
the pik3ca h1047r variant was called by vendor c but missed by vendor b ( fig 2a ) ; it was present in vendor bs raw data but not reported . 
the variant demonstrated high signal - to - noise ratio and similar numbers of reads ( ve for vendor c and four for vendor b ) but was apparently below vendor bs bioinformatic calling threshold . 
concordant true - positive variants ( light blue ) and discordant variants , both false positive ( red ) and false negative ( teal ) , are plotted by the log of the allele fraction ( af )  . 
af is approximated for false - negative calls by averaging the corresponding true - positive calls for that variant . 4 2019 by american society of clinical oncology the patient level we examined variant concordance at ( fig 4 )  . 
five of the 24 patients were tissue - plasma concordant , three were plasma - plasma concordant , two were partially concordant , and 14 had non - shedding tumors ( fig 4 ; table 1 )  . 
the lack of detectable concordant variants in non - shedding plasma samples was consistent with derivation from early - stage cancers ( stage i , n = 8 ; stage ii , n = 13 ; stage iii , n = 2 ; and stage iv , n = 1 ; table 1 )  . detailed analysis of two patients samples the replicate design of this study offered the opportunity to gain more insight into the variant discordance factors by comparing the results generated by four vendors . 
vendor c found all three variants ; vendor b found both the pik3ca and tp53 variants , and the tert variant was not covered by this vendors panel ; vendors a and d missed the pik3ca and tert variants because of elevated background noise ; and vendor d found the tp53 v173l variant also reported by vendor a but assigned it to the wrong patient . 
some ctdna assays claim sensitivity to 0.1% vaf ; however , it is apparent that increasing proportions of fp and fn were apparent in our study and other studies9 , 16 at vaf of less than 1% . 
inspection of the raw sequencing data revealed elevated background noise to be an issue for vendors a and d , whereas vendors b and c had little to no background noise . 
vendors were individually informed about discordant results and , in several cases , they adjusted their variant pipelines , which modestly improved reports ( data not shown )  . cluster analyses of germline snps conrmed that returned ngs datasets corresponded with the correct patients of origin except for eight discrepant samples processed by vendor a . 
the corrected results increased vendor as ppv from 55% to 73% and sensitivity from 38% to 44% ; however , we presented only the returned reports because no other laboratory mixed up samples . 
mixing up patient results during the testing process is a serious issue to be addressed by improved quality control and other best practices . of the 24 patients , 14 ( 58% ) were classied as having nonshedding tumors , because they lacked detected concordant variants matching tumor or a replicate plasma . 
this proportion is higher than the published 20% to 30% rate of non - shedding samples , 16 , 17 likely because 88% ( 21 of 24 ) of patients in our study had stage i and ii tumors ( table 1 )  . stage i and ii tumors generally shed less dna into the bloodstream than more advanced and metastatic tumors16 , 18 ; however , there is variability in sample vafs ( fig 1 )  . 
furthermore , 50% ( 22 of 44 ) of all tp somatic variants had a vaf of less than 1% , underscoring the importance of analytically validated assays with sensitivity below 1% vaf . the set of plasma samples analyzed allowed ctdna ngs methods to be evaluated under challenging conditions such as limited ctdna levels ( sensitivity ) and non - shedding tumors ( ppv ) ; however , the approach may have resulted in a higher - than - expected discrepancy rate . 
according to our results and those of others , 16 application of ctdna assays to early detection analyses may be confounded by fn and fp rates . most discordance was due to assay technical variation , but biologic factors such as clonal hematopoiesis of determinate potential ( chip ) 19 and tumor heterogeneity were responsible for some discordance . 
all vendors were concordant for the tp53 and cdkn2a variants ( fig 4 ) , but vendor c had an fn call ( fig 2b ) for brca2 and vendor a had an fn call for pdgfra . 
this likely represents tumor heterogeneity , because the esr1 variant was not detected in the tumor and , if so , would have been present at approximately 0.3% vaf , well below the detection limit of the tissue assay . discussion many studies have compared ctdna results and tumor analysis , nding substantial discordance that affects which assay to use for patient management . 
by our novel replicate plasma design , we compared sequencing data from four commercial plasma tests with one another and with tumor ndings and determined that technical factors are a major source of variation . 
furthermore , we propose insights for identifying fp variants . to our knowledge , this is the rst study to use replicate tumor - matched plasmas and multiple orthogonal assays to examine the factors affecting tumor - plasma concordance . it is important to note that this design enabled the identication of variants missed when comparing one plasma assay with a tumor9 or another plasma15 assay . 
the replicate plasmas were essential for identifying fn calls and increased the condence of tp calls , particularly for variants found in plasma but not tumor . sensitivity and ppv varied among vendors and was lower for all below 1% vaf . 
although our study demonstrated that the majority of tumor - plasma discordance is a result of technical factors , with continuous improvement over time , ngs should approach the state of genotyping technology in which the majority of discordance is attributable to biologic factors such as tumor heterogeneity and chip.19 all vendor assays performed well above 10% vaf ( none or one discordant results per vendor ) , but below 1% vaf , most variant calls were discordant . 
we determined that lowvaf calls ( fig 1 ) , mutational biases ( data supplement ) , and novel somatic variants ( data supplement ) were the main characteristics of discordant calls . 
in our study , half of tps , and most fn and fp calls , were found below 1% vaf , demonstrating the need for improved assay performance below this threshold . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . daniel stetson employment : astrazeneca stock and other ownership interests : astrazeneca ambar ahmed employment : astrazeneca stock and other ownership interests : astrazeneca xing xu employment : solvebio stock and other ownership interests : solvebio stock options ( inst ) ; gilead sciences , merck ( i ) , celgene ( i ) , bausch health ( i ) barrett r.b. 
dougherty employment : astrazeneca stock and other ownership interests : astrazeneca ( inst ) no other potential conicts of interest were reported . acknowledgment we thank david gross , david caplan , and jeff hull from solvebio for informatics assistance and technical discussions , as well as hilary north scheler from axon communications for providing medical writing assistance funded by astrazeneca . references id = p160045 2017 2016 roche molecular systems : cobas egfr mutation test v2 ( 2015 )  . 
nat rev cancer 17 : 223 - 238 , leary rj , kinde i , diehl f , et al : development of personalized tumor biomarkers using massively parallel sequencing . 
nat med 20 : 548 - 554 , 2014 thress ks , brant r , carr th , et al : egfr mutation detection in ctdna from nsclc patient plasma : a cross - platform comparison of leading technologies to support the clinical development of azd9291 . 
lung cancer 90 : 509 - 515 , 2015 kuderer nm , burton ka , blau s , et al : comparison of 2 commercially available next - generation sequencing platforms in oncology . 
jama oncol 3 : 996 - 998 , 2017 jovelet c , ileana e , le deley mc , et al : circulating cell - free tumor dna analysis of 50 genes by next - generation sequencing in the prospective moscato trial . clin cancer res 22 : 2960 - 2968 , 2016 11 . 
 exceptional response to cabozantinib in a patient with multiply relapsed wilms tumor introduction wilms tumors ( wts ) account for a majority of pediatric renal tumors and have a high cure rate with surgery , radiation , and chemotherapy.1 high - risk features in wts include anaplastic histology , blastemal predominance , bilateral presentation , and loss of heterozygosity of 1p and 16q , and patients who have these features typically have worse outcomes and are treated with more aggressive therapies . 
despite the risk - stratified approach to therapy , outcomes remain poor for the subgroup of patients with wts who relapse.2 better understanding of the biology of wt could help identify risk factors associated with disease relapse . 
 in november 2005 , 3.5 years after completion of therapy , the patient experienced a widespread relapse that was detected on routine surveillance and was treated with cyclophosphamide , etoposide , carboplatin , vincristine , and radiation . two years after therapy for the relapse , a second recurrence was detected on imaging ; he underwent surgical resection and received treatment consisting of ifosfamide / etoposide , cyclophosphamide / topotecan , and autologous stem cell transplantation . 
the patients stem cell transplantation was complicated by severe bk viral hemorrhagic cystitis infection , which necessitated a prolonged stay in the intensive care unit . in october 2010 , 2 years after transplantation , the patient began experiencing back pain and was found to have a third relapse , which was treated with vincristine , adriamycin , and irinotecan . 
approximately 1 year after this therapy was completed , a fourth relapse was treated with eight cycles of sorafenib , which led to stable disease , after which disease progression was detected and the patient began radiation therapy . 
in july 2013 , the patient was then treated with cabozantinib , and he experienced a partial response lasting close to 2 years ( fig 1 )  . after his eventual progression on cabozantinib , the patient advanced through several other therapies , including additional radiation , lorvotuzumab mertansine , axitinib , ramucirumab , and pazopanib . 
mody author affiliations and support information ( if applicable ) appear at the end of this article . none of the sponsors played a role in the design of the study ; collection , management , analysis , and interpretation of the data ; preparation , review , or approval of the manuscript ; or decision to submit the manuscript for publication . 
 the patient experienced a 33% reduction in combined target lesion tumor size by response evaluation criteria in solid tumors ( recist )  . mycn mutations are monoallelic events with no evidence of loss of heterozygosity in the tumor . 
 in addition , transcriptome sequencing ( rna sequencing ) uncovered various overexpressed tyrosine kinases ( ret , met , and tek / tie2 ) when compared with the rest of our institutions compendium of patients with wt ( table 1 )  . 
a review of the family history showed that the mother was positive for melanoma ( age of onset , 37 years ) , and the paternal grandfather had basal cell cancer ( age of onset , 64 years )  . discussion prognosis for relapsed wt continues to be poor in spite of high - dose intensive therapy . 
genetic aberrations discovered through ics can help identify new prognostic markers , can help researchers better understand tumor biology , and can sometimes lead to targeted therapies.3 somatic mutations in max ( p.r60q ) and mycn ( p.p44l ) were identified in our multiply relapsed patient with unquestionably refractory disease . 
the myc proto - oncogene has been implicated in the pathogenesis of many human tumors and was found to be overexpressed and / or activated in more than half of human cancers.7 , 8 myc coordinates changes in the tumor microenvironment , including the activation of angiogenesis.9 in patients with wt , mycn is associated with the high - risk histology features of anaplasia and blastemal predominance as well as poorer relapse - free survival and overall survival . 
this suggests the potential for a poor prognostic implication of combined max and mycn mutations in this case , but further functional studies are required to validate this interaction . cabozantinib is an oral small molecule inhibitor of numerous tyrosine kinase receptors with activity toward vegfr2 ( kdr ) and met ( hepatocyte growth factor receptor )  . 
it also targets important mediators of tumor cell survival , metastasis , and tumor angiogenesis , including ret ( rearranged during transfection ) , kit ( mast / stem cell growth factor receptor ) , axl ( anexelekto ) , tie2 ( angiopoietin receptor ) , and flt3 ( fms - like tyrosine kinase ) .4 , 14 cabozantinib has been approved by the us food and drug administration to treat advanced renal cell carcinoma ( arcc ) and has shown efficacy against medullary thyroid cancer.15 sorafenib targets many of the same kinases as cabozantinib and also targets craf , braf , vegfr3 ( flt4 ) , and pdgfr - .16 because of the patients exceptional response to cabozantinib , prolonged stable disease while receiving sorafenib , and seemingly no genetic mutations or amplifications that could be targeted , we looked at the expression of various kinases that are inhibited by both agents using rna sequencing ( table 1 )  . 
it is known that ret plays a role in the development of the kidney , and expression of high levels of ret in wt has been reported ; therefore , this overexpression may play a role in the development of the disease.17 in addition , the patients tumor was likely highly vascularized as a result of his somatic mycn mutation.9 we hypothesize that inhibition of the vegf signaling pathway enacted by both sorafenib and cabozantinib helps devascularize the tumor , leading to sustained response . 
 previous research in arcc substantiates our experience , which shows cabozantinib to be superior to other multi - tkis such as sorafenib and axitinib , leading to longer progression - free survival.18 further evidence in arcc suggests that increased expression of met and axl are associated with a poor prognosis and that their inhibition may help overcome resistance to vegf pathway inhibition.19 , 20 the rare hoxb13 germ line variant uncovered in this patient has been inconsistently reported to increase cancer risk . 
 with tumor angiogenesis and disease progression.21 there are no reported cases of this germ line mutation in wt , but it seems to be associated with an increased risk of prostate cancer , particularly in whites.22 , 23 thus , this finding led to genetic counseling for the patient and his family . to our knowledge , this is the first report of a patient with wt who had aberrations in both max and mycn genes ; in fact , if the myc / max interaction plays a significant prognostic role in wt , agents are being developed that could potentially target this interaction.24 generally , overexpression is not considered a targetable finding in precision oncology , but if there are no other tangible aberrations , targeting overexpression may be reasonable . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . rama jasty - rao no relationship to disclose yi - mi wu no relationship to disclose trisha paul no relationship to disclose dan robinson no relationship to disclose rajen j . 
grundy p , breslow n , green dm , et al : prognostic factors for children with recurrent wilms tumor : results from the second and third national wilms tumor study . 
yakes fm , chen j , tan j , et al : cabozantinib ( xl184 ) , a novel met and vegfr2 inhibitor , simultaneously suppresses metastasis , angiogenesis , and tumor growth . 
wilhelm sm , carter c , tang l , et al : bay 43 - 9006 exhibits broad spectrum oral antitumor activity and targets the raf / mek / erk pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis . 
wegert j , ishaque n , vardapour r , et al : mutations in the six1 / 2 pathway and the drosha / dgcr8 mirna microprocessor complex underlie high - risk blastemal type wilms tumors . 
dela cruz fs , diolaiti d , turk at , et al : a case study of an integrative genomic and experimental therapeutic approach for rare tumors : identification of vulnerabilities in a pediatric poorly differentiated carcinoma . 
kim a , widemann bc , krailo m , et al : phase 2 trial of sorafenib in children and young adults with refractory solid tumors : a report from the childrens oncology group . 
amzal b , fu s , meng j , et al : cabozantinib versus everolimus , nivolumab , axitinib , sorafenib and best supportive care : a network meta - analysis of progression - free survival and overall survival in second line treatment of advanced renal cell carcinoma . 
choueiri tk , escudier b , powles t , et al : cabozantinib versus everolimus in advanced renal cell carcinoma ( meteor ) : final results from a randomised , open - label , phase 3 trial . 
zhu jy , sun qk , wang w , et al : high - level expression of hoxb13 is closely associated with tumor angiogenesis and poor prognosis of hepatocellular carcinoma . 
cai q , wang x , li x , et al : germline hoxb13 p.gly84glu mutation and cancer susceptibility : a pooled analysis of 25 epidemiological studies with 145 , 257 participants . 
 circulating tumor dna analysis in colorectal cancer : from dream to reality carlotta antoniotti , md1 , 2 ; filippo pietrantonio , md3 , 4 ; salvatore corallo , md3 ; filippo de braud , md3 , 4 ; alfredo falcone , md1 , 2 ; and chiara cremolini , md , phd1 , 2 liquid biopsy is a minimally invasive approach to obtain circulating materials that originate from tumor cells through the sampling of body uids , mainly peripheral blood . 
because of its abilities to detect tumor - derived nucleic acids and proteins and to characterize tumor - specic genomic abnormalities , liquid biopsy has emerged as an approach to orient care of patients with colorectal cancer ( crc )  . 
second , the reliability of studies that evaluate the concordance of ras testing between tissue and plasma samples is impaired by the adoption of assays with heterogeneous analytical sensitivity and coverage of genomic regions . 
there are now several commercially available methods for ctdna assessment and technology platforms based on digital polymerase chain reaction or next - generation sequencing approacheseach one with specic sensitivity , specicity , throughput , gene coverage , costs , and potential clinical applications.12 among them , three test kits are ce - marked in vitro diagnostic devices for detection of ras and braf mutations on ctdna in crc.13 - 16 third , some clinicopathologic variables are likely to affect the amount of tumor - released ctdna.5 , 6 , 8 , 10 , 11 whereas liver involvement and tumor burden are positively associated with the ras mutant allele fraction ( the proportion of mutant dna fragments at a given locus ) , peritoneal , nodal , and lung metastases and mucinous histology are linked to low ras inuence ctdna ctdna detection . 
 antoniotti et al ras wild - type cases , as assessed on ctdna , negligible . is not the parallel assessment of the mutant allele fractions of other key genomic tumor alterations may help solve challenging cases that have undetectable ras mutations and ctdna levels at or lower than the analytic sensitivity of adopted assays . 
to overcome these issues , preanalytic procedures should be standardized , a threshold of detectable mutation rate that confers intrinsic resistance to egfr inhibition should be set and prospectively validated , investigation to understand when plasma and additional and tissue tests are interchangeable and to improve assay sensitivities is warranted . meanwhile , the ras status assessment to address the use of anti - egfr agents must be performed as the gold standard on tumor specimens.3 , 4 , 17 , 18 only when tissuebased testing is technically or logistically unfeasible could it be replaced by ctdna analysis.4 ctdna to estimate prognosis and detect minimal residual disease retrospective studies have focused on the prognostic impact of the quantitative analysis of ctdna . 
in the metastatic setting , a correlation between ctdna concentration and survival has been described , but the independent weight of ctdna quantication when the relative impact of other well - known prognostic factors is taken into account has not been claried ( appendix table a2 )  . the role of the quantitative assessment of ctdna in monitoring response during treatment must be dened in the light of its potential added value when compared with easily available and well - established markers , including carcinoembryonic antigen or early radiologic disease reassessment . 
recently , ctdna has been proposed to detect , measure , and monitor residual disease after radical interventions . a series of proof - of - concept studies reported that liquid biopsy could disclose the persistence of minimal residual disease ( mrd ) through the detection of ctdna in patients with crc who underwent potentially curative surgery ( resection of primary tumor in early - stage crc or radical resection of metastases ) even in the absence of clinical or radiologic signs of residual disease ( appendix table a2 )  . by identifying incomplete eradication of disease after a curative treatment , detectable ctdna predicts an increased risk of relapse regardless of the exposure to an adjuvant treatment . 
the development of such a sensitive tool might improve the risk estimation of disease relapse after a curative intervention to properly stratify clinical trials in early - stage crc and to accordingly drive the therapeutic management . theoretically , two applications of the detection of mrd in this setting may be foreseen : to offer chemotherapy to all postoperative ctdna - positive patients , including those with no histopathologic risk factors to reduce their risk of progressionindeed , ctdna positivity invariably means residual diseaseand to avoid useless adjuvant therapies in postoperative ctdna - negative patients . 
in early - stage crc , ctdna is detected at a lower rate than in the advanced disease.1 therefore , highly sensitive techniques are needed to achieve appropriate accuracy to detect mrd . most available data have been achieved through a twothe identication of specic somatic step procedure : abnormalities in tissue samples , followed by the search for the same alteration in ctdna . 
only trials that aim to optimize the treatment of all postoperative ctdna - positive patients , independent of traditional histopathologic factors , are ethically acceptable . ctdna to track tumor response and resistance to therapy whereas tissue biopsies catch single snapshots of the tumor in a specic spatiotemporal fragment , liquid biopsy may more comprehensively depict the intrinsic and dynamic intratumoral heterogeneity . 
the heterogeneity and dynamism of the tumor clonal evolution under the pressure of targeted treatments are conrmed by the emergence of multiple alterations in the mitogenactivated protein kinase pathway effectors , including ras and mek mutations and kras , brafv600e , and met amplication , during the treatment with braf / egfr , braf / mek and braf / egfr / mek inhibitors in patients with brafv600e - mutant mcrc.23 - 27 two novel ntrk1 mutations have been detected as a potential mechanism of acquired resistance to entrectinib , a tyrosine kinase receptor inhibitor , in a patient with lmna - ntrk1rearranged mcrc.34 nevertheless , some steps should be covered to translate liquid biopsy from the investigational setting into clinical practice . available data indicate that the frequency of molecular alterations in ctdna at the time of disease progression is inconsistent among different series , even when the same methods for plasma ctdna analysis is applied ( table 1 )  . this inconsistency impairs the reliability of adopted techniques and the reproducibility of the ndings . 
setting and validation of a quantitative threshold to dene the clinical relevance of each detectable molecular alteration as clearly associated with a lack of benet from ongoing therapies may be relevant to biologically guide therapeutic decisions . currently , no prospective data are available about usefulness of discontinuing ongoing therapy and initiation of a tailored treatment when signals of acquired resistance emerge in ctdna before clinical or imaging - based disease progression is noted . the co - occurrence of multiple and / or subclonal molecular alterations at the time of acquired resistance highlights an increase in tumor heterogeneity , which complicates the denition of clinical value of each identied alteration as therapeutic target for subsequent tailored strategies . 
in other words , how to therapeutically target the heterogeneous mechanisms of resistance and the subclonal patterns of tumor cell populations that emerge upon drug selection is today still challenging . 
in this proof - ofconcept study , patients who are candidates for anti - egfr rechallenge are eligible only if a notable decrease in ras fractional mutational abundance occurs from the time of disease progression after a rst - line anti - egfrcontaining therapy to the time of rechallenge . liquid biopsy has emerged as a minimally invasive tool to genotype tumors , to assess patient prognosis and detect mrd , to monitor treatment efcacy , and to track the dynamism of clonal evolution over time and therapies . 
sci transl med 6 : 224ra24 , 2014 denis ja , guillerm e , coulet f , et al : the role of beaming and digital pcr for multiplexed analysis in molecular oncology in the era of next - generation sequencing . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american pathologists joint review . 
j clin oncol 36 : 1631 - 1641 , 2018 baraniskin a , van laethem jl , wyrwicz l , et al : clinical relevance of molecular diagnostics in gastrointestinal ( gi ) cancer : european society of digestive oncology ( esdo ) expert discussion and recommendations from the 17th european society for medical oncology ( esmo ) / world congress on gastrointestinal cancer , barcelona . 
ann oncol 29 : 1211 - 1219 , 2018 siravegna g , mussolin b , buscarino m , et al : clonal evolution and resistance to egfr blockade in the blood of colorectal cancer patients . 
nat med 21 : 795 - 801 , 2015 schmiegel w , scott rj , dooley s , et al : blood - based detection of ras mutations to guide anti - egfr therapy in colorectal cancer patients : concordance of results from circulating tumor dna and tissue - based ras testing . 
mol oncol 11 : 208 - 219 , 2017 jones fs , edelstein d , wichner k , et al : performance of standardized beaming platform for detecting ras mutations in the blood of metastatic colorectal cancer ( mcrc ) patients . 
grasselli j , elez e , carat `u g , et al : concordance of bloodand tumor - based detection of ras mutations to guide anti - egfr therapy in metastatic colorectal cancer . 
normanno n , esposito abate r , lambiase m , et al : ras testing of liquid biopsy correlates with the outcome of metastatic colorectal cancer patients treated with rst - line folfiri plus cetuximab in the capri - goim trial . 
sepulveda ar , hamilton sr , allegra cj , et al : molecular biomarkers for the evaluation of colorectal cancer : guideline from the american society for clinical pathology , college of american pathologists , association for molecular pathology , and the american society of clinical oncology . 
khan kh , cunningham d , werner b , et al : longitudinal liquid biopsy and mathematical modeling of clonal evolution forecast time to treatment failure in the prospect - c phase ii colorectal cancer clinical trial . 
pietrantonio f , oddo d , gloghini a , et al : met - driven resistance to dual egfr and braf blockade may be overcome by switching from egfr to met inhibition in braf - mutated colorectal cancer . 
misale s , di nicolantonio f , sartore - bianchi a , et al : resistance to anti - egfr therapy in colorectal cancer : from heterogeneity to convergent evolution . 
montagut c , dalmases a , bellosillo b , et al : identication of a mutation in the extracellular domain of the epidermal growth factor receptor conferring cetuximab resistance in colorectal cancer . 
misale s , arena s , lamba s , et al : blockade of egfr and mek intercepatients heterogeneous mechanisms of acquired resistance to anti - egfr therapies in 37 . 
newhall k , price t , peeters m , et al : frequency of s492r mutations in the epidermal growth factor receptor : analysis of plasma dna from metastatic colorectal cancer patients treated with panitumumab or cetuximab monotherapy . 
raghav k , morris v , tang c , et al : met amplication in metastatic colorectal cancer : an acquired response to egfr inhibition , not a de novo phenomenon . oncotarget 7 : 54627 - 54631 , 2016 41 . 
siena s , sartore - bianchi a , garcia - carbonero r , et al : dynamic molecular analysis and clinical correlates of tumor evolution within a phase ii trial of panitumumab - based therapy in metastatic colorectal cancer . 
takegawa n , yonesaka k , sakai k , et al : her2 genomic amplication in circulating tumor dna from patients with cetuximab - resistant colorectal cancer . oncotarget 7 : 3453 - 3460 , 2016 45 . 
tsuji y , shitara k , yamanaka t , et al : reverce : randomized phase ii study of regorafenib followed by cetuximab versus the reverse sequence for metastatic colorectal cancer patients previously treated with uoropyrimidine , oxaliplatin , and irinotecanbiomarker analysis . 
montagut c , argil es g , ciardiello f , et al : efcacy of sym004 in patients with metastatic colorectal cancer with acquired resistance to anti - egfr therapy and molecularly selected by circulating tumor dna analyses : a phase 2 randomized clinical trial . 
santini d , vincenzi b , addeo r , et al : cetuximab rechallenge in metastatic colorectal cancer patients : how to come away from acquired resistance ? ann oncol 23 : 4602 - 4616 , 2017 23 : 2313 - 2318 , 2012 49 . 
parseghian cm , loree jm , morris vk , et al : anti - egfr resistant clones decay exponentially after progression : implications for anti - egfr re - challenge . 
cremolini c , rossini d , dellaquila e , et al : rechallenge for patients with ras and braf wild - type metastatic colorectal cancer with acquired resistance to rstline cetuximab and irinotecan : a phase 2 single - arm clinical trial . 
bauer , md4 ; young kwang chae , md , mph , mba5 ; gary sherrill , md6 ; paul fanta , md , ms7 ; axel grothey , md8 ; andrew hendifar , md , mph9 ; david henry , md10 ; daruka mahadevan , md , phd11 ; mohammad amin nezami , md12 ; benjamin tan , md13 ; zev a . 
wainberg , md14 ; richard lanman , md2 ; scott kopetz , md , phd1 ; and van morris , md1 purpose gene fusions are established oncogenic drivers and emerging therapeutic targets in advanced colorectal cancer . 
this study aimed to detail the frequencies and clinicopathological features of gene fusions in colorectal cancer using a circulating tumor dna assay . methods circulating tumor dna samples in patients with advanced colorectal cancer were analyzed at 4 , 581 unique time points using a validated plasma - based multigene assay that includes assessment of fusions in fgfr2 , fgfr3 , ret , alk , ntrk1 , and ros1 . 
relative frequencies of genomic alterations were compared between fusion - present and fusion - absent cases using an unpaired t test . results forty - four unique fusions were identied in 40 ( 1.1% ) of the 3 , 808 patients with circulating tumor dna detected : ret ( n = 6 ; 36% of all fusions detected ) , fgfr3 ( n = 2 ; 27% ) , alk ( n = 10 , 23% ) , ntrk1 ( n = 3 ; 7% ) , ros1 ( n = 2 ; 5% ) , and fgfr2 ( n = 1 ; 2% )  . 
mutations associated with a previously reported antiepidermal growth factor receptor ( anti - egfr ) therapy resistance signature ( subclonal ras and egfr mutations ) were found with fusions in fgfr3 ( 10 of 12 patients ) , ret ( nine of 16 patients ) , and alk ( seven of 10 patients )  . 
for the 27 patients with available clinical histories , 21 ( 78% ) had egfr monoclonal antibody treatment before fusion detection . conclusion diverse and potentially actionable fusions can be detected using a circulating tumor dna assay in patients with advanced colorectal cancer . 
the 68 - gene panel included alk , ret , ros1 , and ntrk1 fusions , and the 70and 73 - gene panels also tested for fgfr2 and fgfr3 fusions ( appendix table a1 )  . 
germline variants were ltered out as previously described.27 the reportable range for single nucleotide variants ( snvs ) , indels , fusions , and amplications is greater than 0.04% per two molecules , greater than 0.02% per one molecule , greater than 0.04% per two molecules , and greater than 2.12 copies , respectively , with a greater than 99.9999% per - position analytic specicity.26 clinical information was obtained from test request forms and conrmed by pathology and medical reports and from treating clinicians when available . this research was approved by the quorum institutional review board for the generation of de - identied data sets for research . 
relative exact frequencies of genomic alterations ( point mutations , indels , and splice variants ) were compared between fusionpresent and fusion - absent cases using an unpaired t test . results occurrence of fusions in a ctdna assay the median age at time of ctdna testing was 59 years ( interquartile range , 50 - 69 years )  . 
 ( * ) indicates fusions with statistically signicant differences in prevalence between tissue and ctdna . partner , the most commonly detected fusions were the fgfr3 - tacc3 ( n = 12 ) and ncoa4 - ret ( n = 9 ) fusions ( appendix table a2 )  . 
of samples tested 3 , 808 3 , 808 3 , 808 3 , 808 abbreviation : ctdna , circulating tumor dna . of ret or ntrk fusions between ctdna and tissue assays ( fig 1c ; table 1 ) .28 genomic proling of fusion - positive patients clinicopathology history was available for a subset of patients ( table 2 ; appendix table a3 )  . 
at least some of the molecular data from tissue testing collected at the time of initial diagnosis was available for 24 of 40 ctdna fusion - positive patients , eight of whom had comprehensive ngs in which the presence of fusions was assessed . 
from the available clinical and tissue data , nine of 27 ( 33% ) were right - sided , tumors were predominantly kras wild type ( n = 23 of 24 ; 96% ) , with no concurrent nras or brafv600e mutations , and three of 22 ( 14% ) were msi - h ( table 2 ; table a3 )  . 
 9 kras overall codon 12 codon 13 codon 61 limited nos unknown nras overall codon 12 codon 13 codon 61 limited nos unknown braf v600e overall fusion overall overall loss of mlh1 / pms2 loss of pms2 clifton et al table 2 . 
cumulatively , the data suggest that a sizable proportion of the ctdna fusion - positive population may have had ras / raf mutations and / or the fusion present at levels below the limit of detection in tissue or in a subclone of the tumor tissue that was not sampled for testing . the frequency of amplications , indels , and snvs in clinically relevant cancer genes detectable using the bloodbased ngs assay were compared between fusion - positive and fusion - negative samples ( fig 2a )  . 
 actionable fusions in crc using a ctdna assay fusion not detected fusion present egfr cdk6 cdk4 fgfr1 brca1 arid1a brca2 braf pik3ca smad4 tp53 odds ratio ( 95% ci ) fig 2 . 
 ( b ) egfr , ras , and braf amplications , indels , and mutations occurring in fusion patients in circulating tumor dna ( ctdna ) and matched tissue samples . 
blank cells indicate no mutation detected ( for circulating free dna [ cfdna ] results ) or not available ( for tissue results and prior antiepidermal growth factor receptor [ egfr ] monoclonal antibody [ mab ] )  . 
mutations associated with this anti - egfr therapy resistance signature were found with fusions in fgfr3 ( 10 of 12 patients ) , ret ( nine of 16 patients ) , and alk ( seven of 10 patients ) , including two of the patients with multiple fusions ( fig 2b )  . 
furthermore , the low rvafs of cooccurring ras , egfr , and brafv600e mutations were consistent with subclonal genomic events occurring later in tumorigenesis ( appendix fig a2 )  . 
among the six patients with an anti - egfr resistance signature who had comprehensive genomic proling results available from tissue , four were wild type at the time of initial diagnosis of crc for the corresponding fusion and / or ras / raf alterations that were later detected in ctdna , consistent with these genomic events being acquired later in tumorigenesis . discussion to our knowledge , this is the largest case series describing fusion - positive cases in crc ( whether in tissue or plasma ) and demonstrates that fusions in patients with crc can be identied using a ctdna assay . 
here , fusions were detected at a prevalence of approximately 1% in patients with advanced crc , similar to fusion prevalence using orthogonal tissue - based assays in separate series of patients with crc.8 , 9 all fusions identied are potentially actionable with available targeted drugs . 
thus , this ctdna approach has the potential to allow clinicians to consider additional studies with novel therapeutic combinations for patients with metastatic crc in future trial settings . our data provide new evidence that fusions , particularly involving fgfr3 or ret , may contribute to anti - egfr therapy resistance in crc . 
on the basis of previously validated genomic signatures in this setting , we hypothesize that fusions may arise as a novel , unreported mechanism with antiegfr therapy resistance , given the clinicopathologic data and frequent co - occurrence with subclonal ras and egfr mutations in ctdna . 
the prole of concomitant egfr mutations and subclonal ras mutations mirrors prior studies that have shown associations between these mutations and post - egfr resistance.29 interestingly , prior series performed in tissue have associated fusions with ras wild - type crc tumors.9 - 12 in our series , 23 of 24 ( 96% ) of the ctdna fusionpositive patients with tissue testing available for ras mutational status were ras wild type , whereas 25 of 44 ( 57% ) of fusion - positive ctdna samples in our series had one or more ras mutations . 
we reconcile these ndings on the basis of the greater sensitivity to detect low allele frequency often not detectable with tissue - based assays . furthermore , tissue specimens are often obtained at surgical resections , before a multiple number of sequential lines of systemic therapy , and therefore before exposure to selective pressures that mediate acquisition of resistance mechanisms . 
the majority of blood samples obtained in this cohort of patients with crc came from treatment - refractory individuals seeking clinical trial options who frequently had been exposed to anti - egfr therapies . 
thus , the occurrence of subclonal resistance alterations in ctdna accounted for differences in the tumor genomic proles of advanced , typically heavily pretreated cancers , relative to the lessmutated genomic proles of the tumor taken before therapy initiation . 
 actionable fusions in crc using a ctdna assay eml4 - alk fusion strn - alk fusion fgfr2 - tacc2 fusion fgfr3 - tacc3 fusion ccdc6 - ret fusion ncoa4 - ret fusion trim24 - ret fusion plekha6 - ntrk1 fusion tpm3 - ntrk1 fusion erc1 - ros1 fusion slc34a2 - ros1 fusion anti - egfr signature - positive c other ras - positive c 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 patient fig 3 . 
such alterations have been previously reported as acquired mechanisms of resistance to anti - egfr therapies in crc.20 , 33 collectively these and our data point to a diverse , heterogeneous landscape of potential resistance mechanisms adapted by ras wild - type crc tumors to overcome egfr blockade . fusions represent a potentially actionable therapeutic target in the anti - egfr resistance setting . 
therefore , although targeting subclonal fusions alone may be only partially successful , multipathway suppression may be a promising avenue of additional investigation , possibly in combination with antiegfr therapies . 
such strategies would need to be highly individualized , given the diversity of resistance mechanisms , and could be informed by comprehensive ctdna testing , especially because serial tissue biopsies are less feasible in patients with advanced cancer . in several previous data sets using tissue - based assays , fusions in patients with crc were associated with msi - h cancers.9 - 12 although rates of msi - h and right - sided tumors in our data set were similar to average rates reported in advanced crcs , a proportion of the fusion - positive patients in this series are suspected to have acquired the fusion after selective pressure from anti - egfr therapy , and therefore the fusion may have been present in the primary tumor at levels too low to be associated with msi - h status . 
 clifton et al this series where the fusion was tested for and detected in tissue , the tumors were found to be msi - h . one of the limitations of this data analysis is that complete clinicopathologic features were not available for all patients , given the retrospective nature of the study , and therefore we were unable to obtain clinical histories from all patients with fusions . 
however , using a previously validated method , 29 the majority of fusion - positive patients had at least one variable , which was highly predictive of prior anti - egfr exposure . 
in addition , among the patients with known treatment history and this signature , the majority were indeed conrmed to have prior anti - egfr therapy , thus internally validating the efcacy of this genomics - rst strategy to identify likely resistance cases . we also did not have matched preand post - treatment tissue and plasma for orthogonal and serial proling to conrm which fusions and other co - occurring mutations were acquired / selected for after anti - egfr therapy versus those present as truncal / clonal events . 
however , genomic events that are acquired during cancer progression tend to have lower relative vaf in ctdna than do early truncal mutations , such as those in tumor suppressor genes or clonal ras mutations.13 in our series , fusions occurring at low rvaf tended to be found in samples containing other genomic mechanisms of anti - egfr therapy resistance , which is consistent with our hypothesis that some fusions in crc occur at subclonal levels that are undetectable in pretreatment tissue but are selected for and become detectable in ctdna after anti - egfr therapy resistance . 
another limitation is that the vaf may be affected by biologic factors , such as the degree of tumor shedding , as well as technical factors , including that fusions are more difcult to detect by ngs and in ctdna samples than snvs . 
taken together , the fusion prevalences and vafs observed in this study may be lower than actual because of these technical reasons . in conclusion , actionable fusions were able to be detected at low frequencies but at similar frequencies to the historical tissue - based ngs approach in a large series of patients with crc using a ctdna assay . 
the distribution of coexisting subclonal mutations in egfr , kras , and nras in fusionpresent crc cases matches genomic proles of crc tumors after progression on prior anti - egfr therapy in tumors initially identied as ras wild type using a less - sensitive tissuebased assay . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . katherine clifton employment : wellcare ( i ) stock and other ownership interests : wellcare ( i ) thereasa a . 
raymond employment : trovagene , guardant health stock and other ownership interests : trovagene , guardant health arvind dasari consulting or advisory role : ipsen , abbvie / stemcentrx , novartis , voluntis , lexicon research funding : novartis , effector therapeutics , eisai , hutchison medipharma , merck , guardant health jonathan m . 
j cell mol med 16 : 237 - 248 , 2012 schram am , chang mt , jonsson p , et al : fusions in solid tumours : diagnostic strategies , targeted therapy , and acquired resistance . 
nat rev clin oncol 14 : 735 - 748 , 2017 drilon a , laetsch tw , kummar s , et al : efcacy of larotrectinib in trk fusion - positive cancers in adults and children . 
n engl j med 378 : 731 - 739 , 2018 yakirevich e , resnick mb , mangray s , et al : oncogenic alk fusion in rare and aggressive subtype of colorectal adenocarcinoma as a potential therapeutic target . 
clin cancer res 22 : 3831 - 3840 , 2016 amatu a , somaschini a , cerea g , et al : novel cad - alk gene rearrangement is drugable by entrectinib in colorectal cancer . 
br j cancer 113 : 1730 - 1734 , 2015 subbiah v , gainor jf , rahal r , et al : precision targeted therapy with blu - 667 for ret - driven cancers . 
cancer discov 8 : 836 - 849 , 2018 subbiah v , velcheti v , tuch bb , et al : selective ret kinase inhibition for patients with ret - altered cancers . 
nat commun 5 : 4846 , 2014 cocco e , benhamida j , middha s , et al : colorectal carcinomas containing hypermethylated mlh1 promoter and wild type braf / kras are enriched for targetable kinase fusions . 
zill oa , banks kc , fairclough sr , et al : the landscape of actionable genomic alterations in cell - free circulating tumor dna from 21 , 807 advanced cancer type braf and kras . 
daly c , castanaro c , zhang w , et al : fgfr3 - tacc3 fusion proteins act as naturally occurring drivers of tumor resistance by functionally substituting for egfr / erk signaling . 
mccoach ce , blakely cm , banks kc , et al : clinical utility of cell - free dna for the detection of alk fusions and genomic mechanisms of alk inhibitor resistance in non - small cell lung cancer . 
piotrowska z , isozaki h , lennerz jk , et al : landscape of acquired resistance to osimertinib in egfr - mutant nsclc and clinical validation of combined egfr and ret inhibition with osimertinib and blu - 667 for acquired ret fusion . 
liang w , he q , chen y , et al : metastatic eml4 - alk fusion detected by circulating dna genotyping in an egfr - mutated nsclc patient and successful management by adding alk inhibitors : a case report . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free 26 . 
odegaard ji , vincent jj , mortimer s , et al : validation of a plasma - based comprehensive cancer genotyping assay utilizing orthogonal tissueand plasma - based circulating tumor dna . 
rankin a , klempner sj , erlich r , et al : broad detection of alterations predicted to confer lack of benet from egfr antibodies or sensitivity to targeted therapy in advanced colorectal cancer . 
parseghian cm , loree jm , morris vk , et al : anti - egfr - resistant clones decay exponentially after progression : implications for anti - egfr re - challenge . 
chae yk , ranganath k , hammerman ps , et al : inhibition of the broblast growth factor receptor ( fgfr ) pathway : the current landscape and barriers to clinical oncol 30 : 243 - 249 , 2019 application . 
 actionable fusions in crc using a ctdna assay appendix fusionpositive patients ( n = 40 ) known treatment history ( n = 27 ) no known treatment history ( n = 13 ) prior anti - egfr exposure ( n = 21 ) no prior anti - egfr exposure ( n = 6 ) antiegfr therapy resistance signature ( n = 24 ) no antiegfr therapy resistance signature ( n = 16 ) known treatment history ( n = 19 ) no known treatment history ( n = 5 ) prior anti - egfr exposure ( n = 16 ) no prior anti - egfr exposure ( n = 3 ) fig a1 . 
ramkissoon , md , phd5 , 6 ; and malak abedalthaga , md7 this case study demonstrates that rst - line treatment of a neurotrophic receptor tyrosine kinase ( ntrk ) fusionpositive infantile glioblastoma with larotrectinib , an ntrk inhibitor ( ntrki ) , was a safe and effective way to achieve tumor regression in our patient . 
an 18 - month - old saudi arabian female presented with a history of right - sided weakness and partial seizures . brain magnetic resonance imaging ( mri ) revealed a large left frontal complex contrast - enhancing mass . craniotomy for gross total resection ( gtr ) was performed , and histologic pathologic analysis revealed a diagnosis of glioblastoma . 
follow - up mri was performed 8 weeks after larotrectinib treatment and showed signicant tumor regression , indicating a positive response to treatment with no reported adverse effects . this patient case highlights the importance of genomic proling for pediatric brain tumors to identify targetable alterations . 
mri showed a large , heterogeneous , complex left frontotemporal mass with internal cystic and necrotic changes with mass effect and a rightward midline shift with enhancing focus at the left posterior thalamic region ( 8 7 9 cm ; fig 1a )  . whole - spine imaging was unremarkable , and the patient underwent craniotomy for gtr . 
the patient recovered well from surgery with no neurologic decits . postoperative mri conrmed gtr but did note a small focus of nodular enhancement in the left insular cortex suggestive of residual tumor ( fig 1b )  . intermixed small neuropathologic analysis of resected tissues revealed features consistent with a high - grade glioma best classied as glioblastoma ( who grade iv )  . 
the neoplastic cells were diffusely immunopositive for vimentin ; were focally positive for s100 and synaptophysin ; showed retained nuclear staining for ini1 ; and were immunonegative for sma , ema , cd99 , cd31 , cd34 , and neun ( figs 2c to 2f )  . 
genomic proling of tumor tissue revealed the presence of an etv6 - ntrk3 fusion , which creates a novel chimeric oncoprotein that results in continuous activation of the ntrk3 kinase . 
the fusion encompassed exons 1 to 5 of etv6 and exons 14 to 20 of ntrk3 , which retains the kinase domain of ntrk3 ( fig 2g )  . as a result of the poor survival rates associated with the patients family declined chemoglioblastoma , therapy or radiation therapy despite extensive counseling on standard - of - care treatment . 
 ( b ) after total surgical resection , postgadolinium axial t1 - weighted mri demonstrated total resection of the left frontal mass . ( c ) at recurrence , postgadolinium axial t1 - weighted mri demonstrated newly appearing mass within the operative bed ( arrow ) showing intermediate diffusion signal with corresponding contrast enhancement indicating recurrent tumor . 
the patient has received continuous larotrectinib therapy since october 2019 and was scheduled for her 6 - month followup mri studies in april 2020 ; however , as a result of the global covid - 19 pandemic , the patients family has deferred further nonemergent hospital visits ( including followup mris ) until the self - isolation restrictions have been lifted . 
therefore , the patient was advanced to a digital clinic visit , where she was noted to be clinically stable and neurologically intact without any evidence of adverse events or toxicities to date . 
we obtained consent from the patients guardian to publish the patients presentation and related images . methods genomic proling next - generation sequencing was performed as previously described8 using the oncomine comprehensive assay v3 system ( thermo fisher scientic , waltham , ma )  . 
assays were performed using the ion s5 system and ion 540 chip ( thermo fisher )  . discussion primary brain tumors are a leading cause of cancer - related morbidity and mortality in children.9 high - grade gliomas ( hggs ) account for approximately 10% of pediatric brain tumors and are the second most common malignant cns tumor after medulloblastoma . 
the most frequent tumors are anaplastic astrocytoma ( who grade iii ) and glioblastoma ( who grade iv ) .10 glioblastoma occurs in both children and adults and is associated with a poor prognosis . 
 ( a ) sheet of highly cellular monomorphic round primitive cells with relative demarcation from adjacent brain tissue ( hematoxylin and eosin [ he ] ; original magnication , 20 )  . 
 ( b ) heterogeneous morphologic features of the neoplasm composed of focal areas of spindle cells arranged in small fascicles and primitive round cells in myxoid background with alternating hyperand hypocellularity ( he ; original magnication , 200 )  . ( c ) higher magnication of the primitive cells in the myxoid background ( he ; original magnication , 200 )  . 
 ( g ) schematic of etv6 - ntrk3 fusion detected in patients tumor . etv6 - ntrk3 5 14 for different n - terminal chemotherapy consisting of vincristine , carboplatin , and temozolomide.12 however , progression - free survival rates remain poor for these patients , highlighting the need for new therapeutic options , including small molecules and / or immunotherapy alone or in combination with chemotherapy regimens.13 although the etiology and genomic drivers of glioblastoma are diverse , 14 - 16 a common nding in pediatric hgg , especially infantile hggs , is the presence of fusions involving ntrk , alk , and ros1 , among others . 
trk fusion proteins are oncogenic drivers that have been reported in a wide range of adult and pediatric tumors that occur at high frequencies ( 90% ) in rare cancer types.17 , 18 gene fusions involving the kinase domain of each of the 3 neurotrophin receptors ( ntrk1 , ntrk2 , and ntrk3 ) fusion partners , were identied in 4% of diffuse intrinsic pontine gliomas and 10% of nonbrain stem ( nbs ) hggs . 
notably , in one study , 4 ( 40% ) of 10 nbs - hggs in children younger than 3 years old harbored an ntrk fusion gene . the high frequency of ntrk fusions in nbs - hggs from children age 3 years and the paucity of additional mutations in these tumors strongly suggest that the fusion genes are potent oncogenic drivers in early postnatal brain tumor development.19 ntrk fusions have been identied at low frequencies in lowgrade pediatric astrocytomas and adult glioblastomas.20 , 21 trk ligands ( commonly nerve growth factor for trka , brainderived growth factor or neurotrophin 4 for trkb , and neurotrophin 3 for trkc ) bind with high afnity to the extracellular domain of the trk receptor.22 , 23 this leads to receptor activation and the induction of signal transduction pathways involved in proliferation , differentiation , and survival ( eg , mapk , pi3k , and pkc pathways ) in both normal and neoplastic cells . the activity of the rst - generation trk - selective inhibitor larotrectinib in pediatric patients with tumors harboring ntrk fusions has been explored in clinical trials , including a phase i / ii trial in pediatric patients ( scout ; clinicaltrials.gov identier : nct02637687 ) and a phase ii trial involving adults and adolescents ( navigate ; clinicaltrials.gov identier : nct02576431 )  . 
larotrectinib is a potent and selective inhibitor of all 3 trks , producing potent and well - tolerated responses in adult and pediatric patients with ntrk - rearranged tumors , with sustained tumor regression in  . 
90% of infants , children , and adolescents with trk fusions at doses of 100 mg / m2 twice a day ( maximum , 100 mg per dose ) .1 - 7 the most common adverse events include mild elevations of liver enzyme levels , cytopenias , and vomiting . 
the high solubility of larotrectinib permit its use in liquid formulations in young patients unable to swallow capsules.5 , 24 increasingly , case reports in the literature demonstrate tolerance and clinical response to ntrki in pediatric patients with hgg , even after chemotherapy and radiation treatment.25 these patient cases highlight the need for clinical trials that assess the sustained durability of response to ntrki and whether these targeted therapies should be used as monotherapy agents or in combination with chemotherapy regimens . our patient case demonstrates that trk inhibitors can be integrated as a rst - line therapy for pediatric hggs harboring trk fusions . 
we also highlight the need for the integration of genomic proling in the routine histopathologic analyses of pediatric patients with malignant primary intracranial tumors to detect any genetic mutations that can be targeted with available therapies . 
bakhsh collection and assembly of data : musa alharbi , nahla ali mobark , malak abedalthaga data analysis and interpretation : musa alharbi , nahla ali mobark , fatmah a . 
ramkissoon employment : foundation medicine stock and other ownership interests : foundation medicine no other potential conicts of interest were reported . references bielack ss , cox mc , nathrath m , et al : rapid , complete and sustained tumour response to the trk inhibitor larotrectinib in an infant with recurrent , chemotherapy - refractory infantile brosarcoma carrying the characteristic etv6 - ntrk3 gene fusion . 
lancet oncol 19 : e187 , 2018 drilon a , laetsch tw , kummar s , et al : efcacy of larotrectinib in trk fusion - positive cancers in adults and children . 
n engl j med 378 : 731 - 739 , 2018 dubois sg , laetsch tw , federman n , et al : the use of neoadjuvant larotrectinib in the management of children with locally advanced trk fusion sarcomas . cancer 124 : 4241 - 4247 , 2018 laetsch tw , dubois sg , mascarenhas l , et al : larotrectinib for paediatric solid tumours harbouring ntrk gene fusions : phase 1 results from a multicentre , open - label , phase 1 / 2 study . 
lancet oncol 19 : 705 - 714 , 2018 landman y , ilouze m , wein s , et al : rapid response to larotrectinib ( loxo - 101 ) in an adult chemotherapy - naive patient with advanced triple - negative secretory breast cancer expressing etv6 - ntrk3 fusion . 
espinoza jc , haley k , patel n , et al : outcome of young children with high - grade glioma treated with irradiation - avoiding intensive chemotherapy regimens : final report of the head start ii and iii trials . 
alharbi m , ali mobark n , almubarak l , et al : durable response to nivolumab in a pediatric patient with refractory glioblastoma and constitutional biallelic mismatch repair deciency . 
nat genet 46 : 444 - 450 , 2014 jones dt , hutter b , j ager n , et al : recurrent somatic alterations of fgfr1 and ntrk2 in pilocytic astrocytoma . 
ziegler ds , wong m , mayoh c , et al : brief report : potent clinical and radiological response to larotrectinib in trk fusion - driven high - grade glioma . 
lee , md1 ; daoqui you , phd1 ; rekha soni1 ; rachna shah1 ; marsha reyngold , md , phd1 ; nora katabi , md1 ; vanessa wu1 ; sean m . 
negative controls included samples from seven patients with hpv - negative head and neck cancers and 20 individuals without cancer . results of 97 patients with nonmetastatic , locoregionally conned oropharyngeal squamous cell carcinoma , 90 patients had detectable hpv16 ctdna and three patients had hpv33 ctdna , indicating an overall sensitivity of 95.6%. 
hpv16 ctdna was detected in 19 of 19 patients with low - volume disease , dened as patients with a single , asymptomatic positive lymph node ( n1 ) or an isolated t1 - 2 asymptomatic primary tumor . 
hpv16 ctdna levels directly corresponded to tumor responses to chemoradiation and surgery . conclusion with an updated understanding of hpv subtypes and sequence variation , hpv ctdna by ddpcr is highly sensitive and specic , identifying hpv16 and hpv33 subtypes in a similar distribution as reported in major genomic proling studies . 
2019 by american society of clinical oncology introduction detection of tumor - derived plasma epstein - barr virus dna has been successfully used in a primary screening study for nasopharyngeal cancer ( npc ) , 1 which demonstrated that npcs could be detected in asymptomatic patients with an earlier disease stage distribution than in the unscreened population . 
viral circulating tumor dna ( ctdna ) as a substrate affords unique technical advantages over single nucleotide variant detection in liquid biopsy assays because of the size of the viral genome and differentness from the human genome and because of the multiple copies of viral genome per tumor genome . 
these features elevate the sensitivity of detection of viral tumor dna , although it is unknown whether viral - associated malignancies other than npc could be similarly detected by liquid biopsy . cancers etiologically driven by human papillomaviruses ( hpvs ) include squamous cell carcinomas of the oropharynx , cervix , vulva , vagina , anal canal , and penis . 
pelvic exams and papanicolaou smears are widely adopted screening tools for early detection of early hpv - associated lesions in the cervix ; however , effective screening approaches for other disease sites are lacking . 
 damerla et al context the human papillomavirus ( hpv ) is a causative agent in cancers of the oropharynx , cervix , vulva , vagina , anal canal , and penis . 
here , we used droplet digital polymerase chain reaction technology to detect the most common hpv strains in hpv - associated oropharyngeal cancer , hpv types 16 and 33 , and achieved near - universal detection of hpv in circulating tumor dna derived from the plasma samples of these patients . 
droplet digital polymerase chain reaction of hpv types 16 and 33 was sensitive enough to detect patients with low disease burden , such as an isolated neck node or t1 - 2 primary tumors . 
these data suggest that this technology could be well suited for screening and disease monitoring . in the study , we sought to determine the clinical utility of hpv ctdna by measuring the sensitivity and specicity of the most sensitive ctdna technology available , droplet digital pcr ( ddpcr ) , 9 in patients with macroscopic gross disease and in various clinical settings after initial presentation . 
to determine the utility of ddpcr in a screening setting , we examine 19 patients in low - volume disease states that are analogous to desired subclinical , screen - detected settings . 
these include patients in whom a surgeon has already removed the primary tumor in the oropharynx with transoral robotic surgery , leaving only a single , asymptomatic lymph node in the neck ( n1 disease )  . 
the clinical characteristics of patients in the series are similar to those of previously published cohorts from our institution , 10 - 12 with stage iva , t2 , and n2b disease as the most common american joint committee on cancer seventh edition cancer stage , t stage , and n stage , respectively ( table 1 )  . study patients plasma specimens from 97 hpv - positive patients with opscc with locoregionally conned disease and eight hpv - positive patients with anal squamous cell carcinoma were accrued ( table 1 )  . 
hpv positivity was dened as p16 overexpression by immunohistochemistry with greater than 70% diffuse nuclear or cytoplasmic staining ( n = 95 ) or a clinically reported positive dnaor rna - based in situ hybridization test for hpv ( n = 53 ) .13 samples from seven patients with hpv - negative head and neck cancers , eight patients with hpv - positive opsccs who had already undergone denitive surgery , and 20 patients without cancer were included as negative controls . 
gross tumor volume was determined from the used radiation plans for which the treating radiation oncologist contoured gross disease and the plan was technically accessible ( n = 84 )  . sample collection and preparation ten milliliters of whole blood were collected from each patient into cell - free dna ( cfdna ) bct tubes ( streck , la vista , ne ) or bd vacutainer k2 edta tubes ( bd biosciences , san jose , ca )  . 
plasma was separated rst though centrifugation at 800 g for 10 minutes , followed by an additional centrifugation at 16 , 500 g for 10 minutes . when samples were collected in bd vacutainer k2 edta tubes , plasma was separated within 1 hour of blood collection and stored at 80c in eppendorf lobind tubes ( eppendorf , hamburg , germany )  . 
at a later date , samples were thawed and cfdna was extracted from 4 to 5 ml of plasma using qiagen circulating nucleic acid kits ( qiagen , germantown , md ) into a 50 - l nal elution volume . 
pretreatment plasma samples were collected a median of 2.4 weeks before the start of chemoradiation . ctdna analysis primers and probes for ddpcr assays and quantitative pcr assay are listed in appendix table a1 . 
of patients * opscc ( n = 97 ) initial presentation anal scc ( n = 8 ) 41 - 82 51 - 71 characteristic age , years median range male female race white asian african american other unknown smoking history never , 10 pack - years  . 
plates were read and analyzed using quantasoft software ( bio - rad ) to assess the number of droplets positive for the target gene , reference gene , both , or neither . 
the assay threshold sensitivity was set at two mutant droplets . pathologic data in a subset of patients , targeted next - generation sequencing ( ngs ) data of pathologic samples were available , obtained through the us food and drug administration approved msk - impact ( memorial sloan kettering cancer center , new york , ny ) sequencing platform.14 to evaluate hpv16 copy number in 10 patients , we extracted genomic dna from formalin - xed parafn - embeddedpreserved pathologic samples . 
one corresponding hematoxylin and eosinstained slide was reviewed by a pathologist ( n.k. ) and used as a template to scrape , collect , and extract genomic dna from regions of other unstained slides with greater than 75% tumor cellularity . 
genomic dna was then used for hpv16 ddpcr , and the hpv16 copy number was considered as the ratio of hpv16 - positive droplets to droplets containing the reference gene eif2c1 . statistical analysis sensitivity was dened as the number of pretreatment cfdna samples that were positive for hpv ctdna divided by the total number of patients ( n = 97 )  . 
specicity was dened as the number of negative control samples that had zero hpv - positive droplets divided by the total number of negative controls ( n = 27 )  . 
to evaluate for any correlation between gross tumor volume or copy number with the initial ctdna levels , spearman correlation coefcients were calculated using prism software by graphpad ( san diego , ca )  . 
 hpv ctdna detection results assay design we rst sought to develop the best possible primer - probe set for ddpcr using the following three criteria : location within the e6 or e7 oncogenes because these sequences are the most highly amplied sequences in tumor genomes15 ; smaller amplicon size , to increase probability of amplication in view of the highly fragmented nature of cfdna ; and primers or probes that perfectly match both european and non - european hpv16 isolates , maximizing universality of the test ( appendix fig a1 )  . 
briey , we used the papillomavirus episteme resource ( pave.niaid.nih. gov ) 16 and identied the 10 sequences representative of hpv16 sublineages ( a1 to a4 , b1 and b2 , c1 , and d1 to d3 ) , spanning european , asian , and african isolates of hpv16 ( appendix fig a1 and appendix table a2 )  . 
we chose primers and probes within e6 and e7 that match these 10 most common sequences exactly and then subsequently compared these sequences with an additional 455 hpv16 isolates found in genbank . 
a primerprobe set in e6 with a length of 97 base pairs met all criteria and demonstrated high pcr efciency and sensitivity down to a single molecule of template hpv16 dna in a ddpcr assay ( appendix fig a2 )  . 
we also tested 20 samples from normal individuals without cancer and seven samples from patients with hpv - negative head and neck cancers , and no droplets were detected in these 27 samples , demonstrating 100% specicity . 
in two of those four patients , the tumor pathology was hpv16 negative , and in both patients , no hpv16 ctdna was detected ( fig 1a , teal )  . in the two other patients , the tumor pathology was hpv16 positive , and in both of these patients , hpv16 ctdna was readily detected in plasma ( fig 1a , red ) , demonstrating 100% concordance ( four of four patients ) between plasma and the tumor specimen in hpv subtyping . 
as an additional control , we collected plasma from eight patients who had already undergone denitive surgery , and hpv was detected in none of these patients , although in two available matched samples , hpv16 ctdna was readily detected before surgery ( fig 1a , dark orange )  . the mean hpv16 ctdna level was 1 , 218 copies / ml ( range , 0 to 13 , 163 copies / ml ) , with no clear cut points for classications into groups . 
we also tested eight plasma samples from patients with anal cancers and detected hpv16 ctdna with a mean of 2 , 151 copies / ml in seven samples , demonstrating that the ddpcr assay can be used in other hpv - positive malignancies ( fig 1a )  . 
we then developed a ddpcr assay for hpv33 , which is the next most common hpv type associated with opscc.17 we observed no hpv33 - positive droplets in 20 samples from normal individuals without cancer . 
after these results , additional pathologic specimens in two of the four hpv16 and hpv33 ctdnanegative patients were genotyped by rna in situ hybridization and found to be negative for hpv16 , suggesting other high - risk hpv subtypes . 
the four negative patients ranged in regard to tumor size and stage and had no distinguishing clinical characteristics ( fig 2b )  . hpv ctdna detection in patients with low disease burden the purpose of liquid biopsy in a screening setting would be to identify subclinical tumors at early disease stages . 
we determined hpv ctdna levels in 19 patients with lowvolume tumor burden , and hpv16 ctdna was readily detectable in all 19 patients by ddpcr ( fig 2a )  . 
in 16 patients with hpv - positive opscc , the primary tumor site in the base of tongue and tonsil had been removed with denitive transoral robotic surgery , and thus , there was no remaining gross disease in the oropharynx at the time of plasma collection . 
in two additional patients , a neck dissection with negative margins had been performed to remove all gross disease , leaving only t1 - 2 primary tumors in the oropharynx . 
in these two patients , low - volume t1 base of tongue and t2 tonsillar primary tumors were imperceptible to the patient , and all gross disease in the neck had been removed before plasma collection . 
because the number of hpv genomic copies per tumor genome can be widely variable , 17 we determined the hpv copy number directly in 10 tumor pathologic samples , which ranged from 0.8 to 57 copies per tumor genome ( fig 2c )  . 
 damerla et al sensitivity : 90 / 97 = 92.8% specificity : 27 / 27 = 100% 10 , 000 1 , 000 undetectable normal individuals ( n = 20 ) hpv - negative hnscc ( n = 7 ) hpv - positive opscc ( n = 97 ) hpv - positive opscc after definitive surgery ( n = 8 ) anal cancers ( n = 8 ) pathology : hpv16 positive pathology : hpv16 negative undetermined hpv subtype matched samples , before and after surgery sensitivity : 93 / 97 = 95.9% 10 , 000 1 , 000 undetectable undetectable normal individuals ( n = 20 ) hpv - positive opsccs ( n = 7 ) pathology : hpv16 negative undetermined hpv subtype , hpv16 ctdna negative hpv - positive opsccs ( n = 97 ) hpv16 ctdna positive hpv33 ctdna positive undetectable hpv16 / 33 ctdna fig 1 . 
 ( a ) hpv16 circulating tumor dna ( ctdna ) detected by the hpv16 ddpcr assay in samples obtained from normal individuals ( n = 20 ) and patients with hpv - negative head and neck squamous cell carcinomas ( hnsccs ; n = 7 ) , hpv - associated oropharyngeal squamous cell carcinomas ( opsccs ; n = 97 ) , and anal squamous cell carcinomas ( n = 8 )  . 
 ( c ) correlation between pretreatment levels of hpv measured by hpv16 droplet digital polymerase chain reaction ( ddpcr ) and hpv copy number per tumor genome . ( d ) correlation between pretreatment levels of hpv measured by hpv16 ddpcr and product of hpv copy number and gross tumor volume . 
figure 3a shows the raw data of one patient during the course of radiotherapy , and the number of droplets positive for hpv16 ( blue ) decreases rapidly , whereas the number of droplets positive for a reference gene ( eif2c1 ) remains unchanged . 
 ( a ) amplitude plots of the ddpcr raw data are shown for crt per week of one patient with oropharyngeal squamous cell carcinoma , starting from pretreatment sample on the left , samples collected per week through 8 weeks of crt , and post - treatment sample on the right . 
 ( b ) graph representing median hpv copies per milliliter at each treatment time point from b along with interquartile range ( dark blue dashed lines ) and minimum and maximum values ( light blue dashed lines ) in patients with complete sets of plasma samples ( 28 patients ) every week . 
 ( c ) median hpv copies per milliliter measures in patient samples after every week of crt are represented as a fraction of the same - patient pretreatment sample ( 28 patients )  . 
ctdna , circulating tumor dna . with multiple samples , hpv16 ctdna was generally cleared by week 7 after the start of chemoradiation , with the exception of three patients in whom hpv16 ctdna levels remained detectable at 10 weeks ( appendix fig a3 )  . 
in this study , we demonstrated that hpv ctdna can be detected in nearly all patients with hpv - positive opscc , a marked improvement over previously reported low - cost pcr - based technologies and likely a result of the following four primary improvements : use of the ddpcr technique , which we found to be superior to quantitative pcr ; design of hpv16 primers and probes that match all hpv16 variants ; use of a combination of hpv16 and hpv33 tests ; and plasma collection and storage procedures optimized for cfdna . 
we demonstrated that the assay was portable to other hpv - associated malignancies because we also detected hpv ctdna in seven of eight anal squamous cell carcinomas . because plasma epstein - barr virus dna can be used to detect subclinical npcs in early stages , we examined whether hpv ctdna can be used to detect tumors with low disease burden . in 19 patients , plasma was collected after the symptomatic site of disease had already been resected , and yet hpv16 ctdna remained detectable . 
early detection by liquid biopsy of patients with t1n0 or t1n1 hpv - positive opsccs would be benecial to patients because these patients may be candidates for curative transoral robotic resections alone , obviating the need for postoperative radiation . 
likewise , patients with early t1n0 anal squamous cell carcinoma are candidates for curative local excisions without any radiotherapy . in addition , the specicity of the test , which is of paramount importance in any screening application , was 100% in 27 negative control samples . 
the plasma hpv16 ctdna signal completely responded to tumor - directed therapies and paralleled plasma levels of driver mutations , indicating that hpv ctdna is highly tumor specic . previous research has shown various degrees of sensitivities for hpv detection for patients with head and neck cancer3 - 6 , 8 and anogenital cancer23 - 25 using pcr - based methods . 
a disadvantage of ddpcr would be the requirement to use limited material for independent hpv16 and hpv33 tests . these two hpv subtypes constitute more than 95% of all oropharyngeal26 , 27 and anal hpv - associated squamous cell carcinomas.28 if a single test were to be used to detect cervical cancer as well , an hpv18 ddpcr test would need to be added . 
although our ctdna results match the distribution of hpv16 and hpv33 frequencies in hpv - associated opscc , we were not able to specically hpv genotype all pathologic specimens that were diagnosed as hpv positive by p16 positivity and / or dna or rna in situ hybridization by standard clinical denitions.13 in addition , it should be noted that although hpv ctdna was detected in all 19 patients with low tumor burden , these patients may not be representative of patients with early subclinical disease because micrometastatic lesions in these patients with locoregional disease could articially boost sensitivity of the assay . 
lee stock and other ownership interests : astrazeneca ( i ) consulting or advisory role : merck , pzer , merck serono , sano research funding : astrazeneca sean m . 
mcbride consulting or advisory role : bristol - myers squibb , janssen nadeem riaz honoraria : peerview speakers ' bureau : illumina research funding : bristol - myers squibb , pzer travel , accommodations , expenses : varian medical systems n . 
damerla are inventors on a provisional patent application led by their institution , memorial sloan kettering cancer center ( inst ) no other potential conicts of interest were reported . acknowledgment we thank erik anderson , luis diaz , jonathan leeman , jeremy setton , and dana tsui for helpful discussions . references chan kca , woo jks , king a , et al : analysis of plasma epstein - barr virus dna to screen for nasopharyngeal cancer . 
n engl j med 377 : 513 - 522 , 2017 chaturvedi ak , engels ea , pfeiffer rm , et al : human papillomavirus and rising oropharyngeal cancer incidence in the united states . 
j clin oncol 29 : 4294 - 4301 , 2011 ahn sm , chan jy , zhang z , et al : saliva and plasma quantitative polymerase chain reaction - based detection and surveillance of human papillomavirus - related head and neck cancer . 
jama otolaryngol head neck surg 140 : 846 - 854 , 2014 cao h , banh a , kwok s , et al : quantitation of human papillomavirus dna in plasma of oropharyngeal carcinoma patients . 
mazurek am , rutkowski t , fiszer - kierzkowska a , et al : assessment of the total cfdna and hpv16 / 18 detection in plasma samples of head and neck squamous 6 . 
wang y , springer s , mulvey cl , et al : detection of somatic mutations and hpv in the saliva and plasma of patients with head and neck squamous cell cell carcinoma patients . 
sci transl med 7 : 293ra104 , 2015 gupta gp , kumar s , marron d , et al : circulating tumor hpv16 dna as a biomarker of tumor genomics and disease control in hpv - associated oropharyngeal squamous cell carcinoma . 
int j radiat oncol biol phys 100 : 1310 - 1311 , 2018 dahlstrom kr , li g , hussey cs , et al : circulating human papillomavirus dna as a marker for disease extent and recurrence among patients with oropharyngeal cancer . 
leeman je , li jg , pei x , et al : patterns of treatment failure and postrecurrence outcomes among patients with locally advanced head and neck squamous cell carcinoma after chemoradiotherapy using modern radiation techniques . 
zumsteg zs , lok bh , ho as , et al : the toxicity and efcacy of concomitant chemoradiotherapy in patients aged 70 years and older with oropharyngeal carcinoma in the intensity - modulated radiotherapy era . 
setton j , caria n , romanyshyn j , et al : intensity - modulated radiotherapy in the treatment of oropharyngeal cancer : an update of the memorial sloan - kettering cancer center experience . 
deshmukh aa , tanner rj , luetke mc , et al : prevalence and risk of penile human papillomavirus infection : evidence from the national health and nutrition examination survey 2013 - 2014 . 
faust h , eldenhed alwan e , roslin a , et al : prevalence of human papillomavirus types , viral load and physical status of hpv16 in head and neck squamous cell carcinoma from the south swedish health care region . 
serup - hansen e , linnemann d , skovrider - ruminski w , et al : human papillomavirus genotyping and p16 expression as prognostic factors for patients with american joint committee on cancer stages i to iii carcinoma of the anal canal . 
reference name source k02718 pph16 af536179 af536179 european ( e ) european ( e ) hq644236 hq644236 european ( e ) af534061 af534061 asian ; e ( as ) af536180 af536180 african - 1 ; afr1a hq644298 hq644298 african - 1 ; afr1b af472509 af472509 african - 2 ; afr2a hq644257 hq644257 north american ( na ) 1 ay686579 ay686579 asian_american ( aa ) 2 af402678 af402678 asian_american ( aa ) 1 aligned with clustalw algorithm ( muscle 3.8 ) limited selection to early genes e6 and e7 of hpv16 genome ( amplified regions during hpv integration ) chose amplicons in e6 and e7 < 100 bp in size cross - referenced with 445 hpv16 variants from ncbi genbank alignment blast against human genome and other hpv strains selected best amplicon by ddpcr performance forward primer probe reverse primer standard curves and testing patient plasma samples fig a1 . 
 ( a - c and f ) standard curves of roche cobas hpv test , hpv16 ddpcr , hpv33 ddpcr , and hpv16 quantitative pcr ( qpcr ) using a serial dilution of hpv16 plasmid template on a plasmid . 
approximately 200 base pairs ( bp ) , likely too large for fragmented ctdna , which has a median size of 176 bp.9 ( e and h ) comparison of hpv16 ddpcr and hpv16 qpcr in the same 12 samples . 
at high levels of hpv16 , the hpv ctdna signal can begin to saturate , as observed in three samples because only 20 , 000 droplets are read on the bio - rad qx200 platforthe higher sensitivity of the ddpcr assay despite a larger amplicon size may be secondary to the partitioning of dna into droplets and reduced inhibitory factors compared with qpcr29 ct , cycle threshold . definitive crt samples 15 , 000 10 , 000 7 , 000 6 , 000 5 , 000 4 , 000 3 , 000 2 , 000 1 , 000 pre - crt sample time ( weeks of crt ) post - crt samples fig a3 . 
noonan , mbbchbao , msc1 - 3 ; sagar sardesai , md1 - 3 ; jeffrey vandeusen , md , phd1 - 3 ; robert wesolowski , md1 - 3 ; nicole williams , md1 - 3 ; clara n . 
we evaluated three prespecied questions to assess patients perceptions of genomic testing . results in all , 100 patients underwent genomic testing , with a median of ve mutations ( range , 0 to 13 mutations ) detected per patient . 
genomic testing revealed one or more potential therapies in 98% of patients ( 98 of 100 ) , and 60% of patients ( 60 of 100 ) had one or more recommended treatments with level i / ii evidence for actionability . 
we did not detect a statistically signicant difference in time - to - treatment failure ( log - rank p = .87 ) or overall survival ( p = .71 ) among patients who had treatment change supported by genomic testing versus those who had no treatment change . 
this study provides a prospective analysis of physician decision making and outcomes of patients receiving tumor sequencing as part of mbc clinical care . knowledge generated genomic testing revealed that most patients ( 60% ) had at least one recommended treatment with level i / ii evidence for actionability ; however , only a subset of patients were given a recommendation for a change in treatment , and even fewer actually changed therapy on the basis of genomic testing results . 
patients whose therapy did not change on the basis of genomic testing had a decrease in condence of treatment success . relevance even though standard of care is improving , relatively few patients with mbc changed therapy on the basis of genomic testing in this prospective study . 
this suggests a need to better understand barriers to implementation of mutation - directed therapy as well as a need for improved understanding of patient perception of somatic genomic testing . ( ia ) , pik3ca hotspot - activating missense mutations ( ia ) , microsatellite instability ( ic ) , ntrk fusions ( ic ) , esr1 hotspot - activating missense mutations ( iia ) , pten loss ( iia ) , akt1 mutations ( iib ) , and erbb2 hotspot - activating missense mutations ( iib ) .16 although genomic testing is promising for improving treatment efcacy , it is unclear how frequently it changes treatment.17 - 19 complex interactions between choice of genomic testing and patient perception of care make it imperative to understand the impact of genetic sequencing in clinical oncology.17 , 20 , 21 to date , we have little understanding of how patients perceive the value or accuracy of genomic testing , and whether changes in treatment recommendation based on genomic testing affect patients perceptions of care . 
there are discrepancies between cancer patient and physician expectations of therapy efcacy ; patients demonstrate greater optimism about therapy efcacy.22 - 25 expectations of patients with mbc and their motivations for undergoing somatic genetic testing have not yet been explored . clinical tumor genomic analyses typically rely on targeted next - generation sequencing ( ngs ) of a panel of specic , typically actionable cancer - related genes to analyze somatic gene alterations from biopsy specimens . 
most commercial targeted panel sequencing approaches , including foundationone cdx ( foundation medicine , cambridge , ma ) in this study , provide treatment suggestions that are based on literature.26 we genomic alterations and available clinical hypothesized that prospective implementation of foundationone cdx testing into clinical care for patients with mbc will identify patient - specic approaches that offer improved patient outcomes and perceptions of care . 
in this study , patients with mbc received foundationone cdx testing when a new treatment was initiated and genomic testing results released to the provider at the next progression event . 
the study was approved by the ohio state university institutional review board and informed consent was obtained from all patients . testing was performed by using foundationone cdx , formalin - xed parafnwhich used ngs on patients embedded tumor tissue . 
the foundationone cdx results were then released to the provider . survey measures physicians received a ve - item questionnaire after the foundationone cdx report was released to assess whether their treatment recommendation should be changed on the basis of those results . 
the questionnaire asked how the physician used the foundationone cdx test ( multiple choice ) and what else they would like to see in the report ( multiple choice plus an open - ended other option )  . 
attitudes toward foundationone cdx testing were assessed with three items : ( 1 ) by having the test , i will feel more condent of my treatments success , ( 2 ) i will trust the test results , and ( 3 ) i think the test results will be accurate . 
patient motivation and expectations for study participation were assessed with 15 questions using a ve - point likert scale that ranged from strongly disagree to strongly agree ( data supplement )  . because of small numbers in each group , these questions were assessed as a three - level outcome ( agree v neutral v disagree ) and as a binary outcome ( agree v neutral and disagree )  . statistical and survival analyses mcnemars test of agreement ( 2 level or 3 level ) was used to compare preand post - test survey responses and preversus post - test survey agreement by foundationone cdxsupported treatment change results from the physician questionnaire . 
the offstudy date was dened as the date of the patients second progression ( rst while on study ) , second treatment change ( rst while on study ) , death , or loss to follow - up . 
patients still alive on december 18 , 2018 , were censored as of this date . all statistical analyses were performed using sas version 9.4 ( sas institute , cary , nc ) and r version 3.4.1 , and survival curves were created using the packhv package.27 results study population a total of 142 patients with mbc provided consent and were assessed for eligibility ( fig 1 )  . 
patients were excluded from analysis if there was no available tissue , insufcient tissue , or poor dna quality ( n = 21 ) , died before the foundationone cdx report was released ( n = 13 ) , did not receive foundationone cdx testing within 10 weeks of starting their current line of therapy ( n = 4 ) , were lost to follow - up ( n = 2 ) , withdrew consent ( n = 1 ) , or had a biopsy that revealed no metastatic disease ( n = 1 )  . 
of these samples , 71% ( 71 of 100 ) were metastatic biopsies and 29% ( 29 of 100 ) were primary tumors ( breast or axillary lymph node )  . 
the study population was predominantly age 55 years or older , white , and collegeeducated with a high income ( table 1 )  . foundationone cdx genomic testing results among the 100 patients with successful foundationone cdx ngs testing , the number of mutations identied per patient ranged from 0 to 13 with a median of ve mutations ( data supplement )  . 
as anticipated , the most common mutations were missense or frameshift mutations in tp53 ( n = 49 ) and pik3ca ( n = 40 ) or amplication of myc ( n = 20 ; fig 2a )  . 
among er - positive / her2 - negative patients , 10 ( 16.7% ) of 63 harbored an esr1 mutation . in terms of therapy recommendations , 98 ( 98.0% ) of 100 patients had at least one potential therapy identied on foundationone cdx testing , with a median of nine potential treatments ( range , 0 to 35 potential treatment ; fig 2b )  . 
the most commonly recommended therapy based on a genomic alteration was an mtor inhibitor ( everolimus or temsirolimus ) for pik3ca mutations ( data supplement )  . among all patients , 60 ( 60% ) of 100 had at least one recommended treatment with level i / ii evidence ( not including erbb2 amplications ) , 16 with a median of one potential treatment with level i / ii evidence ( range , 0 to 2 potential treatments ; fig 2b )  . potential germline alterations in somatic tumor testing a known challenge in somatic tumor sequencing is potential identication of germline alterations.28 - 32 among the 100 total patients , 14 had alterations identied in brca1 ( n = 4 ) , brca2 ( n = 8 ) , or palb2 ( n = 2 )  . 
of these six patients with no prior knowledge of germline mutations , two had germline alterations conrmed ( one brca1 and one brca2 ) and one underwent germline testing in which no alterations were identied ( presumed somatic brca2 mutation )  . 
the six patients whose foundationone cdx reports were not therapy without proreleased remained on their initial gression as of september 1 , 2018 ; ve of these patients had er - positive / her2 - negative and one had er - positive / her2 - positive breast cancer . 
across the study population , 10 ( 10.0% ) of 100 total patients or 10 ( 11.5% ) of 87 patients with physician questionnaire data experienced a treatment change supported by foundationone cdx data . 
physicians described not changing treatment because the patient could not be enrolled on a clinical trial ( ie , the recommended trial closed , the patient did not want to enroll , or the patient was ineligible ; n = 10 ) , patient hospitalization ( n = 3 ) , insurance denied treatment charges ( n = 2 ) , patient transferred care to other provider ( n = 1 ) , or patient pursued a nonrecommended trial ( n = 1 ; data supplement )  . genomic alterations and survival among 76 patients evaluable for ttf analysis , there was no signicant difference in ttf ( log - rank p = .87 ) by foundationone cdx - supported treatment change status ( fig 3a )  . 
despite a high frequency of mutation , there was no signicant difference in os by pik3ca mutation status among er - positive / her2 - negative patients ( p = .18 ) or tnbc patients ( p = .66 ) , or when evaluating alterations that could activate the pi3k pathway ( pik3ca mutation , pten loss , or akt1 mutation ) among er - positive / her2 - negative patients ( p = .60 ) or tnbc ( p = .81 ) patients ( data supplement )  . 
in the msk - impact study ( clinicaltrials.gov identier : nct01775072 ) , hrpositive / her2 - negative patients with detected tumor mutations in esr1 , map kinase pathway , or myc or other transcription factors genes had a worse response to aromatase inhibitor these alterations.15 we evaluated the association of these alterations with os and found no signicant difference among the four groups ( fig 3d )  . therapy than patients without patients perceptions of genetic testing a total of 58 ( 58.0% ) of the 100 evaluable patients completed at least a portion of the survey at enrollment ( pretest ) , and 40 ( 40.0% ) completed at least a portion of the survey at study conclusion ( post - test )  . 
in the pretest survey , most patients strongly agreed or somewhat agreed that by having the genetic testing they would feel more condent in their treatments success ( 36 [ 65.5% ] of 55 ) , whereas at study completion , a minority of patients ( 12 [ 30.8% ] of 39 ) felt more condent in their treatments success by having the genetic testing . 
there was no signicant difference in patients trust of test results or thinking the results were accurate ( mcnemars p = .06 for both questions )  . foundationone cdx - supported treatment change status ( fig 3b )  . 
by subtype , there were signicant differences in os ( log - rank p = .01 ) ; patients who had tnbc had worse os than er - positive / her2 - negative patients , as anticipated ( data supplement )  . among genomic alterations , we evaluated several mutations known to be prognostic ( esr1 mutations ) or potentially predictive of outcomes ( pik3ca )  . 
in msk - impact ( a targeted tumor - sequencing assay developed at memorial sloan kettering cancer center [ msk ] termed integrated mutation proling of actionable cancer targets [ impact ] ) , there was evidence that patient response to aromatase inhibitor therapy was signicantly worse for patients whose tumors harbored one of several alterations.15 among we then assessed whether pretest and post - test survey results were associated with treatment change on the basis of foundationone cdx results in patients who completed both the pretest and post - test survey questions of interest . for treatment condence , we found that patients who did not have a treatment change supported by foundationone cdx data were signicantly more likely to change their response from agree to neutral or disagree ( mcnemars test of agreement p = .001 ; fig 4 )  . 
of potential therapies with level i / ii evidence 50 40 30 20 10 0 % mutant tp53 pik3ca cdh1 znf703 esr1 ccnd1 fgfr1 fgf4 fgf19 gata3 fgf3 map2k4 znf217 pten mcl1 brca2 erbb2 map3k1 arid1a smad4 myst mdm2 mutations or potential therapies for each patient fig 2 . 
comut plot created by using the genvisr package.47 ( b ) for each patient ( n = 100 ) , the graph shows number of detectable mutations , number of potential therapies identied , and number of potential therapies with level i / ii evidence for actionability in breast cancer based on condorelli , et al.16 ( * ) indicates patients whose treatment was changed based on genomic testing results . treatment ( data supplement )  . 
 ( a ) kaplan - meier plot of time to treatment failure stratied by patients with treatment change based on genomic testing ( red line ) v no treatment change . 
time to treatment failure dened as time from release of foundationone cdx results to the off - study date , dened as date of second progression or treatment change ( rst during the study period ) , death , or loss to follow - up . 
 ( c ) kaplan - meier plot of overall survival for patients with estrogen receptor ( er ) positive / human epidermal growth factor receptor 2 ( her2 ) negative metastatic breast cancer , stratied by presence or absence of mutation in esr1 . 
we prospectively evaluated the impact of ngs on mbc treatment decisions and potential associations with patient outcomes and their perceptions of genomic testing . in this study , more than 60% of patients had a genomic alteration associated with level i or ii evidence for actionability ( not including her2 amplication ) based on a recent consensus panel16an impressive number that emphasizes the potential for widespread use of ngs . 
a total of 37 patients answered the question both before ( pre - genomic test survey ) and after ( post - genomic test survey ) genomic test results were shared with physicians . 
this is consistent with a previous study that identied barriers such as trial ineligibility , distance to trial , and physical / emotional exhaustion.18 treatment change was not signicantly associated with ttf or os ; however , the small number of patients experiencing treatment change limits the conclusions that can be drawn from these analyses . this is the rst study to our knowledge to prospectively evaluate the inuence of somatic ngs on patient perception of care and it provides important initial data , but the lower patient survey response rate limits denitive conclusions . 
when comparing study surveys at admission to surveys at conclusion , patients seemed less condent in their treatment , particularly those whose treatment was not changed by foundationone cdx results . 
this is consistent with other studies that suggest genetic testing may increase negative emotions in patients with metastatic cancer , 18 although most studies evaluated effects from germline testing.36 - 40 our data preliminarily suggests that there may be ramications of somatic genomic testing for patients despite its known utility in selecting therapy for mbc . because negative emotions are associated with decreased quality of life and potentially with survival , 41 - 44 the possible negative implications of broadly integrating ngs into clinical practice should be considered . furthermore , pretest survey data suggest that patients have misconceptions about what somatic mutation testing can inforwe found that patients had overly optimistic expectations for somatic tumor ngs : almost half believed the test would tell them which medication to take whereas only 11.5% of the total patients switched therapy on the basis of foundationone cdx reports . 
this is similar to previous studies that demonstrate that patients have greater optimism regarding treatment efcacy than physicians do , 24 possibly because physicians provide patients with more optimistic information than their true assessment.45 in addition , most patients incorrectly believed the test would predict future genetic mutations , assess childrens disease risk , or estimate treatment success , despite the informed consent stating that participants would rarely benet personally or therapeutically from this study . 
therefore , this supports further exploration of the readability of consent forms , an area currently under investigation.46 we hypothesize that patients limited knowledge of genetics may explain their decreased condence after genomic testing , 17 - 19 and we also hypothesize that improved decision support for genomic testing for patients will improve condence in their treatment , particularly in the context of increases in available targeted treatments for mbc . there are known limitations to our study that prevent us from making denitive conclusions . 
furthermore , unlike the methodologies in the existing literature , we used a unique prospective and longitudinal methodology to evaluate patient perceptions . future studies may benet by including patient interviews to assess the experience of somatic ngs . 
furthermore , the potential treatments identied by foundationone cdx test results reect drugs approved for any indication , not necessarily for mbc , and therapies available at the time of the test . 
for example , both everolimus and temsirolimus were reported as potential therapies for patients with alterations in nf1 , pten , ak1 , pik3r1 , rptor , akt3 , fbxw7 , kit , pdgfra , stk11 , and vhl ; however , only everolimus is approved by the us food and drug administration for mbc . 
in addition , since this study was completed , multiple new therapies approved by the us food and drug administration have not been considered , suggesting that clinical implications of ngs should be assessed regularly . in conclusion , our ndings suggest that ngs by tools such as foundationone cdx may provide valuable insight for physicians and patients when determining the best treatment option for patients with mbc upon disease progression . 
there are clear clinical challenges involved with integrating ngs into the framework of mbc care , including barriers to preferred treatment options and complex interactions between genomic testing and patient perceptions , which warrant further study . affiliations 1the ohio state university college of medicine , columbus , oh 2the ohio state university comprehensive cancer center , columbus , oh 3stefanie spielman comprehensive breast center , columbus , oh 4the ohio state university , columbus , oh 5foundation medicine , cambridge , ma 6the ohio state university college of public health , columbus , oh 7mt . 
chen consulting or advisory role : novartis , immune design , syapse speakers bureau : novartis , foundation medicine research funding : eisai patents , royalties , other intellectual property : matchtx , a genomics software package that helps researchers , oncologists , and clinical trial managers identify the full set of biomarkers that collectively predict the outcome of patients with cancer to treatment siraj mahamed ali employment : foundation medicine leadership : incysus stock and other ownership interests : exelixis , blueprint medicines , agios , genocea biosciences consulting or advisory role : revolution medicines , azitra ( i ) , princeps tx ( i ) patents , royalties , other intellectual property : patents via foundation medicine and via seres health on microbiome in non - neoplastic disease ( i ) anne m . 
noonan consulting or advisory role : helsinn healthcare , qed therapeutics sagar sardesai consulting or advisory role : novartis speakers bureau : immunomedics travel , accommodations , expenses : novartis jeffrey vandeusen stock and other ownership interests : immunomedics robert wesolowski consulting or advisory role : pzer ( inst ) , puma biotechnology research funding : acerta pharma , astrazeneca travel , accommodations , expenses : pzer , puma biotechnologybhuvaneswari ramaswamy consulting or advisory role : pzer maryam b . 
lustberg consulting or advisory role : tempus , pledpharma other relationship : hologic / cynosure no other potential conicts of interest were reported . references oshaughnessy j : extending survival with chemotherapy in metastatic breast cancer . 
condorelli r , mosele f , verret b , et al : genomic alterations in breast cancer : level of evidence for actionability according to esmo scale for clinical actionability 17 . 
gray sw , park er , najita j , et al : oncologists and cancer patients views on whole - exome sequencing and incidental ndings : results from the canseq study . of molecular targets ( escat )  . 
gollust se , gray sw , carere da , et al : consumer perspectives on access to direct - to - consumer genetic testing : role of demographic factors and the testing 21 . 
meropol nj , weinfurt kp , burnett cb , et al : perceptions of patients and physicians regarding phase i cancer clinical trials : implications for physician - patient communication . 
lux mp , bayer cm , loehberg cr , et al : shared decision - making in metastatic breast cancer : discrepancy between the expected prolongation of life and treatment efcacy between patients and physicians , and inuencing factors . 
parsons dw , roy a , plon se , et al : clinical tumor sequencing : an incidental casualty of the american college of medical genetics and genomics recommendations for reporting of incidental ndings . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
tanioka m , fan c , parker js , et al : integrated analysis of rna and dna from the phase iii trial calgb 40601 identies predictors of response to trastuzumabbased neoadjuvant chemotherapy in her2 - positive breast cancer . 
bakos ad , hutson sp , loud jt , et al : brca mutation - negative women from hereditary breast and ovarian cancer families : a qualitative study of the brca37 . 
hay jl , meischke hw , bowen dj , et al : anticipating dissemination of cancer genomics in public health : a theoretical approach to psychosocial and behavioral negative experience . 
collins vr , meiser b , ukoumunne oc , et al : the impact of predictive genetic testing for hereditary nonpolyposis colorectal cancer : three years after testing . genet med 9 : 290 - 297 , 2007 40 . 
hendriks ks , hendriks mm , birnie e , et al : familial disease with a risk of sudden death : a longitudinal study of the psychological consequences of predictive testing for long qt syndrome . 
chida y , hamer m , wardle j , et al : do stress - related psychosocial factors contribute to cancer incidence and survival ? nat clin pract oncol 5 : 466 - 475 , 2008 42 . 
giese - davis j , collie k , rancourt km , et al : decrease in depression symptoms is associated with longer survival in patients with metastatic breast cancer : a secondary analysis . 
perni s , rooney mk , horowitz dp , et al : assessment of use , specicity , and readability of written clinical informed consent forms for patients with cancer undergoing radiotherapy . 
 leiomyosarcoma ( lms ) arises from smooth muscle , most often in the uterus or retroperitoneu localized disease is treated surgically ; unfortunately , relapse occurs in 40% to 70% of patients.1 , 2 advanced lms is treated with chemotherapy . 
the most active regimens , doxorubicin and olaratumab or gemcitabine and docetaxel , are associated with response rates of 15% to 35% and progression - free survival of 4 to 7 months.3 - 6 the cytotoxic agent trabectedin and the tyrosine kinase inhibitor pazopanib are used after chemotherapy.7 , 8 clinical trials with immune checkpoint blockade have been disappointing , possibly owing to a tumor microenvironment populated by immunosuppressive macrophages.9 , 10 the alkylating agent dacarbazine ( dtic ) , one of the oldest chemotherapeutics used in sarcoma , is often used as a therapy of last resort , but it has durable activity in some patients with lms . 
we also discuss the implications of this alteration for the dna damage response to alkylating agents and review emerging data , which suggest that lms harbors characteristic defects in the homologous recombination ( hr ) dna damage response pathway . a 51 - year - old woman presented to her family physician in december 2008 with abdominal distension . 
by november 2012 , she had further disease recurrence manifested by a 16 - cm abdominal mass , which was also resected . in march 2013 , she experienced multifocal recurrence with at least 12 mesenteric masses . 
a 51 - year - old woman developed multifocal recurrent uterine leiomyosarcoma manifested by numerous intra - abdominal masses as demonstrated by representative computed tomography images obtained in march 2013 ( left )  . 
study - related computed tomography scans every 3 months demonstrated incremental regression , and by february 2014 , she had no radiographic evidence of malignancy ( fig 1 ) and has continued to receive dtic . 
 genomic sequencing of the november 2012 tumor revealed homozygous deletion of brca2 , several variants of unknown significance , copy number gain of cyclin e1 , epidermal growth factor receptor , fibroblast growth factor receptor , platelet - derived growth factor receptor alpha , and copy number loss of phosphate and tensin homolog ( pten )  . 
 o6 - meg is efficiently repaired by the mgmt enzyme , which removes the methyl adduct from o6 - guanine , restoring the normal guanine base.11 o6 alkylating agents are most effective in cancers with low mgmt expression , such as melanoma and glioblastoma , in which o6 - meg cannot be efficiently repaired . 
approximately 40% of glioblastoma patients harbor mgmt promoter methylation , and these patients derive greatest benefit from tmz.12 - 14 the normal cellular response to o6 - meg highlights several distinct but interrelated signaling pathways that make up the dna damage response . 
different types of damage , whether inflicted by environmental insults or chemotherapy , provoke specific repair mechanisms.15 the initial response to o6 - meg requires mgmt ( fig 2 )  . 
the mismatch repair pathway normally attempts to repair this mismatch but is unable to effectively do so , and multiple futile repair attempts ultimately induce a double - stranded dna break ( dsb )  . 
 the homologous recombination ( hr ) pathway is the preferred response to dsbs because hr uses a template strand to return dna to the original sequence , whereas other pathways are less conservative and lead to accumulation of mutations , genomic instability , and cell death . 
tumors with loss of brca2 are defective in hr and highly sensitive to dsb - inducing agents such as platinum chemotherapy and poly ( adp - ribose ) polymerase ( parp ) inhibitors.18 the anticancer efficacy of o6 alkylating agents such as dtic and tmz was traditionally felt to depend on the status of the mgmt and mismatch repair pathway ; cells with deficient mgmt and intact mismatch repair should accumulate dsbs and demonstrate sensitivity to these drugs . 
however , a critical role for hr has been highlighted in recent preclinical studies primarily in glioblastoma models , in which tmz is the standard of care.19 here , disruption of the hr pathway profoundly potentiated the antitumor activity of tmz.20 - 22 furthermore , resistance to tmz and dtic was not mediated by re - expression of mgmt or acquired mismatch repair deficiency but by adaptive tumor cell augmentation of hr repair.16 , 23 , 24 recently , a model capturing the combined status of multiple repair pathways was found to be much more effective in predicting cytotoxicity from o6 alkylating agents than assessing mgmt alone , and hr pathway status was of major import.25 because the hr pathway ultimately determines whether tmzand dtic - induced dsbs are repaired , defective hr , such as that induced by brca2 loss , may sensitize patients to treatment with these drugs and warrants further evaluation as a predictive biomarker . 
the failure to observe mgmt promoter methylation in this case ( confirmed on repeat testing ) is curious because intact mgmt might be expected to repair the upstream o6 - meg lesion before downstream dsbs are created . 
our findings suggest that dysfunctional hr alone may be sufficient to sensitize tumors to alkylating agents in certain contexts . emerging - omics studies suggest that lms frequently harbors defects in hr , of which our patients brca2 loss is emblematic . 
in a whole exome and transcriptomic sequencing study of 49 lms tumors , mutations in tp53 ( 49% ) , retinoblastoma ( 27% ) , and atrx ( 24% ) were most common but are not actionable.27 moreover , the tumor mutational burden was low , suggesting that immune checkpoint inhibitors are of limited benefit . 
interestingly , deleterious alterations in hr pathway genes were observed in most lms tumors and included pten ( 57% ) , brca2 ( 53% ) , fanca ( 27% ) , atm ( 22% ) , chek1 ( 22% ) , xrcc3 ( 18% ) , chek2 ( 12% ) , brca1 ( 10% ) , and rad51 ( 10% )  . 
an alexandrovcatalogue of somatic mutations in cancer ( cosmic ) signature for defective hr contributed to the mutational signature in 98% of lms tumors.27 a separate analysis of 80 patients with lms that used the cancer genome atlas revealed different gene expression signatures between soft tissue and uterine lms , with the latter harboring a higher dna damage response score.28 unfortunately , the frequency of germ line alterations in hr genes among patients with lms remains uncerta in a comprehensive molecular study of 500 patients with various solid tumors , pathogenic germ line mutations were found in 12% , and 75% of these were in dna repair genes , highlighting the importance of the germ line analysis , which is often not performed in clinical next - generation sequencing assays.29 a defective hr phenotype is of therapeutic relevance . 
several approaches to targeting cancer related vulnerabilities in hr - deficient tumors are being explored.15 several of these approaches incorporate parp inhibitors , a novel class of dna - damaging anticancer therapeutics . 
schwartz collection and assembly of data : all authors data analysis and interpretation : all authors manuscript writing : all authors final approval of manuscript : all authors julia e . 
schwartz honoraria : daiichi sankyo , darwinhealth , biomotiv , human longevity , bionaut labs , champions oncology , karyopharm therapeutics , puretech health , ellipses pharma , array biopharma , ptc therapeutics , bayer healthcare pharmaceuticals consulting or advisory role : daiichi sankyo , puretech health , biomotiv , human longevity , darwinhealth , karyopharm therapeutics , champions oncology , bionaut labs , array biopharma , ellipses pharma , ptc therapeutics , bayer healthcare pharmaceuticals matthew a . 
coindre jm , terrier p , guillou l , et al : predictive value of grade for metastasis development in the main histologic types of adult soft tissue sarcomas : a study of 1240 patients from the french federation of cancer centers sarcoma group . 
hensley ml , blessing ja , mannel r , et al : fixed - dose rate gemcitabine plus docetaxel as firstline therapy for metastatic uterine leiomyosarcoma : a gynecologic oncology group phase ii trial . 
sutton g , blessing j , hanjani p , et al : phase ii evaluation of liposomal doxorubicin ( doxil ) in recurrent or advanced leiomyosarcoma of the uterus : a gynecologic oncology group study . 
seddon b , scurr m , jones rl , et al : a phase ii trial to assess the activity of gemcitabine and docetaxel as first line chemotherapy treatment in patients with unresectable leiomyosarcoma . 
tap wd , jones rl , van tine ba , et al : olaratumab and doxorubicin versus doxorubicin alone for treatment of soft - tissue sarcoma : an open - label phase 1b and randomised phase 2 trial . 
van der graaf wt , blay jy , chawla sp , et al : pazopanib for metastatic soft - tissue sarcoma ( palette ) : a randomised , double - blind , placebo - controlled phase 3 trial . 
demetri gd , von mehren m , jones rl , et al : efficacy and safety of trabectedin or dacarbazine for metastatic liposarcoma or leiomyosarcoma after failure of conventional chemotherapy : results of a phase iii randomized multicenter clinical trial . 
burgess ma , bolejack v , van tine ba , et al . : multicenter phase ii study of pembrolizumab ( p ) in advanced soft tissue ( sts ) and bone sarcomas ( bs ) : final results of sarc028 and biomarker analyses . 
kostine m , briaire - de bruijn ih , cleven ahg , et al : increased infiltration of m2 - macrophages , t - cells and pd - l1 expression in high grade leiomyosarcomas supports immunotherapeutic strategies . 
hegi me , liu l , herman jg , et al : correlation of o6 - methylguanine methyltransferase ( mgmt ) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate mgmt activity . 
paz mf , yaya - tur r , rojas - marcos i , et al : cpg island hypermethylation of the dna repair enzyme methyltransferase predicts response to temozolomide in primary gliomas . 
short sc , giampieri s , worku m , et al : rad51 inhibition is an effective means of targeting dna repair in glioma models and cd133 + tumor - derived cells . 
roos wp , nikolova t , quiros s , et al : brca2 / xrcc2 dependent hr , but not nhej , is required for protection against o ( 6 ) - methylguanine triggered apoptosis , dsbs and chromosomal aberrations by a process leading to sces . 
zhang n , wu x , yang l , et al : foxm1 inhibition sensitizes resistant glioblastoma cells to temozolomide by downregulating the expression of dna - repair gene rad51 . 
most patients present with either metastatic pdac ( 50% - 60% ) or locally advanced tumors ( 30% - 40% ) , for which the median survival is 5 - 9 months after diagnosis.1 , 2 outcomes are still suboptimal for the small subset ( 10%20% ) of patients who present with resectable tumors conned to the pancreas ; , 50% of these patients survive 5 years after surgery despite modern adjuvant chemotherapy.3 moreover , at autopsy , the disease is metastatic for 88% of recurrences , and  . 
80% have more than 10 distinct metastatic lesions4 that are genetically related to the primary tumor5 and other metastases.6 these observations indicate that majority of patients with pdac harbor nonradiographically evident , micrometastatic disease after resection and that remnant tumor cells evadeor acquire resistance toadjuvant chemotherapy . 
although tropism to specic organs during metastatic spread is still poorly understood , patients with recurrence in the liver or peritoneum7 survive signicantly shorter than patients with recurrent lung metastases.8 - 10 pdac tumors are commonly classied into two major subtypes : one that shares some features with adenosquamous tumors ( squamoid and / or basal ) and the other that retains a differentiated , glandular and / or ductal morphology ( ductal and / or classical )  . 
squamoid pdac tumors are more glycolytic , 11 , 12 more hypoxic , 13 more inammatory likely broblasts , 16 and yield a poorer prognosis than ductal tumors.17 - 21 classical subtype tumors are associated with more tumor - inltrating leukocytes , denser collagen , and better outcomes , 15 , 22 although signicant subtype heterogeneity within tumors13 complicates the relationship between subtype and outcome . to metastasize , 14 , 15 recruit more cytolytic t cells inltrate pdac tumors than most other tumors , 23 but they are ultimately insufcient to control the cancer . 
density of tumor - inltrating t cells is highly variable among pdac tumors , 24 and abundant cd8 + t cells ( along with a high number of neoantigens ) can support exceptionally long - term survival.25 however , t cellmediated tumor immunity may be restricted by several mechanisms : tumor immunosuppressive cell downregulation of hla , leukocytes , t cell proximity to tumor cells , and t cell exhaustion.26 - 30 effective t cellmediated tumor immunity is enhanced by a high diversity of functional t cell clones , tumor - specic recruitment of effector t cells , and abundant neoantigens.23 , 25 , 31 quantifying the phenotypes , functions , and locations of leukocytes is critical to identify specic tumor immunity required for exceptional positive outcomes . rare , exceptional control of pdac progression may be mediated by equally rare , idiosyncratic patient genetics , tumor - specic somatic alterations and gene expression , and / or stochastically generated tumor immunity . 
in this report , we present the cases of pt1 , who survived more than 46 months with occult , chemotherapy - resistant metastatic pdac , and her niece ( pt2 ) who succumbed to progressing metastatic disease despite aggressive treatment . 
however , during adjuvant chemotherapy , the lung lesion increased to 23 mm 18 mm , and a ct and / or pet was suspicious for malignancy with a standardized uptake value ( suv ) of 4.5. 
ca19 - 9 test results are not shown becausefor both patientsthey were reproducibly approximately 90% lower than the 37 u / ml threshold for normal , even when the primary tumor was present . 
pt1 chose to receive no further treatment and was radiographically disease - free for over 2 years ( fig 1 ) until an isolated , recurrent lung metastasis was identied by ct and fdg - pet ( suv of 9 ) and then treated by 50 gy in ve fractions using stereotactic body radiotherapy . 
pt2 died 19 months after metastatic disease recurrence . treatment a shared germline prss1 mutation and somatic alterations common to pdac we identied germline prss1 p.a16v in both patients and somatic alterations in the two most common pdac driver geneskras and tp53in both tumors from each patient ( table 1 )  . 
each scale bar is 0.5 m ( c ) pdac subtyping of each patients primary and metastatic tumors . left panel : spearman correlation coefcients for tumors from pt1 and pt2 compared with the published rank order of pdassigner genes for classical ( y - axis ) and quasimesenchymal ( x - axis ) tumors . 
both tumors from pt1 aligned well with the classical subtype , whereas the tumors from pt2 were more quasimesenchymal and / or basal with the metastasis scoring more basal than the primary tumor . leukocyte types associated with immunity in tumors from patient 1 we used a multiplex immunohistochemistry ( mihc ) workow34 to quantify the densities of tumor cells , broblasts , and 10 leukocyte subsets ( appendix table a1 )  . 
we selected regions of interest ( rois ) within each section that contained both krt + epithelial or tumor cells and cd45 + immune cells ( appendix fig a1 ) , followed by quantitative assessment of each roi . 
a detailed analysis of leukocyte subsets revealed that cd8 + t cells and cd4 + t cells were signicantly denser in the metastasis from pt1 compared with the primary tumor from pt2 ( fig 3b )  . 
 ( a ) mean cell density per roi for mutually exclusive populations of ki67 + tumor or epithelial cells , ki67neg tumor or epithelial cells , broblasts , and leukocytes . 
among subsets of cd4 + t cells , we found more pro - inammatory th1 t cells in the metastasis from pt1 than either primary tumor ( fig 3c )  . 
within cd4 + t cells , we found a greater density of both cytolytic granzyme b + ( grzb ) cells and proliferative ki67 + cells in the metastasis from pt1 than the primary tumor from pt2 and a higher density of proliferative ki67 + cells in pt1s primary tumor compared with pt2s primary tumor ( fig 3d )  . consistent with the abundant th1 - like macrophages in pt1s primary tumor , we found that the ratio of th1 - like to th2 - like macrophages was signicantly greater in pt1s primary tumor relative to the other 2 tumors ( fig 3e )  . 
a high granulocyte / neutrophil to lymphocyte ratio is associated with poor outcomes.35 , 36 consistent with this , the primary tumor from pt2 had a signicantly higher granulocyte to cd8 + t cell ratio than either tumor from pt1 ( fig 3e )  . 
in addition , across rois , there was no correlation between the densities of cd8 + t cells and granulocytes in the tumors from pt1 , but there was a signicant positive correlation in pt2s primary tumor ( p , .03 , fig 4a )  . 
furthermore , the abundant granulocytes in pt2s tumor were proximal to tumor cells and appeared within lumens ( fig 4b ) where they may contribute to pathogenesis by occluding ducts.37 we also observed tertiary lymph structures ( tlss ) at the border of both primary tumors , but these were more prevalent in the primary tumor from pt1 ( appendix fig a1 )  . 
tlss from pt1 also contained signicantly more b cells , cd4 + t cells , and cd8 + t cells ( appendix fig a2 ) as previously reported for patients with relatively positive outcomes.24 , 38 tissue acquisition and patient consent human tissues were obtained with informed consent in accordance with the declaration of helsinki and were acquired through the oregon pancreas tissue registry under oregon health & science university irb protocol #3609 . discussion we investigated multiple modalities and tumor characteristics of pdac between two related patients . 
we found several similarities between both patients cancers , cluding genes commonly altered in aggressive pdac ( kras , tp53 , and smad439 , 40 ) and a shared prss1 a16v germline mutation that may predispose to pancreatitis41 - 44 but is variably penetrant , 45 raising the possibility that this specic alteration leads to subclinical pancreatitis that accelerates tumorigenesis , consistent with pt2s early onset of disease.41 , 46 the disease course for pt1 was exceptional in the context of occult , chemotherapy - resistant metastatic disease for nearly 4 years . 
an fanca mutation in the metastasis from pt1 might have sensitized that tumor to adjuvant gemcitabine and xeloda and later to sbrt ; however , pt1s lung metastasis progressed during adjuvant chemotherapy , and no adjuvant treatment was given after surgical resection of the lung metastasis , suggesting that the indolent disease might have also been controlled by other factors . 
both tumors from pt2 contained an alteration in cdkn2a , and the metastasis from pt2 had a copy number loss of pten ; alterations in these genes are associated with poor outcomes and drug resistance.47 - 49 pt1s classical subtype tumors and metastases limited to the lungs are both favorable prognostic factors . 
specically , more prevalent tlss at the periphery of the primary tumor ( appendix fig a1 ) and denser cd8 + t cells ( fig 3b ) that did not correlate with the density of granulocytes24 , 50 ( fig 4a ) , opening the possibility that tumor cells and / or cd4 + t cells in pt1s tumors recruit fundamentally different leukocyte cell types from pt2s tumors.51 additionally , granulocytes typically promote immunosuppression in late - stage tumors , 52 consistent with the relatively low number of t cells in pt2s tumor . classical subtype pdac with lung metastases and unconventional tumor immunity may lead to exceptional outcomes . 
sears , phd , brenden - colson center for pancreatic care , oregon health and science university , 2730 s moody ave , portland , or 97201 ; e - mail : searsr@ohsu.edu. support supported by the brenden - colson foundation and nci grant u01 ca224012 to r.c. 
coussens employment : oregon health &amp ; science university ( ohsu ) honoraria : prospect creek foundation , lustgarten foundation for pancreatic cancer research , syndax pharmaceuticals , inc : external advisory board , carisma therapeutics inc : scientic advisory board , verseau therapeutics , inc , scientic advisory board , zymeworks , inc , scientic advisory board , cytomx therapeutics , inc , kineta inc , ( p30 ) koch institute for integrated cancer research , massachusetts inst . 
of tech , ( p30 ) salk institute cancer center , bloomberg - kimmel institute for cancer immunotherapy , sidney kimmel comprehensive cancer center at johns hopkins , dana farber cancer center breast spore , ( p30 ) dana farber / harvard cancer center , ( p30 ) university of california , san diego moores cancer center , starr cancer consortium , lustgarten foundation for pancreatic cancer research , therapeutics working group , nih / nci - frederick national laboratory advisory committee ( fnlac ) , susan g komen foundation , komen scholar consulting or advisory role : cell signaling technologies , pharmacyclics , cytomx therapeutics , syndax , carisma therapeutics , verseau therapeutics , zymeworks , kineta , inc , abbvie , shasqi inc research funding : syndax pharmaceuticals inc , pharmacyclics , cell signaling technologies , innate pharma , deciphera travel , accommodations , expenses : cell signaling technologies , astrazeneca , pharmacyclics , verseau , carisma therapeutics , cytomx therapeutics , zymeworks other relationship : prospect creek foundation , lustgarten foundation for pancreatic cancer research , ( p30 ) koch institute for integrated cancer research , massachusetts inst . 
n engl j med 379 : 2395 - 2406 , 2018 iacobuzio - donahue ca , fu b , yachida s , et al : dpc4 gene status of the primary carcinoma correlates with patterns of failure in patients with pancreatic cancer . j clin oncol 27 : 1806 - 1813 , 2009 yachida s , jones s , bozic i , et al : distant metastasis occurs late during the genetic evolution of pancreatic cancer . 
makohon - moore ap , zhang m , reiter jg , et al : limited heterogeneity of known driver gene mutations among the metastases of individual patients with pancreatic cancer . 
nat genet 49 : 358 - 366 , 2017 katz mhg , wang h , fleming jb , et al : long - term survival after multidisciplinary management of resected pancreatic adenocarcinoma . 
ann surg oncol 16 : 836 - 847 , 2009 downs - canner s , zenati m , boone ba , et al : the indolent nature of pulmonary metastases from ductal adenocarcinoma of the pancreas . 
j surg oncol 112 : 80 - 85 , 2015 deeb a , haque su , olowokure o : pulmonary metastases in pancreatic cancer , is there a survival inuence ? j gastrointest oncol 6 : e48 - e51 , 2015 10 . 
rashid nu , peng xl , jin c , et al : purity independent subtyping of tumors ( purist ) , a clinically robust , single - sample classier for tumor subtyping in pancreatic 34 . 
zhou y , wei q , fan j , et al : prognostic role of the neutrophil - to - lymphocyte ratio in pancreatic cancer : a meta - analysis containing 8252 patients . 
giakoustidis a , neofytou k , costa neves m , et al : identifying the role of neutrophil - to - lymphocyte ratio and platelets - to - lymphocyte ratio as prognostic markers in patients undergoing resection of pancreatic ductal adenocarcinoma . 
herman jm , jabbour sk , lin sh , et al : smad4 loss correlates with higher rates of local and distant failure in pancreatic adenocarcinoma patients receiving adjuvant chemoradiation . 
schubert s , traub f , brakensiek k , et al : cftr , spink1 , prss1 , and ctrc mutations are not associated with pancreatic cancer in german patients . 
mcwilliams rr , maisonneuve p , bamlet wr , et al : risk factors for early - onset and very - early - onset pancreatic adenocarcinoma : a pancreatic cancer casecontrol consortium ( panc4 ) analysis . 
kim m , shah a , zhang j , et al : gemcitabine resistance in cultured pancreatic tumor cells is associated with down regulation of pten and a mesenchymal phenotype . 
ferrer m , de winter jp , mastenbroek dc , et al : chemosensitizing tumor cells by targeting the fanconi anemia pathway with an adenovirus overexpressing dominant - negative fanca . 
ye k , schulz mh , long q , et al : pindel : a pattern growth approach to detect break points of large deletions and medium sized insertions from paired - end short reads . 
exome sequencing libraries were prepared with a kapa hyper prep kit , enriched using the agilent sureselectxt clinical research exome , and paired - end sequenced with an illumina hiseq 2500 to a high depth of 300  . 
rna sequencing was performed as previously reported.62 briey , rna was extracted using an rneasy kit ( qiagen ) and evaluated by the ohsu massively parallel sequencing shared resource ( mpssr ) core facility for library construction with the truseq rna access library prep kit ( illumina )  . 
30% as measured using an agilent 2100 bioanalyzer . rna was sequenced on an illumina hiseq 2500 / 4000 to generate 100 bp paired - end reads for a minimum depth of 40 million reads per sample . 
we followed the published protocol for purist subtyping33 and used the same data set as for pdassigner subtyping . tissue processing and immunostaining tumor tissue samples were xed in 10% neutral buffered formalin for 24 hours and embedded in parafn ( ffpe )  . 
ffpe sections were stained with hematoxylin and eosin , and krt5 was detected with a rabbit monoclonal antibody ( ep1601y , abcam ) and polyclonal goat anti - rabbit antibodies ( invitrogen )  . 
exposure times and signal thresholds were normalized across all images . multiplex immunohistochemistry , image acquisition , and processing multiplex ihc was performed on 5 m ffpe sections using an adapted protocol based on methodology we previously described.34 briey , slides were deparafnized and stained with hematoxylin ( s3301 , dako , santa clara , ca ) , followed by whole slide scanning at 20 magnication on an aperio at2 ( leica biosystems , wetzlar , germany )  . 
slides were digitally scanned following each chromogen development . scanned images were registered in matlab version r2018b using the surf algorithm in the computer vision toolbox ( the mathworks , inc , natick , ma )  . 
image processing and cell quantication were performed using fiji ( fiji is just imagej ) cellproler version 3.5.164 and fcs express 6 image cytometry ruo ( de novo software , glendale , ca )  . aec signal was extracted for quantication and visualization in fiji using a custom macro for color deconvolution . 
 ( a ) krt ( magenta ) and cd45 ( teal ) expression and selected rois ( yellow rectangles ) used for mihc analyses of tumors ( scale bars represent 5 mm )  . 
 o estimated cost of anticancer therapy directed by comprehensive genomic profiling in a single - center study purpose comprehensive genomic profiling ( cgp ) detects several classes of genomic alterations across numerous genes simultaneously and can match more patients with genomically targeted therapies than conventional molecular profiling . 
the current study estimated the costs of anticancer drugs and overall survival ( os ) for patients who were treated with matched and unmatched therapy . methods costs were estimated for patients with complete data ( 188 of 500 patients ) from a prospective , nonrandomized study of patients with diverse refractory cancers who underwent cgp and were treated with matched or unmatched therapy . 
incremental increases in ttf ( + 1.9 months v + 1.2 months ) and observed os ( + 2.5 months v + 2.1 months ) between matched and unmatched therapies were larger for those who underwent cgp in earlierversus later - line therapy . 
incremental increases in drug costs between matched and unmatched therapies were lower for earliercompared with later - line therapy ( + $27 , 000 v + $43 , 000 , respectively )  . conclusion matched therapy was associated with longer ttf , increased os , and manageable incremental cost increases compared with unmatched therapy . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction patients with metastatic cancer who have experienced failure with standard therapy often have few therapeutic options remaining ; however , in recent years , the development of personalized medicine has shifted the oncology treatment landscape . 
this includes the use of next - generation sequencing ( ngs ) platforms that vastly improve dna sequencing efficiency by allowing millions of reactions to occur in parallel.1 - 4 many cancers have been shown to contain genomic alterations that can be targeted by specific therapies . 
most recently , these targeted therapies have become a major area of focus in anticancer drug development.5 - 15 in 2017 , for example , the us food and drug administration ( fda ) added five new targeted drugs and biologics to its list of more than 200 approved anticancer agents.16 , 17 furthermore , recent national comprehensive cancer network clinical practice guidelines in oncology for melanoma and nonsmall - cell lung cancer recommend broad molecular profiling to identify genomic alterations for matched anita chawla filip janku jennifer j . 
 therapy or to appropriately counsel patients with metastatic disease regarding the availability of clinical trials.18 , 19 in addition , the recently developed precision medicine initiative includes the national cancer institute molecular analysis for therapy choice clinical trial , which seeks to determine the effectiveness of genomically targeted therapies.20 furthermore , several prospective pantumor studies using comprehensive genomic profiling ( cgp ) have demonstrated improvements in clinical outcomes for patients who were treated with genomically matched therapy compared with those who received unmatched therapy.7 , 12 , 13 , 21 recent evidence has also suggested that genomic testing at the time of diagnosis of metastatic disease , rather than after one or more lines of systemic therapy , may improve the chances for patients to receive clinically beneficial matched treatment before they undergo a rapid functional decline as they experience disease progression.22 the fda issued its first approval of an ngsbased diagnostic test in december 2016 , the foundationfocus cdxbrca test ( foundation medicine , cambridge , ma ) , which matches patients with advanced ovarian cancer who have either germline ( inherited ) and / or somatic ( acquired ) brca1 / 2 mutation types to treatment with the poly ( adp - ribose ) polymerase inhibitor , rucaparib.23 in addition to testing for individual genomic alterations , use of cgp allows for the detection of four major classes of dna alterations , including base substitutions , copy number alterations , insertions and deletions , and rearrangements . 
collectively , the breadth and accuracy of cgp allow several potential targets for matched therapies to be identified in parallel regardless of anatomic site and without repeated site - specific tests.3 , 4 , 24 therefore , cgp matches more patients with targeted therapies and may mitigate some of the key limitations of conventional molecular testing , including a lack of sufficient tissue because of the need for sequential testing , delays in receiving matched therapy , and an inability to detect actionable alterations.25 - 30 in 2017 , foundationone cdx ( foundation medicine ) , an ngs - based test that detects alterations in 324 genes , select rearrangements , and genomic signatures , such as microsatellite instability ( msi ) and tumor mutational burden ( tmb ) , became the first fda approved broad companion diagnostic for solid tumors . 
although this diagnostic test is newly approved , there is a robust evidence base for the foundationone laboratory - developed test , and the evidence that supports the latter test can be considered transferrable to the fda - approved foundationone cdx . although the clinical utility of genomically matched therapy has been established , questions remain about the value of cgp and the associated costs of targeted therapy . 
the objective of the current study was to estimate anticancer drug costs , time to treatment failure ( ttf ) , and overall survival ( os ) among patients with refractory cancers who had undergone cgp and were either matched or unmatched to targeted therapies . 
 methods patient population our study is a post - hoc retrospective analysis of patients with diverse refractory tumors who were enrolled in a prospective , nonrandomized , phase i oncology center study . 
detailed inclusion criteria have been described elsewhere.10 this study ( clinicaltrials.gov identifier : nct02437617 ) was approved by the md anderson cancer center internal review board , and all patients gave informed consent . 
information collected by study investigators included age , sex , eastern cooperative oncology group performance status , tumor type , number of metastatic sites , and number of prior therapies . 
a drug was considered matched if the half - maximal inhibitory concentration impacted the target at low nanomolar rangefor small - molecule inhibitorsor if the target was the primary one recognized by an antibody . 
additional details on matching definitions are available in the primary clinical study manuscript.10 outcomes we assessed clinical outcomes ttf and os during the observation period as described previously.10 for this retrospective economic analysis , ttf and os were calculated as observed casesthat is , not statistically adjusted . 
mean monthly drug costsadjusted to 2016 usd were imputed for the first regimen used after cgp on the basis of unit costs of the individual drugs or representative agents of the drug classes and their label - based dosing schedules . 
investigational drugs assumed no cost ; administration costs were assumed to be $136.41 on the basis of healthcare common procedure coding system code 96413 for 1 - hour infusion using centers for medicare & medicaid services physician fee schedule . 
total drug and administration costs were calculated as the product of mean monthly drug and administration costs and ttf . statistical analysis the goal of this economic study was to calculate drug - related costs and their association with observed periods of treatment and survival to better understand cost drivers . 
differences between these groups were assessed using t tests . to assess the drivers of drug cost differences between matched and unmatched therapies , the difference was partitioned into two components : the contribution of differences in treatment duration , calculated as the difference in mean durations for matched versus unmatched therapy multiplied by the monthly cost of unmatched therapy , and the contribution of differences in monthly treatment costs , calculated as the difference in monthly drug and administration costs for matched versus unmatched therapy multiplied by the mean duration of matched therapy . 
analyses were conducted for the overall patient and population and also stratified by line of therapy of the first regimen after cgpthat is , earlier - line ( one to three ) versus later - line ( four or later ) therapy . results patient population a total of 500 patients were enrolled . 
 adapted from wheler et al10 with permission from the american association for cancer research . patients who consented to molecular profiling ( n = 500 ) did not undergo molecular profiling insufficient / no tissue died / entered hospice before tissue could be obtained failed report / sequencing commercial ngs performed and therefore excluded not willing to be treated withdrew from study ( n = 161 ) ( n = 111 ) ( n = 37 ) ( n = 6 ) ( n = 4 ) ( n = 2 ) ( n = 1 ) patients who underwent molecular profiling ( n = 339 ) patients with one are more molecular alteration ( n = 322 ) patients with no molecular alteration ( n = 17 ) patients excluded from analysis never received new evaluable treatment after consent hospice / died before treatment could be initiated still on prior therapy lost to follow - up refused watchful waiting only prior immunotherapy action of drug unclear stem - cell transplantation ( n = 134 ) ( n = 124 ) ( n = 79 ) ( n = 32 ) ( n = 8 ) ( n = 4 ) ( n = 1 ) ( n = 6 ) ( n = 3 ) ( n = 1 ) patients included in analysis ( n = 188 ) matched therapy ( n = 122 ) unmatched therapy ( n = 66 ) mostly for reasons of insufficient / no tissue , death , or referral to hospice before tissue could be obtained . 
of the 339 patients who underwent molecular profiling , 322 had at least one genomic alteration detected317 had one or more potentially actionable alterationand 188 received subsequent treatment with either matched ( n = 122 ; 65% ) or unmatched ( n = 66 ; 35% ) therapy10 ( fig 1 )  . 
of the 134 patients with at least one alteration who were not included in the analysis , 124 never received a new evaluable treatment after providing consent , six were excluded as they had received prior immunotherapy , three received a drug with an unclear action , and one underwent stem - cell transplantation.10 median age of treated patients was 59 years ( range , 19 to 82 years ) , with 55% of patients age 60 years and 45% of patients older than age 60 years in both the matched and unmatched therapy groups . 
increased drug treatment costs for matched therapy were largely attributable to a longer duration of therapy ( 66.3% of costs ) , which was associated with longer ttf , rather than higher monthly drug costs ( 33.7% of costs ; fig 2 )  . 
a total of 16 patientsnine who received matched therapy and seven who were unmatchedreceived an investigational agent that was assumed to have no cost ; however , three of these patients received the agent in combination with either one or two costed drugs . earlierversus later - line therapy . 
in addition , the incremental increases in drug costs between matched and unmatched therapies were lower for earlier - compared with later - line therapy ( approximately $27 , 000 v $43 , 000 ; table 2 )  . 
this was a result , in part , of the use of more costly regimens for later - line therapy , including more frequent use of combination therapies ( 77.8% in later line therapy v 58.5% in earlier - line therapy )  . most of the incremental increases in drug treatment costs between matched and unmatched therapies were attributable to a longer time on treatment and survival in both earlierand later line therapies ( 87% and 58% of costs , respectively ; fig 2 )  . 
in addition , the contributions of monthly drug costs to overall anticancer drug costs were much lower in earlierversus later line therapies ( 13% v 42% , respectively )  . discussion to our knowledge , this is the first study to report anticancer drug costs for matched and unmatched therapies in patients with refractory cancer who have undergone cgp . 
patients who were treated with matched therapy generally had a longer time on treatment , greater observed os , and higher anticancer drug costs compared with those on unmatched therapy . 
it is also notable that patients in the matched group experienced these improved outcomes despite having an approximately 6 - month lower median ttf on their prior therapy compared with the unmatched group , which suggests that their disease had been comparatively less responsive to the last line of treatment these patients had received . 
the benefits of matched therapy may therefore outweigh the increased costs . in addition , our analysis found more promising outcomes for matched therapies when administered to a patient population undergoing cgp in earlier lines of therapy . 
similar to the analysis conducted on the full sample , most of the incremental increases in drug treatment costs were attributable to longer ttf and survival in both earlierand later - line therapies . 
because incremental costs were numerically lower per patient in the earlierversus later - line group , despite their increased os and ttf , these findings suggest that earlier use of cgp could enable matching to effective treatment options that have lower costs than those used in later lines . higher anticancer drug costs for matched therapy have been reported elsewhere.32 in this study , however , the observed high costs may have been a result of patients undergoing cgp in lateras opposed to earlier - line therapies , as more than 93% of the patient population had previously received chemotherapy or a targeted therapy . 
 furthermore , the study authors did not detail the individual factors that underpinned anticancer costs , such as a longer time on treatment , which represented 66.3% of the cost increase in our study , rather than higher monthly drug costs . limitations although this retrospective study estimates the anticancer drug costs associated with matched and unmatched therapies , its contribution must be balanced with the limitations of the data . 
it is also important to note that care at a phase i clinic at a premier care center may not represent the level of drug treatment accessible to patients in community practice , especially those who receive later lines of therapy . 
of note , tmb , which can be assessed using foundationone and foundationone cdx , has recently been shown to be predictive of outcomes among patients who are administered checkpoint inhibitors.35 indeed , both tmb and msi were not well - established as markers for targeted therapy during the time period of the phase i study , 10 whereas pembrolizumab is now approved for patients with unresectable or metastatic solid tumors with msi high or mismatch repair deficiency . 
 ( c ) patients with four or more lines of prior therapy . 80 , 000 60 , 000 40 , 000 20 , 000 80 , 000 60 , 000 40 , 000 20 , 000 80 , 000 60 , 000 40 , 000 20 , 000 30 , 664 12 , 825 25 , 240 68 , 729 unmatched therapy monthly drug costs ( matched therapy ) longer duration of therapy matched therapy 34 , 527 3 , 568 23 , 745 61 , 840 unmatched therapy monthly drug costs ( matched therapy ) longer duration of therapy matched therapy 17 , 954 29 , 323 24 , 939 72 , 216 unmatched therapy monthly drug costs ( matched therapy ) longer duration of therapy matched therapy increased clinical utility as more patients have the opportunity to become matched to targeted therapy . in addition , this analysis included phase i drugs that may not have engaged the target or had poor pharmacokinetics , and patients who received all dose levels were also included , which may reduce the positive effect of treatment.10 furthermore , drug acquisition and administration costs were estimated on the basis of list prices and durations of therapy , and costs for medical services were not included . 
our analysis was also limited by the relatively high number of patients who could not be administered therapy during the study as a result of the unpredictable and rapid disease course of refractory cancers . 
among included patients , it was not possible to separate out the possible impacts of line of therapy and performance status on the differential os and ttf findings.10 finally , patients were assumed to take the dose of each drug according to the prescribing information , which would not account for potential dose reduction in combination therapies . in conclusion , cgp is useful for selecting patients with refractory tumors to receive matched therapy . 
miller , rachel anhorn , zhou zhou , james signorovitch manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors vincent a . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . stock and other ownership interests : foundation medicine travel , accommodations , expenses : foundation medicine rachel anhorn employment : foundation medicine , pfizer stock and other ownership interests : foundation medicine zhou zhou employment : analysis group james signorovitch employment : analysis group acknowledgment the analysis presented in this article was conducted by analysis group , which received consulting fees from foundation medicine . 
we thank tim peoples , marcia reinhart , cody patton , and erica birk of analysis group for medical writing and editorial assistance . anita chawla employment : analysis group leadership : cytrx corporation stock and other ownership interests : cytrx corporation filip janku stock and other ownership interests : trovagene consulting or advisory role : deciphera , trovagene , novartis , sequenom , foundation medicine , guardant health , immunome research funding : novartis , biomed valley discoveries , roche , agios , astellas pharma , deciphera , symphony evolution , plexxikon , piqur , fujifilm other relationship : bio - rad support funded by foundation medicine . prior presentation references presented at the 2017 asco annual meeting , chicago , il , june 2 - 6 , 2017 . affiliations anita chawla , zhou zhou , and james signorovitch , analysis group , boston ; vincent a . 
singh rr , patel kp , routbort mj , et al : clinical validation of a next - generation sequencing screen for mutational hotspots in 46 cancer - related genes . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
von hoff dd , stephenson jj jr , rosen p , et al : pilot study using molecular profiling of patients tumors to find potential targets and select treatments for their refractory cancers . 
tsimberidou am , wen s , hong ds , et al : personalized medicine for patients with advanced cancer in the phase i program at md anderson : validation and landmark analyses . 
jardim dl , schwaederle m , wei c , et al : impact of a biomarker - based strategy on oncology drug development : a meta - analysis of clinical trials leading to fda approval . 
johnson db , dahlman kh , knol j , et al : enabling a genetically informed approach to cancer medicine : a retrospective evaluation of the impact of comprehensive tumor profiling using a targeted next - generation sequencing panel . 
schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
levy bp , chioda md , herndon d , et al : molecular testing for treatment of metastatic non small - cell lung cancer : how to implement evidence - based recommendations . 
ali sm , hensing t , schrock ab , et al : comprehensive genomic profiling identifies a subset of crizotinib - responsive alk - rearranged non - small cell lung cancer not detected by fluorescence in situ hybridization . 
drilon a , wang l , arcila me , et al : broad , hybrid capture - based next - generation sequencing identifies actionable genomic alterations in lung adenocarcinomas otherwise negative for such alterations by other genomic testing approaches . 
rankin a , klempner sj , erlich r , et al : broad detection of alterations predicted to confer lack of benefit from egfr antibodies or sensitivity to targeted therapy in advanced colorectal cancer . 
schrock ab , frampton gm , herndon d , et al : comprehensive genomic profiling identifies frequent drug - sensitive egfr exon 19 deletions in nsclc not identified by prior molecular testing . 
pags a , foulon s , zou z , et al : the cost of molecular - guided therapy in oncology : a prospective cost study alongside the moscato trial . 
haslem ds , van norman sb , fulde g , et al : a retrospective analysis of precision medicine outcomes in patients with advanced cancer reveals improved progression - free survival without increased health care costs . 
handorf ea , mcelligott s , vachani a , et al : cost effectiveness of personalized therapy for firstline treatment of stage iv and recurrent incurable adenocarcinoma of the lung . 
 appendix tumor type adenoid cystic breast colorectal cup esophageal gi , anus gi , stomach gi , other gyn , other hepatocellular head and neck lung melanoma neuroendocrine ovarian prostate renal sarcoma thymic thyroid uterine other table a1 . 
 biallelic deletion of palb2 occurs across multiple tumor types and suggests responsiveness to poly ( adp - ribose ) polymerase inhibition background with brca1 / 2 , palb2 ( a partner and localizer of brca2 ) plays a critical role in the dna - damage response pathway . 
palb2 is required for homologous recombination repair , and formation of the brca1 - palb2 - brca2 complex is essential for recruitment of rad51 to sites of dna damage.1 - 3 palb2 stabilizes brca2 and promotes its localization within the nucleus . 
dna repair pathways are disrupted in palb2 - deficient cells , thereby accounting for the tumor suppressor gene properties of palb2.1 , 2 monoallelic loss - of - function mutations in palb2 increase the risk of breast and pancreatic cancers . deleterious mutations in palb2 have been reported in 1% to 2% of familial breast and in 2% to 5% of familial pancreatic cancer syndromes.4 - 8 mutations in palb2 are associated with increased risk of male breast cancer , 9 prostate cancer , 10 gastric cancer , 11 and ovarian cancer.12 biallelic mutations in palb2 are less common and are linked to fanconi anemia.13 although heterozygous mutations in palb2 have been well characterized , homozygous deletions of palb2 have not been previously described . to our knowledge , we present the first published series of palb2 biallelic gene deletions across multiple tumor types . 
in absence of a standard of care , tumor tissue was submitted for comprehensive genomic profiling ( cgp ) .14 two genomic alterations were identified , including a biallelic palb2 gene deletion involving exons 8 to 10 as well as apc p.h785fs * 1 ( table 1 ; fig 3 )  . after providing informed consent , the patient was treated off label with olaparib ( lynparza ; astrazeneca , london , united kingdom )  . 
he was treated in accordance with the declaration of helsinki . restaging ct after 3 months revealed resolution of the primary pancreatic mass and significant reduction in size and number of hepatic metastases ( figs 2b and 2e )  . 
1 year after treatment initiation . palb2 genomic loss across tumor types to identify additional patients who may benefit from parp inhibition , we examined a large genomic sequencing database containing  . 
liver metastases at presentation were refractory to chemoembolization ( d ) but demonstrated durable response to olaparib ( e and f )  . gene deletions involving exons 1 to 13 ( fig 3 )  . 
the remaining 12 tumor samples harbored partial exonic deletions ( fig 3 ; table 1 )  . the majority of samples ( 77% ) involved the c - terminal wd40 domain of palb2 , which mediates interaction between palb2 and brca2 . there were insufficient numbers to assess cooccurrence or mutual exclusivity ; the most common associated gene defects involved tp53 ( 33% , six of 18 ) , cdkn2a ( 28% , five of 18 ) , and cdkn2b ( 22% , four of 18 )  . 
interestingly , the median tumor mutational burden ( tmb ) was low , at 3.47 mutations per dna megabase , among patients in our series ( table 1 )  . 
all homozygous deletions would be predicted to result in loss of function of palb2 . these mutations were previously detected in the cancer genome atlas but have not been specifically reported in the literature . discussion herein , to our knowledge , we report the first series to examine large genomic deletions in palb2 and provide indirect clinical evidence for responsiveness to parp - inhibitor therapy . 
 ( c ) the location of biallelic deletions observed across 18 samples identified by comprehensive genomic profiling are shown . 1 2 3 10 11 binding chan binding mrg15 wd40 methods such as multiplex ligation - dependent probe amplification , quantitative polymerase chain reaction , or comparative genomic hybridization.24 cgp has been demonstrated to be an effective means of identifying large genomic rearrangements.12 the functional domains of palb2 have been well characterized , with loss - of - function mutations linked to multiple cancer types.7 , 12 , 23 the recruitment of brca2 to sites of dna damage is dependent on the presence of a normally functioning palb2 . 
the c - terminal wd40 domain of palb2 , encoded by exons 7 to 13 , mediates the protein protein interactions among palb2 , brca2 , and rad51c ( fig 3 ) .25 truncation or frameshift mutations of the wd40 domain of palb2 disrupts this interaction and is associated with increased genomic instability.2 , 10 , 17 large - scale deletions of these exons is predicted to manifest similarly as a loss of function . although rare , biallelic palb2 deletions involving regions encoding important functional domains may have important clinical implications . orthogonal preclinical support for this observation includes palb2 - deficient tumor cells exhibiting diminished activity of homologous recombination and impaired dna damagerepair pathways.26 loss of functional palb2 is associated with sensitivity to dna - damaging agents1 , 27 and parp inhibitors.3 , 28 functional short interfering rna mediated palb2 knockdown is known to sensitize cell lines to parp inhibitors.3 , 26 the efficacy of olaparib in homologous recombinationdeficient cells is postulated to result from synthetic lethality , in which parp inhibition induces single - strand dna breaks . 
cells deficient in homologous recombination are more susceptible to induced dna damage , thereby leading to selective cell death of tumor cells.29 clinical data supporting the role of palb2 as a predictive biomarker are limited . 
durable benefit from olaparib in a patient with metastatic prostate cancer and heterozygous palb2 gene deletion30 and a case of palb2 - mutant pancreatic cancer treated with mitomycin c have been reported.31 because it is not yet known , further study is warranted to determine how closely the palb2 alterations described thus far share traits with cancers defined by brcaness . 
similarly , palb2 alterations are not part of current homologous recombination deficiency assays . pseudopapillary neoplasm of the pancreas is a rare epithelial tumor comprising 1% to 2% of exocrine pancreatic neoplasms.32 these tumors generally present as large encapsulated masses , although metastatic disease has been reported . 
 tumor - specific and actionable drug targets for uncommon neoplasms . would be needed to address the functional relevance of lower tmb in our data set . the median tmb in our series was low , at 3.47 mutations per dna megabase , which is an unanticipated finding . 
an analysis of ovarian cancer cases from the cancer genome atlas revealed a slightly higher tmb among brca1 / 2 - mutant tumors compared with wild - type tumors , although a subset of brca - mutant , low - tmb patients was observed.36 increased tmb is hypothesized to result from an increased propensity for error in cells deficient in homologous recombination . the genomic characteristics of homologous recombination deficiency are a broader loss of heterozygosity and aneuploidy , whereas tmb is broadly derived from counting short insertions and deletions ( indels )  . 
unfortunately , other patients in our series did not receive parp - inhibitor therapy , and clinical responses tmb is a putative predictive biomarker for immune checkpoint inhibitor responsiveness , and preclinical work suggests synergy between immunotherapy and veliparib in brca - deficient models.37 , 38 although more prospective study is needed , deleterious biallelic palb2 loss may identify a subset of patients for whom parp - inhibitor monotherapy and / or combinatorial strategies with radiation ( eg , nct01908478 ) , immunotherapies ( eg , nct02571725 ) , and / or synthetic lethal dnadamaging agents ( eg , nct02308072 ) is warranted . our series provides support for incorporating cgp in the therapeutic algorithm for patients for whom standard - of - care therapies are lacking . 
ali employment : foundation medicine stock and other ownership interests : exelixis , foundation medicine , epizyme patents , royalties , other intellectual property : patents via foundation medicine ; patents via seres health on the microbiome in non - neoplastic disease ( i ) samuel j . 
klempner honoraria : foundation medicine consulting or advisory role : eli lilly , boston biomedical , celgene speakers bureau : foundation medicine travel , accommodations , expenses : eli lilly 1 . 
buisson r , dion - c ote a - m , coulombe y , et al : cooperation of breast cancer proteins palb2 and piccolo brca2 in stimulating homologous recombination . 
couch fj , hart sn , sharma p , et al : inherited mutations in 17 breast cancer susceptibility genes among a large triplenegative breast cancer cohort unselected for family history of breast cancer . 
ding yc , steele l , kuan cj , et al : mutations in brca2 and palb2 in male breast cancer cases from the united states . breast cancer res treat 126 : 771 - 778 , 2011 10 . 
lu c , xie m , wendl mc , et al : patterns and functional implications of rare germline variants across 12 cancer types . nat commun 6 : 10086 , 2015 12 . 
walsh t , casadei s , lee mk , et al : mutations in 12 genes for inherited ovarian , fallopian tube , and peritoneal carcinoma identified by massively parallel sequencing . 
pylkas k , erkko h , nikkila j , et al : analysis of large deletions in brca1 , brca2 and palb2 genes in finnish breast and ovarian cancer families . 
janatova m , kleibl z , stribrna j , et al : the palb2 gene is a strong candidate for clinical testing in brca1and brca2 - negative hereditary breast cancer . 
blanco a , de la hoya m , balma~na j , et al : detection of a large rearrangement in palb2 in spanish breast cancer families with male breast cancer . 
tischkowitz md , sabbaghian n , hamel n , et al : analysis of the gene coding for the brca2 - interacting protein palb2 in familial and sporadic pancreatic cancer . 
foulkes wd , ghadirian p , akbari mr , et al : identification of a novel truncating palb2 mutation and analysis of its contribution to early - onset breast cancer in french - canadian women . 
shen y , rehman fl , feng y , et al : bmn 673 , a novel and highly potent parp1 / 2 inhibitor for the treatment of human cancers with dna repair deficiency . 
villarroel mc , rajeshkumar nv , garrido - laguna i , et al : personalizing cancer treatment in the age of global genomic analyses : palb2 gene mutations and the response to dna damaging agents in pancreatic cancer . 
ji s , xu j , zhang b , et al : management of a malignant case of solid pseudopapillary tumor of pancreas : a case report and literature review . 
huang j , wang l , cong z , et al : the parp1 inhibitor bmn 673 exhibits immunoregulatory effects in a brca1 ( - / - ) murine model of ovarian cancer . 
2 , 3 we used our recently reported in silico tool ( dpyd - varifier ) 7 to predict that p.t132a would be deleterious to function , which was subsequently validated using ex vivo and in vitro approaches . 
these methodologies permitted us to determine an activity score for the patient that was used to calculate a safe adjusted dose of fu for adjuvant therapy . this study was approved by the mayo clinic institutional review board . 
dpyd - varifier was used to predict the function of p.t132a.7 the effect of p.t132a on dpd activity was measured in vitro as previously described.5 - 7 total rna was isolated from the patients blood sample using the paxgene blood rna kit ( preanalytix , hombrechtikon , switzerland ) , and cdna was reverse transcribed using oligo ( dt ) 15 primers ( thermo fisher scientific , waltham , ma )  . 
the full - length dpyd open reading frame was amplified ( primers : 5 - gtttgtcactggcagactcg - 3 , 5 - ttcacagcaactgtttcacaaa - 3 ) using q5 high fidelity dna polymerase ( new england biolabs , ipswich , ma )  . 
the polymerase chain reaction ( pcr ) product was cloned into the pjet1.2 vector , and 20 cloned constructs were sequenced at the mayo clinic gene analysis shared resource to determine the cis or trans conformation of the variants . 
peripheral blood mononuclear cells ( pbmcs ) were isolated and assessed ex vivo for dpd activity as previously described.9 , 10 additional pcr and quantitative pcr reactions were performed with primers specific to canonically ( e13 / 14 : 5 - ctcttgataaggacattgtgacaaa - 3 , 5 - tttgcagctcttgcgatgc - 3 ) and alternatively spliced dpyd ( e13 / 15 : 5 - ctcttgataagattgtgattgctagc - 3 , 5 - tttgcagctcttgcgatgc - 3 )  . 
 patient presentation ( bowel function alteration and red blood cell per rectum ) colonoscopy pathology staging ct scan 1st cycle began toxicity symptoms dose reduction 2nd cycle radiotherapy only radiotherapy completed 1st cycle began ( reduced mfolfox6 ) 46 - hour infusion 2nd cycle 4th cycle ( dose increased ) 5th cycle ( initial dose ) 8th cycle 10th cycle in silico prediction , ex vivo , and in vitro dpd assay , clonal rna sequencing dpyd sequencing ( mayo medical lab ) blood sample collection surgical resection diagnostic tests neoadjuvant cape + radio variants testing adjuvant mfolfox6 30 34 543 635 no . 
a 32 - year - old male with a history of gastroesophageal reflux and hyperlipidemia presented with altered bowel function and bright red blood per rectu colonoscopy revealed a polyp in the cecum and a mass approximately 10 cm within the rectosigmoid colon . 
a staging computed tomography scan of the chest , abdomen , and pelvis showed no evidence of distal metastases , which suggested stage iii disease . the patient was treated shortly after diagnosis with neoadjuvant therapy that consisted of capecitabine ( 825 mg m2 for 5 days each week ) and radiotherapy ( 50.4 gy in 28 fractions )  . 
 during hospitalization , absolute neutrophil count decreased to 700 cells / l ( normal reference range , 1 , 500 to 8 , 000 cells / l ) , which prompted discontinuation of therapy late in the second week of treatment . 
one and a half months after surgery , the patient was scheduled to begin modified infusional fu , leucovorin , and oxaliplatin ( mfolfox6 ) adjuvant therapy . because of the severe toxicity during the initial neoadjuvant therapy , genetic testing was ordered . 
dpd activity in the patients pbmcs was reduced by 64% ( 92.4 pmol fu min1 mg1 ) compared with previously reported reference values obtained from individuals who do not carry a deleterious dpyd variant10 ( 254.0 pmol fu min1 mg1 ; fig 2a )  . 
to determine whether allelic imbalance of dpyd was occurring , allele - specific pcr primers e13 / 14 and e13 / 15 were used to amplify canonical and alternatively spliced dpyd ( fig 2b , top )  . 
 ( a ) dihydropyrimidine dehydrogenase ( dpd ) enzyme activity was measured in peripheral blood mononuclear cells isolated from the patient and was compared with reference values previously published by our laboratory for peripheral blood mononuclear cells from individuals who do not carry known deleterious variants in dpyd ( noncarriers ) and those heterozygous for * 2a ( * 2a / wild type [ wt ] ) .10 ( b ) polymerase chain reaction primers were designed as indicated ( arrows ) to amplify across the exon 13 / 14 and 13 / 15 junctions to detect the presence of alternative splicing in the patient and a heterozygous carrier of * 2a . 
this level of reduction is similar to that observed for the well - studied p.d949v variant5 , 7 ; thus , the dpd activity score would be expected to be similar . 
finally , the decision was made to administer two additional cycles of therapy that did not contain oxaliplatin because of accumulating grade 1 neuropathy ( fig 1 , gold box )  . 
the patient has completed the 10th cycle of adjuvant therapy and has responded well to the treatment with no severe toxicity and no physical evidence of cancer progression . discussion this report outlines the effective management of toxicity in a patient with rectal cancer treated with an fu - based regimen using a precision oncology approach . 
to date , genetic testing of dpyd variants is not mandated in the united states , and complete dpd deficiency is only listed as a contraindication to fu treatment.11 many studies have increasingly performed functional characterizations of dpyd variants , 3 , 5 - 7 , 9 , 10 , 12 , 13 and the clinical pharmacogenetics implementation consortium has published activity - based fu dosing guidelines for many of the reported dpyd variants.8 however , a paucity of information exists with respect to fu dose adjustments in compound heterozygous variant carriers . in this case report , dpyd genotype and variant function data were used to provide guidance for adjuvant fu dosing in a patient who carries a complex dpyd genotype that includes a novel variant of unknown significance . 
the 38% reduction in in vitro dpd function attributed to p.t132a also is consistent with the approximately 36% activity measured in the patients pbmcs because the p.t132a variant in the patients genome is present in a compound complex heterozygous state ( trans ) with dpyd * 2a . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . shikshya shrestha no relationship to disclose colbren ( scout ) trogstad - isaacson no relationship to disclose timothy j . 
lee am , shi q , pavey e , et al : dpyd variants as predictors of 5 - fluorouracil toxicity in adjuvant colon cancer treatment ( ncctg n0147 )  . 
meulendijks d , henricks lm , sonke gs , et al : clinical relevance of dpyd variants c.1679t > g , c.1236g > a / hapb3 , and c.1601g > a as predictors of severe fluoropyrimidine - associated toxicity : a systematic review and meta - analysis of individual patient data . 
offer sm , wegner nj , fossum c , et al : phenotypic profiling of dpyd variations relevant to 5 - fluorouracil sensitivity using real - time cellular analysis and in vitro measurement of enzyme activity . 
lee am , shi q , alberts sr , et al : association between dpyd c.1129 - 5923 c > g / hapb3 and severe toxicity to 5 - fluorouracil - based chemotherapy in stage iii colon cancer patients : ncctg n0147 ( alliance )  . 
 genomic and proteomic alterations in desmoplastic small round bluecell tumors purpose desmoplastic small round blue - cell tumors ( dsrcts ) are sarcomas that contain the t ( 11 ; 22 ) ( p13 ; q12 ) translocation ews - wt1 fusion protebecause this is a rare tumor type , prospective clinical trials in dsrct are challenging . 
patients are treated in a manner similar to those with ewing sarcoma ; however , differences in prognosis and clinical presentation suggest fundamental differences in biology and potentially different therapeutic implications . 
 methods thirty - five dsrct tumors were assessed using next - generation sequencing , protein expression ( immunohistochemistry ) , and gene amplification ( chromogenic in situ hybridization or fluorescence in situ hybridization )  . 
independent analysis by rna sequencing confirmed higher ar expression from an independent data set of ews - wt1 fusionpositive dsrcts compared with ewing sarcoma and a pan - cancer analysis . 
dsrcts had somatic mutations that were identified in tp53 and foxo3 , averaged five mutations per megabase , and no programmed death - ligand 1 expression was detected in any dsrct samples . 
2018 by american society of clinical oncology ajaz bulbul john paul shen joanne xiu pablo tamayo hatim husain author affiliations and support information ( if applicable ) appear at the end of this article . corresponding author : hatim husain , md , university of california san diego , 3855 health sciences #3011 , la jolla , ca 92093 ; e - mail : hhusain@ucsd.edu. introduction desmoplastic small round blue - cell tumors ( dsrcts ) originate from a cell with multilineage potential and are defined by a molecular hallmark with ews - wt1 reciprocal translocation.1 dsrcts are rare , highly aggressive mesenchymal tumors , with only 200 to 450 affected patients with the disease described to date.1 , 2 ewing sarcoma and dsrcts are treated in a similar manner in the clinic ; however , dsrcts have been represented in limited numbers in sarcoma studies . 
the clinical presentations of these diseases are different , with important comparisons being the site of disease presentation and poor prognosis in dsrct groups . despite aggressive therapy , median survival ranges from 17 to 25 months , 2 , 3 and the 5 - year survival rate remains around 15%3 with higher survival reported among those who underwent removal of at least 90% of the tumor with an absence of extraperitoneal metastasis.4 almost all of these tumors contain the t ( 11 ; 22 ) ( p13 ; q12 ) translocation that fuses ews with wt1 leading to production of a chimeric protein with transcriptional regulatory activity . 
 and pelvic cavity of young adult men in 88% to 97% of cases , 2 , 5 whereas ewing sarcoma commonly occurs in the axial skeleton.7 despite similar histologic morphology , differences in disease presentation and prognosis likely reflect the impact of different biologic factors , including differences in the transcriptional effects of the ews - wt1 fusion relative to the ews - fli1 fusion observed in ewing sarcoma . although there is no standard clinical management strategy for dsrct , treatment usually includes neoadjuvant high - dose cyclophosphamide , doxorubicin , and vincristine , alternating with ifosfamide and etoposide chemotherapy combined with aggressively attempted r0 resection.4 , 8 , 9 recent accelerated us food and drug administration ( fda ) approval in october 2016 of the compound olaratumab , a platelet - derived growth factor receptor a inhibitor , was based on a highly significant improvement in overall survival of 26.5 months in soft tissue sarcomas of histologic subtypes for which an anthracycline containing regimen may be appropriate . 
however , only one patient in the phase ii olaratumab study had round blue - cell sarcoma , and that patient was negative for the ews translocation.10 patients with dsrcts are poorly represented in clinical trials as a result of the rare nature of the disease . 
a list of ongoing clinical trials in this disease is provided in the data supplement . the aim of the current study was to understand the molecular characteristics of dsrcts by analyzing 35 patients with molecularly and immunohistochemically profiled tumors and to compare these with ewing sarcoma to explore therapeutic options and potential oncogenic vulnerabilities for this extremely rare and aggressive cancer type . 
dna from formalin fixed , paraffin - embedded samples was sequenced using the nextseq ( 592 genes selected on the basis of the cosmic database ; agilent sure select xt ; illumina , san diego , ca ) and miseq ( 47 genes ; truseq ) , to evaluate mutation and gene amplification . 
the full list of markers surveyed is available.11 twenty - four tumors were sequenced with the 45 - gene panel , and 11 tumors were sequenced with the 592 - gene panel . 
additional data for ar and epidermal growth factor receptor ( egfr ) ihc in other tumors , including prostate cancer , lung cancer , breast cancer , kidney cancer , endometrial cancer , and glioblastoma , were obtained from a larger cohort of 127 , 000 pan - cancer tumor samples analyzed . we used 2 tests for comparison , and statistical significance was determined as p < .05. 
 ( a ) enrichment scores ( single - sample gene set enrichment analysis ) of selected transcriptional signatures across alveolar rhabdomyosarcoma , desmoplastic small round blue cell tumors , ewing sarcoma , and synovial sarcoma . 
desmoplastic small round blue - cell tumor ( dsrct ) sarcomas are clearly overenriched in androgen receptor ( ar ) , epidermal growth factor receptor ( egfr ) , and ews - wt1 ( kts positive / negative ) signatures compared with those of other sarcomas . 
arms , alveolar rhabdomyosarcoma ; es , ewing sarcoma ; ss , synovial sarcoma . the degree to which the genes in a particular gene set are coordinately upregulated or downregulated within each sample . 
in this manner , ssgsea projects a single sample gene expression profile from the space of single genes onto the space of gene sets . the signature for ar is the hallmark_ androgen_response gene set from the molecular signatures database hallmark subcollection.13 the egfr signature corresponds to the egfr_up.v1_up / dn gene sets from the molecular signatures database subcollection c6.14 the ews - wt1 ( kts positive / negative ) signatures were generated from the geo data set gse53301.15 the ews - fli1 signature was made from the 20 genes listed in figure 1 from mendiola et al.16 transcriptional data sets the multiple sarcoma data set shown in figure 1a is a subset of the data set from filion et al.17 sarcoma samples in figure 1b are the dsrct subset from filion et al.17 prostate samples , shown in figure 1b , consisted of 22 primary prostate cancer samples from the geo data set gse32269.18 endometrial samples are 30 samples from the endometrial cancer data set from raeder et al.19 glioblastoma multiforme samples consisted of a subset of 30 samples from the glioblastoma multiforme data set from verhaak et al.20 results demographic data from the 35 patients whose tumors underwent clinical molecular testing are provided in the data supplement . 
 ( a ) thirty - five desmoplastic small round blue - cell tumors ( dsrcts ) went through molecular profiling using a commercial , multiplatform profiling service ( caris life sciences )  . 
specific tests performed included sequencing ( next - generation sequencing [ seq ] ) , protein expression ( immunohistochemistry [ ihc ] ) , and gene amplification ( chromogenic in situ hybridization or fluorescence in situ hybridization [ ish ] )  . 
 patients with ewing sarcoma had lung and bone involvement , which is consistent with the known distribution pattern of these tumors.21 in the 35 dsrcts , increased expression of top2a was observed in 63% of cases , topo1 in 63% , pten in 62% , ar in 59% , and egfr in 42% of tumors . 
increased expression of tubb3 was observed in 56% of tumors , mgmt in 55% , ts in 52% , rrm1 in 43% , and ercc1 in 24% ( fig 2a )  . 
comparing dsrcts with ewing sarcoma tumors , we found that both ar ( 59% v 3% ; p = 1.7e10 ) and tubb3 ( 56% v 29% ; p = .03 ) were expressed in significantly more dsrcts ( fig 2b )  . 
a full list of all of the patients with ihc staining is provided in the data supplement , which includes a description of staining thresholds . given that both the ar and egfr pathways can be effectively targeted with fda - approved drugs , we sought to further evaluate the activity of these pathways in dsrcts . 
we obtained gene expression profiles for 28 dsrcts as well as profiles for ewing sarcoma , alveolar rhabdomyosarcomas , and synovial sarcoma for comparison with other primitive sarcomas.17 performing ssgsea , we found that the majority of dsrcts was enriched for the ar signature , which indicated that these tumors coordinately upregulated genes that are known to be highly expressed when the ar pathway is activated ( fig 1a )  . 
as expected , ewing sarcoma tumors were enriched for the ews - fli1 signature , and dsrcts were enriched for the ews - wt1 signature , which is consistent with the translocation event pathognomonic for each tumor . to evaluate the significance of ar pathway activation in dsrcts , we made comparisons with tumors that are known to be driven by the ar pathway . 
compared with 59% ar expression in dsrct , ihc results indicated that ar expression was most frequent in prostate cancer ( 94% [ n = 1 , 340 tumors ] ) ; however , dsrct was greater than any other tumor type , including breast cancer ( 50% [ n = 1 , 962 tumors ] ) , kidney cancer ( 30% [ n = 40 tumors ] ) , and the pan - cancer analysis , which demonstrated < 30% expression ( n = 127 , 000 )  . 
a pan - cancer analysis of egfr expression demonstrated a percentage of expression ( 53% [ n > 20 , 000 ] ) that was similar to the 42% observed in dsrct . 
furthermore , tubb3 expression ( 51% [ n = 66 , 000 ] ) and top2a ( 69% [ n = 77 , 000 ] ) was similar to that observed in dsrct . pd - l1 expression ( sp142 ) was not detected in tumors tested ( zero of four ) , whereas programmed death 1 ( pd - 1 ) staining on tumor infiltrating lymphocytes ( tils ) was found in 25% of tumors tested ( two of eight )  . 
pd - l1 expression on tumor cells in ewing sarcoma was absent ( zero of 10 ) , and pd - 1 expression on tils was observed in 32% ( six of 19 ) of ewing sarcoma tumors ( fig 2 )  . 
tumor mutational load was calculated in 11 ewing sarcoma tumors , and mean tumor mutational load was five mutations per megabase ( range , three to eight mutations )  . next - generation sequencing revealed a synonymous tp53 g245g mutation and a foxo3 mutation ( l382fs ) in dscrt , whereas six tp53 mutations ( 13% ) , two apc mutations ( l1129s and i1307k ) , one brca1 ( c.301 + 1g > a ) , and one ctnnb1 ( t41a ) mutation were identified in ewing sarcoma . 
there was no copy number amplification identified in dsrct samples for cmet , her2 ( human epidermal growth factor receptor 2 ) , and egfr with in situ hybridization ( data supplement )  . discussion our understanding of the biology of dsrcts and their distinction from other sarcomas is evolving . 
 apart from the canonical ews - wt1 fusion , some major differences between dsrct and ewing sarcoma reflect those between ar and egfr expression . significantly higher ar expression in dsrcts compared with ewing sarcoma tumors suggest the possibility of a mechanistic link between ews - wt1 activation and androgen receptor expression . 
various ews - wt1 proteins regulate the promoter activity of egr - 1 , a zinc finger transcription factor that contributes to ar pathway activation.25 , 26 egr - 1 knockdown by small interfering rna inhibited activity of the ar signaling pathway.27 evidence for ar activation in dsrcts was observed by ihc as well as the coordinate regulation of the ar - responsive transcriptome . 
the degree of ar enrichment in dsrcts was higher than that of many other tumors , and additional work is needed to understand the clinical sensitivity of these tumors to ar inhibition . fda - approved novel ar inhibitors , including enzalutamide , and cyp17a1 inhibitors , including abiraterone , will need to be explored and provide a potential opportunity for continued drug expansion in this space . 
the three patients who had benefit from complete androgen blockade ( cab ) had normal testosterone levels before cab , whereas the three who did not experience a response had baseline castrate levels of testosterone.28 in vitro studies that evaluated the functional status of ar using western blot analysis and the stimulation of growth by dihydrotestosterone in mtt calorimetric assays in 27 patient tumors suggested that ar in dsrcts is functional , and inhibition of basal and testosterone - stimulated growth by flutamide could be achieved.28 a phase ib study in prostate cancer has combined treatment with enzalutamide and docetaxel chemotherapy and has resulted in a 50% decline from baseline in prostate - specific antigen in 19 of 20 patients , and a phase ii study is ongoing in prostate cancer ( clinicaltrials.gov identifier : nct02685267 ) .29 given the poor outcomes found with conventional therapy , the role of combinatorial therapy with ar inhibitors alone or in combination with chemotherapy in dsrtc deserves additional clinical investigation . tumor mutational load may affect response rates to immunotherapy in melanoma and nonsmallcell lung cancers.30 unfortunately , single - agent antipd - 1 antibodies , to date , have had limited efficacy across sarcomas , and an ongoing phase ii study ( sarc028 ) is evaluating the role of pembrolizumab across various sarcoma histologies ( clinicaltrials.gov identifier : nct02301039 ) .31 none of the patients in our cohort had identifiable tumoral pd - l1 expression by sp142 antibody testing , and the significance of pd - 1 positive tils is unclear at this time . in conclusion , there are distinguishing molecular characteristics between dsrcts and ewing sarcoma . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . ajaz bulbul consulting or advisory role : pfizer , astrazeneca travel , accommodations , expenses : pfizer , astrazeneca joanne xiu employment : caris life sciences pablo tamayo no relationship to disclose hatim husain consulting or advisory role : astrazeneca , abbvie , foundation medicine speakers ' bureau : astrazeneca , merck , bristol - myers squibb research funding : pfizer ( inst ) travel , accommodations , expenses : astrazeneca , merck , bristol - myers squibb , foundation medicine , abbvie affiliations ajaz bulbul , john paul shen , pablo tamayo , and hatim husain , university of california san diego , la jolla , ca ; and joanne xiu , caris life sciences , phoenix , az . funded by university of california san diego moores cancer center institutional funds and national institutes of health grants no . 
honor c , amroun k , vilcot l , et al : abdominal desmoplastic small round cell tumor : multimodal treatment combining chemotherapy , surgery , and radiotherapy is the best option . 
tap wd , jones rl , van tine ba , et al : olaratumab and doxorubicin versus doxorubicin alone for treatment of soft - tissue sarcoma : an open - label phase 1b and randomised phase 2 trial . 
creighton cj , hilger am , murthy s , et al : activation of mitogen - activated protein kinase in estrogen receptor alpha - positive breast cancer cells in vitro induces an in vivo molecular phenotype of estrogen receptor alpha - negative human breast tumors . 
mendiola m , carrillo j , garca e , et al : the orphan nuclear receptor dax1 is up - regulated by the ews / fli1 oncoprotein and is highly expressed in ewing tumors . 
raeder mb , birkeland e , trovik j , et al : integrated genomic analysis of the 8q24 amplification in endometrial cancers identifies atad2 as essential to myc - dependent cancers . 
verhaak rg , hoadley ka , purdom e , et al : integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in pdgfra , idh1 , egfr , and nf1 . 
gerald wl , rosai j , ladanyi m : characterization of the genomic breakpoint and chimeric transcripts in the ews - wt1 gene fusion of desmoplastic small round cell tumor . 
sukhatme vp , cao xm , chang lc , et al : a zinc finger - encoding gene coregulated with c - fos during growth and differentiation , and after cellular depolarization . 
fine rl , shah ss , moulton ta , et al : androgen and c - kit receptors in desmoplastic small round cell tumors resistant to chemotherapy : novel targets for therapy . 
morris mj , rathkopf de , novotny w , et al : phase ib study of enzalutamide in combination with docetaxel in men with metastatic castration - resistant prostate cancer . 
burgess ma , crowley j , reinke dk , et al : sarc 028 : a phase ii study of the anti - pd1 antibody pembrolizumab ( p ) in patients ( pts ) with advanced sarcomas . 
 c case of metastatic extramammary paget disease of the vulva treated successfully with trastuzumab emtansine introduction extramammary paget disease ( empd ) is a rare adenocarcinoma in situ that originates in skin that contains apocrine glands . 
empd is associated with invasive cancer in 4% to 20% of patients.1 in a series of 1 , 439 patients with invasive empd , 80.4% had localized disease , 17.1% had locoregional spread , and only 2.5% presented with distant disease.2 treatment is primarily surgical , but empd frequently is multicentric , and relapses are common regardless of treatment modality . 
since 2002 , she underwent six excisional procedures , three laser ablations , several courses of topical imiquimod treatment , external beam vulvar radiation to a dose of 56 gy to the perineum and the left - side inguinal nodes , and interstitial brachytherapy to 30 gy . 
her treatment was complicated by urethral swelling and eventually obstruction , requiring placement of a temporary suprapubic catheter and urethral reconstruction in late 2014 . in early 2016 , the patient noted worsening urinary retention , and a computed tomography ( ct ) urogram demonstrated two centrally hypodense liver lesions that measured up to 2.2 cm , multiple enlarged retroperitoneal lymph nodes , and a circumferential soft tissue mass that infiltrated the cervix and upper vagina . 
on examination under anesthesia , a paget lesion obliterated the perineum , including the urethra , vulva , and perianal area , and vaginoscopy was required to visualize the cervix . 
vulvar biopsy specimens confirmed invasive , poorly differentiated adenocarcinoma that had arisen from empd , with extensive lymphovascular space invasion and 3 + her2 immunostaining in > 10% of cells ( fig 1 )  . 
on the basis of high concordance between dna sequencingbased methods and fluorescent in situ hybridization ( fish ) in detecting erbb2 amplification , 5 fish was foregone . the patient underwent six cycles of carboplatin area under the curve of 5 mg ml / min and paclitaxel 175 mg / m2 chemotherapy every 21 days , gillian l . 
 ( a ) nests of extramammary paget disease in the epithelium ( black arrows ) with underlying invasive adenocarcinoma in the dermis ( red arrow ; hematoxylin - eosin stain magnification , 10 )  . 
 ( b ) strong , circumferential membranous staining of human epidermal growth factor receptor 2 ( her2 ) in extramammary paget disease and invasive tumor ( her2 immunohistochemical stain magnification , 10 )  . 
 ( c ) invasive adenocarcinoma ( hematoxylin and eosin stain magnification , 10 ) with ( d ) 3 + membranous staining of her2 ( her2 immunohistochemical stain magnification , 10 )  . with trastuzumab added from cycles 2 to 6 . 
repeat pet / ct scanning after 3 months of maintenance therapy revealed an intensely fdg - avid recurrent cervical and vaginal tumor and mildly enlarged and fdg - avid left - side external iliac nodes . 
treatment was well tolerated , with toxicity limited to grade 1 fatigue and grade 1 bleeding , including intermittent vaginal bleeding , hematuria , epistaxis , and gum bleeding , which started after cycle 2 . 
follow - up pet / ct scans were notable for markedly decreased size and metabolic activity of the primary pelvic lesion after cycle 3 and for complete resolution of disease on imaging after cycle 6 ( fig 2 )  . 
positron emission tomography / computed tomography ( a ) axial fusion , ( b ) sagittal fusion , and ( c ) maximum intensity projection images before ( top ) and after treatment ( bottom )  . 
 a hypermetabolic cervical mass ( arrow ) was seen with associated hydrometra , which had complete treatment response to chemotherapy . and hormonal suppression with letrozole.6 , 7 although insufficient data exist on the application of the current breast cancer asco - cap her2 testing guidelines8 to other cancer types , the amplification of her2 has been described in many cases of invasive empd ( table 1 )  . 
 multiple large , multicenter trials have firmly established the benefit of trastuzumab in the treatment of metastatic her2 - positive breast cancer , 9 which has led to consideration of trastuzumab in the treatment of invasive metastatic her2 - amplified empd . retrospective data have suggested that her2 overexpression is higher in patients with invasive empd than with noninvasive empd.19 furthermore , a significant correlation has been demonstrated among the presence of invasion , lymph node metastasis , and her2 overexpression.24 several case reports have demonstrated partial or complete response of widely metastatic empd on trastuzumab with or without paclitaxel ( table 2 )  . the mechanisms that underlie trastuzumabs clinical activity have not been completely table 1 . 
although tdm1 has been shown to be efficacious in patients with breast cancer with her2 - overexpressed malignancy unresponsive to trastuzumab , disease response has not been consistently demonstrated with other tumor types . 
 in her2 - positive unresectable gastric or gastroesophageal junction cancers with prior her2 - targeted therapy.36 the current patients favorable response indicates that in her2 positive invasive empd , tdm1 may be considered a viable treatment option . 
in addition , in hormone receptor and her2 - positive breast cancer , dual targeting of both signaling pathways may improve outcomes significantly.37 in breast cancer , bidirectional cross talk between the estrogen receptor and her2 pathways likely contributes to anti - her2 therapy resistance . 
 preclinical models with breast cancer cell lines have demonstrated a benefit to simultaneous hormone and her2 therapy over her2 - targeted therapy alone , but no phase ii or iii trials have compared the two.38 the tandem trial demonstrated a 2.4 - month survival benefit in postmenopausal patients with metastatic breast cancer who received anastrazole and trastuzumab versus hormonal therapy with anastrazole alone.39 hormone receptor immunohistochemistry was not examined in the current patient , but dual inhibition may be a theoretically superior strategy for treating hormone receptor and her2 - positive invasive empd . one limitation to this study is that the asco - cap her2 testing guidelines have not been routinely adopted or validated for tissues other than breast carcinoma ; thus , the her2 immunohistochemistry results may not be consistent or reliable in this tumor type . 
molecular subtyping has been used increasingly to find appropriate targeted therapies and may be more reliable than immunohistochemistry.23 vornicova et al7 reported a partial response that lasted 12 months in a male with adenocarcinoma with paget disease and negative her2 testing by both immunohistochemistry and fish , but received anti - her2 therapy on the basis of tumor somatic mutation testing alone ( table 2 )  . in light of the poor prognosis with metastatic epmd , paucity of treatment options , high incidence of her2 amplifications in empd , and favorable adverse effect profile of tdm1 , we suggest her2 testing and consideration of tdm1 treatment in patients with her2 overexpressed invasive empd who experience treatment failure with trastuzumab . 
ohara k , fujisawa y , yoshino k , et al : a proposal for a tnm staging system for extramammary paget disease : retrospective analysis of 301 patients with invasive primary tumors . 
vornicova o , hershkovitz d , yablonski - peretz t , et al : treatment of metastatic extramammary pagets disease associated with adnexal adenocarcinoma , with anti - her2 drugs based on genomic alteration erbb2 s310f . 
tchrakian n , flanagan l , harford j , et al : new asco / cap guideline recommendations for her2 testing increase the proportion of reflex in situ hybridization tests and of her2 positive breast cancers . 
ogawa t , nagashima y , wada h , et al : extramammary pagets disease : analysis of growth signal pathway from the human epidermal growth factor receptor 2 prote hum pathol 36 : 12731280 , 2005 16 . 
plaza ja , torres - cabala c , ivan d , et al : her - 2 / neu expression in extramammary paget disease : a clinicopathologic and immunohistochemistry study of 47 cases with and without underlying malignancy . 
villada g , farooq u , yu w , et al : extramammary paget disease of the vulva with underlying mammary - like lobular carcinoma : a case report and review of the literature . 
tanaka r , sasajima y , tsuda h , et al : concordance of the her2 protein and gene status between primary and corresponding lymph node metastatic sites of extramammary paget disease . 
tessier - cloutier b , asleh - aburaya k , shah v , et al : molecular subtyping of mammary - like adenocarcinoma of the vulva shows molecular similarity to breast carcinomas . 
barth p , dulaimi al - saleem e , edwards kw , et al : metastatic extramammary pagets disease of scrotum responds completely to single agent trastuzumab in a hemodialysis patient : case report , molecular profiling and brief review of the literature . 
arnould l , gelly m , penault - llorca f , et al : trastuzumab - based treatment of her2 - positive breast cancer : an antibody - dependent cellular cytotoxicity mechanism ? br j cancer 94 : 259 - 267 , 2006 31 . 
lewis phillips gd , li g , dugger dl , et al : targeting her2 - positive breast cancer with trastuzumab - dm1 , an antibody - cytotoxic drug conjugate . 
krop ie , kim sb , martin ag , et al : trastuzumab emtansine versus treatment of physicians choice in patients with previously treated her2 - positive metastatic breast cancer ( th3resa ) : final overall survival results from a randomised open - label phase 3 trial . 
thuss - patience pc , shah ma , ohtsu a , et al : trastuzumab emtansine versus taxane use for previously treated her2 - positive locally advanced or metastatic gastric or gastro - oesophageal junction adenocarcinoma ( gatsby ) : an international randomised , open - label , adaptive , phase 2 / 3 study . 
lousberg l , collignon j , jerusalem g : resistance to therapy in estrogen receptor positive and human epidermal growth factor 2 positive breast cancers : progress with latest therapeutic strategies . 
kaufman b , mackey jr , clemens mr , et al : trastuzumab plus anastrozole versus anastrozole alone for the treatment of postmenopausal women with human epidermal growth factor receptor 2 - positive , hormone receptor - positive metastatic breast cancer : results from the randomized phase iii tandem study . 
 braf mutations occur infrequently in ovarian cancer but suggest responsiveness to braf and mek inhibition introduction ovarian cancer ( oc ) is the leading cause of death from gynecologic malignancies in the developed world.1 although substantial efforts have been devoted to identifying potentially actionable oncogenic driver mutations in oc , only a few genes are frequently mutated , particularly in high - grade serous oc ( hgsoc ) , the most common histologic subtype . 
the cancer genome atlas reported p53 mutations in almost all primary hgsoc tumors ( 96% ) but a low prevalence of recurrent somatic mutations in additional genes including brca1 ( 12% ) , brca2 ( 11% ) , nf1 ( 4% ) , and cdk12 ( 3% )  . 
2 in the cosmic database , additional infrequent mutations such as kras ( 6% ) , pik3ca ( 2% ) , and braf ( 2% ) have been identified in hgsoc.3 we believe that some of these mutations , although rare , are important drivers in hgsoc . 
for example , mutations in braf lead to constitutive activation of downstream mek1 and mek2 and may regulate proliferation and survival in ovarian tumor cells as in many other cancers.4 the most commonly observed braf mutation , v600e , accounts for 90% of the braf mutations found in all patients with cancer.3 treatment with braf inhibitors such as vemurafenib or dabrafenib in patients with advanced brafv600 - mutated melanoma has shown objective tumor responses in approximately half of the patients.5 recent clinical studies have suggested that concurrent inhibition of the braf and mek kinases of the mitogen - activated protein kinase ( mapk ) pathway with trametinib or cobimetinib can decrease mapk - driven acquired resistance , resulting in greater efficacy and a decrease in the cutaneous toxicities observed from paradoxical mapk pathway activation with braf inhibitor monotherapy.6 here , we report a patient with recurrent hgsoc who had experienced treatment failure with an extensive number of prior treatment regimens and whose tumor was found to harbor a brafv600e mutation . 
of note , this patient responded extremely well to treatment with a braf inhibitor ( vemurafenib ) and subsequently to a braf inhibitor in combination with a mek inhibitor ( dabrafenib and trametinib )  . 
 to further underscore this treatment rationale , we studied the growth - inhibitory effects of the braf inhibitor vemurafenib and the mek inhibitor trametinib across a panel of 32 oc cell lines that were each characterized for mutational status of braf , kras , and nf1 ( fig 1 )  . 
 cell lines , assays , and sequencing methods have been described earlier.7 in brief , cells were plated into 24 - well tissue culture plates and grown with or without increasing concentrations of inhibitors . 
she developed recurrent disease to the left paracolic gutter region , which was not amenable to surgery but treated with carboplatin plus paclitaxel and , after further progression , by liposomal doxorubic subsequent treatments included topotecan , pemetrexed , gemcitabine plus carboplatin , maira p . 
preclinical activity of ( a ) vemurafenib and ( b ) trametinib across a large panel of 32 ovarian cancer ( oc ) cell lines characterized for braf , kras , and nf1 mutations . 
growth inhibition was calculated as a function of the number of generations inhibited in the presence of vemurafenib or trametinib versus the number of generations over the same time course in the absence of the drug as reported previously.7 in brief , the log of the fractional growth inhibition was plotted against the log of the drug concentration , and the half - maximal inhibitory concentration ( ic50 ) values were interpolated from the resulting linear regression curve fit . 
in the absence of a therapeutic standard of care , tumor tissue was submitted to foundation medicine for comprehensive genomic profiling revealing an activating brafv600e mutation as well as asxl1 ( k580fs * 2 ) and runx1t1 ( r520h ) mutations . 
cp , carboplatin and paclitaxel ; gp , carboplatin and gemcitabine ; pld , liposomal doxorubicin . 10 , 000 1 , 000 cg / cp topotecan pembrolizumab / epacadostat vemurafenib pemetrexed nanoparticle albumin - bound paclitaxel dabrafenib / trametinib 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 up to a 3 - log - fold difference in the half - maximal inhibitory concentration values , but were most pronounced in cell lines that harbored braf , kras , or nf1 mutations ( fig 1b )  . 
after providing informed consent , the patient was treated in accordance with the declaration of helsinki with the braf inhibitor vemurafenib at the us food and drug administrationapproved dose of 960 mg twice a day . 
despite the dose reduction , she had to stop treatment after 13 days as a result of a profound diffuse maculopapular skin rash , which significantly improved with systemic corticosteroid medication . 
despite the brevity of treatment , the patients ca - 125 levels decreased from 4 , 495 to 2 , 119 u / ml within 21 days of initiating therapy , and a ct scan of the abdomen and pelvis revealed a partial response ( pr ) 29 days after commencing vemurafenib therapy.3 while the patient remained off therapy awaiting insurance approval for dabrafenib plus trametinib , the ca - 125 level increased again from 1 , 394 to 2 , 345 u / ml with increasing symptoms of abdominal bloating and distension as well as moderate pelvic pact scans of the abdomen and pelvis done before starting new treatment showed renewed progression ( fig 3 )  . 
after starting combination therapy with dabrafenib ( 150 mg twice a day ) and trametinib ( 2 mg daily ) , the ca - 125 levels decreased from 2 , 345 to 12 u / ml ( normal range , < 35 u / ml ) within a 10 - week period . 
in the following weeks , the patient developed mild lower extremity edema and moderate hypertension , requiring a decrease in the doses of dabrafenib from 150 mg to 75 mg twice a day and of trametinib from 2 mg to 1 mg daily . 
after 10 months of combination therapy , the ca - 125 levels remained in the normal range , and updated ct scans demonstrated an ongoing pr with continued reduction in size of her retroperitoneal lymphadenopathy ( fig 2 )  . 
to identify additional patients who might benefit from braf / mek inhibition , we analyzed a subset of 2 , 983 ovarian tumors from the foundation medicine database of more than 125 , 000 clinical cases that had undergone hybrid capturebased next - generation sequencing . 
brafv600 mutations were found in 46 ( 1.5% ) of 2 , 983 serous ocs , in three ( 4.2% ) of 71 ovarian mucinous cancers , in one ( 3.6% ) of 28 ocs with neuroendocrine differentiation , in four ( 5% ) of 80 low - grade serous ocs , and in two ( 13.3% ) of 15 serous borderline tumors ( table 1 )  . discussion we present a patient diagnosed with recurrent hgsoc who had received an extensive number of prior chemotherapy regimens and whose tumor was found to harbor a brafv600e mutation . 
treatment with the braf inhibitor vemurafenib and subsequently the braf inhibitor dabrafenib and mek inhibitor trametinib led to a persistent radiographic pr with normalization of ca - 125 and marked symptomatic improvement . 
the rapid development and widespread availability of ngs provides the opportunity to select an appropriate targeted therapy.9 however , matching patients with the optimum therapy is often challenging , particularly as driver events are generally infrequent and comprehensive clinical response data are rarely available for the specific cancer type , especially with noncanonical variants . 
for example , a left periaortic lymph node is ( a ) initially seen measuring 11 14 mm at baseline and ( b ) subsequently decreased to 7 8 mm after brief treatment with vemurafenib . 
 ( c ) after holding vemurafenib , ct scans of the abdomen and pelvis showed renewed progression of the lymphadenopathy with the left periaortic node measuring 10 11 m ( d ) dabrafenib and trametinib treatment again led to a significant decrease in size of the abdominal and retroperitoneal lesions with a decrease in size of the left periaortic node to 4 6 m ( e ) after further combination therapy , the ca - 125 level remains in the normal range and updated ct scans demonstrate an ongoing partial response with continued reduction in size of the patients left periaortic lymph node now measuring 2 3 m ( f ) ca - 125 tumor marker levels are shown during treatment while on chemotherapy , followed by the braf inhibitor vemurafenib given as a single agent , followed by combination treatment with the braf inhibitor dabrafenib and the mek inhibitor trametinib . cancer type , basket trials have been initiated that categorize patients cancers on the basis of the sequencing of the tumor , rather than the organ of origin.10 nevertheless , individual case reports , such as the present description , may provide important additional evidence to support further table 1 . 
frequency of brafv600e mutations in 2 , 983 ovarian tumors brafv600positive tumors tumor type serous ovarian cancer ovarian mucinous cancer ovarian cancer with neuroendocrine differentiation low - grade serous ovarian cancer serous borderline tumors ( low malignant potential ) total no . 
similarly , mek inhibition with trametinib led to growth inhibition in es - 2 cells ; however , oc cell lines with kras , nf1 , and other braf mutations also seemed to be sensitive to mek inhibition , suggesting potentially a broader role of mek inhibitors in oc . 
 although it is clear that ngs - based technologies are already having an impact on patient care , for broad , widespread use to occur , several factors will need to be addressed in the coming years . 
in addition to expanding basket - type trials , we need further development of data - sharing consortia that are focused on generating an evidence base for precision cancer medicine by integrating robustly validated , broad - based genomic profiling data with clinical outcome data . 
konecny manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors stock and other ownership interests : personal genome diagnostics , ignyta consulting or advisory role : quintiles , janssen diagnostics , ignyta , personal genome diagnostics patents , royalties , other intellectual property : receive royalties for discoveries our group has made at johns hopkins that have been licensed by qiagen ; receive royalties for discoveries our group has made at johns hopkins that have been licensed by myriad ; receive royalties for discoveries our group has made at johns hopkins that have been licensed by personal genome diagnostics ; receive royalties for discoveries our group has made at johns hopkins that have been licensed by genzyme ; receive royalties for discoveries our group has made at johns hopkins that have been licensed by roche jennifer l . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author = center . dylan conklin no relationship to disclose dorothy hallberg no relationship to disclose dennis j . 
konecny stock and other ownership interests : foundation medicine speakers ' bureau : clovis oncology , astrazeneca , tesaro research funding : merck travel , accommodations , expenses : novartis affiliations maira p . 
kujak , rolling oaks radiology , thousand oaks , ca ; victor velculescu and dorothy hallberg , sidney kimmel comprehensive cancer center , johns hopkins university school of medicine , baltimore , md ; and julia elvin , foundation medicine , cambridge , ma . supported by the miriam and sheldon g . 
ihnen m , zu eulenburg c , kolarova t , et al : therapeutic potential of the poly ( adp - ribose ) polymerase inhibitor rucaparib for the treatment of sporadic human ovarian cancer . 
ross js , ali sm , wang k , et al : comprehensive genomic profiling of epithelial ovarian cancer by next generation sequencing - based diagnostic assay reveals new routes to targeted therapies . 
the precise impact of these panels on clinical decision making is not well understood . methods here , we report our institutional experience with a targeted 40 - gene panel ( myeloseq ) that is used to generate a report for both genetic variants and variant allele frequencies for the treating physician ( the limit of mutation detection is approximately one aml cell in 50 )  . results in total , 346 sequencing reports were generated for 325 patients with suspected hematologic malignancies over an 8 - month period ( august 2018 to april 2019 )  . 
to determine the inuence of genomic data on clinical care for patients with acute myeloid leukemia ( aml ) , we analyzed 122 consecutive reports from 109 patients diagnosed with aml and surveyed the treating physicians with a standardized questionnaire . 
inuences were often nuanced and extended beyond identifying actionable genetic variants with us food and drug administrationapproved drugs . conclusion this study provides insights into how physicians are currently using multigene panels capable of detecting relatively rare aml cells . 
the most inuential way to integrate these tools into clinical practice will be to perform prospective clinical trials that assess patient outcomes in response to genomically driven interventions . jco precis oncol 5 : 191 - 203 . 
some quires variant single - gene tests and two multitarget panels are currently us food and drug administration ( fda ) approved and have associated companion diagnostics that designate a specic cancer subtype and indication.2 however , the vast majority of variant identication and annotation is performed by laboratory developed tests ( ldts ) , 3 for which results are provided as a clinical report . 
these reports typically require a certain level of genomic literacy to understand and implement.4 , 5 lack of report standardization , inadequate genomic training , 6 , 7 limited infrastructure to support oncologists , 1 , 8 and limited resourcesfor example , low reimbursement rates and lack of access to clinical trialsare thought to hinder the impact of sequencing data on clinical care.9 , 10 the result is a discrepancy between the identication of clinically actionable variants11 and implementation of change in treatment decisions.1 , 12 acute myeloid leukemia ( aml ) sequencing data are particularly complex because of the genetic and clonal heterogeneity of aml cases . 
 barnell et al context key objective whereas physicians have increased the use of targeted sequencing panels to inform treatment decisions for patients with hematologic malignancies , the impact and utility of sequencing data on clinical decision making is not well understood . 
through standardized surveys , this study captured the nuanced impact of sequencing data on clinical decision making for patients with acute myeloid leukemia . knowledge generated physicians self - reported that 50 of 114 sequencing reports ( 44% ) inuenced clinical care decisions with 56 total inuences reported , 38 of which were related to therapeutic options , 10 related to risk stratication in rst clinical remission , and eight involving measuring persistent molecular disease . 
in 11% of all patients , the panel was ordered at multiple timepoints during the disease course for persistent molecular disease monitoring . relevance this study demonstrates that physicians are integrating sequencing data obtained at various points in the acute myeloid leukemia disease course into clinical decision making , mostly to determine therapy choices , stratify relapse risk , and measure persistent disease . signicance may depend on cooperating covariants in multiple other genes.14 for example , european leukemianet / national comprehensive cancer network guidelines specically mention six genes / mutations in their risk stratication guidelines , all of which must be evaluated in parallel for risk stratication.15 , 16 in addition , whereas pathogenic variants within three genesflt3 , idh2 , and idh1have been used as predictive markers for fda - approved targeted therapeutics , 17 - 19 ongoing clinical trials have demonstrated predictive utility for other pathogenic variants that do not yet have approved companion diagnostics.20 next - generation sequencing ( ngs ) data can provide information beyond therapeutic sensitivity / resistance that is not currently captured by fda - approved diagnostics . specically , quantication of the variant allele frequency ( vaf ) of tumor - associated variants has been used to detect persistent molecular disease ( pmd ) after therapies are administered , and predict both relapse risk21 and survival outcomes.22 - 24 although multipanel ldt diagnostics are being used on patients with aml to supplement treatment decisions , the utility of these reports is not well described . this study sought to better understand the extent to which genomic reports inuence clinical decision making for patients with aml . 
this paper describes physician experience with a targeted gene panel ( myeloseq ) that terrogates 40 recurrently mutated genes or gene hotspots and returns gene variant and vaf data to the treating physician . 
we determined usage patterns over an 8 - month period and surveyed treating physicians regarding how they used the panel results in clinical care . methods study design and patient eligibility this study was conducted at washington university school of medicine ( human research protection ofce #201801112 )  . 
the panel was a targeted sequencing assay that evaluated 40 genes and gene hotspots that are recurrently mutated in myeloid malignancies ( data supplement ) .25 for each patient who was found to have a denitive diagnosis of aml , a survey was sent to the treating physician . 
patients with acute promyelocytic leukemia ( m3 aml subtype ) were excluded.26 the treating physician was asked to complete a ninequestion survey to determine how the physician used the panel to inform treatment decisions ( data supplement )  . panel design and processing the panel uses an amplicon capture - based enrichment with unique molecular identiers for ultra - high variant sensitivity that targets an approximately 98 - kb space ( haloplex target enrichment system ; agilent technologies , santa clara , ca )  . 
this included evaluating bone marrow morphology , standard panels of ow cytometric markers , and a panel of routine cytogenetic and molecular marker studies , including uorescence in situ hybridization probes . target enrichment , variant calling strategies , and variant annotation methods are described in the data supplement . the median number of failed genes across all reports was two ( range , 0 to 24 genes )  . 
wt1 ( n = 211 cases ) , cux1 ( n = 197 cases ) , and cebpa ( n = 111 cases ) were genes that most frequently failed coverage requirements . 
if physicians did not respond to the initial contact , reminders were provided to increase the response rate . ethics , consent , permissions , and consent to publish this study was conducted at washington university school of medicine and was approved by the washington university human research protection ofce ( #201801112 )  . 
summary of patient , report , and physician characteristics characteristic ( n = 109 ) value median time from accessioning to report sign out , daysa 12 ( 4 - 36 ) patient ( n = 109 ) median age , years male female report ( n = 122 ) physician ( n = 14 ) mean time in practice , yearsb academic rank professor associate professor assistant professor degree type md / phd md / mba male female there were 124 reports from patients with aml . 
the reports from patients with aml , after removing patients with apl subtype ( n = 122 samples ) , were derived from 109 unique patients who were under the care of 14 physicians ( table 1 )  . 
only 13 samples had no observed variants , eight of which were acquired from patients who were determined to be in clinical remission using bone marrow biopsy . reports were ordered at various points in the disease course . 
the next most common timepoint was during disease assessment ( n = 28 of 122 ; [ 22.9% ] ) , followed by potential relapse ( n = 19 of 122 [ 15.6% ] ; fig 3a )  . 
for reports ordered during disease response assessments , seven were ordered post - induction , six were ordered after allogeneic hematopoietic cell transplant ( allohct ) , ve were ordered during ongoing treatment with a hypomethylating agent , two were ordered after consolidation with high - dose ara - c , and seven were ordered during or after salvage therapy ( fig 3b )  . 
on the basis of these data , the sequencing report was a factor in determining therapy decisions in 50 ( 43.8% ) of 114 cases ( fig 4a )  . 
the patient accepted the treatment plan 93% of the time ( n = 38 of 41 cases )  . predictive inuences in 35 of 50 reports , physicians indicated that they recommended at least one therapy on the basis of the variants identied by the sequencing panel ( fig 4b )  . 
on the basis of results35 reports with 38 corresequencing panel sponding inuencesphysicians reported recommending a hypomethylating agent in 14 patients for a tp5320 or tet228 , 29 variant , kinase inhibitors ( midostaurin or gilteritinib ) in 12 patients with a flt3 variant30 and one patient with a kit variant , 31 and idh1 / idh2 inhibitors were recommended for eight patients.32 there were three patients for whom a clinical trial was recommended on the basis of observed variants or persistent disease . 65 ( 23 - 90 ) 58 ( 53.2 ) 51 ( 46.8 ) 22 ( 8 - 44 ) 5 ( 36 ) 4 ( 29 ) 5 ( 36 ) 8 ( 57 ) 5 ( 36 ) 1 ( 7 ) 11 ( 79 ) 3 ( 21 ) note . 
of cases ( n = 122 ) flt3 internal tandem duplication splice acceptor variant frameshift variant missense variant flt3 tyrosine kinase domain splice donor variant inframe insertion negative no flt3 variant flt3 status stop gained variant type inframe deletion percentage of cases fig 2 . 
in 16 of 29 eligible cases ( two patients refused treatment ) , physicians reported the following inuences : 12 used the ngs panel to prescribe an flt3 inhibitor and four inuences were independent of flt3 status . 
in three of these four cases , patients had an flt3 inhibitor prescribed based on a previously known result from a different genetic test . there were 13 cases in which the patient had an flt3 variant , but the physician reported no inuence . 
of these 13 cases , three patients died before treatment initiation and eight had an flt3 inhibitor prescribed based on a previously known result from a different genetic test ( data supplement )  . risk stratication for transplant in first clinical remission physicians identied 10 reports that inuenced patient risk assessment and allohct recommendation ( fig 4b )  . specically , allohct was not recommended in rst complete remission in six patients , based on the presence of bialleleic cebpa variants ( n = 2 ) , kit variant status ( n = 1 ) , lack of high - risk variants ( n = 1 ) , or differential clearance of co - occurring variants after induction therapy ( n = 1 )  . 
for the latter patient , three variants were detected at diagnosis ( dnmt3a [ vaf = 42% ] , npm1 [ vaf = 38% ] , and rad21 [ vaf = 43% ] )  . 
these recommendations were based on tp53 variant status ( n = 2 ) , dnmt3a variant status ( n = 1 ) , and co - occurrence of multiple variants ( dnmt3a , npm1 , and flt3 internal tandem duplication ; n = 1 ) .14 assessment for pmd there were eight cases in which the sequencing panel was used to assess for pmd ( fig 4b )  . 
in ve cases , the physician indicated that the reports conrmed relapse / progression of aml , including one case of bone marrow negative , extramedullary relapse in the esophagus ( fig 5d )  . there were three patients who were being assessed for pmd while in an apparent clinical remission . 
two of the three patients did not show persistent aml via pmd assessment . the third patient showed three residual variants at low vaf ( cebpa [ vaf = 4% ] , smc1a [ vaf = 4% ] , and wt1 [ vaf = 3% ] ) , suggesting persistent disease . patients with no change in treatment regimens in response to sequencing panels there were 64 cases for which the physician noted no change in therapeutic plan on the basis of the report . 
the total number of eligible surveys completed by each physician and the total number of cases where the physician changed his or her plan is shown in figure 4c . the sequencing panel reports the vaf and thus can detect possible inherited variants . 
one patient was found to have a gata2 variant observed at a vaf of 52% , indicative of a possible germline variant , which prompted an action by the treating physician . 
a subsequent skin biopsy determined that the patient did not have a germline predisposition for disease . multiple sequencing panels for longitudinal disease monitoring and response therapy , there were 12 patients for whom the treating physician ordered multiple reporters during the disease course21 ( fig 5 and data supplement )  . 
after induction , despite some variants demonthe idh2 vaf of strating response to initial 39% ( 46% at diagnosis ) resulted in initiation of enasidenib . figure 5b shows a patient for whom the physician obtained three sequencing panels for longitudinal pmd monitoring . the rst report was ordered at relapse post - transplant and showed ve variants . 
a month later , the third report showed the re - emergence of three variants despite no excess blasts on the bone marrow biopsy , which resulted in a second allohct . 
figure 5c shows how a physician used sequential reports to observe the patients molecular response to a donor lymphocyte infusion and gilteritinib . figure 5d shows how the reports diagnosed extramedullary relapse in a patient with negative bone marrow biopsy ndings . 
the extramedullary disease showed the presence of some original variants ( nf1 and ezh2 ) , loss of other variants ( dnmt3a ) , and three novel variants ( cux1 , ptpn11 , and wt1 ) , some of which had clinical implications.34 , 35 additional cases with multiple reports are provided in the data supplement . reimbursement and clinical care access one of the biggest challenges for the widespread clinical implementation of ngs - based diagnostics has been inconsistent reimbursement from payers . 
coverage and reimbursement for myeloseq was divided into inpatient testing ( 26% ) and outpatient testing ( 74% ) , and 90% of outpatient testing was reimbursed across all payers . 
of these 124 cases , four were excluded ( acute promyelocytic leukemia [ apl ] subtype [ n = 2 ] ; no survey returned [ n = 2 ] )  . 
in 64 cases , physicians reported that the results did not inform decision making . ( c ) there were 14 physicians who contributed at least one survey to this study . was no specic pattern in terms of the diagnosis of rejected claims ( data supplement )  . discussion this study reports our institutions experience using a targeted capture sequencing panel to inform clinical care in patients with aml . 
 a addition of targeted therapy after lack of response to induction therapy persistent molecular disease monitoring barnell et al cebpa k302e smc1a r96h wt1 a38gfs * 69 wt1 r370pfs * 15 ezh2 k400del nf1 t2507i dnmt3a w297s dnmt3a f755 * smc1a / cepba ( 4% ) wt1 ( 3% ) wt1 ( 0% ) ( itd ) cux1 g1328s ptpn11 a72v wt1 r370pfs * 15 ezh2 k400del nf1 t2507i flt3 status enasidenib treatment ( itd ) gilteritinib new diagnosis of aml ( d0 diagnosis ) 22% blasts ( bmbx ) post 7 + 3 induction ( d49 post - diagnosis ) 3% blasts ( bmbx ) relapse ( d360 sct ) 44% blasts ( bmbx ) remission ( d426 sct ) < 1% blasts ( bmbx ) disease assessment ( d475 sct ) 0% blasts ( bmbx ) persistent molecular disease monitoring post - dli molecular diagnosis of extramedullary relapse bone marrow esophagus asxl1 g646wfs * 12 idh2 r140q runx1 l112cfs * 10 srsf2 p95h stag2 r146 * flt3 status treatment bcor t1129pfs * 30 bcorl1 q459 * dnmt3a r882h tet2 s1448n u2af1 s34f flt3 status flt3 status treatment treatment ( itd ) ( itd ) gilteritinib flt3 status treatment relapsed aml ( 1 month post - dli ) 21% blasts ( bmbx ) relapsed aml ( 3 months post - dli ) 6% blasts ( bmbx ) bone marrow ( ~ 10 yr sct ) 1% blasts ( bmbx ) esophageal tissue ( ~10 yr sct ) 1% blasts ( bmbx ) fig 5 . 
 ( b ) physician reported that the observation of persistent molecular disease at 475 days ( d ) poststem - cell transplantation ( sct ) indicated the need for a second allogeneic stem - cell transplantation despite clinical remission ( 0% blasts on bone marrow biopsy [ bmbx ] )  . 
 ( d ) extramedullary relapse was conrmed by comparing sequencing results of the original tumor and the periesophageal lesion . diagnosis , during disease assessment , and / or at multiple timepoints during the disease course . this study suggests that physicians are increasingly relying on the evaluation of multiple co - occurring variants in parallel ; therefore , simultaneous analysis of these genes is necessary . 
furthermore , pmd monitoring after cytotoxic induction therapy and transplantation21 , 36 has prognostic value ; therefore , the vafs reported on the gene panel studied here were being used to supplement existing standard - of - care mechanisms for residual disease assessment . 
although it is clear that physicians are using this test to measure and assess pmd , this test was not designed for measurable residual disease testing , and the limit of detection is not within the european leukemianet guidelines for measurable residual disease assessment.15 , 37 the prognostic information provided by the detection of persistent variants , the optimal depth of clearance , and the required limit of detection are all areas of active investigation requiring additional study in prospective clinical trials . there were several limitations associated with this study . reports were evaluated over an 8 - month period at a single academic institution among physicians with experience interpreting genomic results . 
 targeted panel inuences decision making for patients with aml were returned contemporaneously and tests were ordered at different timepoints during the disease course ( fig 3 )  . whereas the proportion of cases with inuences was not signicantly different among timepoints ( data supplement ) , inuence types might be different between timepoints and should be evaluated further to understand the impact of disease timepoint on treatment decisions . 
finally , each physician had a varied number of patients ( range , one to 18 patients ) with a large range in the number of cases reported to be inuential ( 0% to 90% )  . 
larger longitudinal , multi - institutional studies are needed to better understand interphysician variability in incorporating genomic data into clinical decision making . this study evaluated reports from the rst 8 months after launch of the diagnostic . 
the physicians were also experienced in interpreting genomic data , having participated in large genomic trials , 20 , 21 , 38 - 41 with many of them being clinical investigators themselves . 
given that the inuence of genomic data varies on the basis of physicians experience with genomic data , 6 , 7 the results presented here might not extrapolate to institutions that are unfamiliar with ordering and interpreting sequencing - based diagnostics . the sequencing panel has several additional limitations . the use of a tumor - only panelthat is , lack of a germline or reference sequenceprevented variants from normal being denitively called somatic . 
in future studies , it will be important to ascertain from physicians if any aspect of the test or the molecular report is unclear , biased , or difcult to interpret . there are several outstanding questions in the eld that could be addressed using a test similar to the one described in this paper . 
the prognostic value of residual dnmt3a , tet2 , and asxl1 mutations after treatment is incompletely understood , 21 , 36 , 42 and the difference between a residual leukemic clone versus rising clonal hematopoiesis43 needs additional study . 
our group is currently conducting a prospective clinical ( clinicaltrials.gov identier : nct02178241 ) to address some of these outstanding questions . trial in summary , the results from this study suggest that variants and associated vafs are being integrated into clinical care to inuence therapy choices , stratify relapse risk , and measure persistent disease . 
barnell employment : geneoscopy stock and other ownership interests : geneoscopy patents , royalties , other intellectual property : inventor of intellectual property in start - up company ( geneoscopy ) travel , accommodations , expenses : geneoscopy kenneth f . 
newcomer employment : geneoscopy ( i ) stock and other ownership interests : geneoscopy ( i ) patents , royalties , other intellectual property : inventor of intellectual property owned by geneoscopy ( i ) zachary l . 
oh consulting or advisory role : incyte , novartis , blueprint medicines , celgene , kartos , cti biopharma corp , pharmaessentia , disc medicine , constellation pharmaceuticals research funding : incyte ( inst ) , gilead sciences ( inst ) , cti biopharma corp ( inst ) , kartos ( inst ) , celgene ( inst ) , sierra oncology ( inst ) , blueprint medicines ( inst ) , constellation pharmaceuticals ( inst ) john s . 
stockerl - goldstein stock and other ownership interests : abbott laboratories , abbvie consulting or advisory role : celgene research funding : glaxosmithkline , takeda , biolinerx , janssen other relationship : cellerant ravi vij consulting or advisory role : bristol myers squibb , celgene , janssen , sano , karyopharm therapeutics , takeda , genentech , abbvie , oncopeptides research funding : takeda , celgene , bristol myers squibb travel , accommodations , expenses : celgene , bristol myers squibb , sano , janssen , davaoncology , karyopharm therapeutics , amgen , takeda , abbvie amanda f . 
abboud stock and other ownership interests : abbvie ( i ) , abbott laboratories ( i ) , gilead sciences ( i ) , bristol myers squibb ( i ) , johnson & johnson ( i ) honoraria : cardinal health , dova pharmaceuticals , nkartatherapeutics , archer biosciences research funding : actinium pharmaceuticals , selvita ( inst ) , allovir ( inst ) , forty - seven ( inst ) armin ghobadi honoraria : kite pharma consulting or advisory role : kite pharma , celgene , amgen , wugen speakers bureau : kite pharma , amgen geoffrey l . 
schroeder consulting or advisory role : astellas pharma , dova pharmaceuticals , flatiron health , glaxosmithkline , incyte , partners therapeutics , partners therapeutics , pzer speakers bureau : abbvie , merck , takeda john f . 
dipersio stock and other ownership interests : magenta therapeutics , wugen honoraria : incyte consulting or advisory role : cellworks , rivervest , magenta therapeutics , incyte research funding : amphivena therapeutics ( inst ) , macrogenics ( inst ) , incyte ( inst ) , wugen ( inst ) , biolinerx ( inst ) , maxcyte ( inst ) , bigelow aerospace ( inst ) patents , royalties , other intellectual property : patents : cd7 and cd2 knockout for cart to cd7 and cdl ; duvelisib for treatment of cytokine release syndrome ; nt - 17 to enhance cart survival ; novel wu mobilizing compounds ( inst ) ; selection of impdh mutant stem cells ; ifn , upregulate mhcii for relapsed aml ; dextran - based molecules to detect car - t cells ; combining integrin inhibitor with chemokine binders , 016131 ; jak and calcineurin inhibition , solid organ transplant ; vla4 , gro - b ; triple combinationcxcr2 , vla - 4 , gro - b ; targeting ifnr / cscr3 in gvhd ; wu / slu compounds vla4 and cxcr2 ( inst ) travel , accommodations , expenses : incyte , macrogenics , magenta therapeutics mary c . 
duncavage stock and other ownership interests : p&v licensing consulting or advisory role : cofactor genomics , bristol myers squibb , eli lilly open payments link : 317379 timothy j . 
additional thanks are also given to the siteman cancer center and the barnesjewish hospital foundation . 11 : 11 , 2016 368 : 2059 - 2074 , 2013 129 : 424 - 447 , 2017 netw 15 : 926 - 957 , 2017 2017 314 : 811 - 822 , 2015 references ray t : survey of precision oncology programs nds agreement on testing , divergence in care delivery . 
precision oncology news , july 19 , 2019 : precisiononcologynews.com / cancer / survey - precision - oncology - programs - nds - agreement - testing - divergence - care - delivery stone rm , mandrekar sj , sanford bl , et al : midostaurin plus chemotherapy for acute myeloid leukemia with a flt3 mutation . 
n engl j med 377 : 454 - 464 , 2017 kim as , bartley an , bridge ja , et al : comparison of laboratory - developed tests and fda - approved assays for braf , egfr , and kras testing . 
jama oncol 4 : 838 - 841 , 2018 li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
bmc med inform decis mak 18 : 107 , 2018 chow - white p , ha d , laskin j : knowledge , attitudes , and values among physicians working with clinical genomics : a survey of medical oncologists . 
j genet couns 27 : 187 - 196 , 2018 gornick mc , ryan ka , scherer am , et al : interpretations of the term actionable when discussing genetic test results : what you mean is not what i heard . j genet couns 28 : 334 - 342 , 2019 statz cm , patterson se , mockus sm : barriers preventing the adoption of comprehensive cancer genomic proling in the clinic . 
au ch , wa a , ho dn , et al : clinical evaluation of panel testing by next - generation sequencing ( ngs ) for gene mutations in myeloid neoplasms . 
marcucci g , mr ozek k , ruppert as , et al : abnormal cytogenetics at date of morphologic complete remission predicts short overall and disease - free survival , and higher relapse rate in adult acute myeloid leukemia : results from cancer and leukemia group b study 8461 . 
chen y , cortes j , estrov z , et al : persistence of cytogenetic abnormalities at complete remission after induction in patients with acute myeloid leukemia : prognostic signicance and the potential role of allogeneic stem - cell transplantation . 
j clin oncol 29 : 2507 - 2513 , 2011 ivey a , hills rk , simpson ma , et al : assessment of minimal residual disease in standard - risk aml . 
loken mr , alonzo ta , pardo l , et al : residual disease detected by multidimensional ow cytometry signies high relapse risk in patients with de novo acute myeloid leukemia : a report from childrens oncology group . 
hou h - a , chou wc , lin li , et al : characterization of acute myeloid leukemia with ptpn11 mutation : the mutation is closely associated with npm1 mutation but 35 . 
gaidzik vi , schlenk rf , moschny s , et al : prognostic impact of wt1 mutations in cytogenetically normal acute myeloid leukemia : a study of the germaninversely related to flt3 / itd . 
blood 113 : 4505 - 4511 , 2009 jongen - lavrencic m , grob t , hanekamp d , et al : molecular minimal residual disease in acute myeloid leukemia . 
schuurhuis gj , heuser m , freeman s , et al : minimal / measurable residual disease in aml : a consensus document from the european leukemianet mrd working party . 
clinical diincluding disease status , was dened by board - certied agnosis , hematopathologists . the panel uses an amplicon capture - based enrichment with unique molecular identiers for ultra - high variant sensitivity that targets an approximately 98 - kg base pair space ( haloplex target enrichment system ; agilent technologies , santa clara , ca )  . 
the panel is generally ordered for one of the following conditions : aml , mds , cytopenia , clonal cytopenia of undetermined signicance , clonal hematopoiesis of indeterminate signicance , myeloproliferative neoplasm , or other myeloid disorders . 
this included evaluating bone marrow morphology , standard panels of ow cytometric markers , and a panel of routine cytogenetic and molecular marker studies , including uorescence in situ hybridization probes . target enrichment for the panel used a commercially available , targeted , next - generation sequencing approach ( haloplexhs ; agilent technologies )  . 
preparation consisted of enzymatic fragmentation ; strand - specic ligation of sequencing primers , sample indexes , 10 - bp degenerate molecular barcodes , and biotin tagging of single dna molecules ; rapid liquid - phase enrichment of target loci using paramagnetic streptavidin - coated beads ; and on - bead polymerase chain reaction amplication . 
sequencing was performed using the illumina miniseq sequencing platform ( target depth = 50 coverage )  . variant calling for the panel was performed using a computational pipeline that employs custom - built tools created at washington university . 
overall reads were required to exceed 1 , 000 , 000 total reads with more than 98% aligned . during this process , unique molecular identier sequences are added to the binary alignment map le . 
once aligned , a custom java - based tool collapsed reads into read families using customized gatk software ( mckenna a , et al : genome res 20 : 1297 - 1303 , 2010 )  . 
collapsed binary alignment maps were used with standard variant calling tools , including varscan2 ( koboldt dc , et al : genome res 22 : 568 - 576 , 2012 ) , platypus ( rimmer a , et al : nat genet 46 : 912 - 918 , 2014 ) , and pindel ( ye k , et al : bioinformatics 25 : 2865 - 2871 , 2009 )  . 
these programs detected single - nucleotide substitutions , insertions or deletions ( indels ) up to 10 bp , and flt3 internal tandem duplication insertions between 21 and 108 bp . 
initial variant annotation was performed using the variant effect predictor tool ( mclaren w , et al : genome biol 17 ( 1 ) : 122 , 2016 )  . 
annotation was format le with variants augmented using a custom variant call identied in the aml the cancer genome atlas data set as well as variants observed in three published sequencing reports that evaluated patients with mds ( haferlach t , et al : leukemia 28 : 241 - 247 , 2014 ; papaemmanuil e , et al : blood 122 : 3616 - 3627 , quiz 3699 , 2013 ; walter mj , et al : leukemia 27 : 1275 - 1282 , 2013 )  . 
variant identication was subject to the following thresholds and cutoffs for reporting purposes : variants must be nonsynonymous , variants must have 2% minimum variant allele frequency and ve variant reads with support on each strand ( flt3 internal tandem duplication alleles require one read on each strand ) , amplicons must have at least ve reads assigned to it during consensus binary alignment map formation , and potential somatic variants must have 0.1% maximum population allele frequency ( max_af across all populations ; 1000 genomes [ v.phase3 ; 1000 genomes project consortium , et al : nature 526 : 6874 , 2015 ] and gnomad database [ v.170228 ; karczewski kj , et al : nature 581 : 434 - 443 , 2020 ] ) or presence in a custom mds / aml variant database . tier 1 ( variants with strong clinical signicance ) and tier 2 variants ( variants with potential clinical signicance ) , four ltered variants ( ie , variant allele frequency , 2% ) , and variants of unknown signicance were manually reviewed for potential clinical relevance . 
 sunitinib in the treatment of thymoma and associated autoimmune neutropenia introduction autoimmune disorders such as myasthenia gravis or pure red cell aplasia ( prca ) are frequent in patients with thymoma . 
thymoma - associated neutropenias , however , are rare ; to the best of our knowledge , only 17 patients have been described.1 - 16 for these patients , the diagnosis of neutropenia occurred simultaneously with that of thymoma or , less often , during the disease course , 2 , 4 - 8 , 10 - 16 and was frequently associated with myasthenia gravis , hypogammaglobulinemia , or anemia.2 - 12 , 14 - 16 granulopoiesis has been described as being completely or almost completely absent or as being arrested at the promyelocyte stage.2 - 7 , 9 , 10 , 12 - 16 bone marrow ( bm ) lymphocytosis is frequent and predominantly the result of t - cell infiltration.2 , 6 , 7 , 10 , 13 in one patient , a monoclonal rearrangement of the t - cell receptor ( tcr ) beta chain was found.8 thymoma - associated neutropenia is likely of autoimmune etiology ( ie , autoimmune neutropenia [ ain ] )  . 
antinuclear antibodies were present4 , 10 - 12 , 14 or not6 , 12 , 15 ; antineutrophil cytoplasmic antibodies were positive in one article , 11 with a decrease in titer upon neutrophil recovery . 
antigranulocyte antibodies were not detected5 , 9 , 12 or present10 ; in the latter case , the antibodies did not react with common human neutrophil antigens.10 studies consistently showed decreased granulocyte - macrophage colony formation in the bm of patients or controls in the presence of patients serum , 5 - 7 , 9 , 10 , 12 whereas formation was not affected by control sera or patients lymphocytes or thymoma cells.5 , 7 , 10 the inhibitory activity of the patients serum was present in the immunoglobulin g fraction.5 , 9 , 10 the treatment of thymoma - associated ain resembles that of prca17 and comprises the use of immunosuppressants ( eg , corticosteroids , 5 , 7 - 13 cyclosporine , 13 - 15 azathioprine , 9 cyclophosphamide , 5 , 10 , 12 mycophenolate , 14 or alemtuzumab14 ) , intravenous immunoglobulins , granulocyte colony - stimulating factor , plasmapheresis , and / or thymectomy.5 , 7 , 9 - 15 neutrophil response varied widely among the patients described . the tyrosine kinase inhibitor sunitinib has recently been shown to be effective against thymic carcinoma and , less frequently , type b thymoma in a phase ii trial and in retrospective analyses.18 , 19 sunitinib inhibits the receptors for the plateletderived growth factor receptor , vascular endothelial growth factor receptor ( vegfr ) , and stem cell factor ( kit ) , as well as other kinases . 
the patient provided written informed consent for the research use of the clinical data and biomaterial in accordance with the declaration of helsinki , as approved by the local ethics committee ( university of freiburg )  . the patient , a 33 - year - old man , was diagnosed with thymoma b2 in masaoka stage iva ( table 1 )  . he received three courses of cisplatin , doxorubicin , and cyclophosphamide ( pac ) chemotherapy followed by total thymectomy and resection of adjacent structures , leaving only microscopic tumor residues . 
shortly thereafter , computed tomography scans revealed new lesions in the right cardiophrenic angle and mediastinuthe patient then agreed to receive radiotherapy . radiotherapy was interrupted after 20 gy because of neutropenia and anemia . 
the neutrophils spontaneously rapidly recovered , but the anemia persisted until corticosteroid therapy was started . after recovery of the blood counts and stopping corticosteroids , and in the light of new lesions , radiotherapy was reinitiated but again stopped heiko becker konrad auman rainer claus nikolas von bubnoff ulrich j . 
diagnosed with thymoma b2 in masaoka stage iva . biopsy negative for variants ( non - synonymous and allele frequency > 10% ) in braf exon 15 ; egfr exons 18 , 19 , 20 , 21 ; kras exons 2 , 3 , 4 ; kit exons 9 , 11 , 13 , 17 ; nras exons 2 , 3 ; pdgfra exon 18 ; pi3kca exons 9 , 20 . 
bold font indicates days being on sunitinib treatment ( v being off treatment )  . abbreviations : gat , granulocyte agglutination test ; gift , granulocyte immunofluorescence test ; nd , not determined . * the interval between the end of cycle 2 and the start of cycle 3 was prolonged by 4 weeks because of patients personal reasons ; during this interval , granulocyte colony - stimulating factor was prophylactically applied once per week . computed tomography scan : progressive disease ; discontinuation of sunitinib treatment after 20 gy because of neutropenia , this time not coinciding with anemia . during the following treatment break , the blood counts completely recovered , but the thymoma lesions slowly progressed , which was associated with increased pain and deterioration of the patients general condition . 
at the same time , the patient developed a severe neutropenia ; the lowest wbc count at presentation was 1.290 3 109 / l with bands accounting for 8% , segmented cells 12% , basophils 2% , monocytes 15% , and lymphocytes 62% . 
immunoglobulin g was decreased ( 420 mg / dl ) ; c - reactive protein and c3d levels were increased , and procalcitonin was repeatedly normal . the patient had not received any thymoma - specific systemic therapy or radiotherapy for approximately fig 1 . 
the patient had taken valproic acid for epilepsy since adolescence . bm examination showed hypoplasia and a marked left shift of the granulopoiesis with almost complete absence of mature granulocytes ( fig 1 ) , which was associated with hyperplasia of the erythropoiesis and megakaryopoiesis , and an interstitial t - cell infiltration ( cd1a , predominantly terminal deoxynucleotidyl transferasenegative )  . in the blood , the cd4 : cd8 and tcr alpha / beta : gamma / delta cell ratios were slightly decreased ( table 2 )  . 
although a low cd4 : cd8 ratio as a result of the relative increase of cd8 + cells has been inconsistently described in patients with thymoma , 24 , 25 our patient had normal cd8 + cell counts . 
no clonal tcr gamma rearrangement was detected . in the search for granulocyte autoantibodies , we performed granulocyte agglutination ( gat ) and granulocyte immunofluorescence tests ( gifts ; table 1 )  . 
in enzyme immunoassays , no reactions were observed against hla class i antigens or human neutrophil antigens cd16b , cd11a , cd11b / cd18 , and cd177 . the neutropenia repeatedly displayed prompt but short - lasting responses to granulocyte colony - stimulating factor application . 
this decision was based on the efficacy of sunitinib against thymoma , 18 , 19 our reluctance to apply chemotherapy during neutropenia , and consideration of the immunomodulatory effect of sunitinib.21 - 23 we decided to use sunitinib at a dose of 25 mg / day because adverse effects were common at higher doses in patients with thymoma18 and in 6 - week cycles ( 4 weeks of treatment followed by 2 weeks without treatment )  . sunitinib treatment was associated with a rapid increase in neutrophil counts and decrease in inflammation parameters and granulocyte antibodies ( table 1 )  . 
moreover , the computed tomography scan showed stable disease . because adverse effects had been minimal during cycle 1 , we increased the dose to 37.5 mg / day in cycle 2 , which resulted in mucositis , sinus arrhythmia , and decreased left ventricular contractility , all of which improved during treatment break . 
from cycle 3 on , we kept the sunitinib dose at 25 mg / day and used an alternative application schedule ( ie , 3 - week cycles consisting of 2 weeks of treatment and 1 week without treatment , in accordance with a current trial [ nct01621568 ; sunitinib for advanced thymus cancer following earlier treatment ] )  . 
subsequent treatments included paclitaxel and gemcitabine . at the last follow - up , approximately 4 months after sunitinib was stopped , the patient remained without neutropenia and no granulocyte antibodies were detected . the rationale for the initial trials of sunitinib in patients with thymoma was the expression and mutation profile of distinct signaling molecules . in retrospective analyses of the thymoma biopsy from the diagnosis , immunohistochemistry showed weak expression of vegfr1 and vegfr2 and no expression of kit . 
in miseq ( illumina , san diego , ca ) - based sequencing of thymoma cells microdissected on the basis of their morphology , no variants were identified in the genes analyzed ( table 1 )  . sunitinib alters the ctla4 expression on cd8 + t cells and pd1 expression on regulatory t cells in patients with thymoma , and low ctla4 and decrease of pd1 are associated with shorter survival among patients with thymoma who are treated with sunitinib.18 although we have no data on pd1 or ctla4 , immunohistochemistry showed moderate to high pd - l1 expression on the thymoma cells . discussion the patient was diagnosed with thymoma b2 in masaoka stage iva ; he developed prca under radiotherapy , which resolved with corticosteroid treatment , and ain under progression of the thymoma , which resolved with sunitinib treatment . although we cannot exclude with certainty another cause for the ain besides the thymoma , the causative relationship is supported by the simultaneous occurrence of the ain and the progression of the thymoma and by the previous reports describing the development of ain in patients with thymoma.1 - 16 considering that the etiology of thymomaassociated ain is a matter of debate , we here demonstrate a clear association between neutrophil counts and the detection of autoantibodies against granulocytes that were present at diagnosis and disappeared under treatment . one may speculate that the neutropenic episodes under radiotherapy that were once associated with prca may have been temporary occurrences of the ain . 
however , bm findings ( ie , hypoplastic erythropoiesis , maturing granulopoiesis ) and the rapid and stable wbc recovery without immunosuppressive therapy suggest that these neutropenic episodes were indeed related to radiotoxicity . to the best of our knowledge , this is the first description of a thymoma - associated autoimmune disorder that resolved under sunitinib . 
in the study by thomas et al , 18 two patients developed prca or hypogammaglobulinemia while receiving sunitinib ; three patients had autoimmune disorders before receiving sunitinib and not during treatment , but it was not specified whether these disorders were present when sunitinib therapy was started ( as opposed to earlier ) and then improved under therapy . because our report is based on clinical observations , we cannot exclude that the neutropenia only coincidentally improved during sunitinib treatment . 
sachs no relationship to disclose konrad aumann research funding : novartis ( inst ) travel , accommodations , expenses : novartis rainer claus honoraria : janssen oncology , roche , novartis consulting or advisory role : roche , janssen oncology speakers bureau : janssen oncology travel , accommodations , expenses : janssen oncology , abbvie , roche nikolas von bubnoff honoraria : novartis , bristol - myers squibb consulting or advisory role : novartis research funding : novartis travel , accommodations , expenses : novartis cornelius f . 
waller consulting or advisory role : mylan , teva , boehringer ingelheim , bristol - myers squibb , astrazeneca , msd , roche travel , accommodations , expenses : bristol - myers squibb acknowledgment we thank ingrid bartsch for flow cytometry analyses and the molecular tumor board ( medical center , university of freiburg , germany ) for the thorough and elaborate discussion of the case . 
schweiz med wochenschr 97 : 1606 - 1611 , 1967 jacobson bm , castleman b : case 1 - 1971anemia , granulocytopenia and terminal sepsis in a 70 - year - old woman . n engl j med 284 : 39 - 47 , 1971 4 . 
nagashima s , yamato h , saito k , et al : cd8 + agranular lymphocyte proliferative disorder with t - cell receptor betachain gene rearrangement associated with thymoma and neutropenia [ in japanese ]  . 
mathieson pw , oneill jh , durrant st , et al : antibody - mediated pure neutrophil aplasia , recurrent myasthenia gravis and previous thymoma : case report and literature review . 
kobayashi m , hasegawa t , iwabuchi s , et al : the effect of thymectomy on myasthenia gravis , thrombocytopenia , and granulocytopenia associated with thymoma : report of a case . 
hirokawa m , sawada k , fujishima n , et al : long - term response and outcome following immunosuppressive therapy in thymoma - associated pure red cell aplasia : a nationwide cohort study in japan by the prca collaborative study group . 
thomas a , rajan a , berman a , et al : sunitinib in patients with chemotherapy - refractory thymoma and thymic carcinoma : an open - label phase 2 trial . 
finke jh , rini b , ireland j , et al : sunitinib reverses type - 1 immune suppression and decreases t - regulatory cells in renal cell carcinoma patients . 
ozao - choy j , ma g , kao j , et al : the novel role of tyrosine kinase inhibitor in the reversal of immune suppression and modulation of tumor microenvironment for immune - based cancer therapies . 
gu y , zhao w , meng f , et al : sunitinib impairs the proliferation and function of human peripheral t cell and prevents t - cell - mediated immune response in mice . 
hoffacker v , schultz a , tiesinga jj , et al : thymomas alter the t - cell subset composition in the blood : a potential mechanism for thymoma - associated autoimmune disease . 
rod rassekh , md , mhsc5 ; rebecca deyell , md5 ; jennifer rauw , md1 ; meg knowling , md2 ; kong khoo , md6 ; ursula lee , md6 ; krista noonan , md6 ; jason hart , md1 ; r . 
taylor2 ; simon chan , msc2 ; karen mungall2 ; eric chuah2 ; yongjun zhao , dvm2 ; andrew mungall , phd2 ; richard moore , phd2 ; howard lim , phd2 ; daniel j . 
jones , phd2 ; marco marra , phd2 ; and janessa laskin , md2 purpose this study investigated therapeutic potential of integrated genome and transcriptome proling of metastatic sarcoma , a rare but extremely heterogeneous group of aggressive mesenchymal malignancies with few systemic therapeutic options . methods forty - three adult patients with advanced or metastatic non - gi stromal tumor sarcomas of various histology subtypes who were enrolled in the personalized oncogenomics program at bc cancer were included in this study . 
fresh tumor tissues along with blood samples underwent whole - genome and transcriptome sequencing . results the most frequent genomic alterations in this cohort are large - scale structural variation and somatic copy number variation . 
outlier rna expression as well as somatic copy number variations , structural variations , and small mutations together suggest the presence of one or more potential therapeutic targets in the majority of patients in our cohort . 
point mutations or deletions in known targetable cancer genes are rare ; for example , tuberous sclerosis complex 2 provides a rationale for targeting the mammalian target of rapamycin pathway , resulting in a few patients with exceptional clinical benet from everolimus . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction adult sarcoma is one of the most difcult cancer types to study in the clinical trial setting , owing to its rarity along with complexity and heterogeneity in histology , tissue origin , and molecular subtypes . 
although improved survival is seen over time in metastatic sarcoma , the prognosis continues to be poor , with a median overall survival of approximately 18 months.1 the genomic prole of adult sarcomas was recently unveiled by a comprehensive genomic study published by the cancer genome atlas ( tcga ) consortium.2 unlike other cancer types , adult sarcomas are characterized by a predominance of copy number variations ( cnvs ) , with relatively few single - nucleotide variants ( snvs ) .2 however , these data were limited to localized primary disease in only six major sarcoma subtypes and did not explore any direct correlation with potential targeted therapies . targeted oncological therapeutics based on genomic proling ( ie , personalized cancer medicine ) , despite many controversies , seem to improve clinical outcome of patients with multiple cancer types . 
 feng et al context key objective using whole - genome and transcriptome sequencing technology , we attempt a systemic analysis of comprehensive proling on a cohort of 43 patients with metastatic non - gi stromal tumor sarcomas to identify therapeutic implication . knowledge generated our data showed large - scale structural variant aberration is the primary mechanism of mutagenesis in metastatic sarcoma . 
in addition , we reported cases of clinical outcomes of personalized oncogenomicsdirected therapy and demonstrated the clinical utility of genomic proling in metastatic sarcomas . furthermore , we reported several novel mutational signatures in metastatic sarcomas , one of which is homologous recombination deciency signature that may associate with superior sensitivity to double - strand dnadamaging agents in several sarcoma subtypes . relevance our results support the use of genomic data in precision medicine and clinical trial design to improve care for patients with metastatic sarcoma . 19 distinct types of advanced metastatic sarcoma as part of the personalized oncogenomics ( pog ) project ( clinicaltrials.gov identier : nct02155621 ) at bc cancer in canada . 
these represent cases refractory to standard therapies that were genomically analyzed with the aim to identify molecular aberrations amenable to a clinically approved targeted therapy or a theoretical targeted therapy under investigation in clinical trials . 
clinical benet of pogdirected therapy was determined by radiographic response on the basis of response evaluation criteria in solid tumors ( recist ; version 1.1 ) and / or progression - free survival of 6 months or more . methods patient samples as previously described , 5 , 6 candidate patients enrolled in pog underwent metastatic tumor biopsies and peripheral blood collection for comparison with detected somatic alterations . 
genome libraries from tumors and blood samples ( normal control ) as well as transcriptome libraries from tumors were constructed using protocols previously described.7 , 8 all patients provided written informed consent for tumor biopsies , blood sample collection , genomic proling , data analysis , and publication of results as part of pog of british columbia approved by the university of british columbia research ethics committee ( reb# h12 - 00137 ) and registered ( clinicaltrials.gov identier : nct02155621 )  . 
raw sequence data and downstream analytics were maintained within a secure computing environment . whole - genome and transcriptome sequencing and bioinformatics paired - end reads from all libraries were generated on illumina hiseq2000 or hiseq2500 sequencer ( illumina , san diego , ca )  . 
determination of somatic snvs , structural variants ( svs ) , cnvs , and gene expression was performed as previously described.5 , 6 transcript outlier expression was determined either by a greater than or a less than two - fold change against tumor illumina bodymap ( arrayexpress id : e - mtab - 513 ) or percentile less than 10% or greater than 90% ( tumor v compendium of tcga ; nih.gov / tcga / )  . 
for gene expression clustering , data were t using the deseq2 package , and hierarchical clustering of log - transformed reads per kilobase of transcript per million mapped reads was performed for genes signicant at an adjusted p value threshold of .01 ( euclidean distance , complete linkage clustering )  . 
the common histologies in this cohort were leiomyosarcoma ( lms ; nongynecologic : n = 7 [ 16% ] and gynecologic : n = 3 [ 7% ] ) , osteosarcoma ( osa ; n = 5 [ 12% ] ) , undifferentiated pleomorphic sarcoma ( ups ; n = 5 [ 12% ] ) , dedifferentiated liposarcoma ( ddlps ; n = 4 [ 9% ] ) , and ewing sarcoma ( n = 3 [ 7% ] ; table 1 )  . 
there were 29 male patients ( 67% ) and 14 female patients ( 33% ) , with a median age at time of metastatic diagnosis of 55 years ( range , 18 to 79 years )  . 
in this cohort , 28 of 43 patients ( 65% ) had received one standard systemic treatment of advanced metastatic disease at the time of sequencing biopsy , and 15 of 43 patients ( 35% ) had received multiple lines ( range , 2 to 7 )  . 
details of standard systemic treatments received in this cohort are described in appendix table a1 . at the time of data analysis , 26 of 43 patients ( 60% ) were deceased , and 17 of 43 ( 40% ) were still alive . potentially targetable alterations in a heterogeneous sarcoma cohort we sought to systematically characterize the genomic landscapes of adult metastatic sarcomas in this heterogeneous cohort and explore how these landscapes relate to therapeutic approaches . 
however , this level of information can potentially be used to inform treatment options for therapies currently available . overall clinical outcome of pog - directed therapy of 42 patients with potentially targetable alterations , 18 ( 43% ) patients attempted pog - directed therapies either in the clinical trial setting or through a compassionate access these , eight of 18 patients ( 44% ) program ( cap )  . 
of derived clinical benet from pog - directed therapy ( table 2 ) and are described in case series of patients who derived clinical benet from pog - directed therapy . response could not be assessed in four cases ( 22% ) owing to short duration ( 2 weeks ) on pog - directed toxicities and / or deterioration of therapies because of performance status ( appendix table a2 )  . 
there were six patients ( 33% ) considered nonresponders to pogdirected therapies , including tyrosine kinase inhibitors , histone deacetylase inhibitors , and immunotherapies ( appendix table a2 )  . 
this result is comparable to a recent study using genomic proling in the clinical setting.15 case series of patients who derived clinical benet from pog - directed therapy infrequent tsc2 mutation / deletion has cases 1 and 2 : therapeutic implications for mammalian target of rapamycin inhibitors . 
case 1 is a 62 - year - old male who presented with recurrent metastatic renal epithelioid angiomyolipoma with lung , liver , peritoneum , and bone metastases ( table 2 )  . 
pog identied a tsc2 pathologic truncating mutation , a relatively well - known event.18 everolimus was given through cap , yielding prolonged partial response ( pr ; appendix fig a3 ) as predicted and continued to show response at 15 months of therapy at the time of analysis . case 2 is a 57 - year - old female who presented with a large right upper lobe mass with mediastinal / hilar lymphadenopathy and pleural effusion and was diagnosed with undifferentiated round cell sarcoma . 
however , the above two cases suggest that the landscape of small mutations and deletions , although rare , has therapeutic relevance in some sarcoma subtypes . our systematic analysis of somatic events based on wholegenome sequencing reveals that the most frequent recurrent alterations in this cohort of adult metastatic sarcomas occur through large - scale structural changes in the genome ( fig 2a ) , rather than through point mutations or small deletions that are frequent in many other cancer types , 20 with a few exceptions of known tumor suppressor genes including tp53 ( approximately 30% ; 13 of 43 ) , atrx ( approximately 12% ; ve of 43 ) , and rb1 ( approximately 7% ; three of 43 ; fig 2b )  . 
 personalized oncogenomics of adult metastatic sarcoma case 3 : targeting common oncogenic amplications such as mouse double minute 2 homolog and cyclin - dependent kinase 4 may have therapeutic implications . 
she subsequently had a pr to a combination of mdm2 and cdk4 inhibitors in a phase ib / ii clinical trial ( clinicaltrials.gov identier : nct02343172 ) and continued to receive therapy for approximately 1 year before death as a result of disease progression . 
although the synergistic effect of mdm2 and cdk4 inhibitors remains controversial22 , 23 pending ongoing clinical trials , the case of this exceptional responder provides rationale for investigating and targeting these genomic abnormalities in this tumor and possibly other sarcoma types . recurrent amplications have been described as a common feature in genomically complex sarcoma subtypes.2 , 24 the canonical 12q13 - 15 amplications targeting mdm2 / cdk4 were observed in ddlps , with an additional case of highgrade osa also harboring this event , suggestive of a dedifferentiated osa arising from a low - grade osa with mdm2 amplication.25 amplication of the oncogene myc on 8q24 was also observed in three of 43 sarcomas . 
the most common region of amplication across this adult sarcoma cohort is 17p11 - 12 altered in 42% ( 18 of 43 ) of cases ( figs 3a and 3b )  . 
strikingly , when we examined the frequency of our recurrent amplication events across tcga cancer types , the amplication on 17p11 - 12 was found to be highly sarcoma specic , which is rarely observed in other tumor types ( fig 3c )  . 
furthermore , the 17p11 - 12 amplicon has been previously described only in osa26 , 27 and lms.28 in our study , this amplicon is observed in a variety of sarcoma subtypes , including ddlps , pleomorphic liposarcoma , osa , lms , chordoma , ups , and undifferentiated endometrial sarcoma ( ues ) , suggesting it harbors an oncogene that is important for sarcoma tumorigenesis . myocd , pmp22 , and cops3 are believed to be potential oncogenic targets within this amplicon in lms and osa.26 - 28 we examined transcriptome expression data for potential oncogenic targets in this region ( fig 3d )  . 
myocd and pmp22 are both within the region most frequently amplied in this cohort , with myocd showing particularly higher expression in amplied samples and pmp22 showing higher expression overall and compared with many other in addition , adora2b was noted as having tumors . amplicon - associated expression ; this gene has not previously been examined as a potential target for this amplicon , but it has been reported to have growth - promoting properties in oral and breast cancers.29 , 30 this analysis suggests that additional functional analysis of genes within this amplicon is required to clearly dene the oncogenic target ( s )  . there are few therapies available that to date , target common amplications in metastatic sarcomas . 
the future development of therapies targeting recurrent amplications such as 17p11 - 12 may be warranted , potentially improving therapeutic options in this disease . case 4 , 5 , and 6 : mutation signatures and homologous recombination deciency may delineate sarcoma subtypes and indicate sensitivity to certain dna - damaging agents . mutation signatures are characteristic patterns of mutation , which may result from specic cellular or environmental pressures and may have implications for therapy.12 the most comprehensive validated set of mutation signatures described to date is reported in cosmic ( sanger.ac.uk / cosmic / signatures )  . 
on the basis of singlenucleotide alterations , we performed mutation signature detection in our sarcoma cohort and identied eight signatures through this de novo process , denoted v1 to v8 ( appendix fig a6 )  . 
comparison of discovered sarcoma signatures to the 30 previously described mutation signatures reported in cosmic31 revealed that sarcoma signatures v1 , v3 , v4 , v5 , v6 , and v8 matched signatures 30 , 5 , 2 , 1 , 8 , and 9 , respectively ( fig 4 )  . 
for example , ddlps possessed similar rearrangement proles , with elevated signature r1 and high mutation burden . signatures v6 and v7 matched signatures 3 and 8 , both of which are associated with hrd signature , 31 also known as brcaness , which has been well described in breast , fig 2 . 
genomic structural complexity is calculated as the average of the proportion of copy segments that are nondiploid and the proportion of the genome represented in nondiploid copy segments , similar to that per the cancer genome atlas research network.2 ( b ) oncoprint of recurrent somatic dna alterations in cancer - related genes observed in sarcomas , grouped by sarcoma subtype . 
types of gene alterations include amplication ( red ) , homozygous deletion ( blue ) , gene fusion ( purple ) , truncating mutation ( black ) , or missense mutation ( green )  . 
 personalized oncogenomics of adult metastatic sarcoma ovarian , and other relatively common cancer types32 including osa.33 we further computed hrd indices and found that in addition to osa , high hrd scores were also seen in lms , ues , and a few other sarcoma subtypes ( fig 4 )  . 
the brcaness phenotype in lms that we observed is consistent with a recent report.34 interestingly , we did not observe deleterious genomic or transcriptomic aberrations of genes such as brca1 and brca2 , which have been described as accounting for the majority of hrd signatures in other tumor types . 
we do observe losses , svs , or downregulated expression of various components implicated in the homologous recombination repair pathway , such as pten , atm , and rad51 ( data not shown ) , suggesting potential alternative contributions to the hrd signature . 
a clear - cut relationship between genetic aberrations and hrd signature is also not observed in other studies.12 , 31 case 4 is a 44 - year - old woman who presented with metastatic ues with lung metastasis . 
the response to ifosfamide was not long - lasting , like all other chemotherapies , and both patients eventually experienced progression , did not respond to any additional treatment , and died as a result of disease progression . 
the hrd signature or brcaness phenotype is associated with therapeutic response to dna - damaging agents , including platinum - based chemotherapies.12 although platinums are not generally used in metastatic soft tissue sarcomas , the dna damaging chemotherapeutic agent ifosfamide has similar , though less - pronounced , dna crosslinking effects . although ifosfamide is an active drug in various sarcoma subtypes , with an overall response rate between 10% and 40% depending on dosing schedule , single - agent response is generally considered low ( , 10% ) , especially in the third - line setting or beyond for sarcomas aside from synovial sarcoma.35 we speculate that the response may relate to partial dna cross - linking property of ifosfamide alkylating agent , which may have therapeutic advantage in hrd tumors . case 6 is 53 - year - old man who presented with recurrent metastatic splenic vein lms with liver and lung metastasis . his disease progressed through docetaxel plus gemcitabine treatment . 
because of a similar hrd signature identied through pog study , he was enrolled in a phase i clinical trial ( clinicaltrials.gov identier : nct02873975 ) investigating an experimental drug ( prexasertib ) targeting hrd tumors . 
these cases support the clinical utility of genomic analysis of hrd and mutation signatures across sarcoma subtypes . case 7 and 8 : potentially targetable aberrations identied solely through transcriptome / expression analysis . 
case 7 is a 77 - year - old male presenting with recurrent multiple intraabdominal ups , which progressed through two cycles of doxorubichis tumors exhibited high rna expression of various tyrosine kinase receptors , such as plateletderived growth factor receptors ; therefore , sunitinib was attempted through cap . 
he then received pog - directed therapy in the form of sunitinib on the basis of his tumor exhibiting high rna expression of fms - like tyrosine kinase 3 and colony - stimulating factor 1 receptor . 
expression in amplicon refers to log ( reads per kilobase of transcript per million mapped reads ) ; increase in amplied samples is measured as expression in amplied samples divided by expression in nonamplied samples in this cohort ; expression percentile is determined relative to all tcga tumors ( see methods )  . 
 ( a ) structural variant ( sv ) mutation signatures ( r1 to r4 ) and single - nucleotide variant ( snv ) mutation signatures ( v1 to v8 ) were deciphered de novo from metastatic sarcomas . 
three dedifferentiated liposarcomas demonstrated high sv and snv burden , with similar r1 rearrangement signature and signatures v4 , v5 , and v6 . signature v4 matched catalogue of somatic mutations in cancer ( cosmic ) signature 2 and is known to be associated with apobec deamination . 
 ( b ) three sarcoma cases ( labeled in red ) with elevated homologous recombination deciency ( hrd ) score and / or the hrd - associated signature 3 demonstrated response to double - strand dnadamaging chemotherapy . 
 ( c ) mutation signatures deciphered de novo from sarcomas were compared against 30 canonical signatures from the cosmic using the cosine similarity metric . signatures that map most closely to a cosmic signature are highlighted . 
 personalized oncogenomics of adult metastatic sarcoma selection who had these two histology subtypes.36 , 37 taken together , these results support the notion that drug efcacy may be dictated by biology measurable through gene expression biomarkers in addition to histology subtype . furthermore , these data support additional research on these putative biomarkers in the context of a complex signaling network , which may help advance the drug development of various tyrosine kinase inhibitors in future sarcoma clinical trials . immune landscape of sarcoma subtypes despite being regarded as a poorly immunogenic tumor because of relatively low mutation burden compared with other cancer types , 2 checkpoint inhibitor immunotherapies have produced promising durable response in a few sarcoma histology subtypes such as ups and alveolar soft part sarcoma thus far.38 , 39 pd - l1 expression has been correlated with efcacy of immunotherapy in some , but not all , cancer types40 and reported as a prognostic marker in soft tissue and bone sarcoma , 41 , 42 but it has not yet been shown to correlate with immunotherapy sensitivity in sarcoma.43 the tumor immune microenvironment plays a key role in determining immunotherapy sensitivity , and emerging data demonstrated a positive correlation between clinical response and preexisting tumor immune microenvironment.44 we examined immune inltration on the basis of gene expression proles ( see methods ) and observed that a subset of tumors show high iiss ( fig 5 ; appendix fig a9 )  . 
we did not observe any correlation between high level of pd - l1 expression and immunotherapy response in a small number of cases ( n = 4 ) within our cohort . interestingly , of the four patients in our cohort treated with immunotherapies , the only patient ( case 8 , lms ) who exhibited some response to immunotherapy also demonstrated an unusually high iis ( fig 5 ; table 2 )  . 
two other cases with high iis , both ups , were not treated with immunotherapy , and two other sarcomas that were treated with immunotherapy , an lms and an osa , had low iis and did not respond . 
overall , our experience suggests that although iis alone may not be sufcient as a predictive biomarker for immunotherapy in investigation , all sarcoma types , particularly in lms . it warrants additional cases 9 and 10 : detection of fusion genes assists in sarcoma diagnosis and therapeutic intervention . 
t - cell inltration score is the sum of all t - cell subtypes excluding regulatory t cells , which are negative modulators . immune inltration score is the sum of scores for all 22 immune cell types proled . 
arms , alveolar rhabdomyosarcoma ; asps , alveolar soft part sarcoma ; ccs , clear cell sarcoma ; chsa , chondrosarcoma ; ddlps , dedifferentiated liposarcoma ; eaml , epithelioid angiomyolipoma ; ews , ewing sarcoma ; mlps , myxoid liposarcoma ; mpnst , malignant peripheral nerve sheath tumor ; msft , malignant solitary brous tumor ; osa , osteosarcoma ; other , groups urs , plps , mlps , ccs , mpnst , eaml , arms , ss , chsa , asps , and sef ; plps , pleomorphic liposarcoma ; sef , sclerosing epithelioid brosarcoma ; ss , synovial sarcoma ; stlms , nongynecologic leiomyosarcoma ; ues , undifferentiated endometrial sarcoma ; ulms , gynecologic leiomyosarcoma ; ups , undifferentiated pleomorphic sarcoma ; urs , undifferentiated round cell sarcoma . sarcoma and change to standard alternating multi - agent chemotherapies with substantial pr . 
her systemic disease also responded to second - line and third - line standard chemotherapies for ewing sarcoma , but she eventually developed brain metastasis and died as a result of disease progression . case 10 is a 68 - year - old man presenting with recurrent metastatic osa ( initial pathologic diagnosis ) of the right femur with lung metastasis . 
 feng et al rapid deterioration shortly after this diagnosis was made from genomic analysis and died as a result of disease progression . within our cohort , in all cases where a fusion would be expected based on pathologic features , we observed a fusion consistent with the sarcoma type ( appendix table a3 )  . the exceptional above two cases , where we identied fusions that had not initially been expected based on pathology , support the utility of genomic proling in rening sarcoma diagnosis and therapeutic intervention , echoing the results of a large retrospective study.17 conclusion our data lend additional support to the current literature data showing that sv and cnv are the primary mechanisms of mutagenesis in sarcomas , as opposed to single - nucleotide alteration . 
we provide anecdotal evidence showing the potential of genomic alterations , such as loss of tsc2 and amplication of mdm2 / cdk4 , to predict exceptional response to targeted treatments . 
overall , we were able to identify potentially actionable genomic alterations in the majority of cases , largely on the basis of outlier rna expression and cnv and sv aberrations . 
renouf , karen gelmon , stephen yip , marco marra , janessa laskin data analysis and interpretation : xiaolan feng , erin pleasance , eric y . zhao , tony ng , jasleen k . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . xiaolan feng honoraria : astrazeneca , nanostring / innomar strategies consulting or advisory role : astrazeneca , ipsen , genomic health , nanostring / innomar strategies , roche tony ng honoraria : nanostring technologies travel , accommodations , expenses : nanostring technologies sara k . 
 personalized oncogenomics of adult metastatic sarcoma krista noonan consulting or advisory role : merck , sano canada , bristol - myers squibb , janssen , pzer , astrazeneca speakers bureau : merck stephen yip consulting or advisory role : bayer , pzer , roche research funding : pzer , bayer , roche , foundation medicine travel , accommodations , expenses : bayer , roche , pzer jason hart honoraria : novartis martin jones employment : qiagen , thermo fisher scientic ( i ) howard lim honoraria : eli lilly / imclone systems , ipsen , merck , roche canada , eisai , taiho pharmaceutical , bristol - myers squibb canada travel , accommodations , expenses : eisai , taiho daniel j . 
renouf honoraria : celgene , servier , taiho pharmaceutical , ipsen , shire research funding : bayer ( inst ) , astrazeneca ( inst ) karen gelmon honoraria : astrazeneca , merck sharp & dohme consulting or advisory role : pzer , novartis , astrazeneca , merck , eli lilly , bristol - myers squibb , nanostring technologies , genomic health , janssen oncology , roche , mylan research funding : pzer ( inst ) , bristol - myers squibb ( inst ) , astrazeneca ( inst ) , roche ( inst ) expert testimony : genentech marco marra travel , accommodations , expenses : roche canada janessa laskin honoraria : boehringer ingelheim , roche canada , astrazeneca , pzer research funding : astrazeneca ( inst ) , roche canada ( inst ) no other potential conicts of interest were reported . acknowledgment we thank katherine mui , jessica nelson , and lindsay zibrik from the personalized oncogenomics ( pog ) program for coordinating our study . this work would not be possible without the participation of our patients and families , the entire pog team , and the generous support of the bc cancer foundation and genome british columbia ( project b20pog )  . 
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the number of potentially targetable alterations was determined by comparing observed somatic mutations and gene expression alterations to a database of events that have clinical or preclinical evidence of potential therapeutic targeting , on the basis of therapeutics currently available or in development . mutation copy number expression only samples with potentially targetable alterations ( % ) fig a2 . 
samples with potentially targetable alterations were determined by comparing observed somatic mutations and gene expression alterations to a database of events that have clinical or preclinical evidence of potential for therapeutic targeting , on the basis of therapeutics currently available or in development . 
 feng et al baseline before standard chemotherapy ( april 2016 ) progression after standard chemotherapy ( august 2016 ) response to pog - directed therapy ( july 2017 ) fig a3 . 
 personalized oncogenomics of adult metastatic sarcoma baseline before standard chemotherapy ( january 2015 ) progression after standard chemotherapy ( may 2015 ) response to pog - directed therapy ( december 2015 ) fig a4 . 
annotation tracks show exposure values calculated from mutation analysis ( structural variant [ sv ] , single - nucleotide variant [ snv ] burden ) , clinical covariates , and histologic properties of the sarcoma cohort . 
 feng et al baseline before standard chemotherapy ( august 2015 ) progression after standard chemotherapy ( july 2016 ) response to pog - directed therapy ( october 2016 ) fig a7 . 
 personalized oncogenomics of adult metastatic sarcoma baseline ( feb 2015 ) progression after standard chemotherapy ( april 2015 ) response to pog - directed therapy ( october 2015 ) fig a8 . 
other than undifferentiated pleomorphic sarcoma ( ups ) , some leiomyosarcoma ( lms ) and dedifferentiated liposarcoma ( ddlps ) and one case of synovial sarcoma ( ss ) seem to have relatively high iis compared with others . 
 impact of genetic ancestry on outcomes in ecog - acrin - 5103 purpose racial disparity in breast cancer outcomes exists between african american and white women in the united states . 
we have evaluated the impact of genetically determined ancestry on disparity in efficacy and therapy - induced toxicity for patients with breast cancer in the context of a randomized , phase iii adjuvant trial . methods this study compared outcomes between 386 patients of african ancestry ( aa ) and 2 , 473 patients of european ancestry ( ea ) in a randomized , phase iii breast cancer trial , ecogacrin - 5103 . 
the primary efficacy end point , invasive diseasefree survival ( dfs ) , and clinically significant toxicities were compared , including anthracycline - induced congestive heart failure , taxane - induced peripheral neuropathy ( tipn ) , and bevacizumab - induced hypertension . results overall , aas had significantly inferior dfs ( p = .002 ; hazard ratio , 1.5 ) compared with eas . 
2017 by american society of clinical oncology introduction african american patients with breast cancer have inferior efficacy outcomes compared with other races.1 , 2 the reason for this imbalance is multifactorial and includes higher stage , higher grade , more triple - negative breast cancer ( tnbc ) , and poorer responsiveness to chemotherapy.3 , 4 these clinical imbalances have previously been attributed to both socioeconomic factors and a different underlying biology of the tumor.2 , 5 patients of african ancestry ( aa ) also experience more adverse drug reactions , including an increase in clinically important toxicities for chemotherapeutic agents . 
although less well characterized , these toxicity disparities are also likely multifactorial and include inherited genetic variation and comorbidities , among other factors.6 - 10 much of the prior work has been based on selfreported race . 
 node - negative breast cancer to intravenous doxorubicin and cyclophosphamide ( ac ) every 2 or 3 weeks ( at discretion of treating physician ) for four cycles followed by 12 weeks of paclitaxel ( 80 mg / m2 once per week ) alone ( arm a ) or to the same chemotherapy with either concurrent bevacizumab ( arm b ) or concurrent plus sequential bevacizumab ( arm c ) ; figure 1a . 
patients with estrogen receptor ( er ) positive disease were ln positive , had a tumor > 5 cm or a tumor measuring 1 to 5 cm , with a recurrence score > 11 . genome - wide association study germline dna from whole blood and companion clinical outcome data were available in 3 , 126 patients . 
if arm c significantly improved dfs relative to arm a , then in the second step , a comparison of arm b with arm a was performed.14 in this correlative study , dfs and overall survival ( os ) were evaluated using kaplan - meier methodology . 
the differences in outcomes between aas and eas were compared with the application of cox proportionalhazards models using either univariate or multivariate analysis , which were corrected with significant covariates . 
to identify the best regression model , a forward and a backward stepwise selection procedure were performed separately to evaluate variable associations , and the akaike information criterion was used to determine the inclusion of potential confounders such as race , menopausal status , age , weight , height , side of cancer involvement , er status , histologic grade , nuclear grade , ln status , type of surgery , types of ac schedule , and dose reductions in ac or paclitaxel . both procedures returned the same model with six predictors , which are listed in the data supplement . dfs was the primary end point of the parent trial ; it was defined as invasive dfs and calculated from the date of random assignment to the date of first treatment failure ( invasive ipsilateral , local / regional invasive , distant recurrence , invasive contralateral breast cancer , invasive nonbreast second primary , or death from any cause [ whichever occurred first ] )  . cases with incomplete follow - up , without a documented invasive dfs event ( including those who developed squamous or basal cell skin cancers or in situ carcinomas of any site as their only event ) were censored at the date of last disease evaluation . 
we previously published biomarker data on each of these toxicities.15 , 17 , 18 in the present study , we sought to compare the frequency of all toxicities between aas and eas and to assess the impact of race on dose modifications . 
statistical analysis was performed using r ( version 3.3.0 ) and the x2 test . odds ratio ( or ) was used to evaluate the significance and magnitude of the differences in the toxicity frequency between aas and eas . chf cases for this study included individuals who had centrally reviewed , cardiologist - adjudicated chf . 
to be selected for inclusion in ecogacrin - 5103 , patients must have had no history of clinically significant cardiovascular disease . cardiovascular health was monitored at the start and during the trial by multigated angiograms or echocardiograms . 
cardiac events included chf , decrease in left ventricle ejection fraction , acute coronary syndrome , supraventricular tachycardia , and myocardial dysfunction diagnosed by a cardiologist . tipn cases for this study were defined as those experiencing either grades 2 to 4 or grades 3 to 4 tipn as assessed by ctcae . 
to serve as a tipn case , the patient must have received at least one dose of paclitaxel , and the neuropathy event must have occurred during treatment or within 3 months of the last dose of therapy . hypertension cases for this study were defined as those experiencing a systolic blood pressure ( sbp )  . 
blood pressure values were collected as part of standard clinical assessment before administration of therapy throughout the conduct of the trial . results genotyped group from ecog - acrin5103 and genetic ancestry a total of 3 , 394 germline dna samples from patients were collected as part of the planned correlative protocol within ecog - acrin5103 ( fig 1b )  . 
the outcomes of the entire genotyped cohort ( all ancestries combined ) used in this correlative study were almost identical to the parent trial4 ( data supplement )  . demographic data and disease comparisons table 1 summarizes the important demographic comparisons between aas and eas . 
we assessed for the differences in the parent trial stratification factors as well as other known outcome predictors , including age , hormone receptor status ( er positive v tnbc ) , ln status , height , weight , type of surgery , and tumor grade . 
there were no significant differences overall in ln status . genetic ancestry as a predictor of efficacy in ecog - acrin - 5103 dfs was the primary efficacy end point of the parent trial . 
an imbalance in associated comorbidities or environmental factors cannot be excluded as a contributing cause , and unfortunately , those data were not collected in ecog - acrin - 5103 . 
dose reductions in paclitaxel also negatively affected dfs for all ancestries ( p = .02 ; data supplement ) ; however , having a dose modification in paclitaxel did not affect dfs for eas , but it did significantly cause an inferior dfs for aas ( fig 4 )  . the difference may have been explained by more severe dose reductions in aas . 
the outcomes for the subgroup genotyped , however , were similar to the parent population , thus minimizing the concern for subgroup bias . we also compared the results of the genetic ancestry with self - reported race , and there was no significant difference in the conclusion . 
work in other disease phenotypes , however , has demonstrated that use of self - reported race as a surrogate for genetic ancestry was not perfect.25 - 27 prior work has demonstrated that  . 
9% of patients cannot supply , or choose not to supply , ancestry information.28 thus , the real impact of accurately self - reported race is probably larger than the 9% that was observed , as the result of misclassification alone . 
although genotype may provide more insight toward underlying biologic diversity , selfreported race still likely represents a reasonable surrogate for genotypic variation in the routine clinical setting for consideration of metabolism and drug toxicity . we previously evaluated genetic markers to predict some of the most clinically important toxicities in ecog - acrin - 5103 : bevacizumabinduced hypertension ( using various definitions ) , tipn , and cardiologist - adjudicated anthracyclineinduced chf.15 , 17 , 18 in this study we compared the likelihood of each of these clinically relevant toxicities between aas and eas . 
these data support prior work from a single - institution retrospective analysis as well as data in pediatric populations that revealed higher likelihood of anthracycline - induced chf in aas . 
similarly , a prior single - institution retrospective analysis demonstrated higher risk of tipn for aas.8 - 10 because aas experience greater toxicity , it is highly unlikely that the imbalance in efficacy is a result of exposure as the result of pharmacokinetic considerations , as the end organs are clearly being affected . 
as expected , all ancestries that had dose reductions in the ac portion of the chemotherapy had inferior dfs ; this difference , however , was present for both eas and aas . 
the significant difference in dfs as the result of paclitaxel dose modifications for aas may have been because of the markedly more severe dose reductions ; this was evidencedby alower meannormalizeddosein thosewho had dose reductions ( p = .03 ) , in large part because table 2 . 
this study did not evaluate socioeconomic factors , which are known to be important variables in outcomes.5 however , in the context of a randomized phase iii trial , many of these inequities are minimized . in conclusion , this study highlights the need to better understand the biologic differences in normal breast biology , tumor biology , and the inherited genetic differences between women of aa and ea . 
it also highlights the need to better personalize counseling when discussing the risk - to - benefit ratio for aas , for whom the disease - specific outcomes are inferior and drug - specific toxicities are higher . 
ecog - 1199 previously demonstrated that the dosing of docetaxel every 3 weeks was as effective but had fewer dose reductions for aas in a similar clinical setting.20 future trials should investigate whether another taxane with less risk of tipn , such as docetaxel , might be more effective for aas in the adjuvant breast cancer setting . 
sparano stock and other ownership interests : metastat consulting or advisory role : genentech , novartis , astrazeneca , celgene , eli lilly , celldex , pfizer , prescient therapeutics , juno therapeutics , merrimack research funding : prescient therapeutics ( inst ) , deciphera ( inst ) , genentech ( inst ) , merck ( inst ) , novartis ( inst ) , merrimack ( inst ) george w . 
sledge jr leadership : syndax stock and other ownership interests : syndax honoraria : symphogen consulting or advisory role : symphogen , coherus biosciences , radius health , peregrine pharmaceuticals , taiho pharmaceutical research funding : genentech ( inst ) travel , accommodations , expenses : nektar , radius health , taiho pharmaceutical kathy d . 
miller consulting or advisory role : nektar , tesaro , merck research funding : genentech ( inst ) , merrimack ( inst ) , entremed ( inst ) , taiho pharmaceutical ( inst ) , macrogenics ( inst ) , medivation ( inst ) , novartis ( inst ) , seattle genetics ( inst ) affiliations support bryan p . 
schneider , fei shen , guanglong jiang , milan radovich , lang li , laura gardner , dongbing lai , tatiana foroud , and kathy d . miller , indiana university school of medicine , indianapolis , in ; anne oneill , dana farber cancer instituteecog - acrin biostatistics center , boston , ma ; joseph a . 
keenan t , moy b , mroz ea , et al : comparison of the genomic landscape between primary breast cancer in african american versus white women and the association of racial differences with tumor recurrence . 
desantis c , jemal a , ward e : disparities in breast cancer prognostic factors by race , insurance status , and education . jama 295 : 2492 - 2502 , 2006 cancer causes control 21 : 1445 - 1450 , 2010 americans . 
kidd rs , curry tb , gallagher s , et al : identification of a null allele of cyp2c9 in an african - american exhibiting toxicity to phenytopharmacogenetics 11 : 803 - 808 , 2001 8 . 
bhatnagar b , gilmore s , goloubeva o , et al : chemotherapy dose reduction due to chemotherapy induced peripheral neuropathy in breast cancer patients receiving chemotherapy in the neoadjuvant or adjuvant settings : a single - center experience . 
cobb rj , thomas cs , laster pirtle wn , et al : self - identified race , socially assigned skin tone , and adult physiological dysregulation : assessing multiple dimensions of race in health disparities research . 
miller k , oneill am , dang ct , et al : bevacizumab ( bv ) in the adjuvant treatment of her2 - negative breast cancer : final results from eastern cooperative oncology group e5103 . 
schneider bp , li l , radovich m , et al : genome - wide association studies for taxane - induced peripheral neuropathy in ecog - 5103 and ecog - 1199 . 
killelea bk , yang vq , wang sy , et al : racial differences in the use and outcome of neoadjuvant chemotherapy for breast cancer : results from the national cancer data base . 
halder i , shriver m , thomas m , et al : a panel of ancestry informative markers for estimating individual biogeographical ancestry and admixture from four continents : utility and applications . 
louwers yv , lao o , fauser bc , et al : the impact of self - reported ethnicity versus genetic ancestry on phenotypic characteristics of polycystic ovary syndrome ( pcos )  . 
mandrekar , phd1 with the launch of the national cancer institutes precision medicine initiative in 2015 , there has been a shift to trial designs that tailor health care solutions to individual patients by using a screening platform and by moving away from the one - trial / one - biomarker - at - a - time approach . 
2019 by american society of clinical oncology introduction precision medicine takes into account the variability of an individual patients genes , environment , and lifestyle when deciding the best approach for disease prevention and treatment.1 since the rst publication of the human genome in february 2001 , 2 scientists have made great progress in understanding inherited differences in genes , making precision medicine a tangible reality . 
even though precision medicine is a relatively new term , tailoring intervention on the basis of a patients specic features is not a new idea . many currently available us food and drug administration ( fda ) approved therapies are based on specic patient subgroups . 
for example , rituximab , the rst monoclonal antibody treatment for cancer , was approved by the fda in 1997 for patients with relapsed or refractory cd20 + , b - cell , low - grade , or follicular non - hodgkin lymphoma . 
the addition of cetuximab to rst - line chemotherapy in patients with kras wild - type metastatic colorectal cancer has been shown to signicantly improve treatment outcomes compared with chemotherapy alone in two randomized clinical trials , crystal ( clinicaltrials.gov identier : nct00154102 ; cetuximab combined with irinotecan in first - line therapy for metastatic colorectal cancer ) 3 and opus ( clinicaltrials.gov identier : nct00125034 ; oxaliplatin and cetuximab in first - line treatment of metastatic colorectal cancer [ mcrc ] ) .4 erlotinib and crizotinib are fda - approved therapies for patients with advanced lung cancer who test positive for an epidermal growth factor receptor ( egfr ) mutation and the alk rearrangement , respectively . 
it was later found that patients with higher programmed death - ligand 1 expression treated with pembrolizumab had longer progression - free survival ( pfs ) and overall survival ( os ) .5 most recently , on may 23 , 2017 , the fda granted accelerated approval to pembrolizumab as the treatment for adult and pediatric patients with unresectable or metastatic microsatellite instability - high or mismatch repair decient solid tumors , 6 which is the rst time that the fda has approved a cancer treatment for an indication on the basis of a common biomarker rather than the primary site or origin ( ie , tissue agnostic ) .7 there are continuing efforts in drug development to move away from the one - size - ts - all approach and toward precision medicine . in this article , we aim to introduce novel trial designs by using case studies as examples . 
 ou et al context key objective to discuss types of clinical trial designs for accelerating development of new drugs in the era of precision medicine . knowledge generated clinical trial designs ( such as umbrella , basket , and subgroup ) that evaluate multiple hypotheses are efcient precision medicine initiatives . 
other designs include enrichment designs for validating the efcacy of an agent in biomarker - driven subgroups and window of opportunity designs to help understand the antitumor activity of an agent in a treatment - naive patient population . relevance the clinical trial designs discussed in this article are practical and relevant for precision medicine trials . 
several case studies are provided to demonstrate the utility of these clinical trial designs . the results of tumor gene sequencing or other testing techniques , such as polymerase chain reaction , ow cytometry , uorescence in situ hybridization ( fish ) , chromogenic in situ hybridization , and immunohistochemistry . 
specic to genotype - driven trials , patients will be matched to trials that are developed on the basis of prior understanding of certain molecular aberrations that may determine sensitivity to certain therapies . 
if a patient is eligible for more than one genotype - driven trial , the trial will be selected by the treating physician on the basis of the totality of information , such as the patients preferences and status , expected benets , and molecular characteristics . 
enrichment designs test selective therapeutic options for matching patients that can result in better patient outcomes.9 the genotype - driven trial mentioned in the case study is one type of enrichment design ; other types of enrichment , such as that based on the epigenome , is also appropriate . 
enrichment trial design is best suited when the rationale for the trial strongly suggests that the agent would be effective in a specic biomarkerdened patients with a specific tumor type with biomarker status unknown may be part of the trial protocol biomarker profiling with targeted biomarker trial enrollment with targeted therapy without targeted biomarker standard of care fig 1 . 
it is particularly advantageous if the biomarker used to dene the patient population is considered soc ; if so , the trial design does not need to take into account the molecular testing because the information would be available for all patients . 
highly specic screening tests are required to identify the low - prevalence molecular prole and to avoid assigning patients who tested false positive to treatment options that may not be efcacious . 
finally , certain molecular proles may have low prevalence and may result in a large proportion of screened patients receiving the soc , which can be inefcient ( eg , if the prevalence of the molecular prole of interest is only 5% , 500 patients will need to be proled to identify 25 patients who are potentially eligible )  . umbrella design umbrella trials include patients with a single tumor type or disease and for which several biomarkers have been identied and used to dene disease subtypes.10 patients are screened for a panel of biochemical , genetic , and / or immunologic markers associated with their disease and , on the basis of the markers detected , assigned to a biomarkerdriven treatment strategy or targeted therapy that is most likely to result in favorable outcomes . 
thus , an umbrella trial consists of multiple substudies , each with independent subgroups of patients receiving different therapies and with the option of assuming different statistical parameters for independent designs . 
the substudies , however , exist under an overarching master protocol that uses a common infrastructure for screening and treatment assignment to reduce the cost and time associated with enrollment to unrelated and often sequential biomarker - informed studies . 
lung - map ( clinicaltrials.gov identier : nct03851445 ; a master screening protocol previously - treated non - small cell lung cancer ) is an example of a phase ii / iii umbrella study in solid tumors.11 lung - map opened to accrual in 2014 for patients with advanced squamous cell carcinoma ( scc ) of the lung . similar to aml , scc of the lung is a biologically complex disease . 
roughly 60% of patients with lung scc harbor genetic abnormalities that might be targeted with therapy.14 lung - map provided a common infrastructure to test patient samples for multiple biomarkers , which informed therapy assignment within 10 to 14 days . 
patients were assigned to a substudy according to a predened algorithm that used specic genetic abnormalities detected via ngs in patients with a specific tumor type biomarker profiling targeted biomarker 1 targeted biomarker 2 targeted biomarker 3 targeted biomarker n no actionable biomarker regimen a regimen b regimen c regimen n standard of care or non - targeted regimen fig 2 . 
a substudy of new therapy versus soc remained open for patients without an actionable biomarker . lung - map was designed to test new therapies against soc for a particular subgroup using multiple , randomized phase ii trials with pfs as the primary end point . 
within a substudy , if a new therapy met criteria that suggested large the substudy was expanded to improvements in pfs , a phase iii study with pfs and os as co - primary end points . patients who were part of the phase ii interim analysis were included in the phase iii assessment . 
with a well - established screening platform in place , an umbrella trial design can be quite efcient in directing patients into different treatment regimens and accelerating the drug development process . 
the concept of umbrella trials can be applied in any disease setting , but it is most appropriate when there is a strong scientic rationale informing which therapies might work best in biomarker - dened subgroups . 
although not used in beat aml or lung - map , adaptive randomization can be applied within a substudy that has multiple arms to assign patients to a therapy that shows the most favorable outcome , given the data already collected.1 , 15 , 16 bayesian posterior probabilities that compare efcacy of experimental therapy with soc can be used in decision making . umbrella trials are exible and have the ability to open or close new substudies with different targeted therapies or statistical designs if appropriate . basket design basket trials are an efcient way of screening experimental therapeutics across patients with a variety of tumor types and / or histology under one master trial . 
basket trials involve several biomarker proles whereby patients are assigned to treatments based on the molecular alterations their tumors contain , regardless of the histologic type of the tumor.17 , 18 the basic premise for this trial design is that the average response to one or more experimental treatments is expected to vary across these different biomarker proles . like umbrella trials , there may be a common genetic screening platform , especially if the cohorts dened by the study are histology - independent and dened only by the presence of a single molecular aberration . 
figure 3 provides a generic representation of a basket trial design schema . basket trial designs may be appropriate when there is reason to believe that one or more experimental treatments may be effective across a range of primary tumor sites or histologies , and in particular , when patients with specic biomarker proles are expected to respond well to a particular treatment , regardless of their specic disease or histology . 
one such design is the bayesian basket design.20 in this design , a bayesian approach is used to model the response probabilities for the various histologic strata , and two hypotheses are considered : ( 1 ) the response probabilities for a particular targeted agent are equal across the corresponding histologic strata , and ( 2 ) the activity of the drug is independent across these strata . 
a hierarchical bayesian design has recently been proposed for randomized phase ii trials with multiple groups ( or baskets ) , allowing for information sharing across baskets while at the same time having the ability to help with decision making for each individual trial or basket.21 within the hierarchical bayesian design framework , the estimate of the treatment effect for each phase ii trial basket within this master protocol shrunk toward the overall mean treatment effect , which alleviates concerns regarding randomness across the trials or baskets.21 , 22 this approach is most powerful when the treatment effects are heterogeneous across the different baskets and inates the type i error in the inferior treatment effect groups or baskets in the case of heterogeneous treatment effects across baskets . 
it has been noted that the results of a basket trial can be heavily tilted toward positive conclusions , 23 even with specication of weak priors in a bayesian setting . 
the goal of this study , as noted in the publication , was to identify molecular biomarkers and determine their frequency and clinical relevance in patients with advanced nonsmall - cell lung cancer , and thymic malignancies and to evaluate the efcacy of multiple targeted therapies in specic molecular subsets of patients.27 ( p1001 ) a limitation of this trial was the lack of the lung cancer , small - cell adaptive trial feature , because seamless addition of new treatment arms for molecular targets or replacing treatments for a particular molecular subtype was not possible . another issue was the timeliness of the availability of some of the core molecular proling results , which had an impact on enrollment to the trial.27 acs e and tapur case studies . 
the french national cancer institutes acs e initiative ( secured access to innovative therapies ) 28 and ascos tapur trial ( clinicaltrials.gov identier : nct02693535 ; testing the use of food and drug administration [ fda ] approved drugs that target a specic abnormality in a tumor gene in people with advanced stage cancer ) 29 are examples of large - scale trials with basket - type design that evaluate the safety and efcacy prole of approved agents in other indications ( ie , tumor types )  . 
the goals of these trials are to provide patients with advanced diseases ( without promising treatment options ) off - label access to approved agents and to evaluate the safety and efcacy of these agents outside their approved indications . 
the acs e - crizotinib trial ( clinicaltrials.gov identier : nct02034981 ; phase 2 study assessing efcacy and safety of crizotinib in patients harboring an alteration on alk , met or ros1 ) is the rst trial coordinated by the acs e prograit includes patients with various solid and hematologic malignancies . 
basket designs test a single agent in different histologic subtypes , which can be efcient if the goal is to screen for antitumor activity of the agent in different disease settings . 
for example , only two ( nsclc with egfr mutations and nsclc with kras or braf mutations ) of the 15 substudies in the custom trial completed accruing ; accrual was unsuccessful for the other 13 rare histologic subtypes . 
 ou et al experimental therapy in the subgroup of patients positive for the biomarker as well as in either the subgroup of patients negative for the biomarker or in all patients . 
with co - primary objectives , the signicance level ( ) is allocated or split between the two objectives to maintain an acceptable overall type i family - wise error rate using a conservative bonferroni correction or a less conservative correction that considers the correlation between the two tests.31 in the case with co - primary objectives dened for the biomarker - positive subgroup and all patients , the design can be subgroup focused or all - population focused.32 a subgroup - focused design is most appropriate when there is evidence that the experimental therapy will be most effective in patients with the biomarker of interest , but it could also have a broad impact in the general disease population . 
a design with an all - population focus is most appropriate when there is less evidence that the experimental therapy will be most effective in patients with the biomarker of interest but could be effective in the general disease population . 
with an accrual goal of 618 patients egfr - positive by fish , there was 92% power to detect a hazard ratio of 0.75 for pfs in the cetuximab arm relative to the control arm with a type i error rate ( ) of 2% . 
with an accrual goal of 1 , 546 total patients , there was 86% power to detect a hazard ratio of 0.83 for os in the cetuximab arm relative to the control arm with a one - sided of 1.5%. 
because of slow accrual and a lower - than - expected percentage of patients who were egfr positive , the study was amended to 400 egfrpositive patients to detect planned differences in pfs with 80% power . 
 case studies for innovative trial designs multiple hypotheses in a subgroup design allows formal testing of efcacy for a biomarker - dened subgroup of patients and the overall patient population . 
by testing multiple hypotheses , the power for testing a specied effect in the overall population is reduced.32 furthermore , in a subgroup - focused design , as the prevalence of a biomarker decreases , the total sample size increases.32 costs also increase because tissue procurement and biomarker testing is required for all patients to reach the target number of subgroup patients.33 these additional resources can be offset by testing for larger treatment effects in the biomarker - dened subgroup.32 window of opportunity design the primary goal of window of opportunity ( woo ) design , is to unalso known as window or phase 0 design , derstand the antitumor activity of an agent in a disease state that is not disrupted by previous or simultaneous treatments . 
these trials occur between patient diagnosis and initiation of standard treatment and typically have a clinical end point such as tumor response or pfs at a predened early time point , often assessed by radiographic imaging ( figure 5 )  . 
woo designs are distinct from neoadjuvant trial settings , in which an investigational agent is commonly given preoperatively along with cytotoxic chemotherapy or hormonal therapy for a longer period of time than in a woo trial . 
woo trials have recently gained attention , specically in the context of evaluating molecular agents or in early advanced or metastatic disease in which the tumor may not be resistant to selective inhibition of a novel cancer target.38 evaluating molecular end points can be hindered by difculties in procuring tumor tissue before and after drug administration and by heterogeneity in previous exposure to cancer therapies . 
woo trials help to overcome these difculties by enrolling treatment - nave patients , including a translational research component , and occurring in a short time frame before the initiation of the standard treatment of the disease . 
the ultimate hope of woo trials is that they can expedite the drug development process by improving understanding of an agents biologic effect early in its development , validating markers that may predict subsets of patients who will benet , and targeting select short courses of experimental treatment diagnosis of disease standard of care ( eg , surgery ) standard treatment option fig 5 . 
it is a design best suited for trials in which the treatment cycles are short , and a short - term outcome ( eg , biomarker change ) is the end point of interest . 
in addition , although it is not yet documented , there is the theoretical possibility that the investigational agent may in fact induce resistance to subsequent therapies . logistically , woo trials require careful coordination among members of a multidisciplinary study team because diagnosis , workup , and study treatment must all be arranged in a short time frame . 
furthermore , tumor heterogeneity can be an issue , and thus it is essential to have the infrastructure capable of performing a centralized and high - quality review of all biomarkers.40 woo trials were initially used to inform a go / no - go decision in development of anticancer agents , to expose a small number of patients to a limited duration and dose of a drug and subsequently proceed to phase i / ii trials on the basis of the results.41 ( p2574 ) woo trials may not be advantageous in settings in which an agent has already been well - studied in other disease setfor which the toxicity prole is already welltings , understood , or even for which little safety data are available regarding the risk of delaying surgery.40 despite these concerns and considerations , woo trials have proved benecial in some settings . 
 ou et al active in untreated patients.42 , 43 woo trials should therefore be considered as a promising alternative trial design . discussion with the ability to simultaneously evaluate the effects of therapies on multiple biomarkers and diseases , umbrella and basket trial designs can help accelerate the discovery of targeted therapy . 
because each substudy of the umbrella and basket trials is essentially an enrichment trial design , we would expect a larger effect size , given the available prior knowledge of certain genotype proles and their sensitivity to therapy . 
woo trials allow investigators to understand the antitumor activity of an agent in the de novo disease setting . trial designs in the precision medicine era require a platform to carry out the biomarker proling . 
given the advances in liquid biopsies , cell - free dna from plasma may provide a minimally invasive alternative to standard tumor biopsies.44 even though we have more options for proling patients , the proling is not without challenge . 
at the same time , a screening cut point will need to be established and validated to ensure the cut point value ( ie , the high and low levels of the biomarker value to predict the response to treatment ) is appropriate.45 in an early work , mandrekar et al46 outlined the criteria for choice of phase ii designs for initial validation of a predictive marker in a review article that considered assay performance , turnaround time , preliminary evidence , and marker prevalence . with the advances in targeted therapy , we now have many therapeutic options for specic targeted biomarkers ; however , mutations that have high prevalence in a certain disease may not be common in another ( for example , human epidermal growth factor receptor 2 overexpression has been reported in approximately 15%47 of patients with breast cancer but only approximately 2% of patients with colorectal cancer ) .48 designing a trial with a biomarker target with extremely low prevalence is a challenge because it is difcult to obtain a sufcient number of patients to properly power the study . 
a statistical trial design that uses small sample sizes is needed to tackle this issue , as well as potentially international partnerships . in this article , we have focused our discussion on trial designs that are better suited for phase ii and iii studies . 
seamless phase i / ii designs , which blur the distinction between dose selection and efcacy evaluation , are one such design . hobbs et al49 provided an overview of the use of seamless designs in rst - in - human studies based on abstracts submitted to asco annual meetings from 2010 to 2017 and provided guidance on the design and conduct of such trials . 
to validate biomarker associations identied from such studies , sufcient statistical rigor is still required , such as clear denition of the primary end point , a prespecied interim and nal analysis plan , and well - dened safety monitoring rules . with immunotherapy gaining traction in oncology , it uncertain whether the current designs can adequately address the needs for these agents . 
if the primary objective is to show efcacy in a biomarker - dened , tumor - specic patient population , then both enrichment design and umbrella design ( ie , as one of the substudies ) are applicable . 
as mentioned in the introduction , pembrolizumab received the rst tissue - agnostic fda approval , and there may be several factors that could have contributed to successfully ling for approval of pembrolizumab.50 first , the tissue collection in the initial randomized pembrolizumab trials allowed investigators to retrospectively test the tissue - agnostic hypothesis . 
second , immunotherapies are designed with a strong foundation of preclinical data . third , multiple prospective clinical trials have veried the hypotheses generated by the retrospective data that contributed to its approval . 
in the case of pembrolizumab , approval was based on the data from ve different trials in which patients were retrospectively identied from two studies and prospectively enrolled in three studies.7 because this design is the rst of its kind , it is unclear which design discussed in this article will be best suited for future agents targeting tissue - agnostic indications . 
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schmitz s , caballero c , locati ld : perspectives on window of opportunity trials in head and neck cancer : lessons from the eortc 90111 - 24111 - noci - hncg and neck ( scchn )  . 
clark pi , slevin ml , joel sp , et al : a randomized trial of two etoposide schedules in small - cell lung cancer : the inuence of pharmacokinetics on efcacy and 44 . 
 successful targeting of an atg7 - raf1 gene fusion in anaplastic pleomorphic xanthoastrocytoma with leptomeningeal dissemination mehdi touat , md1 ; nadia younan , md1 ; philipp euskirchen , md1 , 2 , 3 , 4 , 5 ; maxime fontanilles , md1 ; karima mokhtari , md1 ; caroline dehais , md1 ; patrick tilleul , pharmd6 ; amithys rahimian - aghda , msc1 ; adam resnick , phd7 ; anne - paule gimenez - roqueplo , md , phd8 , 9 , 10 ; h el `ene blons , pharmd , phd8 , 9 , 11 ; kh e hoang - xuan , md , phd1 ; jean - yves delattre , md , phd1 ; ahmed idbaih , md , phd1 ; pierre laurent - puig , md , phd8 , 9 , 11 ; and marc sanson , md , phd1 introduction pleomorphic xanthoastrocytoma ( pxa ) is an uncommon primary brain tumor occurring primarily in children and young adults.1 although pxa is typically considered a relatively indolent entity , some tumors are accompanied by anaplastic features associated with high rates of recurrence after local treatment and unfavorable outcome.2 accordingly , anaplastic pxa ( who grade 3 ) has been added to the 2016 who classication of cns tumors as a distinct entity.1 there is no standard management for patients with anaplastic pxa that recurs after surgery ; neither chemotherapy nor radiotherapy have demonstrated clinical benet in this disease.2 , 3 the identication of activating in recent years , brafv600e mutations in 50% of patients with pxa has rened our understanding of these disorders as malignancies driven by aberrant activation of the mitogenactivated protein kinase ( mapk ) signaling pathway.1 , 3 - 5 this was further supported by the observation of both preclinical and clinical activity of vemurafenib , a selective brafv600e inhibitor , in brafv600e - mutant gliomas.6 , 7 however , patients with braf wild - type pxa are not candidates for treatment with selective brafv600e inhibitors , and little is known about the spectrum of molecular alterations occurring in this population . 
recent studies showed that a subset of braf wild - type pxa might harbor rare braf or raf1 fusions , 8 , 9 which have been associated with exquisite sensitivity to mapk signaling inhibition in preclinical models.10 it remains unknown whether patients who have glioma with braf or raf1 gene fusions might respond to therapies targeting mapk signaling . 
histopathologic analysis showed a tumor composed of pleomorphic glial cells with the presence of mitoses , necrosis , and microvascular proliferation , consistent with who grade 3 anaplastic pxa ( fig 2 )  . 
although rhabdoid morphology was observed in some sectors , both epithelial membrane antigen and p53 immunostainings were negative , and we did not observe loss of ini1 , features that were previously associated with rhabdoid glioblastomas and cns atypical teratoid / rhabdoid tumors , respectively . 
furthermore , targeted sequencing of the tumor did not nd mutations in braf ( exons 11 and 15 ) , idh1 , idh2 , h3f3a , hist1h3b , or smarcb1 . 
array comparative genomic hybridization revealed a 9p deletion involving cdkn2a and cdkn2b , which have been previously associated with poor outcome in brafv600e - mutant gliomas.3 , 11 , 12 given the unfavorable prognosis , she was rst treated with adjuvant chemoradiotherapy ( 60 gy in 30 fractions over 6 weeks along with once - per - day temozolomide 75 mg / m2 ) followed by temozolomide ( 200 mg / m2 ) for 6 cycles until november 2015 ( fig 1a )  . in february 2016 , routine surveillance mri scans showed increased contrast enhancement in the right occipital lobe . 
 touat et al context key objective to report the successful treatment of a patient with refractory braf - wild - type anaplastic pxas harboring an in - frame atg7raf1 fusion with the mek inhibitor cobimetinib . knowledge generated we report for the rst time , to our knowledge , that raf1 gene fusions are actionable molecular events in high - grade gliomas . this report highlights the oncogenic role of mapk - activating alterations including raf1 rearrangements and brafv600e mutations in a subset of pediatric and adult gliomas . relevance cobimetinib can achieve therapeutic exposure within the cns , including the cerebrospinal uid in patients with raf1translocated high - grade gliomas . showed leptomeningeal enhancement in the posterior cerebral fossa ( fig 1b )  . 
neoplastic meningitis was conrmed by lumbar puncture showing 42 white blood cells per l ( 84% lymphocytes ) , hyperproteinorachia ( 0.55 g / l ) , and the presence of tumor cells in the cerebrospinal uid ( csf ; fig 1b )  . 
intravenous thiotepa ( 45 mg / m2 ) was begun , the treatment was discontinued after one infusion because of grade 4 thrombopenia and febrile neutropenia with infectious pneumonia , which required intravenous antibiotics . the patient was enrolled in the precision medicine program exorare for tumor molecular characterization using rna sequencing and matched tumor or normal whole - exome sequencing . 
reverse transcriptase - polymerase chain reaction and sanger sequencing conrmed the expression of an in - frame atg7raf1 fusion transcript in the recurrent tumor ( second surgery , 2016 ; fig 3b )  . 
interestingly , no additional molecular alteration was found by whole - exome among 475 genes previously associated with cancer , which suggested that the atg7 - raf1 fusion was the main oncogenic driver . moreover , previous reports showed that fusions involving exon 8 of raf1 are recurrent events in cancer including pxa8 , 9 , 13 and can result in constitutive activation of the tyrosine kinase domain and downstream mapk signaling through the loss of the n - terminal autoinhibitory domain of raf1.14 treatment with sorafenib ( 200 mg twice per day ) was then started in october 2016 through off - label use after informed consent . 
unfortunately , her clinical condition rapidly deteriorated with partial seizures , confusion , cerebellar ataxia , diplopia , hypoacousia , headaches , and diffuse pain in the lower limbs , for which treatment with intravenous morphine , steroids , and midazolam was begun . on the basis of preclinical data suggesting that mek inhibition in astrocytomas harboring raf1 fusions has superior efcacy , 10 sorafenib was discontinued and treatment with off - label cobimetinib ( 60 mg per day , 3 weeks on and 1 week off ) was started in november 2016 , after family members provided informed consent ( fig 1a )  . 
the patient dramatically improved after a few days of treatment , with complete regression of the confusion and headaches and partial regression of the cerebellar ataxia and pain in the lower limbs . 
subsequent lumbar puncture showed complete cytologic response in the csf ( fig 1b ) with regression of both pleocytosis ( 1 white blood cell per l ) and hyperproteinorachia ( 0.15 g / l ; fig 1b )  . 
 ( b ) top : serial contrast - enhanced t1 - weighted images of brain magnetic resonance imaging ( mri ) and cerebrospinal uid ( csf ; may - gr unwald - giemsa stain ) cytology at ( left ) initial presentation , ( middle ) pretreatment , and ( right ) during treatment with cobimetinib showing the right occipital lesion , the leptomeningeal enhancement on the brain mri ( white arrows ) , and the presence of neoplastic cells in the csf ( black arrowheads ) before treatment with cobimetinib was started . 
the observation of clinical activity with both braf and mek inhibitors suggest that brafand raf1 - driven gliomas , even in patients who have been heavily pretreated , display a high level of mapk pathway dependency and possibly less molecular heterogeneity than the majority of gliomas driven by other alterations such as egfr variants.20 - 22 despite previous reports indicating activity of sorafenib in treating patients with cancer who harbor mutations of braf or araf ( both paralogs of raf1 ) , 23 , 24 our patient did not respond to treatment with sorafenib . 
 ( a ) hematoxylin - eosin and ( b ) reticulin colorations showing a pleomorphic tumor proliferation , with the presence of xanthomatous cells with nuclear atypia , reticulin bers , microvascular proliferation , and necrosis . 
 ( c - f ) immunohistochemical stains showing tumor cell immunoreactivity for ( c ) glial brillary acidic protein , ( d ) cd34 , and ( e ) oligodendrocyte transcription factor 2 . 
 ( f ) high mitotic activity was noted on mib - 1 staining . alterations is currently ongoing ( nct02639546 ; safety and pharmacokinetics of cobimetinib in pediatric and young adult participants with previously treated solid tumors [ imatrixcobi ] ) , although adult patients are not yet eligible for this study . 
furthermore , novel mek inhibitors optimized for achieving higher brain exposure might have a role in these diseases.28 in conclusion , our report provides evidence for the oncoincluding raf1 reargenic role of mapk alterations , in pediatric and adult brain tumors and rangements , suggests that raf1 fusions may represent potential therapeutic targets in a subset of patients with braf wild - type high - grade gliomas . 
a recent report of a patient with melanoma with an ano10raf1 fusion reported long - lasting clinical improvement after treatment with the mek inhibitor trametinib , 29 suggesting that patients with raf1 fusiondriven tumors might respond to mek inhibition regardless of histology . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . mehdi touat consulting or advisory role : agios pharmaceutical , taiho oncology travel , accommodations , expenses : merck sharp & dohme maxime fontanilles travel , accommodations , expenses : roche patrick tilleul honoraria : takeda , astellas pharma , roche , biogen , evidera , baxter , gr unenthal group , chiesi , bristol - myers squibb , msd , sandoz - novartis , pzer , astrazeneca h el `ene blons honoraria : astrazeneca , bristol - myers squibb , msd oncology kh e hoang - xhuan travel , accommodations , expenses : abbvie ahmed idbaih travel , accommodations , expenses : carthera pierre laurent - puig honoraria : amgen , astrazeneca , boehringer ingelheim , merck serono , merck , roche , sano consulting or advisory role : merck , bristol - myers squibb , boehringer ingelheim patents , royalties , other intellectual property : inventor of mir31 - 3p ( license to integragen ) travel , accommodations , expenses : roche , merck marc sanson consulting or advisory role : abbvie , genenta travel , accommodations , expenses : abbvie no other potential conicts of interest were reported . acknowledgment the authors thank ines detrait and delphine le corre for tissue preparation and acid nucleic extractions , and dr yvan bieche and integragen for the genomic analyses performed in this patient . references louis dn , ohgaki h , wiestler od , et al : world health organization histological classication of tumours of the central nervous system , 4th edition revised . lyon , france , international agency for research on cancer , 2016 ida cm , rodriguez fj , burger pc , et al : pleomorphic xanthoastrocytoma : natural history and long - term follow - up . 
brain pathol 25 : 575 - 586 , 2015 lassaletta a , zapotocky m , mistry m , et al : therapeutic and prognostic implications of braf v600e in pediatric low - grade gliomas . 
j clin oncol 35 : 2934 - 2941 , 2017 dias - santagata d , lam q , vernovsky k , et al : braf v600e mutations are common in pleomorphic xanthoastrocytoma : diagnostic and therapeutic implications . plos one 6 : e17948 , 2011 schindler g , capper d , meyer j , et al : analysis of braf v600e mutation in 1 , 320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma , ganglioglioma and extra - cerebellar pilocytic astrocytoma . 
clin cancer res 17 : 7595 - 7604 , 2011 kaley t , touat m , subbiah v , et al : braf inhibition in brafv600 - mutant gliomas : results from the ve - basket study . 
brain pathol 29 : 85 - 96 , 2018 jain p , fierst tm , han hj , et al : craf gene fusions in pediatric low - grade gliomas dene a distinct drug response based on dimerization proles . 
huillard e , hashizume r , phillips jj , et al : cooperative interactions of brafv600e kinase and cdkn2a locus deciency in pediatric malignant astrocytoma as a basis for rational therapy . 
 cobimetinib in anaplastic pleomorphic xanthoastrocytoma with raf1 fusion jones dt , hutter b , j ager n , et al : recurrent somatic alterations of fgfr1 and ntrk2 in pilocytic astrocytoma . 
nat genet 45 : 927 - 932 , 2013 johnson a , severson e , gay l , et al : comprehensive genomic proling of 282 pediatric lowand high - grade gliomas reveals genomic drivers , tumor mutational burden , and hypermutation signatures . 
reinhardt a , stichel d , schrimpf d , et al : anaplastic astrocytoma with piloid features , a novel molecular class of idh wildtype glioma with recurrent mapk pathway , cdkn2a / b and atrx alterations . 
szerlip nj , pedraza a , chakravarty d , et al : intratumoral heterogeneity of receptor tyrosine kinases egfr and pdgfra amplication in glioblastoma denes subpopulations with distinct growth factor response . 
cancer discov 4 : 956 - 971 , 2014 imielinski m , greulich h , kaplan b , et al : oncogenic and sorafenib - sensitive araf mutations in lung adenocarcinoma . 
casadei gardini a , chiadini e , faloppi l , et al : efcacy of sorafenib in braf - mutated non - small - cell lung cancer ( nsclc ) and no response in synchronous braf wild type - hepatocellular carcinoma : a case report . 
choo ef , ly j , chan j , et al : role of p - glycoprotein on the brain penetration and brain pharmacodynamic activity of the mek inhibitor cobimetinib . 
gampa g , kim m , cook - rostie n , et al : brain distribution of a novel mek inhibitor e6201 : implications in the treatment of melanoma brain metastases . 
malignant cells have a high proliferative fraction and typically express b - cell lymphoma 2 ( bcl2 ) , interferon regulatory factor 4 ( irf4 ) , and b - cell lymphoma 6 ( bcl6 ) on immunohistochemistry . gene expression proling is associated with constitutive nuclear factor ( nf ) - b pathway activation and up - regulation of apoptosis - inhibiting genes , similar to ndings in abc dlbcl . 
mutations of myd88 , resulting in constitutive nf - b activation , are reported in pbdlbcl - lt in up to 69% of cases6 , 7 and are associated with a worse clinical outcome.7 apoptosis gene expression proling in pcdlbcl - lt has shown not only high expression levels of antiapoptotic genes but also concomitant downstream inhibition of the intrinsic apoptosis pathway , as reported in chemotherapy - refractory nodal abc dlbcl.8 , 9 treatment of relapsed / refractory pcdlbcl - lt remains a signicant challenge , with many patients succumbing to uncontrolled disease.3 current therapeutic options , including access to clinical trials , are limited because of both the rarity of disease and the often frail , comorbid patient population . 
small case series of patients with myd88 - mutated pcdlbcl - lt have demonstrated responses to inhibition of b cell receptor ( bcr ) signaling via bruton 's tyrosine kinase ( btk ) inhibition , although responses are of short duration ( 4 to 40 weeks ) and associated with rapid emergence of resistance - conferring mutations.10 , 11 the efcacy of the specic bcl2 inhibitor venetoclax in pcdlbcl - lt remains unexplored . 
this study was approved by the university hospitals of leicester nhs trust ( uk ; 06 / q2501 / 122 )  . research and development written informed consent was obtained for publication purposes . expression of cd20 , cd79a , paired box 5 ( pax5 ) , bcl2 , bcl6 , irf4 , and cd10 , with a ki67 fraction of 90% ( figs 1a - 1d )  . 
 ( a ) hematoxylin and eosin staining , ( b ) interferon regulatory factor 4 ( irf4 ) , ( c ) b - cell lymphoma 2 ( bcl2 ) , and ( d ) ki67 expression . 
in 2010 , he relapsed again with left leg cutaneous involvement only , adjacent to the previously treated sites , and received additional treatment with four cycles of r - chop and radiotherapy ( 40 gy in 15 fractions ) , again attaining clinical response . 
two additional relapses in the left leg with progressively shorter disease - free intervals were treated with radiotherapy alone in 2011 and 2012 ( 4 gy , single fraction )  . 
subsequently , in 2016 , an additional relapse , treated with rituximab , gemcitabine , cyclophosphamide , vincristine , and prednisolone , was refractory to chemotherapy . because of high expression of bcl2 and the development of chemorefractory disease , in january 2017 we obtained venetoclax through the uk abbvie compassionate access scheme . 
repeat staging by ct and 18f - labeled uorodeoxyglucosepositron emission tomography / ct scans conrmed that disease remained localized to the left lower leg , as shown in figure 1e . 
the dose of venetoclax was escalated to 1 , 200 mg because of published data in dlbcl and follicular lymphoma conrming safety and tolerability of this dose.12 however , 1 , 200 mg was not tolerated because of persistent grade 2 diarrhea and grade 2 fatigue , and thus 800 mg was adopted as a maintenance dose . 
biopsy after 2 weeks of treatment showed a complete histologic and immunohistochemical response , although ighv polymerase chain reaction showed persistence of clonal b cells ( data not shown )  . 
treatment with venetoclax was stopped 1 month after demonstration of metabolic remission . however , within 4 weeks of stopping venetoclax , disease recurred , once again conned to the left lower leg , but at different anatomic sites . 
an additional biopsy was performed , which conrmed relapsed disease and showed persistent high - level bcl2 expression ( data not shown )  . venetoclax was recommenced at a dose of 400 / 800 mg on alternate days , with only mild gastrointestinal ( gi ) toxicity ( grade 1 common terminology criteria for adverse events )  . eighteen months later , with continued daily venetoclax , his disease remains in complete remission . whole - exome dna sequencing was performed only on the last relapse sample because of a lack of suitable material from prior biopsies . 
call thresholds were a minimum of 10 variant reads , depth 25 reads , variant allele frequency 10% , scale - invariant feature transform score less than 0.35 , polyphen greater than 0.7. 
potential genetic drivers of this malignancy included previously described arid1a and notch2 mutations ( table 1 ) , as well as biallelic deletion of rb1 and cdkn2a and monoallelic loss of tp53 ( fig 2 )  . 
 ( a , b ) somatic copy number analysis of the tumor biopsy was carried out using varscan and sequenza programs against patient germline dna . frequency of copy number ( log2 ratio ) obtained by pairwise comparison of read depths along the sequenced exome . 
dyer honoraria : roche pharma ag , abbvie , sandoz speakers ' bureau : roche research funding : roche ( inst ) , gilead sciences ( inst ) , astex pharmaceuticals ( inst ) , bioinvent ( inst ) travel , accommodations , expenses : abbvie no other potential conicts of interest were reported . references sokol l , naghashpour m , glass lf : primary cutaneous b - cell lymphomas : recent advances in diagnosis and management . 
blood 105 : 3768 - 3785 , 2005 grange f , beylot - barry m , courville p , et al : primary cutaneous diffuse large b - cell lymphoma , leg type : clinicopathologic features and prognostic analysis in 60 cases . 
arch dermatol 143 : 1144 - 1150 , 2007 vermeer mh , geelen fa , van haselen cw , et al : primary cutaneous large b - cell lymphomas of the legs . 
arch dermatol 132 : 1304 - 1308 , 1996 senff nj , noordijk em , kim yh , et al : european organization for research and treatment of cancer and international society for cutaneous lymphoma consensus recommendations for the management of cutaneous b - cell lymphomas . 
 successful treatment with single - agent venetoclax pham - ledard a , cappellen d , martinez f , et al : myd88 somatic mutation is a genetic feature of primary cutaneous diffuse large b - cell lymphoma , leg type . j invest dermatol 132 : 2118 - 2120 , 2012 pham - ledard a , beylot - barry m , barbe c , et al : high frequency and clinical prognostic value of myd88 l265p mutation in primary cutaneous diffuse large b - cell lymphoma , leg - type . 
jama dermatol 150 : 1173 - 1179 , 2014 cillessen sa , hess cj , hooijberg e , et al : inhibition of the intrinsic apoptosis pathway downstream of caspase - 9 activation causes chemotherapy resistance in diffuse large b - cell lymphoma . 
clin cancer res 13 : 7012 - 7021 , 2007 van galen jc , hoefnagel jj , vermeer mh , et al : proling of apoptosis genes identies distinct types of primary cutaneous large b cell lymphoma . 
fox lc , yannakou ck , ryland g , et al : molecular mechanisms of disease progression in primary cutaneous diffuse large b - cell lymphoma , leg type during 11 . 
davids ms , roberts aw , seymour jf , et al : phase i rst - in - human study of venetoclax in patients with relapsed or refractory non - hodgkin lymphoma . 
li l , pongtornpipat p , tiutan t , et al : synergistic induction of apoptosis in high - risk dlbcl by bcl2 inhibition with abt - 199 combined with pharmacologic loss 14 . 
paulli m , lucioni m , maf a , et al : primary cutaneous diffuse large b - cell lymphoma ( pcdlbcl ) , leg - type and other : an update on morphology and treatment . 
little is known about the experiences of individuals tested for cdh1 variants in the multigene panel testing era . methods participants recruited from the prospective registry of multiplex testing completed a cross - sectional self - report survey regarding cdh1 genetic testing experiences , medical management , and psychosocial adaptation . results discordance existed in interpretations of cdh1 results ; 13.3% of cases had disagreements in variant classications among commercial laboratories , and 21.4% had disagreements between participant self - report and clinvar classication . 
2019 by american society of clinical oncology introduction hereditary diffuse gastric cancer syndrome ( hdgc ) , attributed to germline cdh1 pathogenic variants ( pv ) , is associated with increased risk for diffuse gastric cancer ( dgc ) and invasive lobular breast cancer ( lbc )  . 
germline cdh1 pv are present in approximately 40% of hdgc families , 1 and 1% to 3% individuals with gastric cancer have a cdh1 pv.2 reported cumulative lifetime dgc risk ranges from 50% to 80%.1 , 3 - 5 prophylactic gastrectomy is a standard management recommendation for individuals with cdh1 pv , given the inability of endoscopic surveillance to reliably identify dgc.5 because of cumulative lifetime lbc risk estimates ranging from 39% to 52% , recommendations for those with cdh1 pv include breast cancer screening via annual mammography , with consideration of breast magnetic resonance imaging beginning at age 30 years ; risk - reducing mastectomy decisions may occur dependent on family history.3 , 6 before the advent of multigene panel testing ( mgpt ) for hereditary cancer susceptibility , candidates for cdh1 genetic testing were evaluated on criteria dened by the international gastric cancer linkage including a conrmed case of dgc consortium , younger than age 40 years , at least two cases of dgc at any age , or the presence of both dgc and lbc younger than age 50 years.5 , 7 thus , individuals undergoing cdh1 gene analysis had a signicant perfamily history of hdgc - associated sonal and / or cancers . 
 hamilton et al context key objective germline cdh1 variants that are associated with hereditary diffuse gastric cancer syndrome ( hdgc ) are rare , but growth in multigene panel testing is leading to patients with limited personal exposure to hdgc being confronted with such results . 
we addressed the question of what are the genetic testing experiences , medical management , and psychosocial adaptation of patients with cdh1 variants identied through multigene panel testing ? knowledge generated discordant interpretations of cdh1 results were observed across testing laboratories , as well as among participants reporting of their results . 
participants with pathogenic cdh1 variants were more likely than those with variants of uncertain signicance to receive recommendations for prophylactic gastrectomy , yet both groups perceived similar , moderate levels of risk for gastric cancer . relevance findings highlight the unique informational and psychosocial needs of patients living with cdh1 variants , suggesting that they may benet from support tailored to the challenges associated with their particular risk status . genetic testing services are increasingly offered outside the cancer genetics specialty , such as via primary care providers , surgeons , and direct - to - consumer companies.10 - 12 given the variability by which preand post - test genetic counseling and testing for cdh1 are currently provided , we sought to characterize the genetic testing experiences , medical management , and psychosocial adaptation of individuals living with cdh1 variants . methods participants participants were recruited from the prospective registry of multiplex testing ( prompt ) , an online genetic registry founded by investigators at memorial sloan kettering , the university of pennsylvania , dana - farber cancer institute , and mayo clinic for individuals age 18 years or older tested for cancer susceptibility genes as part of any commercial mgpt . 
on self - selected enrollment in prompt , individuals consent to participate in the registry ; complete a baseline questionnaire of personal / family history , genetic testing results , and demographic information ; and share their testing report with prompt staff . 
a centralized institutional review board ( chesapeake irb ) approved this research . procedure study data were collected using redcap software13 through an online cross - sectional survey e - mailed to prompt participants with a self - reported cdh1 variant . invitations including a link to the survey ( with two reminders for nonresponders ) were e - mailed to 135 eligible individuals from september to december , 2017 . 
participants reported their understanding of their cdh1 result ( response options : positive genetic test result , likely positive genetic test result , variant of uncertain signicance , negative genetic test result , my test results are not clear to me ) and the year when they learned their result . 
we assessed personal ( yes , no ) and family history ( yes , no , uncertain ) of breast and gastric cancer , and details ( eg , surgery date ) regarding personal cancer treatment . 
recommendation by a health care provider for risk - reducing mastectomy , meeting with a gastroenterologist , and prophylactic gastrectomy , as well as adoption of breast magnetic resonance imaging , risk - reducing mastectomy , meeting with a gastroenterologist , upper endoscopy , and prophylactic gastrectomy , were assessed ( yes , no , unsure )  . 
genetic knowledge was assessed with 21 items regarding basic genetic concepts and aspects of mgpt selected or modied from the university of north carolina genomic knowledge scale.15 cdh1 knowledge was assessed with six investigator - designed items regarding cancer risks and inheritance of cdh1 variants . item response options were true , false , and don ' t know . 
items adapted from the national cancer institute health information national trends survey assessed absolute perceived cancer risks ( how likely are you to get [ breast / gastric ] cancer in your lifetime ? ; 1 = very unlikely to 5 = very likely ) and affective perceived cancer risks ( i feel like i could easily get [ breast / gastric ] cancer in my lifetime ; 1 = i feel very strongly that this will not happen to 5 = i feel very strongly that this will happen ) .16 decision - making experiences . 
ten items selected or modied from quality of a published measure21 assessed positive ( example : my cdh1 test result allows me to take control of my health ; = 0.75 ) and negative ( example : because of my cdh1 test result , i feel awed and stigmatized ; = 0.79 ) effects of cdh1 genetic testing on quality of life . 
three investigator - designed items assessed whether patients shared their cdh1 results with family , why this information was not shared with family , and family interest in genetic testing . statistical analysis seventy - one individuals responded to the survey ( 53% response rate ; appendix figure a1 ) ; however , three respondents did not have a cdh1 result and were excluded from all analyses . 
conicting interpretation was dened as either disagreement among one or more commercial laboratories in variant classications ( evaluable participants : n = 60 ) , or disagreement between participant self - report and clinvar classication ( evaluable participants : n = 56 )  . 
 medical and psychosocial experiences after cdh1 variant detection second , to determine how individuals understanding of their cdh1 results were associated with their medical and psychosocial experiences , we evaluated the survey data of the 60 respondents who self - reported either a pv ( ie , positive genetic test result or likely positive genetic test result ) or variant of uncertain signicance ( vus )  . 
data were examined for missing values ; patients missing responses to more than 20% of variables were excluded ( n = 3 ) , and average values on the basis of existing items were computed for scales missing less than or equal to 20% of items . 
of concern , three of these cases ( 25% of cases with participant / clinvar disagreement ; 5.4% of evaluable sample ) involved participant self - report of pv ( ie , positive genetic test result or likely positive genetic test result ) but clinvar and test report classication of vus . 
dashes indicate that a p value was not computed because of inadequate cell size . ||individuals with a complete stomach at the time of survey completion only . abbreviations : pv , pathogenic variant ; vus , variant of uncertain signicance . * responses of uncertain or unsure were combined with responses of no for analyses . females with at least one healthy breast at the time of learning their cdh1 results only . females with at least one healthy breast at the time of survey completion only . individuals with a complete stomach at the time of learning their cdh1 results only . least one healthy breast at the time of learning their cdh1 results , a minority ( 25.0% ) reported that a health recommended risk - reducing care provider had ever mastectomy ; no differences in recommendation receipt were observed based on cdh1 result . 
experiences with genetic testing among participants with cdh1 variants experience pv ( n = 16 ) vus ( n = 41 ) total ( n = 57 ) ever offered cancer genetic counseling yes , pros and cons discussed but i didn ' t 0 ( 0 ) unsure ever received cancer genetic counseling was given options to have different numbers of genes tested for hereditary cancer don ' t know was sufciently informed about pros and cons of having different numbers of genes tested for hereditary cancer yes , given sufcient information yes , pros and cons discussed but more information would have been helpful understand no , pros and cons not discussed not sure i felt sufciently informed would have preferred more time to decide about how many genes to have tested don ' t know was informed about possibility of identifying vus when deciding whether to undergo testing yes , i was informed and i understood yes , i was informed but it was not clear what that meant informed after i received results no , i was never informed not sure i was informed would have preferred more time to discuss options with family , friends , or health care providers before having genetic testing don ' t know no . 
approximately 39% of the sample recalled being given options to have different numbers of genes tested for hereditary cancer ; of these they received sufcient participants , most ( 68.2% ) felt information about the pros and cons of having different numbers of genes tested . 
specically , those with pv and vus both reported mean scores for the measures of perceived gastric cancer risks consistent with scale midpoints ( ie , absolute perceived gastric cancer risk : neither unlikely nor likely ; affective perceived gastric cancer risk : i feel i am just as likely to get cancer as i am to not get cancer )  . 
regarding emotional outcomes , participants with pv reported greater distress ( p .001 ) and were more likely to be worried about future discrimination on the basis of their cdh1 result ( p = .05 ) than those with vus . participants reported low decisional regret ( mean , 13.13 , 0 to 100 scale ) regarding cdh1 testing , irrespective of their result . 
consistent with a separate analysis of laboratory interpretations of variants identied by hereditary cancer mgpt , 27 discrepancies were not 13.3% of cases , laboratories differed in their interpretation of cdh1 variant pathogenicity . 
dashes indicate that a p value was not computed because of inadequate cell size . abbreviations : pv , pathogenic variant ; vus , variant of uncertain signicance . * missing response ( n = 1 )  . asked only among those who had not shared cdh1 results with all family ( n = 19 ) and participants could select more than one response . free - text responses included i ' m adopted , so my results don ' t apply to them , and most family members would not nd meaningful . asked only among those who shared with ( some ) family , and responses of yes . 
future research should examine how and why such misunderstandings arise and their implications for patients and families . time , differential provider the survey data indicated that , consistent with clinical guidelines , 5 only participants with a pv reported ever receiving a recommendation to undergo prophylactic gastrectomy . 
however , fewer than half of these individuals recalled receiving this recommendation , and receipt of this recommendation was not associated with clinical factors relevant to the timing of this surgical intervention , such as family gastric cancer history or patient age . 
it is unclear why certain individuals with cdh1 pv have been advised to pursue prophylactic gastrectomy and others have not . although there is speculation that the lifetime risk of gastric cancer may be lower in individuals with cdh1 pv and no family history of gastric cancer ( compared with those with a family history ) , at the current time a discussion of gastrectomy remains standard of care.26 , 28 their despite growing access to genetic testing through nongenetics health care providers , most participants were offered and received genetic counseling . 
 medical and psychosocial experiences after cdh1 variant detection patients understand the risks and benets and feel prepared to make an informed decision about mgpt . results also demonstrate that individuals with cdh1 pv and vus each have unique informational and emotional needs . 
those with vus were also less satised with their health care providers knowledge about the result ; however , it is not possible to discern whether this reects a lack of provider familiarity or existing limitations in scientic knowledge about risks and appropriate management of these variants . 
furthermore , although those with pv accurately perceived themselves to be at greater risk for breast cancer than did those with vus , these individuals had similar perceived risks for gastric cancer , which on average reected scale midpoints . 
risk perceptions are important for promoting cancer prevention behaviors , 30 , 31 including management of hdgc8 ; research is needed to understand why some with cdh1 pv seem to hold inaccurate risk perceptions . 
participants with pv did experience greater distress ; however , levels of distress were modest in this sample , consistent with a recent analysis of patient experiences with mgpt for hereditary breast and ovarian cancer.14 this study has several limitations . 
consequently , these ndings may not be generalizable to the individuals with cdh1 variants . broader population of furthermore , analyses used self - reported data regarding individuals medical and genetic testing experiences . 
nonetheless , this research extends beyond past work , which has largely focused on cdh1 mutation carriers prophylactic surgical decision making and adjustment , 8 , 32 - 34 to provide novel insight into the experiences of individuals with cdh1 variants in the era of mgpt . in conclusion , although many with cdh1 pv or vus are satised with their decision to pursue genetic testing , these individuals have unique informational and psychosocial needs . 
low levels of reported recommendations for prophylactic gastrectomy and perceived gastric cancer risks among those with cdh1 pv are particularly worrisome , suggesting a need for strategies ( eg , innovative applications of risk communication principles , 35 - 37 enhanced provider genetics education38 ) to more effectively engage and educate patients and providers about relevant medical management and cancer risks . 
long employment : depuy synthes companies ( i ) stock and other ownership interests : depuy synthes companies ( i ) travel , accommodations , expenses : depuy synthes companies ( i ) fergus j . 
couch consulting or advisory role : astrazeneca research funding : grail travel , accommodations , expenses : grail , qiagen , ambry genetics research funding : novartis ( i ) , ambry genetics , susan g . 
komen for the cure ( i ) , aacr , diana helis henry medical foundation ( i ) , james p . wilmot foundation ( i ) , adrienne helis malvin medical research foundation ( i ) , global biological standards institute ( i ) , breast cancer research foundation mark e . 
robson honoraria : astrazeneca , pzer consulting or advisory role : mckesson , astrazeneca research funding : astrazeneca ( inst ) , myriad genetics ( inst ) , invitae ( inst ) , pzer ( inst ) , abbvie ( inst ) , tesaro ( inst ) , medivation ( inst ) travel , accommodations , expenses : astrazeneca susan m . 
jama oncol 1 : 23 - 32 , 2015 corso g , marrelli d , pascale v , et al : frequency of cdh1 germline mutations in gastric carcinoma coming from highand low - risk areas : metanalysis and systematic review of the literature . 
bmc cancer 12 : 8 , 2012 pharoah pd , guilford p , caldas c : incidence of gastric cancer and breast cancer in cdh1 ( e - cadherin ) mutation carriers from hereditary diffuse gastric cancer families . 
gastroenterology 121 : 1348 - 1353 , 2001 kaurah p , macmillan a , boyd n , et al : founder and recurrent cdh1 mutations in families with hereditary diffuse gastric cancer . 
jama 297 : 2360 - 2372 , 2007 van der post rs , vogelaar ip , carneiro f , et al : hereditary diffuse gastric cancer : updated clinical guidelines with an emphasis on germline cdh1 mutation carriers . 
national comprehensive cancer network : nccn clinical practice guidelines in oncology ( nccn guidelines ) : genetic / familial high - risk assessment : breast and ovarian , version 1.2019. 
hallowell n , badger s , richardson s , et al : an investigation of the factors effecting high - risk individuals decision - making about prophylactic total gastrectomy and surveillance for hereditary diffuse gastric cancer ( hdgc )  . 
fam cancer 15 : 665 - 676 , 2016 garland sn , lounsberry j , pelletier g , et al : how do you live without a stomach ? : a multiple case study examination of total gastrectomy for palliation or prophylaxis . 
hamilton jg , abdiwahab e , edwards hm , et al : primary care providers cancer genetic testing - related knowledge , attitudes , and communication behaviors : a systematic review and research agenda . 
harris pa , taylor r , thielke r , et al : research electronic data capture ( redcap ) - - a metadata - driven methodology and workow process for providing translational research informatics support . 
cella d , hughes c , peterman a , et al : a brief assessment of concerns associated with genetic testing for cancer : the multidimensional impact of cancer risk assessment ( micra ) questionnaire . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
balmaa j , digiovanni l , gaddam p , et al : conicting interpretation of genetic variants and cancer risk by commercial laboratories as assessed by the prospective registry of multiplex testing . 
robson me , bradbury ar , arun b , et al : american society of clinical oncology policy statement update : genetic and genomic testing for cancer susceptibility . oncol genomic med 4 : 232 - 236 , 2016 j clin oncol 33 : 3660 - 3667 , 2015 30 . 
katapodi mc , lee ka , facione nc , et al : predictors of perceived breast cancer risk and the relation between perceived risk and breast cancer screening : a meta - analytic review . 
hallowell n , lawton j , badger s , et al : the psychosocial impact of undergoing prophylactic total gastrectomy ( ptg ) to manage the risk of hereditary diffuse gastric cancer ( hdgc )  . 
worster e , liu x , richardson s , et al : the impact of prophylactic total gastrectomy on health - related quality of life : a prospective cohort study . 
odoherty k , suthers gk : risky communication : pitfalls in counseling about risk , and how to avoid thej genet couns 16 : 409 - 417 , 2007 36 . 
wolfe cr , reyna vf , widmer cl , et al : efcacy of a web - based intelligent tutoring system for communicating genetic risk of breast cancer : a fuzzy - trace theory approach . 
 appendix hamilton et al sent survey invitation ( n = 135 ) survey respondents ( n = 71 ) excluded : test report indicated no cdh1 result ( n = 3 ) survey respondents with self - reported understanding of their cdh1 result ( n = 68 ) excluded : respondent did not provide site - specific variant or test report respondent did provide sitespecific variant but it could not be found within the cdh1 gene in clinvar ( n = 5 ) ( n = 3 ) excluded : respondent self - reported understanding of their cdh1 result as my test results are not clear to me respondent self - reported understanding of their cdh1 result as negative genetic test result respondent missing responses to greater than 20% of study variables ( n = 4 ) * ( n = 3 ) ( n = 3 ) evaluable sample for analysis of disagreement among commercial laboratories in variant classifications ( n = 60 ) evaluable sample for analysis of how pv or vus cdh1 results are associated with medical and psychosocial experiences ( n = 57 ) excluded : respondent self - reported understanding of their cdh1 result as my test results are not clear to me ( n = 4 ) * evaluable sample for analysis of disagreement between participant self - report and clinvar classification ( n = 56 ) fig a1 . 
study diagra ( * ) indicates that these excluded cases are the same ; these cases are repeated in the gure to more clearly explain the derivation of the samples used in the study analyses . 
yeager , md1 ; jill fitch , md1 ; joseph stanek , ms1 ; deipanjan nandi , md1 ; and susan vear , md1 introduction ( fu ) nasopharyngeal carcinoma ( npc ) is a rare pediatric malignancy . 
npc in pediatric patients has an incidence of 0.5 to 2 per 100 , 000 persons per year , with only 1% occurring in patients younger than 19 years.1 most cases are treated with chemotherapy such as anthracyclines or pyrimidine uorouracil conjunction with radiation . 
cardiotoxicity , both primary and secondary to severe coronary vasospasm , is an infrequent but well - reported adverse effect of fu.2 life - threatening cardiotoxicity secondary to fu is reported at a frequency of less than 1% across all age groups.3 continuous infusion and concomitant use of cisplatin increases fu cardiotoxicity risk.2 , 4 , 5 pharmacogenomic studies have associated dihydropyrimidine dehydrogenase ( dpyd ) deciency with an increased risk of fu cardiotoxicity.6 - 8 dpyd is the initial and rate - limiting enzyme in the catabolism of fu . 
variants in thymidylate synthase ( tyms ) have been associated with up to a 2.5 - fold risk of toxicity in some studies , whereas others have shown no association with toxicity.9 - 11 to date , to our knowledge , there has been one reported case of a patient with severe cardiomyopathy who had variants in both dpyd and tyms.12 uridine triacetate ( ut ) is a safe and effective antidote when administered quickly after fu overdose , 13 but there are few reports of its use in children . 
on day 3 of her rst 96 - hour fu infusion , the patient developed sudden onset of acute tachycardia , and chest pain , shortness of breath , hemodynamic instability . 
her troponin level was initially mildly elevated at 0.764 ng / ml and ecg demonstrated changes consistent with coronary ischemia , including new q waves , st segment elevation , and t - wave inversions . 
fu infusion was stopped after the administration of a total of 4 , 800 mg ( 1 , 000 mg / m2 / d dosing ) following the administration of 75 mg / m2 cisplatin on day 1 of her therapy . symptoms resolved within a 10 - hour period , and troponin levels decreased to 0.566 ng / ml with improved perfusion and acidosis . 
st segment changes and t - wave inversions initially noted on the patients ecg improved on repeated ecg ( appendix fig a1 )  . given this improvement and stable clinical status , the patients treatment was transitioned to milrinone and she was weaned off dobutamine . 
epinephrine was required to maintain normotension and her chest pain and respiratory distress persisted . given the known association of fu with direct cardiotoxicity and coronary vasospasm with resultant angina and dysfunction , a nitroglycerin drip was initiated . 
ut was started 48 hours after her last dose of fu and within 24 hours after the repeated echocardiograut was dosed at 6.2 g / m2 by mouth every 6 hours for a total of 20 doses . 
because ut is only available in enteral form , it was not started earlier because of worsening respiratory distress and the need for noninvasive positive pressure ventilation , as well as initial concerns for poor gi perfusion . 
symptoms improved and she was weaned off milrinone and nitroglycerine . her troponin levels continued to increase while receiving ut , despite her improved symptoms and echocardiogram ndings ( fig 1 ) ; they eventually normalized weeks later . 
 belsky et al context key objective knowledge generated relevance to consider the effects of variants in dpyd and tyms in association with pyrimidine uorouracil ( fu ) cardiotoxicity in a pediatric patient . 
this case involving these variants adds to the literature evidence of successful uridine triacetate administration in a pediatric patient . the unique genotype of dpyd and tyms in a pediatric cancer patient with cardiotoxicity secondary to fu is presented . 
this case demonstrates the successful use of uridine triacetate in a pediatric patient with heart failure associated with fu treatment . these ndings can be applied to patients with cardiac failure who are receiving fu . 
pharmacogenomics is a rapidly changing eld and as medicine becomes more personalized , clinicians should be aware of variants associated with possible cardiac toxicity before administering fu . symptoms and cardiac dysfunction , the patient was weaned from nitroglycerin and milrinone and maintained on carvedilol . she was discharged home 10 days after her initial clinical decompensation . because of the unique nature of her cardiotoxicity , after consulting with our institutes clinical pharmacology and pharmacogenomics advisory committee , a fu pharmacogenomics panel was sent to arup laboratories ( salt lake city , ut ) after the patient underwent chemotherapy and recovery of wbcs to normal range . 
pharmacogenomic testing revealed two polymorphisms in tyms : heterozygous deletion in the 3 ( cid : 1 ) region ( rs16430 ) , associated with intermediate expression , and a g  . 
the patient was ut start ut stop 1 , 200 mg of fu administered ejection fraction , % 132 96 60 24 12 time since initial ut ( hours ) 84 120 fig 1 . 
ut was approved by the us food and drug administration in december 2015 as a safe and highly effective antidote when administered within 96 hours after fu or capecitabine overdose.13 in one clinical trial , 96% of 135 patients with fu toxicity recovered after treatment , compared with 10% in a historical control group.14 from a pharmacogenomics perspective , variants of tyms have been associated with up to a 2.5 - fold risk of toxicity with fu in some studies but were not associated with increased toxicity in others . 
adult case reports have suggested the importance of pharmacogenomic testing for tyms and dpyd mutations to help toxicity in identied personalize therapy and prevent patients.13 to date , there have been published data on dosing guidelines for fu based on dpyd genotype ; however , no dose adjustment guidelines exist for tyms variants . with these data , we opted to change therapy from fu to gemcitabine and the patient had successful remission of her npc . 
also , given the quick resolution of symptoms with primary vasodilator and scavenger use as well as rapid resolution of cardiac function , an acute cardiomyopathy is thought to be less likely . although the patient experienced relief of symptoms coinciding with the administration of ut , the denitive cause of her decompensation cannot be determined . 
although additional studies would be helpful to better identify risk factors for cardiotoxicity in this patient population and determine the need for pharmacogenomic testing in pediatric patients who receive fu , it is unlikely that such studies would be feasible because of the rarity of presentation . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / author - center . no potential conicts of interest were reported . references fandi a , cvitkovic e : biology and treatment of nasopharyngeal cancer . 
j clin pharmacol 33 : 1060 - 1070 , 1993 polk a , vaage - nilsen m , vistisen k , et al : cardiotoxicity in cancer patients treated with 5 - uorouracil or capecitabine : a systematic review of incidence , manifestations and predisposing factors . 
cancer treat rev 39 : 974 - 984 , 2013 l evy e , piedbois p , buyse m , et al : toxicity of uorouracil in patients with advanced colorectal cancer : effect of administration schedule and prognostic factors . j clin oncol 16 : 3537 - 3541 , 1998 van kuilenburg ab , meinsma r , zoetekouw l , et al : high prevalence of the ivs14 + 1g.a mutation in the dihydropyrimidine dehydrogenase gene of patients with severe 5 - uorouracil - associated toxicity . 
pharmacogenetics 12 : 555 - 558 , 2002 saif mw : dihydropyrimidine dehydrogenase gene ( dpyd ) polymorphism among caucasian and non - caucasian patients with 5 - fuand capecitabine - related toxicity using full sequencing of dpyd . 
cancer genomics proteomics 10 : 89 - 92 , 2013 campbell jm , bateman e , peters md , et al : fluoropyrimidine and platinum toxicity pharmacogenetics : an umbrella review of systematic reviews and metaanalyses . 
pharmacogenomics 17 : 435 - 451 , 2016 shahrokni a , rajebi mr , saif mw : toxicity and efcacy of 5 - uorouracil and capecitabine in a patient with tyms gene polymorphism : a challenge or a dilemma ? clin colorectal cancer 8 : 231 - 234 , 2009 10 . 
saif mw , smith m , maloney a : the rst case of severe takotsubo cardiomyopathy associated with 5 - uorouracil in a patient with abnormalities of both dihydropyrimidine dehydrogenase ( dpyd ) and thymidylate synthase ( tyms ) genes . 
ma ww , saif mw , el - rayes bf , et al : emergency use of uridine triacetate for the prevention and treatment of life - threatening 5 - uorouracil and capecitabine 14 . 
santos c , morgan bw , geller rj : the successful treatment of 5 - uorouracil ( 5 - fu ) overdose in a patient with malignancy and hiv / aids with uridine triacetate . pharmacogenomics j 8 : 278 - 288 , 2008 100 : 1549 - 1557 , 2009 toxicity . 
initial ekg with t wave inversions in inferior leads ( ii , iii , avf ) , q waves in v1 , v2 , and st segment elevation in v3 , v4 . these appear to be stable and / or improving in follow - up ekg no.1 , although st segment elevation is noted again v2 and v3 in follow - up ekg no.2. 
the latest innovations in clinical trials have followed with master protocols , which are dened by inclusive eligibility criteria and devised to interrogate multiple therapies for a given tumor histology and / or multiple histologies for a given therapy under one protocol . 
conventionally conducted within the phase ii setting , basket designs have become popular as drug developers seek to effectively evaluate and identify preliminary efcacy signals among clinical indications identied as promising in preclinical study . 
the latest innovations in clinical trials have followed with master protocols , which are dened by inclusive eligibility criteria and devised to interrogate multiple therapies for a given tumor histology and / or multiple histologies for a given therapy under one protocol.1 , 2 the use of master protocols for oncology has become more common with the desire to improve the efciency of clinical research and accelerate overall drug development . 
the lung - map study , for example , used a master protocol for a phase ii / iii comparative trial designed to evaluate biomarkermatched therapies in patients with previously treated advanced squamous nonsmall - cell lung cancer.3 shortly after initiation , the us food and drug administration approved a new immunotherapy for the same population . 
the designs exibility facilitated subsequent modication of the standard of care , which preserved the relevance of the study.1 a second example includes the create study master protocol , which evaluated the efcacy of crizotinib in patients with alk or met mutations among six different tumor histologies , where each histology constituted a subtrial of alk / met - positive or alk / met - negative tumors.2 emerging cancer therapies may target relatively rare molecular subtypes , which poses practical challenges to completing a clinical trial devised to enroll a single clinical indication . 
second , biomarkerguided treatment selection supersedes traditional clinical indicators for the studied populations such that the presence of a molecular marker predicts response to a targeted therapy irrespective of tumor histology.2 , 4 , 5 thus , basket trials are designed to enroll multiple clinical subpopulations in whom it is assumed that the therapy in question offers benecial efcacy in the presence of the targeted molecular prole . 
most often conducted within the phase ii setting , basket designs have become popular as drug developers seek to effectively evaluate and identify preliminary efcacy signals among clinical indications identied as promising in preclinical study.6 - 8 in certain circumstances , however , evidence acquired from a basket trial has served as the basis for regulatory approval.9 , 10 the framework also poses new challenges to statistical analysis . 
 kaizer et al context key objective describe recent developments in the design of basket trials for oncology studies and elucidate several issues that pertain to their statistical analysis and design . knowledge generated basket trials have evolved from their initial use in exploratory contexts to conrmatory , randomized , and platform trials . 
the context of each trial requires consideration of statistical properties for trial design ( strength and type of type i error control and implications for power ) and methods for analysis ( to pool clinical indications with a common target or accommodate the potential for heterogeneous activity among tumor histologies )  . relevance advances in genomics , disease pathways , and drug discovery have progressed into clinical cancer research . 
innovations in trial design with basket trials facilitate interrogations of multiple therapies for a given tumor histology and / or multiple histologies for a given therapy - target combination that bridges aspects of translational and clinical research paradigms . 
molecularly targeted treatment strategies may not offer acceptable efcacy to all putatively promising clinical indications . proliferation rates of tumor cells are known to vary within and between tumor types.11 moreover , contemporary sequencing methodologies have revealed extensive genetic variation among different types of cancer . 
as drug developers pursue drug approvals with broad labels ( eg , pembrolizumab for which indication was expanded in 2017 to include any unresectable or metastatic solid tumors with microsatellite instability , 12 larotrectinib in 2018 for adults and children who harbor ntrk gene fusion without a known acquired resistance mutation13 ) , trials will evaluate multiple disease indications for agnostic effects . 
for these trials , subpopulation analysis becomes central to the study design and should be considered in the evaluation of trial operating characteristics . trials , discusses this article reviews designs of basket several issues that arise with their implementation , and examines the statistical criteria considered in the development of such trials . 
this section discusses examples of exploratory trials designed as basket trials and introduces recent extensions that convey the evolving landscape of basket trial design in oncology . exploratory designs most basket trials have been conducted within exploratory settings to evaluate agent - specic estimates of tumor response . 
cunanan et al5 described three studies implemented in oncology settings that extend the basic formulation of a basket trial to multiple targets and / or agent combinations . the nci - match trial used a master protocol to enroll participants into drugand mutation - specic baskets while also incorporating genetic screening . 
a basket trial approach was selected to accommodate the rare disease setting , which is limited by traditional large prospective trials.14 the custom trial was a phase ii study with a two - stage design that aimed to identify molecular biomarkers and determine their clinical relevance in patients expected to benet from multiple targeted therapies.15 these trials facilitated the study of less common types of malignancies . 
in addition , these studies were devised to identify potential baskets in which the respective therapies demonstrated promising efcacy and , thus , were exploratory . extensions to randomized , conrmatory designs the methodology for basket trials has been extended to accommodate a wide variety of potential motivations beyond exploratory studies . 
one such extension includes randomized design such that participants are randomly assigned to either a standard - of - care arm without regard to specic genetic mutations or to an arm where a specic mutation is hypothesized to be susceptible to targeted treatment.16 , 17 these designs test the hypothesis that biomarker - guided selection demonstrates benecial treatment efcacy . 
 basket designs : statistical considerations for oncology trials fusion of basket and platform trials may accommodate exploratory and conrmatory study.18 one example of this approach is the signature program , which included a series of eight agent - specic , open - label , phase ii basket trials under a single overarching master protocol.21 the signature program was tissue agnostic and driven by molecular indications with the intention to demonstrate the feasibility of rapid signal nding across multiple tumors . 
molecularly targeted treatment strategies , however , may not offer acceptable efcacy to all putatively promising clinical trials pose challenges to the traditional paradigm for trial design and analysis , which assumes that individual patients who enroll in the same clinical study represent exchangeable units that can be averaged . 
imatinib , for example , was shown to be efcacious in treating malignancies , such as philadelphia chromosomepositive chronic myelogenous leukemia , through the dysregulation of imatinib - sensitive protein tyrosine kinases.23 to determine whether other malignancies with similar kinases respond to treatment , a single - arm , open - label , phase ii study was conducted with inclusive enrollment across 40 different pathologic diagnoses.24 neither the number of indications was constrained nor the design devised to examine subpopulation - specic effects . 
the study represents an example of the challenges posed by indication heterogeneity with conventional study design ( eg , see lacombe et al25 ) , the prediction of which may not be feasible in advance of a studys implementation . the opposite end of the spectrum is dened by studies that evaluate the effectiveness of a treatment strategy for a given subpopulation independent of the evidence acquired from patients in differing patient subsets . 
subpopulation - specic inference also fails to delineate which patient subtypes should be considered nonexchangeable on the basis of the observed data . for example , upon establishing that melanomas that exhibit braf v600 mutations respond to vemurafenib , 26 a basket trial was designed to evaluate the effectiveness of vemurafenib in patients without melanoma , with baskets dened by primary tumor origthe study was devised to interrogate the effectiveness of vemurafenib independently among patients who harbor braf v600 mutations within several prespecied tumor organ sites.5 , 9 despite having designed the study with success criteria that were invariant by tumor indication , the studys analysis plan evaluated evidentiary measures of effectiveness independently for each basket . 
with some baskets having observed low accrual and others yielding one or no response , overall conclusions are difcult to ascertain.27 the polarities of independence and pooling require strong a priori assumptions , which yield poor statistical designs when violated . 
these include multistage designs that merge subtypes at interim analyses28 or bayesian adaptive designs with hierarchical modeling strategies.29 hobbs and landin30 adapted the multisource exchangeability model ( mem ) , 31 which facilitates bayesian inference with respect to all possible pairwise exchangeability relationships among the studied subpopulations . 
the design enables the identication of disjointed subpopulations that comprise meta - subtypes or singleton subtypes on the basis of accumulating evidence during the course of study . design criteria and operating characteristics a basket designs operating characteristics should be determined by the trials intention and phase of development . control of falsely concluding that an inferior experimental therapy offers benet to any clinical indication should be emphasized for conrmatory trials that enroll multiple indications . 
statistical designs of clinical studies are conventionally devised in consideration of distributions of test statistics that measure the extent of evidence acquired against a prespecied null hypothesis upon repeated sampling of the trial data for a given decision rule . 
for hypotheses of superiority , null hypotheses dene value ( s ) of statistical parameters for which the underlying true state of nature is characterized by the absence of treatment effectiveness . 
for one - sample the null often is represented by a xed , prestudies , specied value that represents an expected response rate considered inadequately low to justify additional investigation . 
the decision rule determines the extent to which the trials conclusions could be explained as a result of chance . in this context , a type i error occurs when the null hypothesis is true but the trial yields data for which the decision rule rejects the null hypothesis in favor of a positive nding . 
for basket trials designed to examine multiple indications , the possibility of committing a type i error may occur for tests of each subpopulation , yielding two types of false conclusions : marginal type i error rates estimate the type i error rate for each indication separately , whereas family - wise type i error rates consider the entire trial violation if a single indication falsely rejects the null hyrepresents stronger pothesis . 
when selecting a statistical design devised to enroll and evaluate multiple indications , trialists must synthesize the trials operating characteristics with respect to a diverse array of possible trial outcomes , which we denote hereafter as scenarios . 
the strongest control is therefore conferred by the scenario with only one null indication . decisions that pertain to statistical approaches for analysis of multiple subpopulations , expected enrollment distribution , type i error method , and the strength of type i error control have implications for a designs statistical power . 
in the absence of heterogeneity among indications , the pooling of data acquired from all indications has the potential to increase statistical power ( identify smaller effect sizes )  . 
in addition , increasing imbalance among subpopulation sample sizes may reduce power and / or inate bias under strategies that enable data pooling . hierarchical models devised to share information across indications in relation to estimates of statistical exchangeability have been proposed by various authors18 , 35 - 38 with the intention of increasing statistical power while controlling bias . 
hierarchical models were calibrated for strong control of type i error such that marginal type i error was controlled under scenarios for which only one ( of ve or 10 ) subpopulation was truly ineffective . 
it should be noted , however , that under scenarios with multiple null subpopulations , the designs that resulted from hierarchical models demonstrated both commensurate power and reduced type i error when compared with subpopulation - specic analyses . 
in some cases , drastic reductions in type i error were observed for hierarchical models , such as the global null scenario , where strong borrowing reduced the type i error rate from a target of 10% to approximately 1% when considering 10 indications.39 moreover , the authors failed to adjust frequentist tests for multiple comparisons , which would have attenuated their power . 
in the presence of limited or imbalanced enrollment , however , one may wish to combine subpopulations or use statistical methods to facilitate information sharing among patient subpopulations.5 , 30 guidelines for evaluating the operating characteristics of subpopulation analyses are needed for both conrmatory and nonconrmatory settings.33 , 34 to provide an illustration of the existing strategies and their trade - offs , a simulation study was completed for a xedsample basket trial with ve indications each enrolling 25 patients . 
type i error , however , inates quickly to unacceptable levels in the presence of basket heterogeneity . the independent analysis approach , which adjusts for multiplicity , maintains family - wise error rates , 5% , but it is at the expense of power ( only 0.66 probability of identifying any active basket as efcacious )  . 
finally , the analysis of each basket independently and failure to adjust for multiple comparisons yielded high power ( 0.91 ) with large familywise error rates for all scenarios except the one with four active baskets ( ie , one null basket )  . the method for calibrating basket results for the bayesian model are provided for three posterior probability decision thresholds to illustrate the exibility of trials of various types . 
calibration 1 maintains a marginal type i error at 10% with no active baskets as well as increased probability for identifying effective baskets relative to independent approaches with two or more active baskets . family - wise error and marginal type i error rates are inated with fewer active baskets , however . 
reported is the basket - specic probability of being declared efcacious for null ( nonbold ) and active ( bold ) baskets for multisource exchangeability models ( mems ) with different pp threshold calibrations . 
this stands in contrast multiplicity ( low power of 0.66 ) or do not adjust for multiplicity ( higher power at the expense of higher family - wise error rates of 40% )  . 
the three calibrations presented were selected to demonstrate acceptable designs for different types of basket trials , with calibrations 1 and 2 formulated for exploratory settings and calibration 3 appropriate for conrmatory trials . discussion subpopulation heterogeneity is intrinsic to evaluations of cancer therapies that emerge within the evolving oncology landscape . 
basket trials provide a natural framework for studying molecularly targeted treatment strategies in several potentially promising clinical subpopulations while acknowledging the possibility of heterogeneous results . initiated in exploratory settings , basket designs have evolved to include randomization , enrichment of the enrolled population , and incorporation to platform trials and even acquire conrmatory evidence . 
further innovations and renements may make it possible to use the framework across the full spectrum of clinical translation with seamless design.19 as our simulation study demonstrates , basket trials should require analysis strategies and designs that acknowledge the potential for subpopulation effects . 
several aspects of basket trials , including the recommendations for weak versus strong control of type i error and adjustment for multiplicity , require additional consideration and guidance from academic and regulatory leaders to promote best practices for each stage of the research process.33 as basket trials evolve from simple exploratory trialists trials to increasingly complex master protocols , should avoid simple replication of designs from prior study and instead adapt the trial to its context with appropriate assumptions for the phase of inquiry . our simulation study demonstrates that nave pooling of all subpopulations results in unacceptable type i error , whereas independent examination of each basket without correction for multiplicity may result in inated type i error or , conversely , decreased power to detect efcacious baskets when correcting for multiplicity . 
the mem was evaluated with three different posterior probability threshold calibrations , each demonstrating appropriate operating characteristics for basket trials devised for exploratory and conrmatory settings . further improving their accessibility is the availability of the basket package in r to implement the symmetric mem approach.41 these results stand in contrast to challenges and limitations to information sharing previously reported.34 , 39 one should note a few limitations , however , that remain unresolved by the basket trial framework . 
patients described by a common subpopulation may exhibit sources of heterogeneity on the basis of other important considerations , such as immune prole , prior treatment histology , informatics that redemographics , or additional clinical quire patient - level data analysis . 
for a single tumor histology , this can yield insufcient efcacy not because the drug is necessarily ineffective for the indication but because the subtype in the trial happened to enroll a disproportionate number of patients with refractory disease.27 in settings where indication heterogeneity is not only known but also well understood , an effective means for addressing this type of heterogeneity is to construct baskets derived from information contained in clinical and auxiliary data sources . 
given sufcient enrollment , an indication can be partitioned into prognostic subpopulations and thereby provide new avenues for delineating drug versus prognostic effects at the subpopulation level without randomization . basket trials , like many master protocols , require tissue analysis and molecular proling before registration , which often limits their scope to a small number of leading academic institutions . 
molecular match rates for trials of targeted therapies have been reported to be as low as 4%.42 a single - institution study of the msk - impact assay evaluated 1 , 932 patients and a span of 49 cancer types in the real - world setting and in a matched found that 13% of patients were enroll study.43 by facilitating a centralized platform for acquiring and processing genomic data in conjunction with their functional and therapeutic implications , basket trials may overcome such deciencies . 
this has prompted interrogations of electronic health recordderived measures of real - world progression as alternatives to the response evaluation criteria in solid tumors ( recist ) .44 with the emergence of data science and machine learning , pathologic and radiologic data may offer new avenues for rapid , noninvasive patient proling . 
innovations in radiomics , for example , have effectively yielded prognostic signatures from informatics acquired from scanning technologies used routinely in clinical settings.45 - 48 to address the potential for heterogeneity within a clinical indication , innovations that facilitate use of noninvasive biomedical informatics and algorithms should be pursued with the aim of developing prognostic models in concert with the trials design . 
koopmeiners patents , royalties , other intellectual property : listed as an inventor for us patent 9 , 858 , 665 , which describes a computer - aided design system for predicting prostate cancer using magnetic resonance imaging satrajit roychoudhury employment : pzer leadership : pzer stock and other ownership interests : pzer , novartis david s . 
hong stock and other ownership interests : molecularmatch , oncorena , presagia honoraria : adaptimmune , baxter , merrimack , bayer ag consulting or advisory role : baxter , bayer ag , guidepoint global , janssen pharmaceuticals , genentech , eisai , glg , alpha insights , axiom biotechnologies , adaptimmune , grouph , merrimack , medscape , numab , pzer , seattle genetics , takeda pharmaceuticals , trieza therapeutics research funding : novartis , genentech , eisai , astrazeneca , pzer , mirna therapeutics , amgen , daiichi sankyo , merck , mirati therapeutics , eli lilly , adaptimmune , abbvie , bayer ag , bristol - myers squibb , genmab , ignyta , innity pharmaceuticals , kite pharma , kyowa hakko kirin , loxo oncology , medimmune , molecular templates , takeda pharmaceuticals , seattle genetics , amgen , fate therapeutics , mologen , nci - ctep travel , accommodations , expenses : loxo oncology , mirna therapeutics , genmab brian p . 
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ann oncol 28 : 34 - 43 , 2017 steuer ce , papadimitrakopoulou v , herbst rs , et al : innovative clinical trials : the lung - map study . 
clin pharmacol ther 97 : 488 - 491 , 2015 redig aj , j anne pa : basket trials and the evolution of clinical trial design in an era of genomic medicine . 
j clin oncol 33 : 975 - 977 , 2015 cunanan km , gonen m , shen r , et al : basket trials in oncology : a trade - off between complexity and efciency . 
nakamura y , komatsu y , kato k , et al : btmb - high basket trial : a multicenter phase ii trial of nivolumab monotherapy in patients with advanced gastrointestinal cancers with high blood tumor mutational burden ( btmb )  . 
j clin oncol 37 , 2019 ( suppl ; abstr tps179 ) diamond el , subbiah v , lockhart ac , et al : vemurafenib for braf v600 - mutant erdheim - chester disease and langerhans cell histiocytosis : analysis of data from the histology - independent , phase 2 , open - label ve - basket study . 
sch offski p , wozniak a , escudier b , et al : crizotinib achieves long - lasting disease control in advanced papillary renal - cell carcinoma type 1 patients with met mutations or amplication . 
lopez - chavez a , thomas a , rajan a , et al : molecular proling and targeted therapy for advanced thoracic malignancies : a biomarker - derived , multiarm , multihistology phase ii basket trial . 
chen c , li x , yuan s , et al : statistical design and considerations of a phase 3 basket trial for simultaneous investigation of multiple tumor types in one study . 
heinrich mc , joensuu h , demetri gd , et al : phase ii , open - label study evaluating the activity of imatinib in treating life - threatening malignancies known to be associated with imatinib - sensitive tyrosine kinases . 
freidlin b , korn el : borrowing information across subgroups in phase ii trials : is it useful ? clin cancer res 19 : 1326 - 1334 , 2013 40 . 
grifth sd , tucker m , bowser b , et al : generating real - world tumor burden endpoints from electronic health record data : comparison of recist , radiologyanchored , and clinician - anchored approaches for abstracting real - world progression in non - small cell lung cancer . 
yu w , tang c , hobbs bp , et al : development and validation of a predictive radiomics model for clinical outcomes in stage i non - small cell lung cancer . 
lacking multivariable specication of relationships among subpopulations , conventional analysis strategies represent the extremes of either pooling all indications or analyzing each indication independently of the evidence acquired for other indications . 
more specically , pooling in this context refers to an analysis strategy devised to evaluate evidence of effectiveness for all indications through statistical inference of a single response rate that is estimated using data from all indications . 
by relying on only one test , marginal and family - wise type i error rates are identical for the pooled approach . at the other extreme , independent analyses infer a total of ve response rates estimated from the data observed within specic indications . 
our simulation study considers the constrained empirical bayes ( ceb ) priors for symmetric mems described by hobbs and landin.30 briey , unconstrained empirical bayes approach to prior specication identies and conditions the analysis with respect to the mem that maximizes the marginal data likelihood . 
while acknowledging the uncertainty about identifying the true mem from the marginal likelihood , the ceb prior constrains shrinkage using an upper bound ( ub ) on the prior probability of inclusion for each source . 
for the basket trial with ve indications , there are six possible scenarios that range from all indications being null ( scenario 1 ) to all indications being alternative ( scenario 6 ) and four intermediate combinations ( scenarios 2 to 5 )  . 
for the bayesian mem approach with ub = 0.10 , posterior probability thresholds were identied that maintained the basket - specic ( marginal ) type i error rate under the global null scenario , the type i error rate for the scenario with one null basket , and the family - wise type i error rate for the global null scenario . 
 o ros1 gene fusion in advanced lung cancer in women : a systematic analysis , review of the literature , and diagnostic algorithm purpose crizotinib , a mesenchymal - epithelial transition / anaplastic lymphoma kinase / c - ros oncogene 1 ( ros1 ) inhibitor , has recently been approved by the us food and drug administration for the treatment of patients with advanced ros1 - positive nonsmall - cell lung cancer ( nsclc )  . 
limited data are available on the prevalence and distribution of ros1 fusions in patients with advanced - stage nsclc . material and methods a series of 727 lung adenocarcinomas from patients with stage iv disease , negative for epidermal growth factor receptor and anaplastic lymphoma kinase alterations , were tested for ros1 fusions by fluorescent in situ hybridization analysis , with confirmation by immunohistochemistry . 
2017 by american society of clinical oncology introduction the identification of oncogene - driven subtypes of nonsmall - cell lung cancer ( nsclc ) has opened the way to clinical application of new classes of anticancer agents directed against specific biologic targets.1 two main molecular aberrations of nsclc were first identified that now have targeted therapies approved for their treatment : mutations in the epidermal growth factor receptor ( egfr ) gene and rearrangements in the anaplastic lymphoma kinase ( alk ) gene.2 , 3 in august 2011 , the us food and drug administration approved crizotinib ( xalkori , pfizer , new york , ny ) to treat locally advanced or metastatic nsclcs that express the abnormal alk fusion protein.4 more recently , another trans - membrane tyrosine kinase receptor gene , the c - ros oncogene 1 po.ascopubs.org jco precision oncology antonio marchetti massimo barberis alessia di lorito maria vittoria pace chiara di lisio lara felicioni elena guerini - rocco andrea vingiani tommaso dantuono marcella liberatore giampaolo filice graziano de luca filippo de marinis antonio passaro luigi guetti luciana irtelli lucio crin `o felice mucilli fiamma buttitta author affiliations appear at the end of this article . corresponding author : fiamma buttitta , md , phd , oncological and cardiovascular molecular medicine unit , cesimet , g . 
 ( ros1 ) , encoding a protein with high homology with alk in its protein kinase domain , has been shown to be altered in a subset of patients with nsclc.5 the genetic alteration occurs in particular gene fusions that render ros1 a constitutively active tyrosine kinase protethe homology between the binding sites of alk1 and ros1 warranted exploration of crizotinib in patients carrying ros1 fusions . 
in a series of clinical studies , patients with advanced nsclc harboring ros1 rearrangements showed great benefit from crizotinib treatment , 6 with an objective response rate of 60%.7 on the basis of the data obtained in these clinical trials , in march 2016 , the food and drug administration expanded the use of crizotinib to include patients with metastatic nsclcs whose tumors have ros1 gene alteration.8genetic changes in ros1 are rare events , affecting approximately 0.5% to 2% of unselected patients with nsclc.5 these prevalence data have been obtained in previous studies that have mainly been conducted retrospectively on resected samples.9 - 13 only a limited number of studies have prospectively investigated ros1 fusions in patients with advanced - stage tumors for treatment selection.14 , 15 ros1 alterations may be detected with a variety of techniques but , currently , most laboratories rely on fluorescence in situ hybridization ( fish ) assays using a dualcolor break - apart probe design . 
given the rarity of ros1 rearrangements in nsclc , screening of tumors by immunohistochemistry ( ihc ) may allow unnecessary fish analyses in patients with ros1 - negative disease to be avoided and thus substantially reduce the cost of testing . although ihc is widely considered a sensitive detection method , it has been shown that in some cases , it can fail in the detection of ros1positive disease . 
given that an efficient assessment of ros1 status in patients with nsclc is important for directing patient care , a combination of different technologies has been suggested to avoid leaving behind patients for treatment . 
however , a multiple technical approach could further increase the costs of diagnosis if indiscriminately applied.16 , 17 the possibility of clinically selecting subclasses of patients with lung cancer with a high probability of harboring ros1 fusions could allow for an accurate evaluation of these patients with multiple diagnostic assays after a screening test . in this study , a large series of advanced - stage lung adenocarcinomas from patients with inoperable disease , selected by clinicians for centralized ros1 testing , were prospectively investigated by fish analysis . 
the study was conducted in accordance with the precepts of the helsinki declaration . ros1 fish and ihc analysis ros1 fish analysis was performed on unstained 4to 5 - micron , formalin - fixed , paraffinembedded tumor tissue sections using the zytolight spec ros1 dual color break apart probe ( zytovysion , bremerhaven , germany ) or the poseidon repeat free ros1 break probe ( kreatech diagnostics , netherlands ) after pretreatment and denaturation steps , according to the manufacturers protocols . 
the orange fluorochrome directlabeled probe hybridizes distal to and the green fluorochrome direct - labeled probe hybridizes proximal to the ros1 break point region at 6q22.1. fish analysis was performed with a slide scanning system under a 603 oil immersion objective with a fluorescence microscope ( olympus bx61 ; olympus corporation , tokyo , japan ) , equipped with appropriate filters , a charge - coupled device camera , and the fish imaging and capturing software solotouch ( bioview duet ; bioview , rehovat , israel )  . 
more than 50 cancer cells per patient were scored , and signals were evaluated with the imaging systetumor samples were considered ros1 fish positive if more than 15% of the tumor cells showed split red and green signals ( signals separated by one or more signal diameters ) and / or single green 39 signal ( deleted green signal ) , in addition to fused and / or broken - apart signals . 
to be included , each study had to meet all of the following criteria : ( 1 ) be performed on series of lung adenocarcinomas investigated for ros1 fusions ; ( 2 ) provided data about the status of the tumor at diagnosis ( early - stage / resectable v latestage / unresectable ) to distinguish between studies performed on patients who underwent surgical resection and studies performed on patients with advanced disease to be selected for pharmacologic treatment ; and ( 3 ) provided the total number of patients and number of patients harboring the ros1 fusion gene in two categorical groups ( positive / negative ) stratified by clinical variables , that is , sex , smoking status , and age at diagnosis . the variables measured in the study were investigated for association using fishers exact test or x2 test , as appropriate . 
the associations of ros1 fusion , as the dependent variable , with sex , age at diagnosis , and smoking history ( smoker or former smoker v nonsmoker ) were also investigated by logistic regression analysis to account for the effect of the different variables . 
in females , ros1 was found to be rearranged in 27 of 266 patients ( 10.2% ) , whereas the prevalence of ros1 fusion in the 461 males was 0.4% ( p = 1.2e - 10 , fishers exact test )  . 
of the 583 smokers , 11 ( 1.9% ) were ros1 positive , whereas 18 of the 144 nonsmokers ( 12.5% ) showed an ros1 rearrangement ( p = 4.05e - 7 , fishers exact test ; table 1 )  . 
the association of ros1 fusion with sex , age , and smoking history was also evaluated by logistic regression analysis to take into consideration the reciprocal effects of the covariables investigated . 
the highest probability of detecting a fusion involving the ros1 gene was in nonsmoking females ( 17% ) , followed by smoking females ( 6.4% ) and nonsmoking males ( 4% )  . 
in the present cohort of patients , 109 ( 15% ) were younger than 50 years of age . nineteen of these 109 patients ( 17% ) showed ros1 fusion , indicating a high prevalence of this genetic alteration in young patients with advanced lung cancer . 
in young female patients , 11 nonsmokers ( 39% ) and seven smokers ( 18% ) were found to be positive for ros1 fusions . our results were compared with those obtained in selected previous studies on ros1 fusions in patients with lung adenocarcinoma . 
on the basis of the inclusion criteria described in the materials and methods section , 13 studies were selected , including 11 studies on resected patients and two on patients with advanced - stage disease . 
 table 3 correlation of clinicopathologic variables with ros1 fusions in patients with resected and advanced - stage lung adenocarcinomas : data from the literature and this study first author no . 
of patients status total ros1 fusion ( n ) status total ( n ) ros1 fusion ( n ) smoking habits mean age ros + ( years ) resected cao18 cha19 chen20 kim21 kim22 lee23 li24 pan26 sholl27 shan16 mescam - mancini25 advanced stage go14 marchetti ( current study ) wu15 total no . 
 sex male female > 50 50 > 50 50 + + + + fig 2 diagram illustrating the risk of carrying an ros1 fusion in patients with late - stage lung adenocarcinomas . 
 , rare ; + , lowprevalence ; + + , medium prevalence ; + + + , high prevalence ; + + + + , very high prevalence . investigated in a conventional clinical workflow for the selection of patients for targeted therapy . 
seven hundred twenty - seven patients , found to be negative for egfr and alk alterations , were evaluated for ros1 gene fusions by fish analysis , with confirmation by ihc . ros1 rearrangements were detected in 29 patients ( 4% )  . 
univariable statistical analysis showed a higher prevalence of ros1 fusions in females ( all but two of 29 patients with positive results were women ) , younger patients , and nonsmokers . 
limited numbers of patients with ros1 - positive disease have been reported in previous studies , and a multivariable analysis taking into account the reciprocal relationship of these three main clinical parameters ( sex , age , and smoking habits ) has never been conducted.9 , 28 - 30 a multivariable logistic regression analysis in our series of patients with advanced lung adenocarcinoma clearly indicated a strong and independent association of ros1 fusions with female sex and younger age . 
the impact of sex , smoking history , and age on the probability of finding an ros1 - positive patient can be easily assessed on the diagrathe highest probability of detecting ros1 gene fusion in patients with lung adenocarcinoma is in nonsmoking females younger than 50 years of age ( high prevalence , near 40% )  . 
nonsmoking females older than 50 years of age showed a medium prevalence of ros1 fusions , together with nonsmoking males younger than 50 years of age . however , for the latter , the number of patients investigated was too low to draw conclusions , and additional data will be necessary to confirm this trend . 
the real - world possibility that ihc and fish , as with other nonin situ techniques ( reverse transcriptase polymerase chain reaction , next - generation sequencing ) , may fail to detect ros1 fusions strengthens the case for confirmation of results by a second if indiscriminately methodology . 
however , applied , a dual technical approach will increase costs and working time.16 , 17 the selection of patients at medium / high / very high risk in the diagram in figure 2 would substantially decrease the number of double assays required for accurate testing . 
the algorithm presented in figure 3 is proposed for an accurate selection of patients to be treated with ros1 inhibitors . for instance , according to this algorithm , a nonsmoking woman who tested negative at the first screening with ihc or fish should be retested with a second technology . our data on patients with advanced lung cancer were compared with data from the literature . because the current study was performed on patients affected by lung adenocarcinomas , only studies performed on patients with lung adenocarcinoma were considered . 
data from 11 independent studies performed on a total of 3 , 190 patients who had undergone resection were compared with those obtained from three studies , including the present one , that analyzed 918 patients with advanced - stage disease ( table 3 )  . 
instead , it clearly emerged that the incidence of ros1 fusions in females and in nonsmokers was markedly higher in patients with advanced disease than in patients with operable disease ; moreover , the mean age at diagnosis was significantly lower in patients with late - state disease . 
 nsclc ( adenocarcinoma or nonsmoker with squamous histology ) ros1 screening by ihc or fish or nonin situ techniques positive negative reconfirmation by a second method others nonsmoking female , patient younger than 50 years old reconfirmation by a second method fig 3 comprehensive algorithm for an accurate selection of patients to be treated with ros1 inhibitors . 
a comparison with data from the literature indicates a different distribution of these three main clinical parameters in patients with ros1 - positive disease at operable and nonoperable disease stages . 
in particular , it is remarkable that among patients with advanced - stage lung adenocarcinoma , there is a large predominance of women in general and women under 50 years old in particular . 
 elena guerini - rocco consulting or advisory role : thermo fisher scientific ( life technologies ) travel , accommodations , expenses : thermo fisher scientific ( life technologies ) andrea vingiani no relationship to disclose tommaso dantuono no relationship to disclose marcella liberatore no relationship to disclose giampaolo filice no relationship to disclose graziano de luca no relationship to disclose filippo de marinis no relationship to disclose antonio passaro no relationship to disclose luigi guetti no relationship to disclose luciana irtelli no relationship to disclose felice mucilli no relationship to disclose fiamma buttitta no relationship to disclose lucio crin `o honoraria : bristol - meyers squibb , astrazeneca , pfizer affiliations antonio marchetti , alessia di lorito , maria vittoria pace , chiara di lisio , lara felicioni , tommaso dantuono , marcella liberatore , giampaolo filice , graziano de luca , luigi guetti , luciana irtelli , felice mucilli , and fiamma buttitta , university of chieti - pescara , chieti ; massimo barberis , elena guerini - rocco , andrea vingiani , filippo de marinis , and antonio passaro , european institute of oncology , milan ; and lucio crin `o , istituto oncologico romagnolo irccs meldola forli , meldola , italy . references 2012 ( suppl 1 ) 1 . 
rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as first - line treatment for european patients with advanced egfr mutation - positive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
warth a , muley t , dienemann h , et al : ros1 expression and translocations in non - small - cell lung cancer : clinicopathological analysis of 1478 cases . 
cheng h , ye l , xue l : detection of ros1 gene rearrangement by fish and analysis of its clinical features in nonsmall cell lung cancer patients [ in chinese ]  . 
go h , kim dw , kim d , et al : clinicopathologic analysis of ros1 - rearranged non - small - cell lung cancer and proposal of a diagnostic algorithj thorac oncol 8 : 1445 - 1450 , 2013 15 . 
wu s , wang j , zhou l , et al : clinicopathological characteristics and outcomes of ros1 - rearranged patients with lung adenocarcinoma without egfr , kras mutations and alk rearrangements . 
shan l , lian f , guo l , et al : detection of ros1 gene rearrangement in lung adenocarcinoma : comparison of ihc , fish and real - time rt - pcr . 
wu j , lin y , he x , et al : comparison of detection methods and follow - up study on the tyrosine kinase inhibitors therapy in non - small cell lung cancer patients with ros1 fusion rearrangement . 
cao b , wei p , liu z , et al : detection of lung adenocarcinoma with ros1 rearrangement by ihc , fish , and rt - pcr and analysis of its clinicopathologic features . 
chen yf , hsieh ms , wu sg , et al : clinical and the prognostic characteristics of lung adenocarcinoma patients with ros1 fusion in comparison with other driver mutations in east asian populations . 
li c , fang r , sun y , et al : spectrum of oncogenic driver mutations in lung adenocarcinomas from east asian never mod pathol 28 : 468 - 479 , 2015 smokers . 
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pan y , zhang y , li y , et al : alk , ros1 and ret fusions in 1139 lung adenocarcinomas : a comprehensive study of common and fusion pattern - specific clinicopathologic , histologic and cytologic features . 
jurmeister p , lenze d , berg e , et al : parallel screening for alk , met and ros1 alterations in non - small cell lung cancer with implications for daily routine testing . 
lung cancer 87 : 122 - 129 , 2015 jin y , sun pl , kim h , et al : ros1 gene rearrangement and copy number gain in non - small cell lung cancer . 
 predicting expected absolute chemotherapy treatment benet in women with early - stage breast cancer using endopredict , an integrated 12 - gene clinicomolecular assay hatem soliman , md1 ; darl d . 
flake ii , phd2 ; anthony magliocco , md1 ; mark robson , md3 ; lee schwartzberg , md4 ; priyanka sharma , md5 ; krystal brown , phd2 ; saskia wehnelt2 ; ralf kronenwett , md , phd6 ; alexander gutin , phd2 ; johnathan lancaster , md , phd2 ; jack cuzick , phd7 ; and william gradishar , md8 purpose previous studies have shown endopredict ( epclin ) , a test that integrates 12 - gene expression data with nodal status and tumor size , to be predictive for risk of distant recurrence in women with estrogen receptorpositive , human epidermal growth factor receptor 2negative early - stage breast cancer . 
here , we modeled expected absolute chemotherapy benet on the basis of epclin test results . methods the effect of chemotherapy was modeled using previously validated 10 - year risk of distant recurrence as a function of epclin score for patients treated without chemotherapy . 
the early breast cancer trialists collaborative group performed a meta - analysis of more than 100 clinical trials in more than 100 , 000 women to evaluate the benet of adjuvant chemotherapy.6 although the in individual benet trials varied considerably , 3 this meta - analysis demonstrated an average 30% relative reduction in distant recurrence among women who received chemotherapy compared with those who did not.6 for many patients with er - positive disease , the risk of distant recurrence is sufciently low that et alone is adequate . 
in these patients , the adverse effects associated with chemotherapy may outweigh any benet provided by a reduced risk of disease recurrence . historically , identication of these low - risk patients has relied on clinicopathologic parameters , such as nodal status , tumor size , and tumor grade . 
 soliman et al context key objective can an integrated , 12 - gene , clinicomolecular assay predict absolute benet from chemotherapy for women with estrogen receptor ( er ) positive , human epidermal growth factor receptor 2 ( her2 ) negative breast cancer ? knowledge generated in this model , patients with low 12 - gene scores showed no difference in recurrence - free survival with or without chemotherapy , whereas those with high 12 - gene scores showed a marked increase in recurrence - free survival with the addition of chemotherapy . 
overall , this study suggests that the 12 - gene clinicomolecular score is able to predict absolute benet of chemotherapy for women with er - positive , her2 - negative breast cancer . relevance accurate risk assessment is critical for determining appropriate treatment of women with breast cancer . 
breast cancer prognostic assays like the 12 - gene clinicomolecular score have been shown to provide more information about risk than standard clinical information alone in predicting recurrence - free survival in the absence of treatment . 
these new data support the ability of the 12 - gene clinicomolecular score to also predict absolute benet from chemotherapy to help to guide chemotherapy decisions for women with er - positive , her2 - negative breast cancer . are inadequate to guide adjuvant chemotherapy decisions reliably.7 thus , when risk is dened using clinicopathologic features alone , a subset of truly low - risk patients receives unnecessary chemotherapy , whereas others at high risk forgo chemotherapy and experience avoidable disease recurrence . to address this clinical dilemma , multigene expression prognostic assays have been developed for patients with er - positive , human epidermal growth factor receptor 2 ( her2 ) negative early - stage breast cancer . 
several such assays are clinically available , with variable performance according to nodal status , and early ( 0 to 5 years ) versus late ( 5 or more years ) recurrence.8 - 11 these assays better quantify residual risk of recurrence after surgery and et than clinicopathologic features alone , which enables physicians to tailor the use of adjuvant therapies ( et alone v et + c ) more accurately . 
to this end , evidence - based practice guidelines now indicate that prognostic assays are appropriate tools to help guide decision making.12 - 14 one such test is endopredict ( myriad genetics , salt lake city , ut ) , which integrates a 12 - gene molecular score with nodal status and tumor size into a combined clinicomolecular score ( epclin )  . 
previous studies have validated that epclin accurately predicts the risk of distant metastases in patients with er - positive , her2 - negative breast cancers.11 , 15 - 18 although this information can reliably identify patients at sufciently low risk so that chemotherapy may be avoided safely , the ability of epclin to predict benet from the addition of chemotherapy has yet to be fully dened . 
ideal approaches to evaluate this are limited because archival samples from previous randomized trials of et 6 c in appropriate patients largely have been exhausted.3 , 5 a prospective trial of epclin that includes an arm randomized to receive no chemotherapy would now be unethical , given the established benet of chemotherapy for patients with high - risk disease . 
alternative study designs , therefore , are required to explore the scope of epclins ability to predict adjuvant chemotherapy benet for patients with er - positive , her2 - negative earlystage breast cancer . so far , classic factors that inuence breast cancer prognosis have not shown an interaction between the relative effect of adjuvant chemotherapy on absolute risk of recurrence.19 furthermore , the absolute benet of chemotherapy depends on the patients baseline risk of developing recurrent disease.19 as such , as we evaluate a biomarker , it is helpful to understand the principal drivers of its chemopredictive ability . 
one possibility is that chemopredictive ability is driven by high prognostic accuracy , that is , the ability of the biomarker to differentiate those patients at highest versus lowest absolute risk of recurrence and , thus , those latter patients for whom chemotherapy cannot yield any clinically meaningful benet . 
alternatively , it is possible that a biomarkers chemopredictive ability is driven by differences in relative chemotherapy benet between higher versus lower biomarker - dened patient groups ( ie , interaction between the biomarker score and relative chemotherapy benet )  . in the current study , we describe a modeling study that uses epclin score , absolute risk of recurrence , and reduction of this risk associated with the addition of chemotherapy to estimate the differences in absolute risk for distant recurrence across epclin scores . 
we demonstrate that for a molecular marker such as epclin , patients with the highest baseline absolute risk for recurrence experience the greatest absolute benet from chemotherapy . furthermore , we establish that the key factor in any biomarkers chemopredictive capacity is the ability to predict accurately the baseline absolute risk of recurrence . 
through this modeling , we estimate the differences in absolute risk of distant recurrence across epclin scores and examine the impact of different amounts of statistical interaction between epclin and chemotherapy on these estimates . methods gene expression assay the 12 - gene mrna expression assay has been previously described in detail.20 - 22 in brief , the expression of three proliferation - related target genes ( birc5 , dhcr7 , ube2c ) , ve hormone receptorrelated target genes ( azgp1 , il6st , mgp , rbbp8 , stc2 ) , and three normalization genes ( calm2 , oaz1 , rpl37a ) were measured by reversetranscription quantitative polymerase chain reaction . 
a 12gene molecular score was calculated as the linear combination of the normalized target gene expression.11 , 22 the clinicomolecular score epclin was calculated by combining the 12 - gene molecular score with tumor size and the number of positive lymph nodes and was reported as a numerical score from 1 to 6.11 , 22 tumors with an epclin score of less than 3.3 are considered low risk for distant recurrence and tumors with scores of 3.3 or greater are considered high risk for distant recurrence.11 , 17 , 18 statistical methods the ability of epclin to provide an estimate of expected chemotherapy benet was modeled , as shown in figure 1 . the primary component was the prognostic value of epclin to predict the rate of distant recurrence in the rst 10 years of follow - up . 
 soliman et al 10 - year distant recurrence in patients treated with et + c ( treated ) was estimated by introducing a relative chemotherapy benet for a particular epclin score , hrtreatment , to the untreated risk according to a proportional hazards model ( fig 1b ) , as in equation 1 : rtreated ( cid : 1 ) 1 1 runtreated ( cid : 1 ) ( cid : 3 ) hrtreatment mathematically , the dependence of the relative chemotherapy benet on the risk of distant recurrence was modeled using a main effect for chemotherapy and an interaction between treatment status and the prognostic signature.23 interactions were modeled as a linear dependence of the logarithm of hr for treatment on epclin score according to equation 2 : ( cid : 3 ) ( cid : 3 ) ( cid : 3 ) ( cid : 1 ) hrtreatment ( cid : 1 ) ( cid : 1 ) ln hroverall ( cid : 1 ) epclin epclinavg in this equation , represents the interaction strength and denes how strongly the relative chemotherapy benet for a patient depends on epclin score , epclinavg is the mean epclin score in the population , and hroverall is the hazard ratio for the chemotherapy benet in the population . 
with the maximum value of determined for an interaction giving no chemotherapy benet at epclinmin , weaker interactions were modeled using a percentage of the maximum value of ( fig 1b )  . finally , the absolute benet ab from chemotherapy for treated patients was determined according to equation 3 ( fig 1c ) : december 8 , 2017 , who received valid test results . formalin - xed parafn - embedded breast resections of treatment - nave er - positive , her2 - negative breast tissue were tested . 
these scores also were used to calculate epclinavg . results we examined the impact of different magnitudes of treatment effect and interaction strengths on the estimated 10 - year risk of distant recurrence ( table 1 )  . 
maximal interaction strength ( max = 0.159 ) was rst determined by assuming that a patients relative chemotherapy benet is 0 at the lowest possible epclin score and maintaining the hroverall of treatment at 0.7. 
the corresponding hrtreatment as a function of epclin score is shown in figure 2 . hrtreatment is 1 for a patient with an epclin score of 1 and 0.45 for a patient with the maximum observed epclin score of 6 . 
the risk of distant recurrence in the treated arm also was evaluated in a conservative scenario that assumed no interaction between chemotherapy benet and epclin score ( ie , 0% interaction strength , constant hrtreatment )  . 
in this scenario , the relative chemotherapy benet was equal to the overall chemotherapy benet ( = 0 ; hrtreatment = 0.7 ; fig 2 )  . after max was determined , risk of distant recurrence was calculated for scenarios with a variety of interaction strengths between epclin and chemotherapy . 
figure 3a shows the relationship between epclin score and predicted risk of distant recurrence by 10 years for untreated and treated patients under interaction strength ( 0% , 50% , and 100% )  . 
absolute chemotherapy benet for patients with low and high epclin scores assuming different overall relative chemotherapy benet ( 20% , 30% , or 40% ) for different relative interaction strengths ( 0% , 50% , or 100% ) absolute chemotherapy benet ( % ) 20% relative chemotherapy benet low epclin high epclin 0% relative interaction ( no interaction ) 50% relative interaction ab ( cid : 1 ) runtreated rtreated 100% relative interaction ( maximal ) 30% relative chemotherapy benet low epclin high epclin absolute chemotherapy benet for the low - risk category was calculated as a mean absolute chemotherapy benet for all low - risk patients , and the same went for the high - risk category . 
 predicting chemotherapy benet in early - stage breast cancer ln ( hrtreatment ) = ln ( hroverall ) ( epclin epclinavg ) 0% interaction strength ( = 0 ) 100% interaction strength ( = 0.159 ) epclin score fig 2 . 
patient benet from chemotherapy ( hrtreatment ) according to epclin score at no interaction ( 0% ) and maximal interaction ( 100% )  . recurrence is low for low epclin scores , which results in small absolute chemotherapy benet . 
for example , the expected absolute benet of chemotherapy treatment for a patient with low epclin scores ranged from 0.3% ( epclin = 1 ) to 2.9% ( epclin = 3.3 ) for 0% interaction strength and from 0% to 3.0% for 100% interaction strength . 
similar results were obtained when overall relative chemotherapy benet was modeled to be lower ( 20% ; fig 3b ) or higher ( 40% ; table 1 ; fig 3c )  . the various risk estimates across all scenarios of interaction strength were applied to a clinical cohort of patients with er - positive , her2 - negative breast resections and representative epclin scores ( n = 2 , 185 )  . 
the full epclin distribution is shown in figure 4 . overall , 1 , 275 samples ( 58% ) were low risk , and 910 ( 42% ) were high risk . 
risk of distant recurrence as a function of epclin score for different interaction strengths under different assumptions about overall relative chemotherapy benet : ( a ) 30% , ( b ) 20% , and ( c ) 40% . 
epclin scores in pretreatment estrogen receptorpositive , human epidermal growth factor receptor 2negative breast resection tissue ( n = 2 , 185 )  . the absolute chemotherapy benet in lowand high - risk patients was calculated as a function of interaction strength ( fig 5 )  . 
indeed , even with the strongest interaction , the absolute chemotherapy benet for a 30% relative benet in high - risk patients is 7.3% , which is not dramatically different from the 5.3% benet modeled in the patients with high ( 3.3 ) epclin score patients with low ( < 3.3 ) epclin score 40% overall benefit 30% overall benefit 20% overall benefit case of no interaction ( table 1 )  . 
similarly , in low - risk patients , the absolute benet is 1.5% when we assume the strongest interaction , which is close to a 1.8% benet in the case of no interaction . discussion previous studies have demonstrated that epclin accurately identies patients at low risk for distant recurrence 10 years after surgery , with improved prognostic performance compared with clinical features alone or other molecular tests.11 , 15 - 18 for these low - risk patients , it is clear that the addition of adjuvant chemotherapy likely will not yield any clinically meaningful risk reduction . 
here , we evaluated the capacity of epclin to identify patients with er - positive , her2 - negative earlystage breast cancer who may benet most from adding adjuvant chemotherapy to et . 
thus , we evaluated the impact by modeling across a range of potential chemotherapy effects sizes and interaction strengths with epclin . to minimize potential error associated with individual trial values , the absolute benet of chemotherapy in women with various epclin scores was modeled using wellestablished estimates of the average relative benet of adjuvant chemotherapy . 
this risk is particularly noteworthy in interventions in early - stage breast studies of adjuvant cancer because event rates may be low and distant recurrence may manifest many years or even decades after the initial diagnosis and treatment . 
as a result , the absolute predicted chemotherapy benet in low - risk patients was small and largely unaffected by average chemotherapy effect or interaction strength , whereas the toxicity of therapy remains the same . 
this association is irrespective of interaction strength between epclin and predicted chemotherapy benet , and the impact of any interaction on absolute benet is much smaller than the ability to accurately estimate absolute risk . as is typical of modeling studies , this study is not without limitations . 
of note , the approximately 30% reduction in distant recurrence was selected on the basis of its association with chemotherapy regimens that are more consistent with modern patterns of care in contrast to more historic regimens . other aspects of the statistical model are also limitations of the study . 
it is assumed that the relative benet of chemotherapy linearly depends on epclin score ( eq 2 ) , which is the simplest mathematical implementation of dependence . linear dependence also was used to demonstrate interaction between chemotherapy and a prognostic score.8 in statistical analysis , the most common implementation of interaction between variables has the same functional forin addition , only positive interaction between chemotherapy benet and epclin score was included in this analysis because it is expected that high expression of the proliferation - related genes in epclin is associated with higher rather than lower relative chemotherapy benet . 
conversely , high expression of the hormone receptorrelated genes is expected to be associated with smaller relative chemotherapy benet because er - negative tumors have a much stronger response to chemotherapy than er - positive tumors . 
this again corresponds to a positive interaction because the hormone receptorrelated genes contribute negatively to the epclin score . in summary , this modeling analysis demonstrates that epclin is able to predict which patients will gain maximum absolute benet from chemotherapy . 
in addition , these data indicate that the predicted absolute impact of chemotherapy on distant recurrence depends much more on the prognostic ability of the biomarker to predict an individual womans risk of recurrence accurately . 
endopredict has been shown in multiple studies to predict risk of distant recurrence accurately in women with er - positive , her2 - negative early - stage breast cancer who receive 5 years of et , with an accuracy similar to the other best breast prognostic tests and substantially better than the 21 - gene recurrence score ( oncotype dx ; genomic health , redwood city , ca ) .18 , 24 this modeling indicates identied a substantial proportion of that endopredict women whose risk of breast cancer recurrence was so low that the addition of adjuvant chemotherapy would not produce a clinically meaningful benet on recurrence risk . conversely , patients with high epclin scores were predicted to experience the greatest benet from chemotherapy regardless of the simulated interaction strength between epclin and predicted chemotherapy benet . 
flake , anthony magliocco , mark robson , lee schwartzberg , priyanka sharma , ralf kronenwett , alexander gutin , johnathan lancaster , jack cuzick , william gradishar collection and assembly of data : darl d . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . hatem soliman consulting or advisory role : celgene , astrazeneca , eli lilly , novartis , pzer , puma biotechnology research funding : amgen ( inst ) travel , accommodations , expenses : astrazeneca , eli lilly darl d . 
flake employment : myriad genetics stock and other ownership interests : myriad genetics patents , royalties , other intellectual property : multiple patents related to the development of tests at myriad genetics anthony magliocco stock and other ownership interests : protean biodiagnostics , the genomic cancer institute , proscia honoraria : bristol - myers squibb , merck , illumina , leica consulting or advisory role : bristol - myers squibb , merck , proscia , roche , illumina speakers bureau : bristol - myers squibb research funding : biotheranostics , roche , genentech patents , royalties , other intellectual property : various patents pending as co - inventor through moftt cancer center travel , accommodations , expenses : menarini silicon biosystems , illumina , bristol - myers squibb , merck , ventana medical systems mark robson honoraria : astrazeneca consulting or advisory role : mckesson , astrazeneca , merck , pzer , daiichi sankyo research funding : astrazeneca ( inst ) , myriad genetics ( inst ) , invitae ( inst ) , abbvie ( inst ) , tesaro ( inst ) , medivation ( inst ) travel , accommodations , expenses : astrazeneca , pzer lee schwartzberg stock and other ownership interests : vector oncology honoraria : pzer , amgen , nanostring technologies , novartis , napo , taiho pharmaceutical , genentech , e.r. 
squibb and sons , helsinn therapeutics , genomic health , myriad genetics , astrazeneca consulting or advisory role : bristol - myers squibb , pzer , helsinn therapeutics , caris life sciences , spectrum pharmaceuticals , tesaro , genomic health , astrazeneca , e.r. 
squibb and sons , amgen , f . hoffmann - la roche , genentech , eli lilly , novartis , merck , biocept , abbvie , biotheranostics , athenex , coherus biosciences speakers bureau : coherus biosciences , puma biotechnology research funding : amgen ( inst ) , glaxosmithkline ( inst ) , spectrum pharmaceuticals ( inst ) , medivation ( inst ) , bayer ag ( inst ) , genentech ( inst ) , pzer ( inst ) , tesaro ( inst ) , sano ( inst ) , e.r. 
n engl j med 319 : 1681 - 1692 , 1988 early breast cancer trialists collaborative group : tamoxifen for early breast cancer : an overview of the randomised trials . 
lancet 351 : 1451 - 1467 , 1998 early breast cancer trialists collaborative group ( ebctcg ) : effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15 - year survival : an overview of the randomised trials . 
lancet 365 : 1687 - 1717 , 2005 peto r , davies c , godwin j , et al : comparisons between different polychemotherapy regimens for early breast cancer : meta - analyses of long - term outcome among 100 , 000 women in 123 randomised trials . 
curr treat options oncol 16 : 23 , 2015 paik s , tang g , shak s , et al : gene expression and benet of chemotherapy in women with node - negative , estrogen receptor - positive breast cancer . 
j clin oncol 24 : 3726 - 3734 , 2006 van de vijver mj , he yd , vant veer lj , et al : a gene - expression signature as a predictor of survival in breast cancer . 
gnant m , filipits m , greil r , et al : predicting distant recurrence in receptor - positive breast cancer patients with limited clinicopathological risk : using the pam50 risk of recurrence score in 1478 postmenopausal patients of the abcsg - 8 trial treated with adjuvant endocrine therapy alone . 
filipits m , rudas m , jakesz r , et al : a new molecular predictor of distant recurrence in er - positive , her2 - negative breast cancer adds independent information to conventional clinical risk factors . 
coates as , winer ep , goldhirsch a , et al : tailoring therapies - - improving the management of early breast cancer : st gallen international expert consensus on the primary therapy of early breast cancer 2015 . 
harris ln , ismaila n , mcshane lm , et al : use of biomarkers to guide decisions on adjuvant systemic therapy for women with early - stage invasive breast cancer : american society of clinical oncology clinical practice guideline . 
dubsky p , filipits m , jakesz r , et al : endopredict improves the prognostic classication derived from common clinical guidelines in er - positive , her2 - negative 17 . 
martin m , brase jc , calvo l , et al : clinical validation of the endopredict test in node - positive , chemotherapy - treated er + / her2breast cancer patients : results early breast cancer . 
buus r , sestak i , kronenwett r , et al : comparison of endopredict and epclin with oncotype dx recurrence score for prediction of risk of distant recurrence after endocrine therapy . 
warf mb , rajamani s , krappmann k , et al : analytical validation of a 12 - gene molecular test for the prediction of distant recurrence in breast cancer . 
sestak i , buus r , cuzick j , et al : comparison of the performance of 6 prognostic signatures for estrogen receptor - positive breast cancer : a secondary analysis of a randomized clinical trial . 
lumbar spine magnetic resonance imaging ( mri ) demonstrated multiple bone lesions , and computed tomography scans revealed a 4 - cm right - sided hilar lung mass , regional thoracic lymphadenopathy , multiple hepatic metastases , a left - sided adrenal metastasis , and several osseous lesions ( fig 1a )  . 
biopsies of the subcarinal lymph node and the left - side adrenal lesion were performed , which confirmed adenocarcinoma of lung origin ( figs 2a and 2b )  . the patient was treated with radiation therapy to the painful vertebral metastasis and stereotactic radiosurgery to two brain lesions . 
200 genes ( foundationone ; foundation medicine , cambridge , ma ) revealed an egfr exon 19 deletion ( del19 ) mutation , a tp53 v173l mutation , an egfr amplification , and an rb1 loss of exons 18 and 19 . oral erlotinib 150 mg daily was initiated in may 2015 , and uniform disease response was evident on restaging scans in july 2015 ( fig 1b )  . 
however , in october 2015 , repeat imaging showed significant growth in a single liver lesion and a new 2.6 - cm lesion in the spleen , with continued response elsewhere , including the brain ( fig 1c )  . repeat biopsy of the enlarging liver lesion in november 2015 revealed nests of highly atypical cells with finely dispersed chromatin , inconspicuous nucleoli , and abundant mitoses ( figs 2c to 2e )  . immunohistochemical stains were positive for synaptophysin and chromogranin and negative for thyroid transcription factor 1 and napsin a . 
the overall features were consistent with small - cell lung cancer ( sclc ) transformation.4 ngs that consisted of targeted hotspot evaluation in 39 genes ( snapshot ngs ; massachusetts general hospital , boston , ma ) confirmed the presence of the original egfr del19 and tp53 mutations and showed additional mutations in pik3ca ( g545l ) , pik3ca ( g726l ) , erbb3 ( g337a ) , and fbxw7 ( l8p )  . 
a patientderived xenograft generated from this biopsy specimen lacked rb protein expression but retained minimal egfr expression and demonstrated activation of the mitogen - activated protein kinase and akt pathways ( fig 3c ) , likely as a result of the presence of an activating pik3ca mutation . the patient was treated with carboplatin and etoposide chemotherapy and ongoing erlotinib . 
scans after four cycles of chemotherapy showed a mixed response with slight regression in the previously biopsied ( sclc - transformed ) liver metastasis , stable brain metastases , and multiple new distinct sites of hepatic progression ( fig 1d )  . 
 ( a to e ) selected radiographic images of the liver illustrate involvement with cancer . treatments and key biopsy ( bx ) results ( tissue or liquid ) are indicated underneath each image in chronologic order . 
of note , the guardant360 assay can detect rb1 inactivating mutations but not allelic losses . we interpreted the emergence of an egfr t790m positive clone as the most likely resistance mechanism within the growing liver nodules . 
the patient discontinued chemotherapy , and the t790m - specific tki osimertinib7 was administered in march 2016 . restaging in june 2016 ( performed with mri because of renal dysfunction ) revealed that the hepatic lesions that had most recently progressed on chemotherapy ( presumed t790m positive ) had stabilized , but the liver lesion biopsied in 2015 ( sclc histology at that time ) had again enlarged with no other sites of systemic or intracranial progression ( fig 1e )  . 
a repeat plasma guardant360 test in june 2016 confirmed that egfr t790m was now undetectable , but increases were found in the mafs of pik3ca e726k ( 50.9% ) , pik3ca e545k ( 54.3% ) , tp53 v173l ( 54.8% ) , and egfr del19 ( 45.5% ; fig 4 )  . 
 ( a ) hematoxylin and eosin ( h&e ) stain and ( b ) thyroid transcription factor 1 ( ttf - 1 ) immunostain of fine - needle aspiration of a subcarinal lymph node at diagnosis show adenocarcinoma with diffuse ttf - 1 expression consistent with a lung primary . 
 ( c ) h&e stain and ( d ) synaptophysin ( syn ) and ( e ) ttf - 1 immunostains of a liver core biopsy at the time of acquired resistance to erlotinib demonstrate small - cell lung cancer with solid nests of highly atypical epithelial cells with finely dispersed chromatin , inconspicuous nucleoli , and brisk mitotic activity . 
 ( a ) summary of all evaluable probes across all chromosomes ( red , genomic gains ; blue , genomic losses ) shows diffuse losses across chromosome 13 , including the rb1 gene locus . 
 ( b ) a magnified view of four specific genes on chromosome 13 shows that all examined loci of rb1 are lost , with only blue signals and complete absence of red signals . 
 ( c ) tissue obtained from the november 2015 liver biopsy ( which shows sclc ) wasusedtogenerateapatient - derivedxenograft ( pdx ) inannsgmouseandsubsequentlypassagedthroughadditionalnsgmice.thepdxtumordemonstrated sclc histologic features consistent with the patient biopsy sample ( not shown )  . 
a western blot demonstrates relative protein levels of egfr , p - akt ( s473 ) , p - erk , rb , p16 , and actin ( loading control ) among pdx tumors ( mgh1529 ; two tumors shown ) with control lung adenocarcinoma ( adenoca ) and sclc samples for comparison . genetic characteristics of the various cell lines ( rb1 , p16 , and egfr exon 19 deletion [ del19 ] mutations [ mut ] ) are shown above the western blot . 
however , one resistant clone with sclc morphology emerged clinically in october 2015 , and genotyping confirmed an additional private pik3ca e545l mutation , which often is seen in sclc - transformed egfr mutant clones.4 during chemotherapy , we believe that the sclc clone diminished , whereas another clone that harbored an acquired egfr t790m mutation and perhaps two other distinct pik3ca mutations ( e545k and e726k ) emerged as observed in plasma ctdna in march 2016 . although a tissue biopsy specimen could not be obtained at that time , on the basis of our prior observation that sclc and adenocarcinoma populations can oscillate in response to specific treatment4 and that t790m is rarely seen in sclc - transformed tissue biopsy specimens , we hypothesize that the t790m - positive clone detected in plasma maintained an adenocarcinoma phenotype . 
the t790m clone was no longer detectable by june 2016 after treatment with osimertinib , but one or more other clones became dominant with increasing mafs , and subsequently , the disease became refractory to therapy ( fig 4 )  . 
this elevation in ctdna and subsequent clinical decline mirror data that demonstrated the correlation of increased mafs and decreased overall survival.9 - 11 in addition , the patient had a tumor with baseline rb1 mutation that was expected to lead to loss of function and is believed to play an essential role in the histologic transformation to sclc among egfr mutant cancers . 
we previously demonstrated that rb1 is universally lost in sclc - transformed cancers , although not sufficiently for transformation.12 recent work has demonstrated that baseline rb1 loss among egfr mutant tumors is a strong predictor for subsequent sclc transformation.13 as the clinical use of ngs panels increases and baseline inactivating rb1 mutations are more frequently detected , more data to understand the implications will be required , including a better understanding of the critical steps that lead to the lineage shift , so that we can develop more - effective treatment strategies.14 finally , this case report illustrates the potential utility of longitudinal molecular profiling during targeted therapy . 
at present , two mutationspecific food and drug administrationapproved plasma tests may be used to select egfr tkis.15 , 16 clinical practice is rapidly evolving , but on the basis of current evidence , it is reasonable to offer plasma genotyping upon progression with frontline egfr tkis to evaluate for t790m . 
 testing is negative for t790m , reflexive tissue biopsy should be performed because approximately 30% of ctdna test results will be false negative.17 the exact role of liquid biopsies for serial monitoring requires more rigorous evaluation , but a key appeal is that tumor biopsy findings may underestimate the full spectrum of resistant clones present at the time of progression.6 , 18 , 19 in practice , we commonly see the type of mixed radiographic response as observed in this patient with regression of some sites but continued growth in others . 
heterogeneity among distinct cancer subclones may explain such disparate responses , and longitudinal plasma testing might be a valuable adjunct to tissue biopsies to understand the dynamic evolution of various clones . for example , although tissue biopsy may offer critical information about the histology and molecular alterations of a specific progressing lesion , it may lack information about other sites of disease . 
conversely , ctdna genotyping could paint a more - complete picture of the competing resistance clones within a patient , although precise determination of which molecular alterations coexist within one clone or in one anatomic site are not currently possible . 
indeed , other studies have demonstrated that multiple resistance mechanisms can be detected within plasma , 18 and we and others have observed that longitudinal ctdna analyses can track the rise and fall of distinct subclones.6 , 20 in summary , this case report illustrates that the relative magnitude of resistant subclones can fluctuate in response to therapy , that liquid biopsies hold great potential to detect and monitor distinct genetic subpopulations within a patient , and that the presence of a baseline rb1 mutation in an egfr mutant cancer and subsequent sclc transformation raises important questions about monitoring such patients . 
for those with egfr mutant lung cancers , both tissue and liquid biopsy specimens can yield critical information to elucidate dominant clones that drive cancer growth at various time points . 
mooradian no relationship to disclose zofia piotrowska consulting or advisory role : boehringer ingelheim , astrazeneca , ariad pharmaceuticals , takeda pharmaceuticals , superdimension research funding : guardant health ( inst ) benjamin j . 
drapkin no relationship to disclose dora dias - santagata no relationship to disclose nicolas marcoux honoraria : bristol - myers squibb konstantinos arnaoutakis honoraria : foundation medicine , precision advisors , ariad pharmaceuticals consulting or advisory role : foundation medicine , precision advisors , ariad pharmaceuticals rebecca j . 
nagy employment : guardant health stock and other ownership interests : guardant health richard lanman employment : guardant health , veracyte leadership : guardant health stock and other ownership interests : guardant health , veracyte research funding : guardant health anthony j . 
farago honoraria : foundation medicine consulting or advisory role : pharmamar , merrimack , takeda pharmaceuticals , abbvie , intervention insights research funding : pharmamar ( inst ) , abbvie ( inst ) , astrazeneca ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) , loxo ( inst ) , ignyta ( inst ) travel , accommodations , expenses : pharmamar , abbvie , stemcentrx mari mino - kenudson consulting or advisory role : merrimack , h3 biomedicine , acd pharmaceuticals , roche , genentech aaron n . 
sequist honoraria : astrazeneca consulting or advisory role : astrazeneca , genentech , roche , bristol - myers squibb , pfizer research funding : boehringer ingelheim ( inst ) , clovis oncology ( inst ) , genentech ( inst ) , merrimack ( inst ) , novartis ( inst ) , astrazeneca ( inst ) , johnson & johnson ( inst ) , pfizer ( inst ) affiliations meghan j . 
sequist lv , yang jc , yamamoto n , et al : phase iii study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with egfr mutations . 
rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as first - line treatment for european patients with advanced egfr mutation - positive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
yu ha , arcila me , rekhtman n , et al : analysis of tumor specimens at the time of acquired resistance to egfr - tki therapy in 155 patients with egfr - mutant lung cancers . 
piotrowska z , niederst mj , karlovich ca , et al : heterogeneity underlies the emergence of egfrt790 wild - type clones following treatment of t790m - positive cancers with a third - generation egfr inhibitor . 
cancer discov 5 : 713 - 722 , 2015 janne pa , yang jc , kim dw , et al : azd9291 in egfr inhibitor - resistant non - small - cell lung cancer . 
schwaederle mc , patel sp , husain h , et al : utility of genomic assessment of blood - derived circulating tumor dna ( ctdna ) in patients with advanced lung adenocarcinoma . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced nonsmall - cell lung cancer . 
suda k , murakami i , sakai k , et al : small cell lung cancer transformation and t790m mutation : complimentary roles in acquired resistance to kinase inhibitors in lung cancer . 
the new era of precision medicine complicates communication even further as a result of our increasing reliance on genomic data and the varying psychological responses to genomic - based treatments and their expected outcomes . 
however , many of the communication issues are actually similar to perennial long - standing communication issues in oncology , which center on providing hope when breaking bad news and ensuring that adequate informed consent to treatments is obtained . 
we highlight these new communication issues that focus on clinical and ethical questions ( ie , informed consent , shared decision making , patient autonomy , and uncertainty in oncologic treatments ) and provide guidance on working with each scenario . 
in this article , we address common reactions of patients to genomic information and provide thoughtful communication suggestions using a shared decision making framework to help patients cope with the inherent distress - provoking uncertainties in oncology practice . introduction the practice of oncology depends on adequate communication between patients and clinicians . although the science of cancer medicine is advancing exponentially , information technology advances are seriously impeding the critical component of face - to - face communication . 
the key communication issues in oncology are always twofold : the delivery of emotionally charged bad news while sustaining hope in the presence of a lifethreatening prognosis and ensuring that patients understand the information that is provided.1 the advent of precision medicine now complicates the discussion of these issues in oncology in significant ways.2 , 3 although there is a growing emphasis on providing humanistic care in oncology ( eg , palliative care initiatives ) , the increasingly complicated science and public hype , coupled with limited time to talk with patients in busy oncology clinics , significantly threaten this goal . 
science and technology have yet again raced ahead of the human element , which depends on communication to foster those critical elements of mutual respect and trust . the application of genomic medicine is now accessible for the majority of oncology patients because the cost of genomic sequencing has decreased 1 millionfold from a decade ago.4 - 6 the development of molecular diagnostic , prognostic , and predictive biomarkers continues to change the landscape of clinical oncology.7 furthermore , technologic advances facilitate the use of largescale databases that store genomic data , such as ascos cancerlinq and ibm watson.8 - 11 also , research trial design in precision oncology is evolving from the randomized controlled trial to designs that harness these large data - collection initiatives and evaluate responses to drugs across multiple cancer subtypes ( ie , basket trial design ) .12 , 13 at the same time that genomic science and technology have created new research and clinical treatment paradigms , social media and drug direct - to - consumer marketing in the united states has brought information to the public with remarkable speed and fervor.14 , 15 although there are advantages to patients being informed consumers , handling this complex information without any interpretation of its meaning is not helpful.16 this breadth and rapidity of information adds an additional strain to the already complicated communication that occurs between clinicians and their patients.17 direct - to - consumer marketing is new and often amplifies drug benefits beyond reality . 
mcfarland , memorial sloan kettering cancer center , west harrison ; and elizabeth blackler , smita banerjee , and jimmie holland , memorial sloan kettering cancer center , new york , corresponding author : daniel c . 
 understand the implications for the overall cancer experience , eg , their satisfaction or dissatisfaction with their oncology care , or how this information influences patients treatment choices.15 its implications may be greatest when considering the delicate end - of - life conversations that are critical to quality oncology care.18 therefore , it is critical that we understand patients experiences in this new technologic age of genomic medicine . 
the shared decision making ( sdm ) model of communication promotes patient activation and engagement in health care and is an optimal model to guide communication tasks in the precision medicine era.19 , 20 the sdm model describes how clinician and patient jointly participate in making a health decision , having discussed the options and their benefits and harms , as well as having considered the patients values , preferences , and circumstances . 
it respects patient autonomy and leads to improved patient satisfaction when patients are faced with clinical options for which there is not a clearly correct choice , as is frequently encountered in the context of precision oncology.21 - 24 in this article , we highlight the psychological , social , and ethical issues that are part of our new treatments , and we provide communication suggestions on the basis of sdm . 
patients emotional reactions should be considered before , during , and after sdm to ensure effective communication . i won the lottery : the lucky biology club member jane was a 58 - year - old teacher who was diagnosed with stage iv adenocarcinoma of the lung that harbored an epidermal growth factor receptor mutation . her cancer responded well to erlotinib . 
after 15 months , her cancer began to grow as it became increasingly resistant to erlotinib , and chemotherapy was required to treat it . jane became extremely angry and frustrated that her cancer had progressed and found it hard to appreciate the benefit that the drug provided in light of its failure . she remained in shock and disbelief and had a difficult time accepting the need to adjust her treatment to the new situation . the lucky biology club members have driver actionable mutations such as epidermal growth factor receptor , anaplastic lymphoma kinase , ros1 , and brafv600e . 
subsequently , discussions about health care proxies , financial planning , and wills are often delayed . in terms of discussion , it is important to acknowledge the implications of disease biology and to corroborate and correct the information that they have from other sources . 
using sdm principles , the clinician could initiate a discussion of options by saying , there are some specific treatment options for you , on the basis of your cancers genomic make - up . 
however , the clinician must also discuss the framework for treatment and prognosis : although the cancer is still not curable , the mutation indicates that we have some additional treatment options . 
the clinician may say , given this news , it seems like a good time to talk about what to do next or we are in a different place now . 
is it okay if we talk more about the other options and next steps ? addressing the underlying emotions and acknowledging fear are important tools that should be dealt with before focusing on sdm . 
sdm can help provide clinical decision transparency and ameliorate disappointments with limited treatment effectiveness . i failed the test : the unlucky biology club member sarah was a 73 - year - old patient with newly diagnosed nonsmall - cell lung cancer who had no actionable mutation . 
at her first visit , she was clutching an article from the new york times and yelling , i want the serial killer ! she had come to memorial sloan kettering cancer center in hopes of enrolling in a clinical trial with first - line immunotherapy . when she did not meet trial entry criteria because of her pd - l1 status , she became enraged at being denied the opportunity that she had heard and read about . 
the article she held on to described how immunotherapy unleashes a so - called serial killer that ravages all cancer cells in the body , which is understandably what she wanted.29 sarah had delayed seeking cancer treatment in hopes of having the right mutation to enroll in a clinical trial . 
the unlucky biology club members feel that their tumor biology has conspired against them ; they are angered by the treatment options that are available for others but not for thethey feel that it is not fair that other patients with the same cancer are treated with more desirable targeted therapies . 
this may resonate with failures in their life before having cancer . it is crucial to validate their emotions of anger , fear , frustration , and worry by saying , i cant imagine what it has been like for you to hear this news ; i can see this is very upsetting . 
is it okay if we talk a bit more about what this means ? in this scenario , it is important to educate and reorient the patient who has been stuck on one idea as a result of their limited information . 
as part of sdm , the clinician should provide information on benefit and risk , such as , this information helps us plan the very best treatment for you . clinicians should be careful about comparing patients and especially about providing unsolicited information . this is an opportunity to re - establish / realign with the patients goals . 
given what you know about your illness , whats most important to you ? or , as you think about the future , are there situations or things that you want to make sure you accomplish or avoid ? explain that treatment options can be re - evaluated and tailored to the patients wishes . 
it also sends a positive message that although one opportunity for treatment did not work out , the team provides realistic hope and direction , despite a disappointment . a new drug has just come out : a novel treatment michael was a 47 - year - old patient with sinonasal undifferentiated carcinoma , a rare form of head and neck cancer . 
although the drug had never been tested with his specific head and neck cancer , sinonasal undifferentiated carcinoma , his clinicians believed that he had to be informed about the new treatment because he was young and otherwise healthy and , if it worked , would possibly provide a durable response . 
he did not want to complicate or disrupt the decision to accept hospice care , yet he felt ethically bound to offer a treatment for which michael had become eligible . this case highlights an ethical dilemma associated with the interface of advancing science and clinical care . 
it illustrates how the emergence of rapidly changing oncology treatments begets new levels of uncertainty in the cancer experience ( eg , no specific data for a cancer subtype , even though pembrolizumab had been approved for head and neck cancers )  . 
clinicians are taught to provide answers , which can be frustrating when new data become available and raise new questions and more uncertainty ( eg , does the drug work in this cancer subtype ? )  . 
medical training teaches clinicians how to handle uncertainty , but it does not teach them how to share its presence in a constructive way for patients.30 a full discussion was undertaken with the patient about the absence of data , the ethical reason for informing him of the new agent , and the inherent complications of considering another new treatment . 
he decided to stay with his decision to begin hospice care . it is important to discuss patients emotions about clinical uncertainty and the various treatment options , framed in the context of their current clinical situation : i realize this is all very complicated ; let me try to explain these options in the setting of your illness . 
also , it is important to frequently determine their level of understanding ( facilitates deliberation ) : can you tell me , in your words , what you understand ? also , it is helpful to review the patients clinical information together as a background to discussing the uncertainty . in situations of greatest clinical uncertainty , it is important to assure the patient that you are available for further discussion of their concerns and questions . 
a partnering statement , conveying the sense of were in this together and i will continue to work with you , is helpful to demonstrate a personal commitment to providing the best care possible despite the clinical uncertainty . 
patients who feel that they are in a partnership with their clinicians are better able to trust them and tolerate clinical uncertainty.31 i know i can beat it : the denier don was a 68 - year - old patient with progressive metastatic colon cancer who had received all conventional lines of cancer treatment . 
don said that he was the exception , frequently finding ways to explain how the negative clinical facts did not apply to hihe made many references to newly available targeted therapies that he believed could be used to treat his cancer . hope is a critical component of coping , especially in a time of great need . 
these offers are often made in the face of overwhelming disease biology . for most advanced cancers , precision oncology finds driver mutations in only a minority of cases , and the goal of most early trial design is still safety and not necessarily efficacy . 
these are difficult concepts to explain to patients who will probably only hear that there is hope . typically , educating the denier will require revisiting the topic several times to try to arrive at a shared understanding of the actual situation : you may be eligible for a trial if your cancer has a certain mutation ; however , taking the drug in this basket trial may not extend your life . 
when the patients condition worsens , the education may have to become more pointed : scans dont always reflect what is going on in terms of tumor growth and a change in functional status is an indication that the cancer is progressing . 
i hear you saying that what is most important to you is and i understand that you want to make sure to avoid the following this demonstrates that they have been heard , ensures that the goal of hope is not being taken away , and helps the patient to evaluate options . 
it is also important to keep in mind that the denier fluctuates in their level of denial and may partially accept the reality of the situation . patients often maintain two levels of awareness and acknowledgment of the situation . 
one is cognitive : i know how ill i athe other is emotional : i simply cant believe i could die . both exist simultaneously in many patients , which accounts for these day - to - day attitudinal changes . an ongoing positive clinician - patient relationship allows these feelings to emerge , and a more realistic discussion can occur about end - of - life planning , for example . 
often , a patients underlying fear will ameliorate in the context of working closely together . i have done my research : the overly or misinformed patient barbara was a 49 - year - old well - educated professional with nonsmall - cell lung cancer metastatic to brain and bone . 
barbara had strong opinions about the new treatment choices for lung cancer and was unwilling to consider therapies that were more appropriate for her type of disease . this case illustrates the complexity of precision oncology and the level of sophisticated genomic knowledge that is required to have a fully informed conversation with a patient . 
she explained the difference between inherited dna mutations , for which olaparib could be used in the presence of an inherited brca2 mutation in ovarian cancer , and the more common somatic mutations present in noninherited cancers such as her brca2 - mutated nonsmall - cell lung cancer . 
in this situation , correcting misinformation and misunderstanding is essential : there are many exciting cancer treatments , but whats important is that we find the right one for you . some patients want all available information and want to actively participate in their treatment decision . 
also , uncovering each patients major concerns is critical : what information would be most helpful for me to know about you ? then , it is important to acknowledge the patients concerns : this must be very upsetting for you . 
this acknowledges the importance of alliance.32 a request for a specific cancer treatment may be driven by an attempt to reach a goal , which may be modifiable in certain cases . 
it is crucial to demonstrate that you clearly understand their request : i hear you saying that whats most important is to continue treating your cancer to ensure you can reach your goal . case continued : a rebiopsy after progression revealed a her2 mutation that made her eligible for a basket trial . the oncologist invited another discussion of her tumor dna mutations and informed her that a newly found her2 mutation would qualify her for a basket trial . 
the previous genomics discussion on inherited and somatic mutations helped her understand that she would be treated with a group of patients with different cancers , who would all have the same somatic tumor dna mutation , to assess safety and perhaps efficacy in accordance with the trial objectives . she understood alternative treatment options and was offered a decision delay to facilitate deliberation and decision making . 
so far , the literature remains limited.7 , 33 , 34 patients acknowledge the promise of precision medicine but also consistently raise concerns about the discovery of incidental findings , information overload , complications from additional biopsies with delay in definitive cancer treatment , and the lack of a clear benefit.35 the risk of ineffective communication is that patients misunderstand the nature and seriousness of their diseases , which may lead to more aggressive and futile cancer treatments at the end of life or to increased fear and anxiety , while increasing stress and burnout in members of the medical team.36 on the other hand , effective communication enhances shared decision making , decreases patient distress , and increases patient satisfaction and trust in the medical team.25 shared decision making is the communication style that is preferred by most patients.37 patients make choices that are guided by the clinicians information and recommendations . 
patients may also have preferences on a spectrum of communication styles that can range from paternalistic ( ie , the patient is a passive recipient of their care ) to the clinician who is a nondirective information provider or educator ( ie , the patient is given choices and decides on management according to his / her preferences ) .30 it can be helpful to simply ask how the patient and family would like to make decisions , and the clinician can discuss this spectrum to gain clarification . 
discussing therapeutic uncertainty is often a difficult task for clinicians and leads to clinician behaviors of not disclosing all of the information , not talking about it , or oversimplifying the information.30 understandably , this can leave patients with a distorted account of their clinical situation . 
they may make sense of the situation by prescribing to alternative introducing so - called explanations or hopes , extramedical values , or seeking other remedies ( eg , alternative treatments ) when presented with clinical uncertainty.30 the idea of effective disclosure has been proposed as a potential goal in discussing uncertainty with patients . 
this idea supposes that patients can tolerate a certain amount of uncertainty and that extra vigilance is required by the clinician to ensure that they are given the tools and information they need to engage in decision making.30 effectively , extra care is taken in those situations of greater uncertainty to provide information and to ensure that the patient understands . 
this protects autonomy and increases patient trust in the long run . also , addressing underlying emotions , hopes , and fears is an effective way to ensure good communication and informed consent . 
a conversation analysis found that patients who are more assertive draw more empathy from their oncologists , whereas passive patients elicited much less emotional reciprocation from the oncologists.39 clinicians may use open - ended questions , such as , id like to switch gears and check in to see how you are feeling about all of this technical information ? or more closed - ended questions , such as , many patients may feel overwhelmed by all of the scientific information weve discussed ; im wondering if that is true for you ? in addition , checking understanding and emotional reactions enhances the clinician - patient relationship and is key in the setting of uncertainty . directly focusing on the therapeutic aspects of the relationshipby assessing communication preference , checking understanding , and searching for underlying emotional issuesdevelops rapport and may bolster patients resilience in the face of clinical uncertainty . 
the clinician may say something such as : im wondering if there is anything that might be difficult for you to discuss with me ? is there anything that i might be able to do to make this better for you ? or i have a sense that you are someone who likes x , y , and z ; please help me understand if i am correct . 
realigning goals may be accomplished by saying something such as : it is my goal to provide you with the best information for us to make a decision that works for you ; please let me know if that is also your goal or if youd like to focus on additional issues . this human connection dimension of medical care is needed to withstand the uncertainty . 
clinicians frequently underestimate the therapeutic power of their relationships with patients.40 a strong therapeutic bond is fundamental , not simply useful.41 patients have varying abilities to trust , hope , and have faith in their clinicians . 
the relationship is strengthened by focusing on agreed - upon common goals and tasks.32 it is always appropriate to discuss the nuts and bolts of the therapeutic relationship , especially when the clinician perceives that it may not be firmly established or upon entering areas of clinical uncertainty that are inherent in the precision medicine era . summary precision medicine presents a new era of communication in oncology . 
this article has presented some of the common patient reactions to precision medicine and basic communication issues in oncology . communication can be used to enhance rapport , trust , and support . 
zachariae r , pedersen cg , jensen ab , et al : association of perceived physician communication style with patient satisfaction , distress , cancer - related self - efficacy , and perceived control over the disease . 
blanchette ps , spreafico a , miller fa , et al : genomic testing in cancer : patient knowledge , attitudes , and expectations . cancer 120 : 3066 - 3073 , 2014 35 . 
politi mc , studts jl , hayslip jw : shared decision making in oncology practice : what do oncologists need to know ? oncologist 17 : 91 - 100 , 2012 38 . 
kenny da , veldhuijzen w , weijden tv , et al : interpersonal perception in the context of doctor - patient relationships : a dyadic analysis of doctor - patient communication . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction as our knowledge of hereditary cancer risk has testing of cancer preevolved , multigene panel disposition genes has become an important component of patient care . 
the national comprehensive cancer network provides gene - specic recommendations for changes in medical management on the basis of the identication of pathogenic variants.1 to this end , accurate and denitive variant classication is a critical component of clinical genetic testing . 
the american college of medical genetics and genomics ( acmg ) and the association for molecular pathology ( amp ) provide guidelines for clinical variant classication2 ; however , there is often insufcient evidence to support a denitive classication of pathogenic or benign when a variant is rst detected . 
as a result , testing laboratories may vary in their clinical terpretation of acmg / amp guidelines for hereditary cancer genetic testing and , ultimately , in their variant classications.3 - 6 with the increased use of multigene panels , number of variants of uncertain signicance ( vuss ) detected has also increased.7 , 8 in addition to a larger number of genes , many cancer predisposition genes have been more recently incorporated into clinical testing , and there is less evidence available to support variant classication . 
 evolution of cancer gene variant reclassication context key objective with the increasing use of hereditary cancer genetic testing and introduction of multigene panels , robust and accurate methods for variant re / classication of cancer predisposition genes are of utmost importance . 
laboratory - developed methods were the most common lines of evidence used in variant reclassication , with pheno analysis ( a clinical history weighting algorithm ) being used in approximately 40% of cases . relevance many tools traditionally used for variant re / classication are not informative for rare variants ; however , rare variants collectively affect millions of patients . 
this was followed by testing for lynch syndrome ( mlh1 , msh2 , msh6 , and pms2 ) and melanoma - pancreatic cancer syndrome ( cdkn2a ) in 2002 and for familial adenomatous polyposis ( fap ) / attenuated fap / mutyh - associated polyposis ( apc and mutyh ) in 2003 . 
in 2013 , a hereditary cancer multigene panel was introduced , which was analytically validated and described in detail previously.9 the panel included 25 genes : apc , atm , bard1 , bmpr1a , brca1 , brca2 , brip1 , cdh1 , cdk4 , cdkn2a ( p16ink4a and p14arf ) , chek2 , epcam , mlh1 , msh2 , msh6 , mutyh , nbn , palb2 , pms2 , pten , rad51c , rad51d , smad4 , stk11 , and tp53 . 
individuals who had single - site or founder mutation testing were reclassication program the reclassication program used by this testing laboratory included a multidisciplinary committee of laboratory directors , genetic counselors , phd - level scientists , and supporting variant specialists . 
variant classication and reclassication followed a stringent , welldocumented protocol as previously described.13 on the basis of acmg / amp guidelines , variants were classied into ve categories of increasing clinical severity : benign ( b ) , likely benign ( lb ) , vus , likely pathogenic ( lp ) , and pathogenic ( p ) .2 , 14 , 15 automated processes proactively monitored and gathered all available evidence to expedite reclassication . 
multiple classication tools could have been used to evaluate the pathogenicity of a variant at the time of original classication and / or if a variant was reclassied , an reclassication ( fig 1 )  . amended report was sent to health care providers for all patients who were carriers of the variant . 
 esterling et al pheno ( n = 56 , 596 ) clinical history weighting algorithm to assess phenotype severity for variant carriers v carriers of pathogenic and benign variants pt / ha ( n = 36 , 314 ) phase testing or haplotype analysis for genes associated with embryonic lethality or severe clinical phenotypes because of homozygosity or compound heterozygosity homozygosity confirmed continuous evidence evaluation threshold reached population frequency ( n = 17 , 664 ) threshold reached allele frequency from exac and gnomad functional or clinical evidence literature ( n = 4 , 509 ) expert review of automated search results and curated repository structure disrupted structural analysis ( n = 850 ) using validated protein structures mco ( n = 12 , 753 ) likelihood of co - occuring pathogenic variants in the same gene or in genes associated with the same hereditary cancer syndrome threshold reached cosegregation in multiple families segregation analysis ( n = 8 , 255 ) published pedigrees and provider communications rna analysis ( n = 610 ) in - house comprehensive rna analysis to directly assess rna splicing effects splicing disrupted splicing analysis ( n = 3 , 148 ) functional data and expert review other ( n = 4 , 598 ) knowledge of similar variants , species conservation in conjunction with structural analysis , additional statistical analysis , and classification / reclassification protocol refinements evidence queuing for review based on variant type and new evidence multiple expert reviews evidence insufficient wait for additional evidence committee discussion variant reclassified amended reports fig 1 . 
phase testing / homozygosity analysis ( pt / ha ) was only applied to genes associated with autosomal dominant cancer risks that may also be associated with embryonic lethality or severe clinical phenotypes in individuals carrying pathogenic variants in the affected gene on different parental chromosomes as a result of either homozygosity or compound heterozygosity . 
common single nucleotide polymorphisms ( snps ) observed in the patient population were used to develop gene - specic haplotypes , which were automatically assigned to determine the variants phase ( ie , in cis or in trans ) , whenever possible . 
mutation co - occurrence ( mco ) analysis was based on an assumption of a low likelihood that an individual will carry two autosomal dominant pathogenic variants that cause an increased risk for the same or a similar cancers.18 mco analysis measured the statistical signicance of a variant co - occurring multiple times with one or more pathogenic variants in the same patient ( in the same gene or in a different gene for the same syndrome )  . 
99.5%. rna splicing analysis and protein structural analysis . assessment of in silico rna splicing predictions was used to initially evaluate the potential effects of a variant on rna splicing.19 published reports of biochemical splicing analysis , which used high - quality data and appropriate controls , were considered for variant classication . 
in some cases where evidence was unavailable or of insufcient quality or detail to be conclusive , quantitative rna splicing analyses were performed in house , using informative snps , to assess potential rna splicing effects resulting from the variant of interest.20 expert protein structural analysis was also used to assess the impact of variants predicted to affect protein structure at the amino acid level . 
cascade testing is the systematic identification of relatives who are at risk for having the same mutation ( at - risk relatives ) and facilitating genetic testing for the usually , these individuals are unaffected , but they may benefit from early identification and risk management interventions . 
 currently , australian best practice guidelines recommend that genetic testing for unaffected individuals be facilitated by an appropriately trained health professional such as a genetic counselor or clinical geneticist.15 the purpose of this case report is to exemplify a collaboration between the departments of oncology and genetics to extend testing for a dpyd mutation to family members after the index case was identified . patient the index patient was diagnosed with metastatic breast cancer at age 59 years . 
 she developed locoregional recurrences in 2011 , bony metastases in 2013 , and further progression with liver , lung , bone , and soft tissue metastases in 2016 after multiple lines of endocrine therapy . 
with normal baseline renal function , she was prescribed capecitabine at 1 , 000 mg / m2 twice per day on days 1 to 14 in a 21 - day cycle . 
four weeks later , she began palliative paclitaxel to treat symptomatic cancer progression . the patients oncology team referred her to the department of genomic medicine at the royal melbourne hospital . 
the patients personal and family history of cancer had been assessed ; it was considered that there was no increased familial risk of breast ( or other ) cancers . the patients first - degree relatives were subsequently offered genetic counseling and testing for this pharmacogenomic mutation . 
communication of genetic results in families is known to be problematic for various reasons , including miscommunication or misunderstanding of genetic information and passive or active nondisclosure.20 - 22 in this case , family members were already in contact with the genetics service around the time of the index testing and were requesting testing themselves . 
a hypothetical pedigree showing the effect of systematic cascade testing for an autosomal dominant condition . to our knowledge , this is the only published case report of cascade genetic testing in the setting of dpyd pharmacogenomics . 
falvella and colleagues23 reported a case of dpyd genotyping before starting chemotherapy in a 38 - year - old man with colorectal cancer in the context of his mother having a known dpyd variant . 
our case differs in that we extended the dpyd genotyping systematically by offering it to all unaffected at - risk family members . fluoropyrimidines are commonly used drugs , and the cancers treated with these drugs are common in the general population . 
even though this family is not at an increased heritable risk of cancers , it is quite possible that members of the family may need this treatment regimen in the future . 
relationships are self - held unless noted . hereditary breast and ovarian cancer syndrome , cascade pharmacogenomics testing would be predicted to have greater utility because of the higher probability of relatives being affected by these cancers . in conclusion , this case provides an example of cascade testing of family members after an individual with dpd deficiency was identified . 
winship , university of melbourne , parkville , australia acknowledgment we acknowledge michael michael , md , consultant medical oncologist at peter maccallum cancer centre , for his input into an earlier version of this manuscript . support supported by a victorian state - funded clinical service . 
iacovelli r , pietrantonio f , palazzo a , et al : incidence and relative risk of grade 3 and 4 diarrhoea in patients treated with capecitabine or 5 - fluorouracil : a meta - analysis of published trials . 
meulendijks d , van hasselt jgc , huitema adr , et al : renal function , body surface area , and age are associated with risk of early - onset fluoropyrimidine - associated toxicity in patients treated with capecitabine - based anticancer regimens in daily clinical care . 
ison g , beaver ja , mcguinn wd jr , et al : fda approval : uridine triacetate for the treatment of patients following fluorouracil or capecitabine overdose or exhibiting early - onset severe toxicities following administration of these drugs . 
ma ww , saif mw , el - rayes bf , et al : emergency use of uridine triacetate for the prevention and treatment of life - threatening 5 - fluorouracil and capecitabine toxicity . 
henricks lm , opdam fl , beijnen jh , et al : dpyd genotype - guided dose individualization to improve patient safety of fluoropyrimidine therapy : call for a drug label update . 
lunenburg catc , henricks lm , guchelaar hj , et al : prospective dpyd genotyping to reduce the risk of fluoropyrimidine - induced severe toxicity : ready for prime time . 
liu d , li j , gao j , et al : examination of multiple ugt1a and dpyd polymorphisms has limited ability to predict the toxicity and efficacy of metastatic colorectal cancer treated with irinotecan - based chemotherapy : a retrospective analysis . 
claes e , evers - kiebooms g , boogaerts a , et al : communication with close and distant relatives in the context of genetic testing for hereditary breast and ovarian cancer in cancer patients . 
mcclaren bj , aitken m , massie j , et al : cascade carrier testing after a child is diagnosed with cystic fibrosis through newborn screening : investigating why most relatives do not have testing . 
their study is large and includes nearly 27 , 000 patients who underwent multigene panel testing ( including cdh1 ) over a 2 - year period at ambry genetics , an established commercial diagnostic laboratory , and  . 
these cases are divided into patients with igclcpartial phenotype , who exhibit some hint of hdgc in their family , such as individuals with gastric cancer , lobular breast cancer at older ages , or even lobular carcinoma in situ ; and patients who are igclcnegative , who have no evidence of gastric cancer or lobular breast cancer in their family . using this nomenclature , one - quarter of both the laboratoryand clinic - based cdh1 carriers were igclc - negative ( although the results from the laboratory - based group must be considered preliminary , because the patients family histories were obtained from the ordering form rather than through genetic counselorderived pedigrees )  . 
thus , we cannot speculate on their potential risk for early gastric cancer , despite their lack of a suggestive family history . however , three of the 10 patients with igclc partial phenotype underwent prophylactic gastrectomy procedures , and all had early gastric cancer ( one patient as young as 39 years )  . 
although this article does not fully answer what to do for the patient who was the subject of our friday afternoon genetics clinic , it does provide important insights into how to approach these difficult decisions and what additional information we need to do so with greater confidence . certainly , those cdh1 mutation carriers with any personal or family history of gastric or lobular breast cancer , even if they do not meet igclc criteria for hdgc , should at least consider a prophylactic gastrectomy . 
however , for those individuals who are truly igclcnegative after a comprehensive family history is taken by a skilled genetic counselor , it is difficult to recommend such an invasive procedure . 
both groups should definitely have regular endoscopic screening with blind biopsies , and proceed to gastrectomy if any malignant cells are identified.4 finally , all of these recommendations go out the window if and when a case of a true igclcnegative cdh1 carrier develops gastric cancer or has a prophylactic gastrectomy that identifies early gastric cancer . 
van der post rs , vogelaar ip , carneiro f , et al : hereditary diffuse gastric cancer : updated clinical guidelines with an emphasis on germline cdh1 mutation carriers . 
raymond , ms3 ; eduardo vilar , md , phd1 ; michael overman , md1 ; bryan kee , md1 ; cathy eng , md1 ; kanwal raghav , md1 ; and scott kopetz , md , phd1 purpose atypical , non - v600 braf ( abraf ) mutations represent a rare molecular subtype of metastatic colorectal cancer ( mcrc )  . 
preclinical data are used to categorize abraf mutations into class ii ( intermediate to high levels of kinase activity , ras independent ) and iii ( low kinase activity level , ras dependent )  . 
among patients with abraf ras wild - type mcrc who received anti - egfr antibodies ( monotherapy , n = 1 ; combination therapy , n = 10 ) , no responses to anti - egfr therapy were reported , and six patients ( four with class iii abraf mutations , one with class ii , and one unclassied ) achieved stable disease as best response . 
in the ctdna cohort , there was an increased prevalence of abraf mutations and subclonal abraf mutations ( p , .001 for both ) among predicted anti - egfr exposed compared with nonexposed patients . conclusion efcacy of anti - egfr therapy is limited in class ii and iii abraf mcrc . 
2019 by american society of clinical oncology introduction brafv600e missense mutations are present in 6% to 10% of patients with metastatic colorectal cancer ( mcrc ) .1 , 2 within the braf kinase domain , substitution of a valine to glutamic acid at position 600 manifests as constitutive activation and oncogenic signaling along the mitogenactivated protein kinase ( mapk ) pathway.1 brafv600e mcrc represents an aggressive molecular subtype of colorectal cancer inherently refractory to standard chemotherapy ; thus , tremendous research focus has been directed toward novel therapeutic development.2 because of increased use of next - generation sequencing ( ngs ) in the management of mcrc , various mutational hotspots of clinical signicance have emerged within interest , such as expanded ras testing . genes of however , with such broad testing including circulating tumor dna ( ctdna ) , alterations of evolving signicance without clear predictive , prognostic , or therapeutic implications have also been identied . 
atypical , non - v600 braf ( abraf ) mutations that occur outside of codon 600 are one example of an emerging molecular subtype in colorectal cancer . abraf mutations were retrospectively studied at two large centers comprehensively describing the clinical , pathologic , and survival implications of these mutations in patients with mcrc.3 a total of 9 , 643 patients with mcrc underwent ngs testing and 208 patients with abraf mutations were identied , representing 2.2% of all patients tested . 
 johnson et al context key objective to determine if atypical , non - v600 braf ( abraf ) mutations , stratied by class , affect outcomes with anti - egfr therapy and , secondarily , if acquisition of abraf mutations imparts resistance to egfr inhibition . knowledge generated clinical outcomes on the basis of functional class and their effect on anti - egfr efcacy for abraf metastatic colorectal cancer ( mcrc ) have not been dened . 
in patients with abraf wild - type ras mcrc who received anti - egfr antibodies , there were no responses among class ii or iii abraf mcrc , with stable disease as best response in our internal cohort . evaluation of a large external cohort of patients with circulating tumor dna interrogated by an anti - egfr exposure score revealed increased prevalence of abraf and subclonal abraf mutations among predicted anti - egfr exposed compared with nonexposed patients , suggesting an acquired mechanism of resistance to egfr inhibition . relevance anti - egfr therapy is limited in abraf mcrc , with class ii abraf mutations emerging as a negative predictive biomarker . detection of abraf mutations in ctdna may represent a novel mechanism of resistance warranting additional investigation . can be used sequentially throughout the course of the disease . although brafv600e mcrc is known to be predictive of poor response to anti - egfr therapy , the clinical utility of egfr inhibition in abraf mcrc remains unclear.4 , 5 of note , previous retrospective work investigating a cohort of 150 patients with refractory mcrc identied seven patients with abraf mutations and reported poor progression - free survival when they were exposed to anti - egfr therapy in comparison with a cohort with ras / raf wild - type ( ras / rafwt ) mcrc.6 preclinical work has highlighted unique biology among traditional brafv600e mutations ( class i ) and abraf mutations ( classes ii and iii ) .7 , 8 brafv600e mutations , designated as class i , result in feedback inhibition of gtp - bound ras , are ras independent , and signal as active monomers . 
in contrast , class ii abraf mutations have intermediate to high levels of kinase activity , are ras independent , signal as constitutive dimers , and are resistant to vemurafenib , whereas class iii abraf mutations have low or absent kinase activity and are primarily ras dependent and sensitive to erk - dependent feedback of ras.7 , 8 these tumors bind more tightly to ras - gtp than do wild - type braf tumors , and binding to wild - type craf is enhanced , resulting in increased erk signaling.8 these stark differences in underlying tumor biology may explain the varying response to therapy , specically the potential limitations to anti - egfr inhibition in abraf mcrc . 
cohort 1 included patients with stage iv colorectal cancer who were seen at mdacc between march 1 , 2012 , and june 27 , 2017 , and underwent targeted exome sequencing . 
all clinical and outcomes data were derived from this cohort . cohort 2 included patients with mcrc who underwent a targeted ngs ctdna assay ( guardant360 ; guardant health , redwood city , ca ) as part of their care between june 1 , 2014 , and december 26 , 2017 , at multiple institutions . 
 abraf mutations as a mechanism of resistance to egfr inhibition frequencies for both cohorts of abraf mutations : predicted anti - egfr exposed and nonexposed . statistical analysis clinicopathologic features were compared by 2 test or fishers exact test for categorical variables and assessment of odds ratios for associations as appropriate , as well as by t test to compare continuous variables . 
matched analysis adjusted by age and sidedness was undertaken to compare os among all molecular classes . 2 or fishers exact tests , as well as t test , were used to evaluate the adequacy of the matching . 
statistical analyses were performed using graphpad prism , version 6.07 ( graphpad software , la jolla , ca ) , and spss , version 23.0 ( ibm , armonk , ny )  . 
among 36 patients with abraf mcrc , 19 ( 53% ) had left - sided primary tumors and 24 ( 67% ) had microsatellite stable ( mss ) disease and were a median age of 56 years ( table 1 )  . 
of note , in contrast to brafv600e mcrc , which is mutually exclusive with ras mutations , 12 patients with abraf mcrc had ras mutations ( class iii , n = 7 of 21 [ 33% ] ; class ii , n = 5 of 10 [ 50% ] )  . 
among these , one patient with class ii abraf mcrc also had microsatellite instabilityhigh ( msi - h ) disease . for a more accurate comparison of survival outcomes among respective molecular classes , we performed a matched analysis by age and sidedness among the 36 patients with abraf mcrc compared with 36 patients with brafv600e mcrc and 36 with ras / rafwt disease ( fig 3 ; appendix table a1 )  . 
 ( % ) unless otherwise indicated . abbreviations : , not applicable ; abraf , atypical , non - v600 braf ; msi - h , microsatellite instabilityhigh ; mss , microsatellite stable ; wt , wild exposed to anti - egfr therapy in their treatment course . among patients with abraf raswt mcrc , 11 ( 50% ) received anti - egfr monoclonal antibodies ( mabs ; monotherapy , n = 1 , combination therapy , n = 10 ; class iii , n = 7 of 14 [ 50% ] , class ii , n = 3 of 5 [ 60% ] , unclassied , n = 1 )  . there were no responses reported , and six patients ( class iii , n = 4 ; class ii , n = 1 ; unclassied , n = 1 ) achieved stable abraf ( n = 36 ) brafv600e ( n = 221 ) wild - type braf ( n = 438 ) time ( months ) fig 2 . 
in line with previous reports , patients with class i or brafv600e mcrc had worse survival outcomes when exposed to egfr inhibition ( fig 4 )  . abraf mutations as a potential mechanism of resistance to anti - egfr therapy to understand our institutional ndings of limited efcacy of anti - egfr therapy in abraf mcrc , we analyzed an external ctdna cohort of 5 , 257 samples ( cohort 2 ) from 4 , 465 patients with mcrc . 
 abraf mutations as a mechanism of resistance to egfr inhibition abraf ( n = 36 ) brafv600e ( n = 36 ) wild - type braf ( n = 36 ) time ( months ) fig 3 . 
abraf , atypical , nonv600 braf . cohort , after excluding ras and brafv600e mutations , we applied a previously validated anti - egfr exposure score to divide patients into predicted prior exposure to anti - egfr ( n = 644 ) versus predicted nonexposed ( n = 2 , 880 ) .10 this revealed that the prevalence of abraf mutations increased from 3.99% among patients predicted to have no prior antiegfr exposure to 8.38% among those with predicted antiegfr exposure ( p , .001 ; fig 5a )  . as a sensitivity analysis , we evaluated the prevalence of abraf mutations among patients with egfr ectodomain mutations , compared with those without , because egfr ectodomain mutations are unequivocally present only after exposure to egfr inhibition . 
class ii activating g469a mutations ( p , .001 ) and class iii d594n ( p , .001 ) were the most common alterations among patients with predicted antiegfr exposure ( fig 5c )  . 
agents such as vemurafenib are not active , because the drug is designed to target the shape of the v600e - mutated protein and is more than 10 - fold less active against nonmutated forms . 
 ( a ) ctdna prevalence of abraf ( 3.99% ) among non - exposed patients ( n = 2 , 880 ) v ctdna prevalence of abraf ( 8.38% ) among predicted anti - egfr exposed ( n = 644 )  . 
schematic gure of the conserved region3 ( cr3 ) of the braf protein , also known as the braf kinase domain ( amino acids 457 to 717 ) , and the location of the class ii and iii hotspot mutations . 
the kinase domain contains the p - loop ( amino acids 464 - 471 ) , the c helix ( amino acids 492 - 504 ) , the dimerization interface ( dif , amino acids 504 - 511 ) , the catalytic loop ( cl , amino acids 574 - 581 ) , the dfg motif ( amino acids 594 - 596 ) , and the activation segment ( as , amino acids 594 - 623 )  . 
class ii , activating g469a mutations ( p , .001 ) and class iii , d594n ( p , .001 ) were the most common , respectively , among predicted anti - egfr exposed patients . 
although previous reports have suggested class iii abraf mcrc may achieve responses with antiegfr therapy owing to ras dependency , our cohort , albeit small , only revealed stable disease as best response.6 , 8 , 12 - 15 , 19 these results support the ndings of the breac study , which revealed no responses in six patients with abraf mcrc ( n = 2 each with class ii , class iii , and unclassied ) .6 of note , the nding of concomitant ras mutations with abraf mcrc in tissue , conrmed in our external ctdna cohort , clearly precludes the use of egfr inhibition for all patients , highlighting the need for novel combination strategies that consider this distinct signaling biology.20 , 21 in our internal and external cohorts , we did not nd a difference between patients with class ii and class iii abraf mcrc in regard to the presence of concurrent ras mutations . 
 abraf mutations as a mechanism of resistance to egfr inhibition in our mdacc internal cohort , four patients had unclassied disease ( y656d , a415v , k752r , f109i ) and had poor survival outcomes . 
in the external ctdna cohort , we found numerous unclassied abraf mutations that are yet to be functionally described ; most were commonly comutated with ras , pik3ca , and egfr . development of acquired ras and subclonal egfr ectodomain mutations are known mechanisms of resistance to anti - egfr therapy in raswt mcrc.22 - 26 considering the limitation of egfr inhibition noted in our internal cohort , the emergence of abraf potentially reects whether a novel mechanism of acquired resistance to egfr inhibition was investigated . 
analysis of our ctdna cohort showed statistically signicant increases in the prevalence of abraf mutations , subclonal abraf mutations , and multiple abraf mutations in individual ctdna samples among patients predicted to be exposed to egfr inhibition versus those predicted to be nonexposed . 
it is important to note that the rate of abraf mutation in our ctdna cohort was higher than in our internal cohort , as well as higher than what has been published in tissue - based testing.3 these ndings highlight the potential of abraf mutations as a novel acquired resistance mechanism to egfr inhibition and warrant additional prospective investigation . of note , in patients with egfr - mutant nonsmall - cell lung cancer , abraf mutations have been reported as a mechanism of acquired resistance to egfr tyrosine kinase inhibition.27 our results underscore the need for innovative combinatorial targeted approaches for abraf mutations exploiting class biology . 
data suggest that downstream signaling with novel inhibitors of mek or erk may be useful , because the pathway is still active.20 furthermore , because both class ii and class iii mutations signal through braf and / or craf target didimers , next - generation raf inhibitors that merization is of interest in combination with these agents as a rationale next step for therapeutic intervention.21 of note , regimen using safety lead - in data for a novel triplet encorafenib , binimetinib , and cetuximab for refractory brafv600e mcrc have been published showing impressive response rates and signicant in survival outcomes.28 subsequent studies will need to investigate whether this novel triplet combination strategy can also enhance optimal mapk inhibition and provide durable benet in patients with abraf mcrc . improvement there are limitations to this study . 
second , considering the rarity of abraf mcrc , we were only able to identify 36 patients in our internal cohort , of whom 30% received egfr inhibition therapy . third , detailed clinical annotation is not available for the external ctdna cohort , and we were unable to conrm that these patients had received prior anti - egfr therapy . therefore , we applied a previously validated and published anti - egfr exposure score to identify patients predicted to have anti - egfr exposure versus patients predicted to be nonexposed to conduct our ctdna analysis . our results suggest selection criteria for the use of antiegfr therapy in abraf - mutated mcrc should be based not only on raswt status but also the functional class of the atypical variant . 
in our cohort , although limited by sample size , ndings continue to generate more hypotheses in that class ii abraf mutations may represent a negative predictive biomarker of egfr inhibition , and exposure to anti - egfr mabs may adversely affect survival outcomes , whereas efcacy of egfr inhibition was limited in patients with class iii abraf mcrc . 
increased subclonal abraf mutations and prevalence of class ii and class iii variants in ctdna among patients with predicted anti - egfr exposure suggest a novel mechanism of resistance warranting additional prospective investigation . also , our data support the clinical utility of comprehensive genomic proling ( both tissue and ctdna ) in the management of advanced mcrc , because standard hotspot polymerase chain reaction testing would not capture emerging clinically signicant alterations . 
loree , kanwal raghav , scott kopetz provision of study material or patients : all authors collection and assembly of data : all authors data analysis and interpretation : benny johnson , alexandre a . 
morris v , overman mj , jiang zq , et al : progression - free survival remains poor over sequential lines of systemic therapy in patients with braf - mutated colorectal cancer . 
clin colorectal cancer 13 : 164 - 171 , 2014 jones jc , renfro la , al - shamsi ho , et al : non - v600 braf mutations dene a clinically distinct molecular subtype of metastatic colorectal cancer . 
j clin oncol 35 : 2624 - 2630 , 2017 douillard jy , oliner ks , siena s , et al : panitumumab - folfox4 treatment and ras mutations in colorectal cancer . 
n engl j med 369 : 1023 - 1034 , 2013 pietrantonio f , petrelli f , coinu a , et al : predictive role of braf mutations in patients with advanced colorectal cancer receiving cetuximab and panitumumab : a meta - analysis . 
eur j cancer 51 : 587 - 594 , 2015 shinozaki e , yoshino t , yamazaki k , et al : clinical signicance of braf non - v600e mutations on the therapeutic effects of anti - egfr monoclonal antibody treatment in patients with pretreated metastatic colorectal cancer : the biomarker research for anti - egfr monoclonal antibodies by comprehensive cancer genomics ( breac ) study . 
br j cancer 117 : 1450 - 1458 , 2017 yao z , torres nm , tao a , et al : braf mutants evade erk - dependent feedback by different mechanisms that determine their sensitivity to pharmacologic inhibition . 
cancer cell 28 : 370 - 383 , 2015 yao z , yaeger r , rodrik - outmezguine vs , et al : tumours with class 3 braf mutants are sensitive to the inhibition of activated ras . 
nature 548 : 234 - 238 , 2017 strickler jh , loree jm , ahronian lg , et al : genomic landscape of cell - free dna in patients with colorectal cancer . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
parseghian cm , loree jm , morris vk , et al : anti - egfr resistant clones decay exponentially after progression : implications for anti - egfr re - challenge . 
cremolini c , di bartolomeo m , amatu a , et al : braf codons 594 and 596 mutations identify a new molecular subtype of metastatic colorectal cancer at favorable prognosis . 
sullivan rj , infante jr , janku f , et al : first - in - class erk1 / 2 inhibitor ulixertinib ( bvd - 523 ) in patients with mapk mutant advanced solid tumors : results of a phase i dose - escalation and expansion study . 
rankin a , klempner sj , erlich r , et al : broad detection of alterations predicted to confer lack of benet from egfr antibodies or sensitivity to targeted therapy in 19 . 
schirripa m , biason p , cortiula f , et al : clinico - pathological and molecular characterization of braf mutant metastatic colorectal cancer ( mcrc ) : are all advanced colorectal cancer . 
yu ha , suzawa k , jordan e , et al : concurrent alterations in egfr - mutant lung cancers associated with resistance to egfr kinase inhibitors and characterization of mtor as a mediator of resistance . 
distribution of abraf ( atypical , non - v600 braf ) mutations in cohort 1 ( n = 36 )  . abraf mutation class appendix johnson et al table a1 . 
 distinct dicer1 hotspot mutations identify bilateral tumors as separate events introduction dicer1 syndrome1 predisposes to a variety of cancers , including pleuropulmonary blastoma ( ppb ) , 2 ovarian sertoli - leydig cell tumor ( slct ) , 3 embryonal rhabdomyosarcoma , 4 and kidney tumors.5 - 8 in dicer1 syndrome , most patients bear a germline null mutation in dicer1 , and the tumors uniformly bear a second - hit missense substitution at one of five hotspot positions ( 1705e , 1709d , 1809g , 1810d , and 1813e )  . a wide range of clinical phenotypes can be seen in dicer1 syndrome5 , 9 ; some patients are asymptomatic , whereas others develop multiple tumors . 
in the classic cases , patients with a germline null mutation develop different somatic hotspot mutations in each tumor.7 , 10 - 16 however , in some patients , multiple tumors arise from germline mosaicism of the hotspot mutation , with subsequent somatic loss of the wild - type allele.17 - 19 here we report a patient with dicer1 syndrome who developed four tumor types at six anatomic sites over the course of 12 years . 
because we found no other mutations to explain her particularly severe clinical course , we speculate that her subsequent tumors were a product of the intense chemotherapy and radiation regimens she received . methods informed consent was obtained from the patient and her guardian before collection of tumor specimens . 
when needed , pcr products were subcloned into pcr2.1topo ( thermo - fisher scientific , waltham , ma ) before sequencing . for tp53 sequencing , coding regions were amplified using the accel - amplicon comprehensive tp53 panel ( swift biosciences , ann arbor , mi )  . 
reads were trimmed using trimmomatic21 in the galaxy project and were aligned to human genome grch38 using bwa - mem22 ; 88% mapped to the tp53 locus , producing a mean depth of 13 , 367x in targeted regions . 
variants were called using the freebayes algorithm v0.9.20 using frequency - based pooled calling instead of simple diploid calling , which allowed the algorithm to detect subclonal mutations.23 results clinical presentation the patient ( cmcw11 ) presented originally at 5 years of age with a left kidney mass . 
she was treated with 10 months of combination chemotherapy ( vincristine , doxorubicin , cyclophosphamide , irinotecan , and etoposide ) targeted at both anaplastic wilms tumor and sarcoma , together with 2 , 100 cgy whole - abdomen irradiation given in 14 fractions over 19 days . when the patient was 10 years old , a new mass arose in her remaining kidney , with a histology similar to her prior tumor , and she was diagnosed with relapsed wilms tumor ( fig 1b )  . 
 on completion , she underwent right radical nephrectomy and started hemodialysis . this patients kidney tumors were diagnosed initially as wilms tumor , but in retrospect their unique histologic features are more consistent with anaplastic sarcoma of the kidney . 
this entity was first described in 2007 , after our patients kidney tumor first arose.24 biallelic dicer1 mutations have been seen in both wilms tumor and anaplastic sarcoma of the kidney , which may represent neoplastic degeneration from cystic nephroma.6 - 8 at 12 years of age , the patient developed multiple nodules throughout her thyroid . 
thyroidectomy revealed follicular adenomas ( fig 1c )  . at 13 years of age , a mass arising from her bladder was diagnosed as embryonal rhabdomyosarcoma with focal cartilaginous differentiation ( fig 1d )  . 
sanger sequencing chromatograms demonstrating the patients ( a ) dicer1 germline frameshift mutation and ( b ) rnase iiib hotspot mutations in her ovarian sertoli - leydig cell tumors ( slcts ) ; ( c ) left ( l ) kidney tumor ; and ( d ) right ( r ) kidney tumor . 
since that time , she has been observed for > 2 years , with no further disease . dicer1 mutations we performed whole - exome sequencing on the patients first kidney tumor and germline dna as table 1 . 
her tumor bore an additional somatic hotspot mutation ( c.5425g > a ; p.g1809r ; fig 2 and table 1 )  . we then sequenced the rnase iii domains of dicer1 in archival specimens from her other tumors . 
to sequence strands separately , we subcloned the pcr amplicon spanning this mutation and found that both single - nucleotide variants occurred on the same strand , making the tumor heterozygous for a single somatic hotspot mutation ( appendix fig a1 )  . 
contrary to our clinical suspicion at the time , her contralateral kidney tumor was , in fact , a second primary . we also sequenced dicer1 from her thyroid follicular adenoma and bladder rhabdomyosarcoma . 
bladder rhabdomyosarcomas have been associated previously with dicer1 syndrome , although specific discussion of their histologic features was not reported.4 her two ovarian tumors were detected at the same time , and it was unclear whether they represented distinct primary tumors or metastatic spread . 
her initial renal tumor bore a p.v274l variant , as we described previously.20 however , her remaining tumors had no additional somatic tp53 variants and did not undergo loss of heterozygosity at tp53 . discussion clinically , the discovery of a second tumor with similar histology can raise the question of whether it is a recurrence or a new primary tumor . 
had we known that her second kidney tumor was , in fact , metachronous , she might have avoided the increased toxicity of chemotherapy regimens designed for relapsed or refractory tumors . 
in fact , the cartilaginous elements of her bladder tumor led us to question whether it could instead represent a distant recurrence of her renal sarcoma , although its distinct dicer1 mutation proved its independence . thus , this case highlights a powerful but often overlooked application of clinical tumor sequencing : better understanding of the biology behind tumor formation . 
as sequencing costs fall , clinicians should be cognizant that tumor sequencing can help distinguish between new primary tumors and recurrences . in this case , sequencing at initial diagnosis would have also had the added benefit of alerting clinicians to her potential dicer1 status . 
in the case of dicer1 syndrome , because second hit mutations occur at such stereotyped positions , sequencing separate tumors could even occur by simple sanger sequencing rather than by next - generation sequencing . 
in patients with dicer1 syndrome , dedicated tests may be developed for identifying these stereotyped mutations , perhaps including circulating tumor dna assays as a noninvasive screening method . this is one of the most severe cases in the literature of multiple distinct somatic dicer1 mutations arising in a patient . 
shukla no relationship to disclose jason wang no relationship to disclose veena rajaram no relationship to disclose gordan vujanic no relationship to disclose tamra slone no relationship to disclose dinesh rakheja no relationship to disclose james f . 
slade i , bacchelli c , davies h , et al : dicer1 syndrome : clarifying the diagnosis , clinical features and management implications of a pleiotropic tumour predisposition syndrome . 
schultz ka , pacheco mc , yang j , et al : ovarian sex cord - stromal tumors , pleuropulmonary blastoma and dicer1 mutations : a report from the international pleuropulmonary blastoma registry . 
schultz ka , harris a , messinger y , et al : ovarian tumors related to intronic mutations in dicer1 : a report from the international ovarian and testicular stromal tumor registry . 
durieux e , descotes f , mauduit c , et al : the co - occurrence of an ovarian sertoli - leydig cell tumor with a thyroid carcinoma is highly suggestive of a dicer1 syndrome . 
klein s , lee h , ghahremani s , et al : expanding the phenotype of mutations in dicer1 : mosaic missense mutations in the rnase iiib domain of dicer1 cause glow syndrome . 
brenneman m , field a , yang j , et al : temporal order of rnase iiib and loss - of - function mutations during development determines phenotype in dicer1 syndrome : a unique variant of the two - hit tumor suppression model . 
vujani gm , kelsey a , perlman ej , et al : anaplastic sarcoma of the kidney : a clinicopathologic study of 20 cases of a new entity with polyphenotypic features . 
pugh tj , yu w , yang j , et al : exome sequencing of pleuropulmonary blastoma reveals frequent biallelic loss of tp53 and two hits in dicer1 resulting in retention of 5p - derived mirna hairpin loop sequences . 
 evolving landscape of molecular prescreening strategies for oncology early clinical trials rodrigo dienstmann , md1 ; elena garralda , md , msc2 ; susana aguilar , phd1 ; gemma sala , msc2 ; cristina viaplana , msc1 ; fiorella ruiz - pace , msc1 ; jenifer gonz alez - zorelle , bsc1 , 3 ; deborah grazia logiacco , phd3 ; zighereda ogbah , bsc3 ; laia ramos masdeu , phd3 ; francesco mancuso , bsc3 ; roberta fasani , md4 ; jose jimenez , bsc4 ; paola martinez , bsc4 ; ana oaknin , md , phd2 ; cristina saura , md , phd2 ; mafalda oliveira , md , phd2 ; judith balmaa , md , phd2 ; joan carles , md , phd2 ; teresa macarulla , md , phd2 ; elena elez , md , phd2 ; maria alsina , md , phd2 ; irene braa , md , phd2 ; enriqueta felip , md , phd2 ; josep tabernero , md , phd2 ; jordi rodon , md , phd2 , 5 ; paolo nuciforo , md , phd4 ; and ana vivancos , phd3 most academic precision oncology programs have been designed to facilitate enrollment of patients in early clinical trials with matched targeted agents . 
we started with a tumor - agnostic hotspot mutation panel plus in situ hybridization and immunohistochemistry of selected markers and subsequently transitioned to tumor - specic amplicon - based next - generation sequencing ( ngs ) tests together with custom copy number , fusion , and outlier gene expression panels . 
in parallel , biomarker - matched trials evolved from a scenario of few targets and large populations ( such as pi3k inhibitors in pik3ca mutants ) to a complex situation with many targets and small populations ( such as multiple targetable fusion events )  . 
the substantial increase of immunotherapy trials had a major impact in target prioritization and guided clinical implementation of new markers , such as tumor mutational burden , with larger exonbased ngs assays and gene expression signatures to capture microenvironment inltration patterns . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction during the last decade , when targeted agents entered the phase i clinical trial arena and next - generation sequencing ( ngs ) became a standard molecular prescreening test to identify actionable alterations , oncologists experienced a major shift toward biomarkerdriven drug development.1 there was a lot of enthusiasm with this approach , which culminated with the approval of many targeted drugs in molecularly selected patients on the basis of nonrandomized data . 
more recently , another important milestone was reached with the approval of tissue - agnostic therapies in biomarker - selected population , rst the immune checkpoint inhibitor pembrolizumab in microsatellite instable ( msi ) tumors and then trk inhibitors larotrectinib and entrectinib in cancers harboring ntrk fusions . 
given these exciting advances , many reference institutions initiated precision oncology programs with longitudinal patient cohorts undergoing tumor molecular proling nested to early clinical trials with matched targeted agents.2 in parallel , as advances in immuno - oncology are changing the standard of care of many cancer types , the phase i oncology community experienced another paradigm shift in drug development , with an unprecedented number of new investigational agents entering clinical testing.3 not only different antipd - 1 / l1 inhibitors but also multiple combination regimens are being investigated in early clinical trials . 
more recently , genomic biomarkers are inclusion criteria to select patients for immune checkpoint inhibitor therapy , such as tumor mutation burden ( tmb ) and mutations in dna damage repair ( ddr ) pathway genes . in this evolving scenario , early clinical trial units had to constantly adapt to new biomarker - drug codevelopment trends . 
because the molecular prescreening program was established at our institution , we customized the techniques and procedures to accurately identify molecular alterations in tumors from patients eligible for early clinical trials . 
the team meets periodically to discuss existing molecular tests and new biomarkers of interest to be added to our prescreening progracancer biologists and genomicists participate in weekly molecular tumor board meetings with medical oncologists to provide guidance on the interpretation of ngs results and discuss new markers for clinical testing in patients eligible for early clinical trials . as detailed in figure 1a , we started with a targeted single base extension mutation assay covering hotspot events in 20 oncogenes and tumor suppressor genes ( sequenom ) plus uorescent in situ hybridization ( fish ) or immunohistochemistry ( ihc ) analysis of selected genes and proteins . 
in 2013 we implemented an ncounter rna platform ( nanostring ) fusions and gene expression ( gex ) analysis of 26 genesfusion and gex ncounter . in 2014 we substituted the hotspot mutation panel with a custom amplicon - based ngs assay covering 59 genes ( miseq ) , and 1 year later we replaced the fish analysis with a targeted copy number ncounter dna panel of 44 genescna ncounter . 
during 2015 we also added brca1 / brca2 genes to the ngs panel , introduced pdl1 ihc and msi testing by ihc ( with polymerase chain reactionbased test in equivocal cases )  . 
in 2018 we developed an exonbased ngs assay ( hiseq ) with 420 genes , encompassing ddr plus epigenetic pathway genes and ne - tuned for tmb quantication and other genomic signatures , such as msi . 
so far , this assay is limited to selected cases of particular interest , as per discussions in weekly molecular tumor boards , but will eventually replace the amplicon ngs and nanocopy dna panels in the coming year . 
to identify patients eligible for antibody - drug conjugates , different ihc assays were developed in collaboration with pharmaceutical companies , which allowed local testing instead of sample shipment for central proling . 
most pharmaceutical companies also accept our in - housedeveloped genomic testing as screening for biomarker - guided trials , but we noticed that a growing number of early clinical trials mandate central ngs companion diagnostic tests , specically in lung , bladder , and biliary tract cancers . all assays were optimized for archived formalin - xed parafn - embedded tumor tissues . 
importantly , ngs assays had frequent modications in gene coverage to capture emerging biomarkers being investigated in clinical trials at our institution , such as the recent additions of notch family genes to the mutation panel , ntrk breakpoints to the fusion assay , and cd274 ( pd - l1 ) to the cna ncounter test . 
in 2018 , the amplicon - based ngs panel was tailored as multiple tumortypespecic panels , with coverage limited to genes that have been previously found to be mutated in each malignancy . 
finally , circulating tumor dna ( ctdna ) mutation detection panels are under development but still limited to patient cohorts with predened tumor types participating in validation studies . each oncologist has the responsibility to dene a patients eligibility for molecular prescreening on the basis of clinical features , tumor type , and disease setting . 
cna ncounter , copy number alteration nanostring panel ; fish , uorescent in situ hybridization ; fusion and gex ncounter , fusion and gene expression nanostring panel ; ihc , immunohistochemistry ; msi , microsatellite instability ; ngs , next - generation sequencing . yield ( combined prevalence of targetable molecular alterations ) exceeds 3% . 
depending on the tumor type , molecular tests are performed up front ( such as in lung cancer when sufcient tissue is available for ngs ) , while patients receive standard - of - care chemotherapies ( such as biliary tract cancer and hormone receptorpositive breast cancer ) , or after progression to approved regimens ( such as cervical cancer )  . 
we educate clinicians not to indicate ngs as a rescue diagnostic test to identify targets for experimental therapies when patients have fast clinical deterioration , but also to avoid the all - comers up - front strategy , given the potential reduced cost effectiveness of this approach for all advanced tumor types . 
 dienstmann et al after the patient signs informed consent for broad tumor molecular proling , requests are submitted to the molecular oncology unit for sample retrieval , qualication , and preparation , and to perform ihc / fish tests . 
turnaround time is , 1 week for ihc or fish tests and approximately 2 weeks for the amplicon - based ngs panel ; ncounter results are reported within 2 - 3 weeks and exon - based ngs panel in 3 - 4 weeks . 
depending on the assays coverage , it serves as a substitute for the internal program . our molecular prescreening program is free of charge to patients and nanced with both internal resources acquired through competitive grants or donations ( institutional patronage ) and external funds from agreements with pharmaceutical companies running early clinical trials with mandatory biomarker matches . 
in the latter case , which represents 15% of total budget , the conancing models can be a molecular prescreening fee for each patient recruited in the trial or a pay - per - test fee while the trial is recruiting patients ( with monthly reports detailing number of tests performed and positive results )  . as shown in figure 1b , close to 2 , 500 molecular tests were performed in 2018 , a 10 - fold increase as compared with 2010 . 
in 2010 , most early therapeutic trials tested pi3k pathway inhibitors ( n = 11 ) , whereas in 2018 , immunotherapy trials represented the great majority ( n = 75 ) , followed by antibody - drug conjugates ( n = 9 ) , epigenetics ( n = 9 ) , fgfr inhibitors ( n = 9 ) , pi3k pathway inhibitors ( n = 8 ) , mek / erk inhibitors ( n = 8 ) , and egfr / erbb2 inhibitors ( n = 7 )  . 
regarding phase i trials with targeted drugs ( fig 2a ) , we observed a gradual increase in the number of targets of interest for clinical development from 2010 to 2016 , including alk / ros1 , fgfr1 - 3 , braf , met , notch1 - 2 , ddr pathways alterations , and others ( hdm2 , kit , pdgfr , idh1 - 2 , ret , ntrk1 - 3 , cyclin , and wnt pathways )  . 
this was accompanied by a major increase in the number of immunooncology trials , which escalated since 2015 ( fig 2b )  . the criteria for patient enrollment in a given trial go beyond genomic markers . 
in cases of borderline evidence of clinical actionability of molecular targets ( enrichment criteria ) or no oncogene alteration , alternative therapies are considered , type and taking into consideration tumor availability of slots in trials with immunotherapies and other drugs . 
more recently , immune - related markers are used as positive selection criteria for clinical trials with novel immune - oncology drugs and combinations . out of 161 early clinical trials actively recruiting patients in 2018 , 57 investigated targeted agents ( fig 2a ) , 24 of them ( 42% ) were combination regimens , and 48 ( 84% ) had mandatory or enrichment molecular inclusion criteria , the most common being fgfr , mapk , pi3k , egfr / erbb2 , and ddr pathway alterations . 
regarding immunotherapy trials , 57 out of 75 ( 76% ) were combination regimens and 32 ( 43% ) had mandatory or enrichment molecular criteria for recruitment , with pd - l1 expression and msi status being the most common biomarkers ( figs 2b and 2d )  . 
we also gradually increased the number of clinical trials testing antibody - drug conjugates , most of them with mandatory ihc analysis for patient selection , and epigenetic targets , rarely recruiting patients on the basis of genomic proling ( as brd4 - nut fusions for bet inhibitors )  . inclusions in clinical trials with targeted agents uctuated from 2010 to 2018 ( fig 2c )  . 
the multiplicity of drug targets under investigation coupled with a growing number of molecular tests being performed during this period allowed recruitment of more patients in phase i including trials assessing novel biomarkers , fgfr1 - 3 , notch1 - 2 alterations , and brca1 - 2 mutations , among others . 
we observed a major decrease in recruitment rates in phase i trials with targeted agents in 2017 , in part related to lower interest in some targets , such as pi3k pathway inhibitors , but also a 3 - fold increase in the number of patients referred to phase ii or iii trials with molecular matches , from 84 in 2016 to 224 in 2018 . 
chemo , chemotherapy ; immuno , immunotherapy ; msi , microsatellite instability . 306 patients recruited in immunotherapy trials during 2018 , 92 ( 30% ) were based on biomarkers . to investigate the clinical utility of our program , we assessed how the results of proling ( both in - house and external ) affected the immediate treatment decision of each patient . 
the most common tumor types undergoing molecular proling in the last 3 years are shown in figure 3a . in 2018 , we noticed an increase in lung , breast , and pancreatobiliary cancers being tested in line with major advances in precision cancer therapy across these tumor types . 
it is important to emphasize that these represent patients not eligible for standard - of - care genomically guided therapy , such as braf inhibitors in brafv600emutated melanomas , or her2 - targeted therapy in erbb2amplied breast cancer . 
 a 2016 ( n = 1 , 281 ) 18 19 26 dienstmann et al 2017 ( n = 1 , 218 ) 15 22 18 1 , 104 2018 ( n = 1 , 416 ) 18 12 18 colorectal lung breast pancreatobiliary gynecologic gastric head and neck other brain urinary sarcoma skin endocrine inner ring no trial immunotherapy nonimmunotherapy matched phase 1 - 3 trial outer ring biomarker mandatory biomarker enrichment fig 3 . 
 ( b ) recruitment in biomarker - guided ( mandatory or enrichment ) and other alternative trials ( immunotherapy or nonimmunotherapy )  . guided targeted agents , antibody - drug conjugates , and immunotherapies . discussion the vall dhebron institute of oncology ( vhio ) molecular prescreening program is constantly adapting to the needs of our early clinical trial portfolio . 
we observed a dramatic change in the landscape of phase i clinical trials , now focused on targeted agents for rare molecular alterations , immunotherapy combinations , antibody - drug conjugates , and epigenetic agents . 
in parallel , different ngs assays were developed for accurate detection of rare actionable mutations , copy number alterations , fusion events , and novel ihc tests were introduced . from the beginning , we followed a personalized prescreening approach , whereby oncologists are empowered to decide which patients are eligible for testing based on clinicopathological features and which molecular tests are indicated in each disease setting . 
by avoiding the one - testfor - all strategy , we were able to optimize the use of our prescreening program and educate the clinicians on molecular epidemiology of each tumor type as well as emerging diagnostic tests . 
the decision to transition from smaller tumor - specic panels to a larger exon - based ngs that covers both mutation and copy number tests is related to the need to accommodate emerging biomarkers in the ddr and epigenetic pathways as well as tmb quantication . 
overall , 1 out of 10 patients proled are ultimately enrolled in matched targeted trials , which is in line with other academic molecular prescreening programs with nested clinical studies.4 regarding germline sequencing , we follow guideline - directed testing and have regular meetings with genetic counselors to discuss secondary incidental ndings in tumor sequencing.5 in the last decade , biomarker - matched trials evolved from a scenario of few targets and large populations to a complex situation with many targets and small populations . 
even with a growing number of targets being investigated in phase i trials , and the multiplicity of trials per target , we observed a relative reduction in the number of patients enrolled in molecularly guided trials in the last years . 
this is the result of shifting interests in drug development and a clear focus on a limited number of promising biomarkers , such as rare fusion events in fgfr1 - 3 , ret , and ntrk1 - 3 genes . 
in this scenario , liquid biopsies for target identication are becoming quite useful , which has guiding our current development of a ctdna ampliconbased ngs test and prompted collaborations with commercial partners for molecular prescreening . the substantial increase in immunotherapy trials had a major impact on trial prioritization . 
inclusion criteria of immuno - oncology phase i trials are frequently limited to tumor type and treatment line , with few trials having a mandatory molecular match , which facilitates patient recruitment . 
we noticed an increase in biomarker - guided immunotherapy trial recruitment in 2018 , which is guiding implementation of a multimodality biomarker strategy that moves beyond tmb quantication , such as immune cytotoxic / suppressive microenvironment signatures measured through the gex ncounter panel , 6 as well as multiplex ihc panels.7 to conclude , we believe that to accelerate progress in precision oncology , clinical trials with adaptive designs to enroll patients on the basis of multiomics enrichment criteria are needed . 
the previous era of molecularly guided targeted agents is restrictive , and larger portfolios of drugs that include immunotherapeutic and antibody - drug conjugates with recruitment guided by molecular tests must be pursued . 
in our view , the future of cancer drug development will encompass sequential genomic proling to guide matched targeted therapies , complex multimodality biomarkers for immune - oncology agents , individualized immunotherapeutics , novel combination regimens with epigenetic drugs , and antibody - drug conjugates . 
as precision oncology becomes a more paved road , clinicians in the community oncology setting must be cognizant of its full potential and reference institutions must establish patient referral networks to increase the access to innovative experimental therapies . 
 dienstmann et al speakers bureau : msd , roche , thermo fisher scientic research funding : novartis , roche ( inst ) , thermo fisher scientic ( inst ) travel , accommodations , expenses : bristol - myers squibb , menarini , glycotope , merck sharp & dohme ana oaknin consulting or advisory role : roche , astrazeneca , pharmamar , clovis oncology , tesaro , immunogen , genmab research funding : abbvie deutschland ( inst ) , ability pharmaceuticals ( inst ) , advaxis ( inst ) , aeterna zentaris ( inst ) , amgen ( inst ) , aprea therapeutics ( inst ) , clovis oncology ( inst ) , eisai ( inst ) , f . 
mandelker d , donoghue m , talukdar s , et al : germline - focussed analysis of tumour - only sequencing : recommendations from the esmo precision medicine working group . 
lu s , stein je , rimm dl , et al : comparison of biomarker modalities for predicting response to pd - 1 / pd - l1 checkpoint blockade : a systematic review and meta - analysis . 
calvo f , apolone g , baumann m , et al : cancer core europe : a european cancer research alliance realizing a research infrastructure with critical mass and programmatic approach to cure cancer in the 21st century . 
thirty - one patients ( 38% ) and 17 patients ( 46% ) underwent germline testing from the automatic pipeline and other referrals , respectively , and of these patients , 23 ( 72% ) and four ( 24% ) had conrmed germline pathogenic variants ( gpvs ) , respectively . 
the majority of conrmed gpvs were in automatic referral genes , with brca2 being most common ( conrmed gpvs in 11 [ 85% ] of 13 patients tested ) , followed by palb2 ( ve [ 67% ] of six patients ) , brca1 ( two [ 40% ] of ve patients ) , msh6 ( two of three patients ) , and mlh1 ( two of two patients )  . 
germline testing was not performed in 50 ( 62% ) of 81 patients identied by automatic referral as a result of poor patient health or death ( 30% ) , lack of follow - up ( 30% ) , and patient refusal ( 30% )  . conclusion of patients undergoing tgp , 5% had somatic ndings triggering referral , and implementation of an automatic referral pipeline based solely on gene versus other clinical or molecular features resulted in a 74% germline conrmation . 
use of such testing has rapidly increased , with further growth anticipated.1 mutations identied on tumor sequencing may be acquired somatically , caused by postzygotic mosaicism , or identication of inherited through the germline . germline mutations potentially inuences treatment of the current cancer , may provide prognostic information about the potential development of future cancers , and has important implications for family members in terms of risk and prevention of disease.2 however , guidelines on both how to determine which somatic ndings may be potentially germline and the optimal clinical practice for referral and germline testing of patients undergoing tumor sequencing are not widely available , and there is no established standard of care.3 genomic testing of tumor tissue can be performed in tandem with germline testing ( via blood or sequence matched normal tissue ) 4 - 6 ; such tumor - germline paired sequencing allows determination of mutation etiology ( inherited v acquired )  . 
 clark et al context key objective as the use of somatic tumor genomic testing rapidly increases , a pipeline for referral of potential germline genetic ndings is critically needed . knowledge generated more than 70% of known cancer predisposition gene mutations identied in patients tumor genomic testing were conrmed to be germline . 
however , less than half of eligible patients underwent germline genetic testing as a result of poor patient health or death , lack of follow - up , and / or patient refusal . relevance our ndings indicate that patients with pathogenic mutations in well - characterized cancer predisposition genes should be referred for consideration of germline genetic testing , although signicant implementation challenges remain . be associated with hereditary cancer syndromes for both the patients and their family members . 
however , potential germline variants on tgp may be under - recognized.8 national comprehensive cancer network ( nccn ) guidelines are evolving ; they currently state that pathogenic variants in cancer susceptibility genes identied on tumor proling of any tumor type should undergo germline conrmation.9 however , the logistics of identifying potentially actionable germline mutations , such as those in brca1 and brca2 , from tgp can be complex , in large part as a result of the coordination of germline genetic testing for ill patients who may be unaware that their tumor testing could reveal inherited mutations . 
somatic pathogenic mutations as dened in cbioportal were downloaded from the provisional data sets of multiple tumor types in july 2015 ( data supplement ) and updated in july 2018 ( table 1 )  . 
original germline mutation rates were averaged from two large studies of patients with metastatic cancer5 , 6 and updated with tcga data , 11 and mutations were dened as reported in the original studies.5 , 6 , 11 the ratio of the germline mutation rate to total mutation rate was determined ( table 1 ; appendix fig a1 )  . 
genes were also annotated with the number of tumor types with somatic mutation rates greater than 10% ( data supplement )  . delineation of criteria for automatic referral for germline testing criteria for automatic referral for germline testing after tgp from june 2016 through june 2018 , the center for personalized diagnostics ( cpd ; college of american pathologists and clinical laboratory improvement amendments approved ) at penn medicine evaluated 2 , 308 unique tgp tests with either a 153 - gene panel ( n = 2 , 087 ) or a 47 - gene panel ( n = 221 ) , depending on the version of the panel available ( data supplement )  . 
a multidisciplinary team , including genetic counselors , medical oncologists , medical geneticists , and molecular pathologists , was established to determine which somatic results warranted automatic referral for germline genetic testing . 
 clark et al was not included in our analysis because of controversy over screening guidelines and its relatively high prevalence.12 implementation of germline testing referral pipeline solid tumor sequencing reports were signed out per clinical practice standards , and variants in the automatic referral pipeline were identied manually by the reviewing cpd attendings . 
language for clinical reports was created stating that additional testing would be necessary to determine whether the identied pathogenic variant on somatic testing was a germline pathogenic variant ( gpv )  . 
an e - mail was sent from the cpd to the ordering oncologist and a designated genetic counselor ( gc ) when a variant meeting automatic referral criteria was identied . 
initial disclosure of the somatic test results , including the possibility of a germline nding , was the responsibility of the ordering provider.13 after patients were made aware of somatic ndings by their oncologist , patients were contacted by the gc for coordination of care . 
molecular pathologists and oncologists also were encouraged to refer patients outside the automatic pipeline on the basis of genotype - phenotype correlation . data collection , chart review , and statistical analysis a university of pennsylvania institutional review board approved retrospective chart review was conducted , with data collected for all referrals within the automatic pipeline and other referrals based on tgp by the academic laboratory between june 2016 and june 2018 ( 81 referrals by automatic pipeline and 37 referrals outside the pipeline )  . data were collected on patient demographics , tumor type tested ( data supplement ) , and somatic and germline testing results . 
continuous variables were compared using the t test , and contingency tables were analyzed using fishers exact test . results referral characteristics from june 2016 to june 2018 , 2 , 308 patients underwent tgp at penn medicines cpd . 
of those patients , 118 ( 5.1% ) were referred for clinical germline genetic testing based on tgp performed at the cpd . referral of 81 patients was triggered by the automatic algorithm , and 37 patients were referred from either the molecular pathologist ( n = 21 ) or the patients physician ( n = 16 ; fig 2a )  . 
the majority of patients were white ( 88% ) and female ( 52% ) , and the mean age at tgp was tumor types 58 years ( table 2 )  . 
thirteen patients were referred for variants with a high likelihood in chek2 of being germline ( founder mutations ) ( n = 11 ) and one each for variants in atm and egfr ( fig 2a ; data supplement )  . 
direct referrals for germline genetic testing by the molecular pathologist and primary oncologists after a variant was found on tgp were largely based on genotype - phenotype concerns ( eg , tp53 mutations in patients with cancer age 45 years or younger or patients with brain tumors or sarcomas ; cdkn2a / cdk4 mutations in patients with melanoma )  . 
referrals of 21 patients by the molecular pathologist were initiated by e - mail to the gc and primary oncologist and included ndings in nine genes , most commonly tp53 ( fig 2a ; data supplement )  . treating physicians referred 16 patients with variants in 12 different genes ( fig 2a , data supplement )  . results of germline conrmation of 118 patients with referrals , 48 ( 41% ) underwent clinical genetic counseling and genetic testing ( fig 2a )  . 
genetic testing rates were similar for referrals from the automatic referral pipeline and those sent by the molecular pathologist ( 38% and 33% , respectively ) , whereas 63% of patients referred by their treating physician underwent genetic testing ( fig 2a )  . 
a majority of patients ( 27 [ 57% ] of 48 patients ) had variants that were conrmed gpvs , with 74% of automatic referrals being conrmed gpvs and 29% and 20% of molecular pathologist and treating physician referrals being conrmed gpvs ( fig 2a )  . 
the majority of patients ( 60% ) had multiplex panel testing ; however , 19% underwent a small custom panel , 17% were only tested for the variant by single - site testing , and 4% had ashkenazi jewish founder testing ( data supplement )  . the 81 patients referred by the automatic referral pipeline , the majority ( n = 59 ) were referred for variants in high - risk genes ( 73% ; fig 2b )  . 
genetic testing rates were highest for high - risk genes ( 28 referrals [ 47% ] underwent genetic testing compared with three referrals [ 14% ] in the other groups )  . 
mutations in the automatic referral pipeline were agged by the tumor testing laboratory , and an e - mail was sent from molecular pathologist to the ordering physician ( md ) and genetic counselor ( gc )  . 
results not included in the automatic referral pipeline but of concern to the molecular pathologist or ordering oncologist to be possibly germline were also e - mailed to the gc . 
the gc called the patient only after the somatic results were discussed with the patient by the ordering md , the patient agreed to be contacted , and the oncologist contacted the gc . 
an inperson cancer risk evaluation appointment was scheduled , and the appropriate germline testing modality was determined by gc at time of visit ( panel or single - site testing )  . 
results were provided by telephone by the gc , and patients with positive testing results along with their relatives were offered in - person follow - up visits for medical management . 
one hundred eighteen patients were referred for germline testing83 by the automatic pipeline , 20 by the testing laboratory at the discretion of the molecular pathologist , and 15 directly from the ordering physician . 
 ( b ) of the 83 patients referred by the automatic pipeline , 61 had mutations in high - risk genes ( brca1 , brca2 , palb2 , mlh1 , msh2 , and msh6 ) , nine had mutations in moderate - risk genes ( brip1 , rad51c , and rad51d ) , and 13 had mutations with high prior probability of being germline ( likely germline )  . 
 ( c ) in the 51 patients referred by the automatic pipeline but who did not undergo testing , patients were not seen as a result of death before scheduling an appointment , refusal of the consult by the patient , absence of referral placed by ordering physician , or other reason . or both ( fig 2c )  . 
 characteristic age , years mean range male race tumor type white black other lung brain breast other melanoma 526 ( 23 ) 676 ( 29 ) 519 ( 22 ) 125 ( 5 ) 59 ( 3 ) 25 ( 1 ) 122 ( 5 ) germline results in tumor - only genomic testing table 2 . 
 ( a ) in patients who presented for conrmatory germline testing , the age at diagnosis for patients with a conrmed germline pathogenic variant ( gpv ) versus somatic pathogenic variants ( spvs ) not conrmed to be in the germline . 
 ( c ) percentage of patients with a conrmed gpv versus an spv for whom the gene is known to be associated with the tumor where the variant was found . 
of 2 , 308 patients who underwent tgp at penn medicines cpd , 149 ( 6.6% ) had variants in the automatic referral genes , and 118 ( 5% ) were referred for clinical germline genetic testing . 
a majority of the variants tested were conrmed gpvs ( 27 [ 54% ] of 50 variants ) , with the highest germline conrmation rate in high - risk genes ( 21 [ 72% ] of 29 genes )  . 
thus , we have demonstrated that an automatic referral algorithm results in a high yield of conrmed germline mutations . in total , 27 ( 1.1% ) of 2 , 308 patients undergoing tgp had a conrmed gpv . 
germline ndings have been found by retrospective analysis in 4% to 12% of prior clinical tumor sequencing cohorts5 , 6 , 14 and in 8% of the tcga cohort , 11 with 2.8% of the tcga cohort having germline mutations in the genes selected for automatic referral in this study . 
if all 149 patients with mutations in automatic referral genes in our cohort had undergone germline testing , we would have expected 107 to have a germline mutation ( based on our 72% germline conrmation rate )  . 
a limitation of this study is that sequencing was not performed on all patients with metastatic disease at the university of pennsylvania ; in addition , germline sequencing was not performed in all dividuals with somatic sequencing . 
thus , we do not know the exact prevalence of germline mutations in our population . genes were chosen for automatic referral based on several factors , but the major determinant was the ratio of germline mutation rate to total ( germline plus somatic ) mutation rate calculated from public databases . 
thus , referring all individuals with mutations identied in such genes ( eg , tp53 and pten ) identied by tgp in the absence of other factors rarely results in a positive germline nding . 
however , other genes have higher mutation rates in the germline compared with somatic only ( eg , brca1 / 2 ) , and known founder mutations would likely always be germline . 
for example , in our study , all three ashkenazi jewish founder mutations in brca1 / 2 that were found on tgp were conrmed gpvs . the automatic referral algorithm did not rely on clinical characteristics or variant af or exclude patients with likely hypermutated tumors . 
the nding that germline af can be lower than the expected range of 50% or greater is consistent with prior data.15 multiple patients had germline mutations identied despite somatic testing done on tumors not generally considered part of the hereditary syndrome . 
the high germline conrmation rate for the automatic referral genes validates our approach of prioritizing the ratio of germline to total mutations of a gene , with lesser emphasis placed on af or tumor type . 
we have updated both germline and somatic mutation rates with current11 data ( table 1 ) , demonstrating that most of these genes have a ratio of germline mutation rate to total mutation rate of greater than 0.25. 
the only genes not included on our original list that t these criteria are atm and chek2 . we are adding all mutations in these genes to our referral list ( rather than restricting to certain mutations with high prior probability of being germline )  . other approaches for gene selection exist ; however , we wished to prioritize the highest impact genes related to prognosis or therapy , clinical trial enrollment ( eg , brca1 / 2 mutations and poly [ adp - ribose ] polymerase inhibitors ) , or effect on family members.16 - 19 in addition , we wished to assess the success of this approach and potential burdens before widening the scope . 
curation of the list of genes in which mutations prompt referral for germline genetic testing will be an ongoing process and will need to reect updates in guidelines from the american college of medical genetics and nccn . 
the automated referral algorithm had a high specicity , as 72% of ndings were conrmed gpvs . in patients for whom somatic sequencing triggered a referral by their oncologist without meeting criteria on the algorithm , only two patients ( 20% ) had conrmed gpvs ( atm and chek2 )  . 
as mentioned earlier , both genes are being added to our automated referral algorithm . criteria for germline genetic testing are rapidly evolving , and routine germline multiplex panel testing may soon become standard for all patients with metastatic disease . 
in our study , of 28 patients who underwent multiplex panels , either testing was negative ( n = 13 ) or only the somatic variant was found ( n = 15 ) , indicating that there may be limited incremental benet to expanded germline testing in patients with metastatic cancer who have undergone tumor testing . 
however , individuals with a high likelihood of having a germline mutation in a cancer susceptibility gene should be considered for germline testing even in the setting of a negative tgp . 
for example , in individuals with a strong family history of breast and ovarian cancer , approximately 10% of brca1 mutations are large genomic rearrangement , which may be more difcult to detect with current tgp assays . the majority of patients ( 61% ) with a somatic nding identied by the automatic referral pipeline were not seen by clinical cancer genetics services . 
published data suggest that patients express great interest in wishing to learn their germline status20 ; therefore , it is likely that patient and provider education and improved infrastructure for referrals are needed . 
 germline results in tumor - only genomic testing cancer therapy and symptom management , simplifying the process by which patients become aware of the possibility of germline ndings arising from tgp and have access to germline conrmation is critical . 
the ongoing communication and education in tumor proling ( comet ) trial ( clinicaltrials.gov identier : nct02823652 ) , a companion study to the national cancer institute molecular analysis for therapy choice ( nci - match ) trial , will provide additional information regarding best practices . genetic counseling challenges in this population of patients with advanced cancer included the substantial burden of patient illness ( including both symptom and appointment burdens , although considerable efforts were made to coordinate appointments with other visits )  . 
there was an appreciable time commitment by the gc that was not billable ( eg , discussions by phone ) , and a number of patients tested did not have insurance coverage because their tumor type did not meet published criteria for germline testing . 
nccn criteria for further genetic risk evaluation ( version 2.2019 ) now include individuals at any age with a known pathogenic variant or likely pathogenic variant in a cancer susceptibility gene found on tumor testing ; however , during the dates of our study , existing criteria included brca1 / 2 only.9 we performed a single - institution study and only evaluated ndings from penn medicines cpd ; thus , generalizability of this approach is not yet known . 
in addition , full somatic sequencing for all of the automatic referral genes was not performed on every patient as a result of availability of specic somatic sequencing panels at different points in time . 
we did not implement approaches such as telegenetics , which may further reduce patient burden ( although germline samples must still be obtained )  . we have demonstrated that an automated referral process for patients with mutations detected in tgp can lead to the identication of germline mutations in cancer susceptibility genes with implications for both patients and their family members . 
 clark et al jacquelyn powers employment : carevive systems honoraria : cureconnect consulting or advisory role : carevive systems travel , accommodations , expenses : hospital of the university of pennsylvania jessica m . 
long employment : depuy companies ( i ) stock and other ownership interests : depuy companies ( i ) travel , accommodations , expenses : depuy companies ( i ) payal shah stock and other ownership interests : johnson & johnson ( i ) , novartis ( i ) , novo nordisk ( i ) , pzer ( i ) , merck ( i ) , amgen , johnson & johnson , novo nordisk consulting or advisory role : tmunity therapeutics jennifer j.d. 
domchek honoraria : astrazeneca , clovis oncology , bristol - myers squibb research funding : astrazeneca ( inst ) , clovis oncology ( inst ) , pharmamar ( inst ) no other potential conicts of interest were reported . references raymond vm , gray sw , roychowdhury s , et al : germline ndings in tumor - only sequencing : points to consider for clinicians and laboratories . 
j pathol 244 : 610 - 615 , 2018 arango np , brusco l , mills shaw kr , et al : a feasibility study of returning clinically actionable somatic genomic alterations identied in a research laboratory . oncotarget 8 : 41806 - 41814 , 2017 4 . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
ann oncol 27 : 795 - 800 , 2016 schrader ka , cheng dt , joseph v , et al : germline variants in targeted tumor sequencing using matched normal dna . 
jama oncol 2 : 104 - 111 , 2016 robson me , bradbury ar , arun b , et al : american society of clinical oncology policy statement update : genetic and genomic testing for cancer susceptibility . j clin oncol 33 : 3660 - 3667 , 2015 catenacci dv , amico al , nielsen sm , et al : tumor genome analysis includes germline genome : are we ready for surprises ? int j cancer 136 : 1559 - 1567 , 2015 9 . 
yurgelun mb , chittenden ab , morales - oyarvide v , et al : germline cancer susceptibility gene variants , somatic second hits , and survival outcomes in patients with resected pancreatic cancer . 
bolton kl , chenevix - trench g , goh c , et al : association between brca1 and brca2 mutations and survival in women with invasive epithelial ovarian cancer . 2014 res 22 : 4087 - 4094 , 2016 jama 307 : 382 - 390 , 2012 33 : 244 - 250 , 2015 18 . 
hamilton jg , shuk e , garzon mg , et al : decision - making preferences about secondary germline ndings that arise from tumor genomic proling among patients with advanced cancers . 
 germline results in tumor - only genomic testing appendix brca1 brca2 palb2 mlh1 msh2 msh6 brip1 rad51c rad51d chek2 tp53 bap1 cdh1 cdkn2a men1 pten smad4 smarca4 stk11 tsc1 tsc2 original updated ratio of germline mutation rate to overall mutation rate fig a1 . 
 metabolic derangements in succinate dehydrogenase bmutated renal - cell carcinomas : more than meets the eye ? see accompanying article doi : succinate dehydrogenase - deficient renal cell carcinomas ( sdh - rccs ) are rare but highly aggressive malignancies associated with mutations in the four subunits ( sdha , sdhb , sdhc , or sdhd ) of the succinate dehydrogenase ( sdh ) enzyme complex . 
in practically all sdh - rcc cases , patients have monoallelic germ - line inactivating mutations in one of the four sdh genes , followed by somatic loss of the second gene allele in the tumor cells . 
sdhc deficiency is the second most common cause of sdh - rcc , whereas sdha and sdhd loss is rarely associated with rccs.1 - 4 because of the rarity of sdh - rccs , there is little guidance on how to best diagnose , monitor , and treat patients with this disease . lee et al5 retrospectively clinically and genomically profiled three patients with sdh - rcc resulting from biallelic sdhb inactivation . 
they showed that the sdhb gene acts as a tumor suppressor on the basis of the identification of germ - lineinactivating sdhb mutations in all three cases , followed by second - hit somatic loss of the short arm of chromosome 1 ( 1p ) where sdhb is located . 
although arm - level chromosomal deletions are certainly not uncommon among solid tumors , they tend to occur more frequently in regions of low gene density that are less likely to contain genes whose deletion will be poorly tolerated by cells.6 chromosome 1p harbors a large number of genes and has accordingly been found to be one of the least frequently deleted chromosome arms across several solid tumor types.6 however , in addition to sdhrccs , somatic loss of heterozygosity is also the preferred mode of second - hit inactivation in other sdhb - associated tumors such as paragangliomas and pheochromocytomas.7 it is therefore possible that loss of the genes in chromosome 1p produces a net positive effect for cell survival when there is biallelic inactivation of sdhb . 
mutyh encodes the enzyme myh glycosylase , which is involved in dna repair and is associated with polyposis when both mutyh alleles are inactivated.8 such molecular pathways may be exploited therapeutically . for example , loss of the chromosome 1p gene urod , encoding for uroporphyrinogen decarboxylase , can increase production of reactive oxygen species and thus sensitize cells to radiation or cisplatin - based therapy.9 , 10 it is worth noting that all three patients were young ( age 19 to 37 years ) and were initially incorrectly diagnosed despite being evaluated by experienced clinicians and pathologists at a large academic referral center.5 indeed , two of three primary tumors in the study lacked the histologic findings commonly associated with sdhbdeficient sdh - rccs . 
of note , all cases of sdh - rcc are associated with immunohistochemical ( ihc ) loss of sdhb expression , regardless of which sdh subunit inactivation led to the development of tumor.1 , 2 , 5 therefore , it may be prudent to first po.ascopubs.org jco precision oncology pavlos msaouel gabriel g . 
in theory , serum concentrations of lactic acid in such patients should reflect tumor burden independently of the associated cancer type ( sdh - rcc , pheochromocytoma , paraganglioma , or gi stromal tumors )  . this should also be true in patients with hereditary leiomyomatosis and rcc ( hlrcc ) , which is associated with loss of fumarate hydratase ( fh ) , another key krebs cycle enzyme.14 it will be interesting to further explore the value of longitudinal monitoring of lactic acid to determine disease burden in patients with these malignancies or as a screening test for individuals with known germ - line sdh or fh mutations . 
notably , the metabolic shift to aerobic glycolysis also leads to more intense fluorodeoxyglucose ( fdg ) uptake in positron emission tomography ( pet ) imaging of sdh - rcc and hlrcc compared with what is usually seen in other rccs.3 , 5 , 15 how can we best use precision oncology to treat sdh - rcc ? as already mentioned , there may be pathways associated with sdhb or chromosome 1p loss that may be amenable to targeted therapeutic strategies . 
because of the retrospective nature of the study by lee et al , 5 no rna had been banked to allow transcriptome profiling , which would have elucidated the impact of sdhb and chromosome 1p loss on gene expression . tumors such as those in sdh - rcc and hlrcc are highly dependent on glucose influx to maintain aerobic glycolysis . 
bevacizumab inhibits angiogenesis and thus blocks the delivery of glucose to the tumor microenvironment.16 in addition , the epidermal growth factor receptor inhibitor erlotinib can interfere with active glucose transport into tumor cells by downregulating glucose transporter 3.17 accordingly , the combination of bevacizumab with erlotinib has been shown to produce high clinical activity in patients with hlrcc.18 sdh - rcc may similarly benefit from this combination therapy , which has not yet been tested in this tumor type . 
in addition to their addiction to glucose , the loss of sdh activity makes sdh - rcc tumor cells more reliant on glutamine for their metabolism , as shown by the increased glutamine uptake on 18fglutamine pet imaging.5 glutamine supplies biosynthetic intermediates that are crucial for amino acid and lipid synthesis in sdh - deficient tumors . 
inhibition of the glutaminase enzyme , which generates glutamate from glutamine , may impair this metabolic pathway resulting in the death of glutamine - dependent tumor cells.19 nevertheless , the patient treated with glutaminase inhibitor cb - 839 in the lee et al5 study ultimately succumbed to his disease . 
it is possible that the tumor cells overcame the glutaminase inhibition by generating intermediates for macromolecule biosynthesis via alternative pathways.20 therefore , glutaminase inhibition may be more beneficial when used as part of a combination treatment regimen to fully block the production of key metabolic intermediates in sdh - rcc . putting it all together , the study by lee et al , 5 in conjunction with earlier reports on sdh - rcc , 1 , 2 shows that clinicians should be acutely aware of the possibility of sdh - rcc when seeing young patients who harbor aggressive kidney tumors with unusual histology and / or high fdg avidity . the evaluation should begin with a thorough personal and family history that looks for occurrences of paragangliomas , pheochromocytomas , gi stromal tumors , or unexplained earlyor lateonset neurodegenerative disease and / or encephalopathy.21 one should keep in mind that a negative family history in patients with sdh - rcc may be the result of de novo germ - line mutation or incomplete penetrance as in patient 1 in the article by lee et al.5 if there is a positive history of sdhrcc or if the suggestion of that disease remains high despite the lack of associated disorders in the patients family , then ihc for sdhb should be attempted . 
malouf honoraria : novartis , roche , glaxosmithkline , janssen oncology consulting or advisory role : novartis , pfizer , bristol - myers squibb research funding : pfizer , novartis travel , accommodations , expenses : amgen , pfizer , janssen , novartis nizar m . 
tannir honoraria : pfizer , novartis , bristol - myers squibb , exelixis , nektar therapeutics , argos therapeutics , calithera biosciences consulting or advisory role : novartis , exelixis , bristolmyers squibb , nektar therapeutics , pfizer , argos therapeutics , calithera biosciences research funding : bristol - myers squibb , novartis , exelixis , epizyme , mirati therapeutics travel , accommodations , expenses : pfizer , novartis , exelixis , nektar therapeutics , bristol - myers squibb , argos therapeutics , calithera biosciences acknowledgment supported by the grant no . 
gill aj , hes o , papathomas t , et al : succinate dehydrogenase ( sdh ) - deficient renal carcinoma : a morphologically distinct entitya clinicopathologic series of 36 tumors from 27 patients . 
ozluk y , taheri d , matoso a , et al : renal carcinoma associated with a novel succinate dehydrogenase a mutation : a case report and review of literature of a rare subtype of renal carcinoma . hum pathol 46 : 1951 - 1955 , 2015 5 . 
lee ch , gundem g , lee w , et al : persistent severe hyperlactatemia and metabolic derangement in lethal succinate dehydrogenase bmutated metastatic kidney cancer : clinical challenges and examples of extreme warburg effect . 
n engl j med 348 : 791 - 799 , 2003 ito e , yue s , moriyama eh , et al : uroporphyrinogen decarboxylase is a radiosensitizing target for head and neck cancer . 
sudarshan s , sourbier c , kong hs , et al : fumarate hydratase deficiency in renal cancer induces glycolytic addiction and hypoxia - inducible transcription factor 1alpha stabilization by glucosedependent generation of reactive oxygen species . 
makinoshima h , takita m , matsumoto s , et al : epidermal growth factor receptor ( egfr ) signaling regulates global metabolic pathways in egfr - mutated lung adenocarcinoma . 
srinivasan r , su d , stamatakis l , et al : mechanism based targeted therapy for hereditary leiomyomatosis and renal cell cancer ( hlrcc ) and sporadic papillary renal cell carcinoma : interim results from a phase 2 study of bevacizumab and erlotinib . 
lussey - lepoutre c , hollinshead ke , ludwig c , et al : loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolisnat commun 6 : 8784 , 2015 21 . 
sartori et al we thank sartori et al1 for their interest in the recently published review , which characterizes the her2 aberrations observed in nonsmall - cell lung cancer ( nsclc ) and summarizes the efcacy of her2targeted therapies in patients with nsclc.2 nsclc with her2 alterations is a highly heterogeneous disease . 
her2 overexpression , her2 amplication , and her2 mutation could exist alone or synchronously . sartori et al1 highlighted the contribution of immunologic features to the response to her2 - targeted therapy . 
as per the authors exploration , a strong antibody - dependent cell - mediated cytotoxicity triggered by the valine / valine ( v / v ) variant of fcriiia rather than sole target inhibition is the possible reason . the back - line application of singleunfortunately , agent immunotherapy to case 1 eventually led to hyperprogression disease ( hpd )  . 
indeed , the denition of hpd remains controversial to date , let alone the solid association between driver gene mutation and hpd.5 recently , our group also reported a patient with erbb1 exon 20 insertion mutation who suffered from hpd after nivolumab monotherapy.6 we speculated that one of the reasonable causes might be myc amplication , whose upregulation could result in cd47 activation and programmed death ligand 1 overexpression , consequently contributing to tumor growth . to the efcacy of with respect immunotherapy in her2 - mutant nsclc , 4 retrospective studies reported the outcomes.7 - 10 a total of 92 patients were treated with single - agent pd - 1 or pd - l1 inhibitor , most of them having experienced disease progression on standard chemotherapy . 
here , we call for in - depth studies of hpd mechanisms , especially the development of screening methods to identify specic subgroups in whom immune treatment would be harmful . up to now , her2 mutation , especially exon 20 insertion , is a potential therapeutic target with accumulated evidence . 
recent preclinical research reported that poziotinib potentiated the antitumor effect of trastuzumab emtansine ( t - dm1 ) by upregulating her2 expression.11 this mechanism ts the logic and the observed encouraging clinical benet from t - dm1 after disease progression on poziotinib in case 2 . 
in addition , a preclinical study conducted by li et al12 found that cotreatment of t - dm1 with irreversible pan - her inhibitors neratinib or lapatinib enhanced her2 receptor ubiquitination and consequently improved t - dm1 internalization and efcacy . 
whether the optimal combination mode is sequential or synchronous warrants further validation . trastuzumab deruxtecan ( t - dxd ) , another antibody - drug conjugate ( adc ) , harbors a different cytotoxic payload , showed better and more durable responses in her2mutant nsclc , and may be still effective after resistance to t - dm1.12 in extension , on top of her2 mutation , her2 amplication may also confer sensitivity to t - dm1 . 
according to recently released clinical trial data , t - dm1 exhibited identical orr in patients with nsclc with her2 amplication , her2 mutation , or concurrent her2 mutation and amplication ( 55% v 50% v 50% ) .12 collectively , her2 alterations in nsclc are rather heterogeneous . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . no potential conicts of interest were reported . references zhao j , xia y : targeting her2 alterations in non - small - cell lung cancer : a comprehensive review . 
precision oncol 4 : 411 - 425 , 2020 krug lm , miller va , patel j , et al : randomized phase ii study of weekly docetaxel plus trastuzumab versus weekly paclitaxel plus trastuzumab in patients with previously untreated advanced nonsmall cell lung carcinoma . cancer 104 : 2149 - 2155 , 2005 xu x , de angelis c , burke ka , et al : her2 reactivation through acquisition of the her2 l755s mutation as a mechanism of acquired resistance to her2 - targeted therapy in her2 + breast cancer . 
clin cancer res 23 : 5123 - 5134 , 2017 jin r , zhao j , xia l , et al : application of immune checkpoint inhibitors in egfr - mutant non - small - cell lung cancer : from bed to bench . 
huang x , xia l , lan f , et al : treatment of nivolumab results in hyperprogressive disease in a patient harboring egfr exon 20 insertion and myc amplication . 
mazieres j , drilon a , lusque a , et al : immune checkpoint inhibitors for patients with advanced lung cancer and oncogenic driver alterations : results from the immunotarget registry . 
ann oncol 30 : 1321 - 1328 , 2019 lai w - cv , feldman dl , buonocore dj , et al : pd - l1 expression , tumor mutation burden and response to immune checkpoint blockade in patients with her2 - mutant lung cancers . 
j clin oncol 36 , 2018 ( suppl 15 ; abstr 9060 ) dudnik e , bshara e , grubstein a , et al : rare targetable drivers ( rtds ) in non - small cell lung cancer ( nsclc ) : outcomes with immune check - point inhibitors ( icpi )  . 
guisier f , dubos - arvis c , vias f , et al : efcacy and safety of anti - pd - 1 immunotherapy in patients with advanced nsclc with braf , her2 , or met mutations or ret translocation : gfpc 01 - 2018 . 
robichaux jp , elamin yy , vijayan rsk , et al : pan - cancer landscape and analysis of erbb2 mutations identies poziotinib as a clinically active inhibitor and enhancer of t - dm1 activity . 
cancer discov 10 : 674 - 687 , 2020 sartori g , belluomini l , trovato r , et al : her2 aberrations in nsclc : so close yet so far . 
 de novo esr1 hotspot mutation in a patient with endometrial cancer treated with an aromatase inhibitor adeline morel , md1 ; julien masliah - planchon , phd1 ; guillaume bataillon , md2 ; v eronique becette , md2 ; claire morel , msc3 ; samantha antonio1 ; elodie girard , md4 ; ivan bi `eche , phd1 , 5 ; christophe le tourneau , md , phd3 , 4 , 6 ; and maud kamal , phd3 introduction endometrial carcinoma is the fourth most frequent cancer in women . 
endometrial carcinoma is classied into two subtypes : type 1 endometrial carcinoma characterized by estrogen receptor ( er ) expression and obesity , and type 2 endometrial carcinoma in nonobese , older women . 
type 1 endometrial carcinoma is associated with a favorable prognosis , whereas type 2 has a poorer prognosis.1 a prognostic genomic classication of endometrial cancers into four groups has been established using exome sequencing ; however , this classication is not used in the clinic.2 standard - of - care treatment of endometrial carcinoma consists of primary hysterectomy , bilateral salpingooophorectomy , and pelvic lymph node dissection followed by adjuvant therapy based on the histologic assessment of the specimen.3 chemotherapy is proposed in the recurrent and / or metastatic setting , whereas hormone therapy represents a treatment option for patients with er expression.4 in breast cancer , esr1 mutations were clearly identied as a mechanism of resistance to aromatase inhibitors.5 - 8 esr1 mutations were reported in 2% of endometrial cancers , 9 yet their potential occurrence following hormonal therapy is unknown . 
the diagnosis of grade 1 endometrioid carcinoma was conrmed , with less than 50% invasion of the myometrial wall thickness , 4 cm in greatest dimension , lymphovascular invasion , bilateral ovarian metastases , and no pelvic lymph node metastases ( pt3an0 f ed eration internationale de gyn ecologie et dobst etrique iiia )  . 
using immunohistochemistry , tumor cells were shown to express er , progesterone receptor , and a wild - type staining of p53 . in addition , tumor cells expressed ck7 and pax8 and did not express ck20 , ttf1 , cdx2 , and wt1 , conrming the endometrial origin of the tumor . ( ie , letrozole )  . 
after nine cycles of chemotherapy , the patient received maintenance therapy with an aromatase inhibitor receiving letrozole for 6 months , the patient experienced disease progression and was subsequently treated with doxorubicin and cyclophosphamide and then carboplatin alone . 
the patient was then biopsied in the framework of the shiva02 trial ( evaluation of the efcacy of targeted therapy based on tumor molecular proling in patients with advanced cancer using each patient as its own control ; clinicaltrials.gov identier : nct03084757 ) , which aimed to identify druggable molecular alterations , and received a fourth line of chemotherapy with gemcitabine ( fig 1 )  . molecular proling was performed on a frozen biopsy of a metastatic lymph node in the shiva02 trial using a dedicated targeted sequencing panel covering 80 genes . 
in patients with breast cancer , esr1 hotspot mutations in the ligand - binding domain were observed exclusively after hormone therapy5 - 8 and have been shown to be associated with poor prognosis.11 after exposure to aromatase inhibitors , the prevalence of esr1 mutations was signicantly higher in patients exposed during the metastatic phase than during the adjuvant phase ( 36% v 6% ) .11 in a recent study , advanced breast cancer tumors harboring esr1 mutations , or alterations in genes involved in the mitogenactivated protein kinase pathway and in the er transcriptional machinery , were shown to barely benet from aromatase inhibitors.5 esr1 amplications were detected in 12% of endometrial carcinomas , 12 whereas esr1 hotspot mutations were less frequent ( 2% ) .9 esr1 amplications were shown to be associated with a poor prognosis and seemed to be an early event in endometrial carcinoma development.12 selective er degraders , such as fulvestrant , were shown to be effective in patients with hormone receptorpositive breast cancer . 
esr1 mutations were not reported to be associated with clinical resistance to fulvestrant.13 to our knowledge , our case report is the rst to report a potential de novo esr1 mutation in a patient with erpositive endometrial carcinoma treated with an aromatase inhibitor . 
curr opin obstet gynecol 29 : 47 - 58 , 2017 kokka f , brockbank e , oram d , et al : hormonal therapy in advanced or recurrent endometrial cancer . 
cochrane database syst rev ( 12 ) : cd007926 , 2010 razavi p , chang mt , xu g , et al : the genomic landscape of endocrine - resistant advanced breast cancers . 
cancer cell 34 : 427 - 438.e6 , 2018 robinson dr , wu y - m , vats p , et al : activating esr1 mutations in hormone - resistant metastatic breast cancer . 
nat genet 45 : 1446 - 1451 , 2013 toy w , shen y , won h , et al : esr1 ligand - binding domain mutations in hormone - resistant breast cancer . 
nat genet 45 : 1439 - 1445 , 2013 bartels s , christgen m , luft a , et al : estrogen receptor ( esr1 ) mutation in bone metastases from breast cancer . 
mod pathol 31 : 56 - 61 , 2018 backes fj , walker cj , goodfellow pj , et al : estrogen receptor - alpha as a predictive biomarker in endometrioid endometrial cancer . 
schiavon g , hrebien s , garcia - murillas i , et al : analysis of esr1 mutation in circulating tumor dna demonstrates evolution during therapy for metastatic breast 12 . 
spoerke jm , gendreau s , walter k , et al : heterogeneity and clinical signicance of esr1 mutations in er - positive metastatic breast cancer patients receiving mol endocrinol 10 : 1388 - 1398 , 1996 cancer . 
tumor biopsies taken prior to neoadjuvant treatment were analyzed by reverse - phase protein arrays ( rppa ) for expression levels of 210 bc - relevant ( phospho - ) proteins . 
similarly , the mrna virp score was validated ( p , .001 ) in an independent phase ii clinical trial ( promix )  . conclusion our virp score , integrating the expression of nine proteins and validated on mrna data both internally and in an independent clinical trial , may be used to increase the likelihood of benet from treatment with bevacizumab combined with chemotherapy in patients with her2 - negative bc . jco precis oncol 5 : 286 - 306 . 
2021 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction treatment of solid tumors using antiangiogenic therapy has been explored for several decades.1 , 2 discovery of vascular endothelial growth factor a ( vegf - a ) as a major culprit in tumor angiogenesis led to the development of bevacizumab , a recombinant humanized monoclonal antibody targeting vegf - a.3 addition of bevacizumab to various chemotherapy regimens has proven highly benecial in patients with several types of advanced solid tumors resulting in a signicant improvement in overall survival ( os ) and / or progression - free survival ( pfs )  . despite good response to bevacizumab therapy in many individual patients with breast cancer ( bc ) , randomly selected treatment populations do not demonstrate improved os.4 , 5 thus , the clinical use of bevacizumab in bc is limited and currently only approved in europe . 
regardless of this , activity is observed in subsets of patients pointing to the urgent need for novel biomarkers to select subpopulations in which adequate clinical benet can be achieved.6 , 7 one of the most obvious and biological plausible biomarkers was the plasma vegf - a level , and although promising in some studies , evidence from the latter meridian trial did not support its use for identifying patients with benet from added bevacizumab.8 other biomarkers at various molecular levels have been investigated such as soluble carbonic anhydrase ix , 9 brca1 / 2 mutations , 10 and dna methylation signatures.11 however , tissue protein expression and protein signatures have not previously been explored , although data at the proteomic level in general have proven highly valuable in drug response prediction models.12 in this study , we sought to establish protein expression features predicting the response to treatment with bevacizumab in combination with chemotherapy . 
 protein signature predicts response to chemotherapy plus bevacizumab context key objective the use of antivascular endothelial growth factor ( vegf ) targeted therapies in combination with chemotherapy in breast cancer ( bc ) is limited because of the lack of a predictive biomarker . 
in this study , protein expression in pretreatment tumor biopsies was used to develop a predictor for patients with large bcs . knowledge generated a nine - protein response predictor was established by lasso regression trained by a continuous response evaluation . 
the protein signature was validated using corresponding mrna expression in the same patient cohort as well as in an independent patient cohort . relevance our study supports using this protein signature as a predictive marker for vegf inhibition in combination with chemotherapy , and represents a novel opportunity for rational use of this therapy in patients with bc . samples collected from patients with bc prior to neotreatment ( nat ) with chemotherapy ( ctx ) or adjuvant chemotherapy in combination with bevacizumab ( bev plus ctx ) , and analyzed expression against treatment effect on tumor size and clinical outcomes . 
patients with human epidermal growth factor receptor 2 ( her2 ) negative , previously untreated , breast carcinomas with size 2.5 cm were included , and 67 and 71 patients randomly assigned to treatment with ctx or bev plus ctx , respectively . 
one hundred nine samples collected prior to treatment were available for protein analysis on rppa in the ctx treatment arm ( n = 55 ) and in the bev plus ctx treatment arm ( n = 54 ) ( appendix fig a1 )  . 
clinicopathologic characteristics of patients , including adverse events , have previously been described.13 in brief , the primary end point of the clinical study , pcr , was dened as pathologic stage ypt0 and ypn0 after end of therapy . 
in the subset of patients with available protein proles ( n = 109 ) , pcr rates were overall higher in the bev plus ctx ( 26 % ) compared with the ctx ( 13 % ) treatment arm , although not signicantly ( p = .094 ) ( appendix fig a2 )  . 
relative tumor size in percent after 24 weeks of nat was calculated as tumor size at the time of surgery ( longest diameter on histopathologic specimen ) relative to tumor size at week 0 ( mri if available or ultrasound or mammography )  . 
tumor cell content in samples was assessed by use of ascat15 as previously reported.16 the study was approved by the institutional protocol review board , the regional ethics committee , the norwegian medicines agency , performed in accordance with the declaration of helsinki , and informed consent was obtained . 
we further used an external cohort with similar treatment characteristics ( promix trial , clinicaltrials.gov identier : nct00957125 ) , with mrna expression data available , 17 for validation of the virp score . reverse phase protein arrays ( rppa ) proling of 210 cancer - relevant proteins of which 54 were in a phosphorylated state ( appendix table a1 ) were performed by the rppa18 core facility at md anderson cancer center ( houston , tx )  . 
tumor protein lysates were serially diluted two - fold for 5 dilutions ( from undiluted to 1 : 16 dilution ) , probed with antibodies , and visualized by dab colorimetric reaction . 
for development of the virp score , the continuous response parameter relative tumor size was used as outcome , whereas the other clinically used response parameters pcr and rcb were used for evaluation of predictive performance . 
relative importance of each model variable was assessed using the r - package relaimpo23 with metrics lmg . virp scores based on mrna data in the neoava and promix study were computed using the intercept and beta - coefcients determined from the protein data in the neoava study . 
the corresponding surrogate mrna scores were determined using quantile - normalized and probeaveraged mrna expressions from the genes corresponding to the proteins in the original protein signature , including the phosphoproteins . results protein prole of tumors before treatment using unsupervised hierarchical clustering on the expression of the 210 proteins in all 109 tumors collected prior to treatment , they cluster into groups signicantly related to tumor shrinkage and to pam50 subtypes , with most estrogen receptor ( er ) - negative patients ( 17 of 21 ) localizing in a separate subcluster ( appendix fig a4 )  . univariate spearman correlation analysis of protein expressions related to relative tumor size visualized by the nonvariable - dependent rst principal component demonstrated a positive yet limited relationship between the two treatment arms , with expression of almost half of proteins ( n = 103 ) being inversely correlated with response . 
the number of proteins signicantly ( p , .05 unadjusted ) related to response was higher in the ctx treatment arm ( n = 54 ) than in the bev plus ctx arm ( n = 38 ) , with only ten in common and of which two had rho - values with opposite signs ( appendix fig a5 and appendix table a2 )  . development of a protein signature score predicting response to treatment with bev plus ctx addition of bevacizumab to standard treatment with chemotherapy demonstrated benet compared with chemotherapy alone , although not signicant ( appendix fig a2 )  . to develop a signature predicting response to treatment with bev plus ctx , protein expression in biopsies taken prior to treatment were used from patients with available relative tumor size after nat ( n = 54 )  . 
low - variance proteins are likely to have low predictive value , thus a certain level of variance in expression should be present in order for clinical markers to be applicable . 
we thus reduced the original panel of 210 proteins by plotting the variance in expression and demonstrated a mixed distribution ( appendix fig a3 ) where only members belonging mainly to the proteins with higher variance ( n = 114 ; appendix table a3 ) were considered for use in the adaptive lasso regression model . 
a second iterative round of lasso on this subset of ten proteins gave the nal intercept and beta coefcients , of which one was shrunk to zero giving a nal signature of nine proteins with corresponding beta coefcients . 
 protein signature predicts response to chemotherapy plus bevacizumab the virp score for each patient was calculated as the sum of the intercept and beta coefcient weighted expression of the nine proteins . 
in univariate analysis , only the virp score , both as a continuous value and dichotomized ( low v high ) , was signicantly associated with pcr ( table 2 )  . 
no signicant association was found for tumor stage , pam50 subtypes , age , pgr , tp53 mutation , and nodal or er status . we next sought to establish and evaluate the corresponding mrna expression score as a surrogate to the virp protein score . 
by taking the corresponding genes from the virp ( including the parent protein for the phosphoprotein antibodies ) and using the predened lasso intercept and beta coefcients , the mrna score for the neoava patients ( mrna virp scores ) was calculated . 
this demonstrated a signicant increase , approximately doubling , in the percentage of both pcr responders ( p = .009 ) and low rcb ( p = .022 ) in the virp score selected patient population . 
use of the virp score for selecting patients to bev plus ctx demonstrated a clear benet with an increased response both by pcr ( p , .001 ) and rcb ( p , .001 ) compared with standard ctx treatment ( fig 3 )  . discussion we have dened a protein signature score named virp for use in large her2 - negative primary bcs that identies good responders to nat with chemotherapy plus bevacizumab . 
use of the virp score for selection to bev plus ctx therapy signicantly enriches for patients responding 290 2021 by american society of clinical oncology to treatment ( pcr or rcb - low )  . 
importantly , the robustness of the virp score was conrmed through validation in an independent clinical cohort ( promix )  . the lasso regression method was used to select proteins for the virp score . 
however , in silico evaluation the virp proteins under stimulus of bevacizumab treatment links them to response on relevant biological processes ( appendix fig a7 )  . ranking the relative importance of each protein in the virp signature is interesting with respect to , for example , the potential to use each or selected proteins as biomarkers separately . 
it appears that the syk protein is of high importance ( appendix fig a8 ) , which also coincides with being the only protein in the signature differently expressed ( p = .002 ) in pcr versus non - pcr patients . 
the syk protein is enriched in immune cells , but also expressed in breast epithelium and endothelial cells where it plays a role in angiogenesis.26 when passing from protein to mrna expression , functional dimension of activating or deactivating phosphorylations is lost . 
this potentially inuences the performance of the mrna virp score when used as surrogate for the original protein score , and could partly explain the observed drop in auc for the roc analysis between the protein and mrna data sets in the neoava trial ( fig 2a and appendix fig a6a )  . 
spearman correlation between expression of all 210 assessed proteins and mrnas showed that of the nine proteins in the signature , the four with the lowest correlation were phosphorylated or extracellularly localized proteins . 
however , the signature members had a signicantly ( p = .039 ) higher correlation compared with all proteins in the original data set ( appendix fig a9 )  . 
although the neoava protein data set is too small to draw conclusions , a previous observation of nearly 10 , 000 proteins in bc showed that established prognostic mrna - centered signatures ( ie , pam50 - ror , oncotype dx , and mammaprint ) in general had higher protein - mrna correlation than average for the human genome.27 we propose that because of the closer relation proteins have to phenotype , biomarker signatures being developed to identify features with clinical , and thus phenotypical impact , will represent genes with high expressional and translational penetrance . in the neoava clinical trial , patient subgroups were divided based on er expression ( using 1% positivity as cutoff ) , and better response ( as assessed by pcr and rcb ) was in general observed for the er - negative group , whereas only the er - positive group had signicant benet of added in univariate analysis , er bevacizumab . 
this is also consistent with the discrepancy seen in large studies regarding the benet of added bevacizumab in the er - positive or er - negative subpopulations of patients.28 - 30 the relative higher number of proteins signicantly correlated with tumor size after treatment in the ctx arm ( n = 54 ) suggested that a similar protein signature predicting response to chemotherapy alone might be developed . however , use of adaptive lasso regression only returned a signature of two proteins with low predictive performance , and not represented in the bev plus ctx virp signature . use of the virp score developed for bev plus ctx treatment applied on the ctx patients did demonstrate a signicantly lower score in the limited number of patients with pcr ( n = 7 ) , but not when assessed for the low - rcb patients ( n = 14 )  . 
this does not exclude the possibility that the proteins in the virp signature also reect sensitivity to the baseline chemotherapy when combined with bevacizumab . bulk tumor samples may have variable tumor cell content , which might inuence the readout of mrna or proteinbased molecular signatures.31 inuence of tumor cell content ( median of 42% ) on the virp signature , or on its individual proteins , did not demonstrate any signicant associations in the bev plus ctx - treated patients ( appendix fig a11 )  . 
furthermore , the virp score should be related to the mechanism of action of the anti - vegf therapy , which involves normal tissue constituents and in particular endothelial cells and vessels.32 thus , a composite tissue is likely required for the score to function . 
 ( a ) fraction of patients obtaining pcr when treated with standard ctx in all neoava patients ( n = 55 ) or bev plus ctx in all neoava patients ( n = 54 ) and selected based on virp score ( n = 22 )  . 
 ( b ) fraction of patients obtaining low rcb ( 0 and i ) when treated with standard ctx in all neoava patients ( n = 55 ) or bev plus ctx in all neoava patients ( n = 54 ) and selected based on virp score ( n = 22 )  . 
pcr , pathologic complete response ; rcb , residual cancer burden ; virp , vegf inhibition response predictor . 100% 100% random population ctx treatment ( low = 14 high = 41 ) random population bev plus ctx treatment ( low = 20 high = 34 ) virp selected population bev plus ctx treatment ( low = 15 high = 7 ) from treatment with bevacizumab in combination with chemotherapy.34 the virp score is predicting responses , but further studies are warranted to assess for impact on survival . although the discovered virp signature was established for patients treated with bev plus ctx in a neoadjuvant setting , it recapitulates biology related to treatment response and could have the potential to also predict response to bev plus ctx therapy in the metastatic setting . 
additionally , studies are initiated to investigate whether the virp signature recapitulates common response biology shared by other solid cancer types for which bevacizumab in combination with chemotherapy is commonly used.35 affiliations 1department of tumor biology , institute for cancer research , division of cancer medicine , oslo university hospital , the norwegian radium hospital , oslo , norway 2department of genetics , institute for cancer research , division of cancer medicine , oslo university hospital , the norwegian radium hospital , oslo , norway 3department of informaticsbiomedical informatics , university of oslo , oslo , norway 4k.g. 
mills , olav engebraaten provision of study materials or patients : ingrid hedenfalk , elin borgen , niklas loman , thomas hatschek , anne - lise brresen - dale , bjrn naume , gordon b . 
mills , olav engebraaten manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors thomas hatschek consulting or advisory role : roche , pzer , pierre fabre research funding : roche , pzer travel , accommodations , expenses : roche anne - lise brresen - dale employment : arctic pharma as , pubgene stock and other ownership interests : arctic pharma as gordon b . 
mills stock and other ownership interests : catena , signalchem , tarveda therapeutics , immunomet honoraria : nuevolution : astrazeneca , tarveda therapeutics , tesaro , symphogen , pdx pharmacy , immunomet , lilly consulting or advisory role : astrazeneca , signalchem , tarveda therapeutics , symphogen , takeda / millennium , pdx pharmacy , immunomet , lilly , turbine , ion pharma , zentalis research funding : adelson medical research foundation , astrazeneca , nanostring technologies , breast cancer research foundation , karus therapeutics , pzer , prospect creek foundation , tarveda therapeutics , ions pharmaceuticals , immunomet patents , royalties , other intellectual property : hrd assay to myriad genetics , dsp technology patent with nanostring travel , accommodations , expenses : astrazeneca , pzer , symphogen , chrysalis biomedical advisors , immunomet , michigan primary care consortium gunhild m . 
mlandsmo patents , royalties , other intellectual property : patent application submitted for a nine - protein / gene panel predicting response to anti vegf therapies in combination with chemotherapy olav engebraaten patents , royalties , other intellectual property : patent application pending for a biomarker for antibody drug conjugates , patent application submitted for a nine - protein / gene panel predicting response to anti vegf therapies in combination with chemotherapy no other potential conicts of interest were reported . references folkman j : anti - angiogenesis : new concept for therapy of solid tumors . 
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maru d , venook ap , ellis lm : predictive biomarkers for bevacizumab : are we there yet ? clin cancer res 19 : 2824 - 2827 , 2013 gampenrieder sp , westphal t , greil r : antiangiogenic therapy in breast cancer . 
miles d , cameron d , bondarenko i , et al : bevacizumab plus paclitaxel versus placebo plus paclitaxel as rst - line therapy for her2 - negative metastatic breast cancer ( meridian ) : a double - blind placebo - controlled randomised phase iii trial with prospective biomarker evaluation . 
eur j cancer 70 : 146 - 155 , 2017 janning m , m uller v , vettorazzi e , et al : evaluation of soluble carbonic anhydrase ix as predictive marker for efcacy of bevacizumab : a biomarker analysis from the geparquinto phase iii neoadjuvant breast cancer trial . 
fasching pa , loibl s , hu c , et al : brca1 / 2 mutations and bevacizumab in the neoadjuvant treatment of breast cancer : response and prognosis results in patients with triple - negative breast cancer from the geparquinto study . 
ali m , khan sa , wennerberg k , et al : global proteomics proling improves drug sensitivity prediction : results from a multi - omics , pan - cancer modeling 13 . 
silwal - pandit l , nord s , von der lippe gythfeldt h , et al : the longitudinal transcriptional response to neoadjuvant chemotherapy with and without bevacizumab 8 : 2278 - 2288 , 2018 approach . 
h oglander ek , nord s , wedge dc , et al : time series analysis of neoadjuvant chemotherapy and bevacizumab - treated breast carcinomas reveals a systemic shift in genomic aberrations . 
lu y , ling s , hegde am , et al : using reverse - phase protein arrays as pharmacodynamic assays for functional proteomics , biomarker discovery , and drug development in cancer . 
robin x , turck n , hainard a , et al : proc : an open - source package for r and s + to analyze and compare roc curves . 
loibl s , denkert c : how much information do we really need after neoadjuvant therapy for breast cancer ? j clin oncol 35 : 1029 - 1030 , 2017 25 . 
kazerounian s , duquette m , reyes ma , et al : priming of the vascular endothelial growth factor signaling pathway by thrombospondin - 1 , cd36 , and spleen subtype . 
nat commun 10 : 1600 , 2019 von minckwitz g , eidtmann h , rezai m , et al : neoadjuvant chemotherapy and bevacizumab for her2 - negative breast cancer . 
earl hm , hiller l , dunn ja , et al : efcacy of neoadjuvant bevacizumab added to docetaxel followed by uorouracil , epirubicin , and cyclophosphamide , for women with her2 - negative early breast cancer ( artemis ) : an open - label , randomised , phase 3 trial . 
garcia j , hurwitz hi , sandler ab , et al : bevacizumab ( avastin ( r ) ) in cancer treatment : a review of 15 years of clinical experience and future outlook . 
robert nj , di eras v , glaspy j , et al : ribbon - 1 : randomized , double - blind , placebo - controlled , phase iii trial of chemotherapy with or without bevacizumab for rst - line treatment of human epidermal growth factor receptor 2 - negative , locally recurrent or metastatic breast cancer . 
delaloge s , p erol d , courtinard c , et al : paclitaxel plus bevacizumab or paclitaxel as rst - line treatment for her2 - negative metastatic breast cancer in a multicenter national observational study . 
 protein signature predicts response to chemotherapy plus bevacizumab appendix patients assigned to treatment with chemotherapy with or without bevacizumab ( n = 138 ) patients randomly assigned to chemotherapy ( ctx ) ( n = 67 ) patients randomly assigned to chemotherapy and bevacizumab ( bev plus ctx ) ( n = 71 ) ineligible ( n = 1 ) ineligible ( n = 2 ) discontinued treatment ( n = 2 ) patients included in primary endpoint analysis ( n = 66 ) patients included in primary endpoint analysis ( n = 66 ) start of treatment ( n = 55 ) start of treatment ( n = 54 ) fig a1 . 
mixed distribution of protein expression variances ( solid green and blue lines ) ; only proteins with variance above intersection ( red line ) were selected for input in lasso regression to determine the virp ( appendix table a3 )  . 
 protein signature predicts response to chemotherapy plus bevacizumab relative tumor size ( p = .003 ) bevacizumab ( p = .815 ) pam50 ( p < .001 ) bevacizumab relative tumor size pam50 basal her2 luma lumb normal 150% fig a4 . 
rho - values derived from the spearman correlation of protein expression to relative tumor size for patients receiving the bev plus ctx combination ( x - axis ) and ctx treatment only ( y - axis ) with rst principal component of relation between the treatment arms ( stitched red line )  . 
in silico simulation with ingenuity pathway analysis ( version 47547484 , qiagen ) was used to predict biologic effects ( lower row ) affected by the virp member proteins ( second bottom row ) when inuenced by bevacizumab treatment ( top row ) through a set of intermediate proteins ( second top row )  . 
 ( c ) virp scores in rcb low ( 0 and i ) and high ( ii and iii ) patients . ( d ) fraction of patients obtaining pcr when treated with standard ctx in all neoava patients ( n = 55 ) or selected based on virp score ( n = 17 )  . 
 ( e ) fraction of patients obtaining low rcb ( 0 and i ) when treated with standard ctx in all neoava patients ( n = 55 ) or selected based on virp score ( n = 17 )  . 
 o validating comprehensive nextgeneration sequencing results for precision oncology : the nct / dktk molecularly aided stratification for tumor eradication research experience amelie lier roland penzel christoph heining peter horak martina frhlich sebastian uhrig jan budczies martina kirchner anna - lena volckmar barbara hutter simon kreutzfeldt volker endris daniela richter stephan wolf katrin pftze olaf neumann ivo buchhalter cristiano m . 
rieke ( continued ) purpose rapidly evolving genomics technologies , in particular comprehensive next generation sequencing ( ngs ) , have led to exponential growth in the understanding of cancer biology , shifting oncology toward personalized treatment strategies . 
2018 by american society of clinical oncology precision oncology and next - generation sequencing precision oncology is based on a profound understanding of tumor biology through comprehensive molecular profiling that can be leveraged to predict response to specific cancer therapies . 
contributed equally to this work . corresponding author : albrecht stenzinger , md , institute of pathology , heidelberg university hospital , im neuenheimer feld 224 , 69120 heidelberg , germany ; e - mail : albrecht . 
 range from the focused sequencing of hotspot regions to whole - exome sequencing ( wes ) / whole - genome sequencing ( wgs ) as well as total rna sequencing ( rna - seq )  . 
whereas the targeted panel sequencing of selected cancer related genes and genomic regions has been widely adopted in routine molecular diagnostics at many institutions worldwide , 1 , 4 - 7 comprehensive molecular profiling in a clinical setting is currently only performed at dedicated centers ; however , as sequencing costs continue to drop and new actionable targets continuously emerge , comprehensive ngs will be increasingly used with clinical intention . 
 request form 4 - 6 work days sample acquisition ( tissue sectioning , h&e staining , biobanking ) histopathologic diagnosis ( tumor cell content ) request form 8 - 14 work days target validation ( sanger sequencing , fish , ihc ) wes / wgs / rna - seq ( target identification ) 15 work days tumor board ( target identification , clinical interpretation , treatment recommendations ) personalized cancer treatment fig 1 . 
 fish , fluorescence in situ hybridization ; h&e , hematoxylin and eosin ; ihc , immunohistochemistry ; rna - seq , rna sequencing ; wes , whole - exome sequencing ; wgs , whole - genome sequencing . study at the following german cancer centers : nct heidelberg , nct dresden , university hospital heidelberg , university hospital dresden , university hospital munich , charit comprehensive cancer center , charit university hospital berlin , comprehensive cancer center tbingen , university hospital tbingen , university hospital freiburg , university cancer center frankfurt , university cancer center mainz , west german cancer center , and university hospital essen . samples were pseudonymized , and tumor histology , including tumor cellularity , was reviewed and assessed for all cases according to current who criteria by the iph before additional processing . 
of the 220 patients , 77.5% were younger than age 51 years ( fig 2b ) , 49% were registered for the master program within the first 2 years after diagnosis ( fig 2c ) , and the male - to - female ratio was close to 1 ( fig 2d )  . 
patients with bone and soft tissue sarcomas represented the largest subgroup ( 24.9% ) of the 220 patients and together with patients with cancer of unknown primary ( 6.6% ) , skin cancer ( 8% ) , neuroendocrine tumors ( 8% ) , and breast cancer ( 6.6% ) accounted for 54.1% of the validation cohort ( fig 2e )  . 
as shown in figure 2f , the number of validation requests increased over time as a result of the extension of the master program to nine additional referring centers within the german cancer research center in march 2016 . validation statistics a total of 349 genetic alterations , which corresponded to an average of 1.6 alterations per patient , were subjected to molecular pathology validation with a mean turnaround of 10 working days ( range , 1 to 24 days ) to avoid delays in reporting and genomics - guided clinical management . 
validation was performed after review of wes and rna - seq data and in parallel to additional clinical work - up of the cases , which takes place before discussion in the mtb . 
af , allele frequency ; amp , amplification ; cup , cancer of unknown primary ; del , deletion ; fus , gene fusion ; ins , insertion ; oex , overexpression . was requested is shown in the data supplement . 
 for validation requests and validation reports , a quality - controlled standard operating procedure and specific forms were established to ensure a standardized flow of information that provides clear and unambiguous results . 
the request form has a mandatory requirement for the following data : request date , patient identifier , tumor type as defined by histopathologic analysis , full name and affiliation of the requesting physician , and a precise description of the target of interest identified by comprehensive ngs , including official gene name , refseq ( national center for biotechnology information ) , human genome version used for alignment ( eg , hg19 [ grch37 ] ) , chromosome , position of the alteration , type of alteration , allele frequency , and annotations on the cdna and protein level . 
to validate fusion genes , the fusion sequence as well as the 5 and 3 fusion partnerschromosome , position , and exonneed to be stated . nonsynonymous snvs were the most frequently requested alterations ( n = 241 ; 69.1% ) , followed by gene fusions ( n = 44 ; 12.6% ) , small indels ( n = 41 [ 31 deletions and 10 insertions ] ; 11.8% ) , gene amplifications ( n = 10 ; 2.9% ) , and overexpression ( n = 13 ; 3.7% ) of the corresponding protein products ( fig 3b )  . 
a more detailed analysis of the nine snvs that were detected by wes that were not confirmed by sanger sequencing revealed that seven ( 77.8% ) showed allele frequencies of less than 10% ( as low as 5% ) , whereas two ( 22.2% ) showed allele frequencies ( 22% and 27% ) above the lod of sanger sequencing , which indicated other causes underlying the discrepant results . 
 among the patients with discordant validation results , bone and soft tissue sarcoma ( 23.3% ) , cancer of unknown primary ( 16.7% ) , pancreatic adenocarcinoma ( 16.7% ) , and neuroendocrine fig 4 . 
as validation efforts were focused on specific mutations of individual genes and did not involve complex biomarkers , such as high tumor mutational burden ( tmb ) , which represents the most common genetic rationale for assigning patients to immunotherapy , validation requests in the ie basket were infrequent ( 0.9% ; fig 4a ) and mainly related to alterations of cd274 ( also called pd - l1 )  . 
 discussion here , we describe the systematic validation of clinically actionable genetic lesions that were detected by combined wes and rna - seq within a multi - institutional precision oncology progra to our knowledge , this study reports on the largest clinically affiliated program worldwide that systematically validates actionable genetic lesions in tumors that are detected by comprehensive ngs . 
whereas some studies on the value of sanger sequencingbased validation of germline - derived ngs data sets and ngs assays that are in use for hereditary cancer testing challenged the need for independent validation approaches , 19 , 23 , 24 our results not only underscore the need for validation of ngs assays for the interrogation of tumor tissue in a clinical setting , but also highlight the value of orthogonal methods for guiding the development of bioinformatics pipelines , 19 , 25 - 27 which are a critical cornerstone and determinant of any ngs result . 
in contrast , the ie basket , with its major biomarkers tmb and pd - l1 amplification / expression , is underrepresented , because validations were confined to single genetic aberrationsfor example , pd - l1 amplification / overexpression and did not include tmb . when comprehensive ngs entered the clinical arena more than 5 years ago , 28 - 31 our understanding of the pros and cons of ngs technology was still evolving . 
it was noted that , compared with sanger sequencing , ngs platforms can have a generally lower quality per base , which results in decreased specificity and sensitivity.17 - 19 it became evident that many parameters , including tumor cellularity , coverage , read depth , and lod thresholds for variant calling , influence clinical - grade ngs results . 
in our study , validation is defined as testing of genetic aberrations identified by wes and rna - seq by orthogonal methods in a running molecular profiling progra sequencing in master is carried out at the german cancer research center , and results are validated by the center for molecular pathology6 , 32 at the iph . 
whereas comprehensive sequencing is performed on a research - grade level , validation of putative targets is performed in a laboratory for molecular diagnostics that is regularly audited by the german accreditation agency33 and that leads and participates in nationwide round robin trials5 of the quality assurance initiative pathology.34 this dual and complementary approach ensures clinical - grade diagnostics . more than 90% of clinically actionable genomic alterations identified by combined wes and rna - seq were successfully validated with an imas of 0.93 , which demonstrates the high concordance that can be reached within a multi institutional comprehensive ngs program and indicates the importance of an implemented validation step within the workflow . the combination of wes and rna - seq can also yield information on gene fusions and associated transcript levels ; however , this approach must be coupled with data analysis tools that provide sufficient sensitivity and specificity to minimize the risk of false - negative or false - positive results.35 , 36 in the context of the current study , the implementation of a new computational strategy for gene fusion detection in 2016 ( com / suhrig / arriba ) substantially improved sensitivity and specificity , and the concordance rate for gene fusion detection increased to greater than 95% with an imas of 0.96 , which equaled the high imas of snv calling results . systematic re - evaluation of histopathologic diagnosis and tumor cellularity revealed that 25% of patients had insufficient tissue quantity and / or quality . 
first , whereas mathematical modeling predicts that most cancers harbor five to eight driver mutations in their genome , 38 the clinical utility of genetic lesions is governed by the number and type of drugs that are currently available . 
as a consequence , on average , 1.6 alterations per patient are currently subjected to validation in the master program , representing the druggable subset of putative biologically relevant genetic lesions per tumor . 
second , although our approach allows for the analysis of concordance levels between gold standard methods and ngs , the data set is not suited to calculate sensitivity and specificity . 
although an experienced observer may still recognize the variant of interest in an optimal settingthat is , low background noise because of high dna quality or low nontumor contaminationone evidently operates at sensitivity limits . 
conversely , comprehensive sequencing approaches , such as wes , also have inherent limitations when it comes to the coverage of genes and read depth per locus , which can become particularly challenging in the context of genetic heterogeneity and tumor evolution . 
 our approach not only aids clinical decision making on the basis of molecular tumor profiles and helps to optimize bioinformatics pipelines and sequencing protocols , but will also serve as a solid reference in the field . 
whereas overall concordance rates between wes and rna - seq and orthogonal methods are high , low abundant variants remain challenging and should be verified by independent methods , ideally by targeted ngs . 
finally , influenced by our study results , we have now implemented combined wgs and rna - seq to further enhance the detection of gene fusions and to analyze intronic regions for putative cancer drivers . 
our validation approach enables the continuous monitoring of results and associated test parameters for maintenance and for the identification of weak spots of a given assay which may require improvement . 
thus , this program does not replace but complements validation measures that were taken before the use of the assay in a clinical setting . next to ngs of dna and rna , it is foreseeable that additional omics approaches will enter the clinic to capture the entire spectrum of epigenetic , proteomic , and metabolic adaptations that cancers use to facilitate growth and metastatic spread . 
 data analysis and interpretation : amelie lier , christoph heining , martina frhlich , sebastian uhrig , jan budczies , barbara hutter , volker endris , olaf neumann , ivo buchhalter , cristiano m . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . amelie lier no relationship to disclose roland penzel no relationship to disclose christoph heining no relationship to disclose peter horak honoraria : astellas pharma martina frhlich no relationship to disclose sebastian uhrig no relationship to disclose travel , accommodations , expenses : astellas pharma jan budczies patents , royalties , other intellectual property : patent application ep1806413a1 ( inst ) , patent application wo2010076322a1 ( inst ) martina kirchner no relationship to disclose anna - lena volckmar consulting or advisory role : astrazeneca barbara hutter no relationship to disclose simon kreutzfeldt consulting or advisory role : novartis , boehringer ingelheim travel , accommodations , expenses : pfizer , boehringer ingelheim , pharmamar , novartis volker endris honoraria : astrazeneca , thermo fisher scientific consulting or advisory role : novartis , pfizer daniela richter no relationship to disclose stephan wolf no relationship to disclose katrin pftze no relationship to disclose olaf neumann no relationship to disclose ivo buchhalter no relationship to disclose cristiano m . 
morais de oliveira no relationship to disclose stephan singer no relationship to disclose jonas leichsenring honoraria : astrazeneca expert testimony : astrazeneca esther herpel no relationship to disclose frederick klauschen no relationship to disclose philipp j . 
jost honoraria : novartis , bristol - myers squibb consulting or advisory role : novartis research funding : boehringer ingelheim ( inst ) , abbvie ( inst ) klaus h . 
the authors thank the german cancer research center ( dkfz ) heidelberg center for personalized oncology ( hipo ) and nct precision oncology program ( pop ) sample processing laboratory , the dkfz genomics and proteomics core facility , and the dkfz - hipo data management group for technical support and expertise . 
the authors also thank roland eils for providing the bioinformatics infrastructure within dkfz - hipo . affiliations amelie lier , roland penzel , peter horak , jan budczies , martina kirchner , anna - lena volckmar , simon kreutzfeldt , volker endris , olaf neumann , ivo buchhalter , cristiano m . 
morais de oliveira , christof von kalle , peter schirmacher , stefan frhling , and albrecht stenzinger , german cancer consortium , heidelberg ; christoph heining , daniela richter , and hanno glimm , national center for tumor diseases dresden ; christoph heining , evelin schrck , gunnar folprecht , and hanno glimm , technische universitt dresden ; christoph heining , daniela richter , evelin schrck , gunnar folprecht , and hanno glimm , german cancer consortium ; gunnar folprecht , university hospital , dresden ; frederick klauschen , charit university hospital ; frederick klauschen and mario lamping , german cancer consortium ; damian t . 
h021 from german cancer research centerheidelberg center for personalized oncology and national center for tumor diseases precision oncology program . support references 30 : 293 - 303 , 307 , 2016 1 . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
endris v , stenzinger a , pfarr n , et al : ngs - based brca1 / 2 mutation testing of high - grade serous ovarian cancer tissue : results and conclusions of the first international round robin trial . 
czink e , heining c , weber tf , et al : durable remission under dual her2 blockade with trastuzumab and pertuzumab in a patient with metastatic gallbladder cancer [ in german ]  . 
grschel s , bommer m , hutter b , et al : integration of genomics and histology revises diagnosis and enables effective therapy of refractory cancer of unknown primary with pdl1 amplification . 
kordes m , rring m , heining c , et al : cooperation of braf ( f595l ) and mutant hras in histiocytic sarcoma provides new insights into oncogenic braf signaling . 
barbano r , pasculli b , coco m , et al : competitive allele - specific taqman pcr ( cast - pcr ) is a sensitive , specific and fast method for braf v600 mutation detection in melanoma patients . 
rennert h , eng k , zhang t , et al : development and validation of a whole - exome sequencing test for simultaneous detection of point mutations , indels and copy - number alterations for precision cancer care . 
roy s , coldren c , karunamurthy a , et al : standards and guidelines for validating next - generation sequencing bioinformatics pipelines : a joint recommendation of the association for molecular pathology and the college of american pathologists . 
jennings lj , arcila me , corless c , et al : guidelines for validation of next - generation sequencingbased oncology panels : a joint consensus recommendation of the association for molecular pathology and college of american pathologists . 
wagle n , berger mf , davis mj , et al : high - throughput detection of actionable genomic alterations in clinical tumor samples by targeted , massively parallel sequencing . 
christopoulos p , endris v , bozorgmehr f , et al : eml4 - alk fusion variant v3 is a high - risk feature conferring accelerated metastatic spread , early treatment failure and worse overall survival in alk + non - small cell lung cancer . 
carrara m , beccuti m , cavallo f , et al : state of art fusion - finder algorithms are suitable to detect transcription - induced chimeras in normal tissues ? bmc bioinformatics 14 : s2 , 2013 ( suppl 7 ) 36 . 
leichsenring j , volckmar al , kirchner m , et al : targeted deep sequencing of effusion cytology samples is feasible , informs spatiotemporal tumor evolution , and has clinical and diagnostic utility . 
dignam , phd2 in this precision oncology era , where molecular proling at the individual patient level becomes increasingly accessible and affordable , more and more clinical trials are now driven by biomarkers , with an overarching objective to optimize and personalize disease management . 
as compared with the conventional clinical development paradigms , where the key is to evaluate treatment effects in histology - dened populations , the choices of biomarker - driven clinical trial designs and analysis plans require additional considerations that are heavily dependent on the nature of biomarkers ( eg , prognostic or predictive , integral or integrated ) and the credential of biomarkers performance and clinical utility . 
here we provide a concise overview of design options for both the setting of singlebiomarker / single - disease and the setting of multiple - biomarker / multiple - disease types . 
we focus on explaining the trial design and practical considerations and rationale of when to use which designs , as well as how to incorporate various adaptive design components to provide additional exibility , enhance logistical efciency , and optimize resource allocation . 
2019 by american society of clinical oncology introduction in the past 10 to 15 years , cancer clinical trials have experienced some important paradigm changes to embrace the era of precision oncology as dened by various biomarkers . 
the central hypothesis driving this movement is that by integrating the right biomarker information we can properly select , or at least enrich , trial cohorts for patients who are most likely to benet from a particular therapy . 
multiple factors collectively contribute to this movementour knowledge about oncogenic pathways has been greatly advanced ; highthroughput screening and other drug discovery developments have made a lot more candidate agents available for evaluation ; and the rapid development of various omics - based technologies , especially the increasing availability of next - generation genomic sequencing , makes it more feasible and affordable to incorporate biomarker information into clinical trials . 
all of these call for novel biomarker - driven trial designs to expedite the clinical development of multiple treatment agents and newly dened patient populations . in this review , we use biomarker to generically describe any characterizations of biologic molecules or diagnostic tests carried out on dna , rna , proteins , and metabolites from blood , body uids , or tissues for diagnosis purposes including disease conrmation , staging , subtyping , and so on . 
under this working denition , the presence of some actionable mutation , such as a mutation in epidermal growth factor receptor ( egfr ) measured at the protein level , is viewed as a single biomarker ; a dna - based multiplex genotyping that simultaneously determines multiple actionable mutations such as egfr , kras , or eml4 - alk is viewed as multiple biomarkers . treatment benet compared with the traditional paradigm , there are even greater consequences of asking the right questions ( or wrong questions ) in these biomarker - driven clinical trials . 
for example , although often there is good biologic rationale to consider biomarker - negative ( m - negative ) patients unlikely or less likely to benet from the new ( targeted ) therapy , the clinical evidence of whether is conned only in the potential biomarker - positive ( m - positive ) patients may or may not be strong . 
moreover , the development of a validated companion biomarker , including establishing a widely accepted partition or cutoff value to determine whether the biomarker is positive or negative , is sometimes lagging behind the development of novel therapeutic agents . 
 hu and dignam context key objective in the past few decades , clinical trials have experienced a major paradigm shift , aiming to incorporate ever - growing tumor biomarker information and evaluate biomarker - dened patient cohorts , while accelerating the clinical development process simultaneously . 
what are the key considerations when designing and conducting biomarker - driven clinical trials ? knowledge generated we present an overview of the rationale , trial types , key design elements and features , and practical considerations for commonly used biomarker - driven clinical trials . 
examples of trials conducted and the lessons learned are also presented . relevance when properly designed and implemented with adequate resources , biomarker - driven clinical trials may efciently and effectively generate evidence on biomarker - based personalized disease management . 
clinicians and clinical trialists should think thoroughly and critically if and how to design and conduct these trials . properly tailor the trial designs and prioritize research questions on the basis of the development stage of the biomarker and the credentials of its clinical utility . the demand for trial development and conduct has become more daunting than ever , as high - throughput molecular proling becomes more accessible . 
rather than mounting separate trials , an important paradigm shift to expedite clinical development is to establish the so - called master protocol or platform triala program of trial development and conduct implemented with an up - front molecular screen and enrollment infrastructure into subtrials across multiple biomarkers and / or multiple disease typesto enhance logistic and regulatory efciency . some important and useful concepts in development of biomarkers for clinical utility are rst briey introduced here and expanded on in the next sections . 
throughout this review , we assume biomarkers of interest already have good analytical validity ( eg , they can be accurately , reliably , and reproducibly measured ) .1 , 2 a biomarker is called prognostic if it is associated with disease prognosis regardless of treatment type and is called predictive when it exerts prognostic inuence differentially according to different treatments . 
it is desirable to have a biomarker ( either prognostic or predictive ) with good clinical utility ( ie , the biomarker can reliably prompt clinical actions that benet patients )  . 
according to dancey et al , 3 biomarkers are integral when they are inherent in the study design from the onset and must be performed in real time to establish eligibility , identify the correct stratum for stratied enrollment , or assign treatment . 
in other words , to make a biomarker usable for clinical trial setting , an integral biomarker should be fully developed and validated ( eg , the cutoff that dichotomizes the continuous marker measurement is xed ) , with a rapid assay turnaround time . 
fast assay turnaround time is desirable but not required for integrated biomarkers . to design more efcient trials and overcome the aforementioned challenges , the idea of adaptive designs has been promoted , and aspects of this approach play an important role in virtually all existing biomarker - driven trials . although modern oncology trial design has promoted adaptive designs as a new aspect of clinical trials , we note that adaptive elements have played a long - standing role in oncology clinical trials , dating back to early multistage design concepts4 , 5 and continuing in the rich development of group sequential monitoring methods that permit early efcacy or futility stopping.6 also , both traditional and newer phase i oncology trial designs are by their nature adaptive to accumulating data . 
we will discuss the rationale and features of established as well as newer adaptive design elements as they arise in respective biomarker - driven trial designs . finally , a new nomenclature that generally describes trial constructs has been introduced , attempting to describe heuristically the structure , particularly when multiple biomarkers and / or treatment agents are considered . 
the term basket trial refers to a trial with an agent tested among multiple disease types sharing a common molecular feature or target identied by biomarker ( s )  . 
in contrast , an umbrella trial describes the case where , for a common disease entity , multiple agents may be investigated in conjunction with specic molecular targets and biomarkers . 
we note that all of these terms lack precision , and thus the specic study features will provide more detail as to the structure . this article aims to provide a concise and up - to - date overview on the current status of biomarker - driven clinical trials , with a focus on the underlying design considerations , rationale , and lessons learned . 
we organize these discussions in terms of the biomarkers role ( integral v integrated ) , number of biomarkers involved ( one v multiple ) , and trial objective ( conrmatory v discovery )  . 
interim monitoring , including but not limited to group sequential methods , provides the statistical framework to allow repeated assessments of treatment efcacy and early stopping as soon as the accumulated data already provide adequate evidence to conclude that the experimental regimen is highly likely ( efcacy monitoring ) or unlikely ( futility monitoring ) benecial . 
for example , phase ii trials traditionally have implemented a single - arm , noncomparative design , with a planned interim futility analysis , such as simons two - stage design , 7 to minimize patient exposure to ineffective regimens . 
in the late - phase setting , integrated randomized phase ii / iii design8 is a useful adaptive design that combines both screening ( phase ii component ) and conrmatory ( phase iii component ) in a single trial . 
as the data based on the the overall phase ii component are used to provisionally test the study hypothesis of the phase iii component , integrated phase ii / iii designs can effectively be viewed as phase iii studies with rather aggressive ( ie , likely to stop ) interim futility analyses . if more than one experimental regimen is of interest in the phase ii component , a plan of selecting treatment arms can also be incorporated when making the go / no - go decision from phase ii to phase iii . more formally speaking , adaptive designs are those that allow prospectively planned modications in both the statistical and scientic aspects of study designs , on the basis of accumulating data while the trials are still progress.9 adaptations to the statistical aspects of study designs arise when the primary estimand ( eg , the target of estimation ) addressing the scientic question of interest10 remains unchanged ; examples include group sequential designs , 6 sample size adaptation , 11 and , more recently ( although the concept dates back several decades ) , outcome / response - adaptive randomization.12 - 14 adaptations to the scientic aspects of study designs occur when the primary estimand does change ( eg , enriching patient population , or selecting new treatment arms or end points while the trial is still ongoing )  . 
among these adaptive design features , group sequential theorybased interim analysis and treatment assignment modication ( such as add or drop arms ) have been used most frequently , whereas other adaptive design features have not been widely adopted in practice . 
if the new agent is expected to have little effect on tumor shrinkage , or must be combined with an active agent , randomized phase ii screening designs17 with end points such as progression - free survival may be considered and provide valuable information on whether a conrmatory , randomized phase iii enrichment design should be performed . 
of note , although enrichment designs should be efcient ( a small sample size ) because we anticipate a large effect size , if the prevalence of mpositive patients is low , many patients must be screened to obtain the necessary sample size ; if the prevalence is too low , the trial may simply be infeasible . when there is strong evidence suggesting the biomarker is predictive with respect to the experimental regimen ( ie , m - positive patients benet ) , but it remains unclear whether the new treatment may also have a clinically meaningful ( but likely smaller ) benet for m - negative patients , the socalled biomarker - stratied design , which randomly assigns all patients with a valid marker result ( m - positive and m - negative ) as a stratication factor , can be considered ( fig 1b )  . 
one can also view this as an umbrella trial ( dened in a later section ) that contains two separate rcts for m - positive and m - negative subgroups . 
the key design consideration for conrmatory trials is if and how to prioritize the multiple hypotheses within the m - positive subgroup , the m - negative subgroup , or the overall population and properly power for each separately as applicable . 
 a biomarker - driven oncology clinical trial designs biomarker assay biomarker assay m - negative m - positive m - negative m - positive off study randomize randomize randomize standard rx new rx standard rx new rx standard rx new rx biomarker assay randomize biomarker assay nonmarkerbased strategy marker - based strategy marker subgroup 1 marker subgroup 2 marker subgroup 3 new rx randomize standard rx randomize new rx v standard rx standard rx m - negative , standard rx m - positive , new rx standard rx new rx fig 1 . 
m - negative , biomarker - negative ; m - positive , biomarker - positive ; rx , treatment . strategy can be considered by rst testing the m - positive subgroup at signicance level and then testing the m - negative subgroup at the same level if and only if the rst test is statistically signicant.20 an alternative approach to reect such prioritization is to split the overall unequally with a bonferroni correction ( eg , 1 = 0.04 and 2 = 0.01 for m - positive and m - negative , respectively )  . ( cid : 129 ) if the effect in the overall population is of secondary interest , the aforementioned sequential procedure needs to be modied , because the treatment effect of the overall population can still seem to be clinically meaningful even when the new treatment only works in the in the m - negative m - positive subpopulation but not subpopulation at all . 
to mitigate the potentially misleading conclusion that treatment works in all comers ( m - positive and m - negative ) , the marker sequential test design21 was proposed , which rst tests the m - positive subgroup at a reduced signicance level 1 ( , ) : if the test yields a statistically signicant result , the m - negative subgroup will be tested at level , whereas the overall population will be tested at 1 if the test among the m - negative subgroup is not signicant . ( cid : 129 ) if there is no convincing evidence suggesting a particular biomarker is predictive , a fallback strategy can be used , which rst tests the overall population at 1 ( , ) : if the result is signicant , one can claim that the treatment is effective in all patients ; if it is not signicant , then the m - positive population must meet 1 for signicance.22 although biomarker - stratied designs are typically used in the context of a conrmatory phase iii setting , randomized phase ii screening designs may still be considered if the overarching goal is to efciently inform whether to conduct a randomized phase iii enrichment design . 
when the primary interest is to evaluate whether the biomarker - based treatment assignment strategy is more effective than nonbiomarker - based treatment assignment strategy , one may consider a biomarker - strategy design , which randomly assigns all patients ( m - positive and m - negative ) to receive treatments either on the basis of or independent of biomarker status ( fig 1c )  . 
this design may also be used to evaluate whether the biomarker is predictive with some efciency loss , 16 , 23 because there can be a signicant portion of patients with the same biomarker status receiving the same treatments in both arms , reducing the treatment effect size that can realistically be specied . biomarker - directed design may be considered when it is desirable to have an integral biomarker evaluation for all patients , and there is compelling existing evidence suggesting a particular biomarker - dened subgroup should receive a certain regimen with satisfactory efcacy and safety proles ( fig 1d )  . 
in this case , like enrichment designs , a biomarker - directed design only randomizes the biomarker - dened subgroup where the biomarkers clinical utility in directing treatment decisions remains unclear ; meanwhile the other biomarker - dened subgroups are treated deterministically . 
for in tailorx ( program for the assessment of example , clinical cancer tests [ pacct - 1 ] : trial assigning individualized options for treatment ) , 24 patients with breast cancer treated with tamoxifen were classied as low , intermediate , and high risk on the basis of a 21 - gene recurrence score ( oncotype dx , genomic health , redwood city , ca )  . 
patients with intermediate risk were randomly assigned to receive hormonal therapy with or without chemotherapy , whereas lowrisk patients and high - risk patients always received only hormonal therapy with chemotherapy , respectively . in these conrmatory integral biomarker - driven trials , interim monitoring , especially futility monitoring , plays an even more critical role to help prioritize the limited resources by dropping subgroups with unpromising or unresponsive treatment benet and / or dropping ineffective experimental regimens if more than one is being investigated . 
for a biomarker classier that is based on a multiplex assay of genomic , proteomic , and transcriptomic data , the adaptive signature design29 was proposed to evaluate whether the experimental regimen is effective in the overall population or a subset of patients only while developing the biomarker classier simultaneously . 
if the treatment comparison for all patients is not signicant in the overall population at 1 ( , ) , we will either split all patients into training and testing subsets or use k - fold cross - validation30 to develop the biomarker and then evaluate efcacy in identied subgroups . 
of note , the cross - validated approach has also been shown to substantially improve the power of identifying the m - positive subgroup that benets from the new treatment . 
by transforming the multiple candidate genes to a binary classier , the approach does not suffer too much with respect to type i error control as the dimension of genes increases . 
when a biomarker or gene signature can be quantied on a continuous or ordinal scale , we can consider the biomarker - adaptive threshold design31 using a similar strategy to identify and validate an optimal cutoff point that separates m - positive and m - negative subgroups . the use of bootstrap resampling for estimation and inference of the threshold , although accounting for the multiplicity issue due to combining the tests for subgroup m - positive and overall population , has been shown to preserve the power to detect a global treatment effect while developing the biomarker . 
comparing with the conventional approaches , these methods have been shown to substantially improve the likelihood of detecting the mpositive subgroup when differential treatment effect does exist , especially when the prevalence of m - positive is low . 
for example , after the approval of vemurafenib for brafv600 mutation - positive metastatic melanoma , a nonrandomized phase ii basket trial of vemurafenib for multiple nonmelanoma cancers with brafv600 mutation was conducted , 33 with objective response as the primary end point . from a trial conduct perspective , basket trials can be more efcient than multiple histology - specic enrichment trials to carry out conducted separately and are convenient because the biomarker can ( although not necessarily ) be assessed locally at participating sites as part of the eligibility criteria screen . 
furthermore , this trial design can be quite appealing to patients because it conveniently provides access to the experimental therapy across multiple disease types , including in settings where our understanding of biomarker - treatment relationship is relatively limited . 
 molecular target a molecular target b molecular target y molecular target z targeted therapy a standard therapy targeted therapy b standard therapy targeted therapy y standard therapy targeted therapy z standard therapy biomarker - driven oncology clinical trial designs targeted therapy x tumor type a tumor type b tumor type y tumor type z biomarker assays phase iii continue to follow for definitive end point accrue additional n2 patients for definitive end point primary analysis treatment effect sufficiently large initiate phase ii trial phase ii accrue n1 patients assess stop insufficient ie activity promising ie activity run - in : randomization ( p1 = p2 ) randomization arm 1 ( p1 ) v arm 2 ( p2 ) assess interim estimate of treatment effect stop and conclude new rx better treatment effect not sufficiently large arm 1 better arm 2 better increase p1 decrease p2 increase p2 decrease p1 fig 2 . 
 hu and dignam from a scientic perspective , the underlying rationale of conducting a basket trial is that the biomarkers presence may independently predict responses attributed to the corresponding targeted therapy , regardless of histology or disease type.34 if this truly holds , it would be reasonable to redene cancer in a histology - agnostic fashion , suggesting an exciting new approach to therapy development . 
for example , in may 2017 , the us food and drug administration approved pembrolizumab for any patients with microsatellite instabilityhigh / decient dna mismatch repair regardless of histology.35 this approval was the rstever histology - agnostic indication and was partly based on a small , proof - of - concept basket trial.36 because basket trials may be viewed as a collection of enrichment trials , they also inherit all of the advantages and disadvantages , with the same practical considerations as elaborated earlier . 
one unique challenge facing basket trials is the balance between feasibility and the exchangeability ( eg , histology agnostic ) hypothesis , given the potential heterogeneity across different disease types . 
that is , if and when can we assume the molecular proling is sufcient to replace histology and pool patients together with the same biomarker ? for many biomarkers , prevalence is so low that it may be infeasible to accrue enough patients and analyze by each disease type . 
meanwhile , pooling all patients with the same biomarker can be questionable if not at all unrealistic , because the approach implies we can completely ignore the prognosis heterogeneity across different histology and assume disease subtype is not prognostic at all . 
in the case of vemurafenib for patients with brafv600 mutations , response to treatment was high when the primary site was melanoma but low when the primary site was colorectal cancer.33 a practical compromise is to combine some disease types for which prevalence is signicantly lower than others . 
in le et al , 36 patients with microsatellite instability high / decient dna mismatch repair were accrued and analyzed by colorectal and noncolorectal cohorts separately , because the prevalence in colorectal cancer is notably higher than other disease types . several novel adaptive designs have been developed to avoid separate analyses and properly share response information across disease types . 
one approach is based on preplanned interim analyses to determine the next steps.37 for example , if there is adequate evidence suggesting some histology - specic cohorts have similar and promising activities , these cohorts will be aggregated to allow more efcient statistical inference with fewer patients ; otherwise , histology - specic cohorts with exceptionally favorable response will remain separate , and cohorts with low responses will be terminated . 
the other approach is based on statistical modeling and bayesian inference , 38 , 39 which explicitly allows information sharing across different histology - specic cohorts and permits early stopping for some cohorts naturally on the basis of posterior probability of histology - specic response rates . 
nonetheless , it was argued that , for sample sizes typically used in phase ii trials and a reasonable number of cohorts / histology types under investigation , these designs that are meant to share information may not be as useful as one would hope , unless there is a strong rationale and evidence indicating uniform responses across cohorts.40 another important consideration when designing discovery basket trials is whether randomization should be used or not . 
the nonrandomized basket trial may be clearly preferred because of its feasibility and close connection to the conventional single - arm phase ii design traditionally used in early - stage development . 
however , nonrandomized basket design practically mandates objective response to be the only choice of the primary end point , because it is generally considered the only interpretable efcacy end point without a comparator . 
even with this end point , concerns may still arise regarding the relevance of historical control in at least some histology cohorts , because the biomarker likely denes a new disease subtype for which there is little or no historical information on prognosis.41 furthermore , if the experimental regimen is to be administered in combination with other active regimens , how to isolate the impacts of background treatments and properly the experimental regimens role can be chalinterpret lenging . 
in this case , because a centralized molecular screening platform is typically used , one may also view them as umbrella trials . from a pure statistical perspective , one could argue that basket trials may have multiplicity issues , because we simultaneously evaluate multiple histology - specic cohorts . pragmatically speaking , this may not be that problematic , because we are more tolerant of a higher type i error for discovery purposes ; the same issue also exists if we conduct separate trials for each cohort , and more practical concerns such as accrual feasibility often outweigh the type i error control . 
a randomized phase ii / iii trial example is alchemist ( adjuvant lung cancer enrichment marker identication and sequencing trial ) for resectable nonsmall - cell lung cancer ( nsclc ) , 42 , 43 which consists of three treatment subtrials , alchemistegfr for patients with egfr mutation ( clinicaltrials.gov identier : nct02193282 ) , alchemist - alk for patients with alk rearrangements ( clinicaltrials.gov identier : nct02201992 ) , and anvil ( clinicaltrials.gov identier : nct02595944 ) for unmatched patients ( eg , squamous histology , or nonsquamous histology and neither egfr mutation nor alk rearrangements )  . an observation cohort is also available for unmatched patients who refuse to participate in anvil . 
another prominent example is lung - map ( clinicaltrials.gov identier : nct02154490 ) , 44 which was initially for squamous lung cancer but recently was amended for all types of advanced nsclc . 
using sequential development features such as phase ii / iii designs and interim analyses that permit adaptation to ndings , these trials adaptively provide the necessary exibility for the umbrella trial objectives as a whole ( fig 2c )  . when there are multiple candidate regimens that are of interest equally for some or all biomarker cohorts , umbrella trials also can be designed solely for discovery objectives . 
for example , in the battle - 1 ( clinicaltrials.gov identier : nct00411632 , nct00409968 , nct00410059 , nct00410189 , nct00411632 , nct00411671 ) trial , patients with chemotherapy - refractory nsclc were assayed for four candidate biomarkers to be allocated to a total of ve marker strata ( including one nonmatched ) and then randomly assigned to one of four drug regimens.15 in nrglu003 ( clinicaltrials.gov identier : nct03737994 ) , an umbrella trial of previously treated patients with alk - positive lung cancer , a total of 10 marker cohorts ( including an unmatched cohort ) and up to seven new - generation alk inhibitors are to be evaluated . 
however , as the total number of possible drug - marker strata increases , the likelihood to accrue enough patients to have a reasonably accurate estimate of efcacy for each drug - biomarker stratum decreases . 
by exploiting the fact that biomarker allocation is not informative for treatment assignments , battle - 1 considered a learn - as - go approach by explicitly using a bayesian hierarchical probit model for information sharing , 45 along with outcome - adaptive randomization , 12 , 46 which allocates more patients to drug - marker strata that are more likely to have exceptional activities , to potentially provide individuals with more efcacious treatments and improve estimation precision ( fig 2d )  . 
in nrg - lu003 , for each biomarker the investigators are able to determine and prioritize the experimental regimens of interest on the basis of preclinical data , which substantially reduces the total number of drug - biomarker strata to be evaluated . 
freidlin and korn40 suggested that under typical phase ii trial design settings , both approaches may require similar resources . one implication of conducting umbrella trials is that an explicit rule governing how to match biomarkers and candidate regimens needs to be specied prospectively . 
therefore , umbrella trials also provide a unique opportunity to evaluate whether a rule - based policy that matches biomarkers and drugs is effective at all and , if so , to what extent across all genomic characterizations . 
to make such evaluation interpretable , the rule - based assignment policy should be as stable as possible during the trial conduct and ideally cover a wide range of biomarker - drug combinations.47 another unique consideration with umbrella trials is that a patient could be eligible for multiple biomarker cohort allocation because the tumor contains multiple biomarkers . 
with the emergent use of molecular proling , basket trials and umbrella trials , as well as extensions discussed here , can be collectively called master protocols or platform trials . 
more importantly , such a protocol provides substantial exibility in terms of discontinuing unpromising investigations , carrying forward favorable early results to denitive testing in a phase ii / iii framework , and introducing new subtrials as targets and agents are identied in a perpetual manner , using the aforementioned adaptive design methods for single - biomarker settings.48 in addition , the infrastructure advantages of conducting master protocols , such as centralized and streamlined trial conduct ( enrollment , informed consent ) , data collection , governance ( institutional review board , data and safety monitoring committee ) , and quality assurance ( clinical monitoring , imaging reading ) , are subtrials stantial as compared with conducting individual separately . 
when it was activated in 2015 , it started with 10 parallel biomarker - based cohorts to evaluate eight different in may 2016 , a preplanned feasibility interim analysis revealed that only 9% of patients had actionable mutations that could be matched with any of the multiple targeted therapies under investigation . 
the lower - than - expected matching success rate led to a major amendment to improve the overall matching rate by adding additional marker - specic cohorts.49 in june 2017 , when the original screening target was met ( n = 6 , 000 ) , it was found that the common molecular subtypes were rarer than expected.50 the study therefore underwent another major amendment by collapsing multiple subtype cohorts and relaxing the screening process . 
to date , the study remains open to accrual , with a total of 19 cohorts to evaluate 13 drugs.51 four lung - map was originally designed in 2014 for advanced squamous nsclc , consisting of targeted therapy subgroups and one nonmatched subgroup , each using a phase ii / iii seamless design , with progression - free survival and overall survival as primary end points , respectively.52 since 2015 , the treatment landscape of advanced nsclc has changed tremendously because of a series of approvals in immunotherapies especially for squamous and nonsquamous nsclc by the us food and drug administration , which fundamentally changed the standard of care and the study control arms . 
after multiple minor amendments for adding and closing biomarker - specic subtrials , in 2018 the study was completely revamped by expanding eligibility to all histology of advanced nsclc , using a new screening protocol and introducing new biomarker - dened subtrials.53 in summary , although conceptually offering efciency and exibility , master protocols are more prone to various factors that are unknown ( eg , low biomarker prevalence ) or cannot be foreseen ( eg , changing treatment landscape ) at the trial inilogistical tiation . 
these trials also come with substantial complications , especially when several sponsors are involved who may have conicting proprietary interests and regulatory concerns . discussion biomarker - driven clinical trials allow us to investigate patient heterogeneity on the basis of molecular proling , which consequently introduces new opportunities and challenges . 
comparing with the conventional paradigm , these trials require even more thorough planning and comprehensive evaluation on the overarching objectives ( discovery or conrmatory ) , the credential of biomarkers clinical utility , choice of adaptive design and analysis plans , knowledge of cancer biology , existing data from preclinical and early clinical trials , prevalence of each subtype population , logistic readiness to conduct immortal clinical trials , and so on . 
the success of any biomarker - driven trials therefore certainly relies on an even closer collaboration involved in advancing cancer care , including among all clinical investigators , statisticians , sponsors , regulators , drug and assay developers , and patient advocates . our discussions have been limited to phase ii and iii trial designs for discovery and conrmatory purposes . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / author - center . chen hu consulting or advisory role : merck sharp & dohme james j . 
j clin oncol 30 : 4223 - 4232 , 2012 pennello ga : analytical and clinical evaluation of biomarkers assays : when are biomarkers ready for prime time ? clin trials 10 : 666 - 676 , 2013 dancey je , dobbin kk , groshen s , et al : guidelines for the development and incorporation of biomarker studies in early clinical trials of novel agents . 
biometrics 38 : 143 - 151 , 1982 gehan ea : the determination of the number of patients required in a preliminary and a follow - up trial of a new chemotherapeutic agent . 
j chronic dis 13 : 346 - 353 , 1961 jennison c , turnbull bw : group sequential methods with applications to clinical applications - to - clinical - trials / jennison - turnbull / p / book / 9780849303166 simon r : optimal two - stage designs for phase ii clinical trials . 
clin cancer res 16 : 691 - 698 , 2010 jiang w , freidlin b , simon r : biomarker - adaptive threshold design : a procedure for evaluating treatment with possible biomarker - dened subset effect . 
freidlin b , korn el : borrowing information across subgroups in phase ii trials : is it useful ? clin cancer res 19 : 1326 - 1334 , 2013 41 . 
le - rademacher j , dahlberg s , lee jj , et al : biomarker clinical trials in lung cancer : design , logistics , challenges , and practical considerations . 
lih cj , sims dj , harrington rd , et al : analytical validation and application of a targeted next - generation sequencing mutation - detection assay for use in treatment assignment in the nci - mpact trial . 
peeters m , jay price tj , cervantes a : randomized phase iii study of panitumumab with uorouracil , leucovorin , and irinotecan ( folfiri ) compared with folfiri alone as second - line treatment in patients with metastatic colorectal cancer . 
herbst rs , redman mw , kim es , et al : cetuximab plus carboplatin and paclitaxel with or without bevacizumab versus carboplatin and paclitaxel with or without bevacizumab in advanced nsclc ( swog s0819 ) : a randomised , phase 3 study . 
spigel dr , ervin tj , ramlau ra , et al : randomized phase ii trial of onartuzumab in combination with erlotinib in patients with advanced non - small - cell lung cancer . 
bradley jd , paulus r , komaki r , et al : standard - dose versus high - dose conformal radiotherapy with concurrent and consolidation carboplatin plus paclitaxel with or without cetuximab for patients with stage iiia or iiib non - small - cell lung cancer ( rtog 0617 ) : a randomised , two - by - two factorial phase 3 study . 
vansteenkiste jf , cho bc , vanakesa t , et al : efcacy of the mage - a3 cancer immunotherapeutic as adjuvant therapy in patients with resected mage - a3positive non - small - cell lung cancer ( magrit ) : a randomised , double - blind , placebo - controlled , phase 3 trial . 
cobo m , isla d , massuti b , et al : customizing cisplatin based on quantitative excision repair cross - complementing 1 mrna expression : a phase iii trial in nonsmall - cell lung cancer . 
 c dramatic response to sequential braf inhibition in braf v600emutant metastatic lung adenocarcinoma introduction lung [ aq1 ] [ aq2 ] cancer is the leading cause of cancer related death.1 targeted therapy directed toward molecular pathways that drive nonsmall - cell lung cancer ( nsclc ) has dramatically improved treatment options . 
some of the oncogenic drivers identified in lung adenocarcinoma involve the ras - raf - mitogen - activated protein kinase ( mapk ) kinase ( mek ) - extracellular regulated kinase ( erk ) pathway ( mapk - erk pathway ) .2 braf is a key protein in mapk - erk signaling . 
 mutations in braf [ aq3 ] are present in 1% to 3% of nsclc and lead to constitutive activation of the mapk - erk pathway , promoting cancer cell proliferation and survival . 
several other mutations of braf have been identified and are grouped as non - v600e mutations.3 patients with nsclc harboring braf v600e mutations are sensitive to braf inhibitors ( brafis ) alone or in combination with mek inhibitors ( mekis ) .4 - 6 after initial response , patients typically experience relapse as a result of various resistance mechanisms . 
imaging revealed a left - side hilar lung mass , enlarged mediastinal lymph nodes , vertebral body sclerotic lesions , small bilateral pleural effusions , and a pericardial effusion consistent with metastatic disease . 
the repeat biopsy specimen was sent for massively parallel dna sequencing using the illumina hiseq2000 platform ( foundation medicine , cambridge , ma ) to characterize base substitutions , short insertions and deletions , copy number alterations , and selected fusions across 287 cancer - related genes . 
several tumor mutations , including braf v600e , mapk1 ( r306h ) , smad4 ( r361c ) , and setd2 ( splice site 65_71 + 13del20 ) , were identified . 
 a through lymphangitic spread ( fig 2 ) that required oxygen supplementation at 5 l / min and showed malignant involvement of the right - side iliopsoas muscle , which caused significant pa after palliative radiation to the right - side pelvic mass for pain control , he began a combination of dabrafenib 150 mg twice daily and trametinib 2 mg daily . 
at the same time , the patient became oxygen independent , and functional status improved , which allowed him to fulfill his desire to travel to his home country to visit family . more than 5 months later , after dose reduction for grade 3 fatigue , his lung mass again increased in size . 
by that point , he had survived 3.5 years since diagnosis of nsclc with a response duration of 12 months on braf targeted therapy . discussion in this era of personalized medicine , targeted therapies against braf v600e and mek have changed the standard of care in the treatment of braf - mutated metastatic melanoma . 
two brafis , dabrafenib and vemurafenib , with or without mekis , trametinib and cobimetinib , are approved for braf v600emutant metastatic melanoma.7 , 8 such targeted medications also have clinical activity in braf v600emutant nsclc.4 - 6 , 9 , 10 in a retrospective analysis of the european euraf cohort , 35 patients with braf - mutant nsclc treated with brafis were identified . 
after first - line therapy with brafis , overall survival ( os ) was 25.3 months in patients with braf v600emutant nsclc ( n = 29 ) and 11.8 months in patients with nonv600e braf mutations ( n = 6 ) .9 response to chest before and after initiation of vemurafenib , at 2 weeks , and at 3 months demonstrated a significant decrease in the size of the rightside upper - lobe mass [ aq4 ] and improvement of the air space disease , with an overall decrease in tumor volume of greater than 90% ( fig 1 )  . 
the dosage of vemurafenib was reduced twice for grade 3 fatigue , recurrent grade 2 skin rash , and transaminitis . after maintaining response for 7 months on vemurafenib , the patients tumor burden escalated fig 1 . 
computed tomography scans of chest demonstrating response to combination dabrafenib and trametinib treatment brafis also was documented in a phase ii basket trial of vemurafenib in braf v600mutant positive nonmelanoma cancers with an overall response rate ( orr ) of 42% in 20 patients with nsclc.10 in a prospective multicenter phase ii clinical trial , planchard et al5 evaluated the safety and efficacy of dabrafenib trametinib in nsclc . 
previously treated patients ( n = 78 ) and treatment - nave patients ( n = 6 ) responded to dabrafenib alone with a median progression - free survival ( pfs ) of 5.5 months , a median os of 12.7 months , and an orr of 33% . 
testing for braf is recommended as part of the initial molecular analysis in patients with lung cancer of adenocarcinoma histology and is considered in some patients with squamous subtype.11 all patients with braf v600emutant nsclc eventually progress after brafis with or without mekis . 
resistance mechanisms are not as well characterized in nsclc compared with braf v600emutant melanoma.12 secondary resistance mechanisms in melanoma include ras mutations , braf splice variants , braf v600 amplifications , mek1 / 2 mutations , and non - mapk pathway alterations.13 in nsclc , lin et al14 described two classes of resistance patterns to vemurafenib using a preclinical model of hcc364 braf v600emutant lung cancer cells . 
in contrast , expression of c - junmediated egfr ligands that promote continued egfr signaling represents another class.14 plx9394 , a novel brafi , evades resistance caused by alternatively spliced truncated braf v600e , but subsequent resistance occurs through egfr - mediated ras - mammalian target of rapamycin signaling.15 in the clinical setting , kras , tp53 , and cdkn2a mutations were acquired in one patient after progression on dabrafenib.16 currently , there is no standard therapy for patients with braf v600emutant nsclc after progression on brafis . 
in a phase i / ii study , the combination of brafi and meki was evaluated in patients with braf v600mutant metastatic melanoma who had disease progression on a single - agent brafi . 
one of these patients had a partial response to dabrafenib monotherapy after prior treatment with vemurafenib.9 another case report showed a similar response ; a patient who progressed on vemurafenib achieved a 3 - month clinical benefit with single - agent dabrafenib.18 our patient attained clinical response for an additional 5 months with an alternative brafi , dabrafenib , combined with the meki trametinib after progression on vemurafenib . 
two prior case studies described the benefit of combination treatment after progression on initial brafi monotherapy.19 , 20 similar to our patient , schmid et al19 reported on a patient who responded to combination dabrafenib and trametinib for 6 months after progression on vemurafenib . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . sushma jonna no relationship to disclose affiliations all authors : georgetown university , washington , dc . clinical trials or have already progressed on other lines of therapy . 
chen d , zhang lq , huang jf , et al : braf mutations in patients with non - small cell lung cancer : a systematic review and meta - analysis . 
planchard d , besse b , groen hjm , et al : dabrafenib plus trametinib in patients with previously treated braf ( v600e ) - mutant metastatic non - small cell lung cancer : an open - label , multicentre phase 2 trial . 
planchard d , kim tm , mazieres j , et al : dabrafenib in patients with braf ( v600e ) - positive advanced non - small - cell lung cancer : a single - arm , multicentre , open - label , phase 2 trial . 
planchard d , smit ef , groen hjm , et al : dabrafenib plus trametinib in patients with previously untreated brafv600e - mutant metastatic non - small - cell lung cancer : an open - label , phase 2 trial . 
lindeman ni , cagle pt , aisner dl , et al : updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors : guideline from the college of american pathologists , the international association for the study of lung cancer , and the association for molecular pathology . 
johnson db , menzies am , zimmer l , et al : acquired braf inhibitor resistance : a multicenter meta - analysis of the spectrum and frequencies , clinical behaviour , and phenotypic associations of resistance mechanisms . 
okimoto ra , lin l , olivas v , et al : preclinical efficacy of a raf inhibitor that evades paradoxical mapk pathway activation in protein kinase braf - mutant lung cancer . 
rudin cm , hong k , streit m : molecular characterization of acquired resistance to the braf inhibitor dabrafenib in a patient with braf - mutant non - small - cell lung cancer . 
johnson db , flaherty kt , weber js , et al : combined braf ( dabrafenib ) and mek inhibition ( trametinib ) in patients with brafv600 - mutant melanoma experiencing progression with single - agent braf inhibitor . 
schmid s , siano m , joerger m , et al : response to dabrafenib after progression on vemurafenib in a patient with advanced braf v600e - mutant bronchial adenocarcinoma . 
 approximately 40% of families fulfilling the clinical criteria for hdgc have germline cdh1 mutations.5 a subset of the remaining families of hdgc , and ones fulfilling the criteria of other familial gc , harbor pathogenic germline mutation in other genes that are associated with hereditary cancer predisposition syndromes.4 hereditary breast and ovarian cancer is one of the best - described inherited cancer predisposition syndromes , caused by pathogenic germline brca1 or brca2 ( brca1 / 2 ) mutations.6 - 9 the increased risks of cancers other than breast and ovarian cancers were observed in the carriers.10 the association between germline brca1 / 2 mutation and increased risk of gc were demonstrated in previous studies for families with hereditary breast and ovarian cancer.11 - 14 regarding fgc , a recent large - scale study demonstrated that germline brca2 mutations were identified in patients who had a family history that fulfilled the criteria of hdgc but were lacking cdh1 mutations.15 therefore , it is possible that germline brca1 / 2 mutations may cause familial predisposition to gc . recent advances of comprehensive genomic analysis enable us to identify the genomic alterations in gc.16 brca1 / 2 mutations were shown in the subset of gc tumor tissues ; however , the association between germline brca1 / 2 mutations and familial predisposition to gc were not fully understood . 
previously , we performed genomic sequencing of 207 japanese gcs using a 435 - gene panel and identified brca1 / 2 mutations in tumor.17 in this study , we conducted brca1 / 2 genetic testing in seven japanese patients with gc whose tumor had brca1 / 2 mutations . 
we identified pathogenic germline brca1 / 2 mutations in three patients who have a familial component of gc . methods among 28 patients whose tumor had brca1 / 2 mutations in our previous study , seven patients who could provide written informed consent were enrolled . 
we assessed the family history using the criteria for referral for genetic risk assessment and fgc defined by international gastric cancer linkage consortium after the enrollment ( table 1 ) .4 clinicopathological data are listed in table 2 . 
this study was approved by the institutional review boards at niigata university and niigata cancer center hospital . results family history of cases 1 to 4 met the criteria for referral to genetic risk assessment , and that of cases 1 , 3 , and 4 met the criteria for fgc ( table 2 )  . 
cases 5 to 7 also had germline brca1 or brca2 mutations ; however , pathogenicity was determined to be uncertacase 4 had no germline mutations in brca1 / 2 ( table 3 )  . 
he had a strong family history of cancers , with three gc and one breast cancer in firstor second - degree relatives ( fig 2a ) , which met the criteria for fgc ( table 1 )  . 
although adjuvant chemotherapy with s - 1 , an oral fluoropyrimidine preparation combining tegafur , gimeracil , and oteracil potassium , was performed , she suffered para - aortic lymph node recurrence 6 months after gastrectomy . 
she had a strong family history of cancers , with two gcs and one case each of breast , lung , and cervical cancers in firstor second - degree relatives , which did not fulfill the criteria for fgc ( fig 2b )  . 
genetic testing detected double pathogenic nonsense germline mutations in brca1 c.188t > a ( p.l63x ) and in brca2 c.6922a > t ( p.k2308x ) , which were identical to the variants detected in tumor ( table 3 )  . case 3 a 56 - year - old man underwent curative gastrectomy with d2 lymphadenectomy for gc . 
genetic testing detected a pathogenic nonsense germline mutation in brca2 c.9310a > t ( p.k3104x ) , which was identical to the variant detected in tumor ( table 3 )  . discussion previous studies have shown that germline brca1 / 2 mutations increase the risk of gc.11 - 14 however , there is a paucity of data regarding the relationship between germline brca1 / 2 mutations and familial predisposition to gc in japan . 
among these , one patient ( case 2 ) had double pathogenic germline mutations in brca1 and brca2 , which were considered to be rare , as shown by a previous study for ashkenazi jewish double - founder mutations.18 to the best of our knowledge , this is the first report of japanese patients with fgc harboring brca1 / 2 mutations . multiple genetic and environmental factors influence the etiology of gc . 
the well - known environmental factors are high salt intake and hp infection , which cause chronic gastritis.19 the development of gc stands out in the family history of our three cases , rather than breast and ovarian cancers ( fig 2 )  . 
hp infection was detected in cases 1 and 3 and intestinal - type gc , which is most associated with chronic gastritis , was observed in cases 1 and 2 ( fig 1 )  . 
these findings suggested that chronic gastritis , which is associated with hp infection and / or environmental factors , may cause the loss of brca1 / 2 function by the second - hit mutations in patients with the germline mutations . 
this synergistic exacerbatory effect confers the familial predisposition to gc in japan , where a high incidence is observed . brca1 / 2 mutation carriers might have a risk of multiple gcs . 
brca1 / 2 mutations are considered risk factors for ipsilateral breast cancer recurrence.20 regarding hdgc caused by cdh1 germline mutation , multiple foci of signet ring cell carcinoma were detected in the stomach of individual patients.21 , 22 prophylactic total gastrectomy is recommended for cdh1 mutation carriers because of the high cumulative incidence and the difficulty of early detection of hdgc by endoscopy.23 in this report , the intestinal type of gc , which is relatively easy to detect at an early stage by endoscopy , predominantly developed in the affected patients . 
brca1 / 2 - mutated ovarian and breast cancers have high sensitivity to platinum chemotherapy and poly ( adenosine diphosphate ribose ) polymerase ( parp ) inhibitors because of defective dna double - strand break repair.24 - 26 the us food and drug administration has approved the parp inhibitor olaparib for brca1 / 2 - mutated advanced ovarian cancer . 
in this report , case 2 , with both brca1 and brca2 germline mutations , showed high response of platinum - based chemotherapy for lymph node recurrence and long - term survival after the recurrence , in a manner similar to brca1 / 2 - mutated ovarian and breast cancers . 
additional clinical studies are required to clarify whether the brca1 / 2 mutations contribute to the good response to platinum - based chemotherapy and parp inhibitors in gc . in conclusion , brca1 / 2 mutations may predispose to familial gc . 
 shujiro okuda no relationship to disclose stephen lyle employment : kew stock and other ownership interests : kew consulting or advisory role : pfizer kazuaki takabe no relationship to disclose acknowledgment we thank our colleagues masaki aizawa and atsushi matsuki from niigata cancer center hospital for collection of the clinicopathological data . affiliations hiroshi ichikawa , toshifumi wakai , masayuki nagahashi , yoshifumi shimada , takaaki hanyu , yosuke kano , yosoke tajima , yusuke muneoka , takashi ishikawa , kazuyasu takizawa , jun sakata , takashi kobayashi , hitoshi kemeyama , and shujiro okuda , niigata university graduate school of medical and dental sciences ; hiroshi yabusaki , satoru nakagawa , nobuaki sato , takashi kawasaki , keiichi homma , niigata cancer center hospital , niigata city , niigata , japan ; stephen lyle , university of massachusetts medical school , worcester , ma ; and kazuaki takabe , roswell park cancer institute , and university at buffalo , the state university of new york , buffalo , ny . supported by funding from niigata prefecture , japan society for the promotion of science ( jsps ) grants - in - aid for scientific research ( kakenhi ) grant no . 
zanghieri g , di gregorio c , sacchetti c , et al : familial occurrence of gastric cancer in the 2 - year experience of a population - based registry . 
van der post rs , vogelaar ip , carneiro f , et al : hereditary diffuse gastric cancer : updated clinical guidelines with an emphasis on germline cdh1 mutation carriers . 
alsop k , fereday s , meldrum c , et al : brca mutation frequency and patterns of treatment response in brca mutation - positive women with ovarian cancer : a report from the australian ovarian cancer study group . 
 in a multivariable regression analysis adjusted for age , smoking , eastern cooperative oncology group performance status , and the presence of tp53 mt ( p = .063 ) and other coexisting mts ( p = .064 ) did not achieve statistical significance . 
2018 by american society of clinical oncology introduction epidermal growth factor receptor ( egfr ) and tumor suppressor tp53 genes are commonly mutated in patients with nonsmall - cell lung cancer ( nsclc ) with independent prognostic implications . 
a second site mutation in exon 20 of egfrt790maccounts for a significant proportion of acquired resistance to firstand second - generation tkis.5 - 7 resistance mechanisms also include other mutations in egfr ( c797s ) , met amplification , and mutations in other downstream oncogenic pathways.6 , 8 tp53 is a tumor suppressor gene that is located on the short arm of chromosome 17 at band charu aggarwal christiana w . 
inactivation of retinoblastoma and p53 has been demonstrated to be an early clinical biomarker for transformation to small - cell lung cancer.22 the primary objective of this study was to determine the prognostic impact of tp53 mutation on survival in patients with egfr - mt nsclc . 
smoking status was defined as never smokers ( fewer than 100 cigarettes lifetime ) , former smoker ( more than 100 cigarettes lifetime ) , and current smoker ( active smoker within 1 year of diagnosis )  . 
we searched the national death index and online obituaries to determine survival status for patients who were lost to follow - up . egfr and tp53 mutation analysis tissue testing solid tumor dna sequencing was performed using amplicon - based next - generation sequencing ( ngs ) to determine the mutation status of exons 18 through 21 of egfr and exons 4 through 10 of tp53 in a college of american pathologists / clinical laboratory improvement amendmentscertified laboratory at our institution , as previously described.24 patient samples were sequenced using one of three different panels that covered the regions of interest and other commonly mutated oncogenes . 
an illumina truseq amplicon cancer panel ( illumina , san diego , ca ) was used to sequence targeted hotspots or larger exonic regions for 47 genes ( appendix table a1 )  . 
samples with poor - quality tissue and / or insufficient dna for the truseq amplicon cancer panel assay input requirements were sequenced on a custom 20 - gene targeted hotspot panel , the penn precision panel ( appendix table a2 ) , and more recently , with an expanded 153 wholegene panel ( appendix table a3 ; haloplex ; agilent technologies , santa clara , ca )  . 
results were analyzed using a clinically validated bioinformatics pipeline using hg19 genome build as the reference genome.25 the limit of detection for mutations in tp53 and egfr is at 4% allele frequency , typically corresponding to at least 8% tumor in the submitted tissue specimen . 
 the expanded 153 - gene panel has full coverage of tp53 , whereas the 47 - gene panel and penn precision panels detect point mutations and small insertions and deletions in select regions with partial coverage of tp53 ( appendix tables a1 to a3 )  . plasma - based cell - free circulating tumor dna testing blood was collected in two 10 - ml streck tubes ( streck , la vista , ne ) and shipped overnight at ambient temperature to guardant health ( redwood city , ca ) , a clinical laboratory improvement amendmentscertified , college of american pathologistsaccredited laboratory facility . 
samples were sequenced on the 68 - gene panel for 12 patients , on the 70 - gene panel for 46 patients , and on the 73 - gene panel for the remaining seven patients.27 the plasma - based gene panel has full coverage of tp53 ( appendix table a4 )  . a coexisting oncogenic mutation was defined as a nonsynonymous variant detected in genes from solid tissue or plasma sequencing that occurred in addition to the identified egfr / tp53 mutations at the time of assessment . 
 only genes that were covered on both the tissue and plasma platforms were included in the analysis . statistical analysis descriptive statistics summarized patient characteristics and included frequencies and percentages for categorical variables , and mean , standard deviation , and standard error of the mean for continuous variables . 
overall , patient demographics and tumor characteristics were similar between tp53 wild - type ( wt ) and egfr / tp53 double - mt groups ( table 1 )  . 
 other oncogenic coexisting mts were present in 36% of patients with tp53 wt and 45.7% of those with egfr / tp53 double mt ( p = .362 ; fig 2c )  . 
a total of 86 oncogenic coexisting mts were observed in these 55 patients , 18.2% of which was in phosphatidylinositol - 4 , 5 - bisphosphate 3 - kinase catalytic subunit ( pik3ca ; fig 2d and appendix table a8 )  . 
consort diagra ( * ) patients had concurrent tissue and plasma ngs ( next - generation sequencing ) performed ( n = 42 ) ; tissue ngs results were used for analyses . 
 ( c ) prevalence of coexisting mts in the entire cohort , and in patients with tp53 mt , 55 patients were found to have coexisting mts , 37 of whom also harbored a tp53 mt . 
in one patient ( age 67 years , smoker ) , t790m mt emergent mt was evident , and in the other patient ( age 45 years , never smoker ) , a new rb1 mutation appeared . 
findings in individual patients at the time of sclc transformation included the disappearance of the emergent t790m , persistence of the rb1 mutation , and maintenance of a pik3ca mutation ( table 3 )  . 
similar to other series , we observed a significant improvement in os in patients who had a t790m mutation , likely related to the availability and effectiveness of third - generation egfr tkis , such as osimertinib . 
 ( a ) kaplan - meier curve representing os of patients with stage iv disease at diagnosis and tumors with tp53 mutation ( mt ; n = 81 ) and tp53 wild - type ( wt ) cohorts ( n = 50 )  . 
 ( b ) kaplan - meier curve representing os of patients with stage iv disease at diagnosis and tumors with coexisting mt ( n = 55 ) and without coexisting mt ( n = 76 )  . 
 ( c ) kaplan - meier curve representing os of patients with stage iv disease at diagnosis and tumors with tp53 exon 8 mt ( n = 18 ) and tp53 mt other than exon 8 ( n = 63 )  . 
a larger study with 136 patients corroborated the finding that tp53 mutationsanalyzed for 123 patientswere associated with a lower response rate and poor prognosis with the use of tkis in egfr - mt nsclc.36 survival was not statistically significant in this study for the overall population , but there was a significant survival difference for a specific subset of patientsthose with exon 8 mutations36 ( n = 12 )  . 
we report on five patients whose tumors transformed to sclc , with paired biopsies showing an increase in tp53 mt allelic fraction , which is suggestive of an increase in genomic heterogeneity at the time of transformation and potentially complete inactivation of p53 . 
to our knowledge , this is the first report of serial analysis of multiple patients where tp53 mutation was detected with routine clinically indicated ngs at the time of transformation to sclc . 
although these mutations were heterogonous and distinct , our findings offer supportive evidence of the role of p53 inactivation in the clonal evolution of egfr - mt nsclc.45 the current study has a number of important limitations . 
carpenter mutations within the introns , the loss of chromosome 17p or the deletion of the tp53 locus would not be detectable by ngs assays that were used for this analysis . 
this analysis also does not account for tumor heterogeneity with low - level mutations in subpopulations that were not detected , either because of mutation below the level of detection in the submitted specimen or regional heterogeneity within a tumor mass or between metastatic sites . 
furthermore , although our study cohort of double mutants81 patients with egfr / tp53 double - mt nsclcis one of the largest series reported to date , it is still relatively small . 
we controlled for confounding factors by using multivariable regression analyses . in conclusion , tp53 mt and other oncogenic coexisting mutations were associated with worse os in patients with egfr - mt nsclc . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . charu aggarwal consulting or advisory role : genentech , bristol - myers squibb , eli lilly , celgene research funding : genentech ( inst ) , incyte ( inst ) , macrogenics ( inst ) , merck sharp & dohme ( inst ) christiana w . 
thompson consulting or advisory role : oncocyte saman ahmed no relationship to disclose seth jeffries no relationship to disclose stephen bagley research funding : incyte , novocure peter gabriel research funding : varian medical systems tracey l . 
evans honoraria : genentech , genentech ( i ) consulting or advisory role : genentech , genentech ( i ) speakers ' bureau : genentech , genentech ( i ) travel , accommodations , expenses : genentech , genentech ( i ) joshua m . 
bauml consulting or advisory role : bristol - myers squibb , merck , astrazeneca , genentech , celgene , boehringer ingelheim , guardant health research funding : merck ( inst ) , carevive systems ( inst ) , novartis ( inst ) , incyte ( inst ) , bayer ( inst ) , janssen pharmaceuticals ( inst ) christine ciunci no relationship to disclose evan alley no relationship to disclose jennifer j.d. 
cohen honoraria : bristol - myers squibb consulting or advisory role : heat biologics , takeda , cerulean pharma , kolltan pharmaceuticals , zymeworks , pfizer research funding : heat biologics ( inst ) , macrogenics ( inst ) , merck ( inst ) , takeda ( inst ) , cleave biosciences ( inst ) , celldex ( inst ) travel , accommodations , expenses : heat biologics , takeda , kolltan pharmaceuticals , cerulean pharma , zymeworks , bristol - myers squibb , pfizer erica l . 
langer honoraria : bristol - myers squibb , genentech , eli lilly , imclone consulting or advisory role : genentech , eli lilly , imclone , merck , abbott biotherapeutics , bayer , onyx pharmaceuticals , clarient , clovis oncology , celgene , cancer support community , bristol - myers squibb , ariad pharmaceuticals , takeda , astrazeneca research funding : merck ( inst ) , advantagene ( inst ) , clovis oncology ( inst ) , celgene ( inst ) , inovio pharmaceuticals ( inst ) , ariad pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , genentech ( inst ) , stem centrx ( inst ) other relationship : eli lilly , amgen , peregrine pharmaceuticals , synta affiliations charu aggarwal , christiana w . 
pao w , miller v , zakowski m , et al : egf receptor gene mutations are common in lung cancers from never smokers and are associated with sensitivity of tumors to gefitinib and erlotinib . 
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molina - vila ma , bertran - alamillo j , gasc a , et al : nondisruptive p53 mutations are associated with shorter survival in patients with advanced nonsmall - cell lung cancer . 
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lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
thompson jc , yee ss , troxel ab , et al : detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next - generation sequencing of cell - free circulating tumor dna . 
cortot ab , younes m , martel - planche g , et al : mutation of tp53 and alteration of p14 ( arf ) expression in egfrand kras - mutated lung adenocarcinomas . 
ozenne p , dayde d , brambilla e , et al : p14 ( arf ) inhibits the growth of lung adenocarcinoma cells harbouring an egfr l858r mutation by activating a stat3 - dependent pro - apoptotic signalling pathway . 
lueers ac , halbfass v , prenzel r , et al : incidence of egfr , kras , braf , alk and p53 alterations in a cohort of 218 consecutively tested patients from a certified lung cancer center : correlation with clinical characteristics and treatment outcome . 
yu ha , jordan e , ni a , et al : concurrent genetic alterations identified by next - generation sequencing in untreated , metastatic egfr - mutant lung cancers . 
lim sm , kim hr , cho ek , et al : targeted sequencing identifies genetic alterations that confer primary resistance to egfr tyrosine kinase inhibitor ( korean lung cancer consortium )  . 
bria e , pilotto s , amato e , et al : molecular heterogeneity assessment by next - generation sequencing and response to gefitinib of egfr mutant advanced lung adenocarcinoma . 
aisner dl , sholl lm , berry ld , et al : the impact of smoking and tp53 mutations in lung adenocarcinoma patients with targetable mutations : the lung cancer mutation consortium ( lcmc2 )  . 
hata a , katakami n , yoshioka h , et al : spatiotemporal t790m heterogeneity in individual patients with egfr - mutant nonsmall - cell lung cancer after acquired resistance to egfr - tki . 
piotrowska z , niederst mj , karlovich ca , et al : heterogeneity underlies the emergence of egfrt790 wild - type clones following treatment of t790m - positive cancers with a thirdgeneration egfr inhibitor . 
mooradian mj , piotrowska z , drapkin bj , et al : clonal evolution and the role of serial liquid biopsies in a case of small - cell lung cancertransformed egfr mutant nonsmall - cell lung cancer . 
an endobronchial biopsy showed an adenocarcinoma consistent with a lung primary ( thyroid transcription factor 1 positive , napsin a positive , and cytokeratin 7 positive by immunohistochemistry ; figs 1a and 1b )  . targeted exon sequencing of 468 genes with memorial sloan kettering integrated mutation proling of actionable cancer targets ( msk - impact ) was negative for known mitogenic drivers , including kras , egfr , braf , met , and erbb2 mutations and alk , ros1 , and ret rearrangements . 
photomicrographs of ( a ) cytology from the patients lung adenocarcinoma and ( b ) positive immunohistochemistry results remarkable for cytoplasmic and membranous staining using a pan - trk antibody are shown . 
this identied a novel activating trk fusion ( in - frame fusion between eps15 exon 21 and ntrk1 exon 10 ; figs 1c and 1d )  . the patient consented to receive larotrectinib in a basket study for trk fusion - positive cancers ( navigate ; clinicaltrials.gov identier : nct02576431 )  . 
a brisk partial response to therapy was achieved at 4 weeks , which was subsequently conrmed at 8 weeks ( 34% by response evaluation criteria in solid tumors [ recist ] version 1.1 ; fig 2 ) , with regression of all areas of disease . 
this included near total resolution of all brain metastases at 8 weeks ( 95% reduction in aggregate tumor volume ) and subsequently , a complete intracranial response by 16 weeks ( fig 3 )  . 
pathology showed a stage iiic ( t1cn3m0 ) multifocal invasive ductal carcinoma ; the largest lesion measured 1.8 cimmunohistochemistry was positive for estrogen and progesterone receptors and negative for human epidermal growth factor receptor 2 . 
she was treated initially with adjuvant dose - dense doxorubicin , cyclophosphamide , plus paclitaxel and radiotherapy , after which she received anastrozole followed by bilateral salpingo - oophorectomy . the patient developed recurrent , metastatic disease approximately 17 months after completing adjuvant radiolymph node biopsy conrmed therapy . 
sequencing of an abdominal lymph node and a subsequently biopsied liver metastasis using msk - impact revealed the previously reported fusion between lmna exons 1 and 20 and ntrk1 exons 11 and 17 , which was veried using targeted multiplex rna sequencing . the patient consented to receive larotrectinib in the same basket study for trk fusion - positive cancers ( navigate )  . at enrollment , her disease was widely metastatic to liver , adrenal gland , lymph nodes , and bone . 
a brisk and conrmed partial response by recist version 1.1 ( 32% , 47% , and 56% at 4 , 8 , and 16 weeks , respectively ) was achieved . 
of note , her cns metastases likewise regressed at the rst follow - up assessment at 4 weeks , with a complete response in the cns at 8 weeks ( fig 4 )  . 
axial contrastenhanced t1 - weighted images at ( a ) baseline ( brain metastases highlighted by circles ) , ( b ) 8 weeks , and ( c ) 16 weeks after treatment with larotrectinib was initiated demonstrate decreased and then resolved metastases in the right - side insula and left - side occipital lobe . 
three - dimensional models at ( d ) baseline and ( e ) post - treatment at 8 weeks and ( f ) post - treatment at 16 weeks conrmed decreased burden of metastatic intracranial disease ( segmented in green voxels and quantied at bottom )  . 
response to larotrectinib in a patient with a trk fusion - positive metastatic breast cancer . baseline axial computed tomography imaging of the brain was performed with the addition of iodinated contrast that showed ( a ) metastatic brain disease ( circles ) and after 8 weeks of treatment with larotrectinib , ( b ) complete response . 
 rosen et al discussion to our knowledge , this report is the rst to showcase the intracranial efcacy of larotrectinib in metastatic trk fusion - positive cancers of any histology . 
these cases demonstrate that larotrectinib can achieve clinically relevant cns exposure , which highlights its role in the management of trk fusion - positive cancers with intracranial disease . beyond nsclcs and breast cancers , other trk fusionpositive tumors have a proclivity for brain metastases.1 the intracranial activity featured here is consistent with prior work that did not identify cns metastasis as a site of progression on larotrectinib across multiple tumor types4 and with a report that showed a response to larotrectinib in a trk fusion - positive glioma.5 additional clinical experience in patients with trk fusion - positive cancers with cns involvement ultimately will delineate the overall activity and durability of larotrectinib within the cns . although this article represents what is to our knowledge the rst report of intracranial responses to a sein patients with trk fusionlective trk inhibitor positive cancers and brain metastases , intracranial disease control also has been reported with multikinase trk inhibitors . 
entrectinib , a multikinase trk , ros1 , and alk inhibitor , displayed activity in a patient with a trk fusion - positive lung cancer and brain metastases6 and in ros1 fusion - positive lung cancers with intracranial disease.7 repotrectinib , a multikinase trk , ros1 , and alk inhibitor , achieved intracranial disease regression in untreated brain metastases in a patient with a ros1 fusion - positive nsclc.8 finally , this report represents the rst description of the novel eps15 - ntrk1 fusion . 
young stock and other ownership interests : agios , alexion pharmaceuticals , biogen , celgene , gilead sciences , karyopharm therapeutics , spark therapeutics , regeneron pharmaceuticals , stemline therapeutics , vertex consulting or advisory role : agios , puma biotechnology , nordicneurolab , icon clinical research research funding : agios ( inst ) jaclyn f . 
shu research funding : celgene ( inst ) , genentech ( inst ) , roche ( inst ) , janssen pharmaceuticals ( inst ) , medimmune ( inst ) travel , accommodations , expenses : genentech , roche nora c . 
 cns efcacy of larotrectinib in trk fusion - positive solid tumors travel , accommodations , expenses : novartis ( i ) , biotheranostics ( i ) , pzer ( i ) , amgen ( i ) , genentech ( i ) , roche ( i ) , astrazeneca ( i ) , macrogenics ( i ) , peregrine pharmaceuticals ( i ) , pierian bioscience ( i ) david m . 
ziegler ds , wong m , mayoh c , et al : brief report : potent clinical and radiological response to larotrectinib in trk fusion - driven high - grade glioma . 
drilon a , siena s , ou s - hi , et al : safety and antitumor activity of the multi - targeted pan - trk , ros1 , and alk inhibitor entrectinib ( rxdx - 101 ) : combined results from two phase 1 trials ( alka - 372 - 001 and startrk - 1 )  . 
doebele r , ahn m , siena s , et al : oa02.01 : efcacy and safety of entrectinib in locally advanced or metastatic ros1 fusion - positive non - small cell lung cancer 8 . 
drilon a , ou si , cho bc , et al : repotrectinib ( tpx - 0005 ) is a next - generation ros1 / trk / alk inhibitor that potently inhibits ros1 / trk / alk solvent - front 9 . 
pathologic examination showed a 2.9 - cm collecting duct carcinoma ( cdc ) with metastases in eight of 15 resected hilar , suprahilar , and periaortic lymph nodes . after surgery , the patient was initiated on therapy with cisplatin and gemcitabine on a dosing schedule of once every 3 weeks . 
cytologic examination of pleural fluid retrieved during thoracentesis confirmed malignant pleural effusions . given the absence of any standard second - line treatment of cdc , comprehensive genomic profiling ( cgp ) was performed using a commercially available assay in a clinical laboratory improvement amendmentscertified , college of american pathologistsaccredited laboratory ( foundation medicine ; cambridge , ma )  . 
formalin - fixed paraffin - embedded tumor derived from the original nephrectomy specimen was analyzed by hybridization capture of all coding exons from 315 cancerrelated genes and select introns of 28 genes commonly rearranged in cancer . 
sequencing and bioinformatics methods for characterization of genomic alterations ( ga ) have previously been described in detail.1 the sole ga observed in this case was homozygous deletion of cdkn2a / b . 
multiple variants of unknown significance were identiincluding bard1 ( p358_s364del ) , brip1 fied , ( r264w ) , crlf2 ( e213k ) , dicer1 ( v594i ) , fgf6 ( n113k ) , igf1r ( p190s ) , mll2 ( r1292c ) , notch1 ( v1232m ) , pdgfrb ( s408c ) , ros1 ( e401k ) , socs1 ( a44_v45insvp ) , and tet2 ( e1405q )  . 
cdkn2a alteration has been suggested to be a potential predictive biomarker for cdk4 / 6 inhibitors.2 , 3 in light of loss of cdkn2a / b , the patient was offered therapy with the cdk4 / 6 inhibitor palbociclib . 
chung , foundation medicine , cambridge ; and toni k . choueiri , dana - farber cancer institute / brigham and womens cancer center , boston , ma corresponding author : sumanta k . 
the genomic landscape of cdc was defined in a previous report from our group.11 potential biomarkers for response to cdk4 / 6 inhibitors such as palbociclib include alterations in genes involved in the regulation of cdk4 / 6cyclin d kinase complexes , 2 , 3 such as cdk4 amplification , and palbociclib has demonstrated encouraging activity in phase 2 studies of patients with liposarcoma who harbor this driver.12 , 13 although preclinical studies of cell lines from multiple cancer types , including rcc , have reported that cdkn2a / p16ink4a loss of function is associated with sensitivity to cdk4 / 6 inhibitors , 14 - 18 there is limited clinical evidence supporting cdkn2a as a predictive biomarker , with one case study reporting that a patient with uterine leiomyosarcoma harboring cdkn2a homozygous deletion and a concurrent nf2 mutation experienced 4 months of stable disease on palbociclib treatment . in contrast , for patients with advanced breast cancer , for which palbociclib is approved with either letrozole or fulvestrant , cdkn2a loss ( assessed by fluorescent in situ hybridization ) was not associated with further benefit from palbociclib in combination with letrozole , in the palbociclib ongoing trials in the management of breast cancer ( paloma ) - 1 trial for patients with er + / her22 breast cancer.19 similarly , in a basket trial of the cdk4 / 6 inhibitor ribociclib , no responses were observed among the 73 patients with cancer whose tumor harbored a cdkn2a ga , as assessed by cgp.20 in this context and that of limited single - agent efficacy of cdk4 / 6 inhibitors in unselected populations , 19 , 20 the partial response to palbociclib experienced here by a patient with metastatic refractory cdc harboring cdkn2a / b homozygous deletion is unusual . 
in a series of 17 patients with cdc assessed by cgp , the most frequently identified gas were in nf2 ( 29% ) , setd2 ( 24% ) , smarcb1 ( 18% ) , and cdkn2a / b ( 12% , including homozygous deletion and mutation )  . 
characteristics for cases with cdkn2a or cdkn2a / b homozygous deletion characteristic total cases , no . total cases with cdkn2a or cdkn2a / b homozygous deletion , no . cases with cdkn2a or cdkn2a / b homozygous deletion and no co - occurring alteration , no . characteristics of patients with cdkn2a or cdkn2a / b homozygous deletion and no co - occurring alteration female male age , years , median ( range ) median tumor mutational burden , no . 
cdkn2a ( or cdkn2a / b ) homozygous deletion was identified as the sole driver alteration in 1.2% ( 16 of 1 , 322 ) rcc cases and also in other disease types , including 15.2% of salivary gland acinic cell tumors , 14.3% of bone giant - cell tumors , and 11.3% of bone chordomas ( table 1 )  . 
it is conceivable , however , that some of these cases with cdkn2a homozygous deletion as the sole known ga may harbor concurrent driver alterations that have not yet been discovered . 
a larger subset of cases ( 16% ) had homozygous deletion of this locus as well as other gas in other genes ( table 1 ) , and additional cases had loss of heterozygosity of cdkn2a with the remaining allele mutated . 
more investigation is also required to understand how responses to cdk4 / 6 inhibitors for patients with cdkn2a alteration may be modulated by the presence or absence of different driver gene alterations . for patients with cdc , additional genomic profiling studies are needed , given the response to palbociclib observed in this study , which suggests that cdk4 / 6 inhibitors should be further explored for patients with cdkn2a - altered cdc . 
more broadly , this study identifies cdkn2a homozygous deletion as the sole known ga in a small subset ( 0.25% ) of advanced cancer cases and at higher frequency in specific tumor types ( table 1 )  . 
 patents , royalties , other intellectual property : patents via foundation medicine , seres health on microbiome research in non - neoplastic disease ( inst ) genentech , eisai , foundation medicine , cerulean pharma , astrazeneca , peloton therapeutics , exelixis , prometheus , alligent jeffrey ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine honoraria : pfizer , emd merck serono research funding : foundation medicine toni k . 
choueiri honoraria : national comprehensive cancer network , uptodate consulting or advisory role : pfizer , bayer , novartis , glaxosmithkline , merck , bristol - myers squibb , roche / research funding : pfizer ( inst ) , novartis ( inst ) , merck ( inst ) , exelixis ( inst ) , tracon pharma ( inst ) , glaxosmithkline ( inst ) , bristol - myers squibb ( inst ) , astrazeneca ( inst ) , peloton therapeutics ( inst ) , roche / genentech ( inst ) , celldex ( inst ) , agensys ( inst ) travel , accommodations , expenses : advisory boards / consultancy jon h . 
curr oncol 20 : e223 - e232 , 2013 von der maase h , hansen sw , roberts jt , et al : gemcitabine and cisplatin versus methotrexate , vinblastine , doxorubicin , and cisplatin in advanced or metastatic bladder cancer : results of a large , randomized , multinational , multicenter , phase iii study . 
miyake h , haraguchi t , takenaka a , et al : metastatic collecting duct carcinoma of the kidney responded to sunitinib . int j clin oncol 16 : 153 - 155 , 2011 10 . 
dickson ma , schwartz gk , keohan ml , et al : progression - free survival among patients with well - differentiated or dedifferentiated liposarcoma treated with cdk4 inhibitor palbociclib : a phase 2 clinical trial . 
dickson ma , tap wd , keohan ml , et al : phase ii trial of the cdk4 inhibitor pd0332991 in patients with advanced cdk4 - amplified well - differentiated or dedifferentiated liposarcoma . 
logan je , mostofizadeh n , desai aj , et al : pd - 0332991 , a potent and selective inhibitor of cyclin - dependent kinase 4 / 6 , demonstrates inhibition of proliferation in renal cell carcinoma at nanomolar concentrations and molecular markers predict for sensitivity . 
young rj , waldeck k , martin c , et al : loss of cdkn2a expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the cdk4 / 6 inhibitor pd0332991 in melanoma cell lines . 
finn rs , crown jp , lang i , et al : the cyclin - dependent kinase 4 / 6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first - line treatment of oestrogen receptor - positive , her2 - negative , advanced breast cancer ( paloma - 1 / trio - 18 ) : a randomised phase 2 study . 
peguero ja , oneil bh , sohal d , et al : genomic mutation profiling ( gmp ) and clinical outcome in patients ( pts ) treated with ribociclib ( cdk4 / 6 inhibitor ) in the signature prograj clin oncol 34 , 2016 ( suppl ; abstr 2528 ) 21 . 
wang j , papanicolau - sengos a , chintala s , et al : collecting duct carcinoma of the kidney is associated with cdkn2a deletion and slc family gene up - regulation . 
the utility of ultra - deep genomic testing to predict and the impact of conditioning intensity to prevent mds relapse are unknown . methods targeted error - corrected dna sequencing was performed on preconditioning blood samples from patients with mds ( n = 48 ) from the blood and marrow transplant clinical trials network 0901 phase iii randomized clinical trial , which compared outcomes by allogeneic hematopoietic cell transplantation conditioning intensity in adult patients with , 5% marrow myeloblasts and no leukemic myeloblasts in blood on morphological analysis at the time of pretransplant assessment . 
clinical end points ( 53 - month median follow - up ) included transplant - related mortality ( trm ) , relapse , relapse - free survival ( rfs ) , and overall survival ( os )  . 
of the 48 patients examined , 14 experienced trm , 23 are relapse - free , and 11 relapsed , of which 7 died . results using a previously described set of 10 gene regions , 42% of patients ( n = 20 ) had mutations detectable before random assignment to reduced intensity conditioning ( ric ) or myeloablative conditioning ( mac )  . 
testing additional genes , including those associated with mds , did not improve prognostication . conclusion this study provides evidence that targeted dna sequencing in patients with mds before transplant can identify those with highest post - transplant relapse rates . 
2021 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction myelodysplastic syndrome ( mds ) , one of the most common hematologic disorders , is a collection of clinically and genetically heterogeneous diseases . 
 dillon et al context key objective allogeneic hematopoetic cell transplantation ( allohct ) is the only curative therapy for myelodysplastic syndrome ( mds ) but is associated with suboptimal rates of transplant - related mortality ( trm )  . 
using samples from a randomized phase iii clinical trial comparing allohct conditioning intensity in patients with mds , this study examined the prognostic signicance of pretransplant genetic markers on post - transplant outcomes . knowledge generated the presence of mutations within a previously identied set of 10 gene regions before allohct was associated with increased rates of relapse and decreased relapse - free survival in patients with mds who received reduced intensity versus myeloablative conditioning . 
library preparation and pairedend 150 - bp sequencing were performed using unique dualsample indices on an hiseq 2500 ( rapid run mode ; illumina , san diego , ca ) as previously described.10 an average of 43 million paired - end reads were acquired per sample ( data supplement )  . 
details are provided in the data supplement . bioinformatics consensus sequences based on umi read families were mapped to human genome version hg19 ( build grch37 )  . de novo variant calls were made using a minimum allele frequency of 0.001 and were ltered using information regarding umi read families , background error rate models , unique start sites , strand - specic priming , and homopolymer runs . 
alternative approaches were used for insertional mutations in npm1 and flt3 internal tandem duplication . details are provided in the data supplement . statistical analysis kaplan - meier estimation and log - rank test were used for analysis of os and relapse - free survival ( rfs ) and grays test for competing risks of trm and relapse . 
details are provided in the data supplement . results patient cohort a total of 54 patients with mds participated in the bmtctn 0901 clinical trial , of which 48 had blood collected after study enrollment before random assignment to mac ( n = 25 ) or ric ( n = 23 ) conditioning regimens , which was used for genomic analysis in this study . 
this subset of patients was well - matched for baseline characteristics ( table 1 ) , and clinical outcomes were aligned with those previously reported ( data supplement )  . 
ngsg10 mutational status served as a strong predictor of relapse in this cohort of patients with mds , predicting 73% of relapses at 24 months ( 100% for those receiving mac ) , with a specicity of 78% ( 100% for those receiving ric ) ( data supplement )  . to ngsg10 mutational status , no signicant in contrast difference in rates of relapse or os was observed when stratifying patients by disease classication , disease risk group , or cytogenetic prognostic group ( data supplement )  . rates of relapse trended higher in patients with poor cytogenetics or categorized as high risk . 
inclusion of patients with poor cytogenetics before transplant with ngsg10 mutational status improved prediction of relapse at 24 months from 73% to 91% ( data supplement ) and was associated with signicantly higher rates of relapse compared with ngsg10 - negative patients ( data supplement )  . presence of mutations pretransplant predicts relapse by conditioning intensity the impact of conditioning intensity and ngsg10 next , mutational status was examined ( data supplement )  . 
inclusion of patients with poor cytogenetics before transplant with ngsg10 mutational status improved prediction of relapse at 24 months in the ric group to 89% , with a specicity of 89% , and is associated with signicantly increased rates of relapse and decreased rfs compared with patients receiving mac ( data supplement )  . screening for additional genes does not improve test performance the mutational spectrum of mds is complex , with many additional genes recurrently mutated beyond the 10 amlassociated genes described above.7 , 11 , 12 therefore , we expanded our testing to also cover regions in an additional 19 genes including those commonly found in mds at diagnosis ( asxl1 , bcor , cbl , cux1 , dnmt3a , etv6 , ezh2 , gata2 , kras , phf6 , ppm1d , ptpn11 , setbp1 , srsf2 , stag2 , tet2 , u2af1 , wt1 , and zrsr2 )  . 
 ( c ) 10 - gene ngspositive patients had signicantly decreased rfs ( 3 - year rfs , 34% v 71% , p = .006 ) and os ( 3year os , 55% v 79% , p = .045 ) compared with ngs - negative patients . 
the inclusion of dta ( dnmt3a , tet2 , and asxl1 ) genes , known to be associated with age - related clonal hematopoiesis , 13 , 14 resulted in reclassication of 21% ( n = 6 ) of ngsg10negative patients to positive . 
similar to what we observed in patients with aml , 10 samples testing positive for only dta mutations showed no difference in relapse rates or os compared with those testing negative . 
no relapses were observed in patients testing positive only for these 16 genes regardless of conditioning intensity , and only one death was observed ( trm in the mac group )  . 
limiting analysis to those testing positive for an ngsg10 mutation at or above 2.5% vaf reduced the sensitivity at 24 months from 73% to 45% ( data supplement )  . 
overall p values ( in bold ) : grays test for transplant - related mortality and relapse ; log - rank test for rfs and os . abbreviations : ngsg10 , next - generation sequencing 10 - gene ; pos , positive ; neg , negative ; mac , myeloablative conditioning ; ric , reduced intensity conditioning ; rfs , relapse - free survival ; freq , frequency ; os , overall survival ; ci , condence interval . on patient outcomes and found no signicant difference in os based on the mutation number ( data supplement ) or number of mutated genes ( data supplement )  . patients with mds with excess blasts although the requirement for inclusion in the bmt - ctn 0901 study was , 5% marrow myeloblasts and no leukemic myeloblasts in blood on morphological analysis at the time of pretransplant assessment , a total of 22 of the 54 patients with mds ( 41% ) treated on the bmt - ctn 0901 trial had an initial diagnosis of refractory anemia with excess blasts ( raeb ) i or ii ( ie , between 5% and 19% blasts )  . 
seven of these samples tested positive for ngs10g , for which survival was 0% for ric ( n = 2 , relapse ) and 60% for mac - treated patients ( with one death from each relapse and transplantrelated mortality )  . 
ngs10g - positive patients accounted for four of six relapses observed in the raeb cohort , and the other two patients had high - risk cytogenetics with a monosomal karyotype that would not be detectable using this ngs assay . discussion here , we present the impact of genetic mutations detected before random assignment to ric or mac , identied without knowledge of the mutations originally present at diagnosis , on transplant outcomes in patients with mds . 
detection of mutations using a dna - sequencing panel covering regions of 10 genes , previously shown to have utility at the same timepoint in patients with aml , 10 was associated with increased rates of relapse and decreased rfs and os . 
 ( a ) differences in rates of relapse were identied between subgroups dened by conditioning intensity ( ric or mac ) and mutational status ( p , .001 ) , with the highest rate occurring in 10 - gene ngspositive ( ngsg10 - positive ) patients with mds receiving ric . 
the prognostic signicance of the remaining mutations on relapse was dependent on conditioning intensity , with higher rates of relapse and lower rfs observed in patients randomly assigned to receive ric compared with mac . this study provides evidence that the benet of mac versus ric in reducing relapse is found in patients with mds with detectable mutations using a 10 - gene region dnasequencing panel before allohct . 
patients are displayed in columns and grouped by conditioning intensity ( ric or mac ] ) and clinical outcome ( relapse , no relapse , or transplant - related mortality [ trm ] )  . 
genes are displayed in rows and sorted by diagnostic panel groupings , including 10 - gene ( prognostic in aml ) , dta ( associated with age - related clonal hematopoiesis ) , and 16 - gene ( commonly mutated in mds )  . 
although the size of this cohort limits extrapolation , it is possible that the prognostic implications of tp53 mutation detection in patients with mds before allohct and the impact of conditioning intensity differ from those reported previously when those variants are found at a level below the limit of detection of routinely used ngs assays . 
in addition to vaf , other factors such as persistence since initial diagnosis , development during therapy , mutation type , functional classication , and zygosity are likely to be important for determining the relapse risk associated with detecting a tp53 mutation.15 - 22 although mutation detection by targeted dna sequencing before allohct was strongly associated with increased relapse and decreased rfs and these outcomes could be improved with mac , unlike in patients10 with aml , we did not detect an os advantage for conditioning intensication in those testing positive . 
this might have been due to limited sample size , predominately nonrelapse causes of death in this mds cohort ( 67% of deaths , compared with 39% in the previously reported aml cohort ) , or other factors . mds is a genetically heterogenous disease.1 , 11 , 12 , 23 as was observed previously , 8 we found that inclusion of a large number of genes resulted in the majority of patients having mutations present before transplant . 
furthermore , mutations in genes associated with clonal hematopoiesis ( dta ) or 16 other mds - associated genes outside of 10 - gene improve test performance . mutational status did not reanalysis of pretransplant mutational status using only the 10 genes reported here , from another study of patients with mds with , 5% myeloblasts before allohct , 8 conrms our nding of signicantly increased relapse rates in patients testing positive and receiving ric versus mac . 
the modest there are several sample size here offers only a partial sampling of the heterogenous genetics associated with mds , whereas differences in pretransplantation disease burden , biology , and prior treatment history further limit exact comparisons . the signicance of mutation detection before allohct might be inuenced by history of prior treatment , and previous work using the same testing in patients with aml included only those treated to cytomorphological remission before transplant . 
 mds relapse after allohct allohct.9 two randomized studies of conditioning intensity in mds have failed to show a difference in survival between mac and ric , 4 , 6 , 25 although we show here a trend toward improved survival with increased conditioning intensity for those with detectable mutations particularly in the raeb group ( ie , biology and treatment history most analogous to aml )  . 
prior work had shown a benet for increased conditioning intensity in patients with mds with ras pathway mutations ( nras , kras , ptpn11 , cbl , nf1 , rit1 , flt3 , and kit ) undergoing allohct.9 as relapse is not primary cause of death in patients with mds undergoing allohct , the choice of conditioning intensity is currently dependent on the estimated ability of a patient to tolerate transplant - related toxicity.26 allohct remains the only curative therapy for mds ; however , given the median age at diagnosis , many patients will not be eligible for myeloablative approaches.27 , 28 as it is now possible to determine the genomic basis of disease before and after transplantation , personalized approaches for allohct of patients with mds are now conceivable with targeted strategies for those with detectable tp53 , ras pathway ( including flt3 ) , immune augmentation , or other therapies potentially able to supplement the impact of chosen conditioning intensity to minimize relapse risk.29 - 31 idh and jak2 mutations , in conclusion , we show that in adult patients with mds with , 5% marrow myeloblasts and no leukemic myeloblasts in blood before allohct , ultra - deep dna sequencing for mutations in 10 gene regions previously shown to be high risk in patients with aml could identify a subset of 42% of patients who experienced the majority of post - transplant relapses . 
the cibmtr registry is supported primarily by the u24 - ca76518 from nhlbi , nci , and the national institute of allergy and infectious diseases and from hhsh234200637015c ( hrsa / dhhs ) to the center for international blood and marrow transplant research . 
blood 122 : 2943 - 2964 , 2013 luger sm , ringden o , zhang mj , et al : similar outcomes using myeloablative vs reduced - intensity allogeneic transplant preparative regimens for aml or mds . bone marrow transpl 47 : 203 - 211 , 2012 shimoni a , hardan i , shem - tov n , et al : allogeneic hematopoietic stem - cell transplantation in aml and mds using myeloablative versus reduced - intensity conditioning : the role of dose intensity . 
leukemia 20 : 322 - 328 , 2006 kroger n , iacobelli s , franke gn , et al : dose - reduced versus standard conditioning followed by allogeneic stem - cell transplantation for patients with myelodysplastic syndrome : a prospective randomized phase iii study of the ebmt ( ricmac trial )  . 
j clin oncol 35 : 2157 - 2164 , 2017 pulsipher ma : reduced intensity for myelodysplastic syndrome : worth the gamble ? j clin oncol 35 : 2106 - 2108 , 2017 scott bl , pasquini mc , logan br , et al : myeloablative versus reduced - intensity hematopoietic cell transplantation for acute myeloid leukemia and myelodysplastic syndromes . 
j clin oncol 35 : 1154 - 1161 , 2017 bejar r , stevenson ke , caughey b , et al : somatic mutations predict poor outcome in patients with myelodysplastic syndrome after hematopoietic stem - cell transplantation . 
n engl j med 379 : 1028 - 1041 , 2018 lindsley rc , saber w , mar bg , et al : prognostic mutations in myelodysplastic syndrome after stem - cell transplantation . 
n engl j med 371 : 2477 - 2487 , jaiswal s , fontanillas p , flannick j , et al : age - related clonal hematopoiesis associated with adverse outcomes . 
montalban - bravo g , kanagal - shamanna r , benton cb , et al : genomic context and tp53 allele frequency dene clinical outcomes in tp53 - mutated 16 . 
coombs cc , zehir a , devlin sm , et al : therapy - related clonal hematopoiesis in patients with non - hematologic cancers is common and associated with adverse 18 . 
al - kali a , zblewski d , foran jm , et al : outcome of myelodysplastic syndromes over time in the united states : a national cancer data base study from 20042013 . 
sorror ml , sandmaier bm , storer be , et al : long - term outcomes among older patients following nonmyeloablative conditioning and allogeneic hematopoietic cell transplantation for advanced hematologic malignancies . 
maslah n , salomao n , drevon l , et al : synergistic effects of prima - 1met ( apr - 246 ) and azacitidine in tp53 - mutated myelodysplastic syndromes and acute myeloid leukemia . 
 entrectinib in trk and ros1 fusion - positive metastatic pancreatic cancer introduction the treatment of patients with metastatic pancreatic cancer ( paca ) has improved in recent years with the development of chemotherapy combinations ; however , the undeniable improvements in survival have been modest , and response rates remain low at approximately 25% to 35%.1 , 2 it is hoped that by identifying subgroups of patients with paca who harbor distinct actionable molecular alterations , major gains can be realized by using targeted therapies . 
for example , patients with paca who have homologous recombination deficiencypositive tumors may derive benefit with poly ( adp ribose ) polymerase inhibitorbased therapy , 3 - 5 and patients with mismatch repairdeficient / microsatellite instabilityhigh disease have demonstrated response to pembrolizumab.6 , 7 entrectinib ( rxdx - 101 ) is an investigational , cns - active , potent , selective inhibitor of the tyrosine kinases trka , trkb , trkc , ros1 , and alk encoded by ntrk1 , ntrk2 , ntrk3 , ros1 , and alk , respectively . 
8 gene fusions ( rearrangements ) of these target kinases have the potential to be oncogenic drivers , and they are present in small percentages across a variety of tumor types.9 in addition , they are mutually exclusive with other drivers.10 two phase i clinical studies ( alka - 372 - 001 [ first - in - human phase 1 study of entrectinib , an oral pan - trk , ros1 , and alk inhibitor , in patients with advanced solid tumors with relevant molecular alterations ] and startrk - 1 [ study of oral rxdx101 in adult patients with locally advanced or metastatic cancer targeting ntrk1 , ntrk2 , ntrk3 , ros1 , or alk molecular alterations ] ) that evaluated entrectinib in patients with extracranial solid tumors who harbored ntrk , ros1 , or alk gene fusions and who were tyrosine kinase inhibitor nave reported a response rate of 79% ( 19 of 24 ) .11 we present three patients with paca who harbor highly actionable gene fusions ( ntrk1 and ros1 ) who were treated in an ongoing phase ii trial of entrectinib ( nct02568267 ; basket study of entrectinib [ rxdx - 101 ] for the treatment of patients with solid tumors harboring ntrk 1 / 2 / 3 [ trk a / b / c ] , ros1 , or alk gene rearrangements [ fusions ] [ startrk - 2 ] )  . 
treatment duration with entrectinib ( 600 mg once per day ) ranged from approximately 7 months to more than 1 year , and one patient is still receiving treatment . patient 1 : tpr - ntrk1 fusion patient 1 is a 51 - year - old man who was diagnosed with stage iv paca in april 2015 , with lung , liver , and adrenal gland metastases . 
the first positron emission tomography / computed tomography ( pet / ct ) scan after the end of the first cycle ( october 18 , 2016 ) showed stable disease michael j . 
 ( b ) the tumors for patients 1 and 2 harbored a tpr - ntrk1 fusion , and ( c ) the tumor for patient 3 harbored an slc4a4 - ros1 fusion . 
 chr , chromosome ; tm , transmembrane domain . slc4a4 ros1 slc4a4 - ros1 fusion ( 9% by response evaluation criteria in solid tumors [ recist ] v1.1 ) along with a decrease in pet standardized uptake value , mainly in the liver ( figs 2a and 2b )  . 
the bone lesions and primary tumor remained stable , and his carbohydrate antigen 19 - 9 ( ca19 - 9 ) fell to 30 ku / l ( fig 2c )  . 
he gained 20 kg over approximately 3.5 months , which is a possible on - target effect of appetite enhancement as a result of trkb inhibition13 and not a manifestation of peripheral edema . 
in the last 2 months , he also developed hypogonadis he was still receiving entrectinib at the time of this report . patient 2 : tpr - ntrk1 fusion patient 2 is a 47 - year - old man who was diagnosed with borderline resectable paca in april 2016 . 
ngs with foundationone revealed a tpr - ntrk1 fusion ( fig 1 ) , cdkn2a / b loss , and kras wild type.14 on december 8 , 2016 , he started treatment with entrectinib ; at that time , he was rapidly losing weight . 
restaging ct scans on february 27 , 2017 , showed a partial response ( 32% by recist v1.1 ) , which was subsequently confirmed on april 24 , 2017 ( 38% ; fig 3a ) ; his ca19 - 9 fell to 9 ku / l by april 2017 ( fig 3b )  . 
treatment continued for approximately 8 months , at which time the ct scan showed continued response in the pancreas and liver and a slight increase in the size of a nontarget lesion in a portal lymph node . 
 ( b ) positron emission tomography / computed tomography scans at the start of entrectinib treatment ( baseline ) , after cycle 1 , and after cycle 2 demonstrating significant reduction of metabolic activity in liver metastases . 
 ( a ) axial images at the start of entrectinib therapy ( baseline ) , after cycle 5 , and after cycle 7 demonstrating a partial response to entrectinib with decreases in the size of the primary tumor in the pancreas and in liver metastases . 
 ( b ) carbohydrate antigen 19 - 9 ( ca19 - 9 ) biomarker levels during entrectinib treatment ; his ca19 - 9 level was 154 ku / l before starting entrectinib . patient 2 ca19 - 9 during entrectinib treatment ( start 8 december 2016 ) dec - 16 jan - 17 feb - 17 mar - 17 apr - 17 may - 17 jun - 17 jul - 17 aug - 17 the last ct scan in august 2017 showed progression in the portal lymph nodes and liver , and the patient discontinued the study . patient 3 : slc4a4 - ros1 fusion patient 3 is a 54 - year - old man who was diagnosed with metastatic paca in march 2016 . 
 previous treatments included a clinical trial of gemcitabine plus nab - paclitaxel with or without a notch inhibitor , folfirinox , and liver directed transarterial chemoembolization ( with gemcitabine , cisplatin , and mitomycin )  . 
testing with msk - impact , 15 a tumor - profiling multiplex panel , revealed that the tumor had several alterations , including a mutation in cdkn2a , somatic mutations in atm and brca2 , deletion of pik3ca , and amplification of erbb2 ( without her2 overexpression ) , but it was kras wild type . 
within 2 weeks of treatment , the patient experienced myalgias and fatigue , which abated with short - dose interruptions , and the patient resumed the highly active lifestyle he had at baseline , with a normal performance status and no loss of appetite or weight . 
he had a minor response ( a reduction of 12.5% ; stable disease per recist v1.1 ) and was considering definitive resection of the primary pancreatic mass , as well as metastasectomy of the liver disease . 
 discussion although paca remains a devastating disease , we are beginning to identify subgroups of patients whose disease harbors highly actionable molecular alterations , and therapies targeting these molecular alterations have the potential to significantly improve outcomes over standard therapy.1 , 2 , 16 here , we presented three patients with paca who harbored gene fusions and who were responsive to single - agent treatment with the investigational agent entrectinib . 
acquired resistance as a result of the development of secondary mutations that disrupt drug binding has been noted for many tyrosine kinase inhibitors.17 it is not known whether resistance mutations developed in these patients . 
 this effect , although it has been documented as an adverse effect of treatment , may be clinically meaningful considering the high incidence of cachexia in patients with paca.18 this is one of the first reports demonstrating such a substantial response in this specific subset of patients with this difficult - to - treat cancer . interestingly , the two patients with ntrk fusion positive tumors harbored tpr - ntrk1 gene fusions . 
this occurrence may be coincidental , because ntrk fusions can involve ntrk1 , ntrk2 , or ntrk3 and , to date , have been shown to combine with more than 25 fusion partners.17 furthermore , a different gene fusion partner for ntrk , etv6 - ntrk3 , was identified in an elderly patient with paca.19 these results strengthen the case for molecular profiling of patients with advanced paca . 
across more than 1 , 000 patients and samples tested , approximately 25% of patients have molecular alterations for which specific therapies are available.22 in a recent survey of 640 patients with paca who underwent molecular profiling , gene fusions were identified in 1% of the patients , most of which were targetable by entrectinib ( ntrk , ros1 , or alk fusions ) ( pishvaian et al , manuscript submitted for publication ) , which is similar to the percentage of patients with mismatch repairdeficient / microsatellite instability high tumors for which pembrolizumab was approved . 
furthermore , although kras mutations are present in > 90% of patients with pancreatic ductal adenocarcinoma ( pdac ) , 23 none of the gene fusionpositive tumors described here or in a previous report of alk fusion positive pdac24 harbored kras mutations . 
 notwithstanding small numbers , these combined observations suggest that fusions of ntrk , ros1 , or alk may be more likely in younger patients ( those younger than age 55 years ) with kras wild type pdac.24 in support of these observations , a recent analysis of 3 , 426 patients with pdac reported that approximately 12% were kras wild type . 
of these , kinase gene fusions were identified in 8% of pdacs that were kras wild type , 25 including five patients with alk fusions and three with ntrk fusions . 
pishvaian consulting or advisory role : ignyta , perthera ignacio garrido - laguna consulting or advisory role : ignyta stephen liu consulting or advisory role : ariad / takeda , genentech / roche , ignyta , pfizer edna chow - maneval employment : ignyta christian rolfo no relationship to disclose acknowledgments we thank the patients who gave consent to present their cases for the benefit of the scientific , medical , and patient communities worldwide ; laurens carp , md , department of nuclear medicine , and bart op de beeck , md , radiology department , antwerp university hospital , for help with imaging ; and meredith rogers , ms , cmpp , and nick cianciola , phd , of the lockwood group for providing editorial support funded by ignyta . 
liu , lombardi comprehensive cancer center , georgetown university , washington , dc ; ignacio garrido - laguna , huntsman cancer institute , university of utah , salt lake city , ut ; pratik s . 
lowery ma , kelsen dp , stadler zk , et al : an emerging entity : pancreatic adenocarcinoma associated with a known brca mutationclinical descriptors , treatment implications , and future directions . 
pishvaian mj , wang h , zhuang t , et al : a phase i / ii study of abt - 888 in combination with 5 - fluorouracil ( 5 - fu ) and oxaliplatin ( ox ) in patients with metastatic pancreatic cancer ( mpc )  . 
diaz la , marabelle a , delord jp , et al : pembrolizumab therapy for microsatellite instability high ( msi - h ) colorectal cancer ( crc ) and non - crc . 
de braud fg , pilla l , niger m , et al : phase 1 open label , dose escalation study of rxdx101 , an oral pan - trk , ros1 , and alk inhibitor , in patients with advanced solid tumors with relevant molecular alterations . 
drilon a , nagasubramanian r , blake jf , et al : a next - generation trk kinase inhibitor overcomes acquired resistance to prior trk kinase inhibition in patients with trk fusion - positive solid tumors . 
drilon a , siena s , ou si , et al : safety and antitumor activity of the multitargeted pan - trk , ros1 , and alk inhibitor entrectinib : combined results from two phase i trials ( alka - 372 - 001 and startrk - 1 )  . 
murphy da , ely ha , shoemaker r , et al : detecting gene rearrangements in patient populations through a 2 - step diagnostic test comprised of rapid ihc enrichment followed by sensitive nextgeneration sequencing . 
perreault m , feng g , will s , et al : activation of trkb with tam - 163 results in opposite effects on body weight in rodents and non - human primates . 
 real - time genomic characterization of metastatic pancreatic neuroendocrine tumors has prognostic implications and identifies potential germline actionability purpose we assessed the usefulness of real - time molecular profiling through next - generation sequencing ( ngs ) in predicting the tumor biology of advanced pancreatic neuroendocrine tumors ( pannets ) and in characterizing genomic evolution . methods patients with metastatic pannets were recruited in the routine clinical practice setting ( between may 2014 and march 2017 ) for prospective ngs of their tumors as well as for germline analysis using the memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) sequencing platfor when possible , ngs was performed at multiple time points . results ngs was performed in 96 tumor samples from 80 patients . 
sequencing of preand post - treatment samples revealed tumor - grade progression ; clonal evolution patterns were also seen ( molecular resistance mechanisms and chemotherapy - associated mutagenesis )  . 
germline genetic analysis identified clinically actionable pathogenic or likely pathogenic variants in 14 of 88 patients ( 16% ) , including mutations in high - penetrance cancer susceptibility genes ( men1 , tsc2 , and vhl )  . conclusion a clinical ngs platform reveals pertubations of biologic pathways in metastatic pannets that may inform prognosis and direct therapies . 
2018 by american society of clinical oncology introduction pancreatic neuroendocrine tumors ( pannets ) represent 1% to 2% of all pancreatic neoplasms , but they have a rising incidence and prevalence.1 - 4 whole - exome and whole - genome sequencing , largely of resected pannets , has defined genomic events and improved our understanding of disease pathogenesis.5 , 6 these efforts have identified changes in chromatin remodeling , telomere maintenance , and mammalian target of nitya raj ronak shah zsofia stadler semanti mukherjee joanne chou brian untch janet li virginia kelly muyinat osoba leonard b . 
in addition to the well - characterized hereditary syndromes associated with pannet development ( multiple endocrine neoplasia 1 , von hippel - lindau syndrome , neurofibromatosis type 1 ) , recent data suggest the presence of germline alterations in dna damage repair genes in some patients.6 , 7 treatments for unresectable pannets include targeted and cytotoxic therapies , as well as liver directed approaches.8 - 13 no data exist to guide therapy for pannets with defined genomic alterations . 
in addition , disease heterogeneity poses a significant challenge in defining the selection and sequencing of therapy . to that end , using an institutional matched tumor - normal platform , we prospectively performed next - generation sequencing ( ngs ) of tumors in patients with metastatic well differentiated ( wd ) pannets . 
 we also hypothesized that pannets evolve over time with a more aggressive behavior that could be characterized biologically through ngs of repeat biopsies after treatment . methods study population written informed consent was obtained from patients with a diagnosis of wd pannet using a prospective , institutional review board approved protocol ( clinicaltrials.gov identifier : nct01775072 )  . 
pathologic features of the tumor samples ( differentiation and grade ) were determined according to the 2017 who classification , which recognizes the existence of a grade 3 ( g3 ) category within wd pannets ; historical values for differentiation and grade were collected through review of the electronic medical records . 
electronic medical records were also reviewed for patient data on demographics , treatments , and survival . sequencing of tumor samples ngs was performed using memorial sloan ketteringintegrated mutation profiling of actionable cancer targets ( msk - impact ) , a matched tumor - normal sequencing platform approved for clinical use by the new york state department of health , to detect base substitutions , small indels , copy number changes , and selected gene rearrangements.14 - 17 updated assay versions were released between 2014 and 2017 , and ngs of tumor tissue consisted of deep sequencing of all exons and selected introns of 275 ( preclinical pilot test ) , 341 ( version 1 ) , 410 ( version 2 ) , or 468 ( version 3 ) cancer - related genes . 
genotyping was performed using the pilot test for two of 96 samples ( 2% ) , version 2 for 16 of 96 samples ( 17% ) , version 3 for 57 of 96 samples ( 59% ) , and version 4 for 21 of 96 samples ( 22% )  . 
actionable genetic alterations were defined as changes that could guide therapy , including standard of care and investigational treatments ( annotated according to oncokb ) .18 germline sequencing germline variants from the .bam file of the normal dna sequence were called using the mutect and gatk haplotype caller as described previously ; all variants with < 1% population frequency in the exome aggregation consortium database were interpreted.19 , 20 germline analysis included 76 genes known to be associated with hereditary cancer predisposition , including all cancer predisposition genes identified in the american college of medical genetics and genomics guidelines.19 , 21 , 22 variants were interpreted on the basis of american college of medical genetics and genomics criteria . loss of heterozygosity loss of heterozygosity ( loh ) was determined through analysis of total , allele - specific , and integer dna copy number genome - wide using facets.23 statistical analysis descriptive statistics were used to characterize the study population and the somatic and germline genetic results . 
overall survival ( os ) , defined as the time from the date of first tissue acquisition ( from which ngs was completed ) until death or last known follow - up , was estimated by kaplan - meier methods . 
all p values were two sided , and p < .05 was considered to indicate statistical significance . results clinical characteristics as of march 2017 , ngs had been performed on 96 tumor samples from 80 patients ( eight patients with two samples analyzed ; four patients with three samples analyzed )  . 
in 29 patients , the primary tumor was sequenced , and in 58 patients , a metastatic tumor was sequenced ; in seven patients , both primary and metastases were sequenced ( in one instance , sequencing took place synchronously )  . 
recurrent somatic alterations occurred in key pathways and functional groups : chromatin remodeling factors ( crfs ) , histone methyltransferases ( hmts ) , and mtor pathway genes . somatic alterations in crfs ( daxx , atrx , pbrm1 , smarcb1 , arid5b , arid2 , arid1b , and arid1a ) were observed in 52 of 80 patients ( 65% )  . 
baseline patient characteristics ( continued ) wd pannets with msk - impact results systemic therapy somatostatin analogs ( octreotide long - acting release or lanreotide ) everolimus sunitinib platinum drug chemotherapy alkylating drug chemotherapy peptide receptor radionuclide therapy hepatic arterial embolization ( bland , radiotherapy , or chemotherapy ) 58 ( 73 ) 29 ( 36 ) 8 ( 10 ) 18 ( 23 ) 46 ( 58 ) 12 ( 15 ) 35 ( 44 ) note . 
 the clinical behavior of these tumors , including the single g1 tumor , was more aggressive . ten tumor samples ( collected from six patients ) harbored braf alterations : two of 10 g1 ( 20% ) , three of 10 g2 ( 30% ) , and five of 10 g3 ( 50% )  . 
 in addition to two tumors with v600e mutations , several non - v600 braf alterations were seen ( k601e , t599k , and t310i ) , and one tumor expressed both braf g596d and e451k mutations . survival with a median follow - up for survivors of 27.6 months , we observed 14 deaths ; median os was 118.9 months ( 95% ci , 99.6% - not achieved )  . 
tp53 alterations were observed in 10 of 80 patients ( 13% ) ; all were characterized as likely oncogenic . somatic loh in 50% of the genome was highly prevalent ; it was identified in 55 of 95 tested samples ( 58% )  . 
loh was observed recurrently in chromosomes 1 , 2 , 3 , 6 , 8 , 10 , 11 , 15 , 16 , 21 , and 22 ( appendix fig a1 )  . ngs results for the first sequenced sample in all patients are summarized in figure 1 . 
in the recurrently altered genes , there were no statistical differences in genetic spectrum on the basis of g1 versus g2 tumors , but a trend was noted of g2 and g3 tumors harboring tp53 , setd2 , and braf alterations . novel recurrently altered genes in this metastatic cohort , we observed a much higher frequency of setd2 alterations ( 14 of 80 patients [ 18% ] ) in comparison with previous investigations , and a novel finding of braf molecular mechanisms of resistance . 
genomic landscape of pancreatic neuroendocrine tumors , including the first sequenced sample for each patient ( n = 80 ) , as identified by the memorial sloan kettering - integrated mutation profiling of actionable cancer targets . 
 loh , loss of heterozygosity ; mb , megabyte ; mtor , mammalian target of rapamycin . post - treatment samples were available for three patients receiving the mtor inhibitor everolimus and for one patient receiving the braf v600e inhibitor vemurafenib . in all three patients receiving everolimus , mtor pathway alterations were identified at progression . 
on disease progression , a biopsy specimen revealed an oncogenic pten mutation ( q298 * ) , and the sampled areas of tumor progressed from g1 to g3 ( appendix figure a2 )  . 
this pten mutation was seen previously at a low allele frequency when the original pancreas tumor was sequenced , reflecting the possible emergence of this clone through targeted therapy . patient 2 received everolimus for 8 weeks , at which point treatment was held for infectious complications . 
a liver lesion increased in size and a biopsy was performed ; ngs identified a hotspot mutation in rheb ( y35s ) .25 subsequent sampling and ngs of this same liver lesion after 16 months of observation demonstrated the rheb alteration at a higher allele frequency . 
the msk - impact version used to sequence the pretreatment sample did not interrogate rheb , so the allele fraction of the rheb mutation is unknown in this sample ; however , it increased notably after treatment with andprogression on everolimus . patient 3 received everolimus for > 9 months before disease progression . 
at disease progression , ngs of a growing liver lesion demonstrated loss of this igf2 alteration , acquisition of a tsc2 splice site mutation ( x161_ splice ) , and an akt2 mutation ( g16d ; appendix figure a4 )  . 
surprisingly , no shared mutations were noted in sequencing of the two samples ; clinically , however , the disease was consistent with a single primary , suggesting the emergence of a previously unrecognized clone through treatment , although the possibility of a second primary cannot be excluded . patient 4 , whose tumor harbored a braf v600e mutation , enrolled in a basket clinical trial with vemurafenib and developed an nras hotspot mutation ( q61r ; appendix figure a5 ) .26 chemotherapy - associated mutagenesis . 
two patients received alkylating agents , and the third received oxaliplat a review of the tumors exposed to alkylating drugs showed that the variants shared by both samples were observed at a higher allele fraction compared with those unique to the individual samples , and the evolutionary pattern demonstrated a predominant c > t signature consistent with alkylating - agent associated mutagenesis ; in addition , consistent with previous observations , acquired alterations were observed in the mismatch repair genes for both tumors ( fig 3 ) .27 - 29 the sampled tumor in both patients increased from g1 to g3 . germline genetic testing germline testing for 76 cancer - associated genes was performed in 88 patients , 78 in the original prospective cohort and an additional 10 with surgically resected pannets . 
fourteen likely pathogenic germline alterations were identified in known cancer susceptibility genes ( 16% ) : three in established high - penetrance genes ( men1 , vhl , and tsc2 [ 3% ] ) , four in moderate - penetrance genes ( chek2 in four patients [ 5% ] ) , and seven in low - penetrance genes ( monoallelic mutyh in four patients and apc i1307k in three patients [ 8% ] )  . 
notably , we identified a germline tsc2 mutation in a 32 - year - old patient who did not exhibit the classic clinical features of tuberous sclerosis complex ( tsc ) and did not meet the criteria for tsc genetic testing ; magnetic resonance imaging of the brain identified tubers and radial bands , typical of tsc ( appendix figure a6 )  . 
among these patients , as best therapy response , two of 12 ( 17% ) had tumor regression , six of 12 ( 50% ) had stable disease , and one of 12 ( 8% ) had progressive disease ; three of 12 ( 25% ) are still receiving treatment . 
in the remaining 46 patients without somatic mtor pathway alterations , 17 of 46 ( 37% ) received everolimus ; five of 17 ( 29% ) had tumor regression , seven of 17 ( 41% ) had stable disease , four of 17 ( 24% ) had progressive disease , and there was an unknown response in one patient ( lost to follow - up )  . 
results from ngs led to the enrollment of three patients into clinical trials , but with little response noted . to our knowledge , this is the first study to evaluate the real - time clinical usefulness of ngs for patients with advanced , metastatic pannets . 
setd2 alterations have been implicated in the pathogenesis of many cancers ( breast , lung , acute lymphoblastic leukemia , renal cell carcinoma , and gliomas ) , supporting a role for setd2 as a tumor suppressor across many diseases.34 - 41 given our findings , one might consider closer monitoring and / or cytotoxic therapies in patients with setd2or table 3 . 
levels 1 and 2a include food and drug administration ( fda ) - recognized and standardof - care biomarkers predictive of response to an fda - approved drug for pancreatic neuroendocrine tumors . 
level 2b includes fda - approved biomarkers predictive of drug response in another cancer , but not fda or national comprehensive cancer network - compendium listed for pancreatic neuroendocrine tumors . 
level 3a includes alterations for which clinical evidence links the biomarker to drug response for pancreatic neuroendocrine tumors , but neither the biomarker nor the drug are standard of care in any cancer type . 
level 3b includes alterations for which clinical evidence links the biomarker to drug response for other cancers , but neither the biomarker nor the drug are standard of care in any cancer type . 
level 4 includes alterations in which preclinical evidence potentially links the biomarker to drug response , but neither the biomarker nor the drug are standard of care in any cancer type . abbreviation : msk - impact , memorial sloan kettering - integrated mutation profiling of actionable cancer targets . high penetrance high penetrance high penetrance moderate penetrance moderate penetrance moderate penetrance low penetrance low penetrance low penetrance recessive allele recessive allele braf - altered tumors and a higher burden of disease earlier in the treatment course . our study identifies pertubations of biologic pathways in metastatic pannets that were found in previous whole - exome and whole genome studies evaluating mostly localized pannets.5 , 6 we observed alterations largely along three pathways : crfs , hmts , and mtor . 
our study clarifies pannet genetics exclusively in the metastatic cohort , the group which gains immediate clinical value from realtime sequencing as we make steps toward precision medicine . in addition , we found that 16% of patients harbored a germline pathogenic or likely pathogenic alteration in high - , moderate - , or low - penetrance cancer predisposition genes . 
in looking at those patients found to have mutations in low or moderate - penetrance genes , it is notable that both chek2 and monoallaleic mutyh were noted in previous germline assessments of patients with pannets.6 notably , the patient in our series with a tsc2 germline mutation did not meet the clinical criteria for tsc genetic testing ; the identification of this mutation was incidental . 
 because mutations in men1 and tsc2 are two of the most common somatic findings in pannets , our finding of germline mutations within these two high - penetrance genes demonstrates the importance of these genes in pannet pathogenesis . 
although the contribution of the other germline alterations to pannet susceptibility has yet to be elucidated , our data suggest that there is likely a larger - than - anticipated germline contribution in this disease . 
given the lack of predictable phenotypic findings in most highand moderate - risk mutation carriers , combined with the relative rarity of this tumor subtype , consideration of genetic risk assessment for all patients with pannets is not unreasonable . 
such germline findings would have a profound impact on long - term cancer surveillance and cascade genetic testing of at - risk family members . in a small number of patients , somatic mutational signatures guided therapy . 
we identified somatic mtor alterations in a much greater proportion of patients ( 43% ) than described previously , but the presence or absence of an mtor pathway mutation was not predictive of response or resistance to treatment.8 our numbers are small , however , so additional work is needed to clarify the role of mtor pathway alterations in predicting response and / or resistance to targeted mtor pathway inhibitors . our series included only patients with wd tumors , and consistent with previous findings , rb1 alterations were essentially absent and were observed in only one post - treatment sample exposed to chemotherapy.43 , 44 wd pannets are often thought of as cancer in slower motion , and we have used our sequencing efforts to help clarify pannet evolutionary patterns over time and through therapy . 
in the 12 patients who underwent ngs of tumor tissue at multiple time points , we noted increased tumor grade in eight patients ( 67% ) , associated with marked genetic evolution after treatment . 
to our knowledge , we are the first to observe this evolutionary pattern in pannets.27 - 29 in conclusion , comprehensive molecular profiling of pannet samples using archival tissue is feasible . 
in a disease in which the natural history is thought to be indolent , we have demonstrated that the cancer transforms with grade and genetic progression over time and as therapy progresses . 
the practical usefulness of this analysis remains limited at this time ; our data suggest a higher - than - expected germline mutation rate , and consideration of genetic risk assessment for all patients with pannets is reasonable . 
in addition , given the heterogeneity of the disease , rapid , readily available , and clinically feasible ngs should be considered in clinical trials to help stratify these patients . 
specifically , our numbers are too small to clarify the genetic predictors of response or resistance to commonly used therapies in pannets , the prognostic effects of the identified genetic alterations , or the patterns of genetic evolution ( because only 12 patients underwent sequencing at multiple time points )  . 
klimstra , diane reidy - lagunes financial support : nitya raj , diane reidy - lagunes administrative support : nitya raj , joanne chou , virginia kelly , diane reidy - lagunes provision of study material or patients : nitya raj , leonard b . 
 data analysis and interpretation : nitya raj , ronak shah , zsofia stadler , semanti mukherjee , joanne chou , brian untch , janet li , muyinat osoba , leonard b . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . nitya raj research funding : novartis ( inst ) ronak shah no relationship to disclose leonard b . 
saltz consulting or advisory role : mcneil ppc ( i ) research funding : taiho pharmaceutical diana mandelker no relationship to disclose marc ladanyi honoraria : merck ( i ) consulting or advisory role : nccn / boehringer ingelheim afatinib targeted therapy advisory committee , national comprehensive cancer network / astrazeneca tagrisso request for proposal advisory committee research funding : loxo ( inst ) michael f . 
berger research funding : illumina zsofia stadler consulting or advisory role : allergan ( i ) ; genentech ( i ) , regeneron pharmaceuticals ( i ) , optos ( i ) , adverum ( i ) david s . 
klimstra stock and other ownership interests : paige.ai consulting or advisory role : wren laboratories , ipsen semanti mukherjee employment : regeneron pharmaceuticals stock and other ownership interests : regeneron pharmaceuticals research funding : regeneron pharmaceuticals diane reidy - lagunes honoraria : novartis consulting or advisory role : ipsen , pfizer , novartis research funding : novartis acknowledgment the authors thank the patients who kindly agreed to participate in this research effort . joanne chou no relationship to disclose brian untch no relationship to disclose support prior presentation affiliations nitya raj , ronak shah , zsofia stadler , semanti mukherjee , joanne chou , brian untch , janet li , virginia kelly , muyinat osoba , leonard b . 
presented , in part , as poster presentations at the 2015 gastrointestinal cancers symposium ( san francisco , ca , january 15 - 17 , 2015 ) and 2016 gastrointestinal cancers symposium ( san francisco , ca , january 21 - 23 , 2016 )  . 
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walter dm , venancio os , buza el , et al : systematic in vivo inactivation of chromatin - regulating enzymes identifies setd2 as a potent tumor suppressor in lung adenocarcinoma . 
tang lh , basturk o , sue jj , et al : a practical approach to the classification of who grade 3 ( g3 ) well - differentiated neuroendocrine tumor ( wd - net ) and poorly differentiated neuroendocrine carcinoma ( pd - nec ) of the pancreas . 
yachida s , vakiani e , white cm , et al : small cell and large cell neuroendocrine carcinomas of the pancreas are genetically similar and distinct from well - differentiated pancreatic neuroendocrine tumors . 
pathology demonstrated metastatic colloid / mucinous adenocarcinoma ( stage pt4a , pn2b , pm1c )  . immunohistologic testing for dna mismatch repair ( mmr ) proteins on tissue biopsy specimen showed preservation of muts protein homolog 2 ( msh2 ) and muts homolog 6 ( msh6 ) , but loss of mutl homolog 1 ( mlh1 ) and mismatch repair endonuclease pms2 ( pms2 ) , consistent with mmr deciency . 
initially , the patient was treated with four doses of uorouracil , leucovorin , oxaliplatin , and irinotecan ( folfoxiri ) until computed tomography scanning revealed an increase in peritoneal masses and liver metastases . 
he then was treated with pembrolizumab on the basis of mmr deciency ; however , this was discontinued after three doses when scans revealed progressive disease , and a doubling of carcinoembryonic antigen since initiation was noted ( 401 ng / ml to 848 ng / ml )  . 
all patient information was curated under an institutional review board protocol ( mcc 19161 ) , which was formally reviewed and granted approval by moftt cancer center ( mcc )  . 
this institutional review board protocol includes a waiver of consent , because no identifying information is reported in the case . a foundationone cdx test ( foundation medicine , cambridge , ma ) was ordered on the diagnosed omental tissue with subsequent review by the personalized medicine clinical service ( pmcs ) at mcc for guidance regarding future therapeutic options ( table 1 )  . 
the molecular tumor board ( mtb ) discussion focused on approaches for targeted therapeutic considerations in a patient with high tumor mutation burden ( tmb ) after disease progression on an immune checkpoint inhibitor . the patient had 93 reported alterations ; the foundationone cdx report provided interpretive content for 26 of them for correlated therapies with clinical benet , clinical trials , or therapies with a lack of response . 
in a patient with high tmb status and microsatellite instability ( msi ) , the likelihood of an individual alteration primarily driving the cancer is difcult to ascertathe pmcs categorized alterations into common repair / signaling pathways , associating the alterations with possible therapies and ranking therapeutic options on the available evidence ( table 1 )  . 
figure 1 and appendix table a1 illustrate the workow taken by the pmcs to identify and prioritize treatment options . of the 26 alterations for which foundationone cdx provided interpretive content , impaired homologous repair ( hr ) was a commonly altered pathway . 
highlight alterations with targeted therapies tmb : high ( 105 muts / mb ) microsatellite status : unstable kras a59t atm r2227c brca2 n319fs * 8 nf1 i679fs * 21 ptch1 r1308fs * 64 akt1 e17k msh6 f1088fs * 2 men1 r612h mtor a469t apc h298fs * 28 apc r1114 * apc v2194fs * 5 arid1a g889fs * 2 asxl1 g646fs * 12 bcorl1 p1681fs * 20 ctcf t204fs * 18 keap1 f478fs * 2 mll2 p2354fs * 30 notch3 a1701v notch3 g2035fs * 60 notch3 p1317fs * 103 pbrm1 w992fs * 23 ptpn11 s502p qki k134fs * 14 setd2 f1650v sox9 v306fs * 77 pold1 r17q flt1 s733del 66 vus 2 . 
categorize by mechanism tmb - high msh6 arid1a brca2 akt1 kras * nf1 * mmr deficiency mmr deficiency mmr deficiency hr deficiency hr deficiency hr deficiency akt activation ras activation ras activation 3 . 
correlate mechanisms to therapies parp or atr inhibitor second line of immunotherapy ( combination therapy or novel agent on trial ) pi3k - akt - mtor inhibitor arid1a brca2 tmb - high msh6 arid1a brca2 akt1 4 . 
 ( 3 ) correlating grouped repair / signaling mechanisms from step 2 with possible targeted therapy options assists collecting and correlating supporting evidence to be considered by a molecular tumor board ( mtb )  . 
 ( 4 ) the mtb reviewed the evidence and prioritized therapy options according to the patients current disease state , current failed line of therapy , patient preference , strength of evidence associated with an alteration and therapy option , and available clinical trials . 
nct03188965 ( first - in - human study of atr inhibitor bay1895344 in patients with advanced solid tumors and lymphomas ) was recruiting patients with atm alterations and was further supported by clinical , in vivo , and in vitro data . 
 ( * ) the kras a59t and nf1 i679fs * 21 alterations produced conicting likelihoods of response to epidermal growth factor receptor ( egfr ) inhibitors and were based on minimal case report data . 
 nelson et al ( parp ) inhibitors or immunotherapy.2 , 3 parp inhibition in the context of brca alterations has demonstrated clinical benet in breast , ovarian , and prostate cancer and has produced discussion of expansion to other ddr deciencyrelated alterations.4 atm encodes a protein kinase that plays a key role in dna repair , cell cycle checkpoints , and apoptosis in response to dna damage.5 the atm r2227c alteration is clinically relevant , because it produces a missense alteration in the frap - atm - trrap ( fat ) domain , has been categorized as deleterious in breast cancer and adenoid cystic carcinoma , and is classied as pathogenic by clinvar ( clinvar / )  . 
in a phase ii trial of patients with ddr - defective metastatic castrate - resistant prostate cancer receiving olaparib , patients with germline or somatic alterations in genes involved in ddr showed signicantly increased activity , with responses occurring in 14 out of 16 patients ( 88% ) .6 , 7 the at - rich interactive domain - containing protein 1a ( arid1a ) g889fs * 2 alteration is likely inactivating ( catalogue of somatic mutations in cancer mutation id : cosm6911235 )  . arid1a is a subunit of the chromatin remodeling complex swi / snf that is involved in facilitating access of proteins to dna , and inactivating alterations are common across various cancer types.8 pathways involved in ddr have been shown to interact with arid1a , including msh2 . 
during dna replication , it has been demonstrated that arid1a recruits msh2 to chromatin and promotes mmr.8 thus , inactivation of arid1a has been shown to impair mmr . arid1a deciency has demonstrated sensitization to parp inhibitors in vitro and in vivo.9 atr , another important genetic component of ddr , has been the target of novel investigational drugs that inhibit atr and arid1a signaling in vitro and in vivo.5 , 10 collectively , arid1a , atm , and brca2 produce a common theme of ddr / hr deciency , which leads to atr or parp inhibition alone or in combination with platinum or another dna - damaging chemotherapy agent in an off - label or ( more preferred ) clinical trial setting were recommended as viable options . akt1 e17k has been reported as a missense alteration that occurs in the pleckstrin homology domain of rac - alpha serine / threonine - protein kinase ( akt1 ) and activates akt signaling leading to inhibition of cell apoptosis and promotion of cellular proliferation.11 this alteration is therefore considered oncogenic and is well described in the literature . 
in a phase i , dose - escalation trial of the pan - akt inhibitor azd5363 , 58 patients with solid tumor found to have somatic akt1 e17k alterations were enrolled . 
the median progression - free survival was 5.5 months for patients with estrogen - positive breast cancer , 6.6 months for patients with gynecologic disease , and 4.2 months for patients with other solid tumors , including two patients with colorectal cancer ( crc ) .12 however , in a patient with a high tmb and msi , the value of this akt1 e17k mutation as a clinically relevant driver is unclear . 
in case the clinical team decided to target akt , clinical trials , including nct02465060 ( nci - match - arm z1k ) , nct03310541 , or nct02761694 , could be considered . finally , the kras a59t alteration is less common than other known activating kras alterations but has been reported as resulting in increased activation of the map kinase and pi3k pathways in vivo.13 interestingly , this alteration may demonstrate an exception to the observation that patients with kras - mutated metastatic crc do not receptor respond to epidermal growth factor ( egfr ) targeted monoclonal antibodies cetuximab or panitumumab . 
there is one case report of a patient with metastatic crc and the kras a59t alteration who was treated with panitumumab plus uorouracil , leucovorin , and irinotecan and experienced a partial radiographic response ( 36% decrease per response evaluation criteria in solid tumors 1.1 ) , stable carcinoembryonic antigen biomarker , and decreased size of metastases for an 8 - month duration.14 the patient showed no other alterations in exons 2 and 3 of kras , nras , and hras genes ; exon 15 of braf ; and exons 2 , 5 , 9 , 10 , 20 , and 21 of pik3ca . this seems to be the rst observed documentation of the a59t allele not producing resistance to egfr - targeting monoclonal antibodies and may provide a potential future consideration for our patient.14 however , there is limited evidence that the nf1 frame shift may predict poorer response to cetuximab or panitumumab therapy on the basis of a small case series of four chinese patients with metastatic crc who were kras g12 and g13 wt but found to have inactivating nf1 alterations . 
the alterations involved in hr deciency ( brca2 , atm , and arid1a inactivation ) provided the most robust combined evidence of predicted response to future therapy options . the akt1 e17k alteration is known to be activating , but its importance in a highly mutated , unstable tumor was less well dened . 
 therapy option prioritization in crc with high mutation burden follow - up of the patients case , unfortunately , showed that after three treatments of pembrolizumab , there was no sign of response , which led the clinical team to refer back to the above options to consider treatment beyond immunotherapy . 
the clinical team discussed the predominant grouping of alterations ( arid1a , atm , and brca2 ) with the common theme of ddr / hr deciency and ultimately decided to enroll the patient in a clinical trial ( clinicaltrials.gov identier : nct03188965 ) investigating the atr inhibitor bay1895344 in patients with solid tumor with atm loss . the lesson learned with this patient was one of categorizing a large list of alterations and prioritizing each according to therapeutic targets on the basis of dominant signaling / repair pathways and strength of available evidence . 
in doing so , the pmcs was able to provide the treating physician with a succinct , prioritized grouping of alterations validating the initial line of therapy and considerations for future therapeutic options , which led to the patient being enrolled in a clinical trial with the greatest anticipated therapeutic benet . 
walko , pharmd , 12902 magnolia dr , tampa , fl 33612 ; twitter : @mofttnews ; e - mail : christine.walko@moftt.org. support supported in part by national cancer institute comprehensive cancer center support grant no . 
mcleod leadership : cancer genetics stock and other ownership interests : cancer genetics , interpares biomedicine honoraria : genentech , illumina consulting or advisory role : gentris , cancer genetics , saladax biomedical , national institutes of health / national cancer institute , admera health , evicore healthcare , pharmazam speakers bureau : genentech christine m . 
walko employment : mission healthcare stock and other ownership interests : alexion pharmaceuticals ( i ) consulting or advisory role : foundation medicine , jackson laboratory for genomic medicine , intermountain precision genomics other relationship : mission hospital no other potential conicts of interest were reported . references cerniglia m , xiu j , grothey a , et al : association of dna damage response and repair genes ( ddr ) mutations and microsatellite instability ( msi ) , pd - l1 expression , tumor mutational burden ( tmb ) in gastroesophageal cancers . 
j clin oncol 37 , 2019 ( suppl 4 ; abstr 60 ) overman mj , mcdermott r , leach jl , et al : nivolumab in patients with metastatic dna mismatch repair - decient or microsatellite instability - high colorectal cancer ( checkmate 142 ) : an open - label , multicentre , phase 2 study . 
lancet oncol 18 : 1182 - 1191 , 2017 ghiringhelli f , richard c , chevrier s , et al : efciency of olaparib in colorectal cancer patients with an alteration of the homologous repair proteworld j gastroenterol 22 : 10680 - 10686 , 2016 pili e pg , gay cm , byers la , et al : parp inhibitors : extending benet beyond brca - mutant cancers . 
clin cancer res 25 : 3759 - 3771 , 2019 shiloh y , ziv y : the atm protein kinase : regulating the cellular response to genotoxic stress , and more . 
n engl j med 373 : 1697 - 1708 , 2015 shen j , ju z , zhao w , et al : arid1a deciency promotes mutability and potentiates therapeutic antitumor immunity unleashed by immune checkpoint blockade . 
nat med 24 : 556 - 562 , 2018 shen j , peng y , wei l , et al : arid1a deciency impairs the dna damage checkpoint and sensitizes cells to parp inhibitors . 
banerji u , dean ej , p erez - fidalgo ja , et al : a phase i open - label study to identify a dosing regimen of the pan - akt inhibitor azd5363 for evaluation in solid tumors and in pik3ca - mutated breast and gynecologic cancers . 
mei z , shao yw , lin p , et al : smad4 and nf1 mutations as potential biomarkers for poor prognosis to cetuximab - based therapy in chinese metastatic colorectal cancer patients . 
ninety - three alterations were reported on the foundationone cdx report ( including variants of known and unknown signicance )  . twenty - six of the alterations had interpretive content for correlated therapies with clinical benet , clinical trials , or therapies with a lack of response . 
twenty - seven alterations had potentially relevant information ( maf , listed in exac , protein domain location , in oncokb and others ) as reported in the initial screening of ndings . 
 c not so typical : development of atypical chronic myeloid leukemia in a patient with chronic myeloid leukemia introduction chronic myeloid leukemia ( cml ) is a myeloproliferative neoplasm characterized by the uncontrolled production and proliferation of mature well - differentiated granulocytes . 
the hallmark of cml is the translocation of the abelson murine leukemia ( abl1 ) gene on chromosome 9 and the breakpoint cluster region ( bcr ) gene on chromosome 22 ( bcr - abl ) , which results in the abnormal chromosome 22 , the philadelphia ( ph ) chromosome . 
since the approval of imatinib in 2001 , 1 cml has been treated with tyrosine kinase inhibitors ( tkis ) , which has improved the survival of patients with cml to close to that of the normal population.2 although cml itself has been associated with an increased risk of secondary primary malignancies ( spms ) suggestive of an inherent stemcell defect , treatment has been implicated in this phenomenon as well.3 the data on the risk for spms among patients treated with tkis have been conflicting.3 - 6 this discrepancy may be due to the limiting retrospective nature of these studies , heterogeneity across patients , and possible lack of a significant effect . 
however , it is interesting to note that most of the reported spms were solid malignancies , with few cases of chronic monomyelocytic leukemia , lymphoid neoplasms , and plasma cell neoplasms . 
she achieved major molecular response at 30 months and complete molecular response ( > 4 log reduction ) at 40 months , and she had remained in hematologic , cytogenetic , and molecular remission since 2007 with no additional karyotypic abnormalities . in october of 2016 , the patient presented with progressive fatigue . 
she was diagnosed with acml on the basis of the who criteria of prominent dysplastic granulopoiesis , > 10% immature myeloid cells in peripheral blood , and no increase in absolute monocytes . 
bm was sent for next - generation sequencing ( performed at genoptix , carlsbad , ca ) to interrogate 40 genes that are known to be recurrently mutated in myeloid malignancies . 
mutational analysis showed alterations in setbp1d868n ( 49% variant allele frequency ) , nrasg12d ( 19% ) , cblc396y ( 17% ) , runx1g12d ( 47% ) , and gata2r396q ( 47% )  . 
no plans for allogenic hematopoietic cell transplantation were made because of her age and frailty . discussion to our knowledge , this is the first case report of acml occurring in a patient with cml who is in complete remission receiving long - term imatinib therapy . 
secondary hematologic neoplasms reported in the previous studies occurred primarily in older patients , suggesting that a possible antecedent clonal hematopoiesis of indeterminate potential ( chip ) progressed while cml was well controlled on tkis.2 , 10 , 11 recent genomic studies have demonstrated the clonal diversity in the evolution of cml and showed that the mutational persistence does not alter the response to tkis . 
these mutations are predominantly in epigenetic machinery ( tet2 , asxl1 , and dnmt3a ) that are commonly attributed to the development of chip.12 a recent report by kim et al10 detected a spectrum of mutations in patients with cml after tki therapy . 
another theory is that the bcr - abl translocation is an acquired event on top of preexisting chip mutations , thereby converting ph - negative clones into ph - positive clones that lead to cml . 
these ph - positive clones are eliminated by tkis , and the ph - negative clones gain additional mutations such as setbp1 to progress to acml.12 on the basis of large population - based studies , chip is seen at an incidence of 10% to 20% in persons age 70 to 80 years.13 unfortunately , our patient did not have prior sequencing performed that could have identified common chip mutations . focused gene sequencing in this case identified gene mutations that support the diagnosis of acml . 
somatic mutations of genes associated with proliferation signaling are frequently mutated in myeloproliferative neoplasms ; setbp1 is involved in dna replication , cbl encodes e3 ubiquitin - protein ligase , nras is involved in growth factor signaling , and runx1 encodes core binding factor and is involved in normal hematopoiesis.14 acml specifically is associated with frequent setbp1 and nras mutations.15 , 16 furthermore , it is distinguished from chronic neutrophilic leukemia by the absent to rare finding of jak2 and csf3r mutations.17 , 18 morgana is another important factor in the pathogenesis of acml . 
interestingly , low levels of morgana were also found in 16% of patients with typical cml and correlated with a poor response to imatinib.20 testing for this specific gene is not widely available and could not be performed for this patient . although there have been reports of rare pedigrees with genetic predisposition to cml , 21 , 22 the literature does not support any familial aggregation in the general population.23 likewise , family history in our patient was noncontributory , and dna germline sequencing was not deemed necessary or cost effective . 
the median survival of patients with acml is 25 months , with progression to acute myeloid leukemia in 40% of patients at a median duration of 18 months.28 leukocytosis , female sex , and older age have been shown to be adverse prognostic factors for overall survival in multiple studies.16 , 25 , 28 in summary , we present here a patient with cml in the chronic phase who responded to tki therapy for nearly 13 years before developing bcr - ablnegative disease that best fits the diagnosis of acml . 
nras and cbl have also been shown to be drivers of malignant transformation and probably occurred as later events.24 setbp1 mutations are associated with leukocytosis , anemia , and thrombocytopenia at presentation and are often associated with cbl in acml . 
presence of setbp1 mutations in patients with acml is associated with worse overall survival in one study15 but not another.25 other mutations ( ie , tet2 , asxl1 ) have been described in acml and can also be seen in healthy individuals26 , 27 but were not detected in our patient . 
more importantly , next - generation sequencing was not routinely done at the time of the patients initial diagnosis , nor is it currently the standard of care at diagnosis . 
druker bj , tamura s , buchdunger e , et al : effects of a selective inhibitor of the abl tyrosine kinase on the growth of bcr - abl positive cells . 
voglova j , muzik j , faber e , et al : incidence of second malignancies during treatment of chronic myeloid leukemia with tyrosine kinase inhibitors in the czech republic and slovakia . 
verma d , kantarjian h , strom ss , et al : malignancies occurring during therapy with tyrosine kinase inhibitors ( tkis ) for chronic myeloid leukemia ( cml ) and other hematologic malignancies . 
au wy , ma sk , kwong yl : the occurrence of philadelphia chromosome ( ph ) negative leukemia after hematopoietic stem cell transplantation for ph positive chronic myeloid leukemia : implications for disease monitoring and treatment . 
miranda mb , lauseker m , kraus mp , et al : secondary malignancies in chronic myeloid leukemia patients after imatinib - based treatment : long - term observation in cml study iv . 
schmidt m , rinke j , schfer v , et al : molecular - defined clonal evolution in patients with chronic myeloid leukemia independent of the bcr - abl status . 
meggendorfer m , bacher u , alpermann t , et al : setbp1 mutations occur in 9% of mds / mpn and in 4% of mpn cases and are strongly associated with atypical cml , monosomy 7 , isochromosome i ( 17 ) ( q10 ) , asxl1 and cbl mutations . 
patnaik mm , barraco d , lasho tl , et al : targeted next generation sequencing and identification of risk factors in world health organization defined atypical chronic myeloid leukemia . 
 palbociclib for the treatment of metastatic nasopharyngeal carcinoma with cdk4 amplication : a case report xiao - dong jiao , md1 ; ke liu , md1 ; bao - dong qin , md , phd1 ; ying wu , md1 ; ming - qin lin , md1 ; jun liu , md1 ; xi he , md1 ; junjun liu , md2 ; han han - zhang , phd2 ; jianxing xiang , phd2 ; hao liu , ms2 ; and yuan - sheng zang , md , phd1 introduction nasopharyngeal carcinoma ( npc ) , which causes approximately 65 , 000 deaths annually , is an epithelial malignancy with unique geographic distribution . 
although rare in western countries , its incidence exceeds 20 cases per 100 , 000 people in the endemic area , including in southern china , southeast asia , and the middle east / north africa.1 , 2 factors that contribute to the pathogenesis of npc include but are not limited to epstein - barr virus infection , host genetics , and environmental factors . 
although optimal local control has been achieved and the overall prognosis has significantly improved in recent years as a result of the development of intensity - modulated radiotherapy and the adoption of concurrent chemotherapy , 3 , 4 distant metastasis remains a major challenge in the management of npc . 
during radiotherapy , rapid onset of a bilateral sensorineural hearing loss occurred but did not progress beyond moderate impairment . complete remission was achieved after the completion of radiotherapy ; however , 6 months later , follow - up positron emission tomography / computed tomography scan revealed multiple hypermetabolic nodules of the bilateral lung , indicating recurrence . 
he was subsequently treated with nimotuzumab and unfortunately experienced disease progression with an enlarged mass with a total diameter of 59.1 mm after 6 weeks ( fig 2a )  . 
this revealed cdk4 amplication ( copy number , 6.62 ) , among other mutations with no targeted therapy available , including copy number variations in kdm5a , etv6 , nfe2l2 , mdm2 , daxx , pnrc1 , tiparp , and tnfaip3 , as well as single - nucleotide variations in nfkbia , pik3cg , bap1 , and rad5 . 
the patient subsequently began palbociclib 125 mg / d on june 12 , 2017 , for 14 consecutive days followed by 7 days off therapy , comprising a complete cycle of 21 days . 
he experienced grade 3 neutropenia , thrombocytopenia , and myelosuppression , which resulted in interruptions of the treatment two times , a total of 14 days during the rst four cycles . 
 jiao et al capture - based targeted sequencing was performed on biopsy of lung tumor tissue and revealed mutations in cdkn2a / 2b , ccnd1 , cyld , bap1 , arid5b , pik3cg , notch1 , and mdm4 . 
the patient was subsequently switched to ribociclib . discussion in this case study , we have presented clinical evidence of palbociclib in an elderly patient with metastatic npc , highlighting the potential of cdk4 inhibitors for the treatment of metastatic npc , a devastating condition with median overall survival that ranges from 5 months to 11 months.20 , 21 currently , combined chemoradiotherapy is the predominant treatment strategy for patients with metastatic disease , but benets are surprisingly modest.21 - 24 as a result of the lack of comprehensive understanding of the molecular pathogenesis associated with npc , targeted therapy is relatively underdeveloped . 
to our knowledge , there is only one other study reporting the efcacy of palbociclib as a salvage treatment in a patient with metastatic ccnd1 - amplied npc who achieved stable disease as a best response with a pfs of 4 months.19 numerous lines of evidence from both in vitro and in vivo perspectives , as well as from the clinical setting , have demonstrated the potential of cdk4 inhibitors for treating npc . 
at the protein level , rb , cyclin d1 , and its regulator cdk4 are overexpressed in 90.6% , 92% , and 71% of respectively.26 numerous studies have npc tissues , demonstrated that npc cell lines and patient - derived xenografts that harbor mutations in the cdk4 pathway are sensitive to cdk4 / 6 inhibitors.19 , 27 in addition , some studies have shown the prognostic value of cdk4 overexpression.28 it has been reported that the serum level of cdk4 is signicantly elevated in patients with lung cancer and head and neck squamous cell carcinoma and is associated with a more aggressive clinical behavior and shorter survival , which suggests its potential as a biomarker in clinical practice.28 in addition , other studies have shown that cdk4 overexpression is an independent prognostic indicator of distal metastasis and local recurrence.14 available data support the idea that amplication is primarily responsible for cdk4 overexpression . in conclusion , we have reported on an elderly patient with cdk4 - amplied npc who was treated with palbociclib and achieved pr with a pfs of 15 months . 
what is the most clinically relevant biomarker for palbociclib ? what is the most appropriate way to select patients for this treatment ? the patient provided written informed consent and gave permission for the use of biopsies and the publication of case details . 
the patient achieved partial remission ( pr ) with considerable shrinkage of lung lesions , which measured 40.5 mm after 6 months of treatment . disease remained as pr until august 2018 when a ct scan revealed enlarged lesions of bilateral lung measuring 64.4 mm , accompanied by the development of malignant pleural effusion with a progression - free survival ( pfs ) of 15 months ( figs 2b and 2c and 3 )  . 
 ( a ) before the initiation of palbociclib , enlarged masses with a total diameter of 59.1 m ( b ) at partial response , a signicant lesions was observed , measuring 40.5 mm after shrinkage of 6 months of palbociclib treatment . 
lancet 387 : 1012 - 1024 , 2016 paul p , deka h , malakar ak , et al : nasopharyngeal carcinoma : understanding its molecular biology at a ne scale . 
 jiao et al pan c - c , lu j , yu j - r , et al : challenges in the modication of the m1 stage of the tnm staging system for nasopharyngeal carcinoma : a study of 1027 cases and review of the literature . 
exp ther med 4 : 334 - 338 , 2012 leung tw , tung sy , sze wk , et al : treatment results of 1070 patients with nasopharyngeal carcinoma : an analysis of survival and failure patterns . 
head neck 27 : 555 - 565 , 2005 lee aw , sze wm , au js , et al : treatment results for nasopharyngeal carcinoma in the modern era : the hong kong experience . 
int j radiat oncol biol phys 61 : 1107 - 1116 , 2005 baujat b , audry h , bourhis j , et al : chemotherapy in locally advanced nasopharyngeal carcinoma : an individual patient data meta - analysis of eight randomized trials and 1753 patients . 
int j radiat oncol biol phys 64 : 47 - 56 , 2006 sherr cj , beach d , shapiro gi : targeting cdk4 and cdk6 : from discovery to therapy . 
cancer res 65 : 854 , 2005 ( suppl ) jiang q , mai c , yang h , et al : nuclear expression of cdk4 correlates with disease progression and poor prognosis in human nasopharyngeal carcinoma . histopathology 64 : 722 - 730 , 2014 14 . 
chen t - j , lee s - w , lin l - c , et al : cyclin - dependent kinase 4 overexpression is mostly independent of gene amplication and constitutes an independent prognosticator for nasopharyngeal carcinoma . 
rugo hs , di eras v , gelmon ka , et al : impact of palbociclib plus letrozole on patient - reported health - related quality of life : results from the paloma - 2 trial . 
hsu c - l , lui k - w , chi l - m , et al : integrated genomic analyses in pdx model reveal a cyclin - dependent kinase inhibitor palbociclib as a novel candidate drug for nasopharyngeal carcinoma . 
zhang l , huang y , hong s , et al : gemcitabine plus cisplatin versus uorouracil plus cisplatin in recurrent or metastatic nasopharyngeal carcinoma : a multicentre , randomised , open - label , phase 3 trial . 
lancet 388 : 1883 - 1892 , 2016 jin y , shi yx , cai xy , et al : comparison of ve cisplatin - based regimens frequently used as the rst - line protocols in metastatic nasopharyngeal carcinoma . j cancer res clin oncol 138 : 1717 - 1725 , 2012 [ erratum : j cancer res clin oncol 141 : 767 , 2015 ] 23 . 
chen l , hu c - s , chen x - z , et al : concurrent chemoradiotherapy plus adjuvant chemotherapy versus concurrent chemoradiotherapy alone in patients with locoregionally advanced nasopharyngeal carcinoma : a phase 3 multicentre randomised controlled trial . 
tan t , lim w - t , fong k - w , et al : concurrent chemo - radiation with or without induction gemcitabine , carboplatin , and paclitaxel : a randomized , phase 2 / 3 trial in locally advanced nasopharyngeal carcinoma . 
hwang c - f , cho c - l , huang c - c , et al : loss of cyclin d1 and p16 expression correlates with local recurrence in nasopharyngeal carcinoma following radiotherapy . 
zhang w , zeng z , zhou y , et al : identication of aberrant cell cycle regulation in epstein - barr virus - associated nasopharyngeal carcinoma by cdna microarray and gene set enrichment analysis . 
wong c - h , ma bby , hui cwc , et al : preclinical evaluation of ribociclib and its synergistic effect in combination with alpelisib in non - keratinizing nasopharyngeal carcinoma . 
bliss , msc1 ; ian smith , md2 ; and mitch dowsett , phd1 , 2 on behalf of the poetic trial management group and trialists purpose although aromatase inhibitor ( ai ) treatment is effective in estrogen receptorpositive postmenopausal breast cancer , resistance is common and incompletely explained . 
scnas to specic genes may drive intrinsic resistance , or high genomic instability may drive tumor heterogeneity , which allows differential response across tumors and surviving cells to evolve resistance to treatment rapidly . 
fifty - six samples from the ai group included 28 poor responders ( prrs , less than 60% reduction in protein encoded by the mki67 gene [ ki - 67 ] ) and 28 good responders ( gdrs , greater than 75% reduction in ki - 67 )  . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction estrogen deprivation is the major treatment strategy for hormone - dependent breast cancer ( bc ) and typically involves agents that inhibit aromatase , the enzyme that catalyzes the conversion of androgens to estrogens . despite near - complete suppression of circulating estrogen levels by aromatase inhibitor ( ai ) treatment , acquired and de novo resistance to ai is common.1 few pretreatment biomarkers exist for ai resistance , and mechanisms of resistance are incompletely understood.2 mutations and somatic copy number alterations ( scnas ) can play important roles in activating oncogenes or inactivating tumor suppressors , and bc is characterized by multiple recurrent scnas and few recurrent mutations.3 we have previously shown that tp53 mutations ( tp53mut ) occur at a higher rate in tumors with poor response to ai treatment , which suggests that these patients received less benet from ai4 but that scnas to specic genes also may play an important role in ai resistance.5 nonspecic genomic alterations , like high genomic instability , are known to be associated with poor prognosis and probably at least partly the result of tumor heterogeneity , which allows some cells to survive and evolve resistance to treatment.6 there is evidence in other solid tumors of an association between high genomic instability and intrinsic resistance to chemotherapy.7 however , few studies of genomic instability and response to endocrine treatment exist . 
 schuster et al context key objective our study was focused on understanding the link between somatic copy number alterations ( scnas ) and intrinsic resistance to aromatase inhibitor ( ai ) therapy , and we observed that tumors with high levels scnas had intrinsic resistance to therapy . knowledge generated a well - established link exists between high genomic instability and tp53 mutations and resistance to cancer treatment ; however , we are the rst to our knowledge to show that primary estrogen receptor ( er ) positive tumors with high genomic instability have an intrinsic resistance to treatment that can be measured after a short , 2 - week ai treatment . 
high genomic instability tumors do not require time to evolve resistance to estrogen deprivation therapy because they already have de novo resistance to treatment . relevance estrogen deprivation therapy with ai treatment is highly effective in er - positive breast cancer , but more than 20% of postmenopausal women with early - stage breast cancer suffer a relapse . 
the perioperative endocrine therapyindividualizing care ( poetic ) phase iii trial with 2 weeks of perioperative ai therapy offers the opportunity to identify mechanisms and biomarkers of intrinsic ai resistance , and in the poetic trial , up to 20% of tumors showed resistance to ai treatment after just 2 weeks of treatment . 
validation of these results in a larger study would provide a framework for better stratifying patients into high risk of ai resistance who are likely to benet from added or alternative treatment . therefore , was to determine whether genome - wide measures of scnas ( ie , genomic instability ) and / or focal scnas are associated with intrinsic resistance to ai treatment . response to ai treatment can be measured by change in the proliferation marker protein encoded by the mki67 gene ( ki - 67 ) after 2 to 4 weeks of presurgical therapy , and ai resistance in primary tumors can be characterized and dened by limited or no ki - 67 response to ai treatment.8 - 10 this change in ki - 67 has been found to predict benet from endocrine therapy better than clinical response.10 we therefore extended our earlier study on the relationship between mutations and resistance to ais in the presurgical perioperative endocrine therapyindividualizing care ( poetic ) trial . 
poor responders ( prrs ; n = 28 ) were dened as having a ki - 67 decrease of less than 60% between baseline and surgery and good responders ( gdrs ; n = 28 ) as having a greater than 75% ki - 67 decrease . 
patients with intermediate ki - 67 decrease between 60% and 75% were not considered . exome sequencing was available for 72 tumors from a previous study.4 samples from 17 patients who received no ai also were analyzed to ensure that changes in scnas ascribed to ai treatment were not artifactual . 
aliquots were taken from 10 tumor dna samples and assessed as technical replicates ( data supplement )  . dna extractions eight - micrometer sections were taken from rnalaterstored core - cuts embedded in optimal cutting temperature compound ( cryo - m - bed , bright instruments , luton , united kingdom ) and stained with nuclear fast red ( 0.1% weight - to - volume ratio )  . 
 genomic instability and resistance to ai treatment blood using the ez1 system ( life technologies , carlsbad , ca )  . snp array analysis human omniexpressexome v.3 beadchip ( illumina , san diego , ca ) was used to generate genotype and intensity data for blood and tumor samples , and allele - specic copy number analysis of tumors ( ascat ) 13 was used for the estimate of ploidy , fraction of tumor cells , and cnas in the tumor samples . 
either the segmentation penalty in ascat was increased ( 22 samples ) or the estimate of ploidy and purity from oncosnp was used in ascat ( four samples ) to generate scna calls that best described the data . 
data have been deposited in the european genome - phenome archive ( egas00001001940 )  . measures of genomic instability chromosomal gains and losses were determined relative to estimates of tumor ploidy by ascat ( sum of major and minor allele calls minus tumor ploidy rounded to the nearest integer )  . 
the median percentage of the genome with scnas was 46% for tumors , with a single representative tumor sample chosen from matched baseline , surgery , or technical replicate samples to calculate the median percentage of scnas . 
the median percentage of the genome with gains relative to tumor ploidy , losses relative to tumor ploidy , and loh was 15% , 16% , and 15% , respectively ( fig 1b )  . highly recurrent scnas ( gains at 1q , 16p , 20q , and 8q and losses / loh at 11q 16q , 17p , and 8p ) occurred in more than 50% of all representative samples . 
the majority of sites with losses overlapped with loh ( data supplement ) , as expected.17 , 18 intratumoral heterogeneity of scnas overlap of scnas between paired core - cuts . 
of note , these samples had the highest genomic instability , with more than 90% of the genome with scnas ( p088 samples )  . overall scna calls in baseline and surgery ai pairs were similar ( data supplement ) , with the median overlap for scnas at 87% and 88% for 33 baseline / surgery ai pairs and 11 no - ai pairs . 
there was no signicant difference between the frequency of discordant snca calls between baseline and surgery ai pairs after correction for multiple testing , and only 4% of 47 , 807 nonoverlapping regions had greater than 10% more events in baseline or surgery samples ( more than four additional scna events in the baseline or surgery samples in the 33 pairs )  . 
for pairs of baseline and surgery samples , the median percentage the genome with discordant scna calls was 5% ( fig 1c ) , and discordance between samples was associated with the percentage of the genome with scnas . there was only one paired set of core - cuts in which discordant scnas were greater than the scnas shared between the pair of samples , which suggests two independently evolved tumors . discordance in prr and gdr paired samples . 
there was a trend for prrs to have more discordant scnas between paired samples than gdrs ( prr average , 10% ; gdr average , 6% ) , but this difference was not signicant . 
however , the variance in the percentage of the genome with discordant scnas was signicantly greater in prrs than in gdrs ( p , .001 , f test ; fig 1d )  . 
 e gdrs ki - 67 > 75% prrs ki - 67 < 60% controls no ai all scnas ( genomic instability ) gains losses schuster et al gdrs prrs controls baseline - surgery tumor pairs gdrs prrs controls fig 1 . 
 ( a ) arrow plot showing the change in the protein encoded by the mki67 gene ( ki - 67 ) between baseline and surgery for good responder ( gdr ) , poor responder ( prr ) , and untreated control samples ( controls as determined by immunohistochemistry scores )  . 
 ( b ) box plot showing percentage of the genome with somatic copy number alterations ( scnas ) , gains relative tumor ploidy , losses relative to tumor ploidy , loss of heterozygosity ( loh ) , and homozygous deletion ( hd ) for 127 tumor samples . 
 ( c ) bar plot and ( d ) box plot showing the average percentage of genome discordance between pairs of core - cuts ( baseline and surgery ) for all scnas . 
there was an enrichment of poor prognosis intrinsic subtypes ( prr nonluminal / luminal b ) in prr samples ( 64% ) compared with gdr samples ( 20% ) ; however , more than 30% of measured prr samples were luminal a subtypes , which suggests that fully capture intrinsic subtyping did not the higher risk of recurrence in these samples ( data supplement )  . comparison between prrs and gdrs in percentage of genome altered . 
given the overall concordance between baseline and surgery core - cuts in scnas and the results of previous observations of minimal impact of ai treatment on mutation counts , 4 we merged all the scna events from multiple samples from the same tumor to represent the scna events in that tumor ( baseline and surgery , 35 events ; baseline , surgery , and technical replicates , nine events ; baseline technical replicates , one event )  . 
 ( a ) box plot showing the difference in genomic instability ( the percentage of genome with somatic copy number alterations [ scnas ] ) between good responder ( gdr ) and poor responder ( prr ) tumors . 
 ( b ) comparisons of protein encoded by the mki67 gene ( ki - 67 ) baseline immunohistochemistry scores with genomic instability ( the percentage of the genome with scnas ) for gdr , prr , and untreated control samples ( controls , blue )  . 
 ( c ) comparisons of ki - 67 surgery immunohistochemistry scores after aromatase inhibitor treatment with genomic instability ( the percentage of the genome with scnas ) for prrs and gdrs . 
the percentage of a chromosomal arm with gains , losses , and loh was calculated , and prrs showed a signicantly higher percentage of gains in 6p ; losses in 5q ; and loh in 10q , 17p , and 19p ( fdr , 0.1 , one - sided mann - whitney u test ; figs 3a to 3c )  . 
the largest difference in percent values ( mean and median ) for arms between gdrs and prrs was observed in loh at 17p ( figs 3d to 3g ) followed by loh in 8p and gains in 8q . 
these regions had approximately 40% more events in prrs ( 10 to 13 more scna events in the 28 prr samples v gdr samples , respectively ) but were not signicant after multiple correction . tp53 alterations occurrence of tp53mut and loh in cohort . 
our previous work from exome sequencing showed prrs and tp53mut associated with a higher mutational mutational load was correlated with ki - 67 levels at surgery after 2 weeks of ai treatment.4 we did not observe a signicant correlation between the percentage of the genome load and that load , but we did observe with scnas and mutational greater genomic instability in tumors with tp53mut ( fig 4e )  . as expected for a tumor suppressor , loh at the tp53 locus in 17p was associated with tp53mut across all tumors ( which drove loss of the functioning copy of the tumor suppressor gene ; p = .004 , fishers exact test )  . 
of the 17 patients with tp53 mutations in baseline or surgery samples , 15 had loh at the tp53 locus ( nine prr , ve gdr , and three control samples )  . 
there was a signicant enrichment of tp53 genomic alterations in prrs ( p = .03 , fishers exact test ) and a signicant difference in the distribution of tp53 genetic alterations between prrs and gdrs ( p = .02 , 2 test ; fig 4a )  . ai resistance and tp53 status . 
 ( a ) percentage of samples with gains relative to tumor ploidy for poor responders ( prrs ; dark red ) and good responders ( gdrs ; light red ) , ( b ) with losses for gdrs ( light blue ) and prrs ( dark blue ) , and ( c ) with loss of heterozygosity ( loh ) for gdrs ( light green ) and prrs ( dark green ) at 47 , 807 segments generated from the somatic copy number alteration ( scna ) analysis of perioperative endocrine therapyindividualizing care ( poetic ) tumor samples . 
 ( d ) percentage of samples with loh ( gdrs , light green ; prrs , dark green ) for chromosome 17 ( chr17 ) , including ( e ) a table for loh events at tp53 and ( f ) the difference in the percentage of samples with loh between prrs and gdrs . 
bar plot showing the percentage of good responder ( gdr ) and poor responder ( prr ) samples with tp53 wild type ( tp53wt ) and no loss of heterozygosity ( loh ) at the tp53 locus , tp53wt and loh at the tp53 locus , tp53 mutation ( tp53mut ) and no loh at the tp53 locus , and tp53mut and loh at the tp53 locus . 
box plots showing ( b ) the percent change in the protein encoded by the mki67 gene ( ki - 67 ) , ( c ) the ki - 67 baseline immunohistochemistry scores , ( d ) the ki - 67 surgery immunohistochemistry score , and ( e ) genomic instability ( the percentage of the genome with somatic copy number alterations [ scnas ] ) for prrs with tp53wt and no loh at the tp53 locus , tp53wt and loh at the tp53 locus , and tp53mut and loh at the tp53 locus . 
the results with her2 - negative samples were similar to those with all samples , with the most signicant differences between prrs and gdrs being loss at 5q and loh at 17p for her2 - negative samples . 
there was also a signicant enrichment of tp53 genomic alterations in prrs ( p = .02 , fishers exact test ) and a signicant difference in the distribution of tp53 genetic alterations between prrs and gdrs in her2 - negative samples ( p = .03 , 2 test )  . discussion our primary goal was to identify global and focal scnas that were associated with the antiproliferative response of erpositive bc to short - term estrogen deprivation using ais . our selection of samples from more than 3 , 000 patients in the ai group from the poetic trial aimed to exploit this large study to understand good / poor response to ai treatment in a general er - positive bc population but not to represent the trial population per se . 
the sampling of tumors before and after 2 weeks of ai treatment allowed the impact of tissue heterogeneity to be assessed , and prior exome sequencing gave the opportunity to integrate the scna and mutation data to better understand intrinsic resistance . 
 schuster et al not change signicantly in the nearly 3 , 000 poetic aitreated patients within the 2 - week treatment window ( data not shown ) , which indicates little opportunity for selection of resistant cells in that time . 
reduced heterogeneity might be observed from longer treatment.20 these data , therefore , indicate that a small biopsy sample before or after short - term ai treatment is likely to be representative of the whole tumor for most bcs ; however , for tumors with high genomic instability and greater heterogeneity , multiple biopsy samples may be necessary to capture all genomic alterations . there is a large body of evidence to associate genomic instability with poor outcomes in solid tumors , 6 and incorporation of genomic instability scores can greatly improve molecular prognostic models for bc.21 , 22 it is not known whether high genomic instability and greater tumor heterogeneity allow the few surviving tumors to evolve resistance to ai treatment or whether there is intrinsic resistance to ai in these tumors . 
loh and mutations in tp53 have been shown to result in worse outcomes , 23 and we have now shown that it is also associated with poor antiproliferation response to ai and intrinsic resistance to treatment . 
even in tumors with high er expression and good prognosis , tp53 genomic alterations can result in worse outcomes . work by other groups has associated mutations in dna repair pathways25 or mismatch repair pathways19 and coamplication of fgfr1 and ccnd15 with resistance to ai treatment , but we have not observed enrichment of these genomic alterations in our prrs . 
this may be the result of small samples sizes in each study and additional differences in how ai resistance is classied : we classied response / resistance on the basis of changes of ki - 67 between baseline and ai - treated tumors because this dynamic assessment relates to benet from treatment . 
others have used the level of residual ki - 67 in ai - treated tumors as the end point to dene resistance , which reects residual risk of recurrence while on ais . 
of note , a patient with a large reduction in proliferation after treatment has clearly beneted from and responded to ai treatment , regardless of her residual risk on the basis of ki - 67 measurements at surgery.26 we conclude that the poor prognosis of er - positive postmenopausal tumors associated with high genomic instability , tp53 loh , and tp53mut is due at least in part to intrinsic resistance of these tumors to ai therapy . 
short , 2 - week ai treatment can reveal poor antiproliferative response in these primary tumors , which indicates that they continue to proliferate in an estrogen - deprived environment and do not require additional evolution to enable the tumor to resist treatment . 
j clin oncol 27 : 1382 - 1387 , 2009 l opez - knowles e , wilkerson pm , ribas r , et al : integrative analyses identify modulators of response to neoadjuvant aromatase inhibitors in patients with early breast cancer . 
nat genet 45 : 1127 - 1133 , 2013 gellert p , segal cv , gao q , et al : impact of mutational proles on response of primary oestrogen receptor - positive breast cancers to oestrogen deprivation . 
nat commun 7 : 13294 , 2016 giltnane jm , hutchinson ke , stricker tp , et al : genomic proling of er + breast cancers after short - term estrogen suppression reveals alterations associated with endocrine resistance . 
j natl cancer inst 99 : 167 - 170 , 2007 ellis mj , suman vj , hoog j , et al : ki67 proliferation index as a tool for chemotherapy decisions during and after neoadjuvant aromatase inhibitor treatment of breast cancer : results from the american college of surgeons oncology group z1031 trial ( alliance )  . 
dowsett m , smith ie , ebbs sr , et al : short - term changes in ki - 67 during neoadjuvant treatment of primary breast cancer with anastrozole or tamoxifen alone or combined correlate with recurrence - free survival . 
l opez - knowles e , gao q , cheang mc , et al : heterogeneity in global gene expression proles between biopsy specimens taken peri - surgically from primary er - positive breast carcinomas . 
haricharan s , punturi n , singh p , et al : loss of mutl disrupts chk2 - dependent cell - cycle control through cdk4 / 6 to promote intrinsic endocrine therapy resistance in primary breast cancer . 
quenel - tueux n , debled m , rudewicz j , et al : clinical and genomic analysis of a randomised phase ii study evaluating anastrozole and fulvestrant in postmenopausal patients treated for large operable or locally advanced hormone - receptor - positive breast cancer . 
haricharan s , bainbridge mn , scheet p , et al : somatic mutation load of estrogen receptor - positive breast tumors predicts overall survival : an analysis of genome sequence data . 
 de novo esr1 hotspot mutation in a patient with endometrial cancer treated with an aromatase inhibitor adeline morel , md1 ; julien masliah - planchon , phd1 ; guillaume bataillon , md2 ; v eronique becette , md2 ; claire morel , msc3 ; samantha antonio1 ; elodie girard , md4 ; ivan bi `eche , phd1 , 5 ; christophe le tourneau , md , phd3 , 4 , 6 ; and maud kamal , phd3 introduction endometrial carcinoma is the fourth most frequent cancer in women . 
endometrial carcinoma is classied into two subtypes : type 1 endometrial carcinoma characterized by estrogen receptor ( er ) expression and obesity , and type 2 endometrial carcinoma in nonobese , older women . 
type 1 endometrial carcinoma is associated with a favorable prognosis , whereas type 2 has a poorer prognosis.1 a prognostic genomic classication of endometrial cancers into four groups has been established using exome sequencing ; however , this classication is not used in the clinic.2 standard - of - care treatment of endometrial carcinoma consists of primary hysterectomy , bilateral salpingooophorectomy , and pelvic lymph node dissection followed by adjuvant therapy based on the histologic assessment of the specimen.3 chemotherapy is proposed in the recurrent and / or metastatic setting , whereas hormone therapy represents a treatment option for patients with er expression.4 in breast cancer , esr1 mutations were clearly identied as a mechanism of resistance to aromatase inhibitors.5 - 8 esr1 mutations were reported in 2% of endometrial cancers , 9 yet their potential occurrence following hormonal therapy is unknown . 
the diagnosis of grade 1 endometrioid carcinoma was conrmed , with less than 50% invasion of the myometrial wall thickness , 4 cm in greatest dimension , lymphovascular invasion , bilateral ovarian metastases , and no pelvic lymph node metastases ( pt3an0 f ed eration internationale de gyn ecologie et dobst etrique iiia )  . 
using immunohistochemistry , tumor cells were shown to express er , progesterone receptor , and a wild - type staining of p53 . in addition , tumor cells expressed ck7 and pax8 and did not express ck20 , ttf1 , cdx2 , and wt1 , conrming the endometrial origin of the tumor . ( ie , letrozole )  . 
after nine cycles of chemotherapy , the patient received maintenance therapy with an aromatase inhibitor receiving letrozole for 6 months , the patient experienced disease progression and was subsequently treated with doxorubicin and cyclophosphamide and then carboplatin alone . 
the patient was then biopsied in the framework of the shiva02 trial ( evaluation of the efcacy of targeted therapy based on tumor molecular proling in patients with advanced cancer using each patient as its own control ; clinicaltrials.gov identier : nct03084757 ) , which aimed to identify druggable molecular alterations , and received a fourth line of chemotherapy with gemcitabine ( fig 1 )  . molecular proling was performed on a frozen biopsy of a metastatic lymph node in the shiva02 trial using a dedicated targeted sequencing panel covering 80 genes . 
in patients with breast cancer , esr1 hotspot mutations in the ligand - binding domain were observed exclusively after hormone therapy5 - 8 and have been shown to be associated with poor prognosis.11 after exposure to aromatase inhibitors , the prevalence of esr1 mutations was signicantly higher in patients exposed during the metastatic phase than during the adjuvant phase ( 36% v 6% ) .11 in a recent study , advanced breast cancer tumors harboring esr1 mutations , or alterations in genes involved in the mitogenactivated protein kinase pathway and in the er transcriptional machinery , were shown to barely benet from aromatase inhibitors.5 esr1 amplications were detected in 12% of endometrial carcinomas , 12 whereas esr1 hotspot mutations were less frequent ( 2% ) .9 esr1 amplications were shown to be associated with a poor prognosis and seemed to be an early event in endometrial carcinoma development.12 selective er degraders , such as fulvestrant , were shown to be effective in patients with hormone receptorpositive breast cancer . 
esr1 mutations were not reported to be associated with clinical resistance to fulvestrant.13 to our knowledge , our case report is the rst to report a potential de novo esr1 mutation in a patient with erpositive endometrial carcinoma treated with an aromatase inhibitor . 
curr opin obstet gynecol 29 : 47 - 58 , 2017 kokka f , brockbank e , oram d , et al : hormonal therapy in advanced or recurrent endometrial cancer . 
cochrane database syst rev ( 12 ) : cd007926 , 2010 razavi p , chang mt , xu g , et al : the genomic landscape of endocrine - resistant advanced breast cancers . 
cancer cell 34 : 427 - 438.e6 , 2018 robinson dr , wu y - m , vats p , et al : activating esr1 mutations in hormone - resistant metastatic breast cancer . 
nat genet 45 : 1446 - 1451 , 2013 toy w , shen y , won h , et al : esr1 ligand - binding domain mutations in hormone - resistant breast cancer . 
nat genet 45 : 1439 - 1445 , 2013 bartels s , christgen m , luft a , et al : estrogen receptor ( esr1 ) mutation in bone metastases from breast cancer . 
mod pathol 31 : 56 - 61 , 2018 backes fj , walker cj , goodfellow pj , et al : estrogen receptor - alpha as a predictive biomarker in endometrioid endometrial cancer . 
schiavon g , hrebien s , garcia - murillas i , et al : analysis of esr1 mutation in circulating tumor dna demonstrates evolution during therapy for metastatic breast 12 . 
spoerke jm , gendreau s , walter k , et al : heterogeneity and clinical signicance of esr1 mutations in er - positive metastatic breast cancer patients receiving mol endocrinol 10 : 1388 - 1398 , 1996 cancer . 
a study of patients with myeloid malignancy compared the response rate of trametinib among patients with ras wild - type ( n = 30 ) and ras - mutated mds or aml ( n = 50 )  . 
five patients with ras - mutated mds or aml achieved a complete response ( cr ) or cr with incomplete platelet recovery compared with no patients with ras wild - type mds or aml.5 herein , we describe a patient with relapsed mds after undergoing allogeneic stem - cell transplant ( allo - sct ) who had next - generation sequencing ( ngs ) identifying map2k2 p298l , for which the patient received trametinib plus decitabine and achieved a durable cr . 
the map2k2 p298l mutation has been identified previously in a patient with lung cancer , but the functional significance at the protein level has not yet been elucidated.6 the patient is a 52 - year - old asian woman with a 12 - year history of jak2 - negative essential thrombocythemia ( et ) treated with anagrelide ( 10 years ) and hydroxyurea ( 2 years )  . 
time points indicate number of months after transplantation each next - generation sequencing panel was obtained . dnmt3a tet2 calr map2k runx1 mutations , notably runx1 s141 * ( mutation allele frequency [ maf ] , 12% ) , zmym3 g49wfs * 13 ( maf , 27% ) , calr k368rfs * 51 ( maf , 39% ) , and map2k2 p298l ( maf , 30% )  . 
after a 1 - month treatment interruption , the mucositis resolved and treatment resumed with decitabine 20 mg / m2 intravenously on days 1 through 3 of a 28 - day cycle and trametinib 2 mg daily for 21 days of a 28 - day cycle . 
after resolution , treatment with decitabine 20 mg / m2 intravenously on days 1 through 5 of a 28 - day cycle and trametinib 0.5 mg daily for 7 days of a 14 - day cycle continued without additional issues . 
instead , a benign pdgfrb t88i ( maf , 47% ) alteration and tet2 h1380y ( maf , 5% ) and dnmt3a r882s ( maf , 15% ) mutations were identified ( fig 1 ; data supplement )  . subsequently , a donor pb sample was analyzed by a clinical laboratory improvement amendments - certified moffitt 54 - genetargeted myeloid ngs panel8 with an average coverage of 11 , 987 reads . 
a benign asxl1 g652s variant was detected in the patient and the related donor ( maf , 49% ) , likely reflecting a heterozygous germline variant detected in both persons as a result of shared inheritance . 
 this acquired mutation in the patient suggests donor - derived acquisition of a chip mutation . at the time of this report , the patient was without gvhd symptoms , received a donor lymphocyte infusion ( dli ) 16 months after trametinib initiation , and remains in cr 18.5 months after trametinib initiation ( 29.5 months after allo - sct )  . 
 the primary oncologist felt that a dli may achieve the best long - term outcomes because there are no long - term efficacy data of trametinib plus a hypomethylating agent ( hma ) in relapsed mds after allo - sct . 
the patient continues to receive treatment with trametinib and decitabine without evidence of dysplasia or relapse . discussion protein function in the presence of map2k2 p298l has not been previously characterized to our knowledge . 
this map2k2 alteration is located in a proline - rich segment of the protein kinase domain , is near a map2k2 phosphorylation site , and has been observed previously in cancer.6 additionally , per the catalog of somatic mutations in cancer , the map2k2 p298l alteration had been identified as somatic and pathogenic per their prediction tool.11 despite not knowing the functional significance of the alteration , a clinical response was observed after the administration of trametinib and decitabine . 
 the 2 - year os was 12.4% and in those achieving a cr after azacitidine administration , the 2 - year os was 48.4%.12 therefore , it is difficult to determine to what degree , if any , the clinical benefit observed could be attributed to trametinib , because hmas with or without dli have demonstrated clinical efficacy in patients with relapsed mds after sct transplant . adverse effects and their management with trametinib plus decitabine has not been previously discussed to our knowledge . 
based on phase ii data , trametinib was not believed to result in the infectious complications experienced in our case.4 trametinib and decitabine were held to evaluate the etiology of the adverse effects of dermatitis and mucositis , for which a biopsy specimen demonstrated gvhd . 
kevin hicks no relationship to disclose lia perez no relationship to disclose eric padron honoraria : incyte , cell therapeutics , cell therapeutics ( inst ) research funding : incyte ( inst ) rami s . 
kim kb , kefford r , pavlick ac , et al : phase ii study of the mek1 / mek2 inhibitor trametinib in patients with metastatic braf - mutant cutaneous melanoma previously treated with or without a braf inhibitor . 
borthakur g , popplewell l , boyiadzis m , et al : activity of the oral mitogen - activated protein kinase kinase inhibitor trametinib in ras - mutant relapsed or refractory myeloid malignancies . 
kahle , phd1 ; ann marie bailey , phd1 ; chetna wathoo , md1 ; kavitha balaji , phd1 ; mehmet esat demirhan , md1 , dong yang , phd1 ; milind javle , md1 ; ahmed kaseb , md1 ; cathy eng , md1 ; vivek subbiah , md1 ; filip janku , md , phd1 ; victoria m . 
mills shaw , phd1 , and funda meric - bernstam , md1 purpose cell - free dna ( cfdna ) next - generation sequencing is a noninvasive approach for genomic testing . 
of these patients , 438 ( 76.2% ) had at least one alteration detected , and 205 ( 35.7% ) had one or more alterations of high potential for clinical action . 
trials were identied in 80% of patients ( 286 of 357 ) with any alteration and in 92% of patients ( 188 of 205 ) with one or more alterations of high potential for clinical action of whom 57.6% ( 118 of 205 ) had 6 or more months of follow - up available . 
of these patients , 10% ( 12 of 118 ) had received genomically matched therapy through enrollment in clinical trials ( n = 8 ) , off - label drug use ( n = 3 ) , or standard of care ( n = 1 )  . 
2019 by american society of clinical oncology introduction molecular proling of tumors through circulating cellfree dna ( cfdna ) has gained signicant traction in recent years because of its noninvasiveness , sensitivity , and quick turnaround time.1 - 4 for patients with advanced cancers , specically those who have either exhausted their solid tumor block or have quickly progressed on compelling intervening therapy , repeat invasive tissue biopsy is necessary to identify driver mutations and / or resistance mechanisms that arise in tumor evolution / response to treatment or natural progression.3 , 5 in addition , a single sample can serve as a global representation of the mutational landscape across multiple metastatic lesions.6 - 8 together , these attributes make cfdna testing especially attractive for patients with advanced cancer where a quick clinical decision is necessary to identify genomically relevant clinical trials . functional annotation , assignment of potential clinical actionability , and clinical trial matching to guide clinical decision making on the basis of cfdna testing . 
in this prospective study of 575 patients with advanced cancers , we provided such postprocessing to evaluate the clinical utility of cfdna testing in identifying clinically actionable genomic alterations . this entailed the delivery of personalized annotation reports , including functional interpretation of variants and retrieval of genomically relevant clinical trials . however , physicians were allowed to order testing and initiate treatments without waiting for results , as appropriate . 
 s anchez et al context key objective what is the frequency of actionable alterations in patients with advanced / metastatic cancer who undergo cell - free dna ( cfdna ) testing ? knowledge generated in this study , of 575 patients who had cfdna testing , 76.2% had at least one alteration detected and 35.7% had one or more alterations of high potential for clinical action . 
criteria for enrollment were active metastatic / local inoperable advanced cancer , consideration of clinical trial enrollment within the next two lines of therapy , and exhausted tissue block or archival tissue older than 1 year or available tissue block but progression on compelling intervening therapy . 
human investigations were performed after approval by the md anderson institutional review board and in accordance with an assurance led with and approved by the us department of health and human services . comprehensive genomic testing in plasma cfdna was extracted from whole blood collected in 10 - ml streck tubes . 
only variants that occurred within an actionable gene were annotated.17 , 18 a gene is considered actionable if it has an established biologic role in cancer and there is a clinically available drug to which the gene confers sensitivity or resistance , where actionability can be applicable to all or select alteration or tumor types . 
of note , other genes are included in the guardant360 panel , such as tumor suppressor genes , which may not be targetable , but clonal driver mutations in the highest allele these genes are usually variants at fractions , which makes them ideal candidates to index the degree of tumor dna shedding into the bloodstream and to determine the relative subclonality of other variants in the same sample . dening a variants potential for clinical action variants reported in an actionable gene were annotated as follows : ( cid : 129 ) functional signicance ( fs ) : activating , activating inferred , inactivating , inactivating inferred , inactivating and neomorphic , unknown , or likely benign ( cid : 129 ) actionable variant call ( avc ; from highest level of evidence [ loe ] to lowest ) : yes : literature - based ( peerreviewed scientic literature ) ; yes : functional genomics ( institute for personalized cancer therapy internal functional genomics platform ) ; yes : inferred ( intermediate loe includes inferences dictated by the type of alteration and is mostly applicable to frameshift / truncation alterations where loss of region is inferred to be activating / inactivating ) ; potentially ( lower loe where there are indirect and / or limited data to support the function of a specic alteration ) ; unknown ; or no ( fs call is likely benign or inactivating alteration in an oncogene ) ( cid : 129 ) actionable for call : the combination of an fs call and avc : therapeutic intervention , resistance to drugs , or enrollment in select clinical trials . finally , gene actionability can be applicable to all , or specic , tumor types . 
although specic food and drug administration approved indications and / or national comprehensive cancer network guidelines are not included within this parameter , the upstream annotation process depends on gene - level actionability . 
the workow for categorizing a variants potential for clinical action is illustrated in the data supplement . clinical trials identied on the basis of patient genomic prole trial retrieval was restricted to variants that were deemed actionable and to trials available at the md anderson cancer center at the time of patient annotation . patient follow - up patients were followed for at least 6 months after the initial annotation to collect treatment decisions , performance status ( ps ) , and trial screening / enrollment ( and reasons for eligibility / ineligibility )  . results patient characteristics we enrolled 575 patients into the study . 
consistent with this , single nucleotide variants were the most commonly reported alteration type , followed by amplications , indels , and fusions . the results reected the genomic landscape reported across tumor types through solid tumor testing19 , 20 and is shown in figure 2a . 
amplication levels detected ranged from positive , strongly positive , to very strongly positive , as previously dened.9 - 14 genes most frequently reported with very strong positive amplication levels were ccne1 , fgfr1 , met , erbb2 , and ar . 
 s anchez et al patients ( n = 575 ) no alterations reported ( n = 137 ) any alteration reported ( n = 438 ) only alterations in nonactionable genes ( n = 81 ) one or more alterations within actionable genes ( n = 357 ) fig 1 . 
a total of 575 patients was enrolled , of whom 76.2% ( n = 438 ) had at least one alteration reported . of these 438 , 81.5% ( n = 357 ) had at least one alteration reported within an actionable gene , of whom in turn , 54.7% ( n = 205 ) had at least one high potential for clinical action alteration . 
again , these results highlight the importance of assessing each alterations biologic fs individually and in the disease context ( fig 2c )  . the actual variant actionability , or potential for clinical action , was used to stratify patients by disease type into three categories : hpca , low potential for clinical action , and not recommended for clinical action . 
among diseases with 10 or more patients enrolled , the proportion of patients who had at least one hpca alteration was as follows : breast , 67.6% ; colorectal , 60% ; lung , 56.5% ; gall bladder , 56.3% ; ovarian , 50% ; head and neck , 43.5% ; cholangiocarcinoma , 43.3% ; prostate , 40% ; hepatocellular , 25.4% ; pancreatic , 19.5% ; neuroendocrine , 15.4% ; sarcoma , 14.9% ; and appendiceal , 10.6% ( fig 4a )  . overall , these ndings demonstrate that cfdna testing is informative across all tumor types included in this study . specically , among tumor types with greater representation ( 10 patients enrolled ) , 50% or more patients had at least one hpca alteration . 
 a genomic alterations in cell - free dna amplification fusion frameshift / truncation indel very strong positive ( + + + ) strong positive ( + + ) positive ( + ) fig 2 . 
 ( b ) identication of clinical trials across all disease types in the context of variants potential for clinical action . alterations ( 143 patients ) , the concordance rate was 44% ( 56 of 127 alterations )  . 
these alterations potentially represent genomic evolution and mechanisms of acquired resistance and included detection of a pik3ca mutation after treatment with a braf inhibitor and braf and met mutations after treatment with akt / mtor inhibitors . on the other hand , among 391 patients with no previous solid tumor testing attempted , 529 alterations in actionable genes were reported on cfdna testing . 
in addition , of 14 patients who had previous solid tumor testing attempted but the tumor genomic assays failed ( eg , because of an insufcient amount of dna obtained from the sample ) , cfdna testing reported 17 alterations in actionable genes , with 10 patients having any alteration detected , seven having at least one alteration in an actionable gene , and two having at least one specic alteration that was actionable at the time of annotation . identication of clinical trials for variants identied through cfdna the utility of cfdna testing depends on the ability to match patients to relevant targeted therapy delivered through clinical trials , standard of care , or off - label use . 
in addition to variant annotation , for patients with hpca variants and / or select low potential for clinical action variants ( ie , fs , unknown ; avc , potentially ; actionable for call , therapeutic intervention ) , the decision support team identied genotype - matched trials for the patients tumor type , which accounted for additional biomarker inclusion / exclusion criteria , where applicable . 
among patients who had at least one hpca alteration , identication of trials was even higher , with potential trials identied for 188 ( 92% ) of 205 patients ( fig 3b )  . clinical course of action after cfdna testing of the 205 patients with at least one hpca variant , 118 had reached the 6 - month follow - up period at the time of data freeze ( march 6 , 2018 )  . 
at this time , 11% of patients ( 13 of 118 ) had been treated with genotype - matched targeted therapy on the basis of cfdna testing , where a match included enrollment into a clinical trial ( n = 9 ) , off - label drug use ( n = 3 ) , or standard of care ( n = 1 )  . 
on the basis of at least 6 months of follow - up , a major contributor to not acting on a genomic alteration was the ps of patients at the time of trial consideration . 
reports from the university of california , los angeles , also found that biopsy samples required for trial eligibility led to delays and decline in ps , which resulted in reduced enrollment by approximately one half.22 similarly , a review of 55 nonsmall - cell lung cancer trials at princess margaret hospital reported a significant reduction in enrollment because of declining ps or insufcient tissue biopsy sample material when required.23 in addition , we evaluated a novel and important factorinitiation of alternative therapy when ordering cfdna testingthat may have reduced the number of trial - eligible patients because of a decline of ps during next - line therapy . 
of note , cfdna testing has a short enough turnaround time to facilitate selection of next - line therapy ( a median of 7 calendar days now is achieved at the high - volume laboratory used herein ) .3 , 24 thus , its use at the point of care may increase its clinical utility before a decline in ps . 
testing earlier , when ps is better , also is likely to optimize patient outcomes from investigational therapies . reports on cfdna testing there have been several utility.3 , 25 - 27 laufer - geva et al28 reported that treatment decisions were changed in 23% to 32% of patients with nonsmall - cell lung cancer dependent on the clinical scenario . 
of note , we accrued very few patients with lung cancer and accrued predominantly tumor types without standard - of - care , genomically informed treatment options . this may have affected overall actionability rates as well as the number of patients who received genomically matched therapy . previous studies have reported higher concordance rates for cfdna results and tumor testing.24 , 29 leighl et al30 reported that in untreated nonsquamous cell carcinoma , food and drug administrationapproved target ( egfr , alk , ros1 , braf ) concordance was greater than 98.2% , with a 100% positive predictive value for cfdna versus tissue ( 34 of 34 patients with egfr - , alk - , or brafpositive mutations )  . 
our study was not designed to compare cfdna and tissue testing ; thus , it had inherent limitations to investigate this issue , including intervening time between sample collections , intervening treatments and resulting tumor evolution , tumor heterogeneity of solid tumor testing versus global representation of primary and metastatic sites from cfdna , and tumor type dependence on the extent of cfdna shedding . 
however , cfdna testing reported potentially clinically relevant alterations that otherwise may have been missed because solid tumor testing was not attempted or not technically feasible because of lack of tissue or did not have sufcient coverage . our study has other limitations . 
although there may have been greater use of cfdna results for treatment selection if patients were prospectively tested and treatment initiated after results , we believe that our study design gives insight into clinical practice patterns . second , we enrolled patients with a variety of tumor types . this design allowed us to determine that the detection of cfdna and the detection of actionable alterations vary by tumor type . 
third , actionability was assessed at the time of testing by a designated decision support team , but trial matching was performed automatically on the basis of gene - drug associations . where there were trial matches , additional clinical exclusion criteria possibly made patients ineligible for selected trials . 
cfdna also can identify actionable resistance mutations , and including those would have potentially enhanced clinical utility.3 in summary , we demonstrated that cfdna testing detects actionable alterations in patients with advanced cancers across a variety of tumor types . 
lanman employment : guardant health , veracyte leadership : guardant health , biolase stock and other ownership interests : guardant health , biolase , forward medical consulting or advisory role : forward medical research funding : guardant health kenna r . 
 s anchez et al references lebofsky r , decraene c , bernard v , et al : circulating tumor dna as a non - invasive substitute to metastasis biopsy for tumor genotyping and personalized medicine in a prospective trial across all tumor types . 
normanno n , cervantes a , ciardiello f , et al : the liquid biopsy in the management of colorectal cancer patients : current applications and future scenarios . cancer treat rev 70 : 1 - 8 , 2018 zill oa , banks kc , fairclough sr , et al : the landscape of actionable genomic alterations in cell - free circulating tumor dna from 21 , 807 advanced cancer patients . 
clin cancer res 24 : 3528 - 3538 , 2018 polivka j jr , pesta m , janku f : testing for oncogenic molecular aberrations in cell - free dna - based liquid biopsies in the clinic : are we there yet ? expert rev mol diagn 15 : 1631 - 1644 , 2015 diaz la jr , bardelli a : liquid biopsies : genotyping circulating tumor dna . 
biochim biophys acta 1775 : 181 - 232 , 2007 suraj s , dhar c , srivastava s : circulating nucleic acids : an analysis of their occurrence in malignancies . 
biomed rep 6 : 8 - 14 , 2017 janku f , zhang s , waters j , et al : development and validation of an ultradeep next - generation sequencing assay for testing of plasma cell - free dna from patients with advanced cancer . 
clin cancer res 23 : 5648 - 5656 , 2017 zill oa , greene c , sebisanovic d , et al : cell - free dna next - generation sequencing in pancreatobiliary carcinomas . 
kim st , lee ws , lanman rb , et al : prospective blinded study of somatic mutation detection in cell - free dna utilizing a targeted 54 - gene next generation sequencing panel in metastatic solid tumor patients . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
spiegel ml , goldman jw , wolf br , et al : non - small cell lung cancer clinical trials requiring biopsies with biomarker - specic results for enrollment provide unique challenges . 
schwaederle mc , patel sp , husain h , et al : utility of genomic assessment of blood - derived circulating tumor dna ( ctdna ) in patients with advanced lung methodologies . 
clin cancer res 23 : 5101 - 5111 , 2017 janku f , angenendt p , tsimberidou am , et al : actionable mutations in plasma cell - free dna in patients with advanced cancers referred for experimental targeted therapies . 
laufer - geva s , rozenblum ab , twito t , et al : the clinical impact of comprehensive genomic testing of circulating cell - free dna in advanced lung cancer . j thorac oncol 13 : 1705 - 1716 , 2018 29 . 
kato s , schwaederle mc , fanta pt , et al : genomic assessment of blood - derived circulating tumor dna in patients with colorectal cancers : correlation with tissue sequencing , therapeutic response , and survival . 
leighl nb , page rd , raymond vm , et al : clinical utility of comprehensive cell - free dna analysis to identify genomic biomarkers in patients with newly diagnosed metastatic non - small cell lung cancer . 
 outcomes by egfr , kras , and alk genotype after combined modality therapy for locally advanced nonsmall - cell lung cancer purpose in 699 patients with locally advanced nonsmall - cell lung cancer ( nsclc ) treated with radiation therapy as part of combined modality therapy , we compared outcomes among genotyped and ungenotyped patients and by tumor genotype status ( egfr , kras , and alk )  . patients and methods genotyping was performed in 250 patients : egfr + ( 19% ) , kras + ( 32% ) , alk + ( 9% ) , and wild type ( wt / / ; 40% )  . 
there was higher freedom from locoregional recurrence ( lrr ) for egfr + tumors and lower freedom from lrr in alk + tumors , compared with kras + and wt / / tumors ( 3 - year : 77% v 38% v 49% v 46% )  . 
 analysis of post - recurrence survival demonstrated that egfr + / alk + patients treated with appropriate tyrosine kinase inhibitors had higher os compared with other groups . conclusion in this series of locally advanced nsclc treated with combined modality therapy , egfr + and alk + were associated with higher os , whereas lrr was lower in egfr + patients , and the risk of distant metastases was high in all subgroups . 
2018 by american society of clinical oncology introduction advances in cancer genomics in the past decade have identified genetically distinct subgroups of lung adenocarcinoma defined by activating genomic alterations in oncogenes such as kras , egfr , and alk.1 - 5 the unique biology of each subgroup has led to the development of genotype - directed therapy in stage iv disease , with level 1 evidence that targeted therapies for egfr - mutant , 6 - 8 and alk - rearranged stage iv nonsmall - cell lung cancer ( nsclc ) 9 improve outcomes when compared with chemotherapy . 
although the 10 - year anniversary of the discovery of egfr mutations in 2014 marked a new era of targeted therapy and precision medicine for genotypic subgroups of stage iv lung adenocarcinoma , the clinical potential of integration of targeted therapies with conventional modalities of curative therapy ( eg , surgery , radiation , and / or chemotherapy ) for earlier - stage disease has not been fully realized . first , the impact of tumor genotype on the natural history and responsiveness to conventional treatment of earlier stages of disease remains understudied and controversial . 
second , although radiation therapy ( rt ) plays a critical role in combined modality therapy ( cmt ) for locally advanced nsclc ( la - nsclc ) , radiation responsiveness and clinical outcomes after thoracic rt for the genotypic subgroups of nsclc are not well elucidated . 
direct sanger sequencing and / or polymerase chain reaction with capillary gel electrophoresis using primers specific for codons 18 to 21 for egfr and codons 12 , 13 , and 61 of the kras gene27 was performed ( data supplement )  . 
alk testing was performed using immunohistochemistry ( n = 13 ) , fluorescent in situ hybridization ( n = 129 ) , or both ( n = 7 )  . because the indications and scope of genotyping changed during the follow - up period ( eg , the development of alk assays in the later period ) , some patients did not have complete genotyping data for all three oncogenes . 
tumors that had genotyping data for all three oncogenes were classified as egfr + , alk + , kras + , or triple wild type ( wt / / ) and were included in a fourway analysis . 
tumors with partial genotyping results were classified as unknown for four - way analyses but were analyzed in comparisons of wt versus mutant for individual oncogenes . covariates patient age , sex , ethnicity , eastern cooperative oncology group performance status , smoking history ( 1 [ never smokers ] : < 100 cigarettes in lifetime ; 2 [ former smokers ] : quit smoking > 1 year before diagnosis ; 3 [ current smokers ] : smoking at time of diagnosis or quit < 1 year before ) , and tumor characteristics ( size , histology , 7th edition american joint committee on cancer stage , and number of nodal stations involved per the international association for the study of lung cancer atlas ) 28 were analyzed . 
 treatment and follow - up the cmt for each patient was selected with a multidisciplinary approach , typically trimodality therapy for stage iiia and concurrent chemoradiation for medically inoperable ii to iiia or n3 disease . 
all patients were treated with rt per institutional norms , with three - dimensional or four - dimensional computed tomography ( ct ) simulation and either 3dcrt or imrt planning . 
the rt target included primary and nodal disease as defined by positron emission tomography ( pet ) - ct , microscopic margin ( 0 to 7 mm ) , and setup margin ( 5 to 10 mm , depending on localization technology at the time ) , and elective nodal irradiation was not performed . patients were observed with chest ct every 3 to 4 months after treatment ( years 1 and 2 ) , then every 6 months ( years 3 to 5 ) , and annually thereafter . outcomes overall survival ( os ) and patterns of failure , including lrr , distant metastases ( dm ) , any recurrence , and progression - free survival ( pfs ; survival with absence of recurrence ) , were calculated from the date of initiation of treatment to the time of first failure . 
out - offield lrr was defined as occurring outside of the planning target volume . statistical analyses all survival and recurrence outcomes were analyzed using the kaplan - meier method and log - rank test . 
because risk of failures may be altered by salvage systemic treatment during the follow - up period , a cox proportional hazards regression model with systemic treatment as a time - dependent covariate was used to analyze patterns of failures . 
to analyze the impact on os of salvage targeted therapies at the time of recurrence , a postrecurrence landmark analysis of os was performed with cutoffs of 3 , 6 , and 9 months after recurrence . 
all the analyses were performed using sas version 9.3 ( carey , nc )  . results tumor genotyping data and genotyped cohort characteristics genotyping was performed as part of routine clinical care in 250 patients , either before rt ( 50% ) or after rt ( 50% )  . 
the majority of patients received definitive chemoradiation , but 35% of patients also underwent surgery as part of cmt . outcomes and patterns of recurrence : all patients with a median follow - up of 57.4 months among all patients ( n = 699 ) , the median os was 26.9 months ( 3 - year estimate : 41% ) and the median pfs was 11.9 months ( 3 - year estimate : 25% )  . 
patient , tumor , and treatment characteristics by genotypic subgroup * ( continued ) characteristic alk + egfr + kras + total 50.4 - 66 45 - 66.6 45 - 66 6 - 68 6 - 68 rt dose per fraction no . 
 ( % ) unless indicated otherwise . abbreviations : 3dcrt , three dimensionalconformal radiation therapy ; adenoca , adenocarcinoma ; fev1 , forced expiratory volume in 1 second ; imrt , intensity modulated radiation therapy ; nsclc , nonsmall - cell lung cancer ; pretx , pre - treatment ; rt , radiation therapy ; scc , squamous cell carcinoma ; suv , standard uptake value ; wt , wild type for egfr , alk , and kras . * among patients with full genotyping available . the 3 - year estimates of freedom from lrr , dm , and any recurrence were 53% , 41% , and 32% , respectively ( data supplement )  . outcomes and patterns of recurrence : egfr + versus alk + versus kras + versus wt / / the median follow - up among genotyped patients was 48.2 months ; outcomes by genotype are listed in table 2 . 
the median os was 55.8 months for egfr + and was not reached for alk + patients , versus 28.0 months for kras + and 33.2 months for wt / / patients ( p = .02 ; fig 1a )  . 
 there was no significant difference in dm and any recurrence ( table 2 )  . univariable and multivariable analysis univariable analysis showed that younger age , female sex , nodal stations involved , surgery , rt dose , and alk + were associated with increased os , and genotypic subgroup ( egfr + v alk + v kras + v wt / / ) was also associated ( data supplement )  . 
pfs and any recurrence were not associated with tumor genotype ( data supplement )  . analysis of post - recurrence outcomes we analyzed post - recurrence os among genotyped patients to assess the impact of salvage tki on the increased os observed in alk + / egfr +  . 
of those who recurred , 88% of egfr + patients ( 21 of 24 ) and 75% of alk + patients ( nine of 12 ) received tki after recurrence ( three egfr + and two alk + patients died as a result of disease progression before tki , and one alk + patient had a complete response to salvage chemotherapy )  . 
among patients with pre - recurrence genotyping , there was a significant difference in post - recurrence os when comparing egfr + / alk + patients who received appropriate tki with those who did not receive tki and with other genotypic subgroups ( table 5 ; data supplement ) , with similar findings on landmark analyses . those without tumor genotyping ( n = 449 )  . 
the median follow - up was 48.2 months in genotyped patients versus 67.4 months in ungenotyped patients , reflecting more recent adoption of clinical genotyping ( data supplement )  . 
first , in univariable analysis , we demonstrate increased os in patients with alk + and egfr + tumors compared with patients with kras + and wt / / tumors . 
our findings of lower lrr in egfr + tumors may suggest increased radiation sensitivity , supporting the findings of previous in vitro studies.30 , 31 however , the existing literature has been mixed ; some series of chemoradiation for la - nsclc20 , 21 and cns metastases treated with whole - brain rt32 have shown increased rt sensitivity in egfr + tumors , whereas other series have not demonstrated a difference in radiation response.22 , 23 moreover , the numbers of patients in these subgroups were small , and these hypothesis generating results must be interpreted with caution and validated in larger data sets . another key finding of this study is that the incidence of dm was high among all patients , including egfr + and alk + ( 3 - year estimates : 58% and 51% , respectively ) , despite treatment with platinum - based doublet chemotherapy in most patients . 
in addition , using a landmark analysis , we demonstrated that post - recurrence os was higher in patients receiving appropriate genotype - directed tki , reaffirming the efficacy of these therapies and the importance of clinical genotyping.29 these two findings together underscore the importance of continuing to test the optimal integration of tki with curative cmt to reduce dm and improve cure rates . 
in comparison , post hoc analyses of the radiant ( clinicaltrials.gov identifier : nct00373425 ) and br.19 trials of adjuvant tki after cmt in unselected patients have not shown pfs or os benefit in egfr + patients ; however , these trials are limited by inadequate power and possible biases.25 , 26 the effects of indication bias ( eg , genotyping at the time recurrence ) , appropriate wt control groups , and the impact of salvage tki in prolonging os are illustrated in our studys unique analysis of outcomes by genotyped versus ungenotyped status . 
 survival , given the uncertainties in therapeutic interactions and cross - resistance . the strengths of this study include ( 1 ) an analysis of outcomes in one of the largest cohorts of la - nsclc with genotype data available on the three most common genotypic subgroups reported to date , ( 2 ) multiple control groups including wt / / and ungenotyped patients to ascertain the effects of testing bias , ( 3 ) contemporary cmt with modern rt techniques representative of current standards of care , and ( 4 ) sufficiently long follow - up ( median follow - up in genotyped patients : 48 months ) to provide a full natural history of the genotypic subgroups after cmt . 
furthermore , this study was conducted in an era when clinical genotyping involved only a limited gene panel ( kras / egfr / alk ) , and thus , commutations in genes such as those critical in the biology of kras + tumors such as lkb1 / p53 could not be analyzed.35 the heterogeneity of kras + nsclc may account in part for our findings of no difference in outcomes by kras status , as opposed to the findings of a previous chemoradiation series showing worse outcomes with kras + .36 similarly , less common genotypic subgroups of nsclc with targetable mutations ( eg , ros1 rearrangements ) could not be analyzed in this cohort . 
larger studies analyzing tumor responsiveness to rt across a more comprehensive set of targetable genes are needed . the findings from this study will have implications for designing future prospective trials incorporating conventional cmt with targeted therapies for genotypic subgroups of la - nsclc . 
using the higher observed median pfs ( egfr + , 15.3 months and alk + , 13.7 months ) from our study for the control group , the revised sample size estimate would be 412 egfr + and 132 alk + patients ( data supplement )  . 
although the pfs point estimates from our study may not be entirely generalizable because of the sample size and the potential biases as outlined , this exercise illustrates the importance of obtaining accurate estimates of pfs for genotypic subgroups for trial design . these challenges underscore the need for cooperation among multiple centers to comprehensively genotype patients with la - nsclc who are undergoing conventional cmt to further understand the natural history and biology of genotypic subtypes of nsclc , and for the development of novel trial designs to integrate targeted therapies with conventional cmt . 
novel trial designs implemented in the metastatic setting , including basket trials such as nci - match ( clinicaltrials.gov identifier : nct02465060 ) , nci - mpact ( clinicaltrials.gov identifier : nct01827384 ) , and custom ( clinicaltrials.gov identifier : nct01306045 ) , 37 have some appeal but may be complicated by uncertainties regarding interactions between targeted therapies and existing curative modalities . 
thus , obtaining a more detailed understanding of clinical outcomes of rare genotypic subgroups treated with conventional therapy off - trial will be critical to inform the optimal combination therapy , in conjunction with preclinical testing of different combinations of targeted and conventional therapies in genetically engineered mouse models in co - clinical trials as described previously.38 , 39 in this series comparing outcomes in patients with egfr + , alk + , and kras + la - nsclc who were treated with rt as part of curative cmt , we observed differences in os ( increased in alk + ) and lrr ( decreased in egfr + )  . 
the risk of dm and progression were high in all subgroups , and post - recurrence os was higher among egfr + / alk + patients receiving appropriate targeted therapies , underscoring the need for additional integration of genotype - directed therapies with curative cmt and routine genotyping for la - nsclc . 
aerts leadership : sphera stock and other ownership interests : sphera consulting or advisory role : sphera , genospace research funding : varian medical systems travel , accommodations , expenses : genospace elizabeth h . 
swanson honoraria : ethicon , covidien / medtronic consulting or advisory role : armada health care research funding : ethicon ( inst ) yu - hui chen employment : constellation pharmaceuticals ( i ) paul catalano consulting or advisory role : eli lilly bruce e . 
johnson stock and other ownership interests : kew honoraria : chugai pharma consulting or advisory role : novartis , kew , merck , eli lilly , chugai pharmaceutical , boehringer ingelheim , amgen , astrazeneca research funding : novartis ( inst ) , glaxosmithkline ( inst ) , genentech / roche ( inst ) , genentech / roche ( inst ) , toshiba ( inst ) patents , royalties , other intellectual property : danafarber cancer institute pasi a . 
jnne stock and other ownership interests : gatekeeper pharmaceuticals consulting or advisory role : pfizer , boehringer ingelheim , astrazeneca , merrimack , ariad pharmaceuticals , chugai pharmaceutical , roche / genentech , loxo , mirati therapeutics , araxes pharmaceuticals , ignyta , eli lilly research funding : astrazeneca , astellas pharma , daiichi sankyo , eli lilly , boehringer ingelheim , puma biotechnology patents , royalties , other intellectual property : i am a co - inventor on a dana - farber cancer instituteowned patent on egfr mutations licensed to laboratory corp . 
 supported in part by a radiological society of north america ( rsna ) seed grant , department of radiation oncology joint center for radiation therapy ( jcrt ) , and clinical - translational and kaye grants . presented at the american society of radiation oncology annual conference , september 25 - 28 , 2016 , boston , ma . support prior presentation references 1 . 
pao w , miller v , zakowski m , et al : egf receptor gene mutations are common in lung cancers from never smokers and are associated with sensitivity of tumors to gefitinib and erlotinib . 
lynch tj , bell dw , sordella r , et al : activating mutations in the epidermal growth factor receptor underlying responsiveness of non - small - cell lung cancer to gefitinib . 
mitsudomi t , morita s , yatabe y , et al : gefitinib versus cisplatin plus docetaxel in patients with non - small - cell lung cancer harbouring mutations of the epidermal growth factor receptor ( wjtog3405 ) : an open label , randomised phase 3 trial . 
loriot y , mordant p , deutsch e , et al : are ras mutations predictive markers of resistance to standard chemotherapy ? nat rev clin oncol 6 : 528 - 534 , 2009 11 . 
cuffe s , bourredjem a , graziano s , et al : a pooled exploratory analysis of the effect of tumor size and kras mutations on survival benefit from adjuvant platinum - based chemotherapy in nodenegative non - small cell lung cancer . 
chaft je , rusch v , ginsberg ms , et al : phase ii trial of neoadjuvant bevacizumab plus chemotherapy and adjuvant bevacizumab in patients with resectable nonsquamous non - smallcell lung cancers . 
shepherd fa , domerg c , hainaut p , et al : pooled analysis of the prognostic and predictive effects of kras mutation status and kras mutation subtype in early - stage resected non - smallcell lung cancer in four trials of adjuvant chemotherapy . 
schiller jh , adak s , feins rh , et al : lack of prognostic significance of p53 and k - ras mutations in primary resected non - small - cell lung cancer on e4592 : a laboratory ancillary study on an eastern cooperative oncology group prospective randomized trial of postoperative adjuvant therapy . 
tsao ms , sakurada a , ding k , et al : prognostic and predictive value of epidermal growth factor receptor tyrosine kinase domain mutation status and gene copy number for adjuvant chemotherapy in non - small cell lung cancer . 
sun hb , ou w , li y , et al : epidermal growth factor receptor mutation status and adjuvant chemotherapy in resected advanced non - small - cell lung cancer . 
yagishita s , horinouchi h , katsui taniyama t , et al : epidermal growth factor receptor mutation is associated with longer local control after definitive chemoradiotherapy in patients with stage iii nonsquamous non - small - cell lung cancer . 
ahn hk , choi yl , han jh , et al : epidermal growth factor receptor mutation and treatment outcome of mediastinoscopic n2 positive non - small cell lung cancer patients treated with neoadjuvant chemoradiotherapy followed by surgery . 
akamatsu h , kaira k , murakami h , et al : the impact of clinical outcomes according to egfr mutation status in patients with locally advanced lung adenocarcinoma who received concurrent chemoradiotherapy . 
mak rh , doran e , muzikansky a , et al : kras mutation is associated with decreased overall survival after thoracic radiation therapy in patients with locally advanced non - small cell lung cancer . 
kelly k , altorki nk , eberhardt we , et al : adjuvant erlotinib versus placebo in patients with stage ib - iiia non - small - cell lung cancer ( radiant ) : a randomized , double - blind , phase iii trial . 
eberhard da , johnson be , amler lc , et al : mutations in the epidermal growth factor receptor and in kras are predictive and prognostic indicators in patients with non - small - cell lung cancer treated with chemotherapy alone and in combination with erlotinib . 
rusch vw , asamura h , watanabe h , et al : the iaslc lung cancer staging project : a proposal for a new international lymph node map in the forthcoming seventh edition of the tnm classification for lung cancer . 
das ak , chen bp , story md , et al : somatic mutations in the tyrosine kinase domain of epidermal growth factor receptor ( egfr ) abrogate egfr - mediated radioprotection in non - small cell lung carcinoma . 
das ak , sato m , story md , et al : non - small - cell lung cancers with kinase domain mutations in the epidermal growth factor receptor are sensitive to ionizing radiation . 
gow ch , chien cr , chang yl , et al : radiotherapy in lung adenocarcinoma with brain metastases : effects of activating epidermal growth factor receptor mutations on clinical response . 
neal jw , pennell na , govindan r , et al : the select study : a multicenter phase ii trial of adjuvant erlotinib in resected epidermal growth factor receptor ( egfr ) mutation - positive nonsmall cell lung cancer ( nsclc )  . 
li n , ou w , ye x , et al : pemetrexed - carboplatin adjuvant chemotherapy with or without gefitinib in resected stage iiia - n2 non - small cell lung cancer harbouring egfr mutations : a randomized , phase ii study . 
yagishita s , horinouchi h , sunami ks , et al : impact of kras mutation on response and outcome of patients with stageiii non - squamous non - small cell lung cancer . 
lopez - chavez a , thomas a , rajan a , et al : molecular profiling and targeted therapy for advanced thoracic malignancies : a biomarker - derived , multiarm , multihistology phase ii basket trial . 
chen z , akbay e , mikse o , et al : co - clinical trials demonstrate superiority of crizotinib to chemotherapy in alk - rearranged non - small cell lung cancer and predict strategies to overcome resistance . 
 programmed death - ligand 1 expression in a large cohort of pediatric patients with solid tumor and association with clinicopathologic features in neuroblastoma purpose programmed death - ligand 1 ( pd - l1 ) expression represents a potential predictive biomarker of immune checkpoint blockade response . 
however , literature about the prevalence of pd - l1 expression in the pediatric cancer setting is discordant . methods pd - l1 expression was analyzed using immunohistochemistry in 500 pediatric tumors ( including neuroblastoma , sarcomas , and brain cancers )  . 
positive cases were further characterized , with cases with weak intensity pd - l1 staining reported as having low pd - l1 expression and cases with a moderate or strong intensity of staining considered to have high pd - l1 expression . results pd - l1positive staining was identified in 13% of cases , whereas high pd - l1 expression was found in 3% of cases . 
2017 by american society of clinical oncology introduction in recent years , the ability of tumor cells to exert an immunosuppressive action in their local microenvironment has been well described.1 escaping the host immune system occurs via multiple strategies , including loss of tumor antigens or reduced expression of major histocompatibility complex molecules , as well as via recruitment of immunosuppressive inflammatory cells or inadequate costimulation of t cells.2 as a consequence , the ability to evade host immunity has been recognized as one of the hallmarks of cancer.3 the expression of immune inhibitory ligands such as programmed death - ligand 1 ( pd - l1 ) is another strategy used by cancer cells to inhibit the immunologic response.4 pd - l1 is a trans - membrane surface glycoprotein overexpressed in many solid tumors , including carcinomas5 - 8 and melanoma.9 moreover , pd - l1 is one of the two known ligands for programmed death ( pd ) - 1 , a receptor expressed by immune system cells ( including activated t cells , regulatory t cells , b cells , natural killer cells , activated monocytes , and dendritic cells ) that negatively regulates their proliferation federica saletta ricardo e . 
antibody - mediated blockade of pd - l1 induced objective response rates in 6% to 17% and prolonged disease stabilization in 12% to 41% of patients with advanced cancers , including nonsmall - cell lung cancer , melanoma , and renal cell cancer.16 similarly , cumulative response rates for pd - 1 mab immunotherapy were achieved in 18% of patients with nonsmall - cell lung cancer , 28% of patients with melanoma , and 27% of patients with renal - cell cancer.13 despite these promising results achieved in adult cancers , the prevalence of pd - l1 expression in the pediatric setting and the potential use of mabs directed against pd - 1 have only started to be explored . 
the 53 analyzed cases included 18 neuroblastomas , eight extracranial germ - cell tumors , seven germinomas , seven hepatoblastomas , four medulloblatomas , four renal tumors , three rhabdomyosarcomas , and two atypical teratoid / rhabdoid tumors . 
in contrast , chowdhury et al20 observed 66 of 115 pd - l1positive samples ( 57% , ranging from 47% to 86% , depending on tumor type ) in a multitumor study including neuroblastoma , rhabdomyosarcoma , ewing sarcoma , and osteosarcoma . 
alveolar rhabdomyosarcoma showed the highest prevalence of pd - l1positive cases ( 12 of 14 [ 86% ] ) and patients with high - risk neuroblastoma were commonly pd - l1 positive ( 31 of 43 [ 72% ] )  . no previous study has reported associations between pd - l1 staining and the features of pediatric patients with cancer . 
here we present the clinicopathologic features of and survival analysis associated with pd - l1 expression in patients with neuroblastoma , with particular focus on the potential relationship between pd - l1 expression and the risk of cancer relapse . 
we identified a subcohort of patients with pd - l1positive staining and poor outcome , suggesting that pd - l1 may be a negative prognostic factor associated with an elevated risk of cancer recurrence . methods patient cohort the retrospective pediatric cohort consisted of 500 patients who underwent diagnostic biopsy or surgical resection at the childrens hospital at westmead , new south wales , australia . 
formalin - fixed paraffin - embedded hematoxylin and eosinstained tumor samples were reviewed by a histopathologist , and approved samples were used to construct tissue microarrays ( tmas ) using a manual tissue arrayer ( beecher mta1 , diagnostic technology , belrose , new south wales , australia ) and a 1 - mm unitma premade recipient block ( ub06 - 1 , diagnostic technology , belrose , new south wales , australia )  . 
 immunohistochemistry each tma was sectioned at 4 mm , and immunohistochemistry was performed using a bondrx automatic stainer ( leica biosystems , macquarie park , new south wales , australia )  . 
slide deparaffinization was performed with bond dewax solution , followed by alcohol dehydration . epitope retrieval was performed using hier2 buffer ( bond , ar9540 ) , and slides were incubated for 60 minutes at room temperature using a rabbit monoclonal antipd - l1 primary antibody ( e1l3n , no.13684 , cell signaling technology , genesearch , arundel , queensland , australia ) at 1 : 500 dilution in primary antibody diluent ( bond , ar9352 )  . 
staining was visualized using a polymer refine detection kit ( bond , ds9800 ) , and nuclei were counterstained with hematoxylin . tumors with > 1% of cells showing pd - l1 membrane staining ( or a combination of membrane and cytoplasmic staining ) of any intensity were considered pd - l1 positive . 
pd - l1 intensity was scored as weak , moderate , or strong by a histopathologist ( r.e.v. ) with specific expertise in pd - l1 staining assessment in patients with melanoma . 
similarly , fibrillary and stromal pd - l1 staining was also observed occasionally but was not scored positively because the significance of nonmembranous pd - l1 staining remains unclear . statistical analyses statistical analyses were undertaken using spss version 20.0 ( spss , chicago , il )  . 
bivariate relationships between pd - l1positive cases and patient features were analyzed with cross - tabulation , and significance was tested with fishers exact test . overall survival and relapse or progression - free survival were assessed using the kaplan - meier method , and significance was established using a log - rank ( mantel - cox ) test . 
statistical significance was set at p , .05 for two - sided tests . results prevalence of pd - l1positive pediatric patients with cancer as summarized in table 1 , in the overall cohort , 65 ( 13.0% ) of 500 cases were found to be pd - l1 positive . 
of these , 50 ( 76.9% ) of 65 showed weak pd - l1 intensity , whereas 15 ( 23.1% ) of 65 showed moderate to strong pd - l1 intensity . 
as such , in the overall cohort , only 15 ( 3.0% ) of 500 cases had moderate or strong pd - l1 positivity . neuroblastoma cases showed pd - l1 expression more commonly ( 48 of 254 [ 18.9% ] ) than did sarcomas or brain tumors . 
of the 48 positive cases , 40 ( 83.3% ) of 48 were weakly positive for pd - l1 , whereas eight ( 16.7% ) of 48 were moderately to strongly pd - l1 positive . 
overall , the distribution of weakly versus moderately or strongly positive pd - l1 staining in the neuroblastoma cohort was 15.7% ( 40 of 254 ) and 3.1% ( eight of 254 ) , respectively . 
in the brain tumor cohort , six ( 4.4% ) of 136 cases stained positively for pd - l1 , of which three ( 50.0% ) of six showed moderate to strong pd - l1 positivity . 
of the 96 sarcoma samples , seven ( 7.3% ) of 96 stained positively for pd - l1 , with only one of 96 cases ( an undifferentiated sarcoma ) being moderately positive . 
of the lymphoma cases , three ( 50% ) of six were pd - l1 positive ; two of these ( two of three [ 66.7% ] ) were moderately or strongly positive . 
one nasopharyngeal carcinoma showed moderate to strong pd - l1 staining . pd - l1 immunohistochemistry staining pattern observed although in this study only membranous pd - l1 staining was considered positive , together with the occasional combination of both membranous and cytoplasmic positive staining ( n = 5 ) , a range of other staining patterns was observed . 
an example of each pattern is presented in figure 1 ; they included membranous staining in the placenta as a positive control ( fig 1a ) , membranous tumor staining of weak , moderate , and strong intensity ( figs 1b , 1c , and 1d , respectively ) , as well as the combination of both membranous and cytoplasmic tumor pd - l1positive staining ( fig 1e )  . 
pd - l1 positive cytoplasmic ( fig 1f ) , fibrillary ( fig 1g ) , and schwannian stromal staining ( fig 1h ) were also observed , together with pd - l1positive macrophage ( fig 1i ) , but they were scored as negative . cytoplasmic pd - l1 staining was detected in 30 ( 11.8% ) of 254 neuroblastoma cases , ranging from weak ( n = 24 [ 80.0% ] ) to moderate or strong ( n = 6 [ 20.0% ] ) ; however , these cases were not considered pd - l1 positive . 
exclusive macrophage pd - l1 staining ( without pd - l1 positivity in tumor cells ) was detected in one ( 5.6% ) of 18 rhabdomyosarcomas , one ( 3.3% ) of 30 osteosarcomas , one ( 0.4% ) of 254 neuroblastomas , and one ( 2.6% ) of 38 lowgrade gliomas . 
the presence of macrophages was verified via hematoxylin and eosin staining observation . association with clinicopathologic features in pd - l1positive neuroblastoma cases information concerning a number of clinicopathologic features of the broader pediatric cohort was collated and tested ( using a two - sided fishers exact test ) for nonrandom associations with pd - l1 staining . 
interestingly , no differences in pd - l1 staining were found between biopsy specimens obtained before and after radiotherapy ( p = .702 ) , but a significant association between pd - l1 positivity and biopsy specimens obtained before chemotherapy was observed ( p = .044 ) , contrary to the suggestion of uehara et al.18 other clinicopathologic features that define patient prognosis in the neuroblastoma population at diagnosis include the mitosis - karyorrhexis index , shimada classification , mycn status , and overall patient risk ( table 2 )  . 
next , we sought to analyze whether pd - l1positive neuroblastoma cases were associated with cancer recurrence or progression ; however , no significant ( p = .939 ) association was observed ( fig 2b )  . 
although we did not find an association ( p = .508 ) between moderate to strong pd - l1 staining and overall survival ( fig 2c ) , the association with poorer relapse - free survival in this subcohort was significant ( p = .002 , fig 2d )  . 
programmed death - ligand 1 ( pd - l1 ) immunohistochemistry staining patterns . representative images of pd - l1 staining ( in brown ) and counterstained nuclei ( in blue )  . 
black arrows highlight examples of ( a ) pd - l1 staining in placenta used as positive control tissue ; pd - l1 membranous staining of ( b ) weak , ( c ) moderate , and ( d ) strong intensity in lymphoma ; and ( e ) pd - l1positive staining in both membrane and cytoplasm in neuroblastoma . 
 ( f ) exclusive cytoplasmic pdl1 staining and ( g ) pdl1positive schwannian stroma in neuroblastoma were considered negative . ( h ) pd - l1positive fibrillary staining in medulloblastoma and ( i ) pd - l1positive macrophages in osteosarcoma were also scored as negative . 
there were no differences in overall survival ( p = .298 ) or relapse - free survival ( p = .507 ) according to pd - l1 positivity of any intensity ( figs 3a and 3b )  . 
when restricting the analysis to the lowand intermediate - risk neuroblastoma cohort with moderate to strong pd - l1 staining , we found no association ( p = .524 ) with overall survival ( fig 3c ) but significantly ( p , .001 ) poorer relapse - free survival ( fig 3d )  . discussion since discovering that pd - 1 / pd - l1 binding promotes cancer tolerance by blocking the t - cell antigen receptor - induced stop signal , 22 substantial research effort has been aimed at targeting this interaction . 
a list of the pd - l1 antibodies and the staining and scoring methods used by other research groups is summarized in table 3 , which highlights the need for standardized immunohistochemistry methodology and a standardized scoring system for pd - l1positive cases . 
 ( a ) and ( b ) show overall survival and relapsefree survival ( in months ) in association with pd - l1 positivity ( of any intensity ) , respectively . 
 ( c ) and ( d ) show overall survival and relapse - free survival ( in months ) in association with moderate or strong pd - l1 staining intensity , respectively . 
for example , in a glioblastoma study , there was no association between fibrillary pd - l1 detection and overall survival in patients with glioblastoma.31 in our cohort , pd - l1 fibrillary staining was observed in only one of 38 medulloblastoma biopsy specimens , whereas pd - l1 schwannian stromal staining was detected in twelve of 254 patients with neuroblastoma . 
associations between programmed death - ligand 1 ( pd - l1 ) status and survival of patients with neuroblastoma ( nb ) with low and intermediate risk . kaplan - meier plots compare patient survival ( in months ) in the lowand intermediate - risk nb cohort . 
 ( a ) and ( b ) show overall survival and relapsefree survival ( in months ) in association with pd - l1 positivity ( of any intensity ) , respectively . 
 ( c ) and ( d ) show overall survival and relapse - free survival ( in months ) in association with moderate or strong pd - l1 staining intensity , respectively . 
previous reports have shown a discordant association between myc oncogene status and pd - l1 expression . in fact , casey et al36 stated that myc regulates the antitumor immune response through cd47 and pd - l1 in melanoma and nonsmall - cell lung cancer cell lines , whereas dondero et al37 showed that pd - l1 expression in neuroblastoma was interferon - gamma dependent and mycn independent . 
because strong pdl1 staining has been shown to correlate with an elevated mutation burden , 26 we can speculate that these cases may share an underlying dna repair deficit that results in an increased presence of neoantigens and particularly high pd - l1 expression . 
more studies are necessary to verify this hypothesis and to correlate mutation burden in children with stronger pd - l1 staining and a higher likelihood of cancer relapse despite favorable clinical features . pd - l1 may represent a novel predictive marker of cancer recurrence that could be used to identify the proportion of children who may benefit from pd - 1 / pd - l1 mab therapy . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . geoffrey mccowage research funding : novartis ( inst ) , merck ( inst ) acknowledgment we thank the patients and families who agreed to donate samples to the childrens hospital at westmead tumour bank . 
we also thank michael krivanek , md , of childrens hospital at westmead for his help with the histologic review of patient samples before tissue coring for tma construction , and jason madore of royal prince alfred hospital for his help in optimizing the immunohistochemistry protocol and staining interpretation . federica saletta no relationship to disclose ricardo e . 
blank c , gajewski tf , mackensen a : interaction of pd - l1 on tumor cells with pd - 1 on tumor - specific t cells as a mechanism of immune evasion : implications for tumor immunotherapy . 
karim r , jordanova es , piersma sj , et al : tumor - expressed b7 - h1 and b7 - dc in relation to pd - 1 + t - cell infiltration and survival of patients with cervical carcinoma . 
thompson rh , kuntz sm , leibovich bc , et al : tumor b7 - h1 is associated with poor prognosis in renal cell carcinoma patients with long - term follow - up . 
madore j , vilain re , menzies am , et al : pd - l1 expression in melanoma shows marked heterogeneity within and between patients : implications for anti - pd - 1 / pd - l1 clinical trials . 
freeman gj , long aj , iwai y , et al : engagement of the pd - 1 immunoinhibitory receptor by a novel b7 family member leads to negative regulation of lymphocyte activation . 
topalian sl , hodi fs , brahmer jr , et al : safety , activity , and immune correlates of anti - pd - 1 antibody in cancer . n engl j med 366 : 2443 - 2454 , 2012 14 . 
chowdhury f , dunn s , mitchell s , et al : pd - l1 and cd8 + pd1 + lymphocytes exist as targets in the pediatric tumor microenvironment for immunomodulatory therapy . 
fife bt , pauken ke , eagar tn , et al : interactions between pd - 1 and pd - l1 promote tolerance by blocking the tcr - induced stop signal . 
saletta f , wadham c , ziegler ds , et al : molecular profiling of childhood cancer : biomarkers and novel therapies . 703 - 718 , 2013 bba clin 1 : 59 - 77 , 2014 26 . 
madore j , strbenac d , vilain r , et al : pd - l1 negative status is associated with lower mutation burden , differential expression of immune - related genes , and worse survival in stage iii melanoma . 
mu cy , huang ja , chen y , et al : high expression of pd - l1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation . 
yang cy , lin mw , chang yl , et al : programmed cell death - ligand 1 expression in surgically resected stage i pulmonary adenocarcinoma and its correlation with driver mutations and clinical outcomes . 
preusser m , berghoff as , wick w , et al : clinical neuropathology mini - review 6 - 2015 : pd - l1 : emerging biomarker in glioblastoma ? clin neuropathol 34 : 313 - 321 , 2015 32 . 
taube jm , klein a , brahmer jr , et al : association of pd - 1 , pd - 1 ligands , and other features of the tumor immune microenvironment with response to anti - pd - 1 therapy . 
 c prolonged partial response to bevacizumab and valproic acid in a patient with glioblastoma introduction glioblastoma is the most common and malignant type of primary cns tumor.1 , 2 the estimated median overall survival ( os ) duration ranges from 14 months to 16 months , whereas the 5 - year os rate is estimated to be 5%.1 , 3 - 5 chemoradiation followed by adjuvant temozolomide remains the standard frontline therapy.3 bevacizumab has been used as a single agent or in combination with other agents at the time of diagnosis or at disease progression.6 - 11 in patients with newly diagnosed glioblastoma , the addition of bevacizumab to standard treatment regimens did not improve os , which has resulted in limited use.11 in recurrent glioblastoma , bevacizumab monotherapy was associated with higher rates of response ( 28% ) and 6 - month progression - free survival ( pfs ; 29% to 43% ) compared with historical data with radiotherapy and other regimens ( 4% to 9% and 9% to 21% , respectively ) .11 in recent years , immunotherapy has demonstrated promising results in selected patients , although long - term os data are unavailable.12 - 16 valproic acid is a histone deacetylase inhibitor ( hdi ) that has antitumor activity associated with antiangiogenic effects.17 in in vitro and in vivo animal models , hdis upregulated p53 and von hippel - lindau and downregulated hif - 1 and vascular endothelial growth factor ( vegf ) , which inhibits angiogenesis.17 hdis also inhibit vegf - stimulated endothelial cells and angiogenesis , and their antitumor activity increases in combination with a vegf receptor tyrosine kinase inhibitor.18 we have previously reported on the safety of bevacizumab and valproic acid in patients with advanced cancer.19 here , we present a patient with progressive glioblastoma in whom prolonged partial response ( pr ) was achieved with bevacizumab and valproic acid . 
magnetic resonance imaging ( mri ) of the brain revealed a 5.1 - cm mass in the left parietal lobe , peripheral enhancement with edema , and mass effect on the left lateral ventricle . 
in july 2011 , the patient underwent a partial tumor resection followed by intensity - modulated radiation therapy ( total dose , 59.4 gy in 33 fractions ; completed september 2011 ) with concurrent administration of temozolamide . 
mri of the brain in september 2011 demonstrated disease progression and temozolamide was discontinued . in november 2011 , the patient referred himself to our phase i program and was enrolled in a clinical trial of a poly ( adp - ribose ) polymerase inhibitor and temozolamide . 
after three cycles , treatment with the phosphatidylinositol - 3 kinase inhibitor was discontinued because of disease progression . in june 2012 , the patient returned to our clinic and was enrolled in a clinical trial of bevacizumab , temsirolimus , and valproic acid ( clinicaltrials . gov identifier : nct01552434 )  . 
bevacizumab 5 mg / kg was administered iv on days 1 and 15 , valproic acid 5 mg / kg orally once per day , and temsirolimus 20 mg iv once per week . 
he required interruption of bevacizumab after cycle 33 because he underwent transurethral prostate resection as a result of an abscess that was not responding to drainage or iv antibiotics and after cycles 44 and 55 because of transient grade 2 proteinuria . 
in june 2018 , molecular profiling was retrospectively performed on the patients tumor tissue obtained in july 2011 using a 595 - gene panel ( table 1 )  . discussion despite various treatment options , the prognosis of patients with advanced glioblastoma is poor.22 the efficacy of standard treatments is suboptimal and patients are encouraged to participate in clinical trials . 
his tumor had multiple alterations , including cyclin - dependent kinase 4 ( cdk4 ) amplification and fgfr3tacc3 chromosomal fusion . glioblastomas harbor several molecular alterations implicated in the activation of oncogenic pathways.23 - 25 amplification of cdk4located at chromosome 12is common in glioblastoma , 26 , 27 and its incidence was 13% in a comprehensive analysis.25 our patient was treated with valproic acid , which suppresses cdk4 , among other proteins , and induces growth inhibition , apoptosis , and senescence in medulloblastoma cell lines.28 valproic acid was demonstrated to inhibit the proliferation of mcf - 7 breast cancer cells via apoptosis and g1 phase arrest , possibly by downregulating cyclin d1 , 29 which , in turn , decreased cdk4 protein levels.30 other investigators demonstrated that valproic acid significantly reduced mrna expression of cyclin d1 and cdk4 in leukemic cells used in a murine xenograft tumor model , which leads to g0 / g1 arrest.31 cdk4 amplification was found in tumors with fgfr3tacc3 fusions and it was more frequent in isocitrate dehydrogenase wild - type gliomas that harbored fgfr3 - tacc3 fusions than in those without the fusion.32 the fgfr3 gene fuses with multiple partners , including the tacc3 gene , which encodes a protein that participates in the stabilization of the mitotic spindle . 
magnetic resonance images of the brain [ t1 - weighted imaging post gadolinium contrast ; axial plane ( a and b ) , coronal ( c ) ] at ( 1 ) baseline and ( 2 ) after first restaging after two cycles of treatment with bevacizumab and valproic acid . 
formalin - fixed , paraffin - embedded samples were sequenced to detect somatic single - nucleotide variants , insertion - deletions , copy number variants , gene rearrangements , and microsatellite instability . 
we also performed a tempus rna whole - transcriptome panel and used this for expanded detection of gene rearrangements , immune infiltration , and gene expression for immune and targeted therapy related genes . abbreviation : cdk4 , cyclin - dependent kinase 4 . regulates cell growth , differentiation , and tumor neoangiogenesis.38 - 40 our patient was also treated with bevacizumab , an anti - vegf agent . 
ostrom qt , gittleman h , liao p , et al : cbtrus statistical report : primary brain and other central nervous system tumors diagnosed in the united states in 2010 - 2014 . 
stupp r , hegi me , mason wp , et al : effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase iii study : 5 - year analysis of the eortc - ncic trial . 
kreisl tn , kim l , moore k , et al : phase ii trial of single - agent bevacizumab followed by bevacizumab plus irinotecan at tumor progression in recurrent glioblastoma . 
taal w , oosterkamp hm , walenkamp am , et al : single - agent bevacizumab or lomustine versus a combination of bevacizumab plus lomustine in patients with recurrent glioblastoma ( belob trial ) : a randomised controlled phase 2 trial . 
omuro a , vlahovic g , lim m , et al : nivolumab with or without ipilimumab in patients with recurrent glioblastoma : results from exploratory phase i cohorts of checkmate 143 . 
van den bent m , french p , eoli m , et al : updated results of the intellance 2 / eortc trial 1410 randomized phase ii study on depatux - m alone , depatux - m in combination with temozolomide ( tmz ) and either tmz or lomustine ( lom ) in recurrent egfr amplified glioblastoma ( nct02343406 )  . 
qian dz , wang x , kachhap sk , et al : the histone deacetylase inhibitor nvp - laq824 inhibits angiogenesis and has a greater antitumor effect in combination with the vascular endothelial growth factor receptor tyrosine kinase inhibitor ptk787 / zk222584 . 
wheler jj , janku f , falchook gs , et al : phase i study of anti - vegf monoclonal antibody bevacizumab and histone deacetylase inhibitor valproic acid in patients with advanced cancers . 
verhaak rg , hoadley ka , purdom e , et al : integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in pdgfra , idh1 , egfr , and nf1 . 
reifenberger g , reifenberger j , ichimura k , et al : amplification of multiple genes from chromosomal region 12q13 - 14 in human malignant gliomas : preliminary mapping of the amplicons shows preferential involvement of cdk4 , sas , and mdm2 . 
li xn , shu q , su jm , et al : valproic acid induces growth arrest , apoptosis , and senescence in medulloblastomas by increasing histone hyperacetylation and regulating expression of p21cip1 , cdk4 , and cmyc . 
ma xj , wang ys , gu wp , et al : the role and possible molecular mechanism of valproic acid in the growth of mcf - 7 breast cancer cells . 
yu b , lane me , pestell rg , et al : downregulation of cyclin d1 alters cdk 4and cdk 2 - specific phosphorylation of retinoblastoma protemol cell biol res commun 3 : 352 - 359 , 2000 31 . 
bao zs , chen hm , yang my , et al : rna - seq of 272 gliomas revealed a novel , recurrent ptprz1 - met fusion transcript in secondary glioblastomas . 
fons p , gueguen - dorbes g , herault jp , et al : tumor vasculature is regulated by fgf / fgfr signaling - mediated angiogenesis and bone marrow - derived cell recruitment : this mechanism is inhibited by ssr128129e , the first allosteric antagonist of fgfrs . 
giavazzi r , sennino b , coltrini d , et al : distinct role of fibroblast growth factor - 2 and vascular endothelial growth factor on tumor growth and angiogenesis . 
 homologous recombination deficiency in breast cancer : a clinical review brca1 and brca2 germline mutationassociated breast cancers are known to be deficient in the process of homologous recombination and often respond favorably to drugs targeting this important dna repair pathway . 
there is emerging evidence that a significant proportion of patients with brca1 / brca2 wild - type breast cancer are also deficient in homologous recombination , and it is hypothesized that these patients may derive similar benefit from drugs targeting this pathway . 
this review outlines the various approaches that researchers have taken to predict homologous recombination deficiency as part of correlative biomarker work in various studies and clinical trials in breast cancer . 
recent evidence demonstrates that parp is important for resolving stalled replication forks , and its inhibition during base excision repair requires brca - dependent hr to resolve.3 , 4 targeting dna damage response pathways has emerged as an attractive strategy to destabilize tumor genomic integrity and trigger genomic catastrophe and cell death in a tumor - specific fashion . the role of brca1 / brca2 in double - strand dna repair via hr has been well documented , 5 and there is mounting evidence that breast cancers arising in brca1 / brca2 germline mutation carriers respond favorably to therapies that target dna repair pathways , such as platinum salts and parp inhibitors ( parpis ) .6 - 8 hereditary brca1 / brca2 mutations account for up to 7% of all unselected breast cancers , 9 increasing to 11% to 15% of unselected individuals with triple - negative breast cancer ( tnbc : estrogen receptor [ er ] , progesterone receptor [ pr ] , human epidermal growth factor receptor 2 [ her2 ] negative ) and as high as 50% with a strong family history.10 - 12 brca1 mutations are most commonly associated with the tnbc clinical subtype , whereas brca2 mutations are more frequently associated with erpositive tumors . 
some sporadic ( ie , brca1 / brca2 wild - type [ wt ] ) breast cancers also harbor defects involving the hr pathway and respond similarly to platinum salts . 
 ( a ) somatic mutations , including substitutions and small insertions / deletions in key hr - related genes , such as brca1 and brca2 , can be associated with hrd . 
these include telomeric allelic imbalance ( tai ; large allelic imbalances extending into a telomere ) , large - scale transition ( lst ; number of transitions between large regions of differing allelic states ) , and loss of heterozygosity ( loh ; large regions displaying somatic loss of one haplotype , which can be copy variable as in deletion loh or copy neutral )  . current research has focused on the development of a companion diagnostic to identify sporadic brca - like tumors that would allow clinicians to identify those patients who may benefit from drugs targeting dna repair pathways and to spare those who are unlikely to benefit . 
driver germline ( inherited ) or somatic ( acquired ) mutations may take the form of sequence or structural variants that generally result in loss of function or aberrant functioning of brca1 / brca2 or other genes encoding members of the hr pathway . 
the genes and their protein products involved in hr are numerous , and their interactions are complex . germline mutations in hr - associated genes in addition to germline brca1 / brca2 mutations , clinical genetic testing panels now include a number of proposed breast cancer predisposition genes , although not all of these genes have definitively been shown to increase breast cancer risk . 
other hereditary predisposition genes involved in hr that are proven to be moderate to high risk include palb2 , atm , and chek2 . more recently , bard1 and rad51d have been shown to increase breast cancer risk , whereas some genes ( nbn , mre11a , rad50 , rad51c , brip1 ) are unlikely or confirmed not to increase breast cancer risk.15 interestingly , some of these hr genes do increase the risk of ovarian cancer , suggesting that the underlying biology of breast and ovarian cancer is different despite the importance of the role of hrd in both of these malignancies . although considered brca - like , it is not clear if breast cancers arising from these germline mutations are as sensitive to dna - damaging therapies as brca1 / brca2 - mutated breast cancers in large part as a consequence of the rarity and relative recent discovery of these mutations.16 incidence and accurate risk estimates are emerging for non - brca germline mutations , but the prognosis and response to anticancer therapy remain unknown.15 , 17 somatic mutations in hr genes somatic mutations may also arise in genes involved in hr . 
as in ovarian cancer , where somatic mutations in brca1 / brca2 occur in up to 7% of patients , 19 somatic brca1 / brca2 mutations constitute up to 3% of unselected patients with breast cancer20 and likely contribute to the brca - like phenotype , where dna - damaging therapy may also be effective . 
the extent to which these hr genes drive tumorigenesis continues to be explored , but it is likely that they too contribute to the brca - like phenotype . looking beyond germline brca1 / brca2 mutations has implications in terms of choosing patients who stand to benefit from dna - damaging therapies , most notably parpis . 
how this can be achieved is not certafor example , in ovarian cancer , the parpi niraparib resulted in a significant increase in progression - free survival irrespective of brca1 / brca2 mutation status or hrd status ( by myriads mychoice hrd , salt lake city , ut ) for patients with recurrent and highly platinum - sensitive disease.22 unlike in ovarian cancer , parpis in nonbrca1 / brca2mutated breast cancer have shown little activity to date , likely highlighting a different underlying biology , although patient selection may also be a factor . 
early - phase clinical trials of parpis in metastatic tnbc and brca - wt her2 - negative breast cancer are underway , with correlative hrd assessment ( clinicaltrials.gov identifiers : nct02401347 and nct00707707 )  . gene panels of hrd as researchers began to understand the complex nature of hrd , with contributions beyond brca1 / brca2 function , the focus shifted to characterization of brca1 - associated gene expression patterns to identify sets of differently expressed genes . 
seventy - seven of the 115 her2 - negative cohort were predicted to have dna repair deficiency by one of these signatures or by the presence of brca1 / brca2 germline mutations ( 56 of 59 tnbc and 21 of 56 er positive )  . 
thus , the genes involved in hr are not yet well defined , and equal weighting may overestimate hrd , as seen in a study of ovarian cancer.25 similarly , predictive model systems based on an empirical set of genes differentially expressed in brca1 / brca2mutated breast cancers are unlikely to clearly define hrd . 
these caveats represent major limitations to the hr panels currently being developed . epigenetic changes brca1 promoter methylation ( pm ) , which is unique to brca1 and mutually exclusive of germline brca1 mutations , 26 has also been implicated in hrd ( fig 1b )  . 
the importance of brca1 pm alone in this study is difficult to interpret , and results may be explained by deficiencies in brca1 as a result of germline mutations or by mrna expression . 
currently , the role of brca1 pm in hrd remains controversial . copy number aberrations copy number aberration / alteration ( cna ) refers to acquired changes in copy number of genes in tissue , such as tumor , whereas copy number variant ( cnv ) refers to changes in copy number of genes in the germline , affecting all cells in an individual . 
cnvs may also be reported in cancer , although they are usually qualified with the term acquired as compared with constitutional , so as to differentiate between the somatic and germline settings . 
in contrast to entire chromosome number gains or losses ( ie , aneuploidy ) , cna / cnvs are on a much smaller , generally submicroscopic , scale , with the size of dna copy - number alterations ( gain or loss ) being  . 
although the extent to which cnas contribute to tumorigenesis is not entirely known , some of the well - established driver events in cancer are cnas ( eg , myc , her2 , cyclin d1 )  . 
furthermore , an increased burden of cnas is associated with higher genomic instability and subsequent malignant transformation.31 using dna microarray technologies , such as array comparative genomic hybridization ( acgh ) , entire genome imbalances can be identified . 
this technique compares test dna and control dna , allowing the relative fluorescent intensity to be quantified , providing information on relative copy number sequences in the test genome versus the control genome.32 clinically , acgh was first reported using a classifier of brca1 - like tumors on the basis of cna in brca1 - mutated breast cancers.33 this study included 230 patients with stage iii her2negative breast cancer randomly assigned to adjuvant high - dose platinum - based or anthracyclinebased chemotherapy . 
all were statistically significant , and test for interaction was positive . studies of nonplatinum - containing regimens have not shown a benefit in patients with brca1 - like signatures identified by this method . a study in patients with tnbc treated with adjuvant ac ; fec ; docetaxel , doxorubicin , cyclophosphamide ; or cyclophosphamide , methotrexate , fluorouracil chemotherapy found that 65% had brca1 - like disease but with no statistically significant difference in 5 - year rfs in any of the treatment arms.35 similarly , a neoadjuvant study of dose - dense ac or capecitabine / docetaxel in 163 her2 - negative breast cancers showed tumors identified as brca1 - like by this method were not predictive of chemotherapy response even though present in 57% of patients with tnbc.36 these studies suggest that brca1 - like tumors , as defined by acgh , are most commonly associated with tnbc , and platinum - containing regimens are beneficial in those with a brca1 - like signature , whereas nonplatinum - containing regimens fail to improve responses over those without a brca1 - like signature . 
however , the above studies used unusual regimens of high - dose platinum and intensified alkylating agents that are not standard of care , and the response seen in the brca1like group may be due to intensified alkylator therapy . 
the acgh classifier also failed to identify some patients with brca1 / brca2 germline mutations , making it a promising but incomplete test of hrd . structural rearrangements inversions , translocations , and recombination change the location or orientation of a dna sequence.14 translocations result in the exchange of dna between nonhomologous regions of dna . 
recombination results in exchange of dna between homologous regions of dna , and this structural rearrangement may lead to loss of heterozygosity ( loh ) , a gross chromosomal event that results in the loss of entire genes ( eg , brca1 , brca2 )  . two types of acquired loh are important to note : deletion loh , where there is a copy number loss ; and copy numberneutral loh , where the absolute copy number remains the same ( fig 1d )  . 
both deletion and copy number neutral loh , as with cnas , lead to allelic imbalance that can be inferred by studying singlenucleotide common variation across the genome ( single - nucleotide polymorphism [ snp ] analysis ) ; this may be in the form of other types of dna microarrays that may be fully or partially based on snp probes across the genome . three tests of structural rearrangements have come to the forefront : telomeric allelic imbalance ( tai ) , large - scale transition ( lst ) , and loh . tai was developed using a snp genotype array platform37 to detect the number of chromosomal regions with allelic imbalance extending to the subtelomere , a common genomic abnormality that leads to an unequal contribution of maternal and paternal dna sequence but does not necessarily change overall dna copy number . 
breakpoints of tai regions were nonrandom and enriched for cnvs , which results in an imbalance and then leads to hrd , which may result in platinum sensitivity in the way that brca1associated cancer responds . 
allelic imbalance was the best predictor of cisplatin sensitivity after identifying associations between a variety of subchromosomal abnormalities and cisplatin sensitivity , and tai predicted higher rates of pcr in 79 patients enrolled in the neoadjuvant cisplatin - 1 ( monotherapy ) and cisplatin - 2 ( cisplatin plus bevacizumab ) tnbc trials . lst measures chromosomal breaks between adjacent chromosomal regions of at least 10 mb . 
lst was developed using snp array analysis on 65 patients with basal - like ( an intrinsic breast cancer subtype that overlaps with approximately 80% of tnbc ) breast cancer to define brca1 - associated genomic instability.38 lst was predictive of brca1 status and identified 85% of proven brca1 - inactivated cases . 
15 mb and less than a whole chromosome and was recognized as a discriminatory assay in two independent data sets of ovarian tumors.39 loh was more frequently identified in tumors with defective brca1 / brca2 and detected hrd regardless of etiology . 
the combination of all three scores has been shown to correlate with brca1 / brca2 deficiency in all breast cancer subtypes and captures more brca1 / brca2 deficiency information than the individual scores.27 in the geparsixto study42 of neoadjuvant paclitaxel / liposomal doxorubicin with or without carboplatin , the unweighted sums of loh , tai , and lst , in addition to brca1 / brca2 mutation in the primary tumor ( n = 193 ) , found hrd in 70.5% of tnbc tumors ; 60.3% without a brca mutation in the primary tumor were hrd high . 
in the phase iii treating to new targets ( tnt ) study , patients with metastatic tnbc were treated with carboplatin or docetaxel.8 the dichotomized score did not select those patients who would be sensitive to carboplatin versus docetaxel . 
the response rate was 38.2% in patients with hrd - high disease treated with carboplatin versus 42.6% in patients with hrd - high disease treated with docetaxel ; response rate was 29.2% in patients with hrdlow disease treated with carboplatin , versus 34.7% in patients with hrd - low disease treated with docetaxel . 
there is a paucity of published data on the ability of this assay to discriminate between hr - defective tumors and hr - intact tumors on the basis of tissue from a metastatic lesion . 
herein lies a possible explanation for the discrepancy of findings from the tnt trial , in that the assay may be measuring a scar of what has been but does not necessarily reflect what is occurring in the cell at present . 
to determine hr function , viable cells first need to be subjected to dna - damaging insults ( eg , radiation ) followed by assessment of hr . formation of rad51 foci is a functional , or live , assessment of a cells ability to perform hr . 
one study showed that low rad51 scores were more common in tnbc and associated with high grade and high ki - 67 and strongly predicted pcr.46 in this study of sporadic breast cancers receiving anthracycline - based chemotherapy , 68 patients underwent biopsies 24 hours post chemotherapy with rad51 focus formation assessed by immunofluorescence . 
in another study , 54 her2negative fresh primary breast tumors underwent ex vivo irradiation followed by analysis of the formation of ionizing radiationinduced foci of rad51 , and those with impaired foci formation underwent further genetic and epigenetic analysis.47 again , this hr defect was significantly associated with tnbc , but only five of 45 tumors with sufficient numbers of proliferating tumor cells were rad51 - formation deficient . 
more recently , functional assessment of rad51 foci formation in response to ex vivo irradiation coupled with whole - exome sequencing to determine structural genomic alterations ( ie , genomic scar ) showed that the combination of deficient rad51 foci formation and elevated lst or dna repair mutational panel may identify genetic alterations of an hr gene as a result of somatic or germline mutation.48 none of these studies reported outcomes , and they are limited by small numbers , but functional assessment of hr is promising and further studies are warranted . discussion the ability to consistently measure clinically meaningful deficiencies in hr has proven a difficult task . 
developing a reliable biomarker will be key to identifying patients with hrd tumors who may benefit from hrd - targeted therapies . studies attempting to use gene panels known to be involved in hr make sense mechanistically , but it is clear that not all genes are equal in the process , and basing a score on a composite of these genes may overestimate hrd . 
similarly , identifying brca1 pm has not consistently been shown to be useful in defining hrd tumors , as highlighted in the study where patients with either germline brca1 / brca2 mutations , low brca1 mrna expression , or brca1 pm were analyzed together . although platinum - based neoadjuvant chemotherapy resulted in improved os for the entire cohort , the impact brca1 pm had on clinical outcomes remains unclear . the acgh classifier was able to show that patients with brca - like tumors had significantly better rfs and os when treated with high - dose platinum - based chemotherapy versus conventional anthracycline - based chemotherapy . 
however , in the subset of patients with tnbc , the acgh classifier failed to identify nearly 40% of patients with a germline brca1 / brca2 mutation , and the experimental high - dose alkylator chemotherapy regimens used in this study are not standard and confound the findings of this study . more recent tests of hrd that are based on snp analysis have been combined in various ways , such as the myriad genetics mychoice hrd assay ( hrd - tai , hrd - loh , hrd - lst )  . 
this assay is the most developed for clinical use , with promising results in platinum - based neoadjuvant clinical trials of breast cancer with the primary end point of pcr . 
even when patients with known germline brca1 / brca2 were removed from analysis , those with high scores had better rates of pcr . in the metastatic setting , the mychoice hrd assay failed to select those patients who may benefit from platinum - based therapy in the tnt study . 
this discrepancy supports emerging evidence that early - stage breast cancer and advanced disease are different entities whereby treatment - resistant clones emerge in advanced disease , resulting in different tumor characteristics that may involve reversions from an hrdeficient state to an hr - efficient state . 
thus , the approach to identify biomarkers will need to take temporal tumor evolution into consideration . hrd assays that have been developed to date and that focus on the genotype are similar in that they measure a scar , a record of what the genome has been . 
it is known that brca1 / brca2 - deficient cells can over time regain their ability to perform hr , and current biomarkers of hrd are limited copy gains copy losses loss of heterozygosity structural variants genome transcriptome fig 2 . 
in addition to cataloging somatic point mutations and small insertions / deletions , the use of wholegenome sequencing enables characterization of large - scale copy - number variation , loss of heterozygosity , and structural variation . 
patient - derived xenografts represent reasonable models of the human breast tumor49 that could be used to explore real - time functional hr versus measures of hr by other methods described in this review and provide further insight into understanding hrd . ultimately , whole - genome sequencing can provide direct or supportive evidence of almost all the information that each of the above assays is intending to measure ( fig 2 )  . 
this approach , combined with brca mutation status , accurately detected all classes of genomic alterations in the assessment of rucaparib in ovarian cancer : phase 2 clinical trial ( ariel2 ) trial of ovarian cancer , 50 and wholegenome sequencing of breast cancers has identified three signatures that seem to be associated with hrd.18 however , until the cost of whole - genome sequencing decreases , scientists and clinicians are left to find less - expensive alternatives . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . meta - summary there is evidence that hrd assays have predictive value for response to platinum and possibly parpis in combination with standard therapy . 
head - to - head comparisons of the different hrd assays have not been performed , and it is not clear which assays or combination of assays is the best predictor of hrd . 
the asco ( 2014 and 2015 ) and san antonio breast cancer symposium ( 2014 to 2016 ) databases were searched at the title and abstract levels using the search terms breast cancer , brca1 , brca2 , and homologous recombination deficiency . 
nucleic acids res 40 : nature 535 : 382 - 387 , 2016 5795 - 5818 , 2012 von minckwitz g , schneeweiss a , loibl s , et al : neoadjuvant carboplatin in patients with triple - negative and her2positive early breast cancer ( geparsixto ; gbg 66 ) : a randomised phase 2 trial . 
sikov wm , berry da , perou cm , et al : impact of the addition of carboplatin and / or bevacizumab to neoadjuvant once - per - week paclitaxel followed by dose - dense doxorubicin and cyclophosphamide on pathologic complete response rates in stage ii to iii triple - negative breast cancer : calgb 40603 ( alliance )  . 
tutt a , ellis p , kilburn l , et al : the tnt trial : a randomized phase iii trial of carboplatin ( c ) compared with docetaxel ( d ) for patients with metastatic or recurrent locally advanced triple negative or brca1 / 2 breast cancer ( cruk / 07 / 012 )  . 
antoniou a , pharoah pd , narod s , et al : average risks of breast and ovarian cancer associated with brca1 or brca2 mutations detected in case series unselected for family history : a combined analysis of 22 studies . 
couch fj , hart sn , sharma p , et al : inherited mutations in 17 breast cancer susceptibility genes among a large triplenegative breast cancer cohort unselected for family history of breast cancer . 
sharma p , klemp jr , kimler bf , et al : germline brca mutation evaluation in a prospective triple - negative breast cancer registry : implications for hereditary breast and / or ovarian cancer syndrome testing . 
akashi - tanaka s , watanabe c , takamaru t , et al : brcaness predicts resistance to taxane - containing regimens in triple negative breast cancer during neoadjuvant chemotherapy . 
couch fj , hu c , lilyquist j , et al : breast cancer risks associated with mutations in cancer predisposition genes identified by clinical genetic testing of 60 , 000 breast cancer patients . 
nik - zainal s , davies h , staaf j , et al : landscape of somatic mutations in 560 breast cancer whole - genome sequences . nature 534 : 47 - 54 , 2016 19 . 
hennessy btj , timms km , carey ms , et al : somatic mutations in brca1 and brca2 could expand the number of patients that benefit from poly ( adp ribose ) polymerase inhibitors in ovarian cancer . 
winter c , nilsson mp , olsson e , et al : targeted sequencing of brca1 and brca2 across a large unselected breast cancer cohort suggests that one - third of mutations are somatic . 
van t veer l , esserman l , sanil a , et al : dna repair deficiency biomarkers identify hr + / her2breast cancer patients who may benefit from veliparib / carboplatin : results from the i - spy 2 trial . 
timms km , abkevich v , hughes e , et al : association of brca1 / 2 defects with genomic scores predictive of dna damage repair deficiency among breast cancer subtypes . 
sharma p , stecklein s , kimler bf , et al : brca1 insufficiency is predictive of superior survival in patients with triple negative breast cancer treated with platinum based chemotherapy . 
bergamaschi a , kim yh , wang p , et al : distinct patterns of dna copy number alteration are associated with different clinicopathological features and gene - expression subtypes of breast cancer . 
vollebergh ma , lips eh , nederlof pm , et al : an acgh classifier derived from brca1 - mutated breast cancer and benefit of high - dose platinum - based chemotherapy in her2 - negative breast cancer patients . 
schouten pc , marme f , aulmann s , et al : breast cancers with a brca1 - like dna copy number profile recur less often than expected after high - dose alkylating chemotherapy . 
popova t , manie e , rieunier g , et al : ploidy and large - scale genomic instability consistently identify basal - like breast carcinomas with brca1 / 2 inactivation . 
telli ml , jensen kc , vinayak s , et al : phase ii study of gemcitabine , carboplatin , and iniparib as neoadjuvant therapy for triple - negative and brca1 / 2 mutation - associated breast cancer with assessment of a tumor - based measure of genomic instability : precog 0105 . 
isakoff sj , mayer el , he l , et al : tbcrc009 : a multicenter phase ii clinical trial of platinum monotherapy with biomarker assessment in metastatic triple - negative breast cancer . 
von minckwitz g , timms k , untch m , et al : prediction of pathological complete response ( pcr ) by homologous recombination deficiency ( hrd ) after carboplatin - containing neoadjuvant chemotherapy in patients with tnbc : results from geparsixto . 
telli ml , mcmillan a , ford jm , et al : homologous recombination deficiency ( hrd ) as a predictive biomarker of response to neoadjuvant platinum - based therapy in patients with triple negative breast cancer ( tnbc ) : a pooled analysis . 
connolly r , elkin e , timms k et al : homologous recombination deficiency ( hrd ) as a predictive biomarker of response to preoperative systemic therapy ( pst ) in tbcrc008 comprising a platinum in her2 - negative primary operable breast cancer . 
powell sn , riaz n , mutter rw , et al : a functional assay for homologous recombination ( hr ) dna repair and whole exome sequencing reveal that hr - defective sporadic breast cancers are enriched for genetic alterations in dna repair genes . 
zhang x , claerhout s , prat a , et al : a renewable tissue resource of phenotypically stable , biologically and ethnically diverse , patient - derived human breast cancer xenograft models . 
mcneish ia , oza am , coleman rl , et al : results of ariel2 : a phase 2 trial to prospectively identify ovarian cancer patients likely to respond to rucaparib using tumor genetic analysis . 
 systemic immunotherapy effective for refractory extramedullary acute myeloid leukemia mansour alfayez , md1 ; doina ivan , md1 ; naveen pemmaraju , md1 ; naval daver , md1 ; and courtney d . 
not letting the exception pass by unnoticed , dr coley harnessed this observation and injected a number of his patients with bacterial toxins ( coleys toxin )  . this toxin represented a powerful superantigen , leading to dramatic immune system upregulation , including massive polyclonal t - cell activation and cytokine release leading to tumor regression in a notable minority of cases . 
although the mechanism was not understood at that time , this observation captures an important concept : cancer is dependent on immune escape.2 immunity for the treatment of hematologic malignancies , including acute myelogenous leukemia ( aml ) , has been leveraged for decades in the form of allogeneic stem - cell transplantation.3 - 5 aside from allogeneic stemtransplantation , other immune - based strategies cell have been evaluated in aml , including bacillus calmettegu erin vaccination , interleukin - 2 , and interferon alfa , all of which have shown inferior results compared with standard chemotherapy.2 , 6 - 10 however , those strategies were not specic and were not directed against specic immune escape pathways used by malignant cells . 
more recently , with the discovery of immune checkpoint inhibitors ( icpi ) and the successful redirection of the immune system for the treatment of various solid tumors , using the same concept in hematologic malignancy is alluring . we herein report on a 68 - year - old man with refractory extramedullary aml who successfully achieved remission using icpi . 
the patient was originally diagnosed at age 65 years with stage iiia melanoma in the left upper posterior arm ( appendix fig a1 ) , which was treated with surgical excision and lymph node resection , followed by radiation therapy in 2014 . 
the patient went into remission without evidence of recurrence on follow - up imaging ( fig 1 )  . one year later ( october 2015 ) , the patient was referred to the leukemia department with pancytopenia . 
bone marrow aspirate and biopsy revealed myelodysplastic syndrome , 2% blasts , with complex cytogenetics and mutation in nucleophosmin 1 ( npm1 ) and dna methyltransferase 3 alpha ( dnmt3a )  . 
he received a total of 19 cycles , with including full hematologic recomplete remission , covery , resolution of dysplastic morphology on subsequent bone marrow examinations , and cytogenetic remission . 
eighteen months after his original myelodysplastic syndrome diagnosis , the patient was noted to have a dark - colored skin lesion on the left upper arm that was suggestive of melanoma recurrence ( fig 2 )  . positron emission tomography / computed tomography scan showed a single uorodeoxyglucose - avid lesion in the skin and subcutaneous region of the left upper arm ( fig 2 )  . 
the patient then received two cycles of intensive cytarabine - based chemotherapy with cladribine 5 mg / m2 , idarubicin 10 mg / m2 , and cytarabine 1 g / m2 days 1 to 3 every 28 days , with follow - up positron emission tomography scan after two cycles showing progressive disease ( fig 3 )  . 
surveillance bone marrow examination showed myelodysplasia with 2% blasts , diploid cytogenetics , and dnmt3a mutation identied , and npm1 mutation no longer detected at variant allele frequency level of sensitivity of approximately 1% . 
given the progressive yet self - contained extramedullary disease to sites within the left upper arm , the patient was then treated with nine fractions of radiotherapy ( 10 gy ) , without evidence of response to radiation therapy . 
the treatment regimen consisted of azacitidine 75 mg / m2 ( days 1 to 7 ) and nivolumab 3 mg / kg ( day 1 and day 14 ) every 28 days , in addition to ipilimumab 1 mg / kg every 42 days . 
 alfayez et al lymph node resection followed by radiation october 15 , 2014 intensive cytarabine - based chemotherapy ( clia ) 2 cycles june 21 , 2017 radiotherapy september 7 , 2017 guadecitabine ( sgi - 110 ) 19 cycles october 26 , 2015 progressive disease september 15 , 2017 remission november 8 , 2017 stage iiia melanoma october 1 , 2014 progressive disease august 17 , 2017 azacitidine plus nivolumab plus ipilimumab september 18 , 2017 leukemia cutis may 26 , 2017 myelodysplastic syndrome october 1 , 2015 fig 1 . 
the patient continued receiving treatment for another cycle , and a skin biopsy after cycle 2 was negative for cd33 myeloid sarcoma , instead demonstrating lymphocyte ( cd3 + ) inltration , with predominant t - cell predominance of cd8 over cd4 + lymphocytes ( including cd4 + histiocytes ; fig 4 )  . 
the patient has continued receiving this combination therapy and so far received six cycles , with continued clinical and radiologic clearance of his skin lesion ( fig 4 ) and ongoing bone marrow response . inhibitors are antibodies directed immune checkpoint against t - cell inhibitory signals to modulate the interaction between cytotoxic t cells and tumor cells . 
activating programmed cell death protein 1 ( pd - 1 ) prevents cd28 / b7 signaling that is crucial for cd8 t - cell activation.11 pd - 1 interactions and programmed death - ligand 1 ( pd - l1 ) inhibited antitumor immune responses in a murine aml model.12 treatment with antipd - 1 antibody improved survival in those models , indicating the importance of the pd - 1 / pd - l1 pathway in immune evasion by aml blasts as well as providing a rationale for clinical trials targeting this pathway in aml ( eg , with nivolumab ) .12 seventeen - color multiparameter ow cytometry was reported on 38 patients with relapsed aml , 36 patients with untreated aml , and eight healthy controls . 
all t - cell subpopulations ( cd3 + , cd4 + , and cd8 + t cells ) had signicantly higher pd1 expression in relapsed aml ( p , .006 ) and untreated aml ( p , .05 ) compared with healthy controls.13 this suggests that pd1 overexpression is an escape route used by leukemia . 
this nding was also supported in other reports.14 - 16 in a report , t - cell receptor ( tcr ) clonotypes that were minimal or undetectable became enriched after nivolumab administration , and one patient had marked reduction in tcr diversity indexes and clearance of measurable residual disease with tcr clone directed against wilms tumor 1 ( wt1 ) .17 in an ongoing phase ib / ii study of nivolumab in combination with azacitidine in relapsed aml ( clinicaltrials.gov identier : nct02397720 ) , 70 evaluable patients were reported with 63% overall response rate , which compared favorably to historical response rate of 15% to 20% treated at the same institute . 
expression of cd86 was reported on aml blasts in more than half of 100 samples tested , and , in some cases , the level of expression was so high that it was equivalent to that of mature monocytes and activated b lymphocytes , 20 which gives a rationale for targeting this pathway ( eg , with ipilimumab )  . 
in a phase i / ib multicenter trial of ipilimumab in patients with relapsed hematologic cancers after allogeneic stem cell transplantation , 12 patients with relapsed aml and another four with extramedullary relapsed disease were included . 
 ( c ) skin lesion at the time of presentation . immunohistochemical studies demonstrating neoplastic cells positivity for ( d ) cd33 , ( e ) myeloperoxidase ( mpo ) , and ( f ) cd43 . locus16 and dose - dependent upregulation of pd - l1 , pd - l2 , pd - 1 , and cytotoxic t - lymphocyte associated protein 4 ( twofold or greater ) .16 there was trend toward increased expression of those checkpoints in hma - resistant patients compared with sensitive patients , suggesting upregulation of inhibitory immune signals as a mechanism of resistance to hmas . 
 ( c ) positron emission tomography / computed tomography post cycle 2 of cladribine , idarubicin , and cytarabine shows previously identied cutaneous nodule within the left posterior arm has increased in number and uorodeoxyglucose activity . 
 ( a - d ) skin immunohistochemical studies post cycle 2 of azacitidine with dual checkpoint inhibition showing negative cd 33 , t - cell lymphocyte ( cd3positive ) inltration , and predominance of cd8 over cd4 + lymphocytes . 
 ( g ) positron emission tomography / computed tomography post cycle 6 of azacitidine with dual checkpoint inhibition demonstrates complete metabolic response to therapy in terms of previously identied multiple cutaneous and subcutaneous nodules in the left arm . the hypothesis that concomitant administration of immune checkpoint inhibitors in combination with hmas may overcome resistance via immune evasion.16 aml with mutant npm1 is considered to be of favorable risk , and , interestingly , one suggested explanation relates to immune responses developed against epitopes derived from the mutated region of npm1 . 
higher pd - l1 expression in npm1 mutated patients , particularly in leukemic stem / progenitor cells , has been noted , potentially making this particular subgroup of aml more likely to benet from icpi treatment.23 - 25 in this particular case , myeloid sarcoma arising from a previous melanoma site , in a patient with complete remission from previously diagnosed myelodysplastic syndrome , highlights important questions , as does the excellent response to icpi . 
we wondered if the local microenvironment has immune - based hosting and homing characteristics ( ie , poor immune surveillance ) , which leads to weak containment of neoplastic cell expansion . 
also , it is interesting to note that , leukemia cutis started as single lesion , then had what we think was an intransit metastasis with multiple sentinel lesions ( fig 3 )  . 
although no clear data exist to date , this case and previous cases of myeloid sarcoma and leukemia cutis suggest icpi therapy may be an effective treatment modality for patients with isolated extramedullary aml . in conclusion , we report on the successful treatment of primary refractory myeloid sarcoma with hypomethylating agent therapy in combination with pdl1 and cytotoxic t - lymphocyte associated protein 4 inhibition . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . research funding : novartis , stemline therapeutics , incyte , samus therapeutics , abbvie , cellectis , affymetrix / thermo fisher scientic , daiichi sankyo , plexxikon travel , accommodations , expenses : stemline therapeutics , celgene , mustang bio naval daver consulting or advisory role : celgene , agios pharmaceuticals , jazz pharmaceuticals , pzer , abbvie , astellas pharma , daiichi sankyo , novartis , bristol - myers squibb research funding : bristol - myers squibb , pzer , immunogen , genentech , nohla therapeutics , abbvie , astellas pharma , servier , daiichi sankyo naveen pemmaraju honoraria : incyte , novartis , lfb biotechnologies , stemline therapeutics , celgene consulting or advisory role : incyte , novartis , lfb biotechnologies , stemline therapeutics , celgene courtney d . 
hall ss : a commotion in the blood : life , death , and the immune systenew york , ny , henry holt , 1997 alfayez m , borthakur g : checkpoint inhibitors and acute myelogenous leukemia : promises and challenges . 
expert rev hematol 11 : 373 - 389 , 2018 schmid c , labopin m , nagler a , et al : donor lymphocyte infusion in the treatment of rst hematological relapse after allogeneic stem - cell transplantation in adults with acute myeloid leukemia : a retrospective risk factors analysis and comparison with other strategies by the ebmt acute leukemia working party . j clin oncol 25 : 4938 - 4945 , 2007 4 . 
blood 75 : 555 - 562 , 1990 sullivan km , weiden pl , storb r , et al : inuence of acute and chronic graft - versus - host disease on relapse and survival after bone marrow transplantation from hla - identical siblings as treatment of acute and chronic leukemia . 
blood 73 : 1720 - 1728 , 1989 powles rl , russell j , lister ta , et al : immunotherapy for acute myelogenous leukaemia : a controlled clinical study 2 1 / 2 years after entry of the last patient . 
int j oncol 2 : 469 - 472 , 1993 goldstone ah , burnett ak , wheatley k , et al : attempts to improve treatment outcomes in acute myeloid leukemia ( aml ) in older patients : the results of the united kingdom medical research council aml11 trial . 
meloni g , vignetti m , andrizzi c , et al : interleukin - 2 for the treatment of advanced acute myelogenous leukemia patients with limited disease : updated experience with 20 cases . 
daver n , basu s , garcia - manero g , et al : dening the immune checkpoint landscape in patients ( pts ) with acute myeloid leukemia ( aml )  . 
andorsky dj , yamada re , said j , et al : programmed death ligand 1 is expressed by non - hodgkin lymphomas and inhibits the activity of tumor - associated t cells . 
yang h , bueso - ramos c , dinardo c , et al : expression of pd - l1 , pd - l2 , pd - 1 and ctla4 in myelodysplastic syndromes is enhanced by treatment with hypomethylating agents . 
liu h , park j - h , fulton n , et al : tcr clonal evolution in aml patients in morphologic remission treated with anti - pd1 antibody , nivolumab . 
daver n , basu s , garcia - manero g , et al : phase ib / ii study of nivolumab in combination with azacytidine ( aza ) in patients ( pts ) with relapsed acute myeloid leukemia ( aml )  . 
greiner j , ono y , hofmann s , et al : mutated regions of nucleophosmin 1 elicit both cd4 ( + ) and cd8 ( + ) t - cell responses in patients with acute myeloid leukemia . 
greiner j , schneider v , schmitt m , et al : immune responses against the mutated region of cytoplasmatic npm1 might contribute to the favorable clinical outcome of aml patients with npm1 mutations ( npm1mut )  . 
greiner j , hofmann s , schmitt m , et al : acute myeloid leukemia with mutated nucleophosmin 1 : an immunogenic acute myeloid leukemia subtype and potential candidate for immune checkpoint inhibition . 
 poly ( adp - ribose ) polymerase inhibitors for de novo brca2 - null small - cell prostate cancer introduction neuroendocrine prostate cancer ( nepc ) represents 0.5% to 2% of all primary prostate cancers ( pcas ) , 1 and primary pure nepc is even more rare . 
pure small - cell prostate carcinoma ( scpc ) , a subtype of nepc , can occur in two different settings : exceptionally , it can develop de novo ; more frequently , small cells are detected during the evolution to castration - resistant prostate cancer ( crpc ) 2 as a consequence of androgen suppressive therapy . scpc is a proliferation of small and round cells with a high mitotic index and immunohistochemistry staining for synaptophysin , chromogranin a ( cha ) , or neuron - specific enolase , 3 whereas prostate - specific antigen ( psa ) and androgen receptor ( ar ) expression often are associated with a tmprss2 - erg fusion detected in 50% of scpcs.2 , 4 in all cases , the neuroendocrine presentation is associated with poor prognosis and frequent visceral , bone , or brain metastases , which contrasts with a low or normal psa . 
whole - body [ 18f ] fluorodeoxyglucose ( fdg ) positron emission tomography ( pet ) / ct scan showed a large left lobe prostatic infiltration and detected bulky retroperitoneal iliac nodes and l2 to l3 abnormal fdg uptake ( fig 1a )  . 
the patient was treated with luteinizing hormone releasing hormone ( lhrh ) agonist because of the rapid rise in psa and focal ar staining on biopsy specimens . initial treatment : cisplatin and etoposide regimen the patient received six courses of cisplatin 33 mg / m2 / d and etoposide 100 mg / m2 / d for 3 days and every 3 weeks and lhrh agonists . 
after three cycles , the symptoms disappeared , and pet / ct scan showed a major metabolic and biologic response ( psa , 0.18 ng / ml ) confirmed after six courses ( figs 1a and 1b )  . 
important adverse effects occurred , and lhrh agonist was maintained alone . second - line treatment : topotecan regimen nine months after the end of platinum therapy , progression was detected on the right - side adrenal gland with abnormal fdg uptake . 
 prostate - specific antigen ( psa ) and neuron - specific enolase ( nse ) concentrations are plotted on the left y - axis and chromogranin a on the right y - axis with the upper limit of normal represented with dotted lines of the corresponding colors . 
 to identify clinically actionable gene mutations and putative druggable pathways , gene copy number aberrations and somatic mutation status were identified by using whole - genome array comparative genomic hybridization and targeted next - generation sequencing , respectively , of 559 genes as previously described5 , 6 ( gene panel provided in the data supplement )  . 
the array comparative genomic hybridization analysis showed a genomic instability associated with a brcalike profile associated with a homologous repair deficiency ( hrd ) score of 197 ( fig 2a ) , which was calculated on the basis of the number of lost regions with intermediate size ( > 15 mb ) known to be more frequently observed in brca defective tumors . 
 from left to right , the chromosome 13 ideogram with its genomic profile exhibits several copy number aberrations ; the zoom - in on the 13q12.2 to q13 heterozygous lost region 13 : 24 , 648 , 264 to 41 , 238 , 009 ( 16.5 mb ) includes brca2 . copy number aberrations showed ar amplification ( > 11 copies ) and regional heterozygous losses , including brca2 ( fig 2b ) and tp53 genes ( data not shown )  . 
gene transition profiles with breakages in both tmprss2 and erg loci suggested an unbalanced translocation and the presence of a tmprss2 - erg fusion ( data not shown )  . 
the germinal brca2 mutation could have come from the patients fathers side , where a breast cancer aggregation had been observed in the patients sister and two of his fathers aunts ( fig a1 )  . 
pathology analysis found the same nepc and pca - related stainings ( fig 3 )  . third - line treatment : parpi three months later , pet / ct scan revealed a new radiologic progression ( vertebrae and retroperitoneum nodes ) with biologic progression ( figs 1b and c )  . 
after 2 months , pet / ct scan showed a complete response , which was maintained after 12 months treatment ( fig 1c )  . in november 2017 , 4 years after initial diagnosis , the patient was free from tumor symptoms , and serum markers were normal . 
optic microscope photographs show positive staining for synaptophysin ( syn ; magnification , 10 ) , androgen receptor ( ar ; magnification , 10 ) , prostate - specific antigen ( psa ; magnification , 20 ) , prostate - specific acid phosphatase ( psap ; magnification , 20 ) , and ets - related gene ( erg , magnification , 20 ) alpha - methylacyl - coa racemase enzyme ( p504s )  . 
 these stainings are typical of both prostate adenocarcinoma and small neuroendocrine cells . psap p504s but because of the rarity and inconsistent definition of the neuroendocrine disease among studies , no gold standard has been established . 
 guidelines recommend the use of aggressive chemotherapy , such as platinum salts and etoposide , for pure de novo scpc , but for advanced pure or mixed scpc that occurs during adenocarcinoma treatment , docetaxel - based regimen with or without anti - androgen usually is used if the patient is fit enough.2 , 9 at diagnosis , the current patient had elevated serum psa and partial immunostaining for tumor psa and ar ; hence , he benefited from lhrh agonist treatment , which suggests that the tumor was at least partly androgen driven . 
in brca - mutated tumors , inhibition of parp increases the genomic instability and leads to cell lethality when unrepaired single - strand breaks evolve to double - strand breaks.15 , 16 a phase ii study reported a good tolerance in 49 patients treated with olaparib alone for progressive nonselected metastatic crpc , and treatment was associated with tumor response on ct scan , psa level reduction , and decreased number of circulating tumor cells in 16 patients ( 33% )  . 
response to treatment was strongly correlated with mutation or deletion in homologous recombination genes.17 in 2016 , olaparib was given a breakthrough therapy designation by the food and drug administration for crpc , 18 and a more systematic screening for dna repair gene mutations for patients with crpc has been proposed.19 , 20 two phase iii studies currently are investigating olaparib ( profound ; clinicaltrials identifier : nct02987543 ) and rucaparib ( triton3 ; clinicaltrials.gov identifier : nct02975934 ) versus a second line of new hormonal therapy ( nht ; enzalutamide or abiraterone ) in advanced crpc with altered dna repair pathway , and one phase iii study is investigating talazoparib plus nht versus placebo plus nht ( talapro - 2 ; clinicaltrials . gov identifier : nct03395197 ) as first - line treatment of crpc , all with a primary end point of improving radiologic progression - free survival . the current study confirms the sensitivity of scpc to platinum drugs as well as parpis in the case of defect in the homologous recombination dna repair system and highlights interest in sequencing tumor samples when dealing with rare disease and limited guidelines . 
 sverine garnier no relationship to disclose nadine carbuccia no relationship to disclose arnaud guille no relationship to disclose nathalie charrier no relationship to disclose jihane pakradouni no relationship to disclose anthony gonalvs research funding : msd ( inst ) , bristol - myers squibb ( inst ) , novartis ( inst ) , cascadian therapeutics ( inst ) , nektar ( inst ) , boehringer ingelheim ( inst ) , eli lilly ( inst ) , abbvie ( inst ) travel , accommodations , expenses : pfizer , novartis , roche , genentech , celgene isabelle brenot - rossi honoraria : 3a pharma , ipsen , astellas pharma consulting or advisory role : janssen pharmaceuticals , astellas pharma , sanofi , 3a pharma travel , accommodations , expenses : 3a pharma franois eisinger honoraria : roche , msd consulting or advisory role : roche research funding : roche , roche ( inst ) travel , accommodations , expenses : roche , sanofi , celgene jochen walz no relationship to disclose graldine pignot honoraria : janssen - cilag , roche , ipsen , astellas pharma consulting or advisory role : janssen - cilag , roche , novartis ( inst ) , msd ( inst ) travel , accommodations , expenses : pfizer , janssen - cilag , ipsen , roche , ferring pharmaceuticals , intuitive surgical franois bertucci no relationship to disclose daniel birnbaum research funding : amgen gwenalle gravis travel , accommodations , expenses : sanofi , janssen pharmaceuticals , bristol - myers squibb , astellas pharma , pfizer , roche affiliations all authors : institut paoli - calmettes , marseille , france . support supported by national cancer institute integrated cancer research site - linstitut national de la sant et de la recherche mdicale grant no . 
wang l , williamson sr , zhang s , et al : increased androgen receptor gene copy number is associated with tmprss2 - erg rearrangement in prostatic small cell carcinoma . 
gonalves a , bertucci f , guille a , et al : targeted ngs , array - cgh , and patient - derived tumor xenografts for precision medicine in advanced breast cancer : a single - center prospective study . 
corn pg , tapia eln , xiao l , et al : confirmatory analysis to determine associations between platinum - sensitivity , molecular signature of combined tumor suppressor defects and aggressive variant prostate carcinomas ( avpc )  . 
flchon a , pouessel d , ferlay c , et al : phase ii study of carboplatin and etoposide in patients with anaplastic progressive metastatic castration - resistant prostate cancer ( mcrpc ) with or without neuroendocrine differentiation : results of the french genito - urinary tumor group ( getug ) p01 trial . 
pomerantz mm , spisk s , jia l , et al : the association between germline brca2 variants and sensitivity to platinum - based chemotherapy among men with metastatic prostate cancer . 
horita n , yamamoto m , sato t , et al : topotecan for relapsed small - cell lung cancer : systematic review and meta - analysis of 1347 patients . 
family analysis shows a breast cancer aggregation on the fathers side and a breast cancer before 50 years of age in the patients sister , which might be related to brca autosomal dominant transmission . 
carl barrett , phd1 ; and jeeyun lee , md3 purpose some gastric cancers harbor met gene amplications that can be targeted by selective met inhibitors to achieve tumor responses , but resistance eventually develops . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction gastric cancer is the fth most common cancer worldwide and the third leading cause of cancerrelated death.1 gastric cancer is a heterogenous disease , the drivers of which remain unclear , making it difcult to treat these patients . 
pembrolizumab was approved for treating gi malignancies with mismatch repair deciencies and for third - line programmed death ligand 1positive gastric cancer based on its recently observed promising antitumor activity.2 , 3 however , it remains urgent to identify a subset of patients with gastric cancer who may benet from molecularly targeted agents . 
one such potential target is met amplication , which is observed in approximately 5% of patients with gastric cancer.3 - 9 savolitinib ( azd6094 , hmpl - 504 , volitinib ) is a potent small - molecule met kinase inhibitor that inhibits met inhibitory concentration kinase at a half - maximal ( ic50 ) of 4 nm and met phosphorylation in tumor cells . savolitinib was found to inhibit cell growth in vitro against tumors with met gene amplication in the absence of hepatocyte growth factor stimulation , with ic50 generally , 10 nm . 
 met inhibitor in met - amplied gastric cancer context key objective we evaluated the mechanisms of acquired resistance to savolitinib in 3 patients with met - amplied gastric cancer who showed a clinical response and then cancer progression using tumor and circulating tumor dna ( ctdna ) sequencing . knowledge generated ctdna is a powerful tool for identifying potential genomic aberrations that emerge during therapy to confer resistance to targeted therapies . 
using a next - generation sequencing 100 - gene panel , we identied the target mechanisms of resistance met d1228v / n / h and y1230c mutations or high copy number met gene amplications that emerge when resistance to savolitinib develops in patients with met - amplied gastric cancer . relevance we demonstrated the utility of ctdna in gastric cancer and conrmed this approach using baseline tumor tissue or rebiopsy . we identied various met mutations previously unidentied in gastric cancer upon progression to savolitinib , as well as met amplications as drivers of savolitinib resistance during monotherapy treatment of patients with met - amplied gastric cancer . circulating tumor dna ( ctdna ) from baseline until progression every 2 cycles to evaluate the tumor biology during treatment with targeted agents . 
the human investigations were performed after approval by a local human investigations committee ( institutional review board ) and in accordance with an assurance led with and approved by the health authority in korea . 
this prospective open - label trial was designed as a single - arm phase ii study at an academic cancer center ( clinicaltrials.gov identier : nct02449551 )  . treatment was administered as follows : savolitinib 800 mg once a day until disease progression or unacceptable toxicity . 
tumor tissue and plasma ctdna samples from 3 patients enrolled in the viktory trial were obtained before treatment , during therapy , and at the time of progression . next - generation sequencing a targeted next - generation sequencing 100 - gene panel was used to analyze matched tumor dna at baseline and progression and longitudinal ctdna to determine the mechanisms of acquired resistance to savolitinib ( az translational genomics labs , boston , ma )  . 
genes included in this panel are provided in appendix figure a1a . sequencing libraries were prepared using the kapa biosystems hyperprep kit ( wilmington , ma ) and roche nimblegen hybridization capture reagents ( basel , switzerland )  . 
raw sequencing data were processed using the bcbio framework.12 the quality of sequencing data was assessed using the multiqc report.13 fastq les were aligned to the reference genome hg38 using bwa mem aligner , 14 yielding a median depth of coverage of 2 , 000 ( range , 1 , 300 - 6 , 600 ) in plasma samples and 1 , 200 ( range , 1 , 100 - 1 , 400 ) in tissue samples . 
variants were called using vardict15 and classied into the following 3 tiers from high to low based on the likelihood of the variant contributing to cancer - relevant processes : known , likely , and unknown.15 copy number analysis of sequencing data were performed using the copy number caller seq2c.16 all plasma samples were analyzed together as a plasma cohort , whereas tissue samples were processed together as a tissue cohort . 
mapping and sequencing statistics for formalin - xed parafn - embedded tumor tissue ( fig a1b ) and ctdna from frozen plasma ( fig a1c ) indicated that  . 90% and 96% of the 100 genes were sequenced with 500 coverage in tumor and ctdna , respectively , providing a quality data set for analysis . et immunohistochemistry and met fluorescent in situ hybridization met immunohistochemistry ( ihc ) was performed using the rabbit monoclonal primary antibody , confirm antitotal met ( sp44 ; ventana medical systems , tucson , az ) according to the manufacturers protocol . 
 frigault et al performed using dual - color dna - specic met / cep7 probes ( abnova , walnut , ca ) , as described previously.5 , 8 results we analyzed the rst 3 patients enrolled in the savolitinib trial , which enrolls patients with gastric cancer with met amplication in the salvage setting . 
patients were screened for the presence of met amplication by tumor sequencing as part of the viktory trial and are conrmed wild type for met.11 the viktory trial screened 715 patients with genomic sequencing , and the incidence of met amplication was 3.5% ( 25 of 715 patients )  . 
in this study , we tested the feasibility of using a 100 - gene panel in patients with met - amplied gastric cancer serially from baseline until disease progression using both matched biopsies and ctdna . 
the patient had microsatellite - stable and her2 - negative gastric cancer ( fig 1a ) and experienced rapid progression after 5 cycles of capecitabine plus oxaliplatin ( initially stable disease ) to extensive dissemination in the lymph nodes ( fig 1b )  . 
the patient responded dramatically to 2 cycles of savolitinib ( fig 1b , middle panel ) , which was concordant with the decrease in the allele frequency of tp53 p190l ( 7% ) , met 1.4 copy number , and myc 3.4 copy number in the ctdna . 
notably , although the patient maintained a radiologic and clinical partial response ( pr ) to savolitinib , low frequencies of met d1228h ( 5% ) , met d1228n ( 5% ) , met d1228v ( 35% ) , and met y1230c ( 3% ) were newly detected . 
met d1228n is a resistant mutation to crizotinib in lung cancer.17 in addition , met d1228v has been shown to induce resistance to type 1 met tyrosine kinase inhibitors in lung cancer.18 finally , met y1230c is associated with resistance to crizotinib in nonsmall - cell lung cancer.19 the high concordance for the copy number level between ctdna 100 - gene sequencing and tumor rebiopsy specimen sequencing supports the usefulness of copy number evaluation from longitudinal plasma ctdna ( appendix fig a2 )  . 
at disease progression , the patient had mostly experienced progression to bone and bone marrow metastases rather than previous lymph nodes , suggesting the emergence of an aggressive clone that spread to the bone and bone marrow at progression . 
our data suggest that , rather than met amplications , met mutations drove the rapid progression observed in this patient ( fig 1c )  . the second patient was a 74 - year - old woman who was diagnosed with gastric cancer with extensive liver metastases at presentation and enrolled in the viktory trial at diagnosis . 
after 4 cycles of savolitinib , with her pr conrmed radiologically , the met copy number was 2.7 and showed a slight increase from diploid , and a tp53 g245d ( 2% ) variant began to emerge as detected in the ctdna . 
after 6 months ( 7 cycles of savolitinib ) , the patient developed radiologic progression . interestingly , in contrast to the newly emerging mutations conferring resistance to savolitinib in patient 1 ( fig 1 ) , the second patient developed a high level of met amplication of 13 copies and cdk6 copy number of 3.9 at progression ( fig 2a )  . 
although both genes are proximal to one another on chromosome 7 , they are not part of the same amplicon because the trap and pik3cg gene loci are between met and cdk6 , and they remained at a normal copy number in both the ctdna and tumor samples at progression ( fig 2b , top panel )  . 
high concordance was observed between the ctdna and tumor tissue dna ( liver metastasis ) with a reincrease in met amplication and cdk6 amplication that was not detected at pr . 
in addition , the ctdna sample at progression indicated that the tp53 g245d substitution containing the clone had expanded , as suggested by an increase from a 2% allele fraction ( at the third follow - up ) to a 22% allele fraction ( at progression )  . 
this was conrmed in the progression rebiopsy , where the same tp53 g245d substitution that was not detected in normal dna had subsequently increased to an 82% allele fraction ( fig 2b , bottom panel )  . the third patient was a 31 - year - old woman who underwent total gastrectomy for gastric cancer in 2013 . 
pathology , clinical assessment , and circulating tumor dna ( ctdna ) monitoring for patient 1 ( b5 - 002 ) identies met d1228 and y1230 mutations as mechanism of resistance to savolitinib . 
 ( a ) hematoxylin and eosin ( he ) , met immunohistochemistry ( ihc ) , and met uorescence in situ hybridization ( fish ; left to right ) from baseline tumor tissue . 
 ( b ) tumor assessment by radiologic assessment at baseline before savolitinib treatment , after 2 cycles at time of partial response ( pr ) , and after 4 cycles at time of progressive disease ( pd )  . 
clinical assessment and circulating tumor dna ( ctdna ) monitoring for patient 2 ( b5 - 001 ) identify on - target high - level met amplication ( amp ) as mechanism of resistance to savolitinib . 
 ( a ) radiologic assessment of tumor at baseline before savolitinib treatment ( follow - up [ fu ] 1 ) ; after 2 cycles ( fu2 ) and 4 cycles ( fu3 ) , both at time of partial response ( pr ) ; and after 7 cycles at time of progressive disease ( pd )  . 
 ( b ) copy number prole of ctdna plasma ( top ) and tumor tissue or normal dna at progression ( bottom ) , where each sample is represented by a different color at baseline ( fu1 ) , after 2 cycles ( fu2 ) and 4 cycles ( fu3 ) , and after 7 cycles at time of progression . patient was administered postoperative chemoradiation therapy with a ts - 1 plus oxaliplatin regimen and developed recurrent metastases to both ovaries 18 months later . 
 ( a ) hematoxylin and eosin ( he ) , met immunohistochemistry ( ihc ) , and met uorescence in situ hybridization ( fish ; left to right ) from baseline tumor tissue . 
 ( b ) tumor assessment by radiologic assessment at baseline before savolitinib treatment , after 2 cycles at time of partial response ( pr ) , and after 8 cycles at time of progressive disease ( pd )  . 
 frigault et al discussion the aim of this exploratory study was to determine whether relapses on savolitinib in patients with gastric cancer after an initial pr resulted from the development of on - target or bypass mechanisms of resistance . 
furthermore , we established the utility of ctdna from plasma as a matrix for monitoring changes in dna alterations during the course of treatment of gastric cancer and demonstrated the concordance of genomic alterations in ctdna to tumor biopsy both before treatment with a targeted therapy and at the time of progression . 
the efcacy of the trial will be reported once the trial completes recruitment ; here , we focused on the utility of ctdna sequencing to identify potential resistance mechanisms in gastric cancer after met inhibition . 
we identied the following two potential mechanisms of acquired resistance at progression following a dramatic response to savolitinib in met - amplied gastric cancer : newly developed met mutations previously only described in lung cancer and an increase in the met copy number at resistance , which decreased during the clinical response but increased again at disease progression . in the rst patient , newly emerging met d1228h ( 31% ) , met d1228n ( 12% ) , met d1228v ( 1% ) , and met y1230c ( 1% ) mutations were detected with the sudden onset of disease progression to multiple bones and the bone marrow . 
this patient died of rapid progression immediately after developing resistance to savolitinib therapy . mutations in met , including at positions d1228 and y1230 , are among the reported mechanisms of acquired resistance in preclinical models21 , 22 and have been reported to confer clinical resistance to met inhibition in lung cancer.17 , 19 the cocrystal structure of the met kinase domain and met kinase inhibitor revealed an important binding interaction of the met inhibitor and y1230 and explains the abrogation of compound binding with the emergence of mutations at this residue . 
in addition , d1228 is engaged in interactions with other residues to maintain the activation loop in a conformation that enables the critical interaction between y1230 and the met inhibitor.21 these results demonstrate that the on - target resistance mutations emerge in met - amplied gastric cancer treated with a selective met kinase inhibitor . 
therefore , upregulation of met ( in a savolitinib - responsive tumor ) similar to her2 ( in a lapatinib - responsive tumor ) may confer resistance via epithelial to mesenchymal transition and by promoting a metastatic phenotype.26 , 27 an on - target met copy number gain as a mechanism of resistance has been reported in lung cancer , where one met exon 14skipping patient became a responder to a met inhibitor in the clinic ; however , at the time of progression , the exon 14 met allele was amplied , but not the wild - type allele of met , 28 suggesting that highlevel amplication of the met gene is a mechanism of resistance to met inhibitors across indications . in 2 of 3 patients studied , ctdna was detectable in the circulation , harboring somatic alterations in the met pathway that changed over time and were conrmed by rebiopsy at recurrence . 
our data agree with this hypothesis ; in the rst patient , tp53 p190l changed from a fraction of 44% at baseline to 7% during pr and then back to 13% at disease progression , which was further conrmed in tumor tissue with a 17% allele fraction harboring the same mutation in tp53 . 
 met inhibitor in met - amplied gastric cancer contained no detectable ctdna tp53 until after 4 cycles , at which point a g245d amino acid substitution was detectable at a 2% allele fraction and increased to 22% at progression . 
the same alteration was identied by rebiopsy at an 82% allele fraction . in 1 of 3 patients studied , no mutations in any of the 100 genes on the high - coverage deep - sequencing gene panel were detected , and therefore , this patients tumors may not be shedding ctdna into circulation . 
this patient demonstrates the limitations of the use of ctdna to the 57% - 87% of patients with gastric cancer who are shedding ctdna from their tumor into circulation.30 , 31 this patient highlights that tumor collection at the time of progression will still be important for identifying resistance mechanisms to targeted treatments in nonshedders and to understand tumor heterogeneity . in summary , ctdna is a powerful tool for identifying potential genomic aberrations that emerge during therapy to confer resistance to targeted therapies . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . aleksandra markovets employment : astrazeneca stock and other ownership interests : astrazeneca barrett nuttall employment : astrazeneca , cvs health ( i ) stock and other ownership interests : astrazeneca , cvs health ( i ) travel , accommodations , expenses : astrazeneca , cvs health ( i ) se hoon park honoraria : merck sharp & dohme , sano - aventis consulting or advisory role : eli lilly , janssen oncology research funding : kura oncology , ono pharmaceutical esha a . 
gangolli employment : clovis oncology stock and other ownership interests : astrazeneca , clovis oncology , novartis , merck , bristol - myers squibb , jounce therapeutics , unum therapeutics , incyte , alcon , guardant health , titan medical travel , accommodations , expenses : clovis oncology peter g.s. 
nat rev dis primers 3 : 17036 , 2017 fuchs c , doi t , jang r , et al : keynote - 059 cohort 1 : efcacy and safety of pembrolizumab ( pembro ) monotherapy in patients with previously treated advanced gastric cancer . 
n engl j med 372 : 2509 - 2520 , 2015 lee j , ou sh , lee jm , et al : gastrointestinal malignancies harbor actionable met exon 14 deletions . 
mod pathol 26 : 1632 - 1641 , 2013 lee j , kim km , kang wk , et al : innovative personalized medicine in gastric cancer : time to move forward . 
nature 513 : 202 - 209 , 2014 lee j , seo jw , jun hj , et al : impact of met amplication on gastric cancer : possible roles as a novel prognostic marker and a potential therapeutic target . 
oncol rep 25 : 1517 - 1524 , 2011 ahn s , brant r , sharpe a , et al : correlation between mek signature and ras gene alteration in advanced gastric cancer . 
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kim st , banks kc , pectasides e , et al : impact of genomic alterations on lapatinib treatment outcome and cell - free genomic landscape during her2 therapy in her2 + gastric cancer patients . 
tiedt r , degenkolbe e , furet p , et al : a drug resistance screen using a selective met inhibitor reveals a spectrum of mutations that partially overlap with activating mutations found in cancer patients . 
kim dc , park kr , jeong yj , et al : resistance to the c - met inhibitor krc - 108 induces the epithelial transition of gastric cancer cells . 
oliveras - ferraros c , corominas - faja b , cuf s , et al : epithelial - to - mesenchymal transition ( emt ) confers primary resistance to trastuzumab ( herceptin )  . 
shi j , li f , yao x , et al : the her4 - yap1 axis promotes trastuzumab resistance in her2 - positive gastric cancer by inducing epithelial and mesenchymal transition . 
awad mm , bahcall m , sholl lm , et al : mechanisms of acquired resistance to met tyrosine kinase inhibitors ( tkis ) in met exon 14 ( metex14 ) mutant non - small cell lung cancer ( nsclc )  . 
 ( b ) sequencing and mapping statistics of tissue samples and ( c ) plasma circulating tumor dna ( ctdna ) describing number of sequenced reads , percentage of aligned reads , percentage of duplicated reads , percentage of reads mapped on targets ( + 200 base pairs ) , percentage of usable reads , average depth of coverage , gc content ( percentage of nitrogenous bases that are either guanine or cytosine ) , average insert size , and percentage of targeted bases with given coverage . 
sartori et al we thank sartori et al1 for their interest in the recently published review , which characterizes the her2 aberrations observed in nonsmall - cell lung cancer ( nsclc ) and summarizes the efcacy of her2targeted therapies in patients with nsclc.2 nsclc with her2 alterations is a highly heterogeneous disease . 
her2 overexpression , her2 amplication , and her2 mutation could exist alone or synchronously . sartori et al1 highlighted the contribution of immunologic features to the response to her2 - targeted therapy . 
as per the authors exploration , a strong antibody - dependent cell - mediated cytotoxicity triggered by the valine / valine ( v / v ) variant of fcriiia rather than sole target inhibition is the possible reason . the back - line application of singleunfortunately , agent immunotherapy to case 1 eventually led to hyperprogression disease ( hpd )  . 
indeed , the denition of hpd remains controversial to date , let alone the solid association between driver gene mutation and hpd.5 recently , our group also reported a patient with erbb1 exon 20 insertion mutation who suffered from hpd after nivolumab monotherapy.6 we speculated that one of the reasonable causes might be myc amplication , whose upregulation could result in cd47 activation and programmed death ligand 1 overexpression , consequently contributing to tumor growth . to the efcacy of with respect immunotherapy in her2 - mutant nsclc , 4 retrospective studies reported the outcomes.7 - 10 a total of 92 patients were treated with single - agent pd - 1 or pd - l1 inhibitor , most of them having experienced disease progression on standard chemotherapy . 
here , we call for in - depth studies of hpd mechanisms , especially the development of screening methods to identify specic subgroups in whom immune treatment would be harmful . up to now , her2 mutation , especially exon 20 insertion , is a potential therapeutic target with accumulated evidence . 
recent preclinical research reported that poziotinib potentiated the antitumor effect of trastuzumab emtansine ( t - dm1 ) by upregulating her2 expression.11 this mechanism ts the logic and the observed encouraging clinical benet from t - dm1 after disease progression on poziotinib in case 2 . 
in addition , a preclinical study conducted by li et al12 found that cotreatment of t - dm1 with irreversible pan - her inhibitors neratinib or lapatinib enhanced her2 receptor ubiquitination and consequently improved t - dm1 internalization and efcacy . 
whether the optimal combination mode is sequential or synchronous warrants further validation . trastuzumab deruxtecan ( t - dxd ) , another antibody - drug conjugate ( adc ) , harbors a different cytotoxic payload , showed better and more durable responses in her2mutant nsclc , and may be still effective after resistance to t - dm1.12 in extension , on top of her2 mutation , her2 amplication may also confer sensitivity to t - dm1 . 
according to recently released clinical trial data , t - dm1 exhibited identical orr in patients with nsclc with her2 amplication , her2 mutation , or concurrent her2 mutation and amplication ( 55% v 50% v 50% ) .12 collectively , her2 alterations in nsclc are rather heterogeneous . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . no potential conicts of interest were reported . references zhao j , xia y : targeting her2 alterations in non - small - cell lung cancer : a comprehensive review . 
precision oncol 4 : 411 - 425 , 2020 krug lm , miller va , patel j , et al : randomized phase ii study of weekly docetaxel plus trastuzumab versus weekly paclitaxel plus trastuzumab in patients with previously untreated advanced nonsmall cell lung carcinoma . cancer 104 : 2149 - 2155 , 2005 xu x , de angelis c , burke ka , et al : her2 reactivation through acquisition of the her2 l755s mutation as a mechanism of acquired resistance to her2 - targeted therapy in her2 + breast cancer . 
clin cancer res 23 : 5123 - 5134 , 2017 jin r , zhao j , xia l , et al : application of immune checkpoint inhibitors in egfr - mutant non - small - cell lung cancer : from bed to bench . 
huang x , xia l , lan f , et al : treatment of nivolumab results in hyperprogressive disease in a patient harboring egfr exon 20 insertion and myc amplication . 
mazieres j , drilon a , lusque a , et al : immune checkpoint inhibitors for patients with advanced lung cancer and oncogenic driver alterations : results from the immunotarget registry . 
ann oncol 30 : 1321 - 1328 , 2019 lai w - cv , feldman dl , buonocore dj , et al : pd - l1 expression , tumor mutation burden and response to immune checkpoint blockade in patients with her2 - mutant lung cancers . 
j clin oncol 36 , 2018 ( suppl 15 ; abstr 9060 ) dudnik e , bshara e , grubstein a , et al : rare targetable drivers ( rtds ) in non - small cell lung cancer ( nsclc ) : outcomes with immune check - point inhibitors ( icpi )  . 
guisier f , dubos - arvis c , vias f , et al : efcacy and safety of anti - pd - 1 immunotherapy in patients with advanced nsclc with braf , her2 , or met mutations or ret translocation : gfpc 01 - 2018 . 
robichaux jp , elamin yy , vijayan rsk , et al : pan - cancer landscape and analysis of erbb2 mutations identies poziotinib as a clinically active inhibitor and enhancer of t - dm1 activity . 
cancer discov 10 : 674 - 687 , 2020 sartori g , belluomini l , trovato r , et al : her2 aberrations in nsclc : so close yet so far . 
 contributed equally to this work . ( continued ) purpose alk ( anaplastic lymphoma kinase ) rearrangements predict for sensitivity to alk tyrosine kinase inhibitors ( tkis ) ; however , responses to alk tkis are generally short lived . 
although multiple studies support the clinical benefits of repeat tissue sampling , the clinical utility of longitudinal circulating tumor dna analysis has not been established in alk - positive lung cancer . patients and methods we used a 566 - gene hybrid - capture next - generation sequencing assay to perform a longitudinal analysis of plasma specimens from 22 alk - positive patients with acquired resistance to alk tkis to track the evolution of resistance during treatment . 
to determine tissueplasma concordance , we compared plasma findings with the results of repeat biopsies . results at disease progression , we detected an alk fusion in plasma from 19 ( 86% ) of 22 patients and identified alk resistance mutations in plasma specimens from 11 patients ( 50% )  . 
alk g1202rthe most frequent plasma mutation detected after progression on a second - generation tkiwas consistently suppressed during treatment with lorlatinib . conclusion plasma genotyping by next - generation sequencing is an effective method for detecting alk fusions and alk mutations in patients who experience disease progression on alk tkis . 
2018 by american society of clinical oncology introduction oncogenic rearrangements that result in the constitutive activation of anaplastic lymphoma kinase ( alk ) define a molecular subtype of nonsmalllung cancer ( nsclc ) that is characterized by sensitivity to alk tyrosine kinase inhibitors ( tkis ) .1 , 2 since the identification of alk rearrangements in nsclc in 2007 , four tkis have become standard therapies for patients with advanced disease.3 - 6 although these tkis have significantly improved clinical outcomes , most patients experience relapse within 1 to 2 years.1 despite a shared molecular driver , the magnitude of benefit from tki treatment varies widely between patients . 
repeat biopsies upon disease progression have been instrumental in elucidating the molecular mechanisms that drive resistance to alk inhibitors , including the distinct spectrum of alk mutations associated with resistance to each tki7 ; however , repeat biopsies may be challenging to obtain and single - site sampling may not capture the spatial heterogeneity of resistance mechanisms . analysis of circulating tumor dna ( ctdna ) is an emerging approach for tumor genotyping . 
shaw , md , phd , department of medicine , massachusetts general hospital , 32 fruit st , boston , ma 02114 ; e - mail : ashaw1@ partners.org. as plasma sampling is minimally invasive and ctdna may be derived from all metastatic sites , longitudinal monitoring of genetic alterations in ctdna may overcome many of the limitations of tissue sampling . 
 libraries were constructed with illumina truseq nano dna library prep kit ( illumina , san diego , ca ) , enriched for a 566 - gene pancancer gene panel ( data supplement ) by using agilent sureselect xt custom baits ( agilent technologies , santa clara , ca ) , and sequenced on an illumina hisequation 2500 sequencer to a median of 105 million reads , which yielded a median coverage of 1 , 195.18 sequence data were aligned to the hg19 reference genome . 
those with at least four alternate reads were retained on the basis of a calculated background mutation rate and false - positive rate of 1e - 4 and 1e - 6 , respectively . 
ctdna insufficient for analysis indicates those samples that were technically successful , but had insufficient tumor dna present in the plasma to reliably estimate underlying tumor genotype . longitudinal ctdna analysis ( n = 79 ) disease progression on study ( n = 29 ) ctdna analysis successful ( n = 22 ) ctdna insufficient for analysis ( n = 7 ) 23 repeat biopsies attempted for 20 patients * repeat biopsy not attempted ( n = 2 ) tissue insufficient ( n = 7 ) tissue sufficient ( n = 16 ) inaccessible site ( n = 1 ) patient preference ( n = 1 ) * three patients underwent tissue sampling twice during plasma surveillance patients with cns - only progression patients with isolated intrathoracic disease progression patients with isolated progression of a liver lesion ( n = 1 ) ( n = 5 ) ( n = 1 ) three of six attempted repeat biopsies sufficient for analysis results patient characteristics that were administered is depicted in the data supplement and figure 2 . plasma was longitudinally collected from 79 patients with metastatic , alk - positive nsclc ( fig 1 )  . 
as this study was primarily focused on investigating the role of ctdna analysis at disease progression , this report is limited to patients who had progressive disease and analyzable plasma specimens . 
of the seven patients who did not have detectable ctdna , one patient experienced cns - only progression , five patients had intrathoracic progression , and one patient had liver oligoprogression ( fig 1 )  . 
baseline characteristics of the 22 patients and their treatment histories are summarized in table 1 , figure 2 , and the data supplement . plasma genotyping was performed on 88 plasma samples from 22 patients . 
the temporal relationship between plasma collection and the treatments concordance between tissue and plasma genotypes we first evaluated concordance between tissue and plasma genotyping for alk fusions , alk mutations , and non - alk alterations . 
 ( % ) of alk tkis before initial plasma collection * abbreviation : alk , anaplastic lymphoma kinase ; tki , tyrosine kinase inhibitor . * the total number of alk tkis on which a patient had experienced progression before the collection of the initial plasma specimen . 
in instances where plasma was collected during sequential treatment with distinct alk inhibitors , the line of therapy at initial plasma collection is represented . ( 94% ; appendix fig a1 )  . 
among the 12 patients for whom the fusion partner was known and an alk fusion was detected in the plasma , there was 100% agreement between tissue and plasma fusion calls ( fig 3a )  . 
point mutations in the alk kinase domain were identified in plasma specimens from 11 patients ( 50% ) , five of whom had paired biopsies at the time of mutation detection ( fig 3b )  . 
three patients had multiple alk mutations . all tissue - detected alk mutations were also identified in corresponding plasma samples , and all plasma - detected alk mutations were also present in paired biopsies . 
 among 17 patients who had plasma collected during progression on a second - generation alk inhibitorthat is , ceritinib , alectinib , or brigatinibplasma alk mutations were identified in eight patients ( 47% ; data supplement ) , including i1171n ( n = 1 ) , g1202r ( n = 5 ) , i1171n / g1202r ( n = 1 ) , and e1210k ( n = 1 )  . 
 the observed frequency of plasma alk mutations is consistent with our previous analysis of resistance biopsies from patients who developed resistance to second - generation inhibitors , which included five patients from the current plasma cohort.7 non - alk alterations . 
as we did not detect any differences in tissue alterations that were identified in serial specimens from the three patients who underwent multiple biopsies , we restricted the concordance assessment to one biopsy per individual . 
although we were not able to obtain tissue after the patient had developed progression on lorlatinib , amplification was not present when a prelorlatinib liver biopsy was assessed using the same 566gene panel . 
despite high - level met amplification ( > 25 copies ) in tissue , we did not detect met amplification in the corresponding plasma specimen ; however , as the maximum allelic frequency ( af ) of detected alterations in the plasma of mgh939 was approximately 2% , the degree of amplification was below the limit of detection of the assay . evolution of resistance during molecular surveillance alk fusion kinetics and correlation with disease status . 
preclinical models and case reports suggest that the presence of a specific alk mutation in a tki - resistant tissue specimen may predict response to subsequent alk tkis1 , 7 , 23 ; therefore , we analyzed plasma specimens from patients with alk mutations who received treatment with additional alk tkis to evaluate the potential of using plasma findings to inform sequential therapies . 
during the follow - up period , seven of these patients ( 88% ) went on to receive treatment with a tki that targeted the alk mutation ( data supplement )  . 
 detected in plasma only detected in plasma and tissue mutation no paired biopsy paired biopsy available detected in plasma only detected in plasma and tissue eml4 - alk variant 1 variant 2 variant 3 variant 5 i1171n f1174c f1174l g1202r d1203n e1210k fig 3 . 
parentheses denote patients for whom the alk mutation was considered the dominant resistance mechanisfor patients without paired biopsies , alk mutations that were detected at the time of progression on an alk tyrosine kinase inhibitor are listed . 
because the mutations were separated by too many bases , the allelic relationship ( cis v trans ) could not be determined for mgh990s alk mutation . in alk i1171n af from 4.5% to undetectable after 6 weeks ( fig 4a )  . 
one patient ( mgh989 ) underwent microwave ablation to treat a liver lesion with a biopsy - proven g1202r mutation and continued treatment with alectinib for an additional 6 months . 
 mgh990 had a radiographic response to treatment , but we could not evaluate the molecular response as we were only able to collect a single plasma specimen . although the g1202r mutation was suppressed in plasma during the treatment with lorlatinib , two of the four patients with plasma - detected g1202r developed progressive disease soon after initiating lorlatinib ( appendix fig a3 )  . 
we were not able to obtain a biopsy , but plasma analysis demonstrated an increase in copy number of gnas and bcl2l1 and a marked increase in the af of mutations that involved other genes during treatment ( fig 5 )  . 
in both cases , eml4 - alk fusion af increased at progression despite the suppression of alk mutations , which supports the notion that resistance was independent of genetic alk alterations . discussion despite a shared molecular driver , the clinical course is variable for patients with alk - positive nsclc treated with alk tkis . 
our findings highlight the potential clinical utility of hybrid - capture ngs for improving our understanding of the molecular drivers of resistance . in our cohort of patients , 76% of plasma samples contained sufficient tumor - derived dna for molecular analysis compared with 65% of biopsy specimens , which confirms that both are reliable approaches . 
 furthermore , our observation that the presence of specific plasma alk mutations correlated with the response and resistance to distinct alk tkis lends support to the emerging practice of using alk mutations to inform the selection of alk tkis . although tissue sampling is the gold standard for molecular analysis , sampling a single site of disease may overestimate the contribution of a particular alteration to the resistant phenotype . 
for example , we detected an alk g1202r mutation at 50% af in a progressing liver lesion , which suggested that it was a major contributor to alectinib resistance in the case of mgh919 . 
in contrast , the plasma g1202r af of 1% was less than the af of other plasma - detected molecular alterations , including multiple non - alk alterations ( fig 5 )  . 
moreover , this case reinforces the notion that the analysis of liquid biopsies may be more informative than tissue - based genotyping in certain situations . to date , the study of resistance to alk therapeutics has primarily focused on alk mutations . 
figure illustrates the change in the allelic fraction of eml4alk and alk mutations during sequential treatment with next - generation alk inhibitors for ( a ) mgh987 and ( b ) mgh087 . 
 ( a ) allelic fraction of alk ( anaplastic lymphoma kinase ) fusion ( blue ) , alk mutations ( red and gold ) , and non - alk alterations ( gray ) during treatment with sequential alk inhibitors . 
despite these shortcomings , our data suggest that the quantitative assessment of structural variants may be clinically useful and complementary to radiographic assessment in some patients . overall , our data confirm that plasma genotyping by using hybrid - capture ngs technology can reliably detect alk fusions and alk resistance mutations in patients with alk - positive nsclc ; however , there are several limitations of this study , including the small size of our cohort , inconsistent sampling intervals that led to variation in the duration of follow - up after and before disease progression , and the inability to obtain pretreatment plasma and paired biopsies for all patients . 
furthermore , as a result of the small contribution of tumor dna to total cellfree dna in most patients , we cannot definitively exclude the presence of undetected amplification events at resistance . 
as identifying alk resistance mutations is emerging as an important consideration for the management of alk - positive nsclc , we anticipate that genotyping plasma to characterize resistance to alk tkis will also become a routine part of patient care . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . ibiayi dagogo - jack consulting or advisory role : boehringer ingelheim honoraria : foundation medicine a . 
campbell employment : novartis , pfizer ( i ) manuscript writing : all authors stock and other ownership interests : novartis final approval of manuscript : all authors accountable for all aspects of the work : all authors jessica j . 
schultz no relationship to disclose jennifer ackil no relationship to disclose sara stevens no relationship to disclose leila dardaei no relationship to disclose satoshi yoda no relationship to disclose harper hubbeling no relationship to disclose subba r . 
sequist honoraria : astrazeneca consulting or advisory role : astrazeneca , genentech , bristol - myers squibb , pfizer research funding : boehringer ingelheim ( inst ) , clovis oncology ( inst ) , genentech ( inst ) , merrimack pharmaceuticals ( inst ) , novartis ( inst ) , astrazeneca ( inst ) , johnson & johnson ( inst ) , merck ( inst ) , pfizer ( inst ) inga t . 
lennes honoraria : blue cross and blue shield of massachusetts consulting or advisory role : kyruus anthony john iafrate stock and other ownership interests : archer biosciences consulting or advisory role : debiopharm group , constellation pharmaceuticals , chugai pharma , roche research funding : blueprint medicines patents , royalties , other intellectual property : archerdx exclusive license to amp technology rebecca s . 
heist consulting or advisory role : boehringer ingelheim research funding : glaxosmithkline , sanofi , abbvie , novartis , roche , incyte , celgene , mirati therapeutics , peregrine pharmaceuticals , exelixis , millennium pharmaceuticals , debiopharm group christopher g . 
azzoli consulting or advisory role : merck , ariad pharmaceuticals , takeda research funding : pharmamar ( inst ) , abbvie ( inst ) , astrazeneca ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) , loxo ( inst ) , ignyta ( inst ) travel , accommodations , expenses : pharmamar , abbvie , stemcentrx jeffrey a . 
engelman employment : novartis stock and other ownership interests : loxo , agios , kura , gatekeeper pharmaceuticals , novartis consulting or advisory role : novartis , agios , loxo , clovis oncology , ventana medical systems , g1 therapeutics , sanofi , chugai pharma , warp drive bio , asana biosciences , emd serono , synta , allostery , genentech , aveo , biodesix , merck , cell signaling technology , endo pharmaceuticals , astrazeneca , glaxosmithkline , amgen , bristol - myers squibb , cancer progress research funding : novartis , jounce therapeutics , sanofi , araxes , astrazeneca , amgen ( inst ) patents , royalties , other intellectual property : coinventor on a patent application that has been licensed to ventana medical system / roche other relationship : third rock ventures jochen k . 
leary employment : novartis stock and other ownership interests : novartis patents , royalties , other intellectual property : patents , pending patents , and a royalty - sharing agreement with johns hopkins university alice t . 
shaw honoraria : pfizer , novartis , genentech , foundation medicine consulting or advisory role : pfizer , novartis , genentech , roche , ariad pharmaceuticals , ignyta , blueprint medicines , daiichi sankyo , emd serono , taiho pharmaceutical , ksq therapeutics research funding : pfizer , novartis , genentech justin f . 
gainor honoraria : merck , incyte , ariad pharmaceuticals consulting or advisory role : novartis , boehringer ingelheim , clovis oncology , genentech , bristol - myers squibb , theravance , loxo research funding : merck , novartis , genentech , bristolmyers squibb , adaptimmune , astrazeneca , ariad pharmaceuticals travel , accommodations , expenses : affymetrix anna f . 
farago honoraria : foundation medicine consulting or advisory role : pharmamar , merrimack pharmaceuticals , takeda , abbvie , intervention insights acknowledgment we also thank jeremy decker for assistance with plasma preparation and circulating tumor dna processing . 
shaw at , yeap by , mino - kenudson m , et al : clinical features and outcome of patients with nonsmall - cell lung cancer who harbor eml4 - alk . 
kim dw , mehra r , tan ds , et al : activity and safety of ceritinib in patients with alk - rearranged nonsmall - cell lung cancer ( ascend - 1 ) : updated results from the multicentre , open - label , phase 1 trial . 
shaw at , gandhi l , gadgeel s , et al : alectinib in alk - positive , crizotinib - resistant , nonsmallcell lung cancer : a single - group , multicentre , phase 2 trial . 
kim dw , tiseo m , ahn mj , et al : brigatinib in patients with crizotinib - refractory anaplastic lymphoma kinase - positive nonsmall - cell lung cancer : a randomized , multicenter phase ii trial . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced nonsmall - cell lung cancer . 
sacher ag , paweletz c , dahlberg se , et al : prospective validation of rapid plasma genotyping for the detection of egfr and kras mutations in advanced lung cancer . 
paweletz cp , sacher ag , raymond ck , et al : bias - corrected targeted next - generation sequencing for rapid , multiplexed detection of actionable alterations in cell - free dna from advanced lung cancer patients . 
schwaederl mc , patel sp , husain h , et al : utility of genomic assessment of blood - derived circulating tumor dna ( ctdna ) in patients with advanced lung adenocarcinoma . 
thompson jc , yee ss , troxel ab , et al : detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next - generation sequencing of cell - free circulating tumor dna . 
curigliano g , gmez pardo p , meric - bernstam f , et al : ribociclib plus letrozole in early breast cancer : a presurgical , window - of - opportunity study . 
ye k , schulz mh , long q , et al : pindel : a pattern growth approach to detect break points of large deletions and medium sized insertions from paired - end short reads . 
solomon b , bauer tm , felip e , et al : safety and efficacy of lorlatinib ( pf - 06463922 ) from the dose - escalation component of a study in patients with advanced alk + or ros1 + non - small cell lung cancer ( nsclc )  . 
 in two of the three patients for whom plasma analysis failed to detect a fusion , an eml4 - alk fusion ( variants 3 and 5 ) was identified in matching tissue . 
kornaga , msc1 , 2 ; cheryl crozier , msc1 ; gun ho jang , phd1 ; lauren bathurst , msc1 , 3 ; irina kalatskaya , phd1 , 4 ; quang m . 
pathways were identied as aberrant if there were copy number aberrations and / or mutations in any of the predetermined pathway genes : ccnd1 / ccnd2 / ccnd3 / cdk4 / cdk6 , fgfr1 / fgfr2 / fgfr2 / fgfr4 , and akt1 / akt2 / pik3ca / pten . results the 390 of 420 samples that passed quality control were analyzed for distant metastasisfree survival between groups . 
these bcas represent the majority of new bca diagnoses ( 70% to 80% ) , and a recent meta - analysis of 62 , 923 women with estrogen receptor ( er ) positive bca , who were disease free after 5 years of endocrine therapy , reported that approximately one in three women will experience a relapse during the next 20 years.1 - 3 although the development of diagnostic tests have helped to inform patients with regard to residual relapse risk , 4 , 5 as well as the identication of those who do not benet from chemotherapy , 6 none have yet to provide insight for those high - risk patients who may benet from the precision medicine approaches offered in the metastatic setting . 
to fully maximize the benet of targeted therapies seen in unselected patients in the advance / metastatic setting , upfront stratication of patients at risk for recurrence to these targeted agents in the early setting would reduce unnecessary treatment and associated toxicities and form a rational basis for treatment selection . 
this retrospective study of 390 early breast cancers from a phase iii clinical trial demonstrates that genomic changes within key genes in the cyclin d / cyclin - dependent kinase , phosphatidylinositol 3 - kinase / protein kinase b , and broblast growth factor receptor pathways could identify patients who might benet most from treatment that targets those deregulated pathways . knowledge generated in this focused approach , aberrations among genes in these pathways were shown to affect risk of distant relapse . relevance the use of this stratication approach , which benets from the interrogation of a limited number of genes , could be implemented effectively by using existing genomic technologies in the clinical laboratory to aid clinicians in identifying patients for whom specic targeted treatments are needed in combination with standard endocrine therapy . and the cancer genome atlas , 13 , 14 translation of these ndings into novel approaches to the management of cancer is slow . in response , we generated data focused on specic signaling pathways derived from the international cancer genome consortium and the cancer genome atlas13 , 14 together with whole - genome copy number analyses . 
the analysis was performed on the basis of an a priori hypothesis related to molecular pathways for which there are existing targeted therapies currently under evaluation in late - stage clinical trials . 
in a case - control study , 420 patients from the tamoxifen versus exemestane adjuvant multinational ( team ) trial pathology cohort ( clinicaltrials . identiers : nct00279448 , nct00032136 , and nct00036270 ) 15 were selected to determine the prognostic ability of copy number events and specic mutational changes that represent the cyclin d / cyclin - dependent kinase ( ccnd / cdk ) , broblast growth factor receptor / broblast growth factor ( fgfr / fgf ) , and phosphatidylinositol 3 - kinase / protein kinase b ( pi3k / akt ) pathways to predict risk of recurrence after anti - endocrine therapy in the early setting . 
distant metastasisfree survival ( dmfs ) was dened as time from random assignment to distant relapse or death as a result of bca.15 this study complies with the declaration of helsinki , institutional ethics committees , and the international conference on harmonization and good clinical practice guidelines . 
patients provided informed consent , and this study was approved by the university of toronto research ethics board ( protocol number 35339 )  . targeted sequencing and copy number analysis forty nanograms of dna extracted from formalin - xed parafn - embedded tissues were processed for targeted sequencing ( appendix )  . 
only variants that were on target and passed ltering criteria were included in the downstream analysis as described by kalatskaya et al.19 for copy number aberration ( cna ) analyses , 200 ng of dna were processed using oncoscan ffpe express 2.0 snp arrays ( affymetrix , santa clara , ca )  . 
 prognostic signatures in early breast cancers highly variable log - r ratio ( lrr ) , unevenly distributed b - allele frequencies , and waviness in lrr were measured using penncnv.20 estimation of tumor ploidy and aberrant tumor fraction was performed , and copy number segments were used by combining segments generated by two algorithms : allele - specic copy number analysis of tumors version 2.121 and circular binary segmentation22 implemented in the dnacopy package of r . 
an algorithm described by boutros et al23 was used to recalibrate tumor ploidy , aberrant tumor fraction , and copy numbers on the basis of lrr and b - allele frequency data , and adjacent segments having the same copy numbers were merged . segment boundaries were adjusted to obtain smaller argument function values . 
analyses were performed using r version 3.1.0. statistical analysis of the 420 patients , 390 passed ltering criteria and were included in subsequent analyses using stata 12.1 software ( statacorp , college station , tx )  . 
genes that represented pathways of interest were identied as aberrant according to the following criteria : ccnd / cdk pathway ( gains / amplications in any of ccnd1 / ccnd2 / ccnd3 / cdk4 / cdk6 ) , fgfr pathway ( gains / amplications in any of fgfr1 / fgfr2 / fgfr3 / fgfr4 ) , and pi3k / atk pathway ( aberrations in any of the following : gain / amplication of akt1 or possession of a mutation in akt1 , gain / amplication of akt2 , gain / amplication of pik3ca , and pten mutation and accompanying pten loss akt1 / akt2 / pik3ca / pten )  . a second model for the pi3k / akt was analyzed where pik3ca mutation data were included alongside pik3ca cnas and alterations in the genes of the dened pathways . kaplan - meier method and log - rank analyses were used to assess differences in dmfs . 
hrs were calculated using cox proportional hazards regression models , and models were adjusted for age ( continuous variable ) , tumor size ( dichotomized at 2 cm ) , tumor grade ( 1 / 2 v 3 ) , lymph node status , and her2 status ( negative v positive )  . 
all p values reported were two - sided , and p , .05 was considered statistically signicant . results changes in copy number of genes associated with signaling pathways are independently linked to high risk or relapse after anti - endocrine therapy our a priori hypothesis identied candidate pathways and specic genomic changes linked to endocrine treatment failure and outcome , namely ccnd / cdk signaling , the pi3k / akt axis , and fgfr / fgf pathway . 
no statistically signicant differences in age , tumor size , grade , lymph node status , or her2 status between patients with a dmfs event and those without were noted ( table 1 )  . 
of the 272 patients , 172 had aberrations in the specied ccnd / cdk genes ( 147 with aberrations in the fgfr genes and 138 with aberrations in pi3k / akt genes )  . 
of the 390 patients , 54 ( 13.8% ) , 34 ( 8.7% ) , and 43 ( 11% ) possessed aberrations in only one pathway ( ccnd / cdk , pi3k / akt , and fgfr , respectively )  . slightly fewer patients possessed aberrations in two pathways : 37 ( 9.5% ) with aberrant ccnd / cdkand pi3k / aktrelated genes , 37 ( 9.5% ) with aberrant ccnd / cdkand fgfr - related genes , and 23 ( 5.9% ) with aberrant pi3k / aktand fgfr - related genes . 
correlation to grade according to the number of pathways showed that patients with lower - grade tumors ( grade 1 / 2 ) had fewer altered pathways than those with grade 3 tumors ( appendix table a2 )  . 
although widely viewed as a good - prognosis subtype , hormone receptorpositive bcas account more than 84% of newly diagnosed early bca and therefore comprise the majority of bca deaths . 
despite recent advances in adjuvant therapy , outcomes for high - risk hormone receptorpositive bca are suboptimal , with an estimated relapse rate of 25% at 5 years.1 , 2 , 28 patients who are disease free at 5 years after diagnosis can still experelapse 5 to 20 years rience a high risk of distant postdiagnosis , 1 , 28 which directly challenges the dogma that such cancers are low risk and responsive to treatment . we tested the hypothesis that aberrations in key signaling pathways , where targeted therapies exist for bca ( in the advanced or metastatic setting ) , are prognostic in the early bca setting . 
aberrations in key genes of the ccnd / cdk pathway were more frequently detected than those in the pi3k / akt or fgfr axes ; however , the majority of patients ( 52% ) had aberrations in more than one pathway , which offers a rationale for incomplete responses to monotargeted therapy . 
kaplan - meier survival curves for aberrant pathways ( a ) cyclin d / cyclin - dependent kinase ( ccnd / cdk ) , ( b ) broblast growth factor receptor ( fgfr ) , ( c ) phosphatidylinositol 3 - kinase / protein kinase b ( pi3k / akt ) in the absence of pik3ca mutation , and ( d ) pi3k / akt pathway with pik3ca mutations . 
patients who possessed ccnd / cdk alterations exhibited signicantly poorer outcomes ( fig 1a ) than patients with fgfr and pi3k / akt aberrations ( figs 1b and 1c , respectively )  . 
in combination , patients with ccnd / cdk and fgfr aberrations were also at higher risk for relapse ( figs 2 and 3 ) possibly because of the proximity of ccnd1 and ligands fgf2 and fgf3 on chromosome 11q and their frequent co - amplication.29 indeed , the outcome for patients with ccnd / cdk and fgfr aberrations was similar to patients with ccnd / cdk aberrations only ( fig 3b )  . 
when pik3ca mutations were included in the stratication , there was no difference in survival between the two groups , consistent with the nding that pik3ca mutations in early bca and in the context of endocrine therapy seems to have no impact on outcome.30 , 31 increasing numbers of aberrant pathways also were associated with higher grade ( appendix tables a1 and a2 ) , consistent with the role of grade as an important prognostic factor32 and its association with gene expression changes that drive proliferation.33 - 35 intrinsic , endocrine resistance , acquired or remains a therapeutic challenge . 
for example , we have shown that up to one in ve early bcas exhibit amplications of cdk2 / 4 / 6 and ccnds39 , 40 and that ccnd1 amplication , but not overexpression , is associated with increased risk of distant relapse in endocrine - treated early bca.40 similarly , the trial study design did not stratify patients upfront by biomarker . 
consistent with published work , our data support evidence for a key role of cnas in this gene pathway.52 , 53 toxicities associated with mtor inhibitors like everolimus remain a concern . 
kaplan - meier curves of distant metastasisfree survival ( dmfs ) according to ( a ) risk order ( no aberrant pathway , any cyclin d / cyclin - dependent kinase [ ccnd / cdk ] , phosphatidylinositol 3 - kinase / protein kinase b [ pi3k / akt ] and broblast growth factor receptor [ fgfr ] , and fgfr only )  . 
 ( b ) frequency of aberration ( no aberrant pathway ; any pi3k / akt , fgfr and ccnd / cdk ; and ccnd / cdk only )  . class of drug would greatly affect patient management . phase iii trials are now under way to evaluate pi3k / akt inhibitors , including pi3kselective inhibitors in combination with fulvestrant in the advanced / metastatic bca setting ( clinicaltrials.gov identiers : nct02437318 and nct02340221 )  . 
phase ii neoadjuvant trials also are being conducted in the early bca setting ( clinicaltrials.gov identiers : nct01923168 and nct02273973 ) , which requires tumor pik3ca mutation status before determination of treatment randomization . 
however , we and others have shown that in early hormone receptorpositive bca , pik3ca mutations represent one of the most frequent mutations ( 35% ) 31 , 54 , 55 but show limited evidence that these mutations are linked to endocrine resistance in the early setting . the fgfr family of genes are recognized to be amplied in 10% to 15% of bcas.56 , 57 although fgfr1 is most frequently amplied , there is evidence for lower frequency amplication of fgfr2 , fgfr3 , and fgfr458 - 60 in early bca . 
evidence has emerged that links fgfrs to endocrine resistance.58 , 60 - 62 in preclinical studies , fgfr1 amplication enhanced pi3k and mitogen - activated protein kinase pathway signaling , which leads to endocrine resistance and could be reversed using rna interference against fgfr1.57 between 30% and 40% of cancers with ccnd1 amplication exhibit fgfr1 co - amplication despite the fact that these genes are mapped within different amplication loci on chromosomes 11 and 8.63 a challenge in targeting both ccnd1 and fgf pathwayamplied tumors is the proximity of the ligands fgf3 and fgf4 to ccnd1 on chromosome 11q13 . 
the role of fgfr signaling in a number of cancer settings suggests that it is a potential driver of cancer progression.58 , 64 , 65 several fgfr - targeted inhibitors are under investigation in phase ii clinical trials , including fgfr1 to 4 selective inhibitors bgj398 identiers : nct01004224 and ( clinicaltrials.gov nct02160041 ) and azd4547 ( clinicaltrials.gov identiers : nct01791985 , nct01795768 , and nct01202591 )  . 
to date , all trials that involve fgfr - targeted inhibitors are for patients with advanced or metastatic disease , with almost all trials requiring molecular prescreening for only fgfr1 or 11q amplication before study entry . issues of early hormone receptorpositive bca critical management must be addressed , 66 - 70 including molecular heterogeneity7 , 8 , 71 and emergent drug resistance.72 , 73 clinically , patients with such features would benet from early treatment with combinatorial therapies . 
in a drug screen of 42 agents , ribociclib was identied as a strong sensitizer to pi3k inhibition in three er - positive bca cell lines with acquired resistance to pi3k inhibitors.74 targeted doublet combinations of cdk4 / 6 or pi3k / mtor inhibitors with endocrine therapy have improved progression - free survival compared with endocrine therapy alone , 37 , 75 , 76 whereas another study showed that palbociclib plus pictilisib and fulvestrant was more effective than doublet combinations.77 we show that early hormone receptorpositive bcas could be stratied using genomic markers representative of pathways linked to targeted therapeutics that predict risk of recurrence after endocrine treatment . 
bartlett provision of study material or patients : daniel rea collection and assembly of data : jane bayani , cheryl crozier , paul c . boutros , melanie spears , john d . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / author - center . jane bayani patents , royalties , other intellectual property : 95 gene signature of residual risk in endocrine - treated early breast cancer ( provisional us patent 62 / 263 , 805 ) daniel rea honoraria : roche , novartis , pzer , eli lilly consulting or advisory role : genomic health research funding : celgene ( inst ) , roche ( inst ) , biotheranostics ( inst ) travel , accommodations , expenses : pzer , daiichi sankyo john m.s. 
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heiskanen m , kononen j , b arlund m , et al : cgh , cdna and tissue microarray analyses implicate fgfr2 amplication in a small subset of breast tumors . 
taylor - harding b , aspuria pj , agadjanian h , et al : cyclin e1 and rtk / ras signaling drive cdk inhibitor resistance via activation of e2f and ets . 
sledge gw jr , toi m , neven p , et al : monarch 2 : abemaciclib in combination with fulvestrant in women with hr + / her2advanced breast cancer who had progressed while receiving endocrine therapy . 
 prognostic signatures in early breast cancers appendix targeted sequencing submission of 101 selected genes resulted in 6 , 318 amplicons , with a median size of 109 base pairs ( bp ; range , 64 to 142 bp ) that covered 612.79 kbp ( unpublished data )  . 
using ampliseq polymerase chain reaction primer panels ( thermo fisher scientic , waltham , ma ) for the gene set developed above , we adapted our current protocol for illumina ( san diego , ca ) sequencing . 
after amplication of dna regions in four separate pools of the total 6 , 318 amplicons ( pool 1 , 1 , 587 amplicons ; pool 2 , 1 , 580 amplicons ; pool 3 , 1 , 578 amplicons ; pool 4 , 1 , 573 amplicons ; 10 ng / pool , 40 ng total dna ) , library polymerase chain reaction products were combined ( per sample ) , barcoded , and pooled in sets of 96 samples . 
t raw reads ( 101 - bp paired - end ) were aligned against the university of california , santa cruz , human reference sequence hg19 ( including random and unknown sequences ) using novoalign version 2.07.14 ( novocraft technologies , selangor , malaysia )  . 
aligned reads were ltered with samtools version 0.1.1816 to remove unmapped reads , nonprimary aligned reads , and reads aligned with mapping quality less than 30 ( ie , nonunique alignments )  . 
because the tamoxifen versus exemestane adjuvant multinational trial ( clinicaltrial . gov identiers : nct00279448 and nct00032136 , and nct00036270 ) cohort did not have matching normal tissue , the conventional mutation calling pipelines were not applicable . 
 c vemurafenib in patients with relapsed refractory multiple myeloma harboring brafv600 mutations : a cohort of the histology - independent ve - basket study noopur raje ian chau david m . 
yee martin kaiser heather landau jean - marie michot antoine hollebecque luisa veronese martina makrutzki bethany pitcher igor puzanov jose baselga author affiliations and support information ( if applicable ) appear at the end of this article . clinical trial information : nct01524978 . corresponding author : noopur raje , md , professional office building 216 , harvard medical school , 55 fruit st , boston , ma 02114 ; e - mail : nraje@mgh.harvard.edu. introduction multiple myeloma ( mm ) is a genetically heterogeneous , complex disease that arises as a result of a variety of mutations in pathways deregulating the intrinsic biology of the plasma cell.1 although immunomodulatory drugs and proteasome inhibitors have improved outcomes in patients with mm , 2 , 3 patients with relapsed or refractory disease have a poor prognosis.4 despite the identification of many of the genetic events involved in the development of mm , no treatments specifically targeting genetic mutations have been developed to date . 
additional study design details are described in the data supplement . patients patients in the mm cohort had to have previously treated , measurable , relapsed or refractory mm with confirmed brafv600 mutation . 
the presence of brafv600 mutations was confirmed retrospectively in a central laboratory using the roche companion diagnostic cobas 4800 braf v600 test or other standard methodology . assessments the following assessments for mm were performed 8 weeks after the start of therapy and every 4 weeks thereafter : serum protein electrophoresis with quantitation of monoclonal protein level ; urine protein electrophoresis using 24 - hour urine protein electrophoresis ; and serum levels of free light chains , lactate dehydrogenase , and - 2 microglobulbone marrow analysis was performed only to confirm complete response ( cr ) after two consecutive immunofixation analyses were negative . efficacy was evaluated using international myeloma working group uniform response criteria.17 , 18 evaluations were performed at baseline , 8 weeks after the start of vemurafenib administration , every 4 weeks thereafter during treatment , and at the end of treatment . 
responses were classed as cr , stringent cr , very good partial response ( vgpr ) , partial response ( pr ) , stable disease ( sd ) , progressive disease , and clinical relapse . 
responders were defined as those patients with pr or better ( ie , cr , stringent cr , pr , or vgpr )  . statistical analysis the primary efficacy end point of ve - basket was the response rate at week 8 . 
for the purposes of this analysis , two response assessments were required with no specified time between these assessments , although the protocol required that these two assessments be 4 weeks apart . an adaptive simon two - stage design was used to minimize the number of patients treated if vemurafenib was deemed ineffective for a specific tumor type . 
assuming response rates as specified in the hypothesis testing , a power of 80% for a high desirable response and 70% for a low desirable response , and a two - sided of 0.1 , the number of patients required in each cohort was seven for stage 1 , and 13 or 19 for stage 2 , depending on the results in stage 1 . 
however , if a clear clinical benefit was observed for patients in a cohort ( eg , the majority of patients recorded sd at week 8 and no cr or pr was recorded ) , then enrollment in stage 2 might be allowed for the cohort after discussion with the sponsor and study steering committee . 
the data presented here are the results of the final analysis . seven patients entered stage 1 of the mm cohort of the ve - basket study , followed by enrollment of an additional two patients because clear clinical benefit was observed , even though the prespecified minimal response rate was not achieved . 
the patient received palliative radiotherapy and eight cycles of lenalidomide , bortezomib , and dexamethasone before undergoing high - dose melphalan therapy with autologous stem - cell transplantation in november 2008 . 
baseline characteristics of patients with brafv600 mutationpositive multiple myeloma treated with vemurafenib patients with multiple myeloma ( n = 9 ) characteristic male female age , years , median ( range ) ecog performance status brafv600 mutation type * monoclonal protein v600e v600k iga kappa iga lambda igg kappa igg lambda risk stratification high standard lines of prior systemic therapies prior systemic therapies * immunomodulators ( lenalidomide , thalidomide , pomalidomide ) corticosteroids ( dexamethasone ) alkylating agents ( cyclophosphamide , melphalan , bendamustine ) proteasome inhibitors ( bortezomib , carfilzomib ) cytotoxic agents ( doxorubicin , idarubicin ) platinum agent ( cisplatin ) topoisomerase inhibitors ( etoposide ) novel antineoplastic agents ( acy - 1215 , cc - 223 ) note . 
 ( % ) unless indicated otherwise . abbreviations : ecog , eastern cooperative oncology group performance status ; ig , immunoglobulin . * on the basis of a manual review of the data . 
 testing was by snapshot ( n = 2 ) , capillary electrophoresis single - strand conformation analysis ( n = 2 ) , direct ( sanger ) sequencing ( n = 1 ) , sequenom ( n = 1 ) , single - strand conformation analysis ( n = 1 ) , 3730 dna analyzer ( n = 1 ) , and immunohistochemistry ( n = 1 )  . 
at the time of closure of the ve - basket study , the patient was still receiving treatment in the extension study . the second patient was diagnosed with smoldering mm in may 2006 and progressed to stage ii active mm in december 2009 , at which time he was treated with lenalidomide , bortezomib , and dexamethasone . 
 ( b ) change from baseline in serum m protein according to best overall response in individual patients with brafv600 mutation positive multiple myeloma treated with vemurafenib ( n = 9 )  . 
one patient with a substantial reduction in serum m protein level had an unconfirmed pr but was considered to have sd because the patient had pd at the subsequent assessment . 
 subsequent relapses were treated with cyclophosphamide , lenalidomide , and dexamethasone ( april 2013 ) ; carfilzomib , thalidomide , and dexamethasone ( august 2013 ) ; pomalidomide , bortezomib , and dexamethasone ( march 2014 ) ; and carfilzomib , pomalidomide , and dexamethasone ( august 2014 )  . 
snapshot testing of bone marrow aspirate identified an additional nras mutation at the time of relapse that had not been seen at study entry and was reported after study closure ; the original brafv600g mutation was also present at this time . one other patient , who had been treated previously with bortezomib and dexamethasone in the first line , the mammalian target of rapamycin inhibitor cc - 223 on relapse , and lenalidomide in the third line , had a vgpr after treatment with vemurafenib . 
one patient achieved an 83% reduction in serum monoclonal protein level and had a pr at one assessment but progressive disease at the next assessment , for a best overall response of sd . 
time to event details and change from baseline in serum monoclonal protein for individual patients are shown in figure 1 . at the time of study closure , disease progression was observed in six patients . 
median investigator assessed pfs was 4.63 months ( 95% ci , 2.89 months to not estimable ) ; the 6 - month pfs rate was 40% ( 95% ci , 6% to 74% ) , and the median time to progression was 4.63 months ( 95% ci , 2.89 months to not estimable )  . 
kaplan - meier plots of pfs and os are shown in the data supplement . safety the median vemurafenib dose intensity was 79% ( range , 51% to 100% )  . 
adverse events occurring in 20% of patients with brafv600 mutationpositive multiple myeloma treated with vemurafenib ( n = 9 ) any grade grade 3 or 4 event alopecia melanocytic nevus anemia hypokalemia keratosis pilaris skin papilloma sunburn acne back pain constipation diarrhea dysphonia fatigue * hyperkeratosis keratosis follicular leukoplakia oral rash papular seborrheic keratosis skin lesion xerosis upper respiratory tract infection blood alkaline phosphatase increase increased alanine aminotransferase note . 
one patient had grade 2 hypercalcemia , which was judged unrelated to vemurafenib treatment and resolved without treatment interruption , and a grade 3 chest infection , also unrelated to treatment , during which treatment was interrupted temporarily . 
the second patient had grade 4 diabetes that was judged unrelated to the study drug but resulted in dose reduction and grade 3 cellulitis that was also unrelated to treatment but required treatment interruption . 
 the final patient had treatment - related grade 4 sepsis that resolved with dose reduction and treatment - related grade 3 skin lesion and grade 2 upper respiratory infection that were not considered serious but resulted in permanent discontinuation of treatment . 
seven patients had at least one adverse event leading to dose reduction or interruption ( infections [ n = 4 ] , skin disorders [ n = 2 ] , anemia [ n = 1 ] , blood alkaline phosphatase increased [ n = 1 ] , keratosis follicular [ n = 1 ] , diabetes mellitus [ n = 1 ] , and acute kidney injury [ n = 1 ] ) ; one patient had a dose interruption for cataract surgery . qt prolongation , squamous cell carcinoma of the skin , and keratoacanthoma were not observed . 
however , identification of the brafv600 mutation as a driver mutation in mm may lead to patients with this mutation benefitting from targeted treatment with brafv600 inhibitors.1 , 7 , 20 in this study , we have shown that vemurafenib has promising efficacy in patients with brafv600 mutationpositive mm : two patients of nine had encouraging and long - lasting responses to treatment that were ongoing at the time of study closure , and an additional patient had a shorter response . 
these patients had been treated previously with bortezomib and lenalidomide , among other agents , suggesting that resistance mechanisms in those patients did not prevent response to vemurafenib . in this study , not all patients with the brafv600 mutation responded to therapy , providing us with the opportunity to study mechanisms of resistance in this patient population in the future . 
clonal evolution of mm cells and changes in the bone marrow environment have been implicated in resistance , 21 and alterations in the erk pathway are present in almost one half of patients with mm . 
interestingly , the patient in our study who relapsed after achieving a pr had acquired an nras mutation , suggesting an escape mechanism and possible resistance via this molecular pathway . 
 hyman , jean - yves blay , josep tabernero , jrgen wolf , igor puzanov , jose baselga provision of study material or patients : all authors collection and assembly of data : all authors data analysis and interpretation : noopur raje , martina makrutzki , bethany pitcher manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors mechanisms , including driver mutations within subclonal populations , may also account for differential responses . 
understanding resistance mechanisms could lead to the development of new therapies acting on the ras / raf pathway , either alone or in combination , or to the use of mek or pan - raf inhibitors . 
this subject is being addressed in an ongoing clinical trial using a braf inhibitor and a mek inhibitor ( dabrafenib and trametinib , respectively ; clinicaltrials.gov identifier : nct03091257 )  . the safety profile of vemurafenib in this patient cohort was broadly similar to that reported previously , 8 , 9 and no new safety signals were identified . 
cutaneous squamous cell carcinoma , which has been reported in vemurafenib treated patients in earlier studies , 8 , 9 was not observed in our patients . in conclusion , vemurafenib may be an appropriate and effective choice for patients with mm in whom a brafv600 mutation has been identified . 
because the ve - basket study included few patients with mm , additional studies are required to establish the role of vemurafenib , whether as monotherapy or in combination with other agents , for earlyor later - stage disease in the treatment of this poor - prognosis population . 
 ian chau honoraria : eli lilly , gilead sciences consulting or advisory role : eli lilly , bristol - myers squibb , msd , merck serono research funding : janssen - cilag ( inst ) , sanofi ( inst ) , merck serono ( inst ) , eli lilly ( inst ) travel , accommodations , expenses : msd , merck serono , sanofi , eli lilly , bristol - myers squibb martin kaiser honoraria : takeda pharmaceuticals , celgene , amgen , janssen oncology consulting or advisory role : janssen oncology , celgene , bristol - myers squibb , takeda pharmaceuticals , amgen , chugai pharmaceutical research funding : celgene travel , accommodations , expenses : takeda pharmaceuticals david m . 
yee consulting or advisory role : takeda pharmaceuticals , dexcel pharma , adaptive biotechnologies , celgene , janssen pharmaceuticals research funding : bristol - myers squibb ( inst ) , celgene ( inst ) bethany pitcher employment : f . 
hoffmann - la roche igor puzanov consulting or advisory role : amgen , roche / genentech , bristol - myers squibb travel , accommodations , expenses : amgen , merck jose baselga leadership : infinity pharmaceuticals , varian medical systems , grail , bristol - myers squibb stock or other ownership : pmv pharma , juno therapeutics , infinity pharmaceuticals , grail , varian medical systems , tango , foghorn , aura biomedical , apogen , northern biologics , bristol - myers squibb honoraria : pmv pharma , juno therapeutics , infinity pharmaceuticals , grail , northern biologics consulting or advisory role : eli lilly , novartis , grail patents , royalties , other intellectual property : combination therapy using pdk1 and pi3k inhibitors . 
hyman , heather landau , and jose baselga , memorial sloan kettering cancer center , new york ; igor puzanov , roswell park cancer institute , buffalo , ny ; vincent ribrag , jean - marie michot , and antoine hollebecque , institut gustave roussy , villejuif ; jean - yves blay , centre leonberard , lyon , france ; josep tabernero and elena elez , vall dhebron university hospital and institute of oncology , universitat autnoma de barcelona , barcelona , spain ; jrgen wolf , university hospital kln , kln , germany ; luisa veronese and martina makrutzki , f . 
hoffmann - la roche funded third - party writing and editorial support , which was provided by miller medical communications . presented in part at the 57th annual meeting of the american society of hematology , orlando , fl , december 5 - 8 , 2015 . support prior presentation references 1 . 
dimopoulos ma , swern as , li js , et al : efficacy and safety of long - term treatment with lenalidomide and dexamethasone in patients with relapsed / refractory multiple myeloma . 
kumar sk , lee jh , lahuerta jj , et al : risk of progression and survival in multiple myeloma relapsing after therapy with imids and bortezomib : a multicenter international myeloma working group study . 
walker ba , boyle em , wardell cp , et al : mutational spectrum , copy number changes , and outcome : results of a sequencing study of patients with newly diagnosed myeloma . 
mcarthur ga , chapman pb , robert c , et al : safety and efficacy of vemurafenib in braf ( v600e ) and braf ( v600k ) mutation - positive melanoma ( brim - 3 ) : extended follow - up of a phase 3 , randomised , open - label study . 
larkin j , del vecchio m , ascierto pa , et al : vemurafenib in patients with braf ( v600 ) mutated metastatic melanoma : an open - label , multicentre , safety study . 
subbiah v , gervais r , riely gj , et al : efficacy of vemurafenib in patients ( pts ) with nonsmall cell lung cancer ( nsclc ) with brafv600 mutation . 
hyman dm , kaley tj , hollebecque a , et al : vemurafenib in patients with brafv600 mutant glioma : a cohort of the histology - independent ve - basket study . 
diamond el , subbiah v , lockhart ac , et al : vemurafenib for brafv600 - mutant erdheimchester disease and langerhans cell histiocytosis : analysis of data from the histology - independent phase 2 open - label ve - basket study . 
rajkumar sv , harousseau jl , durie b , et al : consensus recommendations for the uniform reporting of clinical trials : report of the international myeloma workshop consensus panel 1 . 
 harnessing clinical sequencing data for survival stratication of patients with metastatic lung adenocarcinomas ronglai shen , phd1 ; axel martin , ms1 ; ai ni , phd1 ; matthew hellmann , md1 ; kathryn c . 
we sought to extend the use of broad - panel sequencing results to survival stratication and clinical outcome prediction . methods by using sequencing results from a cohort of 1 , 054 patients with advanced lung adenocarcinomas , we developed oncocast , a machine learning tool for survival risk stratication and biomarker identication . results with oncocast , we stratied this patient cohort into four risk groups on the basis of tumor genomic prole . 
2019 by american society of clinical oncology introduction with the growth of precision medicine programs driven by genomic testing as well as recent us food and drug administration clearance of next - generation sequencing ( ngs ) platforms for clinical use , there has been rapid growth in the availability of broad - panel sequencing data for patients with cancer . 
zehir et al1 delineated the molecular landscape of 10 , 000 metastatic cancers in a pretreated real - world cohort sequenced by the memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) platform , a hybridization capture - based ngs panel that can detect mutations and copy number alterations in 341 or more cancer - associated genes.2 this study showed that 37% of patients harbored at least one therapeutically actionable alteration , and 11% were matched to genome - directed clinical trials . in patients with lung adenocarcinoma , tumor genotyping is now an essential step in routine clinical decision making . 
to determine treatment , patients with lung adenocarcinomas are currently categorized on the basis of the presence of mutated driver oncogenes ( eg , egfr , alk , ros1 , and braf )  . 
a multiinstitutional study characterized genetic aberrations across 10 genes in 733 tumor samples and identied an oncogenic driver in 64% of the patients.3 by using broad - panel sequencing , jordan et al4 reported on a single - institution experience of 860 patients with metastatic lung adenocarcinoma . 
more than 37% of patients received a matched therapy , and the use of matched therapy was strongly inuenced by the level of pre - existent clinical evidence that the mutation identied predicts the drug response . although the focus of tumor genotyping has been on ascertaining mutations that identify therapeutic targets , there is considerable unexplained variability in clinical outcomes , even within specic molecular subsets of patients with metastatic cancer . 
 shen et al death - ligand 1 ( pd - 1 / pd - l1 ) inhibitors.5 however , additional markers are needed to predict durable benet and long - term survival among these patients . 
some studies have explored the effects of single co - occurring alterations on outcome in patients with egfr - mutant and kras - mutant lung adenocarcinoma , and they observed that the presence or absence of pairs of co - occurring events could be used to identify those patients with a poor prognosis most in need of novel therapeutic approaches.3 , 6 - 8 however , a systematic approach is needed to additionally improve our understanding of survival and treatment outcome of patients . thus , we developed oncocast , a computational tool for survival stratication , and applied it to a large clinical series of patients with metastatic lung adenocarcinomas to improve the understanding of heterogeneity in clinical outcome for these patients and the mutation and comutational patterns that underlie such heterogeneity . 
such real - world evidence can supplement information from randomized clinical trials in that the results are more generalizable to patients treated outside randomized clinical trials , and the larger sample sizes of real - world data sets allow subset analysis that clinical trials are not powered for . 
the open - source computational pipeline that we have developed can facilitate the application of statistical and machine learning approaches for clinico - genomics analysis of precision medicine data sets . methods the oncocast method is described in the data supplement . 
electronic medical records were used to identify patient clinical factors as well as survival outcomes as previously described.4 overall survival ( os ) was dened as the time from date of diagnosis of advanced disease ( stage iv or recurrent cancer ) until date of death or last follow - up . 
however , a fraction ( 21% ) of the tumors were sampled and sequenced more than 6 months from the date of metastatic disease , with 16% sequenced at more than 1 year and 8% sequenced at more than 2 years , which represents older samples used for sequencing analysis . 
those patients with older samples which had been taken at initial diagnosis of from their initial advanced lung cancer were immortal sampling time to the time of referral for msk - impact sequencing . 
this interval can be long for a small fraction of patients ( 8% with a delayed interval of more than 2 years , as mentioned earlier ) , which introduces survival bias . 
details of lefttruncation analysis are described in the data supplement . the most frequently mutated genes were tp53 ( 55.1% ) , kras ( 30% ) , egfr ( 29.4% ) , stk11 ( 17.7% ) , and keap1 ( 17.7% ; data supplement )  . 
among the frequently comutated gene pairs , stk11 and keap1 were comutated in 10% of the tumors , and kras and stk11 were comutated in 9% of the tumors ( data supplement )  . prognostic relevance and clonality of cancer genes to dene the prognostic signicance of msk - impact panel genes that were sequenced , we developed oncocast , a machine learning tool for survival risk stratication and biomarker identication by implementing a lassopenalized proportional hazards regression for deriving prediction rules for os and feature selection ( data supplement )  . 
oncocast uses an ensemble learning strategy by repeatedly splitting the cohort into training and test sets that generate an ensemble of classiers with varying selection of genes and gene combinations ( data supplement )  . the median number of prognostic genes selected was 19 ( range , 4 - 67 genes ) , with a total of 169 cancer genes selected at least once by the ensemble learner . 
the relative prognostic importance of each gene was measured by how often it was selected ( fraction of the models containing the gene ) and its average regression coefcients , which determined the corresponding weights for individual genes in the scoring rule . 
circle size is proportional to mutation frequency . targeted and immunotherapies than subclonal events.9 - 11 we thus used the high depth of coverage afforded by our sequencing data ( mean coverage , 758 ) to determine the clonality of the gene alterations found to be associated with prognosis in the analysis we outlined earlier . 
 shen et al oncocast : an integrated prognostic scoring system based on ngs tumor proling in addition , improvement . oncocast aggregates prognostic effects across the sequenced cancer genes to derive a genomic risk score for each patient ( scaled from 0 to 10 ) , with a higher score indicating a greater likelihood of shorter survival . 
there was a difference of more than 6 units in average risk score between the highand low - risk groups . the oncocast classication substantially outperformed all of the individual genes as a predictor of os ( fig 3b )  . 
oncocast risk score remained a highly signicant predictor after adjusting for clinical variables and treatment types as potential confounding factors in a multivariable cox regression model ( data supplement )  . to conrm the validity of the oncocast survival stratication , we applied it to a separate data set of patients with mostly early - stage nonsmall - cell lung cancer obtained from the cancer genome atlas lung adenocarcinoma analysis . 
stk11 is a tumor suppressor gene that encodes for the serine / threonine kinase lkb1 that functions as a negative regulator of mammalian target of rapamycin ( mtor ) signaling . 
to examine the status of the other allele in such cases , we performed allele - specic cna using the fraction and allele - specic copy number estimates from tumor sequencing ( facets ) algorithm.15 strikingly , more than 90% of stk11 mutant tumors had evidence of biallelic inactivation through loss of heterogeneity ( loh ) of chromosome 19p ( data supplement )  . 
the majority of keap1 mutations were missense , and the mutant copy was frequently duplicated ( present as copy - neutral loh and uniparental gains ) as reected in the average total copy number . we also explored whether other tumor characteristics such as tumor mutational signature or intratumor heterogeneity were prognostic and associated with oncocast risk score . previously dened smoking - associated and apolipoprotein b mrna editing enzyme , catalytic polypeptide - like ( apobec ) signatures were the most prevalent mutational signatures in this cohort . 
however , mutation burden did not provide additional prognostic value beyond oncocast risk score in a multivariable cox model ( data supplement )  . clonal diversity was calculated for each tumor by summarizing the cancer cell fraction for all somatic mutations using the shannon index . 
 ( a ) kaplan - meier plot of survival curves for the four risk subgroups ( low , intermediate - low [ int - low ] , intermediate - high [ int - high ] , and high )  . 
the average risk score ( avg rs ) , median overall survival ( os ) , and 1 - year and 3 - year survival probabilities are reported for each group . 
 ( b ) forest plot of hazard ratios ( hrs ) for age , sex , smoking status , and individual driver gene alterations compared with the oncocast integrated scoring approach . genotypes associated with risk groups to better understand the association between targetable cancer driver mutations and oncocast risk score , we explored the distribution of genotypes within four major risk categories ( fig 5 )  . 
the dening characteristic for the patients in the low - risk group without egfr , alk , ret , and ros1 alterations was an absence of any of the poor prognostic gene alterations . 
this suggests that stk11 and keap1 comutation denes a cohort of patients with resected lung adenocarcinoma at higher risk of disease progression and cancerspecic mortality . survival stratication in specic treatment subsets we then sought to demonstrate the ability of the oncocast technique to explore tumor genomic predictor outcomes in last to death or the subset of patients with mutated driver oncogenes treated with kinase inhibitors . 
the model , oncocast - tr ( targeted therapies model ) , was derived using the genomic proles and survival outcomes from the start of therapy for the 387 patients who received targeted therapies ( egfr , alk , ros )  . 
tp53 is strongly associated with worse survival outcome ( fig 6c ) , with 96% of patients in the tr2 group harboring concurrent tp53 mutations and 0% concurrent tp53 mutations in the tr1 group . 
arid1a also showed association with poor survival with 11 ( 78% ) of 14 arid1a mutations in tr2 . interactive tool for visualizing and exploring mutation pattern and survival to facilitate clinical translation and research use of the data , we created an interactive web application ( data that allows for visualization of mutation supplement ) ros1 fusion , 6% ret fusion , low - intermediate tp53 , egfr , smarca4 , 2% tp53 , ret fusion , 1% tp53 , alk fusion , 1% tp53 , ros1 fusion , 2% tp53 , smarca4 , 3% egfr , 40% tp53 , kras , tp53 , egfr , tp53 , egfr , alk fusion , other , 30% kras , 17% tp53 , 24% high - intermediate keap1 , 2% stk11 , 5% tp53 , kras , stk11 , 6% tp53 , kras , keap1 , 6% tp53 , keap1 , kras , keap1 , kras , stk11 , stk11 , keap1 , smarca4 , 7% tp53 , kras , stk11 , keap1 , 8% stk11 , keap1 , 8% high tp53 , stk11 , keap1 , smarca4 , 6% kras , stk11 , keap1 , 28% tp53 , stk11 , keap1 , 25% tp53 , stk11 , 10% kras , stk11 , keap1 , smarca4 , 16% fig 5 . 
 ( c ) gene importance plot for the oncocast - tr model . patterns and individualized prediction of os to be generated on the basis of a user - dened mutational prole and clinical characteristics . 
along with a patients clinical prole , the application outputs the predicted survival probabilities and 95% cis . although the tool was built using the largest metastatic lung adenocarcinoma cohort to date , it was designed to be dynamically updated as new data are incorporated into the model . discussion tumors from patients with lung adenocarcinoma have a high frequency of actionable oncogenic drivers.16 , 17 the introduction of targeted therapy has transformed the clinical care of patients with lung adenocarcinoma by incorporating tumor genotyping into therapeutic decision making . 
by using an ensemble learning approach , we demonstrated the prognostic utility of data from a large panel ngs assay interrogating 341 cancer - associated genes in 1 , 054 patients with metastatic lung adenocarcinoma treated with currently available therapies . 
we show that it is a practical approach to molecular stratication in metastatic lung adenocarcinoma and provides the ability to identify biomarkers that predict survival outcome . overlaying the oncocast risk score with the tumor genomic landscape revealed novel biologic and clinical insights . 
the major prognostic genes associated with poor survival included stk11 , keap1 , tp53 , kras , and smarca4 . remarkably , comutations of both stk11 and keap1 dened an exceptionally high - risk prole with a short median os of 7.3 months and an increase of more than 6 units in risk score compared with a low - risk group , corresponding to an hr of 4.6. 
some of the favorable prognostic genes may reect the availability of effective targeted therapies in the case of egfr , alk , and ros1 . however , in patients with these targetable oncogenes , there is heterogeneity in the additional mutations their tumors harbor . 
for example , our model revealed that mutations in tp53 and arid1a dene a high - risk subgroup with shorter survival in the tyrosine kinase inhibitortreated patient cohort . is novel in that conversely , some of the unfavorable prognostic genes may reect negative associations with treatment responses ( eg , the recent observation that stk11 inactivation is associated with poor responses to immunotherapy18 )  . 
mutations in keap1 are associated with chemotherapy resistance and poor survival . some studies have suggested that targeting nrf2 may enhance chemotherapy sensitization.19 , 20 mtor is a kinase downstream of lkb1 ( stk11 ) , and mtor inhibitors have been proposed as a potential therapeutic approach in stk11 - mutant tumors.21 smarca4 was also identied as a major driver of a poor prognosis factor in metastatic lung adenocarcinoma in our study . 
 shen et al complexes that regulate genomic instability and dna repair . smarca4 was mutated in 9.4% of lung adenocarcinomas in the msk - impact cohort , with 42% of the mutations being truncating and residing in regions of loh . 
with increasing sample sizes , we will be poised to identify outcome associations with rare alterations , and we will be better able to explain the heterogeneity in clinical outcomes for patients with advanced lung adenocarcinoma . in addition , the oncocast risk score described here provides a valuable tool for more accurately determining the prognosis of patients enrolled in clinical trials or included in real - world data sets . 
although conventional clinical factors such as age , sex , and performance status have long been used , our data indicate that we can signicantly improve the description of patient populations by incorporating the genomic risk scores in our understanding to allow better comparisons of groups of patients enrolled in clinical trials or included in real - world data sets . with the growth of precision medicine programs driven by genomic testing as well as recent us food and drug administration clearance of ngs platforms for clinical use , there will be a rapid growth in the availability of broad sequencing data for patients with a variety of cancers and growing integration with clinical data through institutional databases and electronic health records . 
riely manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors matthew hellmann stock and other ownership interests : shattuck labs honoraria : astrazeneca , bristol - myers squibb consulting or advisory role : bristol - myers squibb , merck , genentech , astrazeneca / medimmune , novartis , janssen , nektar therapeutics , syndax pharmaceuticals , mirati therapeutics , shattuck labs research funding : bristol - myers squibb ( inst ) patents , royalties , other intellectual property : a patent has been led by memorial sloan kettering ( pct / us2015 / 062208 ) for the use of tumor mutation burden for prediction of immunotherapy efcacy , which is licensed to personal genome diagnostics ( inst ) travel , accommodations , expenses : astrazeneca , bristol - myers squibb kathryn c . 
rudin consulting or advisory role : bristol - myers squibb , abbvie , seattle genetics , harpoon therapeutics , genentech , astrazeneca , ascentage pharma , bicycle therapeutics , celgene , daiichi sankyo , ipsen , loxo oncology , pharmamar , elucida oncology research funding : abbvie / stemcentrx ( inst ) , viralytics ( inst ) , merck ( inst ) , daiichi sankyo ( inst ) michael f . 
solit stock and other ownership interests : loxo oncology consulting or advisory role : pzer , loxo oncology , illumina , intezyne technologies , vivideon therapeutics travel , accommodations , expenses : merck maria arcila consulting or advisory role : astrazeneca travel , accommodations , expenses : astrazeneca , invivoscribe technologies , raindance technologies marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network / astrazeneca tagrisso request for proposal advisory committee , takeda pharmaceuticals , bristol - myers squibb , bayer ag , merck ( i ) research funding : loxo oncology ( inst ) , helsinn therapeutics gregory j . 
riely research funding : novartis ( inst ) , genentech ( inst ) , millennium pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , pzer ( inst ) , innity pharmaceuticals ( inst ) , ariad pharmaceuticals ( inst ) patents , royalties , other intellectual property : patent application submitted covering pulsatile use of erlotinib to treat or prevent brain metastases ( inst ) travel , accommodations , expenses : merck sharp & dohme no other potential conicts of interest were reported . references zehir a , benayed r , shah rh , et al : mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10 , 000 patients . 
nat med 23 : 703 - 713 , 2017 cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
j mol diagn 17 : 251 - 264 , 2015 kris mg , johnson be , berry ld , et al : using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs . 
jama 311 : 1998 - 2006 , 2014 jordan ej , kim hr , arcila me , et al : prospective comprehensive molecular characterization of lung adenocarcinomas for efcient patient matching to approved and emerging therapies . 
hellmann md , nathanson t , rizvi h , et al : genomic features of response to combination immunotherapy in patients with advanced non - small - cell lung cancer . cancer cell 33 : 843 - 852.e4 , 2018 arbour kc , jordan e , kim hr , et al : effects of co - occurring genomic alterations on outcomes in patients with kras - mutant non - small cell lung cancer . 
clin cancer res 24 : 334 - 340 , 2018 yu ha , suzawa k , jordan e , et al : concurrent alterations in egfr - mutant lung cancers associated with resistance to egfr kinase inhibitors and characterization of mtor as a mediator of resistance . 
clin cancer res 24 : 3108 - 3118 , 2018 yu ha , sima cs , shen r , et al : prognostic impact of kras mutation subtypes in 677 patients with metastatic lung adenocarcinomas . 
j thorac oncol 10 : 431 - 437 , 2015 de bruin ec , mcgranahan n , mitter r , et al : spatial and temporal diversity in genomic instability processes denes lung cancer evolution . 
rizvi h , sanchez - vega f , la k , et al : molecular determinants of response to anti - programmed cell death ( pd ) - 1 and anti - programmed death - ligand 1 ( pd - l1 ) blockade in patients with non - small - cell lung cancer proled with targeted next - generation sequencing . 
azzoli cg , temin s , giaccone g : 2011 focused update of 2009 american society of clinical oncology clinical practice guideline update on chemotherapy for stage iv non - small - cell lung cancer . 
directors challenge consortium for the molecular classication of lung adenocarcinoma , shedden k , taylor jm , et al : gene expression - based survival prediction in lung adenocarcinoma : a multi - site , blinded validation study . 
ren d , villeneuve nf , jiang t , et al : brusatol enhances the efcacy of chemotherapy by inhibiting the nrf2 - mediated defense mechanisproc natl acad sci u 21 . 
tagal v , wei s , zhang w , et al : smarca4 - inactivating mutations increase sensitivity to aurora kinase a inhibitor vx - 680 in non - small cell lung cancers . 
 o intrahepatic cholangiocarcinoma : genomic heterogeneity between eastern and western patients jingyu cao , md1 ; jing hu , phd2 ; siqin liu , phd3 ; funda meric - bernstam , md4 ; reham abdel - wahab , md5 ; junjie xu , md6 ; qiang li , md7 ; maolin yan , md8 ; yujie feng , phd1 ; jianzhen lin , md9 ; songhui zhao , msc3 ; jian wang , ms3 ; lawrence n . 
borad , md10 ; kai wang , md , phd3 ; milind javle , md4 ; and haitao zhao , md9 purpose intrahepatic cholangiocarcinoma ( ihcca ) , a global health problem , is increasing in incidence and has differing etiologies worldwide . 
ngs data from asia , where ihcca is most prevalent , are limited . methods comprehensive genomic proling of formalin - xed parafn - embedded tumor tissue from 164 asian and 283 western patients with ihcca was performed using ngs . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction cholangiocarcinoma ( cca ) , a global health problem , constitutes about 10% - 20% of all primary liver cancers worldwide . 
in western countries , including the united states , metabolic syndrome , inammatory bowel disease , primary sclerosing cholangitis , hepatitis c , and alcohol abuse are the main cca risk factors , and the age - standardized incidence rate is 1.6 per 100 , 000.1 the incidence in southeast asia is extremely high , up to 71.3 per 100 , 000 in certain parts of asia , and is associated with liver uke and hepatitis b virus ( hbv ) infection.2 notably , there is an increasing incidence of intrahepatic cholangiocarcinoma ( ihcca ) worldwide , and its etiology is unclear.3 surgical resection is the only potentially curative treatment , but the majority of patients present with an advanced disease stage and have a limited therapeutic options . 
ihcca , in particular , is enriched with a relatively high number of actionable mutations , and early reports indicate several promising leads with targeted therapies for broblast growth factor receptor 2 fusion ( fgfr ) , isocitrate dehydrogenase - 1 ( idh1 ) , braf v600e mutations , and her2 / neu amplication.2 nevertheless , ngs data from asia , where cca is most prevalent , are limited . 
patients with high tmb had alterations in direct dna repair genes and caretaker genes , especially in the asian population . relevance these results may reect differences in disease etiology and are relevant for targeted therapy and immunotherapy trials for patients with intrahepatic cholangiocarcinoma . methods comprehensive genomic proling of formalin - xed parafn - embedded ( ffpe ) tumor tissue blocks from the primary tumor or metastatic lesions obtained from patients with ihcca was performed in 283 patients from a single center in the united states and in 164 patients from china . in the united states , all genomic proling analysis was performed using a clinical laboratory improvement amendments ( clia ) - validated platform ( foundation medicine , cambridge , ma )  . 
in china , genomic proling was performed by using another clia - validated targeted sequencing platform ( origimed , shanghai , china ) .5 a bridging study comparing the 2 platforms indicated high concordance ( 96.3% ; 26 / 27 ) for the same study samples ( results indicated in appendix table a1 )  . 
all ffpe tissue blocks were sectioned and stained with hematoxylin and eosin and reviewed by an expert pathologist to conrm the diagnosis and presence of least 20% of the dna derived from tumor cells . 
this research was approved by the institutional review boards at md anderson cancer center and peking union medical college . hybrid selection and sequencing a custom hybridization capture panel including over 23 , 660 individually synthesized 5 ( cid : 3 ) - biotinylated dna 120 bp oligonucleotides was used to target the exons of cancer - related genes , as well as selected introns of genes frequently rearranged in cancer . 
hybridization capture followed the protocol of hybridization capture of dna libraries using xgen lockdown probes and reagents ( version 3 ; integrated dna technologies , san diego , ca )  . postcapture libraries were mixed together , denatured , diluted , and then sequenced . 
for the purpose of estimation of sequencing error rate , a phix spike - in was added as an external control to measure the percentage of reads with 0 - 4 mismatches , following the method described by manley et al.6 bioinformatics pipeline all types of genetic alterations , including single - nucleotide variant ( snv ) , short and long indels , copy number alterations ( cnas ) , and gene rearrangement , were called using a suite of bioinformatics pipelines.5 analysis of snvs and indels began with the alignment of raw reads to the human genome reference sequence ( hg19 ) with the burrows - wheeler aligner ( v0.62 ; bwa , cambridge , ma ) , followed by polymerase chain reaction ( pcr ) duplicates removal using markduplicates algorithm from picard ( version 1.47 ; cambridge , ma )  . 
local realignment and base quality recalibration for snvs were performed using gatk ( v3.1 - 1 ; cambridge , ma ) and subsequently called by mutect ( v1.7 ; cambridge , ma )  . 
the cnas included : ( 1 ) amplication , dened as an increase in the number of gene segment copies by 8 , and ( 2 ) homozygous deletion , dened as decrease of complete loss of gene segment copies in samples with  . 
2 , 000 or zero bp were collected and used as discordant reads , that is , paired - end reads that could not be closely mapped to a genome reference , with each read of paired reads aligned to the same chromosomes or different chromosomes . 
the breakpoints were double conrmed by blat , and the resulting chimeric gene candidates were annotated . oncogenic genetic mutations statistical analysis was only based on oncogenic genetic variants and the variants of unknown signicance ; lowfrequency ( variant allele frequency , .01 ) variants were excluded . 
data are % unless otherwise specied . abbreviations : fhx , family history ; hav , hepatitis a virus ; hbv , hepatitis b virus ; hcv , hepatitis c virus ; hx , history ; sd , standard deviation . studies exist on the mutations situated on the same genome loci and structural disrupting mutations on tumor suppressor genes , such as truncations , splicing sites , and frameshifts ; ( 3 ) conrmed somatic : somatic mutations recorded on the public genomic database , such as cosmic , and detected at least once in any tumor types . tumor mutation burden and known germline alterations in dbsnp were not counted . 
we classied our patients according to the tmb scores into : ( 1 ) tmb low ( tmb - l ) if the number of mutations per megabase ( mut / mb ) was , 6 , ( 2 ) tmb inthe number of mutations per termediate ( tmb - i ) megabase was between 6 and 9 , and ( 3 ) tmb high ( tmb - h ) if the number of mutations per megabase was 10 . 
the tmb cutoff was dened per prior lung cancer trials.7 , 8 tumor mutation burden ( tmb ) was estimated for 157 us patients and 164 chinese patients with ihcca by counting its somatic mutations , including coding snvs and indels per megabase of the sequence examined . 
 cao et al msi score , we classied the samples as msi high ( msi - h ) , dened as instability in 2 microsatellite loci ; msi low , dened as instability in only 1 loci ; and microsatellite stable , dened as absence of any evidence of microsatellite loci instability . 
furthermore , the chinese patients had a signicantly higher rate of well - differentiated adenocarcinoma compared with their us counterparts ( p = .002 ; table 1 )  . western versus asian ngs comprehensive genomic proling comprehensive genomic proling identied 1 , 007 genetic aberrations ( gas ) in chinese patients compared with 971 gas in us patients ( appendix table a2 )  . 
each patient harbored at least 1 ga , with an average of 6.1 ( range , 1 to approximately 20 ) and 3.4 ( range , 1 to approximately 16 ) gas per tumor in chinese and us patients , respectively . 
as indicated in figure 1 , the most commonly reported gas in chinese patients were tumor protein 53 ( tp53 ; 41.5% ) , kirsten rat sarcoma viral oncogene homolog ( kras ; 28.7% ) , at - rich interactive domain - containing protein 1a ( arid1a ; 18.3% ) , chinese patients ( n = 164 ) us patients ( n = 283 ) dna repair genes china substitution / indel gene amplification gene homozygous deletion fusion / rearrangement truncation fig 1 . 
 ( b ) the afliated chart indicates the alterations among direct and caretaker dna repair genes . mutations are colored according to mutation types of substitution / indel , gene amplication , gene homozygous deletion , truncation , and fusion / rearrangement . 
in our cohorts , we dened dna repair genes to include the 20 most frequent previously reported dna repair gene gas , including direct dna repair genes ( atm , atr , brca1 , brca2 , fanca , fancd2 , mlh1 , msh2 , msh6 , palb2 , pold1 , pole , prkdc , rad50 , and slx4 ) and caretaker genes ( bap1 , cdk12 , kmt2c / mll3 , tp53 , and blm ) .9 remarkably , chinese patients harbored more dna repair gas compared with us patients with ihcca , with predominant gas in tp53 , brca1 / 2 , kmt2c , and rad50 . 
furthermore , there was no signicant difference in the median values of tmb - h , tmb - i , and tmb - l groups among the chinese ( 14 , 8 , and 2 mut / mb , respectively ) and us patients with ihcca ( 19 , 7 , and 3 mut / mb , respectively )  . 
moreover , we identied that the rate of msi - h in chinese and us patients was 1.8% ( n = 3 ) and 0.6% ( n = 1 ) , respectively , and all msi - h patients were associated with high tmb values ( fig 2 )  . we explored the association between dna repair gene gas and tmb , and identied a signicantly higher rate of tmb - i and tmb - h in patients who had combined direct and caretaker dna repair gas compared with patients without dna repair gas among the chinese ( p , .001 ) and us ( p = .05 ) patients with ihcca . 
the advent of ngs offers promising breakthroughs with targeted therapy and immunologic interventions . however , it is important to identify genomic heterogeneity between populations because this will have a signicant impact on precision medicine approaches for this cancer . in our study , we performed comprehensive molecular proling of 164 chinese and 283 us patients with ihcca to explore genomic heterogeneity between populations . 
mutation types are presented in the color key at the bottomss , microsatellite stable ; mut / mb , mutations per megabase . noted important differences between asian and western patients with ihcca . 
through the results , chinese patients had signicantly higher frequency of tp53 as well as kmt2c , brca1 / 2 , ddr , tert , tgfbr2 , rbm10 , nf1 , spta1 , and rb1 gas . 
 ( b ) the correlation between tmb status and dna repair genetic aberrations ( gas ) by presenting the rates of tmb levels in different existence patterns of direct dna repair gas and caretaker gas . genetic diversity could be attributed to variations in the in western underlying disease risk factors ( table 2 )  . countries , cca is associated with metabolic syndrome , inammatory bowel disease , primary sclerosing cholangitis , and hepatolithiasis , whereas liver uke and hbv are important risk factors in asian countries . 
in this study , high tmb was associated with longer progression - free survival.13 also , pembrolizumab has been approved by the food and drug administration for the dna mismatch repair decient ( mmr - d ) cancers based on a phase ii clinical trial that showed a 40% objective response rate and 78% progression - free survival rate in patients with colorectal cancer with mmr - d compared with 0% and 11% , respectively , for mmr - procient patients.14 similar results have been achieved in noncolorectal cancers that included 4 patients ( 44% ) with biliary tract cancer . 
the role of combined poly ( adp - ribose ) polymerase inhibitors and pd - 1 inhibitors in advanced solid tumor is currently under investigation and may be applicable to this subgroup . our study has important limitations . 
in these panels : ( 1 ) the content genes had some dissimilarities ; to address this issue , we ltered out differential genes and only included the 320 overlapped genes into the analysis , thereby ensuring the comparability ; ( 2 ) sequencing platforms were illumina hiseq2500 ( illumina , san diego , ca ) for foundationone and novaseq ( illumina ) for origimed450 , with effective depths of 500 and 700 , both reecting the accuracy of sequencing results17 ; ( 3 ) the tmb algorithm of origimed450 was based on the published algorithm of foundationone , and the genomic alterations were selected similarly18 ; ( 4 ) the origimed450 platform uses blood samples from individual patients as their own control for eliminating the impact of germline polymorphisms , whereas foundationone uses the somatic - germline - zygosity algorithm and databases of dbsnp and exac for the same , and nally ; and ( 5 ) we conducted a bridging study comparing the 2 platforms and showed a high degree of concordance . 
furthermore , we have examined the clinical characteristics of the chinese and us patients and demonstrated that demographic differences between these populations were minor , with most patients having an advanced disease stage . 
there was a higher proportion of hepatitis b exposure in the asian cohort , and the pathophysiologic relationship between the viral infection and somatic mutations in cancer is unknown at this time . future studies to investigate the genomic proling in different populations , taking into consideration the disease risk factors , are warranted . 
data regarding pd - 1 and pd - l1 expression were not available ; therefore , we could not identify variations in immune biomarker expression between the 2 cohorts in this study . 
borad , kai wang , milind javle , haitao zhao administrative support : jingyu cao , jing hu , maolin yan provision of study materials or patients : qiang li , jianzhen lin , lawrence n . 
kwong , haitao zhao collection and assembly of data : jingyu cao , jing hu , siqin liu , reham abdel - wahab , qiang li , maolin yan , yujie feng , jianzhen lin , lawrence n . 
kwong , jinwei hu , fernando carapeto , milind javle , haitao zhao data analysis and interpretation : siqin liu , funda meric - bernstam , reham abdel - wahab , qiang li , songhui zhao , jian wang , lawrence n . 
 ihcca : genomic heterogeneity between eastern and western patients funda meric - bernstam employment : md anderson cancer center honoraria : sumitomo group , dialectica consulting or advisory role : genentech , inection biosciences , pieris pharmaceuticals , clearlight diagnostics , darwin health , samsung bioepis , spectrum pharmaceuticals , aduro biotech , origimed , xencor , debiopharm group , mersana , seattle genetics , silverback therapeutics , immunomedics , ibm watson health , f . 
hoffman - laroche , ibm watson health , pact pharma , effector therapeutics speakers bureau : chugai biopharmaceuticals research funding : novartis ( inst ) , astrazeneca ( inst ) , taiho pharmaceutical ( inst ) , genentech ( inst ) , calithera biosciences , debiopharm group ( inst ) , bayer ( inst ) , aileron therapeutics ( inst ) , puma biotechnology ( inst ) , cytomx therapeutics ( inst ) , jounce therapeutics ( inst ) , zymeworks ( inst ) , curis ( inst ) , pzer ( inst ) , effector therapeutics ( inst ) , abbvie ( inst ) , boehringer ingelheim ( i ) , guardant health ( inst ) , daiichi sankyo ( inst ) , glaxosmithkline ( inst ) , seattle genetics ( inst ) travel , accommodations , expenses : taiho pharmaceutical , seattle genetics , beth israel deaconess medical center songhui zhao employment : origimed lawrence n . 
borad stock and other ownership interests : gilead sciences , aveo , intercept pharmaceuticals , spectrum pharmaceuticals consulting or advisory role : g1 therapeutics , fujilm ( inst ) , agios ( inst ) , insys therapeutics ( inst ) , novartis ( inst ) , arqule ( inst ) , celgene ( inst ) , inspyr therapeutics , halozyme ( inst ) , pieris pharmaceuticals ( inst ) , taiho pharmaceutical ( inst ) , immunovative therapies , exelixis , lynx group , genentech , western oncolytics , klus pharma , denovo , merck , imvax research funding : boston biomedical ( inst ) , mirna therapeutics ( inst ) , senhwa biosciences ( inst ) , medimmune ( inst ) , biolinerx ( inst ) , agios ( inst ) , halozyme ( inst ) , celgene ( inst ) , threshold pharmaceuticals ( inst ) , toray industries ( inst ) , dicerna ( inst ) , sillajen ( inst ) , eisai ( inst ) , taiho pharmaceutical ( inst ) , emd serono ( inst ) , isis pharmaceuticals ( inst ) , incyte ( inst ) , sun biopharma ( inst ) , ariad ( inst ) , imclone systems ( inst ) , qed therapeutics ( inst ) , incyte ( inst ) , puma biotechnology ( inst ) , adaptimmune ( inst ) , merck serono ( inst ) , redhill biopharma ( inst ) , basilea ( inst ) travel , accommodations , expenses : arqule , celgene , astrazeneca kai wang employment : origimed leadership : origimed milind javle consulting or advisory role : qed , oncosil , incyte , mundi other relationship : rafael pharmaceuticals , incyte , pieris pharmaceuticals , merck , merck serono , novartis , seattle genetics , beigene , qed therapeutics , bayer no other potential conicts of interest were reported . references chun ys , javle m : systemic and adjuvant therapies for intrahepatic cholangiocarcinoma . 
curr treat options oncol 17 : 58 , 2016 saha sk , zhu ax , fuchs cs , et al : forty - year trends in cholangiocarcinoma incidence in the u.s. : intrahepatic disease on the rise . 
nat genet 44 : 690 - 693 , 2012 cao j , chen l , li h , et al : an accurate and comprehensive clinical sequencing assay for cancer targeted and immunotherapies . 
n engl j med 378 : 2093 - 2104 , 2018 rizvi na , mazi `eres j , planchard d , et al : activity and safety of nivolumab , an anti - pd - 1 immune checkpoint inhibitor , for patients with advanced , refractory squamous non - small - cell lung cancer ( checkmate 063 ) : a phase 2 , single - arm trial . 
lancet oncol 16 : 257 - 265 , 2015 chae yk , anker jf , bais p , et al : mutations in dna repair genes are associated with increased neo - antigen load and activated t cell inltration in lung adenocarcinoma . 
oncotarget 9 : 7949 - 7960 , 2017 jusakul a , cutcutache i , yong ch , et al : whole - genome and epigenomic landscapes of etiologically distinct subtypes of cholangiocarcinoma . 
chan - on w , nairism agi ml , ong ck , et al : exome sequencing identies distinct mutational patterns in liver uke - related and non - infection - related bile duct cancers . 
clarke b , tinker av , lee ch , et al : intraepithelial t cells and prognosis in ovarian carcinoma : novel associations with stage , tumor type , and brca1 loss . 
ali sm , yao m , yao j , et al : comprehensive genomic proling of different subtypes of nasopharyngeal carcinoma reveals similarities and differences to guide pathol 22 : 393 - 402 , 2009 gynecol oncol 141 : 293 - 302 , 2016 targeted therapy . 
the respected blocks were sectioned at 5 mm ; the tumor content size and were conrmed by a trained pathologist , and a minimum of 5 unstained slides were submitted for conrmation using the origimed platforinstitutional review board approval was obtained for this study . 
the discussion mainly focuses on inference for a beneting subpopulation , that is , a characterization of a group of patients who benet from the treatment under consideration more than the overall population . 
when the selection of the interesting subpopulation is carried out as the trial proceeds , the design becomes an adaptive clinical trial design , using subgroup analysis to inform the randomization and assignment of treatments to patients . 
that is , subgroup analysis is concerned with the question of whether a conclusion for the entire eligible patient population in a clinical trial remains valid for subpopulations of interest . 
to be specic , consider a two - arm randomized trial that is carried out to learn about the effectiveness of an experimental therapy versus a control , and assume that no signicant treatment effect can be established in the overall population . 
the question is closely related to personalized optimal treatment selection as it is needed , for example , in precision oncology . the investigation of hypotheses related to the notion that certain subgroups may benet from certain therapies more than others necessitates a disciplined way to identify such subgroups and to quantify the benet to these groups . 
dangers that investigators face in setting up subgroup analyses include studies being underpowered to detect signicant effects in subgroups , the issue of multiple testing , and the possibility for data dredging . we focus on a bayesian perspective because it naturally addresses multiplicities through the use of priors and because of its alignment with decision theoretic approaches , 1 which provide an elegant way to address competing aims in subgroup analysis . 
first , we discuss in general terms some of the questions that arise in subgroup analysis and then present some approaches that have been developed in the bayesian literature and compare them in a small simulation . 
 nugent et al context key objective review and comparison of subgroup analysis approaches and discussion of subgroup enrichment designs for clinical trials . knowledge generated we demonstrate how subgroup analysis can be carried out by different bayesian approaches and discuss how these different approaches identify potentially different subpopulations on the basis of the interests and objectives in practice . relevance subgroup analysis and subgroup enrichment designs are a focus point of precision oncology . 
this may help guide future implementation of these novel statistical methods in real - world trials . parsimony can be induced by restricting the model space . for example , the model space might only include models dened by one or two binary covariates , thereby only allowing subgroups dened by at most two covariates . alternatively , parsimony can be induced through the use an explicit utility function . 
for patient i , let yi denote an outcome that is assumed to be some measure of efcacy , let ti denote a treatment indicator , and let x i ( cid : 1 ) ( xi1 , , xij ) denote a set of covariates . 
the covariates could potentially dene subgroups . regression the earliest discussions of bayesian subgroup one of analysis is by dixon and simon , 3 who approach subgroup analysis as inference on regression coefcients , specically the coefcient for the interaction between a treatment indicator ( ti ) and a covariate ( xij )  . 
although this shrinks the covariates for variables with no effect toward zero , it does not introduce sparsity . sparsity could be enforced by putting alternate priors on these coefcients , such as spike and slab4 - 7 or nonlocal priors.8 model selection here , subgroups are dened by competing models , where the covariates included in the winning model dene the subgroup . 
the general setup starts with a list of possible models and then compares models in a principled manner . in general , bayesian model selection induces sparsity through prior distributions that are specied for each model . one can argue that the use of prior probability models implicitly introduces a complexity penalty by spreading out probability mass for additional parameters.9 , 10 by including the model choice in the prior specication , the approach naturally considers the entire model space at once , making all models directly comparable . we consider two methods that use model selection to perform subgroup analysis.11 , 12 in the method of berger et al , 11 the model space is restricted to models that can be written as a tree dened by one or two covariates . 
in this gure , the enhanced treatment effects for the winning subgroups are circled in red , and the subgroups are underlined . construction , this only allows for subgroups dened by up to two covariates , enforcing a minimum level of sparsity . rather than trees , sivaganesan et al12 use clustering to dene a model space . 
because only models within a family are directly comparable , the approach introduces a reasonable , but ad hoc , rule on the basis of thresholds for selecting models to report as formal subgroups . 
the thresholds can be tuned to control desired frequentist properties of the procedure , such as the rate of falsely choosing a model with a common overall treatment effect over a subgroup model . 
because the algorithm may select multiple subgroups corresponding to different families , it can potentially be difcult to interpret the results , especially because models in different families are not directly comparable . 
in particular , if there is a signicant amount of correlation between covariates , the algorithm is not designed to pick a clear winner . potential outcomes although many approaches to subgroup analysis implicitly include a notion of potential outcomes , some explicitly use potential outcomes as the input to the search for subgroups . 
the authors rst use a random forest to estimate the potential outcomes ( y ( 0 ) ) for each patient and then input this into another tree - based regression model to identify an estimate b ( cid : 1 ) { x : ( x )  . 
here ( x ) is the unknown true difference in expected potential outcomes . subgroups are identied as the paths that lead to leaves of the tree that have an estimated treatment effect above zero or above a specied threshold . , y ( 1 ) decision problems some approaches focus on the nature of a subgroup report as a decision problem and introduce a utility function , u ( a , , y ) , to characterize the preference for a decision a , in this case the beneting subgroup , under data y and a hypothetical truth , represented by the parameters , in an assumed sampling model . 
the utility usually includes a preference for parsimony but can also account for other aims , such as a preference for patient populations that are not well served by currently available treatments . 
although we introduce here a decision theoretic choice of a subgroup report as an alternative to other methods , it could also be argued that it can be included as an additional step in any of the earlier considered approaches . the approach taken by morita and m uller14 denes a utility function that favors a subgroup with a large difference in treatment effect relative to the overall population , a large subgroup that includes many potential patients , and a subgroup that can be parsimoniously described by only a few covariates . 
besides type i and type ii errors related to selecting an overall null or an overall alternative hypothesis , meaning the absence of any treatment effect or the presence of a common treatment effect , respectively , several other frequentist summaries are of interest . 
for example , under a scenario with a true subgroup , morita and m uller14 report a false subgroup rate as the probability of reporting a subgroup effect that is different from the true subgroup effect and a true subgroup rate as the probability of correctly reporting the true subgroup . subgroup as a random quantity the nature of the true subgroup ( b ) as a function of the unknown parameters ( ie , as a random variable ) is the focus in the article by schnell et al.15 although b itself is unknown , bounds on b can be calculated . 
 nugent et al outside of e ( ie , in e c ) have a low probability of benetting . in this way , they dene a credible region , bounded by s and e , that contains the true benetting subgroup ( b ) with a prespecied probability . 
the discussion in schnell et al16 includes a straightforward algorithm to determine ( s , e )  . a strength , and perhaps at the same time a limitation , is that s and e could be complicated subsets of the covariate space that are not necessarily easy to communicate , and the joint report of ( s , e ) could be difcult to interpret . 
we refer to the approaches by the following acronyms : ds for dixon and simon3 ; vt13 ; bw for berger and wang11 ; pu for polya urn12 ; bapofi for bayesian population nding14 ; cs - i for credible subgroups ( inclusive subset ) 15 ; and cs - e for credible subgroups ( exclusive subset ) .15 setup of the simulation study consider a patient population where three biomarkers of interest have been recorded for each patient . 
we consider two scenarios , one that has a predictive effect for biomarker 3 and another that has a predictive effect for biomarker 3 as well as a prognostic effect for biomarker 1 . 
under the simulation truth , patients with the following baseline covariates [ x ( cid : 1 ) ( x1 , x2 , x3 ) ] should be agged : { ( 0 , 0 , 1 ) , ( 1 , 0 , 1 ) , ( 0 , 1 , 1 ) , ( 1 , 1 , 1 ) }  . in addition to identifying subsets of beneting patients , some methods allow a report of no treatment effect ( h0 ) , meaning the treatment is not effective for anyone in the population , or an overall treatment effect ( h1 ) , meaning that the treatment is equally effective for everyone in the population regardless of the presence or absence of a biomarker . 
because h1 was never chosen in this simulation , it is excluded from tables 2 and 3 ( data supplement )  . interpretation the results in tables 2 and 3 show that bapofi and cs - i performed the best in both scenarios . 
bapofi , in particular , correctly identied the beneting subgroup , and only the beneting subgroup , 100% of the time for both scenarios . all methods correctly identied the beneting subgroup the vast majority of the time in both scenarios . 
this table shows the probability ( under repeat simulation ) of x2 b for scenario 1 , that is , a future patient with the indicated baseline covariates x being included in the reported subgroup . 
for berger and wang11 ( bw ) , the rst number refers to all patients who would be recommended for treatment , and the number in parentheses refers to the patients who would be designated as beneting most . 
the table shows the probability ( under repeat simulation ) of x2 b for scenario 2 , that is , a future patient with the indicated baseline covariates x being included in the reported subgroup . 
for berger and wang11 ( bw ) , the rst number refers to all patients who would be recommended for treatment , and the number in parentheses refers to the patients who would be designated as beneting most . 
the column e ( n ) shows the expected number of patients with the respective covariates according to the simulation truth . abbreviations : bapofi , bayesian population nding ; cs - e , credible subgroups ( exclusive subset ) ; cs - i , credible subgroups ( inclusive subset ) ; ds , dixon and simon3 ; pu , polya urn . * covariate combinations that correspond to an enhanced treatment effect under the simulation truth . some of the methods were more prone to identifying a larger subset . 
this is probably because it allows all possible combinations and does not include any penalty for identifying small or awkwardly described groups or for lack of parsimony . as noted , some of the methods explicitly only allow for subgroups dened by one covariate ( bw ) , combinations of subgroups dened by one covariate ( pu ) , or two covariates ( bapofi )  . 
when evaluating their performance , keep in mind that the simulation truth for both scenarios happens to fall within the restricted scope of these methods , with the exception of bw for scenario 2 ; the version of bw implemented in this simulation does not take into account prognostic effects , although it could be set up to do so . because of the nature of the models that are considered in bw , the model that usually wins is the model that nds an enhanced treatment effect for both patients with x13 ( cid : 1 ) 1 and for patients with x13 ( cid : 1 ) 0 . 
bapofi avoided this problem by explicitly including a penalty for complexity . all of these methods can easily be extended to include more covariates and can be modied to include interaction structures . 
however , because each method is geared toward a slightly different aim , it becomes difcult to directly compare them when the data become more complicated . as a result , it becomes more important to specify the aim of the analysis . 
it is useful to distinguish different types of subgroup - based designs , as shown in figure 2 . traditional two - step process the standard subgroup - driven clinical development consists of at least two clinical trialsan exploratory trial for subgroup nding and a conrmatory trial for efcacy conrmation in the identied subgroup . 
the rst is a one - stage subgroup enrichment design , 25 in which one would prospectively specify the subgroups that the new therapy is expected to benet . such subgroup enrichment designs recruit patients from prespecied subpopulations and do not modify the inclusion criteria during the trial . 
however , a sponsor may be primarily interested in a large at - risk population and may consider a biomarker - stratied design that recruits patients from a larger subpopulation , or even the whole patient population ( denoted as f ) , and then includes patient strata ( denoted as s )  . 
the objective of the biomarker - stratied designs is to demonstrate the efcacy of the treatment in f and , if the rst objective fails , to demonstrate the efcacy of in s . 
because there are multiple tests , the treatment a multiple testing procedure can be used to control the the null hypotheses of nofamily - wise error treatment effect in s and in f .26 - 28 rate for conrmatory adaptive subgroup enrichment designs to improve the probability of success in drug development and to speed up the process , several authors have proposed conrmatory adaptive subgroup enrichment designs for phase ii and iii trials.18 - 20 , 29 - 31 in such designs , the trial is divided into several stages with possible subpopulation enrichment after each interim analysis , such as restricting future enrollment to only those subgroups that seemed to be benetting from the experimental therapy . 
on the basis of the results of each interim analysis , the trial may continue as initially planned , be stopped early , or continue with adaptive modications ( eg , revision of recruitment clusion criteria or sample size reestimation )  . 
in the absence inference is conducted to test of enrichment , statistical whether there is a signicant treatment effect in the whole population or in any of the predetermined subgroups . 
the family - wise error rate is controlled by closed testing principles in the strong sense . bayesian tools can be used in the go / no - go decision for the original population or specic subgroups . 
for example , brannath et al19 developed a decision tool on the basis of predictive probabilities for rejecting certain null hypotheses to determine which of two populations , the full population or subpopulation , should be further investigated in the second stage . 
this approach has been applied to an oncology phase ii / iii trial.32 seamless exploratory and conrmatory trials one may also consider combining the exploratory trial and the conrmatory trial seamlessly . 
the subgroups and decision rule be prespecied . in simon and simon , 21 a class of adaptive subgroup enrichment designs is proposed that adaptively update the eligibility criteria without prespecied subgroups . 
this test preserves the type i error regardless of the method used for making enrichment decisions and regardless of , possibly data dependent , time trends in the characteristics of the patients . subgroup estimation will be reported at the nal analysis for regulatory labeling . 
simon and simon22 extended their previous designs21 by incorporating bayesian decision tools into the interim decision making for enrichment and bayesian inference for the treatment effect in the estimated subgroup . 
although the hypothesis testing is statistically sound in both approaches by simon and simon , 21 , 22 the the overall null hypothesis is rejected , treatment effect in the estimated subgroup may be subject to a resubstitution bias . 
to correct for the resubstitution bias in estimating treatment efcacy for the selected subgroup , zhang et al33 propose cross - validation and bootstrap methods . exploratory adaptive enrichment designs some recent literature has developed exploratory adaptive enrichment designs23 , 24 with response - adaptive randomization . 
xu et al23 proposed a bayesian subgroupbased adaptive design ( suba ) using a random partition model for continuous biomarkers ( x ) and binary outcome ( y )  . 
for an example of suba applied to a real - world trial , see the article by simon et al.34 this is an ongoing trial at northshore biomarker 2 biomarker 1 biomarker 1 round 1 biomarker 2 d 2 biomarker 2 round 3 biomarker 2 ss11 sl11 ll12 ls12 ll12 lss121 lsl121 biomarker 1 biomarker 1 fig 3 . 
 nugent et al university health system , which adopts suba for adaptive allocation of patients to the best treatment arm on the basis of posterior predictive probabilities . another adaptive enrichment design for a basket trial of targeted therapies across multiple cancers is discussed in the article by xu et al.35 the design is based on an approach similar to that of morita and m uller , 14 with subgroups dened by binary covariates that include biomarkers and tumor types . discussion we have reviewed several approaches to bayesian subgroup analysis . 
alternatively , an investigator might be mainly interested in an informed individualized treatment choice , in which case an approach that allows for more complex subgroups , such as the potential outcomes approach , might be preferred . 
focusing on the nature of the subgroup as a random quantity enables investigators to explicitly describe the uncertainty of the subgroup report and to make an informed choice to be more or less conservative . a common context for subgroup analysis is the planning of future trials or the closely related problem of adaptation in a current trial . 
an interesting aspect of this application is that one can use bayesian inference to nd an interesting subpopulation but can then proceed with a purely frequentist design for the trial . planning for future trials is not the only application . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . wentian guo employment : laiya consulting stock and other ownership interests : laiya consulting travel , accommodations , expenses : laiya consulting peter m uller consulting or advisory role : novartis yuan ji stock and other ownership interests : laiya consulting , laiya consulting ( i ) consulting or advisory role : astellas pharma no other potential conicts of interest were reported . references robert c : the bayesian choice . 
new york , ny , springer - verlag , 2007 jones he , ohlssen di , neuenschwander b , et al : bayesian models for subgroup analysis in clinical trials . 
biom j 43 : 581 - 589 , 2001 jenkins m , stone a , jennison c : an adaptive seamless phase ii / iii design for oncology trials with subpopulation selection using correlated survival endpoints . pharm stat 10 : 347 - 356 , 2011 30 . 
martn m , chan a , dirix l , et al : a randomized adaptive phase ii / iii study of buparlisib , a pan - class i pi3k inhibitor , combined with paclitaxel for the treatment of her2 - advanced breast cancer ( belle - 4 )  . 
simon kc , tideman s , hillman l , et al : design and implementation of pragmatic clinical trials using the electronic medical record and an adaptive design . jamia open 1 : 99 - 106 , 2018 35 . 
 wide and cystic brain metastases reveal ret - rearranged nonsmall - cell lung cancers francesco facchinetti , md , msc1 , 2 ; francesca bozzetti , md1 ; letizia gnetti , md1 ; roberta minari , phd1 ; pellegrino crafa , md1 ; sara elena rebuzzi , md1 ; roberto ferrara , md3 ; elisa gruppioni , phd4 ; elisa capizzi , phd4 ; ermanno giombelli , md1 ; girolamo crisi , md1 ; annalisa altimari , phd4 ; and marcello tiseo , md , phd1 , 5 introduction the detection of specic molecular activations ( eg , egfr , alk , ros1 , braf , met , ret ) is the basis of effective targeted treatments in nonsmall - cell lung cancer ( nsclc ) .1 in this eld , novel selective inhibitors extend survival outcomes , coupling global prolonged disease control and meaningful activity against metastases developing in the cns.2 lung tumors harboring egfr mutations or alk , ros1 , or ret rearrangements have a tendency toward more frequent cns spreading compared with wild - type ( wt ) nsclc.3 - 5 peculiar neuroradiologic characteristics have been reported for brain metastases from alkpositive nsclc , in terms of cystic aspects acquired after crizotinib treatment.6 - 8 such features can even be misleading , because intracranial cystic lesions from alk - rearranged tumors may be attributed to infectious diseases.9 here we report the histories of two patients presenting with wide cystic brain lesions that were neurosurgically removed and discovered to be metastases from ret - rearranged nsclc . 
brain computed tomography ( ct ) scan and magnetic resonance imaging ( mri ) were performed , documenting a unique bulky cystic - like lesion in the frontal region of the left hemisphere , the morphologic and functional characteristics of which were pathognomonic for an anaplastic astrocytoma ( fig 1 )  . 
a chest x - ray revealed a parahilar lesion of the left lung . diagnosis and staging neurosurgery was then performed with the radical removal of the wide cerebral lesion , the intraoperative macroscopic aspects of which were still in keeping with an anaplastic astrocytoma . 
the postoperative course was uneventful , with a rapid neurologic improvement . the histologic examination revealed a large ( 40 25 20 mm ) lesion , conrming the cystic structure that was evident at sampling ( fig 2a )  . 
ct and pet scans revealed a mass in the left pulmonary hilum and ipsilateral and contralateral lymph nodes ( ct4n3m1b according to tnm staging classication [ eighth edition ] ; fig 3 )  . 
considering the radiologic , pathologic , and molecular elements , which were clearly in line with a diagnosis of advanced nsclc , biopsies of the lung and nodal lesions were not performed . molecular analyses neither the molecular analyses nor ihc performed on the brain metastasis detected egfr , braf , or kras mutations or alk or ros1 rearrangements ; programmed death - 1 ligand ( clone sp263 ) was expressed by 10% of tumor cells . 
next - generation sequencing with a 52 - gene panel ( ion torrent oncomine focus assay kit ; thermo fisher scientic , waltham , ma ; gene list provided in data supplement ) was performed , and a kif5b - ret fusion was found ( figs 4a and 4b )  . 
ret rearrangement was conrmed by uorescence in situ hybridization analysis in 52% of tumor cells ( fig 4c )  . treatment received rst - line chemotherapy with the patient cisplatin and pemetrexed . 
postoperative stereotactic brain radiotherapy ( sbrt ) to the resection cavity was not performed , because available evidence supporting sbrt for better local control has not yet indicated a survival benet.10 , 11 brain mri after 4 months revealed normal surgical outcomes , with no residual disease . 
ct scan after four courses of cisplatin plus pemetrexed showed partial disease response , so the patient was switched to pemetrexed maintenance , four cycles of which led to further reduction of lymph node size , with continuing absence of brain disease on mri . 
no evidence is available concerning ret - rearranged nsclc , an entity of emerging interest given the current development of selective ret inhibitors , also active against cns disease . knowledge generated here we report the similar clinical histories of two patients with ret - positive nsclc , whose diagnoses were obtained on pathologic and molecular analyses performed on wide , cystic , and symptomatic brain metastases with a peculiar neuroradiologic presentation . relevance a characteristic behavior of cns lesions from ret - rearranged nsclc was recognized . 
the current or upcoming availability of selective , cns - active ret inhibitors strongly reinforces the importance of wide molecular screening of cns lung metastases , even more so in the presence of the reported neuroradiologic and pathologic features of brain lesions . the abrupt onset of dizziness , worsening headache , difculty with handgrip , and right homonymous hemianopsia . the observation of a large and cystic - like brain lesion in the left parietal - occipital region was followed by a contrastenhanced mri ( fig 5 )  . 
the brain imaging was suggestive for metastases from an extracerebral tumor . diagnosis and staging the whole - body ct scan documented a mass in the upper left lung lobe , with ipsilateral and contralateral nodal volvement in the hilar - mediastinal and retro - clavicular regions ( fig 6 ) , with clinical staging determined as ct2an3m1c . given the persisting neurologic symptoms , the patient underwent neurosurgery . the pathologic examination conrmed the radiologic hypothesis of a cavitated cystic ( fig 2f ) brain metastasis ( 40 30 20 mm ) from a lung tumor ( figs 2g to 2i )  . 
 ( a ) t1 weighted imaging ( wi ) and ( b ) t2 wi sequences show a well - delineated ( a ) hypointense and hyperintense mass in left frontal lobe cortex involving the subcortical area and deep white matter , ( c ) without dense dystrophic calcication or hemosiderin staining on t2 * gre . 
 ( d ) ttf - 1 ( 40 ) and ( e ) napsin a ( 40 ) immunohistochemistry showed diffuse ( d ) nuclear and ( e ) cytoplasmic positivity . 
 ( a ) a left parahilar lesion conditioning atelectasis was detected , together with ( b ) ipsilateral and ( c ) contralateral pathologic lymph nodes . elements supporting the diagnosis of nsclc , biopsies of the primary tumor and lymph nodes were not proposed . molecular analyses status of egfr , alk , ros1 , kras , braf , and met was found to wt , and ihc showed programmed death - 1 ligand ( clone sp263 ) negative status . 
again , targeted ngs was performed on the brain metastasis ( ion torrent oncomine focus assay kit ; thermo fisher scientic ; data supplement ) , and kif5b - ret fusion was revealed , conrmed by uorescence in situ hybridization analysis in 40% of tumor cells ( figs 4a to 4b and 4d )  . treatment because of the lack of trials with ret inhibitors in the rstline setting and the substantial benet potentially driven by pemetrexed , 12 pemetrexed was combined with cisplatin and administered as a rst - line treatment , once postsurgical recovery was complete . 
again , sbrt to the resection cavity was discussed but not performed , for the same reasons reported in the rst patient case15 , 16 and because of the presence of multiple brain metastases . 
 ( b ) next - generation sequencing analysis report showing the ret fusion with kif5b gene ( ion reporter software and irgv visualization [ thermo fisher scientic , waltham , ma ] ) detected in brain samples from patient cases 1 and 2 . 
t1 weighted imaging ( wi ) and t2 wi sequences show a well - delineated ( a ) hypointense and ( b ) hyperintense mass in left occipital lobe cortex , with a linear internal septum , involving the subcortical area and deep white matter , ( c ) without dense dystrophic calcication or hemosiderin staining on t2 * gre . 
 ( d ) axial t1 postcontrast imaging shows a large bilobed cystic - like supratentorial mass with ring enhancement , sharply delineated borders , and well - dened internal septu ( e ) the content is liquid , with no intralesional levels and with high diffusivity ( apparent diffusion coefcient , 0.00269 mm2 per second v csf , 0.00290 mm2 per second )  . 
similar radiologic appearance is shown by the following : ( g ) t1 wi before gadolinium administration , ( h ) t1 wi after gadolinium administration , and ( i ) t2 wi . imaging documented , in addition to the regular outcomes of the surgical intervention , signs of response in the two residual lesions ( data not shown )  . 
nevertheless , this was not conrmed in another series of ros1 - rearranged lung cancer with baseline brain involvement ; in that series , the ros1 partner gene was known in 17 patients for whom mri results were available.14 in ret - driven lung cancer , the most frequent fusion partner gene is kif5b , as observed in the two patient cases reported here . 
although no associations between ret partner genes and brain metastasis incidence have been anticipated , drilon et al5 reported that intracranial disease response to multitarget kinase inhibitors seemed to occur specically in nonkif5b - ret patient cases ( n = 2 of 5 ) , with no responses in the kif5bret patient cases ( n = 0 of 9 ) .5 at least ve cns responses to the selective ret inhibitor loxo - 292 were observed in ret - rearranged lung tumors ( n = 4 of 4 in the phase i study ) 15 , 16 harboring either kif5b ( n = 2 ) or non - kif5b ( clip - 1 ; n = 1 ) ret partner genes.15 - 17 because of the descriptive nature of our patient cases , no conclusion can be drawn with regard to a novel specic pattern of ret - rearranged lung tumors , potentially associated with large and cystic - like brain metastases at diagnosis . nevertheless , besides providing atypical and intriguing radiologic ndings , the surprising similarity between these two reports , together with the rarity of ret rearrangements in nsclc , suggests a characteristic behavior of cns lesions from this molecular subclass of tumors . 
these observations reinforce the importance of screening for nsclc molecular activations in addition to the canonical and frequent ones , with particular reference to ret fusions in the presence of atypical features of brain metastases . 
 ( a - c ) the lesion was not dissociable and was not completely distinguishable from an obstructive inammatory / atelectasic consolidation involving the entire lingula and part of the upper left lobe . 
 ( c , d ) bilateral hilar , mediastinal , and retro - clavicular lymph nodes were reported ( white arrows )  . involvement , with special regard to novel , specic agents , such as loxo - 292 and blu - 667.5 , 15 - 18 we hope that our two patients will not require further systemic treatment , meaning that their disease is controlled with the current chemotherapy and potential administration of radiotherapy . 
 large and cystic brain metastases from ret - rearranged nsclcs references reck m , rabe kf : precision diagnosis and treatment for advanced non - small - cell lung cancer . 
n engl j med 377 : 849 - 861 , 2017 remon j , besse b : brain metastases in oncogene - addicted non - small cell lung cancer patients : incidence and treatment . 
front oncol 8 : 88 , 2018 tan l , wu y , ma x , et al : a comprehensive meta - analysis of association between egfr mutation status and brain metastases in nsclc . 
pathol oncol res 25 : 791 - 799 , 2019 gainor jf , tseng d , yoda s , et al : patterns of metastatic spread and mechanisms of resistance to crizotinib in ros1 - positive nonsmall - cell lung cancer . 
j clin oncol 32 : e122 - e124 , 2014 saraceni c , li pm , gainor jf , et al : cystic brain metastases in nsclc harboring the eml4 - alk translocation after treatment with crizotinib . 
narayanan v , honce mj , mehrotra s , et al : cystic brain metastases occurring in anaplastic lymphoma kinase gene rearranged non - small - cell lung cancer patients receiving crizotinib . 
clin lung cancer 17 : 85 - 90 , 2016 kim sh , hyun jw , kim hj , et al : de novo cystic brain lesions mimicking neurocysticercosis in alk - positive lung cancer . 
mahajan a , ahmed s , mcaleer mf , et al : post - operative stereotactic radiosurgery versus observation for completely resected brain metastases : a single - centre , randomised , controlled , phase 3 trial . 
patil t , smith de , bunn pa , et al : the incidence of brain metastases in stage iv ros1 - rearranged non - small cell lung cancer and rate of central nervous system progression on crizotinib . 
drilon a , subbiah v , oxnard gr , et al : a phase 1 study of loxo - 292 , a potent and highly selective ret inhibitor , in patients with ret - altered cancers . 
oxnard g , subbiah v , park k , et al : clinical activity of loxo - 292 , a highly selective ret inhibitor , in patients with ret fusion + non - small cell lung cancer . 326 , 2013 27 : 1286 - 1291 , 2016 j thorac oncol 13 : 987 - 995 , 2018 oncol 36 , 2018 ( suppl ; abstr 102 ) j thorac oncol 13 : s349 - s350 , 2018 17 . 
a computed tomography ( ct ) scan of the abdomen revealed a 7.5 - cm head of pancreas mass encasing the celiac and superior mesenteric arteries and the portal vasculature , with dilation of the pancreatic and common bile ducts ( fig 1a )  . 
completion staging with a ct scan of the chest was negative . the patient , with locally advanced unresectable pancreatic ductal adenocarcinoma ( pdac ) , was administered a chemotherapy regimen of fluorouracil , folinic acid , irinotecan , and oxaliplatin on april 8 , 2016.1 after 4 cycles of treatment , restaging ct showed progression of the primary tumor ( fig 1b ) with an increase in cancer antigen 19 - 9 from 202 to 410 ( fig 3 ) and clinical deterioration requiring duodenal stent and gastrostomy tube placement . 
given his young age , his otherwise good health and lack of expected risk factors , and his family history , comprehensive multiomic molecular analysis was performed by perthera , a precision oncology company in mclean , va , that facilitates molecular testing and provides patients with a list of therapy options that match their molecular profile , the specific characteristics of their tumor , and their treatment history . 
all molecular testing , which includes multiomic analysis , is performed at college of american pathologist ( cap ) and clinical laboratory improvement amendment ( clia ) accredited and licensed laboratories and is commercially available ( foundation medicine , cambridge , ma , for nextgeneration sequencing ( ngs ) analysis and caris life sciences ( irving , tx ) for multiplexed proteomic immunohistochemistry [ ihc ] and fluorescent in situ hybridization [ fish ] analysis )  . before providing its services to this patient , perthera obtained informed consent under an institutional review boardapproved registry protocol . as part of the perthera analysis , a clia - certified / cap - accredited commercial ngs - based test ( foundationone ; foundation medicine ) revealed the anaplastic lymphoma kinase ( alk ) rearrangement , recurrent striatin ( strn ) - alk . 
in addition , a clia - certified and capaccredited commercial ihc test ( caris molecular intelligence ; caris life sciences ) revealed overexpression of the alk protein ( fig 2d ) , which confirmed genomic alteration at the protein level . fish testing was not performed . as a result of this finding , treatment options included standard second - line chemotherapies with or without radiation , as well as the possibility of adding crizotinib . 
after conferring with multiple colleagues , it was decided to treat the patient with intensity - modulated radiotherapy ( 60 gy in 2 - gy fractions , from june 23 , 2016 , to august 5 , 2016 ) , concurrent gemcitabine ( 600 mg / m2 / wk ) for 6 weeks , and crizotinib ( 250 mg twice a day ) for the duration of radiotherapy . 
joseph bender emanuel petricoin jonathan brody nicholas nissen richard tuli , simon lo , jaimie koo , and nicholas nissen , cedars - sinai medical center , los angeles , ca ; and michael pishvaian , r . 
computed tomography of the abdomen ( a ) with venous phase contrast pretreatment ; ( b ) after first - line therapy with fluorouracil , folinic acid , irinotecan , and oxaliplatin ; ( c ) 6 weeks after gemcitabine , radiotherapy , and crizotinib ; and ( d ) after 3 months of additional crizotinib . 
restaging ct on september 26 , 2016 , showed significant partial response of tumor ( fig 1c ) with a commensurate decline in cancer antigen 19 - 9 to 25 ( fig 3 )  . 
cancer antigen ( ca ) 19 - 9 values ( a ) after first - line therapy with fluorouracil , folinic acid , irinotecan , and oxaliplatin ; ( b ) after gemcitabine , radiotherapy , and crizotinib ; and ( c ) after approximately 5 months of additional crizotinib . status , suggesting a persistent response to crizotinib combination and single - agent therapies . 
one such targetable oncogenic driver is the alk rearrangement identified in 3% to 5% of nsclcs.4 initially identified by soda et al , 5 a small inversion within chromosome 2p resulted in the formation of a fusion gene comprising portions of the echinoderm microtubule - associated protein - like 4 gene and the alk gene in nsclc . 
archival analysis of 140 pdacs for alk1 expression using ihc and fish was negative in all cases , suggesting that alk is an unlikely therapeutic target in pdac.6 using panel - based multiplexed genomic and proteomic profiling , we were able to identify this actionable mutation previously undetected using standard ihc / fish ( fig 2d )  . 
if the strn gene is a preferred fusion partner of alk in pdac , this may provide an explanation for the negative fish results in the study by graham et al.6 therefore , any pdac case without an identifiable kras driver mutation should be considered for multiplexed sequencingbased testing to identify potential additional drivers , including alk rearrangements . functional alk rearrangements have been found largely to be mutually exclusive of oncogenic driver mutations such as egfr and kras mutations in nsclc.4 although kras mutations serve as oncogenic drivers in 90% of pdacs , our patients tumor did not harbor this mutation . 
in addition , clinical and pathologic variables unique to alkrearranged nsclc include young age , prominent mucinous cells ( adenocarcinoma subtype ) , and never - smokers.8 the young age of our patient in the absence of any risk factors is perhaps suggestive of alk - mediated tumorigenesis , especially given that the median age of patients with pdac is 71 years . patients with alk - rearranged nsclcs are extremely responsive to the tyrosine kinase inhibitors crizotinib and ceritinib.4 the recent phase 3 profile 1014 study ( a clinical trial testing the efficacy of crizotinib versus standard chemotherapy pemetrexed plus cisplatin or carboplatin with alk positive non squamous cancer of the lung ) established crizotinib as standard first - line therapy for patients with metastatic nonsquamous nsclc , with an objective response rate of 74% and progressionfree survival of 11 months , both of which were significantly higher than in standard platinumbased doublet chemotherapy.9 despite a relatively durable response to tyrosine kinase inhibitors , patients ultimately develop resistance to therapy . 
in addition to kras - activating mutations , inactivation of tumor suppressors tp53 , cdkn2a , and smad4 occur at 50% frequency , followed by aberration of genes related to dna damage repair , chromatin remodeling , and others at a much lower frequency.10 using whole - genome sequencing and copy number variation , a recent study identified an average of 119 variations in chromosomal structure per pancreatic cancer , further highlighting this diversity.11 indeed , this diverse intertumoral heterogeneity warrants genomic profiling to increase the likelihood of identifying druggable targets , as noted in the described case . 
although kras is a difficult driver mutation to target therapeutically , many other drivers , such as the alk fusion detected here , have been demonstrated to sensitize tumors to therapies . 
taken together with our previous observation that kras wild - type tumors are enriched for braf mutations and erbb2 amplifications , this suggests that kras wildtype pdac cases have a high potential for sensitivity to molecularly targeted therapies . our patient harbored fusion of strn - alk , which has been characterized previously in multiple other malignancies , including colon , breast , renal , and lung cancers , among others.12 - 14 an alk fusion has been observed in one case of pdac , but with a different fusion partner ( dctn1 ) .15 notably , the case with the dctn1alk fusion also lacked a kras mutation . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . richard tuli no relationship to disclose simon lo no relationship to disclose jamie koo no relationship to disclose michael pishvaian stock and other ownership interests : perthera honoraria : caris life sciences , celgene , sirtex medical consulting or advisory role : caris life sciences , perthera , celgene , sirtex medical research funding : genentech ( inst ) , celldex ( inst ) , merck ( inst ) , daiichi sankyo ( inst ) , glaxosmithkline ( inst ) , medimmune ( inst ) , pfizer ( inst ) , gilead sciences ( inst ) , regeneron pharmaceuticals ( inst ) , novartis ( inst ) travel , accommodations , expenses : caris life sciences , sirtex medical , perthera r . 
 emanuel petricoin leadership : perthera , ceres nanosciences stock and other ownership interests : perthera , ceres nanosciences , d3d consulting or advisory role : perthera , ceres nanosciences , zymeworks research funding : ceres nanosciences ( inst ) , glaxosmithkline ( inst ) patents , royalties , other intellectual property : national institutes of health patents licensing fee distribution or royalty , university - assigned patent licensing fee or royalty travel , accommodations , expenses : perthera , ceres nanosciences jonathan brody employment : perthera stock and other ownership interests : perthera consulting or advisory role : perthera research funding : american association for cancer research , national institutes of health expert testimony : king and spalding llp travel , accommodations , expenses : perthera nicholas nissen no relationship to disclose references med 364 : 1817 - 1825 , 2011 1 . 
vogelzang nj , benowitz si , adams s , et al : clinical cancer advances 2011 : annual report on progress against cancer from the american society of clinical oncology . 
von hoff dd , ervin t , arena fp , et al : increased survival in pancreatic cancer with nab - paclitaxel plus gemcitabine . n engl j med 369 : 1691 - 1703 , 2013 4 . 
gainor jf , varghese am , ou sh , et al : alk rearrangements are mutually exclusive with mutations in egfr or kras : an analysis of 1 , 683 patients with non - small cell lung cancer . 
kelly lm , barila g , liu p , et al : identification of the transforming strn - alk fusion as a potential therapeutic target in the aggressive forms of thyroid cancer . 
 first case report of a dramatic radiographic response to a checkpoint inhibitor in a patient with proficient mismatch repair gene expressing metastatic colorectal cancer metastatic colorectal cancer ( mcrc ) remains the second most common cause of cancer death in the united states , and therapeutic options are limited . 
recently , the checkpoint inhibitor pembrolizumab was given the food and drug administration breakthrough therapy designation for the treatment of patients with mcrc whose tumors demonstrate deficient mismatch repair gene ( dmmr ) expression ( as evidenced by microsatellite instabilityhigh [ msi - h ] )  . 
the designation was based on a phase ii study showing that in patients with dmmr , an objective response rate of 40% was seen , whereas in patients with proficient mismatch repair gene mcrcs , the response rate was 0% . 
because this patients tumor harbored amplification of both the pd - l1 and pd - l2 genes , the observed response was consistent with the presumed mechanism of action of checkpoint inhibitors . 
pathology showed stage iiib ( t4bn1mx ) colonic adenocarcinoma , and a computed tomography ( ct ) scan of the chest , abdomen , and pelvis in december 2014 showed no evidence of metastatic disease . 
 ( pd - l1 ) and the pdcd1lg2 ( pd - l2 ) gene , normal sequence of mlh1 , msh2 , msh6 , and pms2 , and no microsatellite instability ( msi )  . 
the msi test performed by foundation medicine has been validated against currently accepted methods of testing msi , including immunochemical ( ihc ) and polymerase chain reaction , with a concordance of 97% in 60 colorectal and endometrial cancers . 
immunohistochemical assay demonstrated retained nuclear expression of mlh1 , msh2 , msh6 , and pms2 ( department of pathology , baptist hospital and wake forest medical school , winston - salem , nc )  . on january 27 , 2016 , the patient began receiving intravenous nivolumab ( 3mg / kg ) every 2 weeks , and after six doses , a repeat ct chest , abdomen , and pelvis scan on april 13 , 2016 , showed not only improvement in the previously radiated area , but also , as the radiologist noted , other multiple previously described lymph nodes as well as multiple subcutaneous soft tissue nodules and a few small pulmonary nodules had either resolved or considerably decreased in size compared with the most recent examination . 
she continued receiving the nivolumab , and a further response was documented in july 2016 when a ct scan showed only one , further diminished , left posterior subcutaneous nodule remaining , and again no new lesions . discussion the programmed death pathway represses a cytotoxic immune response to tumors . 
checkpoint inhibitors are thought to activate that cytotoxic pathway by inhibiting the programmed death pathway ligands ( pd - l1 and pd - l2 ) .4 , 5 in non small - cell lung cancer , the expression of these ligands predicts responsiveness . 
as a result , the food and drug administration ( fda ) has approved different ihc assays for pd - l1 expression to be used in deciding whether to treat patients using the checkpoint inhibitors approved for lung cancer treatment.6 in mcrc , pd - l1 or pd - l2 expression or gene amplification have not been studied as a marker of responsiveness to the checkpoint inhibitors , although pd - l1 and pd - l2 overexpression has been assessed in deficient mismatch repair gene ( dmmr ) expression and proficient mismatch repair gene ( pmmr ) colon cancers and are associated with prognosis in these cancers.7 yet the basis for the recent fda breakthrough designation of the checkpoint inhibitor pembrolizumab was the result of a study in which the investigators hypothesized that dmmr mcrc would respond to checkpoint inhibitors because these tumors are known to overexpress pd - l1 and pd - l2 far more commonly . 
in fact , in this study , pdl - 1 expression occurred only in dmmr tumors , not in pmmr tumors . le et al8 used msi - h as a surrogate for dmmr ; if msi - h is identified , the same colorectal tumor nearly always demonstrates dmmr , 9 still , it is known that pmmr crcs ( colorectal cancers ) may overexpress pd - l1 or pd - l2 , and that certain clinical features ( eg , tumors with vascular invasion ) might predict this pattern.5 , 10 expression may correlate more closely with the mutation frequency in the tumors , which is characteristically far higher in dmmr crc cases compared with pmmr crc cases.1 pd - l1 or pd - l2 amplification ( as in this case ) would not necessarily result in overexpression . 
however , given the mechanism of action of the checkpoint inhibitors and the dramatic response , and given that this patient had had progressive mcrc despite prior standard therapy , it seems reasonable to conclude that inhibition of pd - l1 and pd - l2 occurred and that that explains the robust response . there are important examples in which gene amplification without wild - type gene product overexpression nonetheless predicts benefit of drugs targeting the gene product . 
 similarly , defining a best test for identifying which patients are likely to benefit from checkpoint inhibitor therapy has been challenging . despite the fact that the results of assays for pdl - 1 and pdl - 2 expression , dmmr ( or msih ) , and mutational burden might not correlate with each other in an individual tumor , all have been shown to predict benefit from checkpoint inhibitor therapy for various malignancies.4 , 6 , 12 given the efficacy of the checkpoint inhibitor seen in our patient , one might predict her tumor to be msi - h ( despite the pmmr ) and / or to demonstrate a high mutational burden . 
regardless of that result , however , reports of tumors with pd - l1 or pd - l2 gene amplification and responsiveness to checkpoint inhibitors would be needed to confirm that gene amplification is another predictor of benefit from the targeting agent , as has been shown to be true for breast cancer , in which her2 ihc is negative but the her2 gene amplification independently predicts benefit . because this is a single case report , it should not be used as confirmation that pdl - 1 and pdl - 2 gene amplification is a confirmed predictor of responsiveness to checkpoint inhibitor therapy in crc . if additional responses of crcs in patients whose tumors show pmmr and pdl - 1 and pdl - 2 gene amplification are reported , consideration should be given to including patients with such tumors in trials aimed at identifying whether this molecular profile might serve as a predictive marker of likely responsiveness to checkpoint inhibitor therapy . in conclusion , the current fda breakthrough designation of checkpoint inhibitor therapy pertains only to mcrcs in patients whose tumors demonstrate dmmr ( as evidenced by msi - h )  . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . steven sorscher consulting or advisory role : merrimack speakers bureau : celgene jamie resnick no relationship to disclose first case report of a dramatic radiographic response to a checkpoint inhibitor in a patient with proficient mismatch repair gene expressing metastatic colorectal cancer the following represents disclosure information provided by authors of this manuscript . 
i = michael goodman stock and other ownership interests : exelixis consulting or advisory role : sanofi , alexion pharmaceuticals research funding : wake forest , veterans administration travel , accommodations , expenses : sanofi , alexion pharmaceuticals , jannsen pharmaceuticals references 1 . 
hall , mj , gowan k , sanford sm , et al : evaluation of microsatellite instability ( msi ) status in gastrointestinal ( gi ) tumor samples tested with comprehensive genomic profiling ( cgp )  . 
cicek ms , lindor nm , gallinger s : quality assessment and correlation of microsatellite instability and immunohistochemical markers among population - and clinic - based colorectal tumors results from the colon cancer family registry . 
droeser ra , hirt c , viehl ct , et al : clinical impact of programmed cell death ligand 1 expression in colorectal cancer . eur j cancer 49 : 2233 - 2242 , 2013 11 . 
gradishar wj , anderson bo , balassanian r , et al : breast cancer guideline in the national comprehensive cancer network guideline , harborside press , huntington , ny , version 2 . 
 targeted error - suppressed detection of circulating paternal dna to establish a diagnosis of gestational trophoblastic neoplasm introduction gestational trophoblastic neoplasms ( gtns ) are a family of pregnancy - related gynecologic malignancies that are derived from invasive placental trophoblast tissue and characterized by their secretion of human choriogonadotropin ( hcg )  . 
these neoplasms cover a wide spectrum of clinical and histologic presentations , from molar pregnancies to metastatic choriocarcinoma.1 whereas presentations vary , gtn is a highly curable disease , even for patients with poor - risk features . 
achieving favorable outcomes largely depends on accurate and timely diagnosis as well as close follow - up of the response to treatment.2 the case presented here illustrates the potential diagnostic challenges that are encountered when outcomes differ from initial expectations . 
clinical diagnosis of gtn was made as an international federation of gynecology and obstetrics stage iii and a risk score of 11term pregnancy , 4 - cm lesion , hcg  . 
as a result of her large burden of lung metastases , the patient received a short course of induction cisplatin and etoposide for 3 days.6 - 9 this led to a drop in hcg and the resolution of hemoptysis and vaginal hemorrhage . 
she then received first - line multiagent chemotherapy with etoposide , methotrexate , actinomycin d , cyclophosphamide , and vincristine ( ema / co ) for nine cycles , with a plateau in hcg that was suggestive of treatment resistance . 
switching to a paclitaxel , cisplatin , and etoposide ( tp / te ) salvage regimen resulted in the normalization of hcg . post - treatment monitoring detected an increase in hcg 4 weeks after the last dose of chemotherapy . induction chemotherapytwo cycles of tp / te followed by two cycles of high - dose chemotherapy with autologous stem - cell rescue were delivered , with the normalization of hcg . 
the patients hcg continued to rise , there was a recurrence of productive cough with blood - tinged sputum , and imaging confirmed the progression of multiple bilateral lung nodules , with no extrapulmonary disease . in the setting of progression after four lines of treatment , the possibility of an alternate diagnosis was entertained . 
the lack of a pathologic correlate opened the possibility of a non - gtn hcg - secreting malignancy , such as an occult gastric or bladder jean - michel lavoie miguel alcaide rosemary a . 
chemotherapy regimen used in the patient case reported regimen schedule dose ( per day ) ema / co ( 14 - day protocol ) etoposide actinomycin d methotrexate folinic acid rescue days 1 and 2 days 1 and 2 day 1 start 24 hours after methotrexate 100 mg / m2 0.5 mg iv bolus 300 mg / m2 15 mg iv every 12 hours vincristine cyclophosphamide day 8 day 8 tp / te ( 28 - day protocol ) paclitaxel days 1 and 15 carboplatin ( replaces day 1 cisplatin ) etoposide hdc / sct carboplatin etoposide autologous hematopoietic stem cells escalated ep ( 14 - day protocol ) etoposide cisplatin day 15 days 1 - 3 day 1 day 7 day 1 day 1 1 mg / m2 600 mg / m2 135 mg / m2 auc 5 150 mg / m2 550 mg / m2 2 , 250 mg / m2 500 mg / m2 60 mg / m2 note . 
escalated etoposide and cisplatin ( ep ) was administered with growth factor support for profilaxis of neutropenia . abbreviations : auc , area under the curve ; ema / co , etoposide , methotrexate , actinomycin d , cyclophosphamide , and vincristine ; hdc / sct , high - dose chemotherapy with autologous stem cell transplantation ; iv , intravenous . targeted hybridization capture and next - generation sequencing we pooled 250 ng of libraries from each partner and enriched them by using a panel of xgen lockdown probes ( integrated dna technologies , coralville , ia ) that targeted the exons of 83 genes according to the hybridization capture protocol provided by the manufacturer . 
the main purpose of this experiment was to select a few genetic polymorphisms to be used as biomarkers for the detection of paternal dna in the patients bloodstreain particular , we were interested in rare , paternally exclusive single - nucleotide polymorphisms ( snps ) or snps that were homozygous for the common allele in the patient , but heterozygous in her partner . 
we selected a panel of five snps ( table 2 ) and performed two successive rounds of hybridization capture using a personalized panel of xgen lockdown probes that targeted each one of the selected snps . enriched , dual - indexed libraries were sequenced in a miseq automated sequencer ( illumina , san diego , ca ) using pe 150 reads and analyzed according to previously reported methods.4 results we detected paternal cell - free dna in four of five markers investigated , all of them located on different chromosomes , at allele frequencies , 1% . all positive loci exhibited duplex sequencing support4 ( fig 3 )  . 
table 2 summarizes the snps that we used to track paternal - derived cell - free dna in the patients bloodstream , minor allele frequency ( according to the exac browser ) , 13 and the genotypes of the patient and her partner at each of the five genomic positions were investigated . given the presence of circulating paternal dna , diagnosis of gtn was confirmed . 
pretreatment hcg was 1 , 300 iu / l ; it briefly became undetectable , then stabilized under 10 iu / l . the patient remained stable on treatment of at least 18 weeks . discussion to our knowledge , this is the first patient in whom paternal dna could be detected at low ( , 1 , 000 iu / l ) hcg levels with high confidence . 
the number of molecules with duplex support and the total number of consensus sequences derived from individual dna strands that support each variant ( in parentheses ) is also indicated . 
mapping consensus sequences from individual dna strands against the reference reveals the presence of paternal - derived cell - free dna in the patients bloodstreatwo molecules with duplex support can be noticed . nucleotide disagreements with respect to the reference ( t ) are colored in green . 
the structure of a circulating tumor dna molecule with duplex support4 is also shown . 100100 210 214 12 - semidegenerate barcode 12 - semidegenerate barcode paternal - derived variant with duplex support strand - specific tags diagnosis of gtn . 
they used 15 primers for the detection of short tandem repeats on 13 different chromosomes , and sequenced cell - free dna in patients serum , maternal genome , paternal genome , and tumor tissue ( when available )  . 
all 14 confirmed cases had hcg levels of > 16 , 326 iu / l , whereas the 11 cases without detectable ctdna had hcg levels of < 14 , 884 iu / l . 
the use of semidegenerate barcoded adapters and biotinylated baits is an attractive option as it is potentially much more sensitive and , therefore , applicable in situations in which genotyping would be unlikely to yield a result . 
similar ultrasensitive methods for rare allele detection that rely on duplex sequencing have been used by kennedy et al14 and newman et al.15 in our case , targeted error - suppressed detection of circulating paternal dna obviated the need for a potentially high - risk biopsy of pulmonary disease , it was sensitive despite low hcg levels , and it was feasible within a clinically relevant timeframe of a few weeks . the advent of multiagent chemotherapy has led to gtn becoming a highly curable malignancy . proper diagnosis is paramount for directing treatment . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . jean - michel lavoie no relationship to disclose miguel alcaide no relationship to disclose rosemary a . 
seckl consulting or advisory role : bristol - myers squibb expert testimony : actavis travel , accommodations , expenses : bristol - myers squibb ryan morin consulting or advisory role : epizyme anna v . 
tinker consulting or advisory role : astrazeneca research funding : astrazeneca , medimmune other relationship : astrazeneca acknowledgment we thank our patient for kindly agreeing to have her case reported . jean - michel lavoie and anna v . 
openshaw mr , harvey ra , sebire nj , et al : circulating cell - free dna in the diagnosis of trophoblastic tumors . ebiomedicine 4 : 146 - 152 , 2015 4 . 
gillessen s , powles t , lim l , et al : low - dose induction chemotherapy with baby - bop in patients with metastatic germ - cell tumours does not compromise outcome : a single - centre experience . 
alifrangis c , agarwal r , short d , et al : ema / co for high - risk gestational trophoblastic neoplasia : good outcomes with induction low - dose etoposide - cisplatin and genetic analysis . 
bolze pa , patrier s , massardier j , et al : pd - l1 expression in premalignant and malignant trophoblasts from gestational trophoblastic diseases is ubiquitous and independent of clinical outcomes . 
 complete response to pd - 1 inhibition in an adolescent with relapsed clear cell adenocarcinoma of the cervix predicted by neoepitope burden and apobec signature anya levinson , md1 ; alex g . 
alejandro sweet - cordero , md1 , 2 ; and elliot stieglitz , md1 , 2 introduction utilization of checkpoint inhibition therapy in the management of malignant diseases is increasing . however , response to these therapies is highly variable , and robust predictive markers remain elusive . identication of predictive biomarkers is crucial considering the toxicities and high nancial cost associated with this approach . 
here , we report an adolescent with relapsed clear cell adenocarcinoma of the cervix ( ccac ) who experienced a complete response to checkpoint blockade despite not exhibiting positive pd - l1 immunohistochemical ( ihc ) staining , microsatellite instability ( msi ) , or a high tumor mutational burden ( tmb )  . 
biopsy of an exophytic mass protruding into the vagina conrmed a human papillomavirus / p16 - negative ccac ( fig 1 )  . family history was notable for paternal clear cell renal carcinoma . 
sixteen months after entering remission , surveillance positron emission tomography ( pet ) computed tomography ( ct ) revealed new hypermetabolic disease , including a new right upper lobe ( rul ) pulmonary nodule , enlarged prevascular right internal mammary , and right paratracheal nodes ( fig 2a ) and a left supraclavicular node . 
biopsy of the supraclavicular node conrmed recurrence of ccac ( fig 3 )  . her relapsed tumor exhibited 0% staining for pd - l1 by ihc , microsatellite stability ( 1 , 149 of 1 , 157 tested microsatellite regions were stable ) , and a low tmb ( 1.9 single nucleotide variants per megabase on wgs , less than the pediatric threshold of 2 ; appendix )  . 
genomic assays performed included targeted dna sequencing , wgs , and rna - seq ( appendix table a1 )  . a targeted institutional dna - seq panel assaying 479 cancer - relevant genes4 showed that both diagnostic and relapsed tumors had a broad approximately 11 - fold amplication on chromosome 20 containing aurora kinase a as a putative driver.5 the diagnostic sample also displayed a chromosome 1p deletion containing arid1a , a gene frequently mutated in cervical cancer , 6 which was absent at relapse . no pathogenic point mutations , insertions / deletions ( indels ) , or germline alterations were detected on this panel at diagnosis or relapse . 
pathologic conrmation of clear cell carcinoma of the cervix . ( a ) primary clear cell carcinoma of the cervix , showing hyperchromatic tumor cell nests in the stroma at left and normal endocervical glandular epithelium at the right ( 40 )  . 
 ( c ) an immunohistochemical stain for hepatocyte nuclear factor 1 - , which is a marker of clear cell carcinoma , shows strong positive nuclear staining in the tumor cells , a positive test result ( 200 )  . the rul pulmonary nodule and the prevascular lymph lymph nodes denode ( fig 2b )  . 
the median age was 53 years , and only three patients were , 30 years of age , 13 making the patient presented here one of the youngest patients ever reported with idiopathic ccac . 
 ( a ) patient developed a new hypermetabolic pulmonary nodule in the right upper lobe ( blue arrow ) , along with prominent hypermetabolic prevascular nodes ( red arrow )  . 
 ( b ) after initial nivolumab therapy , patients right upper lobe nodule decreased in size ( blue arrow ) , and the prevascular node had nearly resolved ( red arrow )  . 
 ( c ) approximately 16 months after initial recurrent disease , the pulmonary nodule has nearly completely resolved ( blue arrow ) , and no new pathologic nodes have developed . known biomarkers for response to checkpoint inhibition are ihc staining for pd - l1 , presence of msi , and high tmb . this patient , who achieved a complete and durable remission from checkpoint inhibition without any of these markers , highlights the need for additional biomarkers . 
we sought to analyze the genomics of her tumors to explain her dramatic response . somatic variations that give rise to amino acid substitutions in tumors yield neoepitopes , or mutated , tumor - specic peptides on the surface of cancer cells that can serve as neoantigens for the adaptive immune system , even if they are not oncogenic . 
determinants of neoantigen tness are the likelihood of their presentation by the major histocompatibility complex and of subsequent t - cell recognition.16 the number of neoepitopes per tumor can be more functionally relevant than the tmb . 
biopsy of supraclavicular node conrms metastatic disease . ( a ) metastatic clear cell carcinoma in a supraclavicular lymph node . the tumor cells line glands or grow in nests and are surrounded by lymphocytes , seen at the lower left ( 200 )  . ( b ) metastatic clear cell carcinoma in a supraclavicular lymph node . 
an immunohistochemical stain for pd - l1 shows minimal staining in the tumor cells and staining of scattered lymphoid cells around the tumor ( 200 )  . patients relapsed tumor , higher than that of available ( although biologically distinct ) comparison cohorts , supporting the idea that high neoepitope burden may predict favorable response to immunotherapy . 
of these , signatures 1 ( aging ) and 3 ( homologous recombination deciency ; appendix fig a4 ) are commonly found in cancer.18 , 19 signatures 2 and 13 represent activity of the aid / apobec family of enzymes.18 , 19 is associated with expression of apobec , a family of zinc - coordinating enzymes that convert cytosines to uracils , has been implicated in mutagenesis of nonsmall - cell lung cancer . 
systematic cancer genomic and transcriptomic association studies have shown that overexpression of one of the family members , apobec3b , immune genes and known immunotherapy response biomarkers such as pd - l1.20 a positive correlation was recently documented between strength of the apobec signature and the neoepitope burden in many cancers , including cervical.21 the authors also found that this mutational signature corresponded to increased abundance of tumorinltrating lymphocytes in some cancers , 21 although these were absent in this case . 
clinically , the apobec mutational signature is enriched in patients with durable clinical benet after immunotherapy , and an apobec signature may be better than tmb in predicting immunotherapy response.22 , 23 finally , there was a small contribution from signature 8 of uncertain etiology ( it is perhaps related to nucleotide excision repair deciency ) .24 the present patient is of interest for several reasons . 
box - and - whisker plots display the interquartile range ( box ) and outlier thresholds ( whiskers ) for the therapeutically applicable research to generate effective treatments neuroblastoma and the cancer genome atlas prostate adenocarcinoma data sets . 
although there may be other factors that contributed to the patients dramatic response , this case supports emerging evidence that a high neoepitope burden and an apobec mutational signature response to are potentially actionable biomarkers of checkpoint blockade . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . jessica van zife employment : adaptive biotechnologies ( i ) stock and other ownership interests : adaptive biotechnologies ( i ) patents , royalties , other intellectual property : adaptive biotechnologies ( i ) benjamin laguna leadership : sira medical stock and other ownership interests : sira medical stanley g . 
leung stock and other ownership interests : illumina , pzer , thermo fisher jacob pfeil employment : abbvie no other potential conicts of interest were reported . acknowledgment we thank the patient and her family for their willingness to share their experience using an unconventional treatment approach . 
naumann rw , hollebecque a , meyer t , et al : safety and efcacy of nivolumab monotherapy in recurrent or metastatic cervical , vaginal , or vulvar carcinoma : results from the phase i / ii checkmate 358 trial . 
european society for medical oncology meeting , barcelona , spain , september 27 - october 1 , 2019 ( abstr lba62 ) kline cn , joseph nm , grenert jp , et al : targeted next - generation sequencing of pediatric neuro - oncology patients improves diagnosis , identies pathogenic germline mutations , and directs targeted therapy . 
curr oncol rep 15 : 566 - 572 , 2013 cho h , kim js , chung h , et al : loss of arid1a / baf250a expression is linked to tumor progression and adverse prognosis in cervical cancer . 
n engl j med 377 : 1345 - 1356 , 2017 rao aa , madejska aa , pfeil j , et al : protect : prediction of t - cell epitopes for cancer therapy . 
reich o , tamussino k , lahousen m , et al : clear cell carcinoma of the uterine cervix : pathology and prognosis in surgically treated stage ib - iib disease in women not exposed in utero to diethylstilbestrol . 
thomas mb , wright jd , leiser al , et al : clear cell carcinoma of the cervix : a multi - institutional review in the post - des era . 
boyd j , takahashi h , waggoner se , et al : molecular genetic analysis of clear cell adenocarcinomas of the vagina and cervix associated and unassociated with diethylstilbestrol exposure in utero . 
turajlic s , litcheld k , xu h , et al : insertion - and - deletion - derived tumour - specic neoantigens and the immunogenic phenotype : a pan - cancer analysis . 28 : 15 - 17 , 2019 lancet oncol 18 : 1009 - 1021 , 2017 18 . 
wang s , jia m , he z , et al : apobec3b and apobec mutational signature as potential predictive markers for immunotherapy response in non - small cell lung cancer . 
chen z , wen w , bao j , et al : integrative genomic analyses of apobec - mutational signature , expression and germline deletion of apobec3 genes , and immunogenicity in multiple cancer types . 
chen pl , roh w , reuben a , et al : analysis of immune signatures in longitudinal tumor samples yields insight into biomarkers of response and mechanisms of resistance to immune checkpoint blockade . 
boichard a , tsigelny if , kurzrock r : high expression of pd - 1 ligands is associated with kataegis mutational signature and apobec3 alterations . oncoimmunology 6 : e1284719 , 2017 jager m , blokzijl f , kuijk e , et al : deciency of nucleotide excision repair is associated with mutational signature observed in cancer . 
the lymph node from the patient was snap frozen and ffpe into optimal cutting temperature ( oct ) mediuall tissues were sectioned to a depth of 5 m and stained with hematoxylin and eossamples were evaluated for tumor content by a certied pathologist . 
rna integrity was quantied with the high sensitivity rna kit dnf - 472 ( advanced analytical technologies , orangeburg , ny ) on the fragment analyzer ( advanced analytical technologies )  . 
immunohistochemical pd - l1 staining was performed on tumor cells with the us food and drug administrationapproved pd - l1 28 - 8 pharmdx antibody for opdivo ( neogenomics laboratories , fort myers , fl )  . whole blood was collected during lymph node metastasectomy in a 4ml edta vacutainer . 
germline dna was extracted using the dneasy blood and tissue kit 69504 ( qiagen )  . dna was quantied using the nanodrop 2000 ( thermo fisher , waltham , ma ) and qubit high sensitivity dsdna assay q32851 ( thermo fisher ) and integrity quantied using the high sensitivity gdna kit dnf488 ( advanced analytical technologies ) on the fragment analyzer ( advanced analytical technologies )  . 
libraries from ffpe samples were made using the truseq rna access kit ( rs - 301 - 2001 ; illumina , san diego , ca ) with an input of 100 ng in accordance with manufacturers instructions . libraries from ffpe samples were made using the truseq stranded mrna kit ( rs - 122 - 2101 ; illumina ) with an input of 400 ng in accordance with manufacturers instructions . 
outliers ( ie , underor overexpression ) were identied using tukey outlier denition . targeted dna sequencing an institutional dna sequencing panel assaying 479 cancer - related genes was used.4 as per kline et al , 4 genomic dna was extracted from peripheral blood and tumor tissue microdissected from ffpe blocks . 
capture - based next - generation sequencing ( ngs ) was performed at the university of california , san francisco ( ucsf ) clinical cancer genomics laboratory , using an assay targeting the coding regions of these genes , tert promoter , select introns from 40 genes ( for detection of gene fusions and other structural variants ) , and intergenic regions at regular intervals along each chromosome ( for chromosomal copy number assessment ) , altogether with a total sequencing footprint of 2.8 mb ( ucsf500 cancer gene panel , appendix fig a2 )  . 
a list of genes included can be found at the following url ( although some genes may have been added or deleted since this patients analysis in 2017 ) : gene - panel - test - ucsf500 - uc500 . whole - genome sequencing libraries were generated using the truseq nano kit fc - 121 - 4001 ( illumina ) with a 350 - bp insert , per manufacturers instructions . 
libraries were made using 200 - ng input gdna , and then quantied on the tapestation 4200 ( agilent , santa clara , ca ) and sequenced on the hiseq x ten ( illumina ) using 2 150 bp paired - end reads . 
nonsynonymous variants were annotated using variant effect predictor ( mclaren w , et al : genome biol 17 : 122 , 2016 )  . predicted tumor neoepitope analysis we used the prediction of t - cell epitopes for cancer therapy ( protect ) pipeline v 2.6.18 to investigate the patients neoepitope burden . 
we compared the patients tumor mutation and neoepitope burden to publicly available protect data for the therapeutically applicable research to generate effective treatments neuroblastoma ( pmid : 23334666 ) and the cancer genome atlas prostate adenocarcinoma data sets ( pmid : 26544944 ) and assessed for signicance using tukeys outlier denition . apobec signature analysis single base substitution mutational signatures were extracted from the samples wgs data using deconstructsigs ( rosenthal r , et al : genome biol 17 : 31 , 2016 ) , with default parameters . 
the patients sample is notable for a signature 3 ( homologous recombination deciency [ hrd ] ) signature . violin plot of the fraction of mutations in each sample from ma et al11 attributed to signature 3 . 
reis - filho , md , phd1 ; diana mandelker , md , phd1 ; and britta weigelt , phd1 purpose endometrial cancer ( ec ) is not considered a component of the hereditary breast and ovarian cancer syndrome but can arise in patients with germline brca1 / 2 ( gbrca1 / 2 ) mutations . 
we sought to determine if ecs in gbrca1 / 2 mutation carriers harbor biallelic alterations and / or features of hrd . methods of 769 patients with ec who underwent germline panel testing , 10 pathogenic gbrca1 / 2 mutation carriers were identied , and their tumorand normal - derived dna was subjected to massively parallel sequencing targeting at least 410 cancer - related genes . 
somatic mutations , copy number alterations , loss of heterozygosity , microsatellite instability ( msi ) , and genomic hrd features were assessed . results of the 13 patients included who had ec , eight harbored pathogenic gbrca1 mutations and ve harbored gbrca2 mutations . 
 smith et al context key objective to determine if endometrial cancers ( ecs ) that arise in patients with pathogenic brca1 or brca2 germline mutations are sporadic cancers or display genomic features of homologous recombination dna repair deciency ( hrd ) , which may guide treatment strategies . knowledge generated ecs in patients with brca1 germline mutations showed biallelic brca1 alterations coupled with genomic features of hrd . 
in contrast , only a subset of ecs in brca2 germline mutation carriers harbored biallelic brca2 alterations ; the remaining cases were likely sporadic cancers . relevance knowledge about the brca1 / 2 allele status and / or hrd features of ecs in patients with germline brca1 / 2 alterations may guide therapeutic decision making , given that tumors with biallelic alterations in brca1 / 2 have been associated with increased response to hr - targeted therapies such as platinum salts and parp inhibitors . brca1 / 2 alterations and show features of hr deciency , including the catalog of somatic mutations in cancer mutational signature 3 , signature multivariate analysis ( sigma ) hr deciency signature sig3 , or large - scale chromosomal breaks in the form of large - scale state transitions ( lsts )  . 
in addition , we assessed the clinicopathologic data of ecs in gbrca1 / 2 mutation carriers and explored the repertoire of somatic mutations present in these tumors . methods case selection all patients with ec and gbrca1 / 2 mutations who consented to germline analysis under an institutional review boardapproved protocol at memorial sloan kettering cancer center and whose tumors were subjected to targeted massively parallel sequencing via memorial sloan ketteringintegrated mutation proling of actionable cancer targets ( msk - impact ) 10 , 11 from july 2015 to may 2019 were identied ( n = 769 sequenced ; n = 10 with pathogenic gbrca1 / 2 mutation )  . 
all gbrca1 / 2 variants were reviewed by a board - certied molecular pathologist ( d.m. ) and classied according to the american college of medical genetics and genomics criteria12 as pathogenic . clinicopathologic data , including age at diagnosis , cancer stage , and past cancer history , were obtained from medical records . 
mlh1 promoter methylation was assessed in the clinical setting on dna obtained from formalin - xed parafn - embedded tumor samples , as previously described.28 statistical analyses fishers exact and mann - whitney u tests were used for comparison of categorical and continuous variables , respectively . 
two - tailed p , 0.05 was considered statistically signicant . results clinicopathologic features of ecs arising in gbrca1 / 2 carriers ten ecs from patients with pathogenic gbrca1 / 2 mutations subjected to msk - impact sequencing ( n = 769 ecs ; 1.3% ) and three subjected to wes by tcga ( n = 232 ecs ; 1.3% ) 13 were included in this study . 
of these 13 gbrca1 / 2associated ecs , eight and ve patients with ec harbored gbrca1 and germline brca2 ( gbrca2 ) mutations , respectively ( table 1 )  . 
the majority of gbrca1 / 2 - associated ecs ( n = 6 of 8 ; 75% ) displayed an endometrioid histology ( n = 2 and 4 international federation of gynecology and obstetrics [ figo ] grades ii and iii , respectively ) but lacked a solid / pseudoendometrioid / transitional cell - like pattern of morphology , which has been associated with gbrca1 / 2 mutations in high - grade serous ovarian cancers.29 , 30 in contrast , ecs from patients with pathogenic gbrca2 mutations were more heterogeneous at the phenotypic level , and included endometrioid ( n = 1 ; figo grade ii ) , carcinosarcoma ( n = 2 ) , high - grade ec not otherwise specied ( nos ; n = 1 ) and serous ( n = 1 ) cancers ( tables 1 and 2 )  . 
of note , none of the gbrca1 / 2 - associated ecs included in this study were of serous histology . gbrca1 / 2 - associated ecs presented at different clinical figo stages ( 2009 staging system31 )  . 
taken together , our data suggest the clinicopathologic features associated with ecs occurring in patients with pathogenic gbrca1 or gbrca2 mutations may be heterogeneous . biallelic brca1 / 2 alterations allele - specic copy number analysis revealed that 77% ( n = 10 of 13 ) of the ecs in patients with pathogenic gbrca1 / 2 mutations displayed biallelic inactivation of brca1 / 2 uniformly through loh of the wild - type allele ( table 2 ; fig 1 )  . 
not all ecs occurring in pathogenic gbrca1 / 2 mutation carriers harbored biallelic brca1 / 2 alterations , which have previously been associated with hr deciency.8 although all ecs in pathogenic gbrca1 mutation carriers ( 100% ) harbored biallelic brca1 inactivation , only two of the ve ecs ( 40% ) from patients with pathogenic gbrca2 mutations displayed biallelic brca2 inactivation ( table 2 ; fig 1 )  . biallelic brca1 / 2 inactivation was found across all histologic subtypes and clinical stages of ec . 
ecs in brca2 mutation carriers lacking loh of the wild - type allele are likely sporadic tumors ; all three were stage iii at diagnosis and were endometrioid grade ii , serous , or high - grade ecs ( table 2 ; fig 1 )  . 
 ( % ) unless otherwise indicated . abbreviations : msk - impact , memorial sloan ketteringintegrated mutation proling of actionable cancer targets ; nos , not otherwise specied ; tcga , the cancer genome atlas ; wes , whole - exome sequencing . * fishers exact and mann - whitney u tests were used for comparison of categorical and continuous variables , respectively . staging information was performed according to the international federation of gynecology and obstetrics system.31 past cancer histories and information regarding radiation exposure were available for the 10 msk - impact cases and reect cancers diagnosed and treated prior to the diagnosis of ec . 13 gbrca1 / 2 - associated ecs was six ( range , 2 to 38 ) , with a median of ve ( range , 2 to 32 ) nonsynonymous mutations ( data supplement )  . 
we observed that all ecs analyzed , irrespective of the presence of monoor biallelic brca1 / 2 alterations , harbored somatic tp53 mutations , of which 12 ( 92% ) were hotspot mutations ( fig 1 ; data supplement )  . 
alterations affecting the pi3k pathway were 4 2019 by american society of clinical oncology common , with pik3ca mutations present in ve ecs ( 38% ) , pik3r1 mutations in two ecs ( 15% ) , and pten mutations / homozygous deletions in three ecs ( 23% ; figs 1 and 2 )  . 
other recurrently altered genes included fat1 , ptch1 , kras , and map3k1 ( each n = 2 ; fig 1 , data supplement )  . we noted that bec - 1 , a likely sporadic ec from a gbrca2 mutation carrier with a monoallelic brca2 alteration , had a high mutational burden with 32 nonsynonymous somatic mutations . 
the ecs with very low levels of gene cnas ( ie , bec - 1 , bec - 6 , and bec - 12 ) had monoallelic brca2 alterations and were likely sporadic , with one ( bec - 1 ) being msi high , as mentioned . 
in addition , we found an nf1 homozygous deletion in the biallelic brca1 grade ii endometrioid tcga - 2 and a smarca4 homozygous deletion in the biallelic brca2 carcinosarcoma bec - 2 . 
of note , smarca4 ( brg1 ) loss is commonly found in unthe endometrium , 32 , 33 and differentiated carcinoma of bec - 2 was a carcinosarcoma with heterologous elements , which , in addition to the carcinoma and sarcomatous components , also displayed an undifferentiated component . 
finally , amplication of bcl6 , tp63 , and eed was found in the biallelic brca1 grade iii endometrioid tcga - 1 ec ( fig 2 )  . taken together , we found that ecs in patients with a gbrca1 / 2 mutation are heterogeneous at the mutational and gene copy number levels . 
we observed that ecs with biallelic brca1 / 2 alterations ( n = 10 ) had high lst scores , whereas ecs with monoallelic brca1 / 2 alterations did not ( table 2 ; fig 3a )  . 
nonsynonymous somatic mutations identied in 410 cancer - related cancer genes in ecs from germline brca1 ( n = 8 ) and germline brca2 ( n = 5 ) mutation carriers ( left ) are shown . the mutation types are color coded according to the legend . 
information on the germline mutation status , somatic loss of heterozygosity of brca1 / 2 , histologic type , microsatellite instability ( msi ) status , and sequencing type is provided in the phenobar below the sample names . 
tcga , the cancer genome atlas . with hr deciency ( fig 3b ) .14 given the limited number of snvs and indels in the ecs subjected to msk - impact sequencing , other genomic hr - deciency features such as indel length , microhomology , or mutational signatures using decomposition algorithms could not be used . therefore , we used sigma , a recently described , likelihoodbased measure signature analysis combined with machinelearning techniques , which can be used to detect the mutational signature sig3 associated with hr deciency from targeted gene panels including msk - impact ( sensitivity range , 48% to 62% for uterine cancers ) .25 consistent with the lst analysis , all six biallelic , brca1 / 2associated ecs subjected to msk - impact and with at least ve snvs displayed either a dominant hr deciency - related sig3 ( n = 2 ) or a dominant clock signature with secondary hr - deciency signatures sig3 ( n = 3 ) or sig834 ( n = 1 ) , to the two sporadic ecs with monoallelic in contrast brca2 alterations , which displayed msi ( sigma ) / signature 6 ( deconstructsigs ) and apobec signatures ( table 2 ; fig 3c )  . 
previous reports of ecs in this population have acknowledged that the prevalence of ec in gbrca1 / 2 mutation carriers is low.5 here , through an in - depth analysis of ecs from patients with pathogenic gbrca1 / 2 mutations , we demonstrate that the majority of these cases ( 77% ) harbored biallelic brca1 / 2 alterations and displayed genomic features of hrd , providing evidence to suggest an etiological link between the pathogenic gbrca1 / 2 mutations and the development of these ecs . 
in fact , this phenomenon was uniformly observed in patients with pathogenic gbrca1 mutations ; conversely , only two of the ve patients with pathogenic gbrca2 mutations had somatic inactivation of the brca2 wild - type allele and genomics features of hr deciency . 
in ovarian cancer , patients with hr - decient tumors have improved overall survival with platinum - based therapy and have better responses to hr deciencydirected treatments such as parp inhibitors than their wild - type counterparts.35 , 36 knowledge of the allele status of brca1 / 2 alterations and / or hr - deciency features could be benecial in therapeutic decision making for patients with gbrca1 / 2 mutations and ec . 
our analysis has demonstrated that not all ecs arising in the context of pathogenic gbrca2 mutations harbor loh of the wild - type allele and genomics features of hr deciency . 
in addition , it revealed that one of these cases was msi high , likely representing a sporadic ( ie , non - brca1 / 2 ) ec arising in a gbrca2 mutation carrier . 
in this context , this patient would potentially benet from immune checkpoint inhibitors , which have been approved for the management of recurrent msi - high / dna mmrdecient cancers.37 in fact , a recent case report highlighted a complete remission after pd - 1 blockade in a patient with mmr - decient ec and a pathogenic monoallelic gbrca1 mutation.38 the majority ( 75% ) of biallelic gbrca1 / 2 - associated ecs were of intermediate / high - grade endometrioid as opposed to serous histology in previous reports.5 , 6 all six biallelic gbrca1 / 2 - associated endometrioid ecs harbored tp53 mutations , had high lst scores , and relatively high levels of cnas ( table 2 ; fig 2 ) , and would likely be of copy - number high ( serous - like ) molecular subtype , as described by tcga.13 it should be noted , however , that of these six endometrioid ecs with biallelic brca1 alterations , four harbored somatic mutations characteristic of endometrioid ecs , including pik3ca mutations , pik3r1 mutations , and pten mutations / homozygous deletions . 
conversely , two of these ecs lacked alterations affecting genes recurrently altered in endometrioid ecs ( fig 1 ) , suggesting that at the these latter two cases resembled serous genetic level , rather than endometrioid carcinomas , and that there is heterogeneity in gbrca1 / 2 - associated ecs . 
carcinosarcomas of the uterus are rare , representing less than 5% of all uterine tumors.39 we noted a high frequency ( 30% ) of carcinosarcomas in the 10 gbrca1 / 2 - associated ecs subjected to msk - impact sequencing , whereas overall , only 10% of the 769 ecs subjected to germline mskimpact sequencing were carcinosarcomas ( remaining cases : 55% endometrioid , 15% serous , 10% endometrial cancer nos , 4% mixed endometrial , 3% clear cell , 1% deor undifferentiated , and 1% other )  . 
interestingly , recurrent somatic brca1 / 2 mutations and brca2 deletions also have been reported in uterine carcinosarcomas.40 - 42 these ndings suggest that ecs arising in gbrca1 / 2 mutation carriers are heterogeneous at the histologic level and are potentially enriched for endometrioid tumors and carcinosarcomas . the data presented here do provide insight into the genomics of ecs in patients with pathogenic gbrca1 / 2 mutations , which may play a role in the tumorigenesis and / or progression of ec in some patients . 
this study has several limitations , however , primarily driven by the small sample size , and it does not resolve the controversy of ec as a feature of hboc syndrome . 
the low incidence of these cases ( 1.3% ; n = 10 of 769 ecs subjected to germline mskimpact testing ) not only limits the sample size of this study but also , on its own , presents a challenge in dening ec as part of the hboc syndrome spectrum of malignancies . 
 brca1 / 2 endometrial cancer lsts mutational signatures ( decontructsigs ) signature 1 ( aging ) signature 3 ( hr defect ) signature 15 ( mmr defect ) signature 19 signature 20 ( mmr defect ) signature 24 signature 25 unknown tcga - 1 tcga - 2 tcga - 3 mlh1 biallelic brca1 / 2 ( n = 10 ) monoallelic brca2 ( n = 3 ) pms2 msh2 msh6 signature 1 aging signature 6 mmr defect signature 21 mmr defect unknown mutational signatures bec - 1 fig 3 . 
 ( a ) large - scale state transition ( lst ) scores in germline ( gbrca1 / 2 ) - associated endometrial cancers with biallelic ( n = 10 ) and monoallelic ( n = 3 ) brca1 / 2 alterations . 
 ( b ) mutational signatures dened by deconstructsigs24 in three gbrca1 / 2 - associated endometrial cancers from the cancer genome atlas ( tcga ) subjected to whole - exome sequencing.13 , 14 mutational signatures are color coded according to the legend on the right . 
 ( c ) immunohistochemical analysis of the ( left ) dna mismatch repair ( mmr ) proteins mlh1 , pms2 , msh2 , and msh6 and ( right ) mutational signatures of case bec - 1 , a monoallelic / sporadic gbrca1 / 2 - associated endometrial cancer . 
bec - 1 shows loss of mlh1 and pms2 expression , mlh1 promoter hypermethylation ( not shown ) , and dominant mutational signatures 6 and 21 associated with defective dna mmr . mutations in patients with ec nor the impact of these mutations on the outcome of patients with ec could be fully dened . 
although we have assessed the genomics features of hr deciency , including lsts and sigma hr deciency signature sig3 in all cases , hr deciency is more accurately assessed with whole - genome sequencing.43 hence , additional studies testing ecs developing in the context of pathogenic gbrca1 / 2 mutations but lacking loss of the wildtype allele by whole - genome sequencing are warranted . despite these limitations , here we demonstrate the importance of germline and somatic genetic characterization of ecs . 
in fact , ecs developing in the context of pathogenic gbrca1 / 2 mutations may harbor genomics features of hr deciency and be causally linked to the loss of function of these tumor suppressor genes . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . nadeem riaz honoraria : peerview speakers bureau : illumina research funding : bristol - myers squibb , pzer travel , accommodations , expenses : varian medical systems mark e . 
robson honoraria : astrazeneca consulting or advisory role : mckesson , astrazeneca research funding : astrazeneca ( inst ) , myriad genetics ( inst ) , invitae ( inst ) , abbvie ( inst ) , tesaro ( inst ) , medivation ( inst ) travel , accommodations , expenses : astrazeneca , pzer other relationship : research to practice , clinical care options , physician education resource robert a . 
assignee : 2015 the regents of the university of california 8 / 18 / 2015 ( inst ) ; application : compositions and methods for the diagnosis and treatment of ovarian cancers that are associated with reduced smarca4 gene expression or protein function ( inst ) ; royalties from published books : cambridge university press and springer publishing jorge s . 
reis - filho consulting or advisory role : roche , invicro , ventana medical systems , volition rx , paige.ai , goldman sachs , novartis britta weigelt consulting or advisory role : roche ( i ) , invicro ( i ) , ventana medical systems ( i ) , volition rx ( i ) , paige.ai ( i ) , goldman sachs ( i ) , novartis ( i ) no other potential conicts of interest were reported . acknowlegement we thank the members of the molecular diagnostics service in the department of pathology , memorial sloan kettering cancer center . references gudmundsdottir k , ashworth a : the roles of brca1 and brca2 and associated proteins in the maintenance of genomic stability . 
oncogene 25 : 5864 - 5874 , 2006 king mc , marks jh , mandell jb : breast and ovarian cancer risks due to inherited mutations in brca1 and brca2 . 
nat rev clin oncol 13 : 41 - 54 , 2016 segev y , iqbal j , lubinski j , et al : the incidence of endometrial cancer in women with brca1 and brca2 mutations : an international prospective cohort study . gynecol oncol 130 : 127 - 131 , 2013 shu ca , pike mc , jotwani ar , et al : uterine cancer after risk - reducing salpingo - oophorectomy without hysterectomy in women with brca mutations . 
jama oncol 2 : 1434 - 1440 , 2016 pennington kp , walsh t , lee m , et al : brca1 , tp53 , and chek2 germline mutations in uterine serous carcinoma . 
cell 173 : 355 - 370.e14 , 2018 riaz n , blecua p , lim rs , et al : pan - cancer analysis of bi - allelic alterations in homologous recombination dna repair genes . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
ashley cw , da cruz paula a , kumar r , et al : analysis of mutational signatures in primary and metastatic endometrial cancer reveals distinct patterns of dna repair defects and shifts during tumor progression . 
geyer fc , li a , papanastasiou ad , et al : recurrent hotspot mutations in hras q61 and pi3k - akt pathway genes as drivers of breast adenomyoepitheliomas . nat commun 9 : 1816 , 2018 e131 , 2016 17 . 
karnezis an , hoang ln , coatham m , et al : loss of switch / sucrose non - fermenting complex protein expression is associated with dedifferentiation in endometrial carcinomas . 
ramalingam p , croce s , mccluggage wg : loss of expression of smarca4 ( brg1 ) , smarca2 ( brm ) and smarcb1 ( ini1 ) in undifferentiated carcinoma of the endometrium is not uncommon and is not always associated with rhabdoid morphology . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
dizon ds , dias - santagata d , bregar a , et al : complete remission following pembrolizumab in a woman with mismatch repair - decient endometrial cancer and a germline brca1 mutation . 
gynecol oncol 137 : 581 - 588 , 2015 jones s , stransky n , mccord cl , et al : genomic analyses of gynaecologic carcinosarcomas reveal frequent mutations in chromatin remodelling genes . 
zhao s , bellone s , lopez s , et al : mutational landscape of uterine and ovarian carcinosarcomas implicates histone genes in epithelial - mesenchymal transition . proc natl acad sci usa 113 : 12238 - 12243 , 2016 jones nl , xiu j , chatterjee - paer s , et al : distinct molecular landscapes between endometrioid and nonendometrioid uterine carcinomas . 
 next - generation sequencing of advanced gi tumors reveals individual treatment options michael bitzer , md1 , 2 , 5 ; leonie ostermann1 ; marius horger , md3 ; saskia biskup , md , phd4 ; martin schulze , dsc4 ; kristina ruhm , msc5 ; franz hilke , msc6 ; oznur oner , msc5 ; konstantin nikolaou , md , mba2 , 3 ; christopher schroeder , md6 ; olaf riess , md6 ; falko fend , md7 , 8 ; daniel zips , md8 , 9 ; martina hinterleitner , md10 ; lars zender , md2 , 8 , 10 ; ghazaleh tabatabai , md , phd2 , 8 , 11 ; janina beha , phd5 ; and nisar p . 
malek , md1 , 2 , 5 , 8 purpose precision oncology connects highly complex diagnostic procedures with patient histories to identify individualized treatment options in interdisciplinary molecular tumor boards ( mtbs )  . 
follow - up data and a response assessment that was based on radiologic imaging were recorded . results ninety - six patients were presented to the mtb of tuebingen university hospital . 
patients with pr or sd in the course of mtb - recommended treatments seemed to benet with respect to pfs and os . jco precis oncol 4 : 258 - 271 . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction the challenge of precision oncology is to connect highly complex diagnostic procedures with individual patient histories to identify optimal treatments . 
detailed data on mtbguided treatment suggestions , and specically the outcomes of patients , have only been reported for nonselected patient groups , including for a variety of different tumors.1 - 4 however , each tumor entity , including gi cancers , harbors unique features that will have to be considered to identify the patients who will benet the most from such an approach.4 recent reports highlight the experiences of mtbs in everyday practice . 
a series from the md anderson cancer center that examined hot - spot regions in 11 to 50 genes for 2 , 000 cancer tissues found actionable mutations for 39% of patients.2 only 11% of these selected patients could be enrolled in genotype - matched clinical trials.2 a report from the mayo clinic identied actionable mutations in 65% of 141 patients , but only 29 ( 21% ) were subsequently treated in clinical trials or with targeted food and drug administration ( fda ) approved drugs , with an objective response or stable disease ( sd ) for 4 months in 10 patients.3 freiburg university reported 104 patients with recommendations that were based on diagnostic tests that ranged from an 8 - gene panel to whole - exome sequencing ( wes ) .1 thirty - three patients received a recommended treatment ; 11 had an objective response , and 8 additional patients reached sd at 10 weeks . 
 molecular tumor board for advanced gi cancers context key objectives academic molecular tumor boards ( mtbs ) bridge the gap between a growing complexity in diagnostic procedures and clinicians at the bedside . 
this retrospective study investigated the course of patients with advanced gastrointestinal ( gi ) cancers that have been considered for personalized treatment options , including the outcome receiving mtb - guided treatment . knowledge generated next - generation sequencing ( ngs ) revealed a relevant number of germline variants , suggesting genetic counseling . 
responding patients with prolonged disease stabilization seemed to derive a meaningful survival benet from cancer genome sequencing and matched treatments . relevance patients with gi cancers can benet from currently available ngs sequencing procedures . 
in perspective , a continued improvement of mtb recommendations and the inclusion of additional complex diagnostic procedures might substantively enhance treatment opportunities in everyday clinical practice for these patients in the near future . the sequencing of matched tumor and normal tissue is an important procedure for the precise identication of potentially actionable genes.5 , 6 this inevitably leads to the detection of germline alterations . 
a recent investigation in 10 , 389 patients across 33 cancer types detected pathogenic or likely pathogenic germline variants in 8% of all patients with cancer and in 8.8% of patients with gi tumors.7 the frequency of germline mutations varied greatly across cancer types in general but also across different gi cancers , ranging from 2.2% for cholangiocarcinoma to 14.1% for pancreatic cancer ( pc ) .7 to our knowledge , our experience with gi tumors in an academic mtb is to date the largest reported series of this group with detailed information of comprehensive sequencing data , subsequent board recommendations , and documented outcome . 
the mtb consists of an interdisciplinary team coordinated by the tuebingen center for personalized medicine and includes experts in clinical and translational oncology , pathology , bioinformatics , molecular biology , radiology , and human genetics . 
an electronic web - based platform ( mtb platform ) was established to introduce patients to the mtb team with all necessary information to prepare and follow up the weekly face - to - face meetings and subsequently document treatment outcomes ( data supplement )  . 
best response assessment was based on radiologic imaging in line with recist 1.1 , for checkpoint inhibitor therapy , irecist , or for hepatocellular carcinomas ( hcc ) , mrecist criteria.8 - 10 genetic tumor / normal characterization tumor and normal tissues were genetically characterized either by ngs panel sequencing of full coding sequences or by wes ( more information in data supplement )  . 
off - label use refers to the administration of an fda / european medicines agency - approved drug outside its approved indication . for recommended off - label therapies , an application for reimbursement was submitted to the patients health individual treatment describes insurance . 
patients treated within a heilversuch have been registered at the local authority , in this case the regierungspr asidium tuebingen . statistical analysis progression - free survival ( pfs ) and overall survival ( os ) were analyzed with the kaplan - meier method dependent on the treatment response ( sd and pr v progressive disease [ pd ] ) compared by log - rank testing . 
btc , biliary tract cancer ; crc , colorectal cancer ; gc , gastric cancer ; hcc , hepatocellular carcinoma ; net , neuroendocrine tumor ; pc , pancreatic cancer ; ugc , upper gi tract cancer . results diagnostic procedure and clinical work - up ninety - six patients received ngs of advanced gi tumors and were presented to the mtb . 
this cohort included 32 colorectal cancer ( crc ) , 22 pc , 19 biliary tract cancer ( btc ) , 11 hcc , 9 upper gi tract cancer ( ugc ) , and 3 neuroendocrine tumors ( net ; fig 1a )  . 
the mean number of systemic anticancer pretreatments was 2.8 ( fig 1b )  . ninety - one patients received a gene panel analysis that examined between 337 and 710 genes , and 5 patients had wes ( fig 1c )  . 
genes represented in the different panels are summarized in the data supplement , together with the total size and the average and median coverage of the 649 and 710 gene panels . germline variants in gi cancers germline ndings were ranked according to a 5 - tiered classication.11 , 12 sixteen patients ( 17% ) had germline variants classied as pathogenic or likely pathogenic ( figs 1d - 1f ) , and in 1 patient with gastric cancer , 2 likely pathogenic variants were detected ( palb2 and fancm )  . five patients ( 5% ) had pathogenic or likely pathogenic germline variants in one of the brca2 , msh6 , or sdhb genes . 
these alterations belong to a list of secondary ndings that should be reported because of the possibility for interventions to signicantly reduce morbidity and mortality.13 in addition , 3 patients showed germline variants in pharmacologically relevant genes , 2 in dpyd and 1 in g6pd ( data supplement ) without showing unusual adverse reactions during chemotherapy . 
this variant has been reported with classications ranging from variant of unknown signicance to pathogenic.14 a germline alteration was directly responsible for mtb recommendations for 5 patients ( 5% ) : brca2 for poly ( adpribose ) - polymerase ( parp ) inhibition ( 3 pc ) , chek2 combined with 2 somatic atm truncations for atr - inhibition and carboplatin ( 1 crc ) , and msh6 and therefore a resulting high tumor mutational burden ( tmb ) for pembrolizumab ( 1 crc )  . 
 molecular tumor board for advanced gi cancers frequency of germline mutations ( n = 96 ) presentation of ngs for gi tumors ( n = 96 ) molecular target identification ( n = 96 ) patients with clinically relevant ( n = 19 [ 20% ] ) germline mutations patients with pathogenic or likely pathogenic ( n = 16 ) variants patients with pharmacogenomic relevant variants ( n = 3 ) patients with at least 1 molecular target ( n = 47 [ 49% ] ) patients without mtb recommendation ( n = 6 ) no progression under last line treatment ( n = 4 ) complete tumor resection ( n = 2 ) patients not treated with recommendation ( n = 16 ) patients with poor performance status ( n = 15 ) patients not evaluable ( n = 5 ) without imaging for response monitoring ( n = 3 ) died within 21 days after treatment start ( n = 2 ) patient not evaluable ( n = 1 ) alternative therapy before response monitoring patients with mtb treatment recommendation ( n = 41 [ 43% ] ) patients received the suggested treatment ( n = 25 [ 26% ] ) patients with colorectal cancer ( n = 11 ) patients with pancreatic cancer ( n = 7 ) patients with biliary tract cancer ( n = 4 ) patients with upper gi tract cancer ( n = 2 ) patient with hepatocellular carcinoma ( n = 1 ) patients evaluable for best response and os ( n = 20 [ 21% ] ) patients pr ( n = 3 [ 15% of 20 patients ] ) patients sd ( n = 6 [ 30% of 20 patients ] ) patients pd ( n = 11 [ 55% of 20 patients ] ) fig 2 . 
 ( b ) frequency of potential target identication by the molecular tumor board ( mtb ) , patients with target identication but no treatment , and patients with mtb - recommended treatment initiation . 
btc , biliary tract cancer ; crc , colorectal cancer ; hcc , hepatocellular carcinoma ; net , neuroendocrine tumor ; pc , pancreatic cancer ; ugc , upper gi tract cancer ; w / o , without . pharyngeal , or bladder cancer ; and the patient with a chek2 mutation had a rst - degree relative with ovarian cancer . 
eight of these 10 patients were also tested for msi , but only 2 patients were found to have a msi - high tumor . tmb is currently regarded as relevant in predicting therapeutic success with checkpoint inhibitors beyond tumors with mismatch repair deciency.15 - 18 this has been shown for patients with crc and ugc , 18 but is less clear for hepatobiliary cancer and pc . 
for tumors known to have a low tmb in general , those with values molecular target identication by the mtb at least 1 potential molecular target was identied in 47 of 96 patients ( 49% ; figs 2b and 3b )  . 
the frequency of target identication varied among tumor types ( fig 3c , table 2 ) , with 74% for btc , 56% for upper gi cancers , 50% for pc , 44% for crc , and 27% for hcc . 
the most frequently altered genes with single - nucleotide variants or copy number variants were cdkn2a / b , brca2 , idh1 , erbb2 , myc , fgf3 , fgf4 , flt3 , fgfr4 , cdk6 , braf , and atm ( table 2 ; data supplement )  . 
target identication per tumor type ( continued ) diagnosis patients tested ( no . ) molecular target frequency erbb2 braf brd4 cdkn2a / b chek2 fgf3 / 4 / 19a flt1 / 3 mlh1 msh6 fgfr2 - bicc1 idh1 braf brca1 brca2 cdkn2a fgfr1 fgfr2b fgfr4 fgfr2 - prkcq fgfr2 - ahcyl2 cdkn2a / b brca2 cdk6 erbb3 nrg3 nrg1 - cdh6 tmem66 - nrg1 cdkn2a / b cdk6 fgf3 / 4 idh1 idh1 abbreviations : btc , biliary tract cancer ; crc , colorectal cancer ; hcc , hepatocellular carcinoma ; pc , pancreatic cancer ; tmb , tumor mutational burden . afgf3 / fgf4 / fgf19 cluster amplication . bcoincident fgfr2 mutation in tumor with fgfr2_ahcyl2 fusion gene . cdkn2a / b alterations , 6 were detected in pc , 2 in ugc , 1 in btc , and 1 in crc . 
three out of 4 brca2 alterations were detected in pc , and 1 in btc ( table 2 )  . outcome and clinical course of patients the course of all patients presented to the mtb is shown in fig 2b . 
mtb recommendations for matched treatments were given for 41 patients ( 43% )  . twenty - ve patients with a mean of 3.4 previous anticancer treatments received the suggested medication ( 61% of patients with mtb recommendation ; 26% of the whole cohort )  . 
the alterations in included cdkn2a / b this subgroup without treatment deletions , braf mutations , brca deletions , flt1 / 3 amplication , fgfr2 fusion , idh mutation , and high tmb ( data supplement )  . treatment was initiated in 11 patients with crc ( 34% of patients with crc ) , 7 patients with pc ( 32% of patients with pc ) , 4 patients with btc ( 21% of patients with btc ) , 2 patients with ugc ( 22% of patients with ugc ) , and 1 patient with hcc ( 9% of patients with hcc )  . 
the applied drugs were either used in - label , used off - label , obtained via a clinical study , or supplied for a matched experimental treatment ( table 1 , drug availability column )  . 
 molecular tumor board for advanced gi cancers patients who reached either sd or pr were treated with pd1 blocking antibodies ( high tmb ) , atr inhibitor with carboplatin ( simultaneous mutations in atm , chek2 , and tp53bp1 ) , trastuzumab with lapatinib ( erbb2 amplication ) , pertuzumab with erlotinib ( nrg1 fusion , high - expression erbb3 and nrg3 ) , idh1 inhibitor bay 1436032 ( idh1 mutation ) , or lenvatinib ( fgfr2 fusion gene ; table 1 )  . 
1 year after treatment initiation reached either sd or pr ( data supplement )  . therefore , as a surrogate end point to estimate whether patients might benet from the mtb - recommended treatment , the achievement of sd for at least 3 months or even pr as best response was compared with pd in a kaplan - meier estimation for pfs and os . 
in several studies , the inclusion of germline ndings enabled a more comprehensive characterization of tumor biology , potential resistances , or even treatment opportunities.22 , 23 in this study , germline variants classied as pathogenic , likely pathogenic , or pharmacogenomic could be identied in 19 patients ( 20% )  . 
these numbers are higher than in a recent investigation that reported pathogenic or likely pathogenic germline predisposition variants in 8.8% of gi tumors , 7 and could be mainly a result of the smaller sample size in our study . 
for example , for brca mutations , the tumor lineage indeed seems to determine the therapeutic benet of parp inhibition.24 conversely , emerging data suggest that patients with germline mutations that are not typically associated with a diagnosed cancer might nevertheless benet from drugs that target this alteration.25 germline variant reporting and the inclusion of experts in clinical genetics , should therefore be a prerequisite for mtbs.22 , 23 , 26 in predicting therapeutic tmb is regarded as relevant outcome with checkpoint inhibitors.15 - 18 the minimum size of tumor panels to reliably calculate tmb was suggested to include at least 300 genes , or more precisely , 1.5 mb of the target region , 21 , 27 , 28 which is met in our approach . 
one patient with crc and high tmb ( 118 var / mb ) who showed progression under pd - 1 inhibition even responded after the addition of ctla4 inhibition on progression . 
in the 4 patients who did not show any response to checkpoint inhibition , the molecular analysis did not reveal one of the currently discussed mechanisms of resistance.29 of note , only a minority of patients with high tmb also had msi - high tumors . 
this observation is in line with an investigation in a broad range of different tumor types , showing that only 16% with high tmb were classied as msi high.21 another study in 6 , 004 patients with crc identied 465 patients with high tmb ; however , only 65% of these could be classied as msi high.30 these observations suggest that a reliable method for tmb estimation should be used if patients with gi tumors are evaluated for personalized treatment options . of 96 patients with advanced gi tumors , mtb treatment recommendations were given for 41 ( 43% ) , which means an additional therapy option beyond established treatment lines for 4 out of 10 patients . 
14 months in our cohort , compared with patients with pd as best response ( fig 4d ) , is remarkable ; however , it has to be interpreted with caution and should be conrmed in larger patient populations with gi or other cancers . 
if this observation could be conrmed , 1 goal in the constant improvement of mtb recommendations should be to gradually increase the percentage of patients who reach disease control of at least 3 months in advanced cancers . early cohorts with molecular - matched therapies have been reported for crc31 and btc.32 in the rst study , 68 out of 254 patients with advanced crc received selected matched therapies to kras / braf / pik3ca mutations , pten , or phosphorylated met expression . 
a subgroup with btc from the moscato - 01 trial reported molecular targets in 23 of 34 patients , with 18 receiving matched therapies ( 53% ) .32 the overall response rate and pfs of 6 months in that study were 33% and 37% , respectively . 
several techniques , such as transcriptome34 , 35 and epigenome36 analysis or wholegenome37 and single - cell sequencing , are expected to improve tumor characterization up front.38 such a constant evolution will require more specialists in these methods to join academic mtbs , which means a new era of terdisciplinary patient care to bridge the gap between a growing complexity in diagnostic procedures and clinicians at the bedside . 
 ( a ) progression - free survival ( pfs ) , dened as the time from start of an mtb - recommended treatment to radiographic progression or death , and ( b ) overall survival ( os ) , dened as the time from start of an mtbrecommended treatment to death as a result of any cause , in days . 
recent examples in crc are pd - l1 and ctla - 4 inhibition in msi - high cancers41 or the combination of a braf inhibitor , mek inhibitor , and antiepidermal growth factor receptor antibody in braf v600e mutated cancers.42 third , a reduction in dropout rates might be achieved with more focused and earlier patient selection for ngs - based diagnostic procedures and a shortening of time intervals to get access to suggested drugs . 
fourth , each patient who is treated according to an mtb recommendation outside clinical trials should be regarded as an n - of - 1 trial.43 this implies a thorough documentation of adverse events and a reliable response assessment along a predened routine . in conclusion , gi tumors are an important group in the eld of personalized medicine . 
malek provision of study material or patients : michael bitzer , falko fend collection and assembly of data : michael bitzer , leonie ostermann , marius horger , saskia biskup , kristina ruhm , oznur oner , christopher schroeder , olaf riess , falko fend , daniel zips , martina hinterleitner , lars zender , janina beha data analysis and interpretation : michael bitzer , leonie ostermann , marius horger , saskia biskup , martin schulze , franz hilke , konstantin nikolaou , christopher schroeder , olaf riess , daniel zips , martina hinterleitner , lars zender , janina beha , nisar p . 
 bitzer et al falko fend consulting or advisory role : roche , eusa pharma daniel zips research funding : elekta ( inst ) , siemens ( inst ) , sennewald ( inst ) travel , accommodations , expenses : elekta ( inst ) martina hinterleitner travel , accommodations , expenses : novartis / ipsen lars zender leadership : heparegenix gmbh consulting or advisory role : boehringer ingelheim research funding : heparegenix gmbh patents , royalties , other intellectual property : patent on mkk4 inhibition for the treatment of acute and chronic liver diseases travel , accommodations , expenses : ipsen ghazaleh tabatabai honoraria : abbvie , bayer , medac , novocure ( inst ) consulting or advisory role : abbvie , bayer travel , accommodations , expenses : novocure ( inst ) nisar p . 
hoefin r , geiler a - l , fritsch r , et al : personalized clinical decision making through implementation of a molecular tumor board : a german single - center experience . 
j clin oncol 33 : 2753 - 2762 , 2015 bryce ah , egan jb , borad mj , et al : experience with precision genomics and tumor board , indicates frequent target identication , but barriers to delivery . oncotarget 8 : 27145 - 27154 , 2017 zehir a , benayed r , shah rh , et al : mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10 , 000 patients . 
nat med 23 : 703 - 713 , 2017 [ erratum : nat med , 2017 ] jones s , anagnostou v , lytle k , et al : personalized genomic analyses for cancer mutation discovery and interpretation . 
sci transl med 7 : 283ra53 , 2015 cheng dt , prasad m , chekaluk y , et al : comprehensive detection of germline variants by msk - impact , a clinical diagnostic platform for solid tumor molecular oncology and concurrent cancer predisposition testing . 
eur j cancer 45 : 228 - 247 , 2009 seymour l , bogaerts j , perrone a , et al : irecist : guidelines for response criteria for use in trials testing immunotherapeutics . 
plon se , eccles dm , easton d , et al : sequence variant classication and reporting : recommendations for improving the interpretation of cancer susceptibility j hepatol 66 : 1166 - 1172 , 2017 genetic test results . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
n engl j med 377 : 2500 - 2501 , 2017 jenkins rw , thummalapalli r , carter j , et al : molecular and genomic determinants of response to immune checkpoint inhibition in cancer . 
fabrizio da , george tj jr , dunne rf , et al : beyond microsatellite testing : assessment of tumor mutational burden identies subsets of colorectal cancer who may respond to immune checkpoint inhibition . 
verlingue l , malka d , allorant a , et al : precision medicine for patients with advanced biliary tract cancers : an effective strategy within the prospective moscato - 01 trial . 
overman mj , lonardi s , wong kym , et al : durable clinical benet with nivolumab plus ipilimumab in dna mismatch repair - decient / microsatellite instabilityhigh metastatic colorectal cancer . 
 duality of purpose : participant and parent understanding of the purpose of genomic tumor profiling research among children and young adults with solid tumors purpose increasing use of genomic tumor profiling may blur the line between research and clinical care . 
we aimed to describe perspectives of research participants about the purpose of genomic tumor profiling research in pediatric oncology . methods we surveyed 45 participants ( response rate , 85% ) in a pilot study of genomic profiling in pediatric solid tumors at four academic cancer centers after the return of sequencing results . 
we defined understanding according to a one - item ( basic ) definition ( recognition that the primary purpose was not to improve the patients treatment ) and a four - item ( comprehensive ) definition ( primary purpose was not to improve patients treatment ; primary purpose was to improve treatment of future patients ; there may not be direct medical benefit ; most likely result of participation was not increased likelihood of cure )  . results sixty - eight percent of respondents ( 30 of 44 respondents ) demonstrated basic understanding of the study purpose ; 55% ( 24 of 44 respondents ) demonstrated comprehensive understanding . 
ninety - three percent of respondents who believed the primary purpose was to improve the patients care simultaneously stated that the research also aimed to benefit future patients . conclusion most participants in pediatric tumor profiling research understand that the primary goal of this research is to improve care for future patients , but many express dual goals when they participate in sequencing research . 
2019 by american society of clinical oncology introduction parents of children with cancer1 , 2 and adults with cancer3 - 5 often fail to understand the purpose of clinical trials in which they participate . 
understanding the distinction between the goals of research and clinical care is of particular importance in early - phase oncology trials , in which response rates approximate 10%.6 , 7 up to 60% of research participants demonstrate evidence of therapeutic misconception , 3 , 4 , 8 , 9 the belief that the primary purpose of research is therapeutic in nature rather than acquisition of generalizable knowledge.10 , 11 the precision medicine era invites new exploration of these findings . 
 among adults16 , 17 and children.17 - 20 although advances in targeted therapeutics generate great excitement , they may also blur the line between research and clinical care.21 , 22 young adult patients and parents of children with cancer have high hopes / expectations for tumor sequencing , 23 , 24 though only a minority experience clinical benefit.25 - 29 this mirrors findings among adult patients with cancer30 - 33 and highlights the need for a deeper understanding of the tumor profiling consent process . 
furthermore , many participants simultaneously identified dual purposes for genomic tumor profiling research in pediatric oncology . relevance consenting clinicians should query and explore participant goals during presequencing counseling to identify both ( 1 ) those who do not recognize that the primary purpose of research sequencing is to generate knowledge to help future patients and ( 2 ) those who report dual purposes for this research . 
additional work is necessary to better understand the perspectives and motivations of those who express this duality and to develop and test interventions to improve equitable understanding of the purpose of genomic tumor profiling research in pediatric oncology . methods we surveyed consenting participants in the icat ( individualized cancer therapy ) pilot study of genomic profiling in children with relapsed , recurrent , and high - risk solid tumors ( clinicaltrials.gov identifier : nct01853345 ) .25 participants were approached at dana - farber / boston childrens cancer and blood disorders center ( boston , ma ) , university of california at san francisco ( san francisco , ca ) , columbia university medical center ( new york , ny ) , and childrens national medical center ( washington , dc )  . 
the study was approved by the institutional review board of all participating institutions . individualized cancer therapy study icat study procedures have been reported previously.25 all patients receiving care at participating institutions were eligible for enrollment if they were age 30 years or younger at enrollment and had a recurrent , refractory , or high - risk ( expected likelihood of cure < 25% ) extracranial solid tumor with sufficient tumor for submission . 
the study consent document described the purpose of the study as follows : to determine how often the panel of experts can [ use tumor sequencing results to ] make an individual treatment recommendation and to use this information to help future patients with cancer . 
 consent discussions were not standardized , nor were data collected on the content of these discussions . enrolled participants underwent tumor profiling via targeted next - generation sequencing and copy number assessment or a sequenom assay . 
survey items for assessment of participant understanding of the purpose of genomic profiling research in pediatric oncology question stem answer choice agree unsure * disagree * the main reason this study was done was to improve the treatment of myself / my child . the main reason this study was done was to improve the treatment of future patients with cancer . there may not have been direct medical benefit to me / my child from participation . what of the following did you think was most likely to happen because of your participation in this research study ? agree * unsure * disagree agree * unsure * disagree i / my child would have a better chance of being cured . doing this testing would give me peace of mind . * doctors would be better able to find cures for future patients . * doctors would be able to learn more about my / my childs cancer . * i / my child would have a greater number of treatment options . * nothing was likely to happen as a result of this research . * i would learn about my / my childs genes . * i would learn about my familys genes . * other * alterations for which a matched targeted therapy was available via clinical trial or us food and drug administrationapproved medication ; the recommendation described actionable alteration ( s ) found and strength of evidence for each treatment recommendation . study population icat participants were offered a self - administered written survey after return of study results to the patients oncologist . 
surveys were offered in english to the consenting individual : the patient if he / she was age 18 years or older at enrollment , or the patients parent / guardian if the patient was younger than age 18 years at enrollment . 
 surveys were not offered if the patient died between the time of enrollment and approach by the study team ( n = 41 ) ; the patient / parent did not understand english sufficiently to complete the survey ( n = 3 ) ; the patient / parent declined additional contact from study investigators after enrollment ( n = 0 ) ; and / or the oncologist did not permit approach by the study team ( n = 4 )  . survey methods survey procedures have been reported previously.24 surveys consisted of 103 items and included scales that addressed participant understanding of the purpose of clinical research , 34 genetic knowledge , 35 and the short form - 36 general health perceptions question . 
secondary outcomes were participant - level predictors of understanding ( demographic characteristics , genetic knowledge , experience with genetics , clinical status , receipt of icat recommendation / targeted therapy )  . 
participants were enrolled between september 2012 and november 2013 ; surveys were administered between september 2014 and july 2015 . participant understanding of the purpose of research sequencing we assessed participant understanding with four independent items ( table 1 )  . 
three items were adapted from the quality of informed consent measurea validated measure to assess understanding of the purpose of oncology clinical trials , 34 by adult patients with cancer and further validated in parents of children with cancer1 ; answer choices were agree , unsure , and disagree.34 the fourth item offered respondents multiple choices about their perceived most likely result of study participation . participants were asked how well they understood conversation ( s ) they had with the doctor about the icat study and the testing involved in it , and responses were collected on a 5 - point likert scale ( responses of extremely well , well , moderately , poorly , extremely poorly )  . 
 to measure patient knowledge about genetics / genomics.24 , 36 , 37 respondents were asked if they had regular exposure to genetics and / or genetic information through their job and if they had ever attended any classes / lectures on genes / genetics . statistical methods understanding of the purpose of the study was defined in two ways . 
 comprehensive understanding was defined as an understanding of all four of the following : ( 1 ) the primary purpose was not to improve their / their childs treatment ; ( 2 ) the primary purpose was to improve treatment of future patients with cancer ; ( 3 ) there may not have been direct medical benefit to them / their child ; and ( 4 ) the most likely result of participation was not an increased likelihood of cure for themselves / their child . 
for example , if a participant identified that the primary purpose of the study was not to improve her childs treatment , he or she demonstrated basic understanding of the studys purpose . 
to be as inclusive as possible , and because of the complexity and uncertainty inherent in tumor profiling research , responses of unsure to any of the first three items were coded as consistent with understanding . 
for the fourth item , only responses that the most likely result of participation in the study was cure were coded as inconsistent with understanding ; all other responses , including answers of other , were coded as understanding . 
missing responses to any of the four understanding items were excluded from analysis of comprehensive understanding ; only those that were missing the first item were excluded from analysis of basic understanding . self - report of degree of understanding of the consent conversation ( s ) was dichotomized as well or extremely well ( coded as good self reported understanding ) versus all others . 
those who answered extremely true or very true to the item about how helpful participation was to them / their child were coded as feeling the study to have been helpful , and those who answered with the other answer choices were coded as feeling it was not . experience with genetics was defined as an affirmative response to questions of regular exposure to genetics or experience with genetics / genetic information and / or enrollment in any classes / lectures on genes or genetics . 
high genetic knowledge was defined as correct answers of all four items from the gki.35 those who answered fewer than four gki items correctly were coded as having low genetic knowledge . respondent demographics and clinical characteristics and understanding of results and the purpose of testing were evaluated using descriptive statistics . 
item nonresponse was less than 10% , and participants with nonresponse to an item were excluded from analyses of that ite all analyses were performed using stata , version 13.1 ( statacorp , college station , tx )  . results respondent characteristics of 101 participants who underwent profiling on the icat study , 53 were eligible for survey administration . 
characteristics of survey respondents are listed in table 2 for the overall cohort and are subdivided into patient ( n = 11 ; 24% ) and parent / guardian ( n = 34 ; 76% ) respondents . 
participant and patient demographics , overall and separately , according to whether the survey was completed by the patients parent / guardian or by the patient himself or herself overall ( n = 45 ) patient respondents ( n = 11 ) no . 
participant and patient demographics , overall and separately , according to whether the survey was completed by the patients parent / guardian or by the patient himself or herself ( continued ) overall ( n = 45 ) patient respondents ( n = 11 ) no . 
sixty - eight percent of respondents ( 30 of 44 respondents ) recognized that the primary reason the study was performed was not to improve the treatment of them / their child , which met our definition of basic understanding of the purpose of the study . 
no significant differences were seen according to respondent age , sex , or race / ethnicity ; according to self - reported health status or likelihood of cure , receipt of an icat treatment recommendation or matched targeted therapy ; or according to participant - identified understanding of what they were told about the study . 
 results were similar when responses of unsure were excluded from analysis ( appendix fig a1 )  . similar results were seen with understanding defined by the composite four - item scale . 
comprehensive understanding of the purpose of genomic profiling research was seen with statistically greater frequency among those with at least a college education ( 73% v 28% ; p = .01 ) and with higher genetic knowledge ( 71% v 23% ; p = .01 ) and among white / non - hispanic respondents ( 68% v 37% ; p = .07 ) , though the comparison by race / ethnicity was not statistically significant . 
similarly , no statistical difference in understanding was seen according to receipt of an icat treatment recommendation or matched targeted therapy or according to whether the respondent reported a good understanding of what they were told about the study / testing . 
among those who mistakenly identified the primary purpose as improvement of their / their childs treatment , 93% ( 13 of 14 respondents ) simultaneously recognized that it aimed to benefit future patients . 
twenty - eight percent ( 12 of 43 respondents ) of those who identified that the primary purpose was to benefit future patients also reported that the primary purpose was to improve their / their childs treatment . discussion in this multi - institutional study that examined the role of molecular profiling of pediatric solid tumors , nearly all participants recognized that the primary purpose was to benefit future patients . 
 however , approximately one third of respondents believed that the primary purpose of the trial was to improve their / their childs treatment , and nearly one fifth expected participation to impart a greater chance of cure . although these responses raise concerns about the quality of informed consent for tumor fig 1 . 
relationship between participant demographics and understanding of purpose of research tumor profiling ( continued ) helpfulness of participating in the study not helpful to myself / my child helpful note . 
 ( % ) 17 ( 71 ) 7 ( 35 ) sequencing , they must be considered in context of a complex technology with an evolving role in clinical care . 
this duality is echoed by the american society of clinical oncology , which states that early - phase clinical trials in oncology simultaneously generate new knowledge and provide participants the opportunity for psychological and clinical benefit.22 , 38 oncologists often balance dual goals for patients by recommending enrollment in a phase i trial while hoping for patient benefit or by simultaneously providing palliative and cancer - directed ( ie , blended ) care.39 in the era of precision cancer medicine , it is reasonable that patients / families might perceive such dualities as well . this duality has important clinical implications . 
however , an initial approach could be to discuss the primary goal of the study as gaining new knowledge to help future patients , followed by an acknowledgment , tempered with realistic expectations , that many patients / parentsand many clinicianshold hope that the child will also benefit from participation . 
in the case of next - generation sequencing , for example , it is important to note that the number of patients who experience direct benefit via receipt of a targeted therapy is quite low , likely in the range of 3% to 19%.25 - 29 our results also underscore the importance of hope among patients and parents of children with cancer in clinical and research settings.40 - 42 hopeful thinking may partially explain why participants who felt the study had helped them / their child and those who were receiving cancer directed therapy at the time of survey completion less frequently demonstrated understanding of the purpose of research tumor sequencing . in this cohort , understanding was observed more frequently in those with at least a college education and those with good genetic knowledge . 
 this finding , also reported elsewhere , 23 is not surprising , given the complexity of these concepts and the expected link between understanding and health literacy / numeracy.43 understanding also varied according to race / ethnicity , consistent with similar work in the pediatric oncology phase i literature , 2 although the difference did not reach statistical significance in this pilot study . 
these disparities underscore the importance of attention to the needs of vulnerable populations when they are counseled about genomic results ; however , the optimal mechanism for such counseling remains unclear.44 prior work in pediatric oncology has identified that refinement of the consent process may improve understanding , 45 but optimal strategies to adequately convey the complexities of tumor sequencing and support fully informed consent for participation in sequencing research are not yet known . 
a follow - up study is in development to examine the benefit of such an intervention for those who demonstrate less than comprehensive understanding , as defined in this cohort . 
patients / parents may have better understood the purpose of profiling closer to the time of consent , but understanding did not vary statistically with time to survey completion in this cohort . 
many study participants died before surveys could be administered ; however , demographic and clinical characteristics of respondents mirrored those of the overall cohort.25 finally , participants were enrolled at four large academic centers , so results may not be generalizable to those from smaller and / or community centers . 
this could , for example , explain the unexpectedly high genetic knowledge and experience seen in this cohort . although some participants misidentified the primary goal of tumor profiling research as therapeutic in nature , participant views are nuanced . 
dubois honoraria : loxo consulting or advisory role : loxo research funding : millennium ( inst ) , merck ( inst ) , novartis ( inst ) , roche ( inst ) , genentech ( inst ) , lilly ( inst ) , curis ( inst ) , loxo ( inst ) travel , accommodations , expenses : loxo , roche , genentech julia glade - bender research funding : bristol - myers squibb ( inst ) , pfizer ( inst ) , novartis ( inst ) , ignyta ( inst ) , amgen ( inst ) , celgene ( inst ) , eisai ( inst ) , lilly ( inst ) , merck ( inst ) travel , accommodations , expenses : novartis , amgen , merck aerang kim no relationship to disclose brian d . 
weinfurt kp , seils dm , lin l , et al : research participants high expectations of benefit in earlyphase oncology trials : are we asking the right question ? j clin oncol 30 : 4396 - 4400 , 2012 6 . 
italiano a , massard c , bahleda r , et al : treatment outcome and survival in participants of phase i oncology trials carried out from 2003 to 2006 at institut gustave roussy . 
druker bj , sawyers cl , kantarjian h , et al : activity of a specific inhibitor of the bcr - abl tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the philadelphia chromosome . 
kantarjian hm , shah np , cortes je , et al : dasatinib or imatinib in newly diagnosed chronicphase chronic myeloid leukemia : 2 - year follow - up from a randomized phase 3 trial ( dasision )  . 
wagle n , grabiner bc , van allen em , et al : activating mtor mutations in a patient with an extraordinary response on a phase i trial of everolimus and pazopanib . 
kushner bh , cheung nv , modak s , et al : a phase i / ib trial targeting the pi3k / akt pathway using perifosine : long - term progression - free survival of patients with resistant neuroblastoma . 
laetsch tw , dubois sg , mascarenhas l , et al : larotrectinib for paediatric solid tumours harbouring ntrk gene fusions : phase 1 results from a multicentre , open - label , phase 1 / 2 study . 
weber js , levit la , adamson pc , et al : reaffirming and clarifying the american society of clinical oncologys policy statement on the critical role of phase i trials in cancer research and treatment . 
oberg ja , ruiz j , ali - shaw t , et al : whole - genome and whole - exome sequencing in pediatric oncology : an assessment of parent and young adult patient knowledge , attitudes , and expectations . 
marron jm , dubois sg , glade bender j , et al : patient / parent perspectives on genomic tumor profiling of pediatric solid tumors : the individualized cancer therapy ( icat ) experience . 
harris mh , dubois sg , glade bender jl , et al : multicenter feasibility study of tumor molecular profiling to inform therapeutic decisions in advanced pediatric solid tumors : the individualized cancer therapy ( icat ) study . 
chang w , brohl as , patidar r , et al : multidimensional clinomics for precision therapy of children and adolescent young adults with relapsed and refractory cancer : a report from the center for cancer research . 
harttrampf ac , lacroix l , deloger m , et al : molecular screening for cancer treatment optimization ( moscato - 01 ) in pediatric patients : a single - institutional prospective molecular stratification trial . 
miller fa , hayeems rz , bytautas jp , et al : testing personalized medicine : patient and physician expectations of next - generation genomic sequencing in late - stage cancer care . 
gray sw , park er , najita j , et al : oncologists and cancer patients views on whole - exome sequencing and incidental findings : results from the canseq study . 
carere da , couper mp , crawford sd , et al : design , methods , and participant characteristics of the impact of personal genomics ( pgen ) study , a prospective cohort study of direct - to - consumer personal genomic testing customers . 
weber js , levit la , adamson pc , et al : american society of clinical oncology policy statement update : the critical role of phase i trials in cancer research and treatment . 
ferrell br , temel js , temin s , et al : integration of palliative care into standard oncology care : american society of clinical oncology clinical practice guideline update . 
sensitivity analyses for the relationship between participant demographics and understanding of the purpose of research tumor profiling basic understanding ( n = 33 ) comprehensive understanding ( n = 26 ) variable survey respondent characteristic no . 
 ( % ) respondent attitudes about icat study understanding of icat information poor self - reported understanding good self - reported understanding helpfulness of participating in icat study not helpful to myself / my child helpful basic understanding ( n = 33 ) comprehensive understanding ( n = 26 ) 10 ( 77 ) 9 ( 45 ) 11 ( 69 ) 8 ( 47 ) 8 ( 80 ) 8 ( 50 ) 10 ( 77 ) 6 ( 46 ) note . 
 erbb3 - activating mutations in small bowel adenocarcinomas purpose functional studies have demonstrated that some mutations of erbb3 , which encodes for human epidermal growth factor receptor ( her ) 3 , are oncogenic via activation of the erbb family signaling pathway . 
this study was designed to report the rate of activating erbb3 mutations in small bowel adenocarcinoma ( sba ) , a rare tumor type in which we previously reported a high rate ( 12% ) of erbb2 - activating mutations . materials and methods dna from 74 sbas , previously characterized for erbb2 mutations and mismatch repair status , was submitted for sequencing of erbb3 exons 3 , 6 , 7 , 8 , and 23 . 
mutant allelic frequencies suggested that both mutations were shared by the same clone rather than being harbored by mutually exclusive tumor subclones . conclusion sbas display a high rate of erbb3 - activating mutations , which have been shown to be targetable by anti - her2 therapies . 
2018 by american society of clinical oncology human epidermal growth factor receptor ( her ) 3 is a tyrosine kinase transmembrane receptor and a member of the her receptor family , which consists of four members : epidermal growth factor receptor ( egfr ) , her2 , her3 , and her4 , which are encoded by the egfr , erbb2 , erbb3 , and erbb4 genes , respectively . 
 unlike other members of this family , her3 has an impaired intracellular kinase domain1 and must heterodimerize with egfr , her2 , or her4 to initiate an intracellular signal , which activates downstream survival signaling such as the phosphoinositide 3 - kinase ( pik3ca ) pathway.2 in 2013 , a first report established that erbb3 displays recurrent hotspot mutations in several cancers , especially in colorectal , gastric , 3 and lobular breast cancer4 ; the most frequent amino acid change ( p.v104m / l ) is related to a single nucleotide variation hotspot located in exon 3 . 
sba is usually treated the same way as colorectal cancer , but few studies have been devoted to this cancer type.8 a first report from our team addressing the specific genomic landscape of sba was published by laforest et al , 9 in which 83 sba samples were subjected to targeted panel sequencing ( 46 genes , ion ampliseq cancer hotspot panel v2 )  . 
this study , which was performed before the discovery of erbb3 activating mutations , reported eight genes that were mutated in more than 5% of sba cases : kras , tp53 , apc , smad4 , pik3ca , erbb2 , braf , and fbxw7 . 
errb2 alterations were highly prevalent : 10 tumors ( 12% ) presented either erbb2 - activating point mutations ( n = 7 ) or erbb2 amplifications ( n = 3 )  . 
2 ethics committee under id - rcb number 2008a01058 - 47 . sample preparation tumor samples were collected as described elsewhere.9 briefly , using a 1.0 - mm punch , paraffin - embedded tissues from all cases were sampled from representative areas containing more than 50% of tumor cells . 
sequencing products were purified with sephadex gel ( ge healthcare , vlizy villacoublay , france ) before running them on a 3500 genetic analyzer ( applied biosystems , foster city , ca )  . 
sequencing data were visualized using finch tv ( geospiza , seattle , wa ) with a detection sensitivity of 10% mutated cells . next - generation sequencing ( ngs ) targeted ngs ( a panel of 39 genes ) was performed on samples with co - occurrence of erbb2 and erbb3 . 
of patients ( % ) 74 ( 100 ) median age in years , range 62 years , 32 - 98 male female tumor stage ( tnm classification ) primary tumor site duodenum jejunum ileum dmmr status msi - h histologic grading 40 ( 54 ) 34 ( 46 ) 1 ( 1 ) 25 ( 34 ) 27 ( 37 ) 19 ( 25 ) 2 ( 3 ) 36 ( 49 ) 26 ( 35 ) 12 ( 16 ) 17 ( 23 ) 57 ( 77 ) 20 ( 27 ) 23 ( 31 ) 30 ( 41 ) 1 ( 1 ) abbreviations : dmmr , mismatch repairdeficient ; msi , microsatellite instability ; mss , microsatellite stable ; na , not available raw reads were aligned on the reference human genome hg19 using the tmap aligner v0.3.7 ( life technologies )  . 
only studies with more than 100 patients and an erbb2 / erbb3 mutation rate higher than 5% ( including putative driver or passenger mutations and at least one erbb3 driver mutation ) were taken into account , with the exception of the mutation rate in the genie cohort . 
after combining erbb3 data with the previous genotyping data , a significant correlation was observed with erbb2 mutation three of the four erbb3 - mutated sba samples also displayed an erbb2 - activating mutation . 
 because the activation of two oncogenes in the same pathway is unusual , this finding was validated by subjecting two samples with sufficient dna to another ngs assay targeting both erbb2 and erbb3 to investigate whether these mutations were restricted to tumor subclones . 
in addition to confirming that erbb2 and erbb3 mutations co - occurred in the same tumor , mafs suggested a clonal distribution , ie , that both mutations were indeed harbored by the same tumor cells . erbb3 mutations , mismatch repair ( mmr ) status , and erbb2 mutations in other tumor types we investigated whether the correlation among erbb3 - activating mutations , erbb2 mutations , and msi - h status was also observed in other tumor types by performing in silico analysis of publicly available data . 
 ( * ) p < .01 between msi - h and microsatellite stable ( mss ) cancers . in 1% to 3% of the following cancer types : lobular breast , gastric , colorectal , endometrial , and cervix adenocarcinoma , together with cholangiocarcinoma and bladder cancer.3 , 4 , 11 - 18 this spectrum of erbb3 - mutated cancers overlaps with that of erbb2 - mutated and erbb2 amplified cancers , supporting the biologic concept of her2 - driven cancer because erbb3 activating mutations act via her2 signaling . 
 because sba is the most common form of erbb2 - mutated cancer ( as initially reported by laforest et al9 ) , we hypothesized that erbb3 sequencing would detect a high rate of erbb3 mutations . 
in more detail , three of the four mutated sba samples harbored the most frequent hotspot mutation of erbb3 ( v104m ) , whereas one sample harbored the e928g mutation , which has been mainly described in lobular breast adenocarcinoma18 and found in only one case in the two largest series of colorectal cancer sequencing.17 , 19 a striking finding of this study was the significant co - occurrence of erbb2 and erbb3 mutations across tumor samples . 
oncogenic mutations that activate the same signaling pathway are usually distributed according to a mutually exclusive mode in a given cancer type , eg , kras , nras , and braf mutations in colorectal cancers.17 in breast cancer , the study of heterogeneous erbb2 - amplified tumors ( a rare phenotype ) has shown that erbb2 - amplified subclones coexisted with her2 - mutated subclones.20 however , the ngs targeted analysis performed in the current study on two of the three cases with both erbb3 and erbb2 mutations did not demonstrate any significant differences between their allelic frequencies . 
the three sba samples with both erbb2 and erbb3 mutations exhibited microsatellite instability and likely had a high mutational load , 21 suggesting a possible causality that has yet to be investigated . more recently , a study performed genomic profiling of 317 sba cases , using colorectal and gastric cancers as controls.22 the reported overall erbb3 mutation rate was 6.3% , similar to that observed in our study , and was significantly higher than that observed in colorectal ( p = .001 ) and gastric ( p = .02 ) cancers in their cohort . 
this previous report provided no data regarding erbb3 and erbb2 mutation co - occurrence or the association between erbb3 mutation and msi - h . in 2017 , the food and drug administration granted accelerated approval to an immune checkpoint inhibitor ( pembrolizumab , an anti pd - 1 antibody ) for the treatment of patients with unresectable or metastatic msi - h solid tumors , regardless of the histology , on the basis of the impressive results observed in these tumors.23 for patients with concomitant msi - h tumors and erbb2 / 3 mutations , anti - her2 and antipd - 1 drugs can therefore both be considered as treatment options . 
in view of the clearly demonstrated benefit of antipd - 1 drugs in this setting , 23 , 24 which can achieve long - lasting complete responses , although anti - her2 / 3 drugs are not currently approved in this setting , anti pd - 1 drugs should probably be preferred . 
studies testing the toxicity and efficacy of immune checkpoint inhibitors and anti - her2 / 3 targeted therapies could be particularly relevant for msi - h erbb2 / 3 - mutated cancers . finally , we acknowledge that our study has several limitations . 
these include the limited sample size resulted in large cis for estimation of mutation rates , the lack of fresh frozen material prevented the performance of more comprehensive whole - exome sequencing , and , in cases with dual erbb2 / erbb3 mutations , the absence of single cell sequencing did not allow the demonstration of epistatic interactions between erbb2 and erbb3 mutations . 
hyman dm , piha - paul sa , rodon j , et al : abstract ct001 : neratinib in her2 or her3 mutant solid tumors : summit , a global , multi - histology , open - label , phase 2 basket study . 
pereira b , chin s - f , rueda om , et al : the somatic mutation profiles of 2 , 433 breast cancers refines their genomic and transcriptomic landscapes . 
ng cky , martelotto lg , gauthier a , et al : intra - tumor genetic heterogeneity and alternative driver genetic alterations in breast cancers with heterogeneous her2 gene amplification . 
 graft - versus - leukemia effect of allogeneic stem - cell transplantation and minimal residual disease in patients with acute myeloid leukemia in first complete remission purpose the detection of minimal residual disease ( mrd ) in patients with acute myeloid leukemia ( aml ) may improve future risk - adapted treatment strategies . 
we assessed whether mrd - positive and mrd - negative patients with aml benefit differently from the graft - versusleukemia effect of allogeneic hematopoietic stem - cell transplantation ( allohsct )  . methods a total of 1 , 511 patients were treated in subsequent dutch - belgian hemato - oncology cooperative group and the swiss group for clinical cancer research aml trials , of whom 547 obtained a first complete remission , received postremission treatment ( prt ) , and had available flow cytometric mrd before prt . 
prt consisted of allohsct ( n = 282 ) , conventional prt by a third cycle of chemotherapy ( n = 160 ) , or autologous hematopoietic stem - cell transplantation ( n = 105 )  . results mrd was positive in 129 patients ( 24% ) after induction chemotherapy before proceeding to prt . 
2017 by american society of clinical oncology introduction acute myeloid leukemia ( aml ) is a heterogeneous malignancy characterized by a variety of underlying cytogenetic and molecular aberrations , which are associated with distinct prognostic features.1 although current treatment approaches induce high percentages of hematologic remission , relapse rates are high and vary according to the underlying risk profile.2 recently , the european leukemianet ( eln ) developed an updated classification on the basis of cytogenetic and molecular aberrancies distinguishing patients with a favorable , intermediate , jurjen versluis burak kalin wendelien zeijlemaker jakob passweg carlos graux markus g . 
therefore , we evaluated whether and to what extent allohsct quantitatively reduces relapse compared with conventional prt in upfront - treated patients with mrd - positive or mrd - negative aml in first cr ( cr1 )  . assessed for eligibility hovon - sakk aml42a hovon - sakk aml92 hovon - sakk aml102 ( n = 1 , 511 ) ( n = 511 ) ( n = 142 ) ( n = 858 ) excluded obtained no cr received no prt no available mrd ( n = 964 ) ( n = 255 ) ( n = 234 ) ( n = 475 ) patients eligible for analysis ( n = 547 ) mrd negative ct auto allo ( n = 418 ) ( n = 118 ) ( n = 89 ) ( n = 211 ) mrd positive ct auto allo ( n = 129 ) ( n = 42 ) ( n = 16 ) ( n = 71 ) methods patients patients participated in three prospective , consecutive dutch - belgian hemato - oncology cooperative group and the swiss group for clinical cancer research ( hovon - sakk ) collaborative group trials ( aml42 , aml92 , and aml102 ; the netherlands trial register numbers ntr230 , ntr1446 , and nrt2187 , respectively ) , for whom assessment of mrd after induction therapy and before prt by allohsct , chemotherapy , or autologous hsct ( autohsct ) was performed.25 , 26 the results of the aml92 trial have not yet been published , but trial information is available in the netherlands trial register ( ntr1446 )  . 
patients were excluded if cr1 did not occur after two induction cycles of chemotherapy ( n = 255 ; 17% ) or the patient did not receive prt after obtaining cr1 ( n = 234 ; 15% )  . 
in addition , a total of 475 patients ( 31% ) received prt in cr1 ; however , their mrd status was not available within a time window of 4 months before prt . 
patients were classified by aml prognostic risk on the basis of the cytogenetic and molecular profile of the underlying aml , according to the eln2017 risk classification.3 molecular analysis was available for the majority of patients , specifically for npm1 ( 93% ) ; fms - like tyrosine kinase 3 internal tandem duplication ( flt3 - itd ; 91% ) , including the flt3 - itd mutant to wild - type ratio ( 86% ) ; evi1 ( 79% ) ; asxl1 ( 83% ) ; runx1 ( 47% ) ; and tp53 ( 47% )  . 
patient characteristics mrd negative ( n = 418 ) mrd positive ( n = 129 ) wbc count at diagnosis , 109 / l cytogenetics of aml cytogenetic abnormalities monosomal karyotype characteristic male female age , years median range < 100  . 
prt included a third cycle of chemotherapy with mitoxantrone and etoposide , high - dose chemotherapy with busulfan and cyclophosphamide followed by autohsct , or allohsct after either myeloablative conditioning or reduced intensity conditioning ( ric )  . 
the myeloablative conditioning regimen contained high - dose cyclophosphamide with at least 8 gy total body irradiation ( tbi ) in 83 patients ( 76% ) ; the remainder of the patients received busulfan with cyclophosphamide . 
although ric regimens varied , the majority contained low - dose ( 2 or 4 gy ) tbi preceded by fludarabine ( n = 126 ; 77% ) , whereas 23% of the patients received fludarabine with busulfan . 
these different prt modalities were applied according to a riskadapted strategy : patients with aml classified as favorable risk , according to cytogenetic and molecular analysis , were planned for a third cycle of chemotherapy ; intermediate - risk patients were preferentially treated with allohsct using an hla - matched sibling donor or a fully hlamatched unrelated donor , if available ; patients with adverse - risk aml proceeded to allohsct using a sibling donor , an unrelated donor , or cord blood grafts ; and patients alternatively received autohsct or a third cycle of chemotherapy if no suitable donor was available.25 - 28 mrd detection and sample selection mrd flow cytometric analysis was performed in a two - step procedure , as previously described.18 in summary , the immunophenotype was determined on blasts defined by cd45 expression with a low sideward scatter . 
mrd percentage was defined as the percentage of laip cells within the wbc compartment multiplied by the correction factor ( 100% divided by the percentage of laippositive blasts at diagnosis )  . 
patient characteristics ( continued ) characteristic year of prt median range mrd negative ( n = 418 ) mrd positive ( n = 129 ) p = .11 2011 2006 - 2014 2010 2006 - 2014 abbreviations : aml , acute myeloid leukemia ; cn - x - y , cytogenetically normal or only loss of x or y chromosome ; cr , complete remission ; eln2017 , european leukemianet 2017 ; hsct , hematopoietic stem - cell transplantation ; iqr , interquartile range ; flt3 - itd , fms - like tyrosine kinase 3 internal tandem duplication ; mrd , minimal residual disease ; npm1 , nucleophosmin - 1 ; prt , postremission treatment . * the cutoff of the flt3 - itd ratio is defined as 0.50. number of induction courses ( ie , i or ii ) was performed to allow the baseline hazard to differ between these two patient groups . 
all analyses were performed with stata software ( release 13.1 ; stata corporation , college station , tx )  . results patient characteristics a total of 547 patients with aml in cr1 and available mrd status proceeded to prt with either allohsct ( n = 282 ) , chemotherapy ( n = 160 ) , or autohsct ( n = 105 )  . 
patients with mutated npm1 were more frequently mrd negative , whereas mrd - positive patients more frequently tended to obtain a late cr1 ( ie , after induction cycle 2 )  . 
the eln2017 risk classification was similarly distributed among the mrd - negative and mrd - positive patients . interestingly , the time interval from cr1 to prt for patients with mrd - positive aml was shorter compared with their negative counterparts , which was mainly apparent in favorablerisk patients with aml . 
no other differences with regard to donor source , conditioning , cytomegalovirus serostatus , and european group for blood and marrow transplantation ( ebmt ) risk score were apparent between mrd - negative and mrdpositive patients . 
patients with a high risk of nrm according to the ebmt risk score32 ( > 3 points ) represented 45% of the patients who underwent transplantation . treatment outcome os and rfs were significantly better in patients without mrd before prt compared with mrdpositive patients ( 65% 6 2% v 50% 6 5% at 4 years ; p = .002 ; and 58% 6 3% v 38% 6 4% ; p , .001 , respectively ; figs 2a and 2b )  . 
the sample with the shortest time interval between prt and the date of collection was selected for analysis . end points the primary end point of the study was the cumulative incidence of relapse . 
the cumulative risks of relapse and nrm over time were calculated as competing risks with actuarial methods , where patients alive in continuing cr1 were censored at the date of last contact . statistical methods a time - dependent analysis of prt was performed as described previously28 , 29 by applying multivariable cox regression with allohsct as the timedependent covariable . 
in both the multivariable analysis and the estimation of the survival curves , these patients were counted as at risk in the chemotherapy group from the start of prt until allohsct and after that as at risk in the allohsct group . 
more detailed outcome estimates according to mrd status , type of prt , and risk of nrm on the basis of the ebmt risk score are presented in the data supplement . the cumulative incidence of relapse was significantly lower in patients without mrd receiving allohsct compared with chemotherapy or autohsct ( 26% 6 3% v 38% 6 3% at 4 years ; p = .027 , respectively ; fig 3a )  . 
the cumulative incidence of relapse in mrd - positive patients is estimated to be 45% 6 6% compared with 66% 6 6% at 4 years ( p = .058 ) for recipients of allohsct compared with recipients of chemotherapy or autohsct , respectively ( fig 3b )  . 
rfs after allohsct proved similar compared with prt with chemotherapy or autohsct in patients without mrd before prt ( 58% 6 4% v 58% 6 4% at 4 years ; p = .99 , respectively ; fig 3c )  . 
rfs after allohsct in mrd - positive patients before prt was 44% 6 6% compared with 31% 6 6% at 4 years ( p = .20 ) after chemotherapy or autohsct , respectively ( fig 3d )  . 
only 46 of relapsing patients ( 22% ) proceeded to allohsct after obtaining a second cr . multivariable analysis the following variables significantly predicted for relapse in the univariable analysis : mrd status , type of prt , age , wbc category , flt3 - itd category , year of prt , time from diagnosis to cr , time from cr to prt , cytogenetics , number of cycles to cr , eln2017 risk classification , npm1 mutation , evi1 overexpression , and cebpa double mutation . 
after forward selection , the multivariable analysis was performed , stratified by the total number of induction courses with adjustment for mrd status , type of prt , age , wbc category at diagnosis , flt3 - itd , eln2017 risk classification , number of cycles to cr , and year of prt ( table 3 )  . 
different variables in the multivariable model were significantly associated with relapse , with the eln2017 risk classification , flt3 - itd mutant to wild - type ratio , number of cycles to reach cr , and type of prt being the most important variables . discussion the development of treatment approaches in patients with aml is increasingly personalized by using genetic and molecular leukemia characteristics at diagnosis and individual treatment response.3 - 6 response and especially mrd , detected by either multiparametric flow cytometry or quantitative polymerase chain reaction , has become an important parameter in a more precise treatment approach for patients with aml.10 - 24 currently , it is unknown whether and how the presence or absence of mrd should guide the application of allohsct as prt . 
recently , the quantitative detection of mutated npm1 has been shown to have high predictive value , and recommendations to tailor the application of allohsct by mrd were made.10 - 12 balsat et al10 suggested refraining from allohsct in npm1 mrdnegative patients and to selectively proceed to allohsct as prt in cr1 in mrd - positive patients or adverse - risk patients on the basis of karyotype or the presence of flt3 - itd . 
kaplan - meier estimates of the cumulative incidence ( ci ) of relapse in ( a ) minimal residual disease ( mrd ) negative patients ; ( b ) ci of relapse in mrdpositive patients ; ( c ) relapse - free survival ( rfs ) in mrd - negative patients ; and ( d ) rfs in mrdpositive patients by type of prt in patients with acute myeloid leukemia in first complete remission from start of prt . 
nevertheless , the advantages of personalized treatment are obvious and continuously being refined by updated and better methods of risk assessment . also , new technologies to better define mrd , such as quantification of leukemic stem - cell content , standardized protocols and antibody panels , and novel software possibilities , are emerging.45 - 47 a personalized approach including mrd identifies patients with a high risk of relapse who qualify for allohsct , but who might benefit from attempts to induce mrd negativity before transplantation . 
previously , a number of investigators reported that patients in cr1 with persistence of mrd before allohsct have worse survival compared with recipients of allohsct with an mrd - negative cr1.17 , 19 - 24 although allohsct is clearly indicated in mrd - positive patients , it is important to study the value of approaches intended to induce mrd negativity before allohsct . 
it has been suggested that continued chemotherapy with one or two consolidation cycles may not be the preferred strategy to obtain an mrd - negative cr before allohsct , 48 but several new drugs are currently being developed and evaluated for aml.49 possible other strategies may include efforts to improve allogeneic immunotherapy by early tapering of immunosuppression and / or preemptive donor lymphocyte infusions , which also could be guided by mrd . 
of events / patients allo ct / auto hr & 95% ci ( ct / auto : allo ) reduction ( sd ) mrd status ( cut - off 0.1 ) negative positive 93 / 207 40 / 58 90 / 211 40 / 71 total 133 / 265 ( 50% ) 130 / 282 ( 46% ) allo better ct / auto better 45% ( 8 ) reduction 2p < .001 collectively , our study shows that the gvl effect was strikingly similar in mrd - positive and mrdnegative patients . 
additional prospective studies are needed to evaluate whether the conversion of mrd positivity into an mrd - negative remission before allohsct further optimizes outcome , and how the gvl effect after allohsct can be optimized . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . jurjen versluis no relationship to disclose burak kalin no relationship to disclose wendelien zeijlemaker patents , royalties , other intellectual property : i have a patent for a laboratory test to identify specific leukemia cells that can be used in patients with acute myeloid leukemia . 
manz consulting or advisory role : amgen , novartis , roche , pfizer marie - christiane vekemans consulting or advisory role : amgen ( inst ) , celgene ( inst ) , bristol - myers squibb ( inst ) , janssen ( inst ) , takeda ( inst ) travel , accommodations , expenses : amgen , bristol - myers squibb , teva , janssen bart j . 
bargetzi no relationship to disclose juergen kuball employment : gadeta stock and other ownership interests : gadeta consulting or advisory role : gadeta , novartis ( inst ) speakers bureau : gadeta research funding : gadeta , miltenyi biotec , novartis patents , royalties , other intellectual property : multiple patents on gamma - delta t cell receptor sequences isolation strategies and targets in combination with gamma - delta t cell receptor sequences travel , accommodations , expenses : gadeta , novartis , miltenyi biotec harry c . 
van der velden consulting or advisory role : celgene ( inst ) research funding : bd biosciences ( inst ) patents , royalties , other intellectual property : patent for euroflow mrd antibody tubes . 
janssen honoraria : bristol - myers squibb netherlands , pfizer europe consulting or advisory role : pfizer europe , novartis research funding : novartis travel , accommodations , expenses : novartis thomas pabst no relationship to disclose bob lowenberg consulting or advisory role : astex pharmaceuticals , clear creek bio , agio pharmaceuticals , celgene mojca jongen - lavrencic no relationship to disclose gerrit jan schuurhuis research funding : becton dickinson ( inst ) gert ossenkoppele honoraria : roche consulting or advisory role : celgene , sunesis pharmaceuticals , janssen , novartis , cti , amgen , pfizer , roche research funding : amgen ( inst ) , janssen ( inst ) , celgene ( inst ) travel , accommodations , expenses : roche jan j . 
cornelissen no relationship to disclose acknowledgment following dutch - belgian hemato - oncology cooperative group and the swiss group for clinical cancer research ( hovon - sakk ) publication rules , coauthorship was offered to centers contributing the highest number of patients . nevertheless , the authors highly appreciate the contribution of many physicians and data managers throughout hovonsakk , who made this analysis possible . affiliations jurjen versluis , burak kalin , bob lowenberg , mojca jongen - lavrencic , and jan j . 
van der velden , erasmus university medical center , rotterdam ; wendelien zeijlemaker , jeroen j.w.m. janssen , gerrit jan schuurhuis , and gert ossenkoppele , vu university medical center ; bart j . 
wijermans , haga hospital , the hague ; mels hoogendoorn , medical center leeuwarden , leeuwarden ; juergen kuball , university medical center utrecht , utrecht ; harry c . schouten , maastricht university medical center , maastricht , the netherlands ; jakob passweg , university hospital basel , basel ; markus g . 
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 c crizotinib failure in a tpm4alkrearranged inflammatory myofibroblastic tumor with an emerging alk kinase domain mutation inflammatory myofibroblastic tumors ( imts ) are a group of rare tumors usually presenting in children and young adults , most commonly in the abdomen , lung , and retroperitoneusurgical excision is the treatment of choice , but the tumors tend to recur locally . 
they show distinct pathologic and molecular features , and approximately one half of them express anaplastic lymphoma kinase ( alk ) protein1 , 2 as a result of alk gene fusions with a variety of partners.3 , 4 inhibition of alk is a valid therapeutic approach for alk - driven malignancies . 
there are currently three tyrosine kinase inhibitors ( tkis ) , crizotinib , ceritinib , and alectinib , approved for alk - positive nonsmall - cell lung cancer and several others tested in clinical trials.5 alk - tkis have shown encouraging results in several published cases of imt with alk rearrangements , 6 - 15 and the national comprehensive cancer network recommends crizotinib and ceritinib in imts with an alk translocation . herein , we present the case of a patient with an alk - positive imt of high - grade malignancy . after two debulking surgeries and hyperthermic intraperitoneal chemotherapy ( hipec ) treatment , the tumor relapsed , and the patient was treated with crizotinib . 
regarding family history , the patients paternal grandfather died at 75 years of pancreatic cancer , and both grandmothers died of colon cancer during their eighth decade . the patient had one debulking surgery at baseline , which was followed by hipec ( fig 1a )  . 
in both cases , histology and immunohistochemistry ( ihc ) were consistent with grade 3 imt expressing alk protein . one month after the second surgery , the ct scan showed a new mass between small intestinal loops . at this point , the patient was admitted to our department . a combined 18f - fluorodeoxyglucose positron emission tomography and ct ( pet / ct ) scan in april 2015 confirmed the presence of a hypermetabolic mass on the left lateral abdominal region ( fig 1b )  . 
fluorescent in situ hybridization ( fish ) was performed retrospectively for the two excised tumors , alk gene rearrangement was demonstrated in both instances , and the patient was started on oral crizotinib 250 mg twice daily ( fig 1a )  . 
green type : additional investigations , including tumor genotyping and phenotyping , germline genotyping , and cell - free dna testing for biologic samples that were obtained at the indicated time points . 
the primary retroperitoneal mass was surgically removed along with two peritoneal implants ; in addition , 27 lymph nodes , the sigmoid colon , and the meckel diverticulum were free of disease . 
on the basis of confirmatory alk immunohistochemistry ( ihc ) and fluorescent in situ hybridization ( fish ) for alk protein expression and gene rearrangement , respectively , the patient received crizotinib and experienced partial response ( 4 )  . 
the tumor grew despite crizotinib , and the patient had a third operation ; except for the mass , 36 cm of adjacent small intestine and 12 lymph nodes free of malignancy were also removed . 
the patient received adjuvant ceritinib , had a negative positron emission tomography ( pet ) / ct scan 14 months later , and remains disease - free ( 6 )  . 
left : strong 18ffluorodeoxyglucose uptake by the relapsed tumor before crizotinib ; middle : subtle uptake after 2.5 months of crizotinib ; right , horizontal and vertical : no uptake after 14 months of ceritinib . 
as of this writing , the patient is in the 21st month of ceritinib without clinical signs of progression or drug - related toxicity . to characterize the alk fusion partner ( figs 2a and 2b ) and to investigate the cause of resistance to crizotinib , the baseline and the postcrizotinib tumors ( fig 1a ) were submitted for genotyping to neo new oncology , cologne , germany . 
dna isolated from paraffin - embedded sections was sheared and subjected to hybrid capturebased next - generation sequencing ( illumina , san diego , ca ) with the neoplus assay ; genomic alterations ( point mutations , small insertions and deletions , copy number changes , and translocations ) were analyzed using neo new oncologys proprietary computational biology analysis pipeline . 
the three vertical microphotograph panels correspond to the three tumors excised from the patient , as indicated . gray stars : tumors submitted for next - generation sequencing genotyping ; tumor cell content at baseline and postcrizotinib was 60% and 80% , respectively . ( a ) hematoxylin and eosin sections showing the initial high - grade sarcoma at baseline , many apoptotic bodies and myxoid stroma after hyperthermic intraperitoneal chemotherapy ( hipec ; inset ) , and morphology similar to baseline and postcrizotinib . 
note the separate green and red dots ( arrows ) , and the multiple copies of the alk locus in occasional nuclei ( arrowheads ; up to five copies per nucleus )  . 
 ( c ) targeted next - generation sequencing revealed the breakpoint ( vertical line ) in intron 8 of tpm4 and intron 19 of alk , and the fusion of the two genes at dna level . 
 ( d ) the graphic representation shows the resulting tpm4 - alk protein ( not to scale ) that was strongly expressed as detected with immunohistochemistry ( clone d5f3 [ ventana , basel , switzerland ] , 1 : 250 ) in all three tumors of the patient . 
the integrative genomics viewer screenshots at the bottom show that the postcrizotinib tumor carried a c.3383g.c substitution at chromosomal position 2 : 29445450 ( grch 37 ) , whereas the same position in the baseline tumor was free of changes ; the reads were of high quality and the position was covered at high depth in both tumors , with assay sensitivity  . 
the substitution localized in exon 21 of the alk gene resulting in the p.g1128a activating mutation ( cosm98475 ) in the kinase domain of the protein ( red star )  . 
in the absence of copy number alterations and at 80% tumor cell content , the observed 13% variant allele frequency for alk c.3383g.c suggested that the new mutation was present in approximately 33% of the neoplastic cells in the crizotinib - resistant tumor . 
 an extremely rare single - nucleotide polymorphism ( snp ; rs750032802 at national center for biotechnology information ; exac minor allele frequency 1 / 106 to 1 / 107 ) of unknown functional significance . 
on the basis of this finding , and also to observe treatment effects on tumor inflammatory infiltrates , we applied ihc for bcl6 , cd138 , cd3 , cd8 , and pd - l1 protein expression on all three tumors ( fig 3 )  . 
in comparison with the baseline tumor , t - cell and cytotoxic t - cell rate declined on hipec and further on crizotinib , whereas pd - l1 was expressed in the postcrizotinib tumor only . on the basis of the patients family history , germline genotyping was performed with the illumina trusight cancer sequencing panel at the molecular diagnostics laboratory , national center for scientific research demokritos . 
this variant lies within the kinase domain but outside the atp - binding pocket , which is targeted by epidermal growth factor receptor tkis ; thus , at present , it appears clinically irrelevant . 
for this sample , the sensitivity of the test was not reached because of low cell - free dna quantity , which was indication of disease considered an additional absence ( no tumor dna shedding )  . discussion apparently , tpm4 - alk is the driving genomic alteration of the present imt , because it was persistent in every tumor this patient developed . although less frequent than tpm3 - alk , tpm4alk fusions are described in imt.3 , 4 the second recurrence in our patient was crizotinib resistant . the most likely resistance mechanism seemed to be the emergence of the alk kinase domain p.g1128a mutation , which is described herein as a novel crizotinib - resistance mutation in imt . 
notably , aside from alk p.g1128a , which was present in approximately 33% of the neoplastic cells , additional resistance mechanism ( s ) may have operated in the present imt . in experimental models , immune escape has been proposed as a mechanism of crizotinib resistance in alk - rearranged lung cancer ( eg , through stat3 activation ) .18 in clinical research , eml4alkrearranged lung adenocarcinomas are reported to express pd - l1 , 19 , 20 which may decrease after crizotinib19 ; these results are , however , not directly comparable to our case , which differs in tumor type and alk translocation . 
here , in three sequential tpm4 - alkrearranged imts from the same patient , we observed a drop in t cell infiltrates and induction of pd - l1 after crizotinib , indicating a different state of immune escape21 in the resistant tumor compared with baseline , which may have contributed to crizotinib resistance as well , together with the emerging alk kinase domain mutation . 
immunohistochemistry pictures are shown for ( a ) bcl6 ( germinal center differentiation factor ; clone pg - b6p [ thermo fisher scientific , paisley , united kingdom ] , 1 : 150 ) ; ( b ) cd138 ( plasma cell marker ; clone mi15 [ thermo fisher scientific ] , 1 : 50 ) ; ( c ) cd3 ( t cells ; clone ps1 [ novocastra , newcastle - upon - tyne , united kingdom ] , 1 : 50 ) ; ( d ) cd8 ( cytotoxic t cells ; clone c8 / 1444b [ dako , glostrup , denmark ] ) ; ( e ) pd - l1 ( immune checkpoint ligand ; clone cal10 [ biocare medical , pacheco , ca ] , ready to use )  . 
as shown in ( a ) , bcl6 protein expression was focally positive in the neoplastic nuclei at baseline ( inset ; overall 15% positive nuclei ) but absent after treatments , indicating that bcl6 p.p139l is not related to the expression of the corresponding prote ( b ) with cd138 , plasma cells were present and positive in all three tumors ; interestingly , 50% of the neoplastic cells at baseline were strongly positive with a diffuse cytoplasmic staining ( inset ) , 25% of the neoplastic cells exhibited intermediate positivity posthyperthermic intraperitoneal chemotherapy ( hipec ) , and a rough granular cytoplasmic pattern was observed in 70% of the neoplastic cells postcrizotinib ( inset )  . 
 ( c , d ) tumor - infiltrating cd3and cd8 - positive t cells constituted 30% and 25% of the tumor tissue population at baseline , respectively ; the rate of both phenotypes decreased to 15% post - hipec and to 2% and 1% postcrizotinib , respectively ( turquoise arrows )  . 
 ( e ) pd - l1 expression followed the opposite pattern as compared with cd3 ; it was completely absent at baseline and post - hipec , but it was present , albeit focally , in neoplastic cells postcrizotinib ( cytoplasmic and membranous staining , inset ) , with an overall positivity of 10% . 
bcl6 and cd138 protein expression by the neoplastic cells are novel reports in imt ; their role , if any , in the biology of these tumors needs to be elucidated . in conclusion , we have presented the disease course of a patient with tpm4 - alkrearranged imt . 
the tumor was locally aggressive and developed resistance to crizotinib through the emergence of an alk kinase domain mutation described for the first time in imts and potentially also through the induction of immune escape . 
cessna mh , zhou h , sanger wg , et al : expression of alk1 and p80 in inflammatory myofibroblastic tumor and its mesenchymal mimics : a study of 135 cases . 
butrynski je , dadamo dr , hornick jl , et al : crizotinib in alk - rearranged inflammatory myofibroblastic tumor . 104 - 108 , 2015 n engl j med 363 : 1727 - 1733 , 2010 7 . 
sasaki t , okuda k , zheng w , et al : the neuroblastoma - associated f1174l alk mutation causes resistance to an alk kinase inhibitor in alk - translocated cancers . 
mosse yp , lim ms , voss sd , et al : safety and activity of crizotinib for paediatric patients with refractory solid tumours or anaplastic large - cell lymphoma : a childrens oncology group phase 1 consortium study . 
lancet oncol 14 : 472 - 480 , 2013 jacob sv , reith jd , kojima ay , et al : an unusual case of systemic inflammatory myofibroblastic tumor with successful treatment with alk - inhibitor . 
kimbara s , takeda k , fukushima h , et al : a case report of epithelioid inflammatory myofibroblastic sarcoma with ranbp2 - alk fusion gene treated with the alk inhibitor , crizotinib . 
subbiah v , mcmahon c , patel s , et al : stump unstumped : anti - tumor response to anaplastic lymphoma kinase ( alk ) inhibitor based targeted therapy in uterine inflammatory myofibroblastic tumor with myxoid features harboring dctn1 - alk fusion . 
ono a , murakami h , serizawa m , et al : drastic initial response and subsequent response to two alk inhibitors in a patient with a highly aggressive alk - rearranged inflammatory myofibroblastic tumor arising in the pleural cavity . lung cancer 99 : 151 - 154 , 2016 14 . 
nishio m , murakami h , horiike a , et al : phase i study of ceritinib ( ldk378 ) in japanese patients with advanced , anaplastic lymphoma kinase - rearranged nonsmall - cell lung cancer or other tumors . 
mansfield as , murphy sj , harris fr , et al : chromoplectic tpm3 - alk rearrangement in a patient with inflammatory myofibroblastic tumor who responded to ceritinib after progression on crizotinib . 
cuy`as e , perez - sanchez a , micol v , et al : stat3 - targeted treatment with silibinin overcomes the acquired resistance to crizotinib in alk - rearranged lung cancer . 
gainor jf , shaw at , sequist lv , et al : egfr mutations and alk rearrangements are associated with low response rates to pd - 1 pathway blockade in nonsmall cell lung cancer : a retrospective analysis . 
koh j , jang jy , keam b , et al : eml4 - alk enhances programmed cell death - ligand 1 expression in pulmonary adenocarcinoma via hypoxia - inducible factor ( hif ) - 1a and stat3 . 
laboratory tests , magnetic resonance imaging ( mri ) , and positron emission tomographycomputed tomography did not indicate involvement of organs other than the skbecause of the limited cutaneous lesions classified as single system lch , topical corticosteroids were used as first - line treatment for 10 weeks , with no improvement . 
treatment with desmopressin ( initial dosage , 0.4 mg daily divided into three doses ) was started , and polyuria and polydipsia improved . function of the anterior pituitary was not affected . targeted next - generation sequencing of the skin biopsy indicated an activating mutation in mek1 ( p.e102_i103delei ; fig 3 )  . 
for this mutation , ex vivo sensitivity to an mek inhibitor ( meki ) has been reported.1a because the skin and the pituitary gland were affected , the disease was now classified as multisystem lch . 
finally , an off - label treatment with trametinib was offered to the patient after interdisciplinary discussion . after detailed explanation and obtaining informed consent , trametinib ( 2 mg once daily ) was started . an mri performed 4 weeks later showed a significant shrinkage of the pituitary enlargement ( fig 3 ) , and skin manifestations resolved both clinically and histologically ( fig 1 )  . 
positron emission tomographycomputed tomography and mri performed in week 12 of treatment showed a slight increase of the pituitary gland in size ; however , the patient had not been receiving trametinib in the previous 3 weeks . 
importantly , the daily dose of desmopressin could be reduced to 0.1 mg / d on trametinib treatment , and anterior pituitary function remained normal . because of the excellent overall response , a drug holiday was recommended , and trametinib treatment was stopped in week 38 . 
hematoxylin and eosin ( h&e ) staining and immunohistochemistry of langerin of a biopsy taken from cutaneous lesions before therapy ( left panels ) and after 4 weeks of therapy ( right panels )  . 
initially , a band - like infiltrate by histiocytes with reniform nuclei and eosinophilic cytoplasm ( upper left , h&e ) reactive with an antilangerin antibody ( brown , lower left ) was observed in the upper dermis and also perifollicular in the lower dermis . 
roughly , clinical subtypes are divided into single system and multisystem ( ms ) forms.3 limited disease usually causes low morbidity , but ms - lch or leukemic forms are a severe threat for patients . organ involvement can cause significant morbidity in patients with lch . 
standard therapies for ms - lch in adults are cytotoxic regimens.4 , 5 radiotherapy is used to treat pituitary lch ; however , this contributes to gradual and often complete loss of anterior pituitary function.6 , 7 the mitogen - activated protein kinase ( mapk ) pathway is critical for cell survival and proliferation . it can be activated by various receptor tyrosine kinases but also by oncogenic mutations in braf or nras found in melanoma and other human malignancies . 
recently , recurrent oncogenic mutations affecting the mapk pathway have been described in lch.1a , 8 these mutations affect braf in approximately 50% and mitogenactivated protein kinase 1 ( map2k1 / mek1 ) in approximately 25% of patients . 
mutations in these genes seem to be mutually exclusive.8 in a multibasket trial for malignancies harboring brafv600 mutations , targeted therapy with the selective braf inhibitor ( brafi ) vemurafenib showed clinical activity in lch.9 besides selective brafi , other small molecules , such as meki , have been evaluated in cancers with oncogenic mapk activation.9 , 10 it has been proposed that activating mek1 mutations is a difficult target in the treatment of lch.3 however , no clinical data on mek inhibition in patients with lch have been reported so far . ( poly ) chemotherapy and radiation are considered standard of care for treatment of ms - lch.3 - 7 high rates of toxicity are observed in adults , and recovery of endocrine function of the pituitary gland is rarely observed with these therapies . 
in week 4 of therapy with trametinib , a significant shrinkage and only a residual contrast enhancement were found . after short - term treatment break , the posterior pituitary gland appeared slightly enlarged again in week 12 . 
in week 28 , size was normalized . representative sagittal images from t1 contrastenhanced magnetic resonance imaging sequences shown ; arrows indicate affected part of the hypophysis . pretreatment after 4 weeks after 12 weeks after 28 weeks harboring a somatic mutation in mek1 . 
radiotherapy and chemotherapy might eventually still have to be considered in these patients . it remains unclear if meki can induce durable responses in lch and non - lch with lasting functional recovery of organs involved . 
 ( b ) the wildtype braf sequencing result is shown with a dotted line and arrow highlighting the c.t1799 location where a braf v600e mutation would be detected if it had been present . 
moeller , dagmar f uhrer , lisa zimmer , antje sucker , dirk schadendorf , elisabeth livingstone , bastian schilling data analysis and interpretation : matina papapanagiotou , klaus g . 
t1799 uwe hillen honoraria : novartis consulting or advisory role : novartis , roche , therakos research funding : novartis ( inst ) travel , accommodations , expenses : novartis tobias t . 
moeller honoraria : pfizer , henning dagmar f uhrer honoraria : ipsen , sanofi , bayer healthcare pharmaceuticals , eisai , genzyme , sobi , pfizer consulting or advisory role : novartis ( inst ) , ipsen , sanofi , bayer , eisai , genzyme , sobi , pfizer research funding : novartis ( inst ) , pfizer ( inst ) , ipsen ( inst ) , novartis , pfizer travel , accommodations , expenses : ipsen , sanofi , bayer , eisai , genzyme , sobi , pfizer lisa zimmer honoraria : novartis , merck sharp & dohme , bristol - myers squibb , roche , merck , glaxosmithkline consulting or advisory role : novartis , bristol - myers squibb , merck sharp & dohme , roche travel , accommodations , expenses : novartis , bristolmyers squibb , amgen , merck sharp & dohme alexander roesch research funding : novartis travel , accommodations , expenses : roche , bristol - myers squibb , merck sharp & dohme , amgen , novartis klaus g . 
 dirk schadendorf honoraria : amgen , array , astra zeneca , bristol - myers squibb , immunocore , merck sharp & dohme , merck , novartis , pfizer , philogen , pierre fabre , regeneron , roche consulting or advisory role : amgen , array biopharma , astrazeneca , bristol - myers squibb , immunocore , merck sharp & dohme , merck , novartis , pfizer , philogen , pierre fabre , regeneron , roche speakers bureau : amgen , bristol - myers squibb , merck sharp & dohme , novartis , pierre fabre , roche research funding : amgen ( inst ) , array ( inst ) , bristol - myers squibb ( inst ) , immunocore , merck sharp & dohme ( inst ) , novartis ( inst ) , philogen ( inst ) , regeneron ( inst ) , roche ( inst ) , bristol - myers squibb , novartis travel , accommodations , expenses : amgen , array , astra zeneca , bristol - myers squibb , immunocore , merck sharp & dohme , merck , novartis , pfizer , philogen , pierre fabre , regeneron , roche elisabeth livingstone no relationship to disclose bastian schilling honoraria : novartis , roche , bristol - myers squibb , merck sharp & dohme consulting or advisory role : roche , bristol - myers squibb speakers bureau : roche , bristol - myers squibb , merck sharp & dohme research funding : bristol - myers squibb ( inst ) , merck sharp & dohme travel , accommodations , expenses : novartis , roche , bristol - myers squibb , amgen matina papapanagiotou , klaus g . 
m oller i , murali r , m uller h , et al : activating cysteinyl leukotriene receptor 2 ( cysltr2 ) mutations in blue nevi . mod pathol 30 : 350 - 356 , 2017 2 . 
girschikofsky m , arico m , castillo d , et al : management of adult patients with langerhans cell histiocytosis : recommendations from an expert panel on behalf of euro - histio - net . 
duan mh , han x , li j , et al : comparison of vindesine and prednisone and cyclophosphamide , etoposide , vindesine , and prednisone as first - line treatment for adult langerhans cell histiocytosis : a single - center retrospective study . 
endocrine 48 : 949 - 956 , 2015 imashuku s , kudo n , kaneda s , et al : treatment of patients with hypothalamic - pituitary lesions as adult - onset langerhans cell histiocytosis . 
hyman dm , puzanov i , subbiah v , et al : vemurafenib in multiple nonmelanoma cancers with braf v600 mutations . 1919 - 1923 , 2010 n engl j med 373 : 726 - 736 , 2015 10 . 
cascade testing is the systematic identification of relatives who are at risk for having the same mutation ( at - risk relatives ) and facilitating genetic testing for the usually , these individuals are unaffected , but they may benefit from early identification and risk management interventions . 
 currently , australian best practice guidelines recommend that genetic testing for unaffected individuals be facilitated by an appropriately trained health professional such as a genetic counselor or clinical geneticist.15 the purpose of this case report is to exemplify a collaboration between the departments of oncology and genetics to extend testing for a dpyd mutation to family members after the index case was identified . patient the index patient was diagnosed with metastatic breast cancer at age 59 years . 
 she developed locoregional recurrences in 2011 , bony metastases in 2013 , and further progression with liver , lung , bone , and soft tissue metastases in 2016 after multiple lines of endocrine therapy . 
with normal baseline renal function , she was prescribed capecitabine at 1 , 000 mg / m2 twice per day on days 1 to 14 in a 21 - day cycle . 
four weeks later , she began palliative paclitaxel to treat symptomatic cancer progression . the patients oncology team referred her to the department of genomic medicine at the royal melbourne hospital . 
the patients personal and family history of cancer had been assessed ; it was considered that there was no increased familial risk of breast ( or other ) cancers . the patients first - degree relatives were subsequently offered genetic counseling and testing for this pharmacogenomic mutation . 
communication of genetic results in families is known to be problematic for various reasons , including miscommunication or misunderstanding of genetic information and passive or active nondisclosure.20 - 22 in this case , family members were already in contact with the genetics service around the time of the index testing and were requesting testing themselves . 
a hypothetical pedigree showing the effect of systematic cascade testing for an autosomal dominant condition . to our knowledge , this is the only published case report of cascade genetic testing in the setting of dpyd pharmacogenomics . 
falvella and colleagues23 reported a case of dpyd genotyping before starting chemotherapy in a 38 - year - old man with colorectal cancer in the context of his mother having a known dpyd variant . 
our case differs in that we extended the dpyd genotyping systematically by offering it to all unaffected at - risk family members . fluoropyrimidines are commonly used drugs , and the cancers treated with these drugs are common in the general population . 
even though this family is not at an increased heritable risk of cancers , it is quite possible that members of the family may need this treatment regimen in the future . 
relationships are self - held unless noted . hereditary breast and ovarian cancer syndrome , cascade pharmacogenomics testing would be predicted to have greater utility because of the higher probability of relatives being affected by these cancers . in conclusion , this case provides an example of cascade testing of family members after an individual with dpd deficiency was identified . 
winship , university of melbourne , parkville , australia acknowledgment we acknowledge michael michael , md , consultant medical oncologist at peter maccallum cancer centre , for his input into an earlier version of this manuscript . support supported by a victorian state - funded clinical service . 
iacovelli r , pietrantonio f , palazzo a , et al : incidence and relative risk of grade 3 and 4 diarrhoea in patients treated with capecitabine or 5 - fluorouracil : a meta - analysis of published trials . 
meulendijks d , van hasselt jgc , huitema adr , et al : renal function , body surface area , and age are associated with risk of early - onset fluoropyrimidine - associated toxicity in patients treated with capecitabine - based anticancer regimens in daily clinical care . 
ison g , beaver ja , mcguinn wd jr , et al : fda approval : uridine triacetate for the treatment of patients following fluorouracil or capecitabine overdose or exhibiting early - onset severe toxicities following administration of these drugs . 
ma ww , saif mw , el - rayes bf , et al : emergency use of uridine triacetate for the prevention and treatment of life - threatening 5 - fluorouracil and capecitabine toxicity . 
henricks lm , opdam fl , beijnen jh , et al : dpyd genotype - guided dose individualization to improve patient safety of fluoropyrimidine therapy : call for a drug label update . 
lunenburg catc , henricks lm , guchelaar hj , et al : prospective dpyd genotyping to reduce the risk of fluoropyrimidine - induced severe toxicity : ready for prime time . 
liu d , li j , gao j , et al : examination of multiple ugt1a and dpyd polymorphisms has limited ability to predict the toxicity and efficacy of metastatic colorectal cancer treated with irinotecan - based chemotherapy : a retrospective analysis . 
claes e , evers - kiebooms g , boogaerts a , et al : communication with close and distant relatives in the context of genetic testing for hereditary breast and ovarian cancer in cancer patients . 
mcclaren bj , aitken m , massie j , et al : cascade carrier testing after a child is diagnosed with cystic fibrosis through newborn screening : investigating why most relatives do not have testing . 
in patients with stage iii or iv melanoma , early changes in ctdna within 1 month after initiation of treatment correctly predicted recist response categories in 19 of 20 patients . 
 braune et al context key objective we evaluated ctdna in stage iii and iv melanoma to detect active disease and to predict response to treatment . knowledge generated ctdna had superior sensitivity to ldh and s100 in detecting active disease in stage iv melanoma . 
written informed consent was obtained from all patients . isolation and quantitation of ctdna from plasma blood samples were collected in edta tubes ( sarstedt , n umbrecht , germany ) and centrifuged twice within the 2 hours after collection . 
the supernatant was stored at 80c . cell - free ctdna was extracted from 2 ml using the qiasymphony circulating dna kit ( qiagen , hilden , germany ) according to the manufacturers instructions and was eluted in 60 l of elution buffer . 
the limit of blank , limit of detection , and lowest detectable copy ratio were determined for each assay ( data supplement )  . imaging analysis imaging and quantitative analyses were performed by a single radiologist blinded to results of the ctdna analyses . 
workows for ct and magnetic resonance imaging are capable of automated segmentation and volume measurements . pet imaging and quantication 18f - uorodeoxyglucose scans were performed with a 64 - slice gemini tf positron emission tomography ( pet ) / ct scanner or a 16 - slice gemini tf big bore pet / ct scanner ( philips healthcare for both ) .17 a contrast - enhanced diagnostic ct ( 120 kvp , 100 , 400 mas , dose modulation applied ) or a low - dose ct ( 120 kvp , 25 mas ) scan was performed for attenuation correction and spatial allocation . 
for each pet study , a quantitative analysis was performed using rover v2.1.12 ( abx , radeberg , germany ) .18 , 19 a threshold of 40% of maximum standardized uptake value was chosen for the determination of the metabolic tumor volume ( mtv ) .20 statistical analysis data analysis was performed separately by stage for all eligible patients . 
correlations were calculated by using a log scale , and ldh and s100 measurements below the standard value were set to the standard value . within - patient correlation was accounted for in the correlation analysis and in the estimation of the progression - free survival ( pfs ) hazard ratio ( hr ) in stage iv patients . 
patient characteristics ( continued ) characteristic 66 ( 28 - 92 ) tissue mutation status braf exon 15 v600e v600k v600r k601n d594g l597s w604c nras q61 q61k q61r total patients female male age , years median ( range ) 60  . 
60 stage stage iii newly diagnosed previous treatment treatment after inclusion surgery surgery radiation systemic treatment relapsed previous treatment of relapse treatment of relapse after inclusion surgery radiation systemic treatment no . 
of previous lines of therapy stage iv mean ( range ) treatment at inclusion braf / mek inhibitor immunotherapy treatment after inclusion immunotherapy braf / mek inhibitor radiotherapy surgery interferon vaccination ( continued in next column ) 1.7 ( 0 - 7 ) * systemic treatment after progression to stage iv . three patients received systemic treatment for unresectable stage iii melanoma , and three patients received systemic treatment after progression to stage iv melanoma . total number of tissue mutations is 64 . 
patient 62 was tissue positive for nras q61r and braf w604c , patient 55 had resection of two different melanomas before inclusion in the study ( one was positive for braf v600e , and the other was positive for nras q61k )  . 
the mean number of samples was 6.8 ( range , 1 to 15 samples ) per patient with stage iii disease , and the mean was 5.8 ( range , 1 to 27 samples ) for stage iv patients . in patients with stage iii disease , 10 ( 83.3% ) of 12 were positive for ctdna at least once ; for patients with stage iv disease , 44 ( 88% ) of 50 patients were positive for ctdna at least once ( table 2 )  . 
s100 and ldh are indicated as being increased when level is above the normal range of , 0.105 mg / l for s100 and 135 to 225 u / l for ldh . 
we identied 10 stage iii and 30 stage iv patients with 56 samples obtained up to 30 days before surgery , initiation of systemic treatment , or change in systemic treatment . 
even when they were combined , ldh and s100 detected measurable disease in only 34 ( 60.7% ) of 56 samples before initiation of treatment ( table 3 ; fig 1c )  . 
thus , ctdna detected a higher proportion of patients with active disease . amount of mutated ctdna correlates with ldh and s100 to compare ctdna with the established biomarkers ldh and s100 , we studied measurement pairs obtained at the same time points . 
ctdna strongly correlated with s100 ( r = 0.64 based on 278 measurement pairs from 62 patients ) and ldh ( r = 0.50 based on 288 measurement pairs from 61 patients )  . ctdna reects tumor volume and metabolic activity we next asked whether mutant ctdna levels correlate with tumor burden . 
analysis was performed according to change in the sum of the diameters of target tumor lesions from baseline to rst ( fig 2a ) and subsequent ( fig 2b ) follow - up imaging scans according to recist 1.1. 
 ( a ) percentage of positive ( ctdna ) or increased ( lactate dehydrogenase [ ldh ] , s100 ) samples from 274 time points with matched ctdna , ldh , and s100 samples for stage iii and stage iv patients , respectively . 
red lines indicate the mean value for ctdna , ldh , or s100 . dotted lines indicate the thresholds of ldh ( 225 u / l ) and s100 ( 0.105 ug / l ) above which values are stated as being increased . 
 ( c ) sensitivity of ctdna , ldh , and s100 to detect disease within 30 days before surgery , initiation of treatment , or change in treatment ( see table 3 for details )  . 
of note , one patient who displayed a new lesion despite favorable ctdna course had an additional braf w604c mutation detectable at baseline that converted from negative at day 43 to positive at day 91 . 
conversely , all nine patients with partial response according to recist had a positive - to - negative conversion , had a decrease in ctdna , or remained ctdna negative . thus , early ctdna dynamics correctly predicted progressive disease or partial response according to recist in 21 of 24 patients . 
the remaining patient had a ctdna - positive sample available 1 day after imaging ( patient 60 ; fig 2c )  . ctdna predicts outcome we analyzed stage iii patients with a ctdna measurement within 30 days after surgery or at initiation of systemic therapy . 
four patients did not meet these criteria because they had no samples within 30 days after surgery ( n = 3 ) or at the initiation of systemic treatment ( n = 1 )  . 
thus , the higher levels of ctdna that appeared early after initiation of treatment or change in treatment indicated inferior pfs . dynamic ctdna proles of individual patients results of biomarker assessment in individual patients demonstrate that ctdna analysis over time is informative with respect to tumor dynamics as shown in the data supplement . 
ctdna demonstrated a superior dynamic signal range compared with ldh and s100 , and ctdna detected progression whereas ldh ( data supplement ) or ldh and s100 ( fig 1c , g ) failed to detect progression . 
of note , the combination of all three biomarkers provides an 8.9% increase in sensitivity to ctdna alone . thus , ctdna might be used together with ldh and s100 in a combined biomarker score . 
we observed a signicant association of ctdna at baseline with ldh , s100 , tumor size , and mtv ( r2 = 0.87 ) , the latter displaying the strongest correlation . 
this is in agreement with two other studies demonstrating a strong correlation between the number of ctdna copies and mtv in melanoma ( r = 0.63 , 23 and r2 = 0.6949 , 24 respectively )  . 
 ( d ) correlation between ctdna and ( continued on following page ) recist 1.1 , ( e ) tumor volume at baseline assessed by radiologic analysis , and ( f ) tumor metabolic activity , assessed by positron emission tomography / computed tomography ( pet / ct ) scan and expressed as the mean total lesion glycolysis ( tlg ) in [ g / ml x ccm ]  . 
the tlg in the equation [ g / ml x ccm ] was calculated by multiplying the lesion volume with the mean standardized uptake value in the volume of interest . 
eleven patients with stage iii and 22 with stage iv disease with a baseline ctdna sample , radiographic imaging before intervention , and a ctdna sample taken within 30 days after initiation of treatment or change in treatment were analyzed . 
 ( a ) waterfall plot showing the change in sum of diameter ( sod ) of target tumor lesions from baseline to rst follow - up imaging according to recist 1.1 in relation to early changes in ctdna . 
t0 denotes baseline ctdna sample , and t1denotes rst sample after baseline at day 8 to 28 after initiation of or change in treatment with ctdna detectable ( + ) or not detectable ( )  . 
dotted lines represent 20% increase in sod , which indicated progressive disease ( pd ) , or 30% decrease , which indicated partial response ( pr ) according to recist 1.1. 
 ( b ) spider plot showing changes in sod from baseline to rst and subsequent follow - up imaging according to early ctdna dynamic changes from baseline to rst sample after baseline taken within 28 days after institution of or change in treatment . 
cr , complete response ; sd , stable disease . importantly , early changes in ctdna levels were superior to ldh and s100 for early assessment of treatment response . of all the patients who achieved partial response , 76.9% had favorable ctdna dynamics as early as 8 to 28 days after initiation of or change in treatment . 
in line with these observations , detectable ctdna as early as within 30 days after initiation of or change to therapy predicted inferior pfs for patients with stage iii or iv disease . 
in stage iv disease , ctdna with more than 10 copies per ml within 30 days after initiation of or change in treatment indicated a population with a particularly short pfs of only 4 months . 
we included patients who were alive and free of progression 1 month after surgery or at the start of systemic therapy and who had a ctdna measurement within 1 month . 
pfs was dened as the time from initiation of treatment to rst reported progression or to the most recent visit . progression events included progression as dened by recist 1.1 , clinical progression , or melanoma - specic death . 
 ( a ) pfs in eight patients with stage iii disease alive and free of progression after 1 month with a ctdna measurement within 1 month after surgery or start of systemic treatment . 
 ( b - c ) pfs in 36 patients with stage iv disease with 46 pfs sections , alive and free of progression at 1 month by the rst ctdna value taken within 30 patients with days ( median , day 22 ; range , days 1 to 30 ) after start of or change in systemic treatment with a ctdna measurement within 1 month , by ( b ) positive v negative , and ( c ) negative or 10 or fewer copies per ml v more than 10 copies per ml . 
our data suggest that ctdna dynamics allow prediction of response and pfs within in the rst month after implementing braf / mek inhibitors or immunotherapy . in contrast to previous reports , 27 - 30 ctdna at baseline was not a prognostic marker for pfs in patients with stage iv disease in our cohort , whereas the level of ctdna at rst measurement after baseline was clearly predictive for pfs , most likely as an expression of response to treatment . notably in the aforementioned studies , most patients were treated with braf / mek inhibitors whereas the majority of our patients received immunotherapy . 
in two other studies of patients with or without melanoma who were receiving pd - 1 antibodies , there was no association between baseline ctdna and pfs.25 , 31 however , subgroup analyses did not reveal signicant differences because this study was not designed to examine the signicance of baseline ctdna in patients treated with braf / mek inhibitors versus immunotherapy . 
 braune et al nras mutations.27 , 32 this is a possible explanation for the increasing copies of nras - mutated ctdna detected in patient 53 after changing the treatment to dabrafenib and trametinib , which suggests selection of a subclonal mutation that mediates resistance to treatment with braf / mek inhibitors . 
nevertheless , previous studies have demonstrated tumor interand intraheterogeneity involving additional genetic events that lead to mapk or pi3k / pten / akt reactivation when disease progresses during treatment with braf inhibitors.33 , 34 few studies address the use of ctdna as a biomarker in stage iii melanoma . 
but in one recent study , ctdna positivity within 12 weeks after surgery indicated adverse disease - free survival and os.35 in our cohort , ctdna positivity after resection of the primary tumor and within 1 month after denitive locoregional treatment or start of systemic therapy predicted a poor pfs . 
with adjuvant therapies showing benet for high - risk stage iii melanoma , 9 , 10 ctdna positivity might help to stratify patients for adjuvant treatment . one limitation of this study is the small sample size . 
validation in a larger prospective trial is required , especially for detection of minimal residual disease in stage iii and to examine whether changes of ctdna early after the start of treatment constitute an independent marker for pfs and os for braf / mek inhibitor treatment versus immunotherapy . 
although mutationspecic assays might be most suitable for monitoring response and minimal residual disease , a broader sequencing approach using next - generation sequencing might be used in previously treated patients to track subclonal mutations to understand treatment resistance and to guide further therapy.34 in addition , including tert promoter mutations present in 70% of melanomas36 could increase coverage when added to brafand nrasspecic ctdna assays.37 in conclusion , our results demonstrate that ctdna is superior to ldh and s100 for detecting active disease and suggest that changes in ctdna early after initiation of treatment predict response to treatment and pfs . 
 ctdna as a biomarker in malignant melanoma dagmar von bubnoff honoraria : roche , iomedico travel , accommodations , expenses : bristol - myers squibb frank meiss honoraria : bristol - myers squibb , novartis , pierre fabre consulting or advisory role : bristol - myers squibb , novartis , pierre fabre , roche pharma ag speakers bureau : bristol - myers squibb travel , accommodations , expenses : novartis , bristol - myers squibb , pierre fabre nikolas von bubnoff honoraria : astrazeneca , amgen , bristol - myers squibb , novartis consulting or advisory role : novartis ( inst ) research funding : novartis ( inst ) no other potential conicts of interest were reported . acknowledgments the authors thank renata koch and aurelia winter for sample collection , patient management , and administrative support . references cancer genome atlas network : genomic classication of cutaneous melanoma . 
cell 161 : 1681 - 1696 , 2015 dummer r , ascierto pa , gogas hj , et al : overall survival in patients with braf - mutant melanoma receiving encorafenib plus binimetinib versus vemurafenib or encorafenib ( columbus ) : a multicentre , open - label , randomised , phase 3 trial . 
lancet oncol 19 : 1315 - 1327 , 2018 long gv , flaherty kt , stroyakovskiy d , et al : dabrafenib plus trametinib versus dabrafenib monotherapy in patients with metastatic braf v600e / k - mutant melanoma : long - term survival and safety analysis of a phase 3 study . 
weber js , gibney g , sullivan rj , et al : sequential administration of nivolumab and ipilimumab with a planned switch in patients with advanced melanoma ( checkmate 064 ) : an open - label , randomised , phase 2 trial . 
lancet oncol 17 : 943 - 955 , 2016 topalian sl , sznol m , mcdermott df , et al : survival , durable tumor remission , and long - term safety in patients with advanced melanoma receiving nivolumab . j clin oncol 32 : 1020 - 1030 , 2014 schadendorf d , hodi fs , robert c , et al : pooled analysis of long - term survival data from phase ii and phase iii trials of ipilimumab in unresectable or metastatic melanoma . 
j clin oncol 33 : 1889 - 1894 , 2015 barbour ap , tang yh , armour n , et al : braf mutation status is an independent prognostic factor for resected stage iiib and iiic melanoma : implications for melanoma staging and adjuvant therapy . 
hofheinz f , p otzsch c , oehme l , et al : automatic volume delineation in oncological pet : evaluation of a dedicated software tool and comparison with manual delineation in clinical data sets . 
krohn t , kaiser hj , gagel b , et al : 3d volume and suv analysis of oncological pet studies : a voxel - based image processing tool with nsclc as example [ in german ]  . 
welsh jl , bodeker k , fallon e , et al : comparison of response evaluation criteria in solid tumors with volumetric measurements for estimation of tumor burden in pancreatic adenocarcinoma and hepatocellular carcinoma . 
gonzalez - cao m , mayo de las casas c , jordana ariza n , et al : early evolution of brafv600 status in the blood of melanoma patients correlates with clinical outcome and identies patients refractory to therapy . 
gonzalez - cao m , mayo - de - las - casas c , molina - vila ma , et al : braf mutation analysis in circulating free tumor dna of melanoma patients treated with braf oncotarget 6 : 42008 - 42018 , 2015 inhibitors . 
santiago - walker a , gagnon r , mazumdar j , et al : correlation of braf mutation status in circulating - free dna and tumor and association with clinical outcome across four brafi and meki clinical trials . 
ascierto pa , minor d , ribas a , et al : phase ii trial ( break - 2 ) of the braf inhibitor dabrafenib ( gsk2118436 ) in patients with metastatic melanoma . 
cabel l , riva f , servois v , et al : circulating tumor dna changes for early monitoring of anti - pd1 immunotherapy : a proof - of - concept study . 
long gv , fung c , menzies am , et al : increased mapk reactivation in early resistance to dabrafenib / trametinib combination therapy of braf - mutant metastatic 28 : 1996 - 2001 , 2017 melanoma . 
mcevoy ac , calapre l , pereira mr , et al : sensitive droplet digital pcr method for detection of tert promoter mutations in cell free dna from patients with metastatic melanoma . 
lum , md1 ; shirley zhu , md , mph1 ; sujay vennam1 ; erna forg o , md1 ; sushama varma , ms1 ; kristen ganjoo , md1 ; trevor hastie , phd3 ; rafck bowen , phd1 ; maria debiec - rychter , md , phd2 ; and matt van de rijn , md , phd1 purpose the preoperative distinction between uterine leiomyoma ( lm ) and leiomyosarcoma ( lms ) is difcult , which may result in dissemination of an unexpected malignancy during surgery for a presumed benign lesion . an assay based on circulating tumor dna ( ctdna ) could help in the preoperative distinction between lm and lms . 
we also proled 36 lm tumor specimens by exome sequencing to develop a panel for targeted detection of point mutations in ctdna of patients with lm . results we identied tumor - derived cnas in the plasma dna of 50% ( six of 12 ) of patients with lm . 
we identied only two recurrently mutated genes in lm tumors ( med12 and acly )  . conclusion our results show that lms do shed dna into the circulation , which provides an opportunity for the development of ctdna - based testing to distinguish lm from lms . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction uterine leiomyomas ( lms ) are common benign smooth muscle tumors that may present with symptoms similar to those associated with uterine leiomyosarcoma ( lms ) , a rare malignant tumor with a poor prognosis.1 with the exception of endometrial lesions , most uterine masses are not biopsied before surgery , and the preoperative distinction between benign and malignant uterine smooth muscle tumors relies primarily on clinical evaluation and imaging . 
as a result , patients may undergo surgery without a denitive distinction between the two entities . it is estimated that one in nine women in the united states will undergo a hysterectomy for benign gynein their lifetime.2 cologic indications , such as lm , power morcellation used to be performed in many in 2014 the us food and drug such cases , until administration discouraged the use of power morcellation for the removal of the uterus or uterine masses , after reporting that this procedure may lead to inadvertent retroperitoneal spread of an unsuspected malignancy in one in 305 patients.3 however , intraabdominal manual morcellation is still performed in patients with large masses diagnosed as lm , and although this is less aggressive than power morcellation , it still carries a risk for dissemination of an unexpected lms . 
risk factors that may favor the diagnosis of lms over lm include postmenopausal status , tamoxifen use , history of retinoblastoma , pelvic irradiation , hereditary leiomyomatosis , and renal cell cancer syndrome , 4 but these factors do not always correlate with a diagnosis of lms . also , the new emerging magnetic resonance imaging techniques present unsatisfactory positive predictive values for the distinction between lm and lms.4 therefore , there is a high need for improved methods for preoperative discrimination between benign lm and malignant tumors . 
in an attempt to improve the distinction between lm and lms , nagai et al5 developed a revised preoperative sarcoma score ( rpress ) based on patients age , serum lactate dehydrogenase ( ldh ) levels , and endometrial cytology ndings . 
 przybyl et al context key objective we sought to explore the feasibility of distinguishing between uterine leiomyoma and leiomyosarcoma based on the analysis of tumor - specic aberrations in circulating tumor dna ( ctdna )  . 
for this purpose , in the current study we aimed to determine whether uterine leiomyomas shed dna into the circulation . knowledge generated we demonstrate that it is feasible to detect ctdna derived from leiomyomas . 
in the limited number of cases analyzed to date , we show that these benign tumors do not carry alterations in tumor suppressor genes that can be detected in the ctdna of most patients with leiomyosarcoma . relevance our ndings are a rst and necessary step toward developing a blood test that , together with clinical and imaging information , ultimately could help clinicians to better evaluate the risk of leiomyosarcoma in a patient presenting with a uterine mass . was developed in a group of 63 patients with lm and lms but has not yet been validated in an independent study . we recently demonstrated that tumor - associated genetic aberrations can be detected in the circulating tumor dna ( ctdna ) of patients with lms.6 in that study , we used two technologies to detect different classes of genomic aberrations in plasma dna : cancer personalized proling by deep sequencing ( capp - seq ) for the analysis of point mutations , 7 , 8 and a genome - wide interrogation of copy number alterations ( cnas ) by shallow whole - genome sequencing.9 , 10 using these two approaches , we were able to detect ctdna in six of seven patients with lms with  . 
the study was approved by the stanford university institutional review board ( irb - 31067 )  . proling plasma cell - free dna cell - free dna was extracted from plasma using qiaamp circulating nucleic acid kit ( qiagen )  . 
sequencing libraries were prepared with the truseq chip preparation kit ( illumina , foster city , ca ) and sequenced using hiseq 2500 instrument ( illumina ) ( data supplement )  . plasma ldh measurement plasma ldh levels were measured on a roche cobas 8000 ( roche , indianapolis , in ) automated platform ( data supplement )  . proling tumor specimens genomic dna extracted from 12 archival lm tumor specimens and germline dna extracted from patient - matched blood leukocytes were used for genome - wide copy number proling with the oncoscan ffpe assay ( thermo fisher scientic / affymetrix , santa clara , ca )  . thirty - six archival lm tumor specimens and normal patientmatched counterpart tissues or peripheral blood leukocytes were used for whole - exome sequencing using the seqcap ez human exome v3.0 library ( roche ) on hiseq 4000 instrument ( illumina ) ( data supplement )  . results tumor - derived copy number aberrations can be detected in plasma dna of patients with lm to explore the possibility of developing a blood - based test for the distinction between lm and lms , we rst determined whether lm can shed dna in the blood . 
we calculated genome - wide segmented z - scores in plasma cell - free dna , and we intersected each genomic region of cnas identied in the eight lm tumor specimens with the segments identied in plasma dna of the matching patients . 
of the 33 cnas detected in the eight tumor specimens , 30 cnas had the same type of copy number alteration in tumor and plasma dna ( ie , matching dna copy number gain or loss ; data supplement )  . 
1.5 in cell - free dna , we found 11 tumor - derived cnas in the plasma dna of six patients with lm ( correlation between log2 ratio in the tumor and z - score in the plasma determined by linear regression was r2 = 0.74 ; p value = .0007 ; table 3 ; appendix fig a1 )  . 
selected large dna cnas identied in lm tumor specimens were not detected in plasma specimens , which is most likely the result of the limit of detection of our method and / or the effect of stochastic sampling of blood specimens . 
we have previously demonstrated that it is possible to detect tumorderived cnas in cell - free dna among patients with lms using the same technology as applied to the patients with lm in the current study.6 we sought to verify whether the aberrations found in ctdna of patients with lm in the present report are specic to these benign tumors or whether the same aberrations could be found also in lms tumor specimens . 
for this purpose , we reanalyzed the previously generated genomic proles of 22 tumors from seven patients with lms using exactly the same criteria as were used for the lm tumor specimens . 
our results show that 10 of 11 genomic aberrations found in the ctdna of patients with lm were also present in the tumor dna of patients with lms analyzed in our previous study , 6 with a median of four aberrations per lms tumor sample ( range , 1 - 8 )  . 
these results demonstrate that the majority of cnas found in lm tumors are not specic for this entity ; therefore , proling these cnas in plasma dna may not be useful to distinguish between lm and lms . 
on the other hand , cnas found in lm specimens did not affect any of the frequently deleted tumor suppressor genes in lms ( eg , tp53 , rb1 , atrx , pten , atm , or cdkn2a )  . 
therefore , the detection of cnas in these tumor suppressor genes in ctdna may favor a diagnosis of lms . patient age and levels of ldh do not allow for distinction between lm and lms previous studies have proposed that ldh level measurements and patient age may be helpful in the distinction between lm and lms.5 , 11 we evaluated the ldh levels in plasma specimens of nine of 12 patients with lm proled for the presence of ctdna and eight untreated patients with uterine lms . 
in the rpress algorithm , the cutoff of serum ldh levels indicative of lms was set at 279 u / l.5 in our group of patients , the sensitivity and specicity values of this test were found to be only 50% and 89% , respectively . 
the mean age of the patients with lm and lms was 48 and 54 years , respectively ( t test two - tailed p value = .19 ; fig 2b )  . 
this performance was poorer than the ndings reported in the rpress study , where the sensitivity , specicity , positive predictive value , and negative predictive value were 93% , 65% , 45% , and 97% , respectively.5 low mutation burden in lm previous genomic studies of lm have identied med12 as the most frequently mutated gene in these lesions , but these studies did not report on other recurrent somatic mutations in these tumors.12 - 14 however , med12 mutations have been reported also in a subset of lms tumors , and therefore these mutations cannot be used for a molecular distinction between lm and lms.15 to investigate whether lm tumors might have recurrently mutated genes other than med12 , we performed whole - exome sequencing on 36 pairs of matched tumor and normal dna specimens from patients with lm . 
mutations in exon 2 of med12 were identied in 39% ( 14 of 36 ) of lm tumor specimens , and the acly gene was mutated in 6% ( two of 36 ) of lm tumor specimens ( data supplement )  . 
mutations in these two genes were present in 15 of 36 patients , with a median of one mutation per tumor ( range , 0 - 2 across all 36 tumors )  . 
all med12 mutations identied in our study were previously reported in the cosmic database ( cosmic v84 ; accessed on november 12 , 2018 ) .16 our results show that recurrent mutations other than in the med12 gene rarely occur in lm . 
importantly , we did not detect in any lm case any of the mutations in tumor suppressor genes that are frequently mutated in lms , such as tp53 , rb1 , pten , atrx , atm , and arid1a.6 this indicates that mutations in these driver genes may be highly specic to malignant tumors such as lms . 
because lms do not carry mutations in these tumor suppressor genes , we propose that detection of these mutations in plasma dna of patients with uncertain diagnosis , using the capp - seq lms - specic panel developed in our previous study , may favor the diagnosis of lms . discussion the clinical utility of ctdna proling has been widely demonstrated in malignant tumors that harbor highly recurrent mutations.7 , 8 , 17 , 18 currently , the two most frequently used sequencing - based approaches for ctdna monitoring include deep targeted sequencing for ultrasensitive quantitative analysis of point mutations , such as capp - seq , and shallow whole - genome sequencing for the detection of cnas . 
histologic appearance of representative leiomyoma ( lm ) cases with ( a ) no detectable dna copy number alterations , ( b ) 20 dna copy number alterations , and ( c ) six dna copy number alterations . 
however , it is not practical to design an lm - specic capture panel for detection of point mutations by deep targeted sequencing of ctdna , because we identied only two recurrently mutated genes in a low percentage of 36 lm tumors . we assume that an approximately two - fold increase of coverage may result in improving the reliability of the calls , by reducing the number of false - positive calls and increasing the number of true - positive calls.22 moreover , most of the patients included in our study had multifocal disease , and we expect that proling all nodules from these patients would reveal a wider spectrum of cnas and thus allow for the detection of overlapping aberrations between plasma and tumor dna in a higher percentage of patients with lm . although ctdna released from malignant tumors has been well characterized in multiple types of cancer , ctdna derived from benign lesions has not been extensively studied . an incidental nding of lm - derived dna in the circulatory system has been reported in pregnant women undergoing noninvasive prenatal testing ( nipt )  . 
dharajiya et al19 reported unexpectedly abnormal nipt proles in 55 of 450 , 000 pregnant women tested for fetal aneuploidy ; 20 of these 55 women were known to have had lm at the time of nipt . 
but the genomic prole of a matching tumor was examined only in a single patient from this group to conrm levels of cell - free dna were indeed that the abnormal derived from this lm.19 although these incidental ndings indicated that lm can shed dna into the circulatory system , the overall sensitivity of shallow whole - genome sequencing for the detection of lm - derived ctdna has not been investigated . 
in the current study , by prospective analysis of plasma and tumor samples of 12 patients with lm , we found ctdna in 50% of these patients , which is a comparable detection rate to the current sensitivity of ctdna detection in many stage i cancers.20 , 21 importantly , the nipt assays used for detecting fetal abnormalities usually focus solely on screening for monosomy or trisomy of chromosomes 13 , 18 , and / or 21 . 
in the current study , instead of focusing only on selected chromosomes , we performed a genome - wide analysis in which we aimed to detect not only chromosome - wide aberrations but also small focal aberrations . 
we performed shallow whole - genome sequencing at approximately 0.1 genomewide coverage ; however , we expect that deeper sequencing would allow for the detection of ctdna in a higher percentage of patients with lm . 
on the basis of previously published studies , it must be noted that the development of a reliable assay for the distinction between lm and lms is challenged by the great difference in prevalence of these two entities . 
 circulating tumor dna in leiomyoma predictive value for any positive result indicating lms would be only 2% , while the negative predictive value of a negative result would be 99.99%. 
on the basis of the genomic proles of lm and lms , we propose that it may be more practical to apply an assay that would rule out the diagnosis of lm . 
this could be possible by applying capp - seq for detection of mutations in tumor suppressor genes that are frequently mutated in lms but have never been reported in lm . 
regardless , given the statistical considerations on the prevalence of lm and lms , validation of such assay in the clinical setting may be challenging and would require a very large prospective study . in summary , we demonstrate in the current study that a substantial portion of lms do shed dna into the circulatory systethese ndings provide an opportunity to develop a noninvasive test for distinction between lm and lms on the basis of ctdna in plasma . 
lyon , iarc , 2014 , in kurman rj , carcangiu ml , herrington cs , et al ( eds ) : who classication of tumours of female reproductive organs . 
nagai t , takai y , akahori t , et al : highly improved accuracy of the revised preoperative sarcoma score ( rpress ) in the decision of performing surgery for patients presenting with a uterine mass . 
nat biotechnol 34 : 547 - 555 , 2016 1 : 814 - 819 , 2015 334 : 252 - 255 , 2011 bayindir b , dehaspe l , brison n , et al : noninvasive prenatal testing using a novel analysis pipeline to screen for all autosomal fetal aneuploidies improves pregnancy management . 
goto a , takeuchi s , sugimura k , et al : usefulness of gd - dtpa contrast - enhanced dynamic mri and serum determination of ldh and its isozymes in the differential diagnosis of leiomyosarcoma from degenerated leiomyoma of the uterus . 
m akinen n , mehine m , tolvanen j , et al : med12 , the mediator complex subunit 12 gene , is mutated at high frequency in uterine leiomyomas . 
wan r , wang z , lee jj , et al : comprehensive analysis of the discordance of egfr mutation status between tumor tissues and matched circulating tumor dna in advanced non - small cell lung cancer . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced nonsmall - cell lung cancer . 
sci transl med van beek dm : detection of copy number variation using shallow whole genome sequencing data to replace array - comparative genomic hybridization analysis [ masters thesis ]  . 
heitzer e , ulz p , belic j , et al : tumor - associated copy number changes in the circulation of patients with prostate cancer identied through whole - genome 27 . 
karczewski kj , francioli lc , tiao g , et al : variation across 141 , 456 human exomes and genomes reveals the spectrum of loss - of - function intolerance across 37 . 
correlation between log2 ratio in tumor dna and z - score in cell - free dna for 11 tumor - derived copy number alterations detected in plasma of leiomyoma patients . 
in their letter , karam et al express concerns about the claims made regarding the inability of rna sequencing ( rna - seq ) to perform allele - specic quantication . however , we believe our statement that rna - seq does not allow the allele - specic quantication of normal full - length transcript is correct . the percent splicing index measures the frequency at which a given splice event occurs compared with others affecting the same site.3 as karam et al1 note , this allows one to quantify reads from aberrant transcripts relative to reference transcripts but it does not provide an allele - specic quantication of splicing . 
however , in the case of intronic variants , rnaseq cannot determine whether any of the full - length transcript is also produced by the variant allele because the variant is not part of the messenger rna sequence . 
in the absence of an additional informative variant in the exon , rna - seq cannot distinguish between a full splice defect ( no full - length transcript is made by the variant allele ) and a partial or leaky splice defect ( some full - length transcript is produced by the variant allele )  . 
as gelli et al4 noted , partial splice defects may not be pathogenic if the variant allele produces enough full - length transcript to support normal protein function . in their letter , karam et al1 highlighted a recent publication by landrith et al3 and indicated that rnaseq was used to identify partial skipping of exon 5 in the brca2 c.426 - 12_426 - 8del variant carrier and control patients . 
of note , the landrith et al denition of partial exon skipping is some portion of the exon is skipped ( ie , alternative acceptor / donor ) , 3 not as a partial splice defect . 
however , other published data do show that brca2 c.426 - 12_426 - 8del causes a partial splice defect.8 our laboratory now classies this variant as benign based on additional phenotypic and mutation co - occurrence data , 2 which suggests that the amount of normal transcript produced by the variant allele does support normal protein function . with increased use of tools such as rna - seq to aid in variant classication , physician understanding of how these tools should be used is critical to appropriate patient care . 
we would like to emphasize that we are not arguing against the use of functional rna studies in producing data to support variant classication . rather , we are promoting the need for improved guidelines for interpretation and use of such data in variant classication . 
sharing of reclassication advancements and variant classication information through publications such as the article by nix et al2 allows for dissemination of high - quality data that has passed peer review . 
nix p , mundt e , manley s , et al : functional rna studies are a useful tool in variant classication but must be used with caution : a case study of one brca2 variant . 
gelli e , colombo m , pinto am , et al : usefulness and limitations of comprehensive characterization of mrna splicing proles in the denition of the clinical relevance of brca1 / 2 variants of uncertain signicance . 
 consistency of brca1 and brca2 variant classifications among clinical diagnostic laboratories purpose genetic tests of cancer predisposition genes , brca1 and brca2 , inform significant clinical decisions for both physicians and patients . 
the clinvar database makes deidentified clinical variant classifications from multiple laboratories publicly available for comparison and review , per recommendations by the american medical association , the american college of medical genetics , the national society for genetic counselors , and other organizations . methods classifications of more than 2 , 000 brca1 / 2 variants in clinvar that represent approximately 22 , 000 patients were dichotomized as clinically actionable or not actionable and compared among as many as seven laboratories . 
clinvar facilitated resolution of many of the discordant variants , and concordance increased to 99.0% per variant and 99.8% per patient when reclassified , but not yet resubmitted , variants and submission errors were addressed . 
most of the remaining discordances seemed to involve either legitimate differences in expert judgment regarding particular scientific evidence or were classifications that predated the availability of important scientific evidence . conclusion significant classification disagreements among professional clinical laboratories represented in clinvar are infrequent yet important . 
2017 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction hereditary breast and ovarian cancer is a cancer predisposition syndrome that results from inherited is , germlineloss - of - function mutations in that brca1 or brca2 genes , collectively , brca1 / 2.such pathogenic , or disease - causing , genetic variants result in a 40% to 80% lifetime risk of developing breast cancer , an 11% to 40% risk of ovarian cancer , and striking increases in the risk of male breast , pancreatic , and prostate cancers.1 , 2 up to 10% of breast cancers are caused by these genes.3 , 4 approximately one in 250 individuals of european descent are born with a pathogenic variant in brca1 / 2 , and prevalence is much higher in certain populationsfor example , ashkenazi jews.5 , 6 decades of clinical testing and research have uncovered tens of thousands of brca1 / 2 genetic variants across the human population.7 the great majority of these variants are benign and confer no increased cancer risk , whereas others are pathogenic . 
to help standardize variant interpretation , the american college of medical genetics ( acmg ) and the association for molecular pathology ( amp ) jointly issued revised guidelines8 for variant classification . 
whereas vus , likely benign , and benign variants in brca1 / 2 are not medically actionable , pathogenic and likely pathogenic variants are actionable , which warrants consideration of additional screening , prevention , or treatment options.6 , 11 , 12 thus , rigorous and consistent variant interpretation is critical to patient care . variant classifications can also potentially conflict if one laboratory has access to proprietary data that are unavailable to others . 
in the 1990s , myriad genetics patented the brca1 / 2 genes and prohibited testing by other laboratories.13 , 14 myriad genetics continued as the sole provider for nearly 20 years until the patents were overturned . 
the company used its monopoly to accumulate a substantial database of variants that it ceased releasing publicly in 2006 and from which it claims a competitive advantage.15 - 17 this practice is contrary to the recommendations of the american medical association ( ama ) , the acmg , the national society for genetic counselors , and other organizations.18 - 20 recognizing that shared knowledge about genetic variants is critical to high - quality medical care , the national institutes of health established clinvar , a public database of clinically observed genetic variants , their pathogenicity classifications from various laboratories , and a summary of the scientific evidence used in those classifications.21 - 25 whereas many commercial and academic laboratories collaboratively submit data to clinvar , others , including myriad , do not . 
nevertheless , a substantial myriad genetics data set has been submitted by ordering clinicians and patients through the sharing clinical reports project ( scrp ) .13 , 26 in this study , we used publicly available data from clinvar to assess agreement among clinical laboratories for classifications of brca1 / 2 variants . experiencedclinical laboratories , and older data sets . 
the complete data set that was used in our analysis is also provided as a data supplement . to compare potential clinical impact , we dichotomized classifications into positive ( pathogenic , likely pathogenic ) or not positive ( benign , likely benign , vus )  . 
although many laboratories exclude benign and likely benign variants from clinical reports , these variants are often submitted to clinvar and many are available for comparison.27 scrp , which is derived directly from clinical reportsand thus benign and likely benign variants are under - represented from this submitter ( table 1 and data supplement ) is an exception . general population allele frequencies for these variants were determined by using the exac database , 28 the 1000 genomes project phase iii database , 29 and the exome variant server.30 common variants were defined as those with allele frequencies greater than 1% in any of these databases . 
these patients variants were also part of the clinvar data set described above . cis were computed by using the wilson method.31 evaluation of the scientific evidence that underlies discordant classifications was performed according to the most recent acmg / amp recommendations.8 our methods for estimating the number of patients who were expected to have discordant variants are detailed in the data supplement . methods classifications of brca1 / 2 variants were extracted from the clinvar may 2016 release . 
variants in clinvar are classified as pathogenic , likely pathogenic , vus , likely benign , or benign , which is consistent with acmg / amp terminology.8 laboratory - specific classification categoriesfor example , deleterious instead of pathogenic or polymorphism instead of benignare mapped to thestandardized nomenclature . 
we thus excluded data from research laboratories , consortia , smallerpossibly less results there were 5 , 124 brca1 / 2 variants submitted to clinvar by seven groups that met our inclusion criteria ( table 1 )  . 
nearly 90% of these variants ( 1 , 769 of 2 , 006 ) were rare , having allele frequencies less than 0.05% in all of the general population databases we examined and less than 0.1% in our clinical database ( fig 1 )  . 
classified variants comparable variants full name in clinvar most recent classification evidence provided note clinvar submitter ambry scrp / myriad genetics invitae genedx counsyl cheo emory total 2 , 792 2 , 327 1 , 998 1 , 216 5 , 124 1 , 613 1 , 351 1 , 367 2 , 006 ambry genetics february 2015 sharing clinical reports december 2015 project invitae genedx counsyl march 2016 october 2015 february 2015 benign and likely benign variants are under - reported no vus submitted molecular genetics dates not provided diagnostic laboratory , childrens hospital of eastern ontario emory genetics laboratory june 2015 note . 
ambry and counsyl had not submitted brca1 / 2 updates for more than 1 year , although all data sets shown here met our study inclusion criteria ( data supplement )  . abbreviation : scrp , sharing clinical reports project ; vus , variants of uncertain significance . variants were a representative subset of 5 , 124 clinvar variants in overall properties , with an expected bias away from rare variants , albeit small in magnitude ( table 2 )  . 
clinvar data were also representative of those observed in clinical practice , with some submitter - specific exceptions ( table 1 and data supplement )  . we compared variant classifications in terms of whether they would or would not potentially affect clinical management ( see methods )  . 
on the basis of the prevalence distribution of variants in clinical testing , we calculated the expected concordance on a per - patient basis to be 99.7% ( fig 3 ; data supplement )  . 
this concordance rate is similar to that reported ( 99.8% ) in a prior study of approximately 1 , 000 prospectively accrued patients that compared acmg / amp - based classifications with those from myriad genetics.32 , 33 a feature of clinvar is that it records the date on which each classification was made , thereby allowing us to consider whether the high concordance we observed could be a result of laboratories being overly influenced by each others prior classifications . 
such influence would be most concerning in the case of myriad genetics classifications submitted by scrp , for which underlying evidence is unavailable for other laboratory directors to evaluate.14 we saw no evidence of such bias , as 99.4% ( 503 of 506 ) of classifications that predated a myriad genetics / scrp entry were concordant with it compared with 99.1% ( 2 , 385 of 2 , 406 ) that postdated it ( data supplement )  . 
these rates are not significantly different . although classifications from the enigma consortium ( evidence - based network from the interpretation of germline mutant alleles ) 34 were not considered in our comparison , they can provide evidence that clinical laboratories use in their classifications . 
although clearly important , rare missense variants are infrequently observed in patients6.3% prevalence in our clinical data set . rare protein truncating and silent variants were also numerous ( 439 of 2 , 006 and 173 of 2 , 006 , respectively ; 30.5% of the data set together ) and were concordantly classified with one exception . other discordant variants were in canonical rna splice sites ( five of 30 ) or an intron ( two of 30 ) or were in - frame deletions ( two of 30 )  . 
finally , two truncating mutations in the last coding exon of brca2 had discordant classifications . to gain insight into the basis for the small number of discordant classifications , we examined all publicly available evidence for and against pathogenicity for each of the 30 discordant variants . 
we also contacted submitting laboratories regarding specific discordant classifications , particularly those for variants with three or more submitters . we found two common explanations for discordant classifications ( data supplement ) : in seven variants , there was a historical difference , meaning that one or more classifications in clinvar was out of date , and although the variant had been reclassified , thus becoming concordant , updates had not yet been submitted to clinvar . 
this mutation causes upregulation of an endogenous alternate rna isoform missing exons 9 and 10 but that seems to provide brca1 functionality.36 this result suggests that laboratory directors should carefully evaluate other mutations in exons 9 and 10 and highlights the complexities of variant classification that laboratory directors must consider in some cases . overall , however , our analysis suggests that a high level of concordance should , perhaps , have been expected . 
nevertheless , the reports significance for clinical management decisions remain similar . furthermore , we explored the likely cause of the few observed discordances and found that approximately one half resulted from out - of - date classifications or submission errors , whereas the remainder were likely expert judgment differences regarding the strength or quality of particular scientific evidence . 
we were pleased that all but one laboratory responded collaboratively to requests for detailed information about their classificationsdespite those requests coming directly from a commercial competitor ( invitae )  . 
this process of identifying and reconciling differences was made possible by shared data in a central and unrestricted public database ( clinvar )  . our findings might at first seem to be at odds with other studies that compared variant classifications . a study by vail et al42 compared the interpretation of approximately 2 , 000 brca1 / 2 variants among a number of public databases and found greater discordance than we report . 
of importance , it incorporated data from research laboratories , older data , and data from curated literature databases that were not classified using modern clinical criteria.8 , 43 , 44 furthermore , it counted differences that would not significantly change management , for example , vus versus likely benign . 
discordances that maxwell et al found were in other cancer genesonly recently incorporated into testsfor which less information is generally available and thus discordance may indeed be higher than it is for brca1 / 2 . separately , balma~na et al46 , 47 examined variants in cancer genes other than brca1 / 2 in the prompt registry.48 the authors found 19 unique variants , which represent 57 of 603 comparable test findings ( 9.5% ) , that had two or more significantly 5 , 000 10 , 000 15 , 000 20 , 000 25 , 000 30 , 000 35 , 000 no . 
other variant typesnotably rare missense and splice - site changesrequire additional experimental or genetic data to classify , but relatively few patients ( , 7% ) carry any such variants , and they are often ( 97.6% ) concordant across laboratories ( table 2 )  . 
when not considered vus , these variants are usually classified as benign , or likely benign , and not pathogenic ( data supplement )  . indeed , a comparison of clinvar releases over the past 2 years shows that most ( 94.5% ) missense vus , when reclassified , are downgraded to benign or likely benign and thus remain not clinically actionable , which is consistent with prior studies.37 discussion in this study , we analyzed publicly available data from the clinvar database and found remarkably few clinically significant discordances in the classifications of more than 2 , 000 variants in two wellcharacterized cancer risk genes , brca1 and brca2 . 
the observation that all discordant variants were rare , although most rare variants remained concordant , suggests that roughly one of 500 patients would be expected to receive results that would significantly change clinical management from the various laboratories in this study . 
percentages indicate the fraction of all variants in this study ( column 2 ) , comparable variants ( those with two or more submitters ; column 3 ) , and concordance for each variant class ( columns 4 and 5 )  . 
clinical prevalence ( column 6 ) indicates the fraction of patients in our clinical database who carry one or more such variants , regardless of pathogenicity ( many are indeed classified benign )  . 
the authors concluded that conflicting interpretation is frequent and may have implications for medical management.48 ( p 46 ) we examined current ( september 30 , 2016 ) clinvar entries for all 19 of these variants and found that six had discordant interpretations only from a nonclinical source ( most commonly , omim49 ) , whereas all clinical laboratories , in fact , agreed with each other.47 one variant was no longer discordant after a 1 - year - old , but more recently submitted , reclassification . 
two low - risk variants had discordance that was attributable to the fact that nomenclature and classification criteria for such variants are not standardized under current acmg guidelines , yet most laboratories still agreed . 
we count 10 variants from balma~na et al , representing 2.2% of findings ( 13 of 603 ) , having a clinically substantial discordance between clinical testing laboratories , 4.3 - fold fewer than the 57 of 603 they report . some of this remaining discordance in non - brca1 / 2 genes seems to be attributable to factors we describe above for brca1 / 2 , for example , older data , although , unfortunately , balma~na et al46 did not contact submitting laboratories to understand the basis of discordance as we did . in another study , the national institutes of health funded clinical sequencing exploratory research consortium performed an experiment in which 99 variants in various genesbiased toward relatively challenging caseswere classified by up to nine laboratories.50 although many classification differences were observed , only a fraction would change management , and only five variants in brca1 / 2 were included . 
of importance , the authors found that sharing classifications among laboratories , thus identifying discordances , enabled discussions that resolved many of the differences and contributed to an overall higher quality than any one laboratory could achieve alone . our study and those mentioned above highlight important best practices in the use of public databases . 
the only classification discordances we observed were in rare variants , which few patients carry , and most rare variants were completely concordant when observed by multiple laboratories ( data supplement )  . sources are valuable and important to centralize and share , database users must apply good judgment and quality control . 
they must pay attention to dates , as variant classifications can become outdated , for example , when new scientific evidence is published . moreover , users must consider whether a classification originates from a clinical laboratory that rigorously follows guidelines - based classification procedures or from a submitter who may have applied a different standard . 
at present , only two of the laboratories included in this study genedx and invitae provide the evidence that supports the classification of specific variants in their clinvar submissions , a situation that we hope will change . 
other laboratories include evidence only in patient reports , but these are not broadly available for both logistical and patient privacy reasons . although our analysis shows that clinically significant disagreements in brca1 / 2 variant classification are infrequent , they are , of course , important to patients and clinicians . 
we believe it is essential for the genetics community to resolve these differences collaboratively , as is standard practice in other areas of oncology , to deliver the best possible patient care.21 - 23 , 38 - 41 , 50 our study supports others in demonstrating that collaborative interaction among laboratories improves the quality of clinical testing.39 - 41 , 50 unlike proprietary databases , clinvar is freely open to all and makes such collaboration possible on a global scale . 
although laboratories with proprietary databases have made claims of superior accuracy , such claims are not subject to detailed and ongoing independent review.14 , 17 indeed , our observation of high concordance across laboratories calls into question some of those claims . 
we note that semipublic databases with restrictive licensing terms , such as brca share , 51 - 53 can present many of the same challenges that are encountered with proprietary databasesfor example , license restrictions prevented the consideration of such data in this study . 
we also note that patient registries , including prompt , although highly valuable for other reasons , do not address the needs that clinvar does.14 , 21 for these reasons , the open sharing of deidentified variant classifications is recommended by the ama , acmg , national society for genetic counselors , and other professional societies . 
in collaboration with international groups , the national institutes of health has recently funded initiatives , including clingen21 , 22 and the brca exchange , 7 that leverage clinvar , the literature , and other resources , to share , compare , and reconcile variant classifications , thus continually improving this important aspect of precision oncology . 
however , at least one major laboratory , myriad genetics , has revised its terms of service to prohibit ordering clinicians from sharing deidentified variant classifications , 54 which is how scrp data used in this study were obtained . 
myriad genetics has also historically resisted requests from patients for their unreported benign variants , prompting legal action by the american civil liberties union.55 such restrictions will make ongoing comparative analyses impossible . 
lincoln employment : invitae stock and other ownership interests : invitae travel , accommodations , expenses : invitae shan yang employment : invitae stock and other ownership interests : invitae melissa s . 
cline no relationship to disclose yuya kobayashi employment : invitae stock and other ownership interests : invitae can zhang no relationship to disclose scott topper employment : invitae stock and other ownership interests : invitae david haussler no relationship to disclose benedict paten stock and other ownership interests : bioturing robert l . 
nussbaum employment : invitae leadership : invitae , personalis , complete genomics honoraria : baxter ( i ) , genzyme , personalis , complete genomics consulting or advisory role : baxter ( i ) , invitae , personals , complete genomics research funding : baxter ( i ) patents , royalties , other intellectual property : national institutes of health , university of california ( inst ) , national institutes of health , university of california expert testimony : ariosa travel , accommodations , expenses : baxter ( i ) , genzyme acknowledgments we are deeply grateful to the laboratories and other groups who are working to advance medical care by submitting data to the clinvar database . 
we particularly thank the staff at scrp , ambry genetics , genedx , counsyl , the cheo molecular genetics laboratory , and the emory genetics laboratory for their hard work on data submissions . 
we thank george riley ( national institutes of health / national center for biotechnology information ) and salina chan ( university of california , san francisco ) for providing updated scrp data . 
we thank olga jarinova and hussein daoud ( cheo molecular genetics laboratory ) , jill dolinsky and tina pesaran ( ambry genetics ) , peter kang ( counsyl ) , and kathryn garber ( emory genetics laboratory ) for clarifying aspects of their data in clinvar . 
whittemore as , gong g , john em , et al : prevalence of brca1 mutation carriers among us non - hispanic whites . cancer epidemiol biomarkers prev 13 : 2078 - 2083 , 2004 6 . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
lindor nm , goldgar de , tavtigian sv , et al : brca1 / 2 sequence variants of uncertain significance : a primer for providers to assist in discussions and in medical management . 
angrist m , cook - deegan r : distributing the future : the weak justifications for keeping human genomic databases secret and the challenges and opportunities in reverse engineering theappl transl genomics 3 : 124 - 127 , 2014 15 . 
pruss d , morris b , hughes e , et al : development and validation of a new algorithm for the reclassification of genetic variants identified in the brca1 and brca2 genes . 
cook - deegan r , conley jm , evans jp , et al : the next controversy in genetic testing : clinical data as trade secrets ? eur j hum genet 21 : 585 - 588 , 2013 18 . 
acmg board of directors : laboratory and clinical genomic data sharing is crucial to improving genetic health care : a position statement of the american college of medical genetics and genomics . 
nucleic acids res 44 ( d1 ) : d862 - d868 , 2016 sharing / sharing - clinical - reports - project - scrp / symp biocomput 22 : 166 - 176 , 2016 285 - 291 , 2016 variation . 
lincoln se , kobayashi y , anderson mj , et al : a systematic comparison of traditional and multigene panel testing for hereditary breast and ovarian cancer genes in more than 1000 patients . 
spurdle ab , healey s , devereau a , et al : enigmaevidence - based network for the interpretation of germline mutant alleles : an international initiative to evaluate risk and clinical significance associated with sequence variation in brca1 and brca2 genes . 
rosenthal et , bowles kr , pruss d , et al : exceptions to the rule : case studies in the prediction of pathogenicity for genetic variants in hereditary cancer genes . 
wong - brown m , mcphillips m , gleeson m , et al : when is a mutation not a mutation : the case of the c.5942a.c splice variant in a woman harbouring another brca1 mutation in trans . 
murray ml , cerrato f , bennett rl , et al : follow - up of carriers of brca1 and brca2 variants of unknown significance : variant reclassification and surgical decisions . 
vail pj , morris b , van kan a , et al : comparison of locus - specific databases for brca1 and brca2 variants reveals disparity in variant classification within and among databases . 
maxwell kn , hart sn , vijai j , et al : evaluation of acmg - guideline - based variant classification of cancer susceptibility and non - cancer - associated genes in families affected by breast cancer . 
balma~na j , digiovanni l , gaddam p , et al : conflicting interpretation of genetic variants and cancer risk by commercial laboratories as assessed by the prospective registry of multiplex testing . 
nussbaum rl , yang s , lincoln se : clinical genetics testing laboratories have a remarkably low rate of clinically significant discordance when interpreting variants in hereditary cancer syndrome genes . 
amberger js , bocchini ca , schiettecatte f , et al : omim.org : online mendelian inheritance in man ( omim ) , an online catalog of human genes and genetic disorders . 
amendola lm , jarvik gp , leo mc , et al : performance of acmg - amp variant - interpretation guidelines among nine laboratories in the clinical sequencing exploratory research consortiuam j hum genet 98 : 1067 - 1076 , 2016 51 . 
caputo s , benboudjema l , sinilnikova o , et al : description and analysis of genetic variants in french hereditary breast and ovarian cancer families recorded in the umd - brca1 / brca2 databases . 
 genetic alterations detected in cell - free dna are associated with enzalutamide and abiraterone resistance in castration - resistant prostate cancer samantha torquato , phd1 ; aparna pallavajjala , ms1 ; alexa goldstein , ms1 ; patricia valda toro , ms1 ; john l . 
hurley , phd1 purpose androgen receptor ( ar ) gene alterations , including ligand - binding domain mutations and copy number ( cn ) gain , have yet to be fully established as predictive markers of resistance to enzalutamide and abiraterone in men with metastatic castration - resistant prostate cancer ( mcrpc )  . 
the goal of this study was to validate ar gene alterations detected in cell - free dna ( cfdna ) as markers of enzalutamide and abiraterone resistance in patients with mcrpc . methods patients with mcrpc ( n = 62 ) were prospectively enrolled between 2014 and 2018 . 
blood was collected before therapiesenzalutamide ( n = 25 ) , abiraterone ( n = 35 ) , or enzalutamide and abiraterone ( n = 2 ) and at disease progression . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction next - generation therapies that target the androgen androgen receptor ( ar ) axis , such as abiraterone and enzalutamide , have improved survival outcomes for men with metastatic castration - resistant prostate cancer ( mcrpc ) , 1 - 4 but both primary and acquired resistance to these drugs continue to be a substantial clinical challenge . 
resistance mechanisms are not fully understood ; however , some forms of resistance likely involve alterations to ar , including amplication and ligand - binding domain ( lbd ) missense mutations . 
although rare in primary prostate cancers , 5 - 7 ar gene alterations are highly prevalent in mcrpc.8 - 13 metastatic tissue biopsies as a sole means to detect and observe changes in ar status is impractical , and thus cell - free dna ( cfdna ) is gaining traction as a minimally invasive and easily obtainable tumor biopsy surrogate . 
previous studies using cfdna from the blood to evaluate the association of ar gene aberrations with resistance to abiraterone and enzalutamide are inclusive.14 - 17 ar copy number ( cn ) gain18 , 19 and / or amplication20 or detection of two or more ar mutations20 have been associated with worse outcomes to such therapies as abiraterone and enzalutamide . 
the ar splice variant ar - v7 is associated with resistance to enzalutamide and abiraterone21 - 23 and is also associated with increased ar cn.24 in addition to ar , alterations in other genes , including tumor protein p53 ( tp53 ) , phosphatase and tensin homolog ( pten ) , and breast cancer gene 2 ( brca2 ) , are enriched in lethal prostate cancer.8 - 11 studies support the idea that lineage plasticity from an ardependent to an ar - independent state through loss of tp53 and retinoblastoma - associated protein 1 ( rb1 ) mediates resistance to ar - targeted therapies.25 - 28 consistent with this , tp53 defects have been shown to be associated with worse outcomes with abiraterone and enzalutamide therapies.17 the role of brca2 and other homology - directed repair ( hdr ) genes in mediating resistance to enzalutamide and abiraterone has not been denitively determined . 
the secondary goal was to determine if alterations in other genes that are enriched in lethal prostate cancer , including tp53 , pten , and brca2 , were associated with response to enzalutamide and abiraterone . in this study , high circulating tumor dna ( ctdna ) burden was signicantly associated with prostate - specic antigen ( psa ) ( pfs ) , and overall survival ( os )  . 
study limitations , including sample size and patient heterogeneity , necessitate larger and prospective validation of the association of plasma ar status with outcomes . response , progression - free survival methods patient information , study end points , sample collection , deep next - generation sequencing ( ngs ) , sequence alignment and analysis of variants , cn variation , estimation of ctdna fraction , and statistical analyses are found in the data supplement . results patient cohort patient characteristics are listed in table 1 . 
clinvarannotated pathogenic or likely pathogenic missense mutations , truncating mutations , and / or cn alterations were detected in cfdna from 89% of patients before therapy initiation and in 92% of patients at disease progression ( figs 1a - 1d and data supplement )  . ctdna total cfdna concentration before therapy was associated with psa ( p = .002 ; data supplement )  . 
nearly all patients ( 61 of 62 ) had detectable cn variation ( s ) and / or mutation ( s ) with an allelic frequency above the 1% cutoff before therapy ( figs 1b and 1c )  . 
multivariable analyses controlled for ctdna high . abbreviations : ar , androgen receptor ; atm , ataxia - telangiectasia mutated gene ; brca1 / 2 , breast cancer gene 1 / 2 ; cn , copy number ; ctdna , circulating tumor dna ; lbd , ligand - binding domain ; or , odds ratio ; pi3k , phosphoinositide 3 - kinase ; psa , prostate - specic antigen ; rb1 , retinoblastoma - associated protein 1 ; tp53 , tumor protein 53 ; wnt , wingless - type mmtv integration site . h875y mutation had psa responses on abiraterone ( fig 2c )  . 
 ( b ) total number of protein - altering genetic changes in 46 genes detected by next - generation sequencing ( ngs ) of cfdna from 62 patients before abiraterone ( abi ) and enzalutamide ( enza ) therapy . 
 ( c ) genetic alterationscopy number ( cn ) status , clinvar pathogenic / likely pathogenic missense and germline mutations , and truncating mutationsin 46 genes detected by ngs of cfdna from 62 patients before abiraterone and enzalutamide therapy in order of best psa response . 
 ( d ) total number of genetic alterationscn , clinvar pathogenic / likely pathogenic missense and germline mutations , and truncating mutationsin 46 genes detected by ngs of cfdna from 62 patients before abiraterone and enzalutamide therapy . 
ar , androgen receptor ; ctdna , circulating tumor dna . defectspathogenic mutations and / or cn losswere not associated with psa response ( p = .602 ) or pfs ( p = .314 ; tables 2 and 3 )  . 
progression - free survival ( pfs ) : pathogenic androgen receptor ( ar ) ligand - binding domain ( lbd ) mutations are associated with a shorter time to progression . 
 ( a ) kaplan - meier method and log - rank test were used to determine median time to progression for patients who had high versus low circulating tumor dna ( ctdna ) before therapy . 
 ( b ) waterfall plot of best prostate - specic antigen ( psa ) response for all patients ( n = 62 ) after therapy as determined by best percentage fold change in psa . 
wingless - type mmtv integration site pathway defects involving genetic alterations in adenomatous polyposis colicn loss and / or truncating mutationsand - catenincn gain and pathogenic missense mutationswere detected in nearly 15% of patients before to therapy ( figs 1c and 1d )  . 
analysis of brca2 , and atm did not increase prognostic signicance . discussion liquid biopsies using cfdna as a tumor analyte are rapidly being developed for cancer diagnostics of solid tumors.35 - 37 when obtained concurrently , plasma - derived cfdna is highly concordant with tissue biopsies for tumor - specic genetic alterations.38 , 39 as a result of their advantages over traditional tissue biopsies , including the ease of accessibility for sequential monitoring of cancer dynamics and recapitulation of tumor heterogeneity , clinical development of cfdna has the potential to advance prostate cancer precision medicine.40 mechanisms of resistance to abiraterone and enzalutamide likely involve alterations to androgenar axis signaling . 
deregulation of these pathways likely mediates resistance to androgenar axis therapies . concurrent tp53 and rb1 defects are highly enriched in ar - independent neuroendocrine mcrpc compared with adenocarcinoma mcrpc.42 combined tp53 and rb1 loss has been shown to promote lineage switching from an ardependent to an ar - independent state41 , 43 , 44 and consequent resistance to ar - targeted therapies . 
similar to tp53 , genetic alterations in pten are enriched in mcrpc compared with metastatic castration - sensitive prostate cancer and localized prostate cancer.11 studies suggest that pten loss may mediate castration resistance by downregulating ar , 25 - 28 thereby supporting a rationale for combined inhibition of pi3k and arin pten - decient mcrpcs.45 , 46 association of pathogenic mutations in hdr genes with response to abiraterone and enzalutamide therapy is conicting . 
a clinical trial in patients with mcrpc suggested that genetic alterations in hdr genes that were detected in metastatic biopsy tissue may be associated with longer pfs when on abiraterone therapy.29 concordant ndings were observed in a second study that supported the idea that patients with mcrpc harboring a germline brca1 / 2 or atm mutation may also have improved outcomes to abiraterone and enzalutamide.30 in contrast , another study showed that truncating mutations in brca2 and atm detected in cfdna were associated with a shorter time to progression on abiraterone and enzalutamide therapies in treatment - nave patients with mcrpc.17 in our study , collective somatic and germline genetic alterations were also not associated with worse outcomes to enzalutamide and abiraterone . 
association differences may reect variables , such as sample size , prior treatment status , disease burden , disease heterogeneity , somatic versus germline , and single versus dual loss . 
certainly , additional prospective investigation is needed to determine the clinical signicance of hdr mutations as predictive markers to abiraterone and enzalutamide therapies . in the current study , many patients had detectable alterations that could serve as potential therapeutic targets . previous studies have shown that patients with mcrpc with either germline or somatic mutations in hdr genes achieved signicant responses to olaparib47 and to abiraterone plus veliparib.29 more than one quarter of patients in our study had a deleterious germline or somatic brca1 , brca2 , or atm mutation detected before therapy or at disease progression , which suggests that these patients may benet from therapies that target poly ( adp - ribose ) polymerase or platinum - based chemotherapy.29 , 47 , 48 in addition , immunotherapy trials have been largely unsuccessful in men with mcrpc49 ; however , rare responders have been reported.50 a seminal clinical trial demonstrated that microsatellite instable cancers caused by mismatch repair ( mmr ) gene deciency were sensitive to programmed death - 1 blockade , perhaps because of the formation of neoantigens resulting from increased mutational burden.51 inactivation of mmr genes and elevated mutational burden have been detected in some men with aggressive prostate cancers.33 , 52 , 53 one patient in our study had a detectable noncanonical mmr gene mutation in his cfdna and a correspondingly high fig 3 . 
 ( a ) kaplan - meier method and log - rank test were used to determine median os for patients who had high versus low circulating tumor dna ( ctdna ) before therapy . 
 ( b ) kaplan - meier method and log - rank test were used to determine median os for patients who had androgen receptor ( ar ) copy number ( cn ) gain before therapy . 
 ( c ) gene schematic illustrating deleterious tp53 mutations detected by deep next - generation sequencing ( ngs ) of cellfree dna ( cfdna ) before abiraterone and enzalutamide therapies and at disease progression while on therapy . 
 ( d ) kaplan - meier method and log - rank test were used to determine median os for patients who had tp53 defectscn loss and or clinvar pathogenic / likely pathogenic mutationsbefore therapy . 
 ( e ) kaplan - meier method and log - rank test were used to determine median os for patients who had both tp53 and retinoblastomaassociated protein 1 ( rb1 ) defects compared with patients who had tp53 defects but were rb1 intactcn loss and or clinvar pathogenic / likely pathogenic mutationsbefore therapy . 
 ( f ) kaplan - meier method and log - rank test were used to determine median os for patients who had pi3k pathway defectscn loss and / or truncating mutations in phosphatase and tensin homolog and / or cn gain of pik3cabefore therapy . association of pi3k defects with os controlled for ctdna burden using multivariable proportional hazards regression modeling . 
 ( g ) kaplan - meier method and log - rank test were used to determine median os for patients who had wingless - type mmtv integration site ( wnt ) pathway defectscn loss and / or truncating mutations in adenomatous polyposis coli and / or cn gain and / or pathogenic missense mutations in - cateninbefore therapy . 
consistent with other reports , patients with prior exposure to abiraterone and enzalutamide experienced worse outcomes.54 - 59 statistical signicance was not reached , likely because of the small overall sample size and the few patients with prior therapy . 
ngs protocols were adjusted on the basis of total input , but for patients with low input the lack of genetic alteration detection was considered indeterminate as opposed to negative . 
in addition , mutations in such genes as including ar - v7 , because of tp53 and atm detected in cfdna at low allelic frequencies may be false positives as a result of clonal hematopoiesis.60 corresponding tissue was not available for all samples to conrm tp53 status , and future studies will examine both prostate tumor tissue and blood leukocytes for genetic alterations . 
a nal limitation was our inability to evaluate ar the resplice variants , quirement of circulating tumor cells or whole - blood rna . the presence of ar - v7 is certainly another established mechanism of primary and acquired resistance to nextgeneration hormonal therapies.21 - 23 future studies should aim to simultaneously analyze the full complement of ar aberrations , including gene mutations , amplications , genomic structural rearrangements , and mrna splice variants , from a single liquid biopsy . in summary , our ndings indicate ctdna burden was highly associated with worse outcomes to enzalutamide and abiraterone . 
eisenberger leadership : veru stock and other ownership interests : veru honoraria : sano , pzer consulting or advisory role : astellas pharma , ipsen , bayer , sano , pzer research funding : sano , tokai pharmaceuticals , genentech travel , accommodations , expenses : bayer , astellas pharma , sano , pzer , veru emmanuel s . 
antonarakis honoraria : sano , dendreon , medivation , janssen biotech , essa pharma , astellas pharma , merck , astrazeneca , clovis oncology consulting or advisory role : sano , dendreon , medivation , janssen biotech , essa pharma , astellas pharma , merck , astrazeneca , clovis oncology research funding : janssen biotech ( inst ) , johnson & johnson ( inst ) , sano ( inst ) , dendreon ( inst ) , aragon pharmaceuticals ( inst ) , exelixis ( inst ) , millennium pharmaceuticals ( inst ) , genentech ( inst ) , novartis ( inst ) , astellas pharma ( inst ) , tokai pharmaceuticals ( inst ) , merck ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , constellation pharmaceuticals ( inst ) patents , royalties , other intellectual property : coinventor of a biomarker technology licensed to qiagen travel , accommodations , expenses : sano , dendreon , medivation bruce j . 
park leadership : loxo pharmaceuticals stock and other ownership interests : loxo pharmaceuticals consulting or advisory role : horizon discovery , foundation medicine , loxo pharmaceuticals , casdin capital , h3 biomedicine , jackson laboratory for genomic medicine , eli lilly speakers ' bureau : astrazeneca research funding : foundation medicine , abbvie , pzer patents , royalties , other intellectual property : royalties paid through inventions at johns hopkins university by horizon discovery travel , accommodations , expenses : eli lilly , loxo pharmaceuticals paula j . 
hurley stock and other ownership interests : loxo pharmaceuticals ( i ) honoraria : foundation medicine ( i ) , roche ( i ) , h3 biomedicine ( i ) , casdin capital ( i ) , loxo pharmaceuticals ( i ) , eli lilly ( i ) consulting or advisory role : loxo pharmaceuticals ( i ) , foundation medicine ( i ) , roche ( i ) , h3 biomedicine ( i ) , casdin capital ( i ) , lilly ( i ) patents , royalties , other intellectual property : cell lines at horizon ( i ) no other potential conicts of interest were reported . acknowledgment we thank the patients who consented to participate in this study . 
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 o identifying a clinically applicable mutational burden threshold as a potential biomarker of response to immune checkpoint therapy in solid tumors purpose an association between mutational burden and response to immune checkpoint therapy has been documented in several cancer types . 
the potential for such a mutational burden threshold to predict response to immune checkpoint therapy was evaluated in several clinical datasets , where mutational burden was measured either by whole - exome sequencing or by using commercially available sequencing panels . anshuman panda anil betigeri kalyanasundaram subramanian jeffrey s . 
johnson gyan bhanot shridar ganesan author affiliations and support information ( if applicable ) appear at the end of this article . corresponding author : shridar ganesan , md , phd , 195 little albany st , new brunswick , nj 08903 ; e - mail : ganesash@ cinj.rutgers.edu. methods whole - exome sequencing and rna sequencing data of 33 solid cancer types from the cancer genome atlas were analyzed to determine whether a robust immune checkpoint activating mutation ( icam ) burden threshold associated with evidence of immune checkpoint activation exists in these cancers that may serve as a biomarker of response to immune checkpoint blockade therapy . results we found that a robust icam threshold , associated with signatures of immune checkpoint activation , exists in eight of 33 solid cancers : melanoma , lung adenocarcinoma , colon adenocarcinoma , endometrial cancer , stomach adenocarcinoma , cervical cancer , estrogen receptorpositive / human epidermal growth factor receptor 2negative breast cancer , and bladder - urothelial cancer . 
in published datasets of melanoma , lung adenocarcinoma , and colon cancer , patients with icam - positive tumors had significantly better response to immune checkpoint therapy compared with those with icamnegative tumors . 
receiver operating characteristic analysis using the cancer genome atlas predictions as the gold standard showed that icam - positive tumors are accurately identifiable using clinical sequencing assays , such as foundationone ( foundation medicine , cambridge , ma ) or strandadvantage ( strand life sciences , bangalore , india )  . 
icam - positive and icamnegative tumors have distinct mutation patterns and different immune microenvironments . conclusion in eight solid cancers , a mutational burden threshold exists that may predict response to immune checkpoint blockade . 
2017 by american society of clinical oncology introduction antiprogrammed death - 1 ( pd - 1 ) and anti cytotoxic t - lymphocyte antigen - 4 ( ctla - 4 ) therapy results in prolonged response in some solid cancers.1 - 5 response to ctla - 4 blockade in melanoma1 , 2 and pd - 1 blockade in lung , 3 colorectal , 4 and urothelial5 cancers is associated with increased mutational burden . 
although this association between mutational burden and response to immune checkpoint therapy is documented in some cancers , 1 - 5 it is unclear whether this relationship exists in other cancers . 
using rna sequencing ( rna - seq ) and whole - exome sequencing data for all solid cancers in the cancer genome atlas ( tcga ) , we asked the following three questions : ( 1 ) is there a mutational burden threshold above which there is a signature of immune activation and immune checkpoint pathway upregulation in some cancers ? ( 2 ) can this threshold be identified using clinical sequencing assays that interrogate a subset of the exome ? ( 3 ) does this threshold identify responders to immune checkpoint therapy ? we found that a robust immune checkpoint activating mutation ( icam ) threshold satisfying the first two conditions exists in eight classes of solid tumors . 
in published datasets where condition ( 3 ) is testable , patients with icam - positive cancers have significantly better clinical response to immune checkpoint therapy than patients with icamnegative cancers . 
analysis of a cohort of patients with melanoma , sequenced using the foundationone assay ( foundation medicine , cambridge , ma ) , 6 demonstrated that patients with icam - positive melanomas had significantly better response to pd - 1 blockade than patients with icam - negative melanomas . 
finally , for these cancer types , distinct patterns of mutations are present in icam - positive versus icam - negative tumors , suggesting potentially distinct underlying disease mechanisms . methods data collection and processing mutation data were obtained from tcga ( maf + files )  . 
all mutation annotation format files current as of january 31 , 2016 ( public and protected ) were downloaded , mapped from hg18 to hg19 using liftover if necessary ( ucsc.edu / cgi - bin / hgliftover ) , re - annotated using oncotator , 7 merged , and de - duplicated . 
for each tumor , rnaseqv2 - scaled estimates from the broad institute broad genome data analysis center ( org ) and tcga data portal were divided by the median value per sample and used to quantify immune infiltration and leukocyte composition by estimate8 and cibersort , 9 respectively . only unambiguous ( p , .05 ) cibersort outputs were used . 
erbb2 focal copy number data and esr1 mrna expression data from the broad genome data analysis center were used to classify breast cancer samples into clinical subtypes ( estrogen receptor [ er ] positive / human epidermal growth factor receptor 2 [ her2 ] negative , er negative / her2 negative , or her2 positive ) , and these subtypes were analyzed separately . 
in addition to cibersort9 p values , two - sided ( p , .05 ) fishers test and log - rank test were used to compare response and survival rates , respectively . 
all bracketed ranges throughout this article correspond to 95% confidence intervals . determination of icam threshold in tcga if nonsynonymous mutational burden is associated with immune checkpoint activation , there should be a mutational burden threshold above which tumors have the following : ( 1 ) cd8 - positive t - cell response ( data supplement ) and ( 2 ) overexpression of immune checkpoint pathways ( data supplement )  . 
for each xi , we tested whether tumors with mutational burden > xi had significantly higher cd8 - positive t - cell activation and pd - 1 / ctla - 4 pathway upregulation compared with tumors with mutational burden , xi ( data supplement )  . 
in eight cancer types , criteria ( 1 ) and ( 2 ) were simultaneously satisfied for several x - values , confirming an association between mutational burden and immune checkpoint activation in those cancer types . 
the presence of tumor - infiltrating lymphocytes was assessed in > 10 fields at the highest magnification , and tumors were graded for lymphocytic density and distribution , on all slides for each case . 
the score per slide was the cumulative grade of density and distribution , with the highest score for each case taken as the final score . melanoma samples used for prospective validation of icam threshold mutation , treatment , and outcome data in a cohort of 196 de - identified samples were obtained under institutional review boardapproved protocols to test the icam threshold for the foundationone assay.6 one hundred eleven samples from the rutgers cancer institute of new jersey and 85 from either the vanderbilt - ingram cancer center or massachusetts general hospital had data from either the 315or 236 - gene foundationone panels . 
one hundred thirteen samples from patients treated with single - agent pd - 1 therapy ( 28 from rutgers cancer institute of new jersey , 85 from vanderbilt - ingram cancer center and massachusetts general hospital ) were stratified by icam status using the mutational burden threshold of either 14 or nine mutations for the foundationone 315and 236 - gene panels , respectively ( data supplement )  . results identification of icam threshold using tcga whole - exome sequencing and rnaseq data for 33 solid cancers , we looked for an icam mutational burden threshold , such that icam - positive tumors ( ie , tumors with mutational burden above the threshold ) had evidence of the following : ( 1 ) immune activation : high cd8a mrna levels , evidence of immune infiltration ( measured by estimate8 ) , and high cd8 - positive t - cell fraction in leukocytes ( measured by cibersort9 ) ; and ( 2 ) upregulation of immune checkpoint pathway gene expression ( ie , pd - 1 , ctla - 4 , and their ligands ; data supplement )  . 
although these tumor features have been previously shown to associate with response to immune checkpoint therapy , 4 , 5 , 12 in this study we used rna - seq data to indirectly measure these criteria . mutational burden has been identified to predict response to immune checkpoint blockade in melanoma , 1 , 2 lung adenocarcinoma , 3 colon adenocarcinoma , 4 and bladder - urothelial cancer.5 we found that , in addition , a robust icam threshold also exists in endometrial cancer , stomach adenocarcinoma , cervical cancer , and erpositive / her2 - negative breast cancers ( data supplement )  . 
a robust mutational burden threshold could not be identified for triple - negative breast cancer and renal cell cancercancer types where immune checkpoint therapy has some clinical activity.13 , 14 as an independent test , a blinded pathologic analysis of high - resolution , whole - slide images of hematoxylin and eosinstained sections from tcga of 15 icam - positive and 15 icam - negative tumors for each cancer type showed that icam - positive tumors had significantly higher lymphocyte infiltration than icam - negative tumors in all eight cancer types ( fig 1c )  . 
for different definitions of mutational burden or different sequencing depths , the thresholds need to be recomputed . icam status predicts response to pd - 1 and ctla - 4 blockade in published data to test the utility of icam , the icam thresholds were evaluated in published datasets for patients treated with ctla - 4 blockade in skin melanoma1 , 2 and pd - 1 blockade in lung adenocarcinoma3 and colon adenocarcinoma4 ( fig 2 ) , with samples stratified into icam - positive and icam - negative classes , as described in the data supplement . 
a robust immune checkpointactivating mutation ( icam ) threshold associated with evidence of immune checkpoint activation can be identified in eight cancer types in the cancer genome atlas ( tcga )  . 
 ( a ) distributions of nonsynonymous mutational burden in eight cancer types ( in log10 scale ) separate the samples into icam - positive ( blue ) and icam - negative ( gold ) subsets . 
 ( b ) immune checkpoint activation criterion ( data supplement ) significantly higher in icam - positive compared with icam - negative is shown in gold for the eight cancer types . 
 ( c ) results from blinded pathologic quantification of lymphocyte infiltration in high - resolution histologic images from tcga of 15 icam - positive and 15 icam - negative tumors in each of the eight cancer types . 
 ( a ) comparison of response in patients with skin melanoma to cytotoxic t - lymphocyte antigen - 4 ( ctla - 4 ) targeted therapy by icam status in two independent datasets.1 , 2 ( b ) comparison of response in patients with lung adenocarcinoma3 and colorectal cancer4 to programmed death - 1 targeted therapy by icam status . 
cb , clinical benefit ; cr , complete response ; dcb , durable clinical benefit ; pd , progressive disease ; pr , partial response . validation of icam threshold in predicting response to immune checkpoint blockade in melanoma using the foundationone assay one hundred ninety - six skin melanoma samples were sequenced using the foundationone assay6 ( data supplement ) and the number of nonsynonymous mutations was calculated per tumor by summing known or likely mutations and variants of unknown significance . 
the icam threshold projected from tcga was estimated as nine or 14 nonsynonymous mutations for foundationone assays that interrogated 236 or 315 genes , respectively ( data supplement )  . 
immune checkpointactivating mutation ( icam ) positive tumors can be identified with high accuracy using routine clinical assays . receiver operating characteristic curves for icam status determination of the cancer genome atlas ( tcga ) samples using only the exons used in foundationone6 ( blue ) and strandadvantage15 ( gold ) assays . 
blca , bladder cancer ; cesc , cervical cancer ; coad , colon adenocarcinoma ; luad , lung adenocarcinoma ; skcm , skin cutaneous melanoma ; stad , stomach adenocarcinoma ; ucec , endometrial cancer . false positive rate ( 1 specificity ) tumors ( but not icam - negative tumors ) had mutations in rnf43 , arid1a , braf , bmpr2 , acvr2a , and rpl22 . 
egfr mutations in lung cancer and apc and tp53 mutations in colon cancer were associated with lower incidences of icampositive tumors , whereas mutations in arid1a in lung cancer and mutations in rnf43 , arid1a , braf , bmpr2 , and acvr2a in colon cancer were associated with higher incidences of icampositive tumors ( data supplement )  . 
tumor suppressor genes rnf43 , 19 acvr2a , 20 and rpl2221 were significantly mutated in icam - positive colon adenocarcinoma ( fig 5 ) , endometrial cancer , and stomach adenocarcinoma ( data supplement ) , but rarely in icam - negative tumors . 
in the other four cancer types , several genes were significantly mutated in icam - positive but not icam - negative tumors and vice versa ( data supplement )  . 
these findings suggest that icam - positive tumors may have distinct underlying driver mutations compared with icam - negative tumors . mutational etiology of icam - positive tumors for each tumor , we estimated the fractional contribution of 30 mutation signatures ( data supplement ) from the catalogue of somatic mutations in cancer22 , 23 ( uk / cosmic / signatures )  . 
compared with icamnegative tumors , icam - positive tumors had a higher contribution from an ultraviolet signature in melanoma and a smoking signature in lung adenocarcinoma ( fig 6a ) , consistent with known pathogenesis of melanoma24 and lung25 cancers . smoking history was also strongly associated with icam status in lung adenocarcinoma ( data supplement )  . in colon adenocarcinoma , endometrial cancer , and stomach adenocarcinoma , icam - positive tumors were enriched in either mismatch repair or pole proofreading defects ( fig 6a )  . 
prospective validation of immune checkpointactivating mutation ( icam ) threshold for melanoma using foundationone6 assay results predicts outcome of immune checkpoint blockade in an independent cohort of patients with melanoma . shown are comparisons of response rates , progression - free survival ( pfs ) , and overall survival in 113 patients with metastatic melanoma treated with single - agent programmed death - 1 ( pd - 1 ) targeted therapy , stratified by icam status using the foundationone assay.6 cr , objective complete response ; pd , progressive disease ; pr , objective partial response ; sd , stable disease ( includes mixed response )  . time ( years ) time ( years ) apolipoprotein b mrna editing enzyme , catalytic polypeptide - like mutation signatures ( fig 6a ) , which builds on the previously reported presence of apolipoprotein b mrna editing enzyme , catalytic polypeptice - like signatures in these cancers.27 in er - positive / her2 - negative breast cancer , approximately 25% of luminal - b tumors were icam positive , whereas , 10% of luminal - a tumors were icam positive ( data supplement )  . 
in all eight cancer types , icam - negative tumors had a higher fractional contribution from deamination of 5 - methylcytosine compared with icam - positive tumors ( fig 6a )  . additional immunologic properties of icam - positive tumors in most of the eight cancer types , icam - positive tumors had significantly higher fractions of natural killer cells in leukocytes and m1 cells in macrophages , and significantly lower fractions of regulatory t cells in t cells ( fig 6b ) , by cibersort9 analysis . 
natural killer cells are involved in immune surveillance , 28 m1 macrophages suppress angiogenesis and induce apoptosis , 29 and regulatory t cells suppress immune response.30 thus icam - positive tumors may have a more favorable immune microenvironment for immune checkpoint therapy . 
these data are consistent with reports suggesting that specific features of immune microenvironments may associate with response to immune checkpoint therapy.31 , 32 sixty - one genes were significantly overexpressed in icam - positive tumors compared with icamnegative tumors in all eight cancer types ( data supplement ) , and approximately 70% of these genes were immune - system related : associated with the interferon - gamma pathway ( ifng , cxcl9 , cxcl10 , and cxcl11 ) and cytotoxic t cells ( cd8a , prf1 , gzma , and gzmb )  . expression of interferon - pathway genes is associated with recruitment of cytotoxic t cells to the tumor microenvironment.33 the immune checkpoint gene lag3 was significantly overexpressed in icam - positive tumors in all eight cancers , suggesting an additional drug target pathway . discussion in eight of 33 solid cancers in tcga , we found a mutational burden threshold ( icam ) that identified tumors with signatures of immune checkpoint activation . 
the icam threshold can be accurately identified using routine clinical sequencing assays . applied to an independent melanoma data set using the foundationone assay , 6 icam - positive status was able to identify responders to singleagent pd - 1 blockade with high accuracy . 
note that these assays do not have germline controls , so some variant calls may be rare germline polymorphisms . however , these should not affect the presence of an icam threshold . 
the thresholds found in this study could be further refined by removing likely germline variants , increasing sequencing depth , analyzing more samples , and conducting a detailed analysis of tumor purity and mutant allele frequency . 
selected significantly mutated genes ( mutsigcv , 17 false discovery rate , 0.1 ) in immune checkpoint activating mutation ( icam ) positive and icam - negative tumors . columns are samples and rows are genes ( white , gene not significantly mutated in that class ; gold , gene significantly mutated in that class but not mutated in that sample ; blue and gray , gene significantly mutated in icam - positive [ blue ] / icam - negative [ gray ] class and mutated in that sample )  . in many cancer types , a robust mutational burden threshold associated with evidence of immune checkpoint activation could not be identified . 
this includes triple - negative breast cancer and renal cell carcinoma , which are cancers with substantial response rates to single - agent immune checkpoint blockade.13 , 14 in these cancers , features other than tumor mutational load are probably responsible for immune checkpoint activation . 
similarly , other features , such as viral etiology , seen in merkel cell cancer , may be strongly immunogenic , yet do not associate with a high nonsynonymous mutational burden.35 , 36 the patterns of mutations present in icampositive tumors reflect the underlying nature of mutagenesis . 
 ( a ) comparison of the fractional contribution of mutation signatures from catalogue of somatic mutations in cancer for etiologies associated with icam - positive versus icamnegative status in the eight cancer types . 
 ( b ) other immune signatures associated with icam status ( natural killer [ nk ] fraction in leukocytes , m1 fraction in macrophages , and regulatory t cell ( t - regs ) fraction in t cells in icam - positive and icam - negative tumors )  . 
similarly , some patients with icam - negative cancers did respond to pd - 1 or ctla - 4 blockade , possibly because of immune responses triggered by mechanisms other than high mutational burden ( eg , expression of exogenous viruses , 35 , 36 expression of endogenous retroviruses40 )  . 
for more information about ascos conflict of interest policy , please refer to or ascopubs.org / po / author - center . anshuman panda no relationship to disclose anil betigeri employment : strand life sciences kalyanasundaram subramanian employment : strand life sciences , syngene international patents , royalties , other intellectual property : patent to predict drug toxicity jeffrey s . 
pavlick employment : foundation medicine stock and other ownership interests : foundation medicine siraj ali employment : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine , patents via seres health on microbiome stuff in non - neoplastic disease ( i ) paul markowski no relationship to disclose howard l . 
kaufman employment : compass therapeutics leadership : compass therapeutics honoraria : amgen , emd serono , merck , celldex , prometheus , turnstone bio , compass therapeutics consulting or advisory role : amgen , merck , merck serono , paometheus , celldex , turnstone bio , bristol - myers squibb , compass therapeutics speakers bureau : merck research funding : amgen ( inst ) , merck ( inst ) travel , accommodations , expenses : emd serono , turnstone bio edmund lattime stock and other ownership interests : johnson & johnson ( i ) patents , royalties , other intellectual property : i am an inventor of an oncolytic virus , for which the patent is held by thomas jefferson university , where i worked from 1989 to 1998 . 
mehnert honoraria : genentech , emd serono consulting or advisory role : merck sharp & dohme , amgen research funding : merck ( inst ) , sanofi ( inst ) , novartis ( inst ) , polynoma ( inst ) , immunocore ( inst ) , amgen ( inst ) , astrazeneca ( inst ) travel , accommodations , expenses : emd serono , merck sharp & dohme other relationship : amgen , emd serono , merck ryan sullivan honoraria : genentech consulting or advisory role : novartis , biodesix , paometheus , amgen , takeda , worldcare clinical , aci clinical , merck , biolinerx research funding : amgen ( inst ) , eli lilly ( inst ) , biomed valley discoveries ( inst ) , merck ( inst ) , deciphera ( inst ) , genentech ( inst ) other relationship : boehringer ingelheim christine m . 
lovly honoraria : novartis , sequenom , qiagen , pfizer , nccn , takeda consulting or advisory role : ariad , clovis oncology , genoptix , novartis , foundation medicine research funding : astrazeneca , novartis travel , accommodations , expenses : pfizer jeffrey sosman honoraria : amgen , merck , array biopharma , bristol - myers squibb consulting or advisory role : amgen , merck , array biopharma , bristol - myers squibb douglas b . 
 shridar ganesan employment : merck ( i ) stock and other ownership interests : ibris , inspirata , merck ( i ) consulting or advisory role : inspirata , novartis patents , royalties , other intellectual property : i hold two patents for digital imaging that may be licensed to ibris and inspirata . travel , accommodations , expenses : inspirata acknowledgment we thank hossein khiabanian , phd , for help with analysis of the foundation medicine mutation data for the rutgers cancer institute of new jersey samples in the prospective cohort . affiliations anshuman panda , ann silk , howard l . 
mehnert , and shridar ganesan , rutgers robert wood johnson medical school , new brunswick ; anshuman panda and gyan bhanot , rutgers university , piscataway , nj ; anil betigeri and kalyanasundaram subramanian , strand life sciences , bangalore , india ; jeffrey s . 
rosenberg je , hoffman - censits j , powles t , et al : atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum - based chemotherapy : a single - arm , multicentre , phase 2 trial . 
koo b - k , spit m , jordens i , et al : tumour suppressor rnf43 is a stem - cell e3 ligase that induces endocytosis of wnt receptors . 
deacu e , mori y , sato f , et al : activin type ii receptor restoration in acvr2 - deficient colon cancer cells induces transforming growth factor - beta response pathway genes . 
rao s , stadanlick je , cai kq , et al : loss of rpl22 promotes tumor progression through regulation of angiogenesis . cancer res 75 , 2015 ( suppl 15 ; abstr 521 ) 22 . 
yuan a , hsiao y - j , chen h - y , et al : opposite effects of m1 and m2 macrophage subtypes on lung cancer progression . sci rep 5 : 14273 , 2015 30 . 
nghiem pt , bhatia s , lipson ej , et al : pd - 1 blockade with pembrolizumab in advanced merkel - cell carcinoma . n engl j med 374 : 2542 - 2552 , 2016 36 . 
kaufman hl , russell j , hamid o , et al : avelumab in patients with chemotherapy - refractory metastatic merkel cell carcinoma : a multicentre , single - group , open - label , phase 2 trial . 
marvel d , gabrilovich di : myeloid - derived suppressor cells in the tumor microenvironment : expect the unexpected . j clin invest 125 : 3356 - 3364 , 2015 39 . 
laboratory tests , magnetic resonance imaging ( mri ) , and positron emission tomographycomputed tomography did not indicate involvement of organs other than the skbecause of the limited cutaneous lesions classified as single system lch , topical corticosteroids were used as first - line treatment for 10 weeks , with no improvement . 
treatment with desmopressin ( initial dosage , 0.4 mg daily divided into three doses ) was started , and polyuria and polydipsia improved . function of the anterior pituitary was not affected . targeted next - generation sequencing of the skin biopsy indicated an activating mutation in mek1 ( p.e102_i103delei ; fig 3 )  . 
for this mutation , ex vivo sensitivity to an mek inhibitor ( meki ) has been reported.1a because the skin and the pituitary gland were affected , the disease was now classified as multisystem lch . 
finally , an off - label treatment with trametinib was offered to the patient after interdisciplinary discussion . after detailed explanation and obtaining informed consent , trametinib ( 2 mg once daily ) was started . an mri performed 4 weeks later showed a significant shrinkage of the pituitary enlargement ( fig 3 ) , and skin manifestations resolved both clinically and histologically ( fig 1 )  . 
positron emission tomographycomputed tomography and mri performed in week 12 of treatment showed a slight increase of the pituitary gland in size ; however , the patient had not been receiving trametinib in the previous 3 weeks . 
importantly , the daily dose of desmopressin could be reduced to 0.1 mg / d on trametinib treatment , and anterior pituitary function remained normal . because of the excellent overall response , a drug holiday was recommended , and trametinib treatment was stopped in week 38 . 
hematoxylin and eosin ( h&e ) staining and immunohistochemistry of langerin of a biopsy taken from cutaneous lesions before therapy ( left panels ) and after 4 weeks of therapy ( right panels )  . 
initially , a band - like infiltrate by histiocytes with reniform nuclei and eosinophilic cytoplasm ( upper left , h&e ) reactive with an antilangerin antibody ( brown , lower left ) was observed in the upper dermis and also perifollicular in the lower dermis . 
roughly , clinical subtypes are divided into single system and multisystem ( ms ) forms.3 limited disease usually causes low morbidity , but ms - lch or leukemic forms are a severe threat for patients . organ involvement can cause significant morbidity in patients with lch . 
standard therapies for ms - lch in adults are cytotoxic regimens.4 , 5 radiotherapy is used to treat pituitary lch ; however , this contributes to gradual and often complete loss of anterior pituitary function.6 , 7 the mitogen - activated protein kinase ( mapk ) pathway is critical for cell survival and proliferation . it can be activated by various receptor tyrosine kinases but also by oncogenic mutations in braf or nras found in melanoma and other human malignancies . 
recently , recurrent oncogenic mutations affecting the mapk pathway have been described in lch.1a , 8 these mutations affect braf in approximately 50% and mitogenactivated protein kinase 1 ( map2k1 / mek1 ) in approximately 25% of patients . 
mutations in these genes seem to be mutually exclusive.8 in a multibasket trial for malignancies harboring brafv600 mutations , targeted therapy with the selective braf inhibitor ( brafi ) vemurafenib showed clinical activity in lch.9 besides selective brafi , other small molecules , such as meki , have been evaluated in cancers with oncogenic mapk activation.9 , 10 it has been proposed that activating mek1 mutations is a difficult target in the treatment of lch.3 however , no clinical data on mek inhibition in patients with lch have been reported so far . ( poly ) chemotherapy and radiation are considered standard of care for treatment of ms - lch.3 - 7 high rates of toxicity are observed in adults , and recovery of endocrine function of the pituitary gland is rarely observed with these therapies . 
in week 4 of therapy with trametinib , a significant shrinkage and only a residual contrast enhancement were found . after short - term treatment break , the posterior pituitary gland appeared slightly enlarged again in week 12 . 
in week 28 , size was normalized . representative sagittal images from t1 contrastenhanced magnetic resonance imaging sequences shown ; arrows indicate affected part of the hypophysis . pretreatment after 4 weeks after 12 weeks after 28 weeks harboring a somatic mutation in mek1 . 
radiotherapy and chemotherapy might eventually still have to be considered in these patients . it remains unclear if meki can induce durable responses in lch and non - lch with lasting functional recovery of organs involved . 
 ( b ) the wildtype braf sequencing result is shown with a dotted line and arrow highlighting the c.t1799 location where a braf v600e mutation would be detected if it had been present . 
moeller , dagmar f uhrer , lisa zimmer , antje sucker , dirk schadendorf , elisabeth livingstone , bastian schilling data analysis and interpretation : matina papapanagiotou , klaus g . 
t1799 uwe hillen honoraria : novartis consulting or advisory role : novartis , roche , therakos research funding : novartis ( inst ) travel , accommodations , expenses : novartis tobias t . 
moeller honoraria : pfizer , henning dagmar f uhrer honoraria : ipsen , sanofi , bayer healthcare pharmaceuticals , eisai , genzyme , sobi , pfizer consulting or advisory role : novartis ( inst ) , ipsen , sanofi , bayer , eisai , genzyme , sobi , pfizer research funding : novartis ( inst ) , pfizer ( inst ) , ipsen ( inst ) , novartis , pfizer travel , accommodations , expenses : ipsen , sanofi , bayer , eisai , genzyme , sobi , pfizer lisa zimmer honoraria : novartis , merck sharp & dohme , bristol - myers squibb , roche , merck , glaxosmithkline consulting or advisory role : novartis , bristol - myers squibb , merck sharp & dohme , roche travel , accommodations , expenses : novartis , bristolmyers squibb , amgen , merck sharp & dohme alexander roesch research funding : novartis travel , accommodations , expenses : roche , bristol - myers squibb , merck sharp & dohme , amgen , novartis klaus g . 
 dirk schadendorf honoraria : amgen , array , astra zeneca , bristol - myers squibb , immunocore , merck sharp & dohme , merck , novartis , pfizer , philogen , pierre fabre , regeneron , roche consulting or advisory role : amgen , array biopharma , astrazeneca , bristol - myers squibb , immunocore , merck sharp & dohme , merck , novartis , pfizer , philogen , pierre fabre , regeneron , roche speakers bureau : amgen , bristol - myers squibb , merck sharp & dohme , novartis , pierre fabre , roche research funding : amgen ( inst ) , array ( inst ) , bristol - myers squibb ( inst ) , immunocore , merck sharp & dohme ( inst ) , novartis ( inst ) , philogen ( inst ) , regeneron ( inst ) , roche ( inst ) , bristol - myers squibb , novartis travel , accommodations , expenses : amgen , array , astra zeneca , bristol - myers squibb , immunocore , merck sharp & dohme , merck , novartis , pfizer , philogen , pierre fabre , regeneron , roche elisabeth livingstone no relationship to disclose bastian schilling honoraria : novartis , roche , bristol - myers squibb , merck sharp & dohme consulting or advisory role : roche , bristol - myers squibb speakers bureau : roche , bristol - myers squibb , merck sharp & dohme research funding : bristol - myers squibb ( inst ) , merck sharp & dohme travel , accommodations , expenses : novartis , roche , bristol - myers squibb , amgen matina papapanagiotou , klaus g . 
m oller i , murali r , m uller h , et al : activating cysteinyl leukotriene receptor 2 ( cysltr2 ) mutations in blue nevi . mod pathol 30 : 350 - 356 , 2017 2 . 
girschikofsky m , arico m , castillo d , et al : management of adult patients with langerhans cell histiocytosis : recommendations from an expert panel on behalf of euro - histio - net . 
duan mh , han x , li j , et al : comparison of vindesine and prednisone and cyclophosphamide , etoposide , vindesine , and prednisone as first - line treatment for adult langerhans cell histiocytosis : a single - center retrospective study . 
endocrine 48 : 949 - 956 , 2015 imashuku s , kudo n , kaneda s , et al : treatment of patients with hypothalamic - pituitary lesions as adult - onset langerhans cell histiocytosis . 
hyman dm , puzanov i , subbiah v , et al : vemurafenib in multiple nonmelanoma cancers with braf v600 mutations . 1919 - 1923 , 2010 n engl j med 373 : 726 - 736 , 2015 10 . 
walsh , md1 , 2 ; rosalba sacca , phd1 ; temima wildman , ms1 ; kimberly amoroso , ms1 ; jennifer kennedy , ms1 ; liying zhang , md , phd1 ; ozge birsoy , phd1 ; diana mandelker , md , phd1 ; zoe steinsnyder1 ; alicia latham , md1 , 2 ; maria i . 
stadler , md1 , 2 ; and kenneth oft , md , mph1 , 2 introduction telomere length assessthe original dyskeratosis congenita , telomere syndrome , was clinically described more than 100 years ago on the basis of individuals who presented with a distinct rash , abnormal nails , and whitening of the tongue.1 from this rare syndrome , our clinical and molecular understanding of telomere syndromes has evolved and has now expanded to include aplastic anemia , myelodysplastic syndrome , and pulmonary brosis.2 - 5 the identication of patients with telomere syndromes is of signicant clinical importance because these patients are exquisitely sensitive to alkylating chemotherapeutic agents and ionizing radiation.6 - 8 precision medicine efforts that deploy tumor - normal sequencing have used various molecular assays and platforms to interrogate and annotate the cancer census genes.9 , 10 tert , a gene that encodes a key protein involved in telomere maintenance , has been analyzed predominantly to identify genomic alterations that occur in tumors.11 , 12 although constitutional telomere syndromes are recognized , they are rarely considered in the oncologists differential diagnosis unless a diagnosis of a telomere syndrome occurred before the patients cancer diagnosis.13 from january 2016 to may 2019 , unselected patients with advanced solid tumors were presented with the option to participate and consent to a memorial sloan kettering cancer center institutional review board approved protocol ( #12 - 245 ; clinicaltrials.gov identier : nct01775072 ) of tumor and germline dna sequencing . 
cancer types in our cohort included four female patients with breast cancer ( ages at diagnosis , 28 , 37 , 46 , and 58 years ) , one male patient with gall bladder adenocarcinoma ( age 33 years ) , one male patient with pancreatic ampullary adenocarcinoma ( age 45 years ) , and one male patient with two malignancies : lymphoma at age 58 years and prostate cancer at age 66 years . 
in the six patients with a solid tumor as their rst malignancy , all were younger than the median age of diagnosis in the general population for their type of cancer24 - 27 ( data supplement )  . clinical histories of three ( 42.8% ) of the seven patients were suggestive of telomere syndromes . 
individual i , diagnosed with breast cancer , endured therapy - induced radiation pneumonitis and refractory thrombocytopenia ; individual iv , diagnosed with ampulla of vater cancer , experienced premature graying and thrombocytopenia that preceded a cancer diagnosis at age 19 years ; and individual vii revealed a history of bone marrow failure in a family member with a segregating tert mutation and showed abnormal pulmonary function testing ( table 1 )  . an understanding of the genetic basis that contributes to therapeutic sensitivities is important for oncology patients who receive chemotherapy , radiation , and other biologic therapeutics . 
patients with telomere syndromes may manifest overt or subtle clinical ndings.5 moreover , for patients with both obvious and subtle telomere syndromes , exquisite treatment sensitivities have been reported.6 , 7 although only one in 1 , 571 individuals in this series of patients with advanced cancer showed a germline tert mutation , this could translate to more than 1 , 000 patients diagnosed with malignancies a year in the united states who may have increased therapeutic sensitivities . 
because telomere syndrome disorders are complex and exhibit anticipation , incomplete penetrance , multiple genes that underpin disease , recognized modiers , and both autosomal dominant and autosomal recessive patterns of inheritance , this estimate will be rened with time . however , until a clearer picture is possible , it seems reasonable for individuals with constitutional germline tert mutations to be considered for monitoring given the potential long - term sequalae from chemotherapy and radiation as well as increased organ - specic damage.7 , 8 , 31 for two individuals , telomere length testing through cytometric uorescence in situ hybridization identied telomere lengths in the 1st percentile or less ; in two additional individuals , telomere lengths were in the 10th percentile or telomere length less ; and in one additional testing failed because of low blood counts that did not recover after chemotherapy ( fig 1 )  . 
somatic mutation analysis of all ve individuals with germline tert mutations and tumor specimens available for analysis individual , integration of germline , somatic , and clinical data for patients in our cohort was also notable for somatic tp53 mutations and a younger age at cancer diagnosis compared with the general population . 
moreover , multiple tert single nucleotide polymorphisms have been shown to be associated with telomere length and breast and ovarian cancer risk.31 all this information together with telomere lengths may provide insights for stratifying patients with regard to age at presentation , outcome , and tumor evolution . lymphocytes granulocytes 99th percentile 50th percentile 90th percentile 99th percentile 50th percentile 90th percentile 1st percentile 10th percentile 1st percentile 10th percentile age ( years ) age ( years ) fig 1 . 
 e walsh et al e113rfs * 79 p308hfs * 43 l392vfs * 145 r486c v694m exon 4 deletion f1084 * telomerase rvt1 1 , 000 1 , 132 aa a86vfs * 55 y163 * m237i m273h / c truncating mutations missense mutations rvt1 i , v / vi fig 2 . 
walsh , md , memorial sloan kettering cancer center , 222 70th st , room 412 , new york , ny 10021 ; e - mail : walshm2@ makcc.org. support supported by the robert and kate niehaus center for inherited cancer genomics and members of the molecular diagnostics service in the department of pathology and the marie - jos ee and henry r . 
also receives grant support from the v foundation , corning fund , and crawford fund for pediatric genomics . all authors at memorial sloan kettering are supported by the memorial sloan kettering cancer center support grant / core grant through national cancer institute grant no . 
walsh , kimberly amoroso , liying zhang collection and assembly of data : rosalba sacca , temima wildman , kimberly amoroso , jennifer kennedy , ozge birsoy , zoe steinsnyder , alicia latham , maria i . 
carlo consulting or advisory role : pzer other relationship : prostate cancer foundation , robert wood johnson foundation karen cadoo research funding : astrazeneca ( inst ) , syndax ( inst ) travel , accommodations , expenses : astrazeneca mark robson honoraria : astrazeneca consulting or advisory role : mckesson , astrazeneca references research funding : astrazeneca ( inst ) , myriad genetics ( inst ) , invitae ( inst ) , abbvie ( inst ) , tesaro ( inst ) , medivation ( inst ) travel , accommodations , expenses : astrazeneca , pzer other relationship : research to practice , clinical care options , physician education resource zsoa k . 
stadler consulting or advisory role : allergan ( i ) , genentech ( i ) , roche ( i ) , regeneron pharmaceuticals ( inst ) , optos ( i ) , adverum ( i ) , biomarin ( i ) , alimera sciences ( i ) , novartis ( i ) , spark therapeutics ( i ) , fortress biotech ( i ) , regenxbio ( i ) no other potential conicts of interest were reported . zinsser f : atrophia cutis reticularis cum pigmentatione , dystrophia unguium et leukoplakia oris . 
n engl j med 361 : 2353 - 2365 , 2009 alder jk , guo n , kembou f , et al : telomere length is a determinant of emphysema susceptibility . 
am j respir crit care med 184 : 904 - 912 , 2011 alder jk , parry em , yegnasubramanian s , et al : telomere phenotypes in females with heterozygous mutations in the dyskeratosis congenita 1 ( dkc1 ) gene . hum mutat 34 : 1481 - 1485 , 2013 armanios m , blackburn eh : the telomere syndromes . 
nat rev genet 13 : 693 - 704 , 2012 [ erratum : nat rev genet 14 : 235 , 2013 ] agrusa je , bertuch aa , dinardo cd , et al : severe therapy - related toxicities after treatment for hodgkin lymphoma due to a pathogenic tert variant and shortened telomeres . 
pediatr blood cancer 66 : e27779 , 2019 de la fuente j , dokal i : dyskeratosis congenita : advances in the understanding of the telomerase defect and the role of stem cell transplantation . 
pediatr transplant 11 : 584 - 594 , 2007 dietz ac , orchard pj , baker ks , et al : disease - specic hematopoietic cell transplantation : nonmyeloablative conditioning regimen for dyskeratosis congenita . bone marrow transplant 46 : 98 - 104 , 2011 cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
rusch m , nakitandwe j , shurtleff s , et al : clinical cancer genomic proling by three - platform sequencing of whole genome , whole exome and transcriptome . nat commun 9 : 3962 , 2018 11 . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
hampel h , bennett rl , buchanan a , et al : a practice guideline from the american college of medical genetics and genomics and the national society of genetic counselors : referral indications for cancer predisposition assessment . 
weitzel jn , blazer kr , macdonald dj , et al : genetics , genomics , and cancer risk assessment : state of the art and future directions in the era of personalized medicine . 
noone am , cronin ka , altekruse sf , et al : cancer incidence and survival trends by subtype using data from the surveillance epidemiology and end results program , 1992 - 2013 . 
bojesen se , pooley ka , johnatty se , et al : multiple independent variants at the tert locus are associated with telomere length and risks of breast and ovarian cancer . 
 mutational landscape in resected periampullary adenocarcinoma : relationship with morphology and clinical outcome sebastian lundgren , md1 ; soe olsson hau , md1 ; jacob elebro , md , phd1 ; margareta heby , md , phd1 ; emelie karnevi , phd1 ; bj orn nodin , phd1 ; jakob eberhard , md , phd1 ; karolina holm , phd1 ; johan staaf , phd1 ; g oran b . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction periampullary adenocarcinoma is a collective term for tumors arising in the area surrounding the ampulla of vater , including the head of the pancreas , the duodenum , and the common bile duct . 
a morphologic classication into intestinal type ( i - type ) and pancreatobiliary type ( pb - type ) has been shown to provide better prognostic information than anatomic origin , with the former having a more favorable clinical outcome.1 - 3 although a plethora of mutations has been documented in pancreatic cancer , 4 - 7 the mutational landscape of other periampullary cancers has been less studied . 
no targeted therapies have proven to be efcient , and adjuvant chemotherapy therefore remains standard of care after resection of these tumors . hence , there is evident need for additional studies on the distribution and clinical signicance of the mutational landscape in the full range of periampullary including pancreatic cancer , adenocarcinomas , enable a better patient stratication and identify potential responders to targeted therapies . 
 lundgren et al context ( cid : 129 ) to what extent does morphology inuence the mutational landscape in periampullary adenocarcinomas , and can we nd novel prognostic and predictive biomarkers that differ by morphology , so as to better characterize this heterogeneous group of tumors in a clinical context ? ( cid : 129 ) the results presented here demonstrate that the distribution and prognostic impact of mutations in key genes indeed differ according to morphology and that emphasis on morphology rather than anatomy is of importance . 
approval for the study was obtained from the ethics committee of lund university ( reference number 445 / 07 ) , whereby the committee waived the need for consent other than the option to opt ; out . next - generation sequencing tissue cores of 1 - mm diameter were taken from tumor cellenriched formalin - xed parafn - embedded tissue . dna extraction was performed using the qiagen generead ( qiagen , hilden , germany ) kit for formalin - xed parafnembedded tissue according to the manufacturers instructions . 
forty - one ( 39.8% ) were classied as i - type and 62 ( 60.2% ) as pb - type . a panel of 70 cancer - associated genes was put together for this study and characterized using illumina truseq custom amplicon assay ( illumina , san diego , ca ) with a miseq instrument according to manufacturers instructions . 
detected mutations were screened against the cosmic and exac databases , to lter single - nucleotide polymorphisms commonly reported in different populations . immunohistochemical analysis of brahma - related gene 1 protein expression immunohistochemical analysis of brahma - related gene 1 ( brg1 ) protein expression , 4 - m tissue microarray sections were automatically pretreated in the pt - link system ( dako , glostrup , denmark ) and stained in an automated immunostainer ( autostainer plus , dako ) using the dako envision flex + detection system , peroxidase / dab , rabbit / mouse with the monoclonal anti - brg1 antibody clone g - 7 ( santa cruz biotechnology , dallas , tx ) , diluted 1 : 25 . brg1 was expressed in the tumor cell nuclei and present in the majority of tumor cells in positive cases . 
kaplanmeier analysis log - rank test was applied to illustrate any difference in 5 - year overall survival ( os ) , and cox regression proportional hazard models were used to estimate hazard ratios ( hrs ) for death within 5 years in both univariable and multivariable analysis . 
multivariable cox regression included adjustment for age ( continuous ) , t - stage ( t1 or t2 v t3 or t4 ) , n - stage ( negative v positive nodal status ) , grade ( well to moderate v poor ) , morphology ( i - type v pb - type ) in the entire cohort , adjuvant chemotherapy ( none v any ) , invasion into vascular and lymphatic structures , and perineural growth . 
the proportional hazard ( ph ) assumption was tested using cox regression with a timedependent covariate analysis , whereby the ph assumption was considered to be satised when the factor time interaction was nonsignicant . 
the ph assumption was also evaluated graphically using log - minus - log plots . in the entire cohort , three cases were excluded in the survival analyses : one patient with a pb - type tumor who was lost to follow - up because of emigration , and two patients with i - type tumors who died as a result of complications from the initial surgical treatment . 
to estimate the interaction treatment and selected bioeffect between adjuvant markers , the following interaction variable was constructed : any adjuvant treatment ( + / ) biomarker ( + / )  . 
all data generated or analyzed during this study are included in this published article ( data supplement )  . results distribution of mutations according to morphologic type there were no signicant differences in the distribution of clinicopathological characteristics between cases with known and unknown mutational status ( data supplement )  . the frequency of mutations according to morphologic type is shown in figure 1 and further outlined in the data supplement . 
the frequency of mutations according to anatomic origin is shown in the data supplement . prognostic impact of the most frequent mutations kaplan - meier analyses demonstrate a signicantly prolonged os for patients with i - type compared with patients with pb - type tumors ( data supplement )  . 
when stratifying for anatomic origin , the survival curves cluster into two groups concordant with morphology ( data supplement )  . of the nine most frequently mutated genes outlined above , only apc , erbb3 , kras , and smarca4 were shown to confer a prognostic value , depending on the context . 
apc mutation was not prognostic in i - type tumors ( data not shown ) , and erbb3 mutation was not prognostic in analyses stratied for morphology ( data not shown )  . kras mutation was the strongest predictor of survival , as visualized in figure 2 . 
when stratifying for ampullary origin , kras mutation remained a prognostic factor in i - type tumors ( p = 0.033 ; fig 2d ) but was not prognostic in pb - type tumors ( fig 2e )  . 
 intestinal type pancreatobiliary type tp53 kras rnf43 smad4 erbb3 smarca4 cdkn2a lundgren et al tp53 kras rnf43 smad4 smarca4 cdkn2a erbb3 genetic alteration no alterations truncating mutation in - frame mutation missense mutation fig 1 . 
the genetic alterations were classied as either truncating , missense , or in - frame mutations . mutation did not differ according to adjuvant chemotherapy in i - type tumors ( data not shown )  . 
in the entire cohort , high brg1 expression was an adverse prognostic factor in patients not receiving adjuvant chemotherapy but was not prognostic in patients receiving adjuvant chemotherapy ( data not shown )  . 
the mutational burden of the analyzed genes did not differ signicantly between i - type and pb - type tumors ( p = .577 ) and was not prognostic in the whole cohort or stratied for morphologic type . discussion morphology is emerging as an important prognostic factor in periampullary adenocarcinoma , 1 , 2 and to our knowledge , this is the rst study to comprehensively map common cancer - associated mutations in the full range of periampullary adenocarcinoma , with particular reference to morphology . 
these ndings are in line with a study on 112 cancers of ampullary origin , demonstrating that the mutational spectrum in i - type tumors resembles that of colorectal cancer , and the mutational spectrum in pb - type tumors resembles that of pancreatic cancer.12 in the current study , mutations in apc but not in cdkn2a were found to be prognostic . 
given that no pb - type tumors harbored apc mutations , it is plausible to assume that the link between these mutations and a favorable outcome in the entire cohort is mainly due to their association with i - type morphology . 
the same line of reasoning applies to the association between erbb3 mutations , being more prevalent in i - type tumors , and a prolonged survival in the entire cohort . 
adding to this , there was no signicant association between her3 expression and erbb3 mutational status , and the clinical utility of assessment of erbb3 mutation status for prognostication purposes needs additional validation . kras mutation was found to signify a signicantly shorter survival in patients with i - type tumors , also when adjusted for established clinical factors , whereas patients with kras wild - type i - type tumors could indeed be classied as long - term survivors . 
as of yet , epidermal growth factor receptor ( egfr ) inhibition has not shown clinical efcacy in pancreatic cancer , 15 but no study has investigated the efcacy of egfr inhibitors in periampullary adenocarcinoma in relation to morphology . although none of the patients in this study had received egfr - inhibiting treatment , the ndings indicate that some ras wild - type periampullary cancers of i - type may indeed benet from such treatment . 
 mutational spectrum and morphology in periampullary cancer kras wt kras mut p = .033 kras wt kras mut p = .018 time since diagnosis ( months ) time since diagnosis ( months ) 2 . 
kaplan - meier curves visualize differences in 5 - year overall survival for patients with kras - mutated ( mut ) and wild - type ( wt ) tumors in ( a ) the entire cohort , intestinal - type tumors , ( c ) pancreatobiliary - type tumors , ( d ) intestinal - type amtumors , and ( e ) pullary pancreatobiliary - type pullary tumors . kras wt kras mut p = .680 kras wt kras mut p = .033 time since diagnosis ( months ) time since diagnosis ( months ) no . 
 lundgren et al smarca4 wt smarca4 mut p = .050 smarca4 wt smarca4 mut p = .803 time since diagnosis ( months ) time since diagnosis ( months ) score 0 score 2 score 3 brg1 low brg1 hight p = .061 brg1 low brg1 high p = .037 time since diagnosis ( months ) time since diagnosis ( months ) no . 
relationship of smarca4 mutation status and brahma - related gene 1 ( brg1 ) protein expression with overall survival according to adjuvant chemotherapy in patients with pancreatobiliary ( pb ) - type tumors . 
 ( a , b ) kaplanmeier curves visualizing differences in 5 - year overall survival in patients with pb - type tumors according to smarca4 mutation status ( a ) without adjuvant chemotherapy , and ( b ) with adjuvant chemotherapy . ( c ) sample immunohistochemical images of brg - 1 expression . 
 ( d , e ) kaplan - meier analyses visualizing differences in 5 - year overall survival in patients with pb - type tumors according to low and high brg1 expression ( d ) without adjuvant chemotherapy , and ( e ) with adjuvant chemotherapy . 
 mutational spectrum and morphology in periampullary cancer despite the lack of a signicant interaction between smarca4 mutation and adjuvant treatment , this observation led us to further explore the expression and prognostic value of brg1 , the protein encoded by smarca4 , in tumors from the full study cohort . 
brg1 was found to be an adverse factor in patients who did not receive adjuvant chemotherapy and , in addition , there was a signicant interaction with adjuvant chemotherapy in pbtype tumors . 
one study on pancreatic cancer ( n = 68 ) failed to demonstrate an association between brg1 expression and gemcitabine response or survival , 17 but the nding of a potential predictive role for smarca4 / brg1 in the current study merits further validation in additional patient cohorts . to our knowledge , this is the rst study to map the prevalence and prognostic signicance of mutations in common cancer - associated genes in the full spectrum of periampullary adenocarcinoma . 
in particular , kras mutation status may be a suitable biomarker for prognostication in patients with i - type tumors , and smarca4 / brg1 expression may add value in the prediction of response to adjuvant chemotherapy treatment in patients with pbtype tumors . 
bmc cancer 8 : 170 , 2008 bronsert p , kohler i , werner m , et al : intestinal - type of differentiation predicts favourable overall survival : conrmatory clinicopathological analysis of 198 periampullary adenocarcinomas of pancreatic , biliary , ampullary and duodenal origbmc cancer 13 : 428 , 2013 kimura w , futakawa n , zhao b : neoplastic diseases of the papilla of vater . 
nature 531 : 47 - 52 , 2016 genet 47 : 1168 - 1178 , 2015 collisson ea , sadanandam a , olson p , et al : subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy . 
nat med 17 : 500 - 503 , 2011 elebro j , jirstr om k : use of a standardized diagnostic approach improves the prognostic information of histopathologic factors in pancreatic and periampullary adenocarcinoma . 
diagn pathol 9 : 80 , 2014 elebro j , heby m , warfvinge cf , et al : expression and prognostic signicance of human epidermal growth factor receptors 1 , 2 and 3 in periampullary adenocarcinoma . 
philip pa , benedetti j , corless cl , et al : phase iii study comparing gemcitabine plus cetuximab versus gemcitabine in patients with advanced pancreatic adenocarcinoma : southwest oncology group - directed intergroup trial s0205 . 
 total number of alterations in liquid biopsies is an independent predictor of survival in patients with advanced cancers peter vu , md1 ; yulian khagi , md1 ; paul riviere , bs1 ; aaron goodman , md2 ; and razelle kurzrock , md1 purpose studies have demonstrated an association between quantity of circulating tumor dna ( ctdna ) and poorer survival . 
our ndings suggest that the total number of alterations in plasma may be an indicator of more aggressive tumor biology and therefore poorer survival . jco precis oncol 4 : 192 - 201 . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction five - year survival rates are incredibly variable among cancer types , ranging from more than 90% in prostate cancer to less than 8% in pancreas cancer , and depend heavily on clinical and pathologic stage.1 although repeat tissue biopsies during the course of treatment or at the time of progression may provide clinically important information , such biopsies are not routinely performed because they can be technically difcult , time consuming , medically invasive , and lead to complications . 
however , liquid biopsies , or cell - free dna ( cfdna ) obtained from blood plasma that contains fragments of circulating tumor dna ( ctdna ) shed from tumor cells into the bloodstream , can identify new actionable alterations and be performed repeatedly with minimal procedural risk.2 - 5 ctdna can then be analyzed using technologies such as digital polymerase chain reaction to detect specic known somatic variants ( eg , egfr t790m ) or next - generation sequencing ( ngs ) that uses massive parallel sequencing to detect up to thousands of somatic and germline alterations in a single run.6 in addition , genomic alterations found on liquid biopsies are often concordant with alterations found on tissue biopsy when obtained within close proximity to one another.7 - 9 a number of studies have demonstrated that there is an association between higher amounts of cfdna or ctdna and poorer survival , perhaps because percent ctdna ( %ctdna ) correlates with tumor burden.10 , 11 for the most part , these reports dichotomized the level of cfdna or ctdna at a cut - point ( often but not always at approximately 5% or 10% ctdna ) .10 , 12 - 18 in the case of surgical candidates , the cut - points may be lower . 
eligible patients included those who had solid tumor malignancies , never received immunotherapy treatment , and were evaluable for clinical correlations , including overall survival ( os ) from ctdna collection date . 
immunotherapytreated patients were omitted because a correlation with blood or tissue tumor mutational burden has been associated with better immunotherapy response and might therefore alter the survival curve.19 , 20 patients with amplications only in ctdna were omitted because the %ctdna for amplications could not be determined . 
in addition to os evaluation , patients data were also collected and analyzed for %ctdna ; the alteration with the highest allele fraction was calculated from all alterations , including variants of unknown signicance ( vuss ) , total number of vuss , and total number of alterations ( which included vuss )  . 
all values for the total number of ctdna alterations and the number of vuss were corrected for the length ( kilobase pairs [ kbp ] ) of dna sequenced based on the date sequencing was performed and multiplied by 100 ( appendix table a1 )  . 
all data were analyzed from the time of ctdna collection from plasma ( two 10 - ml blood tubes )  . this ctdna assay has a sensitivity and specicity of  . 
hazard ratios ( hrs ) for survival were calculated by comparing os above and below cutoffs and performed from the time of ctdna collection ; dichotomization for each variable ( ie , total number of alterations , total number of vuss , %ctdna ) was performed at the median . 
multivariate analyses were conducted using the wald 2 test from a cox proportional hazards model that included all variables with p .05 in univariate analyses ( ie , sex , age , total number of alterations , %ctdna ) , with the exception of vuss because these alterations are already encompassed within the total number of alterations variable . 
associations between %ctdna and total number of alterations were determined using spearmans rank - order correlation . bootstrapping using random sampling with replacement ( n = 1 , 000 bootstrap samples ) and multiple logistic regression analysis were performed , permitting the data of the sample study to be used as a surrogate for a larger population to validate the model . 
intermediate to high %ctdna was dichotomized at 5% because it had been found to be signicant in prior studies.10 the %ctdna for each patient was calculated using the alteration with the highest allele fraction , including variants of unknown signicance validation cohort , as was the case in our study.22 statistical analyses were carried out using prism version 7.0 ( graphpad , san diego , ca ) and r version 3.5 ( r foundation for statistical computing , vienna , austria )  . results patient characteristics this study included 418 patients who had ngs performed on plasma - derived ctdna and did not receive immunotherapy treatment . 
after correcting for the kbp length of dna sequenced for each sample , the median total number of ctdna alterations ( including vuss ) per patient was 1.46 ( range , 0 - 78.8 ) ; the median total number of vus alterations per patient was 0.66 ( range , 0 - 64.2 ) ; and the median %ctdna was 0.4% ( range , 0% - 80.3% ; table 1 )  . 
 total number of alterations in liquid biopsies is prognostic regression model showed that age , sex , and the total number of alterations were independently prognostic for survival ( table 1 )  . 
among these characteristics , only total number of alterations was signicantly associated with survival ( p , .0001 ; table 1 )  . discussion liquid biopsies have been incorporated into clinical practice as a means to obtain noninvasive molecular proling to identify specic oncogenic driver mutations or other alterations that can guide treatment selection . 
in this study , we evaluated the relationship between the total number of alterations and the %ctdna detected by liquid biopsy and survival outcomes in 418 patients with advanced cancers . 
in addition , we intentionally excluded patients who subsequently received immunotherapy treatment , because several studies have suggested that the use of immune checkpoint inhibitors may alter the survival curve in patients with increased tumor mutational burden.19 , 20 , 23 we demonstrate that both the total number of alterations and the %ctdna have prognostic value and correlate with one another , but only an intermediate to high ( 1.46 ) total number of alterations / kbp ( and not high %ctdna ) was independently associated with worse survival outcomes in multivariate analysis in patients with gi tumors ( table 2 ) , as well as in patients with a diverse group of advanced cancers ( table 1 )  . 
it is also plausible that the higher number of alterations and accompanying aggressive biology results in a higher tumor burden that yields a higher %ctdna ( rather than vice versa )  . a strength of our study is that we used sequencing technology that allows for the detection of %ctdna at a low level with high sensitivity and high specicity.21 , 24 in comparison , some prior studies have used low - depth sequencing , which is less capable of detecting ctdna . 
as a result , these studies were only able to conclude that the presence of ctdna was associated with worse outcomes compared with the absence of detectable ctdna.14 , 18 , 25 , 26 indeed , yang et al27 proposed that the presence or absence of ctdna should be added to the tnm staging classication of tumors because it has diagnostic , therapeutic , and prognostic value . 
when greater depth of ctdna sequencing was used , prior studies have reported that %ctdna is correlated with worse survival and also with increased tumor volume.10 , 11 , 14 we also demonstrated that %ctdna correlates with survival measured from the time of blood draw ( fig 1 ) , which suggests that the association between %ctdna and outcomes may be more reective of tumor burden . limitations to our ndings , given the there are several retrospective nature of the analysis . 
although our study used a relatively large sample of 418 patients , we included a diverse group of advanced cancers and , therefore , our ndings may not be applicable to certain tumor types . despite this , the variety of tumor types in our study may make our ndings more generalizable across advanced total no . 
in addition , 112 patients in this study had no detectable %ctdna , which may be due to low disease burden or due to limitations of the ctdna sequencing technique . 
it should also be noted that there was a different number of subgroups in the analysis of %ctdna and number of ctdna alterations ; hence , the conclusion that the total number of alterations and %ctdna have prognostic value and correlate with one another but that only the total number of alterations was independently associated with survival outcomes will need to be further examined and validated . 
also , we do not know whether this patient population is comparable with those who were not analyzed for ctdna because physicians chose not to perform the analysis or with patients who were lost to follow - up early and hence were inevaluable . 
finally , patients had a diverse array of prior treatments , some of which could have confounded the results ; patients treated with immunotherapy were excluded because cancers with higher mutational burden / number appear to do better on this modality . in conclusion , to our knowledge , this is the rst demonstration that the total number of alterations and %ctdna are highly correlated and have prognostic value . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . support supported in part by the joan and irwin jacobs fund and by national cancer institute grant no . 
 total number of alterations in liquid biopsies is prognostic aaron goodman consulting or advisory role : mitsubishi tanabe pharma , gerson lehrman group , jazz pharmaceuticals , speakers bureau : seattle genetics razelle kurzrock leadership : curematch stock and other ownership interests : curematch , idbydna , soluventis , actuate therapeutics , loxo , xbiotech , neo - med , roche , gaido , soluventis , pzer , merck speakers bureau : roche research funding : guardant health ( inst ) , sequenom ( inst ) , merck serono ( inst ) , genentech ( inst ) , pzer ( inst ) , foundation medicine ( inst ) , incyte ( inst ) , konica minolta ( inst ) , grifols ( inst ) , omniseq ( inst ) , debiopharm group ( inst ) , boerhinger ingelheim ( inst ) no other potential conicts of interest were reported . references siegel rl , miller kd , jemal a : cancer statistics , 2018 . 
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intermediate to high %ctdna was dichotomized at 5% because it had been found to be signicant in prior studies.10 the %ctdna for each patient was calculated using the alteration with the highest allele fraction , including variants of unknown signicance . time ( months ) fig a2 . 
gazzaniga et al we are grateful for your comments on our recent publication1 that highlighted that nearly 50% of the patients with ras - mutated metastatic colorectal cancer and prior chemotherapy could display wildtype ras status in the circulating tumoral dna ( ctdna ) , and benet from the late introduction of an antiepidermal growth factor receptor ( egfr ) .2 we indeed acknowledge that your team had worked in a similar direction , and appropriately cited the report that your group published in 2019.3 this article also includes results from preliminary ndings in abstract forms . 
both of our works , as well as that from moati et al , 4 conrm the principle of expected efcacy from targeting egfr in the late stage of treatment of patients with ras - mutated colorectal cancer , if their liquid biopsy does not identify any ras mutation in the ctdna.2 - 4 however , there is a broad discrepancy in the rates of patients without ras mutation detection in plasma between these studies , ranging from 9 / 16 ( 56% ) , 2 through 5 / 11 ( 45% ) 3 to 8 / 36 ( 22% ) , 4 as also discussed in your opinion paper.5 a critical issue involves the conrmation of circulating tumoral dna in the absence of ras mutation , which could be reached through the detection of a persistent somatic mutation3 or the occurrence of wif1 / npy methylation.4 however , we concur and admit that these and other methods have intrinsic limitations , such that no standard one can currently ascertain the presence of circulating tumor dna in the absence of detected ras mutation.5 moreover , although their application drastically reduced the ras mutation conversion rate down to 6.6% - 14% , 3 , 4 this did not impair the fact that the introduction of an antiegfr apparently extended median progression - free survival to nearly 6 months in previously treated patients from both studies.2 , 3 therefore , we feel that two important issues need to be jointly addressed to advance the current guidelines for anti - egfr indication for metastatic colorectal cancer . 
gazzaniga p , nicolazzo c , caponnetto s , et al : about ras mutation clearance in plasma ctdna from ras - mutant colorectal cancer patients . jco precis oncol , 2021 2 . 
bouchahda m , saffroy r , karaboue a , et al : undetectable ras - mutant clones in plasma : possible implication for anti - egfr therapy and prognosis in patients with ras - mutant metastatic colorectal cancer . 
raimondi c , nicolazzo c , belardinilli f , et al : transient disappearance of ras mutant clones in plasma : a counterintuitive clinical use of egfr inhibitors in ras mutant metastatic colorectal cancer . 
nicolazzo c , belardinilli f , caponnetto s , et al : why the therapeutic impact of ras mutation clearance in plasma ctdna deserves to be further explored in metastatic colorectal cancer . 
 eye - tracking study to enhance usability of molecular diagnostics reports in cancer precision medicine purpose we conducted usability studies on commercially available molecular diagnostic ( mdx ) test reports to identify strengths and weaknesses in content and form that drive clinical decision making . 
this work aims to evaluate effectiveness of mdx reports in facilitating cancer treatment planning . methods fourteen clinicians at an academic tertiary care medical facility , with a wide range of experience in oncology and in the use of molecular testing , participated in this study . 
three commercially available , widely used , clinical laboratory improvement amendments ( clia ) certified , college of american pathologists ( cap ) accredited test reports ( labeled laboratories a , b , and c ) were used . 
eye tracking , surveys , and thinkaloud protocols were used to collect usability data for these mdx reports focusing on ease of comprehension and actionability results clinicians found two primary areas in molecular diagnostic reports most useful for patient care : therapy options with benefit or lack of benefit to patients , including enrolling clinical trials ; and pathogenic tumor molecular anomalies detected . 
however , all reports had usability and comprehension issues in these areas and could be improved . conclusion focused usability studies can help drive our understanding of the clinical workflow for use of molecular diagnostic tests in cancer care . 
2018 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license : introduction academic and community clinics are increasingly conducting genomic testing to inform treatment decisions for patients with cancer . 
accumulating research indicates significant potential to improve outcomes by effectively using this rich source of data in treatment planning , especially for clinical trial selection , by matching a patients molecular characteristics to trial eligibility criteria . currently , results from molecular diagnostic ( mdx ) testing are usually provided to clinicians and tumor boards as a multipage pdf document with a one - page summary highlighting any clinically actionable anomalies and treatment vishakha sharma allan fong robert a . 
 standards exist for classification , although they are not always adhered to by the testing vendors at this time.4 subsequent pages contain literature evidence associating the biomarkers to treatment and clinical trial options . 
biomarkers in mdx reports must be evaluated on the basis of their strength of evidence of clinical utility and actionability.5 , 6 mdx reports often provide an evaluation of the evidence and / or links to publications , but the evidence representation is not standardized . 
the increasing availability of mdx tests has caused concern , given the complexity of information presented.7 , 8 there is a clear need to enhance usability of patient - specific , clinically actionable molecular profiling information presented in mdx reports . electronic health record usability is a concern for health care vendors , because poor usability can lead to frustration and safety risks and can keep users from achieving their goals in routine work.9 , 10 the evaluation of cancer care and research products for usability and comprehension has been emphasized recently.3 , 11 - 13 usability techniques , such as surveys , thinkaloud protocols , semistructured interviews , and eye tracking , have been widely used in health care system design.9 , 14 - 21 usability evaluation of oncology electronic medical records and clinical practice guidelines have been performed using survey questionnaires and think - aloud protocols.22 - 26 eye tracking helps elucidate how users visually search and integrate information and has been used to understand the visual search patterns of anesthesiologists and emergency medicine physicians.27 , 28 methods experimental design we invited 43 oncology fellows and attending physicians ( 21 men , 22 women ) to participate in this study from the medstar health syste fourteen volunteered to participate ( 11 women , three men )  . 
six of 14 participants were excluded from the eye - tracking analysis because of low - quality eye - tracking data ( insufficient data for at least 75% of the session ) .28 eye - tracking studies with < 10 participants are common in anesthesiology and radiology studies.29 , 30 studies on usability are generally less concerned with sex variations in the population , unless the specific display requires knowledge that only a certain sex may have . 
 each participant reviewed three mdx reports in a randomly assigned three - period crossover design , which has the advantage of improved efficiency , because each participant serves as his / her own control.32 the reports chosen represent the three most commonly used clia - certified cap - accredited testing laboratories across the georgetown - medstar health system in the washington , dcbaltimore region . 
characteristics of the study participants all participants ( n = 14 ) eye tracking only ( n = 8 ) characteristic highest degree md , msc md , phd position fellow female male attending physician current specialty blood / marrow transplant oncology / breast oncology / gi oncology / hematology oncology / lung and brain years in practice weekly mdx reports weekly patients 5 - 10 10 - 15 20 - 30 5 - 10 0 - 10 10 - 25 25 - 50 50 - 100 abbreviation : mdx , molecular diagnostic . molecular test findings : gene / protein names , alterations , positive or negative test results ; and potential therapies : drug options and clinical trials . 
aois serve as meaningful regions in eye - tracking studies to correlate the eye movements with the interpretations provided by the clinicians from think - aloud recordings and survey answers . 
images of the mdx reports with aois as well as detailed descriptions of the similarities and differences between reports are provided in the data supplement . eye - tracking data collection eye - tracking was used to capture the visual scanning patterns of participants . 
we measured four common eye - tracking measures for all participants33 : fixation count : frequency of fixations within an aoi ; fixation duration : amount of time a participant fixated at an aoi ; refixation count : frequency of return fixations for an aoi ; and fixation transition : frequency of fixation transitions between aois . think - aloud protocol and recording in addition to eye tracking , each session involved a think - aloud protocol that was audio - video recorded . 
 recruit participants conduct eye - tracking study collect data outcomes analyze data for aois clinicians laboratory a laboratory b laboratory c three mdx reports from three laboratories were shown in randomized order eye - tracking survey questionnaire think - aloud protocol aois think - aloud data transcription ranked aois driving cancer treatment planning fig 1 . 
what if anything should be added to this patient report to help you make a treatment decision for this patient ? results comparison of mdx reports information and layout commonalities and variations in information content , location , and variation in labels and descriptive language used across the mdx reports and aois from laboratory a , b , and c were identified , analyzed , and categorized . 
six of 14 participants were excluded from the eye - tracking analysis because of insufficient eye - tracking data for at least 75% of the session.28 the survey results from eight clinicians with eye - tracking data were consistent with the results for the 14 in the relative numbers that agreed or disagreed on a question . 
survey results for just the eight with eye tracking and answers for all s5 suggestions for improvements are included in the data supplement . eye - tracking data analysis table 2 presents the eye movement measures for the three reports , fixation counts , average fixation durations , and refixations from eight participants . 
both higher fixation counts and longer fixation durations are indicative of deeper perceptual processing.34 , 35 the refixation count is how many times an aoi is returned to and shows transitions between different areas . 
more refixations and transitions may indicate inefficient search.36 although it is well understood that these trends are task specific , our results work to bridge this theoretical understanding of eye movement data to the processing of mdx reports . 
 strongly agree agree disagree strongly disagree strongly agree agree disagree strongly disagree laboratory a laboratory b laboratory c laboratory a laboratory b laboratory c laboratory a laboratory b laboratory c laboratory a laboratory b laboratory c strongly agree agree disagree strongly disagree strongly agree agree disagree strongly disagree fig 2 . 
it is easy to find the therapy class ( such as us food and drug administration approved off - label , on - label , clinical trials ) in this patient report . 
it is easy for you to decide on a treatment of this patient on the basis of this report . were 29 instances when participants looked at lack of benefit after clinical trials . think - aloud protocol and video analysis the data supplement summarizes the verbal feedback received from clinicians from the thinkaloud protocol transcription and video analysis . 
think - aloud and video analysis helped gain insight into improvements needed in the mdx reports and helps confirm the survey and eye - movement data . discussion molecular diagnostic reports try to distill the results of genetic tests with therapy and clinical trial options into a single - page summary to help overburdened oncologists make appropriate treatment decisions for their patients . 
perception and comprehension of information are two processes important to understanding.37 what we perceive ( visually through our eyes as captured by the eye tracker ) and how we comprehend it ( captured by our think aloud ) are two necessary components to understanding and fundamental to the usability evaluation . 
the survey showed that all the participants agreed with s2 ( it is easy to find the therapeutic implications [ benefit , lack of benefit ] in this patient report ) , but a participant said , under the indeterminate benefit , i do not know what those mean . 
detailed descriptions of the aois are in the data supplement . abbreviations : aoi , area of interest ; mdx , molecular diagnostic . clinicians were voicing about certain content , their eyes were fixated on a different section , perhaps indicating where their comprehension was heading next . 
hence , we noticed it was important to summarize results from both techniques and call out areas where results from one technique validated those from the other method . the eye - movement analysis ( tables 2 and 3 ) shows that participants fixated most in regions that described therapy options . 
however , each laboratory had a different approach to this information ( data supplement ) , and there were clear differences in how laboratories presented and clinicians responded to the presentation . 
however , for s3 ( it is easy to find the therapy class , such as fda approved off - label , on - label , and clinical trials ) , the majority disagreed , and the larger number of eye fixations and refixations in the therapy sections suggests more cognitive difficulty in interpretation . 
 laboratory a laboratory b laboratory c potential benefit genomic alteration ranked therapy transition count transition count transition count clinical trials lack of benefit results summary therapy genomic findings potential benefit clinical trials lack of benefit genomic alteration results therapy ranked therapy summary genomic findings fig 3 . 
in the laboratory b report , there were 26 instances when participants looked at results after genomic alteration , and in the laboratory c report , there were 19 instances when participants looked at summary after ranked therapy . 
for laboratory c , the majority agreed with s2 , that therapeutic implications were easy to find , but by the same margins they disagreed with s3 , that it is easy to find the therapy class.... 
 unfortunately , participants found this system confusing , as indicated in several comments in the think - aloud transcripts and suggestions . the genomic anomaly results were of high interest as measured by fixations and refixations to the aois , think - aloud comments , and s5 suggestions . 
laboratory a , for s1 ( is it easy to find the molecular anomalies ) did the worst , with half the participants agreeing and half disagreeing with comments about the lack of detail . 
laboratory a did not have a section specific to genomic anomalies ; it only showed gene symbols in the therapy sections and not the actual variants detected , implying that there were anomalies in the gene . 
laboratory cs anomalies section had lower fixation counts , fixation duration , and refixations than laboratory b , suggesting the display was more clear and efficient . how can these results be applied to design improved mdx summary reports ? some simple suggestions include the following : diagnosis should be clearly labeled at or near top of a report . 
 reports should try to work in grayscale and not be too reliant on color , because reports are still often faxed , and approximately 4% to 8% of the population is colorblind.38 genomic anomalies should be clearly labeled with some detail ( laboratory c did best in this area )  . 
therapy sections are the most important and need to distinctly relate anomalies to therapies , with a clear delineation between fda - approved , national comprehensive cancer network guideline , and experimental treatments and appropriate clinical trials . 
a ranking system to summarize complex clinical information on the basis of standardization of clinical evidence seems to be needed ( laboratory c included a ranking , but it was not clear to the participants )  . our current understanding of the science of interpreting variants and treatment options is in flux , and any ranking system would need to be understandable , flexible , and adapt quickly as our knowledge of actionable variants evolves . 
 ( p12 ) this is most of the information that i would care about . ( p01 ) under ranked therapy , i am not really sure what it means when it says molecular profile and there is a number there . 
 ( p15 ) the biggest advantage of this is , its actually making a clear - cut recommendation for oncologists who does not know how to interpret these results ( p13 ) i dont see what mutations the patient has . 
 ( p05 ) they are not really showing the molecular anomalies , just tells you the therapies that they are recommending , ( .. ) so i can infer that braf is negative because they are saying braf has lack of benefit . 
average duration indicates average fixation duration . abbreviations : aoi , area of interest ; fda , us food and drug administration ; nccn , national comprehensive cancer network ; p , physician . are beginning to revise their reports and present additional evidence on request from clinicians who ordered these tests . the sex difference is an unfortunate limitation ; however , it is not uncommon in human factor studies . 
many studies use three to five participants , which provides saturation when looking at specific usability and cognitive aspects of a display or product , and no additional data collection or analysis is necessary after saturation has been attained.31 , 39 recruiting busy expert participants is an ongoing challenge for clinical usability studies . 
 than the other laboratory reports , this study showed numerous issues with presentation of information in laboratory as report indicating that bias , if present , was not a large issue . 
the participants expressed great interest in optimizing the information presented to them to augment their day - to - day clinical work , indicating a clear need for improvement in mdx reports to support decision making . these findings revealed that participants prefer mdx reports with clear therapeutic implications supported by biomarker implications . 
a summary on a tablet or a web site dashboard with options for additional details on trials , drugs , or implicated biomarkers might be the personalized medicine coalition conducted a nationwide survey and found that although most people had not heard about personalized medicine , those who did , or had it explained , were excited about the potential benefits.40 similar usability studies with patients will benefit laboratories that are beginning to generate patient - targeted reports to improve patient understanding of treatment options and enable informed discussions with their clinicians . 
efforts such as sync for science41 and blue button42 allow individuals to access their health data to support the goals of the precision medicine initiative and allow individuals to have control over their personal health information . 
beckman employment : onco - mind stock and other ownership interests : johnson & johnson consulting or advisory role : astrazeneca ; emd serono patents , royalties , other intellectual property : two patents for dynamic precision medicine , a novel approach to precision medicine , have been granted in japan and taiwan and transferred to onco - mind . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . vishakha sharma no relationship to disclose allan fong no relationship to disclose simina m . 
1u01hg008390 - 01 , 2015 - 2016 . presented in part at the american medical informatics association translational summits , san francisco , ca , march 27 - 30 , 2017 , and american society for clinical pharmacology & therapeutics annual meeting , washington , dc , march 21 - 24 , 2018 . support prior presentation references 1 . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
madhavan s : barriers to implementation of precision medicine for cancer treatment in the us healthcare systejournal of precision medicine may / june : 48 - 56 , 2016 8 . 
ratwani rm , fairbanks rj , hettinger az , et al : electronic health record usability : analysis of the user - centered design processes of eleven electronic health record vendors . 
clark ln , guarrera tk , mcgeorge nm , et al : usability evaluation and assessment of a novel emergency department it system developed using a cognitive systems engineering approach . proceedings of the international symposium on human factors and ergonomics in health care 3 : 76 - 80 , 2014 14 . 
fonda sj , paulsen ca , perkins j , et al : usability test of an internet - based informatics tool for diabetes care providers : the comprehensive diabetes management progradiabetes technol ther 10 : 16 - 24 , 2008 21 . 
kushniruk aw , triola mm , borycki em , et al : technology induced error and usability : the relationship between usability problems and prescription errors when using a handheld application . 
sasaki t , kato y , sato a , et al : a questionnaire survey of pharmacists regarding the clinical practice guidelines for the appropriate use of granulocyte - colony stimulating factors . 
phillips p , boone d , mallett s , et al : method for tracking eye gaze during interpretation of endoluminal 3d ct colonography : technical description and proposed metrics for analysis . 
helbren e , halligan s , phillips p , et al : towards a framework for analysis of eye - tracking studies in the three dimensional environment : a study of visual search by experienced readers of endoluminal ct colonography . 
irwin de : fixation location and fixation duration as indices of cognitive processing , in henderson jm , ferriera f ( eds ) : the interface of language , vision , and action : eye movements and the visual world . 
in this study , metex14 ( c.2899g.a ) was detected by targeted next - generation sequencing with memorial sloan kettering integrated mutation proling of actionable cancer targets ( msk - impact ) in a case of advanced lung adenocarcinoma driven by egfr l858r mutation , after 12.5 months of frontline egfrtyrosine kinase inhibitor ( tki ) treatment . 
interestingly , metex14 co - occurred with met amplication and egfr t790m at the time of progression , as revealed by digital polymerase chain reaction on cell - free dna ( but not conrmed on tumor tissue )  . 
earlier , we described a patient whose primary resection sample tested positive for both egfr l858r and metex14 ( c3028 + 1g.t ) , but not for met amplication , as assessed by next - generation sequencing.2 in the current study , although the patient did not respond to met - targeted therapy alone , a combination of crizotinib ( met - tki ) and osimertinib ( third - generation egfr - tki ) led to durable disease control.1 however , we believe that additional key points should be discussed in more detail . 
targeted sequencing did not assess unknown potential mechanisms of resistance . it would have been interesting to learn which other pathways were abnormally activated in this tumor , such as fas or components of the nf - b pathway that specically enhanced cell death induced by rstgeneration egfr - tki erlotinib and caused acquired resistance to third - generation egfr - tki rociletinib in egfr - mutant lung cancer cells.3 , 4 met amplication should be further investigated . 
two recent studies reported a response to a combination of osimertinib and crizotinib in patients with nonsmallcell lung cancer ( nsclc ) harboring egfr l858r , while a rebiopsy showed met amplication after disease progression on erlotinib.5 , 6 similarly , a dramatic response was achieved using crizotinib and erlotinib in a case of met amplication emerging in the setting of egfr l858r mutation , suggesting the potential role of erlotinib and crizotinib combination , 5 which was not tested in vitro . 
in this model , crizotinib monotherapy was unable to modulate cell growth and switch off the egfr downstream signaling . although the drugs were used at a low concentration and no analysis was performed to assess pharmacointeractions , 7 crizotinib restored sensitivity to logical osimertinib in both pc9 and h1975 metex14 models . mechanistically , it was shown that the combination inhibited activation of egfr , met , as well as downstream akt and erk.1 these ndings underline the effectiveness of different tki combinations in inhibition of multiple signaling cascades to overcome cancer cell resistance.8 in particular , met signaling plays a crucial role in driving egfr - tki resistance in oncogene - addicted nsclc , and met inhibition can be a successful strategy to recover sensitivity in cases of met aberrations . 
despite the striking clinical activity of the combination in the current study , there is often a large gap between preclinical evaluations of tki combinations and clinical outcome.9 thus , the identication of predictive biomarkers for response could be useful for proper selection of patients who would likely benet from the therapeutic combination . 
we recently identied p - her3 and p - pras40 as potential candidates to assess the synergy between erlotinib and crizotinib in vitro.10 erlotinib and crizotinib showed remarkable synergy in the ludlu squamous - cell lung cancer ( scc ) cell line , which does not harbor egfrsensitizing mutations , met amplication , or metex14 . interestingly , other investigated scc cell lines ( h1703 and skmes - 1 ) , which were also wild - type for egfr and met , reacted differently ( but still additive ) , suggesting a role for the differential expression of p - her3 and p - pras40 expression in this synergy . 
because we observed a synergistic effect of the combination treatment of erlotinib and crizotinib only in scc cells that showed concordant expression of p - her3 and p - pras40 , we assume that p - her3 and p - pras40 may be candidate biomarkers for effective egfr + met blockade in patients with scc.10 taking into account our ndings , other components of the egfr and met pathways , including p - her3 and p - pras40 , should be investigated in tissues obtained from patients receiving a combination of egfr - tki and met - tki . in conclusion , we are indebted to suzawa et al1 for their elegant study . 
 correspondence alessandro leonetti , md amsterdam umc , vu university medical center , cancer center amsterdam , amsterdam , the netherlands , and university hospital of parma , parma , italy marcello tiseo , md , phd university hospital of parma and university of parma , parma , italy christian rolfo , md , phd , mba university of maryland greenebaum comprehensive cancer center , baltimore , md nele van der steen , phd amsterdam umc , vu university medical center , cancer center amsterdam , amsterdam , the netherlands , and center for oncological research ( core ) , university of antwerp , wilrijk , belgium godefridus j . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . marcello tiseo consulting or advisory role : astrazeneca , bristol - myers squibb , msd , boehringer ingelheim , takeda research funding : astrazeneca christian rolfo consulting or advisory role : mylan speakers bureau : novartis , msd , boehringer ingelheim , guardant health travel , accommodations , expenses : astrazeneca godefridus j . 
peters consulting or advisory role : taiho pharmaceutical , clear creek bio , ellipses pharma research funding : rexahn pharmaceuticals ( inst ) travel , accommodations , expenses : rexahn pharmaceuticals , taiho pharmaceutical no other potential conicts of interest were reported . references suzawa k , ofn m , schoenfeld aj , et al : acquired met exon 14 alteration drives secondary resistance to epidermal growth factor receptor tyrosine kinase inhibitor in egfr - mutated lung cancer . 
clin lung cancer 19 : 35 - 41 , 2018 bivona tg , hieronymus h , parker j , et al : fas and nf - b signalling modulate dependence of lung cancers on mutant egfr . 
nature 471 : 523 - 526 , 2011 galvani e , sun j , leon lg , et al : nf - b drives acquired resistance to a novel mutant - selective egfr inhibitor . 
oncotarget 6 : 42717 - 42732 , 2015 giroux - leprieur e , dumenil c , chinet t : combination of crizotinib and osimertinib or erlotinib might overcome met - mediated resistance to egfr tyrosine kinase inhibitor in egfr - mutated adenocarcinoma . 
j thorac oncol 13 : e232 - e234 , 2018 zhu vw , schrock ab , ali sm , et al : differential response to a combination of full - dose osimertinib and crizotinib in a patient with egfr - mutant nonlung cancer and emergent met amplication . 
lung cancer small cell ( auckl ) 10 : 21 - 26 , 2019 bijnsdorp iv , giovannetti e , peters gj : analysis of drug interactions . methods mol biol 731 : 421 - 434 , 2011 tong cws , wu wkk , loong hhf , et al : drug combination approach to overcome resistance to egfr tyrosine kinase inhibitors in lung cancer . cancer lett 405 : 100 - 110 , 2017 yang z , tam ky : combination strategies using egfr - tki in nsclc therapy : learning from the gap between pre - clinical results and clinical outcomes . 
van der steen n , leonetti a , keller k , et al : decrease in phospho - pras40 plays a role in the synergy between erlotinib and crizotinib in an egfr and cmet wild - type squamous non - small cell lung cancer cell line . 
 next - generation sequencing reveals potentially actionable alterations in the majority of patients with lymphoid malignancies purpose next - generation sequencing ( ngs ) identifies potentially targetable alterations by us food and drug administration ( fda ) approved drugs and / or by available experimental agents that may not have otherwise been contemplated . 
many targeted drugs have been developed for diverse solid cancers ; a smaller number of genomically targeted drugs have been approved for lymphoid malignancies . materials and methods we analyzed ngs results from 60 patients with various lymphoid malignancies and found 224 alterations ( median per patient , three alterations )  . results forty - nine patients ( 82% ) had potentially actionable alterations with the use of fdaapproved drugs and / or experimental therapies ; only 11 patients ( 18% ) had no theoretically actionable alterations . 
only three patients ( 5% ) had an alteration for which an approved drug in the disease is available ( on label ) ; 45 patients ( 75% ) had an alteration for which an approved drug is available for another disease ( off label )  . 
of note , 19 ( 32% ) of 60 patients had intermediate to high tumor mutational burden , which may predict response to certain immunotherapy agents . conclusion ngs identifies alterations that may be pharmacologically tractable in most patients with lymphoid malignancies , albeit with drugs that have usually been developed in the context of solid tumors . 
goodman michael choi matthew wieduwilt carolyn mulroney caitlin costello garrett frampton vincent miller razelle kurzrock author affiliations and support information ( if applicable ) appear at the end of this article . corresponding author : aaron m . 
goodman , md , moores cancer center , university of california san diego , 3855 health science dr , la jolla , ca 92093 ; e - mail : a1goodman@ucsd.edu. introduction the lymphoid malignancies have diverse biologic and clinical behavior and typically are treated with multiagent chemotherapy . 
many therapeutic regimens for b - cell non - hodgkin lymphomas and leukemias also incorporate the anti - cd20 monoclonal antibody rituximab , which has improved patient outcomes.1 treatment of metastatic solid tumors , like lymphoid malignancies , has also relied heavily on the use of cytotoxic chemotherapy . however , over the past decade , the treatment paradigm for metastatic solid tumors has shifted away from chemotherapy toward matching oncogenic driver mutations with targeted therapy ( precision medicine ) .2 - 4 for example , in patients with braf - mutated metastatic melanoma , the braf inhibitor vemurafenib markedly increases overall survival compared with chemotherapy.5 numerous targeted therapies are now approved by the us food and drug administration ( fda ) for patients with metastatic solid tumors across a wide array of histologies ( data supplement )  . 
the first example of targeted therapeutic efficacy is the hematologic disorder chronic myelogenous leukemia , which has been transformed by the use of agents that affect bcr - abl kinase activity . despite the rapid success in development , approval , and use of small - moleculetargeted agents in patients with solid malignancies , a paucity of such therapies approved for use in lymphoid malignancies remains ( data supplement )  . 
 small - moleculetargeted therapeutics are not an available option . the rapid technological advances in nextgeneration sequencing ( ngs ) have allowed oncologists to sequence tumor exomes in a clinically meaningful period of time.6 - 8 some studies have shown that patients with solid malignancies treated with matched therapy have improved outcomes , 9 and meta - analyses in approximately 85 , 000 patients have supported this finding.10 - 12 the targeting of alterations such as braf can result in responses across a wide variety of cancers , including lymphoid malignancies ( eg , hairy cell leukemia ) .13 , 14 although not all malignancies that harbor braf mutations will respond equally well to braf inhibition , the strategy of crosscancer basket trials has been established as highly worthwhile . ngs accurately identifies substitutions , indels , copy number alterations , and gene fusions in hematologic malignancies.15 in this report , we use this technology to analyze the genomic alterations in a cohort of 60 patients with various lymphoid malignancies to estimate the frequency of theoretically actionable alterations . 
this study was performed and consents obtained in accordance with university of california san diego institutional review board guidelines . tumor samples from tissue ( table 1 ) or peripheral blood were collected from 60 patients and submitted for ngs to foundation medicine , a laboratory improvement amendments clinical certified laboratory for ngs . 
the foundationone heme panel was used , which is a hybrid capturebased ngs test.16 the methods used in this assay have been described in detail in previous reports.7 , 15 the foundationone heme assay simultaneously detects all genomic alterations , including base pair substitutions , indels , copy number alterations , and select gene rearrangements , in 405 cancer - related genes . 
for tumor mutational burden ( tmb ) , the number of somatic mutations detected on ngs are quantified , and that value was extrapolated to the whole exome by using a validated algorithm described in detail in earlier publications.17 , 18 alterations with known and likely effects on functional status are not counted . definition of actionable alteration an alteration was defined as potentially actionable if its protein product is a component of a molecularly defined pathway for which there is at least one available fda - approved drug or investigational drug that may affect the function of the protein product of the alteration or the immediate downstream effectors of the protein product or that differentially recognizes the protein in tumor versus normal cells . 
the protein products of genomic alterations were considered to be functional the genomic alterations have been previously identified as relevant to cancer in the cosmic database , 19 which catalogs recurrent somatic alterations in cancer . 
novel base substitution , indel , and rearrangement alterations that result in truncations and homozygous copy number deletions that occur in tumor suppressor genes were considered to have likely functional implications . 
novel genomic alterations that occur at the same position as known alterations as well as alterations with conflicting evidence with regard to implication for function were subject to review by an internal panel of subject matter experts to determine functional status of the relevant protein product on the basis of all available evidence , including , but not limited to , the exac , dbsnp , and clinvar databases.20 - 22 data and statistical analyses patient characteristics were obtained through electronic medical record review . 
descriptive statistics were used , including medians , ranges , and frequencies . results patient characteristics sixty patients ( 35 men [ 58% ] and 25 women [ 42% ] ) with lymphoid malignancies were identified ( table 1 )  . 
the most common malignancy in the cohort was chronic lymphocytic leukemia ( cll ; 32% ) , followed by acute lymphoblastic leukemia ( all ; 20% ) , multiple myeloma ( 18% ) , diffuse large b - cell lymphoma ( dlbcl ; 10% ) , follicular lymphoma ( 8% ) , and other lymphoid neoplasms ( 5% )  . 
however , the actual alteration in nras differed between the two patients ( nras g13d v nras q61r )  . the median number of alterations detected per patient was three ( range , zero to 14 )  . 
as demonstrated in figure 1b , seven patients ( 12% ) had no reportable alterations , 10 ( 17% ) had one alteration , and 43 ( 71% ) had two or more alterations . 
the maximum number of alterations identified was 14 , which was observed in two patients ( 3% ) , one with cll , the other with dlbcl . of note , all patients with dlbcl had five or more alterations . actionable alterations potentially actionable alterations were identified in all disease subtypes ( table 2 )  . 
all disease subtypes , except marginal zone lymphoma , had alterations targetable by fda - approved drugs . forty - nine patients ( 82% ) had potentially actionable alterations with fda - approved drugs and / or experimental therapies ( clinical trials ) , whereas 11 patients ( 18% ) had no theoretically actionable alterations . depicted in figure 2 is the number of patients with potentially fda actionable alterations per disease group . 
only three patients had an alteration for which an approved drug in the disease is available ( on label ) , whereas 45 patients ( 75% ) had an alteration for which an approved drug is available in another disease ( off label )  . 
eleven patients had no targetable alterations ; these patients included seven with no detected genomic alterations . two patients with b - cell all ( b - all ) and one with waldenstr om macroglobulinemia had alterations targetable with on - labelapproved drugs ( fig 3 )  . 
high tmb was seen in five patients ( 8% ) , intermediate in 14 ( 23% ) , and low in 40 ( 67% )  . tmb was not available for one patient ( 2% )  . intermediate to high tmb was noted across almost all histologies ( except for marginal zone lymphoma , natural killer / t - cell lymphoma , and cutaneous t - cell lymphoma )  . 
three patients with multiple myeloma had an intermediate level of tmb . discussion this study demonstrates that in the majority of patients ( 82% ) with lymphoid malignancies whose disease was interrogated by ngs , alterations were found that might be pharmacologically tractable , a percentage similar to the 77% reported by he et al15 for diverse hematologic malignancies . 
another study reported that 70% of patients with various solid tumors had an alteration that was theoretically actionable with an approved drug.24 this finding is similar for lymphoid malignancies , with 75% of the current study patients having an alteration potentially actionable by an approved drug . 
table 3 lists therapeutics with their corresponding targets that were identified in the patient cohort . one of the major obstacles to performing comprehensive genomic profiling of clinical specimens is the necessity for an adequate tumor sample . 
unlike solid malignancies where invasive biopsy is almost always required to obtain a tissue sample , comprehensive genomic profiling of lymphoid malignancies often can be performed with the use of peripheral blood and / or bone marrow . in this cohort , 54% of patients had specimens obtained from blood and / or bone marrow . tumors can acquire new mutations as they progress , which underscores the importance of obtaining new tissue when available for sequencing at the time of therapeutic decision making . 
 other not actionable , 2 mm not actionable , 2 other actionable , actionable , actionable , cll not actionable , fl not actionable , 2 actionable , 3 actionable , dlbcl actionable , fig 2 . 
in one study , 42% of patients did not have metastatic disease at the time of their biopsy.24 lymphoid malignancies are more amenable to repeat biopsy , with more than one half of the current cohort having tissue available from the blood and / or bone marrow . 
this accessibility facilitates repeat ngs to guide therapy . over the past decade , 26 orally administered targeted therapies have been fda approved for the treatment of 13 different solid malignancies ( data supplement )  . 
over the same period , only five orally administered targeted therapies have been approved for the treatment of lymphoid malignancies ( philadelphia chromosomepositive all , cll , follicular lymphoma , mantle cell lymphoma , and waldenstr om macroglobulinemia )  . the current data demonstrate that 45 patients ( 75% ) had an alteration that theoretically could have been targeted with an approved , albeit offlabel , drug , whereas only three patients ( 5% ) had an alteration for which an approved on - label drug was available . 
in a similar study in patients with solid malignancies , 20% had an alteration targeted by on - label agents , whereas 67% had an alteration targetable by off - label use.24 ( of note , the study used the same definitions for actionability as were used in this analysis . ) currently , a paucity of orally administered targeted therapy is available for onlabel use in lymphoid malignancies . 
however , as our findings suggest , the majority of cases likely will have potentially targetable alterations . the mutational landscape for numerous lymphomas has now been well characterized by several whole - exome sequencing studies.46 , 47 for example , mll2 , crebbp , and tp53 alterations in dlbcl , as described by pasqualucci et al , 46 were some of the most common recurrent mutations in the current cohort of patients with dlbcl . 
this alteration was also the most common ( 12% ) in a cohort of patients with cll sequenced by puente et al.47 furthermore , 82% of our patients had alterations that were pharmacologically tractable , similar to the 77% reported by he et al15 in diverse hematologic malignancies . 
these similar findings in other cohorts provide further support of the generalizability of our findings to other large cohorts of lymphoid malignancies . myd88 , an adapter protein used by toll - like receptors , was shown to be mutated ( myd88 l265p ) in  . 
90% of patients with waldenstr om macroglobulinemia ( lymphoplasmacytic lymphoma ) .48 this mutation not only is relatively specific for the disease but also predicts clinical presentation and survival.49 myd88 signaling is important in lymphomagenesis and signals through btk.50 in a phase ii trial , ibrutinib produced an overall response rate of 91% in a group of previously treated patients with waldenstr om macroglobulinemia , which led to its fda approval.37 furthermore , response rates to ibrutinib were significantly higher in patients with myd88 mutations versus wild - type myd88.51 in the current cohort , myd88 l265p mutation was found in the one patient with waldenstr om macroglobulinemia . 
however , myd88 alterations were also identified in one patient with dlbcl ( myd88 l265p ) , one with follicular lymphoma ( myd88 s219c ) , and one with primary cns lymphoma ( myd88 l265p )  . 
myd88 mutations have been discerned in marginal zone b - cell lymphoma , 52 cll , 53 dlbcl , 54 and primary cns lymphoma.55 preliminary data from a phase i trial of single - agent ibrutnib in four patients with cns lymphoma demonstrated responses in two of the three patients evaluated . 
in a phase i trial of 17 patients with relapsed mantle cell lymphoma treated with single - agent palbociclib , five ( 18% ) achieved progression - free survival of  . 
1 year , with one complete and two partial responses.58 the high percentage of cdkn2a / b alterations in the current study population gives further rationale for designing trials with cdk inhibitors in lymphoid malignancies . activating mutations in braf were found in three patients with cll ( two with braf g469a and one with braf v600e ) and one patient with multiple myeloma ( braf v600e )  . 
these mutations are potentially targetable by the braf inhibitors vemurafenib and dabrafenib and the mek inhibitors trametinib and cobimetinib.5 , 27 braf alterations have been identified as a driver mutation and as a biomarker for sensitivity to braf inhibition in both hairy cell leukemia and erdheim - chester disease.59 - 61 braf alterations have been found in approximately 3% of patients with cll.62 in the current study , two patients had mutations that led to an alanine substitution for a glycine at position 469 . 
ngs readily identified philadelphia chromosomelike b - all and can assist in selecting patients for clinical trials of targeted agents aimed at improving the poor outcome of these patients.15 higher neoantigen burden , which may be largely predicted by tmb , has been associated with higher objective response rates and progressionin patients treated with pd - 1 free survival blockade.67 , 68 melanoma , lung cancer , and renal cell carcinoma , all of which are highly responsive to pd - 1 blockade , have been shown to have a high degree of mutational burden from wholegenome and - exome sequencing studies.69 in the current cohort , tmb ranged from one or fewer to 140 ( data supplement )  . 
intermediate to high tmb was noted across almost all histologies ( except for marginal zone lymphoma , natural killer / t - cell lymphoma , and cutaneous t - cell lymphoma )  . 
the majority of the patients had cll , multiple myeloma , and all , whereas other nonhodgkin lymphomas were less well represented . although the majority of patients had actionable mutations theoretically targeted by fda - approved drugs , in practice , insurance approval for off - label drug use often is difficult to obtain.73 in addition , no standard definition exists for a targetable alteration , and the level of evidence needed to support this is a matter of debate . 
the number of actionable alterations discussed in this article may be overestimated , but even so , these patients should still be directed toward clinical trials that target these alterations so that the responsiveness or lack thereof can be determined . 
numerous additional clinical trials with a standardized definition of what constitutes a targetable alteration are needed to determine the extent to which patients respond when an alteration is theoretically druggable . 
currently , a number of such trials are ongoing ( clinicaltrials.gov identifiers nct02534675 , nct00851032 , and nct02465060 )  . in conclusion , we found that most patients with lymphoid malignancies have unique and complex molecular portfolios . 
goodman administrative support : caitlin costello , razelle kurzrock provision of study materials or patients : michael choi , matthew wieduwilt , carolyn mulroney , caitlin costello collection and assembly of data : aaron m . 
 novartis , merck , roche / genetech , genentech mypath , foundation medicine , pfizer , guardant health , merck serono patents , royalties , other intellectual property : four patents travel , accommodations , expenses : win , emd serono , gateway research advisory committee , icrp , win 2015 , caris centers of excellence , aacr , association of american cancer institutes , emd serono & quintiles , global biomarkers consortium , guardant health , global source ventures / novena therapeutics , ascpt , meyers consulting , fda - ocra , genentech , orbimed / global source ventures , sylvester cancer center , journal of precision medicine , curematch , lynx group , mayo clinic cancer center , kaiser permanente , pancan , cedars - sinai , medimmune / jk associates medical communications group affiliations aaron m . 
sehn lh , donaldson j , chhanabhai m , et al : introduction of combined chop plus rituximab therapy dramatically improved outcome of diffuse large b - cell lymphoma in british columbia . 
helsten t , kato s , schwaederle m , et al : cell - cycle gene alterations in 4 , 864 tumors analyzed by next - generation sequencing : implications for targeted therapeutics . 
wheler j , lee jj , kurzrock r : unique molecular landscapes in cancer : implications for individualized , curated drug clin can res 22 : 5497 - 5505 , 2016 mutation . 
nat biotechnol 31 : 1023 - 1031 , 2013 janku f , wheler jj , naing a , et al : pik3ca mutation h1047r is associated with response to pi3k / akt / mtor signaling pathway inhibitors in early - phase clinical trials . 
schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
jardim dl , schwaederle m , wei c , et al : impact of a biomarker - based strategy on oncology drug development : a meta - analysis of clinical trials leading to fda approval . 
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he j , abdel - wahab o , nahas mk , et al : integrated genomic dna / rna profiling of hematologic malignancies in the n engl j med 373 : 726 - 736 , 2015 clinical setting . 
rosenberg je , hoffman - censits j , powles t , et al : atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum - based chemotherapy : a single - arm , multicentre , phase 2 trial . 
spigel dr , schrock ab , fabrizio d , et al : total mutation burden ( tmb ) in lung cancer ( lc ) and relationship with response to pd - 1 / pd - l1 targeted therapies . 
samadder nj , neklason dw , boucher km , et al : effect of sulindac and erlotinib vs placebo on duodenal neoplasia in familial adenomatous polyposis : a randomized clinical trial . 
robert c , karaszewska b , schachter j , et al : improved overall survival in melanoma with combined dabrafenib and n engl j med 374 : 311 - 322 , 2016 trametinib . 
emadi a , faramand r , carter - cooper b , et al : presence of isocitrate dehydrogenase mutations may predict clinical response to hypomethylating agents in patients with acute myeloid leukemia . 
furumoto y , gadina m : the arrival of jak inhibitors : advancing the treatment of immune and hematologic disopin investig drugs 21 : 637 - 655 , 2012 orders . 
infante jr , fecher la , falchook gs , et al : safety , pharmacokinetic , pharmacodynamic , and efficacy data for the oral mek inhibitor trametinib : a phase 1 dose - escalation trial . 
migden mr , guminski a , gutzmer r , et al : treatment with two different doses of sonidegib in patients with locally advanced or metastatic basal cell carcinoma ( bolt ) : a multicentre , randomised , double - blind phase 2 trial . 
said r , hong ds , warneke cl , et al : p53 mutations in advanced cancers : clinical characteristics , outcomes , and correlation between progression - free survival and bevacizumab - containing therapy . 
schwaederle m , lazar v , validire p , et al : vegf - a expression correlates with tp53 mutations in non - small cell lung cancer : implications for anti - angiogenesis therapy . 
koehler k , liebner d , chen jl : tp53 mutational status is predictive of pazopanib response in advanced sarcomas . ann oncol 27 : 539 - 543 , 2016 44 . 
von bubnoff n , schneller f , peschel c , et al : bcr - abl gene mutations in relation to clinical resistance of philadelphia - chromosome - positive leukaemia to sti571 : a prospective study . 
treon sp , cao y , xu l , et al : somatic mutations in myd88 and cxcr4 are determinants of clinical presentation and overall survival in waldenstr om macroglobulinemia . 
finn rs , crown jp , lang i , et al : the cyclin - dependent kinase 4 / 6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first - line treatment of oestrogen receptor - positive , her2 - negative , advanced breast cancer ( paloma - 1 / trio - 18 ) : a randomised phase 2 study . 
nadeu f , delgado j , royo c , et al : clinical impact of clonal and subclonal tp53 , sf3b1 , birc3 , notch1 , and atm mutations in chronic lymphocytic leukemia . 
woyach ja , furman rr , liu t - m , et al : resistance mechanisms for the brutons tyrosine kinase inhibitor ibrutinib . n engl j med 370 : 2286 - 2294 , 2014 66 . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
from march to december 2015 , a local oncology practice administered 10 cycles of the recommended systemic chemotherapy with liposomal pegylated doxorubicin ( 30 to 35 mg / m2 once every 28 days ) , which resulted in complete disappearance of cutaneous lesions . 
at this time , the sarcoma lesions were markedly progressive and caused deep ulcerations that led to constant bleeding and a massively increased need for potent analgesics ( fig 1 )  . 
under this treatment , ulcerations worsened and the patient required red blood cell transfusions once per week because of the continuous bleeding from his sarcomatous lesions ( fig 1 )  . a histopathologic re - examination of the biopsy taken at recurrence revealed expression of programmed death - ligand 1 ( pd - l1 ) on sarcoma cells in 10% of the sections ( fig 2 ) by using the pd - l1 ihc 22c3 pharmdx assay ( dako agilent , waldbronn , germany ) and the monoclonal 28 - 8 antibody ( abcam , cambridge , uk ; staining on dako omnis platform , waldbronn , germany )  . 
of note , the tumor showed a high level of immune cell infiltrates with formation of lymph follicles and germinal centers as well as focal infiltration of cd8 + t cells ( fig 2 )  . 
after five cycles of pembrolizumab , a very good clinical response of all sarcoma lesions was observed , and the patient required rainer hamacher dietrich kmpfe kirsten reuter - jessen christoph pttgen lars e . 
clinical macroscopic course during pembrolizumab treatment course of preauricular and submandibular tumor on the left side and preauricular tumor on the right side before at baseline ( october and november 2016 ) , after five cycles ( march 2017 ) , and after 10 cycles ( august 2017 ) of treatment with 2 mg / kg pembrolizumab in a 3 - week cycle . no more analgesics ( fig 1 )  . 
 moreover , the patient experienced a marked with cas in the head and neck region , those occurrences represent less than 0.15% of all head and neck malignancies.2 , 3 the incidence is highest in elderly white patients with a mean age of 73 years , and there is no difference between sexes.2 , 4 aside from sporadic occurrences , long - standing lymphedema and prior radiation pd - l1 pd - l1 pd - l1 fig 2 . 
three representative photos of immunohistochemical stainings with anti - pd - l1 ( on the left ) and one photo with anti - cd8 ( on the right ) of the re - examined local recurrence of the lesion obtained between 2014 and 2016 . 
 disease followed by salvage surgery of local recurrence.6 , 7 after progression , he received a standard sequence of palliative systemic therapies , 7 - 9 and he exhibited good but transient responses . 
subsequent treatments for his highly symptomatic disease , chosen on the basis of prior reports of clinical activity , 10 - 14 were poorly tolerated or showed no efficacy . overall , patients with cas have a poor prognosis with a high local recurrence rate and 5 - year survival of only 30%.1 , 4 for cas of the scalp , the median overall survival ranges from 6 to 24 months with a 2 - year survival rate of less than 15% in patients with advanced disease.15 although lung metastases are frequent , patients quality of life is often massively compromised by local recurrence with highly mutilating , ulcerating , and disfiguring lesions . 
the interaction of pd - 1 ( a receptor on t cells ) and its ligands ( pd - l1 and pd - l2 ) on target cells is one dominant immune checkpoint pathway that represses cytotoxic immune response . 
whereas the physiologic function of pd - l1 is controlling immune homeostasis , upregulation of pd - l1 by cancer cells and cells of the microenvironment has been described as a mechanism of immune escape.16 , 17 development of monoclonal antibodies blocking either pd - 1 ( such as pembrolizumab or nivolumab ) or pd - l1 ( such as atezolizumab , durvalumab , or avelumab ) have opened novel treatment approaches that show improved overall survival in several cancers , including melanoma , non - small - cell lung cancers ( nsclcs ) , and renal cell cancer ; the list of additional susceptible cancer entities is expanding rapidly.16 , 17 soft tissue sarcomas are not yet in the immunotherapy spotlight , and there is a surprisingly low number of clinical trials , most of which studied cellular therapies that target tumor antigens or sarcoma - specific gene products from oncogenic transcripts.18 - 20 nevertheless , several fusion studies found evidence that sarcomas suppress immune response and have also discovered additional characteristics that provide a rationale for immune checkpoint blockade.18 , 20 , 21 preliminary results of the recent sarc028 phase ii trial reported early responses on pembrolizumab in undifferentiated pleomorphic sarcoma and in a low number of osteosarcoma , liposarcoma , and high - grade or dedifferentiated chondrosarcoma.22 in contrast , ipilimumab , a monoclonal antibody targeting ctla - 4 ( which has been approved for immunotherapy of melanoma ) , showed little activity in a group of 17 adolescent patients with sarcoma.23 numerous studies investigating the role of immunotherapy in soft tissue sarcoma are currently ongoing , but many exclude rare subtypes such as angiosarcoma . predictive biomarkers for icis have not yet been identified in rare tumor types . 
studies in other cancer types ( eg , ncslc ) indicate that intratumoral pd - l1 expression , lymphoid infiltration , and mutational burden are associated with improved therapeutic response.17 two recent retrospective studies reported expression of pd - l1 in 30% to 40% of patients with cas and found an inverse correlation between prognosis and expression levels of pd - l1 and infiltrating t cells.24 , 25 we observed expression of pd - l1 in our patient in 10% of the sarcoma cells in his recurrent lesion ( fig 2 )  . 
notably , a patient with cas who had similar intratumoral pd - l1 expression more than 5% has recently been reported with response to pembrolizumab.26 in addition to pd - l1 expression , we observed a high rate of tumor - infiltrating immune cells , notably with the formation of intratumoral lymph follicles and infiltration by cd8 + cells ( fig 2 )  . 
although single patients preclude making any sort of generalization , the relatively high level of pd - l1 expression could serve as one explanation for the response , especially in light of the generally low pd - l1 expression rates found in sarcomas.22 the recent sarc study used a cutoff of 1% pd - l1 expression resulting in 4% positive tumors when using this threshold in this cohort.22 this cutoff is useful in nsclc , in which pd - l1 positivity of more than 1% is associated with a higher overall response rate.17 , 27 , 28 moreover , we observed an infiltration with lymphoid cells and the presence of lymph follicles , which could be a consequence of chronic inflammation resulting from the ulceration but may also indicate that the tumor has elicited an immune response , which might be useful to explore in a larger cohort of patients . 
 interestingly , other studies indicate sensitivity to immunotherapy of other kinds of skin malignancy beyond melanoma , such as basal cell , cutaneous squamous cell , and merkel cell carcinoma , that is associated with high exposure to ultraviolet light.29 , 30 given the devastating consequences and the limitations of currently available treatments , we believe the unusual response in our report and in one other case report26 strongly warrants further studies in cas . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . rainer hamacher travel , accommodations , expenses : eli lilly , pharmamar , novartis dietrich kmpfe no relationship to disclose kirsten reuter - jessen honoraria : zytovision , ah diagnostics travel , accommodations , expenses : zytovision , ah diagnistics , astrazeneca honoraria : roche pharma ag , boehringer ingelheim speakers bureau : roche pharma ag , boehringer ingelheim christoph pttgen no relationship to disclose lars e . 
dill , md , for providing paraffin blocks of the patients tumor , and the members of the bauer and schildhaus laboratory for expert technical assistance , helpful discussions , and critical reading . 
podleska , farhad farzaliyev , and hans - ulrich steinau , university hospital essen , university duisburg - essen ; martin schuler and sebastian bauer , german cancer consortium , partner site university hospital essen , essen ; dietrich kmpfe , hematology and oncology practice , ldenscheid ; and kirsten reuter - jessen and hans - ulrich schildhaus , university hospital gttingen , gttingen , germany . support supported by sarkomtour ( s.b. ) references 39 : 258 - 263 , 2015 1 . 
albores - saavedra j , schwartz am , henson de , et al : cutaneous angiosarcoma : analysis of 434 cases from the surveillance , epidemiology , and end results program , 1973 - 2007 . 
schlemmer m , reichardt p , verweij j , et al : paclitaxel in patients with advanced angiosarcomas of soft tissue : a retrospective study of the eortc soft tissue and bone sarcoma group . 
kollr a , jones rl , stacchiotti s , et al : pazopanib in advanced vascular sarcomas : an eortc soft tissue and bone sarcoma group ( stbsg ) retrospective analysis . 
le cesne a , ray - coquard i , duffaud f , et al : trabectedin in patients with advanced soft tissue sarcoma : a retrospective national analysis of the french sarcoma group . 
kim jr , moon yj , kwon ks , et al : tumor infiltrating pd1 - positive lymphocytes and the expression of pd - l1 predict poor prognosis of soft tissue sarcomas . 
tawbi ha , burgess m , bolejack v , et al : pembrolizumab in advanced soft - tissue sarcoma and bone sarcoma ( sarc028 ) : a multicentre , two - cohort , single - arm , open - label , phase 2 trial . 
honda y , otsuka a , ono s , et al : infiltration of pd - 1 - positive cells in combination with tumor site pd - l1 expression is a positive prognostic factor in cutaneous angiosarcoma . 
gandini s , massi d , mandal m : pd - l1 expression in cancer patients receiving anti pd - 1 / pdl1 antibodies : a systematic review and meta - analysis . 
dmmr / microsatellite high ( msi - h ) breast cancers were among the responding tumors that led to the recent us food and drug administration accelerated approval of pembrolizumab for any patient with a dmmr / msi - h metastatic solid tumor.2 the authors conclude that they would like to make a case for msi testing in patients with metastatic cancer beyond typical lynch - associated cancers.1 i also propose the study of whether patients now identified with dmmr breast cancers might harbor germline mismatch repair ( mmr ) - mutated genes and , therefore , would be molecularly defined as lynch syndrome family members . the us food and drug administration accelerated approval and suggestion by kok et al represent an opportunity to clarify whether breast cancer should be considered a lynch - associated cancer . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . steven sorscher honoraria : celgene , pfizer , puma biotechnology speakers bureau : celgene , pfizer , puma biotechnology travel , accommodations , expenses : celgene , pfizer , puma biotechnology 1 . 
dinardo , md1 introduction germline predispositions to hematologic malignancies in adult patients presenting with a diagnosis of myelodysplastic syndrome ( mds ) or acute myeloid leukemia ( aml ) are commonly underestimated . 
through next - generation sequencing ( ngs ) , many genes involved in hereditary mds or aml are now evaluated , including runx1 , cebpa , gata2 , tp53 , etv6 , and ddx41.1 , 2 germline gata2 mutations are a common cause for predisposition to mds or aml.3 patients with gata2 deciency can have manifestations ranging from mild cytopenias to life - threatening infections and myeloid neoplasia . 
the incidence of gata2 deciency is still unknown ; however , gata2 mutations may account for approximately 75% of adolescent patients with monosomy 7 mds.4 approximately 20% to 30% of gata2 mutations are inherited in an autosomal dominant fashion , whereas others are sporadic or de novo mutations.5 because molecular testing for mds and aml has become more commonplace , we recognize that some clinicians may incorrectly equate a negative ngs panel as ruling out any potential inherited malignancy predisposition . 
in addition , promoter mutations ( ie , ankrd26 ) and intronic mutations ( ie gata2 ) may often be missed if the ngs panel is restricted to exonic sequences . 
specic testing for fanconi anemia and dyskeratosis congenita when clinically indicated should be performed , for example , diepoxybutane or mitomycin cinduced chromosomal breakage assay and telomere length owuorescence in situ hybridization analysis , respectively . 
publically available registries and genomic data sets such as clinvar and gnomad are increasingly useful to provide insight into clinical signicance of identied variants . here , we report on two patients with a history of immunodeciency and myeloid neoplasia . 
1 is a 24 - year - old hispanic man who presented for evaluation of pancytopenia , mainly leukopenia associated with recurrent extensive warts on bilateral hands and recurrent panniculitis . 
his blood counts were as follows : white blood cell count , 3.2 109 / l ; hemoglobin , 14.1 g / dl ; and platelet count , 186 109 / l . 
concurrent ow cytometry analysis demonstrated a small population of aberrant myeloblasts , lack of hematogones , virtual absence of monocytes , and markedly reduced natural killer ( nk ) cells . 
a peripheral blood sample was obtained for ow cytometry study that showed no monocytes , nk cell lymphopenia ( 0.4% of lymphocyte gate ; fig 1b ) , and decreased b cells ( 3.8% of lymphocyte gate )  . 
a targeted exome sequence analysis of the following genes was performed on cultured skin broblasts : ankrd26 , cebpa , ddx41 , etv6 , gata2 , runx1 , srp72 , terc , tert , and tp53 . 
her blood counts showed a white blood cell count of 2.9 109 / l , with 75% neutrophils , 21% lymphocytes , 1% monocytes , hemoglobin of 10.1 g / dl , and platelet count of 164 109 / l . 
her past medical history was unremarkable , and her family history was negative for any immunologic or hematologic diseases . because of persistent ebv viremia , she received autologous ebv - specic cytotoxic t - lymphocyte cells . 
she received intensive cytarabine - based chemotherapy , and her induction course was complicated by an infection with mycobacterium fortuituflow cytometry analysis showed that she achieved complete remission with negative minimal residual disease . 
before proceeding with allogeneic hematopoietic stem - cell transplantation ( hsct ) from her sibling , the patient was referred to our hereditary hematologic malignancy clinic.6 a skin biopsy was performed for cultured skin broblasts , and genetic testing demonstrated a heterozygous deletion that included at least exon 2 of the gata2 gene . 
top : prehsct , the bone marrow shows a near absence of monocytes on ( left ) cd45 / ssc and ( middle ) cd14 ( thick arrows ) ; ( right ) no stage i or stage ii hematogones or mature b cells ( thin arrows )  . 
this variant has been previously reported in at least 29 other patients.4 , 10 - 14 the gata2 mutation detected in patient no . 2 was a large deletion involving at least exon 2 . 
to our knowledge , this is the rst time this specic deletion has been reported , although other in - frame or full gene gata2 deletions have been described.4 , 15 gata2 deciency can have various clinical presentations.11 , 16 patient no.1 presented initially with dermatologic problems , including extragenital warts and recurrent severe panniculitis ; mild cytopenias and hypocellular mds occurred later in his disease course . 
laboratory ndings include monocytopenia , dendritic cell cytopenias , b and nk lymphocytopenia , relative increase in t cells with an inverted cd4 : cd8 ratio , lack of hematogones , and various degrees of dysplasia.13 bm cellularity may vary from hypocellular marrow with severe cytopenias to normoor hypercellular marrow . 
 abou dalle et al stem cell exhaustion.3 the median age at diagnosis of 380 younger patients was approximately 20 years ( range , 12 to 35 years ) , with a high risk of progression to aml in 75% of carriers . 
complex cytogenetics and del ( 5q ) were not usually observed.5 patients with immunodeciency and recurrent infections along with a new diagnosis of mds at a younger age should be screened for an underlying germline mutation . 
genetic testing for germline mutations should sequence the most common genes responsible for familial predisposition to mds or aml , including ankrd26 , cebpa , ddx41 , etv6 , gata2 , runx1 , srp72 , samd9 , samd9l , terc , tert , and tp53 using nonhematopoietic tissue , usually cultured skin broblasts . gata2 sequencing should involve deletion and duplication analysis and intron 5 of the gata2 gene to detect all possible pathogenic variants . 
dinardo , md , department of leukemia , the university of texas md anderson cancer center , 1515 holcombe blvd , unit 0428 , houston , tx 77030 ; e - mail : cdinardo@mdanderson.org. support supported in part by specialized programs of research excellence grant no . 
cortes consulting or advisory role : bristol - myers squibb , biolinerx , novartis , pzer , amphivena therapeutics , daiichi sankyo , bio - path holdings , astellas pharma , takeda pharmaceuticals research funding : bristol - myers squibb ( inst ) , novartis ( inst ) , pzer ( inst ) , celgene ( inst ) , arog pharmaceuticals ( inst ) , astellas pharma ( inst ) , ambit biosciences ( inst ) , celator pharmaceuticals ( inst ) , immunogen ( inst ) , sun pharmaceutical industries ( inst ) , takeda pharmaceuticals ( inst ) alessandra ferrajoli research funding : celgene , genentech , acerta pharma dimitrios p . 
kontoyiannis employment : md anderson cancer center honoraria : gilead sciences , astellas pharma , t2 biosystems , mylan consulting or advisory role : merck , astellas pharma , fast track to growth , pzer , astellas pharma sa a . 
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kazenwadel j , secker ga , liu yj , et al : loss - of - function germline gata2 mutations in patients with mds / aml or monomac syndrome and primary lymphedema reveal a key role for gata2 in the lymphatic vasculature . 
we demonstrated previously that pemetrexed - based chemotherapy can achieve an objective response rate of 45% and a median progression - free survival ( pfs ) of 19 months.3 furthermore , the activity of targeted therapy has improved dramatically with the introduction of selective ret inhibitors to the clinic . 
in early - phase testing , objective response rates with loxo - 2924 and blu - 6675 are 68% ( 26 of 38 ) and 50% ( seven of 14 ) , respectively . 
these outcomes exceed the modest activity observed previously with multikinase inhibitors such as cabozantinib6 and vandetanib.7 in contrast , the activity of immunotherapy in retrearranged lung cancers has not been well characterized . 
this represents a clear unmet need , given that all prior regulatory approvals of immune checkpoint inhibitors , either alone or in combination with chemotherapy , and in stage iii or iv disease , have technically included patients with ret - rearranged lung cancers.8 , 9 furthermore , although increasing levels of programmed death - ligand 1 ( pd - l1 ) expression and high tumor mutational burden ( tmb ) have been associated with benet from immune checkpoint blockade , 10 the immunophenotype of ret - rearranged lung cancers and the role of pd - l1 and tmb status in relation to benet with immunotherapy remain poorly described . 
we set out to characterize these factors . methods in this retrospective study , patients from memorial sloan kettering cancer center with ret - rearranged lung cancers diagnosed between 2009 and 2017 were identied under an institutional review board approved waiver . 
patients who received immunotherapy , dened as a monoclonal antibody against programmed cell death protein 1 ( pd - 1 ) or pd - l1 , were included in an analysis of treatment history . ret rearrangements were identied using targeted next - generation sequencing of dna ( memorial sloan ketteringintegrated mutation proling of actionable cancer targets [ msk - impact ] or foundation one ) or rna ( anchored multiplex polymerase chain reaction [ pcr ] ; memorial sloan kettering solid fusion panel ) in more contemporary samples , and uorescence in situ hybridization ( 10q11 and 6q22 break apart probe , reversemetasystems , altussheim , germany ) or transcriptase pcr in older samples.11 , 12 pd - l1 immunohistochemistry was evaluated using the e1l3n antibody ( cell signaling technology , danvers , ma ) , our institutional standard , which has been validated against a 22c3 kit performed in a commercial laboratory with comparable results.13 for uniformity , tmb ( reported as the number of nonsynonymous mutations per megabase ) was analyzed only for samples sequenced using msk - impact.14 , 15 the median tmb of ret - rearranged was compared with that of ret wildtype nsclcs ( mann - whitney u test )  . in patients with both baseline and serial on - treatment imaging , the best objective response to therapy ( response evaluation criteria in solid tumors [ recist ] v1.1 ) was determined by a study radiologist . 
time to treatment discontinuation ( ttd ) was dened as the time from therapy initiation to the last dose.16 pfs was dened as the time from therapy initiation to radiologic progression or death . 
ret rearrangement was identied as follows : dna - based next - generation sequencing ( 80% [ n = 59 ] ) , rnabased anchored multiplex pcr ( 1% [ n = 1 ] ) , uorescence in situ hybridization ( 15% [ n = 11 ] ) , and reverse - transcriptase pcr ( 4% [ n = 3 ] )  . 
clinicopathologic features of ret - rearranged lung cancers : summary of demographics and tumor molecular features of 74 patients with ret - rearranged lung cancers feature all patients ( n = 74 ) age , years median range sex , no . 
in 44 patients with sufcient tissue for tmb analysis , the median tmb was 1.75 mutations / mb ( range , 0 to 9.65 mutations / mb ) , signicantly lower ( p , .0001 ) than the median tmb of 5.27 mutations / mb ( range , 0 to 164.20 mutations / mb ) in 3 , 631 patients with ret wild - type nsclcs ( fig 1b )  . the clinical outcomes of immunotherapy in patients with advanced ret - rearranged lung cancers are summarized in table 2 . 
 ( a ) the programmed death - ligand 1 ( pd - l1 ) expression ( e1l3n , cell signaling ) of 26 ret - rearranged lung cancers with sufcient tissue for testing is shown . 
 ( b ) the tumor mutational burden ( tmb ) of 44 ret - rearranged lung cancers is displayed ( left ) relative to the tmb of 3 , 631 ret wild - type lung cancers ( right )  . 
for ease of representation , three outlier ret wild - type lung cancer samples with tmb greater than 75 mutations / mb that were included in the statistical analysis were excluded in this plot . nivolumab ( n = 6 ) , atezolizumab ( n = 2 ) , durvalumab ( n = 1 ) , or ipilimumab plus nivolumab ( n = 1 )  . 
dashes represent tumor samples in which tissue was insufcient for pd - l1 or tmb testing . abbreviations : cr , complete response ; pd , progressive disease ; pd - l1 , programmed death - ligand 1 ; pfs , progression - free survival ; sd , stable disease ; non - cr / non - pd , not cr and not pd in nontarget lesions ; tmb , tumor mutational burden . * treatment discontinued for toxicity . a total of 13 patients with ret - rearranged lung cancers were assessed for clinical and / or radiologic response . response to immunotherapy was not observed . 
progression of disease was observed in 62% of cases ( n = eight of 13 ; table 2 ) ; disease progression involved new or worsening brain metastases in three patients ( cases 4 , 6 , and 15 )  . 
stable disease was achieved in 23% ( three of 13 ) and non - cr / non - pd ( not complete reponse and not progressive disease in nontarget lesions ) in 15% ( two of 13 )  . 
 ret - rearranged nsclc : immunophenotype and immunotherapy response these ndings are consistent with a growing body of evidence uncovering poor outcomes with immune checkpoint inhibition in select oncogene - addicted lung in egfr - mutant and alk - rearranged lung cancers . cancers , early data on the decreased activity ( compared with unselected cancers ) of immunotherapy resulted in the exclusion of patients with these tumors in registrationenabling studies . 
furthermore , we showed previously that met exon 14altered nsclcs had low tmb and poor outcomes with immunotherapy.17 finally , an ongoing global registry ( immunotarget ) and independent series have shown similarly low response rates and short median pfs with single - agent immune checkpoint inhibition in lung cancers with oncogenic drivers.18 immunotarget had 16 patients in the ret - rearranged cohort and reported a response rate of 6% and median pfs of 2.1 months , comparable to the ndings in our study . the clinical implications of these observations relate to the sequence with which immunotherapy is used and the type of immunotherapy strategy selected . 
regarding the former , it is becoming increasingly clear that specic , targeted therapies ( selective ret inhibitors ) 2 , 4 , 19 and chemotherapy agents ( pemetrexed - containing regimens ) 3 can achieve superior outcomes compared with immunotherapy in this series . 
thus , is reasonable to consider the use of checkpoint inhibition only after select targeted therapies and platinum doublet - containing chemotherapy have been administered . note that high pd - l1 expression ( 50% or more ) was uncommon in ret - rearranged lung cancers in this study . only one case treated with immunotherapy highly expressed pd - l1 ( 50% ) and , despite this , still responded poorly to dual immune checkpoint inhibitor therapy ( case 16 )  . 
a recommendation regarding the use of pembrolizumab in treatment - nave , advanced ret - rearranged lung cancers with high pd - l1 expression cannot be made on the basis of our ndings , although its use should be approached with caution . 
in met exon 14altered lung cancers , tmb is similarly low ; although a higher proportion of tumors have high pd - l1 expression , this was not associated with increased benet with single - agent immunotherapy.17 finally , no patients in this series received the combination chemotherapy and immunotherapy that is currently approved for the treatment of egfr and alk wild - type advanced nsclc . 
prospective trials of immunotherapy combinations should incorporate comprehensive molecular proling to establish prospective data in driver - positive subpopulations of patients . in summary , most ret - rearranged lung cancers have low pd - l1 expression and low tmb , and response to immunotherapy was not observed in this series . 
although this study is limited by a small sample size , given the rarity of ret - rearranged nsclcs , these ndings remain meaningful and support evolving literature showing that outcomes with immune checkpoint inhibition are poor in select oncogene - addicted nsclcs . 
contributed equally to this work . support supported in part by the national cancer institute of the national institutes of health ( t32 ca009207 , p30 ca008748 )  . provision of study material or patients : gregory j . 
rudin consulting or advisory role : bristol - myers squibb , abbvie , seattle genetics , harpoon therapeutics , genentech / roche , astrazeneca , ascentage pharma , bicycle therapeutics , celgene , daiichi sankyo , ipsen , loxo , pharmamar , elucida oncology research funding : abbvie / stemcentrx ( inst ) , viralytics ( inst ) , merck ( inst ) , daiichi sankyo ( inst ) gregory j . 
riely research funding : novartis ( inst ) , roche / genentech ( inst ) , millennium pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , pzer ( inst ) , innity pharmaceuticals ( inst ) , ariad pharmaceuticals ( inst ) , mirati therapeutics ( inst ) , merck ( inst ) patents , royalties , other intellectual property : patent application submitted covering pulsatile use of erlotinib to treat or prevent brain metastases ( inst ) travel , accommodations , expenses : merck sharp & dohme matthew d . 
hellmann stock and other ownership interests : shattuck labs honoraria : astrazeneca , bristol - myers squibb consulting or advisory role : bristol - myers squibb , merck , genentech , astrazeneca / medimmune , novartis , janssen pharmaceuticals , nektar , syndax pharmaceuticals , mirati therapeutics , shattuck labs research funding : bristol - myers squibb ( inst ) patents , royalties , other intellectual property : a patent has been led by memorial sloan kettering ( pct / us2015 / 062208 ) for the use of tumor mutation burden for prediction of immunotherapy efcacy , and which is licensed to personal genome diagnostics ( inst ) travel , accommodations , expenses : astrazeneca , bristol - myers squibb maria arcila honoraria : invivoscribe consulting or advisory role : astrazeneca travel , accommodations , expenses : astrazeneca , invivoscribe , raindance technologies mark g . 
li consulting or advisory role : roche , biosceptre international , thermo fisher scientic , mersana , guardant health , hengrui therapeutics research funding : roche / genentech ( inst ) , illumina ( inst ) , biomed valley discoveries ( inst ) , astrazeneca ( inst ) , grail ( inst ) , daiichi sankyo ( inst ) , hengrui therapeutics ( inst ) , guardant health ( inst ) marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network / astrazeneca tagrisso request for proposal advisory committee , takeda pharmaceuticals , bristol - myers squibb , bayer ag , merck ( i ) research funding : loxo ( inst ) , helsinn therapeutics alexander drilon honoraria : medscape , onclive , peervoice , physicians education resources , targeted oncology , more health , research to practice , foundation medicine , peerview consulting or advisory role : ignyta , loxo , tp therapeutics , astrazeneca , pzer , blueprint medicines , genentech / roche , helsinn therapeutics , beigene , hengrui therapeutics , exelixis , bayer ag , tyra biosciences , verastem , takeda pharmaceuticals / ariad pharmaceuticals / millenium pharmaceuticals , bergenbio , more health patents , royalties , other intellectual property : wolters kluwer ( royalties for pocket oncology ) other relationship : merck , glaxosmithkline , teva pharmaceuticals industries , taiho pharmaceutical no other potential conicts of interest were reported . references stransky n , cerami e , schalm s , et al : the landscape of kinase fusions in cancer . 
nat commun 5 : 4846 , 2014 drilon a , hu zi , lai ggy , et al : targeting ret - driven cancers : lessons from evolving preclinical and clinical landscapes . 
nat rev clin oncol 15 : 151 - 167 , 2018 drilon a , bergagnini i , delasos l , et al : clinical outcomes with pemetrexed - based systemic therapies in ret - rearranged lung cancers . 
ann oncol 27 : 1286 - 1291 , 2016 oxnard gr , subbiah v , park k , et al : clinical activity of loxo - 292 , a highly selective ret inhibitor , in patients with ret fusion + non - small cell lung cancer : an update from asco 2018 . 
toronto , canada , september 23 - 26 , 2018 subbiah v , taylor m , lin j , et al : abstract ct043 : highly potent and selective ret inhibitor , blu - 667 , achieves proof of concept in a phase i study of advanced , ret - altered solid tumors . 
drilon a , rekhtman n , arcila m , et al : cabozantinib in patients with advanced ret - rearranged non - small - cell lung cancer : an open - label , single - centre , phase 2 , single - arm trial . 
lancet oncol 17 : 1653 - 1660 , 2016 lee sh , lee jk , ahn mj , et al : vandetanib in pretreated patients with advanced non - small cell lung cancer - harboring ret rearrangement : a phase ii clinical trial . 
ann oncol 28 : 292 - 297 , 2017 gandhi l , rodrguez - abreu d , gadgeel s , et al : pembrolizumab plus chemotherapy in metastatic non - small - cell lung cancer . 
n engl j med 378 : 2078 - 2092 , 2018 reck m , rodrguez - abreu d , robinson ag , et al : pembrolizumab versus chemotherapy for pd - l1 - positive non - small - cell 375 : 1823 - 1833 , 2016 lung cancer . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
rizvi h , sanchez - vega f , la k , et al : molecular determinants of response to anti - programmed cell death ( pd ) - 1 and anti - programmed death - ligand 1 ( pd - l1 ) blockade in patients with non - small - cell lung cancer proled with targeted next - generation sequencing . 
ofn m , rizvi h , tenet m , et al : tumor mutation burden and efcacy of egfr - tyrosine kinase inhibitors in patients with egfr - mutant lung cancers . 
sabari jk , leonardi gc , shu ca , et al : pd - l1 expression , tumor mutational burden , and response to immunotherapy in patients with met exon 14 altered lung 18 . 
mazieres j , drilon ae , mhanna l , et al : efcacy of immune - checkpoint inhibitors ( ici ) in non - small cell lung cancer ( nsclc ) patients harboring activating molecular alterations ( immunotarget )  . 
 ofn et al immunotherapy naive ( n = 46 ) immunotherapy administered ( n = 16 ) log - rank p = .35 time since metastasis diagnosis ( years ) no . 
overall survival from the diagnosis of metastatic disease was compared between patients who received an immune checkpoint inhibitor ( n = 16 ) and those who did not ( n = 46 )  . 
 genetic testing and clinical management practices for variants in non - brca1 / 2 breast ( and breast / ovarian ) cancer susceptibility genes : an international survey by the evidence - based network for the interpretation of germline mutant alleles ( enigma ) clinical working group sarah m . 
domchek ros eeles anna efremidis ( continued ) purpose to describe a snapshot of international genetic testing practices , specifically regarding the use of multigene panels , for hereditary breast / ovarian cancers . 
we conducted a survey through the evidence - based network for the interpretation of germline mutant alleles ( enigma ) consortium , covering questions about 16 non - brca1 / 2 genes . methods data were collected via in - person and paper / electronic surveys . 
additional information was collected via country networks in the united kingdom and in italy . results responses from 61 cancer genetics practices across 20 countries showed that 16 genes were tested by > 50% of the centers , but only six ( palb2 , tp53 , pten , chek2 , atm , and brip1 ) were tested regularly . 
us centers tested the genes most often , whereas united kingdom and italian centers with no direct enigma affiliation at the time of the survey were the least likely to regularly test the most centers tested the 16 genes through multigene panels ; some centers tested tp53 , pten , and other cancer syndromeassociated genes individually . 
gene - specific guidelines for breast and ovarian cancer risk management were limited and differed among countries , especially with regard to starting age and type of imaging and risk - reducing surgery recommendations . conclusion currently , a small number of genes beyond brca1 / 2 are routinely analyzed worldwide , and management guidelines are limited and largely based on expert opinion . 
pedersen maria piane marianna puzzo mark robson maria rossing maria christina sini angela solano jana soukupova gianluca tedaldi manuel teixeira mads thomassen maria grazia tibiletti amanda toland therese trngren erica vaccari liliana varesco ana vega yvonne wallis barbara wappenschmidt jeffrey weitzel amanda b . 
currently , several multigene panels are available that include from < 10 to > 100 known or candidate cancer susceptibility genes , which are tested for diagnostic or research purposes . 
some panels are targeted at diverse cancers ( pan - cancer panels ) , whereas others target specific cancers only ( disease specific panels )  . the ability to run multigene panels at affordable prices has expanded the eligibility criteria and increased the demand for testing.1 - 5 however , the rapid pace at which candidate risk genes are moving from research based to clinical diagnostic testing has its drawbacks . 
the rate of variants of uncertain significance ( vus ) has increased proportionally to the extent of the sequenced genome.5 - 7 moreover , many genes currently included on multigene panels have imprecise cancer risk estimates , and there is no consensus on when to test for a given gene or how to manage a reported ( likely ) pathogenic variant.8 , 9 the aim of this study was to describe a snapshot of the landscape of international genetic testing practices and risk management approaches for bc and boc susceptibility genes beyond brca1 and brca2 . 
a survey was conducted among members of the evidence - based network for the interpretation of germline mutant alleles ( enigma ) , an international consortium focused on determining the clinical significance of variants in brca1 , brca2 , and other ( ascertained or suspected ) bc and boc susceptibility genes , providing expertise to global database and classification initiatives , and exploring optimal avenues of communication of such information at the provider and patient levels . 
additional information was collected via country networks in the united kingdom and in italy , from centers that were not directly involved in enigma research at the time of study initiation . in total , respondents represented cancer genetic experts from 61 centers across 20 countries . 
 to our knowledge , this is the first study to describe international testing practices and risk management guidelines for non - brca1 / 2 genes implicated in bc and boc susceptibility . methods this study was submitted for approval to the ethics committees of the two coordinating sites , the university of chicago and maastricht university . 
a survey about genetic testing practices for nonbrca1 / 2 bc and boc susceptibility genes was developed by enigma clinical working group ( cwg ) leaders during 2016 ( appendix table a1 )  . 
enigma members were invited to complete the survey if they had a clinical genetic testing or diagnostic laboratory affiliation and were involved in ordering , performing , or interpreting dna tests for inherited susceptibility to bc / boc at their center . 
an enigma member is currently defined as a researcher or research group ( consortium ) who is willing to work collaboratively toward classification of variants by contributing data from families and / or conducting statistical analysis or laboratory - based assays within a working group framework . 
there is no requirement for enigma members to state their primary role ( clinician , genetic counselor , laboratory scientist , basic researcher ) , but all members by definition have a research interest in the topic of gene / variant classification . individuals from the same center could work on the survey together or choose a designated representative to complete it , so only one survey per center was counted . 
in brief , an in - person survey of members of the cwg , consisting mainly of laboratory and clinical scientists from academic centers , was conducted during the enigma consortium meeting in limassol , cyprus , in january 2017 . 
 a total of 30 centers from 17 countries participated . a more detailed version of the survey was then distributed by e - mail ( paper / electronic survey ) to the same 30 centers that participated in the in - person survey and to additional enigma affiliated centers worldwide . 
participants were sent a copy of their answers and asked to verify them or to clarify any discrepancies . notably , in italy and in the united kingdom , the paper / electronic version of the survey was also distributed , via country networks , to centers that were not actively involved in enigma research . 
the effort comprised both the enigma - affiliated fondazione istituto di ricovero e cura a carattere scientifico istituto nazionale dei tumori ( milan ) and the santa chiara university hospital ( pisa ) , which were counted among the 38 participating enigma centers , and 14 additional centers , which were not directly affiliated with enigma at the time of the survey ( henceforth referred to as non - enigma ; fig 1 , lower right )  . 
of the 14 italian non - enigma centers , five were dedicated to diagnostic testing only , and nine were dedicated to both diagnostics and research ; moreover , half of them were university affiliated , and half were not . 
 completed the survey for her own enigma affiliated center ( one of the 38 participating enigma centers ) and also coordinated , with the assistance of y.w. , the distribution of the survey through surveymonkey via the association for clinical genetic science mailing list to cancer genetic leads from diagnostic laboratories providing genetic testing for the publicly funded national health service ( nhs )  . 
the recruitment flowchart and the global distribution of participants are illustrated in figure 1 . clinical utility to get a preliminary idea of the participants opinions about the clinical utility of the 16 genes on which the survey focused , the cwg members present at the 2017 enigma meeting in cyprus were asked to answer the following questions relating to each of them : should every patient with bc / oc who qualifies for ( brca1 / 2 ) genetic testing ( by criteria that we recognize may differ by country / center ) be tested for the gene ? and do you agree that the cancer risk associated with ( pathogenic variants in ) the gene is high enough to inform clinical management ? all participants ( n = 23 at this specific session ) stated that they would test every qualifying patient with bc ( as defined in the question ) for palb2 and every qualifying patient with oc ( as defined ) for brip1 , rad51c , and rad51d . 
results for the other nine genes were variable ( appendix fig a1a )  . with regard to clinical management , all participants agreed that palb2 , tp53 , cdh1 , pten , and stk11 along with brip1 , rad51c , and rad51d were associated with high enough ( bc or oc ) risk to alter clinical management . 
please note that 95% cis for this figure and for all the following figures are provided in appendix ( tables a2 - a10 )  . testing practices participants were also asked ( via in - person and / or paper / electronic surveys ) if and how frequently they tested each gene , the method ( single gene v gene panel ) and purpose of testing ( clinical v research ) , and the practices of reporting ( likely ) pathogenic variants and vus to patients . 
of centers that responded to the question varied by gene ( range , 29 to 38 centers ) .regularly was defined as ordered for > 50% of eligible patients ( ie , those who qualified for genetic testing , by criteria that we recognize may differ by center / country )  . 
even though each gene was tested by > 50% of the centers ( range , 52% to 100% ) , only palb2 , tp53 , pten , chek2 , atm , and brip1 were tested regularly by > 50% of centers . testing in a research setting in addition to the clinical setting was common for enigma centers ( appendix fig a2 )  . 
the genes that were most frequently tested ( ie , tested by at least > 30% of centers ) for research purposes only were : nbn , bard1 , rad50 , and mre11a . 
 single - gene testing was performed by a number of centers , varying from one to 21 , for : tp53 , pten , cdh1 , stk11 , palb2 , chek2 , nf1 , atm , men1 , and nbn ( in decreasing order of frequency ) , often based on a specific phenotype ( eg , pten hamartoma syndrome or neurofibromatosis type 1 ) , or these genes were tested as a reflex only when brca1 / 2 testing was noninformative . 
seven centers from four countries ( belgium , brazil , the netherlands , and spain ) testing chek2 only tested for the 1100delc variant . regarding the types of gene panels used , us respondents typically ordered broad cancer panels from commercial laboratories , although the specific panels varied depending on patient preferences , insurance considerations , and clinical scenarios . 
separately , the nine united kingdom nhs laboratories were asked , if you currently only report brca genes but might report broader panels in the future , what issues are major barriers / problems to overcome ? responses were chosen from a menu of nine options plus other , and the four main reasons selected ( by half or more of respondents ) were no request by the oncologists ( of note , nhs oncologists can ask directly for brca1 and brca2 testing but not for multigene panels ) , lengthy and laborious process of variant interpretation , lack of standardization of reporting , and lack of demand for testing . reporting practices and cascade testing . 
for genes analyzed through clinical testing , > 90% of enigma centers reported ( likely ) pathogenic variants to patients ( for chek2 and nbn , the percentages were slightly lower , at 88% and 71% , respectively ; fig 4 )  . 
some centers reported these variants only if the patient met criteria for the associated syndrome ( eg , hereditary diffuse gastric cancer for cdh1 , neurofibromatosis type 1 for nf1 )  . 
almost all centers ( 67% to 81% for nbn , rad50 , mre11a , and bard1 and > 90% for the other genes ) offered cascade testing to family members if a ( likely ) pathogenic variant was identified ( data not shown )  . 
notably , participants from the netherlands reported that they only tested first - degree relatives for chek2 1100delc variant when the estimated risk based on family history was lower than the risk conferred by having the variant , so testing for the variant had clinical utility because it would change surveillance recommendations.11 a high percentage ( 50% to 82% ) of enigma centers reported vus to patients ( fig 4 )  . 
 variant classification systems all respondents reported using the international agency for research on cancer five - tier classification system , 12 and many also used american college of medical genetics and genomics13 classification criteria . 
 the three methods are not mutually exclusive ; notably , the center in kuwait performs whole - genome sequencing for all cases , which is not represented in the figure . 
enigma , evidence - based network for the interpretation of germline mutant alleles . clinical management practices and guidelines most enigma centers ( 80% ) had risk management guidelines for a majority of non - brca1 / 2 genes considered reportable to patients ( fig 5 )  . 
 exceptions were bard1 , rad50 , and mre11a , for which 30% of centers had guidelines . although most enigma centers reported having some type of management guidelines for all genes except bard1 , rad50 , and mre11a , after review , only 10 of 20 countries had national guidelines for ( some of ) these genes ( table 1 )  . 
 furthermore , in some countries ( denmark and germany ) , the national guidelines were not gene specific ( ie , they were broken down by highand moderate - risk categories rather than by specific gene )  . 
ten countries had national guidelines for high - risk cancer syndromeassociated genes such as tp53 , cdh1 , and pten ( with the exception of belgium not having guidelines for cdh1 )  . 
the primary differences between countries were the starting age and type of diagnostic imaging ( mammography v magnetic resonance imaging [ mri ] v sonography ) and the policy on risk - reducing mastectomy . 
for instance , there was no consensus on the age to begin mammograms / mri for carriers of pathogenic variants in nf1 , men1 , palb2 ( age 25 v 30 years ) , or tp53 ( age 20 v 25 years )  . 
 the united kingdom guidelines differed from all others in that breast mri was not the standard imaging technique for carriers of pathogenic variants in other gene carriers ( except for tp53 )  . 
guidelines for risk - reducing mastectomy in carriers of palb2 pathogenic variants ranged among accepted ( n = 1 ) , consider depending on personal / family history ( n = 5 ) , and not enough evidence to recommend ( n = 1 )  . 
for pten and cdh1 , the guidelines that commented on preventive surgery ( four of the seven and five of the eight national guidelines , respectively ) mentioned risk - reducing mastectomy as a possible option . 
 subanalyses : enigma - us versus enigma - other centers and versus non - enigma centers discussion responses from the seven enigma centers in the united states ( enigma - us ) were compared with those of the other 31 enigma centers ( enigma - other )  . 
in addition , responses from 14 non - enigma centers in italy and nine non - enigma laboratories in the united kingdom were compared with those from 38 enigma centers across all countries . results of these comparisons are summarized in appendix figs a3 and a4 . 
a much smaller proportion of non - enigma centers from italy and the united kingdom tested each gene compared with enigma - affiliated centers ( appendix fig a3 )  . management guidelines were more likely to be available in the us - based enigma centers compared with the other enigma centers for all genes except bard1 , rad50 , mre11a , and men1 . 
of centers that responded varied by gene ( range , 12 to 36 centers responding about reporting pathogenic variants ; range , four to 20 centers responding about reporting vus )  . 
our global survey demonstrated that only a few genes are routinely analyzed beyond brca1 / 2 ; most centers clinically test them through multigene panels and report ( likely ) pathogenic variants ( and vus , to a slightly lesser extent ) to patients ; and gene - specific guidelines for bc and oc risk management are limited and differ between countries , especially in regard to starting age and type of imaging and risk - reducing surgery recommendations . with falling costs of sequencing and more genes being identified that are associated with increased bc and boc risk , multigene ( panel ) testing is becoming the northe results of our survey confirm this trend , showing that genes that are commonly offered on commercial panels were tested by > 50% of the surveyed centers . nevertheless , the value of multigene panel testing continues to be debated in the context of three main areas : limited additional yield of pathogenic variants in genes other than brca1 / 2 coupled with significantly increased interpretation workload , reliability of penetrance estimates for moderateor uncertain risk genes ( clinical validity ) , and evidence for informing management recommendations to improve patient outcomes ( clinical utility ) .9 our international survey demonstrates that the use of panel testing varies widely among countries . 
moreover , differences were observed when comparing enigma - affiliated centers with non - enigma italian and united kingdom centers ( with the latter testing nonbrca1 / 2 genes less than one third of the time )  . 
if management guidelines were available , centers were asked to specify the source of such guidelines ( local , national , or international , such as national comprehensive cancer network or national institute for health and care excellence )  . the genes included on panels was the most common concern raised by the participating centers . 
easton et al8 asserted that a genomic test should not be offered until its clinical validity is established8 ( p2 ) ; however , the utility of a gene needs to be continuously reconsidered as more data become available , and this can only be done by analyzing results from large cohorts of individuals who have been tested . 
concerns about the rates of vus were frequently expressed by the study participants , but just as variant rates have significantly decreased over the years for brca1 / 2 as a result of concerted classification efforts , the same trend will likely occur for other susceptibility genes , arguably at a faster pace as ( and provided that ) more laboratories worldwide contribute their testing data to population and peer - reviewed databases.5 , 28 , 29 despite the establishment of such databases , survey participants felt that robust , constantly updated international databases and global data sharing are still lacking . 
this is a worthy goal , but expert judgment in variant classification methods is still required , because fully automated approaches to variant classification that apply guidelines are not ready for clinical practice.6 at a basic level , some centers reported validation of the testing method as a barrier . 
therefore , it is important to recognize the technologic barriers in certain countries , although the transition to massively parallel sequencing is ultimately expected to increase throughput and optimize diagnosis without significantly elevating costs.30 there were also nonmedical barriers to implementing routine testing of many of these surveyed genes . 
insurance can be a major barrier in the united states , where , for example , medicare ( a us federal health insurance program for people who are age 65 years and for certain younger people with disabilities ) will only cover testing for individuals with a bc or oc diagnosis , and many insurers will not cover multigene panel testing if the patient has already had prior genetic testing . 
in many other countries , particularly those with national ( ie , universal ) health care , testing is approved on a gene - by - gene basis or as a package if research - derived evidence is considered robust enough to change clinical management . in terms of risk magnitudes , palb2 and tp53 are the only bc genes , in addition to brca1 / 2 , that consistently fall into the high - risk category across studies ( ie , confer levels of risk greater than four times that in the general population ) 8 ; the remainder have conflicting evidence regarding the risk category into which they fit.8 , 9 , 31 - 33 our survey confirmed that enigma centers test palb2 and tp53 relatively frequently and regard them as clinically actionable genes . 
 these two genes were tested much less consistently by non - enigma centers , evidencing the lack of consensus , even for genes that are generally regarded as high risk . 
 brip1 , rad51c , and rad51d are ever more accepted as oc but not bc risk predisposition genes ( two to five times the risk compared with the general population ) .15 , 32 notably , many respondents agreed that every patient with oc should be tested for these three genes ( in addition to brca1 / 2 )  . 
although there is currently no indication that oc treatment for a carrier of a pathogenic variant in one of these three genes would differ from that for a noncarrier , carriers may benefit from rrso at menopause . the uncertainties and inconsistencies regarding risk and testing practices are magnified when it comes to syndromic cancer genes like pten , cdh1 , stk11 , nf1 , nbn , and men1 , as well as genes conferring an uncertain risk such as bard1 , rad50 , and mre11a . 
although there is significant evidence for elevated bc risk and lobular bc risk in carriers of pathogenic variants in pten and in cdh1 , respectively , 34 - 36 it is likely that these bc risks ( and those from the other syndromic genes ) are overestimated and therefore unreliable , because they were derived from patients whose histories were consistent with these rare syndromes rather than from unselected patients.8 more robust and replicable penetrance estimates from large - cohort and population studies are certainly needed to further define risks . 
however , on the basis of both the evidence available from the literature and the results of our survey , which incorporate an international clinical perspective , the 16 genes can be grouped into five categories : high bc risk : palb2 , tp53 , pten , and cdh1 ; moderate bc risk : atm and chek2 ; bc risk of unclear magnitude ( but established risk for other cancer types ) : stk11 , nf1 , nbn , and men1 ; moderate oc risk : brip1 , rad51c , and rad51d ; and insufficient evidence for bc or oc risk : bard1 , rad50 , and mre11a . the clinical utility of multigene panel testing is assessed based on the improved outcomes of those managed by evidence - based surveillance or prevention approaches . 
a framework for management of moderate - risk bc / boc genes has been extensively reviewed by tung et al9 and includes a comparison of surveillance guidelines among the united states , united kingdom , and germany . 
there are limited national guidelines available even for genes such as palb2 , brip1 , rad51c , and rad51d , which most participants felt should always be tested because they importantly , are clinically actionable . 
this explains why the guidelines often differ in important aspects such as indication for risk - reducing surgery and type of diagnostic imaging recommendations . our study was initiated to provide a snapshot of enigma clinical practice for non - brca1 / 2 genes . 
because panel testing is currently being implemented in large regions of the world like asia , africa , and south america , similar surveys will need to be redistributed once more countries have established testing protocols . 
even at the time of the survey , testing protocols and surveillance recommendations were in flux in some countries , and broader gene panels were expected to be offered within a short time . 
 we acknowledge that our sampling of nonenigma centers was limited , and we aim to survey a more diverse collection of us , canadian , and other worldwide regional or community practices in future studies . massively parallel sequencing represents a transformational technology that we must learn to apply appropriately in health care . 
although the number of genes , other than brca1 / 2 , associated with bc / boc risk is growing , only a small subset of them have clinical utility at the moment . 
the goal of this study was to highlight the differences across countries and to determine what additional information and infrastructure are still needed to move toward more uniform testing practices and management guidelines internationally . our collected evidence suggests that the clinical usefulness of multigene panel testing for bc / boc predisposition can be improved by a better definition of the cancer risks associated with genetic variation in cancer susceptibility genes and by the availability of evidence - based management guidelines . 
to this end , it is key that clinicians share clinical and genetic data , through enigma and / or other international consortia focused on the clarification of the bc and oc risk associated with genetic variation , and that tested individuals are encouraged to participate in initiatives that collate genetic testing data and in long - term follow - up studies that evaluate intervention strategies . 
palmero , maria piane , marianna puzzo , maria christina sini , angela solano , manuel teixeira , mads thomassen , amanda toland , therese trngren , liliana varesco , jeffrey weitzel , encarna b . 
pedersen , maria piane , marianna puzzo , mark robson , maria rossing , maria christina sini , angela solano , jana soukupova , gianluca tedaldi , manuel teixeira , mads thomassen , maria grazia tibiletti , amanda toland , therese trngren , erica vaccari , liliana varesco , ana vega , barbara wappenschmidt , jeffrey weitzel , arcangela de nicolo , encarna b . 
 rien blok no relationship to disclose akira hirasawa research funding : astrazeneca maria adelaide caligo travel , accommodations , expenses : technogenetics mariarosaria calvello no relationship to disclose gabriele lorenzo capone no relationship to disclose pietro cavalli no relationship to disclose t.l. 
couch consulting or advisory role : astrazeneca research funding : grail other relationship : ambry genetics miguel de la hoya no relationship to disclose simona de toffol no relationship to disclose orland diez no relationship to disclose susan m . 
domchek honoraria : astrazeneca , clovis oncology , bristol - myers squibb research funding : astrazeneca ( inst ) , clovis oncology ( inst ) , pharmamar ( inst ) ros eeles honoraria : janssen - cilag speakers bureau : janssen - cilag anna efremidis no relationship to disclose florentia fostira no relationship to disclose david goldgar no relationship to disclose andreas hadjisavvas no relationship to disclose thomas v.o. 
hansen no relationship to disclose claude houdayer no relationship to disclose petra kleiblova no relationship to disclose sophie krieger no relationship to disclose conxi lzaro no relationship to disclose maria loizidou no relationship to disclose siranoush manoukian no relationship to disclose arjen r . 
 mark robson honoraria : astrazeneca erica vaccari consulting or advisory role : color genomics consulting or advisory role : mckesson , astrazeneca research funding : astrazeneca ( inst ) , myriad genetics ( inst ) , invitae ( inst ) , pfizer ( inst ) travel , accommodations , expenses : astrazeneca liliana varesco consulting or advisory role : pfizer maria rossing no relationship to disclose maria christina sini no relationship to disclose angela solano no relationship to disclose jana soukupova no relationship to disclose gianluca tedaldi no relationship to disclose manuel teixeira no relationship to disclose mads thomassen no relationship to disclose maria grazia tibiletti no relationship to disclose amanda toland no relationship to disclose therese trngren honoraria : pfizer , astrazeneca ana vega no relationship to disclose yvonne wallis no relationship to disclose barbara wappenschmidt no relationship to disclose jeffrey weitzel no relationship to disclose amanda b . 
domchek , university of pennsylvania , philadelphia , pa ; anna efremidis , athens medical center ; florentia fostira , national centre for scientific research demokritos , athens , greece ; david goldgar , university of utah school of medicine , salt lake city , ut ; andreas hadjisavvas and maria loizidou , cyprus institute of neurology and genetics , nicosia , cyprus ; thomas v.o. 
pedersen , aalborg university hospital , aalborg , denmark ; akira hirasawa , keio university school of medicine , tokyo , japan ; claude houdayer , universit paris descartes and unicancer genetic group , paris ; sophie krieger , normandy university , cancer center f . 
palmero , barretos cancer hospital , barretos , so paulo , brazil ; mark robson , memorial sloan kettering cancer center , new york , ny ; angela solano , university of buenos aires , buenos aires , argentina ; manuel teixeira , instituto portugus de oncologia do porto francisco gentil , porto , portugal ; amanda toland , ohio state university , columbus , oh ; therese trngren , lund university , lund , sweden ; erica vaccari , dana - farber cancer institute , boston , ma ; barbara wappenschmidt , university hospital cologne and german consortium of hereditary breast and ovarian cancer , cologne , germany ; and jeffrey weitzel , city of hope , duarte , ca . support supported by grant no . 
couch fj , hart sn , sharma p , et al : inherited mutations in 17 breast cancer susceptibility genes among a large triple - negative breast cancer cohort unselected for family history of breast cancer . 
laduca h , stuenkel aj , dolinsky js , et al : utilization of multigene panels in hereditary cancer predisposition testing : analysis of more than 2 , 000 patients . 
maxwell kn , hart sn , vijai j , et al : evaluation of acmg - guideline - based variant classification of cancer susceptibility and non - cancer - associated genes in families affected by breast cancer . 
domchek sm , bradbury a , garber je , et al : multiplex genetic testing for cancer susceptibility : out on the high wire without a net ? j clin oncol 31 : 1267 - 1270 , 2013 8 . 
richards s , aziz n , bale s , et al : acmg laboratory quality assurance committee : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
janatov m , boreck m , soukupov j , et al : palb2 as another candidate gene for genetic testing in patients with hereditary breast cancer in czech republic [ in czech ]  . 
robays j , stordeur s , hulstaert f , et al : oncogenetic testing and follow - up for women with hereditary breast / ovarian cancer , li fraumeni syndrome and cowden syndrome . 
robays j , stordeur s , hulstaert f , et al : oncogenetic testing , diagnosis and follow - up in birt - hoggdub syndrome , familial atypical multiple mole melanoma syndrome and neurofibromatosis 1 and 2 . 
national institute for health and care excellence : familial breast cancer : classification , care and managing breast cancer and related risks in people with a family history of breast cancer . 
lincoln se , kobayashi y , anderson mj , et al : a systematic comparison of traditional and multigene panel testing for hereditary breast and ovarian cancer genes in more than 1000 patients . 
castra l , krieger s , rousselin a , et al : next - generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes . 
pharoah pd , guilford p , caldas c : incidence of gastric cancer and breast cancer in cdh1 ( e - cadherin ) mutation carriers from hereditary diffuse gastric cancer families . 
questions included in the surveys ( by mode of distribution ) ( continued ) question testing methods and setting which method is used to test for gene x ? in - person survey only which genes do you agree should be tested for every bc or oc patient eligible for genetic testing ? describe the gene panels currently used ( if any ) and if they are used in the diagnostic or research setting if you are not currently using gene panels but may in the future , what do you think is required before starting to use them ? ii variant classification classification system ii - 1 ii - 2 reporting and cascade testing of variants clinical i . 
questions included in the surveys ( by mode of distribution ) ( continued ) in - person survey only question ii - 3 variant interpretation in - person and paper surveys paper survey only surveymonkey * who takes responsibility for interpreting the clinical significance of the identified variants ? for cancer susceptibility genes : who takes responsibility for variant interpretation and reporting ? clinical scientist clinical geneticist genetic counselor oncologist ( medical / surgical ) other ( specify ) who takes responsibility for discussing the clinical significance / utility of an identified variant ? ( same choices as above ) are there clinical guidelines for managing patients who carry a pathogenic or likely pathogenic variant in a bc susceptibility gene ? iii risk management guidelines for which genes do you agree that the cancer - associated risks are high enough to alter clinical practice / management ? are management guidelines available at your center for patients with ( likely ) pathogenic variants in these genes ? yes , national guidelines yes , local guidelines or local adaptations of national guidelines no , guidelines are not currently available if clinical management guidelines are available at your center for the specified genes , please provide digital copy , reference , or web site link note . 
questions i - 4 and iii of the in - person survey were asked at a different time compared with the remainder of the survey ; therefore , answers were collected from only 23 centers . 
 these two questions were asked at a different time ( during evidence - based network for the interpretation of germline mutant alleles meeting in cyprus in january 2017 compared with survey questionnaire )  . 
the centers that tested each gene through research only were compared with the proportion of centers that tested the gene only clinically and proportion of those that tested the gene for both clinical and research purposes . 
genes tested regularly by evidence based network for the interpretation of germline mutant alleles ( enigma ) centers in the united states ( enigma - us ) versus other enigma centers ( enigma - other ) versus italian and united kingdom non - enigma centers . 
 of centers testing given gene regularly ( defined as ordered for > 50% of patients eligible for genetic testing , by criteria that we recognize may differ by center / country ) is shown above each bar . 
of centers that answered this question was four to seven , depending on the gene ; of the 31 enigma - other centers , a range of 22 to 30 centers answered this question . 
mcgowan , phd1 , 2 purpose although analyzing germline and tumor samples concurrently provides the best opportunity for differentiating between germline and somatic mutations , tumor - only sequencing is becoming increasingly common in clinical care . 
with limited professional society guidance on how to manage pathogenic mutations identied via tumor - only sequencing , this study contemplates the professional knowledge and skills necessary to have represented on an mgtb to interpret these results in context of potential germline ndings . methods qualitative interviews with mgtb members and an ethnographic case study of a breast cancer mgtb at a national cancer institute cancer center were examined . results this mgtb discussed 34 cases of women with advanced - stage breast cancer over 13 months . interviews and observations of mgtb meetings indicated that members of the mgtb contemplated whether variants were germline or somatic and potential for identication of germline cancer predisposition . 
however , the mgtb only referred 11 patients ( 61% ) for additional germline testing , and the remaining seven patients ( 39% ) were not referred , raising questions about the kind of genomic expertise needed on an mgtb to optimize results interpretation and referrals . conclusion to ensure adequate interpretation , recommendation , and communication of tumor sequencing results , an mgtb should include professionals with knowledge and experience in clinical translation of tumor sequencing , testing methodology , molecular pathology , cancer biology , genomic pathways , germline variant interpretation , evaluation of family history , and application of professional recommendations for germline testing after tumor - only sequencing . 
 fishler et al context key objective : how are tumor - only sequencing results interpreted by a multidisciplinary genomic tumor board ( mgtb ) ? which professional expertise ought to be represented when discussing these results and recommendations ? knowledge generated : commercial laboratories provision of tumor - only sequencing without germline reference samples can complicate the ability of an mgtb to interpret patients genomic sequencing results and make recommendations for targeted treatment and referrals for additional genetic testing . 
providers on an mgtb questioned whether a known or likely pathogenic tumor sequencing variant was germline or somatic , sometimes overlooked known / likely pathogenic mutations in cancer susceptibility genes other than brca1 / 2 , and recognized the necessity of varied professional expertise when interpreting tumor - only sequencing results . relevance : to ensure appropriate interpretation of tumor - only sequencing and identication of patients who ought to be referred to genetics , an mgtb should include individuals with expertise in clinical translation of tumor sequencing , testing methodology , molecular pathology , cancer biology and genomic pathways , germline variant interpretation , evaluation of personal and family history , and application of professional guidelines for germline testing . 
to mitigate provider deliberation and genomic knowledge necessary to determine if additional germline testing is necessary , genetics professional societies and clinical laboratories ought to consider whether tumor - only sequencing should be routinely paired with germline samples to facilitate standardized reporting of germline variants . the basis of tumor - only sequencing results , even when combined with their family history . a previously published study from a breast cancer mgtb revealed that decisions about which patients received genetics referrals were not standardized.10 this prior analysis did not explore the factors the mgtb used to determine which patients received genetics referrals after tumor - only sequencing . 
our study aimed to identify these factors and to explore which professionals ought to be involved on an mgtb to ensure adequate interpretation , clinical application , and referral for additional germline testing when appropriate . each interview ranged in length from 25 to 49 minutes . 
all mgtb members signed a written informed consent , which allowed for recording , transcription , and analysis of the meeting minutes and interview data . data were collected using standard qualitative research including ethnography and in - depth inmethods , interviews and mgtb meetings were audio terviews . recorded and transcribed verbatim by a medical transcriptionist . 
participants in this study included 22 members of a breast cancer mgtb at one urban national cancer institute cancer center . methods included an ethnographic case study of mgtb meetings and interviews with mgtb members . 
this group met monthly from october 2013 to november 2014 to discuss results of tumor - only sequencing ordered for patients with advanced - stage breast cancer in a clinical setting . mgtb members were invited to participate in a semistructured interview regarding their opinions about a tumor board approach to interpreting tumor sequencing results . in total , 12 members of the board were interviewed by the research team , including treating physicians ( n = 6 ) , bioinformaticians ( n = 3 ) , a pathologist ( n = 1 ) , a medical geneticist ( n = 1 ) , and an administrator ( n = 1 ) .10 all patients discussed by this mgtb were women with advanced - stage breast cancer and tumor - only sequencing performed as part of their clinical care ( n = 34 )  . 
the mgtb discussed all of the hospitals patients with breast cancer who underwent tumor sequencing during the study period . all patient samples were sent to the same commercial laboratory for sequencing and analysis . the laboratory - generated tumor sequencing report suggested on - label / off - label treatments and clinical trial options on the basis of known / likely pathogenic mutations . pathogenic and likely pathogenic results were listed separately from vus results . 
as published in the preliminary analysis , mgtb recommendations included genetics referrals for suspected germline variants , clinical trial recommendations , us food and drug administrationapproved on - label therapies , androgen receptor testing , and repeat human epidermal growth factor receptor 2 testing . 
 underlying germline variants in tumor - only sequencing leaderships commitment to adherence with evidencebased medicine and bioethical principles of benecence and nonmalecence.10 of the 34 patients tumor sequencing results that the mgtb reviewed during the study period , 18 patients met nccn criteria for additional germline testing on the basis of their personal history , including type of breast cancer , age of onset , and / or the identication of a known / likely pathogenic tumor mutation in a cancer susceptibility gene . 
the remaining seven patients ( 39% ) represent missed opportunities for additional germline testing on the basis of the patients personal history , prior germline testing , and / or tumor - only sequencing result ( table 2 )  . 
five of these patients were missed on the basis of a combination of these factors ; two were missed on the basis of personal history alone . through analysis of mgtb member interview transcripts and mgtb meetings , three themes emerged : provider confusion about whether a variant identied by tumoronly sequencing was germline or somatic , potential for tumor - sequencing identication of germline cancer predisposition , and genetics expertise necessary for inclusion on an mgtb . provider confusion about whether a mutation was germline or somatic one key question raised by mgtb members was whether the variants listed on tumor sequencing reports were germline or somatic . 
on the basis of the way mgtb members managed the referral process , it became apparent that they disagreed about their role as evaluators of tumor - only sequencing results , including determining whether follow - up with additional germline testing was necessary . 
for example , one medical oncologist mentioned that all mutations relevant treatment were listed on the rst page of the patients report and that if we dont have a lot of time , we may not ip through the rest to see what else might be listed there . in contrast , another oncologist advocated for a more active interpretation : we cant role in report take what [ commercial vendor ] recommends as gospel truth and say okay , this is what were going to go with , because they recommended itthe interpretation needs to happen from our end . just result in addition to differing perspectives about terpretation , there was confusion among the mgtb members regarding whether the reported mutations were somatic or germline . 
a discussion about a patient with a reported mutation in fancc highlights this confusion , which affected their thoughts about whether the patient should receive a genetics referral ( table 3 )  . 
of times reason was discussed during the mgtb meetings patient referrals for additional germline testing on the basis of personal history triple - negative cancer and age of diagnoses , 60 years diagnosis of bilateral breast cancer on the basis of results of tumor - only sequencing report for parp inhibitors identication of known / likely pathogenic fancc mutation identication of known / likely pathogenic chek2 mutation identication of known / likely pathogenic mlh1 mutation identication of known / likely pathogenic nf1 mutation identication of known / likely pathogenic mutation or vus in brca1 / 2 to determine eligibility on the basis of personal history and of tumor - only sequencing report re - evaluation of brca1 / 2 status for parp inhibitors because of prior negative germline results and negative tumor sequencing results for brca1 / 2 identication of known / likely pathogenic tp53 mutation on tumor sequencing and age of diagnoses note . 
missed opportunities for referral for additional germline testing ( n = 7 ) fishler et al missed opportunity on the basis of personal history on the basis of tumor - only sequencing report identication of known / likely pathogenic pten variant on the basis of personal history and tumor - only sequencing report triple - negative breast cancer , rst breast cancer diagnoses , 60 years , and no prior germline genetic testing personal diagnosis of lobular breast cancer and identication of known / likely pathogenic cdh1 variant on the basis of previous genetic test results and tumor - only sequencing report negative brca1 / 2 germline testing before availability of bart testing , no mutation in brca1 / 2 gene identied on tumor negative brca1 / 2 germline testing before availability of bart testing , pten mutation identied on tumor sequencing sequencing total no . 
the tumor - only report does not distinguish variants that are reported pathogenic from variants that are reported likely pathogenic . they are reported as one category on the front page of the report . 
seven patients out of 18 discussed at the mgtb did not receive a genetics referral for additional germline testing , although it would have been indicated by nccn criteria or a known / likely pathogenic mutation identied in a cancer susceptibility gene by tumor - only sequencing . 
at the time of the study of this mgtb , nccn criteria recommended that patients with negative brca1 / 2 sequencing before 2006 have bart testing and women with triple - negative breast cancer and their rst breast cancer diagnoses before age 60 years have brca1 / 2 germline testing . 
in addition , the american college of medical genetics and genomics recommended that pathogenic germline mutations associated with hereditary cancer syndromes be returned to patients ( such as pten and cdh1 )  . abbreviations : bart , brac analysis rearrangement test ; mgtb , multidisciplinary genomic tumor board ; nccn , national comprehensive cancer network . testing , some of the medical oncologists were reluctant , because the gene was not directly related to hereditary breast and ovarian cancer syndrome . 
mgtb interviewees indicated lack of education regarding medical genetics as a limitation of the mgtb , which is a recognized barrier to autonomous use of genetic testing by ordering physicians.2 , 12 potential for identication of germline cancer predisposition consistent with the mgtbs focus on identifying treatment options , 10 members of this mgtb were most concerned about whether a patient had a brca1 / 2 mutation to determine their eligibility for treatment with poly ( adp - ribose ) table 3 . 
vignette : confusion about somatic versus germline mutations vignette medical oncologist 7 : it says in the report that germline mutations in fancc cause fanconi anemia , a clinical heterogeneous disorder , and that appropriate clinical testing [ to detect ] the presence of a germline mutation is recommended . 
whenever we see a mutation , we have to make sure that we realize its one test and we would need it to be validated , but does it have relevance for treatment ? ( talking about prior treatment responses and discussion about parp inhibitor recommendations ) medical oncologist 7 : and she was germline brca tested and all thats negative ? medical oncologist 1 : yeah , she was brca negative . medical oncologist 2 : should we refer to genetics about this fancc thing ? medical oncologist 1 : you think we should refer ? is there a reason to think about it ? medical oncologist 7 : the only way you can test it is on those panels . 
every genetics has a panel where you can test for fanconi anemia genesi dont know how much breast cancer risk or other risks [ are associated with variants in this gene ]  . 
 underlying germline variants in tumor - only sequencing polymerase inhibitors , which gained traction as a mutationguided treatment option during the study period.13 , 14 thus , all patients with tumor - only sequencing results that included a vus or known / likely pathogenic mutation in brca1 / 2 were referred to genetics ( n = 4 )  . patients with known / likely pathogenic mutations in other cancer susceptibility genes ( such as pten ) were not routinely referred to genetics ( table 2 )  . 
at the time of this study , american college of medical genetics and genomics recommended that when analyzing a tumor and paired germline sample , such results should be conrmed and communicated to patients as secondary ndings , because of their association with a hereditary cancer syndrome . however , nccn guidelines did not include a recommendation for additional germline testing for mutations in genes on the secondary ndings list , 7 which may have contributed to inconsistent referral patterns , especially given that the mgtb was reviewing tumor - only sequencing results . two additional patients who met nccn guidelines for germline testing on the basis of their personal history were not referred for germline testing ( table 2 )  . 
at the time of the study of this mgtb , nccn recommended bart ( brac analysis rearrangement test ) testing for patients who had received negative brca1 / 2 germline test results before 2006.7 two patients with negative brca1 / 2 tumor - only sequencing results and negative brca1 / 2 germline test results before the availability of bart testing were not referred to genetics , although a genetics referral would have been indicated by nccn guidelines . 
in discussing how well individual disciplines were represented on the mgtb , a medical oncologist who co - led the mgtb recognized the value of genetics expertise in interpreting whether certain mutations were common or rare and how to manage referrals on the basis of a variants likelihood of being germline . 
explicitly , she noted that knowledge and experience with specic gene pathways enhanced the variant interpretation and productivity of the discussions about treatment options and result interpretation during the mgtb meetings . 
she also described disappointment that a medical geneticist and genetic counselors were invited , but that a medical geneticist only attended one mgtb meeting over the 13 - month study period and no genetic counselors participated . 
however , the other medical oncologist who co - led the mgtb mentioned that scheduling posed a barrier to genetic counselors attendance . mgtb members discussed the limitations of the test throughout the entire study period . 
the pathologist cautioned during her interview that not all providers who order tumor - only sequencing may understand the limitations of the test in terms of identifying germline variants : its hard to speak for other doctors , other community doctors . 
furthermore , at one meeting , the medical geneticist explained sequencing methodology , utility of bart testing for patients who have previously tested negative for brca1 / 2 , and guidelines for germline testing on the basis of a patients personal history , including age and triple - negative diagnosis . 
this information shifted the conversation from discussion of clinical trial options for a patient with triple - negative breast cancer who previously tested negative for mutations in brca1 / 2 to clarication of other germline testing options including bart testing , in accordance with nccn recommendations.7 during his interview , the medical geneticist stressed the value of genetics professionals who can address psychosocial concerns with patients during result return when a germline mutation is suspected . 
he noted that this skill was outside of his scope of practice and was one of the most difcult parts of result return . discussion for patients discussed by this mgtb , tumor sequencing was performed as part of clinical care . 
members of this mgtb , including medical oncologists , questioned whether the results of tumor sequencing were somatic or germline and queried what resources were available to them to clarify this information . 
the medical geneticist was the only mgtb member who was board certied in medical genetics . however , he was not present at the majority of mgtb meetings to provide clarication regarding whether a patient discussed on the mgtb should receive additional germline genetic testing . 
other mgtb members without medical genetics board certication may have qualied as a commission on cancer ( coc ) dened genetics professional by providing cancer risk assessment on a regular basis.15 ( p51 ) a key question that emerged through this analysis is whether these criteria are sufcient to ensure optimal implementation and interpretation of tumor sequencing by an mgtb and how best to support clinicians as they move into using new genomic technologies in their practices . nccn currently recommends genetic risk evaluation for patients with a known / likely pathogenic variant identied in a cancer susceptibility gene via tumor sequencing . 
perceived value of types genetics expertise on an mgtb perceived value by mgtb members member illustrative quote medical oncologists identication of clinical trials evaluation of treatment options medical oncologist : i can look and see if that [ three prior investigational studies ] will exclude her from [ clinical trials ] in the area , but that would sort of be my preference would be to put her on the phase i while looking for regional studies , and ill contact [ name ] and look on clinicaltrials.gov. 
and i guess were not recommending treatment off study with one of these m2r inhibitors ? basic scientists evaluation of functional studies radiation oncologist : someone who has more expertise in the basic science stuffwith better knowledge about genomic pathways understanding of mechanisms of the different mutations and how that may or may not translate into something that the drug might actually work for . medical evaluation of testing geneticists methodology variant classication genetic evaluation of family history counselors variant classication psychosocial counseling during result return medical geneticist : it is still not clear using next - generation sequencing whether a negative result is [ a true negative ] , because you have to look at the coverage across each position and the number of reads theyre reporting . 
second , rearrangements can be missed , so wed want to have her get bartshe should get the [ germline ] testingjust given her age of diagnosis and the fact that its triple - negative . medical geneticist : the most challenging patient encounters ive had is telling a mom that shes passed [ the mutation ] along to her daughter , and having the daughter understand that her mom just passed along a gene that basically assures her that shell get breast cancer in her lifebut it is very challenging , the psychosocial aspect . abbreviations : bart , brac analysis rearrangement test ; mgtb , multidisciplinary genomic tumor board . family history to determine whether people with known / likely pathogenic mutations in cancer susceptibility genes should receive additional germline testing . 
when clinical sequencing laboratories do not compare variants identied in a tumor sample with a germline sample , ordering providers are responsible for lling this gap in professional recommendations by recognizing when clinical germline testing is indicated on the basis of the combination of personal history , tumor sequencing results , and family history . currently , there are no guidelines or recommendations for which professionals or types of genomic expertise should be included by an mgtb to ensure adequate interpretation , recommendation , and communication of genomic test results . 
the coc denes certain roles within a cancer center and provides guidelines for the frequency and format of multidisciplinary tumor boards.15 however , it does not dene what disciplines should be included to constitute an mgtb , nor does it specify guidelines for tumor boards that evaluate tumor - only sequencing . 
the coc denes a genetics professional in a cancer center as someone who provides cancer risk assessment on a regular basis15 ( p51 )  . although multiple clinicians on this mgtb may have met this coc denition , many expressed confusion regarding benets and limitations of genetic testing and genetic risk assessment as well as knowledge deciencies surrounding testing methodology and result interpretation . 
this suggests that the genetics expertise pertinent for interpreting tumor - only sequencing results may be partial or unevenly distributed among those who were tapped to lend their expertise to an mgtb . on the basis of the results of this single - site case study , when considering how denitions of a genetic professional may be applied to mgtbs , providing cancer risk assessment on a regular basis is insufcient for conferring expertise for implementing tumor sequencing in clinical care . to ensure appropriate interpretation of tumor sequencing and identication of patients who ought to be referred to genetics , an mgtb should include individuals with expertise in clinical translation of tumor sequencing , testing methodology , molecular pathology , cancer biology and genomic pathways , germline variant interpretation , evaluation of personal and family history , and application of professional guidelines for germline testing . 
 underlying germline variants in tumor - only sequencing societies and organizations and clinical laboratories ought to consider whether tumor - only sequencing should be routinely paired with germline samples to facilitate standardized reporting of germline variants . given that the mgtb under study was primarily focused on treatment and clinical trial recommendations on the basis of results of tumor sequencing , it may not have been the optimal setting to discuss a patients personal history , including prior genetic testing and / or potentially pathogenic variants in cancer susceptibility genes lacking treatment implications . 
however , to ensure comprehensive referrals for additional germline sequencing after tumor - only sequencing , ordering providers ought to interpret the patients personal history in combination with their tumor - only sequencing results , as discussed by the mgtb . 
for two patients with negative brca1 / 2 tumor - only sequencing results and negative brca1 / 2 germline testing before the availability of bart testing , negative brca1 / 2 tumor - only sequencing results may have been falsely reassuring for these providers , especially considering the possibility that other cancer susceptibility genes may have been responsible for the cause of their cancer . is possible that provider deliberation about whether certain patients should be referred to genetics on the basis of these factors , not specic to the purpose of convening the mgtb the mgtb , may have occurred outside of meetings and would not have been captured by this analysis . 
in a climate where there is a paucity of genetic counselors and medical geneticists , creative strategies for engaging genetics professionals formally and informally in mgtb deliberations should be considered to optimize interpretation of tumor sequencing results . 
this study was limited by the information discussed as part of the mgtb meetings focused on patients with advanced breast cancer and interviews with mgtb members at a single national cancer institutedesignated cancer center . 
thus , possible that there were additional patients with known / likely pathogenic variants in genes associated with an inherited cancer predisposition syndrome that were not captured by this analysis , or that geneticists or genetic counselors may have been consulted about interpretation of laboratory reports outside of the mgtb context . 
fishler research funding : illumina ( inst ) lauren walters - sen employment : invitae stock and other ownership interests : invitae consulting or advisory role : ischemia care no other potential conicts of interest were reported . acknowledgment we thank roselle ponsaran , tracie baker , rebecca sisson , and the multidisciplinary genomic tumor board members for their time and contribution to this study . 
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mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
mcgowan ml , ponsaran rs , silverman p , et al : a rising tide lifts all boats : establishing a multidisciplinary genomic tumor board for breast cancer patients with advanced disease . 
catenacci dv , amico al , nielsen sm , et al : tumor genome analysis includes germline genome : are we ready for surprises ? int j cancer 136 : 1559 - 1567 , 2015 13 . 
 poly ( adp - ribose ) polymerase inhibitor activity in prostate cancers harboring mutations in dna repair genes : who benets ? shridar ganesan , md , phd1 and judy garber , md , mph2 the seminal nding that cancers harboring mutations in brca1 or brca2 are often exquisitely sensitive to poly ( adp - ribose ) polymerase ( parp ) inhibition has led to the successful development of parp inhibitors as effective targeted therapies for brca1 / 2 - mutated ovarian , breast , prostate , and pancreatic cancers.1 - 7 because many other genes are indirectly or directly involved in the brca1 / 2 - associated homologous recombination ( hr ) mediated dna repair ( hrd ) pathway , there has been a great deal of interest in rationally expanding the use of parp inhibitors to include cancers harboring mutations in these related dna repair genes.8 , 9 these include genes intimately associated with the brca1 / 2 hrd pathway , such as palb2 , bard1 , brip1 , and the rad51 paralogs , as well as genes less directly involved in hrd , such as atm and chek2 . 
is there evidence of biallelic loss of function of the gene in the tumor ? both of these criteria would presumably be required for a tumor to manifest a defect in hr - mediated repair sufciently profound to lead to sensitivity to parp inhibitors . 
the second criterion is especially important because , for many dna repair genes , the heterozygous state has at most a subtle repair phenotype that would not impart sensitivity to parp inhibitors . 
even if a lossof - function mutation in a critical dna repair gene is detected in a tumor , there must also be loss of the wildtype allele or , rarely , a dominant negative heterozygous mutation or evidence of epigenetic silencing of the wildtype allele for the tumor to have a profound dna repair defect . 
similarly , for each gene considered as a biomarker of sensitivity to parp inhibitors , data regarding the functional effect of biallelic loss on the hrd pathway and subsequent relative sensitivity to parp inhibitors need to be carefully evaluated . this leads us to the recently published randomized phase iii profound study ( clinicaltrial.gov identier : nct02987543 ) of the efcacy and safety of olaparib in men with metastatic castration - resistant prostate cancer harboring germline mutations in a set of dna repair genes who experienced disease progression on initial hormonal treatment.10 all patients had an alteration in one of 15 genes ( brca1 , brca2 , atm , brip1 , bard1 , cdk12 , chek1 , chek2 , fancl , palb2 , ppp2r2a , rad51b , rad51c , rad51d , and rad54l ) and were randomly assigned in a 2 : 1 fashion to receive either the parp inhibitor olaparib or physicians choice of second - line hormonal therapy with enzalutamide or abiraterone . 
the trial was divided into two cohorts , with cohort a ( n = 245 ) having an alteration in brca1 , brca2 , or atm , and cohort b ( n = 142 ) having alterations in any of the other 12 genes . 
the key question is whether the benet of olaparib treatment seen in both cohort a and cohorts a and b overall is driven primarily by benet in patients with brca2 mutations in both instances . 
here again , analysis of high - depth sequencing data may be able to identify which brca1mutant prostate cancers have undergone loss of heterozygosity with loss of wild - type brca1 allele and are thus truly sensitive to parp inhibitors.13 some individual gene analyses were presented as exploratory analyses as well . 
cancers with mutations in rad51b and rad54l , which are genes directly involved in hrd , had some evidence of benet , but numbers are small ( see supplementary figure s6 in de bono et al10 )  . 
the rest of the genes , including palb2 , bard1 , brip1 , fancl , chek1 , rad51c , and rad51d , conferred eligibility on fewer than ve randomly assigned patients each and cannot be assessed . these data suggest that brca2 - mutant prostate cancers denitely benet from olaparib treatment , and this is a clear advance in the treatment of this subset of prostate cancers . however , it may well be that the benet seen in the brca2mutant population is driving the overall benet seen in both cohort a and the combined group of cohorts a and b in this study . 
the reason for lack of a clear signal in atm - mutant prostate cancer needs to be further investigated , and analysis of both allelic status of the mutations and molecular evidence of patterns of genomic instability associated with hrd may help resolve these issues . 
several reports have shown that atm - mutant breast cancers may not respond to parp inhibitors , 14 , 15 suggesting that atm mutation may not confer clinical sensitivity to parp inhibitors . 
different therapeutic strategies may be required to effectively treat atm - mutant prostate cancers , and other rational treatments , such as atr inhibitors , need to be investigated.16 the lack of clear signal of benet in prostate cancers harboring brca1 mutations may be a result of the small numbers and the fact that , unlike breast , ovarian , and pancreatic cancers , many brca1 - mutant prostate tumors often do not show evidence of biallelic alteration.11 , 17 although there are hints of benet in cancers harboring mutations in other genes critical for hr , including the rad51 paralogs , the numbers in this trial are too small to make any denitive conclusion . 
for example , palb2 and rad51 paralogs clearly play a central role in hrd , and there are data demonstrating that palb2 mutation in breast and prostate cancer and rad51 paralog mutation in ovarian cancer are associated with benet from parp inhibitors.14 , 15 , 18 - 20 evaluation of the landscape of other hrd gene mutations in different cancer types and data regarding the settings in which there is strong evidence of biallelic loss may help guide development of these genes as biomarkers for parp inhibitors . in designing future trials that rationally target specic dna repair defects in cancer , simply identifying a pathogenic mutation in a broad set of genes in the tumor or in germline may not be sufcient to dene the population likely to benet . 
clear evidence that loss of function of each gene of interest confers high - level sensitivity to the drugs being evaluated needs to be presented , and the allelic status of the gene in the tumor may need to be assayed . 
in some settings , such as ovarian and breast cancer , the nding of a pathogenic brca1 or brca2 mutation , either germline or somatic , is almost always associated with evidence of biallelic mutations , but in other cancers and with other dna this may not necessarily be the case.17 repair genes , evaluation of allelic status can be approached by computational methods , but there may be settings , including epigenetic silencing and dominant negative alleles , where this approach will need to be complemented by functional assays of genomic instability . 
here , assays using wholegenome sequencing or targeted sequencing may also be informative in determining whether the presence of a dna repair gene mutation is accompanied by mutation patterns consistent with a profound defect in hrd.21 - 23 the presence of reversion mutations or mutations in compensating genes also can affect sensitivity to parp inhibitors and may need to be considered.24 , 25 in summary , a more sophisticated approach to analysis of dna repair status of cancers , which includes not only identifying the presence of a pathogenic mutation , but also demonstrating that there is loss of the wild - type allele , lack of reversion mutation , and / or genomic evidence of a profound dna repair defect , may be required to optimally identify cancers that may benet from parp inhibitors and related dna - damaging agents . 
grouping together a set of related genes for pooled analysis may , given differences in prevalence of individual mutations and effect size of benet for different genes , lead to settings where the overall benet in the pool may be driven only by a subset of genes . 
if this possibility is not considered , pooled analysis could lead to approval of therapies for gene alterations when these therapies are ineffective and prevent or delay development of more effective targeted therapies for those patients . 
gene - level analysis may make trial design more difcult , but ultimately , it may lead to more precise use of therapies that target specic dna repair defects or other loss - of - function alterations in cancer . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / authors / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . shridar ganesan employment : merck ( i ) stock and other ownership interests : ibris , inspirata , merck ( i ) consulting or advisory role : inspirata , novartis , roche , foghorn therapeutics , foundation medicine , merck sharp & dohme research funding : m2gen ( inst ) patents , royalties , other intellectual property : i hold 2 patents for digital imaging that may be licensed to ibris and inspirata other relationship : national cancer institute / national institutes of health judy garber consulting or advisory role : novartis ( i ) , gtx ( i ) , helix biopharma , konica minolta , aleta biotherapeutics ( i ) , h3 biomedicine ( i ) , kronos bio ( i ) research funding : novartis ( i ) , ambry genetics , invitae genetics , myriad genetics other relationship : susan g . 
n engl j med 361 : 123 - 134 , 2009 gonz alez - martn a , pothuri b , vergote i , et al : niraparib in patients with newly diagnosed advanced ovarian cancer . 
n engl j med 381 : 2391 - 2402 , 2019 kaufman b , shapira - frommer r , schmutzler rk , et al : olaparib monotherapy in patients with advanced cancer and a germline brca1 / 2 mutation . 
moore kn , secord aa , geller ma , et al : niraparib monotherapy for late - line treatment of ovarian cancer ( quadra ) : a multicentre , open - label , single - arm , phase 2 trial . 
lancet oncol 20 : 636 - 648 , 2019 litton jk , rugo hs , ettl j , et al : talazoparib in patients with advanced breast cancer and a germline brca mutation . 
n engl j med 379 : 753 - 763 , 2018 golan t , hammel p , reni m , et al : maintenance olaparib for germline brca - mutated metastatic pancreatic cancer . 
sokol es , pavlick d , khiabanian h , et al : pan - cancer analysis of brca1 and brca2 genomic alterations and their association with genomic instability as measured by genome - wide loss of heterozygosity . 
sakamoto s , iijima k , mochizuki d , et al : homologous recombination repair is regulated by domains at the nand c - terminus of nbs1 and is dissociated with 13 . 
khiabanian h , hirsheld km , goldnger m , et al : inference of germline mutational status and evaluation of loss of heterozygosity in high - depth , tumor - only atm functions . 
gruber jj , afghahi a , hatton a , et al : talazoparib beyond brca : a phase ii trial of talazoparib monotherapy in brca1 and brca2 wild - type patients with advanced her2 - negative breast cancer or other solid tumors with a mutation in homologous recombination ( hr ) pathway genes . 
tung nm , robson me , ventz s , et al : tbcrc 048 : a phase ii study of olaparib monotherapy in metastatic breast cancer patients with germline or somatic mutations in dna damage response ( ddr ) pathway genes ( olaparib expanded )  . 
yap ta , ocarrigan b , penney ms , et al : phase i trial of rst - in - class atr inhibitor m6620 ( vx - 970 ) as monotherapy or in combination with carboplatin in patients with advanced solid tumors . 
kondrashova o , nguyen m , shield - artin k , et al : secondary somatic mutations restoring rad51c and rad51d associated with acquired resistance to the parp inhibitor rucaparib in high - grade ovarian carcinoma . 
chandran ea , kennedy i : signicant tumor response to the poly ( adp - ribose ) polymerase inhibitor olaparib in heavily pretreated patient with ovarian carcinosarcoma harboring a germline rad51d mutation . 
timms km , abkevich v , hughes e , et al : association of brca1 / 2 defects with genomic scores predictive of dna damage repair deciency among breast cancer subtypes . 
telli ml , timms km , reid j , et al : homologous recombination deciency ( hrd ) score predicts response to platinum - containing neoadjuvant chemotherapy in patients with triple - negative breast cancer . 
poly ( adp - ribose ) polymerase inhibitors ( parpi ) represent one approach , as investigated in previous studies using olaparib.5 - 7 we describe a patient with an unresectable mgmt unmethylated , idh wild - type gbm . 
the patient was enrolled in an observational clinical study ( sponsored by strata through which wholeoncology , ann arbor , mi ) , exome and rna sequencing on formalin - xed parafn - embedded tumor tissue revealed loss - offunction mutations in atm and tumor protein 53 ( tp53 )  . 
the tumor was classied as tumor mutational burden ( tmb ) high , programmed death - ligand 1 ( pd - l1 ) low , and microsatellite stable ( mss ; table 1 )  . 
after completion of therapy , additional review of patients records revealed a germline msh6 loss - offunction mutation , which conrmed lynch syndrome . the genomic ndings and high likelihood of lynch syndrome , along with early - phase data suggesting safety and brain penetration , provided rationale for the treating oncologist to initiate olaparib with standard chemoradiation . 
she was also treated with alternating tumor - treating elds ( optune ; novocure , st helier , jersey ) , an externally applied , low - intensity electromagnetic eld treatment shown to improve survival by 4.9 months over maintenance temozolomide alone when used 18 hours per day.8 device compliance was limited , and treatment was discontinued after 1 month . 
outside hospital records included this nding on germline testing performed by ambry genetics ( aliso viejo , ca )  . msh6 is involved in dna mismatch repair ( appendix table a1 )  . 
most mutations are somatic , found almost exclusively after temozolomide therapy.11 in this setting , they are associated with resistance to alkylating agents , hypothesized to be the dominant mechanism of acquired temozolomide resistance after therapy , likely because of failure of temozolomideinduced dna damage to result in apoptosis in mismatch repairdecient ( dmmr ) cells.12 - 24 when msh6 mutations in treatment - nave patients with mgmt are present methylated , otherwise chemosensitive tumors , treatment response is markedly attenuated.13 beyond treatment resistance , ongoing temozolomide exposure after msh6 inactivation leads to a hypermutator phenotype and tumor progression.14 - 16 parpi restore sensitivity to temozolomide in dmmr cells.25 dmmr cells may also have increased resistance to radiotherapy.26 - 28 lynch syndrome was not identied by somatic testing . often , lynch syndrome is discovered after a tumor is found to be microsatellite instability high ( msi - h ) or dmmr on immunohistochemistry . 
msh6 - mutated brain tumors , however , are often not msi - h by standard polymerase chain reaction testing , and immunohistochemistry is not standardly performed.29 in this case , mss status was determined from next - generation sequencing ( ngs ) on the basis of length variant allele counts at multiple microsatellite loci . 
while alternate ngs methods have demonstrated sensitivity and specicity in brain tumors , this specic methodology in gbm is performance of unknown.30 , 31 while decient msh6 immunohistochemical staining would conrm a pathogenic mutation , intact staining would not rule out dmmr because some mutations result in intact expression of dysfunctional protein.32 - 35 the msh6 mutation was not reported by somatic testing because of its presence in a stretch of repeating cytosines , known as a homopolymer region . 
380 cns tumors associated with protein loss of function.45 as previously mentioned , p53 - decent cells may be particularly vulnerable to dna - damaging treatment when an atm mutation is present . 
conversely , the combination of dmmr and p53 - decient cells worsens response because of failed phosphorylation of p53 and , thus , failed cell arrest after treatment - induced dna damage.46 in general , cancers with mutant p53 have reduced sensitivity to chemotherapy and radiation ; however , there are many instances where mutant p53 has no effect or even enhances treatment effect.47 tmb high tmb was determined from ngs and included noncoding and coding , synonymous and nonsynonymous , and singlenucleotide and multinucleotide variants present at  . 
while frequently performed through immunohistochemistry , classication was based on sequencing results in this patient , using a score derived from the percent of maximum pd - l1 expression across tested tumor samples . 
this method is validated in a lung cancer cohort , but accuracy in gbm is less certapd - l1 is expressed on the surface of most glioma cells , with increased frequency in high - grade gliomas such as gbm , and variable detection is based on technique.52 - 54 of note , several studies demonstrated high pd - l1 association with worse survival gbm.55 there are no currently approved drugs that target pd - l1 in gbm , although several trials are ongoing . 
given the efcacy of programmed death 1 ( pd - 1 ) / pd - l1 blockade in dmmr tumors , immunotherapy could be considered.56 the pd - 1 inhibitor pembrolizumab is approved for all msi - h tumors , making it a treatment option for patients with lynch syndromeassociated cancers , which are typically msi - h . rationale for olaparib parp is involved in single - strand dna break and base excision repair . 
 file , morgan , and khagi expression in gbm.57 olaparib is a parpi that impairs the ddr , increasing treatment - induced chromosomal instability , cell cycle arrest , and apoptosis.58 in patients with germline brca1 / 2 mutations that impair double - strand dna break repair by homologous recombination , parpi cause synthetic lethality with signicant clinical benet.59 - 62 parpi also have efcacy in tumors with mutations in ddr including atm , and in patients with neither genes , a germline brca mutation nor other homologous recombination deciency.63 , 64 parpi have been used as monotherapy and in combination with radiation and chemotherapy to prevent the repair of treatment - induced dna breaks , thereby promoting tumor cell death . 
parpi increase sensitivity to temozolomide in cell and xenograft models of gbm.65 - 67 this effect persists in mgmt unmethylated tumors.68 parpi also restore sensitivity to temozolomide in dmmr cells.25 , 69 in addition , exposure to temozolomide before or concurrently with a parpi increases the magnitude of dna damage and led to complete regression of gbm cells in one study.70 this treatment - sensitizing effect is not present in patients with temozolomide resistance , which suggests optimal corporation in newly diagnosed gbm.71 there have been 3 phase i trials of olaparib with temozolomide and / or radiation in gbm . 
the oparatic trial conrmed tumor penetration and dosing schedule , with promising early results.5 , 7 paradigm - 2 investigated olaparib plus radiotherapy with or without temozolomide.6 , 72 these studies support the addition of olaparib to temozolomide and radiation as safe , well tolerated , and potentially radiosensitizing.5 discussion this patient had an excellent , durable response despite many factors that predict a poor prognosis . 
in addition , somatic msh6 loss - of - function mutations contribute to temozolomide resistance , glioma recurrence , and tumor progression , with similar effects expected from a deleterious germline mutation . 
while atm mutations improve treatment sensitivity , particularly with concurrent tp53 mutations , it is unlikely that this would result in a sustained , near - complete response with standard chemoradiation alone . 
it is also unlikely that tumor - treating elds improved clinical outcome given short duration of use . in addition to the general chemoand radiosensitizing properties of parpi , the ability for parpi to restore sensitivity to temozolomide in dmmr and mgmt unmethylated tumors , as well as efcacy of parpi in ddr - decient tumors , strongly suggests that olaparib was an essential component of the treatment regimen . 
the likelihood of olaparib - induced synthetic lethality is high , through impairment of single - stranded dna break repairs in a tumor already decient in base - base substitution , single - base insertion , and single - base deletion mismatch repair as well as double - stranded dna break repair . genomic sequencing allows identication of patients with targetable mutations who may benet from currently available treatments , which are increasing rapidly.73 this is particularly important for patients predicted to have poor outcomes with standard treatment and limited access to clinical trials . novel treatment approaches in the rst - line setting are needed . 
morgan honoraria : medscape , pharmacy times continuing education travel , accommodations , expenses : medscape , pharmacy times continuing education simon khagi research funding : lambda technologies no other potential conicts of interest were reported . acknowledgment we acknowledge jason merker , md , phd ; william kim , md ; shetal patel md , phd ; amber cipriani , pharmd ; and ashlynn messmore , ms , for their contributions to the university of north carolina multidisciplinary mtb . we also acknowledge carlos zamora , md , phd , for review of the radiographic images and scott tomlins , md , phd , co - founder and chief medical ofcer of strata oncology , for providing descriptions of the sequencing methodology . 
we thank the patient for allowing us to share a part of her story . references stupp r , mason wp , van den bent mj , et al : radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma . 
n engl j med 352 : 987 - 996 , 2005 kreth fw , thon n , simon m , et al : gross total but not incomplete resection of glioblastoma prolongs survival in the era of radiochemotherapy . 
n engl j med 360 : 765 - 773 , 2009 chalmers aj , short s , watts c , et al : phase i clinical trials evaluating olaparib in combination with radiotherapy ( rt ) and / or temozolomide ( tmz ) in glioblastoma patients : results of oparatic and paradigm phase i and early results of paradigm - 2 . 
j clin oncol 36 , 2018 ( suppl ; abstr 2018 ) fulton b , short sc , james a , et al : paradigm - 2 : two parallel phase i studies of olaparib and radiotherapy or olaparib and radiotherapy plus temozolomide in patients with newly diagnosed glioblastoma , with treatment stratied by mgmt status . 
halford se , cruickshank g , dunn l , et al : results of the oparatic trial : a phase i dose escalation study of olaparib in combination with temozolomide ( tmz ) in patients with relapsed glioblastoma ( gbm )  . 
j clin oncol 35 , 2017 ( suppl ; abstr 2022 ) stupp r , taillibert s , kanner a , et al : effect of tumor - treating elds plus maintenance temozolomide vs maintenance temozolomide alone on survival in patients with glioblastoma : a randomized clinical trial . 
jama 318 : 2306 - 2316 , 2017 edelmann w , yang k , umar a , et al : mutation in the mismatch repair gene msh6 causes cancer susceptibility . 
cahill dp , levine kk , betensky ra , et al : loss of the mismatch repair protein msh6 in human glioblastomas is associated with tumor progression during temozolomide treatment . 
nguyen sa , stechishin od , luchman ha , et al : novel msh6 mutations in treatment - nave glioblastoma and anaplastic oligodendroglioma contribute to temozolomide resistance independently of mgmt promoter methylation . 
liu l , markowitz s , gerson sl : mismatch repair mutations override alkyltransferase in conferring resistance to temozolomide but not to 1 , 3 - bis ( 2 - chloroethyl ) 1068 , 2008 [ erratum : nature 494 : 506 , 2013 ] nitrosourea . 
pepponi r , marra g , fuggetta mp , et al : the effect of o6 - alkylguanine - dna alkyltransferase and mismatch repair activities on the sensitivity of human melanoma cells to temozolomide , 1 , 3 - bis ( 2 - chloroethyl ) 1 - nitrosourea , and cisplatj pharmacol exp ther 304 : 661 - 668 , 2003 19 . 
hochhauser d , glynne - jones r , potter v , et al : a phase ii study of temozolomide in patients with advanced aerodigestive tract and colorectal cancers and methylation of the o6 - methylguanine - dna methyltransferase promoter . 
aquilina g , ceccotti s , martinelli s , et al : n - ( 2 - chloroethyl ) - n ( cid : 2 ) - cyclohexyl - n - nitrosourea sensitivity in mismatch repair - defective human cells . 
stark am , doukas a , hugo hh , et al : the expression of mismatch repair proteins mlh1 , msh2 and msh6 correlates with the ki67 proliferation index and survival in patients with recurrent glioblastoma . 
koi m , umar a , chauhan dp , et al : human chromosome 3 corrects mismatch repair deciency and microsatellite instability and reduces n - methyl - n ( cid : 2 ) - nitro - nnitrosoguanidine tolerance in colon tumor cells with homozygous hmlh1 mutation . 
curtin nj , wang lz , yiakouvaki a , et al : novel poly ( adp - ribose ) polymerase - 1 inhibitor , ag14361 , restores sensitivity to temozolomide in mismatch repairdecient cells . 
yan t , schupp je , hwang hs , et al : loss of dna mismatch repair imparts defective cdc2 signaling and g ( 2 ) arrest responses without altering survival after ionizing radiation . 
gylling ah , nieminen tt , abdel - rahman wm , et al : differential cancer predisposition in lynch syndrome : insights from molecular analysis of brain and urinary 30 . 
trabucco se , gowen k , maund sl , et al : a novel next - generation sequencing approach to detecting microsatellite instability and pan - tumor characterization of 1000 microsatellite instability - high cases in 67 , 000 patient samples . 
dudley b , brand re , thull d , et al : germline mlh1 mutations are frequently identied in lynch syndrome patients with colorectal and endometrial carcinoma demonstrating isolated loss of pms2 immunohistochemical expression . 
am j surg pathol 39 : 1114 - 1120 , 2015 iv ady g , madar l , dzsudzs ak e , et al : analytical parameters and validation of homopolymer detection in a pyrosequencing - based next generation sequencing systebmc genomics 19 : 158 , 2018 36 . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
int j radiat oncol biol phys 50 : 511 - 523 , 2001 19 : 3189 - 3200 , 2013 18 : 1899 - 1908 , 2019 jiang h , reinhardt hc , bartkova j , et al : the combined status of atm and p53 link tumor development with therapeutic response . 
tchelebi l , ashamalla h , graves p : mutant p53 and the response to chemotherapy and radiation , in deb s , deb s ( eds ) : mutant p53 and mdm2 in cancer . subcellular biochemistry , volume 85 . 
salem m , grothey a , kim e , et al : impact of mlh1 , pms2 , msh2 , and msh6 alterations on tumor mutation burden ( tmb ) and pd - l1 expression in 1 , 057 microsatellite instability - high ( msi - h ) tumors . 
tentori l , leonetti c , scarsella m , et al : systemic administration of gpi 15427 , a novel poly ( adp - ribose ) polymerase - 1 inhibitor , increases the antitumor activity of temozolomide against intracranial melanoma , glioma , lymphoma . 
boulton s , pemberton lc , porteous jk , et al : potentiation of temozolomide - induced cytotoxicity : a comparative study of the biological effects of poly ( adpribose ) polymerase inhibitors . 
delaney ca , wang lz , kyle s , et al : potentiation of temozolomide and topotecan growth inhibition and cytotoxicity by novel poly ( adenosine diphosphoribose ) polymerase inhibitors in a panel of human tumor cell lines . 
miknyoczki sj , jones - bolin s , pritchard s , et al : chemopotentiation of temozolomide , irinotecan , and cisplatin activity by cep - 6800 , a poly ( adp - ribose ) base excision repair . 
clarke mj , mulligan ea , grogan pt , et al : effective sensitization of temozolomide by abt - 888 is lost with development of temozolomide resistance in glioblastoma xenograft lines . 
lesueur p , lequesne j , grellard jm , et al : phase i / iia study of concomitant radiotherapy with olaparib and temozolomide in unresectable or partially resectable glioblastoma : ola - tmz - rte - 01 trial protocol . 
 translating in vivo metabolomic analysis of succinate dehydrogenasedeficient tumors into clinical utility purpose mutations in the mitochondrial enzyme succinate dehydrogenase ( sdh ) subunit genes are associated with a wide spectrum of tumors , including pheochromocytomas and paragangliomas , gi stromal tumors , renal cell carcinomas , and pituitary adenomas . 
our aim was to investigate the potential clinical applications of proton - 1 magnetic resonance spectroscopy ( 1h - mrs ) in a range of suspected sdh related tumors . patients and methods fifteen patients were recruited to this study . 
sequential imaging in a patient with a metastatic abdominal paraganglioma demonstrated loss of the succinate peak after four cycles of [ 177lu ] dotatate , with a corresponding biochemical response in normetanephrine . conclusion this study has demonstrated the translation into clinical practice of in vivo metabolomic analysis using 1h - mrs in patients with sdh - deficient tumors . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction the succinate dehydrogenase ( sdh ) enzyme is composed of four subunits ( a to d ) and plays a key role in the krebs cycle and oxidative phos phorylation.1 in the past two decades , germ line mutations in the genes encoding the four sdh subunits ( sdha , sdhb , sdhc , and sdhd ) , collectively known as sdhx , have emerged as an important cause of human neoplasia and a par adigm for the role of disordered cellular meta bolism in oncogenesis.27 sdhx mutations were described initially in association with head and neck paragangliomas ( pgls ; derived from para sympathetic ganglia ) and in pheochromocytomas and pgls ( ppgls ; derived from sympathetic ganglia and often secreting catecholamines ) .2 , 3 it is now recognized that approximately 40% of patients with ppgls harbor germ line muta tions in inherited ppgl genes , and sdhx mutations comprise the most common cause of ppgl predisposition.8 in addition , germ line sdhb mutations are associated with a high risk of malignancy in ppgls.9 other tumor types associated with sdhx mutations include gi stromal tumors ( gists ) and renal cell car cinomas ( rccs ) .1013 gists are mesenchymal tumors of the gi tract and in adults are usually associated with somatic activating mutations in ruth t . 
 the kit or pdgfra gene.4 , 11 however , gists without kit or pdgfra gene mutations , 4 known as wildtype gists ( wtgists ) , account for 15% of adult and 85% of pediatric gists , and recent studies suggest that up to 88% of wtgists are sdh deficient.11 a wtgist with sdh deficiency may harbor a germ line sdhx mutation ( 75% of cases ) or an sdhc gene epi mutation with hypermethylation of the pro moter region.11 only approximately one third of patients with sdhdeficient wtgists achieve disease stabilization with imatinib therapy , 12 and the risk of metastatic disease is higher with sdhdeficient gists compared with conven tional gists.11 , 12 sdhx - associated rcc may present in patients with a personal or family his tory of ppgl or may present with an rcconly phenotype.13 finally , germ line sdhx mutations have been described in rare patients with pitu itary adenomas.10 despite recent advances in the understanding of the sdhx genes , there are many areas of unmet clinical need , including a lack of robust biomarkers to predict aggressive biologic behavior and inform clinical surveil lance and management.14 succinate has been shown to be elevated by 100fold in sdhxmutated ppgls ex vivo compared with nonsdhxmutated ppgls.15 recently , in vivo detection of succinate by mag netic resonance spectroscopy ( mrs ) was reported in two patient cohorts with sdhdeficient ppgls.16 , 17 similarly , the noninvasive detec tion of 2hydroxyglutarate with proton1 mrs ( 1hmrs ) has been demonstrated within glio mas in patients with gainoffunction mutations in another citric acid cycle enzyme , isocitrate dehydrogenase 1.18 the ability to measure succi nate in vivo has a number of important potential clinical applications , including early identifica tion of sdh deficiency , which could enable tai lored patient surveillance and management . 
suitable patients were identi fied based on sdhx germ line status , suspicious clinical phenotype ( metastatic ppgl , pgl , or wtgist ) , and / or immunohistochemistry of tumor tissue showing absent sdhb immuno staining . 
all participants provided written informed consent , and the study was approved by the cambridge south research ethics committee . mrs analysis both sage ( ge healthcare , waukesha , wi ) and lcmodel ( sprovencher.com ) 19 spectros copy analysis programs were used to recon struct , analyze , and display spectra . 
 ( c ) axial fused [ 18f ] fluorodeoxyglucose ( [ 18f ] fdg ) positron emission tomography ( pet ) / computed tomography ( ct ) image demonstrating an [ 18f ] fdgavid carotid body paraganglioma after sdh deficiency was demonstrated on proton1 mr spectroscopy ( 1hmrs )  . 
three patients had multicentric primary tumors , including patient 5 , who presented with a metastatic wtgist and was subsequently diagnosed with a 1.9cm carotid body pgl ( fig 1d ) ; patient 9 , with an abdominal pgl and a small leftsided 1.5cm carotid pgl ( fig 2b ) ; and patient 8 , with a large leftsided glomus pgl and a 2cm prolactin secreting pituitary adenoma ( data supplement )  . 
 genotype a germ line mutation in an sdhx gene was iden tified in nine ( 60% ) of 15 patients : five in sdhb ( four missense variants and one truncating vari ant ) and four in sdha ( one missense and three truncating )  . 
two additional patients were found to have somatic sdhc epimutations ( table 1 )  . 1h - mrs succinate analysis the 1hmrs characteristics of the 15 patients are listed in the data supplement . 
the liver was the most common site to be assessed ( n = 6 ) , but goodquality spectra were also obtained from a pituitary tumor ( n = 1 ) and ppgls ( n = 5 )  . 
patient 4 had a met astatic wtgist with no detectable germ line sdhx mutation and preserved sdhb protein expression in the tumor tissue ; choline was con fidently fitted on lcmodel , but no succinate was seen . 
patient 6 demonstrated a goodquality spectrum from the remnant pituitary adenoma ; choline was detected on lcmodel and sage processing , but no succinate was detected , and this finding was consistent with the preserva tion of sdhb protein expression in the pitu itary tumor by immunohistochemistry ( fig 4 )  . 
patient 12 demonstrated no reliable detection of succinate or choline because of motion artifact and a low signaltonoise ratio ( snr ) , which probably resulted from inconsistent breathing , because the voxel was at the edge of the liver . 
a metastasis on the edge of the liver was imaged in patient 14 , where again incon sistent respiration probably led to displace ment of the voxel into adjacent adipose tissue . 
 ( a ) t2weighted magnetic resonance image from patient 1 and ( b ) t1weighted image from patient 3 demonstrating liver metastases from which spectra were acquired in the locations indicated by the white arrows . 
after four cycles of treatment , a repeat 1hmrs examina tion on the same pelvic nodal metastases revealed a choline peak but no succinate peak ( fig 5c )  . 
 although the magnetic resonance imaging fea tures of the metastatic lesions were unchanged pre and posttreatment , the loss of a succi nate peak was correlated with a reduction in plasma normetanephrine levels ( from 1 , 861 to 1 , 193 pmol / l ) and tumor avidity on [ 18f ] fluo rodeoxyglucose ( [ 18f ] fdg ) positron emission tomography ( pet ) / computed tomography ( ct ; standardized uptake value : pretreatment , 16.1 ; posttreatment , 9.3 ; figs 5d to 5f )  . 
the detec tion of choline on the acquired spectra both before and after treatment indicates that tumor necrosis is unlikely to account for the absent suc cinate peak posttreatment . a sequential 1hmrs study was performed for patient 5 because of evidence of progressive disease on surveillance ct , despite treatment with a multikinase inhibitor , regorafenib . 
the results for scr were almost identical in these two patients , suggesting good test reproducibility ( data supplement )  . discussion this proofofprinciple study demonstrates that detection of a succinate peak and an increased scr was specific for a variety of sdhdeficient tumor types . 
all six tumors with a positive suc cinate peak and elevated scr were associated with a germ line sdhx mutation ( n = 4 ) or an sdhc epimutation ( n = 2 )  . 
 ( c ) succinate dehydrogenase b ( sdhb ) immunohistochemistry demonstrating preserva tion of the sdhb protein performed on a section of tumor tissue debulked from the pituitary tumor . adequate 1hmrs demonstrated preservation of sdhb expression in the tumors analyzed . 
our findings are complementary to a previous study in which 1hmrs was applied in nine patients with pgls , and succinate peaks were detected in all five with sdhx mutations but not in the four patients without mutations.16 we demonstrate for the first time to our knowledge that 1hmrs can also be used to determine the sdh status of gists and pituitary adenomas and that succinate peaks can be detected in sdhdeficient tumors with epigenetic inactivation of sdhc . 
potential diagnostic applications of this new approach include : assessing the pathoge nicity of patients with germ line sdhx variants of uncertain significance and potentially sdh related tumors ; investigating possible metastatic lesions ( eg , in the liver ) in patients with germ line sdhx mutations and primary sdhdeficient tumors ; assessing patients with multiple pri mary tumors to determine if all are sdh defi cient ; identifying patients without detectable germ line sdhx mutations who might benefit from specialist genetic investigations , such as sdhc promoter methylation status ; and assess ing sdh tumor status preoperatively , particu larly for patients with possible wtgists , because standard adjuvant treatment with imatinib has proven to be less effective in patients with sdh deficient disease.12 notably , here we use the presence of a choline signal as an internal control for viable tissue to discriminate technical failures from a negative finding . 
 this trend is the opposite of what would be expected if necrosis were artificially lowering the overall succinate levels in large tumors and therefore suggests that the method is measuring real differences in succinate , which are indepen dent of tumor size . 
 ( b ) spectra acquired before treatment illustrating succi nate accumulation at 2.4 pp ( c ) spectra ac quired after four cycles of [ 177lu ] dotatate with no detectable succinate peak at 2.4 pp ( d ) plasma metanephrine and methoxytyramine levels before and after treatment with [ 177lu ] dotatate . 
as a consequence , sdhdeficient tumors demonstrate epigenetic abnormalities and activated hypoxic gene responses , and more recently , evidence has suggested that succinate may have a paracrine effect on stromal tis sue.2022 understanding the molecular mech anisms of sdhrelated tumorigenesis provides a rationale for novel therapeutic interventions such as reversing the epigenetic abnormalities or exploiting metabolic vulnerabilities , similar to the recent discovery that tumoral 2hydroxyglutarate accumulation may increase responsiveness to olapa rib , a poly ( adpribose ) polymerase inhibitor.23 the availability of sensitive noninvasive bio markers would greatly facilitate precision medicinebased clinical trials . 
1hmrs is highly specific and allows in vivo detection of individual metabolites without the use of ionizing radiation ; however , 1hmrs is significantly less sensitive than pet , which could limit the detection of low levels of succinate , and it can be challenging to differen tiate intracellular from extracellular metabolites . 
 in the future , 1hmrs may be complemented by other techniques , such as hyperpolarized car bon13 mrs , which can increase 13mrs snr by several orders of magnitude , allowing assess ment of enzyme flux in vivo.26 we show that 1hmrs could be a valuable tool for the assessment of tumor response in the con text of radionuclide and other therapies , because alterations in succinate levels were detected despite stable appearance of the tumor diam eters . 
this important application of 1hmrs could be expanded to include other tumors with specific metabolic defects , including fuma rate hydratasedeficient tumors , 27 idh1mutant tumors , 18 and the recently identified malate dehydrogenase 2deficient tumors.28 however , important limitations of in vivo metabolomic analysis using 1hmrs were also revealed by our study ; for example , spectral quality was poor in close proximity to metal dental work , in areas adjacent to air spaces such as the lung , and in bone metastases and was susceptible to motion artifact . 
in this study , the technical failure rate was 26% , which is similar to the failure rate reported in previous studies using 1hmrs.16 importantly , no patient cases were excluded from this prospective study , with the intention that this would inform the translation of this imaging modality into clinical practice . 
on the basis of the evidence from this exploratory study , we recommend that tumors be selected for 1hmrs analysis based on the following : ideally the largest tumor deposit but at least > 2 cm in size ; tumors located close to bone or lung should be avoided ; tumors with significant necrosis or hemorrhage should be avoided ; superficial tumor deposits should be selected preferentially ; and respiratorytriggered acquisition should be used for tumors in the upper abdomen , such as hepatic metastases . although the use of 1hmrs as a diagnos tic tool is likely to be limited to specialist cen ters , the number of scan averages in our study during spectral acquisition was less than half those reported in a previous study16 ( 200 v 512 ) , without demonstrating a reduction in sensi tivity . 
furthermore , this imaging modal ity could be used to investigate other metaboli cally driven tumors . in conclusion , this study is the largest to date to our knowledge to evaluate 1hmrs in patients with sdh deficiency . 
it reveals that 1hmrs has the potential to be used as a noninvasive biomarker in the precision management of sdhdeficient disease and could have a role as a biomarker of successful treatment response . 
mclean stock and other ownership interests : veryan medical patents , royalties , other intellectual property : patent for stent design for arterial bypass graft basetti madhu no relationship to disclose benjamin g . 
challis no relationship to disclose rogier ten hoopen no relationship to disclose thomas roberts no relationship to disclose consulting or advisory role : bayer healthcare pharmaceuticals , ariad pharmaceuticals speakers bureau : pfizer travel , accommodations , expenses : pierre fabre , teva pharmaceuticals europe helen l . 
bulusu honoraria : pierre fabre kieran allinson no relationship to disclose lisa happerfield no relationship to disclose soo - mi park no relationship to disclose alison marker no relationship to disclose olivier giger stock and other ownership interests : cambridge pathology eamonn r . 
xekouki p , stratakis ca : succinate dehydrogenase ( sdhx ) mutations in pituitary tumors : could this be a new role for mitochondrial complex ii and / or krebs cycle defects ? endocr relat cancer 19 : c33c40 , 2012 7 . 
boikos sa , pappo as , killian jk , et al : molecular subtypes of kit / pdgfra wildtype gastrointestinal stromal tumors : a report from the national institutes of health gastrointestinal stromal tumor clinic . 
holdsworth ch , badawi rd , manola jb , et al : ct and pet : early prognostic indicators of response to imatinib mesylate in patients with gastrointestinal stromal tumor . 
 signicant and durable clinical response to sorafenib and radiation therapy for a patient with stage iv hepatocellular carcinoma and lrrk2 mutation linlin yang , md1 ; patrick wald , md1 ; sameek roychowdhury , md , phd1 ; anne m . 
the level was elevated to alpha fetoprotein ( afp ) 57 , 400 ng / ml , and ct - guided biopsy of the large hepatic lesion revealed stage iv hcc . 
because of radiographic concern for disease progression and invasion into the transverse colon , sorafenib was discontinued and radiation therapy was in addition , started 1 week after sorafenib was discontinued . 
the two large masses were irradiated to a dose of 40 gy in 16 fractions using a conformal radiation plan ( fig 1d )  . repeat ct scans 1 month later showed a large decrease in the size of not only the two radiated extrahepatic lesions but also the unirradiated hepatic lesions , and it was decided to restart the patient on maintenance sorafenib ( 200 mg twice daily ) 6 weeks after radiation . 
on the basis of this impressive response , sorafenib doses were held , and the patient underwent laparoscopic partial left hepatectomy of segment 4b with removal of additional primary liver and extrahepatic omental masses . 
at abdominal masses showed no residual 3 weeks after surgery , sorafenib was restarted at 200 mg twice daily but was discontinued after 2 more months because of sorafenib - related pulmonary toxicity . 
after this exceptional response , we performed next - generation sequencing of whole blood lymphocytes and tumor dna from the diagnostic biopsy specimen ( table 1 ) , which revealed mutations in lrrk2 ( k1316n ) , lrp1b ( v4250a , y4256c ) , tp53 ( v157insa , a nonframeshift insertion of three base pairs : cgc ) , and notch3 ( g501s )  . 
 yang et al started sorafenib 400 mg twice a day sorefenib stopped and rt delivered started maintenance sorafenib 200 mg twice a day surgery sorafenib 200 mg twice a day sorafenib discontinued 9 10 11 12 14 47 52 time ( months ) 180000 160000 140000 120000 100000 80000 60000 40000 20000 fig 1 . 
 ( right ) disappearance of intrahepatic lesions noted in fig 1b . lrrk2 we turned our attention to lrrk2 , a gene that has been linked to the pathogenesis of parkinson disease with known roles in the antioxidant response.2 , 3 ionizing radiation generates reactive oxygen species ( ros ) , which are critical for the ability of radiation to promote tumor cell kill through dna damage . 
the degree of ros generation correlates with serum levels of advanced oxidation protein products and clinical effectiveness in sorafenib - treated patients with hcc.4 because ros are in the response to both radiation and sorafenib , critical through promotion of dna damage , we hypothesized that this mutation in lrrk2 could alter the response to both therapies . 
we observed adequate transfected wild - type ( wt ) and k1316n expression of leucine - rich repeat kinase 2 ( lrrk2 ) protein in multiple cell lines ( appendix fig a3 for rna and protein ; primer sequences intable a1 )  . 
we also tested genetic silencing of lrrk2 with small interfering rna ( sirna ) and found that lrrk2 depletion likewise sensitized hcc cells to sorafenib ( figs 2c and 2d )  . 
in addition , we found that expression of the lrrk2 k1316n mutation in both hela and hepg2 cells resulted in notable radiation sensitization in radiation clonogenic assays , whereas wt lrrk2 had no radiosensitizing effects ( figs 2e and 2f )  . because of the previously identied role of lrrk2 in ros , we assessed whether wt lrrk2 or the k1316n mutant altered levels of ros in cancer cells . 
 ( a , b ) overexpression of lrrk2 k1316n signicantly increases sorafenib sensitivity on hcc cells . ( c , d ) genetic silencing of lrrk2 with sirna sensitizes hcc cells to sorafenib treatment . 
sorafenib sensitivity on hcc cells was determined by alamarblue assay ( thermofisher , waltham , ma ) 72 hours after transfection , whereas radiation sensitivity was determined by clonogenic assay . 
similarly , we found that lrrk2 k1316n greatly increased hydrogen peroxide ( h2o2 ) levels , another known mediator of ros damage , and this increase was reversed by treatment of cells with ebselen , an h2o2 scavenger ( figs 3d , 3e , and 3f )  . 
finally , the addition of the superoxide scavenger tiron greatly attenuated the radiation sensitivity induced by lrrk2 k1316n in radiation clonogenic assays ( figs 4a and 4b )  . discussion in this case of a patient with an exceptional response to sorafenib and radiation , we identied a novel lrrk2 k1316n mutation that induced sorafenib and radiation sensitivity in various hcc cell lines , consistent with our clinical observations . 
 ( * ) signicant difference from corresponding tiron or ebselen treatment group ; ( ) signicant difference from empty vector ; ( ) signicant difference from lrrk2 wild type ( wt )  . 
hepg2 and hela cells transfected with wild - type ( wt ) lrrk2 and lrrk2 k1316n were preincubated with dmso or tiron ( 1 mm ) and then exposed to 4 gy of radiation . 
thus , we hypothesize that this lrrk2 mutation increases lrrk2 kinase activity and enhances sorafenib and radiation - induced apoptotic cell death signaling via increased p53 and p21 activity . our in vitro data suggest that this lrrk2 k1316n mutation like other canonical lrrk2 mutations increases ros , observed in parkinson disease . 
thus , another potential hypothesis to explain the radiosensitivity of disease in this patient is that increased lrrk2 kinase activity in lrrk2 mutants may render cells more susceptible to oxidative stress imparted by radiotherapy through creation of ros , such as oxygen free radicals.6 multiple publications show that lrrk2 mutations induce synergistic or additive cell killing in the presence of oxidative stress.7 , 8 taken together , the presence of increased oxidative stress imparted by an activating lrrk2 mutation could prime tumor cells to increased radiation - mediated cell death . the near - complete pathologic response and more than 4 - year disease - free interval since completion of all therapy are remarkable results from a low to moderate dose of radiation and a drug that rarely induces complete response ( 0% in the sharp trial )  . 
it is also interesting to note the dramatic radiographic responses seen in lesions that were not directly radiated , which suggests an abscopal effect from the combination of sorafenib and radiotherapy . 
certainly , we cannot overlook that the dramatic responses observed may have been due in part to the development of an antitumor immune response , which has been previously attributed to both sorafenib and radiation.9 , 10 indeed , the radiologic progression initially noted after sorafenib started may be related to pseudoprogression and development of an early immune response or apoptosis / necrosis of the tumor , despite the observation of the most dramatic clinical and radiographic response with postradiation sorafenib . in conclusion , these data support a role for a novel , previously unidentied lrrk2 mutation that promotes sorafenib and radiation sensitivity through an ros - dependent mechanism . in addition , the combination of radiation and sorafenib also may have induced an antitumor immune response . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . patrick wald employment : riverside radiation oncology sameek roychowdhury stock and other ownership interests : johnson & johnson ( i ) consulting or advisory role : incyte , abbvie , qed therapeutics research funding : takeda , ignyte anne m . 
noonan consulting or advisory role : helsinn healthcare no other potential conicts of interest were reported . references llovet jm , ricci s , mazzaferro v , et al : sorafenib in advanced hepatocellular carcinoma . 
n engl j med 359 : 378 - 390 , 2008 goldwurm s , di fonzo a , simons ej , et al : the g6055a ( g2019s ) mutation in lrrk2 is frequent in both early and late onset parkinsons disease and originates from a common ancestor . 
j med genet 42 : e65 , 2005 loefer da , klaver ac , coffey mp , et al : increased oxidative stress markers in cerebrospinal uid from healthy subjects with parkinsons diseaseassociated lrrk2 gene mutations . 
front aging neurosci 9 : 89 , 2017 coriat r , nicco c , ch ereau c , et al : sorafenib - induced hepatocellular carcinoma cell death depends on reactive oxygen species production in vitro and in vivo . mol cancer ther 11 : 2284 - 2293 , 2012 5 . 
ho dh , kim h , kim j , et al : leucine - rich repeat kinase 2 ( lrrk2 ) phosphorylates p53 and induces p21 ( waf1 / cip1 ) expression . 
gene 532 : 18 - 23 , 2013 yang d , li t , liu z , et al : lrrk2 kinase activity mediates toxic interactions between genetic mutation and oxidative stress in a drosophila model : suppression by curcumneurobiol dis 47 : 385 - 392 , 2012 8 . 
ho v , lim ts , lee j , et al : tlr3 agonist and sorafenib combinatorial therapy promotes immune activation and controls hepatocellular carcinoma progression . oncotarget 6 : 27252 - 27266 , 2015 10 . 
stable post - operative changes are seen , and there is no evidence of disease . 1 / 30 / 14 : afp decrease to 26 , 343 ng / ml but radiographic progression seen . sorafenib radiation surveillance 2 / 17 / 14 - 3 / 10 / 14 : received 40 gy in 16 fractions . 4 / 21 / 14 - 6 / 20 / 14 : sorafenib restarted at 200 mg twice daily . 
quantitative polymerase chain reaction ( qpcr ) analysis ( a ) and western blot ( b ) conrmed overexpression of wild - type ( wt ) lrrk2 and lrrk2 - k1316n ( mutant ) on four cell lines 72 hours after transfection . 
finally , we characterize the in vitro use of entrectinib against a common alk fusion gatekeeper resistance mutation . methods genomic profiling we performed broad , hybrid capturebased next - generation sequencing using the integrated mutational profiling of actionable cancer targets assay and hisequation 2500 ( illumina , san diego , ca ) .14 alk immunohistochemistry and fluorescence in situ hybridization alk immunohistochemistry was performed using the ventana alk ( d5f3 ) cdx assay ( ventana medical systems , tucson , az )  . 
response , assessed by response evaluation criteria in solid tumors ( recist ) version 1.1 , was monitored by computed tomography ( ct ) imaging performed at baseline , 4 weeks after treatment initiation , and approximately every 8 weeks thereafter.15 treatment was administered until the patient experienced disease progression or unacceptable toxicity . 
permission to publish the patients case was obtained from his health care proxy . generation of engineered ba / f3 cells expressing vcl - alk fusion proteins cdnas that encode vcl - alk and vcl - alk l1196m fusion proteins were inserted into lentiviral vector pvlpuro - ef1a - mcs ( biosettia , san diego , ca )  . 
 transduced ba / f3 cells were selected in puromycin in rpmi 1640 media that contained 10% fetal bovine serum and 10 ng / ml mouse il - 3 ( thermo fisher scientific , waltham , ma ) for 2 weeks . 
 antiproliferative activities of entrectinib and crizotinib in engineered ba / f3 cells results cells seeded at 5 , 000 cells per well in 96 - well plates ( corning , corning , ny ) were treated with serial dilutions of entrectinib ( ignyta , san diego , ca ) and crizotinib ( selleckchem , houston , tx ) added in duplicate to the wells . 
data are averaged from at least two independent experiments and with an r2 of more than 0.9. western blot analysis ten million cells were treated with drug at the indicated concentrations for 2 hours , washed with phosphate - buffered saline , and lysed in 1 radioimmunoprecipitation assay buffer ( emd millipore , billerica , ma ) with benzonase , protease inhibitor , and phosphatase inhibitor cocktails ( emd millipore )  . 
exons 1 to 16 of vcl are fused to exons 20 to 26 of alk , which include the kinase domain . tpm3 eml4 rad51ap2 eml4 22 exons 26 exons exon 1 exon 16 exon 20 - 26 identification of rccs harboring alk fusions a total of 561 samples from 517 patients with rcc were sequenced using broad , hybrid capture based next - generation sequencing ( integrated mutational profiling of actionable cancer targets assay ) .14 , 16 , 17 three ( 0.6% ) of 517 patients with renal cell cancer had tumors that harbored alk fusions . in renal cell cancer , several fusion partners with alk have been previously identified , including vcl , tpm3 , and eml412 , 18 , 19 ( fig 1a )  . 
 the three alk fusions identified in our data set are represented in figure 1b and consist of unclassified - type rcc harboring an vcl - alk fusion developing in a patient with sickle cell trait , chromophobe - type rcc harboring an rad51ap2 - alk fusion , and unclassified - type rcc with an eml4 - alk fusion ( table 1 )  . of these three patients , one was eligible for therapy with a targeted agent against alk , as described below . 
he underwent a right radical nephrectomy , revealing a stage iii ( pt3an1 ) high - grade ( nuclear grade iv ) rcc with rhabdoid and pleomorphic features and nine of 23 positive lymph nodes . 
ct scan 2 months later demonstrated no evidence of recurrence . next - generation sequencing of the primary tumor identified a vcl - alk translocation generating a fusion between exon 16 of vcl and exon 20 of alk ( fig 1c )  . 
clinicopathologic characteristics and therapeutic interventions in patients with alk fusionpositive renal cell carcinoma case age at diagnosis , years pathology fusion additional alterations systemic therapy received vcl - alk none entrectinib renal cell carcinoma , unclassified type renal cell carcinoma , chromophobe type alkrad51ap2 * tert promoter variant pazopanib malt1 h257r nivolumab + bevacizumab nivolumab + lenvatinib lenvatinib + everolimus eml4 - alk tp53 d257y everolimus + bevacizumab renal cell carcinoma , unclassified type erbb4 t194s mst1r f803s dot1l g1358v nivolumab cabozantib abbreviations : alk , anaplastic lymphoma kinase ; f , female ; m ; male . * next - generation sequencing performed on the sarcomatous component of a metastasis . tumor and the age of the patient , hemoglobin electrophoresis was performed , discovering the presence of sickle cell trait.20 entrectinib demonstrates superior in vitro target inhibition and antitumor effect compared with crizotinib twelve months after nephrectomy , surveillance imaging demonstrated confluent lymphadenopathy in the right retrocrural space and an enlarged left superior mediastinal metastatic node . 
given evidence that suggested activity of entrectinib , a multikinase inhibitor of alk , ros1 , trka , trkb , and trkc , against common alk resistance mutations and improved cns penetration compared with crizotinib , the patient enrolled in a clinical trial of entrectinib ( rxdx - 101 ; clinicaltrials.gov identifier : nct02097810 ) .21 four weeks after commencing entrectinib 600 mg per day , a ct scan demonstrated a 31.4% decrease in disease , or partial response . 
the patient maintained excellent performance status throughout treatment , tolerating entrectinib well with the exception of grade 1 peripheral lower extremity edema and grade 2 weight gahis response to entrectinib continued for 19 months , after which imaging revealed radiographic disease progression with increased mediastinal adenopathy , which led to the discontinuation of entrectinib . we interrogated the activity of entrectinib against alk fusions and its common resistance mutations with ba / f3 cell lines that were engineered to overexpress vcl - alk and vclalk l1196m fusion proteins . 
vcl - alk l1196m , a key gatekeeper mutation in the atp binding pocket , confers resistance to crizotinib.22 , 23 for ba / f3 cells that harbor the vcl - alk fusion , entrectinib was more potent than crizotinib ( ic50 = 90.8 nm for entrectinib ; ic50 = 253.0 nm for crizotinib ; fig 3a )  . 
entrectinib and crizotinib both resulted in reduced levels of phospho - alk and the inhibition of phosphorylation of downstream targets , phospholipase c , extracellular signalregulated kinase , and signal transducer and activator of transcription 3 ( fig 3b )  . 
the introduction of the alk l1196m mutation to the vcl - alk construct ( ba / f3 - vcl - alk l1196m ) conferred significantly reduced sensitivity to crizotinib , with an ic50 proliferation value of more than 1 , 000 nm ; however , ba / f3 - vcl - alk l1196m cells remained sensitive to entrectinib , with an ic50 value that was only modestly increased compared with that of ba / f3 - vcl - alk cells ( ic50 = 111.9 nm ; fig 3a )  . 
recently , response to alectinib was reported in patients with metastatic papillary rcc.24 the case detailed here of durable clinical response in a patient with rccadditionally underscores the utility of alk inhibition in alk - rearranged rcc and the potential of targeting alk fusions irrespective of histology . large - scale sequencing efforts have increased the ability to identify alk fusions . 
given their low frequency , single analyte testing for specific alterations is impractical ; however , the increasingly widespread availability of multiplexed next - generation sequencing enhances the feasibility of detecting targetable fusions . 
screening efforts can lead to the detection of other actionable mutations in rcc , such as alterations that involve met , pten , bap1 , mtor , pik3ca , and birc7.25 , 26 clinical outcomes for nonclear - cell rcc ( ncrcc ) remain poor compared with clear - cell rcc . 
despite the small proportion of patients with alk fusion positive rcc , failure to screen in this patient would have prevented the delivery of a well tolerated therapy with a durable response that lasted 19 months . 
 ( b ) treatment of wt ba / f3 vcl - alk and resistant baf3 vcl - alk l1196m cell lines with entrectinib leads to more complete inhibition of downstream targets phosphophospholipase c ( plc ) and phosphosignal transducer and activator of transcription 3 ( stat3 ) compared with crizotinib . 
erk , extracellular regulated kinase . observed in other fusions led to the opening of a multicenter , phase ii basket trial of entrectinib ( clinicaltrials.gov identifier : nct02568267 )  . ultimately , as with this patient , resistance to therapy almost inevitably develops . 
 arcila , hikmat al - ahmadie , gary li , alexander drilon roopal patel employment : ignyta provision of study materials or patients : chung - han lee stock and other ownership interests : ignyta collection and assembly of data : jessica j . 
tao no relationship to disclose ge wei stock and other ownership interests : ignyta , halozyme research funding : ignyta patents , royalties , other intellectual property : ignyta , halozyme travel , accommodations , expenses : ignyta patrick fagan employment : ignyta stock and other ownership interests : ignyta travel , accommodations , expenses : ignyta xueli hao no relationship to disclose julia a . 
arcila consulting or advisory role : astrazeneca travel , accommodations , expenses : astrazeneca , invivoscribe , raindance technologies hikmat al - ahmadie consulting or advisory role : bristol - myers squibb , emd serono chung - han lee consulting or advisory role : exelixis , eisai research funding : pfizer ( inst ) , eisai ( inst ) , bristol - myers squibb ( inst ) , calithera biosciences ( inst ) travel , accommodations , expenses : eisai gary li employment : ignyta alexander drilon consulting or advisory role : ignyta , loxo , tp therapeutics , astrazeneca , pfizer , blueprint medicines , genentech , takeda , helsinn therapeutics , beigene affiliations jessica j . 
arcila , hikmat al - ahmadie , chung - han lee , and alexander drilon , memorial sloan kettering cancer center ; alexander drilon , weill cornell medical center , new york , ny ; ge wei , roopal patel , patrick fagan , and gary li , ignyta , san diego , ca ; xueli hao , st louis pathology associates , mercy hospital , st louis , mo ; and julia a . 
cui jj , tran - dub m , shen h , et al : structure based drug design of crizotinib ( pf - 02341066 ) , a potent and selective dual inhibitor of mesenchymal - epithelial transition factor ( c - met ) kinase and anaplastic lymphoma kinase ( alk )  . 
lin e , li l , guan y , et al : exon array profiling detects eml4 - alk fusion in breast , colorectal , and nonsmall - cell lung cancers . 
sugawara e , togashi y , kuroda n , et al : identification of anaplastic lymphoma kinase fusions in renal cancer : large - scale immunohistochemical screening by the intercalated antibody - enhanced polymer method . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
hyman dm , solit db , arcila me , et al : precision medicine at memorial sloan kettering cancer center : clinical next - generation sequencing enabling next - generation targeted therapy trials . 
debelenko lv , raimondi sc , daw n , et al : renal cell carcinoma with novel vcl - alk fusion : new representative of alk - associated tumor spectrumod pathol 24 : 430 - 442 , 2011 19 . 
smith ne , deyrup at , mario - enriquez a , et al : vcl - alk renal cell carcinoma in children with sickle - cell trait : the eighth sickle - cell nephropathy ? am j surg pathol 38 : 858 - 863 , 2014 21 . 
drilon a , siena s , ou si , et al : safety and antitumor activity of the multitargeted pan - trk , ros1 , and alk inhibitor entrectinib : combined results from two phase i trials ( alka - 372 - 001 and startrk - 1 )  . 
lee j - l , ahn j - h , lim hy , et al : multicenter phase ii study of sunitinib in patients with non clear cell renal cell carcinoma . 
tannir nm , jonasch e , albiges l , et al : everolimus versus sunitinib prospective evaluation in metastatic nonclear cell renal cell carcinoma ( espn ) : a randomized multicenter phase 2 trial . 
ardini e , menichincheri m , banfi p , et al : entrectinib , a pan - trk , ros1 , and alk inhibitor with activity in multiple molecularly defined cancer indications . 
 o detection of circulating tumor dna in patients with leiomyosarcoma with progressive disease purpose leiomyosarcoma ( lms ) is a soft - tissue sarcoma characterized by multiple copy number alterations ( cnas ) and without common recurrent single - nucleotide variants . 
 we evaluated the feasibility of detecting circulating tumor dna ( ctdna ) with next generation sequencing in a cohort of patients with lms whose tumor burden ranged from no evidence of disease to metastatic progressive disease . patients and methods we evaluated cell - free dna in plasma samples and paired genomic dna from resected tumors from patients with lms by ultra - low passage whole genome sequencing . 
sequencing reads were aligned to the human genome and cnas that were identified in cell - free dna and tumor dna by ichorcna software to determine the presence of ctdna . 
clinical data were reviewed to assess disease burden and clinicopathologic features . results we identified lms ctdna in 11 ( 69% ) of 16 patients with disease progression and total tumor burden greater than 5 csixteen patients with stable disease or low disease burden at the time of blood draw were found to have no detectable ctdna . 
ctdna levels declined after resection of progressive disease in one case and became detectable upon disease relapse in another individual patient . conclusion these results suggest that ctdna , assayed by a widely available sequencing approach , may be useful as a biomarker for a subset of patients with uterine and extrauterine lms . 
2018 by american society of clinical oncology introduction leiomyosarcoma ( lms ) is a malignant neoplasm derived from smooth muscle that represents one of the most common subtypes of soft - tissue sarcoma.1 , 2 although the majority of lms cases are sporadic , predisposing factors include li fraumeni syndrome , hereditary retinoblastoma , and radiation exposure.3 , 4 there are no oncogenic single - nucleotide variants ( snvs ) that characterize lms , although loss of tumor suppressors , including tp53 , rb1 , and pten , are commonly observed , as are multiple copy number alterations ( cnas ) .5 - 8 lms is frequently a clinically aggressive disease , and patients are at high risk for local and metastatic relapse after initial complete resection.9 , 10 efforts to improve outcomes for patients would benefit from more reliable indicators of high - risk disease and biomarkers of response to therapy . detection of circulating tumor dna ( ctdna ) has emerged as a new approach for identifying oncogenic mutations , measuring disease burden , clinical prognostication , and assessing tumor response to therapy.11 , 12 most ctdna assays matthew l . 
 have been developed to detect snvs that are highly recurrent in many types of carcinomas.13 although the lack of recurrent snvs in lms limits efforts at targeted sequencing , the numerous cnas that are characteristic of this disease represent an ideal target for detection . 
 a next - generation sequencing approach using ultra - low passage whole - genome sequencing ( ulp - wgs ) can detect cnas in cell - free dna , which indicates the tumor fraction of ctdna present in blood.14 , 15 previous studies have shown that ctdna can be detected in cell - free dna samples that are sequenced at a minimum coverage of 0.1 across the whole genome.14 sequencing for ulp - wgs uses the standard illumina next - generation sequencing platform without modifications or special adaptions ( illumina , san diego , ca )  . 
the ichorcna algorithm is used to detect megabase - scale cnas from ulp - wgs data in which ctdna comprise as little as 3% of the total cell - free dna extracted from a plasma sample.14 in the current study , we evaluated plasma from patients with uterine and extrauterine lms for the presence of ctdna using ulp - wgs . 
paired resected tumors from each patient were also sequenced when available ( 29 of 30 patients ) , which enabled the identification and comparison of cnas between primary tumors and ctdna . 
high - molecular - weight dna contamination of cell - free dna was determined by bioanalyzer ( agilent technologies , santa clara , ca ) and size selection was performed if necessary ( ampure xp beads ; beckman coulter , brea , ca )  . 
up to 40 ng of cell - free dna , 100 ng of dna from fresh frozen tissue , and 200 ng of dna from ffpe tissue were used for kapa hyper library preparation ( kapa biosystems , wilmington , ma )  . 
 after bioruptor sonication ( diagenode ) of formaldehyde - fixed fresh frozen sample , with library preparation using a thruplex dna sequencing kit ( rubicon ) and sequencing on an illumina nextseq 500 . 
this higher sequencing coverage did not alter downstream analyses or data interpretation . sequencing results were demultiplexed , aligned , and processed using picard , burrows - wheeler alignment tool , 17 and genome analysis toolkit.18 , 19 to assess for cnas in tumor and cellfree dna and determine tumor fraction or percentage of ctdna in cell - free dna , we used ichorcna software14 with manual curation of results as necessary to confirm tumor percentages . 
previous studies have demonstrated that this technique can be used to identify and quantitate ctdna that constitutes as little as 3% of the cell - free dna in a sample.14 copy number analysis copy number segments and estimates of tumor fraction and ploidy were generated by ichorcna . 
this analysis was performed separately on tumor resection samples , including the eight samples for which plasma was not available , and on plasma samples that were positive for detectable levels of ctdna . statistical analysis group comparisons between patients with lms with active or indolent disease at the time of plasma collection was performed by nonparametric mann - whitney test , and we performed pearson correlation coefficient between tumor fraction or cell - free dna and tumor burden using graphpad prism ( version 7.0 ; graphpad software , la jolla , ca )  . 
one was added to integer values before log2 transformation to generate non - negative values for data representation . results detection of lms ctdna we retrospectively identified 30 patients who were diagnosed with metastatic lms who had plasma banking performed at variable time points in their treatment history between january 2007 and november 2017 . 
median age at diagnosis was 51 years , most patients were female , the most common primary tumor location was the uterus ( n = 16 ) followed by the retroperitoneum ( n = 8 ) , and most tumors were originally of intermediate or high grade ( table 1 )  . 
twenty - nine of 30 patients had a tumor resection sample available for comparison with cell - free dna . in review of clinical data at the time of plasma banking , the cases could be divided into active disease or indolent disease groups on the basis of tumor volume and evidence of disease progression . 
the active disease group consisted of 16 patients and was defined by having a total tumor burden greater than 5 cm in greatest diameter and progressive disease at the time of blood draw on the basis of imaging or clinical determination . 
in contrast , 16 patients in the indolent disease group had stable disease at the time of blood draw and / or tumor burden less than 5 camong the active disease group , 11 ( 69% ) of 16 patients had detectible ctdna , including patients with both uterine and extrauterine primary tumors . 
none of the samples obtained from the indolent disease group had detectible ctdna ( fig 1a )  . when comparing the amount of ctdna measured as a fraction of total cell - free dna in the plasma sample to the volume of tumor burden reported by computed tomography scan , there was a significant association between higher ctdna levels and increasing tumor burden ( fig 1b )  . 
 abbreviations : a , abdomen ; b , bone ; c , chest ; ctdna , circulating tumor dna ; f , female ; hpf , high - power field ; l , liver ; m , male ; na , not available ( data not reported during pathology review ) ; p , pelvis ; st , soft tissue , including subcutaneous , muscle , and paraspinal locations . cna concordance between lms tumors and ctdna in the 11 plasma samples with detectible levels of ctdna , there was a high concordance of cnas between the tumor sample and ctdna ( figs 2a - 2d ) , with blood collection occurring less than 2 years apart from resection of the matched tumor specimen . 
the active disease group consists of patients with tumors > 5 cm in size and with progressive disease at the time of blood draw as indicated by computed tomography ( ct ) scan or clinical report . 
labels indicate the active disease subgroup ( red ) with tumor size > 5 cm and progressive disease ( n = 16 ) , or indolent disease subgroups ( gray ) with tumor size > 5 cm and stable disease ( n = 2 , diamond ) , tumor size < 5 cm and progressive disease ( n = 4 ; triangle ) , and tumor size < 5 cm and stable disease ( n = 10 ; circle )  . 
overall , recurrent cnas from tumors in this cohort are remarkably similar to those found in prior studies.5 , 8 the most significantly amplified genomic region was found on chromosome 17p , which includes the transcriptional regulator myocd ( copy gained in 20 of 37 tumors )  . 
this gene has previously been reported as significantly amplified in lms and is associated with smooth muscle differentiation.22 additional putative oncogenic and lms associated genes that were recurrently amplified include hdgf in 20 , myc in 14 , cthrc1 in 17 , tnfrsf19 in 10 , and myh2 in 16 of 37 tumors ( fig 3a )  . 
copy gains in these genes were also observed in a portion of cell - free dna samples with detectable ctdna ( myocd in four , hdgf in six , myc in eight , cthrc1 in nine , tnfrsf19 in three , and myh2 in four of 11 ctdna - positive samples ) ; however , the number of evaluable samples was too small for any copy gains of these genes to reach statistical significance ( fig 3b )  . 
from tumor samples , recurrent deletions in tumor suppressors were found , which are characteristic of lms , 5 , 8 , 23 including pten deletions in 27 , rb1 in 27 , and tp53 in 11 of 37 tumors profiled ( fig 3c )  . 
deletions that involved these genes were also detected in ctdna ( pten in eight , rb1 in six , and tp53 in two of 11 ctdna - positive samples ) , but sample size was too small for many of these deletions to reach significance ( fig 3d )  . 
 thus , ulp - wgs is capable of detecting recurrent cnas characteristic of lms , and these methods may be helpful in identifying genelevel cnas in ctdna . ctdna longitudinally correlates with disease status to determine whether ctdna levels change in agreement with longitudinal disease status , we evaluated serial plasma samples in two patients after either resection of localized lms or with disease recurrence after surgery . 
in contrast , in the patient with disease recurrence 32 months after surgical resection , cell - free dna tumor fraction increased from undetectable postoperatively to detectable at the time of disease recurrence ( fig 4b )  . 
 ( a - e ) copy number plots generated from ultra - low passage whole - genome sequencing of five representative tumor surgical ( left panels ) and plasma ( right panels ) sample pairs . 
tumor fraction is indicated for each plot . discussion in the current study , we analyzed cell - free dna from the plasma of patients with lms for evidence of ctdna . 
a significant association between tumor burden and the amount of ctdna was identified , which suggests that larger and actively growing tumors are more likely to release tumor dna into circulation . 
 ( a - d ) gistic2.0 analysis identifying recurrent focal amplified ( a and b ) and deleted ( c and d ) regions in lms tumors ( n = 37 ; left panels ) and cell - free dna samples with detectable circulating tumor dna ( n = 11 ; right panels )  . 
the green line indicates an fdr of 0.25. tumor cell - free dna amplifications tnfrsf19 myocd myh2 amplifications cthrc1 6 7 8 9 10 chromosome 12 14 16 18 20 x 12 14 16 18 20 6 7 8 9 10 chromosome deletions hdlbp deletions cdkn2aip brca2 tp53 pten tsc1 ypel3 12 14 16 18 20 x 12 14 16 18 20 x 6 7 8 9 10 chromosome 6 7 8 9 10 chromosome 10 - 1 0.25 10 - 2 10 - 1 0.25 less than 5 cm in size , regardless of evidence of disease progression , which may represent a technologic sensitivity threshold . 
we further found that ctdna levels decline with resection of disease and increase with disease recurrence in individual patients , which indicates that these methods may be useful for supporting a diagnosis of disease recurrence or response to therapy . sequencing of snvs in cell - free dna has proven clinical utility in detecting resistance mutations to targeted therapies and directing treatment strategies.24 emerging use of whole - genome sequencing to identify and quantify cnas from tumor - derived dna circulating in patients with cancer has been evaluated in a number of cancer types.14 , 25 - 27 this noninvasive approach can be useful in the genomic characterization of malignancy and provide diagnostic and prognostic information relevant for clinical care.15 , 21 , 28 in a recent study of patients with lms , a highly customized lms - specific assay ( cancer personalized profiling by deep sequencing , or cappseq ) was designed to detect selected snvs and combined with cna analysis to identify and quantify ctdna in patients with progressive disease.29 in this study , the investigators found that approximately 20% of patients ( two of nine ) had tumors that did not have somatic events that were detectable by the assay . 
in contrast , we found that tumors from 100% of cases had cnas that were detectable by ulp - wgs , which suggests that ulp - wgs may be more broadly applicable for patients with lms . 
 the interval between surgical date and cell - free dna collection is indicated . in six of seven baseline samples of patients who were eligible for study ( 86% ) and had a sensitivity of 68% among all samples tested from their cohort . 
although these numbers are small , this suggests that capp - seq may have a higher sensitivity for ctdna , which might be expected given the deep sequencing coverage used in this assay . 
for example , our recent study in osteosarcoma demonstrated that pretreatment ctdna levels detected by ulpwgs are prognostic , 28 whereas a more sensitive assay , such as capp - seq , may be advantageous in the setting of disease surveillance.29 there are several potential clinical uses of ctdna in the diagnosis and management of patients with lms . 
crompton strategies for uterine tumors that could potentially harbor lms.32 second , there is significant clinical uncertainty regarding which patients with lms derive benefit from adjuvant chemotherapy and radiation.10 , 33 , 34 should ctdna levels bear prognostic significance for tumors that are at highest risk of recurrence or identify the presence of residual disease , their measurement may help guide clinical decisions regarding adjuvant therapies . 
finally , ctdna levels may be a useful indicator of response to systemic therapy and provide an early indication for switching or intensifying treatment regimens used in this disease.3 , 35 together with technologic improvements in sensitivity and throughput , these initial reports that identify ctdna in lms may quickly evolve to transform clinical practice . 
andersen no relationship to discloses edwin thai no relationship to discloses suzanne george stock and other ownership interests : abbott laboratories , abbvie ( i ) , allergan ( i ) consulting or advisory role : blueprint medicines , deciphera , astrazeneca , blueprint medicines , bayer , eli lilly research funding : pfizer ( inst ) , novartis ( inst ) , bayer ( inst ) , ariad pharmaceuticals ( inst ) , blueprint medicines ( inst ) , deciphera ( inst ) patents , royalties , other intellectual property : uptodate expert testimony : bayer other relationship : research to practice brian d . 
crompton , broad institute of massachusetts institute of technology and harvard , cambridge , ma ; and gavin ha , fred hutchinson cancer research center , seattle , wa . supported by the american society of oncology conquer cancer foundation young investigator award ( m.l.h. ) , national institutes of health grant no . 
toro jr , travis lb , wu hj , et al : incidence patterns of soft tissue sarcomas , regardless of primary site , in the surveillance , epidemiology and end results program , 1978 - 2001 : an analysis of 26 , 758 cases . 
ducimetire f , lurkin a , ranchre - vince d , et al : incidence of sarcoma histotypes and molecular subtypes in a prospective epidemiological study with central pathology review and molecular testing . 
hensley ml , ishill n , soslow r , et al : adjuvant gemcitabine plus docetaxel for completely resected stages i - iv high grade uterine leiomyosarcoma : results of a prospective study . 
oxnard gr , paweletz cp , kuang y , et al : noninvasive detection of response and resistance in egfr - mutant lung cancer using quantitative next - generation genotyping of cell - free plasma dna . 
klega k , imamovic - tuco a , ha g , et al : detection of somatic structural variants enables quantification and characterization of circulating tumor dna in children with solid tumors . 
mermel ch , schumacher se , hill b , et al : gistic2.0 facilitates sensitive and confident localization of the targets of focal somatic copy - number alteration in human cancers . 
stover dg , parsons ha , ha g , et al : association of cell - free dna tumor fraction and somatic copy number alterations with survival in metastatic triple - negative breast cancer . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced nonsmall - cell lung cancer . 
van roy n , van der linden m , menten b , et al : shallow whole genome sequencing on circulating cell - free dna allows reliable noninvasive copy - number profiling in neuroblastoma patients . 
heitzer e , ulz p , belic j , et al : tumor - associated copy number changes in the circulation of patients with prostate cancer identified through whole - genome sequencing . 
shulman ds , klega k , imamovic - tuco a , et al : detection of circulating tumour dna is associated with inferior outcomes in ewing sarcoma and osteosarcoma : a report from the childrens oncology group . 
hensley ml , wathen jk , maki rg , et al : adjuvant therapy for high - grade , uterus - limited leiomyosarcoma : results of a phase 2 trial ( sarc 005 )  . 
reed ns , mangioni c , malmstrm h , et al : phase iii randomised study to evaluate the role of adjuvant pelvic radiotherapy in the treatment of uterine sarcomas stages i and ii : an european organisation for research and treatment of cancer gynaecological cancer group study ( protocol 55874 )  . 
bowles , md2 , 3 introduction fibroblast growth factor receptors ( fgfrs ) 1 through 4 are transmembrane tyrosine kinase receptors that play a key role in cell survival , differentiation , migration , angiogenesis , and carcinogenesis.1 uncontrolled activation of fgfr signaling has been described in almost every type of malignancy and typically is the result of amplication or activating mutation affecting the fgfr genes.2 these aberrations can one of function as cancer drivers but also may inuence prognosis and sensitivity to treatment.3 - 5 only two fgfr - specic inhibitors , erdatinib and pemigatinib , have been approved by the us food and drug administration , though a number of multikinase kinase inhibitors ( mkis ) have some degree of fgfr2 inhibiting properties . although fgfr1 and fgfr3 mutations are more frequent overall , activation of fgfr2 is common in patients with endometrial adenocarcinoma , cholangiocarcinoma , gastric adenocarcinoma , and ameloblastoma , a rare and difcult - to - treat odontogenic tumor.2 , 6 - 9 here , we present the case of a patient with ameloblastoma harboring an activating fgfr2 y375c mutation who had signicant tumor regression in response to treatment with lenvatinib . 
a specimen from a repeated biopsy was sent for nextgeneration sequencing using cancerselect 125 ( personal genome diagnostics , baltimore , md )  . analysis revealed clonal fgfr2 y375c , an activating mutation in the transmembrane domain causing constitutive receptor dimerization through the formation of intermolecular disulde bonds.12 although fgfr2 aberrations are common in ameloblastoma , this point mutation appears not to have been identied previously . 
first characterized in bearestevenson cutis gyrate syndrome , y375c is rare in cancers but has been described in adenoid cystic carcinoma and endometrial carcinoma.13 , 14 a second potential driver mutation affecting the hedgehog signaling pathway , smo l412f , was also found at a lower allele fraction . 
frequently seen in ameloblastoma as well as basal cell carcinoma , medulloblastoma , and a subset of meningiomas , this activating mutation alters the drug - binding pocket of smo , conferring resistance to smo inhibitors such as vismodegib and itraconazole.6 a third mutation , palb2 h786y , was reported but is of unclear signicance , having never been associated with human disease . 
although sensitivity to fgfr inhibition varies by type of mutation , drug - binding site , and degree of fgfr specicity , 15 , 16 cancer cell lines harboring fgfr2 y375c mutations were previously found to be responsive to nonselective pan - fgfr inhibition in vitro.17 because the fgfr - specic inhibitors were not yet commercially available , lenvatinib was selected because of its favorable half maximal inhibitory concentration for fgfr2 ( 27 nmol / l ) and efcacy in other malignancies . the patient started receiving the standard dose for radioactive - iodine refractory thyroid cancer of 24 mg daily . his dose was reduced to 20 mg daily after 1 month , then 20 mg alternating with 10 mg daily after an additional month . 
he experienced typical adverse effects of grade 2 hypertension , grade 2 hypothyroidism , grade 1 diarrhea , and grade 2 weight loss . on follow - up imaging at 6 months , he experienced a partial response with 40% shrinkage , per recist , version 1.1 ( fig 1 )  . 
as of this writing , the patient has been receiving this treatment for 13 months and has a continued partial response . discussion ameloblastoma is a locally invasive , typically benign distransformation with ease that can undergo malignant regional or distant metastasis , sometimes occurring decades after initial presentation.18 - 23 surgery is the mainstay of treatment , although conservative enucleation and curettage are associated with recurrence rates as high as 60% to 90%.24 radical surgery with wide en bloc resection improves these rates but at the cost of disgurement and high morbidity . 
overall , guidance regarding use of adjuvant treatment modalities is lacking . given the dearth of effective and tolerable treatment options , reports of targetable mutations dominating the genetic landscape of ameloblastoma were met with great interest.6 - 8 a large percentage of ameloblastomas , especially those arising from the mandible , have braf v600e mutation . 
ameloblastomas harboring braf v600e mutation are sensitive to braf inhibition in vitro at clinically relevant drug concentrations.6 multiple case reports have demonstrated durable clinical response to braf inhibition or combined braf / mek inhibition , on the basis of braf status.26 - 30 a second , and typically mutually exclusive , molecular phenotype featured mutations in fgfr2 , including c382r and v395d in the transmembrane domain and n549k in the kinase domatogether , braf and fgfr2 mutations are present in approximately 80% of ameloblastomas , with additional somatic mutations in smo , cttnb1 , pik3ca , and smarcb1 . interestingly , the patient we report on here had nearly 90% allele frequency of fgfr2 y375c , suggesting this may have been the initial truncal mutation . 
the implications of clonality for fgfr point mutations and fusions are not well understood , but truncal amplication of fgfr2 is an important predictor of drug response in gastric cancer.31 in a small trial of the selective fgfr inhibitor azd4547 in fgfr2 - amplied gastroesophageal cancer , robust , singleagent clinical response was restricted to high copy - number amplication events with associated high expression of fgfr2 mrna and fgfr2 protethe mechanism for this fig 1 . 
 case report effect was thought to be transactivation of alternate receptor tyrosine kinases resulting in a distinct oncogene addiction phenotype characterized by fgfr - dependent phosphatidylinositol - 3 - kinase and mammalian target of rapamycin signaling . although prospective trials in ameloblastoma continue to insight into be limited by low case numbers , additional treatment of this disease may be drawn from other , molecularly similar cancer types , including endometrial and gastric adenocarcinomas . 
fgfr2 mutations occur in approximately 10% of both tumor types and are mutually exclusive , with kras mutations suggesting a shared signaling mechanism.9 , 32 , 33 in vitro data from endometrial cancer conrmed fgfr2 activation results in increased mapk signaling that is abrogated by fgfr inhibition.34 , 35 these preclinical data prompted a phase ii trial of lenvatinib in advanced endometrial cancer , which reported an objective response rate of 14% and a clinical benet rate of 37% without selection for fgfr status.36 conrmatory trials of fgfr inhibition in endometrial cancer , both alone and in combination with pembrolizumab or other checkpoint blockade , are ongoing.37 subgroup analysis of biomarkerselected populations in studies like these may be a helpful surrogate for utility in rare cancers such as ameloblastoma . complicating the generalization of fgfr inhibitor trial data are the multiple mechanisms of fgfr dysregulation . 
although studies typically lump together patients with any fgfr aberration , the type of mutationfgfr overexpression including gene amplication , increased ligandbinding afnity or ligand expression , or ligand - independent receptor activationresults in differential effects on fgfr signaling . 
using mkis as an example , brivanib primarily affects cells with fgfr1 amplication , dovitinib inhibits fgfr1 - 3 kinase activity in a predominately liganddependent fashion , and ponatinib targets fgfr1 - 4 with three patterns of signaling alteration we have described.17 although the exibility of some nonselective mkis in targeting multiple fgfr aberrations may broaden their use , it comes at the cost of increased toxicity . 
in contrast , early data from selective fgfr tkis demonstrated a favorable adverse effect prole but more limited efcacy.38 , 39 characterizing drug - susceptibility phenotypes will be crucial for the continued development of these agents and other approaches to fgfr inhibition , including monoclonal antibody or peptide inhibitors and fgf ligand traps . although lenvatinib was chosen in this case to target fgfr2 , it is possible the patients response was due to inhibition of other targets , such as vascular endothelial growth factor receptors , platelet - derived growth factors , or kit . 
assessment of phosphorylated - fgfr2 by immunohistochemistry before and after therapy could clarify if his response was due to on - target or off - target effects , but he declined a repeated biopsy procedure . 
lenvatinib has been effective in thyroid , renal , and hepatocellular carcinomas without identiable mutation or overexpression of these kinases , suggesting the mechanism underlying drug sensitivity is incompletely understood.37 most clinical trials of lenvatinib have been in solid tumor types selected on the basis of response to other inhibitors of angiogenesis , but these agents have not been investigated in ameloblastoma . more studies are needed to clarify the role of fgf signaling in drug response and identify predictive biomarkers for lenvatinib . 
clin cancer res 22 : 259 - 267 , 2016 donnem t , al - shibli k , al - saad s , et al : prognostic impact of broblast growth factor 2 in non - small cell lung cancer : coexpression with vegfr - 3 and pdgf - b predicts poor survival . 
j thorac oncol 4 : 578 - 585 , 2009 turner n , pearson a , sharpe r , et al : fgfr1 amplication drives endocrine therapy resistance and is a therapeutic target in breast cancer . 
ware ke , hinz tk , kleczko e , et al : a mechanism of resistance to getinib mediated by cellular reprogramming and the acquisition of an fgf2 - fgfr1 autocrine growth loop . 
nat genet 46 : 722 - 725 , 2014 [ erratum : nat genet 47 : 97 , 2015 ] kurppa kj , cat on j , morgan pr , et al : high frequency of braf v600e mutations in ameloblastoma . 
j pathol 232 : 492 - 498 , 2014 brown na , rolland d , mchugh jb , et al : activating fgfr2 - ras - braf mutations in ameloblastoma . 
clin cancer res 20 : 5517 - 5526 , 2014 dienstmann r , rodon j , prat a , et al : genomic aberrations in the fgfr pathway : opportunities for targeted therapies in solid tumors . 
byron sa , chen h , wortmann a , et al : the n550k / h mutations in fgfr2 confer differential resistance to pd173074 , dovitinib , and ponatinib atp - competitive 17 . 
gozgit jm , wong mj , moran l , et al : ponatinib ( ap24534 ) , a multitargeted pan - fgfr inhibitor with activity in multiple fgfr - amplied or mutated cancer 18 . 
brunet m , khalifa e , italiano a : enabling precision medicine for rare head and neck tumors : the example of braf / mek targeting in patients with metastatic 27 . 
faden dl , algazi a : durable treatment of ameloblastoma with single agent brafi re : clinical and radiographic response with combined braf - targeted therapy in stage 4 ameloblastoma . 
byron sa , gartside m powell ma , et al : fgfr2 point mutations in 466 endometrioid endometrial tumors : relationship with msi , kras , pik3ca , ctnnb1 mutations and clinicopathological features . 
plos one 7 : e30801 , 2012 [ erratum : plos one 7 , 2012 doi : 10.1371 / annotation / 0bfaecca - 0f87 - 43fe - 97ccf2ae3ddeb6d5 ] 33 . 
konecny ge , kolarova t , obrien na , et al : activity of the broblast growth factor receptor inhibitors dovitinib ( tki258 ) and nvp - bgj398 in human endometrial cancer cells . 
chae yk , ranganath k , hammerman ps , et al : inhibition of the broblast growth factor receptor ( fgfr ) pathway : the current landscape and barriers to clinical application . 
differences in the relative fractions of immune cell subpopulations between immune - rich er - positive and tnbcs3 suggested disparate immunotherapy strategies in the two immune - rich subsets . concerning the study of tnbc immune microenvironment , we recently observed the existence of at least three immune - clusters ( im ) in tnbcs by computing transcriptional - based deconvolution algorithms.4 ima , considered hot or t cell - inamed , shows greater enrichment of cytotoxic t cells , inferred by deconvolutions tools or by histologic til counts , and accounts for approximately 30% of the tnbcs analyzed . 
finally , imc was described as warm tumors , because intrinsic immune activities are effectively mounted , but tumors are able to escape and suppress such responses , as indicated by the greater abundance of tregs and the lower expression of pdl - 1 and pd - 1 compared with ima . 
on the basis of our ndings , we would like to comment that besides the heterogeneity of tnbcs in terms of immune - cell inltration , the immune - rich subtype represents immune - related transcriptional heterogeneity , with signicant implications for potential immunotherapeutic strategies . consistent with the genomic characterization of immune - cell inltration obtained in our tnbc cohort , the incidence of immune - enriched ima in tcga and metabric data sets does not exceed 30% ( fig 1a ) , supporting the reproducibility of our immune clusters , especially for hot tumors . 
 ( a ) proportion of immuno - clusters ( im ) previously reported in romero et al4 with the ital - mex , molecular taxonomy of breast cancer international consortium ( metabric ) , and the cancer genome atlas ( tcga ) datasets . 
 ( b ) donut plot of the percentage of immuno - clusters among tumors classied as immune - rich or non - immune - rich from the tcga and metbric cohorts . 
by applying cluster analysis of our immune clusters to tcga and metabric data sets , we found that immune - rich tnbcs ( high til metagene described by omeara et al1 ) are composed mainly of ima ( n = 160 , 62% ) , followed by imc ( n = 54 , 21% ; fig 1b ) , with cases categorized as imb ( n = 44 , 17% ) also present . 
when considering the distribution of high til metagenes ( top 25th percentile ) applied by omeara et al1 in our cohort ( ital - mex , n = 158 ) and classied according to immune - related clusters , we conrmed that immune - rich cases ( ima ) are almost exclusively integrated by high til metagene tumors ( n = 41 , 94% )  . however , we also observed that a relevant percentage of high til cases are also distributed among imc ( n = 17 , 32% ) and less represented in the cold imb immune cluster ( n = 11 , 18% ; fig 1c )  . 
omeara t , marczyk m , qing t , et al : immunological differences between immune - rich estrogen receptor - positive and immune - rich triple - negative breast cancers . 
 identifying erbb2 activating mutations in her2 - negative breast cancer : clinical impact of institute - wide genomic testing and enrollment in matched therapy trials pedro exman , md1 ; ana c . 
our primary aim was to describe the proportion of patients with a qualifying erbb2 mutation identied by our institutional genomic panel ( oncomap or oncopanel ) who enrolled in the trial . 
associations were calculated using fishers exact test . results we identied a total of 1 , 045 patients with metastatic breast cancer without erbb2 amplication who had available genomic testing results . 
of these , 42 patients were found to have erbb2 mutation and 19 patients ( 1.8% ) were eligible for the trial on the basis of the presence of an activating mutation , 18 of which were identied by oncopanel testing . 
fifty - eight percent of potentially eligible patients were approached , and 33.3% of eligible patients enrolled in the trial guided exclusively by oncopanel testing . conclusion more than one half of eligible patients were approached for trial participation and , signicantly , one third of those were enrolled in nct01670877 . 
 exman et al context key objective although tumor genomic sequencing has become more accessible , bridging the gap between patients with targetable mutations and recruitment to specic trials remains a challenge . 
this phase ii study has been active at dfci since 2013 , with consistent slot availability during this time . patients we included 1 , 817 patients in the profile cohort with mbc who had available genomic proling results between august 1 , 2011 , and june 1 , 2017 . 
from the ccpm cohort , we included patients with metastatic her2 - negative ( per ihc and / or fish ) breast cancer in whom targeted genomic panel results were available between june 22 , 2015 , and june we collected data on those patients who consented to be in the profile cohort and who had available oncomap or oncopanel . 
clinical data abstraction was performed via medical record review and included age and stage at initial diagnosis , date of recurrence , histologic features , and date of death or last follow - up . 
clinical her2 status was dened by the 2013 asco / college of american pathologists guidelines.7 survival data were collected through a combination of medical record review and linkage to the national death index . 
all data are stored in a custom redcap database . genomic analysis genomic testing was performed in a clia - certied environment within the center for advanced molecular diagnostics at brigham and womens hospital . 
 excluded patients with her2 - positive disease ( n = 772 ) profile cohort patients with mbc with at least one dfci visit and available oncopanel results between 2011 and 2017 ( n = 1 , 817 ) patients with er - positive / her2negative mbc with oncopanel result ( n = 1 , 045 ) patients with erbb2 mutation previously detected by other genomic test modality ( n = 1 ) erbb2 activating mutations in her2 metastatic breast cancer ccpm cohort patients with mbc with at least one dfci visit and available oncopanel results between 2015 and 2017 ( n = 194 ) excluded patients with her2 - positive disease ( n = 28 ) patients with her2negative mbc with oncopanel result ( n = 166 ) patients with erbb2 mutations ( n = 42 ) patients with eligible erbb2 mutations diagnosed by profile ( n = 18 ) patients approached by trial team after oncopanel result ( n = 11 ) patients approached by trial team after ccpm result ( n = 1 ) patients with eligible erbb2 mutations diagnosed by ccpm ( n = 6 ) patients with erbb2 mutations ( n = 10 ) patients enrolled on the basis of oncopanel results ( n = 6 ) patients enrolled on the basis of ccpm result ( n = 1 ) fig 1 . 
dfci , danafarber cancer institute ; er , estrogen receptor ; her2 , human epidermal growth factor receptor 2 ; mbc , metastatic breast cancer . muther trial this multi - institutional phase ii clinical trial ( clinicaltrials.gov identier : nct 01670877 ) evaluates the efcacy of neratinib in patients with her2 - negative ( per ihc and / or fish ) , erbb2 - mutant mbc . 
the study opened to accrual at dfci on september 20 , 2013 , and it remains open as of december 18 , 2018 . trial matching in 2016 , we instituted several programs , such as the ending metastatic breast cancer for everyone ( embrace ) program for patients with mbc , 11 the matchminer platform , 12 and an interactive web - based trial - matching tool13 ( data supplement ) to assist physicians in trial selection . 
through these programs , key data from oncopanel ( derived either from the profile or ccpm protocols ) , as well as results from other research testing conducted in a clia - environment , were collated into a central database . the data supplement presents details of the embrace prograa custom report ( data supplement ) was shared with the medical oncologist by a designated research coordinator at the time of a patients visit to facilitate realtime trial matching . 
in addition , a custom query for erbb2 alterations was created within the matchminer platform and allowed for the identication of all patients with breast cancer with an erbb2 alteration at dfci . 
this list was used to reach out directly to treating physicians as an extra outreach method to capture patients who might not have a return visit to dfci scheduled . statistical analysis the primary objective of our study was to describe the proportion of patients in the prespecied profile cohort with eligible erbb2 mutations detected by genomic analysis who enrolled in the selected trial . 
secondary objectives included describing the proportion of patients approached for trial screening , the median time from genomic testing results to trial enrollment , the median time from genomic testing to death , and overall survival ( os )  . 
os was dened as the time from diagnosis of metastatic disease ( by imaging or biopsy ) to death from any cause and was estimated using the kaplan - meier method . 
between august 2011 and june 2017 , 1 , 045 patients with her2 - negative mbc consented to the profile protocol and had successful genomic testing . oncopanel was performed in most of the patients of this cohort ( 98.8% , n = 1 , 032 of 1 , 045 )  . 
forty - two patients ( 4.0% ) were found to have erbb2 mutations in their tumors , and of these , 19 ( 1.8% ) had mutations eligible for the trial ( 18 detected by oncopanel , none by oncomap , and one by commercial panel )  . 
between june 2015 and june 2017 , 166 patients with her2 - negative mbc underwent successful oncopanel testing of a fresh tumor biopsy as part of the ccpm initiative ( fig 1 )  . 
in terms of our prespecied primary end point , the proportion of patients with eligible erbb2 mutations who enrolled in the selected neratinib trial on the basis of only oncopanel results was 33.3% ( six of 18 ) , representing 0.5% of the total tested population ( six of 1 , 045 )  . 
one additional patient with a previously known erbb2 mutation detected through commercial testing enrolled in the neratinib trial ; another patient enrolled in the trial on the basis of testing of a fresh metastatic specimen collected in the ccpm protocol ( fig 1 )  . all patients who enrolled in the trial had other multiple mutations detected by oncopanel ( data supplement ) , but none received additional treatment guided by another specic mutation . 
for patients with eligible erbb2 mutations , the median os was not reached ( data supplement )  . among 1 , 045 patients with her2 - negative mbc with available genomic results between 2011 and 2017 and included in our profile cohort , we identied a total of 19 patients with erbb2 mutations that rendered them eligible for the neratinib trial . 
although the total number of enrolled patients ( n = 8 ) at dfci was low , our site was the second - highest accruing site nationally , reecting the low overall prevalence of erbb2 mutations in breast cancer . we observed that the conversion rate from genomic result to trial enrollment was numerically higher in the profile cohort compared with the ccpm cohort , although the patients in the ccpm cohort were not approached because they had stable disease on current therapy . 
patients who enrolled in the trial had received a median of four ( range , one to six ) lines of systemic treatment in the metastatic ( mets ) setting before oncopanel results were available . 
in addition , 75% ( six of eight ) of the registered patients were enrolled after the rst progression after the oncopanel result was available and 25% ( two of eight ) after the second progression . 
 erbb2 activating mutations in her2 metastatic breast cancer mutated and eligible mutated not mutated test technical lack of an adequate archival sample , insufcient archival material , or failure ( the last being uncommon )  . 
because patients were not randomly assigned to undergo testing , we cannot denitively comment on the impact of enterprise - wide testing versus an approach of more selective testing at the time of clinical need . 
the potential for referral bias , given the long median interval between initial metastatic diagnosis and oncopanel testing , and the possibility that only more t patients were able to travel to our site later in their disease course , could have diluted differences in survival by erbb2 mutation status . 
in addition , 20% of patients with eligible mutations were lost to follow - up . the rarity of the patient population meant that only 0.5% of the total tested population ultimately enrolled in the neratinib study . 
it is notable that the median time from metastatic diagnosis to oncopanel result in mutated patients was 7.0 months , and in large part , this reected patients being referred to our institution after having lived with a diagnosis of mbc for some time before their initial visit . our data illustrate the hurdles involved in identifying and enrolling patients with rare molecular alterations in clinical trials . 
we believe that our data suggest that broad - based testing , the development of systems with the ability to query genomic testing results across a clinic population , and robust strategies to approach patients will be critical to the success of studies enrolling rare subsets . 
1 , 000 women to enroll eight patients in the selected trial ; however , this testing simultaneously identied patients for multiple other trials , in a tissueand timeefcient manner . one half of the patients enrolled in the neratinib trial were also potentially eligible for other simultaneous trials on the basis of targetable mutations such as in palb2 or pik3ca ( data supplement )  . 
a direct assessment of the drug activity of neratinib ( with or without fulvestrant ) will be performed in the muther trial , and progression - free survival will be evaluated as a secondary end point.17 a strength of our study is the selection of a specic clinical trial with wide slot availability over an extended period to evaluate the conversion rate from biomarker result to trial enrollment . 
a slightly lower conversion rate was observed by wheler et al , 5 who prospectively evaluated 500 patients ( only 16% with breast cancer ) ; 22% of them received directly guided treatment after tumor molecular proling . 
meric - bernstam et al18 evaluated 2 , 000 patients with different cancer types ; 7% of patients with potentially actionable alterations were enrolled in a specic mutation - driven trial , and 4% were treated in a trial with a molecular rationale . 
the safir - 01 study enrolled 423 patients with advanced breast cancer , and 13% of them were considered eligible for a mutationguided clinical trial.3 our study has several limitations . 
although all new patients to dfci were approached for participation in the overarching protocol allowing for tumor testing , we did not prospectively collect the decline rate , nor did we collect reasons for decline . 
not all consented patients with mbc underwent tumor testing , for a variety of reasons , including affiliations 1dana - farber cancer institute , boston , ma 2washington university school of medicine , st . 
freedman research funding : puma biotechnology ( inst ) , eisai ( inst ) lorenzo trippa consulting or advisory role : galera therapeutics romualdo barroso - sousa consulting or advisory role : lilly speakers bureau : roche , bristol - myers squibb travel , accommodations , expenses : roche simona di lascio honoraria : roche / genentech ethan cerami stock and other ownership interests : blueprint medicines honoraria : merck travel , accommodations , expenses : merck margaret s . 
tolaney consulting or advisory role : novartis , pzer , merck , lilly , nektar , nanostring technologies , astrazeneca , puma biotechnology , genentech , eisai , immunomedics , sano , celldex , bristol - myers squibb , paxman , seattle genetics research funding : genentech / roche ( inst ) , merck ( inst ) , exelixis ( inst ) , pzer ( inst ) , lilly ( inst ) , novartis ( inst ) , bristol - myers squibb ( inst ) , eisai ( inst ) , astrazeneca ( inst ) , nanostring technologies ( inst ) , cyclacel ( inst ) , nektar ( inst ) , immunomedics ( inst ) travel , accommodations , expenses : astrazeneca , lilly , merck , nektar , novartis , pzer , genentech / roche , immunomedics , eisai , nanostring technologies , puma biotechnology , celldex ian e . 
krop employment : amag pharmaceuticals ( i ) leadership : amag pharmaceuticals ( i ) stock and other ownership interests : amag pharmaceuticals ( i ) honoraria : genentech / roche consulting or advisory role : genentech / roche , daiichi sankyo , context therapeutics , macrogenics , taiho pharmaceutical research funding : genentech / roche ( inst ) , seattle genetics ( inst ) , pzer ( inst ) , daiichi sankyo ( inst ) ron bose honoraria : genentech , foundation medicine consulting or advisory role : genentech research funding : puma biotechnology ( inst ) bruce e . 
johnson research funding : novartis ( inst ) , toshiba ( inst ) , novartis ( inst ) , novartis ( inst ) patents , royalties , other intellectual property : dana - farber cancer institute cynthia x . 
ma consulting or advisory role : pzer , novartis , syndax , lilly , biovica , tempus , seattle genetics , agendia , myriad genetics , lilly research funding : pzer ( inst ) , eisai ( inst ) , puma ( inst ) travel , accommodations , expenses : syndax , lilly , agendia , pzer deborah a . 
winer stock and other ownership interests : verastem honoraria : genentech / roche , tesaro , genomic health consulting or advisory role : leap therapeutics , seattle genetics , jounce therapeutics , glaxosmithkline , carrick therapeutics , lilly research funding : genentech ( inst ) , novartis ( inst ) , merck ( inst ) nikhil wagle stock and other ownership interests : foundation medicine honoraria : lilly consulting or advisory role : novartis , lilly research funding : novartis , puma biotechnology nancy u . 
 erbb2 activating mutations in her2 metastatic breast cancer acknowledgment we thank kaitlyn bifolck for her editorial support . references 5 , 2010 ] andre f , mardis e , salm m , et al : prioritizing targets for precision cancer medicine . 
gynecol oncol 148 : 585 - 590 , 2018 andr e f , bachelot t , commo f , et al : comparative genomic hybridisation array and dna sequencing to direct treatment of metastatic breast cancer : a multicentre , prospective trial ( safir01 / unicancer )  . 
lancet oncol 15 : 267 - 274 , 2014 schwaederle m , zhao m , lee jj , et al : impact of precision medicine in diverse cancers : a meta - analysis of phase ii clinical trials . 
cancer res 76 : 3690 - 3701 , 2016 sholl lm , do k , shivdasani p , et al : institutional implementation of clinical tumor proling on an unselected cancer population . 
wolff ac , hammond me , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
plos one 4 : e7887 , 2009 [ erratum : plos one garcia ep , minkovsky a , jia y , et al : validation of oncopanel : a targeted next - generation sequencing assay for the detection of somatic variants in cancer . 
ramkissoon sh , bandopadhayay p , hwang j , et al : clinical targeted exome - based sequencing in combination with genome - wide copy number proling : precision medicine analysis of 203 pediatric brain tumors . 
hughes me , frank es , merrill ms , et al : embrace ( ending metastatic breast cancer for everyone ) : a comprehensive approach to improve the care of patients with metastatic breast cancer . 
lindsay j , del vecchio fitz c , zwiesler z , et al : matchminer : an open source computational platform for real - time matching of cancer patients to precision medicine clinical trials using genomic and clinical criteria . 
collins jr , md1 a 61 - year - old man was diagnosed with acute myeloid leukemia ( aml ) associated with trisomy 8 cytogenetic abnormality and flt3 itd mutation . 
post - transplantation , he showed no morphologic or ow cytometric evidence of disease and his bone marrow chimerism was more than 95% donor cells at several timepoints post - transplantation , including 24 months after the transplant . 
he received tacrolimus post - transplantation and developed mild chronic graft - versus - host disease that was limited to the skthe patient remained on tacrolimus . twenty - nine months after transplantation , he presented with abdominal pacomputed tomography scan of the abdomen and pelvis revealed a known , unchanged ventral abdominal hernia , but also showed a new lytic lesion on the inferior pubic ramus that was concerning for a new malignancy ( fig 1a )  . 
flow cytometry of the lesion demonstrated a predominance of cd117bright positive , cd34 - negative myeloid cells suspected to be mast cells that did not aberrantly express cd2 or cd25 ( fig 3 )  . 
these cells showed atypical expression of fcri ( cd64 ) , which is seen upon mast cell activation . immunohistochemical analysis demonstrated that the cells were positive for cd117 and weakly positive for mast cell tryptase and negative for cd2 , cd25 , and cd34 ( fig 4 )  . 
altogether , supportive of a diagnosis of mast cell sarcoma ( mcs )  . the tumor next - generation sequencing ( foundationone heme panel ; foundation medicine , cambridge , ma ) on the biopsy was signicant for kit y503_f504insay , idh2 r140q , and srsf2 p95h mutations , but was negative for flt3 mutations . 
the particular kit mutation detected has been reported to be responsive to imatinib.1 - 4 the idh2 mutation has not previously been identied in mast cell neoplasms.5 the patient was treated with imatinib 400 mg per day in addition to radiation to the sacrum and pubic ramus4 , 500 cgy in 25 fractions . 
he remains without evidence of aml recurrence 71 months after hematopoietic sct . the original diagnosis of aml and subsequent bone marrow evaluations were reviewed in light of the development of mcs . 
cytogenetic studies revealed 47xy , + 8 [ 13 ] / 46 , xy [ 7 ] and uorescence in situ hybridization was positive for an extra signal for runx1t1 ( 8q22 ) in 59 of 200 interphase cells . 
sanger sequencing on dna from a saliva sample demonstrated wild - type germline idh2 and srsf2 . identical idh2 and srsf2 mutations implied a clonal relationship between the mcs and aml ( fig 5 )  . 
 brown et al context and mast cell sarcoma ? can tools , such as next - generation sequencing ( ngs ) , delineate a clonal relationship in a patient with acute myeloid leukemia we identied a probable clonal relatedness between the two neoplasms , with both sharing identical idh2 and srsf2 mutations . 
this report provides anecdotal evidence of a clonal relationship between acute myeloid leukemia and mast cell sarcoma that is developmentally feasible and describes a unique scenario in which ngs was effective at directing therapy . 
clinicians should consider performing ngs in patients with two neoplasms , particularly if a progenitor cell is feasible , as well as in patients with mast cell disorders and a neoplasm . the clonal progenitor that harbored the idh2 and srsf2 mutations persisted . 
shared mutations between aml and mcs support the existence of a preleukemic clonal progenitor that developed into aml initially and later into mcs . mcs was rst described in 1986 and is one of many diseases arising from the spectrum of mastocytosis.11 - 13 it typically presents as a solitary mass that may arise anywhere in the body and has intensely avid uorodeoxyglucose uptake on petcomputed tomography scan.9 - 11 , 14 - 20 diagnosis depends on the synthesis of pathologic and immunophenotypic that are features in a patient with an aggressive , initially sarcoma - like tumor.16 , 17 although initially localized , these tumors typically metastasize fig 1 . 
 ( a ) computed tomography ( ct ) scan of the abdomen and pelvis obtained for evaluation of abdominal pain incidentally revealed a destructive lesion of the left inferior pubic ramus that measured approximately 3 cm 2 cm , worrisome for malignancy . 
survival ranges from 2 months to 8 years , with a median survival of 6 months.10 , 12 , 15 , 16 , 18 - 21 cells are usually more atypical than those observed in systemic mastocytosis , with an epithelioid appearance with abundant granular cytoplasm and bilobed or multilobed nucleoli.15 , 22 mcs can be misdiagnosed on initial pathologic review given its rarity and resemblance to other tumor types , including lymphomas . 
these cells often lose markers that are characteristic for mast cells , which further complicates the diagnosis.20 , 23 evaluation of markers that are helpful to establish the diagnosis include cd2 , cd25 , cd30 , cd33 , cd34 , cd45 , cd117 , and mast cell tryptase.12 , 18 absence of cd117 positivity should call into question the diagnosis of a mast cell disorder . 
mast cell tryptase is highly specic for mast cell lineage , but may only show variable positivity in mcs.12 , 23 other immunohistochemical markers that overlap with melanoma , lymphomas , or other histiocytic processes have been reported.12 in the absence of another concurrent myeloid neoplasm , serum tryptase elevation strongly supports the diagnosis of a mast cell disorder.12 , 23 mcs have possessed kit mutations in eight of 15 cases that have reported on kit mutation testing , including the present case.10 , 11 , 19 , 20 cells with kit mutations fig 2 . 
flow cytometry of pubic ramus lesion detected a population of medium - sized to large cells , which accounted for approximately 60% of the total events , with high orthogonal light scatter indicative of high granularity / internal complexity ( red , abnormal cells ; blue , lymphocytes )  . 
abnormal cells were cd15 negative , cd117 bright positive , cd7 equivocal , cd13 + , cd2 , cd25 , cd36 , and cd64 + ( variable intensity )  . 
by immunohistochemistry , tumor cells were positive for cd117 and mast cell tryptase ( weakly ; top ) but negative for cd2 and cd25 ( bottom )  . cd25 remain cd117 bright on immunohistochemistry and are unaffected by changes induced by these mutations.24 kit mutations do not affect the ow cytometric detection of cd117 . therapy of mcs is poorly dened but typically involves surgery and radiation to localized lesions ; chemotherapy is usually ineffective.19 , 20 we would suggest that all patients with suspected mcs have kit sequenced . 
results of next - generation sequencing on both mcs and aml mutated allele genomic alteration frequency , % detected fda - approved therapy for patients tumor kit ( y503_ f504insay ) idh2 ( r140q ) srsf2 ( p95h ) flt3 ( s451f ) idh2 ( r140q ) ptpn11 d61h - subclonal n308dsubclonal srsf2 ( p95h ) imatinib none * none none none none enasidenib ( 2017 ) common myeloid progenitor cell idh2 srsf2 idh2 srsf2 flt3 ptpn11 mast cell idh2 srsf2 note . 
enasidenib was approved by the fda in 2017 , several years after the patient had aml . abbreviations : aml , acute myeloid leukemia ; fda , us food and drug administration ; mcs , mast cell sarcoma . * although enasidenib is not fda approved for mcs , it is conceivable that the drug , which targets mutant idh2 , may have activity in this patients case . and thus remains sensitive to imatinib.28 mcs with d816v mutations may be sensitive to other tyrosine kinase inhibitors , including avapritinib , which selectively targets the d816v mutation and is well tolerated.29 , 30 of interest , the idh2 mutation in our patients case is shared between aml and mcs , which suggests that it was present in a lessdifferentiated common progenitor cell . 
collins jr employment : university of texas southwestern medical centersimmons cancer center honoraria : optumhealth research funding : agios , arog , bristol - myers squibb , celgene no other potential conicts of interest were reported . references 2011 guo t , hajdu m , agaram np , et al : mechanisms of sunitinib resistance in gastrointestinal stromal tumors harboring kitay502 - 3ins mutation : an in vitro mutagenesis screen for drug resistance . 
br j haematol 173 : 323 - 326 , 2016 cancer genome atlas research network ; ley tj , miller c , et al : genomic and epigenomic landscapes of adult de novo acute myeloid leukemia . 
cell 168 : 1041 - 1052.e18 , 2017 yamashita a , saito t , akaike k , et al : mast cell sarcoma of the sternum , clonally related to an antecedent germ cell tumor with a novel d579del kit mutation . virchows arch 470 : 583 - 588 , 2017 10 . 
georgin - lavialle s , aguilar c , guieze r , et al : mast cell sarcoma : a rare and aggressive entityreport of two cases and review of the literature . 
krauth mt , f odinger m , rebuzzi l , et al : aggressive systemic mastocytosis with sarcoma - like growth in the skeleton , leukemic progression , and partial loss of mast cell differentiation antigens . 
verstovsek s , tefferi a , cortes j , et al : phase ii study of dasatinib in philadelphia chromosome - negative acute and chronic myeloid diseases , including systemic j cancer 42 : 1093 - 1103 , 2006 mastocytosis . 
brunner am , neuberg ds , wander sa , et al : isocitrate dehydrogenase 1 and 2 mutations , 2 - hydroxyglutarate levels , and response to standard chemotherapy for patients with newly diagnosed acute myeloid leukemia . 
schuurhuis gj , heuser m , freeman s , et al : minimal / measurable residual disease in aml : a consensus document from the european leukemianet mrd working party . 
we identied , for the rst time , an epigenomic factor that can potentially complement dna mutation status in discriminating patients with lung adenocarcinoma who are less likely to benet from egfr - tki treatment , thereby leading to improved patient management in precision medicine . jco precis oncol 5 : 418 - 431 . 
inhibition of epidermal growth factor receptor ( egfr ) kinase activity by egfr - tyrosine kinase inhibitors ( tkis ) , such as erlotinib , getinib , and afatinib , was effective in patients with nsclc with egfr - activating mutations . however , despite the remarkable clinical success , the treatment efcacy was still limited to 50% - 80%.10 , 11 the sizable percentage of nonresponders ( 20% - 30% ) suggested intrinsic tki resistance and substantial heterogeneity among tumors , even among egfrmutant tumors , highlighting the need for reliable predictive biomarkers . the comprehensive molecular proling of pretreatment lung adenocarcinoma to identify inherently tkiresistant cases can aid the development of potential strategies to manage such cases . 
 dna methylation proling for egfr - tki responses context key objective a sizable portion ( 20% - 30% ) of patients with epidermal growth factor receptor ( egfr ) mutant nonsmall - cell lung cancer have no good initial clinical response to egfr - tyrosine kinase inhibitors ( tkis ) , and how to predict intrinsic drug resistance accurately is challenging . 
global dna methylation landscape of tumors from patients with lung adenocarcinoma before tki treatment was analyzed to investigate the association with egfr - tki responses . knowledge generated a total of 216 tki responseassociated methylated sites were identied , and functional analysis revealed the enrichment of homeobox genes . 
in particular , increased methylation of hoxb9 correlated with higher rate of intrinsic resistance ( ir ) to egfr - tki . relevance dna methylation provides a different dimension to complement the dna mutationbased markers for understanding the mechanism of ir . 
evaluation of the methylation level on hoxb9 may be incorporated in the management of lung adenocarcinoma to aid the prediction of egfr - tki response . egfr - tki14 suggest many dna - based biomarkers for ir prediction . 
on the other hand , tumor suppressor genes involved in the alternative mechanisms of ir may be inactivated by epigenetic mechanisms that result in phenotypic or functional changes.15 however , although studies have reported that epigenetic changes in tumor participate in the evolution of acquired drug resistance through regulating gene expression patterns , 16 epigenomic data associated with ir to tki are lacking . 
modication of methylation on dna is stable and abnormal methylation represents an early event for cancer diagnosis , making methylation aberrations equally suitable candidates for recurrence detection and prediction of patient survival.17 - 21 therefore , we undertook a genome - wide approach to investigate dna methylation patterns associated with ir to tki . dna methylation that occurs at cytosines of cpg dinucleotides , especially within cpg islands in the promoter region , can lock genes in off status , resulting in a transcriptionally silent state.22 , 23 although dna methylation is an important mechanism for maintaining normal development and cellular homeostasis , aberrant dna methylationmediated silencing of tumor suppressor genes has been reported to be associated with cell survival and progression in cancer.24 dna methylation proling of tumors , including those of glioma , acute myeloid leukemia , and colorectal and lung cancers , has aided the identication of cancer subtypes correlated with clinical outcomes.25 - 29 in this study , we aimed to identify epigenetic markers for predicting drug efcacy in patients with lung adenocarcinoma . 
we conducted genome - wide dna methylation proling of tumors from patients before their egfr - tki therapy and established a dna methylation landscape to elucidate the association of dna methylation with the egfr - tki response status of patients via a pipeline of statistical analysis , gene ontology ( go ) , and bioinformatics analysis . 
detailed accounts of sample collection and protocols for dna extraction , bisulte conversion , dna methylation analysis , pyrosequencing along with statistical analysis , bioinformatics analysis , and data availability are presented in the data supplement . results clinicopathologic features of the patients table 1 lists the clinicopathologic features of the two cohorts . 
the validation cohort consisted of 163 egfrmutant patients and the majority ( 85.28% ) were at stage iv . the tki response assessmentprogressive disease ( pd ) , stable disease ( sd ) , partial response ( pr ) , or complete response ( cr ) was determined according to the recist guidelines.30 the pd group , dened at the rst scan done at 8 weeks following the start of egfr - tki , is considered as patients with ir . 
using the top 5% coefcient of variation as the cutoff , 24 , 121 probes with the greatest variability were analyzed , and 391 probes were found correlated with egfr - tki response . 
the majority of the probes ( 203 ) had higher dna methylation in patients with a poor response than in those with a favorable response ; hypermethylation correlated with poor response . 
a global view of the dna methylation distribution contrasting the patients with pd against those with pr showed that all but 13 probes were located above the diagonal line , elucidating a clear pattern of methylation gain in patients with pd ( fig 2b - d )  . 
the tumors of patients who were more likely to be resistant to egfr - tkis tended to have higher pretreatment methylation levels . chromosomal context analysis of candidate cpg sites showed enrichment in cpg islands and gene promoter regions the cpg sites were assigned to the annotated categories according to their chromosome positions relative to the nearby transcription start sites and the closest cpg islands ( data supplement )  . 
we examined the changes in the proportion of each category during our probe selection and found an increasing trend in cpg islands and the gene promoter region tss1500 ( between 1 , 500 bp and 200 bp upstream of the transcription start site )  . 
for the 37 transcription - repressive sites , the enrichment pattern in cpg islands was retained and that in tss1500 was increased to 27.03% ( fig 2e )  . identication of transcription - repressive methylation sites we investigated the potential of the 216 methylation sites in cis - regulation of gene expression by correlating publicly accessible mrna gene expression data ( gse60644 ) with dna methylation data ( gse56044 ) in lung adenocarcinoma ( data supplement )  . 
a total of 37 sites were identied as transcription - repressive sites ( data supplement )  . functional enrichment analysis of transcript - linked methylation showed eight probes linked to tfs ( go : 0003677 ) , to evaluate the molecular function of the genes mapped by the 37 transcription - repressive sites , go enrichment analysis was conducted . 
furthermore , using animal transcription factor database ( animaltfdb ) , we found that eight of the 37 sites were annotated to ve tfs : ikzf1 , hoxb9 , sp8 , lass4 , and vax2 ( table 2 and data supplement )  . figure 2g shows the f statistic and the corresponding p value from analysis of variance for each of the eight sites , along with the location and chromosome context information . 
we found that seven of the eight tf - linked sites are located in the context of cpg islands and ve in the transcription start site ( tss ) region ( table 2 )  . 
the two vax2 methylation sites also showed a moderate ability to predict the egfr - tki response ( data supplement )  . we further performed stratication analysis by classifying patients as those with egfr - activating mutations and those increased methylation of hoxb9 without ( figs 3d - f )  . ( cg13643585 ) was observed in pd patients . 
for the egfr wild - type group , the or ( 7.59 ) was comparable , but the p value was .09 , likely because of the small sample size ( fig 3e )  . 
the data in table 3 conrmed the pattern of increased hoxb9 methylation in the egfr - tkiresistant ( pd ) group compared with the disease control group ( cr , pr , or sd )  . 
the pd group was ranked rst in each quartile , and the one - sided rank - sum test for between - group differences indicated a signicant difference ( p = .036 ) ( data supplement )  . in addition , analysis of the auroc showed that hoxb9 methylation statistically signicantly increased the predictive precision of egfr - tki resistance for the dcr ( table 3 )  . 
although signicant advances have been made in understanding acquired resistance ( ar ) , the causes of ir remain unclear.12 in addition to being classied as ar versus ir , resistance mechanisms can be classied in terms of on - target versus off - target , 31 suggesting the activation of collateral signaling . 
the patients characteristics , smoking behavior ( p1 ) , sex ( p2 ) , egfr status ( p3 ) , and egfr - tki response ( p4 ) , are shown by the bars at the top of the heatmap . 
for each cpg probe , the average beta value across patients with pd was plotted against that across patients with pr and is shown as a smooth kernel scatter plot . 
in the gradient scale , red represents the densest region , whereas purple represents the sparsest region ; pr ( red ) , sd ( yellow ) , and pd ( blue )  . 
the probe distributions in the cpg context and the gene context are shown on the left and right , respectively , for all probes in the array ( top ) , for the most variably methylated probes ( upper middle ) , for the tki responseassociated methylation sites ( lower middle ) , and for the transcription - repressive sites ( bottom )  . 
moreover , where genomic resistance has been found , epigenomic modulation has been proposed as the potential mechanism . changes in resistant phenotypes , including epithelialtransition ( emt ) and cancer stemness mesenchymal shift , have been found to be driven by epigenetic remodeling . 
tki - induced dna methylation changes have been reported in advanced egfr - mutated lung cancer.33 decitabine , the dna methyl transferase inhibitor , could reverse the sensitivity of egfr - tkiresistant nsclc cell line pc9 / gr through demethylation of rassf1a and gadd45.34 the combination of tkis with epigenetic drugs has shown in preclinical and clinical promise as a treatment studies.33 - 35 in this study , we conducted clinical oncological investigation on the potential role of dna methylation in mediating ir to egfr - tki treatment in patients with advanced lung adenocarcinoma . 
aberrant dna methylation is one of the most classical events that occurs during lung cancer development.36 many studies have shown altered methylation patterns in lung cancer , indicating roles of epigenetic biomarkers and therapeutic targets.37 - 39 earlier study by zhu et al40 focused on the methylation patterns of wnt antagonists , showing the association of methylated sfrp5 with shortened progression - free survival under egfr - tki treatment , but not with ir to tki . 
interestingly , two 428 2021 by american society of clinical oncology of those ve genes , axl1 and hoxa9 , are homeobox tfs . our pursuit of the primary egfr - tkiresistant methylation markers also identied enrichment of homeobox genes . among the 30 tki - associated methylation probes annotated to tfs , 11 accounted for nine homeobox genes ( data supplement )  . we identied and conrmed the correlation of hoxb9 dna methylation with an increased rate of ir to egfr - tkis . hoxb9 is involved in cell development and proliferation43 and is suggested to function as a tf that can induce the expression of emt genes and several angiogenic factors , such as vegf , il - 8 , and tgf , resulting in the activation of egfr and erbb2.44 - 46 egfr signaling is connected to the nf - b pathway , giving the role in ir or ar to egfr inhibitors.47 however , the molecular mechanisms by which hoxb9 contributes to carcinogenesis are debated.48 , 49 the overexpression of hoxb9 can suppress the akt / nf - b / snail pathway and inhibit the proliferation of gastric carcinoma cells.50 we analyzed the correlation between egfr signaling and nf - bdependent pathways ( gse60644 ) and found that expression of hoxb9 negatively associated with that of kiaa1199 ( cell migrationinducing hyaluronidase 1 ; data supplement )  . 
through protein - protein interaction ( ppi ) analysis , we found that hoxb9 might cross talk with both ir and ar to egfr - tki through ezh2 , sirt1 , and egr2 ( data supplement )  . 
therefore , regulation of hoxb9 is crucial in the cooperated oncogenic loops.12 our data suggested that hoxb9 hypermethylation may be a novel tumor cellular state that is useful for precise categorization of tumor heterogeneity in the study of intrinsic egfr - tki resistance via off - target effects such as redundant or compensating signaling . pattern was consistent between patients with egfr - activating mutations and patients with wild - type egfr , implying that the regulatory effect of dna methylation of hoxb9 may be independent of egfr activity . in addition , dna methylation changes can be accurately detected in tumors and liquid biopsies . 
such detection is promising for the development of biomarkers for cancer screening.51 dna methylation in distal regulatory sites , such as enhancer regions , plays important roles in gene regulation through the binding of cell typespecic tfs and interaction with promoters.52 - 54 we validated a dna methylation site in the enhancer region of hoxb9 that can help the prediction of nonresponse to egfr - tki . 
in cancer , aberrant dna methylation at enhancers couples with recruitment of coactivators or corepressors , forming networks of cancer - associated tfs and their targeted genes.55 - 57 stone et al58 dened hypermethylation enhancers that correlate with sensitivity to endocrine therapy , suggesting the impact of enhancer status on the drug treatment response . 
 dna methylation proling for egfr - tki responses the combination of genetic aberrations , gene expression , and dna methylation highlights the potential of the identied candidates in the development of biomarkers for tumor diagnosis or prognosis . 
in this study , with a focus on medical actionability , we discovered that hoxb9 methylation could be a biomarker useful for discriminating patients with tki resistance from those with tki sensitivity , especially patients whose tumors harbor egfr - activating mutations . 
although improving the sensitivity and specicity of hoxb9 methylation is recommended , our work provided a preliminary proof of concept on the usefulness of hoxb9 methylation for opening up more clinical options to manage lung adenocarcinoma . 
for example , in accordance with the current clinical standard of treating egfr - mutant patients with egfr - tki , for patients with hoxb9 hypermethylation , combination treatment such as egfr - tki plus antiangiogenic therapy59 or egfr - tki plus chemotherapy60 may be another option to overcome the resistance and improve the response rate . 
 acknowledgment the authors thank all the patients and research staff who participated in this work . su et al references siegel rl , miller kd , jemal a : cancer statistics , 2019 . 
mitsudomi t , morita s , yatabe y , et al : getinib versus cisplatin plus docetaxel in patients with non - small - cell lung cancer harbouring mutations of the epidermal growth factor receptor ( wjtog3405 ) : an open label , randomised phase 3 trial . 
lancet oncol 11 : 121 - 128 , 2010 zhou c , wu yl , chen g , et al : erlotinib versus chemotherapy as rst - line treatment for patients with advanced egfr mutation - positive non - small - cell lung cancer ( optimal , ctong - 0802 ) : a multicentre , open - label , randomised , phase 3 study . 
lancet oncol 12 : 735 - 742 , 2011 rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as rst - line treatment for european patients with advanced egfr mutationpositive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
wu yl , zhou c , liam ck , et al : first - line erlotinib versus gemcitabine / cisplatin in patients with advanced egfr mutation - positive non - small - cell lung cancer : analyses from the phase iii , randomized , open - label , ensure study . 
ann oncol 26 : 1883 - 1889 , 2015 yang jch , sequist lv , geater sl , et al : clinical activity of afatinib in patients with advanced non - small - cell lung cancer harbouring uncommon egfr mutations : a combined post - hoc analysis of lux - lung 2 , lux - lung 3 , and lux - lung 6 . 
lancet oncol 16 : 830 - 838 , 2015 yang jch , wu yl , schuler m , et al : afatinib versus cisplatin - based chemotherapy for egfr mutation - positive lung adenocarcinoma ( lux - lung 3 and luxlung 6 ) : analysis of overall survival data from two randomised , phase 3 trials . 
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lee ck , davies l , wu yl , et al : getinib or erlotinib vs chemotherapy for egfr mutation - positive lung cancer : individual patient data meta - analysis of overall survival . 
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zhu j , wang y , duan j , et al : dna methylation status of wnt antagonist sfrp5 can predict the response to the egfr - tyrosine kinase inhibitor therapy in nonsmall cell lung cancer . 
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zhang l , wu q , he c , et al : hoxb9 inhibits proliferation in gastric carcinoma cells via suppression of phosphorylated - akt and nf - b - dependent snail expression . 
taberlay pc , statham al , kelly tk , et al : reconguration of nucleosome - depleted regions at distal regulatory elements accompanies dna methylation of enhancers and insulators in cancer . 
h saito , t fukuhara , n furuya , et al : erlotinib plus bevacizumab versus erlotinib alone in patients with egfr - positive advanced non - squamous non - small - cell lung cancer ( nej026 ) : interim analysis of an open - label , randomised , multicentre , phase 3 trial . 
hosomi y , morita s , sugawara s , et al : getinib alone versus getinib plus chemotherapy for non - small - cell lung cancer with mutated epidermal growth factor receptor : nej009 study . 
kornaga , msc1 , 2 ; cheryl crozier , msc1 ; gun ho jang , phd1 ; lauren bathurst , msc1 , 3 ; irina kalatskaya , phd1 , 4 ; quang m . 
pathways were identied as aberrant if there were copy number aberrations and / or mutations in any of the predetermined pathway genes : ccnd1 / ccnd2 / ccnd3 / cdk4 / cdk6 , fgfr1 / fgfr2 / fgfr2 / fgfr4 , and akt1 / akt2 / pik3ca / pten . results the 390 of 420 samples that passed quality control were analyzed for distant metastasisfree survival between groups . 
these bcas represent the majority of new bca diagnoses ( 70% to 80% ) , and a recent meta - analysis of 62 , 923 women with estrogen receptor ( er ) positive bca , who were disease free after 5 years of endocrine therapy , reported that approximately one in three women will experience a relapse during the next 20 years.1 - 3 although the development of diagnostic tests have helped to inform patients with regard to residual relapse risk , 4 , 5 as well as the identication of those who do not benet from chemotherapy , 6 none have yet to provide insight for those high - risk patients who may benet from the precision medicine approaches offered in the metastatic setting . 
to fully maximize the benet of targeted therapies seen in unselected patients in the advance / metastatic setting , upfront stratication of patients at risk for recurrence to these targeted agents in the early setting would reduce unnecessary treatment and associated toxicities and form a rational basis for treatment selection . 
this retrospective study of 390 early breast cancers from a phase iii clinical trial demonstrates that genomic changes within key genes in the cyclin d / cyclin - dependent kinase , phosphatidylinositol 3 - kinase / protein kinase b , and broblast growth factor receptor pathways could identify patients who might benet most from treatment that targets those deregulated pathways . knowledge generated in this focused approach , aberrations among genes in these pathways were shown to affect risk of distant relapse . relevance the use of this stratication approach , which benets from the interrogation of a limited number of genes , could be implemented effectively by using existing genomic technologies in the clinical laboratory to aid clinicians in identifying patients for whom specic targeted treatments are needed in combination with standard endocrine therapy . and the cancer genome atlas , 13 , 14 translation of these ndings into novel approaches to the management of cancer is slow . in response , we generated data focused on specic signaling pathways derived from the international cancer genome consortium and the cancer genome atlas13 , 14 together with whole - genome copy number analyses . 
the analysis was performed on the basis of an a priori hypothesis related to molecular pathways for which there are existing targeted therapies currently under evaluation in late - stage clinical trials . 
in a case - control study , 420 patients from the tamoxifen versus exemestane adjuvant multinational ( team ) trial pathology cohort ( clinicaltrials . identiers : nct00279448 , nct00032136 , and nct00036270 ) 15 were selected to determine the prognostic ability of copy number events and specic mutational changes that represent the cyclin d / cyclin - dependent kinase ( ccnd / cdk ) , broblast growth factor receptor / broblast growth factor ( fgfr / fgf ) , and phosphatidylinositol 3 - kinase / protein kinase b ( pi3k / akt ) pathways to predict risk of recurrence after anti - endocrine therapy in the early setting . 
distant metastasisfree survival ( dmfs ) was dened as time from random assignment to distant relapse or death as a result of bca.15 this study complies with the declaration of helsinki , institutional ethics committees , and the international conference on harmonization and good clinical practice guidelines . 
patients provided informed consent , and this study was approved by the university of toronto research ethics board ( protocol number 35339 )  . targeted sequencing and copy number analysis forty nanograms of dna extracted from formalin - xed parafn - embedded tissues were processed for targeted sequencing ( appendix )  . 
only variants that were on target and passed ltering criteria were included in the downstream analysis as described by kalatskaya et al.19 for copy number aberration ( cna ) analyses , 200 ng of dna were processed using oncoscan ffpe express 2.0 snp arrays ( affymetrix , santa clara , ca )  . 
 prognostic signatures in early breast cancers highly variable log - r ratio ( lrr ) , unevenly distributed b - allele frequencies , and waviness in lrr were measured using penncnv.20 estimation of tumor ploidy and aberrant tumor fraction was performed , and copy number segments were used by combining segments generated by two algorithms : allele - specic copy number analysis of tumors version 2.121 and circular binary segmentation22 implemented in the dnacopy package of r . 
an algorithm described by boutros et al23 was used to recalibrate tumor ploidy , aberrant tumor fraction , and copy numbers on the basis of lrr and b - allele frequency data , and adjacent segments having the same copy numbers were merged . segment boundaries were adjusted to obtain smaller argument function values . 
analyses were performed using r version 3.1.0. statistical analysis of the 420 patients , 390 passed ltering criteria and were included in subsequent analyses using stata 12.1 software ( statacorp , college station , tx )  . 
genes that represented pathways of interest were identied as aberrant according to the following criteria : ccnd / cdk pathway ( gains / amplications in any of ccnd1 / ccnd2 / ccnd3 / cdk4 / cdk6 ) , fgfr pathway ( gains / amplications in any of fgfr1 / fgfr2 / fgfr3 / fgfr4 ) , and pi3k / atk pathway ( aberrations in any of the following : gain / amplication of akt1 or possession of a mutation in akt1 , gain / amplication of akt2 , gain / amplication of pik3ca , and pten mutation and accompanying pten loss akt1 / akt2 / pik3ca / pten )  . a second model for the pi3k / akt was analyzed where pik3ca mutation data were included alongside pik3ca cnas and alterations in the genes of the dened pathways . kaplan - meier method and log - rank analyses were used to assess differences in dmfs . 
hrs were calculated using cox proportional hazards regression models , and models were adjusted for age ( continuous variable ) , tumor size ( dichotomized at 2 cm ) , tumor grade ( 1 / 2 v 3 ) , lymph node status , and her2 status ( negative v positive )  . 
all p values reported were two - sided , and p , .05 was considered statistically signicant . results changes in copy number of genes associated with signaling pathways are independently linked to high risk or relapse after anti - endocrine therapy our a priori hypothesis identied candidate pathways and specic genomic changes linked to endocrine treatment failure and outcome , namely ccnd / cdk signaling , the pi3k / akt axis , and fgfr / fgf pathway . 
no statistically signicant differences in age , tumor size , grade , lymph node status , or her2 status between patients with a dmfs event and those without were noted ( table 1 )  . 
of the 272 patients , 172 had aberrations in the specied ccnd / cdk genes ( 147 with aberrations in the fgfr genes and 138 with aberrations in pi3k / akt genes )  . 
of the 390 patients , 54 ( 13.8% ) , 34 ( 8.7% ) , and 43 ( 11% ) possessed aberrations in only one pathway ( ccnd / cdk , pi3k / akt , and fgfr , respectively )  . slightly fewer patients possessed aberrations in two pathways : 37 ( 9.5% ) with aberrant ccnd / cdkand pi3k / aktrelated genes , 37 ( 9.5% ) with aberrant ccnd / cdkand fgfr - related genes , and 23 ( 5.9% ) with aberrant pi3k / aktand fgfr - related genes . 
correlation to grade according to the number of pathways showed that patients with lower - grade tumors ( grade 1 / 2 ) had fewer altered pathways than those with grade 3 tumors ( appendix table a2 )  . 
although widely viewed as a good - prognosis subtype , hormone receptorpositive bcas account more than 84% of newly diagnosed early bca and therefore comprise the majority of bca deaths . 
despite recent advances in adjuvant therapy , outcomes for high - risk hormone receptorpositive bca are suboptimal , with an estimated relapse rate of 25% at 5 years.1 , 2 , 28 patients who are disease free at 5 years after diagnosis can still experelapse 5 to 20 years rience a high risk of distant postdiagnosis , 1 , 28 which directly challenges the dogma that such cancers are low risk and responsive to treatment . we tested the hypothesis that aberrations in key signaling pathways , where targeted therapies exist for bca ( in the advanced or metastatic setting ) , are prognostic in the early bca setting . 
aberrations in key genes of the ccnd / cdk pathway were more frequently detected than those in the pi3k / akt or fgfr axes ; however , the majority of patients ( 52% ) had aberrations in more than one pathway , which offers a rationale for incomplete responses to monotargeted therapy . 
kaplan - meier survival curves for aberrant pathways ( a ) cyclin d / cyclin - dependent kinase ( ccnd / cdk ) , ( b ) broblast growth factor receptor ( fgfr ) , ( c ) phosphatidylinositol 3 - kinase / protein kinase b ( pi3k / akt ) in the absence of pik3ca mutation , and ( d ) pi3k / akt pathway with pik3ca mutations . 
patients who possessed ccnd / cdk alterations exhibited signicantly poorer outcomes ( fig 1a ) than patients with fgfr and pi3k / akt aberrations ( figs 1b and 1c , respectively )  . 
in combination , patients with ccnd / cdk and fgfr aberrations were also at higher risk for relapse ( figs 2 and 3 ) possibly because of the proximity of ccnd1 and ligands fgf2 and fgf3 on chromosome 11q and their frequent co - amplication.29 indeed , the outcome for patients with ccnd / cdk and fgfr aberrations was similar to patients with ccnd / cdk aberrations only ( fig 3b )  . 
when pik3ca mutations were included in the stratication , there was no difference in survival between the two groups , consistent with the nding that pik3ca mutations in early bca and in the context of endocrine therapy seems to have no impact on outcome.30 , 31 increasing numbers of aberrant pathways also were associated with higher grade ( appendix tables a1 and a2 ) , consistent with the role of grade as an important prognostic factor32 and its association with gene expression changes that drive proliferation.33 - 35 intrinsic , endocrine resistance , acquired or remains a therapeutic challenge . 
for example , we have shown that up to one in ve early bcas exhibit amplications of cdk2 / 4 / 6 and ccnds39 , 40 and that ccnd1 amplication , but not overexpression , is associated with increased risk of distant relapse in endocrine - treated early bca.40 similarly , the trial study design did not stratify patients upfront by biomarker . 
consistent with published work , our data support evidence for a key role of cnas in this gene pathway.52 , 53 toxicities associated with mtor inhibitors like everolimus remain a concern . 
kaplan - meier curves of distant metastasisfree survival ( dmfs ) according to ( a ) risk order ( no aberrant pathway , any cyclin d / cyclin - dependent kinase [ ccnd / cdk ] , phosphatidylinositol 3 - kinase / protein kinase b [ pi3k / akt ] and broblast growth factor receptor [ fgfr ] , and fgfr only )  . 
 ( b ) frequency of aberration ( no aberrant pathway ; any pi3k / akt , fgfr and ccnd / cdk ; and ccnd / cdk only )  . class of drug would greatly affect patient management . phase iii trials are now under way to evaluate pi3k / akt inhibitors , including pi3kselective inhibitors in combination with fulvestrant in the advanced / metastatic bca setting ( clinicaltrials.gov identiers : nct02437318 and nct02340221 )  . 
phase ii neoadjuvant trials also are being conducted in the early bca setting ( clinicaltrials.gov identiers : nct01923168 and nct02273973 ) , which requires tumor pik3ca mutation status before determination of treatment randomization . 
however , we and others have shown that in early hormone receptorpositive bca , pik3ca mutations represent one of the most frequent mutations ( 35% ) 31 , 54 , 55 but show limited evidence that these mutations are linked to endocrine resistance in the early setting . the fgfr family of genes are recognized to be amplied in 10% to 15% of bcas.56 , 57 although fgfr1 is most frequently amplied , there is evidence for lower frequency amplication of fgfr2 , fgfr3 , and fgfr458 - 60 in early bca . 
evidence has emerged that links fgfrs to endocrine resistance.58 , 60 - 62 in preclinical studies , fgfr1 amplication enhanced pi3k and mitogen - activated protein kinase pathway signaling , which leads to endocrine resistance and could be reversed using rna interference against fgfr1.57 between 30% and 40% of cancers with ccnd1 amplication exhibit fgfr1 co - amplication despite the fact that these genes are mapped within different amplication loci on chromosomes 11 and 8.63 a challenge in targeting both ccnd1 and fgf pathwayamplied tumors is the proximity of the ligands fgf3 and fgf4 to ccnd1 on chromosome 11q13 . 
the role of fgfr signaling in a number of cancer settings suggests that it is a potential driver of cancer progression.58 , 64 , 65 several fgfr - targeted inhibitors are under investigation in phase ii clinical trials , including fgfr1 to 4 selective inhibitors bgj398 identiers : nct01004224 and ( clinicaltrials.gov nct02160041 ) and azd4547 ( clinicaltrials.gov identiers : nct01791985 , nct01795768 , and nct01202591 )  . 
to date , all trials that involve fgfr - targeted inhibitors are for patients with advanced or metastatic disease , with almost all trials requiring molecular prescreening for only fgfr1 or 11q amplication before study entry . issues of early hormone receptorpositive bca critical management must be addressed , 66 - 70 including molecular heterogeneity7 , 8 , 71 and emergent drug resistance.72 , 73 clinically , patients with such features would benet from early treatment with combinatorial therapies . 
in a drug screen of 42 agents , ribociclib was identied as a strong sensitizer to pi3k inhibition in three er - positive bca cell lines with acquired resistance to pi3k inhibitors.74 targeted doublet combinations of cdk4 / 6 or pi3k / mtor inhibitors with endocrine therapy have improved progression - free survival compared with endocrine therapy alone , 37 , 75 , 76 whereas another study showed that palbociclib plus pictilisib and fulvestrant was more effective than doublet combinations.77 we show that early hormone receptorpositive bcas could be stratied using genomic markers representative of pathways linked to targeted therapeutics that predict risk of recurrence after endocrine treatment . 
bartlett provision of study material or patients : daniel rea collection and assembly of data : jane bayani , cheryl crozier , paul c . boutros , melanie spears , john d . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / author - center . jane bayani patents , royalties , other intellectual property : 95 gene signature of residual risk in endocrine - treated early breast cancer ( provisional us patent 62 / 263 , 805 ) daniel rea honoraria : roche , novartis , pzer , eli lilly consulting or advisory role : genomic health research funding : celgene ( inst ) , roche ( inst ) , biotheranostics ( inst ) travel , accommodations , expenses : pzer , daiichi sankyo john m.s. 
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finn rs , crown jp , lang i , et al : the cyclin - dependent kinase 4 / 6 inhibitor palbociclib in combination with letrozole versus letrozole alone as rst - line treatment of oestrogen receptor - positive , her2 - negative , advanced breast cancer ( paloma - 1 / trio - 18 ) : a randomised phase 2 study . 
n engl j med 375 : 1925 - 1936 , 2016 jensen lb , bartlett jms , witton cj , et al : frequent amplications and deletions of g1 / s - phase transition genes , ccnd1 and myc in early breast cancers : a potential role in g1 / s escape . 
miller tw , hennessy bt , gonz alez - angulo am , et al : hyperactivation of phosphatidylinositol - 3 kinase promotes escape from hormone dependence in estrogen receptor - positive human breast cancer . 
baselga j , semiglazov v , van dam p , et al : phase ii randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor - positive breast cancer . 
loi s , michiels s , baselga j , et al : pik3ca genotype and a pik3ca mutation - related gene signature and response to everolimus and letrozole in estrogen 49 . 
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sabine vs , sims ah , macaskill ej , et al : gene expression proling of response to mtor inhibitor everolimus in pre - operatively treated post - menopausal women with oestrogen receptor - positive breast cancer . 
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beelen k , opdam m , severson tm , et al : pik3ca mutations , phosphatase and tensin homolog , human epidermal growth factor receptor 2 , and insulin - like growth factor 1 receptor and adjuvant tamoxifen resistance in postmenopausal breast cancer patients . 
cancer res 70 : 2085 - 2094 , 2010 jaakkola s , salmikangas p , nylund s , et al : amplication of fgfr4 gene in human breast and gynecological cancers . 
heiskanen m , kononen j , b arlund m , et al : cgh , cdna and tissue microarray analyses implicate fgfr2 amplication in a small subset of breast tumors . 
taylor - harding b , aspuria pj , agadjanian h , et al : cyclin e1 and rtk / ras signaling drive cdk inhibitor resistance via activation of e2f and ets . 
sledge gw jr , toi m , neven p , et al : monarch 2 : abemaciclib in combination with fulvestrant in women with hr + / her2advanced breast cancer who had progressed while receiving endocrine therapy . 
 prognostic signatures in early breast cancers appendix targeted sequencing submission of 101 selected genes resulted in 6 , 318 amplicons , with a median size of 109 base pairs ( bp ; range , 64 to 142 bp ) that covered 612.79 kbp ( unpublished data )  . 
using ampliseq polymerase chain reaction primer panels ( thermo fisher scientic , waltham , ma ) for the gene set developed above , we adapted our current protocol for illumina ( san diego , ca ) sequencing . 
after amplication of dna regions in four separate pools of the total 6 , 318 amplicons ( pool 1 , 1 , 587 amplicons ; pool 2 , 1 , 580 amplicons ; pool 3 , 1 , 578 amplicons ; pool 4 , 1 , 573 amplicons ; 10 ng / pool , 40 ng total dna ) , library polymerase chain reaction products were combined ( per sample ) , barcoded , and pooled in sets of 96 samples . 
t raw reads ( 101 - bp paired - end ) were aligned against the university of california , santa cruz , human reference sequence hg19 ( including random and unknown sequences ) using novoalign version 2.07.14 ( novocraft technologies , selangor , malaysia )  . 
aligned reads were ltered with samtools version 0.1.1816 to remove unmapped reads , nonprimary aligned reads , and reads aligned with mapping quality less than 30 ( ie , nonunique alignments )  . 
because the tamoxifen versus exemestane adjuvant multinational trial ( clinicaltrial . gov identiers : nct00279448 and nct00032136 , and nct00036270 ) cohort did not have matching normal tissue , the conventional mutation calling pipelines were not applicable . 
 radiogenomics analysis of intratumor heterogeneity in a patient with high - grade serous ovarian cancer britta weigelt , phd1 ; hebert alberto vargas , md1 ; pier selenica1 ; felipe c . 
reis - filho , md , phd1 ; and evis sala , md , phd1 , 3 introduction the overall survival of patients with high - grade serous ovarian cancer ( hgsoc ) has not improved during the past 20 years.1 studies have revealed that genomic intratumor heterogeneity correlates with poor survival2 , 3 and specic patterns of malignant cell spread in hgsoc.4 the physical distribution of malignant clones across the peritoneal cavity may be nonrandom , because some sites harbor genetically diverse clones.5 these region - specic properties may invasion and expansion , modulate malignant cell thereby shaping evolutionary selection.3 , 6 our understanding of genomic heterogeneity in hgsoc is limited to single biopsies ; little is known about individual spatial and temporal variation across various tumor sites.2 - 5 hence , developing imaging methods for guiding tissue sampling to physiologically and metabolically distinct tumor habitats is desirable . 
such approaches will be vital for new clinical trials , especially those combining immunologic and genomically targeted therapies . here we use lesion - specic three - dimensional ( 3d ) molds for phenotypic image - guided tumor sampling to ensure spatial colocation of imaging , histology , and genomic data , critical for understanding tumor biology . phenotypic imaging maps of heterogeneity ( ie , imaging habitats ) of two hgsoc sites were obtained by combining perfusion , diffusion , and metabolic maps derived from multiparametric imaging . 
four days before primary cytoreductive surgery the patient underwent same - day multiparametric magnetic resonance imaging ( mpmri ; discovery mr750 3t mri system , ge healthcare , chicago , il ) of the abdomen and pelvis and 18f - uorodeoxyglucose ( fdg ) positron emission tomography ( pet ) / computed tomography ( ct ; discovery pet / ct system , ge healthcare )  . 
details of image acquisition , analysis , and mr - pet / ct coregistration are described in the appendix and in appendix table a1 . cluster - guided specimen sampling imaging habitats were identied using k - means clustering of the standardized uptake value ( suv ) , diffusion coefcient ( d ) , perfusion fraction ( f ) , and transfer constant ( dynamic contrast - enhanced parameter ktrans ) voxels , with the number of clusters ( k ) being xed to k = 3.7 custom - made 3d molds of the lesions were printed using a 3d printer ( makerbot replicator 2 ; makerbot , brooklyn , ny ) on the basis of manual segmentation of the right ovarian tumor and metastatic implant on the axial t2 - weighted mri ( appendix )  . 
tissue samples of each imaging cluster were obtained , cut in half , and formalin - xed and parafn - embedded for histopathologic analysis or snap - frozen for genomic analysis ( fig 1a )  . histologic review and immunohistochemistry the three right ovarian mass hgsoc imaging - based habitats ( labeled blue , yellow , or green ) and the omental implant ( blue ) were reviewed by two pathologists blinded to the habitat assignment . 
 ( a ) phenotypically distinct areas of a highgrade serous ovarian cancer ( hgsoc ) were identied by k - means clustering of imaging features derived from magnetic resonance imaging ( mri ) and positron emission tomography ( pet ) / computed tomography . 
the distinct imaging habitats ( labeled blue , yellow , and green ) were sampled from the surgically removed primary hgsoc and metastatic implant using a three - dimensional ( 3d ) mold . 
half of each imaging - based tissue area was formalin - xed parafnembedded for histopathologic review and immunohistochemical analysis , and the other half was ash - frozen for whole - exome sequencing analysis . 
 ( b ) micrographs of representative hematoxylin and eosin ( h and e ) stained sections of the imaging - based hgsoc areas ( top row ) , and immunohistochemical analysis of cd31 , ki - 67 , ca - ix , and hif - 1 . 
 ( c ) imaging features associated with the distinct color areas as dened by k - means clustering of standardized uptake values ( suv ) , diffusion coefcient ( d ) , dynamic contrast - enhanced dce parameter ( ktrans ) , and water volume fraction owing through microvessels ( f ) are plotted on top , and the histopathologic features of the distinct imaging - based tumor areas are shown at the bottocolor - coding according to the legend . dw , diffusion - weighted ; fdg , 18f - uorodeoxyglucose ; til , tumor - inltrating lymphocyte . nonsynonymous somatic mutations ( 74% ) not detected in all tumor areas by wes were independently validated using a custom hybrid - capture targeted massively parallel sequencing assay . 
the clusters were labeled blue ( lowest ktrans , highest suv , highest f ) , yellow ( highest d , lowest f ) , or green ( lowest d , highest ktrans , lowest suv )  . 
the right ovarian cancer contained all three distinct imagingbased clusters , whereas the omental metastatic implant was entirely composed of one cluster ( blue ; fig 1a ) , suggestive of phenotypic imaging heterogeneity in the primary hgsoc . tissue from the distinct imaging - based clusters for immunohistochemical and genetic analyses was obtained through a custom - made 3d mold ( fig 1a )  . imaging - based clusters are underpinned by distinct growth patterns and expression of hypoxia - related markers phenotypic imaging - based clusters were associated with distinct histopathologic growth patterns . 
 ( d ) unsupervised hierarchical clustering of the somatic mutations ( left ) and of the gene copy number alterations ( right ) identied not to be shared by all four imaging - based hgsoc areas using wards algorithm and euclidean distance . left , genes affected by mutations are shown in black ; right , amplication , gain , loss , and deletion are shown in dark red , red , blue , and dark blue , respectively . 
the values on the edges of the clustering are derived from pvclust analysis to assess the stability of the clusters ; both the approximatelyunbiased and bootstrap - probability p values were 100% . 
 weigelt et al signature 1 signature 3 other signatures mutant wild type somatic mutations copy number alterations amplification no change deletion gain loss ovary green ovary yellow ovary blue omentum blue ovary green ovary yellow ovary blue omentum blue 7q11 amp 2q11 amp ovary yellow ovary green tp53 pot1 16p12 amp 6p21 amp ovary blue fig 2 . 
the expression levels and patterns of the hypoxia - marker hif - 1 and its downstream target ca - ix correlated with the distinct areas dened by multiparametric imaging ( fig 1c )  . 
consistent with the imaging ndings , which demonstrated that the ovary blue and omentum blue areas displayed the highest f , an mri marker of tissue vascularity , cd31 immunohistochemical assessment revealed a higher density of tumor neovascularization ( 4 + ) in the ovary blue and omentum blue areas than in the ovary green and ovary yellow areas ( 2 + ; fig 1c )  . 
furthermore , distinct patterns and levels of ca - ix expression were observed among these areas , with ovary yellow and green having h - scores of 80 , compared with 230 and 195 in ovary blue and omentum blue , respectively . 
for hif - 1 , a reverse pattern was found , with ovary yellow and ovary green areas displaying high levels of hif - 1 expression ( h - scores , 115 ) compared with ovary blue and omentum blue ( h - scores , 75 and 60 , respectively ; figs 1b and 1c )  . imaging - based clusters are underpinned by distinct repertoires of genetic alterations high - depth wes of microdissected tumor and normal samples revealed 50 nonsynonymous somatic mutations , including a tp53 p.p47tfs * 5 frameshift mutation , which were shared among all four imaging - based tumor areas ( fig 2 )  . 
twenty - eight nonsynonymous somatic mutations were shared exclusively between ovary blue and omentum blue , and 46 between the ovary green and ovary yellow components ( fig 2a )  . 
although all imaging habitats displayed genomics features of homologous recombination dna repair deciency ( dominant mutational signature 3 , high large - scale transition scores ; fig 2c ) , hierarchical clustering of the 146 nonsynonymous somatic mutations or the 718 gene copy number alterations not shared among all four imagingbased areas revealed that both at the mutational and gene copy number levels , ovary blue and omentum blue clustered together and were distinct from ovary yellow and ovary green ( fig 2d )  . 
several somatic mutations found to be subclonal in ovary blue , including mutations affecting auts2 , eif2ak4 , and trim41 , became clonal in omentum blue ( fig 2a ) , and a subset of genes affected by copy number losses in ovary blue became homozygously deleted in omentum blue ( eg , chromosome 18q23 ; fig 2d )  . discussion several studies have demonstrated that hgsocs display spatial and temporal genetic heterogeneity , 2 - 6 , 22 with tumors composed of genetically distinct clones and exhibiting distinct evolutionary trajectories and mechanisms of therapy resistance.4 , 23 , 24 our observations support the contention that phenotypic and genetic analysis of single biopsy or single tumor samples may not provide a sufciently accurate representation of hgsoc heterogeneity.4 this proof - of - principle study suggests that multiparametric imaging assessment may provide a noninvasive surrogate for intratumor genetic heterogeneity and may guide precise tissue sampling . 
given the evidence supporting the impact of intratumor genetic heterogeneity on tumors metastatic ability and resistance to therapeutic interventions , 25 the results of this proof - of - principle observation provide the basis for studies to dene the type of sampling required for the assessment of preand posttreatment hgsocs . 
it should be noted , however , that multiparametric imaging for tumor heterogeneity and tumor evolution assessment requires not only manual segmentation of the imaging data but also standardization of the image acquisition protocols across different imaging platforms . targeted therapy and immunotherapy are being progressively applied earlier in the treatment of patients with cancer , and certain agents are anticipated to become part of front - line therapy . 
minimally invasive methods for disease monitoring in the form of circulating cell - free plasma dna hold great promise in the assessment of genetic heterogeneity within a patient.26 - 28 our imaging habitatbased method would constitute a noninvasive and complementary approach to such endeavors , given that it may allow for the sampling of selected image habitats without having to analyze every section of every tumor , an impossible task particularly in the metastatic and recurrent settings . 
for more information about asco 's conict of interest policy , please refer to or po.ascopubs.org / site / ifc . britta weigelt consulting or advisory role : genentech ( i ) , invicro ( i ) , ventana medical systems ( i ) , volitionrx ( i ) , paige.ai ( i ) , goldman sachs ( i ) felipe c . 
 weigelt et al niamh conlon employment : abbvie ( i ) stock and other ownership interests : merck ( i ) alexandra snyder employment : adaptive biotechnologies , merck stock and other ownership interests : merck consulting or advisory role : driver group research funding : bristol - myers squibb travel , accommodations , expenses : genentech , bristol - myers squibb robert a . 
assignee : the regents of the university of california august 18 , 2015 ( inst ) ; application : compositions and methods for the diagnosis and treatment of ovarian cancers that are associated with reduced smarca4 gene expression or protein function ( inst ) ; royalties from published books : cambridge university press and springer publishing dennis s . 
chi leadership : csurgeries stock and other ownership interests : bovie medical , verthermia , intuitive surgical , transenterix consulting or advisory role : bovie medical , verthemia ginger j . 
reis - filho consulting or advisory role : genentech , invicro , ventana medical systems , volitionrx , paige.ai , goldman sachs evis sala honoraria : siemens healthineers speakers ' bureau : siemens healthineers travel , accommodations , expenses : siemens healthineers no other potential conicts of interest were reported . references siegel rl , miller kd , jemal a : cancer statistics , 2018 . 
ca cancer j clin 68 : 7 - 30 , 2018 schwarz rf , ng ck , cooke sl , et al : spatial and temporal heterogeneity in high - grade serous ovarian cancer : a phylogenetic analysis . 
cell 173 : 1755 - 1769.e22 , 2018 bashashati a , ha g , tone a , et al : distinct evolutionary trajectories of primary high - grade serous ovarian cancers revealed through spatial mutational proling . j pathol 231 : 21 - 34 , 2013 5 . 
nat genet 48 : 758 - 767 , 2016 jim enez - s anchez a , memon d , pourpe s , et al : heterogeneous tumor - immune microenvironments among differentially growing metastases in an ovarian cancer patient . 
magn reson med 51 : 542 - 551 , 2004 guerini - rocco e , hodi z , piscuoglio s , et al : the repertoire of somatic genetic alterations of acinic cell carcinomas of the breast : an exploratory , hypothesisgenerating study . 
j pathol 237 : 166 - 178 , 2015 al - ahmadie ha , alden d , qin lx , et al : carbonic anhydrase ix expression in clear cell renal cell carcinoma : an immunohistochemical study comparing 2 antibodies . 
salgado r , denkert c , demaria s , et al : the evaluation of tumor - inltrating lymphocytes ( tils ) in breast cancer : recommendations by an international tils 12 . 
dowsett m , nielsen to , ahern r , et al : assessment of ki67 in breast cancer : recommendations from the international ki67 in breast cancer working group . int j gynecol pathol 34 : 437 - 444 , 2015 working group 2014 . 
detre s , saclani jotti g , dowsett m : a quickscore method for immunohistochemical semiquantitation : validation for oestrogen receptor in breast carcinomas . j clin pathol 48 : 876 - 878 , 1995 110 : 1030 - 1034 , 2018 15 . 
schultheis am , ng ck , de filippo mr , et al : massively parallel sequencing - based clonality analysis of synchronous endometrioid endometrial and ovarian cancer res 72 : 5454 - 5462 , 2012 carcinomas . 
the examination was performed on a 3t magnetic resonance imaging ( mri ) whole - body scanner unit ( discovery mr750 ; ge healthcare , chicago , il ) using a dedicated multichannel torso phased - array coil as the receive coil . the standard protocol included the following sequences acquired in two - dimensional t2 - weighted fast spin - echo , t1an axial plane : weighted gradient - echo . 
single - shot incoherent motion intravoxel ( ivim ) based diffusion - weighted images were obtained using echoplanar imaging with a pair of motion - probing gradients along three orthogonal axes , using a chemical shift selective fat - saturation technique . 
dynamic contrast - enhanced ( dce ) images were acquired before and after the intravenous injection of gadopentetate dimeglumine ( magnevist ; berlex laboratories , montville , nj ; 0.1 mmol per kilogram of body weight at a rate of 2 ml / s ) using an automatic injector ( medrad , bayer , pittsburgh , pa )  . 
the patient was scanned on a dedicated positron emission tomography ( pet ) / computed tomography ( ct ) system ( discovery 690 ; ge healthcare , chicago , il )  . 
a standard - of - care acquisition protocol was applied with an intravenous injection of approximately 400 to 455 mbq 18f - uorodeoxyglucose after at least 6 hours of fasting and documentation of blood glucose less than 200 mg / dl followed by a 60 - minute uptake period . 
subsequently , a low - dose , attenuation - correction ct scan ( 120 to 140 kv , approximately 80 ma ) was acquired with the patient in the supine position , followed by acquisition of pet emission images of the whole body ( 3 minutes per bed position , ve to six bed positions )  . imaging analysis and mr - pet coregistration . 
using anatomic landmarks available on axial t2 - weighted mr images and on the axial pet and low - dose ct images , the tumor regions outlined on mri were coregistered with pet . 
this was achieved by calculating the mean location ( corresponding to the center of mass of the tumor ) on the mri image ( t2w ) and the corresponding mean location on the pet / ct image . 
rigid registration was selected to ensure that the geometry ( and the voxel count ) of the region of interest ( roi ) for each slice was maintained between mri and pet . 
the translational matrix was applied to the roi for each slice to determine the displacement of the roi . the ivim biexponential model proposed by le bihan et al ( le bihan d , et al : radiology 168 : 497 - 505 , 1988 ; le bihan d , et al : radiology 161 : 401 - 407 , 1986 ; le bihan d , et al : magn reson med 10 : 324 - 337 , 1989 ) was used to estimate the diffusion parameters for each lesion outlined , including diffusion coefcient ( d ) and the volume fraction of the water owing through the microvessels ( f )  . 
the segmented analysis procedure ( callot v , et al : magn reson med 50 : 531 - 540 , 2003 ; yao l , et al : acad radiol 7 : 27 - 32 , 2000 ) was used to estimate ivim parameters on a voxel - wise basis . 
pharmacokinetic analysis of the dce data were carried out using a one - compartment tofts model ( tofts ps : j magn reson imaging 7 : 91 - 101 , 1997 )  . 
for each outlined lesion , voxel - wise estimates of the volume transfer constant between the blood plasma and the extravascular extracellular space ( ktrans [ min1 ] ) was calculated . 
arterial input function was calculated using a biexponent model as previously described ( weinmann hj , et al : physiol chem phys med nmr 16 : 167 - 172 , 1984 ; priest an , et al : magn reson med 63 : 1044 - 1049 , 2010 )  . gaussian smoothing was applied to all raw mr images before processing with variance of half a voxel , to smooth the parameters . 
for voxels that had estimated parameters that exceeded the bounds set by the tting curve , the values were set to nan ( not - a - number )  . 
once all voxels were tted , the voxels with nan value were replaced by an average of the surrounding voxels so that each voxel had an estimated parameter for the purpose of clustering . the standardized uptake values ( suv ) of the voxels contained within each lesion on 18f - uorodeoxyglucose pet was calculated based on the standard expression given by kinahan et al ( kinahan pe , et al : transl oncol 2 : 223 - 230 , 2009 )  . three - dimensional printing methodology . 
custom made threedimensional ( 3d ) molds were printed based on manual segmentation of the ovarian primary tumor and metastatic implants on the preoperative axial t2 - weighted mr images . 
the 3d model was then compared with the mr images and manually adjusted to resolve any discrepancies using meshlab ( meshlab , visual computing labisti - cnr ) , an extensible mesh processing system , aimed at editing and rendering unstructured 3d triangular meshes . 
the nal 3d models of each lesion were imported into openscad ( openscad , the openscad developers ) , 3d cad modeling software , which was used to create an internal cavity that exactly shaped each lesion according to the mri shape and contour . 
the slits for slicing each lesion were designed into the molds at 5 - mm intervals , which corresponded to the slice thickness and locations of the axial t2w fast relaxation fast spin echo mr images . 
carlo , md1 ; nabeela khan , md1 ; ahmet zehir , phd1 ; sujata patil , phd1 ; yasser ged , mbbs1 ; almedina redzematovic , ms1 ; devyn t . 
voss , md1 purpose nonclear - cell renal cell carcinoma ( nccrcc ) encompasses approximately 20% of renal cell carcinomas and includes subtypes that vary in clinical and molecular biology . 
compared with clear cell renal cell carcinoma , nccrcc demonstrates limited sensitivity to conventional vascular endothelial growth factor and mammalian target of rapamycindirected agents , indicating a need for better therapies . 
microsatellite instability ( msi ) in the tumor was investigated . results of 116 patients included in the analysis , 57 ( 49% ) presented with de novo metastatic disease , and 59 ( 51% ) presented with localized disease that later metastasized . 
of all tumors , 15 ( 13% ) had putative driver somatic alterations amenable to targeted therapies , including alterations in met , tsc1 / 2 , and an alk translocation . 
of 115 available tumors for analysis , two ( 1.7% ) had high and six ( 5% ) had intermediate msi status . conclusion the mutation proles of metastatic nccrcc vary by subtype . 
2019 by american society of clinical oncology introduction there are 63 , 000 estimated new patients diagnosed with renal cell carcinoma ( rcc ) annually in the united states and 14 , 000 deaths per year from advanced disease.1 the most common subtype of rcc is clear cell rcc ( ccrcc ) , which makes up approximately 75% to 80% of all rccs , with dened roles for targeted systemic therapy in localized and metastatic disease.2 the remaining histologic variants are collectively termed nonclear - cell rcc ( nccrcc ) and include papillary , chromophobe , collecting duct , translocationassociated , and unclassied types.3 comparing outcomes to ccrcc , cancer - specic survival is more favorable in patients with nccrcc with localized disease but tends to be worse in the metastatic setting.4 , 5 better treatments for metastatic nccrcc are clearly needed . 
a limited number of phase ii randomized trials have specically studied nccrcc . the espn ( everolimus versus sunitinib prospective evaluation in metastatic nonclear - cell renal cell carcinoma ) and aspen ( everolimus versus sunitinib for patients with metastatic nonclear - cell renal cell carcinoma ) trials compared the antivascular endothelial growth factor ( vegf ) agent sunitinib to the mammalian target of rapamycin ( mtor ) inhibitor everolimus as rst - line treatment of patients with nccrcc.6 , 7 per primary end point analyses , everolimus was not superior to sunitinib , but the heterogeneity of the study populations and the small sizes of variant subgroups limit the applicability of these data . 
these studies have highlighted the need for novel therapeutic approaches and a better understanding of how rcc biology differs among subtypes . increased knowledge of genomics may help identify novel treatment strategies . 
 carlo et al context ( nccrcc ) ? key objective what is the frequency of clinically actionable mutations in patients with advanced nonclear - cell renal cell carcinoma knowledge generated somatic mutation proles vary by nccrcc subtype . 
sequencing the tumor and germline and tumor microsatellite instability analysis reveal clinically actionable mutations in 22% of patients with advanced disease . relevance metastatic nccrcc can have a poor prognosis , and treatment is often extrapolated from clear cell renal cell carcinoma as a result of few studies that include this rare group of cancers . 
routine sequencing of nccrcc yields alterations such as high microsatellite instability , which may affect clinical management . marked responses to several targeted therapies when selected by molecular tumor features , rather than by tumor type.8 , 9 similarly , tumor microsatellite instability ( msi ) status is predictive of response to the antiprogrammed cell death protein 1 inhibitor pembrolizumab.10 indeed , msi status and defective mismatch repair are the rst molecular features linked to a regulatory agency approval irrespective of underlying disease histology . 
this study was approved by the mskcc institutional review board . ngs analysis tumor samples from either primary or metastatic sites were reviewed by genitourinary pathologists and microdissected for maximum tumor content for dna extraction . 
tumor and blood samples were sequenced using msk - impact , an ngs assay developed at mskcc that achieves hybridization capture with target - specic probes from exons of at least 341 cancer - associated genes , as described previously.13 mean depth of sequencing for the cohort was 660 . 
the gene panel is available in appendix table a1 . msi was quantied using msisensor.14 , 15 germline variants for 76 genes associated with cancer predisposition included in msk - impact were analyzed ( appendix table a1 )  . 
variants were classied by a clinical molecular geneticist or molecular pathologist as pathogenic , likely pathogenic , of uncertain signicance , likely benign , or benign , according to american college of medical genetics criteria.16 only pathogenic or likely pathogenic variants ( associated with disease causation and henceforth referred to as pathogenic variants ) are included in this analysis . somatic mutation clinical annotation somatic alterations were annotated through oncokb ( oncokb.org ) , a curated precision oncology knowledge base that stores information about the biologic function and therapeutic implications of individual gene alterations in a tumor typespecic manner . 
gene alterations that act as predictive biomarkers of response to specic targeted therapies are assigned a specic oncokb level of evidence . 17 levels 1 and 2a are us food and drug administration ( fda ) recognized or national comprehensive cancer network ( nccn ) listed biomarkers predictive of response to an fda - approved targeted therapeutic . 
msi - high ( msi - h ) status in solid cancers is considered oncokb level 1 , based on the fda approval of pembrolizumab.10 level 2b is a standard of care biomarker in another indication ( but not in rcc )  . 
mutations with level 4 assignment are those that are considered hypothetical biomarkers for which there is compelling biologic evidence that supports their being predictive of response to a drug and hence their potential use as eligibility criteria for clinical trials . statistical analysis clinical characteristics , somatic and germline results , and msisensor score are presented using descriptive statistics . objective response rate was dened as the best response according to response evaluation criteria in solid tumors ( recist ) , version 1.1. 
overall survival , dened as the time from diagnosis of metastatic disease to death or last known follow - up , was estimated using the kaplan - meier method . statistical analyses were performed using r version 3.5. results patient characteristics a total of 116 patients with metastatic nccrcc were included in this cohort ; demographics are listed in table 1 . median age was 53 years ( range , 12 to 77 years ) , and 67% of patients were male . 
patients with unclassied and papillary rcc had similar characteristics : most patients were male ( 78% and 89% , respectively ) and were diagnosed initially with localized disease , followed by subsequent recurrence . in comparison , most patients with chromophobe and translocation - associated rcc were female ( 53% and 69% , respectively )  . 
the median overall survival time for the cohort from time of diagnosis of metastatic disease was 34 months ( 95% ci , 24 to 59 months ) , with a median follow - up of 18 months . somatic mutation frequencies and clinically actionable somatic alterations most patients ( 57% ) had sequencing of the primary renal tumor . 
in comparison , in papillary tumors , the most frequent mutations were tert promoter mutations ( n = 12 ; 50% ) , followed by alterations in met ( n = 9 ; 38% ) and nf2 ( n = 2 ; 17% )  . chromophobe tumors had a distinct prole , with a high frequency of mutations in tp53 ( n = 10 ; 63% )  . translocation - associated tumors all had tfe3 translocations conrmed by uorescence in situ hybridization or ngs but had a paucity of other somatic alterations . we classied somatic alterations as potentially actionable per denitions previously established through oncokb.17 as summarized in table 2 , tumors of 15 patients ( 13% ) harbored an actionable , putative driver somatic mutation assigned an oncokb level of evidence of 2a , 2b , 3a , or 3b ( fig 1 and appendix table a3 )  . 
ten patients had mtor pathway alterations , including in pik3ca ( level 3 ; predictive of response to phosphatidylinositol 3 - kinase inhibitors ) , tsc1 / 2 ( level 2b ; predictive of response to nccn - listed everolimus ) , and mtor ( hypothetical predictor of response to mtor inhibitors ) .18 of these , six patients received an mtor inhibitor alone or in combination with bevacizumab or lenvatinib ( table 3 ) .19 an additional patient with a pten mutation ( level 4 ) was treated with bevacizumab and everolimus . 
median duration on treatment with an mtor inhibitor for the seven patients was 16 months ( range , 1 to 42 months ) , with one patient still on treatment ; three patients experienced a partial response ( all with predicted driver mtor pathway mutations ) , one had stable disease , and three were not evaluable . three patients had amplications in met ( level 3 ; possibly predictive of response to cabozantinib ) , and ve patients had additional driver mutations ; of these , two patients received the vegf and met inhibitor cabozantinib as second - line therapy and were evaluable for response . 
one patient had an alk translocation ( level 2b ; predictive of response to alk inhibitors in lung cancer ) , was enrolled onto a clinical trial with a multitarget alk inhibitor , and , as previously reported , achieved a partial response with 19 months on therapy.20 twenty - eight patients ( 24% ) had a level 4 alteration ( hypothetical biomarker , not proven to including mutations in pten , be clinically actionable ) , smarcb1 , cdkn2a , and atm ( figs 1b and 1c )  . fourteen patients had both the primary tumor and a metastatic lesion sequenced ( and one additional patient had two metastatic sites sequenced )  . 
of these , four patients ( 27% ) had a primary or metastatic site tumor sample with an actionable somatic alteration ( three with tsc2 and one with alk translocation ; appendix table a4 )  . 
additional patients received rst - line treatment elsewhere and are not included in this section . germline mutation analysis of the 116 patients , 45 ( 39% ) underwent germline mutation testing with a panel of 76 cancer - associated genes ; some of these patients were included in a previous report.21 in total , 11 ( 24% ) of 45 patients harbored germline events in cancer - associated genes . 
the most frequently germline mutated gene was fh ( n = 6 ; 13% ) , followed by one patient each who carried mutations in met , atm , chek2 , palb2 , and apc . 
 ( b ) percentages of tumors with somatic alterations by oncokb level of evidence , of tumors with oncogenic drivers without known potentially actionable alterations , and of tumors with no known driver . 
the tcga analyzed papillary and chromophobe tumors in two separate efforts.11 , 12 in contrast with the tcga analyses , where only primary tumors were processed , the patients in our study had primary and metastatic sites sequenced , perhaps better representing the limitations seen in clinic , where primary tumor tissue is either not available or not sampled . 
similar to these authors , 26 we found a predominance of met , tert promoter , and nf2 mutations in papillary tumors ( although unlike the pal et al26 study , we did not distinguish between type 1 and type 2 papillary tumors )  . 
this is consistent with a previous study that showed that nf2 loss in unclassied rcc may be a driver of tumorigenesis and associated with worse clinical outcome.27 the comparatively high proportion of unclassied patients , many with molecular similarities to patients with papillary rcc , reects the evolving denitions of rcc variant , with rccs previously termed high - grade papillary rcc now termed unclassied . using the oncokb classication to annotate individual mutations for their therapeutic potential , we found that 15% of tumors harbored potentially actionable somatic mutations . 
unlike in other cancers such as melanoma or nonsmall - cell lung cancer , in rcc , there are no fdarecognized or nccn - listed biomarkers predictive of response to therapies ( ie , level 1 oncokb ) ; the only exception is high tumor msi , which is predictive of response to pembrolizumab in any cancer type . 
we did nd 10 patient tumors ( 9% ) with a level 2 alteration , indicating a standard care biomarker predictive of response in an fda - approved drug in rcc or another indication . 
oncoprint of the most frequently mutated genes overall and microsatellite instability ( msi ) in tumors of 116 patients with advanced nonclear - cell renal cell carcinoma ( rcc )  . 
genetic alteration type , including germline pathogenic mutations , is indicated at the bottom of the oncoprint . included an alk translocation , met amplications , and tsc1 or tsc2 alterations . 
level 3 indicates compelling clinical evidence supporting the biomarker as predictive of response ( in this or other indication )  . * high microsatellite instability is classied as oncokb level 1 . alterations in mtor and pik3ca . 
most alterations were level 4 , which are hypothetical biomarkers potentially predictive of response on the basis of preclinical data . although these are not considered clinically actionable , they may be biomarkers and may be used as eligibility criteria for early - phase clinical trials . we explored the efcacy of targeted therapies in patients with alterations considered potentially actionable who had received matched treatment . 
although the number of patients was small , 33% of patients had an objective response , and an additional 25% had stable disease , which is notable in a group of diseases for which there are few effective therapies . 
patients with met alterations ( amplications or driver missense mutations ) who received cabozantinib and patients with mtor pathway driver alterations who received everolimus seemed to benet from therapy , such as a patient with a pik3ca driver mutation who had a partial response for more than 42 months on the combination of everolimus and bevacizumab . 
actionable somatic alterations and response to targeted treatment mutation predicted function effect * therapy length on therapy ( months ) best response patient ncc049 ncc108 ncc055 ncc024 ncc031 ncc112 ncc022 ncc051 ncc014 ncc019 ncc035 ncc115 met amplication met missense mtor missense pik3ca missense pik3ca missense tsc1 missense tsc1 truncating tsc2 missense tsc2 truncating alk translocation pten truncating pten truncating driver driver driver driver driver unknown driver unknown driver driver driver driver everolimus + bevacizumab cabozantinib cabozantinib lenvatinib + everolimus everolimus + bevacizumab everolimus + bevacizumab everolimus + bevacizumab everolimus everolimus entrectinib everolimus + bevacizumab everolimus + lenvatinib not evaluable not evaluable not evaluable not evaluable not evaluable note . 
two patients in our study , one with medullary rcc and one with chromophobe rcc , had high msi scores ; neither had received immunotherapy at the time of analysis . 
of interest , 29% of chromophobe tumors were msi - h ( msisensor score of 10 or greater ) or msi - i ( msisensor score between 3 and 9 )  . 
previous analyses of prevalence of msi - h in limited subtypes of nccrcc showed a 1% or lower prevalence of msi - h ; however , in these cohorts , less than 10% of patients had metastatic disease.11 , 12 , 30 , 31 additional investigation to characterize larger cohorts of chromophobe , medullary , and other nccrcc tumors , especially in patients with metastatic disease , is warranted . 
the overall low rate of msi - h in nccrcc should not preclude the possibility of benet from checkpoint inhibitors ; despite msi - h being rare in ccrcc , these tumors are clearly responsive to checkpoint hibitors.32 , 33 in addition , retrospective series in nccrcc table 4 . 
 ( % ) tumor subtype unclassied papillary chromophobe translocation other 1 ( 2 ) 1 ( 4 ) 4 ( 24 ) 1 ( 6 ) 1 ( 8 ) have suggested benet from antiprogrammed cell death protein 1 therapy.34 notably , consistent with previous reports , there was a high proportion of patients with germline mutations , some which could direct therapy.21 of 45 patients receiving germline genetic testing through our protocol , 13% had mutations in fh , which is diagnostic of the syndrome hereditary leiomyomatosis and rcc ( hlrcc ) , which in turn is associated with nccrcc and cutaneous and uterine leiomyomas.21 , 35 identication of patients with hlrcc has become particfor treatment decisions since the 2018 ularly relevant nccn guidelines added two bevacizumab - containing combination therapies ( bevacizumab plus everolimus and bevacizumab plus erlotinib ) as potential treatment for patients with hlrcc.36 in our exploratory analysis of response to bevacizumab and everolimus , most patients had an objective response , with a median progression - free survival nearing 12 months . 
in a phase ii study of a dual met / vegf receptor 2 inhibitor in patients with papillary rcc , the presence of a germline met mutation was highly predictive of response.37 one patient in our cohort harbored a met germline mutation , but although met - directed therapy with cabozantinib was initiated , the patient was subsequently lost to follow - up . of note , there was a high percentage of black patients with nccrcc ( 21% ) in this cohort . 
 carlo et al potential tumor genomic differences.38 , 39 these observations merit additional investigation in larger cohorts . this approach could have missed other nonexonic as well as epigenetic changes although this study represents a large cohort of patients with several subtypes of nccrcc , it is important to recognize its limitations . 
finally , our analysis was limited to targeted exome sequencing , and in summary , in this detailed genomic analysis of a wide array of metastatic nccrcc tumors , matched germline and tumor analyses revealed recurrent genomic events across rcc variants and suggest that a relevant proportion of patients harbor potentially actionable alterations . 
there is a need for additional collaborative studies to characterize metastatic nccrcc further so that targeted therapies can be pursued rationally , exploiting such information . affiliation 1memorial sloan kettering cancer center , new york , ny this manuscript . 
hyman consulting or advisory role : atara biotherapeutics , chugai pharma , cytomx therapeutics , boehringer ingelheim , astrazeneca , pzer , bayer , debiopharm group , genentech research funding : astrazeneca , puma biotechnology , loxo travel , accommodations , expenses : genentech , chugai pharma marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network / astrazeneca tagrisso rfp advisory committee , takeda , bristol - myers squibb , bayer , merck ( i ) research funding : loxo ( inst ) , helsinn therapeutics mark robson honoraria : astrazeneca , pzer consulting or advisory role : mckesson , astrazeneca , merck research funding : astrazeneca ( inst ) , myriad genetics ( inst ) , invitae ( inst ) , pzer ( inst ) , abbvie ( inst ) , tesaro ( inst ) , medivation ( inst ) travel , accommodations , expenses : astrazeneca chung - han lee consulting or advisory role : exelixis , eisai research funding : pzer ( inst ) , eisai ( inst ) , bristol - myers squibb ( inst ) , calithera biosciences ( inst ) , exelixis ( inst ) travel , accommodations , expenses : eisai darren r . 
motzer consulting or advisory role : pzer , novartis , eisai , exelixis , merck , genentech , incyte research funding : pzer ( inst ) , bristol - myers squibb ( inst ) , eisai ( inst ) , novartis ( inst ) , genentech ( inst ) martin h . 
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muller m , ferlicot s , guillaud - bataille m , et al : reassessing the clinical spectrum associated with hereditary leiomyomatosis and renal cell carcinoma syndrome in french fh mutation carriers . 
choueiri tk , vaishampayan u , rosenberg je , et al : phase ii and biomarker study of the dual met / vegfr2 inhibitor foretinib in patients with papillary renal cell carcinoma . 
 gene ctcf ctla4 ctnnb1 cul3 cxcr4 cyld cysltr2 daxx dcun1d1 ddr2 dicer1 * dis3 dnajb1 dnmt1 dnmt3a dnmt3b dot1l drosha dusp4 e2f3 egfl7 egfr * eif1ax ago2 eif4a2 eif4e elf3 ep300 epas1 epcam * epha3 epha5 epha7 ephb1 erbb2 erbb3 erbb4 ercc2 ercc3 ercc4 ercc5 oncogenomics of metastatic nonclear - cell renal cell carcinoma table a1 . 
this study aimed to describe the hr pathway mutations and hrd status and determine their association with treatment response and outcome in patients with pdac . patients and methods we performed a retrospective analysis of tumor samples from patients treated at indiana university for locally advanced or metastatic pdac . 
patients were included if they received gemcitabine plus nanoparticle albumin - bound paclitaxel ( control ) or fluorouracil , oxaliplatin , leucovorin , and irinotecan ( folfirinox ) and had adequate follow - up to assess survival and response to therapy . 
tumor analysis generated a three - biomarker hrd score and mutation data for 44 genes . results ninety - one samples met inclusion criteria , and 78 samples ( formalin - fixed paraffinembedded , n = 15 ; fine - needle aspiration , n = 63 ) generated mutation data . 
hrd analysis was successful for 57 samples ( hrd score : median , 18 ; range , 5 to 61 ) ; the primary cause of failure was low tumor cellularity . 
2018 by american society of clinical oncology introduction pancreatic ductal adenocarcinoma ( pdac ) is the fourth leading cause of cancer - related mortality in the united states.1 most patients present with advanced disease where palliative chemotherapy is minimally effective with high rates of toxicity , and the lack of meaningful benefit associated with targeted therapy continues to be a challenge.2 gemcitabine plus nanoparticle albumin - bound ( nab ) paclitaxel ( g + nab - p ) and fluorouracil , oxaliplatin , leucovorin , and irinotecan ( folfirinox ) are commonly used first - line treatment regimens for advanced disease , with objective response rates of 23% to 31% and median overall survival ( os ) times of , 1 year.3 , 4 kras and tp53 mutations remain the most common molecular abnormalities in pdac , 5 in the absence of effective therapy targeting these mutations . 
genomic analysis has revealed the presence of low - frequency , potentially actionable mutations in patients with pdac6 ; 15% of patients with pdac may carry a mutation in the safi shahda kirsten m . 
therefore , a score of 42 was not chosen as a cutoff in this study ; instead , we decided to evaluate the range of scores and correlate outcome based on hrd scores greater than and less than the median , without intending to establish the median as the cutoff . data collection data were retrieved from a retrospective database ( under a separate irb - approved protocol ) of patients who were diagnosed with pdac and who were partially or completely observed at our institution . 
the data were housed in an oncore ( forte research systems , madison , wi ) database per institutional guidelines . determination of radiographic response and outcome the principal investigator reviewed all images of eligible patients and analyzed response to therapy per response evaluation criteria in solid tumors ( recist ) v1.1. 
median follow - up time for the 16 patients without progression was 9 months ( 11 months for the six patients without progression who were alive at end of study )  . 
pfs and os were calculated from the time of initiating second - line therapy . tissue samples the direct smears prepared from the pancreatic fine - needle aspirates ( fna ) were screened by a cytopathologist ( a.a.i. ) to assure adequacy for molecular testing . 
 formalin - fixed paraffin - embedded ( ffpe ) cell blocks were prepared in a subset of the fnas . the hematoxylin and eosinstained sections cut from those blocks were also examined by a cytopathologist ( a.a.i. ) , and the block was considered acceptable for molecular testing if the estimated tumor cellularity was at least 20% . 
either the entire paraffin cell block or sections cut from the block ( one 4to 5 - mm hematoxylin and eosin slide followed by five consecutive 10 - mm unstained slides ) were submitted for analysis . tissue processing and dna extraction ffpe blocks and fna smears from pretreatment samples were retrieved . 
mutations identified were only included in the analysis if they were classified as deleterious or suspected deleterious on the basis of previously described criteria.21 to calculate the hrd score for samples analyzed by custom hybridization sequencing assay , reads covering snp positions were used to generate allelic imbalance profiles as described previously.14 hrd score was defined as the unweighted sum of loh , tai , and lst scores ( hrd = loh + tai + lst )  . statistical analysis clinical characteristics were compared between treatment groups using nonparametric methods ( wilcoxon rank sum or fishers exact test )  . 
response was coded as a binary variable ( complete response [ cr ] + partial response [ pr ] v stable disease [ sd ] + progressive disease [ pd ] or cr + pr v pd , unless otherwise noted ) , and logistic regression analysis was used to test association with clinical and molecular variables . 
hrd score was dichotomized at the median of 18 ( score , or > 18 ) , and median survival times were estimated using kaplan - meier curves compared by log - rank tests . 
all p values are two - sided , with significance set at p = .05. study approval and ethical considerations this was a retrospective analysis , and at the time of analysis , most patients had died as a result of the advanced nature of their illness . 
patients received care at the discretion of their treating oncologist , with genetic counseling and testing as deemed appropriate . results study population ninety - one patients met inclusion criteria , of whom seven had inadequate tissue for analysis after a second screening process by a pathologist from myriad genetics , 14 had ffpe tissue , and 70 had fna ( fig 1 )  . patients characteristics and treatment patient characteristics are listed in table 1 . 
summary of sample screening and number of samples used in the final analysis . hrd , homologous recombination deficiency . tumor samples ( n = 91 ) final clinical data merged with molecular results ( n = 84 ) inadequate tissue for molecular analysis ( n = 7 ) mutation screen and hrd score failed ( n = 6 ) mutation screen passed ( n = 78 ) brca1 / 2 mutation ( n = 6 ) brca1 / 2 wild type ( n = 72 ) hrd score failed ( n = 1 ) hrd score passed ( n = 5 ) hrd score failed ( n = 20 ) hrd score passed ( n = 52 ) mutant with no hrd score ( n = 1 ) mutants with hrd score ( n = 5 ) wild type with no hrd score ( n = 20 ) wild type with hrd score ( n = 52 ) 5 to 61 , with a median score of 18 . 
six brca1 / 2 mutants were detected in this cohort , and hrd score analysis was successful in five of six patients , with scores ranging from 13 to 61 and a median score of 44 . 
loss of the second allele of the mutant locus via either loh or a second deleterious mutation was observed in three of the six mutant samples , with hrd scores of 43 , 52 , and 53 , respectively . 
in comparison , the median hrd score observed in brca1 / 2 mutation carriers with breast or ovarian tumors was 65 , suggesting that the distribution of hrd scores may differ between pdac and these other tumor types , consistent with our preclinical observations . description of brca mutation functionality and pathogenicity six patients harbored brca mutations , four brca1 mutations and two brca2 mutations . individual mutations and hrd scores associated with those mutations are listed in table 2 . association of hrd with response to folfirinox for the purpose of this study we explored the following two scenarios , which were disease response defined as cr + pr versus sd + pd or cr + pr versus pd ( fig 2 )  . 
the composite variables that were assessed were any pathogenic mutation regardless of the status of the second allele and any brca1 / 2 mutation regardless of the status of the second allele against response , pfs , and os . 
in a subgroup analysis of metastatic status , with or without adjusting for treatment , hrd score was not significant for any of the outcomes ; however , the sample size was not large enough to test whether hrd behaves differently in these two groups . discussion this is the first study to our knowledge to evaluate the relationship between hr gene mutations , hrd score , and response to chemotherapy in patients with pdac . 
higher hrd score was correlated with brca1 / 2 mutations but was not associated with the presence of other hr brca1 mutation brca2 mutation brca1 / 2 wild type brca1 mutation brca2 mutation brca1 / 2 wild type fig 2 . 
 ( a ) complete response ( cr ) or partial response ( pr ) versus stable disease or progressive disease ( pd ) , and ( b ) cr or pr versus pd . 
distribution of homologous recombination deficiency ( hrd ) scores across samples of locally advanced ( la ) and metastatic pancreatic ductal adenocarcinoma ( pdac )  . max , maximum ; min , minimum ; qu , quartile ; sd , standard deviation . time from start of metastatic therapy ( months ) pathway mutations . 
one possible explanation for the absence of an elevated hrd score despite the presence of an hr pathway mutation is the retained functional allele in the affected genes . although conducted retrospectively , our study demonstrates the feasibility of performing genomic analysis on fna samples available from patients with pdac . 
on the contrary , however , this study also highlights the challenge of conducting correlative studies in patients with pdac ; most patients with pancreatic malignancies are diagnosed based on endoscopic ultrasound - guided fna only.22 in addition , high nontumor content , likely as a result of abundant desmoplastic stroma , often led to low dna content and thus a high assay failure rate . interestingly , we experienced a higher failure rate associated with core needle biopsy samples as opposed to fna samples , as a result of higher levels of nontumor dna content , even after macrodissection . 
 despite the low incidence of dna repair deficiency in pdac , it continues to represent an attractive target for drug development.23 several clinical trials using single agents or drug combinations are ongoing . 
in addition , patients with pdac tend to have a higher symptom burden and experience rapid progression after first - line therapy , frequently prohibiting second - line therapy.3 , 4 therefore , the optimal timing for testing such methods to select patients for subsequent therapies will likely be early in the disease process to inform secondline therapy.24 although our study does not address the presence of heterogeneity between the primary and metastatic lesions , some literature exists regarding hrd heterogeneity in other tumor types . for example , a study of patients with triplenegative breast cancer compared hr score in three different areas from the same primary tumor.25 overall , there was no significant difference among these samples , and the authors concluded that heterogeneity for hrd metrics was small and did not influence hrd score or status from three samples in the same tumor . whether hrd is more common in metastatic sites remains unknown in pdac ; however , a higher rate of germline mutations associated with dna repair genes ( without examining hrd score ) was found in metastatic prostate cancer when compared with localized disease.26 this study has limitations . 
it is a single - institution , nonrandomized , retrospective study comparing two commonly used chemotherapy regimens and including patients who had locally advanced and metastatic disease , which may carry different biology and represent a challenge in assessing response to therapy in locally advanced pdac . 
we had a relatively high assay failure rate in both fna and ffpe , although it was higher in the ffpe samples . in conclusion , this study did not demonstrate a correlation between hrd score and radiographic response to platinum - based therapy in patients with pdac . 
although hr gene mutations or hrd score may represent targets for drug development in pdac , larger prospective studies may be needed to evaluate the utility of these markers and their correlation with response to dna - damaging agents . 
for more information about ascos conflict of interest policy , please refer to or ascopubs.org / po / author - center . safi shahda consulting or advisory role : merrimack , bayer research funding : myriad genetics kirsten m . 
timms employment : myriad genetics stock and other ownership interests : myriad genetics patents , royalties , other intellectual property : austria ( e840080 , 3012329 ) , china pr ( zl 201280070358.0 ) , denmark , european patent convention , finland , france , great britain , ireland , the netherlands , spain , sweden , and switzerland ( 2582847 , 3012329 ) , germany ( 602011031723.7 , 3012329 ) , italy ( 502016000131029 , 3012329 ) , japan ( 6117194 ) , new zealand ( 625468 ) , united states ( 9 , 574 , 229 ; 9 , 279 , 156 ; 9 , 388 , 427 ) travel , accommodations , expenses : myriad genetics ashley a . 
 milan radovich no relationship to disclose sulfikar ibrahim no relationship to disclose brian allen employment : myriad genetics , grail stock and other ownership interests : myriad genetics , grail bert h . 
oneil , indiana university school of medicine , indianapolis ; ashley a . ibrahim and sulfikar ibrahim , indiana university health ball memorial hospital , muncie , in ; and kirsten m . 
moore mj , goldstein d , hamm j , et al : erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer : a phase iii trial of the national cancer institute of canada clinical trials group . j clin oncol 25 : 1960 - 1966 , 2007 3 . 
von hoff dd , ervin t , arena fp , et al : increased survival in pancreatic cancer with nab - paclitaxel plus gemcitabine . med 364 : 1817 - 1825 , 2011 n engl j med 369 : 1691 - 1703 , 2013 jones s , zhang x , parsons dw , et al : core signaling pathways in human pancreatic cancers revealed by global genomic analyses . 
risch ha , mclaughlin jr , cole de , et al : population brca1 and brca2 mutation frequencies and cancer penetrances : a kin - cohort study in ontario , canada . 
yang d , khan s , sun y , et al : association of brca1 and brca2 mutations with survival , chemotherapy sensitivity , and gene mutator phenotype in patients with ovarian cancer . 
mccabe n , turner nc , lord cj , et al : deficiency in the repair of dna damage by homologous recombination and sensitivity to poly ( adp - ribose ) polymerase inhibition . 
timms km , abkevich v , hughes e , et al : association of brca1 / 2 defects with genomic scores predictive of dna damage repair deficiency among breast cancer subtypes . 
telli ml , timms km , reid j , et al : homologous recombination deficiency score predicts response to platinumcontaining neoadjuvant chemotherapy in patients with triple - negative breast cancer . 
mills gb , timms km , reid je , et al : homologous recombination deficiency score shows superior association with outcome compared with its individual score components in platinum - treated serous ovarian cancer . 
 future of evidence synthesis in precision oncology : between systematic reviews and biocuration precision medicine refers to the tailoring of interventions to patients using approaches that go beyond traditional clinical characteristics ( eg , age , sex , disease , symptoms , and medical history ) by considering biomarkers consisting of genetic characteristics or molecular profiles.1 in particular , precision oncology ( po ) 2 includes both the development of novel cancer therapies targeting specific changes that occur in some individuals but not others ( eg , inherited mutations or somatic mutations that arise in the carcinogenesis process ) and the stratification of individuals according to existing interventions ( eg , via screening programs tiered by genetic risk or chemotherapies predicted to work for certain molecular profiles ) , which is expected to show improved outcomes in the resulting subgroups . 
 herein , we discuss two approaches for synthesizing evidence for or against the use of specific po interventions , namely , systematic reviews ( srs ) and biocuration , and argue that their engagement with each other could facilitate the timely delivery of appropriate po interventions . cancer is known to be highly heterogeneous , and clinical responses differ among patients . 
 po thus includes a wide variety of applications , including both the use of targeted therapies against particular changes occurring in individual tumorssuch imatinib against the bcr - abl fusion in chronic myeloid leukemia , 3 vemurafenib4 and dabrafenib5 against specific braf mutations in melanoma , and trastuzumab against her2 - postive breast cancer6and the assignment between existing therapy classes via biomarkers that are not direct drug targets . 
this can be based either on the activation of certain pathways , as in the case of egfr inhibitors in kras - wild type colon cancer , 7 , 8 or on gene expression signatures or other molecular characteristics ( ie , oncotype dx test for breast cancer , used to predict recurrence and likely benefit of chemotherapy ) .9 , 10 these biomarkers are well - established and have been approved by the us food and drug administration for specific cancer types . the biomarkers discussed in the previous paragraph are classified as having the highest level of evidence for clinical use in society guidelines ( eg , those jointly put forward by the association for molecular pathology , asco , and the college of american pathologists ) .11 to be meaningful for clinical decision making , biomarkers should have high predictive performance ( ie , they can be used to stratify patients into treatment groups with differential outcomes )  . 
most research to date , however , has assessed whether biomarkers are prognostic ( ie , are associated with a clinical outcome for untreated or standardof - care patients ) .12 it is often the case that the supporting literature includes few if any randomized control trials.13 as a result , lower levels of evidence may be assigned , eg , to biomarkers that predict response to a treatment based on well - powered studies evaluated by expert consensus or that predict response in a different tumor type than that being studied . many conceptual frameworks have been proposed to facilitate clinical decision making by helping end users ( especially patients and clinicians ) assess the evidence for or against the use of medical tests , including biomarkers.14 , 15 with minor variations , the different frameworks involve a stepwise approach that starts with analytic validity ( laboratory reproducibility ) , proceeds to clinical validity ( predictive accuracy , including sensitivity , specificity , and positive and negative predictive values ) and clinical utility ( use of the biomarker leads directly to improvements in outcomes like survival and quality of life ) , and eventually assesses the cost effectiveness of the biomarker . 
comparison of key features of systematic reviews and biocuration feature systematic reviews biocuration overall approach top down ; starts with a key scientific question and develops evidence base around it either top down or bottom up ; starts with a number of scientific questions or allows individual articles to be curated and slotted into categories evidence assessment exhaustive literature searches and systematic multiple frameworks exist assessment of evidence presence of bias systematic assessment of the risk of bias higher chance for bias by selecting studies with delivery approach peer - reviewed article or whitepaper research approach team usually assembled at the start of the project often relies on crowdsourcing and expert review of updating significant lag closer to real time , depending on availability of authors or curators multidisciplinary teams , often exclude or reduce role of primary study authors or subject matter experts due to risk of bias scientists and clinicians , who are frequently subject matter experts , from a variety of fields , and often with an expert panel to resolve conflicting interpretations significant findings database of entries may include summary of findings and links to original studies and other resources entries curators as with all tests in clinical practice , tests related to biomarkers are delivered within given health care settings , and currently , little structured evidence exists in regard to how po can successfully be implemented for particular sets of patients , with different degrees of access to health care , across diverse health care systems . 
the first is the traditional evidence appraisal that applies the well - established concepts of evidence - based medicine ( ebm ) .17 , 18 ebm integrates a physicians clinical experience with scientific evidence that has undergone an sr , which involves extensive surveying of the literature followed by a synthesis of the primary studies.19 the second is biocuration , which refers to the distillation and integration of biologic information from scientific literature and large data sets using databaseor research field specific , controlled vocabularies or ontologies ; it is a cornerstone of bioinformatics.20 both disciplines have the same overarching goal of bringing to patients only the interventions proven to be effective by carefully balancing benefits and harms , doing so in different ways ; srs start with a particular question and systematically develop the evidence base around it , whereas biocuration may be either top - down or bottom - up and usually involves more real - time updating . 
we compare key aspects of these two approaches in table 1 and summarize important feature of specific curated databases in appendix table a1 . the goal of srs is to allow a comprehensive and global view of the available evidence base on a particular question of interest by analyzing primary studies that meet prespecified eligibility criteria via explicit , reproducible methodologies that minimize bias . 
the typical steps involved in an sr include identification of the clinical question , specification of eligibility criteria , systematic search for all studies that meet these criteria , extraction of evidence from eligible studies and assessment of their methodologic rigor , and analysis and qualitative and / or quantitative synthesis ( meta - analysis ) of the extracted data.19 an sr is typically structured around the picots elements : the population toward which the findings will be applicable , the intervention , the comparisons being performed , the clinical outcomes , the timing of the eligible studies , and the clinical setting . 
during the course of decades , a toolbox of methods has been developed in srs and ebm to evaluate key elements of published studies including systematic biases , statistical precision , applicability to a target clinical setting , and others.21 - 23 evidence for specific interventions on the basis of supporting srs is generally considered strong . 
 clinical practice guidelines on the basis of srs that support the use of a genomic test as tier one ( ready to implement in clinical practice24 ) and those that are not based on srs as tier two ( may be useful in the context of informed clinical decision making24 )  . in general , sr teams are multidisciplinary , consisting of clinicians , content experts , methodologists , statisticians , and librarians . 
because researchers may often be influenced by their own investment in the field when interpreting the evidence base , 25 ideally , parties involved in a sr should have no personal vested interests in the topic of the sr . 
although content experts are critical for the clinical interpretation of primary studies and for putting them in context with the broader evidence , their role as authors in srs has been debated.26 however , since srs have various degrees of complexity , relevant sponsors often make decisions on a case - by - case basis by thoroughly reviewing the potential conflicts of individual researchers . 
for example , the agency for healthcare and research quality evidence - based practice centers program has a set of guidelines for how different types of conflicts should be handled and alleviated.27 given that precision medicine is a relatively new and rapidly evolving field with potentially more complex statistical analyses and contextual questions compared to traditional studies , including and relying on content experts is more critical . synthesis of evidence from po studies encounters a number of specific challenges . 
consider for instance a cochrane review of first - line treatments in individuals with egfr - mutated noncurable stage iiib to iv nonsquamous nonsmall - cell lung cancer , which included 19 rcts.28 the overall conclusion was that the tyrosine kinase inhibitor therapies ( erlotinib , gefitinib , and afatinib ) led to improved progression - free survival but not to improved overall survival , whereas the monoclonal antibody cetuximab did not show any improved outcomes . 
while this study included 19 rcts total , comparing targeted therapies to chemotherapy or best supportive care , the number of rcts conducted for each therapy was between two and eight and the study designs , outcome measures , and analyses performed and reported varied . 
srs have also identified existing evidence gaps for other prevision medicine interventions , such as the lack of robust evidence for tailoring smoking cessation interventions on the basis of germline genetic variation.29 many issues with the sr of evidence for po interventions will be solved in time , as more studies accumulate , but it is important to note that the desire for new therapies may be at a historical high given the stated promise of precision medicine and po in particular . 
however , another challenge that will linger is that the definition of precision medicine will continue to make it difficult to satisfy the picots framework as , for example , more drugs are tested separately in different subsets of patients ( eg , if the same biomarker is considered for different tumor types ) or molecular tests rapidly evolve to add more biomarkers or change how they are tested ( eg , protein expression , gene expression , or dna amplification ) .13 new rct designs such as umbrella and basket trials are now being implemented to meet some of these challenges . 
although other study designs may provide additional valuable information on specific treatments , the question of clinical utility , in particular , is difficult to answer in the absence of evidence from rcts . biocuration identifies and summarizes biomedical results , including potentially those from srs , into bioinformatic databases , often by using controlled vocabularies and prespecified standards.20 a text summary of the evidence related to specific scientific questions may also be included , along with links to the original studies or other resources . 
in particular , medical curation focuses on disease associations and genetic factors and includes efforts like the omim ( online mendelian inheritance in man ) database30 as well as specialized databases for particular genes , variants , and diseases . 
clinvar , a public archive of relationships among medically important germline and somatic variants and human phenotypes , was launched in april 2013.31 biocuration generally provides more immediate updating than formal srs , but the lack of a unified systematic framework may lead to only partial or inconsistent results . 
 curators , resulting in a wealth of information but also in issues such as contradictory interpretations and many variants of uncertain significance.32 the clingen ( clinical genome resource ) initiative aims to resolve some of these problems and answer the critical questions of clinical validity , disease causality ( pathogenicity ) , and clinical actionability by standardizing data collection and sharing and implementing an approach for consensus among expert curators.32 many other cancer - specific databases now exist for synthesizing evidence for po approaches , 33 - 37 leading clingen to recently develop recommendations and guidelines for defining cancer somatic variants on the basis of their diagnostic , prognostic , and predictive roles , using evidence of their significance and clinical utility.38 in a translational field such as po , where molecular genomics and clinical practice come together , a particular challenge for biocuration is that each content area has its own detailed terminology ( eg , specific diseases and syndromes can be described using icd - 10 codes39 and snomed terms40 )  . 
however , mapping these terminologies to specific causal mutations , as well as to biomarkers and tratments , can often be a challenge . biocurators come from a wide variety of backgrounds , usually in biology , biochemistry , or medical genetics , and increasingly have interdisciplinary training that includes computer and information sciences , with subject matter experts playing an important role in the field of biocuration.20 , 41 a growing number of clinical laboratories in hospitals and community clinics are now also conducting molecular diagnostic testing to identify sequence variants and inform treatment decisions for patients , in which case the curation is often performed by molecular pathologists as well as clinicians . 
the expert panels used by resources such as clingen to resolve conflicting interpretations and curate variants of unknown significance include medical professionals , medical geneticists , clinical laboratory diagnosticians and molecular pathologists that have a long standing scope of work in the disease gene in question . an example of a curated cancer database is the recently released civic ( clinical interpretation of variants in cancer ) database , 36 which provides associations between drugs and genes or variants in specific cancer types . 
however , the authors state the presence of a bias toward positive associations with treatment outcomes ( 91% of records show support for the use of a therapy ) , which illustrates the absence of a systematic , unbiased framework for identifying the evidence base . 
given that civic considers many different types of studies and that studies of cancer drugs may have many different designs , it is true that it would be extremely challenging to have a fixed list of terms that would be applicable to all studies and easily translatable into picots terminology . in general , literature searches for srs are typically more exhaustive and thorough than for curated databases , where eligible studies tend to be identified by experts in a given field . 
 although currently this may have little direct impact on identifying relevant studies for biocuration , given the small number of clinical po publications , this will eventually become a major issue in the next years , because the field will need to deal with increasing amounts of published and unpublished evidence . 
srs also tend to have more well - defined a priori eligibility criteria that a study has to meet to be considered in the evidence synthesis ; in contrast , biocuration approaches show more variability and rely more on individual experts in the field . 
although biocuration currently lacks a standardized and widely accepted framework to assess the quality of published evidence , several efforts are under way to change this.38 , 42 - 44 beyond trying to develop standardized frameworks for biocuration , a number of other approaches for improving curated databases have been considered , including the use of specific incentives . 
the emphasis on a systematic survey of the literature which includes non - significant results that is present in srs is also not an explicit part of the biocuration philosophy . 
for example , if the ebm community engages the biocuration community , they may find ways of incorporating more elements of the picots framework into existing and future databases . there is often a tension between systematic , unbiased syntheses and assessments versus timely and accessible curated information , and the highly promising field of po is a clear example that shows the difficulties in finding a good solution that considers both of these dimensions . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . approaches , and products , instead of continuing to proceed on parallel paths . 
curated databases will continue to be an initial first stop for many researchers and clinicians , especially because srs may lag years behind current studies , and decisions often need to be made in cases where srs are lacking . 
 however , more training is needed for users to understand that , for example , a higher risk of bias exists for finding more significant associations when consulting a curated po database than when using a sr . 
obrien sg , guilhot f , larson ra , et al ; iris investigators : imatinib compared with interferon and low - dose cytarabine for newly diagnosed chronic - phase chronic myeloid leukemia . 
hauschild a , grob j - j , demidov lv , et al : dabrafenib in braf - mutated metastatic melanoma : a multicentre , open - label , phase 3 randomised controlled trial . 
slamon dj , leyland - jones b , shak s , et al : use of chemotherapy plus a monoclonal antibody against her2 for metastatic breast cancer that overexpresses her2 . 
de roock w , piessevaux h , de schutter j , et al : kras wild - type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab . 
calonge n , klein rd , berg js , et al : recommendations from the egapp working group : does the use of oncotype dx tumor gene expression profiling to guide treatment decisions improve outcomes in patients with breast cancer ?  . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
panagiotou oa , ioannidis jp : primary study authors of significant studies are more likely to believe that a strong association exists in a heterogeneous meta - analysis compared with methodologists . 
greenhalgh j , dwan k , boland a , et al : first - line treatment of advanced epidermal growth factor receptor ( egfr ) mutation positive non - squamous non - small cell lung cancer . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
hunter je , irving sa , biesecker lg , et al : a standardized , evidence - based protocol to assess clinical actionability of genetic disorders associated with genomic variation . 
strande nt , riggs er , buchanan ah , et al : evaluating the clinical validity of gene - disease associations : an evidence - based framework developed by the clinical genome resource . 
uniprot is not disease focused ; omim , clinvar , and clingen are disease agnostic ; the remaining databases are specific to cancer . abbreviations : asco , american society of clinical oncology ; civic , clinical interpretation of variants in cancer ; clingen , clinical genome resource ; cosmic , catalogue of somatic mutations in cancer ; fda , us food and drug administration ; nccn , national comprehensive cancer network ; nih , national institutes of health ; omim , online mendelian inheritance in man ; po , precision oncology . 
nucleic acids res 43 : d204 - d212 , 2015 . landrum mj , lee jm , benson m , et al : clinvar : public archive of interpretations of clinically relevant variants . 
nucleic acids res 44 : d862 - d868 , 2016 forbes sa , beare d , boutselakis h , et al : cosmic : somatic cancer genetics at high - resolution . 
nucleic acids res 45 : d777 - d783 , 2017 . taylor ad , micheel cm , anderson ia , et al : the path ( way ) less traveled : a pathway - oriented approach to providing information about precision cancer medicine on my cancer genome . 
riedlinger , md , phd1 ; aleksander chojecki , md1 , 2 ; hana aviv , phd1 ; david weissmann , md1 ; sonali joshi , ms1 ; susan m . 
hirsheld , md , phd1 , 3 ; and shridar ganesan , md , phd1 introduction the 2016 revision to the who classication of myeloid neoplasms and acute leukemia added myeloid or lymphoid neoplasms with pcm1 - jak2 as provisional entities.1 the t ( 8 ; 9 ) ( p22 ; p24.1 ) translocation was rst described in a patient in 1990 , 2 and this cytogenetic abnormality was subsequently found to result a pcm1 - jak2 fusion gene in 2005.3 approximately 50 patients with lymphoid or myeloid neoplasms associated with t ( 8 ; 9 ) ( p22 ; p24.1 ) pcm1 - jak2 have been reported , and these neoplasms have had an array of presenting diagnoses.4 , 5 common features associated with this translocation are a male predominance , hepatosplenomegaly , and eosinophilia . 
one of the initial reports in the literature describing the t ( 8 ; 9 ) ( p22 ; p24 ) translocation was found in the cutaneous t - cell lymphoma of a patient who initially was reported to have lymphomatoid papulosis , followed by a diagnosis of hodgkin disease , with evidence that all three of these diseases were derived from a common t - cell clone.6 here , we report what we believe is the second patient harboring the pcm1 - jak2 fusion with a history of cutaneous t - cell lymphoma who was later diagnosed with classic hodgkin lymphoma . 
the patient had a left axillary lymph node biopsy performed in may 2010 , which was diagnosed as dermatopathic lymphadenopathy , with t - cell receptor rearrangement studies detecting a clonal t - cell population suggestive of minimal involvement by t - cell lymphoma despite unremarkable morphologic and immunophenotypic ndings . 
computed tomography scan at this time showed multiple enlarged lymph nodes in the chest , abdomen , and pelvis . skin biopsy of the right abdomen during this workup was highly suggestive for a cutaneous t - cell lymphoma , with the vast majority of cells positive for cd3 and cd4 and virtually negative for cd7 and cd8 and a clonal t - cell population detected by t - cell rearrangement studies . 
the patient received eight cycles of bleomycin , etoposide , doxorubicin , cyclophosphamide , vincristine , procarbazine , and prednisone chemotherapy from december 2012 to july of 2013 and did not receive any radiation therapy . 
in june 2015 , the skin condition worsened and became pruritic with a cervical lymph node with increased uptake noted on positron emission tomography scan . two lymph nodes from the right axillary region were biopsied , with one lymph node showing a nodular lymphoid inltrate divided by thick collagenous bands and numerous large atypical cd30 + cells but only a few with reed - sternberg cells or mononuclear variants ( fig 1 )  . 
 ( d ) a 2015 left back skin biopsy showing a brotic , partially necrotic nodule with overlying skin with a dense subepidermoid lymphoid inltrate consistent with cutaneous t - cell lymphoma . biopsy performed at that time showing cutaneous t - cell lymphoma showed the same t - cell clone . 
after completing this therapy , the patient was felt to be stable for approximately 11 months before possible recurrence of transformation of disease with adenopathy in the inguinal region in july 2017 . the patients last scan was september 2017 , which revealed stable disease . 
tissues from the axillary node from december 2012 diagnosed as classic hodgkin lymphoma , nodular sclerosis subtype ; from the axillary lymph node from june 2015 with large atypical cells favored to be cutaneous t - cell lymphoma ; and from the skin from the axillary region in june 2015 diagnosed as cutaneous t - cell lymphoma were all sent for comprehensive genomic proling in june and july of 2015 ( foundation medicine , cambridge , ma )  . 
all three samples showed pcm1 - jak2 fusions and identical single nucleotide variants of unclear function in six genes ( prkdc a1680v , brca2 t2515i , nfe2l2 a480v , cux1 q191p , epha5 g409d , and flt1 s722del ) , which supports these being different manifestations of the same disease process . 
the pcm1 - jak2 fusion is reported to be sensitive to jak inhibitors in vitro , and there is a report of a patient with chronic eosinophilic leukemia harboring this fusion who experienced complete cytogenetic remission after treatment with ruxolitinib.7 the genomic proling results from this patient were presented at our institutional molecular tumor board , with the recommendation for treatment with a jak inhibitor on disease progression . subsequent uorescence in situ hybridization studies performed on the specimen diagnosed as classic hodgkin lymphoma and specimen with large atypical cells favored to be cutaneous t - cell lymphoma showed separation of a jak2 probe indicating a translocation had taken place ( appendix fig a1 )  . 
fish performed in april 2018 showed jak2 rearrangement . completed 16 cycles of brentuximab . august 2016 right axillary lymph node 1 favored to be large - cell transformation of t - cell lymphoma . 
genomic profiling performed in july 2015 with pcm1 - jak2 . fish performed in april 2018 showed jak2 rearrangement . stopped bexarotene and started brentuximab . right axillary lymph node 2 suggestive of involvement of cutaneous t - cell lymphoma . 
clonal t - cell receptor . genomic profiling performed in july 2015 with pcm1 - jak2 rearrangement . inguinal region adenopathy , but otherwise stable disease . subsequently lost to follow - up . 2000 2009 may 2010 may 2014 june 2015 july 2017 fig 2 . 
beacopp , bleomycin , etoposide , doxorubicin , cyclophosphamide , vincristine , procarbazine , and prednisone ; fish , uorescence in situ hybridization ; puva , psoralen and ultraviolet a . the right axillary lymph node atypical nodular lymphoid inltrate and skin biopsy showing cutaneous t - cell lymphoma in june 2015 ( fig 3 )  . 
in total , these results argue that the same underlying disease process was present beginning in may 2010 and potentially earlier and was responsible for all of the patients subsequent lymphoproliferative disorders , including the specimen diagnosed as classic hodgkin lymphoma in december 2012 , which instead seems to have been a cd30 + t - cell lymphoma . discussion comprehensive genomic proling is now routinely performed at major academic medical centers with a major identifying clinically actionable alterations.8 goal of another benet of genomic proling is that repeat testing of specimens from a patient can indicate if they the same underlying clonal process , are a result of possibly with tumor evolution as a result of resistance to therapy , or are the result of separate primary clonal disorders.9 - 11 classic hodgkin lymphoma is dened by the presence of reed - sternberg cells . 
nodular sclerosing is the most common subtype and is characterized by the presence of rare , scattered lacunar reed - sternberg cells , which often compose less than 1% of the tumor cell mass , in a background of reactive lymphocytes , eosinophils , plasma cells , and brosis . 
reed - sternberg cells in classic hodgkin disease are generally believed to originate from b cells , but there are reports that a small fraction may arise from t cells , incidence as low as 1% to 2%.12 , 13 inwith an overall terestingly , several reports in the literature describing hodgkin lymphoma of t - cell derivation report an association with cd30 + lymphoproliferative disorders , whose atypical cells often have a similar morphology to reedsternberg cells.6 , 14 , 15 in addition to the pcm1 - jak2 fusion , two cases of classic hodgkin lymphoma that lacked the b - cell lineage marker pax5 and were positive for tia1 have been reported to harbor an sec31a - jak2 fusion . 
the sec31a - jak2 fusion caused a lethal t - cell lymphoblastic lymphoma and myeloid phenotype in a murine model.16 additional uncharacterized jak2 rearrangements have been reported in lymphomas diagnosed histologically as classic hodgkin lymphoma.17 it is possible that a small fraction of tumors histologically classied as hodgkin disease actually are cd30 + t - cell lymphomas , which may have different optimal treatment strategies and prognosis than standard hodgkin disease . 
in addition to pcm1 - jak2 , sec31a - jak2 is also reported to respond to jak inhibitors , and other jak2 rearrangements may also be targetable with these agents . 
studies of t - cell receptor gene rearrangement were performed on the ( a ) 2010 left axillary lymph node , ( b ) 2012 left axillary lymph node biopsy diagnosed as nodular sclerosing classic hodgkin lymphoma , ( c ) 2012 right abdomen skin biopsy , ( d ) 2015 right axillary lymph node favored to be nodal involvement of the patients t - cell lymphoma , and ( e ) 2015 left back skin biopsy involved by cutaneous t - cell lymphoma . 
a similar clonal product of approximately 263 base pairs ( bp ) was detected using the t - cell receptor v beta and j beta 1 / 2 primer set in all of the samples except the 2010 specimen and the 2012 classic hodgkin lymphoma , which were polyclonal ( left column )  . 
riedlinger , md , phd , rutgers cancer institute of new jersey , 195 little albany st , new brunswick , nj 08901 ; e - mail : gr338@ cinj.rutgers.edu. support supported by national institutes of health grant no . 
riedlinger honoraria : mjh healthcare holdings , gerson lehrman group consulting or advisory role : personal genome diagnostics hana aviv patents , royalties , other intellectual property : patent for markers to detect transformation of barrett esophagus into esophageal adenocarcinoma ( inst ) kim m . 
hirsheld employment : merck stock and other ownership interests : merck travel , accommodations , expenses : merck shridar ganesan employment : merck ( i ) stock and other ownership interests : ibris , inspirata , merck ( i ) consulting or advisory role : inspirata , novartis , roche , foghorn therapeutics , foundation medicine patents , royalties , other intellectual property : i hold two patents for digital imaging that may be licensed to ibris and inspirata travel , accommodations , expenses : inspirata no other potential conicts of interest were reported . references arber da , orazi a , hasserjian r , et al : the 2016 revision to the world health organization classication of myeloid neoplasms and acute leukemia . 
blood 127 : 2391 - 2405 , 2016 stewart k , carstairs kc , dub e id , et al : neutrophilic myelobrosis presenting as philadelphia chromosome negative bcr non - rearranged chronic myeloid leukemia . 
cancer res 65 : 2662 - 2667 , 2005 bain bj , ahmad s : should myeloid and lymphoid neoplasms with pcm1 - jak2 and other rearrangements of jak2 be recognized as specic entities ? br j haematol 166 : 809 - 817 , 2014 tang g , sydney sir philip jk , weinberg o , et al : hematopoietic neoplasms with 9p24 / jak2 rearrangement : a multicenter study . 
mod pathol 32 : 490 - 498 , 2019 davis th , morton cc , miller - cassman r , et al : hodgkins disease , lymphomatoid papulosis , and cutaneous t - cell lymphoma derived from a common t - cell clone . 
n engl j med 326 : 1115 - 1122 , 1992 lierman e , selleslag d , smits s , et al : ruxolitinib inhibits transforming jak2 fusion proteins in vitro and induces complete cytogenetic remission in t ( 8 ; 9 ) ( p22 ; p24 ) / pcm1 - jak2 - positive chronic eosinophilic leukemia . 
oncologist vignot s , frampton gm , soria jc , et al : next - generation sequencing reveals high concordance of recurrent somatic alterations between primary tumor and metastases from patients with non - small - cell lung cancer . 
foss hd , reusch r , demel g , et al : frequent expression of the b - cell - specic activator protein in reed - sternberg cells of classical hodgkins disease provides further evidence for its b - cell origblood 94 : 3108 - 3113 , 1999 13 . 
willenbrock k , ichinohasama r , kadin me , et al : t - cell variant of classical hodgkins lymphoma with nodal and cutaneous manifestations demonstrated by single - cell polymerase chain reaction . 
 ( a ) a 2012 left axillary lymph node biopsy diagnosed as nodular sclerosing classic hodgkin lymphoma showed translocation involving the jak2 gene on chromosome 9p24.1 in 62% of 100 interphase cells . 
 ( b ) in the 2015 right axillary lymph node favored to be nodal involvement of the patients t - cell lymphoma , jak2 translocation was detected in 13% of 100 interphase cells . 
with the jak2 break - apart probe , the normal pattern appears as fusion of red and green ( yellow ) , whereas a translocation shows up as red and green separated on one or more chromosomes 9 . 
 temporal dynamics of genomic alterations in a brca1 germline mutated pancreatic cancer with low genomic instability burden but exceptional response to fluorouracil , oxaliplatin , leucovorin , and irinotecan hui - li wong eric y . 
this approach helped to reconcile the following paradoxical findings : genomic stability and low hrd mutation signature despite a germline brca1 mutation and exceptional response to fluorouracil , oxaliplatin , leucovorin , and irinotecan ( folfirinox )  . 
the primary tumor had been resected previously , followed by 6 months of adjuvant cisplatin and gemcitabine chemotherapy , before detection of liver metastases 12 months after surgery and within 6 months after discontinuing chemotherapy . 
the patient had an excellent response to treatment , with ca19 - 9 halving in 2 months and a complete positron emission tomography response within 4 months ( fig 1a )  . 
renouf , md , mph , british columbia cancer agency , 600 w 10th ave , vancouver , bc v5z 4e6 , canada ; twitter : @zhaoez , @bccancerfdn , @ubcmedicine ; e - mail : drenouf@bccancer.bc.ca. methods mutation signature and timing analysis sample processing , sequencing , and bioinformatics tissue from the primary pancreatic tumor and liver metastasis was sequenced along with matched normal blood . 
the primary samples low tumor content necessitates careful interpretation of variant calls along with orthogonal validation of key findings . patterns of somatic structural variants ( svs ) , single nucleotide variants ( snvs ) , and copy number variants ( cnvs ) were interrogated to determine the contribution of hrd to mutagenesis . 
timing started folfirinox stopped oxaliplatin ( 16 cycles ) complete response on pet week 1 week 20 time since diagnosis of metastatic pdac ( weeks ) 0 1 2 0 1 2 8 6 7 8 6 7 fig 1 . 
within 4 weeks of commencing fluorouracil , oxaliplatin , leucovorin , and irinotecan ( folfirinox ) , ca19 - 9 decreased by > 50% ; positron emission tomography ( pet ) complete response was seen at 20 weeks . 
verification of signit signatures and temporal dissection was performed using deconstructsigs with temporal partioning of mutations into early and late categories ( data supplement ) as described previously.16 data availability wgs data ( binary alignment map files ) have been submitted to the european genome - phenome archive17 under the study accession number egas00001001159 and sample accession egad00001003029 . results brca1 loss in the primary and metastasis both the primary tumor and the metastasis demonstrated low sv burden , which falls into the stable category defined by waddell et al , 18 and possessed < 50 sv events ( figs 1b to 1c )  . 
to our knowledge , this is the first biologic validation of a mutation timing inference model using multiple sequencing time points . metastasis and the 17th of 41 events ( 0 = 0.40 ) in the primary ( figs 2c and 2d )  . 
tumor content in the pancreatic primary was insufficient to estimate clonality . evolution of mutation signatures from primary to metastasis the relative contributions of 30 previously described mutation signatures12 were determined from 5 , 683 snvs in the primary and 8 , 315 in the metastasis . 
signature 3 and , to a lesser extent , signature 8 have been associated with hrd.20 , 21 mutations associated with signature 3 rose by 1 , 593 in the metastasis , and signature 8 rose by 1 , 424 , more than any other signature ( fig 3a )  . 
furthermore , of new somatic mutations ( present in the metastasis but absent in the primary tumor ) , 26% were associated with the hrd signature ( fig 3b ) , which suggests major involvement of hrd - associated mutagenesis in the evolution of this pdac . 
strong signature bleed was observed between signatures 3 and 8 ( data supplement ) , but there was little bleed between hrd and other signatures . evolution of orthogonal hrd - associated mutational signatures the presence of recently described genomic signatures associated with hrd21 was investigated in the primary and metastasis . 
rearrangement signatures 3 and 5 ( fig 4a ) and the fraction of indels with microhomology ( fig 4b ) were low but rose between the primary and the metastasis . 
 the hrd composite score , combining loh , telomeric allelic imbalance , and large - scale transition , increased from 30 in the primary to 38 in the metastasis ( fig 4c )  . mutation signature timing the cnloh event on chromosome 17 that resulted in homozygosity of brca1 was the ninth earliest of 30 events ( 0 = 0.15 ) in the analysis with signit revealed the evolution of mutation signatures between two tumor subpopulations . 
 ( a ) inferred timing of copy change events in the primary tumor and metastasis were strongly correlated , with a slope of 0.45 , intercept of 0.02 , r = 0.7 , and p < .001. 
the loss of heterozygosity event encompassing brca1 has been highlighted , and is the 17th earliest of 41 inferred events in the primary and ninth of 30 events in the metastasis . 
these early mutations accounted for 36% of mutations , whereas later mutations made up 64% . the early subpopulation was dominated by signature 1 , which accounted for 59% of early mutations , whereas signatures 3 , 8 , 9 , and 16 were active in the later subpopulation . 
 ( b ) the complete catalog of new somatic snvs ( present in metastasis but absent in primary tumor ) , with snvs categorized into 96 classes on the basis of variant type and 3 / 5 context , was matched against 30 predefined mutation signatures . 
 ( c ) temporal dissection by signit revealed two tumor subpopulations , which revealed a drop in signature 1 and a rise in signatures 3 and 8 . signatures 3 , 8 , and 9 from the primary to the metastatic sample . 
these findings provide additional evidence that hrd remains an active mutation - causing process in the metastatic tumor , despite the overall low sv burden and moderate hrd signature exposure . discussion previous studies have reported platinum sensitivity in pdacs with brca1 / 2 mutations , rampant svs , and strong mutation signature.18 we explored the genomic evolution of a brca1 germlinemutated pdac with paradoxically low hrd mutation signature and sv burden . 
on the basis of temporal analysis of mutational signatures , we postulate that hrd onset may have occurred too recently or gradually to produce a heavy burden of genomic instability . 
however , rising hrd signature exposure suggests that hrd remains a currently active process , which may explain the patients excellent and sustained response to folfirinox . this analysis has some limitations . 
the archival primary sample had low tumor content ( 25% ) , which is known to limit the accuracy of mutation calling , and thus required analytical validation of the major findings . 
snvs were called by strelka , which is designed to operate under low cellularity.22 despite the low tumor content , timing of cnvs was concordant between the primary and metastasis , and temporal dissection of the metastasis corroborated mutation signature evolution patterns . of note , the patient did not exhibit a dramatic response to adjuvant cisplatin and gemcitabine therapy in the primary setting . 
another explanation is mechanistic differences between cisplatin and oxaliplatin action , 23 which may limit cross - resistance between them as corroborated by differing activities of the two agents across cancer types . 
should the disease progress on folfirinox , we intend to perform a follow - up screening for brca1 reversion27 and associated hrd dynamics . there is growing evidence that hrd may predict response to platinum - based therapy and poly ( adp - ribose ) polymerase inhibitors . 
 ( c ) the total hrd score rose from 30 to 38 , largely driven by a rise in loss of heterozygosity ( loh ) and large - scale transitions ( lsts )  . 
morin consulting or advisory role : epizyme janessa laskin honoraria : boehringer ingelheim , roche canada , astrazeneca , pfizer research funding : astrazeneca ( inst ) , roche canada ( inst ) marco a . 
renouf honoraria : celgene , shire consulting or advisory role : celgene , shire research funding : bayer ag ( inst ) , astrazeneca ( inst ) acknowledgment we thank the patient and his family for participating in the personalized oncogenomics program of british columbia . 
is supported by a bcca - cihr - ubc / phd studentship , a university of british columbia 4 - year doctoral fellowship , and a canadian institutes of health research vanier canada graduate scholarship . references res 18 : 99 - 113 , 2008 nature 408 : 429 - 432 , 2000 1 . 
tutt a , tovey h , cheang mcu , et al : carboplatin in brca1 / 2 - mutated and triple - negative breast cancer brcaness subgroups : the tnt trial . 
laskin j , jones s , aparicio s , et al : lessons learned from the application of whole - genome analysis to the treatment of patients with advanced cancers . 
timms km , abkevich v , hughes e , et al : association of brca1 / 2 defects with genomic scores predictive of dna damage repair deficiency among breast cancer subtypes . 
ha g , roth a , khattra j , et al : titan : inference of copy number architectures in clonal cell populations from tumor whole - genome sequence data . 
boot a , huang mn , ng awt , et al : in - depth characterization of the cisplatin mutational signature in a human cell lines and in esophageal and liver tumors . 
 sequential her2 - targeted therapy in salivary ductal carcinoma with her2 / neu overexpression and a concomitant erbb2 mutation introduction salivary duct carcinoma ( sdc ) is a rare malignancy arising from the major and minor salivary glands.1 , 2 though curative intent surgery followed by radiation is the treatment of choice , locoregional recurrences and distant metastases are frequently reported.1 , 3 cytotoxic chemotherapeutic agents such as platinum analogs , anthracyclines , and taxanes have been effective in treating sdc but there is no established standard of care.4 overexpression of her2 / neu is frequently seen ; thus , trastuzumab can be an effective treatment option.3 however , the role of other her2 - targeting agents after trastuzumab failure is unclear . 
this involved a multiplex polymerase chain reaction using the ion ampliseq cancer hotspot primer pool and the ion ampliseq library kit ( both from thermo fisher scientific , waltham , ma )  . 
 data files were analyzed using the ion torrent variant caller software and integrated genomic viewer ( thermo fisher scientific ) using hg19 reference sequence and nextgene software ( softgenetics , state college , pa )  . 
in addition , three possible germline polymorphisms ( met n375s , tp53 p72r , and flt c.1310 3t > c ) were also detected in the tumor specimen . the patient was treated postoperatively with concurrent radiation to the parotid bed and right neck ( 6 , 600 cgy in 33 fractions over 48 days ) , with high - dose cisplatin ( 100 mg / m2 every 3 weeks for two cycles ) as radiation sensitizer . 
 he underwent a left modified radical neck dissection followed by concurrent radiation ( 6 , 000 cgy in 30 fractions over 42 days to the left neck and 6 , 600 cgy in 33 fractions over 48 days to the axilla ) with weekly carboplatin ( area under the curve , 2 ) because of marginal renal function . 
he had a possible anaphylactic reaction to pertuzumab and hence received trastuzumab monotherapy for seven additional cycles and had a partial response . the patient was disease free for 6 months and then a biopsy - proven recurrence was found on his sk lapatinib treatment was restarted and continued for 9 months ; there was a complete clinical response in his sk the patient is currently on a drug holiday , with no obvious recurrence . 
biopsy - proven skin recurrence of salivary duct carcinoma with complete resolution with lapatinib treatment . two years after the initial recurrence , the patient underwent a wedge resection of a solitary metastasis in the right lower lobe of the lung . nine months later , disease progression was detected in the right axillary and mediastinal lymph nodes . 
 however , because of the response of the sdc to her2 - targeted therapies.3 her2 overexpression is associated with higher recurrence risk , shorter survival , and aggressive clinical behavior with early regional and distant metastasis.2 taxanes and trastuzumab have been used anecdotally in metastatic sdc with her2 / neu overexpression with a good response , 7 but the role of targeted therapy remains unclear . in this case report , we have demonstrated the successful sequential use of her2 - targeted therapy in her2 - overexpressing metastatic sdc . 
after an initial response to trastuzumab docetaxel combination therapy , her2 - targeted agents , with differing mechanisms of action , were used sequentially with good response and prolonged disease control . 
the patient received , sequentially , trastuzumab ( which binds to the extracellular segment of the her2 receptor , neutralizing it and also producing antibody dependent cellular cytotoxicity ) , lapatinib ( which binds to the atp - binding pocket of the her2 protein kinase domain and inhibits tyrosine kinase activity ) , and ado - trastuzumab emtansine ( an antibody - drug conjugate in which trastuzumab inhibits the her2 receptor and the cytotoxic maytansinoid portion of the conjugate is delivered intracellularly and produces cell death )  . 
also , to the best of our knowledge , this is only the second report of ado trastuzumab emtansine use in sdc8 and the first describing sequential her2 targeting in this disease . sequential her2 - directed therapy is well established in metastatic her2 - positive breast cancer , with frontline chemotherapy along with dual her2 - targeted therapy with trastuzumab and pertuzumab followed by other her2 targeted agents.9 a higher absolute erbb2 gene copy number results in shorter time to metastasis in trastuzumab - untreated early breast cancer but longer progression - free survival upon initiation of trastuzumab - based treatment.10 though her2 - positive tumors are highly sensitive to the receptor blockade , resistance has been shown to develop through three main mechanisms : ( 1 ) incomplete blockade as a result of redundant ligands and receptors that enable alternative dimerization patterns ; ( 2 ) reactivation of pathway signaling at or downstream of the receptor layer , such as activating mutations in the pik3ca pathway ; and ( 3 ) use of pre existing or acquired alternate pathways , such as hormone - receptor pathways.11 interestingly , the patient in this case report had an erbb2 v777l mutation in a metastatic specimen before exposure to her2 - targeted therapy . 
the incidence of erbb2 mutations in sdc is uncommon , with a recent series reporting a mutation in four of 37 patients studied.12 all these mutations were in exons 17 through 19 ( namely , i767m , d769y , g776v , and v842i ) , but this series did not identify the v777l mutation . 
the coexistence of her2 amplification and her2 mutation is extremely uncommon because most of the her mutations reported in breast cancer have been seen in patients with her2 neu amplification - negative disease.13 there are limited data on the role of this mutation in breast cancer . 
v777l is present in the proximity of the kinase domain dfg motif of the erbb2 gene ( exon 20 ) and functions as an activating mutation.13 the effects of the erbb2 v777l mutation on response to her2 - targeted therapy are less clear . 
some in vitro studies suggest that this mutation did not affect the sensitivity to her2 - directed therapies in proliferation assays13 ; however , a recent report suggests that the presence of this mutation confers resistance to trastuzumab.14 because the coexistence of her2 amplification and erbb2 mutation is extremely rare , the effect of this combination on response to her2 - targeted therapy is unknown . 
 however , it is possible that the combination of these two events may have led to the excellent response seen in the patient in this report . though this is a single report , we believe that testing for the presence of an erbb2 mutation may be indicated in patients with sdc who have her2 amplification . 
sequential her2 targeting , similar to the breast cancer paradigm , may be a feasible approach in her2 - overexpressing sdcs , especially in those with a concomitant activating erbb2 mutation . 
locati ld , perrone f , cortelazzi b , et al : clinical activity of androgen deprivation therapy in patients with metastatic / relapsed androgen receptor - positive salivary gland cancers . 
van boxtel w , boon e , weijs wlj , et al : combination of docetaxel , trastuzumab and pertuzumab or treatment with trastuzumab - emtansine for metastatic salivary duct carcinoma . 
borley a , mercer t , morgan m , et al : impact of her2 copy number in ihc2 + / fish - amplified breast cancer on outcome of adjuvant trastuzumab treatment in a large uk cancer network . 
 o intrahepatic cholangiocarcinoma : genomic heterogeneity between eastern and western patients jingyu cao , md1 ; jing hu , phd2 ; siqin liu , phd3 ; funda meric - bernstam , md4 ; reham abdel - wahab , md5 ; junjie xu , md6 ; qiang li , md7 ; maolin yan , md8 ; yujie feng , phd1 ; jianzhen lin , md9 ; songhui zhao , msc3 ; jian wang , ms3 ; lawrence n . 
borad , md10 ; kai wang , md , phd3 ; milind javle , md4 ; and haitao zhao , md9 purpose intrahepatic cholangiocarcinoma ( ihcca ) , a global health problem , is increasing in incidence and has differing etiologies worldwide . 
ngs data from asia , where ihcca is most prevalent , are limited . methods comprehensive genomic proling of formalin - xed parafn - embedded tumor tissue from 164 asian and 283 western patients with ihcca was performed using ngs . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction cholangiocarcinoma ( cca ) , a global health problem , constitutes about 10% - 20% of all primary liver cancers worldwide . 
in western countries , including the united states , metabolic syndrome , inammatory bowel disease , primary sclerosing cholangitis , hepatitis c , and alcohol abuse are the main cca risk factors , and the age - standardized incidence rate is 1.6 per 100 , 000.1 the incidence in southeast asia is extremely high , up to 71.3 per 100 , 000 in certain parts of asia , and is associated with liver uke and hepatitis b virus ( hbv ) infection.2 notably , there is an increasing incidence of intrahepatic cholangiocarcinoma ( ihcca ) worldwide , and its etiology is unclear.3 surgical resection is the only potentially curative treatment , but the majority of patients present with an advanced disease stage and have a limited therapeutic options . 
ihcca , in particular , is enriched with a relatively high number of actionable mutations , and early reports indicate several promising leads with targeted therapies for broblast growth factor receptor 2 fusion ( fgfr ) , isocitrate dehydrogenase - 1 ( idh1 ) , braf v600e mutations , and her2 / neu amplication.2 nevertheless , ngs data from asia , where cca is most prevalent , are limited . 
patients with high tmb had alterations in direct dna repair genes and caretaker genes , especially in the asian population . relevance these results may reect differences in disease etiology and are relevant for targeted therapy and immunotherapy trials for patients with intrahepatic cholangiocarcinoma . methods comprehensive genomic proling of formalin - xed parafn - embedded ( ffpe ) tumor tissue blocks from the primary tumor or metastatic lesions obtained from patients with ihcca was performed in 283 patients from a single center in the united states and in 164 patients from china . in the united states , all genomic proling analysis was performed using a clinical laboratory improvement amendments ( clia ) - validated platform ( foundation medicine , cambridge , ma )  . 
in china , genomic proling was performed by using another clia - validated targeted sequencing platform ( origimed , shanghai , china ) .5 a bridging study comparing the 2 platforms indicated high concordance ( 96.3% ; 26 / 27 ) for the same study samples ( results indicated in appendix table a1 )  . 
all ffpe tissue blocks were sectioned and stained with hematoxylin and eosin and reviewed by an expert pathologist to conrm the diagnosis and presence of least 20% of the dna derived from tumor cells . 
this research was approved by the institutional review boards at md anderson cancer center and peking union medical college . hybrid selection and sequencing a custom hybridization capture panel including over 23 , 660 individually synthesized 5 ( cid : 3 ) - biotinylated dna 120 bp oligonucleotides was used to target the exons of cancer - related genes , as well as selected introns of genes frequently rearranged in cancer . 
hybridization capture followed the protocol of hybridization capture of dna libraries using xgen lockdown probes and reagents ( version 3 ; integrated dna technologies , san diego , ca )  . postcapture libraries were mixed together , denatured , diluted , and then sequenced . 
for the purpose of estimation of sequencing error rate , a phix spike - in was added as an external control to measure the percentage of reads with 0 - 4 mismatches , following the method described by manley et al.6 bioinformatics pipeline all types of genetic alterations , including single - nucleotide variant ( snv ) , short and long indels , copy number alterations ( cnas ) , and gene rearrangement , were called using a suite of bioinformatics pipelines.5 analysis of snvs and indels began with the alignment of raw reads to the human genome reference sequence ( hg19 ) with the burrows - wheeler aligner ( v0.62 ; bwa , cambridge , ma ) , followed by polymerase chain reaction ( pcr ) duplicates removal using markduplicates algorithm from picard ( version 1.47 ; cambridge , ma )  . 
local realignment and base quality recalibration for snvs were performed using gatk ( v3.1 - 1 ; cambridge , ma ) and subsequently called by mutect ( v1.7 ; cambridge , ma )  . 
the cnas included : ( 1 ) amplication , dened as an increase in the number of gene segment copies by 8 , and ( 2 ) homozygous deletion , dened as decrease of complete loss of gene segment copies in samples with  . 
2 , 000 or zero bp were collected and used as discordant reads , that is , paired - end reads that could not be closely mapped to a genome reference , with each read of paired reads aligned to the same chromosomes or different chromosomes . 
the breakpoints were double conrmed by blat , and the resulting chimeric gene candidates were annotated . oncogenic genetic mutations statistical analysis was only based on oncogenic genetic variants and the variants of unknown signicance ; lowfrequency ( variant allele frequency , .01 ) variants were excluded . 
data are % unless otherwise specied . abbreviations : fhx , family history ; hav , hepatitis a virus ; hbv , hepatitis b virus ; hcv , hepatitis c virus ; hx , history ; sd , standard deviation . studies exist on the mutations situated on the same genome loci and structural disrupting mutations on tumor suppressor genes , such as truncations , splicing sites , and frameshifts ; ( 3 ) conrmed somatic : somatic mutations recorded on the public genomic database , such as cosmic , and detected at least once in any tumor types . tumor mutation burden and known germline alterations in dbsnp were not counted . 
we classied our patients according to the tmb scores into : ( 1 ) tmb low ( tmb - l ) if the number of mutations per megabase ( mut / mb ) was , 6 , ( 2 ) tmb inthe number of mutations per termediate ( tmb - i ) megabase was between 6 and 9 , and ( 3 ) tmb high ( tmb - h ) if the number of mutations per megabase was 10 . 
the tmb cutoff was dened per prior lung cancer trials.7 , 8 tumor mutation burden ( tmb ) was estimated for 157 us patients and 164 chinese patients with ihcca by counting its somatic mutations , including coding snvs and indels per megabase of the sequence examined . 
 cao et al msi score , we classied the samples as msi high ( msi - h ) , dened as instability in 2 microsatellite loci ; msi low , dened as instability in only 1 loci ; and microsatellite stable , dened as absence of any evidence of microsatellite loci instability . 
furthermore , the chinese patients had a signicantly higher rate of well - differentiated adenocarcinoma compared with their us counterparts ( p = .002 ; table 1 )  . western versus asian ngs comprehensive genomic proling comprehensive genomic proling identied 1 , 007 genetic aberrations ( gas ) in chinese patients compared with 971 gas in us patients ( appendix table a2 )  . 
each patient harbored at least 1 ga , with an average of 6.1 ( range , 1 to approximately 20 ) and 3.4 ( range , 1 to approximately 16 ) gas per tumor in chinese and us patients , respectively . 
as indicated in figure 1 , the most commonly reported gas in chinese patients were tumor protein 53 ( tp53 ; 41.5% ) , kirsten rat sarcoma viral oncogene homolog ( kras ; 28.7% ) , at - rich interactive domain - containing protein 1a ( arid1a ; 18.3% ) , chinese patients ( n = 164 ) us patients ( n = 283 ) dna repair genes china substitution / indel gene amplification gene homozygous deletion fusion / rearrangement truncation fig 1 . 
 ( b ) the afliated chart indicates the alterations among direct and caretaker dna repair genes . mutations are colored according to mutation types of substitution / indel , gene amplication , gene homozygous deletion , truncation , and fusion / rearrangement . 
in our cohorts , we dened dna repair genes to include the 20 most frequent previously reported dna repair gene gas , including direct dna repair genes ( atm , atr , brca1 , brca2 , fanca , fancd2 , mlh1 , msh2 , msh6 , palb2 , pold1 , pole , prkdc , rad50 , and slx4 ) and caretaker genes ( bap1 , cdk12 , kmt2c / mll3 , tp53 , and blm ) .9 remarkably , chinese patients harbored more dna repair gas compared with us patients with ihcca , with predominant gas in tp53 , brca1 / 2 , kmt2c , and rad50 . 
furthermore , there was no signicant difference in the median values of tmb - h , tmb - i , and tmb - l groups among the chinese ( 14 , 8 , and 2 mut / mb , respectively ) and us patients with ihcca ( 19 , 7 , and 3 mut / mb , respectively )  . 
moreover , we identied that the rate of msi - h in chinese and us patients was 1.8% ( n = 3 ) and 0.6% ( n = 1 ) , respectively , and all msi - h patients were associated with high tmb values ( fig 2 )  . we explored the association between dna repair gene gas and tmb , and identied a signicantly higher rate of tmb - i and tmb - h in patients who had combined direct and caretaker dna repair gas compared with patients without dna repair gas among the chinese ( p , .001 ) and us ( p = .05 ) patients with ihcca . 
the advent of ngs offers promising breakthroughs with targeted therapy and immunologic interventions . however , it is important to identify genomic heterogeneity between populations because this will have a signicant impact on precision medicine approaches for this cancer . in our study , we performed comprehensive molecular proling of 164 chinese and 283 us patients with ihcca to explore genomic heterogeneity between populations . 
mutation types are presented in the color key at the bottomss , microsatellite stable ; mut / mb , mutations per megabase . noted important differences between asian and western patients with ihcca . 
through the results , chinese patients had signicantly higher frequency of tp53 as well as kmt2c , brca1 / 2 , ddr , tert , tgfbr2 , rbm10 , nf1 , spta1 , and rb1 gas . 
 ( b ) the correlation between tmb status and dna repair genetic aberrations ( gas ) by presenting the rates of tmb levels in different existence patterns of direct dna repair gas and caretaker gas . genetic diversity could be attributed to variations in the in western underlying disease risk factors ( table 2 )  . countries , cca is associated with metabolic syndrome , inammatory bowel disease , primary sclerosing cholangitis , and hepatolithiasis , whereas liver uke and hbv are important risk factors in asian countries . 
in this study , high tmb was associated with longer progression - free survival.13 also , pembrolizumab has been approved by the food and drug administration for the dna mismatch repair decient ( mmr - d ) cancers based on a phase ii clinical trial that showed a 40% objective response rate and 78% progression - free survival rate in patients with colorectal cancer with mmr - d compared with 0% and 11% , respectively , for mmr - procient patients.14 similar results have been achieved in noncolorectal cancers that included 4 patients ( 44% ) with biliary tract cancer . 
the role of combined poly ( adp - ribose ) polymerase inhibitors and pd - 1 inhibitors in advanced solid tumor is currently under investigation and may be applicable to this subgroup . our study has important limitations . 
in these panels : ( 1 ) the content genes had some dissimilarities ; to address this issue , we ltered out differential genes and only included the 320 overlapped genes into the analysis , thereby ensuring the comparability ; ( 2 ) sequencing platforms were illumina hiseq2500 ( illumina , san diego , ca ) for foundationone and novaseq ( illumina ) for origimed450 , with effective depths of 500 and 700 , both reecting the accuracy of sequencing results17 ; ( 3 ) the tmb algorithm of origimed450 was based on the published algorithm of foundationone , and the genomic alterations were selected similarly18 ; ( 4 ) the origimed450 platform uses blood samples from individual patients as their own control for eliminating the impact of germline polymorphisms , whereas foundationone uses the somatic - germline - zygosity algorithm and databases of dbsnp and exac for the same , and nally ; and ( 5 ) we conducted a bridging study comparing the 2 platforms and showed a high degree of concordance . 
furthermore , we have examined the clinical characteristics of the chinese and us patients and demonstrated that demographic differences between these populations were minor , with most patients having an advanced disease stage . 
there was a higher proportion of hepatitis b exposure in the asian cohort , and the pathophysiologic relationship between the viral infection and somatic mutations in cancer is unknown at this time . future studies to investigate the genomic proling in different populations , taking into consideration the disease risk factors , are warranted . 
data regarding pd - 1 and pd - l1 expression were not available ; therefore , we could not identify variations in immune biomarker expression between the 2 cohorts in this study . 
borad , kai wang , milind javle , haitao zhao administrative support : jingyu cao , jing hu , maolin yan provision of study materials or patients : qiang li , jianzhen lin , lawrence n . 
kwong , haitao zhao collection and assembly of data : jingyu cao , jing hu , siqin liu , reham abdel - wahab , qiang li , maolin yan , yujie feng , jianzhen lin , lawrence n . 
kwong , jinwei hu , fernando carapeto , milind javle , haitao zhao data analysis and interpretation : siqin liu , funda meric - bernstam , reham abdel - wahab , qiang li , songhui zhao , jian wang , lawrence n . 
 ihcca : genomic heterogeneity between eastern and western patients funda meric - bernstam employment : md anderson cancer center honoraria : sumitomo group , dialectica consulting or advisory role : genentech , inection biosciences , pieris pharmaceuticals , clearlight diagnostics , darwin health , samsung bioepis , spectrum pharmaceuticals , aduro biotech , origimed , xencor , debiopharm group , mersana , seattle genetics , silverback therapeutics , immunomedics , ibm watson health , f . 
hoffman - laroche , ibm watson health , pact pharma , effector therapeutics speakers bureau : chugai biopharmaceuticals research funding : novartis ( inst ) , astrazeneca ( inst ) , taiho pharmaceutical ( inst ) , genentech ( inst ) , calithera biosciences , debiopharm group ( inst ) , bayer ( inst ) , aileron therapeutics ( inst ) , puma biotechnology ( inst ) , cytomx therapeutics ( inst ) , jounce therapeutics ( inst ) , zymeworks ( inst ) , curis ( inst ) , pzer ( inst ) , effector therapeutics ( inst ) , abbvie ( inst ) , boehringer ingelheim ( i ) , guardant health ( inst ) , daiichi sankyo ( inst ) , glaxosmithkline ( inst ) , seattle genetics ( inst ) travel , accommodations , expenses : taiho pharmaceutical , seattle genetics , beth israel deaconess medical center songhui zhao employment : origimed lawrence n . 
borad stock and other ownership interests : gilead sciences , aveo , intercept pharmaceuticals , spectrum pharmaceuticals consulting or advisory role : g1 therapeutics , fujilm ( inst ) , agios ( inst ) , insys therapeutics ( inst ) , novartis ( inst ) , arqule ( inst ) , celgene ( inst ) , inspyr therapeutics , halozyme ( inst ) , pieris pharmaceuticals ( inst ) , taiho pharmaceutical ( inst ) , immunovative therapies , exelixis , lynx group , genentech , western oncolytics , klus pharma , denovo , merck , imvax research funding : boston biomedical ( inst ) , mirna therapeutics ( inst ) , senhwa biosciences ( inst ) , medimmune ( inst ) , biolinerx ( inst ) , agios ( inst ) , halozyme ( inst ) , celgene ( inst ) , threshold pharmaceuticals ( inst ) , toray industries ( inst ) , dicerna ( inst ) , sillajen ( inst ) , eisai ( inst ) , taiho pharmaceutical ( inst ) , emd serono ( inst ) , isis pharmaceuticals ( inst ) , incyte ( inst ) , sun biopharma ( inst ) , ariad ( inst ) , imclone systems ( inst ) , qed therapeutics ( inst ) , incyte ( inst ) , puma biotechnology ( inst ) , adaptimmune ( inst ) , merck serono ( inst ) , redhill biopharma ( inst ) , basilea ( inst ) travel , accommodations , expenses : arqule , celgene , astrazeneca kai wang employment : origimed leadership : origimed milind javle consulting or advisory role : qed , oncosil , incyte , mundi other relationship : rafael pharmaceuticals , incyte , pieris pharmaceuticals , merck , merck serono , novartis , seattle genetics , beigene , qed therapeutics , bayer no other potential conicts of interest were reported . references chun ys , javle m : systemic and adjuvant therapies for intrahepatic cholangiocarcinoma . 
curr treat options oncol 17 : 58 , 2016 saha sk , zhu ax , fuchs cs , et al : forty - year trends in cholangiocarcinoma incidence in the u.s. : intrahepatic disease on the rise . 
nat genet 44 : 690 - 693 , 2012 cao j , chen l , li h , et al : an accurate and comprehensive clinical sequencing assay for cancer targeted and immunotherapies . 
n engl j med 378 : 2093 - 2104 , 2018 rizvi na , mazi `eres j , planchard d , et al : activity and safety of nivolumab , an anti - pd - 1 immune checkpoint inhibitor , for patients with advanced , refractory squamous non - small - cell lung cancer ( checkmate 063 ) : a phase 2 , single - arm trial . 
lancet oncol 16 : 257 - 265 , 2015 chae yk , anker jf , bais p , et al : mutations in dna repair genes are associated with increased neo - antigen load and activated t cell inltration in lung adenocarcinoma . 
oncotarget 9 : 7949 - 7960 , 2017 jusakul a , cutcutache i , yong ch , et al : whole - genome and epigenomic landscapes of etiologically distinct subtypes of cholangiocarcinoma . 
chan - on w , nairism agi ml , ong ck , et al : exome sequencing identies distinct mutational patterns in liver uke - related and non - infection - related bile duct cancers . 
clarke b , tinker av , lee ch , et al : intraepithelial t cells and prognosis in ovarian carcinoma : novel associations with stage , tumor type , and brca1 loss . 
ali sm , yao m , yao j , et al : comprehensive genomic proling of different subtypes of nasopharyngeal carcinoma reveals similarities and differences to guide pathol 22 : 393 - 402 , 2009 gynecol oncol 141 : 293 - 302 , 2016 targeted therapy . 
the respected blocks were sectioned at 5 mm ; the tumor content size and were conrmed by a trained pathologist , and a minimum of 5 unstained slides were submitted for conrmation using the origimed platforinstitutional review board approval was obtained for this study . 
 o tissue / site - agnostic study of ribociclib for tumors with cyclin dcdk4 / 6 pathway genomic alterations : a phase ii , open - label , single - arm basket study julio peguero , md1 ; davendra p . 
grilley - olson , md6 ; vivek subbiah , md7 ; lincoln nadauld , md , phd8 ; das purkayastha , phd9 ; erica stealey , ms9 ; alejandro d . 
kang , phd9 ; and joseph paul eder , md10 purpose as part of the novartis signature program , this study evaluated the efcacy of ribociclib ( selective cyclin - dependent kinase 4 / 6 [ cdk4 / 6 ] inhibitor ) in patients with cyclin dcdk4 / 6 pathwayaberrant tumors . methods this was a phase ii , single - arm , signal - seeking study in patients with advanced malignancies that had progressed on or after standard treatment . 
clinical benet ( dened as the proportion of patients with response or stable disease at 16 weeks ) was the primary end point . results from 61 centers in the united states , 106 patients ( median age , 62.5 years ) were enrolled across multiple malignancies . 
in patients with solid tumors , the clinical benet rate was 18.1% ( n = 19 of 105 ) and the overall response rate was 2.9% ( n = 3 of 105 ) ; three partial responses occurred in patients with adenocarcinoma ( unknown primary ) , soft tissue sarcoma , and urothelial carcinoma . 
2019 by american society of clinical oncology introduction cyclin - dependent kinases 4 and 6 ( cdk4 / 6 ) , in conjunction with their protein regulator cyclin d1 ( encoded by ccnd1 ) , phosphorylate the retinoblastoma ( rb ) protein and regulate g1 cell - cycle progression . 
the p16 ( cdkn2a ) and p15 ( cdkn2b ) tumor suppressors and negaproteins are critical tive regulators of this pathway , mediating cellular senescence.1 , 2 arf ( p19 ) plays an important role in the p53 / mouse double minute 2 homolog ( mdm2 ) - dependent checkpoint ; arf expression leads to activation and stabilization of p53 by promoting mdm2 degradation.3 , 4 pathway alteration can result including from multiple mechanisms , rb mutation or loss , increased signaling through cdk4 and cdk6 amplication , cyclin d1 overexpression , and loss of inhibitors.1 cdk pathway alterations contribute to tumor proliferation and genomic instability , and have been linked to poorer clinical outcomes in multiple cancers . 
cdk4 / 6 and cdkn2a / b abnormalities were reported in 22.8% of patients with various cancers who underwent molecular proling by targeted next - generation sequencing.2 ribociclib is a selective cdk4 / 6 inhibitor that induces dose - dependent rb dephosphorylation , reducing cell proliferation via g1 arrest . 
ribociclib is approved in combination with an aromatase inhibitor as initial endocrine - based therapy for postmenopausal women with hormone receptorpositive / human epidermal growth factor receptor 2 ( her2 ) - negative advanced or metastatic breast cancer . 
 peguero et al context key objective to determine if tumor cyclin dcyclin - dependent kinase 4 / 6 ( cdk4 / 6 ) pathway alterations are an effective signal of efcacy for treatment with ribociclib , a selective cdk4 / 6 inhibitor . knowledge generated in this phase ii , signal - seeking study , 105 patients with heavily pretreated cyclin dcdk4 / 6 pathwayaberrant solid tumors received ribociclib treatment . 
although the primary end point of the study was not met , the clinical benet rate was 18.1% , with partial responses observed in three patients with single gene amplications in the cyclin dcdk4 / 6 pathway . relevance a deeper understanding of the molecular context of tumors is required to accurately match patients to ribociclib , either as a monotherapy or in combination with agents that target interconnected signaling pathways . we present ndings from a signal - seeking study that assessed ribociclib as part of the novartis signature program ( clinicaltrials.gov identier : nct02187783 ) , comprising a series of signal - nding basket protocols in which different single - agent targeted therapies were investigated in a tissueor site - agnostic indication , mutation - specic manner.6 , 7 in this study , patients preidentied as having cdk4 / 6 pathwayaberrant tumors were matched to ribociclib monotherapy . methods study design this was a phase ii , open - label , singletreatment arm , basket study in patients with solid tumors or hematologic malignancies with cdk4 / 6 pathway alterations and whose disease had progressed on or after standard treatment . patients received ribociclib 600 mg , once daily , on a 3week - on / 1 - week - off schedule until disease progression ( by investigator assessment ) , unacceptable toxicity , death , or discontinuation from study treatment ( eg , consent withdrawal , starting a new antineoplastic therapy , investigator decision )  . 
ribociclib dose was reduced to 400 mg , and then to 200 mg if an additional dose reduction was required ; ribociclib dose could not be reescalated . the primary end point was the clinical benet rate assoinvestigator asciated with ribociclib treatment by local sessment , dened as the proportion of patients achieving a complete response ( cr ) , partial response ( pr ) , or stable disease ( sd ) at 16 weeks or later by response evaluation criteria in solid tumors 1.1. 
patients who discontinued treatment before week 16 for reasons other than progressive disease ( pd ) were considered nonevaluable ; patients who had pd before week 16 were considered to have no clinical benet . 
overall response rate ( orr ; ie , cr or pr ) by local investigator assessment was the key secondary end point . patient eligibility criteria patients at least 18 years old with solid tumors or hematologic malignancies with cdk4 / 6 mutations or amplications , cyclin - d1 / d3 gene amplications , or p16 gene mutations or loss were enrolled . 
patients had at least one prior treatment of their recurrent , metastatic , and / or locally advanced disease and had no remaining standard therapy options expected to result in durable response . 
because of overlapping disease - oriented studies , the following tumor types were excluded : breast cancer ( except triple negative ) , liposarcoma , teratoma , castrate - resistant prostate cancer , melanoma , and mantle cell lymphoma . 
all patients gave written informed consent before treatment , according to federal and local stitutional guidelines . genomic proling gene aberrations were assessed in a local clinical laboratory improvement amendmentscertied laboratory before patient consent . 
once a patient was identied , vestigators contacted novartis study enrollment consideration.8 post hoc broad molecular proling using a next - generation dna sequencing panel of more than 280 cancerassociated genes9 was performed centrally on pretreatment tumor biopsy specimens from all patients . 
 mutation - specic , tissue / site - agnostic study of ribociclib statistical analysis tumor cohorts were formed when four or more patients with a particular tumor type were enrolled . 
the number of patients enrolled per tumor type was based on a patientsparing , adaptive design , allowing early closure of nonresponding groups and / or groups with early success . clinical benet was evaluated using a bayesian adaptive statistical design that allowed dynamic borrowing of information across cohorts on the basis of their degree of similarity.8 the statistical design used a hierarchical model that allowed dynamic borrowing of information between groups . 
this design allowed analyses with small sample sizes for each cohort . at least 10 patients were required to evaluate early futility , and at least 15 to evaluate early success and make a go or no - go decision for each tumor type cohort . 
if there was an 80% or greater probability that the response rate exceeded the historical rate , the group was considered a success . orr , pr , or cr on the basis of local investigator assessment were summarized , and 95% exact cis using the clopperpearson method were also reported.8 correlation between a specic genomic ( somatic ) dna prole , based laboratory analysis , and clinical benet was on central explored . 
in addition , ribociclib safety and tolerability were reported . results patients in total , 106 patients from 61 centers in the united states were enrolled across multiple malignancies between august 25 , 2014 , and may 4 , 2015 . 
of the 105 patients with solid tumors , 19 ( 18.1% ) achieved clinical benet ( primary end point ) ; the orr ( key secondary end point ) was 2.9% ( three prs ; table 2 )  . 
the rst patient , a 59 - year - old man with poorly differentiated adenocarcinoma of unknown primary with cdk6 amplication , achieved a pr ( 100% reduction in target lesions ) after the fourth cycle of ribociclib and was continuing therapy at last data cutoff with a duration of response of + 796 days . 
the second patient , a 25year - old man with poorly differentiated , round blue cell soft tissue sarcoma not otherwise specied with cdk4 amplication , had a pr ( 100% reduction ) after two cycles of ribociclib , with a duration of response of 330 days . 
the third patient , a 53 - year - old man with urothelial carcinoma of the bladder with ccnd1 amplication , also experienced a pr ( 37.5% reduction ) after two cycles of ribociclib , with a duration of response of 254 days . 
his bladder cancer had not responded to either rst - line cisplatin plus gemcitabine combination or apatorsen ( ogx - 427 ) an experimental antisense inhibitor targeting heat shock protein 27 . 
one patient with papillary serous ovarian carcinoma with cdk6 mutation ( not conrmed by central review ) experienced a tumor burden reduction of at least 30% , without subsequent conrmation , and had sd for 239 days . 
 ( % ) unless otherwise indicated . abbreviations : ecog , eastern cooperative oncology group ; ps , performance status . * gastroesophageal junction ( n = 4 ) , head and neck nonsquamous cell carcinoma ( n = 4 ) , unknown primary site ( n = 4 ) , uterus ( n = 4 ) , esophagus ( n = 3 ) , ovarian ( n = 3 ) , skin nonmelanoma ( n = 3 ) , cholangiocarcinoma ( n = 2 ) , colorectal ( n = 2 ) , renal cell carcinoma ( n = 2 ) , liver ( n = 2 ) , neuroendocrine ( n = 2 )  . 
other histologies ( n = 10 ) only included one each . based on local molecular proling . a patient can be counted in multiple categories ; therefore , the overall count can be  . 
100. 4 2019 by american society of clinical oncology there was no association between any specic genomic ( somatic ) dna prole and clinical benet per central analysis ( fig 3 )  . 
however , 18 of 19 patients ( 94.7% ) with clinical benet had tumors with a single hit in the cyclin d - cdk4 / 6 pathway and one patient with clinical benet had multiple mutational events ; patients with a pr had tumors with cdk4 / 6 or ccnd1 amplication . 
in total , 86 patients had a single cyclin dcdk4 / 6 pathway mutation and 10 patients had multiple pathway mutations ; central mutation analysis was not available in nine patients . 
the most common aes suspected to be related to the study drug included fatigue ( 31.1% ) , neutropenia ( 30.2% ; decreased neutrophils , 15.1% ) , and nausea ( 29.2% ; table 3 )  . 
of these , fatigue and nausea were typically mild . the most common grade 3 / 4 ae suspected to be related to the study drug was neutropenia ( 18.9% ; n = 20 ; decreased neutrophils , 7.5% , n = 8 ) ; only one incident of febrile neutropenia ( grade 3 ) occurred , but it was effectively managed with supportive care and antibiotics . 
 mutation - specic , tissue / site - agnostic study of ribociclib screened ( n = 176 ) enrolled ( n = 106 ) excluded * unacceptable laboratory value unacceptable test result unacceptable medical history intercurrent medical event did not meet diagnostic / severity criteria use of excluded medications patient withdrew consent other ( n = 70 ) ( n = 22 ) ( n = 15 ) ( n = 6 ) ( n = 6 ) ( n = 7 ) ( n = 8 ) ( n = 2 ) ( n = 10 ) still receiving study drug ( n = 1 ) discontinued disease progression patient / guardian decision adverse event death protocol deviation physician decision ( n = 105 ) ( n = 80 ) ( n = 9 ) ( n = 8 ) ( n = 3 ) ( n = 3 ) ( n = 2 ) consented for survival follow - up ( n = 89 ) death lost to follow - up ( n = 58 ) ( n = 31 ) fig 1 . 
as such , more patients have been identied with potentially actionable pathway activation , raising the hope of personalized medicine through targeted drug treatment.10 , 11 the design of basket trials enables molecularly driven enrollment and treatment assignment , with simultaneous testing of a single targeted agent in tumors of different histologies that harbor genetic alterations in the targeted pathway.12 - 14 the signature program was built on this approach and included eight agentspecic , signal - seeking phase ii basket trials of various targeted therapies in a histology - agnostic , mutation - specic manner , matching patients with potentially actionable mutations to the corresponding agent . 
the aim of the signature program was to support or refute the hypothesis that driver mutations determine therapeutic response to treatment in a histology - agnostic manner . this tissueor site - agnostic approach was recently evaluated in patients with solid tumors and led to the us food and drug administration granting accelerated approval to pembrolizumab for the rst tissueor site - agnostic indication.15 indeed , the prospects for the tissueor siteagnostic approach in oncology have never looked brighter , as the successful stories of larotrectinib and entrectinib continue to unfold.16 moreover , the food and drug administration has also recently granted marketing approval to several diagnostics based on next - generation sequencing : the praxis extended ras panel ( illumina , san diego , ca ) , the oncomine dx target test ( thermo fisher scientic , waltham , ma ) , and the foundationone cdx ( foundation medicine , cambridge , ma ) .17 - 19 these tests , and others under clinical investigation , could identify which patients with pathway - aberrant tumors may benet from targeted treatment options . 
thus , the aim for the present ribociclib signal - seeking study was to match patients with cdk4 / 6 pathway - aberrant tumors with the selective cdk4 / 6 inhibitor ribociclib . the primary end point of the study was the rate of clinical benet associated with ribociclib monotherapy , based on local investigator assessment . 
prs were observed in three patients , and 16 patients had sd for 16 weeks or longer . two patients with pr had the maximum percentage reduction from baseline in target lesions ( 100% )  . 
unlike the mutations affecting rb activation , cdkn2a mutation or loss concomitantly decreases the critical function of the p53 - stabilizing effect of the arf protein on the ubiquitin ligase mdm2 , mimicking many mutations in tp53 . 
other selective cdk4 / 6 inhibitors , palbociclib and abemaciclib , have shown low - rate monotherapy activity outside of breast cancer.20 - 23 similarly , a few extraordinary long - term responses have been observed with these agents in tumors with ccnd1cdk4 / 6 amplications . 
these observations may be partly due to the molecular context of the tumor . cyclin dcdk4 / 6 pathway activity is regulated by a number of mitogenic signals , including epidermal growth factors , signaling through the her2 and estrogen receptors , and the phosphoinositide 3 - kinase ( pi3k ) akt pathway.24 studies have suggested that response to targeted therapy may be affected by the presence of tumor mutations in related signaling pathways . 
 mutation - specic , tissue / site - agnostic study of ribociclib chromosome head and neck nonsquamous - cell carinoma head and neck squamous - cell carinoma adrenals bladder breast , triple negative cervix cholangiocarcinoma chordoma colorectal esophagus gallbladder ducts gastroesophageal junction gist kidneys liver lung , nonsmall - cell nonadenocarcinoma lung , nonsmall - cell adenocarcinoma lung , nonsmall - cell squamous lymphoma mesothelioma neuroendocrine ovarian pancreas penile prostate salivary gland sarcoma skin , nonmelanoma thyroid unknown primary uterus shrinkage mutation amplication loss rearrangement short variant fig 3 . 
the most common grade 3 / 4 aes suspected to be related to ribociclib was neutropenia ( 18.9% ) and decreased neutrophils ( 7.5% ) ; only one incident of febrile neutropenia ( grade 3 ) was reported , which was effectively managed with supportive care and antibiotics . 
no deaths due to aes were suspected to be related to study drug per the investigator . one study limitation was the restriction of cohorts by tumor type rather than molecular subtype . 
a second limitation was the exclusion of the most obvious indications for single - agent cdk4 / 6 inhibition : hormone - sensitive breast cancer , liposarcoma , teratoma , castrate - resistant prostate cancer , melanoma , and mantle cell lymphoma . 
this exclusion was due to overlapping disease - oriented studies in these indications , which already were in place by the time this tissueor site - agnostic study started . in conclusion , although no tumor - type cohort graduation was observed , preliminary activity was seen in tumors with single - hit ccnd1cdk4 / 6 amplications . 
these ndings investigation and suggest that in the warrant additional absence of ccnd1cdk4 / 6 amplications or in patients with multiple mutational events , cdk4 / 6 inhibition may require combination with other agents that provide additive or synergistic effects . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . julio peguero employment : oncology consultants leadership : oncology consultants davendra p . 
sohal honoraria : foundation medicine consulting or advisory role : perthera , ability pharma research funding : novartis ( inst ) , celgene ( inst ) , oncomed ( inst ) , bayer ( inst ) , genentech ( inst ) , bristol - myers squibb ( inst ) , agios ( inst ) , incyte ( inst ) , loxo ( inst ) bert h . 
oneil employment : eli lilly consulting or advisory role : bristol - myers squibb , merck karen kelly honoraria : merck consulting or advisory role : astrazeneca , roche , eli lilly , regeneron , abbvie , janssen , emd serono , merck , pzer , astrazeneca research funding : emd serono ( inst ) , genentech ( inst ) , abbvie ( inst ) , five prime therapeutics ( inst ) , regeneron ( inst ) patents , royalties , other intellectual property : author royalties for uptodate travel , accommodations , expenses : astrazeneca , roche , merck , regeneron , eli lilly , abbvie , emd serono , merck juneko e . 
grilley - olson consulting or advisory role : bayer , chimerix research funding : nanocarrier ( inst ) , genentech ( inst ) , seattle genetics ( inst ) , medimmune ( inst ) , pzer ( inst ) vivek subbiah consulting or advisory role : medimmune research funding : novartis ( inst ) , glaxosmithkline ( inst ) , nanocarrier ( inst ) , northwest biotherapeutics ( inst ) , roche ( inst ) , berg pharma ( inst ) , bayer ( inst ) , incyte ( inst ) , fujilm ( inst ) , pharmamar ( inst ) , d3 oncology solutions ( inst ) , pzer ( inst ) , amgen ( inst ) , abbvie ( inst ) , multivir ( inst ) , blueprint medicines ( inst ) , loxo ( inst ) , vegenics ( inst ) , takeda ( inst ) , alfasigma ( inst ) , agensys ( inst ) , idera ( inst ) , boston biomedical ( inst ) , inhibrx ( inst ) , exelixis ( inst ) travel , accommodations , expenses : pharmamar , bayer lincoln nadauld stock and other ownership interests : toma biosciences , citizen corporation consulting or advisory role : roche speakers bureau : genentech patents , royalties , other intellectual property : navican travel , accommodations , expenses : roche , chugai pharma das purkayastha employment : novartis pharmaceuticals erica stealey employment : novartis stock and other ownership interests : novartis alejandro d . 
clin cancer res 21 : 2905 - 2910 , 2015 kato s , schwaederle m , daniels ga , et al : cyclin - dependent kinase pathway aberrations in diverse malignancies : clinical and molecular characteristics . 
cell cycle 14 : 1252 - 1259 , 2015 zhang y , xiong y , yarbrough wg : arf promotes mdm2 degradation and stabilizes p53 : arf - ink4a locus deletion impairs both the rb and p53 tumor suppression pathways . 
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n engl j med 375 : 1738 - 1748 , 2016 kang bp , slosberg e , snodgrass s , et al : the signature program : bringing the protocol to the patient . 
n engl j med 377 : 62 - 70 , 2017 slosberg ed , kang bp , peguero j , et al : signature program : a platform of basket trials . 
oncotarget 9 : 21383 - 21395 , 2018 frampton gm , fichtenholtz a , otto ga , et al : development and validation of a clinical cancer genomic proling test based on massively parallel dna sequencing . 
lopez - chavez a , thomas a , rajan a , et al : molecular proling and targeted therapy for advanced thoracic malignancies : a biomarker - derived , multiarm , multihistology phase ii basket trial . 
patnaik a , rosen ls , tolaney sm , et al : efcacy and safety of abemaciclib , an inhibitor of cdk4 and cdk6 , for patients with breast cancer , non - small cell lung cancer , and other solid tumors . 
goldman jw , shi p , reck m , et al : treatment rationale and study design for the juniper study : a randomized phase iii study of abemaciclib with best supportive care versus erlotinib with best supportive care in patients with stage iv non - small - cell lung cancer with a detectable kras mutation whose disease has progressed after platinum - based chemotherapy . 
dickson ma , schwartz gk , keohan ml , et al : progression - free survival among patients with well - differentiated or dedifferentiated liposarcoma treated with cdk4 inhibitor palbociclib : a phase 2 clinical trial . 
edelman mj , redman mw , albain ks , et al : a phase ii study of palbociclib ( p ) for previously treated cell cycle gene alteration positive patients ( pts ) with stage iv squamous cell lung cancer ( scc ) : lung - map sub - study swog s1400c . 
ther adv med oncol 10 : 1 - 15 , 2018 janku f , hong ds , fu s , et al : assessing pik3ca and pten in early - phase trials with pi3k / akt / mtor inhibitors . 
kim s , tiedt r , loo a , et al : the potent and selective cyclin - dependent kinases 4 and 6 inhibitor ribociclib ( lee011 ) is a versatile combination partner in preclinical cancer models . 
carbone , md , phd4 immunotherapy , the advent of including immune checkpoint inhibitors ( icis ) , has contributed to improved survival in patients with lung cancer.1 three antiprogrammed death - 1 ( pd - 1 ) / programmed death ligand 1 ( pd - l1 ) therapies , nivolumab , pembrolizumab , and atezolizumab , are approved by the us food and drug administration ( fda ) for the treatment of patients with advanced nonsmall - cell lung cancer ( nsclc ) in the rst - line setting.2 - 4 despite the improvements in survival seen with icis , there remains a subset of patients who do not benet from ici monotherapy or combination treatments.5 as a result , many research efforts have been initiated to identify biomarkers predictive of response to treatment with icis . 
predictive biomarkers that have entered clinical practice include pd - l1 expression on tumor and immune cells , and tumor mutational burden.2 - 4 , 6 , 7 however , the accuracy of predictions of patient response based on current biomarkers leaves ample room for improvement . 
successful attempts will likely rely on integrated models combining multiple features . the scientic to tackle such complex challenges , community has become increasingly interested in the possibilities offered by data sharing and crowdsourcing as a framework for mobilizing groups of people with shared interests around the world and bringing forward augmented solutions to the existing problems while encouraging collaboration . 
in this context , data sharing can be executed according to two paradigms.8 in the data - to - modeler paradigm , both training and validation data sets are provided to participants . 
the model - to - data paradigm , where participants submit containerized models to organizers , allows validation data sets to remain hidden from participants , which both protects condential data and helps ensure unbiased assessment of predictions . 
these approaches are instrumental broadening our understanding of tumor biology and the tumor microenvironment , which will ultimately drive precision medicine with immunotherapy . to enable such collaboration among scientists and physicians , dialogue on reverse engineering and assessment methods ( dream ) challenges engage experts from around the world and offer novel solutions to biological problems.9 since the rst dream challenges were initiated in 2006 , more than 60 have completed , with analysis results generated through virtual collaborative interactions.10 in oncology , crowdsourcing approaches have been used in multiple tumor types since 2012 for augmenting the work of teams generating data , developing protocols for randomized controlled trials , developing prognostic models , and assessing healthcare behaviors on a large scale.11 more specically , three key dream challenges in oncology have demonstrated the value of crowdsourcing for the development of prognosis models or the renement of diagnostic methods . 
 vincent et al following on from these successful dream challenges , experts in lung cancer have shown interest in accessing primary clinical trial data to explore alternative biomarkers to pd - l1 expression and tumor mutational burden . 
the antipd - 1 response prediction challenge will be the rst dream challenge focused on the prediction of response to immuno - oncology therapies using data from a large phase iii clinical trial , with the goal of addressing the need for additional predictive biomarkers to use with icis in patients with nsclc . 
moreover , this initiative will also enhance the understanding of tumor biology and provide further insights into the potential mechanisms underpinning treatment resistance to icis . challenge objectives the antipd - 1 response prediction challenge will focus on developing models that are predictive of benet from ici treatment , specically nivolumab monotherapy , using patient samples obtained during the conduct of a clinical trial . clinical trial data related to nivolumab , including data related to pd - l1 expression , tumor mutational burden , and tumor transcriptomics , will be used to assess the performance of predictive models submitted in a crowdsourced challenge setting . 
the testing data set will use deidentied data from checkmate 026 ( clinicaltrials.gov identier : nct02041533 ) , a phase iii trial that compared nivolumab with platinum - based chemotherapy in patients with metastatic or recurrent nsclc and pd - l1 tumor expression 5%.15 critically , the data set from checkmate 026 is large , randomized , mature , and well annotated , allowing it to support robust testing of predictive models . 
this challenge also lays the foundation for potential future challenges as the immunooncology landscape is rapidly evolving . the antipd - 1 response prediction challenge aims to assess the accuracy of models for predicting clinical outcomes after nivolumab monotherapy , including efcacy end points such as overall survival , progression - free survival , and best overall response , applying clinical and genomic data from checkmate 026 . 
the challenge will comprise three subchallenges , and their objectives are summarized in figure 1 . challenge design framework has been developed to receive and evaluate dockerized models , which are in silico packages comitself plus necessary software compoprising the model nents to run the model as developed by the participant team . the challenge is open to anyone who will be able to construct models for submission , subject to specications in the challenge rules , including experts and innovators in genomics , computational biology , and translational biomarker development . 
participants can sign up on the synapse website.16 to facilitate data ingestion , participants will have access to a small synthetic data set with the same formatting as the validation data set . 
the full synthetic data set can be downloaded on the synapse website.17 in the rst phase , participants will construct models using any data available to thethe iatlas platform , 18 a database supporting the study of interactions between cancer and the immune microenvironment , hosts harmonized genomics data and associated clinical annotations from published immuno - oncology studies . 
participants can use these as training data sets or to augment other training data already available to them.19 participants could also use professor shirley lius labs tide resources , 20 a computational framework that integrated and modeled data from 189 human cancer studies , comprising 33 , 197 samples.21 more resources to consider are listed on the antipd - 1 response prediction challenge website.16 participants dockerized models will be applied to the checkmate 026 data and evaluated during the competitive phase . 
performance metrics , such as area under the receiver operating characteristic or precision - recall curves and harrells c - index , will be used to assess the predictive performance of participants models in each arm of the checkmate 026 trial . top - performing teams will be invited to participate in the collaborative model - development phase , during which data will be analyzed and interpreted to deepen the understanding of the submitted models . 
eventually , opportunities for additional subchallenges and potential projects will be dened based on the learnings from the current challenge , and teams submitting the best - performing models will have the opportunity to co - author the planned publication in accordance with medical publication standards and best practices . the challenge will rely on a partnership between sage the organization supporting the dream bionetworks , challenge initiatives , and bristol myers squibb and will comprise three phases : competitive , collaborative , and nal . 
the anticipated outcome and impact the identied models are expected to identify potential biomarkers of response to nivolumab monotherapy and may then be applied to predict which patients are more likely to derive clinical benet from icis . 
nsclc , nonsmallcell lung cancer ; os , overall survival ; pd , progressive disease ; pd - 1 , programmed death - 1 ; pfs , progression - free survival . dream challenge challenge question 1 predict response to nivolumab , in terms of pfs , via an immune checkpointspecific model using clinical , demographic , and gene expression data challenge question 2 predict response to nivolumab , in terms of os , via an immune checkpointspecific model using clinical , demographic , and gene expression data challenge question 3 predict which patients will have a best overall response of pd checkmate 026 mature data set from a large randomized phase iii clinical trial in patients with nsclc nivolumab chemotherapy dream challenge impact ( cid : 129 ) provide biomarkers with predictive value for patients via a transparent and collaborative approach ( cid : 129 ) lay the foundation for understanding the utility of biomarkers in the setting of combination therapy patients . 
it is anticipated that some of the models may also have prognostic value for patients with nsclc . phenotype , thereby increasing their responsiveness to ici therapies . the challenge results should also provide a deeper understanding of biological mechanisms resulting in tumor resistance to treatment and assist in understanding why patients with advanced nsclc receiving nivolumab had similar efcacy outcomes to patients receiving chemotherapy in checkmate 026 . 
an improved understanding of the biological mechanisms behind resistance could pave the way to therapeutic interventions that immunologically activate tumors that do not display an immune - inltrated successful completion of this dream challenge will provide a model for future collaborations among industry , academia , and citizen scientists to discover determinants of response or resistance to immunotherapy , facilitating rapid development of clinically actionable biomarkers . 
szustakowski employment : bristol myers squibb stock and other ownership interests : bristol myers squibb research funding : bristol myers squibb patents , royalties , other intellectual property : i am an employee of bristol myers squibb , and an inventor on one or more pending patent applications , including applications for tmb as a predictive biomarker of immunotherapy travel , accommodations , expenses : bristol myers squibb parul doshi employment : bristol myers squibb stock and other ownership interests : bristol myers squibb travel , accommodations , expenses : bristol myers squibb justin guinney consulting or advisory role : astrazeneca research funding : celgene , genentech / roche , bristol myers squibb david p . 
carbone manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors references american cancer society : facts & figures 2020 reports largest one - year drop in cancer mortality . 
sambi m , bagheri l , szewczuk mr : current challenges in cancer immunotherapy : multimodal approaches to improve efcacy and patient response rates . j oncol 2019 : 4508794 , 2019 bristol myers squibb : yervoy ( ipilimumab ) [ package insert ]  . 
nat biotechnol 36 : 391 - 392 , 2018 saez - rodriguez j , costello jc , friend sh , et al : crowdsourcing biomedical research : leveraging communities as innovation engines . 
guinney j , wang t , laajala td , et al : prediction of overall survival for patients with metastatic castration - resistant prostate cancer : development of a prognostic model through a crowdsourced challenge with open clinical trial data . 
carbone dp , reck m , paz - ares l , et al : first - line nivolumab in stage iv or recurrent non - small - cell lung cancer . 
immunity 48 : 812 - 830.e814 , 2018 jiang p , gu s , pan d , et al : signatures of t cell dysfunction and exclusion predict cancer immunotherapy response . 
 whole - genome and whole - exome sequencing in pediatric oncology : an assessment of parent and young adult patient knowledge , attitudes , and expectations purpose the complexity of results generated from whole - genome sequencing ( wgs ) and whole - exome sequencing ( wes ) adds challenges to obtaining informed consent in pediatric oncology . 
little is known about knowledge of wgs and wes in this population , and no validated tools exist in pediatric oncology . methods we developed and psychometrically evaluated a novel wgs and wes knowledge questionnaire , the precision in pediatric sequencing knowledge questionnaire ( pipseqkq ) , to identify levels of understanding among parents and young adult cancer survivors ( 18 years old ) , off therapy for at least 1 year from a single - institution pediatric oncology outpatient clinic . 
of the total cohort , 77 ( 69% ) were female , 63 ( 57% ) self - identified as white , and 74 ( 67% ) self - identified as non - hispanic . 
 overall , participants felt the benefits of wgs and wes outweighed the potential risks . conclusion parents and young adult cancer survivors have some genetics knowledge , but they lack knowledge about wgs and wes . 
the pipseqkq is a reliable tool that can identify deficits in knowledge , identify perceptions of risks and benefits of wgs and wes , and help clinicians tailor their consent discussions to best fit families . 
2018 by american society of clinical oncology introduction advances in next - generation sequencing , particularly whole - genome sequencing ( wgs ) and whole - exome sequencing ( wes ) have led to a deeper understanding of the pathogenesis of cancer and offer promising avenues for clinical applications.1 , 2 in pediatric oncology , wgs and wes is being used to identify targetable mutations , clarify diagnosis , provide risk stratification , and identify pharmacogenomic modifiers.3 - 6 although these genome - scale sequencing methods yield a wide spectrum of potential results , only a few are medically actionable ; a large number are classified as variants of unknown significance ( vus )  . 
furthermore , wgs and wes of matched tumornormal tissue can identify pathogenic germline mutations not related to the indication for which testing was ordered , commonly referred to as secondary findings , and jennifer a . 
the american college of medical genetics has defined a list of secondary findings that include noncancerous germline disease - causing mutations and cancer susceptibility mutations recommended for return by clinical laboratories.7 , 8 such findings may be of clinical significance not only to the patient but also to family members not directly involved in testing.9 - 13 an understanding of these complexities is necessary for parents and patients to make informed decisions about consent to genomic sequencing.14 , 15 for most pediatric cancers , standard treatment protocols are highly effective , and wgs and wes results infrequently alter the initial treatment plan.16 however , among parents of pediatric patients with cancer , the hope that genomic sequencing will lead to a cure often exceeds the expected results of testing.17 , 18 thus , parents may be more inclined to participate in wgs and wes without fully understanding the range of results that may be returned , including vus and secondary findings.17 , 18 even within the general public , including those who are well educated , there is a relatively low understanding of genetic concepts.19 research to examine knowledge held by parents and young adult patients about the utility and limitations of genome sequencing is scarce , although there is evidence to suggest that participants with lower educational attainment or those from racial / ethnic minority groups may possess less knowledge.14 within pediatric oncology , there are almost no data related to parent and young adult patient knowledge about genetics and wgs and wes , and there are no validated tools to assess these concepts in pediatric cancer . as part of our clinical sequencing program at columbia university medical center ( cumc ) , we developed the precision in pediatric sequencing knowledge questionnaire ( pipseqkq ) to assess knowledge , attitudes , and expectations of parents and young adult patients about wgs and wes . 
the objectives of the study were to describe the development and psychometric properties of the pipseqkq and to identify preliminary levels of understanding within this population . methods participants participants were recruited from the pediatric oncology outpatient clinic at cumc between august 2015 and june 2016 . 
wgs and wes in pediatric oncology at cumc commenced in january 2014 , and 1 year off therapy was chosen as a cutpoint to minimize the likelihood that participants had undergone or had been exposed to the concepts of wgs and wes in the clinic setting . 
a total of 35 items and three knowledge domains were generated : general genetic concepts ( n = 7 items ) , genetic concepts associated with general health and cancer ( n = 12 items ) , and wgs and wes concepts ( n = 16 items )  . 
a multidisciplinary team of experts from medical genetics , psychiatry , pediatric oncology , molecular pathology , nursing , and behavioral science reviewed the items and domains for content and clarity and to ensure good content validity across the three domains.26 response categories included true , false , and not sure . 
health literacy was assessed with the newest vital sign.27 the six - question validated tool was scored as follows : 4 or greater ( adequate literacy ) , 2 to 3 ( possibly limited literacy ) , and 0 to 1 ( limited literacy )  . 
 numeracy was evaluated with an abbreviated ( five - question ) version of a validated objective numeracy scale , 28 which assessed the ability to comprehend risks and probabilities of disease . 
both measures were available in english and spanish . exploratory analyses were conducted by using two - tailed independent sample t tests to examine differences in pipseqkq scores for select dichotomized demographic variables : race / ethnicity ( white non - hispanic v others ) and educational attainment ( less than college / college v graduate / postgraduate )  . 
participants were contacted 2 weeks later by e - mail or standard post to complete the second pipseqkq ( required to be completed within 4 weeks after the first administration )  . 
participants who completed both surveys received a $15 gift card . data analysis pipseqkq responses were examined for high levels of nonresponse , which indicated an issue with an ite descriptive statistics were examined for all items . 
a floor or ceiling effect was present if more than 10% of participants achieved the results participants of the 135 eligible participants , 11 ( 8% ) declined participation and 13 ( 10% ) were not approached because of timing conflicts ( eg , rapid triage , radiology scans ) during their clinic visits . 
 ( % ) of patients ( n = 111 ) 34 ( 31 ) 77 ( 69 ) 35 ( 32 ) 76 ( 68 ) 103 ( 93 ) 8 ( 7 ) characteristic unknown health literacy limited possibly limited adequate numeracy limited adequate no . 
main health literacy and numeracy data are presented as mean ( standard deviation ) scores . * hematologic conditions consisted primarily of hematologic malignancies ( 61 of 65 )  . for race , other , 17 of 25 self - identified as dominican . pipseqkq scale and item descriptive statistics item response distributions that showed items for which knowledge and uncertainty were highest and lowest are displayed in figure 1 . 
 only a ceiling effect was detected for three items , which were retained for their importance in evaluation of general genetic knowledge and genetics knowledge related to health and cancer . 
test - retest reliability from the 75 participants who completed the pipseqkq twice was 0.82. pipseqkq scores figure 2 shows the proportion of correct and incorrect responses for the pipseqkq . 
internal and test - retest reliability knowledge domain general genetic concepts genetic concepts , general health , and cancer whole - genome / whole - exome sequencing concepts total score no . 
ggc , general genetic concepts ; gghc , general genetic concepts related to health and cancer ; sc , sequencing concepts . ggc1 ggc2 ggc3 ggc4 ggc5 ggc6 ggc7 gghc8 gghc9 gghc10 gghc11 gghc12 gghc13 gghc14 gghc15 gghc16 gghc17 gghc18 gghc19 sc20 sc21 sc22 sc23 sc24 sc25 sc26 sc27 sc28 sc29 sc30 sc31 sc32 sc33 sc34 sc35 correct incorrect not sure frequency of responses ( mean , 4.11 ; sd , 1.41 ; range , 1 to 7 of 7 )  . 
the two items with the most incorrect or not sure responses were the definition of an exome ( n = 79 ; 71% ) and that statement about blood containing a full copy of your genes ( n = 60 ; 53% )  . genetic concepts related to general health and cancer . 
the median domain score for genetic concepts related to general health and cancer was 8.0 ( mean , 8.07 ; sd , 2.37 ; range , 1 to 12 of 12 )  . 
the most frequent items answered incorrectly or not sure were wgs / wes in children with cancer performed at diagnosis will commonly guide initial treatment ( n = 97 ; 88% ) ; there are laws that prevent the use of genetic information to determine eligibility for life insurance , disability insurance , and long - term care insurance ( n = 102 ; 93% ) ; and wgs / wes will have no impact on cancer care for most children with cancer ( n = 98 ; 89% )  . 
 greater than 50% of participants were uncertain about the number of genes that can be tested with wgs and wes ( ie , a small subset v the complete set of genes ) , whether wgs and wes compares the dna of cancer cells to the dna of normal cells , whether wgs and wes can find changes in genes related to diseases other than cancer , and what vus means . 
last , although 60% understood that secondary findings also may provide information about present and future health risks of other family members , 36% were unsure of the correct answer . perceptions of risks and benefits of wgs and wes . 
furthermore , content validity was high , because item generation focused on areas of knowledge necessary to make informed decisions about wgs and wes . consistent with studies in other populations , 14 , 24 , 33 participants had a basic understanding of genetics and heredity . 
lower knowledge has been associated with racial and ethnic minority groups , 14 and we observed this for knowledge related to genetic concepts associated with health and cancer . the strengths and limitations of wgs and wes were not well understood . 
deficits in knowledge were observed for items related to the likelihood of wgs and wes to guide initial treatment or to have an effect on cancer care for most children and whether laws existed to prevent discrimination for certain types of insurance on the basis of genetic information . 
similar to tolusso et al , 39 we found that participants were uncertain about the ability of wgs and wes to examine the complete set of genes , compare tumor and normal dna , find changes in genes related to diseases other than cancer , and identify vus . although there is strong parent / patient interest in genomic sequencing , 17 , 40 - 43 its potential benefit in cancer care was overestimated . 
in a recent study of adult patients with advanced cancer , 64% who underwent genomic testing had a strong belief that testing would significantly improve their cancer care.42 similarly , results from the dana farber cancer institute44 revealed that parents and patients express significant hopes for molecular testing , particularly regarding increased number of treatment options ( 72% ) and increased hope for a cure ( 59% ) .17 in our study , 61% felt that even a small chance that their child would benefit outweighed all other risks of wgs and wes . 
thus far , relatively few pediatric patients with cancer who undergo genomic testing are matched to a targeted therapy.3 , 4 , 6 , 44 these findings reflect two salient issues in pediatric oncology . 
many of the newer molecularly targeted drugs lack efficacy data in pediatric diseases or safety data in children and , therefore , are not approved for administration in this population . 
 consequently , families should be provided with realistic information during consent discussions to manage expectations when they consider genomic testing . our previous work indicates that families strongly desire more education and improved modalities to communicate wgs and wes information before consent for genomic sequencing.18 similarly , parents have reported that the standard consent process for genome - wide sequencing was not adequate and have requested availability of more resources to aid their understanding.45 the pipseqkq is a valid tool that can help identify knowledge deficits and guide the development of patient education materials to improve understanding of wgs and wes . 
our center is testing the effectiveness of a web - based tool in english and spanish as an educational resource to improve patient education and informed consent for clinical wgs and wes . this study had a number of limitations to consider , which represent important areas for future research . 
 second , those surveyed had children who were off therapy for more than 1 year , and their prolonged exposure to treatment in a pediatric oncology center may have biased their knowledge of the concepts presented here , including their assessment of risk and benefit . 
third , we did not have sufficient sample size to validate the pipseqkq spanish version , evaluate cultural differences for non - english speaking participants , or conduct factor analyses or item - response theory analyses . 
last , a factor not addressed by the pipseqkq that is being investigated through ancillary studies relates to evaluation of the potential cost to families for clinical wgs and wes . 
insurance companies provide inconsistent coverage and variable payment for wgs and wes , and families will need to understand the financial responsibility they may incur from testing . in conclusion , as wgs and wes become more ubiquitous in pediatric oncology , medical providers will need to help families understand the benefits and limitations of testing , including the potential financial cost to the family for testing and the possible identification of secondary findings . 
this may be especially advantageous for providers who lack the expertise or time to fully engage with their patients about testing . our findings highlight that , although participants understand that there are risks and benefits of wgs and wes , many would accept any risk for the possibility of a small benefit , and their hope for a cure often exceeds the expected results from sequencing . 
the pipseqkq is a valid tool that can aid providers in precision oncology to identify levels of knowledge and tailor their informed consent discussions to best fit the patient and family . 
kung consulting or advisory role : darwin health , mi bioresearch , imago bioscience patents , royalties , other intellectual property : royalty from licensing agreements with mi bioresearch wendy k . 
glade bender research funding : bristol - myers squibb ( inst ) , pfizer ( inst ) , novartis ( inst ) , ignyta ( inst ) , amgen ( inst ) , celgene ( inst ) , eisai ( inst ) , lilly ( inst ) , merck ( inst ) travel , accommodations , expenses : novartis , amgen , merck jennifer m . 
worst bc , van tilburg cm , balasubramanian gp , et al : next - generation personalised medicine for high - risk paediatric cancer patients : the inform pilot study . 
marron jm , dubois sg , glade bender j , et al : patient / parent perspectives on genomic tumor profiling of pediatric solid tumors : the individualized cancer therapy ( icat ) experience . 
morren m , rijken m , baanders an , et al : perceived genetic knowledge , attitudes towards genetic testing , and the relationship between these among patients with a chronic disease . 
streicher sa , sanderson sc , jabs ew , et al : reasons for participating and genetic information needs among racially and ethnically diverse biobank participants : a focus group study . 
kaphingst ka , blanchard m , milam l , et al : relationships between health literacy and genomicsrelated knowledge , self - efficacy , perceived importance , and communication in a medically underserved population . 
langer mm , roche mi , brewer nt , et al : development and validation of a genomic knowledge scale to advance informed decision - making research in genomic sequencing . 
calsbeek h , morren m , bensing j , et al : knowledge and attitudes towards genetic testing : a two year follow - up study in patients with asthma , diabetes mellitus and cardiovascular disease . 
miller fa , hayeems rz , bytautas jp , et al : testing personalized medicine : patient and physician expectations of next - generation genomic sequencing in late - stage cancer care . 
mccullough lb , slashinski mj , mcguire al , et al : is whole - exome sequencing an ethically disruptive technology ? perspectives of pediatric oncologists and parents of pediatric patients with solid tumors . 
harris mh , dubois sg , glade bender jl , et al : multicenter feasibility study of tumor molecular profiling to inform therapeutic decisions in advanced pediatric solid tumors : the individualized cancer therapy ( icat ) study . 
chinnaiyan , md , phd4 , 5 case description in january 2018 , a 79 - year - old man was referred to our genitourinary medical oncology clinic for management of his prostate adenocarcinoma metastatic to multiple bones . 
his prostate cancer spread to additional bony sites , which were likewise treated with radiation , including radiation to the thoracic spine in july 2017 and the proximal humerus in august 2017 , rather than any systemic in december 2017 , he had prophylactic therapy . nipple irradiation to prevent gynecomastia in preparation for noncastrating medical therapy with singleagent bicalutamide . 
on meeting the patient for the rst time in january 2018 , we elected to continue with the plan for single - agent bicalutamide 50 mg per day , given the patients preference and the uncertain prognosis from his metastatic melanoma . 
his prostatespecic antigen ( psa ) was 30.8 ng / ml at the time of treatment initiation and responded rapidly to bicalutamide , reaching a nadir of 1.1 ng / ml in september 2018 ( fig 1a )  . his melanoma was diagnosed from a shave biopsy of the right superior lateral lower back in august 2015 as localized ulcerated malignant melanoma , unclassied , with nevoid features . 
he subsequently had a microstaging excision and a right back excision with a negative sentinel lymph node in the right groin 2017 , the patient noticed a new lump on his right lower back scar in 2017 . 
in november 2017 , excisional biopsies of his right lower back and right groin both showed metastatic melanoma , as conrmed by immunohistochemistry ( ihc ) showing positive staining for sox - 10 and mart - 1 . 
a 7 - mm lesion was also detected on his posterior scalp and was also visible on magnetic resonance imaging of his brain and metabolically active on positron emission tomography ( pet ) / computed tomography ( ct ) ; hence , it was diagnosed as stage iv melanoma . 
he began treatment of his metastatic melanoma with pembrolizumab 200 mg intravenous every 3 weeks and received four treatments before the pembrolizumab was stopped because of the development of pneumonitis . 
his pneumonitis was treated with a taper of prednisone , which was reduced to physiologic dosing by august 2018 . however , despite evidence of response in his melanoma by physical examination , there was concern for progression on pet / ct in june 2018 , with fdg update in the descending colon / small bowel wall , perisplenic region , a mildly fdg - avid left internal iliac lymph node , and a right external iliac nodal conglomerate encasing the right ureter , 4.1 cm in diameter and with an suv max of 11.7. 
 case report systemic therapy : gem - carbo docetaxel bicalutamide pembro bicalutamide leuprolide 1.1 ureter stent and pelvic mass biopsy abdominal wall excisional biopsy psa : ( ng / ml ) 30.8 event : date : 1 / 2018 6 / 2018 1 / 2019 6 / 2019 1 / 2020 6 / 2020 before gem - carbo after gem - carbo before docetaxel after docetaxel fig 1 . 
psa , prostate - specic antigen . pelvic mass was judged to be atypical for either prostate cancer or melanoma , it was biopsied by pelvic laparoscopy in november 2018 and was found on surgical pathology assessment to be consistent with a high - grade carcinoma with squamous features ( fig 2a )  . 
we performed exome sequencing using the university of michigan oncoseq panel ( mi - oncoseq ) and whole transcriptome analysis on formalin - xed parafn - embedded tissue from this biopsy . 
all patients enrolled in the mi - oncoseq study provided written informed consent approved by the university of michigan institutional review board . consent is inclusive of publishing information and / or images from participants ( or their designate )  . 
because the pelvic mass was causing pain and urinary obstruction , we elected to treat it with a platinum doublet active in urothelial carcinoma and carcinoma of unknown primary , despite not knowing the tissue of origin january 2019 , we began carboplatin area under the curve 5 mg / ml / minute on day 1 and gemcitabine 1 , 000 mg / m2 on days 1 and 8 of 21 - day cycles , and we completed six cycles before stopping because of progressive fatigue . 
 ( d ) ihc for erg of the primary prostate tumor showing strong labeling . ( e ) h&e histology of the abd wall nodule biopsy specimen used for molecular analysis . 
 ( f ) ihc for erg of the abd wall nodule showing much weaker labeling . in june 2019 , while being treated only with bicalutamide , the patient was noted on ct scan as having a 1.4 - cm nodule in the supercial anterior abdominal wall , which subsequently became easily palpable and caused skin together with smaller adjacent erythema . 
this nodule , nodules , was removed by excisional biopsy in july 2019 , where surgical pathology assessment demonstrated tumor features consistent with metastatic squamous cell carcinoma ( fig 2b )  . 
a fresh portion of this specimen was sent for analysis using the mi - oncoseq platforthis specimen showed a much higher tumor content of 54% , allowing the discovery of additional molecular alterations . precision medicine tumor board discussion results were discussed at the university of michigan precision medicine tumor board in september 2019 . somatic aberrations detected in the previous sample were detected in the new biopsy specimen , suggesting clonal relatedness , and additional alterations consistent with the increased tumor content were noted as well ( table 2 )  . 
for prostate adenocarcinoma , the addition of medical castration , likely in addition to either abiraterone and prednisone or a nonsteroidal second - generation antiandrogen , would have been a reasonable next line of therapy . 
however , the transcriptomics data of the mi - oncoseq platform showed low expression of the androgen receptor and androgen responsive genes klk2 , klk3 ( psa ) , tmprss2 , acpp , and slc45a3 ( fig 3b )  . 
therefore , we did not plan chemotherapy with a regimen such as carboplatin and etoposide , which is active against small - cell neuroendocrine carcinomas . we examined the remainder of the molecular results in an effort to nd alternative , clinically actionable molecular targets . 
however , overall , the results for mtor inhibitors in prostate cancer have been disappointing.3 some randomized data support the use of pi3k inhibitors in metastatic castration - resistant prostate cancer.4 however , this was in combination with abiraterone , which made pi3k inhibitors less attractive in this case , given the patients near total lack of androgen signaling . 
however , we decided against this option because cdk4 / 6 inhibitors have thus far shown disappointing results in prostate cancer.5 therefore , we elected to treat with docetaxel , the most common cytotoxic chemotherapy drug for castration - resistant prostate cancer . at this point , the patients lung nodules , which were previously indeterminate , had enlarged greatly ( fig 1d )  . 
we started treatment with docetaxel at 75 mg / m2 for one cycle , then decreased the dose to 60 mg / m2 because of fatigue , and completed three more cycles . 
ct completed after three cycles showed an interval decrease in the size of multiple bilateral lung nodules , and a decrease in size of his right pelvic mass , compatible with a therapeutic response ( fig 1e )  . discussion in this case , we used next - generation sequencing to determine that the patients squamous cell carcinoma was actually of prostate origin , because of the presence of a gene fusion between tmprss2 and erg . 
 case report prostate cancer subtypes has become more common since the development of abiraterone and nonsteroidal secondgeneration antiandrogens , 6 possibly as a means to escape continual selective pressure against androgen signaling , a phenomenon that had been described rarely in the past.9 some of these double - negative prostate cancer ( dnpc ) tumors seem to be driven by broblast growth factor ( fgf ) alterations , and trials of fgf inhibitors have recently begun in advanced prostate cancer.10 our patient did not have an fgf abnormality . 
in the current case , despite having squamous differentiation , this patient had only stable disease in response to platinum - based chemotherapy . after using whole exome sequencing and rna sequencing to identify this tumor as a squamous neoplasm of prostate origin , we elected to treat him with an agent approved for prostate cancer ( docetaxel ) , an agent we would not have elected to use without knowing the tissue of origwe elected not to pursue the therapies that could target molecular alterations in his tumor because of the known survival benet of docetaxel in men with advanced prostate cancer , but therapies targeting his tumors molecular alterations remain options down the road if his disease progresses . 
alumkal consulting or advisory role : merck sharp & dohme , dendreon research funding : aragon pharmaceuticals ( inst ) , astellas pharma ( inst ) , zenith epigenetics ( inst ) , gilead sciences ( inst ) travel , accommodations , expenses : astellas pharma , merck sharp & dohme other relationship : astellas pharma arul chinnaiyan stock and other ownership interests : oncopia , tempus , esanik , oncofusion therapeutics , medsyn consulting or advisory role : tempus patents , royalties , other intellectual property : co - inventor on a patent on the detection of ets gene fusions in prostate cancer issued to the university of michigan . no other potential conicts of interest were reported . acknowledgment we thank the mi - oncoseq clinical sequencing team , leslie fecher , md , jeff montgomery , md , and michael sabel , md , for collaborative clinical care and discussion ; alex hopkins for assistance with gures ; and jyoti athanikar for assistance with scientic editing and manuscript preparation . 
cell 161 : 1215 - 1228 , 2015 [ erratum : cell 162 : 454 , 2015 ] statz cm , patterson se , mockus sm : mtor inhibitors in castration - resistant prostate cancer : a systematic review . 
target oncol 12 : 47 - 59 , 2017 de bono js , de giorgi u , rodrigues dn , et al : randomized phase ii study evaluating akt blockade with ipatasertib , in combination with abiraterone , in patients with metastatic prostate cancer with and without pten loss . 
clin cancer res 25 : 928 - 936 , 2019 clinicaltrials.gov : a phase ii study of androgen deprivation therapy with or without palbociclib in rb - positive metastatic prostate cancer . 
gov / ct2 / show / nct02059213 bluemn eg , coleman im , lucas jm , et al : androgen receptor pathway - independent prostate cancer is sustained through fgf signaling . 
cancer cell 32 : 474 - 489.e6 , 2017 labrecque mp , coleman im , brown lg , et al : molecular proling straties diverse phenotypes of treatment - refractory metastatic castration - resistant prostate cancer . 
j clin invest 129 : 4492 - 4505 , 2019 parwani av , kronz jd , genega em , et al : prostate carcinoma with squamous differentiation : an analysis of 33 cases . 
weindorf sc , taylor as , kumar - sinha c , et al : metastatic castration resistant prostate cancer with squamous cell , small cell , and sarcomatoid elementsa clinicopathologic and genomic sequencing - based discussion . 
 major response to carboplatin in a patient with metastatic triple - negative breast cancer with somatic mutation of brca1 and loss of rad51b lor `ene seguin , md1 ; max chaffanet , phd , hdr1 ; s everine garnier , phd1 ; jos e ad elade , msc1 ; nadine carbuccia , msc1 ; arnaud guille , msc1 ; jihane pakradouni , pharmd , phd2 ; renaud sabatier , md , phd1 ; cornel popovici , md , phd2 ; daniel birnbaum , md , phd1 ; franois bertucci , md , phd1 ; and anthony goncalves , md , phd1 background triple - negative breast cancer ( tnbc ) , clinically dened as not expressing estrogen receptor , progesterone receptor , and human epidermal growth factor receptor 2 tyrosine kinase receptor , accounts for approximately 15% of breast cancers and is associated with a poor prognosis and a lack of recognized molecular targets for therapeutics.1 some 15% to 20% of tnbcs arise in patients with brca1 germline mutations , and tnbc is the most common molecular subtype in this context.2 breast cancer type 1 susceptibility protein ( brca1 ) and many other proteins , such as the dna repair protein rad 51 homolog 1 ( rad51 ) paralogs , are involved in the dna homologous recombination ( hr ) repair pathway.3 breast cancers associated with germline brca mutations display dna double - strand break repair defects , which is believed to render them particularly sensitive to dna repairtargeting drugs such as poly ( adp ) ribose polymerase ( parp ) inhibitors , 4 but also to dna - damaging agents , such as platinum analogs.5 although the presence of germline brca1 / 2 mutations was recently shown to predict benet of platinum compounds , 6 little is known about other determinants of efcacy of this class of cytotoxics . 
clinical procedures performed after molecular tumor board decision were done according to routine clinical management . in july 2015 , a 72 - year - old woman presented with a ct2n0m0 tumor of the right breast . 
biopsy showed scarff - bloom - richardson grade 3 , triple - negative invasive carcinoma , which was treated by breastconserving surgery and axillary lymph node dissection ( fig 1 )  . 
pathologic examination of surgical specimen revealed a 5 - cm invasive ductal breast carcinoma , nuclear grade 3 , with lymphovascular invasion and two of 16 axillary lymph nodes invaded by metastasis , one with extracapsular spread . immunohistochemistry did not show any expression of estrogen receptor , progesterone receptor , human epidermal growth factor receptor 2 , or cytokeratins 5 and 6 ; ki67 antigen was expressed by 50% of malignant cells . 
then , because of positive margins , a mastectomy was done , without pathologic residual tumor , followed by adjuvant radiotherapy started on february 2016 . in april 2016 , while the patient was ending radiotherapy , she presented visual troubles , dizziness , and gait disorders . 
at this time , the disease progressed in the lung and liver , with stable cranial disease , and she received three cycles of pembrolizumab between september 2016 and november 2016 in the context of a clinical trial . 
the disease progressed at both cranial and extracranial levels , and she was enrolled in november of 2016 in ( clinicaltrials.gov identier : our permed - 01 trial nct02342158 ) , whose aim is to identify targetable molecular alterations in locally advanced and metastatic tumors . 
2018 adjuvant adj rt brain relapse nodes liver , lung , nodes , brain liver , lung , nodes , brain liver , lung , nodes , brain cr ( nodes , lung , and liver ) pd in brain fec100 - wptx wbrt cape pembro eribulin re - xrt brain , carboplatin diagnosis surgery fig 1 . 
adj , adjuvant ; cape , capecitabine ; cr , complete response ; ct , computed tomography ; fec100 , uorouracil - epirubicine 100mg / m - cyclophosphamide ; mst , mastectomy ; pd , progressive disease ; pembro , pembrolizumab ; rt , radiotherapy ; wbrt , whole - brain radiation therapy ; wptx , weekly paclitaxel ; xrt , radiation therapy . genomic copy number aberrations and somatic mutation status were identied on metastatic tissue by using wholegenome array - comparative genomic hybridization ( acgh ) and targeted next - generation sequencing of 559 actionable genes , as previously described7 , 8 ( data supplement )  . 
analysis of germline dna revealed no brca mutation . in may 2017 , after an initial partial response to eribulin , the disease spread to brain ( multifocal ) , lung , mediastinum lymph nodes , and liver ( figs 3 and 4 ) , and a molecular tumor board recommended treatment with platinum derivatives . the patient started single - agent carboplatin ( area under the curve = 5 mg / ml / min ) in june 2017 . 
after three cycles of carboplatin in august 2017 , a striking partial response was observed at the extracranial level ( fig 3 ) , and a signicant partial response was observed at the brain level in december 2017 after seven cycles of carboplatin ( fig 4 )  . 
the patient was seen on february 2018 , after only one additional cycle of carboplatin ( in january 2018 ) , because of thrombocytopenia ; a complete response was still observed in lung , mediastinum , and liver . 
in april 2018 , a persistent , nearly complete response was noted at the extracranial level ( fig 3 ) , but the disease progressed in the brain , with deterioration of the patients clinical status . 
because of the small sample size of this analysis , however , relevant conclusions cannot be drawn from this small subset . germline palb2 mutations may also be associated with platinum sensitivity.10 similarly , proofs of efcacy of parp inhibitors in patients with advanced breast cancer have been restricted to patients with germline brca mutations , 11 , 12 and no clinical data exist demonstrating efcacy of these drugs in patients with somatic brca mutations . 
in addition to germline or somatic brca mutations , brca1 / 2 promoter methylation , as well as other numerous non - brca genomic alterations , may also drive hrd and the so - called brcaness phenotype . 
computed tomography scan was obtained before ( may 2017 , left panel ) and after carboplatin ( august 2017 , middle panel ; april 2018 , right panel ) showing a nearly complete response at the extracranial level . been associated with predisposition to breast cancer.13 the recruitment of rad51 to dna double - strand breaks is dependent on rad51 paralogs ( ie , rad51b , rad51c , rad51d , xrcc2 , and xrcc3 ) as well as on rad52 , the deciency of which worsens the phenotype of brca1 , brca2 , or palb2 alterations.14 interestingly , preclinical evidence shows that inactivation of rad51b increases sensitivity to dna - damaging agents , including platinum , because of both alteration in the cell cycle checkpoint response and impairment in hr repair.15 in addition , recent data indicate that the functionality of hr , as measured by rad51 foci , may predict sensitivity to parp inhibitors in brca - mutated breast cancer.16 thus , in the case presented here , loss of rad51b and somatic brca1 mutation may have cooperated to induce a brcaness phenotype , as suggested by the high hrd score identied by acgh . of note , despite experiencing initial response , the disease ultimately progressed at the brain level only , in the context of chemotherapy delay due to persistent carboplatinrelated thrombocytopenia . 
yet , at the time of this case report , parp inhibitors were not routinely available for patients with breast cancer in france . to estimate the clinical relevance of rad51b alterations , we examined their frequency in the permed trial as well as in other public databases , either alone or in combination with brca mutations . 
these results are discussed in the appendix . in conclusion , we have described a major response to carboplatin in a heavily pretreated patient with tnbc with somatic alterations in dna repair genes , suggesting that this class of potent and inexpensive cytotoxics could be used as an authentic targeted therapeutic in a precision fig 2 . 
arrowheads indicate lesions subjected to stereotactic radiation ; arrows show subcentimetric lesions not subjected to radiation but regressive after systemic treatment . to its medicine perspective using biomarkers pertinent mechanisms of action . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . renaud sabatier honoraria : novartis , astrazeneca , pzer research funding : novartis ( inst ) , eisai ( inst ) , astrazeneca ( inst ) travel , accommodations , expenses : amgen , pzer cornel popovici travel , accommodations , expenses : astrazeneca anthony goncalves research funding : msd ( inst ) , bristol - myers squibb ( inst ) , novartis ( inst ) , cascadian therapeutics ( inst ) , nektar ( inst ) , boehringer ingelheim ( inst ) , eli lilly ( inst ) , abbvie ( inst ) , genentech ( inst ) , astrazeneca ( inst ) , roche ( inst ) travel , accommodations , expenses : pzer , novartis , genentech , celgene no other potential conicts of interest were reported . acknowledgment we thank the computing facilities disc ( datacenter it and scientic computing , centre de recherche en canc erologie de marseille ) for their technical support . 
n engl j med 363 : 1938 - 1948 , 2010 bianchini g , qi y , alvarez rh , et al : molecular anatomy of breast cancer stroma and its prognostic value in estrogen receptor - positive and - negative cancers . j clin oncol 28 : 4316 - 4323 , 2010 3 . 
oncogene 22 : 7265 - 7279 , 2003 tutt a , tovey h , cheang mcu , et al : carboplatin in brca1 / 2 - mutated and triple - negative breast cancer brcaness subgroups : the tnt trial . 
nat med 24 : 628 - 637 , 2018 bertucci f , finetti p , guille a , et al : comparative genomic analysis of primary tumors and metastases in breast cancer . 
oncotarget 7 : 27208 - 27219 , 2016 gonalves a , bertucci f , guille a , et al : targeted ngs , array - cgh , and patient - derived tumor xenografts for precision medicine in advanced breast cancer : a single - center prospective study . 
oncotarget 7 : 79428 - 79441 , 2016 abkevich v , timms km , hennessy bt , et al : patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer . 
br j cancer 107 : 1776 - 1782 , 2012 isaac d , karapetyan l , tamkus d : association of germline palb2 mutation and response to platinum - based chemotherapy in metastatic breast cancer : a case series . 
lok bh , carley ac , tchang b , et al : rad52 inactivation is synthetically lethal with deciencies in brca1 and palb2 in addition to brca2 through rad51mediated homologous recombination . 
cruz c , castroviejo - bermejo m , guti errez - enrquez s , et al : rad51 foci as a functional biomarker of homologous recombination repair and parp inhibitor resistance in germline brca - mutated breast cancer . 
lin nu , claus e , sohl j , et al : sites of distant recurrence and clinical outcomes in patients with metastatic triple - negative breast cancer : high incidence of central nervous system metastases . 
 platinum in tnbc with brca1 somatic mutation and loss of rad51b appendix can this observation be extended to other patients of our trial or other institutions ? another patient with refractory triple - negative breast cancer ( tnbc ) enrolled in the permed - 01 trial displayed rad51b homozygous deletion with high homologous recombination deciency ( hrd ) score in the biopsy of a skin metastasis ; yet , at the time of the manuscript writing , this patient did not receive platinum salt or poly ( adp ) - ribose polymerase inhibitor . 
because of its potential therapeutic interest , we assessed the frequency of rad51b homozygous deletion in the the cancer genome atlas ( tcga ; abouttcga / overview ) and genomics evidence neoplasia information exchange , version 4 ( genie ; aacr project genie consortium : cancer discov 7 : 818 - 831 , 2017 ) public databases . 
this alteration was rare , with only 21 ( approximately 0.2% ; 21 of 10 , 837 ) and 35 ( approximately 0.1% ; 35 of 35 , 498 ) cases in each database , respectively ( data supplement )  . 
in breast cancer , rad51b homozygous deletion was observed in 0.09% of cases in both databases ( one case out of 1 , 079 in tcga and four out of 4 , 421 in genie ; data supplement ) , which are exclusively or mostly composed of primary tumor samples ( appendix fig a1a )  . 
although remaining quite uncommon , the detection of this alteration in two tnbc samples out of the rst set of 183 patients with advanced breast cancer enrolled in the permed - 01 trial , including 64 tnbcs , suggests that rad51b homozygous deletion may be enriched this subtype . 
we also examined public in metastatic samples of databases for the concomitant presence of brca mutation and rad51b deletion and found it at variable frequencies in cancer : rad51b homozygous deletion combined with brca mutation was only observed in two cases , a bladder urothelial cancer ( tcga ) and a breast cancer ( genie ) , both involving brca2 and never brca1 ( data supplement )  . 
however , among 8 , 851 patients with various cancers from tcga , 8% ( 709 of 8 , 851 ) of tumor genomic proles exhibited the double event combining heterozygous mutation or loss of rad51b with heterozygous mutation or loss of brca . the top ve frequencies were 28% in ovarian serous cystadenocarcinomas ( 163 of 579 ) , 25% in uterine carcinosarcomas ( 14 of 56 ) , 19% in cholangiocarcinomas , 14% in sarcomas ( 37 of 256 ) and mesotheliomas ( 12 of 87 ) , as well as 11% in breast invasive carcinoma ( 118 of 1 , 074 ; appendix fig a1b )  . 
moreover , a similar highly signicant association was observed between the concomitant presence of double heterozygous ( rad51b loss or mutation and brca loss or mutation ) events and hrd ( p , .001 ; appendix fig a2b , right )  . 
altogether , these data suggest that combination of genomic alterations in the dna repair pathway , including heterozygous alterations , may increase hrd and thus expand the population of patients who are candidates for therapeutic targeting of this pathway . 
 ( a ) hrd score was calculated as described in abkevich9 and is presented as box plots in patients with ( left ) and without ( right ) rad51b homozygous deletion . 
 high expression of fgd3 , a putative regulator of cell morphology and motility , is prognostic of favorable outcome in multiple cancers purpose identification of single - gene biomarkers that are prognostic of outcome can shed new insights on the molecular mechanisms that drive breast cancer and other cancers . methods exploratory analysis of 20 , 464 single - gene messenger rnas ( mrnas ) in the molecular taxonomy of breast cancer international consortium ( metabric ) discovery cohort indicates that low expression of fgd3 mrna is prognostic for poor outcome . 
2017 by american society of clinical oncology licensed under the creative commons attribution 4.0 license scooter willis yuliang sun mark abramovitz teng fei brandon young xiaoqian lin min ni justin achua meredith m . 
goldstein roberto salgado sherene loi giancarlo pruneri giuseppe viale myles brown brian leyland - jones author affiliations and support information ( if applicable ) appear at the end of this article . the contents are solely the responsibility of the authors and do not necessarily represent the official views of the national cancer institute . ( continued ) introduction an increasing collection of breast cancer cohorts have been molecularly profiled on affymetrix and illumina platforms , so it is now feasible to conduct an exploratory analysis to identify single - gene biomarkers in which messenger rna ( mrna ) expression is prognostic of outcome.1 by limiting the exploratory analysis to a single gene , we intended to identify novel gene ( s ) that might provide insight into biological mechanisms that drive breast cancer metastasis.2 the starting point for this analysis was the molecular taxonomy of breast cancer international consortium ( metabric ) data set , which contains clinical traits , expression data , copy number variation profiles , and single nucleotide polymorphism genotypes derived from breast tumors collected from participants in the metabric trial.3 we also used the genomic data commons , which incorporates the cancer genome atlas ( tcga ) cancer cohorts and currently spans 21 cancer types suitable for survival analysis . 
fgd3 and susd34 cell motility genes were compared as a single - gene biomarker with proliferation genes mki67 , 5 , 6 aurka , 7 - 10 and pcna11 , 12 in six distinct breast cancer cohorts and as a pan - cancer biomarker in tcga cancer cohorts . 
fgd3 mrna expression as a biomarker for an immune response was evaluated by using tumor - infiltrating lymphocyte cells in tcga breast cancer and breast international group 1 - 98 ( big 1 - 98 ; letrozole or tamoxifen in treating postmenopausal women with breast cancer ) cohorts . 
esr1 transcriptional regulation of fgd3 mrna expression in the breast cancer cell line zr - 75 - 1 was confirmed . methods cohorts detailed description of each cohort is provided in the data supplement . survival analysis to illustrate the outcome benefit of low versus high expression , the kaplan - meier method13 was used to estimate the distribution of timeto - disease outcome end points by gene expression status bifurcated on the cohort mean and median ( data supplement )  . 
the wald - test p values from cox proportional hazards models for the association of cancer outcomes with gene expression as a continuous variable for each cohort were used to determine the stouffer weighted z - test p value . 
tumor - infiltrating lymphocytes ( tils ) were called in 389 samples from tcga breast cancer high - resolution slide images using methods previously defined by the international til working group 2014.20 tils were called using a similar methodology in 725 samples from the big 1 - 98 dasl cohort , and correlation to fgd3 mrna expression was determined by using pearsons correlation coefficient ( r ) .21 fgd3 protein expression analysis fgd3 protein expression in tumor samples was determined by immunohistochemical ( ihc ) staining . 
breast cancer samples were provided by avera cancer institute , and breast cancer tmas ( br1504a , br1505b , hbre - duc068bch - 01 , and br20837 ) were purchased from us biomax ( rockville , md )  . fgd3 protein expression was quantitatively determined in the range of 0 to 4 . 
the us biomax metadata indicates whether the patient had no regional lymph node metastasis ( n0 ) or degrees of metastasis in regional lymph nodes ( n1 to n3 )  . fgd3 expression levels for samples with n0 designation ( n = 135 ) and samples with n1 to n3 designations in metastatic tissue ( n = 98 ) were compared by using an unpaired t test . 
tissues for matched lymph node metastasis ( n = 103 ) were compared with primary tumor tissues using unpaired t test , and figures were generated using graphpad prism 7.0. 
additional details on analysis can be found in the data supplement . fgd3 mrna expression regulated by estrogen receptors breast cancer cell line zr - 75 - 1 was grown in rpmi - 1640 medium with 10% fetal bovine serufor the treatment of estrogen , cells were deprived of hormone for 3 days in phenol - free rpmi - 1640 medium with 5% charcoal - stripped fetal bovine serum and then treated with either ethanol ( vehicle ) or 1 nm 17b - estradiol ( estrogen ) for 16 hours . 
reverse transcriptase - quantitative polymerase chain reaction ( rt - qpcr ) was performed and is described in the data supplement . mrna expression of genes of interest with published data the expression atlas web site22 was used to query rna sequencing ( rna - seq ) expression levels in breast cancer cell lines . 
 used to illustrate differences in differential expression of the proliferation genes using prism 7.0. continuous and categorical variables , kaplanmeier figures split on cohort means , and expression profiles can be found in the data supplement . results discovery of fgd3 we undertook an exploratory analysis of 20 , 464 possible single - gene biomarkers as categorical variables split on the mean in the metabric discovery cohort and identified fgd3 mrna expression as the highest ranked prognostic gene based on the p value for overall survival ( os ) , which we subsequently verified as being prognostic in the metabric validation cohort ( data not shown )  . 
the hungarian academy of science ( has ) breast cancer cohort represents a collection of all publicly available breast cancer cohorts profiled on the affymetrix platforthe kaplanmeier plotter web interface allows selection of has cohorts on the basis of er status using the mrna expression level . 
hungarian academy of science breast cancer cohort kaplanmeier plots for recurrencefree survival splitting on fgd3 median messenger rna ( mrna ) expression in which estrogen receptor ( er ) positive status is derived from mrna using the kaplan - meier plotter web interface to generate graphs . 
to determine whether fgd3 was a prognostic biomarker in a specific prediction analysis of microarray 50 ( pam50 ) subtype , the has cohort was subdivided via the web interface for the kaplan - meier plotter . 
hungarian academy of science breast cancer cohort kaplan - meier plot for recurrence - free survival splitting on fgd3 median messenger rna ( mrna ) expression in the prediction analysis of microarray 50 subtype . 
seven of the 21 tcga cohorts did not have a single - gene biomarker from the list ( fgd3 , susd3 , mki67 , pcna , aurka ) that was prognostic . 
by using the biomarker p value and the number of samples in each cohort , the stouffer25 p value was calculated to rank the prognostic value as a pan - cancer biomarker . 
by using the tcga cohort , tils were called from high - resolution images , and fgd3 mrna expression did not correlate with tils evaluated on hematoxylin and eosin slides defined by using a previously published method26 in which tils represented a pre - existing antitumor t - cell response ( unpublished data )  . 
in addition , til calls from the big 1 - 98 dasl cohort did not correlate with fgd3 mrna expression . to further investigate whether fgd3 mrna expression is a feature of the tumor , breast cancer tmas were purchased from us biomax , and ihc was used to determine fgd3 protein expression levels ( scored from 0 to 4 )  . 
by using metadata provided by us biomax , fgd3 protein levels in tumors ( n = 135 ) with no regional lymph node metastasis ( n0 ) were compared with tumors with lymph node metastasis ( n1 to n3 ) and corresponding matched metastatic tissues ( fig 4 )  . an unpaired t test comparing n0 with n1 to n3 suggests that lymph node metastasis is associated with lower fgd3 protein levels ( p , 1e - 4 )  . 
benign tumors ( fig 5a ) and breast adenocarcinomas in lower stages ( fig 5b - c ) showed strong expression of fgd3 , whereas late - stage breast adenocarcinomas in higher stages ( fig 5d - f ) showed mild to weak expression . 
stage iia invasive breast cancer ( data supplement ) showed strong fgd3 expression compared with its matched metastatic carcinoma ( data supplement ) , in which fgd3 was weakly expressed . 
this is a significant finding in the tcga cohorts , considering the median survival time in these cohorts was typically less than 2 years . fgd3 has a putative guanine nucleotide exchange factor that targets cell division control protein 42 homolog ( cdc42 ) 31 and shares high sequence similarity with fgd1 in their dbl homology ( 70% ) and pleckstrin homology ( 60.6% ) domains ; however , fgd3 lacks the n - terminal proline - rich domain found in fgd1 . 
the proline - rich domain plays a crucial role in the response to extracellular signal - responsive translocation of fgd1 to the leading - edge membrane in cells facing toward a wound during the wound - healing process.32 through a highly conserved recognized destruction motif ( dsgids ) in both fgd3 and fgd1 as well as in other unrelated proteins including ikbs and b - catenin , downregulation occurs through the ubiquitin / proteasome system via phosphorylation by gsk - 3b of the serine residues in the dsgids motif.33 thus , both fgd3 and fgd1 intracellular levels are tightly regulated by the same destruction pathway . 
functionally , fgd1 is involved in the regulation of cellular structures required for invadopodia biogenesis and extracellular matrix degradation in an invasive cell model by modulating cdc42 activation.34 - 37 in addition , mutations in fgd1 are responsible for the x - linked disorder known as faciogenital dysplasia , and fgd1 is highly expressed during bone growth and mineralization.36 using the hela tet - off cell system , hayakawa et al31 showed that notwithstanding their similarity , fgd3 and fgd1 played quite different roles in regulating cellular functions . 
furthermore , inducible expression of fgd3 resulted in significant morphologic changes that included the formation of broad sheet - like protrusions known as lamellipodia when gtpbound cdc42 levels were significantly increased by the inducible expression of fgd3 , whereas high expression of fgd1 led to the formation of filopodia , which are found in migrating cells.31 thus , cell motility seemed to be inversely regulated by fgd3 and fgd1 : fgd3 inhibited cell migration and fgd1 stimulated it . the fgd3 - susd3 metagene ( these genes share the same chromosomal location directly adjacent to each other at chr9q22.31 ) was ranked with the highest concordance index38 in the sage bionetworks - dream breast cancer prognosis challenge and was a key biomarker presented by ascopubs.org / journal / po jco precision oncology 7 n1 - n3 metastatic lymph node metastatic fgd3 protein expression is inversely associated with stage . published esr1 chromatin immunoprecipitation sequencing data in breast cancer cell lines mcf - 727 and zr - 75 - 128 identified a conserved esr1 binding site within the gene locus of fgd3 ( data supplement )  . 
by using rt - qpcr , we also determined that estradiol stimulation substantially increased the mrna expression level of fgd3 , as well as the known esr1 targeted gene tff1 in the zr - 75 - 1 cell line ( data supplement )  . 
in a previously published esr1 knockdown experiment in the mcf - 7 erpositive breast cancer cell line , fgd3 was downregulated by 2.9 and susd3 by 1.3 on a log2 scale ( data supplement ) .29 cbioportal was used to query co - expression relationships in the metabric cohort , which showed a tendency toward cooccurrence with p , .001 and a log odds ratio of 1.52. the tcga breast cohort showed a tendency toward co - occurrence with p , .001 and a log odds ratio of 2.3 in tma expression data . 
surprisingly , fgd3 mrna expression in bt - 20 , a triple - negative breast cancer cell line , was upregulated to 1.99 and esr1 was upregulated to 0.47 on a log2 scale when treated with an egfr inhibitor ( data supplement ) .30 discussion the exploratory analysis of 20 , 464 possible singlegene biomarkers in the metabric discovery cohort identified fgd3 as a highly prognostic biomarker . 
 ( a ) strong fgd3 expression in benign tumor and ( b - c ) at lower cancer stages i and iia , ( d - e ) mild fgd3 expression at stages iib and iiia , and ( f ) weak fgd3 expression at stage iiib . the group submitting the winning model.39 using the metabric data set , 3 they determined that the expression value of two genes , fgd3 and susd3 , was the most prognostic molecular metagene marker associated with a good prognosis , and they referred to it as a protective metagene because it displayed a ci that was significantly less than 0.5. 
the prognostic significance of fgd3 and susd3 as single gene prognostic biomarker using cox regression models in a large collection of breast cancer cohorts and tcga cohorts has not been published . in a follow - up study , the group developed a breast cancer prognostic test , breast cancer attractor metagenes ( bcam ) , which had several molecular features , including the breast cancerspecific fgd3 - susd3 metagene , as well as tumor size , and number of positive lymph nodes.41 notably , ou yang et al41 went on to suggest that breast cancer subtype classification as well as hormone receptor and her2 status did not add additional prognostic information when expression levels of the fgd3susd3 metagene and the attractor metagenes were known and taken into consideration . in a similar manner , susd3 expression was found to be regulated by esr1 in mcf - 7 cells through direct interaction of e2 with its regulatory region . experiments in mcf - 7 cells further showed that susd3 was implicated in e2 - mediated cell proliferation , adhesion , and migration.4 however , as with fgd3 , the role that susd3 plays in er - positive breast cancer has not been fully established . on the basis of normal tissue expression profiles , fgd3 is highly expressed in t cells , natural killer cells , monocytes associated with immune response , and myeloid whole blood cells . 
a characteristic of these cell types is that they are mobile , and evidence from the hela tet - off wound healing assay suggests that high expression of fgd3 limits cell mobility.31 fgd3 mrna expression did not correlate with tils in the big 1 - 98 dasl and tcga breast cancer cohorts , suggesting that the prognostic value of fgd3 is not indicating immune cells in patients tumors . ihc data from breast cancer tmas indicates that fgd3 protein expression is a feature of the tumor , and low expression indicates a higher chance of cell migration to lymph nodes . 
big 1 - 98 , breast international group 1 - 98 [ trial ] ; e2197 , combination chemotherapy in treating women with breast cancer [ trial ] ; er , estrogen receptor ; has , hungarian academy of science ; her2 - e , human epidermal growth factor receptor 2 subtype e ; metabric , molecular taxonomy of breast cancer international consortium ; os , overall survival ; rna - seq , rna sequencing . wide range of treatment options . 
comparing the prognostic value of fgd3 in breast cancer with the prognostic value of genes associated with proliferation such as mki67 , pcna , and aurka indicates that fgd3 may offer superior disease progression metrics in all clinically relevant breast cancer subtypes ( fig 6a )  . 
overall , aurka is the most prognostic gene in the tcga cancer cohorts in which the median time of 2 years suggests that it is an indicator of early relapse as measured by os ( fig 6b )  . 
a pubmed search for fgd3 and cancer mentioned in the abstract resulted in one publication.41 repeating the pubmed search for the other proliferation genes resulted in the following publication metrics ( mki67 , 92 ; ki67 , 3 , 104 ; pcna , 2 , 741 ; and aurka , 284 )  . the key differentiator of fgd3 as a putative biomarker is that high expression indicates favorable outcome ; for other established proliferation biomarkers , high expression of mki67 , ki67 , pcna , and aurka are prognostic of poor outcome . the availability of a growing collection of cancer cohorts with outcome data has allowed for the confirmation of the clinical importance of fgd3 as a prognostic biomarker and implications that can guide treatment . 
regan , myles brown , brian leyland - jones financial support : myles brown , brian leyland - jones administrative support : roswitha kammler , joseph a . sparano , brian leyland - jones provision of study materials or patients : joseph a . 
sparano , brian leyland - jones collection and assembly of data : scooter willis , mark abramovitz , teng fei , brandon young , xiaoqian lin , min ni , justin achua , meredith m . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . scooter willis no relationship to disclose yuliang sun no relationship to disclose mark abramovitz no relationship to disclose teng fei no relationship to disclose brandon young no relationship to disclose xiaoqian lin no relationship to disclose min ni no relationship to disclose justin achua employment : avera mckennan hospital meredith m . 
regan consulting or advisory role : merck , ipsen ( inst ) research funding : veridex ( inst ) , oncogenex ( inst ) , pfizer ( inst ) , ipsen ( inst ) , novartis ( inst ) , merck ( inst ) , ferring ( inst ) , celgene ( inst ) , astrazeneca ( inst ) , pierre fabre ( inst ) , ipsen ( inst ) kathryn p . 
gray stock and other ownership interests : mdgn robert gray research funding : abbott molecular , agios , amgen , astrazeneca , bristol - myers squibb , boehringer ingelheim , celgene , genentech , genomic health , genzyme , glaxosmithkline , imclone systems , janssen - ortho , kanisa , millennium , nodality , onyx , osi pharmaceuticals , pfizer , sanofi , sequenta , syndax , novartis victoria wang no relationship to disclose bradley long no relationship to disclose roswitha kammler no relationship to disclose joseph a . 
goldstein honoraria : genentech , roche pharma ag , puma biotechnology , pfizer , glenmark consulting or advisory role : genentech , domp e farmaceutici , roche pharma ag , puma biotechnology , pfizer , merck , astrazeneca research funding : merck ( inst ) , genentech ( inst ) other relationship : roche pharma ag roberto salgado travel , accommodations , expenses : roche sherene loi research funding : genentech ( inst ) , pfizer ( inst ) , novartis ( inst ) , merck ( inst ) , puma biotechnology ( inst ) , bristol - myers squibb ( inst ) patents , royalties , other intellectual property : pi3k pathway gene signature granted by the european and us patent offices ( inst ) myles brown consulting or advisory role : novartis , gtx research funding : novartis patents , royalties , other intellectual property : : as part of my work at the dana - farber cancer institute i have made invention disclosures and the institute is filing patents on technology related to endocrine resistance in breast cancer and improved crispr dual sgrna library design . travel , accommodations , expenses : gtx brian leyland - jones stock and other ownership interests : catalyst pharmaceuticals , progenix , puma biotechnology , sucampo pharmaceuticals , ariad , zogenix consulting or advisory role : glaxosmithkline , amgen speakers bureau : genentech , exelixis research funding : takeda , tesaro expert testimony : amgen giancarlo pruneri no relationship to disclose giuseppe viale honoraria : msd oncology consulting or advisory role : dako , genentech , astrazeneca , bristol - myers squibb , astellas pharma travel , accommodations , expenses : roche , celgene scooter willis , yuliang sun , mark abramovitz , brandon young , xiaoqian lin , justin achua , casey williams , and brian leyland - jones , avera cancer institute , sioux falls , sd ; teng fei , meredith m . 
gray , robert gray , victoria wang , and myles brown , dana - farber cancer institute , boston , ma ; min ni , childrens medical center research institute , university of texas southwestern medical center , dallas , tx ; bradley long , molecular core , scripps florida , jupiter , fl ; roswitha kammler , international breast cancer study group , bern , switzerland ; joseph a . 
goldstein , fox chase cancer center , philadelphia , pa ; roberto salgado , breast cancer translational research laboratory / institut jules bordet , brussels , belgium ; sherene loi , peter maccallum cancer centre , east melbourne , vic , australia ; and giancarlo pruneri and giuseppe viale , european institute of oncology , university of milan , milan , italy . affiliations support supported by public health service grants ca23318 , ca66636 , ca21115 , ca49883 , ca14958 , ca39229 , ca16116 , ca27525 , and ca114737 ( to the e2197 study ) from the breast cancer research foundation , national cancer institute ( nci ) , national institutes of health , department of health and human services ; by the susan g . 
bullwinkel j , baron - l uhr b , l udemann a , et al : ki - 67 protein is associated with ribosomal rna transcription in quiescent and proliferating cells . 
nouri m , ratther e , stylianou n , et al : androgen - targeted therapy - induced epithelial mesenchymal plasticity and neuroendocrine transdifferentiation in prostate cancer : an opportunity for intervention . 
lv q , zhang j , yi y , et al : proliferating cell nuclear antigen has an association with prognosis and risks factors of cancer patients : a systematic review . 
salgado r , denkert c , demaria s , et al : the evaluation of tumor - infiltrating lymphocytes ( tils ) in breast cancer : recommendations by an international tils working group 2014 . 
petryszak r , keays m , tang ya , et al : expression atlas update : an integrated database of gene and protein expression in humans , animals and plants . 
gy } orffy b , surowiak p , budczies j , et al : online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non - small - cell lung cancer . 
gyorffy b , lanczky a , szallasi z : implementing an online tool for genome - wide validation of survival - associated biomarkers in ovarian - cancer using microarray data from 1287 patients . 
denkert c , wienert s , poterie a , et al : standardized evaluation of tumor - infiltrating lymphocytes in breast cancer : results of the ring studies of the international immuno - oncology biomarker working group . 
hayakawa m , matsushima m , hagiwara h , et al : novel insights into fgd3 , a putative gef for cdc42 , that undergoes scf ( fwd1 / beta - trcp ) - mediated proteasomal degradation analogous to that of its homologue fgd1 but regulates cell morphology and motility differently from fgd1 . 
oshima t , fujino t , ando k , et al : proline - rich domain plays a crucial role in extracellular stimuli - responsive translocation of a cdc42 guanine nucleotide exchange factor , fgd1 . 
ayala i , giacchetti g , caldieri g , et al : faciogenital dysplasia protein fgd1 regulates invadopodia biogenesis and extracellular matrix degradation and is up - regulated in prostate and breast cancer . 
daubon t , buccione r , genot e : the aarskog - scott syndrome protein fgd1 regulates podosome formation and extracellular matrix remodeling in transforming growth factor b - stimulated aortic endothelial cells . 
egorov mv , capestrano m , vorontsova oa , et al : faciogenital dysplasia protein ( fgd1 ) regulates export of cargo proteins from the golgi complex via cdc42 activation . 
laetsch author affiliations and support information ( if applicable ) appear at the end of this article . creative commons attribution non commercial no derivatives 4.0 license clinical trial information : nct02637687 . corresponding author : theodore w . 
in normal development , these genes regulate the growth , differentiation , and survival of neurons.14 , 15 gene fusions that involve one of these genes ( trk fusions ) have been described in a range of pediatric and adult cancers.16 - 25 in these fusions , the 3 region of the ntrk gene , which includes the kinase domain , is joined downstream of the promoter and 5 region of an unrelated gene . 
most reported fusions in extra cranial solid tumors to date have involved either ntrk1 or ntrk3 , with ntrk2 fusions more common in primary cns tumors.26 larotrectinib is the first highly selective inhibitor of all three trk kinases to enter clinical development . 
after two cycles of chemotherapy , disease progression continued , and an aortic pseudo aneurysm developed within the mass , which led to displacement of the inferior vena cava and ureteral impingement ( fig 1b )  . 
 ( e ) after 14 months of larotrectinib treatment . the patient was subsequently referred to our institution , where surgical intervention to debulk the tumor and repair the pseudo - aneurysm was offered . 
histologically , the tumor consisted of sheets of epithelioid cells with large eccentrically placed nucleus , occasional nucleoli , and abundant eosinophilic cytoplasm , mimicking rhabdoid cells ( fig 3 )  . 
immunohistochemically , the cells showed retained ini - 1 nuclear positivity and scattered cells positive for s - 100 protein and cd34 . postoperatively , gross residual tumor remained in the vertebral bodies . 
next generation sequencing ( ngs ) of her tumor was performed using foundationoneheme ( foundation medicine , cambridge , ma ) , a combined dna and rna sequencing hybrid capture - based assay that has been validated for clinical use without requirement for an orthogonal confirmatory assay.28 sequencing revealed a novel strn - ntrk2 fusion in which the kinase domain of ntrk2 is joined in frame to strn ( figs 3e and 3f )  . 
copy number loss of cdkn2b also was identified . two months after surgery , after informed consent , the patient was enrolled in the pediatric phase i trial of larotrectinib.29 she was treated with dose level 1 ( 100 - mg adult equivalent dose by simcyp modeling [ simcyp , sheffield , uk ] ) and received larotrectinib 75 mg twice a day continuously on the basis of her age and body surface area . 
pharmacokinetic assessment after the first dose of larotrectinib revealed an estimated 24 - hour area under the plasma concentration time curve ( auc0 - 24 ) of 2 , 980 ng h / ml , which was less than the protocol - specified threshold of 3 , 500 ng h / ml that would allow intrapatient dose escalation . 
pharmacokinetics at this dose demonstrated an estimated auc0 - 24 of 3 , 830 ng h / ml . at the time of initiation of larotrectinib , the patients lansky play - performance score ( lps ) was 60 . 
by 14 days after initiation of larotrectinib , the patient was noticeably less fatigued ( lps of 70 ) , the ascites had resolved by physical examination , and her back pain had resolved ( fig 4 )  . 
 ( d ) excised tumor specimen , including the aortic bifurcation . ureter position of vertebral bodies 2 , demonstrated resolution of pet avidity of all tumors , resolution of ascites , and significant reduction in the size of the residual pulmonary metastases and retroperitoneal tumors consistent with a partial response by response evaluation criteria in solid tumors ( recist ) version 1.1 ( figs 1d and 1e )  . 
five months after starting larotrectinib , the patient underwent uncomplicated closure of her colostomy and removal of her denver shunt . despite heavy pretreatment , the patient has tolerated larotrectinib well , with the only drug related adverse events consisting of mild ( common terminology criteria for adverse events [ version 4 ] grade 1 ) fatigue and intermittent grade 1 to 2 cytopenias . 
her response continues to deepen with complete resolution of her pulmonary metastasis and only residual pet - negative tissue at the site of tumor invasion of the vertebral bodies after 22 months of larotrectinib therapy as of february 2018 . 
 currently , the patients lps is 90 , and she has returned to school . discussion we describe a patient in whom tumor sequencing led to the identification of a novel ntrk2 fusion in a chemotherapy - refractory , metastatic , unresectable , undifferentiated sarcoma and successful treatment with larotrectinib . 
the identification of an ntrk2 fusion is consistent with other reports of oncogenic fusions in this histology.13 , 30 - 39 a wide range of tumors that harbor trk fusions has been reported to respond to larotrectinib.27 the activity of larotrectinib is striking in children , with a 93% confirmed objective response rate and all patients with trk fusions experiencing tumor regression.29 this patients tumor harbored a fusion of ntrk2 , and she was the sole patient in the initial 55 - patient data set to have a fusion of this gene.27 other rare ntrk2 fusions have been described in gliomas , specifically with vcl , agbl4 , qki , and nacc2 , 20 , 40 but to our knowledge , this report is the first of an ntrk2 fusion in a pediatric extracranial solid tumor . 
 ( b ) hematoxylin and eosin stain that shows epithelioid / rhabdoid cells with eccentric , pleomorphic nuclei ; occasional nucleoli and abundant eosinophilic cytoplasm ; and a single mitosis . 
 ( f ) rna sequencing reads that support the strn - ntrk2 fusion . ntrk3 suggests that these sarcomas should be evaluated for fusions of all three ntrk genes.41 strn on chromosome 2p22.2 encodes striatin , a ubiquitously expressed calmodulin - binding protein thyroid cancer , it is a common 5 fusion partner for alk also located on 2p.42 however , strn has not previously been reported as an ntrk fusion partner , which is consistent with the broad diversity of 5 fusion partners seen in trk fusion - positive cancers27 and is an important consideration when designing testing strategies for trk fusions . 
tests such as reverse transcriptase polymerase chain reaction only detect known fusion partners and are likely to miss tumors with uncommon or unique ntrk fusion genes , which can respond to trk inhibition . 
testing that uses hybrid capturebased ngs has the potential to detect a diversity of ntrk fusion partners , but careful attention must be paid to the initial substrate ( dna v rna ) , the target enrichment strategy ( hybrid capture v amplicon v anchored multiplex polymerase chain reaction ) , and the detailed probe design . the profound clinical response seen in this strn - ntrk2 fusion patient to larotrectinib establishes that the trk fusion is the key driver for this patients tumor . 
in the latter , despite high initial response rates , nearly all patients treated with vemurafenib or dabrafenib experience disease progression within 12 to 18 months of starting therapy.43 , 44 durable responses also have been observed in patients with fusions that involve ntrk1 and ntrk3 treated with larotrectinib.27 in conclusion , ngs of the undifferentiated sarcoma in our patient was critical in identifying a highly actionable kinase fusion ( strn - ntrk2 ) , which resulted in a profound and durable clinical response on matched treatment . 
laetsch manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors veena rajaram no relationship to disclose dean c . 
miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center lee a . 
cox employment : bayer ag , loxo oncology , merck , amgen stock and other ownership interests : loxo oncology , bayer ag , merck , amgen patents , royalties , other intellectual property : us patent 62 / 318 , 041 issued to loxo oncology ( inst ) theodore w . 
laetsch , the university of texas southwestern medical center ; tara pavlock , alison patterson , anne post , caitlyn ambrose , veena rajaram , andrew martin , steve megison , and theodore w . 
ferrari a , sultan i , huang tt , et al : soft tissue sarcoma across the age spectrum : a populationbased study from the surveillance epidemiology and end results database . 
pappo as , devidas m , jenkins j , et al : phase ii trial of neoadjuvant vincristine , ifosfamide , and doxorubicin with granulocyte colony - stimulating factor support in children and adolescents with advanced - stage nonrhabdomyosarcomatous soft tissue sarcomas : a pediatric oncology group study . 
antonescu cr , suurmeijer aj , zhang l , et al : molecular characterization of inflammatory myofibroblastic tumors with frequent alk and ros1 gene fusions and rare novel ret rearrangement . 
simon mp , pedeutour f , sirvent n , et al : deregulation of the platelet - derived growth factor b - chain gene via fusion with collagen gene col1a1 in dermatofibrosarcoma protuberans and giant - cell fibroblastoma . 
moss yp , voss sd , lim ms , et al : targeting alk with crizotinib in pediatric anaplastic large cell lymphoma and inflammatory myofibroblastic tumor : a childrens oncology group study . 
laetsch tw , roy a , xu l , et al : undifferentiated sarcomas in children harbor clinically - relevant oncogenic fusions and gene copy - number alterations : a report from the childrens oncology group . 
bourgeois jm , knezevich sr , mathers ja , et al : molecular detection of the etv6 - ntrk3 gene fusion differentiates congenital fibrosarcoma from other childhood spindle cell tumors . 
knezevich sr , garnett mj , pysher tj , et al : etv6 - ntrk3 gene fusions and trisomy 11 establish a histogenetic link between mesoblastic nephroma and congenital fibrosarcoma . 
el demellawy d , cundiff ca , nasr a , et al : congenital mesoblastic nephroma : a study of 19 cases using immunohistochemistry and etv6 - ntrk3 fusion gene rearrangement . 
laetsch tw , dubois sg , mascarenhas l , et al : larotrectinib for paediatric solid tumours harbouring ntrk gene fusions : phase 1 results from a multicentre , open - label , phase 1 / 2 study . 
italiano a , sung ys , zhang l , et al : high prevalence of cic fusion with double - homeobox ( dux4 ) transcription factors in ewsr1 - negative undifferentiated small blue round cell sarcomas . 
yamada y , kuda m , kohashi k , et al : histological and immunohistochemical characteristics of undifferentiated small round cell sarcomas associated with cic - dux4 and bcor - ccnb3 fusion genes . 
li ws , liao ic , wen mc , et al : bcor - ccnb3 - positive soft tissue sarcoma with round - cell and spindle - cell histology : a series of four cases highlighting the pitfall of mimicking poorly differentiated synovial sarcoma . 
omeara e , stack d , phelan s , et al : identification of an mll4 - gps2 fusion as an oncogenic driver of undifferentiated spindle cell sarcoma in a child . 
kao yc , sung ys , zhang l , et al : recurrent bcor internal tandem duplication and ywhaenutm2b fusions in soft tissue undifferentiated round cell sarcoma of infancy : overlapping genetic features with clear cell sarcoma of kidney . 
sugita s , arai y , aoyama t , et al : nutm2a - cic fusion small round cell sarcoma : a genetically distinct variant of cic - rearranged sarcoma . 
kelly lm , barila g , liu p , et al : identification of the transforming strn - alk fusion as a potential therapeutic target in the aggressive forms of thyroid cancer . 
hauschild a , grob jj , demidov lv , et al : dabrafenib in braf - mutated metastatic melanoma : a multicentre , open - label , phase 3 randomised controlled trial . 
 evaluation of commercial circulating tumor dna test in metastatic prostate cancer sinja taavitsainen , msc1 , 2 ; matti annala , msc1 , 2 ; elisa ledet , phd3 ; kevin beja , msc1 ; patrick j . 
each sample was analyzed using guardant360 and a research panel encompassing 73 prostate cancer genes . concordance of somatic mutation and copy number calls was evaluated between the two approaches . results targeted sequencing independently conrmed 94% of somatic mutations identied by guardant360 at an allele fraction greater than 1% . 
the research approach detected several clinically relevant dna repair gene alterations not reported by guardant360 , including four germline truncating brca2 / atm mutations , two somatic atm stop gain mutations , one brca2 biallelic deletion , 11 brca2 stop gain reversal mutations in a patient treated with olaparib , and a hypermutator phenotype in a patient sample with 42 mutations per megabase . conclusion guardant360 accurately identies somatic ctdna mutations in patients with metastatic prostate cancer , but low allele frequency mutations should be interpreted with caution . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction molecular stratication is poised to guide treatment of metastatic prostate cancer ( mpc ) , but tissue biopsies are difcult to acquire , because many patients lack soft tissue metastases . 
fortunately , circulating tumor dna ( ctdna ) can be detected in peripheral blood cellfree dna ( cfdna ) from most patients with progressive disease.1 - 4 numerous research , industry , and commercial cfdna sequencing assays have arisen , each aiming to provide a practical alternative to metastatic tissue biopsy for tumor molecular stratication . unlike many other solid cancers , where ctdna assays can rely on detection of recurrent clinically relevant mutations , 5 mpc is characterized by a low mutation rate and high prevalence of large structural rearrangements , including deletions and translocations.6 furthermore , up to 30% of patients with mpc harbor deleterious germline and / or somatic defects in dna damage repair genes , such as brca2.7 the clinical laboratory improvement amendments certied guardant360 ctdna test ( guardant health , redwood city , ca ) identies single - nucleotide variants ( snvs ) in 73 genes , insertions / deletions ( indels ) in 23 genes , copy number amplications ( cnas ) in 18 genes , and gene fusions in six genes . 
 taavitsainen et al context key objective to evaluate a commercial circulating tumor ( ctdna ) test by comparing it against a prostate cancerspecic research panel in matched same - day plasma samples from men with metastatic prostate cancer . knowledge generated high concordance between the approaches was observed for ar gene copy number calls and somatic mutations at an allele fraction greater than 1% . 
the commercial test did not report several clinically actionable dna repair defects , including brca2 somatic homozygous deletions , structural rearrangements , and germline truncating mutations . relevance our results suggest that clinicians should use caution when interpreting commercial ctdna test results , particularly in the context of low allele fraction mutations . 
test utility in prostate cancer is limited by the lack of reporting on germline mutations , somatic deletions , and structural variants . specimens to guardant360 ctdna testing and foundationone ( foundation medicine , cambridge , ma ) tumor tissue testing and also found high discordance , particularly for mutations reported at allele frequencies lower than 1% . 10 together , these results raise signicant concerns about the accuracy of ctdna testing in precision oncology and are consistent with recent consensus statements on insufcient evidence of clinical validity for ctdna assays in advanced cancer11 and lack of support for routine ctdna testing in mpc.12 given the increasing use of commercial ctdna testing , we sought to assess the strengths and limitations of guardant360 in mpc . 
raw guardant360 sequencing data were not available for bioinformatic analysis , and there was no research agreement with guardant health . matched same - day samples from the 24 patients with mpc sent for guardant360 testing were subjected to targeted cfdna and wbc sequencing using a previously published academic approach ( the vancouver panel ) .2 , 13 targeted cfdna and wbc sequencing and data analysis were performed and nalized before examination of guardant360 reports . 
approval for this study was granted by the tulane university institutional review board ( certicate m0600 ) and the university of british columbia clinical research ethics board ( certicate h18 - 00944 )  . 
the af was required to be at least 20 times higher than the average af across all wbc samples and at least three times higher than the af in the paired wbc sample . 
 evaluation of commercial ctdna test in metastatic prostate cancer and the average distance of the mutant allele from the nearest read end was required to be at least 15 bases . 
our mutation analysis pipeline was previously validated in matched tissue - cfdna cohorts13 , 18 and in dilution series experiments.2 protein - level consequences of variants were predicted using annovar.19 ctdna fractions were estimated based on somatic mutation afs as previously described.2 mutations were considered subclonal if their af was less than 0.25 times the ctdna fraction ( for autosomes ) or less than 0.5 times the ctdna fraction ( for chromosome x )  . 
this conservative threshold was determined by halving the expected af for truncal mutations . two tp53 missense mutations in the cohort had an af greater than 1% in cfdna and a similar af in the matched wbc sample . 
one of these mutations ( a tp53 p.r273h missense mutation in patient 5 ) was reported by guardant360 and is included in figures 1a and 1b and the data supplement . analysis of deleterious germline variants germline variants were called in wbc samples by searching for variants with an alternate af of at least 15% and at least eight supporting reads . 
gc fraction was calculated for all target regions , and a scatter plot was created for each sample showing target regions as dots , with coverage log ratio relative to golden reference on the y - axis and gc fraction on the x - axis . 
these conservative thresholds were determined empirically by studying a plot of coverage log ratios and heterozygous snp afs in samples with and without detectable ctdna . categorization of putative pc driver alterations the following somatic mutations detected in the cohort were considered putative pc drivers : tp53 missense and truncating mutations ; ar missense mutations in amino acids 702 , 742 , 875 , 878 , and 893 ; akt1 p.e17k missense mutations ; apc truncating mutations ; braf missense mutations in amino acids 600 and 601 ; pten missense mutations in amino acid 35 ; pik3ca missense mutations in amino acids 378 , 391 , and 1047 ; atm truncating mutations ; ctnnb1 missense mutations in amino acids 32 to 45 ; and rb1 truncating mutations and missense mutations in amino acid 661 . 
putative driver mutations were determined from review of recently published large castration - resistant mpc sequencing studies.6 , 20 , 21 analysis of serial blood collections in addition to the matched 24 same - day samples subjected to guardant360 testing and vancouver panel sequencing , guardant360 reports were available for an additional 86 blood collections from the same patients collected between august 2015 and july 2018 . 
note that throughout the course of the study time period , three versions of the guardant360 assay ( 68 - , 70 - , and 73 - gene panels ) were used , with expanding coverage of genes and alterations . 
all somatic mutations identied as discordant in this study were conrmed to have been tested with the guardant360 73 - gene panel . results concordance of somatic mutation calls our objective was to compare the guardant360 test against a pc - specic ctdna research assay ( vancouver panel ) that identies snvs , indels , amplications , deletions , and rearrangements in the exonic regions of 73 mpc - relevant genes ( data supplement ) .2 , 13 , 22 between september 2016 and april 2018 , we sent plasma from 24 patients with clinically progressive mpc for guardant360 testing and performed targeted sequencing on matched same - day samples using the vancouver panel ( median depth , 1 , 435 ; data deposited to european genome - phenome archive under accession egas00001003352 ; data supplement )  . 
across the remaining 20 patients , ctdna fractions estimated by the vancouver panel ranged between undetectable ( , 2% ) and 80% ( median , 23% ; data supplement )  . focusing on 26 genes covered by both approaches , 30 ( 94% ) of 32 somatic mutations identied by guardant360 with an af greater than 1% were independently conrmed by the vancouver panel ( figs 1a and 1b ; data supplement )  . 
 a subclonal 1 - 100 known prostate cancer driver not a known prostate cancer driver not called in mutation analysis known prostate cancer driver not a known prostate cancer driver not called in mutation analysis * * * * * * taavitsainen et al not reported by guardant360 ; allele fraction is unknown * * * * * * * atm q2414x stop gain tp53 r273h missense ( clonal hematopoiesis ) atm q2593x stop gain subclonal highest allele percentage at other time points cg > tg substitution other base substitution insertion / deletion 1 2 5 6 7 8 9 10 11 12 13 15 16 17 patient fig 1 . 
it is plausible that some mutations labeled as subclonal had a nonprostate orig ( c ) plot showing all mutations ( dots ) identied by guardant360 in the cell - free dna ( cfdna ) time points that were also analyzed with the vancouver panel , grouped by allele fraction . 
patient 6 displays a hypermutation signature enriched for somatic cg.tg transitions and insertions / deletions , consistent with an underlying mismatch repair defect . supporting reads for 24 ( 86% ) of these mutations in cfdna , but nine of the mutations were found at a similar af in matched wbcs ( which are not analyzed by guardant360 ; figs 1a and 1b )  . 
furthermore , 82% of mutations with an af lower than 1% were found to be subclonal ( figs 1a and 1b ; criteria described in patients and methods )  . 
to explore further , we examined serial guardant360 test results from the same patients and found that 96% of mutations detected at an af lower than 1% did not reach an af greater than 2% at any time point ( fig 1c ) and did not track with overall ctdna fraction ( data supplement )  . 
these data suggest that many low af mutations identied by guardant360 represent subclonal passenger events or rare somatic clones of nonprostate origin . guardant360 did not report seven of 39 somatic mutations identied by the vancouver panel at allele fractions between 6% and 14% ( median , 10% ; data supplement )  . 
 evaluation of commercial ctdna test in metastatic prostate cancer vancouver panel also detected a high somatic mutation burden ( 42 mutations per mb ) in patient 6 ( fig 1d ) , accompanied by msh2 and msh6 monoallelic losses . guardant360 does not report silent mutations or total mutation burden . 
this patient had a complete remission ( 9 months at last follow - up ) after pembrolizumab treatment . sensitivity toward somatic changes in ar the ar gene is altered in most treatment - resistant mpcs , and ar status is associated with therapy response.1 , 2 , 6 , 23 in this cohort , ar amplications were identied in 11 of 24 patients , with perfect concordance between assays . 
somatic hotspot mutations in the androgen receptor ( ar ) ligand - binding domain were also highly concordant , except for three ar mutations detected by guardant at an af lower than 0.5% ( fig 2a )  . 
these mutations had supporting reads in our assay and were likely subclonal based on the presence of other mutations at higher afs ( data supplement )  . guardant360 does not report structural variants within the ar gene . 
we identied two patients with ar ligand - binding domain rearrangements predicted to yield a constitutively active ar protein ( fig 2b ) .24 identication of actionable dna repair gene alterations deleterious alterations in homologous recombination repair genes are associated with response to poly ( adp - ribose ) polymerase ( parp ) inhibitors and platinum - based chemotherapy.7 , 25 the vancouver panel identied six patients with homologous recombination repair defects : four patients with a germline brca2 or atm truncating mutation ( independently veried by commercial germline testing ) , two patients with somatic atm stop gain mutations , and one patient with a somatic brca2 biallelic deletion ( figs 3a and 3b )  . 
 ( a ) bar plot showing allele fractions of somatic ar hotspot mutations detected by the two assays . ar amplication status is indicated by blue and red tiles in the middle . 
 ( c ) eleven brca2 stop gain reversing somatic alterations detected with the vancouver panel in cfdna from patient 17 after treatment with poly ( adp - ribose ) polymerase inhibitor olaparib . 
all deletions overlapping the stop gain mutation are multiple of 3 bp in length and therefore remove the stop gain mutation while avoiding frameshistop gainreversing base substitutions are shown in the embedded table . patient 17 was sampled after olaparib resistance in march 2018 , and his cfdna revealed 11 cancer cell populations with somatic deletions or mutations reversing the germline brca2 stop gain ( fig 3c ) .26 these alterations were not reported by guardant360 , likely because it only reports short indels . concordance of gene amplication calls amplication of chromosome 7 genes met , braf , and cdk6 is rare in mpc , although low - level gain of chromosome 7 is common.6 , 21 met , braf , and / or cdk6 was reported amplied by guardant360 in 13 patients . 
guardant360 additionally reported 12 amplications in pik3ca , ccnd1 , and myc , nine of which showed evidence consistent with single copy gaonly 11 of 40 reported non - ar amplications displayed evidence for more than a single copy gain ( fig 4b )  . 
guardant360 also reported 11 amplications in genes egfr , pdgfra , kit , and fgfr1 , which are rarely altered in tissue - based mpc studies6 and are not assessed by the vancouver panel . guardant360 does not search for alterations in mpc genes spop , cdk12 , pik3r1 , foxa1 , msh2 , msh6 , tmprss2 , and erg and does not report large somatic deletions or complex structural variants in any genes . in total , we identied potentially clinically relevant alterations that were not reported by guardant360 in 12 of 24 patients , including a tp53 inactivating rearrangement , a cdkn1b biallelic deletion , intragenic ar rearrangements in two patients , three spop mutations , and dna repair defects in seven patients ( data supplement )  . discussion the guardant360 commercial ctdna assay has improved access to genomic testing across academic and nonacademic practitioners , especially in settings where tissue biopsy is not feasible . 
as with any narrow pan - cancer assay , there are compromises for individual cancer types . overall , we found excellent concordance between guardant360 and the vancouver pc panel for high af somatic mutations . 
copy numbers were estimated based on sequencing coverage log ratio and circulating tumor ( ctdna ) fraction ( described in patients and methods ) and represent the average gene copy numbers in ctdna - shedding cancer cells . 
 ( b ) sequencing coverage log ratios of genes met , braf , cdk6 , pik3ca , myc , ccnd1 , and ar , quantied with the vancouver panel in all 24 cfdna samples . 
numbers above bars indicate the average gene copy numbers in ctdna - shedding cancer cells that would produce the observed coverage log ratio in the vancouver panel data , correcting for presence of normal cfdna ( patients and methods )  . 
first , a vast majority of these mutations were subclonal and therefore potentially poor biomarkers for therapies aiming at broad antitumor effect , although they may still be of relevance for detecting emerging resistant clones . 
 taavitsainen et al somatic alterations falsely attributed to pc - derived ctdna if the wbc fraction were not analyzed.3 because guardant360 only analyzes cfdna , the test cannot reject somatic cfdna mutations that are simultaneously detected in the blood lineage . 
consistent with these three points , a vast majority of low af mutations did not track with overall ctdna fraction and never rose to a signicant clonal fraction in longitudinal sampling . 
finally , earlier studies comparing commercial ctdna assays found large discordances in reported low af somatic mutations.10 together , these results suggest that guardant360 should consider differential reporting of low af mutations , especially for mpc . somatic changes to the ar gene , including mutations , amplications , and rearrangements resulting in constitutive activation , are primary drivers of resistance to systemic therapies targeting the androgen axis in pc24 and are under development as predictive cfdna biomarkers.2 , 29 , 30 we found near - perfect concordance between guardant360 and the vancouver panel for ar amplications and hotspot mutations , with the only limitation being that guardant360 does not currently report intragenic ar rearrangements . the vancouver panel identied two patients with a potentially ligand - independent ar in this cohort . guardant360 does not report germline alterations , somatic copy number deletions , or large structural variants in the dna damage repair genes included in its panel . 
in mpc , these classes of variants are common , especially across brca2 , and can have both prognostic and predictive implications.31 of particular relevance , biallelic brca2 defects can sensitize to parp inhibitors , whereas deletions overlapping the site of a pre - existing mutation ( ie , so - called reversion mutations ) can drive parp inhibitor resistance . 26 , 32 one quarter of the patients proled here had biologically and clinically relevant dna damage repair gene alterations identied via sequencing with the vancouver panel . 
because nongenomic specialists may not be aware of the limits of commercial assays , we caution that negative results from guardant360 should be interpreted critically ; absence of reported somatic mutation in a gene does not preclude other types of alterations ( eg , somatic biallelic brca2 deletion , rearrangement , or germline truncating mutation )  . in conclusion , we show that guardant360 and targeted ctdna sequencing with a research assay ( ie , the vancouver panel ) are highly concordant for high af mutations in mpc . however , the guardant360 test has potential limitations in its reporting of indels , rearrangements , and germline variants . 
in the future , optimal ctdna commercial assays for mpc should identify and report on all types of including deletions and rearrangesomatic alterations , include mpc - specic genes , such as foxa1 , ments ; spop , and erg ; and identify deleterious germline alterations . 
limitations of this study include the absence of matched tissue to adjudicate discordances and the lack of a standardized cohort to draw clinical outcome correlates . affiliations 1university of british columbia , vancouver , british columbia , canada 2tampere university , tampere , finland 3tulane university , new orleans , la 4british columbia cancer agency , vancouver , british columbia , canada data analysis and interpretation : sinja taavitsainen , matti annala , matti nykter , oliver sartor , alexander w . 
 evaluation of commercial ctdna test in metastatic prostate cancer research funding : bayer healthcare pharmaceuticals ( inst ) , johnson & johnson ( inst ) , sano ( inst ) , endocyte ( inst ) , innocrin pharma ( inst ) , merck ( inst ) , invitae ( inst ) , constellation pharmaceuticals ( inst ) , advanced accelerator applications ( inst ) expert testimony : sano travel , accommodations , expenses : bayer healthcare pharmaceuticals , johnson & johnson , sano , astrazeneca , progenics alexander w . 
wyatt consulting or advisory role : genzyme speakers bureau : janssen research funding : janssen no other potential conicts of interest were reported . acknowledgment we acknowledge the csc - it center for science , espoo , finland , for providing computational resources . references romanel a , gasi tandefelt d , conteduca v , et al : plasma ar and abiraterone - resistant prostate cancer . 
sci transl med 7 : 312re10 , 2015 annala m , vandekerkhove g , khalaf d , et al : circulating tumor dna genomics correlate with resistance to abiraterone and enzalutamide in prostate cancer . cancer discov 8 : 444 - 457 , 2018 3 . 
hovelson dh , liu c - j , wang y , et al : rapid , ultra low coverage copy number proling of cell - free dna as a precision oncology screening strategy . 
genome med 10 : 85 , 2018 8 : 89848 - 89866 , 2017 corcoran rb , chabner ba : application of cell - free dna analysis to cancer treatment . 
n engl j med 379 : 1754 - 1765 , 2018 quigley da , dang hx , zhao sg , et al : genomic hallmarks and structural variation in metastatic prostate cancer [ erratum : cell 175 : 889 , 2018 ]  . 
eur urol 71 : 417 - 425 , 2017 zill oa , banks kc , fairclough sr , et al : the landscape of actionable genomic alterations in cell - free circulating tumor dna from 21 , 807 advanced cancer patients . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american pathologists joint review . 
scher hi , graf rp , schreiber na , et al : assessment of the validity of nuclear - localized androgen receptor splice variant 7 in circulating tumor cells as a predictive biomarker for castration - resistant prostate cancer . 
quigley d , alumkal jj , wyatt aw , et al : analysis of circulating cell - free dna identies multiclonal heterogeneity of brca2 reversion mutations associated with resistance to parp inhibitors . 
cancer discov 7 : 999 - 1005 , 2017 jaiswal s , fontanillas p , flannick j , et al : age - related clonal hematopoiesis associated with adverse outcomes . 
conteduca v , wetterskog d , sharabiani mta , et al : androgen receptor gene status in plasma dna associates with worse outcome on enzalutamide or abiraterone for castration - resistant prostate cancer : a multi - institution correlative biomarker study . 
a study of patients with myeloid malignancy compared the response rate of trametinib among patients with ras wild - type ( n = 30 ) and ras - mutated mds or aml ( n = 50 )  . 
five patients with ras - mutated mds or aml achieved a complete response ( cr ) or cr with incomplete platelet recovery compared with no patients with ras wild - type mds or aml.5 herein , we describe a patient with relapsed mds after undergoing allogeneic stem - cell transplant ( allo - sct ) who had next - generation sequencing ( ngs ) identifying map2k2 p298l , for which the patient received trametinib plus decitabine and achieved a durable cr . 
the map2k2 p298l mutation has been identified previously in a patient with lung cancer , but the functional significance at the protein level has not yet been elucidated.6 the patient is a 52 - year - old asian woman with a 12 - year history of jak2 - negative essential thrombocythemia ( et ) treated with anagrelide ( 10 years ) and hydroxyurea ( 2 years )  . 
time points indicate number of months after transplantation each next - generation sequencing panel was obtained . dnmt3a tet2 calr map2k runx1 mutations , notably runx1 s141 * ( mutation allele frequency [ maf ] , 12% ) , zmym3 g49wfs * 13 ( maf , 27% ) , calr k368rfs * 51 ( maf , 39% ) , and map2k2 p298l ( maf , 30% )  . 
after a 1 - month treatment interruption , the mucositis resolved and treatment resumed with decitabine 20 mg / m2 intravenously on days 1 through 3 of a 28 - day cycle and trametinib 2 mg daily for 21 days of a 28 - day cycle . 
after resolution , treatment with decitabine 20 mg / m2 intravenously on days 1 through 5 of a 28 - day cycle and trametinib 0.5 mg daily for 7 days of a 14 - day cycle continued without additional issues . 
instead , a benign pdgfrb t88i ( maf , 47% ) alteration and tet2 h1380y ( maf , 5% ) and dnmt3a r882s ( maf , 15% ) mutations were identified ( fig 1 ; data supplement )  . subsequently , a donor pb sample was analyzed by a clinical laboratory improvement amendments - certified moffitt 54 - genetargeted myeloid ngs panel8 with an average coverage of 11 , 987 reads . 
a benign asxl1 g652s variant was detected in the patient and the related donor ( maf , 49% ) , likely reflecting a heterozygous germline variant detected in both persons as a result of shared inheritance . 
 this acquired mutation in the patient suggests donor - derived acquisition of a chip mutation . at the time of this report , the patient was without gvhd symptoms , received a donor lymphocyte infusion ( dli ) 16 months after trametinib initiation , and remains in cr 18.5 months after trametinib initiation ( 29.5 months after allo - sct )  . 
 the primary oncologist felt that a dli may achieve the best long - term outcomes because there are no long - term efficacy data of trametinib plus a hypomethylating agent ( hma ) in relapsed mds after allo - sct . 
the patient continues to receive treatment with trametinib and decitabine without evidence of dysplasia or relapse . discussion protein function in the presence of map2k2 p298l has not been previously characterized to our knowledge . 
this map2k2 alteration is located in a proline - rich segment of the protein kinase domain , is near a map2k2 phosphorylation site , and has been observed previously in cancer.6 additionally , per the catalog of somatic mutations in cancer , the map2k2 p298l alteration had been identified as somatic and pathogenic per their prediction tool.11 despite not knowing the functional significance of the alteration , a clinical response was observed after the administration of trametinib and decitabine . 
 the 2 - year os was 12.4% and in those achieving a cr after azacitidine administration , the 2 - year os was 48.4%.12 therefore , it is difficult to determine to what degree , if any , the clinical benefit observed could be attributed to trametinib , because hmas with or without dli have demonstrated clinical efficacy in patients with relapsed mds after sct transplant . adverse effects and their management with trametinib plus decitabine has not been previously discussed to our knowledge . 
based on phase ii data , trametinib was not believed to result in the infectious complications experienced in our case.4 trametinib and decitabine were held to evaluate the etiology of the adverse effects of dermatitis and mucositis , for which a biopsy specimen demonstrated gvhd . 
kevin hicks no relationship to disclose lia perez no relationship to disclose eric padron honoraria : incyte , cell therapeutics , cell therapeutics ( inst ) research funding : incyte ( inst ) rami s . 
kim kb , kefford r , pavlick ac , et al : phase ii study of the mek1 / mek2 inhibitor trametinib in patients with metastatic braf - mutant cutaneous melanoma previously treated with or without a braf inhibitor . 
borthakur g , popplewell l , boyiadzis m , et al : activity of the oral mitogen - activated protein kinase kinase inhibitor trametinib in ras - mutant relapsed or refractory myeloid malignancies . 
hung , md , phd1 ; dora dias - santagata , phd1 ; maristela onozato , md1 ; nikunj shah , bs2 ; eric severson , md2 ; daniel duncan , md2 ; brendan j . 
we characterized molecular and clinicopathologic features of the pertinent fusion - positive patient cases . results we identied 11 patients with thymomas harboring a gene fusion of kmt2a and mastermind - like transcriptional coactivator 2 ( maml2 )  . 
the frequency of kmt2a - maml2 fusion was 4% of all thymomas ( 10 of 242 ) and 6% of thymomas of b2 or b3 histology ( 10 of 169 )  . conclusion kmt2a - maml2 represents the rst recurrent fusion described in type b thymoma . 
the identication of this fusion offers insights into the biology of thymoma and may have clinical relevance for patients with disease refractory to conventional therapeutic modalities . jco precis oncol 4 : 109 - 115 . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction thymoma , a neoplasm that arises from or exhibits thymic epithelial differentiation , is the most common tumor of the adult thymus.1 thymoma has a strong association with autoimmune diseases , particularly myasthenia gravis , which typically resolve upon successful tumor resection . 
we aim to identify recurrent genetic alterations present in this tumor . knowledge generated a subset of type b2 and b3 thymomas harbor recurrent gene fusions between lysine methyltransferase 2a ( kmt2a ) and mastermind - like transcriptional coactivator 2 ( maml2 )  . 
aside from rare case reports of hematologic neoplasms in the literature , the kmt2a - maml2 rearrangement seems specic to type b2 and b3 thymomas among human tumors . relevance the nding of recurrent kmt2a - maml2 fusion provides insights into tumor biology and potential therapeutic targets in type b2 and b3 thymomas , which are clinically aggressive with limited curative options . solid fusion assay analysis ( mgh solid fusion assay ; mgh , boston , ma ; data supplement provides list of gene targets ) and single - nucleotide variant and insertion / deletion analysis ( snapshot dna - based assay ; thermo fisher scientic , waltham , ma ; data supplement provides list of gene targets ) .7 the solid fusion assay used rna - based fusiontargeted anchored multiplex polymerase chain reaction ( archerdx primers , boulder , co ) and illumina ( san diego , ca ) sequencing . 
in brief , 60 ng of dna was extracted from 255 , 008 cancer specimens , including 242 thymoma specimens , in 40 m of formalin - xed , parafn - embedded tissue blocks . 
the samples were assayed by cgp using adaptor ligation , and hybrid capture was performed for all coding exons from 287 ( version 1 ) to 315 ( version 2 ) cancer - related genes plus select introns from 19 ( version 1 ) to 28 ( version 2 ) genes frequently rearranged in cancer ( data supplement )  . 
gtf2i included on any list of cancer - related genes . was not various samples were similarly assayed but performed in dna on 406 genes and selected introns of 31 genes involved in rearrangements ( data supplement ) and in rna on 265 genes commonly rearranged in cancer . 
sequences were analyzed for all classes of genomic alterations , including short variant alterations ( base substitutions , insertions , and deletions ) , copy - number alterations ( focal amplications and homozygous deletions ) , and select gene fusions or rearrangements by methods previously described.8 - 10 thymomas harboring a kmt2a - maml2 the cohort of fusion comprised 10 patient cases assayed with cgp ( foundation medicine ) during clinical care at other institutions . 
seven years later , the patient developed recurrent thymoma , with direct invasion of the lung , pericardium , and distal left main pulmonary artery , as well as metastasis to paratracheal lymph nodes ( fig 1a )  . the patient received induction chemotherapy and underwent surgical resection ( radical thymectomy , radical pericardiectomy , and left pneumonectomy ) , followed by paratracheal lymph node excision and radiotherapy . 
 ( a ) computed tomography of the chest from the patients rst recurrence demonstrates a 7.8 - cm anterior mediastinal mass ( arrow ) in contact with the pericardium , aorta , and portions of the main and left pulmonary arteries . 
 ( b ) histopathologic examination of the tumor shows sheets of large epithelioid cells lacking signicant lymphocytic inltration , consistent with type b3 thymoma ( hematoxylin and eosin stain ; magnication 400 )  . 
 ( d ) ihc for terminal deoxynucleotidyl lineage thymocytes associated with the tumor ( magnication 400 )  . transferase highlights scattered nuclei of t - cell recurrence of myasthenia gravis . 
the patient died as a result of complications of her thymoma 15 years after initial diagnosis . given the aggressive nature of this thymoma , molecular genetic assays were performed on the initial recurrence to lung , in an attempt to identify potentially targetable genetic alterations . 
this fusion assay result was reproduced on the patients subsequent paratracheal lymph node excision . kmt2a - maml2 fusion occurs in aggressive histologic subtypes of thymoma to determine the frequency of kmt2a - maml2 fusion in a cohort specically enriched in clinically more aggressive cases of thymoma , we reviewed a set of 242 patient cases of thymoma from the foundation medicine archives . 
the patient case was reviewed , and the original diagnosis was conrmed . the kmt2a - maml2 fusion was reamong thymomas , stricted to the most aggressive histologic thymoma subtypes , being present in 10 ( 5.9% ) of 169 thymomas that included b2 or b3 components , but 0 ( 0% ) of 64 of the remaining thymomas ( types a , ab , and b1 )  . 
no other known or likely pathogenic alterations were identied in 7 of 10 patient cases with a kmt2a - maml2 fusion , whereas a concurrent mutation in tp53 , arid1a , or sf3b1 was identied in 1 patient case each . 
of patients age at diagnosis , years final staging ( modied masaoka ) median range male female unknown histology b2 + b3 b3 + c 112 2020 by american society of clinical oncology discussion in the 243 patient cases of thymoma evaluated , we discovered a recurrent fusion of kmt2a and maml2 in 11 . this fusion seems to be highly specic to thymomas with aggressive histologic features , because all fusion - positive patient cases contained type b2 and / or b3 histology . 
the striking restriction of kmt2a - maml2 fusion to thymomas was underscored by the absence of the fusion in approximately 255 , 000 patient cases of diverse tumor types , including 366 thymic carcinomas , with the exception of a single patient case of plasmacytoma . kmt2a , rst described in 1991 , was initially termed mixedlineage leukemia - 1 because of its frequent appearance as a translocation partner in acute myeloid and lymphoid leukemias.12 the 36 - exon gene is located on chromosome 11q23 . 
the encoded protein binds dna and methylates histone h3 at lysine - 4 to regulate other genes , including several homeobox ( hox ) genes.13 kmt2a has been found to be a promiscuous translocation partner , with  . 
80 unique fusion partners identied.14 currently , there is no unifying theory on how kmt2a rearrangements lead to neoplasia.14 maml2 is a 5 - exon gene residing on chromosome 11q21 . 
maml2 and other maml family proteins are involved in notch pathwaymediated transcriptional activation.15 recurrent gene rearrangements involving maml2 have been described in mucoepidermoid carcinoma , in which fusion of the rst exon of the camp response element - binding proteinregulated transcription coactivator - 1 ( crtc1 ) with maml2 exons 2 to 5 leads to notch pathway disruption.16 the kmt2a - maml2 gene fusion results from inv ( 11 ) ( q21q23 ) , a cytogenetic abnormality rst reported in 1998 by obama et al17 in a patient with therapy - related acute myeloid leukemia . subsequent reports of the fusion are exceptionally rare and no . 
to our knowledge , only 8 patient cases have been reported , including 2 of acute myeloid leukemia , 2 of myelodysplastic syndrome , and 4 of acute lymphoblastic leukemia.17 - 22 reported fusion proteins in these patient cases involved regions similar to those identied in our cohort , with the exception of 2 patient cases reported by metlzer et al21 with maml2 breakpoints in introns 2 and 3 . prior functional studies of the kmt2a - maml2 construct have shown evidence of disrupted notch pathway signaling . 
in addition to describing the fusion in patient cases therapy - related myeloid neoplasms , nemoto et al19 the kmt2ademonstrated via luciferase assay that maml2 fusion suppresses promoter activation of the notch1 target gene hes1.19 gene expression proles from 2 patient cases of kmt2a - maml2positive t - cell acute lymphoblastic leukemia showed differential expression patterns relative to controls , suggesting activation of genes downstream of notch1.21 another study demonstrated oncogenic activity by kmt2a - maml2 fusion inserted into cell lines with sleeping - beauty vectors.23 the sum of these studies has demonstrated oncogenic function of kmt2a - maml2 fusion , likely via disruption of notch signaling . 
further study is warranted to examine the impact of this fusion on notch signaling . in patients with malignancies not amenable to traditional surgical or chemoradiotherapy protocols , targeted therapy offers an additional potential opportunity for disease control . 
early data suggest disease refractory to initial that maml2 fusionpositive mucoepidermoid carcinoma may respond to targeted therapeutics , including epidermal growth factor receptor inhibitors such as getinib.24 - 26 given the low tumor mutational burden seen in thymoma , identication of this small but genomically distinctive subset of kmt2a - maml2rearranged tumors may provide a therapeutic target in patients not responsive to traditional therapy . 
hasserjian , abner louissaint jr , erik a . williams manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors nikunj shah employment : foundation medicine eric severson employment : foundation medicine , partners healthcare stock and other ownership interests : foundation medicine daniel duncan employment : foundation medicine jeffrey s . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . dora dias - santagata consulting or advisory role : rarecyte ( i ) maristela onozato patents , royalties , other intellectual property : patent pending for multiplex fish assay , led on october 25 , 2019 ( no direct compensation related to this patent ) jo - anne vergilio employment : foundation medicine stock and other ownership interests : foundation medicine , roche valentina nardi consulting or advisory role : thermo fisher scientic ( i ) , cell signaling technology ( i ) robert p . 
hasserjian stock and other ownership interests : avanos medical , danaher , henry schein , hologic , idexx laboratories , johnson & johnson , kimberly clary , medtronic , stryker , cooper companies , thermo fisher scientic consulting or advisory role : jazz pharmaceuticals , promedior erik a . 
crit rev oncol hematol 99 : 332 - 350 , 2016 radovich m , pickering cr , felau i , et al : the integrated genomic landscape of thymic epithelial tumors . 
cancer cell 33 : 244 - 258.e10 , 2018 petrini i , meltzer ps , kim ik , et al : a specic missense mutation in gtf2i occurs at high frequency in thymic epithelial tumors . 
front oncol 4 : 103 , 2014 thomas a , rajan a , berman a , et al : sunitinib in patients with chemotherapy - refractory thymoma and thymic carcinoma : an open - label phase 2 trial . 
lancet oncol 16 : 177 - 186 , 2015 [ erratum : lancet oncol 16 : e105 , 2015 ] petrini i , rajan a , pham t , et al : whole genome and transcriptome sequencing of a b3 thymoma . 
nat med 20 : 1479 - 1484 , 2014 frampton gm , fichtenholtz a , otto ga , et al : development and validation of a clinical cancer genomic proling test based on massively parallel dna sequencing . 
ziemin - van der poel s , mccabe nr , gill hj , et al : identication of a gene , mll , that spans the breakpoint in 11q23 translocations associated with human leukemias . 
k ochert k , ullrich k , kreher s , et al : high - level expression of mastermind - like 2 contributes to aberrant activation of the notch signaling pathway in human lymphomas . 
tonon g , modi s , wu l , et al : t ( 11 ; 19 ) ( q21 ; p13 ) translocation in mucoepidermoid carcinoma creates a novel fusion product that disrupts a notch signaling pathway . 
obama k , furukawa y , tara m , et al : secondary monocytic leukemia with rearrangement of the mll gene occurring during the course of adult t - cell leukemia . int j hematol 68 : 323 - 326 , 1998 18 . 
mariani ra , silva m , caparelli e , et al : inv ( 11 ) ( q21q23 ) : kmt2a - maml2 , a recurrent genetic abnormality in t - cell therapy - related acute lymphoblastic leukemia . 
nemoto n , suzukawa k , shimizu s , et al : identication of a novel fusion gene mll - maml2 in secondary acute myelogenous leukemia and myelodysplastic syndrome with inv ( 11 ) ( q21q23 )  . 
tang g , lu x , wang sa , et al : homozygous inv ( 11 ) ( q21q23 ) and mll gene rearrangement in two patients with myeloid neoplasms . 
metzler m , staege ms , harder l , et al : inv ( 11 ) ( q21q23 ) fuses mll to the notch co - activator mastermind - like 2 in secondary t - cell acute lymphoblastic leukemia . 
chen z , chen j , gu y , et al : aberrantly activated areg - egfr signaling is required for the growth and survival of crtc1 - maml2 fusion - positive mucoe26 . 
li s , zhang z , tang h , et al : pathological complete response to getinib in a 10 - year - old boy with egfr - negative pulmonary mucoepidermoid carcinoma : a pidermoid carcinoma cells . 
bryce purpose genomic testing has increased the quantity of information available to oncologists . unfortunately , many identified sequence alterations are variants of unknown significance ( vuss ) , which thus limit the clinicians ability to use these findings to inform treatment . 
we applied a combination of in silico prediction and molecular modeling tools and laboratory techniques to rapidly define actionable vuss . materials and methods exome sequencing was conducted on 308 tumors from various origins . most single nucleotide alterations within gene coding regions were vuss . 
a subset of receptor tyrosine kinase vuss was characterized by laboratory comparison of each vus versus its wild - type counterpart in terms of expression and signaling activity . results the study identified 4 , 327 point mutations of which 3 , 833 were vuss . 
three vuss ( fgfr2 f276c , fgfr4 r78h , and kdr g539r ) showed increased basal or ligand - stimulated erk phosphorylation compared with their wild - type counterparts , which suggests that they support transformation . 
for example , braf v600e is well accepted as a therapeutic target in metastatic melanoma.7 however , a current challenge in genomic oncology care is the evaluation of the potential therapeutic significance of large numbers of uncharacterized , nonsynonymous sequence alterations referred to as variants of unknown significance ( vuss ) in potentially oncogenic proteins . when novel vuss are identified , the clinical team must try to draw conclusions about whether the variant is a driver mutation or has no significance for cancer pathogenesis . 
although some recurrent mutations within oncogenic proteins have been characterized as having an effect on protein function and thus , the promotion of transformation , novel vuss identified through clinical genomic testing often are lacking functional information . the definition of the therapeutic value of vuss is a current unmet need.8 we demonstrate how in silico analysis and experimental laboratory studies can rapidly determine the potential therapeutic value of a vus . 
by using bioinformatic analysis of exome sequencing results , a large number of potentially deleterious vuss that are therapeutically targetable were identified with a high frequency of occurrence in kinases . 
the mayo clinic irb approved the in vitro functional studies of somatic mutations identified in tumors of patients enrolled in the cim oncology service ( irb 15 - 003386 )  . 
redcap ( research electronic data capture ) hosted at the mayo clinic was used to collect and store clinical follow - up data.10 results vuss are the most common findings in tumor exome sequencing analysis targeted and exome sequencing were performed on heterogeneous solid ( 57% ) and hematologic ( 43% ) malignancies ( data supplement )  . 
vuss , which constitute mutations that are functionally uncharacterized or previously unreported in the cosmic ( catalogue of somatic mutations in cancer ) database , 11 comprised the majority ( 89% ) of the point mutations observed in this cohort ( fig 1a )  . 
obstacles to clinical use of these data resulted from large numbers of vuss identified in each patient , uncertainty whether identified vuss are potential drivers of transformation , and whether encoded proteins were therapeutically targetable . to begin to address these difficulties , vuss were filtered into a subgroup of 905 in genes that encode proteins therapeutically targetable by food and drug administrationapproved drugs or by investigational agents available through clinical trials . 
protein class analysis demonstrated that rtks and serine / threonine kinases represent the largest therapeutically targetable functional classes , with mutations in both hematologic and solid tumors ( fig 1b )  . these 905 therapeutically targetable vuss were then evaluated in silico , and those determined to be benign by two prediction tools were removed , which left 522 potentially deleterious , therapeutically targetable vuss in 226 patients ( data supplement ) or 12% of the total variants detected ( fig 1a )  . 
the distribution of protein classes represented in this deleterious , therapeutically targetable subgroup ( fig 1c ) was similar to that of the therapeutically targetable vus group ( fig 1b )  . 
 a significant findings , 11% targetable , deleterious , vuss , 12% other vuss , receptor tyrosine kinase serine / threonine kinase notch receptor dna endonuclease endocytosis nonreceptor tyrosine kinase tumor suppressor pi3k family ubiquitin ligase apoptotic regulator growth inhibition receptor tyrosine kinase serine / threonine kinase notch receptor endocytosis nonreceptor tyrosine kinase dna endonuclease pi3k family histone deacetylase tumor suppressor hormone receptor fgf receptor ubiquitin ligase growth inhibition hematologic solid no . 
 ( a ) frequency of significant findings and uncharacterized / unreported variants of unknown significance ( vuss ) in 4 , 327 point mutations reported in 308 patient tumors , including solid and hematologic malignancies . 
 ( b ) frequency of the most commonly observed protein classes in 905 therapeutically targetable vuss . with pymol software ( schrodinger , new york , ny ; data supplement ) , which predicted potentially altered function on the basis of their location within the proteins kinase domain ( data supplement )  . in silico and functional characterization of rtk vuss identified a subset of new targetable mutations ten rtk vuss from varied solid and hematologic malignancies ( data supplement ) and in reinterest were selected for gions of functional additional evaluation by in vitro testing . 
in addition , sequence alignment across species demonstrated that the wt residue that corresponded to each of the 10 vuss was completely conserved among diverse species , including mouse , rat , bovine , and human proteins . 
biochemical studies of r78h demonstrated no significant differences from wt fgfr4 in overall expression levels when expressed in kmch - 1 cholangiocarcinoma cells ( figs 2b and 2c )  . 
cellular localization of wt and r78h fgfr4 was also similar and exhibited mainly a plasma membrane distribution with intracellular labeling of probable golgi membranes ( fig 2e )  . however , r78h fgfr4 exhibited a small , but significantly elevated fgf2 - stimulated phosphoerk ( perk ) level compared with wt ( figs 2b and 2d )  . two kdr vuss , g55e and g539r , are located in the extracellular domain in proximity to amino acids that form disulfide bonds ( c53 and c530 ) ; thus , these amino acid substitutions may potentially affect neighboring disulfide bonds . 
 ( c ) frequency of the most commonly observed protein classes in 522 therapeutically targetable , potentially deleterious vuss . cells that expressed g55e kdr ( figs 3a and 3c )  . kdr variants and wt exhibited a similar punctate distribution by immunofluorescence ( fig 3d )  . vuss g251e , v484m , and t643m of pdgfra and variants v258l and v316m of pdgfrb were investigated . 
all variants fall in the extracellular domain of their respective receptor except t643m , which occurs within the pdgfrb kinase domav258l was predicted to be functionally benign but was of interest for in vitro study because of its ig3 location . 
pdgfr vuss were generally similar to their wt counterparts in terms of expression , distribution of polypeptide species on western blots , pdgf - stimulated perk levels , and cellular localization ( data supplement ) , except that pdgfra v484m was significantly lower in expression and pdgfstimulated perk levels than wt ( data supplement ) and pdgfrb v316m was significantly lower in expression than wt ( data supplement )  . f276c mutant is a new , constitutively active form of fgfr2 although predicted in silico to be benign , fgfr2 k41e , identified in an acute myeloid leukemia , was selected for additional study because of its location in the extracellular ig1 of fgfr2 and its unknown effect on ligand binding in nearby ig2 and ig3 ( data supplement )  . 
the fgfr2 f276c mutation identified here from a cholangiocarcinoma was also in a single cholangiocarcinoma in the cosmic database but has not been characterized.11 , 12 f276c is located in an extracellular , ig - like c2 - type 3 domain13 where ligand binding occurs.14 a different amino acid substitution at the same residue , f276v , has been reported in crouzon syndrome.15 modeling of wt and f276c fgfr2 showed that the extracellular receptor of fgfr2 contains an intrinsic disulfide bond between c278 and c342 in ig3 ( fig 4a , shown in gold )  . 
these data suggest that the f276c variant is functionally altered relative to the wt form . when fgfr2 proteins were expressed in kmch - 1 cells , immunofluorescence microscopy showed that the wt and k41e fgfr2 proteins were localized mainly to the cell surface and occurred on intracellular structures , likely endosomes and the golgi apparatus ( fig 4c )  . 
in contrast , the f276c variant exhibited an endoplasmic reticulumlike appearance and bright golgi - like intracellular structures in most cells , with only a minority of cells showing an obvious plasma membrane distribution ( fig 4c )  . 
each protein was expressed as an approximately 130 - kda polypeptide as detected by western blot ( fig 4d )  . however , the f276c variant was expressed at a higher level and the k41e variant at a lower level compared with wt ( fig 4d ) , although cells were transfected with equal amounts of dna for the different fgfr2 constructs . functional characteristics of fgfr2 proteins were assessed by studying perk signaling . 
because the f276c fgfr2 variant is expressed at higher levels than wt when equal amounts of dna were used , a lower ratio of f276c construct dna was used for transfection in the following experiments so that resulting levels of wt and f276c fgfr2 proteins were comparable ( figs 4e and 4f ) : kmch - 1 cells transfected with fgfr2 constructs were treated for 16 hours in serum - free media with fgf2 , lysed , and analyzed by western blot . 
the expression of wt or k41e fgfr2 in the absence of fgf2 increased perk levels beyond control levels , and treatment with fgf2 led to approximately fivefold increases in perk compared with control for both wt and k41e . 
however , expression of f276c fgfr2 significantly increased the perk level in the absence of fgf2 compared with wt fgfr2 , with little increase upon treatment with fgf2 ( figs 4e and 4g )  . 
in summary , f276c fgfr2 has high expression , altered cellular distribution , and increased constitutive activity compared with wt . finally , the sensitivity of wt and f276c fgfr2 activities to treatment with the fgfr inhibitor bgj398 were compared . 
at concentrations between 0 and 100 nm , erk phosphorylation was similarly partially inhibited by bgj398 in cells that expressed wt or f276c , and both fgfr2 forms were completely inhibited by bgj398 at 200 nm ( figs 5a and 5b )  . 
right panel : after 1 day , the cells were incubated with and without 20 ng / ml fgf2 in 0.1% bovine serum albumin / dmem for 16 hours at 37c . 
values are mean 6 se normalized to wt ( + fgf2 ) levels . bracket indicates groups within treatment types ( 2fgf or + fgf ) among fgfr4 - transfected samples that were significantly different ( p , .05 ) from each other in twotailed t tests . 
 ( e ) hucct - 1 cells were transfected with flag - tagged wt and r78h fgfr4 for 2 days and then processed for immunofluorescence by using an anti - flag antibody . 
he was originally treated in a clinical trial with gemcitabine , cisplatin , and silmitasertib , which resulted in a partial response . he remained on this treatment of 10 months , at which time his disease progressed and he was switched to capecitabine and oxaliplattwo months later , the disease became refractory to that regimen . 
after 1 day , the cells were serum starved in 0.1% bovine serum albumin / dmem overnight and then incubated with and without 25 ng / ml vascular endothelial growth factor ( vegf ) for 10 min at 37c . 
cell samples were then lysed and subjected to western blot analysis for flag - kdr , phospho - erk ( perk ) , total erk , and vinculequal amounts of total protein were loaded per lane . 
g55e expression of full - length receptor ( approximately 200 kda ) was decreased compared with wt , with the appearance of approximately 70to 80 - kda fragments , which suggested decreased stability / increased degradation of the g55e forperk was barely detectable in g55e samples and not increased by vegf treatment . 
brackets indicate groups within treatment types ( 2vegf or + vegf ) among kdr - expressing samples that were significantly different ( p , .05 ) from one another in two - tailed t tests . 
 ( d ) hucct - 1 cells were transfected with flag - tagged wt and variants of unknown significance kdr for 2 days and then processed for immunofluorescence with an anti - flag antibody . 
on the basis of this finding , the patient started on bgj398 ( clinicaltrials.gov identifier : nct02150967 ) , achieved a partial response to therapy after 2 months ( figs 5c and 5d ) , and maintained the response for an additional 4 months , at which time new lesions developed that led to discontinuation of bgj398 . 
as a result of our in vitro studies of f276c , a mechanism of action has now been correlated with this observed clinical response of the tumor to bgj398 . discussion the volume of new data from individual and group sequencing efforts ( eg , the cancer genome atlas ) has rapidly expanded the understanding of the incidence and frequency of mutations in disparate cancers . 
few mutations have extensive preclinical and clinical evidence that support the effectiveness of targeted therapies , and genomic testing often reveals that tumors have numerous vuss , including variant sequences for which no functional data are available . mutations that have not been functionally characterized present a significant and growing challenge to the treating physician . 
the extracellular receptor of fgfr2 contains an intrinsic disulfide bond between c278 and c342 in the immunoglobulin - like domain 3 ( ig3 ; shown in gold )  . residue f276 is highlighted in gray and is proximal to the disulfide bridge . 
the fgfr2 f276c variant ( highlighted in red ) may lead to the introduction of aberrant disulfide bonds that could alter the activation state of the prote ( b ) sequence alignment shows that residue f276 is highly conserved among the fgfr2 family from zebrafish to humans ( sequence alignment performed by using clustal omega [ embl - ebi , wellcome genome campus , uk ] ; uniprotkb entry numbers are shown )  . 
 ( c ) hucct - 1 cholangiocarcinoma cells transfected with the wt , k41e , and f276c fgfr2 forms for 2 days were fixed , permeabilized , and processed for immunofluorescence by using the flag antibody . 
kmch - 1 cells were transfected with fgfr2 forms by using a 3.5 / 5 ratio of f276c / wt and k41e dna to adjust expression of the f276c protein to similar levels as the wt forafter 1 day , cells were switched to serum - free medium with or without 20 ng / ml fgf2 and incubated an additional 16 hours at 37c before lysis . 
lysates were analyzed by western blot for expression of fgfr2 - flag , phospho - erk ( perk ) , total erk , and vincul ( f ) quantitation of fgfr2 levels in western blots as in ( e )  . 
brackets indicate groups within treatment types ( 2fgf or + fgf ) among fgfr2 - expressing samples that were significantly different ( p , .05 ) from one another in two - tailed t tests . 
 ( a ) kmch - 1 cells were transfected with f276c and wild type ( wt ) by using a 3.5 / 5 dna ratio , respectively , to normalize expression levels . 
values are mean 6 se and expressed as percent inhibition of perk signal compared with cells with no bgj398 . response of fgfr2 f276ccontaining tumor to bgj398 in ( c ) september 2015 ( pretreatment , with magnetic resonance imaging showing a 30.1mm tumor diameter [ red line ] ) and ( d ) october 2015 ( postinitiation of treatment , with pan - fgfr inhibitor bgj398 magnetic resonance imaging showing tumor shrinkage to an 18.2 - mm diameter [ red line ] )  . fgfr2flag perk fgfr2flag perk f276c bgj398 ( nm ) 50 100 200 f276c bgj398 ( nm ) becomes how to quickly assess the potentially deleterious effect of vuss that occur in therapeutically targetable genes . 
with multiple uncharacterized / unreported mutations returned for each patient , a method of prioritizing which variants to study in depth is necessary . we addressed this problem by developing an approach that uses an in silico filtering process to prioritize mutations of the highest biologic and clinical interest . 
of these 10 variants , seven were found to be altered in expression or activity relative to the wt protein , and three of these ( fgfr2 f276c , fgfr4 r78h , and kdr g539r ) demonstrated greater activity than their wt counterparts ( table 1 ) , which suggests that these mutations play a role in promoting oncogenesis . 
in contrast , four vuss ( fgfr4 k41e , kdr g55e , pdgfra v484m , and pdgfrb v316m ) exhibited reduced expression compared with their wt counterparts , and thus were unlikely to be involved in oncogenesis in the tumors where they occurred . 
these findings not only support the strength of our in silico analysis in predicting whether vuss are functionally altered but also point to the inability to distinguish among activating , deactivating , and destabilizing mutations . optimization of appropriate cellular models is important in methods development for functional evaluation of vuss . 
for more understanding of the functional significance of vuss , these studies should be followed by experiments that use cell types that match the vus tumor of origin and evaluate end points such as cell growth and viability . fgfr2 f276c was identified as a vus of potential interest because of the presence of several factors , including prediction of a deleterious effect by two algorithms , 3d modeling that suggests an increase in activity on the basis of its location in the ligand - binding ig3 , and proximity to a key disulfide bond . 
dashes ( ) indicate no change versus wild - type protein . abbreviation : vus , variant of unknown significance . * increases or decreases in activity ( phospho - erk levels ) indicate changes seen versus wild type upon stimulation with ligand , except for fgfr2 f276c , which exhibited increased constitutive activity . constitutive receptor dimerization and activation , which lead to a variety of skeletal and craniosynostosis disorders ( eg , crouzon and pfeiffer syndromes ) .16 - 18 our molecular modeling combined with the demonstration that f276c fgfr2 is more highly expressed and constitutively active than the wt receptor suggests that this mutation alters disulfide bonds , which alters receptor dimerization and activity similarly to the fgfr2 mutations seen in craniosynostosis syndromes . 
by using in vitro studies , we show that the f276c fgfr2 variant is sensitive to bgj398 , a panfgfr inhibitor , which was also reflected clinically in the response of a patients tumor when treated with bgj398 as part of a clinical trial ( fig 5 )  . 
however , additional studies , such as the testing of bgj398 effectiveness in impeding growth of organoids or xenografts that express wt versus f276c , are needed to confirm that fgfr2 f276c is actionable . the ability to identify potentially actionable vuss from numerous vuss for each patient tumor would simplify therapeutic choices . 
in silico analysis requires only hours to conduct and as demonstrated here , can yield a subset of vuss that encode therapeutically targetable proteins likely to be altered in function by their variant sequence . in vitro functional studies are more time consuming and may not fit practically within a patients progression timeline , but they may yield moredefinitive findings . 
gawryletz no relationship to disclose sikander ailawadhi consulting or advisory role : amgen , takeda pharmaceuticals , novartis research funding : pharmacyclics ( inst ) travel , accommodations , expenses : amgen , takeda pharmaceuticals , novartis stephen m . 
ansell honoraria : webmd , research to practice research funding : bristol - myers squibb ( inst ) , celldex therapeutics ( inst ) , seattle genetics ( inst ) , merck ( inst ) , affimed ( inst ) , trillium therapeutics ( inst ) kelly k . 
halfdanarson consulting or advisory role : lexicon ( inst ) , ipsen ( inst ) , merrimack ( inst ) research funding : esanex ( inst ) , ipsen ( inst ) , boston biomedical ( inst ) thai h . 
 research funding : merck , bristol - meyers squibb , roche ( inst ) , genentech ( inst ) , x4p pharmaceuticals ( inst ) , amgen ( inst ) prashant kapoor consulting or advisory role : sanofi ( inst ) research funding : amgen ( inst ) , takeda pharmaceuticals ( inst ) aaron s . 
mansfield consulting or advisory role : trovagene ( inst ) , genentech ( inst ) , bristol - myers squibb ( inst ) travel , accommodations , expenses : bristol - myers squibb nathalie meurice no relationship to disclose amulya a . 
nowakowski consulting or advisory role : celgene ( inst ) , morphosys ( inst ) , genentech ( inst ) research funding : celgene ( inst ) animesh pardanani no relationship to disclose sameer a . 
feldman consulting or advisory role : infinity pharmaceuticals patents , royalties , other intellectual property : inventor of technology for which the mayo clinic holds an unlicensed patent or has submitted a patent application ( inst ) ramesh k . 
ramanathan honoraria : taiho pharmaceutical , cerulean pharma , pharmacyclics , insys therapeutics , novocure consulting or advisory role : cerulean pharma , novocure , insys therapeutics , pharmacyclics research funding : abbvie ( inst ) , celgene ( inst ) , merck ( inst ) , schering plough ( inst ) , merrimack ( inst ) , boston biomedical ( inst ) , berg ( inst ) , superlab far east ( inst ) steven i . 
robinson travel , accommodations , expenses : tricon pharmaceuticals raoul tibes research funding : novartis ( inst ) , merck ( inst ) , seattle genetics ( inst ) , boehringer ingelheim ( inst ) , astellas pharma ( inst ) , astex pharmaceuticals ( inst ) heidi d . 
mcwilliams consulting or advisory role : merrimack ( inst ) research funding : prism biolab ( inst ) , genentech ( inst ) , novartis ( inst ) , newlink genetics ( inst ) , eli lilly ( inst ) , aduro biotech ( inst ) , pfizer ( inst ) , sanofi ( inst ) travel , accommodations , expenses : astrazeneca mrinal m . 
wieben patents , royalties , other intellectual property : champions oncologylicensed ip - targeted therapy with abiraterone acetate ( $0 received ) , wuxi appteclicensed ip - breast cancer xenografts ( $12 , 737 to mayo clinic ; inst ) , affymetrix - licensed ip on several variants in drug processing enzymes ( $0 in past 2 years ) , life technologieslicensed ip on several variants in drug processing enzymes ( $0 in past 2 years ) tammy m . 
kennedy patents , royalties , other intellectual property : patent on unrelated work ( us patent 9 , 458 , 189 ) for ligation of stapled polypeptides issued october 4 , 2016 eric w . 
borad stock and other ownership interests : glaxosmithkline , gilead sciences consulting or advisory role : g1 therapeutics , td2 , fujifilm ( inst ) , agios ( inst ) , insys therapeutics ( inst ) , novartis ( inst ) , arqule ( inst ) , celegene ( inst ) , inspyr therapeutics , halozyme therapeutics ( inst ) research funding : boston biomedical ( inst ) , mirna therapeutics ( inst ) , senhwa biosciences ( inst ) , medimmune ( inst ) , bioline ( inst ) , agios ( inst ) , halozyme therapeutics ( inst ) , celgene ( inst ) , threshold pharmaceuticals ( inst ) , toray ( inst ) , dicerna pharmaceuticals ( inst ) , sillajen ( inst ) , eisai ( inst ) , taiho pharmaceuticals ( inst ) , emd serono ( inst ) , isis pharmaceuticals ( inst ) , incyte ( inst ) , sun biopharma ( inst ) , ariad pharmaceuticals ( inst ) , imclone systems ( inst ) travel , accommodations , expenses : arqule , celgene , astrazeneca aminah jatoi research funding : entera health , boston biologics martin e . 
kennedy , university of georgia , athens , ga . supported by the mayo clinic center for individualized medicine . presented at the science of team science 2016 conference , phoenix , az , may 16 - 19 , 2016 . support prior presentation references 35 : 49 - 62 , 2016 1 . 
horak p , fr ohling s , glimm h : integrating next - generation sequencing into clinical oncology : strategies , promises onco targets ther 9 : 7355 - 7365 , 2016 and pitfalls . 
harris pa , taylor r , thielke r , et al : research electronic data capture ( redcap ) a metadata - driven methodology and workflow process for providing translational research informatics support . 
steinberger d , vriend g , mulliken jb , et al : the mutations in fgfr2 - associated craniosynostoses are clustered in five structural elements of immunoglobulin - like domain iii of the receptor . 
lajeunie e , heuertz s , el ghouzzi v , et al : mutation screening in patients with syndromic craniosynostoses indicates that a limited number of recurrent fgfr2 mutations accounts for severe forms of pfeiffer syndrome . 
ratisoontorn c , fan gf , mcentee k , et al : activating ( p253r , c278f ) and dominant negative mutations of fgfr2 : differential effects on calvarial bone cell proliferation , differentiation , and mineralization . 
 o circulating braf v600e cell - free dna as a biomarker in the management of anaplastic thyroid carcinoma purpose anaplastic thyroid cancer ( atc ) is a deadly form of thyroid cancer . 
we explored the ability of droplet digital polymerase chain reaction ( ddpcr ) to identify this mutation in circulating cell - free dna ( cfdna ) in plasma . materials and methods the ddpcr assay was evaluated for its sensitivity , specificity for detection of braf v600e cfdna , and concordance with tumor tissue . 
the assay also was used to evaluate its potential role as a biomarker of response . results forty - four patients with atc who were tested for the braf mutation by tumor tissue dna sequencing or immunohistochemistry were included . 
in addition , cfdna levels by ddpcr were predictive of treatment response in 71% of samples . conclusion cfdna detection by ddpcr is highly sensitive , specific , and concordant with mutation status on atc tumors . 
2018 by american society of clinical oncology introduction anaplastic thyroid carcinoma ( atc ) is responsible for one third of thyroid cancer deaths and has a high mortality rate.1 , 2 braf v600e is the most common actionable driver mutation in atc , which is seen in approximately 45% of these cancers.3 , 4 indeed , targeted treatment strategies are extending patient survival , 5 - 8 and the braf plus mek inhibitor combination dabrafenib plus trametinib was recently approved by the food and drug administration for braf v600emutated atc.9 , 10 diagnosis of atc usually is made by biopsy . 
 unlike differentiated tumors , the lack of sufficient viable tissue hampers molecular diagnostic testing.2 , 11 , 12 given the rapid progression of atc , rapid testing is needed for timely therapeutic intervention . 
liquid biopsy ( mutation testing performed on blood samples ) provides a potential alternative to tissue biopsy in the management of certain malignancies.13 - 15 the detection of mutations in circulating cell - free dna ( cfdna ) , such as braf v600e , can serve as a surrogate for mutation testing on tissue biopsy , and its overall levels provide a novel marker in predicting thyroid cancer burden.16 , 17 the need for molecular testing on tissue before treatment with selective braf inhibitors may substantially delay the time to initiation of a beneficial treatment in patients with braf - mutated atc.8 , 18 - 22 we have demonstrated previously priyanka c . 
 patients with atc prospectively enrolled ( n = 58 ) excluded ( n = 14 ) did not have atc ( n = 6 ) mutation on tumor not tested ( n = 2 ) no evidence of active atc ( n = 2 ) no guardant sample drawn simultaneously ( n = 4 ) ( n = 44 ) correlation of cfdna braf status by ddpcr with tissue braf - mutated cfdna as a biomarker of response ( n = 44 ) ( n = 16 ) fig 1 . 
 in the absence of readily available tumor mutation profiling , we obtain both braf v600e by ihc on tissue biopsy and cfdna on plasma by ngs simultaneously before initiation of treatment . 
the ihc analysis antibody is anti - braf p.v600e ( clone ve1 , dilution 1 : 50 ; spring bioscience , pleasanton , ca ) and is performed on a ventana benchmark ultra autostainer ( ventana medical systems , tucson , az )  . patients who had a cfdna analysis by ngs more than 2 weeks before the research ddpcr sample draw were excluded from concordance testing . 
however , if patients tested positive for the braf v600e mutation on their tumor tissue and were receiving treatment , serial plasma samples were collected for ddpcr assay during their treatment to examine the correlation of cfdna levels with response to treatment . study population for correlation of changes in cfdna levels with response to treatment patients with tissue - positive braf v600e mutation were followed prospectively during their respective treatments to evaluate whether levels of circulating braf v600e cfdna by ddpcr could serve as a biomarker for monitoring response to therapy . 
blood samples for cfdna analysis were drawn at routinely scheduled laboratory appointments . braf v600e mutation analysis tumor tissue was tested for braf mutation status by either ihc or ngs at our center ( 50gene somatic mutation analysis panel , 25 solid tumor genomics assay version 1 that examines 134 genes , 26 or foundationone genomic test27 ) , depending on the quantity of viable tumor tissue available for testing . 
of patients median age , years ( range ) male female stage at diagnosis braf status on tumor braf v600e mutation * braf wild type method of braf analysis on tumor ihc without ngs baseline liquid biopsy sample by ngs drawn not drawn no . 
 abbreviations : ihc , immunohistochemistry ; ngs , next - generation sequencing . * of these patients , 17 ( 85% ) had tumor tissue tested by ngs and three ( 15% ) by ihc . of the five patients who were tested for the presence of the braf v600e protein by ihc , one had testing for and detection of the same mutation by single - gene polymerase chain reaction at an outside facility before referral to our center . 
two patients who had tumor tissue tested only by ihc did not have additional testing by ngs because of insufficient tumor tissue . sequencing test28 ( guardant health , redwood city , ca )  . evaluation of response to treatment a single independent radiologist reviewed the images at baseline and at the time of restaging while patients were receiving treatment . 
 we determined clinical responses by calculating the percent change ( from baseline and after / during treatment ) in the size of target lesions identified using response evaluation criteria in solid tumors ( recist ) version 1.1 , 29 but we modified slightly the definitions of response . 
for the purpose of our study , an increase in tumor size was defined as an increase in the sum of target lesions greater than or equal to 10 mrecist progression in nontarget lesions was assigned an additional 20 mm to the sum of target lesions . 
a decrease in tumor size was defined as a decrease in sum of target lesions greater than or equal to 10 m stable tumor size was defined as a change in tumor burden between 9 and 9 mm . data analysis testing for concordance between circulating v600e cfdna by ddpcr and tumor tissue and cfdna by ngs and tumor tissue . 
a false positive was defined by the presence of a mutation in cfdna when tumor tissue tested negative , and a false negative was indicated when the mutation was not detected in cfdna but tumor tissue tested positive for braf v600e mutation . concordance between braf v600e mutation in cfdna by ddpcr with braf mutation status on tumor tissue by ngs , ihc , and pcr was calculated as number of concordant samples / ( number of concordant samples + number of discordant samples )  . 
concordance also was calculated for the detection of braf mutation in cfdna samples by ngs and tumor tissue . testing for correlation of change in circulating v600e ddpcr levels with changes in tumor size . 
 in patients who were followed prospectively , concordance was calculated between the change in the level of braf - mutated cfdna by ddpcr and change in tumor size on restaging scans during treatment . 
any increase in the level of cfdna was called concordant if that correlated with an increase in tumor size of greater than or equal to 10 m similarly , any decrease in the level of cfdna was called concordant if it correlated with a decrease in tumor size of greater than or equal to 10 mif there was no change in the level of cfdna correlating with 9 to 9 mm , the sample was called concordant . 
concordance between detection of circulating braf v600e cell - free dna ( cfdna ) by droplet digital polymerase chain reaction ( ddpcr ) and next - generation sequencing ( ngs ) on liquid biopsy with mutation status on tumor . 
npv , negative predictive value ; ppv , positive predictive value . statistical tests sensitivity and specificity of detection of braf - mutated cfdna by ddpcr and by ngs were calculated and reported with exact 95% cis . 
fishers exact test was conducted to calculate concordance between cfdna levels and the corresponding radiologic events , specifically , increase in tumor size , tumor shrinkage , and stable tumor size . results fifty - eight patients with a suspected atc diagnosis were prospectively enrolled in the study . 
of the remaining 44 patients , 16 who had a braf v600e positive tumor were followed for correlation changes in cfdna with response to treatment . detection of braf v600e cfdna on liquid biopsy the lowest allelic fraction ( af ) for braf v600e detected by ddpcr was 0.07% and for cfdna by ngs , 0.1%. 
of the 20 patients who tested positive for braf mutation on tissue , 17 ( 85% ) were positive for braf v600e cfdna by ddpcr and 16 ( 80% ) were positive for cfdna by ngs ( fig 2 )  . 
detection of braf - mutated cfdna by ddpcr was 93% concordant with tumor testing , whereas detection of cfdna by ngs was 91% ( 95% ci , 8% to 14% )  . 
forty - three ( 98% ) also had blood samples sent for cfdna by ngs . correlation between changes in braf v600e cfdna by ddpcr and changes in tumor size sixteen patients whose tumors tested positive for braf mutation were prospectively followed and had plasma samples drawn during scheduled laboratory appointments before the clinic visit . 
in the three patients who tested false negative by ddpcr , tumor burden at baseline ranged from 31 to 33.3 m these patients had had their primary tumor treated with surgery ( thyroidectomy in two patients and hemithyroidectomy in one patient )  . 
the braf v600e cfdna was detectable in tumors larger than 52.5 mm , with the exception of a single patient who had an 8 - mm lung nodule that presented while he was receiving adjuvant radiation to the thyroid bed with chemotherapy . of 36 time points , 27 ( 75% ) provided concordant findings between changes in cfdna levels and response to therapy ( table 3 )  . 
we included the three patients who tested false negative by ddpcr in this group because the braf v600e cfdna levels continued to remain undetectable , which correlated with these patients continued response to treatment with braf inhibitor . 
one patient with braf positivity on tumor did not get testing done by next - generation sequencing ( ngs ) on liquid biopsy ; therefore , the analysis was done on 43 patient samples . 
because all 24 of the patients without braf mutation tested negative for cell - free dna by droplet digital polymerase chain reaction ( ddpcr ) and ngs , the receiver operating characteristic curve is degenerate . 
one patient showed an increase in cfdna levels 2 weeks after starting braf inhibitor therapy , which was consistent with the increased tumor size observed on his first restaging scans 2 months after starting this treatment . 
figure a1 shows an example of the trends in cfdna levels that correlated with changes in tumor size on imaging in a patient who was initially treated with nab - paclitaxel followed by braf and mek inhibitor therapy and then with the addition of immunotherapy ( pembrolizumab )  . 
the levels of braf cfdna trended upward despite the addition of pembrolizumab because the patient was not able to swallow the braf inhibitor , which led to nonadherence and eventual progression . discussion the braf v600e mutation is the most common actionable mutation in atc tumors . 
 recent studies have shown promising results with dabrafenib plus trametinib ( braf and mek inhibitors ) in terms of clinical response and survival , 9 , 18 , 30 and this combination is now food and drug administration approved for braf - mutated atc . 
hence , identification of this mutation for timely initiation of treatment is important . in addition to the ability to identify an actionable mutation for initiation of therapy , development of biomarkers is needed to predict and assess treatment response in atc . 
of patients median age , years ( range ) male female stage at diagnosis treatment received * surgery radiation sensitizing chemotherapy systemic therapy braf inhibitor + mek inhibitor braf inhibitor + mek inhibitor + immunotherapy no . 
patients with a braf mutation on their tumor tissue had liquid biopsy samples drawn prospectively for assessing correlation of changes in levels of circulating braf v600e cell - free dna detected by droplet digital polymerase chain reaction with response to treatment . * patients received more than one treatment modality . ( tg ) levels are a biomarker of response to therapy . 
given the successes observed for other cancers where tumor - specific biomarkers do not exist , we believe that changes in braf v600e cfdna could provide a specific biomarker for monitoring response to treatment in patients with atc.19 although response can be assessed by imaging , a marker to predict response allows sufficient time to change the treatment plan before the patient becomes too ill . in this study , we show that cfdna by ddpcr is a sensitive and highly specific technique for determining braf status in patients with atc . 
braf cfdna and rna in liquid biopsy have been shown to be useful biomarkers in patients with dtc as well as in a murine model of atc.31 , 32 the current study is the first in our knowledge to evaluate ddpcr to detect braf - mutated cfdna in the circulation of patients with atc . 
in a phase ii clinical trial in radioiodine - refractory dtc , braf v600e cfdna was detectable by ddpcr in only 35% of patients , 33 whereas in our study , it was detected in 85% of patients with atc . 
 therefore , we suggest that in patients with low measurable disease , reliance solely on cfdna for identifying the braf mutation may not be advisable , and molecular testing on tumor tissue should be awaited . a murine model of braf - mutated atc circulating rna levels has been shown to be a useful biomarker of response to therapy.32 in the current study , we show that ddpcr is a useful technique for serial monitoring of cfdna levels as a predictive biomarker in patients with atc table 3 . 
increase in tumor size was defined as any change in the sum of target lesions 10 mprogression in nontarget lesions ( ie , nonmeasurable disease ) was assigned an additional 20 mm to the sum of target lesions . 
these findings were similar to that observed in patients with melanoma.34 , 35 in patients whose braf v600e cfdna levels rise after initiating braf and mek inhibitor therapy , resistance to treatment should be considered , prompting early restaging scans and consideration of alternative treatment options upon confirmation . 
one possible explanation is that targeted treatment indeed led to a reduction in the braf v600eharboring tumor cells but led to clonal expansion of tumor cells that harbored other driver mutations as a mechanism of resistance . 
however , the small sample size limited us from evaluating the association of the af of the braf v600e cfdna with prognosis and overall survival . in conclusion , ddpcr is a novel , noninvasive technique for detecting braf v600e cfdna in patients with atc and should be considered in routine standard - of - care testing . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . ramona dadu research funding : astrazeneca , merck , eisai , genentech , bristol - myers squibb mark zafereo no relationship to disclose naifa l . 
 renata ferrarotto consulting or advisory role : ayala research funding : oncomed pharmaceuticals ( inst ) , g1 therapeutics ( inst ) , astrazeneca ( inst ) , medimmune ( inst ) , emd serono ( inst ) , genentech ( inst ) , roche ( inst ) , merck serono ( inst ) vivek subbiah consulting or advisory role : medimmune research funding : novartis ( inst ) , glaxosmithkline ( inst ) , nanocarrier ( inst ) , northwest biotherapeutics ( inst ) , genentech ( inst ) , roche ( inst ) , berg ( inst ) , bayer ag ( inst ) , incyte ( inst ) , fujifilm ( inst ) , pharmamar ( inst ) , d3 oncology solutions ( inst ) , pfizer ( inst ) , amgen ( inst ) , abbvie ( inst ) , multivir ( inst ) , blueprint medicines ( inst ) , loxo oncology ( inst ) , vegenics ( inst ) , takeda pharmaceuticals ( inst ) , alfasigma ( inst ) , agensys ( inst ) , idera ( inst ) , boston biomedical ( inst ) , inhibrx ( inst ) , exelixis ( inst ) travel , accommodations , expenses : pharmamar , bayer neil gross honoraria : intuitive surgical consulting or advisory role : pds biotechnology , verb surgical ( inst ) research funding : regeneron pharmaceuticals , medimmune ( inst ) , genentech ( inst ) brandon g . 
cabanillas consulting or advisory role : loxo oncology , blueprint medicines research funding : kura oncology , eisai , roche , genentech , exelixis acknowledgment we thank the patients with atc who agreed to provide samples , karen bohannon - johnson for phlebotomy support , and clinical colleagues who provided access to their patients for this study . affiliations all authors : the university of texas md anderson cancer center , houston , tx . supported by an endocrine fellows foundation grant ( p.c.i. ) and national cancer institute grant no . 
takahashi s , kiyota n , yamazaki t , et al : phase ii study of lenvatinib in patients with differentiated , medullary , and anaplastic thyroid cancer : final analysis results . 
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cradic kw , milosevic d , rosenberg am , et al : mutant braf ( t1799a ) can be detected in the blood of papillary thyroid carcinoma patients and correlates with disease status . 
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schreuer m , meersseman g , van den herrewegen s , et al : quantitative assessment of braf v600 mutant circulating cell - free tumor dna as a tool for therapeutic monitoring in metastatic melanoma patients treated with braf / mek inhibitors . 
chen g , mcquade jl , panka dj , et al : clinical , molecular , and immune analysis of dabrafenibtrametinib combination treatment for braf inhibitor - refractory metastatic melanoma : a phase 2 clinical trial . 
 cfdna declined at the time of response to therapy correlated with tumor shrinkage from baseline , and an increase in cfdna levels at the time of progression correlated with a radiologic increase in tumor burden . - 25c nab - paclitaxel dabrafenib + trametinib + pembrolizumab time ( months ) isolation of cell - free dna for droplet digital polymerase chain reaction after obtaining patient consent , approximately 8 to 10 ml of blood were drawn into each of two edta tubes . 
plasma was separated from the cell components after centrifuging twice at 2 , 000 rpm for 10 minutes at 4c and then stored at 80c to allow for batch isolation of cell - free dna ( cfdna )  . 
the amount of dna was quantified using a qubit fluorometer ( thermo fisher scientific , waltham , ma )  . droplet digital polymerase chain reaction the detection of the braf v600e mutation was performed in a research laboratory setting using the qx200 droplet digital pcr system ( bio - rad , hercules , ca ) and the validated primepcr ddpcr mutation assay ( bio - rad ) , which was multiplexed with an assay for detection of braf unmutated codon ( wild type )  . 
the cfdna yields using this protocol ranged from 2 to 132 ng / ml when measured by qubit fluorometer , which allowed a median estimated input of 5 ng per assay . 
droplets were counted with the qx200 droplet reader , and data were analyzed using quantasoft software ( bio - rad ) , with thresholds for fam and hex detection manually set on the basis of results from the no - template control . 
 therapy - related erythroleukemia in a man with metastatic ewing sarcoma : a clinical role for advanced molecular diagnostics introduction therapy - related leukemia may be challenging to diagnose when a patient has cytopenia during treatment of another malignancy , particularly when there are few circulating leukemia cells . 
 here , we report a case of a 55 - year - old man with refractory ewing sarcoma who presented with thrombocytopenia and tumor lysis syndrome ( tls ) ; he was found to have pure erythroid leukemia ( pel ) , likely resulting from prior alkylating agent and / or topoisomerase ii inhibitor chemotherapy . 
furthermore , when patients with metastatic solid tumor have cytopenia , they may not undergo bone marrow ( bm ) evaluation if the cytopenia has already been attributed to therapies or the prior malignancy . in this case , initial diagnostics and the poorly differentiated morphology seen in the bm biopsy specimen did not readily distinguish between ewing sarcoma and acute leukemia . 
he was diagnosed with ewing sarcoma in 2011 , after a known right - side lower - lobe lung mass demonstrated increased size and malignant radiologic features on computed tomography ( ct ) imaging . 
the patient underwent right - side lower lobectomy and metastasectomy of bilateral pectoral lesions , radiation ( xrt ) to his rib , and additional chemotherapy with dactinomycin , vincristine , and cyclophosphamide , followed by xrt to his pectoral surgical bed . 
he started a trial of olaparib and temozolomide in august 2015 , which continued until october 2016 , when he was removed from protocol after ct imaging showed disease progression . 
cumulatively , he received 31 cycles of chemotherapy and four treatments of xrt . one month after stopping therapy , the patient presented with fatigue , dyspnea , fevers , bruising , and petechial rash , with no hepatosplenomegaly . 
peripheral smear revealed left - shifted myeloid cells and large basophilic cells with vacuolated cytoplasm ; these cells constituted 9% of blood leukocytes and were interpreted as either reactive atypical lymphocytes , circulating ewing sarcoma cells , or circulating blasts . 
zmynd8 exons 1 through 22 fuse with rela exons 3 through 11 to form the fusion gene zmynd8 - rela , which has been described in erythroleukemia.2 zmynd8 exons 1 - 22 zmynd8 - rela fusion chromosome 20 exons 3 - 11 rela chromosome 11 cbc counts at the ewing sarcoma diagnosis in 2011 were unavailable , cbc counts in 2013 were normal ( wbc , 4.9 103 / l ; hemoglobin , 14.0 g / dl ; platelets , 207 103 / l )  . he underwent bm biopsy and aspiration , and began treatment of tls with rasburicase , allopurinol , and normal saline . 
however , treatment was complicated by persistent cytopenia and hallucinations , likely related to ifosfamide . the bm biopsy specimen was hypercellular and revealed replacement by primitive , discohesive cells , which , by flow cytometry , were negative for cd45 , cd34 , myeloperoxidase , cd13 , and cd56 . 
by immunohistochemistry , cells were negative for cytokeratin , cd43 , and cd56 , but were positive for cd99 ( present in up to 98% of ewing sarcoma but also expressed in other cancers , including leukemia ) .3 - 6 preliminary bm karyotype identified an 11q13 abnormality , but no ewsr1 rearrangement . 
interphase fluorescent in situ hybridization study also did not reveal ewsr1 rearrangement . molecular testing was performed via heme snapshot assay , an institutional ngs platform using illumina truseq amplicon technology ( san diego , ca ) for single nucleotide variant and insertion / deletion detection in genomic dna.7 snapshot results are descriptive and cannot determine clonality , tumor - stromal percentage , or percentage of cells carrying mutations . 
 analysis was undertaken using a sequencing library generated with an oligo pool focusing on approximately 141 kb of genomic content consisting of 568 amplicons covering 15 full genes and exonic hotspots in 39 genes ( trusight myeloid sequencing panel ; illumina )  . testing identified three tp53 mutations with the following allelic frequencies : r337p , 38% ; i255f , 38% ; and y220c , 10% . 
these findings confirmed diagnosis of therapy - related pel , rather than progressive ewing sarcoma . the patient transiently improved after ifosfamide / etoposide treatment , but the leukemia ultimately progressed , at which point he opted for comfort care . 
he died 2 months after pel diagnosis . methods this anchored multiplex fusion assay is an ngs method that amplifies small amounts of nucleic acid and detects fusions without targeting specific rearrangements . 
two heme - nested pcr reactions are then performed to create a fully functional sequencing library targeting specific genes and exons ( fusionplex solid tumor kit ; archerdx , boulder , co )  . 
illumina misequation 2 147 base pairend sequencing results are aligned to the hg19 human genome reference , and a laboratory - developed algorithm is used for fusion transcript detection and annotation . 
the resulting product can then be processed for illumina or ion torrent ( thermo fisher scientific , waltham , ma ) sequencing.10 discussion this case highlights how the evolving field of molecular genetic diagnostics can help distinguish specific cancer diagnoses . 
advanced molecular testing using ngs and an rna - based solid fusion assay ultimately clarified this patients diagnosis . ewing sarcoma describes a family of cancers with fusion oncogenes involving the ewsr1 gene.13 there is a high correlation between bony metastases and bm involvement , and , to our knowledge , pel has not been described in refractory ewing sarcoma before this case report.14 - 17 therapy - related leukemia is a rare adverse effect of alkylating agents , radiotherapy , and topoisomerase ii inhibitors . 
tumor cells were positive for e - cadherin and cd71 ( not shown )  . erythroid precursors with 30% proerythroblasts.22 patients typically present with sequelae of pancytopenia from erythroid precursor proliferation . pel also has a very complex karyotype compared with other leukemias and can be difficult to diagnose , given its negativity for other markers typically expressed in aml , such as cd45 , cd13 , myeloperoxidase , and cd34.20 , 22 , 23 cd45 is commonly used to screen for hematologic neoplasms on initial pathologic staining . 
 ( megakaryoblastic anemia is another cd45 - negative leukemia . ) e - cadherin , expressed in most pel cases , helped lead us toward the correct diagnosis , as did demonstration of the rare zmynd8 - rela fusion . 
the latter is hypothesized to promote oncogenesis via constitutive nf - b activation.1 by definition , aml after exposure to cytotoxic chemotherapy or radiation is therapy - related aml , of which there are two primary etiologic subtypes : alkylating agent / radiotherapyrelated type and topoisomerase ii inhibitorrelated type.24 , 25 alkylating agents and radiotherapy exhibit dose - dependent risk of disease effected via centromeric breakage and partial or complete chromosome loss , particularly at chromosomes 5 and 7 . 
the leukemia had several balanced translocations , including t ( 11 ; 20 ) ( q12 ; q13.1 ) , and a partial deletion of chromosome 4 . mutations in tp53 , as seen in this case , are common in t - aml . 
interestingly , recent data suggest that tp53 - mutated clones may exist before the exposure to chemotherapy and expand under the selective pressure of treatment of another malignancy.27 this is the first reported case of greater than two tp53 mutations in t - aml.9 , 27 regardless of the inciting agents , t - aml is associated with dismal outcomes , such as were experienced by this patient . in patients with advanced cancers treated with alkylating agents , radiotherapy , or topoisomerase ii inhibitors , t - aml should be considered as a cause of refractory cytopenia . 
angelini df , ottone t , guerrera g , et al : a leukemia - associated cd34 / cd123 / cd25 / cd99 + immunophenotype identifies flt3 - mutated clones in acute myeloid leukemia . 
dias - santagata d , akhavanfard s , david ss , et al : rapid targeted mutational analysis of human tumours : a clinical platform to guide personalized cancer medicine . 
ok cy , patel kp , garcia - manero g , et al : tp53 mutation characteristics in therapy - related myelodysplastic syndromes and acute myeloid leukemia is similar to de novo diseases . 
green dc , deharvengt sj , de abreu fb , et al : use of anchored multiplex pcr enrichment for detection of gene fusions in solid tumors by next generation sequencing . 
thiel u , wawer a , von luettichau i , et al : bone marrow involvement identifies a subgroup of advanced ewing sarcoma patients with fatal outcome irrespective of therapy in contrast to curable patients with multiple bone metastases but unaffected marrow . 
cuneo a , van orshoven a , michaux jl , et al : morphologic , immunologic and cytogenetic studies in erythroleukaemia : evidence for multilineage involvement and identification of two distinct cytogenetic - clinicopathological types . 
kasyan a , medeiros lj , zuo z , et al : acute erythroid leukemia as defined in the world health organization classification is a rare and pathogenetically heterogeneous disease . 
olney hj , mitelman f , johansson b , et al : unique balanced chromosome abnormalities in treatment - related myelodysplastic syndromes and acute myeloid leukemia : report from an international workshop . 
in their letter , karam et al express concerns about the claims made regarding the inability of rna sequencing ( rna - seq ) to perform allele - specic quantication . however , we believe our statement that rna - seq does not allow the allele - specic quantication of normal full - length transcript is correct . the percent splicing index measures the frequency at which a given splice event occurs compared with others affecting the same site.3 as karam et al1 note , this allows one to quantify reads from aberrant transcripts relative to reference transcripts but it does not provide an allele - specic quantication of splicing . 
however , in the case of intronic variants , rnaseq cannot determine whether any of the full - length transcript is also produced by the variant allele because the variant is not part of the messenger rna sequence . 
in the absence of an additional informative variant in the exon , rna - seq cannot distinguish between a full splice defect ( no full - length transcript is made by the variant allele ) and a partial or leaky splice defect ( some full - length transcript is produced by the variant allele )  . 
as gelli et al4 noted , partial splice defects may not be pathogenic if the variant allele produces enough full - length transcript to support normal protein function . in their letter , karam et al1 highlighted a recent publication by landrith et al3 and indicated that rnaseq was used to identify partial skipping of exon 5 in the brca2 c.426 - 12_426 - 8del variant carrier and control patients . 
of note , the landrith et al denition of partial exon skipping is some portion of the exon is skipped ( ie , alternative acceptor / donor ) , 3 not as a partial splice defect . 
however , other published data do show that brca2 c.426 - 12_426 - 8del causes a partial splice defect.8 our laboratory now classies this variant as benign based on additional phenotypic and mutation co - occurrence data , 2 which suggests that the amount of normal transcript produced by the variant allele does support normal protein function . with increased use of tools such as rna - seq to aid in variant classication , physician understanding of how these tools should be used is critical to appropriate patient care . 
we would like to emphasize that we are not arguing against the use of functional rna studies in producing data to support variant classication . rather , we are promoting the need for improved guidelines for interpretation and use of such data in variant classication . 
sharing of reclassication advancements and variant classication information through publications such as the article by nix et al2 allows for dissemination of high - quality data that has passed peer review . 
nix p , mundt e , manley s , et al : functional rna studies are a useful tool in variant classication but must be used with caution : a case study of one brca2 variant . 
gelli e , colombo m , pinto am , et al : usefulness and limitations of comprehensive characterization of mrna splicing proles in the denition of the clinical relevance of brca1 / 2 variants of uncertain signicance . 
rna - seq allows the use of a method called the percent splicing index to quantify allelic expression by mapping rna - seq reads from alternate or aberrant transcripts relative to reference ( normal ) transcripts as described by schafer et al.7 we have recently published data demonstrating that the variant brca2 c.426 - 12_426 - 8delgtttt not only results in partial skipping of exon 5 ( brca2 r.426_475del ) , but this splicing event was also seen in 20 of 345 control patients.8 laboratories should proceed with caution when weighting functional evidence in variant curation , because interpretation of these results can be complex . 
in this specic case , the rna - seq results and the new clinical data prompted reassessment and resulted in reclassication of this brca2 variant . the article by nix et al6 highlights the need for laboratories to increase collaboration , particularly when siloed clinical information can shed light onto discrepant classications . 
submission of a classication and its justication to dynamic databases such as clinvar is a critical step in this process , 9 because data sharing among clinical diagnostic laboratories improves the resolution of variant interpretation differences.10 in summary , we believe that innovation , such as introduction of rna - seq to hereditary cancer clinical genetic testing as well as participation in multi - institutional efforts designed to increase transparency between laboratories , is of utmost importance for advancing variant classication that will ultimately benet patients . rachid karam , md , phd ; holly laduca , ms ; marcy e . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . rachid karam employment : ambry genetics research funding : ambry genetics travel , accommodations , expenses : ambry genetics holly laduca employment : ambry genetics stock and other ownership interests : ambry genetics marcy e . 
richardson employment : ambry genetics tina pesaran employment : ambry genetics stock and other ownership interests : ambry genetics travel , accommodations , expenses : ambry genetics elizabeth chao employment : ambry genetics no other potential conicts of interest were reported . references cummings bb , marshall jl , tukiainen t , et al : improving genetic diagnosis in mendelian disease with transcriptome sequencing . 
sci transl med 9 : eaal5209 , 2017 fr esard l , smail c , ferraro nm , et al : identication of rare - disease genes using blood transcriptome sequencing and large control cohorts . 
nat med 25 : 911 - 919 , 2019 gonorazky hd , naumenko s , ramani ak , et al : expanding the boundaries of rna sequencing as a diagnostic tool for rare mendelian disease . 
am j hum genet 104 : 466 - 483 , 2019 lee h , huang ay , wang l - k , et al : diagnostic utility of transcriptome sequencing for rare mendelian diseases . 
genet med 22 : 490 - 499 , 2020 karam r , conner b , laduca h , et al : assessment of diagnostic outcomes of rna genetic testing for hereditary cancer . 
nix p , mundt e , manley s , et al : functional rna studies are a useful tool in variant classication but must be used with caution : a case study of one brca2 variant . 
jco precis oncol 4 : 730 - 735 , 2020 schafer s , miao k , benson cc , et al : alternative splicing signatures in rnaseq data : percent spliced in ( psi )  . 
curr protoc hum genet 87 : 11.16.111.16.14 , 2015 landrith t , li b , cass aa , et al : splicing prole by capture rna - seq identies pathogenic germline variants in tumor suppressor genes . 
the precise impact of these panels on clinical decision making is not well understood . methods here , we report our institutional experience with a targeted 40 - gene panel ( myeloseq ) that is used to generate a report for both genetic variants and variant allele frequencies for the treating physician ( the limit of mutation detection is approximately one aml cell in 50 )  . results in total , 346 sequencing reports were generated for 325 patients with suspected hematologic malignancies over an 8 - month period ( august 2018 to april 2019 )  . 
to determine the inuence of genomic data on clinical care for patients with acute myeloid leukemia ( aml ) , we analyzed 122 consecutive reports from 109 patients diagnosed with aml and surveyed the treating physicians with a standardized questionnaire . 
inuences were often nuanced and extended beyond identifying actionable genetic variants with us food and drug administrationapproved drugs . conclusion this study provides insights into how physicians are currently using multigene panels capable of detecting relatively rare aml cells . 
the most inuential way to integrate these tools into clinical practice will be to perform prospective clinical trials that assess patient outcomes in response to genomically driven interventions . jco precis oncol 5 : 191 - 203 . 
some quires variant single - gene tests and two multitarget panels are currently us food and drug administration ( fda ) approved and have associated companion diagnostics that designate a specic cancer subtype and indication.2 however , the vast majority of variant identication and annotation is performed by laboratory developed tests ( ldts ) , 3 for which results are provided as a clinical report . 
these reports typically require a certain level of genomic literacy to understand and implement.4 , 5 lack of report standardization , inadequate genomic training , 6 , 7 limited infrastructure to support oncologists , 1 , 8 and limited resourcesfor example , low reimbursement rates and lack of access to clinical trialsare thought to hinder the impact of sequencing data on clinical care.9 , 10 the result is a discrepancy between the identication of clinically actionable variants11 and implementation of change in treatment decisions.1 , 12 acute myeloid leukemia ( aml ) sequencing data are particularly complex because of the genetic and clonal heterogeneity of aml cases . 
 barnell et al context key objective whereas physicians have increased the use of targeted sequencing panels to inform treatment decisions for patients with hematologic malignancies , the impact and utility of sequencing data on clinical decision making is not well understood . 
through standardized surveys , this study captured the nuanced impact of sequencing data on clinical decision making for patients with acute myeloid leukemia . knowledge generated physicians self - reported that 50 of 114 sequencing reports ( 44% ) inuenced clinical care decisions with 56 total inuences reported , 38 of which were related to therapeutic options , 10 related to risk stratication in rst clinical remission , and eight involving measuring persistent molecular disease . 
in 11% of all patients , the panel was ordered at multiple timepoints during the disease course for persistent molecular disease monitoring . relevance this study demonstrates that physicians are integrating sequencing data obtained at various points in the acute myeloid leukemia disease course into clinical decision making , mostly to determine therapy choices , stratify relapse risk , and measure persistent disease . signicance may depend on cooperating covariants in multiple other genes.14 for example , european leukemianet / national comprehensive cancer network guidelines specically mention six genes / mutations in their risk stratication guidelines , all of which must be evaluated in parallel for risk stratication.15 , 16 in addition , whereas pathogenic variants within three genesflt3 , idh2 , and idh1have been used as predictive markers for fda - approved targeted therapeutics , 17 - 19 ongoing clinical trials have demonstrated predictive utility for other pathogenic variants that do not yet have approved companion diagnostics.20 next - generation sequencing ( ngs ) data can provide information beyond therapeutic sensitivity / resistance that is not currently captured by fda - approved diagnostics . specically , quantication of the variant allele frequency ( vaf ) of tumor - associated variants has been used to detect persistent molecular disease ( pmd ) after therapies are administered , and predict both relapse risk21 and survival outcomes.22 - 24 although multipanel ldt diagnostics are being used on patients with aml to supplement treatment decisions , the utility of these reports is not well described . this study sought to better understand the extent to which genomic reports inuence clinical decision making for patients with aml . 
this paper describes physician experience with a targeted gene panel ( myeloseq ) that terrogates 40 recurrently mutated genes or gene hotspots and returns gene variant and vaf data to the treating physician . 
we determined usage patterns over an 8 - month period and surveyed treating physicians regarding how they used the panel results in clinical care . methods study design and patient eligibility this study was conducted at washington university school of medicine ( human research protection ofce #201801112 )  . 
the panel was a targeted sequencing assay that evaluated 40 genes and gene hotspots that are recurrently mutated in myeloid malignancies ( data supplement ) .25 for each patient who was found to have a denitive diagnosis of aml , a survey was sent to the treating physician . 
patients with acute promyelocytic leukemia ( m3 aml subtype ) were excluded.26 the treating physician was asked to complete a ninequestion survey to determine how the physician used the panel to inform treatment decisions ( data supplement )  . panel design and processing the panel uses an amplicon capture - based enrichment with unique molecular identiers for ultra - high variant sensitivity that targets an approximately 98 - kb space ( haloplex target enrichment system ; agilent technologies , santa clara , ca )  . 
this included evaluating bone marrow morphology , standard panels of ow cytometric markers , and a panel of routine cytogenetic and molecular marker studies , including uorescence in situ hybridization probes . target enrichment , variant calling strategies , and variant annotation methods are described in the data supplement . the median number of failed genes across all reports was two ( range , 0 to 24 genes )  . 
wt1 ( n = 211 cases ) , cux1 ( n = 197 cases ) , and cebpa ( n = 111 cases ) were genes that most frequently failed coverage requirements . 
if physicians did not respond to the initial contact , reminders were provided to increase the response rate . ethics , consent , permissions , and consent to publish this study was conducted at washington university school of medicine and was approved by the washington university human research protection ofce ( #201801112 )  . 
summary of patient , report , and physician characteristics characteristic ( n = 109 ) value median time from accessioning to report sign out , daysa 12 ( 4 - 36 ) patient ( n = 109 ) median age , years male female report ( n = 122 ) physician ( n = 14 ) mean time in practice , yearsb academic rank professor associate professor assistant professor degree type md / phd md / mba male female there were 124 reports from patients with aml . 
the reports from patients with aml , after removing patients with apl subtype ( n = 122 samples ) , were derived from 109 unique patients who were under the care of 14 physicians ( table 1 )  . 
only 13 samples had no observed variants , eight of which were acquired from patients who were determined to be in clinical remission using bone marrow biopsy . reports were ordered at various points in the disease course . 
the next most common timepoint was during disease assessment ( n = 28 of 122 ; [ 22.9% ] ) , followed by potential relapse ( n = 19 of 122 [ 15.6% ] ; fig 3a )  . 
for reports ordered during disease response assessments , seven were ordered post - induction , six were ordered after allogeneic hematopoietic cell transplant ( allohct ) , ve were ordered during ongoing treatment with a hypomethylating agent , two were ordered after consolidation with high - dose ara - c , and seven were ordered during or after salvage therapy ( fig 3b )  . 
on the basis of these data , the sequencing report was a factor in determining therapy decisions in 50 ( 43.8% ) of 114 cases ( fig 4a )  . 
the patient accepted the treatment plan 93% of the time ( n = 38 of 41 cases )  . predictive inuences in 35 of 50 reports , physicians indicated that they recommended at least one therapy on the basis of the variants identied by the sequencing panel ( fig 4b )  . 
on the basis of results35 reports with 38 corresequencing panel sponding inuencesphysicians reported recommending a hypomethylating agent in 14 patients for a tp5320 or tet228 , 29 variant , kinase inhibitors ( midostaurin or gilteritinib ) in 12 patients with a flt3 variant30 and one patient with a kit variant , 31 and idh1 / idh2 inhibitors were recommended for eight patients.32 there were three patients for whom a clinical trial was recommended on the basis of observed variants or persistent disease . 65 ( 23 - 90 ) 58 ( 53.2 ) 51 ( 46.8 ) 22 ( 8 - 44 ) 5 ( 36 ) 4 ( 29 ) 5 ( 36 ) 8 ( 57 ) 5 ( 36 ) 1 ( 7 ) 11 ( 79 ) 3 ( 21 ) note . 
of cases ( n = 122 ) flt3 internal tandem duplication splice acceptor variant frameshift variant missense variant flt3 tyrosine kinase domain splice donor variant inframe insertion negative no flt3 variant flt3 status stop gained variant type inframe deletion percentage of cases fig 2 . 
in 16 of 29 eligible cases ( two patients refused treatment ) , physicians reported the following inuences : 12 used the ngs panel to prescribe an flt3 inhibitor and four inuences were independent of flt3 status . 
in three of these four cases , patients had an flt3 inhibitor prescribed based on a previously known result from a different genetic test . there were 13 cases in which the patient had an flt3 variant , but the physician reported no inuence . 
of these 13 cases , three patients died before treatment initiation and eight had an flt3 inhibitor prescribed based on a previously known result from a different genetic test ( data supplement )  . risk stratication for transplant in first clinical remission physicians identied 10 reports that inuenced patient risk assessment and allohct recommendation ( fig 4b )  . specically , allohct was not recommended in rst complete remission in six patients , based on the presence of bialleleic cebpa variants ( n = 2 ) , kit variant status ( n = 1 ) , lack of high - risk variants ( n = 1 ) , or differential clearance of co - occurring variants after induction therapy ( n = 1 )  . 
for the latter patient , three variants were detected at diagnosis ( dnmt3a [ vaf = 42% ] , npm1 [ vaf = 38% ] , and rad21 [ vaf = 43% ] )  . 
these recommendations were based on tp53 variant status ( n = 2 ) , dnmt3a variant status ( n = 1 ) , and co - occurrence of multiple variants ( dnmt3a , npm1 , and flt3 internal tandem duplication ; n = 1 ) .14 assessment for pmd there were eight cases in which the sequencing panel was used to assess for pmd ( fig 4b )  . 
in ve cases , the physician indicated that the reports conrmed relapse / progression of aml , including one case of bone marrow negative , extramedullary relapse in the esophagus ( fig 5d )  . there were three patients who were being assessed for pmd while in an apparent clinical remission . 
two of the three patients did not show persistent aml via pmd assessment . the third patient showed three residual variants at low vaf ( cebpa [ vaf = 4% ] , smc1a [ vaf = 4% ] , and wt1 [ vaf = 3% ] ) , suggesting persistent disease . patients with no change in treatment regimens in response to sequencing panels there were 64 cases for which the physician noted no change in therapeutic plan on the basis of the report . 
the total number of eligible surveys completed by each physician and the total number of cases where the physician changed his or her plan is shown in figure 4c . the sequencing panel reports the vaf and thus can detect possible inherited variants . 
one patient was found to have a gata2 variant observed at a vaf of 52% , indicative of a possible germline variant , which prompted an action by the treating physician . 
a subsequent skin biopsy determined that the patient did not have a germline predisposition for disease . multiple sequencing panels for longitudinal disease monitoring and response therapy , there were 12 patients for whom the treating physician ordered multiple reporters during the disease course21 ( fig 5 and data supplement )  . 
after induction , despite some variants demonthe idh2 vaf of strating response to initial 39% ( 46% at diagnosis ) resulted in initiation of enasidenib . figure 5b shows a patient for whom the physician obtained three sequencing panels for longitudinal pmd monitoring . the rst report was ordered at relapse post - transplant and showed ve variants . 
a month later , the third report showed the re - emergence of three variants despite no excess blasts on the bone marrow biopsy , which resulted in a second allohct . 
figure 5c shows how a physician used sequential reports to observe the patients molecular response to a donor lymphocyte infusion and gilteritinib . figure 5d shows how the reports diagnosed extramedullary relapse in a patient with negative bone marrow biopsy ndings . 
the extramedullary disease showed the presence of some original variants ( nf1 and ezh2 ) , loss of other variants ( dnmt3a ) , and three novel variants ( cux1 , ptpn11 , and wt1 ) , some of which had clinical implications.34 , 35 additional cases with multiple reports are provided in the data supplement . reimbursement and clinical care access one of the biggest challenges for the widespread clinical implementation of ngs - based diagnostics has been inconsistent reimbursement from payers . 
coverage and reimbursement for myeloseq was divided into inpatient testing ( 26% ) and outpatient testing ( 74% ) , and 90% of outpatient testing was reimbursed across all payers . 
of these 124 cases , four were excluded ( acute promyelocytic leukemia [ apl ] subtype [ n = 2 ] ; no survey returned [ n = 2 ] )  . 
in 64 cases , physicians reported that the results did not inform decision making . ( c ) there were 14 physicians who contributed at least one survey to this study . was no specic pattern in terms of the diagnosis of rejected claims ( data supplement )  . discussion this study reports our institutions experience using a targeted capture sequencing panel to inform clinical care in patients with aml . 
 a addition of targeted therapy after lack of response to induction therapy persistent molecular disease monitoring barnell et al cebpa k302e smc1a r96h wt1 a38gfs * 69 wt1 r370pfs * 15 ezh2 k400del nf1 t2507i dnmt3a w297s dnmt3a f755 * smc1a / cepba ( 4% ) wt1 ( 3% ) wt1 ( 0% ) ( itd ) cux1 g1328s ptpn11 a72v wt1 r370pfs * 15 ezh2 k400del nf1 t2507i flt3 status enasidenib treatment ( itd ) gilteritinib new diagnosis of aml ( d0 diagnosis ) 22% blasts ( bmbx ) post 7 + 3 induction ( d49 post - diagnosis ) 3% blasts ( bmbx ) relapse ( d360 sct ) 44% blasts ( bmbx ) remission ( d426 sct ) < 1% blasts ( bmbx ) disease assessment ( d475 sct ) 0% blasts ( bmbx ) persistent molecular disease monitoring post - dli molecular diagnosis of extramedullary relapse bone marrow esophagus asxl1 g646wfs * 12 idh2 r140q runx1 l112cfs * 10 srsf2 p95h stag2 r146 * flt3 status treatment bcor t1129pfs * 30 bcorl1 q459 * dnmt3a r882h tet2 s1448n u2af1 s34f flt3 status flt3 status treatment treatment ( itd ) ( itd ) gilteritinib flt3 status treatment relapsed aml ( 1 month post - dli ) 21% blasts ( bmbx ) relapsed aml ( 3 months post - dli ) 6% blasts ( bmbx ) bone marrow ( ~ 10 yr sct ) 1% blasts ( bmbx ) esophageal tissue ( ~10 yr sct ) 1% blasts ( bmbx ) fig 5 . 
 ( b ) physician reported that the observation of persistent molecular disease at 475 days ( d ) poststem - cell transplantation ( sct ) indicated the need for a second allogeneic stem - cell transplantation despite clinical remission ( 0% blasts on bone marrow biopsy [ bmbx ] )  . 
 ( d ) extramedullary relapse was conrmed by comparing sequencing results of the original tumor and the periesophageal lesion . diagnosis , during disease assessment , and / or at multiple timepoints during the disease course . this study suggests that physicians are increasingly relying on the evaluation of multiple co - occurring variants in parallel ; therefore , simultaneous analysis of these genes is necessary . 
furthermore , pmd monitoring after cytotoxic induction therapy and transplantation21 , 36 has prognostic value ; therefore , the vafs reported on the gene panel studied here were being used to supplement existing standard - of - care mechanisms for residual disease assessment . 
although it is clear that physicians are using this test to measure and assess pmd , this test was not designed for measurable residual disease testing , and the limit of detection is not within the european leukemianet guidelines for measurable residual disease assessment.15 , 37 the prognostic information provided by the detection of persistent variants , the optimal depth of clearance , and the required limit of detection are all areas of active investigation requiring additional study in prospective clinical trials . there were several limitations associated with this study . reports were evaluated over an 8 - month period at a single academic institution among physicians with experience interpreting genomic results . 
 targeted panel inuences decision making for patients with aml were returned contemporaneously and tests were ordered at different timepoints during the disease course ( fig 3 )  . whereas the proportion of cases with inuences was not signicantly different among timepoints ( data supplement ) , inuence types might be different between timepoints and should be evaluated further to understand the impact of disease timepoint on treatment decisions . 
finally , each physician had a varied number of patients ( range , one to 18 patients ) with a large range in the number of cases reported to be inuential ( 0% to 90% )  . 
larger longitudinal , multi - institutional studies are needed to better understand interphysician variability in incorporating genomic data into clinical decision making . this study evaluated reports from the rst 8 months after launch of the diagnostic . 
the physicians were also experienced in interpreting genomic data , having participated in large genomic trials , 20 , 21 , 38 - 41 with many of them being clinical investigators themselves . 
given that the inuence of genomic data varies on the basis of physicians experience with genomic data , 6 , 7 the results presented here might not extrapolate to institutions that are unfamiliar with ordering and interpreting sequencing - based diagnostics . the sequencing panel has several additional limitations . the use of a tumor - only panelthat is , lack of a germline or reference sequenceprevented variants from normal being denitively called somatic . 
in future studies , it will be important to ascertain from physicians if any aspect of the test or the molecular report is unclear , biased , or difcult to interpret . there are several outstanding questions in the eld that could be addressed using a test similar to the one described in this paper . 
the prognostic value of residual dnmt3a , tet2 , and asxl1 mutations after treatment is incompletely understood , 21 , 36 , 42 and the difference between a residual leukemic clone versus rising clonal hematopoiesis43 needs additional study . 
our group is currently conducting a prospective clinical ( clinicaltrials.gov identier : nct02178241 ) to address some of these outstanding questions . trial in summary , the results from this study suggest that variants and associated vafs are being integrated into clinical care to inuence therapy choices , stratify relapse risk , and measure persistent disease . 
barnell employment : geneoscopy stock and other ownership interests : geneoscopy patents , royalties , other intellectual property : inventor of intellectual property in start - up company ( geneoscopy ) travel , accommodations , expenses : geneoscopy kenneth f . 
newcomer employment : geneoscopy ( i ) stock and other ownership interests : geneoscopy ( i ) patents , royalties , other intellectual property : inventor of intellectual property owned by geneoscopy ( i ) zachary l . 
oh consulting or advisory role : incyte , novartis , blueprint medicines , celgene , kartos , cti biopharma corp , pharmaessentia , disc medicine , constellation pharmaceuticals research funding : incyte ( inst ) , gilead sciences ( inst ) , cti biopharma corp ( inst ) , kartos ( inst ) , celgene ( inst ) , sierra oncology ( inst ) , blueprint medicines ( inst ) , constellation pharmaceuticals ( inst ) john s . 
stockerl - goldstein stock and other ownership interests : abbott laboratories , abbvie consulting or advisory role : celgene research funding : glaxosmithkline , takeda , biolinerx , janssen other relationship : cellerant ravi vij consulting or advisory role : bristol myers squibb , celgene , janssen , sano , karyopharm therapeutics , takeda , genentech , abbvie , oncopeptides research funding : takeda , celgene , bristol myers squibb travel , accommodations , expenses : celgene , bristol myers squibb , sano , janssen , davaoncology , karyopharm therapeutics , amgen , takeda , abbvie amanda f . 
abboud stock and other ownership interests : abbvie ( i ) , abbott laboratories ( i ) , gilead sciences ( i ) , bristol myers squibb ( i ) , johnson & johnson ( i ) honoraria : cardinal health , dova pharmaceuticals , nkartatherapeutics , archer biosciences research funding : actinium pharmaceuticals , selvita ( inst ) , allovir ( inst ) , forty - seven ( inst ) armin ghobadi honoraria : kite pharma consulting or advisory role : kite pharma , celgene , amgen , wugen speakers bureau : kite pharma , amgen geoffrey l . 
schroeder consulting or advisory role : astellas pharma , dova pharmaceuticals , flatiron health , glaxosmithkline , incyte , partners therapeutics , partners therapeutics , pzer speakers bureau : abbvie , merck , takeda john f . 
dipersio stock and other ownership interests : magenta therapeutics , wugen honoraria : incyte consulting or advisory role : cellworks , rivervest , magenta therapeutics , incyte research funding : amphivena therapeutics ( inst ) , macrogenics ( inst ) , incyte ( inst ) , wugen ( inst ) , biolinerx ( inst ) , maxcyte ( inst ) , bigelow aerospace ( inst ) patents , royalties , other intellectual property : patents : cd7 and cd2 knockout for cart to cd7 and cdl ; duvelisib for treatment of cytokine release syndrome ; nt - 17 to enhance cart survival ; novel wu mobilizing compounds ( inst ) ; selection of impdh mutant stem cells ; ifn , upregulate mhcii for relapsed aml ; dextran - based molecules to detect car - t cells ; combining integrin inhibitor with chemokine binders , 016131 ; jak and calcineurin inhibition , solid organ transplant ; vla4 , gro - b ; triple combinationcxcr2 , vla - 4 , gro - b ; targeting ifnr / cscr3 in gvhd ; wu / slu compounds vla4 and cxcr2 ( inst ) travel , accommodations , expenses : incyte , macrogenics , magenta therapeutics mary c . 
duncavage stock and other ownership interests : p&v licensing consulting or advisory role : cofactor genomics , bristol myers squibb , eli lilly open payments link : 317379 timothy j . 
additional thanks are also given to the siteman cancer center and the barnesjewish hospital foundation . 11 : 11 , 2016 368 : 2059 - 2074 , 2013 129 : 424 - 447 , 2017 netw 15 : 926 - 957 , 2017 2017 314 : 811 - 822 , 2015 references ray t : survey of precision oncology programs nds agreement on testing , divergence in care delivery . 
precision oncology news , july 19 , 2019 : precisiononcologynews.com / cancer / survey - precision - oncology - programs - nds - agreement - testing - divergence - care - delivery stone rm , mandrekar sj , sanford bl , et al : midostaurin plus chemotherapy for acute myeloid leukemia with a flt3 mutation . 
n engl j med 377 : 454 - 464 , 2017 kim as , bartley an , bridge ja , et al : comparison of laboratory - developed tests and fda - approved assays for braf , egfr , and kras testing . 
jama oncol 4 : 838 - 841 , 2018 li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
bmc med inform decis mak 18 : 107 , 2018 chow - white p , ha d , laskin j : knowledge , attitudes , and values among physicians working with clinical genomics : a survey of medical oncologists . 
j genet couns 27 : 187 - 196 , 2018 gornick mc , ryan ka , scherer am , et al : interpretations of the term actionable when discussing genetic test results : what you mean is not what i heard . j genet couns 28 : 334 - 342 , 2019 statz cm , patterson se , mockus sm : barriers preventing the adoption of comprehensive cancer genomic proling in the clinic . 
au ch , wa a , ho dn , et al : clinical evaluation of panel testing by next - generation sequencing ( ngs ) for gene mutations in myeloid neoplasms . 
marcucci g , mr ozek k , ruppert as , et al : abnormal cytogenetics at date of morphologic complete remission predicts short overall and disease - free survival , and higher relapse rate in adult acute myeloid leukemia : results from cancer and leukemia group b study 8461 . 
chen y , cortes j , estrov z , et al : persistence of cytogenetic abnormalities at complete remission after induction in patients with acute myeloid leukemia : prognostic signicance and the potential role of allogeneic stem - cell transplantation . 
j clin oncol 29 : 2507 - 2513 , 2011 ivey a , hills rk , simpson ma , et al : assessment of minimal residual disease in standard - risk aml . 
loken mr , alonzo ta , pardo l , et al : residual disease detected by multidimensional ow cytometry signies high relapse risk in patients with de novo acute myeloid leukemia : a report from childrens oncology group . 
hou h - a , chou wc , lin li , et al : characterization of acute myeloid leukemia with ptpn11 mutation : the mutation is closely associated with npm1 mutation but 35 . 
gaidzik vi , schlenk rf , moschny s , et al : prognostic impact of wt1 mutations in cytogenetically normal acute myeloid leukemia : a study of the germaninversely related to flt3 / itd . 
blood 113 : 4505 - 4511 , 2009 jongen - lavrencic m , grob t , hanekamp d , et al : molecular minimal residual disease in acute myeloid leukemia . 
schuurhuis gj , heuser m , freeman s , et al : minimal / measurable residual disease in aml : a consensus document from the european leukemianet mrd working party . 
clinical diincluding disease status , was dened by board - certied agnosis , hematopathologists . the panel uses an amplicon capture - based enrichment with unique molecular identiers for ultra - high variant sensitivity that targets an approximately 98 - kg base pair space ( haloplex target enrichment system ; agilent technologies , santa clara , ca )  . 
the panel is generally ordered for one of the following conditions : aml , mds , cytopenia , clonal cytopenia of undetermined signicance , clonal hematopoiesis of indeterminate signicance , myeloproliferative neoplasm , or other myeloid disorders . 
this included evaluating bone marrow morphology , standard panels of ow cytometric markers , and a panel of routine cytogenetic and molecular marker studies , including uorescence in situ hybridization probes . target enrichment for the panel used a commercially available , targeted , next - generation sequencing approach ( haloplexhs ; agilent technologies )  . 
preparation consisted of enzymatic fragmentation ; strand - specic ligation of sequencing primers , sample indexes , 10 - bp degenerate molecular barcodes , and biotin tagging of single dna molecules ; rapid liquid - phase enrichment of target loci using paramagnetic streptavidin - coated beads ; and on - bead polymerase chain reaction amplication . 
sequencing was performed using the illumina miniseq sequencing platform ( target depth = 50 coverage )  . variant calling for the panel was performed using a computational pipeline that employs custom - built tools created at washington university . 
overall reads were required to exceed 1 , 000 , 000 total reads with more than 98% aligned . during this process , unique molecular identier sequences are added to the binary alignment map le . 
once aligned , a custom java - based tool collapsed reads into read families using customized gatk software ( mckenna a , et al : genome res 20 : 1297 - 1303 , 2010 )  . 
collapsed binary alignment maps were used with standard variant calling tools , including varscan2 ( koboldt dc , et al : genome res 22 : 568 - 576 , 2012 ) , platypus ( rimmer a , et al : nat genet 46 : 912 - 918 , 2014 ) , and pindel ( ye k , et al : bioinformatics 25 : 2865 - 2871 , 2009 )  . 
these programs detected single - nucleotide substitutions , insertions or deletions ( indels ) up to 10 bp , and flt3 internal tandem duplication insertions between 21 and 108 bp . 
initial variant annotation was performed using the variant effect predictor tool ( mclaren w , et al : genome biol 17 ( 1 ) : 122 , 2016 )  . 
annotation was format le with variants augmented using a custom variant call identied in the aml the cancer genome atlas data set as well as variants observed in three published sequencing reports that evaluated patients with mds ( haferlach t , et al : leukemia 28 : 241 - 247 , 2014 ; papaemmanuil e , et al : blood 122 : 3616 - 3627 , quiz 3699 , 2013 ; walter mj , et al : leukemia 27 : 1275 - 1282 , 2013 )  . 
variant identication was subject to the following thresholds and cutoffs for reporting purposes : variants must be nonsynonymous , variants must have 2% minimum variant allele frequency and ve variant reads with support on each strand ( flt3 internal tandem duplication alleles require one read on each strand ) , amplicons must have at least ve reads assigned to it during consensus binary alignment map formation , and potential somatic variants must have 0.1% maximum population allele frequency ( max_af across all populations ; 1000 genomes [ v.phase3 ; 1000 genomes project consortium , et al : nature 526 : 6874 , 2015 ] and gnomad database [ v.170228 ; karczewski kj , et al : nature 581 : 434 - 443 , 2020 ] ) or presence in a custom mds / aml variant database . tier 1 ( variants with strong clinical signicance ) and tier 2 variants ( variants with potential clinical signicance ) , four ltered variants ( ie , variant allele frequency , 2% ) , and variants of unknown signicance were manually reviewed for potential clinical relevance . 
newberg , phd3 ; meagan montesion , phd3 ; and peter mu - hsin chang , md , phd1 , 2 introduction salivary gland tumors comprise a rare group of neoplasms that arise from salivary ducts , most of which are benign.1 the parotid gland is the most frequently involved site and comprises more than 80% of salivary tumors.2 of the parotid tumors , approximately 75% are benign , whereas the remaining 25% are malignant.3 the most frequent subtype of malignant salivary gland cancer is mucoepidermoid carcinoma , followed by adenoid cystic carcinoma.4 salivary gland tumors have been linked to exposure to high ultraviolet radiation.5 salivary gland cancer has a poor prognosis ( 5 - year overall survival , 50% ) and a high propensity for recurrence.6 the mainstay of treatment of salivary gland cancers is surgery in patients with early and locally advanced disease.7 in metastatic or recurrent disease , consensus is lacking on the optimal regimen for systemic chemotherapy.8 the low prevalence of this disease precludes large - scale randomized trials , and current clinical data are mostly derived from small clinical studies . 
current data , although dated , support cisplatin , anthracyclines , uorouracil , and cyclophosphamide as active agents against salivary gland cancer.9 - 11 in general , response to chemotherapy is limited ( a review of response to chemotherapy was published by laurie and licitra , 12 with response rates mostly ranging from 10% to 30% ) , and chemotherapy is mostly for palliative control.8 , 12 experts have urged a coordinated effort for clinical trials to further elucidate the role of chemotherapy in this patient group , 12 but this proves to be difcult , owing to the rarity of this cancer . the success of epidermal growth factor receptor ( egfr ) inhibitors in head and neck cancers13 and lung cancers14 has sparked hope for the possibility of use in salivary gland tumors because egfr is sometimes upregulated in this disease group.15 however , attempts to use egfr inhibitors , such as getinib and cetuximab , have largely been disappointing , with limited response.16 , 17 multiple mechanisms have been proposed for the lack of response to egfr inhibition in salivary gland cancers.18 to gain additional insight into the egfr expression pattern in salivary gland cancers , the dna and protein status of more than 800 tumor samples were analyzed for egfr gene alteration and protein levels in a retrospective study.19 the study revealed a high frequency of egfr low prevalence of egfr gene protein expression , mutation , and , more interestingly , no occurrence of egfr gene amplication . this is signicantly different from studies of head and neck squamous cell carcinomas , in which the prevalence of egfr amplication has been reported to be 17%20 and was linked to worse clinical outcome . 
interestingly , studies in colorectal cancer have shown that egfr gene amplication is linked to better outcomes and response to cetuximab.21 , 22 conversely , egfr amplication is proposed as an intrinsic resistance mechanism in lung cancer , 23 although other studies report conicting results , demonstrating egfr amplication associated with better outcomes with tyrosine kinase inhibitors ( tkis ) in patients with advanced nonsmall - cell lung cancer.24 therefore , the exact role of egfr amplication in response to egfr inhibition therapy remains to be claried . in this case report , we describe an unusual patient with a salivary gland cancer , refractory to initial chemotherapy agents but demonstrating signicant response to afatinib , an egfr and human epidermal growth factor receptor 2 tki . 
 ( c ) a computed tomography scan was performed 3 months after starting afatinib , showing marked decrease in size of the tumor ( right )  . pathology studies conrmed the diagnosis of high - grade salivary ductal carcinoma with inltration to the nontumor portion of the parotid gland . 
copy number ( cn ) plot ( top ) and minor allele frequency plot ( bottom ) showing marked increase in copy number of epidermal growth factor receptor . after discussion with the patient , the decision was made to initiate a therapeutic regimen of the egfr inhibitor afatinib ( 40 mg , every other day )  . 
written informed consent for publication of this case report was obtained from the patient . discussion afatinib is a tki that irreversibly inhibits the human egfr and human epidermal growth factor receptor 2 kinases.26 it has proven clinical activity and is approved for treatment of nonsmall - cell lung cancer with mutant egfr27 ( as shown in the lux - lung 3 and lux - lung 6 trials )  . 
interestingly , afatinib showed signicant activity against tumors harboring the deletion of exon 19 in egfr , while lacking activity against the common l858r point mutation , 27 , 28 highlighting the differences in biology between different egfr mutation types.29 afatinib was shown to be effective in advanced head and neck cancers , with benet progression - free survival30 , 31 ; however , little data exist for afatinib in salivary gland cancers . although some subtypes of salivary gland tumors exhibit egfr overexpression , 15 it is generally established that egfr inhibitors such as cetuximab are ineffective against salivary gland tumors . 
mechanistically , egfr amplication has been linked to higher spatial density of the receptor and thus linked to heightened dimerization and signaling.32 interestingly , as described in a recent case report , a patient with triple - negative breast cancer with egfr amplication was also successfully treated with cetuximab.33 a recent study retrospectively investigated a subgroup of esophageal cancers with egfr amplication.34 although previous studies have failed to demonstrate efcacy of egfr inhibition in esophageal cancers , 35 this study demonstrated a high response rate ( objective response rate , 58% ; disease control rate , 100% ) in this patient population.34 the ndings in our case report are in line with the aforementioned studies , highlighting the prognostic value of egfr amplication with treatment with egfr inhibitors . 
our patient is unique in that egfr amplication is extremely rare in salivary gland tumors , 19 compared with higher prevalence in triple - negative breast cancer ( up to 92% ) 36 or in esophageal cancer ( 7% ) .37 these reports that egfr amplication may indicate a favorsuggest able response to egfr inhibitors , regardless of the tumor site . 
this highlights the importance of genetic proling of individual tumors to determine personalized treatment of patients . in conclusion , we report a patient with a high copy number of egfr who demonstrated good response to egfr inhibition therapy . 
newberg employment : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : im on a patent stemming from research in my graduate school days related to identifying location biomarkers using imaging techniques . 
head neck surg 8 : 177 - 184 , 1986 liu y , li h , qin l , et al : prognostic factors in malignant sublingual salivary gland tumors . 
j oral maxillofac surg 75 : 1542 - 1548 , 2017 spitz mr , sider jg , newell gr , et al : incidence of salivary gland cancer in the united states relative to ultraviolet radiation exposure . 
head neck surg 10 : 305 - 308 , 1988 johnston ml , huang sh , waldron jn , et al : salivary duct carcinoma : treatment , outcomes , and patterns of failure . 
head neck 38 : e820 - e826 , 2016 ( suppl 1 ) lewis ag , tong t , maghami e : diagnosis and management of malignant salivary gland tumors of the parotid gland . 
otolaryngol clin north am 49 : 343 - 380 , 2016 aleri s , granata r , bergamini c , et al : systemic therapy in metastatic salivary gland carcinomas : a pathology - driven paradigm ? oral oncol 66 : 58 - 63 , 2017 licitra l , cavina r , grandi c , et al : cisplatin , doxorubicin and cyclophosphamide in advanced salivary gland carcinoma . 
proc natl sci counc repub china b 25 : 90 - 96 , 2001 jakob ja , kies ms , glisson bs , et al : phase ii study of getinib in patients with advanced salivary gland cancers . 
chen z , chen j , gu y , et al : aberrantly activated areg - egfr signaling is required for the growth and survival of crtc1 - maml2 fusion - positive mucoepidermoid carcinoma cells . 
temam s , kawaguchi h , el - naggar ak , et al : epidermal growth factor receptor copy number alterations correlate with poor clinical outcome in patients with head and neck squamous cancer . 
hirsch fr , herbst rs , olsen c , et al : increased egfr gene copy number detected by uorescent in situ hybridization predicts outcome in non - small - cell lung cancer patients treated with cetuximab and chemotherapy . 
nukaga s , yasuda h , tsuchihara k , et al : amplication of egfr wild - type alleles in non - small cell lung cancer cells confers acquired resistance to mutationselective egfr tyrosine kinase inhibitors . 
cappuzzo f , hirsch fr , rossi e , et al : epidermal growth factor receptor gene and protein and getinib sensitivity in non - small - cell lung cancer . 
sk alov a a , vanecek t , sima r , et al : mammary analogue secretory carcinoma of salivary glands , containing the etv6 - ntrk3 fusion gene : a hitherto undescribed salivary gland tumor entity . 
solca f , dahl g , zoephel a , et al : target binding properties and cellular activity of afatinib ( bibw 2992 ) , an irreversible erbb family blocker . 
yang jc , wu yl , schuler m , et al : afatinib versus cisplatin - based chemotherapy for egfr mutation - positive lung adenocarcinoma ( lux - lung 3 and lux - lung 6 ) : analysis of overall survival data from two randomised , phase 3 trials . 
kato t , yoshioka h , okamoto i , et al : afatinib versus cisplatin plus pemetrexed in japanese patients with advanced non - small cell lung cancer harboring activating egfr mutations : subgroup analysis of lux - lung 3 . 
kaneda t , yoshioka h , tamiya m , et al : differential efcacy of cisplatin plus pemetrexed between l858r and del - 19 in advanced egfr - mutant non - squamous non - small cell lung cancer . 
machiels jp , haddad ri , fayette j , et al : afatinib versus methotrexate as second - line treatment in patients with recurrent or metastatic squamous - cell carcinoma of the head and neck progressing on or after platinum - based therapy ( lux - head & neck 1 ) : an open - label , randomised phase 3 trial . 
clement pm , gauler t , machiels jp , et al : afatinib versus methotrexate in older patients with second - line recurrent and / or metastatic head and neck squamous cell carcinoma : subgroup analysis of the lux - head & neck 1 trial . 
suntharalingam m , winter k , ilson d , et al : effect of the addition of cetuximab to paclitaxel , cisplatin , and radiation therapy for patients with esophageal cancer : the nrg oncology rtog 0436 phase 3 randomized clinical trial . 
 parents , health care professionals , and scientists experiences of a precision medicine pilot trial for patients with high - risk childhood cancer : a qualitative study janine vetsch , phd1 , 2 ; claire e . 
ziegler , md , phd1 , 2 purpose children with high - risk cancers have low survival rates because current treatment options are limited . precision medicine trials are designed to offer patients individualized treatment recommendations , potentially improving their clinical outcomes . 
therefore , we wanted to gain an in - depth understanding of the experiences of all stakeholders involved in a new precision medicine pilot trial called target , including parents , their childs hcps , and the scientists who conducted the laboratory research and generated the data used to make treatment recommendations . methods we conducted semistructured interviews with all participants and analyzed the interviews thematically . results we interviewed 15 parents ( 9 mothers ; 66.7% bereaved ) , 17 hcps , and 16 scientists . 
we identied the following themes in parents interviews : minimal understanding and need for more information , hope as a driver of participation , challenges around biopsies , timing , and drug access , and few regrets . 
 vetsch et al context key objective what are the experiences of parents , health care professionals , and scientists participating in a precision medicine pilot trial for patients with high - risk childhood cancer ? knowledge generated parents had a minimal understanding of the trial aims . 
health care professionals and scientists embraced new technologies and collaborations but faced challenges managing families expectations , timing of testing , and drug access . relevance educating families will help them to better understand the potential benets and limitations of precision medicine trials , increase satisfaction with their childs care , and ameliorate additional long - term psychosocial burden for families already impacted by high - risk childhood cancer . 
health care professionals and scientists will also benet from further educational support so that they can prepare for the challenges of the rapidly emerging eld of precision medicine . typically securing responsible informed consent.15 previous studies suggested that poorer understanding is associated with lower socioeconomic status and having an ethnic background.6 some hcps have difculty explaining the purpose of experimental trials , which highlights the importance of ensuring that consent discussions are appropriate for parents with different educational backgrounds , languages , and cultural contexts.6 , 16 , 17 despite the fact that some hcps are concerned that parents understanding of clinical trials may be limited , hcps often share parents therapeutic optimism.17 hcps may nd it difcult to manage families expectations , including maintaining hope , while the likelihood of cure.11 , 18 for being realistic about scientists , precision medicine trials can be challenging because they represent a new way of conducting research , including adding a sense of accountability to families who are awaiting results . 
to our knowledge , scientists experiences of working on a precision medicine trial have not been assessed previously . the psychosocial with childhood cancer precision medicine trials being implemented worldwide , implications for families , hcps , and scientists need to be explored in depth . 
although some studies have examined the impact of pediatric genetic testing on families and hcps , 19 no studies have assessed the experiences of parents , hcps , and scientists participating in a precision medicine trial for patients with high - risk childhood cancer . 
therefore , we aimed to explore the experiences of parents , hcps , and scientists participating in the target study . methods participants the following three participant groups were eligible : parents ( including bereaved parents ) , hcps , and scientists . 
when families enrolled in target , they were informed that the primary objective of the trial was to assess the feasibility of a new precision medicine protocol and that personalized therapies ( if identied ) might not benet their child . 
with study progression and the establishment of the platform , results of actionable ndings were fed back to families . parents were eligible for this qualitative study if their child was enrolled in target , they provided informed consent , and they could speak conversational english . 
treating oncologists assessed all potential families for eligibility and excluded patients who were deemed to be experiencing severe mental health difculties or other conditions that may have impacted their study participation . hcps were eligible if they had been involved in the care of any target patient . 
we audio - recorded and transcribed the interviews with participants permission ( the average length of interviews was 35 minutes for parent , 27 minutes for hcps , and 31 minutes for scientists )  . 
we interviewed hcps and scientists to gain an understanding of their professional and personal experiences with target ( data supplement )  . we collected demographic data including age , sex , and education level from all participants . 
we collected clinical data from the target database ( table 1 )  . data analysis we used descriptive statistics to summarize participants demographic information using spss v25 ( ibm , armonk , ny )  . 
most children enrolled in target had a cns tumor ( seven families ) , and parents were , on average , 3.3 years from their childs initial cancer diagnosis at the time of the interview . 
numbers do not always add up as a result of missing values . abbreviation : sd , standard deviation . * includes students and homemakers . parents experiences we identied several terviews ( table 3 )  . important themes in parents minimal understanding and need for more information . parents generally recalled signing the target consent forhowever , they commonly could not recall the study aims or what their childs enrollment in target involved . of the parents who could not recall signing the consent form , they expressed confusion about the difference between target and other trials their child had participated in because [ everything ] just bleeds into one ( bereaved mother , id010 )  . 
some parents were not sure whether an additional biopsy or tissue sample was taken for target . other parents spoke of the target drug , not understanding that target was an analysis platform and not one specic drug . 
parents described their the target capacity to understand information about study as impeded by stressit just ew over the top of our heads ( bereaved father , id015 )  . 
parents also desired developmentally tailored information for their child because most parents included their children in treatment decisions . however , other parents reported not wanting any additional study information because it was too overwhelming and reminded them that their child had a terminal illness . hope as a driver of participation in target . 
target offered a sense of control to parents with minimal options , allowing them to take some sort of control of the uncontrollable ( bereaved mother , id008 )  . 
some parents whose children died found solace in the idea that their participation in target might help future patients . challenges around biopsies , timing , and drug access . parents reported feeling challenged by several ( uncontrollable ) factors , including the need for additional biopsy and conicting priorities as a result of limited available tumor tissue . 
parents queried why biopsies were not obtained and stored at the time of their childs diagnosis , and they expressed hope that this process would be in place for future patients . 
one mother explained that there was 4 2019 by american society of clinical oncology competing priority between using the biopsy for research and exploring other treatment options interstate or overseas . 
at the same time , parents were often willing for their child to undergo as many biopsies as needed to enhance their childs treatment options and contribute to research ( including being willing to consent to an autopsy after their childs death )  . parents also described challenges around timing . 
some parents expressed regret that the study was not offered earlier , particularly when they received the results either when their child was too unwell to start a new treatment or after their child had died . 
as one mother explained , its very frustrating , but youve missed an opportunity to better understand the tumor and what could reduce it ( id008 )  . another challenge described by parents was accessing the drugs identied through testing and recommended by target . 
although some parents described willingness to pay for drug costs , they were commonly advised to access the drug through an open clinical trial so that outcomes could be monitored , but this sometimes further slowed access . 
hcps valued that target gave them potential access to additional therapies not likely to be considered without the testing platforhcps highly valued that they were able to offer individualized therapies and state - of - the - art care ( hcp , their site , assisting families to avoid traveling overseas to access experimental therapies . id020 ) at challenges managing expectations , unexpected ndings , and drug access . 
im trying to remember , i dont actually remember what they said the objectives were , to be honest . i believe that the other treatment was utilising drugs that were used for a different type of leukaemia that if an all can actually sometimes look like or it will mimic , so i think the hypothesis was that the aml drugs may actually prove to respond better for [ childs ] s type of all , the infant all , and the main goal of the study was to investigate that . i know that it focuses on studying the genetic makeup of the tumor and  . 
what sort of treatment can help address genetic makeup of the tumor that she has . how long had they had the results and if they had them sooner , why the hell didnt they say , weve got the results because my thing is that if they can somehow have them on their target treatments and radiation as quickly as possible , maybe theres going to be a better result . i would quite like to have seen that all laid out with some bubbles with some arrows . 
so i could at least understand visually how it was . i just think [ sharing results ] should be one - on - one ; i didnt need ve bereaved father people there . 
 , no more resections , no more other drugs , no more radiation so might as well [ participate ]  . we enrolled [ child ] to the program not because of anything but because it was the only option we had . the hope is helping out future families . 
would be awesome if we can bring any value to anybody else whos been going through what weve been through . some drug will [ help ] so thats how you live its really important to have as much data as possible . 
i think its really important for families to participate . i think it was keep our child alive as long as we can in the hope that bereaved mother there is still hope  . 
there is still hope , mother there is still hope . challenges involving biopsies , timing , and drug access i think it would be seriously helpful though for newly diagnosed families to make sure that we get the tumor from that rst surgery . even , you know , and i know theres limited resources available to look at tumors , but theres always ways to nd somebody else to look at tumors at a cost . 
themes with representative parent quotes ( continued ) theme and quotes precisely , because the sheer quantity of tests and biopsies and treatments and scans and everything they go through already is beyond comprehension . 
so , the idea of agreeing to research that has the potential to increase that or add additional invasive procedures or negative experiences , then that would be , for me , that would be my deal breaker . 
at that point i would say , no more . so i dont know what it is , why it takes 8 weeks , if thats just literally , you know , in practical terms , how long it takes to grow and run the tests or whether its just a case of if you have more people and resources , you could do all that faster . so i didnt appreciate when she came off that drug how quickly things would move from there . 
so that would be something i would be warning people about . it was only another probably 3 weeks after [ surgery ] , that the cancer came back again anyway ! so , yeah , poor bugger had his brain surgery for nothing . it would be awesome to have more trials , more trials in australia . where you dont have to sell the house , re - mortgage the house , to go to germany or go for america and pay the bills over there just to try and get yourself enrolled in a trial that might help . 
some hcps were concerned that oncologists did not have good connections with pharmaceutical companies , which might limit access to recommended drugs for their patients . scientists experiences we identied the following themes from interviews with scientists ( illustrative quotes are provided in table 5 )  . embracing new collaborations and knowledge . 
scientists valued new interdisciplinary collaborations with clinicians and being able to learn and generate new knowledge . scientists described feeling a connection to real patients and felt that their work was meaningful , with the potential for real - life impact on families . 
themes with representative hcp quotes theme and quotes embracing new technology and collaboration so for families , i found that its made a huge difference to be able to offer them the possibility of targeted therapy , knowing that we may not nd anything for their particular child , or their particular tumor , but its made it so that we can offer something where there may , have not been anything else to be able to be offered . i have got one patient with a relapsed tumor where having access to the target has allowed us to use different drugs and those different drugs have put the patient into remission . challenges drug access if you havent got contacts within the pharmaceutical industry then thats also very difcult so at the moment im trying to get access to a drug which i cant get access to in australia so i think thats also difcult because the patient now knows theres a potential target and we wont have anything that we can give . also turnaround timesso many patients are rapidly progressing  . 
we need to still be optimistic but realistic at the same time and maybe this could give them an excess of hope , maybe thats what i consider as a problem . this has the potential to give other options and even if it doesnt give other options for them it gives potential options for the future , and as a i said , the disadvantage is that even if you were offering  . 
so its hard to give consent . theyre hoping that their child will have the one - in - a million where you nd a cure , based on their genetics . 
but from what , more than that , the vast majority of kids we either nd something thats adjuvant , like can be added in , but may not be curative , and in some patients we havent found anything thats targetable and the disappointment that comes with that . 
i dont think is a fault of the study , but comes with the territory of what it is that youve found . unexpected ndings one family who had a germline mutation , so i think that is something unexpected and it just adds another level of complexity . 
scientists expressed that target was unique compared with other studies they had worked on because of the potential for this personal psychological impact . discussion we found that many parents could not recall the specic aims of target and that hope commonly motivated participation . 
most parents desired additional , more accessible , written information about the study and valued regular updates from the treating physician . despite reporting challenges , parents did not regret participating in target . 
with this opportunity , i can interact into bigger communities across different research groups and also with some research clinicians from sydney childrens hospital . it was good , it was a really good learning experience to get a knowledge base and foundational skills in personalized medicine and research . its so rewarding doing what we do and nding things , i get a kick out of nding things that were missed by every other test before it . finding things that youd never think to look for , i think thats cool . . 
. conversely , when we get the biopsies , not getting enough sample . and we get blamed for that , and its like well were not the one that got the sample . there had been a few instances where patients had passed away and there would be an email sent out to everybody and seeingwhen you work in this sort of industry or this sort of workand you see the same names pop up , youll notice that sometimes therell be a positive reaction to treatment and its noted somewhere , but then a few months down the track , you see the names pop up again and youd be like , this persons obviously gotten worse again . there was an expectation that we would have [ the testing platform ] set up very rapidly , so that was a particular challenge to try meet those expectations and manage those expectations . 
that was quite tricky at times . drug access we always hope that something might come up but just sometimes you do get that type of result and theres nothing we can do to help with the kids and we dont  . 
scientists were challenged by time pressure and a new sense of responsibility to families , but at the same time , they valued this new connection with patients . echoing previous studies examining parents understandings of genomic tumor proling and experimental trials , 8 , 21 , 22 parents understanding of target was low . 
our results conrm that parents of children participating in clinical trials may experience therapeutic optimism.22 there is some evidence that ( potentially unrealistic ) optimism for a personal benet might be positively associated with patients mental health status throughout treatment23 ; however , long - term outcomes are unclear . 
echoing recent studies , some parents participation had a dual purpose , hoping for a benet for their child and for future patients.21 , 24 ensuring that families can make informed decisions about precision medicine will be critical for its integration into routine clinical care . 
hcps in our study reported challenges managing families expectations and avoiding false hope . the primary goal of this precision medicine trial , similar to other trials , was to benet future patients and research , with only a small proportion of current patients likely to gain medical benet.25 , 26 there is evidence that low literacy or educational attainment is associated with poorer understanding of precision medicine6 , 8 , 21 and may be more common in ethnic or racial minority groups , 6 highlighting the importance of easily understandable resources in different languages potentially supported with infographics or owcharts of trial steps.8 clinicians could consider providing families with information about the study multiple times over the course of treatment as well as offering multiple opportunities to have discussions with their treating team . although emerging literature has reected on the implications of cross - disciplinary processes inherent to precision medicine ( eg , sharing of big data sets between clinicians and researchers , the coming together of laboratories and clinics ) , 27 the experience of different stakeholders has received little attention . 
with more precision medicine trials being implemented worldwide , biopsy at diagnosis for up - front next - generation sequencing analysis will likely become standard of care for future patients with childhood cancer.28 timing was also challenging for scientists when children died before analysis of their data was completed . 
scientists should be given the opportunity to debrief and discuss their emotional reactions within the study team . a study strength was the inclusion of multiple stakeholders . parents response rate was moderate ; however , many parents were recently bereaved and were perhaps not yet ready to share their experience . 
we recruited participants from one hospital and only included english - speaking parents , which limits the generalizability of our results . participating hcps were highly interested in precision medicine , potentially limiting the generalizability of our hcp ndings . 
in addition , the change in practice over the study course needs to be considered when interpreting our results . future larger prospective trials may want to assess stakeholders experiences over the course of a precision medicine trial . parents willingly enroll their children in cancer precision medicine trials to explore all avenues of potential treatment . however , they may overestimate the likely benet for their child . 
easy - to - understand educational resources in multiple languages explaining the main concepts of precision medicine trials are needed . parents expectations should be monitored by hcps throughout their trial participation . 
tucker travel , accommodations , expenses : astrazeneca uncompensated relationships : astrazenica david malkin consulting or advisory role : bayer no other potential conicts of interest were reported . acknowledgment we thank janice yung , lianne wong , jessica deng , julie van , josh weisel , and pirathat techakesari for their contribution to this research . references eur j cancer 65 : 91 - 101 , 2016 tannock if , hickman ja : limits to personalized cancer medicine . 
harris mh , dubois sg , glade bender jl , et al : multicenter feasibility study of tumor molecular proling to inform therapeutic decisions in advanced pediatric solid tumors : the individualized cancer therapy ( icat ) study . 
jama oncol 2 : 608 - 615 , 2016 lau l , byrne j , ekert pg , et al : pilot study of a comprehensive precision medicine platform for children with high - risk cancer . 
n engl j med 372 : 855 - 862 , 2015 cousino mk , zyzanski sj , yamokoski ad , et al : communicating and understanding the purpose of pediatric phase i cancer trials . 
j clin oncol 30 : 4367 - 4372 , 2012 alderfer ma , zelley k , lindell rb , et al : parent decision - making around the genetic testing of children for germline tp53 mutations . 
cancer 121 : 286 - 293 , 2015 oberg ja , ruiz j , ali - shaw t , et al : whole - genome and whole - exome sequencing in pediatric oncology : an assessment of parent and young adult patient knowledge , attitudes , and expectations . 
beranger a , bouazza n , de haut de sigy a , et al : parents and childrens comprehension and decision in a paediatric early phase oncology trial : a prospective study . 
kass n , taylor h , fogarty l , et al : purpose and benets of early phase cancer trials : what do oncologists say ? what do patients hear ? j empir res hum res 14 . 
estlin ej , cotterill s , pratt cb , et al : phase i trials in pediatric oncology : perceptions of pediatricians from the united kingdom childrens cancer study group and the pediatric oncology group . 
marron jm , cronin am , dubois sg , et al : duality of purpose : participant and parent understanding of the purpose of genomic tumor proling research among children and young adults with solid tumors . 
harttrampf ac , lacroix l , deloger m , et al : molecular screening for cancer treatment optimization ( moscato - 01 ) in pediatric patients : a single - institutional prospective molecular stratication trial . 
chang w , brohl as , patidar r , et al : multidimensional clinomics for precision therapy of children and adolescent young adults with relapsed and refractory cancer : a report from the center for cancer research . 
although several inhibitors have been created , duvelisib and idelalisib have moved far in the clinic1 ; in fact , the latter in combination with rituximab is an approved agent for treatment of relapsed / refractory cll.2 , 3 duvelisib targets the dand g - isoforms , whereas idelalisib inhibits only the d - isoform.1 hence , we hypothesized that these inhibitors may have similar effects in downstream target proteins . 
these observations were in circulating cells after duvelisib therapy ( ie , after peripheral blood lymphocytosis ) .6 idelalisib showed a similar phenomenon during phase i , single - agent investigations.7 to test our hypothesis , we evaluated these cll lymphocytes from the peripheral blood before and after idelalisib treatment . fifteen samples from five patients were assessed ( table 1 )  . 
all these patients with cll were previously untreated and enrolled in a dana - farber cancer institute clinical trial of upfront idelalisib for 2 months followed by the addition of ofatumumab . 
all received idelalisib 150 mg twice per day as a single agent during the time these samples were collected . rppa assays were performed from these samples to determine the protein expression of the bcl - 2 family . 
the bcl - 2 family proteins are mcl - 1 , bclxl , bcl - 2 , and bcl - 2 - a1 among the antiapoptotic members ; bax and bak ( multidomain proapoptotic members ) ; and bim , bid , puma , and bad.ps155 among the bh3 - only members . 
close to 300 proteins were included in the rppa assay , which included 10 members ( blue symbols ) of the bcl - 2 family ( figs 1a and 1b )  . 
the treatment duration at the time of measurement varied , starting as early as 15 days and as late as cycle 3 day 1 ( ie , 56 days )  . 
a volcano plot demonstrates that 40 proteins after 15 or 28 days ( post - treatment t1 ; fig 1a ) and 35 proteins after two cycles ( 56 days ) of idelalisib ( post - treatment t2 ; fig 1b ) were significantly modulated . 
brown varsha gandhi qingshan yang , prexy modi , anil korkut , and varsha gandhi , the university of texas md anderson cancer center , houston , tx ; stacey m . fernandes , john hanna , and jennifer r . 
primary cll cells from five patients were isolated fresh from peripheral blood samples before treatment ( baseline , designated as d0 [ day 0 ] ) and d15 ( t1 ) or d28 ( t1 ) and d56 ( t2 ) after the start of oral idelalisib 150 mg twice per day . 
reverse phase protein array was done after lysis of cells at md anderson cancer center . the assay contained 300 proteins.8 lysates were diluted and samples probed with antibodies and visualized by colorimetric reaction . 
relative protein levels for each sample were determined by interpolation of each dilution curve from the standard curve of the slide . all data were normalized to protein loading and transformed to a linear value . 
 ( a ) volcano plot of the fold change of protein and statistical significance between pretreatment and post - treatment t1 ( d15 and d28 after start of idelalisib therapy )  . 
 ( b ) volcano plot of the fold change of protein and statistical significance between pretreatment and post - treatment t2 ( ie , d56 after start of idelalisib treatment )  . 
among the four evaluated antiapoptotic proteins , bcl - 2 was consistently increased in all patient samples at every time point ( fig 1c )  . bcl - 2 - a1 and bcl - xl generally decreased but varied . 
among proapoptotic bh3 - only domain proteins , bid remained the same , but bim was consistently increased at both evaluation times ( p 5 .01 ; fig 1c )  . 
idelalisib therapy results in an increase in bcl - 2 and bim and a decrease in mcl - 1 protebar graph of three proteins that show significant changes . primary chronic lymphocytic leukemia cells from patients were isolated fresh from peripheral blood before and after therapy and subjected to reverse phase protein array analysis as described in figure 1 . 
 ( a ) changes in the level of bcl - 2 protein after idelalisib therapy ( t1 5 15 or 28 days and t2 5 56 days after treatment )  . 
as shown in figs 2a to 2c , bcl - 2 , mcl - 1 , and bim levels were modulated in every sample , which were similar to those obtained with duvelisib.6 in general , these data suggest that bcr pathway inhibition leads to high bcl - 2 and bim protein levels and a decline in mcl - 1 protein levels in cll cells , which was also previously reported with ibrutinib therapy.11 given that the bcr axis inhibitors do not induce overt apoptosis of cll cells , strategies to combine venetoclax with idelalisib , duvelisib , or ibrutinib should result in apoptosis and removal of cll cells from peripheral blood . 
relationships are self - held unless noted . i 5 immediate family member , inst 5 my institution . relationships may not relate to the subject matter of this manuscript . for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . the following represents disclosure information provided by authors of this manuscript . 
 prexy modi no relationship to disclose anil korkut stock and other ownership interests : vivoz biolabs ( i ) patents , royalties , other intellectual property : drug combinations for treatment of melanoma and other cancers , c . 
brown honoraria : janssen pharmaceuticals , abbvie consulting or advisory role : gilead sciences , pharmacyclics , janssen pharmaceuticals , roche , genentech , celgene , sun pharma , infinity pharmaceuticals , astrazeneca , redx pharma , astellas pharma , abbvie research funding : gilead sciences varsha gandhi research funding : gilead sciences acknowledgment supported in part by an md anderson cancer center support grant , a cll global research foundation alliance grant , and sponsored research agreement from gilead to v.g. 
yang q , modi p , newcomb t , et al : idelalisib : first - in - class pi3k delta inhibitor for the treatment of chronic lymphocytic leukemia , small lymphocytic leukemia , and follicular lymphoma . 
miller bw , przepiorka d , de claro ra , et al : fda approval : idelalisib monotherapy for the treatment of patients with follicular lymphoma and small lymphocytic lymphoma . 
herman se , gordon al , wagner aj , et al : phosphatidylinositol 3 - kinase - d inhibitor cal - 101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals . 
hoellenriegel j , meadows sa , sivina m , et al : the phosphoinositide 39 - kinase delta inhibitor , cal - 101 , inhibits b - cell receptor signaling and chemokine networks in chronic lymphocytic leukemia . 
patel vm , balakrishnan k , douglas m , et al : duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocytic leukemia cells to venetoclax ( abt - 199 )  . 
brown jr , byrd jc , coutre se , et al : idelalisib , an inhibitor of phosphatidylinositol 3 - kinase p110d , for relapsed / refractory chronic lymphocytic leukemia . 
roberts aw , seymour jf , brown jr , et al : substantial susceptibility of chronic lymphocytic leukemia to bcl2 inhibition : results of a phase i study of navitoclax in patients with relapsed or refractory disease . 
ramkissoon , md , phd5 , 6 ; and malak abedalthaga , md7 this case study demonstrates that rst - line treatment of a neurotrophic receptor tyrosine kinase ( ntrk ) fusionpositive infantile glioblastoma with larotrectinib , an ntrk inhibitor ( ntrki ) , was a safe and effective way to achieve tumor regression in our patient . 
an 18 - month - old saudi arabian female presented with a history of right - sided weakness and partial seizures . brain magnetic resonance imaging ( mri ) revealed a large left frontal complex contrast - enhancing mass . craniotomy for gross total resection ( gtr ) was performed , and histologic pathologic analysis revealed a diagnosis of glioblastoma . 
follow - up mri was performed 8 weeks after larotrectinib treatment and showed signicant tumor regression , indicating a positive response to treatment with no reported adverse effects . this patient case highlights the importance of genomic proling for pediatric brain tumors to identify targetable alterations . 
mri showed a large , heterogeneous , complex left frontotemporal mass with internal cystic and necrotic changes with mass effect and a rightward midline shift with enhancing focus at the left posterior thalamic region ( 8 7 9 cm ; fig 1a )  . whole - spine imaging was unremarkable , and the patient underwent craniotomy for gtr . 
the patient recovered well from surgery with no neurologic decits . postoperative mri conrmed gtr but did note a small focus of nodular enhancement in the left insular cortex suggestive of residual tumor ( fig 1b )  . intermixed small neuropathologic analysis of resected tissues revealed features consistent with a high - grade glioma best classied as glioblastoma ( who grade iv )  . 
the neoplastic cells were diffusely immunopositive for vimentin ; were focally positive for s100 and synaptophysin ; showed retained nuclear staining for ini1 ; and were immunonegative for sma , ema , cd99 , cd31 , cd34 , and neun ( figs 2c to 2f )  . 
genomic proling of tumor tissue revealed the presence of an etv6 - ntrk3 fusion , which creates a novel chimeric oncoprotein that results in continuous activation of the ntrk3 kinase . 
the fusion encompassed exons 1 to 5 of etv6 and exons 14 to 20 of ntrk3 , which retains the kinase domain of ntrk3 ( fig 2g )  . as a result of the poor survival rates associated with the patients family declined chemoglioblastoma , therapy or radiation therapy despite extensive counseling on standard - of - care treatment . 
 ( b ) after total surgical resection , postgadolinium axial t1 - weighted mri demonstrated total resection of the left frontal mass . ( c ) at recurrence , postgadolinium axial t1 - weighted mri demonstrated newly appearing mass within the operative bed ( arrow ) showing intermediate diffusion signal with corresponding contrast enhancement indicating recurrent tumor . 
the patient has received continuous larotrectinib therapy since october 2019 and was scheduled for her 6 - month followup mri studies in april 2020 ; however , as a result of the global covid - 19 pandemic , the patients family has deferred further nonemergent hospital visits ( including followup mris ) until the self - isolation restrictions have been lifted . 
therefore , the patient was advanced to a digital clinic visit , where she was noted to be clinically stable and neurologically intact without any evidence of adverse events or toxicities to date . 
we obtained consent from the patients guardian to publish the patients presentation and related images . methods genomic proling next - generation sequencing was performed as previously described8 using the oncomine comprehensive assay v3 system ( thermo fisher scientic , waltham , ma )  . 
assays were performed using the ion s5 system and ion 540 chip ( thermo fisher )  . discussion primary brain tumors are a leading cause of cancer - related morbidity and mortality in children.9 high - grade gliomas ( hggs ) account for approximately 10% of pediatric brain tumors and are the second most common malignant cns tumor after medulloblastoma . 
the most frequent tumors are anaplastic astrocytoma ( who grade iii ) and glioblastoma ( who grade iv ) .10 glioblastoma occurs in both children and adults and is associated with a poor prognosis . 
 ( a ) sheet of highly cellular monomorphic round primitive cells with relative demarcation from adjacent brain tissue ( hematoxylin and eosin [ he ] ; original magnication , 20 )  . 
 ( b ) heterogeneous morphologic features of the neoplasm composed of focal areas of spindle cells arranged in small fascicles and primitive round cells in myxoid background with alternating hyperand hypocellularity ( he ; original magnication , 200 )  . ( c ) higher magnication of the primitive cells in the myxoid background ( he ; original magnication , 200 )  . 
 ( g ) schematic of etv6 - ntrk3 fusion detected in patients tumor . etv6 - ntrk3 5 14 for different n - terminal chemotherapy consisting of vincristine , carboplatin , and temozolomide.12 however , progression - free survival rates remain poor for these patients , highlighting the need for new therapeutic options , including small molecules and / or immunotherapy alone or in combination with chemotherapy regimens.13 although the etiology and genomic drivers of glioblastoma are diverse , 14 - 16 a common nding in pediatric hgg , especially infantile hggs , is the presence of fusions involving ntrk , alk , and ros1 , among others . 
trk fusion proteins are oncogenic drivers that have been reported in a wide range of adult and pediatric tumors that occur at high frequencies ( 90% ) in rare cancer types.17 , 18 gene fusions involving the kinase domain of each of the 3 neurotrophin receptors ( ntrk1 , ntrk2 , and ntrk3 ) fusion partners , were identied in 4% of diffuse intrinsic pontine gliomas and 10% of nonbrain stem ( nbs ) hggs . 
notably , in one study , 4 ( 40% ) of 10 nbs - hggs in children younger than 3 years old harbored an ntrk fusion gene . the high frequency of ntrk fusions in nbs - hggs from children age 3 years and the paucity of additional mutations in these tumors strongly suggest that the fusion genes are potent oncogenic drivers in early postnatal brain tumor development.19 ntrk fusions have been identied at low frequencies in lowgrade pediatric astrocytomas and adult glioblastomas.20 , 21 trk ligands ( commonly nerve growth factor for trka , brainderived growth factor or neurotrophin 4 for trkb , and neurotrophin 3 for trkc ) bind with high afnity to the extracellular domain of the trk receptor.22 , 23 this leads to receptor activation and the induction of signal transduction pathways involved in proliferation , differentiation , and survival ( eg , mapk , pi3k , and pkc pathways ) in both normal and neoplastic cells . the activity of the rst - generation trk - selective inhibitor larotrectinib in pediatric patients with tumors harboring ntrk fusions has been explored in clinical trials , including a phase i / ii trial in pediatric patients ( scout ; clinicaltrials.gov identier : nct02637687 ) and a phase ii trial involving adults and adolescents ( navigate ; clinicaltrials.gov identier : nct02576431 )  . 
larotrectinib is a potent and selective inhibitor of all 3 trks , producing potent and well - tolerated responses in adult and pediatric patients with ntrk - rearranged tumors , with sustained tumor regression in  . 
90% of infants , children , and adolescents with trk fusions at doses of 100 mg / m2 twice a day ( maximum , 100 mg per dose ) .1 - 7 the most common adverse events include mild elevations of liver enzyme levels , cytopenias , and vomiting . 
the high solubility of larotrectinib permit its use in liquid formulations in young patients unable to swallow capsules.5 , 24 increasingly , case reports in the literature demonstrate tolerance and clinical response to ntrki in pediatric patients with hgg , even after chemotherapy and radiation treatment.25 these patient cases highlight the need for clinical trials that assess the sustained durability of response to ntrki and whether these targeted therapies should be used as monotherapy agents or in combination with chemotherapy regimens . our patient case demonstrates that trk inhibitors can be integrated as a rst - line therapy for pediatric hggs harboring trk fusions . 
we also highlight the need for the integration of genomic proling in the routine histopathologic analyses of pediatric patients with malignant primary intracranial tumors to detect any genetic mutations that can be targeted with available therapies . 
bakhsh collection and assembly of data : musa alharbi , nahla ali mobark , malak abedalthaga data analysis and interpretation : musa alharbi , nahla ali mobark , fatmah a . 
ramkissoon employment : foundation medicine stock and other ownership interests : foundation medicine no other potential conicts of interest were reported . references bielack ss , cox mc , nathrath m , et al : rapid , complete and sustained tumour response to the trk inhibitor larotrectinib in an infant with recurrent , chemotherapy - refractory infantile brosarcoma carrying the characteristic etv6 - ntrk3 gene fusion . 
lancet oncol 19 : e187 , 2018 drilon a , laetsch tw , kummar s , et al : efcacy of larotrectinib in trk fusion - positive cancers in adults and children . 
n engl j med 378 : 731 - 739 , 2018 dubois sg , laetsch tw , federman n , et al : the use of neoadjuvant larotrectinib in the management of children with locally advanced trk fusion sarcomas . cancer 124 : 4241 - 4247 , 2018 laetsch tw , dubois sg , mascarenhas l , et al : larotrectinib for paediatric solid tumours harbouring ntrk gene fusions : phase 1 results from a multicentre , open - label , phase 1 / 2 study . 
lancet oncol 19 : 705 - 714 , 2018 landman y , ilouze m , wein s , et al : rapid response to larotrectinib ( loxo - 101 ) in an adult chemotherapy - naive patient with advanced triple - negative secretory breast cancer expressing etv6 - ntrk3 fusion . 
espinoza jc , haley k , patel n , et al : outcome of young children with high - grade glioma treated with irradiation - avoiding intensive chemotherapy regimens : final report of the head start ii and iii trials . 
alharbi m , ali mobark n , almubarak l , et al : durable response to nivolumab in a pediatric patient with refractory glioblastoma and constitutional biallelic mismatch repair deciency . 
nat genet 46 : 444 - 450 , 2014 jones dt , hutter b , j ager n , et al : recurrent somatic alterations of fgfr1 and ntrk2 in pilocytic astrocytoma . 
ziegler ds , wong m , mayoh c , et al : brief report : potent clinical and radiological response to larotrectinib in trk fusion - driven high - grade glioma . 
 graft - versus - host diseasefree antitumoral signature after allogeneic donor lymphocyte injection identied by proteomics and systems biology xiaowen liu , phd1 , 2 ; zongliang yue , ms3 ; yimou cao , ms1 ; lauren taylor , md2 ; qing zhang , ms4 ; sung w . 
chen , phd3 ; huanmei wu , phd1 ; and sophie paczesny , md , phd2 purpose as a tumor immunotherapy , allogeneic hematopoietic cell transplantation with subsequent donor lymphocyte injection ( dli ) aims to induce the graft - versus - tumor ( gvt ) effect but often also leads to acute graftversus - host disease ( gvhd )  . 
our novel six - step systems biology analysis involved removing common proteins and gvhd - specic proteins , creating a protein - protein interaction network , calculating relevance and penalty scores , and visualizing candidate biomarkers in gene networks . 
we then performed a second proteomics experiment in a validation set of patients who experienced gvt without acute gvhd after dli for comparison with the proteome of patients before dli . 
an independent experiment with single - cell proling in tumor antigenactivated t cells from a patient with posthematopoietic cell transplantation relapse was performed . results the approach provided a list of 46 proteins in the training set , and 30 proteins in the validation set were associated with gvt without gvhd . 
finally , the single - cell proling in activated t cells found 43 of the 61 genes . novel markers , such as rpl23 , ilf2 , cd58 , and crtam , were identied and could be extended to other antitumoral responses . conclusion our multiomic analysis provides , to our knowledge , the rst human plasma signature for gvt without gvhd . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction allogeneic hematopoietic cell transplantation ( hct ) is one of the most effective forms of tumor immunotherapy available to date . 
the lymphocytes in the donor graft recognize and eliminate residual tumoral cells through the graft - versus - tumor ( gvt ) effect , and thus , donor lymphocyte injection ( dli ) often is used at the time of relapse post - hct to induce gvt . 
despite the correlation of gvt indirect evidence for a gvt reaction and gvhd , separate from gvhd has been reported in large cohorts of hct patients or when dli is administered to induce remission in hct patients who have experienced a relapse.1 , 2 gvt activity can be increased by targeted therapy , as has been shown with sorafenib in flt3 internal tandem duplicationmutant leukemia cells.3 the gvt effect is mediated by minor histocompatibility antigens ( mihags ) on recipient leukemic cells that are recognized by donor cd4 + and cd8 + t cells.4 mihags can mediate gvt without inducing gvhd if they are only expressed by the recipient hematopoietic cells ( ie , minor h antigen [ ha ] - 1 , ha - 2 , bcl2a1 , and hb - 1 ) .5 , 6 nonhistocompatibility antigen proteins expressed by tumor cells , called tumor - associated antigens ( survivin , wilms tumor 1 , proteinase 3 ) also can mediate gvt activity.7 cytokines ( ie , interleukin - 15 [ il - 15 ] ) 3 ; checkpoint proteins , such as pd - 1 / pd - l18 ; and t - cell trafcking modulators9 are other possible mediators of gvt . our study aim was to develop a proteomic signature to identify gvt without gvhd after allogeneic dli . 
we attempted to do so through plasma proteomics and systems biology analyses of patients in relapse after hct who received donor lymphocytes as immunotherapy . knowledge generated our novel six - step systems biology analysis involved the removal of common proteins and gvhd - specic proteins , creation of a protein - protein interaction network , calculation of relevance and penalty scores , and visualization of candidate biomarkers in gene networks to dene a unique , biologically relevant 61 - protein signature of gvhd - free gvt . 
forty - three of the 61 genes also were found in an independent experiment using massive single - cell proling of tumor antigenactivated t cells from a patient who experienced post - hct relapse . relevance this multiomic analysis provides the rst human plasma signature for gvhd - free gvt to our knowledge . 
risk stratication on the basis of the gvt without gvhd protein signature would allow for customized treatment plans after hct . of 61 proteins that are signicantly expressed in the plasma of hct patients who received dli for tumor relapse . 
the methodology for this study is described in the data supplement . results plasma proteomics of gvhd - free gvt in a training set our initial approach to identifying gvt - specic biomarkers was to undertake a proteomics analysis . 
we reasoned that the best setting to observe a gvt effect without any effect of chemotherapy or preparative conditioning regimen would be after dli is given for relapse of malignant disease . 
our rst proteomics experiment ( ipas01 ) was designed to distinguish proteomes that predict gvhd - free gvt from those that predict gvt with gvhd after dli in a training set . 
we compared a pool of plasma samples taken approximately 30 days after dli from ve patients without relapse and without gvhd ( gvt - positive , gvhd - negative ) labeled with the heavy isotope and compared them with a pool of plasma samples at matched time points from 11 patients without relapse but with concomitant gvhd ( gvt - positive , gvhd - positive ) labeled with the light isotope ( fig 1 )  . 
no signicant differences between groups were observed for patient age , disease status ( all but one with morphologic relapse , and only one patient did not receive chemotherapy ) , dli donor type , and dli dose , and all patients were off immunosuppression . 
 gvhd - free antitumoral signature after donor lymphocyte injection gvt - positive , and gvhd - positive post - dli ( ipas - 01 ) gvt - positive , and gvhd - negative pre - dli ( ipas - 02 ) gvt - positive , and gvhd - negative post - dli ( ipas - 01 and - 02 ) isotopic labeling light isotopic labeling heavy immunodepletion six most abundant proteins acrylamide alkylation samples mixed fig 1 . 
the intact - protein analysis system ( ipas ) compared graft - versus ( gvt ) positive and graft - versus - host disease tumor ( gvhd ) negative postdonor lymphocyte injection ( dli ; heavy isotope ) with gvt - positive and gvhd - positive postdli ( light isotope ) samples in a training set ( ipas - 01 ) , and gvt - positive and gvhd - negative post - dli ( heavy isotope ) with gvt - positive and gvhd - negative pre - dli ( light isotope ) samples in a validation set ( ipas - 02 )  . anion - exchange chromatography ( n = 8 ) reversed - phase chromatography ( n = 60 ) liquid chromatography - ms / ms software tool ( data supplement )  . 
after this data processing , the ipas - 01 experiment provided a list of 46 proteins associated with a gvt signature without gvhd post - dli in the training set ( table 1 )  . 
although we started with ipas proteins with a heavy / light ratio greater than 1.2 , the systems biology process may have resulted in a nal score of less than 1 ; however , because all these proteins are gvt specic , they all are included in the nal gvhd - free gvt signature . 
the rst node is centered around cd8 ( cd8a ) and includes cd58 , il1a , ly75 , fas , and gpnmb and a secondary node centered around raf1 and containing guk1 , ephb4 , and cxcl12 . 
our data with the cd8a - centered node strongly suggest that gvt is majorly mediated by cd8 t cells and gvhd less so , which provides an opportunity to separate gvt and gvhd by manipulating these two t - cell subsets differently . 
as a proof of concept , we performed the experiment without applying these proteins to show that their nal scores have not been signicantly changed by the use of penalty scores ( table 1 ; data supplement )  . plasma proteomics of gvhd - free gvt in a validation set our second proteomics experiment ( ipas - 02 ) used a validation set that was designed to compare a pool of samples from ve gvt - positive , gvhd - negative ( labeled with the heavy isotope ) patients with a pool of samples from the same patients taken before dli when the patients were in relapse and thus gvt negative ( labeled with the light isotope ; fig 1 )  . 
a major cluster is constituted by centromere protein m ( cenpm ) , nup160 , wapal , dhx37 , stx7 , and il enhancer binding factor 2 ( ilf2 ) , which are all proteins implicated in the activation of cytotoxic t cells consistent with a gvt response ( table 2 )  . 
for example , cenpm ( also called pane1 ) is a known mihag expressed on b - lymphoid cells that is highly relevant to gvt - mediated post - dli.12 the protein encoded by ilf2 is a transcription factor needed for t - cell expression of il - 2.13 the second cluster is similar to the one found in ipas - 01 centered on map3k6 and dbnl . final 61 - protein gvhd - free gvt signature after dli the next step was to combine the proteins found in the training and validation sets . 
combined analysis of the two ipas experiments yielded 61 proteins : 49 with a nal combined score of greater than 1 and 12 with a nal combined score of less than 1 , all specic to gvt without gvhd ( table 1 )  . 
these include one similar to that seen in ipas - 01 centered on cd8a , one similar to that seen in ipas - 02 centered on cenpm - wapal and also containing dhx37 , and one found in both experiments and centered on map3k6 and dbnl . 
this additional cluster is centered around cse1l , which is a ras - related nuclear protein with a potential role in ras / raf / mapk signaling in t cells . 
some potential important novel markers , such as cytotoxic and regulatory t - cell molecule ( crtam ) , which has been shown to determine the cd4 + cytotoxic t - lymphocyte lineage , 14 are not part of a cluster . 
 ( a ) intact - protein analysis system ( ipas ) - 01 : geneterrain visualization shown in 2d for the graftversus - tumor ( gvt ) signature obtained by comparing gvt - positive , graft - versus - host disease ( gvhd ) negative with gvt - positive , gvhd - positive postdonor lymphocyte injection ( dli ) samples . 
a mean reads number of 381 , 079 per cell and median gene number of 1 , 091 per cell were analyzed for an average of 1 , 436 cells per condition . 
among the 61 genes identied in the proteomic gvhd - free gvt signature , 11 were more highly expressed in prame - specic t cells compared with cmvspecic t cells or nonreactive t cells . 
the expression prole of four representative gvt markers in the single - cell rna sequencing analysis of prame - specic t cells ( rpl23 , ilf2 , cd58 , and crtam ) is shown in figure 3 . 
rpl23 is a component of the 60s ribosomal subunit and has been shown to link the oncogenic ras signaling to p53 - mediated tumor suppression.15 t - cell responses to rpl23 also are increased in autoimmune diseases , 16 which suggests a role for the ras / raf / mapk pathway in t cells that is currently underexplored . 
the cd58 / cd2 axis is the primary costimulatory pathway for cd8 + t cells that lack cd28 , which suggests an alternate activation mechanism during gvt for exhausted t cells.17 crtam is upregulated in cd4 + and cd8 + t cells and encodes a type 1 transmembrane protein with v and c1 - like immunoglobulin domains.18 it has been shown to negatively regulate zeb1 ( zinc nger e - boxbinding homeobox 1 ) in t cells19 and to determine the cd4 + cytotoxic t - lymphocyte lineage , 14 and it might determine the cd8 + cytotoxic t - lymphocyte lineage as well . 
expression prole of four representative graft - versus - tumor ( gvt ) markers in single - cell rna sequencing analysis of prameand cytomegalovirus ( cmv ) pp65specic t cells . 
one systems biology novelty of our approach was the performance of a one - layer extension on ppi data using interactions one node away from the gene in the original list that are called outer genes . 
a strength of our approach is that we ltered out nonrelevant proteins using a penalty score , which led to a more - specic list of candidate proteins and avoided contaminants . 
this study favored a large - scale proteomics approach as opposed to a hypothesis - driven candidate approach.21 we have shown that for gvhd markers , this method is efcient in discovering new candidate markers.10 , 11 compared with our previous studies , we experimentally removed the gvhd proteins by assigning them the light isotope . 
of note , this approach showed that the proteins identied and their ratio have not been inuenced much by the implementation of penalty scores . in an independent experiment with single - cell proling of t cells from a patient with relapse after hct that were activated in vitro , 43 of the 61 proteomic genes also were found in activated t cells , which suggests that the proteins identied are biologically relevant in different antitumoral responses . the biology of the gvt markers not yet described is as follows . 
the function of trpc4ap ( transient receptor potential cation channel subfamily c member 4associated protein ) has been shown to be involved in the ubiquitination of e3 ligase skp222 and the activation of c - jun nh ( 2 ) terminal kinase and transcription factor ap - 1.23 guanylate kinase 1 ( guk1 ) is an enzyme that catalyzes the transfer of a phosphate group from atp to guanosine monophosphate ( gmp ) to form guanosine diphosphate ( gdp ) and is believed to be a good target for cancer chemotherapy.24 its expression on tumor - specic t cells was not previously reported . 
pin1 ( peptidylprolyl cis / trans isomerase , nimainteracting 1 ) catalyzes the cis / trans isomerization of peptidyl - prolyl peptide bonds and thus catalytically regulates the postphosphorylation conformation of its substrates and is involved in the regulation of t - cell biology . 
in particular , its implication has been shown in systemic lupus erythematosus and t - cell acute lymphoblastic leukemia progression.25 - 27 wapl cohesin release factor has been shown to restrict chromatin loop extension.28 of note , it was part of a microrna - mrna network in allogeneic t - cell responses.29 fermitin family member 3 ( fermt3 ) a member of the kindlins that mediates ppi involved in integrin activation . 
mutations in this gene cause the auadhesion deciency tosomal leukocyte syndrome - 3.30 its role in t cells has not been studied . cd160 is another surface protein tightly expressed on peripheral cytotoxic cd8 t lymphocytes and natural killer cells , 31 and soluble cd160 enhances cd8 + t cells , which results in increased interferon - , il - 2 , and tumor necrosis recessive factorsecretion as well as cytolysis against tumor cells in vitro and in vivo.32 not surprisingly , serglycin ( srgn ) , which serves as a mediator of granule - mediated apoptosis through the macromolecular complex of granzymes and perforin and determines the secretory granule repertoire of cytotoxic t lymphocytes , 33 was also upregulated in this study . 
the fas cell surface death receptor ( fas ) that plays a critical role in the activation of the death - inducing signaling complex with fas - associated death domain protein and triggers a downstream caspase cascade that leads to apoptosis34 also was upregulated . the chemokine cxcl12 has been proposed to be able to distinguish immune cells that induce gvt going to the bone marrow from immune cells that induce gvhd.35 it is one of the rare proteins for which the nal score was modied more than twice by the penalty score but is still included overall as a gvt proteseveral of the tripartite motif ( trim ) members encode for mihags.21 although trim42 , 22 , and 37 are located on chromosomes 3 , 11 , and 17 , respectively , trim39 is on chromosome 6 in the major histocompatability class i region and , in our study , showed global expression on activated t cells . 
raf1 is the cellular homolog of viral raf proto - oncogene ( v - raf ) and is also a map3k that functions downstream of the ras family of membrane - associated gtpases to which it binds directly . 
cse1l is a ras - related nuclear protein that binds strongly to nuclear localization signal - free importin - , and this complex is then released in the cytoplasm by the combined action of ranbp1 and rangap1 . 
one particular protein of nont - cell origin , il - 1 , was found to be elevated in the plasma of patients with gvt response and may play a signicant role in antitumor effects . 
of note , il - 1engineered tumor cells rarely develop into tumors , and if they do , the tumors are quickly destroyed through a mechanism that involves cd8 + t cells and natural killer cells , and il - 1 tumor immunoediting microenvironment.36 although our approach identied several proteins for gvhd - free gvt , there are limitations in this study . 
third , the systems biology pipeline relies on knowledge from the published domain , which makes the application of this method to diseases that have not been well studied , such as gvt , difcult . 
chen travel , accommodations , expenses : wuxi apptec sophie paczesny patents , royalties , other intellectual property : inventor on a patent entitled methods of detection of graft - versus - host disease ( us 20130115232a1 , wo 2013066369a3 ) no other potential conicts of interest were reported . acknowledgment the raw mass spectrometry data are publicly available at massive ( massive.ucsd.edu ) with the identication number msv000081057 . 
we thank gregory yanik , md , for providing the patient samples through a university of michigan - indiana university material transfer agreement . references ringd en o , labopin m , gorin nc , et al : is there a graft - versus - leukaemia effect in the absence of graft - versus - host disease in patients undergoing bone marrow transplantation for acute leukaemia ? br j haematol 111 : 1130 - 1137 , 2000 bar m , sandmaier bm , inamoto y , et al : donor lymphocyte infusion for relapsed hematological malignancies after allogeneic hematopoietic cell transplantation : prognostic relevance of the initial cd3 + t cell dose . 
mathew nr , baumgartner f , braun l , et al : sorafenib promotes graft - versus - leukemia activity in mice and humans through il - 15 production in flt3 - itdmutant leukemia cells . 
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leuk lymphoma 45 : 2229 - 2237 , 2004 koestner w , hapke m , herbst j , et al : pd - l1 blockade effectively restores strong graft - versus - leukemia effects without graft - versus - host disease after delayed adoptive transfer of t - cell receptor gene - engineered allogeneic cd8 + t cells . 
blood 117 : 1030 - 1041 , 2011 kim ym , sachs t , asavaroengchai w , et al : graft - versus - host disease can be separated from graft - versus - lymphoma effects by control of lymphocyte trafcking with fty720 . 
vander lugt mt , braun tm , hanash s , et al : st2 as a marker for risk of therapy - resistant graft - versus - host disease and death . 
brickner ag , evans am , mito jk , et al : the pane1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in b - lymphoid 13 . 
kao pn , chen l , brock g , et al : cloning and expression of cyclosporin aand fk506 - sensitive nuclear factor of activated t - cells : nf45 and nf90 . 
cancer res 76 : 5030 - 5039 , 2016 ito y , hashimoto m , hirota k , et al : detection of t cell responses to a ubiquitous cellular protein in autoimmune disease . 
leitner j , herndler - brandstetter d , zlabinger gj , et al : cd58 / cd2 is the primary costimulatory pathway in human cd28 - cd8 + t cells . 
yeh jh , sidhu ss , chan ac : regulation of a late phase of t cell polarity and effector functions by crtacell 132 : 846 - 859 , 2008 19 . 
bachireddy p , hainz u , rooney m , et al : reversal of in situ t - cell exhaustion during effective human antileukemia responses to donor lymphocyte infusion . blood 123 : 1412 - 1421 , 2014 21 . 
tissue antigens 81 : 183 - 193 , 2013 jamal a , swarnalatha m , sultana s , et al : the g1 phase e3 ubiquitin ligase truss that gets deregulated in human cancers is a novel substrate of the s - phase e3 ubiquitin ligase skp2 . 
soond sm , terry jl , riches dw : truss , a tumor necrosis factor receptor - 1 - interacting protein , activates c - jun nh ( 2 ) - terminal kinase and transcription factor ap - 1 . 
ruppert r , moser m , sperandio m , et al : kindlin - 3 - mediated integrin adhesion is dispensable for quiescent but essential for activated hematopoietic stem cells . j exp med 212 : 1415 - 1432 , 2015 31 . 
kotsiou e , davies jk : new ways to separate graft - versus - host disease and graft - versus - tumour effects after allogeneic haematopoietic stem cell transplantation . br j haematol 160 : 133 - 145 , 2013 36 . 
we identied , for the rst time , an epigenomic factor that can potentially complement dna mutation status in discriminating patients with lung adenocarcinoma who are less likely to benet from egfr - tki treatment , thereby leading to improved patient management in precision medicine . jco precis oncol 5 : 418 - 431 . 
inhibition of epidermal growth factor receptor ( egfr ) kinase activity by egfr - tyrosine kinase inhibitors ( tkis ) , such as erlotinib , getinib , and afatinib , was effective in patients with nsclc with egfr - activating mutations . however , despite the remarkable clinical success , the treatment efcacy was still limited to 50% - 80%.10 , 11 the sizable percentage of nonresponders ( 20% - 30% ) suggested intrinsic tki resistance and substantial heterogeneity among tumors , even among egfrmutant tumors , highlighting the need for reliable predictive biomarkers . the comprehensive molecular proling of pretreatment lung adenocarcinoma to identify inherently tkiresistant cases can aid the development of potential strategies to manage such cases . 
 dna methylation proling for egfr - tki responses context key objective a sizable portion ( 20% - 30% ) of patients with epidermal growth factor receptor ( egfr ) mutant nonsmall - cell lung cancer have no good initial clinical response to egfr - tyrosine kinase inhibitors ( tkis ) , and how to predict intrinsic drug resistance accurately is challenging . 
global dna methylation landscape of tumors from patients with lung adenocarcinoma before tki treatment was analyzed to investigate the association with egfr - tki responses . knowledge generated a total of 216 tki responseassociated methylated sites were identied , and functional analysis revealed the enrichment of homeobox genes . 
in particular , increased methylation of hoxb9 correlated with higher rate of intrinsic resistance ( ir ) to egfr - tki . relevance dna methylation provides a different dimension to complement the dna mutationbased markers for understanding the mechanism of ir . 
evaluation of the methylation level on hoxb9 may be incorporated in the management of lung adenocarcinoma to aid the prediction of egfr - tki response . egfr - tki14 suggest many dna - based biomarkers for ir prediction . 
on the other hand , tumor suppressor genes involved in the alternative mechanisms of ir may be inactivated by epigenetic mechanisms that result in phenotypic or functional changes.15 however , although studies have reported that epigenetic changes in tumor participate in the evolution of acquired drug resistance through regulating gene expression patterns , 16 epigenomic data associated with ir to tki are lacking . 
modication of methylation on dna is stable and abnormal methylation represents an early event for cancer diagnosis , making methylation aberrations equally suitable candidates for recurrence detection and prediction of patient survival.17 - 21 therefore , we undertook a genome - wide approach to investigate dna methylation patterns associated with ir to tki . dna methylation that occurs at cytosines of cpg dinucleotides , especially within cpg islands in the promoter region , can lock genes in off status , resulting in a transcriptionally silent state.22 , 23 although dna methylation is an important mechanism for maintaining normal development and cellular homeostasis , aberrant dna methylationmediated silencing of tumor suppressor genes has been reported to be associated with cell survival and progression in cancer.24 dna methylation proling of tumors , including those of glioma , acute myeloid leukemia , and colorectal and lung cancers , has aided the identication of cancer subtypes correlated with clinical outcomes.25 - 29 in this study , we aimed to identify epigenetic markers for predicting drug efcacy in patients with lung adenocarcinoma . 
we conducted genome - wide dna methylation proling of tumors from patients before their egfr - tki therapy and established a dna methylation landscape to elucidate the association of dna methylation with the egfr - tki response status of patients via a pipeline of statistical analysis , gene ontology ( go ) , and bioinformatics analysis . 
detailed accounts of sample collection and protocols for dna extraction , bisulte conversion , dna methylation analysis , pyrosequencing along with statistical analysis , bioinformatics analysis , and data availability are presented in the data supplement . results clinicopathologic features of the patients table 1 lists the clinicopathologic features of the two cohorts . 
the validation cohort consisted of 163 egfrmutant patients and the majority ( 85.28% ) were at stage iv . the tki response assessmentprogressive disease ( pd ) , stable disease ( sd ) , partial response ( pr ) , or complete response ( cr ) was determined according to the recist guidelines.30 the pd group , dened at the rst scan done at 8 weeks following the start of egfr - tki , is considered as patients with ir . 
using the top 5% coefcient of variation as the cutoff , 24 , 121 probes with the greatest variability were analyzed , and 391 probes were found correlated with egfr - tki response . 
the majority of the probes ( 203 ) had higher dna methylation in patients with a poor response than in those with a favorable response ; hypermethylation correlated with poor response . 
a global view of the dna methylation distribution contrasting the patients with pd against those with pr showed that all but 13 probes were located above the diagonal line , elucidating a clear pattern of methylation gain in patients with pd ( fig 2b - d )  . 
the tumors of patients who were more likely to be resistant to egfr - tkis tended to have higher pretreatment methylation levels . chromosomal context analysis of candidate cpg sites showed enrichment in cpg islands and gene promoter regions the cpg sites were assigned to the annotated categories according to their chromosome positions relative to the nearby transcription start sites and the closest cpg islands ( data supplement )  . 
we examined the changes in the proportion of each category during our probe selection and found an increasing trend in cpg islands and the gene promoter region tss1500 ( between 1 , 500 bp and 200 bp upstream of the transcription start site )  . 
for the 37 transcription - repressive sites , the enrichment pattern in cpg islands was retained and that in tss1500 was increased to 27.03% ( fig 2e )  . identication of transcription - repressive methylation sites we investigated the potential of the 216 methylation sites in cis - regulation of gene expression by correlating publicly accessible mrna gene expression data ( gse60644 ) with dna methylation data ( gse56044 ) in lung adenocarcinoma ( data supplement )  . 
a total of 37 sites were identied as transcription - repressive sites ( data supplement )  . functional enrichment analysis of transcript - linked methylation showed eight probes linked to tfs ( go : 0003677 ) , to evaluate the molecular function of the genes mapped by the 37 transcription - repressive sites , go enrichment analysis was conducted . 
furthermore , using animal transcription factor database ( animaltfdb ) , we found that eight of the 37 sites were annotated to ve tfs : ikzf1 , hoxb9 , sp8 , lass4 , and vax2 ( table 2 and data supplement )  . figure 2g shows the f statistic and the corresponding p value from analysis of variance for each of the eight sites , along with the location and chromosome context information . 
we found that seven of the eight tf - linked sites are located in the context of cpg islands and ve in the transcription start site ( tss ) region ( table 2 )  . 
the two vax2 methylation sites also showed a moderate ability to predict the egfr - tki response ( data supplement )  . we further performed stratication analysis by classifying patients as those with egfr - activating mutations and those increased methylation of hoxb9 without ( figs 3d - f )  . ( cg13643585 ) was observed in pd patients . 
for the egfr wild - type group , the or ( 7.59 ) was comparable , but the p value was .09 , likely because of the small sample size ( fig 3e )  . 
the data in table 3 conrmed the pattern of increased hoxb9 methylation in the egfr - tkiresistant ( pd ) group compared with the disease control group ( cr , pr , or sd )  . 
the pd group was ranked rst in each quartile , and the one - sided rank - sum test for between - group differences indicated a signicant difference ( p = .036 ) ( data supplement )  . in addition , analysis of the auroc showed that hoxb9 methylation statistically signicantly increased the predictive precision of egfr - tki resistance for the dcr ( table 3 )  . 
although signicant advances have been made in understanding acquired resistance ( ar ) , the causes of ir remain unclear.12 in addition to being classied as ar versus ir , resistance mechanisms can be classied in terms of on - target versus off - target , 31 suggesting the activation of collateral signaling . 
the patients characteristics , smoking behavior ( p1 ) , sex ( p2 ) , egfr status ( p3 ) , and egfr - tki response ( p4 ) , are shown by the bars at the top of the heatmap . 
for each cpg probe , the average beta value across patients with pd was plotted against that across patients with pr and is shown as a smooth kernel scatter plot . 
in the gradient scale , red represents the densest region , whereas purple represents the sparsest region ; pr ( red ) , sd ( yellow ) , and pd ( blue )  . 
the probe distributions in the cpg context and the gene context are shown on the left and right , respectively , for all probes in the array ( top ) , for the most variably methylated probes ( upper middle ) , for the tki responseassociated methylation sites ( lower middle ) , and for the transcription - repressive sites ( bottom )  . 
moreover , where genomic resistance has been found , epigenomic modulation has been proposed as the potential mechanism . changes in resistant phenotypes , including epithelialtransition ( emt ) and cancer stemness mesenchymal shift , have been found to be driven by epigenetic remodeling . 
tki - induced dna methylation changes have been reported in advanced egfr - mutated lung cancer.33 decitabine , the dna methyl transferase inhibitor , could reverse the sensitivity of egfr - tkiresistant nsclc cell line pc9 / gr through demethylation of rassf1a and gadd45.34 the combination of tkis with epigenetic drugs has shown in preclinical and clinical promise as a treatment studies.33 - 35 in this study , we conducted clinical oncological investigation on the potential role of dna methylation in mediating ir to egfr - tki treatment in patients with advanced lung adenocarcinoma . 
aberrant dna methylation is one of the most classical events that occurs during lung cancer development.36 many studies have shown altered methylation patterns in lung cancer , indicating roles of epigenetic biomarkers and therapeutic targets.37 - 39 earlier study by zhu et al40 focused on the methylation patterns of wnt antagonists , showing the association of methylated sfrp5 with shortened progression - free survival under egfr - tki treatment , but not with ir to tki . 
interestingly , two 428 2021 by american society of clinical oncology of those ve genes , axl1 and hoxa9 , are homeobox tfs . our pursuit of the primary egfr - tkiresistant methylation markers also identied enrichment of homeobox genes . among the 30 tki - associated methylation probes annotated to tfs , 11 accounted for nine homeobox genes ( data supplement )  . we identied and conrmed the correlation of hoxb9 dna methylation with an increased rate of ir to egfr - tkis . hoxb9 is involved in cell development and proliferation43 and is suggested to function as a tf that can induce the expression of emt genes and several angiogenic factors , such as vegf , il - 8 , and tgf , resulting in the activation of egfr and erbb2.44 - 46 egfr signaling is connected to the nf - b pathway , giving the role in ir or ar to egfr inhibitors.47 however , the molecular mechanisms by which hoxb9 contributes to carcinogenesis are debated.48 , 49 the overexpression of hoxb9 can suppress the akt / nf - b / snail pathway and inhibit the proliferation of gastric carcinoma cells.50 we analyzed the correlation between egfr signaling and nf - bdependent pathways ( gse60644 ) and found that expression of hoxb9 negatively associated with that of kiaa1199 ( cell migrationinducing hyaluronidase 1 ; data supplement )  . 
through protein - protein interaction ( ppi ) analysis , we found that hoxb9 might cross talk with both ir and ar to egfr - tki through ezh2 , sirt1 , and egr2 ( data supplement )  . 
therefore , regulation of hoxb9 is crucial in the cooperated oncogenic loops.12 our data suggested that hoxb9 hypermethylation may be a novel tumor cellular state that is useful for precise categorization of tumor heterogeneity in the study of intrinsic egfr - tki resistance via off - target effects such as redundant or compensating signaling . pattern was consistent between patients with egfr - activating mutations and patients with wild - type egfr , implying that the regulatory effect of dna methylation of hoxb9 may be independent of egfr activity . in addition , dna methylation changes can be accurately detected in tumors and liquid biopsies . 
such detection is promising for the development of biomarkers for cancer screening.51 dna methylation in distal regulatory sites , such as enhancer regions , plays important roles in gene regulation through the binding of cell typespecic tfs and interaction with promoters.52 - 54 we validated a dna methylation site in the enhancer region of hoxb9 that can help the prediction of nonresponse to egfr - tki . 
in cancer , aberrant dna methylation at enhancers couples with recruitment of coactivators or corepressors , forming networks of cancer - associated tfs and their targeted genes.55 - 57 stone et al58 dened hypermethylation enhancers that correlate with sensitivity to endocrine therapy , suggesting the impact of enhancer status on the drug treatment response . 
 dna methylation proling for egfr - tki responses the combination of genetic aberrations , gene expression , and dna methylation highlights the potential of the identied candidates in the development of biomarkers for tumor diagnosis or prognosis . 
in this study , with a focus on medical actionability , we discovered that hoxb9 methylation could be a biomarker useful for discriminating patients with tki resistance from those with tki sensitivity , especially patients whose tumors harbor egfr - activating mutations . 
although improving the sensitivity and specicity of hoxb9 methylation is recommended , our work provided a preliminary proof of concept on the usefulness of hoxb9 methylation for opening up more clinical options to manage lung adenocarcinoma . 
for example , in accordance with the current clinical standard of treating egfr - mutant patients with egfr - tki , for patients with hoxb9 hypermethylation , combination treatment such as egfr - tki plus antiangiogenic therapy59 or egfr - tki plus chemotherapy60 may be another option to overcome the resistance and improve the response rate . 
 acknowledgment the authors thank all the patients and research staff who participated in this work . su et al references siegel rl , miller kd , jemal a : cancer statistics , 2019 . 
mitsudomi t , morita s , yatabe y , et al : getinib versus cisplatin plus docetaxel in patients with non - small - cell lung cancer harbouring mutations of the epidermal growth factor receptor ( wjtog3405 ) : an open label , randomised phase 3 trial . 
lancet oncol 11 : 121 - 128 , 2010 zhou c , wu yl , chen g , et al : erlotinib versus chemotherapy as rst - line treatment for patients with advanced egfr mutation - positive non - small - cell lung cancer ( optimal , ctong - 0802 ) : a multicentre , open - label , randomised , phase 3 study . 
lancet oncol 12 : 735 - 742 , 2011 rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as rst - line treatment for european patients with advanced egfr mutationpositive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
wu yl , zhou c , liam ck , et al : first - line erlotinib versus gemcitabine / cisplatin in patients with advanced egfr mutation - positive non - small - cell lung cancer : analyses from the phase iii , randomized , open - label , ensure study . 
ann oncol 26 : 1883 - 1889 , 2015 yang jch , sequist lv , geater sl , et al : clinical activity of afatinib in patients with advanced non - small - cell lung cancer harbouring uncommon egfr mutations : a combined post - hoc analysis of lux - lung 2 , lux - lung 3 , and lux - lung 6 . 
lancet oncol 16 : 830 - 838 , 2015 yang jch , wu yl , schuler m , et al : afatinib versus cisplatin - based chemotherapy for egfr mutation - positive lung adenocarcinoma ( lux - lung 3 and luxlung 6 ) : analysis of overall survival data from two randomised , phase 3 trials . 
lee ck , wu yl , ding pn , et al : impact of specic epidermal growth factor receptor ( egfr ) mutations and clinical characteristics on outcomes after treatment with egfr tyrosine kinase inhibitors versus chemotherapy in egfr - mutant lung cancer : a meta - analysis . 
lee ck , davies l , wu yl , et al : getinib or erlotinib vs chemotherapy for egfr mutation - positive lung cancer : individual patient data meta - analysis of overall survival . 
santoni - rugiu e , melchior lc , urbanska em , et al : intrinsic resistance to egfr - tyrosine kinase inhibitors in egfr - mutant non - small cell lung cancer : differences and similarities with acquired resistance . 
blakely cm , watkins tbk , wu w , et al : evolution and clinical impact of co - occurring genetic alterations in advanced - stage egfr - mutant lung cancers . 
niu x , liu f , zhou y , et al : genome - wide dna methylation analysis reveals gabbr2 as a novel epigenetic target for egfr 19 deletion lung adenocarcinoma with induction erlotinib treatment . 
nelson hh , marsit cj , christensen bc , et al : key epigenetic changes associated with lung cancer development : results from dense methylation array proling . epigenetics 7 : 559 - 566 , 2012 37 . 
zhu j , wang y , duan j , et al : dna methylation status of wnt antagonist sfrp5 can predict the response to the egfr - tyrosine kinase inhibitor therapy in nonsmall cell lung cancer . 
rauch t , wang z , zhang x , et al : homeobox gene methylation in lung cancer studied by genome - wide analysis with a microarray - based methylated cpg island recovery assay . 
zhang l , wu q , he c , et al : hoxb9 inhibits proliferation in gastric carcinoma cells via suppression of phosphorylated - akt and nf - b - dependent snail expression . 
taberlay pc , statham al , kelly tk , et al : reconguration of nucleosome - depleted regions at distal regulatory elements accompanies dna methylation of enhancers and insulators in cancer . 
h saito , t fukuhara , n furuya , et al : erlotinib plus bevacizumab versus erlotinib alone in patients with egfr - positive advanced non - squamous non - small - cell lung cancer ( nej026 ) : interim analysis of an open - label , randomised , multicentre , phase 3 trial . 
hosomi y , morita s , sugawara s , et al : getinib alone versus getinib plus chemotherapy for non - small - cell lung cancer with mutated epidermal growth factor receptor : nej009 study . 
 response to entrectinib in differentiated thyroid cancer with a ros1 fusion introduction the detection of gene fusions in cancer provides insight into tumorigenesis and in some cases reveals potential therapeutic targets.1 there are now a multitude of small - molecule inhibitors that effectively target tumors harboring specific gene fusions . 
gene fusions in ros1 , for example , predict sensitivity to the oral tyrosine kinase inhibitor crizotinib and have led to the approval of crizotinib for the treatment of nsclc with an ros1 fusion.2 ros1 fusions have also been detected in multiple other cancer types , including glioblastoma multiforme , 3 gastric cancer , 4 and acral lentiginous melanoma , 5 but response to ros1 tyrosine kinase inhibitors in tumors other than lung cancer is not well characterized . 
over the next 8 years , she was treated with radioactive iodine ( 200 mci after a radioactive iodine scan showed hilar uptake ) , stereotactic radiosurgery , and surgical resection ( including a left pneumonectomy ) , which confirmed metastatic papillary thyroid carcinoma . 
imaging then showed metastases in the thorax , rib , liver , and brashe began firstline systemic therapy with sorafenib , which she continued for 18 months , achieving a best response of stable disease . 
her thyroglobulin levels were detectable but low and did not change notably during treatment . with no established second - line agents for metastatic papillary thyroid cancer , the patient then participated in prescreening for the phase ii basket study of entrectinib , which facilitated genomic testing of her tumor sample . 
as part of that testing , mrna was isolated from a formalin - fixed paraffinembedded sample from the resected brain metastasis and a sequencing library prepared using a custom - designed anchored multiplex pcr kit ( archerdx , boulder , co ) targeting fusions in the ntrk1 / 2 / 3 , ros1 , alk , and ret genes.10 sequencing was performed on an illumina miseqdx system ( illumina , san diego , ca )  . 
this assay identified an ezr ( exon 10 ) ros1 ( exon 34 ) fusion . the patient provided informed consent , enrolled in the phase ii study of entrectinib , and started treatment in august 2016 . 
at baseline , positron emission tomography / computed tomography imaging revealed a left periaortic soft tissue nodule ( figs 1a and 1d ) and a solitary liver metastasis not well visualized with computed tomography alone ( figs 2a and 2d )  . 
after 4 weeks of therapy , the periaortic nodule was no longer visualized ( figs 1b and 1e ) and remained absent 6 months after starting therapy ( figs 1c and 1f )  . 
there is a growing body of evidence illustrating the activity of targeted agents across cancer types , underscoring the relevance of a genomic diagnosis.11 in nsclc , the presence of a pathogenic ros1 rearrangement identifies candidates for treatment with crizotinib , and targeting ros1 has been a promising strategy across fusion partners.2 , 12 - 14 ros1 gene rearrangements create fusion proteins with constitutively active kinase domains that activate downstream signaling pathways , leading to oncogenic properties in cells , including uncontrolled proliferation and resistance to cell death with prolonged tumor - cell survival . 
these pathways include rasextracellular regulated kinase for cellular proliferation and the janus kinasesignal transducers and activators of transcription and phosphatidylinositol 3 - kinase / akt pathways , which regulate cell survival ( antiapoptosis ) and proliferation . 
cancers that have these pathways activated tend to be more aggressive , with invasion and metastasis leading to poor patient survival.15 with increasing access to genomic profiling of tumors , ros1 rearrangements are now being identified in many other cancers . 
here , we report another unique event : an ezr - ros1 fusion , previously described only in nsclc.16 , 17 although identification of these genomic aberrations across disease types is of interest , there is greater value in documenting clinical response to ros1 inhibition . 
liver metastasis response to entrectinib . axial and coronal images at ( a , d ) baseline , ( b , e ) 4 weeks , and ( c , f ) 6 months . the liver metastasis ( red arrow ) was not well visualized on computed tomography images alone but was [ 18f ] fluorodeoxyglucose avid at baseline ( standardized uptake value , 12.02 ) but was not visualized at 4 weeks and remains absent . is clinically relevant only if it reveals a viable therapeutic target . 
liu consulting or advisory role : genentech , boehringer ingelheim , pfizer , ariad pharmaceuticals , celgene , eli lilly , taiho pharmaceutical , bristol - myers squibb , astrazeneca , ignyta , takeda pharmaceuticals research funding : genentech , pfizer , threshold pharmaceuticals , clovis oncology , corvus pharmaceuticals , esanex , bayer healthcare pharmaceuticals , oncomed , ignyta , merck , medimmune , lycera , astrazeneca laura a . 
imran no relationship to disclose jason christiansen employment : ignyta stock and other ownership interests : ignyta edna chow - maneval employment : ignyta zachary hornby employment : ignyta leadership : ignyta stock and other ownership interests : ignyta travel , accommodations , expenses : ignyta pratik s . 
ardini e , menichincheri m , banfi p , et al : entrectinib , a pan - trk , ros1 , and alk inhibitor with activity in multiple molecularly defined cancer indications . 
murphy da , ely ha , shoemaker r , et al : detecting gene rearrangements in patient populations through a 2 - step diagnostic test comprised of rapid ihc enrichment followed by sensitive next - generation sequencing . 
bansal p , osman d , gan gn , et al : recent advances in targetable therapeutics in metastatic non - squamous nsclc . front oncol 6 : 112 , 2016 12 . 
bos m , gardizi m , schildhaus hu , et al : complete metabolic response in a patient with repeatedly relapsed non - small cell lung cancer harboring ros1 gene rearrangement after treatment with crizotinib . 
subbiah v , hong ds , meric - bernstam f : clinical activity of ceritinib in ros1 - rearranged non - small cell lung cancer : bench to bedside report . 
li h , pan y , wang r , et al : response to crizotinib observed in metastatic mediastinum lymph node from a non - small cell lung cancer patient harboring ezr - ros1 fusion . 
kasi , mbbs , md , ms1 ; judong yang , md , ms2 ; phani keerthi surapaneni , mbbs1 ; tanios bekaii - saab , md3 ; daniel h . 
borad , md3 purpose recent advances in molecular diagnostic technologies have allowed for the evaluation of solid tumor malignancies via noninvasive blood sampling , including circulating tumor dna ( ctdna ) proling . 
after excluding variants of unknown signicance , therapeutically relevant alterations were observed in 76 patients ( 55% ) , including braf mutations , erbb2 amplications , fgfr2 fusions , fgfr2 mutations , and idh1 mutations seen in 21% of patients . 
a different spectrum of alterations was observed in patients with early - onset btc ( younger than age 50 years ) compared with older patients ( older than age 50 years )  . conclusion data on ctdna in btc is currently limited . 
intact circulating tumor cells and cell - free dna ( cfdna ) from both leukocytes and tumors ( circulating tumor dna [ ctdna ] ) can now be isolated and analyzed using advanced sequencing methods . 
ctdna has been shown to carry tumor - specic genetic or epigenetic alterations , such as point mutations , copy number variations , chromosomal rearrangements , and dna methylation patterns.1 - 4 the testing approaches in this eld are two dominant polymerase chain reaction ( pcr ) based ( digital pcr ) and next - generation sequencing ( ngs )  . 
ngs approaches have the ability to investigate a larger these number of genes simultaneously ; however , techniques have historically been limited by the need for high sensitivity and specicity and the cost associated with sequencing . 
this approach allows for agnostic analysis of large portions of the genome and can identify multiple mutations with increased sensitivity.5 evaluation of ctdna is particularly attractive as it enables the assessment of patient - specic tumoral genetic / epigenetic alterations while allowing for serial monitoring of tumor genomes in a noninvasive , convenient , and accurate manner . 
 mody et al biliary tract cancers ( btcs ) are an uncommon malignancy arising from epithelial cells that line the biliary tree and have a rapidly fatal course , with 5 - year survival rates of less than 10%.1 , 2 although relatively rare in western countries , btcs are more prevalent in southeast asia.3 in the united states , btcs account for approximately 2% of all new cancer diagnoses and have been rising in incidence during the past few decades.4 - 10 patients with cholangiocarcinoma stand to gain immensely from the use of ctdna given that diagnosis currently is more often made at advanced stages of disease ; recurrences are common , despite the pursuit of potentially curable treatments , such as surgery in a subset of patients ; biopsies are not always obtained or often yield suboptimal quantities of tumor cells for tissue - based genomic proling ; and multiple genomic alterations , which are potential therapeutic targets , are known to occur in btcs . 
genetic alterations that have been identied include activating kras mutations , tp53 mutations / deletions , idh1 and idh2 mutations , fgfr2 gene fusions , cdkn2a / b mutations / deletions , and , less commonly , ntrk gene fusions and alterations in erbb2 ( her2 ) , met , braf , nras , and pik3ca.6 , 7 therefore , the purpose of the current study was to examine the results of ctdna testing from a large cohort of patients with btc at a national cancer institute comprehensive cancer center , acquired in the course of clinical practice , and to characterize the mutational landscape of btcs using this technology . 
data analysis from this patient cohort was reviewed and approved by the mayo clinic institutional review board . demographic information and the date of blood collection were available for all patients . 
additional patient information was collected from the mayo clinic electronic medical record and included conrmation of btc diagnosis ; risk factors , including history of hepatitis a , b , or c , nonviral chronic liver disease , diabetes , obesity , or primary sclerosing cholangitis ; and pertinent clinical information , such as treatment status at the time of ctdna testing and history of liver - directed therapy . comprehensive genomic testing in plasma cfdna was extracted from whole blood collected in 10 - ml streck tubes . 
samples were shipped to a clinical laboratory improvement actcertied , college of american pathologistsaccredited laboratory ( guardant health )  . after double ultracentrifugation , 5 to 30 ng cfdna was isolated for digital sequencing . cfdna fragments , both leukocyte and tumor derived , were simultaneously sequenced . 
analytical sensitivity allowed for the detection of one to two mutant fragments in a 10 - ml blood sample ( 0.1% limit of detection ) with analytic specicity of more than 99.9999%. twelve copy number alterations were reported as the absolute gene copy number in plasma . 
as most cfdna is leukocyte derived , the gene copy number is generally 2.0. tumor - derived dna shed into the bloodstream raises this value , but , because of the relative proportions of tumorderived versus leukocyte - derived cfdna , is typically a minor contributor . 
in the 68 - gene panel , eight genes were retired from the single - nucleotide variation gene set , whereas coverage of gene amplications expanded from three to 16 genes , and the addition of detection of fusions in four genes and insertions or deletion of bases ( indels ) in one gene was made . 
the 70 - gene panel included all national comprehensive cancer network somatic genomic targets , cluding complete or critical exon coverage in 30 and 40 genes , respectively , amplications in 14 genes , fusions in six genes , and indels in three genes . 
the majority of samples in this study ( n = 122 ) were tested using the 73 - gene panel . to determine if an alteration was therapeutically relevant , we dened therapeutically relevant as any gene alteration with an oncokb level of evidence of 1 , 2a , 2b , 3a , 3b , or r1.8 oncokb is a precision oncology knowledge base that contains information about the effects and treatment implications of specic cancer gene alterations.8 oncokb contains detailed information about specic alterations in 477 cancer genes compiled from various sources , cluding guidelines from the us food and drug administration ( fda ) , national comprehensive cancer network , asco , clinicaltrials.gov , and the scientic literature . 
level 4 includes those variants that are considered predictive of response to targeted agents on the basis of compelling biologic , nonclinical evidence . by these standards , a gene is considered therapeutically relevant if there is supporting evidence that the gene is a driver of tumorigenesis , and wherein actionability of the gene can refer to either sensitivity and / or resistance to a drug ( s )  . 
in addition , there must be a clinically available agent , including clinical trials , that targets the gene / protein for a specic agent , and there must be at least preclinical evidence that supports its role in targeting the specic gene / protein . on the basis of the aforementioned criteria , the following genes were considered actionable : akt1 , alk , araf , atm , braf , brca1 , brca1 , ccnd1 , ccnd2 , cdk4 , cdk6 , cdkn2a , ctnnb1 , egfr , erbb2 , esr1 , fgfr1 , fgfr2 , gnas , idh1 , idh2 , jak2 , kras , met , map2k1 , mlh1 , mtor , myc , ntrk1 , ntrk3 , pdgfra , pik3ca , raf1 , ptpn11 , stk11 , tsc1 , and vhl . 
finally , variants found in actionable genes that did not alter the amino acid sequencethat is , synonymous mutationsand / or those that lacked any supporting functional evidence of pathogenicity were excluded . statistical analysis the distribution of each continuous variable is summarized by its mean , standard deviation , and range . 
the relationship between gene types with regard to mutation and amplication , as well as synonymous and targetable status , was evaluated using spearmans rank correlation and was displayed in the correlation matrix in which nonsignicant correlations are marked with a blank in the graph . 
a small subset of patients had underlying chronic liver disease , such as primary sclerosing cholangitis ( 8% ) or other chronic liver disease ( 6% )  . landscape of alterations among a total of 138 ctdna samples , 105 ( 76% ) of these included one or more alterations when variants of unknown signicance were excluded . 
tp53 and kras were the two most common alterations in all subtypes of disease , followed by fgfr2 in 7% ( intrahepatic subtype ) , arid1a ( extrahepatic subtype ) and cdk6 , apc , and smad4 ( gallbladder subtype )  . 
in the more prevalent of the subtypes ( intrahepatic ) in our sampleaside from fgfr2pik3ca and idh1 were fairly commonly altered . a different spectrum of alterations was observed in patients with early - onset btc ( younger than age 50 years ) compared with older patients ( older than age 50 years ) , although overall the differences were not statistically signicant ( p = .55 ; fig 3 )  . 
other alterations which were more common in younger patients included pik3ca , met , and braf . therapeutically relevant alterations after excluding variants of unknown signicance , therapeutically relevant alterations were seen in 55% ( n = 76 ) of the total cohort of samples . 
several risk factors for btc have been described , with most etiologies resulting in long - standing inammation.9 , 12 the only standard systemic therapy for the disease is gemcitabine and cisplatthere is no standard second - line therapy that has been shown to unequivocally improve survival for patients who experience disease progression on gemcitabine / cisplatin.13 btcs are traditionally classied by location as intrahepatic , extrahepatic , and gallbladder on the basis of their presumed site of origin within the biliary duct systethis 4 2019 by american society of clinical oncology mody et al 65 ( 28 - 87 ) 85 ( 69 ) 22 ( 18 ) 16 ( 13 ) 60 ( 48 ) 63 ( 52 ) 42 ( 35 ) 38 ( 30 ) 43 ( 35 ) 21 ( 17 ) 14 ( 12 ) 10 ( 8 ) 2 ( 1 ) 6 ( 5 ) 15 ( 11 ) 30 ( 22 ) 26 ( 19 ) 19 ( 14 ) 48 ( 35 ) anatomic classication has been effective in differentiating btcs with regard to epidemiology , etiology , clinical presentation , and treatment.9 early diagnosis is ideal given that surgical resection or liver transplantationfor perihilar tumorsoffers the best chance at cure ; however , majority of patients are diagnosed with advanced - stage disease precluding surgical management . 
of particular importance , pathologic tissue - based conrmation of btc can be challenging given that clinical yield is often suboptimal and insufcient , especially for dna extraction to enable genomic proling . 
this is primarily a result of the more desmoplastic nature of many btc tumors . with the availability of next - generation genomic proling in recent years , a number of genomic subtypes of btc have been described , many of which are dened by therapeutically targetable genomic alterations.7 , 14 - 18 for example , fgfr fusion rearrangements and idh1 and idh2 mutations have emerged as signicant driver mutation genomic subtypes . 
at present , several targeted drugs are in clinical development , with encouraging results thus far . for the above reasons , the potential for use of ctdna in the management of btc is of particular interest . 
investigations of the use of ctdna in cca have previously been hampered by the rarity of the disease , the relatively incomplete understanding of the genetics of this cancer , and the lack of a validated and widely used ctdna assay . 
this has now changed with recent increased focus on the disease ; recent characterization of the btc genome by several studies , including that from the cancer genome atlas ; and also with the availability of a number of commercially available ctdna assays in clinical practice.14 , 15 , 19 thus far , data on ctdna in cholangiocarcinoma have been sparse . 
andersen and jakobsen20 have demonstrated the early feasibility of a ctdna assay in btc , using a multiplex digital pcr assay to screen for 31 mutations in kras , nras , braf , and pik3ca . 
the authors then performed the assay on serum from ve patients with btc with known tumor mutations and six patients who were known to be wild type for the assayed mutations . 
the assay correctly identied the ve known mutations , whereas none of the six wild - type samples had mutations identied in cfdna . goyal and colleagues21 demonstrated the usefulness of ctdna in serial monitoring of disease and for the detection of resistance mechanisms in the setting of targeted therapy . among 32 patients enrolled in a phase ii study of the fgfr inhibitor bgj398 , nine patients ( 28% ) had fgfr2 fusions detected and four patients were enrolled in the trial . 
pivotal trials exploring inhibitors of fgfr and idh in the management of btc specically are ongoing ( clinicaltrials.gov identiers : nct02924376 , nct03230318 , nct02052778 , and nct02989857 )  . other data have highlighted the application of erbb2 and braf targeted therapies in the disease.22 - 27 for these reasons , it is critically important to identify therapeutically relevant alterations in patients with cholangiocarcinoma . we demonstrate , for the rst time to our knowledge , the ability to detect a broad array of therapeutically relevant alterations using ctdna testing at a signicant rate ( 55% ) in patients with btc . 
whereas the oncokb system is an extensive , comprehensive , and curated precision oncology knowledge base that is capable of supporting precision oncology treatment decisions , should be noted that universally accepted denitions of actionable alterations are not currently available.8 more practically , a total of 21% of patients in our cohort were cohort ; fig 3 . 
 circulating tumor dna in biliary tract cancer identied as having actionable alterations in braf , erbb2 , fgfr2 , and idh1 , for which there are dedicated clinical trials available for patients with btc or for which currently available data exist on the treatment of patients with btc with existing targeted therapies . limitations , our study does have other including the moderate sample size , its retrospective nature , and the fact that patients are derived from a single institution . 
we also have limited clinical data as a result of the retrospective nature of the study . ctdna in btc has been an underexplored area , but our study for the rst time to our knowledge demonstrates the true feasibility of ctdna testing in this disease . 
we believe there is a rationale for ctdna to be incorporated in cholangiocarcinoma trials going forward so as to further explore the technology as a means for identifying patients with actionable genomic alterations , as a biomarker of response , and for serial monitoring of disease and detection of resistance mechanisms , as well as for prognostic purposes . 
borad manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors policy , please refer to or ascopubs.org / po / authorcenter . kabir mody consulting or advisory role : celgene , genentech , merrimack pharmaceuticals , eisai , astrazeneca , vicus therapeutics research funding : fibrogen , senhwa biosciences , ariad pharmaceuticals , tracon pharma , medimmune , agios , arqule , taiho pharmaceutical pashtoon m . 
kasi consulting or advisory role : taiho pharmaceutical ( inst ) , ipsen ( inst ) , bristol - myers squibb ( inst ) , advanced accelerator applications ( inst ) , array biopharma ( inst ) , celgene ( inst ) tanios bekaii - saab consulting or advisory role : amgen ( inst ) , glenmark , ipsen ( inst ) , eli lilly ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) , bayer ( inst ) , celgene ( inst ) , genentech ( inst ) , abbvie , incyte ( inst ) , imugene , immuneering other relationship : exelixis , merck , sillajen , armo biosciences daniel h . 
ahn consulting or advisory role : celltrion ( i ) , merrimack pharmaceuticals , astellas pharma , eisai , cardinal health , paradigm , lexicon amit mahipal research funding : taiho pharmaceutical jason s . 
nat med 20 : 548 - 554 , 2014 sia d , hoshida y , villanueva a , et al : integrative molecular analysis of intrahepatic cholangiocarcinoma reveals 2 classes that have different outcomes . gastroenterology 144 : 829 - 840 , 2013 valle jw , lamarca a , goyal l , et al : new horizons for precision medicine in biliary tract cancers . 
shaib yh , davila ja , mcglynn k , et al : rising incidence of intrahepatic cholangiocarcinoma in the united states : a true increase ? j hepatol 40 : 472 - 477 , 2004 11 . 
chan - on w , kuwahara k , kobayashi n , et al : cholangiocarcinomas associated with long - term inammation express the activation - induced cytidine deaminase and germinal center - associated nuclear protein involved in immunoglobulin v - region diversication . 
j gastrointest oncol 7 : 797 - 803 , 2016 javle m , bekaii - saab t , jain a , et al : biliary cancer : utility of next - generation sequencing for clinical management . 
goyal l , saha sk , liu ly , et al : polyclonal secondary fgfr2 mutations drive acquired resistance to fgfr inhibition in patients with fgfr2 fusion - positive cholangiocarcinoma . 
j clin oncol 33 , 2017 ( suppl ; abstr e15137 ) javle m , churi c , kang hc , et al : her2 / neu - directed therapy for biliary tract cancer . 
kocsis j , aroksz all asi a , andr as c , et al : combined dabrafenib and trametinib treatment in a case of chemotherapy - refractory extrahepatic braf v600e mutant cholangiocarcinoma : dramatic clinical and radiological response with a confusing synchronic new liver lesion . 
loaiza - bonilla a , clayton e , furth e , et al : dramatic response to dabrafenib and trametinib combination in a braf v600e - mutated cholangiocarcinoma : implementation of a molecular tumour board and next - generation sequencing for personalized medicine . 
golan t , raitses - gurevich m , kelley rk , et al : overall survival and clinical characteristics of brca - associated cholangiocarcinoma : a multicenter retrospective a brief review . 
oncogenic met activation results from a hijacking of various aspects of the canonical met pathway otherwise normally active in development and wound healing.2 the canonical pathway consists of hgf ligand - induced dimerization , tyrosine kinase domain activation , and the subsequent activation of a downstream signaling cascade resulting in the met - driven oncophenotype . 
shouldnt a targeted therapy be directed toward a susceptible cancer cell that harbors the target ? but what was the biomarker to reliably signify that a tumor was truly met dependent and would derive substantial benet from anti - met therapy ? the leading biomarker candidate was , and still is , met amplication , which has severalfold higher protein expression as observed by mass spectrometry compared with nonamplied tumors.8 although rare , amounting to only approximately 5% of gea ( the highest incidence of any cancer ) , met amplication has been the most consistent and reliable biomarker both of overall poor prognosis and predictive benet from agents targeting the receptor.3 , 4 , 12 - 14 however , other potential predictive biomarkers have also been proposed , including met tyrosine kinase phosphorylation ( difcult to measure ) , 15 met activating mutation or fusion ( rare ) , met exon 14 skipping or deletion ( mostly lung cancer ) , 16 high hgf tumor and / or serum levels ( not consistently demonstrated ) , and met overexpression ( in the absence of met amplication ) .17 focus on the latter overexpression ensued , likely because this was generally than the more stringent genomic more prevalent denition of amplication , making this more expedient and lucrative to study . 
what exactly is the denition of met overexpression ? met expression by immunohistochemistry ( ihc ) has many descriptions , including cellular distribution ( membranous , cytoplasmic , and / or nuclear staining ) , extensity ( the percentage of tumor cells actually staining ) , and intensity ( the semiquantied staining strength from 0 to 3 + )  . 
 catenacci death ligand 1 ( pd - l1 ) , various alterations in the scoring criteria can change a tumor from met negative to met positive , or vice versa , and dramatically alter positivity incidence with slight scoring modications . 
benet in the met - high population ( 64% incidence in that study ) hinged on a small retrospective subgroup of 47 and 21 patients receiving rilotumumab and placebo , respectively.18 unfortunately , the competition to be rst to market and , therefore , the rush to initiate phase iii studies with rilotumumab in rilomet - 1 ( international phase iii multicenter , randomized , double - blind , placebocontrolled trial of rilotumumab plus epirubicin , cisplatin , and capecitabine as first - line therapy in patients with advanced met - positive gastric or gastroesophageal junction adenocarcinoma ) and onartuzumab in metgastric ( fluorouracil , leucovorin , and oxaliplatin with or without onartuzumab in her2 - negative , met - positive gastroesophageal adenocarcinoma ) , without more attention to these issues , resulted in a fundamental lack of biomarker understanding . 
because the questions were unasked , the answers were unknown . importantly , thus , the market - based engines steamed on ahead with the following two bits of data : one patient who responded to an lbab monotherapy5 and one retrospective small subgroup analysis of a phase ii randomized study.18 unfortunately , ultimately both lbab phase iii studies were negative.10 , 11 regrettably , neither study successfully enriched for a population who would benet from met lbabs . 
notably , for the rilotumumab studies , the initial antibody prototype assay and , the actual scoring system used in the phase ii study were changed for the prospective phase iii study to ofcially codevelop a companion diagnostic assay . the assay in rilomet - 1 likely inadequately selected any optimal patient population for treatment , with most patients ( 81% ) screened considered positive for met expression using the lax criteria of greater than or equal to 1 + intensity at 25% or greater extensity staining ( hardly selecting ) and most patients having low - level expression.11 prospectively testing formalin - xed parafn - embedded ( ffpe ) freshly cut samples from recently diagnosed patients in rilomet - 1 compared with a retrospective batched archival ffpe analysis in the phase ii study may also have contributed to the incidence discrepancy between the studies , because ihc sensitivity is decreased with older ffpe samples and , when still positive in light of this , would represent those indeed , true benet randomized phase ii samples likely having the highest levels of expression to begin with . 
conversely , the metgastric study had only the slightly more stringent criteria of positivity for eligibility of greater than or equal to 1 + intensity at 50% or greater extensity staining ( ie , more cells needed to be expressing at least low - level amounts of protein )  . 
notably , in this latter underpowered subgroup analysis , the hr for overall survival was 0.64 in favor of onartuzumab ( p = .062 ) , and the median survival increased from 9.7 months to 11 months with onartuzumab.10 could this have been statistically signicant with the originally intended number of patients ? because the question was unasked ( study terminated early ) , the answer is unknown . 
in the end , the optimal met biomarker assay , scoring , and positivity criteria of expression for lbabs , if any , remain undened . nevertheless , even if we could rewind and do things differently with this hindsight ( could we perform prospective , adequately accrued , randomized phase ii and / or iii studies with only the highest ihc criteria amounting to approximately 20% to 30% of all patients with gea ? ) , the marginal absolute benets that could be realistically achieved with lbab in an optimal expressing subgroup , as exemplied by the metgastric subgroup analysis hr of 0.64 , lend pause to question whether this would really provide signicant clinical improvement . 
yet , frustratingly , met amplication , now serving as a driver oncogene in this scenario , is the biomarker subset to likely derive the most benet from targeted met receptor inhibitors . 
in comparison , pd - l1 checkpoint blockade monotherapies , despite the hysteria , ultimately demonstrated an approximate 10% response rate in the third - line or higher setting in gea all - comers , with a median progression - free survival time of 2 months , and only a 13.3% response rate ( 19 of 143 patients ) in the subset of pd - l1positive , microsatellite stable ( mss ) selected patients , with a median dor of less than 8 months ( only ve patients , or ve [ 28% ] of 19 responders or ve [ 3% ] of 143 pd - l1positive / mss patients , had dor greater than 12 months ) .25 yet on the basis of these results , pembrolizumab was recently successfully pursued for accelerated approval for the entire pd - l1positive / mss subset by the us food and drug administration.25 , 26 similar clinical outcomes are consistently reported in patients with met - amplied gea ( with comparable incidence of responses lasting greater than 12 months )  . 
in the face of being an uncommon genomic event , this discussion still matters for patients with gea with tumors harboring met amplication . this amg337 example , along with other met inhibitors , has well - elucidated reasons for perceived failure . 
these include underappreciating the general poor prognosis of met - amplied tumors in single - arm , late - line studies , intrapatient heterogeneity with regions harboring and not harboring met amplication , and the presence of concurrent genomic aberrations in some met - amplied cells . all of these factors could lead to relatively quick progression on monotherapy either outright or shortly after an initial response in most , but importantly not all , responding patients . 
however , the ideal randomized and combination therapy studies have been elusive to date because of the infeasibility of classic clinical study designs and the requirement of extraordinarily high numbers of patients screened to identify the met - amplied subset , with the consequent abandonment of additional investigation by pharma because of this apparently high - risk and low - yield scenario . 
with met , supportive evidence abounds that there are patients who derive benet from met inhibitors , particularly those with met - amplied tumors , yet to date , no met - specic inhibitor is approved by the us food and drug administration in any met - specic indication for any tumor . 
lack of progress can be attributed to a number of issues including inadequate validation of biomarker cut - offs , along with the inherent hurdles to developing drugs in lowincidence biomarker populations , coupled with complex intrapatient heterogeneous biology rendering monotherapy in late - line therapy less likely to impress , 34 and certainly so for accelerated approval paths . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . daniel catenacci honoraria : genentech , eli lilly , amgen , oncoplex diagnostics , foundation medicine , taiho pharmaceutical , genmab , nantomics , guardant health , merck , bristol - myers squibb , gritstone oncology , five prime therapeutics , astellas pharma consulting or advisory role : genentech , amgen , merck , eli lilly , taiho pharmaceutical , bristol - myers squibb , astellas pharma speakers ' bureau : guardant health , foundation medicine , genentech , eli lilly , merck references giordano s , di renzo mf , ferracini r , et al : p145 , a protein with associated tyrosine kinase activity in a human gastric carcinoma cell line . 
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hong ds , lorusso pm , hamid o , et al : phase 1 study of amg 337 , a highly selective small - molecule met inhibitor , in patients with advanced solid tumors . 
van cutsem e , karaszewska b , kang yk , et al : a multicenter phase 2 study of amg 337 in patients with met - amplied gastric / gastroesophageal junction / esophageal adenocarcinoma and other solid tumors . 
fuchs cs , doi t , jang rw , et al : safety and efcacy of pembrolizumab monotherapy in patients with previously treated advanced gastric and gastroesophageal junction cancer : phase 2 clinical keynote - 059 trial . 
catenacci dvt , park h , uronis he , et al : margetuximab ( m ) plus pembrolizumab ( p ) in erbb2 - amplied pd - l1 + gastroesophageal adenocarcinoma ( gea ) post trastuzumab ( t )  . 
lancet oncol 16 : 1276 - 1278 , 2015 joshi ss , maron sb , lomnicki s , et al : personalized antibodies for gastroesophageal adenocarcinoma ( pangea ) : a phase ii precision medicine trial ( nct02213289 )  . 
j natl cancer inst 97 : 249 - 250 , 2005 jardim dl , groves es , breitfeld pp , et al : factors associated with failure of oncology drugs in late - stage clinical development : a systematic review . 
hamid o , gajewski tf , frankel ae , et al : epacadostat plus pembrolizumab in patients with advanced melanoma : phase 1 and 2 efcacy and safety results from echo - 202 / keynote - 037 . 
long gv , dummer r , hamid o , et al : epacadostat ( e ) plus pembrolizumab ( p ) versus pembrolizumab alone in patients ( pts ) with unresectable or metastatic melanoma : results of the phase 3 echo - 301 / keynote - 252 study . 
gene amplication , or increased gene dosage , leads to increased messenger rna expression , which leads to exponential increase in the amount of met protein receptor ( green ) expression at the membrane . 
most patients present with either metastatic pdac ( 50% - 60% ) or locally advanced tumors ( 30% - 40% ) , for which the median survival is 5 - 9 months after diagnosis.1 , 2 outcomes are still suboptimal for the small subset ( 10%20% ) of patients who present with resectable tumors conned to the pancreas ; , 50% of these patients survive 5 years after surgery despite modern adjuvant chemotherapy.3 moreover , at autopsy , the disease is metastatic for 88% of recurrences , and  . 
80% have more than 10 distinct metastatic lesions4 that are genetically related to the primary tumor5 and other metastases.6 these observations indicate that majority of patients with pdac harbor nonradiographically evident , micrometastatic disease after resection and that remnant tumor cells evadeor acquire resistance toadjuvant chemotherapy . 
although tropism to specic organs during metastatic spread is still poorly understood , patients with recurrence in the liver or peritoneum7 survive signicantly shorter than patients with recurrent lung metastases.8 - 10 pdac tumors are commonly classied into two major subtypes : one that shares some features with adenosquamous tumors ( squamoid and / or basal ) and the other that retains a differentiated , glandular and / or ductal morphology ( ductal and / or classical )  . 
squamoid pdac tumors are more glycolytic , 11 , 12 more hypoxic , 13 more inammatory likely broblasts , 16 and yield a poorer prognosis than ductal tumors.17 - 21 classical subtype tumors are associated with more tumor - inltrating leukocytes , denser collagen , and better outcomes , 15 , 22 although signicant subtype heterogeneity within tumors13 complicates the relationship between subtype and outcome . to metastasize , 14 , 15 recruit more cytolytic t cells inltrate pdac tumors than most other tumors , 23 but they are ultimately insufcient to control the cancer . 
density of tumor - inltrating t cells is highly variable among pdac tumors , 24 and abundant cd8 + t cells ( along with a high number of neoantigens ) can support exceptionally long - term survival.25 however , t cellmediated tumor immunity may be restricted by several mechanisms : tumor immunosuppressive cell downregulation of hla , leukocytes , t cell proximity to tumor cells , and t cell exhaustion.26 - 30 effective t cellmediated tumor immunity is enhanced by a high diversity of functional t cell clones , tumor - specic recruitment of effector t cells , and abundant neoantigens.23 , 25 , 31 quantifying the phenotypes , functions , and locations of leukocytes is critical to identify specic tumor immunity required for exceptional positive outcomes . rare , exceptional control of pdac progression may be mediated by equally rare , idiosyncratic patient genetics , tumor - specic somatic alterations and gene expression , and / or stochastically generated tumor immunity . 
in this report , we present the cases of pt1 , who survived more than 46 months with occult , chemotherapy - resistant metastatic pdac , and her niece ( pt2 ) who succumbed to progressing metastatic disease despite aggressive treatment . 
however , during adjuvant chemotherapy , the lung lesion increased to 23 mm 18 mm , and a ct and / or pet was suspicious for malignancy with a standardized uptake value ( suv ) of 4.5. 
ca19 - 9 test results are not shown becausefor both patientsthey were reproducibly approximately 90% lower than the 37 u / ml threshold for normal , even when the primary tumor was present . 
pt1 chose to receive no further treatment and was radiographically disease - free for over 2 years ( fig 1 ) until an isolated , recurrent lung metastasis was identied by ct and fdg - pet ( suv of 9 ) and then treated by 50 gy in ve fractions using stereotactic body radiotherapy . 
pt2 died 19 months after metastatic disease recurrence . treatment a shared germline prss1 mutation and somatic alterations common to pdac we identied germline prss1 p.a16v in both patients and somatic alterations in the two most common pdac driver geneskras and tp53in both tumors from each patient ( table 1 )  . 
each scale bar is 0.5 m ( c ) pdac subtyping of each patients primary and metastatic tumors . left panel : spearman correlation coefcients for tumors from pt1 and pt2 compared with the published rank order of pdassigner genes for classical ( y - axis ) and quasimesenchymal ( x - axis ) tumors . 
both tumors from pt1 aligned well with the classical subtype , whereas the tumors from pt2 were more quasimesenchymal and / or basal with the metastasis scoring more basal than the primary tumor . leukocyte types associated with immunity in tumors from patient 1 we used a multiplex immunohistochemistry ( mihc ) workow34 to quantify the densities of tumor cells , broblasts , and 10 leukocyte subsets ( appendix table a1 )  . 
we selected regions of interest ( rois ) within each section that contained both krt + epithelial or tumor cells and cd45 + immune cells ( appendix fig a1 ) , followed by quantitative assessment of each roi . 
a detailed analysis of leukocyte subsets revealed that cd8 + t cells and cd4 + t cells were signicantly denser in the metastasis from pt1 compared with the primary tumor from pt2 ( fig 3b )  . 
 ( a ) mean cell density per roi for mutually exclusive populations of ki67 + tumor or epithelial cells , ki67neg tumor or epithelial cells , broblasts , and leukocytes . 
among subsets of cd4 + t cells , we found more pro - inammatory th1 t cells in the metastasis from pt1 than either primary tumor ( fig 3c )  . 
within cd4 + t cells , we found a greater density of both cytolytic granzyme b + ( grzb ) cells and proliferative ki67 + cells in the metastasis from pt1 than the primary tumor from pt2 and a higher density of proliferative ki67 + cells in pt1s primary tumor compared with pt2s primary tumor ( fig 3d )  . consistent with the abundant th1 - like macrophages in pt1s primary tumor , we found that the ratio of th1 - like to th2 - like macrophages was signicantly greater in pt1s primary tumor relative to the other 2 tumors ( fig 3e )  . 
a high granulocyte / neutrophil to lymphocyte ratio is associated with poor outcomes.35 , 36 consistent with this , the primary tumor from pt2 had a signicantly higher granulocyte to cd8 + t cell ratio than either tumor from pt1 ( fig 3e )  . 
in addition , across rois , there was no correlation between the densities of cd8 + t cells and granulocytes in the tumors from pt1 , but there was a signicant positive correlation in pt2s primary tumor ( p , .03 , fig 4a )  . 
furthermore , the abundant granulocytes in pt2s tumor were proximal to tumor cells and appeared within lumens ( fig 4b ) where they may contribute to pathogenesis by occluding ducts.37 we also observed tertiary lymph structures ( tlss ) at the border of both primary tumors , but these were more prevalent in the primary tumor from pt1 ( appendix fig a1 )  . 
tlss from pt1 also contained signicantly more b cells , cd4 + t cells , and cd8 + t cells ( appendix fig a2 ) as previously reported for patients with relatively positive outcomes.24 , 38 tissue acquisition and patient consent human tissues were obtained with informed consent in accordance with the declaration of helsinki and were acquired through the oregon pancreas tissue registry under oregon health & science university irb protocol #3609 . discussion we investigated multiple modalities and tumor characteristics of pdac between two related patients . 
we found several similarities between both patients cancers , cluding genes commonly altered in aggressive pdac ( kras , tp53 , and smad439 , 40 ) and a shared prss1 a16v germline mutation that may predispose to pancreatitis41 - 44 but is variably penetrant , 45 raising the possibility that this specic alteration leads to subclinical pancreatitis that accelerates tumorigenesis , consistent with pt2s early onset of disease.41 , 46 the disease course for pt1 was exceptional in the context of occult , chemotherapy - resistant metastatic disease for nearly 4 years . 
an fanca mutation in the metastasis from pt1 might have sensitized that tumor to adjuvant gemcitabine and xeloda and later to sbrt ; however , pt1s lung metastasis progressed during adjuvant chemotherapy , and no adjuvant treatment was given after surgical resection of the lung metastasis , suggesting that the indolent disease might have also been controlled by other factors . 
both tumors from pt2 contained an alteration in cdkn2a , and the metastasis from pt2 had a copy number loss of pten ; alterations in these genes are associated with poor outcomes and drug resistance.47 - 49 pt1s classical subtype tumors and metastases limited to the lungs are both favorable prognostic factors . 
specically , more prevalent tlss at the periphery of the primary tumor ( appendix fig a1 ) and denser cd8 + t cells ( fig 3b ) that did not correlate with the density of granulocytes24 , 50 ( fig 4a ) , opening the possibility that tumor cells and / or cd4 + t cells in pt1s tumors recruit fundamentally different leukocyte cell types from pt2s tumors.51 additionally , granulocytes typically promote immunosuppression in late - stage tumors , 52 consistent with the relatively low number of t cells in pt2s tumor . classical subtype pdac with lung metastases and unconventional tumor immunity may lead to exceptional outcomes . 
sears , phd , brenden - colson center for pancreatic care , oregon health and science university , 2730 s moody ave , portland , or 97201 ; e - mail : searsr@ohsu.edu. support supported by the brenden - colson foundation and nci grant u01 ca224012 to r.c. 
coussens employment : oregon health &amp ; science university ( ohsu ) honoraria : prospect creek foundation , lustgarten foundation for pancreatic cancer research , syndax pharmaceuticals , inc : external advisory board , carisma therapeutics inc : scientic advisory board , verseau therapeutics , inc , scientic advisory board , zymeworks , inc , scientic advisory board , cytomx therapeutics , inc , kineta inc , ( p30 ) koch institute for integrated cancer research , massachusetts inst . 
of tech , ( p30 ) salk institute cancer center , bloomberg - kimmel institute for cancer immunotherapy , sidney kimmel comprehensive cancer center at johns hopkins , dana farber cancer center breast spore , ( p30 ) dana farber / harvard cancer center , ( p30 ) university of california , san diego moores cancer center , starr cancer consortium , lustgarten foundation for pancreatic cancer research , therapeutics working group , nih / nci - frederick national laboratory advisory committee ( fnlac ) , susan g komen foundation , komen scholar consulting or advisory role : cell signaling technologies , pharmacyclics , cytomx therapeutics , syndax , carisma therapeutics , verseau therapeutics , zymeworks , kineta , inc , abbvie , shasqi inc research funding : syndax pharmaceuticals inc , pharmacyclics , cell signaling technologies , innate pharma , deciphera travel , accommodations , expenses : cell signaling technologies , astrazeneca , pharmacyclics , verseau , carisma therapeutics , cytomx therapeutics , zymeworks other relationship : prospect creek foundation , lustgarten foundation for pancreatic cancer research , ( p30 ) koch institute for integrated cancer research , massachusetts inst . 
n engl j med 379 : 2395 - 2406 , 2018 iacobuzio - donahue ca , fu b , yachida s , et al : dpc4 gene status of the primary carcinoma correlates with patterns of failure in patients with pancreatic cancer . j clin oncol 27 : 1806 - 1813 , 2009 yachida s , jones s , bozic i , et al : distant metastasis occurs late during the genetic evolution of pancreatic cancer . 
makohon - moore ap , zhang m , reiter jg , et al : limited heterogeneity of known driver gene mutations among the metastases of individual patients with pancreatic cancer . 
nat genet 49 : 358 - 366 , 2017 katz mhg , wang h , fleming jb , et al : long - term survival after multidisciplinary management of resected pancreatic adenocarcinoma . 
ann surg oncol 16 : 836 - 847 , 2009 downs - canner s , zenati m , boone ba , et al : the indolent nature of pulmonary metastases from ductal adenocarcinoma of the pancreas . 
j surg oncol 112 : 80 - 85 , 2015 deeb a , haque su , olowokure o : pulmonary metastases in pancreatic cancer , is there a survival inuence ? j gastrointest oncol 6 : e48 - e51 , 2015 10 . 
rashid nu , peng xl , jin c , et al : purity independent subtyping of tumors ( purist ) , a clinically robust , single - sample classier for tumor subtyping in pancreatic 34 . 
zhou y , wei q , fan j , et al : prognostic role of the neutrophil - to - lymphocyte ratio in pancreatic cancer : a meta - analysis containing 8252 patients . 
giakoustidis a , neofytou k , costa neves m , et al : identifying the role of neutrophil - to - lymphocyte ratio and platelets - to - lymphocyte ratio as prognostic markers in patients undergoing resection of pancreatic ductal adenocarcinoma . 
herman jm , jabbour sk , lin sh , et al : smad4 loss correlates with higher rates of local and distant failure in pancreatic adenocarcinoma patients receiving adjuvant chemoradiation . 
schubert s , traub f , brakensiek k , et al : cftr , spink1 , prss1 , and ctrc mutations are not associated with pancreatic cancer in german patients . 
mcwilliams rr , maisonneuve p , bamlet wr , et al : risk factors for early - onset and very - early - onset pancreatic adenocarcinoma : a pancreatic cancer casecontrol consortium ( panc4 ) analysis . 
kim m , shah a , zhang j , et al : gemcitabine resistance in cultured pancreatic tumor cells is associated with down regulation of pten and a mesenchymal phenotype . 
ferrer m , de winter jp , mastenbroek dc , et al : chemosensitizing tumor cells by targeting the fanconi anemia pathway with an adenovirus overexpressing dominant - negative fanca . 
ye k , schulz mh , long q , et al : pindel : a pattern growth approach to detect break points of large deletions and medium sized insertions from paired - end short reads . 
exome sequencing libraries were prepared with a kapa hyper prep kit , enriched using the agilent sureselectxt clinical research exome , and paired - end sequenced with an illumina hiseq 2500 to a high depth of 300  . 
rna sequencing was performed as previously reported.62 briey , rna was extracted using an rneasy kit ( qiagen ) and evaluated by the ohsu massively parallel sequencing shared resource ( mpssr ) core facility for library construction with the truseq rna access library prep kit ( illumina )  . 
30% as measured using an agilent 2100 bioanalyzer . rna was sequenced on an illumina hiseq 2500 / 4000 to generate 100 bp paired - end reads for a minimum depth of 40 million reads per sample . 
we followed the published protocol for purist subtyping33 and used the same data set as for pdassigner subtyping . tissue processing and immunostaining tumor tissue samples were xed in 10% neutral buffered formalin for 24 hours and embedded in parafn ( ffpe )  . 
ffpe sections were stained with hematoxylin and eosin , and krt5 was detected with a rabbit monoclonal antibody ( ep1601y , abcam ) and polyclonal goat anti - rabbit antibodies ( invitrogen )  . 
exposure times and signal thresholds were normalized across all images . multiplex immunohistochemistry , image acquisition , and processing multiplex ihc was performed on 5 m ffpe sections using an adapted protocol based on methodology we previously described.34 briey , slides were deparafnized and stained with hematoxylin ( s3301 , dako , santa clara , ca ) , followed by whole slide scanning at 20 magnication on an aperio at2 ( leica biosystems , wetzlar , germany )  . 
slides were digitally scanned following each chromogen development . scanned images were registered in matlab version r2018b using the surf algorithm in the computer vision toolbox ( the mathworks , inc , natick , ma )  . 
image processing and cell quantication were performed using fiji ( fiji is just imagej ) cellproler version 3.5.164 and fcs express 6 image cytometry ruo ( de novo software , glendale , ca )  . aec signal was extracted for quantication and visualization in fiji using a custom macro for color deconvolution . 
 ( a ) krt ( magenta ) and cd45 ( teal ) expression and selected rois ( yellow rectangles ) used for mihc analyses of tumors ( scale bars represent 5 mm )  . 
 predisposing germline mutations in young patients with squamous cell cancer of the oral cavity introduction squamous cell carcinoma ( scc ) of the oral cavity ( oscc ) , and especially scc of the oral tongue ( otscc ) , is a malignancy associated with poor survival rates , with established risk factors considered to include advanced age , male gender , human papillomavirus ( hpv ) infection , and tobacco and alcohol abuse.1 , 2 scc of the oral cavity has been described as part of the phenotypic spectrum of rare bone marrow failure syndromes , that is , dyskeratosis congenita ( dc ) and fanconi anemia ( fa )  . 
 in particular , among patients with fa carrying biallelic mutations in a number of genes , as well as among patients with dc carrying mutations in genes that are involved in telomere maintenance , head and neck scc ( hnscc ) is the most frequent solid tumor diagnosed.3 , 4 patients with fa , in particular , face a 100 - fold risk for scc , compared with the general population.5 , 6 although there are few reports describing familial aggregation and patients with hereditary oscc , the actual genetic predisposition of oscc has never been thoroughly assessed , especially in individuals who lack the traditional risk factors . 
 considering that early age at cancer diagnosis is a hallmark of genetic predisposition in most cancer types , we sought to elucidate the possible germline gene defects by implementation of a comprehensive cancer gene panel in a highly selected cohort of young patients diagnosed with oscc . 
the yocc study was approved by the review board of attikon university hospital and evangelismos general hospital , and the bioethics committee of the national center for scientific research demokritos in compliance with the 1975 helsinki declaration . 
patient clinical characteristics and survival status are listed in table 1 . genetic analysis genomic dna was used to produce indexed libraries to target the sequence of 94 cancer predisposing genes using the trusight cancer panel ( illumina , san diego , ca ) following manufacturers instructions . 
odds ratios were calculated using fishers exact test . results prevalence of germline loss - of - function mutations germline loss - of - function mutations in three genes , namely , cdkn2a , sdhb , and recql4 , were identified in 13.3% ( four of 30 ) of the individuals tested . 
patients were followed for a median of 40.1 months , with one third of them ( 10 of 30 ) having died or being diagnosed with a relapse . cdkn2a mutations were the most prevalent because they were detected in two nonrelated patients . 
moreover , sdhb protein expression was retained on the tumor sample of patient ot - 2 after immunohistochemistry staining , also indicating that sdhb mutation was not directly related to his cancer diagnosis . associations between detected loss - of - function alleles per gene were compared with the exac data , as already described ( results are listed in table 3 )  . 
 although our results reached statistical significance , the cis were quite wide , mainly because of a small number of patients in our study ; therefore , these associations should be considered with caution . 
its design aimed to look beyond the usual risk factors for oscc , thus investigating a selected cohort composed of young , non alcohol drinking , nonhpv - infected patients with otscc . 
approximately , one in seven patients in our cohort carried a loss - of - function mutation in genes ( cdkn2a , sdhb , and recql4 ) known to be associated with various forms of cancer , the most prevalent being cdkn2a mutations . 
although not surprising , because none of the individuals included in the study had a clinical diagnosis of fa and therefore a prior diepoxybutane induced chromosome breakage test had not been ordered , it is possible that monoallelic mutations in the fa - associated genes are not associated with higher head and neck cancer incidence . individuals with sdhb mutations are known to face a lifetime risk of developing paragangliomas reaching 70% by the age of 80 years , 9 whereas they develop renal cell carcinomas , gastrointestinal stromal tumors , and pheochromocytomas.10 however , biallelic recql4 mutations cause rothmund - thomson syndrome , mainly characterized by poikiloderma , skeletal abnormalities , and increased risk of osteosarcoma.11 to our knowledge , mutations in these genes have never been reported before in patients with oscc and of course it is likely that these findings are purely incidental . 
although a direct association with tumorigenesis cannot be made , the genomic instability caused by the malfunction of these genes can be considered of importance . germline cdkn2a mutation carriers are known to have an elevated lifetime risk of melanoma and pancreatic cancer.12 although there have been previous case reports describing individuals diagnosed with hnscc in families carrying cdkn2a mutations , 13 a clear association , followed by clinical guidelines , has never been made . 
the high rate of somatic cdkn2a mutations and the high frequency of cdkn2a disruption detected in hnscc , 14 , 15 along with its critical role in tumor suppression , further support a potential role in hnscc initiation and development , while highlighting cdkn2a as an ideal candidate for hnscc susceptibility . 
interestingly , none of the cdkn2a carriers identified in this study seem to have a local or distant recurrence after approximately 5 and approximately 8 years of follow - up . 
there is a definite association of the initial tumor staging ( both t1n0 ) with improved survival , but an association of mutation status can also be assumed , which should be investigated further . the main limitation of our study lies in the small number of patients . 
furthermore , the lack of evaluation of genes known to be associated with dc , which might be of importance in this cohort of highly selected patients , is also considered a limiting factor . this study suggests a possible genetic predisposition in a small but notable fraction of young , apparently sporadic , oral patients with scc . 
 although , through the findings of this study , cdkn2a is proposed as a candidate gene , further studies are essential to establish its possible causative role and its inclusion in genetic analysis , whereas larger - scale analyses are imperative for defining the susceptibility gene spectra for oral scc . 
patel sc , carpenter wr , tyree s , et al : increasing incidence of oral tongue squamous cell carcinoma in young white women , age 18 to 44 years . 
goldstein am , chan m , harland m , et al : high - risk melanoma susceptibility genes and pancreatic cancer , neural system tumors , and uveal melanoma across genomel . 
ricketts cj , forman jr , rattenberry e , et al : tumor risks and genotype - phenotype - proteotype analysis in 358 patients with germline mutations in sdhb and sdhd . 
gaal j , stratakis ca , carney ja , et al : sdhb immunohistochemistry : a useful tool in the diagnosis of carney - stratakis and carney triad gastrointestinal stromal tumors . 
reed al , califano j , cairns p , et al : high frequency of p16 ( cdkn2 / mts - 1 / ink4a ) inactivation in head and neck squamous cell carcinoma . 
hoang , md1 , 2 ; rui yang , md1 , 2 ; daniel weiser , md2 ; jonathan morris , md3 ; richard gorlick , md4 ; jonathan b . 
we have identied a single patient who was initially diagnosed with a benign osteoblastoma and subsequently presented years later with a high - grade osteosarcoma in the same anatomic region . 
a biopsy sample of the mass demonstrated conventional - type osteoblastoma with focal epithelioid features for which the patient underwent intralesional curettage ( fig 2 )  . subsequent imaging demonstrated a persistent abnormal signal in the sacrum , and a plan of continued surveillance was agreed upon . 
over the ensuing 4 - year imaging failed to demonstrate appreciable period , radiographic change , and the patients clinical course remained stable . five years later , the patient returned with increasing paimaging demonstrated an increase in the size of the mass , and an urgent open sacral biopsy was performed ( fig 3 )  . 
staging studies performed at that time were negative for distant disease . neoadjuvant chemotherapy was started in accordance with childrens oncology group protocol aost0331 ; however , because of severe pain , patient required semi - urgent surgical intervention and did not complete planned neoadjuvant treatment . 
the decision was made to manage conservatively , and as of july 2019 , surveillance demonstrated no evidence of disease . methods including the osteoblastoma , patient consent and institutional review board approval were obtained before study initiation . 
sequencing and data analysis were performed by novogene technology ( beijing , china ) to evaluate for structural variants , copy number variation , single nucleotide variants , and insertions / deletions . 
germline - matched comparison from each tumor was undertaken to evaluate for somatic changes . to evaluate for pathogenic variants , somatic analysis on small variants focused on changes that would elicit a coding or splice site change and were not common polymorphisms ( allele frequency , 0.01 in 1 , 000 genomes and exac databases )  . 
furthermore , we evaluated whether any variants were found in cancer mutational hotspots ( recurrence of more than ve in cosmic [ catalog of somatic mutations in cancer ] ) or were a truncating variant in a known tumor suppressor gene ( cancer gene census )  . 
 geller et al context key objective does genomic analysis of a patient who was initially diagnosed with a benign osteoblastoma and subsequently presented years later with a high - grade osteosarcoma in the same anatomic region demonstrate evidence of malignant transformation ? knowledge generated there was near - zero overlap in the somatic small variants , somatic copy number variation pattern , and predicted structural variants in the osteoblastoma compared with the osteosarcoma . 
findings from this study argue against malignant transformation as an evolving or stepwise process and conversely support two distinct neoplasms with dissimilar genetic makeups . relevance this study performed an in - depth genetic characterization of two distinct tumors that historically have been believed to be along the same spectrum of disease . 
in addition , a germline mutation in brip1 was discovered , which lends itself to additional investigation . pathogenic or likely pathogenic for a cancer predisposition syndrome . results whole - genome sequencing resulted in high - quality data , which generated an average 63 million reads per tumor , with more than 98% usable on the basis of quality metrics . 
after ltering and duplicate removal , mapping to hg19 resulted in an average sequencing depth of 29 and 32 for the benign and malignant tumors , respectively , and at least 4 coverage in more than 99% of the genome . somatic variant analysis revealed approximately 98 , 000 and 141 , 000 high - condence small variants in the benign and malignant tumor , respectively , which corresponded to somatic mutational burdens of 33 and 47 mutations / megabase ( mb )  . 
in the osteosarcoma , a single variant recurrent in cosmic of unclear oncologic signicance , znf429 p.n426k , was found as well as truncating mutations in two tumor suppressor genes , daxx and ncor1 . structural and copy number variation analysis revealed signicant alterations in both tumors . 
a striking number of somatic inversion events were found in both tumors ( approximately 1 , 750 per tumor ) , particularly compared with other structural changes , such as translocations , deletions , or tandem duplications ( each approximately 10 to 30 per tumor )  . 
 ( a ) a 2015 hematoxylin and eosinstained low - power ( 10 ) micrograph demonstrates ndings consistent with high - grade osteosarcoma , including high cellularity , multiple foci of tumor necrosis , and osteoid formation . 
 ( b ) a 2015 hematoxylin and eosinstained high - power ( 40 ) micrograph that demonstrates pleiomorphic and bizarre cellular features . overlap in the predicted structural variants in the osteoblastoma compared with the osteosarcoma . germline analysis using the methods described identied a single pathogenic germline mutation , brip1 r798x , identied with high condence in all three samples . 
a summary of genomic ndings is listed in table 1 . discussion the notion that osteoblastoma can undergo malignant transformation to osteosarcoma dates back to an original report in 1967.1 since then , 24 cases have appeared in the literature , none of which investigated the transformation event on a genomic level1 - 20 ( table 2 )  . 
given the lack of objective genetic data and short time to transformation in some cases , there is a distinct possibility that some of these reports represent coincidence or diagnostic error . a few prior reports have attempted to document malignant transformation through a limited analysis of sequential biopsies samples.4 , 18 , 21 however , these tumors were characterized only in terms of ploidy , thereby limiting understanding of the transformation process.4 , 18 , 21 although these reports suggested that transformation is associated with an increase in dna content , only one patients tumor showed an observable change in dna content over time . moreover , in the current study , we have demonstrated the benign and malignant tumors to have distinct genomic proles with almost no overlap in somatic changes . 
whether these tumors are driven in part by an unrecognized anatomic predisposition remains speculative . we also identied a germline mutation in the brip1 tumor suppressor gene , a member of the recq helicase family associated with brca1.22 , 23 brip1 mutations are associated with a predisposition to breast cancer , ovarian cancer , and fanconi anemia , 22 , 24 , 25 and brip1 is known to play a critical role in dna repair , with knockdown leading to chromosomal instability.26 of note in our patient , both the benign and the malignant tumors exhibited a higher - thantypical somatic mutation rate compared with most pediatric malignancies27 as well as a high number of structural variants with a preponderance of inversion events , which suggests a functional role of this germline nding . 
we have speculated that these changes may have accumulated because of brip1 partial dysfunction that led to chromoinstability and impaired dna crosslink repair.26 somal admittedly , denitive conclusions cannot be drawn on the basis of a single patient , and additional investigation seems warranted . although osteoblastoma infrequently has been associated with mutations involving rb , tp53 , fos , fosb , d - jun , and mdm2 , the genetic landscape of this tumor is still largely unknown.28 - 31 the osteoblastoma in this report is similar to previous reports in that we observed many structural table 1 . 
many osteosarcomas demonstrate chromosomal abnormalities that commonly involve tumor suppressor genes or dna helicases.32 , 33 however , osteosarcoma generally is recognized as exhibiting tremendous genetic complexity with no clearly identiable genetic driver . this heterogeneity may stem from chromothripsis , 34 - 36 markers of which have been described in 2% to 3% of all cancers and up to 33% of osteosarcomas . 
although the current case did not exhibit evidence of chromothripsis , it demonstrates an otherwise typical genomic prole for osteosarcoma with a high degree of chromosomal instability , increased ploidy , and truncating point mutations in several tumor suppressor genes . in some cases , in conclusion , although malignant transformation of osteoblastoma to osteosarcoma historically has been accepted , review of the literature has revealed a paucity of convincing evidence , and controversy has persisted . 
to our knowledge , this study is the rst to report an in - depth genetic characterization of two distinct tumors within the same patient and the same anatomic location . 
findings from this study argue against malignant transformation as an evolving or stepwise process and conversely supports two distinct neoplasms with dissimilar genetic makeups . this report also has uncovered a germline truncating mutation in brip1 in this patient , which raises the question of whether this serves as an underlying predisposition for both tumors . 
geller , md , monteore greene medical arts pavilion , 3400 bainbridge ave , bronx , ny 10467 ; e - mail : dgeller@monteore.org. support supported by clinical and translational science awards catalytic seed grant ul1 tr001073 ( d.s.g. ) from the national center for advancing translational sciences , a component of the national institutes of health . the content is solely the responsibility of the authors and does not necessarily represent the ofcial views of the national institutes of health . 
j bone joint surg am 57 : 424 - 426 , 1975 stutch r : osteoblastomaa benign entity ? orthopaed rev 4 : 27 , 1975 grace j , mccarthy s , stankovic r , et al : malignant transformation of osteoblastoma : study using image analysis microdensitometry . 
 genomic analysis does not support malignant transformation kunze e , enderle a , radig k , et al : aggressive osteoblastoma with focal malignant transformation and development of pulmonary metastases . 
sun x , brieo - enrquez ma , cornelius a , et al : fancj ( brip1 ) loss - of - function allele results in spermatogonial cell depletion during embryogenesis and altered processing of crossover sites during meiotic prophase i in mice . 
weber - lassalle n , hauke j , ramser j , et al : brip1 loss - of - function mutations confer high risk for familial ovarian cancer , but not familial breast cancer . 
radig k , schneider - stock r , mittler u , et al : genetic instability in osteoblastic tumors of the skeletal systepathol res pract 194 : 669 - 677 , 1998 31 . 
lorenz s , bary t , sun j , et al : unscrambling the genomic chaos of osteosarcoma reveals extensive transcript fusion , recurrent rearrangements and frequent novel tp53 aberrations . 
 contributed equally to this work . ( continued ) purpose alk ( anaplastic lymphoma kinase ) rearrangements predict for sensitivity to alk tyrosine kinase inhibitors ( tkis ) ; however , responses to alk tkis are generally short lived . 
although multiple studies support the clinical benefits of repeat tissue sampling , the clinical utility of longitudinal circulating tumor dna analysis has not been established in alk - positive lung cancer . patients and methods we used a 566 - gene hybrid - capture next - generation sequencing assay to perform a longitudinal analysis of plasma specimens from 22 alk - positive patients with acquired resistance to alk tkis to track the evolution of resistance during treatment . 
to determine tissueplasma concordance , we compared plasma findings with the results of repeat biopsies . results at disease progression , we detected an alk fusion in plasma from 19 ( 86% ) of 22 patients and identified alk resistance mutations in plasma specimens from 11 patients ( 50% )  . 
alk g1202rthe most frequent plasma mutation detected after progression on a second - generation tkiwas consistently suppressed during treatment with lorlatinib . conclusion plasma genotyping by next - generation sequencing is an effective method for detecting alk fusions and alk mutations in patients who experience disease progression on alk tkis . 
2018 by american society of clinical oncology introduction oncogenic rearrangements that result in the constitutive activation of anaplastic lymphoma kinase ( alk ) define a molecular subtype of nonsmalllung cancer ( nsclc ) that is characterized by sensitivity to alk tyrosine kinase inhibitors ( tkis ) .1 , 2 since the identification of alk rearrangements in nsclc in 2007 , four tkis have become standard therapies for patients with advanced disease.3 - 6 although these tkis have significantly improved clinical outcomes , most patients experience relapse within 1 to 2 years.1 despite a shared molecular driver , the magnitude of benefit from tki treatment varies widely between patients . 
repeat biopsies upon disease progression have been instrumental in elucidating the molecular mechanisms that drive resistance to alk inhibitors , including the distinct spectrum of alk mutations associated with resistance to each tki7 ; however , repeat biopsies may be challenging to obtain and single - site sampling may not capture the spatial heterogeneity of resistance mechanisms . analysis of circulating tumor dna ( ctdna ) is an emerging approach for tumor genotyping . 
shaw , md , phd , department of medicine , massachusetts general hospital , 32 fruit st , boston , ma 02114 ; e - mail : ashaw1@ partners.org. as plasma sampling is minimally invasive and ctdna may be derived from all metastatic sites , longitudinal monitoring of genetic alterations in ctdna may overcome many of the limitations of tissue sampling . 
 libraries were constructed with illumina truseq nano dna library prep kit ( illumina , san diego , ca ) , enriched for a 566 - gene pancancer gene panel ( data supplement ) by using agilent sureselect xt custom baits ( agilent technologies , santa clara , ca ) , and sequenced on an illumina hisequation 2500 sequencer to a median of 105 million reads , which yielded a median coverage of 1 , 195.18 sequence data were aligned to the hg19 reference genome . 
those with at least four alternate reads were retained on the basis of a calculated background mutation rate and false - positive rate of 1e - 4 and 1e - 6 , respectively . 
ctdna insufficient for analysis indicates those samples that were technically successful , but had insufficient tumor dna present in the plasma to reliably estimate underlying tumor genotype . longitudinal ctdna analysis ( n = 79 ) disease progression on study ( n = 29 ) ctdna analysis successful ( n = 22 ) ctdna insufficient for analysis ( n = 7 ) 23 repeat biopsies attempted for 20 patients * repeat biopsy not attempted ( n = 2 ) tissue insufficient ( n = 7 ) tissue sufficient ( n = 16 ) inaccessible site ( n = 1 ) patient preference ( n = 1 ) * three patients underwent tissue sampling twice during plasma surveillance patients with cns - only progression patients with isolated intrathoracic disease progression patients with isolated progression of a liver lesion ( n = 1 ) ( n = 5 ) ( n = 1 ) three of six attempted repeat biopsies sufficient for analysis results patient characteristics that were administered is depicted in the data supplement and figure 2 . plasma was longitudinally collected from 79 patients with metastatic , alk - positive nsclc ( fig 1 )  . 
as this study was primarily focused on investigating the role of ctdna analysis at disease progression , this report is limited to patients who had progressive disease and analyzable plasma specimens . 
of the seven patients who did not have detectable ctdna , one patient experienced cns - only progression , five patients had intrathoracic progression , and one patient had liver oligoprogression ( fig 1 )  . 
baseline characteristics of the 22 patients and their treatment histories are summarized in table 1 , figure 2 , and the data supplement . plasma genotyping was performed on 88 plasma samples from 22 patients . 
the temporal relationship between plasma collection and the treatments concordance between tissue and plasma genotypes we first evaluated concordance between tissue and plasma genotyping for alk fusions , alk mutations , and non - alk alterations . 
 ( % ) of alk tkis before initial plasma collection * abbreviation : alk , anaplastic lymphoma kinase ; tki , tyrosine kinase inhibitor . * the total number of alk tkis on which a patient had experienced progression before the collection of the initial plasma specimen . 
in instances where plasma was collected during sequential treatment with distinct alk inhibitors , the line of therapy at initial plasma collection is represented . ( 94% ; appendix fig a1 )  . 
among the 12 patients for whom the fusion partner was known and an alk fusion was detected in the plasma , there was 100% agreement between tissue and plasma fusion calls ( fig 3a )  . 
point mutations in the alk kinase domain were identified in plasma specimens from 11 patients ( 50% ) , five of whom had paired biopsies at the time of mutation detection ( fig 3b )  . 
three patients had multiple alk mutations . all tissue - detected alk mutations were also identified in corresponding plasma samples , and all plasma - detected alk mutations were also present in paired biopsies . 
 among 17 patients who had plasma collected during progression on a second - generation alk inhibitorthat is , ceritinib , alectinib , or brigatinibplasma alk mutations were identified in eight patients ( 47% ; data supplement ) , including i1171n ( n = 1 ) , g1202r ( n = 5 ) , i1171n / g1202r ( n = 1 ) , and e1210k ( n = 1 )  . 
 the observed frequency of plasma alk mutations is consistent with our previous analysis of resistance biopsies from patients who developed resistance to second - generation inhibitors , which included five patients from the current plasma cohort.7 non - alk alterations . 
as we did not detect any differences in tissue alterations that were identified in serial specimens from the three patients who underwent multiple biopsies , we restricted the concordance assessment to one biopsy per individual . 
although we were not able to obtain tissue after the patient had developed progression on lorlatinib , amplification was not present when a prelorlatinib liver biopsy was assessed using the same 566gene panel . 
despite high - level met amplification ( > 25 copies ) in tissue , we did not detect met amplification in the corresponding plasma specimen ; however , as the maximum allelic frequency ( af ) of detected alterations in the plasma of mgh939 was approximately 2% , the degree of amplification was below the limit of detection of the assay . evolution of resistance during molecular surveillance alk fusion kinetics and correlation with disease status . 
preclinical models and case reports suggest that the presence of a specific alk mutation in a tki - resistant tissue specimen may predict response to subsequent alk tkis1 , 7 , 23 ; therefore , we analyzed plasma specimens from patients with alk mutations who received treatment with additional alk tkis to evaluate the potential of using plasma findings to inform sequential therapies . 
during the follow - up period , seven of these patients ( 88% ) went on to receive treatment with a tki that targeted the alk mutation ( data supplement )  . 
 detected in plasma only detected in plasma and tissue mutation no paired biopsy paired biopsy available detected in plasma only detected in plasma and tissue eml4 - alk variant 1 variant 2 variant 3 variant 5 i1171n f1174c f1174l g1202r d1203n e1210k fig 3 . 
parentheses denote patients for whom the alk mutation was considered the dominant resistance mechanisfor patients without paired biopsies , alk mutations that were detected at the time of progression on an alk tyrosine kinase inhibitor are listed . 
because the mutations were separated by too many bases , the allelic relationship ( cis v trans ) could not be determined for mgh990s alk mutation . in alk i1171n af from 4.5% to undetectable after 6 weeks ( fig 4a )  . 
one patient ( mgh989 ) underwent microwave ablation to treat a liver lesion with a biopsy - proven g1202r mutation and continued treatment with alectinib for an additional 6 months . 
 mgh990 had a radiographic response to treatment , but we could not evaluate the molecular response as we were only able to collect a single plasma specimen . although the g1202r mutation was suppressed in plasma during the treatment with lorlatinib , two of the four patients with plasma - detected g1202r developed progressive disease soon after initiating lorlatinib ( appendix fig a3 )  . 
we were not able to obtain a biopsy , but plasma analysis demonstrated an increase in copy number of gnas and bcl2l1 and a marked increase in the af of mutations that involved other genes during treatment ( fig 5 )  . 
in both cases , eml4 - alk fusion af increased at progression despite the suppression of alk mutations , which supports the notion that resistance was independent of genetic alk alterations . discussion despite a shared molecular driver , the clinical course is variable for patients with alk - positive nsclc treated with alk tkis . 
our findings highlight the potential clinical utility of hybrid - capture ngs for improving our understanding of the molecular drivers of resistance . in our cohort of patients , 76% of plasma samples contained sufficient tumor - derived dna for molecular analysis compared with 65% of biopsy specimens , which confirms that both are reliable approaches . 
 furthermore , our observation that the presence of specific plasma alk mutations correlated with the response and resistance to distinct alk tkis lends support to the emerging practice of using alk mutations to inform the selection of alk tkis . although tissue sampling is the gold standard for molecular analysis , sampling a single site of disease may overestimate the contribution of a particular alteration to the resistant phenotype . 
for example , we detected an alk g1202r mutation at 50% af in a progressing liver lesion , which suggested that it was a major contributor to alectinib resistance in the case of mgh919 . 
in contrast , the plasma g1202r af of 1% was less than the af of other plasma - detected molecular alterations , including multiple non - alk alterations ( fig 5 )  . 
moreover , this case reinforces the notion that the analysis of liquid biopsies may be more informative than tissue - based genotyping in certain situations . to date , the study of resistance to alk therapeutics has primarily focused on alk mutations . 
figure illustrates the change in the allelic fraction of eml4alk and alk mutations during sequential treatment with next - generation alk inhibitors for ( a ) mgh987 and ( b ) mgh087 . 
 ( a ) allelic fraction of alk ( anaplastic lymphoma kinase ) fusion ( blue ) , alk mutations ( red and gold ) , and non - alk alterations ( gray ) during treatment with sequential alk inhibitors . 
despite these shortcomings , our data suggest that the quantitative assessment of structural variants may be clinically useful and complementary to radiographic assessment in some patients . overall , our data confirm that plasma genotyping by using hybrid - capture ngs technology can reliably detect alk fusions and alk resistance mutations in patients with alk - positive nsclc ; however , there are several limitations of this study , including the small size of our cohort , inconsistent sampling intervals that led to variation in the duration of follow - up after and before disease progression , and the inability to obtain pretreatment plasma and paired biopsies for all patients . 
furthermore , as a result of the small contribution of tumor dna to total cellfree dna in most patients , we cannot definitively exclude the presence of undetected amplification events at resistance . 
as identifying alk resistance mutations is emerging as an important consideration for the management of alk - positive nsclc , we anticipate that genotyping plasma to characterize resistance to alk tkis will also become a routine part of patient care . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . ibiayi dagogo - jack consulting or advisory role : boehringer ingelheim honoraria : foundation medicine a . 
campbell employment : novartis , pfizer ( i ) manuscript writing : all authors stock and other ownership interests : novartis final approval of manuscript : all authors accountable for all aspects of the work : all authors jessica j . 
schultz no relationship to disclose jennifer ackil no relationship to disclose sara stevens no relationship to disclose leila dardaei no relationship to disclose satoshi yoda no relationship to disclose harper hubbeling no relationship to disclose subba r . 
sequist honoraria : astrazeneca consulting or advisory role : astrazeneca , genentech , bristol - myers squibb , pfizer research funding : boehringer ingelheim ( inst ) , clovis oncology ( inst ) , genentech ( inst ) , merrimack pharmaceuticals ( inst ) , novartis ( inst ) , astrazeneca ( inst ) , johnson & johnson ( inst ) , merck ( inst ) , pfizer ( inst ) inga t . 
lennes honoraria : blue cross and blue shield of massachusetts consulting or advisory role : kyruus anthony john iafrate stock and other ownership interests : archer biosciences consulting or advisory role : debiopharm group , constellation pharmaceuticals , chugai pharma , roche research funding : blueprint medicines patents , royalties , other intellectual property : archerdx exclusive license to amp technology rebecca s . 
heist consulting or advisory role : boehringer ingelheim research funding : glaxosmithkline , sanofi , abbvie , novartis , roche , incyte , celgene , mirati therapeutics , peregrine pharmaceuticals , exelixis , millennium pharmaceuticals , debiopharm group christopher g . 
azzoli consulting or advisory role : merck , ariad pharmaceuticals , takeda research funding : pharmamar ( inst ) , abbvie ( inst ) , astrazeneca ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) , loxo ( inst ) , ignyta ( inst ) travel , accommodations , expenses : pharmamar , abbvie , stemcentrx jeffrey a . 
engelman employment : novartis stock and other ownership interests : loxo , agios , kura , gatekeeper pharmaceuticals , novartis consulting or advisory role : novartis , agios , loxo , clovis oncology , ventana medical systems , g1 therapeutics , sanofi , chugai pharma , warp drive bio , asana biosciences , emd serono , synta , allostery , genentech , aveo , biodesix , merck , cell signaling technology , endo pharmaceuticals , astrazeneca , glaxosmithkline , amgen , bristol - myers squibb , cancer progress research funding : novartis , jounce therapeutics , sanofi , araxes , astrazeneca , amgen ( inst ) patents , royalties , other intellectual property : coinventor on a patent application that has been licensed to ventana medical system / roche other relationship : third rock ventures jochen k . 
leary employment : novartis stock and other ownership interests : novartis patents , royalties , other intellectual property : patents , pending patents , and a royalty - sharing agreement with johns hopkins university alice t . 
shaw honoraria : pfizer , novartis , genentech , foundation medicine consulting or advisory role : pfizer , novartis , genentech , roche , ariad pharmaceuticals , ignyta , blueprint medicines , daiichi sankyo , emd serono , taiho pharmaceutical , ksq therapeutics research funding : pfizer , novartis , genentech justin f . 
gainor honoraria : merck , incyte , ariad pharmaceuticals consulting or advisory role : novartis , boehringer ingelheim , clovis oncology , genentech , bristol - myers squibb , theravance , loxo research funding : merck , novartis , genentech , bristolmyers squibb , adaptimmune , astrazeneca , ariad pharmaceuticals travel , accommodations , expenses : affymetrix anna f . 
farago honoraria : foundation medicine consulting or advisory role : pharmamar , merrimack pharmaceuticals , takeda , abbvie , intervention insights acknowledgment we also thank jeremy decker for assistance with plasma preparation and circulating tumor dna processing . 
shaw at , yeap by , mino - kenudson m , et al : clinical features and outcome of patients with nonsmall - cell lung cancer who harbor eml4 - alk . 
kim dw , mehra r , tan ds , et al : activity and safety of ceritinib in patients with alk - rearranged nonsmall - cell lung cancer ( ascend - 1 ) : updated results from the multicentre , open - label , phase 1 trial . 
shaw at , gandhi l , gadgeel s , et al : alectinib in alk - positive , crizotinib - resistant , nonsmallcell lung cancer : a single - group , multicentre , phase 2 trial . 
kim dw , tiseo m , ahn mj , et al : brigatinib in patients with crizotinib - refractory anaplastic lymphoma kinase - positive nonsmall - cell lung cancer : a randomized , multicenter phase ii trial . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced nonsmall - cell lung cancer . 
sacher ag , paweletz c , dahlberg se , et al : prospective validation of rapid plasma genotyping for the detection of egfr and kras mutations in advanced lung cancer . 
paweletz cp , sacher ag , raymond ck , et al : bias - corrected targeted next - generation sequencing for rapid , multiplexed detection of actionable alterations in cell - free dna from advanced lung cancer patients . 
schwaederl mc , patel sp , husain h , et al : utility of genomic assessment of blood - derived circulating tumor dna ( ctdna ) in patients with advanced lung adenocarcinoma . 
thompson jc , yee ss , troxel ab , et al : detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next - generation sequencing of cell - free circulating tumor dna . 
curigliano g , gmez pardo p , meric - bernstam f , et al : ribociclib plus letrozole in early breast cancer : a presurgical , window - of - opportunity study . 
ye k , schulz mh , long q , et al : pindel : a pattern growth approach to detect break points of large deletions and medium sized insertions from paired - end short reads . 
solomon b , bauer tm , felip e , et al : safety and efficacy of lorlatinib ( pf - 06463922 ) from the dose - escalation component of a study in patients with advanced alk + or ros1 + non - small cell lung cancer ( nsclc )  . 
 in two of the three patients for whom plasma analysis failed to detect a fusion , an eml4 - alk fusion ( variants 3 and 5 ) was identified in matching tissue . 
 c acquired resistance to immune checkpoint inhibitor therapy through outgrowth of cells lacking cd274 and pdcd1lg2 amplification introduction the recent introduction of immune checkpoint inhibitor ( icpi ) therapies has expanded effective treatment options for many patients with metastatic solid tumors , although typically acquired resistance develops over time . 
here , we present a patient with metastatic colon cancer in whom next - generation sequencing ( ngs ) demonstrated amplification of both cd274 , or programmed cell death ligand 1 ( pd - l1 ) , and pdcd1lg2 , or programmed cell death ligand 2 ( pd - l2 )  . 
ngs testing of the progressed tumor no longer demonstrated cd274 ( pd - l1 ) and pdcd1lg2 ( pd - l2 ) amplification but did not identify any new genomic alterations compared with the initial tumor profile . 
 histologic examination revealed a stage iiib ( t4bn1mx ) colon adenocarcinoma , and a computed tomography ( ct ) scan of the chest , abdomen , and pelvis showed no evidence of metastatic disease . 
she received 12 doses of adjuvant fluorouracil plus leucovorin plus oxaliplatin chemotherapy . a repeat ct scan in july 2015 revealed development of a retroperitoneal soft tissue nodule , which was confirmed by biopsy to be metastatic colon adenocarcinoma . 
 in december 2015 , a ct scan showed interval enlargement of extraperitoneal tumor implants as well as numerous subcutaneous and muscular metastases . a commercial ngs assay ( foundation medicine , cambridge , ma ) was performed on the pretreatment adenocarcinoma sample and revealed gene amplification of both cd274 ( pd - l1 ) and pdcd1lg2 ( pd - l2 )  . 
the total mutation burden was 11 mutations / mb , which falls in the intermediate burden category . in january 2016 , the patient began receiving intravenous nivolumab ( 3 mg / kg ) every 2 weeks . 
 after six doses , a repeat ct scan ( chest , abdomen , and pelvis ) in april 2016 showed resolution or considerable decrease in size of all nodules , both within and outside of the radiation field . 
a left supraclavicular lymph node core biopsy showed a poorly differentiated carcinoma consistent with spread from the patients known adenocarcinoma . ngs ( foundation medicine ) performed on the november 2017 supraclavicular lymph node biopsy specimen showed a similar genomic mutation profile as the 2014 primary tumor ; however , this sample lacked amplification of both cd274 ( pd - l1 ) and pdcd1lg2 ( pd - l2 ) as well as flt3 amplification and stag - 2 p.w85l. 
no new alterations were reported , and the total mutation burden was 11 mutations / mb . discussion in 2017 , we first reported this patient with proficient dna mismatch repair metastatic colorectal cancer whose tumor responded dramatically to an icpi.2 icpis are now routinely used as firstor second - line metastatic disease therapies for patients with a variety of cancer types , including deficient mismatch repair expressing microsatellite instabilityhigh colorectal cancer , where the response rate is roughly 40%.3 there is existing literature that pd - l1 gene amplification can be a predictive marker for checkpoint inhibitor therapy efficacy.4 ikeda et al5 reported a patient with metastatic basal cell carcinoma to the liver who had an exceptional response to nivolumab . 
in addition , in patients with nonsmall - cell lung cancer , clav et al6 demonstrated pd - l1 gene amplification resulting in high pd - l1 expression , a confirmed biomarker for effective checkpoint inhibitor therapy . 
pd - l1 expression seen at the time of initiation of the effective checkpoint inhibitor therapy and no longer demonstrated in the progressive disease would be supportive evidence that pd - l1 gene amplification was not only a biomarker of efficacy but also related causally to the efficacy of the checkpoint inhibitor , given the mechanism of action of the checkpoint inhibitor used ( inhibiting the programmed death pathway ligand )  . we reasoned that our patients tumor responded to an icpi , because ngs testing demonstrated amplification of both cd274 ( pd - l1 ) and pdcd1lg2 ( pd - l2 ) , and the mechanism for this therapy is to elicit a cytotoxic immune response by inhibiting the programmed death pathway ligands ( pd - l1 and pd - l2 )  . 
unlike pd - l1 protein expression , amplification of pd - l1 and pd - l2 in cancer is rare and most likely to be detected by routine clinical ngs testing rather than fluorescent in situ hybridization assays . 
routine screening for pd - l1 and pd - l2 amplification would be costly from a time , financial , and tissue perspective , only to show a low rate of cases with gene amplifications . 
the ability of any ngs assay to detect gene amplifications is dependent on the bait set design of the assay , quality of extracted dna , adequacy of tumor content , and depth of coverage . 
in cases where pd - l1 / pd - l2 amplifications are suspected but ngs quality metrics are suboptimal , fluorescent in situ hybridization testing should be considered as an orthogonal assay to confirm gene amplifications . 
for example , overman et al8 recently reported that in patients with deficient mismatch repair expressing metastatic colorectal cancers , indirect comparisons suggest combination therapy provides improved efficacy relative to anti - program death 1 monotherapy.8 ( p773 ) studies of secondary or acquired resistance to icpi therapy have been reported . 
the authors of a recent review wrote that mechanisms of resistance to icpis include somatic mutations in antigen processing and presentation as well as upregulation of genes involved in cell adhesion , angiogenesis , and extracellular matrix remodeling . 
they also note the potential to target mechanisms of resistance such as an angiogenic phenotype and defects in interferon signaling pathways.9 it seems likely that our patients cancer that progressed in the supraclavicular area was due to the selection and outgrowth of a small population of tumor cells ( subclones ) that did not harbor cd274 ( pd - l1 ) and pdcd1lg2 ( pd - l2 ) amplification . 
during the 8 months of checkpoint inhibitor therapy , those subclones became the predominant population because , on repeat ngs testing , no additional genomic alterations were identified , and the cd274 ( pd - l1 ) and pdcd1lg2 ( pd - l2 ) amplifications were not detected . because there has been no tumor progression outside the supraclavicular area , it is possible that the supraclavicular area was the only site of subclones lacking cd274 ( pd - l1 ) and pdcd1lg2 ( pd - l2 ) amplifications . 
finally , we hypothesize that combination therapies targeting both cd274 ( pd - l1 ) and pdcd1lg2 ( pd - l2 ) amplified and nonamplified cell populations might have resulted in a more durable response . in conclusion , on the basis of ngs testing of our patients progressive disease after icpi treatment , resistance to icpi therapy was likely due to the selection and outgrowth of subclones lacking cd274 ( pd - l1 ) and pdcd1lg2 ( pd - l2 ) amplifications . 
sorscher s , resnick j , goodman m : first case report of a dramatic radiographic response to a checkpoint inhibitor in a patient with proficient mismatch repair gene expressing metastatic colorectal cancer . 
inoue y , yoshimura k , mori k , et al : clinical significance of pd - l1 and pd - l2 copy number gains in non - small - cell lung cancer . 
ikeda s , goodman am , cohen pr , et al : metastatic basal cell carcinoma with amplification of pd - l1 : exceptional response to anti - pd1 therapy . 
lee ck , kim s , lee js , et al : next - generation sequencing reveals novel resistance mechanisms and molecular heterogeneity in egfr - mutant non - small cell lung cancer with acquired resistance to egfr - tkis . 
overman mj , lonardi s , wong kym , et al : durable clinical benefit with nivolumab plus instability - high metastatic in dna mismatch repair - deficient / microsatellite ipilimumab colorectal cancer . 
chen pl , roh w , reuben a , et al : analysis of immune signatures in longitudinal tumor samples yields insight into biomarkers of response and mechanisms of resistance to immune checkpoint blockade . 
 discordance in human epidermal growth factor receptor 2 ( her2 ) phenotype between primary tumor and circulating tumor cells in women with her2 - negative metastatic breast cancer purpose discordance in human epidermal growth factor receptor 2 ( her2 ) status between primary tumor and metastases might have important implications for treatment response and therapy decisions . 
here , we evaluate both the frequency of circulating tumor cells ( ctcs ) and the factors predicting her2 discordance between primary tumor and ctcs as a potential surrogate for tumor biology and tumor heterogeneity in patients with metastatic breast cancer . patients and methods the number of ctcs in 7.5 ml of peripheral blood and her2 status were evaluated in 1 , 123 women with her2 - negative metastatic breast cancer . 
2017 by american society of clinical oncology introduction circulating tumor cells ( ctcs ) are observed in early and metastatic breast cancer ( mbc ) , and their prognostic relevance has been demonstrated in both early and advanced disease.1 , 2 according to previous studies , approximately 65% to 85% of patients with mbc have detectable ctcs , and approximately half of these patients have five or more ctcs in 7.5 ml of peripheral blood.2 , 3 in addition to the prognostic value of ctcs as assessed before treatment , monitoring ctc dynamics during therapy provides important information on therapy response.4 - 7 however , valuable information with regard to therapy response and suitability of additional treatment options may be provided not only by the prevalence and changes in the number of ctcs but also by the phenotype of ctcs . 
friedl jens huober christoph scholz nikolaus de gregorio brigitte rack elisabeth trapp marianna alunni - fabbroni sabine riethdorf volkmar mueller andreas schneeweiss klaus pantel franziska meier - stiegen bernadette jaeger andreas hartkopf florin - andrei taran peter a . 
assessing ctc phenotype may be especially important in mbc , because it can be used as a noninvasive liquid biopsy to characterize the metastatic disease , thus reflecting tumor heterogeneity and possible discordance in hormone receptor and / or human epidermal growth factor receptor 2 ( her2 ) expression with potentially far - reaching implications in terms of follow - up treatment and additional targeted therapies . although discordance in her2 status among primary tumor , distant metastases , and ctcs has been described in several studies , 3 , 8 - 10 it has not been implemented in clinical practice to guide therapy decisions . 
the study concept was in accordance to the declaration of helsinki , adhered to good clinical practice and german pharmaceutical law , and was approved by the local ethic committees of the participating centers . assessment of ctc presence and her2 phenotype on ctcs from all patients , 7.5 - ml peripheral blood samples were collected , and presence and phenotype of ctcs were assessed as previously described in detail , 11 using the semiautomatic and standardized cellsearch system ( janssen diagnostics , raritan , nj ) , which is the only us food and drug administrationapproved method for the enumeration of ctcs . 
for her2 assessment , cells were also labeled with her2 antibodies , and ctcs were considered as her2 positive only if they showed a strong ihc staining intensity for her2 ( ihc score , 3 + )  . 
analyses for determining ctc prevalence and her2 phenotype of ctcs were performed by trained scientists with a peer - review system at four independent reference laboratories . statistical calculations descriptive statistics for the categorical data are provided in terms of absolute and relative frequencies . 
the non - normally distributed continuous variables age ( in years ) and time interval between primary diagnosis and screening ( in months ) are described by medians and ranges . 
associations between presence of ctcs or presence of her2 - positive ctcs and patient or tumor characteristics were evaluated using mann - whitney u tests for age and time interval between primary diagnosis and screening , and x2 tests were used for all other categorical variables . 
to assess which factors predicted discordance in the her2 phenotype between primary tumor and ctcs , we used a multivariable logistic regression model with discordance in her2 status ( yes v no ) as a binary response variable and a stepwise backward selection procedure ( significance level cutoff for exclusion , .05 [ likelihood ratio test ] )  . 
demographic and primary tumor characteristics of patients with her2negative mbc screened for ctcs within the detect study program ( n = 1 , 123 ) characteristic age at screening , years total no . 
 ( % ) of patients time between primary diagnosis and screening , months median range median range menopausal status at screening premenopausal postmenopausal unknown tumor stage unknown nodal stage unknown histologic grade unknown histologic type invasive ductal invasive lobular other hormone receptor status negative positive unknown 26 - 89 0 - 450 136 ( 12.1 ) 734 ( 65.4 ) 253 ( 22.5 ) 308 ( 27.4 ) 503 ( 44.8 ) 114 ( 10.2 ) 125 ( 11.1 ) 13 ( 1.2 ) 60 ( 5.3 ) 363 ( 32.3 ) 378 ( 33.7 ) 147 ( 13.1 ) 136 ( 12.1 ) 99 ( 8.8 ) 47 ( 4.2 ) 576 ( 51.3 ) 406 ( 36.2 ) 94 ( 8.4 ) 751 ( 66.9 ) 199 ( 17.7 ) 173 ( 15.4 ) 171 ( 15.2 ) 886 ( 78.9 ) 66 ( 5.9 ) had hormone receptorpositive invasive ductal tumors . 
median number of ctcs detected in ctc - positive patients was seven ( interquartile range , two to 30 ; range , one to 35 , 078 ctcs )  . 
frequency distribution of the number of ctcs detected is shown in appendix figure a1 . presence of at least one ctc was significantly associated with nodal stage and histologic type of the primary tumor but not with other patient or primary tumor characteristics ( table 2 )  . 
in 158 patients ( 22.2% ) , at least one ctc with an her2 2 + ihc score , but no ctcs with an her2 3 + ihc score , was detected ; these patients were not considered to have her2 - positive ctcs in the blood ( as described in patients and methods )  . 
patients with grade 3 primary tumors were less likely to have her2 - positive ctcs than patients with grade 1 or 2 primary tumors ( p = .028 ; fig 3a )  . 
patients with invasive lobular primary tumors were more likely to have her2positive ctcs than patients with other primary tumor histologic types ( p , .001 ; fig 3b ) , and patients with hormone receptorpositive primary tumors were more likely to have her2 - positive ctcs than patients with hormone receptor negative ( ie , triple negative ) primary tumors ( p , .001 ; fig 3c )  . 
of her2 - positive ctcs 6 - 10 11 - 50 > 50 predictors of discordance in her2 status between primary tumor and ctcs to evaluate which factors can independently predict discordance in her2 status between primary tumor and ctcs , we used a multivariable logistic regression model with discordance in her2 status ( yes v no ) as a binary response variable . 
patient and primary tumor characteristics included as independent factors were patient age in years , time since primary diagnosis in months , tumor stage ( pt ) , nodal stage ( pn ) , histologic grade , histologic type , and hormone receptor status . 
to account for the association between number of ctcs detected and presence of her2 - positive ctcs , we included number of ctcs ( one to four v > five ctcs ) as an additional explanatory variable . 
please note that the or for age listed in table 3 ( ie , 0.977 ) corresponded to the change of the odds of showing discordance in her2 phenotype associated with a 1 - year increase in age . discussion to our knowledge , the combined ctc screening of the detect study program provides the largest worldwide cohort of patients with mbc tested with regard to both rate of discordance in her2 phenotype between primary tumor and ctcs and possible predictors of her2 discordance . 
our analyses revealed an overall ctc prevalence of 63.3% in 1 , 123 patients with her2 - negative mbc , which is within the range reported in other studies on mbc.2 , 3 , 12 discordance in her2 phenotype between primary tumor and ctcs was observed in 18.8% of 711 ctc - positive patients and was independently predicted by histologic type and hormone receptor status of the primary tumor . 
of ctcs 11 - 50 > 50 6 - 10 11 - 25 > 25 her2 - positive ctcs ( % ) her2 status was more frequent in patients with five or more ctcs compared with patients with fewer than five ctcs . some previous studies have reported even higher discordance rates , although with considerably smaller sample sizes . 
lobular carcinomas are less cohesive tumors that are characterized by impaired cell - cell adhesion with reduced expression of the adhesion molecule e - cadherin.19 , 20 reduced cell and tissue adhesion may lead to an easier spread of tumor cells and could be one explanation for the association of presence of ctcs with lobular carcinomas . 
in general , her2 is rarely amplified or overexpressed in lobular carcinomas21 ; thus , the fact that we observed an association between presence of her2 - positive ctcs and lobular carcinomas is surprising and not easily explained . there is some evidence that lobular breast cancer shows a higher frequency of her2 mutations compared with ductal breast cancer.22 - 24 therefore , it could be speculated that the increased rate of her2 discordance observed in lobular carcinomas might be related to her2 mutations that affect her2 expression and membrane staining . the fact that hormone receptorpositive primary tumors exhibit higher rates of her2 discordance than triple - negative tumors is also a new finding that must be explored in more detail . clearly , additional investigations of tumor biology and pathogenesis of ctcs are necessary to help us understand these results . regardless of the underlying mechanisms , our findings may have implications for clinical practice . 
data on changes in her2 status during tumor progression represent crucial information for adequate patient therapy , because they may lead to modification of systemic and / or addition of targeted treatments . 
on the basis of our results , we suggest physicians consider attempting to obtain additional biopsies of metastatic lesions for detection of potential changes in her2 phenotype , especially in patients with lobular carcinomas or hormone receptorpositive primary tumors , because these are risk factors for her2 discordance identified in our study . 
prevalence of at least one human epidermal growth factor receptor 2 ( her2 ) positive circulating tumor cell ( ctc ; ie , discordance rate of her2 status [ % ] ) according to tumor characteristics of the primary tumor . 
 ( a ) prevalence of at least one her2 - positive ctc according to histologic grade of the primary tumor in 654 ctc - positive patients with her2 - negative metastatic breast cancer ( mbc ) screened for the detect study program ( for 57 patients , histologic grade of the primary tumor was unknown ; table 2 )  . 
 ( b ) prevalence of at least one her2 - positive ctc according to histologic type of the primary tumor in 711 ctc - positive patients with her2 - negative mbc screened for the detect study progra ( c ) prevalence of at least one her2positive ctc according to hormone receptor status of the primary tumor in 670 ctc - positive patients with her2 - negative mbc screened for the detect study program ( for 41 patients , hormone receptor status of the primary tumor was unknown ; table 2 )  . therapy in patients with histologically her2positive mbc revealed discordance of her2 status between tissue and ctcs in 62% of patients , and negative her2 status of ctcs was significantly associated with shorter progressionfree survival after the new line of anti - her2 therapy.25 another recent study indicated that her2 - targeted therapy with lapatinib was effective in decreasing the number of her2positive ctcs in patients with mbc.26 if additional research confirms that ctcs can be used as an easily accessible liquid biopsy to assess changes in tumor biology and her2 status of metastases , detection of her2 - positive ctcs in patients with mbc with her2negative primary tumors might even trigger the addition of her2 - targeted therapies without the need for a confirmatory biopsy of metastatic lesions . 
although primary tumors were considered her2 positive if more than 10% of tumor cells showed protein overexpression , her2 - positive ctc status was defined as presence of at least one ctc with an her2 3 + ihc score in the blood . 
furthermore , because this study was based on the initial screening results of the detect study program , and only a subsample of screened patients was recruited in one of the detect clinical trials , associations between ctc prevalence or presence of her2 - positive ctcs with metastatic disease patterns and patient outcomes could not be analyzed at this stage . 
ctc detection was based on an epithelial cell adhesion molecule selection process ; therefore , ctcs undergoing epithelial - tomesenchymal transition with phenotypic changes in terms of lacking epithelial cell adhesion molecule expression and gaining mesenchymal and / or stem - cell marker characteristics might have been missed , which could have led to falsely low rates of ctc positivity . 
furthermore , her2 status of ctcs was assessed by ihc only and was not confirmed using fish . in conclusion , discordance in her2 status between primary tumor and ctcs was observed in 18.8% of patients with her2 - negative mbc and was associated with lobular carcinomas , hormone receptorpositive primary tumors , and high ctc counts . 
moreover , the concept of liquid biopsy using ctcs as a real - time noninvasive monitoring tool to evaluate tumor biology , progression , and heterogeneity as a basis for more personalized treatment decisions should be tested in prospective randomized clinical trials . 
fasching provision of study material or patients : amelie de gregorio , jens huober , nikolaus de gregorio , brigitte rack , elisabeth trapp , andreas schneeweiss , klaus pantel , andreas hartkopf , peter a . 
fasching , tanja fehm collection and assembly of data : amelie de gregorio , christoph scholz , nikolaus de gregorio , brigitte rack , elisabeth trapp , sabine riethdorf , volkmar mueller , andreas schneeweiss , klaus pantel , franziska meier - stiegen , bernadette jaeger , andreas hartkopf , florin - andrei taran , peter a . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . amelie de gregorio consulting or advisory role : roche pharma ag thomas w.p. 
 jens huober honoraria : novartis , roche consulting or advisory role : novartis , roche travel , accommodations , expenses : novartis , roche christoph scholz consulting or advisory role : roche pharma ag research funding : novartis ( inst ) , roche pharma ag ( inst ) , janssen oncology ( inst ) nikolaus de gregorio consulting or advisory role : roche travel , accommodations , expenses : astrazeneca , tesaro brigitte rack consulting or advisory role : novartis speakers bureau : roche , pfizer research funding : sanofi ( inst ) , novartis ( inst ) , eli lilly ( inst ) , astrazeneca ( inst ) , chugai pharma ( inst ) , janssen diagnostics ( inst ) travel , accommodations , expenses : pfizer elisabeth trapp no relationship to disclose marianna alunni - fabbroni no relationship to disclose volkmar mueller consulting or advisory role : astrazeneca , celgene , daiichi sankyo , eisai , nektar , genentech , novartis travel , accommodations , expenses : pfizer , roche pharma ag andreas schneeweiss honoraria : roche pharma ag , celgene , astrazeneca , pfizer , novartis , amgen , roche pharma ag ( inst ) , pfizer ( inst ) research funding : roche pharma ag ( inst ) , celgene ( inst ) travel , accommodations , expenses : roche , celgene , amgen klaus pantel honoraria : agena bioscience consulting or advisory role : wntresearch , roche , novartis , sanofi research funding : janssen diagnostics franziska meier - stiegen consulting or advisory role : amgen bernadette jaeger no relationship to disclose andreas hartkopf no relationship to disclose florin - andrei taran no relationship to disclose peter a . 
fasching honoraria : roche , amgen , novartis , pfizer , celgene research funding : novartis ( inst ) wolfgang janni honoraria : janssen diagnostics research funding : janssen diagnostics tanja fehm consulting or advisory role : roche , novartis , amgen research funding : novartis acknowledgment we thank all the patients for participating in this study and donating their blood samples for research purposes . 
friedl , jens huober , christoph scholz , nikolaus de gregorio , and wolfgang janni , university hospital ulm , ulm ; brigitte rack , elisabeth trapp , and marianna alunni - fabbroni , hospital of ludwig - maximilians - university , munich ; sabine riethdorf , volkmar mueller , and klaus pantel , university hospital hamburg - eppendorf , hamburg ; andreas schneeweiss , university hospital heidelberg , heidelberg ; franziska meier - stiegen , bernadette jaeger , and tanja fehm , heinrichheine - university duesseldorf , duesseldorf ; andreas hartkopf and florin - andrei taran , university hospital tuebingen , tuebingen ; and peter a . 
fasching , university hospital erlangen , erlangen , germany . affiliations support the detect study program is supported by the investigator - initiated study program of janssen diagnostics , with clinical trials also supported by pierre fabre pharma , teva pharmaceuticals industries , amgen , novartis pharma , and eisai ; study medications vinorelbine , nonpegylated liposomal doxorubicin , lapatinib and everolimus , eribulin , and pertuzumab were provided free of charge by pierre fabre pharma , teva pharmaceuticals industries , novartis pharma , eisai , and roche , respectively . the funding sources had no role in study design ; data collection , analysis , or interpretation ; or writing of the report . 
bidard fc , peeters dj , fehm t , et al : clinical validity of circulating tumour cells in patients with metastatic breast cancer : a pooled analysis of individual patient data . 
fehm t , m uller v , aktas b , et al : her2 status of circulating tumor cells in patients with metastatic breast cancer : a prospective , multicenter trial . 
hayes df , cristofanilli m , budd gt , et al : circulating tumor cells at each follow - up time point during therapy of metastatic breast cancer patients predict progression - free and overall survival . 
pierga jy , hajage d , bachelot t , et al : high independent prognostic and predictive value of circulating tumor cells compared with serum tumor markers in a large prospective trial in first - line chemotherapy for metastatic breast cancer patients . 
simmons c , miller n , geddie w , et al : does confirmatory tumor biopsy alter the management of breast cancer patients with distant metastases ? ann oncol 20 : 1499 - 1504 , 2009 10 . 
ligthart st , bidard fc , decraene c , et al : unbiased quantitative assessment of her - 2 expression of circulating tumor cells in patients with metastatic and non - metastatic breast cancer . 
schramm a , friedl tw , schochter f , et al : therapeutic intervention based on circulating tumor cell phenotype in metastatic breast cancer : concept of the detect study prograarch gynecol obstet 293 : 271 - 281 , 2016 12 . 
tewes m , aktas b , welt a , et al : molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer : an option for monitoring response to breast cancer related therapies . 
wallwiener m , hartkopf ad , riethdorf s , et al : the impact of her2 phenotype of circulating tumor cells in metastatic breast cancer : a retrospective study in 107 patients . 
houssami n , macaskill p , balleine rl , et al : her2 discordance between primary breast cancer and its paired metastasis : tumor biology or test artefact ? insights through meta - analysis . 
aurilio g , disalvatore d , pruneri g , et al : a meta - analysis of oestrogen receptor , progesterone receptor and human epidermal growth factor receptor 2 discordance between primary breast cancer and metastases . 
sinn hp , helmchen b , heil j , et al : lobular neoplasms and invasive lobular breast cancer [ in german ] pathologe 35 : 45 - 53 , 2014 20 . 
gomes ds , porto ss , rocha rm , et al : usefulness and limitations of e - cadherin and b - catenin in the classification of breast carcinomas in situ with mixed pattern . 
ross js , wang k , sheehan ce , et al : relapsed classic e - cadherin ( cdh1 ) - mutated invasive lobular breast cancer shows a high frequency of her2 ( erbb2 ) gene mutations . 
zhang s , li l , wang t , et al : real - time her2 status detected on circulating tumor cells predicts different outcomes of anti - her2 therapy in histologically her2 - positive metastatic breast cancer patients . 
 appendix results additional data regarding both the association between hormone receptor status and histologic type and rates of human epidermal growth factor receptor 2 ( her2 ) discordance in relation to the combined hormone receptor and tumor type status are as follows : hormone receptor status and histologic type of primary tumors were significantly associated with each other ( p , .001 ; n = 670 ; 41 patient cases with missing values for hormone receptor status )  . 
frequency distribution of the number of circulating tumor cells ( ctcs ) detected in 7.5 - ml peripheral blood samples from 1 , 123 patients with human epidermal growth factor receptor 2negative metastatic breast cancer screened for the detect study program ( cutoff for ctc positivity , > one ctc )  . 6 - 10 11 - 50 > 50 no . 
 olaparib monotherapy for brip1 - mutated high - grade serous endometrial cancer kohei nakamura , md , phd1 , 2 ; eriko aimono , md1 ; shigeki tanishima , md , phd3 ; mitsuho imai , md , phd1 ; akiko kawano nagatsuma , md , phd1 ; hideyuki hayashi , md , phd1 ; yuki yoshimura , md4 ; kentaro nakayama , md , phd4 ; satoru kyo , md , phd4 ; and hiroshi nishihara , md , phd1 introduction treatment the prognosis for women with high - grade endometrial cancers is poor , with little improvement in the last two decades.1 the mainstay of is surgery lymphadenectomy . ( hysterectomy ) with or without although adjuvant radiotherapy is considered standard for high - risk endometrial cancers , the added value of chemotherapy has been the subject of recent trials . 
recently , other mutations were reported in ovarian cancers , such as those in fanconi anemia genes ( eg , palb2 [ fancn ] , brip1 [ fancj ] , and rad51c ) and other genes involved in hrr ( eg , atm , bard1 , nbn , cdk12 , tp53 , and chek2 ) .2 , 3 poly ( adp - ribose ) polymerase ( parp1 ) is a dna repair enzyme involved in base excision and single - stranded break repair pathways.4 parp1 inhibition in hrrdecient cells blocks single - stranded dna break repair pathways , leaving only double - stranded dna break repair pathways functional . 
this results in synthetic lethality from the loss of homologous recombination and base excision repair , thus activating highly error - prone dna repair pathways , eventually causing cell death.5 , 6 parp inhibitors also trap parp - dna complexes at the replication fork , increase toxic nonhomologous end joining in parp1 - decient cells , and block parp1 / pol - mediated alternative end joining.7 the us food and drug administration ( fda ) has approved three parp inhibitors ( olaparib , rucaparib , and niraparib ) as monotherapies for breast and ovarian cancers . 
after surgery , the patient received six cycles of carboplatin and paclitaxel . there was also evidence of after rst - line chemotherapy , ct indicated stable disease based on recist 1.0 criteria , with only improvements in the cardiophrenic swollen lymph node ( fig 1 )  . 
after six cycles , ct revealed essentially stable disease without iliac in the para - aortic or left improvement swollen lymph nodes ( fig 1 )  . lateral targeted next - generation sequencing of the patients blood and resected specimen was performed using an in - house assay during treatment . 
a tp53 somatic point mutation ( p.r175h ) and somatic frameshift brip1 ( p.q554hfs * 35 ) alterations were detected as pathogenic variants in the tumor ; detailed information is provided in appendix figures a1 and a2 . 
secondary germ line examination found no american college of medical genetics and genomics recommended genes for testing . given her somatic brip1 mutation and ndings indicating loh high status , the potential efcacy of the parp inhibitor olaparib was discussed with the patient . 
9 months of olaparib and showed complete response on her most recent ct . discussion endometrial cancers are categorized into two main histologic types.8 type i endometrial tumors show endometrioid histology and typically express estrogen ( er ) and progesterone ( pr ) receptors . 
in contrast , type ii endometrial tumors do not exhibit endometrioid histology ( predominantly serous histology ) and are associated with poorer prognoses , with a 5 - year overall survival rate of 55%.1 type ii tumors neither express er / pr nor respond to endocrine therapy . 
meanwhile , type i tumors exhibit signicantly more pi3k pathway alterations ( primarily pten and pik3ca mutations ) .9 , 10 in our case , next - generation target panel sequencing results reected the molecular events observed in serous endometrial cancers , such as tp53 mutations . whereas brip1 is implicated in double - stranded dna break repair via hrr pathways , the frequency of brip1 mutations in type ii endometrial tumors remains unclear . in a previous study by heeke et al , 11 the prevalence of recombinationrelated gene mutations homologous across multiple cancer types was investigated in 52 , 426 malignant tumors . 
this has promoted clinical studies of parp inhibitors in contexts other than ovarian cancer . moreover , their potential therapeutic applications might be extended from germ line brca mutations to target a more diverse group of sporadic tumors , such as those with epigenetic disruption of brca1 / 2 function or genetically or epigenetically acquired aberrations in other important hrr pathway constituents.12 there is clinical evidence to support this theory , with olaparib approved by the fda as an alternative for patients with germ line or somatic brca1 / 2 mutations and as maintenance therapy after platinumbased chemotherapy in platinum - sensitive recurrent epithelial ovarian carcinoma , regardless of brca mutation status.13 hrr alterations are associated with brca1 / 2 mutations in high - grade serous endometrial cancers ; however , parp inhibitors await fda approval for this indication , and there are currently no trials of parp inhibitors in patients with endometrial cancers with brca or other hrr - associated genealterations . accumulating evidence indicates that patients with brip1 mutations have hrr deciency and are consequently hypersensitive to parp inhibition.14 brip1 is a brca1interacting protein ( the brip1 - brca1 interaction is important for hrr ) and associates with brca1 as cells progress through the s phase of the cell cycle.15 , 16 brip1 is a dna helicase that interacts with the cooh - terminal brct repeat of brca1 . 
brip1 is associated with the gm1 / 2 checkpoint , as well as the activation of chk1 , regulation of entry into the s phase , and maintenance of genomic stability.16 thus , if the complex involving brca1 and brip1 is involved in tumor suppression , mutations in the genes that encode these proteins should be associated with altered cancer risk . 
recently , three independent dnabased measures of genomic instability reecting underlying tumor homologous recombination dna repair deciency were developed based on loh , telomeric allelic imbalance , and large - scale state transitions.17 - 19 however , we could not determine the homologous recombination deciency score , because the relevant test is not covered by insurance in japan , and therefore , we could not prove this . 
however , we can hypothesize that the brip1 mutation results in hrr deciency and subsequently causes a high loh frequency and scattered allelic imbalance , potentially resulting in good response to parp inhibitors . in conclusion , we describe the case of a 70 - year - old woman exhibiting a durable clinical radiographic response to olaparib . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . support supported by the japan agency for medical research and development under grant no . 
lancet 387 : 1094 - 1108 , 2016 somyajit k , subramanya s , nagaraju g : rad51c : a novel cancer susceptibility gene is linked to fanconi anemia and breast cancer . 
carcinogenesis 31 : 2031 - 2038 , 2010 sigurdsson s , van komen s , bussen w , et al : mediator function of the human rad51b - rad51c complex in rad51 / rpa - catalyzed dna strand exchange . genes dev 15 : 3308 - 3318 , 2001 4 . 
morales j , li l , fattah fj , et al : review of poly ( adp - ribose ) polymerase ( parp ) mechanisms of action and rationale for targeting in cancer and other diseases . crit rev eukaryot gene expr 24 : 15 - 28 , 2014 farmer h , mccabe n , lord cj , et al : targeting the dna repair defect in brca mutant cells as a therapeutic strategy . 
nature 434 : 917 - 921 , 2005 ashworth a : a synthetic lethal therapeutic approach : poly ( adp ) ribose polymerase inhibitors for the treatment of cancers decient in dna double - strand break repair . 
j clin oncol 26 : 3785 - 3790 , 2008 konstantinopoulos pa , ceccaldi r , shapiro gi , et al : homologous recombination deciency : exploiting the fundamental vulnerability of ovarian cancer . 
gynecol oncol 15 : 10 - 17 , 1983 kandoth c , schultz n , cherniack ad , et al : cancer genome atlas research network : integrated genomic characterization of endometrial carcinoma . 
sato k , koyasu m , nomura s , et al : mutation status of rad51c , palb2 and brip1 in 100 japanese familial breast cancer cases without brca1 and brca2 oncogene 26 : 2157 - 2165 , 2007 mutations . 
dong y , hakimi ma , chen x , et al : regulation of brcc , a holoenzyme complex containing brca1 and brca2 , by a signalosome - like subunit and its role in 16 . 
genomic testing was performed on a plessision internal clinical sequencing apparatus ( keio university , tokyo , japan ) , which is used for all genome sequencingrelated analyses in our hospital ( keio university hospital )  . 
this apparatus was used to extract genomic dna from tumor samples and peripheral blood mononuclear cells extracted from patients with cancer , after the provision of consent to undergo comprehensive genomic testing . 
this study was conducted in accordance with the declaration of helsinki and title 45 , us code of federal regulations , part 46 , protection of human subjects , effective december 13 , 2001 . dna quality was checked by calculating the dna integrity number ( din ) using an agilent 2000 tapestation ( agilent technologies , waldbronn , germany ) before conducting targeted amplicon exome sequencing of the 160 genes implicated in cancer using the illumina miseq sequencing platform ( illumina , san diego , ca )  . 
cancer - specic changes in somatic genes , including single - nucleotide variants , insertions / deletions , and copynumber variations were detected and used to determine the tumor mutation burden . 
these preclinical observations suggest a role for the targeted inhibitors of the bcr pathway in the treatment of dlbcl.1 dlbcl is most commonly present as malignant inltration of lymph nodes . 
the use of btk inhibitors is now incorporated into standard therapy for these conditions , and the genetic basis of acquired resistance in cll , dominated by acquired mutation of btk or plcg2 , has been well - studied.6 , 7 by contrast , the role of bcr inhibition in dlbcl remains less clear and little is known about the genetics of acquired resistance . a 75 - year - old male presented with rapidly enlarging cutaneous nodules . 
clinical examination revealed cutaneous nodules up to 25 mm in diameter affecting the feet , legs , arms , and abdomen . a repeat biopsy showed features identical to his original diagnostic biopsy ( fig 1a ) , and computed tomography scan conrmed exclusively cutaneous disease . 
the patient was managed with palliative radiotherapy . the rapid clinical response and prolonged remission , followed by later re - emergence of tumor , suggested the acquisition of new genetic alterations driving resistance to bcr inhibition . 
this study was approved by the east of england cambridge south research ethics committee ( approval reference number 07 / mre05 / 44 )  . full variant and copy number data are presented in the data supplement . 
 ( a ) histology and immunohistochemistry from tumor biopsy showing a diffuse inltration by large atypical cells with predominantly centroblast - like morphology expressing cd20 , mum1 , bcl2 , weak bcl6 , no cd10 , and mib1 proliferation fraction 90% . 
 ( b ) images of selected lesions on the forearm , hand , popliteal fossa , and abdomen taken prior to therapy ( top row ) , at 28 days ( middle row ) , and at 6 months ( bottom row ) of b cell receptortargeted therapy . 
 ( a ) summary of whole - exome sequencing of samples taken from three different timepoints ; diagnosis , prior to bcr - targeted therapy , and at the time of relapse . 
gray indicates no mutation or copy number change identied . ( b ) a simplied schematic showing the critical signaling components of the bcr pathway that converge onto activation of canonical nf - b . components targeted pharmacologically in this patient are indicated . 
we manually examined the loci of plcg2 and btk , mutations that are commonly acquired in btki - resistant cll , and conrmed both genes to be wild type at all timepoints . 
 case report in the bcr pathway to activate canonical nf - b signaling.1 activating mutation in the coiled - coil domain of card11 was previously proposed as a mechanism of resistance to bcr inhibition in mantle cell lymphoma8 and primary resistance in dlbcl.9 the acquired k215t mutation in our patient is located in the coiled - coil domain and has been previously shown to activate nf - b.10 we conrmed the near - clonal presence of the card11 k215t mutation at relapse ( variable allele frequency 40% ) but zero mutant reads at this position in either of the two pretreatment biopsies ( read depth , 180 and 210 , appendix fig a3 )  . 
taken together , these data strongly suggest that card11 mutation was the genetic driver of the acquired resistance in this case . the limited information about acquired resistance to bcr inhibition in dlbcl suggests potential differences compared with that of cll . 
the latter is predominantly associated with mutation of btk and plcg2.6 , 7 card11 identied in 10% - 15% of dlbcl at mutation is presentation11 but is rarely identied ( , 1% ) in cll.12 this suggests that the bcr signal in cll and dlbcl may be qualitatively different and therefore that genetic mechanisms of resistance to bcr inhibition may also differ . however , establishing the genetic basis of resistance to bcr inhibition in dlbcl presents specic challenges that contrast with the situation in cll . 
first , patients with dlbcl receiving bcr inhibitors are typically treated simultaneously with multiagent immunochemotherapy regimens , making it hard to establish which mutations provide resistance to which agent , or indeed if sensitivity to the bcr inhibitor ever existed in the rst place . 
combined with the biopsy - accessible cutaneous location of disease , this provided a rare opportunity to study genetic mechanisms of resistance to bcr inhibition in abc dlbcl . indeed , we have found only one other case describing the genetic basis of acquired resistance to bcr inhibition in dlbcl . 
this also involved a case of pcdlbcl - lt and also reported acquisition of card11 mutation ( interesting also k215 mutant ) in a patient who relapsed following an initial response to a btk inhibitor.13 however , the concurrent nding of nfkbie mutation and igh - irf8 translocation reported in that study might also have contributed to resistance , leaving the role of the card11 mutation uncertain . our ndings of acquired activating card11 mutation , the absence of any likely alternative genetic explanation , and the demonstrated ability of the k215t mutation to affect btki resistance in vitro strongly suggest that card11 mutation is the dominant driver of bcr independence in our case . as we move toward the introduction of precision medicine in dlbcl and the real - time monitoring of clonal evolution , it will become increasingly important to understand genetic mechanisms of drug resistance . 
our study uses the unique features of this case of cutaneous dlbcl to highlight the importance of card11 mutation as a driver of acquired resistance to bcr inhibition in dlbcl . 
hodson provision of study materials or patients : nimish shah collection and assembly of data : rebecca caeser , ieuan walker , jie gao , nimish shah , livia rasso - barnett , jose - ezequiel martin , daniel j . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . rebecca caeser consulting or advisory role : karus therapeutics nimish shah consulting or advisory role : abbvie speakers bureau : roche , janssen travel , accommodations , expenses : abbvie livia rasso - barnett leadership : cambridge pathology stock and other ownership interests : cambridge pathology honoraria : cambridge pathology consulting or advisory role : cambridge pathology daniel j . 
semin hematol 52 : 77 - 85 , 2015 swerdlow sh , campo e , pileri sa , et al : the 2016 revision of the world health organization classication of lymphoid neoplasms . 
blood 127 : 2375 - 2390 , 2016 grange f , beylot - barry m , courville p , et al : primary cutaneous diffuse large b - cell lymphoma , leg type : clinicopathologic features and prognostic analysis in 60 cases . 
mareschal s , pham - ledard a , viailly pj , et al : identication of somatic mutations in primary cutaneous diffuse large b - cell lymphoma , leg type by massive parallel sequencing . 
j invest dermatol 137 : 1984 - 1994 , 2017 pham - ledard a , prochazkova - carlotti m , andrique l , et al : multiple genetic alterations in primary cutaneous large b - cell lymphoma , leg type support a common lymphomagenesis with activated b - cell - like diffuse large b - cell lymphoma . 
j clin oncol 35 : 1437 - 1443 , 2017 ahn ie , underbayev c , albitar a , et al : clonal evolution leading to ibrutinib resistance in chronic lymphocytic leukemia . 
fox lc , yannakou ck , ryland g , et al : molecular mechanisms of disease progression in primary cutaneous diffuse large b - cell lymphoma , leg type during ibrutinib therapy . 
nagel d , spranger s , vincendeau m , et al : pharmacologic inhibition of malt1 protease by phenothiazines as a therapeutic approach for the treatment of aggressive abc - dlbcl . 
 case report chr1 chr3 chr5 chr11 chr13 chr15 chr17 chr19 chr21 chrx prdm1 tnfaip3 chr7 cdkn2a chr9 rb1 gna13 chr2 chr4 chr6 chr8 chr10 chr12 chr14 chr16 chr18 chr20 chr22 chry mir17 - 92 bcl2 malt1 tcf4 cd79a spib mir17 - 92 bcl2 malt1 tcf4 cd79a spib chr1 chr3 chr5 chr7 chr9 chr11 chr15 chr17 chr19 chr21 chrx chr2 chr4 chr6 chr8 chr10 chr12 chr14 chr16 chr18 chr20 chr22 chry prdm1 tnfaip3 rb1 chr13 gna13 appendix relapse pre - treatment diagnosis chr1 chr3 chr5 chr7 chr9 chr11 chr13 chr15 chr17 chr19 chr21 chrx chr2 chr4 chr6 chr8 chr10 chr12 chr14 chr16 chr18 chr20 chr22 chry fig a1 . 
 o genetic alterations detected in cell - free dna are associated with enzalutamide and abiraterone resistance in castration - resistant prostate cancer samantha torquato , phd1 ; aparna pallavajjala , ms1 ; alexa goldstein , ms1 ; patricia valda toro , ms1 ; john l . 
hurley , phd1 purpose androgen receptor ( ar ) gene alterations , including ligand - binding domain mutations and copy number ( cn ) gain , have yet to be fully established as predictive markers of resistance to enzalutamide and abiraterone in men with metastatic castration - resistant prostate cancer ( mcrpc )  . 
the goal of this study was to validate ar gene alterations detected in cell - free dna ( cfdna ) as markers of enzalutamide and abiraterone resistance in patients with mcrpc . methods patients with mcrpc ( n = 62 ) were prospectively enrolled between 2014 and 2018 . 
blood was collected before therapiesenzalutamide ( n = 25 ) , abiraterone ( n = 35 ) , or enzalutamide and abiraterone ( n = 2 ) and at disease progression . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction next - generation therapies that target the androgen androgen receptor ( ar ) axis , such as abiraterone and enzalutamide , have improved survival outcomes for men with metastatic castration - resistant prostate cancer ( mcrpc ) , 1 - 4 but both primary and acquired resistance to these drugs continue to be a substantial clinical challenge . 
resistance mechanisms are not fully understood ; however , some forms of resistance likely involve alterations to ar , including amplication and ligand - binding domain ( lbd ) missense mutations . 
although rare in primary prostate cancers , 5 - 7 ar gene alterations are highly prevalent in mcrpc.8 - 13 metastatic tissue biopsies as a sole means to detect and observe changes in ar status is impractical , and thus cell - free dna ( cfdna ) is gaining traction as a minimally invasive and easily obtainable tumor biopsy surrogate . 
previous studies using cfdna from the blood to evaluate the association of ar gene aberrations with resistance to abiraterone and enzalutamide are inclusive.14 - 17 ar copy number ( cn ) gain18 , 19 and / or amplication20 or detection of two or more ar mutations20 have been associated with worse outcomes to such therapies as abiraterone and enzalutamide . 
the ar splice variant ar - v7 is associated with resistance to enzalutamide and abiraterone21 - 23 and is also associated with increased ar cn.24 in addition to ar , alterations in other genes , including tumor protein p53 ( tp53 ) , phosphatase and tensin homolog ( pten ) , and breast cancer gene 2 ( brca2 ) , are enriched in lethal prostate cancer.8 - 11 studies support the idea that lineage plasticity from an ardependent to an ar - independent state through loss of tp53 and retinoblastoma - associated protein 1 ( rb1 ) mediates resistance to ar - targeted therapies.25 - 28 consistent with this , tp53 defects have been shown to be associated with worse outcomes with abiraterone and enzalutamide therapies.17 the role of brca2 and other homology - directed repair ( hdr ) genes in mediating resistance to enzalutamide and abiraterone has not been denitively determined . 
the secondary goal was to determine if alterations in other genes that are enriched in lethal prostate cancer , including tp53 , pten , and brca2 , were associated with response to enzalutamide and abiraterone . in this study , high circulating tumor dna ( ctdna ) burden was signicantly associated with prostate - specic antigen ( psa ) ( pfs ) , and overall survival ( os )  . 
study limitations , including sample size and patient heterogeneity , necessitate larger and prospective validation of the association of plasma ar status with outcomes . response , progression - free survival methods patient information , study end points , sample collection , deep next - generation sequencing ( ngs ) , sequence alignment and analysis of variants , cn variation , estimation of ctdna fraction , and statistical analyses are found in the data supplement . results patient cohort patient characteristics are listed in table 1 . 
clinvarannotated pathogenic or likely pathogenic missense mutations , truncating mutations , and / or cn alterations were detected in cfdna from 89% of patients before therapy initiation and in 92% of patients at disease progression ( figs 1a - 1d and data supplement )  . ctdna total cfdna concentration before therapy was associated with psa ( p = .002 ; data supplement )  . 
nearly all patients ( 61 of 62 ) had detectable cn variation ( s ) and / or mutation ( s ) with an allelic frequency above the 1% cutoff before therapy ( figs 1b and 1c )  . 
multivariable analyses controlled for ctdna high . abbreviations : ar , androgen receptor ; atm , ataxia - telangiectasia mutated gene ; brca1 / 2 , breast cancer gene 1 / 2 ; cn , copy number ; ctdna , circulating tumor dna ; lbd , ligand - binding domain ; or , odds ratio ; pi3k , phosphoinositide 3 - kinase ; psa , prostate - specic antigen ; rb1 , retinoblastoma - associated protein 1 ; tp53 , tumor protein 53 ; wnt , wingless - type mmtv integration site . h875y mutation had psa responses on abiraterone ( fig 2c )  . 
 ( b ) total number of protein - altering genetic changes in 46 genes detected by next - generation sequencing ( ngs ) of cfdna from 62 patients before abiraterone ( abi ) and enzalutamide ( enza ) therapy . 
 ( c ) genetic alterationscopy number ( cn ) status , clinvar pathogenic / likely pathogenic missense and germline mutations , and truncating mutationsin 46 genes detected by ngs of cfdna from 62 patients before abiraterone and enzalutamide therapy in order of best psa response . 
 ( d ) total number of genetic alterationscn , clinvar pathogenic / likely pathogenic missense and germline mutations , and truncating mutationsin 46 genes detected by ngs of cfdna from 62 patients before abiraterone and enzalutamide therapy . 
ar , androgen receptor ; ctdna , circulating tumor dna . defectspathogenic mutations and / or cn losswere not associated with psa response ( p = .602 ) or pfs ( p = .314 ; tables 2 and 3 )  . 
progression - free survival ( pfs ) : pathogenic androgen receptor ( ar ) ligand - binding domain ( lbd ) mutations are associated with a shorter time to progression . 
 ( a ) kaplan - meier method and log - rank test were used to determine median time to progression for patients who had high versus low circulating tumor dna ( ctdna ) before therapy . 
 ( b ) waterfall plot of best prostate - specic antigen ( psa ) response for all patients ( n = 62 ) after therapy as determined by best percentage fold change in psa . 
wingless - type mmtv integration site pathway defects involving genetic alterations in adenomatous polyposis colicn loss and / or truncating mutationsand - catenincn gain and pathogenic missense mutationswere detected in nearly 15% of patients before to therapy ( figs 1c and 1d )  . 
analysis of brca2 , and atm did not increase prognostic signicance . discussion liquid biopsies using cfdna as a tumor analyte are rapidly being developed for cancer diagnostics of solid tumors.35 - 37 when obtained concurrently , plasma - derived cfdna is highly concordant with tissue biopsies for tumor - specic genetic alterations.38 , 39 as a result of their advantages over traditional tissue biopsies , including the ease of accessibility for sequential monitoring of cancer dynamics and recapitulation of tumor heterogeneity , clinical development of cfdna has the potential to advance prostate cancer precision medicine.40 mechanisms of resistance to abiraterone and enzalutamide likely involve alterations to androgenar axis signaling . 
deregulation of these pathways likely mediates resistance to androgenar axis therapies . concurrent tp53 and rb1 defects are highly enriched in ar - independent neuroendocrine mcrpc compared with adenocarcinoma mcrpc.42 combined tp53 and rb1 loss has been shown to promote lineage switching from an ardependent to an ar - independent state41 , 43 , 44 and consequent resistance to ar - targeted therapies . 
similar to tp53 , genetic alterations in pten are enriched in mcrpc compared with metastatic castration - sensitive prostate cancer and localized prostate cancer.11 studies suggest that pten loss may mediate castration resistance by downregulating ar , 25 - 28 thereby supporting a rationale for combined inhibition of pi3k and arin pten - decient mcrpcs.45 , 46 association of pathogenic mutations in hdr genes with response to abiraterone and enzalutamide therapy is conicting . 
a clinical trial in patients with mcrpc suggested that genetic alterations in hdr genes that were detected in metastatic biopsy tissue may be associated with longer pfs when on abiraterone therapy.29 concordant ndings were observed in a second study that supported the idea that patients with mcrpc harboring a germline brca1 / 2 or atm mutation may also have improved outcomes to abiraterone and enzalutamide.30 in contrast , another study showed that truncating mutations in brca2 and atm detected in cfdna were associated with a shorter time to progression on abiraterone and enzalutamide therapies in treatment - nave patients with mcrpc.17 in our study , collective somatic and germline genetic alterations were also not associated with worse outcomes to enzalutamide and abiraterone . 
association differences may reect variables , such as sample size , prior treatment status , disease burden , disease heterogeneity , somatic versus germline , and single versus dual loss . 
certainly , additional prospective investigation is needed to determine the clinical signicance of hdr mutations as predictive markers to abiraterone and enzalutamide therapies . in the current study , many patients had detectable alterations that could serve as potential therapeutic targets . previous studies have shown that patients with mcrpc with either germline or somatic mutations in hdr genes achieved signicant responses to olaparib47 and to abiraterone plus veliparib.29 more than one quarter of patients in our study had a deleterious germline or somatic brca1 , brca2 , or atm mutation detected before therapy or at disease progression , which suggests that these patients may benet from therapies that target poly ( adp - ribose ) polymerase or platinum - based chemotherapy.29 , 47 , 48 in addition , immunotherapy trials have been largely unsuccessful in men with mcrpc49 ; however , rare responders have been reported.50 a seminal clinical trial demonstrated that microsatellite instable cancers caused by mismatch repair ( mmr ) gene deciency were sensitive to programmed death - 1 blockade , perhaps because of the formation of neoantigens resulting from increased mutational burden.51 inactivation of mmr genes and elevated mutational burden have been detected in some men with aggressive prostate cancers.33 , 52 , 53 one patient in our study had a detectable noncanonical mmr gene mutation in his cfdna and a correspondingly high fig 3 . 
 ( a ) kaplan - meier method and log - rank test were used to determine median os for patients who had high versus low circulating tumor dna ( ctdna ) before therapy . 
 ( b ) kaplan - meier method and log - rank test were used to determine median os for patients who had androgen receptor ( ar ) copy number ( cn ) gain before therapy . 
 ( c ) gene schematic illustrating deleterious tp53 mutations detected by deep next - generation sequencing ( ngs ) of cellfree dna ( cfdna ) before abiraterone and enzalutamide therapies and at disease progression while on therapy . 
 ( d ) kaplan - meier method and log - rank test were used to determine median os for patients who had tp53 defectscn loss and or clinvar pathogenic / likely pathogenic mutationsbefore therapy . 
 ( e ) kaplan - meier method and log - rank test were used to determine median os for patients who had both tp53 and retinoblastomaassociated protein 1 ( rb1 ) defects compared with patients who had tp53 defects but were rb1 intactcn loss and or clinvar pathogenic / likely pathogenic mutationsbefore therapy . 
 ( f ) kaplan - meier method and log - rank test were used to determine median os for patients who had pi3k pathway defectscn loss and / or truncating mutations in phosphatase and tensin homolog and / or cn gain of pik3cabefore therapy . association of pi3k defects with os controlled for ctdna burden using multivariable proportional hazards regression modeling . 
 ( g ) kaplan - meier method and log - rank test were used to determine median os for patients who had wingless - type mmtv integration site ( wnt ) pathway defectscn loss and / or truncating mutations in adenomatous polyposis coli and / or cn gain and / or pathogenic missense mutations in - cateninbefore therapy . 
consistent with other reports , patients with prior exposure to abiraterone and enzalutamide experienced worse outcomes.54 - 59 statistical signicance was not reached , likely because of the small overall sample size and the few patients with prior therapy . 
ngs protocols were adjusted on the basis of total input , but for patients with low input the lack of genetic alteration detection was considered indeterminate as opposed to negative . 
in addition , mutations in such genes as including ar - v7 , because of tp53 and atm detected in cfdna at low allelic frequencies may be false positives as a result of clonal hematopoiesis.60 corresponding tissue was not available for all samples to conrm tp53 status , and future studies will examine both prostate tumor tissue and blood leukocytes for genetic alterations . 
a nal limitation was our inability to evaluate ar the resplice variants , quirement of circulating tumor cells or whole - blood rna . the presence of ar - v7 is certainly another established mechanism of primary and acquired resistance to nextgeneration hormonal therapies.21 - 23 future studies should aim to simultaneously analyze the full complement of ar aberrations , including gene mutations , amplications , genomic structural rearrangements , and mrna splice variants , from a single liquid biopsy . in summary , our ndings indicate ctdna burden was highly associated with worse outcomes to enzalutamide and abiraterone . 
eisenberger leadership : veru stock and other ownership interests : veru honoraria : sano , pzer consulting or advisory role : astellas pharma , ipsen , bayer , sano , pzer research funding : sano , tokai pharmaceuticals , genentech travel , accommodations , expenses : bayer , astellas pharma , sano , pzer , veru emmanuel s . 
antonarakis honoraria : sano , dendreon , medivation , janssen biotech , essa pharma , astellas pharma , merck , astrazeneca , clovis oncology consulting or advisory role : sano , dendreon , medivation , janssen biotech , essa pharma , astellas pharma , merck , astrazeneca , clovis oncology research funding : janssen biotech ( inst ) , johnson & johnson ( inst ) , sano ( inst ) , dendreon ( inst ) , aragon pharmaceuticals ( inst ) , exelixis ( inst ) , millennium pharmaceuticals ( inst ) , genentech ( inst ) , novartis ( inst ) , astellas pharma ( inst ) , tokai pharmaceuticals ( inst ) , merck ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , constellation pharmaceuticals ( inst ) patents , royalties , other intellectual property : coinventor of a biomarker technology licensed to qiagen travel , accommodations , expenses : sano , dendreon , medivation bruce j . 
park leadership : loxo pharmaceuticals stock and other ownership interests : loxo pharmaceuticals consulting or advisory role : horizon discovery , foundation medicine , loxo pharmaceuticals , casdin capital , h3 biomedicine , jackson laboratory for genomic medicine , eli lilly speakers ' bureau : astrazeneca research funding : foundation medicine , abbvie , pzer patents , royalties , other intellectual property : royalties paid through inventions at johns hopkins university by horizon discovery travel , accommodations , expenses : eli lilly , loxo pharmaceuticals paula j . 
hurley stock and other ownership interests : loxo pharmaceuticals ( i ) honoraria : foundation medicine ( i ) , roche ( i ) , h3 biomedicine ( i ) , casdin capital ( i ) , loxo pharmaceuticals ( i ) , eli lilly ( i ) consulting or advisory role : loxo pharmaceuticals ( i ) , foundation medicine ( i ) , roche ( i ) , h3 biomedicine ( i ) , casdin capital ( i ) , lilly ( i ) patents , royalties , other intellectual property : cell lines at horizon ( i ) no other potential conicts of interest were reported . acknowledgment we thank the patients who consented to participate in this study . 
n engl j med 364 : 1995 - 2005 , 2011 ryan cj , smith mr , de bono js , et al : abiraterone in metastatic prostate cancer without previous chemotherapy . 
n engl j med 368 : 138 - 148 , 2013 scher hi , fizazi k , saad f , et al : increased survival with enzalutamide in prostate cancer after chemotherapy . 
n engl j med 367 : 1187 - 1197 , 2012 beer tm , armstrong aj , rathkopf de , et al : enzalutamide in metastatic prostate cancer before chemotherapy . 
cancer cell 18 : 11 - 22 , 2010 grasso cs , wu ym , robinson dr , et al : the mutational landscape of lethal castration - resistant prostate cancer . 
nature 487 : 239 - 243 , 2012 kumar a , coleman i , morrissey c , et al : substantial interindividual and limited intraindividual genomic diversity among tumors from men with metastatic prostate cancer . 
abida w , armenia j , gopalan a , et al : prospective genomic proling of prostate cancer across disease states reveals germline and somatic alterations that may affect clinical decision making . 
azad aa , volik sv , wyatt aw , et al : androgen receptor gene aberrations in circulating cell - free dna : biomarkers of therapeutic resistance in castration - resistant prostate cancer . 
annala m , vandekerkhove g , khalaf d , et al : circulating tumor dna genomics correlate with resistance to abiraterone and enzalutamide in prostate cancer . cancer discov 8 : 444 - 457 , 2018 18 . 
salvi s , casadio v , conteduca v , et al : circulating cell - free ar and cyp17a1 copy number variations may associate with outcome of metastatic castrationresistant prostate cancer patients treated with abiraterone . 
conteduca v , wetterskog d , sharabiani mta , et al : androgen receptor gene status in plasma dna associates with worse outcome on enzalutamide or abiraterone for castration - resistant prostate cancer : a multi - institution correlative biomarker study . 
qu f , xie w , nakabayashi m , et al : association of ar - v7 and prostate - specic antigen rna levels in blood with efcacy of abiraterone acetate and enzalutamide treatment in men with prostate cancer . 
antonarakis es , lu c , luber b , et al : clinical signicance of androgen receptor splice variant - 7 mrna detection in circulating tumor cells of men with metastatic castration - resistant prostate cancer treated with rstand second - line abiraterone and enzalutamide . 
cancer res 67 : 6535 - 6538 , 2007 jiao j , wang s , qiao r , et al : murine cell lines derived from pten null prostate cancer show the critical role of pten in hormone refractory prostate cancer development . 
hussain m , daignault - newton s , twardowski pw , et al : targeting androgen receptor and dna repair in metastatic castration - resistant prostate cancer : results from nci 9012 . 
antonarakis es , changxue l , luber b , et al : germline dna - repair gene mutations and outcomes in men with metastatic castration - resistant prostate cancer receiving rst - line abiraterone and enzalutamide . 
salvi s , casadio v , conteduca v , et al : circulating ar copy number and outcome to enzalutamide in docetaxel - treated metastatic castration - resistant prostate cancer . 
lallous n , volik sv , awrey s , et al : functional analysis of androgen receptor mutations that confer anti - androgen resistance identied in circulating cell - free dna from prostate cancer patients . 
na r , zheng sl , han m , et al : germline mutations in atm and brca1 / 2 distinguish risk for lethal and indolent prostate cancer and are associated with early age at death . 
de bono js , de giorgi u . , massard c , et al : pten loss as a predictive biomarker for the akt inhibitor ipatasertib combined with abiraterone acetate in patients with metastatic castration - resistant prostate cancer ( mcrpc )  . 
brahmer jr , drake cg , wollner i , et al : phase i study of single - agent anti - programmed death - 1 ( mdx - 1106 ) in refractory solid tumors : safety , clinical activity , pharmacodynamics , and immunologic correlates . 
kwon ed , drake cg , scher hi , et al : ipilimumab versus placebo after radiotherapy in patients with metastatic castration - resistant prostate cancer that had progressed after docetaxel chemotherapy ( ca184 - 043 ) : a multicentre , randomised , double - blind , phase 3 trial . 
loriot y , bianchini d , ileana e , et al : antitumour activity of abiraterone acetate against metastatic castration - resistant prostate cancer progressing after docetaxel and enzalutamide ( mdv3100 )  . 
brasso k , thomsen fb , schrader aj , et al : enzalutamide antitumour activity against metastatic castration - resistant prostate cancer previously treated with docetaxel and abiraterone : a multicentre analysis . 
maughan bl , luber b , nadal r , et al : comparing sequencing of abiraterone and enzalutamide in men with metastatic castration - resistant prostate cancer : a retrospective study . 
emamekhoo h , barata pc , edwin nc , et al : evaluation of response to enzalutamide consecutively after abiraterone acetate / prednisone failure in patients with metastatic castration - resistant prostate cancer . 
craig lockhart , md17 ; cristiana sessa , md18 ; thomas zander , md19 ; matthew ng , md , phd20 ; giuseppe curigliano , md , phd21 , 22 ; jennifer bendiske , mba23 ; xueying chen , phd23 ; somesh choudhury , phd23 ; diana graus - porta , phd24 ; nancy lewis , md23 ; jose manuel perez garcia , md , phd25 ; and mara jos e de miguel - luken , md , phd26 purpose concurrent pik3ca mutations and broblast growth factor receptor ( fgfr ) alterations occur in multiple cancer types , including estrogen receptorpositive breast cancer , bladder cancer , and endometrial cancer . 
archival tumor samples were sequenced to explore genomic correlates of response . results in combination , both agents were escalated to full , single - agent recommended doses ( alpelisib , 300 mg per day continuously ; ingratinib , 125 mg per day 3 weeks on followed by 1 week off )  . 
the toxicity prole of the combination was consistent with the established safety prole of each agent , although 71% of all patients required at least one treatment interruption or dose reduction . 
molecularly selected dose expansions in breast cancer and other solid tumors harboring pik3ca mutations , alone or in combination with fgfr alterations , identied sporadic responses , predominately in tumor types and genotypes previously dened to have sensitivity to these agents . conclusion the combination of alpelisib and ingratinib can be administered at full single - agent doses , although the high rate of dose interruption or reduction suggests long - term tolerability may be challenging . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction genome - driven oncology has been focused primarily , to date , on the targeting of individual genomic alterations present in each tumor.1 although this approach has led to the development of many highly effective therapies , 2 - 6 we now understand that most oncogenes exist within a complex genomic landscape characterized by additional alterations that may , themselves , be targetable or modify sensitivity to therapy.7 even in instances in which the paradigm of targeting an individual genomic alteration has been successful , adaptation of the tumor eventually leading to acquired resistance ultimately limits effectiveness . 
 hyman et al context key objective to determine if the - elective pi3k inhibitor alpelisib can be combined safely with the selective broblast growth factor receptor ( fgfr ) inhibitor ingratinib . knowledge generated alpelisib and ingratinib were successfully escalated in combination to full single - agent doses ; however , high rates of treatment interruption and dose reduction suggest long - term tolerability may be challenging . 
in exploratory signal - seeking cohorts of patients harboring dual pik3ca and fgfr1 - 3 alterations , no clear evidence of synergistic activity was observed . relevance additional studies are necessary to determine the potential role of combined pi3k and fgfr inhibition in treatment of various advanced solid tumors with alterations in pik3ca and / or fgfr1 - 3 . suggested that the combined inhibition of these oncogenes might be benecial.14 moreover , in breast cancer , pik3ca mutations often co - occur with fgfr1 gene amplications , indicating pik3ca and fgfr1 could cooperate as potential dual oncogenic drivers in this tumor type.12 , 15 similarly , concurrent activating pik3ca and fgfr2 mutations have been observed in endometrial carcinomas , with 90% or more fgfr2 - mutant endometrial cancers also exhibiting genomic activation of the pi3k pathway.16 - 18 taken together , these data provided a rationale for evaluating the feasibility and preliminary activity of combined fgfr and pik3ca inhibition in the clinic . to evaluate this therapeutic strategy , we studied the combination of alpelisib ( byl719 ) and ingratinib ( bgj398 )  . 
alpelisib is a selective class i pi3k inhibitor that recently demonstrated a statistically signicant and clinically relevant improvement in progression - free survival in a phase iii study ( solar - 1 ) of patients with pik3camutant , er + metastatic breast cancer.19 ingratinib is an orally bioavailable , selective pan - fgfr kinase inhibitor that has demonstrated single - agent activity in fgfr2 / 3altered bladder cancers and fgfr2 fusion - positive cholangiocarcinomas.20 , 21 we hypothesized that the combination of a selective fgfr and pik3ca inhibitors would be safe and would synergize in tumors with concurrent alteration of these oncogenes . 
dose escalation was guided by a bayesian logistic regression model with escalation with overdose control.22 in total , three arms in the expansion cohort were planned : dual pik3camutant and fgfr - altered breast ( arm 1 ) and nonbreast ( arm 2 ) cancers and pik3ca - mutant solid tumors ( arm 3 )  . because of the slow rate of accrual and overall safety prole , the study was closed before the target enrollment was completed . the primary objective was to determine the maximum tolerated dose and / or recommended dose for expansion of the combination of ingratinib and alpelisib . 
the secondary objectives included assessment of safety and tolerability of the combination , characterization of pharmacokinetic ( pk ) proles , assessment of preliminary antitumor activity as measured by overall response rate ( orr ) , and progressionfree survival ( pfs ) , per recist , version 1.1. study drug administration eligibility patients were at least 18 years of age and had advanced solid tumors with pik3ca mutation with or without fgfr genetic alteration in which standard therapy was unsuccessful . 
patients remained in the study until disease progression , unacceptable adverse investigator events ( aes ) , or withdrawal at patient or discretion . study assessments tumor responses were measured by recist , version 1.1 ( investigator assessed ) , using computed tomography or magnetic resonance imaging.23 assessments were performed at screening and every 8 weeks after rst treatment until disease progression . 
safety was monitored throughout institute the study according to the national cancer common terminology criteria for adverse events , version 4.03.24 ecgs were assessed and independently reviewed by a central laboratory . pharmacokinetic analysis during dose escalation , blood samples were collected to measure alpelisib and ingratinib plasma concentrations . samples were collected predose and 0.5 , 1 , 2 , 3 , 4 , 6 , 8 , and 24 hours postdose on cycle 1 day 1 , cycle 1 day 15 , and cycle 2 day 1 . 
drug plasma concentrations ( and active metabolites ) were measured using validated liquid chromatographytandem mass spectrometry with a lower limit of quantication of 1 ng / ml . genomic sequencing genomic alterations potentially associated with fgfr and pik3ca signaling were assessed from archival tumor specimens of fresh , pretreatment tumor biopsy specimens . for samples with sufcient tissue material ( n = 41 ) , nextgeneration sequencing ( ngs ) analysis was performed in a clinical laboratory improvement amendmentscertied laboratory ( foundation medicine , cambridge , ma ) using previously published methods.25 statistical analysis the data cutoff for all statistical analyses was august 23 , 2016 . 
the sample size for each expansion cohort was selected primarily for continued safety evaluation and was not powered for any specic efcacy end point . results patient demographics a total of 62 patients were enrolled in the study between october 2013 and august 2016 across 22 clinical centers in 12 countries . 
the median age of patients at the time of study entry was 60 ( range , 30 to 78 ) years , and all patients had an eastern cooperative oncology group performance status of 1 or less . 
patients were heavily pretreated , having received a median of three prior lines of systemic therapy . safety and tolerability dose - limiting toxicities were reported in three patients ( 10.7% ) who were included in the dose - escalation cohort ( n = 38 ) during the rst 28 days of treatment . 
dose - limiting toxicities included grade 4 hyperglycemia in one patient receiving alpelisib 300 mg and ingratinib 40 mg ; and grade 3 stomatitis in two patients ( one receiving alpelisib 300 mg and ingratinib 75 mg ; and the other receiving alpelisib 300 mg and ingratinib 100 mg )  . 
the recommended dose for expansion was determined to be alpelisib 300 mg once per day ( administered continuously ) and ingratinib 125 mg once per day ( administered on a schedule of 3 weeks on followed by 1 week off )  . overall , the safety prole of alpelisib and ingratinib was similar to the known safety proles of each agent ; no new major aes were noted ( table 2 )  . 
serious aes suspected to be drug related were reported in 12.9% of patients ( n = 8 of 62 ) overall and 16.1% ( n = 5 of 31 ) treated at the recommended dose for expansion . 
a total of 13 deaths ( 21% ) were reported during the study , including three ( 4.8% ) deaths resulting from disease progression while the patient was receiving study treatment . 
similarly , hyperglycemia , which is a biomarker of on - target pi3k inhibition , was also observed in 38.7% of all patients treated with alpelisib 200 mg or 300 mg , consistent with prior experience with this agent.27 for ingratinib , 37 patients ( 59.7% ) reported at least one dose interruption and 23 patients ( 37.1% ) reported at least one dose reduction . 
there was no or negligible pk interaction between ingratinib and alpelisib . antitumor efcacy in total , 52 patients were evaluable for response and 10 ( n = 6 in the dose escalation study ; n = 4 in the dose expansion study ) were nonevaluable on the basis of discontinuing therapy before the rst disease assessment ( table 3 )  . 
among 60 patients with a locally annotated pik3ca mutation reported , sequencing with high condence coverage was obtained centrally in 35 samples and the locally reported pik3ca variant conrmed in 80% patients ( n = 28 of 35 )  . 
 ( % ) , except where n = 0 . abbreviations : cr , complete response ; pd , progressive disease . * for patients who only had nontarget lesions at baseline and did not experience either a cr or pd , response is reported as non - cr / non - pd . patient with unknown best overall response due to ( 1 ) no valid postbaseline assessment , or ( 2 ) stable disease occurred too early ( , 6 weeks ) dened as patients who have best overall response cr , partial response , or stable disease , in which stable disease needs to occur at least 6 weeks after the rst dosing . squamous anal cancer with pik3ca e545k mutation . 
interestingly , in the patient with melanoma who had a pr , the only genomic activation of either pathway identied by central sequencing was an fgfr2 m722i mutation , an alteration not known to be recurrent or of biologic signicance.27 we also explored whether genetic alterations of the mapk pathway co - occurred with mutations in the fgfr and / or pi3k pathways and may account for lack of response , at least partially , in those settings . 
overall , ngs studies showed no clear association of alterations in the fgfr , pi3k , or mapk pathways with response . discussion to our knowledge , this is the rst study to evaluate the safety and preliminary efcacy of combined selective pi3k and fgfr inhibition in patients . 
we dened a recommended dose for expansion of alpelisib 300 mg once per day and ingratinib 125 mg once per day , 3 weeks on followed by 1 week off , which is the full single - agent dose and schedule of each drug . 
although this combination did not raise new specic safety concerns , 71% of patients required a dose interruption of alpelisib and 60% of patients reported at least one dose interruption of ingratinib during the course of treatment . 
of note , dose interruptions in the range of 50% to 70% have previously been reported with each agent when used individually , again suggesting that their combination may not be associated with signicant additive toxicity over the component parts but that long - term tolerance each drug alone can exhibit issues.20 , 28 , 29 no drug - drug interactions were reported between ingratinib and alpelisib . this study does have some important limitations . 
the relatively small number of patients treated with relevant concurrent alterations , as well as the heterogeneity of the population , do not permit a denitive assessment of synergistic efcacy . as such , any efcacy data presented from these expansions should be considered hypothesis generating only . second , we note that in the subset of 58% of patients ( n = 35 of 60 ) with tumor tissue in which successful central sequencing was completed , the pik3ca mutations were centrally conrmed in 80% of patients . 
as a result , some patients may have been enrolled on the basis of pik3ca mutations characterized by subclonality , spatial or temporal heterogeneity , or even test failures , as has been observed in prior genome - driven studies.7 finally , enrollment in the expansion cohorts for patients harboring concurrent pik3ca and fgfr1 - 3 alterations was not restricted to variants functionally characterized as activating but to regions of the affected genes that are recurrently the target of alteration . 
efcacy by ( a ) a waterfall plot depicting best percent change in the target tumor burden from baseline according to cohort and ( b ) a swimmer plot depicting progression - free survival by patients according to cohort . 
integrated genomic analysis of efcacy according to mutations in the fgfr , pi3k , and mapk pathways by central next - generation sequencing . ( * ) missing response data , as a result of discontinuation before rst response assessment . 
indel , insertion and / or deletion . fgfr amplication is partially dependent on the degree of amplication.30 both the inclusion of variants of unknown signicance as well as copy number amplications with a specic fold - change requirement may have adversely affected the observed orr by enrolling tumors without true pathway dependency on fgfr . despite these important limitations , a relatively low orr of only 9.7% across the entire study suggests that these two classes of agents may not provide synergistic activity . moreover , responses were generally observed in tumors arising from anatomic sites and harboring genetic alterations previously described as sensitizing to either alpelisib or ingratinib . 
it is also noteworthy that a signicant proportion of patients did not respond , or had stable disease as their best response , despite harboring pik3ca mutations alone or in combination with fgfr alterations . 
 hyman et al implementation has been identication of targeted therapy combinations have often been cited as a means of overcoming both intrinsic and acquired resistance , but a number of scientic , technical , and logistic impediments have made successful implementation challenging.31 , 32 perhaps the most difcult barrier to successful right drug combinations . 
enrichment for concurrent alterations of two oncogenes in one or multiple cancer types has served as one basis for selecting potential drug combinations and formed the basis of the hypothesis evaluated in this clinical study . 
recently , ndings were reported from two precision medicine studies , i - predict and winther , suggesting that a higher degree of molecular matching between somatic alterations detected at baseline and the therapy administered was associated with improved outcomes.33 , 34 although these results imply that targeting multiple oncogenic alterations with polytherapy may be a worthwhile strategy in select circumstances , the data presented here demonstrate that more rigorous evaluation of specic biomarker and drug combinations is required before the approach is more widely embraced . in conclusion , the combination of pik3ca and fgfr inhibition with alpelisib and ingratinib was feasible , although overall tolerability was limited by each agent . 
schellens , mario campone , cristiana sessa , giuseppe curigliano , jennifer bendiske , xueying chen , somesh choudhury , nancy lewis , jose manuel perez garcia , mara jos e de miguel - luken provision of study material or patients : luis paz - ares , jan h . 
craig lockhart , cristiana sessa , thomas zander , matthew ng , giuseppe curigliano , jose manuel perez garcia , mara jos e de miguel - luken collection and assembly of data : david m . 
craig lockhart , cristiana sessa , thomas zander , giuseppe curigliano , xueying chen , somesh choudhury , nancy lewis , jose manuel perez garcia , mara jos e de miguel - luken data analysis and interpretation : david m . 
schellens employment : modra pharmaceuticals stock and other ownership interests : modra pharmaceuticals honoraria : debiopharm group consulting or advisory role : debiopharm group research funding : dutch cancer society ( inst ) patents , royalties , other intellectual property : patent on oral taxanes philippe l . 
bedard research funding : bristol - myers squibb ( inst ) , sano ( inst ) , astrazeneca ( inst ) , roche ( inst ) , servier ( inst ) , glaxosmithkline ( inst ) , novartis ( inst ) , signalchem ( inst ) , ptc therapeutics ( inst ) , nektar ( inst ) , merck ( inst ) , seattle genetics ( inst ) , mersana ( inst ) , immunomedics ( inst ) , eli lilly ( inst ) mario campone honoraria : novartis , eli lilly consulting or advisory role : novartis ( inst ) , servier ( inst ) , menarini , sano ( inst ) , eli lilly ( inst ) , pzer ( inst ) , astrazeneca / medimmune ( inst ) , abbvie ( inst ) , pierre fabre ( inst ) , accord ( inst ) , sandoz - novartis ( inst ) speakers bureau : novartis , amgen research funding : novartis ( inst ) travel , accommodations , expenses : novartis , astra zeneca , pzer other relationship : roche philippe a . 
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nature 554 : 189 - 194 , 2018 [ erratum : nature 2019 ; 566 : e11 - e12 ] baselga j , campone m , piccart m , et al : everolimus in postmenopausal hormone - receptor - positive advanced breast cancer . 
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corcoran rb , atreya ce , falchook gs , et al : combined braf and mek inhibition with dabrafenib and trametinib in braf v600mutant colorectal cancer . j clin oncol 33 : 4023 - 4031 , 2015 11 . 
hortobagyi gn , chen d , piccart m , et al : correlative analysis of genetic alterations and everolimus benet in hormone receptorpositive , human epidermal growth factor receptor 2negative advanced breast cancer : results from bolero - 2 . 
nogova l , sequist lv , perez garcia jm , et al : evaluation of bgj398 , a broblast growth factor receptor 1 - 3 kinase inhibitor , in patients with advanced solid tumors harboring genetic alterations in broblast growth factor receptors : results of a global phase i , dose - escalation and dose - expansion study . 
pal sk , rosenberg je , hoffman - censits jh , et al : efcacy of bgj398 , a broblast growth factor receptor 13 inhibitor , in patients with previously treated advanced urothelial carcinoma with fgfr3 alterations . 
neuenschwander b , matano a , tang z , et al : bayesian industry approach to phase i combination trials in oncology , in zhao w , yang h , eds : statistical methods in drug combination studies . 
w ohrle s , bonny o , beluch n , et al : fgf receptors control vitamin d and phosphate homeostasis by mediating renal fgf - 23 signaling and regulating fgf - 23 expression in bone . 
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cancer discov 8 : 174 - 183 , 2018 javle m , lowery m , shroff rt , et al : phase ii study of bgj398 in patients with fgfr - altered advanced cholangiocarcinoma . 
scarlett uk , chang dc , murtagh tj , et al : high - throughput testing of novel - novel combination therapies for cancer : an idea whose time has come . 
indeed , triple - negative breast cancer ( tnbc ) , the most lethal breast cancer subgroup , has been reported to occur at a higher incidence in japan than in the united states . 
here , we sought to elucidate the geographic and ethnic diversity of breast cancer by examining actionable driver alterations in tnbc tumors from japanese patients and comparing them with the cancer genome atlas ( tcga ) database , which gathers data primarily from non - asian patients . materials and methods we performed comprehensive genomic profiling , including an analysis of 435 known cancer genes , among japanese patients with tnbc ( n = 53 ) and compared the results with independent data obtained from tcga ( n = 123 )  . results driver alterations were identified in 51 ( 96% ) of 53 japanese patients . 
by american society of clinical oncology introduction although breast cancer is one of the most frequently diagnosed cancers among women in the united states and japan , 1 , 2 its biologic characteristics may differ among different geographic or ethnic populations . 
indeed , breast cancer incidence , peak age of highest incidence , and mortality are different between women in the united states and japan.3 importantly , triple - negative breast cancer ( tnbc ) , the most lethal subgroup of breast cancer and characterized as estrogen receptor negative and progesterone receptor negative with no overexpression of human epidermal growth factor receptor 2 / neu , 4 is known to be more prevalent in japan than in the united states.5 , 6 large - scale genomic studies based on next generation sequencing ( ngs ) , such as the cancer genome atlas ( tcga ) and the international cancer genome consortium , have revealed important driver gene alterations in many types of solid tumors , 7 - 14 which have led to new targeted therapeutic strategies for precision cancer medicine.15 - 19 these studies have also highlighted the geographic and ethnic diversity in the cancer genome.20 , 21 however , most ngs - based studies have been conducted in western countries , where the asian population is a minority , and the resulting genomic information may not truly represent this population.8 for instance , only 5.5% of participants have identified themselves as asians in the tcga cohort . 
tnbc tumors in both the japanese cohort and the tcga database were defined according to previous reports.23 , 24 sequencing library preparation archival tissue in the formalin - fixed , paraffin embedded ( ffpe ) tumor obtained during routine biopsy or resection of primary tumor was used for analysis . 
all sample preparation , comprehensive genomic sequencing , and analytics were performed in a clinical laboratory improvement amendments / college of american pathologistsaccredited laboratory ( kew , cambridge , ma ) as described previously.19 comprehensive genomic sequencing ffpe genomic dna ( 50 to 150 ng ) was used to construct libraries that were enriched for 435 genes using cancerplex ( kew ) as described previously.19 the term gene alterations is used when there are genetic aberrations such as amplifications in addition to mutations . 
to compare the mutational burden of the 435 - gene panel with that of the tcga whole - exome sequencing ( wes ) data , the data set of single - nucleotide polymorphisms was downsampled to the 435 genes in the panel , and the panel mutation rate was determined , calculated as mutations per megabase , as described previously.19 results genetic alterations in japanese patients with tnbc the clinicopathologic characteristics of the patients with tnbc in the japanese and tcga cohorts are summarized in the data supplement . 
the japanese cohort included more patients with m1 disease ( 11% ) compared with the tcga cohort ( 1% ; p < .01 ) , which seemed to affect the difference in stage among the two cohorts ( data supplement )  . 
after performing comprehensive genomic sequencing on all japanese tnbc samples , we found at least one alteration in 82 of 435 cancer - associated genes ( fig 1 ; data supplement )  . 
although a total of 186 alterations were found in the 53 patients , there were only three frequently altered genes ( seen in > 10% of patients ) : tp53 , pik3ca , and pten ( fig 1 )  . 
among them , biopsy specimens of primary tumors before neoadjuvant chemotherapy were used for analysis in 11 patients , and surgical specimens of remnant primary tumors after neoadjuvant chemotherapy were used in 24 patients ( data supplement )  . 
 ( * ) p < .05 for alteration frequencies between japanese patients and the tcga cohort . genes including those for tp53 , pik3ca , and pten , were comparable between the two groups ( data supplement )  . 
of note , there were two distinct categories in the tcga cohort : tnbc based on pathologic subtyping and basal - like breast cancer based on pam50 analysis , with some overlap between the two groups . 
interestingly , the tp53 alteration rate in tnbc ( 65 [ 52.8% ] of 123 ) was relatively low in the tcga cohort , although the rate in basal - like breast cancer ( 73 [ 78.5% ] of 93 ) was much higher in the same cohort ( data supplement )  . 
generally , the gene alteration rates were comparable between japanese patients and the tcga cohort ; however , the alteration frequencies in two genes ( myc and ptk2 ) were significantly different between the two cohorts ( fig 1 ; data supplement )  . similar to that of the tcga cohort ( figs 2a to 2j )  . 
however , there were some exceptions , such as significantly more amplification of myc in the tcga cohort ( 28% ) than in japanese patients ( 6% ; p < .01 ; fig 2 )  . we found that eight patients ( 15% ) showed brca1 / 2 alterations , including five brca1 and three brca2 alterations ( figs 2a to 2j )  . 
moreover , bioinformatic analysis revealed that seven of eight alterations were possible germ line alterations , based on the allelic fraction and alteration site compared with a database , such as clinvar ( table 1 )  . 
we did not confirm the germ line alteration using patients normal tissue . genomic alterations in cancer signaling pathways tumor mutational burden and microsatellite instability in patients with tnbc we next assessed genomic alterations in major oncogenic pathways involving phosphatidylinositol 3 - kinase , the cell cycle , dna double strand break ( dsb ) repair , erbb2 / kras , and - catenin / wnt signaling and determined whether there were differences between the two cohorts ( figs 2a to 2j )  . 
overall , the gene alteration spectrum of the japanese cohort was a high tumor mutational burden ( tmb - h ) tumor is defined as a tumor with a high rate of somatic mutation . 
 ( a - e ) japanese patients ( n = 53 ) and ( f - j ) the cancer genome atlas ( tcga ) cohort ( n = 123 ) were evaluated for gene alterations in key cancer pathways : ( a , f ) phosphatidylinositol 3 - kinase ( pi3k ; 45% and 25% of patients , respectively ) , ( b , g ) cell cycle ( 26% and 29% of patients , respectively ) , ( c , h ) dna double - strand break ( dsb ) repair ( 75% and 58% of patients , respectively ) , erbb2 / kras ( 17% and 11% of patients , respectively ) , and ( e , j ) - catenin / wnt ( 11% and 29% of patients , respectively )  . 
this classification was based on our previous studies of colorectal and gastric cancers , which included a number of patients with microsatellite instability , although no microsatellite instability was observed in any of the 53 japanese patients with tnbc ( fig 3a )  . 
 ( a ) mutation rate and microsatellite instability ( msi ) in patients with triple - negative breast cancer in japan ( n = 53 ) ; three ( 5.7% ) of 53 hypermutated . 
 ( b ) mutation rate data from whole - exome sequencing ( wes ) for the cancer genome atlas cohort ( n = 123 ) were downsampled to the content of the 435 - gene panel platform ; six ( 4.9% ) of 123 hypermutated . 
cluster one ( n = 83 ; 47.2% ) , in which no common actionable alterations were seen , was the largest of the 10 identified clusters , and cluster nine ( n = 23 ; 13.1% ) , in which pik3ca alterations were common , was the second largest cluster ( fig 4b )  . 
cluster seven ( n = 16 ) was characterized by pten alterations , cluster eight ( n = 12 ) was characterized by cdkn2a and cdkn2b alterations , and clusters six ( n = 6 ) and three ( n = 6 ) were characterized by brca1 and brca2 alterations , respectively . 
moreover , cluster two ( n = 7 ) contained fgfr1 alterations , cluster four ( n = 7 ) contained ddr2 alterations , and cluster five ( n = 10 ) contained ccnd2 / 3 alterations . 
 a d0578 d0575 d0588 d0600 tcgaewa1p701 tcgaewa1p401 d0605 d0601 d0606 d0607 d0611 d0610 d0614 d0616 d0717 d0716 d0719 d0735 d0741 d0739 tcgaa2a0d001 tcgaa2a0d201 tcgaa2a0t201 tcgaa2a0st01 tcgaa2a1g601 tcgaa7a26g01 tcgaana0fx01 tcgaa8a07c01 tcgaana0g001 tcgaaoa03u01 tcgaaoa12801 tcgaaoa0j401 tcgaara0ts01 tcgaara0u101 tcgab6a0iq01 tcgaara1ay01 tcgab6a0re01 tcgab6a0rs01 tcgab6a0wx01 tcgab6a0ru01 tcgabha0av01 tcgabha0b901 tcgabha0rx01 tcgabha0bl01 tcgabha1fc01 tcgac8a26y01 tcgad8a27f01 tcgad8a1jl01 tcgad8a27m01 tcgae2a14r01 tcgae2a15801 tcgae2a14x01 tcgae2a1b601 tcgae2a1l701 tcgae2a1lk01 tcgae2a1li01 tcgae2a1ls01 tcgaewa1p101 tcgad8a1jf01 tcgaara1aq01 d0597 tcgad8a1xk01 tcgabha18t01 tcgae2a15001 tcgabha18q01 tcgad8a1jg01 tcgac8a13401 d0595 tcgaewa1p801 tcgaa7a0da01 tcgaara0tu01 tcgac8a27b01 tcgaaoa0jl01 tcgac8a26x01 d0625 d0609 tcgaa7a26i01 tcgaa8a09x01 tcgad8a14201 tcgaa7a26f01 tcgaana0fl01 tcgaa2a04q01 d0581 tcgaara1ar01 d0574 tcgaa2a0ym01 tcgabha0e601 tcgaara25601 tcgae2a1lh01 tcgae2a1lg01 tcgaana04d01 d0718 tcgaara1ai01 d0577 tcgaa7a0ce01 d0586 tcgab6a0rt01 tcgaa2a0cm01 tcgac8a1hj01 tcgad8a14701 tcgaa8a07o01 tcgabha0e001 tcgaaoa12901 tcgaa2a04t01 tcgaa1a0sk01 tcgae2a1az01 tcgae9a22g01 d0584 tcgaa1a0sp01 d0585 tcgaana0ar01 tcgaewa1ow01 tcgaaoa12f01 d0736 d0594 tcgaana0xu01 tcgabha18g01 tcgaa1a0so01 tcgabha0wa01 tcgaaoa0j201 d0738 tcgae2a15901 tcgae9a1nd01 d0580 tcgabha0b301 d0598 tcgaara0u001 d0579 d0599 tcgaewa1ov01 tcgaewa1pb01 tcgaa2a0ye01 d0590 tcgad8a13z01 tcgaa2a0t001 tcgabha0bg01 d0589 tcgabha0bw01 tcgaana0al01 d0740 tcgabha18v01 tcgaana0at01 d0604 tcgaara0u401 tcgaaqa04j01 tcgae2a1ll01 tcgaa2a04u01 tcgad8a1xq01 tcgabha1ew01 tcgad8a27h01 tcgaa2a0sx01 tcgaaoa12401 tcgac8a13101 d0596 tcgaa2a04p01 d0592 d0591 d0576 d0733 d0638 d0583 d0742 tcgaa8a08r01 tcgab6a0rg01 tcgab6a0ie01 tcgac8a12v01 tcgab6a0rn01 tcgab6a0ik01 tcgad8a14301 d0572 d0587 tcgae2a14n01 tcgaaoa0j601 d0615 d0573 d0582 donors fig 4 . 
 ( a ) a cluster analysis was performed on 176 cases of triple - negative breast cancer ( tnbc ; 53 tumors from japanese patients and 123 tumors from the cancer genome atlas [ tcga ] cohort ) by using euclidean distance and wards clustering method ( distance based on common mutated genes are colored blue to yellow )  . 
ado - trastuzumab , emtansine , lapatinib , pertuzumab , trastuzumab japanese , 3 ( 5.7% ) of 53 ; tcga , 6 ( 4.9% ) of 123 kit , pdgfr , abl imatinib japanese , 2 ( 3.8% ) of 53 ; tcga , 15 ( 12.2% ) of 123 jak1 / 2 . 
belinostat , panobinostat , vorinostat japanese , 0 ( 0% ) of 53 ; tcga , 6 ( 4.9% ) of 123 ceritinib , crizotinib , alectinib japanese , 0 ( 0% ) of 53 ; tcga , 5 ( 4.1% ) of 123 vegfr2 . 
genetic alterations in japanese patients with triple - negative breast cancer and patients in the cancer genome atlas ( tcga ) cohort potentially targetable by us food and drug administration ( fda ) approved cancer therapies . 
interestingly , we similarly analyzed the tcga cohort and found that , as with the japanese cohort , 66.7% of cases had at least one potentially actionable alteration associated with fda - approved drugs . for japanese patients , genes associated with mammalian target of rapamycin ( mtor ) , poly ( adp - ribose ) polymerase ( parp ) , and cdk4 / 6 inhibitors showed the three highest alteration rates ( fig 5 )  . 
seventeen percent of japanese patients and 22% of tcga cases had alterations in actionable genes related to the cell - cycle pathway , which could be treated by cdk4 / 6 inhibition . 
moreover , other fda - approved drugs , including multitargeted tyrosine kinase inhibitors , mek inhibitors , antihuman epidermal growth factor receptor 2 therapies , and jak inhibitors , were indicated as potential targeted therapies for japanese patients with tnbc , with even more drugs suggested for tcga cases ( fig 5 )  . 
of note , oncoprint analysis showed coalterations in many cases of tnbc in both cohorts , which may be associated with resistance to the targeted therapies ( fig 5 )  . 
however , the overall alteration spectrum of the japanese patients was similar to that of the tcga cohort.8 importantly , 66% of japanese patients , as well as 67% of tcga cases , had at least one genomic alteration in actionable genes , providing potentially new treatment strategies . we demonstrated significant differences in the frequencies of alterations in two genes : ptk2 and myc . 
it has been previously reported that myc is one of the most important driver genes , and there are known ethnic differences in myc amplification frequency.30 although we found a myc amplification rate of 5.7% in our cohort , high incidence ( 30% to 35% ) of myc amplification has been repeatedly reported from us groups , similar to tcga rates.31 - 33 a unique patient population comprises the cohort described in our study , which could be useful given the sparse genomic data from asian tnbc populations . we analyzed genomic alterations in ffpe tumor tissue from a cohort of japanese patients with tnbc by comprehensive ngs - based sequencing of a panel of 435 cancer - relevant genes , at an average 500 depth . 
method of preservation is critical for dna analysis using ffpe tissue samples , as we reported previously.34 the quality of all dna samples used in this study was confirmed to be adequate for accurate ngs analysis by strict quality control checks . 
indeed , most of the genes altered in both the japanese and tcga cohorts showed similar frequencies , consistent with previous reports , despite the different sequencing techniques used . previous comprehensive genomic profiling studies have reported that tnbc is a heterogeneous disease involving diverse genomic alterations.8 , 35 , 36 our results also demonstrated the heterogeneity of tnbc , with only three genes mutated in > 10% of patients and most of the genetic alterations differing from one another . 
 more comprehensive approach , such as the one used in this study , will be required to find actionable alterations in each patient with tnbc . to explore the clinical utility of targeted sequencing with a large panel for tnbc , we identified actionable genes that can be treated by fda - approved drugs and found that 35 japanese patients ( 66% ) had at least one such actionable gene alteration . 
we identified several signaling pathways for molecular targeted therapies , including the pi3k pathway for mtor inhibitors , the dsb repair pathway for parp inhibitors , and the cell - cycle pathway for cdk4 / 6 inhibitors ( fig 5 )  . 
although none of these patients received immune checkpoint inhibitor therapy , there is a high likelihood that these patients may have responded , given clinical data demonstrating efficacy in tmb - h patients . in our study , we found that seven ( 13% ) of 53 japanese patients with tnbc had possible germ line brca1 / 2 alterations based on bioinformatic analysis of tumor dna . 
although specific genetic tests are needed to confirm the frequency of germ line brca1 / 2 alterations , this estimated frequency is in agreement with previous reports.40 recently , germ line alteration of brca1 / 2 has attracted increasing attention because of the use of parp inhibitors . 
therefore , we considered 53 as a sufficient number for the japanese cohort , at least to the extent that the alteration rate of more than one gene was significantly different between the japanese and tcga cohorts . another limitation is that we included patients receiving neoadjuvant chemotherapy , and we cannot exclude the possibility that the neoadjuvant chemotherapy affected genomic alterations . 
however , when we compared the gene alterations between the chemotherapy - nave tissue samples ( n = 29 ) and chemotherapy affected tissue samples ( n = 24 ) , we found that gene alteration frequencies , including those for tp53 , pik3ca , and pten , were comparable between the two groups ( data supplement )  . 
 moreover , considering that we did not see major differences in genomic alterations between the japanese and tcga cohorts except for myc , it is unlikely that neoadjuvant chemotherapy dramatically influenced the landscape of driver alterations . 
although it is not easy yet in japan to recruit a large number of patients with tnbc for genomic sequencing , additional studies with a greater number of patients are warranted to confirm our findings in the future . despite these limitations , to our knowledge , this is the largest cohort of japanese patients with tnbc to undergo comprehensive genomic profiling to date , and our data provide important insights into the molecular heterogeneity underlying this aggressive disease from an ethnogeographic perspective . 
although our study indicates the possibility of targeted therapies for patients with tnbc , an accumulation of clinical evidence will be required to reveal the usefulness of each therapy for this disease . 
 are required to reveal potential treatments for patients with tnbc . in conclusion , our study revealed actionable driver alterations in japanese patients with tnbc that could potentially be treated with existing drugs . 
tsuchida j , nagahashi m , rashid om , et al : at what age should screening mammography be recommended for asian women ? cancer med 4 : 1136 - 1144 , 2015 4 . 
barnes dm , harris wh , smith p , et al : immunohistochemical determination of oestrogen receptor : comparison of different methods of assessment of staining and correlation with clinical outcome of breast cancer patients . 
wolff ac , hammond me , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
rizvi na , hellmann md , snyder a , et al : cancer immunology : mutational landscape determines sensitivity to pd - 1 blockade in non - small cell lung cancer . 
parsons ha , beaver ja , cimino - mathews a , et al : individualized molecular analyses guide efforts ( image ) : a prospective study of molecular profiling of tissue and blood in metastatic triple - negative breast cancer . 
radovich m , clare se , atale r , et al : characterizing the heterogeneity of triple - negative breast cancers using microdissected normal ductal epithelium and rna - sequencing . 
 high detection rate of myd88 mutations in cerebrospinal fluid from patients with cns lymphomas jun watanabe , md1 ; manabu natsumeda , md , phd1 ; masayasu okada , md , phd1 ; daiki kobayashi , ms1 ; yu kanemaru , md1 ; yoshihiro tsukamoto , md , phd1 ; makoto oishi , md , phd1 ; akiyoshi kakita , md , phd1 ; and yukihiko fujii , md , phd1 purpose biopsy is the gold standard for the diagnosis of primary cns lymphoma ( pcnsl )  . 
furthermore , we performed dna sequencing of myd88 in 21 cases with available surgically obtained formalin - xed parafn - embedded ( ffpe ) tissue and compared the results . results the median cfdna amount extracted from 1 ml csf was 219 ng / ml ( 25th to 75th percentile , 129 to 333 ng / ml )  . 
2019 by american society of clinical oncology introduction primary cns lymphomas ( pcnsls ) are highly malignant neoplasms ; the incidence of pcnsl has been increasing , and it accounts for 4% of all primary brain tumors.1 , 2 stereotactic or open biopsies are the gold the diagnosis of pcnsl.3 however , standard for pcnsl often occurs in deep brain structures , such as the basal ganglia and brain stem , and hemorrhage as a result of biopsy may cause serious morbidity or even mortality . 
therefore , a more minimally invasive , reliable , and rapid diagnostic method is needed . there has been growing interest in cancer diagnosis using cell - free dna ( cfdna ) as a source for tumor biomarkers in cancers , including malignant lymphomas.4 , 5 systemic diffuse large b - cell lymphomas ( dlbcls ) are known to be clinically and morphologically heterogeneous.6 hans et al6 classied dlbcl into the activated b - cell type ( abc type ) and germinal center b cell type ( gcb type )  . 
in abc - type dlbcl , somatic mutations are frequent in genes in the b - cell receptor signaling pathway , the toll - like receptor / interleukin - 1 receptor pathway , and the nuclear factor - b pathway.7 of these mutations , ngo et al7 has shown that myeloid differentiation primary response 88 gene ( myd88 ) mutations are the driver mutations for dlbcl and are related to activation of nuclear factor - b pathway and janus kinase - signal transducer and activator of transcription 3 ( jakstat3 ) signaling . 
the majority of pcnsls were reported to be of abc type.8 furthermore , the prevalence of myd88 mutations is reported to be more common in pcnsl than in systemic dlbcl.9 - 15 therefore , detecting these mutations may serve as a genetic marker for the diagnosis of pcnsl and a potential basis for molecularly targeted therapy . 
 watanabe et al context key objective to date , it is not known whether myd88 mutation , an important driver mutation of primary cns lymphoma , can be reliably detected from cell - free dna ( cfdna ) taken from cerebrospinal uid ( csf )  . 
in patients with primary cns lymphoma , detection of cfdna from the blood has been reported ; however , the detection rate is low . knowledge generated we showed that myd88 mutations were detected in cfdna extracted from 1 ml of csf from 76.9% ( 20 of 26 patients ) by combination of sanger sequencing and droplet digital polymerase chain reaction . 
detection of mutation was 100% concordant with that of tumor dna taken from matched tissue in 21 available cases . relevance these results showed that tumor cfdna is abundant in the csf , and tumor cfdna taken from the csf can be used as a lessinvasive molecular diagnostic and monitoring tool . 
in the near future , patients may not need surgery for the diagnosis . to date , the detection of cfdna from cerebrospinal uid ( csf ) in pcnsl has been reported in only two cases.18 , 19 the aim of this study is to determine whether reliable detection of myd88 mutations is possible in csf taken from patients with cns lymphomas . 
this study was approved by the institutional review board , and written informed consent to use csf and resected tissue for research purposes was obtained from patients ( #g20180008 )  . 
dna was extracted from formalin - xed parafn - embedded ( ffpe ) tissue in 21 cases using the qiaamp ffpe tissue kit ( qiagen , valencia , ca )  . 
sections were incubated with the working dilution of each antibody raised against the following antigens as previously described20 : cd10 ( clone 56c6 ; leica , vienna , austria ; diluted 1 : 100 ) , bcl - 6 ( clone pg - b6p ; dako , glostrup , denmark ; diluted 1 : 50 ) , mum1 ( clone mum1p ; dako ; diluted 1 : 200 )  . 
tumors were subclassied according to the expression pattern of gcb and abc markers , using the methods published by hans et al.6 determination of tumor volume post - contrast t1 - weighted sequences were used to dene the tumor volume as the area of contrast enhancement including central necrotic tissue . 
to determine the volume , we measured the tumor area in each slice using a region - ofinterest method ( brainlab software ; brainlab , feldkirchen , germany )  . 
although volumetrics is well established , this method is highly subjective and likely underestimates the total tumor volume . statistical analysis the sample size of generated data was small , so the data were considered to have a nonparametric distribution . 
the kaplan - meier method was used to estimate progressionfree survival ( pfs ) and overall survival ( os ) , and log - rank test was used to test for signicance . 
pfs was calculated from the day of diagnosis until magnetic resonance imagingconrmed tumor progression or end of follow - up . os was dened as the interval from the day of diagnosis until death or the end of follow - up . 
l265p in exon5 was the most frequent myd88 mutation found in 19 of 20 ( 95.0% ) cases ( fig 1b ) , and s219c in exon 3 was detected in one case ( fig 1c )  . 
likewise , myd88 mutation was not detected in the six patients with myd88wild - type tumor , yielding a 100% match of myd88 detection between csf and tumor - derived biopsy specimen . comparison of sanger sequencing and ddpcr first , we performed sanger sequencing in cfdna extracted from csf . 
mutations for l265p were detected in 15 cases , and s219c mutation was detected in one case ( fig 2 )  . however , in 10 cases we found the overlapping peaks to be small or not detectable , making the determination of mutant or wild type by sanger sequencing difcult . 
in two out of four ( 50% ) cases in which mutation could be detected only by ddpcr , magnetic resonance images showed little to no contrast - enhancing lesions localized to the brainstem ( figs 4a - 4c )  . 
after incubation at 4c for 2 hours , the amount of cfdna decreased to approximately one - third compared with rapid processing ( appendix fig a3 ) , and myd88 mutation was difcult to detect even by ddpcr . definite l265p s219c ( n = 15 ) ( n = 1 ) nondefinite ( n = 10 ) discussion mutant ( n = 4 ) myd88 mutant type l265p s219c ( n = 20 ) ( n = 19 ) ( n = 1 ) ddpcr wild type ( n = 6 ) wild type ( n = 6 ) in this study , a high detection rate of myd88 mutations in csf from patients with cns dlbcls was achieved . 
in the 21 cases with sufcient surgically obtained ffpe tissue for the detection of myd88 mutation from dna analysis , cfdna was 100% consistent with that of tumor - derived dna from ffpe tissue . 
therefore , we used sanger sequencing to screen for myd88 mutations and subsequently performed ddpcr to conrm specic mutations . furthermore , we found that obtaining high concentrations of cfdna is important . 
as has already been reported in glioma and metastatic brain tumors , tumor - derived dna could be found in csf located in the proximity of neoplastic lesions.23 , 24 especially in pcnsl , in which the primary site fig 2 . 
in two cases , we analyzed the myd88 l265p mutation before and during chemotherapy . patients in cases 5 and 14 received methotrexate , procarbazine , and vincristine.21 the amount of cfdna and number of mutant droplets serially decreased with treatment ( appendix fig a1 )  . pfs and os were compared between myd88 - mutant ( n = 12 ) versus myd88wild - type cases ( n = 2 ) , and no difference was detected ( pfs : p = .831 ; os : p = .464 ; logrank test )  . 
likewise , no difference in pfs and os were detected between high ( n = 7 ) versus low ( n = 7 ) cfdna amount ( pfs : p = .252 ; os : p = .824 ; appendix fig a2 )  . because the number of newly diagnosed pcnsl cases is small ( n = 14 ) and the observation period is short , it is still difcult to draw any conclusions . cfdna stability in csf to study the stability of cfdna , we examined the concentration of cfdna and the time from csf collection to storage at 80c in two cases . 
magnetic resonance imaging showed a tumor on the left side of the midbra ( a - c ) diffusion - weighted images ( a ) and uid attenuated inversion recovery ( b ) showed high signal intensity ; however , contrast enhancement was not observed in post - contrast t1 - weighted images ( c )  . 
 ( d , e ) l265p mutation was detected by sanger method in formalin - xed parafn - embedded ( ffpe ) tissue ( d ) , whereas l265p mutation was not detected in csf by sanger sequencing ( e )  . 
 ( f ) several l265p - mutant droplets were conrmed in csf by droplet digital polymerase chain reaction ( ddpcr )  . is still controversial , future studies directly comparing matched plasma and csf cfdna concentrations are needed . 
in addition , lumbar puncture has a possibility of blood contamination . this is certainly relevant in patients with systemic lymphoma . we should also consider the clearance of cfdna in addition to release of cfdna . 
it was reported that clearance of fetal cfdna from the plasma of pregnant women had a mean half - life of 16 to 60 minutes.25 , 26 although there has been no prior study of the clearance of cfdna in csf , the present analysis showed that promptly centrifuging and storing the supernatant is important to obtain a sufcient amount of cfdna . 
as previously reported , 28 we found that isolation efciency of cfdna by the maxwell rsc machine was superior to that of traditional methods using other dna extraction kits ( data not shown )  . 
myd88 mutations have been reported so far in only b - cell malignancies such as dlbcl , waldenstrom macroglobulinemia , and igm monoclonal gammopathy.32 it has also been conrmed that myd88 mutations do not appear in gliomas , which proves useful because gliomas are often considered for differential diagnosis.13 according to a large - scale study , myd88 l265p mutation was found in only one out of 12 , 380 healthy individuals.33 myd88 mutation in dlbcl has also been reported as a negative prognostic factor for patients undergoing concurrent methotrexate - based polychemotherapy.34 , 35 with the introduction of targeted therapies , it is anticipated that myd88 mutation will become a positive predictive factor in the future.36 the bruton tyrosine kinase inhibitor ibrutinib was found to be effective in abc - type systemic dlbcl , especially in cases with myd88 l265p mutation.36 furthermore , gromme et al37 and lionakis et al38 reported that ibrutinib was also effective for relapsed pcnsl . 
detection of myd88 mutations will enable the timely commencement of treatment in mutationpositive cases and might be used for monitoring treatment response and detecting early relapse , thus potentially contributing to an improved outcome in patients with pcnsl . in conclusion , the current study shows that reliable detection of myd88 mutations in patients with cns dlbcls was possible . 
crit rev oncol hematol 113 : 97 - 110 , 2017 scherer f , kurtz dm , newman am , et al : distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor dna . 
sci transl med 8 : 364ra155 , 2016 rossi d , diop f , spaccarotella e , et al : diffuse large b - cell lymphoma genotyping on the liquid biopsy . 
hans cp , weisenburger dd , greiner tc , et al : conrmation of the molecular classication of diffuse large b - cell lymphoma by immunohistochemistry using a tissue microarray . 
nature 470 : 115 - 119 , 2011 kreher s , j ohrens k , strehlow f , et al : prognostic impact of b - cell lymphoma 6 in primary cns lymphoma . 
neuro - oncol 17 : 1016 - 1021 , 2015 gonzalez - aguilar a , idbaih a , boisselier b , et al : recurrent mutations of myd88 and tbl1xr1 in primary central nervous system lymphomas . 
yamada s , ishida y , matsuno a , et al : primary diffuse large b - cell lymphomas of central nervous system exhibit remarkably high prevalence of oncogenic myd88 and cd79b mutations . 
nakamura t , yamashita s , fukumura k , et al : genome - wide dna methylation proling identies primary central nervous system lymphoma as a distinct entity different from systemic diffuse large b - cell lymphoma . 
zheng m , perry am , bierman p , et al : frequency of myd88 and cd79b mutations , and mgmt methylation in primary central nervous system diffuse large 16 . 
fontanilles m , marguet f , bohers e , et al : non - invasive detection of somatic mutations using next - generation sequencing in primary central nervous system 17 . 
hattori k , sakata - yanagimoto m , suehara y , et al : clinical signicance of disease - specic myd88 mutations in circulating dna in primary central nervous b - cell lymphoma . 
hiemcke - jiwa ls , minnema mc , radersma - van loon jh , et al : the use of droplet digital pcr in liquid biopsies : a highly sensitive technique for myd88 p . ( l265p ) detection in cerebrospinal uid . 
zorofchian s , lu g , zhu jj , et al : detection of the myd88 p.l265p mutation in the csf of a patient with secondary central nervous system lymphoma . 
aoki h , ogura r , tsukamoto y , et al : advantages of dose - dense methotrexate protocol for primary central nervous system lymphoma : comparison of two different protocols at a single institution . 
de mattos - arruda l , mayor r , ng cky , et al : cerebrospinal uid - derived circulating tumour dna better represents the genomic alterations of brain tumours 24 . 
wang y , springer s , zhang m , et al : detection of tumor - derived dna in cerebrospinal uid of patients with primary tumors of the brain and spinal cord . 
fernando mr , chen k , norton s , et al : a new methodology to preserve the original proportion and integrity of cell - free fetal dna in maternal plasma during sample processing and storage . 
sorber l , zwaenepoel k , deschoolmeester v , et al : a comparison of cell - free dna isolation kits : isolation and quantication of cell - free dna in plasma . 
schroers r , baraniskin a , heute c , et al : detection of free immunoglobulin light chains in cerebrospinal uids of patients with central nervous system diagn 19 : 162 - 168 , 2017 lymphoma . 
schroers r , baraniskin a , heute c , et al : diagnosis of leptomeningeal disease in diffuse large b - cell lymphomas of the central nervous system by ow cytometry and cytopathology . 
hattori k , sakata - yanagimoto m , okoshi y , et al : myd88 ( l265p ) mutation is associated with an unfavourable outcome of primary central nervous system 35 . 
takano s , hattori k , ishikawa e , et al : myd88 mutation in elderly predicts poor prognosis in primary central nervous system lymphoma : multi - institutional lymphoma . 
 detection of myd88 mutations from csf in cns lymphoma appendix direct sequencing for myd88 mutations twenty nanograms of cell - free dna ( cfdna ) were amplied using primers for the detection of the myd88 mutations . 
next , we performed sequencing of other known myd88 point mutations ( s219c , m232t , v233m , d239n , p267s , i266v , t285i , c293y , t294i , t200i ) in l265p wild - type cases . the rst set of primers within exon 5 had a product of 383 bp : ( 1 ) forward 5 - gctgttgttaaccctggggttgaag - 3 and ( 1 ) reverse 5gacgtgtctgtgaagttggcatctc - 3 . 
the exon 3 primers had a product size of 310 bp : ( 3 ) forward 5 - aagccttcccatggagctctgaccac - 3 and ( 3 ) reverse 5 - gctaggaggagatgcccagtatctg - 3 . 
the 20l pcr mix , composed of 10 l of 2 ddpcr supermix for probes ( no deoxyuridine triphosphate ; bio - rad ) , 1 l ddpcr mutation assay ( bio - rad ) , and 9 l cfdna was loaded into sample wells of an eightchannel disposable droplet generator cartridge ( bio - rad )  . 
after droplet generation , droplets were transferred into a 96 - well pcr plate and proceeded to thermal cycling . amplication was carried out on the 20 - l reaction mixture on the qx200 ddpcr system ( bio - rad )  . 
after pcr , the 96 - well pcr plate was subjected to the qx - 200 droplet reader ( bio - rad ) , and data were analyzed by quantasoft analysis software ( bio - rad )  . 
myd88 l265p mutation - specic signals were generated in the 6 - uorescein amidite channel , and myd88 wild - type signals were generated in the hexachloro - uorescein channel . 
the cfdna amount at baseline and after methotrexate , procarbazine , and vincristine ( mpv ) two courses from case 5 ( b , c ) and case 14 ( e , f )  . 
this study was performed to characterize mutagenesis and mutational target genes underlying lung carcinogenesis in patients with uip . patients and methods a cohort of 691 japanese patients with lung adenocarcinoma ( ladc ) , of whom 54 had uip and 637 did not , was studied for driver oncogene aberrations . 
2018 by american society of clinical oncology introduction smoking is a major risk factor for lung cancer , 1 and its mutagenic process and mutational targets , including kras and tp53 , have been elucidated.2 smoking is also a pathogenic factor for idiopathic pulmonary fibrosis ( ipf ) , which is pathologically diagnosed as usual interstitial pneumonia ( uip ) , 3 characterized by destruction of alveolar architecture in association with fibrosis.4 uip is a strong risk factor for lung cancer independent of smoking ( relative risk , 8.25 ) 5 and increases the cumulative incidence rate of lung cancer within 10 years to 54.7%.6 notably , deleterious germline mutations in pulmonary surfactant system genes ( pssgs ) , including nkx2 - 1 / ttf1 , sftpa1 , sftpa2 , sftpb , and sftpc , have been implicated in familial neonatal respiratory failure and fibrotic lung disease , 7 as well as in lung cancer development.8 , 9 therefore , loss of function of pssgs in morphogenesis / maintenance of alveolar structure7 might also play a role in uip and subsequent development of lung cancer in individuals with germline pssg mutations . 
however , such tumors are rarely subjected to surgical resections ( 3.1% ; 1 , 300 of 41 , 742 ) , 10 largely because of the risk of acute exacerbation of interstitial pneumonia and an associated high mortality rate.10 consequently , no comprehensive genomic analysis of uip - associated lung cancers has been published to date . 
one of the few genetic characteristics of these cancers reported previously is that egfr oncogene mutations are relatively infrequent in uip - associated lung adenocarcinomas ( ladcs ) .11 - 13 in this study , we performed comparative and comprehensive genomic characterization of ladcs with and without uip . 
ladc was chosen for comparison for several reasons : it is the most common histologic type of lung cancer and frequently develops in the lungs of patients suffering from uip11 ; the genetic aberrations responsible for carcinogenesis , represented by driver oncogene aberrations , 14 - 16 have been well documented ; and mutational signature studies17 have revealed the mutational events underlying this disease , which include dna damage / adduct formation as a result of smoking , spontaneous deamination of 5 - methylcytosine , 18 and the action of apolipoprotein b mrna editing enzyme catalytic polypeptide - like cytidine deaminases.19 to deduce the mutagenic processes and elucidate commonly affected genes , we examined driver oncogene aberrations in a cohort of 691 patients with ladc , including 54 patients who were uip positive and 637 patients who were uip negative , and performed whole - exome sequencing ( wes ) analysis in 296 ladcs ( 51 uip positive and 245 uip - negative )  . 
of those , 679 were consecutive patients who underwent surgical resection between 1997 and 2008 at the national cancer center ( ncc ) hospital ( ncch ) , tokyo , and for which snap - frozen cancerous and noncancerous lung tissues suitable for genome analysis were available in the ncc biobank . 
 surgical specimens of these patients were classified as uip negative and uip positive ( 637 and 42 cases , respectively ) on the basis of previously described pathologic criteria.20 uip positivity was defined as the presence of the uip pattern ( on the basis of the 2002 american thoracic society / european respiratory society4 and 2011 ipf criteria3 )  . 
uip - positive cases were defined as those having tumorous lesions located within or adjacent to uip lesions ; tumors discontinuous to the uip lesions were excluded ( appendix )  . 
 to increase the number of uip - positive cases , an additional 12 patients who underwent surgical resection between 2011 and 2014 at ncch or tokyo medical and dental university hospital , tokyo were also studied . 
thus , the total number of uip - positive cases was 54 ( 42 + 12 )  . all patients were diagnosed according to the seventh tnm classification of malignant tumors.21 information regarding age , sex , smoking history , and clinical stage was retrospectively collected . 
 this study was approved by the institutional review boards of both the ncc and tokyo medical and dental university . driver gene aberration screening all 691 cases were screened for egfr , kras , braf , and her2 hotspot mutations by high resolution melting analysis or target sequencing analysis using the ion ampliseq cancer hotspot panel v2 ( life technologies , rockville , md )  . 
 alk , ret , and ros1 fusions were examined by reverse transcriptase polymerase chain reaction ( rt - pcr ) or molecular counting assay as previously described.15 , 22 , 23 wes analysis wes was performed for 245 uip - negative and 51 uip - positive ladcs ( 296 cases in total )  . 
 of these , 211 of the uip - negative cases and all 51 uip - positive cases were selected from the 691 patients with ladc used for driver gene aberration screening on the basis that sufficient amounts of dna were available for comprehensive genomic profiling . 
 wes was performed for cancerous and noncancerous genomic dnas using the sureselect human all exon exome capture kit ( version 4 or 5 ; agilent technologies japan , tokyo , japan ) on the illumina hisequation 2000 platform ( illumina , san diego , ca )  . 
in this condition , 10 randomly chosen snvs and 47 indels were verified at high confidence by mass - spectrometric genotyping on a massarray system ( agena bioscience , san diego , ca ) : 10 of 10 ( 100% ) for snvs and 45 of 47 ( 95.7% ) for indels , respectively ( appendix )  . 
genes recurrently affected by somatic snv and indel mutations in their coding regions were identified by mutsigcv analysis25 according to the following criterion : q value < 0.1 after adjusting for gene length and applying the benjamini - hochberg correction . 
 more detailed information about immunohistochemistry procedures is provided in the appendix . statistical analysis statistical analyses of differences were analyzed by student t test , mann - whitney u test , pearsons 2 test , or fishers exact test . 
for association studies , clinicopathological factors and mutational signatures were coded as binary or ordinal explanatory variables , and association between these signatures and clinical background was analyzed by fishers exact test and logistic regression analysis using the jmp 8.0 software ( sas institute japan , tokyo , japan )  . 
survival analysis was performed using the kaplan - meier method , and groups were compared by log - rank test using the graphpad prism 7 software ( graphpad , la jolla , ca )  . 
this result is consistent with previous reports of patients with uip3 and patients with ladc with uip.11 - 13 baseline characteristics of patients with uip - negative and - positive ladc were similar to those of previously described asian cohorts.16 , 27 , 28 driver oncogene aberrations in uip - negative cases , approximately 50% of which were egfr mutations , were similar to those of overall ladcs in east asians.16 , 28 on the other hand , the prevalence of oncogene aberrations clearly differed between uip - positive and uip - negative cases ( p < .001 by pearsons 2 ; fig 1a ; table 1 )  . 
however , the rate of egfr mutation was markedly lower in uip - positive cases than in uip - negative cases ( 1.9% [ one of 54 ] v 49.9% [ 318 of 637 ] ; p < .001 [ fishers exact test ] ) , even in heavy smokers , who have a low egfr mutation frequency30 ( 25.3% ; 38 of 150 ; p < .001 [ fishers exact test ] )  . 
 ( % ) unless otherwise noted . abbreviations : ladc , lung adenocarcinoma ; uip , usual interstitial pneumonia . * p value was derived from the comparison between the non - uip cohort and uip - ladc by mann - whitney u test , fishers exact test , or pearsons 2 test , as appropriate . invasive mucinous adenocarcinoma and colloid , fetal , and enteric carcinoma were categorized as variants of invasive adenocarcinoma , as in travis et al.29 mutational events underlying carcinogenesis deduced by mutational signatures next , we performed a genome - wide mutational analysis to deduce the mechanisms of mutagenesis during lung carcinogenesis . 
patient characteristics and driver oncogene aberrations of the uip negative cases were similar to those of the 637 uip - negative cases described above ( appendix table a1 )  . 
the median snv rates in uip positive and - negative cases were 1.43 / mb and 0.97 / mb , respectively ( p = .0044 [ mann whitney u test ] ) , whereas the median indel mutation rates in uip - positive and - negative cases were 0.25 / mb and 0.080 / mb , respectively ( p < .001 [ mann - whitney u test ] )  . 
therefore , more mutational events , including indel mutations , occurred in the genomes of uip - positive cancers . among the 296 patients , we observed cosmic signatures 4 ( smoking associated ) , 2 ( apolipoprotein b mrna editing enzyme , catalytic polypeptide - like associated ) , 1 ( age associated ) , and 16 ( unknown etiology ; appendix fig a1 )  . 
uip - negative cases were subcategorized based on smoking status : never smoker , ever smoker , and heavy smoker ( 40 pack - years [ py ] )  . 
these findings indicated that mutational events involved in the development of uip positive ladc are largely caused by smoking . significantly mutated genes in uippositive ladc mutsigcv analysis of all 296 cases revealed frequent mutations in several genes reported in previous genome - wide mutation analysis of ladc14 : egfr , kras , tp53 , stk11 , cdkn2a , ctnnb1 , and smad4 ( appendix table a3 )  . 
mutsigcv analysis of the 51 uip - positive cases revealed that tp53 ( 20 of 51 ; 39.2% ) and kras ( 10 of 51 ; 19.6% ) were the two most frequently mutated genes ( fig 1b ; appendix table a3 )  . 
mutations in other genes reported as mutated in ladc14 were also detected in patients with uip - positive ladc , albeit at lower frequencies than tp53 , kras , and nkx2 - 1 . deleterious pssg mutations in a subset of ladc the above observation prompted us to investigate whether other pssgs are mutated in ladcs . 
we found that four other pssgs , sftpa1 , sftpa2 , sftpb , and sftpc , were mutually exclusively mutated in 10 of 296 ( 3.3% ) of all ladc cases and three of 51 ( 5.9% ) of uip - positive cases ( fig 2a )  . 
most of the mutations in these pssgs were indels in the 3untranslated regions ( utrs ; fig 2b ) , consistent with a recent report that indel mutations occur preferentially in lineage - defining genes in human cancers.32 comparative genotyping of dna and cdna isolated from cancerous and noncancerous tissues revealed that the 3 - utr indel mutant alleles of sftpa1 , sftpa2 , and sftpc were expressed at significantly lower levels than the wild - type alleles ( appendix fig a2 )  . in total , 21 of 296 ladcs ( 7.1% ) had mutations in five pssgs ( nkx2 - 1 , sftpa1 , sftpa2 , sftpb , and sftpc ) , with the mutations in individual genes occurring almost mutually exclusively . 
the prevalence of indels in all cases ( 15 of 296 ; 5.1% ) and uip - positive ladc cases ( six of 51 ; 11.8% ) indicated that pssgs are a major target for indel mutagenesis ( fig 2a )  . pssg mutations define a clinically distinct subset of ladcs next , we examined the characteristics of 21 patients with ladc with pssg mutations ( table 2 )  . 
notably , 11 cases ( 52.3% ) exhibited histology corresponding to mucinous differentiation , 29 with a frequency much higher than in ladcs overall ( 6.1% to 16%29 , 33 , 34 ; appendix table a4 ; representative cases in fig 3a )  . 
most tumors with mutations in nkx2 - 1 and all tumors with mutations in sftpa1 and sftpa2 lost expression of the corresponding proteins , whereas tumors with sftpb mutations retained surfactant protein ( sp ) - b protein expression . 
patients who were uip positive with pssg mutations had shorter median overall survival : 24 months versus 125 months for patients who were uip positive without such mutations ( p = .0080 ; fig 3b )  . 
however , no such association was observed for patients with uip - negative ladc ( appendix fig 3a and 3b )  . discussion we present here the first comprehensive comparative genome profiles , to our knowledge , of ladcs with and without uip . 
 ( % ) unless otherwise noted . abbreviations : ladc , lung adenocarcinoma ; pssg , pulmonary surfactant system gene ; uip , usual interstitial pneumonia . * p value was derived from the comparison between cases with or without pssg mutation by mann - whitney u test , fishers exact test , or pearsons 2 test , as appropriate . invasive mucinous adenocarcinoma and colloid , fetal , and enteric carcinoma were categorized as variants of invasive adenocarcinoma , as in travis et al.29 profiles distinct from those of uip - negative ladcs , characterized by infrequent egfr mutations . 
furthermore , focal genome copy number gain associated with abundant transcript expression is a mode of activation of the egfr oncogene.37 uip - positive ladcs did not exhibit significant copy - number gains at egfr , whereas several uip - positive ladcs did ( appendix fig a4a - a4c )  . 
rna sequencing of representative uip - positive cases did not reveal overexpression of egfr ( appendix fig a4d - a4f ) or oncogenic egfr fusions , which have been reported as driver events in ladc.38 these results suggested that the egfr oncogene infrequently contributes to the development of uip - positive ladc . 
ladcs in east asian individuals frequently harbor egfr mutations , both in smokers and nonsmokers.39 therefore , our findings show that uip affects the molecular mechanism underlying lung carcinogenesis by changing the relative dependence of oncogene aberrations . mutational signature analysis revealed that the mutational events involved in uip - positive ladc are largely due to smoking . 
indel mutations were also frequent in uip - positive ladcs , consistent with their prevalence in smoking associated cancers.31 importantly , our results newly identify pssgs as major targets for indel mutations . 
pssg deficiency had been implicated in lung carcinogenesis on the basis of the association between deleterious germline mutations in these genes and severe familial lung diseases , such as interstitial pneumonia and lung cancer development.7 - 9 noncancerous lung tissues of patients with germline sftpb mutation and sftpc - deficient mice develop interstitial fibrosis and alveolar type ii cell dysplasia , 40 , 41 suggesting that pssg deficiency perturbs normal pneumocytic differentiation . 
 ( a ) pathologic features of pssg - mutated lung adenocarcinoma , including invasive mucinous ( case 12 ) and acinar - predominant with a mucinous cribriform pattern ( case 14 )  . 
nk2 homeobox1 ( nkx2 - 1 ) / thyroid transcription factor 1 ( ttf1 ) and surfactant protein ( sp ) - a were detected in normal pneumocytes but were undetectable in tumors with the corresponding sftpa and / or nkx2 - 1 mutations . 
hematoxylin and eosin staining ( he , upper right ) and immunohistochemistry for nkx2 - 1 / ttf1 ( lower left ) and sp - a ( lower right ) proteins , with 3 , 3 - diaminobenzidine visualization ( magnification 200 )  . 
 ( b ) kaplan - meier curve showing overall survival ( os ) of patients with usual interstitial pneumonia ( uip ) positive ladc with pssg mutation ( mutant ) and without pssg mutation ( wild type )  . 
inflammation contributes not only to tumor development but also to tumor progression , by increasing cell invasion and expanding the fraction of undifferentiated cells.43 , 44 however , pulmonary surfactant proteins play important roles in epithelial integrity and cell differentiation in the lung.7 , 45 therefore , deficiency in pulmonary surfactant protein caused by pssg mutations might increase inf lammation - driven tumor progression by disturbing epithelial integrity / differentiation . 
 pssg mutations were more likely to be present in ladcs of smokers and in egfr mutation negative ladcs , which are both associated with poor prognosis.46 , 47 in addition , the number of cases with pssg mutations was small . 
the roles of aberrations of minor driver oncogenes and pssgs should also be studied in a larger cohort , including patients with squamous cell carcinoma , to more fully elucidate lung carcinogenesis associated with uip . 
the 3 - utrs of genes play regulatory roles in the stability and abundance of mrna , and oncogenic mutations have been reported in the 3 - utrs of kras and tp53 genes.48 , 49 accordingly , functional analysis of the 3 - utr mutations in sftps is warranted . in summary , we elucidated the genomic profile of uip - ladcs and observed a predominance of smoking - related mutations in these cancers . 
 kyohei masai no relationship to disclose hirohiko totsuka employment : stagen noriko motoi honoraria : agilent , msd , novartis , astrazeneca , bristolmyers squibb japan consulting or advisory role : bristol - myers squibb , miraca life sciences , astrazeneca , chugai pharmaceuticals , research funding : roche ( inst ) yoko shimada no relationship to disclose ayaka otsuka no relationship to disclose yae kanai no relationship to disclose kenichi okubo no relationship to disclose shun - ichi watanabe no relationship to disclose masashi kobayashi no relationship to disclose takumi akashi no relationship to disclose sachiyo mimaki no relationship to disclose katsuya tsuchihara no relationship to disclose suenori chiku no relationship to disclose kouya shiraishi no relationship to disclose support references 2005 koji tsuta speakers ' bureau : msd , roche naohiko inase no relationship to disclose takashi kohno research funding : daiichi sankyo , sysmex acknowledgment we thank y . 
sakaguchi for excellent technical assistance with immunohistochemistry . affiliations takayuki honda , kouya shiraishi , yoko shimada , ayaka otsuka , and takashi kohno , national cancer center research institute , chuo - ku ; takayuki honda , hiroyuki sakashita , masashi kobayashi , takumi akashi , kenichi okubo , and naohiko inase , tokyo medical and dental university , bunkyo - ku ; kyohei masai , noriko motoi , and shun - ichi watanabe , national cancer center hospital , chuo - ku ; kyohei masai and yae kanai , keio university school of medicine , sinjuku - ku ; hirohiko totsuka , stagen , taito - ku ; suenori chiku , mizuho information and research institute , chiyoda - ku , tokyo ; sachiyo mimaki and katsuya tsuchihara , epoc , national cancer center , kashiwa , chiba ; and koji tsuta , kansai medical university , hirakata , osaka , japan . supported by the japan agency for medical research and development grant no . 
raghu g , collard hr , egan jj , et al : an official ats / ers / jrs / alat statement : idiopathic pulmonary fibrosis : evidence - based guidelines for diagnosis and management . 
fujimoto d , tomii k , otoshi t , et al : preexisting interstitial lung disease is inversely correlated to tumor epidermal growth factor receptor mutation in patients with lung adenocarcinoma . 
masai k , tsuta k , motoi n , et al : clinicopathological , immunohistochemical , and genetic features of primary lung adenocarcinoma occurring in the setting of usual interstitial pneumonia pattern . 
goldstraw p , crowley j , chansky k , et al : the iaslc lung cancer staging project : proposals for the revision of the tnm stage groupings in the forthcoming ( seventh ) edition of the tnm classification of malignant tumours . 
sunami k , furuta k , tsuta k , et al : multiplex diagnosis of oncogenic fusion and met exon skipping by molecular counting using formalin - fixed paraffin embedded lung adenocarcinoma tissues . 
travis wd , brambilla e , noguchi m , et al : international association for the study of lung cancer / american thoracic society / european respiratory society international multidisciplinary classification of lung adenocarcinoma . 
yatabe y , koga t , mitsudomi t , et al : ck20 expression , cdx2 expression , k - ras mutation , and goblet cell morphology in a subset of lung adenocarcinomas . 
tang x , kadara h , behrens c , et al : abnormalities of the titf - 1 lineage - specific oncogene in nsclc : implications in lung cancer pathogenesis and prognosis . 
hirsch fr , varella - garcia m , bunn pa jr , et al : epidermal growth factor receptor in non - smallcell lung carcinomas : correlation between gene copy number and protein expression and impact on prognosis . 
wallot m , wagenvoort c , demello d , et al : congenital alveolar proteinosis caused by a novel mutation of the surfactant protein b gene and misalignment of lung vessels in consanguineous kindred infants . 
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fujisawa t , iizasa t , saitoh y , et al : smoking before surgery predicts poor long - term survival in patients with stage i non - small - cell lung carcinomas . 
takano t , fukui t , ohe y , et al : egfr mutations predict survival benefit from gefitinib in patients with advanced lung adenocarcinoma : a historical comparison of patients treated before and after gefitinib approval in japan . 
 appendix histologic diagnosis surgically resected specimens ( ie , tissues remaining after snap - frozen tissues were set aside ) were formalin fixed by transbronchial or transpleural injection ( in an inflated state ) , cut serially into 5 - mmthick sections , and macroscopically examined . 
the reads were aligned to the reference human genome ( university of california , santa cruz , human genome 19 ; hg19 ) with the burrows wheeler aligner multi - vision software package . 
because duplicate reads were generated during the polymerase chain reaction ( pcr ) amplification process , duplicated paired - end reads that aligned to the same genomic positions were marked using picard . 
depth of coverage at capture targets in tumor samples was counted using read coverage spanning a target segment with the total number of aligned reads and proportionally calibrated to estimate the copy ratio using depths observed in a panel of normal ( noncancer ) diploid genomes . 
thresholds were as follows : board length cutoff , 0.5 chromosome arms ; confidence interval , 90% . validation of candidate mutations using mass - spectrometric genotyping validation and quantification of candidate mutations were performed by mass - spectrometric ( ms ) genotyping as previously described ( su ky , et al : j clin oncol 30 : 433 - 440 , 2012 ) according to the user instructions for the massarray system ( agena bioscience , san diego , ca )  . 
extracted dna ( 10 ng ) was amplified using specific primers ; unincorporated nucleotides were inactivated by shrimp alkaline phosphatase ; a single - base extension reaction was performed using extension primers that hybridized immediately adjacent to the mutations . 
salts were removed by addition of cation exchange resafter cleanup with spectroclean resin , samples were loaded onto the spectrochip matrix using a nanodispenser and then analyzed by matrix - assisted laser desorption ionizationtime - of - flight mass spectrometry on a bruker autoflex instrument . 
 mutational signature analysis mutational signature in this study was analyzed using nonnegative matrix factorization ( nmf ) , which was applied to the 96 possible substitutions in a trinucleotide context including the bases 5 and 3 of each substitution site , as previously described.17 , 26 nmf was applied to the 96 - substitution pattern using published software from the wellcome trust sanger institute.9 at least 2 , 400 iterations of nmf were run , and each nmf run was iterated to convergence ( 10 , 000 iterations without change ) or until 1 million iterations were performed . 
nmf runs with various numbers of signatures were tested , from one to 10 ; four was best , because signature stability remained high and the reconstruction error converged at a low value ( appendix fig a2a )  . 
 one microgram of total rna was reverse - transcribed using superscript iii first - strand synthesis system for reverse transcriptase ( rt ) pcr ( thermo fisher scientific , waltham , ma ) after dnase treatment . 
ten nanograms of genomic dna and cdna corresponding to 50 ng of rna were prepared from both cancerous and noncancerous tissues . for ms genotyping , the experimental procedures described above were applied to both genomic dna and cdna using the same primer , and mutant allele frequency ( represented as peak heights ) was calculated using the typer4 software ( agena bioscience )  . for fragment analysis , regions including mutations were amplified by pcr from genomic dna and cdna using the same primers according to the manufacturers instructions ( applied biosystems , foster city , ca )  . 
pcrs were performed in triplicate with the following conditions : 35 cycles of denaturation at 95c for 1 minute , annealing at 60c for 1 minute , and extension at 72c for 1 minute . 
mutant allele frequency ( represented as peak heights ) was calculated using the abi genemapper software ( applied biosystems )  . differences in the sftpa1 , sftpa2 , and sftpc gene expression between cancerous and noncancerous tissue were analyzed using quantitative rt - pcr . 
triplicate samples were used for all experiments described in this paragraph . rna sequencing rna - sequencing libraries were prepared from 200 ng of total rna using the truseq rna sample prep kit ( illumina )  . 
the resultant libraries were subjected to paired - end sequencing of 75 - bp reads on a hisequation 2000 ( illumina ) as previously described ( nakaoku t , et al : clin cancer res 20 : 3087 - 3093 , 2014 )  . 
egfr fusion transcripts were identified using the tophat - fusion algorithm ( kim d , et al : genome biol 12 : r72 , 2011 ) .12 expression values were normalized against the corresponding levels of a housekeeping gene ( tbp , encoding tata - binding protein ) .19 immunohistochemistry studies immunohistochemistry studies were performed on 4 - mthick , formalin - fixed , paraffin - embedded tissue sections derived from representative tumor blocks . 
3 , 3 - diaminobenzidine was used as the chromogen , and hematoxylin was used as the counterstareactions that labeled at least 5% of cells were considered positive for thyroid transcription factor 1 ( nuclear )  . 
 ( a ) mass - spectrometric genotyping of genomic dna and cdna from cancerous tissues with sftp - 3 untranslated region ( utr ) indel mutation ( mut ) and noncancerous tissue ( wt )  . 
 ( a ) kaplan - meier curve of overall survival ( os ) for patients with usual interstitial pneumonianegative lung adenocarcinoma with pulmonary surfactant system gene ( pssg ) mutation ( mutant ) and without pssg mutation ( wild type )  . 
 ( a ) copy - number analysis of usual interstitial pneumonia ( uip ) positive lung adenocarcinomas ( ladcs ; n = 51 ) in comparison with uip - negative ladcs ( n = 245 ) ; ( b ) uip - negative ladcs with egfr mutation ( n = 130 ) ; and ( c ) uip - negative ladcs without egfr mutation ( n = 115 )  . 
 ( d ) expression levels of egfr in three uip - positive ladcs in comparison with uip - negative ladcs ( n = 138 ) ; ( e ) uip - negative ladcs with egfr mutation ( n = 35 ) ; and ( f ) uip - negative ladcs without egfr mutation ( n = 103 )  . 
expression levels are shown as fold changes in fragments per kilobase of exon per milllion reads mapped , normalized against the gene encoding tata - binding protein ( tbp )  . 
identification of biomarkers predictive of a benefit from trastuzumab will minimize overtreatment and lower health care costs . methods to identify possible single - gene biomarkers , an exploratory analysis of 3 , 669 gene probes not expected to be expressed in normal breast tissue was conducted . 
 disease - free survival ( dfs ) was used as the end point in a cox regression model , with the interaction term between c8a mrna and treatment as a categorical variable split on the cohort mean . results a significant interaction between c8a mrna and treatment was detected ( p < .001 ) , indicating a predictive response to trastuzumab treatment . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction breast cancer is the leading cause of cancer death among women worldwide.1 it is a heterogeneous disease , so to maximize benefit to the patient by individualizing treatment regimens , clinicians need a sound classification syste clinicopathologic variables , including the estrogen receptor 1 , progesterone receptor , and human epidermal growth factor receptor 2 ( her2 ) , are used to classify tumors as hormone positive , her2 positive ( her2 + ) , or triple negative and to guide treatment decisions . 
pam50 , oncotype dx , and mammaprint were developed using retrospective analysis of mrna gene expression data from breast cancer cohorts . identification of single genes or gene signatures that can be used to classify a breast cancer tumor as a subtype with known treatment strategies can improve outcomes and minimize unnecessary treatment . 
the identification that 15% to 25% of breast cancer tumors overexpress the her2 protein , a transmembrane tyrosine kinase that regulates growth and cell survival , resulted in the development of monoclonal antibody therapy that targets her2 . 
 the exploratory analysis identified c8a , a member of the membrane attack complex and part of the innate immune system , which was prognostic of outcome in the observation arm and predictive of benefit from trastuzumab . 
we also characterized c8a protein expression in stable cancer cell lines . methods study population the hera trial was an international , intergroup , open - label , phase iii randomized study.5 a total of 5 , 102 women with her2 + primary breast cancer ( after a minimum of four courses of standard chemotherapy ) were enrolled and assigned randomly to one of the following three treatment arms : observation ( no trastuzumab ) and 1 or 2 years of adjuvant trastuzumab administered intravenously every 3 weeks . 
an interim analysis5 showed that patients assigned to the 1 - year trastuzumab arm experienced a significantly lower hazard of a dfs event than those in the observation arthis finding resulted in a protocol amendment that allowed for observation patients to selectively crossover to trastuzumab treatment ( 1 or 2 years ) , with the restriction of being alive and disease - free as of may 16 , 2005 . 
the transhera tissue resource consisted of 1 , 203 blocks that originated from 15 countries and were processed at royal marsden hospital in the united kingdo in addition , an extra 600 - m core was taken from each block . 
sample rna was extracted using the expressart ffpe rnaready isolation kit ( amptec , hamburg , germany ) , which is optimized for isolation of total rna specifically from ffpe tissue samples , and eluted into a volume of 20 l . 
the biotinylated cdna is then immobilized to a streptavidin - coated solid support and annealed to a dasl assay pool of gene - specific oligonucleotides for extension and ligation followed by polymerase chain reaction amplification with a biotinylated and a fluorophore - labeled universal primer . 
 control ( liver and brain ) rna samples were included for each processed batch of 48 samples to ensure that rna processing was successful and for quality control and normalization of data between assay batches . 
cubic spline has been shown to combine the positive effects of quantile normalization and to avoid the drawbacks of discontinuous mapping of intensity values and no - rank preservation.11 analysis information from the hera database , with a clinical cutoff date of april 12 , 2012 , and 8 years of median follow - up , was used in the current study.7 the primary end point was dfs , defined as the time from random assignment to first occurrence of any of the following dfs events : recurrence of breast cancer at any site ; the development of ipsilateral or contralateral breast cancer , including ductal carcinoma in situ but not lobular carcinoma in situ ; second nonbreast malignant disease other than basal - cell or squamous - cell carcinoma of the skin or carcinoma in situ of the cervix ; or death as a result of any cause without documentation of a cancer related event . exploratory analysis of possible predictive biomarkers in this exploratory analysis , 3 , 669 gene mrna probes on the illumina humanht - 12 v3 array , which are based on normal breast tissue mrna expression profiles not expected to be expressed in breast tissue , 10 were used to conduct an exploratory analysis of possible predictive biomarkers.1 each probe was used in a cox proportional hazards regression predictive model as a categorical variable bifurcated on the mean of gene mrna expression . 
the list of probes ranked by interaction p value were then used to identify c8a as a gene of interest because it is not normally expressed in breast tissue and is a member of the membrane attack complex and part of the innate immune systea volcano plot of the exploratory analysis is shown in figure 1 . 
exploratory analysis of treatment interaction p value as a categorical variable split on probe mrna expression of 3 , 669 gene probes in the herceptin adjuvant cdna - mediated annealing , selection , extension , and ligation cohort that have zero expected expression in normal breast tissue . 
in the early events analysis , only early disease - defining events were taken into account ( ie , at 2.2 years of median follow - up time ) , when almost no patient had switched from observation to trastuzumab . 
 cox models were adjusted for several clinicopathologic factors : age , pathologic tumor size , progesterone receptor local , estrogen receptor local , tumor grade , menopausal status , nodal status , prior ( neo ) adjuvant chemotherapy , eastern cooperative oncology group performance status , race , and region . 
the weights for the ipw analysis were estimated using a time - varying covariate and the following two baseline covariates : age at study entry and prior ( neo ) adjuvant chemotherapy . 
follow - up time of each eligible patient was divided into 1 - month intervals , and the time - varying covariate was estimated as the within - interval cumulative time of eligibility to cross over . prognostic her2 + gastric cancer cohort we used the publicly available data from the hungarian academy of science ( has ) gastric cancer cohort to further explore c8a as a possible biomarker in her2 + gastric cancer.12 the has gastric cancer cohort is a collection of publicly available cohorts with outcome data profiled on the affymetrix platform ( santa clara , ca )  . 
molecular signature database ( version 4.0 ) gene sets tested included immunologic signatures ( n = 1 , 910 ) , canonical pathways ( n = 1 , 320 ) , molecular gene ontology terms ( n = 1 , 454 ) , microrna ( n = 221 ) , chromosome positions ( n = 326 ) , transcription factors ( n = 615 ) , chemical genetic perturbations ( n = 3 , 402 ) , and oncogenic signatures ( n = 189 )  . c8a mrna normal and cancer tissue expression profile the gtex web portal17 was used to generate a figure for the c8a mrna expression in normal tissues . 
figures that illustrate c8a mrna expression from cancer cell lines were generated using the cancer cell line portal.18 the cancer cell line encyclopedia was used to determine the c8a mrna mean and standard deviation in 1 , 036 cancer cell lines . 
tumor cell lines , hepatocellular carcinoma ( tma809 ) , lung cancer ( dms454 ) , triple negative breast cancer ( mda - 231 ) , her2 normal breast cancer ( mda - 175 ) , and her2 + breast cancer ( sk - br - 3 ) were used for c8a ihc staining . 
 the mrna expression profile suggests that for approximately 70% of the human epidermal growth factor 2positive cohort , c8a mrna was not expressed , and 30% had high expression of c8a mrna . 
kaplan - meier curve for disease - free survival ( dfs ) in the herceptin adjuvant cdna - mediated annealing , selection , extension , and ligation cohort , where the c8a mrna split on the mean represents c8a - low and c8a - high categories . 
the c8a phenotype is illustrated in the gsea histogram shown in figure 4 , with the top 50 genes enriched in the c8a - low group versus the top 50 genes enriched in the c8ahigh group . 
c8a is part of the innate immunity and membrane attack complex and clearly differentiated the enrichment of gene sets associated with immunologic response . c8a mrna expression the c8a mrna on / off expression profile in our analysis cohort suggests that c8a may be expressed in other cancer cell lines . 
on the basis of the results presented in the data supplement , we see that c8a is highly expressed in distinct ovarian , liver , stomach , pancreatic , lung , and hematopoietic / lymphoid cancer types . 
to verify that c8a mrna results in c8a protein expression , c8a antibody was used to stain available cancer cell lines , and the results are the data supplement . discussion identification of single genes or gene signatures that can be used to classify a breast cancer tumor as a subtype with known treatment strategies can improve outcomes and minimize unnecessary treatments . 
 the immune gene list from molecular signature database ( version 4.0 ) on the right represents the top 50 enriched immune genes in c8a - low versus c8ahigh samples and the top 50 enriched immune genes in c8a - high versus c8a - low samples . 
the cancer cell line encyclopedia data show that dms454_lung , ov90_ovary , and snu719_stomach are examples that have high expression of c8a and that c8a is a feature of those immortal cancer cell lines . c8a protein is a critical component of the membrane attack complex that inserts itself into the outer membrane of target cells and anchors the recruitment of c9 proteins to form a pore that leads to cell lysis and death.20 , 21 the membrane attack complex is a component in the complement system , which is part of the innate immune system found in plant and animals and evolved before adaptive immunity.22 the innate immune system can work independently of or be triggered by the adaptive immune system and plays a role in the monitoring of host cells that are damaged or have died and should be cleared.23 the complement system proteins are synthesized by the liver and normally circulate in the blood until stimulated by complement activation . 
molecular characterization of c8a high her2 + tumors may indicate that it is a new cancer phenotype that can escape the immune response and become an important mechanism in cancer that affects survival . 
goldhirsch a , gelber rd , piccart - gebhart mj , et al : 2 years versus 1 year of adjuvant trastuzumab for her2 - positive breast cancer ( hera ) : an open - label , randomised controlled trial . 
skedgel c , rayson d , younis t : is adjuvant trastuzumab a cost - effective therapy for her - 2 / neu - positive t1bn0 breast cancer ? ann oncol 24 : 1834 - 1840 , 2013 10 . 
benita y , cao z , giallourakis c , et al : gene enrichment profiles reveal t - cell development , differentiation , and lineage - specific transcription factors including zbtb25 as a novel nf - at repressor . 
gyrffy b , lanczky a , eklund ac , et al : an online survival analysis tool to rapidly assess the effect of 22 , 277 genes on breast cancer prognosis using microarray data of 1 , 809 patients . 
gyorffy b , lnczky a , szllsi z : implementing an online tool for genome - wide validation of survival - associated biomarkers in ovarian - cancer using microarray data from 1287 patients . 
gyrffy b , benke z , lnczky a , et al : recurrenceonline : an online analysis tool to determine breast cancer recurrence and hormone receptor status using microarray data . 
said ea , dupuy fp , trautmann l , et al : programmed death - 1 - induced interleukin - 10 production by monocytes impairs cd4 + t cell activation during hiv infection . 
 c entrectinib in two pediatric patients with inflammatory myofibroblastic tumors harboring ros1 or alk gene fusions introduction inflammatory myofibroblastic tumor ( imt ) is a rare , indolent spindle cell tumor that typically affects children and young adults.1 , 2 imts occur predominantly in visceral soft tissue and are categorized as an intermediate malignancy because they have a potential for local recurrence but rarely progress to distant metastases.2 , 3 approximately 50% of imts harbor alk rearrangements , although gene fusions involving ros1 , ntrk3 , and pdgfr have also been identified.2 , 4 - 6 previously , the presence of alk gene fusions was typically identified using immunohistochemistry7 ; however , newer techniques such as hybridization capture - based next - generation sequencing ( ngs ) and ngs - based anchored multiplex polymerase chain reaction ( pcr ) testing can now be used to detect gene fusions.8 - 10 identifying patients with actionable gene fusions is important , given the availability of promising therapeutic strategies.2 , 4 surgical resection is the standard of care for patients with a solitary imt , 11 but patients with unresectable or advanced disease have limited treatment options . 
the current national comprehensive cancer network guidelines for soft tissue sarcoma recommend the use of alk inhibitors for patients with alk gene fusions12 ; however , there are no targeted agents approved specifically for use in fusion - positive imts . 
 entrectinib is an investigational , cns - active , potent , and selective inhibitor of trk , ros1 , and alk.8 , 13 , 14 in phase i trials , entrectinib demonstrated clinical efficacy in patients with different tumor types harboring ntrk , ros1 , or alk fusions , regardless of the fusion partner gene.8 a majority of the responses to entrectinib occurred in a rapid and durable manner , and some patients remained on study because they received clinical benefit , even after experiencing disease progression.8 here , we present two patients with fusion positive imts ( dctn1 - alk and tfg - ros1 ) who were enrolled in the ongoing startrk - ng phase i / ib trial ( nct02650401 : study of rxdx 101 in children with recurrent or refractory solid tumors and primary cns tumors , with or without trk , ros1 , or alk fusions ) and treated with entrectinib . 
further molecular testing using a hybrid capture - based targeted exome sequencing assay15 revealed a dctn1 - alk fusion ( fig 1a ) , which determined eligibility to enroll in the startrk - ng trial . 
 ( a ) the dctn1alk rearrangement identified in patient 1 is a deletion that results in the fusion of dctn1 exons 1 - 26 with alk exons 20 - 29 . 
 ( d ) axial t1 postcontrast image shows complete response of the tumor to entrectinib treatment ( no measurable tumor present , red arrow )  . the patient provided informed consent , and entrectinib was started in may 2017 at a dose of 550 mg / m2 taken orally once per day ( 1 cycle was 28 days )  . 
 at the time of this report , she continues daily entrectinib and has no limitations in academic or athletic activities . patient 2 is a 10 - year - old girl who presented with daily high - grade fevers , cough , weight loss , and changes in physical activity over a 2 - month period . 
hybrid capture - based targeted exome sequencing and anchored multiplex pcr as described above15 , 16 identified a tfg - ros1 gene fusion ( fig 2a ) , and she was subsequently enrolled in the startrk - ng trial . the patient provided informed consent and entrectinib was started in may 2017 at a dose of 550 mg / m2 orally once per day . 
in addition , by month 4 of treatment , the residual mass was primarily nonenhancing on contrast - enhancing magnetic resonance imaging , suggesting relative lack of activity ( fig 2b - 2c )  . 
while on treatment , she experienced constipation , mild bloating , nausea , and weight gashe also experienced nighttime urinary incontinence that was potentially related to treatment and improved with simple changes in her nighttime routine . 
both patients exhibited clinically significant responses to the investigational agent entrectinib , which was well tolerated , and the observed adverse effects were consistent with the reported safety profile of entrectinib.8 these cases are in agreement with previous reports demonstrating the clinical efficacy and tolerability of entrectinib in patients with solid tumors harboring ntrk , ros1 , or alk gene fusions , regardless of fusion partner.8 , 17 - 20 entrectinib seems to be well tolerated , as demonstrated in 203 patients treated at the recommended phase ii dose of 600 mg once per day.21 the most common treatment - related adverse events ( traes ) of any grade were dysgeusia ( 38% ) , fatigue ( 29% ) , constipation ( 23% ) , and dizziness ( 23% )  . 
in preclinical studies , entrectinib was a potent inhibitor of trk - driven neuroblastoma , 22 and it has demonstrated clinical activity in a 20 - month - old patient with infantile fibrosarcoma harboring an ntrk fusion.23 the benefit observed in the two patients reported here along with published data on the benefit of entrectinib in trk fusion - positive infantile fibrosarcoma highlight the potential of entrectinib in pediatric tumors harboring trk , ros1 , or alk fusions . 
the startrk - ng trial is currently enrolling pediatric patients with cancer , including primary cns tumors , neuroblastoma , and other non - neuroblastoma extracranial solid tumors harboring gene fusions in ntrk , ros1 , or alk , or harboring alk molecular alterations.23 these cases add to the growing body of evidence that demonstrates the diversity of gene fusion drivers in the formation of imt and underscore the importance of screening patients with imts for ntrk , ros1 , and alk gene fusions by using the best available diagnostic techniques . 
 the gene fusions present in these patients were identified by using a workflow that sequentially uses two different diagnostic platforms : a hybrid capture - based targeted exome sequencing assay ( msk - impact ) and anchored multiplex pcr ( archer fusionplex )  . 
 these cases also highlight the importance of treating patients with imts who harbor trk , ros1 , or alk fusions with drugs , such as entrectinib , that bind and inhibit the kinase domains of these fusion proteins . 
imt is often characterized by the presence of alk rearrangements ; however , additional gene fusions have also been identified in this rare cancer , including ros1 , ntrk3 , and pdgfr , 2 , 4 , 5 including a patient with etv6 - ntrk3 fusion - positive imt who is being considered for treatment with entrectinib.27 in patients with alk fusion positive imt , there have been varying efficacy results reported when patients are treated with alk inhibitors , including crizotinib , ceritinib , and alectinib.28 - 32 in addition , a recent report noted that a patient with imt harboring a tfg - ros1 fusion exhibited a poor response to crizotinib.6 there remains an unmet need in this patient population for safe and effective therapies that can treat patients across a wide range of gene fusion drivers . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . the potential benefit of entrectinib in that it inhibits select targeted kinases with high potency , allowing for the treatment of multiple distinct patient populations with one therapy . we have demonstrated clinically significant improvement in two patients with imts who harbor actionable gene fusions treated with entrectinib in the startrk - ng trial . 
basu no relationship to disclose acknowledgments the authors thank the patients who granted us consent to present their cases for the benefit of the scientific , medical , and patient communities worldwide and nick cianciola , of the lockwood group , for providing medical writing support funded by ignyta . accountable for all aspects of the work : all authors stock and other ownership interests : ignyta affiliations srikanth r . 
basu , memorial sloan kettering cancer center , new york , ny ; and edna chow - maneval , ignyta , san diego , ca . support supported by ignyta ; funded by national institutes of health national cancer institute grant no . 
antonescu cr , suurmeijer aj , zhang l , et al : molecular characterization of inflammatory myofibroblastic tumors with frequent alk and ros1 gene fusions and rare novel ret rearrangement . 
drilon a , siena s , ou si , et al : safety and antitumor activity of the multitargeted pan - trk , ros1 , and alk inhibitor entrectinib : combined results from two phase i trials ( alka - 372 - 001 and startrk - 1 )  . 
loke bn , lee vkm , sudhanshi j , et al : novel exon - exon breakpoint in cic - dux4 fusion sarcoma identified by anchored multiplex pcr ( archer fusionplex sarcoma panel )  . 
menichincheri m , ardini e , magnaghi p , et al : discovery of entrectinib : a new 3 - aminoindazole as a potent anaplastic lymphoma kinase ( alk ) , c - ros oncogene 1 kinase ( ros1 ) , and pantropomyosin receptor kinases ( pan - trks ) inhibitor . 
ardini e , menichincheri m , banfi p , et al : entrectinib , a pan - trk , ros1 , and alk inhibitor with activity in multiple molecularly defined cancer indications . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
green dc , deharvengt sj , de abreu fb , et al : use of anchored multiplex pcr enrichment for detection of gene fusions in solid tumors by next generation sequencing . 
drilon a , li g , dogan s , et al : what hides behind the masc : clinical response and acquired resistance to entrectinib after etv6 - ntrk3 identification in a mammary analogue secretory carcinoma ( masc )  . 
sartore - bianchi a , ardini e , bosotti r , et al : sensitivity to entrectinib associated with a novel lmna - ntrk1 gene fusion in metastatic colorectal cancer . 
ahn mj , cho bc , siena s , et al : entrectinib in patients with locally advanced or metastatic ros1 fusion - positive non - small cell lung cancer ( nsclc )  . 
rangaraju s , li g , christiansen j , et al : pediatric phase 1 / 1b study of entrectinib in patients with primary brain tumors , neuroblastoma , and ntrk , ros1 , or alk fusions . 
vendrell ja , taviaux s , bganton b , et al : detection of known and novel alk fusion transcripts in lung cancer patients using next - generation sequencing approaches . 
subbiah v , mcmahon c , patel s , et al : stump unstumped : anti - tumor response to anaplastic lymphoma kinase ( alk ) inhibitor based targeted therapy in uterine inflammatory myofibroblastic tumor with myxoid features harboring dctn1 - alk fusion . 
ono a , murakami h , serizawa m , et al : drastic initial response and subsequent response to two alk inhibitors in a patient with a highly aggressive alk - rearranged inflammatory myofibroblastic tumor arising in the pleural cavity . 
mansfield as , murphy sj , harris fr , et al : chromoplectic tpm3 - alk rearrangement in a patient with inflammatory myofibroblastic tumor who responded to ceritinib after progression on crizotinib . 
 large - cell neuroendocrine carcinoma of the lung : a focused analysis of braf alterations and case report of a braf non - v600 mutated tumor responding to targeted therapy purpose in advanced stages , large - cell neuroendocrine carcinoma of the lung ( l - lcnec ) mimics small - cell lung cancer despite its traditional classification as a non small - cell lung cancer . 
here we present a focused analysis of braf mutations in this population . patients and methods comprehensive genomic profiling of tumor tissues was performed from a cohort of 300 patients with biopsy - proven l - lcnec . 
the importance of biomarker - driven therapy is subsequently highlighted with our case of a 69 - year - old man diagnosed with metastatic l - lcnec who did not respond to cisplatin plus etoposide . 
a significant durable response was then demonstrated with therapy targeted toward a braf non - v600e activating mutation ( g469r ) associated with biomarker response identified through circulating cell - free tumor dna analysis . 
giles author affiliations and support information ( if applicable ) appear at the end of this article . corresponding author : young kwang chae , md , mph , mba , northwestern medicine developmental therapeutics institute , 645 n . 
the 2015 who classification modified this outline and instead included all lung nets as a single entity , acknowledging that there exist major histologic , epidemiologic , and clinical differences between the carcinoids and high - grade l - lcnec and sclc.3 l - lcnec remains both rare and difficult to treat given the discord between its histologic and clinical characteristics . 
 chemotherapy regimens used for the treatment of sclc should be used ( etoposide and platinum combinations ) , which is reflected in national clinical cancer network ( nccn ) guidelines . 
 in the era of next - generation sequencing ( ngs ) , it is interesting to note that this strategy has yet to be adapted for the category of nonadenocarcinomas and nonsquamous cell carcinomas , which is certainly limiting in our ability to further identify these difficult - to - classify malignancies . 
nccn guidelines only mention ngs for adenocarcinomas . here we summarize the current landscape of l - lcnec and explore in detail the specific genomic alterations pertaining to braf , a member of the serine / threonine kinase raf family , in the largest cohort of patients with l - lcnec . 
 mutations and copy - number gains in the phosphatidylinositol 3 - kinase / akt / mammalian target of rapamycin pathway were detected in 15% of the tumors compared with 17% of sclc tumors.6 in another study using ngs in a cohort of 45 patient cases of l - lcnec , the most commonly altered genes similarly included tp53 ( 78% ) , rb1 ( 38% ) , stk11 ( 33% ) , keap1 ( 31% ) , and kras ( 22% )  . 
l - lcnecs with coaltered rb1 and tp53 ( 40% ) were identified as sclc - like , whereas tumors without this pattern ( 56% ) were identified as nsclc - like . 
as a whole , braf mutations are detected in 2% to 4% of lung cancers and have primarily been identified in adenocarcinomas.8 interestingly , although v600 mutations comprise the majority of braf alterations in melanoma , the v600e mutation is accountable for approximately 50% of braf mutations identified in nsclcs and up to 80% in late stages.8 as recently as late 2015 , the prognostic and biologic implications of braf mutations in nsclc were limited and controversial when evaluating the existing case series . 
subsequent studies were positive for cytokeratin 7 , thyroid transcription - 1 , synaptophysin , and chromogranin , thereby resulting in the diagnosis of metastatic , high - grade neuroendocrine carcinoma ( l - lcnec ) of lung primary ( fig 1 ; appendix ) .11 additional evaluation with brain magnetic resonance imaging and positron emission tomography ct identified a 1.4 - cm nodule in the superior segment of the left lower lobe , likely the site of the primary tumor . 
three months after rt , ct scans showed that the left hemipelvic mass had decreased in size to 3.5 4 cm , with a central area of necrosis ; however , the left lower lobe lung nodule was noted to have grown to 2 cm . ngs of tumor tissue ( foundationone ; foundation medicine , cambridge , ma ) had been performed at diagnosis and revealed mutations in braf ( g469r ) , erbb4 ( a118s ) , stk11 ( l282fs * 3 ) , and tp53 ( r248l ) ; myc amplification was also identified . 
given these molecular findings describing an activating braf mutation , treatment with trametinib ( 2 mg orally once daily ) and dabrafenib ( 150 mg orally twice daily ) was initiated at this time . 
trametinib is a mek inhibitor that prevents raf - dependent mek phosphorylation.8 dabrafenib is a braf serine / threonine kinase inhibitor that has high selectivity for braf v600emutant proteins.8 cell - free dna ( guardant360 ; guardant health , redwood city , ca ) analysis performed at diagnosis demonstrated concordance in detecting mutations in braf ( g469r ) and tp53 ( r248l ) , as well as myc amplification . 
 histology of needle core biopsy : ( c ) hematoxylin stain , 600 ; immunostains for ( d ) prostate - specific antigen , ( e ) cytokeratin 7 , ( f ) chromogranin , ( g ) synaptophysin , ( h ) thyroid transcription - 1 , and ( i ) cdx2 , 600 . expression of the patients braf g469r mutation was demonstrated , from 36.8% at time of diagnosis to nondetectable at both 5 months ( which may also have been in part a result of rt and subsequent tissue necrosis ) and 10 months after initiation of dabrafenib and trametinib ( fig 2 )  . there was an increase in other detectable alterations , which included synonymous mutations in brca1 , arid1a , and smo as well as vuss in apc , pten , fgfr1 , and pik3ca ( figs a1 - a3 )  . 
 presently , a full 15 months since initiating therapy with dabrafenib and trametinib , the patient is continuing with therapy and continues to demonstrate an eastern cooperative oncology group performance status of 0 . 
his latest ct scans ( 18 months after rt and 15 months after initiating targeted therapy ) show no residual pelvic mass and a stable left lower lobe nodule measuring 1.7 cm ( fig 3 )  . 
samples were pulled from an ngs database as part of a deidentified pool with only basic demographic information available ( required for processing the ngs order ) from many institutions . 
an inherent limitation of this pool is that we have to assume the histology of the malignancy was confirmed at each local institution . the atypical variants of braf in l - lcnec are identified as g469a , k601n , and g469r , which are all known to be activating mutations.14 - 17 there also exists g466v , which is an oncogenic kinase - impaired alteration.18 the remainder includes n581i and vus alterations e220k , w210l , g9d , and p149t . 
n581i is a known recurrently somatic braf alteration , but its effect has not been characterized ; otherwise , to our knowledge , these remaining braf mutations have not been previously identified.19 our analysis is summarized in table 1 . discussion current understanding of braf v600 mutations originated in melanoma , where they are the most commonly identified oncogenic mutations and are seen in nearly 50% of cutaneous melanomas.21 braf is an upstream effector of the mitogen - activated protein kinase pathway ( ras / raf / mek / extracellular regulated kinase ) , which plays a central role in cell growth , division , and differentiation . 
ninety percent of braf - mutated melanomas result from a substitution of glutamic acid for valine at codon 600.22 the braf v600e mutations show a 500 - fold increase in catalytic activity compared with the wild type.23 initially , selective braf inhibitors such as vemurafenib and dabrafenib were evaluated as monotherapy in patients with metastatic melanoma harboring either braf v600e or v600k mutations . 
contrast - enhanced computed tomography scans of the ( a , c , e , g ) abdomen and ( b , d , f , h ) chest of the patient ( a , b ) at diagnosis , ( c , d ) approximately 1 month after chemotherapy , ( e , f ) 3 months after radiation therapy ( rt ) directed at the left hemipelvic mass , and ( g , h ) after 15 months of targeted therapy with dabrafenib and trametinib . 
 melanoma , colorectal cancer , or nsclc , investigators concluded that kinase activity is likely to be elevated in 35% of braf mutations , impaired in 15% , and unknown in 10%.18 known nonv600e activating variants of braf that have been identified in patients with nsclc include g464v , g466a , g469a / v / r / s , and k601e . 
 mutations with impaired activity have been reported to be g466r / v , d594a / e / g / h / n / v , and g596c / r.20 interestingly , it has been identified that concomitant kras or nras mutations are more likely occur with the kinase - impaired braf mutants than the kinase - activated braf mutants.18 preclinical data have demonstrated sensitivity to treatment with dabrafenib and trametinib in hek293t cells , lung epithelial cellular models ( beas - 2b ) , and human cancer cell lines harboring non - v600 braf mutations ( both activating and kinase impaired )  . 
this same study also identified that the kinase activity of the g469s variant was comparable to that seen with the braf v600e mutation.20 this further provides a basis for the ongoing exploration of targeted therapy in non - v600 braf - mutant lung cancers . with regard to lung cancer , nccn guidelines for nsclc delineate emerging targeted therapy options for patients with genetic alterations.25 vemurafinib has been studied in multiple nonmelanoma cancers with braf v600 mutations in a basket trial design ( clinicaltrials.gov identifier nct01524978 ) ; in a cohort of 20 patients with nsclc , eight achieved partial responses with targeted braf inhibition.26 in a clinical trial studying dabrafenib plus trametinib in patients with previously treated braf v600emutant metastatic nsclc ( clinicaltrials.gov identifier nct01336634 ) , investigators reported an overall response rate of 63.2% in a cohort of 57 patients.27 this led to the recent approval of dabrafenib with trametinib as a treatment option for patients with advanced or metastatic nsclc with a braf v600 mutation by the us food and drug administration in june 2017.28 interestingly , there is minimal guidance available for non - v600 braf - mutated lung malignancies and even less information specifically focused on sclc or nets . 
remarkably when using ngs ( a broad and sensitive detection tool ) , non - v600e mutations comprise a substantial portion of the braf mutations in different tumors ( nsclc , 86% ; melanoma , 34% ; and colorectal cancer , 23% ) , as assessed in a cohort of more than 800 patients.17 this is a higher proportion of non - v600e braf mutations than identified in earlier retrospective studies with cohorts of 30 to 50 patients . 
in a retrospective study detailing the characteristics of nearly 700 patients with lung adenocarcinoma , braf mutations were present in 3% of patients ( v600e , 50% ; g469a , 39% ; and d594g , 11% )  . 
 interestingly , all of these patients were current or former smokers.29 currently , ngs assays demonstrate the best - known sensitivity and breadth of analysis for the clinical detection of braf mutations including non - v600e alterations.17 in our patient , of course , we must acknowledge the confounding variable of rt directed at the left hemipelvis in the reduction in size of the mass in this location . 
given this , in addition to the steep drop - off seen in braf circulating tumor dna , we suspect that this is a durable response to therapy . it is worth mentioning that cosmic presents information related to adenocarcinoma and squamous cell carcinoma but not l - lcnec . 
this does not reflect the scenario of a real - world stage iv malignancy . although not common , l - lcnec does seem to contain activating and thus actionable alterations as evidenced by our patient 's remaining stable with therapy focused at braf inhibition . 
 in particular , given the scope of non - v600 braf alterations , the basket trial design is ideal for furthering our understanding of the functionality of these various mutations in a histology - agnostic manner . 
the nci - match ( national cancer institute molecular analysis for therapy choice ) trial is currently under way and includes a nonv600 arm in which patients will receive trametinib . 
giles jon chung employment : foundation medicine administrative support : young kwang chae provision of study material or patients : young kwang chae , vincent miller collection and assembly of data : young kwang chae , keerthi b . 
 for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . stock and other ownership interests : foundation medicine xiaoqi lin no relationship to disclose vincent miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center siraj m . 
ali employment : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine ; patents via seres health on microbiome stuff in non - neoplastic disease ( i ) young kwang chae consulting or advisory role : foundation medicine , boehringer ingelheim , biodesix , counsyl , astrazeneca , guardant health speakers bureau : merck , genentech / roche travel , accommodations , expenses : hanmi francis j . 
tamragouri no relationship to disclose consulting or advisory role : novartis travel , accommodations , expenses : medimmune , novartis , foundation medicine affiliations young kwang chae , keerthi b . 
lurie comprehensive cancer center , northwestern university ; xiaoqi lin , northwestern memorial hospital , northwestern university , chicago , il ; and jon chung , vincent miller , and siraj m . 
yao jc , hassan m , phan a , et al : one hundred years after carcinoid : epidemiology of and prognostic factors for neuroendocrine tumors in 35 , 825 cases in the united states . 
travis wd , brambilla e , nicholson ag , et al : the 2015 world health organization classification of lung tumors : impact of genetic , clinical and radiologic advances since the 2004 classification . 
rekhtman n , pietanza mc , hellmann md , et al : next - generation sequencing of pulmonary large cell neuroendocrine carcinoma reveals small cell carcinoma - like and non - small cell carcinoma - like subsets . 
lin x , saad rs , luckasevic tm , et al : diagnostic value of cdx - 2 and ttf - 1 expressions in separating metastatic neuroendocrine neoplasms of unknown orig appl immunohistochem mol morphol 15 : 407 - 414 , 2007 12 . 
noeparast a , teugels e , giron p , et al : non - v600 braf mutations recurrently found in lung cancer predict sensitivity to the combination of trametinib and dabrafenib . 
planchard d , besse b , groen hjm , et al : dabrafenib plus trametinib in patients with previously treated braf ( v600e ) - mutant metastatic non - small cell lung cancer : an open - label , multicentre phase 2 trial . 
 fine - needle aspiration and core - needle biopsy percutaneous fine - needle aspiration ( fna ) and core - needle biopsy ( cnb ) were performed under ultrasound guidance using a 22 - gauge needle for fna and a 20 - gauge core biopsy device for cnb.10 one fna pass was used for localization . 
airdried fna smear was stained with diff - quik stain for on - site evaluation of adequacy and immediate interpretation by a board - certified cytopathologist ( x.l. ) , and alcohol - fixed fna smear was stained with papanicolaou statwo cnb passes were obtained for histologic examination . 
immunohistochemical stains for prostate - specific antigen ( a0562 ; dakocytomation , carpinteria , ca ) , cytokeratin 7 ( ck7 ; a7018 ; dakocytomation ) , chromogranin ( 760 - 2519 ; ventana , tucson , az ) , synaptophysin ( 336a - 76 ; cell marque , rocklin , ca ) , thyroid transcription - 1 ( ttf - 1 ; 343m - 98 ; cell marque ) , and cdx2 ( mu392a - uc ; biogenex , san ramon , ca ) were performed in an automated immunostainer with appropriate positive and negative controls following manufacturers instructions , meaning that positive controls were stained positive and negative controls were stained negative . pathology results fna smears showed loosely cohesive nests or three - dimensional clusters of pleomorphic epithelial cells with focal rosette pattern ( figs a1a and a1b )  . 
the tumor cells were positive for neuroendocrine immunomarkers ( chromogranin and synaptophysin ; fig a1f and a1g ) , ck7 ( fig a1e ) , and ttf - 1 ( a marker for lung adenocarcinoma , lung neuroendocrine neoplasm , 11 and thyroid neoplasm ; fig a1h )  . 
the tumor cells were negative for prostate - specific antigen ( fig a1d ) and cdx2 ( an immunomarker for gi adenocarcinoma and neuroendocrine neoplasm arising from gi tract ; fig a1i ) .11 on the basis of the morphology and immunoprofile , a large - cell neuroendocrine carcinoma diagnosis was rendered . 
the tumor cells were also positive for ttf - 1 and ck7 and negative for cdx2 , suggesting lung origin.11 appendix sample submission time point oct - 16 ( % ) aug - 15 ( % ) pten ( q214 * ) erbb2 ( r143q ) braf ( g469r ) 36.8 mar - 17 ( % ) fig . 
miller , md4 purpose next - generation sequencing ( ngs ) of tumor and germline dna is foundational for precision oncology , with rapidly expanding diagnostic , prognostic , and therapeutic implications . 
although few question the importance of ngs in modern oncology care , the process of gathering primary molecular data , integrating it into electronic health records , and optimally using it as part of a clinical workow remains far from seamless . numerous challenges persist around data standards and interoperability , and clinicians frequently face difculties in managing the growing amount of genomic knowledge required to care for patients and keep up to date . methods this review provides a descriptive analysis of genomic data workows for ngs data in clinical oncology and issues that arise from the inconsistent use of standards for sharing data across systems . 
questions about their provenance and timeliness of updating remapotentially useful standards for sharing genomic data , such as hl7 fhir and mcode , remain primarily in the research and / or development stage . 
2019 by american society of clinical oncology introduction next - generation sequencing ( ngs ) of tumor and inherited ( germline ) genomes has revolutionized and rened cancer treatment during the past two decades and is now vital for evaluating therapeutic opportunities in many solid and hematologic malignancies.1 currently , ngs panels including sets of genes are the most widespread method of rapidly identifying sequence variation in patients with cancer . 
ngs panels provide information for a variety of purposes , cluding diagnostics ( eg , determination of sarcoma subtype ) , hereditary risk assessment ( eg , lynch syndrome ) , prognosis ( eg , kras mutations in lung adenocarcinoma ) , and treatment selection ( eg , biomarkers for immunotherapy responsiveness , such as tumor mutational burden and microsatellite instability ; therapeutic selection for clinically actionable alterations , such as braf v600e in melanoma ; and biomarkers of resistance , such as loss of b2m for immunotherapy ) .2 , 3 as a metric of the signicance of ngs in oncology care , 29 of the 43 national comprehensive cancer network clinical practice guidelines denote specic sequence - based biomarkers important for clinical care ( table 1 )  . 
of these 29 guidelines for treatment of cancer by site , 12 include both somatic and germline biomarkers , 16 include somatic only , and one includes germline only . recently , there has been an increased focus on performing ngs on matched normal samples ( either adjacent tissue or blood ) to compare with tumor biopsies . 
 conway et al context key objective the amount of molecular knowledge required for patient care is exploding , but the minimal adoption of genomic data standards in electronic health records compounds the challenges facing oncologists . 
using examples from asco cancerlinq , this review covers the current status of next - generation sequencing , describes relevant data standards , highlights the pilot of the genomic smart - on - fhir application , and provides a call to action for laboratories and electronic health record vendors to ensure that the chain of data custody for structured genomic content remains unbroken and reaches the clinician . knowledge generated several genomic knowledge bases for variant interpretation are available to support clinicians . 
nonetheless , transmitting nextgeneration sequencing results via pdf hinders the use of decision support tools intended for clinical care , including trial selection , and blocks the downstream reuse of genomic data . relevance the consistent use of evolving standards ( eg , hl7 fhir and mcode ) should improve genomic data interoperability and care quality . 
however , the full integration of genotype and phenotype will require an innovative and socially driven commitment by all oncologic ecosystem participants , beginning with sequencing laboratories , to better leverage technologic solutions . also embedded in guidelines and reimbursement for certain treatments ( eg , poly [ adp - ribose ] polymerase inhibitors for ovarian cancer )  . 
multiple molecularly targeted agents have now been incorporated into standard patient management and reected in numerous guidelines . clinical quality measures the 2019 merit - based incentive payment system ( mips ) of the centers for medicare and medicaid services quality payment program contains 24 general oncology measures , two of which involve genetic testing ( kras and nras in patients with colorectal cancer ) .14 in addition , a number of clinical quality measures in the asco quality oncology practice initiative reference ngs testing , including nonsmall - cell lung cancer ( nsclc ) measures evaluating the use and turnaround time of molecular testing in patients with adenocarcinoma histology.15 as clinical quality measurement evolves from clinicianfocused process measures to more patient - centric outcome measures , it is reasonable to expect that more measures will incorporate results from ngs panels and potentially circulating tumor - free dna testing to foster appropriate diagnostic testing and therapy selection that affect patient outcomes . molecular testing has become essential to oncology care.12 survey data from 2017 showed that more than 75% of oncologists used ngs tests to guide cancer treatment . 
inclusion of genetic testing in nccn cancer site guidelines s , g , b , or n cancer subtype details cancer site acute lymphoblastic leukemia ( adult and aya ) acute lymphoblastic leukemia ( pediatric ) acute myeloid leukemia aids - related kaposi sarcoma anal carcinoma bladder cancer bladder cancer upper tract tumors urothelial carcinoma of the prostate primary carcinoma of the urethra ewing sarcoma family of tumors giant cell tumor of the bone bone chondrosarcoma chordoma osteosarcoma breast cancer cns cancer cervical cancer lymphoma chronic myeloid leukemia colon / rectal cancer colon cancer rectal cancer chronic lymphocytic leukemia / small lymphocytic esophageal and esophagogastric junction cancers gastric cancer gestational trophoblastic neoplasia hairy cell leukemia head and neck cancers hepatobiliary cancers hodgkin lymphoma kidney cancer malignant pleural mesothelioma melanoma cutaneous melanoma uveal melanoma multiple myeloma / other plasma cell neoplasms multiple myeloma systemic light chain amyloidosis waldenstr om macroglobulinemia / lymphoplasmacytic lymphoma myelodysplastic syndromes myeloproliferative neoplasms ( continued on following page ) efciently assess eligibility criteria . 
barriers include insufcient clinician knowledge , training , and condence regarding the use of ngs results.20 , 21 although many clinicians may take laboratorybased interpretations at face value , numerous knowledge bases have been created to aid oncologists and clinical researchers in the curation and interpretation of ngs data . these were developed largely because of the recognition that institutional ( local ) knowledge based on a limited sampling of the cancer population , including that possessed by testing laboratories , would not be sufcient to type , capture the genomic diversity of any cancer common or rare . 
perhaps a surprise to clinicians , many commonly tested genes , such as brca1 and brca2 , show considerable disagreement by interpreting laboratories about the pathogenicity of genetic variants.22 one of the earliest resources to address this issue was mycancergenome , 23 created at vanderbilt university in 2009 . 
since that time , there have been numerous entrants into an increasingly crowded eld , with the most well - known publicly available resources for variant interpretation being civic , 24 oncokb , 25and clinvar , 26 the latter of which is the rst fda - recognized public genetic variant database , housing clingen expert - curated data.27 each of these efforts was developed in relative isolation and with slightly different goals , leading to a divergence of data models , evidentiary models , and user experiences . 
the variant interpretation in cancer consortium , a driver project of the global alliance for genomics and health , 28 was founded with the mission statement to help unify and harmonize the disparate interpretation in cancer data sources . 
inclusion of genetic testing in nccn cancer site guidelines ( continued ) cancer site neuroendocrine and adrenal tumors s , g , b , or n cancer subtype details neuroendocrine tumors of the gi tract , lung , and thymus ( carcinoid tumors ) neuroendocrine tumors of the pancreas adrenal gland tumors pheochromocytoma / paraganglioma men1 men2 non - hodgkins lymphomas chronic lymphocytic leukemia / small lymphocytic lymphoma b - cell lymphomas primary cutaneous b - cell lymphomas t - cell lymphomas nonmelanoma skin cancers basal cell skin cancer dermatobrosarcoma protuberans merkel cell carcinoma squamous cell skin cancer nonsmall - cell lung cancer occult primary ( cancer of unknown primary ) ovarian cancer epithelial ovarian cancer ( including fallopian tube cancer and primary peritoneal cancer ) pancreatic adenocarcinoma penile cancer prostate cancer small - cell lung cancer soft tissue sarcoma systemic mastocytosis testicular cancer thyroid carcinoma uterine neoplasms endometrial carcinoma uterine sarcoma vulvar cancer thymomas and thymic carcinomas note . 
categorization of national comprehensive cancer network ( nccn ) guideline44 by cancer site showing whether the guideline recommends somatic testing ( s ) , germline testing ( g ) , both ( b ) , or neither ( n )  . 
genetic testing refers to short variations , structural sequence variations , cytogenetic analyses , and mismatch repair assays . abbreviations : aya , adolescent or young adult ; men1 , multiple endocrine neoplasia type 1 ; men2 , multiple endocrine neoplasia type 2 . several challenges associated with knowledge bases must be considered . 
although the aforementioned knowledge bases are free to users , they 4 2019 by american society of clinical oncology are generally not integrated into the clinical workow.30 the second is trustworthiness and culpability . 
can the assertions made by knowledge bases be trusted ? if advice is erroneous , the primary responsibility clearly lies with the managing clinician , but can knowledge bases also be held culpable ? furthermore , advice can become outdated ; this must be expected to some degree , given the exponential growth of the eld and the lag between the announcement of trial results , publication of ndings , fda labeling , and revision of variant interpretations ; however , poor recency of data in knowledge bases will diminish trust . 
many knowledge bases spend much effort on attractive so - called skins and search abilities , but the degree to which clinicians nd these interfaces useful has not been extensively studied . technological challenges of data integration : opportunities with fhir and mcode integration of ngs results into the clinical workow via tackle several ehr interfaces must technologic challenges . interpreted genomic results are in particular , generally reported in pdf format , which cannot support electronic search , clinical decision support ( cds ) , or secondary use.31 moreover , the lack of a common data model for genomic testing impedes all use cases by hampering data storage and interoperability between ehr systems and / or data warehouses , thereby hindering the use of cds tools intended to assist clinicians in caring for their patients . these issues have prompted the emergence of initiatives aimed at improving genomic data standards and facilitating genomic data interoperability . 
the top section shows total count of prognostic or predictive assertions related to this gene - variant combination , the breakdown by data source ( ve are shown ) , and the breakdown by assertion type per association for molecular pathology ( amp ) / asco / college of american pathologists ( cap ) guidelines.46 the middle section cross - references drugs and diseases with the gene of interest ( rst column ) , as well as potentially related genes ( adjacent columns ) , which are automatically determined ; heatmap colors indicate the quantity of evidence for a drug - gene or diseasegene correlation . 
the vicc meta - knowledgebase user guide is available online.47 auspices of sync for genes.36 these reports were generated from proprietary pipelines used for the fmi foundationone and foundationact ngs panels . 
although customers typically receive a pdf of the ndings and interpretations , fmi also sends xml les , based on a custom internal standard used to generate the pdfs , upon request . 
these xml les include more detailed information than that presented in the pdf , some of which is for research use only , and can be parsed to generate information relevant for fhir resources and elements ( eg , variant allele fraction , sequencing depth and functional effect for mutations , absolute copy number and copy - number ratio for copy - number events , number of supporting read pairs and identity of both genes involved for rearrangements , and genomic location for all alterations )  . 
briey , clinical elds such as patient , sample , and physician information could be mapped to patient , specimen , and physician fhir resources , respectively . genomic elds such as alterations ( eg , braf p.v600e , amplication , rearrangement ) , therapies , and clinical trials were mapped to elements of the observation genetics resource , with more detailed genetic information ( eg , chromosome , start position , end position , variant allele frequency , coverage , and variant type ) mapped to the molecular sequence resource . 
the entire fmi report was represented as a diagnostic report resource , which included references to the patient , physician , specimen , observation genetics , and molecular sequence resources . 
the modular design ( fig 2 ) was architected with four major goals : assist in clinical interpretation of variants within context , provide links to external resources , facilitate point - of - care physician - patient conversations , and demonstrate that smart - on - fhir technology can be used for a clinicogenomic use case . 
cancerlinq aggregates structured and unstructured data from ehrs and other sources via direct feeds and processes the data in a series of cloud - based databases where it normalized and deidentied for secondary reuse.40 database the cancerlinq database , now representing more than 1.5 million patients , contains vast amounts of detailed longitudinal clinical data , such as demographics , diagnoses , laboratory results , and medications . 
however , a signicant volume of the data is not computable , because many critical concepts ( cancer staging , biomarkers , disease progression , and adverse events ) reside primarily in text notes and other unstructured documents and not in discrete elds . 
smart ( sustainable medical apps , reusable technology ) precision cancer medicine ( pcm ) architecture diagrathe application is designed to be modular and extensible , particularly in the ability to add presentation modules and interfaces to external data sources . 
unique text strings are shown for two well - known egfr gene variations abstracted during curation of unstructured genomic reports that were obtained from electronic health records of practices participating in cancerlinq . 
although natural language processing technology itself has not yet demonstrated sufcient precision or recall to be used as a standalone solution , it has been successfully embedded in the curation workbench used by the data abstractors , where it functions to surface putative data elements from unstructured text that can then be conrmed or disregarded . curation of genomic data from ngs panels in cancerlinq as noted , ngs panels are typically brought into the ehr as pdfs or scanned faxes and therefore are not computable . however , since 2017 , cancerlinq has been extracting high - value genomic information via user interfaceassisted data abstraction . 
cancerlinq has also obtained and processed structured genomic reports in xml format and is evaluating automated processes to scan and extract data from reports with standardized formats . the magnitude of the genomic data gap in native ehrs is considerable and shows how poorly precision oncology is supported . 
similarly , for advanced nsclc , 85.3% of curated patient records had epidermal growth factor receptor tests , but structured epidermal growth factor receptor data were found in native ehrs for only 1.7% of all records for patients with advanced nsclc . it is worth noting that although cancerlinq has developed various technical solutions to structuring genomic data as it is housed in ehrs , all ngs data are originally generated as structured data by molecular diagnostics laboratories . 
the data capture of the various chemistries that underlie ngs sequencing , as well as the components of bioinformatic pipelines ( sequence aligners , variant callers ) and even clinical report production , comprises machine - readable , structured genomic data . 
the technical solutions cancerlinq has painstakingly developed would not be necessary at all if the originally structured data were provided . a stark example of how data quality is diminished when optical character recognition , needed to parse genomic reports , is applied , particularly to scanned images ( eg , faxed reports ) and tables , is listed in table 2 . 
 conway et al of national comprehensive cancer network guidelines as well as payment incentives ( eg , mips ) and quality programs ( eg , quality oncology practice initiative )  . 
clinical trial eligibility in oncology increasingly incorporates genomic biomarkers , and the fda has been modernizing its policies and review processes for cancer , resulting in an avalanche of new agents and approved indications for targeted therapies.41 there is no dearth of cds tools to help translate patient data into quality care , nor is there a lack of technical solutions to transmit , harmonize , curate , store , or otherwise manage data . 
these problems are not primarily technical , and solving them requires a will to action , with social incentives and disincentives . for example , ngs test results that are natively structured are sent to ordering physicians and stored in their ehrs , but in a manner that requires costly and data - lossy curation to restructure back into usable information . 
considering that the genomic results are reimbursed through federal and private insurers , but the reporting format impedes their utility to clinicians , we call for all molecular diagnostic laboratories to provide structured data as part of routine reporting at the request of ordering physicians and their practices . 
this would help laboratories meet the denition of interoperability by the 21st century cures act34 to facilitate health information exchange without special effort on the part of the user while avoiding the act 's prohibition of information blocking . it is notable that on the basis of curated records , structured brca1 or brca2 gene test results are extremely underrepresented among the 10 ehrs used by the more than 50 cancerlinq practices . 
it is possible to integrate genomic and clinical data from apis externally to native ehrs , but greater cooperation is needed from vendors to export clinical data for cds use and to provide a seamless experience for the clinician , whose attention is tethered to the ehr . adoption of data standards , including hl7 fhir and mcode , by all the entities that generate , transmit , and receive health information would also facilitate the implementation of precision oncology . 
the endless customizability of ehrs is at odds with health information technology interoperability and suboptimally supports automated quality measure reporting as required by mips.42 , 43 the dearth of coding for cancer staging , laboratory data , and medications within ehrs could be rectied by adopting widely used standard vocabularies , which would facilitate the computable representation of patients oncology records as envisioned by mcode . 
miller , md , cancerlinq , american society of clinical oncology , 2318 mill rd , suite 800 , alexandria , va 22314 ; e - mail : robert.miller@asco.org. support supported by award no . 
 ngs data challenges and solutions references freedman an , klabunde cn , wiant k , et al : use of next - generation sequencing tests to guide cancer treatment : results from a nationally representative survey of oncologists in the united states . 
mol oncol 8 : 859 - 873 , 2014 kamps r , brando rd , bosch bj , et al : next - generation sequencing in oncology : genetic diagnosis , risk prediction and cancer classication . 
int j mol sci 18 : e308 , 2017 jones s , anagnostou v , lytle k , et al : personalized genomic analyses for cancer mutation discovery and interpretation . 
sci transl med 7 : 283ra53 , 2015 cibulskis k , lawrence ms , carter sl , et al : sensitive detection of somatic point mutations in impure and heterogeneous cancer samples . 
nat biotechnol 31 : 213 - 219 , 2013 carter sl , cibulskis k , helman e , et al : absolute quantication of somatic dna alterations in human cancer . 
nat biotechnol 30 : 413 - 421 , 2012 robson me , bradbury ar , arun b , et al : american society of clinical oncology policy statement update : genetic and genomic testing for cancer susceptibility . j clin oncol 33 : 3660 - 3667 , 2015 karow j : tumor - only sequencing may misguide therapy but many labs omit matched control . 
may - misguide - therapy - many - labs - omit - matched - control damodaran s , berger mf , roychowdhury s : clinical tumor sequencing : opportunities and challenges for precision cancer medicine . 
saigi m , alburquerque - bejar jj , sanchez - cespedes m : determinants of immunological evasion and immunocheckpoint inhibition response in non - small cell patterns of carcinoma evolution . 
gao p , zhang r , li j : comprehensive elaboration of database resources utilized in next - generation sequencing - based tumor somatic mutation detection . biochim biophys acta rev cancer . 
j clin oncol 32 : 1317 - 1323 , 2014 johnson l - m , valdez jm , quinn ea , et al : integrating next - generation sequencing into pediatric oncology practice : an assessment of physician condence and understanding of clinical genomics . 
lawler m , siu ll , rehm hl , et al : all the worlds a stage : facilitating discovery science and improved cancer care through the global alliance for genomics and 29 . 
hughes ks , ambinder ep , hess gp , et al : identifying health information technology needs of oncologists to facilitate the adoption of genomic medicine : recommendations from the 2016 american society of clinical oncology omics and precision oncology workshop . 
li m , datto m , duncavage e : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
ali , md2 ; luke juckett , ms2 ; funda meric - bernstam , md1 ; and vivek subbiah , md1 introduction one to two percent of nonsmall - cell lung cancers ( nsclcs ) harbor ros1 gene rearrangements.1 - 3 ros1 gene rearrangement leads to constitutive activation receptor tyrosine kinase that activates downstream mitogen - activated protein kinase , phosphoinositide - 3 kinase , signal transducer and transcription 3 , and other pathways activator of leading to oncogenesis.4 because ros1 shares 49% amino acid sequence homology with anaplastic lymphoma kinase ( alk ) in the kinase domain , ros1 rearranged ( ros1 - positive ) nsclc tends to be sensitive to alk inhibitors.5 crizotinib , a mesenchymal epithelial transition factor ( met ) / alk / ros1 tyrosine kinase inhibitor ( tki ) , is the only us food and drug administrationapproved drug for ros1 - positive nsclc.6 ceritinib and brigatinib , second - generation alk inhibitors , have demonstrated activity in crizotinib - nave ros1 - positive nsclc but lacked activity in patients who were crizotinib resistant in anecdotal cases.7 , 8 however , the efcacy of both ceritinib and brigatinib in patients who are crizotinib resistant remains unclear . 
fludeoxyglucose positron emission tomographyct scan after completing four cycles of chemotherapy showed increasing pleural nodularity . comprehensive next - generation sequencing of tumor tissue obtained revealed a cd74 - ros1 rearrangement ( fig 1b )  . 
each of the two cerebellar lesions was treated with gamma knife radiosurgery ( 20 gy to 50% isodose ) , and the patient was enrolled in an anticytotoxic t - cell lymphocyte - 4 antibody ipilimumab and radiation trial ( clinicaltrials . gov identier : nct02239900 )  . 
as per trial protocol , he received external beam radiotherapy to the rll lesion ( 60 gy in 10 fractions ) with concurrent ipilimumab . there was disease progression noted in mediastinal lymph nodes and pleural metastasis while on the aforementioned trial . 
he was then enrolled in the modular phase ii identier : basket signature ( clinicaltrials.gov nct02186821 ) trial with ceritinib for ros1 - aberrant cancers at 750 mg orally daily.11 restaging scans after two cycles and once again after four cycles conrmed a partial response ( pr ; 56% decrease ) per response evaluation criteria in solid tumors ( recist ) 1.1. 
in addition , mri showed reduction in brain metastasis . he continued to have pr while receiving ceritinib for 8 months , until it had to be held for grade 3 elevation in ast and alt . 
 ( a ) the various treatments the patient received for metastatic ros1 - positive nonsmall - cell lung cancer , along with the duration of and best response to each treatment . 
 ( c ) computed tomography and magnetic resonance images of the patients right anterior diaphragmatic lymph node , right lower lobe nodule , and left cerebellar metastases before and at the indicated times after he initiated treatment with brigatinib . 
a radiologic unconrmed partial response by response evaluation criteria in solid tumors 1.1 was achieved after 3 months in the target lesion ( right lower lobe nodule ) , with concurrent response in the right anterior diaphragmatic lymph node and stable cerebellar metastases . cr , complete response ; pd , progressive disease ; pr , partial response ; upr , unconrmed partial response ; sd , stable disease . ceritinib continued to clinically benet the patient , demonstrating both systemic and cns activity for another 17 months . 
eventually , he experienced progression in brain metastases , mediastinal lymph nodes , and rll lesions . plasma cell - free dna ( cfdna ) testing ( guardant panel , guardant health , redwood , ca ) showed notch s2435s , tp53 g245a , and p190t , as well as fbxw7 g477s mutations but no cd74 - ros1 fusions or other ros1 pathway aberrations . 
ct scan of the chest also showed 59% decrease in target lesions by recist 1.1 criteria ( unconrmed pr ) , with decrease in rll lesions and resolution of mediastinal lymphadenopathy ( fig 1c )  . 
however , owing to a signicant decline in performance status ( eastern cooperative oncology group score , 4 ) due to other comorbidities and prolonged hospitalization , he was transitioned to hospice care . discussion to our knowledge , this is the rst report of crizotinibresistant ros1 - positive nsclc responding for an extended period of time ( 25 months ) , to ceritinib9 and subsequently to brigatinib . 
mechanisms of resistance to crizotinib in ros1 - positive nsclc include mutations involving the ros1 kinase domain and activation of bypass signaling pathways.13 - 15 the most common of the ros1 resistance mutations is the g2032r solvent front mutation that causes steric interference to crizotinib binding.15 , 16 the g2032r mutation occurs in close to 50% of crizotinibresistant nsclc.15 in vitro studies have demonstrated that both ceritinib and brigatinib are unable to overcome the common g2032r resistance mutation.12 , 17 various other mutations in the ros1 kinase domain confer resistance to crizotinib.18 - 21 of these , the l2026m gatekeeper mutation maintains sensitivity to ceritinib and brigatinib.12 , 20 brigatinib is a tki that has preclinical activity against ros1.12 in an in vitro kinase inhibitory screen of more than 300 kinases , alk ( half maximal inhibitory concentration [ ic50 ] ; 0 . 6 nm ) was the only kinase inhibited with an ic50 less than 1 viability potently inhibited brigatinib nm . 
another possibility is the re - emergence of the cd74 - ros1 fusion during ipilimumab plus radiation and the subsequent 3 - month treatment hiatus because of lack of selection pressure in the absence of crizotinib , rendering the tumor sensitive to ceritinib . 
however , the lapse between ceritinib and brigatinib therapy was only 12 days , making such a phenomenon less likely . one of the major limitations of this study is the lack of onand post - progression biopsies , which are critical to understanding response and / or resistance mechanisms . these were not performed because the patient declined biopsy , considering advanced age , comorbidities , clinical treatment at progression , and the risk of urgency of pneumothorax . instead , we obtained cfdna testing ( guardant panel ) at disease progression , which was nondiagnostic , either because of ros1 cfdna suppression by ongoing ceritinib treatment or limited sensitivity of cfdna platform in detecting the ros1 fusion . 
although cns activity of brigatinib in ros1 - positive nsclc is not documented , it is likely superior to crizotinib and possibly even ceritinib , given the ndings in alk - rearranged nsclc . 
our patient was refractory to ipilimumab and radiation but responded promptly to subsequent ros1directed therapy , once again highlighting the lack of benet of second - line immune checkpoint monotherapy in patients with certain subsets of oncogene - driven nsclc.27 , 28 several ros1 - directed tkis are being studied in clinical trials ( table 1 )  . 
hong stock and other ownership interests : molecularmatch , oncoresponse , presagia honoraria : adaptimmune , baxter , merrimack pharmaceuticals , bayer consulting or advisory role : alpha insights , axiom , adaptimmune , baxter , bayer , genentech , glg pharma , group h , guidepoint global , innity , janssen pharmaceuticals merrimack , medscape , numab , pzer , seattle genetics , takeda , trieza therapeutics research funding : abbvie , adaptimmune , amgen , astra - zeneca , bayer , bms , daiichi - sankyo , eisai , fate therapeutics , genentech , genmab , ignyta , innity , kite , kyowa , lilly , loxo , merck , medimmune , mirati , mirna , molecular templates , mologen , nci - ctep , novartis , pzer , seattle genetics , takeda travel , accommodations , expenses : loxo oncology , mirna therapeutics siraj m . 
ann oncol 24 : 1822 - 1827 , 2013 yoshida a , kohno t , tsuta k , et al : ros1 - rearranged lung cancer : a clinicopathologic and molecular study of 15 surgical cases . 
am j surg pathol 37 : 554 - 562 , 2013 chin lp , soo ra , soong r , et al : targeting ros1 with anaplastic lymphoma kinase inhibitors : a promising therapeutic strategy for a newly dened molecular subset of non - small - cell lung cancer . 
j thorac oncol 7 : 1625 - 1630 , 2012 ou s - hi , tan j , yen y , et al : ros1 as a druggable receptor tyrosine kinase : lessons learned from inhibiting the alk pathway . 
n engl j med 371 : 1963 - 1971 , 2014 lim sm , kim hr , lee j - s , et al : open - label , multicenter , phase ii study of ceritinib in patients with non - small - cell lung cancer harboring ros1 rearrangement . j clin oncol 35 : 2613 - 2618 , 2017 gettinger sn , bazhenova la , langer cj , et al : activity and safety of brigatinib in alk - rearranged non - small - cell lung cancer and other malignancies : a singlearm , open - label , phase 1 / 2 trial . 
lancet oncol 17 : 1683 - 1696 , 2016 subbiah v , hong ds , meric - bernstam f : clinical activity of ceritinib in ros1 - rearranged non - small cell lung cancer : bench to bedside report . 
zhang s , anjum r , squillace r , et al : the potent alk inhibitor brigatinib ( ap26113 ) overcomes mechanisms of resistance to rstand second - generation alk inhibitors in preclinical models . 
gainor jf , tseng d , yoda s , et al : patterns of metastatic spread and mechanisms of resistance to crizotinib in ros1 - positive non - small - cell lung cancer . 
drilon a , somwar r , wagner jp , et al : a novel crizotinib - resistant solvent - front mutation responsive to cabozantinib therapy in a patient with ros1 - rearranged lung cancer . 
song a , kim tm , kim d - w , et al : molecular changes associated with acquired resistance to crizotinib in ros1 - rearranged non - small cell lung cancer . 
schrock ab , pavlick d , klempner sj , et al : hybrid capture - based genomic proling of circulating tumor dna from patients with advanced cancers of the gastrointestinal tract or anus . 
clark ta , chung jh , kennedy m , et al : analytical validation of a hybrid capture - based next - generation sequencing clinical assay for genomic proling of cell - free circulating tumor dna . 
patil t , smith de , bunn pa , et al : the incidence of brain metastases in stage iv ros1 - rearranged non - small cell lung cancer and rate of central nervous system progression on crizotinib . 
camidge dr , kim d - w , tiseo m , et al : exploratory analysis of brigatinib activity in patients with anaplastic lymphoma kinase - positive nonsmall - cell lung cancer and brain metastases in two clinical trials . 
gainor jf , shaw at , sequist lv , et al : egfr mutations and alk rearrangements are associated with low response rates to pd - 1 pathway blockade in non - small cell lung cancer : a retrospective analysis . 
lee ck , man j , lord s , et al : checkpoint inhibitors in metastatic egfr - mutated non - small cell lung cancer : a meta - analysis . 
zou hy , li q , engstrom ld , et al : pf - 06463922 is a potent and selective next - generation ros1 / alk inhibitor capable of blocking crizotinib - resistant ros1 30 . 
facchinetti f , loriot y , kuo ms , et al : crizotinib - resistant ros1 mutations reveal a predictive kinase inhibitor sensitivity model for ros1and alk - rearranged mutations . 
drilon a , ou si , cho bc , et al : repotrectinib ( tpx - 0005 ) is a next - generation ros1 / trk / alk inhibitor that potently inhibits ros1 / trk / alk solvent - front 32 . 
ardini e , menichincheri m , ban p , et al : entrectinib , a pan - trk , ros1 , and alk inhibitor with activity in multiple molecularly dened cancer indications . 
 enrichment of fgfr3 - tacc3 fusions in patients with bladder cancer who are young , asian , or have never smoked purpose fgfr3 - tacc3 ( fibroblast growth factor receptor 3transforming acidic coiled coil - containing protein 3 ) fusions have recently been identified as driver mutations that lead to the activation of fgfr3 in bladder cancer and other tumor types and are associated with sensitivity to tyrosine kinase inhibitors . 
we examined the clinical and molecular characteristics of patients with fgfr3 - tacc3 fusions and hypothesized that they are enriched in a subset of patients with bladder cancer . materials and methods we correlated somatic fgfr3 - tacc3 fusions with clinical and molecular features in two cohorts of patients with bladder cancer . 
the second cohort consisted of patients with mibc or high - grade nonmibc at the dana - farber cancer institute that had targeted capture sequencing of a selected panel of cancer genes ( n = 356 )  . 
the clinical response of one patient with fgfr3 - tacc3 bladder cancer to an fgfr3 inhibitor was investigated . results overall , 751 patients with high - grade bladder cancer without fgfr3 - tacc3 fusions and 17 with fgfr3 - tacc3 fusions were identified in the pooled analysis of the data sets from the cancer genome atlas and the dana - farber cancer institute . 
 fgfr3 - tacc3 fusions were enriched in patients age 50 years versus age 51 to 65 years versus those older than 65 years ( pooled , p = .002 ) , and were observed in four ( 12% ) of 33 patients age 50 years in the pooled analysis . 
in 2017 , 79 , 030 new cases of bladder cancer are expected to be diagnosed , and approximately 16 , 870 deaths are predicted to occur from the disease in the united states.1 , 2 compared with other cancer subtypes , advances in the management of bladder cancer have been limited in the past three decades , and there is an unmet need to develop novel therapeutic agents that target potentially actionable alterations.3 , 4 genomic alterations in fibroblast growth factor receptors ( fgfrs ) are among the most frequent events during bladder cancer development . 
fgfrs are receptor tyrosine kinases that orchestrate various cellular processes , including cell proliferation , differentiation , and survival.5 fgfr mutations lead to developmental syndromes when present in the germline , and contribute to cancer growth when acquired somatically.6 fgfr fusions with an intact kinase domain have been identified in several cancer amin h . 
consort diagram for 850 patients with bladder cancer . clinical cohort ( n = 850 ) dana - farber cancer institute ( n = 438 ) the cancer genome atlas ( n = 412 ) whole - exome sequencing ( n = 412 ) paired tumor - normal muscle - invasive bladder cancer rna sequencing for structural variant calls assessment of clinical and molecular characteristics in fgfr3 - tacc3positive versus negative bladder tumors institutional targeted next - generation sequencing assay oncopanel ( n = 438 ) computational workflow ( n = 365 ) mutational calling ( mutect ) copy number variations ( viscap - cancer ) breakmer algorithm ( structural variants ) exclusions low - grade nonmuscle invasive histology ( n = 73 ) exclusions low tumor purity ( < 20% ; n = 9 ) final analysis ( n = 356 ) assessment of clinical and molecular characteristics in fgfr3 - tacc3positive versus negative bladder tumors types , including cervical cancer , bladder carcinoma , glioblastoma multiforme , squamous lung carcinoma , and head and neck cancer.7 - 15 fgfr3 , a member of this family , has been reported to be involved in fusions with several genes in bladder carcinoma , including tacc3 ( transforming acidic coiled coil - containing protein 3 )  . 
tacc3 normally is thought to mediate the stabilization and organization of the mitotic spindle during mitosis.14 in the cancer genome atlas ( tcga ) muscle invasive bladder cancer ( mibc ) cohort , in - frame activating fgfr3 - tacc3 fusionsobserved in 10 ( 2.4% ) of 412 patientswere the most common gene fusions identified.7 fgfr3 - tacc3 fusion proteins consist of the immunoglobulin , transmembrane , and tyrosine kinase domains of fgfr3 , fused to the coiled - coil domain of tacc3 . 
through the promotion of dimerization , these fusions lead to a constitutively active fgfr3 kinase protein that has been demonstrated to promote cell proliferation in vivo and in vitro.7 - 9 , 13 phase i and ii trials of fgfr inhibitors have reported promising antitumor activity in patients with fgfr genetic alterations , especially bladder cancer.16 certain genetic alterations , particularly gene fusion events , are enriched in clinical subsets of patients with cancer . 
 patients with mibc and high - grade non - mibc were pooled together in the dfci cohort as there is substantial evidence that the two subtypes are biologically and genomically similar.21 - 23 overall , seven patients with fgfr3 - tacc3 fusions were identified in the dfci cohort using an institutional targeted next - generation sequencing assay24 ( oncopanel )  . 
figure 1 shows the sample inclusion and exclusion criteria and workflow . tissue collection and dna extraction tumor specimens and clinicopathologic information were collected with institutional review board approval at dfci . 
tumor areas that contained at least 20% of tumor cells ( mean tumor purity , 58% ; range , 20% to 100% ) were isolated from normal tissue and chosen for dna extraction . 
dna was quantified by nanodrop and pico - green assays . targeted sequencing two hundred nanograms of genomic dna from each sample was subjected to targeted exon capture and sequencing using oncopanel_v1 to v3 cancer gene panels at brigham and womens hospital ( boston , ma )  . 
the oncopanel gene panel includes capture probes for 275 to 560 cancer - associated genes , as well as intronic portions of 60 genes for rearrangement detection , including fgfr3.24 sample dna was captured using oncopanel_v1 to v3 bait sets using a solution - phase agilent sureselect hybrid capture kit ( agilent technologies , santa clara , ca )  . 
single - nucleotide variants and small indels were analyzed using mutect version 1 0.27200 ( display / cgatools / mutect ; accessed may 2013 ) and annotated by oncotator ( broadinstitute.org / oncotator ; accessed may 2013 )  . 
 copy number alterations were analyzed using a custom r - based tool26 , 27 ( viscap - cancer )  . mean depth of read coverage for the targeted genes was 283 . 
mean , median , and range of percentage of target bases with read depth > 30 was 98% , 99% , and 78% to 99% , respectively . identification of rearrangements and analysis of genomic breakpoints fgfr3 fusion sequences were identified using the breakmer algorithm28 and were manually reviewed using integrated genomic viewer29 to exclude sequencing or alignment artifacts . 
all analyses of sequencing data and mutation and fusion calls were performed blinded to clinical data . clinical response to anti - fgfr3 therapy one patient with fgfr3 - tacc3 mibc received anti - fgfr3 therapy along with docetaxel and the clinical response was monitored . statistical analysis we used fisher 's exact test for categorical data and the wilcoxon rank - sum test for quantitative data . 
 ( % ) , unless otherwise noted . abbreviations : dfci , dana - farber cancer institute ; fgfr3 , fibroblast growth factor receptor 3 ; hg , high grade ; nd , not determined ; nmibc , nonmuscle invasive bladder cancer ; tacc3 , transforming acidic coiled - coil - containing protein 3 ; tcga , the cancer genome atlas . fusion partner fgfr3 - tacc3 fgfr3 - tacc3 fgfr3 - tacc3 fgfr3 - tacc3 fgfr3 - tacc3 fgfr3 - tacc3 fgfr3 - tacc3 fgfr3 - lmnb2 fgfr3 - tnip2 fgfr3 - fam184b fgfr3 - jakmip1 fgfr3 4 : 1808826 ; 4 : 1737404 4 : 1808771 ; 4 : 1740268 4 : 1808744 ; 4 : 1740297 4 : 1808806 ; 4 : 1741100 4 : 1808737 ; 4 : 1741297 4 : 1808690 ; 4 : 1741336 4 : 1808937 ; 4 : 1732863 4 : 1808556 ; 19 : 2436887 4 : 1808750 ; 4 : 2752213 4 : 1809003 ; 4 : 17691454 4 : 1808936 ; 4 : 6091989 fig 2 . 
schematic representation of the genomic rearrangements observed in 11 tumor samples that harbor fibroblast growth factor receptor 3 ( fgfr3 ) fusion variants identified using the oncopanel assay in the dana - farber cancer institute cohort . 
fam184b , family with sequence similarity 184 member b ; lmnb2 , lamin b2 ; jakmip1 , janus kinase and microtubule interacting protein 1 ; tacc3 , transforming acidic coiled - coil - containing protein 3 ; tnip2 , tnfaip3 interacting protein 2 . results formalin - fixed , paraffin - embedded tumor specimens were obtained from 438 patients at dfci . 
 we excluded 82 tumors from the analysis because they were of low - grade nonmuscle invasive histology ( n = 73 ) or had low ( < 20% ) tumor purity ( n = 9 )  . 
we mapped the genomic breakpoints of fgfr3 and its corresponding fusion partners that were identified in the dfci cohort , which included four non - tacc3 fusions ( fig 2 )  . 
 ( % ) , unless otherwise noted . abbreviations : dfci , dana - farber cancer institute ; fgfr3 , fibroblast growth factor receptor 3 ; tacc3 , transforming acidic coiled - coil - containing protein 3 ; tcga , the cancer genome atlas . patients age 50 harboring a fusion ( p = .03 ; table 2 )  . 
fgfr3 - tacc3 fusions in tcga were also more frequent in asians ( six [ 14% ] of 44 patients ) compared with other races ( p < .001 ) , as well as in never smokers ( eight [ 7.2% ] of 111 patients ) compared with ever smokers ( p < .001 ; table 2 )  . 
similarly , fgfr3 - tacc3 fusions were more common in dfci patients age 50 years ( one [ 12% ] of eight patients ) compared with other age groups ( p = .001 ; table 2 )  . 
eleven ( 65% ) of 17 patients with fgfr3tacc3 fusions were associated with least one of these three clinical characteristics , and three ( 18% ) of the 17 patients were asian never smokers age 50 years . we next examined whether tumors with fgfr3 - tacc3 fusions had molecular features that distinguished them from other tumors . 
as the oncopanel analysis was performed on tumor samples only , we excluded variants that were observed at any frequency in the exome aggregation consortium database , 30 as they were considered likely germline variants . 
the 17 patients whose tumors harbored fgfr3 - tacc3 fusions were enriched for cdkn1a mutations ( 5 [ 29% ] of 17 v 76 [ 10% ] of 751 ; p = .03 ; table 3 )  . 
conversely , fgfr3 - tacc3 fusion - positive tumors had significantly fewer tp53 mutations ( p = .02 ) , and none had rb1 mutations ( p = .054 ; table 3 )  . 
 somatic copy number alterations were also analyzed in both cohorts using criteria for loss , deletion , gain , and amplification that were developed and applied independently in the two cohorts ( table 3 )  . 
dfci cohort : amplification : log2 ( copy ratio ) > 1.8 , gain : 1.1 log2 ( copy ratio ) < 1.8 ; deletion : log2 ( copy ratio ) < 2 , loss : 2 log2 ( copy ratio ) < 1 . abbreviations : cnv , copy number variation ; dfci , dana - farber cancer institute ; fgfr3 , fibroblast growth factor receptor 3 ; snv , single nucleotide variant ; tacc3 , transforming acidic coiled - coil - containing protein 3 ; mdm2 , murine double minute 2 ; tcga , the cancer genome atlas . mdm2 ( murine double minute 2 ) gain ( p = .04 ) , deletion of pten ( p = .02 ) , and deletion of cdk2na ( p = .0033 ; table 3 )  . as a result of differences in the extent of genome sequencing in the tcga and dfci cohorts , we analyzed the overall mutational burden in each cohort separately . 
in the tcga cohort , the nonsynonymous somatic mutation rate across 18 , 862 genes was significantly higher in patients without fgfr3 - tacc3 fusions compared with those with fusions ( median 224 v 128 ; p = 0.04 ; table 3 )  . 
 in addition , there were no significant differences in the frequency of somatic copy number alterations in either the tcga or dfci cohorts ( table 3 )  . one patient who harbored the fgfr3 - tacc3 fusion in mibc in the dfci cohort was treated with an fgfr3 inhibitor and docetaxel and experienced complete remission for approximately 10 months . discussion our results demonstrate that patients with bladder cancer with fgfr3 - tacc3 fusions have distinct clinical and molecular features compared with the general population of patients with bladder cancer . 
in addition , fgfr3tacc3 fusions were associated with a low frequency of tp53 and rb1 mutations and a higher frequency of cdkn1a mutations , fgfr3 and mdm2 amplifications , and pten deletions . 
 because fgfr3 - tacc3 fusion - positive tumors can be sensitive to fgfr inhibitors , 9 , 31 , 32 these observations suggest that molecular testing to detect fgfr3 - tacc3 fusions in bladder cancer should be prioritized for patients who are young ( age 50 years ) , of asian race , and / or who have never smoked . 
most strikingly , we observed that all patients with bladder cancer who were asian never smokers younger than age 50 years ( n = 3 ) had fgfr3 - tacc3 fusions . we emphasize that our study has significant limitations as a result of the small number of patients with fgfr3 - tacc3 fusions included ( n = 17 ) , which reflects that this is a relatively rare molecular subset of bladder cancer . 
most importantly , we strongly advocate additional studies of this association to extend and confirm these findings . in conclusion , fgfr3 - tacc3 fusion - positive bladder cancer is highly enriched in asians , never smokers , and those age 50 years . 
nassar no relationship to disclose kevin lundgren no relationship to disclose mark pomerantz honoraria : bayer eliezer van allen stock and other ownership interests : synapse , tango therapeutics , genome medical consulting or advisory role : synapse , roche , third rock ventures , takeda , novartis , genome medical , invitae speakers ' bureau : illumina research funding : bristol - myers squibb , novartis lauren harshman consulting or advisory role : medivation , astellas pharma , pfizer , genentech , theragene , kew , corvus pharmaceuticals , merck , exelixis , bayer research funding : medivation , astellas pharma ( inst ) , bayer ( inst ) , sotio ( inst ) , genentech ( inst ) , dendreon ( inst ) , bristol - myers squibb ( inst ) , takeda ( inst ) , merck ( inst ) , janssen oncology ( inst ) , pfizer ( inst ) travel , accommodations , expenses : bayer atish d . 
sonpavde honoraria : uptodate consulting or advisory role : bayer , genentech , sanofi , merck , novartis , pfizer , argos therapeutics , agensys , eisai , astrazeneca , janssen pharmaceuticals , amgen , bristolmyers squibb , exelixis speakers ' bureau : clinical care options , national comprehensive cancer network , physician education resource , onclive , research to practice research funding : onyx pharmaceuticals ( inst ) , bayer ( inst ) , boehringer ingelheim ( inst ) , celgene ( inst ) , merck ( inst ) , pfizer ( inst ) other relationship : boehringer ingelheim , astrazeneca affiliations amin h . 
1p01ca120964 : molecular pathogenesis of the hamartoma syndromes . a directly related abstract was accepted as a poster presentation in the 2018 genitourinary cancer symposium of the american society of oncology , san francisco , ca , february 8 - 10 , 2018 . support prior presentation references 1 . 
acquaviva j , he s , zhang c , et al : fgfr3 translocations in bladder cancer : differential sensitivity to hsp90 inhibition based on drug metabolis mol cancer res 12 : 1042 - 1054 , 2014 4 . 
nogova l , sequist lv , perez garcia jm , et al : evaluation of bgj398 , a fibroblast growth factor receptor 1 - 3 kinase inhibitor , in patients with advanced solid tumors harboring genetic alterations in fibroblast growth factor receptors : results of a global phase i , dose - escalation and dose - expansion study . 
shaw at , yeap by , mino - kenudson m , et al : clinical features and outcome of patients with nonsmall - cell lung cancer who harbor eml4 - alk . 
humphrey pa , moch h , cubilla al , et al : the 2016 who classification of tumours of the urinary system and male genital organs - part b : prostate and bladder tumours . 
balbs - martnez c , sagrera a , carrillo - de - santa - pau e , et al : recurrent inactivation of stag2 in bladder cancer is not associated with aneuploidy . 
lindgren d , frigyesi a , gudjonsson s , et al : combined gene expression and genomic profiling define two intrinsic molecular subtypes of urothelial carcinoma and gene signatures for molecular grading and outcome . 
garcia ep , minkovsky a , jia y , et al : validation of oncopanel : a targeted next - generation sequencing assay for the detection of somatic variants in cancer . 
tabernero j , bahleda r , dienstmann r , et al : phase i dose - escalation study of jnj - 42756493 , an oral pan - fibroblast growth factor receptor inhibitor , in patients with advanced solid tumors . 
in cohort b , the orr and disease control rate were 75% and 75% . conclusion capmatinib in combination with erlotinib demonstrated safety proles consistent with prior studies . we observed efcacy in specic patient populations . 
early studies evaluated onartuzumab ( an anti - met monoclonal antibody ) in combination the phase iii with erlotinib in recurrent nsclc.16 , 17 in the phase ii study , patients with met 2 + or 3 + by immunohistochemistry ( ihc ) demonstrated a signicant improvement in survival end points compared with low met expression . 
despite this , trial was negative.18 tivantinib , a non - atp competitive small molecule met inhibitor , was evaluated in combination with erlotinib in a phase ii trial of unselected pretreated patients . 
the combination did not meet its overall efcacy goal ; however , a planned subset analysis showed a trend toward prolonged progression - free survival ( pfs ) in patients with met amplication.19 this lack of efcacy may be due to the lack of specicity of tivantinib for the met pathway compared with other met inhibitors.20 capmatinib ( inc280 ) is a highly potent and selective oral met inhibitor that has recently been approved for the treatment of tumors with met exon 14 mutations.21 in the phase i study , antitumor activity was observed in pretreated patients with egfr wild - type ( wt ) tumors with met dysregulation . 
because of overlapping signaling pathways , we sought to determine whether the combination of capmatinib with erlotinib would be safe and demonstrate an efcacy signal in patients with egfror metaltered tumors . knowledge generated we demonstrated that the combination of capmatinib and erlotinib is safe . 
however , it restored sensitivity to erlotinib and promoted apoptosis in nsclc models rendered erlotinib resistant by hgf.23 on the basis of the data available during trial development , we propose that the combination of capmatinib plus erlotinib would be safe and show efcacy in patients with egfr - mutated tumors experiencing disease progression on erlotinib due to met activating bypass pathways . 
the initial phase of the study was a dose escalation to determine the maximum tolerated dose ( mtd ) of capmatinib plus erlotinib ; there was no intrapatient dose escalation . 
patients in cohort a must have an egfr activating mutation and a biopsy at the time of progression that shows evidence of met positivity . treatment capmatinib capsules were administered orally every 12 hours of a 28 - day cycle at 100 - 600 mg . 
a dose - limiting toxicity ( dlt ) was dened as per the study protocol ( data supplement )  . pk blood samples were collected for patients in the doseescalation cohort on cycle 1 , day 15 ; cycle 2 , days 1 and 15 ; and cycles 3 and 4 , day 1 . 
analysis of hgf levels was conducted in duplicate by enzyme - linked immunosorbent assay ( r&d systems , minneapolis , mn ; cat dhg00 ) as per the manufacturers instructions . statistical methods all patients who had at least one dose of study therapy were included in the analyses . 
twelve and ve patients were enrolled in cohorts a and b , respectively ( fig 1 )  . patient demographics and disease characteristics patient demographics and disease characteristics are listed in table 1 . 
in the doseescalation cohort and cohort a , 67% ( patients 1 and 313 ) and 92% ( all except patient 29 ) had prior treatment with at least one egfr tki . 
swimmers plot demonstrating the cohort , met , and epidermal growth factor receptor ( egfr ) status of each patient and corresponding response status and progression - free survival of each patient . 
patient 30 was found to have an egfr t790m mutation on a biopsy sample taken after egfr tyrosine kinase inhibitor ( tki ) treatment ; however , on immediate pretreatment biopsy this mutation was not detected . 
a waterfall plot of best response for all evaluable patients is shown in figure 3 . the aes that were possibly , probably , or denitely attributed to the study drugs are shown in table 2 for all dose levels . 
the most common aes of any grade were acneiform rash ( 62.9% ) , fatigue ( 51% ) , nausea ( 45.7% ) , diarrhea , lower extremity edema , vomiting , and hypoalbuminemia ( 37% each )  . 
in patients who received the recommended phase ii dose ( rp2d ) , the most common aes were acneiform rash ( 65% ) , fatigue and nausea ( both 60% ) , vomiting ( 55% ) , and hypoalbuminemia and edema ( both 50% ) ; additional aes are detailed in appendix table a1 . there were no grade 5 toxicities related to the drug combination . dlt and mtd in dose level 5 , one patient developed grade 3 neutropenia possibly related to treatment . 
dose level 6 served as the expansion of dose level 5 , given that 600 - mg capsules were previously shown to be pharmacokinetically equivalent to 400 - mg tablets . 
six patients received dose modications of capmatinib due to nausea ( 2 ) , alt abnormalities ( 1 ) , edema ( 1 ) , low neutrophil count ( 1 ) , and elevated amylase ( 1 )  . 
five patients received dose modications of erlotinib due to paronychia ( 2 ) , acneiform rash ( 1 ) , creatinine increase ( 1 ) , and diarrhea ( 1 )  . 
four patients received dose modications of both drugs for lipase elevation ( 1 ) , creatinine increase ( 1 ) , lymphopenia ( 1 ) , and lung inammation / pneumonitis ( 1 )  . the pk properties of erlotinib and capmatinib were examined during dose escalation on cycle 1 , day 15 at multiple time points after drug administration . 
the same 400mg dose of capmatinib in the tablet formulation demonstrates a higher drug exposure with greater variation ( appendix table a2 ) compared with the capsule formulation , 182 2021 by american society of clinical oncology although it was not statistically signicant . in addition , erlotinib showed a dose - dependent change of systemic drug exposure ( 150 mg v 100 mg ; appendix fig a1b and appendix table a2 ) ; coadministration with capmatinib did not have signicant impact on erlotinib pk ( appendix table a2 )  . 
overall , the range of erlotinib exposures during dose escalation was similar to previously published results.28 efcacy across all evaluable patients , the orr was 31% ( 8 / 26 ; fig 3 )  . 
among patients with pr or cr , the responses were prolonged , from patient 24 , who discontinued study after apart 3 months because of pneumonia / pneumonitis ; vestigators could not rule out possible drug - related pneumonitis , and thus the patient was discontinued from study . two responders were treatment nave ( 20 and 24 ) ; the remainder had at least one prior regimen ( table 1 )  . dose - escalation cohort fifteen of the 18 patients in the dose - escalation cohort were evaluable . 
two patients ( patients 21 and 23 ) withdrew from the study by individual choice , one patient ( patient 33 ) developed new brain metastasis and withdrew , and one patient ( patient 30 ) had a prolonged hospitalization for a cerebrovascular event deemed unrelated to study drugs . 
efcacy data across dose - escalation cohort dose level median cycles ( range ) 2 ( 2 - 4 ) 5 ( 4 - 16 ) 2 ( 1 - 7 ) 2 ( 2 - 8 ) 4 ( 1 - 10 ) 4 ( 4 - 29 ) inc280 , mg twice a day erlotinib , mg once daily evaluable patients complete response partial response stable disease progressive disease overall response rate abbreviation : inc280 , capmatinib . 1 ( 33% ) 3 ( 100% ) disease control rate 2 ( 67% ) 2 ( 67% ) 1 ( 33% ) 2 ( 67% ) 1 ( 33% ) five egfr - wt patients were enrolled in cohort b ; one patient ( patient 25 ) was not evaluable because of death during cycle 1 deemed unrelated to the study drugs . 
efcacy data across dose - expansion cohorts dosing and response groups cohort a median cycles ( range ) 2 ( 1 - 33 ) 3 ( 1 - 45 ) inc280 , mg twice daily erlotinib , mg once daily evaluable patients complete response partial response stable disease progressive disease overall response rate , % disease control rate , % cohort b abbreviations : inc280 , capmatinib ; ne , not evaluable . at the recommended phase ii dosing , responses were seen in patients with met amplication by ngs and / or 3 + ihc expression and those with met exon 14 mutations , but not in patients with 2 + ihc expression . 
a study in chinese patients with getinib plus capmatinib also reported that 2 + ihc expression was not predictive of response unless it was accompanied by a gene copy number of 5.29 the met / cen7 ratios were not fully reported in this study , but a ratio  . 
serial blood hgf levels were measured to evaluate baseline levels and treatment - related changes in patients over time ; however , no overt correlations with baseline hgf levels and treatment outcomes were observed ( data not shown )  . the patient population with the greatest potential to benet from the combination treatment was cohort a , which enrolled egfr - positive patients who had experienced progression on egfr tkis . 
additional evaluation of an egfr - tki plus a met - tki to overcome this common resistance mechanism is warranted . affiliations 1helen diller family comprehensive cancer center , university of california , san francisco , ca 2university of california davis comprehensive cancer center , sacramento , ca 3mount sinai tisch cancer institute , new york , ny subject matter of this manuscript . 
mccoach honoraria : novartis , genentech , guardant health consulting or advisory role : astrazeneca speakers bureau : novartis research funding : novartis , revolution medicine travel , accommodations , expenses : loxo , eli lilly , takeda pharmaceuticals aiming yu stock and other ownership interests : johnson & johnson patents , royalties , other intellectual property : patents : ( 1 ) yu am , wang wp , chen qx , li mm . 
gandara honoraria : astrazeneca consulting or advisory role : astrazeneca ( i ) , guardant health ( i ) , oncocyte ( i ) , amgen , io biotech ( i ) , merck , roche / genentech , boehringer ingelheim , inivata , novartis research funding : roche / genentech ( i ) , merck ( i ) , amgen ( i ) travel , accommodations , expenses : boehringer ingelheim jonathan w . 
lara consulting or advisory role : janssen research funding : aragon pharmaceuticals ( i ) , janssen biotech ( i ) , tracon pharma ( i ) , merck ( i ) , pharmacyclics ( i ) , incyte ( i ) , taiho pharmaceutical ( i ) matthew gubens consulting or advisory role : bristol myers squibb , roche / genentech , astrazeneca , heron , boehringer ingelheim , takeda pharmaceuticals , beyondspring pharmaceuticals , inivata research funding : celgene ( i ) , merck ( i ) , novartis ( i ) , roche / genentech ( i ) , oncomed ( i ) philip c . 
mack honoraria : guardant health consulting or advisory role : astrazeneca , guardant health , amgen research funding : boehringer ingelheim consulting or advisory role : roche / genentech , regeneron , abbvie , emd serono , merck , pzer , astrazeneca , novartis , symphogen , inivata , bristol myers squibb , takeda pharmaceuticals , eli lilly , amgen , genmab , targeted oncology , genentech research funding : emd serono ( i ) , genentech ( i ) , abbvie ( i ) , five prime therapeutics ( i ) , regeneron ( i ) , astellas pharma ( i ) , tizona therapeutics , ( i ) eli lilly ( i ) , novartis ( i ) , amgen ( i ) patents , royalties , other intellectual property : author royalties for uptodate , an evidence - based , peer - reviewed information resource , available via the web , desktop , and pda travel , accommodations , expenses : astrazeneca , roche / genentech , merck , regeneron , eli lilly , abbvie , emd serono no potential conicts of interest were reported . acknowledgment we thank patients and their families for participation in this study . karen kelly honoraria : merck references finocchiaro g , toschi l , gianoncelli l , et al : prognostic and predictive value of met deregulation in non - small cell lung cancer . 
ann transl med 3 : 83 , 2015 go h , jeon yk , park hj , et al : high met gene copy number leads to shorter survival in patients with non - small cell lung cancer . 
j thorac oncol 5 : 305 - 313 , 2010 dimou a , non l , chae yk , et al : met gene copy number predicts worse overall survival in patients with non - small cell lung cancer ( nsclc ) ; a systematic review and meta - analysis . 
plos one 9 : e107677 , 2014 tong jh , yeung sf , chan aw , et al : met amplication and exon 14 splice site mutation dene unique molecular subgroups of non - small cell lung carcinoma with poor prognosis . 
clin cancer res 22 : 3048 - 3056 , 2016 gherardi e , birchmeier w , birchmeier c , et al : targeting met in cancer : rationale and progress . 
nat rev cancer 12 : 89 - 103 , 2012 [ erratum : nat rev cancer 12 : 637 , 2012 ] engelman ja , zejnullahu k , mitsudomi t , et al : met amplication leads to getinib resistance in lung cancer by activating erbb3 signaling . 
science 316 : 1039 - 1043 , 2007 bean j , brennan c , shih jy , et al : met amplication occurs with or without t790m mutations in egfr mutant lung tumors with acquired resistance to getinib or erlotinib . 
proc natl acad sci usa 104 : 20932 - 20937 , 2007 cappuzzo f , j anne pa , skokan m , et al : met increased gene copy number and primary resistance to getinib therapy in non - small - cell lung cancer patients . ann oncol 20 : 298 - 304 , 2009 piotrowska z , thress ks , mooradian m , et al : met amplication ( amp ) as a resistance mechanism to osimertinib . 
le x , puri s , negrao mv , et al : landscape of egfr - dependent and - independent resistance mechanisms to osimertinib and continuation therapy beyond progression in egfr - mutant nsclc . 
drilon a , cappuzzo f , ou si , et al : targeting met in lung cancer : will expectations nally be met ? j thorac oncol 12 : 15 - 26 , 2017 15 . 
spigel dr , ervin tj , ramlau ra , et al : randomized phase ii trial of onartuzumab in combination with erlotinib in patients with advanced non - small - cell lung 18 . 
spigel dr , edelman mj , obyrne k , et al : results from the phase iii randomized trial of onartuzumab plus erlotinib versus erlotinib in previously treated stage iiib or iv non - small - cell lung cancer : metlung . 
sequist lv , von pawel j , garmey eg , et al : randomized phase ii study of erlotinib plus tivantinib versus erlotinib plus placebo in previously treated non - smallcell lung cancer . 
calles a , kwiatkowski n , cammarata bk , et al : tivantinib ( arq 197 ) efcacy is independent of met inhibition in non - small - cell lung cancer cell lines . 
liu x , wang q , yang g , et al : a novel kinase inhibitor , incb28060 , blocks c - met - dependent signaling , neoplastic activities , and cross - talk with egfr and her22 . 
bang y - j , su w - c , nam d - h , et al : phase i study of the safety and efcacy of inc280 in patients with advanced met - dependent solid tumors . 
lara ms , holland ws , chinn d , et al : preclinical evaluation of met inhibitor inc - 280 with or without the epidermal growth factor receptor inhibitor erlotinib in non - small - cell lung cancer . 
paik pk , drilon a , fan pd , et al : correction : response to met inhibitors in patients with stage iv lung adenocarcinomas harboring met mutations causing exon 14 skipping . 
paik pk , drilon a , fan pd , et al : response to met inhibitors in patients with stage iv lung adenocarcinomas harboring met mutations causing exon 14 skipping . 
cancer discov 5 : 842 - 849 , 2015 jorge se , schulman s , freed ja , et al : responses to the multitargeted met / alk / ros1 inhibitor crizotinib and co - occurring mutations in lung adenocarcinomas with met amplication or met exon 14 skipping mutation . 
fukudo m , ikemi y , togashi y , et al : population pharmacokinetics / pharmacodynamics of erlotinib and pharmacogenomic analysis of plasma and cerebrospinal uid drug concentrations in japanese patients with non - small cell lung cancer . 
wu yl , zhang l , kim dw , et al : phase ib / ii study of capmatinib ( inc280 ) plus getinib after failure of epidermal growth factor receptor ( egfr ) inhibitor therapy in patients with egfr - mutated , met factor - dysregulated non - small - cell lung cancer . 
wolf j , overbeck tr , han j , et al : capmatinib in patients with high - level met - amplied advanced nonsmall cell lung cancer ( nsclc ) : results from the phase 2 geometry mono - 1 study . 
camidge dr , otterson ga , clark jw , et al : crizotinib in patients ( pts ) with met - amplied non - small cell lung cancer ( nsclc ) : updated safety and efcacy ndings from a phase 1 trial . 
caparica r , yen ct , coudry r , et al : responses to crizotinib can occur in high - level met - amplied non - small cell lung cancer independent of met exon 14 33 . 
wolf j , seto t , han j , et al : results of the geometry mono - 1 phase ii study for evaluation of the met inhibitor capmatinib ( inc280 ) in patients with metex14 mutated advanced non - small cell lung cancer . 
yu h , ahn m , kim s , et al : ct032 tatton phase ib expansion cohort : osimertinib plus savolitinib for patients ( pts ) with egfr - mutant , met - amplied nsclc after progression on prior rst / second - generation epidermal growth factor receptor ( egfr ) tyrosine kinase inhibitor ( tki )  . 
sequist l , lee js , han j , et al : ct033 tatton phase ib expansion cohort : osimertinib plus savolitinib for patients ( pts ) with egfr - mutant , met - amplied nsclc after progression on prior third - generation epidermal growth factor receptor ( egfr ) tyrosine kinase inhibitor ( tki )  . 
treatment - related adverse events by cohort and dose level ( continued ) dose level 1 dose level 2 dose level 3 dose level 4 dose level 5 dose level 6 expansion adverse event gr 1 / 2 gr 3 gr 1 / 2 gr 3 gr 1 / 2 gr 3 gr 1 / 2 gr 3 gr 1 / 2 gr 3 gr 1 / 2 gr 3 gr 1 / 2 gr 3 total gastroesophageal reux disease oral mucositis hyponatremia stroke proteinuria back pain peripheral sensory neuropathy abbreviations : gr , grade ; inr , international normalized ratio ; sae , signicant adverse event . 
 r fundamental concepts in the application of plasma genotyping ( liquid biopsy ) to egfr mutation detection in nonsmall - cell lung cancer plasma genotyping has rapidly evolved from an investigational technology into a standard - of - care tool used to direct therapy in metastatic nonsmall - cell lung cancer ( nsclc )  . 
the optimal use and interpretation of plasma genotyping requires understanding of cell - free dna biology , the assay characteristics of the available testing technologies , and the application of testing in each clinical scenario . 
 current recommendations for plasma genotyping in metastatic nsclc are limited to patients with newly diagnosed disease and those with acquired resistance to targeted therapy , in particular , epidermal growth factor receptor ( egfr ) kinase inhibitors . 
in newly diagnosed metastatic nsclc , under certain circumstances , plasma genotyping is useful for the detection of targetable genomic alterations or nontargetable driver alterations ( eg , kras ) that are mutually exclusive with targetable alterations . 
in patients with acquired resistance to therapy , such as egfr t790m - mediated acquired resistance to egfr kinase inhibitors , plasma genotyping may detect resistance mutations missed by standard tissue genotyping because of tumor heterogeneity . 
in both scenarios , the high specificity and positive predictive value of validated plasma genotyping assays allow for the reliable selection of therapy on the basis of a positive plasma genotyping result . 
this technology has become commonplace in prenatal research as well as cancer diagnostics and biomarker research.1 - 6 liquid biopsy has emerged as the colloquial term for the specific use of cfdna genotyping analysis to interrogate plasma , urine , and csf.7 , 8 this technology has been piloted in multiple solid tumor types to detect specific genomic alterations.2 , 4 , 5 its potential utility has been best demonstrated in lung cancer , where the rapid detection of targetable genomic alterations is key to selecting optimal systemic therapy.9 , 10 liquid biopsy technology has inarguably entered the realm of standard care in the treatment of nonsmall - cell lung cancer ( nsclc ) .11 , 12 multiple platforms for performing liquid biopsy have been evaluated for the detection of targetable genomic alterations in metastatic nsclc.9 , 10 , 12 - 20 the most rigorous of these studies have used either prospective sample testing or batched testing of samples collected from prospective clinical trials compared with standard tissue genotyping . 
 ( fda ) approval of a liquid biopsy assay using the cobas epidermal growth factor receptor ( egfr ) mutation test v2 ( roche molecular systems ) platform for the detection of egfr exon 19 del / l858r mutations as well as the egfr t790m resistance mutation constitutes the best example of the rapid transition of this technology from a research assay to a tool for guiding clinical care.21 furthermore , the commercial availability of multiple liquid biopsy assays for the detection of increasingly large panels of genomic alterations underscores the growing clinical role of this technology.22 , 23 the reliability , interpretation , and optimal use of liquid biopsy technology in lung cancer are potentially confusing , given the long history of the field and the number of testing platforms available . 
this hypotheses was confirmed by the detection of known mutations from a patients tumor in cfdna isolated from matched patient plasma.31 tumor - derived cfdna constitutes only a minor and potentially variable fraction of cfdna in a patient with an active malignancy , with the majority of the cfdna originating from normal tissue and blood cells.1 , 30 , 32 the careful isolation of cfdna from plasma as opposed to serum can potentially limit additional contamination with nontumor dna derived from the lysis of white blood cells during specimen processing.33 technical aspects of cfdna isolation , including prompt plasma processing and the use of dna - preservation tubes , may also reduce contamination with nontumor dna ( see specimen type and handling section ) .34 however , the relatively low frequency of tumor - derived cfdna present in the blood as well the short fragment length historically constituted key technical barriers to the reliable detection of tumor genomic alterations in cfdna . the advent of highly sensitive genotyping technologies now allows for the reliable genotyping of the tumor - derived component of plasma cfdna . 
a variety of highly sensitive validated platforms can detect specific genomic alterations in the small fraction of plasma cfdna that is derived from tumor cells ( see technology section ) .9 , 10 , 12 - 20 however , the fundamental limitation of any highly sensitive plasma genotyping assay is the degree to which a given tumor sheds cfdna into the peripheral circulation . 
the determinants of tumor cfdna shed are incompletely understood at present but are hypothesized to relate to both tumor burden and site of disease with tumor characteristics , which include histology , mitotic rate , and tumor vascularization.27 , 28 , 30 , 35 in studies of quantitative plasma genotyping of patients with metastatic nsclc , increasing disease burden , as measured by an increase in metastatic sites , as well as the presence of bone or liver metastases correlate with the concentration of tumor - derived cfdna.35 in all cases , the fraction of tumor - derived cfdna relative to total cfdna , and thus the degree of tumor cfdna shed , must be inferred from the detection of mutant cfdna relative to wild - type cfdna at a given genetic locus . 
the distinction between a shedding and nonshedding tumor is key to the understanding and interpretation of the results of any plasma genotyping assay in nsclc . technology the development of highly sensitive genotyping platforms propelled the analysis of the tumor - derived portion of cfdna from a laboratory curiosity to a potentially reliable clinical tool . 
however , both the presence and rate of cfdna shed vary among patients and directly affect the diagnostic sensitivity of plasma genotyping assays , which can contribute to false - negative results with these assays . circulating cell - free normal dna circulating tumor dna bloodstream normal tissue metastatic nsclc . 
however , the ability of individual plasma genotyping assays to detect specific types of genomic alterations is highly dependent on the testing platform , with the identification of complex rearrangements and copy number alterations being more difficult than the detection of point mutations . 
 analytical sensitivity and specificity refer to the technical ability of a genotyping assay to detect low levels of a given genomic alteration and distinguish it from other alterationsa metric that is most relevant for the development of laboratory tests and procedures . 
this review focuses primarily on clinical diagnostic sensitivity and specificity , which refer to the ability of an assay to accurately identify patients who have or do not have a given genomic alteration compared with a reference standardthe key criteria for using a test for making treatment decisions . cobas egfr mutation test and polymerase chain reactionbased approaches the cobas egfr mutation test is a real - time polymerase chain reaction ( pcr ) based assay originally developed for the purpose of detecting egfr sensitizing and t790m resistance mutations in tumor tissue . 
it is presently the only plasma genotyping assay with an fda indication for the detection of egfr mutations in metastatic nsclc for the purpose of initiating treatment with an egfr kinase inhibitor in both the initial and acquired resistance setting.21 the cobas assay was evaluated for these indications through retrospective testing of prospectively collected plasma samples from the ensure trial compared with standard tissue genotyping . 
 tissue biopsy negative t790m test metastatic site 1 metastatic site 2 blood draw positive t790m test egfrm - expressing tumor cell egfr - t790m - expressing tumor cell circulating cell - free normal dna t790m - negative ctdna t790m - positive ctdna circulating tumor dna circulating cell - free normal dna circulating tumor dna fig 2 . 
the presence of egfr t790m has been demonstrated to exhibit heterogeneity across metastatic sites , potentially resulting in false - negative results when performing tissue genotyping on a rebiopsy specimen taken from a nonrepresentative metastatic site . 
pooled data from these studies demonstrated a sensitivity of 61% ( 95% ci , 57% to 66% ) and specificity of 79% ( 95% ci , 70% to 85% ) for the detection of the egfr t790m mutation.38 a similar comparison was performed using prospectively collected samples from the aura3 study that randomly assigned patients with metastatic nsclc with egfr t790m mediated acquired resistance to egfr kinase inhibitors to either standard platinum - based chemotherapy or osimertinib.39 the study of the plasma cobas assay for egfr t790m in aura3 demonstrated a lower sensitivity of 51% ( 95% ci , 46% to 57% ) but similar specificity of 77% ( 95% ci , 71% to 83% ) .40 interestingly , both studies demonstrated near - perfect specificity for the detection of egfr exon 19 del and l858r , leading to suspicion that the lower specificity was secondary to heterogeneity of resistance mechanisms between metastatic sites within the same patient , thus rendering tumor tissue genotyping a nondefinitive reference standard . 
this hypothesis is supported by the finding that patients who are plasma - positive for egfr t790m exhibit clinical benefit similar to those who are tissue genotypingpositive and that repeat tissue biopsy of patients with acquired resistance led to discordant tissue genotyping results for egfr t790m but not egfr sensitizing mutations ( fig 2 ) .20 , 35 the cobas assay is the best - validated example of the use of pcr - based plasma genotyping assay to assess specific genetic hotspots corresponding to potentially targetable genomic alterations . 
 multiple other plasma genotyping assays have been evaluated for the detection of genomic alterations in metastatic nsclc in studies ranging in scope from analysis of prospectively collected banked plasma samples from large clinical trials to small retrospective series . 
these studies have demonstrated a consistent trend of high specificity but more modest sensitivity.9 , 41 , 42 plasma genotyping using pcr - based approaches is readily able to detect point mutations as well as defined stereotypical insertions and deletions . 
 however , the detection of complex rearrangements and copy number alterations using these platforms is challenging . droplet digital pcr and beads , emulsion , amplification , and magnetics genotyping emulsion digital pcr - based assays for plasma genotyping afford the ability to detect mutant cfdna in a highly sensitive but also quantitative manner . 
 study demonstrated that this assay was able to detect egfr exon 19 del and l858r mutations with a sensitivity of 82% and 74% , respectively , and a specificity of 100% . 
similar assay characteristics were reported for the detection of kras codon 12 mutations ( sensitivity 64% , specificity 100% ) .35 interestingly , similar sensitivity ( 77% ) but more modest specificity ( 63% ) was noted for the detection of egfr t790m . 
a subset of five patients initially reported as being falsely positive for egfr t790m by plasma genotyping were found to be truly positive on repeat tissue biopsy and genotyping of an alternative metastatic sitea finding not observed for any of the aforementioned driver mutations.35 this finding underscores the potentially heterogeneous nature of resistance mutations such as egfr t790m , whereby only growing sites of disease may possess the resistance mutation of interest . 
 tissue genotyping thus has the potential to be falsely negative when testing for an acquired resistance mutation , in contrast to plasma genotyping , which allows for the detection across metastatic sites within the same patient . beads , emulsion , amplification , and magnetics ( beaming ) based plasma genotyping assays have similarly been evaluated for the detection of egfr sensitizing and resistance mutations in patients with metastatic nsclc with acquired resistance to egfr kinase inhibitors . 
despite the rapid and quantitative nature of emulsion - based technologies , they are somewhat limited in terms of panel size and ability to multiplex . next - generation sequencing multiple next - generation sequencing ( ngs ) based platforms for plasma genotyping have been evaluated for the detection of genomic in nsclc.16 , 17 , 22 , 43 ngs - based alterations plasma genotyping assays have a greater ability to detect complex genomic alterations , such as complex rearrangements and high copy number alterations , that may be difficult to identify using other methods . 
a recent retrospective study of plasma samples collected in the biocast / ifct - 1002 study compared an ngsbased ( iontorrent , thermo fisher scientific , waltham , ma ) plasma genotyping assay to standard tissue genotyping in 68 matched samples of never - smokers with metastatic nsclc . 
this study demonstrated a sensitivity of 58% ( 95% ci , 43% to 71% ) and specificity of 87% ( 95% ci , 62% to 96% ) for the detection of mutations in key genes , including egfr , her2 , kras , braf , and pik3ca.16 a more recent retrospective study of 48 patients with metastatic nsclc comparing an ngs - based plasma genotyping assay performed on a miseq platform ( resolution bioscience ) to both ddpcr - based plasma genotyping and standard tissue genotyping revealed comparable sensitivity ( 77% , 48 of 62 positive cases detected ) and specificity ( 100% ; 95% ci , 90% to 100% ) , as well as the ability to reliably detect complex alterations , including alk and ros1 rearrangements.43 commercial plasma genotyping assays that use ngs - based platforms with expansive gene panels have been compared with tissue genotyping reference standard across multiple tumor types , and concordance rates between the two sample types are approximately 85% ( guardant360 , foundationact ) .22 , 23 other methods a multitude of other testing platforms have been used to develop plasma genotyping assays . 
 testing platforms are beyond the scope of this article and have been recently reviewed elsewhere . specimen type and handling careful and standardized methods for the isolation of cfdna from plasma are essential to optimize assay sensitivity and reproducibility . 
the collection of blood in a dna preservation tube , rapid sample processing , and the careful isolation of plasma in a fashion that minimizes cellular contamination ( centrifugation with double spin and disabled brake ) have been previously demonstrated to optimize sample quality for subsequent plasma genotyping.35 the use of plasma is preferable to serum , because the level of nontumor cfdna contamination is lower and less variable in plasma . 
the total cfdna level is frequently higher in serum samples , presumably because of white blood cell rupture during standard serum isolation , which requires allowing the blood sample to clot . 
plasma genotyping results can be available significantly faster compared with standard tissue genotyping for metastatic nsclc in both patients with newly diagnosed disease and those with disease progression while receiving initial targeted therapy.35 however , the optimal use and interpretation of plasma genotyping results hinges on specific details of the clinical scenario in which it is used . clinical actionability plasma genotyping assays consistently demonstrate high specificity and positive predictive value but modest sensitivity . 
the detection of a targetable genomic alteration in genes , including egfr , alk , ros1 , braf , met , ret , and her2 , provides useful information that may be directly used to select therapy . 
for instance , the detection of an egfr exon 19 del mutation in a patient with newly diagnosed metastatic nsclc may be used to initiate therapy with a firstor second - generation egfr kinase inhibitor ( eg , erlotinib , gefitinib , or afatinib )  . 
as a corollary , the detection of mutually exclusive , nontargetable driver genomic alterations ( eg , kras mutation ) is also useful because it provides reassurance that one of the aforementioned targetable genomic alterations is not present and that targeted therapy is not a therapeutic option.48 , 49 thus , plasma genotyping panels must be designed to detect clinically actionable genomic alterations as well as known nontargetable driver mutations . treatment status the established utility of plasma genotyping for the purpose of treatment selection in metastatic nsclc is limited to a small number of clinical scenarios . 
the best - evaluated use of plasma genotyping is for the selection of therapy in patients with newly diagnosed metastatic nsclc.15 , 37 validated plasma genotyping assays have been demonstrated to yield high sensitivity as well as near - perfect specificity for the detection of targetable genomic alterations in patients with newly diagnosed disease . 
plasma genotyping also has the capability to yield genotyping results with improved turnaround time and obviates the need for repeat biopsies in patients with newly diagnosed disease whose initial diagnostic biopsy samples are insufficient for tissue genotyping . patients with a targetable genomic alteration and acquired resistance to initial targeted therapy represent an additional clinical scenario in which plasma genotyping can guide therapy . 
the best - characterized example is the egfr t790m resistance mutation in metastatic nsclc that confers acquired resistance to firstand second - generation egfr kinase inhibitors but sensitivity to third - generation egfr kinase inhibitors . 
potentially targetable acquired resistance mutations , such as egfr t790m , emerge in a heterogeneous fashion in metastatic nsclc ; as a result , not all sites of disease may harbor a given resistance mutation , and thus the detection of such genomic alterations is inherently complex . plasma genotyping has also been evaluated with regard to monitoring the emergence of treatment resistance in patients already receiving therapy.50 , 51 the use of plasma genotyping in this context to detect early disease progression , in advance of clinical or radiographic evidence of progression , is presently investigational and should not be used to change treatment in the absence of objective evidence of disease progression . clinical stage the use of plasma genotyping has demonstrated utility for guiding therapy in metastatic nsclc but is investigational in nonmetastatic disease . 
the optimal use of this technology and interpretation of its results hinges on careful synthesis of three key concepts : cfdna biology , specific assay technology , and the individual clinical oncology of the individual patient ( fig 3 )  . patients with newly diagnosed disease plasma genotyping in a patient with newly diagnosed metastatic nsclc is most informative when a targetable genomic alteration ( eg , egfr , alk , ros1 , braf , met ) or a nontargetable mutually exclusive driver alteration ( eg , kras ) is found . 
the use of an fda - approved and rigorously validated plasma genotyping assay allows for immediate initiation of appropriate targeted or systemic therapy in the instance of a positive result , given the high specificity and positive predictive value of these assays . 
negative plasma genotyping results where no driver alteration is detected are not useful in guiding therapy , given the modest sensitivity of such assays , and should prompt appropriate tissue genotyping if not already initiated . 
importantly , caution must be exercised in interpreting low - level positive results near the limit of detection , particularly of nondriver alterations detected on poorly validated panels , as there exists the risk of assay artifact and a false - positive result . acquired resistance plasma genotyping should be used as the initial genotyping method in the setting of acquired resistance to targeted therapy in metastatic nsclc . 
this approach has the ability to obviate the need for repeat biopsy in many instances but also detects resistance mechanisms that may be missed on repeat tissue genotyping because of heterogeneity of resistance mechanisms . 
the absence of an identifiable resistance mutation should prompt repeat tissue biopsy and genotyping to rule out a false - negative result , as well as examine for alternative resistance mechanisms that are not easily identified through plasma genotyping , such as small - cell transformation or met amplification . 
as a corollary , a plasma genotyping result that fails to detect the original targetable alteration in a patient with acquired resistance should be considered indicative of the absence of tumor cfdna shed , and the result invariably a false negative . 
importantly , plasma testing for acquired resistance as well as repeat biopsy should only be initiated in patients with clinically or radiographically significant disease progression , to avoid inadvertently labeling a patients disease t790m negative because of testing before the emergence of clinical resistance . 
a recent retrospective study demonstrated improved sensitivity with the use of combined urine and plasma genotyping in a cohort of patients with metastatic resistant egfr - mutant nsclc.52 this finding supports the hypothesis that the use of multiple orthogonal testing strategies may be effective in enhancing overall diagnostic sensitivity . 
furthermore , the combination of plasma and tissue genotyping alone has the potential to surmount the modest sensitivity in the context of acquired resistance . the potential utility of serial quantitative plasma genotyping for the purpose of monitoring response to therapy , as well as the evolution of acquired resistance , constitutes another promising area of research . 
 therapeutic response in clinical trials of novel agents may allow for real - time monitoring of response to therapy as well as early prediction of potential mechanisms of acquired resistance . 
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thress ks , paweletz cp , felip e , et al : acquired egfr c797s mutation mediates resistance to azd9291 in non - small cell lung cancer harboring egfr t790m . 
sacher a , alden r , oconnell a , et al : a prospective study of rapid plasma genotyping utilizing sequential ddpcr and ngs in newly diagnosed advanced nsclc patients . 
 cholangiocarcinoma with fgfr genetic aberrations : a unique clinical phenotype purpose fgfr genetic aberrations ( gas ) occur in an estimated 10% to 16% of intrahepatic cholangiocarcinomas ( ccas )  . 
the natural history of cca with fgfr gas , the prognostic role of coexisting gas , and the outcome with fgfr - targeted inhibitors are unknown . patients and methods patients with cca with fgfr gas were identified using next generation sequencing or fluorescence in situ hybridization from four tertiary cancer centers and compared with fgfr wild - type counterparts . 
fishers exact test , kaplan - meier plots , and log - rank tests were used for statistical analysis . results three hundred seventy - seven patients with cca were identified , and 95 had fgfr gas . 
bap1 mutation was the most common coexisting mutation without prognostic impact , whereas tp53 ( p = .04 ) and cdkn2a / b ( p = .04 ) were correlated with a shorter conclusion cca with fgfr gas represents a unique subtype occurring in younger patients with an indolent disease course . 
2018 by american society of clinical oncology introduction biliary tract cancers including intrahepatic and extrahepatic cholangiocarcinoma ( cca ) and gallbladder cancers are uncommon tumors that typically present at an advanced disease stage and are characterized by an aggressive disease course and poor clinical outcome . 
the incidence of these cancers is hard to estimate accurately because some intrahepatic ccas ( ihccas ) are still classified as cancer of unknown primary or included into the larger group of primary liver cancer . 
in 2016 , 40 , 710 primary liver cancers ( which include hepatocellular cancer and ihcca ) and 11 , 740 gallbladder and extrahepatic biliary tract cancers were diagnosed in the united states.1 the incidence of these cancers , particularly ihcca , is increasing in the western world.2 higher incident rates of cca occur in the southeast asian population as compared with the western population ( > 80 per 100 , 000 population in northeast thailand v 0.3 per 100 , 000 population in canada ) , 3 whereas gallbladder cancer occurs more commonly in south asia and latin america . 
this geographic difference can at least partly be attributed to etiologic factors such as fluke infection endemic to certain parts of asia.4 not only are there major etiologic and epidemiologic differences between the three types of apurva jain mitesh j . 
with emerging technolincluding next - generation sequencing ogies , ( ngs ) , scientists have identified actionable mutations in the isocitrate dehydrogenase 1 / 2 ( idh1 / 2 ) , fgfr2 , braf , and her2 / neu genes for targeted therapeutics in cca and gallbladder cancer.5 - 8 these studies indicate that the incidence of specific genetic differences may vary according to biliary cancer subtype . 
idh1 / 2 , fgfr2 , and braf genetic aberrations ( gas ) occur with significantly higher incidence in ihcca , whereas kras , tp53 , and cdkn2a / b are more common in extrahepatic cca and her2 / neu amplifications are more likely to occur in gallbladder cancer . 
idh1 mutations and fgfr2 fusions seem to be mutually exclusive.9 , 10 ongoing trials are investigating the impact of targeted agents against these actionable mutations with promising early results . the fibroblast growth factor receptor ( fgfr ) family consists of four transmembrane receptors ( fgfr1 to fgfr4 ) , 18 fgf ligands , and a heparan sulfate proteoglycan that stabilizes and sequesters the fgfs . 
the ligand - receptor combination is responsible for the activation of downstream ras / raf / mek , jak / stat , and pi3k / akt pathways.11 gas such as activating mutations , amplifications , or chromosomal translocations / fusions in the fgfr pathway contribute to malignant transformation . 
in ihcca , fgfr2 fusions have been observed in 10% to 16% of patients using next - generation tumor dna sequencing methods.12 , 13 however , rna sequencing studies suggest that fgfr gas occur at a higher rate of 45% in surgically resectable disease stage.14 the natural history , prognostic impact , response to fgfr - directed therapy , and clinical phenotype of cca with fgfr gas have not yet been described in detail . 
a better understanding of this subset may enable precision medicine approaches for the management of these rare , fatal cancers . university cancer center , mayo clinic , and university of californiasan francisco helen diller family comprehensive cancer center . 
 the 95 patients included in the study were those who had pathologically confirmed biliary cancer between january 2000 and december 2015 , had at least 3 months of follow up , and had fgfr gas ( fgf19 , fgfr1 , fgfr2 , fgfr3 , or fgfr4 ) on molecular testing . 
our comparison group included 282 patients with the same cancers from the same institutions without any known fgfr gas on molecular profiling with ngs using a common platform ( foundationone ; foundation medicine , cambridge , ma ) , as described in the next section . 
these data included demographic characteristics , clinical history , diagnosis and tumor location , tumor stage and grade at diagnosis , surgical procedures , radiation and systemic treatments received , and current status . targeted ngs pretreatment formalin - fixed , paraffin - embedded tissue from biopsies of primary or metastatic tumor sites or surgically resected specimens were sent for targeted ngs . 
finally , fgfr2 break apart fish probe mix ( abbott molecular diagnostics , des plaines , il ) , containing spectrumorange ( spanning 353.6 kb targeting the centromeric part of gene ) and spectrumgreen ( spanning 491.3 kb targeting the telomeric part of gene ) probes in hybridization buffer , was then applied to the etched area of the slide , and a cover slip was applied . 
these studies were performed as a part of standard management of patients with cca , and the methods have been described in detail elsewhere.13 , 16 statistical methods categorical variables such as patient sex , histologic tumor grade , and stage at diagnosis were summarized as total counts and frequencies . 
we used the log - rank test for discrete variables and the cox proportional hazards model for continuous variables to test the associations of clinical / genetic features with os and to estimate hazard ratios ( hrs ) and 95% cis . 
the median age was 61 years ( range , 22 to 85 years ) in patients with fgfr gas and 56 years ( range , 22 to 82 years ) in patients without fgfr gas . 
no differences were noted in the percentage of patients who received surgical and radiation therapy . no genetic aberration ( n = 282 ; median os , 20 months [ 95% ci , 17 to 26 months ] ) genetic aberration present ( n = 59 ; median os , 30 months [ 95% ci , 20 to 97 months ] ) time ( months ) no . 
 in patients with advanced disease ( stage iii or iv ) , patients with fgfr gas had a longer median os compared with the fgfr wt group ( 24 v 17 months , respectively ; p < .004 ; appendix fig a1 ) , suggesting that advanced cca with fgfr gas may have a more indolent course . 
kras , idh1 , and braf mutations did not coexist with the fgfr2 fusions , suggesting that these may be mutually exclusive . table 2 lists the univariable analysis results in patients with fgfr gas ( n = 95 )  . 
patients who received fgfr - directed treatment had a significantly improved os ( 44.8 months ) compared with patients who received standard treatment ( 24.3 months ; p = .01 ; fig 3 )  . 
 the majority of the patients enrolled onto clinical trials had fgfr2 fusion ( n = 27 , 75% ) ; other gas included fgfr3 ( n = 2 ) and fgf19 ( n = 1 )  . 
 our results suggest that fgfr gas may be associated with a relatively indolent disease course ( confirming earlier reports from our group ) 5 and responsiveness to targeted fgfr inhibitors . 
furthermore , the relationship between fgfr gas with tumor location ( primary tumor v metastases ) and uncommon histologies such as undifferentiated cancers and hepatocholangiocarcinoma deserves further study . several fgfr - targeted inhibitors are currently in clinical trials . 
these include multitargeted tyrosine kinase inhibitors , such as ponatinib , nintedanib , and dovitinib , and more specific fgfr - directed small - molecule tyrosine kinase inhibitors , such as bgj398 , jnj425756493 , prn1371 , arq087 , and tas - 120 . 
the overall response rate was 15% , disease control rate was 95% , and pfs was 6 months , indicating the potential of this therapy for ccas with fgfr gas.17 the more recent third - generation therapy options , including fgfr isoformspecific fgfr inhibitors ( eg , rg744 / mgfr1877s , fgf401 ) and fgfr trap molecules ( eg , gsk3052230 ) , need further investigation.18 these agents are currently being investigated in the second - line setting after prior chemotherapy . 
further investigation of these drugs in the first - line setting and concurrently with chemotherapy is required . although the majority of our patients had fgfr2 fusions ( 77% ) , the rest of the patients with other fgfr gas exhibited a clinical course similar to that of patients with fgfr2 fusions and showed response or prolonged stability ( eg , with fgf19 amplifications )  . 
bap1 mutation is associated with a poor prognosis in uveal melanoma and renal and colorectal cancer.19 - 21 coexisting bap1 mutations occurred frequently in patients with fgfr gas in our series ( n = 16 , 22% )  . 
however , these cancers in the advanced disease setting can be misclassified on radiology alone , and it is possible that the number of patients with ihcca may be higher in our study . 
furthermore , the ngs platforms have become more comprehensive with time , leading to the possibility that some patients with cca investigated using older platforms were false negative for table 3 . 
 shroff , milind javle financial support : funda meric - bernstam , milind javle administrative support : funda meric - bernstam , milind javle provision of study material or patients : robin kate kelley , funda meric - bernstam , ahmed omar kaseb , tanios bekaii - saab , rachna t . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . apurva jain no relationship to disclose tertiary care centers with availability of clinical trials of targeted therapies . despite these limitations , this study is important , as it is , to our knowledge , the first clinical and molecular characterization of a large cohort of patients with cca harboring fgfr gas . 
 funda meric - bernstam honoraria : dialecta honoraria : sumitomo group , genentech , inflection biosciences , pieris pharmaceuticals , clearlight diagnostics , darwin health , grail thomas deleon no relationship to disclose andrea grace bocobo no relationship to disclose research funding : novartis , astrazeneca , taiho pharmaceutical , genentech , calithera biosciences , debiopharm group , bayer , aileron therapeutics , puma biotechnology , cytomx therapeutics , jounce therapeutics , zymeworks , effective pharmaceuticals , curis , pfizer tanios bekaii - saab consulting or advisory role : amgen , celgene , national comprehensive cancer network , genentech , bayer , boehringer ingelheim , research to practice , merrimack , glenmark , ipsen ( inst ) , eli lilly , bristol - myers squibb ( inst ) keith a . 
shroff consulting or advisory role : celgene , codiak biosciences , amgen , agios , halozyme research funding : eli lilly , celgene , agios , halozyme travel , accommodations , expenses : celgene , codiak biosciences , amgen , agios , halozyme daniel h . 
ahn consulting or advisory role : celltrion ( i ) , merrimack , astellas pharma , eisai , cardinal health , paradigm milind javle no relationship to disclose affiliations apurva jain , ying wang , reham abdel - wahab , funda meric - bernstam , keith a . 
borad and thomas deleon , mayo clinic , scottsdale , az ; robin kate kelley and andrea grace bocobo , university of californiasan francisco helen diller family comprehensive cancer center , san francisco , ca ; reham abdel - wahab , assiut university hospital , assiut , egypt ; and daniel h . 
blum , michael lu , and patricia maxin funds for cholangiocarcinoma research and the international cholangiocarcinoma research network . presented , in part , at the clinical science symposium of the 52nd annual meeting of the american society of clinical oncology , chicago , il , june 3 - 7 , 2016 . support prior presentation references 1 . 
goyal l , govindan a , sheth ra , et al : prognosis and clinicopathologic features of patients with advanced stage isocitrate dehydrogenase ( idh ) mutant and idh wild - type intrahepatic cholangiocarcinoma . 
di stefano al , fucci a , frattini v , et al : detection , characterization , and inhibition of fgfr - tacc fusions in idh wild - type glioma . 
borad mj , champion md , egan jb , et al : integrated genomic characterization reveals novel , therapeutically relevant drug targets in fgfr and egfr pathways in sporadic intrahepatic cholangiocarcinoma . 
javle mm , shroff rt , zhu a , et al : a phase 2 study of bgj398 in patients ( pts ) with advanced or metastatic fgfr - altered cholangiocarcinoma ( cca ) who failed or are intolerant to platinum - based chemotherapy . 
hierro c , rodon j , tabernero j : fibroblast growth factor ( fgf ) receptor / fgf inhibitors : novel targets and strategies for optimization of response of solid tumors . 
progression - free survival ( pfs ) kaplan - meier curves for patients with and without fibroblast growth factor receptor ( fgfr ) genetic aberrations ( gas ) who received first - line chemotherapy for systemic disease . 
ileana dumbrava , md1 ; kavitha balaji , phd1 , 2 ; kanwal raghav , md1 ; kenneth hess , phd1 ; milind javle , md1 ; mariela blum - murphy , md1 ; jaffer ajani , md1 ; scott kopetz , md , phd1 ; russell broaddus , md , phd1 ; mark routbort , md , phd1 ; mehmet demirhan , md1 ; xiaofeng zheng , phd1 ; shubham pant , md1 ; apostolia m . 
piha - paul , md1 ; and funda meric - bernstam , md1 purpose human epidermal growth factor receptor 2 ( her2 ) is an effective therapeutic target in breast and gastric and gastroesophageal junction cancers . 
overexpression of her2 protein occurs through her2 gene amplication or through translational mechanisms3 other and can result in the formation of spontaneous receptor homodimers , resulting in the initiation of downstream signaling cascades and malignant transformation.4 , 5 her2 amplication is a prognostic transcriptional or biomarker for worse survival in the absence of antiher2 therapy.6 , 7 her2 is a compelling therapeutic target in patients with breast6 , 8 - 10 and gastric or gastroesophageal junction ( gej ) cancers.11 for her2 - overexpressing or her2 - amplied breast cancer , several her2 - targeted therapies are approved for use in the adjuvant and metastatic settings , including trastuzumab ( metastatic and adjuvant ) , pertuzumab ( metastatic and adjuvant ) , lapatinib ( metastatic ) , ado - trastuzumab emtansine ( metastatic ) , and neratinib ( adjuvant )  . 
 dumbrava et al context key objective our study focused on assessment of erbb2 ( her2 ) amplication in patients with solid tumors , excluding breast cancer , who underwent next - generation sequencing . 
we evaluated the clinical benet of her2 - targeted therapy by measuring the timedependent overall survival from the genomic testing results , progression - free survival ( pfs ) , and pfs during her2 - targeted therapy compared with pfs during prior therapy . knowledge generated we showed that her2 amplication is present in a clinically relevant proportion of tumors and in a variety of tumor types and that her2 - targeted therapy may confer clinical benet , with increased survival in patients with tumor types other than those for which her2 inhibitors are approved . relevance her2 is an established effective therapeutic target in breast , gastric , and gastroesophageal junction cancers ; however , less is known about the prevalence of her2 amplication and efcacy of her2 - targeted treatment in other tumors . 
validation of these results in a larger study could focus on determining the associations of copy number , simultaneous her2 mutations , and other coalterations with response to her2 - targeted therapies . agents are in development , such as the bispecic her2 antibody zw25 and the antibody - drug conjugate ds - 8201.12 , 13 targeted therapy with pfs during prior therapy . 
we also compared the overall survival ( os ) of patients who received her2 - targeted therapy with the os of patients who did not . the main mechanism of her2 overexpression is her2 gene amplication , which occurs in 18% to 20% of patients with breast cancer14 , 15 and 7% to 34% of patients with gastric or gej cancers.11 , 16 , 17 asco and the college of american pathologists recommended testing in breast and gastric or gej cancers using immunohistochemistry ( ihc ) for her2 protein expression or in situ hybridization ( uorescence in situ hybridization [ fish ] , chromogenic in situ hybridization , or silver in situ hybridization ) for her2 gene amplication.14 , 18 other techniques , such as comparative genomic hybridization , can also be used to detect copy number variations.19 - 21 however , with the development and integration of next - generation sequencing ( ngs ) in cancer care and the increasing capacity of ngs to determine copy number variations concurrently with other alterations such as mutations , ngs has become a more cost - effective and tissue - efcient alternative to current single - gene assessment methods.22 her2 amplication also occurs in other carcinomas at differing frequency.23 - 25 although relatively little is known about the role of her2 in other tumor types , emerging data indicate that her2 - targeted therapy may have efcacy in other her2 - positive tumors.26 we hypothesized that her2 - targeted therapy could be associated with clinical benet in tumor types other than breast and gastric or gej cancers . 
for the current study , the ngs analysis was performed using four platforms , including the oncomine comprehensive assay ( thermofisher , waltham , ma ) or ion ampliseq comprehensive cancer panels ( thermofisher ) performed at the university of texas md anderson cancer center molecular diagnostic laboratory , 27 foundationone or foundationone heme ( foundation medicine , cambridge , ma ) tumor testing , or guardant360 ( guardant health , redwood city , ca ) circulating cell - free dna ( cfdna ) testing . 
the genomic testing results were annotated by the precision oncology decision support system at the university of texas md anderson cancer center.28 the patients relevant clinical and molecular characteristics were collected from electronic medical records and prospectively maintained institutional databases ( table 1 )  . 
of prior lines of treatment 57 ( 11 ) 29 - 79 64 ( 52 ) 58 ( 48 ) 42 ( 34 ) 80 ( 66 ) 15 ( 12 ) 107 ( 88 ) 98 ( 80 ) 17 ( 14 ) 7 ( 6 ) abbreviations : fish , uorescent in situ hybridization ; her2 , human epidermal growth factor receptor 2 ; ihc , immunohistochemistry ; sd , standard deviation . * some patients had multiple genomic testing . for her2 amplication , was performed in some patients and was also reviewed in this study . the her2 - targeted clinical trials had been individually approved and conducted at the university of texas md anderson cancer center in accordance with institutional review board guidelines , and this reported analysis was conducted under an institutional review boardapproved protocol . her2 amplication and overexpression analysis her2 amplication determined by ngs was dened according to each platforms analytic pipeline , was based on the resulting reports and validation , and varied between greater than ve to greater than seven estimated copy numbers for reporting high - condence amplication.29 , 30 for patients who underwent cfdna analysis , digital sequencing was performed by guardant health , using a 54gene panel ( guardant360 )  . 
to determine the clinical benet of her2 - targeted therapy , we measured pfs during matched her2 - targeted therapy ( pfs2 ) and compared it with pfs during prior therapy ( pfs1 ) .32 , 33 pfs was dened as the time from the start of treatment until disease progression or death . 
response to treatment and progression were determined using response evaluation criteria in solid tumors ( recist ) version 1.1 , as measured by radiologists or investigators.34 patients who received her2 - targeted therapy as the rst systemic treatment were excluded from the pfs2 - to - pfs1 analysis . we also evaluated the os of patients who received her2targeted therapy and compared it with the os of patients who did not receive her2 - targeted therapy . 
os was calculated as a time - dependent indicator variable in both the kaplan - meier and cox proportional hazards analyses from the genomic testing result until death from any cause . 
the royal marsden hospital prognostic score for predicting survival in phase i trials35 ( including albumin , lactate dehydrogenase [ ldh ] , and number of metastatic sites ) , number of prior lines of treatment , disease stage , and eastern cooperative oncology group ( ecog ) performance status at the time of genomic testing were also analyzed . statistical analysis we used descriptive statistics to summarize the characteristics of patients with her2 amplications . 
concordance between ngs and ihc and between ngs and fish tests was calculated by dividing the number of samples that had concordant results by the total number of samples . univariable and multivariable cox proportional hazards models were t to assess the association between prognostic factors and os , in which the prognostic factors included her2 - targeted therapy , sex , age , histology , ecog performance status , number of prior therapies , number of metastatic sites , disease stage , ldh , albumin , and number of metastatic sites at time of genomic testing . 
one hundred six patients were found to have her2 amplication on tumor tissue analysis on the foundationone , oncomine comprehensive assay , or ion torrent ampliseq comprehensive cancer platforms , and 24 patients were found to have her2 amplications on cfdna analysis using guardant360 technology ( fig 1 )  . 
the most frequent her2 - amplied tumor types included gastric or gej , esophageal , endometrial , bladder , biliary or gallbladder , salivary gland , colorectal , and cervical tumors ( fig 2 )  . 
of the two patients with discordant results , one had low - level ( 1 + ) her2 amplication on cfdna , and the other had equivocal amplication on foundationone . twenty - four patients had positive her2 amplication on the guardant360 platform for cfdna . 
among these 11 patients , ve also had ihc testing , all with concordant positive her2 protein expression , and her2 amplication was conrmed in all three patients who had fish testing ( appendix table a1 )  . clinical benet of her2 - targeted therapy we studied the clinical actionability of her2 amplication and clinical benet of her2 - targeted therapy in 122 evaluable patients who had the molecular testing done more than 6 weeks from the current analysis . 
response to treatment was determined by recist version 1.1 , except in three patients who had clinical progression without radiologic documentation of progressive disease . forty - two ( 34% ) of 122 patients with her2 amplications on ngs also underwent her2 ihc testing . 
of these 42 patients , 31 ( 74% ) had her2 protein overexpression ( 3 + ) , four ( 9% ) had equivocal expression ( 2 + ) , two ( 5% ) had low expression ( 1 + ) , and ve ( 12% ) had no her2 expression . forty patients with other tumor types than the ones for which her2 inhibitors are approved ( 38% ) received at least one line of her2 - targeted therapy , with eight patients receiving more than one line of her2 - targeted therapy . most patients ( 93% ) received trastuzumab in combination foundationone or foundationone heme panels ( n = 2 , 086 ) oncomine comprehensive assay panel ( n = 2 , 494 ) ion ampliseq comprehensive cancer panel ( n = 153 ) guardant 360 panel ( n = 269 ) fig 1 . 
of patients cancer type % colorectal biliary gastric 11.9 lung endometrial bladder esophageal ovarian pancreatic salivary gland hnscc liver cervical melanoma peritoneal prostate urachal 28.6 small intestine unknown primary vulvar 10.0 6 / 116 6 / 64 5 / 307 4 / 172 4 / 86 3 / 106 3 / 173 2 / 69 1 / 346 1 / 38 1 / 192 1 / 36 1 / 58 1 / 10 foundationone oncomine cms400 guardant360 fig 2 . 
cms400 , ion ampliseq comprehensive cancer panel ; her2 , human epidermal growth factor receptor 2 ; hnscc , head and neck squamous cell carcinoma ; oncomine , oncomine comprehensive assay panel . with chemotherapy or other targeted therapies . 
across different lines of treatment , 27 patients received trastuzumab with other targeted therapies such as pertuzumab , 14 patients received trastuzumab and chemotherapy , three patients received small - molecule inhibitors targeting her2 , three patients received antibody - drug conjugates or bispecic antibodies against her2 , and two patients received trastuzumab alone . 
other factors associated with a longer os were ecog performance status of 0 or 1 , a royal marsden hospital prognostic score of 0 or 1 ( normal albumin and ldh levels and two or fewer metastatic sites ) , and colorectal cancer tumor type as compared with other histologies ( table 2 )  . among 37 patients with cancers other than gastric , gej , or esophageal cancers in whom her2 - targeted treatment was given in the second line or later and for whom previous treatment the pfs2 - to - pfs1 ratio was 1.3 or greater in 21 patients ( 57% ) , and the information was available , median pfs2 and pfs1 times were 24 and 13 weeks , respectively ( p , .001 ; fig 4 )  . twelve ( 30% ) of 40 patients with tumor types other than gastric cancer achieved an objective response as dened by complete or partial response per recist version 1.1 , with seven patients receiving trastuzumab and pertuzumab , four patients receiving trastuzumab with chemotherapy , and one patient receiving an her2 antibody - drug conjugate . 
of prior lines of treatment : 0 - 2 v 3 - 7 determinants of enrollment on her2 - targeted therapy the median time between ngs showing an her2 amplication and start of her2 - targeted therapy was 16 weeks . we investigated the reasons why 68 patients did not receive her2 - targeted therapy after their ngs results showed her2 amplication . 
the leading cause was noneligibility for an her2 - targeted clinical trial ( n = 28 ; 41% ) because of equivocal her2 amplication results , insurance denial , or clinical issues such as poor performance status , chronic tumor - related bleeding , or inadequate organ function ( appendix fig a1 )  . discussion in a large cohort of patients who underwent targeted ngs to facilitate personalized cancer treatment , we found her2 amplication in tumor types other than breast and gastric or gej cancers . 
ngs has been shown to meet the sensitivity of detection for mutations used in clinical trials , permitting simultaneous testing of copy number variations in hundreds of genes.28 , 40 , 43 currently , there are several ongoing clinical trials evaluating prevalence of her2 alterations and the benet of targeting her2 in different tumor types ( eg , clinicaltrials.gov identiers : nct02465060 , nct02675829 , nct02091141 , nct02693535 )  . 
the recently published results from the mypathway trial26 , 45 studying treatment with trastuzumab and pertuzumab in colorectal cancer showed an overall response rate of 40% in patients without kras mutations , conrming preliminary data that her2 testing could be integrated in future guidelines for biomarker testing in other tumor types , such as colorectal , 44 , 46 salivary , bladder , and biliary cancers . 
on the basis of these ndings , the national comprehensive cancer network colorectal cancer guidelines were updated recently to include pertuzumab plus trastuzumab and trastuzumab plus lapatinib as category 2b recommendations for her2 - positive colorectal cancer.47 in this study , ngs identied her2 amplication in 2.4% of patients across 20 tumor types . 
although her2 amplications were found in many different epithelial cancers , positive results were rare , and often nonexistent , in malignancies of nonepithelial origthis nding was consistent with the her2 overexpression results reported by yan et al42 in 37 , 992 patients . 
 her2 amplication identied by ngs in contrast with breast cancer , for which her2 - targeted therapies have been established for a long time with ve treatment options approved by the us food and drug administration , for gastric , gej , or esophageal cancers , less is known about the prognostic role of her2 , and therapeutic options are limited to trastuzumab in combination with chemotherapy.26 our results suggest there is a clinical benet in patients with indications beyond gastric or gej cancers . in tissue samples , the thresholds for reporting are higher for ngs than for fish ; therefore , the tissue - based ngs test used in our study may have underestimated the rate of her2 amplication . 
our results on testing for her2 status by ngs compared with ihc and fish are consistent with a previous report of high concordance between ihc and fish in colorectal cancer.49 , 50 many patients were not eligible for her2 - targeted therapies , highlighting the importance of patient selection for genomic testing . 
however , as evidence for actionability of her2 increases , her2 testing should be considered earlier in the treatment course for tumor types in which her2 is more frequently amplied ( eg , colorectal cancer )  . sequential testing by ihc and fish and further mutation analyses may lead to tissue exhaustion before the completion of all necessary testing . 
thus , early incorporation of ngs into clinical practice for diseases with frequent actionable genomic alterations has the advantage of screening for multiple therapeutic options simultaneously while sparing tissue . limit limitations that might our study has several generalizability of our ndings . 
although ngs has many advantages , samples with low tumor content , heterogeneity , and low levels of amplication may result in false - negative results where her2 amplication might have been detected on fish20 ; thus , we are likely underestimating the frequency of her2 amplication.51 a higher prevalence of her2 amplication on liquid biopsies could be , at least in part , related to a selection bias and may be consistent with emergence of her2 amplication as a mechanism of rereceptortargeted sistance to epidermal growth factor therapy , 52 - 54 explaining the higher discordance rates when compared with gold standard tissue - based tests . 
however , patients with her2 amplication detected on ngs may have higher levels of amplication and therefore may have greater benet from her2 - targeted therapy . ngs reveals her2 amplication in a clinically relevant proportion of tumors and in a variety of tumor types , and her2 - targeted therapy may confer clinical benet in tumor types beyond those for which her2 inhibitors are approved . 
further studies are needed to conrm these results and to determine the associations of copy number , simultaneous her2 mutations , and other coalterations with response to her2 - targeted therapies . 
the association of her2 amplications with genomic alterations in other oncogenic drivers provides rationale for novel therapeutic combinations . affiliations 1the university of texas md anderson cancer center , houston , tx 2lexicon pharmaceuticals , houston , tx translational sciences grant no . 
ileana dumbrava , kavitha balaji , milind javle , mariela blum - murphy , scott kopetz , russell broaddus , mark routbort , mehmet demirhan , xiaofeng zheng , vivek subbiah , david s . 
tsimberidou honoraria : covance , genentech consulting or advisory role : roche research funding : emd serono ( inst ) , baxter ( inst ) , foundation medicine ( inst ) , onyx ( inst ) , bayer ( inst ) , boston biomedical ( inst ) , placon ( inst ) , immatics ( inst ) , karus therapeutics ( inst ) , stem cells ( inst ) , obi pharma ( inst ) patents , royalties , other intellectual property : parker institute for cancer immunotherapy ( inst ) vivek subbiah consulting or advisory role : medimmune research funding : novartis ( inst ) , glaxosmithkline ( inst ) , nanocarrier ( inst ) , northwest biotherapeutics ( inst ) , genentech ( inst ) , berg pharma ( inst ) , bayer ( inst ) , incyte ( inst ) , fujilm ( inst ) , pharmamar ( inst ) , d3 oncology solutions ( inst ) , pzer ( inst ) , amgen ( inst ) , abbvie ( inst ) , multivir ( inst ) , blueprint medicines ( inst ) , loxo ( inst ) , vegenics ( inst ) , takeda ( inst ) , alfasigma ( inst ) , agensys ( inst ) , idera ( inst ) , boston biomedical ( inst ) , inhibrx ( inst ) , exelixis ( inst ) travel , accommodations , expenses : pharmamar , bayer david s . 
hong stock and other ownership interests : molecularmatch , oncorena , presagia honoraria : adaptimmune , baxter , merrimack , bayer consulting or advisory role : baxter , bayer , guidepoint global , janssen , genentech , eisai , glg , alpha insights , axiom biotechnologies , adaptimmune , grouph , merrimack , medscape , numab , pzer , seattle genetics , takeda , trieza therapeutics research funding : novartis , genentech , eisai , astrazeneca , pzer , mirna therapeutics , amgen , daiichi sankyo , merck , mirati therapeutics , eli lilly , adaptimmune , abbvie , bayer , bristol - myers squibb , genmab , ignyta , innity pharmaceuticals , kite pharma , kyowa hakko kirin , loxo , medimmune , molecular templates , takeda , seattle genetics , amgen , fate therapeutics , mologen , national cancer institute cancer therapy evaluation program travel , accommodations , expenses : loxo , mirna therapeutics , genmab jordi rodon consulting or advisory role : novartis , eli lilly / imclone , servier , orion pharma , peptomyc , kelun pharmaceuticals , merck sharp & dohme , spectrum pharmaceuticals , pzer research funding : novartis , bayer kenna m . 
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wolff ac , hammond meh , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
press mf , sauter g , buyse m , et al : her2 gene amplication testing by uorescent in situ hybridization ( fish ) : comparison of the asco - college of american pathologists guidelines with fish scores used for enrollment in breast cancer international research group clinical trials . 
yeh i - t , martin ma , robetorye rs , et al : clinical validation of an array cgh test for her2 status in breast cancer reveals that polysomy 17 is a rare event . 
ross ds , zehir a , cheng dt , et al : next - generation assessment of human growth factor receptor 2 ( erbb2 ) amplication status : clinical validation in the context of a hybrid capture - based , comprehensive solid tumor genomic proling assay . 
zhao m , wang q , wang q , et al : computational tools for copy number variation ( cnv ) detection using next - generation sequencing data : features and perspectives . 
yoon hh , sukov wr , shi q , et al : her - 2 / neu gene amplication in relation to expression of her2 and her3 proteins in patients with esophageal adenocarcinoma . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors on the basis of molecular proles : results from mypathway , an open - label , phase iia multiple basket study . 
singh rr , patel kp , routbort mj , et al : clinical massively parallel next - generation sequencing analysis of 409 cancer - related genes for mutations and copy number variations in solid tumours . 
frampton gm , fichtenholtz a , otto ga , et al : development and validation of a clinical cancer genomic proling test based on massively parallel dna se24 : 2719 - 2731 , 2018 quencing . 
press mf , pike mc , hung g , et al : amplication and overexpression of her - 2 / neu in carcinomas of the salivary gland : correlation with poor prognosis . 
gatzemeier u , groth g , butts c , et al : randomized phase ii trial of gemcitabine - cisplatin with or without trastuzumab in her2 - positive non - small - cell lung 39 . 
serrano - olvera a , dueas - gonz alez a , gallardo - rinc on d , et al : prognostic , predictive and therapeutic implications of her2 in invasive epithelial ovarian 40 . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
sartore - bianchi a , trusolino l , martino c , et al : dual - targeted therapy with trastuzumab and lapatinib in treatment - refractory , kras codon 12 / 13 wild - type , her2 - positive metastatic colorectal cancer ( heracles ) : a proof - of - concept , multicentre , open - label , phase 2 trial . 
meric - bernstam f , hurwitz h , raghav kps , et al : pertuzumab plus trastuzumab for her2 - amplied metastatic colorectal cancer ( mypathway ) : an updated report from a multicentre , open - label , phase 2a , multiple basket study . 
richman sd , southward k , chambers p , et al : her2 overexpression and amplication as a potential therapeutic target in colorectal cancer : analysis of 3256 patients enrolled in the quasar , focus and piccolo colorectal cancer trials . 
seo an , kwak y , kim dw , et al : her2 status in colorectal cancer : its clinical signicance and the relationship between her2 gene amplication and 50 . 
takezawa k , pirazzoli v , arcila me , et al : her2 amplication : a potential mechanism of acquired resistance to egfr inhibition in egfr - mutant lung cancers that lack the second - site egfrt790m mutation . 
raghav kps , overman mj , yu r , et al : her2 amplication as a negative predictive biomarker for anti - epidermal growth factor receptor antibody therapy in metastatic colorectal cancer . 
negative results were dened as those with no staining ( score , 0 ) or faint or barely perceptible membranous staining ( score , 1 + ) in less than 10% of the invasive tumor cells . 
equivocal results were dened as those with weak to moderate complete , basolateral , or lateral membranous reactivity in at least 10% of invasive tumor cells ( score , 2 + )  . her2 amplication by fluorescent in situ hybridization her2 uorescent in situ hybridization was also performed in some patients , and her2 amplication was dened as an overall ratio of 2.0 or greater with an average her2 copy number of greater than 4.0 signals per cell ; an overall ratio of 2.0 or greater with an average her2 copy number of less than 4.0 signals per cell ; an average ratio of 2.0 or greater with an average her2 copy number of 4.0 or more but less than 6 signals per cell ; or an average ratio of less than 2.0 with an average her2 copy number of 6.0 or more signals per cell . the patient died before return of results insurance denial patient elected to receive a different treatment the patient was not seen back at mdacc after genomic testing 16% not eligible to enroll in her2 - targeted clinical trials 40% possible future option equivocal result fig a1 . 
dignam , phd2 in this precision oncology era , where molecular proling at the individual patient level becomes increasingly accessible and affordable , more and more clinical trials are now driven by biomarkers , with an overarching objective to optimize and personalize disease management . 
as compared with the conventional clinical development paradigms , where the key is to evaluate treatment effects in histology - dened populations , the choices of biomarker - driven clinical trial designs and analysis plans require additional considerations that are heavily dependent on the nature of biomarkers ( eg , prognostic or predictive , integral or integrated ) and the credential of biomarkers performance and clinical utility . 
here we provide a concise overview of design options for both the setting of singlebiomarker / single - disease and the setting of multiple - biomarker / multiple - disease types . 
we focus on explaining the trial design and practical considerations and rationale of when to use which designs , as well as how to incorporate various adaptive design components to provide additional exibility , enhance logistical efciency , and optimize resource allocation . 
2019 by american society of clinical oncology introduction in the past 10 to 15 years , cancer clinical trials have experienced some important paradigm changes to embrace the era of precision oncology as dened by various biomarkers . 
the central hypothesis driving this movement is that by integrating the right biomarker information we can properly select , or at least enrich , trial cohorts for patients who are most likely to benet from a particular therapy . 
multiple factors collectively contribute to this movementour knowledge about oncogenic pathways has been greatly advanced ; highthroughput screening and other drug discovery developments have made a lot more candidate agents available for evaluation ; and the rapid development of various omics - based technologies , especially the increasing availability of next - generation genomic sequencing , makes it more feasible and affordable to incorporate biomarker information into clinical trials . 
all of these call for novel biomarker - driven trial designs to expedite the clinical development of multiple treatment agents and newly dened patient populations . in this review , we use biomarker to generically describe any characterizations of biologic molecules or diagnostic tests carried out on dna , rna , proteins , and metabolites from blood , body uids , or tissues for diagnosis purposes including disease conrmation , staging , subtyping , and so on . 
under this working denition , the presence of some actionable mutation , such as a mutation in epidermal growth factor receptor ( egfr ) measured at the protein level , is viewed as a single biomarker ; a dna - based multiplex genotyping that simultaneously determines multiple actionable mutations such as egfr , kras , or eml4 - alk is viewed as multiple biomarkers . treatment benet compared with the traditional paradigm , there are even greater consequences of asking the right questions ( or wrong questions ) in these biomarker - driven clinical trials . 
for example , although often there is good biologic rationale to consider biomarker - negative ( m - negative ) patients unlikely or less likely to benet from the new ( targeted ) therapy , the clinical evidence of whether is conned only in the potential biomarker - positive ( m - positive ) patients may or may not be strong . 
moreover , the development of a validated companion biomarker , including establishing a widely accepted partition or cutoff value to determine whether the biomarker is positive or negative , is sometimes lagging behind the development of novel therapeutic agents . 
 hu and dignam context key objective in the past few decades , clinical trials have experienced a major paradigm shift , aiming to incorporate ever - growing tumor biomarker information and evaluate biomarker - dened patient cohorts , while accelerating the clinical development process simultaneously . 
what are the key considerations when designing and conducting biomarker - driven clinical trials ? knowledge generated we present an overview of the rationale , trial types , key design elements and features , and practical considerations for commonly used biomarker - driven clinical trials . 
examples of trials conducted and the lessons learned are also presented . relevance when properly designed and implemented with adequate resources , biomarker - driven clinical trials may efciently and effectively generate evidence on biomarker - based personalized disease management . 
clinicians and clinical trialists should think thoroughly and critically if and how to design and conduct these trials . properly tailor the trial designs and prioritize research questions on the basis of the development stage of the biomarker and the credentials of its clinical utility . the demand for trial development and conduct has become more daunting than ever , as high - throughput molecular proling becomes more accessible . 
rather than mounting separate trials , an important paradigm shift to expedite clinical development is to establish the so - called master protocol or platform triala program of trial development and conduct implemented with an up - front molecular screen and enrollment infrastructure into subtrials across multiple biomarkers and / or multiple disease typesto enhance logistic and regulatory efciency . some important and useful concepts in development of biomarkers for clinical utility are rst briey introduced here and expanded on in the next sections . 
throughout this review , we assume biomarkers of interest already have good analytical validity ( eg , they can be accurately , reliably , and reproducibly measured ) .1 , 2 a biomarker is called prognostic if it is associated with disease prognosis regardless of treatment type and is called predictive when it exerts prognostic inuence differentially according to different treatments . 
it is desirable to have a biomarker ( either prognostic or predictive ) with good clinical utility ( ie , the biomarker can reliably prompt clinical actions that benet patients )  . 
according to dancey et al , 3 biomarkers are integral when they are inherent in the study design from the onset and must be performed in real time to establish eligibility , identify the correct stratum for stratied enrollment , or assign treatment . 
in other words , to make a biomarker usable for clinical trial setting , an integral biomarker should be fully developed and validated ( eg , the cutoff that dichotomizes the continuous marker measurement is xed ) , with a rapid assay turnaround time . 
fast assay turnaround time is desirable but not required for integrated biomarkers . to design more efcient trials and overcome the aforementioned challenges , the idea of adaptive designs has been promoted , and aspects of this approach play an important role in virtually all existing biomarker - driven trials . although modern oncology trial design has promoted adaptive designs as a new aspect of clinical trials , we note that adaptive elements have played a long - standing role in oncology clinical trials , dating back to early multistage design concepts4 , 5 and continuing in the rich development of group sequential monitoring methods that permit early efcacy or futility stopping.6 also , both traditional and newer phase i oncology trial designs are by their nature adaptive to accumulating data . 
we will discuss the rationale and features of established as well as newer adaptive design elements as they arise in respective biomarker - driven trial designs . finally , a new nomenclature that generally describes trial constructs has been introduced , attempting to describe heuristically the structure , particularly when multiple biomarkers and / or treatment agents are considered . 
the term basket trial refers to a trial with an agent tested among multiple disease types sharing a common molecular feature or target identied by biomarker ( s )  . 
in contrast , an umbrella trial describes the case where , for a common disease entity , multiple agents may be investigated in conjunction with specic molecular targets and biomarkers . 
we note that all of these terms lack precision , and thus the specic study features will provide more detail as to the structure . this article aims to provide a concise and up - to - date overview on the current status of biomarker - driven clinical trials , with a focus on the underlying design considerations , rationale , and lessons learned . 
we organize these discussions in terms of the biomarkers role ( integral v integrated ) , number of biomarkers involved ( one v multiple ) , and trial objective ( conrmatory v discovery )  . 
interim monitoring , including but not limited to group sequential methods , provides the statistical framework to allow repeated assessments of treatment efcacy and early stopping as soon as the accumulated data already provide adequate evidence to conclude that the experimental regimen is highly likely ( efcacy monitoring ) or unlikely ( futility monitoring ) benecial . 
for example , phase ii trials traditionally have implemented a single - arm , noncomparative design , with a planned interim futility analysis , such as simons two - stage design , 7 to minimize patient exposure to ineffective regimens . 
in the late - phase setting , integrated randomized phase ii / iii design8 is a useful adaptive design that combines both screening ( phase ii component ) and conrmatory ( phase iii component ) in a single trial . 
as the data based on the the overall phase ii component are used to provisionally test the study hypothesis of the phase iii component , integrated phase ii / iii designs can effectively be viewed as phase iii studies with rather aggressive ( ie , likely to stop ) interim futility analyses . if more than one experimental regimen is of interest in the phase ii component , a plan of selecting treatment arms can also be incorporated when making the go / no - go decision from phase ii to phase iii . more formally speaking , adaptive designs are those that allow prospectively planned modications in both the statistical and scientic aspects of study designs , on the basis of accumulating data while the trials are still progress.9 adaptations to the statistical aspects of study designs arise when the primary estimand ( eg , the target of estimation ) addressing the scientic question of interest10 remains unchanged ; examples include group sequential designs , 6 sample size adaptation , 11 and , more recently ( although the concept dates back several decades ) , outcome / response - adaptive randomization.12 - 14 adaptations to the scientic aspects of study designs occur when the primary estimand does change ( eg , enriching patient population , or selecting new treatment arms or end points while the trial is still ongoing )  . 
among these adaptive design features , group sequential theorybased interim analysis and treatment assignment modication ( such as add or drop arms ) have been used most frequently , whereas other adaptive design features have not been widely adopted in practice . 
if the new agent is expected to have little effect on tumor shrinkage , or must be combined with an active agent , randomized phase ii screening designs17 with end points such as progression - free survival may be considered and provide valuable information on whether a conrmatory , randomized phase iii enrichment design should be performed . 
of note , although enrichment designs should be efcient ( a small sample size ) because we anticipate a large effect size , if the prevalence of mpositive patients is low , many patients must be screened to obtain the necessary sample size ; if the prevalence is too low , the trial may simply be infeasible . when there is strong evidence suggesting the biomarker is predictive with respect to the experimental regimen ( ie , m - positive patients benet ) , but it remains unclear whether the new treatment may also have a clinically meaningful ( but likely smaller ) benet for m - negative patients , the socalled biomarker - stratied design , which randomly assigns all patients with a valid marker result ( m - positive and m - negative ) as a stratication factor , can be considered ( fig 1b )  . 
one can also view this as an umbrella trial ( dened in a later section ) that contains two separate rcts for m - positive and m - negative subgroups . 
the key design consideration for conrmatory trials is if and how to prioritize the multiple hypotheses within the m - positive subgroup , the m - negative subgroup , or the overall population and properly power for each separately as applicable . 
 a biomarker - driven oncology clinical trial designs biomarker assay biomarker assay m - negative m - positive m - negative m - positive off study randomize randomize randomize standard rx new rx standard rx new rx standard rx new rx biomarker assay randomize biomarker assay nonmarkerbased strategy marker - based strategy marker subgroup 1 marker subgroup 2 marker subgroup 3 new rx randomize standard rx randomize new rx v standard rx standard rx m - negative , standard rx m - positive , new rx standard rx new rx fig 1 . 
m - negative , biomarker - negative ; m - positive , biomarker - positive ; rx , treatment . strategy can be considered by rst testing the m - positive subgroup at signicance level and then testing the m - negative subgroup at the same level if and only if the rst test is statistically signicant.20 an alternative approach to reect such prioritization is to split the overall unequally with a bonferroni correction ( eg , 1 = 0.04 and 2 = 0.01 for m - positive and m - negative , respectively )  . ( cid : 129 ) if the effect in the overall population is of secondary interest , the aforementioned sequential procedure needs to be modied , because the treatment effect of the overall population can still seem to be clinically meaningful even when the new treatment only works in the in the m - negative m - positive subpopulation but not subpopulation at all . 
to mitigate the potentially misleading conclusion that treatment works in all comers ( m - positive and m - negative ) , the marker sequential test design21 was proposed , which rst tests the m - positive subgroup at a reduced signicance level 1 ( , ) : if the test yields a statistically signicant result , the m - negative subgroup will be tested at level , whereas the overall population will be tested at 1 if the test among the m - negative subgroup is not signicant . ( cid : 129 ) if there is no convincing evidence suggesting a particular biomarker is predictive , a fallback strategy can be used , which rst tests the overall population at 1 ( , ) : if the result is signicant , one can claim that the treatment is effective in all patients ; if it is not signicant , then the m - positive population must meet 1 for signicance.22 although biomarker - stratied designs are typically used in the context of a conrmatory phase iii setting , randomized phase ii screening designs may still be considered if the overarching goal is to efciently inform whether to conduct a randomized phase iii enrichment design . 
when the primary interest is to evaluate whether the biomarker - based treatment assignment strategy is more effective than nonbiomarker - based treatment assignment strategy , one may consider a biomarker - strategy design , which randomly assigns all patients ( m - positive and m - negative ) to receive treatments either on the basis of or independent of biomarker status ( fig 1c )  . 
this design may also be used to evaluate whether the biomarker is predictive with some efciency loss , 16 , 23 because there can be a signicant portion of patients with the same biomarker status receiving the same treatments in both arms , reducing the treatment effect size that can realistically be specied . biomarker - directed design may be considered when it is desirable to have an integral biomarker evaluation for all patients , and there is compelling existing evidence suggesting a particular biomarker - dened subgroup should receive a certain regimen with satisfactory efcacy and safety proles ( fig 1d )  . 
in this case , like enrichment designs , a biomarker - directed design only randomizes the biomarker - dened subgroup where the biomarkers clinical utility in directing treatment decisions remains unclear ; meanwhile the other biomarker - dened subgroups are treated deterministically . 
for in tailorx ( program for the assessment of example , clinical cancer tests [ pacct - 1 ] : trial assigning individualized options for treatment ) , 24 patients with breast cancer treated with tamoxifen were classied as low , intermediate , and high risk on the basis of a 21 - gene recurrence score ( oncotype dx , genomic health , redwood city , ca )  . 
patients with intermediate risk were randomly assigned to receive hormonal therapy with or without chemotherapy , whereas lowrisk patients and high - risk patients always received only hormonal therapy with chemotherapy , respectively . in these conrmatory integral biomarker - driven trials , interim monitoring , especially futility monitoring , plays an even more critical role to help prioritize the limited resources by dropping subgroups with unpromising or unresponsive treatment benet and / or dropping ineffective experimental regimens if more than one is being investigated . 
for a biomarker classier that is based on a multiplex assay of genomic , proteomic , and transcriptomic data , the adaptive signature design29 was proposed to evaluate whether the experimental regimen is effective in the overall population or a subset of patients only while developing the biomarker classier simultaneously . 
if the treatment comparison for all patients is not signicant in the overall population at 1 ( , ) , we will either split all patients into training and testing subsets or use k - fold cross - validation30 to develop the biomarker and then evaluate efcacy in identied subgroups . 
of note , the cross - validated approach has also been shown to substantially improve the power of identifying the m - positive subgroup that benets from the new treatment . 
by transforming the multiple candidate genes to a binary classier , the approach does not suffer too much with respect to type i error control as the dimension of genes increases . 
when a biomarker or gene signature can be quantied on a continuous or ordinal scale , we can consider the biomarker - adaptive threshold design31 using a similar strategy to identify and validate an optimal cutoff point that separates m - positive and m - negative subgroups . the use of bootstrap resampling for estimation and inference of the threshold , although accounting for the multiplicity issue due to combining the tests for subgroup m - positive and overall population , has been shown to preserve the power to detect a global treatment effect while developing the biomarker . 
comparing with the conventional approaches , these methods have been shown to substantially improve the likelihood of detecting the mpositive subgroup when differential treatment effect does exist , especially when the prevalence of m - positive is low . 
for example , after the approval of vemurafenib for brafv600 mutation - positive metastatic melanoma , a nonrandomized phase ii basket trial of vemurafenib for multiple nonmelanoma cancers with brafv600 mutation was conducted , 33 with objective response as the primary end point . from a trial conduct perspective , basket trials can be more efcient than multiple histology - specic enrichment trials to carry out conducted separately and are convenient because the biomarker can ( although not necessarily ) be assessed locally at participating sites as part of the eligibility criteria screen . 
furthermore , this trial design can be quite appealing to patients because it conveniently provides access to the experimental therapy across multiple disease types , including in settings where our understanding of biomarker - treatment relationship is relatively limited . 
 molecular target a molecular target b molecular target y molecular target z targeted therapy a standard therapy targeted therapy b standard therapy targeted therapy y standard therapy targeted therapy z standard therapy biomarker - driven oncology clinical trial designs targeted therapy x tumor type a tumor type b tumor type y tumor type z biomarker assays phase iii continue to follow for definitive end point accrue additional n2 patients for definitive end point primary analysis treatment effect sufficiently large initiate phase ii trial phase ii accrue n1 patients assess stop insufficient ie activity promising ie activity run - in : randomization ( p1 = p2 ) randomization arm 1 ( p1 ) v arm 2 ( p2 ) assess interim estimate of treatment effect stop and conclude new rx better treatment effect not sufficiently large arm 1 better arm 2 better increase p1 decrease p2 increase p2 decrease p1 fig 2 . 
 hu and dignam from a scientic perspective , the underlying rationale of conducting a basket trial is that the biomarkers presence may independently predict responses attributed to the corresponding targeted therapy , regardless of histology or disease type.34 if this truly holds , it would be reasonable to redene cancer in a histology - agnostic fashion , suggesting an exciting new approach to therapy development . 
for example , in may 2017 , the us food and drug administration approved pembrolizumab for any patients with microsatellite instabilityhigh / decient dna mismatch repair regardless of histology.35 this approval was the rstever histology - agnostic indication and was partly based on a small , proof - of - concept basket trial.36 because basket trials may be viewed as a collection of enrichment trials , they also inherit all of the advantages and disadvantages , with the same practical considerations as elaborated earlier . 
one unique challenge facing basket trials is the balance between feasibility and the exchangeability ( eg , histology agnostic ) hypothesis , given the potential heterogeneity across different disease types . 
that is , if and when can we assume the molecular proling is sufcient to replace histology and pool patients together with the same biomarker ? for many biomarkers , prevalence is so low that it may be infeasible to accrue enough patients and analyze by each disease type . 
meanwhile , pooling all patients with the same biomarker can be questionable if not at all unrealistic , because the approach implies we can completely ignore the prognosis heterogeneity across different histology and assume disease subtype is not prognostic at all . 
in the case of vemurafenib for patients with brafv600 mutations , response to treatment was high when the primary site was melanoma but low when the primary site was colorectal cancer.33 a practical compromise is to combine some disease types for which prevalence is signicantly lower than others . 
in le et al , 36 patients with microsatellite instability high / decient dna mismatch repair were accrued and analyzed by colorectal and noncolorectal cohorts separately , because the prevalence in colorectal cancer is notably higher than other disease types . several novel adaptive designs have been developed to avoid separate analyses and properly share response information across disease types . 
one approach is based on preplanned interim analyses to determine the next steps.37 for example , if there is adequate evidence suggesting some histology - specic cohorts have similar and promising activities , these cohorts will be aggregated to allow more efcient statistical inference with fewer patients ; otherwise , histology - specic cohorts with exceptionally favorable response will remain separate , and cohorts with low responses will be terminated . 
the other approach is based on statistical modeling and bayesian inference , 38 , 39 which explicitly allows information sharing across different histology - specic cohorts and permits early stopping for some cohorts naturally on the basis of posterior probability of histology - specic response rates . 
nonetheless , it was argued that , for sample sizes typically used in phase ii trials and a reasonable number of cohorts / histology types under investigation , these designs that are meant to share information may not be as useful as one would hope , unless there is a strong rationale and evidence indicating uniform responses across cohorts.40 another important consideration when designing discovery basket trials is whether randomization should be used or not . 
the nonrandomized basket trial may be clearly preferred because of its feasibility and close connection to the conventional single - arm phase ii design traditionally used in early - stage development . 
however , nonrandomized basket design practically mandates objective response to be the only choice of the primary end point , because it is generally considered the only interpretable efcacy end point without a comparator . 
even with this end point , concerns may still arise regarding the relevance of historical control in at least some histology cohorts , because the biomarker likely denes a new disease subtype for which there is little or no historical information on prognosis.41 furthermore , if the experimental regimen is to be administered in combination with other active regimens , how to isolate the impacts of background treatments and properly the experimental regimens role can be chalinterpret lenging . 
in this case , because a centralized molecular screening platform is typically used , one may also view them as umbrella trials . from a pure statistical perspective , one could argue that basket trials may have multiplicity issues , because we simultaneously evaluate multiple histology - specic cohorts . pragmatically speaking , this may not be that problematic , because we are more tolerant of a higher type i error for discovery purposes ; the same issue also exists if we conduct separate trials for each cohort , and more practical concerns such as accrual feasibility often outweigh the type i error control . 
a randomized phase ii / iii trial example is alchemist ( adjuvant lung cancer enrichment marker identication and sequencing trial ) for resectable nonsmall - cell lung cancer ( nsclc ) , 42 , 43 which consists of three treatment subtrials , alchemistegfr for patients with egfr mutation ( clinicaltrials.gov identier : nct02193282 ) , alchemist - alk for patients with alk rearrangements ( clinicaltrials.gov identier : nct02201992 ) , and anvil ( clinicaltrials.gov identier : nct02595944 ) for unmatched patients ( eg , squamous histology , or nonsquamous histology and neither egfr mutation nor alk rearrangements )  . an observation cohort is also available for unmatched patients who refuse to participate in anvil . 
another prominent example is lung - map ( clinicaltrials.gov identier : nct02154490 ) , 44 which was initially for squamous lung cancer but recently was amended for all types of advanced nsclc . 
using sequential development features such as phase ii / iii designs and interim analyses that permit adaptation to ndings , these trials adaptively provide the necessary exibility for the umbrella trial objectives as a whole ( fig 2c )  . when there are multiple candidate regimens that are of interest equally for some or all biomarker cohorts , umbrella trials also can be designed solely for discovery objectives . 
for example , in the battle - 1 ( clinicaltrials.gov identier : nct00411632 , nct00409968 , nct00410059 , nct00410189 , nct00411632 , nct00411671 ) trial , patients with chemotherapy - refractory nsclc were assayed for four candidate biomarkers to be allocated to a total of ve marker strata ( including one nonmatched ) and then randomly assigned to one of four drug regimens.15 in nrglu003 ( clinicaltrials.gov identier : nct03737994 ) , an umbrella trial of previously treated patients with alk - positive lung cancer , a total of 10 marker cohorts ( including an unmatched cohort ) and up to seven new - generation alk inhibitors are to be evaluated . 
however , as the total number of possible drug - marker strata increases , the likelihood to accrue enough patients to have a reasonably accurate estimate of efcacy for each drug - biomarker stratum decreases . 
by exploiting the fact that biomarker allocation is not informative for treatment assignments , battle - 1 considered a learn - as - go approach by explicitly using a bayesian hierarchical probit model for information sharing , 45 along with outcome - adaptive randomization , 12 , 46 which allocates more patients to drug - marker strata that are more likely to have exceptional activities , to potentially provide individuals with more efcacious treatments and improve estimation precision ( fig 2d )  . 
in nrg - lu003 , for each biomarker the investigators are able to determine and prioritize the experimental regimens of interest on the basis of preclinical data , which substantially reduces the total number of drug - biomarker strata to be evaluated . 
freidlin and korn40 suggested that under typical phase ii trial design settings , both approaches may require similar resources . one implication of conducting umbrella trials is that an explicit rule governing how to match biomarkers and candidate regimens needs to be specied prospectively . 
therefore , umbrella trials also provide a unique opportunity to evaluate whether a rule - based policy that matches biomarkers and drugs is effective at all and , if so , to what extent across all genomic characterizations . 
to make such evaluation interpretable , the rule - based assignment policy should be as stable as possible during the trial conduct and ideally cover a wide range of biomarker - drug combinations.47 another unique consideration with umbrella trials is that a patient could be eligible for multiple biomarker cohort allocation because the tumor contains multiple biomarkers . 
with the emergent use of molecular proling , basket trials and umbrella trials , as well as extensions discussed here , can be collectively called master protocols or platform trials . 
more importantly , such a protocol provides substantial exibility in terms of discontinuing unpromising investigations , carrying forward favorable early results to denitive testing in a phase ii / iii framework , and introducing new subtrials as targets and agents are identied in a perpetual manner , using the aforementioned adaptive design methods for single - biomarker settings.48 in addition , the infrastructure advantages of conducting master protocols , such as centralized and streamlined trial conduct ( enrollment , informed consent ) , data collection , governance ( institutional review board , data and safety monitoring committee ) , and quality assurance ( clinical monitoring , imaging reading ) , are subtrials stantial as compared with conducting individual separately . 
when it was activated in 2015 , it started with 10 parallel biomarker - based cohorts to evaluate eight different in may 2016 , a preplanned feasibility interim analysis revealed that only 9% of patients had actionable mutations that could be matched with any of the multiple targeted therapies under investigation . 
the lower - than - expected matching success rate led to a major amendment to improve the overall matching rate by adding additional marker - specic cohorts.49 in june 2017 , when the original screening target was met ( n = 6 , 000 ) , it was found that the common molecular subtypes were rarer than expected.50 the study therefore underwent another major amendment by collapsing multiple subtype cohorts and relaxing the screening process . 
to date , the study remains open to accrual , with a total of 19 cohorts to evaluate 13 drugs.51 four lung - map was originally designed in 2014 for advanced squamous nsclc , consisting of targeted therapy subgroups and one nonmatched subgroup , each using a phase ii / iii seamless design , with progression - free survival and overall survival as primary end points , respectively.52 since 2015 , the treatment landscape of advanced nsclc has changed tremendously because of a series of approvals in immunotherapies especially for squamous and nonsquamous nsclc by the us food and drug administration , which fundamentally changed the standard of care and the study control arms . 
after multiple minor amendments for adding and closing biomarker - specic subtrials , in 2018 the study was completely revamped by expanding eligibility to all histology of advanced nsclc , using a new screening protocol and introducing new biomarker - dened subtrials.53 in summary , although conceptually offering efciency and exibility , master protocols are more prone to various factors that are unknown ( eg , low biomarker prevalence ) or cannot be foreseen ( eg , changing treatment landscape ) at the trial inilogistical tiation . 
these trials also come with substantial complications , especially when several sponsors are involved who may have conicting proprietary interests and regulatory concerns . discussion biomarker - driven clinical trials allow us to investigate patient heterogeneity on the basis of molecular proling , which consequently introduces new opportunities and challenges . 
comparing with the conventional paradigm , these trials require even more thorough planning and comprehensive evaluation on the overarching objectives ( discovery or conrmatory ) , the credential of biomarkers clinical utility , choice of adaptive design and analysis plans , knowledge of cancer biology , existing data from preclinical and early clinical trials , prevalence of each subtype population , logistic readiness to conduct immortal clinical trials , and so on . 
the success of any biomarker - driven trials therefore certainly relies on an even closer collaboration involved in advancing cancer care , including among all clinical investigators , statisticians , sponsors , regulators , drug and assay developers , and patient advocates . our discussions have been limited to phase ii and iii trial designs for discovery and conrmatory purposes . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / author - center . chen hu consulting or advisory role : merck sharp & dohme james j . 
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lih cj , sims dj , harrington rd , et al : analytical validation and application of a targeted next - generation sequencing mutation - detection assay for use in treatment assignment in the nci - mpact trial . 
peeters m , jay price tj , cervantes a : randomized phase iii study of panitumumab with uorouracil , leucovorin , and irinotecan ( folfiri ) compared with folfiri alone as second - line treatment in patients with metastatic colorectal cancer . 
herbst rs , redman mw , kim es , et al : cetuximab plus carboplatin and paclitaxel with or without bevacizumab versus carboplatin and paclitaxel with or without bevacizumab in advanced nsclc ( swog s0819 ) : a randomised , phase 3 study . 
spigel dr , ervin tj , ramlau ra , et al : randomized phase ii trial of onartuzumab in combination with erlotinib in patients with advanced non - small - cell lung cancer . 
bradley jd , paulus r , komaki r , et al : standard - dose versus high - dose conformal radiotherapy with concurrent and consolidation carboplatin plus paclitaxel with or without cetuximab for patients with stage iiia or iiib non - small - cell lung cancer ( rtog 0617 ) : a randomised , two - by - two factorial phase 3 study . 
vansteenkiste jf , cho bc , vanakesa t , et al : efcacy of the mage - a3 cancer immunotherapeutic as adjuvant therapy in patients with resected mage - a3positive non - small - cell lung cancer ( magrit ) : a randomised , double - blind , placebo - controlled , phase 3 trial . 
cobo m , isla d , massuti b , et al : customizing cisplatin based on quantitative excision repair cross - complementing 1 mrna expression : a phase iii trial in nonsmall - cell lung cancer . 
 poly ( adp - ribose ) polymerase inhibitor activity in prostate cancers harboring mutations in dna repair genes : who benets ? shridar ganesan , md , phd1 and judy garber , md , mph2 the seminal nding that cancers harboring mutations in brca1 or brca2 are often exquisitely sensitive to poly ( adp - ribose ) polymerase ( parp ) inhibition has led to the successful development of parp inhibitors as effective targeted therapies for brca1 / 2 - mutated ovarian , breast , prostate , and pancreatic cancers.1 - 7 because many other genes are indirectly or directly involved in the brca1 / 2 - associated homologous recombination ( hr ) mediated dna repair ( hrd ) pathway , there has been a great deal of interest in rationally expanding the use of parp inhibitors to include cancers harboring mutations in these related dna repair genes.8 , 9 these include genes intimately associated with the brca1 / 2 hrd pathway , such as palb2 , bard1 , brip1 , and the rad51 paralogs , as well as genes less directly involved in hrd , such as atm and chek2 . 
is there evidence of biallelic loss of function of the gene in the tumor ? both of these criteria would presumably be required for a tumor to manifest a defect in hr - mediated repair sufciently profound to lead to sensitivity to parp inhibitors . 
the second criterion is especially important because , for many dna repair genes , the heterozygous state has at most a subtle repair phenotype that would not impart sensitivity to parp inhibitors . 
even if a lossof - function mutation in a critical dna repair gene is detected in a tumor , there must also be loss of the wildtype allele or , rarely , a dominant negative heterozygous mutation or evidence of epigenetic silencing of the wildtype allele for the tumor to have a profound dna repair defect . 
similarly , for each gene considered as a biomarker of sensitivity to parp inhibitors , data regarding the functional effect of biallelic loss on the hrd pathway and subsequent relative sensitivity to parp inhibitors need to be carefully evaluated . this leads us to the recently published randomized phase iii profound study ( clinicaltrial.gov identier : nct02987543 ) of the efcacy and safety of olaparib in men with metastatic castration - resistant prostate cancer harboring germline mutations in a set of dna repair genes who experienced disease progression on initial hormonal treatment.10 all patients had an alteration in one of 15 genes ( brca1 , brca2 , atm , brip1 , bard1 , cdk12 , chek1 , chek2 , fancl , palb2 , ppp2r2a , rad51b , rad51c , rad51d , and rad54l ) and were randomly assigned in a 2 : 1 fashion to receive either the parp inhibitor olaparib or physicians choice of second - line hormonal therapy with enzalutamide or abiraterone . 
the trial was divided into two cohorts , with cohort a ( n = 245 ) having an alteration in brca1 , brca2 , or atm , and cohort b ( n = 142 ) having alterations in any of the other 12 genes . 
the key question is whether the benet of olaparib treatment seen in both cohort a and cohorts a and b overall is driven primarily by benet in patients with brca2 mutations in both instances . 
here again , analysis of high - depth sequencing data may be able to identify which brca1mutant prostate cancers have undergone loss of heterozygosity with loss of wild - type brca1 allele and are thus truly sensitive to parp inhibitors.13 some individual gene analyses were presented as exploratory analyses as well . 
cancers with mutations in rad51b and rad54l , which are genes directly involved in hrd , had some evidence of benet , but numbers are small ( see supplementary figure s6 in de bono et al10 )  . 
the rest of the genes , including palb2 , bard1 , brip1 , fancl , chek1 , rad51c , and rad51d , conferred eligibility on fewer than ve randomly assigned patients each and cannot be assessed . these data suggest that brca2 - mutant prostate cancers denitely benet from olaparib treatment , and this is a clear advance in the treatment of this subset of prostate cancers . however , it may well be that the benet seen in the brca2mutant population is driving the overall benet seen in both cohort a and the combined group of cohorts a and b in this study . 
the reason for lack of a clear signal in atm - mutant prostate cancer needs to be further investigated , and analysis of both allelic status of the mutations and molecular evidence of patterns of genomic instability associated with hrd may help resolve these issues . 
several reports have shown that atm - mutant breast cancers may not respond to parp inhibitors , 14 , 15 suggesting that atm mutation may not confer clinical sensitivity to parp inhibitors . 
different therapeutic strategies may be required to effectively treat atm - mutant prostate cancers , and other rational treatments , such as atr inhibitors , need to be investigated.16 the lack of clear signal of benet in prostate cancers harboring brca1 mutations may be a result of the small numbers and the fact that , unlike breast , ovarian , and pancreatic cancers , many brca1 - mutant prostate tumors often do not show evidence of biallelic alteration.11 , 17 although there are hints of benet in cancers harboring mutations in other genes critical for hr , including the rad51 paralogs , the numbers in this trial are too small to make any denitive conclusion . 
for example , palb2 and rad51 paralogs clearly play a central role in hrd , and there are data demonstrating that palb2 mutation in breast and prostate cancer and rad51 paralog mutation in ovarian cancer are associated with benet from parp inhibitors.14 , 15 , 18 - 20 evaluation of the landscape of other hrd gene mutations in different cancer types and data regarding the settings in which there is strong evidence of biallelic loss may help guide development of these genes as biomarkers for parp inhibitors . in designing future trials that rationally target specic dna repair defects in cancer , simply identifying a pathogenic mutation in a broad set of genes in the tumor or in germline may not be sufcient to dene the population likely to benet . 
clear evidence that loss of function of each gene of interest confers high - level sensitivity to the drugs being evaluated needs to be presented , and the allelic status of the gene in the tumor may need to be assayed . 
in some settings , such as ovarian and breast cancer , the nding of a pathogenic brca1 or brca2 mutation , either germline or somatic , is almost always associated with evidence of biallelic mutations , but in other cancers and with other dna this may not necessarily be the case.17 repair genes , evaluation of allelic status can be approached by computational methods , but there may be settings , including epigenetic silencing and dominant negative alleles , where this approach will need to be complemented by functional assays of genomic instability . 
here , assays using wholegenome sequencing or targeted sequencing may also be informative in determining whether the presence of a dna repair gene mutation is accompanied by mutation patterns consistent with a profound defect in hrd.21 - 23 the presence of reversion mutations or mutations in compensating genes also can affect sensitivity to parp inhibitors and may need to be considered.24 , 25 in summary , a more sophisticated approach to analysis of dna repair status of cancers , which includes not only identifying the presence of a pathogenic mutation , but also demonstrating that there is loss of the wild - type allele , lack of reversion mutation , and / or genomic evidence of a profound dna repair defect , may be required to optimally identify cancers that may benet from parp inhibitors and related dna - damaging agents . 
grouping together a set of related genes for pooled analysis may , given differences in prevalence of individual mutations and effect size of benet for different genes , lead to settings where the overall benet in the pool may be driven only by a subset of genes . 
if this possibility is not considered , pooled analysis could lead to approval of therapies for gene alterations when these therapies are ineffective and prevent or delay development of more effective targeted therapies for those patients . 
gene - level analysis may make trial design more difcult , but ultimately , it may lead to more precise use of therapies that target specic dna repair defects or other loss - of - function alterations in cancer . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / authors / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . shridar ganesan employment : merck ( i ) stock and other ownership interests : ibris , inspirata , merck ( i ) consulting or advisory role : inspirata , novartis , roche , foghorn therapeutics , foundation medicine , merck sharp & dohme research funding : m2gen ( inst ) patents , royalties , other intellectual property : i hold 2 patents for digital imaging that may be licensed to ibris and inspirata other relationship : national cancer institute / national institutes of health judy garber consulting or advisory role : novartis ( i ) , gtx ( i ) , helix biopharma , konica minolta , aleta biotherapeutics ( i ) , h3 biomedicine ( i ) , kronos bio ( i ) research funding : novartis ( i ) , ambry genetics , invitae genetics , myriad genetics other relationship : susan g . 
n engl j med 361 : 123 - 134 , 2009 gonz alez - martn a , pothuri b , vergote i , et al : niraparib in patients with newly diagnosed advanced ovarian cancer . 
n engl j med 381 : 2391 - 2402 , 2019 kaufman b , shapira - frommer r , schmutzler rk , et al : olaparib monotherapy in patients with advanced cancer and a germline brca1 / 2 mutation . 
moore kn , secord aa , geller ma , et al : niraparib monotherapy for late - line treatment of ovarian cancer ( quadra ) : a multicentre , open - label , single - arm , phase 2 trial . 
lancet oncol 20 : 636 - 648 , 2019 litton jk , rugo hs , ettl j , et al : talazoparib in patients with advanced breast cancer and a germline brca mutation . 
n engl j med 379 : 753 - 763 , 2018 golan t , hammel p , reni m , et al : maintenance olaparib for germline brca - mutated metastatic pancreatic cancer . 
sokol es , pavlick d , khiabanian h , et al : pan - cancer analysis of brca1 and brca2 genomic alterations and their association with genomic instability as measured by genome - wide loss of heterozygosity . 
sakamoto s , iijima k , mochizuki d , et al : homologous recombination repair is regulated by domains at the nand c - terminus of nbs1 and is dissociated with 13 . 
khiabanian h , hirsheld km , goldnger m , et al : inference of germline mutational status and evaluation of loss of heterozygosity in high - depth , tumor - only atm functions . 
gruber jj , afghahi a , hatton a , et al : talazoparib beyond brca : a phase ii trial of talazoparib monotherapy in brca1 and brca2 wild - type patients with advanced her2 - negative breast cancer or other solid tumors with a mutation in homologous recombination ( hr ) pathway genes . 
tung nm , robson me , ventz s , et al : tbcrc 048 : a phase ii study of olaparib monotherapy in metastatic breast cancer patients with germline or somatic mutations in dna damage response ( ddr ) pathway genes ( olaparib expanded )  . 
yap ta , ocarrigan b , penney ms , et al : phase i trial of rst - in - class atr inhibitor m6620 ( vx - 970 ) as monotherapy or in combination with carboplatin in patients with advanced solid tumors . 
kondrashova o , nguyen m , shield - artin k , et al : secondary somatic mutations restoring rad51c and rad51d associated with acquired resistance to the parp inhibitor rucaparib in high - grade ovarian carcinoma . 
chandran ea , kennedy i : signicant tumor response to the poly ( adp - ribose ) polymerase inhibitor olaparib in heavily pretreated patient with ovarian carcinosarcoma harboring a germline rad51d mutation . 
timms km , abkevich v , hughes e , et al : association of brca1 / 2 defects with genomic scores predictive of dna damage repair deciency among breast cancer subtypes . 
telli ml , timms km , reid j , et al : homologous recombination deciency ( hrd ) score predicts response to platinum - containing neoadjuvant chemotherapy in patients with triple - negative breast cancer . 
 specifying the trueand falsepositive rates in basket trials in the clinical evaluation of anticancer therapies , after identification of a recommended dose , investigators typically seek evidence of drug efficacy in populations of patients hypothesized to benefit from it . 
the purpose of these signal - finding studies is to prioritize additional development and to determine whether drugs should be tested in randomized phase iii trials and , if so , in which populations of patients . 
because these identified populations usually are small and efficacy often is substantially better than that for available therapies , conclusions from single - arm trials of this nature can be used to inform off - label treatment decisions and sometimes lead to inclusion in treatment guidelines or regulatory approvals . 
an understanding of the performance characteristics of the novel clinical trial designs commonly used in this setting is critical to avoid inappropriate clinical decision making on the basis of false - positive clinical trial results . the group of patients in whom a therapy is hypothesized to have activity can be identified in a number of ways , including genomic and proteomic profiles , and activity of the drug can be influenced by histology or the primary tumor site . 
the combination of a hypothesized mechanism of action and uncertainty around the populations of patients who would likely benefit has encouraged a trend toward more - complex earlyphase trials.1 a major challenge is the desire to study the effects of the drug simultaneously in patients with different primary sites of disease or histologies with the goal of evaluating the efficacy of the drug in these contexts , often referred to as baskets.2 - 9 a related recent trend in clinical trial design has been increasing enthusiasm for the use of adaptive designs , whereby design parameters can be changed dynamically as the trial progresses and evidence about efficacy gradually emerges.10 a final important trend has been the use of bayesian design as a tool to evaluate the emerging evidence in a formal statistical model.11 - 15 many of these methods are predicated on the underlying expectation of broadly similar efficacy across baskets because the statistical model allows the sharing of information across baskets with the purpose of completing the trial in a shorter time frame with fewer patients than a traditional strategy whereby each basket is regarded as an independent phase ii trial . 
this combination of design complexity with sophisticated statistical modeling has led to situations in which important clinical trials are being launched without a broad understanding of the implications of the novel methods used , an issue that has been identified previously in the context of novel phase i study designs.16 in this commentary , we examine the properties of a particular bayesian adaptive design that increasingly is being used in the context of basket trials , and we use the clinical setting of a completed basket trial to demonstrate that the design is heavily tilted toward positive conclusions about the efficacy of the drug.17 the complexity of these modern methods makes having a clear understanding of the properties of design , characterized by easily interpretable measures , essential when planning a new clinical trial . although bayesian statistical analyses generally focus on reporting the probability that an individual drug works ( the so - called posterior probability ) , we believe that the evaluation of properties of designs in terms of the familiar metrics used in the context of clinical trials is important . 
these properties are the true - positive rate ( the probability that a truly effective agent will be shown to be effective [ often referred to as power ] ) and the falsepositive rate ( the probability that an ineffective agent is erroneously judged to be effective [ often referred to as the type i error ] )  . 
we generally like to keep the false - positive rate low because we do not want to encourage additional study of a drug that does not work , and we want the true - positive rate to be high because we want to be confident that if the drug truly works , its effectiveness will be recognized . 
therefore , to fully understand the implications of these data - sharing methods , we must calculate a more - elaborate set of trueand false - positive rates , specifically when the drug does not work at all , when it only works in one kristen m . 
 basket , when it only works in two baskets , and so forth . as an illustration of the clinical setting , we refer to the findings of a recent basket trial that investigated the effects of vemurafenib , a selective oral inhibitor of the braf kinase.2 the trial assessed efficacy in five disease - specific baskets : nonsmallcell lung cancer , colorectal cancer , cholangiocarcinoma , erdheim - chester disease or langerhans cell histiocytosis , and anaplastic thyroid cancer . 
the investigators concluded that the drug shows promising activity in the nonsmall - cell lung cancer basket and the erdheim - chester disease or langerhans cell histiocytosis basket but that it is inactive in colorectal cancer . 
although this trial did not use the bayesian hierarchical design proposed by berry et al , 17 we use the clinical setting of the vemurafenib trial to investigate the performance of the berry design . the bayesian hierarchical model is structured to capture the correlation between the anticipated efficacies of the drug across various baskets . 
this aggregation of evidence is rooted in a key feature of bayesian methods , the prior distribution of the between - basket variability , an entity that is prespecified by the analyst . 
in the context of a basket trial , this prior distribution essentially titrates the extent to which emerging evidence of drug efficacy from one basket can be used to bolster the evidence in other baskets . 
however , they presented a sensitivity analysis that concluded that the properties are actually relatively insensitive to this choice . we evaluated the false - positive and false - negative error rates for a hypothetical trial similar in structure to the vemurafenib trial , with five baskets and null and alternative response rates of 15% and 45% , respectively . 
we simulated trials by following the bayesian hierarchical design and conducted interim analyses after the first 10 patients were accrued and then after every additional five patients until a maximum of 19 patients per basket were enrolled . 
early stopping rules terminated accrual to individual baskets if the basket - specific posterior probability that the true response rate is greater than a midlevel response rate of 30% was , 5% ( stop for futility ) or  . 
at the end of the trial , the treatment was declared efficacious in a basket if the posterior probability that the response rate that exceeded the null value of 15% was  . 
for example , in the second row ( one active basket ) , basket 1 is the one in which the drug is truly active , whereas in baskets 2 to 5 , the drug is not active . 
in this scenario , we see that a 79% chance for observing a true - positive result exists in the active basket , whereas for each nonactive basket , the false - positive rate is 18% . 
the next column on the right captures the probability that one or more of the nonactive baskets is a false - positive finding , a term usually referred to as the family - wise error rate , a key criterion for evaluating multiple hypotheses in clinical trials.18 , 19 we see that this overall false - positive rate is 37% when the drug truly works in only one basket and rises to 57% when the drug does not work in only one basket . 
although the maximum trial size was set to 95 patients , the expected trial size ranged from 59 to 84 patients , depending on the true efficacy configuration of the baskets . 
the table also lists the family - wise error rates for the setting in which each basket is regarded as an entirely independent clinical trial wherein the false - positive rate is 10% . 
each basket displays the observed response rate ( rr ) from the vemurafenib trial for a particular disease ( sample sizes in parentheses )  . ( * ) patients received combination therapy ( vemurafenib 1 cetuximab )  . 
statistical properties of bayesian hierarchical design probability drug will be declared efficacious , % family - wise error rate , % * basket 1 basket 2 basket 3 basket 4 basket 5 hierarchical independent no . 
bold represents baskets in which the drug is active ; no bold represents baskets in which the drug is not active . * the family - wise error rate represents the probability that the drug has false - positive efficacy in any one or more of the nonactive baskets ( ie , the overall false - positive rate of the design given the number of baskets in which the drug is truly active )  . 
this is shown for both the bayesian hierarchical design and the setting in which all five baskets are considered to be independent clinical trials . close examination of table 1 demonstrates two complementary results from using the bayesian design . 
although the targeted overall false - positive rate of 10% has been achieved for the setting in which the drug has no efficacy ( ie , the drug works in none of the baskets as shown in the first row ) , this standard rapidly erodes as we investigate settings in which the drug works in some baskets but not in others . for example , if we look at the two active baskets row of the table , we see a high probability ( 94% ) of declaring each of the two active baskets to be efficacious . 
consider the most extreme case ( the four active baskets row of the table ) where we see that the nonactive basket has a false - positive rate of 57% . 
in this setting , the evidence typically is dominated by strong positive results from the four baskets in which the drug is truly active , and the imposition of the prior distribution encourages the model to interpret the results from all five baskets as strongly correlated ( ie , similar in efficacy ) , which leads to a high probability that the inactive basket will be classified incorrectly as active . 
in related work , we explored in depth options for modifying the bayesian design used in our simulations by examining various choices of prior distributions and of decision rules used , demonstrating that trials can be designed on the basis of hierarchical modeling with much better control of the familywise error rates.20 we believe that straightforward reporting of the various trueand false - positive rates , as listed in table 1 , are fundamental to understanding the merits of any proposed basket study design . seemingly innocuous choices , such as the use of a prior distribution that berry et al17 characterized as weak , which allows the data to shape the amount of borrowing , can actually have a substantial influence on the real risks of obtaining false - positive results . 
hyman consulting or advisory role : atara biotherapeutics , chugai pharmaceutical , cytomx therapeutics , boehringer ingelheim , astrazeneca research funding : astrazeneca , puma biotechnology , loxo oncology gregory j . 
riely consulting or advisory role : genentech research funding : novartis ( inst ) , roche ( inst ) , genentech ( inst ) , millennium pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , pfizer ( inst ) , infinity pharmaceuticals ( inst ) , ariad pharmaceuticals ( inst ) patents , royalties , other intellectual property : patent application submitted covering pulsatile use of erlotinib to treat or prevent brain metastases ( inst ) travel , accommodations , expenses : novartis , merck sharp & dohme kristen m . 
begg no relationship to disclose affiliations all authors : memorial sloan kettering cancer center , new york , ny . supported in part through national cancer institute awards ca008748 and ca163251 . support references 33 : 975 - 977 , 2015 1 . 
hyman dm , puzanov i , subbiah v , et al : vemurafenib in multiple nonmelanoma cancers with braf v600 mutations . n engl j med 373 : 726 - 736 , 2015 3 . 
eortc network of core institutions : cross - tumoral phase 2 clinical trial exploring crizotinib ( pf - 02341066 ) in patients with advanced tumors induced by causal alterations of alk and / or met ( create ) , 2013 . 
lopez - chavez a , thomas a , rajan a , et al : molecular profiling and targeted therapy for advanced thoracic malignancies : a biomarker - derived , multiarm , multihistology phase ii basket trial . 
burris ha , hurwitz h , perez ea , et al : mypathway : an open - label phase iia study of trastuzumab / pertuzumab , erlotinib , vemurafenib , and vismodegib in patients who have advanced solid tumors with mutations or gene expression abnormalities targeted by these agents . 
iasonos a , g onen m , bosl gj : scientific review of phase i protocols with novel dose - escalation designs : how much information is needed ? j clin oncol 33 : 2221 - 2225 , 2015 17 . 
chen c , li n , shentu y , et al : adaptive informational design of confirmatory phase iii trials with an uncertain biomarker effect to improve the probability of success . 
 precision medicine for relapsed multiple myeloma on the basis of an integrative multiomics approach alessandro lagan itai beno david melnekoff violetta leshchenko deepu madduri dennis ramdas larysa sanchez scot niglio deepak perumal brian a . 
contributed equally to this work . corresponding author : alessandro lagana , 770 lexington avenue , 14 - 12 new york , ny 10065 ; twitter : @alagana1 ; e - mail : alessandro.lagana@mssm.edu. licensed under the creative commons attribution 4.0 license purpose multiple myeloma ( mm ) is a malignancy of plasma cells , with a median survival of 6 years . 
current approaches in precision oncology aim at matching specific dna mutations to drugs , but incorporation of genome - wide rna profiles has not yet been clinically assessed . methods we have developed a novel computational platform for precision medicine of relapsed and / or refractory mm on the basis of dna and rna sequencing . 
we tested our approach in a pilot precision medicine clinical trial with 64 patients with relapsed and / or refractory mm . results we generated treatment recommendations in 63 of 64 patients . 
of these , 11 received a drug that was based on rna findings , eight received a drug that was based on dna , and two received a drug that was based on both rna and dna . 
sixteen of the 21 evaluable patients had a clinical response ( ie , reduction of disease marker 25% ) , giving a clinical benefit rate of 76% and an overall response rate of 66% , with five patients having ongoing responses at the end of the trial . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction multiple myeloma ( mm ) is a mostly incurable malignancy of terminally differentiated plasma cells that affects 6.5 per 100 , 000 people per year in the united states , making it the second most common hematologic malignancy.1 typically , the trajectory of mm is characterized by a pattern of recurrent remissions and relapses , with patients becoming increasingly refractory to treatment . 
 hallmarks of mm include chromosomal translocations and copy number alterations ( cna ) .2 however , the causal drivers of mm pathogenesis are still unclear , and treatment is administered empirically on the basis of recurrence risk rather than genetic events . 
high - throughput dna sequencing of patients with mm has revealed wide and remarkable heterogeneity of the mutational spectrum across patients and a complex subclonal structure , 3 , 4 suggesting that the use of a personalized therapeutic approach is likely to improve the outcomes for myeloma.5 in the past 4 years , we have focused on the design and development of a computational platform for personalized therapy of relapsed and / or refractory mm , on the basis of a comprehensive patient profile generated from dna and rna sequencing . 
this is leading to a landmark paradigm shift in cancer therapy , in which treatment may be administered on the basis of the specific genomic alterations observed in a patients tumor , rather than on the tumor histology or tissue type . our approach to precision medicine of relapsed mm critically incorporates rna sequencing based drug repurposing . 
importantly , although our pipeline is designed and tailored for the specific needs of mm therapy , it can provide a general framework for incorporating rna sequencing based drug repurposing in oncology . methods protocol approvals and patient enrollment the patients were physician referred . 
enrollment criteria , which included relapsed myeloma , lack of food and drug administration ( fda ) approved therapeutic options , and a prognosis of 6 months of survival , were approved by the mount sinai institutional review board ( irb )  . 
the study was concluded in september 2017 . sample processing and sequencing bone marrow ( bm ) aspirates and peripheral blood ( pb ) were obtained from patients with mm in the study . 
raw sequencing data are available at national center for biotechnology information sequence read archive ( accession number : prjna474747 )  . data processing and bioinformatics analysis we designed and implemented a software framework for the definition and execution of data analysis workflows . 
extended methods are provided in the data supplement . results overview of the study and patient characteristics we developed a computational platform for personalized therapy of relapsed and / or refractory mm , on the basis of a comprehensive patient profile generated from dna sequencing and rnaseq data ( fig 1 )  . 
we evaluated the feasibility and effectiveness of our approach in an irb approved precision medicine clinical trial with 64 participants treated at mount sinai ( see methods and table 1 )  . 
 overall , the patients had received a median of seven lines of therapy , with 13 patients ( 20% ) having received > 10 lines of therapy . genomic landscape of patients with relapsed mm and actionable findings we obtained dna data , either wes or targeted sequencing or both , for 55 patients ( 86% ; data supplement )  . 
the pipeline identified a total of 21 , 166 somatic mutations in 10 , 403 genes in 54 of the 55 patients with dna data available ( data supplement )  . 
whole - exome and / or targeted panel sequencing ( seq ) is performed , and the obtained reads are mapped to the reference genome and analyzed for the identification of somatic mutations and copy number alterations , which are then prioritized on the basis of their actionability . 
we used the clinical interpretations of variants in cancer database ( org ) to identify actionable mutations ( ie , those associated with sensitivity to one or more drugs in one or more cancer types6 ; fig 2c ; data supplement )  . 
the other actionable mutations detected were associated with other hematologic malignancies or with solid cancers . cna constitute another important category of abnormalities observed frequently in mm , as well as in other cancers . 
overall , we found a total of 3 , 541 genes affected by cna in at least one patient and identified 31 actionable alterations ( fig 2d ; data supplement )  . 
we assessed concordance of wes - based cna with fluorescent in situ hybridization and cytogenetics for detection of the most recurrent and clinically relevant cna in mm ( del [ 1p ] , del [ 13q ] , del [ 17p ] , and gain [ 1q21 ] ) and observed a 69% overlap . analysis of the mm transcriptome for the identification of actionable variations rnaseq data were available for 60 patients ( 94% ) and were used to determine outlier genes and pathway activation and to perform rna drug repurposing ( data supplement )  . 
we then calculated pathway activation by single - sample gene set variation analysis7 on a set of actionable pathways relevant in mm : xbp1s activation , mammalian target of rapamycin signaling , histone deacetylase ( hdac ) , dna repair , interleukin - 6 signaling , pi3k / akt activation , hedgehog signaling , fibroblast growth factor receptor 3 , and mitogen activated protein kinase ( mapk )  . 
 ( % ) unless indicated otherwise . abbreviations : asct , autologous stem cell transplant ; iss , international staging system . ranged from 113 to 1 , 423 , with an average of 527.5 mutations per patient ( fig 2a )  . 
mutations were distributed among 14 different categories according to their nature and position in the genome , the majority of them being located in introns ( 57% ; fig 2b )  . 
 ( a ) distribution of mutational burden ( ie , number of total mutations per megabase [ mb ] detected ; n = 41 patients with whole exome sequencing [ wes ] data available )  . 
fgfr3 , fibroblast growth factor receptor 3 ; hdac , histone deacetylase ; il - 6 , interleukin - 6 ; mapk , mitogen - activated protein kinase ; mtor , mammalian target of rapamycin ; p13k akt , phosphoinositide 3 - kinase ; xbp1s , xbox binding protein 1 spliced . the l1000cds2 method , which is based on characteristic direction signatures.8 , 9 the rationale behind this approach is that a drug inducing a gene expression profile that is opposite to a patients profile might be able to revert the disease - associated signature and the phenotype . 
this approach has been demonstrated successfully in several published cases.10 - 12 figure 3d presents the distribution of fda - approved cancer drugs selected by our analysis in the cohort . 
the data supplement summarizes fda - approved noncancer drugs options that were selected for at least one patient . implemented recommendations and response to therapy our pipeline generated recommendations for 63 of 64 patients ( 98% )  . 
trametinib was recommended because of mutations in either nras or kras ; venetoclax was recommended because of high bcl2 expression in the context of all the patients analyzed ( data supplement ) ; panobinostat was recommended on the basis of rnaseq analysis indicating activation of the hdac pathway and / or by rna - based drug repurposing selecting the pan - hdac inhibitor vorinostat . 
 a - 100 ismms56 ismms43 ismms42 ismms41 ismms40 ismms36 ismms34 ismms27 ismms26 ismms25 ismms23 ismms21 ismms20 ismms19 ismms10 ismms09 ismms08 ismms07 ismms05 ismms02 ismms01 dna based rna based dna + rna based treatment ongoing ( patient still responding ) patients disease progression physicians choice adverse event remission achieved death additional drugs used treatment ongoing other therapy trametinib venetoclax panobinostat dabrafenib etoposide time ( days ) fig 4 . 
each color represents a different drug , coded as follows : trametinib ( light blue ) , venetoclax ( dark gold ) , panobinostat ( dark blue ) , dabrafenib ( salmon ) , and etoposide ( dark red )  . 
triangle indicates discontinuation of treatment , where reasons are color coded as follows : disease progression ( gold ) , physicians choice ( green ) , adverse event ( pink ) , and remission achieved ( white )  . 
of these , 11 ( 52% ) received a drug on the basis of rna profiling , eight ( 38% ) on the basis of dna profiling , and two ( 10% ) on the basis of both rna and dna profiling . 
 five patients received our recommended drugs alone , whereas for 16 patients , the drugs were either added to previous treatment ( n = 10 ) or administered in combination with other drugs on the basis of physician discretion ( n = 6 )  . 
in particular , of the 21 evaluable patients , one ( 5% ) achieved cr ; three ( 14% ) , very good partial response ; and 10 ( 47% ) , pr , to give an overall response rate of 66% . 
five patients had still ongoing responses at the end of the study ( september 1 , 2017 ) , with response durations of 235 , 182 , 150 , 172 , and 99 days , respectively ( fig 4b )  . 
significant ( grade 3 ) nonhematologic toxicities were seen in five patients ( 24% ) ; these included neutropenic fever , diarrhea , fatigue , and cardiomyopathy ( table 2 )  . case studies case 1 : patient ismms05 ( rna - based recommendation : panobinostat plus venetoclax )  . 
on the basis of these findings , she was administered venetoclax 400 mg po once daily , the hdac inhibitor panobinostat 20 mg monday , wednesday , and friday , 2 weeks on , 1 week off , and , in addition , pomalidomide 2 mg monday to friday , 3 weeks on , 1 week off . 
after relapsing after receiving multiple treatments including pomalidomide 2 mg ( immediate preceding regimen ) , his cd138 + cells and pb samples were sent for sequencing ( data supplement )  . 
before treatment , his iga and free kappa light chains measured 661 mg / dl and 576 mg / l , respectively ( free kappa / lambda ratio , 1932 )  . 
three months after treatment began , his iga had reached a nadir of 94 mg / dl , whereas his free kappa light chains had decreased to 109 mg / l . 
the patient relapsed 5 months later , with free kappa light chains rising to 390 mg / l ( iga , 187 mg / dl ; data supplement )  . case 3 : patient ismms40 ( wes + rna - based recommendation : trametinib plus venetoclax )  . 
the patient was initially administered lenalidomide and dexamethasone , which resulted in relapse and , after multiple failed regimens , his cd138 + cells and pb were sent for sequencing ( data supplement )  . 
after relapse , the patient was challenged with venetoclax 400 mg monday to friday , trametinib 2 mg monday , wednesday , friday , and carfilzomib 20 / 27 mg / m2 . 
the patient has been continuing this regimen for 3 months ( data supplement )  . discussion here , we reported our integrated multiomics approach for personalized therapy of mm and its application in a pilot precision medicine clinical trial with 64 patients with relapsed mm seen at mount sinai . 
the results of our study show how a precision medicine approach incorporating rna sequencing may identify viable and effective therapeutic options beyond the current fda - approved armamentarium in mm . 
our pipeline generated recommendations from a larger pool of fda - approved cancer drugs ( mm and non - mm ) for 63 of 64 patients in the trial ( 98% ; fig 5 )  . treatment was implemented in 40% of patients , and 81% of these were evaluable ( table 2 )  . 
 two drugs that we repurposed successfully from other cancers in our study were trametinib and venetoclax , the former selected on the basis of dna and the latter on the basis of rna findings . 
trametinib is a mek1 / 2 inhibitor approved by the fda in combination with dabrafenib for unresectable or metastatic melanoma and nonsmall - cell lung cancer carrying mutations in braf ( v600e / v600k )  . 
recent studies have shown activating mutations in nras , kras , and braf in mm , making the mapk pathway a significant therapeutic target also in mm.3 - 5 a retrospective study has demonstrated clinical activity of trametinib in patients with mm with ras - mutated tumors.14 eleven of 21 evaluable patients in our study received trametinib , either alone or in combination with other drugs . 
six of them had clinical response , with a median progression - free survival of 110 days , and two patients had ongoing responses at the end of the study . 
 bone marrow rnaseq ( 94% ) wes ( 64% ) targeted dna panel ( 53% ) analysis pipeline rx 1 rx 2 rx n drug recommendation genomics transcriptomics n = 64 pts relapsed and / or refractory mm peripheral blood n = 16 pts ( 76% ) responded ( mr ) n = 5 pts ( 24% ) did not respond ( sd ) n = 21 pts ( 81% ) evaluable dna - based ( n = 8 [ 38% ] ) rna - based ( n = 10 [ 48% ] ) dna + rna - based ( n = 3 [ 14% ] ) n = 26 pts ( 40% ) treatment implemented n = 38 pts ( 60% ) treatment not implemented no drugs no drugs identied ( 3% ) insurance denial ( 11% ) pt progression before implementation or lost to follow - up ( 86% ) possible problems potential solutions clonal heterogeneity bone marrow microenvironment bad predictions wes - based clonality assessment scrnaseq cytof scrnaseq of microenvironment in silico validation ( from proling ) in vitro / in vivo validation fig 5 . 
we obtained rna sequencing ( rnaseq ) for 94% , whole - exome sequencing ( wes ) for 64% , and targeted dna panel data for 53% of the pts . 
 according to imwg criteria , 76% of the pts had a clinical response ( mr and above , where mr = minimal response and corresponds to a 25% reduction of disease marker ) , whereas 24% of the pts had stable disease ( sd ) or worse . 
problems to address to improve recommendations include assessment of clonal heterogeneity , analysis of bone marrow microenvironment , and extension of reference data to include mm - specific drug profiles from in vitro and in vivo models . 
treatment was not implemented in 60% of the pts , because either no drugs were identified , because insurance denied the proposed drugs , or because of rapid progression of the patient before the results of sequencing were available . 
cytof , mass cytometry ; scrna , single - cell rna . progression - free survival of 161 days , and five patients had ongoing responses at the end of the study . 
overall , these results compare favorably to the efficacy seen in several novel agents for mm , including pomalidomide , daratumumab , and elotumumab , as well as targeted precision medicine approaches such as the nci - match ( national cancer institute molecular analysis for therapy choice ) trial , in which patients with specific solid tumors received treatment on the basis of actionable dna mutations.19 our trial also identified the challenges in implementing ngs - based recommendations in a real - world setting . 
in 60% of the patients with recommendations , treatment was not implemented , either because of insurance denial of the drug or because of rapid progression of disease before sequencing results were available . 
this may be more expensive initially but may become cost effective with greater use . of the 21 treated patients who were evaluable , five did not respond to the recommended therapy . 
 populations may evolve by selective pressure of therapy.5 , 20 , 21 we have recently started to investigate the impact of clonal heterogeneity on therapy selection , by extending our analysis to include clonality assessment on the basis of wes and / or single - cell rna sequencing ( scrnaseq )  . 
integration of both wes and scrnaseq may provide a more comprehensive profile of a patients tumor , enabling drug repurposing at the subclonal level . another key factor in optimal therapy prediction is the bm microenvironment . 
mm cells are strongly dependent on the surrounding microenvironment , which promotes their homing , growth , survival , and migration , as well as their resistance to drugs.22 therapies targeting both the cellular and the noncellular bm compartments are available and are currently used in mm , although not in a targeted manner . 
profiling the microenvironment by scrnaseq and / or mass cytometry , which allows simultaneous measurements of up to 50 markers at single - cell resolution , may help dissect the nontumor compartment and better inform targeted therapy . 
 we are currently investigating the feasibility of including this feature in the next generation of our platforin particular , the use of scrnaseq in clinical precision oncology is limited by several challenges , including increased cost compared with bulk sequencing ( approximately 10 times higher ) , high sensitivity to sample purity , and dropouts ( ie , undetected transcripts due to low amounts of rna sequenced within individual cells ) , as well as data processing and interpretation . 
dudley , samir parekh provision of study materials or patients : ajai chari , hearn - jay cho , deepu madduri , bart barlogie , sundar jagannath , samir parekh collection and assembly of data : alessandro lagan , violetta leshchenko , deepu madduri , dennis ramdas , larysa sanchez , scot niglio , deepak perumal , ajai chari , hearn jay cho , bart barlogie , sundar jagannath data analysis and interpretation : alessandro lagan , itai beno , david melnekoff , brian a . 
kidd , riccardo miotto , jane houldsworth , rita shaknovich manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors technology that will likely complement current precision medicine strategies in the near future . a third key factor to consider concerns the accuracy of the predictions . 
we are working on extending our knowledge base to incorporate mm specific data , including transcriptional effects of novel agents , to quickly and effectively translate robust research findings into clinical practice . 
to further improve drug recommendations , we are implementing validation of the in silico findings using both in vitro ( micro - c3 ) and in vivo ( pdx ) models.23 , 24 this will also contribute to reducing both the costs and the toxicities associated with ineffective treatments . 
finally , we are going to equip our platform with a machine learning flow , which will implement interactive learning techniques to refine the predictions on the basis of therapy outcome and physicians opinion . in conclusion , here we have described our sequencing - based precision medicine platform for relapsed and / or refractory mm and reported the results of a pilot clinical trial to assess the feasibility and usefulness of this approach . 
the trial has allowed us to test and reveal the accuracy of our platform and to understand the pitfalls and limitations of the approach , laying the foundation for our next - generation precision medicine framework . 
 david melnekoff no relationship to disclose violetta leshchenko no relationship to disclose deepu madduri employment : roivant sciences ( i ) leadership : roivant sciences ( i ) honoraria : abbvie consulting or advisory role : abbvie speakers ' bureau : baxalta / shire stock and other ownership interests : roivant sciences ( i ) patents , royalties , other intellectual property : roivant sciences ( i ) dennis ramdas employment : eli lilly ( i ) stock and other ownership interests : eli lilly ( i ) speakers ' bureau : janssen pharmaceuticals , sanofi , takeda pharmaceuticals ( i ) larysa sanchez no relationship to disclose scot niglio no relationship to disclose deepak perumal no relationship to disclose brian a . 
kidd no relationship to disclose riccardo miotto no relationship to disclose rita shaknovich employment : cgix leadership : cgix stock and other ownership interests : cgix consulting or advisory role : astrazeneca travel , accommodations , expenses : astrazeneca ajai chari consulting or advisory role : array biopharma , celgene , novartis , millennium pharmaceuticals , amgen , janssen oncology , adaptive biotechnologies , bayer ag , seattle genetics research funding : array biopharma , celgene , millennium pharmaceuticals , novartis , onyx pharmaceuticals , janssen pharmaceuticals , pharmacyclics , acetylon pharmaceuticals ( inst ) , biotest ( inst ) , bristol - myers squibb ( inst ) travel , accommodations , expenses : takeda pharmaceuticals , celgene , novartis , amgen , janssen oncology , bristol - myers squibb hearn jay cho honoraria : genentech / roche consulting or advisory role : janssen research & development , genentech , bristol - myers squibb research funding : agenus , janssen research & development , bristol - myers squibb travel , accommodations , expenses : roche / genentech , bristol - myers squibb bart barlogie no relationship to disclose sundar jagannath honoraria : celgene , karyopharm therapeutics consulting or advisory role : celgene , bristol - myers squibb , janssen pharmaceuticals , novartis , karyopharm therapeutics research funding : celgene ( inst ) , karyopharm therapeutics ( inst ) , bristol - myers squibb ( inst ) joel t . 
dudley consulting or advisory role : janssen pharmaceuticals , allergan research funding : astrazeneca ( inst ) samir parekh consulting or advisory role : foundation medicine acknowledgment we thank the genomics core facility at the icahn institute and the department of genetics and genomic sciences at mount sinai , the mount sinai hematological malignancies tissue bank ( hmtb ) , and the human immune monitoring core ( himc ) for their support . 
we also thank scientific computing at the icahn school of medicine at mount sinai for their computational resources and staff expertise . affiliations alessandro lagan , itai beno , david melnekoff , violetta leshchenko , deepu madduri , dennis ramdas , larysa sanchez , scot niglio , deepak perumal , brian a . 
touzeau c , dousset c , le gouill s , et al : the bcl - 2 specific bh3 mimetic abt - 199 : a promising targeted therapy for t ( 11 ; 14 ) multiple myeloma . 
kumar s , kaufman jl , gasparetto c , et al : efficacy of venetoclax as targeted therapy for relapsed / refractory t ( 11 ; 14 ) multiple myeloma . 
moreau p , chanan - khan a , roberts aw , et al : promising efficacy and acceptable safety of venetoclax plus bortezomib and dexamethasone in relapsed / refractory mm . 
pak c , callander ns , young ew , et al : microc ( 3 ) : an ex vivo microfluidic cis - coculture assay to test chemosensitivity and resistance of patient multiple myeloma cells . 
mody , md , ms2 introduction somatic mutations in b - cell lymphoma 6 ( bcl6 ) corepressor ( bcor ) are reported in solid and hematologic malignancies in adults and children . 
bcor mediates transcriptional repression through epigenetic modication believed to cause oncogenesis through both overexpression or loss of tumor suppressor function.1 it is found to affect multiple diverse downstream gene targets and plays a role in development and function across many tissue lineages , especially the cns.2 , 3 in pediatric malignancies , bcor - ccnb3 fusion is prominent in undifferentiated round cell sarcoma ( uds ) , which , until recently , was treated similarly to ewing sarcoma ( ews )  . 
now considered its own genetic entity , bcor - ccnb3 fusions are typically identied in uds using transcriptome sequencing ( rna - seq ) and are present in up to 14% of uds cases.4 , 5 internal tandem duplications in bcor are found in pediatric cns neuroepithelial tumors and clear cell sarcoma of the kidney , 6 , 7 and mutations are reported in rhabdomyosarcoma and diffuse intrinsic pontine glioma ( dipg ) .8 , 9 however , uncertainty remains regarding the genomic landscape and signicance of bcor alterations in pediatric malignancies . in this article , we describe bcor alterations identied in our integrative clinical sequencing ( ics ) cohort in patients with sarcoma and high - grade glioma ( hgg ) these alterations in and explore the landscape of publicly available databases . 
furthermore , we examine their histopathologic features and co - occurring genomic aberrations and propose prospective targeted treatment strategies . methods pediatric and young adult patients ( , 26 years ) described were consented and enrolled in a prospective , institutional review boardapproved ( hum# : ics trial pediatricmichigan oncology 00056496 ) sequencing ( peds - mioncoseq ) at c.s. 
details of the sequencing procedures and bioinformatics analyses have been previously described.10 briey , somatic mutations , copy number variants / alterations , insertions and deletions , gene fusions , and gene expression are analyzed.11 , 12 investigation of clinical relevance of somatic variants uses an integrated approach incorporating technical considerations , variant and patient - specic information , and published data . st judes pediatric cancer data portal ( pecan ) was used ( accessed january 2019 ) to explore bcor alterations in a larger cohort of pediatric patients . 
the pecan database is a pediatric cancer genome repository containing whole - genome sequencing , wholeexome sequencing , and rna - seq data on more than 4 , 300 patients . 
 mankuzhy et al of the 86 sarcoma cases sequenced at the time of this report , 18 osteosarcomas and ve malignant peripheral nerve sheath tumors did not contain bcor alterations . twenty - seven of the remaining 63 cases contained clinically relevant diagnostic fusions , one ( 3.7% ) of which ( pax3 - foxo1positive alveolar rhabdomyosarcoma ) also had a bcor alteration . 
of the 15 patients with dipg sequenced , three ( 20% ) harbored mutations in bcor . pathologic evaluation of the bcor - mutant sarcoma cases showed both round - cell ( ewing - like ) morphology and the recently recognized variant with spindle - cell sarcoma morphology.17 figure 1 shows noncohesive sheets of malignant round to ovoid cells , with primitive - appearing chromatin , high mitotic activity , and necrosis . 
there were interlacing short fascicles with whorled pattern and foci of somewhat epithelioid congurations around larger vessels , mimicking synovial sarcoma and malignant peripheral nerve sheath tumor . mitotic gures were also identied . 
vimentin immunostain was diffusely strongly positive , and cd99 was negative . review of the pecan data set revealed 16 patients with tumors harboring nonsynonymous bcor alterations , ranging in age from 2 to 21 years ( table 2 )  . 
of the nine hggs , six contained concurrent gfr alterations , including two egfr and four platelet - derived growth factor receptor a mutations . the frequency of bcor mutations of primary adult cancers in tcga was 15 of 393 ( 3.8% ) in sarcoma and two of 273 ( 0.7% ) in hgg , particularly gbm . 
in the mi - oncoseq data set with advanced or refractory adult patients , sarcoma had a bcor mutation frequency of three of 199 ( 1.5% ) and gbm one of 49 ( 2% )  . discussion through integrative analysis of a cohort of high - risk pediatric malignancies and review of other large genomic databases , our study further describes the frequency and distribution of bcor alterations in pediatric sarcoma and high - grade glial malignancies . 
we recognized a higher overall prevalence of bcor alterations in these pediatric malignancy subtypes compared with their adult counterparts , both in primary cases ( tcga ) and refractory / advanced cases ( mi - oncoseq )  . 
this could be explained by bcors role in embryonic stem - cell differentiation , hematopoiesis , and regulation of vertebrate laterality and germline bcor mutations causing lenz microphthalmia and oculofaciocardiodental syndromes , which display fig 1 . 
 ( d ) cd99 immunostain ( 20 ) is negative . phenotypically overlapping skeletal , cns , and ocular effects , supporting its pleotropic developmental function.1 , 3 , 18 our study shows relatively high prevalence of bcor alterations in pediatric hgg ( 17% ) and in pediatric sarcomas , which are negative for fusions and other pathognomonic molecular alterations ( 11% ) , emphasizing the need for ics analysis to better dene molecular subtypes of these cases . 
our study also conrms earlier observations of bcor alterations in fusion - positive and - negative alveolar rhabdomyosarcoma.19 , 20 however , our ics cohort did not detect any bcor alterations in classic ewsr1 - rearranged ews or osteosarcoma , as seen in pecan . previous research has suggested that the prognostic signicance of bcor alterations could be tissue and diagnosis specic , with bcor - mutant leukemia being associated with a worse prognosis and no survival difference detected when comparing bcor - ccnb3 and ews.4 , 21 , 22 however , because of our cohorts small sample size and a lack of availability of outcomes data in larger data sets , additional investigation is needed to truly dene prognostic signicance of bcor alterations , specically in hgg . a novel nding in our cohort is the high frequency ( 57% ) of concurrent bcor and gfr alterations . 
gfrs are typically expressed on cell surfaces and play important roles in cell growth , survival , angiogenesis , and metastasis.23 the concurrent gfr mutations seen in our cohort are all known to be gain - of - function mutations resulting in constitutive activation of the gfr pathway and increased cell proliferation.24 - 27 bcl6 depletion results in downregulation of kinases in the receptor tyrosine kinase pathway , suggesting a potential link between bcl6 and related proteins such as bcor with receptor tyrosine kinase signaling that could be further explored to determine the signicance of these mutations.28 the bcl6 / bcor previous research has suggested that complex could serve as a potential therapeutic target . reported downstream targets of bcor activity include bcl6 targets , hoxa cluster genes , notch homolog 1 targets , and sonic hedgehog effectors such as gli1 and gli2.2 , 29 - 31 bcor can interact with specic histone deacetylase of both class i and ii , which increases the repression of transcriptional activity.32 the effect of histone deacetylase inhibitors on tumors with bcor mutations warrants additional investigation to assess the biologic relevance of these interactions , particularly in patients with dipg who also harbor histone mutations . 
 bcor alterations in pediatric solid tumors molecular and genomic characterization of tumors can complement histology - based classications of human cancers.35 recognizing which alterations are clinically meaningful and developing effective targeted treatment strategies can further contribute to dening this new cancer routine genomic proling can taxonomy . identify potential drivers of oncogenesis and possible targetable aberrations that can stimulate development of novel investigations of therapeutic approaches and additional in addition , molecular mechanisms of cancer . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . chandan kumar patents , royalties , other intellectual property : royalty for um agr #3199.101 recurrent gene fusions in prostate cancer licensed to gen - probe , inc . no other potential conicts of interest were reported . references hum mol genet 16 : 1773 - 1782 , 2007 1 . 
hilton en , manson fdc , urquhart je , et al : left - sided embryonic expression of the bcl - 6 corepressor , bcor , is required for vertebrate laterality determination . gearhart md , corcoran cm , wamstad ja , et al : polycomb group and scf ubiquitin ligases are found in a novel bcor complex that is recruited to bcl6 targets . mol cell biol 26 : 6880 - 6889 , 2006 3 . 
plos one 3 : e2814 , 2008 kao yc , owosho aa , sung ys , et al : bcor - ccnb3 fusion positive sarcomas : a clinicopathologic and molecular analysis of 36 cases with comparison to morphologic spectrum and clinical behavior of other round cell sarcomas . 
am j surg pathol 42 : 604 - 615 , 2018 peters tl , kumar v , polikepahad s , et al : bcor - ccnb3 fusions are frequent in undifferentiated sarcomas of male children . 
mod pathol 28 : 575 - 586 , 2015 argani p , kao y - c , zhang l , et al : primary renal sarcomas with bcor - ccnb3 gene fusion : a report of 2 cases showing histologic overlap with clear cell sarcoma of kidney , suggesting further link between bcor - related sarcomas of the kidney and soft tissues . 
am j surg pathol 41 : 1702 - 1712 , 2017 sturm d , orr ba , toprak uh , et al : new brain tumor entities emerge from molecular classication of cns - pnets . 
cell 164 : 1060 - 1072 , 2016 cramer sl , miller al , pressey jg , et al : pediatric anaplastic embryonal rhabdomyosarcoma : targeted therapy guided by genetic analysis and a patient - derived xenograft study . 
mackay a , burford a , carvalho d , et al : integrated molecular meta - analysis of 1 , 000 pediatric high - grade and diffuse intrinsic pontine glioma . 
tirode f , surdez d , ma x , et al : genomic landscape of ewing sarcoma denes an aggressive subtype with co - association of stag2 and tp53 mutations . 
 mankuzhy et al juckett lt , lin di , madison r , et al : a pan - cancer landscape analysis reveals a subset of endometrial stromal and pediatric tumors dened by internal tandem duplications of bcor . 
li w - s , liao i - c , wen m - c , et al : bcor - ccnb3 - positive soft tissue sarcoma with round - cell and spindle - cell histology : a series of four cases highlighting the pitfall of mimicking poorly differentiated synovial sarcoma . 
shern jf , chen l , chmielecki j , et al : comprehensive genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic axis in fusion - positive and fusion - negative tumors . 
puls f , niblett a , marland g , et al : bcor - ccnb3 ( ewing - like ) sarcoma : a clinicopathologic analysis of 10 cases , in comparison with conventional ewing sarcoma . 
bellus ga , spector eb , speiser pw , et al : distinct missense mutations of the fgfr3 lys650 codon modulate receptor kinase activation and the severity of the skeletal dysplasia phenotype . 
de gunst mm , gallegos - ruiz mi , giaccone g , et al : functional analysis of cancer - associated egfr mutants using a cellular assay with yfp - tagged egfr 28 . 
proc natl acad sci usa 114 : 3981 - 3986 , 2017 [ erratum : proc intracellular domamol cancer 6 : 56 , 2007 natl acad sci usa 114 : e4317 , 2017 ] 29 . 
tiberi l , bonnefont j , van den ameele j , et al : a bcl6 / bcor / sirt1 complex triggers neurogenesis and suppresses medulloblastoma by repressing sonic hedgehog signaling . 
schnidar h , eberl m , klingler s , et al : epidermal growth factor receptor signaling synergizes with hedgehog / gli in oncogenic transformation via activation of the mek / erk / jun pathway . 
 ras mutations are associated with inferior progression - free survival and overall survival of patients with mcrc compared with patients with nonmutated tumors.1 it is conceivable that ras mutations are negative prognostic factors per se ; however , the availability of only one actionable molecular target , that is , the inhibition of angiogenesis , in patients with mutated tumors certainly affects survival in this subgroup of patients . 
recent studies have demonstrated that the analysis of circulating tumor dna ( ctdna ) in blood samples , through its ability to recapitulate tumor heterogeneity , is a remarkable surrogate of tumor biopsy for mutation detection.2 extensive research on liquid biopsy led to significant achievements in the comprehension of the biologic reasons behind acquired resistance to anti - egfr therapies.3 to date , studies with liquid biopsy have been selectively concentrated on the appearance of ras - mutant clones in the blood of patients with ras wild - type primary crc , as biomarkers of anti - egfr therapy resistance . 
we here report that ctdna analysis , over the course of antiangiogenic treatment , might reveal a therapeutically exploitable window of opportunity , characterized by the relative prevalence of ras wild - type clones over time . 
a whole - body contrast enhanced computed tomography ( ct ) scan revealed a left supraclavicular lymphadenopathy , transverse colon thickening ( 3 cm ) , multiple chest and abdominal lymphadenopathies , and peritoneal carcinomatosis . 
mutational analysis of the lymph node metastasis performed through real - time polymerase chain reaction ( pcr ; real - time oncoscreen nras ) revealed exon 4 nras a146t mutation at 29 cycles . 
serum tumor markers significantly decreased , with rapid negativization ( carcinoembryonic antigen [ cea ] , 1.31.23 ; ca 19 - 9 , 71.839 ; ca - 125 , 8226 )  . 
at that time , serum tumor markers increased again ( ca 19 - 9 , 530 ; cea , 9 ) , with clinical evidence of bilateral axillary lymph node enlargement . 
concurrently , the patient was enrolled in an observational study open at our institution , which aimed to serially monitor the mutational status of ctdna in patients with ras - mutant mcrc disease , before starting any new line of treatment and in course of therapy until progression . methods blood samples were prospectively collected after obtaining informed consent with ethical committee approval . 
idylla ( biocartis , nv , mechelen , belgium ) , a fully automated , realtime pcr - based molecular diagnostics system , was used to investigate ras mutational profile from plasma . 
the first allows the detection of 21 mutations in codons 12 , 13 , 59 , 61 , 117 , and 146 of the kras gene , and the second allows the detection of 18 mutations in codons 12 , 13 , 59 , 61 , 117 , and 146 of the nras gene , five mutations in codon 600 of the braf gene , and two mutations in codon 492 of the egfr gene from 1 ml of plasma . among the parameters describing the generated pcr curves , the cq value is calculated as the difference between the quantification cycle value ( cq ) of the gene control signal and the cq of the mutant signal . 
according to the manufacturer 's instructions , a cq value of the nras control of 35.5 or higher indicates that a low amount of cell - free dna is present in the sample . 
in such cases , low - frequency mutations may not be detected . results in july 2017 , a ctdna mutational analysis was performed and revealed the absence of any clinically relevant mutation in kras , nras , and braf genes . 
this new insight into the biology of the disease convinced us to take this window of therapeutic opportunity , which was not guaranteed to remain open indefinitely ; in july 2017 , the treatment was changed to the second - line combination of irinotecan and cetuximab . 
as early as after the first cycle of chemotherapy , an objective complete clinical response of the bilateral axillary adenopathy was documented , and this was confirmed at ultrasound assessment . 
restaging ct scan after eight cycles of therapy showed a partial response , with significant reduction of pleural effusion and pulmonary lymphangitic carcinomatosis and stability of peritoneal metastases ( fig 1 )  . 
a second ctdna analysis was performed on november 2017 , confirming the ras genes wild - type status ( cq of nras total : 35.5 , which might be due to a reduction in ctdna amount )  . 
 june 2017 ( before folfiri - cetuximab ) october 2017 ( after 3 months of folfiri - cetuximab ) pleural effusion pulmonary lymphangitic carcinomatosis malignant ascites peritoneal carcinosis fig 1 . 
axial computed tomography scan after eight cycles of fluorouracil , leucovorin , and irinotecan ( folfiri ) plus cetuximab shows a partial response , with significant reduction of pleural effusion and pulmonary lymphangitic carcinomatosis and stability of peritoneal metastases . 
 sequence of treatments , clinical and radiologic response to the different schedules adopted ( partial response [ pr ] is indicated by blue arrow , and progressive disease [ pd ] is indicated by red arrow ) , and molecular tests performed on tumor tissue and plasma at different time points . 
bev , bevaci zumab ; cet , cetuximab ; ctdna , circulating tumor dna ; folfiri , fluoro uracil , leucovorin , and irinotecan ; folfoxiri , fluorouracil , leucovorin , oxaliplatin , and irinotecan ; fu , fluorouracil . 
nevertheless , a146t results in increased nras activity and downstream signaling and is transforming in cell culture resulting from increased nras guanosine diphosphate / guanosine triphosphate exchange rate , 4 and consequently patients who harbor mutations in nras also have significantly inferior anti - egfr treatment outcomes benefit compared with those without any ras mutations.5 it is recommended that patients with crc being considered for anti - egfr therapy must receive ras mutational testing , including kras and nras codons 12 and 13 of exon 2 , 59 , and 61 of exon 3 and 117 and 146 of exon 4 , and that anti - egfr should only be prescribed for patients with mcrc who are wild type for all known ras - activating mutations.6 in patients with ras - mutant primary colorectal cancers , ineligible for egfr inhibitors , the importance of the egfr pathway , which indeed sustains the disease , is counterintuitive . 
international guidelines currently recommend treatment of ras mutant mcrc with bevacizumab first line , followed by chemotherapy backbone change or aflibercept / ramucirumab at disease progression.7 preclinical and clinical data , however , demonstrate that tumor angiogenesis inhibition induces biologic changes in tumor - stroma interactions , mainly derived from hypoxia . 
preclinical observations suggest that hypoxia exerts a negative selection against ras - mutant clones through a mechanism known as secretory senescence , in which ras - mutant senescent cells operate in a paracrine manner to support the growth of surrounding ras wild - type clones , leading to their relative prevalence over time.8 we here report that ctdna analysis , under table 1 . 
 antiangiogenic treatment , might reveal a therapeutically exploitable window of opportunity , characterized by the relative prevalence of ras wild - type clones , which can be converted in a clinically meaningful benefit for patients . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . paola gazzaniga no relationship to disclose cristina raimondi no relationship to disclose federica urbano no relationship to disclose the following represents disclosure information provided by authors of this manuscript . 
modest dp , ricard i , heinemann v , et al : outcome according to kras - , nrasand brafmutation as well as kras mutation variants : pooled analysis of five randomized trials in metastatic colorectal cancer by the aio colorectal cancer study group . 
sorich mj , wiese md , rowland a , et al : extended ras mutations and anti - egfr monoclonal antibody survival benefit in metastatic colorectal cancer : a meta - analysis of randomized , controlled trials . 
sepulveda ar , hamilton sr , allegra cj , et al : molecular biomarkers for the evaluation of colorectal cancer : guideline summary from the american society for clinical pathology , college of american pathologists , association for molecular pathology , and american society of clinical oncology . 
long terra lucas shreshtha madaan kristin mattie danielle mckenna susan montgomery sarah nielsen jacquelyn powers kim rainey christina rybak michelle savage christina seelaus ( continued ) purpose multigene panels ( mgps ) are increasingly being used despite questions regarding their clinical utility and no standard approach to genetic counseling . 
how frequently genetic providers use mgp testing and how patient - reported outcomes ( pros ) differ from targeted testing ( eg , brca1 / 2 only ) are unknown . methods we evaluated use of mgp testing and pros in participants undergoing cancer genetic testing in the multicenter communication of genetic test results by telephone study ( clinicaltrials.gov identifier : nct01736345 ) , a randomized study of telephone versus in - person disclosure of genetic test results . 
genetic providers offered targeted or mgp testing based on clinical assessment . results since the inclusion of mgp testing in 2014 , 395 patients ( 66% ) were offered mgp testing . 
being offered mgp testing was significantly associated with not having ashkenazi jewish ancestry , having a history of cancer , not having a mutation in the family , not having made a treatment decision , and study site . 
after demographic adjustment , patients offered mgp testing had lower general anxiety ( p = .04 ) , state anxiety ( p = .03 ) , depression ( p = .04 ) , and uncertainty ( p = .05 ) pre - disclosure compared with patients offered targeted testing . 
there was a greater increase in change in uncertainty ( p = .04 ) among patients who underwent mgp testing . conclusion mgp testing was more frequently offered to patients with lower anxiety , depression , and uncertainty and was associated with favorable outcomes , with the exception of a greater increase in uncertainty compared with patients who had targeted testing . 
delivery of genetic testing results by telephone is one of several adaptations to the traditional genetic counseling practice model that have been studied in an effort to improve counseling access through the more efficient use of resources.3 - 5 beginning in 2012 , as part of the national cancer institute funded multicenter communication of genetic test results by telephone ( cogent ) clinical trial , patients eligible for brca1 / 2 genetic testing for hereditary breast or ovarian cancer risk assessment were recruited and randomly assigned to in - person or telephone disclosure of genetic test results . 
primary outcomes of the cogent study6 have demonstrated that telephone disclosure is noninferior to in - person counseling across all primary and secondary short term outcomes . a robust literature on patient - reported outcomes ( pros ) of brca1 / 2 and targeted genetic testing exists , including two randomized studies of telephone delivery of both pretesting and post - testing genetic services for patients considering genetic testing for these genes.3 , 5 cogent is , to our knowledge , the first randomized study to examine uptake of mgp tests in the population of patients presenting for risk assessment to a tertiary high - risk clinic , as well as to report on pros among patients receiving traditional targeted gene counseling and testing compared with patients receiving counseling and testing adapted to mgps . 
 in the current study , trends in testing choices with the advent of mgp testing and across the cogent study time period and participating sites are reported , and pros previously reported in aggregate are analyzed by counseling and testing modality selected for the patient . methods study design , setting , and participants the cogent study was a multicenter , randomized , noninferiority trial comparing the psychosocial and behavioral outcomes of phone versus in - person disclosure of genetic test results . 
 initially designed to include targeted brca1 / 2 testing only , the study eligibility was adapted in may 2014 to add all clinical germline genetic testing for hereditary breast , gynecologic , and / or gi cancer syndromes , allowing individuals receiving either targeted testing or mgp testing . 
participants were recruited after in - person pretest counseling with a genetic counselor and completed pros at the time of enrollment ( after pretest counseling [ t0 ] ) and after results disclosure ( t1 )  . random assignment after completion of the survey given after pretest counseling , participants were assigned to either the in - person arm or telephone ar random assignment was stratified by study site and sex only . 
participants who did not want to receive results by telephone and thus were not willing to be randomly assigned were permitted to self - select in - person disclosure and were analyzed separately . all 22 genetic counseling professionals used a tiered and binned counseling model for pretest sessions where mgp testing was offered.7 , 8 all disclosures were made using standardized communication protocols and visual aids.7 - 9 both a genetic counseling professional and a medical provider were present when results were provided in person , whereas individuals who received results by telephone were recommended to return to their institution to meet with a medical provider . 
patients responded on a seven - item likert scale and could mark not applicable . statistical analysis for baseline analyses , we characterized variables using means , proportions , standard deviations , and ranges for age . 
variables included in the tables and regressions included age , sex , race ( white v nonwhite ) , education ( college graduate v less than a college degree ) , jewish ethnicity ( no or missing v yes ) , number of firstand second - degree relatives with cancer , known cancer mutation , treatment decision , random assignment or self - selecting for in - person disclosure arm , and study site . 
we did not include in the imputation analyses individuals missing both t0 and t1 response data because we felt that such individuals had too much missing data to reliably impute data . 
the criterion for statistical significance was p < .05. results use and predictors of mgp testing versus targeted testing in total , 600 participants were offered genetic testing after inclusion of mgp testing ; 62.7% of participants ( 376 of 600 participants ) underwent mgp testing , whereas 37.3% of participants underwent targeted testing ( fig 1 )  . 
 targeted testing was most frequently offered to patients with a known familial mutation ( 86% ) and to patients of ashkenazi jewish ancestry ( 67% ) , a population in which three founder mutations in brca1 / 2 are found at high frequency ( prevalence of approximately 1 : 40 )  . 
of note , of 395 patients offered mgp testing by the genetic counselor , a small fraction ( 19 [ 4.8% ] of 395 patients ) declined mgp testing and chose targeted testing . 
characteristics associated with being more likely to be offered mgp testing were older age , female sex , nonashkenazi jewish heritage , personal history of cancer , no known familial mutation , nonrandomization status ( ie , preference for in - person disclosure ) , and study site . the percentage of patients offered mgp testing increased over time ( from 57% in 2014 to 66% in 2015 ; p = .02 ) and was found to vary significantly by study site ( range , 46% to 81% of all tests offered ; fig 2 )  . 
identification of carriers and true - negative results were higher in the targeted testing group , which is likely secondary to a higher number of patients with a known familial mutation ( table 1 )  . 
the frequency of uncertain variants was higher in the mgp group ( 25% v 2% with targeted testing )  . pros assessed by offer of targeted testing versus mgp testing in full models , adjusting for baseline variables , pros were assessed and compared by whether patients were offered mgp testing or targeted testing . 
characteristics of participants in the cogent study after adaptation for mgp by type of testing offered offered targeted testing only ( n = 205 ) offered multigene panel testing ( n = 395 ) characteristic had mgp testing , no . 
finally , there were no significant differences in knowledge at any time point . discussion in the largest study to date of pros after mgp testing , we found that patients eligible for hereditary cancer risk assessment and offered mgp , compared with targeted testing , had similar predisclosure and postdisclosure knowledge levels but lower predisclosure anxiety , depression , and uncertainty . 
these results demonstrate that genetic counselors can incorporate mgp testing into their practice without compromising patient understanding , even when results are delivered by telephone.6 one of the concerns commonly raised with mgp testing is the possibility of knowledge deficits as a result of the complexity of information communicated . 
 the potential negative impact of information overload from mgp counseling or testing on psychological outcomes is not supported by our findings , because we found that postdisclosure anxiety and depression were similar between the mgp and targeted testing groups . 
when the lower levels of predisclosure anxiety and depression among participants offered mgp are considered , our results also suggest that counselors may be more likely to offer complex mgp testing to patients they feel are at lower risk of negative psychological outcomes . 
in this way , these findings suggest that counselors are more than just gatekeepers to genetic testing and that comprehensive counseling includes both knowledge exchange and shared decision making with patients about concerns and preferences for genetic evaluation . 
 in the higher rates of selection of in - person disclosure among patients offered mgp testing , we observe counselors and patients counterbalancing a patients ability to manage the complexity and uncertainty of mgp testing with the standard approach of in - person results disclosure . these findings suggest that telephone disclosure is readily scalable to mgp testing and thus has an important role in the future of genomic medicine . 
although other studies have previously supported the efficacy of alternative telephone - based modalities of genetics services delivery , 3 , 5 these studies have not included mgp testing and have , notably , not reported detailed pros . 
one study recruited women at increased risk of hereditary breast and / or ovarian cancer and randomly assigned participants to in - person or telephone counseling , whereas the second study randomly assigned women with at least a 10% risk of having a brca1 / 2 mutation but without cancer to telephone counseling versus usual care . 
in both studies , telephone counseling was noninferior to in - person counseling for the selected outcomes ( which included anxiety , cancer - specific distress , perceived control , and decisional conflict in one study5 and satisfaction , distress , physical functioning , decisional conflict , and knowledge at 3 months and 1 year in the other study3 ) , although these results are specific to brca1 / 2 testing . 
although pros were not ascertained , the rate of preference for and receipt of mastectomy was similar regardless of whether a brca1 / 2 pathogenic variant or a pathogenic variant in a gene other than brca1 / 2 was identified by mgp , arguing that mgp may lead to more mastectomies over time . 
pros have not been explored in other areas of medicine where mgp testing has entered clinical care . lower postdisclosure anxiety and distress among patients offered mgp testing is consistent with differences seen pre - disclosure and reflect counselorand patient - driven selection of less anxious patients toward mgp testing . 
for others , high pretest risk was secondary to a known familial mutation ; 50% of the targeted testing population had a known familial mutation compared with 4% of the mgp testing population . 
similarly , patients tested by mgp had lower pretest likelihood of a pathogenic mutation , although the risk of identifying uncertain variants was higher as a result of the larger number of genes sequenced . although no other randomized studies have been conducted involving mgp , lumish et al19 performed a post - test survey measuring pros in patients undergoing testing for hereditary breast or ovarian cancer and in whom mgp testing was performed . 
they found older age , nonwhite race or non - hispanic ethnicity , lower educational attainment , and lower knowledge to be associated with higher anxiety and adverse psychological effects from counseling . 
analyses by cancer status ( affected or unaffected ) and stratified by result also found differential impact on psychological outcomes.19 nonetheless , it remains uncertain how the differences would have changed had mgp testing been replaced by targeted testing . 
 intolerance related to genomic testing21 have shown that uncertainty intolerance is associated with impaired decision making , feelings of vulnerability , and other adverse psychological outcomes.22 overall , we found that use of mgp testing increased over the study period.23 several factors are likely driving this change , including the need for more efficient delivery of genetic services . 
 the declining costs and the competitive marketplace for commercial testing established by the us supreme courts decision against myriad genetic laboratories and gene patenting have improved access to genetic testing . 
regional and community hospitals may be particularly disadvantaged when it comes to access to genetic counseling services and thus could be the earliest beneficiaries of models that incorporate remote counseling and testing options . 
however , after accounting for nonrandom differences between the targeted and mgp testing groups , short - term postdisclosure pros were generally similar , suggesting that the benefits of counseling rest in the live conversation between the counselor and patient and not in their proximity . 
time and resource limitations have undermined the traditional rigorous preand post - test counseling model used in cogent ; for example , katz et al25 found that 47% of patients did not receive counseling . 
in addition , few cogent participants were tested to guide a breast cancer treatment decision , and a recent population - based study found that mgp testing was associated with lower rates of testing among patients with breast cancer who had preoperative testing.18 finally , it also possible that patients choosing an mgp test versus targeted testing do so with family and family cancer risks in mind , so there may be additional information on testing selection gained from studying patterns of communication of results with relatives . in summary , the world of clinical genetic testing is changing rapidly , and use of mgp testing has increased over time . 
post - test knowledge , general anxiety , and depression were no worse and state anxiety was lower among patients who received mgp testing compared with those who received targeted testing . 
 bradbury manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors the following represents disclosure information provided by authors of this manuscript . 
 stock and other ownership interests : canceriq , tempus travel , accommodations , expenses : hospital of the university of pennsylvania research funding : novartis ( inst ) other relationship : tempus , color genomics , genentech , myriad genetics , bio ventures for global health kim rainey no relationship to disclose linda j . 
domchek honoraria : astrazeneca , clovis oncology , bristol - myers squibb research funding : astrazeneca ( inst ) , clovis oncology ( inst ) , pharmamar ( inst ) kristin mattie no relationship to disclose danielle mckenna no relationship to disclose susan montgomery no relationship to disclose sarah nielsen no relationship to disclose jacquelyn powers employment : carevive systems honoraria : cureconnect consulting or advisory role : carevive systems christina rybak no relationship to disclose michelle savage no relationship to disclose christina seelaus no relationship to disclose jessica stoll no relationship to disclose jill e . 
olopade employment : canceriq ( i ) leadership : canceriq dominique fetzer no relationship to disclose amanda brandt no relationship to disclose rachelle chambers no relationship to disclose dana f . 
clark no relationship to disclose rikki gaber honoraria : canceriq cassandra gulden no relationship to disclose janice horte no relationship to disclose andrea forman consulting or advisory role : invitae , astrazeneca , ambry speakers ' bureau : astrazeneca , ambry genetics , invitae jessica m . 
daly , andrea forman , susan montgomery , kim rainey , christina rybak , and michelle savage , fox chase cancer center , temple university health system ; susan m . 
olopade , center for clinical cancer genetics and global health , the university of chicago ; pamela ganschow , rikki gaber , terra lucas , and christina seelaus , the john h . 
hospital of cook county ; rachelle chambers , cassandra gulden , shreshtha madaan , sarah nielsen , and jessica stoll , the university of chicago , chicago , il ; and generosa grana , dana f . 
clark , janice horte , kristin mattie , and xinxin ( shirley ) yao , md anderson cancer center at cooper , camden , nj . support supported by national cancer institute grant no . 
kinney ay , steffen le , brumbach bh , et al : randomized noninferiority trial of telephone delivery of brca1 / 2 genetic counseling compared with in - person counseling : 1 - year follow - up . 
bradbury ar , patrick - miller lj , egleston bl , et al : randomized noninferiority trial of telephone vs in - person disclosure of germline cancer genetic test results . 
bradbury ar , patrick - miller lj , egleston bl , et al : patient feedback and early outcome data with a novel tiered - binned model for multiplex breast cancer susceptibility testing . 
bradbury ar , patrick - miller l , long j , et al : development of a tiered and binned genetic counseling model for informed consent in the era of multiplex testing for cancer susceptibility . 
kelly k , leventhal h , marvin m , et al : cancer genetics knowledge and beliefs and receipt of results in ashkenazi jewish individuals receiving counseling for brca1 / 2 mutations . 
cella d , hughes c , peterman a , et al : a brief assessment of concerns associated with genetic testing for cancer : the multidimensional impact of cancer risk assessment ( micra ) questionnaire . 
pieterse ah , van dulmen am , beemer fa , et al : cancer genetic counseling : communication and counselees post - visit satisfaction , cognitions , anxiety , and needs fulfillment . 
biesecker bb , klein w , lewis kl , et al : how do research participants perceive uncertainty in genome sequencing ? genet med 16 : 977 - 980 , 2014 22 . 
identication and monitoring the molecular mechanisms of acquired resistance to cetuximab or panitumumab in crc is important to anticipate the failure of anti - egfr antibodies and design strategies for additional treatments . several mechanisms of acquired resistance to antiegfr have been described in metastatic crc ( mcrc )  . 
 maurel et al context key objective the key objective was to evaluate the predictive and prognostic value of ras and braf mutation dynamics in plasma from patients with metastatic colorectal cancer treated with antiepidermal growth factor receptor ( anti - egfr ) therapy within two clinical trials ( posiba and pulse )  . knowledge generated baseline mutant ras / braf circulating tumor ( ct ) dna was a negative predictive marker of response to anti - egfr therapy , as well as a poor prognostic factor . 
the emergence of ctdna ras / braf mutations had limited relevance to the time to progression to anti - egfr therapy but conferred poor prognosis . relevance this work conrms the clinical utility of ctdna for ras / braf molecular testing to guide treatment decisions in patients with metastatic colorectal cancer ; however , clinical impact of ras / braf dynamics deserves further characterization . of ras , braf , or egfr mutations that drive acquired resistance in approximately 50% of patients.5 , 6 the use of circulating tumor ( ct ) dna for genotyping the tumor ( liquid biopsy ) offers clear advantages compared with repeated tumor tissue biopsies , including the minimally invasive method and no need for a tumor lesion accessible to biopsy . 
our objective was to determine the role of ctdna ras and braf mutations at the time of progression to anti - egfr therapy . methods study design and patient population the pulse and posiba studies were both prospective biomarker - design trials . 
patients were recruited into the pulse trial from november 2010 to april 2013 in 24 spanish centers and treated with infusional uorouracil , leucovorin , and oxaliplatin ( folfox ) - 6 plus panitumumab ( 6 mg / kg )  . 
patients were recruited into the posiba trial from july 2011 to may 2015 in 28 spanish centers and treated with folfox6 or uorouracil , leucovorin , and irinotecan ( folfiri ; at the investigators choice ) plus biweekly cetuximab ( 500 mg / m2 )  . in both trials , cytotoxic drugs were administered for 6 months , and anti - egfr monotherapy was continued until pd or unacceptable toxicity occurred . 
extended ras mutational analysis ( including kras / nras exons 2 , 3 , and 4 ) started in october 2013 in the pulse trial and in october 2015 in the posiba trial after protocol amendments . 
competitive allele - specic taqman pcr ( castpcr ; thermo fisher scientic , waltham , ma ) and an ultrasensitive sequencing technique ( digital polymerase chain reaction [ pcr ] ) were used to reanalyze tissue ras determination in patients with discrepancies between basal liquid biopsy ( mutation ) and formalin - xed parafn - embedded ( ffpe ) specimens ( wt )  . ctdna ras and braf mutational analysis this was an exploratory translational study with a primary objective of investigating measurement of ras and braf v600 - mutant ctdna in patients treated in the posiba and pulse trials using the idylla ctras and ctbraf mutation assay ( biocartis , jersey city , nj )  . 
plasma samples were collected basally , and serum samples were collected every 2 months in the pulse trial and every 3 months in the posiba trial , until pd was detected according to response evaluation criteria in solid tumors ( recist ) v1.1. 
two blood samples were analyzed for each patient for this study : a baseline plasma sample and a serum sample collected at the time of the last available determination . integrated ctdna purication and allele - specic quantitative pcr followed by automated data analysis was performed using the idylla ctkras and ctnras mutation assays to detect ras exon 2 , 3 , and 4 mutations and braf v600 mutations using 0.5 to 1 ml of plasma or serum per assay . 
the investigators who performed plasma genotyping were blinded to the clinical patient information . statistical analysis imaging was performed every 8 weeks in the pulse trial and every 12 weeks in the posiba trials and response was based on recist v1.1 as assessed by the investigators . progression - free survival ( pfs ) was dened as time from enrollment to disease progression ( recist criteria ) , death , or end of follow - up , whichever came rst . 
concordance was assessed using the kappa statistic and the characteristics of the diagnostic test using the sensitivity , specicity , and positive and negative predictive values by collapsing ras plus braf versus wt . 
statistical analysis was performed with spss statistics 22 ( spss , chicago , il ) and sas 9.4 software ( sas institute , cary , nc )  . results patient characteristics and concordance of baseline ras and braf mutation determination in plasma versus tissue the study ow chart is presented in figure 1 . 
kaplan - meier estimates of ( a ) progression - free survival and ( b ) overall survival , on the basis of ( c ) solid and ( d ) liquid biopsies . 
blood extraction was performed in group 1 ( between blood extraction and pd ) with a median ( p25 to p75 ) of + 7 days ( 1 to + 23 days ) and in group 2 with a median of 157 days ( 76 to 330 days ) , with the date of progression as the index date . the emergence of ras / braf mutations in ctdna was observed in 15 patients after initiation of therapy ( 11% ; appendix table a3 , online only )  . 
however , one of 11 patients ( 9% ) with acquired mutations in ctdna received anti - egfr compounds after disease progression compared with 22 of 79 ( 27.8% ) of those who remained free of mutations , potentially contributing to improved os . discussion our study indicates that ras and braf testing in baseline if not better , pfs and os ctdna results in a similar , discrimination compared with tissue testing in patients treated with rst - line chemotherapy plus anti - egfr therapy . 
 maurel et al 20116 20601 10019 10129 10140 10039 10027 10149 23209 10090 10153 20804 10131 10082 20401 10004 10159 20602 10006 10182 20306 10124 10115 21119 10122 22008 10167 22901 20611 10174 10126 10189 10147 22001 mutational status ( os bar ) : negativitzation no change overall response rate ( pfs bar ) : metastasectomy end of treatment liquid biopsy 1 , 000 1 , 200 1 , 400 time ( days ) fig 4 . 
progression - free survival ( pfs ) and overall survival ( os ) in patients with mutant ras and braf on basal liquid biopsy ( n = 34 )  . 
in a recent study with 43 patients , oncobeam demonstrated 72.1% concordance with tissue results , whereas idylla demonstrated 46.5% concordance with kras tissue results.15 finally , we observed that the subgroup of patients who had ras or braf mutations on tissue and wt on ctdna was enriched with patients with low tumor burden ( ldh , 1 uln )  . 
our results analyzing plasma samples at baseline and serum samples at progression differ with the analysis from the aspecct third - line phase iii panitumumab study , which showed that patients with emergent ras mutations at progression had similar pfs and os versus patients whose status remained wt at progression.19 in our study , patients with ras and braf with acquired mutations that were initially 2 wt showed signicantly poorer survival than those patients who remained wt at progression ; also , patients with disappearance of mutations showed better os . 
we have to note that patients in our study had been treated in rst - line therapy ( compared with third line in the aspecct trial ) and , therefore , second - line egfr therapies in truly sensitive patients ( those with liquid biopsies that remain wt ) would affect survival . 
moreover , we could not rule out that at pd , in patients with a poor prognosis ( identied by a high level of ldh at disease progression ) , we would have more chance to detect acquired mutations in ctdna . 
our study addresses most of the potential limitations from previous analyses , such as low number of patients , treatment heterogeneity ( including type of treatment and mixed rst , second , and third lines ) , and lack of a control group ( analysis of ras and braf in samples of patients without progression or before pd )  . 
 clinical impact of circulating tumor ras and braf mutations with rst - line therapy with currently accepted schedules of chemotherapy ( folfox and folfiri ) and approved antiegfr compounds ( cetuximab and panitumumab ) to establish differences between both groups ( 14% and 8% , respectively ; p = .47 ) , and global mutation acquisition was observed in only 11% of patients . 
this suggests that other mechanisms beyond ras and braf acquired mutations will probably play an important role , not only in the subset of patients without acquired ras and braf mutations , but also in the group of patients with acquired ras and braf mutations . 
n engl j med 360 : 1408 - 1417 , 2009 van cutsem e , lenz hj , k ohne ch , et al : fluorouracil , leucovorin , and irinotecan plus cetuximab treatment and ras mutations in colorectal cancer . 
j clin oncol 33 : 692 - 700 , 2015 douillard jy , siena s , cassidy j , et al : randomized , phase iii trial of panitumumab with infusional uorouracil , leucovorin , and oxaliplatin ( folfox4 ) versus folfox4 alone as rst - line treatment in patients with previously untreated metastatic colorectal cancer : the prime study . 
j clin oncol 28 : 4697 - 4705 , 2010 douillard jy , oliner ks , siena s , et al : panitumumab - folfox4 treatment and ras mutations in colorectal cancer . 
montagut c , dalmases a , bellosillo b , et al : identication of a mutation in the extracellular domain of the epidermal growth factor receptor conferring cetuximab resistance in colorectal cancer . 
montagut c , argil es g , ciardiello f , et al : efcacy of sym004 in patients with metastatic colorectal cancer with acquired resistance to anti - egfr therapy and molecularly selected by circulating tumor dna analyses : a phase 2 randomized clinical trial . 
jama oncol 4 : e175245 , 2018 pietrantonio f , vernieri c , siravegna g , et al : heterogeneity of acquired resistance to anti - egfr monoclonal antibodies in patients with metastatic colorectal cancer . 
clin cancer res 23 : 2414 - 2422 , 2017 diaz la jr , williams rt , wu j , et al : the molecular evolution of acquired resistance to targeted egfr blockade in colorectal cancers . 
siena s , sartore - bianchi a , garcia - carbonero r , et al : dynamic molecular analysis and clinical correlates of tumor evolution within a phase ii trial of panitumumab - based therapy in metastatic colorectal cancer . 
normanno n , esposito abate r , lambiase m , et al : ras testing of liquid biopsy correlates with the outcome of metastatic colorectal cancer patients treated with rst - line folfiri plus cetuximab in the capri - goim trial . 
grasselli j , elez e , carat `u g , et al : concordance of bloodand tumor - based detection of ras mutations to guide anti - egfr therapy in metastatic colorectal cancer . 
ann oncol 29 : 1211 - 1219 , 2018 jacobs b , claes b , pomella v , et al : analytical and clinical validation of the idylla ( cid : 1 ) ctkras and ctnras - braf liquid biopsy tests identies mcrc patient groups with high and low ctdna shedding . 
ras and braf mutational status in baseline plasma sample and secondary extraction serum sample : characteristics of patients with ctdna ras / braf mutation in baseline and second liquid biopsy plasma liquid biopsy ( basal ) serum liquid biopsy ( secondary ) , no . ras mutated braf mutated patient primary side basal liquid biopsy site of metastasis response surgery of second liquid metastasis biopsy maurel et al mutated braf mutated 2 wt total rectum kras ( a146p / t / v ) plus nras ( g13r / v ) liver left colon nras ( g13r / v ) liver , lung sigmoid kras ( g12d ) liver , lung sigmoid nras ( g13r / v ) liver , peritoneum sigmoid kras ( g12v ) node right side kras ( a146p / t / v ) local relapse sigmoid kras ( k117n ) liver , lung left colon kras ( g12v ) peritoneum rectum kras ( g12r ) liver , lung rectum kras ( k117n ) liver , lung right side braf ( v600e / d ) liver , peritoneum rectum nras ( q61h ) rectum braf ( v600e / d ) right side braf ( v600e / d ) kras ( g12d ) liver liver liver liver rectum kras ( a146p / t / v ) liver , node , suprarenal  . 
ras and braf mutational status in baseline plasma sample and secondary extraction serum sample : characteristics of patients with ctdna ras / braf mutation in baseline and second liquid biopsy ( continued ) serum liquid biopsy ( secondary ) , no . 22008 10122 10115 10006 20602 10004 10082 10153 22001 10189 10174 21119 10131 10090 rectum nras ( g61k ) liver , lung sigmoid nras ( g61r ) liver , lung right side kras ( a59t / e / g ) liver rectum kras ( a146p / t / v ) liver , lung left colon nras ( g61h ) liver , node , peritoneum  . 
 no evidence of increased risk of breast cancer in women with lynch syndrome identied by multigene panel testing jessica stoll , ms , cgc1 ; eric rosenthal , scm , phd2 ; shelly cummings , ms , cgc2 ; jamie willmott , ms , cgc2 ; ryan bernhisel , mstat2 ; and sonia s . 
kupfer , md1 purpose prior estimates of breast cancer risk in women with lynch syndrome ( ls ) range from population risk to 18 - fold increased risk with reported differences by gene . 
standardized incidence ratios ( sirs ) and 95% cis of breast cancer were calculated compared with age - matched incidence in the general us female population and with women negative for pvs stratied by the test indication . results in total , 0.8% of women ( 30 , 362 of 441 , 966 women ) carried mmr gene pvs . 
women with pvs in pms2 and msh6 were tested more frequently for hboc , whereas those with pvs in mlh1 and msh2 / epcam were tested more frequently for ls . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction breast cancer risk in lynch syndrome ( ls ) has been debated , with published data indicating anywhere from no increased risk1 - 4 up to 18 - fold increased risk for women with a pathogenic variant ( pv ) in a mismatch repair ( mmr ) gene.5 - 9 a recent laboratorybased study reported a 2to 3 - fold increased incidence of breast cancer in women with pvs in msh6 and pms2 compared with the general us female population , but not in mlh1 or msh2 carriers.10 these authors used us female population data from the seer registry ; however , comparing cancer histories in a population heavily weighted toward women being assessed for hereditary breast and ovarian cancer ( hboc ) to cancer histories in the general population could result in ascertainment bias.11 - 13 specically , this approach likely increases the proportion of the sample with a personal diagnosis of breast cancer , resulting in elevated breast cancer rates regardless of mmr gene status . 
this ascertainment bias might be less evident for women with pvs in the higher penetrance mmr genes ( ie , mlh1 , msh2 , and epcam ) who are more likely to be ascertained for testing on the basis of personal or family history of early - onset colorectal or endometrial cancer or other ls - associated cancers . therefore , to properly estimate cancer risk , women with pvs should be compared with pv - negative women within the same testing population . quantifying breast cancer risk in women with ls has signicant implications for screening and risk reduction , especially for patients with ls caused by pvs in msh6 and pms2 , which are more prevalent than previously thought . 
 stoll et al context key objective using a large , laboratory - based database , we determined whether women with lynch syndrome ( ls ) are at increased risk of breast cancer compared with the general population and to a population undergoing genetic testing . knowledge generated women with ls are not at increased risk of breast cancer . 
these ndings are in line with results from the prospective ls database but contrast with other laboratory - based studies . relevance these ndings support average - risk breast cancer screening in women with ls . ascertained for suspicion of hboc and / or ls but were not found to have a pv by multigene panel testing . methods study population women tested with a single commercial laboratorys ( myriad genetic laboratories , salt lake city , ut ) multigene panel between september 2013 and november 2018 were included unless they resided in a state prohibiting use of deidentied genetic or clinical information for research . 
clinical and demographic data for participants were obtained from test request forms submitted by ordering health care providers . ls - associated cancers included colorectal , endometrial , ovarian , pancreatic , stomach , gastric , ureter , brain , small intestine , and biliary tract cancers . 
hboc - associated cancers included breast , ovarian , prostate , and pancreatic cancers . testing criteria individuals met providers indicated on the test requisition forms whether testing was ordered for suspicion of ls or hboc . 
for hboc , 2013 national comprehensive cancer network ( nccn ) guidelines were used , 14 but the contribution of prostate cancer was excluded because gleason score or metastatic disease information was not collected reliably . these exclusions were not expected to substantially inuence results but may have resulted in an underestimate of the number of women meeting hboc criteria . 
to directly evaluate rates against the recent publication that established a 2to 3fold elevated breast cancer incidence for msh6 and pms2 , 10 the us female population from the seer registry17 was used as one comparator group . 
to compensate for the ascertainment bias expected in a population selected for testing on the basis of suspicion of hereditary cancer risk , we also calculated the sirs using the following 3 additional comparator populations : women who tested negative for any pv who were ascertained for suspicion of hboc or ls ; women who were ascertained for hboc only ; and women who were ascertained for ls only . 
all analyses were performed using sas software ( sas institute , cary , nc )  . results demographic and clinical characteristics in total , 441 , 996 women met inclusion criteria ( appendix table a1 )  . 
among these , 3 , 362 women ( 0.8% ) were found to carry a single pv in an mmr gene ( mlh1 , msh2 , msh6 , pms2 , or epcam ) , and 438 , 634 women ( 99.2% ) were negative for pvs in any of the other genes included on the panel . 
epcam deletions were identied in only 11 women and therefore were included with msh2 for all analyses . demographic and clinical characteristics for women with mmr gene pv and all women tested are listed in table 1 . for women with an mmr gene pv , median age at testing was 47 years , and 56.8% of women reported white or caucasian ancestry . 
demographic and clinical characteristics of women with mmr gene pvs and all women undergoing multigene panel testing all women tested ( n = 441 , 996 ) women with mmr gene pv ( n = 3 , 362 ) characteristic age at testing , yearsa mean ( sd ) range mean ( sd ) range age at bc diagnosis , yearsb self - reported ancestry , no . 
similar differences in mutation spectrum were found for women meeting nccn testing criteria for ls versus hboc ( fig 1 )  . the majority of mlh1 and msh2 / epcam mutation carriers were ascertained for clinical suspicion of ls , whereas msh6 and pms2 mutation carriers were more commonly ascertained for suspicion of hboc ( fig 2 )  . phenotypic spectrum the majority of women with pvs in mlh1 and msh2 / epcam were affected by cancer , whereas most pms2 carriers were unaffected . 
for msh2 / epcam carriers , endometrial and colorectal cancers were the most common cancers reported ( n = 162 [ 24.8% ] and n = 150 [ 23.0% ] , respectively )  . 
carriers of mlh1 and msh2 / epcam pvs had lower rates of breast cancer , whereas there was no difference in sirs for msh6 or pms2 carriers compared with seer population breast cancer rates ( table 2 )  . table 3 lists sirs for women with mmr gene pvs compared with women with negative panel testing . 
when all women ascertained for hboc or ls were analyzed , breast cancer rates were lower in women with pvs in mmr genes . specically , breast cancer rates ranged from 22% to 50% lower than expected among the various mmr genes . 
similarly , when compared with women ascertained for suspicion of ls , breast cancer rates also did not differ between women with mmr gene pvs compared with pv - negative women . 
distribution of pathogenic variants ( pvs ) by mismatch repair gene on the basis of ascertainment for testing indicated by ordering provider and testing criteria met ( mlh1 , n = 462 ; msh2 / epcam , n = 653 ; msh6 , n = 985 ; pms2 , n = 1 , 262 )  . 
prevalence of mismatch repair pathogenic variants by ascertainment for lynch syndrome ( ls ) and hereditary breast and ovarian cancer syndrome ( hboc ) testing as indicated by ordering provider ( mlh1 , n = 462 ; msh2 / epcam , n = 653 ; msh6 , n = 985 ; pms2 , n = 1 , 262 )  . discussion data from multigene panel testing in ls have called into question the unresolved issue of whether breast cancer risk is increased in ls . 
although there is a wealth of existing literature on breast cancer in ls , data from these studies are conicting , with risk estimates ranging from no increased risk1 - 4 up to 18 - fold increased risk.5 - 9 recent studies report an increased breast cancer risk in women with pvs in msh6 and pms2 , but not in mlh1 and msh2 / epcam , 10 or an increased risk of breast cancer exclusive to msh6.19 in the present laboratory - based study , including a large sample of msh6 and pms2 pv carriers , previously published data showing an increased breast cancer risk in women with pvs in msh6 and / or pms2 were not replicated when the same methodology was used.10 moreover , the rate of breast cancer was found to be lower than expected among mlh1 and msh2 / epcam carriers . 
findings from the current study are in line with a recent report from the prospective ls database.20 the ndings in the current study likely differ from previous studies showing increased breast cancer risk in ls for several reasons . 
first , the current study includes the largest number of mmr gene pv carriers reported to date , including large numbers of msh6 and pms2 carriers , thereby increasing precision in breast cancer risk estimates compared with more moderately sized studies . 
moreover , this sample included a large proportion of women unaffected by cancer , which appropriately shifts the breast cancer estimates closer to those of the general us female population . colon endometrial breast ovarian other * unaffected mlh1 msh2 / epcam msh6 pms2 no mutation fig 3 . 
in these analyses , no difference in breast cancer incidence was found among women with or without mmr gene pvs whether they were ascertained for suspicion of hboc or ls . 
these results support current guidelines to follow average - risk breast cancer screening recommendations in all women with ls.22 consistent with ndings from other laboratory and population - based studies , these data demonstrate that pvs in msh6 and pms2 account for a much higher proportion of ls than previously estimated.10 , 23 also similar to previous studies , 23 , 24 this study found that the current ls testing criteria perform reasonably well for women with pvs in mlh1 and msh2 but miss close to half of women with pvs in msh6 and pms2 . 
 no increased breast cancer risk in lynch syndrome these results suggest that current clinical criteria are insufcient for identication of the full spectrum of patients with ls who would benet from appropriate surveillance.25 one limitation inherent to utilization of a laboratory - based testing population is that clinical information was limited to that which was included on the test requisition form as provided by ordering clinicians and therefore may not be wholly inclusive of individuals personal and family histories . 
however , we obtained essentially the same ndings through comparisons between different populations within this study , suggesting that underor overreporting of cancer history in any of these populations did not affect the ndings . 
although the study was designed specically to address ascertainment bias , the population included only women undergoing genetic testing for clinical suspicion of a hereditary cancer syndrome and may not be representative of the more general population with ls . however , this was at least in part mitigated by the fact that the comparator population was from the same testing population to ensure that any population differences between women with and without pvs were valid . in the largest study of mmr gene pv carriers identied by a multigene panel through a commercial laboratory , there was no evidence of increased breast cancer risk in women with pvs in mmr genes compared with the general us female population or with woman undergoing multigene panel testing . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . jessica stoll consulting or advisory role : invitae travel , accommodations , expenses : invitae eric rosenthal employment : myriad genetic laboratories stock and other ownership interests : myriad genetic laboratories shelly cummings employment : myriad genetics stock and other ownership interests : myriad genetics travel , accommodations , expenses : myriad genetics jamie willmott employment : myriad genetics stock and other ownership interests : myriad genetics research funding : myriad genetics travel , accommodations , expenses : myriad genetics ryan bernhisel employment : myriad genetics sonia s . 
kupfer honoraria : advance medical no other potential conicts of interest were reported . references baglietto l , lindor nm , dowty jg , et al : risks of lynch syndrome cancers for msh6 mutation carriers . 
j natl cancer inst 102 : 193 - 201 , 2010 pande m , wei c , chen j , et al : cancer spectrum in dna mismatch repair gene mutation carriers : results from a hospital based lynch syndrome registry . 
mller p , sepp al a tt , bernstein i , et al : cancer risk and survival in path_mmr carriers by gene and gender up to 75 years of age : a report from the prospective lynch syndrome database . 
gut 67 : 1306 - 1316 , 2018 therkildsen c , ladelund s , smith - hansen l , et al : towards geneand gender - based risk estimates in lynch syndrome ; age - specic incidences for 13 extracolorectal cancer types . 
 stoll et al oliveira ferreira f , napoli ferreira cc , rossi bm , et al : frequency of extra - colonic tumors in hereditary nonpolyposis colorectal cancer ( hnpcc ) and familial colorectal cancer ( fcc ) brazilian families : an analysis by a brazilian hereditary colorectal cancer institutional registry . 
fam cancer 3 : 41 - 47 , 2004 scott rj , mcphillips m , meldrum cj , et al : hereditary nonpolyposis colorectal cancer in 95 families : differences and similarities between mutation - positive and mutation - negative kindreds . 
win ak , young jp , lindor nm , et al : colorectal and other cancer risks for carriers and noncarriers from families with a dna mismatch repair gene mutation : a prospective cohort study . 
j clin oncol 30 : 958 - 964 , 2012 goldberg m , bell k , aronson m , et al : association between the lynch syndrome gene msh2 and breast cancer susceptibility in a canadian familial cancer registry . 
harkness ef , barrow e , newton k , et al : lynch syndrome caused by mlh1 mutations is associated with an increased risk of breast cancer : a cohort study . j med genet 52 : 553 - 556 , 2015 genet med 20 : 1167 - 1174 , 2018 10 . 
2018 : is breast cancer truly caused by msh6 and pms2 variants or is it simply due to a high prevalence of these variants in the population ? genet med 21 : 256 - 257 , 2019 12 . 
evans dg , woodward er , lalloo f , et al : are women with pathogenic variants in pms2 and msh6 really at high lifetime risk of breast cancer ? genet med , 2018 13 . 
dominguez - valentin m , sampson jr , sepp al a tt , et al : cancer risks by gene , age , and gender in 6350 carriers of pathogenic mismatch repair variants : findings from the prospective lynch syndrome database . 
giardiello fm , allen ji , axilbund je , et al : guidelines on genetic evaluation and management of lynch syndrome : a consensus statement by the us multisociety task force on colorectal cancer . 
 patterns of metastatic spread and mechanisms of resistance to crizotinib in ros1 - positive nonsmall - cell lung cancer purpose the ros1 tyrosine kinase is activated through ros1 gene rearrangements in 1% to 2% of nonsmall - cell lung cancers ( nsclcs ) , which confer sensitivity to treatment with the anaplastic lymphoma kinase ( alk ) / ros1 / mesenchymal - epithelial transition factor inhibitor crizotinib . 
currently , insights into patterns of metastatic spread and mechanisms of crizotinib resistance among patients with ros1 - positive disease are limited . patients and methods we reviewed clinical and radiographic imaging data of patients with ros1and alk - positive nsclc to compare patterns of metastatic spread at initial metastatic diagnosis . 
to determine molecular mechanisms of crizotinib resistance , we analyzed repeat biopsy specimens from a cohort of patients with ros1 - positive disease who progressed on crizotinib . results we identified 39 and 196 patients with advanced ros1and alk - positive nsclc , respectively . 
ros1 mutations included g2032r ( 41% ) , d2033n ( 6% ) , and s1986f ( 6% )  . conclusion compared with alk rearrangements , ros1 rearrangements are associated with lower rates of extrathoracic metastases , including fewer brain metastases , at initial metastatic diagnosis . 
2017 by american society of clinical oncology introduction treatment paradigms for advanced nonsmallcell lung cancer ( nsclc ) continue to evolve and reflect a growing understanding of the genetic underpinnings of the disease . 
subsequent work has shown that such rearrangements are present in 1% to 2% of nsclcs and define a distinct molecular subgroup.2 - 4 of note , ros1 is phylogenetically related to the anaplastic lymphoma kinase ( alk ) receptor tyrosine kinase.5 thus , like alk rearrangements in nsclc , 6 , 7 ros1 fusions confer sensitivity to the alk / ros1 / mesenchymal - epithelial transition factor inhibitor crizotinib.3 in profile 1001 ( study of oral pf - 02341066 , a c - met / hepatocyte growth factor tyrosine kinase inhibitor , in patients with advanced cancer ) , crizotinib produced an objective response rate justin f . 
shaw author affiliations and support information ( if applicable ) appear at the end of this article . presented at the 2016 american society of clinical oncology annual meeting , chicago , il , june 3 - 7 , 2016 . corresponding author : alice t . 
on the basis of this activity , the us food and drug administration expanded crizotinibs approval to include patients with advanced , ros1 - positive nsclc.12 consistent with these reports , we observed several prolonged responses to crizotinib among patients with ros1 - positive nsclc at our institution . specifically , we observed particularly durable responses among patients with intrathoracic - only disease ( ie , m1a disease ) , 3 which led us to hypothesize that differences in malignant phenotypes between ros1 and alk rearrangements , including differences in patterns of metastatic spread , affect therapeutic outcomes . 
in one early study , doebele et al13 reported that alk rearrangements are associated with pericardial , pleural , and liver metastases compared with an egfr / kras / alk wild - type cohort . 
outside this report , however , few studies have evaluated patterns of metastatic spread according to molecular genotype in nsclc , and none have focused on ros1.14 , 15 in this study , we investigated the distribution of malignant disease at initial metastatic diagnosis and evaluated associations with clinical outcomes in ros1 - positive nsclc . 
data were updated as of january 2016 . all studies were performed under an institutional review boardapproved protocol . pfs was measured from the time of crizotinib initiation to investigator - assessed , radiographic progression or death in the absence of documented progression . 
overall survival ( os ) was measured from initiation of crizotinib until death . patients without a known date of death were censored at the time of last follow - up . in addition , patients with ros1 - positive nsclc who underwent repeat biopsies / resection of progressing lesions on crizotinib between july 2012 and december 2016 were identified . 
age and lines of therapy were analyzed by wilcoxon rank sum test . the kaplan - meier method was used to estimate pfs and os medians and probabilities , and the log - rank test was used to compare their differences between groups . 
no significant differences in baseline clinicopathologic characteristics were observed between groups ( data supplement ) , including no difference in the median number of prior lines of therapy before crizotinib ( median , 1 ; data supplement )  . 
to address whether this difference was confounded by prior therapy , we examined pfs among patients who received second - line crizotinib ( ie , after one prior therapy ) because this was the most common line of crizotinib in both cohorts ( ros1 , 60% ; alk , 42% )  . 
of note , 101 patients with alk - positive nsclc ( 58% ) received secondor thirdgeneration alk inhibitors after crizotinib , whereas only seven patients with ros1 - positive nsclc ( 23% ) received additional ros1 inhibitors ( p , .001 ; data supplement )  . 
thus , the longer median pfs with crizotinib among patients with ros1 - positive disease may have been partly offset by greater access to next - generation targeted therapies among patients with alk - positive disease . sites of metastatic disease to determine whether these clinical outcomes may have also been affected by distribution of metastatic disease , we compared sites of disease at initial metastatic diagnosis among patients with alkand ros1 - positive nsclc . 
metastatic involvement of a given anatomic site was determined on the basis of clinical staging ( tnm seventh edition ) .30 positron emission tomography - computed tomography ( ct ) and ct scans of the chest , abdomen , and pelvis were performed in 72% and 26% of patients with alk - positive nsclc and 82% and 18% of patients with ros1 - positive nsclc , respectively ( table 1 )  . 
brain magnetic resonance imaging ( alk , 76% ; ros1 , 82% ) or a head ct scan with intravenous contrast ( alk , 9% ; ros1 , 10% ) were also performed in most patients . 
in addition to having a lower rate of brain metastases at initial diagnosis , fewer patients with ros1 - positive nsclc developed brain metastases over time compared with those with alk - positive nsclc . 
the cumulative incidence of brain metastasis among patients with alkand ros1 - positive disease was 73% and 34% , respectively , at 5 years after initial metastatic diagnosis ( p , .001 ; fig 3a )  . 
percentages may not add to 100 because of rounding . abbreviations : ct , computed tomography ; ecog , eastern cooperative oncology group ; mri , magnetic resonance imaging ; pet , positron emission tomography . * includes one patient with adenosquamous histology . on the basis of performance status at initial metastatic presentation or at the first documented assessment . 
performance status was not available in four patients with alk - positive disease and one patient with ros1 - positive disease . systemic staging and neuroimaging studies were not done or were not available for review in five and 35 patients with alk - positive disease , respectively . 
four patients with alk - positive disease had both a brain mri and a head ct scan . neuroimaging studies were not done or not available for review in three patients with ros1 - positive disease . time was also greater among those with alkpositive disease ( 56% v 22% at 5 years ; p = .001 ; fig 3b )  . 
of note , 101 patients with alk - positive nsclc ( 58% ) received next - generation alk inhibitors ( ceritinib , 40% ; alectinib , 27% ; brigatinib , 3% ) after crizotinib ( data supplement ) ; however , 93% of these patients received such agents after the initial development of brain metastases . 
collectively , these findings suggest that alk - positive cancers have an increased tropism for the brain compared with ros1 - positive nsclcs . next , we evaluated outcomes of crizotinib treatment according to the presence of extrathoracic metastases ( ie , m1a v m1b disease )  . 
thus , this difference in pfs for crizotinib treatment outcomes between patients with alkand ros1 - positive nsclc in the overall study population may have been partly driven by the higher frequency of m1a disease among those with ros1 - positive disease because this group appears to derive greater benefit with crizotinib . ros1 : crizotinib resistance despite the effectiveness of crizotinib in alkand ros1 - positive nsclc , most patients acquire resistance . 
to investigate potential mechanisms of resistance to crizotinib in ros1 - positive nsclc , we analyzed postprogression biopsy specimens from patients with ros1 - positive disease who progressed on crizotinib treatment . 
all patients underwent tissue sampling within 7 days of crizotinib discontinuation . persistence of the original ros1 rearrangement was observed in nine ( 100% ) of nine postcrizotinib specimens available for testing ( repeat fish [ n = 2 ] , rt - pcr [ n = 5 ] , or ngs [ n = 2 ] )  . 
 ( d ) os as measured from the time of crizotinib initiation in the second - line setting . time since crizotinib initiation ( months ) time since crizotinib initiation ( months ) no . 
thus , ros1 resistance mutations were detected in a majority ( 10 [ 62.5% ] of 16 ) of patients with crizotinib resistance . because crizotinib has limited blood - brain barrier penetration , 31 we analyzed crizotinib - resistant samples by site of disease . 
among non - cns specimens ( n = 14 ) , the frequency of ros1 resistance mutations was 64% ( appendix fig a2 ) , with 50% of specimens harboring the ros1 g2032r mutation . 
among six specimens that underwent targeted ngs with the mgh snapshot platform , none was found to have high - level copy number changes in any analyzed gene ( data supplement )  . 
 a mgh047 - 5 ros1 g2032r mgh9018 - 1 ros1 wt biopsy ros1 wt ros1 wt ros1 g2032r ros1 g2032r ros1 g2032r ros1 s1986f ros1 g2032r * ros1 wt ros1 wt ros1 wt * mgh070 - 1 mgh096 - 1 mgh077 - 1 mgh080 - 2 mgh998 - 1 mgh508 - 1 mgh095 - 1 mgh996 - 1 mgh943 - 1 mgh995 - 1 mgh933 - 1 mgh081 - 1 mgh968 - 1 mgh986 - 1 fig 4 . 
mgh , massachusetts general hospital ; wt , wild type . g2032r ros1 d2033n ros1 wt ( 2 sites ) ros1 g2032r s1986f d2033n progression - free survival ( months ) ros1 g2032r discussion we observed that despite shared clinical features between ros1and alk - positive nsclcs , these genetic alterations were associated with different patterns of metastatic spread . 
although the shorter pfs with crizotinib treatment among patients with alk - positive nsclc may have been an influence in this series , we observed significantly higher rates of brain metastases at initial diagnosis ( ie , before exposure to crizotinib ) in this group . together , these observations suggest that alkrearranged nsclcs have a greater tropism for the cns . 
the majority of patients with alkpositive disease who developed brain metastases did so before receiving more cns penetrable , next - generation alk inhibitors , which provides an additional rationale for investigating the upfront use of next - generation alk inhibitors to prevent or delay the emergence of brain metastases . 
several ongoing first - line studies are comparing crizotinib with second - generation alk inhibitors ( eg , alex [ study comparing alectinib with crizotinib in treatment - naive anaplastic lymphoma kinasepositive advanced nonsmall cell lung cancer participants ] , alta - 1l [ phase iii study of brigatinib versus crizotinib in alk - positive advanced non small - cell lung cancer patients ] ) and have generally incorporated secondary end points focused on intracranial disease activity . one limitation of this analysis is that we observed a relatively higher frequency of baseline brain metastases in the alk - positive cohort than had other studies.6 , 13 although this observation may reflect differences in local imaging practices or referral patterns , such factors would have likely affected both alk and ros1 cohorts . 
nonetheless , several other studies have shown a comparable pfs to our series.8 - 11 of note , although patients with ros1 - positive disease had a longer median pfs with crizotinib treatment than patients with alk - positive disease , no difference in os was found between cohorts . 
we cannot exclude the possibility that this was due to unaccounted - for differences in patient characteristics , although the higher rates of next - generation targeted therapy use among patients with alk - positive disease may have offset the longer pfs with crizotinib treatment among patients with ros1positive disease . despite the significant activity of crizotinib , acquired resistance remains a challenge for patients with alkand ros1 - positive nsclc alike.16 , 43 - 45 thus far , insights into the mechanisms of resistance to crizotinib among those with ros1 - positive disease have been limited and generally consist of isolated case reports , small series , and preclinical evaluations.16 - 19 , 46 to date , seven different ros1 resistance mutations have been described.16 - 19 , 46 in addition , upregulation of bypass signaling pathways ( eg , egfr , ras , kit ) have also been reported.20 - 22 , 47 to our knowledge , we present the largest study of crizotinib resistance in ros1 - positive nsclc to date , which has found ros1 resistance mutations in a majority of specimens . 
 emerged through selection of pre - existing clones or through genetic evolution of drugtolerant cells.48 despite similarities between alk and ros1 , each has a different frequency and spectrum of on - target mechanisms of crizotinib resistance . 
indeed , patients with ros1 - positive nsclc have a much higher frequency of on - target resistance mutations , but such mutations are concentrated in a narrower segment of the kinase , which perhaps reflects the greater potency of crizotinib for ros1 than for alk . potential limitations of this analysis are that several different genotyping platforms were used . 
thus , we may have overlooked ros1 mutations outside this region . however , all specimens that underwent sanger sequencing and / or deep sequencing ( n = 8 ) were evaluated for mutations across the entire ros1 kinase domaanother important limitation is that we were unable to identify a mechanism of crizotinib resistance in all patients . 
thus , additional studies are necessary to elucidate other potentially target - independent mechanisms of crizotinib resistance . in alk - positive nsclc , second - generation alk inhibitors demonstrate significant activity in the crizotinib - resistant setting largely as a result of their greater potency against alk compared with crizotinib . 
beyond cabozantinib and tpx - 0005 , several other agents with ros1 activity are being evaluated , including entrectinib ( clinicaltrials.gov identifier nct02097810 ) , 53 ds - 6051b ( clinicaltrials.gov identifier nct02279433 ) , and lorlatinib ( clinicaltrials.gov identifier nct01970865 ) , 50 , 54 but these agents have not demonstrated clinical activity against ros1 g2032r to date ( data supplement )  . in summary , despite a shared susceptibility to crizotinib , alk and ros1 rearrangements differ in distributions of metastatic disease . 
nonetheless , acquired resistance to crizotinib remains a significant challenge for both molecular subsets . among patients with ros1 - positive nsclc , ros1 mutations ( most notably g2032r ) are the predominant mechanisms of resistance to crizotinib , which underscores the need for clinical development of next - generation ros1 inhibitors with activity against this mutation . 
provisional application to the us patent and trademark office ( application number 61817229 ) satoshi yoda no relationship to disclose ibiayi dagogo - jack no relationship to disclose luc friboulet no relationship to disclose jessica j . 
hubbeling no relationship to disclose leila dardaei no relationship to disclose james hardwick employment : pfizer stock and other ownership interests : pfizer travel , accommodations , expenses : pfizer donghui huang employment : pfizer stock and other ownership interests : pfizer travel , accommodations , expenses : pfizer mari mino - kenudson consulting or advisory role : merrimack , h3 biomedicine , acd pharmaceuticals , roche , genentech a . 
john iafrate stock and other ownership interests : archer biosciences consulting or advisory role : debiopharm group , constellation pharmaceuticals , chugai pharma , roche research funding : blueprint medicines patents , royalties , other intellectual property : archerdx exclusive license to amp technology anna f . 
farago honoraria : foundation medicine consulting or advisory role : pharmamar , merrimack , takeda pharmaceuticals , abbvie , intervention insights travel , accommodations , expenses : pharmamar , abbvie , stemcentrx aaron n . 
ferris no relationship to disclose zofia piotrowska consulting or advisory role : boehringer ingelheim , astrazeneca , ariad pharmaceuticals , takeda pharmaceuticals , superdimension research funding : guardant health ( inst ) alice t . 
shaw honoraria : pfizer , novartis , roche , genentech , foundation medicine consulting or advisory role : pfizer , novartis , genentech , roche , ariad pharmaceuticals , ignyta , blueprint medicines , daiichi sankyo , emd serono , taiho pharmaceutical , ksq therapeutics research funding : pfizer , novartis , roche , genentech acknowledgment we thank cyril h . 
shaw at , kim dw , nakagawa k , et al : crizotinib versus chemotherapy in advanced alk - positive lung cancer . med 371 : 2167 - 2177 , 2014 n engl j med 368 : 2385 - 2394 , 2013 8 . 
moro - sibilot d , faivre l , zalcman g , et al : crizotinib in patients with advanced ros1 - rearranged nonsmall - cell lung cancer ( nsclc ) : preliminary results of the acse phase ii trial . 
goto k , yang j , kim d , et al : phase ii study of crizotinib in east asian patients ( pts ) with ros1 - positive advanced nonsmall - cell lung cancer ( nsclc )  . 
russo a , franchina t , picone a , et al : different metastatic pattern according to the egfr mutational status in a cohort of lung adenocarcinomas ( adcs ) : a single - institution report . 
facchinetti f , loriot y , kuo ms , et al : crizotinib - resistant ros1 mutations reveal a predictive kinase inhibitor sensitivity model for ros1and alk - rearranged lung cancers . 
drilon a , somwar r , wagner jp , et al : a novel crizotinib - resistant solvent - front mutation responsive to cabozantinib therapy in a patient with ros1 - rearranged lung cancer . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
shaw at , yeap by , mino - kenudson m , et al : clinical features and outcome of patients with nonsmall - cell lung cancer who harbor eml4 - alk . 
goldstraw p , crowley j , chansky k , et al : the iaslc lung cancer staging project : proposals for the revision of the tnm stage groupings in the forthcoming ( seventh ) edition of the tnm classification of malignant tumours . 
gainor jf , ou sh , logan j , et al : the central nervous system as a sanctuary site in alk - positive nonsmall - cell lung j clin oncol 29 : e443 - e445 , 2011 cancer . 
soria jc , tan ds , chiari r , et al : first - line ceritinib versus platinum - based chemotherapy in advanced alkrearranged nonsmall - cell lung cancer ( ascend - 4 ) : a randomised , open - label , phase 3 study . 
gadgeel sm , shaw at , govindan r , et al : pooled analysis of cns response to alectinib in two studies of pretreated patients with alk - positive nonsmall - cell lung cancer . 
crin `o l , ahn mj , de marinis f , et al : multicenter phase ii study of whole - body and intracranial activity with ceritinib in patients with alk - rearranged nonsmall - cell lung cancer previously treated with chemotherapy and crizotinib : results from ascend - 2 . 
woo cg , seo s , kim sw , et al : differential protein stability and clinical responses of eml4 - alk fusion variants to various alk inhibitors in advanced alk - rearranged nonsmall cell lung cancer . 
huber kv , salah e , radic b , et al : stereospecific targeting of mth1 by ( s ) - crizotinib as an anticancer strategy . nature 508 : 222 - 227 , 2014 42 . 
shaw a , camidge d , engelman j , et al : clinical activity of crizotinib in advanced nonsmall - cell lung cancer ( nsclc ) harboring ros1 gene rearrangement . 
zou hy , li q , engstrom ld , et al : pf - 06463922 is a potent and selective next - generation ros1 / alk inhibitor capable of blocking crizotinib - resistant ros1 mutations . 
cui j , rogers e , zhai d , et al : ending the endless acquired tyrosine kinase resistance mutationsdesign of tpx0005 , a multi - target alk / ros1 / trk inhibitor with broad spectrum activity against wild - type and mutants including alk g1202r , ros1 g2032r , and trka g595r . 
drilon a , siena s , ou si , et al : safety and antitumor activity of the multi - targeted pan - trk , ros1 , and alk inhibitor entrectinib ( rxdx - 101 ) : combined results from two phase i trials ( alka - 372 - 001 and startrk - 1 )  . cancer discov 7 : 400 - 409 , 2017 54 . 
solomon b , bauer t , felip e , et al : safety and efficacy of lorlatinib ( pf - 06463922 ) from the dose - escalation component of a study in patients with advanced alk + or ros1 + nonsmall - cell lung cancer ( nsclc )  . 
progressionfree survival ( pfs ) of alkand ros1 - positive patients with ( a ) m1a and ( b ) m1b disease . time since crizotinib initiation ( months ) time since crizotinib initiation ( months ) no . 
fish , phd1 , 3 , 4 introduction immune checkpoint inhibitors ( icis ) have signicantly improved advanced cancer outcomes.1 , 2 although icis unleash antitumor immune responses , negating natural immune inhibitory mechanisms can result in the development of self - reactive immune cells and an array of immune - related adverse events ( iraes ) .3 although reports of serious myocarditis have emerged , 4 - 9 data on long - term outcomes following an episode of ici - associated myocarditis are limited and molecular analyses on surviving patients are lacking . 
here , we report a case of recurrent myocarditis in a patient previously treated with a programmed death ligand 1 ( pd - l1 ) inhibitor ( durvalumab ) , an ici that blocks the binding of pd - l1 to programmed cell death protein 1 and cd80.10 molecular investigations revealed cytokine modulations suggestive of a sustained t - helper 1 ( th1 ) - like cellular immune response as well as unique antigen - bound serum autoantibodies , highlighting a induction of potential autoantibody production in iraes . role for th1 - cell - dependent case initial presentation a 67 - year - old woman presented to the emergency department with severe sudden - onset chest pain 2 days after receiving the 12th cycle of pd - l1 inhibitor ( 48 weeks after starting ici ) for the treatment of metastatic urothelial carcinoma ( fig 1a )  . 
she was previously treated for remote triple - negative right - sided breast invasive ductal carcinoma with surgery ; cyclophosphamide , epirubicin , and 5 - uorouracil chemotherapy ; and radiation . 
her treatment was largely uneventful , with only a noted diagnosis of hypothyroidism after receiving the 7th cycle of pd - l1 inhibitor , which was treated with hormone replacement therapy without interruption of ici . although the patient was hemodynamically stable , cardiac laboratory investigations revealed high - sensitivity troponin i levels were elevated at 4 , 114 ng per l ( reference range , , 26 ng per l ) , and brain natriuretic peptide levels ( bnp ) were elevated at 2 , 275 pg per ml ( reference range , , 100 pg per ml ) ( figs 1b and 1c , data supplement )  . 
2d echocardiogram demonstrated severely decreased biventricular systolic function , with a left ventricular ejection fraction ( lvef ) of 20% , right ventricular ( rv ) fraction area change of 22% , and a left ventricular ( lv ) thrombus ( fig 1d and video 1 )  . 
with a history of ici treatment , together with clinical , ecg ( fig 2a ) , and imaging ndings , a diagnosis of ici - associated myocarditis was made . 
treatment with intravenous methylprednisolone ( 2 mg / kg / d ) , furosemide , betablocker , angiotensin - converting enzyme inhibitor , and low - molecular - weight heparin was initiated . 
cardiovascular magnetic resonance ( cmr ) imaging conrmed severe biventricular dysfunction , mild pericardial effusion , and demonstrated late gadolinium enhancement ( lge ) involving the basal and apical segments predominantly in a subepicardial distribution ( video 2 , data supplement )  . 
follow - up ecg remained abnormal with low - voltage qrs complex as well as persistent st elevation ( fig 2b )  . the patient was discharged home in stable condition on 90 mg oral prednisone with a plan to taper and discontinue in 8 weeks . 
 echocardiogram ( apical the patient demonstrating four chamber view ) of biventricular dysfunction as well as a focused apex view with contrast showing a thrombus ( admission )  . case report evidence of pulmonary emboli , whereas whole - body computed tomography scanning showed no evidence of metastatic disease . 
there was signicant clinical recovery 1 month after discharge , with echocardiography revealing partial recovery of lv function ( lvef = 40% ; global longitudinal strain = 17.1% ) ; however , rv systolic function remained severely reduced ( rv fraction area change 17% , data supplement )  . 
ecg remained abnormal with low - voltage qrs , however , now with right bundle branch block ( fig 2c )  . recurrent presentation four months after the initial event , and without resumption of ici treatment , the patient presented to the cardiology clinic with extreme fatigue . 
she was admitted and a subsequent cmr conrmed the severity of lv dysfunction , multiple new lv thrombi , larger pericardial effusion , and a mild interval worsening of nonischemic pattern lge ( data supplement )  . a cardiac biopsy ( figs 2e and 2f ) conrmed the diagnosis of myocarditis with predominantly t cells ( fig 2g ) and macrophages ( fig 2h ) ; polymerase chain reaction was negative for cardiotropic viruses.11 given the biopsy result , she was diagnosed with recurrence of ici - associated myocarditis . 
she deteriorated and subsequently succumbed to heart failure a year - and - a - half later . methods the patient was enrolled in this research ethics boardapproved case report at the university health network after obtaining informed written consent . 
next - generation sequencing ( immunoseq ) , cytokine multiplex assays , targeted microrna proling , and a mass spectrometry - based assay to identify serum autoantibody complexes with their cognate antigens were performed ( for a detailed description of the methods , see the data supplement )  . results analysis of the distribution , clonality , and diversity of t - cell receptors pre - recurrence ( january ) and during treatment for recurrence of myocarditis ( march onward ) with immunosuppressants revealed limited clonal modulation ( data supplement )  . 
multiplex analysis of cytokines ( luminex , austin , tx ) in serum revealed remarkably high concentrations of t - cellderived cytokines during recurrence despite withdrawal of ici therapy and initiation of immunosuppressants . 
substratifying by t - helper ( th ) cell subtype revealed that there was an apparent dominance of th1 - cellenriched cytokines over th2 - cellenriched cytokines ( figs 3a and 3b ; data supplement )  . quantication of 92 micrornas ( fluidigm , san francisco , ca ) with known inammatory and cardiac roles revealed two micrornas ( mir - 130a - 3p and mir - 122 - 5 ) that displayed consistent upregulation and downregulation , respectively , in parallel with circulating cytokine abundance . 
increases in mir - 130a - 3p ( figs 3b and 3c ) appeared to correlate with declines in interleukin ( il ) - 4 , 12 whereas decreases in mir122 - 5p , a noted negative regulator of il - 1 , appeared to be associated with elevations in il - 1 ( figs 3a and 3c ) .13 principal component and pathway analysis of differentially regulated micrornas ( cross - timepoint comparison ) revealed modulation during treatment course ( data supplement )  . to assess the downstream effects of a potentially aberrant th1 cell response , we examined antigen - bound serum autoantibodies . 
it was noteworthy that the patient experienced hypothyroidism after receiving ici as we identied antithyroglobulin autoantibody across all sample collection points ( roche electrochemiluminescent assay ) ( data supplement )  . 
a mass spectrometry - based assay14 - 16 to identify serum autoantibodies complexed with their cognate antigens detected thirteen autoantibodies related to cardiac and coagulative pathway ( fig 3d ; data supplement )  . 
by contrast , the recurrent presentation , although displaying low ejection fraction and elevated bnp ( c and d ) , did not display markedly elevated troponin levels ( b )  . 
follow - up ecg after initial presentation shows persistence of st elevation and development of low - voltage ecg ( b )  . ecg one month after initial presentation shows low - voltage ecg and development of right bundle branch block ( rbbb , c )  . 
myocardial biopsy ( e ) demonstrated mixed inammation ( yellow arrow ) with some associated myocyte damage ( orange arrow ) and some healing injury ( green star ) near relatively uninvolved myocardium ( cyan star ) ; higher - power image is also shown ( f ) ; hematoxylin and eosin staining ; digital acquisition ; scale bars as shown = 100 m . immunohistochemistry demonstrated that most of this inltrate was composed of cd3positive t cells ( g , stained cells in brown ) and cd68 - positive macrophages ( h , stained cells in brown )  . 
avr , augmented vector right ; avl , augmented vector left ; avf , augmented vector foot . cmr showing the presence of severe biventricular dysfunction , mild pericardial effusion , and a short axis late gadolinium enhancement ( lge ) stack showing lge in a subepicardial distribution involving the basal and apical segments ( admission )  . although iraes often develop within the rst few weeks to months after initiation of treatment , these events can present at any time , including after cessation of ici therapy . 
luminex - based analysis of circulating inammatory cytokines revealed consistently elevated responses enriched in th1 cell cytokines ( a ) in contrast to th2 cell cytokines ( b )  . two mirs related to cardiac function and inammation displayed consistent trends in expression with mir - 130 - 3p trending upward and mir - 122 - 5p trending downward ( c )  . 
serum autoantibody complexes were observed consistently in the programmed cell death protein - 1 ligand 1induced fulminant myocarditis patient compared to ageand sex - matched control patients ( n = 7 ) , and a patient with myocarditis from systemic lupus erythematosus ( n = 1 ; d )  . serum autoantibody complex data are displayed as relative expression ( average protein intensity ) with data presented as the mean 6 the standard deviation . 
 case report surveillance after an episode of myocarditis , and the necessity for rapid and effective immunosuppression.4 treatment with high - dose glucocorticoids and mmf appeared to improve symptomology ; however , it may have failed to affect the underlying etiology as bnp levels remained marginally abnormal while high levels of inammatory cytokines and autoantibodies persisted . 
it is possible that in addition to generating autoreactive t cells against cardiac epitopes ( a proposed cause of ici - mediated myocarditis ) , icis may also induce th1 - dependent pathogenic autoantibody production ( data supplement )  . patients presenting with symptoms suggestive of ici - related myocarditis present a signicant diagnostic challenge . cardiac troponins can be negative in cases of myocarditis.20 indeed , in this case , troponins were only intermittently and mildly elevated during myocarditis recurrence . 
the diagnostic value of echocardiography and ecg is often limited , with ndings suggestive of myocarditis either absent initially or lacking specicity.4 , 6 the addition of cmr may be to detect benecial owing to its potential inammation , edema , and brosis within myocardial tissue . 
however , lge alone has low sensitivity and accuracy in diagnosing chronic or recurrent myocarditis , and cmr is difcult in hemodynamically unstable patients.21 although the recovery of lv function and resolution of symptoms followed by a separate recurrence of symptoms consistent with acute myocarditis is suggestive of a distinct myocarditis event , one other possibility could include the persistence of subclinical inammation between episodes , leading to smouldering myocarditis and subsequent heart raises awareness that serious iraes can occur after ici discontinuation , and without re - exposure , reinforcing the importance of cardiovascular surveillance after an episode of ici - associated myocarditis along with detailed diagnostic work - ups . 
fish provision of study materials or patients : nazanin aghel , paaladinesh thavendiranathan collection and assembly of data : nazanin aghel , dakota gustafson , ashley di meo , milena music , michael a . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . support preparation of this paper used the resources of the toronto general hospital research institute , university health network , toronto , canada . 
hansen consulting or advisory role : merck , glaxosmithkline , bristol - myers squibb , eisai research funding : karyopharm therapeutics , merck , bristol - myers squibb , boehringer ingelheim , glaxosmithkline , roche / genentech , janssen , astrazeneca / medimmune , astellas pharma , macrogenics paaladinesh thavendiranathan honoraria : amgen no other potential conicts of interest were reported . acknowledgment the authors would like to thank the peter munk cardiac centre cardiovascular biobank for the assistance provided with the clinical sample storage , maintenance , and retrieval . references antonia sj , villegas a , daniel d , et al : durvalumab after chemoradiotherapy in stage iii non - small - cell lung cancer . 
n engl j med 377 : 1919 - 1929 , 2017 larkin j , chiarion - sileni v , gonzalez r , et al : combined nivolumab and ipilimumab or monotherapy in untreated melanoma . 
n engl j med 373 : 23 - 34 , 2015 puzanov i , diab a , abdallah k , et al : managing toxicities associated with immune checkpoint inhibitors : consensus recommendations from the society for immunotherapy of cancer ( sitc ) toxicity management working group . 
j am coll cardiol 71 : 1755 - 1764 , 2018 l aubli h , balmelli c , bossard m , et al : acute heart failure due to autoimmune myocarditis under pembrolizumab treatment for metastatic melanoma . j immunother cancer 3 : 11 , 2015 8 . 
mir h , alhussein m , alrashidi s , et al : cardiac complications associated with checkpoint inhibition : a systematic review of the literature in an important emerging area . 
hsu sh , wang b , kota j , et al : essential metabolic , anti - inammatory , and anti - tumorigenic functions of mir - 122 in liver . 
ohyama k , yoshimi h , aibara n , et al : immune complexome analysis reveals the specic and frequent presence of immune complex antigens in lung cancer patients : a pilot study . 
in this article , we cover topics related to the analysis of prognostic factors , centering on factors that are both relevant at the time of diagnosis or initial treatment and during treatment . 
we then review the state - of - the art methods for dynamic prediction and compare the strengths and limitations of these methods . although static models will continue to play an important role in oncology , developing and validating dynamic models of clinical outcomes need to take a higher priority . 
2019 by american society of clinical oncology introduction the identication of prognostic factors and building of risk assessment prognostic models will continue to play a major role in 21st century medicine in patient management and decision making.1 prognostic factors in oncology associate host and tumor variables treatment.2 with clinical outcomes independent of gospodarowicz et al3 classied factors as either tumor related , host , or environmental . 
tumor - related factors are variables related to the tumor and reveal the tumor biology and pathology , such as size of tumor , lymph node involvement , presence of metastasis , and molecular markers ( overexpression of pten gene , presence of androgen receptor variant ar - v7 )  . 
finally , environmental factors are external to the patient , such as access to health care.3 prognostic models are increasingly used in the design , conduct , and analysis of clinical trials . for example , trials of prostate cancer , randomization was stratied by the predicted survival probability determined by the prognostic model of overall survival.4 - 7 in the tailorx trial , oncotype dx , a 21 - gene score that predicts the likelihood of in several recurrence , is used to classify women with breast cancer by their risk group of recurrence.8 prognostic factors also have been used for enriching the patient population in trials with targeted therapies . 
for example , in the toga trial , patients with human epireceptor 2positive gastric dermal growth factor cancer were randomly assigned to either trastuzumab plus chemotherapy or chemotherapy alone.9 in this article , we cover topics that are related to the analysis of prognostic factors , centering on factors that are relevant both at the time of diagnosis or initial treatment and during treatment or follow - up . 
we make a distinction between static and dynamic predictive models and provide a review of the state - of - the - art methods for dynamic predictive models to promote them for future use . knowledge generated to date , most risk assessment models in oncology have been based on static prognostic models . 
in patients with advanced cancer , the disease has substantially evolved race , age , performance status , body mass index , host factors tumor factors psa , ldh , alkaline phosphatase , p53 , etc clinical outcomes overall survival progression - free survival objective response rate prediction window all available information up to t timeline study baseline landmark time horizon time fig 1 . 
 developing and validating risk assessment models and has a heterogeneous presentation within the patient.10 the interand intrapatient variability should be taken into account in statistical modeling.11 , 12 dynamic prediction incorporates time - dependent covariates so that risk prediction would be continually updated with new observations to reect the patients prognosis . we dene terminology that is typically used in dynamic risk prediction . 
standard variable selection approaches , such as forward selection , backward selection , and so forth , with logistic regression for binary end points , 13 and proportional hazards regression for timeto - event end points , 14 have been applied . 
criticism for the stepwise variable selection has been well documented.15 , 16 of note , classication trees , such as recursive partitioning for both binary and time - to - event end points , 17 - 20 frequently have been used.2 , 21 - 26 we concentrate on penalized methods that t and shrink p predictors and in doing so , reduce the variance of the coefcient estimates.27 thus , these methods would improve the accuracy of the model.28 the least absolute shrinkage and selection operator ( lasso ) and adaptive lasso ( alasso ) have been widely used to develop prognostic models of clinical outcomes.29 , 30 we will briey describe ridge regression and penalized methods . 
ridge regression minimizes the residual sum of squares function , but a caveat is that it does not reduce all the coefcients exactly to 0.28 let yi be the response , xij the jth covariate value ( j = 1 , 2 , , p ) corresponding to the ith individual , j the regression coefcient jth covariate , and a tuning parameter . 
similar to ridge regression , the rst term in lasso is the residual sum of squares , and lasso minimizes this function subject to the l1 penalty ( eq 1 ) : ( cid : 1 ) i ( cid : 1 ) 1 yi ( cid : 1 ) j ( cid : 1 ) 1 j xij ( cid : 2 ) + ( cid : 1 ) ( cid : 2 ) j ( cid : 2 ) ( cid : 2 )  . j ( cid : 1 ) 1 a large tuning parameter causes the coefcient estimates to be equal to 0 , thus the lasso will have the sparsity property.28 lasso is an improvement over ridge regression , although it has the main limitation of tending to select too many unimportant variables , and it performs poorly in situations when p  . 
the alasso enjoys the oracle property.30 , 34 elastic net regression uses a combination of l1 penalty and ridge l2 penalty and is a compromise between lasso and ridge regression . 
n is that it retains more than n variables in the model.35 hastie et al28 provided a thorough comparison of these shrinkage techniques . we applied lasso and alasso from calgb 90401 , a phase iii clinical trial in advanced prostate cancer , with the overall goal of building a model of overall survival.5 we had 22 variables that were common between calgb 90401 and the enthuse trial ( external data set ) .36 because of missing data in the covariates , we used methods to impute them as described by white and royston.37 the regressions estimates from the cox proportional hazards model , lasso , and alasso , are listed in table 1 . 
we considered lasso and alasso and applied both the akaike information criterion and the bayesian information criterion to choose the optimal model of overall survival . lasso and alasso selected eight and nine variables , respectively ( table 1 )  . 
figure 2 shows the solution lactate depath for alasso , and we observe that hydrogenase ( ldh ) greater than the upper limit of normal and eastern cooperative oncology group ( ecog ) performance status were selected early in the l1 path compared with the other variables . 
this is followed by visceral disease , alkaline phosphatase , albumin , hemoglobin , pain , bone metastases , and then psa ( the bayesian information criterion stopped at psa )  . 
the nal model selected the following prognostic factors : ldh greater than the upper limit of normal , ecog performance status , metastatic site ( presence of visceral disease , presence of bone metastases ) , psa , alkaline phosphatase , albumin , hemoglobin , and analgesic opioid use . we have focused on variable selection when the number of predictors is small relative to the sample size . 
in recent years , a few statistical studies extended the penalized method for the joint modeling of longitudinal data and survival outcomes.42 , 43 the general idea is to postulate the joint likelihood linking the two submodels through latent random variables and to add shrinkage operators to select xed and random effects . 
while these methods have not been implemented in oncology , the 4 2019 by american society of clinical oncology heterogeneity of treatment effect ( hte ) is another important area to consider when building prognostic models . hte is the nonrandom , explainable variability in the direction and magnitude of treatment effects for individuals within a population.44 there are different sources for hte , and hte may arise from an underlying causal mechanism , artifacts , measurements , or methods.45 , 46 one main goal of hte analyses is to predict whether a patient might benet from a treatment . 
when we turned to our prognostic model of overall survival in prostate cancer , we computed a risk score from the estimated regression coefcients and the predicted survival at 24 months using the baseline cumulative hazard . 
we observe that patient 2 had a worse predicted survival probability at 24 months than patient 1 . another important task in static predictive modeling is to construct prognostic risk groups , which can be formed on the basis of their quantiles from the estimated linear predictor . 
t , we can update the risk prediction by calculating i * ( ut ( cid : 5 ) )  . there are two general dynamic risk prediction frameworks : joint modeling and landmark analysis . 
joint modeling comprises two linked submodels , one for the longitudinal process and one for the time - to - event data , and both depend on a common set of latent random variables.54 , 55 in particular , the longitudinal data usually are modeled by a linear mixed - effects model . 
the cox regression coefcient quanties the association between the latent longitudinal process and the hazard rate at time t . the longitudinal process and the event time process are assumed to be independent given the latent random effects , and the joint likelihood can be derived . 
the model can be estimated either using a frequentist approach that attains maximum likelihood through an expectationmaximization algorithm54 , 56 - 58 or a bayesian approach that uses markov chain monte carlo to obtain posterior means.59 - 61 while assuming that the parameters are readily estimated from the observed data , the conditional probability i * ( ut ) can be computed . 
a monte carlo estimate of i * ( ut ) can be obtained by sampling the random effects and the parameters from the corresponding distributions.62 on the other hand , landmarking63 - 66 consists of a series of related cox regression models , where each one is dened at a distinct landmark time t.63 - 66 for each pair of { u , t } , a separate model is tted to the individuals who remain in the study and have not yet experienced the event of interest . 
joint modeling models the dual distribution of the longitudinal process and the failure times and hence satises the consistency conditions for dynamic prediction.68 moreover , it exploits the full information of collected data and takes into account the measurement error of the longitudinal data . 
joint modeling also takes a considerable amount of effort to estimate the parameters , and the computational cost is high because it involves complicated joint distribution and numerical integration . landmarking circumvents the aforementioned model assumptions and computational burden , but it is not a comprehensive probability model of in contrast , the longitudinal process and failure times and , as such , does not satisfy the consistency conditions . 
another major shortcoming is that landmarking only considers the patients at risk at the landmark time and does not fully explore the information . several articles have focused on the comparison of joint modeling and landmarking . 
ferrer et al71 compared the two approaches in the case of model misspecication , and they aimed for predicting competing risks of prostate cancer from psa history . dynamic risk prediction ( joint modeling ) relies on model assumptions , and hence , its performance suffers from model misspecication . 
we have previously explored whether psa decline at different landmark times is prognostic for overall survival in patients with advanced prostate cancer.79 fontein et al80 developed a dynamic model for predicting overall survival in patients with breast cancer . 
other applications of dynamic models have been implemented to prostate cancer82 - 85 and colorectal cancer.86 we demonstrated the application of dynamic risk prediction using the datatop study , 87 a clinical trial that examined the benets of deprenyl and - tocopherol in slowing the progression of parkinson disease ( pd ) .88 multiple longitudinal biomarkers were collected in the datatop study , including unied pd rating scale total score , modied hoehn and yahr scale , and schwab and england activities of daily living . 
 developing and validating risk assessment models trajectories ( fig 3a ) and risk of functional disability ( fig 3b )  . patient 169 , who had more severe disease with earlier development of functional disability , and patient 718 , who had less severe disease , were selected to illustrate the patient - specic predictions at clinically relevant future time points conditional on their available longitudinal measurements . 
 ( a ) predicted unied parkinson disease rating scale ( updrs ) trajectories and ( b ) predicted conditional failure probabilities for patient 169 ( top rows ) and patient 718 ( bottom rows )  . 
 halabi , li , and luo joint modeling , our major interest was to predict probability of functional disability after visits at time t given the patients longitudinal proles and event - free status up to time t . 
overtting occurs when a high predictive accuracy is estimated from a model that has been applied to the training set but has low accuracy when assessed in an independent data set.90 a good example of overtting is provided in halabi and owzar.2 the validation of a prognostic model is considered a critical step after a risk assessment model has been built . 
there are two types of validation : external and internal.16 , 89 , 90 external validation , where the frozen model from the training data is applied to an independent data set , is the most rigorous approach . 
of note , other types of resampling methods , such as cross - validation , bootstrapping , and bootstrapping using 0.632 + , are considered appropriate approaches to model validation.32 , 53 , 91 , 92 investigators plot assessment of the models performance usually is conducted by examining the calibration and discriminative ability of the model . 
using data from two phase iii clinical trials , we evaluated the overall survival model for calibration at different landmarks.36 , 93 figure 4a shows that the predicted survival probabilities at 18 months in the enthuse 33 trial were close to the proportion of patients who survived 18 months . 
the rst two points ( circles ) show that the model overpredicted the proportion of patients who survived 12 months , whereas the third and fth data points show that the model underpredicted the proportion of patients who survived 12 months . discrimination describes the ability of a prognostic model to distinguish between patients with and without the outcome of interest.16 several metrics are used to report the performance of a model . 
these models are anticipated to be implemented in both the design and the conduct of future trials . although static models will continue to play an important role in oncology , developing and validating dynamic models of clinical outcomes need to take a higher priority . 
one of the limitations is that modelers may be constrained by the lack of access to the longitudinal biomarker data ; therefore , the next generation of risk assessments are highly recommended to take into consideration the longitudinal biomarker data and outcomes so that predictions are updated . in summary , risk assessment will remain an important research task in precision oncology . 
calibration of the overall survival model for observed and predicted survival probability at ( a ) 18 months and ( b ) 12 months . pertinent to patient outcomes , dene the primary end point a priori , justify the sample size , and describe the appropriate methods for variable selection and model assessment . 
lastly , they should be validated using external data sets if available . an understanding of the longitudinal relationship between host and tumor - related factors and their impact on clinical outcomes is critical . 
we expect to see an upsurge in dynamic risk assessments in oncology , and as such , the american joint committee on cancer and the transparent reporting of a multivariable prediction model for individual prognosis or diagnosis guidelines should be extended to dynamic predictive modeling . 
regardless of whether static or dynamic modeling is the primary objective , we envision that this review will bridge gaps in knowledge and motivate investigators to take risk assessment as a discipline by itself . 
 halabi , li , and luo references osullivan b , brierley j , gospodarowicz m : prognosis and classication of cancer , in osullivan b , brierley jd , dcruz ak , et al : uicc manual of clinical oncology ( ed 9 )  . 
halabi s , lin cy , kelly wk , et al : updated prognostic model for predicting overall survival in rst - line chemotherapy for patients with metastatic castrationresistant prostate cancer . 
j clin oncol 32 : 671 - 677 , 2014 kelly wk , halabi s , carducci m , et al : randomized , double - blind , placebo - controlled phase iii trial comparing docetaxel and prednisone with or without bevacizumab in men with metastatic castration - resistant prostate cancer : calgb 90401 . 
morris mj , heller g , bryce ah , et al : alliance a031201 : a phase iii trial of enzalutamide ( enz ) versus enzalutamide , abiraterone , and prednisone ( enz / aap ) for metastatic castration resistant prostate cancer ( mcrpc )  . 
j clin oncol 37 , 2019 ( suppl ; abstr 5008 ) sparano ja , gray rj , makower df , et al : adjuvant chemotherapy guided by a 21 - gene expression assay in breast cancer . 
n engl j med 379 : 111 - 121 , 2018 bang yj , van cutsem e , feyereislova a , et al : trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of her2 - positive advanced gastric or gastro - oesophageal junction cancer ( toga ) : a phase 3 , open - label , randomised controlled trial . 
miller kd , oneill a , gradishar w , et al : double - blind phase iii trial of adjuvant chemotherapy with and without bevacizumab in patients with lymph nodepositive and high - risk lymph node - negative breast cancer ( e5103 )  . 
rudloff u , jacks lm , goldberg ji , et al : nomogram for predicting the risk of local recurrence after breast - conserving surgery for ductal carcinoma in situ . 
fizazi k , higano cs , nelson jb , et al : phase iii , randomized , placebo - controlled study of docetaxel in combination with zibotentan in patients with metastatic castration - resistant prostate cancer . 
halabi sp , pi l : statistical considerations for developing and validating prognostic models of clinical outcomes , in kelly do , kevin w , halabi s ( eds ) : oncology clinical trials : successful design , conduct , and analysis ( ed 2 )  . 
gabler nb , duan n , liao d , et al : dealing with heterogeneity of treatment effects : is the literature up to the challenge ? trials 10 : 43 , 2009 46 . 
dane a , spencer a , rosenkranz g , et al : subgroup analysis and interpretation for phase 3 conrmatory trials : white paper of the efspi / psi working group on 51 . 
kattan mw , hess kr , amin mb , et al : american joint committee on cancer acceptance criteria for inclusion of risk models for individualized prognosis in the practice of precision medicine . 
rizopoulos d , hateld la , carlin bp , et al : combining dynamic predictions from joint models for longitudinal and time - to - event data using bayesian model averaging . 
yao f , m uller hg , clifford aj , et al : shrinkage estimation for functional principal component scores with application to the population kinetics of plasma folate . biometrics 59 : 676 - 685 , 2003 74 . 
yan f , lin x , li r , et al : functional principal components analysis on moving time windows of longitudinal data : dynamic prediction of times to event . 
halabi s , conaway mr , small ej , et al : serum prostate specic antigen as a predictor of survival in prostate cancer patients treated with second - line hormonal 80 . 
proust - lima c , taylor jm : development and validation of a dynamic prognostic tool for prostate cancer recurrence using repeated measures of posttreatment psa : a joint modeling approach . 
s `ene m , taylor jm , dignam jj , et al : individualized dynamic prediction of prostate cancer recurrence with and without the initiation of a second treatment : development and validation . 
kr ol a , ferrer l , pignon jp , et al : joint model for left - censored longitudinal data , recurrent events and terminal event : predictive abilities of tumor burden for cancer evolution with application to the ffcd 2000 - 05 trial . 
moons kg , altman dg , reitsma jb , et al : transparent reporting of a multivariable prediction model for individual prognosis or diagnosis ( tripod ) : explanation checklist . 
petrylak dp , vogelzang nj , budnik n , et al : docetaxel and prednisone with or without lenalidomide in chemotherapy - naive patients with metastatic castrationresistant prostate cancer ( mainsail ) : a randomised , double - blind , placebo - controlled phase 3 trial . 
halabi s , pi l , lin c - y : developing and validating prognostic models of clinical outcomes , in halabi s , michiels s ( eds ) : textbook of clinical oncology : a statistical perspective . 
 c acquired ctnnb1 mutation drives immune checkpoint inhibitoracquired resistance in a microsatellite instabilityhigh gastroesophageal adenocarcinoma with brain metastases introduction the landscape of advanced gastric and esophageal adenocarcinomas continues to evolve , partly owing to improved molecular characterization identifying genomically defined subgroups that may benefit from matched therapies.1 - 3 the keynote - 059 and attraction - 2 trials have demonstrated efficacy for the antiprogrammed death - 1 ( pd - 1 ) antibodies pembrolizumab and nivolumab in gastroesophageal adenocarcinomas ( geas ) after two or more prior lines of therapy.3 , 4 the keynote - 059 study population included seven known patients with microsatellite instabilityhigh ( msi - h ) tumors , among whom the response rate was 57% . 
outside of msi - h tumors , changes in neoantigen landscape , acquired jak1 mutations , mhc - i loss , beta - 2microglobulin mutations affecting antigen presentation , and expression of alternate checkpoints have been shown to mediate acquired resistance to immune checkpoint blockade.9 - 12 with the tumor agnostic approval of pembrolizumab for msi - h tumors in 2017 , a better understanding of acquired resistance is increasingly important . 
 standard - of - care human epidermal growth factor receptor 2 immunohistochemical testing was negative , and he received first - line fluorouracil and oxaliplatin with palliative radiotherapy to a painful bone metastasis with a best response of stable disease . 
at the time of first - line progression , his liver biopsy was screened for microsatellite instability , found to be msi - h , and he was enrolled in a clinical trial of ipilimumab 1 mg / kg and nivolumab 3 mg / kg ( table 1 )  . 
after complete radiographic response of distant disease lasting 18 months , he received palliative radiotherapy ( 0.414 gy ) concurrent with nivolumab every 2 weeks for endoscopically improved , but residual , disease and remained in the trial . 
genomic alterations with preclinical and / or clinical evidence supporting resistance to immune checkpoint inhibitors are shown in bold . abbreviations : gej , gastroesophageal junction ; msi - h , microsatellite instabilityhigh ; mut , mutation ; tmb , tumor mutation burden ; wt , wild type . the resection specimen revealed persistence of an 8.5 - cm segment of poorly differentiated gej adenocarcinoma positive for lymphovascular invasion . all lymph nodes were negative , and evidence of significant treatment effect was seen ( tumor regression grade 2 ) , overall staged as ypt3ypn0 with negative margins . 
the surgical specimen was subjected to hybrid - capture comprehensive genomic profiling using a commercial assay ( foundationone , foundation medicine , cambridge , ma ) , which confirmed msi - h ( table 1 )  . 
during the treatment time encompassing stereotactic brain radiotherapy and nivolumab , washout biopsy samples were taken from a stable subcutaneous scalp metastasis , and an intramuscular rectus femoris lesion reappeared on restaging imaging ( table 1 )  . 
in an effort to explore putative resistance mechanisms after excellent durable response to ipilimumab and nivolumab , the samples were all subjected to the same hybrid - capture comprehensive genomic profiling assay ( table 1 ; fig 1 )  . 
after stereotactic brain radiotherapy , the patient went on to enroll in a third - line trial of paclitaxel in combination with the anti - dkk1 monoclonal antibody dkn - 01 ( clinicaltrials . gov identifier : nct02013154 ) and achieved stable disease lasting 4 months , ultimately , with progression in the brain and extracranial sites concurrently . 
clonal divergence and acquired ctnnb1 in a patient with microsatellite instabilityhigh ( msi - h ) gastroesophageal junction adenocarcinoma with disease progression while receiving ipilimumab in combination with nivolumab after a durable response . 
however , the sampled site of cns progression contained private alterations known or suspected to mediate immuno oncology resistance not seen at other disease sites ( fig 1 )  . 
the ctnnb1 t41a and d32y mutations are known ( t41a ) or predicted ( d32y ) stabilizing mutations that lead to t - cell exclusion and resistance to antiprogrammed death - ligand 1 therapies in preclinical models.17 although less well studied , the d32y mutation should increase stability owing to proximity to the cki and gsk3b phosphorylation sites and is a part of the recognition motif for b - trcp ( e3 ligase for b - catenin )  . 
the additional ctnnb1 n387k is not well characterized functionally , although there is some evidence for it leading to increased b - catenin activity in liver cancer.18 stabilizing or gain - of - function ctnnb1 alterations are recurrent in gea and other tumor types and may be associated with a worse outcome , although clinical immunotherapy - specific outcome data are lacking ( fig 3 ) .2 , 19 , 20 it is also possible that the blood - brain barrier may have reduced the efficacy of checkpoint blockade , allowing for the tissue - specific development of the ctnnb1 alterations we observed . 
however , because our methods are limited to dna - based assessment , we cannot exclude nongenomic mediators of acquired resistance that are known to exist.24 overall , our patient may add early support to the possibility of tissue - contextspecific acquired resistance under selective pressure of immune checkpoint inhibitors , although we cannot draw significant conclusions from a single patient . 
in an analogy to oncogene - driven gea , intertumoral heterogeneity may emerge as an acquired resistance mechanism in msi - driven gea.2 in fact , a recent correlative article from a phase ii trial of pembrolizumab in advanced gea identified intratumoral msi heterogeneity in a patient refractory to therapy.6 if further validated , our observation may have implications beyond gea and inform rational , and perhaps individualized , salvage strategies for msi - h tumors progressing while the patient is taking checkpoint inhibitor therapy . 
klempner stock and other ownership interests : tp therapeutics speakers ' bureau : foundation medicine research funding : leap therapeutics ( inst ) , astellas pharma ( inst ) travel , accommodations , expenses : eli lilly alexa b . 
ali employment : foundation medicine leadership : incysus stock and other ownership interests : exelixis , blueprint medicines , agios , genocea biosciences patents , royalties , other intellectual property : patents via foundation medicine , patents via seres health on microbiome stuff in non - neoplastic disease ( i ) charlotte d . 
kubicky consulting or advisory role : bristol - myers squibb , eisai , array biopharma , loxo , bayer , arqule , blueprint medicines , novartis speakers ' bureau : bristol - myers squibb , eisai consulting or advisory role : eli lilly , boston biomedical , astellas pharma matthew h . 
fuchs cs , doi t , jang rw , et al : safety and efficacy of pembrolizumab monotherapy in patients with previously treated advanced gastric and gastroesophageal junction cancer : phase 2 clinical keynote - 059 trial . 
kang yk , boku n , satoh t , et al : nivolumab in patients with advanced gastric or gastrooesophageal junction cancer refractory to , or intolerant of , at least two previous chemotherapy regimens ( ono - 4538 - 12 , attraction - 2 ) : a randomised , double - blind , placebo - controlled , phase 3 trial . 
janjigian yy , bendell j , calvo e , et al : checkmate - 032 study : efficacy and safety of nivolumab and nivolumab plus ipilimumab in patients with metastatic esophagogastric cancer . 
overman mj , lonardi s , wong kym , et al : durable clinical benefit with nivolumab plus instability - high metastatic in dna mismatch repair - deficient / microsatellite ipilimumab colorectal cancer . 
shitara k , zgrolu m , bang yj , et al : pembrolizumab versus paclitaxel for previously treated , advanced gastric or gastro - oesophageal junction cancer ( keynote - 061 ) : a randomised , openlabel , controlled , phase 3 trial . 
smyth ec , wotherspoon a , peckitt c , et al : mismatch repair deficiency , microsatellite instability , and survival : an exploratory analysis of the medical research council adjuvant gastric infusional chemotherapy ( magic ) trial . 
this study was performed to characterize mutagenesis and mutational target genes underlying lung carcinogenesis in patients with uip . patients and methods a cohort of 691 japanese patients with lung adenocarcinoma ( ladc ) , of whom 54 had uip and 637 did not , was studied for driver oncogene aberrations . 
2018 by american society of clinical oncology introduction smoking is a major risk factor for lung cancer , 1 and its mutagenic process and mutational targets , including kras and tp53 , have been elucidated.2 smoking is also a pathogenic factor for idiopathic pulmonary fibrosis ( ipf ) , which is pathologically diagnosed as usual interstitial pneumonia ( uip ) , 3 characterized by destruction of alveolar architecture in association with fibrosis.4 uip is a strong risk factor for lung cancer independent of smoking ( relative risk , 8.25 ) 5 and increases the cumulative incidence rate of lung cancer within 10 years to 54.7%.6 notably , deleterious germline mutations in pulmonary surfactant system genes ( pssgs ) , including nkx2 - 1 / ttf1 , sftpa1 , sftpa2 , sftpb , and sftpc , have been implicated in familial neonatal respiratory failure and fibrotic lung disease , 7 as well as in lung cancer development.8 , 9 therefore , loss of function of pssgs in morphogenesis / maintenance of alveolar structure7 might also play a role in uip and subsequent development of lung cancer in individuals with germline pssg mutations . 
however , such tumors are rarely subjected to surgical resections ( 3.1% ; 1 , 300 of 41 , 742 ) , 10 largely because of the risk of acute exacerbation of interstitial pneumonia and an associated high mortality rate.10 consequently , no comprehensive genomic analysis of uip - associated lung cancers has been published to date . 
one of the few genetic characteristics of these cancers reported previously is that egfr oncogene mutations are relatively infrequent in uip - associated lung adenocarcinomas ( ladcs ) .11 - 13 in this study , we performed comparative and comprehensive genomic characterization of ladcs with and without uip . 
ladc was chosen for comparison for several reasons : it is the most common histologic type of lung cancer and frequently develops in the lungs of patients suffering from uip11 ; the genetic aberrations responsible for carcinogenesis , represented by driver oncogene aberrations , 14 - 16 have been well documented ; and mutational signature studies17 have revealed the mutational events underlying this disease , which include dna damage / adduct formation as a result of smoking , spontaneous deamination of 5 - methylcytosine , 18 and the action of apolipoprotein b mrna editing enzyme catalytic polypeptide - like cytidine deaminases.19 to deduce the mutagenic processes and elucidate commonly affected genes , we examined driver oncogene aberrations in a cohort of 691 patients with ladc , including 54 patients who were uip positive and 637 patients who were uip negative , and performed whole - exome sequencing ( wes ) analysis in 296 ladcs ( 51 uip positive and 245 uip - negative )  . 
of those , 679 were consecutive patients who underwent surgical resection between 1997 and 2008 at the national cancer center ( ncc ) hospital ( ncch ) , tokyo , and for which snap - frozen cancerous and noncancerous lung tissues suitable for genome analysis were available in the ncc biobank . 
 surgical specimens of these patients were classified as uip negative and uip positive ( 637 and 42 cases , respectively ) on the basis of previously described pathologic criteria.20 uip positivity was defined as the presence of the uip pattern ( on the basis of the 2002 american thoracic society / european respiratory society4 and 2011 ipf criteria3 )  . 
uip - positive cases were defined as those having tumorous lesions located within or adjacent to uip lesions ; tumors discontinuous to the uip lesions were excluded ( appendix )  . 
 to increase the number of uip - positive cases , an additional 12 patients who underwent surgical resection between 2011 and 2014 at ncch or tokyo medical and dental university hospital , tokyo were also studied . 
thus , the total number of uip - positive cases was 54 ( 42 + 12 )  . all patients were diagnosed according to the seventh tnm classification of malignant tumors.21 information regarding age , sex , smoking history , and clinical stage was retrospectively collected . 
 this study was approved by the institutional review boards of both the ncc and tokyo medical and dental university . driver gene aberration screening all 691 cases were screened for egfr , kras , braf , and her2 hotspot mutations by high resolution melting analysis or target sequencing analysis using the ion ampliseq cancer hotspot panel v2 ( life technologies , rockville , md )  . 
 alk , ret , and ros1 fusions were examined by reverse transcriptase polymerase chain reaction ( rt - pcr ) or molecular counting assay as previously described.15 , 22 , 23 wes analysis wes was performed for 245 uip - negative and 51 uip - positive ladcs ( 296 cases in total )  . 
 of these , 211 of the uip - negative cases and all 51 uip - positive cases were selected from the 691 patients with ladc used for driver gene aberration screening on the basis that sufficient amounts of dna were available for comprehensive genomic profiling . 
 wes was performed for cancerous and noncancerous genomic dnas using the sureselect human all exon exome capture kit ( version 4 or 5 ; agilent technologies japan , tokyo , japan ) on the illumina hisequation 2000 platform ( illumina , san diego , ca )  . 
in this condition , 10 randomly chosen snvs and 47 indels were verified at high confidence by mass - spectrometric genotyping on a massarray system ( agena bioscience , san diego , ca ) : 10 of 10 ( 100% ) for snvs and 45 of 47 ( 95.7% ) for indels , respectively ( appendix )  . 
genes recurrently affected by somatic snv and indel mutations in their coding regions were identified by mutsigcv analysis25 according to the following criterion : q value < 0.1 after adjusting for gene length and applying the benjamini - hochberg correction . 
 more detailed information about immunohistochemistry procedures is provided in the appendix . statistical analysis statistical analyses of differences were analyzed by student t test , mann - whitney u test , pearsons 2 test , or fishers exact test . 
for association studies , clinicopathological factors and mutational signatures were coded as binary or ordinal explanatory variables , and association between these signatures and clinical background was analyzed by fishers exact test and logistic regression analysis using the jmp 8.0 software ( sas institute japan , tokyo , japan )  . 
survival analysis was performed using the kaplan - meier method , and groups were compared by log - rank test using the graphpad prism 7 software ( graphpad , la jolla , ca )  . 
this result is consistent with previous reports of patients with uip3 and patients with ladc with uip.11 - 13 baseline characteristics of patients with uip - negative and - positive ladc were similar to those of previously described asian cohorts.16 , 27 , 28 driver oncogene aberrations in uip - negative cases , approximately 50% of which were egfr mutations , were similar to those of overall ladcs in east asians.16 , 28 on the other hand , the prevalence of oncogene aberrations clearly differed between uip - positive and uip - negative cases ( p < .001 by pearsons 2 ; fig 1a ; table 1 )  . 
however , the rate of egfr mutation was markedly lower in uip - positive cases than in uip - negative cases ( 1.9% [ one of 54 ] v 49.9% [ 318 of 637 ] ; p < .001 [ fishers exact test ] ) , even in heavy smokers , who have a low egfr mutation frequency30 ( 25.3% ; 38 of 150 ; p < .001 [ fishers exact test ] )  . 
 ( % ) unless otherwise noted . abbreviations : ladc , lung adenocarcinoma ; uip , usual interstitial pneumonia . * p value was derived from the comparison between the non - uip cohort and uip - ladc by mann - whitney u test , fishers exact test , or pearsons 2 test , as appropriate . invasive mucinous adenocarcinoma and colloid , fetal , and enteric carcinoma were categorized as variants of invasive adenocarcinoma , as in travis et al.29 mutational events underlying carcinogenesis deduced by mutational signatures next , we performed a genome - wide mutational analysis to deduce the mechanisms of mutagenesis during lung carcinogenesis . 
patient characteristics and driver oncogene aberrations of the uip negative cases were similar to those of the 637 uip - negative cases described above ( appendix table a1 )  . 
the median snv rates in uip positive and - negative cases were 1.43 / mb and 0.97 / mb , respectively ( p = .0044 [ mann whitney u test ] ) , whereas the median indel mutation rates in uip - positive and - negative cases were 0.25 / mb and 0.080 / mb , respectively ( p < .001 [ mann - whitney u test ] )  . 
therefore , more mutational events , including indel mutations , occurred in the genomes of uip - positive cancers . among the 296 patients , we observed cosmic signatures 4 ( smoking associated ) , 2 ( apolipoprotein b mrna editing enzyme , catalytic polypeptide - like associated ) , 1 ( age associated ) , and 16 ( unknown etiology ; appendix fig a1 )  . 
uip - negative cases were subcategorized based on smoking status : never smoker , ever smoker , and heavy smoker ( 40 pack - years [ py ] )  . 
these findings indicated that mutational events involved in the development of uip positive ladc are largely caused by smoking . significantly mutated genes in uippositive ladc mutsigcv analysis of all 296 cases revealed frequent mutations in several genes reported in previous genome - wide mutation analysis of ladc14 : egfr , kras , tp53 , stk11 , cdkn2a , ctnnb1 , and smad4 ( appendix table a3 )  . 
mutsigcv analysis of the 51 uip - positive cases revealed that tp53 ( 20 of 51 ; 39.2% ) and kras ( 10 of 51 ; 19.6% ) were the two most frequently mutated genes ( fig 1b ; appendix table a3 )  . 
mutations in other genes reported as mutated in ladc14 were also detected in patients with uip - positive ladc , albeit at lower frequencies than tp53 , kras , and nkx2 - 1 . deleterious pssg mutations in a subset of ladc the above observation prompted us to investigate whether other pssgs are mutated in ladcs . 
we found that four other pssgs , sftpa1 , sftpa2 , sftpb , and sftpc , were mutually exclusively mutated in 10 of 296 ( 3.3% ) of all ladc cases and three of 51 ( 5.9% ) of uip - positive cases ( fig 2a )  . 
most of the mutations in these pssgs were indels in the 3untranslated regions ( utrs ; fig 2b ) , consistent with a recent report that indel mutations occur preferentially in lineage - defining genes in human cancers.32 comparative genotyping of dna and cdna isolated from cancerous and noncancerous tissues revealed that the 3 - utr indel mutant alleles of sftpa1 , sftpa2 , and sftpc were expressed at significantly lower levels than the wild - type alleles ( appendix fig a2 )  . in total , 21 of 296 ladcs ( 7.1% ) had mutations in five pssgs ( nkx2 - 1 , sftpa1 , sftpa2 , sftpb , and sftpc ) , with the mutations in individual genes occurring almost mutually exclusively . 
the prevalence of indels in all cases ( 15 of 296 ; 5.1% ) and uip - positive ladc cases ( six of 51 ; 11.8% ) indicated that pssgs are a major target for indel mutagenesis ( fig 2a )  . pssg mutations define a clinically distinct subset of ladcs next , we examined the characteristics of 21 patients with ladc with pssg mutations ( table 2 )  . 
notably , 11 cases ( 52.3% ) exhibited histology corresponding to mucinous differentiation , 29 with a frequency much higher than in ladcs overall ( 6.1% to 16%29 , 33 , 34 ; appendix table a4 ; representative cases in fig 3a )  . 
most tumors with mutations in nkx2 - 1 and all tumors with mutations in sftpa1 and sftpa2 lost expression of the corresponding proteins , whereas tumors with sftpb mutations retained surfactant protein ( sp ) - b protein expression . 
patients who were uip positive with pssg mutations had shorter median overall survival : 24 months versus 125 months for patients who were uip positive without such mutations ( p = .0080 ; fig 3b )  . 
however , no such association was observed for patients with uip - negative ladc ( appendix fig 3a and 3b )  . discussion we present here the first comprehensive comparative genome profiles , to our knowledge , of ladcs with and without uip . 
 ( % ) unless otherwise noted . abbreviations : ladc , lung adenocarcinoma ; pssg , pulmonary surfactant system gene ; uip , usual interstitial pneumonia . * p value was derived from the comparison between cases with or without pssg mutation by mann - whitney u test , fishers exact test , or pearsons 2 test , as appropriate . invasive mucinous adenocarcinoma and colloid , fetal , and enteric carcinoma were categorized as variants of invasive adenocarcinoma , as in travis et al.29 profiles distinct from those of uip - negative ladcs , characterized by infrequent egfr mutations . 
furthermore , focal genome copy number gain associated with abundant transcript expression is a mode of activation of the egfr oncogene.37 uip - positive ladcs did not exhibit significant copy - number gains at egfr , whereas several uip - positive ladcs did ( appendix fig a4a - a4c )  . 
rna sequencing of representative uip - positive cases did not reveal overexpression of egfr ( appendix fig a4d - a4f ) or oncogenic egfr fusions , which have been reported as driver events in ladc.38 these results suggested that the egfr oncogene infrequently contributes to the development of uip - positive ladc . 
ladcs in east asian individuals frequently harbor egfr mutations , both in smokers and nonsmokers.39 therefore , our findings show that uip affects the molecular mechanism underlying lung carcinogenesis by changing the relative dependence of oncogene aberrations . mutational signature analysis revealed that the mutational events involved in uip - positive ladc are largely due to smoking . 
indel mutations were also frequent in uip - positive ladcs , consistent with their prevalence in smoking associated cancers.31 importantly , our results newly identify pssgs as major targets for indel mutations . 
pssg deficiency had been implicated in lung carcinogenesis on the basis of the association between deleterious germline mutations in these genes and severe familial lung diseases , such as interstitial pneumonia and lung cancer development.7 - 9 noncancerous lung tissues of patients with germline sftpb mutation and sftpc - deficient mice develop interstitial fibrosis and alveolar type ii cell dysplasia , 40 , 41 suggesting that pssg deficiency perturbs normal pneumocytic differentiation . 
 ( a ) pathologic features of pssg - mutated lung adenocarcinoma , including invasive mucinous ( case 12 ) and acinar - predominant with a mucinous cribriform pattern ( case 14 )  . 
nk2 homeobox1 ( nkx2 - 1 ) / thyroid transcription factor 1 ( ttf1 ) and surfactant protein ( sp ) - a were detected in normal pneumocytes but were undetectable in tumors with the corresponding sftpa and / or nkx2 - 1 mutations . 
hematoxylin and eosin staining ( he , upper right ) and immunohistochemistry for nkx2 - 1 / ttf1 ( lower left ) and sp - a ( lower right ) proteins , with 3 , 3 - diaminobenzidine visualization ( magnification 200 )  . 
 ( b ) kaplan - meier curve showing overall survival ( os ) of patients with usual interstitial pneumonia ( uip ) positive ladc with pssg mutation ( mutant ) and without pssg mutation ( wild type )  . 
inflammation contributes not only to tumor development but also to tumor progression , by increasing cell invasion and expanding the fraction of undifferentiated cells.43 , 44 however , pulmonary surfactant proteins play important roles in epithelial integrity and cell differentiation in the lung.7 , 45 therefore , deficiency in pulmonary surfactant protein caused by pssg mutations might increase inf lammation - driven tumor progression by disturbing epithelial integrity / differentiation . 
 pssg mutations were more likely to be present in ladcs of smokers and in egfr mutation negative ladcs , which are both associated with poor prognosis.46 , 47 in addition , the number of cases with pssg mutations was small . 
the roles of aberrations of minor driver oncogenes and pssgs should also be studied in a larger cohort , including patients with squamous cell carcinoma , to more fully elucidate lung carcinogenesis associated with uip . 
the 3 - utrs of genes play regulatory roles in the stability and abundance of mrna , and oncogenic mutations have been reported in the 3 - utrs of kras and tp53 genes.48 , 49 accordingly , functional analysis of the 3 - utr mutations in sftps is warranted . in summary , we elucidated the genomic profile of uip - ladcs and observed a predominance of smoking - related mutations in these cancers . 
 kyohei masai no relationship to disclose hirohiko totsuka employment : stagen noriko motoi honoraria : agilent , msd , novartis , astrazeneca , bristolmyers squibb japan consulting or advisory role : bristol - myers squibb , miraca life sciences , astrazeneca , chugai pharmaceuticals , research funding : roche ( inst ) yoko shimada no relationship to disclose ayaka otsuka no relationship to disclose yae kanai no relationship to disclose kenichi okubo no relationship to disclose shun - ichi watanabe no relationship to disclose masashi kobayashi no relationship to disclose takumi akashi no relationship to disclose sachiyo mimaki no relationship to disclose katsuya tsuchihara no relationship to disclose suenori chiku no relationship to disclose kouya shiraishi no relationship to disclose support references 2005 koji tsuta speakers ' bureau : msd , roche naohiko inase no relationship to disclose takashi kohno research funding : daiichi sankyo , sysmex acknowledgment we thank y . 
sakaguchi for excellent technical assistance with immunohistochemistry . affiliations takayuki honda , kouya shiraishi , yoko shimada , ayaka otsuka , and takashi kohno , national cancer center research institute , chuo - ku ; takayuki honda , hiroyuki sakashita , masashi kobayashi , takumi akashi , kenichi okubo , and naohiko inase , tokyo medical and dental university , bunkyo - ku ; kyohei masai , noriko motoi , and shun - ichi watanabe , national cancer center hospital , chuo - ku ; kyohei masai and yae kanai , keio university school of medicine , sinjuku - ku ; hirohiko totsuka , stagen , taito - ku ; suenori chiku , mizuho information and research institute , chiyoda - ku , tokyo ; sachiyo mimaki and katsuya tsuchihara , epoc , national cancer center , kashiwa , chiba ; and koji tsuta , kansai medical university , hirakata , osaka , japan . supported by the japan agency for medical research and development grant no . 
raghu g , collard hr , egan jj , et al : an official ats / ers / jrs / alat statement : idiopathic pulmonary fibrosis : evidence - based guidelines for diagnosis and management . 
fujimoto d , tomii k , otoshi t , et al : preexisting interstitial lung disease is inversely correlated to tumor epidermal growth factor receptor mutation in patients with lung adenocarcinoma . 
masai k , tsuta k , motoi n , et al : clinicopathological , immunohistochemical , and genetic features of primary lung adenocarcinoma occurring in the setting of usual interstitial pneumonia pattern . 
goldstraw p , crowley j , chansky k , et al : the iaslc lung cancer staging project : proposals for the revision of the tnm stage groupings in the forthcoming ( seventh ) edition of the tnm classification of malignant tumours . 
sunami k , furuta k , tsuta k , et al : multiplex diagnosis of oncogenic fusion and met exon skipping by molecular counting using formalin - fixed paraffin embedded lung adenocarcinoma tissues . 
travis wd , brambilla e , noguchi m , et al : international association for the study of lung cancer / american thoracic society / european respiratory society international multidisciplinary classification of lung adenocarcinoma . 
yatabe y , koga t , mitsudomi t , et al : ck20 expression , cdx2 expression , k - ras mutation , and goblet cell morphology in a subset of lung adenocarcinomas . 
tang x , kadara h , behrens c , et al : abnormalities of the titf - 1 lineage - specific oncogene in nsclc : implications in lung cancer pathogenesis and prognosis . 
hirsch fr , varella - garcia m , bunn pa jr , et al : epidermal growth factor receptor in non - smallcell lung carcinomas : correlation between gene copy number and protein expression and impact on prognosis . 
wallot m , wagenvoort c , demello d , et al : congenital alveolar proteinosis caused by a novel mutation of the surfactant protein b gene and misalignment of lung vessels in consanguineous kindred infants . 
oshima h , nakayama m , han t - s , et al : suppressing tgf signaling in regenerating epithelia in an inflammatory microenvironment is sufficient to cause invasive intestinal cancer . 
fujisawa t , iizasa t , saitoh y , et al : smoking before surgery predicts poor long - term survival in patients with stage i non - small - cell lung carcinomas . 
takano t , fukui t , ohe y , et al : egfr mutations predict survival benefit from gefitinib in patients with advanced lung adenocarcinoma : a historical comparison of patients treated before and after gefitinib approval in japan . 
 appendix histologic diagnosis surgically resected specimens ( ie , tissues remaining after snap - frozen tissues were set aside ) were formalin fixed by transbronchial or transpleural injection ( in an inflated state ) , cut serially into 5 - mmthick sections , and macroscopically examined . 
the reads were aligned to the reference human genome ( university of california , santa cruz , human genome 19 ; hg19 ) with the burrows wheeler aligner multi - vision software package . 
because duplicate reads were generated during the polymerase chain reaction ( pcr ) amplification process , duplicated paired - end reads that aligned to the same genomic positions were marked using picard . 
depth of coverage at capture targets in tumor samples was counted using read coverage spanning a target segment with the total number of aligned reads and proportionally calibrated to estimate the copy ratio using depths observed in a panel of normal ( noncancer ) diploid genomes . 
thresholds were as follows : board length cutoff , 0.5 chromosome arms ; confidence interval , 90% . validation of candidate mutations using mass - spectrometric genotyping validation and quantification of candidate mutations were performed by mass - spectrometric ( ms ) genotyping as previously described ( su ky , et al : j clin oncol 30 : 433 - 440 , 2012 ) according to the user instructions for the massarray system ( agena bioscience , san diego , ca )  . 
extracted dna ( 10 ng ) was amplified using specific primers ; unincorporated nucleotides were inactivated by shrimp alkaline phosphatase ; a single - base extension reaction was performed using extension primers that hybridized immediately adjacent to the mutations . 
salts were removed by addition of cation exchange resafter cleanup with spectroclean resin , samples were loaded onto the spectrochip matrix using a nanodispenser and then analyzed by matrix - assisted laser desorption ionizationtime - of - flight mass spectrometry on a bruker autoflex instrument . 
 mutational signature analysis mutational signature in this study was analyzed using nonnegative matrix factorization ( nmf ) , which was applied to the 96 possible substitutions in a trinucleotide context including the bases 5 and 3 of each substitution site , as previously described.17 , 26 nmf was applied to the 96 - substitution pattern using published software from the wellcome trust sanger institute.9 at least 2 , 400 iterations of nmf were run , and each nmf run was iterated to convergence ( 10 , 000 iterations without change ) or until 1 million iterations were performed . 
nmf runs with various numbers of signatures were tested , from one to 10 ; four was best , because signature stability remained high and the reconstruction error converged at a low value ( appendix fig a2a )  . 
 one microgram of total rna was reverse - transcribed using superscript iii first - strand synthesis system for reverse transcriptase ( rt ) pcr ( thermo fisher scientific , waltham , ma ) after dnase treatment . 
ten nanograms of genomic dna and cdna corresponding to 50 ng of rna were prepared from both cancerous and noncancerous tissues . for ms genotyping , the experimental procedures described above were applied to both genomic dna and cdna using the same primer , and mutant allele frequency ( represented as peak heights ) was calculated using the typer4 software ( agena bioscience )  . for fragment analysis , regions including mutations were amplified by pcr from genomic dna and cdna using the same primers according to the manufacturers instructions ( applied biosystems , foster city , ca )  . 
pcrs were performed in triplicate with the following conditions : 35 cycles of denaturation at 95c for 1 minute , annealing at 60c for 1 minute , and extension at 72c for 1 minute . 
mutant allele frequency ( represented as peak heights ) was calculated using the abi genemapper software ( applied biosystems )  . differences in the sftpa1 , sftpa2 , and sftpc gene expression between cancerous and noncancerous tissue were analyzed using quantitative rt - pcr . 
triplicate samples were used for all experiments described in this paragraph . rna sequencing rna - sequencing libraries were prepared from 200 ng of total rna using the truseq rna sample prep kit ( illumina )  . 
the resultant libraries were subjected to paired - end sequencing of 75 - bp reads on a hisequation 2000 ( illumina ) as previously described ( nakaoku t , et al : clin cancer res 20 : 3087 - 3093 , 2014 )  . 
egfr fusion transcripts were identified using the tophat - fusion algorithm ( kim d , et al : genome biol 12 : r72 , 2011 ) .12 expression values were normalized against the corresponding levels of a housekeeping gene ( tbp , encoding tata - binding protein ) .19 immunohistochemistry studies immunohistochemistry studies were performed on 4 - mthick , formalin - fixed , paraffin - embedded tissue sections derived from representative tumor blocks . 
3 , 3 - diaminobenzidine was used as the chromogen , and hematoxylin was used as the counterstareactions that labeled at least 5% of cells were considered positive for thyroid transcription factor 1 ( nuclear )  . 
 ( a ) mass - spectrometric genotyping of genomic dna and cdna from cancerous tissues with sftp - 3 untranslated region ( utr ) indel mutation ( mut ) and noncancerous tissue ( wt )  . 
 ( a ) kaplan - meier curve of overall survival ( os ) for patients with usual interstitial pneumonianegative lung adenocarcinoma with pulmonary surfactant system gene ( pssg ) mutation ( mutant ) and without pssg mutation ( wild type )  . 
 ( a ) copy - number analysis of usual interstitial pneumonia ( uip ) positive lung adenocarcinomas ( ladcs ; n = 51 ) in comparison with uip - negative ladcs ( n = 245 ) ; ( b ) uip - negative ladcs with egfr mutation ( n = 130 ) ; and ( c ) uip - negative ladcs without egfr mutation ( n = 115 )  . 
 ( d ) expression levels of egfr in three uip - positive ladcs in comparison with uip - negative ladcs ( n = 138 ) ; ( e ) uip - negative ladcs with egfr mutation ( n = 35 ) ; and ( f ) uip - negative ladcs without egfr mutation ( n = 103 )  . 
expression levels are shown as fold changes in fragments per kilobase of exon per milllion reads mapped , normalized against the gene encoding tata - binding protein ( tbp )  . 
 o programmed cell death 1 ( pd - 1 ) ligand ( pd - l1 ) expression in solid tumors as a predictive biomarker of benefit from pd - 1 / pd - l1 axis inhibitors : a systematic review and meta - analysis monica khunger adrian v . 
pennell james stevenson sanjay mukhopadhyay kurt schalper vamsidhar velcheti monica khunger , sagar rakshit , sanjay mukhopadhyay , cleveland clinic ; adrian v . hernandez , university of connecticut / hartford hospital , universidad peruana de ciencias aplicades ; vinay pasupuleti , case western reserve university school of medicine ; nathan a . pennell , james stevenson and vamsidhar velcheti , taussig cancer center , cleveland , oh ; and kurt schalper , yale university school of medicine , new haven , ct . corresponding author : vamsidhar velcheti , md , facp , center for immuno - oncology research , cleveland clinic , cleveland , oh 44195 ; e - mail : velchev@ ccf.org. purpose drugs targeting the programmed cell death 1 ( pd - 1 ) / programmed cell death ligand 1 ( pd - l1 ) pathway show significant clinical activity across several tumor types . 
use of biomarker assays to predict response to these agents is an active area of research ; however , the predictive value of pd - l1 immunohistochemistry ( ihc ) assays is largely inconsistent across clinical trials . 
in this meta - analysis of clinical trials of pd - 1 / pd - l1targeted agents , we evaluate the predictive value of a tumor and tumor - infiltrating immune cell pd - l1 ihc assay as a biomarker for objective response to pd - 1 / pd - l1 inhibitors . methods we searched databases ( pubmed , medline , asco abstracts , european society for medical oncology abstracts , and scopus ) up until december 2016 for clinical trials using pd - 1 / pd - l1 inhibitors with reported pd - l1 biomarker data . 
objective response rates ( primary end point ) from all phase i to iii trials investigating nivolumab , pembrolizumab , atezolizumab , durvalumab , and avelumab in advanced solid tumors were collected . 
odds ratios ( ors ) for response in pd - l1positive patients compared with pd - l1negative patients were calculated using the dersimonian - laird random effects model to combine trials . 
we performed metaanalysis as per the preferred reporting items for systematic reviews and meta - analyses guidelines . results forty - one distinct trials with 6 , 664 patients were identified . 
2017 by american society of clinical oncology introduction tumors often evade the immune system by either ineffective antigen presentation or by using mechanisms to thwart an effector response.1 - 3 programmed cell death 1 ( pd - 1 ) is a negative regulator or checkpoint receptor on t cells that is a key determinant of induction of immune tolerance in the tumor microenvironment . 
pd - l1 and pd - l2 expression in the normal tissues is a major mechanism of physiologic peripheral immune tolerance to dampen tissue autoimmune responses after a sustained inflammatory response to tissue damage . 
pdl1 is upregulated in various tumor types including melanoma , nonsmall - cell lung cancer ( nsclc ) , and squamous cell head and neck carcinomas and is a major mechanism of immune evasion . 
in early trials with these agents , pd - l1 expression on tumor cells ( tcs ) , measured by immunohistochemistry ( ihc ) assay , was thought to be an important biomarker for predicting response to these agents.4 however , subsequent trials , particularly atezolizuincorporated both tc and tumormab trials , infiltrating immune cell ( ic ) pd - l1 expression , and this combined pd - l1 expression was also associated with significantly higher objective response rates ( orrs ) in clinical trials . 
pharmaceutical companies have developed different pd - l1 ihc assays , and their comparability and equivalence are the subject of active research.5 , 6 however , most such studies have included relatively small numbers of patients and have not used response or outcome as an end point . 
the databases searched included pubmed , medline , embase , asco abstracts , european society for medical oncology abstracts , google scholar , and scopus . the dates searched were from the inception of each database to december 4 , 2016 . 
the search terms included the following keywords : pd - 1 , pd - l1 , cd274 , programmed cell death receptor 1 , programmed cell death receptor ligand , immune checkpoint inhibitor , nivolumab , bms936558 , pembrolizumab , mk - 3475 , mpdl3280a , atezolizumab , avelumab , msb0010718c , durvalumab , medi - 4736 , predictive biomarker , and cancer immunotherapy . 
the following information was extracted : study design , study population and setting , mean patient age , type of cancer , type of treatment , pd - l1 expression cutoff values , and follow - up time . 
pd - l1 clones used for the evaluation of pd - l1 expression varied across different trials ; 16 studies used 28 - 8 , seven studies used 22c3 , 10 studies used sp142 , three studies used sp263 , and five studies used dako clone 73 - 10 ( dako , carpinteria , ca )  . 
among all of the included studies , 10 were randomized clinical trials , whereas the rest were single - arm trials . data synthesis and analysis study quality analysis some degree of heterogeneity was expected , and random - effects meta - analyses were performed using dersimonian - laird random effects models.10 to consider the sources of heterogeneity , the following two subgroup meta - analyses were prespecified : type of cancer and type of pd - l1 ihc assay . we used the inverse variance method to calculate pooled odds ratios ( ors ) and their 95% cis . 
i2 values of 30% to 60% represented a moderate level of heterogeneity . we used review manager ( revman 5.3 ; cochrane collaboration , copenhagen , denmark ) for our statistical analyses . results eligible studies our search retrieved 3 , 542 publications . 
the majority of the studies had high risk of bias on performance bias and detection bias . meta - analyses of the association between pd - l1 expression and orr no evidence of publication bias was observed in the funnel plot ( eggers test p = .4 ; appendix fig a2 , online only )  . 
there was low heterogeneity of effects across studies in all categories ( fig 2 )  . we performed a subgroup analysis across all nsclc trials simultaneously assessing cutoff values of 1% , 5% , and 10% for pd - l1positive tcs to identify the optimal ihc cutoff threshold . this analysis included the following four trials : brahmer et al , 14 borghaei et al , 15 rizvi et al , 17 and gettinger et al.20 all of these trials included patients with nsclc ( both squamous and nonsquamous histology ) treated with nivolumab and used the dako 28 - 8 antibody for pd - l1 ihc . 
there was limited heterogeneity of effects across studies in all subgroups ( figs 3a - c )  . an additional subgroup analysis was performed across different pd - l1 ihc assays ( fig 4 )  . 
forest plots representing odds ratios of objective response in programmed cell death ligand 1 ( pd - l1 ) positive patients compared with pd - l1negative patients across different cancer types . 
however , because of the paucity of data and power limitations , we could not compare tc and ic subgroups separately . a major objective of our study was to identify the optimal pd - l1 cutoff value associated with maximum objective response . 
this was achieved by performing a subgroup analysis of four trials simultaneously reporting objective response data for three different cut points ( 1% , 5% , and 10% ) in nsclc.14 , 15 , 17 , 20 we restricted the analysis to these four trials to remove heterogeneity associated with tumor type , treatment agents , and pdl1 antibody used for ihc assay . 
all four trials assessed patients with nsclc ( both squamous and nonsquamous histology ) treated with nivolumab and used the 28 - 8 ( dako ) clone for pd - l1 ihc . 
unfortunately , because of power limitations , we were unable to assess the optimal ihc cutoff points of the us food and drug administrationapproved pd - l1 antibodies 22c3 and sp142 . 
these issues may be significant and require additional study . a standardized biomarker such as tc and ic pdl1 expression can identify patients who will derive maximum benefit from these novel agents , sparing others from potentially harmful immune - related toxicities and elevated costs . 
 a vexing issue is the finding that , in some studies , patients respond to pd - 1 / pd - l1 axis inhibitors despite negative tumor pd - l1 expression . 
this assumes special importance in the current scenario of synergistic treatment combinations to enhance therapeutic response to antipd - 1 therapy . some other limitations to using tumor pd - l1 as a standardized biomarker include different collection times of the pathology specimen and the dynamic nature of the tumor microenvironment . 
pd - l1 staining in most cases is performed on archival formalin - fixed , paraffinembedded tissue , because fresh biopsies just before or close to treatment are often not available . 
it has been suggested that previous lines of therapy might alter tumor pd - l1 expression and may explain some of the confounding results across different trials.11 however , we believe this may not have been a significant confounder in our meta - analysis because , more recently , pd - 1 / pd - l1 inhibitor trials have included fresh biopsy specimens ( within 6 months of starting treatment ) , especially many of the recent trials with pd - 1 / pd - l1 inhibitors in the front - line setting.58 tumor pd - l1 expression  . 
the response rate and progressionfree survival ( primary end point of the study ) were higher in the pembrolizumab group than in the chemotherapy group ( orr , 44.8% in pembrolizumab arm v 27.8% in chemotherapy arm )  . 
this is in stark contrast to the results of the checkmate - 26 trial ( nivolumab v platinum - based chemotherapy in the front - line setting ) , which included treatment - nave patients with advanced nsclc and allowed a more liberal inclusion criterion of  . 
these studies further highlight the importance of incorporating pd - l1 expression into future trials of pd - 1 / pd - l1 moabs , especially in the front - line setting where patients have multiple treatment options . we used objective response as the primary end point of our analysis to ensure inclusion of maximum patients , because mature survival data were not available for many of the recent trials . 
for instance , in the checkmate 17 , checkmate 57 , poplar , and oak trials , objective response did not improve as much as the os in the pd - 1 / pd - l1 arm , suggesting postprogression survival advantage.14 , 15 , 49 , 50 however , this should not significantly affect our aim of establishing the validity of pd - l1 expression as a biomarker because orr is commonly used as a surrogate biomarker for clinical benefit in oncology . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . vinay pasupuleti no relationship to disclose sagar rakshit no relationship to disclose nathan a . 
pennell consulting or advisory role : boehringer ingelheim , astrazeneca , eli lilly , regeneron research funding : genentech ( inst ) , newlink genetics ( inst ) , clovis oncology ( inst ) , astex pharmaceuticals ( inst ) , celgene ( inst ) , astrazeneca ( inst ) , pfizer ( inst ) , merck james stevenson research funding : merck sharp & dohme ( inst ) , bayer ( inst ) , bristol - myers squibb ( inst ) sanjay mukhopadhyay research funding : philips healthcare other relationship : phillips kurt schalper honoraria : takeda research funding : vasculox , tesaro , onkaido therapeutics , genoptix , takeda vamsidhar velcheti honoraria : novartis , foundation medicine , merck , bristolmyers squibb , genentech consulting or advisory role : clovis oncology , genentech , bristol - myers squibb , merck , celgene , foundation medicine , astrazeneca / medimmune , genoptix research funding : genentech ( inst ) , trovagene ( inst ) , eisai ( inst ) , oncoplex diagnostics ( inst ) , alkermes ( inst ) , nantomics ( inst ) , genoptix ( inst ) , altor bioscience ( inst ) , merck ( inst ) , bristol - myers squibb ( inst ) , atreca ( inst ) , heat biologics ( inst ) , leap therapeutics ( inst ) travel , accommodations , expenses : astrazeneca / medimmune , eisai 1 . 
hirsch f , mcelhinny a , stanforth d , et al : pd - l1 immunohistochemistry assays for lung cancer : results from phase 1 of the blueprint pd - l1 assay comparison project . 
gettinger sn , horn l , gandhi l , et al : overall survival and long - term safety of nivolumab ( anti - programmed death 1 antibody , bms - 936558 , ono - 4538 ) in patients with previously treated advanced nonsmall - cell lung cancer . 
borghaei h , paz - ares l , horn l , et al : nivolumab versus docetaxel in advanced nonsquamous nonsmall - cell lung 2018 - 2028 , 2015 in cancer patients . 
antonia s , kim s , spira a , et al : safety and clinical activity of durvalumab ( medi4736 ) , an anti - pd - l1 antibody , in treatment - nave patients with advanced nonsmall - cell lung cancer . 
rizvi na , mazi`eres j , planchard d , et al : activity and safety of nivolumab , an anti - pd - 1 immune checkpoint inhibitor , for patients with advanced , refractory squamous nonsmall - cell lung cancer ( checkmate 063 ) : a phase 2 , single - arm trial . 
verschraegen c , chen f , spigel d , et al : avelumab ( msb0010718c ; anti - pd - l1 ) as a first - line treatment for patients with advanced nsclc from the javelin solid tumor phase 1b trial : safety , clinical activity , and pd - l1 expression . j clin oncol 34 , 2016 ( suppl ; abstr 9036 ) 19 . 
herbst rs , baas p , kim d - w , et al : pembrolizumab versus docetaxel for previously treated , pd - l1 - positive , advanced nonsmall - cell lung cancer ( keynote - 010 ) : a randomised controlled trial . 
weber js , dangelo sp , minor d , et al : nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti - ctla - 4 treatment ( checkmate 037 ) : a randomised , controlled , open - label , phase 3 trial . lancet oncol 16 : 375 - 384 , 2015 22 . 
larkin j , chiarion - sileni v , gonzalez r , et al : combined nivolumab and ipilimumab or monotherapy in untreated med 372 : 320 - 330 , 2015 melanoma . 
hamid o , sosman ja , lawrence dp , et al : clinical activity , safety , and biomarkers of mpdl3280a , an engineered pd - l1 antibody in patients with locally advanced or metastatic melanoma ( mm )  . 
hodi fs , kluger h , sznol m , et al : long - term survival of ipilimumab - nave patients ( pts ) with advanced melanoma ( mel ) treated with nivolumab ( anti - pd - 1 ; bms - 936558 , ono - 4538 ) in a phase 1 trial . 
ribas a , puzanov i , dummer r , et al : pembrolizumab versus investigator - choice chemotherapy for ipilimumabrefractory melanoma ( keynote - 002 ) : a randomised , controlled , phase 2 trial . 
daud ai , wolchok jd , robert c , et al : programmed death - ligand 1 expression and response to the anti - programmed death 1 antibody pembrolizumab in melanoma . 
topalian sl , hodi fs , brahmer jr , et al : safety , activity , and immune correlates of anti - pd - 1 antibody in cancer . 2521 - 2532 , 2015 n engl j med 366 : 2443 - 2454 , 2012 30 . 
choueiri tk , fishman mn , escudier bj , et al : immunomodulatory activity of nivolumab in previously treated and untreated metastatic renal cell carcinoma ( mrcc ) : biomarker - based results from a randomized clinical trial . 
mcdermott df , sosman ja , sznol m , et al : atezolizumab , an antiprogrammed death - ligand 1 antibody , in metastatic renal cell carcinoma : long - term safety , clinical activity , and immune correlates from a phase ia study . 
plimack e , bellmunt j , gupta s , et al : pembrolizumab ( mk - 3475 ) for advanced urothelial cancer : updated results and biomarker analysis from keynote - 012 . 
apolo ab , infante jr , hamid o , et al : safety , clinical activity , and pd - l1 expression of avelumab ( msb0010718c ) , an anti - pd - l1 antibody , in patients with metastatic urothelial carcinoma from the javelin solid tumor phase ib trial . 
massard c , gordon ms , sharma s , et al : safety and efficacy of durvalumab ( medi4736 ) , an anti - programmed cell death ligand - 1 immune checkpoint inhibitor , in patients with advanced urothelial bladder cancer . 
rosenberg je , hoffman - censits j , powles t , et al : atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum - based chemotherapy : a single - arm , multicentre , phase 2 trial . 
balar av , galsky md , rosenberg je , et al : atezolizumab as first - line treatment in cisplatin - ineligible patients with locally advanced and metastatic urothelial carcinoma : a single - arm , multicentre , phase 2 trial . 
sharma p , callahan mk , bono p , et al : nivolumab monotherapy in recurrent metastatic urothelial carcinoma ( checkmate 032 ) : a multicentre , open - label , two - stage , multi - arm , phase 1 / 2 trial . 
segal n , ou s , balmanoukian a , et al : safety and efficacy of medi4736 , an anti - pd - l1 antibody , in patients from a squamous cell carcinoma of the head and neck ( scchn ) expansion cohort . 
le dt , bendell jc , calvo e , et al : safety and activity of nivolumab monotherapy in advanced and metastatic ( a / m ) gastric or gastroesophageal junction cancer ( gc / gec ) : results from the checkmate - 032 study j clin oncol 34 , 2016 ( suppl 4s ; abstr 6 ) 43 . 
nishina t , shitara k , iwasa s , et al : safety , pd - l1 expression , and clinical activity of avelumab ( msb0010718c ) , an anti - pd - l1 antibody , in japanese patients with advanced gastric or gastroesophageal junction cancer . 
chung hc , arkenau h - t , wyrwicz l , et al : safety , pd - l1 expression , and clinical activity of avelumab ( msb0010718c ) , an anti - pd - l1 antibody , in patients with advanced gastric or gastroesophageal junction cancer . 
kaufman hl , russell j , hamid o , et al : avelumab in patients with chemotherapy - refractory metastatic merkel cell carcinoma : a multicentre , single - group , open - label , phase 2 trial . 
nghiem pt , bhatia s , lipson ej , et al : pd - 1 blockade with pembrolizumab in advanced merkel - cell carcinoma . n engl j med 374 : 2542 - 2552 , 2016 47 . 
antonia sj , l opez - martin ja , bendell j , et al : nivolumab alone and nivolumab plus ipilimumab in recurrent small - cell lung cancer ( checkmate 032 ) : a multicentre , open - label , phase 1 / 2 trial . 
wakelee h , patel jd , heist r , et al : phase ii trial of atezolizumab for patients with pd - l1 - selected advanced nsclc ( birch ) : updated efficacy and exploratory biomarker results : topic : medical oncology . 
rittmeyer a , barlesi f , waterkamp d , et al : atezolizumab versus docetaxel in patients with previously treated nonsmall - cell lung cancer ( oak ) : a phase 3 , open - label , multicentre randomised controlled trial . 
fehrenbacher l , spira a , ballinger m , et al : atezolizumab versus docetaxel for patients with previously treated nonsmall - cell lung cancer ( poplar ) : a multicentre , open - label , phase 2 randomised controlled trial . 
spiegel dr , chaft je , gettinger sn , et al : clinical activity and safety from a phase ii study ( fir ) of mpdl3280a ( anti - pdl1 ) in pd - l1selected patients with non - small cell lung cancer ( nsclc )  . 
carbognin l , pilotto s , milella m , et al : differential activity of nivolumab , pembrolizumab and mpdl3280a according to the tumor expression of programmed death - ligand - 1 ( pd - l1 ) : sensitivity analysis of trials in melanoma , lung and genitourinary cancers . 
aguiar pn jr , santoro il , tadokoro h , et al : the role of pd - l1 expression as a predictive biomarker in advanced nonsmall - cell lung cancer : a network meta - analysis . 
socinski m , creelan b , horn l , et al : nsclc , metastaticcheckmate 026 : a phase 3 trial of nivolumab vs investigators choice ( ic ) of platinum - based doublet chemotherapy ( pt - dc ) as first - line therapy for stage iv / recurrent programmed death ligand 1 ( pd - l1 ) - positive nsclc . 
 efcacy of lorlatinib in primary crizotinib - resistant adult neuroblastoma harboring alk y1278s mutation antoine vasseur , md1 ; luc cabel , md1 ; romain geiss , md1 ; gudrun schleiermacher , md , phd1 ; ga elle pierron , phd1 ; maud kamal , phd1 ; nina jehanno , md1 ; guillaume bataillon , md1 ; jean - marc guinebretiere , md1 ; and laurence bozec , md1 introduction anaplastic lymphoma kinase ( alk ) encodes a highly conserved receptor tyrosine kinase described as an oncogene in anaplastic large - cell lymphoma and other cancers.1 alk fusions ( mostly alk - eml4 ) represent 3% to 7% of all nonsmall - cell lung cancers ( nsclcs ) , and these tumors are generally sensitive to crizotinib ( a tyrosine kinase inhibitor [ tki ] targeting alk ) as rstline therapy in the metastatic setting.2 neuroblastoma harbors activating somatic alk mutations in 8% to 9% of patients ( up to 14% of high - risk neuroblastomas ) , with mutations occurring in the tyrosine kinase domain at three key positions ( f1174 , f1245 , and r1275 ) , which account for approximately 85% of all alk mutations in neuroblastoma.3 activation of alk may also occur by genomic amplication in 3% to 4% of patients , and it has been suggested that patients with neuroblastoma who have genetic alterations of alk could also be treated with crizotinib.4 in this article , we report , to our knowledge for the rst time , the case of an adult patient with ganglioneuroblastoma harboring a somatic alk y1278s mutation with primary resistance to crizotinib , but which was highly sensitive to lorlatinib , a third - generation tki targeting alk genomic alterations . 
computed tomography ( ct ) the chest , abdomen , and pelvis showed scan of disseminated lymph node and vertebral bone lesions . 123i - metaiodobenzylguanidine ( 123i - mibg ) scintigraphy was positive with bone and lymph node metastases . 
laboratory tests showed normal lactate dehydrogenase levels and increased urinary catecholamines ( normetanephrine , 40 , 825 nmol / l ; metanephrine , 572 nmol / l )  . the patient received rst - line induction chemotherapy according to the rapid cisplatin , vincristine , carboplatin , etoposide , and cyclophosphamide ( cojec ) regimen.5 after eight cycles , stable disease was observed in lymph nodes , and progression was observed in bone lesions . 
four months after chemotherapy , clinical disease progression was observed with bone pain , and urinary catecholamines were increased ( normetanephrine , 104 , 651 nmol / l ; metanephrine , 1 , 208 nmol / l ) despite stable mibg scintigraphy and ct scan . a lymphadenectomy ( left subclavicular lymph node ) was performed before second - line therapy with topotecan and cyclophosphamide was started for complementary molecular and immunohistochemistry analyses . immunohistochemistry revealed expression of alk by 100% of tumor cells . 
targeted next - generation in alk , sequencing found a pathogenic variant ptyr1278ser , with an allelic frequency of 23% ( fig 1 )  . the patient was included in the french acs e protocol ( nct02034981 ; phase 2 study assessing efcacy and safety of crizotinib in patients harboring an alteration on alk , met or ros1 ) , and crizotinib was initiated in june 2017 ( 250 mg taken orally twice per day )  . 
treatment was stopped in october 2017 after rapid progression of symptomatic bone metastasis conrmed by mibg scintigraphy , with marked general physical deterioration ( eastern cooperative oncology group [ ecog ] performance status 3 ) , asthenia , anorexia , and weight loss . 
lorlatinib was then obtained from a pzer compassionate - use program and was initiated ( 100 mg taken orally once per day ) in november 2017 , with a signicant clinical benet after 2 weeks of treatment . after 2 months , the patient was asymptomatic with marked improvement of performance status ( from ecog 3 to ecog 0 )  . 
alk positivity by ( a ) immunochemistry and ( b ) next - generation sequencing . partial reossication of bone metastases in favor of treatment response ( left subclavicular lymph node , largest lymph node target lesion ; fig 2 )  . 
after 10 months , 123i - mibg scintigraphy re - evaluation of treatment showed a good partial metabolic response of the left supraclavicular lymph node inltration as seen on the single photon emission computed tomography scan associated with partial response of bone inltration . 
international society of pediatric oncology europe neuroblastoma group ( siopen ) score for bone extension6 , 7 was 30 on baseline scintigraphy , which decreased to a siopen score of 15 on the re - evaluation scintigraphy ( 50% decrease ; relative mibg score of 0.5 ; fig 3 )  . 
overall response according to the international neuroblastoma response criteria consensus was assessed as a partial response.8 after 12 months ( last evaluation in november 2018 ) , the clinical benet was maintained ( ct scan showed a partial reduction of 33% in tumor size [ recist1.1 ] corresponding to the nadir ; left subclavicular lymph node ; fig 2 )  . 
no toxicity was reported during treatment with lorlatinib except for grade 1 hypertriglyceridemia and grade 1 edema of the lower limbs ( common terminology criteria for adverse events [ ctcae ] v5 )  . discussion to our this case report of adult neuroblastoma shows , knowledge for the rst time , that lorlatinib , unlike crizotinib , can be effective in patients harboring an alk y1278s mutation . 
the most common resistance mutations after crizotinib therapy are the l1196m ( 5% to 10% ) and g1269a ( , 5% ) mutations , 9 and many other mutations have also been reported . 
these mutations confer various mechanisms of resistance , such as catalytic domain alteration , which causes atp competitive inhibitor resistance , as t790m mutation in the epidermal growth factor receptor ( egfr ) mutation alters the conformation and / or the atp - binding afnity of the kinase.10 lorlatinib , a third - generation alk inhibitor that also targets ros1 , is an attractive tki in patients with nsclc pretreated with rstor second - generation alk inhibitors because of its activity against most alk resistance mutations , such as g1202r mutations11 ( fig 4b )  . 
lorlatinib has demonstrated high clinical activity in alkor ros1 - rearranged nsclc in treatment - naive or crizotinib pretreated patients.12 in nsclc , the y1278s alk mutation has not been described in the clinical setting , and the efcacy of lorlatinib in nsclc remains unknown . 
however , a different mutation at the same position , y1278h , has been reported in nsclc cell lines that have the alk - eml4 fusion exposed to accelerated mutagenesis , 13 which was resistant to crizotinib . neuroblastoma is the most common solid tumor in children , but adult patients are exceptional , because fewer than 0.3 patients are diagnosed per million people per year.14 crizotinib has been shown to be less effective in neuroblastoma than in nsclc , in which alk mutations , rather than alk fusions , are a major oncogenic event in nearly 10% of the patients , 15 and alk is the most common somatically mutated gene . 
in a phase i / ii trial ( nct00939770 ; crizotinib in treating younger patients with relapsed or refractory solid tumors or anaplastic large cell lymphoma ) , 11 patients with alk - mutated neuroblastoma were treated with crizotinib . 
crystal structure of ( a ) crizotinib / alk and ( b ) lorlatinib / alk . in neuroblastoma reported that y1278s is oncogenic ( ligand - independent activation ) and that crizotinib is inlines effective in vitro and in vivo in neuroblastoma cell harboring the alk y1278s mutation , whereas lorlatinib seemed to be highly effective.19 y1278s is located in the the alk activation loop . 1278 - yrasyy - 1283 motif of donella - deana et al20 demonstrated the important role of initial phosphorylation of tyrosine y1278 for the a - loop activation in alk - npm fusion . 
a study of the crystal structure of alk conrmed this role and highlighted a tight interaction in the inactive form of alk between the unphosphorylated y1278 and a cysteine at position 1097.21 in our patient , a y1278 mutation from tyrosine to serine the phosphorylation and thus the alk should inhibit activation , 20 but guan et al19 showed in a preclinical study that y1278 phosphorylation in neuroblastomas was not for alk activation ( unlike phosphorylation of essential y1283 , which is essential )  . 
however , this hypothesis was not conrmed , so the mechanism of alk activation with y1278s mutation is controversial.19 the y1278s mutation seems to be a rare mutation , but it has been reported in six neuroblastoma patients ( catalogue of somatic mutation in cancer [ cosmic ] database ) .17 , 22 - 24 no clinical data regarding the efcacy of alk inhibitors were reported in these six tumors harboring a y1278s alk mutation , but in two patients , preclinical data showed an in vitro resistance to crizotinib ( high concentration that inhibits 50% [ ic50 ] )  . 
note that a clinical trial of lorlatinib for patients with alk - driven relapsed or refractory neuroblastoma is ongoing as part of the new approaches to neuroblastoma therapy ( nant ) consortium ; this trial ( nct03107988 ; study of lorlatinib [ pf - 06463922 ] , an oral small molecule inhibitor of alk / ros 1 , for patients with alk - driven relapsed or refractory neuroblastoma ) is not yet recruiting in europe , and results are pending . in conclusion , we show that lorlatinib is effective against the alk y1278s mutation in neuroblastoma , whereas this mutation confers primary resistance to crizotinib . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . gudrun schleiermacher honoraria : bristol - myers squibb research funding : bristol - myers squibb ( inst ) , pzer ( inst ) , msdavenir ( inst ) , roche ( inst ) travel , accommodations , expenses : roche no other potential conicts of interest were reported . references chiarle r , voena c , ambrogio c , et al : the anaplastic lymphoma kinase in the pathogenesis of cancer . 
nat rev cancer 8 : 11 - 23 , 2008 solomon bj , mok t , kim dw , et al : first - line crizotinib versus chemotherapy in alk - positive lung cancer . 
moss e yp , lim ms , voss sd , et al : safety and activity of crizotinib for paediatric patients with refractory solid tumours or anaplastic large - cell lymphoma : a childrens oncology group phase 1 consortium study . 
lancet oncol 14 : 472 - 480 , 2013 pearson ad , pinkerton cr , lewis ij , et al : high - dose rapid and standard induction chemotherapy for patients aged over 1 year with stage 4 neuroblastoma : a randomised trial . 
lancet oncol 9 : 247 - 256 , 2008 lewington v , lambert b , poetschger u , et al : 123i - mibg scintigraphy in neuroblastoma : development of a siopen semi - quantitative reporting , method by an international panel . 
matthay kk , shulkin b , ladenstein r , et al : criteria for evaluation of disease extent by ( 123 ) i - metaiodobenzylguanidine scans in neuroblastoma : a report for the international neuroblastoma risk group ( inrg ) task force . 
br j cancer 102 : 1319 - 1326 , 2010 park jr , bagatell r , cohn sl , et al : revisions to the international neuroblastoma response criteria : a consensus statement from the national cancer institute clinical trials planning meeting . 
shaw at , felip e , bauer tm , et al : lorlatinib in non - small - cell lung cancer with alk or ros1 rearrangement : an international , multicentre , open - label , singlearm rst - in - man phase 1 trial . 
yoda s , lin jj , lawrence ms , et al : sequential alk inhibitors can select for lorlatinib - resistant compound alk mutations in alk - positive lung cancer . 
cancer discov 8 : 714 - 729 , 2018 3 : 108ra114 , 2011 cell 26 : 682 - 694 , 2014 infarinato nr , park jh , krytska k , et al : the alk / ros1 inhibitor pf - 06463922 overcomes primary resistance to crizotinib in alk - driven neuroblastoma . 
donella - deana a , marin o , cesaro l , et al : unique substrate specicity of anaplastic lymphoma kinase ( alk ) : development of phosphoacceptor peptides for the assay of alk activity . 
lee cc , jia y , li n , et al : crystal structure of the alk ( anaplastic lymphoma kinase ) catalytic domabiochem j 430 : 425 - 437 , 2010 22 . 
de brouwer s , de preter k , kumps c , et al : meta - analysis of neuroblastomas reveals a skewed alk mutation spectrum in tumors with mycn amplication . 
clin cancer res 16 : 4353 - 4362 , 2010 janoueix - lerosey i , lequin d , brugi `eres l , et al : somatic and germline activating mutations of the alk kinase receptor in neuroblastoma . 
cgp altered physician treatment selection in 25% of evaluable patients ( n = 7 of 28 ) and was associated with improved progression - free survival . conclusion to our knowledge , this is the largest technical evaluation of the performance of cgp in sarcoma . cgp was effectively performed in the vast majority of sarcoma samples and altered physician treatment selection . 
2020 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction sarcomas are uncommon , highly morbid cancers that constitute approximately 1% of all adult malignancies . currently , more than 70 recognized histologic subtypes exist , which makes them extremely challenging to accurately diagnosis and treat.1 sarcomas have multiple genomic alterations , including copy number changes , point mutations , insertions and deletions , and fusions.2 - 4 thus , detailed rna and dna sequence analysis is key for diagnosis as well as for treatment decision - making.5 is not surprising , that comprehensive therefore , genomic proling ( cgp ) is increasingly being used in the evaluation and management of bone and soft tissue sarcomas . 
cgp is the sequencing of dna and rna from tumor samples , which enables the identication of known and novel alterations that may drive inherent oncogenicity.6 - 9 cgp may rene the histologic tumor diagnosis , with its management.10 , 11 indeed , in a study by groisberg et al1 specic to sarcomas , the authors found 61% of 102 patients in a retrospective cohort had a potentially actionable genomic target . ramications despite the potential role that cgp may have in the treatment of sarcoma , the impact of dnaand rnabased cgp on physician decision - making is poorly documented.12 , 13 similarly , it is equally unclear what factors may contribute to the success or failure of cgp in an outpatient clinical setting . 
 challenges in sarcoma comprehensive genomic proling as mentioned previously is unknown ; neither are the relative contributions of biopsy site ( bone v soft tissue ) and methods ( ie , computed tomography [ ct ] - guided core needle biopsy v open excisional biopsy )  . 
to answer these questions , we herein report the results of two studies : the rst is a prospective clinical trial examining the impact of dnaand rna - based cgp on physician decision - making . the second is a retrospective analysis of the success and failure rates of cgp in an even larger patient cohort , which includes the aforementioned patients . methods sarcoma decision impact clinical trial study design . 
to examine the effects of cgp on physician decision - making , a prospective clinical trial ( the ohio state university [ osu ] institutional review board approval no . osu - 12067 ) was performed in collaboration with foundation medicine . 
the primary objectives of the study were to assess the feasibility and logistics associated with a clinical trial using a commercially available cgp platform ( foundationoneheme ; foundation medicine , cambridge , ma ) in an academic clinical setting and to determine the proportion of patients who would receive a cancer - related therapy based on cgp results . 
progression - free survival ( pfs ) was summarized using kaplan - meier survival analysis . the ohio state retrospective sarcoma study data following : date of sample procurement , method of specimen procurement ( eg , excisional biopsy , needle core biopsy , ne needle aspiration ) , biopsy site , specimen source ( hospital ) , and pathology classication by osu . 
the foundation medicine records were interrogated for all sarcoma samples sent by osu and the following information was compiled : specimen type ( formalin - xed parafn - embedded tissue blocks v unstained slides ) , morphologic tumor purity , dna and rna extraction yield , sequencing metrics , and the nature of the released report ( ie , pass , fail , or qualied )  . 
a qualied report indicates a sample for which it was unable to complete the entire cgp process ( eg , rna extraction , low tumor purity ) but for which some genomic information was obtained . in contrast , a passed report indicates a sample for which dna and rna proling was completed successfully . 
2 analysis was used to calculate the signicance of intergroup alterations . results physician decision - making was prospectively altered for 25% of patients with sarcoma who underwent cgp as part of a decision impact trial , patients with sarcoma whose disease had not yet progressed on the current line of therapy were approached to undergo cgp as part of the study . 
treating oncologists were blinded to cgp results time of progression ( based on imaging results )  . until treating oncologists rst documented their treatment decision for the patient with sarcoma without cgp results . cgp data were then released for review and the treating oncologist would note whether their treatment decision had been altered by the cgp results . 
 a hay et al patients with sarcoma diagnosis ( n = 34 ) treatment recommendations chosen before cgp patients with liposarcoma ( n = 15 ) patients with nonliposarcomatous sarcoma ( n = 19 ) no data on whether recommendation changed ( n = 3 ) did not switch therapy on the basis of cgp results ( n = 8 ) switched therapy on the basis of cgp results ( n = 4 ) no data on whether recommendation changed ( n = 3 ) did not switch therapy on the basis of cgp results ( n = 13 ) switched therapy on the basis of cgp results ( n = 3 ) patients assessed for eligibility ( n = 493 ) excluded ( n = 31 ) misdiagnosed or miscategorized tumors excluded ( n = 16 ) duplicates or no clinical data available excluded ( n = 41 ) samples that failed initial cgp remaining individuals evaluated ( n = 408 ) patients included in analysis ( n = 392 ) patients with preliminary reports ( n = 351 ) total no . 
of patients for whom full cgp analysis was completed ( n = 297 ) qualified ( n = 54 ) samples for which cgp could not be completed fig 1 . 
 ( % ) male female mean age ; median ( range ) , years data 14 ( 50 ) 14 ( 50 ) 57 ; 62 ( 2480 ) no change ( n = 12 ) changed ( n = 5 ) p = .03 altered , six patients received the selected treatment ; one patient died before initiating therapy . 
as an exploratory end point , we noted that the median pfs in the cgp - selected group was 124 days versus 54 days in the noncgpselection group ( p = .03 ; fig 2 )  . cgp was performed on almost 400 patients with sarcoma a total of 392 patients representing 413 unique samples were proled over 3 years . 
l - type sarcomas ( ie , leiomyosarcoma , liposarcoma ) constituted the greatest proportion of cases , and this is indicative by the preponderance of tp53 , rb1 alterations and mdm2 and cdk4 amplications ( table 3 )  . fifty - six of the 413 samples ( 13.6% ) had reports qualied for low tumor purity , failed rna extraction , or suboptimal sequencing metrics , whereas 10.7% ( n = 44 of 413 ) samples failed testing . 
examining sarcoma subtypes individually , leiomyosarcomas ( 74.0% ; n = 57 of 77 ) and liposarcomas ( 79.4% ; n = 50 of 63 ) had proportionally higher pass rates than did bone - based sarcomas . 
chordomas had the lowest successful initial genomic proling rate , with a 43% initial pass rate ( table 3 )  . biopsy site and method inuence successful cgp completion biopsy sites . 
the other category encompassed an array of sites , including , but not limited to , brain , eye , epicardium , aorta , lymph node , submandibular gland , bladder , prostate , testes , ovary , cervix , and vagina . 
this was partially due to nding duplicate patients ( ie , some data were obtained with patients identities removed , because they were included in the decision impact trial , which was blinded ; in unblinding , we found some individuals had been included twice )  . 
other reasons included being unable to locate individuals within ihis ( ohio state universitys electronic medical record ) and data necessary for additional analysis ( including biopsy location and method ) were unavailable . 
breakdown of the most common sarcomas subtypes proled sarcoma subtype qualied passed failed total bone - based chondrosarcoma osteosarcoma ewing chordoma soft tissue based leiomyosarcoma liposarcoma spindle cell liposarcoma myxoid liposarcoma pleomorphic liposarcoma well - differentiated liposarcoma dedifferentiated liposarcoma gist synovial myxobrosarcoma angiosarcoma fibromatosis dscrt epithelioid epithelioid hemangioendothelioma solitary brous tumor abbreviations : dscrt , desmoplastic small round cell tumor ; gist , gi stromal tumor . 180 2020 by american society of clinical oncology in conclusion , to our knowledge , this study is the only prospective evaluation of dnaand rna - based cgp in sarcoma on physician decision - making and the largest indepth evaluation of success of cgp in sarcoma to date . 
we note in this cohort that cgp in sarcoma is successful in a large majority of patients and alters physician treatment decision - making in an estimated 25% of patients . 
furthermore , this percentage may have been driven by the fact that the study data were from the era when cdk4 inhibitors were just starting to be considered for use off - label in adipocytic tumors , and conrmation of cdk4 amplication was critical for this drug selection . 
we would anticipate , as time progresses , the types of tumor for which there are dened targetable alterations will increase and thus there would be an increase in physician treatment - decision changes . 
a classic example would be that of gi tumors for which kit / pdgfr sequencing is now considered standard of care for determining whether a patient has these alterations and , if so , whether the patient has resistance alterations.17 a developing example would be that in leiomyosarcoma , where we have previously reported that homologous recombination alterations are a common feature of uterine leiomyosarcomas and may be amenable to parp inhibition.18 tumor location and tissue subtype signicantly clearly , inuence proling success , likely secondary to preanalytic variables that inuence quality of dna and rna , such as decalcication , tumor cellularity , and available tissue volume . 
of note , of the 36 bone biopsy samples that were collected in this study , pathology reports for only 13 mentioned the sample underwent decalcication ; however , no specications regarding the decalcication agent used or the xation times were documented in the report . 
protocols to prevent decalcication during sample processing may aid in yielding higher cgp success rates.19 , 20 in addition , obtaining excisional biopsy specimens rather than imaged - guided biopsy specimens seems to lead to greater likelihood of cgp success . in these studies , it should be mentioned that germline aberrations were not evaluated , because foundation medicine does not test for these . 
 challenges in sarcoma comprehensive genomic proling causes of sample failure causes of sample qualification low cellularity rna failure dna and rna failure dna failure unknown contamination rna failure failed rna metrics low tumor purity noisy cna data contamination rna failure and noisy cna data failed rna metrics and contamination low dna coverage unknown n = 44 samples n = 56 samples fig 3 . 
also , many of our samples are often obtained at outside hospitals , and there is no standardization of pathology report contents . in summary , sarcoma cgp appears to alter physician decision - making regarding treatment and may affect the ultimate outcome of the patient . 
chen consulting or advisory role : novartis , immune design , syapse speakers bureau : novartis , foundation medicine , eisai patents , royalties , other intellectual property : matchtx . no other potential conicts of interest were reported . references groisberg r , hong ds , holla v , et al : clinical genomic proling to identify actionable alterations for investigational therapies in patients with diverse sarcomas . oncotarget 8 : 39254 - 39267 , 2017 abeshouse a , adebamowo c , adebamowo sn , et al : comprehensive and integrated genomic characterization of adult soft 171 : 950 - 965.e28 , 2017 yakirevich e , madison r , fridman e , et al : comprehensive genomic proling of adult renal sarcomas provides insight into disease biology and opportunities for targeted therapies . 
cancer res 76 : 3690 - 3701 , 2016 fang w , ma y , yin jc , et al : comprehensive genomic proling identies novel genetic predictors of response to anti - pd - ( l ) 1 therapies in non - small cell lung cancer . 
clin cancer res 25 : 5015 - 5026 , 2019 chung jh , dewal n , sokol e , et al : prospective comprehensive genomic proling of primary and metastatic prostate tumors . 
jco precis oncol 3 : 1 - 23 , 2019 al - rohil rn , tarasen aj , carlson ja , et al : evaluation of 122 advanced - stage cutaneous squamous cell carcinomas by comprehensive genomic proling opens the door for new routes to targeted therapies . 
ross js , gay lm , wang k , et al : comprehensive genomic proles of metastatic and relapsed salivary gland carcinomas are associated with tumor type and reveal new routes to targeted therapies . 
rodriguez - rodriguez l , hirsheld km , rojas v , et al : use of comprehensive genomic proling to direct point - of - care management of patients with gynecologic 14 . 
thway k , rockcliffe s , gonzalez d , et al : utility of sarcoma - specic fusion gene analysis in parafn - embedded material for routine diagnosis at a specialist 16 . 
thway k , wren d , lee j , et al : evaluation of the optimal provision of formalin - xed , parafn - embedded material for reverse transcription - pcr in soft - tissue 21 : 1315 - 1325 , 2016 cancers . 
singh vm , salunga rc , huang vj , et al : analysis of the effect of various decalcication agents on the quantity and quality of nucleic acid ( dna and rna ) recovered from bone biopsies . 
choi s - e , hong sw , yoon so : proposal of an appropriate decalcication method of bone marrow biopsy specimens in the era of expanding genetic molecular study . 
this article describes medical oncologists condence with genomic testing and the association between genomic condence and test use . methods we used data from the 2017 national survey of precision medicine in cancer treatment to characterize oncologists condence with genomic testing . 
genomic condence was examined separately by type of test user : next - generation sequencing ( ngs ) only , gene expression ( ge ) only , both ngs and ge , or nonuser . predictors of genomic condence were examined with multinomial logistic regression . 
the association between genomic condence and test use was examined with multivariable linear regression . results more than 75% of genomic test users were either moderately or very condent about using results from multimarker tumor panel tests to guide patient care . 
condence with using multimarker tumor panel tests was highest among both ngs and ge test users , with 60.1% very condent in using test results , and lowest among ngs - only test users , with 38.2% very condent in using test results . 
oncologists were most condent in using single - gene tests and least condent in using whole - genome or - exome sequencing to guide patient care . genomic condence was positively associated with self - reported test use . 
in adjusted models , training in genomics , larger patient volume , and treating patients with solid tumors predicted higher genomic condence . onsite pathology services and receipt of electronic medical record alerts for genomic testing predicted lower genomic condence . conclusion oncologists condence varies by testing platform , patient volume , genomic training , and practice infrastructure . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction the landscape of cancer care has changed dramatically over the past decade as therapies are increasingly personalized to the molecular characteristics of a patients tumor.1 whereas genomic testing in oncology started with testing for single mutations in specic genes , advances in molecular proling has spurred the development and now widespread use of multimarker tumor panels.2 , 3 these panels assess different types of genomic alterations commonly detected through nextgeneration sequencing ( ngs ) and gene expression ( ge ) proles . 
such testing can identify therapeutic targets that can be treated with particular anticancer agents and determine when individuals are at higher risk for recurrence and may need therapy.3 , 4 although not standard practice as of yet , whole - genome or whole - exome sequencing is a newer technology that shows promise in informing patient care.4 the rapid proliferation of genomic testing has outpaced the development of practice guidelines on their appropriate use.5 , 6 multimarker tumor panel tests generate vast data about the biology of a patients tumor . 
in cases where the tumor can be successfully sequenced , an actionable target is identied for 39%90% of patients , a wide range that varies by patient population and the size of the panel test.7 - 10 although testing reports typically highlight multimarker panel potentially benecial therapies and relevant clinical trials , individual providers must navigate the process of communicating results to their patients and selecting an appropriate targeted therapy . 
furthermore , multimarker panel testing frequently identies variants of unknown signicance or germline variants , which can further complicate interpretation and care management.11 - 13 very few studies have examined providers attitudes about genomic testing . 
 oncologist condence in genomic testing context key objective this article provides unique data about oncologists in the united states and their condence in using results from commercially available multimarker tumor panel tests to guide decisions about patient treatment and management . knowledge generated oncologists who used both next - generation sequencing ( ngs ) and gene expression tests reported the highest condence in using multimarker tumor panel results to guide patient care ; oncologists who only used ngs tests had the lowest condence . 
oncologists with higher condence were more likely to report using results from multimarker tumor panels to inform patient care . relevance these ndings underscore the importance of genomic training and suggest other providerand practice - level factors that can be leveraged to build oncologists condence in using genomic testing . providers lack condence in using new genomic tests , which could limit the extent to which new tests are appropriately applied in practice.5 , 6 , 14 , 15 for example , providers report less condence in using multimarker tumor panels compared with single - gene tests , which are well established for certain patient populations.6 in addition , providers vary in their understanding of how to interpret and act upon results from whole - genome sequencing , which is not available outside of clinical trials.16 given the potential of precision oncology , there is a need to better understand oncologists attitudes and experiences with genomic testing and to identify factors that foster genomic testing condence . this article builds upon previous studies by describing oncologists condence with genomic testing and the association between condence and genomic test use over the past 12 months . 
oncologists condence in using multimarker tumor panels was rst examined in relation to their condence in using single - gene somatic tests and whole - genome or - exome sequencing . 
oncologists condence was then further characterized by exploring the individualand practice - level characteristics associated with condence and assessing whether their condence was associated with their use of multimarker tumor panels in practice . methods data and study population this study uses data from the national survey of precision medicine in cancer treatment , a nationally representative study of how oncologists use genomic testing in practice . eligible providers were identied from the american medical association physician masterle and selected using multistage probability sampling . 
additional information about the study design and methods have been published elsewhere.17 the survey is available upon request . measures the national survey of precision medicine in cancer treatment collected detailed data about oncologists condence in using genomic testing ; their genomic test use ; and demographics , specialty , and practice characteristics . 
condence was assessed for genomic tests for individual genes or chromosomal alterations ( referred to as single - gene tests ) , commercially available multimarker tumor panel tests , and whole - genome or - exome sequencing . 
response options were very condent , moderately condent , a little condent , or not at all condent . type of genomic test user was determined on the basis of oncologists reported use of 18 specic multimarker tumor panels in the past 12 months . 
the multimarker tumor panel tests included commercially available ge tests ( eg , oncotype dx breast ; genomic health , redwood city , ca ) , commercially available ngs tests ( eg , foundationone ; foundation medicine , cambridge , ma ) , and noncommercial panels performed at an academic medical center . 
training in genomics was also captured and dened as any formal training ( eg , instruction during residency / fellowship , professional lectures or seminars , symposiums , conferences , continuing medical education ) in the use of genomic testing . practice setting information was assessed with questions about the number of unique patients with cancer treated per month . 
responses were categorized as academic medical center or medical school and nonacademic setting . infrastructure to support precision medicine was assessed with a series of questions about whether the respondents primary practice had the following genomic testing services : onsite pathology , internal policies or protocols for use of genomic and biomarker testing , an electronic medical record ( emr ) that alerts when a genomic test is recommended , or a genomic / molecular tumor board . 
response options were categorized as yes and no or do not know . analysis weighted percentages were calculated to describe the sample and provider condence in using single - gene tests , multimarker tumor panel tests , and whole - genome or - exome sequencing . 
two sets of models were examined : one that adjusted for type of genomic test user and a second that simultaneously adjusted for type of genomic test user and the other physician and practice characteristics in the model . 
results are presented as odds ratios and adjusted percentages , also referred to as predicted margins.19 multivariable linear regression was then used to assess whether condence was associated with the percentage of patients for whom multimarker panel test results guided patient care in the past 12 months . 
multivariable models adjusted for years since graduation , sex , race , census region , specialty , training in genomics , practice setting , patient volume , and infrastructure for precision medicine . 
results are presented as adjusted percentages.19 all analyses were conducted using sudaan release 11.0 statistical software ( rti international , research triangle park , nc ) and weighted to account for the complex sampling approach and survey nonresponse . results sample characteristics the sample comprised 1 , 281 oncologists ( table 1 )  . 
participants reported a range of institutional resources to support precision medicine , with onsite pathology and internal policies or protocols for use of genomic and biomarker testing reported most frequently . genomic test use was reported by 95.4% of the sample , with 58.6% using both ngs and ge tests , 26.4% using only ngs tests , and 10.3% using only ge tests . condence using genomic tests in practice condence using multimarker tumor panel tests to guide decisions about patient treatment and management was highest among ngs and ge users , with 60.1% very condent and 35.6% moderately condent in using test results . 
even among oncologists who did not use multimarker tumor panel tests in the past 12 months , 27.1% were very condent and 37.8% were moderately condent in using test results ( fig 1b )  . in contrast to multimarker tumor panel tests , oncologists were more condent in using somatic single - gene tests and less condent in using whole - genome or - exome sequencing ( figs 1a and 1c )  . 
30 female male race white asian other region midwest northeast south west specialty solid and hematologic malignancies hematologic malignancies only solid tumors only training in genomics practice setting practice afliation academic medical center or medical school other practice settingb no . 
percentages may not sum to 100 because of missing data . bother settings include a medical center not afliated with a medical school , community hospital , ofce - based practice , health maintenance organization or integrated health care system , and other . guide decisions about patient treatment and management . in the fully adjusted models ( table 2 ) and in models that only adjusted for the type of genomic test user ( appendix table a1 ) , oncologists with training in genomics had higher condence than those who did not . 
likewise , oncologists who treated 100 patients per month had higher condence than those who treated , 49 patients per month . compared with oncologists who treated both solid and hematologic malignancies , those who only treated solid tumors were more condent , whereas those who treated only hematologic malignancies were less condent in using results of multimarker panel tests to guide patient care . oncologists who practice in institutions with onsite pathology or emr alerts for recommended genomic tests had lower condence than those who practice in institutions without onsite pathology or emr alerts . 
for example , ngs and ge users who were very condent used multimarker panel test results to inform care for 30.97% of their patients ( who had received testing ) compared with 18.44% among ngs and ge users who were not at all or a little condent . 
oncologists condence in using results from ( a ) single - gene tests , ( b ) commercially available multimarker somatic panels , and ( c ) whole - genome or - exome sequencing to guide decisions about patient treatment and management ( n = 1 , 281 )  . 
on the basis of 2 tests for independence , overall differences in condence among 4 types of genomic test users were statistically signicant at p , .001 for single - gene tests , multimarker tumor panels , and whole - genome or - exome sequencing . discussion the current study adds to the literature on provider attitudes about genomic testing . 
most ge panel tests are used to inform the treatment of patients with breast cancer , and there are clear guidelines for test results.20 , 21 for users of other multimarker tumor panel tests , the application of test results to patient management is guideline endorsed for a limited set of clinical indications . testing and the interpretation of tumor panel tests to inform care is more complicated than using single - gene tests . 
at the time the survey was conducted , there were only consensus recommendations for multimarker tumor panel testing in treating nonsmall - cell lung cancer.23 despite this , providers condence using multimarker tumor panel tests was relatively high , with  . 75% of genomic test users reporting that they were either moderately or very condent in using results to guide patient care . 
these ndings are consistent with other studies of provider condence.15 , 24 - 26 oncologists condence also varied by test type , which is consistent with previous research.6 single - gene tests have been used for several decades as companion diagnostics to targeted therapies ; thus , clinical appropriateness and utility are well prescribed.22 even among genomic test nonusers , more than half reported being very condent in using single - gene tests to guide patient care . 
presented are adjusted percentages from multivariable linear regression models that were adjusted for years since graduation , sex , race , census region , specialty , training in genomics , practice setting , and infrastructure for precision medicine . 
historically , providers who treat patients with a hematologic malignancy have technologies ( eg , ow cytometry for relied on different immunophenotyping ) or single - gene somatic tests to identify relevant targeted therapies.27 in contrast , use of multimarker tumor panel tests has been more common in the treatment of solid tumors.23 the importance of experience was also highlighted in our ndings that providers who treat  . 
100 patients per month had higher condence than providers who treat , 50 . providers in high - volume clinics have more opportunity to encounter patients for whom multimarker tumor panel tests are warranted . 
indeed , patient volume has been associated with better treatment outcomes in other settings.28 providers in lower - volume clinics may benet from additional resources , including clinical decision support , to strengthen evidence - based use of genomic testing in treatment planning.29 our ndings underscore the importance of genomic training to increase provider condence in using multimarker panel tests to guide patient care . 
training was dened broadly and included any instruction during residency or fellowship , professional lectures , conferences , or continuing medical education in the use of genomic testing . additional research should identify the specic competencies needed by different types of oncologists and be tailored to support providers who demonstrate relatively low condence in using genomic testing in practice . 
several categories of training could be pursued , including revision of the curricula of medical and nursing schools to provide training in genomics ; advanced training of physicians and other practitioners in genomics ; and continuing medical education to help practicing clinicians to stay up to date on advances in the eld.30 , 31 strategies such as case - based learning , clinical decision - support tools , administrative support for prior authorization , and clinical trial matching may be helpful in strengthening provider condence around genomic testing at the point of care and supporting successful implementation of precision medicine programs.29 genomic testing services available in an oncologists primary practice were either not associated with provider condence or , paradoxically , associated with lower condence . 
oncologists who reported that their practice had onsite pathology spent a lower percentage of time in delivering patient care ( data not shown ) , which could have inuenced their condence in using genomic test results to guide treatment decisions . 
oncologists in settings with onsite pathology could also have been more likely to have a research focus outside of genomics , although that is not something we can explore with our data . in addition , it is possible that other features of the practice setting or availability of appropriate training were more important drivers of provider condence . 
this work should address both the knowledge and the skills to use genomic testing appropriately and to act on test results as well as system - level resources to support precision oncology . our study results should be considered in light of a few limitations . 
first , providers were asked generally about their condence in using genomic test results to guide patient care ; we do not know how condence with a particular testing approach may vary on the basis of specic patient subgroups or clinical scenarios . 
however , participants are representative of practicing oncologists in the united states in terms of age , sex , and geographic location , and all analyses were weighted to adjust estimates for survey nonresponse . 
de moor , phd , mph , healthcare delivery research program , division of cancer control and population sciences , national cancer institute , 9609 medical center dr , room 3e438 , rockville , md 20850 ; e - mail : demoorjs@mail.nih.gov. support supported by the national cancer institute ( nci ) , national human genome research institute , and american cancer society through nci contract no . 
gray stock and other ownership interests : magenta therapeutics ( i ) consulting or advisory role : grail industries , magenta therapeutics ( i ) expert testimony : riley and associates no other potential conicts of interest were reported . references kalia m : biomarkers for personalized oncology : recent advances and future challenges . 
morganti s , tarantino p , ferraro e , et al : complexity of genome sequencing and reporting : next generation sequencing ( ngs ) technologies and implementation of precision medicine in real life . 
crit rev oncol hematol 133 : 171 - 182 , 2019 el - deiry ws , goldberg rm , lenz hj , et al : the current state of molecular testing in the treatment of patients with solid tumors , 2019 . 
bmc health blazer kr , nehoray b , solomon i , et al : next - generation testing for cancer risk : perceptions , experiences , and needs among early adopters in community healthcare settings . 
genet test mol biomarkers 19 : 657 - 665 , 2015 tan o , shrestha r , cunich m , et al : application of next - generation sequencing to improve cancer management : a review of the clinical effectiveness and costeffectiveness . 
j clin oncol serv res 9 : 131 , 2009 33 : 2753 - 2762 , 2015 johnson db , dahlman kh , knol j , et al : enabling a genetically informed approach to cancer medicine : a retrospective evaluation of the impact of comprehensive tumor proling using a targeted next - generation sequencing panel . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
freedman an , klabunde cn , wiant k , et al : use of next - generation sequencing tests to guide cancer treatment : results from a nationally representative survey of oncologists in the united states . 
duffy mj , harbeck n , nap m , et al : clinical use of biomarkers in breast cancer : updated guidelines from the european group on tumor markers ( egtm )  . 
godin g , b elanger - gravel a , eccles m , et al : healthcare professionals intentions and behaviours : a systematic review of studies based on social cognitive theories . 
implement sci 3 : 36 , 2008 johnson lm , valdez jm , quinn ea , et al : integrating next - generation sequencing into pediatric oncology practice : an assessment of physician condence and understanding of clinical genomics . 
allegretti m , fabi a , buglioni s , et al : tearing down the walls : fda approves next generation sequencing ( ngs ) assays for actionable cancer genomic 33 . 
pennell na , mutebi a , zhou z - y , et al : economic impact of next - generation sequencing versus single - gene testing to detect genomic alterations in metastatic nonsmall - cell lung cancer using a decision analytic model . 
unadjusted models of provider and practice characteristics associated with condence in using genomic test results to guide patient management not at all or a little condent moderately condent very condent moderately v not at all or a little condent very condent v not at all or a little condent total wtd % 95% ci wtd % 95% ci wtd % 95% ci 95% ci 95% ci overall p characteristic provider years since graduation 10 11 - 20 21 - 30  . 
unadjusted multinomial logistic regression models controlled for type of genomic test use , which was dened according to the multimarker somatic panel tests each oncologist used in the past 12 months . 
 about ras mutation clearance in plasma ctdna from ras - mutant colorectal cancer patients bouchahda et al1 recently published in your journal . authors conducted a pilot study aimed to investigate the efcacy and safety of antiegfr - targeted therapy added to chemotherapy in patients with unresectable metastatic colorectal cancer with raswild - type circulating tumor dna ( ctdna ) but ras - mutant primary tumor . 
we would like to emphasize the following . in 2017 , our group for the rst time started to investigate the clearance of ras - mutant clones in plasma , supporting an unexpected negative selection of rasmutant clones during the clonal evolution of rasmutant metastatic colorectal cancer ( mcrc ) .2 one year later , we reported that ve patients with rasmutant ras ( ras - mt ) mcrc who switched to wildtype ras ( wt - ras ) in plasma ctdna received egfr inhibitors as a second - line treatment , achieving a durable clinical benet , 3 thus suggesting that ctdna analysis might reveal a therapeutically exploitable window of opportunity , characterized by the prevalence of wt - ras clones , which can be converted in a clinically meaningful benet for patients . 
we published a detailed case report4 in which we described the study of a patient with a primary n - ras - mutant colorectal cancer , who received second - line treatment with antiegfr , after failure of rst - line triplet chemotherapy plus bevacizumab , according to the absence of any clinically relevant mutation of ras genes in blood , achieving a partial response . 
with this background , we designed the currently ongoing kairos trial ( eudract number : 2019 - 001328 - 36 ) , aimed to investigate the efcacy and safety of second - line cetuximab plus chemotherapy treatment in initially ras - mt mcrc patients who converted to raswt at the time of rst progression.3 , 4 we regret having to note that bouchahda et al1 have not cited neither our publications nor have they named the kairos trial in their entire discussion . 
in fact , the authors state that the team of raimondi et al5 recently reported the disappearance of ras - mt clones in ctdna after tumor progression while receiving bevacizumab and chemotherapy in four patients with ras - mt mcrc . 
this last information is incorrect in that we prospectively enrolled mcrc patients at the time of progression of disease and the mutational status of ctdna was assessed before initiating a further line of therapy ( as far as we underthe same inclusion criteria as reported by stand , bouchahda et al1 in their study )  . 
furthermore , bouchahda et al strongly limited the discussion of our data to the number of patients who converted to wt - ras in plasma , omitting to specify that these patients were then treated with egfr blockade based on the results obtained in ctdna . 
in these patients , the introduction of anti - egfr agents , which would have not been supported by international guidelines , allowed to achieve a 12 - month , 10 - month , and 6 - month pfs in three patients treated in second - line setting and a pfs of 4 months in patients treated in fourth - line.5 these data should have been included and discussed . 
finally , the authors state that studies with liquid biopsy have been to date selectively focused on the early detection of the appearance of ras - mt clones in tumor deposits by analyzing ctdna in blood samples from patients with raswt primary tumors . 
bouchahda m , saffroy r , karaboue a , et al : undetectable rasmutant clones in plasma : possible implication for anti - egfr therapy and prognosis in patients with ras - mutant metastatic colorectal cancer . 
gazzaniga p , raimondi c , urbano f , et al : second line egfr - inhibitors in ras mutant metastatic colorectal cancer : the plasma ras wild type window of opportunity . 
gazzaniga p , raimondi c , urbano f , et al : egfr inhibitor as second - line therapy in a patient with mutant ras metastatic colorectal cancer : circulating tumor dna to personalize treatment . 
raimondi c , nicolazzo c , belardinilli f , et al : transient disappearance of ras mutant clones in plasma : a counterintuitive clinical use of egfr inhibitors in ras mutant metastatic colorectal cancer . 
nicolazzo c , belardinilli f , caponnetto s , et al : why the therapeutic impact of ras mutation clearance in plasma ctdna deserves to be further explored in metastatic colorectal cancer . 
 o prospective feasibility study for using cell - free circulating tumor dnaguided therapy in refractory metastatic solid cancers : an interim analysis purpose retrospective studies have demonstrated that cell - free circulating tumor dna ( ctdna ) hotspot testing predicts matched therapy response to firstand second - line therapies in patients with advanced nonsmall - cell lung cancer ( nsclc )  . 
however , no prospective outcomes studies have evaluated ctdna - guided matched therapy decision making on the basis of comprehensive plasma genomic testing including all four major classes of alterations . 
here , we report the clinical utility of this approach in advanced solid tumor cancers . patients and methods we conducted a multiple parallel cohort , open - label , clinical trial using ctdna - guided matched therapy when tissue was insufficient or unobtainable for next - generation sequencing . 
patients with prespecified targetable alterations in metastatic nsclc , gastric cancer ( gc ) , and other cancers were matched to several independent targeted agent trials at a tertiary academic center . results somatic alterations were detected in 59 patients with gc ( 78% ) , and 25 patients ( 33% ) had targetable alterations ( erbb2 , n = 11 ; met , n = 5 ; fgfr2 , n = 3 ; pik3ca , n = 6 )  . 
2017 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction the national comprehensive cancer network guidelines recommend genotyping in seven solid tumor cancers ( nonsmall - cell lung cancer [ nsclc ] , breast , gastric , esophageal , colorectal , melanoma , and gi stromal tumors ) for 11 genomic targets ( gts ) to inform targeted therapy selection.1 - 6 however , biopsy specimens can be inadequate for comprehensive profiling in 25% to 50% of patients , 7 - 10 leading to incomplete genotyping or repeat invasive biopsy to obtain more tissue . 
repeat biopsy is also recommended at progression in patients with breast cancer and nsclc to capture targetable genomic changes such as erbb2 ( human epidermal growth factor receptor 2 [ her2 ] ) copy number amplification ( cna ) or egfr and alk resistance mutations , respectively.4 , 5 , 11 comprehensive ctdna testing covering point mutations , insertions / deletions ( indels ) , fusions , seung tae kim kimberly c . 
 and cna may obviate the need for repeat invasive biopsies for genotyping when tissue is of insufficient quantity or unobtainable at initial diagnosis or at progression.12 , 13 in general , next - generation sequencing ( ngs ) seems to detect more actionable variants in target genes than non - ngs methods ( hotspot testing ) such as polymerase chain reaction ( pcr ) , immunohistochemistry ( ihc ) , or fluorescence in situ hybridization.14 - 18 beyond the benefits of invasive biopsy avoidance and higher sensitivity compared with nonngs methods , comprehensive ctdna ngs may provide a global summary of multiple lesions , whereas tissue genotyping of small biopsies may fail to capture intraand intertumor heterogeneity.19 - 21 retrospective studies in nsclc using ctdna genotyping for egfr mutations in the firstline ( egfrl858r / exon19del ) 22 and second - line ( egfrt790m ) 23 , 24 settings have produced response rates similar to studies of therapies directed by tissue - based genotyping . 
the study was conducted in accordance with the current ethical principles outlined in the declaration of helsinki and good clinical practice guidelines . patients eligible patients were older than age 20 years with histologically confirmed metastatic cancer , who had sufficient tumor tissue to test cancer - specific biomarkers but not to undergo comprehensive genomic profiling ( ngs )  . 
rr and disease control rate ( dcr = rr + stable disease ) were centrally adjudicated in accordance with response evaluation criteria in solid tumors ( recist ) version 1.1.28 statistical analysis descriptive statistics were calculated for demographics , ctdna alteration detection rate , and substudy matching . 
after double ultracentrifugation , 5 to 30 ng of cfdna was isolated for digital sequencing as previously described.12 , 26 , 29 all exons in 30 genes and critical exons ( those known to harbor somatic mutations ) of 40 genes were completely sequenced . 
sequencing data were analyzed using a custom bioinformatics pipeline to identify single nucleotide variants ( snvs ) in 70 genes ( 150 - kb panel footprint ) , cnas in 18 genes , indels in three genes ( egfr and erbb2 exons 19 and 20 ; met exon 14 ) , and alk , ret , ros1 , ntrk1 , fgfr2 , and fgfr3 fusions ( appendix fig a2 )  . 
tumor - derived dna shed into the bloodstream increases this value but , as a result of the relative proportions of tumorderived versus leukocyte - derived cfdna , typically a minor contributor . 
90th percentiles , respectively , of all cna calls in the guardant360 database . results patient enrollment and demographics from august 2014 to february 2016 , informed consent was obtained from 210 consecutive patients with metastatic cancer whose tissue was available for cancer - specific biomarker testing , but insufficient for ngs , at initial diagnosis or at progression . 
sixteen patients were lost to follow - up or withdrew consent , leaving 194 patients molecularly profiled by ctdna ngs ( appendix fig a3 )  . median age was 60 years ( range , 28 to 78 years ) for nsclc and 57 years ( range , 23 to 82 years ) for gc , melanoma , and other cancers ; 43% , 58% , 56% , and 89% of patients with these cancers were male , respectively ( table 1 )  . 
newly diagnosed ( firstline ) patients composed 29% of patients with nsclc , 37% of those with gc , 68% of those with melanoma , and 11% of those with other table 1 . 
erbb2 ( her2 ) cna was identified in two patients at progression ( one co - occurring with egfrt790m and one with the erbb2 g778_p780dup ) , and met was amplified in four patients at progression ( one with erbb2 insertion and three with egfrt790m )  . 
however , cnas in nsclc were not prespecified gts . on the basis of rolling substudy availability , inclusion criteria , and patient comorbidities , 10 ( 40% ) of the 25 patients with gc ( erbb2 , n = 6 ; met , n = 1 ; fgfr2 , n = 1 ; and pik3ca , n = 2 ) and 17 ( 50% ) of the 34 patients with nsclc ( egfr , n = 7 ; egfrt790m , n = 7 ; and alk , n = 1 ) with prespecified gts were matched to a molecularly targeted therapy ( tables 2 and 3 )  . 
one patient with gc and two patients with nsclc were lost to follow - up , leaving nine patients ( 90% ) and 15 patients ( 88% ) evaluable for response , respectively . ctdna - guidable targeted therapies and response by cancer type in gc , cnas in erbb2 ( n = 5 ) , fgfr2 ( n = 1 ) , and met ( n = 1 ) and snvs in pik3ca ( n = 2 ) were targeted with one patient achieving complete response ( cr ) , five partial response ( pr ) , and three stable disease ( sd ) for an rr of 67% ( 95% ci , 31% to 91% ) and dcr of 100% ( 95% ci , 63% to 100% ; table 4 , fig 2a )  . 
of patients ctdna alterations detected patients with prespecified second line of therapy third or greater line of therapy treated with matched therapy evaluable for response treated with matched therapy evaluable for response treated with matched therapy evaluable for response treated with matched therapy evaluable for response abbreviations : ctdna , circulating tumor dna ; gt , genomic target . erbb2 amplification amplification fgfr2 amplification pik3ca mutation no . 
one patient with ctdna - detected erbb2 ( her2 ) amplification ( + + + ) achieved complete remission after six cycles of capecitabine , oxaliplatin , and lapatinib ( fig 2b )  . in nsclc , egfrexon19del ( n = 5 ) , egfrl858r ( n = 2 ) , egfrt790m ( n = 7 ) , and alk fusion ( n = 1 ) were targeted , with 13 patients achieving pr and two sd for a rr of 87% ( 95% ci , 58% to 98% ) and a dcr of 100% ( 95% ci , 75% to 100% ; table 4 , fig 2c )  . 
of the seven patients receiving first - line epidermal growth factor receptor inhibitors ( egfri ) , six achieved pr on afatinib , erlotinib , or gefitinib , whereas the one patient with sd received rociletinib . 
similarly , six egfrt790m patients achieving pr were treated with osimertinib or olmutinib , whereas the patient with sd was treated with afatinib plus insulin - like growth factor ligand monoclonal antibody . 
% clinical status at time of ctdna collection new diagnosis treated with matched therapy evaluable for response second line of therapy treated with matched therapy evaluable for response treated with matched therapy evaluable for response treated with matched therapy evaluable for response abbreviations : ctdna , circulating tumor dna ; gt , genomic target . third or greater line of therapy all patients discussion to our knowledge , this is the first prospective ctdna - guided molecular testing program with objective response evaluated in solid tumors . 
this program guided patients in whom biopsy was not readily available or in whom tumor material was not sufficient for comprehensive sequencing to genomically matched therapies available in practice or clinical trials . 
of 194 patients , 30 ( 15.5% ) were successfully enrolled onto one of the ongoing matched therapy clinical trials , a rate comparable to tumor sequencing - based trials . responses to ctdna - guided matched therapy in gc and nsclc were similar to those published in tissue - based matched therapy studies , although the sample sizes here are modest . in gc , cnas were found in erbb2 ( her2 ) , met , and fgfr2 in 31% of our patients , split evenly between newly diagnosed and pretreated patients , consistent with previous primary tumor estimates of these cnas at 20% to 22%.30 , 31 significantly , four ( 80% ) of five patients with erbb2 ( her2 ) - amplified gc responded , including one cr ( fig 2a ) , with all achieving clinical benefit ( cr , pr , or sd )  . 
the one patient with gc with erbb2 cna without pr ( but with sd ) was on lapatinib monotherapy , raising the question of whether chemotherapy produced most of the benefit here . 
however , addition of lapatinib to chemotherapy did produce a significant overall survival benefit in asian patients in the lapatinib optimization study in her2 - positive gastric cancer ( logic ) study.32 in addition , a patient with refractory colon cancer with erbb2 cna achieved sd as best response ( table 4 )  . 
matched therapy response and disease control rate by cancer cohort response gc ( n = 78 ) nsclc ( n = 73 ) melanoma ( n = 34 ) other ( n = 9 ) no . 
thus , an advantage of ctdna over tissue genotyping is that quantitation of the relative vafs can provide an indication of the subclonality and potentially predict treatment response , in contrast to a binary positive or negative result . 
however , a ratio , 10% may be misleading if there is focal amplification of the egfr driver mutation and not egfrt790m.42 beyond t790m , recent reports suggest that comprehensive profiling at progression may be important in nsclc given the multiple other resistance mechanisms after egfri therapy.43 these include non - egfrt790m on - target point mutations , as well as bypass mutations in braf , kras , mek , and pik3ca ; cnas in met and erbb2 ; fusions in alk ; or rb1 inactivation heralding epithelial to mesenchymal cell transition.34 , 44 - 47 because egfrt790m is the resistance mechanism in only half of patients experiencing progression on firstline egfri , a comprehensive ctdna ngs test covering all major types of genomic alterations is particularly relevant . small sample sizes for targeted therapy in melanoma and colon cancer limit the conclusions that can be drawn in those cohorts ; however , the rr cis in gc and nsclc are consistent with tissue - guided matched therapy rrs . 
single - arm objective rrs exceeding 30% have led to us food and drug administration regulatory approval of matched therapies.48 , 49 the rrs to ctdna - detected alterations in this interim analysis ( 67% [ 95% ci , 31% to 91% ] for gc and 87% [ 95% ci , 58% to 98% ] for nsclc ) support clinical utility for guardant360 in patients with advanced nsclc and gc in whom tissue is insufficient or inaccessible and build upon previous validation studies of the diagnostic test used herein.12 , 26 because this study was not randomized , its primary limitation is the potential for selection bias to enroll patients more likely to benefit . 
not all patients with targetable alterations could receive matched therapy because of the various requirements of the multiple parallel matched therapy substudy protocols , performance status , or loss to follow - up . 
future studies should examine ctdnaguided matched therapy outcomes in more racially diverse cohorts . to our knowledge , this is the first prospective study to examine the clinical utility of comprehensive ctdna genomic testing to guide matched therapy selection . 
lanman , amirali talasaz , keunchil park , jeeyun lee financial support : young suk park , amirali talasaz administrative support : young suk park , amirali talasaz provision of study materials or patients : seung tae kim , se hoon park , joon oh park , young suk park , ho yeong lim , won ki kang , amirali talasaz , keunchil park , jeeyun lee collection and assembly of data : seung tae kim , kimberly c . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . ho yeong lim no relationship to disclose won ki kang no relationship to disclose seung tae kim no relationship to disclose kimberly c . 
banks employment : guardant health stock and other ownership interests : guardant health se - hoon lee honoraria : astrazeneca / medimmune , roche , bristol - myers squibb , merck consulting or advisory role : pfizer , novartis , astrazeneca , bristol - myers squibb travel , accommodations , expenses : novartis kyung kim no relationship to disclose joon oh park honoraria : celgene consulting or advisory role : celgene speakers bureau : celgene research funding : celgene , astrazeneca se hoon park no relationship to disclose young suk park no relationship to disclose richard b . 
lanman employment : guardant health , veracyte leadership : guardant health stock and other ownership interests : guardant health , veracyte research funding : guardant health amirali talasaz employment : guardant health leadership : guardant health stock and other ownership interests : guardant health research funding : guardant health patents , royalties , other intellectual property : guardant health travel , accommodations , expenses : guardant health keunchil park consulting or advisory role : astellas pharma , astrazeneca , boehringer ingelheim , clovis oncology , eli lilly , hanmi , kyowa hakko kirin , novartis , ono pharmaceutical , roche speakers bureau : boehringer ingelheim research funding : astrazeneca jeeyun lee no relationship to disclose affiliations support seung tae kim , se hoon park , kyung kim , joon oh park , se - hoon lee , young suk park , ho yeong lim , won ki kang , keunchil park , and jeeyun lee , samsung medical center , sungkyunkwan university school of medicine , seoul , korea ; and kimberly c . 
j natl compr canc netw 13 : 448 - 475 , 2015 von mehren m , randall rl , benjamin rs , et al : gastrointestinal stromal tumors , version 2.2014. 
sundaresan tk , sequist lv , heymach jv , et al : detection of t790m , the acquired resistance egfr mutation , by tumor biopsy versus noninvasive blood - based analyses . 
thompson jc , yee ss , troxel ab , et al : detection of therapeutically targetable driver and resistance mutations in lung cancer patients by next - generation sequencing of cell - free circulating tumor dna . 
novello s , barlesi f , califano r , et al : metastatic non - small - cell lung cancer : esmo clinical practice guidelines for diagnosis , treatment and follow - up . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
lebofsky r , decraene c , bernard v , et al : circulating tumor dna as a non - invasive substitute to metastasis biopsy for tumor genotyping and personalized medicine in a prospective trial across all tumor types . 
lim sm , kim ey , kim hr , et al : genomic profiling of lung adenocarcinoma patients reveals therapeutic targets and confers clinical benefit when standard molecular testing is negative . 
schrock ab , frampton gm , herndon d , et al : comprehensive genomic profiling identifies frequent drug - sensitive egfr exon 19 deletions in nsclc not identified by prior molecular testing . 
ali sm , hensing t , schrock ab , et al : comprehensive genomic profiling identifies a subset of crizotinib - responsive alkrearranged non - small cell lung cancer not detected by fluorescence in situ hybridization . 
rozenblum ab , ilouze m , dudnik e , et al : clinical impact of hybrid capture - based next - generation sequencing on changes in treatment decisions in lung cancer . 
karachaliou n , mayo - de las casas c , queralt c , et al : association of egfr l858r mutation in circulating free dna with survival in the eurtac trial . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced non - small - cell lung cancer . 
liang dh , ensor je , liu z - b , et al : cell - free dna as a molecular tool for monitoring disease progression and response to therapy in breast cancer patients . 
kim st , lee w - s , lanman rb , et al : prospective blinded study of somatic mutation detection in cell - free dna utilizing a targeted 54 - gene next generation sequencing panel in metastatic solid tumor patients . 
zill oa , mortimer sa , banks kc , et al : somatic genomic landscape of over 15 , 000 patients with advanced - stage cancer from clinical next - generation sequencing analysis of circulating tumor dna . 
zill oa , greene c , sebisanovic d , et al : cell - free dna next - generation sequencing in pancreatobiliary carcinomas . cancer discov 5 : 1040 - 1048 , 2015 30 . 
deng n , goh lk , wang h , et al : a comprehensive survey of genomic alterations in gastric cancer reveals systematic patterns of molecular exclusivity and co - occurrence among distinct therapeutic targets . 
hecht jr , bang y - j , qin sk , et al : lapatinib in combination with capecitabine plus oxaliplatin in human epidermal growth factor receptor 2 - positive advanced or metastatic gastric , esophageal , or gastroesophageal adenocarcinoma : trio - 013 / logica randomized phase iii trial . 
arrieta o , cardona af , martn c , et al : updated frequency of egfr and kras mutations in nonsmall - cell lung cancer in latin america : the latin - american consortium for the investigation of lung cancer ( clicap )  . 
rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as first - line treatment for european patients with advanced egfr mutation - positive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
sequist lv , yang jc - h , yamamoto n , et al : phase iii study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with egfr mutations . 
vallee a , audigier - valette c , herbreteau g , et al : rapid clearance of circulating tumor dna during treatment with azd9291 of a lung cancer patient presenting the resistance egfr t790m mutation . 
marchetti a , palma jf , felicioni l , et al : early prediction of response to tyrosine kinase inhibitors by quantification of egfr mutations in plasma of nsclc patients . 
piotrowska z , niederst mj , karlovich ca , et al : heterogeneity underlies the emergence of egfrt790 wild - type clones following treatment of t790m - positive cancers with a third - generation egfr inhibitor . 
liang w , he q , chen y , et al : metastatic eml4 - alk fusion detected by circulating dna genotyping in an egfrmutated nsclc patient and successful management by adding alk inhibitors : a case report . 
stewart el , tan sz , liu g , et al : known and putative mechanisms of resistance to egfr targeted therapies in nsclc patients with egfr mutations : a review . 
 discordance in human epidermal growth factor receptor 2 ( her2 ) phenotype between primary tumor and circulating tumor cells in women with her2 - negative metastatic breast cancer purpose discordance in human epidermal growth factor receptor 2 ( her2 ) status between primary tumor and metastases might have important implications for treatment response and therapy decisions . 
here , we evaluate both the frequency of circulating tumor cells ( ctcs ) and the factors predicting her2 discordance between primary tumor and ctcs as a potential surrogate for tumor biology and tumor heterogeneity in patients with metastatic breast cancer . patients and methods the number of ctcs in 7.5 ml of peripheral blood and her2 status were evaluated in 1 , 123 women with her2 - negative metastatic breast cancer . 
2017 by american society of clinical oncology introduction circulating tumor cells ( ctcs ) are observed in early and metastatic breast cancer ( mbc ) , and their prognostic relevance has been demonstrated in both early and advanced disease.1 , 2 according to previous studies , approximately 65% to 85% of patients with mbc have detectable ctcs , and approximately half of these patients have five or more ctcs in 7.5 ml of peripheral blood.2 , 3 in addition to the prognostic value of ctcs as assessed before treatment , monitoring ctc dynamics during therapy provides important information on therapy response.4 - 7 however , valuable information with regard to therapy response and suitability of additional treatment options may be provided not only by the prevalence and changes in the number of ctcs but also by the phenotype of ctcs . 
friedl jens huober christoph scholz nikolaus de gregorio brigitte rack elisabeth trapp marianna alunni - fabbroni sabine riethdorf volkmar mueller andreas schneeweiss klaus pantel franziska meier - stiegen bernadette jaeger andreas hartkopf florin - andrei taran peter a . 
assessing ctc phenotype may be especially important in mbc , because it can be used as a noninvasive liquid biopsy to characterize the metastatic disease , thus reflecting tumor heterogeneity and possible discordance in hormone receptor and / or human epidermal growth factor receptor 2 ( her2 ) expression with potentially far - reaching implications in terms of follow - up treatment and additional targeted therapies . although discordance in her2 status among primary tumor , distant metastases , and ctcs has been described in several studies , 3 , 8 - 10 it has not been implemented in clinical practice to guide therapy decisions . 
the study concept was in accordance to the declaration of helsinki , adhered to good clinical practice and german pharmaceutical law , and was approved by the local ethic committees of the participating centers . assessment of ctc presence and her2 phenotype on ctcs from all patients , 7.5 - ml peripheral blood samples were collected , and presence and phenotype of ctcs were assessed as previously described in detail , 11 using the semiautomatic and standardized cellsearch system ( janssen diagnostics , raritan , nj ) , which is the only us food and drug administrationapproved method for the enumeration of ctcs . 
for her2 assessment , cells were also labeled with her2 antibodies , and ctcs were considered as her2 positive only if they showed a strong ihc staining intensity for her2 ( ihc score , 3 + )  . 
analyses for determining ctc prevalence and her2 phenotype of ctcs were performed by trained scientists with a peer - review system at four independent reference laboratories . statistical calculations descriptive statistics for the categorical data are provided in terms of absolute and relative frequencies . 
the non - normally distributed continuous variables age ( in years ) and time interval between primary diagnosis and screening ( in months ) are described by medians and ranges . 
associations between presence of ctcs or presence of her2 - positive ctcs and patient or tumor characteristics were evaluated using mann - whitney u tests for age and time interval between primary diagnosis and screening , and x2 tests were used for all other categorical variables . 
to assess which factors predicted discordance in the her2 phenotype between primary tumor and ctcs , we used a multivariable logistic regression model with discordance in her2 status ( yes v no ) as a binary response variable and a stepwise backward selection procedure ( significance level cutoff for exclusion , .05 [ likelihood ratio test ] )  . 
demographic and primary tumor characteristics of patients with her2negative mbc screened for ctcs within the detect study program ( n = 1 , 123 ) characteristic age at screening , years total no . 
 ( % ) of patients time between primary diagnosis and screening , months median range median range menopausal status at screening premenopausal postmenopausal unknown tumor stage unknown nodal stage unknown histologic grade unknown histologic type invasive ductal invasive lobular other hormone receptor status negative positive unknown 26 - 89 0 - 450 136 ( 12.1 ) 734 ( 65.4 ) 253 ( 22.5 ) 308 ( 27.4 ) 503 ( 44.8 ) 114 ( 10.2 ) 125 ( 11.1 ) 13 ( 1.2 ) 60 ( 5.3 ) 363 ( 32.3 ) 378 ( 33.7 ) 147 ( 13.1 ) 136 ( 12.1 ) 99 ( 8.8 ) 47 ( 4.2 ) 576 ( 51.3 ) 406 ( 36.2 ) 94 ( 8.4 ) 751 ( 66.9 ) 199 ( 17.7 ) 173 ( 15.4 ) 171 ( 15.2 ) 886 ( 78.9 ) 66 ( 5.9 ) had hormone receptorpositive invasive ductal tumors . 
median number of ctcs detected in ctc - positive patients was seven ( interquartile range , two to 30 ; range , one to 35 , 078 ctcs )  . 
frequency distribution of the number of ctcs detected is shown in appendix figure a1 . presence of at least one ctc was significantly associated with nodal stage and histologic type of the primary tumor but not with other patient or primary tumor characteristics ( table 2 )  . 
in 158 patients ( 22.2% ) , at least one ctc with an her2 2 + ihc score , but no ctcs with an her2 3 + ihc score , was detected ; these patients were not considered to have her2 - positive ctcs in the blood ( as described in patients and methods )  . 
patients with grade 3 primary tumors were less likely to have her2 - positive ctcs than patients with grade 1 or 2 primary tumors ( p = .028 ; fig 3a )  . 
patients with invasive lobular primary tumors were more likely to have her2positive ctcs than patients with other primary tumor histologic types ( p , .001 ; fig 3b ) , and patients with hormone receptorpositive primary tumors were more likely to have her2 - positive ctcs than patients with hormone receptor negative ( ie , triple negative ) primary tumors ( p , .001 ; fig 3c )  . 
of her2 - positive ctcs 6 - 10 11 - 50 > 50 predictors of discordance in her2 status between primary tumor and ctcs to evaluate which factors can independently predict discordance in her2 status between primary tumor and ctcs , we used a multivariable logistic regression model with discordance in her2 status ( yes v no ) as a binary response variable . 
patient and primary tumor characteristics included as independent factors were patient age in years , time since primary diagnosis in months , tumor stage ( pt ) , nodal stage ( pn ) , histologic grade , histologic type , and hormone receptor status . 
to account for the association between number of ctcs detected and presence of her2 - positive ctcs , we included number of ctcs ( one to four v > five ctcs ) as an additional explanatory variable . 
please note that the or for age listed in table 3 ( ie , 0.977 ) corresponded to the change of the odds of showing discordance in her2 phenotype associated with a 1 - year increase in age . discussion to our knowledge , the combined ctc screening of the detect study program provides the largest worldwide cohort of patients with mbc tested with regard to both rate of discordance in her2 phenotype between primary tumor and ctcs and possible predictors of her2 discordance . 
our analyses revealed an overall ctc prevalence of 63.3% in 1 , 123 patients with her2 - negative mbc , which is within the range reported in other studies on mbc.2 , 3 , 12 discordance in her2 phenotype between primary tumor and ctcs was observed in 18.8% of 711 ctc - positive patients and was independently predicted by histologic type and hormone receptor status of the primary tumor . 
of ctcs 11 - 50 > 50 6 - 10 11 - 25 > 25 her2 - positive ctcs ( % ) her2 status was more frequent in patients with five or more ctcs compared with patients with fewer than five ctcs . some previous studies have reported even higher discordance rates , although with considerably smaller sample sizes . 
lobular carcinomas are less cohesive tumors that are characterized by impaired cell - cell adhesion with reduced expression of the adhesion molecule e - cadherin.19 , 20 reduced cell and tissue adhesion may lead to an easier spread of tumor cells and could be one explanation for the association of presence of ctcs with lobular carcinomas . 
in general , her2 is rarely amplified or overexpressed in lobular carcinomas21 ; thus , the fact that we observed an association between presence of her2 - positive ctcs and lobular carcinomas is surprising and not easily explained . there is some evidence that lobular breast cancer shows a higher frequency of her2 mutations compared with ductal breast cancer.22 - 24 therefore , it could be speculated that the increased rate of her2 discordance observed in lobular carcinomas might be related to her2 mutations that affect her2 expression and membrane staining . the fact that hormone receptorpositive primary tumors exhibit higher rates of her2 discordance than triple - negative tumors is also a new finding that must be explored in more detail . clearly , additional investigations of tumor biology and pathogenesis of ctcs are necessary to help us understand these results . regardless of the underlying mechanisms , our findings may have implications for clinical practice . 
data on changes in her2 status during tumor progression represent crucial information for adequate patient therapy , because they may lead to modification of systemic and / or addition of targeted treatments . 
on the basis of our results , we suggest physicians consider attempting to obtain additional biopsies of metastatic lesions for detection of potential changes in her2 phenotype , especially in patients with lobular carcinomas or hormone receptorpositive primary tumors , because these are risk factors for her2 discordance identified in our study . 
prevalence of at least one human epidermal growth factor receptor 2 ( her2 ) positive circulating tumor cell ( ctc ; ie , discordance rate of her2 status [ % ] ) according to tumor characteristics of the primary tumor . 
 ( a ) prevalence of at least one her2 - positive ctc according to histologic grade of the primary tumor in 654 ctc - positive patients with her2 - negative metastatic breast cancer ( mbc ) screened for the detect study program ( for 57 patients , histologic grade of the primary tumor was unknown ; table 2 )  . 
 ( b ) prevalence of at least one her2 - positive ctc according to histologic type of the primary tumor in 711 ctc - positive patients with her2 - negative mbc screened for the detect study progra ( c ) prevalence of at least one her2positive ctc according to hormone receptor status of the primary tumor in 670 ctc - positive patients with her2 - negative mbc screened for the detect study program ( for 41 patients , hormone receptor status of the primary tumor was unknown ; table 2 )  . therapy in patients with histologically her2positive mbc revealed discordance of her2 status between tissue and ctcs in 62% of patients , and negative her2 status of ctcs was significantly associated with shorter progressionfree survival after the new line of anti - her2 therapy.25 another recent study indicated that her2 - targeted therapy with lapatinib was effective in decreasing the number of her2positive ctcs in patients with mbc.26 if additional research confirms that ctcs can be used as an easily accessible liquid biopsy to assess changes in tumor biology and her2 status of metastases , detection of her2 - positive ctcs in patients with mbc with her2negative primary tumors might even trigger the addition of her2 - targeted therapies without the need for a confirmatory biopsy of metastatic lesions . 
although primary tumors were considered her2 positive if more than 10% of tumor cells showed protein overexpression , her2 - positive ctc status was defined as presence of at least one ctc with an her2 3 + ihc score in the blood . 
furthermore , because this study was based on the initial screening results of the detect study program , and only a subsample of screened patients was recruited in one of the detect clinical trials , associations between ctc prevalence or presence of her2 - positive ctcs with metastatic disease patterns and patient outcomes could not be analyzed at this stage . 
ctc detection was based on an epithelial cell adhesion molecule selection process ; therefore , ctcs undergoing epithelial - tomesenchymal transition with phenotypic changes in terms of lacking epithelial cell adhesion molecule expression and gaining mesenchymal and / or stem - cell marker characteristics might have been missed , which could have led to falsely low rates of ctc positivity . 
furthermore , her2 status of ctcs was assessed by ihc only and was not confirmed using fish . in conclusion , discordance in her2 status between primary tumor and ctcs was observed in 18.8% of patients with her2 - negative mbc and was associated with lobular carcinomas , hormone receptorpositive primary tumors , and high ctc counts . 
moreover , the concept of liquid biopsy using ctcs as a real - time noninvasive monitoring tool to evaluate tumor biology , progression , and heterogeneity as a basis for more personalized treatment decisions should be tested in prospective randomized clinical trials . 
fasching provision of study material or patients : amelie de gregorio , jens huober , nikolaus de gregorio , brigitte rack , elisabeth trapp , andreas schneeweiss , klaus pantel , andreas hartkopf , peter a . 
fasching , tanja fehm collection and assembly of data : amelie de gregorio , christoph scholz , nikolaus de gregorio , brigitte rack , elisabeth trapp , sabine riethdorf , volkmar mueller , andreas schneeweiss , klaus pantel , franziska meier - stiegen , bernadette jaeger , andreas hartkopf , florin - andrei taran , peter a . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . amelie de gregorio consulting or advisory role : roche pharma ag thomas w.p. 
 jens huober honoraria : novartis , roche consulting or advisory role : novartis , roche travel , accommodations , expenses : novartis , roche christoph scholz consulting or advisory role : roche pharma ag research funding : novartis ( inst ) , roche pharma ag ( inst ) , janssen oncology ( inst ) nikolaus de gregorio consulting or advisory role : roche travel , accommodations , expenses : astrazeneca , tesaro brigitte rack consulting or advisory role : novartis speakers bureau : roche , pfizer research funding : sanofi ( inst ) , novartis ( inst ) , eli lilly ( inst ) , astrazeneca ( inst ) , chugai pharma ( inst ) , janssen diagnostics ( inst ) travel , accommodations , expenses : pfizer elisabeth trapp no relationship to disclose marianna alunni - fabbroni no relationship to disclose volkmar mueller consulting or advisory role : astrazeneca , celgene , daiichi sankyo , eisai , nektar , genentech , novartis travel , accommodations , expenses : pfizer , roche pharma ag andreas schneeweiss honoraria : roche pharma ag , celgene , astrazeneca , pfizer , novartis , amgen , roche pharma ag ( inst ) , pfizer ( inst ) research funding : roche pharma ag ( inst ) , celgene ( inst ) travel , accommodations , expenses : roche , celgene , amgen klaus pantel honoraria : agena bioscience consulting or advisory role : wntresearch , roche , novartis , sanofi research funding : janssen diagnostics franziska meier - stiegen consulting or advisory role : amgen bernadette jaeger no relationship to disclose andreas hartkopf no relationship to disclose florin - andrei taran no relationship to disclose peter a . 
fasching honoraria : roche , amgen , novartis , pfizer , celgene research funding : novartis ( inst ) wolfgang janni honoraria : janssen diagnostics research funding : janssen diagnostics tanja fehm consulting or advisory role : roche , novartis , amgen research funding : novartis acknowledgment we thank all the patients for participating in this study and donating their blood samples for research purposes . 
friedl , jens huober , christoph scholz , nikolaus de gregorio , and wolfgang janni , university hospital ulm , ulm ; brigitte rack , elisabeth trapp , and marianna alunni - fabbroni , hospital of ludwig - maximilians - university , munich ; sabine riethdorf , volkmar mueller , and klaus pantel , university hospital hamburg - eppendorf , hamburg ; andreas schneeweiss , university hospital heidelberg , heidelberg ; franziska meier - stiegen , bernadette jaeger , and tanja fehm , heinrichheine - university duesseldorf , duesseldorf ; andreas hartkopf and florin - andrei taran , university hospital tuebingen , tuebingen ; and peter a . 
fasching , university hospital erlangen , erlangen , germany . affiliations support the detect study program is supported by the investigator - initiated study program of janssen diagnostics , with clinical trials also supported by pierre fabre pharma , teva pharmaceuticals industries , amgen , novartis pharma , and eisai ; study medications vinorelbine , nonpegylated liposomal doxorubicin , lapatinib and everolimus , eribulin , and pertuzumab were provided free of charge by pierre fabre pharma , teva pharmaceuticals industries , novartis pharma , eisai , and roche , respectively . the funding sources had no role in study design ; data collection , analysis , or interpretation ; or writing of the report . 
bidard fc , peeters dj , fehm t , et al : clinical validity of circulating tumour cells in patients with metastatic breast cancer : a pooled analysis of individual patient data . 
fehm t , m uller v , aktas b , et al : her2 status of circulating tumor cells in patients with metastatic breast cancer : a prospective , multicenter trial . 
hayes df , cristofanilli m , budd gt , et al : circulating tumor cells at each follow - up time point during therapy of metastatic breast cancer patients predict progression - free and overall survival . 
pierga jy , hajage d , bachelot t , et al : high independent prognostic and predictive value of circulating tumor cells compared with serum tumor markers in a large prospective trial in first - line chemotherapy for metastatic breast cancer patients . 
simmons c , miller n , geddie w , et al : does confirmatory tumor biopsy alter the management of breast cancer patients with distant metastases ? ann oncol 20 : 1499 - 1504 , 2009 10 . 
ligthart st , bidard fc , decraene c , et al : unbiased quantitative assessment of her - 2 expression of circulating tumor cells in patients with metastatic and non - metastatic breast cancer . 
schramm a , friedl tw , schochter f , et al : therapeutic intervention based on circulating tumor cell phenotype in metastatic breast cancer : concept of the detect study prograarch gynecol obstet 293 : 271 - 281 , 2016 12 . 
tewes m , aktas b , welt a , et al : molecular profiling and predictive value of circulating tumor cells in patients with metastatic breast cancer : an option for monitoring response to breast cancer related therapies . 
wallwiener m , hartkopf ad , riethdorf s , et al : the impact of her2 phenotype of circulating tumor cells in metastatic breast cancer : a retrospective study in 107 patients . 
houssami n , macaskill p , balleine rl , et al : her2 discordance between primary breast cancer and its paired metastasis : tumor biology or test artefact ? insights through meta - analysis . 
aurilio g , disalvatore d , pruneri g , et al : a meta - analysis of oestrogen receptor , progesterone receptor and human epidermal growth factor receptor 2 discordance between primary breast cancer and metastases . 
sinn hp , helmchen b , heil j , et al : lobular neoplasms and invasive lobular breast cancer [ in german ] pathologe 35 : 45 - 53 , 2014 20 . 
gomes ds , porto ss , rocha rm , et al : usefulness and limitations of e - cadherin and b - catenin in the classification of breast carcinomas in situ with mixed pattern . 
ross js , wang k , sheehan ce , et al : relapsed classic e - cadherin ( cdh1 ) - mutated invasive lobular breast cancer shows a high frequency of her2 ( erbb2 ) gene mutations . 
zhang s , li l , wang t , et al : real - time her2 status detected on circulating tumor cells predicts different outcomes of anti - her2 therapy in histologically her2 - positive metastatic breast cancer patients . 
 appendix results additional data regarding both the association between hormone receptor status and histologic type and rates of human epidermal growth factor receptor 2 ( her2 ) discordance in relation to the combined hormone receptor and tumor type status are as follows : hormone receptor status and histologic type of primary tumors were significantly associated with each other ( p , .001 ; n = 670 ; 41 patient cases with missing values for hormone receptor status )  . 
frequency distribution of the number of circulating tumor cells ( ctcs ) detected in 7.5 - ml peripheral blood samples from 1 , 123 patients with human epidermal growth factor receptor 2negative metastatic breast cancer screened for the detect study program ( cutoff for ctc positivity , > one ctc )  . 6 - 10 11 - 50 > 50 no . 
the aim is to characterize the availability and scope of economic evidence . materials and methods we searched medline ( pubmed ) , embase ( ovid ) , and web of science databases for english - language full - text peer - reviewed articles published between 2000 and 2016 . 
we found testing was frequently used to predict prognosis ( 67% ) , to diagnose patients ( 24% ) , or to identify targeted therapeutic options ( 7% )  . 
deterministic and probabilistic analyses were typically used to characterize parameter and decision uncertainty ( 91% and 75% )  . conclusion although the availability of economic evidence examining precision oncology increased over time , methods used often did not align with current guidelines . 
regier , ma , phd , bc cancer research centre , 675 west 10th ave , vancouver , british columbia , canada , v5z 1l3 ; e - mail : dregier@bccrc.ca. introduction precision oncology is transforming routine cancer care . 
historically , cancers have been classified and treated according to their anatomic site of origin.1 now , precision oncology is shifting focus to the individual by using information on a patients genomic profile to better prevent , treat , and prognosticate disease.2 - 4 precision oncology tests range from single - gene tests to more comprehensive strategies involving next - generation sequencing ( ngs ) technologies . 
although recent scientific advances have resulted in improvements in speed and throughput of ngs technologies , 5 the state of current economic evidence supporting resource allocation decisions in cancer control is unknown . past studies reviewing economic evaluations surrounding personalized medicine found that few tests had been formally evaluated for their cost effectiveness as of 2013.6 - 8 the majority of reviewed studies examined the trade off between costs and effectiveness of older single - gene tests . 
 determine expression profiles for large numbers of genes . proponents of precision oncology report that genomic testing has the potential to reduce health system costs and improve patients health through targeted therapies and treatment stratification.11 , 12 yet , genomic testing will also involve significant expenditures that may challenge the affordability of adopting ngs technologies in routine practice . 
in text : precision * or personalized * or individualized * or sequenc * or next generation or genetic * or genom * or companion diag * or predictive testing or gene or genotype or mutation and 3 . 
in text : oncology or cancer these search terms were developed on the basis of relevant articles identified in exploratory searches and search terms used in prior systematic reviews of economic evaluations of personalized medicine and ngs technologies.6 - 8 after initial identification , we imported all articles into endnote x6 ( reuters , new york )  . 
 quality assessment of the studies was restricted to analyzing transparency . given the differences across included studies , we summarized cost - effectiveness findings according to ngs technology , test strategy , and cancer type . 
we compared incremental cost effectiveness , expressed in terms of cost per quality adjusted life - year ( qaly ) gained separately from incremental cost effectiveness expressed in terms of cost per life - year gained ( lyg )  . 
all monetary outcomes were converted to 2016 us dollars using country - specific inflation and exchange rates . results search results the database search identified 1 , 350 records , of which 577 were duplicates . 
ngs , next - generation sequencing . records identified through database searching ( n = 1 , 350 ) additional records identified through other sources ( n = 6 ) records after duplicates removed ( n = 779 ) records excluded ( conference abstracts ; n = 238 ) records screened for eligibility ( n = 541 ) records excluded ( unrelated to ngs or evaluation incomplete ; n = 446 ) full - text articles assessed for eligibility ( n = 95 ) studies included in qualitative synthesis ( n = 55 ) full - text articles excluded ( n = 40 ) unable to access or not in english ( n = 5 ) evaluation incomplete ( n = 10 ) not ngs ( n = 24 ) reported duplicate results ( n = 1 ) most common reasons for exclusion were that studies did not focus on ngs technologies ( n = 24 ) or did not include full economic evaluations comparing costs and health consequences of two or more competing technologies ( n = 10 )  . 
approximately 20% to 25% metastasize to distant sites and are generally associated with poor response to treatment.1 outcomes with systemic chemotherapy have historically been poor.2 with improved understanding of molecular biology of these tumors , we are beginning to identify promising potential therapeutic targets . in particular , the human epidermal growth factor receptor 2 / neu ( her2 / neu ) oncogene , one of the tyrosine kinase receptors , is overexpressed in salivary duct carcinomas ( 44% ) , salivary gland adenocarcinomas ( 21% ) , and adenoid cystic carcinomas ( 2% ) , among other subtypes.3 trastuzumab , a monoclonal antibody that binds to the extracellular domain of her2 / neu , inhibits the proliferation of tumor cells that overexpress her2 / neu.4 patients with her2 / neu - overexpressing sgns are often treated with trastuzumab - containing regimens with encouraging results , but disease progression is inevitable , even with a favorable initial response . 
the antibody - drug conjugate adotrastuzumab emtansine ( t - dm1 ) is currently approved for treating her2 - positive metastatic breast cancer that has progressed on therapy with trastuzumab . this antibody - drug conjugate selectively directs the cytotoxic drug emtansine to tumor cells with her2 / neu expression , thereby minimizing systemic toxicities.5 results favorable given the seen with adotrastuzumab emtansine in patients with breast cancer , it is logical to consider administering it to patients with sgns that overexpress her2 / neu after they have progressed on trastuzumab with or without chemotherapy . 
sequential her2 / neu - directed therapy has been successfully used with sustained clinical benet , albeit in a single patient.6 here we report on the clinical outcomes of seven patients with metastatic sgns who were treated at our institution with adotrastuzumab emtansine ( table 1 ) , and we summarize the clinical course and response to therapy of one patient . a 45 - year - old man presented with a painless right neck mass in late 2013 . 
fine - needle aspiration revealed a high - grade carcinoma and positron emission tomography / ct ( pet / ct ) showed multiple uorodeoxyglucose ( fdg ) - avid lymph nodes in the right cervical region . 
surgical pathology revealed a 3.9 - cm high - grade salivary duct carcinoma that was positive for androgen receptor ( ar ) and her2 / neu ( 3 + ) by immunohistochemistry ( ihc ) staining . 
because of the high - risk features of extracapsular nodal extension and close margins , he received adjuvant chemotherapy with cisplatin and radiation therapy once per week in october 2014 . 
 ( c ) restaging scan in june 2018 after ado - trastuzumab emtansine shows a complete metabolic response of previously noted metastatic lymphadenopathy . currently a void in the therapeutic guidelines for managing this disease . 
standard treatment of sgns involves surgical resection with or without adjuvant radiation therapy . locoregional recurrence and metastases are common , at which time palliative chemotherapy is considered , but response rates are generally poor , and the duration of response is brief without a clear favorable impact on survival . 
given the aggressive nature of these tumors and the limited treatment options upon relapse and metastasis , there has been an increased interest in developing targeted therapies . two such targets , the ar and her2 / neu receptor tyrosine kinase , are currently used in clinical practice with variable results.7 a relatively large retrospective case series has reported favorable outcomes using androgen deprivation therapy in ar - positive sgns with good tolerability and an overall survival benet.8 the success of her2 / neu - directed therapy in breast cancer has resulted in multiple case reports that show benet from using trastuzumab - based regimens in treating sgns.4 , 9 despite initial success with targeted therapy , however , progression is inevitable . mechanisms of resistance have not been extensively explored . 
therefore , it seems reasonable to consider the use of ado - trastuzumab emtansine upon progression on trastuzumab , drawing on its demonstrated activity and clinical benet in the analogous clinical scenario in patients with advanced her2 / neu - positive breast cancer.10 by contrast to the previous reports , we describe one of the largest single - institution experiences with her2 / neutargeted therapy in patients with metastatic sgns . 
most of our patients ( n = 6 of 7 ) experienced progression after trastuzumab , which highlights the role of sequential her2 / neu therapy described by almquist et al.6 all of our patients tumors demonstrated her2 / neu overexpression , and most had a high degree of her2 / neu receptor positivity . in our retrospective case series , there was variability in the methods for her2 / neu testing that reect the natural evolution of institutional testing practices . 
nevertheless , clinical benet was seen regardless of the method of testing diagnosed with high - grade right parotid salivary carcinoma solitary liver metastasis retroperitoneal lymph node metastases progression of retroperitoneal lymph node metastases surgical resection with modied radical neck dissection left hepatic lobectomy cisplatin and rt bicalutamide and leuprolide carboplatin and paclitaxel ado - trastuzumab emtansine trastuzumab and pertuzumab maintenance time ( months ) fig 2 . 
 swed , cohen , and aggarwal ( ihc , uorescence in situ hybridization , or next - generation sequencing ) , conrming the role of a her2 / neu - targeted approach . 
for example , the patient discussed here initially experienced a complete metabolic and radiographic response and derived a sustained clinical benet with a progression - free survival of 29 months that is ongoing . progression - free survival in all of our patients ranged from 6 to 29 months , and overall survival ranged from 9 to 33 months . 
all of the patients experienced varying degrees of neuropathy , which proved to be dose - limiting in patient 2 . thrombocytopenia was another adverse effect of therapy , which led to interruption of therapy in one patient . 
cohen honoraria : bristol - myers squibb consulting or advisory role : heat biologics , takeda pharmaceuticals , cerulean pharma , kolltan pharmaceuticals , zymeworks , pzer research funding : heat biologics ( inst ) , macrogenics ( inst ) , merck ( inst ) , takeda pharmaceuticals ( inst ) , cleave biosciences ( inst ) , celldex ( inst ) travel , accommodations , expenses : heat biologics takeda pharmaceuticals , kolltan pharmaceuticals , cerulean pharma , zymeworks , bristol - myers squibb , pzer charu aggarwal consulting or advisory role : genentech , bristol - myers squibb , eli lilly , celgene , medimmune research funding : genentech ( inst ) , incyte ( inst ) , macrogenics ( inst ) , merck sharp & dohme ( inst ) , astrazeneca / medimmune ( inst ) no other potential conicts of interest were reported . references bradley pj : distant metastases from salivary glands cancer . 
otolaryngol head neck surg 154 : 1041 - 1046 , 2016 alotaibi am , alqarni ma , alnobi a , et al : human epidermal growth factor receptor 2 ( her2 / neu ) in salivary gland carcinomas : a review of literature . 
j clin diagn res 9 : ze04 - ze08 , 2015 limaye sa , posner mr , krane jf , et al : trastuzumab for the treatment of salivary duct carcinoma . 
oncologist 18 : 294 - 300 , 2013 girish s , gupta m , wang b , et al : clinical pharmacology of trastuzumab emtansine ( t - dm1 ) : an antibody - drug conjugate in development for the treatment of her2 - positive cancer . 
cancer chemother pharmacol 69 : 1229 - 1240 , 2012 almquist d , umakanthan jm , ganti ak : sequential her2 - targeted therapy in salivary ductal carcinoma with her2 / neu overexpression and a concomitant erbb2 mutation . 
head neck pathol 12 : 95 - 104 , 2018 boon e , van boxtel w , buter j , et al : androgen deprivation therapy for androgen receptor - positive advanced salivary duct carcinoma : a nationwide case series of 35 patients in the netherlands . 
head neck 40 : 605 - 613 , 2018 van boxtel w , boon e , weijs wlj , et al : combination of docetaxel , trastuzumab and pertuzumab or treatment with trastuzumab - emtansine for metastatic salivary duct carcinoma . 
baron jm , boster bl , barnett cm : ado - trastuzumab emtansine ( t - dm1 ) : a novel antibody - drug conjugate for the treatment of her2 - positive metastatic breast cancer . 
hyman , md1 , 2 purpose matching patients to investigational therapies requires new tools to support physician decision making . we designed and implemented precision insight support engine ( precise ) , an automated , just - in - time , clinical - grade informatics platform to identify and dynamically track patients on the basis of molecular and clinical criteria . 
median time from sequencing to enrollment was 163 days ( interquartile range , 66 to 357 days ) , and from precise identication to enrollment 87 days ( interquartile range , 37 to 180 days )  . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction genomic data are increasingly used to guide both routine and investigational treatment decisions for patients with cancer . 
simultaneously , clinically validated broad next - generation sequencing platforms that permit the detection of multiple potentially actionable alterations are now accessible at the point of care.1 , 2 these improvements , however , have not been accompanied by commensurate advancements in the systems that are available to help physicians interpret and act on these data . indeed , previous experience with patients with advanced solid tumor who undergo genome proling suggests that only a minority5% to 24% , depending on the breadth of sequencing and practice settingare subsequently enrolled in genome - matched trials.3 - 8 numerous challenges exist in identifying and enrolling patients in appropriate genome - driven clinical trials . clinicians must correctly interpret sequencing data not only at the time of the initial results , but also longitudinally , as our understanding of genomic biomarkers and associated clinical evidence continuously evolves.9 enrolling patients in trials also requires access to , and up - to - date knowledge of , a changing portfolio of studies , each with its own study - specic eligibility criteria and dynamic slot availability . 
finally , all of this information must be readily accessible to the clinician at critical decision points in a patients care . to address these challenges , multiple strategies have emerged to facilitate matching patients to genomedriven clinical trials ( appendix table a1 )  . 
 tao et al context key objective new strategies to facilitate matching patients to genome - driven trials are needed to support physician decision making . precision insight support engine ( precise ) is an automated , just - in - time , clinical - grade informatics platform that identies and dynamically tracks patients on the basis of molecular and clinical criteria . 
to our knowledge , this represents the rst effort to evaluate outcomes of a bioinformatics patienttrial matching platform . knowledge generated precise identied nearly one half ( 43% ) of all enrollment in early - phase , genome - driven studies across a wide variety of tumor types and molecular alterations , with a 5 - month median time from sequencing to enrollment . 
a major area for improving matching efcacy is better integration of nonstructured , clinical eligibility criteria . relevance use of automated bioinformatics platforms represents an important means by which to increase clinical trial accrual and deliver precision oncology care to patients . straightforward of these approaches involves directly annotating molecular sequencing reports at initial sign - out to indicate the clinical signicance of each alteration and list potentially suitable clinical trials . 
multiple initiatives are underway to harmonize the annotation of individual variants and incorporate multitiered levels of evidence for actionability.10 - 12 some commercial laboratories and academic institutions have created on - demand molecular tumor boards that review molecular sequencing reports to make treatment recommendations and manually curate existing clinical trials for alterations of interest.13 all of these approaches have potential limitations . 
similarly , molecular tumor boards are time consuming , difcult to scale , and potentially inuenced by participants knowledge base and anecdotal experience . in response to these limitations and to address the ongoing unmet need of matching patients to clinical trials at our center , we created precision insight support engine ( precise ) , an automated , just - in - time , clinical - grade informatics platform to identify and dynamically track patients on the basis of molecular and clinical criteria . precise was designed to empower clinical investigators to proactively identify and recruit optimal candidates to their genome - driven trials . 
initially , limited proling data were generated via a mass spectrometrybased hotspot assay covering eight genes ( massarray ; sequenom , san diego , ca ) or an amplicon - based next - generation sequencing test covering 48 genes ( truseq amplicon cancer panel ; illumina , san diego , ca ) .14 a customized hybrid capture - based next - generation sequencing assay ( mskimpact ) was later performed per previously published methods.15 msk - impact can identify all classes of genomic alterationssingle - nucleotide variants , indels , copy number alterations , and select structural rearrangementsin up to 468 genes , depending on the assay version . 
these data are used for clinical report sign - out and delivery to the data warehouse that precise uses for patient identication.16 , 17 sequenced patients had cancer types for which proling was considered routine at the time performed or underwent clinical testing under a prospective institutional review boardapproved protocol that was designed to evaluate the utility of proling in these cancer types ( clinicaltrials.gov identier : nct01775072 )  . precise platform the precise platform was available to any principal investigator of an irb - approved therapeutic study that included molecular eligibility criteria . 
 outcomes of a physician support system for genome - driven oncology the functionality of the precise platform evolved during the study period on the basis of user feedback ( fig 1 )  . 
beginning in 2014 , precise was enhanced to offer notications , customized per study , of potentially clinically important events that could prompt a treatment change , such as upcoming appointments or restaging scans . 
for most of the study duration , precise notications were provided directly to the research team , which empowered them to further screen potential patients for appropriateness and notify treating physicians accordingly . 
use of this direct - to - primary oncologist notication function was at the discretion of the research teaall notications to the research team and the primary oncologist were generated as emails and did not directly integrate with the electronic medical record . cohort eligibility , data collection , and statistical analysis all precise cohorts for early - phase studiespilot , phase i , and phase i and iithat included at least one genomic eligibility criterion and that were created after april 16 , 2014 , were included for analysis . 
an internally developed mpath application was used to obtain the date that sequencing data was signed out and entered into the medical record for each patient , as well as the specic qualifying genomic alteration present . the msk institutional review board evaluated and approved a retrospective research protocol to evaluate precise platform outcomes . 
primary outcome measure was to determine what proportion of patients who were enrolled in the evaluated genome - driven studies was facilitated by precise . for the purpose of this analysis , enrollment was considered to be facilitated if precise identied the patient as eligible and generated a notication to either the research team or the primary oncologist before the enrollment date . 
to identify areas for future precise functionality enhancement , reasons for the failure of precise identify eligible patients before study enrollment were also explored through manual record review . in the relevant results study characteristics a total of 41 therapeutic trials used precise for genomic matching during the study period ( table 1 )  . 
the initial iteration ( version 1 ) of precise involved generating cohorts on the basis of complex genetic and clinical criteria dened by the studys principal investigator ( pi ) , which could then be sent to the pi at dened intervals . 
the capability of precise was later enhanced ( version 2 ) to enable pi notications triggered by certain events of interest , such as an upcoming patient appointment or computed tomography scan . 
present day ( version 3 ) precise can also incorporate a patients prior treatment history and allows for direct notication of the patients treating oncologist that a patient may be eligible for a study , often prompting an exchange between the treating oncologist and pi that initiates the patients future enrollment . 
of investigational agents antibody ( nonimmunotherapy ) therapeutic class * small molecule immunotherapy tumor type eligibility multiple breast lung ( nonsmall cell ) lymphoma mesothelioma glioma sarcoma prostate thoracic breast 5 - 10 10 - 15  . 
eight of 41 trials used the direct - to - oncologist notication system . principal investigator characteristics the 41 therapeutic trials were led by 19 unique principal investigators ( pis ) who individually led between one and ve studies ( table 1 )  . 
median time since the completion of terminal subspecialty training for pis was 7 years ( range , 2 to 27 years )  . patient matching during the study period , a total of 755 patients who were treated primarily by 150 unique oncologists were enrolled in 41 trials . 
precise prospectively identied 43% ( 327 of 755 ) of cases before patient enrollment , successfully notifying study investigators and / or the primary oncologist of the potential match ( fig 2 )  . 
reecting eligibility criteria among the trials , breast cancer ( 76 [ 23% ] of 327 ) and lung cancer ( 55 [ 17% ] of 327 ) were the most common tumor types among enrolled patients identied by precise . 
accounting for patients with more than one eligible molecular alteration , erbb2 ( 71 [ 21% ] of 335 ) was the most common genomic alteration , followed by pik3ca ( 47 [ 14% ] of 335 )  . at the individual protocol level , the percent of patients identied by precise before enrollment ranged from 0% to 100% . 
patients in whom the genomic alteration that ultimately led to enrollment did not meet the pre - established precise molecular criteria , as dened by the investigator , accounted for 33% ( 140 of 428 ) of missed cases . 
a lack of internal sequencing at the time of accrual accounted for 30% ( 127 of 428 ) of missed cases , predominantly among patients who enrolled on the basis of genomic proling that was performed outside the institution and was therefore not available for parsing by precise . 
another 23% ( 100 of 428 ) of cases was missed because precise cohort criteria that would have included the patient were amended only after the time of patient enrollment . 
several other technical and clinical reasons accounted for the remaining 14% ( 61 of 428 ) of cases , the majority of which ( 50 of 61 ) consisted of patients who did not meet clinical criteria available for * the total adds up to more than 41 as studies may include more than one agent class . twelve principal investigators had dual afliations with a diseasespecic group and the phase i group . one half ( 22 [ 54% ] of 41 ) of studies were phase i , and 61% ( 25 of 41 ) included multiple tumor types . 
of diseasespecic studies , the most common tumor types included breast cancer ( ve [ 12% ] of 41 ) and nonsmall - cell lung cancer ( four [ 10% ] of 41 )  . 
 ( b ) each column depicts patient enrollments by study principal investigator , with patient enrollment facilitated by precise shaded in blue and patient enrollment not facilitated by precise shaded in red . 
 ( c ) each column represents a unique study , with the absolute number of patients in each category labeled above ( non - precise ) and below ( precise enrollment ) each column . capture by precise , such as presence of triple - negative breast cancer , which was not always readily documented in the medical record . to further understand how physicians and patients use tumor genomic data to guide investigational treatment decisions , we evaluated the timing of enrollment relative to two important milestones : the completion of sequencing and the initial precise identication . 
upon evaluation of the 327 patients who were identied by precise and successfully enrolled in a genome - driven study , there was signicant variability in time intervals between these three events at the individual patient level . 
median time from sequencing to therapeutic enrollment was 163 days ( interquartile range , 66 to 357 days ; range , 5 to 1 , 281 days ) and from precise identication to enrollment 87 days ( interquartile range , 37 to 180 days ; range , 1 to 850 days ; fig 3 )  . 
reasons for delay from sequencing and precise identication to study enrollment included the availability of alternative routine therapy or a lack of need for treatment among patients without evidence of active disease . causes of patient attrition before enrollment to better understand the reasons why patients who were identied by precise did not subsequently enroll in the relevant therapeutic study , a representative cohort involving a multitumor phase i and ii expansion study of a targeted small molecule was selected for manual record review . 
of these 98 patients , 22 were immediately determined to be permanently ineligible or excluded as a result of a static characteristic that rendered the patient ineligible for the trial indenitely . 
these included having a second primary cancer ( n = 6 ) , a nonqualifying malignancy ( n = 6 ) , being deceased but not listed as such in the medical record ( n = 5 ) , and other reasons ( n = 5 )  . 
another 45% ( 10 of 22 ) involved a data element that was sometimes availablesecond primary cancer , prohibited prior therapy , and hiv / aidsfor use but not under all circumstances . 
for example , exclusions on the basis of prior therapy will miss agents administered at other medical centers or some oral anticancer agents , especially if dispensed by third - party pharmacies . after permanent exclusions , 76 potentially eligible patients remained . 
upon manual review , 78% ( 59 of 76 ) of patients did not immediately qualify for treatment at the time of the initial precise identication on the basis of not requiring active therapy ( n = 44 ) or ongoing response to current therapy ( n = 15 )  . 
collectively , 84% ( 59 of 70 ) of these temporary exclusion criteria are not consistently available for use by precise , primarily because the current disease status of the patient is not captured as a structured data element in the medical record . 
 a 2 , 000 1 , 500 1 , 000 500 1 , 000 1 , 500 outcomes of a physician support system for genome - driven oncology 1 , 400 1 , 200 1 , 000 sequencing to enrollment identification to enrollment patient sequencing identified by precise enrollment fig 3 . 
each series of three dots on a single vertical axis represents a single patients course , from initial sequencing ( blue dot ) to identication by precise ( precision insight support engine ; red dot ) to enrollment in the study ( gray dot )  . 
 ( b ) box and whisker plot showing the interval of time from sequencing to enrollment in the study ( left ; median , 163 days ) and from identication by precise to enrollment in the study ( right ; median , 87 days )  . therapies , rapidly deteriorating , or deemed inappropriate because of other issues , such as prior nonadherence . 
reasons for permanent or temporary exclusion from study qualication are depicted in blue ( criteria that is available for use by precise ) , red ( criteria that is sometimes available and could potentially be captured as an extractible structured data element ) , or teal ( criteria that is not yet available for use by precise )  . 
to our knowledge , this report represents the rst effort to evaluate real - world outcomes of an automated , just - in - time , clinicalgrade informatics platform to facilitate patient matching . we also discovered that a signicant time interval often elapses between the initial generation of tumor genomic data and subsequent enrollment in a matched study . specically , among those who ultimately accrue to a matched study , median duration from data generation to enrollment was 5 months , with some outliers enrolling up to 42 months later . 
these data emphasize the importance of supporting physician decision making longitudinally through a patients entire treatment . although these pilot data are encouraging , our analysis also identied areas of ongoing challenge for such matching systems as precise . 
of importance , nearly three quarters of initially identied patients were not immediately eligible on the basis of clinical factors that were not readily available for use by precise , most commonly related to challenges in algorithmically dening patient disease status . 
the result is that precise had a high false - positive rate that may ultimately limit the utility of this system for some indications , particularly for recruiting patients with more prevalent genomic alterations . 
indeed , a previous analysis of overall match rates at our institution that was conducted during roughly the same time period found a match rate of only 11% , despite 37% of patients harboring a potentially actionable alteration.5 taken together , these data demonstrate that even with the use of a sophisticated decision support system , there is an ongoing need for additional improvement in the methodologies to match patients to clinical trials on the basis of tumor genomic and clinical information . 
this requires developing agreed - upon standards , including discrete , structured criteria , for extracting clinical data from the electronic medical record . in the future , leveraging natural language processing and information extraction technologies may also enhance the ability to accurately capture unstructured data . this analysis has some important limitations . 
foremost , we considered any patient about whom precise successfully notied the pi or treating physician of potential eligibility before enrollment as an accrual that was potentially facilitated by the systehowever , we cannot determine exactly how many of these enrollments might have occurred without the use of this systeindeed , our study was retrospective in design and we cannot denitively make conclusions on the incremental value of precise . 
moreover , this analysis represents real - world use of precise by each pi , who were responsible for setting his or her own cohort criteria and using the results as he or she felt best complimented the practice . 
additional evaluation is needed to determine how the utility of this system is affected by recent features , such as full automation of direct - to - treating physician alerts triggered by critical events , like scans that show progression or rising tumor markers . precise is not the only system designed to address the emerging need of matching patients to relevant clinical trials on the basis of the patients genomic prole . 
several other centers have developed strategies by which to achieve this goal that range from on - site or virtual molecular tumor boards to automated matching platforms13 , 18 - 23 ( appendix table a1 )  . 
potential advantages of an automated informatics approach include scalability and the ability to track patients longitudinally and respond to changing molecular and clinical data . in summary , this pilot study of real - world use of precise to guide enrollment in genome - driven studies suggests that this type of real - time decision support system can meaningfully facilitate patient enrollment . 
harding consulting or advisory role : bristol - myers squibb , cytomx therapeutics , eli lilly , eisai research funding : bristol - myers squibb ( inst ) , pzer ( inst ) , eli lilly ( inst ) , novartis ( inst ) , incyte ( inst ) , calithera biosciences ( inst ) , polaris ( inst ) lillian m . 
smyth honoraria : astrazeneca , pzer consulting or advisory role : astrazeneca , genentech research funding : astrazeneca ( inst ) , genentech ( inst ) , puma biotechnology ( inst ) travel , accommodations , expenses : pzer , genentech , puma biotechnology research funding : novartis ( inst ) , genentech ( inst ) , debio pharmaceuticals ( inst ) , adc therapeutics ( inst ) , pzer ( inst ) , novita pharmaceuticals ( inst ) , clovis oncology ( inst ) , eli lilly ( inst ) , zymeworks ( inst ) travel , accommodations , expenses : taiho pharmaceutical , jounce therapeutics , pzer , astrazeneca other relationship : novartis , pzer , taiho pharmaceutical , jounce therapeutics alexander drilon honoraria : medscape , onclive , peervoice , physicians education resources , targeted oncology , more health , research to practice , foundation medicine , peerview consulting or advisory role : ignyta , loxo , tp therapeutics , astrazeneca , pzer , blueprint medicines , genentech , helsinn therapeutics , beigene , hengrui therapeutics , exelixis , bayer , tyra biosciences , verastem , takeda , ariad pharmaceuticals , millennium pharmaceuticals , bergenbio , more health , eli lilly research funding : foundation medicine patents , royalties , other intellectual property : wolters kluwer ( royalties for pocket oncology ) other relationship : merck , glaxosmithkline , teva pharmaceuticals , taiho pharmaceutical , pzer , pharmamar , puma biotechnology marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network / astrazeneca tagrisso rfp advisory committee , takeda , bristol - myers squibb , bayer , merck ( i ) research funding : loxo ( inst ) , helsinn therapeutics david b . 
solit stock and other ownership interests : loxo consulting or advisory role : pzer , loxo , illumina , intezyne technologies , vivideon therapeutics travel , accommodations , expenses : merck michael f . 
jhaveri consulting or advisory role : novartis , pzer , spectrum pharmaceuticals , astrazeneca , taiho pharmaceutical , jounce therapeutics , adc therapeutics , synthon , intellisphere , bristol - myers squibb acknowledgment the authors thank alisa pinkhasik for assistance with study data . 
for providing inspiration for this report . references 33 : 2753 - 2762 , 2015 schram am , berger mf , hyman dm : precision oncology : charting a path forward to broader deployment of genomic proling . 
plos med 14 : e1002242 , 2017 cheng ml , berger mf , hyman dm , et al : clinical tumour sequencing for precision oncology : time for a universal strategy . 
nat rev cancer 18 : 527 - 528 , 2018 jordan ej , kim hr , arcila me , et al : prospective comprehensive molecular characterization of lung adenocarcinomas for efcient patient matching to approved and emerging therapies . 
j clin oncol zehir a , benayed r , shah rh , et al : mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10 , 000 patients . 
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mantripragada kc , olszewski aj , schumacher a , et al : clinical trial accrual targeting genomic alterations after next - generation sequencing at a non - national cancer institute - designated cancer prograj oncol pract 12 : e396 - e404 , 2016 8 . 
massard c , michiels s , fert e c , et al : high - throughput genomics and clinical outcome in hard - to - treat advanced cancers : results of the moscato 01 trial . cancer discov 7 : 586 - 595 , 2017 schram am , reales d , galle j , et al : oncologist use and perception of large panel next - generation tumor sequencing . 
taylor ad , micheel cm , anderson ia , et al : the path ( way ) less traveled : a pathway - oriented approach to providing information about precision cancer medicine on my cancer genome . 
mateo j , chakravarty d , dienstmann r , et al : a framework to rank genomic alterations as targets for cancer precision medicine : the esmo scale for clinical actionability of molecular targets ( escat )  . 
ann oncol 29 : 1895 - 1902 , 2018 johnson a , zeng j , bailey am , et al : the right drugs at the right time for the right patient : the md anderson precision oncology decision support platfordrug discov today 20 : 1433 - 1438 , 2015 14 . 
rekhtman n , paik pk , arcila me , et al : clarifying the spectrum of driver oncogene mutations in biomarker - veried squamous carcinoma of lung : lack of egfr / kras and presence of pik3ca / akt1 mutations . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
lindsay j , fitz cdv , zwiesler z , et al : matchminer : an open source computational platform for real - time matching of cancer patients to precision medicine clinical trials using genomic and clinical criteria . 
tao j , eubank mh , pamer e , et al : precise : a clinical - grade automated molecular eligibility screening and just - in - time ( jit ) physician decision support solution for molecularly - selected oncology trials . 
notications are generated from scheduled queries and use an open - source java e - mail package implemented as a database user - dened function to send emails from the sql statement . 
a web application using open - source javascript libraries allows system administrators to capture cohort logic and settings and provides end users the ability to annotate patient status for their study . 
 ( a ) each column depicts patient enrollments by study pi , with pis with the most years in practice on the left and the fewest years in practice on the right . 
 ntrk alterations in pediatric highrisk malignancies identied through european clinical sequencing programs constitute promising drug targets over the last 2 years , two neurotrophic - tropomyosin receptor tyrosine kinase ( ntrk ) inhibitors have been approved for a tissue - agnostic indication in adults and children , namely , larotrectinib and entrectinib both in the united states and europe . 
although ntrkfusionpositive malignancies are rare , patients with tumors harboring this alteration may greatly benet from those innovative targeted therapies . molecular characterization platforms have been set up in europe over the last 5 years to identify targetable alterations in tumors of individual pediatric patients and to guide therapeutic prioritization . 
mappyacts , coordinated in france ( molecular proling for pediatric and young adult cancer treatment stratication ; clinicaltrials.gov identier : nct02613962 ) , and in ( individualized form , coordinated in germany therapy for relapsed malignancies in childhood ; eudract number : 2018 - 000127 - 14 ) , are the largest european efforts , offering real - time clinical sequencing to relapsed or refractory or very high - risk pediatric patients from 13 european countries.1 - 3 they are part of the innovative therapies for children with cancer ( itcc ) precision medicine and new drug development program , matching patients tumor molecular proles to new drugs in early clinical trials . alterations of the ntrk 1 - 3 were recently investigated in approximately 3 , 500 pediatric tumor samples across tumor types , with fusions involving these genes being detected in 0.34% of samples and amplication , mutation , and mrna overexpression occurring less frequently.4 although ntrk fusions are druggable by selective ( eg , larotrectinib and entrectinib ) and less specic small molecules , 4 - 11 reports on response of nonfusion ntrk alterations to ntrk inhibitors ( ntrki ) are lacking to date.4 since its initiation in january 2016 until july 2020 , approximately 690 patients with relapsed , refractory , or very high - risk pediatric cancer have been screened through mappyacts , whereas approximately 1 , 100 patients were screened through the inform registry from february 2015 until july 2020 . 
apart in mappyacts patients inform who were included because of being very high risk at rst diagnosis , all other tumor samples were obtained at relapse or progression ( table 1 )  . ntrk fusions were the most frequent alterations ( n = 25 ) , with ntrk1 , 2 , 3 being involved in 9 / 25 , 8 / 25 , and 8 / 25 patients , respectively . 
even for previously not described fusions , the similar structure to known ntrk fusions , with conservation of the tyrosine kinase domain , supports the hypothesis of an activation of the tyrosine kinase domain of ntrk . single nucleotide variants ( snvs ) were detected in 14 tumors ( 3 ntrk1 , 7 ntrk2 , and 4 ntrk3 ) , with six snvs being located in the kinase domain of the respective protein , one snv each in the immunoglobulin i - set domain and the leucine - rich domain , respectively , and the remaining six snvs falling outside of annotated functional domains . two cases harbored a focal amplication of ntrk1 genomic locus . a total of 17 patients with tumors harboring an ntrk fusion were treated with the specic ntrki larotrectinib . 
of these , four patients from germany , four patients from sweden , four patients from france , and one patient from poland were enrolled in an early phase clinical trial . 
complete resection of the tumor , stabilization with conventional therapy , missing availability of the drug ( before the initiation of pediatric trials ) , and loss to follow - up were stated as reasons not to treat with ntrki . 
one patient with ntrk1 amplication as nonfusion ntrk alteration received larotrectinib in a clinical trial setting . through this pan - european initiative , we demonstrate the feasibility and value of next - generation sequencing to identify children and adolescents with an ntrk tumor alteration - harboring malignancy ( across all types ) who may be eligible for ntrki therapy . addition , it allows the capture of all potential ntrk fusion partners and novel snvs in functionally relevant domains of ntrk . 
the current development of additional pediatric sequencing programs ( ie , smpaeds in the united kingdom , ither in the netherlands , and the danish program ) as part of the itcc strategy will provide an expanded access to targeted agents for children and adolescents across europe . elke pfaff , md hopp childrens cancer center heidelberg ( kitz ) , heidelberg , germany ; pediatric glioma research group , german cancer research center ( dkfz ) , heidelberg , germany ; department of pediatric oncology , hematology and immunology , heidelberg university hospital , heidelberg , germany tiphaine adam de beaumais , md clinical research department , gustave roussy cancer center , villejuif , france antonin marchais , phd bioinformatics and inserm u1015 , gustave roussy cancer center , university paris - saclay , villejuif , france cornelis m . 
van tilburg , md hopp childrens cancer center heidelberg ( kitz ) , heidelberg , germany ; department of pediatric oncology , hematology and immunology , heidelberg university hospital , heidelberg , germany ; clinical cooperation unit pediatric oncology , german cancer research center ( dkfz ) , heidelberg , germany german cancer consortium ( dktk ) , germany ; kitz clinical trial unit , hopp childrens cancer center heidelberg ( kitz ) , german cancer research center ( dkfz ) and heidelberg university hospital , heidelberg , germany mirjam blattner - johnson , phd hopp childrens cancer center heidelberg ( kitz ) , heidelberg , germany ; pediatric glioma research group , german cancer research center ( dkfz ) , heidelberg , germany uta dirksen , md german cancer consortium ( dktk ) , partner essen , germany ; university hospital essen , pediatrics iii , hematology and oncology , west german cancer centre , essen , germany ingrid ra , md , phd department of pediatric oncology and hematology , karolinska university hospital , solna , sweden ; skane university hospital , lund , sweden birgit geoerger , md , phd department of pediatric and adolescent oncology , gustave roussy cancer center , inserm u1015 , university parissaclay , villejuif , france gudrun schleiermacher , md equipe siric rtop recherche translationelle en oncologie p ediatrique , institut curie , paris , france ; siredo : care , innovation and research for children , adolescents and young adults with cancer , institut curie , paris , france stefan m . 
 correspondence hematology and immunology , heidelberg university hospital , heidelberg , germany ; division of pediatric neurooncology , german cancer consortium ( dktk ) and german cancer research center ( dkfz ) , heidelberg , germany data analysis and interpretation : elke pfaff , antonin marchais , ingrid ra , birgit geoerger , gudrun schleiermacher , stefan m . 
the inform project is nancially supported by the german consortium for translational cancer research ( dktk ) , german cancer aid , the german childhood cancer foundation , the german cancer research center ( dkfz ) , bild e.v. 
pster provision of study materials or patients : elke pfaff , uta dirksen , ingrid ra , birgit geoerger , olaf witt collection and assembly of data : elke pfaff , tiphaine adam de beaumais , antonin marchais , cornelis m . 
van tilburg consulting or advisory role : novartis , bayer uta dirksen consulting or advisory role : lilly , ipsen ingrid ra consulting or advisory role : bayer birgit geoerger consulting or advisory role : roche or genentech , merck kgaa , boehringer ingelheim , bayer , azd , novartis gudrun schleiermacher honoraria : bristol - myers squibb research funding : bristol - myers squibb , pzer , msdavenir , roche travel , accommodations , expenses : roche stefan m . 
pster research funding : lilly , bayer , roche , pharmamar , pzer patents , royalties , other intellectual property : patent on utilizing dna methylation proling for tumor classication olaf witt consulting or advisory role : novartis , astrazeneca , janssen research & development , bristol - myers squibb , roche , bayer , dayonetx research funding : biomed valley discoveries david t.w. 
worst bc , van tilburg cm , balasubramanian gp , et al : next - generation personalised medicine for high - risk paediatric cancer patients : the inform pilot study . 
harttrampf ac , lacroix l , deloger m , et al : molecular screening for cancer treatment optimization ( moscato - 01 ) in pediatric patients : a singleinstitutional prospective molecular stratication trial . 
 correspondence berlanga p , pierron g , lacroix l , et al : can pediatric and adolescent patients with recurrent tumors benet from a precision medicine program ? the european mappyacts experience . 
j clin oncol 37 , 2019 ( suppl ; abstr 10018 ) okamura r , boichard a , kato s , et al : analysis of ntrk alterations in pan - cancer adult and pediatric malignancies : implications for ntrk - targeted therapeutics . 
cancer discov 7 : 400 - 409 , 2017 laetsch tw , dubois sg , mascarenhas l , et al : larotrectinib for paediatric solid tumours harbouring ntrk gene fusions : phase 1 results from a multicentre , open - label , phase 1 / 2 study . 
lancet oncol 19 : 705 - 714 , 2018 konicek bw , capen ar , credille km , et al : merestinib ( ly2801653 ) inhibits neurotrophic receptor kinase ( ntrk ) and suppresses growth of ntrk fusion bearing tumors . 
oncotarget 9 : 13796 - 13806 , 2018 van tilburg cm , dubois sg , albert cm , et al : larotrectinib efcacy and safety in pediatric trk fusion cancer patients . 
j clin oncol 37 , 2019 ( suppl ; abstr 10010 ) robinson gw , gajjar aj , gauvain km , et al : phase 1 / 1b trial to assess the activity of entrectinib in children and adolescents with recurrent or refractory solid tumors including central nervous system ( cns ) tumors . 
hong ds , dubois sg , kummar s , et al : larotrectinib in patients with trkfusionpositive solid tumours : a pooled analysis of three phase 1 / 2 clinical trials . 
steensma , md6 ; amy dezern , md7 ; gail roboz , md8 ; guillermo garcia - manero , md9 ; harry erba , md , phd10 ; benjamin l . 
maciejewski , md , phd1 purpose we developed an unbiased framework to study the association of several mutations in predicting resistance to hypomethylating agents ( hmas ) in patients with myelodysplastic syndromes ( mds ) , analogous to consumer and commercial recommender systems in which customers who bought products a and b are likely to buy c : patients who have a mutation in gene a and gene b are likely to respond or not respond to hmas . methods we screened a cohort of 433 patients with mds who received hmas for the presence of common myeloid mutations in 29 genes that were obtained before the patients started therapy . 
the association between mutations and response was evaluated by the apriori market basket analysis algorithrules with the highest condence ( condence that the association exists ) and the highest lift ( strength of the association ) were chosen . we validated our biomarkers in samples from patients enrolled in the s1117 trial . results among 433 patients , 193 ( 45% ) received azacitidine , 176 ( 40% ) received decitabine , and 64 ( 15% ) received hma alone or in combination . 
the median number of mutations per sample was three ( range , zero to nine ) , and 176 patients ( 41% ) had three or more mutations per sample . 
these molecular signatures were present in 30% of patients with three or more mutations / sample with an accuracy rate of 87% in the training cohort and 93% in the validation cohort . conclusion genomic biomarkers can identify , with high accuracy , approximately one third of patients with mds who will not respond to hmas . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction the hypomethylating agents azacitidine ( aza ) and decitabine ( dac ) have been approved by the food and drug administration and the european medicine agency for patients with myelodysplastic syndromes ( mds ) .1 - 4 although treatment with these agents is well tolerated , only 30% to 40% of patients will respond to therapy , with the majority achieving hematologic improvement in blood counts and only a minority ( 10% to 15% ) achieving a complete response , the response criterion most reliably associated with improvement in overall survival ( os ) .1 - 4 more importantly , it may take up to six cycles of treatment for patients to achieve their best response.5 given the limited number of patients who benet from these agents and the long administration of identifying biotheir treatment , markers that can predict resistance is essential , because it can prevent prolonged exposure to ineffective therapy and unnecessary toxicities and treatment costs can be avoided . because clinical variables and patient characteristics have not consistently predicted response to hypomethylating agents ( hmas ) , molecular data represent a biologic opportunity6 - 8 to enhance patient response rates and outcomes . 
because identifying a single gene or comutated genes is unlikely to yield an understanding of how these mutations dene disease biology or phenotype , an unbiased approach is needed to study the relationship of these abnormalities to each other and to mds biology . in this study , we used unbiased , machine learning approaches ( a recommender system similar to that used by netix or amazon.com ) to assess the impact of molecular data on resistance to hmas in a large cohort of patients treated with hmas at different academic institutions , and we validated our results in a population treated in a contemporary prospective clinical trial of hma therapy15 of aza alone and in combination . methods patients for the training cohort , patients treated with either azaor dac - based regimens were included in this study : 230 patients were treated at cleveland clinic ( between 2002 and 2012 ) ; 203 were from other academic institutions ( dana - farber cancer institute [ 2003 to 2010 , n = 42 ] and md anderson cancer center [ 2003 to 2010 , n = 103 ] ) or were part of the daco - 020 clinical trial ( adopt [ 2005 to 2006 , n = 58 ] ) .11 all patients consented to blood or bone marrow samples at each institution under institutional review boardapproved protocols in accordance with each institution policy and the declaration of helsinki . 
more information regarding the patient cohort , response criteria , and validation cohort is included in the data supplement . dna sequencing and mutational analysis a panel of 29 genes that are commonly mutated in mds and myeloid malignancies was evaluated ( data supplement )  . 
more information regarding sequencing method is included in the data supplement . statistical analyses test variables were compared using the wilcoxon rank sum test and fishers exact for continuous and categorical variables , respectively . 
os was calculated from the date of diagnosis to the date of last follow - up or death ( whichever came rst ) , and survival curves were constructed using the kaplan - meier method and compared using the log - rank test . 
mutation distribution among responders versus nonresponders total responders non - responders nazha et al mutations asxl1 tet2 srsf2 runx1 dnmt3a sf3b1 tp53 u2af1 bcor ezh2 nras idh2 phf6 npm1 jak2 zrsr2 etv6 prpf8 idh1 prpn11 suz12 kras gata2 cebpa received dac ( 176 [ 86% ] alone and 29 [ 14% ] in combination with other agents )  . 
cytogenetic analyses per international prognostic scoring system ( ipss ) revised ( r ) criteria16 included 234 patients ( 54% ) with very good or good risk , 66 ( 15% ) with intermediate risk , 33 ( 8% ) with poor risk , 66 ( 15% ) with very poor risk , and 34 ( 8% ) not documented ( table 1 )  . 
the frequency of these mutations in our patient cohort was similar to those identied in other mds cohorts , with the exception of asxl1 , which was slightly higher because it was overrepresented in one cohort11 ( 203 patients from other academic institutions )  . 
patterns of mutation association were also similar to those in previous reports ( fig 1 )  . asxl1 mutations were commonly commutated with tet2 in 42 patients ( 10% ) , and with srsf2 38 ( 9% ) , runx1 35 ( 8% ) , u2af1 24 ( 6% ) , and dnmt3a 21 ( 5% ; fig 1 )  . overall , 367 patients ( 85% ) in the training cohort had a mutation in at least one gene . 
the graph is separated to show the spectrum of mutations in responders compared with nonresponders . ( 4% ) partial remission ( pr ) , 59 ( 14% ) hematologic improvement ( hi ) , 142 ( 33% ) stable disease , and 107 ( 24% ) progressive disease . in general , clinical characteristics such as age , cytopenias , and treatment regimens did not affect response , with the exception of the median blast percentage in the bone marrow , which was higher among responders compared with nonresponders ( 9% v 2% , p = .02 ; table 1 )  . 
risk stratications per ipss and ipss - r did not affect the overall response rate ( table 1 )  . idh1 and ezh2 , no single gene mutation was signicantly associated with response and resistance to hmas in univariate analyses , with the exception of respectively ( table 2 )  . 
the number of mutations per sample also did not affect response , with patients with three or more mutations having similar response rates to those with fewer than three mutations ( table 1 )  . 
genomic biomarkers dened by the recommender system association rules for resistance to hmas asxl1 , nf1 asxl1 , ezh2 , tet2 asxl1 , ezh2 , runx1 ezh2 , srsf2 , tet2 asxl1 , ezh2 , srsf2 asxl1 , runx1 , srsf2 asxl1 , tet2 , srsf2 asxl1 , bcor , runx1 the impact of mutations on response was assessed after controlling for clinical variables such as age and ipss - r scoring system , using logistic regression analyses . 
no mutation was associated with overall resistance or response to hmas , even after adjustment for clinical variables ( age , ipss - r , and sex ; data supplement )  . recommender system genomic biomarkers that predict response to build strong association rules ( associations between genes and outcomes [ response v no response ] ) , we used strict criteria to identify rules with the highest support ( how many times the rules appeared in the data set ) , high condence ( the condence of the algorithm in the association rule was set at 95% ) , and higher lift ( a measure that is reected in the strength of the association : the higher the lift is , the stronger is the association ) in the training cohort . 
when the rules were applied to our patient cohort , they predicted resistance to hmas correctly in 46 of 53 patients ( 87% ) with relevant molecular mutations . among the 105 patients with lower - risk disease by ipss - r ( low and very low risk groups ) , 62 ( 59% ) did respond to hmas . 
the difference in the presence of the biomarkers in lowerversus higher - risk mds is related to the higher percentage of patients with three or more mutations / sample in the higher - risk ( 42% ) versus the lower - risk ( 30% ) group . association with overall survival survival data were available for 375 patients from the training cohort . 
genomic biomarkers of resistance to aza were present in 14 of 39 samples ( 35% ) with three or more mutations ; 13 of 14 of these patients ( 93% ) did not respond to therapy . discussion predicting response or resistance to our currently available standard hma therapy in mds remains a signicant clinical challenge . 
identifying patients up front who may not respond to hmas can potentially improve outcome , decrease unnecessary adverse effects , and save money , especially when current recommendations are for a minimum of 6 months of treatment before deeming it a failure . 
although it is tempting to identify an isolated molecular abnormality or a pair of mutations that can predict hma resistance , this approach does not allow for the complexity and evolution of the genomic landscape in mds . in this study , we developed an unbiased framework using a machine learning , recommender system algorithm to identify highly sensitive genomic associations ( molecular signatures or genomic biomarkers ) that can predict resistance to hmas with high accuracy . 
these associations were validated in an independent cohort in samples from patients enrolled in a randomized phase ii / iii clinical trial . although our biomarkers were identied in only 25% of patients , their presence predicted resistance in almost all patients who had these mutations . 
by denition , a biomarker can be present in a small subset of patients , but when present can predict , with high accuracy and reliability , response or resistance to a therapy . 
detecting these biomarkers in 29% of patients suggests that other biologic mechanisms ( eg , changes in gene expression or epigenetic changes ) or clinical characteristics may contribute more to hma response and failure than does genomics . 
our ndings conrm that genomic associations may lead to different gene expressions and / or epigenetic changes that contribute to the response or resistance and thus , identifying one or two genes that can predict response may not be sufcient to build reliable and predictable models . although we included patients who received hmas in combination with other investigational agents , these combinations did not affect the response or resistance rate or os ; thus , their impact on the output of our recommender system algorithms is negligible . 
furthermore , neither ipss nor ipss - r predicted response or resistance to hmas in our study in accordance with prior reports.1 , 15 prior studies have attempted to use genomic data to predict response or resistance to hmas . 
for example , some studies have shown that tp53 mutations may predict response to hmas , whereas others did not conrm that nding.13 , 17 in a small study of 84 patients with acute myeloid leukemia ( aml ) and mds treated with a 10 - day dac course , a small subset of patients with tp53 mutations had a higher response rate to dac compared with tp53 wild - type patients . furthermore , the median os for tp53 mutated patients who received dac and underwent an allogeneic stem - cell transplantation was similar to that of patients with wild - type tp5317 . 
contrary to this nding , in a study of 71 patients with aml , there was no difference in overall response rate and survival among patients who received 5 days of dac compared with those who had a 10 - day schedule . 
 genomic biomarkers for resistance in mds with variant allele frequency greater than 10% may predict response to hmas , especially in patients with wild - type asxl1 mutations , but this nding was not validated in our study.11 the discrepancy in the results of these studies could be related to sample size , the number of genes tested , and the statistical approach that was used to analyze the data . 
it is also possible that genomic changes in themselves are not the drivers of response to hmas but rather , changes in the gene expression and methylation prole that are derived from the combination of these mutations . 
in a study of whole - genome sequencing , rna sequencing , and methylation prole of samples from patients with chronic myelomonocytic leukemia , a serial sequencing demonstrates that the response to hypomethylating agents is associated with changes in dna methylation and gene expression , without any decrease in the mutation allele burden or prevention of new genetic alteration occurrence.18 if eligible , directly , without this study includes several areas of innovation . 
for example , if a patient with mds with higher - risk disease carries one of these biomarkers , he or she should be encouraged to enroll in a clinical trial with a novel therapy or to proceed with an allogeneic stem - cell transplantation , the use of hmas , because the response to such therapy is predicted to be low . 
although all patients with mds should be encouraged to enroll in a clinical trial with novel therapies , having biomarkers that accurately predict resistance may ease the conversation with some patients who are hesitant to try newer approaches and prefer food and drug administrationapproved therapies.19 alternatively , patients with higher - risk disease and a high blast percentage may consider intensive , aml - type chemotherapy before allogeneic stem - cell transplantation , as opposed to an hma that is predicted to do little to affect the disease in the absence of other biomarkers that could predict resistance to chemotherapy , such as complex karyotype cytogenetics , and the presence of tp53 mutations and the absence of targetable mutations such as idh1 and idh2 . 
because the optimal timing for patients with mds with lower - risk disease can be challenging and because these genomic biomarkers predicted poor outcome even in patients with lower - risk disease . 
these biomarkers could be used as a justication to proceed with allogeneic stem - cell transplantation early in the disease course , especially if the patient has a lower blast percentage . 
in addition , identifying , with high accuracy , patients who may or may not respond to therapy can prevent prolonged exposure to ineffective therapy and can lead to lower cost without decreasing value or changing patient outcomes . 
introducing these biomarkers into normal hematopoietic cells using crispr / cas9 may offer an opportunity to model and understand hma resistance to develop novel drugs to overcome this resistance . our study highlights the importance of machine learning algorithms such as the recommender system in translating genomic data into useful clinical tools that can be used by physicians in the clinic.20 nevertheless , some limitations to our approach exist . 
it is possible that the response to hmas is derived mainly from epigenetic changes and is not dependent on the genomic changes that we studied here . in summary , our study identied genomic abnormalities that predict response or resistance to hmas in patients with mds , and we validated our results in an independent patient cohort treated in a randomized clinical trial . 
identication of biomarkers that can provide personalized treatment approaches that can predict response or resistance to cancer therapy remains an important clinical challenge , and future drug development should focus on identifying the subgroup of patients who may benet the most from any given cancer therapy . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . open payments is a public database containing information reported by companies about payments made to us - licensed physicians open payments . aziz nazha honoraria : dci consulting or advisory role : karyopharm therapeutics , tolero pharmaceuticals speakers bureau : novartis , incyte research funding : jazz pharmaceuticals mikkael a . 
sekeres consulting or advisory role : celgene , millennium pharmaceuticals , syros pharmaceuticals research funding : takeda pharmaceuticals ( inst ) , pzer ( inst ) rafael bejar honoraria : celgene , alexion pharmaceuticals , abbvie / genentech , astex pharmaceuticals , neogenomics laboratories , daiichi sankyo , forty seven consulting or advisory role : celgene , foundation medicine , neogenomics laboratories , abbvie / genentech , astex pharmaceuticals , daiichi sankyo research funding : celgene , takeda pharmaceuticals travel , accommodations , expenses : celgene megan othus consulting or advisory role : celgene , glycomimetics , cascadia laboratories rami s . 
komrokji stock and other ownership interests : abbvie consulting or advisory role : celgene , novartis , daiichi sankyo , pzer , janssen pharmaceuticals , agios , incyte speakers bureau : novartis , alexion pharmaceuticals , jazz pharmaceuticals travel , accommodations , expenses : celgene , incyte , alexion pharmaceuticals , novartis , jazz pharmaceuticals , daiichi sankyo david p . 
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blood 117 : 403 - 411 , 2011 takahashi k , patel k , bueso - ramos c , et al : clinical implications of tp53 mutations in myelodysplastic syndromes treated with hypomethylating agents . oncotarget 7 : 14172 - 14187 , 2016 9 . 
sekeres ma , othus m , list af , et al : randomized phase ii study of azacitidine alone or in combination with lenalidomide or with vorinostat in higher - risk myelodysplastic syndromes and chronic myelomonocytic leukemia : north american intergroup study swog s1117 . 
short nj , kantarjian hm , loghavi s , et al : treatment with a 5 - day versus a 10 - day schedule of decitabine in older patients with newly diagnosed acute myeloid leukaemia : a randomised phase 2 trial . 
we sought to identify the molecular alterations associated with mpmri - invisible tumors and determine whether mpmri visibility is associated with pca prognosis . methods discovery and validation cohorts included patients who underwent mpmri before radical prostatectomy and were found to harbor both mpmri - visible ( prostate imaging and reporting data system 3 to 5 ) and - invisible ( prostate imaging and reporting data system 1 or 2 ) foci on surgical pathology . 
next - generation sequencing was performed to determine differential gene expression between mpmri - visible and - invisible foci . a genetic signature for tumor mpmri visibility was derived in the discovery cohort and assessed in an independent validation cohort . 
its association with long - term oncologic outcomes was evaluated in a separate testing cohort . results the discovery cohort included 10 patients with 26 distinct pca foci on surgical pathology , of which 12 ( 46% ) were visible and 14 ( 54% ) were invisible on preoperative mpmri . 
a nine - gene signature ( composed largely of cell organization / structure genes ) associated with mpmri visibility was derived ( area under the curve = 0.89 ) , and the signature predicted mri visibility with 75% sensitivity and 100% specicity ( area under the curve = 0.88 ) in the validation cohort . 
recent efforts to overcome this have involved the development and optimization of several diagnostic strategies , including multiparametric magnetic resonance imaging ( mpmri )  . identication of areas that are mpmri permits visual suggestive for intermediate to high - grade cancer . 
the emergence of various mri / ultrasound fusion biopsy platforms has led to increased detection of aggressive pca by facilitating targeted biopsy of visible lesions.1 - 6 as a result , mpmri is now widely used in guiding treatment decisions in men with clinically localized disease , especially when selecting patients suitable for active surveillance or potentially focal therapy.7 - 10 the prevailing view is that only mpmri - visible cancers require clinical action . however , use of mpmri in the evaluation of men with pca is limited by cancer multifocality and interfocal disease heterogeneity . 
individual patients are known to harbor multiple spatially distinct pca foci with varying clinical , radiographic , and pathologic characteristics.11 - 15 up to 55% of all pca foci and 35% of clinically signicant foci are not visible on mpmri.3 , 16 , 17 furthermore , more than 35% of lesions 1 cm or larger are missed by mpmri.17 although some studies have demonstrated that up to 50% of mpmriinvisible pca may harbor relevant genomic alterations , the clinical and prognostic signicance of mpmriinvisible pca remains unknown.18 an improved understanding of the molecular characteristics and clinical trajectories of mpmri - visible and - invisible cancers could facilitate more optimal treatment allocation . 
 salami et al context key objective what is the molecular basis for prostate cancer visibility on multiparametric magnetic resonance imaging ( mpmri ) , and do mpmri - invisible tumors harbor any clinical or biologic signicance compared with visible tumors ? knowledge generated mpmri - visible tumors demonstrated underexpression of genes associated with cellular organization and structure . 
using a novel genetic signature for tumor visibility on mpmri , patients with predicted mpmri - visible and - invisible tumors did not experience signicant differences in biochemical recurrence , distant metastasis , or cancer - specic mortality during followrelevance prostate cancers that are mpmri invisible have similar clinical behavior to mpmri - visible tumors . 
negative mpmri seems insufcient to rule out clinically relevant prostate cancer , and patients at increased risk should be considered for additional testing or systematic prostate biopsy . to be biologically indolent , those with a known diagnosis of low - grade disease and a negative mpmri could be directed toward active surveillance . 
in addition , we test the prognostic signicance of our mpmri - derived genomic signature after radical prostatectomy ( rp )  . methods study design the study used three independent patient populations : discovery , validation , and testing cohorts . 
first , we identied patients with clinically localized disease who the university of underwent preoperative mpmri at michigan in 2015 to 2016 and were subsequently found to harbor multifocal pca at rp . 
the validation cohort from cedars - sinai medical center included patients with either mpmri - visible or - invisible foci , as previously described.19 the testing cohort was composed of patients from the decipher grid pca database treated at johns hopkins medical institute and mayo clinic ( clinicaltrials.gov identier : nct02609269 ) who underwent genome - wide expression proling after rp.20 , 21 preoperative prostate mpmri and pathologic evaluation in the discovery and validation cohorts , mpmri comprising t2 - weighted imaging , diffusion - weighted imaging , and dynamic contrast - enhanced imaging was obtained . 
all mpmri results were re - reviewed and coregistered with whole - mount formalin - xed parafn - embedded rp specimens to delineate mpmri - visible ( prostate imaging and reporting data system [ pi - rads ] version 2 ; score , 3 to 5 ) and - invisible foci . 
our targeted ngs assays were designed to assess relevant pca genomic and transcriptomic alterations and derive clinically available prognostic tests.15 the details of rna sequencing in the validation cohort and genome - wide expression proling in the testing cohorts have been previously described.19 , 24 bioinformatic analysis of the discovery cohort ngs data analysis was performed using torrent suite ( 4.2.0 ; thermo fisher scientc , waltham , ma ) and the coverage analysis plug - ins v.5.0.4. 
details regarding targeted ngs techniques , quality control parameters , dna copy number alterations and variant calls , fusion isoform and partner level analysis , androgen receptor ( ar ) and arsplice variants detection , and prognostic scores derivation have been previously described and summarized in the data supplement.15 , 25 , 26 differential gene expression analysis of mpmri - visible and - invisible cancer foci to determine gene expression differences between mpmri - visible and - invisible tumors , we analyzed rnaseq data from the discovery cohort using two approaches differential expression ( de ) analysis and random forest ( rf ) classieras described in the data supplement . 
 ( a ) cartoon depicting multifocal prostate cancer ( pca ) with both multiparametric magnetic resonance imaging ( mpmri ) visible ( solid black , left ) and invisible ( gray with black discontinuous borders , right ) lesions . 
 salami et al prognostic signicance of mpmri - based gene expression signature 55 ( 14.7% ) developed metastasis , and 28 ( 7.5% ) died as a result of pca ( data supplement )  . a total of 375 patients with genome - wide expression proles were pooled from two independent case - cohort studies20 , 21 to constitute the testing cohort . 
kaplanmeier curves and cox proportional hazard regression were used to evaluate the performance of this signature in predicting oncological outcomes : biochemical recurrencefree survival ( bfs ) , distant metastasisfree survival ( dmfs ) , and pca - specic mortality ( pcsm )  . 
multivariable analyses were performed to evaluate this signature as an independent predictor of oncological outcomes after adjusting for relevant clinicopathological variables , including preoperative prostate - specic antigen , pathologic grade group ( gg ) , surgical margins , extraprostatic extension , seminal vesicle invasion , and lymph node invasion . spearman correlation analysis was performed to measure the association of the gene signature with cellular organization pathway activity . 
among the 14 mpmri - invisible foci ( 54% ) , ve ( 36% ) were gg2 and the remainder were gg1 ( fig 1c and data supplement )  . 
there were 16 patients in the validation cohort , of whom eight ( 50% ) had mpmri - invisible cancer lesions , and two of these ( 25% ) were gg2 ( data supplement )  . 
during follow - up , 136 ( 36.3% ) patients experienced biochemical recurrence , detection of mutations and copy number alterations in the discovery cohort we detected high - condence mutations in 14 of 26 ( 54% ) tumor foci ; six ( 43% ) of the mutations were identied in mpmri - invisible lesions . 
we detected pten one copy number loss in 25% ( three of 12 ) and 14.3% ( two of 14 ) of mpmri - visible and - invisible foci , respectively ( fig 1c )  . discovery and validation of a nine - gene expression signature for mpmri visibility of the 26 total tumor foci in the discovery cohort and 306 amplicons on the rnaseq panel , 24 samples and 74 amplicons , respectively , passed quality control parameters and underwent de analysis ( data supplement )  . 
using de analysis ( data supplement ) and rf classier ( data supplement ) to identify candidate differentially expressed genes , we interrogated four separate logistic regression models for predicting mpmri tumor visibility status using the 19 de analysis genes , 20 rf genes , 11 shared genes between the de analysis and rf gene sets , and 11 shared genes combined with the mutually exclusive genes ( data supplement )  . 
a multivariable rnaseq - based logistic regression model with the best performance for predicting mpmri visibility status , comprising a nine - gene expression signature , was developed from the intersection of the de analysis and rf gene sets ( fig 2a ; data supplement )  . 
this signature correctly predicted seven ( 70% ) of the mpmrivisible and 13 ( 93% ) of the mpmri - invisible foci in the discovery cohort , yielding an area under the curve of 0.89. the optimal probability cutoff for predicting mpmri - visible tumor was greater than 0.46 , with a sensitivity and specicity of 80% and 86% , respectively , in the discovery cohort ( figs 2a and 2b )  . 
we observed underexpression of seven of the nine genes in mpmri - visible tumors , the majority of which were stromal , cellular organization , and structure genes ( fig 2a ; data supplement )  . the nine - gene expression signature was then evaluated in the independent validation cohort ( cedars - sinai medical center ) using the predetermined optimal probability cutoff ( from the discovery cohort ) to predict mpmri visibility status . 
pten one copy number loss was observed in 25% ( three of 12 ) and 14.3% ( two of 14 ) of mpmri - visible and invisible cancer foci , respectively ( false discovery rate , less than 5% )  . 
comparisons of observed ( rst row ) versus predicted ( second row ) mpmri visibility using the nine - gene signature are shown in the annotation , as well as international society of urological pathology grade group . 
 ( b ) receiver operating characteristic curves for the signature in the discovery ( university of michigan [ um ] ) versus the validation ( cedars - sinai medical center [ csmc ] ) cohorts . 
the optimal probability cutoff for predicting mpmri - visible tumor was greater than 0.46 , with a sensitivity and specicity of 75% and 100% in the validation cohort , respectively . for predicting mpmri visibility status were 75% and 100% , respectively , in the validation cohort . 
notably , the signature correctly predicted two gg2 cancers that were mpmri invisible in the validation cohort . prognostic signicance of the nine - gene mpmri visibility expression signature the distribution of each gene composing the nine - gene signature in the normalized microarray data ( testing cohort ) from the decipher grid mapped to the cancer genome atlas prostate adenocarcinoma ( tcga - prad ) rnaseq closely resemble that of the discovery cohort ( data supplement )  . 
prognostic signicance of predicted multiparametric magnetic resonance imaging ( mpmri ) visibility status . patients ( n = 375 ) in the testing cohort were pooled from two independent case - cohort studies ( johns hopkins medical institute [ n = 260 ] and mayo clinic [ n = 235 ] ) to test the capacity of predicted mpmri visibility status to predict ( a ) biochemical recurrence - free survival ( bfs ) , ( b ) distant metastasis - free survival ( dmfs ) , and ( c ) prostate cancerspecic mortality ( pcsm )  . 
normalization was performed to match the distribution of the genomic data from this cohort to the cancer genome atlas prostate adenocarcinoma rnaseq data , as described in methods , to facilitate testing of the rnaseq - based nine - gene signature to predict mpmri - visible tumors ( data supplement )  . 
similar results were obtained using the affymetrix microarray data that were not matched to the distribution of the the cancer genome atlas prostate adenocarcinoma rnaseq data ( data supplement )  . underexpression of seven of the nine genes in mpmrivisible tumors , the majority of which were stromal , cellular organization , and structure genes ( fig 2a ; data supplement )  . 
multivariable analysis to assess the prognostic signicance of predicted multiparametric magnetic resonance imaging ( mpmri ) visibility status . using data from the testing cohort described in figure 3 ( affymetrix microarray data matched to the distribution of the the cancer genome atlas prostate adenocarcinoma rnaseq data ; n = 375 ) , multivariable cox proportional hazard models were developed to assess the capacity of predicted mpmri visibility status to predict : ( a ) biochemical recurrence - free survival ( bfs ) , ( b ) distant metastasisfree survival ( dmfs ) , and ( c ) prostate cancerspecic mortality ( pcsm ) , adjusting for relevant clinicopathological variables . 
epe , extraprostatic extension ; gg , grade group ; hr , hazard ratio ; lni , lymph node invasion ; psa , prostate - specic antigen ; sm , surgical margins ; svi , seminal vesical invasion . using a targeted multiplex ngs approach . 
using robust biostatistic methods , we developed and validated a novel nine - gene signature to predict pca mpmri visibility status . interrogation of this signature in a distinct cohort with longterm follow - up revealed no signicant association with bfs , dmfs , or pcsm . 
taken together , these ndings indicate that mpmri - invisible cancer foci harbor many of the same aggressive molecular features as mpmrivisible foci and may also be clinically signicant . the molecular basis of pca visibility on mpmri is poorly understood . 
 ( a ) box plots of derived prolaris cell cycle progression ( mxccp ) score , oncotype dx genomic prostate score ( mxgps ) , and decipher genomic classier ( mxgc ) stratied by mpmri visibility status in the discovery cohort ( n = 10 patients ; 26 cancer foci )  . 
box plots of derived mxgps androgen , cellular organization , and stromal submodules stratied by mpmri visibility status are shown , with each point representing an individual cancer focus colored according to gg . 
only the cellular organization submodule had a signicant difference in mean expression ( p = .014 ) , suggesting that at the rna expression level mpmri visibility is related to underlying cellular organization of the tumor . explain cancer visibility on mpmri.28 in a recent report , li et al19 observed signicant fold changes of differentially expressed genes on the basis of mpmri visibility regardless of gleason score or tumor size , including genes involved in cytoskeleton organization . 
 molecular basis and prognosis of mri - invisible prostate cancer mpmri - visible foci in the discovery cohort demonstrated pten one copy number loss compared with 14% in mpmri - invisible foci . 
although pca is multifocal and mpmri may miss up to 35% of intermediateto high - grade pca , the absence of visible lesions on mpmri has been proposed as a reason to defer conrmatory biopsy when considering active surveillance.1 , 3 , 16 , 17 in addition , mpmri is increasingly being used to identify the index or dominant cancer foci for focal therapy . 
to be sure , although size and grade are believed to be important , how best to dene the biologically dominant cancer in multifocal disease is not known.35 to date , no study has demonstrated the clinical trajectory of mpmri - visualized lesions . 
such a study would be a challenge to perform , given the long duration of follow - up required and the multifocal nature of pca , with frequent coexistence of mpmri - visible and - invisible cancers within the same gland.31 salmasi et al36 reported that the pi - rads ( a grading system for mpmri lesion visibility ) was not a signicant predictor of adverse pathology at the time of rp . 
similarly , parry et al18 found that 50% of mpmri - invisible cancers harbored one or more genetic alterations commonly observed in metastatic castrate - resistant pca , suggesting that mpmri - invisible tumors may be as important as visible ones . 
in the study by li et al , 19 a four - gene signature comprising genes differentially expressed between mpmri - visible and - invisible pca was shown to predict bfs in two external data sets . 
however , this signature was not developed as a predictor of or a surrogate for mpmri cancer visibility , but rather it was selected on the basis of their common association with mpmri visibility and metastasis . 
by contrast , in this rst study of its kind to our knowledge , using a validated novel mpmri - based rnaseq signature as a surrogate instrument for mpmri visibility status , we have demonstrated that predicted mpmri visibility status was not associated with bfs , dmfs , or pcsm during long - term follow - up . 
put another way , mpmriinvisible pca does not seem to represent purely indolent disease ; mpmri - invisible lesions may be just as clinically relevant as mpmri - visible disease . 
first , in the diagnostic setting , these data corroborate ndings from several institutions indicating that a negative mpmri does not rule out the presence of clinically signicant pca3 , 17 , 31 and should therefore not preclude a prostate biopsy without consideration of clinical risk.37 , 38 second , in the setting of active surveillance , our ndings underscore the potential mpmri - invisible cancer foci to harbor similar biologic trajectories as mpmri - visible disease . 
although additional studies are needed to delineate the utility of mpmri in reducing the frequency of surveillance biopsies , the current literature supports systematic in addition to targeted biopsies in men undergoing active surveillance.3 , 6 last , for men considering focal therapy , our data demonstrate that mpmri alone is not sufcient to rule out the presence of a potentially lethal , nondominant cancer focus . our study has several limitations . 
however , our novel rnaseq signature demonstrated high accuracy for predicting mpmri visibility in the validation cohort , including 19% gg5 lesions . moreover , gg4 and 5 lesions are generally mpmri visible , and such patients routinely undergo whole - gland therapy . fourth , the discovery cohort was made up of a relatively small sample , with low proportion of erg - positive tumors . nonetheless , we similarly observed high test performance in the validation cohort with erg overexpression in 31% of samples . 
to facilitate reproducibility , all lesions in the current study were scored according to the validated pi - rads v2 systepi - rads 1 and 2 lesions were classied as mpmri invisible , and pi - rads 3 to 5 were classied as mpmri visible . 
thus , additional studies are needed to delineate the prognostic signicance of mpmri - invisible pca in a prospective clinical cohort . discerning aggressive from indolent disease remains a signicant clinical challenge in the evaluation and management of men with primary pca . 
our ndings indicate that mpmri - invisible cancers were no less likely to harbor lethal biologic potential than visible tumors , highlighting the limitation of using mpmri alone to guide patient management or delineate specic index cancer foci for ablative therapy . 
morgan consulting or advisory role : myriad genetics , terumo bct research funding : myriad genetics ( inst ) , mdxhealth ( inst ) , genomedx ( inst ) arvin k . 
tomlins employment : strata oncology leadership : strata oncology stock and other ownership interests : strata oncology consulting or advisory role : abbvie , janssen , astellas medivation , strata oncology , sano , almac diagnostics research funding : astellas medivation ( inst ) , genomedx ( inst ) patents , royalties , other intellectual property : i am a coauthor on a patent issued to the university of michigan on ets gene fusions in prostate cancer . 
the diagnostic eld of use has been licensed to hologic / genprobe , who has sublicensed some rights to ventana medical systems / roche . travel , accommodations , expenses : strata oncology hyung l . 
palapattu stock and other ownership interests : nantkwest consulting or advisory role : janssen scientic affairs research funding : minomic patents , royalties , other intellectual property : implantable nanotechnology for long - term testosterone delivery functionalized ducial marker for drug delivery no other potential conicts of interest were reported . references kasivisvanathan v , rannikko as , borghi m , et al : mri - targeted or standard biopsy for prostate - cancer diagnosis . 
n engl j med 378 : 1767 - 1777 , 2018 ahmed hu , el - shater bosaily a , brown lc , et al : diagnostic accuracy of multi - parametric mri and trus biopsy in prostate cancer ( promis ) : a paired validating conrmatory study . 
lancet 389 : 815 - 822 , 2017 filson cp , natarajan s , margolis dja , et al : prostate cancer detection with magnetic resonance - ultrasound fusion biopsy : the role of systematic and targeted biopsies . 
cancer 122 : 884 - 892 , 2016 salami ss , ben - levi e , yaskiv o , et al : in patients with a previous negative prostate biopsy and a suspicious lesion on magnetic resonance imaging , is a 12 - core biopsy still necessary in addition to a targeted biopsy ? bju int 115 : 562 - 570 , 2015 salami ss , vira ma , turkbey b , et al : multiparametric magnetic resonance imaging outperforms the prostate cancer prevention trial risk calculator in predicting clinically signicant prostate cancer . 
cancer 120 : 2876 - 2882 , 2014 siddiqui mm , rais - bahrami s , turkbey b , et al : comparison of mr / ultrasound fusion - guided biopsy with ultrasound - guided biopsy for the diagnosis of prostate cancer . 
jama 313 : 390 - 397 , 2015 ahmed hu , hindley rg , dickinson l , et al : focal therapy for localised unifocal and multifocal prostate cancer : a prospective development study . 
j urol 196 : 68 - 75 , 2016 ahmed hu , dickinson l , charman s , et al : focal ablation targeted to the index lesion in multifocal localised prostate cancer : a prospective development study . eur urol 68 : 927 - 936 , 2015 10 . 
guillaumier s , peters m , arya m , et al : a multicentre study of 5 - year outcomes following focal therapy in treating clinically signicant nonmetastatic prostate cancer . 
cooper cs , eeles r , wedge dc , et al : analysis of the genetic phylogeny of multifocal prostate cancer identies multiple independent clonal expansions in neoplastic and morphologically normal prostate tissue . 
j urol 193 : 87 - 94 , 2015 johnson dc , raman ss , mirak sa , et al : detection of individual prostate cancer foci via multiparametric magnetic resonance imaging . 
ross ae , johnson mh , youse k , et al : tissue - based genomics augments post - prostatectomy risk stratication in a natural history cohort of intermediateand 21 . 
karnes rj , bergstralh ej , davicioni e , et al : validation of a genomic classier that predicts metastasis following radical prostatectomy in an at risk patient high - risk men . 
hovelson dh , mcdaniel as , cani ak , et al : development and validation of a scalable next - generation sequencing system for assessing relevant somatic variants in solid tumors . 
erho n , crisan a , vergara ia , et al : discovery and validation of a prostate cancer genomic classier that predicts early metastasis following radical prostatectomy . plos one 8 : e66855 , 2013 466 : 297 - 311 , 2015 25 . 
palapattu gs , salami ss , cani ak , et al : molecular proling to determine clonality of serial magnetic resonance imaging / ultrasound fusion biopsies from men on active surveillance for low - risk prostate cancer . 
klein ea , cooperberg mr , magi - galluzzi c , et al : a 17 - gene assay to predict prostate cancer aggressiveness in the context of gleason grade heterogeneity , tumor multifocality , and biopsy undersampling . 
truong m , hollenberg g , weinberg e , et al : impact of gleason subtype on prostate cancer detection using multiparametric magnetic resonance imaging : correlation with nal histopathology . 
vargas ha , akin o , shukla - dave a , et al : performance characteristics of mr imaging in the evaluation of clinically low - risk prostate cancer : a prospective study . radiology 265 : 478 - 487 , 2012 30 . 
hurrell sl , mcgarry sd , kaczmarowski a , et al : optimized b - value selection for the discrimination of prostate cancer grades , including the cribriform pattern , using diffusion weighted imaging . 
salmasi a , khoshnoodi p , felker er , et al : a 17 - gene genomic prostate score assay provides independent information on adverse pathology in the setting of combined multiparametric magnetic resonance imaging fusion targeted and systematic prostate biopsy . 
a recent retrospective analysis found a clear association of improved survival with continuation of endocrine therapy upon diagnosis of brain metastases.10 remarkably , the cdk4 / 6 - inhibitor abemaciclib had a clinical benet rate of 25%.11 preclinical models of bc brain metastases suggest pi3k pathway inhibition may be effective for treatment of brain metastases.12 notably , both sandpiper and solar - 1 excluded patients with untreated or active cns metastases6 , 7 ; however , the precursor to alpelisib , buparlisib , did have brain penetration , which was thought to be the cause for the higher incidence of mood disorders.13 an increase in depression has distinctly not been seen with the - specic pi3kinhibitor alpelisib and , in preclinical animal models with intact blood - brain barrier , no signicant distribution into the brain was seen ( unpublished data )  . thus , the activity of alpelisib in brain metastases is unknown . herein , we report a case series of 4 patients with er + / progesterone receptorpositive ( pr + ) / her2 metastatic bc with progressive brain metastases ( fig 1 ) treated with alpelisib . 
all patients provided consent to publish their information and images . case 1 a 55 - year - old woman presented with a large breast ulceration , biopsy specimendiagnosed invasive ductal carcinoma ( idc ) , grade 3 , er + / pr + / her2 . computed tomography ( ct ) scan revealed pulmonary nodules , osseous lesions , and hypodense lesions within the right hepatic lobe . 
brain magnetic resonance imaging ( mri ) showed a 10 - mm mass in the left cerebellar hemisphere ; this was treated with stereotactic radiosurgery with initial shrinkage to 8 mm and stabilization on follow - up . 
brain mri showed an increase of the left cerebellar lesion to 12 mm , judged by neuroradiology and radiation oncology to be more compatible with progression than with radiation - induced tissue necrosis . 
the patients body mass index ( bmi ) was 25 ( calculated as kilograms divided by square of height in meters ) and eastern cooperative oncology group ( ecog ) performance status ( ps ) score of 0 . 
brain mri after 6 weeks showed a reduction in the left cerebellar lesion to 9 mm ( 62% reduction of bidimensional areas , 35% reduction of sum of longest distances )  . 
follow - up mri 2 months later revealed stability of the cns lesion . subsequent brain mri at 3 , 4 , and 6 months showed stable disease without changes in measurements . 
this was compatible with a partial response per response in neuro - oncology brain metastases assessment ( rano - bm ) criteria.14 case 2 a 71 - year - old woman was diagnosed in 2010 with pt2n0 idc of the right breast , grade 3 , er + / pr + / her2 . 
in 2017 , she had a recurrence in the right breast , pleural effusion , and bone metastases and was treated with multiple regimens : letrozole and palbociclib , fulvestrant and palbociclib , and capecitabine and paclitaxel . 
she was serially treated with fulvestrant and palbociclib , radiation to the base of skull to treat cranial nerve symptoms , and with exemestane and everolimus , capecitabine , and abemaciclib . 
for the rst time , brain mri demonstrated multiple parenchymal metastases and she was treated with hippocampal - sparing wbrt . subsequently , treatment was changed from abemaciclib to liposomal doxorubicin for progression in the brain , followed by gemcitabine . 
brain mri 6 weeks later demonstrated a 14% reduction in the sum of longest distances of measurable brain metastases ( 24% reduction in bidimensional areas ) and regressions of nonmeasurable brain metastases . 
the most recent brain mri at the 6 - month mark revealed progressive parenchymal disease requiring change of therapy . case 4 a 70 - year - old woman was diagnosed in 2013 with t1n1 idc of the right breast , grade 3 , er + / pr + / her2 . 
she was treated with breast - conserving surgery , adjuvant docetaxel plus cyclophosphamide , radiation , and anastrozole . 2018 , metastatic disease developed to the lung , liver , and bone while she received adjuvant anastrozole . 
follow - up mri demonstrated at least 15 new intraparenchymal lesions and several progressing dural - based lesions , so the patient proceeded to undergo wbrt and restarted capecitabine therapy . 
examination of a liquid biopsy specimen revealed multiple pi3k mutations ( pik3ca : h1047r , e81k , and e563k ) and she was administered alpelisib ( 300 mg ) table 1 . 
restaging after 6 weeks of therapy revealed substantial disease regression in the lungs and liver as well as interval resolution of punctate cerebellar and cerebral metastasis and reduction of a dural metastasis ( appendix fig a1 ) , compatible with stable disease per rano - bm criteria.14 repeated brain mri at the 3and 5month marks showed stable disease . discussion activating pik3ca mutations occur early in breast carcinogenesis and are typically not lost or acquired during clonal evolution in later stages of the diseasefeatures that suggest these are driver mutations . 
table 1 provides a summary of mutational prole and previous treatments . cases 1 and 2 harbored the activating mutation pik3ca h1047r in the coding exon 20 , a kinase - activating mutation and the most common mutation in bc.14 case 1 also had pik3c2b amplication , which occurs in 13% to 25% of patients.15 pik3c2b is a class ii pi3 - kinase.16 whether its amplication in conjunction with pik3ca mutation deepens or attenuates pi3k - pathway dependence of cancer and whether pik3c2bs kinase activity is inhibited by alpelisib is unknown . 
however , understanding this relationship appears to be important because activating pik3ca mutations and copy number gain of pik3c2b do co - occur in bc.15 case 3 harbored the pik3ca e545k mutation , affecting the helical domain of p110that activates signaling because of its detachment from the inhibitory p85 subunit of pi3k.17 helical domain mutations are the second most frequent in bc , with an incidence of 6.4%.14 case 4 had 3 mutations in pik3ca ; e81k and h1047r are activating mutations and e563k is novel . 
this combination of a major ( h1047r ) and a minor ( e81k ) pik3ca mutation is thought to amplify pi3k signaling and predict for responsiveness to pi3k inhibition.18 this patient had a comutation of pik3ca mutations and esr1 . 
the earlier pan - pi3k inhibitor buparlisib was tested in 4 patients with treatment - refractory cns lymphoma , 1 of whom achieved a partial remission.23 in animal models , systemically administered buparlisib showed activity against bc brain metastases.24 - 26 patients with active brain metastases were excluded from the pivotal solar - 1 and sandpiper studies.6 , 7 conclusions from our observations are limited by the small number of patients selected on the basis of their surprising response . 
the rano - bm criteria consider lesions , 10 mm as nonmeasurable.14 here , we show examples with resolution of small lesions , which are clinically meaningful but classied as stable disease per the criteria . 
additional investigation to prospectively evaluate alpelisib in patients with bc and cns involvement may be justied . in conclusion , we have described cases of regression or stabilization of progressive cns lesions in patients with hr + / her2 metastatic bc treated with the pi3k inhibitor alpelisib . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . stacy moulder research funding : oncothyreon ( inst ) , pzer ( inst ) , novartis ( inst ) , genentech ( inst ) , takeda ( inst ) , bayer ( inst ) , emd serono ( inst ) , genentech ( inst ) eric p . 
winer stock and other ownership interests : verastem honoraria : roche , genomic health consulting or advisory role : leap therapeutics , seattle genetics , jounce therapeutics , glaxosmithkline , carrick therapeutics , eli lilly , genomic health , g1 therapeutics research funding : genentech ( inst ) , novartis ( inst ) hope s . 
rugo consulting or advisory role : ionis , celtrion research funding : macrogenics ( inst ) , obi pharma ( inst ) , eisai ( inst ) , pzer ( inst ) , novartis ( inst ) , eli lilly ( inst ) , genentech ( inst ) , merck ( inst ) , immunomedics ( inst ) , odonate therapeutics ( inst ) , daiichi sankyo ( inst ) , seattle genetics ( inst ) travel , accommodations , expenses : pzer , puma biotechnology , mylan , amgen , astrazeneca , macrogenics , daiichi sankyo , merck , novartis , obi pharma open payments link : nancy u . 
lin consulting or advisory role : roche , seattle genetics , puma biotechnology , novartis , daiichi sankyo research funding : genentech , pzer , seattle genetics , merck patents , royalties , other intellectual property : royalties for chapter in upto - date regarding management of breast cancer brain metastases , royalties from jones & bartlett gerburg m . 
wulf stock and other ownership interests : selecta biosciences ( i ) research funding : merck patents , royalties , other intellectual property : pin1 as a marker for abnormal cell growth ( patent no . : 8129131 ) no other potential conicts of interest were reported . references cancer genome atlas network : comprehensive molecular portraits of human breast tumours . 
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genes chromosomes cancer 52 : 69 - 80 , 2013 ramirez - ardila de , helmijr jc , look mp , et al : hotspot mutations in pik3ca associate with rst - line treatment outcome for aromatase inhibitors but not for tamoxifen . 
breast cancer res treat 139 : 39 - 49 , 2013 kalinsky k , jacks lm , heguy a , et al : pik3ca mutation associates with improved outcome in breast cancer . 
clin cancer res 15 : 5049 - 5059 , 2009 baselga j , cortes castan j , de laurentiis m , et al : sandpiper : phase iii study of the pi3 - kinase ( pi3k ) inhibitor taselisib ( gdc - 0032 ) plus fulvestrant in patients ( pts ) with oestrogen receptor ( er ) - positive , her2 - negative locally advanced or metastatic breast cancer ( bc ) enriched for pts with pik3ca - mutant tumours . 
ann oncol 27 : vi99 , 2016 ( suppl 6 ) andr e f , ciruelos e , rubovszky g , et al : alpelisib for pik3ca - mutated , hormone receptor - positive advanced breast cancer . 
n engl j med 380 : 1929 - 1940 , 2019 adamo b , deal am , burrows e , et al : phosphatidylinositol 3 - kinase pathway activation in breast cancer brain metastases . 
breast cancer res 13 : r125 , 2011 fitzgerald dm , muzikansky a , pinto c , et al : association between pik3ca mutation status and development of brain metastases in hr + / her2metastatic breast cancer . 
bergen es , berghoff as , medjedovic m , et al : continued endocrine therapy is associated with improved survival in patients with breast cancer brain metastases . clin cancer res 25 : 2737 - 2744 , 2019 11 . 
tolaney sm , lin nu , thornton d , et al : abemaciclib for the treatment of brain metastases ( bm ) secondary to hormone receptor positive ( hr + ) , her2 negative breast cancer . 
j clin oncol 35 : 1019 , 2017 ( 15 suppl ) ippen fm , alvarez - breckenridge ca , kuter bm , et al : the dual pi3k / mtor pathway inhibitor gdc - 0084 achieves antitumor activity in pik3ca - mutant breast cancer brain metastases . 
campone m , im s - a , iwata h , et al : buparlisib plus fulvestrant versus placebo plus fulvestrant for postmenopausal , hormone receptor - positive , human epidermal growth factor receptor 2 - negative , advanced breast cancer : overall survival results from belle - 2 . 
takeshita t , yamamoto y , yamamoto - ibusuki m , et al : analysis of esr1 and pik3ca mutations in plasma cell - free dna from er - positive breast cancer patients . 
study_list = brca_igr_2015%2cbrca_mbcproject_wagle_2017&case_set_id = all&data_priority = 0&gene_list = esr1%253amut%250apik3ca%253amut& geneset_list = %20&prolefilter = 0&tab_index = tab_visualize juric d , castel p , grifth m , et al : convergent loss of pten leads to clinical resistance to a pi ( 3 ) k inhibitor . 
grommes c , pentsova e , nolan c , et al : phase ii study of single agent buparlisib in recurrent / refractory primary ( pcnsl ) and secondary cns lymphoma 24 . 
maira s - m , pecchi s , huang a , et al : identication and characterization of nvp - bkm120 , an orally available pan - class i pi3 - kinase inhibitor . 
 germline and somatic smoothened mutations in nonsmall - cell lung cancer are potentially responsive to hedgehog inhibitor vismodegib introduction smoothened ( smo ) is a seven - membrane spanning receptor involved in the hedgehog signaling pathway and is directly inhibited by patched ( ptch ) .1 in the absence of ptch inhibition , smo activates gli family transcription factors and subsequently regulates stemand progenitor cell differentiation . 
dysregulation and activation of the hedgehog pathway are associated with carcinogenesis.2 smo has been identified as an oncogene with activating mutations in sporadic basal cell carcinomas ( bccs ) 1 and medulloblastomas.3 activating smo mutations are highly relevant targets for therapy , because oral smo inhibitors , such as vismodegib ( erivedge ; genentech , san francisco , ca ) , have had proven success in antitumor activity.4 , 5 in nonsmall - cell lung cancer ( nsclc ) , neither somatic nor germline smo mutations have previously been characterized . 
 we discovered a novel germline smo p641a mutation in a patient with refractory nsclc who responded to vismodegib therapy for several months . the patient was a 69 - year - old nonsmoking man ( patient a ) with a prior fully resected bcc who developed widely metastatic nsclc . 
 none of the other first - degree relatives reported a malignancy history , and none carried the p641a mutation . we subsequently identified two additional patients ( patients b and c ) with metastatic nsclc who had smo mutations by our routine genetic profile of 50 genes via the md anderson cancer center molecular diagnostics laboratory and treated them with vismodegib . 
patient b ( adenocarcinoma ) was also found to carry the germline smo p641a mutation ; however , despite initial stabilization of his rapidly growing disease , he stopped vismodegib at 14 weeks because of grade 3 fatigue , experienced rapid disease progression , and died . 
 ( a ) radiographic images of patient a at baseline with images of lung and liver metastases and restaging chest and abdominal imaging of lung metastases after 6 weeks of vismodegib . 
all patient and family members signed informed consent for participation in this analysis within institutional review boardapproved protocol pa2014 - 0455 . results we determined the incidence of somatic smo mutations to be 2.6% in the lung adenocarcinoma cohort and found one case ( d209y ) in the squamous cohort from tcga ( n = 230 and 178 , respectively )  . 
eight germline smo p641a mutation cases ( variant frequency , 0.11% ) were identified in various cancer types , whereas none of the control germline cohort individuals had smo p641a mutation . 
similar results were obtained using additional lung cancer cell lines and the hedgehog inhibitor cyclopamine ( appendix fig a1 , online only )  . we hypothesized that ptch1 may interact with smo in the cytoplasmic domains and that smo p641a mutation disrupts smo / ptch1 interactions and releases smo suppression . 
although multiple models propose how ptch1 suppresses smo activity , 8 the binding affinity of ptch1 to smo is approximately 2 nm.9 on the basis of our in silico models of the cytoplasmic domain structures of both ptch1 and smo using our previously described approach of homology modeling and structural refinement , 10 - 12 we determined that smo p641 is located in the cytoplasmic domain on a loop between two beta sheets , in the area where smo and ptch1 interact ( fig 3 )  . computational alanine scanning and mutabind13 suggest that smo p641 destabilizes ptch1 binding with loss of binding energy and is a hotspot residue ( g > 1.5 kcal / mol )  . 
loss of the proline - aromatic interactions ( pro641 v phe1264 ) , which are critical in protein stability and protein - protein interactions , partly contributes to the binding reduction.14 , 15 once mutated to alanine , the smo loop can change conformation easily and potentially cause steric hindrance with another hotspot residue : val639 of smo ( g = 3.7 kcal / mol )  . 
computational alanine scanning showed that smo p641a mutation disfavors smo and ptch1 binding , leading to loss of binding energy of 2.3 kcal / mol , suggesting smo p641 is a hotspot residue ( g > 1.5 kcal / mol ) on the binding interface . 
this reduction in binding is partially because of the loss of proline - aromatic interactions ( pro641 v phe1264 ) , which are critical in protein stability and protein - protein interactions.14 , 15 in addition , the prolyl ring restricts and maintains the smo loop conformation and drives formation of the ring - ring stacking interactions of pro641 and phe1264 . 
also , smo p641a causes the side chain of ptch1 phe1264 to swing away because of the loss of interaction with proline , and this new position causes some steric hindrance with another hotspot residue : val639 of smo ( g = 3.7 kcal / mol )  . 
multiple proteins , including 2cz9 , 2no4 , and 4b3h structures , were used as the template for smo , and 2hg4 , 2q7v , 4xyc , and 5bwi were used for ptch1 . 
our structural modeling of smo p641a suggests that the conformational changes prevent or significantly limit ptch binding to smo , which leads to constitutive activation of downstream hedgehog signaling and dysregulated growth . 
this model does not preclude the possibility of catalytic regulation of smo by ptch1 via diffusion of a second messenger molecule , 8 where the diminished interaction would reduce smo translocation to the membrane for activation . in our analysis of the tcga databases , somatic smo mutations are rare , occurring in 1.7% of patients with nsclc . 
germline smo p641a mutations were not observed in patients with nsclc in the tcga database , but in the overall tcga database they were observed in 0.11% of patients with cancer , compared with 0% in individuals without cancer . 
 larger studies are needed to further investigate this hypothesis . it is possible that germline smo p641a mutation may be a precursor mutation that requires an additional alteration , such as tp53 mutation , to induce full carcinogenesis . 
in support of this hypothesis , both our patients with smo p641amutated nsclc had concomitant ( but different ) somatic tp53 mutations in their lung cancers , but not in their germline analyses . 
for example , it was reported16 , 17 that hedgehog pathway signaling via mutant smo constitutive activation can prevent p53 accumulation by enhancing p53 binding to mdm2 for ubiquitination , thus potentially leading to acceleration of carcinogenesis . 
additional studies will be needed to investigate the possible interactions between these pathways and determine whether , in the context of hedgehog pathway activation from smo mutations , p53 loss or mutation may serve as a protumorigenic second hit . in conclusion , we identified and treated a patient with a germline smo p641a mutation with vismodegib and achieved a significant response . 
 for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . evidence that smo mutations may be a heritable driver not only of bcc but potentially of nsclc as well . 
however , the specific role of germline and somatic smo alterations in promoting carcinogenesis , interactions with p53 , and other oncogenic alterations and the responsiveness of different smo mutations to hedgehog inhibitors are important questions that merit further investigation . 
 lixia diao no relationship to disclose jing wang no relationship to disclose vassiliki papadimitrakopoulou consulting or advisory role : clovis oncology , genentech , merck , biothera , eli lilly , janssen , gensignia life sciences , astrazeneca , ariad pharmaceuticals , nektar research funding : merck ( inst ) , novartis ( inst ) , celgene ( inst ) , clovis oncology ( inst ) , bayer healthcare pharmaceuticals ( inst ) , bristol - myers squibb ( inst ) , astrazeneca ( inst ) , pfizer ( inst ) , janssen oncology ( inst ) , acea biosciences ( inst ) myvizhi esai selvan no relationship to disclose zeynep h . 
heymach stock and other ownership interests : cardinal spine , bio - tree consulting or advisory role : astrazeneca , abbvie , boehringer ingelheim , bristol - myers squibb , medivation , ariad pharmaceuticals , synta , oncomed , novartis , genentech , calithera biosciences research funding : astrazeneca ( inst ) acknowledgment we thank fred de sauvage , md , emily roarty , phd , li xu , and robert klein for contributing to the project . ximing tang no relationship to disclose wei lu no relationship to disclose humam kadara no relationship to disclose dimitry n . 
tsao , ignacio wistuba , dianren xia , lauren byers , lixia diao , jing wang , vassiliki papadimitrakopoulou , ximing tang , wei lu , zhi tan , shuxing zhang , monique nilsson , and john v . 
heymach , the university of texas md anderson cancer center , houston , tx ; humam kara , american university of beirut , beirut , lebanon ; and dimitry n . 
chen l , du - cuny l , moses s , et al : novel inhibitors induce large conformational changes of gab1 pleckstrin homology domain and kill breast cancer cells . 
 appendix mutation and sequencing analysis archival tumor tissue dna was extracted using the generead dna formalin - fixed , paraffin - embedded ( ffpe ) kit ( qiagen , hilden , germany )  . 
surveying of mutations in hotspot regions of 46 cancer - related genes including smo was performed via ion ampliseq cancer panel ( life technologies , carlsbad , ca )  . 
the details for these methods using ampliseq cancer panel have been described previously ( singh rr , et al : j mol diagn 15 : 607 - 622 , 2013 )  . 
from the barcoded library , a manual emulsion polymerase chain reaction was performed , followed by sequencing of eight multiplexed samples using the ion personal genome machine on ion 318 chips . 
 torrent suite software ( version 2.0 ; thermo fisher ) was used for analysis of the sequence generated . for the germline and validation tumor analyses , a custom ampliseq panel targeting the coding regions of smo and p53 was designed on ampliseq.cothe smo / p53 panel was then used to prepare libraries for 24 dna samples from ffpe tissue , saliva , and blood with a modified protocol of the ion ampliseq library kit 2.0 ( life technologies )  . 
the target amplification step for each of the two primer pools was carried out in a half - volume reaction and combined after thermal cycling to create a single - volume reaction used in the rest of the preparation . 
the barcoded libraries were quantitated with quantitative polymerase chain reaction using the ion library taqman quantitation kit ( life technologies )  . the libraries were then pooled for 12 samples per chip , and the equivalent of 7 l of a 100 - pm solution was used to prepare template with the ion pi template ot2 200 kit ( version 3 ; life technologies ) according to manufacturer protocol . 
the template was then loaded onto an ion pi chip ( version 2 ; life technologies ) and sequenced on an ion proton sequencer with the sequencing reagents provided in the ion pi sequencing 200 kit ( version 3 ; life technologies ) as per manufacturer instructions . a bed file provided from ampliseq.com for the smo / p53 panel was used to generate a sequencing report in the ion torrent browser ( version 4.2 ) for use in assessing the quality of the libraries , chips , and reagents during the sequencing process . 
it was deparaffinized with deparaffinization solution at 56c for 3 minutes and digested with proteinase k at 56c for 1 hour ; nucleic acid was reverse formaldehyde modified at 90c for 1 hour , and artificially induced uracils were removed with the enzyme uracil - n glycosilase at 50c for 1 hour in a thermomixer . 
dna was quantified with nanodrop ( nd1000 spectrophotometer ; wilmington , de ) and the qubit dsdna hs assay ( life technologies )  . dna isolated from saliva saliva was collected with the ogr - 500 kit ( dna genotek ) , and dna was extracted with the prepit.l2p kit ( dna genotek )  . 
clear supernatant was transferred to a fresh 15 - ml centrifuge tube , to which 1.2 volume of room - temperature 100% ethanol was added , and mixed ; the mixture was then left to stand at room temperature for 10 minutes . 
these rare smo variants were scored for functional deleteriousness with combined annotation dependent depletion ( kircher m , et al : nat genet 46 : 310 - 315 , 2014 ) and kept if phred score 20 for subsequent variant frequency estimates . cell viability assays tumor cells were plated at a ratio of 2 , 000 cells per well in 96 - well plates and were treated with 10% fetal bovine serum media with increasing concentrations of vismodegib or cyclopamine . 
 ( a ) western blot demonstrating expression of gli1 and ptch1 / 2 in h1437 ( left ) and hcc4006 ( right ) nonsmall - cell lung cancer ( nsclc ) cells expressing smo wild type ( wt ) or smo p461a . 
giannetta , md1 ; gianluca tomasello , md1 ; stefano stabile , phd1 ; valentina motta , phd1 ; spyridon alexiadis , md1 ; francesco scaglione , md , phd1 , 2 ; angelo vanzulli , md1 , 2 ; massimo torre , md1 ; emanuela bonoldi , md1 ; silvio m . 
veronese , phd1 ; salvatore siena , md1 , 2 ; and andrea sartore - bianchi , md1 , 2 introduction epidermal growth factor receptor ( egfr ) gene mutations are strong oncogenic drivers in a subset of nonsmall - cell lung cancer ( nsclc )  . 
their inhibition with specic tyrosine kinase inhibitors ( tkis ) represents a successful example of precision medicine.1 nevertheless , disease progression almost invariably occurs after 9 - 14 months of treatment with either getinib , erlotinib , or afatinib ( rstand second - generation tkis ) 2 - 4 and after 19 months with osimertinib ( a thirdgeneration tki ) 5 because of the development of acquired therapeutic resistance . several mechanisms of tki resistance have been reported , with different mechanisms for rst - , second - , 6 and third - generation tkis.7 the acquisition of t790m mutation in exon 20 of egfr represents the most frequent mechanism ( more than 50% of patients ) of disease progression with getinib , erlotinib , or afatinib , which can be overcome by osimertinib and other thirdgeneration inhibitors . 
however , a frequent cause of third - generation tki resistance is the development of mutations at the egfr c797 codon , because they prevent binding to the egfr active site.8 , 9 in these patients , preclinical studies have shown that c797s mutation can be found in the same ( in cis ) or different ( in trans ) t790m - mutated alleles , or in other patients the t790m mutation can be lost.10 in clinical practice , resistant mechanisms to different tkis can be detected on disease progression through tissue rebiopsy . 
the isolation and analysis of circulating tumor dna ( ctdna ) through liquid biopsy is also recommended and widely used for identication of acquired resistance mutations of egfr.11 however , not only target - dependent resistance mechanisms can occur . 
after 9 months , the patient experienced disease progression in the liver ( data supplement ) , and a liquid biopsy revealed the acquisition of egfr t790m mutation ( fig 1b ; table 1 )  . 
almost 12 months later , dyspnea worsened , with evidence of increased right pleural effusion and disease progression in the lung , lymph nodes , and liver ( data supplement )  . a new liquid biopsy identied persistence of t790m plus the acquisition of c797s in trans conformation ( table 1 ; fig 1 - c )  . 
synopsis of therapeutic history and histologic and molecular analysis of the present patient showing tumor evolution toward small - cell lung cancer ( sclc ) transformation and accumulation of epidermal growth factor receptor ( egfr ) genetic abnormalities . 
in the case of sclc transformation , a favorable response to etoposide and platinum can be expected25 ; therefore , a rebiopsy should be performed when a histologic transformation is suspected ( eg , aggressive progression )  . in this case report , which describes a representative patient , different subclones harboring distinct resistance mechanisms coexisted . 
the initial ratio of t790m / activating mutation was below the threshold of 0.05 and , according to the ndings of chabon et al , 24 the response to osimertinib was limited to a stable disease by recist criteria . 
this ratio deeply decreased ( table 1 ) at progression after 12 months of therapy with osimertinib when c797s appeared concomitantly with a small - cell transformation , after a total of 20 months of treatment with tkis . 
at this time point , we observed a reduction of t790m / del19 ratio in ctdna , which was similar to the c797s / del19 ratio ( table 1 )  . 
 pizzutilo et al in conclusion , our case report underlines the importance of monitoring the evolution of tissue rebiopsy for identifying heterogeneous resistance mechanisms , especially in patients with aggressive progression , the disease and of to better dene potential treatment modications . 
pizzutilo , calogero lauricella , giulio cerea , massimo torre , salvatore siena , andrea sartore - bianchi financial support : salvatore siena provision of study materials or patients : elio g . 
giannetta , stefano stabile , valentina motta , spyridon alexiadis , angelo vanzulli , emanuela bonoldi , silvio m . veronese , salvatore siena , andrea sartore - bianchi data analysis and interpretation : elio g . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . angelo vanzulli travel , accommodations , expenses : bracco diagnostics , ge healthcare emanuela bonoldi consulting or advisory role : bayer silvio m . 
mitsudomi t , morita s , yatabe y , et al : getinib versus cisplatin plus docetaxel in patients with non - small - cell lung cancer harbouring mutations of the epidermal growth factor receptor ( wjtog3405 ) : an open label , randomised phase 3 trial . 
lancet oncol 11 : 121 - 128 , 2010 rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as rst - line treatment for european patients with advanced egfr mutationpositive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
lancet oncol 13 : 239 - 246 , 2012 sequist lv , yang jc - h , yamamoto n , et al : phase iii study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with egfr mutations . 
j clin oncol 31 : 3327 - 3334 , 2013 soria j - c , ohe y , vansteenkiste j , et al : osimertinib in untreated egfr - mutated advanced non - small - cell lung cancer . 
 case report thress ks , paweletz cp , felip e , et al : acquired egfr c797s mutation mediates resistance to azd9291 in non - small cell lung cancer harboring egfr t790m . nat med 21 : 560 - 562 , 2015 10 . 
niederst mj , hu h , mulvey he , et al : the allelic context of the c797s mutation acquired upon treatment with third - generation egfr inhibitors impacts sensitivity to subsequent treatment strategies . 
rolfo c , mack pc , scagliotti gv , et al : liquid biopsy for advanced non - small cell lung cancer ( nsclc ) : a statement paper from the iaslc . 
tan dsw , yom ss , tsao ms , et al : the international association for the study of lung cancer consensus statement on optimizing management of egfr mutation - positive non - small cell lung cancer : status in 2016 . 
j thorac oncol 11 : 946 - 963 , 2016 jukna a , montanari g , mengoli mc , et al : squamous cell carcinoma transformation concurrent with secondary t790m mutation in resistant egfr - mutated adenocarcinomas . 
yu ha , arcila me , rekhtman n , et al : analysis of tumor specimens at the time of acquired resistance to egfr - tki therapy in 155 patients with egfr - mutant 75ra26 , 2011 lung cancers . 
oxnard gr , hu y , mileham kf , et al : assessment of resistance mechanisms and clinical implications in patients with egfr t790m - positive lung cancer and acquired resistance to osimertinib . 
yang z , yang n , ou q , et al : investigating novel resistance mechanisms to third - generation egfr tyrosine kinase inhibitor osimertinib in non - small cell lung cancer patients . 
leone a : c797s and t790m egfr mutations in non - small cell lung cancer : in trans or in separate clones ? j thorac oncol 13 : e21 - e22 , 2018 19 . 
wang z , yang j - j , huang j , et al : lung adenocarcinoma harboring egfr t790m and in trans c797s responds to combination therapy of rstand thirdgeneration egfr tkis and shifts allelic conguration at resistance . 
zhou z , zhao y , shen s et al : durable clinical response of lung adenocarcinoma harboring egfr 19del / t790m / in trans - c797s to combination therapy of rstand third - generation egfr tkis . 
minari r , bordi p , del re m , et al : primary resistance to osimertinib due to sclc transformation : issue of t790m determination on liquid re - biopsy . 
piotrowska z , niederst mj , karlovich ca , et al : heterogeneity underlies the emergence of egfrt790 wild - type clones following treatment of t790m - positive cancers with a third - generation egfr inhibitor . 
marcoux n , gettinger sn , okane g , et al : egfr - mutant adenocarcinomas that transform to small - cell lung cancer and other neuroendocrine carcinomas : clinical outcomes . 
the patient experienced progression on trametinib 0.5 mg per day , which is 25% of the clinically recommended dose and a dose likely insufficient to inhibit erk signaling . a previous publication2 and our experiences in the clinic suggest that pulsatile dosing may maintain the mek inhibitor dose to inhibit erk signaling sufficiently in the tumor and allow normal tissues to recover from toxic effects . 
in addition , preclinical data with raf inhibitors in melanoma models suggest that intermittent treatment may extend the period of response and forestall the development of lethal drug - resistant disease.3 we have treated two patients with colorectal tumors with the triplet combination of raf , mek , and egfr inhibitors who experienced dose - limiting drug toxicity attributable to mek inhibitors and continued to have difficulty tolerating treatment despite a reduction of one dose level . 
one patient had a partial response to vemurafenib , cobimetinib , and cetuximab lasting 16 months while taking cobimetinib on a dose schedule of 40 mg once per day , with 1 week on and 3 weeks off , as a result of grade 2 myalgias and grade 3 creatine phosphokinase elevations ( creatine phosphokinase > 1 , 000 iu / l ) ; the reduction of the cobimetinib dose to 40 mg occurred 2 weeks after initiation of treatment . 
the other patient has had ongoing stable disease for 12 months on dabrafenib , trametinib , and panitumumab , with a trametinib dose of 1 mg once daily , with 4 days on and 3 days off , as a result of intolerable , grade 2 acneiform rash ; the dose modification of the mek inhibitor occurred 4 weeks after the initiation of treatment . 
 one of our patients with melanoma treated from the start with an intermittent schedule of both dabrafenib and trametinib continues treatment after 3 years with dabrafenib 150 mg twice per day ( 2 weeks on and 2 weeks off ) and trametinib 8 mg once per day for the first 3 days of every other 4 - week cycle ; this patient has tolerated treatment well and experienced a sustained complete response of his tumor . 
kim kb , semrad r , schrock ab , et al : significant clinical response to a mek inhibitor therapy in a patient with metastatic melanoma harboring an raf1 fusion . 
 exceptional response to 177lutetium prostate - specic membrane antigen in prostate cancer harboring dna repair defects megan crumbaker , mbbs1 , 2 , 3 ; louise emmett1 , 3 ; lisa g . 
psma - targeted small molecules bound to 177lutetium ( 177lu ) , a medium - energy b - emitter , have been administered as a targeted therapy for metastatic prostate cancer ; the psma - targeted small molecule binds to psma on the cancer cell surface and is internalized , leading to delivery of a potent but targeted dose of radiation to the cell . 
a small molecule commonly used for this purpose is psma - 617 , a human psma - targeting ligand that can be conjugated to 177lu . 177lupsma - 617 therapy has shown great promise in early - phase trials , with a prostate - specic antigen ( psa ) response rate of 57% in a phase ii prospective study , 1 and randomized trials are currently recruiting internationally . de novo and acquired resistance are common , however , and biomarkers are needed to guide patient selection and rationalize combinatorial approaches . 177lupsma - 617 induces cell death through doublestrand dna breaks.2 mechanisms to repair doublestrand dna breaks are decient or absent in cells with mutations in homologous repair genes such as brca1 or brca2 , and such mutations are increasingly recognized in metastatic castration - resistant prostate cancer ( mcrpc ) .3 , 4 thus , a deciency in homologous repair may render a tumor more sensitive to 177lupsma - 617 and be relevant in crpc . 
postoperative radiotherapy was withheld because of a history of colorectal carcinoma treated with surgical resection followed by adjuvant radiotherapy 25 years prior . soon after his radical prostatectomy , he biochemically relapsed and was treated with intermittent androgen deprivation therapy . 
he eventually developed overt metastatic disease , with a perihilar mass found on a computed tomography ( ct ) scan of the chest , abdomen , and pelvis after 10 years of hormone manipulation . a nuclear medicine bone scan was negative , but an 18f - labeled uorodeoxyglucose positron emission tomography ( pet ) scan showed low - grade uptake in the mass , which was conrmed to be metastatic prostate cancer on biopsy . 
a choline pet was undertaken to assess the extent of his disease and revealed uptake in his left hilar nodes with inltration in the lingula and involvement of right paratracheal nodes . given the extent of disease across both hemithoraces , he commenced enzalutamide . 
his psa continued to increase ( fig 1 ) despite 8 weeks of treatment , and additional radiologic progression with impending bronchial obstruction prompted radiotherapy to his hilar disease . his psa initially decreased after radiotherapy but began to increase soon after . 
he then received two cycles of cabazitaxel , after which a restaging ct scan showed enlargement of his liver metastases and development of new pelvic nodal metastases . having progressed through all standard lines of therapy , this patient was enrolled in a clinical trial of 177lupsma - 617 therapy and received 4 6.5 gigabecquerel doses once every 6 weeks ( anzctr identier : actrn12615000912583 )  . 
plot of prostate - specic antigen ( psa ) values over time . treatment regimens : cabazi , cabazitaxel ; doce , docetaxel ; enz , enzalutamide ; lupsma , 177lutetium prostate - specic membrane antigen ; rt , radiotherapy . time ( months ) the return of his right upper quadrant abdominal pahe was consented for prescreening of his circulating tumor dna ( ctdna ) for a poly adp - ribose polymerase ( parp ) inhibitor trial , but while awaiting this result , his pain increased signicantly , with derangement of his liver function tests and a further increase in his psa . 
he was approved for an additional two compassionate 7.0 - gigabecquerel doses of 177lupsma - 617 ; he received these with clinical and radiologic responses associated with a psa response from 169 to 43 after the rst dose . a resolution bioscience targeted ctdna analysis for dna repair defects revealed abnormalities in three homologous fig 2 . 
to assess his germline , we performed targeted sequencing of coding regions and splice sites of atm , brca1 , brca2 , brip1 , cdh1 , cdkn2a , chek2 , hoxb13 , palb2 , pten , rad51c , rad51d , stk11 , and tp53 on dna extracted from blood ; this conrmed a frameshift mutation in exon 11 of the brca2 gene . 
up - front resistance and early relapses are common , however , signifying the need for strategies to improve patient selection and / or test rational combination approaches . as a form of ionizing radiation , 177lupsma - 617 induces double - strand dna breaks , which are believed to be the main lethal event in most exposed cells . 
preclinical and clinical studies have identied an association between cancers with homologous recombination defects and increased radiation sensitivity.8 germline mutations in genes that encode and mediate key enzymes for dna repair pathways , such as brca2 and atm , occur in approximately 11.8% of men with metastatic prostate cancer and somatic mutations in at least 23% of patients with mcrpc , 3 , 4 rendering many prostate cancers homologous recombination decient . homologous recombination deciency has been associated with responses to parp inhibition and platinum - based chemotherapy , 9 , 10 but these treatments may cause signicant toxicity . this patients germline mutation likely predisposed him to more aggressive disease , 11 and he was resistant to not only enzalutamide but also two lines of taxane - based chemotherapy . 
after his 177lupsma - 617 treatment , he also responded to carboplatin chemotherapy and a parp inhibitor . is biologically plausible that this gentlemans aberrations in homologous repair pathways enhanced his susceptibility to the radiation effects of 177lupsma - 617 therapy . 
we hypothesize that patients with mutations in dna repair pathway genes would be especially responsive to peptide receptor radionuclide therapy because of their inability to overcome dna breaks induced by the treatment . 
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eur urol 73 : 656 - 661 , 2018 emmett l , willowson k , violet j , et al : lutetium 177 psma radionuclide therapy for men with prostate cancer : a review of the current literature and discussion of practical aspects of therapy . 
pomerantz mm , spis ak s , jia l , et al : the association between germline brca2 variants and sensitivity to platinum - based chemotherapy among men with 11 . 
castro e , goh c , olmos d , et al : germline brca mutations are associated with higher risk of nodal involvement , distant metastasis , and poor survival outcomes metastatic prostate cancer . 
 s use of real - world data for the research , development , and evaluation of oncology precision medicines although randomized controlled trials remain the scientific ideal for determining the efficacy and safety of new treatments , they are sometimes insufficient to address the evidentiary requirements of regulators and payers . 
this is particularly the case when it comes to precision medicines because trials are often small , deliver incomplete insights into outcomes of most interest to policymakers ( eg , overall survival ) , and may fail to address other complex diagnostic and treatment - related questions . 
a number of modified earlyand latephase trial designs have been proposed to better support earlier biomarker validation , patient identification , and selection for regulatory studies , but there is still a need for confirmatory evidence from real - world data sources . 
these data are usually provided through observational , postapproval , phase iiib and iv studies , which rely heavily on registries and other electronic data setsmost notably data from electronic health records . 
notable examples include imatinib for the treatment of chronic myeloid leukemia , gefitinib for egfr mutationpositive nonsmall - cell lung cancer , and olaparib for patients with germline brca mutationpositive ovarian cancer . however , it is not always easy to assess the impact of new targeted cancer medicines and determine what , if any , benefit they have over existing therapies.1 - 3 table 1 lists some of the challenges of generating evidence in precision oncology . in part , this difficulty is because the types of large randomized trials that have successfully generated evidence to support blockbuster drugs are not well suited to generating evidence about targeted therapies . 
this raises a number of significant challenges to the generation and assessment of evidence . first , simply by virtue of their size , trials of precision medicines frequently do not generate the kind of evidence about overall survival that regulators and payers may demand.3 the difficulty in assessing overall survival is exacerbated by the ethical imperative to allow crossover among biomarker - positive patients , particularly when targeted agents are being assessed as alternatives to more toxic chemotherapy treatments . these challenges were evident in phase iii studies of imatinib in chronic myeloid leukemia , 4 of gefitinib5 and erlotinib6 in nonsmall - cell lung cancer , and more recently , of olaparib in advanced breast cancer in women with a known germline brca1 or brca2 gene mutation.7 the olaparib study , which recently reported improved progression - free survival with olaparib compared with chemotherapy , has thus far been unable to demonstrate an overall survival advantage . 
although this may be because such an advantage does not exist , it may also be because of crossover of patients receiving chemotherapy to olaparib , both during active monitoring and after completion of the study . 
challenges of generating evidence in precision oncology challenges small and geographically spread study populations ethical imperative to allow crossover within studies epidemiologic characterization of biomarker - defined populations evolution of disease biology leading to changes in molecular targets complexity , resource intensity , and cost of studies , particularly with adaptive trial designs failure to capture some clinically significant outcome measures need to simultaneously evaluate companion diagnostics conduct of studies only in high - income / resource - rich settings and countries unclear generalizability of data from study to target populations introduced by investigator behaviors ( eg , more frequently approaching patients with family histories or disease characteristics with known links to a biomarker ) may obscure their true prevalence and distribution . 
furthermore , more detailed knowledge about the true distribution of biomarkers by age , sex , response to prior therapies , geographies , and ethnicity is similarly difficult to obtain if the only data generated comes from screening limited numbers of patients as part of enrollment onto a single or small number of randomized controlled trials . closely aligned with this challenge is that of simultaneously assessing companion diagnostics that will eventually be used for the identification of biomarker - positive patients . 
adaptive trials may , for example , assign patients to randomly selected initial treatment groups , followed by additional testing for relevant biomarkers and appropriate stratification over the course of a trial.18 the advantages of these adaptive methods is that they can accommodate emerging information about biomarkers , changes in disease biology , and different patterns of treatment responsiveness among subsets of patients.19 two examples of adaptive studies that have been used in the develinclude the opment of precision medicines biomarker - integrated approaches of targeted therapy for lung cancer elimination ( battle ) trial , a nonsmall - cell lung cancer umbrella trial with four phase ii protocols , and i - spy2 , a breast cancer trial comparing novel drugs in combination with standard chemotherapy to standard therapy alone . and small size of trials of precision medicines may also make it more difficult to detect infrequent but serious adverse effects.8 second , although all trials face problems of generalizability ( as a result of , for example , the limited size of trial populations , effects of selection criteria , close monitoring of compliance , and frequency of clinical monitoring ) , 9 trials of targeted cancer therapies raise additional issues because patients enrolled onto such trials may be selected on the basis of biomarkers that are not routinely used in clinical practice . 
furthermore , because trials of precision medicines are expensive , sponsors may frequently choose to recruit the smallest numbers of patients required to deliver a statistically significant result , raising additional questions about the clinical significance of trial results.10 third , as in any trial , the statistical power assigned secondary end points is often considerably less than that given to primary objectives . 
this is exacerbated when secondary end points are used in trials of precision medicines because of their size , their complexity ( eg , allowing crossover ) , and the impact of both known and unknown differences between study and real - world populations . 
in the absence of postapproval follow - up studies involving large numbers of patients , drug labels are likely to be highly restricted . finally , even if a targeted therapy can be shown to be sufficiently efficacious and safe in a small trial , it can be extremely difficult to determine which patients should subsequently be screened for biomarkers and potentially offered treatment . 
 however , adaptive trials are methodologically and logistically complex and rely upon close collaboration between clinician - researchers and industry across multiple sites and geographic regions.20 they also do not address the full range of challenges associated with evidence development in precision medicine . 
in this context , physicians often prescribe medicines off - label ( ie , for indications other than those approved by the regulator ) or seek individual patient access to unregistered medicines on compassionate grounds , particularly when these seem promising based on phase i and ii studies . the problem with using off - label prescribing and patient - by - patient compassionate access as means of providing access to new medicines is that they tend to be ad hoc , inequitable , and driven more by compassion and desperation than by evidence . 
in an effort to overcome these problems , numerous formal approaches to accelerated access have been developed around the world ( eg , by the fda , the european medicines agency , health canada , and singapores health sciences authority ) .24 these programs both speed up access and enable approval based on limited data . 
examples of cancer medicines that have recently been approved in the united states on the basis of phase ii data include olaparib , in 2014 , for the management of metastatic ovarian cancer and brigatinib , in 2017 , for patients with nonsmall - cell lung cancer who have experienced progression after initially responding to crizotinib , which was itself approved via accelerated approval in 2011.25 the difficulty with accelerated access programs is that they generally mitigate against the collection of high - quality , standardized data , in part because there is no financial incentive on the part of industry and clinicians to generate additional data once a medicine is on the market and in part because patients are unwilling to enroll onto phase iii trials when they have other , guaranteed means of access and ongoing supply.10 in the long run , this creates sustained uncertainties and may place patients at risk ; we know that medicines approved through accelerated access schemes are more likely to have health warnings related to unanticipated toxicities and more likely to be subsequently withdrawn.26 , 27 accelerated access may also encourage more widespread off - label prescribing , as in the case of vemurafenib , a kinase inhibitor indicated for the treatment of metastatic melanoma with activating braf v600e mutation , which has been used off - label for refractory braf v600e mutation positive hairy cell leukemia in the absence of formal phase iii studies of its use for this indication.28 given the challenges associated with accelerated access , regulators and funders often request additional evidence of safety and / or effectiveness after registration or subsidization . 
these enable approval subject to ongoing data collection and , ideally , review of registration and subsidization decisions based on these data.2 evidence to support accelerated access and managed entry schemes may be generated through controlled trials , cohort studies , registries , and the like , all of which depend to a significant degree on the ongoing collection of clinical data regarding patient outcomes using real - world data ( rwd ) sources . 
in part , this is because they contain a vast array of different types of data including quantitative data ( eg , laboratory values ) , qualitative data ( eg , text - based documents and demographics ) , and transactional data ( eg , records of medication delivery ) , 29 all of which can be repurposed for clinical and health services research . 
 databases , pharmacies , insurance claims , disease registries , bio - repositories , public health databases , census records , and social media , which can be additional sources of data . importantly , data from ehrs may be used to assess whether the results of randomized controlled trials are generalizable to real - world populations who may differ in many ways from study populations ( eg , according to age , comorbidity , concurrent therapies ) 30 ; may identify other factors that modify clinical outcomes , including treatment availability and clinician preferences ; and may reveal patterns in treatment responsiveness and adverse effects.31 , 32 furthermore , large volumes of electronic clinical information may offer insights into potential new indications for drugs.33 in addition , where linked bio - specimens are available , data from ehrs may help to characterize genotype - phenotype associations.34 if , for example , one wished to gain a greater understanding of the efficacy and safety of poly ( adp - ribose ) polymerase inhibitors in women with breast cancer , one could ask how and when olaparib might be used with routinely available treatments , such as tamoxifen in the management of hormone receptor and brca gene mutationpositive breast cancer , or whether comorbidities that typically exclude patients from participating in regulatory phase iii clinical trials , such as liver or renal impairment , really do influence the clinical effectiveness of treatment or increase toxicities . recognition of the importance of rwd in general , and ehrs in particular , is evident in recent initiatives such as the asco - sponsored cancerlinq and the international registry to improve outcomes in men with advanced prostate cancer ( ironman )  . the asco 2017 report on the state of oncology practice in the united states , for example , notes that : learning health care systems that are based on big sets of routine clinical practice data offer great potential for improving both patient care and research , and the benefits of these systems may have particular importance for cancer care . 
the fda appears to have taken this on board , with asco recently announcing : under the new collaboration , fda plans to use cancerlinqs repository of real - world evidence on patient treatment and outcomes to track how genomically targeted drugs and newer immunotherapies are used in clinical practice , comparing their impact to what has been seen in clinical trials , and tracing their use by physicians either according to , or in spite of , their labelling information.37 enthusiasm for rwd has led numerousprofessional , commercial , and not - for - profit organizations38 - 45 to develop expertise in the collection , linkage , and use of rwd to support cancer research , and there are now examples of cancer studies that have used rwd to understand biomarker prevalence and influence on clinical outcomes , 46 - 48 validate standards of care , 49 assess the generalizability of randomized controlled trial outcomes , 50 and conduct comparative effectiveness research.51 however , despite the promise of using rwd to support research , there are considerable epistemic , social , and ethical challenges to its effective and responsible use . 
potential opportunities and challenges associated with the use of ehr - generated real - world data identification of clinically relevant study compatibility of ehr platforms for data populations exchange potential opportunity access to large populations , including via linkage of ehrs to administrative and research data sets inclusion of patients usually excluded from rcts ( eg , older patients , patients with hepatic impairment ) identification and accurate determination of biomarker prevalence among patient ( as opposed to study ) populations ability to account for differing treatment lines and sequences of therapies ability to collect outcome data relating to standard - of - care treatments not selected as comparators in rcts ability to quantify survival impact that may be obscured by crossover , either within clinical trials or after poststudy exposure to an investigational agent potential for long - term follow - up challenges data quality and compatibility issues , including accuracy , completeness , clarity , provenance , detail , and consistency in methods of measurement generation and harmonization of standards for data collection and analytical methodologies funding and resources for establishment , maintenance , and use of ehrs systematic exclusion of countries and settings that lack ehr systems data custodianship and patient privacy consent to secondary use in research of clinical data acceptance of data and methodologies among professional groups , regulators , and payers abbreviations : ehr , electronic health record ; rct , randomized controlled trial . defined rwd , can be challenging because , although data might be more complete and fit for purpose , the very process of controlling and cleaning data sets can compromise the authenticity and applicability of the research.66 , 67 although there is no doubt that it would help to build consensus about methods of data collection , curation , and analysis , 45 it may never be possible to completely standardize rwd because requirements for each study may be profoundly different and it is impossible to predict the data requirements for every potential study . 
different interventions will require specific and disparate evidence needs and thus demand the collection of different types of data , and each may require unique study protocols , clinical report forms , participant information and consent forms , and so on . 
standardization is also challenged by the nuances and complexity of multidisciplinary cancer management.68 for these reasons , asco has supported development of a framework for oncology data exchange based on a document markup standard known as high level 7 clinical document architecture ( hl7 cda )  . 
the position that has underpinned research ethics for more than 50 years is that consent from patients is required for research of any kind , and lawmakers and health care service providers generally hold the same views . 
the issues in relation to rwd are whether , and to what extent , patients should have control over information about their health care and whether accessing information without consent would constitute a breach of privacy . 
although autonomy is highly valued , the need to obtain explicit patient consent for the use of routine clinical data can be resource intensive and lead to biases as a result of differences between consenters and nonconsenters , 89 including those related to sex , socioeconomic status , or health status.90 this is particularly relevant where studies seek to assess the survival of a particular group of patients for future comparison with newer treatments in similar clinical settings . assessment of survival is likely to be further confounded if data can only be collected from living patients . 
this public opinion seems to support the view that respect for autonomy and privacy is not absolute and that public benefit is important enough and institutions are trustworthy enough to allow individuals health - related data to be used in specific situations either without consent or with broad consent . 
a study conducted after the establishment of the united kingdom national cancer registry , for example , suggested that the public does not necessarily consider the confidential use of personal information for public health research and surveillance to be an invasion of privacy.91 other studies have demonstrated public support for data linkage , with the caveat of adequate measures being in place to protect privacy.92 new policies and guidelines for researchers and human ethics committees are providing direction on whether consent is required before the use of rwd for research . 
in general , the need for consent depends on the level of risk to patients privacy , whether data can be anonymized , and the reason provided for analyzing patient data . 
additional considerations include practical difficulties associated with obtaining consent , the epidemiologic necessity for comprehensive data , the importance of the research ( public interest ) , and whether the researchers wanting access to data have commercial interests ( in most cases , waivers of consent do not apply when commercial entities seek access to data )  . it is also generally accepted that where consent cannot be waived , open - ended ( as distinct from repeated , project - specific ) consent is sufficient.93 importantly , consent may be sought not simply for use by a single researcher or research group but also for data sharing with additional researchers or research networks concurrently or in the future . research that encompasses long - term storage and recurring use of personal data will thus inevitably raise issues about access , including access by commercial entities and those located internationally.76 , 94 , 95 the issue here is that those who collect data lose control and may have to relinquish custodianship altogether . 
the sharing of data also brings to the fore questions about the degree of personal control people should have over their data and how this control should be realized.95 - 100 innovative models of consent , such as dynamic consent , supported by web - based information technologies , may help to facilitate the use of information from ehrs in ways that provide a range of ethical safeguards . 
 are being considered include open consent ( obtained once from consenters for all future research uses of genetic and other data ) , meta consent ( for use of retrospective and prospective data after being informed about the types of research these data might be used to conduct ) , and portable legal consent ( a system through which users can donate their data to databases that remove identifying details , such as name and e - mail address ) .77 , 87 , 88 , 101 , 102 more effective and efficient data linkage and networking of databases and information from other sources ( eg , biobanks , registries , and clinical trials ) , along with greater regulatory harmonization , may also improve processes for using rwd in the development of precision medicines . 
existing processes for regulatory oversight might be complemented by the establishment of independent authorities with appropriate legal , scientific , and technical expertise who could work as coordinators , receivers , and custodians of rwd . maintaining patient confidentiality and privacy the size , complexity , possible linkage , long - term storage , and potential commercialization of ehrgenerated data add significant complexity to efforts to ensure confidentiality of health - related information . 
lewis data analysis and interpretation : all authors manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors to the research question being asked but might have clinical significance for the research participant . 
the dilemma here is that , although informing participants of such findings might enable them to prevent or respond more rapidly to disease , the clinical significance of findings is not always obvious . 
the problem with promising to return individual results is that their clinical significance is often unclear , the clinical validity of findings generated in research laboratories is often uncertain , and people might not want to receive such information no matter how clinically significant it might be . 
regulators and payers have begun to acknowledge the need to establish new ways of generating and evaluating evidence , 110 and systems have developed to facilitate alternative , including real - world , methods of data collection and evidence generation . 
although these methods have the potential to transform hospitals and health care systems into learning health care systems , they raise their own technical , scientific , organizational , economic , ethical , and legal issues . 
lewis employment : astrazeneca ian kerridge no relationship to disclose wendy lipworth employment : msd animal health ( i ) affiliations all authors : sydney health ethics , university of sydney , sydney , new south wales , australia . references lewis j , lipworth w , kerridge i : ethics , evidence and economics in the pursuit of personalized medicine . 
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lewis jrr , lipworth w , kerridge i , et al : dilemmas in the compassionate supply of investigational cancer drugs . intern med j 44 : 841 - 845 , 2014 23 . 
alsop k , fereday s , meldrum c , et al : brca mutation frequency and patterns of treatment response in brca mutation - positive women with ovarian cancer : a report from the australian ovarian cancer study group . 
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abbing - karahagopian v , kurz x , de vries f , et al : bridging differences in outcomes of pharmacoepidemiological studies : design and first results of the protect project . 
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coloma pm , schuemie mj , trifir `o g , et al : combining electronic healthcare databases in europe to allow for largescale drug safety monitoring : the eu - adr project . 
suissa s , henry d , caetano p , et al : cnodes : the canadian network for observational drug effect studies . open med 6 : e134 - e140 , 2012 45 . 
garrison lp jr , neumann pj , erickson p , et al : using real - world data for coverage and payment decisions : the ispor real - world data task force report . 
wang li , shewade a , lambert p , et al : characteristics of advanced non - small cell lung cancer ( ansclc ) patients ( pts ) receiving molecular diagnostic ( md ) testing in us routine clinical practice . 
he ky , zhao y , mcpherson ew , et al : pathogenic mutations in cancer - predisposing genes : a survey of 300 patients with whole - genome sequencing and lifetime electronic health records . 
feinberg ba , kish jk , garofalo d , et al : evaluation of real - world electronic medical record ( emr ) treatment outcome data compared to clinical trial data for an advanced breast cancer treatment . 
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n engl j med 370 : 2161 - 2163 , 2014 van staa t - p , dyson l , mccann g , et al : the opportunities and challenges of pragmatic point - of - care randomised trials using routinely collected electronic records : evaluations of two exemplar trials . 
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hemkens lg , contopoulos - ioannidis dg , ioannidis jpa : agreement of treatment effects for mortality from routinely collected data and subsequent randomized trials : meta - epidemiological survey . 
science 346 : 1054 - 1055 , 2014 ioannidis jpa , trikalinos ta : early extreme contradictory estimates may appear in published research : the proteus phenomenon in molecular genetics research and randomized trials . 
sboner a , mu xj , greenbaum d , et al : the real cost of sequencing : higher than you think ! genome biol 12 : 125 , 2011 75 . 
hill em , turner el , martin rm , et al : lets get the best quality research we can : public awareness and acceptance of consent to use existing data in health researcha systematic review and qualitative study . 
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makady a , ham rt , de boer a , et al : policies for use of real - world data in health technology assessment ( hta ) : a comparative study of six hta agencies . 
van gent , phd1 , 2 ; and agnes jager , md , phd3 purpose biomarkers that predict response to poly ( adp - ribose ) polymerase inhibitors ( parpis ) are required to detect parpi sensitivity beyond germline brca - mutated ( gbrcam ) cancers and parpi resistance among reverted gbrcam cancers . 
therefore , we previously developed the repair capacity ( recap ) test , a functional homologous recombination ( hr ) assay that exploits the formation of rad51 foci in proliferating cells after ex vivo irradiation of fresh primary breast cancer tissue . 
the aim of the current study was to validate the feasibility of this test on histologic biopsy specimens from metastatic breast cancer and to explore the utility of the recap test as a predictive tool for treatment with dna - damaging agents , such as parpis . methods fresh tissue biopsies from easily accessible metastatic lesions from patients with locally advanced or metastatic breast cancer were irradiated with 5 gy and cultured for 2 hours followed by detection of rad51 foci presence ( hr procient ) or absence ( hr decient [ hrd ] )  . 
although hrd was detected in 13 ( 32% ) of 41 specimens , only ve showed a gbrcain three patients with gbrcam , post - treatment recap tests showed hr phenotype reversion after in vivo progressive disease on platinum and parpi treatment , which was explained in one patient by a secondary brca1 mutation . conclusion the recap test , which reects real - time hr status regardless of brca mutations , is feasible in metastatic breast cancer biopsy specimens . 
2019 by american society of clinical oncology introduction tumors developed among germline brca1 / 2 mutation ( gbrcam ) carriers have a defect in homologous recombination ( hr ) and are therefore highly sensitive to poly ( adp - ribose ) polymerase inhibitors ( parpis ) and dna double - strand break ( dsb ) inducing chemotherapies.1 - 4 recently , food and drug administration approval was obtained for the use of olaparib in advanced gbrcam breast cancer.3 until now , gbrcam status is the only predictive biomarker for response to parpi therapy . 
however , evidence is emerging that parpis also are effective in cancers with hr deciency ( hrd ) caused by other mechanisms than gbrcam , such as somatic brca1 / 2 mutations or mutations of other hr genes.5 several different hrd tests have been designed to identify hrd tumors in addition to gbrcam tumors to enlarge the population of patients with breast cancer who could benet from treatments that target the hr pathway.6 - 10 however , a gold standard test for predicting response to parpi therapy is not yet available . 
most hrd tests are based on specic genomic or transcriptomic patterns derived from gbrcam tumors , which also occur in sporadic cancers ( brcaness ) .6 - 10 these tests measure the accumulation of mutations and chromosomal aberrations over time ; thus , they do not necessarily reect the real - time hr status of the tumor . 
 meijer et al context key objective to determine whether the repair capacity ( recap ) test is feasible in histologic biopsy specimens from metastatic breast cancer and to explore the predictive value of the recap test for double - strand break ( dsb ) inducing therapies . knowledge generated the recap test is feasible in metastatic biopsy specimens . 
although 13 ( 32% ) of 41 specimens were found to be homologous recombination ( hr ) decient , only ve had a germline brca mutation ( gbrcam )  . 
in three patients with gbrcam with progressive disease on dsb - inducing therapies , post - treatment recap tests showed hr status reversion , which was explained by a secondary brca1 mutation in one of these patients . relevance the recap test reects real - time hr status regardless of brca mutations . 
therefore , recap is expected to predict sensitivity to dsb - inducing treatment more precisely than germline mutation analysis only . proliferating cells after ex vivo irradiation of fresh breast cancer tissue , was developed previously for primary breast cancer ( n = 148 ) .12 , 13 in the current study , we aimed to show feasibility of this test on biopsy specimens from metastatic breast cancer lesions to enhance the diagnostic potential and clinical applicability of the recap test . 
next , we aimed to nd a molecular explanation for the observed hrd phenotype , and we explored the utility of the recap test as a predictive tool for treatment with dsb - inducing agents , such as parpis , in this setting . methods biopsy specimens patients with locally advanced or metastatic breast cancer with metastatic lesions amenable for biopsy and who were planned to start systemic treatment were eligible . 
patients with pulmonary and / or bone metastasis only were excluded because of the risk of pneumothorax and technical difculties with experimental procedures caused by calcications ( data supplement )  . 
patients were to have a bilirubin level less than 1.5 the upper limit of normal and both ast and alt levels less than ve times the upper limit of normal in case a liver biopsy was scheduled . 
platelets were to be greater than 100 109 / l and international normalize ratio less than 1.5 , unless platelet and international normalized ratio values were not necessary according to local protocols or after consent of the interventional radiologist for that particular biopsy site ( eg , breast )  . 
after registration and written informed consent , each patient was scheduled for a biopsy performed by a ( interventional ) radiologist according to local protocols . for distant metastases , a core needle biopsy with a minimum of an 18 - gauge and maximum of a 12 - gauge needle was performed under imaging guidance . 
in brief , presence of rad51 foci was determined in geminin ( gmn ) positive , least 30 gmn - expressing cells were counted per tumor sample . a cell was considered rad51 positive when at least ve rad51 foci were detected . 
tumors were classied as hr procient ( hrp ) , hrd , or hr intermediate ( hri ) when more than 50% , less than 20% , or 20% to 50% of gmnpositive cells showed ve or more rad51 foci , respectively . thus actively proliferating , cells only . 
at brca1 / 2 analyses and rad51 assay on cell lines see data supplement . statistical analysis the aim of the study was to show feasibility of the recap test on biopsy specimens from metastatic breast cancer . the primary end point was the proportion of patients with metastatic breast cancer with a successful recap test result . 
in total , 47 biopsy specimens were obtained from 44 patients , of which three did not contain tumor cells , resulting in 44 representative specimens . the recap test could be performed successfully in 41 ( 93% ; 95% ci , 86% to 100% ) of the 44 biopsied metastatic lesions ( p , .001 ; fig 1a )  . 
the reason for unsuccessful testing in three biopsy specimens was an insufcient number of proliferating ( ie , gmn - positive ) tumor cells ( fewer than 30 gmn - positive in m189 , m250 , and m332 )  . 
m250 and m332 contained six and 19 gmn - positive cells , respectively , of which all were rad51 positive . biopsy specimens were derived from several metastatic sites ( fig 1b )  . 
the recap test could be performed on core needle biopsy ( n = 37 ) as well as on punch biopsy ( n = 4 ) specimens of skin metastases . 
in 19 specimens ( four triplenegative breast cancer [ tnbc ] , four human epidermal growth factor receptor 2positive , and 11 estrogen receptor [ er ] / progesterone receptor positive ) , we determined whether standard er , progesterone receptor , and human epidermal growth factor receptor 2 immunohistochemistry analyses could be performed on the recap biopsy specimen . 
in one specimen , which was originally scored as tnbc , we found a 10% er expression in the recap specimen , which is just above the threshold for er positivity ( ie , greater than or equal to 10% in the netherlands )  . among the 41 successful recap test results , we identied 13 hrd , two hri , and 26 hrp tumor samples ( fig 1 )  . recap status was independent of histopathologic characteristics of the tumors , biopsy type , and localization of the metastatic lesion ( data supplement )  . molecular defects in hrd tumors the 13 hrd tumors were analyzed further to nd a molecular explanation for the observed hrd phenotype ( table 1 )  . 
five ( 38% ) of 13 of hrd tumors harbored gbrcam of which one harbored a brca2 variant of unknown signicance that was functionally validated for affecting hr.14 in two hrd tumors ( 15% ) , somatic brca2 variants of unknown signicance were found , which are predicted to be nonpathogenic . 
brca1 promotor hypermethylation was identied in one hrd tumor , and brca1 rna was indeed absent in this tumor as assessed by rna in situ hybridization ( data supplement )  . 
thus , the hrd phenotype was explained by a brca defect in six tumors ( 46% ) , which left seven ( 54% ) of the 13 hrd tumors to be non - brca related . 
one non - brcarelated hrd tumor harbored a germline mutation in palb2 . change of hrd phenotype in response to dsb - inducing therapy next , we explored the added value of the recap test as a predictive biomarker to select patients for parpi treatment in the metastatic setting . 
the patient was treated consecutively with capecitabine , carboplatin and gemcitabine , and low - dose cisplatin in combination with nivolumab , on which she developed pd after six cycles . 
the second specimen was very heterogeneous , harboring both hrd ( rad51 - negative ) and hrp ( rad51 - positive ) cells , which overall resulted in an hri phenotype . 
 hrd reversal after dna damaging chemotherapy patient 1 : brca2 vus 06 - 2014 primary brc 12 - 2014 lumpectomy 02 - 2016 07 - 2016 12 - 2016 02 - 2017 11 - 2015 mastitis carcinomatosa / metastasis 4x ddac 12x paclitaxel capecitabine carboplatin + gemcitabine 2x cisplatin + 6x nivolumab olaparib biopsy m222 rad51 negative biopsy m298 rad51 intermediate patient 2 : brca1 mutation 03 - 2014 primary brc lumpectomy 03 - 2016 locoregional recurrence 02 - 2017 04 - 2017 3x fec 3x docetaxel carboplatin + paclitaxel and veliparib capecitabine biopsy m242 rad51 negative biopsy m303 rad51 positive patient 3 : brca2 mutation 05 - 2007 primary brc ablation 05 - 2013 bone metastases 04 - 2015 01 - 2016 03 - 2017 5x fec + tamoxifen 3x fac + anastrozol tamoxifen carboplatin + paclitaxel and veliparib biopsy m234 rad51 negative biopsy m319 rad51 positive m222 m242 m234 pretreatment post - treatment dapi geminin rad51 overlay dapi geminin rad51 overlay m298 15 m m303 m319 10 m fig 2 . 
 ( a ) an additional point mutation ( ofcially , 4327_ 4329delinstgg ) was acquired next to the original 4327c.t mutation , which resulted in restoration of the open reading frame disrupted by the original mutation and , consequently , full - length brca1 protein production . 
 ( b ) fluorescence in situ hybridization analysis showed that both preand posttreatment biopsy specimens harbored two copies of brca1 , which indicates that the original mutated allele was duplicated . 
of note , both tumors developed an hrp phenotype after they had developed resistance in response to the combination of carboplatin , paclitaxel , and veliparib ( parpi ) within a clinical trial . 
these three patients had been previously treated with anthracycline - based therapy , which also induces dsb but did not lead to hr phenotype reversion . preand post - treatment biopsy specimens , loss of heterozygosity was observed . 
fluorescence in situ hybridization analysis showed that both preand post - treatment specimens harbored two copies of brca1 , which indicates that the original mutated allele was duplicated ( fig 3b )  . thus , the tumor became hrp as a result of reversion of one mutant brca1 copy . to unravel the molecular mechanisms that lead to resistance in these patients , we searched for secondary brca1 / 2 mutations in the post - treatment tumor biopsy specimens . 
patient 2 acquired an additional point mutation , that is , 4329a.g ( ofcially , 4327_4329delinstgg ) next to the original 4327c.t mutation , which resulted in restoration of the open reading frame disrupted by the original mutation and , consequently , full - length brca1 protein production ( fig 3a )  . 
other mechanisms for parpi resistance have been proposed , such as loss of 53bp1 proteloss of 53bp1 was examined but not found in any resistant tumor ( data supplement )  . 
brc , breast cancer ; dapi , 4 ( cid : 2 ) , 6 - diamidino - 2 - phenylindole ; edu , 5 - ethynyl - 2 ( cid : 2 ) deoxyuridine ; fac , uorouracil , doxorubicin , and cyclophosphamide ; ir , irradiated ; loh , loss of heterozygosity ; pd , progressive disease . 20 m when this patient showed pd after 5 months of carboplatin treatment ; however , pretreatment hr and paclitaxel status remains unknown because a fresh pretreatment biopsy specimen was not available for this patient ( fig 4 )  . 
because this tumor might have developed reversal of the hrd phenotype , both preand posttreatment tumor material ( 70% tumor ) were sequenced . the germline brca2 mutation was present at an maf of 41% and 34% , respectively , and secondary mutations were not present . 
 meijer et al brca2 mutation suggested that the brca2 wild - type allele was still present in the tumor , which was conrmed by uorescence in situ hybridization ( fig 4b )  . 
chek2 is a known breast cancer susceptibility gene , but the direct effect of chek2 mutations on hr status remains elusive . therefore , the hr status of sum102pt cells , which harbor the exact same c.1100delc mutation in chek2 as found in this patient , was determined , and the chek2 c.1100delc mutation did not cause hrd ( fig 4d )  . 
she showed pd after the rst response evaluation at 8 weeks , which corresponded to the outcome of the recap test ( hrp ; fig 4a )  . discussion this study shows that the recap test is feasible ( 93% successful ) in metastatic breast cancer biopsy specimens , which greatly enhances its diagnostic potential and clinical applicability . 
the functional recap test is robust , and its applicability extends from core needle biopsy specimens of different metastatic sites to punch biopsy specimens of skin metastases , and test results are available within 1 week . hrd was detected in 13 ( 32% ) of 41 tumors . 
among 13 hrd tumors , only ve showed a gbrcam , which indicates that the recap test may identify twice as many candidates for parpi treatment as gbrca analysis . we believe that this study provides the rst evidence that the hr phenotype , measured by a functional hr test , changes over time in patients with gbrcam as a result of previous therapies . 
within the current cohort , reversion of the hr phenotype was detected in three patients with gbrcam tumors after developing in vivo resistance to dsb - inducing therapies ( carboplatin and cisplatin or parpi )  . 
another gbrcam patient with a brca - procient tumor showed resistance to dsb - inducing agents and was refractory to olaparib , a nding concordant with the result of the recap test ( hrp )  . 
these ndings highlight the need for hr tests that are based on the phenotype , such as the recap test , especially in the advanced setting . the molecular mechanism that leads to resistance and reversion of the hrd phenotype was explained in one of three patients by a secondary mutation in brca1 . 
this is unique because for breast cancer , most reversion mutations are described within brca2 , and a brca1 reversion has been described only once before.15 the incidence of brca1 / 2 secondary mutations within resistant tumors is not yet clear because most are described in case reports or studies with small sample sizes . 
in 26 patients who had platinum - resistant recurrent ovarian cancer , the incidence of brca1 / 2 secondary mutations was 46.2%.16 in two ( 40% ) of ve patients with gbrcam with platinum and parpi therapy - resistant metastatic breast cancer , secondary brca2 mutations were detected in circulating tumor dna.17 several case reports have highlighted that brca1 / 2 secondary mutations are a universal resistance mechanism for various tumor types ( eg , prostate , pancreatic ) with germline or somatic loss - of - function mutations.18 - 21 however , because not all resistant patients harbor secondary mutations , detection of parpi - resistant tumors by screening for secondary brca mutations would lead to an underestimation and only identify a subgroup of resistant tumors . 
the recap test , however , detects resistant tumors regardless of the exact underlying resistance mechanism . we show a case of coincidence of both germline brca2 and chek2 c.1100delc mutations in a patient with an hrp breast tumor . 
the driver mutation of this tumor was chek2 instead of brca2 , which highlights two important issues : first , breast cancer that arises in a patient with gbrcam is not necessarily brca associated , and second , the chek2 c.1100delc mutation does not cause hrd . 
furthermore , the recap test does not depend on high tumor percentage in a sample because the microscopic readout allows differentiation between tumor and stromal cells . another advantage is its broad applicability : where many hrd tests focus specically on tnbcs , the recap test detects hrd regardless of histologic grade and subtype . because recap procedures ( irradiation and 2 hours of inuence standard hormone receptor culturing ) do not analyses , the fresh biopsy specimen used for recap testing also could be used for diagnostic pathology analyses , which thereby limits the amount of tissue needed . 
first , functional testing requires fresh ( instead of parafn - embedded ) tumor specimens , and for the recap test in particular , ex vivo dna damage is induced by x - ray or gamma irradiation . 
 hrd reversal after dna damaging chemotherapy moreover , reversion of the hr phenotype is detected by recap , which identies patients with gbrcam who will no longer benet from parpi or dsb - inducing agents as a result of resistance . 
the functional recap test , which determines the real - time hr status independent of gbrcam status , shows great potential as a predictive biomarker for parpi treatment of metastatic breast cancers . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . hendrikus - jan dubbink consulting or advisory role : pzer , personal genome diagnostics research funding : astrazeneca winand n.m. 
dinjens consulting or advisory role : amgen , bristol - myers squibb , astrazeneca ( inst ) , roche no other potential conicts of interest were reported . acknowledgment we thank the medical oncologists of erasmus mc for informing patients about the trial and other activities that led to patient enrollment . 
hollestelle , phd , for providing the t47d , mm463 , and sum102pt cell lines . references audeh mw , carmichael j , penson rt , et al : oral poly ( adp - ribose ) polymerase inhibitor olaparib in patients with brca1 or brca2 mutations and recurrent ovarian cancer : a proof - of - concept trial . 
lancet 376 : 245 - 251 , 2010 ledermann j , harter p , gourley c , et al : olaparib maintenance therapy in patients with platinum - sensitive relapsed serous ovarian cancer : a preplanned retrospective analysis of outcomes by brca status in a randomised phase 2 trial . 
lancet oncol 15 : 852 - 861 , 2014 robson m , im sa , senkus e , et al : olaparib for metastatic breast cancer in patients with a germline brca mutation . 
n engl j med 377 : 523 - 533 , 2017 tutt a , robson m , garber je , et al : oral poly ( adp - ribose ) polymerase inhibitor olaparib in patients with brca1 or brca2 mutations and advanced breast cancer : a proof - of - concept trial . 
lancet 376 : 235 - 244 , 2010 den brok wd , shrader ka , sun s , et al : homologous recombination deciency in breast cancer : a clinical review . 
br j cancer 107 : 1776 - 1782 , 2012 birkbak nj , wang zc , kim jy , et al : telomeric allelic imbalance indicates defective dna repair and sensitivity to dna - damaging agents . 
cancer discov 2 : 366 - 375 , 2012 davies h , glodzik d , morganella s , et al : hrdetect is a predictor of brca1 and brca2 deciency based on mutational signatures . 
nat med 23 : 517 - 525 , 2017 joosse sa , van beers eh , tielen ih , et al : prediction of brca1 - association in hereditary non - brca1 / 2 breast carcinomas with array - cgh . 
konstantinopoulos pa , spentzos d , karlan by , et al : gene expression prole of brcaness that correlates with responsiveness to chemotherapy and with outcome in patients with epithelial ovarian cancer . 
j clin oncol 28 : 3555 - 3561 , 2010 incorvaia l , passiglia f , rizzo s , et al : back to a false normality : new intriguing mechanisms of resistance to parp inhibitors . 
afghahi a , timms km , vinayak s , et al : tumor brca1 reversion mutation arising during neoadjuvant platinum - based chemotherapy in triple - negative breast cancer is associated with therapy resistance . 
weigelt b , comino - m endez i , de bruijn i , et al : diverse brca1 and brca2 reversion mutations in circulating cell - free dna of therapy - resistant breast or ovarian j clin oncol 29 : 3008 - 3015 , 2011 cancer . 
carneiro ba , collier ka , nagy rj , et al : acquired resistance to poly ( adp - ribose ) polymerase inhibitor olaparib in brca2 - associated prostate cancer resulting from biallelic brca2 reversion mutations restores both germline and somatic loss - of - function mutations . 
cheng hh , salipante sj , nelson ps , et al : polyclonal brca2 reversion mutations detected in circulating tumor dna after platinum chemotherapy in a patient with metastatic prostate cancer . 
banda k , swisher em , wu d , et al : somatic reversion of germline brca2 mutation confers resistance to poly ( adp - ribose ) polymerase inhibitor therapy . 
wolff ac , hammond me , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
zhu xd , k uster b , mann m , et al : cell - cycle - regulated association of rad50 / mre11 / nbs1 with trf2 and human telomeres . 
voss ( continued ) purpose with prospective clinical sequencing of tumors emerging as a mainstay in cancer care , an urgent need exists for a clinical support tool that distills the clinical implications associated with specific mutation events into a standardized and easily interpretable format . 
to this end , we developed oncokb , an expert - guided precision oncology knowledge base . methods oncokb annotates the biologic and oncogenic effects and prognostic and predictive significance of somatic molecular alterations . 
potential treatment implications are stratified by the level of evidence that a specific molecular alteration is predictive of drug response on the basis of us food and drug administration labeling , national comprehensive cancer network guidelines , disease - focused expert group recommendations , and scientific literature . results to date , > 3 , 000 unique mutations , fusions , and copy number alterations in 418 cancerassociated genes have been annotated . 
with the shift from single analyte tests and small hotspot panels to larger gene panels and whole - exome and - genome platforms , interpretation of the clinical significance of the increasing number of genomic alterations identified in individual tumors has become a challenge . 
a smaller subset are known or suspected functionally significant mutations with no clear therapeutic implications , and the smallest subset consists of known driver mutations that are clinically actionable . the information that discriminates whether an alteration is clinically actionable can reside in various silos , including us food and drug administration ( fda ) labeling , national comprehensive cancer network ( nccn ) guidelines , conference proceedings , disease - focused expert group recommendations , and the scientific literature . 
therefore , an urgent need exists for a clinical support tool that distills this information into a standardized and easily interpretable format that democratizes its access to clinicians of all knowledge levels and at all centers . 
to address these limitations , we describe oncokb , a comprehensive precision oncology knowledge base that offers oncologists detailed , evidence - based information about individual somatic mutations and structural alterations present in patient tumors to support optimal treatment decisions . 
oncokb content is supervised by a dedicated panel of physicians and cancer biologists who review and edit biomarker - associated investigational therapeutic strategies ( data supplement )  . through continual dialogue with the scientific and medical community , oncokb integrates clinical best practices as defined by institutionwide , multidisciplinary disease management teams ( fig 1 )  . 
oncokb communicates information on the biomarker - guided use of fdaapproved therapies and investigational agents under evaluation in clinical trials and highlights negative clinical results to discourage off - label use of expensive targeted therapies shown to be ineffective in specific mutational contexts . methods oncokb includes biologic , clinical , and therapeutic information curated from multiple unstructured information resources , including guidelines and recommendations derived from fda labeling , nccn guidelines , other diseasespecific expert and advocacy group recommendations , and the medical literature ( fig 1 )  . with recognition that clinical implications vary substantially on the basis of specific alterations within a gene and tumor context , information in oncokb is hierarchically organized by gene , alteration , tumor type , and clinical implication ( fig 1 )  . 
oncokb information is publicly available through an interactive web site19 and incorporated into the cbioportal for cancer genomics , 20 - 22 where patient genomic alterations are annotated with information from oncokb and their biologic effects and clinical implications are summarized , which facilitates cancer researcher and clinician interpretation of complex genomic data ( fig 1 )  . 
3 , 000 alterations in 418 cancer - associated genes ( table 1 )  . levels of evidence to communicate the clinical utility of individual mutant alleles consistently , a level of evidence classification system was developed ( fig 2 ) that takes into account the site of tumor origin by recognizing that the effects of targeted inhibitors vary by tumor lineage , even in cancers that share the same mutant allele ( fig 3 )  . 
potentially actionable alterations in a specific cancer type are assigned to one of four levels that are based on the strength of evidence that the mutation is a predictive biomarker of drug sensitivity to fdaapproved or investigational agents for a specific indication . 
level 1 includes genes for which specific alterations have been recognized by the fda as predictive of response to an fda - approved drug in a particular disease context ( data supplement )  . examples include braf v600e and either vemurafenib or dabrafenib as monotherapy or in combination with the mek inhibitors cobimetinib or trametinib in melanoma ; egfr l858r and erlotinib , afatinib , or gefitinib in nonsmall - cell lung cancer ( nsclc ) ; and mutations in exons 9 and 11 of kit and imatinib , sunitinib , and regorafenib in gi stromal tumors . 
in total , 82 alterations in 12 genes are considered level 1 ( data supplement )  . in recognition that some alterations in what would be considered a level 1 gene are intrinsically resistant to currently available fda - approved drugs or fall outside explicit fda approval , oncokb assigns individual alterations to a level of evidence as opposed to the entire gene . 
 variant databases gene alteration tumor type clinical implications incorporation of feedback onc kb oncokb.org web site statistical recurrence treatment guidelines akt1 scientific literature data sources breast cancer ovarian cancer lung cancer e17k e40k l52r q79k amplification prevalence prognosis nccn guidelines standard therapy clinical genomics annotation committee oncokb api cbioportal msk clinical reports investigational therapy curation review variant curation oncokb access fig 1 . 
data sources : alterations are identified by their recurrence18 from public variant databases ( cbioportal , cosmic [ catalogue of somatic mutations in cancer ] , the memorial sloan kettering [ msk ] impact internal clinical sequencing cohort ) , and by prior knowledge available in the literature . biologic and clinical therapeutic implications of alterations are curated from several public resources , including disease - specific treatment guidelines ; abstracts from major conference proceedings , such as asco , european society of medical oncology , and american association for cancer research ; and the scientific literature through pubmed . 
level 2a includes alterations that are not fda - recognized biomarkers but are considered standard care predictive biomarkers of response to an fda - approved therapy in specific cancer types . 
these alterations are highlighted in expert panel guidelines , such as the nccn compendium and asco clinical practice guidelines . several level 2a associations involve rare cancer types ( eg , braf v600e mutant histiocytosis ) or small subpopulations of common cancers ( eg , braf v600e mutant lung cancer ) , and therefore , accrual to a prospective randomized phase iii clinical study may not be feasible . 
because the total number of patients affected often is small , an application for an fda indication may never be filed , but disease experts consider the biomarker - drug association as a standard off - label use . 
in total , 11 genes with 85 alterations are currently considered level 2a ( data supplement )  . as an example , met exon 14 alterations are present in approximately 3% of nsclc27 and represent a distinct , molecularly defined subpopulation of lung adenocarcinomas that are mutually exclusive with tumors that harbor activating mutations in egfr and kras and fusions of alk , ros1 , and ret.28 - 30 an adequately powered randomized trial that compares the met inhibitor crizotinib with other standard approaches , such as chemotherapy and immunotherapy , has not yet been performed partly because of the rarity of these alterations . 
however , durable complete or partial responses to crizotinib and cabozantinib in patients with met exon 14altered lung cancers have been reported.27 , 31 although widely available as a result of its fda approval for use in alk fusionpositive nsclc , crizotinib is not explicitly fda approved in the setting of met exon 14altered nsclc . 
because the nccn guidelines consider off - label prescription of crizotinib a standard treatment approach for patients with lung cancer with met exon 14 alterations , 32 they are classified as level 2a ( fig 2 )  . 
although vemurafenib is not fda approved for use in patients with these specific braf v600e mutant indications , the off - label use of vemurafenib is a well - supported treatment option included in the nccn guidelines on the basis of compelling clinical data33 - 35 ( fig 3 )  . level 2b includes alterations that are standard predictive biomarkers of drug sensitivity in other tumor types but for which data in the tumor in question are either lacking or negative to date . 
for example , braf v600e mutations have been identified in several cancer types , including urothelial carcinomas and germ cell tumors , 36 , 37 for which no clinical response data are reported in the literature . 
in these tumors , the use of raf inhibitors in patients with braf mutant tumors remains investigational , and braf v600e is therefore classified as level 2b36 ( fig 3 )  . 
in patients with braf v600e mutant colorectal cancer , braf inhibitors , such as vemurafenib , have been tested with disappointing results.38 such negative data are referenced in oncokb and argue against the use of raf inhibitor monotherapy in patients with braf v600e colorectal cancers . 
level 3a includes mutations that are candidate predictive biomarkers of drug response on the basis of off - label use of fdaapproved drugs or investigational agents not yet fda approved for any indication . 
for the former , the evidence that supports the predictive value of the alteration is not considered sufficient to warrant a change in standard clinical practice , and disease experts would consider the use of the fda - approved drug in this context to be investigational . 
 into oncokb in real time . oncokb access : oncokb data are available for public use through an interactive web site19 and the cbioportal for cancer genomics22 and are used internally to annotate msk clinical reports . 
api , application program interface ; nccn , national comprehensive cancer network . activity has been reported , and the mutation - drug association is classified as level 3b in all other tumor types . 
fifty - five alterations in 25 genes are considered level 3 ( data supplement )  . a representative example of a level 3 alteration is akt1 e17k ( fig 3 )  . 
promising clinical activity consistent with preclinical studies of this compound has been reported with the investigational panakt inhibitor azd5363 in patients with akt1 e17k mutant breast , lung , cervical squamous , and endometrial cancers.40 - 42 on the basis of these emerging clinical data , akt1 e17k is classified as a level 3a mutation in breast , cervical , endometrial , ovarian , and lung cancers . 
a more complex example of level 3 alterations are erbb2 missense mutations that are present in a minority of a broad range of human cancers43 and that often arise in patients without erbb2 amplification or human epidermal growth factor receptor 2 ( her2 ) protein overexpression.44 these erbb2 mutants demonstrate varying degrees of sensitivity to her2 - selective kinase inhibitors , such as lapatinib and neratinib44 ( fig 3 )  . 
level 4 alterations are candidate predictive biomarkers of response to either fdaapproved or investigational agents on the basis of compelling laboratory data and an absence of substantiating compelling clinical data . 
although anecdotal responses to targeted agents may have been demonstrated in individual patients whose tumor harbored a level 4 alteration , the data are not sufficiently robust to indicate that the presence of the mutation is associated with significantly greater activity than tumors that lack the alteration . 
for example , although studies in mouseand patient - derived xenograft models have suggested that mammalian target of rapamycin or akt - targeted inhibitors may be effective in pten - null tumors , 46 the clinical data that support pten loss as a predictive biomarker of response to phosphatidylinositol 3 - kinase , akt , or mammalian target of rapamycin inhibitors in patients are limited and conflicting.47 - 52 therefore , classification of loss - of - function pten alterations as a level 4 alteration indicates that patients with pten - deficient tumors would be rational candidates for a clinical trial of investigational phosphatidylinositol 3 - kinase pathway inhibitors alone or in combination with other agents , but that the use of such agents outside the context of a clinical trial is not yet supported by the sum of the clinical data . 
additional examples of level 4 alterations include nf1 inactivating alterations , which may be predictive of response to mek1 / 2 inhibitors , 53 and egfr exon 20 insertions in lung adenocarcinomas that respond poorly to erlotinib54 , 55 but which may be sensitive to ap32788 , an investigational inhibitor of egfr and her256 ( fig 3 )  . 
because oncokb levels are dynamic , mutations currently classified as level 4 may be reclassified as level 3 or higher if additional compelling clinical data emerge . levels of resistance ( levels r1 to r3 )  . 
oncokb classifies mutations that have been shown to confer resistance to specific targeted therapies into one of three levels on the basis of the strength of the evidence that the mutation is predictive of treatment resistance ( fig 2 )  . 
level r1 includes mutational events for which there is sufficient evidence to recommend routine testing for the mutation to identify , with a high likelihood , patients who will not respond to a standard therapy . identification of r1 mutations , therefore , would lead to a recommendation that the associated therapy be withheld in patients whose tumors harbor the mutation . 
by definition , level r1 mutations predict for resistance to fda - approved drugs , and testing for such mutations is typically recommended by expert guidelines , such as those published by the nccn . 
level r1 alterations include activating ras mutations in colorectal cancer , which predict for resistance to the egfr - targeted monoclonal antibodies cetuximab and panitumumab57 , 57a ; egfr t790m mutations in nsclc , which predict for intrinsic and acquired resistance to the egfr tyrosine kinase inhibitors ( tkis ) erlotinib , afatinib , and gefitinib58 ; and pdgfra d842v , which predicts for oncokb annotation metric no . 
 level level level level level level level level level fda - recognized biomarker predictive of response to an fdaapproved drug in this indication standard care biomarker predictive of response to an fdaapproved drug in this indication * standard care biomarker predictive of response to an fdaapproved drug in another indication but not standard care for this indication compelling clinical evidence supports the biomarker as being predictive of response to a drug in this indication , but neither biomarker nor drug is standard care compelling clinical evidence supports the biomarker as being predictive of response to a drug in another indication , but neither biomarker nor drug is standard care compelling biologic evidence supports the biomarker as being predictive of response to a drug , but neither biomarker nor drug is standard care standard care biomarker predictive of resistance to an fda - approved drug in this indication compelling clinical evidence supports the biomarker as being predictive of resistance to a drug , but neither biomarker nor drug is standard care compelling biologic evidence supports the biomarker as being predictive of resistance to a drug , but neither biomarker nor drug is standard care standard therapeutic implications * includes biomarkers that are recommended as standard care by the nccn or other expert panels but not necessarily fda recognized for a particular indication investigational therapeutic implications possibly directed to clinical trials hypothetical therapeutic implications on the basis of preliminary , nonclinical data standard therapeutic implications hypothetical therapeutic implications on the basis of preliminary , nonclinical data fig 2 . 
standard therapeutic implications include food and drug administration ( fda ) recognized biomarkers that are predictive of response to an fda - approved drug in a specific indication ( level 1 ) and standard care biomarkers that are predictive of response to an fda - approved drug in a specific indication ( level 2a )  . 
investigational therapeutic implications include fda - approved biomarkers predictive of response to an fdaapproved drug detected in an off - label indication ( level 2b ) , fdaor nonfda - recognized biomarkers that are predictive of response to novel targeted agents that have shown promising results in clinical trials ( level 3a ) , and nonfdarecognized biomarkers that are predictive of response to novel targeted agents on the basis of compelling biologic data ( level 4 )  . 
nccn , national comprehensive cancer network . resistance to imatinib in patients with gi stromal tumors.59 alterations classified as levels r2 and r3 have hypothetical therapeutic implications and include alterations that are predictive of drug resistance on the basis of clinical and biologic data , respectively , but their use in guiding treatment decisions is considered investigational ( fig 2 )  . 
for example , although multiple strategies to reverse the oncogenic effects of tp53 loss have been explored in laboratory studies and early - phase clinical trials , 61 , 62 the agents tested do not directly target tp53 , their activity is typically not restricted to tp53 mutant models , and clinical trials that test these agents either have been aborted because of lack of efficacy or do not use tp53 status as a selection criterion.63 thus , although genomic alterations in tp53 are typically oncogenic , oncokb does not consider them therapeutically actionable . 
90% of alterations in oncokb have curated biologic effects and are classified as oncogenic but are not associated with actionability . actionable alterations across cancer types although targeted inhibitors have been shown to improve clinical outcomes in melanoma and lung cancer among others , 64 the broader clinical utility of large panel or whole - exome testing remains undefined . 
implicit in the designation of a level of evidence for each branch is whether the biomarker is food and drug administration ( fda ) recognized standard care or investigational and whether it is predictive of response to a drug that is fda approved or currently being tested in clinical trials . 
90% of samples harbored at least one known oncogenic mutation , only 41% had one or more alterations for which compelling clinical data currently exist to justify the use of a standard or an investigational agent ( levels 1 to 3b ) ( fig 4a )  . 
level 1 and 2a alterations were most common in melanoma ( 44% ) , ovarian cancer ( 21% , which includes 65 of 312 samples that have either germline or somatic brca1or brca2 - inactivating mutations ) , soft tissue sarcomas ( 19% on the basis of cdk4 amplifications , which predict response to palbociclib in welldifferentiated and dedifferentiated liposarcomas but not in other soft tissue sarcomas ) , nsclc ( 14% ) , esophagogastric cancer ( 13% ) , and breast cancer ( 12% )  . 
however , whereas 44% of melanomas had mutations that predict for clinical benefit with standard therapies in patients with melanoma , the majority of actionable alterations in lgg were associated with only investigational implications , with the most common mutation being idh1 r132c ( 77% of lgg samples ) , a level 3b alteration that is based on promising clinical data with the idh1 inhibitor ag - 120 in patients with acute myeloid leukemia ( figs 4a and 4b )  . 
in total , just over 10% of all samples had a level 3a mutation as their highest actionable alteration , a cohort of patients for which enrollment in a clinical trial would represent a compelling treatment option after standard treatments . 
in addition , approximately 15% of samples had level 3b alterations as their highest actionable event , ie , alterations for which promising clinical data have been observed in an investigational setting in another cancer type . on average , there were approximately three oncogenic mutations per sample , and the number of known oncogenic mutations per sample in tumor types was independent of overall mutation burden ( fig 4c )  . 
for example , although renal cell cancers had , on average , one oncogenic mutation per sample , these mutations were typically inactivating in tumor suppressors , such as vhl and pbrm1 , which are not clinically actionable at this time . 
in contrast , although thyroid cancers also on average had approximately one oncogenic mutation per sample ( fig 4c ) , these alterations were typically actionable , such as ret fusions and braf and nras mutations . 
frequencies of level of evidence 1 to 3 assignments in the cancer genome atlas cohorts . patient samples from 19 cancer types ( the cancer genome atlas ) are classified by the alteration that carries the highest level of evidence . 
 ( a ) inset pie chart : fraction of samples across all cancer types that carry a mutation considered actionable according to the levels of evidence , oncogenic but not actionable , or variants of unknown significance ( vus )  . 
cell color as shown in the key for the inset pie chart ( a )  . columns indicate sample tumor type , rows indicate gene alteration present in sample , and numbers indicate the percentage of samples per tumor type that harbor an alteration in each gene . 
 ( c ) each patient sample was classified by the number of oncogenic alterations or the number of actionable alterations . shown is the mean number of actionable ( black ) , oncogenic ( dark gray ) , or total ( gray ) mutations per sample per tumor type . 
 ( d ) each tumor type was evaluated for the percentage of samples that carry zero , one , two , three , or four or more actionable mutations per sample ( indicated in shades of blue )  . 
 mutations ( 31% , 25% , and 22% , respectively ) , which is consistent with data that demonstrate that these tumor types are driven by multiple oncogenic mutations in nonredundant pathways65 - 68 ( fig 4d ) and may explain why targeted monotherapies have shown disappointing results to date in some of these cancer types.69 , 70 discussion since the introduction of imatinib for chronic myeloid leukemia more than a decade ago , 71 , 72 a growing number of drugs that target specific genetic alterations required for tumor initiation and progression have been shown to significantly improve outcomes in molecularly defined populations of patients with cancer.64 , 73 - 74a although tumor genetic testing is now part of routine patient care in an increasing number of tumor types , interpretation of variants remains an important challenge , and in major academic cancer centers , a significant proportion of physicians report low confidence in their ability to make optimal treatment recommendations on the basis of genomic information.75 although multiple classification systems exist for the annotation of germline variants , 76 , 77 efforts to define the clinical utility of somatic alterations have been limited to established biomarkers , 78 - 80 and prior efforts often have classified actionability as a binary variable that results in the grouping of biomarkers that are fda recognized with those that are nonactionable but oncogenic . 
to this end , we assigned each mutation to one of four levels that are based on available clinical and laboratory data that support the use of the mutation as a predictive biomarker . 
standard therapeutic implications are classified as either level 1 or 2a to recognize that not all mutation - drug associations used in standard practice have been recognized by the fda . 
moreover , there are cases where off - label use of an fda - approved drug is explicitly not warranted because of existing data that argue against the use of a targeted agent in a specific cancer type . 
for example , although the braf inhibitor vemurafenib is a standard treatment option for patients with braf v600e mutant melanoma or nsclc , robust clinical reports from multiple independent centers demonstrated that patients with braf v600e mutant colorectal cancer do not respond to raf inhibitors , at least as monotherapy.38 , 84 similarly , although erbb2 amplification predicts for the clinical utility of her2 - targeted therapies in breast and esophagogastric cancers , the clinical activity of trastuzumab in erbb2 - amplified lung cancers has been disappointing.85 braf v600e and erbb2 amplification are , therefore , level 2b when detected in colorectal cancers . 
 individual mutant alleles within a single gene may be functionally distinct , with different predictive value and , therefore , individual therapeutic implications , which complicates the development of clinical decision support tools , particularly in the context of often - vague fda labeling and expert guidelines , such as those provided by the nccn , that may not define at a granular level whether specific mutations within a gene are predictive of drug response . 
to address this complexity , oncokb groups mutations in level 1 genes , such as egfr and kit , according to whether they are biologically active , whether there are preclinical data to suggest that the allele is sensitive or resistant to the matched targeted agents , and whether there are clinical data to suggest clinical sensitivity or intrinsic resistance to the approved targeted therapy . 
the challenge of rare drug - sensitive variants in level 1 genes also highlights the need for consortia efforts , such as the american association for cancer research project genie ( genomics evidence neoplasia information exchange ) , which should allow for the collection of clinical response data for rare alleles to help to guide the treatment of patients with these less common mutations . although new laboratory and clinical data are continually generated , fda labels and professional guidelines are updated at irregular intervals . 
for example , although explicit fda approval of crizotinib in ros1 - rearranged nsclc did not occur until march 2016 , offlabel use of crizotinib in patients with ros1 fusion - positive lung cancers has been considered standard of care by several expert groups for some time.86 - 88 as another example , whereas kras was initially considered a level 3a alteration in nsclc on the basis of promising data from the randomized phase ii study that supplemented standard chemotherapy with a mek inhibitor , 89 the subsequent phase iii trial showed no survival benefit with this combination , 90 which is consistent with negative data associated with mek inhibitor use in kras mutant pancreatic and colorectal cancers.91 , 92 nonetheless , anecdotal clinical and compelling preclinical data support the use of kras as a predictive biomarker of sensitivity to novel mek and erk inhibitors alone or in combination with other agents.93 - 97 oncokb thus recognizes and reassigns the level of evidence of mutational events , if appropriate , on the basis of newer , moredefinitive , and negative randomized clinical data , which take precedence over prior preliminary clinical findings . to incorporate new clinical and research findings , we have made the oncokb annotation available publically through an interactive web site19 and through the cbioportal for cancer genomics.22 both systems include a comment feature to facilitate crowdsourcing curation of this knowledge base . 
user suggestions are evaluated by the scientific team and incorporated into oncokb through periodic updates . oncokb is also an active member in efforts to promote harmonization of variant annotation across existent knowledge bases and is participating in both clingen and the global alliance for genomic health through the variant interpretation cancer consortium . in the future , we will curate information about mutational signatures , such as overall mutation burden and the possible link to immunotherapy , mutational clonality , and the impact on drug sensitivity of co - occurrence of specific oncogenic and actionable mutations . 
rudolph , ederlinda paraiso collection and assembly of data : debyani chakravarty , jianjiong gao , sarah phillips , ritika kundra , hongxin zhang , tara soumerai , moriah h . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . debyani chakravarty no relationship to disclose jianjiong gao no relationship to disclose sarah phillips no relationship to disclose ritika kundra no relationship to disclose hongxin zhang no relationship to disclose jiaojiao wang no relationship to disclose julia e . 
chang no relationship to disclose rona yaeger consulting or advisory role : glaxosmithkline , advaxis sarat chandarlapaty honoraria : chugai pharmaceutical , foresite capital , astrazeneca , sermonix pharmaceuticals , macrogenics , agendia research funding : eli lilly ( inst ) , novartis ( inst ) travel , accommodations , expenses : chugai pharmaceutical , macrogenics , astrazeneca tiffany a . 
traina consulting or advisory role : genentech , roche , eisai , mundipharma , medivation , pfizer , astrazeneca , bayer ag , immunomedics , merck research funding : medivation , eisai , pfizer , novartis , myriad genetics , innocrin pharmaceuticals , astrazeneca paul k . 
paik honoraria : celgene , bristol - myers squibb , eli lilly , ariad pharmaceuticals consulting or advisory role : celgene , eli lilly , ariad pharmaceuticals , bristol - myers squibb , celgene research funding : celgene , emd serono travel , accommodations , expenses : emd serono alan l . 
ho consulting or advisory role : oncology consortium , bristol - myers squibb , eisai , genzyme , merck , novartis , sun pharmaceutical industries , kura oncology speakers bureau : medscape , omniprex america , eli lilly , genentech , roche , astrazeneca , bayer ag , kura oncology , kolltan pharmaceuticals , eisai , astrazeneca travel , accommodations , expenses : janssen pharmaceuticals , merck , kura oncology feras m . 
hantash employment : quest diagnostics stock and other ownership interests : quest diagnostics patents , royalties , other intellectual property : patents on molecular methods , but not related to manuscript andrew grupe employment : quest diagnostics stock and other ownership interests : quest diagnostics patents , royalties , other intellectual property : patents or patent applications travel , accommodations , expenses : quest diagnostics shrujal s . 
danila honoraria : astellas pharma , angle , bayer ag , janssen pharmaceuticals consulting or advisory role : angle , bayer ag research funding : prostate cancer foundation , genentech , janssen pharmaceuticals ( inst ) patents , royalties , other intellectual property : gene expression profile associated with prostate cancer travel , accommodations , expenses : cambridge healthtech institute , prostate cancer foundation , angle , bayer ag , american austrian foundation open medical institute , global technology community , janssen pharmaceuticals , oncology education mrinal gounder honoraria : amgen , daiichi sankyo , karyopharm therapeutics , tracon pharmaceuticals , amgen consulting or advisory role : daiichi sankyo , karyopharm therapeutics , epizyme speakers bureau : amgen travel , accommodations , expenses : amgen travel , accommodations , expenses : daiichi sankyo , karyopharm therapeutics james j . 
harding no relationship to disclose dana rathkopf consulting or advisory role : janssen pharmaceuticals research funding : janssen pharmaceuticals ( inst ) , medivation ( inst ) , celgene ( inst ) , takeda pharmaceuticals ( inst ) , millennium pharmaceuticals ( inst ) , ferring pharmaceuticals ( inst ) , novartis ( inst ) , taiho pharmaceutical ( inst ) , astrazeneca ( inst ) , genentech ( inst ) , roche ( inst ) , tracon pharmaceuticals ( inst ) alexander n . 
shoushtari consulting or advisory role : vaccinex , castle biosciences , immunocore research funding : bristol - myers squibb , immunocore travel , accommodations , expenses : bristol - myers squibb neerav shukla no relationship to disclose martin h . 
voss honoraria : novartis consulting or advisory role : novartis , calithera biosciences , natera , glaxosmithkline , exelixis , pfizer , alexion pharmaceuticals research funding : pfizer , bristol - myers squibb , genentech , roche travel , accommodations , expenses : novartis , takeda pharmaceuticals matthew d . 
hellmann consulting or advisory role : bristol - myers squibb , merck , genentech , astrazeneca , medimmune , novartis , janssen pharmaceuticals research funding : bristol - myers squibb , genentech , roche ederlinda paraiso no relationship to disclose ahmet zehir no relationship to disclose gopa iyer no relationship to disclose yelena y . 
levine stock and other ownership interests : critical outcome technologies consulting or advisory role : clovis oncology , mtrap , biodesix , tesaro maeve lowery consulting or advisory role : agios , celgene antonio omuro consulting or advisory role : stemline therapeutics , juno therapeutics , bristol - myers squibb , oxigene , alexion pharmaceuticals , astrazeneca , inovio pharmaceuticals , merck michael a . 
postow honoraria : bristol - myers squibb , merck consulting or advisory role : amgen , bristol - myers squibb , novartis research funding : bristol - myers squibb ( inst ) , novartis ( inst ) , array biopharma ( inst ) , infinity pharmaceuticals ( inst ) travel , accommodations , expenses : bristol - myers squibb leonard b . 
riely consulting or advisory role : novartis , genentech research funding : novartis ( inst ) , roche ( inst ) , genentech ( inst ) , millennium pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , pfizer ( inst ) , infinity pharmaceuticals ( inst ) , ariad pharmaceuticals ( inst ) patents , royalties , other intellectual property : patent application submitted that covers pulsatile use of erlotinib to treat or prevent brain metastases ( inst ) travel , accommodations , expenses : novartis marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network , boehringer ingelheim , astrazeneca research funding : loxo oncology ( inst ) david m . 
 jos e baselga leadership : infinity pharmaceuticals , varian medical systems , grail , foghorn stock or other ownership : pmv pharma , juno therapeutics , infinity pharmaceuticals , grail , varian medical systems , tango , foghorn , aura biomedical , apogen , northern biologics honoraria : pmv pharma , juno therapeutics , infinity pharmaceuticals , grail , northern biologics consulting or advisory role : eli lilly , novartis , grail patents , royalties , other intellectual property : combination therapy using pdk1 and pi3k inhibitors . 
may 16 travel , accommodations , expenses : roche / genentech paul sabbatini research funding : bristol - myers squibb ( inst ) , ludwig institute for cancer research ( inst ) david b . 
solit honoraria : loxo oncology , pfizer consulting or advisory role : pfizer , loxo oncology nikolaus schultz no relationship to disclose acknowledgment we thank the following individuals for serving as oncokb curators : andrew intlekofer , eric smith , piro lito , jaclyn hechtman , dmitriy zamarin , wassim abida , mythili koneru , weiyi toy , pedram razavi , philip iaquinta , byron lee , martin dalin , matthew jones , elizabeth adams , karuna ganesh , olga guryanova , carolyn jackson , william terry , yu chen , ping chi , eduard reznik , aphrothiti hanrahan , sevin turcan , philip watson , neeman mohibullah , elena goldberg , aaron viny , emily foley , samuel kaffenberger , andrew winer , connie batlevi , helen won , lindsay saunders , kinisha gala , philip jonsson , fiona brown , eneda toska , i~nigo landalopez , and tripti shrestha - bhattarai . affiliations debyani chakravarty , jianjiong gao , sarah phillips , ritika kundra , hongxin zhang , jiaojiao wang , julia e . 
j am med inform assoc 10.1093 / jamia / ocw148 [ epub ahead of print on october 27 , 2016 ] institute of precision medicine : welcome to the precision medicine knowledgebase . 
kim kb , kefford r , pavlick ac , et al : phase ii study of the mek1 / mek2 inhibitor trametinib in patients with metastatic braf - mutant cutaneous melanoma previously treated with or without a braf inhibitor . 
paik pk , drilon a , fan pd , et al : response to met inhibitors in patients with stage iv lung adenocarcinomas harboring met mutations causing exon 14 skipping . 
ma pc , jagadeeswaran r , jagadeesh s , et al : functional expression and mutations of c - met and its therapeutic inhibition with su11274 and small interfering rna in non - small cell lung cancer . 
ma pc , kijima t , maulik g , et al : c - met mutational analysis in small cell lung cancer : novel juxtamembrane domain mutations regulating cytoskeletal functions . 
frampton gm , ali sm , rosenzweig m , et al : activation of met via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to met inhibitors . 
planchard d , besse b , groen hj , et al : dabrafenib plus trametinib in patients with previously treated braf ( v600e ) mutant metastatic non - small cell lung cancer : an open - label , multicentre phase 2 trial . 
davies br , greenwood h , dudley p , et al : preclinical pharmacology of azd5363 , an inhibitor of akt : pharmacodynamics , antitumor activity , and correlation of monotherapy activity with genetic background . 
hyman dm , smyth l , bedard pl , et al : abstract b109 : azd5363 , a catalytic pan - akt inhibitor , in akt1 e17k mutation positive advanced solid tumors . 
hyman d , piha - paul s , rod on j , et al : abstract pd5 - 05 : neratinib for erbb2 mutant , her2 non - amplified , metastatic breast cancer : preliminary analysis from a multicenter , open - label , multi - histology phase ii basket trial . cancer res 76 : pd5 - 05 , 2016 46 . 
lin j , sampath d , nannini ma , et al : targeting activated akt with gdc - 0068 , a novel selective akt inhibitor that is efficacious in multiple tumor models . 
clin cancer res 19 : 1760 - 1772 , 2013 de bono js , de giorgi u , massard c , et al : pten loss as a predictive biomarker for the akt inhibitor ipatasertib combined with abiraterone acetate in patients with metastatic castration - resistant prostate cancer ( mcrpc )  . 
ma cx , luo j , naughton m , et al : a phase i trial of bkm120 ( buparlisib ) in combination with fulvestrant in postmenopausal women with estrogen receptor - positive metastatic breast cancer . 
rodon j , bra~na i , siu ll , et al : phase i dose - escalation and - expansion study of buparlisib ( bkm120 ) , an oral panclass i pi3k inhibitor , in patients with advanced solid tumors . 
yang l , clarke mj , carlson bl , et al : pten loss does not predict for response to rad001 ( everolimus ) in a glioblastoma orthotopic xenograft test panel . 
naidoo j , sima cs , rodriguez k , et al : epidermal growth factor receptor exon 20 insertions in advanced lung adenocarcinomas : clinical outcomes and response to erlotinib . 
yasuda h , park e , yun ch , et al : structural , biochemical , and clinical characterization of epidermal growth factor receptor ( egfr ) exon 20 insertion mutations in lung cancer . 
gonzalvez f , zhu x , huang w - s , et al : abstract 2644 : ap32788 , a potent , selective inhibitor of egfr and her2 oncogenic mutants , including exon 20 insertions , in preclinical models . 
druker bj , sawyers cl , kantarjian h , et al : activity of a specific inhibitor of the bcr - abl tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the philadelphia chromosome . n engl j med 344 : 1038 - 1042 , 2001 72 . 
druker bj , talpaz m , resta dj , et al : efficacy and safety of a specific inhibitor of the bcr - abl tyrosine kinase in chronic myeloid leukemia . 
tsimberidou am , wen s , hong ds , et al : personalized medicine for patients with advanced cancer in the phase i program at md anderson : validation and landmark analyses . 
macarthur dg , manolio ta , dimmock dp , et al : guidelines for investigating causality of sequence variants in oncol 32 : 1317 - 1323 , 2014 human disease . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
simon rm , paik s , hayes df : use of archived specimens in evaluation of prognostic and predictive biomarkers . j natl cancer inst 101 : 1446 - 1452 , 2009 81 . 
conti rm , bernstein ac , villaflor vm , et al : prevalence of off - label use and spending in 2010 among patentprotected chemotherapies in a population - based cohort of medical oncologists . 
langer cj , stephenson p , thor a , et al : trastuzumab in the treatment of advanced non - small - cell lung cancer : is there a role ? focus on eastern cooperative oncology group study 2598 . 
j clin oncol 22 : 1180 - 1187 , 2004 85a . jordan ej , kim hr , arcila me , et al : prospective comprehensive molecular characterization of lung adenocarcinomas for efficient patient matching to approved and emerging therapies . 
cell 131 : 1190 - 1203 , 2007 janne pa , shaw at , pereira jr , et al : selumetinib plus docetaxel for kras - mutant advanced non - small - cell lung cancer : a randomised , multicentre , placebo - controlled , phase 2 study . 
clin lung cancer 17 : e1 - e4 , 2016 infante jr , fecher la , falchook gs , et al : safety , pharmacokinetic , pharmacodynamic , and efficacy data for the oral mek inhibitor trametinib : a phase 1 dose - escalation trial . 
rinehart j , adjei aa , lorusso pm , et al : multicenter phase ii study of the oral mek inhibitor , ci - 1040 , in patients with advanced non - small - cell lung , breast , colon , and pancreatic cancer . 
hu - lieskovan s , mok s , homet moreno b , et al : improved antitumor activity of immunotherapy with braf and mek inhibitors in braf ( v600e ) melanoma . 
kakavand h , wilmott js , menzies am , et al : pd - l1 expression and tumor - infiltrating lymphocytes define different subsets of mapk inhibitor - treated melanoma patients . 
loi s , dushyanthen s , beavis pa , et al : ras / mapk activation is associated with reduced tumor - infiltrating lymphocytes in triple - negative breast cancer : therapeutic cooperation between mek and pd - 1 / pd - l1 immune checkpoint inhibitors . 
yoon yk , kim hp , han sw , et al : kras mutant lung cancer cells are differentially responsive to mek inhibitor due to akt or stat3 activation : implication for combinatorial approach . 
 o androgen receptor immunohistochemistry as a companion diagnostic approach to predict clinical response to enzalutamide in triple - negative breast cancer purpose the androgen receptor ( ar ) is increasingly recognized as a potential biomarker for identifying a subset of patients with possible hormonally driven triple - negative breast cancer ( tnbc )  . 
thus , we evaluated ar expression by immunohistochemistry in patients with advanced tnbc before treatment with the ar inhibitor enzalutamide . methods we optimized and validated immunohistochemistry assays in breast and prostate cancer cell lines and tissues using two commercial ar monoclonal antibodies ( sp107 and ar441 )  . 
ar expression was then examined in patients with advanced tnbc enrolled in a phase ii study of enzalutamide ( clinicaltrials.gov identifier : nct01889238 ) on archived or fresh tissue before treatment . 
association with clinical response was assessed by sensitivity , specificity , positive predictive value ( ppv ) , drop - out rate , and survival . results ar expression was detected in 80% and 63% of breast cancer tissue using sp107 and ar441 , respectively . 
2017 by american society of clinical oncology introduction breast cancer is a heterogeneous disease characterized by distinct molecular and genetic subtypes.1 , 2 recent progress in gene expression studies has identified major breast cancer subtypesthe estrogen receptor ( er ) or progesterone receptor ( pgr ) positive ( er + or pgr + ) luminal a and luminal b , human epidermal growth factor receptor 2 ( her2 ) positive ( her2 + ) , and basal - like breast cancers , most of which express neither er , pgr , nor her2 and are referred to as triple - negative breast cancer ( tnbc ) .1 - 4 in clinical settings , targeted therapies play a critical role in the treatment of patients diagnosed with er + or pgr + and her2 + breast cancer . 
more than 77% of invasive breast tumors are defined as ar positive ( ar + ) when 10% or more ar nuclear staining is present in cancer cells as shown by immunohistochemistry ( ihc ) 9 , 10 ; however , ar oncogenic activity in breast cancer is poorly understood.11 , 12 enzalutamide significantly inhibited tumor growth in a xenograft model13 and , compared with other antiandrogen agents , also showed stable disease and antitumor response in clinical settings.14 - 16 in this study , we evaluated the use of the ar as a putative biomarker , using ihc to identify a subset of patients with tnbc who could benefit from enzalutamide . targeted ar therapy requires the appropriate selection of patients with breast cancer . 
breast carcinoma cell lines ( ar strongly positive t - 47d and ar + mcf7 ) and prostate carcinoma cell lines ( ar + lncap and ar - negative [ ar ] du145 ) were used to confirm staining procedures . 
accuracy , sensitivity , and specificity were calculated for both antibodies . tissue evaluation for protocol optimization was performed on commercial breast tissue ( normal and tumor ) and prostate tumors . 
formalin - fixed paraffin - embedded blocks of 4to 10 - mm sections on slides were shipped in kits provided by a central laboratory ( covance , princeton , nj ) from multiple clinical trial sites to clarient diagnostic services . 
all stained slides were evaluated for nuclear staining and were scored for ar expression by a trained pathologist using bright field microscopy ( data supplement )  . all ihc staining resulted in two scores : a staining intensity ranging from 0 to 3 ( 0 = not detectable , 1 = translucent , 2 = opaque , 3 = solid ) and the percentage of cells staining positively at each level of staining intensity . 
in addition to the total staining score , we evaluated other ihc scoring functions , including the h score , to determine whether they may perform better in classifying responders . 
the h score is a widely used , semiquantitative measure that considers both the cell staining intensity and the percentage of cells stained positively.17 the score is obtained by the equation , 3 3 the percentage of solid - staining nuclei ( score at 3 ) + 2 3 the percentage of opaque - staining nuclei ( score at 2 ) + the percentage of translucent - staining nuclei ( score at 1 ) , yielding results in a range of 0 to 300 , which are then normalized to a scale of 0% to 100% . to validate the optimized procedures , 93 independent breast carcinoma tissue samples and 10 independent prostate carcinoma tissue samples in a microarray format were then stained with the optimized protocol for sp107 . 
slides stained using the optimized sp107 and ar441 procedures and clia - compliant ar441 reference assay were scored for ar expression by the pathologist , using a standard bright field microscope ( appendix table a1 )  . phase ii clinical trial mdv3100 - 11 was a simon two - stage , single - arm , open - label phase ii trial to evaluate the efficacy and safety of enzalutamide in patients with locally advanced or metastatic ar + tnbc ( clinicaltrials.gov identifier : nct01889238 ) .18 the study was conducted in accordance with the principles of the declaration of helsinki and good clinical practice . 
all patients provided written informed consent before any study - related procedures . the intention - to - treat patients with advanced tnbc , defined as , 1% staining by ihc for er and pgr and 0 to 1 + by ihc for her2 or negative for her2 amplification by in situ hybridization for 2 + ihc , and 0.05% or more ar + staining were enrolled and treated with enzalutamide ( 160 mg / d )  . 
the association between ar expression and progression - free survival ( pfs ) and overall survival ( os ) was analyzed using cox proportional hazards regression models and was presented as kaplan - meier estimates with hazard ratio ( hr ) and 95% ci . 
for sp107 , antigen retrieval was optimized for 64 minutes at standard high ph ( appendix fig a2 ) , and primary antibody incubation was optimized without dilution for 24 minutes . 
the primary antibody was incubated at a 1 : 800 dilution for 30 minutes . the optimized ihc conditions were then verified on breast carcinoma tissue , normal breast tissue , and two breast carcinoma cell lines known to express the ar ( t - 47d and mcf719 )  . 
the staining patterns clearly indicated that sp107 displayed more intense ar expression than did ar441 ( fig 1 and appendix fig a3 )  . breast and prostate carcinoma tissues microarrays were used to optimize the ihc staining procedures . 
however , we observed that sp107 had a more robust staining signal compared with ar441 regardless of whether slides were collected after a shorter or a longer time ( appendix tables a2 and a3 )  . interestingly , when comparing the incidence of ar positivity ( defined as expression in > 10% of cells ) with the rate reported in the literature , ar + detection rates with ar441 were lower overall for breast and prostate carcinoma . 
for example , in prostate cancer samples , ar incidence was 80% ( eight of 10 ) and 44% ( four of nine ) with sp107 and ar441 , respectively . to further assess the performance of the ihc conditions , we compared the results from the current ihc methods with an ihc assay validated in a clia - certified laboratory to determine accuracy , sensitivity , and specificity . 
the optimized sp107 conditions were used to stain 46 breast carcinoma tissues in microarray format , and the optimized ar441 conditions ( table a1 ) were used to stain 43 breast carcinoma tissues in microarray format . 
the sensitivity was 100% for both , and the specificity was 42% and 57% for sp107 and ar441 , respectively . sp107 had higher positivity rates of 78% compared with ar441 , which had 70% positivity rates . 
importantly , the positivity rates for these optimized conditions were much higher than the rates from the clia - certified assay , which were 48% and 47% in the sp107 and ar441 sample sets , respectively . 
rate of ar expression in breast and prostate carcinoma compared with literature 10% of cells ar + , n of n ( % ) literature reports tissue type tnbc sp107 ar441 antibody clone ( source ) 9 of 26 ( 35 ) 6 of 15 ( 40 ) ar441 ( thermo scientific ) ar27 ( novocastra ) ar441 ( dako ) er + and / or pgr + breast carcinoma 50 of 50 ( 100 ) 19 of 25 ( 76 ) ar441 ( thermo scientific ) her2 + breast carcinoma 15 of 17 ( 88 ) 10 of 16 ( 63 ) ar27 ( novocastra ) breast carcinoma , any subtype 74 of 93 ( 80 ) 35 of 56 ( 63 ) ar411 ( dako ) prostate carcinoma 8 of 10 ( 80 ) 4 of 9 ( 44 ) ar441 ( dako ) cutoff for positivity ( % ) > 10  . 
of these prescreened patients , 118 with advanced tnbc met the other eligibility criteria and were enrolled in the trial . clinical outcomes from the phase ii trial have been reported.18 with clinical benefit rate at 16 weeks as the primary end point , 29 of 118 enrolled intentionto - treat patients ( 24.6% ) were responders to enzalutamide . 
the maximum ppv of 39.6% was reached at a high cutoff value of 80% , with a dr of 55.1% and a lost responder rate of 27.6% ( eight of 29 )  . 
importantly , a lower cutoff decreased ppv from 40% to 30% , but it increased the sensitivity and reduced the dr substantially , suggesting that a lower threshold may be favorable . we also explored the h score to evaluate the number of patients with ar + tnbc , responders , ppv , and dr at different thresholds of 0% , 10% , 30% , 50% , and 80% ( methods , appendix fig a6a , and appendix table a4 )  . 
the 10% cutoff has the potential to capture 56% of total patients with tnbc ( appendix fig a7 ) , which may translate to nearly 138 , 000 patients globally . 
in addition , not all ar + tnbc tumors accurately represent the tumor molecular biology of an ar - driven disease . thus , a genomic signaturebased approach independent of ar expression level may better identify patients . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . iulia cristina tudor employment : medivation , pfizer stock and other ownership interests : medivation , pfizer travel , accommodations , expenses : medivation amy peterson employment : beigene , medivation leadership : beigene stock and other ownership interests : beigene , medivation , loxo patents , royalties , other intellectual property : patent for identifying patients with tnbc who may benefit from enzalutamide travel , accommodations , expenses : beigene , medivation hirdesh uppal employment : medivation stock and other ownership interests : medivation patents , royalties , other intellectual property : patents related to patient selection varun kumar no relationship to disclose jianjun yu employment : predicine , novartis ( i ) , medivation / pfizer stock and other ownership interests : medivation / pfizer , predicine , novartis ( i ) vernon phan employment : medivation / pfizer stock and other ownership interests : medivation / pfizer travel , accommodations , expenses : medivation / pfizer affiliations all authors : medivation , san francisco , ca . acknowledgment medical writing and editorial support was provided by stephanie vadasz and paula stuckart of ashfield healthcare communications . 
the sponsors were responsible for the study design , data collection and analysis , decision to publish , and preparation of the manuscript . support supported by medivation and astellas pharma , the co - developers of enzalutamide . 
collins lc , cole ks , marotti jd , et al : androgen receptor expression in breast cancer in relation to molecular phenotype : results from the nurses health study . 
gucalp a , tolaney s , isakoff sj , et al : phase ii trial of bicalutamide in patients with androgen receptor - positive , estrogen receptor - negative metastatic breast cancer . 
phan vt , protter aa , peterson ac , et al : a novel diagnostic androgen receptor gene signature links clinical outcomes and preclinical response to enzalutamide , paclitaxel or the combination in triple - negative breast cancer [ abstract ]  . cancer res 76 : p2 - 07 - 04 , 2016 17 . 
traina ta , miller k , yardley da , et al : results from a phase 2 study of enzalutamide ( enza ) , an androgen receptor ( ar ) inhibitor , in advanced ar + triple - negative breast cancer ( tnbc )  . 
magklara a , brown tj , diamandis ep : characterization of androgen receptor and nuclear receptor co - regulator expression in human breast cancer cell lines exhibiting differential regulation of kallikreins 2 and 3 . 
anestis a , karamouzis mv , dalagiorgou g , et al : is androgen receptor targeting an emerging treatment strategy for triple negative breast cancer ? cancer treat rev 41 : 547 - 553 , 2015 27 . 
hickey te , robinson jl , carroll js , et al : minireview : the androgen receptor in breast tissues : growth inhibitor , tumor suppressor , oncogene ? mol endocrinol 26 : 1252 - 1267 , 2012 29 . 
process flow diagraclia , clinical laboratory improvement amendments . dako ( ar441 ) ventana ( sp107 ) protocol optimization protocol verification with cell lines reproducibility and repeatability studies to compare both ventana and dako methods stability studies to compare both methods correlation analysis with clia - compliant ar441 - based method fig a2 . 
white lines indicate medians , and gray dotted line indicates average age of all patients . the average age of responders is approximately 9.5% higher than the average age of nonresponders . 
 ( a ) bar plot of the number of diagnostic positives and true - positives for selected h - score thresholds . positive predictive value ( ppv ; gray ) and drop - out rate ( dr ; red ) were plotted as lines . 
the overall prevalence rate of ar + patients is approximately 56% at an optimal 10% threshold selected in this study ( ar , > 10% )  . color by 10% cutoff 30% cutoff 50% cutoff 80% cutoff sp107 total nuclear ar % sp107 nuclear h score sum sp107 nuclear ar 3 + , 2 + sum sp107 nuclear 3 + table a1 . 
other listed variants were reported by foundation medicine as variants of unknown signicance . abbreviations : chip , clonal hematopoiesis of indeterminate potential ; mtb , molecular tumor board ; total depth , total depth of sequencing reads . tumor , whereas the pten variant was at an allele frequency compatible with being homozygous in the tumor as a result of loss of heterozygosity or gene conversion to inactivate the second wild - type allele . 
these observations , in addition to the kit mutation having an allele fraction of 0.23% , raised the possibility that the lower allele fraction alterations may be arising from the patients melanoma ( fig 2 )  . 
several studies have shown that ctdna correlates with tumor burden , and it may be that the only melanoma tissue mass in this patient was in his brain.2 , 3 the reex testing of the patients colon cancer and melanoma was performed using the illumina trusight tumor 15 gene panel ( illumina , san diego , ca ) , with results masked to only report out specic genes depending on the indication ( ie , kras , nras , and braf for colon cancer and braf , nras , and kit for melanoma )  . 
studies have shown that clonal mutations in the hematopoietic compartment may be detected on tumor tissue testing.4 , 5 the results suggest that ctdna analysis in this patient identied not only clonal dna from two separate solid tumors , but also an additional third clonal mutation in the hematopoietic compartment , as has been recently described.6 the patient was evaluated at rutgers cancer institute of new jersey and provided informed consent to participate in a prospective study trial for tumor genomic proling ( clinicaltrials.gov identier : nct02688517 )  . 
the results of genomic proling , clinical course , and pathology were reviewed at the rutgers cancer institute of new jersey molecular tumor board ( mtb ) , which was approved by the rutgers university new brunswick health sciences institutional review board ( pro2012002075 )  . 
because the alteration occurred in a homopolymer region and brca2 is a large gene , this may have been a passenger alteration as a result of the tumor being decient in mmr . 
 detection of three distinct clonal populations using ctdna determine whether both alleles were inactivated , the mtb suggested that platinum drugs or poly ( adp - ribose ) polymerase inhibitors could be effective . 
recent work has shown that cdk12 regulates dna damage response genes , including brca1 , and is sensitive to dna - damaging agents and poly ( adpribose ) polymerase inhibitors.11 , 12 the mtb suggested that these agents may be an option with the potential to target both the brca2 truncating mutation and the cdk12 inactivating mutation . 
in addition , during the mtb meeting , accumulating evidence suggesting that loss of genes involved in dna damage repair , such as brca2 or cdk12 , may have a role in sensitivity to immune checkpoint inhibitors was presented.13 , 14 it is unusual that three truncating apc mutations that likely arose from the patients colon cancer at heterozygous levels were identied . 
apc mutations are typically correlated with microsatellite - stable colon cancer , whereas one mutation of each allele would be sufcient to inactivate the proteit may be that the colon cancer initially developed along the microsatellite - stable pathway with biallelic inactivation of apc before acquiring an additional frameshift mutation as a result of loss of mmr function , with apc being particularly susceptible to being damaged by a loss of mmr because it is a large gene . 
shortly after , the patient became septic , developed endocarditis , and ultimately died during an operation to remove the aortic valve . after the mtb meeting , pms2 ihc was performed on the melanoma from the craniotomy , which showed intact pms2 ( fig 1b )  . 
therefore , the ve lower allele frequency variants showing alterations in homopolymer regions most likely represent subclonal alterations in the colon cancer as a result of the defect in mmr . 
also after the mtb meeting , sequencing of a lymph node negative for tumor from the patients original sentinel lymph node dissection on the illumina trusight tumor 15 gene panel was performed . 
in addition , because the lymph node was removed 18 months before the patients blood was sequenced , the tp53 mutation may not have even been present at that time or present at even lower levels . 
in both cases , tp53 was negative in tumor cells , although melanin and occasional tp53 - positive cells were observed in the melanoma ( figs 1a and 1b )  . 
tp53 somatic missense mutations in cancer are generally reported to be positive by ihc , and p.g244c was reported to be positive in 17 of 18 samples.15 in total , the negative sequencing and ihc the tp53 results strongly suggest p.g244c mutation having arisen in the hematopoietic compartment . the possibility of in summary , ctdna proling identied alterations at different allele frequencies that seemed to correspond with mutations present in his colon cancer and melanoma . remarkably , there was a tp53 mutation identied in the liquid biopsy , with subsequent sequencing and ihc results indicating it was unlikely to be present in either of the solid tumors and suggesting a third clonal mutation in the hematopoietic compartment . 
when ctdna analysis is performed , knowledge of genomic alterations in the primary cancer may not be available , and occasionally , there may be a second unknown primary cancer . 
mehnert , hossein khiabanian , shridar ganesan administrative support : hossein khiabanian provision of study materials or patients : elizabeth poplin , roman groisberg , hossein khiabanian collection and assembly of data : gregory m . 
mehnert honoraria : emd serono , pzer consulting or advisory role : amgen , boehringer ingelheim , array biopharma , merck sharp & dohme research funding : merck ( inst ) , sano ( inst ) , novartis ( inst ) , polynoma ( inst ) , immunocore ( inst ) , amgen ( inst ) , astrazeneca ( inst ) , incyte ( inst ) , macrogenics ( inst ) , bristol - myers squibb ( inst ) travel , accommodations , expenses : emd serono , merck sharp & dohme , array biopharma , bristol - myers squibb other relationship : amgen , emd serono , merck , boehringer ingelheim , array biopharma roman groisberg honoraria : guidepoint global shridar ganesan employment : merck ( i ) stock and other ownership interests : ibris , inspirata , merck ( i ) consulting or advisory role : inspirata , novartis , roche , foghorn therapeutics , foundation medicine patents , royalties , other intellectual property : i hold two patents for digital imaging that may be licensed to ibris and inspirata travel , accommodations , expenses : inspirata no other potential conicts of interest were reported . references khiabanian h , hirsheld km , goldnger m , et al : inference of germline mutational status and evaluation of loss of heterozygosity in high - depth , tumor - only sequencing data . 
j clin oncol 32 : 579 - 586 , 2014 dawson sj , tsui dw , murtaza m , et al : analysis of circulating tumor dna to monitor metastatic breast cancer . 
n engl j med 368 : 1199 - 1209 , 2013 severson ea , riedlinger gm , connelly cf , et al : detection of clonal hematopoiesis of indeterminate potential in clinical sequencing of solid tumor specimens . blood 131 : 2501 - 2505 , 2018 riedlinger g , hadigol m , khiabanian h , et al : association of jak2 - v617f mutations detected by solid tumor sequencing with coexistent myeloproliferative neoplasms . 
clin cancer res 24 : 4437 - 4443 , 2018 overman mj , lonardi s , wong kym , et al : durable clinical benet with nivolumab plus ipilimumab in dna mismatch repair - decient / microsatellite instabilityhigh metastatic colorectal cancer . 
hodi fs , corless cl , giobbie - hurder a , et al : imatinib for melanomas harboring mutationally activated or amplied kit arising on mucosal , acral , and chronically sun - damaged skj clin oncol 31 : 3182 - 3190 , 2013 9 . 
blazek d , kohoutek j , bartholomeeusen k , et al : the cyclin k / cdk12 complex maintains genomic stability via regulation of expression of dna damage response joshi pm , sutor sl , huntoon cj , et al : ovarian cancer - associated mutations disable catalytic activity of cdk12 , a kinase that promotes homologous recombination repair and resistance to cisplatin and poly ( adp - ribose ) polymerase inhibitors . 
ahluwalia , md5 ; howard colman , md , phd6 ; geoffrey fell , ms1 ; evanthia galanis , md7 ; john de groot , md8 ; jan drappatz , md9 ; andrew b . 
wen , md1 purpose adequately prioritizing the numerous therapies and biomarkers available in late - stage testing for patients with glioblastoma ( gbm ) requires an efcient clinical testing platforwe developed and implemented insight ( individualized screening trial of innovative glioblastoma therapy ) as a novel adaptive platform trial ( apt ) to develop precision medicine approaches in gbm . methods insight compares experimental arms with a common control of standard concurrent temozolomide and radiation therapy followed by adjuvant temozolomide . 
at the initiation of insight , three experimental arms ( neratinib , abemaciclib , and cc - 115 ) , each with a proposed genomic biomarker , are tested simultaneously . 
as the trial progresses , randomization probabilities adapt on the basis of accumulating results using bayesian estimation of the biomarker - specic probability of treatment impact on progression - free survival . treatment arms may drop because of low probability of treatment impact on overall survival , and new arms may be added . 
detailed information on the statistical model and randomization algorithm is provided to stimulate discussion on trial design choices more generally and provide an example for other investigators developing apts . conclusion insight ( nct02977780 ) is an ongoing novel biomarker - based , bayesian apt for patients with newly diagnosed unmethylated gbm . 
clinical trials in the era of precision medicine must consider how to develop biomarker data during the trials , make efcient use of multiplexed biomarker screening , and develop assignment algorithms for patients positive for more than one biomarker . 
 alexander et al master protocols and adaptive platform trials ( apts ) have been proposed as attractive solutions to efciently address multiple therapeutic and biomarker hypotheses.5 - 7 we developed insight ( individualized screening trial of innovative glioblastoma therapy ) , a multisite investigator - initiated phase ii screening apt , as a solution to precision medicine development challenges for patients with gbm . 
insight was specically designed to enable efcient use of randomly assigned controls , generate information to support genomic biomarker development , and leverage the xed cost of trial development across more experimental therapies . methods eligibility patients are eligible for insight if they have newly diagnosed gbm with unmethylated o6 - methylguanine dna methyltransferase ( mgmt ) gene promoters and negative idh1 r132h mutationspecic immunohistochemistry . 
the marginal benet of temozolomide ( tmz ) in patients with unmethylated mgmt promoters8 offers the opportunity to test experimental therapies without combinations with tmz.9 , 10 this potentially accelerates the overall time for drug development ( by not needing prior separate phase i studies with tmz ) and eliminates the potential for subtherapeutic dose of the experimental agent as a result of overlapping toxicity with tmz.2 , 3 insight therefore can support experimental arms with tmz combinations or with the experimental agent alone . 
experimental therapies may also replace tmz in the concurrent radiation therapy ( rt ) portion ( if there is a compelling radiosensitizing hypothesis ) , the adjuvant portion , or both . a pure radiosensitizing agent or experimental rt regimen could theoretically replace standard rt / tmz while keeping the adjuvant tmz intact . eligibility for insight requires sufcient genotyping data to dene the predetermined biomarker categories for arms currently in the trial . 
additional details regarding the initial experimental arms and biomarkers are included in the data supplement . statistical considerations operating characteristics , and secondary analyses use the proportional hazards ( ph ) model for both pfs and os . there are several arguments to support the use of different outcomes for adaptive randomization ( pfs ) and nal efcacy evaluation ( os )  . 
the relationship between accrual rate and event timing is crucial for response - adaptive trials , because effective variation of randomization probabilities requires rapid generation of treatment effect estimates on the basis of an adequate number of individual outcomes . 
in addition , treatment effects may have a stronger signal on pfs , a relationship illustrated previously.12 finally , potential issues with pseudoprogression and pseudoresponse13 , 14 are mitigated by preserving os as the foundation for stopping rules and nal efcacy analyses . that is , promising early results of an experimental treatment accelerate the accrual rate of the corresponding arm without reducing the nal sample size of the other experimental arms . group - specic adaptive randomization probabilities . 
patients are randomly assigned using an adaptive algorithm that updates randomization probabilities for the various arms in each biomarker group monthly.15 the algorithm uses available information generated by insight ( the individual biomarker groups combined with individual pfs ) to determine the randomization probabilities that will be used to allocate patients for the subsequent month . 
the hazard function for each patient assigned to an experimental arm is modeled , rescaling the baseline by a factor that depends on treatment ( main effect ) , biomarker - specic coefcients , and biomarker - treatment interaction terms . 
we previously described the use of ph models for the computation of randomization probabilities.17 we indicate the individual prole withx ( cid : 1 ) ( x1 , x2 , x3 )  . 
the hazard function x , a ( t ) for the timeto - event outcome of a patient with biomarker prole x , randomly assigned to experimental arm a , is proportional to the baseline ( t )  . 
insight will randomly assign a maximum of 70 patients to each experimental arthe sample size for the initial set of arms is 70 patients per arm across four arms , for a total of 280 patients , but there are early stopping rules for futility in each arm , and other arms may be added as the trial is ongoing . 
such an increase will depend on the number of experimental arms added and the time at which they will start enrollment . the later new arms start enrolling , the larger the corresponding sample size expansion will be . 
indeed , when a new experimental arm is added , the design increases the control - specic sample size to guarantee enrollment of 70 patients in the control after the addition of a novel arwe previously described the adjustment of the control sample size with the addition of novel arms and potential futility early stopping of experimental arms in platforms.18 operating characteristics of outcome adaptive randomization are related to the accrual rate relative to the event rate , because information from events is used to alter probability of random assignment for newer patients . 
biomarker frequencies were assumed on the basis of data from prior genomic proling.19 monthly updates of the randomization probabilities are combined with a sequential decision rule that drops experimental arms when there is insufcient preliminary evidence to warrant additional investigation of the treatment based on the primary end point ( os )  . 
we describe a linear boundary that provides thresholds for predictive probabilities to dene the decision rule . 2 of the main effect prior normal distributions were used for the regression parameters  . 
they are a priori independent , and the variance a a is set to have the 80th and 20th percentiles that correspond to ( log [ 2 ] ; duplication of the hazard ) and ( log [ 1 / 2 ] )  . 
 alexander et al whereas for a hypothetic arm added later during the study , it increases with the number of patients randomly assigned to the novel arm and the concomitant random assignments to the control aradaptive randomization intervenes only after the initial burn - in period . as we discussed previously , 17 , 20 to preserve the power of detecting treatment effects , it is important to guarantee sufcient enrollment in the control arrandomization probabilities for the control are obtained by matching ( under the hypothesis that all the active arms will complete accrual and will not be stopped for futility ) the expected number of patients randomly assigned to the control and the planned sample size specic to the control . 
for example , if after 10 enrollments two patients have been assigned to the control , then the 11th patient is assigned to the control with a probability of ( 70 2 ) / ( 280 10 )  . 
the same approach is used if one experimental arm is dropped for futility or a novel arm is added , with the consequence of an expansion of the control sample size.18 burn - in randomization . 
this is an important difference with respect to other adaptive designs , which include a component of competition and negative correlation with the nal number of patients enrolled by different experimental arms . 
the accrual can be stopped earlier for an experimental arm ( before this maximum is reached ) only when the likelihood of a positive nal result becomes insufcient and triggers the early stopping on the basis of futility . 
as a consequence , the adaptive algorithm has a substantial impact on the duration the arm - specic accrual period , which tends to be shortened for the arms with positive treatment effects , with little effect on other operating characteristics . 
bayesian randomization can only modify the enrollment rate and accelerate accrual to the most promising arms , particularly for the patients who are more likely to benet from these treatments . 
we also predict future biomarker proles x using a standard dirichlet conjugate model . monthly , bayesian sampling is used to generate nal trial data from the predictive distribution , including the enrollment of future patients , and pfs and os outcomes , both for patients previously enrolled and for those who have not yet been enrolled . 
using bayesian terminology , we sample multiple times from the predictive distribution every month . these data sets , including the actual data generated up to a time point by the trial and a complementary component of probabilistically imputed data , describe expectation and uncertainty on how we predict the data to look at completion of the study ( including censored data points ) on the the available information . 
these computations basis of assume that the open arms will reach the nal sample size of 70 patients and allow us to derive a single predictive probability of interest for each experimental arm , at each interim analysis , of a well - dened event . 
prediction ( the probability that the arm - specic result will be signicant ) is used monthly to decide either to continue or to discontinue the study of the experimental ara key step of the process is the simulation of the baseline and the ph model parameters from the posterior at each interim analysis . 
these steps are iterated so that the relative frequency of generated arm - specic p values below the signicance threshold approximates the targeted prediction probability . the arm is stopped if the prediction probability of a positive result becomes small . 
a linear boundary that increases with the number of enrollments from 0 at the onset of the study up to 0.1 is used to dene monthly the cutoff for discontinuing or proceeding with the study of the experimental treatment . 
in the comparison of these simulations with a balanced design , keeping the described early stopping mechanism for futility , enrollment in arms with positive treatment effects was completed faster , with an average time reduction between 16% and 27% across simulation scenarios . a similar power analysis like that for pfs was repeated for os . 
although the power to reject the null was of course considerably the power to detect a signicant biomarker / reduced , treatment interaction ( null hypothesis : no treatment effect in the cdk - positive group ) in the secondary analyses was higher ( 0.66 ; table 2 )  . 
the noncompeting maximum number of patients ( ie , 70 ) per arm maintained the power of detecting a positive treatment effect for each experimental arm stable with respect to the presence of treatment effects on the remaining arms . sensitivity to pfs and os correlation seemed limited . 
we considered both different magnitudes of pfs and os hrs contrasting an experimental treatment and a control ( appendix table a2 ) and various pfs - os correlation degrees at the individual level . 
additional sensitivity analyses , including the probability of reporting a positive treatment effect for the biomarker - positive group under selected scenarios ( appendix table a4 ) , the power for arms added later during the course of the platform trial ( appendix table a5 ) , and the power assuming different possible accrual rates ( appendix table a6 ) , are included in the appendix . 
in contrast to alternative response - adaptive designs , the low correlation of accrual rate with nal armspecic sample size ( which , as described in methods , can be fewer than 70 patients only as a result of early stopping for futility ) induces little variations of power estimates across table 2 . 
in the middle column , only a single arm has positive treatment effects restricted to a single biomarker - positive subpopulation ( prevalence , 0.5 ) and hazard ratios ( hrs ) of 1 for the rest of the patients . 
accrual rate therefore correlates with time required to complete the arm evaluation , but it does not correlate with power . discussion clinical trials under master protocols have been proposed as a methodologic innovation to more efciently answer therapeutic development questions.6 , 7 , 22 such innovations are particularly important in the era of precision medicine , where biomarker testing adds complexity by increasing the testable hypotheses . 
platform trials under number of master protocols are intended to study multiple targeted therapies in the context of a single disease in a perpetual manner , with therapies allowed to enter or leave the platform on the basis of a decision algorithm.7 ( p63 ) potential efciencies include the conservation of control arms , multiplexed biomarker screening data , and reduced downtime because the trial infrastructure is maintained as treatment arms enter or leave the trial . 
platforms also enable innovative statistical approaches to increase efciency.23 - 27 insight is the rst biomarker - based apt designed to apply these general solutions to specic problems in therapeutic development for gbm.28 late - stage clinical trial failures are a major issue for therapeutic development in general1 and in gbm specically.2 , 3 failure in phase iii may be linked to erroneous go / no - go decisions on the basis of phase ii results that have different end points than the desired pivotal trial , overestimate treatment effects on the basis of comparisons with historical controls , or both . 
prior data have shown that effects on imaging - based end points such as response rate and pfs may not translate to effects on os.4 , 13 , 29 - 31 furthermore , comparison of end points like pfs and os with historical controls may overestimate treatment effects through selection bias , temporal drift , and failure to account for control variability.4 for these reasons , we chose to use os as the primary end point of in unmethylated gbm , the trial . survival postprogression is generally short , and there are no proven effective therapies at recurrence that increase the chance of detecting a true therapeutic impact on os.12 we included a randomly assigned control arm to avoid the pitfalls associated with comparison with historical controls for os in gbm.4 the platform design affords considerable efciency by using a single control arm for comparison against multiple therapies and offers patients a higher probability of being randomly assigned to an experimental arfurthermore , we use bayesian rar17 based on accumulating pfs results to increase the probability of random assignment to arms that showed more promise.28 , 32 we had previously shown that rar using an os end point was possible for recurrent gbm17 and additionally supported this approach in our simulations during the development of insight . 
however , to increase efciency , we opted to use pfs , because earlier end point assessment can more rapidly inuence randomization.16 if we were to nd a discordance between therapeutic impact on pfs and os , randomization probability would be altered , but decision making on the primary end point would remain unchanged . another advantage of platform trials is the efcient use of multiplexed genomic biomarker data for treatment assignments . 
gbm is characterized by redundant and overlapping alterations in several molecular pathways rather than mutually exclusive driver mutations.33 as such , patients can be positive for multiple genomic biomarkers . some platform trials like nci - match ( national cancer institute molecular analysis for therapy choice ) , 34 lung map ( lung cancer master protocol ) , 35 battle ( biomarkerintegrated approaches of targeted therapy for lung cancer elimination ) , 36 and n2m2 ( national center for tumor diseases neuro master match ) 37 generally deal with this situation through an assignment algorithm on the basis of relative evidence of biomarker - specic accrual and / or therapeutic efcacy . i - spy 2 ( investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2 ) , in contrast , uses biomarker subgroupspecic randomization probabilities to allow data generated during the trial to drive the biomarker specicity of arm assignments.38 insight does the latter , starting with equal randomization among the experimental arms and allowing the rar procedure to prioritize randomization in a biomarker - specic way . 
for example , if only patients with egfr amplication in the neratinib arm were living longer than those in the control , the randomization algorithm would increase the probability of egfr - amplied patients assigned to neratinib ( regardless of their other biomarkers ) while potentially reducing assignment to neratinib for egfr wild - type patients . in fact , this situation occurred in i - spy 2 , where the adaptive randomization algorithm stopped assigning patients with human epidermal growth factor receptor 2 ( her2 ) negative / hormone receptorpositive cancer and those with her2negative / hormone receptornegative cancer to neratinib during the course of the trial , even as it reached the prespecied efcacy threshold in the her2 - positive / hormone receptor negative signature.39 this strategy may be optimal when there are limited pretrial data or a weak hypothesis suggesting a biomarker - specic effect . 
in these situations , more biomarker specicity may be desired from the start of the trial , and we have suggested ways to integrate this into a platform design.40 more generally , adding future biomarkers specic to additional experimental arms increases design complexity and requires appropriate denitions of the randomization probabilities . 
even though insight used randomization , we queried clinically annotated genomic data33 , 43 to investigate the possibility that the biomarkers we used had prognostic signicance or a variable relationship between pfs and os and found no such associations.19 although the perpetual clinical provided by the apt framework can provide signicant efciencies , it can create additional challenges as well . maintaining ongoing operations requires both nancial framework that trial models that support ongoing concerns and active pipeline development to maintain a regular ow of experimental arms . 
more complex bayesian designs require engaged clinical and statistical investigators and new ways of determining operating characteristics through simulation.44 reporting of trial results is complicated because of the separation between the master protocol and the armspecic data . 
arms that leave the trial because of success or failure need to be reported while the overall trial is still ongoing , which does not lend itself to traditional trial reporting best practices like consort . 
two recent publications from i - spy 2 are examples.39 , 45 conversely , reporting on the overall master protocol does not have a natural time point , although most groups publish a general description of the overall trial structure without results.35 , 36 , 38 , 41 in conclusion , insight is an ongoing novel biomarkerbased bayesian apt for patients with newly diagnosed unmethylated gbm . 
ahluwalia stock and other ownership interests : mimivax honoraria : prime oncology , elsevier , itamar medical ( i ) consulting or advisory role : monteris medical , astrazeneca , bristol - myers squibb , abbvie , cbt pharmaceuticals , kadmon , vbi vaccines research funding : novartis , tracon pharma , novocure , spectrum pharmaceuticals , eli lilly / imclone , boehringer ingelheim , astrazeneca howard colman honoraria : merck sharp & dohme consulting or advisory role : genentech , roche , novocure , omniox , insys therapeutics , abbvie research funding : newlink genetics , plexxikon , kadmon , orbus therapeutics , merck , dnatrix , abbvie , beigene evanthia galanis consulting or advisory role : genentech / roche ( inst ) , abbvie ( inst ) , oncorus research funding : genentech / roche ( inst ) , merck ( inst ) john de groot employment : helsinn therapeutics ( i ) , ziopharm oncology ( i ) leadership : ziopharm oncology ( i ) stock and other ownership interests : gilead sciences , ziopharm oncology ( i ) consulting or advisory role : celldex , deciphera , vascular biogenics , foundation medicine , genentech / roche , omniox , oxigene , abbvie , novogen , kadmon , merck , five prime therapeutics , insys therapeutics , astrazeneca , boston biomedical , gw pharmaceuticals , carthera research funding : deciphera , novartis , eli lilly , sano , emd serono , mundipharma patents , royalties , other intellectual property : sano , research support ; astrazeneca , research support ; emd serono , research support ; eli lilly , research support ; novartis , research support ; deciphera pharmaceuticals , research support jan drappatz employment : via oncology stock and other ownership interests : exelixis , bristol - myers squibb honoraria : uptodate , via oncology consulting or advisory role : oncorus , immunomix patents , royalties , other intellectual property : uptodate andrew b . 
burt nabors consulting or advisory role : bristol - myers squibb speakers bureau : merck / schering plough sandro santagata consulting or advisory role : rarecyte david schiff consulting or advisory role : orbus therapeutics , monteris medical , cavion ( inst ) research funding : bayer healthcare pharmaceuticals ( inst ) mary r . 
wen consulting or advisory role : abbvie , genentech / roche , agios , astrazeneca , karyopharm therapeutics , vivus , monteris medical , aurora biopharma , vascular biogenics , kadmon , ziopharm oncology , gw pharmaceuticals , eli lilly , immunomic therapeutics speakers bureau : merck , agios ( inst ) , abbvie ( inst ) , angiochem ( inst ) , genentech / roche ( inst ) , glaxosmithkline ( inst ) , astrazeneca ( inst ) , immunocellular therapeutics ( inst ) , karyopharm therapeutics ( inst ) , merck ( inst ) , novartis ( inst ) , oncoceutics ( inst ) , sano ( inst ) , ariad pharmaceuticals ( inst ) , vascular biogenics ( inst ) , eli lilly no other potential conicts of interest were reported references thomas dw , burns j , audette j , et al : clinical development success rates 2006 - 2015 . 
j clin oncol 35 : 2402 - 2409 , 2017 vanderbeek am , rahman r , fell g , et al : the clinical trials landscape for glioblastoma : is it adequate to develop new treatments ? neuro oncol 20 : 1034 - 1043 , 2018 grossman sa , schreck kc , ballman k , et al : point / counterpoint : randomized versus single - arm phase ii clinical trials for patients with newly diagnosed glioblastoma . 
nat rev clin oncol 9 : 199 - 207 , 2011 berry sm , connor jt , lewis rj : the platform trial : an efcient strategy for evaluating multiple treatments . 
n engl j med 377 : 62 - 70 , 2017 stupp r , hegi me , mason wp , et al : effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase iii study : 5 - year analysis of the eortc - ncic trial . 
alexander bm , trippa l : getting it rst versus getting it right : weighing the value of and evidence for progression - free survival as a surrogate endpoint for overall survival in glioblastoma . 
han k , ren m , wick w , et al : progression - free survival as a surrogate endpoint for overall survival in glioblastoma : a literature - based meta - analysis from 91 trials . neuro - oncol 16 : 696 - 706 , 2014 30 . 
herbst rs , gandara dr , hirsch fr , et al : lung master protocol ( lung - map ) : a biomarker - driven protocol for accelerating development of therapies for squamous cell lung cancerswog s1400 . 
pfaff e , kessler t , balasubramanian gp , et al : feasibility of real - time molecular proling for patients with newly diagnosed glioblastoma without mgmt promoter hypermethylation : the nct neuro master match ( n2m2 ) pilot study . 
the phosphatidylinositol 3 - kinase ( pi3k ) / akt / mtor signaling axis plays a central role in cell growth , survival , motility , and metabolism in a variety of cancers ( fruman da , et al : nat rev drug discov 13 : 140 - 156 , 2014 ; engelman ja : nat rev cancer 9 : 550 - 562 , 2009 ) , including glioblastoma ( gbm ; brennan cw , et al : cell 155 : 462 - 477 , 2013 )  . 
dna - dependent protein kinase is a serine / threonine kinase involved in the repair of dna double - strand breaks ( collis sj , et al : oncogene 24 : 949 - 961 , 2005 ) , which are considered to be the most lethal dna lesions and the main driver of cellular death after treatment with ionizing radiation therapy ( rt )  . 
therefore , beyond its hypothesized growth - inhibitory effect as monotherapy , cc - 115 has the potential to be a radiation - sensitizing agent in the treatment of gbm ( zhao y , et al : cancer res 66 : 5354 - 5362 , 2006 )  . 
one phase ia / ib multicenter open - label clinical study established 10 mg twice per day as the recommended dose for cohort expansion and phase ii with near - maximal inhibition of phosphorylated akt and partial inhibition of phosphorylated 4ebp ( munster pn , et al : j clin oncol 34 , 2016 [ suppl ; abstr 2505 ] )  . 
because this dose has never been combined with rt , the treatment arm will start with a safety lead - in with the combination before expansion to the full phase ii setting . abemaciclib retinoblastoma ( rb ) protein is a key tumor suppressor that inhibits progression through the g1 checkpoint ( sherr cj : science 274 : 16721677 , 1996 )  . 
abemaciclib is a highly specic atp - competitive cdk4 / 6 inhibitor that induces reversible g1 phase cell - cycle arrest in rb - procient tumor models and is approved for hormone receptorpositive , human epidermal growth factor receptor 2 ( her2 ) negative advanced or metastatic breast cancer . orally dosed abemaciclib achieved brain exposures in excess of the concentrations required for cdk4 / 6 inhibition in an orthotopic rat gbm model and signicantly increased survival alone or in combination with tmz ( raub tj , et al : drug metab dispos 43 : 1360 - 1371 , 2015 )  . 
neratinib , an orally available potent irreversible small - molecule panerbb family tyrosine kinase inhibitor that targets the intracellular tyrosine kinase domains in egfr , is approved for her2 - positive breast cancer ( martin m , et al : lancet oncol 18 : 1688 - 1700 , 2017 ; cancer discov 7 : of1 , 2017 ) 39 and has shown activity in controlling and delaying cns progression of breast cancer metastases ( awada a , et al : jama oncol 2 : 1557 - 1564 , 2016 )  . 
in preclinical gbm studies , neratinib was shown to selectively cause cell death in cell lines harboring genetic activation of egfr and was more effective than other egfr inhibitors in lines harboring the extracellular domain mutations seen in gbm ( vivanco i , et al : cancer discov 2 : 458 - 471 , 2012 )  . 
neratinib has also been shown to exhibit potential for potent inhibition of amplied egfrvii and egfrviii in gbm patient - derived cell - line models ( francis jm , et al : cancer discov 4 : 956 - 971 , 2014 )  . 
neratinib is signicantly more potent than lapatinib in limiting the growth of primary gbm cell lines , and this increased potency is an attractive feature , given that negative clinical trials for lapatinib have been attributed to inadequate tumor concentrations of the drug ( vivanco i , et al )  . 
neratinib will be administered in place of tmz after standard concurrent rt / tmz . biomarkers prospective patients may have biomarker data already available from academic or commercial sources , or they may take advantage of a companion consortium ( abc2 allele consortium ) that generates free portable genotyping data using whole - exome sequencing and copy array testing performed in a clinical laboratory improvement amendmentscertied clinical laboratory for patient use in clinical trials . 
the biomarkers determined are as follows : egfr positive dened as patients with egfr amplication or mutation ; pi3k positive dened as patients with pik3ca mutation / amplication , pik3r1 mutation , akt3 amplication , pik3c2b  . 
one copy gain , or pten dual allele inactivation through either homozygous deletion or deletion plus an inactivating mutation ; and cdk positive dened as patients with rb1 wild type and cdk4 amplication , cdk6 amplication , or cdkn2a or cdkn2b  . 
one copy loss , or cdkn2a or cdkn2b one copy loss plus an inactivating mutation . for amplications listed , the genotyping report must state clear gene amplication and not gain , which is typically greater than a log2 ratio of + 2.0. 
copy number losses would be values of , 0.3 , and more than single copy deletions are inferred relative to baseline for the chromosome on which they are located ( eg , single copy chromosome 9 loss with additional loss of cdkn2a / b below this level in focal region )  . 
ve prior events reported in cosmic or are well - established hotspots known to be activating or inactivating mutations through experimental data or novel mutations that are predicted to be clearly inactivating , such as nonsense and frameshift mutations . 
 reliable pan - cancer microsatellite instability assessment by using targeted next - generation sequencing data purpose microsatellite instability ( msi ) / mismatch repair ( mmr ) status is increasingly important in the management of patients with cancer to predict response to immune checkpoint inhibitors . 
we determined msi status from large - panel clinical targeted next - generation sequencing ( ngs ) data across various solid cancer types . methods the msi statuses of 12 , 288 advanced solid cancers consecutively sequenced with memorial sloan kettering - integrated mutation profiling of actionable cancer targets clinical ngs assay were inferred by using msisensor , a program that reports the percentage of unstable microsatellites as a score . 
of 9 , 591 non - crc / uec tumors with mss msisensor status , 456 ( 4.8% ) had slightly elevated scores ( 3 and < 10 ) of which 96.6% with available material were confirmed to be mss by msi pcr . 
msisensor correctly scored all 15 polymerase ultra - mutated cancers as negative for msi . conclusion msi status can be reliably inferred by msisensor from large - panel targeted ngs data . 
these sites are prone to dna replication errors as a result of dna polymerase slippage , which is effectively corrected through the mismatch repair ( mmr ) systedeficiencies in mmr result in increased variation at genomic loci with mononucleotide repeats . 
 drug approved for any solid tumor with a specific genetic feature ( msi - high [ msi - h ] status ) on the basis of new data that confirm its activity across 12 different cancer types , with complete responses observed in 21% of patients.5 until now , the gold standard for assessment of msi , a reliable screen for functional mmr status , has been concurrent analysis of patient tumor and normal dna for five mononucleotide microsatellite loci with pcr . 
in recent years , reports have shown that next - generation sequencing ( ngs ) facilitates identification of patients with deficiencies in the mmr pathway by comparing sequencing reads around microsatellite regions in the tumor and the matched normal or by counting mutations identified in exons . 
hause et al6 identified msi / mmr across a wide spectrum of tumor types surveyed by the cancer genome atlas but with limited validation data available in only a subset of crc / uecs and stomach cancers . 
msk - impact is an ngs assay that uses tumor and matched normal dna to identify somatic mutations , structural variants , and copy number alterations in all coding regions and select introns of 341 ( version 1 ) , 410 ( version 2 ) , or 468 ( version 3 ) cancer - related genes.7 tumor purity ( tp ) was estimated with a combination of median variant allele frequency of mutations identified in each sample and microscopic analysis of hematoxylin and eosinstained specimens . sufficient coverage in a tumor / normal pair where it identifies deletion length variation . 
x2 test is used to identify the significantly varied loci , and the percentage of unstable loci , after multiple testing correction is performed on the p values , is reported as an msisensor score ; additional details have been previously published.9 this score was used to infer msi / mmr status from ngs data in the current study . 
non - neoplastic colonic mucosa and colorectal tumors known to be deficient of mlh1 , msh2 , msh6 , and pms2 were used as external positive and negative controls , respectively . 
retained expression of each protein was defined by nuclear ihc reactivity of tumor cells , whereas loss of expression for each protein was defined by the complete absence of nuclear ihc reactivity of tumor cells . 
for the purposes of this study , all tumors with fewer than two unstable loci were interpreted as microsatellite stable ( mss )  . msisensor score analysis and cutoff determination proper detection of msi - h / mmr - d status from ngs data by using msisensor depends on whether the tumor is analyzed against its matched normal sample , because analysis with an unmatched sample results in significant inflation of the msisensor scores ( p , .001 ; appendix fig a1 )  . 
alignment files generated from the msk - impact pipeline in binary alignment map format were analyzed by using msisensor on all matched tumor - normal pairs and then separately on all unmatched tumor samples to generate the score . 
msisensor was run at the following successive dilutions : 100% tumor dna , 50% tumor dna and 50% normal dna , 25% tumor dna and 75% normal dna , 12% tumor dna and 88% normal dna , and 6% tumor dna and 94% normal dna . 
we used a total tp threshold of > 25% for this application of msisensor . mutation burden and mutational signatures tumor mutation burden ( tmb ) was calculated by dividing the total number of reported mutations by the genomic area where mutations were reported for each sample . 
these mutations were resampled 1 , 000 times and then subjected to decomposition analysis in which the kullback - leibler divergence is minimized between the sample signature and the approximation built up from 30 signatures such that each signature is assigned a weight that corresponds to the percentage of mutations explained by each given signature . 
40% of observed mutations were attributable to that signature.10 survival analysis time from procedure date to last follow - up was used for survival analysis among patients whose biopsy date and sequencing date were within 1 year of each other . 
the survival package in r version 3.2 was used for the survival analysis . results validation of msisensor in crc and uec samples one hundred thirty - eight crc and 40 uec samples with matched normals were selected for orthogonal msi / mmr status by pcr / ihc and used as the training data set for msisensor score threshold determination . 
 ( a ) green points indicate samples where orthogonal methodology indicated microsatellite stable ( mss ) phenotype ; red points indicate samples where a microsatellite instabilityhigh ( msi - h ) phenotype is observed . 
 ( c ) a poorly differentiated crc ( hematoxylin and eosin [ he ] stain ) with ( d ) mlh1 and ( e ) pms2 loss only had one unstable locus , ( f ) mono - 27 ( arrows ) , and was not msi - h by polymerase chain reaction , whereas ( g ) msisensor detected msi - h status with > 10% of loci with instability . 
from 10 , 900 patients , 11 , 553 samples had sufficient coverage of 2003 and tp of > 25% [ as described in analytic ( technical ) sensitivity ] and matched normal samples for analysis . 
among the patients without crc / uec , bladder cancer ( 11 [ 3.1% ] of 355 ) , esophagogastric carcinoma ( seven [ 2.5% ] of 282 ) , and prostate cancer ( 12 [ 1.7% ] of 722 ) had msi - h incidence . 
forty - six were concordant ( pcr , n = 39 ; ihc , n = 7 ) , whereas three were msi low ( msi - l ) by pcr but were mmr - d by ihc ( figs 1c - 1g ) , including a carcinoma of unknown primary with 16 mutations , a squamous cell carcinoma with 65 mutations , and a prostate carcinoma with 44 mutations . 
of note , use of the msisensor on targeted panel ngs data had higher clinical sensitivity as a screen for mmr - d than msi pcr . exome data and validated at a cutoff score of 3.5 from an average of 10 , 000 evaluable microsatellites per sample.9 msk - impact covers less of the genome with higher coverage , with approximately 1 , 000 evaluable microsatellites per sample . this number has increased with the addition of genes to the msk - impact panel ( appendix fig a3 )  . 
with consideration of the different cutoffs between the present and previous studies , we also evaluated the 456 ( 4.8% ) of 9 , 591 msk - impact non - crc / uecs with slightly elevated msisensor scores ( > 3 and , 10 )  . 
of note , three samples ( a germ cell tumor with two mutations , a cancer of unknown primary with 16 mutations , and a uterine sarcoma with 19 mutations ) that were msi - h by both msisensor and pcr had , 20 exonic mutations in mskimpact , a cutoff shown to be highly predictive of msi - h in crc samples.9 analysis of msi status in unmatched tumors because most clinical laboratories that offer ngs testing use a pooled unrelated normal rather than a matched normal , we investigated whether and at what cutoff msisensor could be used for unmatched tumors . 
 colorectal cancer ( n = 1 , 030 ) endometrial cancer ( n = 270 ) prostate cancer ( n = 820 ) bladder cancer ( n = 369 ) esophagogastric carcinoma ( n = 302 ) cancer of unknown primary ( n = 254 ) nonsmall - cell lung cancer ( n = 1 , 619 ) small bowel cancer ( n = 32 ) head and neck carcinoma ( n = 210 ) pancreatic cancer ( n = 578 ) skin cancer , nonmelanoma ( n = 133 ) biliary cancer ( n = 242 ) germ cell tumor ( n = 298 ) uterine sarcoma ( n = 99 ) gi neuroendocrine tumor ( n = 46 ) mesothelioma ( n = 127 ) renal cell carcinoma ( n = 380 ) small - cell lung cancer ( n = 94 ) soft tissue sarcoma ( n = 478 ) thyroid cancer ( n = 238 ) breast carcinoma ( n = 1 , 498 ) ovarian cancer ( n = 240 ) melanoma ( n = 420 ) adrenocortical carcinoma ( n = 30 ) glioma ( n = 628 ) gestational trophoblastic disease ( n = 13 ) gi stromal tumor ( n = 165 ) meningothelial tumor ( n = 33 ) osteosarcoma ( n = 56 ) salivary carcinoma ( n = 116 ) chordoma ( n = 20 ) embryonal tumor ( n = 91 ) ependymomal tumor ( n = 18 ) hepatocellular carcinoma ( n = 115 ) histiocytosis ( n = 22 ) nerve sheath tumor ( n = 21 ) sellar tumor ( n = 7 ) sex cord stromal tumor ( n = 21 ) validation status validated msi - h / mmr - d validated mss / mmr - p not validated msisensor score fig 2 . 
validation showed high concordance , with only two cancers with msi - h / mismatch repair deficiency status by polymerase chain reaction / ihc that were , 10 by msisensor . 
mmr - d , mismatched repair deficient ; mmr - p , mismatched repair proficient ; mss , microsatellite stable . known hotspot mutations in pole12 ( table 2 )  . 
our analysis of survival status for msi - h compared with mss in cancer types where we identified at least 10 patients with msi - h status showed no significant difference after adjusting for age ( fig 4 )  . 
 ( a ) for cancers with high tmb and microsatellite stable results on the basis of msisensor , ( b ) other signatures were identified , including uv exposure , apobec deficiency , smoking , temozolomide ( tmz ) treatment , and polymerase e ( pole ) deficiency . 
 ( c ) on the other hand , mismatch repair - deficient ( mmr - d ) / microsatellite instabilityhigh ( msi - h ) samples showed similar rates of tmb . 
although colorectal cancer ( crc ) showed a trend of better survival for patients with msi - h status ( p = .057 ) , other tumor types did not show a significant association between msi status and survival . 
although the bias in our data set may underestimate the total number of msi - h tumors across all patients with cancer , it gives a more accurate estimate of the proportion of patients with advanced solid cancers who may be eligible for immune checkpoint inhibition on the basis of msi - h status . although mutation rate has been used to infer msi status in crc , 10 , 14 various other hypermutation signatures ( pole , uv , smoking , temozolomide ) also are associated with an increased mutation rate in other tumor types . 
klimstra provision of study material or patients : liying zhang , justyna sadowska , jinru shia collection and assembly of data : sumit middha , liying zhang , khedoudja nafa , gowtham jayakumaran , justyna sadowska , deborah f . 
delair , ahmet zehir , jaclyn f . hechtman data analysis and interpretation : sumit middha , liying zhang , khedoudja nafa , gowtham jayakumaran , donna wong , hyunjae r . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . sumit middha no relationship to disclose liying zhang no relationship to disclose khedoudja nafa no relationship to disclose gowtham jayakumaran no relationship to disclose donna wong no relationship to disclose hyunjae r . 
berger consulting or advisory role : cancer genetics , sequenom zsofia stadler consulting or advisory role : allergan ( i ) , genentech ( i ) , roche ( i ) , regeneron pharmaceuticals ( i ) , optos ( i ) , adverum biotechnologies ( i ) david s . 
klimstra stock and other ownership interests : paige.ai consulting or advisory role : wren laboratories , ipsen marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network / boehringer ingelheim afatinib targeted therapy advisory committee , national comprehensive cancer network / astrazeneca tagrisso rfp advisory committee research funding : loxo ( inst ) ahmet zehir no relationship to disclose jaclyn f . 
hechtman jf , middha s , stadler zk , et al : universal screening for microsatellite instability in colorectal cancer in the clinical genomics era : new recommendations , methods , and considerations . 
ribic cm , sargent dj , moore mj , et al : tumor microsatellite - instability status as a predictor of benefit from fluorouracil - based adjuvant chemotherapy for colon cancer . 
le dt , durham jn , smith kn , et al : mismatch repair deficiency predicts response of solid tumors to pd - 1 blockade . 2520 , 2015 science 357 : 409 - 413 , 2017 6 . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
stadler zk , battaglin f , middha s , et al : reliable detection of mismatch repair deficiency in colorectal cancers using mutational load in next - generation sequencing panels . 
metrics table with number of tumors , median msisensor score , and median number of exonic mutations all samples ( 10 ) mss ( msisensor < 10 ) msi - h ( msisensor 10 ) general tumor type frequency median exonic mutation median no . 
metrics table with number of tumors , median msisensor score , and median number of exonic mutations ( continued ) all samples ( 10 ) mss ( msisensor < 10 ) msi - h ( msisensor 10 ) general tumor type frequency median exonic mutation median no . 
 ras mutations are associated with inferior progression - free survival and overall survival of patients with mcrc compared with patients with nonmutated tumors.1 it is conceivable that ras mutations are negative prognostic factors per se ; however , the availability of only one actionable molecular target , that is , the inhibition of angiogenesis , in patients with mutated tumors certainly affects survival in this subgroup of patients . 
recent studies have demonstrated that the analysis of circulating tumor dna ( ctdna ) in blood samples , through its ability to recapitulate tumor heterogeneity , is a remarkable surrogate of tumor biopsy for mutation detection.2 extensive research on liquid biopsy led to significant achievements in the comprehension of the biologic reasons behind acquired resistance to anti - egfr therapies.3 to date , studies with liquid biopsy have been selectively concentrated on the appearance of ras - mutant clones in the blood of patients with ras wild - type primary crc , as biomarkers of anti - egfr therapy resistance . 
we here report that ctdna analysis , over the course of antiangiogenic treatment , might reveal a therapeutically exploitable window of opportunity , characterized by the relative prevalence of ras wild - type clones over time . 
a whole - body contrast enhanced computed tomography ( ct ) scan revealed a left supraclavicular lymphadenopathy , transverse colon thickening ( 3 cm ) , multiple chest and abdominal lymphadenopathies , and peritoneal carcinomatosis . 
mutational analysis of the lymph node metastasis performed through real - time polymerase chain reaction ( pcr ; real - time oncoscreen nras ) revealed exon 4 nras a146t mutation at 29 cycles . 
serum tumor markers significantly decreased , with rapid negativization ( carcinoembryonic antigen [ cea ] , 1.31.23 ; ca 19 - 9 , 71.839 ; ca - 125 , 8226 )  . 
at that time , serum tumor markers increased again ( ca 19 - 9 , 530 ; cea , 9 ) , with clinical evidence of bilateral axillary lymph node enlargement . 
concurrently , the patient was enrolled in an observational study open at our institution , which aimed to serially monitor the mutational status of ctdna in patients with ras - mutant mcrc disease , before starting any new line of treatment and in course of therapy until progression . methods blood samples were prospectively collected after obtaining informed consent with ethical committee approval . 
idylla ( biocartis , nv , mechelen , belgium ) , a fully automated , realtime pcr - based molecular diagnostics system , was used to investigate ras mutational profile from plasma . 
the first allows the detection of 21 mutations in codons 12 , 13 , 59 , 61 , 117 , and 146 of the kras gene , and the second allows the detection of 18 mutations in codons 12 , 13 , 59 , 61 , 117 , and 146 of the nras gene , five mutations in codon 600 of the braf gene , and two mutations in codon 492 of the egfr gene from 1 ml of plasma . among the parameters describing the generated pcr curves , the cq value is calculated as the difference between the quantification cycle value ( cq ) of the gene control signal and the cq of the mutant signal . 
according to the manufacturer 's instructions , a cq value of the nras control of 35.5 or higher indicates that a low amount of cell - free dna is present in the sample . 
in such cases , low - frequency mutations may not be detected . results in july 2017 , a ctdna mutational analysis was performed and revealed the absence of any clinically relevant mutation in kras , nras , and braf genes . 
this new insight into the biology of the disease convinced us to take this window of therapeutic opportunity , which was not guaranteed to remain open indefinitely ; in july 2017 , the treatment was changed to the second - line combination of irinotecan and cetuximab . 
as early as after the first cycle of chemotherapy , an objective complete clinical response of the bilateral axillary adenopathy was documented , and this was confirmed at ultrasound assessment . 
restaging ct scan after eight cycles of therapy showed a partial response , with significant reduction of pleural effusion and pulmonary lymphangitic carcinomatosis and stability of peritoneal metastases ( fig 1 )  . 
a second ctdna analysis was performed on november 2017 , confirming the ras genes wild - type status ( cq of nras total : 35.5 , which might be due to a reduction in ctdna amount )  . 
 june 2017 ( before folfiri - cetuximab ) october 2017 ( after 3 months of folfiri - cetuximab ) pleural effusion pulmonary lymphangitic carcinomatosis malignant ascites peritoneal carcinosis fig 1 . 
axial computed tomography scan after eight cycles of fluorouracil , leucovorin , and irinotecan ( folfiri ) plus cetuximab shows a partial response , with significant reduction of pleural effusion and pulmonary lymphangitic carcinomatosis and stability of peritoneal metastases . 
 sequence of treatments , clinical and radiologic response to the different schedules adopted ( partial response [ pr ] is indicated by blue arrow , and progressive disease [ pd ] is indicated by red arrow ) , and molecular tests performed on tumor tissue and plasma at different time points . 
bev , bevaci zumab ; cet , cetuximab ; ctdna , circulating tumor dna ; folfiri , fluoro uracil , leucovorin , and irinotecan ; folfoxiri , fluorouracil , leucovorin , oxaliplatin , and irinotecan ; fu , fluorouracil . 
nevertheless , a146t results in increased nras activity and downstream signaling and is transforming in cell culture resulting from increased nras guanosine diphosphate / guanosine triphosphate exchange rate , 4 and consequently patients who harbor mutations in nras also have significantly inferior anti - egfr treatment outcomes benefit compared with those without any ras mutations.5 it is recommended that patients with crc being considered for anti - egfr therapy must receive ras mutational testing , including kras and nras codons 12 and 13 of exon 2 , 59 , and 61 of exon 3 and 117 and 146 of exon 4 , and that anti - egfr should only be prescribed for patients with mcrc who are wild type for all known ras - activating mutations.6 in patients with ras - mutant primary colorectal cancers , ineligible for egfr inhibitors , the importance of the egfr pathway , which indeed sustains the disease , is counterintuitive . 
international guidelines currently recommend treatment of ras mutant mcrc with bevacizumab first line , followed by chemotherapy backbone change or aflibercept / ramucirumab at disease progression.7 preclinical and clinical data , however , demonstrate that tumor angiogenesis inhibition induces biologic changes in tumor - stroma interactions , mainly derived from hypoxia . 
preclinical observations suggest that hypoxia exerts a negative selection against ras - mutant clones through a mechanism known as secretory senescence , in which ras - mutant senescent cells operate in a paracrine manner to support the growth of surrounding ras wild - type clones , leading to their relative prevalence over time.8 we here report that ctdna analysis , under table 1 . 
 antiangiogenic treatment , might reveal a therapeutically exploitable window of opportunity , characterized by the relative prevalence of ras wild - type clones , which can be converted in a clinically meaningful benefit for patients . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . paola gazzaniga no relationship to disclose cristina raimondi no relationship to disclose federica urbano no relationship to disclose the following represents disclosure information provided by authors of this manuscript . 
modest dp , ricard i , heinemann v , et al : outcome according to kras - , nrasand brafmutation as well as kras mutation variants : pooled analysis of five randomized trials in metastatic colorectal cancer by the aio colorectal cancer study group . 
sorich mj , wiese md , rowland a , et al : extended ras mutations and anti - egfr monoclonal antibody survival benefit in metastatic colorectal cancer : a meta - analysis of randomized , controlled trials . 
sepulveda ar , hamilton sr , allegra cj , et al : molecular biomarkers for the evaluation of colorectal cancer : guideline summary from the american society for clinical pathology , college of american pathologists , association for molecular pathology , and american society of clinical oncology . 
genomic profiling was performed on 703 appendiceal cancer specimens to compare the mutation profiles of appendiceal subtypes to crc and other cancers , with the ultimate aim to identify potential biomarkers and novel therapeutic targets . methods tumor specimens were submitted to a clinical laboratory improvement amendmentscertified laboratory ( foundation medicine , cambridge , ma ) for hybrid - capturebased sequencing of 3 , 769 exons from 315 cancer - related genes and 47 introns of 28 genes commonly rearranged in cancer . 
kras ( 35% to 81% ) and gnas ( 8% to 72% ) were the most frequent alterations in epithelial cancers ; apc and tp53 mutations were significantly less frequent in appendiceal cancers relative to crc . 
the mutation status of gnas and tp53 strongly predicted os ( median , 37.1 months for tp53 mutant v 75.8 gnas - tp53 wild type v 115.5 gnas mutant ; log - rank p = .0031 ) and performed as well as grade in risk stratifying patients . conclusion epithelial appendiceal cancers and goblet cell carcinoids show differences in kras and gnas mutation frequencies and have mutation profiles distinct from crc . 
2018 by american society of clinical oncology introduction the rarity of appendiceal neoplasms has made it difficult to conduct prospective or randomized clinical trials to guide therapy for these tumors . 
 the small number of appendiceal tumors that are detected , in many cases as an incidental finding in < 1% of appendectomy specimens , 1 comprise multiple histopathologic subtypes , including noninvasive mucinous neoplasms , mucinous and nonmucinous adenocarcinomas , carcinoids , goblet cell carcinoids ( gccs , now also called goblet cell tumors ) , and signet ring cell carcinomas.2 early - stage cancers can be treated definitively with surgery , and selected patients derive long - term benefit from cytoreductive surgery and heated intraperitoneal chemotherapy ( hipec ) .3 however , there is no standard of care for the systemic treatment of advanced , unresectable disease . in the absence of randomized phase iii data , the majority of medical oncologists use colorectal cancer ( crc ) chemotherapy regimens for the treatment of unresectable epithelial appendiceal neoplasms , as is currently recommended by the national comprehensive cancer network guidelines . 
clinical and demographic patient characteristics by subtype median age ( years ) sex ratio m : f subtype mucinous adenocarcinoma adenocarcinoma goblet cell carcinoid pseudomyxoma peritonei signet ring carcinoma 43 : 57 42 : 58 36 : 64 46 : 54 49 : 51 mutation analysis revealed kras to be the most frequently mutated gene in mad ( 77% ) , ad ( 56% ) , and pmp ( 81% ) and the second most frequently mutated gene in srcc ( 35% )  . 
in contrast , kras mutations were significantly less frequent in gccs ( 13% ; 2 p < .001 ) , where tp53 ( 33% ) was the most frequently mutated gene ( fig 1b ; appendix fig a1a )  . 
braf , brca1 , cdkn1b , cdkn2a , myc , pten , and tgfbr2 mutations were present in < 10% of cases across all subtypes ( table 2 )  . 
gnas and kras were the only gene pair significantly comutated ( odds ratio , 6.8 ; bonferroni corrected p = 8.6x10 - 17 ) ; gnas and tp53 were the only gene pair significantly mutually exclusive ( odds ratio , 0.20 ; bonferroni corrected p = 6.7x10 - 13 ; fig 1c ; data supplement )  . pathway - based analysis of mutation profiles genetic aberrations were subsequently grouped by signaling pathway ( appendix table a1 )  . 
 components of the ras / raf signaling pathway ( ie , braf , hras , kras , and nras ) were the most frequently altered genes in epithelial appendix cancers , occurring in > 80% of mads and pmps , 60% of ads , but only 33% of gccs ( 2 p < .001 ; fig 1d )  . 
alterations in homologous recombination deficiency genes were observed in > 50% of all subtypes but were most prevalent in srcc ( 80% ) fluorouracil , leucovorin , and oxaliplatin ; fluorouracil , leucovorin , and irinotecan ; and targeted agents in appendiceal adenocarcinomas compared with crc , 8 , 9 it is known that appendiceal neoplasms have a better prognosis after cytoreductive surgery with hipec treatment.10 there is also a growing body of data showing that there are clear molecular differences between appendiceal and colorectal cancers.11 - 14 here we present a cohort of 703 molecularly profiled appendiceal neoplasms , the largest such cohort to date in this rare disease . 
comparing the mutational landscapes across histologic subtypes we find significant differences in kras , gnas , and fat3 mutation prevalence and confirm that mutational profiles of appendiceal neoplasms are distinct from crc and other gi cancers . 
 in addition , we identify that patients can be risk stratified using the combined mutation status of gnas and tp53 and that outcomes are favorable for patients with kras wild - type disease when treated with irinotecan . results mutation landscape of appendiceal neoplasms the 703 cases were categorized into four different histopathological subtypes consistent with the recently updated consensus classification from the peritoneal surface oncology group international ; in addition , cases of pseudomyxoma peritonei ( pmp ) were included , because this syndrome usually arises from the appendix.2 the majority were either mucinous adenocarcinomas ( mad , 46% ) or adenocarcinomas ( ad , 30% ) , with the rest being gccs ( 12% ) , pmps ( 7.7% ) , or signet ring cell carcinoma ( srcc , 5.3% ; appendix ; table 1 )  . 
the majority of specimens submitted for sequencing came from intraperitoneal metastatic deposits , although there were also primary appendiceal tumors and a small number of lung , liver , and bone metastases ( fig 1a )  . 
data presented as % frequency . abbreviations : ad , adenocarcinoma ; crc , colorectal cancer ; gcc , goblet cell carcinoid ; mad , mucinous adenocarcinoma ; msi - h , microsatellite instability - high ; pdac , pancreatic ductal adenocarcinoma ; pmp , pseudomyxoma peritonei . comparison of genomic aberrations in appendix , colorectal , and pancreas cancers given the clinical practice of treating metastatic appendiceal cancers with crc regimens , we compared genomic alteration profiles of the appendiceal subtypes with those of 10 , 000 crcs profiled by the same laboratory ( table 2 )  . 
the high frequency of kras mutations observed in multiple appendiceal subtypes prompted inquiry into possible parallels with ( pdac ) , pancreatic ductal adenocarcinoma which harbors kras mutations in up to 95% of cases.16 sequencing of 2 , 800 pancreatic tumors revealed kras mutations in 87% of pdacs . 
there tended to be a higher frequency of microsatellite unstable or tumor mutational burdenhigh srccs and ads compared with mads ( table 2 ; appendix fig a1e )  . histopathologic and molecular features predictive of survival to determine the influence of histologic and molecular features on clinical outcomes , a retrospective review of a single - institution case series was performed . 
similar to the full 703 - patient cohort , the majority of cases were either ads ( n = 17 ) or mads ( n = 33 ) , with fewer low - grade appendiceal mucinous neoplasms that manifested as pmps ( n = 13 ) and few srccs ( n = 9 )  . 
data for chemotherapy treatment were available for 60 patients , showing that the majority were treated with a fluoropyrimidine and either oxaliplatin or irinotecan ( appendix ; table 3 )  . overall survival ( os ) , determined from time of initial diagnosis , was similar for ad and mad ( logrank p = .29 ; appendix fig a2a ) , so these groups were combined in subsequent analyses . 
 ( % ) unless otherwise noted . abbreviations : egfr , epidermal growth factor receptor ; hipec , heated intraperitoneal chemotherapy ; lamn , low - grade appendiceal mucinous neoplasms ; pd1 , programmed cell death protein 1 ; pd - l1 , programmed death ligand 1 ; pmp , pseudomyxoma peritonei ; sd , standard deviation ; vegf , vascular endothelial growth factor . for srccs ( p = .11 ; fig 2a )  . 
 however , use of irinotecan in any line of therapy was associated with a survival advantage in kras wild - type tumors ( log - rank p = .041 ; fig 2f ) but not in kras mutant tumors ( log - rank p = .32 ; appendix fig a2b )  . 
low - grade tumors were enriched for gnas mutations ( 72% v 18% for high grade ; 2 p < .001 ; appendix fig a3a ) , consistent with our prior report , 14 whereas highgrade tumors were enriched for tp53 mutations ( 56% v 6.9% for low grade ; 2 p < .001 ; appendix fig a3b )  . to assess the relative contributions of mutation and grade to the observed os differences , a cox proportional hazard analysis was performed including age , sex , kras , gnas , tp53 , and grade as covariates . 
however , the effect of tp53 mutation on survival was independent of grade , with the rare low - grade , tp53 - mutant tumors having os similar to other tp53 - mutant tumors ( median , 24.6 months ; fig 3b )  . 
at risk : irinotecan 8 no irinotecan 15 time ( months ) the mutational profile of 703 appendix neoplasms provides insight into the molecular aberrations that differentiate histologic subtypes and identifies putative prognostic and predictive biomarkers that may help guide treatment in this rare malignancy . 
compared with ad , mad , and pmp , kras and gnas mutations were much less frequent in gccs , whereas mutations in fat3 and arid1a were more frequent ( figs 1b and 1c )  . 
 these differences were also seen in the pathway analysis , in which gccs had less - frequent alterations of the ras / raf pathway relative to epithelial appendiceal cancers ( fig 1d )  . 
at risk : gnas mut 28 tp53 mut 25 neither 18 high - gnas mutant moderate - gnas mutant p = .011 low - gnas mutant time ( months ) no . 
these data also show that all of the appendiceal subtypes are molecularly distinct from crc , with more frequent gnas mutation and significantly lower prevalence of apc and tp53 mutations , which are key pathogenic alterations in crc . 
 the mutation profiles of ad and srcc bore the most resemblance to crc , with ad and crc sharing similar frequencies of mutation in kras , smad4 , and arid1a ; of note , appendiceal ads have been referred to as colonic - type adenocarcinoma , given clinical behavior more similar to crc.20 , 27 in this series , srcc had the worst prognosis of the epithelial appendiceal tumors and also has a clinical course similar to crc.28 the frequent comutation of kras and gnas in mad , ad , and pmp parallels the molecular profile of intraductal papillary mucinous neoplasms.14 , 29 notably , both intraductal papillary mucinous neoplasms and , in particular , gnas mutant appendiceal cancers are characterized by mucin production and a generally indolent clinical course . 
the differences in mutation profiles between the epithelial appendiceal tumors can be at least partially explained by grade , with significant association of gnas mutation with low - grade tumors and tp53 mutation with highgrade tumors ( appendix figs a3a and a3b )  . 
 consistent with this is the higher incidence of tp53 mutation and lower incidence of gnas mutation in srccs , which are by definition all high grade . histologic grade was also a strong predictor of survival ( fig 2b ) , consistent with prior reports.17 , 30 we did not observe a significant difference in os between ad and mad ; however , the distribution of tumor grade was similar between these two subtypes in our study . 
 conversely , tp53 mutation was an independent predictor of poor survival , with low - grade , tp53 - mutant tumors having survival similar to that of high - grade tumors . 
in addition , because appendiceal tumors are so rare , they are difficult to diagnose pathologically and are frequently overinterpreted by community pathologists.31 although gnas mutation is not an independent predictor of survival , because gnas and tp53 mutations occur mutually exclusive of each other and are associated with lowand high - grade tumors , respectively , the two genes can be substituted for grade to predict survival . 
the survival stratification achieved with the gnas - tp53 biomarker is similar to grade , an important observation , given that it is now much easier to obtain a mutation profile than an expert pathology review in the community oncology setting . the absence of a gnas mutation in the majority of high - grade tumors and the mutual exclusivity of tp53 and gnas mutations both strongly suggest that most high - grade appendiceal tumors occur de novo , rather than progressing from low - grade tumors , confirming , on a larger scale , our previous observations.14 however , there were a minority of tumors with both tp53 and gnas mutation ( n = 41 ; 6.7% ) , suggesting that transformation from low grade to high grade can occur . 
serial biopsy or serial measurement of circulating tumor dna would be needed to confirm that these tp53 mutations did in fact occur after the formation of a low - grade , gnas mutant tumor . 
however , given the low propensity for appendiceal tumors to spread beyond the abdominal cavity , there may be limited tumor dna in circulation , potentially making bloodbased tumor detection difficult . 
indeed , three of the four university of california , san diego , patients who underwent circulating tumor dna sequencing had no reportable alterations . regarding predictive biomarkers , kras wildtype status was associated with better survival in the subset of patients treated with irinotecan . 
a retrospective study in metastatic crc reported better response to irinotecan in patients with wild - type versus mutant plasma kras.32 however , a larger prospective study found that mutant kras was associated with poor survival but not with response to irinotecan in crc.33 regarding targeted therapies , there are unfortunately few clinically actionable mutations in appendiceal cancers , although the ras / raf signaling pathway is frequently altered in epithelial appendiceal cancers . 
data on therapeutic targeting of the ras / raf cascade in appendiceal tumors are limited , although a recent case report described clinical benefit in a patient with appendiceal mad harboring a gnas r201h mutation who was treated with trametinib.34 because only eight patients in this cohort received an anti - egfr antibody , we were unable to assess interactions between kras mutation status and response to these agents . a major limitation of this study is its retrospective design . 
with regard to the 703 - patient cohort , clinical information such as precise tnm stage was not available , but the fact that > 80% of specimens submitted came from metastases indicates that the majority of patients had stage iv disease . 
although specimens were independently reviewed by pathologists to confirm the diagnosis before undergoing sequencing , subtype definitions are potentially subject to variability and overlap , because there was no consensus classification system for appendiceal neoplasms and pmp until recently.2 for example , pmp is an inherently imprecise term used to describe the clinical syndrome of mucinous peritoneal dissemination from an appendiceal neoplas pmp encompasses a spectrum of both highand low - grade lesions but does not reference the histopathologic characteristics of the appendiceal primary from which it arises . 
 newer classification schemes separate pmps with low - grade and high - grade features ( also known as disseminated peritoneal adenomucinosis and peritoneal mucinous carcinomatosis , respectively35 ) and pmp with signet ring cells.2 because this study did not distinguish between these subtypes , we are unable to report on genomic differences associated with grade in the larger 70 - patient cohort . 
in addition , analysis of interactions between genotype and specific drugs are confounded by the fact that most patients received multiple lines of therapy . this study of unprecedented size in this rare disease highlights important molecular differences between different subtypes of appendix cancer and identifies gnas and tp53 mutation status as a prognostic biomarker . 
this comprehensive portrait of the molecular landscape of appendix cancer will help with the design of future clinical studies to develop and test therapeutic strategies specific to this disease . in conclusion , appendiceal neoplasms have molecular profiles that are distinct from crc and are characterized by frequent gnas and kras mutations , especially in low - grade tumors . 
ross employment : foundation medicine leadership : foundation medicine financial support : john paul shen , paul fanta , trey ideker administrative support : john paul shen , paul fanta stock and other ownership interests : foundation medicine provision of study material or patients : john paul shen , jeffrey s . 
miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center miriam t . 
ali employment : foundation medicine leadership : incyte stock and other ownership interests : exelixis , blueprint medicines , agios pharmaceuticalas , genocea biosciences patents , royalties , other intellectual property : patents via foundation medicine and seres health joel baumgartner research funding : merck andrew lowy consulting or advisory role : halozyme , merck research funding : mitsubishi tanabe pharma , syros pharmaceuticals expert testimony : merck ascopubs.org / journal / po jco precision oncology 9 justin k . 
 paul fanta no relationship to disclose trey ideker leadership : data4cure consulting or advisory role : ideaya biosciences , genetic modifiers newco , merck , la jolla institute for allergy and immunology research funding : pfizer travel , accommodations , expenses : sage bionetworks sherri z . 
chen , david xu , joel baumgartner , andrew lowy , paul fanta , trey ideker , and olivier harismendy , university of california , san diego , la jolla , ca ; jeffrey s . 
carr nj , cecil td , mohamed f , et al : a consensus for classification and pathologic reporting of pseudomyxoma peritonei and associated appendiceal neoplasia : the results of the peritoneal surface oncology group international ( psogi ) modified delphi process . 
sugarbaker ph , bijelic l , chang d , et al : neoadjuvant folfox chemotherapy in 34 consecutive patients with mucinous peritoneal carcinomatosis of appendiceal origj surg oncol 102 : 576581 , 2010 5 . 
farquharson al , pranesh n , witham g , et al : a phase ii study evaluating the use of concurrent mitomycin c and capecitabine in patients with advanced unresectable pseudomyxoma peritonei . 
garin l , corbinais s , boucher e , et al : adenocarcinoid of the appendix vermiformis : complete and persistent remission after chemotherapy ( folfox ) of a metastatic case . 
tejani ma , ter veer a , milne d , et al : systemic therapy for advanced appendiceal adenocarcinoma : an analysis from the nccn oncology outcomes database for colorectal cancer . 
johncilla m , stachler m , misdraji j , et al : mutational landscape of goblet cell carcinoids and adenocarcinoma ex goblet cell carcinoids of the appendix is distinct from typical carcinoids and colorectal adenocarcinomas . 
borazanci e , millis sz , kimbrough j , et al : potential actionable targets in appendiceal cancer detected by immunohistochemistry , fluorescent in situ hybridization , and mutational analysis . 
alakus h , babicky ml , ghosh p , et al : genome - wide mutational landscape of mucinous carcinomatosis peritonei of appendiceal orig genome med 6 : 43 , 2014 [ erratum : genome med 6 : 53 , 2014 ] 15 . 
reid md , basturk o , shaib wl , et al : adenocarcinoma ex - goblet cell carcinoid ( appendicealtype crypt cell adenocarcinoma ) is a morphologically distinct entity with highly aggressive behavior and frequent association with peritoneal / intra - abdominal dissemination : an analysis of 77 cases . 
singhi ad , davison jm , choudry ha , et al : gnas is frequently mutated in both low - grade and high - grade disseminated appendiceal mucinous neoplasms but does not affect survival . 
mccusker me , cot tr , clegg lx , et al : primary malignant neoplasms of the appendix : a population - based study from the surveillance , epidemiology and end - results program , 19731998 . 
asare ea , compton cc , hanna nn , et al : the impact of stage , grade , and mucinous histology on the efficacy of systemic chemotherapy in adenocarcinomas of the appendix : analysis of the national cancer data base . 
 appendix tumor tissue from 703 patients with appendiceal cancer was submitted to a clinical laboratory improvement amendments certified laboratory ( foundation medicine , cambridge , ma ) for dna sequencing and variant calling . 
a minimum of 50 ng of dna was extracted and a hybrid - capture method used to capture 3 , 769 exons from 315 cancer - related genes and 47 introns of 28 genes commonly rearranged in cancer ; this material was then sequenced to high ( average , 756x ) uniform coverage allowing for evaluation of genomic alterations , including base substitutions , indels , amplifications , copy number alterations , and fusions / rearrangements . 
clinical characteristics and outcomes , including tumor histology , grade , stage , overall survival ( os ) , and chemotherapy given were determined from review of the electronic medical record . 
sequencing failed quality control in two cases , and two patients with only blood - based cell - free dna sequencing were excluded , leaving 76 patients available for analysis . 
because there were only four goblet cell carcinoids , these were removed from survival analysis . for mutual exclusivity and comutation analysis , goblet cell carcinoid tumors and microsatellite instability - high tumors were removed , and a fishers exact test was performed for all gene combinations , followed by bonferroni multiple hypothesis correction . 
 inherited mutations in men undergoing multigene panel testing for prostate cancer : emerging implications for personalized prostate cancer genetic evaluation see accompanying editorial doi : purpose multigene panels are commercially available for the evaluation of prostate cancer ( pca ) predisposition , which necessitates tailored genetic counseling ( gc ) for men . 
here we describe emerging results of genetic evaluation of men , prospective multigene testing study in pca to inform personalized genetic counseling , with emerging implications for referrals , cancer screening , and precision therapy . patients and methods eligibility criteria for men affected by or at high risk for pca encompass age , race , family history ( fh ) , and pca stage / grade . 
 pca20 - 24 along with risk for multiple additional cancers , including colorectal , endometrial , ovarian , and pancreatic.25 , 26 in addition , a recurrent mutation in hoxb13 has been identified to predispose to hereditary pca ( hpc ) , and confers an approximate twoto eightfold increased risk for pca and twoto 10 - fold increased risk for earlyonset disease , 27 - 34 consistent with the longdescribed phenotype of hpc.35 thus , inherited pca may be suspected on the basis of an fh of pca , fh of multiple other cancers , or young age at diagnosis of pca . 
this multigene testing capability raises the importance of tailoring genetic counseling ( gc ) for men and their families to make informed decisions about proceeding with genetic testing.39 , 40 because genetic testing guidelines for pca are limited , lack of clinical genetic data exists on potential rates of mutations , family cancer risks relevant to both sexes , variant of uncertain significance ( vus ) rates , concordance of mutations with personal / fh , and cancer screening impact , all of which are important to gc and informed genetic testing.39 , 40 robust data from clinical genetic testing , therefore , are needed to address this gap in gc practice and responsibly implement multigene panel testing for the evaluation of inherited pca . the genetic evaluation of men ( gem ) study was developed with three main goals : implement prospective multigene panel testing for inherited pca in the context of gc ; characterize the genetic variant spectrum in clinically available cancer risk genes to inform men and their families about cancer risks , screening , and precision treatment ; and develop a registry of men with and without pca that encompasses detailed personal / fh of cancer , diet , exercise , and exposures that are not usually available from commercial genetic testing laboratory databases . 
the study has plans to recruit additional sites pending institutional review board approvals . referrals and recruitment referrals come from treatment clinics for men with pca and men without pca enrolled in screening efforts . 
at the sidney kimmel cancer center , referrals also come from the genitourinary multidisciplinary clinic where men with pca receive multidisciplinary evaluation , treatment planning , and an opportunity to engage in gc.41 eligibility criteria eligibility criteria for gem were intended to be broad to encompass inclusion of a spectrum of risk for pca ( young age of cancer diagnoses , 19 , 26 , 42 , 43 african american race , 44 - 46 fh of pca , 47 fh of hcs - associated cancers , 19 , 25 , 26 and fh that meets strict criteria for hcss19 , 35 , 48 ) and are modeled on evaluation of other cancers.19 , 26 personal / fh of cancers associated with specific hcss include hboc ( breast , ovarian , pancreatic , and prostate cancers and melanoma ) , ls ( colorectal , ovarian , endometrial , upper gi , pancreatic , and upper urinary tract cancers and sebaceous carcinoma ) , and hpc ( pca )  . 
furthermore , with emerging insights into germline mutations in metastatic pca , 36 , 37 eligibility criteria also included metastatic pca as well as higher gleason score and t3 disease to explore mutation rates in advanced / high - risk disease . 
prap eligibility criteria include age < 69 years and any of the following : fdr with pca , two sdrs with pca on the same side of the family , and african american race . 
individuals who consented to prap participation were offered genetic evaluation of men study participation . genetic test multigene panel testing initially included 25 cancer risk genes : apc , brca1 , brca2 , mlh1 , msh2 , pms2 , msh6 , cdh1 , pten , stk11 , tp53 , atm , bard1 , bmpr1a , brip1 , cdk4 , cdkn2a , chek2 , epcam , mutyh , nbn , palb2 , rad51c , rad51d , and smad4 . 
subsequently , three more genes related to colon polyposis were added to the panel : pold1 , pole , and grem1 ( appendix table a1 ; details for variant detection provided in the appendix )  . 
discussion includes an overview of medical history and a review of pedigrees , test results , and personalized recommendations about cancer risk , screening , and personalized management for each participant and his blood relatives . post - test genetic disclosure post - test discussion focuses on cancer screening , risk reduction strategies , and further gc / testing in the family on the basis of test results ( positive for pathogenic variant , negative for any pathogenic variants , or vus [ full details provided in the appendix ] ; appendix table a1 )  . 
participants with vus also receive information for the prospective registry of multiplex testing ( promptstudy.info ) for contributing to the clarification of vus.49 statistical methods mutation rates were summarized by eligibility criteria . 
in such cases , the participant is included in the mutation rate calculation for multiple eligibility criteria . fh was summarized as the number of men who reported a history of cancer in first - degree relatives ( fdrs ) , second - degree relatives , and thirddegree relatives as well as the number of affected family members . 
furthermore , fh was summarized for five specific cancer sites of primary interestprostate , breast , ovarian , colon , and pancreaticinvolved in hcs and in which pca has been implicated . 
hcss were classified based on the participant or fh meeting criteria for hboc , ls , or hpc.19 , 35 , 48 in addition to fh , demographic information ( race , hispanic ethnicity , ashkenazi jewish ancestry ) and pca information for affected men ( stage , gleason score , age at diagnosis ) was collected . unadjusted mutation rates were compared across fh in fdrs , fh in any relatives , presence of hcs , and pca status for all men using fishers exact test . 
an examination of broader fh showed that participants with an fh of any relative with breast cancer had a higher rate of mutations ( 10.3% ) than those with no fh of breast cancer ( p = .004 ; table 3 )  . 
concordance was defined as having a personal history or any fdr , second - degree relative , or third - degree relative with a cancer in the spectrum linked with the gene mutation ( descriptive examples provided in the appendix )  . 
of the nine mutation carriers , 44.4% received their pca diagnosis at age younger than 60 years , 66.7% had a gleason score > 7 , and 44.4% had a gleason score > 8 . 
all chek2 ( ile157thr ) carriers had a personal / fh of pca , and three of four had an fh of breast and / or colon cancers consistent with the current chek2 - associated cancer spectrum . precision gc model for pca in current gc for pca , a void of information exists with regard to expected mutation and vus rates from multigene testing , potential findings and concordance of mutations , and cancer risks for patients and their families ; such information is needed for informed decision making about genetic testing.39 , 40 the current emerging results are being used to tailor gc for men concerned about inherited pca ( table 5 )  . discussion implications for precision gc the findings of this study begin to fill a practice gap for gc to inform men about potential multigene test results before proceeding with genetic testing , which is important to report at this time because of the current commercial availability of pca gene panels ( table 5 )  . 
the gem study implements multigene panel testing for evaluation of inherited pca and was intended to include a broad group of men to comprehensively uncover and characterize mutations and variants to inform genetic evaluation strategies in this underdeveloped area pertinent to pca . 
the cohort is heterogeneous and includes men with and without pca who also have personal or familial features of risk for pca , including young age at cancer diagnosis , african american race , metastatic pca , and fh of cancer . 
the chance of a genetic finding not consistent with a personal / fh also needs to be discussed due to cancer screening and management implications for patients and their families . the results reveal a proportionately higher rate of germline mutations in dna repair genes among men with a strong fh of cancer , particularly for breast cancer , with mutations identified in men without evidence of metastatic disease . 
precision genetic counseling for prostate cancer finding highlights the importance of germline genetic testing to be considered not only for men with metastatic pca but also for those with early - stage disease , particularly with a strong fh of cancer . 
oncologic , urologic , and primary care providers must be aware of the need for full intake of fh ( maternal and paternal ) , including all cancers for appropriate referrals of men for genetic evaluation . the overall mutation rate of 5.5% is comparable to non - brca mutation rates in breast and ovarian cancers where guidelines exist for genetic if we include chek2 testing.19 , 50 however , ( ile157thr ) as pathogenic , the overall prevalence of mutations in the current cohort approaches 7.5%. 
we chose to include mutation counts as stringently as possible that are based only on clinical report interpretation of a variant as pathogenic , thus excluding chek2 ( ile157thr ) at this time from mutation prevalence estimation . in castration - resistant pca and in the setting of deleterious germline brca2 mutations.51 , 52 in addition , a phase ii clinical trial suggested that a common subset of metastatic pcas , characterized by defects in dna repair , can be molecularly stratified for treatment with parp inhibitors , with a notable response seen in patients whose tumors had atm alterations and brca2 loss.38 therefore , the current findings become particularly important for men with advanced or metastatic disease who may be eligible for targeted therapy with parp inhibition . 
furthermore , as dna repair germline mutations are identified in early - stage disease , there may be an added rationale to develop trials of targeted / tailored management of these individuals , which widens the role of genetic testing for treatment and management decision making along the pca stage spectrum . implications for unaffected men at high - risk for pca implications for men with pca the identification of dna repair germline mutations in men with pca , particularly those with metastatic disease , is of high clinical importance . data suggest a potential role for parp inhibitors current national comprehensive cancer network guidelines only address pca screening in the context of brca1 / 2 mutations , 19 with a need to expand genetically informed pca screening . 
risks for colon cancer , pancreatic cancer , melanoma , and male breast cancer in men with or at risk for pca have been identified , which necessitates guidelines - based screening / management or referral for discussion of screening options.19 , 25 , 26 furthermore , male and female blood relatives of all participants now have insight into which specific mutations may predispose to cancer risk in their family and can pursue site - specific mutation testing through guidelines - based cancer screening approaches.19 , 25 , 26 consideration of study results there are some limitations when considering study results . 
although genetic mutations identified in this study inform risks for several cancers , the majority of mutations ( with the exception of brca1 and brca2 ) currently provide limited information about pca penetrance and highlight the need for additional functional studies of their role in pca predisposition . in conclusion , this study supports a comprehensive approach to genetic evaluation for pca and highlights the need to consider multiple types of cancers in families of men concerned about inherited pca risk . 
hegarty no relationship to disclose susan montgomery no relationship to disclose andrea forman consulting or advisory role : invitae speakers bureau : invitae ruth bingler no relationship to disclose william k . 
chen employment : tenet healthcare ( i ) stock and other ownership interests : pfizer ( i ) consulting or advisory role : argos therapeutics travel , accommodations , expenses : argos therapeutics costas d . 
gomella patents , royalties , other intellectual property : nuview ( inst ) , nuview travel , accommodations , expenses : abbvie , bayer ag , astellas pharma , janssen pharmaceuticals acknowledgment myriad genetics provided genetic testing free of cost to study participants . 
giusti rm , rutter jl , duray ph , et al : a twofold increase in brca mutation related prostate cancer among ashkenazi israelis is not associated with distinctive histopathology . 
leongamornlert d , mahmud n , tymrakiewicz m , et al : germline brca1 mutations increase prostate cancer risk . br j cancer 106 : 1697 - 1701 , 2012 10 . 
eerola h , pukkala e , pyrh onen s , et al : risk of cancer in brca1 and brca2 mutation - positive and - negative breast cancer families ( finland )  . 
castro e , goh c , olmos d , et al : germline brca mutations are associated with higher risk of nodal involvement , distant metastasis , and poor survival outcomes in prostate cancer . 
edwards sm , kote - jarai z , meitz j , et al : two percent of men with early - onset prostate cancer harbor germline mutations in the brca2 gene . 
engel c , loeffler m , steinke v , et al : risks of less common cancers in proven mutation carriers with lynch syndrome . j clin oncol 30 : 4409 - 4415 , 2012 553 - 557 , 2014 oncol 31 : 1713 - 1718 , 2013 21 . 
ryan s , jenkins ma , win ak : risk of prostate cancer in lynch syndrome : a systematic review and meta - analysis . cancer epidemiol biomarkers prev 23 : 437 - 449 , 2014 24 . 
xu j , lange em , lu l , et al : hoxb13 is a susceptibility gene for prostate cancer : results from the international consortium for prostate cancer genetics ( icpcg )  . 
shang z , zhu s , zhang h , et al : germline homeobox b13 ( hoxb13 ) g84e mutation and prostate cancer risk in european descendants : a meta - analysis of 24 , 213 cases and 73 , 631 controls . 
laitinen vh , wahlfors t , saaristo l , et al : hoxb13 g84e mutation in finland : population - based analysis of prostate , breast , and colorectal cancer risk . 
kote - jarai z , mikropoulos c , leongamornlert da , et al : prevalence of the hoxb13 g84e germline mutation in british men and correlation with prostate cancer risk , tumour characteristics and clinical outcomes . 
powell ij , bock ch , ruterbusch jj , et al : evidence supports a faster growth rate and / or earlier transformation to clinically significant prostate cancer in black than in white american men , and influences racial progression and mortality disparity . 
balma~na j , digiovanni l , gaddam p , et al : conflicting interpretation of genetic variants and cancer risk by commercial laboratories as assessed by the prospective registry of multiplex testing . 
sandhu sk , omlin a , hylands l , et al : poly ( adp - ribose ) polymerase ( parp ) inhibitors for the treatment of advanced germline brca2 mutant prostate cancer . 
sandhu sk , schelman wr , wilding g , et al : the poly ( adp - ribose ) polymerase inhibitor niraparib ( mk4827 ) in brca mutation carriers and patients with sporadic cancer : a phase 1 dose - escalation trial . 
bancroft ek , page ec , castro e , et al : corrigendum to targeted prostate cancer screening in brca1 and brca2 mutation carriers : results from the initial screening round of the impact study [ eur urol 2014 ; 66 : 489 - 99 ]  . 
eur urol 67 : e126 , 2015 before the genetics appointment , participants receive questionnaires on medical history , family history ( fh ) , and risk factors for clinical and study purposes . 
genetic counseling ( gc ) involves a detailed intake of medical history , prostate cancer ( pca ) features ( eg , age at diagnosis , gleason score , treatment ) , fh of cancer , and cancer - related risk factors . 
the discussion involves basic principles of disease inheritance , genetic test options ( single - gene testing versus multigene testing ) , genespecific cancer risks , types of test results ( pathogenic variant , variant of uncertain significance [ vus ] , or no pathogenic variant ) , potential management implications , genetic discrimination laws , and reproductive implications . 
pretest counseling is done in person by a cancer genetics provider ( sidney kimmel cancer center ) and by a video session designed for this study by a genetic counselor ( a.f. ) at fox chase cancer center with support by study staff . 
the myrisk test initially included 25 cancer - risk genes : apc , brca1 , brca2 , mlh1 , msh2 , pms2 , msh6 , cdh1 , pten , stk11 , tp53 , atm , bard1 , bmpr1a , brip1 , cdk4 , cdkn2a , chek2 , epcam , mutyh , nbn , palb2 , rad51c , rad51d , and smad4 . 
per laboratory protocol , all genes are sequenced as well as assessed for deletion / duplication except for epcam ( large rearrangement only ) , pole and pold1 ( sequencing of select regions ) , and grem1 ( large rearrangement analysis )  . 
additional discussion includes psychosocial supports and connection of family members with gc services as needed . individual characteristics of mutation carriers detailed characteristics for each participant with regard to mutations identified , pca status and features , fh of cancer , and concordance of mutation with personal / fh were assessed to inform gc ( appendix table a3 )  . 
concordance was defined as a personal history or any first - , second - , or third - degree relative with a cancer in the spectrum linked with the aforementioned gene mutations . 
in contrast , participant 5 had a brip1 mutation but no fh of ovarian cancer , which is the only cancer in the brip1 cancer - associated spectrum at this time . 
current myrisk panel from myriad genetics . abbreviations : br , breast ; co , colon ; en , endometrial ; fap , familial adenomatous polyposis ; ga , gastric ; hboc , hereditary breast and ovarian cancer ; hdgs , hereditary diffuse gastric cancer syndrome ; hht , hereditary hemorrhagic telangiectasia ; jps , juvenile polyposis syndrome ; ls , lynch syndrome ; lfs , li - fraumeni syndrome ; map , myh - associated polyposis ; mcs , melanoma cancer syndrome ; me , melanoma ; m - pcs , melanoma - pancreatic cancer syndrome ; oc , other cancers ; ov , ovarian ; pa , pancreatic ; phts , pten hamartoma tumor syndrome ; pjs , peutz - jeghers syndrome ; pr , prostate . 
 significant clinical response to a mek inhibitor therapy in a patient with metastatic melanoma harboring an raf1 fusion introduction systemic therapy for metastatic melanoma has changed dramatically since the development of targeted drugs for braf - mutant melanoma and immune checkpoint inhibitors . 
the overall survival duration of those with advanced melanoma has improved significantly with these novel drugs , with the median survival duration now reaching nearly 2 years.1 - 3 the checkpoint inhibitors ( anti cytotoxic t - cell lymphocyte - 4 antibodies and anti - programmed cell death - 1 [ pd - 1 ] antibodies ) produce antitumor response through their ability to activate cytotoxic t - lymphocytes . 
although many of these clinical responses are durable , the median progression - free survival durations are approximately 2 to 3 months and 5 to 6 months with anti - ctla4 antibody and ipilimumab , respectively.4 - 7 a combination of nivolumab and ipilimumab improves the overall response rate and progression - free survival compared with anti - ctla4 antibody alone.7 for patients with metastatic v600 braf - mutant melanoma , combinations of a braf inhibitor and a mek inhibitor have been established as a standard therapy because of a high response rate and superior survival over single - agent braf inhibitors.1 - 3 however , approximately 50% of melanomas do not harbor a v600 braf mutation and therefore do not benefit from braf inhibitor therapy.8 for these patients with wildtype braf , the identification of relevant and actionable genetic alterations and the development of effective matched therapies are urgently needed . 
recently , next - generation sequencing ( ngs ) analyses have revealed a multitude of less frequent and less well - characterized genetic alterations in melanoma , and these findings could lead to the development of effective treatments . 
in october 2014 , he had recurrent melanoma in the right cervical lymph node , and by december 2014 , his disease had progressed in the cervical and intrathoracic lymph nodes and the liver . 
in january 2015 , the patient started treatment with the combination of nivolumab and ipilimumab , but the treatment was discontinued after two doses because of the development of diabetic ketoacidosis . 
from august 2015 to march 2016 , he was treated with single - agent nivolumab and subsequently nivolumab in combination with intralesional talimogene laherparepvec injection to the right cervical nodes , but by march 2016 , the disease had progressed in the right neck nodes and liver . 
 in april 2016 , he underwent a debulking radical right neck lymph node dissection for a palliative purpose . in october 2016 , the patient developed a painful right neck mass , and his performance status worsened ( eastern cooperative oncology group performance status of 2 )  . 
he was found to have rapid disease progression in the right supraclavicular , mediastinal , gastrohepatic , and portocaval lymph nodes ; the liver ; the spleen ; and the t - 12 spine . 
 melanoma specimen from the right neck node was analyzed for genetic alterations using a hybrid - capturebased ngs assay ( foundationone ) .9 the results were wild type for braf , nras , and kit ; however , a number of genetic alterations , including a ano10 - raf1 fusion , flt1 r281q , notch3 splice site 4404 - 1g > a , arid2 q760fs * 7 , pbrm1 loss , spta1 q1720 * , and tert promoter - 146c > t , were detected . 
because of the development of significant skin toxicity , the dose was reduced to 1.5 mg / d after 4 weeks and further down to 1 mg / d in january 2017 . 
the patients pain in the right neck and shoulder improved dramatically with the treatment , his performance status improved , and a computed tomography scan performed at 10 weeks showed significant regression of the metastatic lesions in the right supraclavicular , mediastinal , gastrohepatic , and portocaval lymph nodes ; the liver ; and the spleen ( fig 1 )  . 
treatment with another mek inhibitor was discussed with the patient , but because of rapid clinical deterioration and the concern for significant skin toxicity , he decided on palliative care , and he died in june 2017 . discussion in this article , we describe , to our knowledge , the first case of clinical benefit from an mek inhibitor in a patient with metastatic melanoma harboring an ano10 - raf1 fusion . 
the patients tumor did not harbor a v600 braf mutation , and therefore , selective braf inhibitors or mek inhibitors were not clinically indicated at the time of the treatment . 
without the comprehensive genomic testing capable of detecting gene fusions and rearrangements in addition to mutations and copy number changes , we could have missed the great response witnessed with the mek inhibitor , underscoring the importance of ngs analysis . 
it is not clear whether the tumor progression was caused by true acquired resistance to mek inhibition or by suboptimal dosing of the treatment because of the intolerable cutaneous toxicity . although a single base substitution at codon 600 is the most common genetic alteration in raf genes in melanoma , other raf gene alterations have been observed , with various functional consequences.10 many of the non - v600 braf mutations do not activate the mitogen - activated protein kinase ( mapk ) signaling pathway in as robust a manner as do v600 mutations , and they are largely insensitive to the braf v600 specific inhibitors dabrafenib and vemurafenib.11 genetic mutations in the other raf genes , such as araf and craf ( raf1 ) , are relatively rare.12 abnormal rearrangements of raf1 , including raf1 fusions , occur in approximately 1% of cutaneous melanomas , 13 and their prognostic significance is not known . 
some of these genetic alterations , such as esrp1 - raf1 fusion , were found to activate the mapk pathway and are capable of transforming cells.10 palanisamy et al10 showed that prostate epithelial cells expressing an esrp1 - raf1 fusion were sensitive to a combination of a raf inhibitor ( sorafenib ) and a mek inhibitor ( u0126 ) in vitro . 
although the function of ano10 - raf1 fusion , as found in our case , has not been described , it is likely that this alteration activates the mapk pathway because mek protein inhibition caused significant tumor regression in our case . 
the breakpoint in this case is in raf1 exon 7 and in the fact that the resulting in - frame fusion retains the raf1 kinase domain while removing the n - terminal auto - inhibitory domain ( fig 2 )  . 
this case , together with ours , strongly suggests the clinical relevance of raf gene fusions in melanoma and offers mek inhibitors as a potential therapeutic option for patients with raf fusionpositive tumors . 
for patients with melanoma harboring a braf fusion , there is a phase ii study of trametinib ( the national cancer institute molecular analysis for therapy choice [ nci - match ] trial [ clinicaltrials . gov identifier : nct02465060 ] ) ; however , at this time there is no specific trial for patients with raf1 fusionpositive melanoma . 
in addition to the clinical investigation of mek inhibitors , clinical studies of pan - raf inhibitors , such as sorafenib and regorafenib , should be encouraged on the basis of their preclinical antitumor activity in raf1 fusionassociated cells . furthermore , a mek inhibitorbased combination approach may lead to more durable disease control . 
several groups have demonstrated potential mechanisms of resistance to mek inhibition , including induction of immunoglobulin transcription factor 2 , 15 induction of signal transducer and activator of transcription 3 , 16 and amplification of cyclin - dependent kinase 4 and 6 , 17 among others . 
 furthermore , there is increasing evidence that mek inhibition leads to antigen - specific cd8 + t cells proliferation within the tumor and pro motes the survival of tumor - infiltrating cd8 + t cells.18 these phenomena were corroborated by experiments in which the addition of an mek inhibitor to an anti pd - 1 ( or pro grammed death - ligand 1 [ pd - l1 ] ) antibody resulted in durable and synergistic tumor regression.18 several clinical trials are evaluating the clinical efficacy of an mek inhibitor in combination with the checkpoint inhibitors in patients with melanoma ( clinicaltrials.gov identifiers : nct03149029 , nct03178851 , and nct02910700 )  . in conclusion , the ngs analysis of the melanoma lesion in our patients tumor was essential in identifying the clinically relevant genetic alteration , ano10 - raf1 fusion , and in selecting a mek inhibitor treatment that resulted in brief , but significant , tumor regression and symptom improvement . 
kim honoraria : genentech , glaxosmithkline , novartis , foundation medicine consulting or advisory role : genentech , glaxosmithkline , novartis , foundation medicine speakers ' bureau : bristol - myers squibb , merck , novartis research funding : glaxosmithkline ( inst ) , novartis ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) , amgen , immune design ( inst ) travel , accommodations , expenses : genentech , bristol - myers squibb , merck , novartis thomas semrad consulting or advisory role : amgen , genentech , onyx pharmaceuticals , celgene , eisai , genzyme ( i ) research funding : eisai ( inst ) , millennium pharmaceuticals ( inst ) , novartis ( inst ) alexa b . 
ali employment : foundation medicine honoraria : emd merck serono research funding : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine ; patents via seres health on microbiome stuff in non - neoplastic disease ( i ) mark singer no relationship to disclose jeffrey s . 
ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine honoraria : pfizer mohammed kashani - sabet stock and other ownership interests : melanoma diagnostics , dnarx honoraria : cepheid consulting or advisory role : castle biosciences research funding : merck patents , royalties , other intellectual property : myriad genetics affiliations kevin b . 
kim , mark singer , and mohammed kashani - sabet , california pacific medical center research institute , san francisco ; thomas semrad , gene upshaw memorial tahoe forest cancer center , truckee , ca ; alexa b . 
long gv , stroyakovskiy d , gogas h , et al : dabrafenib and trametinib versus dabrafenib and placebo for val600 braf - mutant melanoma : a multicentre , double - blind , phase 3 randomised controlled trial . 
weber js , dangelo sp , minor d , et al : nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti - ctla - 4 treatment ( checkmate 037 ) : a randomised , controlled , open - label , phase 3 trial . 
bid hk , kibler a , phelps da , et al : development , characterization , and reversal of acquired resistance to the mek1 inhibitor selumetinib ( azd6244 ) in an in vivo model of childhood astrocytoma . 
ebert pjr , cheung j , yang y , et al : map kinase inhibition promotes t cell and anti - tumor activity in combination with pd - l1 checkpoint blockade . 
at result reporting , the variant had not been described previously and was interpreted as nonactionable , despite the well - described role of fgfr2 fusion proteins in cholangiocarcinoma1 , 2 and preliminary evidence of efcacy of fgfr2 inhibitors in this context.3 , 4 by the time he was evaluated in the clinic , even greater evidence that an fgfr2 inhibitor was likely to be effective had emerged.2 , 3 , 5 - 9 enrollment into the national cancer institute ( nci ) match ( molecular analysis for therapy choice ) fgfr arm ( subprotocol k - 2 ) and matching with erdatinib was sought . 
because the fgfr2 - bend3 fusion had not previously been described , approval of the variant as a novel actionable mutation of interest ( amoi ) was required by the nci before enrollment . 
numerous logistical issues ensued , summarized in figure 1 . discussion of the case an overwhelming minority of tumors have amois , 10 - 13 and few amois would not have been identied as part of standard molecular subtyping workup ( eg , egfr , alk , ros , met in nonsmall - cell lung cancer ) .14 in the rare circumstances where amois are identied by nonstandard testing , the expected marginal benet of targeted therapy over conventional chemotherapy to most patients is not of great magnitude.15 others have posited that even exceptional responses to off - label targeted therapy may be as a result of a tumors inherent susceptibility to all therapies.16 - 20 finally , the value of whole - genome or exome sequencing to into our knowledge been dividual patients has not stringently assessed by , for example , value of information analysis.21 - 23 on the basis of the available data , then , it is difcult to justify broad sequencing of tumors as a cost - effective approach at any line in therapy , even in an age of rapidly declining costs of sequencing.24 , 25 despite the absence of evidence , the heuristic of population - level prospective sequencing and matching generates excitement , is used with increasing frequency , and carries cachet in the eld , driven by the hope that maximal precision in diagnostics will lead to accuracy in therapeutics . precision in the modern era reects an underlying desire to proceed with maximal exactitude . 
in the case of diagnosis , this means demonstrating the molecular lesion ( s ) driving a given cancers growth , and in the case of therapeutics , to specically inhibit this offending mechanism with a treatment . 
in doing so , the goal is to meaningfully affect the natural history of diseasethe sine qua non of accurate treatment . although it is tempting to linearly connect precision with accuracy , in practice the two concepts must be kept distinct.14 precise treatments are not always accurate . 
highly precise body surface areabased dosing of chemotherapeutic agents lacks evidence demonstrating benet or reduction in harms when compared with less - precise dosing approaches.26 , 27 accurate therapies do not always require precision , even in the era of targeted agents : in certain contexts , the off - target effects ( imprecision ) of pantyrosine kinase inhibitors are a feature rather than a bug , such that these accurate treatments do not require or even benet from precise diagnosis . furthermore , therapies that are both precise and accurate do not necessarily require precise diagnosis . 
given the overwhelming pretest probability of philadelphia chromosome positivity , chronic myelogenous leukemia could be treated with inhibitors of the bcr - abl fusion protein tyrosine kinase without molecular testing . 
a successful therapeutic trial is diagnostic , and the efcacy of philadelphia chromosomenegative chronic myelogenous leukemia treatments is limited at best.28 - 30 finally , the most likely result of an individual sequencing panel is discovering an absence of amois or identifying molecular drivers that cannot be targeted , 31 , 32 situations in which the genetic test has failed to provide diagnostic or therapeutic value . 
unclear at this time whether the rejection is on scientific or logistical grounds . back - channel communication with high - ranking nih official : the entire problem in this case is that [ referring laboratory ] cannot provide the actual breakpointsthis is not an isolated incident and all referring labs know that we need this data to be able to enter the patient . " contact chief medical officer of referral laboratory requesting exact breakpoint sequences . referral laboratory : needs legal clearance ( new roi ) to provide breakpoint data . referral laboratory sends fgfr2 and bend3 breakpoints . 
we must use our usual channels as directed by the ncimatch protocol . referral laboratory : in summary , it is [ referral laboratory ] 's understanding that were waiting on the determination decision from the nci . referral laboratory : we have just received clarification on what genomic data the nci needs to proceed with the review of this case . 
timeline of novel actionable mutation of interest ( amoi ) approval processes of national cancer institute ( nci ) match ( molecular analysis for therapy choice ) and asco targeted agent and proling utilization registry ( tapur ) in the documented case . 
in both cases , patients are exposed to a matched therapy if their tumor contains any one putative driver mutation from a preapproved list of amois . there is no comparison group in either trial . 
the outcome measures of interest are different between the two trials . match aims to maximize each arms response rate.10 , 34 as such , the question being addressed in match is , using the example of our patient with fgfr2 fusion , do patients whose advanced cancers harbor particular fgfr2 mutations and fusions respond to treatment with erdatinib at a rate sufcient to generate interest for subsequent randomized study ? success in the study is achieving ve of 31 responses ( 95% ci , 5% to 34% ) , and anything less is failure . 
given the recent history of larotrectinibs fast - tracked approval , it is possible , although remote , that drugs could become approved by the us food and drug administration for tissue - agnostic indications on the basis of match . 
in contrast , tapur aims to identify signals by maximizing the number of patients who benet from matched therapy.35 the question thus addressed in tapur is , as in our example , are there responses to sunitinib among patients whose advanced biliary carcinomas harbor fgfr2 mutation or amplication ? success in the study is not so rigorously dened , with maximization of the total number of responseshelping patientsbeing the overarching goal . 
 precision and accuracy in basket trials what of the situations in which an moi seems pathogenic but is not on the list ? in the context of a clinical trial , reproducible methods to evaluate and approve or reject novel amois , then , are critical to the scientic integrity and generalizability of these trials . 
not surprisingly , approval processes are a function of the trials purpose . as detailed in figure 1 , in the case of match this means in - depth centralized review of data not available to patients or treating physicians and provided only by partner private referral laboratories . 
clearly , as our experience shows , the goals and , consequently , the designs of precision medicine basket trials are not equal , but our hope is that all can agree on a shared set of values . certain principles ought to be adhered to in any novel amoi approval process . 
statistical purists could trials eligibility credibly say that changing a clinical criteria as it progresses limits validity and generalizability a shaky foundation from which to launch a large , expensive randomized study . 
even when a signal is seen in a given match basket ( which , curiously , sometimes consists of both mutations and fusions ) , delineation of the responding amois in a subsequent randomized study would be required . 
in tapur , increased heterogeneity of the study population would not be a fatal aw and may even enhance the possibility of identifying new signals . second , if we fail to account for the known unknowns of a cancerits temporal and spatial heterogeneity36then even the best - intentioned and well - reasoned therapy may fall short of the mark . 
nonstandard information is needed for approval of a novel amoi in match , and neither match nor tapur incorporates quantication of heterogeneity or subsequent evaluation of genetic biomarkers . returning to the case of fgfr2 fusion in cholangiocarcinoma , if investigators had previously identied divergent signals within the fgfr2 arm of the study , then there would be cause for requesting specic fusion breakpoints before attempting therapy . finally , if novel amois are to be included in a precision medicine basket trial , then the referral process should be transparent . 
an open referral process maximizes the opportunity for benecence and minimizes injustices by reducing the chance of two mutationally equivalent prospective enrollees having different clinical outcomes strictly as a result of the differential data - sharing behaviors of private companies . 
in the case of match , by virtue of the private - public partnership that now serves as the foundation of the trial , the process is heterogeneous , and the subsequent analysis of nucleotide - level data is not transparent . 
expertise is needed to interpret tumor sequencing data , but there is no evidence that a group of experts , even at high - volume centers , is better than a single individual physician who is experienced in reviewing sequencing reports and the relevant literature regarding targeted therapies . 
there are not , to our knowledge , data demonstrating that mtbs improve patient outcomes.37 , 38 moreover , there are real questions about the reproducibility of mtbs from institution to institution . 
ultimately , the novel amoi approval process matters , insofar as it is a manifestation of the research question and a litmus test for a basket trials prioritization of accuracy and precision . 
rigor in our case also applies to the original biomarker , fgfr2 , and combining fusions and point mutations fails to yield generalizable conclusions or provide reassuring negative truth claims . our elds bias toward conating precision and accuracy carries risks , especially in the context of precision medicine trials . 
as early precision medicine trials begin to read out , we must be willing to entertain the possibilities that an overemphasis on precision in design fails to generate knowledge useful for the individual patient case and risks making false - negative conclusions . 
going forward , we recommend sober consideration of the goals of a clinical trialthe best - intentioned attempts to open trials to novel amois can inherently defeat the overarching purpose of a trial . 
finally , in the spirit of understanding that each patient represents a unique opportunity for learning , we should ask ourselves the bigger question : is an essentially hypothesisfree basket trial the best use of nite resources , rare genetic events , and patient effort ? the case , continued at the rst sign of logistical issues , the patient was referred , in parallel , to a local nonacademic center with access to the tapur study , for consideration of sunitinib through that studys fgfr2 arsix weeks after presentation to the developmental therapeutics clinic , the novel amoi was approved by the nci . 
 strohbehn and ratain had been evaluated at the tapur referral site and enrolled in a different clinical trial of fgfr2 inhibitor tas - 120 ( foenix - 101 ; clinicaltrials.gov identier : nct02052778 ) the day prior , within 10 days of his presentation to the tapur referral site . 
ratain consulting or advisory role : ascentage pharma , cyclacel , aptevo therapeutics , shionogi research funding : dicerna ( inst ) , abbvie ( inst ) , genentech ( inst ) patents , royalties , other intellectual property : royalties related to ugt1a1 genotyping for irinotecan , royalties related to ugt1a1 genotyping for irinotecan ( inst ) , pending patent related to a genomic prescribing system , pending patent related to a genomic prescribing system ( inst ) expert testimony : testimony and patent litigation consulting on behalf of multiple generic pharmaceutical companies other relationship : value in cancer care consortium , beigene no other potential conicts of interest were reported . references arai y , totoki y , hosoda f , et al : fibroblast growth factor receptor 2 tyrosine kinase fusions dene a unique molecular subtype of cholangiocarcinoma . hepatology 59 : 1427 - 1434 , 2014 jusakul a , cutcutache i , yong ch , et al : whole - genome and epigenomic landscapes of etiologically distinct subtypes of cholangiocarcinoma . 
cancer discov 7 : 1116 - 1135 , 2017 javle m , lowery m , shroff rt , et al : phase ii study of bgj398 in patients with fgfr - altered advanced cholangiocarcinoma . 
mazzaferro a , el - rayes bf , cotsoglou c , et al : arq 087 , an oral pan - broblast growth factor receptor ( fgfr ) inhibitor , in patients ( pts ) with advanced intrahepatic cholangiocarcinoma ( icca ) with fgfr2 genetic aberrations . 
j clin oncol 35 , 2017 ( suppl 15 , abstr 4017 ) deleon tt , ahn dh , bogenberger jm , et al : novel targeted therapy strategies for biliary tract cancers and hepatocellular carcinoma . 
future oncol 14 : 553 - 566 , 2018 egan jb , marks dl , hogenson tl , et al : molecular modeling and functional analysis of exome sequencing - derived variants of unknown signicance identify a novel , constitutively active fgfr2 mutant in cholangiocarcinoma . 
clin cancer res 24 : 847 - 857 , 2018 sia d , losic b , moeini a , et al : massive parallel sequencing uncovers actionable fgfr2 - pphln1 fusion and araf mutations in intrahepatic cholangiocarcinoma . 
wang y , ding x , wang s , et al : antitumor effect of fgfr inhibitors on a novel cholangiocarcinoma patient derived xenograft mouse model endogenously expressing an fgfr2 - ccdc6 fusion protecancer lett 380 : 163 - 173 , 2016 10 . 
tuxen iv , rohrberg ks , oestrup o , et al : copenhagen prospective personalized oncology ( coppo ) - clinical utility of using molecular proling to select patients 13 . 
von hoff dd , stephenson jj jr , rosen p , et al : pilot study using molecular proling of patients tumors to nd potential targets and select treatments for their 33 : 2753 - 2762 , 2015 to phase i trials . 
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bellmunt j , mullane sa , werner l , et al : association of pd - l1 expression on tumor - inltrating mononuclear cells and overall survival in patients with urothelial carcinoma . 
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wimberly h , brown jr , schalper k , et al : pd - l1 expression correlates with tumor - inltrating lymphocytes and response to neoadjuvant chemotherapy in breast 21 . 
carlson jj , thariani r , roth j , et al : value - of - information analysis within a stakeholder - driven research prioritization process in a us setting : an application in cancer . 
phillips ka , ann sakowski j , trosman j , et al : the economic value of personalized medicine tests : what we know and what we need to know . 
bins s , ratain mj , mathijssen rh : conventional dosing of anticancer agents : precisely wrong or just inaccurate ? clin pharmacol ther 95 : 361 - 364 , 2014 27 . 
ratain mj : body - surface area as a basis for dosing of anticancer agents : science , myth , or habit ? j clin oncol 16 : 2297 - 2298 , 1998 28 . 
giri s , pathak r , martin mg , et al : characteristics and survival of bcr / abl negative chronic myeloid leukemia : a retrospective analysis of the surveillance , epidemiology and end results database . 
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ghazani aa , oliver nm , st pierre jp , et al : assigning clinical meaning to somatic and germ - line whole - exome sequencing data in a prospective cancer precision board . 
however , the interpretation of unexpected cdh1 mutations identified in patients who do not meet igclc criteria or do not have phenotypes suggestive of hereditary diffuse gastric cancer is clinically challenging . 
this study aims to describe phenotypes of cdh1 mutation carriers identified through multigene panel testing ( mgpt ) and to offer informed recommendations for medical management . patients and methods this cross - sectional prevalence study included all patients who underwent mgpt between march 2012 and september 2014 from a commercial laboratory ( n = 26 , 936 ) and an academic medical center cancer genetics clinic ( n = 318 ) to estimate cdh1 mutation prevalence and associated clinical phenotypes . 
cdh1 mutation carriers were classified as igclc positive ( met criteria ) , igclc partial phenotype , and igclc negative . results in the laboratory cohort , 16 ( 0.06% ) of 26 , 936 patients were identified as having a pathogenic cdh1 mutation . 
all three cdh1 mutation carriers who had risk - reducing gastrectomy had pathologic evidence of diffuse gastric cancer despite not having met igclc criteria . conclusion the majority of cdh1 mutations identified on mgpt are unexpected and found in individuals who do not fit the accepted diagnostic testing criteria . 
2017 by american society of clinical oncology introduction mutations in the cdh1 gene cause hereditary diffuse gastric cancer ( hdgc ) , which confers up to an 80% lifetime risk of diffuse gastric cancer ( dgc ) and up to a 60% lifetime risk of female invasive lobular carcinoma ( ilc ) , with the average age at diagnosis being 38 and 53 years , respectively.1 - 3 these high risks , coupled with the difficulty of diagnosing early - stage dgc and current clinical recommendations to consider prophylactic gastrectomy in cdh1 carriers , 4 , 5 highlight the importance and clinical challenges of identifying and managing individuals with cdh1 mutations ( cdh1 + )  . traditionally , genetic testing for cancer susceptibility is performed after the development of a differential diagnosis , including mendelian syndromes caused by a few highly penetrant genes suggested by personal / family history . 
with the advent of multigene panel testing ( mgpt ) , clinicians are able to analyze multiple genes simultaneously , including those lower on the differential diagnosis or not considered at all . 
the international gastric cancer linkage consortium ( igclc ) published guidelines in 2010 ( updated in 2015 ) for identifying individuals as appropriate for cdh1 gene testing4 , 5 ( table 1 ) ; however , multigene panels are now used to test increasing numbers of individuals for cdh1 . we describe a cross - sectional prevalence study of cdh1 + individuals identified by mgpt . 
a retrospective review of two cohorts was performed : the laboratory cohort included all probands , unrelated to our knowledge , who underwent mgpt for cdh1 ( genes analyzed , five to 43 ) at ambry genetics between march 16 , 2012 , and september 30 , 2014 ( n = 26 , 936 ) , and the clinic cohort consisted of all patients who underwent mgpt with cdh1 included in the panel ( genes analyzed , five to 110 ) from april 15 , 2013 , to may 29 , 2014 , at usc ( n = 318 )  . 
for all the patients in the clinic cohort , mgpt was ordered because one or more of the genes included in the panel was the primary target and part of the differential diagnosis . 
no overlap existed between the cohorts in patients who tested positive for cdh1 , and patients who tested negative for cdh1 mutations shared by usc and ambry were excluded from ambrys count . 
for the laboratory cohort , data were abstracted from test requisition forms and by contacting the ordering provider to obtain further pathology , personal and family history , testing information on family members , and outcomes data . 
subsequent blinded classifications were repeated by clinicians at ambry and usc . cdh1 + cases were classified into three categories on the basis of the 2010 igclc guidelines4 : igclc positive ( iglcc - pos ) was defined as mutation carriers who met igclc criteria ; igclc partial phenotype ( igclc - pp ) was defined as mutation carriers who did not meet igclc criteria , but hdgc was in their differential diagnosis because of the presence of gastric cancer , age older than 40 years , or ilc at any and igclc negative ( igclc - neg ) was defined as mutation carriers who did not meet igclc criteria and had no gastric cancer or ilc present in the family . 
myrisk manufactured by myriad genetics ( salt lake city , ut ) ; colonext , brcaplus , breastnext , and cancernext manufactured by ambry genetics ( aliso viejo , ca )  . abbreviations : bc , breast cancer ; bil breast , bilateral breast cancer ; crc , colorectal cancer ; gf , grandfather ; ggf , great grandfather ; ggm , great grandmother ; gm , grandmother ; gyn , gynecologic cancer ; h / n , head and neck cancer ; idc , invasive ductal breast cancer ; igclc , international gastric cancer linkage consortium ; lcis , lobular carcinoma in situ ; mat , maternal ; neg , negative ; nos , not otherwise specified ; pat , paternal ; pos , positive ; pp , partial phenotype ; rcc , renal cell carcinoma ; ua , unaffected . * family members are untested unless otherwise indicated . 
unaffected relatives who tested negative were excluded from this table . case was further reviewed on the basis of the 2015 guidelines.5 e - cadherin immunohistochemistry ( ihc ) staining was conducted on invasive ductal carcinoma ( idc ) tissue when feasible . results in the laboratory cohort , 0.06% ( 16 of 26 , 936 ) of patients were cdh1 +  . 
case cc4 was classified as igclc - pos as a result of family history ; however , the probands presentation was atypical because she was given a diagnosis of idc at age 44 years . 
 a breast 25 colon / rectum 70 ovary 29 breast 48 stomach 66 cdh1 + stomach 52 stomach stomach 71 ovary 40 colon / rectum 71 breast 44 cdh1 + cdh1 + breast 44 cdh1 + prostate 44 stomach 46 stomach 38 colon / rectum 43 cdh1 + clinic case 1 ( cc1 ) classification : igclc - neg result : cdh1 del exons 1 - 2 medical management : recommended prophylactic gastrectomy deferred until completion of breast cancer treatment pathology : ductal breast cancer ihc : expression of cdh1 clinic case 2 ( cc2 ) classification : igclc - pp result : cdh1 c.2164 + 1g > a pathology : lobular breast cancer medical management : prophylactic gastrectomy identified stage i diffuse gastric cancer clinic case 4 ( cc4 ) classification : igclc - pos result : cdh1 c.504del medical management : recommended prophylactic gastrectomy not completed initial pathology : ductal breast cancer ihc : lack of expression of cdh1 ; breast cancer reclassified as lobular e - cadherin immunostain : tumor cells strongly positive for e - cadherin e - cadherin immunostain : negative in tumor cells with positive internal control fig 1 . 
two cases were igclc - pp , and one was igclc - neg because the proband had idc ( confirmed on ihc ) , no family history of cancer , and complete family structure ( fig 1 )  . overall , 20 pathogenic cdh1 mutations were identified . 
one of the probands was unaffected . of patients with cancer , 42% ( n = 8 ) presented with ilc and 21% ( n = 4 ) with gastric cancer consistent with the expected phenotype ; however , 21% ( n = 4 ) presented with idc , 5% ( n = 1 ) with idc with lobular features , 5% ( n = 1 ) with breast neoplasm not otherwise specified , and 5% ( n = 1 ) with colon cancer ( fig 2a )  . 
cases were reviewed to determine whether they met the 2015 guidelines , and case lc17 , initially classified as igclc - pp , met the revised testing criteria as a result of a diagnosis of bilateral ilc at age 45 years.5 therefore , 65% of cases did not meet the 2015 guidelines for consideration of testing . mutation description sixteen distinct pathogenic / likely pathogenic cdh1 alterations were detected in 20 cdh1 + probands , with three recurrent mutations identified ( fig 3 ; data supplement )  . 
a variety of mechanisms lead to pathogenicity , including nonsense - mediated decay6 of mrnas that contain premature termination codons.7 , 8 for cdh1 , nonsense - mediated decay has been shown to cause downregulation of alleles , which results in loss of function.7 of the mutations described , seven are novel to our knowledge ( fig 3 )  . 
both canonical variants are predicted to abolish the native donor splice site by four different splicing prediction tools ( human splicing finder [ hsf ] , maxentscan , berkeley drosophila genome project [ bdgp ] , and esefinder ) , and were classified as likely pathogenic as a result of lack of additional clinical evidence . the six previously described mutations were classified as pathogenic . 
the four different splicing prediction tools ( hsf , maxentscan , bdgp , and esefinder ) predict that these two variants will abolish the native donor site , but we cannot exclude that the use of alternative cryptic donor sites may affect the expressivity of the phenotype in these cases . we detected four multiexon deletions : three ( 59utr_in2del and ex1_2del twice ) include the transcription and translation start sites , and the fourth ( in2_ex5del ) is predicted to include 1 , 237 nucleotides of coding sequence and to be out of frame . 
two different deletions encompassing exons 1 to 2 were previously reported in three families with hdgc ; however , we cannot confirm whether the deletions of this region in the current cohort have the same breakpoints as those previously reported.18 to our knowledge , these deletions contain the only functional translation start sites in the gene and therefore are predicted to abrogate protein synthesis . 
the two families in this cohort with the deletion of exons 1 to 2 were classified as igclc - neg , the family with the deletion that spans from the 59utr through intron 2 was classified as igclc - pp , and the family with the intron 2 through exon 5 deletion was classified as igclc - pos ( data supplement )  . clinical follow - up all cdh1 + clinic cohort patients underwent a discussion of management consistent with hdgc guidelines , 5 and the testing of at - risk family members for the cdh1 mutation was recommended ( subsequent family testing data listed in table 2 )  . one of the four clinic cohort patients elected to proceed with prophylactic gastrectomy . 
for the other three clinical cases , two patients declined prophylactic surgery at this time , and one elected to wait until cancer treatment was completed to make a decision about prophylaxis . 
for one of the laboratory cohort patients who underwent a prophylactic gastrectomy , a family member was seen at usc after identification of the mutation , which allowed additional information to be obtained ( lc12 )  . 
in 15% of patients ( three of 20 [ cc2 , lc12 , and lc6 ] ) , either the proband or the family member is known to have pursued prophylactic gastrectomy and were found to have dgc ( table 3 )  . 
patient lc12 ( proband had idc at age 36 years ) was initially classified as igclc - neg on the basis of a family history of lobular carcinoma in situ in a seconddegree relative ( maternal aunt ) with unknown age of onset and no documented family history of gastric cancer . 
although some of the mutations in the current study have been documented in highly penetrant families , others have not and , therefore , may represent less - penetrant mutations . 
because the threshold for testing for cdh1 changes over time , it is reasonable to anticipate that the cancer risks will have a wider range as families who present outside the high - risk criteria are ascertained , which is consistent with what has been derived in other well - studied cancer predisposition syndromes , such as hereditary breast and ovarian cancer syndrome.25 variable expressivity for cdh1 mutations has been demonstrated in studies of cdh1 + patients with ilc and / or a family history of ilc in the absence of dgc.9 , 14 , 26 consideration of ilc was added to the revised guidelines published in 20155 ( table 1 )  . 
detailed family histories that require deep investigation and retrieval of pathology reports from distant or deceased relatives are helpful when available , and post hoc review of family histories in this study identified previously unconfirmed gastric cancer and lobular breast cancers that changed the classification of risk for some patients who had undergone testing . current management guidelines encourage prophylactic gastrectomy between ages 20 and 30 years and annual breast magnetic resonance imaging starting at age 30 years.4 , 5 these recommendations are based on the cancer risk estimates derived from high - penetrance cdh1 + families . the unexpected cdh1 mutations may reflect an table 3 . 
counseling and support balanced by considerations of risks , benefits , and costs are integral to partnering with these patients for shared decision making about optimal management strategies and their timing . 
the option of gastrectomy in igclc - pp families typically is framed in the context of the family history of cancer , which underscores the importance of obtaining a complete family history and review of pathology reports when available . 
as genomics is further integrated into clinical practice , the gathering of families into research registries is important for long - term follow - up to expand the clinical understanding of cdh1 mutations , including range of cancer risk , and appropriate medical management across the phenotypic spectrum . in conclusion , mgpt identifies cdh1 + individuals who do not meet criteria for cdh1 testing . 
mgpt may provide an opportunity to identify individuals with an increased risk for a highly morbid cancer ( dgc ) before they present with advanced disease , even in the absence of suggestive family history . many of these families presented with cancer histories more suggestive of hereditary breast and ovarian cancer because of early - onset idc , and testing previously may have been limited to the brca genes , which raises the question of whether genetic testing criteria should be broadened to identify more cdh1 + patients . 
at this time , whether the cancer risks in the incidentally identified carriers match the levels reported for classic hdgc families is unclear , although in this series , 100% of cdh1 + patients who underwent gastrectomy had gastric cancer . 
in the absence of comprehensive data on penetrance , we currently recommend classic hdgc management guidelines for all cdh1 mutation carriers.4 , 5 published online on po.ascopubs.org on march 29 , 2017 . immediate family member , inst = my institution . 
espenschied employment : ambry genetics duveen sturgeon no relationship to disclose charit e ricker no relationship to disclose rachid karam employment : ambry genetics research funding : ambry genetics holly laduca employment : ambry genetics julie o . 
 elizabeth chao employment : ambry genetics leadership : ambry genetics stock and other ownership interests : ambry genetics consulting or advisory role : premier genomics julia sturgeon no relationship to disclose virginia speare employment : ambry genetics yanling ma no relationship to disclose kerry kingham no relationship to disclose affiliations marilena melas no relationship to disclose gregory e . 
pharoah pd , guilford p , caldas c : incidence of gastric cancer and breast cancer in cdh1 ( e - cadherin ) mutation carriers from hereditary diffuse gastric cancer families . 
j med genet 47 : 436 - 444 , 2010 [ erratum : j med genet 48 : 216 , 2011 ] van der post rs , vogelaar ip , carneiro f , et al : hereditary diffuse gastric cancer : updated clinical guidelines with an emphasis on germline cdh1 mutation carriers . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
karam r , carvalho j , bruno i , et al : the nmd mrna surveillance pathway downregulates aberrant e - cadherin transcripts in gastric cancer cells and in cdh1 mutation carriers . 
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suriano g , yew s , ferreira p , et al : characterization of a recurrent germ line mutation of the e - cadherin gene : implications for genetic testing and clinical management . 
norton ja , ham cm , van dam j , et al : cdh1 truncating mutations in the e - cadherin gene : an indication for total gastrectomy to treat hereditary diffuse gastric cancer . 
kim s , chung jw , jeong td , et al : searching for e - cadherin gene mutations in early onset diffuse gastric cancer and hereditary diffuse gastric cancer in korean patients . 
brooks - wilson ar , kaurah p , suriano g , et al : germline e - cadherin mutations in hereditary diffuse gastric cancer : assessment of 42 new families and review of genetic screening criteria . 
antoniou a , pharoah pd , narod s , et al : average risks of breast and ovarian cancer associated with brca1 or brca2 mutations detected in case series unselected for family history : a combined analysis of 22 studies . 
 evolving landscape of molecular prescreening strategies for oncology early clinical trials rodrigo dienstmann , md1 ; elena garralda , md , msc2 ; susana aguilar , phd1 ; gemma sala , msc2 ; cristina viaplana , msc1 ; fiorella ruiz - pace , msc1 ; jenifer gonz alez - zorelle , bsc1 , 3 ; deborah grazia logiacco , phd3 ; zighereda ogbah , bsc3 ; laia ramos masdeu , phd3 ; francesco mancuso , bsc3 ; roberta fasani , md4 ; jose jimenez , bsc4 ; paola martinez , bsc4 ; ana oaknin , md , phd2 ; cristina saura , md , phd2 ; mafalda oliveira , md , phd2 ; judith balmaa , md , phd2 ; joan carles , md , phd2 ; teresa macarulla , md , phd2 ; elena elez , md , phd2 ; maria alsina , md , phd2 ; irene braa , md , phd2 ; enriqueta felip , md , phd2 ; josep tabernero , md , phd2 ; jordi rodon , md , phd2 , 5 ; paolo nuciforo , md , phd4 ; and ana vivancos , phd3 most academic precision oncology programs have been designed to facilitate enrollment of patients in early clinical trials with matched targeted agents . 
we started with a tumor - agnostic hotspot mutation panel plus in situ hybridization and immunohistochemistry of selected markers and subsequently transitioned to tumor - specic amplicon - based next - generation sequencing ( ngs ) tests together with custom copy number , fusion , and outlier gene expression panels . 
in parallel , biomarker - matched trials evolved from a scenario of few targets and large populations ( such as pi3k inhibitors in pik3ca mutants ) to a complex situation with many targets and small populations ( such as multiple targetable fusion events )  . 
the substantial increase of immunotherapy trials had a major impact in target prioritization and guided clinical implementation of new markers , such as tumor mutational burden , with larger exonbased ngs assays and gene expression signatures to capture microenvironment inltration patterns . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction during the last decade , when targeted agents entered the phase i clinical trial arena and next - generation sequencing ( ngs ) became a standard molecular prescreening test to identify actionable alterations , oncologists experienced a major shift toward biomarkerdriven drug development.1 there was a lot of enthusiasm with this approach , which culminated with the approval of many targeted drugs in molecularly selected patients on the basis of nonrandomized data . 
more recently , another important milestone was reached with the approval of tissue - agnostic therapies in biomarker - selected population , rst the immune checkpoint inhibitor pembrolizumab in microsatellite instable ( msi ) tumors and then trk inhibitors larotrectinib and entrectinib in cancers harboring ntrk fusions . 
given these exciting advances , many reference institutions initiated precision oncology programs with longitudinal patient cohorts undergoing tumor molecular proling nested to early clinical trials with matched targeted agents.2 in parallel , as advances in immuno - oncology are changing the standard of care of many cancer types , the phase i oncology community experienced another paradigm shift in drug development , with an unprecedented number of new investigational agents entering clinical testing.3 not only different antipd - 1 / l1 inhibitors but also multiple combination regimens are being investigated in early clinical trials . 
more recently , genomic biomarkers are inclusion criteria to select patients for immune checkpoint inhibitor therapy , such as tumor mutation burden ( tmb ) and mutations in dna damage repair ( ddr ) pathway genes . in this evolving scenario , early clinical trial units had to constantly adapt to new biomarker - drug codevelopment trends . 
because the molecular prescreening program was established at our institution , we customized the techniques and procedures to accurately identify molecular alterations in tumors from patients eligible for early clinical trials . 
the team meets periodically to discuss existing molecular tests and new biomarkers of interest to be added to our prescreening progracancer biologists and genomicists participate in weekly molecular tumor board meetings with medical oncologists to provide guidance on the interpretation of ngs results and discuss new markers for clinical testing in patients eligible for early clinical trials . as detailed in figure 1a , we started with a targeted single base extension mutation assay covering hotspot events in 20 oncogenes and tumor suppressor genes ( sequenom ) plus uorescent in situ hybridization ( fish ) or immunohistochemistry ( ihc ) analysis of selected genes and proteins . 
in 2013 we implemented an ncounter rna platform ( nanostring ) fusions and gene expression ( gex ) analysis of 26 genesfusion and gex ncounter . in 2014 we substituted the hotspot mutation panel with a custom amplicon - based ngs assay covering 59 genes ( miseq ) , and 1 year later we replaced the fish analysis with a targeted copy number ncounter dna panel of 44 genescna ncounter . 
during 2015 we also added brca1 / brca2 genes to the ngs panel , introduced pdl1 ihc and msi testing by ihc ( with polymerase chain reactionbased test in equivocal cases )  . 
in 2018 we developed an exonbased ngs assay ( hiseq ) with 420 genes , encompassing ddr plus epigenetic pathway genes and ne - tuned for tmb quantication and other genomic signatures , such as msi . 
so far , this assay is limited to selected cases of particular interest , as per discussions in weekly molecular tumor boards , but will eventually replace the amplicon ngs and nanocopy dna panels in the coming year . 
to identify patients eligible for antibody - drug conjugates , different ihc assays were developed in collaboration with pharmaceutical companies , which allowed local testing instead of sample shipment for central proling . 
most pharmaceutical companies also accept our in - housedeveloped genomic testing as screening for biomarker - guided trials , but we noticed that a growing number of early clinical trials mandate central ngs companion diagnostic tests , specically in lung , bladder , and biliary tract cancers . all assays were optimized for archived formalin - xed parafn - embedded tumor tissues . 
importantly , ngs assays had frequent modications in gene coverage to capture emerging biomarkers being investigated in clinical trials at our institution , such as the recent additions of notch family genes to the mutation panel , ntrk breakpoints to the fusion assay , and cd274 ( pd - l1 ) to the cna ncounter test . 
in 2018 , the amplicon - based ngs panel was tailored as multiple tumortypespecic panels , with coverage limited to genes that have been previously found to be mutated in each malignancy . 
finally , circulating tumor dna ( ctdna ) mutation detection panels are under development but still limited to patient cohorts with predened tumor types participating in validation studies . each oncologist has the responsibility to dene a patients eligibility for molecular prescreening on the basis of clinical features , tumor type , and disease setting . 
cna ncounter , copy number alteration nanostring panel ; fish , uorescent in situ hybridization ; fusion and gex ncounter , fusion and gene expression nanostring panel ; ihc , immunohistochemistry ; msi , microsatellite instability ; ngs , next - generation sequencing . yield ( combined prevalence of targetable molecular alterations ) exceeds 3% . 
depending on the tumor type , molecular tests are performed up front ( such as in lung cancer when sufcient tissue is available for ngs ) , while patients receive standard - of - care chemotherapies ( such as biliary tract cancer and hormone receptorpositive breast cancer ) , or after progression to approved regimens ( such as cervical cancer )  . 
we educate clinicians not to indicate ngs as a rescue diagnostic test to identify targets for experimental therapies when patients have fast clinical deterioration , but also to avoid the all - comers up - front strategy , given the potential reduced cost effectiveness of this approach for all advanced tumor types . 
 dienstmann et al after the patient signs informed consent for broad tumor molecular proling , requests are submitted to the molecular oncology unit for sample retrieval , qualication , and preparation , and to perform ihc / fish tests . 
turnaround time is , 1 week for ihc or fish tests and approximately 2 weeks for the amplicon - based ngs panel ; ncounter results are reported within 2 - 3 weeks and exon - based ngs panel in 3 - 4 weeks . 
depending on the assays coverage , it serves as a substitute for the internal program . our molecular prescreening program is free of charge to patients and nanced with both internal resources acquired through competitive grants or donations ( institutional patronage ) and external funds from agreements with pharmaceutical companies running early clinical trials with mandatory biomarker matches . 
in the latter case , which represents 15% of total budget , the conancing models can be a molecular prescreening fee for each patient recruited in the trial or a pay - per - test fee while the trial is recruiting patients ( with monthly reports detailing number of tests performed and positive results )  . as shown in figure 1b , close to 2 , 500 molecular tests were performed in 2018 , a 10 - fold increase as compared with 2010 . 
in 2010 , most early therapeutic trials tested pi3k pathway inhibitors ( n = 11 ) , whereas in 2018 , immunotherapy trials represented the great majority ( n = 75 ) , followed by antibody - drug conjugates ( n = 9 ) , epigenetics ( n = 9 ) , fgfr inhibitors ( n = 9 ) , pi3k pathway inhibitors ( n = 8 ) , mek / erk inhibitors ( n = 8 ) , and egfr / erbb2 inhibitors ( n = 7 )  . 
regarding phase i trials with targeted drugs ( fig 2a ) , we observed a gradual increase in the number of targets of interest for clinical development from 2010 to 2016 , including alk / ros1 , fgfr1 - 3 , braf , met , notch1 - 2 , ddr pathways alterations , and others ( hdm2 , kit , pdgfr , idh1 - 2 , ret , ntrk1 - 3 , cyclin , and wnt pathways )  . 
this was accompanied by a major increase in the number of immunooncology trials , which escalated since 2015 ( fig 2b )  . the criteria for patient enrollment in a given trial go beyond genomic markers . 
in cases of borderline evidence of clinical actionability of molecular targets ( enrichment criteria ) or no oncogene alteration , alternative therapies are considered , type and taking into consideration tumor availability of slots in trials with immunotherapies and other drugs . 
more recently , immune - related markers are used as positive selection criteria for clinical trials with novel immune - oncology drugs and combinations . out of 161 early clinical trials actively recruiting patients in 2018 , 57 investigated targeted agents ( fig 2a ) , 24 of them ( 42% ) were combination regimens , and 48 ( 84% ) had mandatory or enrichment molecular inclusion criteria , the most common being fgfr , mapk , pi3k , egfr / erbb2 , and ddr pathway alterations . 
regarding immunotherapy trials , 57 out of 75 ( 76% ) were combination regimens and 32 ( 43% ) had mandatory or enrichment molecular criteria for recruitment , with pd - l1 expression and msi status being the most common biomarkers ( figs 2b and 2d )  . 
we also gradually increased the number of clinical trials testing antibody - drug conjugates , most of them with mandatory ihc analysis for patient selection , and epigenetic targets , rarely recruiting patients on the basis of genomic proling ( as brd4 - nut fusions for bet inhibitors )  . inclusions in clinical trials with targeted agents uctuated from 2010 to 2018 ( fig 2c )  . 
the multiplicity of drug targets under investigation coupled with a growing number of molecular tests being performed during this period allowed recruitment of more patients in phase i including trials assessing novel biomarkers , fgfr1 - 3 , notch1 - 2 alterations , and brca1 - 2 mutations , among others . 
we observed a major decrease in recruitment rates in phase i trials with targeted agents in 2017 , in part related to lower interest in some targets , such as pi3k pathway inhibitors , but also a 3 - fold increase in the number of patients referred to phase ii or iii trials with molecular matches , from 84 in 2016 to 224 in 2018 . 
chemo , chemotherapy ; immuno , immunotherapy ; msi , microsatellite instability . 306 patients recruited in immunotherapy trials during 2018 , 92 ( 30% ) were based on biomarkers . to investigate the clinical utility of our program , we assessed how the results of proling ( both in - house and external ) affected the immediate treatment decision of each patient . 
the most common tumor types undergoing molecular proling in the last 3 years are shown in figure 3a . in 2018 , we noticed an increase in lung , breast , and pancreatobiliary cancers being tested in line with major advances in precision cancer therapy across these tumor types . 
it is important to emphasize that these represent patients not eligible for standard - of - care genomically guided therapy , such as braf inhibitors in brafv600emutated melanomas , or her2 - targeted therapy in erbb2amplied breast cancer . 
 a 2016 ( n = 1 , 281 ) 18 19 26 dienstmann et al 2017 ( n = 1 , 218 ) 15 22 18 1 , 104 2018 ( n = 1 , 416 ) 18 12 18 colorectal lung breast pancreatobiliary gynecologic gastric head and neck other brain urinary sarcoma skin endocrine inner ring no trial immunotherapy nonimmunotherapy matched phase 1 - 3 trial outer ring biomarker mandatory biomarker enrichment fig 3 . 
 ( b ) recruitment in biomarker - guided ( mandatory or enrichment ) and other alternative trials ( immunotherapy or nonimmunotherapy )  . guided targeted agents , antibody - drug conjugates , and immunotherapies . discussion the vall dhebron institute of oncology ( vhio ) molecular prescreening program is constantly adapting to the needs of our early clinical trial portfolio . 
we observed a dramatic change in the landscape of phase i clinical trials , now focused on targeted agents for rare molecular alterations , immunotherapy combinations , antibody - drug conjugates , and epigenetic agents . 
in parallel , different ngs assays were developed for accurate detection of rare actionable mutations , copy number alterations , fusion events , and novel ihc tests were introduced . from the beginning , we followed a personalized prescreening approach , whereby oncologists are empowered to decide which patients are eligible for testing based on clinicopathological features and which molecular tests are indicated in each disease setting . 
by avoiding the one - testfor - all strategy , we were able to optimize the use of our prescreening program and educate the clinicians on molecular epidemiology of each tumor type as well as emerging diagnostic tests . 
the decision to transition from smaller tumor - specic panels to a larger exon - based ngs that covers both mutation and copy number tests is related to the need to accommodate emerging biomarkers in the ddr and epigenetic pathways as well as tmb quantication . 
overall , 1 out of 10 patients proled are ultimately enrolled in matched targeted trials , which is in line with other academic molecular prescreening programs with nested clinical studies.4 regarding germline sequencing , we follow guideline - directed testing and have regular meetings with genetic counselors to discuss secondary incidental ndings in tumor sequencing.5 in the last decade , biomarker - matched trials evolved from a scenario of few targets and large populations to a complex situation with many targets and small populations . 
even with a growing number of targets being investigated in phase i trials , and the multiplicity of trials per target , we observed a relative reduction in the number of patients enrolled in molecularly guided trials in the last years . 
this is the result of shifting interests in drug development and a clear focus on a limited number of promising biomarkers , such as rare fusion events in fgfr1 - 3 , ret , and ntrk1 - 3 genes . 
in this scenario , liquid biopsies for target identication are becoming quite useful , which has guiding our current development of a ctdna ampliconbased ngs test and prompted collaborations with commercial partners for molecular prescreening . the substantial increase in immunotherapy trials had a major impact on trial prioritization . 
inclusion criteria of immuno - oncology phase i trials are frequently limited to tumor type and treatment line , with few trials having a mandatory molecular match , which facilitates patient recruitment . 
we noticed an increase in biomarker - guided immunotherapy trial recruitment in 2018 , which is guiding implementation of a multimodality biomarker strategy that moves beyond tmb quantication , such as immune cytotoxic / suppressive microenvironment signatures measured through the gex ncounter panel , 6 as well as multiplex ihc panels.7 to conclude , we believe that to accelerate progress in precision oncology , clinical trials with adaptive designs to enroll patients on the basis of multiomics enrichment criteria are needed . 
the previous era of molecularly guided targeted agents is restrictive , and larger portfolios of drugs that include immunotherapeutic and antibody - drug conjugates with recruitment guided by molecular tests must be pursued . 
in our view , the future of cancer drug development will encompass sequential genomic proling to guide matched targeted therapies , complex multimodality biomarkers for immune - oncology agents , individualized immunotherapeutics , novel combination regimens with epigenetic drugs , and antibody - drug conjugates . 
as precision oncology becomes a more paved road , clinicians in the community oncology setting must be cognizant of its full potential and reference institutions must establish patient referral networks to increase the access to innovative experimental therapies . 
 dienstmann et al speakers bureau : msd , roche , thermo fisher scientic research funding : novartis , roche ( inst ) , thermo fisher scientic ( inst ) travel , accommodations , expenses : bristol - myers squibb , menarini , glycotope , merck sharp & dohme ana oaknin consulting or advisory role : roche , astrazeneca , pharmamar , clovis oncology , tesaro , immunogen , genmab research funding : abbvie deutschland ( inst ) , ability pharmaceuticals ( inst ) , advaxis ( inst ) , aeterna zentaris ( inst ) , amgen ( inst ) , aprea therapeutics ( inst ) , clovis oncology ( inst ) , eisai ( inst ) , f . 
mandelker d , donoghue m , talukdar s , et al : germline - focussed analysis of tumour - only sequencing : recommendations from the esmo precision medicine working group . 
lu s , stein je , rimm dl , et al : comparison of biomarker modalities for predicting response to pd - 1 / pd - l1 checkpoint blockade : a systematic review and meta - analysis . 
calvo f , apolone g , baumann m , et al : cancer core europe : a european cancer research alliance realizing a research infrastructure with critical mass and programmatic approach to cure cancer in the 21st century . 
we then used polymerase chain reaction to quantify gene expression in diagnostic biopsy tissue across a prospectively designed archival cohort of 754 consecutive thinand intermediate - thickness primary cutaneous melanomas . 
a penalized maximum likelihood estimation algorithm was used to train logistic regression models in a repeated cross - validation scheme to predict the presence of sln metastasis from molecular , clinical , and histologic variables . results expression of genes with roles in epithelial - to - mesenchymal transition ( glia - derived nexin , growth differentiation factor 15 , integrin - 3 , interleukin 8 , lysyl oxidase homolog 4 , transforming growth factorreceptor type 1 , and tissue - type plasminogen activator ) and melanosome function ( melanoma antigen recognized by t cells 1 ) were associated with sln metastasis . 
per current guidelines ( table 1 ) , slnb is not recommended if the risk of nodal metastasis is , 5% , as in melanoma with a breslow thickness of , 0.8 mm and no adverse features . 
the key objective of this study was to identify primary melanoma clinicopathologic ( cp ) variables and a gene expression prole ( gep ) that associate with a low risk of sln metastasis . knowledge generated cp variables in combination with an eight - gene gep tied to epithelial - to - mesenchymal transition as a biologic process inherent to metastasis effectively stratied melanoma according to its likelihood of sln metastasis . relevance our cp - gep model promises to work as an sln biopsy reduction tool . 
10% risk of shortand long - term complications , including bleeding , infection , lymphocele , lymphatic stula , pain , neuropathy , and lymphedema , 7 as well as an up to 5% risk of hospital readmission within 30 days because of postsurgical complications.8 better methods are needed to identify patients whose risk of nodal metastasis is so low that they may safely forgo slnb . 
here , we report the design of a model that combines established clinicopathologic ( cp ) variables with a gene expression prole ( gep ) to identify patients who have , on average , a risk of nodal metastasis of , 5% . 
of the 754 patients in this cohort , 373 were included in a previously published cohort.10 all specimens were analyzed by quantitative polymerase chain reaction ( pcr ) between february 2018 and october 2018 . eligibility was determined on the basis of histopathology data derived from patient medical records and established by two or more board - certied mayo clinic dermatopathologists . inclusion was determined by the ajcc 7th edition on the basis of institutional practice guidelines of the mayo clinic for recommending slnb , which were based on breslow thickness , ulceration , mitoses , and age . 
 molecular model to assess sentinel lymph node metastasis risk exclusion criteria were m1 disease within 90 days of primary diagnosis ; insufcient primary tumor diagnostic biopsy tissue ; inadequate rna harvested ; and , for minnesota , denial of access to medical records for research purposes ( per minnesota state law )  . 
because there is an ongoing debate about the relevance of , 0.1 mm metastasis in sln ( ie , isolated tumor cells [ itcs ] and cell clusters , 0.1 mm in diameter ) , patients with , 0.1 mm metastasis were excluded from model development . 
isolated benign melanocytes and histiocytic melanophages can be present elsewhere in the sln and mimic itcs.13 some authors cautioned against hidden tumor burden in , 0.1 mm metastatic sln and highlighted the need for enhanced pathology assessment protocols.14 , 15 others found that , 0.1 mm metastasis has no impact on prognosis compared with negative slns.16 , 17 enrollment of patients and inclusion and exclusion criteria are summarized in appendix figure a1 . 
this study was approved by the mayo clinic institutional review board . gene expression by quantitative pcr see the appendix for details . statistical methods logistic regression and least absolute shrinkage and selection operator . 
features with a tolerance 0.15 were removed from the input data set ( the tolerance represents the fraction of variance in the kth feature that cannot be accounted for by other features )  . 
the output of logistic regression models estimated the probability of sln metastasis and was converted into binary results : samples with a probability of metastasis greater than the cutoff were classied as positive , whereas samples with a probability lower than the cutoff were classied as negative . 
the performance metrics of the classiers are listed in appendix table a2 and are cutoff specic , except the area under the receiver operating characteristic curve ( auc )  . double - loop cross - validation . 
however , splitting the available data just once into a training set and a test set may be viewed as inefcient.19 a better solution is to estimate the average performance of the model by repeated cross - validation or bootstrapping . 
here , we opted for a repeated crossvalidation scheme ( ie , double - loop cross - validation [ dlcv ] ) .20 the key idea of dlcv is to get a reliable estimate of the outof - sample performance of a classier by averaging the performance of multiple classiers trained in cross - validation a number of times ( appendix fig a2 )  . 
see the appendix for details . memorial sloan kettering cancer center nomograsee the appendix for details . results epithelial - to - mesenchymal transition in high - risk melanoma to identify candidate genes tied to biologic processes inherent to metastasis and differentially expressed between metastatic and nonmetastatic melanoma , we rst reviewed rna sequencing data obtained previously.10 genes with a false discovery rate of , 0.01 in a comparison of either benign nevi and cutaneous melanoma or cutaneous melanoma with and without sln metastasis were selected for further qualication . 
a total of 194 candidate biomarkers and 3 control genes were screened for performance in breslow thickness and age - matched case - control studies by quantitative pcr ( appendix table a3 )  . 
we noted that genes predictive of nodal metastasis had been associated with epithelial - to - mesenchymal transition ( emt ) , a biologic process known to promote metastasis in primary cutaneous melanoma.21 our prospectively designed archival cohort22 comprised 754 patients with thinand intermediate - thickness primary cutaneous melanoma who underwent an slnb within 90 days of diagnosis ( table 2 )  . 
of 754 patients , 128 ( 17% ) were sln positive , in agreement with the typical prevalence in an slnb - eligible population.3 our approach was to develop models of the likelihood of sln metastasis on the basis of either cp variables ( cp models ) or geps of the primary tumor ( gep models ) and then to assess the performance of a combined model of cp and gep factors ( cpgep models )  . 
 ( % ) characteristic other mixed not documented negative ( n = 626 ) positive ( n = 128 ) 4 of 626 ( 0.6 ) 6 of 626 ( 1.0 ) 31 of 626 ( 5.0 ) 2 of 128 ( 1.6 ) 6 of 128 ( 4.7 ) abbreviations : sd , standard deviation ; slnb , sentinel lymph node biopsy . acomparisons of patients with slnb - negative and - positive outcomes were performed using the 2 test for categorical variables , the twosample t test for patient age , and the wilcoxon rank sum test for all other variables . using gene expression to predict sln metastasis dlcv and lasso were used to identify a gep dened from 11 genes that differentiated the patients with and without nodal metastasis detected by slnb within 90 days of primary diagnosis : adam metallopeptidase domain 12 ( adam12 ) , interleukin 8 ( cxcl8 ) , growth differentiation factor 15 ( gdf15 ) , integrin - 3 ( itgb3 ) , galectin 1 ( lgals1 ) , lysyl oxidase like 4 ( loxl4 ) , melanoma antigen recognized by t cells 1 ( mlana ) , tissue - type plasminogen activator ( plat ) , protein kinase c ( prkcb ) , glia - derived nexin ( serpine2 ) , and transforming growth factor ( tgf ) receptor 1 ( tgfbr1 )  . 
finally , logistic regression modeling was used to develop a novel model combining cp factors ( ie , breslow thickness and patient age ) and a gep . the combined cp - gep model was based on the expression of mlana , a melanosome marker , 23 and seven genes functionally linked to emt and with specic roles in angiogenesis / hypoxia and coagulation : gdf15 , 24 - 26 cxcl8 , 27 , 28 loxl4 , 29 , 30 tgfbr1 , 31 , 32 itgb3 , 33 - 36 plat , 37 , 38 and serpine239 , 40 ( table 3 )  . 
the cp - gep model , therefore , promised to work as an slnb reduction tool : patients with a negative test may forgo slnb because their risk of nodal metastasis is , on average , , 5% , a reduction from the pre - test probability41 ( table 1 )  . for a predictor of sln status to be clinically relevant , it must change the pretest probability within each t category of melanoma . 
the high slnb reduction rate for t1b melanoma is particularly meaningful in light of the increasing incidence of thinner melanoma , 42 for which cp variables are less predictive.5 to further dene the clinical relevance of the cp - gep model , we compared cp - gep performance to the wellknown memorial sloan kettering cancer center ( mskcc ) nomogram for predicting sln metastasis . 
shown are the models that are based on the mskcc nomogram , clinicopathologic variables ( cp model ) , and gene expression prole ( gep ) and cp variables combined ( cp - gep model )  . 
curves are averages over 100 repeats obtained by concatenating the threefold crossvalidation test results . that the mskcc nomogram performed similarly to the cp model but was outperformed by the cp - gep model in auc ( appendix fig a5 ) and slnb reduction rate ( fig 2 )  . discussion while completion lymphadenectomy for which slnb was a key determinant has fallen out of favor , 43 , 44 slnb continues to determine patient eligibility for adjuvant therapy . unfortunately , the majority of slnb procedures performed today are negative , which conrms only the low - risk nature of the primary tumor without inuencing decision making toward adjuvant therapy . 
here , we present a model that considers gene expression and cp variables ( ie , breslow thickness and patient age ) to assess the likelihood of sln metastasis in patients diagnosed with thinand intermediate - thickness primary cutaneous melanoma . 
the ability to characterize melanoma at the molecular level reduces the need for slnb , a surgical procedure that carries a risk of complications.7 our approach of combining cp factors with molecular proling better identies patients who may forgo the slnb procedure because of their low risk of metastasis . for melanoma with a 5% - 10% chance of sln metastasis ( breslow thickness , 0.8 - 1 mm ) , slnb is optional but should be discussed with the patient.45 even though slnb in this risk group is optional ,  . 
50% of affected patients in the united states undergo slnb.46 the majority of these patients have negative slnb ndings , which highlights our current dilemma with melanoma risk stratication and the limitations of histopathology alone as a predictor of regional metastasis . 
multivariable models have used breslow thickness , tumor ulceration , and patient age to predict sln status , with age being a negative predictor and breslow thickness as well as tumor ulceration being strong positive predictors.47 - 50 angiolymphatic invasion was also found to positively correlate with sln metastasis in some models.5 , 51 the most ambitious cp models , such as those developed from a large bi - institutional series , achieved slnb reduction rates of 18% - 30% , with a negative predictive value table 4 . 
in comparison , the cp plus molecular model developed here showed an slnb reduction rate of 42% at a negative predictive value of 96% ( appendix table a4 )  . 
there seems to be a clear limit in the ability of cp factors to predict sln metastasis . to improve the performance of predictive models , we developed a gep from primary diagnostic biopsy tissue . gep has been used successfully in breast cancer to individualize therapy.52 previous research on gene expression in invasive breast cancer , 53 prostate cancer , 54 colon cancer , 55 melanoma , 10 and other solid cancers56 has consistently demonstrated the upregulation of adhesion receptors and secreted factors that remodel the tumor microenvironment and are involved in emt.10 , 53 - 57 here , we have conrmed this upregulation and found genes involved in emt with specic roles in angiogenesis ( growth differentiation factor 15 , 25 interleukin 8 , 28 lysyl oxidase homolog 4 , 58 tgfreceptor type 1 , 32 and integrin 334 ) and coagulation ( tissue - type plasminogen activator , 38 and gliaderived nexin40 ) as well as the melanosome biogenesis marker melanoma antigen recognized by t cells 123 to be associated with sln metastasis ( table 3 )  . 
the functional roles of these genes have been demonstrated by genetic approaches32 , 34 , 38 , 59 and pharmacologic efcacy studies where the inhibition of integrin - 3 by cyclic peptide , 60 tgfreceptor type 1 by kinase inhibitor , 61 and interleukin 8 by neutralizing antibody62 reduced tumor angiogenesis , tumor growth , and metastasis . 
tumor vascularity in melanoma diagnostic biopsy tissue is well known to associate with nodal and distant metastasis but has been difcult quantify in the past.63 likewise , constitutive brinolytic activity in tumor tissue has been described as early as 191164 and attributed largely to plasminogen activators38 and other serine proteases , such as glia - derived nexin , 65 which promote metastasis , 66 disseminated intravascular coagulation , and bleeding in patients with metastatic cancer.67 a drawback of the simultaneous selection of cp variables and genes by our feature selection algorithm is the absence of established variables easily recognizable by clinicians , such as ulceration , in the cp - gep model . 
moreover , we excluded t4 lesions ( ie , melanoma with a breslow thickness , 4 mm ) because the pretest probability of regional metastasis for these patients is very high . 
for example , 21 ( 70% ) of 30 patients with t4 lesions in our cohort presented with regional metastasis , which is well above the recommend threshold for recommending slnb . 
finally , eligibility of patients with t1 melanoma was determined by the mayo clinic institutional practice guidelines for recommending slnb , which select for higher - risk patients , such as those , 40 years of age and with t1b melanoma . 
however , slnb to slnb metastasis risk was only , 5% if cohorts were enriched for a priori low - risk cases , such as by including t1a melanoma or restricting the analysis to patients  . 
previous attempts to develop molecular risk factors have been limited by small cohort sizes , incomplete tnm staging data , or limited clinical utility of the resulting models.49 , 69 , 70 our approach of combining cp factors and gene expression variables improved the performance of cp factors alone by outperforming current clinical practice and important benchmarks , such as the mskcc nomograthe combined cp - gep model maintained an average negative predictive value of  . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . domenico bellomo employment : skylinedx stock and other ownership interests : synlogic , skylinedx patents , royalties , other intellectual property : gene signatures for predicting metastasis of melanoma julia s . 
van vliet employment : skylinedx stock and other ownership interests : skylinedx patents , royalties , other intellectual property : named inventor on patents in the multiple myeloma eld jvalini dwarkasing employment : skylinedx stock and other ownership interests : skylinedx alexander meves research funding : skylinedx patents , royalties , other intellectual property : mayo clinic has led several patent applications on which i am listed as an inventor ; technologies are in the area of wound healing and skin cancer ( inst ) open payments link : 478139 no other potential conicts of interest were reported . acknowledgment we thank vera j . 
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zhang m , kleber s , r ohrich m , et al : blockade of tgfsignaling by the tgfr - i kinase inhibitor ly2109761 enhances radiation response and prolongs clin cancer res 11 : 6270 - 6279 , 2005 survival in glioblastoma . 
huang s , mills l , mian b , et al : fully humanized neutralizing antibodies to interleukin - 8 ( abx - il8 ) inhibit angiogenesis , tumor growth , and metastasis of human 63 . 
boulaftali y , ho - tin - noe b , pena a , et al : platelet protease nexin - 1 , a serpin that strongly inuences brinolysis and thrombolysis . 
 molecular model to assess sentinel lymph node metastasis risk appendix methods quantitative polymerase chain reaction ( pcr ) was performed as previously described.10 rna purication was from formalin - xed parafn - embedded tissue ( qiagen , hilden , germany )  . 
gene expression was corrected by the mean of housekeeping genes ( rlp0 , rlp8 , and - actin ) using the ct method . statistical methods double - loop cross - validation . 
in the inner loop ( 10 - fold cross - validation ) , we optimized the parameter by determining the number of features ( ie , the weight of the least absolute shrinkage and selection operator penalty term ) , and in the outer loop ( threefold cross - validation ) , we designed a classier on the training set ( two of the three folds )  . 
next , we assessed the performance of the trained classier on the remaining fold ( test set ) , with the parameter xed to the value estimated in the training set . 
the nal classiers were trained on the entire data set using the average ? parameter over 300 runs . memorial sloan kettering cancer center nomograthe majority of online tools for melanoma provide prognostic information47 ( zabor et al : ann surg oncol 25 : 2172 - 2177 , 2018 )  . 
the memorial sloan kettering cancer center ( mskcc ) nomogram , in contrast , is a tool specically designed to predict the probability of primary cutaneous melanoma metastasis to sln.23 the nomogram corresponds to a logistic regression model that is based on ve clinicopathologic variables : age ( range , 20 - 95 years ) , breslow thickness ( range , 0.1 - 10 mm ) , clark level ( ii , iii , iv , or v ) , biopsy location ( trunk , extremity , or head and neck ) , and tumor ulceration ( yes or no )  . 
while attempting to apply the nomogram to our cohort , we could not calculate the probability of sln metastasis for 16 patients because of missing values or because the values were outside the allowable range for the nomograsix patients were , 20 years of age , seven had a missing clark level ( one of whom was also , 20 years of age ) , and four did not have ulceration status available . 
study ow diagrathe gure depicts the enrollment of patients , inclusion and exclusion criteria , and other potential sources of retrospective bias such as the absence of research consent , the unavailability of tissue for molecular analysis , or failed gene expression proling ( gep )  . 
 molecular model to assess sentinel lymph node metastasis risk x 10 cvs x 100 repeats x 3 cvs train train test train test train average test performance test train train select optimal for lasso penalty fig a2 . 
the dlcv consists of two nested cross - validation loops : in the inner loop ( tenfold cross - validation ) , we estimate the optimal parameter , namely , the weight of the lasso penalty term ( ie , optimal feature selection ) ; in the outer loop ( threefold cross - validation ) , we assess the performance of the classier on each test set , with the parameter as determined in the training set . 
moreover , in each training set of the outer loop , we choose and x an operating point on the receiver operating characteristic curve , and we assess the performance of the classier at that operating point in the corresponding test set . 
the cross - validation procedure has been repeated 100 times , and unless otherwise stated , we reported the average performance over 300 test sets ( three test sets per outer loop , repeated 100 times )  . 
lasso , least absolute shrinkage and selection operator . cp1 ; auc , 0.78 cp2 ; auc , 0.78 cp3 ; auc , 0.78 cp4 ; auc , 0.77 cp5 ; auc , 0.77 cp6 ; auc , 0.77 false - positive rate fig a3 . 
 bellomo et al cp model ; auc , 0.78 gep model ; auc , 0.78 combined cp - gep model ; auc , 0.82 false - positive rate fig a4 . 
blue line , model for predicting sentinel based on cp variables ( cp model ) ; red line , model based on gene expression prole ( gep model ) ; teal line , model based on combined gep and cp variables ( cp - gep model )  . 
curves are averages over 300 double loop crossvalidationgenerated test sets . mskcc nomogram ; auc , 0.77 cp model ; auc , 0.78 combined cp - gep model ; auc , 0.82 false - positive rate fig a5 . 
orange line , model based on mskcc nomogram ; blue line , model based on cp variables ( cp model ) ; teal line , model based on gene expression and cp variables combined ( cp - gep model )  . 
clinicopathologic variables available for statistical modeling clinicopathologic variable age categorical biopsy location biopsy location mskcc breslow thickness breslow thickness categorical mitotic rate categorical clark level ulceration regression tumor - inltrating lymphocytes microsatellitosis angiolymphatic invasion histology type abbreviation : mskcc , memorial sloan kettering cancer center . head and neck , trunk , upper extremity , lower extremity , acral value female , male years 15 - 39 years , 40 - 59 years , 60 years head and neck , trunk , extremity millimeters , 1 mm , 1 - 2 mm , 2 - 4 mm 0 / mm2 , 1 - 6 / mm2 ,  . 
6 / mm2 , not determined ii , iii , iv , v yes , no , not determined yes , no , not determined absent , nonbrisk , brisk , not determined yes , no , not determined yes , no , not determined supercial spreading , nodular , desmoplastic , lentigo maligna , acral lentiginous , spindled , dermal , spitzoid , nevoid , unclassied , other , mixed , not determined table a2 . 
average performance of the cp , gep , and combined cp - gep models were trained in double - loop cross - validation 300 times . performance is shown for the 754 - patient cohort assembled on the basis of inclusion criteria and devoid of patients with equivocal sln metastasis ( ie , patients with , 0.1 mm metastatic disease )  . 
 o evaluation of the her / pi3k / akt family signaling network as a predictive biomarker of pathologic complete response for patients with breast cancer treated with neratinib in the i - spy 2 trial purpose in the i - spy 2 trial ( investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2 ) , the panerythroblastic oncogene b inhibitor neratinib was available to all hormone receptor ( hr ) / human epidermal growth factor receptor 2 ( her2 ) subtypes and graduated in the hr - negative / her2 - positive signature . 
we hypothesized that neratinib response may be predicted by baseline her2 epidermal growth factor receptor ( egfr ) signaling activation / phosphorylation levels independent of total levels of her2 or egfr proteins . materials and methods complete experimental and response data were available for between 130 and 193 patients . 
in qualifying analyses , which used logistic regression and treatment interaction analysis , 18 protein / phosphoprotein , 10 mrna , and 12 dna biomarkers that related to her family signaling were evaluated . 
 exploratory analysis of her family signaling in patients with triple - negative ( tn ) disease found that activation of egfr y1173 ( p = .005 ) and her2 y1248 ( pher2 ) ( p = .019 ) were positively associated with pathologic complete response . 
exploratory analysis in this pegfr / pher2activated tn subgroup identified elevated levels of estrogen receptor ( p < .006 ) in these patients . conclusion activation of her family phosphoproteins associates with response to neratinib , but only egfr y1173 and stmn1 appear to add value to the graduating signature . 
 introduction the i - spy 2 trial ( investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2 ; clinicaltrials.gov identifier : nct01042379 ) is a phase ii , adaptive neoadjuvant therapy trial in which the primary goal was to determine the predictive probabilities of phase iii trial success for various targeted therapeutics . 
patients with locally advanced , high - risk breast cancer had their diseases assigned to one of eight subtypes on the basis of hormone receptor ( hr ) , human epidermal growth factor receptor 2 ( her2 ) , and mammaprint - based high1 / ( ultra ) high2 risk statuses.1 - 3 neratinib , a pan - erythroblastic oncogene b ( erbb ) inhibitor , was available to patients with all tumor subtypes in the i - spy 2 trial , and the agent availability was graduated in the hr negative / her2 - positive signature.4 neratinib has shown activity against her2 - positive metastatic breast cancer , and there is also evidence for activity against her2 - negative tumor cells in vitro.5 , 6 because neratinib was available to all patients in the trial , this study provided an opportunity to test the efficacy of the drug in patients with her2 - negative tumors . cell linebased preclinical studies have implicated alterations in her / akt / mtor family genes on the dna , mrna , or protein level as predictive of neratinib response.7 - 11 however , recent her2 therapybased clinical trials in which her family biomarkers at the total protein and / or mrna level were analyzed for response prediction found that none provided predictive value compared with the conventional , us food and drug administrationapproved immunohistochemistry ( ihc ) / fluorescence in situ hybridizationbased her2 / estrogen receptor ( er ) testing methods.12 - 14 previous work with the i - spy 1 trial cohort revealed that her2 phosphorylation was highly concordant with her2 expression.15 that study identified a subpopulation of patients who had her2 negative disease by standard testing yet had her2 phosphorylation levels similar to patients with her2 - positive disease . 
the her2 activation seen in these patients in the her2 - negative subtype appeared coincident with epidermal growth factor receptor ( egfr ) activation . on the basis of the mechanism of action of neratinib as a potent her family kinase inhibitor combined with observations of i - spy 1 trial results , we postulated that her2 and egfr activation / phosphorylation may be predictive of neratinib response independent of total her2 status in the i - spy 2 trial ; this hypothesis was motivation for an exploratory analysis within the her2 - negative and triple - negative ( tn ) subpopulation . 
our analysis of her / phosphoinositide - 3 - kinase ( pi3k ) / akt signaling family components in pretreatment samples from the neratinib and concurrent control arms of the i - spy 2 trial was performed at a unique multiomic level . 
we report results from the following : ( 1 ) assessment of selected mutation / copy number alterations by exome sequencing , ( 2 ) mrna expression levels by expression microarrays , and ( 3 ) analysis of protein / phosphoprotein levels by reverse phase protein arrays ( rppas ) as specific biomarkers of neratinib response . 
rppa analysis assessed the ability of 18 protein / phosphoproteins that comprise the known drug targets of neratinib ( egfr , her2 ) and downstream effector molecules , such as shc transforming protein 1 ( shc ) and mammalian target of rapamycin ( mtor ) / ak thyoma ( akt ) signaling components to predict complete pathologic response ( pcr ) to neratinib . 
 patients randomly assigned to the experimental arm were treated with neratinib in addition to standard chemotherapy ( neratinib + t > ac ) .4 in patients with her2 - positive disease , neratinib was administered in place of trastuzumab ( appendix fig a1 )  . 
consort diagram that outlines the number of patients included in the neratinib and control arms of the i - spy 2 trial ( investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2 ) and those included in the subsequent analyses . 
cnv , copy number variation ; rppa , reverse phase protein array . primary efficacy analysis cohort neratinib concurrent control pretreatment expression data available neratinib concurrent control ( n = 193 ) ( n = 115 ) ( n = 78 ) ( n = 193 ) ( n = 115 ) ( n = 78 ) withdrew consent for use of tissue ( n = 2 ) pretreatment samples available for additional biomarker assessment ( n = 191 ) neratinib concurrent control ( n = 115 ) ( n = 76 ) insufficient material ( n = 23 ) insufficient material ( n = 61 ) rppa data available ( n = 168 ) neratinib ( n = 106 ) concurrent control ( n = 62 ) somatic mutation and cnv data available neratinib concurrent control ( n = 168 ) ( n = 78 ) ( n = 52 ) expression by using agilent 44k expression arrays ( agendia , irvine , ca ) , signaling protein activation by rppa , and dna sequencing ( approximately 2 , 000 - gene mini - cancer genome ; utrecht , the netherlands )  . 
details of sample preparation and data processing are provided in the data supplement . in our prespecified analysis plan , 16 logistic regression was used to assess association with pcr in the control and neratinib treated populations individually . 
relative biomarker performance between arms ( biomarker - by - treatment interaction ) was assessed with a logistic model ( pcr approximately equaled treatment + biomarker + [ treatment biomarker ] )  . 
 if a continuous biomarker did not follow a normal distribution , we applied a nonparametric method that discretized the score by using a series of cut points , and we applied logistic modeling for the dichotomized biomarker at each cut point ; significance was assessed by permutation testing . 
for significant biomarkers , bayesian logistic regression analysis was performed , as previously described , with the following model : pcr hr + her2 + biomarker + treatment + ( treatment hr ) + ( treatment her2 ) + ( treatment biomarker ) .16 in additional analyses , parametric t tests ( for normally distributed data ) or nonparametric wilcoxon rank sum tests ( for non - normally distributed data ) were used to assess association of protein end points with pcr in the control and neratinib - treated populations individually . 
hr , hormone receptor ; tn , triple negative . curves were generated for end points associated with pcr in the neratinib - treated arm of the trial to identify potential cut points for biomarker positivity rates within selected patient subtypes . 16 patients with mutated pik3ca achieved pcr in the neratinib arm compared with 24 ( 39% ) of 62 patients with wild - type pik3ca . 
however , pik3ca mutation status did not show a significant biomarker - by - treatment interaction . results evaluation of her family gene point mutations and dna amplification / loss as predictors of neratinib sensitivity we evaluated somatic mutations and copy number variations ( cnvs ) in 12 her family linked signaling pathway genesegf , egfr , her2 , nrg1 , igf1r , pik3ca , akt1 , pten , stmn1 , and mtorby targeting exome sequencing across all evaluable patients . 
 consistent with publications that link pik3ca mutation with resistance to neratinib and other her2 - targeted agents , 14 , 17 - 19 only two ( 13% ) of association of her family gene expression with response to neratinib we evaluated 10 predefined her family signaling genes as expression biomarkers of neratinib response in all evaluable patients ( appendix table a1 )  . 
her2 expression was significantly associated with sensitivity to neratinib combination therapy ( and in the her2 - positive subpopulation ) , and igf1r , to resistance ; neither gene was associated with response in the control arm of the trial ( appendix tables a1 and a2 )  . 
within the her2 negative subset , stmn1 was associated with response to neratinib ( p = .0023 ) and not control , and this result showed a significant biomarker - by - treatment interaction ( p = .0036 ; fig 3a ; appendix table a3 )  . 
 2 neratinib arm : her2 control arm : her2 hr + her2 no pcr no pcr her2 subset stmn1 - low stmn1 - high unselected her2 her2 / stmn1 - high pcr probability pcr probability fig 3 . 
 ( d ) bayesian estimated pcr probability distributions in ( left ) the unselected her2 - negative subset and ( right ) the her2 - negative / stmn1 - high subset in the control ( blue ) and neratinib ( gold ) arms . of patients with her2 - negative disease ( 29 of 106 patients ) had stmn1 - high status ( fig 3b - c )  . 
by using bayesian modeling , the estimated pcr rate of patients with her2 negative / stmn1 - high status in the treatment arm was 57% compared with 17% in the control arm ( fig 3d ) ; the predictive probability of phase iii success in this subset was 97% . her family protein signaling activation as predictors of neratinib sensitivity we evaluated 18 her family signaling proteins / phosphoproteins as biomarkers of neratinib response by using rppa data from pretreatment , laser capture microdissection ( lcm ) purified tumor epithelia across all evaluable patients in the neratinib and concurrent control arms . 
six of the 18 her family biomarkers tested ( egfr y1068 , egfr y1173 , egfr y992 , her2 total , her2 y1248 , and shc y317 ) were associated with pcr in neratinib - treated patients but not in concurrent control - arm patients in the trial ( table 1 )  . 
we dichotomized patients by their egfr y1173 intensity values into high and low groups ( optimal cut point of 4 , 501 , which maximized the significance of the biomarker - by - treatment interaction term ) , and we evaluated the distribution of pcr rates ( table 2 )  . 
bold indicates p < 0.05. the predictive probability of phase iii success ( appendix table a4 )  . comparison of her family gene expression , protein , phosphoprotein , and mutation data unsupervised clustering revealed that total protein levels of her - family genes clustered with its gene expression level , whereas phosphoprotein levels were more highly correlated to one another than to their respective gene or total protein expression levels ( fig 4 )  . 
pik3ca mutations ( 28 of 130 patients ) were not associated with altered levels of pik3ca expression of protein / phosphoprotein ; however , they were associated with lower levels of stmn1 and egfr gene expression and higher levels of the phosphoproteins pten s380 and akt s473 ( data not shown )  . exploratory analysis of her family signaling in patients with tn disease although neratinib graduated in the hr negative / her2 - positive signature , this signature was characterized by ihc / f luorescence in situ hybridizationbased analysis of total her2 expression and did not measure her2 or egfr phosphorylation . 
pcr rates for egfr y1173 and her2 y1248 biomarker high / low groups by hr / her2 subtype in neratinib - treated and control groups subtype neratinib ( n = 106 ) control ( n = 62 ) egfr y1173 ( riu > 4501 cutoff ) egfr y1173 high egfr y1173 low egfr y1173 high egfr y1173 low no . 
two - way plots of egfr y1173 and her2 y1248 for neratinib treated and concurrent controls demonstrated that nine ( 82% ) of 11 patients with tn disease who exhibited elevated phospho - egfr ( pegfr ) and phospho - her2 ( pher2 ) levels experienced a pcr in response to neratinib treatment compared with four ( 36% ) of 11 with tn disease among the concurrent controls ( table 2 ; fig 5a - b )  . 
bayesian evaluation of egfr y1173 and her2 y1248 as biomarkers for neratinib response in the tn population revealed a comparable probability of success in a phase iii trial to that of the graduated hr negative / her2 - positive population ( fig 5c ; appendix tables a4 and a5 )  . 
in this pegfr / pher2 - high group of tn tumors , 16 ( 76% ) of 21 patients had erlevels greater than the median value for the tn population compared with 11 ( 37% ) of 30 patients in the rest of the tn population ( fig 5d )  . 
in the neratinib - treated population alone , 11 ( 35% ) of 31 patients had pegfr / pher2 - high status , and nine ( 82% ) of 11 patients had total er levels greater than the tn population median . discussion as precision cancer medicine evolves into concomitant processes of discovery , validation , and development of biomarkers predictive of therapeutic response alongside the clinical assessment of drug efficacy , there is increasing emphasis on identification of predictive biomarker candidates as early as possible . 
the subset of biomarkers represented include phosphoproteins associated with response to neratinib , and their associated mrna and total protein levels ( scaled to a median of 0 and standard deviation of 1 )  . 
 egfr , epidermal growth factor receptor ; erbb , erythroblastic oncogene b ; her2 , human epidermal growth factor receptor 2 ; hr , hormone receptor ; na , not available ; shc , shc transforming protein ; tn , triple negative . neratinib arm control arm treatment receptor subtype pten loss pik3ca mut egf mut her2 mut egfr total egfr mrna stmn1 mrna erbb3 total erbb3 mrna egfr y1068 egfr y1173 her2 y1248 shc y317 egfr y992 erbb3 y1289 her2 total her2 mrna no pcr no pcr treatment receptor subtype mutation status heatmap color key neratinib control hr / her2 + hr + / her2 + hr + / her2 mutated wild type 3 2 1 0 1 2 3 value proteomic / phosphoproteomic biomarkers were measured in all pretreatment biopsy samples . 
given that her2 total protein levels do not strictly correlate with her2 phosphorylation in the her2 - negative setting , 15 and given that the phosphorylation status of her2 protein ( compared with total her2 ) provides significant information on breast cancer survival , 23 - 27 we postulated that phosphorylation levels of the neratinib drug targets her2 and egfr in pretreatment biopsy specimens would correlate with pcr in both her2 - positive and her2 negative tumors . our data revealed that coactivation of her2 and egfr correlated with pcr in both her2 positive and tn tumors . 
the pcr rate in the pegfr / pher2 - high tn population , according to an optimal cut point for both markers , was 82% ( nine of 11 patients ) in the treatment arm , compared with 36% ( four of 11 patients ) with pcr in the control ar although these data are exploratory , this pcr rate was higher than the 55% of patients who had a complete response observed in the graduated hr negative / her2 - positive arm for neratinib.4 in keeping with these results , in which phosphorylated egfr levels rather than total egfr were predictive for pcr , phosphorylated egfr has been shown to be a potential predictive marker for anti - egfr therapies in other tumors , such as nonsmall - cell lung cancer.28 , 29 also , our expression analysis produced only one predictive biomarker for neratinib sensitivity , stmn1 , a gene implicated in her2 pathway signaling and chemotherapy sensitivity in non - her2 amplified cell lines.30 - 33 although our data indicate that phosphorylation levels of her2 and egfr correlate with pcr in neratinib - treated patients with breast cancer , a number of caveats in the study limit generalizability . 
the adaptive design of the trial allowed for efficient and quick graduation ( or termination ) of agent / marker combinations on the basis of their estimated likelihood of phase iii success . 
analysis of epidermal growth factor receptor ( egfr ) y1173 and human epidermal growth factor receptor 2 ( her2 ) y1248 as biomarkers for neratinib response in patients with triple - negative ( tn ) disease enrolled in the i - spy 2 trial ( investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2 )  . 
 ( a ) and ( b ) two - way plots of egfr y1173 and her2 y1248 intensities for tn ( a ) control group and ( b ) neratinib - treated patients . 
other studies with her family inhibitors have shown that neratinib , in combination with insulin - like growth factor receptor ( igfr ) inhibitors had antiproliferative effects in her2 non - overexpressing breast cancer cell lines , and pilot clinical studies of lapatinib treatment in various metastatic cancers showed a correlation of pher2 with lapatinib response.37 , 38 what is the basis for her2 and egfr activation in the tn subpopulation ? the frequency of her2 and egfr mutations in the neratinib cohort is too small ( n = 4 and 3 , respectively ) to be a contributing factor . 
in the absence of genomic alterations related to her family signaling when her activation is seen , a logical postulate is that the process is mediated by ligand - driven events . 
recently , it has been shown that neuregulin 1 ( nrg1 or heregulin ) and her2 phosphorylation coincidentally occurred in a subset of her2 - negative tumors and that inhibition of egfr or her2 or both receptors reduced breast cancer stem cell survival and self - renewal.39 although we evaluated nrg1 in our study and did not find any correlation with pcr or her2 activation status ( table 1 ) , we did explore the possibilities of other ligand - driven events underpinning the her2 and egfr activation observed in tn tumors , namely estrogen signaling . 
estrogen can exert nongenomic activity called membrane - initiated steroid signaling through binding with er at low concentrations40 ; er may exist in a signalsome complex with a variety of receptor tyrosine kinases , such as igfr , 41 egfr , 42 , 43 or her2 , 44 , 45 and lead to activation of egfr , her2 , and igfr121 in the absence of gene transcription . 
 we found that erlevels in the tn cohort were higher in pegfr / pher2 - high tumors than in tumors that were not her2 / egfr activated ( fig 5 )  . 
this result could provide a potential explanation for the paradoxical finding of her2 / egfr activation in tn cancers in the absence of her2 genomic alterations and must be confirmed in independent study sets . the lcm - rppa workflow used in this study provides a powerful and unique approach to quantitatively measure the activated signaling architecture of a large number of cancer - related pathways , including the her family , from microscopic quantities of tissue . 
this technology and workflow is especially well suited for clinical sample assessment.46 , 47 past studies have revealed the need for lcm to accurately assess phosphorylated and total protein levels , and to facilitate biomarker evaluation in the context of high and low tumor cell content.48 moreover , unlike ihc - based approaches that can be adversely effected by choice of antigen retrieval method , the rppa technique utilizes fully denatured protein in which phospho - epitopes are fully linearized and recognized by the cognate primary antibody . patients with tn breast cancer have a paucity of targeted therapeutic options available . 
given the pcr rate observed and biomarker positive prevalence we found in the tn setting , we believe these data provide a strong molecular rationale to consider prospective validation of the findings in patients with tn disease who received neratinib . 
to our knowledge , this study is the first of its kind to quantitatively assess activated her family signaling in the context of clinical response to her - directed therapies in a tn population . 
 the biomarker findings , although prespecified , ultimately are based on small numbers of patients and must be confirmed with larger patient populations in independent clinical studies to validate fig 5 . 
wulfkuhle honoraria : dava oncology patents , royalties and other intellectual property : coinventor on filed george mason universityassigned patents related to phosphorylated her2 and egfr response predictors for her family directed therapeutics . 
gallagher no relationship to disclose lamorna brown - swigart no relationship to disclose gillian hirst no relationship to disclose laura esserman consulting or advisory role : blue cross blue shield association research funding : merck travel , accommodations , expenses : blue cross blue shield association donald berry employment : berry consultants leadership : berry consultants stock and other ownership interests : berry consultants consulting or advisory role : berry consultants travel , accommodations , expenses : berry consultants minetta liu research funding : eisai ( inst ) , seattle genetics ( inst ) , novartis ( inst ) , roche ( inst ) , genentech ( inst ) , grail ( inst ) , merck ( inst ) , janssen diagnostics ( inst ) travel , accommodations , expenses : grail , mreck , celgene , agena bioscience , menarini silicon biosystems , cynvenio biosystems john w . 
park stock and other ownership interests : merrimack consulting or advisory role : genentech ( inst ) speakers ' bureau : genentech , pfizer , agendia ( i ) lodewyk f.a. 
petricoin iii leadership : perthera , ceres nanosciences stock and other ownership interests : perthera , ceres nanosciences , avant diagnostics consulting or advisory role : perthera , ceres nanosciences , azgen , avant diagnostics research funding : ceres nanosciences ( inst ) , glaxosmithkline ( inst ) , abbvie ( inst ) , symphony evolution ( inst ) patents , royalties , other intellectual property : national institutes of health patents licensing fee distribution / royalty . 
6 - 10 , 2016 , and the annual meeting of the american society for clinical oncology , chicago , il , may 29 - june 2 , 2015 . support prior presentation references 1 . 
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loibl s , majewski i , guarneri v , et al : pik3ca mutations are associated with reduced pathological complete response rates in primary her2 - positive breast cancer : pooled analysis of 967 patients from five prospective trials investigating lapatinib and trastuzumab . 
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wang f , wang s , wang z , et al : phosphorylated egfr expression may predict outcome of egfr - tkis therapy for the advanced nsclc patients with wild - type egfr . 
saal lh , johansson p , holm k , et al : poor prognosis in carcinoma is associated with a gene expression signature of aberrant pten tumor suppressor pathway activity . 
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chakraborty a , hatzis c , digiovanna mp : co - targeting the her and igf / insulin receptor axis in breast cancer , with triple targeting with endocrine therapy for hormone - sensitive disease . 
lee cy , lin y , bratman sv , et al : neuregulin autocrine signaling promotes self - renewal of breast tumor - initiating cells by triggering her2 / her3 activation . 
arpino g , wiechmann l , osborne ck , et al : crosstalk between the estrogen receptor and the her tyrosine kinase receptor family : molecular mechanism and clinical implications for endocrine therapy resistance . 
song rx , barnes cj , zhang z , et al : the role of shc and insulin - like growth factor 1 receptor in mediating the translocation of estrogen receptor to the plasma membrane . 
pietras rj , arboleda j , reese dm , et al : her - 2 tyrosine kinase pathway targets estrogen receptor and promotes hormone - independent growth in human breast cancer cells . 
akbani r , becker kf , carragher n , et al : realizing the promise of reverse phase protein arrays for clinical , translational , and basic research : a workshop report : the rppa ( reverse phase protein array ) society . 
i - spy2 trial ( investigation of serial studies to predict your therapeutic response with imaging and molecular analysis 2 ) schema for patients in the control and experimental therapy arms . 
 molecular characterization and clinical outcomes of primary gleason pattern 5 prostate cancer after radical prostatectomy pedro isaacsson velho , md1 ; david lim , ms1 ; hao wang , phd1 ; jong chul park , md1 ; harsimar b . 
lotan , md1 purpose very high - risk prostate cancer ( pc ) is associated with poor response to local and systemic treatments ; however , few cases have been molecularly proled . 
clinicopathologic and genomic parameters were correlated with biochemical relapse , metastasis - free survival , time to castration resistance , and overall survival using cox proportional hazards models . results of patients with somatic sequencing data and clinical follow - up , 34% had dna repair gene mutations , including 22% ( 11 of 49 ) with homologous recombination and 12% ( six of 49 ) with mismatch repair gene alterations . 
these data are retrospective and hypothesis generating . conclusion potentially actionable homologous recombination and mismatch repair alterations are observed in a signicant proportion of patients with very high - risk pc at the time of radical prostatectomy . 
2019 by american society of clinical oncology introduction the national comprehensive cancer network very high - risk prostate cancer ( pc ) subset includes patients with t3b to t4 disease or more than four biopsy cores with gleason score 8 to 10 ( grade group 4 / 5 ) or those presenting with gleason score 5 + 5 = 10 or 5 + 4 = 9 ( primary gleason pattern 5 [ pg5 ] ; grade group 5 ) .1 these patients have the poorest oncologic outcomes , with higher relapse rates , frequent development of distant metastasis , poor response to systemic treatments , and short survival.2 - 4 previous studies have suggested that the proportion of gleason pattern 5 tumor on biopsy or surgical specimen denes a subset with independent prognostic implications and distinct outcomes after local and systemic treatments.5 - 8 although we have a wealth of data substantiating the poor prognosis of patients with predominant or pg5 , the molecular we know surprisingly little about alterations in these poorly differentiated tumors . among the 333 primary prostate tumors proled for the cancer genome atlas ( tcga ) , only eight patient cases ( 2% ) had pg5.9 because the prevalence of targetable alterations is relatively increased in other aggressive histologic subtypes of pc , 10 - 12 it is critically important to ll in these gaps in our molecular characterization of this disease . evolving data on metastatic castration - resistant pc ( mcrpc ) have demonstrated that druggable genomic alterations are not uncommon . 
 isaacsson velho et al context key objective we performed an integrated clinical and genomic analysis of prostate tumors with primary gleason pattern 5 at radical prostatectomy to identify potentially actionable alterations . knowledge generated in this very high - risk patient subset , adverse pathologic ndings and common molecular alterations , such as tp53 mutation , pten loss , and erg expression , were associated with poor outcomes . 
importantly , one third of patients had pathogenic mutations in dna repair genes , including in the homologous repair and mismatch repair pathways . relevance these data support the notion that aggressive subsets of primary prostate cancer are enriched for actionable genomic alterations and could inform the design of prospective clinical trials in this population . ( pd - 1 ) inhibitors.18 yet how these results in mcrpc might translate into treatment for clinically localized disease remains unclear . 
several clinical trials evaluating perioperative therapies in patients with high - risk localized including androgen - deprivation therapy ( adt ) , 19 antiandrogens , 20 and docetaxel , 21 have failed to demonstrate relevant benet . 
however , the utility of neoadjuvant and / or adjuvant parp and pd - 1 inhibitors has not yet been studied , in large part because of the relative rarity of these alterations in primary pc cohorts . here , we assembled a consecutive cohort of clinically localized prostate tumors with pg5 and long - term clinical follow - up after radical prostatectomy ( rp )  . 
to maximize duration of oncologic follow - up with the most recent pathologic specimens possible to preserve dna quality , we queried the pathology database for consecutive rps with pg5 from 2005 to 2015 and identied 60 patient cases with available tissue and clinical follow - up ( appendix fig a1 )  . immunohistochemistry tissue microarrays were constructed as previously reported.11 immunostaining for erg , pten , and p53 status was performed using previously reported and genetically validated rabbit monoclonal antibodies and staining and scoring protocols.22 - 26 punches from regions with the highest percentage of gleason pattern 5 tumor . 
dna was extracted from formalin - xed parafn - embedded material as previously described.11 next - generation tumor sequencing targeted next - generation deep sequencing to evaluate 262 cancer - related genes at a 500 average depth was performed using uw - oncoplex ( university of washington , seattle , wa ) as previously described.27 reported alterations were limited to those deemed pathogenic or likely pathogenic in the clinvar database . 
germline mutations were inferred by expert molecular pathologist review through cross - referencing against the clinvar database and by variant allele fraction in the context of tumor content , ploidy , and loss of heterozygosity status.28 , 29 two patients ( two of 49 ) did not have tumor dna available for uwoncoplex but had prior somatic sequencing performed using a clinical platform for clinical care purposes ( pgdx ; foundation medicine , cambridge , ma , and baltimore , md ) and were included in the analysis . 
patient cases in which tumor purity and / or dna quality was not high enough to exclude false - negative results are listed in the data supplement . germline sequencing altogether , 55% patient cases ( 27 of 49 ) underwent germline sequencing ( data supplement )  . 
 ( % ) a conrmatory test , 31 and metastasis - free survival ( mfs ) , dened as the time from rp to the rst detection of distant metastasis on imaging or death resulting from any cause , whichever occurred rst . 
time to castration resistance ( tcr ) , dened as the time from adt to two consecutive elevations in psa at least 4 weeks apart , and overall survival ( os ) , dened as the time from rp to death , were also assessed . 
univariable and multivariable cox proportional hazards models were used to estimate hazard ratios ( hrs ) and corresponding 95% cis and test for the association of the variables with clinical outcomes . 
because this study was hypothesis generating , we did not perform corrections for multiple comparisons . results baseline characteristics demographic , clinical , and pathologic characteristics of the pg5 cohort ( n = 60 ) at rp are listed in table 1 . 
at surgery , most patients had pathologic evidence of locally advanced disease , including nonorgan - conned ( pt3 / t4 ) tumors ( 54 [ 90% ] of 60 ) , seminal vesicle invasion ( svi ) ( 35 [ 58% ] of 60 ) , positive lymph nodes ( 16 [ 27% ] of 60 ) , and positive surgical margins ( 28 [ 47% ] of 60 )  . 
of the 37 patients who received either salvage or adjuvant radiation therapy , 27 ( 73% ) also received adjuvant adt . also , of the 16 patients who had positive lymph nodes ( pn + ) , 11 ( 69% ) received adjuvant adt . prevalence of gene mutations overall , 81.7% ( 49 of 60 ) of patients had available sequencing data on somatic genomic alterations ( data supplement ) , and 35% ( 17 of 49 ) of these had at least one pathogenic mutation in a gene involved in dna repair ( table 2 ; data supplement )  . 
overall , 55% ( 27 of 49 ) had germline sequencing results available , including six patients with presumed germline homologous recombination alterations on the basis of somatic sequencing , all of which were conrmed on germline sequencing ( brca2 , n = 3 ; brca1 , chek2 , and palb2 , n = 1 each )  . 
in addition to these alterations in homologous recombination genes , mmr deciency was found in 12% ( six of 49 ) of patients ( all in msh2 and included in our previous immunohistochemistry [ ihc ] study11 )  . other mutations common in pc were also identied ( data supplement )  . 
of 57 patients who had tumors evaluated by ihc , 51% ( 29 of 57 ) had pten protein loss . foxa1 mutations were seen in 12% ( six of 49 ) of patients , and spop mutations were seen in 8% ( four of 49 ) of patients . 
several pathologic characteristics were associated with increased risk of bcr on univariable analysis , including svi ( appendix table a1 ; appendix fig a2a ) and presence of ductal or intraductal histology ( appendix table a1 ; appendix fig a2b )  . 
multivariable models were t separately for each genomic alteration , adjusting for ductal or intraductal histology and svi ; however , none of the genomic alterations remained signicantly associated with bcr ( appendix table a2 )  . overall , 43% ( 26 of 60 ) of the cohort developed metastasis , with a median mfs of 86.4 months ( 95% ci , 40.7 months to not reached )  . 
visceral disease was seen in 31% of patients who developed metastasis , with liver ( 23% ) and lung ( 19% ) the most common sites . among clinicopathologic factors on univariable analysis , presence of svi ( table 3 ; fig 1a ) and ductal or intraductal histology ( table 3 ; fig 1b ) , along with patient age , psa , and nodal status , were both associated with increased risk of metastasis ( fig 1b )  . 
kaplan - meier analysis of metastasis - free survival in primary gleason pattern 5 cohort by ( a ) seminal vesicle invasion ( svi ) status , ( b ) ductal or intraductal ( doi ) histology status , ( c ) tp53 mutation status , and ( d ) pten protein status using immunohistochemistry . 
we chose a subset of patients with established unfavorable clinical outcomes who were diagnosed before evidence of metastatic disease and received the same initial local treatment to minimize potential biases associated with extent of disease . 
almost all patients had nonorganconned disease , more than half had svi , and a quarter had node - positive disease , demonstrating the aggressive behavior of these poorly differentiated tumors . the prevalence of genomic abnormalities in the dna repair pathway in clinically localized pg5 seems to be comparable to or perhaps even higher than that in patients with mcrpc . pritchard et al13 showed that 12% of patients with mcrpc table 5 . 
abnormalities in homologous recombination and mmr genes could have potential therapeutic implications for the treatment of all stages of pc , including the design of adjuvant and neoadjuvant trials in the pg5 population . 
in addition , our ndings have important implications for the families of patients with very high - risk disease , given that 82% of patients with homologous recombination mutations in this study had apparent underlying germline mutations . 
these ndings clearly support the recent national comprehensive cancer network guidelines , which recommend genetic testing in all patients with high - risk or very high - risk pc . our results show that there are pathologic and molecular features that are useful for risk stratication after rp . 
patients with svi or ductal or intraductal histology have more than four times greater risk of metastasis , and tp53 mutation , pten loss , and erg expression were associated with increased risk of biochemical failure and metastasis . 
notably , our study validates current clinicopathologic variables used for risk stratication and suggests that pathologic ndings after rp may be more signicant for risk prediction than molecular alterations in multivariable models . 
however , where full pathologic data are not available ( eg , in patients who do not undergo rp ) , molecular alterations could be of added value . future studies should evaluate prospectively the utility of tp53 mutation and pten loss in well - designed biomarkerdriven clinical trials cohorts , particularly in the biopsy setting . in addition , although we believe that our data are unique and informative , these results should be viewed as preliminary because of relatively the small numbers and multiplicity of clinical , pathologic , and molecular data points , which limit the power of the observations with regard to outcomes . 
it is likely that a prospective evaluation with additional numbers and follow - up time will allow for a more precise study of the prognostic signicance of clinicopathologic and molecular parameters in this group of patients . the pivotal role of adt in pc with gleason scores 9 to 10 has been called into question in recent years.8 a recent large retrospective study of patients undergoing external - beam radiation therapy in combination with adt compared outcomes of patients with gleason score 8 versus 9 to 10 , suggesting that those in the higher gleason group may derive less benet than patients with gleason score 8.8 recent studies have shown that the addition of either docetaxel21 , 32 or abiraterone33 , 34 improves os in the locally advanced or metastatic hormone - sensitive setting , with higher benet among high - risk patients , including those with high disease burden , 32 t3 to t4 disease , 21 , 34 psa greater than 40 ng / ml , 21 , 34 and gleason score 8 to 10.21 , 33 , 34 our data suggest that compared with lower - grade tumors , pg5 tumors may be less dependent on androgens and potentially more dependent on alternative oncogenic drivers , including defects in dna repair pathways . limitations of the study include the fact that it was retrospective , and clinical follow - up and dna sequencing were available only for a subset of patients , although the clinicopathologic parameters of patients with and without follow - up and sequencing were not signicantly different ( appendix fig a1 )  . 
also , the quality of the archival dna and tumor purity were not uniformly high , and false negatives at sequencing could not be excluded in all patient cases , suggesting that our results may actually underestimate the prevalence of dna repair alterations among pg5 tumors . 
 isaacsson velho et al in conclusion , we performed the rst integrated clinical and genomic analysis to our knowledge of pg5 prostate tumors at rp , a subset that has not been well represented in other sequencing efforts . 
clinical outcomes and response to conventional treatment are worse compared with lower - risk groups , and adverse pathologic ndings and common molecular alterations such as tp53 mutation , pten loss , and erg expression are associated with poor outcomes . 
the relatively high incidence of hrd and mmr deciency observed herein could inform the design of multimodal clinical trials employing parp inhibitors or pd - 1 / pd - 1 ligand monoclonal antibodies in very high - risk patients with clinically localized disease . 
contributed equally to this work . support supported by the patrick walsh research fund ; by national cancer institute ( nci ) , national institutes of health , prostate specialized program of research excellence grant no . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . travel , accommodations , expenses : astrazeneca , astellas pharma , pzer , merck serono , merck michael a . 
carducci consulting or advisory role : astellas pharma , abbvie , roche / genentech , pzer , foundation medicine research funding : bristol - myers squibb ( inst ) , pzer ( inst ) , astrazeneca ( inst ) , gilead sciences ( inst ) , emd serono ( inst ) , effector therapeutics ( inst ) samuel r . 
antonarakis honoraria : sano , dendreon , medivation , janssen biotech , essa , astellas pharma , merck , astrazeneca , clovis oncology consulting or advisory role : sano , dendreon , medivation , janssen biotech , essa , astellas pharma , merck , astrazeneca , clovis oncology research funding : janssen biotech ( inst ) , johnson & johnson ( inst ) , sano ( inst ) , dendreon ( inst ) , aragon pharmaceuticals ( inst ) , exelixis ( inst ) , millennium pharmaceuticals ( inst ) , genentech ( inst ) , novartis ( inst ) , astellas pharma ( inst ) , tokai pharmaceuticals ( inst ) , merck ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , constellation pharmaceuticals ( inst ) patents , royalties , other intellectual property : coinventor of biomarker technology licensed to qiagen travel , accommodations , expenses : sano , dendreon , medivation mario a . 
eisenberger leadership : veru stock and other ownership interests : veru honoraria : sano , pzer consulting or advisory role : astellas pharma , ipsen , bayer healthcare pharmaceuticals , sano , pzer research funding : sano , tokai pharmaceuticals , genentech travel , accommodations , expenses : bayer healthcare pharmaceuticals , astellas pharma , sano , pzer , veru pedro isaacsson velho honoraria : bayer healthcare pharmaceuticals speakers bureau : astrazeneca , pzer , bristol - myers squibb research funding : bristol - myers squibb , pzer ( inst ) expert testimony : bayer healthcare pharmaceuticals tamara l . 
prostate cancer 2018 : 151 epstein ji , zelefsky mj , sjoberg dd , et al : a contemporary prostate cancer grading system : a validated alternative to the gleason score . 
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clin cancer res 23 : 6863 - 6874 , 2017 isaacsson velho p , silberstein jl , markowski mc , et al : intraductal / ductal histology and lymphovascular invasion are associated with germline dna - repair gene mutations in prostate cancer . 
powell ij , tangen cm , miller gj , et al : neoadjuvant therapy before radical prostatectomy for clinical t3 / t4 carcinoma of the prostate : 5 - year followup , phase ii southwest oncology group study 9109 . 
vale cl , burdett s , rydzewska lhm , et al : addition of docetaxel or bisphosphonates to standard of care in men with localised or metastatic , hormone - sensitive prostate cancer : a systematic review and meta - analyses of aggregate data . 
tosoian jj , almutairi f , morais cl , et al : prevalence and prognostic signicance of pten loss in african - american and european - american men undergoing 23 . 
lotan tl , wei w , ludkovski o , et al : analytic validation of a clinical - grade pten immunohistochemistry assay in prostate cancer by comparison with pten radical prostatectomy . 
maughan bl , guedes lb , boucher k , et al : p53 status in the primary tumor predicts efcacy of subsequent abiraterone and enzalutamide in castration - resistant prostate cancer . 
pritchard cc , salipante sj , koehler k , et al : validation and implementation of targeted capture and sequencing for the detection of actionable mutation , copy number variation , and gene rearrangement in clinical cancer specimens . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
na r , zheng sl , han m , et al : germline mutations in atm and brca1 / 2 distinguish risk for lethal and indolent prostate cancer and are associated with early age 31 . 
cookson ms , aus g , burnett al , et al : variation in the denition of biochemical recurrence in patients treated for localized prostate cancer : the american urological association prostate guidelines for localized prostate cancer update panel report and recommendations for a standard in the reporting of surgical outcomes . 
n engl j med 377 : 352 - 360 , 2017 james nd , de bono js , spears mr , et al : abiraterone for prostate cancer not previously treated with hormone therapy . 
pathology database query for consecutive radical prostatectomies ( rps ) with primary gleason score 5 ( pg5 ) from 2005 to 2015 , where 60 patient cases were identied with available tissue and clinical follow - up . 
this model combines the expression of eight genes in primary melanoma with breslow thickness and patient age to identify patients who may forgo a sentinel lymph node biopsy ( slnb ) because of their low risk of nodal metastasis . 
cp - gep can guide physicians on slnb referrals and reduce the frequency of slnb surgeries.4 the authors of both commentaries acknowledge the need for a tool to reduce the frequency of slnb procedures . 
bartlett et al1 appreciated our approach of incorporating cp variables into our model because it ensures transparency on the added value of the gep , and they nd the comparison of cp - gep and cp models appropriate . 
the authors of both commentaries reected on the evidence that supports the utility of the cp - gep model in clinical practice and elaborated on some methodologic considerations . both commentaries rightfully emphasized the need for adequately sized external validation and additional clinical utility studies before widespread clinical adoption of cp - gep . 
we agree that extensive validation in diverse patient populations and different geographical contexts is critical . for example , we recently validated the cp - gep model in a dutch retrospective patient cohort ( n = 211 ) 5 in which patient selection , surgical procedures , and pathologic assessment of slnb are signicantly different from clinical practice in the united states . 
cp - gep achieved an slnb reduction rate ( rr ) of 41% in t1 / t2 melanoma at a negative predictive value ( npv ) of 90.7%.5 additional studies are underway to elucidate cp - gep as an slnb reduction tool . 
slnb recommendations were made on the basis of individualized considerations and within the framework of institutional practice guidelines . we , in turn , derive institutional practice guidelines from established national and international guidelines . 
moreover , in terms of net benet , the cp - gep model is consistently superior to the cp model across a range of clinically reasonable risk thresholds with a more substantial gain for higher risk thresholds . varey et al2 rightfully observe that the npv is a function of the prevalence of slnb positivity in the population and , in particular , that npv decreases as the prevalence increases . 
second , the risk of metastasis to the sentinel lymph node ( sln ) ; as reected by npv and positive predictive value ( ppv ) upon taking a test is a clinically relevant metric in the process of making a decision for or against an slnb , as indicated by national comprehensive cancer network guidelines . 
furthermore , in a previous publication by mocellin et al , 9 team members thompson and scolyer themselves recommended building predictive models for sln metastasis with the aim of maximizing the npv and reducing the rate of slnb procedures through minimizing the error rate . 
in a consensus statement on the development of gep in cutaneous melanoma , grossman et al10 recently commented that gep - based tests should add to the npv of current models while minimizing false - negative predictions . bartlett et al discuss the stratication of the results by t categories . 
after consulting with dermatopathologists at mayo clinic , we concluded that the 140 excluded patients could results stratied by t category , and we hope that our data will enable physicians to critically assess the potential of cp - gep . 
knowing model performance within each t category ( as opposed to overall performance ) is likely to gain importance because the distribution of melanoma t categories is expected to change over time with more melanomas diagnosed at earlier t categories . 
we further sought to limit variability in our data by requiring slnb to be performed within 90 days of the diagnosis of primary melanoma.3 from our point of view , combining end points such as slnb status and regional recurrence can be problematic , because they occur on different time scales and vary in their methods of detection . 
the relatively high rate of patients who develop regional recurrences after negative slnb , however , highlights the need for better , more sensitive staging tools , such as the cp - gep model . 
accordingly , and to avoid missing stage iii disease , too many patients undergo invasive and costly slnb , which highlights the need for an slnb reduction tool . varey et al2 from the melanoma institute australia ( mia ) recently published a cp nomogram to predict the risk of sln metastasis.12 the mia nomogram is based on six cp variables : patient age , breslow thickness , ulceration , histologic subtype , mitotic rate , and lymphovascular invasion . in their commentary , the authors speculate that the mia nomogram outperforms the cp - gep model . 
therefore , since the mia nomogram is available online , we sought to assess the performance of the mia nomogram in our cohort.3 we found that the mia nomogram risk score could not be calculated for approximately 19% of the patients ( 143 of 754 ) in our cohort . 
of the 143 patients excluded , 140 were not suitable for the nomogram because of histologic type , and three patients were excluded because they were younger than age 18 years at biopsy . 
shown are models based on the mia nomogram , clinicopathologic variables ( cp model ) , and cp variables and a gene expression prole ( cp - gep )  . 
 correspondence not be consistently reclassied into the ve histologic types available for the mia nomograit is likely that in clinical practice , we would encounter a similar number of patients who are difcult to classify for the mia nomograit is important to note that 16.4% ( 23 ) of the 140 excluded patients showed sln metastasis . 
more data are needed to characterize our model fully , and to this end , we are currently implementing a comprehensive validation plan . domenico bellomo , phd skylinedx , rotterdam , the netherlands alina g . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . domenico bellomo employment : skylinedx stock and other ownership interests : synlogic , skylinedx patents , royalties , other intellectual property : gene signatures for predicting metastasis of melanoma tina j . 
hieken research funding : genentech ( inst ) alexander meves research funding : skylinedx ( inst ) patents , royalties , other intellectual property : mayo clinic has led several patent applications on which i am listed as an inventor . 
mulder eeap , dwarkasing jt , hollestein d , et al : validation of a clinicopathological and gene expression prole ( cp - gep ) model for sentinel lymph node metastasis in primary cutaneous melanoma . 
bioinformatics 21 : 3755 - 3762 , 2005 steyerberg ew , harrell fe jr , borsboom gj , et al : internal validation of predictive models : efciency of some procedures for logistic regression analysis . 
mocellin s , thompson jf , pasquali s , et al : sentinel node status prediction by four statistical models : results from a large bi - institutional series ( n = 1132 )  . 
 analysis of ntrk alterations in pan - cancer adult and pediatric malignancies : implications for ntrk - targeted therapeutics purpose fusions that involve neurotrophic - tropomyosin receptor kinase ( ntrk ) genes are known drivers of oncogenesis . 
among 31 adult samples carrying ntrk fusions , co - alterations occurred often and usually involved the downstream phosphoinositide - 3 - kinase signaling pathway , cell - cycle machinery , other tyrosine kinase receptors , and mitogen - activated protein kinase signals . conclusion whereas ntrk fusions are exceedingly rare , other ntrk abnormalities affect 14% of patients with cancer . 
2018 by american society of clinical oncology introduction ntrk1 , ntrk2 , and ntrk3 genes encode the neurotrophic - tropomyosin receptor tyrosine kinases ( ntrks ) trka ( ntrk1 ) , trkb ( ntrk2 ) , and trkc ( ntrk3 )  . 
 nerve growth factor ( ngf ) binds to ntrk1 ; brain - derived neurotrophic factor ( bdnf ) and neurotrophin - 4 ( nt - 4 ) and nt - 5 bind to ntrk2 ; and nt - 3 binds both ntrk1 and ntrk3.1 binding of neurotrophic factors to their receptors activates the downstream effectors of ntrk : phospholipase c - , mitogen - activated protein kinase ( mapk ) , and phosphatidylinositol 3 - kinase ( pi3k ) / akt pathways . 
p75ntr is a positive regulator of the ngf / ntrk1 system that reduces ligand induced receptor ubiquitination and delays receptor internalization and degradation.2 ntrk receptors promote the proliferation and survival of neuronal cells3 - 8 ( fig 1 )  . 
the ligands nerve growth factor ( ngf ) , brain - derived neurotrophic factor ( bdnf ) , neurotrophin 3 ( nt - 3 ) , and nt - 4 bind to their receptors , namely ntrk1 ( tropomyosin receptor kinase a or trka ) , ntrk2 ( tropomyosin receptor kinase b or trkb ) , and ntrk3 ( tropomyosin receptor kinase c or trkc )  . 
several signaling cascades are further activatedphospholipase c ( plc - ) , mitogen - activated protein kinase ( mapk ) , and phosphoinositide - 3 - kinase ( pi3k ) and are converging to protumorigenic cell processes , such as proliferation , survival invasion , or differentiation . 
the hyperactivation of the ntrk signaling pathway induced by ntrk alterations fusions or point mutations can be overcome by the use of ntrk antagonists ( eg , ana - 12 and cyclotraxin b ) or small - molecule tyrosine kinase inhibitors ( eg , larotrectinib and entrectinib )  . 
these drive ntrk mrna and protein overexpression , which further leads to constitutive activation of downstream signaling.12 the prevalence of ntrk fusions is low , but can reach more than 80% in some rare tumors , such as mammary - analog secretory carcinoma of the salivary gland , secretory breast carcinoma , and infantile congenital fibrosarcoma.12 - 20 ntrk fusions are also found in 40% of pediatric non - brainstem high - grade glioma.21 among all alterations in ntrk genes , transcript fusions are currently the best characterized and the most pharmacologically tractable . 
nonfusion ntrk alterationsfor example , mutation or amplificationhave been associated with a lack of response with some ntrk inhibitors.22 because ntrk fusions are rare , the number of patients who can benefit from drugs that target ntrk receptors is relatively low , but the antitumor activity of such agents is remarkable.23 , 24 indeed , larotrectinib , a pan - ntrk inhibitor , demonstrated a response rate of 76% in patients with ntrk fusionpositive tumors ( 17 cancer types ) .15 , 18 tumor regression has been maintained for more than 1 year in 71% of patients . 
entrectinib , an oral pan - ntrk , ros1 , and alk inhibitor demonstrated a 79% objective response in patients with ntrk , ros1 , or alk fusions.22 in may 2017 , a new precedent was set when an immune checkpoint inhibitorpembrolizumab was approved by the us food and drug administration ( fda ) for use in a tissue - agnostic fashion on the basis of a genomic biomarker ( mismatch repair gene deficiency ) .25 ntrk - selective inhibitors represent another pharmacology class that has been developed on the sole basis of somatic molecular patterns . 
all data used in this study respected the tcgas human subjects protection and data access policies31 and the st jude cloud terms of use.32 lists of significant variants were generated using whole - genome somatic mutation data and the mutsig2cv algorithm ( org / cancer / cga / mutsig ) , taking into account the somatic background mutation rate for each gene and its neighbor genes.33 focal copy number variations that correspond to genome - wide single - nucleotide polymorphism array data were normalized and assessed at the gene level using the gistic2 protocol , 34 where a deep loss was documented by the value ( 2 ) , a single - copy loss by the value ( 1 ) , a low - level gain by the value ( + 1 ) , and an amplification by the value ( + 2 )  . 
only ntrk - related gene amplifications were kept for the analysis . sequencing - based mrna expression signals were integrated and normalized for each gene per sample using the rna - sequencing by expectation maximization protocol . 
the standard score ( z - score ) for each gene per sample was calculated using the mean values and standard deviation found in all similar tumors same tumor typethat are diploid for the said gene . 
in adults , co - alterations within signaling cascades , such as tp53 , mapk , pi3k , tyrosine kinase receptor , or cell - cycle signaling pathways , were curated from tcga . 
all nonsynonymous missense , nonsense , nonstop , deletion / insertions , frameshift , or splicing site mutations within the genes of interest , as well as deep losses or amplifications and mrna under or overexpressions , were kept for analysis . results prevalence of ntrk fusions in tcga ( adult ) and st jude pecan ( pediatric ) databases fusion frequency in adults of the 9 , 966 adult tumor samples in the tcga database , 0.31% ( n = 31 samples ) presented an ntrk fusion . 
 ( there were 5 , 023 patient samples with these 22 ntrk fusion negative tumor types [ samples per tumor type = 36 to 541 ]  . ) ntrk3 fusions were the most common ( n = 16 ) , followed by ntrk1 ( n = 9 ) and ntrk2 ( n = 6 ) fusions in adults ( table 1 )  . fusion frequency in children of the 3 , 501 pediatric tumor samples ( st jude pecan database ) , 0.34% ( n = 12 ) presented an ntrk fusion . 
frequency of ntrk receptor transcript fusions in tcga ( n = 9 , 966 adult tumor samples ) and st jude pediatric cancer database ( n = 3 , 501 pediatric tumor samples ) , and specific tumors with high incidence of ntrk fusions in the literature no . 
sixteen molecules are currently being evaluated in clinical trials , with the most advanced being larotrectinib ( loxo oncology , stamford , ct ; ic50 for ntrk1 , ntrk2 , and ntrk3 fusions ranging from 4 to 9 nm )  . 
the new drug application was submitted to the fda in december 2017 and granted priority review status on the basis of remarkable clinical activity23 ( table 2 )  . types of ntrk - related alterations in adult and pediatric tumors and sensitivity to ntrk inhibitors to understand the potential benefits of selective ntrk inhibitors for the treatment of adult and pediatric patients with cancer , we first aimed to describe the prevalence and type of ntrk activating pathway alterations , including point mutations , gene copy number amplifications , and mrna overexpression of ntrk receptors , co - receptor , and ligands , within a large cohort of pan - cancer samples ( figs 1 and 2 )  . 
the number of samples with comprehensive data for this analysis was 11 , 621 ( 9 , 966 adults and 1 , 655 children )  . alterations in ntrk receptors and ligands genomic and / or transcriptomic ntrk receptor alterations were found in 14.2% ( 1 , 648 of 11 , 621 ) of samples , with gene amplification and mrna overexpression being the most frequent alterations . 
transcript fusions were observed in ntrk receptor genes only , with the exception of two samples that presented one transcript fusion of bdnf ligand and one transcript fusion of p75ntr ( positive regulator of the ngf / ntrk1 machinery ; fig 2 )  . - transcript fusion types ntrk - transcript fusions that were observed in the pan - cancer cohort and / or described in the literature are listed in table 3 . 
this variant is a known biomarker of sensitivity to larotrectinib and entrectinib.71 , 72 variants tpm3ntrk1 ( 0.04% ) , irf2bp2 - ntrk1 ( < 0.01% ) , and sqstm1 - ntrk1 ( < 0.01% ) are also sensitive to larotrectinib ; however , the sensitivity of the remaining 22 unique variants observed in the pan - cancer cohort is not currently known . 
nine rearrangements previously described in the literature were not found in the tcga and st jude pecan databases ( table 3 )  . point mutations several point mutations are acquired resistant variants to first - generation ntrk inhibitors ( larotrectinib or entrectinib ) , but not to loxo - 195 , specifically designed to overcome secondary resistance . 
these variants , namely ntrk1 g595r , ntrk1 g667c , ntrk3 g696a , and ntrk3 g623r , were not observed in any of the 13 , 467 combined adult and pediatric tumors reviewed ( treatment - nave samples ; table 3 )  . co - alterations observed in ntrk fusionpositive adult tumor samples among 31 adult tumors presenting ntrk fusions , 61.3% ( 19 of 31 ) harbored one or more co alteration that activated the downstream pi3k signaling pathway ; 58.1% ( 18 of 31 ) harbored one or more co - alteration within cell - cycleassociated genes ; 58.1% ( 18 of 31 ) harbored one or more co - alteration within other tyrosine kinase receptors ; 32.2% ( 10 of 31 ) harbored one or more co - alteration within the mapk signaling pathway ; and 35.5% ( 11 of 31 ) harbored one or more co - alteration within tp53 - associated genes . 
 nf2 - activating mutations were associated with ntrk fusions in 42% ( 13 of 31 ) of samples , and tp53 ( 10 of 31 ) , rb1 ( six of 31 ) and cdkn2a ( five of 31 ) occurred in more than 15% of the ntrk fusionpositive samples ( fig 3 and appendix table a1 )  . 
 ( adequate data to comprehensively assess co - alteration data in children was not available . ) samples bearing ntrk fusions were significantly associated with ntrk mrna overexpression compared with samples without the fusion ( appendix fig a1 )  . 
distribution of molecular alterations leading to the hyperactivation of the neurotrophic - tropomyosin receptor tyrosine kinase ( ntrk ) signaling pathway in human tumors ( n = 11 , 621 samples with comprehensive molecular data )  . 
all samples that presented a nonsilent mutation , gene copy amplification , gene fusion , or mrna overexpression of ntrk receptors ( ntrk1 , ntrk2 , and ntrk3 ) , co - receptor ( p75ntr ) , or ligands ( nerve growth factor [ ngf ] , brain - derived neurotrophic factor [ bdnf ] , neurotrophin 3 [ nt - 3 ] , and nt - 4 ) were retrieved from a large adult and pediatric pan - cancer cohort ( the cancer genome atlas and st judes pecan databases ; n = 11 , 621samples )  . 
among the ntrk fusion cases ( n = 31 from tcga cohort ) , four cases had concomitant alteration within the genes that code the ntrk pathway membersligands , co - receptor , and receptorsas follows : low - grade glioma , ntrk3 fusion plus ntf3 amplification ( n = 1 ) ; low - grade glioma , ntrk1 fusion plus ntrk1 amplification ( n = 1 ) ; glioblastoma , ntrk1 fusion plus ntrk1 amplification ( n = 1 ) ; head and neck squamous cell carcinoma , ntrk3 fusion plus ntrk3 amplification ( n = 1 )  . we reviewed data from 13 , 467 tumor samples in the tcga ( adult tumors ) and st jude pecan ( pediatric tumors ) databases and found ntrk fusions in 0.3% of pan - cancer tumors ( table 1 )  . 
 although the prevalence of these alterations is low , ntrk fusions are more often found in specific and rare tumors , such as mammary - analog secretory carcinoma of the salivary gland ( 93% to 100% of tumors presenting an etv6 - ntrk3 fusion ) , secretory breast carcinoma ( etv6ntrk3 fusions in 92% of tumors ) , infantile congenital fibrosarcoma ( etv6 - ntrk3 fusions in 86% to 91% of tumors ) , and pediatric non brainstem high - grade glioma14 - 21 ( 40% of tumors presenting an ntrk fusion ; table 1 )  . of importance , various ntrk inhibitors are in clinical development and have differential activities ( table 2 )  . 
drugs with established clinical trial data and the ability to affect ntrk1 , ntrk2 , and ntrk3 fusions at low ic50 include , but are not limited to , larotrectinib ( 76% response rate in diverse malignancies bearing ntrk fusions ) and entrectinib , which also affects alk and ros1 rearrangements ( 79% response rate ) , and some of these responses are durable and occur with remarkable speed22 , 23 ( table 2 )  . 
furthermore , five of these small inhibitors are already approved by the fda for other indications : cabozantinib ( ic50 against ntrk2 , 7 nm ) , crizotinib ( ic50 against ntrk1 and ntrk2 , 1 nm ) , midostaurin ( ic50 against ntrk1 , - 2 , and - 3 ranging from 11 to 51 nm ) , nintedanib ( ic50 against ntrk1 , - 2 , amd - 3 ranging from 17 to 264 nm ) , and regorafenib ( ic50 against ntrk1 , 74 nm )  . 
 ntrk mutations that are associated with larotrectinib or entrectinib resistance include ntrk1 f589l g595r , g667c , g667s , v573m , and ntrk3 g696a , g623r ( table 3 )  . 
 ( these mutations were not detected in tcga , likely because these patients had not been previously treated with ntrk inhibitors . ) the resistant alterations are targetable with loxo - 195 , a next - generation , selective ntrk inhibitor with promising preliminary clinical activity50 ( fig 2 )  . 
in the current study , ntrk - associated co - alterations were commonly discerned in genes that are involved in pi3k signaling ( 61% of patient samples ) , tyrosine kinase families ( 58% of patient samples ) , cell - cycle machinery ( 58% of patient samples ) , and mapk pathways ( 32% of patient samples ; fig 3 and appendix table a1 )  . 
frequencies of alterations were computed using a large adult and pediatric pan - cancer cohort ( the cancer genome atlas and st judes pecan databases ; n = 13 , 467 samples )  . 
sensitivity and resistance criteria presented in this table correspond to objective clinical responses or nonresponses observed in fusion - positive or mutation - positive patients who received the drug . abbreviations : na , not available ; ntrk , neurotrophic - tropomyosin receptor tyrosine kinase . drilon et al , 50 wei et al77 drilon et al , 50 hyman et al , 71 russo et al78 drilon et al , 50 russo et al78 drilon et al , 50 wei et al77 drilon et al , 50 wei et al77 drilon et al50 drilon et al , 50 , 79 hyman et al71 fusions were significantly associated with ntrk mrna overexpression ( appendix fig a1 ) , which is consistent with a previous report.101 of interest , in the adult cohort , ntrk fusionpositive samples were significantly associated with a lower mutational burden compared with fusion - negative tumors ( p < .001 ; appendix fig a2 )  . 
this observation echoes a previous report that demonstrated that tumors harboring a driver fusion tend to have a lower number of point mutations.101 in contrast , high microsatellite unstable metastatic colorectal tumors have been shown to preferentially bear alk , ros1 , or nrtk fusions.102 in our cohort , three ntrk fusionpositive colon cancer samples were observed and two of them presented with microsatellite instability - high status ( data not shown )  . 
finally , we found that nonfusion ntrk gene alterations , such as mutation , amplification , and mrna overexpression , were found in approximately 14% of pan - cancer samples ( fig 2 )  . 
all samples that presented a gene fusion of ntrk receptorsntrk1 , ntrk2 , and ntrk3were retrieved from a large adult pan cancer cohort ( the cancer genome atlas database ; n = 9 , 966 samples )  . 
despite these limitations , the current report provides a comprehensive portrait of the genomic landscape of ntrk alterations among pan - cancer tumors using large databases . in conclusion , ntrk fusions were observed in 0.31% ( 31 of 9 , 966 ) of adult tumors and 0.34% ( 12 of 3 , 501 ) of pediatric cancers , mostly in ntrk3 ( 0.16% of adult tumors ) and ntrk1 ( 0.14% of pediatric tumors ) ; however , some tumor types had more frequent ntrk fusions ( table 1 )  . 
genomic co alterations were commonly observed in ntrk fusionpositive cancers , including in genes involved in pi3k signaling , tyrosine kinase families , cell - cycleassociated regulators , and the mapk pathway ( fig 3 )  . 
furthermore , it would be of interest to determine whether the salutary effects of ntrk inhibitors in patients who harbor cancers with ntrk fusions can be extended via rational compound design to any of the more common ntrk alterations , such as mutation , amplification , and overexpression . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . ryosuke okamura no relationship to disclose amlie boichard no relationship to disclose jason k . 
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drilon a , li g , dogan s , et al : what hides behind the masc : clinical response and acquired resistance to entrectinib after etv6 - ntrk3 identification in a mammary analogue secretory carcinoma ( masc )  . 
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 predictive biomarkers in metastatic colorectal cancer : a systematic review juan ruiz - baobre , md1 , 2 , 3 ; raju kandimalla , phd2 ; and ajay goel , phd2 purpose the development and use of predictive biomarkers to guide treatment decisions are paramount not only for improving survival in patients with metastatic colorectal cancer ( mcrc ) , but also for sparing them from unnecessary toxicity and reducing the economic burden of expensive treatments . 
we conducted a systematic review of published studies and evaluated the predictive biomarker landscape in the mcrc setting from a molecular and clinical viewpoint . methods studies analyzing predictive biomarkers for approved therapies in patients with mcrc were identied systematically using electronic databases . 
of all the biomarkers analyzed , 1.4% ( two of 148 ) were explored in a prospective manner , whereas 98.6% ( 146 of 148 ) were evaluated in retrospective studies . 
of the latter group , 78.8% ( 115 of 146 ) were not tested in subsequent phases , 9.6% ( 14 of 146 ) were tested in other retrospective cohorts , 8.9% ( 13 of 146 ) were retrospectively tested in at least one or more randomized cohorts , and only 2.7% ( four of 146 ) were prospectively tested in a clinical trial . 
2019 by american society of clinical oncology introduction colorectal cancer ( crc ) remains the third leading cause of cancer - related deaths in the western world . despite ongoing efforts aimed at increased population screening and improved early detection strategies , approximately 20% of patients still present with metastatic disease at diagnosis , and approximately 35% of those who undergo curative surgeries for a localized disease relpase.1 during the past three decades , the median overall survival ( os ) of patients with metastatic crc ( mcrc ) has gradually increased because of the implementation of combined chemotherapy regimens as well as targeted molecular therapies against egfr and angiogenic factors.2 since the identication of ras mutations as a negative predictive marker , antiegfr therapy has had the greatest impact on the management of patients with mcrc ; nonetheless , the response rates of these treatments remain only approximately 40% to 60% . 
in addition , the recognition landscape for immunotherapy in the treatment patients with microsatellite instability - high ( msi - h ) or dna decient mismatch - repair ( dmmr ) mcrc has been encouraging for this subset of patients . 
crc , colorectal cancer ; mcrc , metastatic colorectal cancer ; neoadjuvant - crt , neoadjuvant chemoradiotherapy . 2 2019 by american society of clinical oncology conventional chemotherapy and triuridine / tipiracil the backbone of treatment in patients with mcrc has historically been chemotherapy , and several chemotherapeutic agents are now approved in this setting : uoropyrimidines ( uorouracil [ fu ] and capecitabine ) , oxaliplatin , irinotecan , and since 2015 , triuridine / tipiracil ( tas - 102 )  . fluoropyrimidine - based chemotherapy and tas - 102 . 
studies of the role of thymidylate synthase ( ts ) in uoropyrimidine - based therapy ( primarily fu plus leucovorin ) in various retrospective and prospective studies have yielded discordant results . 
in this regard , multiple studies have shown that low levels of ts expression in metastatic tumor tissues generally correlate with higher overall response rate ( orr ) .3 - 6 surprisingly , such a correlation was not evident when ts levels were measured in primary tumor tissues.4 , 7 similarly , low levels of ts and dihydropyrimidine dehydrogenase in metastatic tumor tissues were associated with a favorable response to fu in patients with mcrc8 ; however , a subsequent report in 2006 did not validate these ndings.9 likewise , the role of thymidine phosphorylase as a predictive biomarker was also investigated , but the results remain inconclusive.10 , 11 in 2009 , a meta - analysis of ve studies examining a total of 861 patients with mcrc concluded that compared with microsatellite - stable patients , msi - h patients did not achieve a statistically signicant better response rate to fu - based chemotherapy.12 similarly , while investigating the relationship between msh2 gene expression and capecitabine efcacy in patients with mcrc , jensen et al13 observed that a higher msh2 expression was associated with a better response . 
in 2015 , hamauchi et al30 found that patients who develop grade 3 or 4 neutropenia during the rst cycle of tas - 102 treatment had a smaller risk of disease progression . 
selected biomarkers the nal analysis . distribution of biomarkers by individual treatment group . ( b ) distribution of biomarkers by study design . antipd - 1 , antiprogrammed cell death - 1 drugs ; fu , uoropyrimidine - based chemotherapy ; irinotecan , irinotecan - based chemotherapy ; msi , microsatellite instability ; oxaliplatin , oxaliplatin - based chemotherapy ; tas - 102 , triuridine / tipiracil . 
 ( * ) predictive biomarker for anti - egfr drugs ( cetuximab / panitumumab ) ; ( ) predictive biomarker for antipd - 1 drugs ( pembrolizumab and nivolumab alone or in combination with ipilimumab )  . testing in 1 randomized cohorts ( n = 13 ; 8.9% ) ras mutations * testing in a clinical trial ( n = 4 ; 2.7% ) msi status oxaliplatin - based chemotherapy . 
the most notable attempts in this regard have been for the ercc1 gene , and as reported from the mavericc ( marker evaluation for avastin research in crc ) trial , intratumoral ercc1 gene expression failed to predict response to oxaliplatin treatment.17 another gene evaluated in this setting was the x - ray repair cross - complementing group 1 ( xrcc1 ) gene , a base excision repair modulator , wherein a polymorphism in this gene ( xrcc1 - 839 arg / gln or gln / gln ) correlated with worse orr to fu / oxaliplatin.18 interestingly , several micrornas ( mirnas ) have been explored for their predictive response potential to fu , leucovorin , and oxaliplatin ( folfox ) or capecitabine plus oxaliplatin ( capeox ) based regimens . 
using a proteomic approach in 2010 , aoyagi et al32 reported that lower levels of plasma soluble vascular endothelial growth factor ( vegf ) receptor 1 were associated with improved disease control in a subset of 46 patients treated with modied fu , leucovorin , and oxaliplatin ( mfolfox6 ) plus bevacizumab . 
in the same year , another study identied a signicant correlation between low angiopoetin - 2 serum levels and better survival outcomes in a cohort of patients with mcrc treated with bevacizumab - based therapy.33 conversely , the correlation between vegf - a levels and the clinical benet of bevacizumab - based chemotherapy is still under evaluation . 
perhaps in the near future , mavericc trial results will offer more insights into the usefulness of plasma vegf - a levels in this setting.17 with regard to the predictive role of ras , in a subset of 230 patients with mcrc treated in a phase iii randomized clinical trial with either irinotecan , fu , and leucovorin ( ifl ) plus placebo , or ifl plus bevacizumab , only the wild - type kras subset of patients obtained a signicantly higher response rate in the bevacizumab arm.35 in addition to the molecular markers listed previously , a few clinical factors have been studied in relation to bevacizumab response . 
two retrospective studies based on the data from the correct ( patients with metastatic colorectal cancer treated with regorafenib or placebo after failure of standard therapy ) and consigna ( regorafenib in subjects with metastatic colorectal cancer who have progressed after standard therapy ) trials have evaluated the usefulness of different computed tomography scan based parameters in predicting the clinical benet of regorafenib therapy . 
one of the rst studies , in 2016 , reported a correlation between improved pfs and lung metastases cavitation before therapy initiation and even at week 8.38 in this study , baseline lung metastases cavitation and changes in the sum of target lesion diameters were deemed to be predictors for improved os in the multivariate analysis . 
a year later , the correct trial also demonstrated a signicant association between survival ( pfs and os ) and several radiologic parameters such as response or stable disease in size and density of lung metastases.39 likewise , highlighting the use of dynamic contrast - enhanced magnetic resonance imaging , a recent study reported that a  . 
these and other potential predictive biomarkers for anti - egfr therapy are summarized in table 3 and in the data supplement . antiprogrammed cell death - 1 drugs : pembrolizumab and nivolumab in may and july of 2017 , the us food and drug administration approved pembrolizumab and nivolumab for the treatment of patients with msi - h mcrc in whom the disease has progressed after treatment with uoropyrimidine , oxaliplatin , and irinotecan therapies . 
almost a year later , in july 2018 , a nivolumab plus ipilimumab combined regimen was approved , which opened up three novel treatment options for patients with msi - h or dmmr mcrc , who represent approximately 5% of all patients with mcrc.23 the belief is that , despite their worse prognosis , a large prolymphocytic inltration and the presence of portion of mutation - associated neoantigens61 confer to patients with msi - positive mcrc the clinical benet they derive from antiprogrammed cell death - 1 ( pd - 1 ) therapy.62 - 65 this exciting discovery has now led to universal msi testing for the management of patients with mcrc . disease monitoring by liquid biopsies recently , liquid biopsies have emerged as powerful tools for monitoring disease evolution and therapeutic response through the analysis of cell - free dna and rna biomarkers in bodily uids . 
one of the rst studies , in 1979 , which reported that a gradual decrease in carcinoembryonic antigen ( cea ) levels during chemotherapy was signicantly associated with better survival rates , was the basis for this concept.66 such a correlation between cea are and improved pfs and os was conrmed a few years later in a subset of 670 patients with mcrc undergoing rst - line chemotherapy.67 although cea is not a crc - specic biomarker , cea monitoring in blood , alone or in addition to ca 19 - 9 , 68 is still performed commonly in routine clinical practice . 
in this context , the accuracy of cea change in predicting disease progression has been demonstrated recently in a study involving 2 , 828 patients from seven rstline clinical trials.69 in addition to this , other analyses of circulating tumor cells70 and endothelial cells71 in mcrc have been undertaken by several groups . 
in 2015 , hansen et al72 reported that circulating levels of mirna - 126 in a subset of 68 patients with mcrc were predictive of tumor response to bevacizumab - based chemotherapy . 
since then , multiple studies have reported distinct genetic alterations in ctdna from patients with primary disease or acquired resistance to antiegfrbased therapies in genes such as kras , nras , met , erbb2 , flt3 , egfr , and map2k1 by droplet digital polymerase chain reaction , beaming , and next - generation sequencing methodologies.74 , 75 using a massively parallel sequencing - based assay in a prospective cohort of 53 patients with mcrc , it was shown that early changes in ctdna during rst - line standard chemotherapy can also predict subsequent radiologic response.76 similarly , 2017 , a study demonstrated a signicant correlation between the decrease in ras mutant clones in blood after 8 weeks of therapy and improved pfs and os in a cohort of patients treated with regorafenib.40 intriguingly , clonal evolution is a dynamic process , yet the emergence of drugresistant clones in circulation increases during treatment , whereas drug withdrawal results in a decrease of such clones . 
the understanding of this fact has paved a path for novel treatment strategies that are already under evaluation as part of the rasintro ( ras mutations in ctdna and anti - egfr reintroduction in mcrc ) study ( clinicaltrials . gov identier : nct03259009 ) and the chronos ( rechallenge with panitumumab driven by ras dynamic of resistance ) trial ( clinicaltrials.gov identier : nct03227926 ) , which are evaluating the predictive impact of ctdna ras mutations on the efcacy of anti - egfr monotherapy rechallenge in patients with ras wild - type mcrc whose disease has progressed after anti - egfrfree chemotherapy . 
in addition , a ve - gene methylation panel for monitoring tumor burden in liquid biopsies using a methylbeaming assay was described recently77 in 182 patients with mcrc treated with chemotherapy and / or targeted therapy , in which the authors discovered a signicant correlation between the dynamics of methylation markers and orr and pfs . discussion despite the tremendous body of effort devoted to the identication of predictive biomarkers for various treatments used in patients with mcrc , thus far only two of such markers have been translated into routine clinical practice . the rst one , the mutations in the ras gene , serves as a negative predictive biomarker that is present in approximately 55% of patients with mcrc78 and correlates with the lack of efcacy of anti - egfr antibody treatments . 
the exciting result of the association between msi - h and response to nivolumab in the rst - in - human clinical trial ( clinicaltrials . gov identier : nct00441337 ) led to two subsequent phase ii clinical trials , which were instrumental in the approval of antipd - 1 drugs ( pembrolizumab or nivolumab ) alone or in combination with ipilimumab ( nivolumab plus ipilimumab ) as a treatment option for patients with msi - h or dmmr mcrc.63 - 65 , 79 other well - described predictive biomarkers used in the management of several tumor types have shown promising usefulness in selecting patients with mcrc for various targeted therapy - based regimens . 
results from two clinical trials in patients with braf v600epositive mcrc have highlighted this mutation as a predictive biomarker for braf inhibitorbased regimens ( data supplement ) .80 , 81 regarding the role of human epidermal growth factor receptor 2 ( her2 ) amplication or overexpression as a predictive biomarker results of two phase ii clinical trials evaluating the dual her2 blockade in a biomarker - selected subset of heavily pretreated patients with mcrc , with either trastuzumab plus lapatinib ( heracles [ her2 amplication for colorectal cancer enhanced stratication ] trial ) or with pertuzumab and trastuzumab ( mypathway trial ) , demonstrate an impressive orr of approximately 30% to 40% ( data supplement ) .82 , 83 for anti - her2based therapies , nonetheless , the discovery and validation of novel predictive biomarkers that can assist in decision making has been a challenging endeavor , resulting in a long list of failed predictive markers . 
in crc , because the use of single - agent chemotherapeutic regimens has shown limited efcacy , and the majority of current treatment options include various combinations of drugs , biomarker discovery for specic drugs is more complicated , not surprisingly , because of the interactions among different cytotoxic agents.84 similar concerns remain regarding developing predictive biomarkers for therapeutic response to bevacizumab , because ( 1 ) it is also not used as a single agent in the clinic , 84 ( 2 ) its mechanisms of action are poorly understood , 85 and ( 3 ) angiogenesis is an intriguingly adaptive process that involves numerous factors.86 presumably , the inherent complexity of angiogenesis has been a substantial hurdle in the attempts to develop responsepredictive biomarkers for other multitargeted antiangiogenic drugs such as aibercept or regorafenib . 
 predictive biomarkers in metastatic colorectal cancer a possible source of discrepancy even when the molecular marker is analyzed in a different region of the same source.88 besides their spatial heterogeneity , tumors are dynamic entities that continue to evolve over time , especially if they are under selective pressure.89 for this reason , the time from sample acquisition to biomarker analysis is of clinical relevance ; however , overlooked in most studies . 
because only approximately 20% of patients with crc present with metastatic disease at the time of diagnosis , it is often the practice or only option available to analyze archival tissues from the primary tumor to identify biomarkers , which is not always optimal.90 this is an issue that patient selection is gaining importance , which is evidenced by the recent initiative , the us national cancer institutes exceptional responder program.91 consideration of extreme phenotypes such as long - term responders and extremely early progressors for biomarker discovery can facilitate successful identication of molecular alterations that better correlate with clinical phenotypes . 
for instance , in the majority of studies presented in this article , there was no consideration of pfs as a selection criterion , and many studies included in the nonresponders patients with stable disease . 
in general , improved orr and longer pfs are superior indicators of the true efcacy of any drug intervention , whereas inclusion of gain in os as a selection feature may inadvertently introduce bias . 
in addition , new biomarker - driven study designs such as basket or umbrella trials , which assign a treatment according to tumor molecular characteristics , not only are going to improve clinical drug development , but also will facilitate improved biomarker validation . 
besides the examples already described previously in the text , new drugs that target tyrosine kinase fusions in genes such as nrtk1 / 2 / 3 , ret , alk , and ros1 are emerging with promising preliminary results in phase i and ii clinical trials that include patients with crc ( data supplement ) .92 - 97 although analysis of clinical specimens with robust followup data from retrospective series or randomized trials are of tremendous value , subsequent prospective clinical cohorts using longitudinally collected specimens are much needed to establish clinically translatable predictive biomarkers . 
in addition , although many surgical specimens are of suitable quality , needle biopsyderived metastatic lesions often yield lower amounts of dna and rna than that required for robust sequencing experiments98 , 99 ; having access to liquid biopsybased predictive markers would be transformative in overcoming this limitation in patients with mcrc . 
furthermore , liquid biopsy biomarkers will improve patient compliance and eliminate the concerns surrounding intratumor heterogeneity associated with tumor or biopsy specimens and may also help in disease monitoring as well as in predicting secondary resistance . the international community has to consolidate initiatives to improve biomarker development studies and , more importantly , undertake conscious efforts to validate the results gathered from retrospective studies in prospective randomized multicenter cohorts . 
last , the implementation of novel high - throughput molecular analytic techniques and the integration of multiomic approaches with clinical and epidemiologic data using machine - learning algorithms will denitely hasten biomarker development in the coming years.100 despite many attempts over the past decades , there remain only two well - established predictive biomarkers , mutations in the ras gene and msi status , that currently guide treatment decisions in patients with mcrc . 
 ruiz - baobre , kandimalla , and goel juan ruiz - baobre travel , accommodations , expenses : bristol - myers squibb , merck sharp & dohme , ipsen , pharmamar no other potential conicts of interest were reported . references siegel rl , miller kd , jemal a : cancer statistics , 2018 . 
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 erbb3 - activating mutations in small bowel adenocarcinomas purpose functional studies have demonstrated that some mutations of erbb3 , which encodes for human epidermal growth factor receptor ( her ) 3 , are oncogenic via activation of the erbb family signaling pathway . 
this study was designed to report the rate of activating erbb3 mutations in small bowel adenocarcinoma ( sba ) , a rare tumor type in which we previously reported a high rate ( 12% ) of erbb2 - activating mutations . materials and methods dna from 74 sbas , previously characterized for erbb2 mutations and mismatch repair status , was submitted for sequencing of erbb3 exons 3 , 6 , 7 , 8 , and 23 . 
mutant allelic frequencies suggested that both mutations were shared by the same clone rather than being harbored by mutually exclusive tumor subclones . conclusion sbas display a high rate of erbb3 - activating mutations , which have been shown to be targetable by anti - her2 therapies . 
2018 by american society of clinical oncology human epidermal growth factor receptor ( her ) 3 is a tyrosine kinase transmembrane receptor and a member of the her receptor family , which consists of four members : epidermal growth factor receptor ( egfr ) , her2 , her3 , and her4 , which are encoded by the egfr , erbb2 , erbb3 , and erbb4 genes , respectively . 
 unlike other members of this family , her3 has an impaired intracellular kinase domain1 and must heterodimerize with egfr , her2 , or her4 to initiate an intracellular signal , which activates downstream survival signaling such as the phosphoinositide 3 - kinase ( pik3ca ) pathway.2 in 2013 , a first report established that erbb3 displays recurrent hotspot mutations in several cancers , especially in colorectal , gastric , 3 and lobular breast cancer4 ; the most frequent amino acid change ( p.v104m / l ) is related to a single nucleotide variation hotspot located in exon 3 . 
sba is usually treated the same way as colorectal cancer , but few studies have been devoted to this cancer type.8 a first report from our team addressing the specific genomic landscape of sba was published by laforest et al , 9 in which 83 sba samples were subjected to targeted panel sequencing ( 46 genes , ion ampliseq cancer hotspot panel v2 )  . 
this study , which was performed before the discovery of erbb3 activating mutations , reported eight genes that were mutated in more than 5% of sba cases : kras , tp53 , apc , smad4 , pik3ca , erbb2 , braf , and fbxw7 . 
errb2 alterations were highly prevalent : 10 tumors ( 12% ) presented either erbb2 - activating point mutations ( n = 7 ) or erbb2 amplifications ( n = 3 )  . 
2 ethics committee under id - rcb number 2008a01058 - 47 . sample preparation tumor samples were collected as described elsewhere.9 briefly , using a 1.0 - mm punch , paraffin - embedded tissues from all cases were sampled from representative areas containing more than 50% of tumor cells . 
sequencing products were purified with sephadex gel ( ge healthcare , vlizy villacoublay , france ) before running them on a 3500 genetic analyzer ( applied biosystems , foster city , ca )  . 
sequencing data were visualized using finch tv ( geospiza , seattle , wa ) with a detection sensitivity of 10% mutated cells . next - generation sequencing ( ngs ) targeted ngs ( a panel of 39 genes ) was performed on samples with co - occurrence of erbb2 and erbb3 . 
of patients ( % ) 74 ( 100 ) median age in years , range 62 years , 32 - 98 male female tumor stage ( tnm classification ) primary tumor site duodenum jejunum ileum dmmr status msi - h histologic grading 40 ( 54 ) 34 ( 46 ) 1 ( 1 ) 25 ( 34 ) 27 ( 37 ) 19 ( 25 ) 2 ( 3 ) 36 ( 49 ) 26 ( 35 ) 12 ( 16 ) 17 ( 23 ) 57 ( 77 ) 20 ( 27 ) 23 ( 31 ) 30 ( 41 ) 1 ( 1 ) abbreviations : dmmr , mismatch repairdeficient ; msi , microsatellite instability ; mss , microsatellite stable ; na , not available raw reads were aligned on the reference human genome hg19 using the tmap aligner v0.3.7 ( life technologies )  . 
only studies with more than 100 patients and an erbb2 / erbb3 mutation rate higher than 5% ( including putative driver or passenger mutations and at least one erbb3 driver mutation ) were taken into account , with the exception of the mutation rate in the genie cohort . 
after combining erbb3 data with the previous genotyping data , a significant correlation was observed with erbb2 mutation three of the four erbb3 - mutated sba samples also displayed an erbb2 - activating mutation . 
 because the activation of two oncogenes in the same pathway is unusual , this finding was validated by subjecting two samples with sufficient dna to another ngs assay targeting both erbb2 and erbb3 to investigate whether these mutations were restricted to tumor subclones . 
in addition to confirming that erbb2 and erbb3 mutations co - occurred in the same tumor , mafs suggested a clonal distribution , ie , that both mutations were indeed harbored by the same tumor cells . erbb3 mutations , mismatch repair ( mmr ) status , and erbb2 mutations in other tumor types we investigated whether the correlation among erbb3 - activating mutations , erbb2 mutations , and msi - h status was also observed in other tumor types by performing in silico analysis of publicly available data . 
 ( * ) p < .01 between msi - h and microsatellite stable ( mss ) cancers . in 1% to 3% of the following cancer types : lobular breast , gastric , colorectal , endometrial , and cervix adenocarcinoma , together with cholangiocarcinoma and bladder cancer.3 , 4 , 11 - 18 this spectrum of erbb3 - mutated cancers overlaps with that of erbb2 - mutated and erbb2 amplified cancers , supporting the biologic concept of her2 - driven cancer because erbb3 activating mutations act via her2 signaling . 
 because sba is the most common form of erbb2 - mutated cancer ( as initially reported by laforest et al9 ) , we hypothesized that erbb3 sequencing would detect a high rate of erbb3 mutations . 
in more detail , three of the four mutated sba samples harbored the most frequent hotspot mutation of erbb3 ( v104m ) , whereas one sample harbored the e928g mutation , which has been mainly described in lobular breast adenocarcinoma18 and found in only one case in the two largest series of colorectal cancer sequencing.17 , 19 a striking finding of this study was the significant co - occurrence of erbb2 and erbb3 mutations across tumor samples . 
oncogenic mutations that activate the same signaling pathway are usually distributed according to a mutually exclusive mode in a given cancer type , eg , kras , nras , and braf mutations in colorectal cancers.17 in breast cancer , the study of heterogeneous erbb2 - amplified tumors ( a rare phenotype ) has shown that erbb2 - amplified subclones coexisted with her2 - mutated subclones.20 however , the ngs targeted analysis performed in the current study on two of the three cases with both erbb3 and erbb2 mutations did not demonstrate any significant differences between their allelic frequencies . 
the three sba samples with both erbb2 and erbb3 mutations exhibited microsatellite instability and likely had a high mutational load , 21 suggesting a possible causality that has yet to be investigated . more recently , a study performed genomic profiling of 317 sba cases , using colorectal and gastric cancers as controls.22 the reported overall erbb3 mutation rate was 6.3% , similar to that observed in our study , and was significantly higher than that observed in colorectal ( p = .001 ) and gastric ( p = .02 ) cancers in their cohort . 
this previous report provided no data regarding erbb3 and erbb2 mutation co - occurrence or the association between erbb3 mutation and msi - h . in 2017 , the food and drug administration granted accelerated approval to an immune checkpoint inhibitor ( pembrolizumab , an anti pd - 1 antibody ) for the treatment of patients with unresectable or metastatic msi - h solid tumors , regardless of the histology , on the basis of the impressive results observed in these tumors.23 for patients with concomitant msi - h tumors and erbb2 / 3 mutations , anti - her2 and antipd - 1 drugs can therefore both be considered as treatment options . 
in view of the clearly demonstrated benefit of antipd - 1 drugs in this setting , 23 , 24 which can achieve long - lasting complete responses , although anti - her2 / 3 drugs are not currently approved in this setting , anti pd - 1 drugs should probably be preferred . 
studies testing the toxicity and efficacy of immune checkpoint inhibitors and anti - her2 / 3 targeted therapies could be particularly relevant for msi - h erbb2 / 3 - mutated cancers . finally , we acknowledge that our study has several limitations . 
these include the limited sample size resulted in large cis for estimation of mutation rates , the lack of fresh frozen material prevented the performance of more comprehensive whole - exome sequencing , and , in cases with dual erbb2 / erbb3 mutations , the absence of single cell sequencing did not allow the demonstration of epistatic interactions between erbb2 and erbb3 mutations . 
hyman dm , piha - paul sa , rodon j , et al : abstract ct001 : neratinib in her2 or her3 mutant solid tumors : summit , a global , multi - histology , open - label , phase 2 basket study . 
pereira b , chin s - f , rueda om , et al : the somatic mutation profiles of 2 , 433 breast cancers refines their genomic and transcriptomic landscapes . 
ng cky , martelotto lg , gauthier a , et al : intra - tumor genetic heterogeneity and alternative driver genetic alterations in breast cancers with heterogeneous her2 gene amplification . 
 response to selective ret inhibition with loxo - 292 in a patient with ret fusion - positive lung cancer with leptomeningeal metastases robin guo , md1 ; mark schreyer , ms1 ; jason c . 
hyman , md1 , 4 ; and alexander drilon1 , 4 introduction the development of leptomeningeal metastases is a poor prognostic factor in patients with advanced cancers.1 - 3 in nonsmall - cell lung cancers ( nsclcs ) , median overall survival of patients from the diagnosis of leptomeningeal disease is 1 to 2 months without treatment and up to 8 months with systemic therapy.4 - 6 furthermore , patients with leptomeningeal disease have historically had limited access to novel therapies in clinical trials . 
recent efforts from many groups , including the european society for medical oncology and the us food and drug administration ( fda ) , have encouraged the inclusion of patients with leptomeningeal metastases in clinical trials , in addition to promoting standardization of intracranial response assessments.7 - 9 these efforts are crucial given that many investigational agents have substantial cns activity and may improve outcomes in driver - positive cancers with leptomeningeal involvement.5 , 10 ret fusions are actionable oncogenic drivers that are identied in 1% to 2% of nsclcs.11 , 12 to date , chemotherapy and / or immunotherapy remain the only approved systemic therapies for these cancers . 
although these agents were found to be active in these patients , outcomes are modest a subset of compared with targeted therapies in other driverpositive lung cancers , and intracranial activity is poor.13 , 14 selective ret inhibitors currently in development , such as loxo - 292 and blu - 667 , have improved outcomes for patients with ret fusion - positive cancers because of increased potency and less offtarget toxicity.15 , 16 in september of 2018 , loxo - 292 received breakthrough therapy designation from the fda for treatment of patients with metastatic ret fusion - positive nsclcs ( as well as ret fusion - positive thyroid cancers and ret - mutant medullary thyroid cancer )  . 
in addition , conrmed intracranial responses and durable disease control have been achieved in patients with brain metastases in an ongoing phase i / ii trial of loxo - 292 for patients with ret fusion - positive cancers.15 its activity in leptomeningeal disease , however , has not previously been characterized . 
computed and positron - emission tomography imaging revealed a hypermetabolic 4.8lobe mass , mediastinal and hilar cm right adenopathy , and osseous metastases involving l1 , the sacrum , and the left anterolateral sixth rib . 
magnetic resonance imaging ( mri ) of the brain showed three subcentimeter enhancing foci in the right precentral lobe . gyrus , right parietal endobronchial biopsy of an r4 lymph node revealed adenocarcinoma with signet ring cell features ( fig 1a )  . tumor cells were positive for ttf - 1 and negative for p40 by immunohistochemistry . 
broad , hybrid capture based next - generation sequencing using the memorial sloan kettering integrated mutation proling of actionable cancer targetsmsk - impactand illumina hiseq 2500 ( illumina , san diego , ca ) 17 identied an eml4 - ret fusion ( fig 1b ) in addition to a tp53 p.p142tfs * 5 frameshift mutation . 
this eml4 - ret fusion was conrmed using a targeted rna - based anchored multiplex polymerase chain reaction archer fusion assay ( archer , boulder , co )  . with identication of the ret fusion , the patient was treated with the investigational anti - ret multikinase inhibitor rxdx - 105.18 , 19 although a conrmed partial response was initially achieved ( a near - complete response in her brain metastases ) , her course was marked by isolated asymptomatic intracranial progression requiring multiple radiation treatments . 
 guo et al eml4 ( nm_019063.3 ) ret ( nm_020630.4 ) 8 9 10 1112 13 14 15 16 1718 1912 13 1415 1617 18 ence eml4 ( + ) nm_019063.3|exon : 19 ret ( + ) nm_020630.4|exon : 12 fig 1 . 
 ( a ) a hematoxylin and eosinstained section from a cell block of a ne - needle aspiration specimen from a lower paratracheal lymph node conrmed a diagnosis of lung adenocarcinoma . 
clusters of malignant epithelial cells with signet - ring cell morphology ( eccentrically placed nuclei , focally prominent nucleoli , and abundant amount of cytoplasm containing grayish - blue mucin ) are shown . 
 ( b ) an in - frame ret fusion containing the ret tyrosine kinase domain was identied in extracted dna from this sample by broad , hybrid capturebased next - generation sequencing using the memorial sloan kettering integrated mutation proling of actionable cancer targetsmsk - impact and illumina hiseq 2500 ( illumina , san diego , ca )  . exon 19 of the 5 ( cid : 1 ) upstream gene partner eml4 was fused to exon 12 of 3 ( cid : 1 ) ret . 
this eml4 - ret fusion was conrmed using an rna - based anchored multiplex polymerase chain reaction ( archer , illumina miseq [ archer , boulder , co ] )  . four months later , the patient developed symptomatic progression of brain metastases and new leptomeningeal disease . 
she presented with left facial , tongue , and upper extremity tingling and worsening neck pathese symptoms were deemed to be secondary to leptomeningeal disease that was identied radiologically in the right hemisphere , predominantly in the right parietal lobe ( fig 2a ; top panel ) , recognizing that nonradiologically apparent disease was likely present in other areas.8 multiple brain metastases had also increased ( largest measuring 2.7 cm in the right frontal lobe ; fig 2a ; bottom panel )  . 
a cant lumbar puncture was recommended , but the patient declined ; a brain biopsy to potentially determine the mechanism of resistance to rxdx - 105 was not deemed safe . extracranial imaging again showed no evidence of disease . given that the patient was highly symptomatic with progressive symptoms , a single - patient use protocol of loxo - 292 was approved by the fda and institutional review board . 
magnetic resonance imaging axial brain images are shown ( a ) at baseline , ( b ) 5 weeks , and ( c ) 21 weeks after the initiation of loxo - 292 therapy in a patient with an eml4ret fusion - positive lung cancer . 
a representative right superior medial and right lower frontal intraparenchymal metastasis ( green arrows ) regressed with loxo - 292 therapy , along with several other metastases followed on serial intracranial response by recist ( response evaluation criteria in solid tumors ) v1.1 and a complete response in leptomeningeal disease by response assessment in neuro - oncology were achieved . 
a conrmed partial patient provided written informed consent before enrollment , and loxo - 292 was administered orally at 160 mg twice daily . this dose was selected based on preliminary safety and efcacy results from an ongoing phase i / ii trial of the drug ( clinicaltrials.gov identier : nct03157128 )  . imaging assessments ( mri of the brain and computed tomography of the chest , abdomen , and pelvis ) were performed every 8 weeks . response was assessed by recist ( response evaluation criteria in solid tumors ) version 1.1.20 response of leptomeningeal metastases was assessed in accordance with response assessment in neuro - oncology criteria.8 additional volumetric three - dimensional imaging was performed on subsequent scans ( sloan kettering advanced imaging lab , sail ; figs 3b and 3c )  . a clinical response to therapy was achieved within the rst week of therapy , with improvement and subsequent resolution of the patients neurologic symptoms . 
in addition , loxo - 292 therapy achieved complete resolution of leptomeningeal in neuroenhancement , with a response assessment oncology leptomeningeal score dropping from 1 at baseline to 0 at 8 weeks . 
volumetric assessment revealed a continued decrease in the total volume of signicant intracranial disease ( leptomeningeal and parenchymal ) , with a maximal shrinkage of 65% ( from 20.1 cm3 at baseline to 7 cm3 ) at 5 months ( fig 3c )  . the patient continues to receive therapy with loxo - 292 at 10.8 months , with ongoing radiologic disease control and no neurologic symptoms . 
volumetric three - dimensional magnetic resonance imaging analyses were performed on serial imaging performed at ( a ) baseline , ( b ) 5 weeks , and ( c ) 21 weeks . 
maximal volumetric disease regression of 65% was achieved at 21 weeks in the graph of total volume over time on loxo - 292 therapy as shown in the bottom panel . 
in nsclcs with leptomeningeal metastases , there is no consensus on the use of wholebrain radiotherapy , because it has not been shown to consistently improve survival.1 , 3 although systemic chemotherapy with more contemporary regimens ( including pemetrexed and / or bevacizumab ) and intrathecal chemotherapy have been shown to improve outcomes in select series , the development of new agents with higher response rates and more durable disease control continues to represent an unmet need for many patients.4 , 6 this report represents the rst description , to our knowledge , of leptomeningeal metastases responding to any systemic therapy in a patient with a ret fusion - positive cancer . 
a brisk and durable ongoing response to loxo - 292 was achieved in a patient with a ret fusion - positive lung cancer who had notable disease progression while 4 2019 by american society of clinical oncology receiving a prior multikinase inhibitor and multiple prior stereotactic radiosurgery treatments . 
in preliminary data from trial ( clinicaltrials.gov identier : an ongoing phase i / ii nct03157128 ) , all four patients with untreated measurable parenchymal metastases had conrmed intracranial responses to therapy accompanied by overall disease control.15 durable intracranial and extracranial disease control was likewise established in several other patients with untreated nonmeasurable brain metastases before loxo - 292 therapy . the activity of loxo - 292 in the cns can be attributed to several factors . 
the drug is active preclinically , with oral dosing in an orthotopic mouse model of a ret fusionpositive patient - derived tumor implanted into the brain.21 , 22 its potency and selectivity for ret are also likely contributory . 
using a highly active agent in the cns is crucial in ret fusion - positive lung cancers , because close to 25% of patients present with intracranial disease at baseline , whereas the lifetime prevalence of brain metastases approaches 50%.14 in addition , loxo - 292 was designed to target potential resistance mechanisms that can emerge from prior multikinase inhibitor use , such as ret v804m / l gatekeeper substitutions . 
 response to loxo - 292 in ret - rearranged leptomeningeal disease fusions are also identied in papillary thyroid , anaplastic thyroid , colorectal , pancreatic , and breast cancers ; spitzoid neoplasms ; and chronic myeloproliferative neoplasms.11 somatic and germline - activating ret mutations are likewise actionable drivers of oncogenesis that are identied in medullary thyroid cancers and potentially other malignancies.33 although the frequency at which intracranial metastases present is lower for many of these other cancers compared with nsclcs , metastatic disease in the cns can occur in some cases.34 in conclusion , selective ret inhibition with loxo - 292 achieved a clinically meaningful and conrmed response in a patient with a ret fusion - positive lung cancer with leptomeningeal disease and heavily pretreated brain metastases . 
although additional conrmation of this activity will help elucidate overall intracranial disease outcomes , this report underscores the potential of selective ret inhibition as a means of treating and preventing the occurrence of disease in the cns in patients with ret - dependent cancers of any histology . affiliations 1memorial sloan kettering cancer center , new york , ny 2loxo oncology , stamford , ct 3new york university langone medical center , new york , ny 4weill cornell medical college , new york , ny s . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . dahlia henry employment : loxo stock and other ownership interests : loxo , allergan , axovant sciences , palatin technologies mark g . 
kris consulting or advisory role : astrazeneca , regeneron , pzer travel , accommodations , expenses : astrazeneca , genentech other relationship : memorial sloan kettering cancer center robert j . 
young stock and other ownership interests : agios , alexion pharmaceuticals , biogen , celgene , gilead sciences , karyopharm therapeutics , spark therapeutics , regeneron , stemline therapeutics , vertex consulting or advisory role : agios , puma biotechnology , nordicneurolab , icon clinical research research funding : agios ( inst ) david m . 
future oncol 14 : 391 - 407 , 2018 omuro am , kris mg , miller va , et al : high incidence of disease recurrence in the brain and leptomeninges in patients with nonsmall cell lung carcinoma after response to getinib . 
cancer 103 : 2344 - 2348 , 2005 lee sj , lee ji , nam dh , et al : leptomeningeal carcinomatosis in non - small - cell lung cancer patients : impact on survival and correlated prognostic factors . j thorac oncol 8 : 185 - 191 , 2013 li ys , jiang by , yang jj , et al : leptomeningeal metastases in patients with nsclc with egfr mutations . 
j thorac oncol 11 : 1962 - 1969 , 2016 park jh , kim yj , lee jo , et al : clinical outcomes of leptomeningeal metastasis in patients with non - small cell lung cancer in the modern chemotherapy era . lung cancer 76 : 387 - 392 , 2012 kim es , bruinooge ss , roberts s , et al : broadening eligibility criteria to make clinical trials more representative : american society of clinical oncology and friends of cancer research joint research statement . 
neuro - oncol 19 : 484 - 492 , 2017 le rhun e , weller m , brandsma d , et al : eano executive board and esmo guidelines committee : eano - esmo clinical practice guidelines for diagnosis , treatment and follow - up of patients with leptomeningeal metastasis from solid tumours . 
liao bc , lee jh , lin cc , et al : epidermal growth factor receptor tyrosine kinase inhibitors for non - small - cell lung cancer patients with leptomeningeal carcinomatosis . 
aisner dl , sholl lm , berry ld , et al : the impact of smoking and tp53 mutations in lung adenocarcinoma patients with targetable mutations - the lung cancer mutation consortium ( lcmc2 )  . 
drilon a , rekhtman n , arcila m , et al : cabozantinib in patients with advanced ret - rearranged non - small - cell lung cancer : an open - label , single - centre , phase 14 . 
oxnard gr , subbiah v , park k , et al : clinical activity of loxo - 292 , a highly selective ret inhibitor , in patients with ret fusion + non - small cell lung cancer : an update from asco 2018 . 
subbiah v , taylor m , lin j , et al : highly potent and selective ret inhibitor , blu - 667 , achieves proof of concept in a phase i study of advanced , ret - altered solid tumors . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
drilon ae , subbiah v , oxnard gr , et al : a phase 1 study of loxo - 292 , a potent and highly selective ret inhibitor , in patients with ret - altered cancers . 
yang jc - h , cho bc , kim d - w , et al : osimertinib for patients ( pts ) with leptomeningeal metastases ( lm ) from egfr - mutant nonsmall cell lung cancer ( nsclc ) : updated results from the bloom study . 
reungwetwattana t , nakagawa k , cho bc , et al : cns response to osimertinib versus standard epidermal growth factor receptor tyrosine kinase inhibitors in patients with untreated egfr - mutated advanced nonsmall - cell lung cancer . 
wu yl , ahn mj , garassino mc , et al : cns efcacy of osimertinib in patients with t790m - positive advanced nonsmall - cell lung cancer : data from a randomized 28 . 
costa db , shaw at , ou sh , et al : clinical experience with crizotinib in patients with advanced alk - rearranged nonsmall - cell lung cancer and brain phase iii trial ( aura3 )  . 
gadgeel s , peters s , mok t , et al : alectinib versus crizotinib in treatment - naive anaplastic lymphoma kinase - positive ( alk + ) non - small - cell lung cancer : cns efcacy results from the alex study . 
shaw at , kim tm , crin `o l , et al : ceritinib versus chemotherapy in patients with alk - rearranged non - small - cell lung cancer previously given chemotherapy and crizotinib ( ascend - 5 ) : a randomised , controlled , open - label , phase 3 trial . 
camidge dr , kim dw , tiseo m , et al : exploratory analysis of brigatinib activity in patients with anaplastic lymphoma kinase - positive nonsmall - cell lung cancer and brain metastases in two clinical trials . 
solomon bj , besse b , bauer tm , et al : lorlatinib in patients with alk - positive non - small - cell lung cancer : results from a global phase 2 study . 
 o genomic resistance patterns to second - generation androgen blockade in paired tumor biopsies of metastatic castration - resistant prostate cancer purpose patients with castration - resistant prostate cancer ( crpc ) receive second - generation androgen - deprivation therapy , but frequently experience relapse or do not respond . 
understanding the genetic mechanisms of resistance will help to identify strategies and biomarkers that are essential for the next line of therapy . patients and methods we analyzed whole exomes of patient - matched preand post - treatment tumors from patients with crpc . 
these patients had received the secondary androgendeprivation therapy agent , abiraterone , which suppresses androgens to below castration levels , or enzalutamide , which competitively inhibits the key androgen signaling effector , androgen receptor . results we observed that abiraterone - resistant tumors harbored alterations in ar and myc , whereas enzalutamide - resistant tumors gained alterations in cell - cycle pathway genes , such as mutation in cyclin - dependent kinase n2a ( cdkn2a ) or amplification of cdk6 . 
experimentally , overexpressing cell - cycle kinases promoted enzalutamide resistance in androgen - sensitive lncap cells that was mitigated via cdk4 / 6 blockadepalbociclib and ribociclib . conclusion cdk4 / 6 - mediated resistance observed in preclinical experiments suggests that cdk4 / 6 amplifications may sufficiently promote enzalutamide resistance in crpc , and that these patients may respond to palbociclib or ribociclib . 
2017 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction prostate cancer is among the most prevalent adult malignancies in men.1 patients with metastatic prostate cancer receive primary androgen - deprivation therapy ( adt ) , and whereas many patients achieve a response , almost all develop castration - resistant prostate cancer ( crpc ; fig 1a )  . 
for patients with crpc , the standard of care includes secondgeneration inhibitors of androgen receptor ( ar ) signaling , including abiraterone2 and enzalutamide.3 these agents effectively prolong survival , but all patients eventually develop resistance . moreover , considering the potential wider usage of abiraterone from recent findings on the benefit of adding abiraterone and prednisone to primary adt in hormone - sensitive advanced prostate cancer , 4 understanding the resistance mechanisms that are specific to these agents is even more critical . previous studies have identified several mechanisms of primary or secondary adt resistance : ar activation , ar bypass , and resistance independent of the ar signaling axis.5 ar - activating alterations include amplifications , 6 mutations , 7 and splicing variants.8 multiple studies of patients with crpc at single time points identified alterations in other pathways , including dna repair9 and cell - cycle pathways , 6 although their specific g . 
patients with resistance to nextgeneration androgendeprivation therapy ( adt ) can be classified according to acquired and intrinsic mechanisms using prostate - specific antigen ( psa ) level change . ( a ) schematic overview of paired ( preand posttreatment ) tumor biopsy collection in the context of next - generation adt , abiraterone , and enzalutamide , followed by whole - exome sequencing and computational analysis to investigate clinical resistance mechanisms . ( b ) change in psa levels between the start of treatment ( at the time of pretreatment biopsy , day 0 ) and the end ( at the time of post - treatment biopsy ) of treatment . 
crpc , castration - resistant prostate cancer . primary prostate crpc germline pretreatment biopsy next - generation ( abiraterone / enzalutamide ) postrelapse biopsy resistant time clinical information psa level patient history radiographic images post whole - exome sequencing identify resistanceassociated alterations clinical resistance mechanisms 6 months patients abi - 1 abi - 2 abi - 3 abi - 4 enza - 1 enza - 2 enza - 3 acquired resistance intrinsic resistance psa50 psa75 psa88 time of treatment ( days ) relationship to treatment resistance has been incompletely characterized . whereas genomic studies of metastatic prostate cancer have demonstrated genomic alterations in ar and its pathway , the genomic characterization of paired biopsy samples from living patients with crpc before secondary adt initiation and at the time of resistance have been limited as a result of the logistical challenges of obtaining repeated tumor biopsies and tumor heterogeneity in metastatic prostate cancer . 
after assessment of pathology and whole - exome sequencing quality metrics ( appendix ) for the 15 patients who were included in this clinical series , results from seven patients were available for analysis ( data supplement )  . 
we also examined clinical information for each patient ( data supplement ) , including prostate - specific antigen ( psa ) levels ( fig 1b ) , treatment history , and radiographic images ( data supplement )  . 
we primarily used therapy duration and changes in psa level to define clinical response.10 we confirmed soft tissue progression using response evaluation criteria in solid tumors ( recist v1.1 ) 11 criteria and bone disease progression using protocol by prostate cancer working group 212 criteria ( appendix )  . 
6 months , with the remaining patients being intrinsically resistant ( fig 1b )  . by this measure , three patients ( abi - 1 , enza - 1 , and enza - 3 ) exhibited acquired resistance , and four patients ( abi - 2 , abi - 3 , abi - 4 , and enza - 2 ) were intrinsically resistant . we then performed mutation , copy number , and phylogenetic analyses of these tumors to nominate putative genetic correlates of resistance by therapeutic class ( appendix )  . 
in abiraterone patients , one patient ( abi - 2 ) , who was clinically classified as intrinsically resistant , harbored a wellcharacterized ar resistance mutation ( l702h ) 13 , 14 in the post - treatment sample that was not detected in the pretreatment sample ( 0 of 62 reads and 17 of 46 reads in preand post - treatment tumors , respectively ; fig 2 )  . 
in two additional intrinsic patients ( abi - 3 and abi - 4 ) , both preand abi - 2 rac1 lmna jak1 tp53 myo5a pretreatment allelic fraction chrx : 66 , 931 , 463 reference : 62 variant : 0 ar p.l702h ( t > a ) reference : 46 variant : 17 fig 2 . 
 ( a and b ) ar , myc amplification , and abiraterone resistance . copy number profile of paired samples of patients abi - 1 , abi - 3 , and abi - 4 across the genome ( top ) are depicted , and the status of chrx and chr8 is displayed for respective patients ( bottom )  . 
vertical bars and alternating colors demarcate the borders between chromosomes . both preand posttreatment samples from abi - 3 and abi - 4 harbored ar amplifications , whereas samples from abi - 1 harbored pre - existing ar amplification but obtained a focal gain of myc in the resistant sample . 
although our observations associate ar with abiraterone resistance , one patient with pre - existing ar focal amplification ( abi - 1 ) demonstrated an initial 50% decrease in psa level response before ultimately developing resistance ( fig 1b )  . 
white line represents the needle used to withdraw tumor tissue . ( b ) in patient enza - 1 , the fraction of mutations unique to the pretreatment and post - treatment states are shown in blue and red , respectively . 
cancer cell fractions ( ccf ) for mutations in the pretreatment ( x - axis ) and post - treatment ( y - axis ) tumors are compared . selected cancer genes are annotated in the ccf plots . ( c ) p81l mutation of cdkn2a , found only in the post - treatment tumor , is visualized in the integrated genomics viewer ( igv ) browser . 
change of nucleotide , amino acid , and number of reads in reference and variant alleles are shown in the box . enza - 1 post enriched in posttreatment tumor tp53 shared by both tumor samples cdkn2a enriched in pretreatment tumor pre - ccf chr9 : 21 , 971 , 116 p81l ( g > a ) reference : 140 variant : 0 p81l ( g > a ) reference : 39 variant : 9 cdkn2a resistance to ar suppression and bicalutamide.15 , 16 our result associates myc with abiraterone resistance independent of ar status , which suggests that genetic changes beyond ar may also contribute to clinical abiraterone resistance . we then examined genetic evolution in the context of clinical resistance to enzalutamide . 
in one patient ( enza - 1 ) with paired biopsy samples that were obtained from the same site ( fig 4a ) , a p81l mutation in cdkn2a was only detected in the resistant tumor ( figs 4b and 4c and data supplement )  . 
this is a clinically observed cancer mutation17 that is also adjacent to a hotspot location ( r80 ) .18 in addition , relative to wild - type cdkn2a , p81l is functionally defective when overexpressed in melanoma cells.19 the posttreatment tumor from a second enzalutamideresistant patient ( enza - 2 ) had chr7q ( spanning cdk6 ) , whereas ar amplification was detected at both time points ( fig 5 )  . 
because the genetic loss of all rb family members promotes the constitutive activation of cdk signaling , we also investigated alterations of rb family proteins ( rb1 , rbl1 , and rbl2 ; data supplement )  . 
in the last acquired - resistance patient ( enza - 3 ) , we did not detect alterations in cell - cycle genes or oncogenic pathways that had been previously associated with adt resistance . the observation of cell - cycle up - regulation specifically from these enzalutamide - resistant patients suggests the activity of cell - cycle kinases in enzalutamide resistance . 
we followed the schematics in figure 6a and used open reading frames that contained cdk4 or cdk6 to overexpress these genes in enzalutamide - sensitive lncap cells.20 , 21 lncap cells were used to examine the efficacy of enzalutamide in preclinical applications , 20 and have recently been used to study acquired resistance to enzalutamide.21 after confirming overexpression by immunoblotting ( data supplement ) , we mimicked adt by first culturing each resulting cell line in media that was supplemented with androgenfree media ( charcoal - stripped serum [ css ] ) for 3 days and , subsequently , in both css and enzalutamide . 
we observed significant differences in adt proliferation , as cdk4 / 6 - expressing cells continued to proliferate , whereas luciferaseexpressing negative control cells failed to do so ( p , .005 ; two - tailed t test ; fig 6b )  . in combination with the estrogen inhibitor letrozole , the cdk4 / 6 inhibitor palbociclib has recently been approved by the us food and drug administration for treatment of estrogen receptorpositive breast cancers.22 another cdk4 / 6 inhibitor , ribociclib , has demonstrated efficacy in rb wildtype23 and ar mutant prostate cancer cells.24 in two clinical trials , cdk4 / 6 inhibition was thought to benefit prostate cancers that express wild - type rb ( clinicaltrials.gov identifiers : nct02059213 and nct02555189 )  . 
copy number profiles of paired samples of patient enza - 2 across the genome ( top ) are depicted , and the status of chr7 is displayed ( bottom )  . 
to test the clinical potential of combining adt with cdk4 / 6 inhibitors specifically in enzalutamide - resistant crpcs with cdk4 / 6 amplifications , we again used our preclinical model in which cdk4 / 6 sufficiently promoted enzalutamide resistance . 
the average of three experiments is plotted and error bars represent standard deviation . ( * ) overexpression of a gene led to a significant difference compared with luciferase control cells . 
 ( ) cell - cycle inhibitor treatment led to a significant reduction of proliferation ( p , .005 , two - tailed t test )  . enzalutamide - resistant metastatic crpcs that have cdk4 / 6 amplifications in patient cases of clinical resistance that are either intrinsic or acquired . discussion in summary , we used a paired biopsy approach and have associated the clinical resistance of abiraterone with ar alterations and , in one patient , with myc . 
two enzalutamide - resistant patients harbored aberrations in cell - cycle pathway genes . our preclinical data demonstrate that cdk4 / 6 overexpression sufficiently drove enzalutamide resistance , but this phenotype was abrogated by cdk4 / 6 inhibitors . 
for enzalutamide - resistant patients , we identify the specific cell - cycle mutations , cdkn2a and cdk4 / 6 , as biomarkers that may predict whether an enzalutamide - resistant patient could benefit from combination therapy that involves cdk4 / 6 inhibition and enzalutamide . 
in this study , we do not disambiguate enzalutamide resistance from general adt resistance ; however , other forms of adt resistance , including ar - splice variants , that promote enzalutamide resistance22 , 25 , 26 and general castration resistance27 demonstrate a challenge in mechanistically discerning treatment - specific or class - wide resistance mechanisms . 
addressing differences via expanded clinical cohorts and diverse preclinical models may determine strategies by which patients with such resistance may be treated . although two patients were classified as intrinsically resistant by clinical parameters , paired analysis demonstrated clinically relevant alterations in ar and cell - cycle genes only in the resistant tumor . 
in addition , difficulties in consistently obtaining anatomically matched biopsies at two time points for a subset of this cohort make definitive conclusions about resistance mechanisms or tumor heterogeneity difficult to tease apart . 
mechanistically , we speculate that adt - resistant clonal evolution can rapidly occur ; therefore , matched tissue and / or blood - based biopsies sampled at more finite intervals , along with rna analysis for ar splicing and transcriptional changes , if available , may inform us of the dynamic selection of resistant subclones and patients who were not explained solely by bulk tumor biopsies . 
hahn , mary - ellen taplin financial support : mary - ellen taplin administrative support : mary - ellen taplin provision of study materials or patients : zhenwei zhang , glenn j . 
balk honoraria : janssen pharmaceuticals consulting or advisory role : sanofi patents , royalties , other intellectual property : license to nkt therapeutics for an antibody expert testimony : astellas medivation travel , accommodations , expenses : janssen pharmaceuticals william c . 
ca cancer j clin 66 : 7 - 30 , 2016 de bono js , logothetis cj , molina a , et al : abiraterone and increased survival in metastatic prostate cancer . 
scher hi , halabi s , tannock i , et al : design and end points of clinical trials for patients with progressive prostate cancer and castrate levels of testosterone : recommendations of the prostate cancer clinical trials working group . j clin oncol 26 : 1148 - 1159 , 2008 13 . 
comstock ces , augello ma , goodwin jf , et al : targeting cell cycle and hormone receptor pathways in cancer . 2016 oncogene 32 : 5481 - 5491 , 2013 24 . 
antonarakis es , lu c , wang h , et al : ar - v7 and resistance to enzalutamide and abiraterone in prostate cancer . n engl j med 371 : 1028 - 1038 , 2014 26 . 
hu r , dunn ta , wei s , et al : ligand - independent androgen receptor variants derived from splicing of cryptic exons signify hormone - refractory prostate cancer . 
cancer res 69 : 16 - 22 , 2009 patients who were eligible for this analysis had metastatic castration - resistant prostate cancer ( crpc ) and were being considered for next - generation androgen - deprivation therapy ( adt ) using abiraterone acetate or enzalutamide . 
all patients provided written informed consent to obtain metastatic tumor biopsies and for molecular profiling of preand post - treatment tumor and germline samples . biopsies and pathology review paired biopsies were collected as outlined in the protocols of the clinical trials described above and in a prior study ( mckay rr , et al : clin cancer res 23 : 935 - 945 , 2017 )  . 
pretreatment baseline biopsies were obtained before starting therapy , and post - treatment progression biopsies were obtained at the time of radiographic progression ( fig 1a ) while the patients were still on treatment . 
matched trios of germline , pre - , and post - treatment tumor samples were obtained for all patients , and seven patients who were treated with abiraterone ( n = 4 ) or enzalutamide ( n = 3 ) among an initial 15 patients passed the quality control . 
clinical responses to therapy were determined using the response evaluation criteria in solid tumors ( recist v1.1 ) 11 for soft tissue progression and prostate cancer working group 212 for bone disease progression . participants were evaluated every 4 weeks by prostate - specific antigen and every 12 weeks by computed tomography and bone scan and were not taken off treatment until documented symptomatic progression or progression by imaging as protocol defined by recist ( v1.1 ) and prostate cancer working group 2 . whole - exome sequencing and sequence data processing whole - exome sequencing was performed on extracted dna using illumina ( san diego , ca ) and agilent technologies ( santa clara , ca ) platforms . 
exome sequencing data were processed using established analytic pipelines at the broad institute of mit and harvard ( cambridge , ma ; van allen em , et al : nat med 20 : 682 - 688 , 2014 )  . 
bam files were produced via the picard pipeline ( picard.sourceforge.net / ) , uploaded , and processed through the firehose pipeline ( cga / firehose )  . bam files are currently in process for submission to the database of genotypes and phenotypes . data availability sequencing quality control exome sequence data processing and analysis were performed using pipelines at the broad institute . 
from the patients we examined , individuals with immediate cessation of therapy , with low coverage ( germline , 350 , tumor , 3100 ) in one or more tumors , or those who experienced the failure of manual copy number profile inspection were filtered out ( data supplement )  . 
furthermore , individuals with tumor purity of > 0.15 for all matched trios of germline , pre - , and posttreatment tumor samples were obtained , which resulted in seven individuals for additional analyses . 
no additional selection process was used to identify the seven individuals for analysis . variant calling and phylogenetic analysis to identify somatic single - nucleotide variants , mutect ( cibulskis k , et al : nat biotechnol 31 : 213 - 219 , 2013 ) was applied and reviewed with integrated genomics viewer ( igv ; robinson jt , et al : nat biotechnol 29 : 24 - 26 , 2011 )  . 
artifacts from dna oxidation during sequencing were removed using a filtered - based method ( costello m , et al : nucleic acids res 41 : e67 , 2013 )  . 
 variants in cancer genes reported from the cosmic cancer gene census ( cancer.sanger.ac.uk / census ; futreal pa , et al : nat rev cancer 4 : 177 - 183 , 2004 ) bona fide cancer genes that are causally implicated in cancersare shown in allelic frequency scatter plots as in figure 1d . probability distributions of possible cancer cell fractions of point mutations were calculated on the basis of local copy number and the estimated sample purity was inferred using absolute ( carter sl , et al : nat biotechnol 30 : 413 - 421 , 2012 )  . copy number analysis copy ratios were segmented using the circular binary segmentation algorithm ( olshen ab , et al : biostatistics 5 : 557 - 572 , 2004 )  . 
rescaled copy numbers for each genomic segment were calculated using absolute ( carter sl , et al : nat biotechnol 30 : 413 - 421 , 2012 ) to correct copy ratios for variations in sample purity and ploidy . 
to classify segments into categories , including high - level amplification , amplification , homozygous deletion , and no alteration , the focality of each genomic segment was calculated as previously described ( brastianos pk , et al : cancer discov 5 : 1164 - 1177 , 2015 )  . 
after the change of copy number alterations was computed , we selected copy number alterations , such as gained high amplification and gained amplification categories , that were present in the post - treatment tumor only . 
after 4 days of selection , cell lysates were collected and expressions of open reading frames were determined using immunoblots with cdk4 and cdk6 antibodies ( cell signaling technology , danvers , ma )  . 
after confirming their respective expression , each cell line was cultured in androgen - free media for 3 days , and vi - cell was used to count and seed 100 , 000 cells in quadruplicates in a 12 - well plate for 14 days in androgenfree media ( charcoal - stripped serum ; atlanta biologicals , atlanta , ga ) with enzalutamide 2.5 mm and either ribociclib 500 nm , palbociclib 500 nm ( selleck chemicals , houston , tx ) , or dmso control . 
lncap cells are not among the cell lines that are easily misidentified as described by the international cell line authentication committee . these cells are purchased directly from american type culture collection ( manassas , va ) , which maintains authenticated cell lines by sequencing and comparing short tandem repeats to parental lncap cells in their database . 
before the experiments , cells were tested for several strains of mycoplasma contamination using mouse / rat comprehensive clear panel w / corynebacterium bovis from charles river laboratories ( wilmington , ma )  . 
 comprehensive analysis of the unfolded protein response in breast cancer subtypes purpose triple - negative breast cancers ( tnbcs ) are associated with a worse prognosis and patients with tnbc have fewer therapeutic options than patients with non - tnbc . 
recently , the ire1a - xbp1 branch of the unfolded protein response ( upr ) was implicated in tnbc prognosis on the basis of a relatively small patient population , suggesting the diagnostic and therapeutic value of this pathway in tnbcs . 
in addition , the ire1a - xbp1 and hypoxia - induced factor 1 a ( hif1a ) pathways have been identified as interacting partners in tnbc , suggesting a novel mechanism of regulation . 
to comprehensively evaluate and validate these findings , we investigated the relative activities and relevance to patient survival of the upr and hif1a pathways in different breast cancer subtypes in large populations of patients . materials and methods we performed a comprehensive analysis of gene expression and survival data from large cohorts of patients with breast cancer . 
the patients were stratified based on the average expression of the upr or hif1a gene signatures . results we identified a strong positive association between the xbp1 gene signature and estrogen receptorpositive status or the hif1a gene signature , as well as the predictive value of the xbp1 gene signature for survival of patients who are estrogen receptor negative , or have tnbc or her2 +  . 
2017 by american society of clinical oncology introduction triple - negative breast cancers ( tnbcs ) represent a breast cancer subpopulation lacking estrogen receptor ( er ) , progesterone receptor ( pr ) , and her2 expression.1 patients with tnbc have a worse prognosis than those with non - tnbc , suggesting more aggressive biologic behavior . 
the unfolded protein response ( upr ) is an adaptive process to alleviate endoplasmic reticulum stress during tumor growth and proliferation.2 one major upr signaling pathway , ire1a leading to activation of xbp1 , is implicated in multiple types of cancers , including breast cancer.3 a recent study reported that tnbc cell lines and primary tumor samples from patients with tnbc express higher levels of the activated form of xbp1 than non - tnbc.4 furthermore , this study also revealed that in tnbc cells , xbp1 coregulates hif1a target genes , and that both xbp1 and hif1a gene signatures were associated with poor prognosis in tnbc , but not in patients with er + breast cancer . 
for example , the expression of esr1 , gata3 , and xbp1 were highly correlated in breast cancer.5 , 6 in addition , xbp1 enhances the transactivation activity of era7 and was identified as an estrogenresponsive gene.8 recently , our laboratory identified that doxorubicin , a widely used chemotherapeutic agent in breast cancer treatment , is a potent inhibitor of xbp1 activation.9 therefore , clarifying the role of xbp1 in breast cancer prognosis would not only improve the application of existing treatment strategy but also would dadi jiang brandon turner jie song ruijiang li maximilian diehn quynh - thu le purvesh khatri albert c . 
using the same four luminal and six basal - like cell lines reported by chen et al , 4 ( a ) average expression of the xbp1 gene signature was higher in the luminal type , although not statistically significant , whereas ( b ) average expression of the atf4 / chop signature was significantly higher in the basal - like type . 
the cell line collection consists of 19 estrogen receptor ( er ) + and 32 er , or 25 triple - negative breast cancer ( tnbc ) and 26 non - tnbc cell lines defined on the basis of mrna / protein levels of er / progesterone receptor / her2 . 
 ( e ) the same analysis on microarray data of patients with breast cancer from the cancer genome atlas database ( n = 404 er + and n = 118 er , or n = 91 tnbc and n = 431 non - tnbc )  . 
 support the preclinical development of therapy on the basis of modulating xbp1 activity.10 , 11 in this study , we comprehensively investigated the activity of xbp1 , its relationship with the er status as well as the hif1a pathway , and its prognostic value in a large group of patients with breast cancer . 
the rnaseq ( n = 1 , 182 ; illumina hiseq ; illumina , san diego , ca ) and microarray ( n = 597 , a subset of the 1 , 182 patients ; agilentg4502a_07_3 array ; agilent technologies , santa clara , ca ) data with the clinical information of the patients with breast cancer were downloaded from the cancer genome atlas ( tcga ) database through the university of california santa cruz cancer browser . 
all signature and expression data were normalized as z - scores with a mean of 0 and standard deviation of 1 . differences in average expression ( microarray or rnaseq ) level of the gene signatures by er and tnbc status were assessed using the nonparametric wilcoxon rank - sum test . 
mrna expression levels of foxa1 , esr1 , and gata3 were first individually normalized ( mean , 0 ; standard deviation , 1 ) and then averaged together before comparing with either the xbp1 gene signature expression or xbp1 gene expression using regression . 
cox proportional hazards regression models were fit to breast cancer survival data , and the nonparametric log - rank test was used to test for significant differences in relapse - free survival ( rfs ) between subtypes of patients with high or low levels of average xbp1 , atf4 / chop , or hif1a gene signature expression . 
patient survival data up to 150 months were used for the analysis . cell culture and viability assay mda - mb - 231 , hs578t , mda - mb - 468 , mcf7 , t47d , and bt474 cells were purchased from american type culture collection ( manassas , va ) and maintained in dmem supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin , and cells were cultured at 37c with 5% co2 . 
after 72 hours of treatment , xtt reagent ( american type culture collection ) was added to the wells , then cells were incubated for 2 hours , and cell viability was calculated with the following formula : absorbance = a475nm ( test ) a475nm ( blank ) a660nm ( test ) using a biotek synergy h1 plate reader ( biotek , winooski , vt )  . results first , we tested the initial conclusion of the previous study4 that basal - like breast cancer cell lines , which consist primarily of tnbc cells , express higher levels of the spliced / activated form of xbp1 compared with those of the luminal type , which consist primarily of er + cells.4 we found the average expression of the same xbp1 gene signature used by the previous investigators to measure xbp1 activity4 was actually higher in the same set of luminal ( n = 4 ) than basal - like ( n = 6 ) cell lines , albeit without statistical significance ( fig 1a )  . 
 2 average expression of the xbp1 gene signature was significantly higher in er + compared with cell lines , consistent with previous reports ( fig 1c ) .5 - 8 however , there was no statistically significant difference in the xbp1 signature between tnbc and non - tnbc cell lines ( fig 1c )  . in contrast , expression of the atf4 / chop sigor tnbc than in er + or nature was higher in er non - tnbc cell lines , respectively ( fig 1c )  . in established cell these observations lines prompted us to evaluate these pathways in tumor samples from patients with breast cancer . 
we performed the same analyses on 1 , 182 patients with breast cancer from tcga , on the basis of rnaseq ( fig 1d ) , 5 or a subset of 597 patients on the basis of microarray ( fig 1e ) , as well as 1 , 809 curated microarray gene expression profiles ( fig 1f ) .14 in each comparison , we found expression of the xbp1 gene signature was significantly higher in er + or non - tnbc than in er or tnbc tumor samples , respectively . 
overall , we identified a strong correlation between the expression of xbp1 , which is also a transcriptional target of activated xbp1 itself , 4 and er pathway genes ( ie , esr1 , gata3 , and foxa1 ; fig 2a and 2b )  . 
this relationship was also significant when we correlated the average expression of the xbp1 gene signature with the average er pathway gene expression ( fig 2c and 2d )  . these data are consistent with a previous report of xbp1 playing a crucial role in 17 - b - estradiolinduced growth of er + breast cancer cells.15 to test the reported coregulation of target gene expression by activated xbp1 and hif1a4 in a large cohort of patients with breast cancer , we performed similar correlation analysis using the tcga dataset . 
overall , we found a strong correlation between the xbp1 and hif1a gene signatures in patients either er + or er , and with non - tnbc or tnbc , who were from tcga ( fig 2e and 2f )  . 
this result confirms the molecular - level interaction between the two pathways with large - scale gene - expression data from patients and suggests the xbp1 and hif1a pathways are highly correlated in breast cancer regardless of the subtypes . to assess the influence of the upr and hif1a pathways on survival of patients with breast cancer and extend the analysis of the previous study4 to larger patient cohorts , we performed survival analyses of the 1 , 809 - patient data set , using the same high - low expression cutoff ( 58th percentile ) as previously reported.4 surprisingly , the xbp1 signature was not associated with worse rfs in the whole population of patients with breast cancer ( fig 3a , left )  . 
however , when the patient population was further stratified into tnbc versus , or her2 + versus non - tnbc , er + versus er , higher expression of the xbp1 signature her2 was associated with worse rfs in tnbc ( fig 3b , ( fig 3c , left ) , or her2 + ( fig 3d , left ) left ) , er patients , but not in patients positive for er ( fig 3c , left ) or negative for her2 ( fig 3d , left )  . 
interestingly , the atf4 / chop signature was not associated with rfs ( fig 3a , middle ) , even after stratification into either tnbc / non - tnbc , er + / 2 her2 + / 2 status ( fig 3b - 3d , middle )  . 
in contrast , the hif1a signature was significantly associated with rfs regardless of how the patients were stratified ( fig 3a - 3d , right )  . to evaluate whether using gene signature expression cutoffs at the extremes would reveal stronger association , we applied quartile ( the highest 25% v the lowest 25% ) cutoff and performed the same survival analysis . 
the atf4 / chop signature became significantly associated with rfs in the whole population ( data supplement ) and the xbp1 signature became significantly associated ( data with rfs in patients who were her2 supplement )  . 
however , the positive association between the xbp1 signature and rfs in the patients with non - tnbc using the 58th percentile cutoff became negative when using the quartile cutoff , which likely was due to the reduced number of patients in the new categories ( data supplement )  . 
nevertheless , the major conclusions of the current study remain unchanged . discussion in summary , on the basis of a comprehensive analysis on large cohorts of patients with breast cancer , our findings confirm the initial observations from chen et al4 that xbp1 activity was associated with a worse prognosis in tnbc , but not er + patients , and extend this relationship to ( fig 3c , left )  . 
correlation of xbp1 mrna expression and the xbp1 gene signature with estrogen receptor ( er ) pathway gene expression and correlation of the xbp1 and hif1a gene signatures in the the cancer genome atlas ( tcga ) breast cancer data set . 
correlation analysis of expression of the xbp1 gene and the average of three er pathway genes ( esr1 , foxa1 , and gata3 ) using ( a ) rnaseq or ( b ) microarray data from 1 , 182 patients with breast cancer in tcga breast cancer database and the same correlation analysis of the average expression of the xbp1 gene signature and the three er pathway genes on the basis of ( c ) rnaseq or ( d ) microarray data in the tcga database . 
 ( e , f ) correlation of the average expression of the xbp1 and hif1a gene signatures in the groups of patients with breast cancer , on the basis of the rnaseq data in the tcga database : ( e ) er + ( n = 600 patients , gold ) and er ( n = 179 patients , blue ) ; and ( f ) triple - negative breast cancer ( tnbc ; n = 125 patients , blue ) and non - tnbc ( n = 647 patients , gold )  . 
kaplan - meier graphs showing relapse - free survival ( rfs ) of the different subtypes of patients with breast cancer stratified by the xbp1 , atf4 / chop , or hif1a gene signatures . 
kaplan - meier graphs of rfs of ( a ) the total of 1 , 640 patients , ( b ) 225 patients with triple - negative breast cancer ( tnbc ) and 1 , 415 with non - tnbc , ( c ) 1 , 286 who are estrogen receptor ( er ) + and 354 who are er with rfs < 150 months separated by high and low ( with the 58th percentile cutoff used previously4 ) levels of the average expression of the xbp1 ( left column ) , atf4 / chop ( middle column ) , or hif1a ( right column ) gene signature . 
this may partially explain those who are her2 why the xbp1 - rfs association is stronger in than in those with tnbc patients who are er ( fig 3b and 3c , left ) , because some of the patients / her2 + are included in the patients who are er but not in the patients with tnbc . who are er however , through analyzing large - scale gene expression profiles , we identified that average expression of the same xbp1 gene signature reported by chen et al4 was higher in er + or non - tnbc or tnbc cell lines or patients , respecthan er tively ( fig 1c - 1f ) , to correct the initial conclusion by chen et al.4 these data are consistent with other previous studies . 
our findings suggest that , albeit with higher xbp1 activities , patients who are er + , and other patients with non - tnbc ( excluding her2 + ) , may not benefit from therapeutic strategies targeting xbp1 activity . 
the design and aims of the current study are summarized in the schematic shown in the data supplement . recently , we have shown that doxorubicin blocks xbp1 activation through the inhibition of ire1a rnase activity.9 these data suggest that pa , and possibly tients with tnbc , who are er those who are her2 + , may benefit the most from anthracycline - based therapies . 
davies mp , barraclough dl , stewart c , et al : expression and splicing of the unfolded protein response gene xbp - 1 are significantly associated with clinical outcome of endocrine - treated breast cancer . 
finlin bs , gau cl , murphy ga , et al : rerg is a novel ras - related , estrogen - regulated and growth - inhibitory gene in breast cancer . 
j biol chem 276 : 42259 - 42267 , 2001 jiang d , lynch c , medeiros bc , et al : identification of doxorubicin as an inhibitor of the ire1a - xbp1 axis of the unfolded protein response . 
jiang d , niwa m , koong ac : targeting the ire1a - xbp1 branch of the unfolded protein response in human diseases . semin cancer biol 33 : 48 - 56 , 2015 11 . 
gy orffy b , lanczky a , eklund ac , et al : an online survival analysis tool to rapidly assess the effect of 22 , 277 genes on breast cancer prognosis using microarray data of 1 , 809 patients . 
sengupta s , sharma cg , jordan vc : estrogen regulation of x - box binding protein - 1 and its role in estrogen induced growth of breast and endometrial cancer cells . 
lacroix m , leclercq g : about gata3 , hnf3a , and xbp1 , three genes co - expressed with the oestrogen receptoralpha gene ( esr1 ) in breast cancer . 
tozlu s , girault i , vacher s , et al : identification of novel genes that co - cluster with estrogen receptor alpha in breast tumor biopsy specimens , using a large - scale real - time reverse transcription - pcr approach . 
andres sa , wittliff jl : relationships of esr1 and xbp1 expression in human breast carcinoma and stromal cells isolated by laser capture microdissection compared to intact breast cancer tissue . 
wang dy , fulthorpe r , liss sn , et al : identification of estrogen - responsive genes by complementary deoxyribonucleic acid microarray and characterization of a novel early estrogen - induced gene : eeig1 . 
karn t , metzler d , ruckhaberle e , et al : data - driven derivation of cutoffs from a pool of 3 , 030 affymetrix arrays to stratify distinct clinical types of breast cancer . 
wirth , md1 ; takashi kohno , phd2 ; hibiki udagawa , md , phd3 ; shingo matsumoto , md , phd3 ; genichiro ishii , md , phd3 ; kevin ebata , phd4 ; brian b . 
michael rothenberg , md , phd4 ; and koichi goto , md , phd3 introduction the ret receptor tyrosine kinase is oncogenically activated by ret gene fusions in 1% to 2% of nonsmall - cell lung cancers ( nsclc ) and by ret gene mutations in most medullary thyroid cancers ( mtc ) .1 although multikinase inhibitors ( mkis ) with nonspecic anti - ret activity are approved for the treatment of mtc irrespective of ret mutation status , their investigational use for patients with ret fusion - positive nsclc has been limited by substantial off - target adverse effects that lead to dose reductions and inadequate ret - specic inhibition.2 - 7 as a result , tumor responses to mkis have been infrequent and short lived , and a comprehensive molecular understanding of mki response and resistance is lacking . in contrast to the mkis , loxo - 292 is a highly selective , small molecule ret tyrosine kinase inhibitor ( tki ) with nanomolar potency against diverse ret alterations , favorable pharmacokinetic ( pk ) properties , and signicant cns penetration.8 in an ongoing , phase i / ii identier : study of loxo - 292 ( clinicaltrials.gov nct03157128 ) , conrmed partial responses ( prs ) were achieved in most ret - altered patients across different tumor histologies and diverse ret gene fusions and mutations . 
a 42 - yearold woman with nsclc had received nine prior systemic therapy regimens , including approved chemotherapy , immunotherapy , and investigational tkis , before the identication of a kif5b - ret fusion in a sample from a recurrent pleural fusion ( fig 1a , data supplement )  . 
she received alectinib and additional chemotherapy and then vandetanib ( fig 1a ) .6 she experienced a conrmed pr by response evaluation criteria in solid tumors ( recist ) 1.1 with vandetanib treatment , with improvement in multiple lung and pleural - based lesions . 
targeted ret gene sequencing of a biopsy specimen from a progressing right lung lesion identied a ret valine - to - leucine ( v804l ) gatekeeper mutation absent from a pretreatment tumor sample ( fig 1a , data supplement )  . v804l allele frequency in the progression sample was 15% by next - generation sequencing ( 120 of 799 reads ) compared with 0% in the pretreatment sample ( 0 of 847 reads ; data supplement )  . the patient received additional systemic chemotherapy with pemetrexed and amrubicin but experienced continued disease progression . 
after providing written informed consent , she started treatment with loxo292 in the phase i portion of the phase i / ii clinical trial at the recommended phase ii dose of 160 mg twice a day . 
 ( a ) various treatments the patient received for metastatic , kif5b - ret fusion - positive nonsmall - cell lung cancer are shown , together with the levels of carcinoembryonic antigen ( cea ; red diamonds ) and the sum of target lesions by response evaluation criteria in solid tumors ( recist ) 1.1 ( blue squares ) over time during treatment . 
the estimated levels of ret target inhibition at the 50% inhibitory concentration ( ic50 ) and 90% inhibitory concentration ( ic90 ) for kif5b - ret and kif5b - ret - v804l were modeled using actual patient human pharmacokinetic parameters ( data supplement )  . 
 ( c ) computed tomographic images of the patients metastatic lung and liver disease before and at the indicated times after initiating treatment with vandetanib ( left three pairs of panels ) or loxo - 292 ( right two pairs of panels )  . 
consistent with this , she experienced a rapid clinical and biochemical response to loxo - 292 , with decreased shortness of breath and reduction in serum carcinoembryonic antigen ( fig 1a )  . repeat imaging after 8 weeks of treatment demonstrated a 48% decrease in measurable tumor burden by recist 1.1 that was maintained after 16 weeks , indicating a conrmed pr ( figs 1a and 1c )  . 
the patient has received loxo - 292 for more than 11 months , is still receiving treatment , and is tolerating therapy well , without dose interruption , and with all treatment - emergent adverse events grade 1 or 2 . case 2 : hereditary ret v804m mtc . 
a 50 - year - old man with hereditary ret v804m multiple endocrine neoplasia 2a and mtc developed primary disease progression despite previous treatment with three anti - ret mkis ( fig 2a )  . before treatment with loxo - 292 , he was highly symptomatic , with abdominal pain from tumor inltration of the liver and severe tumor - related diarrhea . 
consistent with this , serum carcinoembryonic antigen and calcitonin levels decreased rapidly , together with resolution of diarrhea , abdominal distension , and abdominal parepeat imaging after 8 weeks of treatment indicated a complete response by recist 1.1 , which was conrmed after 12 weeks of treatment , with complete resolution of target lymph node lesions and nontarget liver memediastinal tastases and normalization of serum tumor markers ( figs 2c and 2d )  . 
the patients dose was increased to 160 mg twice a day as allowed by the protocol , with doseproportional increases in exposure and continuous more than ic90 ret v804m target coverage ( fig 2b ) , continued complete response , and continued normalization of tumor markers ( fig 2c )  . 
the patient experienced three adverse events : transient grade 3 altered mental status ( sedation ) , judged by the investigator to be related to concomitant treatment with anxiolytic medications and unrelated to loxo - 292 ; grade 1 constipation ; and grade 2 nausea . 
he remains in complete response and is still receiving treatment at more than 20 months after the start of loxo - 292 . loxo - 292 but not anti - ret mkis maintain inhibitory activity against ret gatekeeper mutations preclinically . 
in contrast to mkis , loxo - 292 is predicted to better accommodate the bulky leucine and methionine side chains in the gatekeeper residues without any steric interactions ( fig 3b )  . to determine the impact of ret kinase domain resistance mutations on inhibitor activity , loxo - 292 , vandetanib , and cabozantinib were tested in vitro against the puried wildtype and mutant ret kinase domains ( fig 3c )  . 
at physiologic ( 1 mm ) atp concentration , loxo - 292 had potent inhibitory activity against wild - type ret , ret v804l , and ret v804m kinases . the anti - ret mkis , in contrast , vandetanib and cabozantinib , displayed signicantly reduced activity against ret v804l / m compared with wildtype ret . 
these data demonstrate that loxo - 292 , but not the mkis vandetanib or cabozantinib , maintain potent inhibitory activity against ret gatekeeper mutations . discussion selective tkis targeting actionable kinase alterations have transformed the treatment landscape for several human cancers . 
although acquired resistance to tkis is universal , a detailed molecular understanding of resistance has led to the development of next - generation tkis that have extended disease control and clinical benet in resistant patients.16 , 17 in several instances , initial treatment with the next - generation inhibitor has led to more durable treatment responses than treatment with the rstgeneration tki and has become standard upfront systemic therapy.18 , 19 loxo - 292 combines features of both rstand nextgeneration tkis used to treat other single kinase - driven cancers . 
it is highly potent against and selective for diverse founder activating ret alterations in human cancers , and in preclinical experiments , it overcomes ret v804 gatekeeper mutations , which have been identied in rare patients with acquired resistance to anti - ret mkis . 
for both patients , inhibiintegration of preclinical ret target tory activity with the actual exposures safely achieved at the recommended phase ii dose of 160 mg twice a day allowed more than ic90 calculated target coverage of both wild - type and gatekeeper - mutated ret in patients ( figs 1b and 2b )  . 
 ( b ) pharmacokinetic analysis at steady state after 8 days of treatment with loxo - 292 at a dose of 80 mg , 120 mg , and 160 mg twice a day . 
the estimated levels of ret target inhibition at the 50% inhibitory concentration ( ic50 ) and 90% inhibitory concentration ( ic90 ) for wild - type ( wt ) ret and ret v804m were modeled as in fig 1b . 
the red arrows indicate diffuse inltration of the liver by tumor . lenvat , lenvatinib ; rt , radiation treatment ; vande , vandetanib . treatment with loxo - 292 after the prior mkis has signicantly extended the period of durable clinical and biochemical response and disease control . 
 wirth et al hibiki udagawa honoraria : astrazeneca , chugai pharmaceutical , bristol - myers squibb , ono pharmaceutical , msd , taiho pharmaceutical , amco , abbvie , boehringer ingelheim , daiichi sankyo consulting or advisory role : abbvie , boehringer ingelheim research funding : msd , abbvie , daiichi sankyo , amgen shingo matsumoto honoraria : pzer , novartis , chugai pharma , astrazeneca research funding : chugai pharma ( inst ) , novartis ( inst ) , lilly ( inst ) , merck serono ( inst ) , merck sharp & dohme ( inst ) kevin ebata employment : loxo oncology stock and other ownership interests : loxo oncology travel , accommodations , expenses : loxo oncology brian b . 
tuch employment : loxo oncology stock and other ownership interests : loxo oncology consulting or advisory role : kezar life sciences patents , royalties , other intellectual property : biomarkers for ntrk inhibition ; biomarkers for proteasome inhibition edward y . 
zhu employment : loxo oncology stock and other ownership interests : loxo oncology travel , accommodations , expenses : loxo oncology michele nguyen employment : loxo oncology stock and other ownership interests : loxo oncology travel , accommodations , expenses : loxo oncology steve smith consulting or advisory role : loxo oncology , various patents , royalties , other intellectual property : various patents and applications travel , accommodations , expenses : various lauren m . 
burkard employment : array biopharma , university of colorado hospital ( i ) stock and other ownership interests : array biopharma , pzer , pzer ( i ) james f . 
blake employment : array biopharma stock and other ownership interests : array biopharma patents , royalties , other intellectual property : i am listed as a co - author on approximately 46 patents ( inst ) kevin r . 
brandhuber employment : loxo oncology stock and other ownership interests : loxo oncology , array biopharma travel , accommodations , expenses : loxo oncology steve andrews employment : loxo oncology leadership : loxo oncology stock and other ownership interests : loxo oncology travel , accommodations , expenses : loxo oncology s . 
michael rothenberg employment : loxo oncology stock and other ownership interests : loxo oncology travel , accommodations , expenses : loxo oncology koichi goto honoraria : bristol - myers squibb , astrazeneca , pzer , chugai pharmaceutical , taiho pharmaceutical , ono pharmaceutical , novartis , eli lilly , boehringer ingelheim , quintiles , merck serono , life technologies , msd , abbvie , riken genesis , nippon kayaku , takeda pharmaceuticals , otsuka pharmaceutical , srl diagnostics consulting or advisory role : otsuka pharmaceutical research funding : msd , astrazeneca , taiho pharmaceutical , chugai pharmaceutical , boehringer ingelheim , ono pharmaceutical , sumitomo dainippon , takeda pharmaceuticals , novartis , daiichi sankyo , kyowa hakko kirin , astellas pharma , eisai , eli lilly , pzer , riken genesis , bristol - myers squibb , merck serono , ignyta , life technologies , research triangle institute d / b / a rti health solutions , janssen , xcoo , loxo oncology no other potential conicts of interest were reported . references ji jh , oh yl , hong m , et al : identication of driving alk fusion genes and genomic landscape of medullary thyroid cancer . 
wells sa jr , robinson bg , gagel rf , et al : vandetanib in patients with locally advanced or metastatic medullary thyroid cancer : a randomized , double - blind phase iii trial . 
j clin oncol 30 : 134 - 141 , 2012 drilon a , rekhtman n , arcila m , et al : cabozantinib in patients with advanced ret - rearranged non - small - cell lung cancer : an open - label , single - centre , phase 2 , single - arm trial . 
lancet oncol 17 : 1653 - 1660 , 2016 lin jj , kennedy e , sequist lv , et al : clinical activity of alectinib in advanced ret - rearranged non - small cell lung cancer . 
j thorac oncol 11 : 2027 - 2032 , 2016 yoh k , seto t , satouchi m , et al : vandetanib in patients with previously treated ret - rearranged advanced non - small - cell lung cancer ( luret ) : an open - label , multicentre phase 2 trial . 
lancet respir med , 5 : 42 - 50 , 2017 lee sh , lee jk , ahn mj , et al : vandetanib in pretreated patients with advanced non - small cell lung cancer - harboring ret rearrangement : a phase ii clinical trial . 
ann oncol 28 : 292 - 297 , 2017 brandhuber bb , haas j , tuch bb , et al : ena - 0490 the development of loxo - 292 , a potent , kdr / vegfr2 - sparing ret kinase inhibitor for treating patients with ret - dependent cancers . 
aacr - nci - eortc international conference on molecular targets and cancer therapeutics , munich , germany , november 29 - december 2 , 2016 oxnard gr , subbiah v , park k , et al : clinical activity of loxo - 292 , a highly selective ret inhibitor , in patients with ret fusion + non - small cell lung cancer : an update from asco 2018 . 
wirth lj , cabanillas me , sherman ej , et al : clinical activity of loxo - 292 , a highly selective ret inhibitor , in patients with ret - altered thyroid cancers : an update from asco 2018 . 
drilon ae , subbiah v , oxnard gr , et al : a phase 1 study of loxo - 292 , a potent and highly selective ret inhibitor , in patients with ret - altered cancers . 
yang , m , cai j , guo s , et al : rapid conversion to resistance , of a colon pdw with ret - fusion , by ponatinib treatment could potentially be attributed to the introduction of the gate keeper mutation , v804m . 
dagogo - jack i , stevens se , lin jj , et al : emergence of a ret v804m gatekeeper mutation during treatment with vandetanib in ret - rearranged nsclc . j thorac oncol 13 : e226 - e227 , 2018 15 . 
nat commun 9 : 625 , 2018 j anne pa , yang jc , kim dw , et al : azd9291 in egfr inhibitor - resistant non - small - cell lung cancer . 
ou sh , ahn js , de petris l , et al : alectinib in crizotinib - refractory alk - rearranged non - small - cell lung cancer : a phase ii gobal study . 
pao w , miller va , politi ka , et al : acquired resistance of lung adenocarcinomas to getinib or erlotinib is associated with a second mutation in the egfr kinase 2016 293 : 876 - 880 , 2001 domaplos med 2 : e73 , 2005 22 . 
katayama r , khan tm , benes c , et al : therapeutic strategies to overcome crizotinib resistance in non - small cell lung cancers harboring the fusion oncogene eml4 - alk . 
 intratumoral cd3 + and cd8 + t - cell densities in patients with dna mismatch repairdecient metastatic colorectal cancer receiving programmed cell death - 1 blockade sakti chakrabarti , md1 ; lucas j . 
sinicrope , md1 introduction the programmed cell death - 1 ( pd - 1 ) pathway represses type 1 t - helper cell cytotoxic immune responses and is upregulated in many tumors and in their microenvironment . 
blockade of this pathway with antibodies to pd - 1 has led to robust clinical responses in patients with many tumor types , including a subset of metastatic colorectal cancers ( mcrcs ) with decient dna mismatch repair ( dmmr ) .1 the clinical benet of pd - 1 blockade by pembrolizumab in dmmr mcrc was rst reported in a phase ii study in which the objective response rate ( orr ) and immunerelated progression - free survival rate at 20 weeks were 40% ( four of 10 patients ) and 78% ( seven of nine patients ) , respectively.2 expansion of this cohort to 40 patients indicated an orr of 52%.1 among dmmr tumors , mechanisms underlying responsiveness or resistance to pd - 1 blockade remain unknown , 1 and a predictive biomarker has not been identied within this tumor subtype . colorectal cancers ( crcs ) with dmmr have high microsatellite instability ( msi - h ) and are associated with a high mutational burden , neoantigen load , and typically a dense lymphocytic inltrate.3 in a recent report , the densities of cd3 + and cd8 + t cells were found to be signicantly more heterogeneous in dmmr versus procient mismatch repair ( pmmr ) colon cancers and were shown to prognostically stratify patients with both dmmr and pmmr tumors.4 , 5 on the basis of these data , we hypothesized that the responsiveness of dmmr mcrcs to immunotherapy may be related to the extent of inltration . 
tumor response was assessed by radiographic studies using response evaluation criteria in solid tumors ( recist ) version 1.1.6 the study was approved by the institutional review board of mayo clinic . informed consent was obtained from all patients before collection of data and tumor specimens for analysis . tumors were analyzed for mmr protein ( mlh1 , msh2 , msh6 , and pms2 ) expression by immunohistochemistry ( ihc ) or using a polymerase chain reactionbased assay for msi.7 in archival primary colorectal tumor tissues , cd3 + and cd8 + t - lymphocyte staining was performed by ihc ( ventana medical systems , tucson , az ) , and t - cell densities were determined in tumor core ( ct ) and invasive margin ( im ) by automated image analysis ( appendix fig a1 )  . 
analyses of cd3 + and cd8 + density scores and programmed death ligand 1 ( pd - l1 ) expression were performed by investigators blinded to clinical characteristics and patient outcomes . 
density values for cd3 + and cd8 + t cells , calculated by dividing the cell count by the area ( square millimeters ) within ct or im compartments , were used to calculate a density score ( 0 to 100 ) for each marker by compartment ( cd3 + im , cd3 + ct , cd8 + im , and cd8 + ct ) , as previously described.5 pd - l1 protein expression in tumor cells and in tumor - inltrating immune cells was determined by ihc using ventana pd - l1 ( sp263 ) rabbit monoclonal antibody and was graded according to the percentage of tumor cells with membranous pdl1 expression or as the percentage of positive immune cells in the inammatory inltrate.8 patient and tumor characteristics , details of prior treatment , and pembrolizumab response data are listed in table 1 . 
box plots illustrating the distribution of cd3 + and cd8 + t - cell density ( score range , 0 to 100 ) at the tumor core ( ct ) and the invasive margin ( im ) among the responders and nonresponders . 
responders are dened as patients with complete responses ( crs ) and partial responses ( prs ) ; nonresponders are dened as those with stable disease ( sd ) or progressive disease ( pd )  . regimens received was one ( range , one to four regimens ; one regimen in seven patients , two regimens in three patients , and four regimens in two patients )  . 
median follow - up time of the study cohort since initiation of pembrolizumab was 19.5 months ( range , 9 to 41 months ) in all patients , 21 months ( range , 16 to 24 months ) in nonresponders , and 16 months ( range , 9 to 41 months ) in responders . patient radiographic response data were as follows : two complete responses ( crs ) , ve partial responses ( prs ) , four patients with stable disease , and one patient with progressive disease . 
among pembrolizumab - treated patients who had a cr or pr ( n = 7 ) , the median time to response was 12 weeks ( range , 6 to 40 weeks )  . to examine the relationship between t - cell density scores and treatment response , patients were divided into responders ( cr and pr , n = 7 ) and nonresponders ( stable disease and progressive disease , n = 5 ) to pembrolizumab . median values and ranges of cd3 + and cd8 + t - cell density scores at the ct ( cd3 + ct and cd8 + ct ) and im ( cd3 + im and cd8 + im ) in each response category are provided in figure 1 and table 2 . 
all median t - cell density scores were higher in responders than in nonresponders , with score for cd3 + ct ( 74 v 52 , differences that were greatest respectively ) and cd8 + ct ( 88 v 37 , respectively ) , as shown in figure 1 . 
all median t - cell density scores were higher in the patient group in whom disease control was achieved for more than 12 months ( n = 8 ) , with the greatest score difference seen for cd8 + ct ( 88 v 36 for patients with disease control for more than 12 months v less than 12 months , respectively )  . median tumor cell pd - l1 expression was 2% ( range , 1% to 40% ) among responders and 1% ( range , 0% to 60% ) among nonresponders . 
median pd - l1 expression in intratumoral immune cells was 5% for both responders ( range , 2% to 25% ) and nonresponders ( range , 1% to 10% )  . 
of the ve nonresponding patients , one patients tumor underwent testing for a jak2 mutation , which was not detected . discussion the ability to distinguish high versus low immunogenic tumors could enable selection of patients who are more likely to benet from immunotherapy . 
we used an automated analysis of cd3 + and cd8 + tumor - inltrating lymphocyte density quantitation that was initially reported in stage ii colon cancers and found to be signicantly prognostic independent of mmr status.9 more recently , we validated this platform in nearly 600 stage iii colon cancers from a clinical trial of folfox - based adjuvant chemotherapy where cd3 + and cd8 + t - cell densities were shown to prognostically stratify patients with both dmmr and pmmr colon cancers.5 we found that both cd3 + and cd8 + t - cell density scores in ct and im were higher in patients with dmmr tumors who beneted from pembrolizumab , both in terms of objective response rate and duration of disease control . despite variability observed in our small patient cohort , median density scores of t - cell markers were higher in responders versus nonresponders and in those in whom disease control was achieved for more than versus less than 12 months . 
conversely , these response data suggest that lower t - cell densities are associated with immunotherapy resistance in dmmr tumors and are consistent with the reported nding that lower t - cell densities are associated with worse survival in patients with dmmr stage iii colon cancers receiving cytotoxic chemotherapy.5 our data are consistent with the ndings that increased intratumoral cd8 + t cells can predict response to immunotherapy in patients with metastatic melanoma10 and in patients with head and neck cancers.11 median follow - up of 21 months among nonresponders exceeded the median follow - up time of 12.5 months that was reported in the largest published series of patients with dmmr mcrcs treated with pd - 1 blockade.1 in that study , the mean time to the best radiographic response was 7 months , 1 suggesting that our study follow - up time was of sufcient duration to detect response outcomes . studies have shown higher expression of tumor pd - l1 protein in dmmr versus pmmr crcs , 3 and our analysis of the cancer genome atlas database revealed higher pd - l1 mrna levels in dmmr versus pmmr primary colon cancers ( unpublished data )  . 
in the current study of patients with dmmr and msi - h crcs , we found similar median levels of pd - l1 expression on tumor cells and on inltrating immune cells in responders versus nonresponders . studies of crc , expression of pd - l1 has not been shown to be a predictive biomarker , in contrast to data in patients with nonsmall - cell lung cancer . 
furthermore , a phase ii study of patients with dmmr mcrcs treated with pembrolizumab found that the expression of pd - l1 was not signicantly associated with progression - free survival or overall survival.2 insight into responses to antipd - 1 blockade in dmmr colon cancers is suggested by functional analysis in a responding patient in which rapid in vivo expansion of neoantigen - specic t - cell clones that were reactive to mutant neopeptides were found in the tumor.1 immune inltration may reect the tumors clonal evolution during treatment . 
potential causes of immunotherapy resistance include 2 - microglobulin and jak1 / 2 mutations , 12 and in our study , only one of ve nonresponders was tested for a jak2 mutation , which was not detected . 
other potential biomarkers in dmmr tumors such as the elimination of neoepitopes and differences in t - cell clonal diversity and tumor microenvironment proles warrant additional study . it is important to recognize that dmmr and msi - h are predictive biomarkers for responsiveness to immune checkpoint inhibitors . 
tumor mutation burden ( tmb ) may be a promising biomarker and has been studied in relationship to a t - cellinamed gene expression prole across 22 tumor types.13 although tmb and t - cell gene expression signature were poorly correlated , their combination jointly predicted responders to pembrolizumab , suggesting that features of neoantigenicity and t - cell activation.13 using tmb as a biomarker for immunotherapy responsiveness has important limitations in that not all mutations generate neoantigens , the correlation of tmb with neoantigen load is variable , and an established cutoff point remains unresolved.14 they may capture distinct in summary , we observed that higher cd3 + and cd8 + t - cell densities were associated with higher objective response rates and duration of disease control in patients with dmmr mcrc treated with pembrolizumab . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . andrea muranyi employment : roche stock and other ownership interests : roche patents , royalties , other intellectual property : roche travel , accommodations , expenses : roche june clements employment : ventana medical systems stock and other ownership interests : ventana medical systems research funding : ventana medical systems shalini singh employment : ventana medical systems stock and other ownership interests : ventana medical systems patents , royalties , other intellectual property : method of identifying treatment - responsive nonsmall - cell lung cancer using alk as a marker ( inst ) joleen m . 
hubbard consulting or advisory role : bayer ( inst ) research funding : boston biomedical ( inst ) , senhwa biosciences ( inst ) , bayer ( inst ) , merck ( inst ) , taiho pharmaceutical ( inst ) , treosbio ( inst ) , effector therapeutics ( inst ) robert r . 
mcwilliams consulting or advisory role : merrimack ( inst ) , zeno pharmaceuticals , newlink genetics ( inst ) research funding : prism biolab ( inst ) , genentech ( inst ) , novartis ( inst ) , newlink genetics ( inst ) , eli lilly ( inst ) , aduro biotech ( inst ) , pzer ( inst ) , sano ( inst ) , merck ( inst ) , bristol - myers squibb ( inst ) kandavel shanmugam employment : ventana medical systems stock and other ownership interests : roche patents , royalties , other intellectual property : patents ( inst ) travel , accommodations , expenses : ventana medical systems frank a . 
sinicrope stock and other ownership interests : illumina honoraria : imedex , american society of clinical oncology consulting or advisory role : roche ( inst ) , bristol - myers squibb ( inst ) , ventana medical systems ( inst ) , haliodx ( inst ) research funding : ventana medical systems ( inst ) travel , accommodations , expenses : ventana medical systems no other potential conicts of interest were reported . references le dt , durham jn , smith kn , et al : mismatch repair deciency predicts response of solid tumors to pd - 1 blockade . 
science 357 : 409 - 413 , 2017 le dt , uram jn , wang h , et al : pd - 1 blockade in tumors with mismatch - repair deciency . 
n engl j med 372 : 2509 - 2520 , 2015 llosa nj , cruise m , tam a , et al : the vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter - inhibitory checkpoints . 
cancer discov 5 : 43 - 51 , 2015 pag `es f , mlecnik b , marliot f , et al : international validation of the consensus immunoscore for the classication of colon cancer : a prognostic and accuracy study . 
lancet 391 : 2128 - 2139 , 2018 yoon hh , shi q , heying en , et al : intertumoral heterogeneity of cd3 + and cd8 + t - cell densities in the microenvironment of dna mismatch - repair - decient colon cancers : implications for prognosis . 
eur j cancer 45 : 228 - 247 , 2009 sinicrope fa , mahoney mr , smyrk tc , et al : prognostic impact of decient dna mismatch repair in patients with stage iii colon cancer from a randomized trial of folfox - based adjuvant chemotherapy . 
j clin oncol 31 : 3664 - 3672 , 2013 el jabbour t , ross js , sheehan ce , et al : pd - l1 protein expression in tumour cells and immune cells in mismatch repair protein - decient and - procient colorectal cancer : the foundation study using the sp142 antibody and whole section immunohistochemistry . 
j clin pathol 71 : 46 - 51 , 2018 gibbs ph , hutchinson r , tran b , et al : immune prole and survival outcomes in stage 2 colon cancer . 
partlov a s , bou cek j , kloudov a k , et al : distinct patterns of intratumoral immune cell inltrates in patients with hpv - associated compared to non - virally induced head and neck squamous cell carcinoma . 
 ( a ) hematoxylin and eosin ( he ) stained whole - tissue sections were manually annotated to outline the entire tumor region ( red line ) and demarcate the invasive margin ( orange line )  . 
 ( b and c ) annotations were transferred onto adjacent cd3 + and cd8 + ihc images with an algorithm outlining the invasive margin ( im ; green line ) and core of the tumor ( ct ; red line )  . 
 repeated abscopal effect with radiotherapy and programmed death 1 blockade in mismatch repairdeficient endometrial cancer introduction radiotherapy for cancer has long been valued for its ability to target specific lesions to achieve tumor regression and improve patient symptoms . 
though the effect of radiotherapy is typically limited to its targeted fields , reports have described response in distant tumors a phenomenon termed the abscopal effect.1 prior studies have demonstrated that this effect may be mediated by an enhanced immune response against tumors , 2 possibly elicited by tumor specific antigens released by cancer cells exposed to radiation.3 interest in leveraging the abscopal effect has been revived by recent advances in immunotherapy.4 inhibitors of immune checkpoints , including cytotoxic t - lymphocyteassociated protein 4 ( ctla - 4 ) , programmed death 1 ( pd - 1 ) , and programmed death ligand 1 ( pd - l1 ) , have been shown to induce a strong clinical response in multiple cancer types.5 - 8 also , the us food and drug administration has approved pd - 1 inhibitors for use against any solid tumors that have deficiencies in dna mismatch repair ( mmr ) .9 these treatments block signaling pathways that normally suppress the immune system and thus can unleash a potent antitumor immune response.8 it has been hypothesized that immunotherapies , given their immune - based mechanisms , might synergize with radiotherapy and induce a more consistent and intense abscopal effect . in one early report , a patient with metastatic melanoma was treated with ipilimumab , a ctla - 4 inhibitor , and treatment resulted in significant regression in multiple areas of cancer after radiation was administered to a paraspinal mass.10 the patient had temporally associated increases in activated cd4 + t cells and antibodies against cancer antigens , which supports an immunologic basis for this effect . 
pathology demonstrated grade 1 endometrial adenocarcinoma with myometrial invasion and abnormal peritoneal washings , consistent with stage iiic2 disease per the international federation of gynecology and obstetrics staging syste of note , immunohistochemical studies showed absent expression of the mmr proteins mlh1 and pms2 ( fig 1 )  . the patient was given adjuvant paclitaxel 175 mg / m2 and an area under the curvebased carboplatin target dose of 5 for six cycles , but a subsequent ct scan revealed progression of disease . 
she received six cycles of doxorubicin ( reduced to 50 mg / m2 per cycle for severe neutropenia ) , but the follow - up ct scan showed enlarged pelvic lymph nodes . 
oh young kwang chae author affiliations and support information ( if applicable ) appear at the end of this article . corresponding author : young kwang chae , md , mph , mba , department of medicine , northwestern university feinberg school of medicine , robert h . 
at this time , immunotherapy was considered because of data to suggest its efficacy in mmr - deficient tumors.14 meanwhile , the patients performance status deteriorated rapidly , and she was no longer able to ambulate without assistance , in part because cutaneous metastases presented as large fungating masses on her left thigh . 
ultimately , she started nivolumab 3 mg / kg every 2 weeks ; the first cycle occurred 23 months after surgery . two months after nivolumab treatment initiation , a ct scan demonstrated a mixed response , with improved pulmonary metastatic disease but increased pelvic lymph nodes and a nodule of the sigmoid mesentery . 
because of worsening leg pain , the patient was referred to a radiation oncologist and received palliative radiation to her left thigh , which consisted of 25 gy in five fractions via en face photon beams . 
notably , she also experienced decreased size of hepatic masses , sigmoid nodule , and retroperitoneal and pelvic lymphadenopathy . ct obtained 2 months afterward concerningly showed progression of the sigmoid nodule and pelvic lymphadenopathy as well as marked growth of a splenic lesion and a 7 - cm l3 vertebral metastasis that was causing significant back pa the patient was again referred for palliative radiotherapy and received 20 gy to the lumbar spine in five fractions using conventional anterior - posterior / posterior - anterior ( appa ) technique . 
clinically , the patients back pain significantly improved ; with the aid of physical therapy , she was able to ambulate independently agahowever , the patient developed recurrent difficulties with walking , and progression of a nodal mass in the left external iliac chain was discovered . 
a repeat ct scan showed reduction in this mass as well as response in the hepatic and splenic masses to near resolution . by this time , it was noted that the patient had exhibited a presumed abscopal effect after each of her radiation treatments ( fig 2 )  . 
 ( a ) metastases targeted by radiotherapy ( rt ) were in the ( 1 ) left thigh ; ( 2 ) l3 vertebra ; ( 3 , 4 ) left external iliac chain ; and ( 5 ) bladder , as identified by red circles on computed tomography ( ct ) images . 
 ( b ) ct images of liver metastases ( gold circle and arrow ) , which were located on an axial plane distinct from any radiation fields , showed response to rt treatments ( numbered as in [ a ] )  . 
the patient did develop hematuria and dysuria consistent with radiation - induced grade 2 cystitis , but she otherwise continues to tolerate nivolumab without high - grade toxicities . discussion we describe here the case of a patient with widely metastatic endometrial cancer who exhibited abscopal responses after multiple rounds of palliative radiotherapy while on nivolumab . 
this patient experienced disease response to five separate radiotherapy treatments with concurrent pd - 1 blockade , which yielded an ongoing survival benefit that lasted longer than 28 months as of this writing . abscopal effects are difficult to clearly identify , because multiple factors affect patient response . 
 for example , tumor shrinkage after the first radiotherapy treatment may have represented a delayed effect of nivolumab , which can cause pseudoprogression before a definite clinical response.15 the repeated benefit with radiotherapy in this case , however , suggests the presence of a true abscopal effect . 
however , one clinical trial showed that the use of ipilimumab with pelvic radiotherapy for prostate cancer did not result in a greater than - expected increase in toxicities.16 a retrospective analysis of 133 patients with various cancer types who received both immunotherapy and radiotherapy similarly did not uncover an increase in major adverse effects.17 thoughtful and judicious use of these treatments , even in a repeated fashion , may be tolerated by many patients . not all patients treated with radiotherapy exhibit an abscopal effect , and such an effect may be dependent on patient - specific immune parameters . 
for example , this patients primary tumor exhibited mmr deficiency , which is a possible sensitizing factor to radiotherapy , 18 presumably because the ability of cancer cells to repair dna damage inflicted by radiation is reduced.19 in addition , mmr deficiency has been associated with an increase in tumor mutations and tumor - specific neoantigens.9 this attribute may be beneficial in the context of a desired abscopal effect , because radiotherapy could amplify the immunogenic stimulus provided by these neoantigens . 
 relevant studies have almost exclusively been performed in preclinical models , but this evidence suggests that concurrent radiotherapy and immunotherapy is more beneficial than sequential administration.20 the ideal dosing regimen to achieve an abscopal effect also remains undetermined , but one study that used mouse models of breast and colon cancer showed that a fractionated strategy was superior to a single large dose.21 although these approaches certainly were effective in our patient , forthcoming data from prospective trials are awaited.22 our report adds to the overall evidence in support of an abscopal effect , and it raises the possibility that immunotherapy can be periodically boosted by radiotherapy ; however , additional studies are needed to validate such an approach . 
sharabi ab , nirschl cj , kochel cm , et al : stereotactic radiation therapy augments antigenspecific pd - 1 - mediated antitumor immune responses via cross - presentation of tumor antigen . 
herbst rs , baas p , kim d - w , et al : pembrolizumab versus docetaxel for previously treated , pdl1 - positive , advanced nonsmall - cell lung cancer ( keynote - 010 ) : a randomised controlled trial . 
rosenberg je , hoffman - censits j , powles t , et al : atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinumbased chemotherapy : a single - arm , multicentre , phase 2 trial . 
kwon ed , drake cg , scher hi , et al : ipilimumab versus placebo after radiotherapy in patients with metastatic castration - resistant prostate cancer that had progressed after docetaxel chemotherapy ( ca184 - 043 ) : a multicentre , randomised , double - blind , phase 3 trial . 
bang a , wilhite tj , pike lrg , et al : multicenter evaluation of the tolerability of combined treatment with pd - 1 and ctla - 4 immune checkpoint inhibitors and palliative radiation therapy . 
martin lm , marples b , coffey m , et al : dna mismatch repair and the dna damage response to ionizing radiation : making sense of apparently conflicting data . 
dewan mz , galloway ae , kawashima n , et al : fractionated but not single - dose radiotherapy induces an immune - mediated abscopal effect when combined with antictla - 4 antibody . 
it also re - examines some of the conventional wisdom that previously dominated clinical trial design and discusses development and internal validation of a predictive biomarker as a new paradigm for optimizing the intended - use subset for a treatment . 
some previous paradigms for clinical trial design can limit the development of more effective methods on the basis of prospectively planned adaptive methods , but useful new methods have been developed for analysis of genome - wide data and for the design of adaptively enriched studies . 
in many cases , the heterogeneity of populations eligible for clinical trials as traditionally dened makes it unlikely that molecularly targeted treatments will be effective for a majority of the eligible patients . 
with the completion of the human genome project and the initiation of large dna sequencing of human tumors , somatic mutations were discovered that accounted for the invasion and progression of cancers . 
it became evident that the traditional tumor classications on the basis of site and histologic type were inadequate for developing therapeutics . the discovery of somatic mutations led to major changes in the development of oncology drugs . 
the focus of oncology drug development switched to developing inhibitors of mutated protein kinases and blocking the protein products of amplied receptors . the new strategies of drug discovery required new strategies for clinical trial design . 
the traditional assumption that qualitative interactions are unlikely was frequently shown to be wrong . at about the same time , new whole - genome assays such as rna transcript microarrays , copy number microarrays , and methylation arrays were becoming available . 
the resulting statistical methods were based on classication and prediction , which enabled the development of classiers on the basis of high - dimensional assays for predicting prognosis and response to therapy for individual patients.3 , 4 this article will highlight some of the important developments in clinical trial design and classication analysis that have been stimulated by the dramatic changes in oncology drug development . 
 simon context key objective to describe new and innovative methods for discovering and using predictive biomarkers to develop effective treatments in the presence of patient heterogeneity . knowledge generated a method was developed for discovering and validating therapeutically relevant patient subsets using a new paradigm on the basis of predictive classication rather than multiple hypothesis testing . 
with the development of drugs targeted to specic genomic alterations , attention has shifted to phase ii studies with eligibility determined by specic genomic alterations rather than histologic type , the so - called basket clinical trial.5 - 9 large basket trials include multiple drugs , each with a targeted genomic alteration . 
patients have their tumors sequenced , and then they are assigned the drug appropriate to the genomic alteration found in their tumor . basket trials have generally been sized by using standard methods either for inference on the basis of all patients with the genomic alteration histologic type or for separate inference for each histologic type . 
recently , bayesian designs have been developed that attempt to share response information among histology baskets for patients whose tumor contains the same genomic alteration.10 some of these are based on hierarchical models of treatment effects . 
however , cunanan11 has shown that the prior distributions for such designs must be chosen carefully or the false - positive error for some histology baskets may be highly inated . 
if the accumulating information indicates homogeneity of response probabilities , then the baskets are essentially pooled and a small sample size sufces . cunanan12 developed a frequentist design on the basis of two - stage sampling . 
otherwise , sampling and inference are conducted independently for the baskets . the platform design is for settings with multiple treatments and multiple candidate biomarker strata.13 the design is based on the assumption that the candidate biomarkers are the same for each drug , which somewhat limits the realm of applications . 
most platform designs are bayesian designs and the term promising is interpreted to mean that the posterior probability of a positive phase iii trial is at least 80% . in a platform design , a patient is initially randomly assigned with equal probability to receive any of the drugs . 
the objective is to nd candidates for phase iii trials with a smaller total sample size than would be required for conducting a separate simon two - stage optimal trial in each of the strata.14 the bayesian models used are generally based on hierarchical modeling.13 , 15 freidlin et al16 developed a simpler phase ii trial design for a single drug with a single binary biomarker . 
 statistical methods for biomarker - driven clinical trials simon and maitournam17 , 18 studied the number of patients needed to screen and randomize for the enrichment design compared with a standard design , in which the assay is not performed . 
they found that the enrichment design generally requires many fewer randomly assigned patients . they showed that the ratio of number of required randomly assigned patients was approximately ( cid : 1 ) nenrichment nstandard ( cid : 1 ) te + + ( 1 ) te ( cid : 3 ) 2 where te + and te are the treatment effects in mutationpositive and mutation - negative patients , respectively , and denotes the proportion of mutation - positive patients . 
this formula indicates that the randomization ratio equals the reciprocal of the square of the treatment effects in the two trial designs . if the treatment effect in mutation - negative patients is zero , then the randomization ratio equals 2 . 
if the treatment effect in the mutation - negative patients equals half that in mutationpositive patients , then the randomization ratio becomes { + ( 1 ) / 2 } 2 , so if = 0.5 , then the randomization ratio equals 9 : 16 , and the enrichment design requires slightly more than one half the number of patients as the standard design . adaptive determination of the intended - use population the efciency of the enrichment design highlights the critical relationship between the required size of a phase iii clinical trial and the target treatment effect . 
simon et al19 developed a focused approach for using such archived tissues called the prospective - retrospective study design , which was used for establishing that patients with advanced colorectal cancer do not benet from anti - egfr antibodies if their tumors contain a mutation in the kras gene.20 this approach was also used in a structured manner in the development of the oncotype dx recurrence score as a prognostic and predictive signature for patients with stage i and stage ii breast cancer.21 in addition to using archived samples from a failed randomized controlled trial ( rct ) , adaptive methods have been developed for prospective design of an rct in a statistically rigorous way to adaptively identify the subset of patients who benet from the test treatment . 
some of these adaptive methods will be discussed , but their use has required regulators and clinical trialists to accept that the intended - use population need not be the full eligible population . by prospectively allocating some of the type i error rate to a planned subset analysis , 22 we do not need to use the convention that subset analysis should be performed only if the all - comers analysis is signicant or if there is a statistically signicant interaction . 
statisticians and clinical investigators have sometimes been slow to shed some of the conventional wisdom of the previous paradigm , such as the requirement that the intended - use population must be the eligible population . adaptive enrichment single binary candidate biomarker . 
the most common type of adaptive enrichment in phase iii trials involves whether to use a binary candidate biomarker to restrict eligibility . initially patients are characterized with regard to the biomarker and then randomly assigned to the test treatment or control , but the biomarker is not used to restrict eligibility . 
at some point during the trial an interim analysis is performed and one of three decisions is made : ( 1 ) the entire trial is closed for futility , ( 2 ) the trial continues accruing without any restriction on eligibility until its originally planned sample size is reached , or ( 3 ) accrual is continued only for biomarker - positive patients . 
in the third case , the total target accrual may be increased to adequately power the nal analysis for biomarker - positive patients . several authors have proposed designs for the binary biomarker case described previously.23 - 32 they contain multiplicity adjustments to account for the fact that inferences are being made for the treatment effect overall and for biomarker - positive patients . 
in many cases , there is a quantitative or semiquantitative biomarker that is thought to be predictive of which patients are most likely to benet . often , previous studies have not reliably established the relationship between biomarker value and likelihood of benet , and there is no adequately determined cut point for positivity . 
a recent example is the programmed deathligand 1 ( pd - l1 ) level of expression on nonsmall - cell lung tumors as a predictor of response to t - cell checkpoint therapy . because of regulators requirements that sponsors demonstrate statistically signicant effectiveness of the treatment of an intended - use population , most sponsors want to have the intended - use population dened at the outset . this may require using a cut point selected on the basis of inadequate phase ii trials . 
 simon of the relationship between biomarker level and treatment effectiveness . biomarkers , not for settings in which whole - genome data are used for classier development . there are only a few cases in which investigators use an adaptive design to determine an optimal cut point on the basis of interim data , perhaps because they are unaware of the new statistical designs that may achieve this in a statistically valid manner . jiang et al34 showed how one could test the null hypothesis of no treatment effect using a minimal p value statistic for which a p value for treatment effect is computed for the cases above each of the candidate cut points . 
their proposed analysis is performed at the end of the trial using the full data set and is based on the null distribution of the min - p - value statistic . 
this approach is easy to implement because it does not require any changes during the trial . the intended - use population is adaptively determined at the end of the trial . is adaptive in that simon and simon35 illustrated the use of their adaptive enrichment approach for modifying eligibility during the trial on the basis of an interim analysis of the relationship of biomarker value to treatment effect . 
the signicance test preserves the type i error , regardless of the strategy used for modifying eligibility . although the method is frequentist , they advocate using a bayesian model for selecting features and building the classier at interim analyses.33 the bayesian model can also be used for selecting an intended - use population . one of the questions about using adaptive enrichment is how to estimate the treatment effect in the adaptively selected intended - use population in an unbiased manner . simon and simon36 described how to use bootstrap resampling in an unbiased manner to estimate treatment effects for the intended - use population using data from periods that also determined how to modify eligibility . 
one approach involves developing separate prognostic scores for the active treatment group ( eg , h ( x ; a ) for the log hazard ratio relative to baseline hazard for a patient with covariate vector x receiving the active treatment ) and the control group ( h ( x ; c ) )  . 
that is , if the hazard of a failure event on treatment a is less than the hazard of that event on the control by a tolerance of at least c for a patient with baseline covariate vector x , then the predictive biomarker is considered to have value 1 , and otherwise to have value 0 . 
instead of using proportional hazards modeling as suggested here , one could use other methods such as random forest modeling37 or any other method of prognostic modeling . a major feature of the new paradigm for subset analysis introduced by the adaptive signature design ( asd ) is that nding an appropriate intent - to - treat subpopulation should be treated as a classication problem , not as a hypothesistesting problefinding a subset with a large apparent treatment effect is of little value because the resubstitution estimate of treatment effect is so often optimistically biased . the asd and cross - validated asd ( cv - asd ) provide unbiased estimates and tests of such treatment effects . simon38 described sensitivity , specicity , positive predictive value ( ppv ) , and negative predictive value ( npv ) for predictive classiers with survival time outcome . 
when the survival time for active treatment versus control has proportional hazards for classier - positive and classier - negative patients , then the ppv can be written38 ppv ( cid : 1 ) 1 + + where + denotes the hazard ratio of control versus active treatment of classier - positive patients . 
us food and drug administration ( fda ) guidance on the use of enrichment strategies describes the asd in a positive manner.42 practical experience in designing a clinical trial using the asd is described by sher et al , 43 simon , 44 and mi.45 where + denotes the prevalence of classier - positive patients . 
the asd and cv - asd to be described in a later section were originally described in terms of a specic kind of classier for gene expression data , but the concept is quite general and will be described here in its generality . 
it is not limited to high - dimension genomic data or signatures of gene expression measurements ; is best used with a moderate number of candidate predictive biomarkers . in fact , at the nal analysis , the patients randomly assigned in the clinical trial are randomly partitioned into a training set and a validation set . 
a predictive classier is developed by using only training set data . with the asd , however , one is permitted to develop only a single predictive classier , and it must be completely specied before the validation set data are used in any way . finally , the patients in the validation set are classied as likely to benet from the active treatment ( f ( x ) = 1 ) or not ( f ( x ) = 0 )  . 
the set of covariate vectors for which f ( x ) = 1 is considered the intended - use subset s : s = { x : f ( x ) = 1 }  . although the asd provides a new paradigm for subset analysis of rcts , its statistical power is limited because of the sample splitting . 
freidlin et al46 introduced an improved method called the cv - asd with improved power establishing the signicance of the treatment effect in the adaptively determined intended - use subset . 
we denote the resulting predictive classier as c ( a , d ) where d denotes the complete data set . the resubstitution estimator of treatment effect is denoted ( c ( a , d ) , d ) where the operator computes the empirical treatment effect on the subset of d for which classier c is positive . 
this new training starts from scratch ; that is , it does not use the feature selection steps of development of the full sample classier c . let ck ( a , dk ) denote the classier developed on dk . 
i is the prevalidation classication of patient i . having developed a single predictive classier using training data , the next step is to evaluate the treatment effect for patients in the validation set with covariate vectors in s . 
outcomes of those who received active treatment are compared with outcomes of those who received the control . because the validation set was not used to develop the classier , this comparison of outcomes is unbiased . after completing all k - folds of the cross - validation , all of the cases have been classied once . 
these { i } are called prevalidated indicators of the intended - use subset.47 let iu = { i : i = 1 } , the intended - use subset on the basis of the prevalidated predictive classications . 
 simon empirical treatment effect in iu , and take that as an approximation for the treatment effect in using the full sample classier c ( a , d ) with new cases in the future . 
simulation studies have shown that this estimator is much better than the resubstitution estimator but somewhat conservative.46 a statistical signicance test for treatment effect among future patients classied positive using c ( a , d ) is approximated by computing the permutation distribution of ( iu )  . for each permutation of the treatment labels , the crossvalidation procedure is repeated giving a new iu , say iu ( cid : 4 ) , and a new value of ( iu )  . 
with many permutations of the treatment labels , one can approximate the null distribution . the null hypothesis here is that there is no subset for which there is a positive treatment effect . 
zhang et al48 describe an alternative approach to de - biasing the resubstitution estimator ( c ( a , d ) , d ) using bootstrap sampling instead of using prevalidated classication . other phase iii designs zhao et al49 describe a method for using the results of a previous clinical trial of a treatment and control to select a subset of patients for a new rct . 
for higher - dimensional data , the authors recommend sample splitting or cross - validation to separate the development of predictive scores from the evaluation of the outcome difference in the selected patients . 
the authors assume that feature selection does not use the clinical trial data because a partial cross - validation approach in which feature selection is not repeated for each resampling is described and can be highly biased when feature selection uses the data.50 foster et al37 describe a method very similar to the cv - asd for binary outcome data in which the random forest method is used for developing prognostic prediction function in the active treatment and control groups . 
they also investigate bootstrap resampling to estimate the treatment effect in the selected subset . cv - asds are quite general with regard to the type of models used for predicting difference in expected outcome between treatment groups as a function of the covariate vector . 
these treatment effect estimates may be biased if the assumed parametric model is misspecied , however . outcome weighted learning is an approach to developing approximately optimum individualized treatment rules that are robust to model misspecication.51 , 52 sequentially adaptive interventions are multistage treatto tailor a sequence of ment strategies that attempt treatment interventions to the characteristics of individual patients and their responses to previous treatments . 
such a score can be based on the difference between a prognostic risk function ft ( x ) trained on the treatment group and a prognostic risk function fc ( x ) trained on the control group , where x denotes the vector of measured covariates . 
using the k - fold cross - validation approach of the cv - asd , these functions can be developed on dk training sets and used to compute prevalidated predictive scores ft ( xi ; dk ) fc ( xi ; dk ) for the cases in the withheld sets dk . 
these prevalidated predictive scores si being quantiles are thus in the range ( 0 , 1 )  . matsui et al55 suggest that these prevalidated scores be used as the independent variable in a proportional hazards model containing a main effect of the treatment and an interaction between treatment and prevalidated predictive score quantile . 
for any test statistic involving the estimated treatment effect scores , the null hypothesis of no treatment effect can be performed by permuting the treatment labels and repeating the cross - validation procedure . 
predictions for future patients require rst computing the predictive index quantile score for the new patient using quantiles of the full data set and then plugging that value into the tted proportional hazards model . cai et al56 described a two - stage procedure for estimating a predictive score . 
for high dimensional their approach is probably best used with covariates , prevalidated predictive scores . discussion the development of personalized oncology has been a consequence of the discovery of somatic driver mutations in tumors . 
development of drugs and companion diagnostics for inhibiting driver mutations has provided many two decades . therapeutic successes during the past complex statistical methods have not been necessary for developing predictive biomarkers in these cases because the presence of is the biomarker . the mutation itself however , most somatic mutations are not driver mutations , and identication of predictive biomarkers in those cases can be much more difcult . 
consequently , the fda developed a regulatory pathway in which the diagnostic was not labeled as distinguishing patients who are likely to benet from the drug from those who are not , but rather for identifying patients who have a tumor containing the mutation that makes them eligible for the approved drug . 
during the past two decades , numerous drugs and companion diagnostics for patients with cancer have been approved on the basis of the enrichment design . the question of whether to restrict eligibility to a phase iii trial on the basis of a binary biomarker or on the basis of the value of a quantitative biomarker frequently arises in the design of pivotal trials . 
having larger phase ii trials would provide better evidence for designing phase iii trials , but often the phase iii design decisions are made on the basis of insufcient phase ii data . 
the adaptive designs described in which eligibility is not initially restricted but may subsequently be restricted to biomarker - positive patients on the basis of a prespecied interim analysis are widely applicable . 
more general adaptive enrichment designs with multiple candidate biomarkers can be effective if markers are sufciently powerful their effects on outcome can be discovered at interim analyses . that the asd and cv - asd are easy to use because they do not require any eligibility changes during the course of the trial . they are adaptive in the sense that the intended - use population is adaptively determined by the trial data . however , because they do not adapt eligibility , the opportunity for power gains is limited . 
the cv - asd has much better power and is appropriate for settings in which strong candidate biomarkers are not known at the start of the trial . analyzing phase iii trials by using a continuous score biomarker that reects relative treatment effectiveness can provide medically useful information without the arbitrariness of specifying a cut point . 
it is important , however , to internally validate the calibration of the scoring function by using resampling methods as described here . trials for nding optimal dynamic regimes for clinical multistage decision making have not yet found much application in oncology but have been used effectively in other areas . some of the conventional wisdom that has guided the design and analysis of phase iii trials is undergoing reevaluation . 
for example , the paradigm of broad eligibility and the use of subset analysis on the basis of multiple hypothesis testing require re - evaluation . is not always for molecularly targeted treatments , reasonable to expect a signicant treatment effect for all eligible patients dened by using conventional histology and stage criteria . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . richard simon consulting or advisory role : abbvie pharmaceuticals , amgen biotechnology , janssen pharmaceuticals , bristol - myers squibb , pzer , onconano medicine travel , accommodations , expenses : amgen references talpaz m , silver rt , drucker bj , et al : imatinib induces durable hematologic and cytogenic responses in patients with accelerated phase chronic myeloid leukemia : results of a phase 2 study . 
blood 99 : 1928 - 1937 , 2002 slamon dj , leyland - jones b , shak s , et al : use of chemotherapy plus a monoclonal antibody against her2 for metastatic breast cancer that overexpresses her2 . 
new york , ny , chapman and hall / crc , 2015 antonijevic z , beckman ra ( eds ) : platform trial designs in drug development : umbrella trials and basket trials . 
new york , ny , chapman and hall / crc , 2018 berry sm , broglio kr , groshen s , et al : bayesian hierarchical modeling of patient subpopulations : efcient designs of phase ii oncology clinical trials . 
clin trials 10 : 720 - 734 , 2013 simon rm : primary site independent clinical trials in oncology , in antonijevic z , beckman ra : platform trial designs in drug development : umbrella trials and basket trials . 
bokemeyer c , kohne c , rougier p , et al : cetuximab with chemotherapy ( ct ) as rst - line treatment for metastatic colorectal cancer ( mcrc ) : analysis of the crystal and opus studies according to kras and braf mutation status . 
paik s , tang g , shak s , et al : gene expression and benet of chemotherapy in women with node - negative , estrogen receptor - positive breast cancer . 
biom j 51 : 358 - 374 , 2009 jenniso c , turnbull bw : conrmatory seamless phase ii / iii clinical trials with hypotheses selection at interim : opportunities and limitations . 
stat med 32 : 2695 - 2714 , 2013 jenkins m , stone a , jennison c : an adaptive seamless phase ii / iii design for oncology trials with subpopulation selection using correlated survival endpoints . pharm stat 10 : 347 - 356 , 2011 30 . 
biostatistics 19 : 27 - 41 , 2018 jiang w , freidlin b , simon r : biomarker - adaptive threshold design : a procedure for evaluating treatment with possible biomarker - dened subset effect . 
bai x , tsiatis aa , lu w , et al : optimal treatment regimes for survival endpoints using a locally - efcient doubly - robust estimator from a classication perspective . 2003 lifetime data anal 23 : 585 - 604 , 2017 52 . 
a recent retrospective analysis found a clear association of improved survival with continuation of endocrine therapy upon diagnosis of brain metastases.10 remarkably , the cdk4 / 6 - inhibitor abemaciclib had a clinical benet rate of 25%.11 preclinical models of bc brain metastases suggest pi3k pathway inhibition may be effective for treatment of brain metastases.12 notably , both sandpiper and solar - 1 excluded patients with untreated or active cns metastases6 , 7 ; however , the precursor to alpelisib , buparlisib , did have brain penetration , which was thought to be the cause for the higher incidence of mood disorders.13 an increase in depression has distinctly not been seen with the - specic pi3kinhibitor alpelisib and , in preclinical animal models with intact blood - brain barrier , no signicant distribution into the brain was seen ( unpublished data )  . thus , the activity of alpelisib in brain metastases is unknown . herein , we report a case series of 4 patients with er + / progesterone receptorpositive ( pr + ) / her2 metastatic bc with progressive brain metastases ( fig 1 ) treated with alpelisib . 
all patients provided consent to publish their information and images . case 1 a 55 - year - old woman presented with a large breast ulceration , biopsy specimendiagnosed invasive ductal carcinoma ( idc ) , grade 3 , er + / pr + / her2 . computed tomography ( ct ) scan revealed pulmonary nodules , osseous lesions , and hypodense lesions within the right hepatic lobe . 
brain magnetic resonance imaging ( mri ) showed a 10 - mm mass in the left cerebellar hemisphere ; this was treated with stereotactic radiosurgery with initial shrinkage to 8 mm and stabilization on follow - up . 
brain mri showed an increase of the left cerebellar lesion to 12 mm , judged by neuroradiology and radiation oncology to be more compatible with progression than with radiation - induced tissue necrosis . 
the patients body mass index ( bmi ) was 25 ( calculated as kilograms divided by square of height in meters ) and eastern cooperative oncology group ( ecog ) performance status ( ps ) score of 0 . 
brain mri after 6 weeks showed a reduction in the left cerebellar lesion to 9 mm ( 62% reduction of bidimensional areas , 35% reduction of sum of longest distances )  . 
follow - up mri 2 months later revealed stability of the cns lesion . subsequent brain mri at 3 , 4 , and 6 months showed stable disease without changes in measurements . 
this was compatible with a partial response per response in neuro - oncology brain metastases assessment ( rano - bm ) criteria.14 case 2 a 71 - year - old woman was diagnosed in 2010 with pt2n0 idc of the right breast , grade 3 , er + / pr + / her2 . 
in 2017 , she had a recurrence in the right breast , pleural effusion , and bone metastases and was treated with multiple regimens : letrozole and palbociclib , fulvestrant and palbociclib , and capecitabine and paclitaxel . 
she was serially treated with fulvestrant and palbociclib , radiation to the base of skull to treat cranial nerve symptoms , and with exemestane and everolimus , capecitabine , and abemaciclib . 
for the rst time , brain mri demonstrated multiple parenchymal metastases and she was treated with hippocampal - sparing wbrt . subsequently , treatment was changed from abemaciclib to liposomal doxorubicin for progression in the brain , followed by gemcitabine . 
brain mri 6 weeks later demonstrated a 14% reduction in the sum of longest distances of measurable brain metastases ( 24% reduction in bidimensional areas ) and regressions of nonmeasurable brain metastases . 
the most recent brain mri at the 6 - month mark revealed progressive parenchymal disease requiring change of therapy . case 4 a 70 - year - old woman was diagnosed in 2013 with t1n1 idc of the right breast , grade 3 , er + / pr + / her2 . 
she was treated with breast - conserving surgery , adjuvant docetaxel plus cyclophosphamide , radiation , and anastrozole . 2018 , metastatic disease developed to the lung , liver , and bone while she received adjuvant anastrozole . 
follow - up mri demonstrated at least 15 new intraparenchymal lesions and several progressing dural - based lesions , so the patient proceeded to undergo wbrt and restarted capecitabine therapy . 
examination of a liquid biopsy specimen revealed multiple pi3k mutations ( pik3ca : h1047r , e81k , and e563k ) and she was administered alpelisib ( 300 mg ) table 1 . 
restaging after 6 weeks of therapy revealed substantial disease regression in the lungs and liver as well as interval resolution of punctate cerebellar and cerebral metastasis and reduction of a dural metastasis ( appendix fig a1 ) , compatible with stable disease per rano - bm criteria.14 repeated brain mri at the 3and 5month marks showed stable disease . discussion activating pik3ca mutations occur early in breast carcinogenesis and are typically not lost or acquired during clonal evolution in later stages of the diseasefeatures that suggest these are driver mutations . 
table 1 provides a summary of mutational prole and previous treatments . cases 1 and 2 harbored the activating mutation pik3ca h1047r in the coding exon 20 , a kinase - activating mutation and the most common mutation in bc.14 case 1 also had pik3c2b amplication , which occurs in 13% to 25% of patients.15 pik3c2b is a class ii pi3 - kinase.16 whether its amplication in conjunction with pik3ca mutation deepens or attenuates pi3k - pathway dependence of cancer and whether pik3c2bs kinase activity is inhibited by alpelisib is unknown . 
however , understanding this relationship appears to be important because activating pik3ca mutations and copy number gain of pik3c2b do co - occur in bc.15 case 3 harbored the pik3ca e545k mutation , affecting the helical domain of p110that activates signaling because of its detachment from the inhibitory p85 subunit of pi3k.17 helical domain mutations are the second most frequent in bc , with an incidence of 6.4%.14 case 4 had 3 mutations in pik3ca ; e81k and h1047r are activating mutations and e563k is novel . 
this combination of a major ( h1047r ) and a minor ( e81k ) pik3ca mutation is thought to amplify pi3k signaling and predict for responsiveness to pi3k inhibition.18 this patient had a comutation of pik3ca mutations and esr1 . 
the earlier pan - pi3k inhibitor buparlisib was tested in 4 patients with treatment - refractory cns lymphoma , 1 of whom achieved a partial remission.23 in animal models , systemically administered buparlisib showed activity against bc brain metastases.24 - 26 patients with active brain metastases were excluded from the pivotal solar - 1 and sandpiper studies.6 , 7 conclusions from our observations are limited by the small number of patients selected on the basis of their surprising response . 
the rano - bm criteria consider lesions , 10 mm as nonmeasurable.14 here , we show examples with resolution of small lesions , which are clinically meaningful but classied as stable disease per the criteria . 
additional investigation to prospectively evaluate alpelisib in patients with bc and cns involvement may be justied . in conclusion , we have described cases of regression or stabilization of progressive cns lesions in patients with hr + / her2 metastatic bc treated with the pi3k inhibitor alpelisib . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . stacy moulder research funding : oncothyreon ( inst ) , pzer ( inst ) , novartis ( inst ) , genentech ( inst ) , takeda ( inst ) , bayer ( inst ) , emd serono ( inst ) , genentech ( inst ) eric p . 
winer stock and other ownership interests : verastem honoraria : roche , genomic health consulting or advisory role : leap therapeutics , seattle genetics , jounce therapeutics , glaxosmithkline , carrick therapeutics , eli lilly , genomic health , g1 therapeutics research funding : genentech ( inst ) , novartis ( inst ) hope s . 
rugo consulting or advisory role : ionis , celtrion research funding : macrogenics ( inst ) , obi pharma ( inst ) , eisai ( inst ) , pzer ( inst ) , novartis ( inst ) , eli lilly ( inst ) , genentech ( inst ) , merck ( inst ) , immunomedics ( inst ) , odonate therapeutics ( inst ) , daiichi sankyo ( inst ) , seattle genetics ( inst ) travel , accommodations , expenses : pzer , puma biotechnology , mylan , amgen , astrazeneca , macrogenics , daiichi sankyo , merck , novartis , obi pharma open payments link : nancy u . 
lin consulting or advisory role : roche , seattle genetics , puma biotechnology , novartis , daiichi sankyo research funding : genentech , pzer , seattle genetics , merck patents , royalties , other intellectual property : royalties for chapter in upto - date regarding management of breast cancer brain metastases , royalties from jones & bartlett gerburg m . 
wulf stock and other ownership interests : selecta biosciences ( i ) research funding : merck patents , royalties , other intellectual property : pin1 as a marker for abnormal cell growth ( patent no . : 8129131 ) no other potential conicts of interest were reported . references cancer genome atlas network : comprehensive molecular portraits of human breast tumours . 
nature 490 : 61 - 70 , 2012 loi s , michiels s , baselga j , et al : correction : pik3ca genotype and a pik3ca mutation - related gene signature and response to everolimus and letrozole in estrogen receptor positive breast cancer . 
genes chromosomes cancer 52 : 69 - 80 , 2013 ramirez - ardila de , helmijr jc , look mp , et al : hotspot mutations in pik3ca associate with rst - line treatment outcome for aromatase inhibitors but not for tamoxifen . 
breast cancer res treat 139 : 39 - 49 , 2013 kalinsky k , jacks lm , heguy a , et al : pik3ca mutation associates with improved outcome in breast cancer . 
clin cancer res 15 : 5049 - 5059 , 2009 baselga j , cortes castan j , de laurentiis m , et al : sandpiper : phase iii study of the pi3 - kinase ( pi3k ) inhibitor taselisib ( gdc - 0032 ) plus fulvestrant in patients ( pts ) with oestrogen receptor ( er ) - positive , her2 - negative locally advanced or metastatic breast cancer ( bc ) enriched for pts with pik3ca - mutant tumours . 
ann oncol 27 : vi99 , 2016 ( suppl 6 ) andr e f , ciruelos e , rubovszky g , et al : alpelisib for pik3ca - mutated , hormone receptor - positive advanced breast cancer . 
n engl j med 380 : 1929 - 1940 , 2019 adamo b , deal am , burrows e , et al : phosphatidylinositol 3 - kinase pathway activation in breast cancer brain metastases . 
breast cancer res 13 : r125 , 2011 fitzgerald dm , muzikansky a , pinto c , et al : association between pik3ca mutation status and development of brain metastases in hr + / her2metastatic breast cancer . 
bergen es , berghoff as , medjedovic m , et al : continued endocrine therapy is associated with improved survival in patients with breast cancer brain metastases . clin cancer res 25 : 2737 - 2744 , 2019 11 . 
tolaney sm , lin nu , thornton d , et al : abemaciclib for the treatment of brain metastases ( bm ) secondary to hormone receptor positive ( hr + ) , her2 negative breast cancer . 
j clin oncol 35 : 1019 , 2017 ( 15 suppl ) ippen fm , alvarez - breckenridge ca , kuter bm , et al : the dual pi3k / mtor pathway inhibitor gdc - 0084 achieves antitumor activity in pik3ca - mutant breast cancer brain metastases . 
campone m , im s - a , iwata h , et al : buparlisib plus fulvestrant versus placebo plus fulvestrant for postmenopausal , hormone receptor - positive , human epidermal growth factor receptor 2 - negative , advanced breast cancer : overall survival results from belle - 2 . 
takeshita t , yamamoto y , yamamoto - ibusuki m , et al : analysis of esr1 and pik3ca mutations in plasma cell - free dna from er - positive breast cancer patients . 
study_list = brca_igr_2015%2cbrca_mbcproject_wagle_2017&case_set_id = all&data_priority = 0&gene_list = esr1%253amut%250apik3ca%253amut& geneset_list = %20&prolefilter = 0&tab_index = tab_visualize juric d , castel p , grifth m , et al : convergent loss of pten leads to clinical resistance to a pi ( 3 ) k inhibitor . 
grommes c , pentsova e , nolan c , et al : phase ii study of single agent buparlisib in recurrent / refractory primary ( pcnsl ) and secondary cns lymphoma 24 . 
maira s - m , pecchi s , huang a , et al : identication and characterization of nvp - bkm120 , an orally available pan - class i pi3 - kinase inhibitor . 
 o next - generation sequencing of advanced gi tumors reveals individual treatment options michael bitzer , md1 , 2 , 5 ; leonie ostermann1 ; marius horger , md3 ; saskia biskup , md , phd4 ; martin schulze , dsc4 ; kristina ruhm , msc5 ; franz hilke , msc6 ; oznur oner , msc5 ; konstantin nikolaou , md , mba2 , 3 ; christopher schroeder , md6 ; olaf riess , md6 ; falko fend , md7 , 8 ; daniel zips , md8 , 9 ; martina hinterleitner , md10 ; lars zender , md2 , 8 , 10 ; ghazaleh tabatabai , md , phd2 , 8 , 11 ; janina beha , phd5 ; and nisar p . 
malek , md1 , 2 , 5 , 8 purpose precision oncology connects highly complex diagnostic procedures with patient histories to identify individualized treatment options in interdisciplinary molecular tumor boards ( mtbs )  . 
follow - up data and a response assessment that was based on radiologic imaging were recorded . results ninety - six patients were presented to the mtb of tuebingen university hospital . 
patients with pr or sd in the course of mtb - recommended treatments seemed to benet with respect to pfs and os . jco precis oncol 4 : 258 - 271 . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction the challenge of precision oncology is to connect highly complex diagnostic procedures with individual patient histories to identify optimal treatments . 
detailed data on mtbguided treatment suggestions , and specically the outcomes of patients , have only been reported for nonselected patient groups , including for a variety of different tumors.1 - 4 however , each tumor entity , including gi cancers , harbors unique features that will have to be considered to identify the patients who will benet the most from such an approach.4 recent reports highlight the experiences of mtbs in everyday practice . 
a series from the md anderson cancer center that examined hot - spot regions in 11 to 50 genes for 2 , 000 cancer tissues found actionable mutations for 39% of patients.2 only 11% of these selected patients could be enrolled in genotype - matched clinical trials.2 a report from the mayo clinic identied actionable mutations in 65% of 141 patients , but only 29 ( 21% ) were subsequently treated in clinical trials or with targeted food and drug administration ( fda ) approved drugs , with an objective response or stable disease ( sd ) for 4 months in 10 patients.3 freiburg university reported 104 patients with recommendations that were based on diagnostic tests that ranged from an 8 - gene panel to whole - exome sequencing ( wes ) .1 thirty - three patients received a recommended treatment ; 11 had an objective response , and 8 additional patients reached sd at 10 weeks . 
 molecular tumor board for advanced gi cancers context key objectives academic molecular tumor boards ( mtbs ) bridge the gap between a growing complexity in diagnostic procedures and clinicians at the bedside . 
this retrospective study investigated the course of patients with advanced gastrointestinal ( gi ) cancers that have been considered for personalized treatment options , including the outcome receiving mtb - guided treatment . knowledge generated next - generation sequencing ( ngs ) revealed a relevant number of germline variants , suggesting genetic counseling . 
responding patients with prolonged disease stabilization seemed to derive a meaningful survival benet from cancer genome sequencing and matched treatments . relevance patients with gi cancers can benet from currently available ngs sequencing procedures . 
in perspective , a continued improvement of mtb recommendations and the inclusion of additional complex diagnostic procedures might substantively enhance treatment opportunities in everyday clinical practice for these patients in the near future . the sequencing of matched tumor and normal tissue is an important procedure for the precise identication of potentially actionable genes.5 , 6 this inevitably leads to the detection of germline alterations . 
a recent investigation in 10 , 389 patients across 33 cancer types detected pathogenic or likely pathogenic germline variants in 8% of all patients with cancer and in 8.8% of patients with gi tumors.7 the frequency of germline mutations varied greatly across cancer types in general but also across different gi cancers , ranging from 2.2% for cholangiocarcinoma to 14.1% for pancreatic cancer ( pc ) .7 to our knowledge , our experience with gi tumors in an academic mtb is to date the largest reported series of this group with detailed information of comprehensive sequencing data , subsequent board recommendations , and documented outcome . 
the mtb consists of an interdisciplinary team coordinated by the tuebingen center for personalized medicine and includes experts in clinical and translational oncology , pathology , bioinformatics , molecular biology , radiology , and human genetics . 
an electronic web - based platform ( mtb platform ) was established to introduce patients to the mtb team with all necessary information to prepare and follow up the weekly face - to - face meetings and subsequently document treatment outcomes ( data supplement )  . 
best response assessment was based on radiologic imaging in line with recist 1.1 , for checkpoint inhibitor therapy , irecist , or for hepatocellular carcinomas ( hcc ) , mrecist criteria.8 - 10 genetic tumor / normal characterization tumor and normal tissues were genetically characterized either by ngs panel sequencing of full coding sequences or by wes ( more information in data supplement )  . 
off - label use refers to the administration of an fda / european medicines agency - approved drug outside its approved indication . for recommended off - label therapies , an application for reimbursement was submitted to the patients health individual treatment describes insurance . 
patients treated within a heilversuch have been registered at the local authority , in this case the regierungspr asidium tuebingen . statistical analysis progression - free survival ( pfs ) and overall survival ( os ) were analyzed with the kaplan - meier method dependent on the treatment response ( sd and pr v progressive disease [ pd ] ) compared by log - rank testing . 
btc , biliary tract cancer ; crc , colorectal cancer ; gc , gastric cancer ; hcc , hepatocellular carcinoma ; net , neuroendocrine tumor ; pc , pancreatic cancer ; ugc , upper gi tract cancer . results diagnostic procedure and clinical work - up ninety - six patients received ngs of advanced gi tumors and were presented to the mtb . 
this cohort included 32 colorectal cancer ( crc ) , 22 pc , 19 biliary tract cancer ( btc ) , 11 hcc , 9 upper gi tract cancer ( ugc ) , and 3 neuroendocrine tumors ( net ; fig 1a )  . 
the mean number of systemic anticancer pretreatments was 2.8 ( fig 1b )  . ninety - one patients received a gene panel analysis that examined between 337 and 710 genes , and 5 patients had wes ( fig 1c )  . 
genes represented in the different panels are summarized in the data supplement , together with the total size and the average and median coverage of the 649 and 710 gene panels . germline variants in gi cancers germline ndings were ranked according to a 5 - tiered classication.11 , 12 sixteen patients ( 17% ) had germline variants classied as pathogenic or likely pathogenic ( figs 1d - 1f ) , and in 1 patient with gastric cancer , 2 likely pathogenic variants were detected ( palb2 and fancm )  . five patients ( 5% ) had pathogenic or likely pathogenic germline variants in one of the brca2 , msh6 , or sdhb genes . 
these alterations belong to a list of secondary ndings that should be reported because of the possibility for interventions to signicantly reduce morbidity and mortality.13 in addition , 3 patients showed germline variants in pharmacologically relevant genes , 2 in dpyd and 1 in g6pd ( data supplement ) without showing unusual adverse reactions during chemotherapy . 
this variant has been reported with classications ranging from variant of unknown signicance to pathogenic.14 a germline alteration was directly responsible for mtb recommendations for 5 patients ( 5% ) : brca2 for poly ( adpribose ) - polymerase ( parp ) inhibition ( 3 pc ) , chek2 combined with 2 somatic atm truncations for atr - inhibition and carboplatin ( 1 crc ) , and msh6 and therefore a resulting high tumor mutational burden ( tmb ) for pembrolizumab ( 1 crc )  . 
 molecular tumor board for advanced gi cancers frequency of germline mutations ( n = 96 ) presentation of ngs for gi tumors ( n = 96 ) molecular target identification ( n = 96 ) patients with clinically relevant ( n = 19 [ 20% ] ) germline mutations patients with pathogenic or likely pathogenic ( n = 16 ) variants patients with pharmacogenomic relevant variants ( n = 3 ) patients with at least 1 molecular target ( n = 47 [ 49% ] ) patients without mtb recommendation ( n = 6 ) no progression under last line treatment ( n = 4 ) complete tumor resection ( n = 2 ) patients not treated with recommendation ( n = 16 ) patients with poor performance status ( n = 15 ) patients not evaluable ( n = 5 ) without imaging for response monitoring ( n = 3 ) died within 21 days after treatment start ( n = 2 ) patient not evaluable ( n = 1 ) alternative therapy before response monitoring patients with mtb treatment recommendation ( n = 41 [ 43% ] ) patients received the suggested treatment ( n = 25 [ 26% ] ) patients with colorectal cancer ( n = 11 ) patients with pancreatic cancer ( n = 7 ) patients with biliary tract cancer ( n = 4 ) patients with upper gi tract cancer ( n = 2 ) patient with hepatocellular carcinoma ( n = 1 ) patients evaluable for best response and os ( n = 20 [ 21% ] ) patients pr ( n = 3 [ 15% of 20 patients ] ) patients sd ( n = 6 [ 30% of 20 patients ] ) patients pd ( n = 11 [ 55% of 20 patients ] ) fig 2 . 
 ( b ) frequency of potential target identication by the molecular tumor board ( mtb ) , patients with target identication but no treatment , and patients with mtb - recommended treatment initiation . 
btc , biliary tract cancer ; crc , colorectal cancer ; hcc , hepatocellular carcinoma ; net , neuroendocrine tumor ; pc , pancreatic cancer ; ugc , upper gi tract cancer ; w / o , without . pharyngeal , or bladder cancer ; and the patient with a chek2 mutation had a rst - degree relative with ovarian cancer . 
eight of these 10 patients were also tested for msi , but only 2 patients were found to have a msi - high tumor . tmb is currently regarded as relevant in predicting therapeutic success with checkpoint inhibitors beyond tumors with mismatch repair deciency.15 - 18 this has been shown for patients with crc and ugc , 18 but is less clear for hepatobiliary cancer and pc . 
for tumors known to have a low tmb in general , those with values molecular target identication by the mtb at least 1 potential molecular target was identied in 47 of 96 patients ( 49% ; figs 2b and 3b )  . 
the frequency of target identication varied among tumor types ( fig 3c , table 2 ) , with 74% for btc , 56% for upper gi cancers , 50% for pc , 44% for crc , and 27% for hcc . 
the most frequently altered genes with single - nucleotide variants or copy number variants were cdkn2a / b , brca2 , idh1 , erbb2 , myc , fgf3 , fgf4 , flt3 , fgfr4 , cdk6 , braf , and atm ( table 2 ; data supplement )  . 
target identication per tumor type ( continued ) diagnosis patients tested ( no . ) molecular target frequency erbb2 braf brd4 cdkn2a / b chek2 fgf3 / 4 / 19a flt1 / 3 mlh1 msh6 fgfr2 - bicc1 idh1 braf brca1 brca2 cdkn2a fgfr1 fgfr2b fgfr4 fgfr2 - prkcq fgfr2 - ahcyl2 cdkn2a / b brca2 cdk6 erbb3 nrg3 nrg1 - cdh6 tmem66 - nrg1 cdkn2a / b cdk6 fgf3 / 4 idh1 idh1 abbreviations : btc , biliary tract cancer ; crc , colorectal cancer ; hcc , hepatocellular carcinoma ; pc , pancreatic cancer ; tmb , tumor mutational burden . afgf3 / fgf4 / fgf19 cluster amplication . bcoincident fgfr2 mutation in tumor with fgfr2_ahcyl2 fusion gene . cdkn2a / b alterations , 6 were detected in pc , 2 in ugc , 1 in btc , and 1 in crc . 
three out of 4 brca2 alterations were detected in pc , and 1 in btc ( table 2 )  . outcome and clinical course of patients the course of all patients presented to the mtb is shown in fig 2b . 
mtb recommendations for matched treatments were given for 41 patients ( 43% )  . twenty - ve patients with a mean of 3.4 previous anticancer treatments received the suggested medication ( 61% of patients with mtb recommendation ; 26% of the whole cohort )  . 
the alterations in included cdkn2a / b this subgroup without treatment deletions , braf mutations , brca deletions , flt1 / 3 amplication , fgfr2 fusion , idh mutation , and high tmb ( data supplement )  . treatment was initiated in 11 patients with crc ( 34% of patients with crc ) , 7 patients with pc ( 32% of patients with pc ) , 4 patients with btc ( 21% of patients with btc ) , 2 patients with ugc ( 22% of patients with ugc ) , and 1 patient with hcc ( 9% of patients with hcc )  . 
the applied drugs were either used in - label , used off - label , obtained via a clinical study , or supplied for a matched experimental treatment ( table 1 , drug availability column )  . 
 molecular tumor board for advanced gi cancers patients who reached either sd or pr were treated with pd1 blocking antibodies ( high tmb ) , atr inhibitor with carboplatin ( simultaneous mutations in atm , chek2 , and tp53bp1 ) , trastuzumab with lapatinib ( erbb2 amplication ) , pertuzumab with erlotinib ( nrg1 fusion , high - expression erbb3 and nrg3 ) , idh1 inhibitor bay 1436032 ( idh1 mutation ) , or lenvatinib ( fgfr2 fusion gene ; table 1 )  . 
1 year after treatment initiation reached either sd or pr ( data supplement )  . therefore , as a surrogate end point to estimate whether patients might benet from the mtb - recommended treatment , the achievement of sd for at least 3 months or even pr as best response was compared with pd in a kaplan - meier estimation for pfs and os . 
in several studies , the inclusion of germline ndings enabled a more comprehensive characterization of tumor biology , potential resistances , or even treatment opportunities.22 , 23 in this study , germline variants classied as pathogenic , likely pathogenic , or pharmacogenomic could be identied in 19 patients ( 20% )  . 
these numbers are higher than in a recent investigation that reported pathogenic or likely pathogenic germline predisposition variants in 8.8% of gi tumors , 7 and could be mainly a result of the smaller sample size in our study . 
for example , for brca mutations , the tumor lineage indeed seems to determine the therapeutic benet of parp inhibition.24 conversely , emerging data suggest that patients with germline mutations that are not typically associated with a diagnosed cancer might nevertheless benet from drugs that target this alteration.25 germline variant reporting and the inclusion of experts in clinical genetics , should therefore be a prerequisite for mtbs.22 , 23 , 26 in predicting therapeutic tmb is regarded as relevant outcome with checkpoint inhibitors.15 - 18 the minimum size of tumor panels to reliably calculate tmb was suggested to include at least 300 genes , or more precisely , 1.5 mb of the target region , 21 , 27 , 28 which is met in our approach . 
one patient with crc and high tmb ( 118 var / mb ) who showed progression under pd - 1 inhibition even responded after the addition of ctla4 inhibition on progression . 
in the 4 patients who did not show any response to checkpoint inhibition , the molecular analysis did not reveal one of the currently discussed mechanisms of resistance.29 of note , only a minority of patients with high tmb also had msi - high tumors . 
this observation is in line with an investigation in a broad range of different tumor types , showing that only 16% with high tmb were classied as msi high.21 another study in 6 , 004 patients with crc identied 465 patients with high tmb ; however , only 65% of these could be classied as msi high.30 these observations suggest that a reliable method for tmb estimation should be used if patients with gi tumors are evaluated for personalized treatment options . of 96 patients with advanced gi tumors , mtb treatment recommendations were given for 41 ( 43% ) , which means an additional therapy option beyond established treatment lines for 4 out of 10 patients . 
14 months in our cohort , compared with patients with pd as best response ( fig 4d ) , is remarkable ; however , it has to be interpreted with caution and should be conrmed in larger patient populations with gi or other cancers . 
if this observation could be conrmed , 1 goal in the constant improvement of mtb recommendations should be to gradually increase the percentage of patients who reach disease control of at least 3 months in advanced cancers . early cohorts with molecular - matched therapies have been reported for crc31 and btc.32 in the rst study , 68 out of 254 patients with advanced crc received selected matched therapies to kras / braf / pik3ca mutations , pten , or phosphorylated met expression . 
a subgroup with btc from the moscato - 01 trial reported molecular targets in 23 of 34 patients , with 18 receiving matched therapies ( 53% ) .32 the overall response rate and pfs of 6 months in that study were 33% and 37% , respectively . 
several techniques , such as transcriptome34 , 35 and epigenome36 analysis or wholegenome37 and single - cell sequencing , are expected to improve tumor characterization up front.38 such a constant evolution will require more specialists in these methods to join academic mtbs , which means a new era of terdisciplinary patient care to bridge the gap between a growing complexity in diagnostic procedures and clinicians at the bedside . 
 ( a ) progression - free survival ( pfs ) , dened as the time from start of an mtb - recommended treatment to radiographic progression or death , and ( b ) overall survival ( os ) , dened as the time from start of an mtbrecommended treatment to death as a result of any cause , in days . 
recent examples in crc are pd - l1 and ctla - 4 inhibition in msi - high cancers41 or the combination of a braf inhibitor , mek inhibitor , and antiepidermal growth factor receptor antibody in braf v600e mutated cancers.42 third , a reduction in dropout rates might be achieved with more focused and earlier patient selection for ngs - based diagnostic procedures and a shortening of time intervals to get access to suggested drugs . 
fourth , each patient who is treated according to an mtb recommendation outside clinical trials should be regarded as an n - of - 1 trial.43 this implies a thorough documentation of adverse events and a reliable response assessment along a predened routine . in conclusion , gi tumors are an important group in the eld of personalized medicine . 
malek provision of study material or patients : michael bitzer , falko fend collection and assembly of data : michael bitzer , leonie ostermann , marius horger , saskia biskup , kristina ruhm , oznur oner , christopher schroeder , olaf riess , falko fend , daniel zips , martina hinterleitner , lars zender , janina beha data analysis and interpretation : michael bitzer , leonie ostermann , marius horger , saskia biskup , martin schulze , franz hilke , konstantin nikolaou , christopher schroeder , olaf riess , daniel zips , martina hinterleitner , lars zender , janina beha , nisar p . 
 bitzer et al falko fend consulting or advisory role : roche , eusa pharma daniel zips research funding : elekta ( inst ) , siemens ( inst ) , sennewald ( inst ) travel , accommodations , expenses : elekta ( inst ) martina hinterleitner travel , accommodations , expenses : novartis / ipsen lars zender leadership : heparegenix gmbh consulting or advisory role : boehringer ingelheim research funding : heparegenix gmbh patents , royalties , other intellectual property : patent on mkk4 inhibition for the treatment of acute and chronic liver diseases travel , accommodations , expenses : ipsen ghazaleh tabatabai honoraria : abbvie , bayer , medac , novocure ( inst ) consulting or advisory role : abbvie , bayer travel , accommodations , expenses : novocure ( inst ) nisar p . 
hoefin r , geiler a - l , fritsch r , et al : personalized clinical decision making through implementation of a molecular tumor board : a german single - center experience . 
j clin oncol 33 : 2753 - 2762 , 2015 bryce ah , egan jb , borad mj , et al : experience with precision genomics and tumor board , indicates frequent target identication , but barriers to delivery . oncotarget 8 : 27145 - 27154 , 2017 zehir a , benayed r , shah rh , et al : mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10 , 000 patients . 
nat med 23 : 703 - 713 , 2017 [ erratum : nat med , 2017 ] jones s , anagnostou v , lytle k , et al : personalized genomic analyses for cancer mutation discovery and interpretation . 
sci transl med 7 : 283ra53 , 2015 cheng dt , prasad m , chekaluk y , et al : comprehensive detection of germline variants by msk - impact , a clinical diagnostic platform for solid tumor molecular oncology and concurrent cancer predisposition testing . 
eur j cancer 45 : 228 - 247 , 2009 seymour l , bogaerts j , perrone a , et al : irecist : guidelines for response criteria for use in trials testing immunotherapeutics . 
plon se , eccles dm , easton d , et al : sequence variant classication and reporting : recommendations for improving the interpretation of cancer susceptibility j hepatol 66 : 1166 - 1172 , 2017 genetic test results . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
n engl j med 377 : 2500 - 2501 , 2017 jenkins rw , thummalapalli r , carter j , et al : molecular and genomic determinants of response to immune checkpoint inhibition in cancer . 
fabrizio da , george tj jr , dunne rf , et al : beyond microsatellite testing : assessment of tumor mutational burden identies subsets of colorectal cancer who may respond to immune checkpoint inhibition . 
verlingue l , malka d , allorant a , et al : precision medicine for patients with advanced biliary tract cancers : an effective strategy within the prospective moscato - 01 trial . 
overman mj , lonardi s , wong kym , et al : durable clinical benet with nivolumab plus ipilimumab in dna mismatch repair - decient / microsatellite instabilityhigh metastatic colorectal cancer . 
ferguson , mbbs , mrcp , frcr4 ; alessandra curioni - fontecedro , md , pd1 ; martin zoche , phd1 ; holger moch , md1 ; and bart vrugt , md , phd1 introduction malignant mesothelioma ( mm ) is a rare neoplasm arising from the mesothelial cell lining of the pleura ( 80% to 90% ) , but it can also develop in the peritoneum ( 10% to 15% ) , pericardium , and tunica vaginalis.1 , 2 because of the long latency of the disease , the age at diagnosis ranges from 54 to 65 years3 - 5 ; thus , mm rarely occurs in young patients and children . the frequency of both pleural and peritoneal mm is almost equal in patients younger than age 40 years . 
in addition , previous asbestos exposure is less common in peritoneal compared with pleural mm.6 peritoneal mesothelioma commonly presents with vague and unspecic symptoms , including abdominal distension and weight loss.7 consequently , the diagnosis of peritoneal mesothelioma generally is made at an advanced stage of the disease , 8 which partly explains the poor clinical outcome , even after optimal oncologic and surgical treatment . 
the median survival is less than 12 months in the majority of patients.9 , 10 patients with mm are not routinely analyzed for the presence of potentially drug - targetable gene mutations , including anaplastic lymphoma tyrosine kinase ( alk )  . 
by using targeted next - generation sequencing of tumor dna and rna , hung et al11 recently identied alk rearrangements with three novel alk fusion partners in a small subgroup of patients with peritoneal mm . 
the alk breakpoint was mapped to intron 19 in all three cases , the strn breakpoint to intron 3 , the atg16l1 breakpoint to intron 2 , and the tpmi1 breakpoint to the beginning of exon 9 . here we report a 13 - year - old girl with peritoneal mesothelioma harboring an strn - alk gene fusion , which was identied by comprehensive genomic proling ( cgp )  . 
images before treatment showed large - volume ascites , bilateral hydrotissue nephrosis , and widespread peritoneal soft deposits particularly within the pelvis and around the liver with scalloping of the liver margin ( fig 1 )  . 
she had no history of asbestos or radiation exposure . peritoneal biopsies were obtained by an explorative laparoscopy and submitted for a histologic and molecular work - up . pathology multiple peritoneal biopsies revealed a tumor with a mixed tubulopapillary and solid growth pattern . immunohistochemistry with positivity for ck5 / 6 and calretinin in the absence of expression of the epithelial marker berep - 4 and claudin - 4 conrmed the mesothelial origin of the tumor . 
even though necrosis and mitotic activity ( less than 1 mitosis per 10 high - power elds ) were not evident , growth pattern , cytonuclear atypia of the tumor cells , and immune prole fullled the criteria for malignant epithelioid mesothelioma ( figs 2a and 2b )  . 
images before treatment demonstrate large - volume ascites , bilateral hydronephrosis , and widespread peritoneal soft tissue deposits particularly within the pelvis ( all ndings marked by green arrows )  . 
 ( b ) schematic representation of the strn - alk fusion showing the 700 - amino - acid ( aa ) - long fusion gene , with 137 aas originating from the n terminus of strn and the remaining 562 aas originating from the c terminus of alk . 
 ( a ) immunohistochemical staining showing strong expression of alk protein ( original magnication 40 )  . ( b ) alk uorescent in situ hybridization break - apart probes showing one fused red - green signal and one red signal in 96% of the tumor cells compatible with 5 ( cid : 1 ) alk deletion ( original magnication 100 )  . for a transient and self - limiting rise in alanine aminotransferase ( alt ) , no adverse effects were noted . discussion we report a dramatic tumor regression after the rst cycle of ceritinib in a 13 - year - old girl with alk - rearranged peritoneal mm . 
the alk receptor can be translocated , mutated , or overexpressed in non - squamous nonsmall - cell lung cancer , inammatory myobroblastic tumors ( imts ) , and anaplastic large - cell lymphomas ( alcls ) .14 it is well known that pediatric patients with imts or alcl can show strong and sustained clinical response to the inhibitory effects of the alk - targeting drug crizotinib.15 peritoneal mm is an extremely rare tumor in children , but an almost complete response to an alk - targeting drug has never been described before . 
recently , loharamtaweethong et al16 reported a 10 - year - old girl with an alk - translocated peritoneal mm detected by fish and immunohistochemistry , but the exact fusion partner was not determined . 
genetic alterations frequently seen in pleural and peritoneal mesothelioma , including cdkn2a , bap - 1 , nf2 , and setd2 , are not detected in patients with alk - rearranged disease.11 even though the prevalence of alk - rearranged peritoneal mesotheliomas is low , alk alterations seem to be restricted to peritoneal mesothelioma only and do not occur in pleural mm . 
this is consistent with our observation , because we were unable to demonstrate alk expression in a tissue microarray containing tumor samples from 221 patients with pleural mesothelioma , including 22 females of whom three were younger than age 40 years ( unpublished data )  . in our patients tumor sample , we identied a rare alk fusion by cgp . 
the platform used in this study can detect all alk alterations ( including atg16l1 and tpm1 fusions ) and has been extensively validated.12 even in alk fishnegative patients , alk rearrangements could be identied by using this method.18 in our patient , we detected an strn - alk fusion with similar breakpoints but with a fusion in the alk - rearranged patient protein identical described by hung et al.11 the strn - alk fusion was an inframe alteration with breakpoints for strn in intron 3 and alk in intron 19 . to that patients with alk - rearranged mm cannot be distinguished from their conventional counterparts , but the differences in clinical presentation ( young patients with no history of asbestos exposure ) and the detection of isolated alk mutations would justify a separate tumor entity of alk - rearranged mesothelioma . 
in the absence of genetic alterations of cdkn2a , bap - 1 , nf2 , and setd2 , it is tempting to speculate that fusions involving the alk gene may represent the predominant oncogenic driver in peritoneal mm in young female patients . 
on the basis of the success of therapeutic alk inhibition in our patient , alk fusion screening by cgp is highly advised in peritoneal mms , especially in young patients . 
the promising response in our patient also stresses the need for investigations involving a larger cohort of young female patients with alkrearranged peritoneal mm to determine the efcacy and durability of alk inhibition . 
r uschoff , elise gradhand , alessandra curioni - fontecedro , martin zoche , holger moch , bart vrugt administrative support : holger moch provision of study materials or patients : elise gradhand , helen rees , martin zoche , bart vrugt collection and assembly of data : elise gradhand , abdullah kahraman , helen rees , jane l . 
hoffmann - la roche holger moch honoraria : roche consulting or advisory role : roche , deniens research funding : roche ( inst ) travel , accommodations , expenses : roche pharma travel , accommodations , expenses : deniens no other potential conicts of interest were reported . references 20 : 935 - 944 , 2009 ray m , kindler hl : malignant pleural mesothelioma : an update on biomarkers and treatment . 
cancer causes control beebe - dimmer jl , fryzek jp , yee cl , et al : mesothelioma in the united states : a surveillance , epidemiology , and end results ( seer ) - medicare investigation of treatment patterns and overall survival . 
eur j gynaecol oncol 18 : 141 - 143 , 1997 asensio ja , goldblatt p , thomford nr : primary malignant peritoneal mesothelioma : a report of seven cases and a review of the literature . 
arch surg 125 : 1477 - 1481 , 1990 thomas a , chen y , yu t , et al : distinctive clinical characteristics of malignant mesothelioma in young patients . 
tumori 89 : 269 - 273 , 2003 kaya h , sezgi c , tanrikulu ac , et al : prognostic factors inuencing survival in 35 patients with malignant peritoneal mesothelioma . 
fabrizio da , george tj jr , dunne rf , et al : beyond microsatellite testing : assessment of tumor mutational burden identies subsets of colorectal cancer who may respond to immune checkpoint inhibition . 
moss e yp , voss sd , lim ms , et al : targeting alk with crizotinib in pediatric anaplastic large cell lymphoma and inammatory myobroblastic tumor : a childrens oncology group study . 
loharamtaweethong k , puripat n , aoonjai n , et al : anaplastic lymphoma kinase ( alk ) translocation in paediatric malignant peritoneal mesothelioma : a case report of novel alk - related tumour spectruhistopathology 68 : 603 - 607 , 2016 17 . 
ali sm , ou shi , he j , et al : identifying alk rearrangements that are not detected by fish with targeted next - generation sequencing of lung carcinoma . 
an endobronchial biopsy showed an adenocarcinoma consistent with a lung primary ( thyroid transcription factor 1 positive , napsin a positive , and cytokeratin 7 positive by immunohistochemistry ; figs 1a and 1b )  . targeted exon sequencing of 468 genes with memorial sloan kettering integrated mutation proling of actionable cancer targets ( msk - impact ) was negative for known mitogenic drivers , including kras , egfr , braf , met , and erbb2 mutations and alk , ros1 , and ret rearrangements . 
photomicrographs of ( a ) cytology from the patients lung adenocarcinoma and ( b ) positive immunohistochemistry results remarkable for cytoplasmic and membranous staining using a pan - trk antibody are shown . 
this identied a novel activating trk fusion ( in - frame fusion between eps15 exon 21 and ntrk1 exon 10 ; figs 1c and 1d )  . the patient consented to receive larotrectinib in a basket study for trk fusion - positive cancers ( navigate ; clinicaltrials.gov identier : nct02576431 )  . 
a brisk partial response to therapy was achieved at 4 weeks , which was subsequently conrmed at 8 weeks ( 34% by response evaluation criteria in solid tumors [ recist ] version 1.1 ; fig 2 ) , with regression of all areas of disease . 
this included near total resolution of all brain metastases at 8 weeks ( 95% reduction in aggregate tumor volume ) and subsequently , a complete intracranial response by 16 weeks ( fig 3 )  . 
pathology showed a stage iiic ( t1cn3m0 ) multifocal invasive ductal carcinoma ; the largest lesion measured 1.8 cimmunohistochemistry was positive for estrogen and progesterone receptors and negative for human epidermal growth factor receptor 2 . 
she was treated initially with adjuvant dose - dense doxorubicin , cyclophosphamide , plus paclitaxel and radiotherapy , after which she received anastrozole followed by bilateral salpingo - oophorectomy . the patient developed recurrent , metastatic disease approximately 17 months after completing adjuvant radiolymph node biopsy conrmed therapy . 
sequencing of an abdominal lymph node and a subsequently biopsied liver metastasis using msk - impact revealed the previously reported fusion between lmna exons 1 and 20 and ntrk1 exons 11 and 17 , which was veried using targeted multiplex rna sequencing . the patient consented to receive larotrectinib in the same basket study for trk fusion - positive cancers ( navigate )  . at enrollment , her disease was widely metastatic to liver , adrenal gland , lymph nodes , and bone . 
a brisk and conrmed partial response by recist version 1.1 ( 32% , 47% , and 56% at 4 , 8 , and 16 weeks , respectively ) was achieved . 
of note , her cns metastases likewise regressed at the rst follow - up assessment at 4 weeks , with a complete response in the cns at 8 weeks ( fig 4 )  . 
axial contrastenhanced t1 - weighted images at ( a ) baseline ( brain metastases highlighted by circles ) , ( b ) 8 weeks , and ( c ) 16 weeks after treatment with larotrectinib was initiated demonstrate decreased and then resolved metastases in the right - side insula and left - side occipital lobe . 
three - dimensional models at ( d ) baseline and ( e ) post - treatment at 8 weeks and ( f ) post - treatment at 16 weeks conrmed decreased burden of metastatic intracranial disease ( segmented in green voxels and quantied at bottom )  . 
response to larotrectinib in a patient with a trk fusion - positive metastatic breast cancer . baseline axial computed tomography imaging of the brain was performed with the addition of iodinated contrast that showed ( a ) metastatic brain disease ( circles ) and after 8 weeks of treatment with larotrectinib , ( b ) complete response . 
 rosen et al discussion to our knowledge , this report is the rst to showcase the intracranial efcacy of larotrectinib in metastatic trk fusion - positive cancers of any histology . 
these cases demonstrate that larotrectinib can achieve clinically relevant cns exposure , which highlights its role in the management of trk fusion - positive cancers with intracranial disease . beyond nsclcs and breast cancers , other trk fusionpositive tumors have a proclivity for brain metastases.1 the intracranial activity featured here is consistent with prior work that did not identify cns metastasis as a site of progression on larotrectinib across multiple tumor types4 and with a report that showed a response to larotrectinib in a trk fusion - positive glioma.5 additional clinical experience in patients with trk fusion - positive cancers with cns involvement ultimately will delineate the overall activity and durability of larotrectinib within the cns . although this article represents what is to our knowledge the rst report of intracranial responses to a sein patients with trk fusionlective trk inhibitor positive cancers and brain metastases , intracranial disease control also has been reported with multikinase trk inhibitors . 
entrectinib , a multikinase trk , ros1 , and alk inhibitor , displayed activity in a patient with a trk fusion - positive lung cancer and brain metastases6 and in ros1 fusion - positive lung cancers with intracranial disease.7 repotrectinib , a multikinase trk , ros1 , and alk inhibitor , achieved intracranial disease regression in untreated brain metastases in a patient with a ros1 fusion - positive nsclc.8 finally , this report represents the rst description of the novel eps15 - ntrk1 fusion . 
young stock and other ownership interests : agios , alexion pharmaceuticals , biogen , celgene , gilead sciences , karyopharm therapeutics , spark therapeutics , regeneron pharmaceuticals , stemline therapeutics , vertex consulting or advisory role : agios , puma biotechnology , nordicneurolab , icon clinical research research funding : agios ( inst ) jaclyn f . 
shu research funding : celgene ( inst ) , genentech ( inst ) , roche ( inst ) , janssen pharmaceuticals ( inst ) , medimmune ( inst ) travel , accommodations , expenses : genentech , roche nora c . 
 cns efcacy of larotrectinib in trk fusion - positive solid tumors travel , accommodations , expenses : novartis ( i ) , biotheranostics ( i ) , pzer ( i ) , amgen ( i ) , genentech ( i ) , roche ( i ) , astrazeneca ( i ) , macrogenics ( i ) , peregrine pharmaceuticals ( i ) , pierian bioscience ( i ) david m . 
ziegler ds , wong m , mayoh c , et al : brief report : potent clinical and radiological response to larotrectinib in trk fusion - driven high - grade glioma . 
drilon a , siena s , ou s - hi , et al : safety and antitumor activity of the multi - targeted pan - trk , ros1 , and alk inhibitor entrectinib ( rxdx - 101 ) : combined results from two phase 1 trials ( alka - 372 - 001 and startrk - 1 )  . 
doebele r , ahn m , siena s , et al : oa02.01 : efcacy and safety of entrectinib in locally advanced or metastatic ros1 fusion - positive non - small cell lung cancer 8 . 
drilon a , ou si , cho bc , et al : repotrectinib ( tpx - 0005 ) is a next - generation ros1 / trk / alk inhibitor that potently inhibits ros1 / trk / alk solvent - front 9 . 
 rationale and design of the targeted agent and profiling utilization registry study purpose case reports and small prospective trials suggest that administering targeted therapies to patients with advanced cancer and an identified genomic target may be associated with clinical benefit . 
the targeted agent and profiling utilization registry ( tapur ) study , a phase ii prospective , nonrandomized , multibasket pragmatic clinical trial , aims to identify signals of drug activity when us food and drug administration approved drugs are matched to prespecified genomic targets in patients with advanced cancer , outside of approved indications . methods patients eligible to participate in tapur are age 12 years and have advanced measurable or evaluable solid tumors , multiple myeloma , or b - cell non - hodgkin lymphoma . 
secondary end points include safety , progression - free survival , and overall survival . results more than 1 , 000 participants have thus far been registered , and more than 800 have been treated with a tapur study drug . 
two study cohorts have permanently closed to enrollment because of lack of antitumor activity , and 12 have expanded to the second stage of enrollment after promising preliminary activity . conclusion the tapur study will describe the efficacy and toxicity of the targeted drugs used outside of their approved indications when matched to a somatic genomic variant . evidence is building through reports of clinical trials , case reports , and clinical anecdotes to suggest that patient outcomes may be improved when a targeted agent is matched to a genomic alteration present in a patients tumor.1 - 6 clinical reports to date suggest that 30% to 80% of advanced solid tumors harbor potentially actionable genomic variants.7 - 10 in a meta - analysis of 570 phase ii studies of new anticancer agents , schwaederle et al11 examined response rate ( rr ) , progression - free survival ( pfs ) , and overall survival ( os ) for 32 , 149 patients who received a personalized treatment strategy versus those who did not . 
 of the md anderson cancer center experience of genomic profiling of patients with solid tumors with advanced disease , the impact ( initiative for molecular profiling and advanced cancer therapy ) study . 
the moscato - 01 ( molecular screening for cancer treatment optimization ; clinicaltrials.gov identifier : nct01566019 ) trial enrolled patients with treatment - resistant progressive metastatic cancers with lesions accessible to biopsy to perform genomic profiling.14 this study compared pfs using therapy based on genomic assessment with pfs for the most recent therapy during which the patient had experienced disease progression . 
le tourneau et al17 published the results of a randomized phase ii trial comparing therapy on the basis of tumor molecular profiling versus physician choice of therapy in patients with refractory cancer . 
this article will describe the rationale and design of the targeted agent and profiling utilization registry ( tapur ) study , a large pragmatic precision medicine basket trial with the overarching goal of describing the efficacy and toxicity of targeted anticancer drugs used outside of their approved indications for treatment of patients with advanced cancer based on a tumor genomic profile . methods objectives the primary objective of tapur is to evaluate the antitumor activity of commercially available targeted anticancer drugs used outside of their fda - approved indications for treatment of patients with advanced solid tumors , multiple myeloma , or b - cell non - hodgkin lymphoma with a genomic alteration known to be a drug target . 
secondary objectives include determination of pfs , os , and safety . design the tapur study is a phase ii prospective , nonrandomized , open - label clinical trial that aims to define signals of drug activity . 
list of available tapur study treatments for adults and pediatric patients study drug population to which available adults and children age 12 - 17 years adults only adults and children age 12 - 17 years adults and children age 16 - 17 years adults and children age 13 - 17 years adults and children age 12 - 17 years adults and children age 16 - 17 years adults only adults and children age 12 - 17 years adults and children age 12 - 17 years adults and children age 12 - 17 years nivolumab + ipilimumab adults only pembrolizumab adults and children age 12 - 17 years axitinib bosutinib cetuximab crizotinib dasatinib erlotinib olaparib palbociclib regorafenib sunitinib temsirolimus trastuzumab + pertuzumab adults only vemurafenib + cobimetinib adults only vismodegib adults and children age 12 - 17 years note . 
the study includes participants as young as 12 years of age with a tumor harboring a genomic alteration known to be a target of or to predict sensitivity to at least one of the 16 therapeutic options available in the study ( table 1 )  . 
eligible participants must have a tumor genomic variant identified on a test performed in a laboratory that has certification under the clinical laboratory improvement amendments and accreditation by the college of american pathologists . 
patients are matched to at least one of the available study treatments through a set of protocol - defined genomic matching rules or after review of a participant case by the tapur molecular tumor board ( mtb )  . 
the study uses simons optimal two - stage design for cohort analysis . tapur was designed independently by asco staff and volunteer leaders , with input from patient advocates , community - based investigators , the initial collaborating pharmaceutical companies , and the asco cancer research committee . 
the study was registered on clinicaltrials.gov ( nct02693535 ) before study launch . a data and safety monitoring board ( dsmb ) , consisting of a group of independent experts not involved in the conduct of tapur , meets biannually to monitor the study data and outcomes . 
 their primary functions include assessing the safety and efficacy of study treatments , safeguarding the interests and safety of trial participants , and ensuring that study results are both credible and reported to the medical community in a timely manner . 
for expanded cohorts , it will review all final cohort analyses and make recommendations regarding release of the cohort data . study population and recruitment the pragmatic approach of tapur allows for broad eligibility criteria , including eastern cooperative oncology group performance status of 0 to 2 , age 12 years , prior malignancies or positive hiv status , and previously treated but clinically stable brain metastases . 
tapur inclusion and exclusion criteria comport fully with recently published recommendations by asco and friends of cancer research for broadening eligibility criteria for cancer clinical trials.23 participating sites include both academic and community centers . study treatment decision once a potential participant is identified , the clinical site obtains informed consent and registers the participant in the tapur electronic data capture ( edc ) platform as shown in figure 1 . 
if a study drug is identified and the drug - specific inclusion and exclusion criteria are met , the clinical site completes the drug specific informed consent process and enrolls the participant in the study . 
 a screening consent general eligibility criteria met registration treatment 1 treatment 2 , continued tapur matching to study drug ( s ) based on genomic criteria ( or approved by mtb ) treatment decision by treating physician target variant 1 target variant 2 screening continued drug - specific eligibility criteria met tumor type 1 tumor type 2 tumor type 1 tumor type 2 enrolled and placed into cohorts enrollment cohort 1 cohort 2 cohort 3 cohort 4 fig 1 . 
the mtb , composed of clinical oncologists , molecular pathologists , and patient advocates , reviews the genomic test results , pathology reports , and clinical histories of submitted patient cases and identifies potential treatment matches among the study treatments . 
of the patient cases reviewed by the mtb , approximately 65% resulted in identification of a study drug option . permitted at the discretion of the treating physician in accordance with the recommended dose modifications contained in the approved prescribing information . 
management of aes is performed according to institutional standards of care , informed by the package insert . tumor measurements and radiologic evaluations and evaluation of clinical disease status are performed every 8 weeks after initiation of treatment for the first 16 weeks , and then every 12 weeks if the treatment continues , and at the end of study treatment , if possible . 
study treatment is continued until progressive disease is documented . after progression of disease during any study treatment , participants may be reassessed to determine eligibility for treatment with another study drug after a minimum of 30 days has elapsed from the last dose of the previous drug and any aes have resolved to grade 2 or stabilized . 
all general and drug - specific eligibility criteria must be reconfirmed , and the participant must sign a new drug - specific consent form . treatment schedule and interval of evaluations ae and sae criteria study treatments are administered according to the recommended starting dose and schedule described in the package insert of each drug . 
events not resolved at the end of study treatment require follow - up every 30 days until the event resolves to ctcae grade 2 . tapur requires reporting of ctcae grade 3 to 4 aes that are possibly , probably , or definitely related to the study treatment , whether expected or not . 
all saes , regardless of grade , relatedness to study drug , or expectedness , must also be reported , including all deaths . end points the primary end point is objective response at 8 weeks or sd documented at 16 weeks . 
tapur study milestones date milestone march 14 , 2016 study launch at 37 clinical site locations with 14 study drugs first participant registered ( consented ) first participant enrolled ; two additional drugs added for total of 16 study drugs additional drug added for total of 17 study drugs 100th participant registered november 2016 100th participant enrolled march 2016 april 2016 may 2016 august 2016 april 2017 june 2017 august 2017 september 2017 november 2017 december 2017 february 2018 march 2018 33 clinical sites added for total of 70 locations 500th participant registered additional therapy added for 19 study drugs first cohort reaches stage one 500th participant enrolled 1 , 000th participant registered 10th cohort reaches stage one 43 clinical sites added for total of 113 locations abbreviation : tapur , targeted agent and profiling utilization registry . the basis of the presence , absence , or unequivocal progression of the lesions . 
participants who have no tumor evaluation beyond baseline and are alive with no signs of progressive disease at the time of leaving the study will be replaced in their cohort with another participant . secondary end points include grade 3 to 5 aes and saes , pfs , response duration , and os . 
the null hypothesis is that the probability of objective response or sd of at least 16 weeks duration is 15% , versus the alternative hypothesis that it is at least 35% . 
after 28 participants in the cohort have been observed for the primary outcome , if seven or more participants achieve objective response or sd of at least 16 weeks duration , the null hypothesis will be rejected , and we will declare that the study drug is active in the cohort of participants defined by tumor type and genomic alteration . 
this design has a 54% chance of early stopping if the null hypothesis is true . estimates of the rr and 95% ci will be presented for each cohort upon closure ( at either stage one or stage two ) .24 the kaplan - meier method will be used to estimate the duration of response , pfs , and os distributions . results table 3 summarizes key study milestones . 
figure 2 also shows the distribution of genomic targets by tumor type ( fig 2b ) and study drug ( fig 2c ) , highlighting the heterogeneity of genomic targets and tumor types , resulting in 336 unique cohorts at the time of this writing . 
targeted agent and profiling utilization registry ( tapur ) study registration , enrollment , and cohort creation by ( a ) month , ( continued on next page ) many cancers that are driven by specific molecular alterations that can be targeted with drugs that inhibit aberrant signaling pathways in tumor cells . 
thus , the targeting of bcr - abl alterations in chronic myeloid leukemia , flt3 alterations in acute myeloid leukemia , egfr mutations and alk translocations in nonsmallcell lung cancer , braf mutations in melanoma and nonsmall - cell lung cancer , and human epidermal growth factor receptor 2 overexpression in breast and gastric cancers , among others , represent examples of the successful application of precision medicine approaches in clinical oncology that have extended the lives of many patients.1 , 25 - 30 interest in precision medicine has been further fueled by reported responses ranging from modest to exceptional when targeted treatments are selected based on large - scale genomic tumor profiling.5 the widespread availability of commercial next generation sequencing tests presents opportunities for patients who have exhausted standard treatment options to pursue treatments with investigational agents or approved drugs prescribed outside of their labeled indications . the tapur study stemmed from the recognition that the rapid dissemination of genomic profiling provides an opportunity to learn from the application of precision cancer medicine in practice while at the same time providing a framework for clinical decision support for clinical oncologists who are struggling to interpret the complex genomic data they now confront in practice . 
 b vegfr3 ( flt4 ) mutation , amplification , or overexpression vegfr1 ( flt1 ) mutation , amplification , or overexpression smo mutation lck mutation prkdc mutation pold1 mutation msh2 mutation vegfr3 ( flt4 ) mutation or amplification ewsr1nrya3 fusion cdk12 mutation ccnd3 bcr - abl mutation alk fusion or mutation msi high status egfr mutation rictor amplification vegfr1 ( flt1 ) mutation or amplification csf1r mutation or amplification vegfr2 ( kdr ) mutation , amplification , or overexpression vhl mutation or amplification ptch1 deletion or inactivating mutation mlh1 mutation tsc2 mutation pdgfra mutation or amplification palb2 mutation stk11 mutation vegfr2 ( kdr ) mutation or amplification ret mutation or amplification pdgfr ? mutation or amplification kit mutation atm mutation tsc1 mutation pole mutation fgfr3 mutation or amplification cdk6 amplification raf1 mutation or amplification pten mutation kit mutation or amplification braf mutation or amplification akt1 mutation flt3 mutation or amplification brca1 / brca2 mutation met amplification or mutation pik3ca mutation mtor mutation fgfr2 mutation or amplification ccnd1 amplification fgfr1 mutation or amplification cdk4 amplification atm mutation or deletion braf_v600e / d / k / r mutation erbb2 / erbb3 mutation , amplification , or overexpression kras , nras , and braf wild type ( all must be wild type ) germline or somatic brca1 / brca2 inactivating mutations high tumor mutational burden cdkn2a loss or mutation total fig 2 . 
 ( a ) april 2018 registration , enrollment , and cohort creation in progress at the time of this report ; ( b , c ) the number in each box indicates the number of patients . 
 c vegfr3 ( flt4 ) mutation , amplification , or overexpression vegfr1 ( flt1 ) mutation , amplification , or overexpression smo mutation lck mutation prkdc mutation pold1 mutation msh2 mutation vegfr3 ( flt4 ) mutation or amplification ewsr1nrya3 fusion cdk12 mutation ccnd3 bcr - abl mutation alk fusion or mutation msi high status egfr mutation rictor amplification vegfr1 ( flt1 ) mutation or amplification csf1r mutation or amplification vegfr2 ( kdr ) mutation , amplification , or overexpression vhl mutation or amplification ptch1 deletion or inactivating mutation mlh1 mutation tsc2 mutation pdgfra mutation or amplification palb2 mutation stk11 mutation vegfr2 ( kdr ) mutation or amplification ret mutation or amplification pdgfr ? mutation or amplification kit mutation atm mutation tsc1 mutation pole mutation fgfr3 mutation or amplification cdk6 amplification raf1 mutation or amplification pten mutation kit mutation or amplification braf mutation or amplification akt1 mutation flt3 mutation or amplification brca1 / brca2 mutation met amplification or mutation pik3ca mutation mtor mutation fgfr2 mutation or amplification ccnd1 amplification fgfr1 mutation or amplification cdk4 amplification atm mutation or deletion braf_v600e / d / k / r mutation erbb2 / erbb3 mutation , amplification , or overexpression kras , nras and braf wild type ( all must be wild type ) germline or somatic brca1 / brca2 inactivating mutations high tumor mutational burden cdkn2a loss or mutation total fig 2 . 
 ( continued ) ( c ) ibrance ( pfizer , new york , ny ) , lynparza ( astrazeneca , wilmington , de ) , sutent ( pfizer ) , keytruda ( merck , kenilworth , nj ) , torisel ( pfizer ) , erbitux ( eli lilly , indianapolis , in ) , perjeta ( genentech , south san francisco , ca ) , herceptin ( genentech ) , cotellic ( genentech ) , zelboraf ( genentech ) , opdivo ( bristol - myers squibb , new york , ny ) , yervoy ( bristol - myers squibb ) , stivarga ( bayer , whippany , nj ) , xalkori ( pfizer ) , sprycel ( bristol - myers squibb ) , erivedge ( genentech ) , inlyta ( pfizer ) , bosulif ( pfizer ) , and tarceva ( genentech )  . 
tapur study initial cohort expansions and closures study drug tumor type variant ovarian cancer kras , nras , and braf wild type colorectal cancer braf_v600e / d / k / r mutation breast cancer colorectal cancer germline or somatic brca1 / brca2 inactivating mutations atm mutation or deletion head and neck cancer soft tissue sarcoma malignant neoplasm of bronchus and lung cdkn2a loss or mutation cdk4 amplification cdkn2a loss or mutation breast cancer colorectal cancer uterine cancer high tumor mutational burden high tumor mutational burden high tumor mutational burden colorectal cancer erbb2 / erbb3 mutation , amplification , or overexpression expansion to stage two cetuximab cobimetinib + vemurafenib olaparib palbociclib pembrolizumab pertuzumab + trastuzumab sunitinib closed at stage one palbociclib breast cancer fgfr1 mutation or amplification pancreatic cancer malignant neoplasm of gallbladder and bile ducts cdkn2a loss or mutation abbreviation : tapur , targeted agent and profiling utilization registry . data collection , and discretion left to the treating physician regarding choice of which tumor biospecimen to interrogate and which genomic profiling test to use . 
however , tapur uses standard treatment response and toxicity criteria for assessment of efficacy and toxicity outcomes , structured data collection , protocol - specified evaluation times , and a standard simons twostage statistical design to assess the primary end point as well as an independent dsmb to provide recommendations on cohort expansion and closure . 
importantly , the study also provides educational opportunities and clinical decision support tools to treating physicians in the form of protocol - specified genomic matching rules and access to an mtb to assist in interpretation of genomic test reports . although study results are not yet available , preliminary information about the status of certain cohorts suggests that the study can provide meaningful information . 
 enrollment rates when the frequency of genomic targets was not well defined for many tumor types , identifying clinical launch sites that routinely used genomic profiling , arranging for drug distribution , and building an experienced clinical trial management team to operate the study . 
mangat no relationship to disclose susan halabi consulting or advisory role : tokai pharmaceuticals consulting or advisory role : eisai , bayer healthcare pharmaceuticals travel , accommodations , expenses : bayer healthcare pharmaceuticals suanna s . 
bruinooge no relationship to disclose elizabeth garrett - mayer stock and other ownership interests : abbott laboratories , abbvie consulting or advisory role : tactical therapeutics , okava pharmaceuticals ajjai alva consulting or advisory role : eisai , astrazeneca , genentech / roche speakers bureau : astrazeneca research funding : genentech ( inst ) , novartis ( inst ) , bristol - myers squibb ( inst ) , bind biosciences ( inst ) , acerta pharma ( inst ) , merck sharp & dohme ( inst ) , prometheus laboratories ( inst ) , covance ( inst ) , mirati therapeutics ( inst ) , united biosource ( inst ) , ariad pharmaceuticals ( inst ) , astrazeneca ( inst ) , pfizer ( inst ) , genentech / roche ( inst ) , hoosier cancer research network ( inst ) , bayer healthcare pharmaceuticals ( inst ) katherine a . 
stella no relationship to disclose emile voest consulting or advisory role : interna , biogeneration ventures research funding : novartis ( inst ) , glaxosmithkline ( inst ) , genentech / roche ( inst ) , pfizer ( inst ) , astrazeneca ( inst ) , eisai ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) kathleen j . 
kim honoraria : celgene , eli lilly , astrazeneca , boehringer ingelheim consulting or advisory role : eli lilly , celgene , astrazeneca , boehringer ingelheim travel , accommodations , expenses : eli lilly , celgene , astrazeneca , boehringer ingelheim richard l . 
kim , carolinas healthcare systems levine cancer institute , charlotte , nc ; ajjai alva , university of michigan ; jane perlmutter , gemini group , ann arbor ; philip j . 
janeway , data - farber / boston childrens cancer and blood disorders center , boston , ma ; emile voest , netherlands cancer institute , amsterdam , the netherlands ; and navin pinto , seattle childrens hospital , seattle , wa . 
wagle n , grabiner bc , van allen em , et al : activating mtor mutations in a patient with an extraordinary response on a phase i trial of everolimus and pazopanib . 
cobain ef , robinson dr , wu y - m , et al : clinical impact of high - throughput sequencing in patients with advanced cancer : lessons learned from the michigan oncology sequencing center . 
jardim dl , schwaederle m , wei c , et al : impact of a biomarker - based strategy on oncology drug development : a meta - analysis of clinical trials leading to fda approval . 
tsimberidou am , wen s , hong ds , et al : personalized medicine for patients with advanced cancer in the phase i program at md anderson : validation and landmark analyses . 
massard c , michiels s , fert c , et al : high - throughput genomics and clinical outcome in hardto - treat advanced cancers : results of the moscato 01 trial . 
haslem ds , van norman sb , fulde g , et al : a retrospective analysis of precision medicine outcomes in patients with advanced cancer reveals improved progression - free survival without increased health care costs . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , openlabel , proof - of - concept , randomised , controlled phase 2 trial . 
cheson bd , fisher ri , barrington sf , et al : recommendations for initial evaluation , staging , and response assessment of hodgkin and non - hodgkin lymphoma : the lugano classification . 
barrington sf , mikhaeel ng , kostakoglu l , et al : role of imaging in the staging and response assessment of lymphoma : consensus of the international conference on malignant lymphomas imaging working group . 
kim es , bruinooge ss , roberts s , et al : broadening eligibility criteria to make clinical trials more representative : american society of clinical oncology and friends of cancer research joint research statement . 
druker bj , talpaz m , resta dj , et al : efficacy and safety of a specific inhibitor of the bcr - abl tyrosine kinase in chronic myeloid leukemia . 
slamon dj , leyland - jones b , shak s , et al : use of chemotherapy plus a monoclonal antibody against her2 for metastatic breast cancer that overexpresses her2 . 
bang yj , van cutsem e , feyereislova a , et al : trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of her2 - positive advanced gastric or gastrooesophageal junction cancer ( toga ) : a phase 3 , open - label , randomised controlled trial . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors on the basis of molecular profiles : results from mypathway , an open - label , phase iia multiple basket study . 
butler , mph ; kuo guo , ms ; molly holoubek , ccrn ; samiha islam ; linda miller , msn , rn , ocn ; shamika ranasinghe , ms ; brittany m . 
precursors and carcinomas were immunohistochemically compared and analyzed for mutations , gene amplications , microsatellite instability , and tumor mutational burden using an next - generation sequencing panel containing 523 cancer - related genes . 
in addition , presence of fusion genes was analyzed using a panel of 55 genes . results sixty percent of neuroendocrine cases ( 6 / 10 ) presented with a precursor lesion , either intracholecystic papillary neoplasm ( n = 3 ) or biliary intraepithelial neoplasia ( n = 3 )  . 
moreover , nearly half of the ne gbcs carried at least one potentially actionable molecular alteration , highlighting the importance of molecular testing in this highly lethal cancer . jco precis oncol 5 : 473 - 484 . 
2021 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction neuroendocrine neoplasms ( nens ) can arise from almost any anatomical site including , but not limited to , the lung , the prostate , and throughout the gi and hepatobiliary tract . 
two major categories of nens have been described : well - differentiated neuroendocrine tumors ( nets ) and poorly differentiated neuroendocrine carcinomas ( necs ) , the latter being high grade by denition.1 morphologically , necs are classied as either pure small - cell or large - cell nec , and they might also be present together with an adenocarcinoma ( ac ) component . 
they nonneuroendocrine neoplasms ( minens ) when both the neuroendocrine component ( generally nec ) and the non - neuroendocrine component ( generally ac ) comprise at least 30% of the neoplasm.2 regardless of the anatomical site , necs and minens are highly aggressive tumors and carry a dismal prognosis . neuroendocrine as mixed classied necs can either arise de novo or during progression of an epithelial neoplasm , for example , because of a therapy resistance mechanism.3 the molecular features of necs have been best described in lung , pancreas , and prostate , but the cell of origin of necs has been a matter of controversy , specically in tumors with a mixed phenotype ( ie , minens )  . 
this study was approved by the radboud university medical center medical ethics committee ( 2018 - 4126 )  . histopathologic review formalin - xed parafn - embedded ( ffpe ) blocks were selected on the basis of pathology reports and histopathologic review . 
histologic typing and grading , including description of a precursor lesion ( either bilin or icpn ) , was performed using the who histologic classication of tumors of the gallbladder ( fth edition ) .2 t classication was performed using the american joint committee on cancer tumor - node - metastasis classication system ( eighth edition ) .12 additionally , presence of vascular , lymphatic , and perineural invasion was assessed . immunohistochemistry immunohistochemistry ( ihc ) was performed on 4 - mm whole - slide ffpe sections semiautomated using the lab vision autostainer ( immunologic , duiven , the netherlands ) for muc2 , muc5ac , muc6 , and ttf - 1 essentially as described before13 or fully automated using tissue - tek genie ( sakura finetek , torrance , ca ) for chromogranin a , cd56 , ck7 , ck20 , ki - 67 , ema ( muc1 ) , p53 , and synaptophysin with ready - to - use reagents . 
discrepancies in scoring were reevaluated until agreement . nucleic acid extraction for molecular analyses , dna and rna were isolated from ffpe - derived tumor tissue and the precancerous lesion if available . 
dna was isolated , quantied , and precipitated manually as described before.14 after precipitation , the nal concentration was determined using the qubit high sensitivity kit ( thermo fisher scientic , waltham , ma )  . rna was isolated using the reliaprep ffpe total rna miniprep system ( promega , madison , wi ) according to the manufacturers protocol , omitting the dnase treatment step . 
 histologic and molecular features of ne gallbladder cancer subsequently , 60 ng of either dna or rna was used as input for the library preparation . library preparation dna and rna library preparations were performed separately using the hybrid capture - based trusight oncology ( tso ) 500 dna and rna library preparation kits , respectively ( illumina , san diego , ca ) according to the manufacturers protocol essentially as described before.15 the tso500 dna assay targets 523 cancer - related genes for assessment of singleand multiple - nucleotide variants , gene amplications , tumor mutational burden ( tmb ) , and microsatellite instability ( msi ) , whereas the rna kit targets 55 genes for fusion gene analyses ( data supplement )  . 
in short , dna samples were fragmented , and rna samples were used for rstand second - strand cdna synthesis . next , samples underwent end - repair and a - tailing , followed by unique molecular identier ligation and barcoding . two target capture and purication steps , allowing for maximal target enrichment , were followed by polymerase chain reaction amplication and purication . 
libraries were quantied and normalized for uniform library representation . sequencing and data analysis sequencing was performed on a nextseq 500 system ( illumina ) , with 10 dna libraries on a nextseq high - output cassette or 16 rna libraries on a nextseq mid - output cassette . 
the number of gene copies in the tumor cells was estimated on the basis of the relative coverage corrected for the percentage of neoplastic cells in the sample . variant annotation was performed as described before via an in - housedeveloped pipeline.17 variants were ltered by exclusion of ( 1 ) variants outside exons and splice site regions ( 8 / + 8 ) except those in the tert promoter region , ( 2 ) synonymous variants , unless present in a splice site region , ( 3 ) variants present with a frequency of  . 
0.1% in the exac ( version 0.2 ) database , ( 4 ) variants with a variant allele frequency ( vaf ) of , 5% , and ( 5 ) variants with , 5 variant reads . 
remaining variants were manually inspected and curated and classied into ve classes on the basis of their level of evidence for pathogenicity , largely on the basis of american college of medical genetics / association for molecular pathology guidelines18 , 19 : class 1 , not pathogenic ; class 2 , unlikely pathogenic ; class 3 , possibly pathogenic ; class 4 , likely pathogenic ; and class 5 , pathogenic . 
potential effects on splicing were evaluated on the basis of the majority vote of splicesitefinder - like , maxentscan , nnsplice , and genesplicer ( all available from alamut visual version 2.13 [ sophia genetics , lausanne , switzerland ] )  . 
details on classes and their interpretation are provided in figure a1 , online only . variant - specic level 1 , 2 , 3 , or 4 evidence for actionability across all tumor types was derived from oncokb.21 level 1 and 2 biomarkers are dened as fda - recognized or standard care biomarkers , respectively , that are predictive of response to fda - approved drugs in specic tumor types . for level 3a biomarkers , there is compelling clinical evidence of drug response in a specic indication , and level 3b alterations are predictive of response to an fdaapproved or investigational drug in another indication . level 4 alterations comprise alterations for which there is compelling biological evidence that predicts response to a drug . 
seven cases ( 70% ) were pure high - grade net or nec , whereas three tumors ( 30% ) showed both ac and nec features and were classied as minens . 
data on type of systemic therapy are not available . tumors ( 50% ) were large - cell nec , four cases ( 40% ) were small - cell nec , and one case ( 10% ) was a net grade 3 , hereafter collectively referred to as ne gbc . 
in 90% of cases , at least one tumor component ( either nec or , if present , ac ) showed angioinvasion ; lymphatic invasion and perineural invasion were observed in 80% and 30% of cases , respectively . neuroendocrine expression with synaptophysin , chromogranin a , and cd56 conrmed neuroendocrine histology and was absent in the precancerous lesions ( table 2 and fig 1 )  . 
tumor characteristics case diagnosis ptnm ( m location ) icpn ( mixed type ) with nec ( sc ) t2anx bilin with nec ( sc ) t3nx nec ( sc ) t3n2m1 ( liver ) icpn ( intestinal type ) with nec ( lc ) t3n1 icpn ( biliary type ) with minen ( lc ) t2an1 bilin with minen t3nx ( lc ) nec ( lc ) nec ( lc ) t2nx t3nxm1 ( peritoneum ) 476 2021 by american society of clinical oncology abbreviations : + ,  . 
neuroendocrine marker expression with synaptophysin was positive in the nec and negative in the icpn and ac ( chromogranin a and cd56 were comparable ; table 2 ) , whereas epithelial marker expression ( ck 7 ) was positive in the icpn and ac and negative in the nec ( ck20 - negative in all three ; table 2 )  . 
since tp53 is frequently altered in necs of other anatomical origin , case 5 served as an exemplary case for which p53 ihc was performed next to a molecular screening , which was done for all cases . 
both the ac and nec components showed aberrant p53 expression , whereas the icpn component showed predominantly a normal p53 expression with only a small region with overexpression ( fig 1 )  . 
 de bitter et al molecular characteristics discussion for each case , dna and rna were isolated from the precursor lesion , the nec component and , if available , the ac component . 
quality control parameters of dna and rna libraries including reference values are listed in the data supplement . for case 1 , both the nec and icpn components did not meet quality control for msi , tmb , and cnv assessment . all likely pathogenic and pathogenic singleand multiplenucleotide variants ( classes 4 and 5 ) were considered potentially clinically relevant and are listed in figure 2 and the data supplement . 
a variety of other genes harbored potentially clinically relevant mutations in individual cases . mutations in tp53 were shared among the different precursor , ac , and nec components in all but one case ( case 8 ) , where two different mutations were observed in the ac versus the nec . 
interestingly , the icpn of case 5 did share the same tp53 mutation with the invasive tumor components , albeit with a low vaf ( 8.7% ) , reecting the limited region with p53 overexpression ( fig 1 )  . 
three cases ( 30% ) harbored potentially actionable variants ( level 1 ) in atm , brca2 , and rad54l , albeit with a low vaf for the atm variant of case 4 . in six cases , gene amplications were observed in a variety of genes including potentially actionable amplications of erbb2 ( level 1 ) and mdm2 ( level 3a ) , the latter being in a case without a tp53 mutation ( fig 2 )  . 
whereas the majority of mutations were shared among different precancerous , ac , and nec components , for gene amplications this was for a cnne1 amplication that not observed , except was shared between the ac and nec , but not the icpn , of case 5 . a fusion involving fgfr3 and tacc3 genes was observed in all three components of case 5 ( fig 2 )  . 
despite these phenotypical differences , the shared tp53 mutation in the different components in ve of six cases strongly suggests a common origin for neuroendocrine and epithelial components of ne gbc . 
in addition , 50% of the cases also had cholelithiasis , which is a well - known risk factor for pure gallbladder ac , 26 and may further reinforce the concept of a common origother studies of digestive system necs also support the lineage transformation hypothesis with molecular analyses.5 , 27 , 28 nearly all cases had multiple pathogenic or likely pathogenic mutations in a variety of genes with tp53 , ctnnb1 , rb1 , and atm being the most frequently involved . 
our ndings are largely in line with observations in lung , prostate , and pancreatic necs , where tp53 and rb1 are altered in roughly 80% of cases.3 interestingly , also pure gallbladder ac is molecularly highly heterogeneous with tp53 as the most frequently mutated gene ( 47.1% - 59% of cases ) .29 however , large - scale molecular data remain scarce and are mainly derived from endemic regions , which could hamper the extrapolation of ndings to nonendemic regions . gene amplications ( n = 6 cases ) were observed in a subset of cases and were in all but one case not shared between the different components . 
this suggests that , in contrast to mutations that are shared between the precursor and invasive components , oncogenic amplications typically occur as a later event in carcinogenesis , which has been observed before in esophageal ac.30 in case 5 , a ccne1 amplication was shared between the ac and nec , but not the icpn . 
potentially clinically relevant mutations ( class 4 and class 5 ) , gene amplications , fusion genes , microsatellite status , and total tumor mutational burden , identied by tso500 . 
another limitation of this study is the limited information on used therapeutic regimens , such as the type and outcome of adjuvant systemic therapy received by three of the patients , because of the retrospective anonymized nature of this study . in conclusion , we provide a comprehensive histopathologic and molecular overview of a very rare and understudied tumor type . 
ligtenberg employment : synthon ( i ) honoraria : astrazeneca ( inst ) , msd ( inst ) consulting or advisory role : astrazeneca ( inst ) , bayer ( inst ) , bristol - myers squibb ( inst ) , janssen ( inst ) , merck sharp and dohme ( inst ) , novartis ( inst ) , roche ( inst ) , lilly ( inst ) research funding : astrazeneca ( inst ) , bristol - myers squibb ( inst ) no other potential conicts of interest were reported . acknowledgment we would like to thank mandy hermsen , lisa plettenburg , and chinook ophelders for technical assistance ; palga and the ncr for providing data ; and pathology laboratories ( pathan , gelre hospitals , olvg , meander medical center , and martini hospital ) for providing tumor tissue . references rindi g , klimstra ds , abedi - ardekani b , et al : a common classication framework for neuroendocrine neoplasms : an international agency for research on cancer ( iarc ) and world health organization ( who ) expert consensus proposal . 
mod pathol 31 : 1770 - 1786 , 2018 klimstra ds , lam ak , paradis v , et al : tumours of the gallbladder and extrahepatic bile ducts , in who classication of tumours , 5th edition : digestive system tumours . 
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j clin oncol 26 : 3063 - 3072 , 2008 sciarra a , missiaglia e , trimech m , et al : gallbladder mixed neuroendocrine - non - neuroendocrine neoplasm ( minen ) arising in intracholecystic papillary neoplasm : clinicopathologic and molecular analysis of a case and review of the literature . 
meguro y , fukushima n , koizumi m , et al : a case of mixed adenoneuroendocrine carcinoma of the gallbladder arising from an intracystic papillary neoplasm associated with pancreaticobiliary maljunction . 
acosta am , hamedani fs , kajdacsy - balla a , et al : primary mixed adenoneuroendocrine carcinoma of the gallbladder in a 55 - year - old female patient : a case report and review of the literature . 
de bitter tjj , van der linden rla , van vliet s , et al : colorectal metastasis to the gallbladder mimicking a primary gallbladder malignancy : histopathological and molecular characteristics . 
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scardoni m , vittoria e , volante m , et al : mixed adenoneuroendocrine carcinomas of the gastrointestinal tract : targeted next - generation sequencing suggests a monoclonal origin of the two components . 
voss mh , hierro c , heist rs , et al : a phase i , open - label , multicenter , dose - escalation study of the oral selective fgfr inhibitor debio 1347 in patients with advanced solid tumors harboring fgfr gene alterations . 
we completely agree with the authors that better ways of selecting patients with melanoma for snb are required , because approximately 80% of patients who currently have an snb are found to be sn negative . 
improved methods of identifying those at low risk of being sn positive are needed so that they are not unnecessarily subjected to the procedure , while those at higher risk are not excluded . bellomo et al1 propose that using their gene expression prole ( gep ) test will achieve greater accuracy , but we are not convinced that this is correct . 
first , we have several concerns about the statistical methodology used in their study to support the claim that a combination of two clinicopathologic ( cp ) features and an eight - gene test ( a cp - gep model ) improves the accuracy of selecting patients for snb . and second , we have evidence that prognostic estimates of sn status equivalent to or better than those provided by the cp - gep model of bellomo et al1 can be achieved using readily available cp parameters only . one of our statistical concerns is that the study had a relatively small sample size of patients who underwent snb ( n = 754 ) and only 128 were sn positive . the small sample size used for model development may explain why there was no apparent improvement in performance when more complex cp models were used compared with the use of breslow thickness and age only . another statistical concern is that the model reported by bellomo et al1 was assessed only by internal crossvalidation , and external validation was not performed . this is important , because there is a high probability that external validation using an unrelated data set will demonstrate a reduced discrimination ( c statistic ) compared with the development set because models tend to overt the data set on which they were developed.2 the apparent gain in discrimination achieved by the cp - gep model relative to the memorial sloan kettering cancer center ( mskcc ) nomogram ( from 77% to 82% ) is therefore likely to be is externally validated . reduced when the model furthermore , the c statistics had considerable overlap , and there is no evidence that there was a statistically signicant improvement . the 95% cis of in addition , the reported snb reduction rate reported by bellomo et al1 is based upon a negative predictive value ( npv ) threshold of 95% . 
however , the npv of any test decreases as the prevalence of the condition increases ; thus , in practice , it cannot be xed.3 therefore , it would be more informative for both clinicians and patients to indicate the rate reductions of snb with individual risk thresholds rather than the npv . the above statistical concerns , regardless of question of whether the gep test improved prognostic accuracy remains . 
it should be noted that the c statistic obtained by using the mskcc nomogram was considerably higher in the report by bellomo et al1 than in both our study and the original mskcc study , 5 which probably reects selection bias in their study sample that excluded thick tumors . 
the 5% absolute improvement in the c statistic reported in their study combining cp parameters ( two only ) and molecular risk factors ( an eight - gene test ) is less than the 6.2% achieved by our new model , which used cp parameters only . for all of the above reasons , we are not convinced that the addition of gep to cp parameters improves the reliability of sn - positivity estimates . 
scolyer , phd melanoma institute australia and faculty of medicine and health , the university of sydney , sydney , and department of tissue oncology and diagnostic pathology , royal prince alfred hospital and nsw health pathology , camperdown , nsw , australia john f . 
scolyer employment : royal prince alfred hospital consulting or advisory role : bristol myers squibb , glaxosmithkline , merck sharp & dohme , neracare , novartis amgen , myriad genetics , qbiotics research funding : the ainsworth foundation ( inst ) , national health and medical research council travel , accommodations , expenses : bristol myers squibb , novartis john f . 
bellomo d , arias - mejias sm , ramana c , et al : model combining tumor molecular and clinicopathologic risk factors predicts sentinel lymph node metastasis in primary cutaneous melanoma . 
lo sn , ma j , scolyer ra , et al : improved risk prediction calculator for sentinel node positivity in patients with melanoma : the melanoma institute australia nomograj clin oncol 38 : 2719 - 2727 , 2020 5 . 
wong sl , kattan mw , mcmasters km , et al : a nomogram that predicts the presence of sentinel node metastasis in melanoma with better discrimination than the american joint committee on cancer staging systeann surg oncol 12 : 282 - 288 , 2005 6 . 
scolyer ra , judge mj , evans a , et al : data set for pathology reporting of cutaneous invasive melanoma : recommendations from the international collaboration on cancer reporting ( iccr )  . 
the authors stated that there are currently no trials of parp inhibitors in endometrial carcinoma , mainly because mutations in brip1 , brca1 , brca2 , and other homologous recombination ( hr ) related genes are too rare , being reported overall in , 5% of cases.2 in fact , the french group gineco ( groupe dinvestigateurs nationaux pour letude des cancers ovariens ) is conducting a national randomized phase ii trial evaluating olaparib as maintenance therapy in platinumsensitive advanced uterine carcinoma ( utola trial ; clinicaltrials.gov identier : nct03745950 ; fig 1 ) versus placebo . 
we anticipate that patients with endometrial tumors might benet from a parp inhibitor , even in cases of no brca or related genetic mutations . the hr - decient ( hrd ) phenotype was initially characterized in approximately 50% of high - grade serous ovarian carcinomas , and 20% to 25% of these had no brca1 or brca2 mutations.3 importantly , this phenotype is associated with clinical activity of platinum and parp inhibitors in platinumsensitive ovarian carcinomas regardless of brca mutations.4 , 5 according to the cancer genome atlas data , the hrd phenotype was also recently reported in 25% of uterine endometrial carcinomas.6 it is almost exclusively found in the serous - like , tp53 - mutated subtype , in which it represents nearly 50% of cases.7 molecular alterations responsible for the hrd phenotype in endometrial carcinoma have not been largely explored and remain to be identied , because brca1 promoter methylation seems uncommon.7 finally , hr deciency could be induced by platinum - based chemotherapy ; a recent study showed decreased expression of rad51 protein , a key partner of hr , after neoadjuvant chemotherapy in a subset of patients with ovarian cancer.8 in tp53 wild - type endometrioid carcinoma , common molecular alterations such as pten or arid1a loss have been also associated in vitro with signicant parp inhibitor activity.9 , 10 according to these preliminary data , inclusion criteria in the utola trial are not restricted to specic molecular alterations ; rather , patients could be enrolled if their disease is platinum sensitive , a well - recognized landmark of the hrd phenotype . 
patients are stratied according to mrr and tp53 immuno - phenotypes , and a large screening of molecular alterations , including hrd prole , will be conducted to determine the best candidates for parp inhibitor maintenance . in conclusion , we agree with nakamura et al1 that parp inhibitors should be explored in patients with advanced endometrial carcinoma , beyond those with rare mutations in brca or related genes . 
the secondary end points are overall survival , response rate , quality of life , safety , time from random assignment to rst and second subsequent therapies , and time from random assignment to second disease progression or death . 
exploratory analyses included homologous recombinationdecient ( hrd ) phenotype and multiple - gene next - generation sequencing , including brca1 and brca2 and other hrd - related genes ( tp53 , pten , pole , and arid1a ) , and microsatellite instability status . 
nature 474 : 609 - 615 , 2011 [ erratum : nature 490 : 298 , 2012 ] ray - coquard i , pautier p , pignata s , et al : olaparib plus bevacizumab as rstline maintenance in ovarian cancer . 
marquard am , eklund ac , joshi t , et al : pan - cancer analysis of genomic scar signatures associated with homologous recombination deciency suggests novel indications for existing cancer drugs . 
biomark res 3 : 9 , 2015 de jonge mm , auguste a , van wijk lm , et al : frequent homologous recombination deciency in high - grade endometrial carcinomas . 
clin cancer res 25 : 1087 - 1097 , 2019 kubelac p , genestie c , auguste a , et al : changes in dna damage response markers with treatment in advanced ovarian cancer . 
cancers ( basel ) 12 : 707 - 719 , 2020 dedes kj , wetterskog d , mendes - pereira am , et al : pten deciency in endometrioid endometrial adenocarcinomas predicts sensitivity to parp inhibitors . 
the patient , a 33 - year - old man , presented with a primary tumor in the pancreatic tail and metastatic disease in regional and distant lymph nodes ( retroperitoneal , hilar , mediastinal , left supraclavicular ) , liver , and lungs . 
a supraclavicular lymph node core biopsy showed sheets and acini composed of epithelial cells that strongly expressed keratins by immunohistochemistry and had periodic acidschiffpositive diastase - resistant apical granularity . zymogen granules were seen with electron microscopy , consistent with pacc . 
some tumor cells expressed synaptophysin and had electron microscopydetected neurosecretory granules , suggesting possible neuroendocrine differentiation , but this was not conrmed because of limited tumor quantity . multiple lines of cytotoxic therapy were trialed over a 3 - month period because of rapid disease progression and a lack of clinical response . 
concurrently , molecular testing of tumor dna involving the targeted sequencing analysis of 386 cancer - related genes was performed with informed consent ( approval , peter maccallum cancer centre human research ethics committee 17 / 94 )  . testing detected a gatm ( exons 1 - 2 ) - raf1 ( exons 10 - 17 ) fusion that was conrmed by reverse transcriptase polymerase chain reaction / sanger quencing . 
concurrent biallelic cdkn2a - cdkn2b deletion was detected . at the time of obtaining the molecular results , mek inhibitors were reported to be effective against pacc with braf fusions in vitro4 and clinically effective in a melanoma with an raf1 fusion in our institute.3 because cytotoxic chemotherapy had proven ineffective , the patient started receiving trametinib 2 mg orally once daily . 
over a period of 4 weeks , no clinical response was observed , and the patient continued to deteriorate . sorafenib was briey trialed , but the patient had entered the terminal phase of his illness and died 5 months after diagnosis . unlike previously reported patients with oncogenic raf1 fusions , where rapid clinical improvement and tumor regression were observed after mek inhibitor treatment , 1 - 3 the response in this patient was transient and partial . 
available research is conned to case reports or retrospective series , and in the metastatic setting , survival outcomes are generally poor.5 , 6 targetable genomic alterations , such as raf fusions , promise to offer new treatment opportunities . a study of 44 paccs identied raf fusions in 10 patients ( 23% ) , 4 and although in vitro susceptibility to mek inhibition was demonstrated , the clinical efcacy of mek inhibitors or sorafenib has not previously been reported . 
to our knowledge , this study is notable as the rst clinical evaluation of an mek inhibitor in a pacc with a previously unreported gatm - raf1 fusion . biallelic loss of cdkn2a was found in this patient and in two previous raf fusion - positive patients who responded to mek inhibitors.2 , 3 concurrent cdkn2acdkn2b deletion and raf fusions were reported in a subset of paccs4 and anaplastic pleomorphic xanthoastrocytomas.7 it seems that raf fusion - driven mapk activation is commonly associated with both cell cycle dysregulation and p53 - dependent apoptosis because of loss of p16ink4a , p15ink4b , and p14arf . despite this , combined mek and cdk4 / 6 inhibition failed to control disease progression in this patient . potential explanations for this patients lack of response include activation of downstream effectors such as erk , 8 a possible component of neuroendocrine carcinoma or ductal adenocarcinoma lacking the gatm - raf1 fusion , the advanced state of the disease , limited medication absorption due to marked gut dysfunction , and potential impact of the gatm fusion partner . 
mcevoy , phd peter maccallum cancer centre , melbourne , victoria , australia damien kee , mbbs , dmedsc peter maccallum cancer centre , melbourne , and the walter and eliza hall institute of medical research , parkville , victoria , australia owen w.j. 
clay , mbbs ( hons ) , dmedsc st john of god subiaco hospital , subiaco , western australia , australia clare scott , md , phd the walter and eliza hall institute of medical research , parkville , victoria , australia anastasia backhouse , mbbs australian clinical labs , subiaco , western australia , australia stephen b . 
clay honoraria : mundipharma , merck serono , novartis , pzer , roche , astrazeneca speakers bureau : merck sharp & dohme , astrazeneca , novartis travel , accommodations , expenses : merck sharp & dohme , astrazeneca , genentech , bristol - myers squibb clare scott research funding : genentech , clovis oncology , sierra oncology , eisai patents , royalties , other intellectual property : royalty agreement for venetoclax ( inst ) expert testimony : astrazeneca travel , accommodations , expenses : astrazeneca other relationship : clovis oncology anastasia backhouse employment : australian clinical laboratories stephen b . 
kim kb , semrad t , schrock ab , et al : signicant clinical response to a mek inhibitor therapy in a patient with metastatic melanoma harboring an raf1 fusion . 
chmielecki j , hutchinson ke , frampton gm , et al : comprehensive genomic proling of pancreatic acinar cell carcinomas identies recurrent raf fusions and frequent inactivation of dna repair genes . 
brunetti o , aprile g , marchetti p , et al : systemic chemotherapy for advanced rare pancreatic histotype tumors : a retrospective multicenter analysis . pancreas 47 : 759 - 771 , 2018 6 . 
corcoran rb , ebi h , turke ab , et al : egfr - mediated re - activation of mapk signaling contributes to insensitivity of braf mutant colorectal cancers to raf inhibition with vemurafenib . 
guideline - aligned biomarker testing rates for ras , braf , and microsatellite instability / mismatch repair deciency over this study period were 41% , 43% , and 51% , respectively . 
patients were more likely to have guideline - aligned testing for ras and braf if they were treated at an academic center , were diagnosed with de novo metastatic disease , and were female . 
of the 177 patients ( 12% of cohort ) who received antiepidermal growth factor receptor therapy , only 50 ( 28% ) had complete guideline - aligned biomarker testing . conclusion despite guideline recommendations and signicant therapeutic implications , overall biomarker testing rates in mcc remain suboptimal . 
2019 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction the promise of precision oncology requires the application of biomarker testing to direct appropriate clinical care . 
among patients with metastatic colorectal cancer ( mcrc ) , genomic proling may identify somatic mutations that are prognostic for outcomes and / or predictive of response to approved treatments . in 2009 , asco and the national comprehensive cancer network ( nccn ) published guidelines recommending that patients with mcrc undergo testing for activating mutations in codons 12 and 13 of kras exon 2 on the basis of data that patients with these mutations do not derive clinical benet from the antiepidermal growth factor receptor ( anti - egfr ) monoclonal antibody ( mab ) therapies cetuximab and panitumumab.1 , 2 kras exon 2 mutation testing allows oncologists to avoid the use of ineffective anti - egfr therapy in approximately 40% of patients with mcrc who may otherwise receive these drugs as second - line therapy.3 over the subsequent 10 years , biomarker testing guidelines have been expanded ( fig 1 )  . 
testing for mutations in kras exons 3 or 4 and nras exons 2 , 3 , or 4 nds 20% more ras mutations than exon 2 testing alone , adding another 8% of patients with mcrc who will have poor responses to anti - egfr therapy ( fig 2 ) .3 , 4 braf mutations , specically v600e , predict lack of response to cetuximab and panitumumab in another 9%.5 , 6 erbb2 ( her2 ) amplication , present in approximately 2% of mcrc , is also a negative predictor of anti - egfr mab response , increasing the percentage of patients with mcrc in whom these ineffective agents could be avoided to almost 60%.7 , 8 in addition to their clinical utility as negative predictors of cetuximab and panitumumab response , braf mutations and her2 overexpression are positive predictors of response to targeted therapy . 
 gutierrez et al context key objective how often are patients with metastatic colon cancer ( mcc ) receiving guideline - aligned biomarker testing ? knowledge generated this retrospective medical record review of patients treated at academic and community centers in the united states demonstrates that only 40% of patients with mcc received guideline - aligned biomarker testing between 2013 and 2017 . patients were more likely to receive guideline - aligned biomarker testing if they were female , diagnosed at , 65 years of age , treated at an academic center , and diagnosed with de novo metastatic disease . relevance biomarker testing plays an increasingly important role in treatment selection in mcc , and exploring new approaches to increase genotyping rates is essential for improved patient outcomes . response and disease control populations.9 - 12 rates in these specic microsatellite instability ( msi ) is an important predictive biomarker for response to immune checkpoint blockade ( icb ) .13 , 14 in 2018 , the tumor - agnostic approval of pembrolizumab for the treatment of patients with unresectable or metastatic msi high ( msi - h ) solid tumors after progression on prior approved therapies established this critical biomarker to guide icb therapy selection.15 it is also an important screening test for the hereditary cancer syndrome , lynch syndrome , and a prognostic marker in stage ii crc . 
50 years of age in 2011 , and then for patients with mcrc at any age in 2015.16 , 17 although biomarker testing has been advocated for a decade in mcrc guidelines , published testing rates remain low . 
20 , 000 patients in the seer database reported only 30% of patients diagnosed with mcrc in 2010 were tested for kras.19 shaikh et al20 reported 28.2% of patients diagnosed with mcrc between 2010 and 2012 had testing for dmmr . we hypothesize that genomic testing rates for guidelinerecommended biomarkers have improved over time . 
at the time of data extraction , the cota database included demographic , diagnostic , treatment , and quality - of - care information for patients with colon cancer abstracted from the electronic health records from 23 practices including 258 oncologists in arkansas , maryland , michigan , new jersey , new york , and tennessee who contribute data under business associate agreements . 
the electronic exempt medical records of each patient ( all clinical progress notes , laboratory reports , and pathology reports ) were reviewed by trained cota abstractors for any mention of biomarker testing ( including results placed in the record in both structured and unstructured formats , such as scanned pdf les )  . 
we dened a patient as receiving extended ras testing if the laboratory or pathology report indicated analysis of exons 2 , 3 , and 4 for both kras and nras and as limited ras testing if analysis was anything less than exons 2 , 3 , and 4 in both kras and nras . 
fu , uorouracil ; ihc , immunohistochemistry ; mmr , mismatch repair ; msi , microsatellite instability ; msi - h , microsatellite instability high ; nccn , national comprehensive cancer network ; ngs , next - generation sequencing ; pts , patients ; wt , wild type . testing for a given biomarker was considered guideline aligned if the nccn guidelines recommended testing for that biomarker for the entire year being analyzed . guideline - aligned testing in 2013 and 2014 included testing for kras by any methodology , in 2015 included extended testing of nras and kras , and in 2016 and 2017 included extended testing of both kras and nras , braf testing by any methodology , and msi / dmmr analysis by any methodology . 
because the cota database captures limited family history data , we did not include msi / dmmr in the guideline - aligned genotyping analysis until it was recommended for all patients regardless of age or family history . results patient demographics a total of 1 , 497 patients with mcc were identied in the observational database ( table 1 )  . 
of these patients , 1 , 325 ( 89% ) had de novo presentation of metastatic disease , and 172 ( 11% ) had been previously diagnosed with earlier stage colon cancer and became metastatic during the study time frame . 
the patients were treated at 23 centers , including 1 , 152 ( 77% ) at academic cancer centers and 345 ( 23% ) at community cancer centers . overall guideline - aligned biomarker testing guideline - aligned biomarker testing was completed in 40% of patients in this study ( 601 of 1 , 497 )  . 
 ( a ) frequency of mutations identied in expanded ras testing.4 , 20 , 21 ( b ) approximately 60% of patients with metastatic colorectal cancer ( mcrc ) will have a genetic alteration that may predict poor response to anti - egfr monoclonal antibody ( mab ) therapies cetuximab and panitumumab . 
most of these nonresponders will have kras exon 2 mutations , but expanded ras , braf , and erbb2 analysis identies an additional 20% of patients who will not benet from therapy . nras testing by any methodology , with 7% of tested patients harboring an nras mutation ( fig 3 )  . braf testing rates guideline - aligned braf testing was completed in 43% of patients ( 235 of 546 ) in 2016 - 2017 when braf testing was guideline recommended . 
although pcr was the most common testing methodology for kras in 2013 , ngs was the dominant testing methodology for kras after 2013 and for nras and braf throughout this study . 
the the entire period of proportion of patients who had testing for kras , nras , and braf by ngs increased each year over the course of the study ( fig 4 )  . use of anti - egfr therapy in this study , 177 ( 11.8% ) patients received cetuximab or panitumumab between 2013 and 2017 . 
of these patients , 50 ( 28% ) had guideline - aligned testing for ras and braf . sixty - three ( 36% ) were tested for kras by any methodology , 37 ( 21% ) were tested for nras by any methodology , and 44 ( 26% ) were tested for braf by any methodology . 
it is not known whether ras testing occurred before or after initiation of therapy . msi and dmmr testing discussion guideline - aligned dmmr testing was completed in 51% of patients ( 276 of 546 ) in 2016 - 2017 when dmmr testing was recommended for all patients with mcc . 
although the proportion of patients tested for individual markers increased , because guidelines signicantly expanded between 2013 and 2017 , the number of patients receiving guideline - aligned genotyping in 2017 was lower than it was at the start of the study . 
 variable patients female male age groupa , 65 years 65 years race white black asian other 2013 2014 2015 2016 2017 0 - iii stage at diagnosis practice type academic community ras , raf , and mmr biomarker testing in metastatic colon cancer table 1 . 
observed undergenotyping rates in this analysis are consistent with previous reports of kras testing rates ( 30% - 48% ) between 2008 and 2013.18 , 19 , 21 undergenotyping for guideline - recommended biomarkers may place patients at risk for receiving ineffective antiegfr mab therapy and / or missing the opportunity to receive appropriate immunotherapy options . 
however , in this cohort , 72% of patients receiving cetuximab and / or panitumumab were undergenotyped , providing evidence against the hypothesis that low testing rates are explained by patients not being considered for anti - egfr therapy . using the observed real - world mutation rates for kras , nras , and braf ( 62% , 7% , 17% ) , we estimated the proportion of patients who would have been considered candidates for anti - egfr therapies had all patients undergone complete biomarker testing . 
giving anti - egfr therapy to patients who are ras / raf positive poses risks to patients , in particular severe infusion reactions , which occur in 3% of patients given cetuximab and 1% of patients given panitumumab.22a in addition , mab therapies are expensive relative to testing costs . 
guideline - aligned testing in 2013 - 2014 included testing for kras by any methodology , in 2015 included extended testing of nras and kras , and in 20162017 included extended testing of both kras and nras , braf testing by any methodology , and microsatellite instability / mismatch repair deciency ( dmmr ) analysis by any methodology . 
mcc , metastatic colon cancer . although nccn guidelines do not specify a preferred methodology for biomarker testing , a sequential approach to testing multiple biomarkers amplies challenges with tissue insufciency , turnaround time , and cost.23 we saw increased use of ngs for biomarker testing between 2013 and 2017 for kras , nras , and braf . 
if every patient in this study who was tested for at least one biomarker had a tissueor plasma - based ngs panel with comprehensive coverage of 2013 2014 2015 2016 2017 extended ras , braf , and msi , we would have observed a nearly 50% increase in the percentage of patients who had guideline - aligned biomarker testing ( from 40% to 59% )  . 
therefore , additional barriers must exist , preventing adoption of testing in mcc . tissue - based biomarker barriers to molecular testing in mcc have not been well turnaround studied but may include tissue availability , time , physician education / knowledge , cost / insurance coverage , patient preference , and patient eligibility for therapy on the basis of performance status and comorbidities.18 , 24 a qualitative study of oncologists perspectives on kras testing in 2010 found all participating clinicians reported ordering kras testing and endorsed the value of testing , but there was a lack of consensus on test timing and confusion about what tissue sample to test ( fresh v archival and primary v metastatic ) .25 a 2018 survey of us oncologists , pathologists , and surgeons about msi / dmmr testing practices in mcrc found 84% were aware of published guidelines for dmmr / msi testing in patients with mcrc , and 78% followed published guidelines . 
although the majority of physicians ( 69% ) stated they perform universal testing for all patients with mcrc , nearly 30% of physicians selected patients for testing on an individualized basis . 
 ras , raf , and mmr biomarker testing in metastatic colon cancer current study , guideline - aligned testing was signicantly more likely if the patient was diagnosed with de novo metastatic disease . 
more complete testing in patients with de novo metastatic disease versus patients who experienced progression from an earlier stage may suggest challenges in obtaining archival tissue specimens or patient or physician resistance to repeat tissue biopsy related to complication risks or nancial pressures . several well - validated plasma - based ngs assays that include comprehensive analysis of biomarkers from circulating tumor dna ( ctdna ) have demonstrated high sensitivity , specicity , and concordance with tissue genotyping . 
as with tissue genotyping , ctdna testing can predict response to targeted therapies ( msi - high , braf v600e mutated , erbb2 amplied , ntrk fusions ) and lack of response to anti - egfr treatment but may also reduce barriers associated with obtaining archival tissue or rebiopsy ( gupta et al , manuscript in review ) .25a , 26 - 35 given the relative ease of use and rapid turnaround time , plasmabased ngs offers one method of overcoming barriers associated with tissue testing , but other strategies to reduce undergenotyping must be explored , including physician education , implementation of testing protocols like universal msi / dmmr screening , and use of electronic health records to identify patients who require genotyping . there are multiple limitations of this study . 
mainly , the study relied on medical record input for database review . patients whose genomic testing was not documented in the medical record could have led to errors of omission . 
it is also possible that patients had biomarker testing before being referred to the centers included in this data set , and this testing was not documented in the record . 
in addition , data were collected only on patients with colon cancer . guideline recommendations for kras , nras , braf , and msi all specify testing for metastatic colorectal cancer . 
prior studies suggest that patients with rectal cancer are tested less frequently than those with colon cancer.19 therefore , true testing rates for mcrc may be lower than what is presented in this mcc study . 
this is an important factor that should be further explored in diverse patient cohorts . mutation - positive rates reported for kras , nras , and braf in this study were higher than previously published literature , including a large cohort of mcrc tumors sequenced by msk - impact , which cites kras , nras , and braf mutation rates of 45% , 4% , and 12% , respectively.36 we acknowledge a potential for positive result bias , with physicians documenting positive results in their clinical notes more often than negative results . 
there may have been factors such as treatment ineligibility or refusal that contributed to undergenotyping rates . the results of this study indicate that adherence to evidence - based biomarker testing guidelines in mcc remains poor in both academic and community settings in the united states , with only 40% of patients completing guideline - aligned biomarker testing . 
 gutierrez et al open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . consulting or advisory role : forward medical research funding : guardant health martin e . 
gutierrez stock and other ownership interests : cota healthcare honoraria : foundation medicine , guardant health consulting or advisory role : exenex speakers bureau : bristol - myers squibb , merck , eli lilly research funding : bristol - myers squibb ( inst ) , merck ( inst ) , incyte ( inst ) , nextcure ( inst ) , pzer ( inst ) , genentech ( inst ) , boehringer ingelheim ( inst ) , gsb pharma ( inst ) , moderna therapeutics ( inst ) , eisai ( inst ) , silenseed ( inst ) , seattle genetics ( inst ) , regeneron ( inst ) , sano ( inst ) , johnson & johnson ( inst ) , medimmune ( inst ) , checkpoint therapeutics ( inst ) travel , accommodations , expenses : guardant health kristin s . 
nagy employment : guardant health stock and other ownership interests : guardant health irfan shah employment : cota healthcare stock and other ownership interests : guardant health michael mulcahy employment : cota healthcare andrew d . 
goldberg employment : cota healthcare stock and other ownership interests : cota healthcare research funding : celator pharmaceuticals ( inst ) , bristol - myers squibb ( inst ) , novartis ( inst ) no other potential conicts of interest were reported . references allegra cj , jessup jm , somereld mr , et al : american society of clinical oncology provisional clinical opinion : testing for kras gene mutations in patients with metastatic colorectal carcinoma to predict response to anti - epidermal growth factor receptor monoclonal antibody therapy . 
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 phase i study of the braf inhibitor vemurafenib in combination with the mammalian target of rapamycin inhibitor everolimus in patients with braf - mutated malignancies vivek subbiah shiraj sen kenneth r . 
are co - first authors and contributed equally to this work . the funders had no role in the design of the study ; the collection , analysis , and interpretation of the data ; the writing of the article ; and the decision to submit the article for publication . corresponding author : vivek subbiah , md , department of investigational cancer therapeutics ( phase i clinical trials program ) , unit ( continued ) purpose parallel activation of the phosphatidylinositol 3 - kinasemammalian target of rapamycin pathway represents a mechanism of primary and acquired resistance to braf - targeted therapy , but the two pathways have yet to be cotargeted in humans . 
we performed a phase i study to evaluate the safety and activity of the braf inhibitor vemurafenib in combination with the mammalian target of rapamycin inhibitor everolimus in braf - mutated advanced solid tumors . patients and methods we performed a 3 + 3 dose - escalation study with escalating doses of both oral ( po ) vemurafenib administered twice a day and po everolimus administered daily . results twenty patients with advanced cancers were enrolled . 
 patients were heavily pretreated with prior braf or mek inhibitors ( n = 11 ) , phase i clinical trial therapy ( n = 10 ) , surgery ( n = 18 ) , radiation therapy ( n = 11 ) , and chemotherapy ( n = 13 )  . 
one of the two pediatric patients initially experienced grade 3 rash , but after dermatologic intervention , the patient remains on trial with partial response and no dose reduction at time of analysis . 
one pediatric patient with pleomorphic xanthroastrocytoma remains on protocol with continued clinical response after 38 cycles . conclusion the combination of vemurafenib 720 mg po twice a day and everolimus 5 mg po daily is safe and well tolerated and has activity across histologies , with partial responses noted in advanced nonsmall - cell lung cancer , melanoma , optic nerve glioma , and xanthroastrocytoma , including patients who previously experienced progression on braf and / or mek inhibitor therapy . 
a recent basket study of vemurafenib demonstrated clinical activity in patients with advanced nonmelanoma cancers.8 unfortunately , many patients exhibited primary and acquired resistance to therapy . reactivation of mapk signaling and activation of alternative parallel signaling pathways such as the phosphatidylinositol 3 - kinase ( pi3k ) mammalian target of rapamycin ( mtor ) pathway have been hypothesized to contribute to primary and acquired resistance to braf - targeted therapy.9 , 10 specifically , akt mutation , 9 pten deficiency , 11 and downstream mtor activation have been implicated in resistance to both vemurafenib and dabrafenib in metastatic melanoma , both clinically and in preclinical models.12 , 13 preclinical studies have demonstrated efficacy in dual targeting of both the mapk and pi3kmtor pathways in various tumor types.14 , 15 we also recently identified a strong association between co - occurring pi3k - mtor pathway aberrations and primary resistance to braf targeted therapy . 
all patients were treated at the clinical center for targeted therapy at the university of texas md anderson cancer center from september 2013 to august 2015 and met all the eligibility criteria . 
patients who had a myocardial infarction within 3 months before starting treatment , who were pregnant or breastfeeding , or who had undergone a major surgical procedure within 28 days were excluded . 
palliative radiation therapy was allowed during the study treatment . study design and treatment this study was a nonrandomized , open - label , standard 3 + 3 dose - escalation study of oral ( po ) vemurafenib , starting at a dose of 720 mg daily , and po everolimus , starting at a dose of 5 mg daily , for 28 days . 
the primary objectives were to determine the safety , mtd , and dose - limiting toxicity ( dlt ) of the combination of vemurafenib and everolimus in patients with braf - mutated advanced cancer . 
 two pediatric patients were enrolled onto this study and were treated with vemurafenib po 480 mg daily and everolimus po 2.5 mg daily , per body surface areabased dose adjustment . if a patient experienced new grade 3 or greater toxicity , treatment was withheld until the condition was addressed and it recovered to grade 1 or baseline . 
the patients continued treatment until they experienced disease progression or intolerable toxicities , until the treating physician or patient felt that it was not in the patients best interest to continue for any reason , or until consent was withdrawn for any other reason . all patients were evaluated for dlts during the first 28 days and evaluated in clinic every 28 days before initiation of each subsequent cycle . 
dlts were treatment - related grade 3 or 4 nonhematologic toxicities , as defined by the national cancer institute common terminology criteria for adverse events version 3.0 , or grade 4 hematologic toxicities lasting > 7 days or accompanied by fever or bleeding during the first 4 weeks of therapy . 
clinical laboratory improvement amendments ( clia ) certified next generation sequencing data were collected for each patient . response to therapy was assessed using response evaluation criteria in solid tumors ( recist ) version 1.1. 
median time to disease progression and os duration were determined using the kaplan - meier method . circulating tumor dna three patients agreed to have an optional blood draw to measure circulating tumor dna ( ctdna ) at baseline and at each restaging . 
at least 8 ng of unamplified cell - free dna were tested with a droplet digital polymerase chain reaction brafv600e mutationspecific assay and / or multiplex brafv600 screening kit ( bio - rad , hercules , ca ) to distinguish the wild - type allele from the three most common mutations ( brafv600e , brafv600k , and brafv600r ) using the qx200 droplet digital pcr system ( bio - rad ) according to the manufacturers standard protocol . 
planned dose schedule in patients who received the combination of vemurafenib and everolimus in a phase i study dose level vemurafenib orally twice a day ( mg ) everolimus orally once a day ( mg ) * body surface area - based dose adjustment was made ( level 1 ) for pediatric patients dose level 1 was the recommended phase ii dose . frequency for the multiplexed screening assay and < 0.1% mutant allele frequency per single well for the mutation - specific assays . results patient characteristics the demographics and clinical characteristics of the 20 patients who received at least one dose of vemurafenib and everolimus and completed the dlt window are listed in table 2 . 
the most common tumor types were melanoma ( n = 7 ) , glioma ( n = 5 ) , and thyroid cancer ( n = 4 ) , and one patient each had appendiceal cancer , colorectal cancer , nsclc , and unknown primary cancer . 
patients were heavily pretreated with prior therapies , including braf or mek inhibitor therapy ( n = 11 ) , prior phase i clinical trial therapy ( n = 10 ) , surgery ( n = 18 ) , radiotherapy ( n = 11 ) , and chemotherapy ( n = 13 )  . 
one patient had measurable disease but not evaluable disease , and a second patient was not evaluable as a result of withdrawing consent before the first restaging , but both patients cleared the toxicity window and therefore are included in the toxicity evaluation but not in the response evaluation . safety all 20 patients were evaluated for dlts . 
 five patients ( 25% ) had everolimus dose reductions throughout their therapy as a result of toxicity , and one patient had the vemurafenib dose reduced as a result of renal insufficiency . 
two patients without dlts withdrew consent before disease progression . response of the 18 patients evaluable for response , 11 patients ( 61% ) had objective tumor volume reduction as best response . 
two patients were not evaluable for response ; one patient had measurable but not evaluable disease and a second patient completed the dlt window but withdrew consent before restaging . prs were seen in patients with nsclc , melanoma , optic nerve glioma , and pleomorphic xanthroastrocytoma . 
of the nine patients who had disease progression on braf or mek inhibitors before enrolling onto this study , two ( 22% ) had prs and five ( 56% ) had stable disease with vemurafenib and everolimus . at the time of analysis , 14 patients ( 78% ) had discontinued therapy because of disease progression . 
 ( b ) representative restaging images of a patient with metastatic melanoma and a pten ( p95s ) mutation who had a partial response on vemurafenib and everolimus after disease progression on vemurafenib and px866 , an investigational pi3k inhibitor . 
astro , anaplastic astrocytoma ; at , anaplastic thyroid ; crc , colorectal cancer ; cup , cancer of unknown primary ; glio , glioblastoma ; glioma , optic nerve glioma ; mel , melanoma ; pt , papillary thyroid . prior braf or mek inhibitor use no prior braf or mek inhibitor use still on trial without progression pleomorphic xanthroastrocytoma remains on protocol with continued clinical response after 38 cycles . genomic profiling all evaluable patients underwent clia - certified clinical next - generation sequencing of at least 50 genes before trial enrollment . 
among the four patients who achieved pr , a patient with metastatic melanoma who had previously experienced progression on vemurafenib plus an investigational pi3k inhibitor ( px866 ) had a concurrent pten ( p95s ) mutation . 
a patient with nsclc whose tumor harbored an idh1 ( r132c ) mutation , arid2 alteration ( splice site 92 + 1 g > a ) , and ppp1r2a mutation ( r183w ) achieved a pr after previously experiencing progression on dabrafenib . 
all concurrent molecular aberrations and best response to therapy for each patient are listed in table 4 . circulating tumor dna patients enrolled onto this study were offered an optional blood draw to measure ctdna throughout the course of the study . 
 a third patient with a brafv600e mutation had detectable ctdna levels , which were measured at four time points throughout therapy and are depicted in figure 2 alongside the patients scans . 
dlts included fatigue ( 20% ) and rash ( 15% ) , but both were present in only one patient at the mtd and recommended phase ii dose of vemurafenib 720 mg twice daily and everolimus 5 mg daily . 
 the patient with glioblastoma who experienced a grade 3 rash had a resolution of his rash after initial everolimus discontinuation and was able to resume everolimus at a dose of 2.5 mg daily without toxicity . 
 the use of nonpharmacologic approaches , such as physical and mindfulness - based exercise therapy , 20 , 21 and pharmacologic approaches , such as corticosteroids22 in treating cancerand therapy related fatigue , has recently demonstrated efficacy in randomized controlled trials and may benefit these patients moving forward . we observed only one metabolic adverse event ( hypertriglyceridemia and hyperglycemia ) , which is attributed to mtor inhibitors . 
overall , most patients at the recommended phase ii dose tolerated the treatment well . our trial also demonstrates that the addition of an mtor inhibitor to everolimus treatment is able to overcome resistance to braf and / or mek inhibition in a subset of patients with braf - mutant advanced cancers . 
graphical depiction of a patients circulating tumor dna level of brafv600e ( shown as copies per milliliter of plasma ) while on treatment with vemurafenib plus everolimus with representative restaging scans and response to therapy , per response evaluation criteria in solid tumors ( recist ) 1.1. 
pd , progression of disease ; pr , partial response . baseline baseline july 16 , 2014 october 14 , 2014 november 12 , 2014 december 10 , 2014 the 20 patients in this study had a pr to therapy . 
 next - generation sequencing performed at the time of initial diagnosis revealed idh1 ( r132c ) , arid2 ( splice site 92 + 1 g > a ) , and ppp1r2a ( r183w ) aberrations . 
unfortunately , no molecular profiling was available at the time the patient experienced progression on dabrafenib immediately before enrolling onto this trial . a second patient with metastatic melanoma and a pten ( p95s ) mutation who had recently had disease progression on vemurafenib and px866 , an investigational pi3k inhibitor , also achieved a pr on our trial ( fig 1b )  . 
in vivo , pten - deficient , braf - mutated mouse models treated with vemurafenib and pi3k inhibitors also demonstrate synergistic antitumor activity.23 interestingly , this patient experienced progression on the vemurafenib and investigational pi3k inhibitor combination but responded with a pr to vemurafenib and everolimus . 
a 78 - year - old patient with metastatic papillary thyroid cancer whose tumor harbored a pik3ca ( h1047r ) mutation had 7 months of stable disease with a 27% reduction in tumor burden before having any disease progression . 
both of these patients had no co occurring genomic alterations identified on their tumors other than the brafv600e mutation , illustrating the fact that although co - occurring alterations may lead to activation of parallel signaling pathways that increase resistance to therapy in some patients , they may render other tumors susceptible to therapy when actionable . we have previously reported our institutions experience with implementing multiplex hotspot testing and the need to enroll patients onto genotype - matched trials when feasible.24 a number of the responders on this trial further support the hypothesis that the identification of actionable alterations can help guide the right patient to the right targeted therapy . 
all nonresponders were either previously treated with braf or mek inhibitor or harbored non - v600 braf mutations . in the patient with metastatic melanoma with measurable ctdna , the level of mutated brafv600e copies per milliliter decreased with clinical response and increased before subsequent restaging that demonstrated disease progression . 
previous studies have identified an association between detection and levels of circulating tumor dna and disease burden and survival across braf and mek inhibitor trials.27 it is plausible that this may explain why only wildtype braf was detectable in the plasma of these two patients . vitro data suggest that the combination of mek and mtor inhibition may be more effective than braf and mtor inhibition in activating bax and caspase - 3 and inducing caspase - dependent apoptosis in melanoma cell lines.31 future studies to investigate the safety and effectiveness of combining everolimus with both braf and mek inhibitors together are being planned . our study has several limitations , including a small patient population and the variety of molecular profiling platforms used . 
although all patients underwent clia - certified next generation sequencing of at least 40 genes , including those in the pi3k - mtor pathway , some patients underwent more extensive 400gene profiling . 
ideally , all patients should have a broader panel of testing to identify all actionable aberrations , both at the time of study initiation and at disease progression , to better understand the clonal evolution of tumors treated with vemurafenib and everolimus . 
 sherman , patrick hwu manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors in conclusion , the combination of vemurafenib and everolimus is safe and well tolerated and has clinical activity across histologies , with prs noted in patients with advanced nsclc , melanoma , optic nerve glioma , and xanthroastrocytoma . 
our results do not support the addition of everolimus based solely on alterations in the pi3k - mtor pathway ; however , given the responders who did harbor concurrent mutations in this pathway , further investigation in a larger cohort of molecularly matched patients with uniform genomic profiling is warranted . 
hess no relationship to disclose filip janku stock and other ownership interests : trovagene consulting or advisory role : deciphera , trovagene , novartis , sequenom , foundation medicine , guardant health , immunome research funding : novartis , biomed valley discoveries , roche , agios , astellas pharma , deciphera , symphony evolution , plexxikon , piqur , fujifilm other relationship : bio - rad david s . 
hong stock and other ownership interests : molecularmatch , oncorena honoraria : adaptimmune , baxter , merrimack , bayer consulting or advisory role : baxter , bayer , guidepoint global , janssen speakers ' bureau : genentech , janssen oncology research funding : novartis , genentech , eisai , astrazeneca , pfizer , mirna therapeutics , amgen , daiichi sankyo , merck , mirati therapeutics , eli lilly , adaptimmune , abbvie , bayer , bristol - myers squibb , genmab , ignyta , infinity pharmaceuticals , kite pharma , kyowa hakko kirin , loxo , medimmune , molecular templates , takeda travel , accommodations , expenses : loxo , mirna therapeutics other relationship : oncorena , eisai soumen khatua no relationship to disclose research funding : phosplatin therapeutics travel , accommodations , expenses : phosplatin therapeutics javier munoz consulting or advisory role : kite pharma , pfizer , pharmacyclics , bayer , alexion pharmaceuticals , bristolmyers squibb , janssen , seattle genetics , gilead sciences , kyowa hakko kirin speakers ' bureau : kite pharma gerald s . 
falchook employment : sarah cannon research institute , healthone research funding : millennium , emd serono , celgene , medimmune , genmab , vegenics , novartis , astrazeneca , incyte , armo biosciences , kolltan pharmaceuticals , 3 - v biosciences , abbvie , aileron therapeutics , delmar pharmaceuticals , effector therapeutics , strategia therapeutics , fujifilm , hutchison medipharma , regeneron , biothera , curegenix , curis , eli lilly , jounce therapeutics , oncomed , precision oncology , syndax , taiho pharmaceutical , tesaro , takeda , beigene , ignyta , merck , rgenix , tarveda therapeutics , tocagen patents , royalties , other intellectual property : handbook of targeted cancer therapy travel , accommodations , expenses : millennium , sarah cannon research institute , emd serono , bristol - myers squibb roman groisberg no relationship to disclose apostolia m . 
tsimberidou consulting or advisory role : roche research funding : emd serono ( inst ) , baxter ( inst ) , foundation medicine ( inst ) , onyx ( inst ) , bayer ( inst ) , boston biomedical ( inst ) , placon ( inst ) , immatics ( inst ) , karus therapeutics ( inst ) , stem cells ( inst ) , obi pharma ( inst ) steven i . 
 support prior presentation presented in part at the 52nd annual meeting of the american society of clinical oncology , chicago , il , june 3 - 7 , 2016 . references 417 : 949 - 954 , 2002 1 . 
turski ml , vidwans sj , janku f , et al : genomically driven tumors and actionability across histologies : braf - mutant cancers as a paradigmol cancer ther 15 : 533 - 547 , 2016 3 . 
planchard d , besse b , groen hjm , et al : dabrafenib plus trametinib in patients with previously treated braf ( v600e ) - mutant metastatic non - small cell lung cancer : an open - label , multicentre phase 2 trial . 
subbiah v , kreitman rj , wainberg za , et al : dabrafenib and trametinib treatment in patients with locally advanced or metastatic braf v600 - mutant anaplastic thyroid cancer . 
nazarian r , shi h , wang q , et al : melanomas acquire resistance to b - raf ( v600e ) inhibition by rtk or n - ras upregulation . 
gopal yn , rizos h , chen g , et al : inhibition of mtorc1 / 2 overcomes resistance to mapk pathway inhibitors mediated by pgc1 and oxidative phosphorylation in melanoma . 
greger jg , eastman sd , zhang v , et al : combinations of braf , mek , and pi3k / mtor inhibitors overcome acquired resistance to the braf inhibitor gsk2118436 dabrafenib , mediated by nras or mek mutations . 
faber ac , coffee em , costa c , et al : mtor inhibition specifically sensitizes colorectal cancers with kras or braf mutations to bcl - 2 / bcl - xl inhibition by suppressing mcl - 1 . 
sen s , meric - bernstam f , hong ds , et al : co - occurring genomic alterations and association with progression - free survival in brafv600 - mutated nonmelanoma tumors . 
subbiah v , westin sn , wang k , et al : targeted therapy by combined inhibition of the raf and mtor kinases in malignant spindle cell neoplasm harboring the kiaa1549 - braf fusion protej hematol oncol 7 : 8 , 2014 20 . 
yennurajalingam s , frisbee - hume s , palmer jl , et al : reduction of cancer - related fatigue with dexamethasone : a double - blind , randomized , placebo - controlled trial in patients with advanced cancer . 
bonnevaux h , lemaitre o , vincent l , et al : concomitant inhibition of pi3k and braf or mek in pten - deficient / braf - mutant melanoma treatment : preclinical assessment of sar260301 oral pi3k - selective inhibitor . 
santiago - walker a , gagnon r , mazumdar j , et al : correlation of braf mutation status in circulating - free dna and tumor and association with clinical outcome across four brafi and meki clinical trials . 
janku f , claes b , huang hj , et al : braf mutation testing with a rapid , fully integrated molecular diagnostics systeoncotarget 6 : 26886 - 26894 , 2015 29 . 
penna i , molla a , grazia g , et al : primary cross - resistance to brafv600e - , mek1 / 2and pi3k / mtor - specific inhibitors in braf - mutant melanoma cells counteracted by dual pathway blockade . 
 o development and validation of a clinical polygenic risk score to predict breast cancer risk elisha hughes , phd1 ; placede tshiaba , ms1 ; shannon gallagher , mph1 ; susanne wagner , phd1 ; thaddeus judkins , ms1 ; benjamin roa , phd1 ; eric rosenthal , phd , ms1 ; susan domchek , md2 ; judy garber , md , mph3 ; johnathan lancaster , md , phd1 ; jeffrey weitzel , md4 ; allison w . 
lanchbury , phd1 ; alexander gutin , phd1 ; and mark robson , md6 purpose women with a family history of breast cancer are frequently referred for hereditary cancer genetic testing , yet , 10% are found to have pathogenic variants in known breast cancer susceptibility genes . 
largescale genotyping studies have identied common variants ( primarily single - nucleotide polymorphisms [ snps ] ) with individually modest breast cancer risk that , in aggregate , account for considerable breast cancer susceptibility . 
candidate polygenic risk scores ( prss ) as predictors of personal breast cancer history were developed through multivariable logistic regression models adjusted for age , cancer history , and ancestry . 
an optimized prs was validated in 2 independent cohorts ( n = 13 , 174 ; n = 141 , 160 )  . results within the training cohort ( n = 24 , 259 ) , 4 , 291 women ( 18% ) had a personal history of breast cancer and 8 , 725 women ( 36% ) reported breast cancer in a rst - degree relative . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction although breast cancer ranks among the cancer types with the highest heritability , 1 characterization of the incomplete . 
pathounderlying genetic causes is still genic variants in high - penetrance genes ( eg , brca1 , brca2 , palb2 ) associated with substantial increases in risk are individually rare in the general population.2 - 4 all known highand moderate - penetrance breast cancer susceptibility genes are estimated to account for a combined 20% of familial relative risk.5 as a result , most women who are referred for hereditary cancer genetic testing are negative for a pathogenic variant and without a clear understanding of the magnitude of their risk.6 genome - wide association studies ( gwas ) have identied several hundred , common , single - nucleotide polymorphisms ( snps ) as breast cancer susceptibility variants.7 - 11 although their individual contribution to risk is minor , polygenic risk scores ( prss ) of such variants can dene variant combinations with potentially actionable clinical risk.9 , 11 together , pathogenic variants in highand moderate - risk genes and panels of snps are estimated to explain up to 40% of the familial risk for developing breast cancer.11 the rst well - characterized prs combined 77 snps ( prs77 ) and was evaluated in  . 
this prs was highly predictive of breast cancer in two independent validation cohorts . relevance prss that aggregate genotypes from common variants have emerged as a new approach to improve breast cancer risk assessment . 
we developed and validated an 86 - snp score that was adjusted for family breast cancer history and was highly predictive of breast cancer risk , making it appropriate for clinical use to identify women at increased risk of developing breast cancer . recently , a polygenic score of 313 snps was deduced from the same gwas and then validated in an independent population cohort.11 the same study also supported previous work indicating snp - based risk scores may have to be optimized depending on breast cancer estrogenreceptor ( er ) status . 
composition and individual contribution of variants of the ideal snp - based breast cancer risk score remain to be dened . family history ( fh ) of breast cancer , a combination of biologic , social , and environmental factors , includes a genetic component that is expected to overlap with snpbased risk.9 , 11 to avoid double counting of risk due to correlation between genetic components of fh and prs , joint modeling of both genetic factors is required . 
here , we describe the development and validation of a prs in which we co - estimate snp - based risk and familial risk to obtain effect sizes independent of fh . 
additional details are provided in the data supplement . snp genotyping amplicons for 94 snp markers published at the time of this study were incorporated into a next - generation sequencing panel.8 , 9 two markers failed design due to their location within repetitive elements , leaving 92 markers for evaluation . 
clinical information from provider - completed test request forms included ancestry , personal and family cancer history , cancer type ( s ) , and age ( s ) at diagnosis . 
 clinical polygenic risk score for breast cancer previous work supports a multiplicative model as the best method for capturing the combined snp effects.9 , 11 we centered the multiplicative model according to generalpopulation allele frequencies so the or for an individual patient represents the fold - change in risk relative to the general population . 
patient clinical characteristics validation study tested the association of the prs with invasive breast cancer after adjusting for age , personal and family cancer history , and ancestry . in exploratory analyses , we assessed the predictive ability of the optimized prs compared with a previously described prs77 by adding both to a multivariable model . 
the observed versus theoretical effect of the prs on breast cancer risk under the multiplicative polygenic model was assessed by comparing ors from the continuous prs with those obtained from analysis of patients binned in categories according to prs percentiles . 
heterogeneity of the prs effect size according to age , ancestry , and fh of breast cancer was evaluated by tting additional interaction terms and by repeating the primary analysis restricted to subcohorts stratied by age , ancestry , and fh severity . 
overall , 8 , 725 patients ( 36% ) reported a breast cancer diagnosis in 1 rst - degree relative and 13 , 230 ( 55% ) had no rst - degree relatives with breast or ovarian cancer . effect sizes and population frequencies were used to rank 92 susceptibility variants by their informativeness and to construct a polygenic score by successive addition of the most informative variant . 
distribution of relative risks , or fold changes , in breast cancer risks due to the 86 - snp score in unaffected women was similar between the rst and second validations ( data supplement figures s1 and s2 )  . 
ors for developing breast cancer by 86 - snp score percentile in each of the two validation cohorts are given in table 2 . consistently , women in the top 95th percentile of the 86snp score distribution had a more than 2 - fold higher risk of breast cancer development than women with an average 80 86 snps ranked by informativeness * fig 1 . 
effect sizes of the 86 - snp score were consistent across subcohorts stratied by age ( table 3 ; data supplement figure s4 ) , ancestry ( data supplement table s3 ) , and fh ( table 4 ; data supplement figure s5 ) in both validation cohorts . 
these results indicate the simple multiplicative model remains the best method for capturing the risk conferred by the combined effects of snps after accounting for family cancer history . discussion prss aggregating genotypes from common variants offer new ways to improve breast cancer risk assessment and can contribute to better understanding of breast cancer risk in all women . 
modication of risk estimates by prs - based stratication has been shown in a variety of clinical settings , including women who carry a pathogenic variant , high - risk women , and population cohorts.11 , 15 - 17 these novel tools , however , are of particular interest to women who present for genetic risk assessment with increased risk due to a fh of breast cancer . 
fh and susceptibility variants contribute independently to risk prediction , yet the approximately 13% reduction in the or for fh when adjusted for the prs77 ( or prs313 ) indicates an overlapping contribution to risk.9 , 11 , 18 adjustment for this correlation between snps and fh can be achieved by co - estimating ors from multivariable models . 
the reported interaction was only signicant in er - positive disease , a subanalysis not available in our data set.11 since the inception of this study , additional breast cancer risk variants have been described , and expanded breast cancer prss have been evaluated and validated in independent data sets.10 , 11 the enlarged prss are estimated to account for a larger fraction of familial risk and promise observed theoretical observed theoretical > 1 - 5 5 - 10 10 - 20 20 - 40 40 - 60 60 - 80 80 - 90 90 - 95 95 - 99 > 99 > 1 - 5 5 - 10 10 - 20 20 - 40 40 - 60 60 - 80 80 - 90 90 - 95 95 - 99 > 99 percentiles of the 86 - snp score percentiles of the 86 - snp score fig 2 . 
the approach described here of co - estimation in multivariate models could be applied to any emerging prs , irrespective of number of variants or ancestral origin . among the strengths of this study are the use of large , contemporary , and fully independent cohorts for development and 2 validation studies . 
thus , to our knowledge , the 86 - snp score is the rst prs with an snp component adjusted for fh and , as such , would be an appropriate addition to conventional clinical models . limitations of the study include the inability to apply the 86snp score to patients of non - european ancestry because changes in genetic structure and linkage disequilibrium may affect the predictive ability of individual risk variants when present in a different ancestral background . 
although association studies with equivalent explanatory power in other ancestries have yet to be described , they are the subject of ongoing research and the expansion of breast cancer prs to other ancestries is a high priority . 
however , previous studies have demonstrated that unbiased risk estimates can be obtained from analysis of a clinical testing population through multivariable adjustment for the factors related to ascertainment.14 , 19 although the 86 - snp score described here adjusted for fh , genetic factors are not the sole contributors to breast cancer risk , even in cohorts with exceptional fh . 
validated clinical risk models incorporate variables such as body mass index , age at menarche , parity , age at rst birth and hormone replacement therapy use , as well as details of fh.20 - 23 other important risk factors , such as mammographic density and diet , are not taken into account here.24 - 26 more studies will be needed to accommodate these additional variables and to determine their dependent contributions . 
lanchbury data analysis and interpretation : elisha hughes , placede tshiaba , shannon gallagher , susanne wagner , benjamin roa , eric rosenthal , susan domchek , judy garber , jeffrey weitzel , allison w . 
komen for the cure ( i ) , american association for cancer research , diane helis henry medical foundation , james p . wilmot foundation ( i ) , adrienne helis malvin medical research foundation ( i ) , breast cancer research foundation , facing our risk of cancer empowered johnathan lancaster employment : myriad genetics , regeneron stock and other ownership interests : myriad genetics , regeneron jeffrey weitzel speakers bureau : astrazeneca allison w . 
jama 315 : 68 - 76 , 2016 peto j , collins n , barfoot r , et al : prevalence of brca1 and brca2 gene mutations in patients with early - onset breast cancer . 
j natl cancer inst 91 : 943 - 949 , 1999 anglian breast cancer study group : prevalence and penetrance of brca1 and brca2 mutations in a population - based series of breast cancer cases . 
 hughes et al collaborative group on hormonal factors in breast cancer : familial breast cancer : collaborative reanalysis of individual data from 52 epidemiological studies including 58 , 209 women with breast cancer and 101 , 986 women without the disease . 
j mammary gland biol neoplasia 9 : 221 - 236 , 2004 desmond a , kurian aw , gabree m , et al : clinical actionability of multigene panel testing for hereditary breast and ovarian cancer risk assessment . 
michailidou k , beesley j , lindstrom s , et al : genome - wide association analysis of more than 120 , 000 individuals identies 15 new susceptibility loci for breast cancer . 
am j hum genet 104 : 21 - 34 , 2019 judkins t , leclair b , bowles k , et al : development and analytical validation of a 25 - gene next generation sequencing panel that includes the brca1 and brca2 genes to assess hereditary cancer risk . 
li h , feng b , miron a , et al : breast cancer risk prediction using a polygenic risk score in the familial setting : a prospective study from the breast cancer family 17 . 
n aslund - koch c , nordestgaard bg , bojesen se : common breast cancer risk alleles and risk assessment : a study on 35 441 individuals from the danish registry and kconfab . 
cuzick j , brentnall ar , segal c , et al : impact of a panel of 88 single nucleotide polymorphisms on the risk of breast cancer in high - risk women : results from two randomized tamoxifen prevention trials . 
lindstr om s , thompson dj , paterson ad , et al : genome - wide association study identies multiple loci associated with both mammographic density and breast cancer risk . 
 high - content biopsies facilitate molecular analyses and do not increase complication rates in patients with advanced solid tumors purpose precision oncology relies on frequent pathologic , molecular , and genomic assessments of tumor tissue to guide treatment selection , evaluate pharmacodynamic effects of novel agents , and determine drug resistance mechanisms . 
it remains unknown how the need for serial biopsies with large numbers of tumor cores relates to tissue yields and biopsy complication rates . materials and methods in this study , we performed a retrospective analysis of 199 focal liver biopsies performed in 143 patients in the setting of oncologic research protocols ( research biopsy group ) over a 4 - year period at a single - intervention oncology service . 
practice patterns and complication rates were compared with those related to 1 , 522 consecutive biopsies performed in 1 , 154 patients in whom two cores were obtained for standard clinical management of patients ( standard biopsy )  . results in the research biopsy group , 1 , 100 tissue cores ( average , 5.5 cores per procedure ) were harvested and distributed to trial sponsors , internal research laboratories , and pathology services . 
in the standard biopsy control group , major complications were observed in 1.4% of procedures ( 22 of 1 , 522 ) and minor complications in 0.2% ( three of 1 , 522 )  . 
2017 by american society of clinical oncology introduction precision oncology aims to adapt treatment decisions based on the molecular characteristics of an individual tumor , thereby increasing the chance for a successful clinical outcome.1 tumors are increasingly being classified into subsets based on molecular profile with corresponding tailored treatment . 
there are substantial data demonstrating metastatic tissue can harbor unique genomic alterations compared with the primary tumor , and heterogeneity can also be seen among different metastatic sites in the same patient.2 , 3 it is generally accepted that the use of genomics and biomarkers substantially increases the success rate of clinical trials while lowering their cost.4 despite the rapid emergence of liquid biopsies ( eg , circulating tumor dna , 5 exosomes , circulating tumor cells ) , image - guided , minimally invasive biopsies remain the gold standard in procuring sufficient malignant tissue for molecular analysis . there are several reasons for sampling increasing amounts of tissue more frequently : the realization that tumors can adapt rapidly to therapeutic pressures and that molecular analysis of progression site biopsy , rather than primary specimen , is crucial to identify mechanisms of resistance ; the nathan e . 
 increasing use of immunotherapy trials where treatment response assessment can be difficult by imaging ( eg , pseudoprogression ) ; the emergence of more sophisticated analytic models , including patient - derived tumor cell lines and xenografts , requiring additional tissue ; the fact that a single biopsy core ( 10 mm3 ) maximally contains 30 million cells but often much fewer because of stroma and other factors ; and as a precautionary method to ensure that genomic analysis is indeed possible , because results can help with potential selection of matched targeted therapy . 
for example , in a large recent federal trial of personalized cancer medicine , 13% of tumor biopsy samples could not be used for genomic analysis.6 a recent trend in image - guided biopsies has therefore been to collect more cores ( four to 10 cores ) than necessary for traditional pathology workup ( eg , one to two cores )  . additional cores can be harvested with the use of coaxial systems that allow repeat 18 - gauge samples to be obtained through a hollow needle placed under image guidance . 
with such an approach , consecutive tissue fragments can then be used for immunohistochemistry , fluorescence in situ hybridization analysis , genomic analysis , protein and phosphoprotein biomarker testing , and establishment of patient - derived models , including cell lines , organoids , and mouse xenografts.7 these multicore biopsies , which we herein term highcontent biopsies ( hcbx ) , are thus attractive in initiating and evaluating genotype - centric drug trials and may be critically important for comprehensive genomic and proteomic analyses . 
with increased core sampling , a critical question remains : is there an increased complication rate associated with more extensive tissue harvesting ? data on the safety of research biopsies are limited , 8 , 9 in part because such data are typically not reported in clinical trials , and there is no standard consensus for the reporting of adverse events.10 the technical success rates of obtaining adequate cores after repeat sampling are largely unpublished , although anecdotal reports describe potential reduction in sample quality after repeat sampling.11 furthermore , performing biopsies within research protocols requires more complex logistics , with dedicated personnel and workflows . the main goal of this study was to evaluate if the increased number of cores obtained for research biopsies was associated with increased complication rates or decreased sample quality . 
to address these questions , we performed a retrospective analysis of focal tumor biopsies in the liver performed in the setting of specific oncologic research protocols at a single institution over a 4 - year period . 
practice patterns and complication rates were compared with those of 1 , 522 consecutive biopsies performed in 1 , 154 patients for standard clinical management during the same time period . 
of these , 199 biopsies were performed in 143 patients in the setting of oncologic research protocols ( research biopsy group ) ; 1 , 522 were performed in 1 , 154 patients for standard clinical management ( standard biopsy group )  . in the standard biopsy group , patients were referred from their oncologists directly to the interventional oncology service . 
these cores were placed in formalin and sent to pathology for standard processing : hematoxylin and eosin staining , immunohistochemistry as needed , and , more recently , molecular testing ( snapshot or foundation medicine [ fm ] , as discussed in pathology analysis ) when requested by referring oncologists . for research biopsies , however , a dedicated staff research coordinator was responsible for coordinating the entire process : arranging for the biopsy scheduling , determining and discussing the number of cores needed with the interventional radiologist , and collecting the samples and distributing them to their appropriate destinations . 
this was performed according to a standard operating procedure ( sop ) associated with each research study in which these patients were enrolled and was prospectively logged in an institutional database . 
for research biopsies , a dedicated staff member ( biopsy coordinator ) is responsible for coordinating the entire process : arranging for the scheduling , discussing the number of cores , and collecting the samples and distributing them to their appropriate destinations . the most common workflow involves obtaining five cores : in formalin for pathology , including mutational analyses ; fixed in formalin for 6 to 24 hours and subsequently transferred to ethanol , to be sent to trial sponsor ; in fetal bovine serum ( fbs ) , for development of patientderived models and drug testing ; in formalin for intrainstitutional research laboratories ; and snap frozen for tissue bank . prior imaging coaxial biopsy research / clinical trial biopsy > 5 cores formalin formalin formalin snap frozen patient 1 - 2 cores analysis of : size location growth approach routine pathology tissue type ihc mutations analysis of : tissue type pathway analysis mutations drug response oncologist interventional radiologist biopsy coordinator the fourth in formalin for intra - institutional research laboratories ; and the fifth , which was snap frozen ( fig 1 )  . 
patients in the research group were consented for additional risk of bleeding and other complications . image - guided biopsy procedures all procedures were performed using 18 - gauge coaxial , semiautomatic needle biopsy cutting needles ( bard mission ; bard biopsy systems , tempe , az ) under imaging guidance . 
50 , 000 / ml and an international normalized ratio of prothrombin time , 1.5 for all patients undergoing liver biopsy . in the research biopsies , 54.3% of the procedures were performed using ultrasound guidance ( supersonic imagine , aix - en - provence , france ; hitachi aloka medical , tokyo , japan ) , and 45.7% were performed using computed tomography guidance ( general electrics , fairfield , ct )  . 
a total of 193 procedures were performed using moderate sedation ( intravenous use of fentanyl and midazolam ) , three biopsies were performed with general anesthesia or monitored anesthesia care , and three procedures were performed with local anesthetic only ( lidocaine )  . complication rates were determined by review of medical notes and imaging studies obtained up to 30 days after the procedure ( research biopsies ) and of a prospectively maintained departmental database ( research and standard biopsies ; hi - iq ; conexsys , lincoln , ri )  . 
complications were classified as major ( including all patients who required more therapy other than clinical support , required hospitalization , or experienced an unplanned increase in the level of care , permanent adverse sequelae , or death ) or minor ( others ) .13 we did not record intraprocedural pain scores , because a majority of patients received conscious sedation in a patient - focused manner and by different care providers . 
all patient cases with negative results were reviewed , and clinical and imaging follow - up was used to determine whether they were true or false negatives . pathology analysis a negative biopsy was defined as 0% tumor cells . when rare tumor cells were present , they were noted , and the diagnosis was positive for malignancy . 
this was performed in house with polymerase chain reaction ( pcr ) based assay ( pcr - snapshot14 ) or targeted nextgeneration sequencing ( ngs ) tumor genotyping assay ( ngs - snapshot ) .15 selected samples were sent to fm for a different comprehensive genomic profiling using ngs.16 statistical analysis descriptive statistics were used to summarize results . 
table 1 summarizes patient demographics . because of heterogeneity among the clinical trial protocols , the mean number of cores obtained in the research cohort varied ( fig 3a )  . 
for a majority of procedures ( n = 186 of 199 ; hcbx ) , a larger number of cores ( range , four to 10 ; mean , 5.7 cores ) was obtained . 
in a smaller characteristic age , years mean range male female cancer type breast lung colorectal other primary stage * complication minor major 57.9 28 - 84 60 ( 30.2 ) 139 ( 69.8 ) 68 ( 34.2 ) 55 ( 27.6 ) 26 ( 13.1 ) 50 ( 25.1 ) 1 ( 0.5 ) 5 ( 2.5 ) 3 ( 1.5 ) 190 ( 95.5 ) 2 ( 1.0 ) 1 ( 0.5 ) * patient cases of stage i to iii disease correspond to cases of intrahepatic cholangiocarcinoma or hepatocellular carcinoma ; patient cases of stage iv disease represent all other neoplasms . number of procedures ( n = 13 of 199 ) , fewer cores ( range , two to three ; mean , 2.8 cores ) were sufficient to satisfy trial requirements and / or research needs . sample processing and analysis the 1 , 100 cores were distributed to their appropriate destination by the research biopsy coordinator according to the sop of each study ( fig 3b ) ; 229 cores ( from 127 procedures ) were sent to trial sponsors , 121 cores ( from 110 procedures ) were sent for development of patient - derived cell lines , 466 cores ( from 192 procedures ) were sent to other in - house research laboratories for various studies , and 284 cores ( from 174 procedures ) were sent to standard pathology . 
among the procedures from which samples were sent to standard pathology ( n = 174 ) , molecular analysis was attempted on 73 : multiplex pcr - based snapshot in 13 , ngs - based snapshot in 39 , and foundationone in 19 ; two samples had insufficient tissue for molecular analysis . the success rates of different analyses for research biopsies are summarized in figure 3c . 
 ( a ) distribution of number of cores per biopsy ( median , five ; mean , 5.5 6 standard deviation , 1.5 ; range , two to 10 cores )  . 
of these , four were shown to be true negatives ( ie , no evidence of metastasis or persistent local response after treatment on followup ) and were excluded from the analyses of success for the different tests . 
of the evaluated parameters ( table 2 ) , none were clearly associated with either satisfactory or nondiagnostic samples . the complication rates in the standard biopsy cohort were similar , with major complications observed in 1.4% of procedures ( 22 of 1 , 522 )  . the major complications were hematomas requiring transfusion or intervention ( n = 15 ) , pneumothorax requiring chest tube ( n = 1 ) , hemobilia requiring endoscopic retrograde cholangiopancreatogram ( n = 1 ) , infection requiring admittance ( n = 1 ) and / or percutaneous drainage ( n = 1 ) , and death ( n = 3 ; all secondary to bleeding )  . 
the minor complication rate was 1.0% ( n = 2 ; both were hematomas managed conservatively : one patient with metastatic breast cancer [ seven cores ] and one with colorectal cancer [ five cores ] )  . image - guided biopsies have long played an important role in the management of cancer . 
these findings are not entirely unexpected , because we used a coaxial biopsy technique that allows for multiple cores to be harvested through a single coaxial needle , therefore minimizing the number of capsular punctures probably a factor for decreasing the risk of complications in biopsies.24 one evident finding of our study is the progressive increase of research biopsies over the study period . this is consistent with the increasing popularity of molecularly targeted trials , as well as the realization that hcbx are technically feasible and safe . 
it has already been reported that genetic analysis is contingent on sufficient material.6 accordingly , different methods of molecular analysis in our data had results that ranged from 79% ( fm ) to 100% ( pcr - based snapshot )  . although ngs can more efficiently detect a wider range of molecular aberrations , more material is needed compared with older pcrbased techniques . to streamline research biopsies and accommodate larger numbers of procedures , we implemented a specific workflow with dedicated staff for efficient communication among clinicians , researchers , trial sponsors , and interventional radiologists , as well as collection and distribution of samples . 
although we did not measure the exact length of time required for the procedures in this study , we had previously determined that additional core sampling only required a few minutes longer , unless the coaxial needle had to be repositioned because of poor sample material ( eg , necrotic core , cystic tumor , fragmented specimen )  . 
interestingly , in our experience , the order in which cores were harvested was not associated with lower success rates for pathology , contrary to prior published reports.11 the samples sent for cell - line creation had a lower success rate . 
although this may have been related to the sample volume , it may also have resulted from other variables beyond our control ( amount of tumoral cells required for successful cell - line creation , timing , distance , or personnel , among others )  . 
interestingly , however , within the samples that were used for cell - line creation , the number of cores was not associated with cellline creation success rate . our study was designed to answer simple questions : how successful are hcbx , and what is their complication rate ? this is of timely interest ; data on the safety of research biopsies are limited , because they are often not reported in clinical trials.10 as such , our study focused on so - called all comers in a large oncologic interventional care setting rather than on specific subsets of patients , biopsy sites , or operators . 
 3% pi3k - akt signaling pathway fgfr1 / 2 / 3 / 4 and fgf familiy mapk / ras signaling pathway cyclin - dependent kinase related tp53 egfr esr1 other negative fig 4 . 
the genomic abnormalities detected in our study were similar to those found in several large studies17 and reflect the study population involving breast , lung , and colorectal cancer and cholangiocarcinoma trials at our institution . 
mapk , mitogen - activated protein kinase ; pi3k , phosphatidylinositol 3 - kinase . thoracic lesions , and similar studies may be warranted for those cohorts . our study has several limitations . 
medical records were reviewed for complications exclusively in the research biopsy group , although this would , if anything , overestimate the relative risk of complications compared with that of standard biopsies . 
only data on liver biopsies were reviewed , and therefore , additional studies are required to verify if the conclusions apply to other organs frequently biopsied , such as lungs , lymph nodes , and bones . 
vadvala , aditya bardia , jeffrey engelman , peter r . mueller , dejan juric , ralph weissleder financial support : dejan juric , ralph weissleder administrative support : dejan juric , ralph weissleder provision of study material or patients : laura spring , dejan juric collection and assembly of data : nathan e . 
frenk , laura spring , alona muzikansky , mari mino - kenudson , amy ly , aditya bardia , dianne finkelstein , dejan juric , ralph weissleder manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors the field of blood - based biomarkers in oncology for disease monitoring and molecular analysis is rapidly evolving . 
liquid biopsies can be used to detect circulating extracellular vesicles25 or tumor cells within a blood specimen , providing a tool to monitor cancer noninvasively.26 similarly , a number of technologies have also been developed to identify and track tumor - specific mutations via circulating free dna in the blood and urine.5 as these technologies are further developed and validated , they might replace some of the tissue biopsies in certain research and clinical settings . however , tissue biopsies allow for sampling of specific lesions and evaluation of tissue architecture and the tumor microenvironment , including proteins , phosphoproteins , and varied cytokines . image - guided biopsies will therefore continue to remain important for phenotyping tumor responses , identifying tumor heterogeneity , identifying therapeutic resistance , and characterizing host responses in the era of immunotherapy and targeted therapies . 
additional advances in image - guided tumor sampling , including lower computed tomography doses , shorter image acquisition times , and systems for acquiring , processing , and analyzing smaller samples may further expand the use of hcbx . 
mueller consulting or advisory role : cook medical patents , royalties , other intellectual property : royalties from cook ( percutaneous catheters ) dejan juric consulting or advisory role : novartis , emd serono , eisai ralph weissleder consulting or advisory role : moderna therapeutics , alivio therapeutics , tarveda therapeutics , lumicell , t2 biosystems , bioanalytix acknowledgment we thank all members of the division of interventional and abdominal radiology who contributed to the review of cases and / or performing of biopsies , in particular steven dawson , md , ashraf thabet , md , ronald arellano , md , suvranu ganguli , md , avinash kambadakone , md , michael zalis , md , and omar zurkiya , md , phd . 
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im h , shao h , park yi , et al : label - free detection and molecular profiling of exosomes with a nano - plasmonic sensor . nat biotechnol 32 : 490 - 495 , 2014 26 . 
 exceptional response to 177lutetium prostate - specic membrane antigen in prostate cancer harboring dna repair defects megan crumbaker , mbbs1 , 2 , 3 ; louise emmett1 , 3 ; lisa g . 
psma - targeted small molecules bound to 177lutetium ( 177lu ) , a medium - energy b - emitter , have been administered as a targeted therapy for metastatic prostate cancer ; the psma - targeted small molecule binds to psma on the cancer cell surface and is internalized , leading to delivery of a potent but targeted dose of radiation to the cell . 
a small molecule commonly used for this purpose is psma - 617 , a human psma - targeting ligand that can be conjugated to 177lu . 177lupsma - 617 therapy has shown great promise in early - phase trials , with a prostate - specic antigen ( psa ) response rate of 57% in a phase ii prospective study , 1 and randomized trials are currently recruiting internationally . de novo and acquired resistance are common , however , and biomarkers are needed to guide patient selection and rationalize combinatorial approaches . 177lupsma - 617 induces cell death through doublestrand dna breaks.2 mechanisms to repair doublestrand dna breaks are decient or absent in cells with mutations in homologous repair genes such as brca1 or brca2 , and such mutations are increasingly recognized in metastatic castration - resistant prostate cancer ( mcrpc ) .3 , 4 thus , a deciency in homologous repair may render a tumor more sensitive to 177lupsma - 617 and be relevant in crpc . 
postoperative radiotherapy was withheld because of a history of colorectal carcinoma treated with surgical resection followed by adjuvant radiotherapy 25 years prior . soon after his radical prostatectomy , he biochemically relapsed and was treated with intermittent androgen deprivation therapy . 
he eventually developed overt metastatic disease , with a perihilar mass found on a computed tomography ( ct ) scan of the chest , abdomen , and pelvis after 10 years of hormone manipulation . a nuclear medicine bone scan was negative , but an 18f - labeled uorodeoxyglucose positron emission tomography ( pet ) scan showed low - grade uptake in the mass , which was conrmed to be metastatic prostate cancer on biopsy . 
a choline pet was undertaken to assess the extent of his disease and revealed uptake in his left hilar nodes with inltration in the lingula and involvement of right paratracheal nodes . given the extent of disease across both hemithoraces , he commenced enzalutamide . 
his psa continued to increase ( fig 1 ) despite 8 weeks of treatment , and additional radiologic progression with impending bronchial obstruction prompted radiotherapy to his hilar disease . his psa initially decreased after radiotherapy but began to increase soon after . 
he then received two cycles of cabazitaxel , after which a restaging ct scan showed enlargement of his liver metastases and development of new pelvic nodal metastases . having progressed through all standard lines of therapy , this patient was enrolled in a clinical trial of 177lupsma - 617 therapy and received 4 6.5 gigabecquerel doses once every 6 weeks ( anzctr identier : actrn12615000912583 )  . 
plot of prostate - specic antigen ( psa ) values over time . treatment regimens : cabazi , cabazitaxel ; doce , docetaxel ; enz , enzalutamide ; lupsma , 177lutetium prostate - specic membrane antigen ; rt , radiotherapy . time ( months ) the return of his right upper quadrant abdominal pahe was consented for prescreening of his circulating tumor dna ( ctdna ) for a poly adp - ribose polymerase ( parp ) inhibitor trial , but while awaiting this result , his pain increased signicantly , with derangement of his liver function tests and a further increase in his psa . 
he was approved for an additional two compassionate 7.0 - gigabecquerel doses of 177lupsma - 617 ; he received these with clinical and radiologic responses associated with a psa response from 169 to 43 after the rst dose . a resolution bioscience targeted ctdna analysis for dna repair defects revealed abnormalities in three homologous fig 2 . 
to assess his germline , we performed targeted sequencing of coding regions and splice sites of atm , brca1 , brca2 , brip1 , cdh1 , cdkn2a , chek2 , hoxb13 , palb2 , pten , rad51c , rad51d , stk11 , and tp53 on dna extracted from blood ; this conrmed a frameshift mutation in exon 11 of the brca2 gene . 
up - front resistance and early relapses are common , however , signifying the need for strategies to improve patient selection and / or test rational combination approaches . as a form of ionizing radiation , 177lupsma - 617 induces double - strand dna breaks , which are believed to be the main lethal event in most exposed cells . 
preclinical and clinical studies have identied an association between cancers with homologous recombination defects and increased radiation sensitivity.8 germline mutations in genes that encode and mediate key enzymes for dna repair pathways , such as brca2 and atm , occur in approximately 11.8% of men with metastatic prostate cancer and somatic mutations in at least 23% of patients with mcrpc , 3 , 4 rendering many prostate cancers homologous recombination decient . homologous recombination deciency has been associated with responses to parp inhibition and platinum - based chemotherapy , 9 , 10 but these treatments may cause signicant toxicity . this patients germline mutation likely predisposed him to more aggressive disease , 11 and he was resistant to not only enzalutamide but also two lines of taxane - based chemotherapy . 
after his 177lupsma - 617 treatment , he also responded to carboplatin chemotherapy and a parp inhibitor . is biologically plausible that this gentlemans aberrations in homologous repair pathways enhanced his susceptibility to the radiation effects of 177lupsma - 617 therapy . 
we hypothesize that patients with mutations in dna repair pathway genes would be especially responsive to peptide receptor radionuclide therapy because of their inability to overcome dna breaks induced by the treatment . 
hofman ms , sandhu s , eu p , et al : 785olutetium - 177 psma ( lupsma ) theranostics phase ii trial : efcacy , safety and qol in patients with castrate - resistant prostate cancer treated with lupsma . 
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eur urol 73 : 656 - 661 , 2018 emmett l , willowson k , violet j , et al : lutetium 177 psma radionuclide therapy for men with prostate cancer : a review of the current literature and discussion of practical aspects of therapy . 
pomerantz mm , spis ak s , jia l , et al : the association between germline brca2 variants and sensitivity to platinum - based chemotherapy among men with 11 . 
castro e , goh c , olmos d , et al : germline brca mutations are associated with higher risk of nodal involvement , distant metastasis , and poor survival outcomes metastatic prostate cancer . 
 circulating tumor dna analysis in colorectal cancer : from dream to reality carlotta antoniotti , md1 , 2 ; filippo pietrantonio , md3 , 4 ; salvatore corallo , md3 ; filippo de braud , md3 , 4 ; alfredo falcone , md1 , 2 ; and chiara cremolini , md , phd1 , 2 liquid biopsy is a minimally invasive approach to obtain circulating materials that originate from tumor cells through the sampling of body uids , mainly peripheral blood . 
because of its abilities to detect tumor - derived nucleic acids and proteins and to characterize tumor - specic genomic abnormalities , liquid biopsy has emerged as an approach to orient care of patients with colorectal cancer ( crc )  . 
second , the reliability of studies that evaluate the concordance of ras testing between tissue and plasma samples is impaired by the adoption of assays with heterogeneous analytical sensitivity and coverage of genomic regions . 
there are now several commercially available methods for ctdna assessment and technology platforms based on digital polymerase chain reaction or next - generation sequencing approacheseach one with specic sensitivity , specicity , throughput , gene coverage , costs , and potential clinical applications.12 among them , three test kits are ce - marked in vitro diagnostic devices for detection of ras and braf mutations on ctdna in crc.13 - 16 third , some clinicopathologic variables are likely to affect the amount of tumor - released ctdna.5 , 6 , 8 , 10 , 11 whereas liver involvement and tumor burden are positively associated with the ras mutant allele fraction ( the proportion of mutant dna fragments at a given locus ) , peritoneal , nodal , and lung metastases and mucinous histology are linked to low ras inuence ctdna ctdna detection . 
 antoniotti et al ras wild - type cases , as assessed on ctdna , negligible . is not the parallel assessment of the mutant allele fractions of other key genomic tumor alterations may help solve challenging cases that have undetectable ras mutations and ctdna levels at or lower than the analytic sensitivity of adopted assays . 
to overcome these issues , preanalytic procedures should be standardized , a threshold of detectable mutation rate that confers intrinsic resistance to egfr inhibition should be set and prospectively validated , investigation to understand when plasma and additional and tissue tests are interchangeable and to improve assay sensitivities is warranted . meanwhile , the ras status assessment to address the use of anti - egfr agents must be performed as the gold standard on tumor specimens.3 , 4 , 17 , 18 only when tissuebased testing is technically or logistically unfeasible could it be replaced by ctdna analysis.4 ctdna to estimate prognosis and detect minimal residual disease retrospective studies have focused on the prognostic impact of the quantitative analysis of ctdna . 
in the metastatic setting , a correlation between ctdna concentration and survival has been described , but the independent weight of ctdna quantication when the relative impact of other well - known prognostic factors is taken into account has not been claried ( appendix table a2 )  . the role of the quantitative assessment of ctdna in monitoring response during treatment must be dened in the light of its potential added value when compared with easily available and well - established markers , including carcinoembryonic antigen or early radiologic disease reassessment . 
recently , ctdna has been proposed to detect , measure , and monitor residual disease after radical interventions . a series of proof - of - concept studies reported that liquid biopsy could disclose the persistence of minimal residual disease ( mrd ) through the detection of ctdna in patients with crc who underwent potentially curative surgery ( resection of primary tumor in early - stage crc or radical resection of metastases ) even in the absence of clinical or radiologic signs of residual disease ( appendix table a2 )  . by identifying incomplete eradication of disease after a curative treatment , detectable ctdna predicts an increased risk of relapse regardless of the exposure to an adjuvant treatment . 
the development of such a sensitive tool might improve the risk estimation of disease relapse after a curative intervention to properly stratify clinical trials in early - stage crc and to accordingly drive the therapeutic management . theoretically , two applications of the detection of mrd in this setting may be foreseen : to offer chemotherapy to all postoperative ctdna - positive patients , including those with no histopathologic risk factors to reduce their risk of progressionindeed , ctdna positivity invariably means residual diseaseand to avoid useless adjuvant therapies in postoperative ctdna - negative patients . 
in early - stage crc , ctdna is detected at a lower rate than in the advanced disease.1 therefore , highly sensitive techniques are needed to achieve appropriate accuracy to detect mrd . most available data have been achieved through a twothe identication of specic somatic step procedure : abnormalities in tissue samples , followed by the search for the same alteration in ctdna . 
only trials that aim to optimize the treatment of all postoperative ctdna - positive patients , independent of traditional histopathologic factors , are ethically acceptable . ctdna to track tumor response and resistance to therapy whereas tissue biopsies catch single snapshots of the tumor in a specic spatiotemporal fragment , liquid biopsy may more comprehensively depict the intrinsic and dynamic intratumoral heterogeneity . 
the heterogeneity and dynamism of the tumor clonal evolution under the pressure of targeted treatments are conrmed by the emergence of multiple alterations in the mitogenactivated protein kinase pathway effectors , including ras and mek mutations and kras , brafv600e , and met amplication , during the treatment with braf / egfr , braf / mek and braf / egfr / mek inhibitors in patients with brafv600e - mutant mcrc.23 - 27 two novel ntrk1 mutations have been detected as a potential mechanism of acquired resistance to entrectinib , a tyrosine kinase receptor inhibitor , in a patient with lmna - ntrk1rearranged mcrc.34 nevertheless , some steps should be covered to translate liquid biopsy from the investigational setting into clinical practice . available data indicate that the frequency of molecular alterations in ctdna at the time of disease progression is inconsistent among different series , even when the same methods for plasma ctdna analysis is applied ( table 1 )  . this inconsistency impairs the reliability of adopted techniques and the reproducibility of the ndings . 
setting and validation of a quantitative threshold to dene the clinical relevance of each detectable molecular alteration as clearly associated with a lack of benet from ongoing therapies may be relevant to biologically guide therapeutic decisions . currently , no prospective data are available about usefulness of discontinuing ongoing therapy and initiation of a tailored treatment when signals of acquired resistance emerge in ctdna before clinical or imaging - based disease progression is noted . the co - occurrence of multiple and / or subclonal molecular alterations at the time of acquired resistance highlights an increase in tumor heterogeneity , which complicates the denition of clinical value of each identied alteration as therapeutic target for subsequent tailored strategies . 
in other words , how to therapeutically target the heterogeneous mechanisms of resistance and the subclonal patterns of tumor cell populations that emerge upon drug selection is today still challenging . 
in this proof - ofconcept study , patients who are candidates for anti - egfr rechallenge are eligible only if a notable decrease in ras fractional mutational abundance occurs from the time of disease progression after a rst - line anti - egfrcontaining therapy to the time of rechallenge . liquid biopsy has emerged as a minimally invasive tool to genotype tumors , to assess patient prognosis and detect mrd , to monitor treatment efcacy , and to track the dynamism of clonal evolution over time and therapies . 
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tsuji y , shitara k , yamanaka t , et al : reverce : randomized phase ii study of regorafenib followed by cetuximab versus the reverse sequence for metastatic colorectal cancer patients previously treated with uoropyrimidine , oxaliplatin , and irinotecanbiomarker analysis . 
montagut c , argil es g , ciardiello f , et al : efcacy of sym004 in patients with metastatic colorectal cancer with acquired resistance to anti - egfr therapy and molecularly selected by circulating tumor dna analyses : a phase 2 randomized clinical trial . 
santini d , vincenzi b , addeo r , et al : cetuximab rechallenge in metastatic colorectal cancer patients : how to come away from acquired resistance ? ann oncol 23 : 4602 - 4616 , 2017 23 : 2313 - 2318 , 2012 49 . 
parseghian cm , loree jm , morris vk , et al : anti - egfr resistant clones decay exponentially after progression : implications for anti - egfr re - challenge . 
cremolini c , rossini d , dellaquila e , et al : rechallenge for patients with ras and braf wild - type metastatic colorectal cancer with acquired resistance to rstline cetuximab and irinotecan : a phase 2 single - arm clinical trial . 
 aggressive - variant microsatellitestable pole mutant prostate cancer with high mutation burden and durable response to immune checkpoint inhibitor therapy the increasing application of next - generation tumor dna sequencing may define molecular subsets of prostate cancer for which precision medicine approaches are enabled . 
transrectal ultrasound guided needle biopsy demonstrated gleason 4 + 5 = 9 ( grade group 5 ) adenocarcinoma in 12 of 12 cores involving 95% to 100% of each core with perineural invasion . 
tumor cells stained positive for psa and negative for chromogranin and synaptophys radiologic staging studies indicated bilateral external iliac , common iliac , and retroperitoneal adenopathy without definitive evidence of bone metastases . 
with persistent bulky disease in the prostate on digital rectal examination at 12 weeks from the start of hormonal therapy , a repeat biopsy was performed , which demonstrated gleason 4 + 5 = 9 adenocarcinoma with focal synaptophysin expression . 
restaging studies showed only modest regression in pelvic adenopathy discordant with the brisk decline in psa , with tumor infiltrating the bladder base and seminal vesicles , and abutting the rectum with a preserved fat plane on magnetic resonance imaging . 
pathologic examination indicated a high - grade tumor with morphologic and immunohistochemical evidence of neuroendocrine differentiation ( synaptophysin and chromogranin expression ) with lymphocytic infiltration ( fig 1 ; appendix fig a1 )  . 
seminal vesicle involvement was absent , but there was extensive extraprostatic extension and multiple positive surgical margins including the bladder neck , and one of eight lymph nodes were involved.the patient did well in the postoperative period , with early recovery of continence ; however , restaging studies showed progressive retroperitoneal and pelvic adenopathy . 
repeat radiologic imaging after an interval on observation again demonstrated progressive pelvic adenopathy ( fig 2a )  . methodology genomic sequencing studies on the primary tumor were performed with the foundationone assay1 ( foundation medicine , cambridge , ma ) , and the tumor mutation burden was estimated as previously described.2 mutations of the androgen receptor , pten , tp53 , and atm , which have been previously implicated in the genomic landscape of prostate cancer , 3 , 4 were identified , along with a pole v511l mutation ( appendix table a1 )  . 
using all nondriver and nongermline missense mutations , we found that 77% of the alterations in the sample were attributable to the pole signature , making this the dominant signature over other lesions identified in the sample that have been implicated in dna repair , including tp53 , atm , or pten ( appendix table a1 )  . 
germline screens for inherited mutations included a screen for brca1 and brca2 ( ambry genetics , aliso viejo , ca ) and a breast - ovarian panel ( genedx , gaithersburg , md )  . 
infiltrating cd4 + and cd8 + lymphocytes were observed in a three to one ratio.the patients family history raised concerns of an autosomal - dominant inherited risk of prostate and breast cancer . 
his father had been diagnosed with high - grade prostate cancer at 76 years of age , and his two brothers had screen - detected low - risk prostate cancer at 54 and 48 years of age , respectively . 
one sister had died of triple - negative metastatic breast cancer at 45 years of age , and another was diagnosed with ductal carcinoma in situ at 55 years of age . 
 was identified in the father in brip1 ( c2392c > t [ p . arg798ter ] ) , along with a brip1 variant of uncertain significance ( c.3710c > a [ p.ser1237tyr ] ) ; the latter alone was found in the patient and two living siblings ( appendix fig a2 )  . 
given the early castration - resistant and chemotherapy - resistant progression of the patients disease and the high tumor mutation burden identified , he was started on immune checkpoint inhibitor blockade with pembrolizumab therapy 200 mg every 3 weeks intravenously . 
sixteen months later , he has remained without evidence of disease progression after 24 cycles of therapy , with residual subcentimeter pelvic nodes , a normal testosterone level without hormonal therapy , and an eastern cooperative oncology group performance score of zero . discussion to our knowledge , this first report in prostate cancer indicates the presence of a mutation in the highly conserved exonuclease domain of dna polymerase ( pole ) 7 in an aggressive - variant metastatic prostate cancer8 that presented with a bulky primary tumor , early castration , and chemotherapy resistance but with an excellent and durable subsequent response to immune checkpoint blockade . 
pole mutant cancers constitute a unique genomic subset of cancers described previously in colorectal and endometrial cancers.9 the somatic v411l mutation of pole has been associated with impaired dna proofreading repair and the generation of ultramutated tumor phenotypes that are microsatellite stable.9 , 10 mutations that could contribute to castration resistance ( androgen receptor , pten ) or dna damage repair insufficiency ( atm , tp53 ) were also identified , consistent with the known genomic landscape of prostate cancer.3 however , two methods of analyzing genomic signatures were used that implicate the pole lesion as the dominant source of the hypermutated phenotype in this case . 
 hypermutated phenotypes in prostate cancer are uncommon and were first identified in 12% of tumors ( 7 of 60 ) sourced from patient - derived xenografts and a rapid autopsy series.11 in this study set , hypermutated tumors , defined as > 300 somatic protein - altering mutations , were universally associated with micro satellite instability largely secondary to msh2 and msh6 structural rearrangements and not mlh1 epigenetic silencing , as is more commonly seen in colorectal and endometrial cancers with dna mismatch repair deficiency . 
 our case report suggests that microsatellite stable pole mutant prostate cancer represents a distinct , albeit exceedingly rare , genomic subset of highmutation - burden prostate cancers with demonstrable potential for high - quality and durable response to immune checkpoint blockade , similar to that reported in endometrial and colorectal pole mutant cancers.12 , 13although responses to immune checkpoint inhibitors in castration resistant prostate cancer have been uncommon to date , 14 - 16 in one study , three of 10 enzalutamide resistant tumors responded to pembrolizumab therapy.17 one evaluable tumor was found to be microsatellite unstable , and a second was microsatellite stable . 
to our knowledge , other that this case report , there are no data that link the control of prostate cancer with immune checkpoint inhibitors to genomically defined highmutation - burden subsets of prostate cancers , whether microsatellite stable or unstable . 
more studies will be required to establish whether high - quality and durable responses to immune checkpoint inhibitors as single agents will be confined to prostate tumors with high mutation burdens . 
hotspot germline genomic sequencing using commercially available assays via clinical genetics consultation did not identify a pathogenic lesion in the patient and two of the living siblings with cancer diagnoses , but his father was identified with a pathogenic brip1 variant along with the brip1 variant of uncertain significance found in his children . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center  . lisa lee no relationship to disclose elizabeth genega no relationship to disclose dallas reed no relationship to disclose ethan sokol no relationship to disclose paul mathew honoraria : exelixis patents , royalties , other intellectual property : patent pending on new therapeutic invention travel , accommodations , expenses : exelixis affiliations lisa lee , elizabeth genega , dallas reed , and paul mathew , tufts medical center , boston ; siraj ali and ethan sokol , foundation medicine , cambridge , references 1 . 
graff jn , puri s , bifulco cb , et al : sustained complete response to ctla - 4 blockade in a patient with metastatic , castration - resistant prostate cancer . 
kwon ed , drake cg , scher hi , et al : ipilimumab versus placebo after radiotherapy in patients with metastatic castration - resistant prostate cancer that had progressed after docetaxel chemotherapy ( ca184 - 043 ) : a multicentre , randomised , double - blind , phase 3 trial . 
kote - jarai z , jugurnauth s , mulholland s , et al : a recurrent truncating germline mutation in the brip1 / fancj gene and susceptibility to prostate cancer . 
 legend prostate cancer squamous cell carcinoma colorectal cancer thyroid cancer melanoma basal cell cancer breast cancer 61 years brip1 c.3710c > a ( p.ser1237tyr ) ; likely benign variant prostate cancer , 59 years gleason 9 60 years breast cancer ( unilateral ) , 54 years brip1 c.3710c > a , p.ser1237tyr ; likely benign variant 53 years prostate cancer , 52 years melanoma 49 years prostate cancer , 48 years basal cell cancer d . 
87 years colorectal cancer , 87 years 85 years brip1 c.2392c > t ( p.arg798ter ) ; pathogenic prostate cancer , 79 years squamous cell carcinoma 83 years thyroid cancer , 40s fig a2 . 
steensma , md6 ; amy dezern , md7 ; gail roboz , md8 ; guillermo garcia - manero , md9 ; harry erba , md , phd10 ; benjamin l . 
maciejewski , md , phd1 purpose we developed an unbiased framework to study the association of several mutations in predicting resistance to hypomethylating agents ( hmas ) in patients with myelodysplastic syndromes ( mds ) , analogous to consumer and commercial recommender systems in which customers who bought products a and b are likely to buy c : patients who have a mutation in gene a and gene b are likely to respond or not respond to hmas . methods we screened a cohort of 433 patients with mds who received hmas for the presence of common myeloid mutations in 29 genes that were obtained before the patients started therapy . 
the association between mutations and response was evaluated by the apriori market basket analysis algorithrules with the highest condence ( condence that the association exists ) and the highest lift ( strength of the association ) were chosen . we validated our biomarkers in samples from patients enrolled in the s1117 trial . results among 433 patients , 193 ( 45% ) received azacitidine , 176 ( 40% ) received decitabine , and 64 ( 15% ) received hma alone or in combination . 
the median number of mutations per sample was three ( range , zero to nine ) , and 176 patients ( 41% ) had three or more mutations per sample . 
these molecular signatures were present in 30% of patients with three or more mutations / sample with an accuracy rate of 87% in the training cohort and 93% in the validation cohort . conclusion genomic biomarkers can identify , with high accuracy , approximately one third of patients with mds who will not respond to hmas . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction the hypomethylating agents azacitidine ( aza ) and decitabine ( dac ) have been approved by the food and drug administration and the european medicine agency for patients with myelodysplastic syndromes ( mds ) .1 - 4 although treatment with these agents is well tolerated , only 30% to 40% of patients will respond to therapy , with the majority achieving hematologic improvement in blood counts and only a minority ( 10% to 15% ) achieving a complete response , the response criterion most reliably associated with improvement in overall survival ( os ) .1 - 4 more importantly , it may take up to six cycles of treatment for patients to achieve their best response.5 given the limited number of patients who benet from these agents and the long administration of identifying biotheir treatment , markers that can predict resistance is essential , because it can prevent prolonged exposure to ineffective therapy and unnecessary toxicities and treatment costs can be avoided . because clinical variables and patient characteristics have not consistently predicted response to hypomethylating agents ( hmas ) , molecular data represent a biologic opportunity6 - 8 to enhance patient response rates and outcomes . 
because identifying a single gene or comutated genes is unlikely to yield an understanding of how these mutations dene disease biology or phenotype , an unbiased approach is needed to study the relationship of these abnormalities to each other and to mds biology . in this study , we used unbiased , machine learning approaches ( a recommender system similar to that used by netix or amazon.com ) to assess the impact of molecular data on resistance to hmas in a large cohort of patients treated with hmas at different academic institutions , and we validated our results in a population treated in a contemporary prospective clinical trial of hma therapy15 of aza alone and in combination . methods patients for the training cohort , patients treated with either azaor dac - based regimens were included in this study : 230 patients were treated at cleveland clinic ( between 2002 and 2012 ) ; 203 were from other academic institutions ( dana - farber cancer institute [ 2003 to 2010 , n = 42 ] and md anderson cancer center [ 2003 to 2010 , n = 103 ] ) or were part of the daco - 020 clinical trial ( adopt [ 2005 to 2006 , n = 58 ] ) .11 all patients consented to blood or bone marrow samples at each institution under institutional review boardapproved protocols in accordance with each institution policy and the declaration of helsinki . 
more information regarding the patient cohort , response criteria , and validation cohort is included in the data supplement . dna sequencing and mutational analysis a panel of 29 genes that are commonly mutated in mds and myeloid malignancies was evaluated ( data supplement )  . 
more information regarding sequencing method is included in the data supplement . statistical analyses test variables were compared using the wilcoxon rank sum test and fishers exact for continuous and categorical variables , respectively . 
os was calculated from the date of diagnosis to the date of last follow - up or death ( whichever came rst ) , and survival curves were constructed using the kaplan - meier method and compared using the log - rank test . 
mutation distribution among responders versus nonresponders total responders non - responders nazha et al mutations asxl1 tet2 srsf2 runx1 dnmt3a sf3b1 tp53 u2af1 bcor ezh2 nras idh2 phf6 npm1 jak2 zrsr2 etv6 prpf8 idh1 prpn11 suz12 kras gata2 cebpa received dac ( 176 [ 86% ] alone and 29 [ 14% ] in combination with other agents )  . 
cytogenetic analyses per international prognostic scoring system ( ipss ) revised ( r ) criteria16 included 234 patients ( 54% ) with very good or good risk , 66 ( 15% ) with intermediate risk , 33 ( 8% ) with poor risk , 66 ( 15% ) with very poor risk , and 34 ( 8% ) not documented ( table 1 )  . 
the frequency of these mutations in our patient cohort was similar to those identied in other mds cohorts , with the exception of asxl1 , which was slightly higher because it was overrepresented in one cohort11 ( 203 patients from other academic institutions )  . 
patterns of mutation association were also similar to those in previous reports ( fig 1 )  . asxl1 mutations were commonly commutated with tet2 in 42 patients ( 10% ) , and with srsf2 38 ( 9% ) , runx1 35 ( 8% ) , u2af1 24 ( 6% ) , and dnmt3a 21 ( 5% ; fig 1 )  . overall , 367 patients ( 85% ) in the training cohort had a mutation in at least one gene . 
the graph is separated to show the spectrum of mutations in responders compared with nonresponders . ( 4% ) partial remission ( pr ) , 59 ( 14% ) hematologic improvement ( hi ) , 142 ( 33% ) stable disease , and 107 ( 24% ) progressive disease . in general , clinical characteristics such as age , cytopenias , and treatment regimens did not affect response , with the exception of the median blast percentage in the bone marrow , which was higher among responders compared with nonresponders ( 9% v 2% , p = .02 ; table 1 )  . 
risk stratications per ipss and ipss - r did not affect the overall response rate ( table 1 )  . idh1 and ezh2 , no single gene mutation was signicantly associated with response and resistance to hmas in univariate analyses , with the exception of respectively ( table 2 )  . 
the number of mutations per sample also did not affect response , with patients with three or more mutations having similar response rates to those with fewer than three mutations ( table 1 )  . 
genomic biomarkers dened by the recommender system association rules for resistance to hmas asxl1 , nf1 asxl1 , ezh2 , tet2 asxl1 , ezh2 , runx1 ezh2 , srsf2 , tet2 asxl1 , ezh2 , srsf2 asxl1 , runx1 , srsf2 asxl1 , tet2 , srsf2 asxl1 , bcor , runx1 the impact of mutations on response was assessed after controlling for clinical variables such as age and ipss - r scoring system , using logistic regression analyses . 
no mutation was associated with overall resistance or response to hmas , even after adjustment for clinical variables ( age , ipss - r , and sex ; data supplement )  . recommender system genomic biomarkers that predict response to build strong association rules ( associations between genes and outcomes [ response v no response ] ) , we used strict criteria to identify rules with the highest support ( how many times the rules appeared in the data set ) , high condence ( the condence of the algorithm in the association rule was set at 95% ) , and higher lift ( a measure that is reected in the strength of the association : the higher the lift is , the stronger is the association ) in the training cohort . 
when the rules were applied to our patient cohort , they predicted resistance to hmas correctly in 46 of 53 patients ( 87% ) with relevant molecular mutations . among the 105 patients with lower - risk disease by ipss - r ( low and very low risk groups ) , 62 ( 59% ) did respond to hmas . 
the difference in the presence of the biomarkers in lowerversus higher - risk mds is related to the higher percentage of patients with three or more mutations / sample in the higher - risk ( 42% ) versus the lower - risk ( 30% ) group . association with overall survival survival data were available for 375 patients from the training cohort . 
genomic biomarkers of resistance to aza were present in 14 of 39 samples ( 35% ) with three or more mutations ; 13 of 14 of these patients ( 93% ) did not respond to therapy . discussion predicting response or resistance to our currently available standard hma therapy in mds remains a signicant clinical challenge . 
identifying patients up front who may not respond to hmas can potentially improve outcome , decrease unnecessary adverse effects , and save money , especially when current recommendations are for a minimum of 6 months of treatment before deeming it a failure . 
although it is tempting to identify an isolated molecular abnormality or a pair of mutations that can predict hma resistance , this approach does not allow for the complexity and evolution of the genomic landscape in mds . in this study , we developed an unbiased framework using a machine learning , recommender system algorithm to identify highly sensitive genomic associations ( molecular signatures or genomic biomarkers ) that can predict resistance to hmas with high accuracy . 
these associations were validated in an independent cohort in samples from patients enrolled in a randomized phase ii / iii clinical trial . although our biomarkers were identied in only 25% of patients , their presence predicted resistance in almost all patients who had these mutations . 
by denition , a biomarker can be present in a small subset of patients , but when present can predict , with high accuracy and reliability , response or resistance to a therapy . 
detecting these biomarkers in 29% of patients suggests that other biologic mechanisms ( eg , changes in gene expression or epigenetic changes ) or clinical characteristics may contribute more to hma response and failure than does genomics . 
our ndings conrm that genomic associations may lead to different gene expressions and / or epigenetic changes that contribute to the response or resistance and thus , identifying one or two genes that can predict response may not be sufcient to build reliable and predictable models . although we included patients who received hmas in combination with other investigational agents , these combinations did not affect the response or resistance rate or os ; thus , their impact on the output of our recommender system algorithms is negligible . 
furthermore , neither ipss nor ipss - r predicted response or resistance to hmas in our study in accordance with prior reports.1 , 15 prior studies have attempted to use genomic data to predict response or resistance to hmas . 
for example , some studies have shown that tp53 mutations may predict response to hmas , whereas others did not conrm that nding.13 , 17 in a small study of 84 patients with acute myeloid leukemia ( aml ) and mds treated with a 10 - day dac course , a small subset of patients with tp53 mutations had a higher response rate to dac compared with tp53 wild - type patients . furthermore , the median os for tp53 mutated patients who received dac and underwent an allogeneic stem - cell transplantation was similar to that of patients with wild - type tp5317 . 
contrary to this nding , in a study of 71 patients with aml , there was no difference in overall response rate and survival among patients who received 5 days of dac compared with those who had a 10 - day schedule . 
 genomic biomarkers for resistance in mds with variant allele frequency greater than 10% may predict response to hmas , especially in patients with wild - type asxl1 mutations , but this nding was not validated in our study.11 the discrepancy in the results of these studies could be related to sample size , the number of genes tested , and the statistical approach that was used to analyze the data . 
it is also possible that genomic changes in themselves are not the drivers of response to hmas but rather , changes in the gene expression and methylation prole that are derived from the combination of these mutations . 
in a study of whole - genome sequencing , rna sequencing , and methylation prole of samples from patients with chronic myelomonocytic leukemia , a serial sequencing demonstrates that the response to hypomethylating agents is associated with changes in dna methylation and gene expression , without any decrease in the mutation allele burden or prevention of new genetic alteration occurrence.18 if eligible , directly , without this study includes several areas of innovation . 
for example , if a patient with mds with higher - risk disease carries one of these biomarkers , he or she should be encouraged to enroll in a clinical trial with a novel therapy or to proceed with an allogeneic stem - cell transplantation , the use of hmas , because the response to such therapy is predicted to be low . 
although all patients with mds should be encouraged to enroll in a clinical trial with novel therapies , having biomarkers that accurately predict resistance may ease the conversation with some patients who are hesitant to try newer approaches and prefer food and drug administrationapproved therapies.19 alternatively , patients with higher - risk disease and a high blast percentage may consider intensive , aml - type chemotherapy before allogeneic stem - cell transplantation , as opposed to an hma that is predicted to do little to affect the disease in the absence of other biomarkers that could predict resistance to chemotherapy , such as complex karyotype cytogenetics , and the presence of tp53 mutations and the absence of targetable mutations such as idh1 and idh2 . 
because the optimal timing for patients with mds with lower - risk disease can be challenging and because these genomic biomarkers predicted poor outcome even in patients with lower - risk disease . 
these biomarkers could be used as a justication to proceed with allogeneic stem - cell transplantation early in the disease course , especially if the patient has a lower blast percentage . 
in addition , identifying , with high accuracy , patients who may or may not respond to therapy can prevent prolonged exposure to ineffective therapy and can lead to lower cost without decreasing value or changing patient outcomes . 
introducing these biomarkers into normal hematopoietic cells using crispr / cas9 may offer an opportunity to model and understand hma resistance to develop novel drugs to overcome this resistance . our study highlights the importance of machine learning algorithms such as the recommender system in translating genomic data into useful clinical tools that can be used by physicians in the clinic.20 nevertheless , some limitations to our approach exist . 
it is possible that the response to hmas is derived mainly from epigenetic changes and is not dependent on the genomic changes that we studied here . in summary , our study identied genomic abnormalities that predict response or resistance to hmas in patients with mds , and we validated our results in an independent patient cohort treated in a randomized clinical trial . 
identication of biomarkers that can provide personalized treatment approaches that can predict response or resistance to cancer therapy remains an important clinical challenge , and future drug development should focus on identifying the subgroup of patients who may benet the most from any given cancer therapy . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . open payments is a public database containing information reported by companies about payments made to us - licensed physicians open payments . aziz nazha honoraria : dci consulting or advisory role : karyopharm therapeutics , tolero pharmaceuticals speakers bureau : novartis , incyte research funding : jazz pharmaceuticals mikkael a . 
sekeres consulting or advisory role : celgene , millennium pharmaceuticals , syros pharmaceuticals research funding : takeda pharmaceuticals ( inst ) , pzer ( inst ) rafael bejar honoraria : celgene , alexion pharmaceuticals , abbvie / genentech , astex pharmaceuticals , neogenomics laboratories , daiichi sankyo , forty seven consulting or advisory role : celgene , foundation medicine , neogenomics laboratories , abbvie / genentech , astex pharmaceuticals , daiichi sankyo research funding : celgene , takeda pharmaceuticals travel , accommodations , expenses : celgene megan othus consulting or advisory role : celgene , glycomimetics , cascadia laboratories rami s . 
komrokji stock and other ownership interests : abbvie consulting or advisory role : celgene , novartis , daiichi sankyo , pzer , janssen pharmaceuticals , agios , incyte speakers bureau : novartis , alexion pharmaceuticals , jazz pharmaceuticals travel , accommodations , expenses : celgene , incyte , alexion pharmaceuticals , novartis , jazz pharmaceuticals , daiichi sankyo david p . 
blood 123 : 829 - 836 , 2014 fenaux p , mufti gj , hellstrom - lindberg e , et al : efcacy of azacitidine compared with that of conventional care regimens in the treatment of higher - risk myelodysplastic syndromes : a randomised , open - label , phase iii study . 
lancet oncol 10 : 223 - 232 , 2009 l ubbert m , suciu s , baila l , et al : low - dose decitabine versus best supportive care in elderly patients with intermediateor high - risk myelodysplastic syndrome ( mds ) ineligible for intensive chemotherapy : final results of the randomized phase iii study of the european organisation for research and treatment of cancer leukemia group and the german mds study group . 
j clin oncol 29 : 1987 - 1996 , 2011 steensma dp , baer mr , slack jl , et al : multicenter study of decitabine administered daily for 5 days every 4 weeks to adults with myelodysplastic syndromes : the alternative dosing for outpatient treatment ( adopt ) trial . 
leuk res 41 : 43 - 47 , 2016 zeidan am , sekeres ma , garcia - manero g , et al : comparison of risk stratication tools in predicting outcomes of patients with higher - risk myelodysplastic syndromes treated with azanucleosides . 
leukemia 30 : 649 - 657 , 2016 itzykson r , th epot s , quesnel b , et al : prognostic factors for response and overall survival in 282 patients with higher - risk myelodysplastic syndromes treated with azacitidine . 
blood 117 : 403 - 411 , 2011 takahashi k , patel k , bueso - ramos c , et al : clinical implications of tp53 mutations in myelodysplastic syndromes treated with hypomethylating agents . oncotarget 7 : 14172 - 14187 , 2016 9 . 
sekeres ma , othus m , list af , et al : randomized phase ii study of azacitidine alone or in combination with lenalidomide or with vorinostat in higher - risk myelodysplastic syndromes and chronic myelomonocytic leukemia : north american intergroup study swog s1117 . 
short nj , kantarjian hm , loghavi s , et al : treatment with a 5 - day versus a 10 - day schedule of decitabine in older patients with newly diagnosed acute myeloid leukaemia : a randomised phase 2 trial . 
 significant tumor response to the poly ( adp - ribose ) polymerase inhibitor olaparib in heavily pretreated patient with ovarian carcinosarcoma harboring a germline rad51d mutation introduction brca1 and brca2 are the best - known examples of proteins that contribute to repair of double - strand dna breaks via the homologous recombination repair ( hrr ) pathway . the rad51 paralogs rad51b , rad51c , and rad51d are proteins that are also involved in the hrr pathway . 
parp inhibitors are rationally designed drugs that inhibit parp1 by trapping the inactivated enzyme onto the single - strand break , generating a potential block for cellular dna replication.5 - 7 trapped parp - dna complexes can lead to the stalling and / or collapse of replication forks , resulting in the generation of more deleterious double - strand breaks.8 in cells unable to carry out hrr because of brca mutation , the added genotoxic stress of inhibition of single - strand repair is cytotoxic and referred to as synthetic lethality.5 , 7 in vitro and mice studies support the use of parp inhibition , not only in the presence of brca mutation but also in malignancy with other genetic defects that affect hrr , such as rad51 deletion.9 given the rarity of rad51 mutations , there is limited clinical evidence in this context . we describe a heavily pretreated patient with ovarian carcinosarcoma , with known germline rad51d mutation , that exhibited a remarkable and durable response to parp inhibitor therapy . 
subsequently the patient experienced remissions of varying degrees and durations , with additional lines of therapy including paclitaxel , cisplatin , ifosfamide , and doxorubictwo years before presentation of this case report , the patient was enrolled in a phase i clinical trial using a novel pd - 1 inhibitor . 
 a writing , the patient continues to take olaparib , with no evidence of disease regrowth . discussion synthetic lethality is a term used to describe the combination of two defects that do not affect cell viability when each occurs individually but are cytotoxic when they occur simultaneously in a single cell.7 , 10 parp inhibitors have been used to exploit synthetic lethality in brca1and brca2 - mutant ovarian , breast , prostate , and other cancers . 
 however , hrr deficiency extends beyond cancers harboring brca1 / 2 mutations , and the term brcaness has been coined to describe the clinical and biologic features of such tumors . 
 this not only includes similar histomorphological features such as basal - like phenotype in breast cancers or high - grade serous morphology in ovarian cancers , but also similar immunophenotypic profile ( eg , triple - negative breast cancers ) , drug sensitivity ( eg , to platinum agents and parp inhibitors ) , as well as prognosis.11 - 13 the brcaness phenotype is seen in tumors with loss - of - function mutations involving other hrr pathway genes , including atm , atr , bard1 , brip1 , mre11a , palb2 , rad50 , rad51d , rad54 , nbs1 , chek1 , and chek2 , as well as components of the fanconi anemia repair pathway.13 , 14 in vitro and mice studies have demonstrated efficacy of parp inhibitors against cancer cells with defects in rad51 . 
separate studies by shah et al , cruz et al , and wang et al found that breast or ovarian cancer cells with absent or low levels of rad51 were sensitive to parp inhibitors.15 - 17 another study observed that parp inhibitor sensitivity was increased by 1 , 000 - fold in cells that are rad51 knockdown.14 cruz et al and wang et al additionally demonstrated that the presence of rad51 conferred resistance to parp inhibitors in these cells.16 , 17 in their experiments on triple - negative breast cancer cells , wang et al postulated that it may be possible to expand the sensitivity of triple - negative breast cancer to parp inhibitors by targeting rad51.17 other studies found increased sensitivity to parp inhibition when rad51 inhibitors were coadministered.10 , 18 interestingly , falchi et al found the addition of a brca2 - rad51 disruptor to parp inhibition to be synergistic , rather than merely additive , which is probably a consequence of fig 1 . 
 ( a ) baseline computed tomography ( ct ) scan of pelvis , demonstrating heterogeneous mass ( arrowhead ) , ( b ) ct scan after 8 weeks on olaparib , showing excellent partial response in pelvic mass ( arrowhead ) , and ( c ) ct scan after 16 weeks on olaparib , showing additional disease response ( arrowhead )  . disease response for 16 months , followed by rapid and significant tumor regrowth ( fig 1a )  . the patient had previously been determined to have a germline mutation in rad51d , evidenced by a heterozygous variant c.263 + 1g > a . 
 this mutation has previously been described in ovarian cancer.4 with the possibility of defective hrr in the patients tumor as the result of loss of the remaining functional rad51d allele , a therapeutic trial of olaparib was initiated . 
we are not aware of other case reports that have demonstrated disease response to parp inhibitors in tumors with an underlying rad51d mutation . our clinical experience is consistent with preclinical models and adds to the current limited clinical evidence of benefit of parp inhibition in the context of rad51 mutations . 
chandran no relationship to disclose ian kennedy no relationship to disclose the simultaneous inhibition of two dna - repair mechanisms.10 a 2016 phase ii study ( ariel2 ; a study of rucaparib in patients with platinum - sensitive , relapsed , high - grade epithelial ovarian , fallopian tube , or primary peritoneal cancer ) used the parp inhibitor rucaparib in recurrent , platinum - sensitive , high - grade ovarian carcinoma . 
 of 206 patients , there were seven with rad51 mutations : one patient with rad51b mutation with stable disease , who was receiving rucaparib ; four patients with rad51c mutations ( three partial responses and one patient with stable disease ) ; and two patients with rad51d mutation , both with stable disease.19 using baseline and postprogression biopsy samples from the same study , kondrashova et al reported that in five of six patients with rad51c and rad51d truncation mutations that subsequently developed resistance to rucaparib , there were one or more secondary mutations restoring the open reading frame . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
song h , dicks e , ramus sj , et al : contribution of germline mutations in the rad51b , rad51c , and rad51d genes to ovarian cancer in the population . 
tan dsp , rothermundt c , thomas k , et al : brcaness syndrome in ovarian cancer : a casecontrol study describing the clinical features and outcome of patients with epithelial ovarian cancer associated with brca1 and brca2 mutations . 
mccabe n , turner nc , lord cj , et al : deficiency in the repair of dna damage by homologous recombination and sensitivity to poly ( adp - ribose ) polymerase inhibition . 
shah mm , dobbin zc , nowsheen s , et al : an ex vivo assay of xrt - induced rad51 foci formation predicts response to parp - inhibition in ovarian cancer . 
cruz c , castroviejo - bermejo m , gutirrez - enrquez s , et al : rad51 foci as a functional biomarker of homologous recombination repair and parp inhibitor resistance in germline brca - mutated breast cancer . 
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 r predictive biomarkers in metastatic colorectal cancer : a systematic review juan ruiz - baobre , md1 , 2 , 3 ; raju kandimalla , phd2 ; and ajay goel , phd2 purpose the development and use of predictive biomarkers to guide treatment decisions are paramount not only for improving survival in patients with metastatic colorectal cancer ( mcrc ) , but also for sparing them from unnecessary toxicity and reducing the economic burden of expensive treatments . 
we conducted a systematic review of published studies and evaluated the predictive biomarker landscape in the mcrc setting from a molecular and clinical viewpoint . methods studies analyzing predictive biomarkers for approved therapies in patients with mcrc were identied systematically using electronic databases . 
of all the biomarkers analyzed , 1.4% ( two of 148 ) were explored in a prospective manner , whereas 98.6% ( 146 of 148 ) were evaluated in retrospective studies . 
of the latter group , 78.8% ( 115 of 146 ) were not tested in subsequent phases , 9.6% ( 14 of 146 ) were tested in other retrospective cohorts , 8.9% ( 13 of 146 ) were retrospectively tested in at least one or more randomized cohorts , and only 2.7% ( four of 146 ) were prospectively tested in a clinical trial . 
2019 by american society of clinical oncology introduction colorectal cancer ( crc ) remains the third leading cause of cancer - related deaths in the western world . despite ongoing efforts aimed at increased population screening and improved early detection strategies , approximately 20% of patients still present with metastatic disease at diagnosis , and approximately 35% of those who undergo curative surgeries for a localized disease relpase.1 during the past three decades , the median overall survival ( os ) of patients with metastatic crc ( mcrc ) has gradually increased because of the implementation of combined chemotherapy regimens as well as targeted molecular therapies against egfr and angiogenic factors.2 since the identication of ras mutations as a negative predictive marker , antiegfr therapy has had the greatest impact on the management of patients with mcrc ; nonetheless , the response rates of these treatments remain only approximately 40% to 60% . 
in addition , the recognition landscape for immunotherapy in the treatment patients with microsatellite instability - high ( msi - h ) or dna decient mismatch - repair ( dmmr ) mcrc has been encouraging for this subset of patients . 
crc , colorectal cancer ; mcrc , metastatic colorectal cancer ; neoadjuvant - crt , neoadjuvant chemoradiotherapy . 2 2019 by american society of clinical oncology conventional chemotherapy and triuridine / tipiracil the backbone of treatment in patients with mcrc has historically been chemotherapy , and several chemotherapeutic agents are now approved in this setting : uoropyrimidines ( uorouracil [ fu ] and capecitabine ) , oxaliplatin , irinotecan , and since 2015 , triuridine / tipiracil ( tas - 102 )  . fluoropyrimidine - based chemotherapy and tas - 102 . 
studies of the role of thymidylate synthase ( ts ) in uoropyrimidine - based therapy ( primarily fu plus leucovorin ) in various retrospective and prospective studies have yielded discordant results . 
in this regard , multiple studies have shown that low levels of ts expression in metastatic tumor tissues generally correlate with higher overall response rate ( orr ) .3 - 6 surprisingly , such a correlation was not evident when ts levels were measured in primary tumor tissues.4 , 7 similarly , low levels of ts and dihydropyrimidine dehydrogenase in metastatic tumor tissues were associated with a favorable response to fu in patients with mcrc8 ; however , a subsequent report in 2006 did not validate these ndings.9 likewise , the role of thymidine phosphorylase as a predictive biomarker was also investigated , but the results remain inconclusive.10 , 11 in 2009 , a meta - analysis of ve studies examining a total of 861 patients with mcrc concluded that compared with microsatellite - stable patients , msi - h patients did not achieve a statistically signicant better response rate to fu - based chemotherapy.12 similarly , while investigating the relationship between msh2 gene expression and capecitabine efcacy in patients with mcrc , jensen et al13 observed that a higher msh2 expression was associated with a better response . 
in 2015 , hamauchi et al30 found that patients who develop grade 3 or 4 neutropenia during the rst cycle of tas - 102 treatment had a smaller risk of disease progression . 
selected biomarkers the nal analysis . distribution of biomarkers by individual treatment group . ( b ) distribution of biomarkers by study design . antipd - 1 , antiprogrammed cell death - 1 drugs ; fu , uoropyrimidine - based chemotherapy ; irinotecan , irinotecan - based chemotherapy ; msi , microsatellite instability ; oxaliplatin , oxaliplatin - based chemotherapy ; tas - 102 , triuridine / tipiracil . 
 ( * ) predictive biomarker for anti - egfr drugs ( cetuximab / panitumumab ) ; ( ) predictive biomarker for antipd - 1 drugs ( pembrolizumab and nivolumab alone or in combination with ipilimumab )  . testing in 1 randomized cohorts ( n = 13 ; 8.9% ) ras mutations * testing in a clinical trial ( n = 4 ; 2.7% ) msi status oxaliplatin - based chemotherapy . 
the most notable attempts in this regard have been for the ercc1 gene , and as reported from the mavericc ( marker evaluation for avastin research in crc ) trial , intratumoral ercc1 gene expression failed to predict response to oxaliplatin treatment.17 another gene evaluated in this setting was the x - ray repair cross - complementing group 1 ( xrcc1 ) gene , a base excision repair modulator , wherein a polymorphism in this gene ( xrcc1 - 839 arg / gln or gln / gln ) correlated with worse orr to fu / oxaliplatin.18 interestingly , several micrornas ( mirnas ) have been explored for their predictive response potential to fu , leucovorin , and oxaliplatin ( folfox ) or capecitabine plus oxaliplatin ( capeox ) based regimens . 
using a proteomic approach in 2010 , aoyagi et al32 reported that lower levels of plasma soluble vascular endothelial growth factor ( vegf ) receptor 1 were associated with improved disease control in a subset of 46 patients treated with modied fu , leucovorin , and oxaliplatin ( mfolfox6 ) plus bevacizumab . 
in the same year , another study identied a signicant correlation between low angiopoetin - 2 serum levels and better survival outcomes in a cohort of patients with mcrc treated with bevacizumab - based therapy.33 conversely , the correlation between vegf - a levels and the clinical benet of bevacizumab - based chemotherapy is still under evaluation . 
perhaps in the near future , mavericc trial results will offer more insights into the usefulness of plasma vegf - a levels in this setting.17 with regard to the predictive role of ras , in a subset of 230 patients with mcrc treated in a phase iii randomized clinical trial with either irinotecan , fu , and leucovorin ( ifl ) plus placebo , or ifl plus bevacizumab , only the wild - type kras subset of patients obtained a signicantly higher response rate in the bevacizumab arm.35 in addition to the molecular markers listed previously , a few clinical factors have been studied in relation to bevacizumab response . 
two retrospective studies based on the data from the correct ( patients with metastatic colorectal cancer treated with regorafenib or placebo after failure of standard therapy ) and consigna ( regorafenib in subjects with metastatic colorectal cancer who have progressed after standard therapy ) trials have evaluated the usefulness of different computed tomography scan based parameters in predicting the clinical benet of regorafenib therapy . 
one of the rst studies , in 2016 , reported a correlation between improved pfs and lung metastases cavitation before therapy initiation and even at week 8.38 in this study , baseline lung metastases cavitation and changes in the sum of target lesion diameters were deemed to be predictors for improved os in the multivariate analysis . 
a year later , the correct trial also demonstrated a signicant association between survival ( pfs and os ) and several radiologic parameters such as response or stable disease in size and density of lung metastases.39 likewise , highlighting the use of dynamic contrast - enhanced magnetic resonance imaging , a recent study reported that a  . 
these and other potential predictive biomarkers for anti - egfr therapy are summarized in table 3 and in the data supplement . antiprogrammed cell death - 1 drugs : pembrolizumab and nivolumab in may and july of 2017 , the us food and drug administration approved pembrolizumab and nivolumab for the treatment of patients with msi - h mcrc in whom the disease has progressed after treatment with uoropyrimidine , oxaliplatin , and irinotecan therapies . 
almost a year later , in july 2018 , a nivolumab plus ipilimumab combined regimen was approved , which opened up three novel treatment options for patients with msi - h or dmmr mcrc , who represent approximately 5% of all patients with mcrc.23 the belief is that , despite their worse prognosis , a large prolymphocytic inltration and the presence of portion of mutation - associated neoantigens61 confer to patients with msi - positive mcrc the clinical benet they derive from antiprogrammed cell death - 1 ( pd - 1 ) therapy.62 - 65 this exciting discovery has now led to universal msi testing for the management of patients with mcrc . disease monitoring by liquid biopsies recently , liquid biopsies have emerged as powerful tools for monitoring disease evolution and therapeutic response through the analysis of cell - free dna and rna biomarkers in bodily uids . 
one of the rst studies , in 1979 , which reported that a gradual decrease in carcinoembryonic antigen ( cea ) levels during chemotherapy was signicantly associated with better survival rates , was the basis for this concept.66 such a correlation between cea are and improved pfs and os was conrmed a few years later in a subset of 670 patients with mcrc undergoing rst - line chemotherapy.67 although cea is not a crc - specic biomarker , cea monitoring in blood , alone or in addition to ca 19 - 9 , 68 is still performed commonly in routine clinical practice . 
in this context , the accuracy of cea change in predicting disease progression has been demonstrated recently in a study involving 2 , 828 patients from seven rstline clinical trials.69 in addition to this , other analyses of circulating tumor cells70 and endothelial cells71 in mcrc have been undertaken by several groups . 
in 2015 , hansen et al72 reported that circulating levels of mirna - 126 in a subset of 68 patients with mcrc were predictive of tumor response to bevacizumab - based chemotherapy . 
since then , multiple studies have reported distinct genetic alterations in ctdna from patients with primary disease or acquired resistance to antiegfrbased therapies in genes such as kras , nras , met , erbb2 , flt3 , egfr , and map2k1 by droplet digital polymerase chain reaction , beaming , and next - generation sequencing methodologies.74 , 75 using a massively parallel sequencing - based assay in a prospective cohort of 53 patients with mcrc , it was shown that early changes in ctdna during rst - line standard chemotherapy can also predict subsequent radiologic response.76 similarly , 2017 , a study demonstrated a signicant correlation between the decrease in ras mutant clones in blood after 8 weeks of therapy and improved pfs and os in a cohort of patients treated with regorafenib.40 intriguingly , clonal evolution is a dynamic process , yet the emergence of drugresistant clones in circulation increases during treatment , whereas drug withdrawal results in a decrease of such clones . 
the understanding of this fact has paved a path for novel treatment strategies that are already under evaluation as part of the rasintro ( ras mutations in ctdna and anti - egfr reintroduction in mcrc ) study ( clinicaltrials . gov identier : nct03259009 ) and the chronos ( rechallenge with panitumumab driven by ras dynamic of resistance ) trial ( clinicaltrials.gov identier : nct03227926 ) , which are evaluating the predictive impact of ctdna ras mutations on the efcacy of anti - egfr monotherapy rechallenge in patients with ras wild - type mcrc whose disease has progressed after anti - egfrfree chemotherapy . 
in addition , a ve - gene methylation panel for monitoring tumor burden in liquid biopsies using a methylbeaming assay was described recently77 in 182 patients with mcrc treated with chemotherapy and / or targeted therapy , in which the authors discovered a signicant correlation between the dynamics of methylation markers and orr and pfs . discussion despite the tremendous body of effort devoted to the identication of predictive biomarkers for various treatments used in patients with mcrc , thus far only two of such markers have been translated into routine clinical practice . the rst one , the mutations in the ras gene , serves as a negative predictive biomarker that is present in approximately 55% of patients with mcrc78 and correlates with the lack of efcacy of anti - egfr antibody treatments . 
the exciting result of the association between msi - h and response to nivolumab in the rst - in - human clinical trial ( clinicaltrials . gov identier : nct00441337 ) led to two subsequent phase ii clinical trials , which were instrumental in the approval of antipd - 1 drugs ( pembrolizumab or nivolumab ) alone or in combination with ipilimumab ( nivolumab plus ipilimumab ) as a treatment option for patients with msi - h or dmmr mcrc.63 - 65 , 79 other well - described predictive biomarkers used in the management of several tumor types have shown promising usefulness in selecting patients with mcrc for various targeted therapy - based regimens . 
results from two clinical trials in patients with braf v600epositive mcrc have highlighted this mutation as a predictive biomarker for braf inhibitorbased regimens ( data supplement ) .80 , 81 regarding the role of human epidermal growth factor receptor 2 ( her2 ) amplication or overexpression as a predictive biomarker results of two phase ii clinical trials evaluating the dual her2 blockade in a biomarker - selected subset of heavily pretreated patients with mcrc , with either trastuzumab plus lapatinib ( heracles [ her2 amplication for colorectal cancer enhanced stratication ] trial ) or with pertuzumab and trastuzumab ( mypathway trial ) , demonstrate an impressive orr of approximately 30% to 40% ( data supplement ) .82 , 83 for anti - her2based therapies , nonetheless , the discovery and validation of novel predictive biomarkers that can assist in decision making has been a challenging endeavor , resulting in a long list of failed predictive markers . 
in crc , because the use of single - agent chemotherapeutic regimens has shown limited efcacy , and the majority of current treatment options include various combinations of drugs , biomarker discovery for specic drugs is more complicated , not surprisingly , because of the interactions among different cytotoxic agents.84 similar concerns remain regarding developing predictive biomarkers for therapeutic response to bevacizumab , because ( 1 ) it is also not used as a single agent in the clinic , 84 ( 2 ) its mechanisms of action are poorly understood , 85 and ( 3 ) angiogenesis is an intriguingly adaptive process that involves numerous factors.86 presumably , the inherent complexity of angiogenesis has been a substantial hurdle in the attempts to develop responsepredictive biomarkers for other multitargeted antiangiogenic drugs such as aibercept or regorafenib . 
 predictive biomarkers in metastatic colorectal cancer a possible source of discrepancy even when the molecular marker is analyzed in a different region of the same source.88 besides their spatial heterogeneity , tumors are dynamic entities that continue to evolve over time , especially if they are under selective pressure.89 for this reason , the time from sample acquisition to biomarker analysis is of clinical relevance ; however , overlooked in most studies . 
because only approximately 20% of patients with crc present with metastatic disease at the time of diagnosis , it is often the practice or only option available to analyze archival tissues from the primary tumor to identify biomarkers , which is not always optimal.90 this is an issue that patient selection is gaining importance , which is evidenced by the recent initiative , the us national cancer institutes exceptional responder program.91 consideration of extreme phenotypes such as long - term responders and extremely early progressors for biomarker discovery can facilitate successful identication of molecular alterations that better correlate with clinical phenotypes . 
for instance , in the majority of studies presented in this article , there was no consideration of pfs as a selection criterion , and many studies included in the nonresponders patients with stable disease . 
in general , improved orr and longer pfs are superior indicators of the true efcacy of any drug intervention , whereas inclusion of gain in os as a selection feature may inadvertently introduce bias . 
in addition , new biomarker - driven study designs such as basket or umbrella trials , which assign a treatment according to tumor molecular characteristics , not only are going to improve clinical drug development , but also will facilitate improved biomarker validation . 
besides the examples already described previously in the text , new drugs that target tyrosine kinase fusions in genes such as nrtk1 / 2 / 3 , ret , alk , and ros1 are emerging with promising preliminary results in phase i and ii clinical trials that include patients with crc ( data supplement ) .92 - 97 although analysis of clinical specimens with robust followup data from retrospective series or randomized trials are of tremendous value , subsequent prospective clinical cohorts using longitudinally collected specimens are much needed to establish clinically translatable predictive biomarkers . 
in addition , although many surgical specimens are of suitable quality , needle biopsyderived metastatic lesions often yield lower amounts of dna and rna than that required for robust sequencing experiments98 , 99 ; having access to liquid biopsybased predictive markers would be transformative in overcoming this limitation in patients with mcrc . 
furthermore , liquid biopsy biomarkers will improve patient compliance and eliminate the concerns surrounding intratumor heterogeneity associated with tumor or biopsy specimens and may also help in disease monitoring as well as in predicting secondary resistance . the international community has to consolidate initiatives to improve biomarker development studies and , more importantly , undertake conscious efforts to validate the results gathered from retrospective studies in prospective randomized multicenter cohorts . 
last , the implementation of novel high - throughput molecular analytic techniques and the integration of multiomic approaches with clinical and epidemiologic data using machine - learning algorithms will denitely hasten biomarker development in the coming years.100 despite many attempts over the past decades , there remain only two well - established predictive biomarkers , mutations in the ras gene and msi status , that currently guide treatment decisions in patients with mcrc . 
 ruiz - baobre , kandimalla , and goel juan ruiz - baobre travel , accommodations , expenses : bristol - myers squibb , merck sharp & dohme , ipsen , pharmamar no other potential conicts of interest were reported . references siegel rl , miller kd , jemal a : cancer statistics , 2018 . 
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cancer discov 7 : 400 - 409 , 2017 amatu a , somaschini a , cerea g , et al : novel cad - alk gene rearrangement is drugable by entrectinib in colorectal cancer . 
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j natl cancer inst 109 ( 12 ) : djx089 , 2017 kris mg , johnson be , berry ld , et al : using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs . 
 clinicopathologic features of non small - cell lung cancer harboring an ntrk gene fusion purpose gene rearrangements that involve ntrk1 / 2 / 3 can generate fusion oncoproteins that contain the kinase domains of trka / b / c , respectively . 
despite the potential benefit of identifying these fusions , the clinicopathologic features of ntrk fusion - positive nsclcs are not well characterized . methods we compiled a database of patients with nsclcs that harbor ntrk fusions . 
 we characterized clinical , molecular , and histologic features with central review of histology . results we identified 11 patients with nsclcs that harbor ntrk gene fusions verified by next - generation sequencing and with available clinical and pathologic data . 
nine patients had adenocarcinoma , including two with invasive mucinous adenocarcinoma and one with adenocarcinoma with neuroendocrine features ; one had squamous cell carcinoma ; and one had neuroendocrine carcinoma . 
2018 by american society of clinical oncology introduction the neurotrophin kinase ( ntrk ) genes ntrk1 , ntrk2 , and ntrk3 encode the tropomyosin receptor tyrosine kinases trka , trkb , and trkc , respectively , which function during normal neuronal development and maintenance . 
of patients male female smoking history , pack - years median pack - years of smoking ( range ) stage at diagnosis ( ajcc seventh edition , criteria ) 5 - 20 > 20 bone lung liver brain histology ( local assessment ) adenocarcinoma squamous cell carcinoma neuroendocrine carcinoma no . 
 ( % ) 48 ( 25 - 86 ) 6 ( 55 ) 5 ( 45 ) 8 ( 73 ) 3 ( 27 ) 0 ( 0 - 58 ) 2 ( 18 ) 1 ( 9 ) 8 ( 73 ) 9 ( 82 ) 1 ( 9 ) 1 ( 9 ) trk signaling in preclinical models results in cell death and tumor regression.4 in early - phase clinical trials , solid tumors that harbor ntrk gene rearrangements have been highly sensitive to selective trk tyrosine kinase inhibitors ( tkis ) , including larotrectinib , which is selective for trka / b / c , and entrectinib , which targets trka / b / c as well as alk and ros1 . 
among four patients with nsclc , response evaluation criteria in solid tumors ( recist ) responses were seen in three , and the fourth had an approximately 20% reduction in tumor size . 
one of four ntrk - rearranged tumors treated with entrectinib in an adult phase i trial was nsclc , and this patient had a partial response , including a complete response in the cns.9 despite the potent activity of trk inhibitors , the clinical and pathologic features of ntrk - rearranged nsclcs are poorly defined . 
 we show that ntrk fusions occur across age and smoking status and suggest that all patients with nsclc be screened for fusions by using a multiplexed next - generation sequencing ( ngs ) assay . methods physicians across seven institutions contributed de - identified patients with nsclc to an ntrk fusion nsclc database . 
assays used identified fusions through a variety of technologies : rna - based fusion - targeted anchored multiplex polymerase chain reaction ( pcr ) and illumina ( san diego , ca ) sequencing10 ( massachusetts general hospital [ mgh ] solid fusion assay , memorial sloan kettering [ msk ] solid fusion assay , archerdx fusionplex performed at caris life sciences [ irving , tx ] ) ; dna hybridization capture with intron tiling and illumina sequencing ( foundationone11 [ foundation medicine , cambridge , ma ] , msk - integrated mutation profiling of actionable cancer targets [ impact ] 12 , 13 ) , total nucleic acid extraction , pcr amplification , and ion torrent sequencing ( pcdx14 ; paradigm , phoenix , az )  . the kaplan - meier method was used to obtain estimates of overall survival from diagnosis of stage iv disease to death or last follow - up ( censored )  . 
data collection and analysis were performed under institutional review boardapproved protocols . results we reached out to physicians at 47 institutions in the united states who were actively participating in a trk inhibitor clinical trial that enrolled adult patients and invited them to contribute data on living or deceased patients with nsclcs that harbored an ntrk gene rearrangement . 
candidate fusions initially were identified by using a combination of rnaand dna - based ngs assays , with validation by one or more of rna - based ngs , fluorescent in situ hybridization , and reverse transcription pcr on a patient - by - patient basis . 
 among these patients , in - frame trk fusions that contained the kinase domain were verified in 11 , which formed the study cohort . of note , three patients were excluded from the study cohort for the following reasons . 
note that exons are drawn at a larger scale than introns and that introns are not drawn to the same scale for each gene ( ntrk1 locus is approximately 21 kilobases [ kb ] , ntrk2 is approximately 358 kb , and ntrk3 is approximately 384 kb )  . 
this group includes patients 1 to 4 , 11 , and s1 to s6 . abbreviations : mgh , massachusetts general hospital ; mskcc , memorial sloan kettering cancer center ; nsclc , nonsmall - cell lung cancer . fusion assay , an rna - based fusion - specific targeted ngs assay that uses anchored multiplex pcr.10 the candidate fusion contained p2ry8 exon 2 fused with ntrk1 exons 1 to 5 . 
the second patient had an ntrk2 intragenic deletion that disrupted the exon 18 3 splice site , which is predicted to disrupt the kinase domain and , therefore , to be inactivating . 
this patient also had a concurrent her2 l755p mutation , which is predicted to be activating.15 the clinical characteristics of the 11 patients in the study cohort are listed in table 1 . 
patient 4 had a candidate ntrk1 fusion detected by msk - impact with an equivocal partner , and the correct fusion partner was determined using the msk solid fusion assay . 
all ntrk fusions couple the kinase domain of ntrk1 or ntrk3 ( with or without the membrane - spanning helix ) to an n - terminal gene fusion partner with domains known or predicted to mediate dimerization or oligomerization ( table 2 ; fig 1 )  . 
in all patients tested , potential oncogenic alterations in the following genes , when interrogated , were not detected : kras ( zero of 10 ) , egfr ( zero of 11 ) , alk ( zero of 11 ) , ros1 ( zero of 11 ) , braf ( zero of 11 ) , pik3ca ( zero of 10 ) , her2 ( zero of eight ) , and met ( zero of eight )  . to estimate the overall frequency of ntrk fusions in nsclc , we reviewed consecutively tested patients with nsclc from mgh and the msk cancer center , where ngs screening of 4 , 872 unique patients identified 11 ntrk fusions ( 0.23% ; table 3 )  . 
five of these patients had available clinical and pathologic data for inclusion in the study cohort , and we report the molecular details of the additional six patients ( s1 to s6 ; appendix table a1 )  . 
we diagram the fusion positions of all 17 of these patients ( study cohort patients 1 to 11 plus patients s1 to s6 ) in figure 1 . we next examined the histologic features of the 11 patients who formed the study cohort . 
among the patients with adenocarcinoma , we observed a range of histologic subtypes , including adenocarcinoma with neuroendocrine features ( patient 2 ; fig 2a ) , poorly differentiated adenocarcinoma with solid pattern and signet ring cells ( patient 1 ; fig 2b ) , and invasive mucinous adenocarcinoma ( patients 4 and 6 ; fig 2c )  . 
 ( a ) patient 2 had an adenocarcinoma with solid growth pattern , diffuse neuroendocrine differentiation , and signet ring cells ; inset shows high magnification ( x400 )  . 
 ( e ) patient 11 had neuroendocrine carcinoma with well - differentiated morphology and increased mitotic activity ( left ) ; high magnification ( x400 ) shown in middle . ttf1 chromogranin mucicarmine ttf1 ttf1 synaptophysin absence of ttf1 ( patient 8 ; fig 2d )  . 
patient 11 ( fig 2e ) had a morphologically well - differentiated neuroendocrine tumor ( equivalent to atypical carcinoid ) with an increased mitotic index of 12 per 10 high - power fields and a brain metastasis ; this tumor was classified as large - cell neuroendocrine carcinoma in accordance with current who criteria.16 although analysis of the cohort is limited by size and the fact that this review is retrospective across multiple institutions , we sought to describe clinical outcomes in these patients . 
the median overall survival of the 10 patients with metastatic disease was 40.8 months ( 95% ci , 0.79 months to not reported ) , with a median follow - up of 52.8 months ( fig 3 )  . three patients had early - stage disease at the time of diagnosis . 
patient 4 had stage iib ( t3n0 ) disease at diagnosis , was treated with surgery followed by adjuvant cisplatin and pemetrexed , and remained recurrence free at the most recent follow - up 30.0 months after initial diagnosis . 
patient 6 had stage iia ( t1bn1 ) disease at diagnosis , was treated with surgery followed by cisplatin and pemetrexed , and developed metastatic disease 24.5 months after initial diagnosis . 
we describe the clinicopathologic features of a cohort of 11 patients with nsclcs harboring ntrk gene rearrangements that resulted in the fusion of the trk tyrosine kinase domain with a dimerization - inducing partner . 
these are predicted or previously have been reported to be activating.4 , 17 the current cohort includes both men and women across a range of ages , histologies , and smoking histories . 
 although the cohort is small , the only defining pattern of clinical characteristics that emerges is the lack of an alternate canonical driver mutation in all patients , similar to others with kinase fusion - positive nsclcs.18 of note , ntrk rearrangements were identified in patients with and without a history of smoking ; although the majority of patients ( eight [ 73% ] of 11 ) had a minimal to never smoking history , three of the 11 had a history of 30 pack - years . 
similarly , alk - , ros1 - , and ret - driven nsclcs are enriched in never - smokers but can be seen in current and former smokers as well.5 , 19 , 20 nine of the 11 patients had adenocarcinoma that tended to be mucinous or poorly differentiated , including one with a tpr - ntrk1 fusion with fig 3 . 
however , other histologies also were observed , including one squamous cell carcinoma with an etv6 - ntrk3 fusion and one neuroendocrine carcinoma with an sqstm1 - ntrk3 fusion . ascertainment bias as a result of selective testing has historically limited an accurate assessment of frequency of ntrk fusions in nsclc . 
 these assays generally are used at the time of tissue diagnosis in both institutions ; therefore , this population likely represents a previously unscreened group in which patients were not already selected to be negative for other known driver mutations in lung cancer . 
we note that cancers selected for molecular testing may be enriched for patients with metastatic disease because no established role exists for targeted therapies in early - stage lung cancer to date . 
 although ntrk fusions are rare in lung cancer , we estimate that with approximately 234 , 000 new nsclc diagnoses annually in the united states , > 500 of these patients may be candidates for highly effective trk inhibitor therapy . 
 significantly more patients with ntrk fusion nsclc may exist when considering the global incidence of lung cancer . the natural history of ntrk fusion nsclcs , compared with nsclcs in general , is not well established . 
although we observed a median overall survival of 40.8 months among the 10 patients with metastatic disease , we acknowledge the small size of this retrospective cohort , among whom eight received at least one trk tki . 
the observation that one of two patients diagnosed at stage ii and one at stage iii developed relapsed metastatic disease is consistent with the natural history of nsclcs in general , although selection bias may have existed against screening patients with early - stage cancer who did not develop metastatic disease . because there seems to be no uniform defining clinical or pathologic feature of ntrk fusion positive nsclcs , we recommend screening all nsclcs for ntrk gene rearrangements . 
 advantages over dna - based methods , including high sensitivity , confident identification of breakpoints and in - frame fusions , and deeper coverage.10 three patients with predicted nonfunctional ntrk alterations also were identified in this study , which emphasizes the added value of ngs - based sequencing and attention to the breakpoints . 
although immunohistochemical assays for the detection of trk expression are in development , 21 allocation of an unstained slide for trk immunohistochemistry may be impractical given the need to test for a wide range of molecular alterations on often - limited tissue samples . 
similarly , given the seeming lack of concurrent canonical driver mutations in these patients , consideration of an initial dna - based ngs for mutational profiling may be reasonable , with reflex multiplexed fusion - targeted rna - based ngs in tumors that lack such a driver . 
however , sequential testing for possible gene alterations can delay the ultimate molecular diagnosis , may be problematic for small samples , and relies on mutual exclusivity of a kinase fusion and oncogenic driver mutation . 
therefore , we favor concurrent ngs - based mutational analysis with multiplexed ngs - based targeted rna sequencing for the identification of gene fusions in nsclc rather than sequential mutation testing or immunohistochemistry , which consumes more time and tissue . 
ou , mari mino - kenudson research funding : pharmamar ( inst ) , abbvie ( inst ) , bristol - myers squibb ( inst ) , merck ( inst ) , loxo oncology ( inst ) , ignyta ( inst ) travel , accommodations , expenses : pharmamar , abbvie , stemcentrx martin s . 
doebele stock and other ownership interests : rain therapeutics honoraria : pfizer , astrazeneca , ariad pharmaceuticals , guardant health , takeda pharmaceuticals , spectrum pharmaceuticals , trovagene consulting or advisory role : pfizer , oncomed pharmaceuticals , trovagene , ignyta , greenpeptide , astrazeneca patents , royalties , other intellectual property : licensing fees from abbott molecular for patent pct / us2013 / 057495 , licensing fees from ignyta for biologic materials ( inst ) travel , accommodations , expenses : ignyta , ariad pharmaceuticals , guardant health provision of study materials or patients : anna f . 
le stock and other ownership interests : archerdx speakers bureau : astrazeneca , roche , genentech , takeda pharmaceuticals travel , accommodations , expenses : astrazeneca , roche , genentech , takeda pharmaceuticals , tp therapeutics shivaani kummar consulting or advisory role : corvus pharmaceuticals , medtree ( i ) , nodus therapeutics research funding : bristol - myers squibb ( inst ) , dynavax technologies ( inst ) , pfizer ( inst ) , loxo oncology ( inst ) , corvus pharmaceuticals ( inst ) , plexxikon ( inst ) , jounce therapeutics ( inst ) , adc therapeutics ( inst ) , advenchen laboratories ( inst ) alexander i . 
spira research funding : roche ( inst ) , astrazeneca ( inst ) , boehringer ingelheim ( inst ) , astellas pharma ( inst ) , medimmune ( inst ) , novartis ( inst ) , newlink genetics ( inst ) , incyte ( inst ) , abbvie ( inst ) , ignyta ( inst ) , lam therapeutics ( inst ) , trovagene ( inst ) , takeda pharmaceuticals ( inst ) , macrogenics ( inst ) , cytomx therapeutics ( inst ) theresa a . 
haura consulting or advisory role : bristol - myers squibb , janssen pharmaceuticals research funding : eli lilly , ignyta , janssen pharmaceuticals , boehringer ingelheim , forma therapeutics patents , royalties , other intellectual property : patent pending on proximity ligation assay technology related to kinase inhibitor sensitivity biomarkers travel , accommodations , expenses : bristol - myers squibb , roche maria e . 
aisner honoraria : astrazeneca , clovis oncology consulting or advisory role : inivata , abbvie , bristolmyers squibb research funding : genentech ( inst ) , roche ( inst ) patents , royalties , other intellectual property : patent pending for pneumatic cell collection device travel , accommodations , expenses : seracare life sciences nora k . 
horick no relationship to disclose consulting or advisory role : archerdx patents , royalties , other intellectual property : coinventor of the anchored multiplex pcr technology , which is licensed to archerdx and receive royalty payments for this patent travel , accommodations , expenses : archerdx a . 
john iafrate stock and other ownership interests : archerdx consulting or advisory role : debiopharm group , chugai pharmaceutical , roche research funding : blueprint medicines patents , royalties , other intellectual property : archerdx exclusive license to amp technology sai - hong i . 
ou stock and other ownership interests : tp therapeutics honoraria : pfizer , roche , genentech , ariad pharmaceuticals , takeda pharmaceuticals , novartis , astrazeneca , foundation medicine consulting or advisory role : pfizer , roche , genentech , novartis , astrazeneca , takeda pharmaceuticals , foundation medicine , tp therapeutics , ignyta speakers bureau : genentech , astrazeneca , takeda pharmaceuticals research funding : pfizer ( inst ) , roche ( inst ) , astrazeneca ( inst ) , medimmune ( inst ) , clovis oncology ( inst ) , ariad pharmaceuticals ( inst ) , ignyta ( inst ) , peregrine pharmaceuticals ( inst ) , glaxosmithkline ( inst ) , astellas pharma ( inst ) , chugai pharmaceutical ( inst ) alice t . 
shaw at , yeap by , mino - kenudson m , et al : clinical features and outcome of patients with nonsmall - cell lung cancer who harbor eml4 - alk . 
drilon a , siena s , ou si , et al : safety and antitumor activity of the multitargeted pan - trk , ros1 , and alk inhibitor entrectinib : combined results from two phase i trials ( alka - 372 - 001 and startrk - 1 )  . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
weiss gj , hoff br , whitehead rp , et al : evaluation and comparison of two commercially available targeted next - generation sequencing platforms to assist oncology decision making . 
rekhtman n , pietanza mc , hellmann md , et al : next - generation sequencing of pulmonary large cell neuroendocrine carcinoma reveals small cell carcinoma - like and non - small cell carcinoma - like subsets . 
ardini e , bosotti r , borgia al , et al : the tpm3 - ntrk1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to trka kinase inhibition . 
gainor jf , varghese am , ou sh , et al : alk rearrangements are mutually exclusive with mutations in egfr or kras : an analysis of 1 , 683 patients with non - small cell lung cancer . 
gautschi o , milia j , filleron t , et al : targeting ret in patients with ret - rearranged lung cancers : results from the global , multicenter ret registry . 
hyman dm , laetsch tw , kummar s , et al : the efficacy of larotrectinib ( loxo - 101 ) , a selective tropomyosin receptor kinase ( trk ) inhibitor , in adult and pediatric trk fusion cancers . 
 therapy - related erythroleukemia in a man with metastatic ewing sarcoma : a clinical role for advanced molecular diagnostics introduction therapy - related leukemia may be challenging to diagnose when a patient has cytopenia during treatment of another malignancy , particularly when there are few circulating leukemia cells . 
 here , we report a case of a 55 - year - old man with refractory ewing sarcoma who presented with thrombocytopenia and tumor lysis syndrome ( tls ) ; he was found to have pure erythroid leukemia ( pel ) , likely resulting from prior alkylating agent and / or topoisomerase ii inhibitor chemotherapy . 
furthermore , when patients with metastatic solid tumor have cytopenia , they may not undergo bone marrow ( bm ) evaluation if the cytopenia has already been attributed to therapies or the prior malignancy . in this case , initial diagnostics and the poorly differentiated morphology seen in the bm biopsy specimen did not readily distinguish between ewing sarcoma and acute leukemia . 
he was diagnosed with ewing sarcoma in 2011 , after a known right - side lower - lobe lung mass demonstrated increased size and malignant radiologic features on computed tomography ( ct ) imaging . 
the patient underwent right - side lower lobectomy and metastasectomy of bilateral pectoral lesions , radiation ( xrt ) to his rib , and additional chemotherapy with dactinomycin , vincristine , and cyclophosphamide , followed by xrt to his pectoral surgical bed . 
he started a trial of olaparib and temozolomide in august 2015 , which continued until october 2016 , when he was removed from protocol after ct imaging showed disease progression . 
cumulatively , he received 31 cycles of chemotherapy and four treatments of xrt . one month after stopping therapy , the patient presented with fatigue , dyspnea , fevers , bruising , and petechial rash , with no hepatosplenomegaly . 
peripheral smear revealed left - shifted myeloid cells and large basophilic cells with vacuolated cytoplasm ; these cells constituted 9% of blood leukocytes and were interpreted as either reactive atypical lymphocytes , circulating ewing sarcoma cells , or circulating blasts . 
zmynd8 exons 1 through 22 fuse with rela exons 3 through 11 to form the fusion gene zmynd8 - rela , which has been described in erythroleukemia.2 zmynd8 exons 1 - 22 zmynd8 - rela fusion chromosome 20 exons 3 - 11 rela chromosome 11 cbc counts at the ewing sarcoma diagnosis in 2011 were unavailable , cbc counts in 2013 were normal ( wbc , 4.9 103 / l ; hemoglobin , 14.0 g / dl ; platelets , 207 103 / l )  . he underwent bm biopsy and aspiration , and began treatment of tls with rasburicase , allopurinol , and normal saline . 
however , treatment was complicated by persistent cytopenia and hallucinations , likely related to ifosfamide . the bm biopsy specimen was hypercellular and revealed replacement by primitive , discohesive cells , which , by flow cytometry , were negative for cd45 , cd34 , myeloperoxidase , cd13 , and cd56 . 
by immunohistochemistry , cells were negative for cytokeratin , cd43 , and cd56 , but were positive for cd99 ( present in up to 98% of ewing sarcoma but also expressed in other cancers , including leukemia ) .3 - 6 preliminary bm karyotype identified an 11q13 abnormality , but no ewsr1 rearrangement . 
interphase fluorescent in situ hybridization study also did not reveal ewsr1 rearrangement . molecular testing was performed via heme snapshot assay , an institutional ngs platform using illumina truseq amplicon technology ( san diego , ca ) for single nucleotide variant and insertion / deletion detection in genomic dna.7 snapshot results are descriptive and cannot determine clonality , tumor - stromal percentage , or percentage of cells carrying mutations . 
 analysis was undertaken using a sequencing library generated with an oligo pool focusing on approximately 141 kb of genomic content consisting of 568 amplicons covering 15 full genes and exonic hotspots in 39 genes ( trusight myeloid sequencing panel ; illumina )  . testing identified three tp53 mutations with the following allelic frequencies : r337p , 38% ; i255f , 38% ; and y220c , 10% . 
these findings confirmed diagnosis of therapy - related pel , rather than progressive ewing sarcoma . the patient transiently improved after ifosfamide / etoposide treatment , but the leukemia ultimately progressed , at which point he opted for comfort care . 
he died 2 months after pel diagnosis . methods this anchored multiplex fusion assay is an ngs method that amplifies small amounts of nucleic acid and detects fusions without targeting specific rearrangements . 
two heme - nested pcr reactions are then performed to create a fully functional sequencing library targeting specific genes and exons ( fusionplex solid tumor kit ; archerdx , boulder , co )  . 
illumina misequation 2 147 base pairend sequencing results are aligned to the hg19 human genome reference , and a laboratory - developed algorithm is used for fusion transcript detection and annotation . 
the resulting product can then be processed for illumina or ion torrent ( thermo fisher scientific , waltham , ma ) sequencing.10 discussion this case highlights how the evolving field of molecular genetic diagnostics can help distinguish specific cancer diagnoses . 
advanced molecular testing using ngs and an rna - based solid fusion assay ultimately clarified this patients diagnosis . ewing sarcoma describes a family of cancers with fusion oncogenes involving the ewsr1 gene.13 there is a high correlation between bony metastases and bm involvement , and , to our knowledge , pel has not been described in refractory ewing sarcoma before this case report.14 - 17 therapy - related leukemia is a rare adverse effect of alkylating agents , radiotherapy , and topoisomerase ii inhibitors . 
tumor cells were positive for e - cadherin and cd71 ( not shown )  . erythroid precursors with 30% proerythroblasts.22 patients typically present with sequelae of pancytopenia from erythroid precursor proliferation . pel also has a very complex karyotype compared with other leukemias and can be difficult to diagnose , given its negativity for other markers typically expressed in aml , such as cd45 , cd13 , myeloperoxidase , and cd34.20 , 22 , 23 cd45 is commonly used to screen for hematologic neoplasms on initial pathologic staining . 
 ( megakaryoblastic anemia is another cd45 - negative leukemia . ) e - cadherin , expressed in most pel cases , helped lead us toward the correct diagnosis , as did demonstration of the rare zmynd8 - rela fusion . 
the latter is hypothesized to promote oncogenesis via constitutive nf - b activation.1 by definition , aml after exposure to cytotoxic chemotherapy or radiation is therapy - related aml , of which there are two primary etiologic subtypes : alkylating agent / radiotherapyrelated type and topoisomerase ii inhibitorrelated type.24 , 25 alkylating agents and radiotherapy exhibit dose - dependent risk of disease effected via centromeric breakage and partial or complete chromosome loss , particularly at chromosomes 5 and 7 . 
the leukemia had several balanced translocations , including t ( 11 ; 20 ) ( q12 ; q13.1 ) , and a partial deletion of chromosome 4 . mutations in tp53 , as seen in this case , are common in t - aml . 
interestingly , recent data suggest that tp53 - mutated clones may exist before the exposure to chemotherapy and expand under the selective pressure of treatment of another malignancy.27 this is the first reported case of greater than two tp53 mutations in t - aml.9 , 27 regardless of the inciting agents , t - aml is associated with dismal outcomes , such as were experienced by this patient . in patients with advanced cancers treated with alkylating agents , radiotherapy , or topoisomerase ii inhibitors , t - aml should be considered as a cause of refractory cytopenia . 
angelini df , ottone t , guerrera g , et al : a leukemia - associated cd34 / cd123 / cd25 / cd99 + immunophenotype identifies flt3 - mutated clones in acute myeloid leukemia . 
dias - santagata d , akhavanfard s , david ss , et al : rapid targeted mutational analysis of human tumours : a clinical platform to guide personalized cancer medicine . 
ok cy , patel kp , garcia - manero g , et al : tp53 mutation characteristics in therapy - related myelodysplastic syndromes and acute myeloid leukemia is similar to de novo diseases . 
green dc , deharvengt sj , de abreu fb , et al : use of anchored multiplex pcr enrichment for detection of gene fusions in solid tumors by next generation sequencing . 
thiel u , wawer a , von luettichau i , et al : bone marrow involvement identifies a subgroup of advanced ewing sarcoma patients with fatal outcome irrespective of therapy in contrast to curable patients with multiple bone metastases but unaffected marrow . 
cuneo a , van orshoven a , michaux jl , et al : morphologic , immunologic and cytogenetic studies in erythroleukaemia : evidence for multilineage involvement and identification of two distinct cytogenetic - clinicopathological types . 
kasyan a , medeiros lj , zuo z , et al : acute erythroid leukemia as defined in the world health organization classification is a rare and pathogenetically heterogeneous disease . 
olney hj , mitelman f , johansson b , et al : unique balanced chromosome abnormalities in treatment - related myelodysplastic syndromes and acute myeloid leukemia : report from an international workshop . 
mcgowan , phd1 , 2 purpose although analyzing germline and tumor samples concurrently provides the best opportunity for differentiating between germline and somatic mutations , tumor - only sequencing is becoming increasingly common in clinical care . 
with limited professional society guidance on how to manage pathogenic mutations identied via tumor - only sequencing , this study contemplates the professional knowledge and skills necessary to have represented on an mgtb to interpret these results in context of potential germline ndings . methods qualitative interviews with mgtb members and an ethnographic case study of a breast cancer mgtb at a national cancer institute cancer center were examined . results this mgtb discussed 34 cases of women with advanced - stage breast cancer over 13 months . interviews and observations of mgtb meetings indicated that members of the mgtb contemplated whether variants were germline or somatic and potential for identication of germline cancer predisposition . 
however , the mgtb only referred 11 patients ( 61% ) for additional germline testing , and the remaining seven patients ( 39% ) were not referred , raising questions about the kind of genomic expertise needed on an mgtb to optimize results interpretation and referrals . conclusion to ensure adequate interpretation , recommendation , and communication of tumor sequencing results , an mgtb should include professionals with knowledge and experience in clinical translation of tumor sequencing , testing methodology , molecular pathology , cancer biology , genomic pathways , germline variant interpretation , evaluation of family history , and application of professional recommendations for germline testing after tumor - only sequencing . 
 fishler et al context key objective : how are tumor - only sequencing results interpreted by a multidisciplinary genomic tumor board ( mgtb ) ? which professional expertise ought to be represented when discussing these results and recommendations ? knowledge generated : commercial laboratories provision of tumor - only sequencing without germline reference samples can complicate the ability of an mgtb to interpret patients genomic sequencing results and make recommendations for targeted treatment and referrals for additional genetic testing . 
providers on an mgtb questioned whether a known or likely pathogenic tumor sequencing variant was germline or somatic , sometimes overlooked known / likely pathogenic mutations in cancer susceptibility genes other than brca1 / 2 , and recognized the necessity of varied professional expertise when interpreting tumor - only sequencing results . relevance : to ensure appropriate interpretation of tumor - only sequencing and identication of patients who ought to be referred to genetics , an mgtb should include individuals with expertise in clinical translation of tumor sequencing , testing methodology , molecular pathology , cancer biology and genomic pathways , germline variant interpretation , evaluation of personal and family history , and application of professional guidelines for germline testing . 
to mitigate provider deliberation and genomic knowledge necessary to determine if additional germline testing is necessary , genetics professional societies and clinical laboratories ought to consider whether tumor - only sequencing should be routinely paired with germline samples to facilitate standardized reporting of germline variants . the basis of tumor - only sequencing results , even when combined with their family history . a previously published study from a breast cancer mgtb revealed that decisions about which patients received genetics referrals were not standardized.10 this prior analysis did not explore the factors the mgtb used to determine which patients received genetics referrals after tumor - only sequencing . 
our study aimed to identify these factors and to explore which professionals ought to be involved on an mgtb to ensure adequate interpretation , clinical application , and referral for additional germline testing when appropriate . each interview ranged in length from 25 to 49 minutes . 
all mgtb members signed a written informed consent , which allowed for recording , transcription , and analysis of the meeting minutes and interview data . data were collected using standard qualitative research including ethnography and in - depth inmethods , interviews and mgtb meetings were audio terviews . recorded and transcribed verbatim by a medical transcriptionist . 
participants in this study included 22 members of a breast cancer mgtb at one urban national cancer institute cancer center . methods included an ethnographic case study of mgtb meetings and interviews with mgtb members . 
this group met monthly from october 2013 to november 2014 to discuss results of tumor - only sequencing ordered for patients with advanced - stage breast cancer in a clinical setting . mgtb members were invited to participate in a semistructured interview regarding their opinions about a tumor board approach to interpreting tumor sequencing results . in total , 12 members of the board were interviewed by the research team , including treating physicians ( n = 6 ) , bioinformaticians ( n = 3 ) , a pathologist ( n = 1 ) , a medical geneticist ( n = 1 ) , and an administrator ( n = 1 ) .10 all patients discussed by this mgtb were women with advanced - stage breast cancer and tumor - only sequencing performed as part of their clinical care ( n = 34 )  . 
the mgtb discussed all of the hospitals patients with breast cancer who underwent tumor sequencing during the study period . all patient samples were sent to the same commercial laboratory for sequencing and analysis . the laboratory - generated tumor sequencing report suggested on - label / off - label treatments and clinical trial options on the basis of known / likely pathogenic mutations . pathogenic and likely pathogenic results were listed separately from vus results . 
as published in the preliminary analysis , mgtb recommendations included genetics referrals for suspected germline variants , clinical trial recommendations , us food and drug administrationapproved on - label therapies , androgen receptor testing , and repeat human epidermal growth factor receptor 2 testing . 
 underlying germline variants in tumor - only sequencing leaderships commitment to adherence with evidencebased medicine and bioethical principles of benecence and nonmalecence.10 of the 34 patients tumor sequencing results that the mgtb reviewed during the study period , 18 patients met nccn criteria for additional germline testing on the basis of their personal history , including type of breast cancer , age of onset , and / or the identication of a known / likely pathogenic tumor mutation in a cancer susceptibility gene . 
the remaining seven patients ( 39% ) represent missed opportunities for additional germline testing on the basis of the patients personal history , prior germline testing , and / or tumor - only sequencing result ( table 2 )  . 
five of these patients were missed on the basis of a combination of these factors ; two were missed on the basis of personal history alone . through analysis of mgtb member interview transcripts and mgtb meetings , three themes emerged : provider confusion about whether a variant identied by tumoronly sequencing was germline or somatic , potential for tumor - sequencing identication of germline cancer predisposition , and genetics expertise necessary for inclusion on an mgtb . provider confusion about whether a mutation was germline or somatic one key question raised by mgtb members was whether the variants listed on tumor sequencing reports were germline or somatic . 
on the basis of the way mgtb members managed the referral process , it became apparent that they disagreed about their role as evaluators of tumor - only sequencing results , including determining whether follow - up with additional germline testing was necessary . 
for example , one medical oncologist mentioned that all mutations relevant treatment were listed on the rst page of the patients report and that if we dont have a lot of time , we may not ip through the rest to see what else might be listed there . in contrast , another oncologist advocated for a more active interpretation : we cant role in report take what [ commercial vendor ] recommends as gospel truth and say okay , this is what were going to go with , because they recommended itthe interpretation needs to happen from our end . just result in addition to differing perspectives about terpretation , there was confusion among the mgtb members regarding whether the reported mutations were somatic or germline . 
a discussion about a patient with a reported mutation in fancc highlights this confusion , which affected their thoughts about whether the patient should receive a genetics referral ( table 3 )  . 
of times reason was discussed during the mgtb meetings patient referrals for additional germline testing on the basis of personal history triple - negative cancer and age of diagnoses , 60 years diagnosis of bilateral breast cancer on the basis of results of tumor - only sequencing report for parp inhibitors identication of known / likely pathogenic fancc mutation identication of known / likely pathogenic chek2 mutation identication of known / likely pathogenic mlh1 mutation identication of known / likely pathogenic nf1 mutation identication of known / likely pathogenic mutation or vus in brca1 / 2 to determine eligibility on the basis of personal history and of tumor - only sequencing report re - evaluation of brca1 / 2 status for parp inhibitors because of prior negative germline results and negative tumor sequencing results for brca1 / 2 identication of known / likely pathogenic tp53 mutation on tumor sequencing and age of diagnoses note . 
missed opportunities for referral for additional germline testing ( n = 7 ) fishler et al missed opportunity on the basis of personal history on the basis of tumor - only sequencing report identication of known / likely pathogenic pten variant on the basis of personal history and tumor - only sequencing report triple - negative breast cancer , rst breast cancer diagnoses , 60 years , and no prior germline genetic testing personal diagnosis of lobular breast cancer and identication of known / likely pathogenic cdh1 variant on the basis of previous genetic test results and tumor - only sequencing report negative brca1 / 2 germline testing before availability of bart testing , no mutation in brca1 / 2 gene identied on tumor negative brca1 / 2 germline testing before availability of bart testing , pten mutation identied on tumor sequencing sequencing total no . 
the tumor - only report does not distinguish variants that are reported pathogenic from variants that are reported likely pathogenic . they are reported as one category on the front page of the report . 
seven patients out of 18 discussed at the mgtb did not receive a genetics referral for additional germline testing , although it would have been indicated by nccn criteria or a known / likely pathogenic mutation identied in a cancer susceptibility gene by tumor - only sequencing . 
at the time of the study of this mgtb , nccn criteria recommended that patients with negative brca1 / 2 sequencing before 2006 have bart testing and women with triple - negative breast cancer and their rst breast cancer diagnoses before age 60 years have brca1 / 2 germline testing . 
in addition , the american college of medical genetics and genomics recommended that pathogenic germline mutations associated with hereditary cancer syndromes be returned to patients ( such as pten and cdh1 )  . abbreviations : bart , brac analysis rearrangement test ; mgtb , multidisciplinary genomic tumor board ; nccn , national comprehensive cancer network . testing , some of the medical oncologists were reluctant , because the gene was not directly related to hereditary breast and ovarian cancer syndrome . 
mgtb interviewees indicated lack of education regarding medical genetics as a limitation of the mgtb , which is a recognized barrier to autonomous use of genetic testing by ordering physicians.2 , 12 potential for identication of germline cancer predisposition consistent with the mgtbs focus on identifying treatment options , 10 members of this mgtb were most concerned about whether a patient had a brca1 / 2 mutation to determine their eligibility for treatment with poly ( adp - ribose ) table 3 . 
vignette : confusion about somatic versus germline mutations vignette medical oncologist 7 : it says in the report that germline mutations in fancc cause fanconi anemia , a clinical heterogeneous disorder , and that appropriate clinical testing [ to detect ] the presence of a germline mutation is recommended . 
whenever we see a mutation , we have to make sure that we realize its one test and we would need it to be validated , but does it have relevance for treatment ? ( talking about prior treatment responses and discussion about parp inhibitor recommendations ) medical oncologist 7 : and she was germline brca tested and all thats negative ? medical oncologist 1 : yeah , she was brca negative . medical oncologist 2 : should we refer to genetics about this fancc thing ? medical oncologist 1 : you think we should refer ? is there a reason to think about it ? medical oncologist 7 : the only way you can test it is on those panels . 
every genetics has a panel where you can test for fanconi anemia genesi dont know how much breast cancer risk or other risks [ are associated with variants in this gene ]  . 
 underlying germline variants in tumor - only sequencing polymerase inhibitors , which gained traction as a mutationguided treatment option during the study period.13 , 14 thus , all patients with tumor - only sequencing results that included a vus or known / likely pathogenic mutation in brca1 / 2 were referred to genetics ( n = 4 )  . patients with known / likely pathogenic mutations in other cancer susceptibility genes ( such as pten ) were not routinely referred to genetics ( table 2 )  . 
at the time of this study , american college of medical genetics and genomics recommended that when analyzing a tumor and paired germline sample , such results should be conrmed and communicated to patients as secondary ndings , because of their association with a hereditary cancer syndrome . however , nccn guidelines did not include a recommendation for additional germline testing for mutations in genes on the secondary ndings list , 7 which may have contributed to inconsistent referral patterns , especially given that the mgtb was reviewing tumor - only sequencing results . two additional patients who met nccn guidelines for germline testing on the basis of their personal history were not referred for germline testing ( table 2 )  . 
at the time of the study of this mgtb , nccn recommended bart ( brac analysis rearrangement test ) testing for patients who had received negative brca1 / 2 germline test results before 2006.7 two patients with negative brca1 / 2 tumor - only sequencing results and negative brca1 / 2 germline test results before the availability of bart testing were not referred to genetics , although a genetics referral would have been indicated by nccn guidelines . 
in discussing how well individual disciplines were represented on the mgtb , a medical oncologist who co - led the mgtb recognized the value of genetics expertise in interpreting whether certain mutations were common or rare and how to manage referrals on the basis of a variants likelihood of being germline . 
explicitly , she noted that knowledge and experience with specic gene pathways enhanced the variant interpretation and productivity of the discussions about treatment options and result interpretation during the mgtb meetings . 
she also described disappointment that a medical geneticist and genetic counselors were invited , but that a medical geneticist only attended one mgtb meeting over the 13 - month study period and no genetic counselors participated . 
however , the other medical oncologist who co - led the mgtb mentioned that scheduling posed a barrier to genetic counselors attendance . mgtb members discussed the limitations of the test throughout the entire study period . 
the pathologist cautioned during her interview that not all providers who order tumor - only sequencing may understand the limitations of the test in terms of identifying germline variants : its hard to speak for other doctors , other community doctors . 
furthermore , at one meeting , the medical geneticist explained sequencing methodology , utility of bart testing for patients who have previously tested negative for brca1 / 2 , and guidelines for germline testing on the basis of a patients personal history , including age and triple - negative diagnosis . 
this information shifted the conversation from discussion of clinical trial options for a patient with triple - negative breast cancer who previously tested negative for mutations in brca1 / 2 to clarication of other germline testing options including bart testing , in accordance with nccn recommendations.7 during his interview , the medical geneticist stressed the value of genetics professionals who can address psychosocial concerns with patients during result return when a germline mutation is suspected . 
he noted that this skill was outside of his scope of practice and was one of the most difcult parts of result return . discussion for patients discussed by this mgtb , tumor sequencing was performed as part of clinical care . 
members of this mgtb , including medical oncologists , questioned whether the results of tumor sequencing were somatic or germline and queried what resources were available to them to clarify this information . 
the medical geneticist was the only mgtb member who was board certied in medical genetics . however , he was not present at the majority of mgtb meetings to provide clarication regarding whether a patient discussed on the mgtb should receive additional germline genetic testing . 
other mgtb members without medical genetics board certication may have qualied as a commission on cancer ( coc ) dened genetics professional by providing cancer risk assessment on a regular basis.15 ( p51 ) a key question that emerged through this analysis is whether these criteria are sufcient to ensure optimal implementation and interpretation of tumor sequencing by an mgtb and how best to support clinicians as they move into using new genomic technologies in their practices . nccn currently recommends genetic risk evaluation for patients with a known / likely pathogenic variant identied in a cancer susceptibility gene via tumor sequencing . 
perceived value of types genetics expertise on an mgtb perceived value by mgtb members member illustrative quote medical oncologists identication of clinical trials evaluation of treatment options medical oncologist : i can look and see if that [ three prior investigational studies ] will exclude her from [ clinical trials ] in the area , but that would sort of be my preference would be to put her on the phase i while looking for regional studies , and ill contact [ name ] and look on clinicaltrials.gov. 
and i guess were not recommending treatment off study with one of these m2r inhibitors ? basic scientists evaluation of functional studies radiation oncologist : someone who has more expertise in the basic science stuffwith better knowledge about genomic pathways understanding of mechanisms of the different mutations and how that may or may not translate into something that the drug might actually work for . medical evaluation of testing geneticists methodology variant classication genetic evaluation of family history counselors variant classication psychosocial counseling during result return medical geneticist : it is still not clear using next - generation sequencing whether a negative result is [ a true negative ] , because you have to look at the coverage across each position and the number of reads theyre reporting . 
second , rearrangements can be missed , so wed want to have her get bartshe should get the [ germline ] testingjust given her age of diagnosis and the fact that its triple - negative . medical geneticist : the most challenging patient encounters ive had is telling a mom that shes passed [ the mutation ] along to her daughter , and having the daughter understand that her mom just passed along a gene that basically assures her that shell get breast cancer in her lifebut it is very challenging , the psychosocial aspect . abbreviations : bart , brac analysis rearrangement test ; mgtb , multidisciplinary genomic tumor board . family history to determine whether people with known / likely pathogenic mutations in cancer susceptibility genes should receive additional germline testing . 
when clinical sequencing laboratories do not compare variants identied in a tumor sample with a germline sample , ordering providers are responsible for lling this gap in professional recommendations by recognizing when clinical germline testing is indicated on the basis of the combination of personal history , tumor sequencing results , and family history . currently , there are no guidelines or recommendations for which professionals or types of genomic expertise should be included by an mgtb to ensure adequate interpretation , recommendation , and communication of genomic test results . 
the coc denes certain roles within a cancer center and provides guidelines for the frequency and format of multidisciplinary tumor boards.15 however , it does not dene what disciplines should be included to constitute an mgtb , nor does it specify guidelines for tumor boards that evaluate tumor - only sequencing . 
the coc denes a genetics professional in a cancer center as someone who provides cancer risk assessment on a regular basis15 ( p51 )  . although multiple clinicians on this mgtb may have met this coc denition , many expressed confusion regarding benets and limitations of genetic testing and genetic risk assessment as well as knowledge deciencies surrounding testing methodology and result interpretation . 
this suggests that the genetics expertise pertinent for interpreting tumor - only sequencing results may be partial or unevenly distributed among those who were tapped to lend their expertise to an mgtb . on the basis of the results of this single - site case study , when considering how denitions of a genetic professional may be applied to mgtbs , providing cancer risk assessment on a regular basis is insufcient for conferring expertise for implementing tumor sequencing in clinical care . to ensure appropriate interpretation of tumor sequencing and identication of patients who ought to be referred to genetics , an mgtb should include individuals with expertise in clinical translation of tumor sequencing , testing methodology , molecular pathology , cancer biology and genomic pathways , germline variant interpretation , evaluation of personal and family history , and application of professional guidelines for germline testing . 
 underlying germline variants in tumor - only sequencing societies and organizations and clinical laboratories ought to consider whether tumor - only sequencing should be routinely paired with germline samples to facilitate standardized reporting of germline variants . given that the mgtb under study was primarily focused on treatment and clinical trial recommendations on the basis of results of tumor sequencing , it may not have been the optimal setting to discuss a patients personal history , including prior genetic testing and / or potentially pathogenic variants in cancer susceptibility genes lacking treatment implications . 
however , to ensure comprehensive referrals for additional germline sequencing after tumor - only sequencing , ordering providers ought to interpret the patients personal history in combination with their tumor - only sequencing results , as discussed by the mgtb . 
for two patients with negative brca1 / 2 tumor - only sequencing results and negative brca1 / 2 germline testing before the availability of bart testing , negative brca1 / 2 tumor - only sequencing results may have been falsely reassuring for these providers , especially considering the possibility that other cancer susceptibility genes may have been responsible for the cause of their cancer . is possible that provider deliberation about whether certain patients should be referred to genetics on the basis of these factors , not specic to the purpose of convening the mgtb the mgtb , may have occurred outside of meetings and would not have been captured by this analysis . 
in a climate where there is a paucity of genetic counselors and medical geneticists , creative strategies for engaging genetics professionals formally and informally in mgtb deliberations should be considered to optimize interpretation of tumor sequencing results . 
this study was limited by the information discussed as part of the mgtb meetings focused on patients with advanced breast cancer and interviews with mgtb members at a single national cancer institutedesignated cancer center . 
thus , possible that there were additional patients with known / likely pathogenic variants in genes associated with an inherited cancer predisposition syndrome that were not captured by this analysis , or that geneticists or genetic counselors may have been consulted about interpretation of laboratory reports outside of the mgtb context . 
fishler research funding : illumina ( inst ) lauren walters - sen employment : invitae stock and other ownership interests : invitae consulting or advisory role : ischemia care no other potential conicts of interest were reported . acknowledgment we thank roselle ponsaran , tracie baker , rebecca sisson , and the multidisciplinary genomic tumor board members for their time and contribution to this study . 
 fishler et al references schwaederle m , parker ba , schwab rb , et al : molecular tumor board : the university of california - san diego moores cancer center experience . 
oncologist 19 : 631 - 636 , 2014 tafe lj , gorlov ip , de abreu fb , et al : implementation of a molecular tumor board : the impact on treatment decisions for 35 patients evaluated at dartmouthhitchcock medical center . 
ann oncol 27 : 795 - 800 , 2016 jones s , anagnostou v , lytle k , et al : personalized genomic analyses for cancer mutation discovery and interpretation . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
mcgowan ml , ponsaran rs , silverman p , et al : a rising tide lifts all boats : establishing a multidisciplinary genomic tumor board for breast cancer patients with advanced disease . 
catenacci dv , amico al , nielsen sm , et al : tumor genome analysis includes germline genome : are we ready for surprises ? int j cancer 136 : 1559 - 1567 , 2015 13 . 
 next - generation rapid autopsies enable tumor evolution tracking and generation of preclinical models purpose patients with cancer who graciously consent for autopsy represent an invaluable resource for the study of cancer biology . 
to advance the study of tumor evolution , metastases , and resistance to treatment , we developed a next - generation rapid autopsy program integrated within a broader precision medicine clinical trial that interrogates preand postmortem tissue samples for patients of all ages and cancer types . materials and methods one hundred twenty - three ( 22% ) of 554 patients who consented to the clinical trial also consented for rapid autopsy . 
this report comprises the first 15 autopsies , including patients with metastatic carcinoma ( n = 10 ) , melanoma ( n = 1 ) , and glioma ( n = 4 )  . whole - exome sequencing ( wes ) was performed on frozen autopsy tumor samples from multiple anatomic sites and on non - neoplastic tissue . 
tissue was also used for the development of preclinical models , including tumor organoids and patient - derived xenografts . results three hundred forty - six frozen samples were procured in total . 
mutational profiles of primary tumors and metastases yielded candidate mediators of metastatic spread and organotropism including cul9 and pigm in metastatic ependymoma and ankrd52 in metastatic melanoma to the lung . 
rna - seq data identified novel gene fusion candidates . conclusion a next - generation sequencingbased autopsy program in conjunction with a premortem precision medicine pipeline for diverse tumors affords a valuable window into clonal evolution , metastasis , and alterations underlying treatment . 
the goal of the caryl and israel englander institute for precision medicine ( ipm ; new york , ny ) is to harness as much information as possible from an individual patients tumor tissue not only in an effort to benefit the patient during life , 1 but also to develop preclinical platforms2 and coclinical trials with the potential to benefit larger populations . 
an important arm of our precision medicine program permits postmortem tissue donation linked to robust downstream applications through a dedicated autopsy program . institutional rapid autopsy programs have been well described1 , 3 - 12 and have previously led to significant advances in cancer research in the areas of prostate , 1 , 3 , 4 , 6 , 7 , 11 pancreas , 10 breast , 8 and pediatric brain tumors.5 , 9 , 12 this program builds on prior autopsy programs by enrolling patients of all ages and cancer types , capturing samples throughout a patients disease course , and leveraging multiple parallel platforms to generate high - throughput sequencing data and robust preclinical tumor models . 
we highlight our initial experience with 15 patients who underwent rapid autopsy and emphasize the following three themes : capturing the molecular status of a diverse set of neoplasms at the end point in disease progression relative to ascopubs.org / journal / po jco precision oncology david j . 
at our institution , the generic hospital autopsy consent form must also be signed by the next of kthis institutional review boardapproved autopsy tissue donor program is based in part on protocols developed at the university of michigan by drs mark a . 
once the treating physician notifies a contact member of a patients death by pager , a robotic calling system is activated to alert the autopsy teaattending and resident autopsy pathologists , an autopsy technical assistant , and two to four members of the ipm laboratory are available at all times . 
total rna was prepared for rna sequencing ( rnaseq ) in accordance with the standard illumina mrna sample preparation protocol ( illumina )  . paired - end rna - seq at read lengths of 50 or 51 base pairs was performed with the hisequation 2000 ( illumina )  . 
tumor samples are represented in a phylogenetic tree as nodes with no children , whereas the internal nodes model inferred tumor cell populations on the basis of observed single nucleotide variants using the parsimony ratchet method.21 , 22 see data supplement for gene clonality assessment . rna - seq data analysis all reads were independently aligned with star_2.4.0f123 for sequence alignment against the human genome build hg19 , downloaded via the university of california santa cruz genome browser . 
for fusion analysis , we used star - fusion ( star - fusion_v0.5.1 ) , 24 , 25 fusioncatcher ( v0.99.3e ) , 26 and fusionseq.27 - 31 nucleic acid extraction dna extraction and whole - exome and wholegenome sequencing . 
sequencing is performed using illumina hisequation 2500 rapid run mode ( 23101bp ; illumina , san diego , ca )  . patient - derived , tumor organoid , and xenograft development platform as previously described , 2 fresh tumor tissue is dissociated before seeding cells into growth factor reduced matrigel ( corning , corning , ny )  . 
 with matrigel in the flanks of nu / nu nude mice ( charles rivers laboratories , wilmington , ma )  . were variably treated with prior surgery , radiation , and / or chemotherapeutic agents ( data supplement )  . results consent one hundred twenty - three ( 22% ) of 554 patients who consented to the clinical trial have also consented for rapid autopsy . 
of those patients for whom we have documented death in addition to premortem consent , we have performed autopsy on approximately 54% ( see materials and methods for details on consent )  . diversity of tumor types and metastatic sites the first 15 autopsies performed comprise a diverse set of neoplasms and metastatic sites ( fig 1 )  . primary tumors were available in 12 ( 80% ) of 15 patients from prior surgical resections and / or from recurrent or residual primary tumors obtained at autopsy . 
in patients with known metastatic disease before death , matched primary and metastatic tumor from individual patients was available in 10 ( 77% ) of 13 patients , and metastatic disease was collected at autopsy in 100% ( n = 13 ) of these patients . 
transport was successfully managed from up to 50 miles from our manhattanbased institution . this initial cohort yielded a total of 346 frozen aliquots over 131 distinctly annotated anatomic sites , of which 310 contained tumor upon histologic review of frozen sections ( fig 1b )  . 
one hundred thirteen distinct samples underwent whole - exome sequencing ( wes ) , including 82 frozen tumor and 10 frozen non - neoplastic control tissue samples obtained at autopsy , six samples derived from peripheral blood , and 15 formalin - fixed paraffinembedded or frozen samples from prior surgical procedures . 
in addition to wes , rnaseq was performed on a subset of samples ( n = 72 )  . data regarding rna concentration , rna integrity number values , 260 / 280 values , and 260 / 230 values over all rna samples are provided in the data supplement . 
despite transporting the patient from an outside institution , our pmi for this patient was only 2 hours , and the effort led to viable cell cultures . tumor clonal architecture and evolution autopsy material in combination with premortem surgical material is crucial to the study of the spatiotemporal evolution of cancer . 
in one illustrative patient ( wcm419 ) , a young girl with recurrent anaplastic ependymoma , material was available from eight distinct surgical resection procedures spanning more than 5 years in addition to material obtained at autopsy ( fig 2 )  . multiple sites of disease were available for study including the posterior fossa ( the primary site ) , metastatic lesions within the spinal cord , and a metastatic deposit involving the lateral ventricle obtained at autopsy ( figs 2a to 2c )  . 
histologic examination of all tumor material showed prominent perivascular pseudorosette formation ( fig 2d )  . wes was performed on formalin - fixed paraffinembedded material derived from prior surgical specimens as well as frozen autopsy tissue from two distinct sites ( figs 2e and 2f )  . 
reconstruction of the evolutionary tree of this tumor revealed a complex branching pattern where several mutations ( erbb3 , dnmt3a , brca1 , notch1 , and runx1t1 ) in the primary tumor samples were not shared at different time points ( 0 to 64 months ) or with descendent clones ( fig 2g )  . 
moreover , we identified two genes , cul9 and pigm , as harboring alterations specific to three or greater metastatic sites of disease and absent from all tissues resected from the primary site of disease . molecular features among multiple metastatic sites even in the absence of a definitive primary site , it is possible to gain insights into the evolutionary dynamics of tumor growth in distinct anatomic compartments . 
a case in point is wcm642 , a 50 - year - old male patient who presented with disseminated disease from a primary tumor of unknown origin ( fig 3 )  . 
prior biopsy demonstrated an epithelioid neoplasm without a definitive diagnosis . at autopsy , tumor was frozen from 15 distinctly annotated anatomic locations including adrenal gland and three distinct lobes of the lungs ( figs 3a and 3b ) , parietal pleura , omentum , lymph nodes ( three distinct sites ) , small intestine , large intestine , liver ( two distinct lobes ) , gallbladder , and pancreas . diagnosis of this tumor was only ascertained at autopsy . 
at autopsy , cells demonstrated both epithelioid and sarcomatoid morphology ( figs 3c and 3d ) and focal positive staining for s100 ( not shown ) , which had been negative in the biopsy . sequencing revealed mutations in nras and idh1 ( fig 3e ) and contributed to the final diagnosis of malignant melanoma . reconstruction of the clonal architecture of eight metastases from this patient demonstrated several mutations that were shared in all samples ( mllt4 , idh1 , aff3 , arid2 , ect2l , and nras ) , as well as mutations restricted to particular metastatic sites ( eg , apc in an lm and smad4 in lung ; fig 3f )  . remarkably , the branch points of the phylogenetic tree reflect the anatomic distribution of metastases , with those encompassing the digestive system grouped separately from those to the adrenal gland and lungs . 
 a wcm419 cul9 pigm runx1t1 notch1 brca1 dnmt3a erbb3 mutated wild type anaplastic ependymoma 1 - 5 : posterior fossa primary 6 : spinal thoracic met 7 : spinal thoracic met 8 : lateral ventricle met 9 : spinal met 10 : spinal met 9 10 runx1t1 shared in more than one sample but not all samples private primary private metastatic notch1 brca1 msh2 dnmt3a erbb3 spec id# months location pf , lv fig 2 . 
multiple sites of disease were available for study , including ( a and b ) the posterior fossa ( primary site ; [ a ] ventral surface of the whole brain with exophytic tumor component indicated by the arrow ; [ b ] metastatic deposits within the supratentorial compartment involving the lateral ventricle and subventricular brain parenchyma , and lesions within the spinal cord ( not shown ) , ( c ) axial cross - section through the pons and cerebellum )  . 
 ( f ) whole - exome sequencing was performed on prior surgical resection formalin - fixed paraffin - embedded specimens including primary tumor in posterior fossa ( samples 1 , 3 , 4 , and 5 ) and metastases to spinal cord ( samples 6 , 7 , 9 , and 10 ) , as well as on frozen autopsy tissue from two distinct sites , including recurrent primary tumor in the posterior fossa ( sample 2 ) and metastasis to the lateral ventricle ( sample 8 )  . 
both the color - coded bar on the right and the phylogenetic tree below indicate whether mutations are private to primary or private to metastases or shared in more than one sample . 
several mutations ( erbb3 , dnmt3a , brca1 , notch1 , and runx1t1 ) in the primary tumor samples are not shared either between them at different time points ( 0 to 64 months ) or with descendent clones . 
in particular , for both of these patients , genes were identified that showed consistent overexpression across metastatic tumors for each patient at the transcriptional level relative to the expression of these genes over a large set of rna - seq samples . 
for wcm772 , these data correlated with evidence of amplification of the met gene derived from wes , whereas for wcm0 , no such correlation was observed ( data supplement )  . additional rna - seq analysis of wcm0 samples identified novel gene fusion candidates such as znf526 - megf8 ( figs 4b to 4d )  . 
in addition , wgs on two lms from this patient yielded additional candidates , including the discovery of a gene fusion involving styk1 , a putative serine / threonine and tyrosine receptor protein kinase of potential clinical significance in castrate - resistant prostate cancer32 ( fig 5 )  . tumor organoid , pdx , and cell line development pdxs and cell lines are the most frequently used models in cancer research and anticancer drug screening . 
 a mutated wild type melanoma 1 : liver right met 2 : lung left upper lobe met 3 : lung left upper lobe met 4 : lung right upper lobe 5 : small intestine met 6 : large intestine met 7 : adrenal left met 8 : pancreas met shared in all samples shared in more than one sample but not all samples private metastatic wcm642 1 2 3 4 5 6 7 8 smad4 mllt4 idh1 aff3 arid2 ect2l nras mllt4 idh1 aff3 arid2 ect2l nras ankrd52 smad4 fig 3 . 
a 50 - year - old male patient presented with disseminated disease from primary tumor of unknown origa previous biopsy ( not shown ) showed an unclassified malignant and epithelioid neoplasm . at autopsy , tissue samples from 17 distinct anatomic locations were snap frozen , including ( a ) adrenal gland and ( b ) lung . 
both the colorcoded bar on the right and the phylogenetic tree below indicate whether mutations are private to any metastatic site or are shared in more than one sample or in all samples . 
few mutations are shared in more than one sample but not all samples ( eg , apc in right liver metastasis ( met ) ; smad4 in left upper lobe of lung )  . 
scale bars = ( a and b ) , 1 cm ; ( c and d ) , 50 mm . in vivo biologic processes and may provide a new avenue for personalized cancer care.33 , 34 as demonstrated in patient wcm331 , despite a delayed pmi of 6.5 hours as a result of transportation from an outside hospital , tumor organoid development of ovarian serous adenocarcinoma was achieved . 
viable organoids histologically comparable to metastatic autopsy tissue were produced and consistent with ovarian serous adenocarcinoma ( figs 6b to 6d )  . wes was performed on a metastatic omental lesion biopsied 2 years before autopsy , on several metastatic lesions obtained at autopsy , and on the tumor organoid material derived from a peritoneal nodule obtained at autopsy . 
reconstruction of the clonal architecture of metastatic samples and tumor organoid demonstrates that ptprb mutation is shared across all samples , whereas foxo1 mutation is present in one metastasis ( to small bowel serosa ) and the tumor organoid only . 
alterations restricted to the tumor organoid material only ( eg , prdm16 and kmt2a ) may reflect either additional tumor heterogeneity or alterations arising in vitro . another example of patient - derived preclinical model development from rapid autopsy tissue is illustrated in a patient with colon adenocarcinoma ( wcm715 ; fig 6g ) for whom both tumor organoid ( figs 6h and 6i ) and downstream pdx models ( fig 6j ) were established . 
 a lm1 coverage [ 0 93 ] lm1 junctions lm2 coverage [ 0 75 ] lm2 junctions pm1 coverage [ 0 48 ] pm1 junctions pm2 coverage [ 0 59 ] pm2 junctions t24 coverage [ 0 53 ] t24 junctions t5 coverage [ 0 85 ] t5 junctions t16 coverage [ 0 50 ] t16 junctions refseq cdkn2c cdkn2c cdkn2c cdkn2c cdkn2c wcm0 wcm772 [ 0 93 ] [ 0 75 ] [ 0 48 ] [ 0 59 ] [ 0 53 ] [ 0 85 ] [ 0 50 ] znf526 megf8 fig 4 . 
 ( a ) gene expression outlier analysis across 300 rna sequencing samples derived from a diversity of tumors within the institute for precision medicine cohort highlights wcm0 ( small - cell carcinoma of prostate ) and wcm772 ( pleomorphic carcinoma of lung ) , two patients who show cdkn2c and met as recurrent outliers in distinct anatomic samples , respectively . 
z scores were calculated for 70 druggable cancer genes , and outliers were selected at a cutoff of z score greater than 2.5 and fragments per kilobase million greater than 50 . 
 ( b ) rna sequencing analysis by fusioncatcher and fusionseq allows for the identification of novel gene fusion candidates . schematic of the fusion between znf526 and megf8 in wcm0 showing the connected exons . 
 a ybx3 chr12 styk1 lm1 coverage lm1 junctions lm2 coverage lm2 junctions pm1 coverage pm1 junctions pm2 coverage pm2 junctions t24 coverage t24 junctions t5 coverage t5 junctions t16 coverage t16 junctions refseq fig 5 . 
a 55 - year - old male patient status post prostatectomy presented with local recurrence , metastases in the pelvis and liver , and cushing syndrome in the month before death . 
overexpression of styk1 may serve as a potential molecular target in castrate - resistant prostate cancer.32 discussion tissue procurement from patients with cancer at autopsy and within the context of a pan - cancer precision medicine clinical trial captures biologic data at a state in disease progression that is rarely studied . 
in contrast to previously reported autopsy programs , we have incorporated data analysis and development of preclinical models using multiple platforms , 2 clinical - grade assays , 13 and computational algorithms , 13 , 35 , 36 leveraging the infrastructure of ipm . 
here , we illustrate a proof of principle in developing a powerful model for interrogating a diverse set of primary tumor types and metastatic sites , including newly generated tumor organoids , pdx models , and cell culture . 
indeed , with just the first 15 autopsies , we have revealed novel genetic candidates as potential mediators of metastatic spread in both ependymoma and melanoma , as well as provided the foundation for additional work investigating treatment effect in metastatic urothelial carcinoma . as a testament to our ability to track patients through their disease course , we analyzed primary tumor samples in 77% of patients for whom metastatic disease was procured at autopsy and in 80% of patients overall . 
 a wcm331 prdm16 kmt2a foxo1 ptprb foxo1 kmt2a prdm16 shared in all samples shared in more than one sample but not all samples private metastatic ptprb serous ovarian carcinoma 1 : omentum met 2 : diaphragm met 3 : small intestine met 4 : organoid from peritoneal met mutated wild type fig 6 . 
 ( d ) cytologic comparison of these tumor organoids with representative sections from metastatic autopsy tissue revealed morphologically similar cells ( diff - quik stain , 3400 original magnification )  . 
 ( e ) whole - exome sequencing was performed on formalin - fixed paraffin - embedded material derived from an omental metastasis biopsied 2 years before autopsy ( sample 1 ) , on frozen material from two metastases obtained at autopsy ( samples 2 and 3 ) , and on tumor organoid material derived from a peritoneal nodule obtained at autopsy ( sample 4 )  . 
both the color - coded bar on the right and the phylogenetic tree below indicate whether mutations are private to the metastatic sites or are shared in more than one sample or in all samples . 
 ( f ) in the absence of material from the primary tumor , partial reconstruction of the clonal evolution of metastatic samples ( including the tumor organoid as surrogate of one of the metastasis at autopsy ) demonstrates that ptprb mutation is shared in all samples and foxo1 mutation is present in two metastases . 
 ( g ) in case wcm715 , an he - stained section of a liver metastasis obtained at autopsy showed metastatic mucinous adenocarcinoma of the colon ( 3200 original magnification )  . 
 ( j ) he - stained section of patient - derived xenograft derived from organoid implantation in mouse demonstrates comparable histomorphology with original liver metastasis and derived tumor organoid ( 3200 original magnification )  . 
for example , analysis of wcm419 revealed alterations in cul9 and pigm that were present in most or all spatiotemporally distinct metastatic sites of ependymoma but absent from multiple resections at the primary site . 
 whereas pigm encodes a mannosyltransferase involved in glycosylphosphatidylinositol - anchor biosynthesis.38 in addition , we have identified gene candidates for further study that may mediate organotropism of metastases in melanoma ; for example , we found that ankrd52 mutations were restricted to lung metastases in a patient with widely metastatic melanoma . 
finally , additional work on two patients from within this cohort ( wcm117 and wcm259 ) has already yielded further insights into chemotherapy - resistant urothelial carcinoma , demonstrating enrichment in clonal alterations including targetable mutations ( eg , l1cam and integrin signaling pathways ) .21 comparison of wes between primary tumors , metastatic sites , and preclinical in vitro models of disease also enables an assessment of the extent to which critical molecular features of a tumor are reiterated , for example , in tumor organoids . 
the latter seem to recapitulate the patients metastatic disease at the exome level , representing an opportunity to develop coclinical trials and other clinically relevant applications ( discussed in additional detail by pauli et al39 )  . 
in addition , investigation of wgs and rna - seq data including gene expression outlier analysis across our precision medicine cohort has allowed the identification of novel gene fusions and overexpression of certain transcripts across metastatic sites with potential clinical relevance . we have set the foundation to investigate several critical questions over a diverse set of neoplasms , including the temporal evolution of tumors , the clonal evolution of tumors with respect to widely disseminated metastatic disease sites , and the production and examination of tumor organoid models derived from these specimens . 
robinson , alexandros sigaras , rema rao , bishoy faltas , rohan bareja , prajwal rajappa , jeffrey greenfield , anne - katrin emde , nicolas robine , olivier elemento , andrea sboner , francesca demichelis , himisha beltran , mark a . rubin , juan miguel mosquera manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors david j . 
robinson stock and other ownership interests : metastat , consulting or advisory role : progenics pharmaceuticals patents , royalties , other intellectual property : methods for diagnosing and treating prostate cancer david m . 
tagawa consulting or advisory role : medivation , astellas pharma , dendreon , janssen , bayer , genentech , sanofi , endocyte , immunomedics speakers bureau : amgen research funding : eli lilly ( inst ) , sanofi ( inst ) , janssen ( inst ) , astellas pharma ( inst ) , progenics ( inst ) , millennium ( inst ) , amgen ( inst ) , bristol - myers squibb ( inst ) , dendreon ( inst ) , rexahn pharmaceuticals ( inst ) , bayer ( inst ) , genentech ( inst ) , newlink genetics ( inst ) , inovio pharmaceuticals ( inst ) , astrazeneca ( inst ) , immunomedics ( inst ) , novartis ( inst ) , aveo ( inst ) , rexahn pharmaceuticals ( inst ) , boehringer ingelheim ( inst ) , merck ( inst ) , stem centrx ( inst ) travel , accommodations , expenses : sanofi jeffrey greenfield no relationship to disclose anne - katrin emde no relationship to disclose nicolas robine no relationship to disclose olivier elemento no relationship to disclose andrea sboner no relationship to disclose alexandros sigaras employment : weill cornell medical college mark a . 
rubin research funding : eli lilly , janssen francesca demichelis patents , royalties , other intellectual property : coinventor on a patent filed by the university of michigan and the brigham and womens hospital covering the diagnostic and therapeutic fields for ets fusions in prostate cancer . 
the diagnostic field has been licensed to gen - probe . himisha beltran consulting or advisory role : bayer , janssen oncology , genzyme research funding : astellas pharma ( inst ) , eli lilly ( inst ) , janssen ( inst ) , millennium ( inst ) , stemcentryx abbvie juan miguel mosquera no relationship to disclose acknowledgment we thank our patients and their families for participation in this study . 
we also acknowledge douglas scherr , md , and christopher barbieri , md , for contributing samples ; to yasmin khakoo , md , and our other referring clinicians ; to our clinical pathology fellows for their assistance during rapid autopsies ; to terra mcnary , loredana puca , mirjam blattner , uday bhanu maachani , nector garcia , anvioris taveras , and yelena churakova for technical assistance ; to funeral home transport personnel ; to autopsy and laboratory personnel involved in this project ; and to rogerio paulo da silva for art design . myriam kossai no relationship to disclose jacqueline fontugne no relationship to disclose robert kim no relationship to disclose rema rao no relationship to disclose danielle pancirer no relationship to disclose bishoy faltas no relationship to disclose rohan bareja no relationship to disclose ana m . 
shah rb , mehra r , chinnaiyan am , et al : androgen - independent prostate cancer is a heterogeneous group of diseases : lessons from a rapid autopsy progracancer res 64 : 9209 - 9216 , 2004 5 . 
zarghooni m , bartels u , lee e , et al : whole - genome profiling of pediatric diffuse intrinsic pontine gliomas highlights platelet - derived growth factor receptor alpha and poly ( adp - ribose ) polymerase as potential therapeutic targets . j clin oncol 28 : 1337 - 1344 , 2010 6 . 
esgueva r , park k , kim r , et al : next - generation prostate cancer biobanking : toward a processing protocol amenable for the international cancer genome consortiudiagn mol pathol 21 : 61 - 68 , 2012 7 . 
udager am , shi y , tomlins sa , et al : frequent discordance between erg gene rearrangement and erg protein expression in a rapid autopsy cohort of patients with lethal , metastatic , castration - resistant prostate cancer . 
prostate 74 : 1199 - 1208 , 2014 juric d , castel p , griffith m , et al : convergent loss of pten leads to clinical resistance to a pi ( 3 ) ka inhibitor . 
xie t , musteanu m , lopez - casas pp , et al : whole exome sequencing of rapid autopsy tumors and xenograft models reveals possible driver mutations underlying tumor progression . 
hoffman lm , dewire m , ryall s , et al : spatial genomic heterogeneity in diffuse intrinsic pontine and midline highgrade glioma : implications for diagnostic biopsy and targeted therapeutics . 
rennert h , eng k , zhang t , et al : development and validation of a whole - exome sequencing test for simultaneous detection of point mutations , indels and copy - number alterations for precision cancer care . 
quail ma , kozarewa i , smith f , et al : a large genome centers improvements to the illumina sequencing systenat methods 5 : 1005 - 1010 , 2008 med genomics 8 : 9 , 2015 2015 17 . 
sboner a , habegger l , pflueger d , et al : fusionseq : a modular framework for finding gene fusions by analyzing paired - end rna - sequencing data . 
derrien t , johnson r , bussotti g , et al : the gencode v7 catalog of human long noncoding rnas : analysis of their gene structure , evolution , and expression . 
chung s , tamura k , furihata m , et al : overexpression of the potential kinase serine / threonine / tyrosine kinase 1 ( styk 1 ) in castration - resistant prostate cancer . 
demichelis f , greulich h , macoska ja , et al : snp panel identification assay ( spia ) : a genetic - based assay for the identification of cell lines . 
 large - cell neuroendocrine carcinoma of the lung : a focused analysis of braf alterations and case report of a braf non - v600 mutated tumor responding to targeted therapy purpose in advanced stages , large - cell neuroendocrine carcinoma of the lung ( l - lcnec ) mimics small - cell lung cancer despite its traditional classification as a non small - cell lung cancer . 
here we present a focused analysis of braf mutations in this population . patients and methods comprehensive genomic profiling of tumor tissues was performed from a cohort of 300 patients with biopsy - proven l - lcnec . 
the importance of biomarker - driven therapy is subsequently highlighted with our case of a 69 - year - old man diagnosed with metastatic l - lcnec who did not respond to cisplatin plus etoposide . 
a significant durable response was then demonstrated with therapy targeted toward a braf non - v600e activating mutation ( g469r ) associated with biomarker response identified through circulating cell - free tumor dna analysis . 
giles author affiliations and support information ( if applicable ) appear at the end of this article . corresponding author : young kwang chae , md , mph , mba , northwestern medicine developmental therapeutics institute , 645 n . 
the 2015 who classification modified this outline and instead included all lung nets as a single entity , acknowledging that there exist major histologic , epidemiologic , and clinical differences between the carcinoids and high - grade l - lcnec and sclc.3 l - lcnec remains both rare and difficult to treat given the discord between its histologic and clinical characteristics . 
 chemotherapy regimens used for the treatment of sclc should be used ( etoposide and platinum combinations ) , which is reflected in national clinical cancer network ( nccn ) guidelines . 
 in the era of next - generation sequencing ( ngs ) , it is interesting to note that this strategy has yet to be adapted for the category of nonadenocarcinomas and nonsquamous cell carcinomas , which is certainly limiting in our ability to further identify these difficult - to - classify malignancies . 
nccn guidelines only mention ngs for adenocarcinomas . here we summarize the current landscape of l - lcnec and explore in detail the specific genomic alterations pertaining to braf , a member of the serine / threonine kinase raf family , in the largest cohort of patients with l - lcnec . 
 mutations and copy - number gains in the phosphatidylinositol 3 - kinase / akt / mammalian target of rapamycin pathway were detected in 15% of the tumors compared with 17% of sclc tumors.6 in another study using ngs in a cohort of 45 patient cases of l - lcnec , the most commonly altered genes similarly included tp53 ( 78% ) , rb1 ( 38% ) , stk11 ( 33% ) , keap1 ( 31% ) , and kras ( 22% )  . 
l - lcnecs with coaltered rb1 and tp53 ( 40% ) were identified as sclc - like , whereas tumors without this pattern ( 56% ) were identified as nsclc - like . 
as a whole , braf mutations are detected in 2% to 4% of lung cancers and have primarily been identified in adenocarcinomas.8 interestingly , although v600 mutations comprise the majority of braf alterations in melanoma , the v600e mutation is accountable for approximately 50% of braf mutations identified in nsclcs and up to 80% in late stages.8 as recently as late 2015 , the prognostic and biologic implications of braf mutations in nsclc were limited and controversial when evaluating the existing case series . 
subsequent studies were positive for cytokeratin 7 , thyroid transcription - 1 , synaptophysin , and chromogranin , thereby resulting in the diagnosis of metastatic , high - grade neuroendocrine carcinoma ( l - lcnec ) of lung primary ( fig 1 ; appendix ) .11 additional evaluation with brain magnetic resonance imaging and positron emission tomography ct identified a 1.4 - cm nodule in the superior segment of the left lower lobe , likely the site of the primary tumor . 
three months after rt , ct scans showed that the left hemipelvic mass had decreased in size to 3.5 4 cm , with a central area of necrosis ; however , the left lower lobe lung nodule was noted to have grown to 2 cm . ngs of tumor tissue ( foundationone ; foundation medicine , cambridge , ma ) had been performed at diagnosis and revealed mutations in braf ( g469r ) , erbb4 ( a118s ) , stk11 ( l282fs * 3 ) , and tp53 ( r248l ) ; myc amplification was also identified . 
given these molecular findings describing an activating braf mutation , treatment with trametinib ( 2 mg orally once daily ) and dabrafenib ( 150 mg orally twice daily ) was initiated at this time . 
trametinib is a mek inhibitor that prevents raf - dependent mek phosphorylation.8 dabrafenib is a braf serine / threonine kinase inhibitor that has high selectivity for braf v600emutant proteins.8 cell - free dna ( guardant360 ; guardant health , redwood city , ca ) analysis performed at diagnosis demonstrated concordance in detecting mutations in braf ( g469r ) and tp53 ( r248l ) , as well as myc amplification . 
 histology of needle core biopsy : ( c ) hematoxylin stain , 600 ; immunostains for ( d ) prostate - specific antigen , ( e ) cytokeratin 7 , ( f ) chromogranin , ( g ) synaptophysin , ( h ) thyroid transcription - 1 , and ( i ) cdx2 , 600 . expression of the patients braf g469r mutation was demonstrated , from 36.8% at time of diagnosis to nondetectable at both 5 months ( which may also have been in part a result of rt and subsequent tissue necrosis ) and 10 months after initiation of dabrafenib and trametinib ( fig 2 )  . there was an increase in other detectable alterations , which included synonymous mutations in brca1 , arid1a , and smo as well as vuss in apc , pten , fgfr1 , and pik3ca ( figs a1 - a3 )  . 
 presently , a full 15 months since initiating therapy with dabrafenib and trametinib , the patient is continuing with therapy and continues to demonstrate an eastern cooperative oncology group performance status of 0 . 
his latest ct scans ( 18 months after rt and 15 months after initiating targeted therapy ) show no residual pelvic mass and a stable left lower lobe nodule measuring 1.7 cm ( fig 3 )  . 
samples were pulled from an ngs database as part of a deidentified pool with only basic demographic information available ( required for processing the ngs order ) from many institutions . 
an inherent limitation of this pool is that we have to assume the histology of the malignancy was confirmed at each local institution . the atypical variants of braf in l - lcnec are identified as g469a , k601n , and g469r , which are all known to be activating mutations.14 - 17 there also exists g466v , which is an oncogenic kinase - impaired alteration.18 the remainder includes n581i and vus alterations e220k , w210l , g9d , and p149t . 
n581i is a known recurrently somatic braf alteration , but its effect has not been characterized ; otherwise , to our knowledge , these remaining braf mutations have not been previously identified.19 our analysis is summarized in table 1 . discussion current understanding of braf v600 mutations originated in melanoma , where they are the most commonly identified oncogenic mutations and are seen in nearly 50% of cutaneous melanomas.21 braf is an upstream effector of the mitogen - activated protein kinase pathway ( ras / raf / mek / extracellular regulated kinase ) , which plays a central role in cell growth , division , and differentiation . 
ninety percent of braf - mutated melanomas result from a substitution of glutamic acid for valine at codon 600.22 the braf v600e mutations show a 500 - fold increase in catalytic activity compared with the wild type.23 initially , selective braf inhibitors such as vemurafenib and dabrafenib were evaluated as monotherapy in patients with metastatic melanoma harboring either braf v600e or v600k mutations . 
contrast - enhanced computed tomography scans of the ( a , c , e , g ) abdomen and ( b , d , f , h ) chest of the patient ( a , b ) at diagnosis , ( c , d ) approximately 1 month after chemotherapy , ( e , f ) 3 months after radiation therapy ( rt ) directed at the left hemipelvic mass , and ( g , h ) after 15 months of targeted therapy with dabrafenib and trametinib . 
 melanoma , colorectal cancer , or nsclc , investigators concluded that kinase activity is likely to be elevated in 35% of braf mutations , impaired in 15% , and unknown in 10%.18 known nonv600e activating variants of braf that have been identified in patients with nsclc include g464v , g466a , g469a / v / r / s , and k601e . 
 mutations with impaired activity have been reported to be g466r / v , d594a / e / g / h / n / v , and g596c / r.20 interestingly , it has been identified that concomitant kras or nras mutations are more likely occur with the kinase - impaired braf mutants than the kinase - activated braf mutants.18 preclinical data have demonstrated sensitivity to treatment with dabrafenib and trametinib in hek293t cells , lung epithelial cellular models ( beas - 2b ) , and human cancer cell lines harboring non - v600 braf mutations ( both activating and kinase impaired )  . 
this same study also identified that the kinase activity of the g469s variant was comparable to that seen with the braf v600e mutation.20 this further provides a basis for the ongoing exploration of targeted therapy in non - v600 braf - mutant lung cancers . with regard to lung cancer , nccn guidelines for nsclc delineate emerging targeted therapy options for patients with genetic alterations.25 vemurafinib has been studied in multiple nonmelanoma cancers with braf v600 mutations in a basket trial design ( clinicaltrials.gov identifier nct01524978 ) ; in a cohort of 20 patients with nsclc , eight achieved partial responses with targeted braf inhibition.26 in a clinical trial studying dabrafenib plus trametinib in patients with previously treated braf v600emutant metastatic nsclc ( clinicaltrials.gov identifier nct01336634 ) , investigators reported an overall response rate of 63.2% in a cohort of 57 patients.27 this led to the recent approval of dabrafenib with trametinib as a treatment option for patients with advanced or metastatic nsclc with a braf v600 mutation by the us food and drug administration in june 2017.28 interestingly , there is minimal guidance available for non - v600 braf - mutated lung malignancies and even less information specifically focused on sclc or nets . 
remarkably when using ngs ( a broad and sensitive detection tool ) , non - v600e mutations comprise a substantial portion of the braf mutations in different tumors ( nsclc , 86% ; melanoma , 34% ; and colorectal cancer , 23% ) , as assessed in a cohort of more than 800 patients.17 this is a higher proportion of non - v600e braf mutations than identified in earlier retrospective studies with cohorts of 30 to 50 patients . 
in a retrospective study detailing the characteristics of nearly 700 patients with lung adenocarcinoma , braf mutations were present in 3% of patients ( v600e , 50% ; g469a , 39% ; and d594g , 11% )  . 
 interestingly , all of these patients were current or former smokers.29 currently , ngs assays demonstrate the best - known sensitivity and breadth of analysis for the clinical detection of braf mutations including non - v600e alterations.17 in our patient , of course , we must acknowledge the confounding variable of rt directed at the left hemipelvis in the reduction in size of the mass in this location . 
given this , in addition to the steep drop - off seen in braf circulating tumor dna , we suspect that this is a durable response to therapy . it is worth mentioning that cosmic presents information related to adenocarcinoma and squamous cell carcinoma but not l - lcnec . 
this does not reflect the scenario of a real - world stage iv malignancy . although not common , l - lcnec does seem to contain activating and thus actionable alterations as evidenced by our patient 's remaining stable with therapy focused at braf inhibition . 
 in particular , given the scope of non - v600 braf alterations , the basket trial design is ideal for furthering our understanding of the functionality of these various mutations in a histology - agnostic manner . 
the nci - match ( national cancer institute molecular analysis for therapy choice ) trial is currently under way and includes a nonv600 arm in which patients will receive trametinib . 
giles jon chung employment : foundation medicine administrative support : young kwang chae provision of study material or patients : young kwang chae , vincent miller collection and assembly of data : young kwang chae , keerthi b . 
 for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . stock and other ownership interests : foundation medicine xiaoqi lin no relationship to disclose vincent miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan kettering cancer center siraj m . 
ali employment : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine ; patents via seres health on microbiome stuff in non - neoplastic disease ( i ) young kwang chae consulting or advisory role : foundation medicine , boehringer ingelheim , biodesix , counsyl , astrazeneca , guardant health speakers bureau : merck , genentech / roche travel , accommodations , expenses : hanmi francis j . 
tamragouri no relationship to disclose consulting or advisory role : novartis travel , accommodations , expenses : medimmune , novartis , foundation medicine affiliations young kwang chae , keerthi b . 
lurie comprehensive cancer center , northwestern university ; xiaoqi lin , northwestern memorial hospital , northwestern university , chicago , il ; and jon chung , vincent miller , and siraj m . 
yao jc , hassan m , phan a , et al : one hundred years after carcinoid : epidemiology of and prognostic factors for neuroendocrine tumors in 35 , 825 cases in the united states . 
travis wd , brambilla e , nicholson ag , et al : the 2015 world health organization classification of lung tumors : impact of genetic , clinical and radiologic advances since the 2004 classification . 
rekhtman n , pietanza mc , hellmann md , et al : next - generation sequencing of pulmonary large cell neuroendocrine carcinoma reveals small cell carcinoma - like and non - small cell carcinoma - like subsets . 
lin x , saad rs , luckasevic tm , et al : diagnostic value of cdx - 2 and ttf - 1 expressions in separating metastatic neuroendocrine neoplasms of unknown orig appl immunohistochem mol morphol 15 : 407 - 414 , 2007 12 . 
noeparast a , teugels e , giron p , et al : non - v600 braf mutations recurrently found in lung cancer predict sensitivity to the combination of trametinib and dabrafenib . 
planchard d , besse b , groen hjm , et al : dabrafenib plus trametinib in patients with previously treated braf ( v600e ) - mutant metastatic non - small cell lung cancer : an open - label , multicentre phase 2 trial . 
 fine - needle aspiration and core - needle biopsy percutaneous fine - needle aspiration ( fna ) and core - needle biopsy ( cnb ) were performed under ultrasound guidance using a 22 - gauge needle for fna and a 20 - gauge core biopsy device for cnb.10 one fna pass was used for localization . 
airdried fna smear was stained with diff - quik stain for on - site evaluation of adequacy and immediate interpretation by a board - certified cytopathologist ( x.l. ) , and alcohol - fixed fna smear was stained with papanicolaou statwo cnb passes were obtained for histologic examination . 
immunohistochemical stains for prostate - specific antigen ( a0562 ; dakocytomation , carpinteria , ca ) , cytokeratin 7 ( ck7 ; a7018 ; dakocytomation ) , chromogranin ( 760 - 2519 ; ventana , tucson , az ) , synaptophysin ( 336a - 76 ; cell marque , rocklin , ca ) , thyroid transcription - 1 ( ttf - 1 ; 343m - 98 ; cell marque ) , and cdx2 ( mu392a - uc ; biogenex , san ramon , ca ) were performed in an automated immunostainer with appropriate positive and negative controls following manufacturers instructions , meaning that positive controls were stained positive and negative controls were stained negative . pathology results fna smears showed loosely cohesive nests or three - dimensional clusters of pleomorphic epithelial cells with focal rosette pattern ( figs a1a and a1b )  . 
the tumor cells were positive for neuroendocrine immunomarkers ( chromogranin and synaptophysin ; fig a1f and a1g ) , ck7 ( fig a1e ) , and ttf - 1 ( a marker for lung adenocarcinoma , lung neuroendocrine neoplasm , 11 and thyroid neoplasm ; fig a1h )  . 
the tumor cells were negative for prostate - specific antigen ( fig a1d ) and cdx2 ( an immunomarker for gi adenocarcinoma and neuroendocrine neoplasm arising from gi tract ; fig a1i ) .11 on the basis of the morphology and immunoprofile , a large - cell neuroendocrine carcinoma diagnosis was rendered . 
the tumor cells were also positive for ttf - 1 and ck7 and negative for cdx2 , suggesting lung origin.11 appendix sample submission time point oct - 16 ( % ) aug - 15 ( % ) pten ( q214 * ) erbb2 ( r143q ) braf ( g469r ) 36.8 mar - 17 ( % ) fig . 
suarez , md1 introduction genomic sequencing of colorectal cancers reveals that 16% have a very high tumor mutation burden ( tmb ) .1 three quarters of such tumors display microsatellite instability ( msi ) associated with silencing or somatic mutation of mismatch repair ( mmr ) genes . 
among advanced colorectal cancers , prevalence of pole mutations remains unknown , but only 3.5% are defective in mmr.2 pole proofreading and mmr act in concert to correct replication errors . because mmr - decient tumors often respond to therapy , 3 - 5 pole - mutated tuimmune checkpoint mors might also respond . 
pathology revealed moderately differentiated adenocarcinoma with two involved lymph nodes , classied as stage iiic disease ( pt4bn1b according to the american joint committee on cancer staging system , eighth edition )  . 
after adjuvant chemotherapy with uorouracil and oxaliplatin , computerized tomography ( ct ) scan showed no evidence of disease . three years later , biopsy of an enlarging left supraclavicular lymph node revealed a kras - mutated mss adenocarcinoma with abundant extracellular mucin and a lack of programmed death ligand 1 ( pd - l1 ) tumor cell expression by both e1l3n and sp263 antibody clones . 
the tumor - inltrating lymphocytes ( tils ) could not be assessed , because this was a lymph node metastasis and the primary tumor was unavailable . the patient received uorouracil and irinotecan plus bevacizumab . 
treatment was complicated by a small bowel stula , which required discontinuation of bevacizumab , partial small bowel resection , takedown of an enterorectal stula , and placement of a permanent rectal tube . after a 3 - month recovery from surgical complications , imaging showed new pulmonary metastases . 
the enlarging left supraclavicular nodal mass led to horner syndrome with ptosis and near syncope , which required palliative radiation . the left supraclavicular lymph node specimen obtained 3 years after surgery displayed approximately 20% tumor purity by histology . 
genomic proling with the stanford solid tumor actionable mutation panel , a hybrid capturebased next - generation sequencing assay , revealed an ultra - high tmb relative to colorectal carcinomas analyzed on the same panel ( fig 1b )  . additional testing demonstrated intact expression of mmr proteins as well as mss by polymerase chain reaction . 
the boxplot shows the median , interquartile range , and standard deviation for the tmb in 82 microsatellite - stable ( mss ) colorectal cancers ( crcs ) detected by the stanford solid tumor actionable mutation panel at the time of patient testing . 
the gray dots indicate tmb for those pole - mutated tumors reported to have responded to programmed death 1 ( pd - 1 ) blockade : two crcs and two endometrial cancers ( ecs )  . 
the box plots show the tmb for a survey of 859 tumors with likely driver mutations in mismatch repair genes that would produce microsatellite instability ( msi ) and 102 tumors with known or likely functional pole mutations ( adapted from chalmers et al16 )  . 
on the basis of the ultra - high tmb , estimated at 200 mutations / mb ( fig 1c ) , our molecular tumor board recommended immunotherapy with an immune checkpoint inhibitor . pembrolizumab was obtained for compassionate use . treatment led to a transient increase in the carcinoembryonic antigen ( cea ) from 2 , 742 ng / ml to a peak of 3 , 727 ng / ml followed by a decline to a plateau of 57 to 83 ng / ml which was maintained through 25 months of treatment and an additional 3 months of follow - up ( fig 2a )  . this was associated with resolution of pain and normalization of performance status from eastern cooperative oncology group status of 2 to 0 . 
bars above the graph indicate periods of treatment with uorouracil and irinotecan ( folfiri ) , uorouracil and oxaliplatin ( folfox ) , regorafenib , triuridine ( tfd ) and tipiracil ( tpi ) , radiation therapy ( xrt ) to the left supraclavicular mass , and the programmed death 1 ( pd - 1 ) inhibitor pembrolizumab . 
the delayed response was consistent with slow clearance of mucin after tumor cell death . the patient experienced a brief episode of localized herpes zoster and later a brief episode of asymptomatic grade 1 transaminitis . 
each episode was addressed by withholding one cycle of pembrolizumab , and each quickly resolved without sequelae . discussion in recent years , immune checkpoint blockade has emerged as a safe and effective treatment of many solid tumors . 
in particular , tumors with high level of msi and mismatch repair deciency have shown dramatic responses to treatment with inhibitors.4 , 5 similarly , accumulating immune checkpoint evidence suggests that tmb alone , independent of mmr status , correlates with response to immune checkpoint blockade for some tumor types.3 , 6 - 9 hence , mss tumors with an ultra - mutated phenotype as a result of mutations in pole or pold110 , 11 represent an intriguing subset of tumors that may also respond to immune checkpoint inhibitors . the case presented here adds to the few reports of polemutated tumors that responded to pd - 1 checkpoint blockade . 
by contrast , the responsive tumors were inconsistent in pd - l1 expression ( fig 1d )  . unlike patients in the other cases , the patient in this report began treatment with the greatest extent of disease and enjoyed the longest sustained response ( which continued beyond 28 months )  . 
radiologic density of tumors failed to change signicantly for 8 months before nally disappearing at 28 months . the dramatic response in this patient shows the potential benet of evaluating mss tumors for pole and possibly pold1 mutations . 
however , it is important to realize that some pole or pold1 mutations , particularly previously uncharacterized mutations , may prove to be passenger alterations with no effect on tmb.10 a prospective analysis of 80 , 853 patients with advanced solid tumors revealed known genomic alterations in pole in only 259 patients ( 0.3% ) , with a median tmb of 31 mutations / mb.17 the most common mutation was p.r446q ( n = 77 ) , which is uncharacterized , associated with low tmb ( less than ve mutations / mb ) , and predominantly germline . 
the two nextmost - common mutations , p.p286r ( n = 41 ) and p.v411l ( n = 29 ) , are both functional , associated with high tmb ( greater than 20 mutations / mb ) , predominantly somatic , and enriched in colorectal cancer and endometrial carcinoma . 
these were the mutations present in the four polemutant tumors that have responded to pd - 1 checkpoint blockade in this and other published reports ( fig 1d )  . however , not all pole - mutated tumors respond to checkpoint blockade . 
two colorectal cancers with the p.p286r mutation showed progressive and stable disease after 1 and more than 10 months of follow - up.15 both cases showed low levels of cd8 + tils . 
lo employment : genentech , roche stock and other ownership interests : genentech , roche gilbert chu patents , royalties , other intellectual property : rapid small volume detection of blood ammonia , patent no . 
nature 487 : 330 - 337 , 2012 koopman m , kortman ga , mekenkamp l , et al : decient mismatch repair system in patients with sporadic advanced colorectal cancer . 
br j cancer 100 : 266 - 273 , 2009 rizvi na , hellmann md , snyder a , et al : mutational landscape determines sensitivity to pd - 1 blockade in nonsmall - cell lung cancer . 
science 348 : 124 - 128 , 2015 le dt , durham jn , smith kn , et al : mismatch repair deciency predicts response of solid tumors to pd - 1 blockade . 
science 357 : 409 - 413 , 2017 le dt , uram jn , wang h , et al : pd - 1 blockade in tumors with mismatch - repair deciency . 
n engl j med 372 : 2509 - 2520 , 2015 snyder a , makarov v , merghoub t , et al : genetic basis for clinical response to ctla - 4 blockade in melanoma . 
n engl j med 371 : 2189 - 2199 , 2014 goodman am , kato s , bazhenova l , et al : tumor mutational burden as an independent predictor of response to immunotherapy in diverse cancers . 
mol cancer ther 16 : 2598 - 2608 , 2017 . kowanetz m , zou w , shames d , et al : oa20.01 : tumor mutation burden ( tmb ) is associated with improved efcacy of atezolizumab in 1l and 2l + nsclc patients . 
santin ad , bellone s , buza n , et al : regression of chemotherapy - resistant polymerase ( pole ) ultra - mutated and msh6 hyper - mutated endometrial tumors with nivolumab . 
wang c , gong j , tu ty , et al : immune proling of microsatellite instability - high and polymerase ( pole ) mutated metastatic colorectal tumors identies predictors of response to antipd - 1 therapy . 
schrock ab , fabrizio d , he y , et al : analysis of pole mutation and tumor mutational burden ( tmb ) across 80 , 853 tumors : implications for immune checkpoint inhibitors ( icpis )  . 
 expanded analysis of secondary germline findings from matched tumor / normal sequencing identies additional clinically signicant mutations ecaterina ileana dumbrava , md1 ; lauren brusco , phd1 ; molly s . 
mills , md1 ; ken chen , phd1 ; and funda meric - bernstam , md1 purpose next - generation sequencing ( ngs ) for tumor molecular proling can reveal secondary germline likely pathogenic and pathogenic variants ( lpv / pv )  . 
the american college of medical genetics and genomics ( acmg ) recommends return of secondary results for a subset of 59 genes , but other genes with evidence of clinical utility are emerging . 
five mutations in highpenetrance hereditary cancer predisposition genes were selected to be returned to patients or their representatives : bap1 , cdh1 , cdkn2a , egfr , and smarca4 . conclusion broader genomic testing is likely to identify additional secondary pathogenic germline alterations , some with potential clinical utility for return to patients and their relatives . 
2019 by american society of clinical oncology introduction with the exponential development of next - generation sequencing ( ngs ) , specically the ability to sequence larger panels of genes in more depth , molecular proling increasingly is being integrated into oncology practice.1 , 2 the main goal of ngs is to identify actionable genomic alterations in the tumor to target by matched drugs in efforts to personalize treatment.3 in addition , ngs can be used for testing of prognostic biomarkers of disease progression and metastasis , testing of cancer predisposition genes , and cancer risk assessment for at - risk asymptomatic family members . sequencing matched tumor and normal tissue samples from the same patient can assist in more - accurate calling of somatic variants.4 - 6 often , germline variants are subtracted and ignored ; however , analysis of secondary germline ndings might identify variants associated with an increased susceptibility to develop cancer or other diseases . 
 ileana dumbrava et al solicit patients preference with regard to the return of pathogenic germline alterations before testing.9 however , the systematic return of germline alterations found during ngs requires implementation of bioinformatics programs for variant detection , curation , and annotation ; such implementation increases the required resources , time , and costs.9 , 10 in this study , we sought to determine whether ngs of matched tumor and normal samples revealed secondary germline pv and likely pathogenic variants ( lpv ) in genes beyond those currently recommended by the acmg . 
patients also were offered possible secondary germline mutation testing in a companion institutional review boardapproved protocol.11 the patients relevant clinical characteristics were collected from electronic medical records and prospectively maintained institutional databases . matched tumor / normal dna sequencing paired tumor / normal dna - targeted exome sequencing of 201 genes was performed in a research laboratory ( data supplement ) .12 tumor samples were acquired as formalinxed parafn - embedded slides in which the tumor area the tumorwas circled to facilitate macrodissection of containing region . 
the key elements of ngs , including dna extraction , library preparation , target enrichment , sequencing , and variant calling , were performed on tumor / normal tissue samples . 
alignment of the sequenced data to human reference assembly hg19 and variant calling of the sequencing reads have been described previously.13 - 15 single nucleotide variants and small indels were called using the genome analysis tool kit ( broad institute , cambridge , ma )  . 
loss of the normal allele in the tumor ( loss of heterozygosity ) was evaluated in the analyzed genes . selection of genes for analysis for secondary germline findings our group previously published the germline lpv / pv results for 18 of 56 genes recommended by acmg that were included in our 201 - gene panel.11 palb2 also was included in a previous secondary germline analysis because of its strong association with hereditary breast cancer.16 after a thorough literature search and evaluation of relevant databases , 17 - 19 we selected 54 additional genes from our panel for which we analyzed germline lpv / pv . 
these genes were selected on the basis of the mode of inheritance and known penetrance for disease phenotype , including genes previously described as hereditary - cancer susceptibility ; genes currently tested in commercially available hereditary panels ; cancer - related genes often tested on matched normal / tumor panels , however with the mode of inheritance nonconsistent with phenotypic expression in patients ; and noncancer - related genes with suggested familial heritance ( data supplement )  . 
on the basis of the mode of inheritance , known penetrance for a disease phenotype , and presence or absence of management guidelines , the identied lpv / pv were grouped into ve categories ( data supplement ) : ( 1 ) established hereditary cancer susceptibility genes ( not in the acmg recommended genes in 2015 , which were previously reported by our group11 ) and smad4 ( which was added to the recommended genes in 2016 ) , ( 2 ) hereditary cancer susceptibility genes with moderate penetrance included in available genetic testing panels with suggested management guidelines ( atm and chek2 ) , ( 3 ) genes wherein somatic variants are associated with cancer but germline mutations are associated with noncancer phenotypes , ( 4 ) other cancer - related genes with unknown clinical validity of pathogenic germline alterations , and ( 5 ) hereditary noncancer susceptibility genes with other possible clinical utility . clinical signicance interpretation of variants variant clinical signicance classication was assigned according to acmg guidelines.20 , 21 the online tools and databases used to classify the clinical signicance of the remaining variants are detailed in figure 1 . 
the clinical signicance annotation of all variants in the 54 additional genes analyzed in this study were independently analyzed using intervar , a bioinformatics software tool that uses an annotated le generated from annovar22 and classies each variant on the basis of the association of molecular pathology / acmg 2015 guidelines , 20 and by a scientist with germline variants annotation expertise from the institute for personalized cancer therapy at the university of texas md anderson cancer center . 
 ileana dumbrava et al recommendation for cancer somatic variant evaluation , we focused on tier i and ii variants , which include lpv / pv.21 furthermore , personal and family history of cancer was reviewed for all patients with identied lpv / pv . determination of which results to return to patients a committee of oncologists , a genetic counselor , molecular pathologists , an ethicist , and behavioral scientists developed criteria for return of results to patients . 
the lpv / pv in wellestablished hereditary cancer predisposition genes with high penetrance for which management recommendations were available were recommended to be returned to patients who expressed interest in knowing about secondary germline ndings . 
variants selected to be returned to patients or their personal representative , were validated with an orthogonal assay in a clinical laboratory improvement amendmentscertied laboratory using the same deidentied research specimen before formal genetic counseling and genetic testing . results study population among the 1 , 000 patients who underwent matched tumor / normal dna sequencing for personalized cancer therapy in the clearinghouse protocol , the most frequent tumor types were breast ( 25.3% ) and colorectal ( 15.6% ) cancers , glioblastoma ( 15.1% ) , melanoma ( 14.3% ) , and sarcoma ( 10.2% ; fig 2 )  . 
four patients had a personal history of a previous malignancy ( data supplement )  . we identied two patients who had two germline lpv / pv ( one mutation from the acmg - recommended return of results list that was found previously and one from the list of genes described here )  . 
the second patient was a 13 - year - old girl who had a solid pseudopapillary neoplasm of the pancreas with two deleterious germline mutations ( msh6 and ercc3 ) , and although she had no relevant family history , previous genetic testing revealed a germline mutation in msh6 suggestive of lynch syndrome . of the 46 patients with lpv / pv , only 11 ( 24% ) had been referred previously for genetic counseling . 
the most frequent lpv / pv were identied in the following genes : 10 chek2 mutations ( 1% ) , ve ar mutations ( 0.5% ) , four atm mutations ( 0.4% ) , four ercc3 mutations ( 0.4% ) , four mitf mutations ( 0.4% ) , four pkhd1 muthree nf1 mutations ( 0.3% ) , and two tations ( 0.4% ) , chek1 mutations ( 0.2% ; fig 3 )  . 
on the basis of currently available drugs ( food and drug administration approved or currently in clinical trials ) , 26 lpv / pv were found in eight potentially therapeutically actionable genes ( chek2 , ar , atm , nf1 , cdkn2a , egfr , hras , ptch1 )  . lpv / pv were found in several tumor types , most frequently breast cancer , colorectal cancer , glioblastoma , melanoma , sarcoma , and ovarian cancer ( table 2 )  . 
among the 10 patients with breast cancer with lpv / pv , 10 variants were observed in eight genes ( data supplement )  . of the 46 lpv / pv , 20 ( 44% ) were known to be pathogenic and previously reported in clinvar23 and related to an increased risk of cancer or other diseases . 
twenty - six variants were identied as lpv on the basis of the effect / location of the genomic change on the protein function or the databases and online tools shown in figure 1 . 
in cases of low median allele fraction , the results were rechecked and validated to be of germline origfour patients ( 8.7% ) presented loss of heterozygosity in the same genes as the identied germline lpv / pv ( two patients with nf1 mutation , one with atm mutation , and one with smarca4 mutation )  . concordance of manual and automatic variant interpretation variant clinical signicance was determined using databases and online tools ( fig 1 )  . 
all variants also were annotated using intervar.24 we compared manual annotations with the automatic annotation tool and observed concordance rates between 88% and 98% , which depended on the type of alteration analyzed ( point v truncating mutations )  . return of results to patients or their personal representative the committee decided that germline lpv / pv in the following established hereditary cancer predisposition genes with high penetrance should be returned to patients : bap1 , cdh1 , cdkn2a , egfr , and smarca4 ( appendix fig a1 )  . all these results were previously unknown to the patient , and none of the ve patients had been previously referred to genetic counseling or underwent prior genetic testing . all ve lpv / pv were conrmed using de - identied samples in a clinical laboratory improvement amendments certied laboratory using a different platform ( 100% concordance obtained )  . 
the patient underwent formal genetic testing and genetic counseling , and the cdkn2a p.g101w mutation was conrmed ; she was enrolled in a pancreatic cancer screening program in addition to dermatologic surveillance because of her history of metastatic melanoma , which is currently without evidence of disease . 
indeed , a recent study showed that more than one half of patients with deleterious secondary germline ndings would not have been tested using current clinical guidelines.27 in the current study , from the additional genes tested , only the nf1 mutations had been identied before the matched normal / tumor dna sequencing , which indicates that important cancer predisposition genes may be missed by current criteria for referral to a genetic counselor and genetic testing . 
furthermore , in addition to colon , breast , and ovarian cancers , we identied lpv / pv in melanoma , glioblastoma , sarcoma , and pancreatic and gastric cancers . the 2016 acmg recommendations changed the terminology to secondary ndings instead of incidental ndings because the genes were intentionally analyzed . 
 secondary germline findings on matched tumor discussion in the return of secondary germline findings committee about which germline lpv or pv should be reported confirmation of the result in clia - approved laboratory ( if initial testing done in a research laboratory ) genetic counseling ( initial visit ) patient or personal representative ( if patient deceased ) informed by genetic counselor and physician of secondary germline findings genetic counseling ( follow - up visit ) discussion of results of the genetic testing ( confirmation or not of germline secondary finding ) formal clinical genetic testing for suspected hereditary syndrome for patients ( after informed consent ) assessment and management of second primary cancer risk family risk assessment and guidance personalized targeted treatment determination establishment of germline knowledge database participant informed consent for : tumor profiling companion germline variants analysis return of results of secondary germline findings tumor and normal sample sequencing somatic molecular profile report germline sequence annotation fig 4 . 
clia , clinical laboratory improvement amendments ; lpv / pv , likely pathogenic and pathogenic variants . likely will evolve continuously as more knowledge about hereditary syndromes is accumulated and as ngs is integrated into oncology practice , which thus will increase the likelihood of detecting secondary germline ndings.8 the cancer risk predictions associated with our gene classications is based on highly penetrant families . 
lpv / pv in hereditary cancer susceptibility genes also are present in the general population at a very low frequency , and whether these ndings will have the same implications in patients without signicant family histories is unknown . although established public knowledgebases of lpv / pv , such as clinvar , 23 are helpful in determining the clinical signicance of germline mutations , this might have conicting results from different sources , and many variants are not yet described . 
to improve these databases , we need to share the results and establish guidelines for return of secondary germline ndings.35 the potential effect of reporting secondary germline ndings in cancer - related genes presents signicant opportunities to assess and manage the risk of second primary cancers , assess familial risk , and provide targeted treatment options . 
although the clinical utility of germline lpv / pv in some genes outside the acmg list is still unknown , rapid advances of biologic knowledge and new drug development could aid in identifying new actionable alterations to serve as the basis for future clinical trial design and personalized cancer therapy.36 , 37 increasing patient interest exists in knowing about secondary germline ndings ; however , which results should be returned remains controversial . 
in this study , a committee of oncologists , a genetic counselor , molecular pathologists , an ethicist , and behavioral scientists believed that in the absence of signicant cancer family history , the clinical utility of pathogenic secondary germline ndings in moderate penetrance genes like chek2 remains with many uncertainties and knowledge gaps ; thus , moderate penetrance genes were not returned . 
this decision was made , in part , because the patients were deceased , which made it difcult to denitively conrm results because they were obtained in the research environment . 
our process for identifying , conrming , and returning secondary germline ndings is summarized in figure 4 . results cases , 40 but this might be overcome with the integration of new variant analysis tools and education programs to ll the knowledge gap among treating cancer care providers and patients . the current study is that one limitation of the patient population was from an academic center where many patients were referred for consideration of clinical trials ; this population may inuence the overall results / detection of germline lpv / pv in particular tumor types . 
in addition , our return - of - results efforts did not include variants of unknown signicance that might have clinical implications in the future with the rapidly evolving eld . important barriers left to be overcome when disclosing germline deleterious variants are psychological outcomes and family communication barriers.38 , 39 in deceased patients , identication and contact of a personal representative and making the information on secondary germline ndings available but allowing the right to decline represent other barriers in return of results.25 currently , only 5% of genetic counselors feel prepared to handle tumor proling in conclusion , secondary germline ndings identied on tumor / normal dna proling could have implications in the assessment and management of second primary cancer risk ; family risk assessment and guidance ; and most importantly , personalized treatment determination . 
most patients would like to know about these secondary germline ndings for themselves and their families , 41 but some barriers remain to be overcome , such as the determination of which results to disclose and how to disclose them as well as the burdens that this process would place on the cancer care prograa systematic analysis of germline variants could increase the cost and time involved in dna sequencing and the interpretation of clinical signicance . 
hong , jordi rodon , scott kopetz , funda meric - bernstam collection and assembly of data : ecaterina ileana dumbrava , lauren brusco , chetna wathoo , kenna r . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . lauren brusco employment : celgene jennifer litton consulting or advisory role : pzer , astrazeneca , medivation speakers bureau : physicians education resource , uptodate , med learning group , medscape research funding : novartis ( inst ) , bristol - myers squibb ( inst ) , genentech ( inst ) , pzer ( inst ) , emd serono ( inst ) , jounce therapeutics ( inst ) , glaxosmithkline ( inst ) , medivation ( inst ) patents , royalties , other intellectual property : uptodate travel , accommodations , expenses : physicians education resource , med learning group , medscape sarina a . 
 secondary germline findings on matched tumor vivek subbiah consulting or advisory role : medimmune research funding : novartis ( inst ) , glaxosmithkline ( inst ) , nanocarrier ( inst ) , northwest biotherapeutics ( inst ) , genentech ( inst ) , roche ( inst ) , berg ( inst ) , bayer ag ( inst ) , incyte ( inst ) , fujilm ( inst ) , pharmamar ( inst ) , d3 oncology solutions ( inst ) , pzer ( inst ) , amgen ( inst ) , abbvie ( inst ) , multivir ( inst ) , blueprint medicines ( inst ) , loxo oncology ( inst ) , vegenics ( inst ) , takeda pharmaceuticals ( inst ) , alfasigma ( inst ) , agensys ( inst ) , idera pharmaceuticals ( inst ) , boston biomedical ( inst ) , inhibrx ( inst ) , exelixis ( inst ) travel , accommodations , expenses : pharmamar , bayer ag david s . 
hong stock and other ownership interests : molecularmatch , oncorena honoraria : adaptimmune , baxter , merrimack , bayer ag consulting or advisory role : baxter , bayer ag , guidepoint global , janssen pharmaceuticals , genentech , eisai , glg pharma research funding : novartis , genentech , eisai , astrazeneca , pzer , mirna therapeutics , amgen , daiichi sankyo , merck , mirati therapeutics , eli lilly , adaptimmune , abbvie , bayer ag , bristol - myers squibb , genmab , ignyta , innity pharmaceuticals , kite pharma , kyowa hakko kirin , loxo oncology , medimmune , molecular templates , takeda pharmaceuticals , seattle genetics , amgen travel , accommodations , expenses : loxo oncology , mirna therapeutics jordi rodon consulting or advisory role : novartis , eli lilly , imclone systems , servier , orion , peptomyc , kelun pharmaceutical , merck sharp & dohme , spectrum pharmaceuticals , pzer research funding : novartis , bayer ag scott kopetz stock and other ownership interests : molecularmatch , navire pharma consulting or advisory role : roche , genentech , emd serono , merck , karyopharm therapeutics , amal therapeutics , biocartis , navire pharma , symphogen research funding : amgen ( inst ) , sano ( inst ) , biocartis ( inst ) , guardant health ( inst ) , array biopharma ( inst ) , genentech ( inst ) , roche ( inst ) , emd serono ( inst ) , medimmune ( inst ) , novartis ( inst ) john mendelsohn stock and other ownership interests : merrimack patents , royalties , other intellectual property : royalty payments from university of california , san diego gordon b . 
mills stock and other ownership interests : catena pharmaceuticals , spindletop capital , immunome , signalchem , tarveda therapeutics honoraria : nuevolution , astrazeneca , tarveda therapeutics , tesaro , symphogen , immunome consulting or advisory role : astrazeneca , catena pharmaceuticals , critical outcome technologies , signalchem , tarveda therapeutics , symphogen , takeda pharmaceuticals , millennium pharmaceuticals , ion pharmaceuticals , immunome research funding : miriam and sheldon g . 
adelson medical research foundation , astrazeneca , nanostring technologies , breast cancer research foundation , karus therapeutics , immunome , ion pharmaceuticals , komipharm , ovarian cancer research foundation , pzer , prospect creek foundation , tarveda pharmaceuticals , millennium pharmaceuticals patents , royalties , other intellectual property : hrd assay to myriad genetics travel , accommodations , expenses : astrazeneca , pzer , immunome , symphogen funda meric - bernstam honoraria : sumitomo group , dialectica consulting or advisory role : genentech , inection biosciences , pieris pharmaceuticals , clearlight diagnostics , darwinhealth , samsung bioepis , spectrum pharmaceuticals , aduro biotech , origimed , xencor , debiopharm group research funding : novartis , astrazeneca , taiho pharmaceutical , genentech , calithera biosciences , debiopharm group , bayer ag , aileron therapeutics , puma biotechnology , cytomx therapeutics , jounce therapeutics , zymeworks , curis , pzer , effector therapeutics , abbvie , boehringer ingelheim ( i ) no other potential conicts of interest were reported . acknowledgment we thank bryan tutt in the department of scientic publications , the university of texas md anderson cancer center , for editorial assistance . references 33 : 2753 - 2762 , 2015 cummings ca , peters e , lacroix l , et al : the role of next - generation sequencing in enabling personalized oncology therapy . 
sci transl med 7 : 283ra53 , 2015 gray pn , vuong h , tsai p , et al : tumornext : a comprehensive tumor proling assay that incorporates high resolution copy number analysis and germline status to improve testing accuracy . 
oncotarget 7 : 68206 - 68228 , 2016 christoforides a , carpten jd , weiss gj , et al : identication of somatic mutations in cancer through bayesian - based analysis of sequenced genome pairs . 
bmc genomics 14 : 302 , 2013 green rc , berg js , grody ww , et al : acmg recommendations for reporting of incidental ndings in clinical exome and genome sequencing . 
genet med 19 : 249 - 255 , 2017 [ erratum : genet med 19 : 484 , 2017 ] robson me , bradbury ar , arun b , et al : american society of clinical oncology policy statement update : genetic and genomic testing for cancer susceptibility . j clin oncol 33 : 3660 - 3667 , 2015 10 . 
hegde m , santani a , mao r , et al : development and validation of clinical whole - exome and whole - genome sequencing for detection of germline variants in inherited disease . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
tung n , lin nu , kidd j , et al : frequency of germline mutations in 25 cancer susceptibility genes in a sequential series of patients with breast cancer . 
kline cn , joseph nm , grenert jp , et al : targeted next - generation sequencing of pediatric neuro - oncology patients improves diagnosis , identies pathogenic germline mutations , and directs targeted therapy . 
boland g , piha - paul s , subbiah v , et al : clinical next generation sequencing to identify actionable alterations in a phase i prograoncotarget 6 : 20099 - 20110 , 37 . 
the patient declined surgery , and the centrally located rll tumor was treated with stereotactic body radiation therapy ( sbrt ) at a dose of 50 gy in five fractions . 
 six months later , the patient presented with a 3 - month history of right hip and left shoulder pain , which was a biopsy - proven recurrence of the rll primary with diffuse bony metastases . 
 the patient consented to an institutional review boardapproved protocol ( #226210 at the university of california , davis )  . tumor and germline genomic sequencing assays plasma ctdna was isolated from 10 ml of whole blood drawn in streck cell - free dna tubes , enriched by hybrid capture to target exons of 70 genes and critical introns of six genes , and sequenced to an average depth of approximately 15 , 000 on an illumina nextseq500 ( guardant360 assay ; guardant health , redwood , ca ) at a clinical laboratory improvement actcertified commercial laboratory.6 germline dna was isolated from the patients pbmcs and enriched for targeted regions using a hybridization - based protocol and was sequenced with 50 or greater depth using illumina technology at a commercial genetic testing laboratory ( invitae , san francisco , ca )  . 
 methods for in vitro studies are described in detail in the data supplement . this patients family history suggests a hereditary predisposition to lung cancer : three other individuals across three generations had a history of lung cancer ( data supplement )  . 
tumor genomic profiling of plasma ctdna with the guardant360 assay identified concurrent low ( 0.3% ) mutation allelic frequency ( maf ) of egfr l858r and high ( 51% ) maf of egfr t790m ( fig 1a - 1b )  . 
 the patient was referred to a genetic counselor , and germline dna sequencing confirmed that this patient carried a germline egfr t790m mutation . we determined the in vitro cytotoxicity of egfr tkis on this patients pbmcs using a permanent epstein barr virustransformed lymphoblastoid cell line , a good surrogate for the patients germline genomic materials , 11 , 12 established before initiation of any systemic treatment . 
we found that the lymphoblastoid cells ( fig 2a ) and the patients pbmcs ( fig 2b ) were resistant to all first - , second - , and third - generation egfr tkis tested . 
consistent with previous reports , 13 h3255 cells ( which harbored only an oncogenic egfr l858r mutation ) were sensitive to all egfr tkis ( fig 2c ) , whereas h1975 cells ( which harbored both oncogenic egfr l858r and t790m mutations ) were resistant to erlotinib but sensitive to afatinib and osimertinib ( fig 2d )  . 
we found that the patients pbmcs and pbmc - derived cells did not express egfr and akt protein , the two key signaling molecules that mediate the downstream oncogenic signaling pathways in h3255 and h1975 cells , by western blots ( fig 2e )  . 
we also confirmed that there was no egfr mrna expression in patients pbmcs or in the patient - derived ebv - transformed lymphoblastoid cell line ( data supplement )  . 
thus , afatinib and osimertinib could be used safely in this patient with a germline egfr t790 mutation . during the in vitro studies , the patient first received palliative radiation for the right hip pa he had stable disease after first - line combination chemotherapy with carboplatin , pemetrexed , and bevacizumab for four cycles , and he experienced tumor progression at the primary rll and metastatic bony lesions after maintenance treatment with pemetrexed and bevacizumab for four cycles . 
a restaging positron emission tomography ( pet ) / ct scan 2 months after afatinib treatment began revealed extensive tumor progression in the primary rll tumor and multiple bone metastases . 
serial guardant360 assays revealed that the changes in the maf of egfr l858r in plasma ctdna correlated with the tumor responses to chemotherapy , afatinib , and osimertinib ; the maf of egfr t790m remained at approximately 50% ( fig 1a - 1b )  . 
time points ( ie , dates ) were selected on the basis of radiographic assessments at baseline and after each treatment according to recist version 1.1 during the entire disease course . 
targeted ngs of malignant cells in pleural effusion by foundationone ( foundation medicine , cambridge , ma ) revealed multiple genomic alterations , including egfr l858r and t790m ( fig 1c )  . 
similar to plasma ctdna analysis , egfr genotyping of the patients tumor dna by droplet digital polymerase chain reaction revealed that the maf of egfr l858r in tumor dna increased with tumor progression , whereas the maf of egfr t790m remained at approximately 60% ( fig 1c )  . 
the patient did not tolerate subsequent treatment on a clinical trial and died on hospice approximately 3 months later . discussion routine clinical application of multiplex tumor genomic profiling has increased the detection rate of rare germline mutations that may affect the patients clinical care and that raise concerns for cancer risks in family carriers.15 , 16 the maf of a somatic mutation in the plasma ctdna is usually much lower than that of archived tumor dna , whereas the maf of germline egfr t790m remains at 50% to 60% in plasma ctdna and tumor dna . 
results from the inherit study ( nct01754025 ) , which provided free genetic counseling and tested for lung cancer in patients and family members with an egfr t790m mutation at diagnosis and at longitudinal surveillance with serial ct scans , 17 are highly anticipated . patients with lung cancer who have a germline egfr t790m usually harbor a distinct spectrum of one or more concurrent oncogenic egfr mutations ( data supplement )  . 
the median progression - free survival was 4.9 months in the pooled analysis of patients ( the data supplement ) , whereas the median progression - free survival was 9 to 13 months in patients with egfr tkisensitive somatic egfr mutations.18 furthermore , data from our patient and the patients in the data supplement also revealed less clinical benefit from chemotherapy and radiation , which suggests tumor resistance to these standard therapeutic strategies . 
stollenwerk , tianhong li provision of study material or patients : debbie lewis , yanhong zhang , jamie hung , jenna welborn , tianhong li data analysis and interpretation : weijie ma , jidong shan , wenwu xiao , yanhong zhang , sixi wei , kit s . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . weijie ma no relationship to disclose jay gong no relationship to disclose jidong shan no relationship to disclose debbie lewis no relationship to disclose wenwu xiao no relationship to disclose elizabeth h . 
moore no relationship to disclose yanhong zhang no relationship to disclose jamie hung no relationship to disclose patents , royalties , other intellectual property : patent for device and method for assessing beam quality in computed tomography by measuring the half - value layer ( inst ) sixi wei no relationship to disclose jeanna welborn no relationship to disclose nicholas s . 
lam leadership : lamnotherapeutics stock and other ownership interests : lamnotherapeutics , lp therapeutics , cardioprobe patents , royalties , other intellectual property : i have more than 20 issued patents , many with the university of california , davis ( no royalty received in the past 2 years )  . tianhong li consulting or advisory role : foundation medicine , takeda , puma research funding : foundation medicine ( inst ) , pfizer ( inst ) , astrazeneca ( inst ) , hengrui ( inst ) acknowledgment we dedicate this work to the late jeannine h . 
mans stock and other ownership interests : concert pharmaceuticals , corcept therapeutics , varex imaging , verasterm , ziopharm oncology affiliations weijie ma , jay gong , wenwu xiao , elizabeth h . 
3 - ( 4 , 5 - dimethylthiazol - 2 - yl ) - 5 - ( 3 - carboxymethoxyphenyl ) - 2 - ( 4 - sulfophenyl ) - 2h - tetrazolium assay for ebv - transformed lymphoblastoid cell line established from ( a ) this patient , ( b ) the patients pbmcs , ( c ) h3255 , and ( d ) h1975 . 
 ( e ) the expression of egfr and downstream signaling molecules in the ebv - transformed lymphoblastoid cells and pbmcs from this patient , h3255 , and h1975 are shown . 
mulloy r , ferrand a , kim y , et al : epidermal growth factor receptor mutants from human lung cancers exhibit enhanced catalytic activity and increased sensitivity to gefitinib . 
lanman rb , mortimer sa , zill oa , et al : analytical and clinical validation of a digital sequencing panel for quantitative , highly accurate evaluation of cell - free circulating tumor dna . 
ancevski hunter k , friedland dm , villaruz lc , et al : first - line osimertinib in patients with treatment - naive somatic or germline egfr t790m - mutant metastatic nsclc . 
hussain t , kotnis a , sarin r , et al : establishment and characterization of lymphoblastoid cell lines from patients with multiple primary neoplasms in the upper aero - digestive tract & healthy individuals . 
li d , shimamura t , ji h , et al : bronchial and peripheral murine lung carcinomas induced by t790m - l858r mutant egfr respond to hki - 272 and rapamycin combination therapy . 
meric - bernstam f , brusco l , daniels m , et al : incidental germline variants in 1 , 000 advanced cancers on a prospective somatic genomic profiling protocol . 
hu y , alden rs , odegaard ji , et al : discrimination of germline egfr t790m mutations in plasma cell - free dna allows study of prevalence across 31 , 414 cancer patients . 
oxnard gr , heng jc , root ej , et al : initial results of a prospective , multicenter trial to study inherited lung cancer risk associated with germline egfr t790m : inherit egfr . 
kris mg , natale rb , herbst rs , et al : efficacy of gefitinib , an inhibitor of the epidermal growth factor receptor tyrosine kinase , in symptomatic patients with non - small cell lung cancer : a randomized trial . 
 oncologists use of genomic sequencing data to inform clinical management purpose to determine whether oncologists intended to change treatment as a result of tumor sequencing , and subsequently , whether patients experienced an alteration of clinical management or derived clinical benefit . patients and methods a prospective survey of oncologists referring adult patients with rare , advanced , or refractory cancer to the michigan oncology sequencing program was conducted from june 2014 to march 2015 to assess the use of and intent to disclose sequencing findings . 
medical records were reviewed retrospectively to determine if clinical management was informed or changed by sequencing results . results oncologists ( response rate , 93% ) referring 112 consecutive patients were surveyed . 
2018 by american society of clinical oncology introduction clinical implementation of tumor genomic sequencing is emerging as a promising strategy to improve patient outcomes in oncology.1 integrating an individuals clinical history with genomic data can inform both diagnostic and treatment strategies , potentially suggesting therapies that are tailored to the patients tumor mutational landscape.2 there are several ongoing national efforts to evaluate the effectiveness of treating cancers with targeted therapies informed by alterations in patients tumors . 
the national cancer institute 's molecular analysis for therapy choice ( match ) is a basket study matching patients with metastatic solid tumor malignancies to food and drug administrationapproved or experimental drugs on the basis of specific tumor alterations.3 the tumor agent and profiling utilization registry study ( tapur ) , sponsored by asco , matches patients to commercially available targeted antineoplastic agents on the basis of potentially actionable tumor events.4 the initiative for molecular profiling in advanced cancer therapy ( impact ) program in the phase i clinic at md anderson cancer center matches targeted agents with tumor molecular alterations in patients with advanced cancer.5 evaluating the evidence for improved outcomes for patients with cancer matched to targeted agents is an essential first step . 
scott roberts author affiliations and support information ( if applicable ) appear at the end of this article . the study funders had no role in the design of the study ; the collection , analysis , or interpretation of the data ; the writing of the manuscript ; or the decision to submit the manuscript for publication . corresponding author : michele c . 
furthermore , there is debate among medical professionals about what should be considered clinically actionable and what types of findings from clinical sequencing should be disclosed to patients.6 - 12 still , it is common for studies that use sequencing results to match patients with targeted therapy to report the frequency of actionable results.13 however , few report what percentages of these actionable results were eventually acted on in clinical practice . currently , most commercially available tumor testing platforms focus on targetable alterations in a limited set of genes . 
in contrast , the michigan oncology sequencing ( mi - oncoseq ) program has adopted a more comprehensive approach by sequencing the genome , exome , and transcriptome of tumors , as well as a matched normal sample , in an effort to characterize novel variants that may increase cancer risk . 
 this has led to the identification of alterations with important clinical implications , such as the activation of esr1 mutations in patients with estrogen receptor - positive metastatic breast cancer refractory to hormonal therapies , implicating these mutations as a likely mechanism of resistance to endocrine therapy.3 furthermore , novel fibroblast growth factor receptor fusions , potentially targetable with fibroblast growth factor receptor inhibitors currently being studied in clinical trials , have been identified across a diverse range of cancer types.4 these discoveries highlight how taking a comprehensive approach to identifying therapeutic targets for individual patients with cancer can expand the molecular taxonomy of cancer . 
to address whether oncologists are using sequencing findings to change a patients clinical management , and to what extent potentially actionable findings are being acted on , we surveyed oncologists referring patients with rare , advanced , or refractory cancer to the mi - oncoseq program , a precision oncology research study at university of michigan comprehensive cancer center.14 we also examined whether there was concordance between an oncologists intention to disclose the finding and the referred patients perception of a disclosure of results . 
detailed information on the program , including patient eligibility criteria , have been reported previously.14 results were generated within approximately 6 to 8 weeks , and potentially actionable findings were discussed at an institutional precision medicine tumor board ( pmtb ) composed of members with expertise in medical oncology , hematology , clinical pathology , cancer genetics and genetic counseling , bioinformatics , and bioethics . 
the mi - oncoseq program received institutional review board approval from the university of michigan . framework for classification of genomic alterations to determine the clinical relevance or potential actionability of comprehensive sequencing results , the study team developed a post hoc clinical tiering system to allow for a uniform and scalable approach for classifying alterations for research purposes ( table 1 )  . tier 1 alterations represent the highest level of clinical evidence and are generally incorporated into national comprehensive cancer net work guidelinebased recommendations for the treatment of malignancies . 
this includes food and drug administrationapproved therapies in the context of a molecular biomarker that predicts therapeutic response to a drug , pathogenic germline alterations conferring increased cancer risk , or sequencing information that contributed to a change in cancer diagnosis for tumors of unknown primary orig tier 2 alterations represent the identification of molecular alterations that meet the enrollment criteria for a clinical trial or registry study of a targeted therapeutic agent . 
potentially actionable genomic alteration classification tier 1 approved biomarker predictive of therapeutic response to an approved drug in that disease for example : brafv600e mutation , vemurafenib , melanoma germline alteration conferring increased cancer risk for example : pathogenic germline brca1 alteration change in cancer diagnosis for example : cup determined to be nsclc tier 2 rationale for enrollment in a clinical trial or registry study ( tapur ) for example : fgfr2 fusion in cholangiocarcinoma , fgfr inhibitor bgj398 tier 3 a . 
alteration in known oncogene or tumor suppressor for example : apc mutation abbreviations : cup , carcinoma of unknown primary ; fgfr , fibroblast growth factor receptor ; nsclc , nonsmall - cell lung cancer ; tapur , tumor agent and profiling utilization registry study ; vegfr , vascular endothelial growth factor receptor . particular molecular alteration with regard to tumor pathogenesis , but are not considered to be clinically actionable . oncologist survey after receiving the pmtb report , oncologists were surveyed about their intended use of sequencing information . 
each oncologist received a survey for every patient he or she referred during the 9 - month period , and therefore , the same oncologist may have completed multiple surveys . 
for example , oncologists were asked , will you make any changes to this patients cancer treatment based on pmtb and / or the mi - oncoseq report ? ( response options were yes , no , not sure . ) if the oncologist selected yes , he or she was asked , what changes will you make ? if the oncologist endorsed no or not sure , he or she was asked , what is your reasoning for not making any changes ? additional items asked about intention to disclose the findings to patients as well as how and when this communication would occur . 
 patient surveys patients enrolled in the mi - oncoseq program were administered surveys after they consented to participate in the study and after the referring oncologist received the pmtb report . 
 at baseline , a 23 - item survey assessed patients knowledge and expectations regarding genomic sequencing , including beliefs about incidental findings , and preference for the return of results . 
 a follow - up survey mailed approximately 2 weeks after the referring oncologist received the results assessed patients fulfillment of expectations , decisional regret , satisfaction with results , and behavioral changes . concordance between oncologist and patient responses to assess gaps in the communication process , we compared items about an oncologists intention to disclose the sequencing results with the patients recollection of a results disclosure discussion . 
 specifically , will you share the genetic sequencing results with this patient ? ( response options were yes , no , not sure . ) similarly , in their follow - up survey , patients were asked , since you began participating in this research study , has your doctor ( s ) discussed one or more of your genome sequencing test results with you ? ( response options were yes , no , not sure . ) next , we conducted a content analysis of patients medical records to find documentation of a disclosure discussion . 
outcomes assessed included whether treatment was altered on the basis of results , the type of treatment or change in management , and the identification of a germline alteration with implications for management of patient or family member care ( eg , cascade testing )  . data analysis descriptive data , including frequencies and percentages , were calculated for demographic variables and responses to the survey questions for both the referring oncologists and the patients . 
whether survey participants characteristics and responses predicted intention to change clinical management and report of a results discussion were compared using fishers exact tests for categorical variables for low cell counts . 
all analyses were conducted using spss version 22 ( spss , chicago , il )  . results oncologist and patient surveys forty - three oncologists referring 112 patients to the mi - oncoseq study between june 2014 and march 2015 completed the survey ( response rate , 93% )  . 
approximately one half ( 52% ) of the oncologists surveyed referred three or more patients ( median , two patients ; range , one to 12 patients )  . of the 112 patients , the mean age was 57 years ( sd , 12.7 years ) , 51.8% were female , 94.6% self - reported as being white , and 32.1% were college graduates ( table 2 )  . 
the oncologists had no plans to change the treatment of 51% of patients ( n = 60 ) , or were not sure if they were going to make any treatment changes for 28 patients . 
 the most frequently endorsed reasons for not changing clinical management were a lack of locally available trials offering therapies relevant to the findings ( 59% ) , findings not actionable ( ie , not enough clinical evidence or results not clinically significant ; 27.4% ) , and the effectiveness of a patients current treatment ( 12% )  . regardless of an intention to change treatment , oncologists intended to share sequencing results with 94 of the 112 patients ( 84% )  . 
several indicated that they would disclose results by telephone ( 20% ) or before the patients next visit ( 24% )  . intention to change treatment and the mode of communication of findings were stratified by the median number of patients referred and the patient diagnosis . 
no variables significantly predicted intention to change treatment ( fig 1 )  . concordance between oncologist and patient responses of the 94 patients for whom an oncologist intended to share results , 57 completed the follow - up survey . 
oncologist and patient survey respondent characteristics respondent year medical training completed , median ( range ) 1993 ( 1969 - 2013 ) hematology and medical oncology oncologist primarily sees patients at an academic medical center no . 
 ( % ) unless indicated otherwise . * includes physicians referring patients to the study between june 2014 and february 2015 ; 94% response rate . and a documented note in their medical record supported their response . 
controlling for referring oncologist or cancer type did not significantly predict report of a results discussion . actionability classification of tumor genomic alterations , and patient outcomes on the basis of the retrospective chart review , nine of 24 patients had actual changes in clinical management informed by sequencing results . 
 on the basis of a review of medical records , these reasons included patient barriers ( eg , unable to travel to referred trial ) , insurance or cost ( eg , phase i clinical trial not covered ) , ineligibility for an identified study ( eg , impaired liver or renal function ) , and loss to follow - up or patient deceased . discussion comprehensive genomic sequencing offers the opportunity to characterize somatic and germline alterations for patients who have exhausted standard therapies and to potentially inform patients of additional treatment options . 
in this study , we found that oncologists reported intentions to make changes to treatment on the basis of genomic findings for 24 of 112 patients with rare or refractory cancers . 
these findings demonstrate both the potential benefit that can result from taking a comprehensive approach to identify targeted therapies tailored to a patients mutational landscape and the barriers to implementation of tumor - related genomic results into clinical management . 
significant obstacles include , but are not limited to , the need for additional educational support for both clinicians and patients , the need to define what constitutes an actionable finding , lack of access to clinical trials and off label therapies , and the need for future research to develop improved patient - provider communication mechanisms . our findings are consistent with those of other studies that have reported notable barriers to the integration of sequencing results into clinical practice.15 , 16 we found several instances that suggested a need for educational resources to support both the patient and the clinician . 
 example , patients with advanced or rare disease , such as the patients in this study , often join multiple research studies , go through multiple treatments regimens in a brief timeframe , and meet with several providers . 
it is reasonable that patients might not remember the specific study being discussed , be fatigued from treatments and appointments , or be confused by complex medical terminology . defining what constitutes an actionable finding is often challenging when multiple stakeholders are involved . 
of cases ( n = 112 ) abbreviations : cmml , chronic myelomonocytic leukemia ; dlbcl , diffuse large b - cell lymphoma ; nsclc , nonsmall - cell lung cancer ; scc , squamous cell carcinoma . the need for improved and better - designed test reports . discordance between an oncologist and a patients perceptions of a disclosure of results suggests the need for improvements in patient provider communication as a point of intervention . 
for referred to mi - oncoseq ( n = 122 ) completed survey ( n = 112 ) planned to share results with patients ( n = 94 ) did not plan or unsure of plan to share results with patients ( n = 18 ) completed follow - up survey ( n = 57 ) loss to follow - up ( n = 19 ) deceased or hospice ( n = 18 ) completed follow - up survey ( n = 7 ) loss to follow - up ( n = 4 ) deceased or hospice ( n = 7 ) had a discussion with physician ( n = 23 ) did not have a discussion with physician ( n = 34 ) had a discussion with physician ( n = 1 ) did not have a discussion with physician ( n = 6 ) medical chart review ( n = 57 ) documented disclosure discussion ( n = 14 ) discussion after follow up survey ( n = 5 ) no documentation of disclosure ( n = 15 ) ascopubs.org / journal / po jco precision oncology 7 fig 1 . 
 actionable can mean different things to different individuals on the care tea therefore , establishing a clear framework for defining actionable findings is critical to the implementation of comprehensive genomic sequencing information in clinical management . 
our attempt to address this barrier was the tiering scalable framework for classification of alterations to be implemented in future test reports . other prominent barriers patients and oncologists faced included uncertainty about locally available clinical trials and the lack of access to off - label therapies . 
these barriers include a lack of information about existing trials and therapies , a lack of resources to identify trial eligibility , inability to obtain approval for compassionate use for off - label drugs , and logistical challenges for the patient ( travel , lack of insurance coverage , or financial constraints )  . our study sample is not representative of oncologists in general given its over - representation of practitioners in an academic medical center . 
in addition , because medical record information is not always complete and may not accurately capture the existence or nature of discussions with patients , the verification of disclosures and outcomes was limited to a subset of individuals . 
le no relationship to disclose natalie bartnik no relationship to disclose elena stoffel research funding : cancer prevention pharmaceuticals ( inst ) scott schuetze consulting or advisory role : emd serono , janssen pharmaceuticals , daiichi sankyo research funding : ab science ( inst ) , janssen pharmaceuticals ( inst ) , amgen ( inst ) , biomed valley discoveries ( inst ) , cytrx ( inst ) , plexxikon ( inst ) , eli lilly ( inst ) , karyopharm therapeutics ( inst ) , adaptimmune ( inst ) moshe talpaz consulting or advisory role : gilead sciences , cti biopharma , nynex research funding : gilead sciences , incyte , cti biopharma , ariad pharmaceuticals , novartis , aptose biosciences , pfizer , sanofi expert testimony : bristol - myers squibb canada arul chinnaiyan consulting or advisory role : tempus j . 
tsimberidou am , wen s , hong ds , et al : personalized medicine for patients with advanced cancer in the phase i program at md anderson : validation and landmark analyses . 
scheuner mt , peredo j , benkendorf j , et al : reporting genomic secondary findings : acmg members weigh genet med 17 : 27 - 35 , 2015 7 . 
brandt ds , shinkunas l , hillis sl , et al : a closer look at the recommended criteria for disclosing genetic results : perspectives of medical genetic specialists , genomic researchers , and institutional review board chairs . 
townsend a , adam s , birch ph , et al : i want to know whats in pandoras box : comparing stakeholder perspectives on incidental findings in clinical whole genomic sequencing . 
middleton a , morley ki , bragin e , et al : attitudes of nearly 7000 health professionals , genomic researchers and publics toward the return of incidental results from sequencing research . 
although survival from any pediatric cancer has improved dramatically during past decades , a number of cancers continue to yield dismal prognoses , which has motivated the continued study of novel therapeutic strategies . 
furthermore , even patients cured of pediatric cancer often experience severe adverse effects of treatment and other long - term health implications , such as cardiotoxicity or loss of fertility . 
fortunately , pediatric cancers are rare overall , but even among patients with the same narrow cancer type , there is often broad heterogeneity in terms of prognosis , molecular features or pathology , current treatment strategies , and scientic objectives . 
as a result , the design of clinical trials in the pediatric cancer setting is challenged by a number of practical issues that must be addressed to ensure trial feasibility for this vulnerable group of patients . 
in this review , we discuss some of the unique trial design considerations often encountered in any rare tumor setting through the lens of our experiences as faculty statisticians for the childrens oncology group , the largest organization in the world dedicated exclusively to pediatric cancer research and clinical trials . these topics include risk stratication within individual trials , relaxation of trial operating characteristics and parameters , use of historical controls , and address of noninferiority - type objectives in small cohorts . 
2019 by american society of clinical oncology introduction overview of pediatric cancer and current directions in the united states , cancer remains the leading cause of disease - related death in children.1 , 2 although the prognosis for most pediatric patients has improved dramatically during the past several decades to achieve an overall 5 - year survival rate of 84% , pediatric cancer truly comprises many heterogenous diseases with a great deal of variability in expected survival outcomes and current scientic objectives.3 , 4 among the childhood cancer types with the best prognosis ( thanks in large part to decades of therapeutic intensication trials for improved survival ) , achievement of an incremental improvement upon current outcomes remains a key objective of most pediatric trials alongside objectives and novel treatment strategies intended to minimize short - term treatment - related toxicities and improve longer - term considerations , such as fertility , cardiac health , and quality of life.5 - 9 for other childhood cancers , such as acute myelogenous leukemia , high - risk neuroblastoma , and brainstem glioma , progress has been more limited , with 50% or more patients eventually succumbing to their disease . 
in those diseases , the study of novel therapeutic strategies to improve overall survival ( os ) remains the highest priority.10 , 11 role of the childrens oncology group the childrens oncology group ( cog ) , a member of the national clinical trials network supported by the national cancer institute , is the worlds largest organization devoted exclusively to childhood and adolescent cancer research . 
the current organizational structure of cog was established in the year 2000 as the result of a voluntary merger of its four predecessor groups : the childrens cancer study group ( ccg ) , pediatric oncology group ( pog ) , intergroup rhabdomyosarcoma study group , and national wilms tumor study group . 
more than 90% of the 14 , 000 children and adolescents newly diagnosed with cancer each year in the united states are cared for at cog these member institutions , with more than half of patients historically enrolling in cog therapeutic clinical trials . 
 renfro et al context challenges . key objective clinical trials in pediatric cancer and other rare diseases often include unique statistical features or design knowledge generated in the pediatric setting , common design components include use of prognostic risk stratication and use of data from historical controls . 
statistical challenges include maintaining power or addressing noninferiority - type hypotheses when only a limited population of patients is available to enroll . relevance we provide an overview of these features and challenges and describe existing approaches in the literature as well as those used in example trials from the childrens oncology group . active follow - up in cog studies at more than 220 leading childrens hospitals , universities , and cancer centers across the united states , canada , australia , and new zealand . pediatric cancer as a rare disease with trial planning implications fortunately , even the most common pediatric cancers occur only rarely in the general population , representing only 1% of new cancer diagnoses in the united states.12 also fortunate is the current availability of effective therapies for most of these patients . 
however , both the rarity of pediatric cancers and their generally favorable outcomes present unique challenges to clinical trial design , where improving outcomes and lessening the burden of treatment remain critically important . in some pediatric malignancies , it is not feasible to conduct a randomized phase iii clinical trial to detect a modest , yet clinically relevant improvement in outcome with high statistical power and a low type i error rate . 
furthermore , with targeted therapies now increasingly regarded as promising treatment strategies for patients whose tumors harbor particular molecular features or mutations , the already small disease populations that currently comprise pediatric cancer are becoming even smaller as a result of additional molecular characterization , thereby exacerbating some of the usual trial design challenges inherent in any rare disease . 
in addition , for those pediatric cancer subtypes where exceptional prognosis or cure can now reasonably be expected with standard treatment , current and future trials likely will be designed to answer questions such as whether historically toxic treatments can be safely reduced or altered in some way without negatively affecting outcomes . these next - generation questions , although important to answer , generally necessitate noninferiority ( ni ) - type trial design , which on average , requires a far greater sample size than a traditional phase iii randomized trial . in this article , we highlight several trial design features and challenges that are common to pediatric cancer trials and , in some cases , are broadly applicable to trials for any rare cancer . 
these include risk stratication as a design component , relaxation of trial operating characteristics to maintain feasibility , alternative approaches to testing nitype hypotheses , and use of historical controls . 
these risk groups might be derived from a number of patient and disease factors , such as age of the patient , size or local stage of the tumor , tumor histology , molecular biomarkers , or other features that have been previously shown to predict patients natural course of disease or response to standard treatments . 
in most cog trials where upfront risk stratication takes place , patients assigned to different strata ( eg , low risk , intermediate risk , high risk ) will go on to receive ( or be randomly assigned to ) treatment strategies of increasing intensity , depending on the severity of their initial risk assignment . 
in some pediatric cancer types , therapeutic deintensication is the main objective for those known to be at low or very low risk of adverse outcomes , such as disease recurrence or progression . challenges for risk stratication in rare diseases although risk stratication offers a logical and feasible approach to studying prognostically different cancer stages or subtypes under a unied protocol , this practice presents some challenges . 
first , it is common in pediatric cancers for stratum - specic risk classications to change over time because knowledge of the individual diseases and their corresponding standard treatments continue to evolve . 
 trial design for precision oncology in rare tumors group of patients considered to be very low risk during one era of treatment trials of a given cancer might be redened to become more inclusive in the next era , whereas discovery of a new negatively prognostic biomarker present across all risk groups might add a layer of upstaging from lowerto higher - risk groups across the board . 
another challenge of risk stratication is that introduction of a new risk factor or threshold to distinguish lower - risk from higher - risk patients might be based on limited patient numbers or incomplete clinical evidence ( eg , a biomarker collected for only a subset of patients ) , which precludes a statistically rigorous evaluation of the risk classication system in question and leaves much to the discretion and instincts of study investigators . 
even the methodology originally used to determine that a particular patient or disease feature is sufciently prognostic to map a patient to a different risk level ( and thus treatment assignment ) , such as whether a patients age is above or below some threshold at the time of diagnosis , might be questioned or re - evaluated after more data become available . 
modications , updates , or other inconsistencies between risk classications from one generation of trials to the next within a single disease type can lead to challenges in dening risk groupspecic historical controls when planning the next generation of trials , particularly when available cohorts of reference patients are already small or systematically different in some other way ( historical controls will be discussed subsequently in this article )  . nonetheless , risk stratication remains central to many pediatric cancers , and thus , the development of optimal approaches for risk stratication in clinical trials remains of critical interest to cog researchers . examples of risk stratication in cog trials hepatoblastoma . 
on the basis of these ndings , cog opened the hepatoblastoma studies int - 009815 in 1989 and p964516 in 1999 , using resectability at exploratory surgery and histology information to dene a risk stratication framework . 
in the early 2000s , the radiologybased pretreatment extent of disease ( pretext ) staging factor in risk stratication system became a dominant because exploratory surgery was no longer routinely supported with the advent of advanced radiographic imaging techniques . 
in 2009 , cog opened a new hepatoblastoma study , ahep0731 that ( clinicaltrials.gov identier : nct00980460 ) , that incorporated pretext and - fetoprotein in the risk stratication . as noted previously , the performance of direct comparisons of patient outcomes from trials or groups that use dissimilar risk classication strategies is challenging , and such was the case in hepatoblastoma . 
to address this issue , the childhood hepatic tumor international consortium established a database that consists of 1 , 605 patients from eight prior studies from cog , the international childhood liver tumors strategy group , the german society for pediatric oncology and haematology , and the japanese study group for pediatric liver tumors.17 the goal was to develop a common risk stratication system for pediatric hepatic tumors . 
figure 1 illustrates the details of this new risk stratication tool , which has been implemented in the pediatric hepatic malignancy international therapeutic trial ( phitt ) with participants from cog , the international childhood liver tumors strategy group , and the japanese study group for pediatric liver tumors . 
acute lymphoblastic leukemia ( all ) is the most common cancer diagnosed in children , with an estimated 3 , 500 diagnoses per year in persons younger than 20 years of age in the united states.18 in the current era , overall cure rates for children with newly diagnosed all are approaching 85%.19 although survival gains during the past four decades are noteworthy , many children predicted to be at low risk of relapse at initial diagnosis ultimately experience treatment failure . 
all other patients were classied as nci standard risk . yes is dened as the presence of double trisomy 4 and 10 or etv6 - runx1 fusion . consists of patients with cns3 , hypodiploidy ( , 44 chromosomes and / or dna index , 0.81 ) , intrachromosomal amplication of chromosome 21 , induction failure ( m3 marrow day 29 ) , or kmt2a ( mll ) rearrangement ( not kmt2a deletion )  . 
bcr - abl1positive patients are eligible for a separate , dedicated philadelphia chromosomepositive acute lymphoblastic leukemia study , aall1122 or aall1631 ( if open )  . includes four nci standard - risk and high - risk patients with testicular disease . group classication protocol aall03b1 , formed the basis for the renement of the risk stratication algorithm subsequently used in aall08b1 . initial registration and risk stratication in after aall08b1 , patients could be deemed eligible for a cog frontline all treatment trial as follows . 
additional studies performed at local and cog reference laboratories at the time of initial diagnosis and at dened time points during induction were used to rene postinduction therapy . patients with national cancer institute standard - risk and high - risk20 b - lymphoblastic leukemia ( b - all ) 21 were then distributed among the following four risk groups after induction : low risk , average risk , high risk , and very high risk ( table 1 )  . 
although a welcome problem is that children with cancer are living longer than ever before , a lower event rate within a given disease setting directly reduces the statistical power of a clinical trial , unless the sample size is increased ( which may or may not be feasible )  . 
during the past 50 years , the 5 - year survival rate for pediatric cancers has risen dramatically from 10% to 83% , which makes future clinical trials in pediatric cancers more challenging to design.24 , 25 taking standard - risk all as an example , the cure rate is 90% or greater for children in the general population.26 yet another challenge in rare and pediatric cancers is the advent of stratied or precision medicine , with an everincreasing number of new targeted therapies available for testing within narrowly dened risk - stratied or molecularly dened cohorts . 
even among pediatric cancers with the highest incidence rates ( eg , all , which accounts for approximately 30% of all cancers in children ) , most trials are conducted within far - smaller cohorts dened by risk stratication systems wherein a given risk stratum has unique objectives and possible treatment assignments , as described in the risk stratication section . 
 renfro et al a setting , even a common cancer usually is studied as a series of essentially independent , smaller trials and is therefore subject to many of the same design challenges as rarer cancers.27 , 28 when layered upon the general rare cancer issues previously described , precision medicine creates an additional urgency at the design stage to strike a balance between what is statistically rigorous and what is acceptable to support feasibility . rationale for relaxing operating characteristics / design parameters and common approaches under current conventions , a typical two - arm randomized phase iii clinical trial that tests a superiority hypothesis often uses a two - sided 5% type i error rate ( which corresponds to a one - sided 2.5% type i error rate ) and power in the range of 80% to 90% to detect a clinically relevant difference in outcome between treatment arms . 
for example , assuming a cancer with a baseline cure rate of 50% where improvement to 65% cure is desired but only 40 patients per year will likely enroll , a two - arm randomized trial would require the recruitment of 360 patients accrued during 9 years to detect the desired improvement with 80% power and a 5% type i error rate.23 in pediatric cancer , an accrual rate of 20 to 50 patients per year is common , such as in the setting of risk - stratied relapsed all . 
although a prolonged trial achieves the typical type i and type ii error rates and increases our condence in the conclusions we obtain from a completed trial , it limits the number of trials we can conduct in the long run and reduces the number of new treatment strategies or agents that we can test . 
in addition , a treatment can become irrelevant after a long duration , and a long trial can become obsolete before it completed.23 , 29 therefore , other designs are considered for clinical trials in small populations.30 at cog and in other groups that study rare diseases , a number of approaches commonly are used to minimize the overall sample size required for a trial and are subject to context - specic constraints . 
when the enrollment in a trial is not sufcient to achieve the sample size that an ideal design would require , one option is to target a larger effect size than the smallest effect that would be clinically relevant ( eg , to detect a hazard ratio [ hr ] of 0.60 [ 40% risk reduction ] rather than a more modest hr of 0.80 [ 20% risk reduction ] )  . 
in doing so , the required trial sample size is reduced as one raises the bar of the expected treatment benet under the alternative hypothesis.27 , 31 - 37 particularly in low - incidence cancer settings where only one novel therapy can be studied at a time ( to avoid splitting the population between competing trials ) , it can be reasonable to power a trial to detect a large treatment effect so long as such an effect is believed to be feasible given the type of treatment under study and the intended population.34 when the cohort to be studied is so small as to preclude formal hypothesis testing , a primary objective may instead be to enroll as many patients as possible within a given time frame and estimate the outcomes of interest ( eg , efs at 3 years )  . 
another sample size reduction approach particularly common in phase ii and some phase iii trials is a relaxation ( an increase ) of the maximum allowable rate of type i error or the probability of a false - positive result.23 , 27 - 29 , 33 - 35 , 38 , 39 a conventional rule of thumb for many clinical studies ( not just clinical trials ) is to specify a two - sided type i error rate of 5% to control the probability false - positive results when the null hypothesis ( no treatment effect ) is true . 
a third approach to reducing sample size ( albeit less common at cog ) is to relax or decrease the amount of power or probability with which the targeted effect size will be detected if it is true ( a power less than 80% usually is not considered )  . 
although each of these adjustments comes with some corresponding statistical sacrice , a compromise among the three objectives of low type i error , high power , and a reasonable targeted effect is often possible and straightforward to implement ; thus , these approaches remain popular in rare disease settings.23 , 27 - 39 guidance for relaxing operating characteristics and trial parameters . 
when trials include most of the patients in the target population , sposto and stram23 investigated the relationship between type i error , sample size , trial duration , patient accrual rate , and therapeutic innovation rate and the increase in treatment efcacy achieved after a series of two - treatment randomized phase iii trials . 
they proposed that a more appropriate view of trial design in lowincidence cancer settings is in the overall context of the research setting and long - term goals rather than in the narrow context of the current single trial . ( p1183 ) sposto and stram concluded that judicious choice of type i error and accrual duration will result in a larger average gain in treatment efcacy as measured by improvements in cure rates over the long term , especially in low - incidence disease , than would use of small type i error and high power for small differences . 
strauss and simon29 considered the optimum allocation of patient resources to maximize success rates in a phase ii context and had similar conclusions ( ie , more frequent , smaller trials are warranted in settings where patient resources are limited )  . 
these articles support the notion that performance of a series of small trials with relaxed operating characteristics over a long - term research horizon leads ( on average ) to larger survival benets . 
an optimal choice of design parameters can be selected to maximize the expected total survival benet by considering factors such as accrual rate , therapeutic innovation rate , and disease severity.23 , 28 , 39 despite the justications , the targeting of a larger effect size or relaxing of type i error has some disadvantages . 
targeting a larger effect size with the type ii error rate of 20% could lead to underpowered studies for new treatments associated with smaller , but still clinically meaningful benets and , hence , increases the risk of missing a moderate or small treatment effect.31 , 33 use of larger type i error rates and / or shorter trials is associated with worse apparent treatment efcacy in the long term and an increased likelihood of ultimately selecting an inferior treatment compared with standard larger trials with lower type i error , 23 and increasing type i error ( one - sided ) above 20% generally is not recommended.23 , 28 , 39 examples of relaxed design constraints and solutions from cog trials at cog , many phase ii and some phase iii randomized studies were designed with relaxed type i error rates ( onesided 5% , 10% , or 15% ) or were powered to target a larger effect size with a reduced statistical power of 80% . 
table 2 lists some of these trials and includes their specic objectives and operating characteristics as examples . among these is the study design for low - risk patients with relapsed b - all enrolled in the cog trial aall1331 ( clinicaltrials.gov identier : nct02101853 )  . 
the cohortspecic objective was to compare disease - free survival ( dfs ) between patients randomly assigned to chemotherapy alone ( control arm ) or chemotherapy plus blinatumomab ( a bispecic single - chain antibody that targets the cd19 antigen ) after re - induction . 
the corresponding study was designed to have 80% power to detect an hr of 0.55 using a log - rank test with 5% one - sided type i error , which corresponds to an increase in the 3 - year dfs rate from 73% in the control arm to 84% in the experimental arthe b - all cohort was expected to accrue during 5.6 years to reach 206 eligible patients , with the analysis requiring 3 years of additional follow - up after completion of enrollment . 
common objectives in randomized pediatric oncology trials include the comparison of drug combinations , altered doses , or adjuvant therapies against standard therapeutic backbones , with trials powered to detect improved outcomes in the experimental arms versus the control aralso common in pediatric trials are situations where the goal is not to prove superiority but to show that a new experimental therapy is not statistically inferior to an active control or standard of care and retains most ( if not all ) of the controls therapeutic benet . 
in this case , factors such as potential improvements in safety , tolerability , cost , convenience , availability , or other secondary factors are used to justify the study of the experimental regimen . 
despite their relevance in situations such as those just described , ni trials are often more challenging to design than superiority trials , and a number of factors must be considered during the planning stages to ensure a feasible trial capable of answering the ni - type question of interest.41 , 42 planning and design considerations for ni trials overview of randomized ni designs using time - to - event end points . 
as such , the results presented usually include a signicance test for differences between groups ( eg , a log - rank test ) and a numerical summary of that difference ( eg , the hr estimated from a cox proportional hazards regression model )  . 
in the randomized ni trial setting with a time - to - event outcome , however , interest lies in determining whether the estimated hr and its ci fall in such a region that inferiority of the experimental arm can be statistically ruled out . 
at the time of the nal analysis , if this condition is met , ni ( or a stronger conclusion such as superiority ) can be claimed for the experimental treatment ( fig 2 )  . general cautions about ni designs . 
first , conclusions about the ni of a new regimen versus an active control are not particularly useful if the control was never established as best care in prior research.43 , 44 in addition , if outcomes in the control group are better than anticipated during trial planning , a resulting ni study could be underpowered ( even when the treatment arms are equivalent ) because of an overall shortage of events among the enrolled patients . 
a typical ni design is powered under the assumption of risk equality between groups , although this can be adjusted in some cases ( see herein the challenges with conducting ni designs in rare disease settings and some ad hoc and literature - based solutions section )  . 
in many cases , futility monitoring can be incorporated to halt the ni trial if it becomes evident that the therapy is truly inferior , although specic experimental testing approaches and decision rules should be considered carefully.45 - 47 challenges with conducting ni designs in rare disease settings and some ad hoc and literature - based solutions as is often the case in rare disease settings , the design of randomized ni trials for cog studies requires extra consideration and some degree of compromise to increase trial feasibility . 
within cog , all aspects of statistical design and supporting clinical rationale ultimately undergo several layers of review , during which the benets and caveats are evaluated carefully with respect to the realistic constraints imposed by treatment options , the patient population , and other resources . relaxation of type i error . 
ideally , ni trials , especially those for regulatory indications , would specify no greater than a one - sided type i error rate of 2.5% by default to retain a high degree of rigor and safeguard against false - positive conclusions.44 , 52 as in the superiority setting , a smaller type i error requires a larger sample size and widens the ci for the hr , making an ni conclusion much less likely to be a false - positive result . 
within cog , ni trials usually are motivated by the desire to reduce treatment intensity in groups of patients with established good prognoses , thereby reducing shortor long - term toxicities among children expected to be cured . 
in this context , there is precedent for conducting ni trials with a one - sided type i error rate of 5% ( or even more relaxed ; see relaxing trial operating characteristics and design parameters section ) , which strikes a compromise between error rate conservation and simple feasibility . literature - proposed solutions for ni hypotheses in rare diseases . 
freidlin et al53 adapted the ni design to allow for sample size calculations in situations where the assumed hr for the power calculation is not equal to 1 ( equivalence ) but reects modest superiority for the experimental treatment ( ie , hr slightly less than 1 )  . 
 renfro et al enhanced when a small improvement in outcome truly exists for the experimental arthis is often a welcome compromise when the experimental arm already offers less toxicity or other advantages . 
in rare disease settings , such as pediatric cancer trials conducted by cog , this design often is considered when small provements are deemed plausible or likely ( see example trials from cog testing ni - type hypotheses section for examples )  . 
in the same article , freidlin et al also described a sequential testing procedure that can be applied when a test for ni followed by a test for superiority ( a higher bar ) is desired . treatment example trials from cog testing ni - type hypotheses one recently opened cog trial with an ni design is aall1631 ( clinicaltrials.gov identier : nct03007147 ) , an international collaborative study of the ni of an experimental , less - toxic postinduction treatment in patients with standard - risk philadelphia chromosomepositive all.54 the 3 - year using historical data during trial planning , dfs rate was predicted to be 70% in the control arm , and m = 1.43 was set to correspond to a minimally reduced 3 - year dfs rate of 60% in the experimental group . assuming 6 years of accrual and a minimum of 3 years of follow - up , random assignment of 475 patients will yield 80% power to conclude ni using a one - sided type i error rate of 5% . 
for patients with low - grade gliomas , the question of treatment de - intensication to avoid harmful adverse effects without sacricing efcacy is highly relevant . however , given the rarity of this patient population , in addition to proposing a larger - than - usual ni margin ( hr of 1.7 ) and somewhat inated type i error rate of 10% , these two studies have used the design proposed by freidlin et al53 and described here in the challenges with conducting ni designs in rare disease settings and some ad hoc and literature - based solutions section to make these rare cancer ni trials more feasible because superiority is possible . 
in addition , availability of individual patient data from the potential historical trials or data sources is key so that the formal cohort that resembles the control arm can be tailored to t the current trials eligibility criteria and other characteristics . 
in cases where large historical control cohorts with adequate patient - level data are available , these designs can serve as an effective alternative to randomized trials in the context of signal nding . 
our sense is that such approaches work best in circumstances where the outcome is dismal , the targeted efcacy signal is large , and the historical cohort available is deemed to be sufciently similar to a cohort that would be enrolled if a concurrent control arm were included in the trial design . review of trial designs using historical controls dixon and simon62 described an initial approach to designing phase ii studies using historical controls ; however , it was subsequently shown by korn and freidlin63 that this method does not adequately protect against type i or ii errors . 
instead , korn and freidlin proposed a two - sample randomized design approach with adjusted allocation to the size of the historical cohort , which optimizes estimation of the median sample size and controls the type i and ii errors effectively . 
a drawback , however , is that the design is conservative in the sense that the number of events in the control cohort is almost always larger because of follow - up in the historical control being longer than what would have been observed with concurrent random assignment to cohorts of equal sample size . another drawback of this approach is that calculation of accumulating end point information during the trial for the purposes of timing interim analyses is not readily possible as with traditional designs . 
both drawbacks are addressed in an article by wu and xiong64 that focused on the median sample size as the primary parameter to be optimized on the basis of the one - sided sequential log - rank test . 
overall survival ( os ) outcomes for historical childrens oncology group studies in newly diagnosed diffuse intrinsic pontine glioma displayed ( a ) by study and ( b ) combined . a transformed information time to accommodate appropriately the information from the historical control and experimental group together to enable interim analyses . work by wu and li ( manuscript submitted for publication ) further extends this method for use with interim analyses on the basis of the alpha and beta spending functions of lan and demets . end point of this trial is progression - free survival , and the efcacy comparison is based on a historical control cohort constructed from prior cog studies , including acns0423 ( clinicaltrials.gov identier : nct00100802 ) and acns0822 ( clinicaltrials.gov identier : nct01236560 ) , as well as global meta - analysis cohorts where tumors received similar molecular annotation.65 example trials from cog using historical controls recommendations for use of historical controls a phase ii study currently in development for newly diagnosed diffuse intrinsic pontine glioma will use pooled historical data from several similar completed studies as the comparison cohort , where the primary end point of interest is os . 
using the methodology described in wu and xiong64 and the empirical kaplan - meier estimate to model the combined os distribution ( fig 3b ) , a reasonable phase ii design will require 45 patients to target an hr of 0.60 with 80% power and maintain less than a 5% type i error rate using a one - sided log - rank test . this hr translates to an approximately 18% net provement in 1 - year os rate ( 42% to 60% )  . 
because the study accrual duration is expected to be 1 to 1.5 years , the design will require 1 year of additional follow - up for all patients after accrual completion . 
a similar approach also has been used for acns1721 ( clinicaltrials.gov identier : nct03581292 ) , which enrolls newly diagnosed patients with high - grade glioma at least 3 years of age whose tumors are risk stratied by molecular criteria at initial study screening . 
the primary although phase ii designs that are based on historical control cohorts have desirable properties that support feasibility in rare disease settings , they are most useful when the available historical control group is relatively large . 
when the sample size and / or the number of events in the historical control cohort are small , the designs outlined here may not be feasible because the sample size in the proposed trial may not be able to compensate for the lack of patients / events supporting the historical cohort . 
on the other hand , if the historical control cohort is very large , then the design essentially reduces to a literature - controlled design where the comparison is against a xed rate at a specied time point . 
more sophisticated approaches for borrowing information from historical controls while simultaneously randomly assigning a small subset of patients to a control arm also have been described.67 discussion in this article , we highlighted some of the most prominent features and challenges that affect clinical trials designed for the pediatric oncology setting . 
renfro , phd , childrens oncology group , 222 e huntington dr , monrovia , ca 91016 ; twitter : @lindsayrenfro ; e - mail : lrenfro@ childrensoncologygroup.org. support supported by the national cancer institute ( 1u10ca180899 - 05 )  . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . arzu onar - thomas honoraria : eli lilly research funding : novartis ( inst ) , apexigen ( inst ) , pzer ( inst ) , celgene ( inst ) , merck ( inst ) travel , accommodations , expenses : eli lilly john a . 
kairalla stock and other ownership interests : johnson & johnson , sophiris bio no other potential conicts of interest were reported . acknowledgment we acknowledge the childrens oncology group committees associated with the examples cited in this article ( acute lymphatic leukemia , cns , neuroblastoma , acute myeloid leukemia , and rare tumors )  . references siegel r , naishadham d , jemal a : cancer statistics , 2012 . 
nat rev cancer 12 : 776 - 782 , 2012 bhatia s , meadows at : long - term follow - up of childhood cancer survivors : future directions for clinical care and research . 
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evans ae , land vj , newton wa , et al : combination chemotherapy ( vincristine , adriamycin , cyclophosphamide , and 5 - uorouracil ) in the treatment of children with malignant hepatoma . 
ortega ja , douglass ec , feusner jh , et al : randomized comparison of cisplatin / vincristine / uorouracil and cisplatin / continuous infusion doxorubicin for treatment of pediatric hepatoblastoma : a report from the childrens cancer group and the pediatric oncology group . 
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clin trials 8 : 270 - 276 , 2011 jung sh , kang sj , mccall lm , et al : sample size computation for two - sample noninferiority log - rank test . 
fangusaro j , onar - thomas a , young - poussaint t , et al : a phase ii prospective study of selumetinib in children with recurrent or refractory low - grade glioma ( lgg ) : a pediatric brain tumor consortium ( pbtc ) study . 
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mackay a , burford a , carvalho d , et al : integrated molecular meta - analysis of 1 , 000 pediatric high - grade and diffuse intrinsic pontine glioma . 
 complete response to pd - 1 inhibition in an adolescent with relapsed clear cell adenocarcinoma of the cervix predicted by neoepitope burden and apobec signature anya levinson , md1 ; alex g . 
alejandro sweet - cordero , md1 , 2 ; and elliot stieglitz , md1 , 2 introduction utilization of checkpoint inhibition therapy in the management of malignant diseases is increasing . however , response to these therapies is highly variable , and robust predictive markers remain elusive . identication of predictive biomarkers is crucial considering the toxicities and high nancial cost associated with this approach . 
here , we report an adolescent with relapsed clear cell adenocarcinoma of the cervix ( ccac ) who experienced a complete response to checkpoint blockade despite not exhibiting positive pd - l1 immunohistochemical ( ihc ) staining , microsatellite instability ( msi ) , or a high tumor mutational burden ( tmb )  . 
biopsy of an exophytic mass protruding into the vagina conrmed a human papillomavirus / p16 - negative ccac ( fig 1 )  . family history was notable for paternal clear cell renal carcinoma . 
sixteen months after entering remission , surveillance positron emission tomography ( pet ) computed tomography ( ct ) revealed new hypermetabolic disease , including a new right upper lobe ( rul ) pulmonary nodule , enlarged prevascular right internal mammary , and right paratracheal nodes ( fig 2a ) and a left supraclavicular node . 
biopsy of the supraclavicular node conrmed recurrence of ccac ( fig 3 )  . her relapsed tumor exhibited 0% staining for pd - l1 by ihc , microsatellite stability ( 1 , 149 of 1 , 157 tested microsatellite regions were stable ) , and a low tmb ( 1.9 single nucleotide variants per megabase on wgs , less than the pediatric threshold of 2 ; appendix )  . 
genomic assays performed included targeted dna sequencing , wgs , and rna - seq ( appendix table a1 )  . a targeted institutional dna - seq panel assaying 479 cancer - relevant genes4 showed that both diagnostic and relapsed tumors had a broad approximately 11 - fold amplication on chromosome 20 containing aurora kinase a as a putative driver.5 the diagnostic sample also displayed a chromosome 1p deletion containing arid1a , a gene frequently mutated in cervical cancer , 6 which was absent at relapse . no pathogenic point mutations , insertions / deletions ( indels ) , or germline alterations were detected on this panel at diagnosis or relapse . 
pathologic conrmation of clear cell carcinoma of the cervix . ( a ) primary clear cell carcinoma of the cervix , showing hyperchromatic tumor cell nests in the stroma at left and normal endocervical glandular epithelium at the right ( 40 )  . 
 ( c ) an immunohistochemical stain for hepatocyte nuclear factor 1 - , which is a marker of clear cell carcinoma , shows strong positive nuclear staining in the tumor cells , a positive test result ( 200 )  . the rul pulmonary nodule and the prevascular lymph lymph nodes denode ( fig 2b )  . 
the median age was 53 years , and only three patients were , 30 years of age , 13 making the patient presented here one of the youngest patients ever reported with idiopathic ccac . 
 ( a ) patient developed a new hypermetabolic pulmonary nodule in the right upper lobe ( blue arrow ) , along with prominent hypermetabolic prevascular nodes ( red arrow )  . 
 ( b ) after initial nivolumab therapy , patients right upper lobe nodule decreased in size ( blue arrow ) , and the prevascular node had nearly resolved ( red arrow )  . 
 ( c ) approximately 16 months after initial recurrent disease , the pulmonary nodule has nearly completely resolved ( blue arrow ) , and no new pathologic nodes have developed . known biomarkers for response to checkpoint inhibition are ihc staining for pd - l1 , presence of msi , and high tmb . this patient , who achieved a complete and durable remission from checkpoint inhibition without any of these markers , highlights the need for additional biomarkers . 
we sought to analyze the genomics of her tumors to explain her dramatic response . somatic variations that give rise to amino acid substitutions in tumors yield neoepitopes , or mutated , tumor - specic peptides on the surface of cancer cells that can serve as neoantigens for the adaptive immune system , even if they are not oncogenic . 
determinants of neoantigen tness are the likelihood of their presentation by the major histocompatibility complex and of subsequent t - cell recognition.16 the number of neoepitopes per tumor can be more functionally relevant than the tmb . 
biopsy of supraclavicular node conrms metastatic disease . ( a ) metastatic clear cell carcinoma in a supraclavicular lymph node . the tumor cells line glands or grow in nests and are surrounded by lymphocytes , seen at the lower left ( 200 )  . ( b ) metastatic clear cell carcinoma in a supraclavicular lymph node . 
an immunohistochemical stain for pd - l1 shows minimal staining in the tumor cells and staining of scattered lymphoid cells around the tumor ( 200 )  . patients relapsed tumor , higher than that of available ( although biologically distinct ) comparison cohorts , supporting the idea that high neoepitope burden may predict favorable response to immunotherapy . 
of these , signatures 1 ( aging ) and 3 ( homologous recombination deciency ; appendix fig a4 ) are commonly found in cancer.18 , 19 signatures 2 and 13 represent activity of the aid / apobec family of enzymes.18 , 19 is associated with expression of apobec , a family of zinc - coordinating enzymes that convert cytosines to uracils , has been implicated in mutagenesis of nonsmall - cell lung cancer . 
systematic cancer genomic and transcriptomic association studies have shown that overexpression of one of the family members , apobec3b , immune genes and known immunotherapy response biomarkers such as pd - l1.20 a positive correlation was recently documented between strength of the apobec signature and the neoepitope burden in many cancers , including cervical.21 the authors also found that this mutational signature corresponded to increased abundance of tumorinltrating lymphocytes in some cancers , 21 although these were absent in this case . 
clinically , the apobec mutational signature is enriched in patients with durable clinical benet after immunotherapy , and an apobec signature may be better than tmb in predicting immunotherapy response.22 , 23 finally , there was a small contribution from signature 8 of uncertain etiology ( it is perhaps related to nucleotide excision repair deciency ) .24 the present patient is of interest for several reasons . 
box - and - whisker plots display the interquartile range ( box ) and outlier thresholds ( whiskers ) for the therapeutically applicable research to generate effective treatments neuroblastoma and the cancer genome atlas prostate adenocarcinoma data sets . 
although there may be other factors that contributed to the patients dramatic response , this case supports emerging evidence that a high neoepitope burden and an apobec mutational signature response to are potentially actionable biomarkers of checkpoint blockade . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . jessica van zife employment : adaptive biotechnologies ( i ) stock and other ownership interests : adaptive biotechnologies ( i ) patents , royalties , other intellectual property : adaptive biotechnologies ( i ) benjamin laguna leadership : sira medical stock and other ownership interests : sira medical stanley g . 
leung stock and other ownership interests : illumina , pzer , thermo fisher jacob pfeil employment : abbvie no other potential conicts of interest were reported . acknowledgment we thank the patient and her family for their willingness to share their experience using an unconventional treatment approach . 
naumann rw , hollebecque a , meyer t , et al : safety and efcacy of nivolumab monotherapy in recurrent or metastatic cervical , vaginal , or vulvar carcinoma : results from the phase i / ii checkmate 358 trial . 
european society for medical oncology meeting , barcelona , spain , september 27 - october 1 , 2019 ( abstr lba62 ) kline cn , joseph nm , grenert jp , et al : targeted next - generation sequencing of pediatric neuro - oncology patients improves diagnosis , identies pathogenic germline mutations , and directs targeted therapy . 
curr oncol rep 15 : 566 - 572 , 2013 cho h , kim js , chung h , et al : loss of arid1a / baf250a expression is linked to tumor progression and adverse prognosis in cervical cancer . 
n engl j med 377 : 1345 - 1356 , 2017 rao aa , madejska aa , pfeil j , et al : protect : prediction of t - cell epitopes for cancer therapy . 
reich o , tamussino k , lahousen m , et al : clear cell carcinoma of the uterine cervix : pathology and prognosis in surgically treated stage ib - iib disease in women not exposed in utero to diethylstilbestrol . 
thomas mb , wright jd , leiser al , et al : clear cell carcinoma of the cervix : a multi - institutional review in the post - des era . 
boyd j , takahashi h , waggoner se , et al : molecular genetic analysis of clear cell adenocarcinomas of the vagina and cervix associated and unassociated with diethylstilbestrol exposure in utero . 
turajlic s , litcheld k , xu h , et al : insertion - and - deletion - derived tumour - specic neoantigens and the immunogenic phenotype : a pan - cancer analysis . 28 : 15 - 17 , 2019 lancet oncol 18 : 1009 - 1021 , 2017 18 . 
wang s , jia m , he z , et al : apobec3b and apobec mutational signature as potential predictive markers for immunotherapy response in non - small cell lung cancer . 
chen z , wen w , bao j , et al : integrative genomic analyses of apobec - mutational signature , expression and germline deletion of apobec3 genes , and immunogenicity in multiple cancer types . 
chen pl , roh w , reuben a , et al : analysis of immune signatures in longitudinal tumor samples yields insight into biomarkers of response and mechanisms of resistance to immune checkpoint blockade . 
boichard a , tsigelny if , kurzrock r : high expression of pd - 1 ligands is associated with kataegis mutational signature and apobec3 alterations . oncoimmunology 6 : e1284719 , 2017 jager m , blokzijl f , kuijk e , et al : deciency of nucleotide excision repair is associated with mutational signature observed in cancer . 
the lymph node from the patient was snap frozen and ffpe into optimal cutting temperature ( oct ) mediuall tissues were sectioned to a depth of 5 m and stained with hematoxylin and eossamples were evaluated for tumor content by a certied pathologist . 
rna integrity was quantied with the high sensitivity rna kit dnf - 472 ( advanced analytical technologies , orangeburg , ny ) on the fragment analyzer ( advanced analytical technologies )  . 
immunohistochemical pd - l1 staining was performed on tumor cells with the us food and drug administrationapproved pd - l1 28 - 8 pharmdx antibody for opdivo ( neogenomics laboratories , fort myers , fl )  . whole blood was collected during lymph node metastasectomy in a 4ml edta vacutainer . 
germline dna was extracted using the dneasy blood and tissue kit 69504 ( qiagen )  . dna was quantied using the nanodrop 2000 ( thermo fisher , waltham , ma ) and qubit high sensitivity dsdna assay q32851 ( thermo fisher ) and integrity quantied using the high sensitivity gdna kit dnf488 ( advanced analytical technologies ) on the fragment analyzer ( advanced analytical technologies )  . 
libraries from ffpe samples were made using the truseq rna access kit ( rs - 301 - 2001 ; illumina , san diego , ca ) with an input of 100 ng in accordance with manufacturers instructions . libraries from ffpe samples were made using the truseq stranded mrna kit ( rs - 122 - 2101 ; illumina ) with an input of 400 ng in accordance with manufacturers instructions . 
outliers ( ie , underor overexpression ) were identied using tukey outlier denition . targeted dna sequencing an institutional dna sequencing panel assaying 479 cancer - related genes was used.4 as per kline et al , 4 genomic dna was extracted from peripheral blood and tumor tissue microdissected from ffpe blocks . 
capture - based next - generation sequencing ( ngs ) was performed at the university of california , san francisco ( ucsf ) clinical cancer genomics laboratory , using an assay targeting the coding regions of these genes , tert promoter , select introns from 40 genes ( for detection of gene fusions and other structural variants ) , and intergenic regions at regular intervals along each chromosome ( for chromosomal copy number assessment ) , altogether with a total sequencing footprint of 2.8 mb ( ucsf500 cancer gene panel , appendix fig a2 )  . 
a list of genes included can be found at the following url ( although some genes may have been added or deleted since this patients analysis in 2017 ) : gene - panel - test - ucsf500 - uc500 . whole - genome sequencing libraries were generated using the truseq nano kit fc - 121 - 4001 ( illumina ) with a 350 - bp insert , per manufacturers instructions . 
libraries were made using 200 - ng input gdna , and then quantied on the tapestation 4200 ( agilent , santa clara , ca ) and sequenced on the hiseq x ten ( illumina ) using 2 150 bp paired - end reads . 
nonsynonymous variants were annotated using variant effect predictor ( mclaren w , et al : genome biol 17 : 122 , 2016 )  . predicted tumor neoepitope analysis we used the prediction of t - cell epitopes for cancer therapy ( protect ) pipeline v 2.6.18 to investigate the patients neoepitope burden . 
we compared the patients tumor mutation and neoepitope burden to publicly available protect data for the therapeutically applicable research to generate effective treatments neuroblastoma ( pmid : 23334666 ) and the cancer genome atlas prostate adenocarcinoma data sets ( pmid : 26544944 ) and assessed for signicance using tukeys outlier denition . apobec signature analysis single base substitution mutational signatures were extracted from the samples wgs data using deconstructsigs ( rosenthal r , et al : genome biol 17 : 31 , 2016 ) , with default parameters . 
the patients sample is notable for a signature 3 ( homologous recombination deciency [ hrd ] ) signature . violin plot of the fraction of mutations in each sample from ma et al11 attributed to signature 3 . 
a biopsy specimen of the liver lesion revealed a poorly differentiated carcinoma , with tumor cells displaying a partly pleomorphic , signet ring or spindle cell appearance , fitting to the metastatic presentation of either a carcinoma of the bile duct or pancreatico - biliary syste additional staging procedures indicated a large mass in the head of the pancreas . 
biopsy of this region identified tumor cells that resembled the previous liver lesion . after comprehensive review of all data , our interdisciplinary tumor board diagnosed a hepatically metastasized pancreatic ductal adenocarcinoma ( pdac ) and recommended palliative chemotherapy with gemcitabine and nanoparticle albumin bound paclitaxel . 
after an additional four cycles of gemcitabine monotherapy , overt progression was observed with only minor growth of the primary tumor in the pancreas but newly diagnosed small metastases in the liver . to obtain information about the molecular profile of the tumor , genomic dna was isolated from the liver metastasis of the pancreatic carcinoma , and a blood sample from the patient was used as a reference . 
the samples were enriched for whole - exome sequencing and sequenced on an illumina hiseq next - generation sequencing platform ( illumina , san diego , ca ) by cegat ( tuebingen , germany )  . 
the most relevant somatic driver mutations were a loss - of - function stop mutation within fancb ( e472 * ) and an activating mutation in braf ( v600e )  . 
the patient also had no germline mutations in known cancer susceptibility genes . because of the remarkably high tmb and to directly interfere with the driver mapk / erk signaling pathway , the patient was offered a personalized off - label therapy with the intravenous immune checkpoint pd - 1 antibody pembrolizumab ( 200 - mg flat dose ) in combination with the oral mek inhibitor trametinib ( 2 mg daily )  . 
 the mapk / erk pathway has been implicated in the immune resistance of tumors , and inhibition of this pathway can lead to an increased infiltration of the tumor by cd8 - positive t cells in preclinical models.1 furthermore , mek inhibition promotes t - cell activity , eliciting a synergistic effect with checkpoint blockage.2 although trametinib therapy had to be terminated after 3 months because of severe adverse effects , including edema and fatigue , the immunotherapy was well tolerated and continued for another 6 months . 
the only relevant immune - related adverse event was a suppression of the endogenous level of cortisol ( hypocortisolism ) , resulting in the need of permanent oral replacement therapy . 
surveillance positron emission tomography computed tomography scans after eight cycles of chemotherapy ( a ) before change to immunotherapy and mek inhibition , and ( b ) 5 months and ( c ) 9 months after change to immunotherapy . 
 ( a ) during chemotherapy , positron emission tomographycomputed tomography scan reveals progression of the primary tumor in the pancreas ( ) and of the hypermetabolic solitary metastasis of the liver ( * )  . 
 ( c ) metabolic activity in both lesions considerably decreased . surveillance positron emission tomography computed tomography scans revealed declining disease according to positron emission tomography response criteria in solid tumors , regarding the size of the masses in the pancreas and liver , as well as their metabolic activity , providing clear evidence for an explicit and sustainable response to the personalized therapy ( figs 1a - 1c )  . to change the therapeutic concept from palliative to potentially curative , exploratory laparotomy was performed 18 months after primary diagnosis . 
instead , the tumor was irradiated with a total dose of 54 gy administered over 6 weeks to promote local control , while pembrolizumab administration was discontinued to avoid additive toxicity to the gi tract in combination with irradiation . histologic analysis of the resected liver tissue led to the diagnosis of a metastasized , poorly differentiated , in parts undifferentiated / anaplastic pancreatic adenocarcinoma , which represents a variant of pdac . 
a moderate number of cd3 - positive t cells were observed within the tumor tissue ( approximately 20 t cells per 100 nucleated cells ) , including equal amounts of cd8 - positive and cd4 - positive cells , with a moderately higher density at the tumor margin ( figs 2e and 2f )  . 
mmr and additional dna repair proteins such as atm , atr , and brca1 were regularly expressed by the tumor cells ( not shown )  . the prognosis of undifferentiated pancreatic adenocarcinoma is usually worse than that of conventional pdac , with distant metastases frequently present at the time of diagnosis.3 however , our patient still is in good condition with no evidence of relapse 2 years after primary diagnosis . discussion in pdac , only minimal progress has been made so far in identifying therapies targeted toward altered signaling pathways , and single - agent therapy with antictla - 4 or antipd - 1 antibodies was found to be ineffective in early clinical trials.4 instead , chemotherapeutic selection on the basis of histopathologic analysis alone remains the standard of care.5 here , we report the case of a patient with a metastasized , undifferentiated pdac with a high tmb who is experiencing durable remission to single - agent checkpoint inhibition . the tmb has already been correlated with clinical benefits of antipd - 1 and antictla - 4 therapy in various tumor types , including melanoma , 6 , 7 nonsmall - cell lung cancer , 8 and several dna repairdeficient tumors.9 - 11 however , no generally agreed - on definition of hypermutation has been postulated yet . 
in a recent study based on > 80 , 000 tumors , the threshold for hypermutation was set to 10 mutations / mb.12 this value coincides with the median tmb of patients previously reported to respond to checkpoint inhibition.8 , 13 in pdac , a high tmb is usually rare . 
 ( a ) moderately to poorly differentiated adenocarcinoma consistent with a metastasis of a pancreatic ductal adenocarcinoma ( hematoxylin and eosin staining [ h&e ] ; magnification , 100 )  . 
 ( b ) tumor heterogeneity : undifferentiated component ( left ) and a highly regressive carcinoma component ( right ) showing fibrosis and myxoid change ( h&e ; magnification , 100 )  . 
it was also shown recently that high levels of microsatellite instability are mostly restricted to tumors in the range of 10 to 100 mutations / mb.12 however , the tumor of the patient in this report was microsatellite stable . 
 instead , we found a loss - of - function stop mutation within the fanconi anemia complementation group b ( fancb ) gene ( e472 * ) and an activating mutation in braf ( v600e )  . 
somatic mutations in the fa gene family can be found in approximately 13% of pancreatic tumors.17 , 18 recently , chalmers et al19 identified > 100 somatic mutations significantly associated with increased tmb , including missense mutations in the fa pathway genes fancd2 and fancj , providing evidence that the loss - offunction fancb mutation might contribute to the high tmb in our patient . the mapk / erk pathway is also often altered in pdac . 
the braf gene is mutated in one - third of pancreatic cancers with known wild - type kras.20 it has been suggested that the kras and braf oncogenes function in a mutually exclusive manner in the transformation and carcinogenesis of pancreatic cancer . 
to block the mutationally activated cellular pathway and to increase the immune effect in the typical desmoplastic stroma within pancreatic cancers , we combined pembrolizumab with the oral mek inhibitor trametinib . 
however , the latter had to be terminated prematurely after 3 months because of intolerable adverse effects ; its relative contribution to the patients response cannot be addressed precisely . in conclusion , our data suggest that whole exome sequencing has the potential to identify a small but clinically meaningful subgroup of pdac with a high tmb . 
liu l , mayes pa , eastman s , et al : the braf and mek inhibitors dabrafenib and trametinib : effects on immune function and in combination with immunomodulatory antibodies targeting pd - 1 , pd - l1 , and ctla - 4 . 
ebert pjr , cheung j , yang y , et al : map kinase inhibition promotes t cell and anti - tumor activity in combination with pd - l1 checkpoint blockade . 
howitt be , shukla sa , sholl lm , et al : association of polymerase e - mutated and microsatelliteinstable endometrial cancers with neoantigen load , number of tumor - infiltrating lymphocytes , and expression of pd - 1 and pd - l1 . 
strickland kc , howitt be , shukla sa , et al : association and prognostic significance of brca1 / 2 - mutation status with neoantigen load , number of tumor - infiltrating lymphocytes and expression of pd - 1 / pd - l1 in high grade serous ovarian cancer . 
the patient subsequently underwent neoadjuvant radiation in addition to receiving weekly carboplatin and paclitaxel , followed by esophagogastrectomy.7 pathology revealed moderately differentiated adenocarcinoma invading the adventitia ( pt3 ) , but none of the 14 lymph nodes were involved ( pn0 )  . 
one and a half years after resection , surveillance imaging revealed new concerning hypodensity in the liver , accompanied by an increase in the carcinoembryonic antigen ( cea ) levels . 
next - generation sequencing ( ngs ) was performed on biopsies from the time of initial and recurrent liver metastasis using the commercially available comprehensive genomic proling ( cgp ) assay from foundation medicine ( cambridge , ma ; table 1 )  . 
serial blood collections were also obtained to determine the presence of ctdna using a tumorinformed multiplex polymerase chain reaction - ngs assay from natera ( san carlos , ca ) designed from tumor genomic alterations ( table 1 )  . 
at the time of disease recurrence in the liver , the tumor - informed assay with an established analytical sensitivity5 revealed elevated levels of ctdna , measured in mean tumor molecules / ml of plasma ( fig 1 )  . the patient underwent the patient approximately 760 days postdiagnosis , received 6 cycles ( c6 ) of uorouracil ( fu ) , leucovorin , and oxaliplatin ( mfolfox6 ) , with radiographic response in the liver lesion . 
after another year ( approximately 1 , 490 days postdiagnosis ) , the patient developed a new fdgavid mass in the ablation cavity , accompanied by an increase in cea level . 
the patient received 1 cycle ( c1 ) of fu and leucovorin ( fu / lv ; approximately 1 , 520 days postdiagnosis ) , complicated by oral mucositis , followed by resection of the liver mass ( approximately 1 , 580 days postdiagnosis ) , and pathologically conrmed to be metastatic eac . 
after metastasectomy , surveillance imaging showed no evidence of recurrence , ctdna levels became negative , and the patient remains in remission , currently 11 months ( approximately 320 days ) postmetastasectomy . 
the use of more sensitive cancer detection methods may help optimize treatment approaches ( escalation or de - escalation of therapies ) in the localized setting . in this case study , the patient experienced oligometastatic recurrence despite aggressive trimodality therapy . 
although we were unable to assess ctdna in the localized setting for this patient , a recent study investigated ctdna in the context of neoadjuvant chemoradiation for localized esophageal cancer and showed that detectable ctdna levels postchemoradiation , along with metabolic imaging analysis , predicted tumor progression with 100% sensitivity versus ctdna alone ( 71% ) and imaging alone ( 57% ) .15 thus , detectable , ctdna could help clarify ambiguous ndings ( as observed in this case study ) and could help identify high - risk patients with mrd who could be upper limit of normal cea 1 , 000 0.01 1 , 370 1 , 523 1 , 677 1 , 826 1 , 219 time since diagnosis ( days ) 1 , 066 no evidence of disease or disease response ctdna - positive ( mtm / ml ) liver metastasis suspicious finding , subsequently resolved or stable ctdna - negative ( mtm / ml ) mfolfox6 suspicious finding , subsequently confirmed cea ( ng / ml ) fluorouracil / leucovorin fig 1 . 
another study evaluated prognosis based on the presence of detectable ctdna after neoadjuvant chemotherapy prior to cystectomy in patients with bladder cancer.3 the preoperative ctdna status was 100% sensitive in predicting pathologic complete response , suggesting that trials could strategize testdeferred surgery in patients with undetectable ctdna postneoadjuvant therapy . in this case study , the initial plan was to proceed with chemotherapy ( mfolfox6 ) , followed by metastasectomy for removal of any residual disease ; however , the latter was delayed because of the ambiguous ndings that showed enlarged pulmonary / soft tissue nodules ( table 2 ; fig 1 ) , requiring further follow - up . 
retrospectively , serial ctdna analysis suggested that chemotherapy was more effective than rfa in reducing the tumor burden , and thus , consideration of consolidation metastasectomy in lieu ofor soon afterrfa could have prevented the second local recurrence . 
 ( * ) brd4 and smad4 have 3 missing data points between september 2017 and june 2018 . mfolfox6 , 6 cycles of uorouracil , leucovorin , and oxaliplatin . this case study illustrates the potential advantage of using ctdna as a conrmatory , adjunctive surveillance tool . 
the early and continued increase in ctdna post - rfa showed the sensitivity and specicity of the ctdna assay in predicting disease recurrence by a lead time of 174 days compared with cea and 350 days before radiographic conrmation . 
cea is known to be a lagging indicator of disease and is relatively insensitive , nonspecic , and can be confounded by acute or chronic inammation , smoking , liver disease , and diabetesall common comorbidities in this patient population.16 in this particular case , the multiple postresection complications confounded the interpretation of an increase in cea , an increase that ultimately attenuated and has not translated into relapse by imaging or ctdna with 11 months of follow - up . 
this could be a faster and more accurate measure than following the standard approach of using radiographic progression - free survival alone . it is important to note that the tumor - informed ctdna assay is a personalized method that requires separate whole - exome sequencing of the tumor tissue to develop patient - specic primers . 
the customized design of this assay targeting a set of clonal mutations known to be present in the specic tumor leads to its high sensitivity and specicity ; it is not intended to be a discovery assay for assessment of actionable genomic targets , but rather , a tool for detecting mrd . 
it was notable to observe that genomic alterations in the primary tumor were detectable with the tumor - informed ctdna assay at the time of relapse , and they all appeared to track in parallel ( fig 2 )  . 
we did not observe evidence of signicant selection for or against clones harboring any of the genomic targets tested during treatment , but we cannot rule out the acquisition of additional subclonal genomic alterations . our study has certain limitations . 
another aspect is how often and how long a patient should be monitored in this setting . although tumor burden may be undetectable by the ctdna assay , the patient is still at risk and needs surveillance . however , given the features described , it is reasonable to test this assays utility in multiple disease settings as part of clinical trials in esophageal and other cancers . 
einstein research funding : trovagene ( inst ) , bristol - myers squibb ( inst ) nathan liang employment : natera stock and other ownership interests : natera meenakshi malhotra employment : natera stock and other ownership interests : natera alexey aleshin employment : natera stock and other ownership interests : natera consulting or advisory role : mission bio solomon moshkevich employment : natera , evidation health ( i ) stock and other ownership interests : natera patents , royalties , other intellectual property : named as inventor on a patent led by natera travel , accommodations , expenses : natera paul r . 
billings employment : natera leadership : trovagene , biological dynamics , omniseq , alveo biotechnologies stock and other ownership interests : natera , trovagene , biological dynamics , omniseq , alveo biotechnologies consulting or advisory role : bethesda group , guidepoint global eirini pectasides consulting or advisory role : clovis oncology research funding : bristol - myers squibb no other potential conicts of interest were reported . references shah m , denlinger cs : optimal post - treatment surveillance in cancer survivors : is more really better ? oncology ( williston park ) 29 : 230 - 240 , 2015 2 . 
natl j maxillofac surg 7 : 17 - 20 , 2016 christensen e , birkenkamp - demtr oder k , sethi h , et al : early detection of metastatic relapse and monitoring of therapeutic efcacy by ultra - deep sequencing of plasma cell - free dna in patients with urothelial bladder carcinoma . 
j clin oncol 37 : 1547 - 1557 , 2019 abbosh c , birkbak nj , wilson ga , et al : phylogenetic ctdna analysis depicts early - stage lung cancer evolution . 
nature 545 : 446 - 451 , 2017 [ erratum : nature 554 : 264 , 2018 ] coombes c , page k , salari r , et al : personalized detection of circulating tumor dna antedates breast cancer metastatic recurrence . 
clin cancer res 25 : 14255 - 4263 , 2019 reinert t , henriksen tv , christensen e , et al : analysis of plasma cell - free dna by ultradeep sequencing in patients with stages i to iii colorectal cancer . 
jama oncol 5 : 1124 - 1131 , 2019 shapiro j , van lanschot jjb , hulshof mccm , et al : neoadjuvant chemoradiotherapy plus surgery versus surgery alone for oesophageal or junctional cancer ( cross ) : long - term results of a randomised controlled trial . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american pathologists joint review . 
arch pathol lab med 142 : 1242 - 1253 , 2018 araujo dv , bratman sv , siu ll : designing circulating tumor dna - based interventional clinical trials in oncology . 
bray f , ferlay j , soerjomataram i , et al : global cancer statistics 2018 : globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries . 
 prospective longitudinal ctdna workow reveals clinically actionable alterations in ovarian cancer jaana oikkonen , phd1 ; kaiyang zhang , msc1 ; liina salminen , md2 ; ingrid schulman1 ; kari lavikka1 ; noora andersson , phd1 ; erika ojanper a1 ; sakari hietanen , md , phd2 ; seija gr enman , md , phd2 ; rainer lehtonen , phd1 ; kaisa huhtinen , phd3 ; olli carp en , md , phd1 , 3 , 4 ; johanna hynninen , md , phd2 ; anniina f arkkil a , md , phd1 , 4 , 5 ; and sampsa hautaniemi , d tech1 purpose circulating tumor dna ( ctdna ) detection is a minimally invasive technique that offers dynamic molecular snapshots of genomic alterations in cancer . 
although ctdna markers can be used for early detection of cancers or for monitoring treatment efcacy , the value of ctdna in guiding treatment decisions in solid cancers is controversial . 
we applied the workow to a prospective cohort consisting of 78 ctdna samples from 12 patients with high - grade serous ovarian cancer before , during , and after treatment . these longitudinal data sets were analyzed using our open - access ctdna - tailored bioinformatics analysis pipeline and in - house translational oncology knowledgebase to detect clinically actionable genomic alterations . 
the alterations were ranked according to the european society for medical oncology scale for clinical actionability of molecular targets . results our results show good concordance of mutations and copy number alterations in ctdna and tumor samples , and alterations associated with clinically available drugs were detected in seven patients ( 58% )  . treatment of one chemoresistant patient was changed on the basis of detection of erbb2 amplication , and this ctdna - guided decision was followed by signicant tumor shrinkage and complete normalization of the cancer antigen 125 tumor marker . conclusion our results demonstrate a proof of concept for using ctdna to guide clinical decisions . 
analysis of circulating tumor dna ( ctdna ) is an approach with the potential of overcoming all three obstacles.1 indeed , ctdna sampling is a clinically attractive , minimally invasive technique that is based on the observation that tumor cells leak dna to the bloodstream , where it can be captured by genomic assays . 
 oikkonen et al context key objective the aim of the study was to identify the clinical usefulness of circulating tumor dna ( ctdna ) in the treatment of high - grade serous ovarian cancer ( hgsoc ) and to provide a clinical workow for reliable detection of ctdna alterations . knowledge generated we show that longitudinal ctdna sampling can be used to detect response to platinum - taxane primary therapy after the rst cycles of chemotherapy and to identify clinically applicable mutations and copy number alterations . 
in one patient with chemoresistant hgsoc , ctdna - guided treatment including trastuzumab was administered during tumor progression and yielded tumor shrinkage . relevance the early detection of poor - responding patients allows the design of alternative treatment strategies in hgsoc . 
especially for platinum - resistant patients who have limited treatment options , rapid discovery of resistant cell populations can provide an early opportunity to interfere with the development of recurrence . 
this is a substantial improvement in the management of recurrent solid cancers , where tumors are not usually sampled either because of the risk of potential intervention complications or simply because the sample could be insufcient or not representative of the disease . ctdna analysistailored bioinformatics pipelines , and a translational oncology knowledgebase to identify clinically actionable mutations and copy number alterations ( cnas )  . 
we demonstrate the usefulness of this approach in high - grade serous ovarian cancer ( hgsoc ) , which is the most common and aggressive form of epithelial ovarian cancer , with a 5 - year survival rate of 43%.7 the current standard - of - care therapy for advanced hgsoc consists of cytoreductive surgery and platinum - paclitaxel chemotherapy.8 although the initial response to standard - of - care therapy is usually good , nearly all patients eventually relapse and have limited treatment options . 
the samples were stored at 80c as 1 to 2 ml aliquots frozen less than 2 hours after collection to avoid lysis of the white blood cells.1 cell - free dna ( cfdna ) was extracted from plasma samples and subjected to 1000 targeted illumina hi - seq sequencing at bgi ( bgi europe a / s , copenhagen , denmark ) using the oseq solid cancer panel with more than 500 clinically actionable genes ( data supplement )  . dna was isolated from 21 liquid ln2 snap - frozen tumor tissue and ascites samples ( data supplement ) , as well as from whole blood ( germline ) buffy coat samples , and was sequenced using the oseq panel with coverage of 200 . mutation and copy number detection mutation detection was performed in the computational ecosystem anduril10 using the ctdna data analysis pipeline , which is based on best practices for sequencing data analysis ( data supplement )  . 
filtering steps included detection of likely spurious mutations by identifying clusters of variants shared between patients but not known in cancer , 12 and functionally irrelevant variants by a combined annotation dependent depletion13 score of less than 15 . 
to estimate the change in mutational proles , we identied the largest proportion of signicantly decreased variants during treatment compared with the pretreatment sample ( p , .01 , fishers exact test )  . 
because of varying numbers of mutations in the pretreatment samples , the proportion of signicantly decreased variants was corrected for mutation counts . pathway analysis was performed for genes with a nondecreasing mutation frequency ( p , .01 , fishers exact test ; data supplement ) using the consensuspathdb15 algoriththe consensuspathdb integrates data from 32 databases and allows comprehensive over - representation analysis for mutations . immunohistochemistry and in situ hybridization potentially clinically actionable alterations were validated through immunohistochemistry ( ihc ) and in situ hybridization for alterations classied as most prominent ( escat , the european society for medical oncology scale for clinical actionability of molecular targets ) 16 and shown to exist in patients tumor tissue ( data supplement )  . results ctdna panel reliably captures mutations and cnas we collected 78 longitudinal ctdna samples and 21 tumor tissue samples from 12 unselected patients with hgsoc ( data supplement )  . 
we applied a sequencing panel of more than 500 genes , followed by variant and cna calling , ltering , and prioritization of clinically relevant aberrations as shown in fig 1a and the data supplement . 
altogether , 265 mutations in 185 genes ( data supplement ) and cna aberrations in 113 genes ( data supplement ) passed our calling and ltering criteria . the median concordance of mutations detected from plasma to tumor tissue samples from the same patient was 79% ( fig 1b ; data supplement )  . 
 oikkonen et al 12 patients with hgsoc variant and cna calling targeted sequencing mutation discovery filtering plasma , sampled longitudinally cancer gene panel cna detection germline dna tumor tissue validation clinical application interpretation prioritization early response monitoring vaf dynamics drug relevance translational oncology knowledgebase tumor burden and mutational composition during therapy plasma only shared tissue only patient 12 months 12 months fig 1 . 
pfi , platinum - free interval . ctdna mutation proles reect long - term treatment response and reveal targetable pathways in patients with aggressive hgsoc ctdna allows several ways to monitor treatment response . for hgsoc , parkinson et al4 suggested the use of tp53 measured from ctdna in disease monitoring . 
we observed that , consistent with their results , tp53 vaf was below 0.5% in all except one patient during primary chemotherapy and was increased in all patients at disease progression ( fig 2 )  . 
these results suggest that the changes in vafs detected from ctdna samples can be used for the early identication of patients with poor response to chemotherapy . we hypothesized that genes whose mutation vafs remained stable or increased during treatment in poor responders could reveal pathways crucial for chemoresistance . 
pathways enriched in the poor - responding patients in comparison with the good - responding patients included transcription , p53 , and chromatin regulatory pathways and dna double - strand break repair pathways ( data supplement )  . 
these alterations are predicted to cause sensitivity to cdk2 / 425 and / or cdk4 / 6 inhibitors.26 regardless of the presence of hr - deciencyconferring alterations , this patient , who had no residual tumor after surgery , progressed 8.9 months after the last platinum cycle . ctdna identies actionable mutations in patients with hgsoc we prioritized and interpreted all 265 mutations and 113 cnas using the translational oncology knowledgebase , which integrates information from 11 databases and 14 scientic articles ( data supplement )  . 
the knowledgebaseprioritized mutations and cnas revealed four major targetable processes : mammalian target of rapamycin ( mtor ; patients eoc429 , eoc49 ) , dna repair ( patients eoc429 , eoc677 , and eoc1067 ) , epidermal growth factor receptor ( egfr ; patients eoc587 , eoc736 , and eoc568 ) , and cyclin dependent kinases ( cdks ; patient eoc1067 )  . the highest allele frequency mutations matching tumor content were detected in pten ( patient eoc49 ) and nf1 ( patient eoc429 ) , which cause overactivation of the mtor pathway.20 , 22 patients with the activated mtor pathway have been shown to be responsive to phosphatidylinositol 3 - kinase / mtor inhibitors.22 we validated overactivity of the mtor pathway in the two patients with ihc from tumor tissue samples ( fig 3 ; data supplement )  . 
in patient eoc49 , pten mutation vaf in omentum ( 8% and 6% for pretreatment and interval samples , respectively ) and plasma matched the pattern of tp53 mutation , indicating that tumor cells and presents an early event in tumor evolution ( data supplement )  . 
pten was detected in a follow - up sample at 8 months after primary treatment with vaf of 0.1% , conrming its persistence during therapy . the pten mutation exists in all we observed mutations and cnas in genes associated with dna repair in three patients . 
in patient eoc677 , we detected mutations in brca2 and stag2 , which potentially confer homologous recombination ( hr ) dna repair deciency and sensitivity to poly - adp ribose polymerase ( parp ) inhibitors . however , the frequencies of these mutations were low in the tumor tissues ( 0.2% for brca2 and 3.1% for stag2 ) , suggesting subclonal events . 
in patient eoc429 , we detected a germline deletion in rad51c from plasma , which has been shown to confer hr deciency and parp inhibitor sensitivity , 21 and , consistent with this , the patient had a prolonged response to platinum - based chemotherapy . 
we also detected a somatic deletion of fanca in patient three patients had mutations or cnas associated with sensitivity to drugs targeting the epidermal growth factor receptor ( egfr ) pathway . 
patient eoc568 had an erbb4 mutation , which is predicted to confer sensitivity to egfr inhibitors.27 patient eoc587 had a mapk mutation and a simultaneous mapk1 amplication , which are associated with resistance to the egfr targeting therapies.28 , 29 we detected a high copycount erbb2 amplication in the ctdna in patient eoc736 . the erbb2 locus is amplied in 3% to 11% of hgsocs30 , 31 and is a predictive biomarker for trastuzumab . 
the erbb2 amplication was validated in primary omentum tissue using snp genotypebased cna detection and in the tumor tissue sample from interval debulking surgery with in situ hybridization and ihc ( figs 4a and 4b )  . patient eoc736 was treated with nact , and she participated in a clinical trial after primary treatment , receiving maintenance bevacizumab and olaparib or placebo ( fig 4c )  . 
although she had a clinical and radiologic complete response to primary therapy , tp53 vaf remained detectable throughout the treatment , which , on the basis of our ndings , indicates a poor response to platinum taxane ( data supplement )  . 
this patient also suffered from severe paclitaxel - induced neuropathy after primary treatment , and therefore , pegylated liposomal doxorubicin ( pld ) was started at the rst relapse instead of dose - dense paclitaxel therapy . 
pld was discontinued after four cycles because of constantly rising ca125 ( from 193 to 769 ) and progression in lymph nodes as evidenced by a computed tomography scan . the erbb2 amplication detected in on the basis of ctdna , treatment of patient eoc736 was changed to trastuzumab 600 mg subcutaneously once every 3 weeks , combined with reduced doses of carboplatin ( auc4 ) and dose - dense paclitaxel ( 80 mg / m2 on days 1 and 8 )  . 
the patient responded to this combination well , and a biochemical complete response ( ca - 125 value reduced from 840 iu / ml to 19 iu / ml ) was achieved after only three treatment cycles ( fig 4c )  . 
pathogenic mutation in nf1 was detected in circulating tumor dna ( ctdna ) , leading to functionally overactive mammalian target of rapamycin ( mtor ) signaling in the tumor tissue . 
 ( a ) mutational frequencies in a good - responding patient ( eoc429 ) during primary treatment and follow - up . variant allele frequency ( vaf ) values are normalized for the frequency level to show relative changes in the levels . 
 ( b ) mtor pathway activation was detected in primary tumor tissue with higher staining for phosphorylated mtor ( pmtor ) and e4 - bp1 compared with normal ovarian tissue . 
scale bar , 100 m ( c ) in addition to having a complete response on the basis of recist 1.1 , the response to primary therapy was good , as indicated by cancer antigen 125 ( ca - 125 ) values that stayed low during treatment and follow - up . 
cadd , combined annotation dependent depletion ; hgsoc , high - grade serous ovarian cancer ; pte , primary treatment end ; 4e - bp , 4e - binding protein . sequencing of more than 500 genes from liquid biopsies , a ctdna data analysis pipeline , and a translational oncology knowledgebase to enable longitudinal analysis of ctdna during therapy and guide personalized cancer treatment decisions . 
the ctdna analysis pipeline and translational oncology knowledgebase are open - source and available with documentation ( data supplement )  . we detected high ctdnatumor congruence , which , together with the absence of alterations from the blood cells used as germline samples and comprehensive ltering of mutations through clustering , maximizes the likelihood that the reported alterations originated from tumor and not from other sources , such as clonal hematopoiesis.1 indeed , our ltering approach using longitudinal samples removed false - positive alterations in all patients . 
rara was within the amplied region in plasma but not in primary omentu ( b ) erbb2 protein was overexpressed in cancer tissue ( arrowheads ) in immunohistochemistry ( ihc ) and the erbb2 gene was amplied in dna in situ hybridization ( ish ) analysis ( scale bars , 20 mm )  . 
the most informative comparisons for predicting treatment outcome are samples at the preoperative stage , after two cycles of chemotherapy , and after debulking surgery , because they provide information on treatment response and on persisting tumor subclones we identied potentially clinically applicable mutations or cnas in more than one half of the patients with hgsoc . these included two patients with mtor pathwayactivating mutations that were validated . 
both patients had a good response to the platinum - based rst - line therapy , but in the case of relapse they could most likely benet from mtor inhibitors alone or in combination with chemotherapy.22 in patient eoc429 , we identied alterations conferring both mtor pathway activation and hr deciency , which suggests potential benet from combination treatment with , for example , mtor inhibition and parp inhibition . 
in addition , two patients had more than one concurrent , potentially targetable alteration . patient eoc1067 progressed after 9 months of platinum - based therapy despite the fanca deletion conferring hr deciency , potentially because of the contribution of the cdk alterations on cell - cycle regulation and tumor progression.32 on the basis of the concurrent ckdn1b and ckdn2b deletions , this patient could potentially benet from a combination targeting both pathways , for instance , parp inhibitor and cdk4 / 6 inhibitor . these results indicate that longitudinal ctdna sampling can detect and monitor a relapse at an early stage and identify potentially effective drug combinations . we identied a high - condence erbb2 amplication in the ctdna of a patient with platinum - resistant hgsoc and used this information to change the patients treatment to include trastuzumab with reduced doses of platinum and paclitaxel . 
thus , rapid response to the ctdna - guided treatment , detected by complete normalization of ca - 125 values within 2 months after starting the combination treatment , was surprising . 
earlier studies that have combined erbb2 inhibitors with platinum and paclitaxel report similar encouraging responses for relapsed patients with advanced hgsoc.31 although the number of patients in these studies and our study remains small because of the low number of patients with erbb2 - amplied hgsoc , these results warrant testing erbb2 amplication from relapsed hgsoc patients with advanced disease . ctdna sampling and sequencing with a large panel offers several benets to physicians . 
for a detailed description of the disclosure categories , or for more information about ascos conict of interest policy , please refer to or po.ascopubs.org / site / ifc sakari hietanen consulting or advisory role : tesaro , astrazeneca travel , accommodations , expenses : roche pharma ag olli carp en stock and other ownership interests : orion corporation honoraria : merck kgaa , roche pharma ag sampsa hautaniemi stock and other ownership interests : nanostring technologies no other potential conicts of interest were reported . acknowledgment we thank the patients who participated in this study , dr tuulia vallius and dr zoltan maliga for critical review of the manuscript , ma tiia pelkonen for proof reading , and the csc - it center for science ltd . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american pathologists joint review . 
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 o duodenal - jejunal flexure gi stromal tumor frequently heralds somatic nf1 and notch pathway mutations purpose gi stromal tumors ( gists ) are commonly associated with somatic mutations in kit and pdgfra . 
we define the anatomic distribution of nf1 alterations in gist . methods we describe the demographic / clinicopathologic features of 177 patients from two institutions whose gists underwent next - generation sequencing of 315 cancer - related genes . results we initially identified six ( 9.7% ) of 62 gists with nf1 genomic alterations from the first cohort . 
of the five djf gists with any nf1 alteration , three ( 60% ) had kit mutations , and three ( 60% ) had notch pathway mutations ( notch2 , maml2 , cdc73 )  . 
we validated these findings in a second cohort of 115 gists , where two ( 40% ) of five unifocal nf1 - mutated gists arose at the djf , and one of these also had a notch pathway mutation ( ep300 )  . conclusion broad genomic profiling of adult gists has revealed that nf1 alterations are enriched in djf gists . 
 ( ie , kras , nras , hras ) by increasing the catalytic conversion of the active form ( ras - gtp ) to the inactive form ( ras - gdp ) .14 pathogenic nf1 variants disrupt the normal function of neurofibromin and result in constitutive ras activation.15 this activation increases downstream signaling through braf / craf - mediated activation of the mitogen - activated protein kinase pathway and hence , facilitates tumor initiation and progression.16 until recently , nf1 mutations in gist were believed to be primarily of germline origin , associated with clinical nf - 1 , and combined with a second somatic mutation in the tumor . 
thus , our understanding of nf1 in gist continues to evolve . approximately 30% of all gists occur in the small intestine , 1 which measures approximately 5 to 6 m in length.18 most small bowel gists are associated with somatic kit mutations , whereas a subset is associated with nf1 mutations.19 the small bowel is divided into three anatomically , histologically , and functionally distinct segments , namely the duodenum , jejunum , and ileum.18 although some studies distinguish duodenal gists from other small bowel gists , most combine them and do not characterize the biology of tumors on the basis of these distinct locations . 
moreover , the exact transition from jejunum to ileum is somewhat arbitrary , but the duodenal - jejunal flexure ( djf ) , also known as the ligament of treitz , represents a clear anatomic site that marks the transition from duodenum to jejunuto date , no study of gists has specifically characterized tumors that arise from the djf . 
the current study , however , started with presumably sporadic gists , which led to the unexpected finding that somatic nf1 mutations in gists are more prevalent than previously suspected and that they are enriched in tumors that arise from the djf . methods primary study population patient demographic and tumor clinicopathologic data were retrospectively collected in an unbiased fashion from every patient with pathologically confirmed gist seen within the university of california , san diego ( ucsd ) , health system from january 1 , 2000 , to april 30 , 2017 , under a ucsd institutional review boardapproved protocol . 
available operating notes or imaging reports were reviewed for these 165 patients to distinguish the primary site of origin of any small bowel gist as duodenal , djf , jejunal , or ileal . comprehensive genomic profiling of the 165 patients with gist in the cohort , 62 underwent next - generation sequencing ( ngs ) of coding regions of cancer - related genes . 
dna was extracted from formalin - fixed paraffin - embedded ( ffpe ) sections that contained a minimum of 20% tumor tissue and were used for comprehensive genomic profiling with hybridization - captured , adaptor ligationbased libraries . 
the number of genes in the foundationone panel has evolved over time as new data on cancer - related genes have been published and currently includes 315 cancerrelated genes plus select introns from 28 genes often rearranged or altered in solid tumor cancers . however , all versions of the assay simultaneously analyze the extracted dna for base substitutions , short insertions and deletions , amplifications and homozygous deletions , and gene rearrangements with > 99% sensitivity.20 specimens submitted to the ucsd clinical genomics laboratory underwent ngs of the exons of 397 genes involved in pathways that control growth or differentiation and are known to be frequently mutated in solid tumors . 
coverage depth ranged from 2833 to 8303 across all sequenced tumor samples from both the foundationone and the ucsd assays . nontumor tissue sequencing patients found to have nf1 mutations in their tumor tissue were also tested for germline nf1 mutation . four samples tested by using dna extracted from ffpe sections of adjacent normal tissue or peripheral blood underwent ngs cancer - related mutation panel testing by the ucsd clinical genomics laboratory as just described . 
one additional sample was tested by using commercially available targeted sequencing of the nf1 gene from peripheral blood by arup laboratories ( salt lake city , ut )  . secondary study population a confirmatory cohort of  . 
1 , 000 patients with pathologically confirmed gists from memorial sloan kettering cancer center ( mskcc ) with retrospectively collected patient demographic and tumor clinicopathologic data also were analyzed under an institutional review boardapproved protocol . 
data included age , sex , primary gist site , tumor size , and pathologic characteristics . one hundred fifteen patients with gists had ngs results available in the msk - impact ( integrated mutation profiling of actionable cancer targets ) platform , which characterized frozen or ffpe tumor specimens for somatic dna mutations , copy number alterations , and select rearrangements of 341 cancer - associated genes.21 statistical analysis all statistical analyses were performed with stata 9.0 software ( statacorp , college station , tx )  . comparisons between groups were performed by using the two - sample test of proportions . 
none of these patients had previously known clinical manifestations of nf - 1 ( eg , cafe au lait spots ; axillary / inguinal freckling ; dermal neurofibromas ; lisch nodules ; iris hamartomas ; nervous system tumors , including malignant peripheral nerve sheath tumors [ mpnsts ] , pancreatic neuroendocrine tumors , pheochromocytomas , or other sarcomas ] ) .10 therefore , the application of expanded ngs panels to characterize the mutational profile of gists identified an unappreciated subset of patients with nf1 genomic alterations in their tumors but no clinical evidence of nf - 1 . nf1 genomic alterations in gist frequently occur at the djf of the six patients with gist for whom we identified nf1 genomic alterations , five ( 83.3% ) had unifocal tumors at the djf , and one ( 16.7% ) had a unifocal tumor in the stomach ( fig 1a )  . 
the demographic , clinicopathologic , and genomic characteristics of these nine patients with djf tumors are listed in table 1 ( n = 7 ngs gists ) and the data supplement ( n = 2 non - ngs gists )  . 
given the infrequency of nf1 genomic alterations in gist , the djf appears to be an over - represented focus of nf1 mutant tumors . nf1 genomic alterations in gist are primarily somatic but can herald mild neurofibromatosis nf1 genomic alterations for each patient are listed in table 2 . 
although none of the patients had known prior clinical evidence of nf - 1 , we next obtained nontumor dna from blood or nontumor tissue to determine whether any of these patients had germline nf1 mutations . 
 ( a ) from a total of 165 patients ( university of california , san diego [ ucsd ] ) with pathologically confirmed gists , 62 had available next generation sequencing ( ngs )  . 
this finding is consistent with the mild phenotype / low penetrance reported in other patients with this germline nf1 mutation.22 nf1 genomic alterations in gists are associated with other cancer - related mutations at the genomic level , the ucsd cohort ngs data generally parallel the known distribution of driver mutations in gists ( fig 2 )  . 
of the 65 driver mutations identified in the 62 gists , oncogenic kit ( 63% ) , pdgfra ( 11% ) , and sdh subunit ( 11% ) mutations were most frequent , whereas braf and kras mutations were less common . moreover , nf1 mutations ( six of 65 ) represented 9% of total genomic alterations . 
the loss of the nf1 tumor suppressor gene often leads to additional chromosomal alterations , but second - hit mutations and / or loss of heterozygosity are necessary to promote tumorigenesis.23 therefore , we analyzed a cohort of five patients with nf1 mutant djf gists for additional cancer - related gene mutations . 
demographic and clinicopathologic characteristics of these two patients are listed in table 3 , with the characteristics of the remaining five patients without djf and / or multifocal gists listed in the data supplement . 
in addition to an nf1 mutation , one patient had a concomitant mutation in ep300 , which encodes a histone acetyltransferase / transcriptional coactivator that has been previously reported to interact with maml1 and maml2 to potentiate notch signaling.24 this supports our earlier observation that unifocal nf1 mutant djf gists also can have impaired notch signaling . 
in summary , 40% of unifocal nf1 mutant gists in the mskcc cohort were localized at the djf , which corroborates the high rate ( 83.3% ) of similar tumors in the ucsd cohort . 
we now show in two single - institution cohorts of patients with gists that 6.1% ( mskcc , seven of 115 ) to 9.7% ( ucsd , six of 62 ) have nf1 alterations . 
if we exclude the 17 patients who overlap with our previous study ( two of whom have nf1 mutations [ ucsd patients 3 and 5 in the current study ] ) , we show that 8.9% ( four of 45 ) of patients with gists have nf1 alterations . 
taken together , nf1 mutation frequencies range from 6% to 10% in gist and are higher than previously appreciated but similar to our recent analysis.7 for the first time in our knowledge , we show that broad genomic profiling of djf or ligament of treitz gists in adults reveals frequent nf1 alterations ( somatic and / or germline ) that occur even in the absence of clinical nf - 1 . 
 yes high high high high pathologic feature tumor resection yes tumor size , cm 13 mitotic index / 5 mm2 high cellular morphology mixed spindle / epithelioid histologic grade g2 immunostain positivity dog - 1 genomic alterations somatic kit ( af ) kit exon somatic nf1 ( af ) ( 30% ) none other somatic alterations ( af ) none table 2 . 
of the 62 patients with gi stromal tumors with available next generation sequencing , 65 known driver mutations were identified : 41 in kit , seven in pdgfra , six in nf1 , seven in succinate dehydrogenase ( sdh ) subunits ( a , b , c , or d ) , two in braf , and two in kras . awareness of the risk of additional tumors ( eg , dermal neurofibromas , nervous system tumors [ including mpnsts , pancreatic neuroendocrine tumors , pheochromocytomas , and other sarcomas ] ) 10 and allows for earlier screening of these nf - 1associated tumors , although whether different malignancies and gists have unique presentation patterns remains to be determined ; and diagnosis of nf - 1 allows for genetic counseling and testing of potentially affected family members . 
of note , individuals can have segmental mosaicism of nf - 1 , which occurs when an nf1 somatic mutation occurs early in embryonic development such that only the tissues derived from the nf1 - mutated cell carry the mutation while the remaining tissues are wild type.26 as with ucsd patient 2 , a clinical genetics work - up may be indicated for patients with nf1 mutations on tumor profiling that could indicate a germline rather than a somatic origin . tumor development in the setting of nf1 genomic alterations is associated with additional second - hit mutations.23 we now have shown that djf tumors with nf1 genomic alteration also can harbor concurrent kit mutations . 
in general , most kit mutations portend imatinib sensitivity , whereas nf1 mutations do not.3 , 27 consistent with this aforementioned drug sensitivity , tumors with a high risk of recurrence are recommended for adjuvant imatinib on the basis of the american college of surgeons oncology group z900128 and scandinavian sarcoma group xviii / aio29 trials where cumulative results demonstrated improved relapse - free survival ( rfs ) and overall survival with 36 months of adjuvant imatinib in patients with high - risk disease . 
in our cohort , three patients with djf gists had a high risk of recurrence by validated risk assessment models.30 , 31 ucsd patient 4 lacked a kit mutation and was predicted to not respond to imatinib therapy ; instead , this patient could experience toxicity . 
this approach was supported by eortc - 62005 and southwest oncology group s0033 / cancer and leukemia group b 150105 phase iii trials that collectively demonstrated improved response rates and rfs in patients with advanced gists and kit exon 9 mutations treated with high - dose imatinib ( 800 mg daily ) compared with standard - dose imatinib ( 400 mg daily ) .32 the patient also had a germline nf1 variant and ultimately developed a local recurrence after 5 years of adjuvant imatinib therapy . ucsd patient 3 had a known kit exon 11 mutation and was treated with dose - escalated imatinib followed by the multikinase inhibitor sunitinib for metastatic disease . 
a brief trial of the mammalian target of rapamycin inhibitor everolimus in combination with imatinib was attempted to block parallel signaling pathways , but the patient rapidly succumbed to the disease . much like patients with metastatic colorectal cancer who are tested for pan - ras mutations before treatment targeting upstream cell surface receptors ( ie , epidermal growth factor receptor ) , 33 , 34 we propose that patients with djf gists be tested for nf1 ( which would activate ras ) and braf v600e genomic alterations ( which two of the patients with djf gists also harbored ) before being offered imatinib therapy , which targets upstream kit . we have recently shown that notch1 can be mutated in both wild - type and nonwild - type gists.7 we now add evidence that implicates the notch signaling pathway in gists . 
we identified another tumor with a mutation in cdc73 , which encodes the rna polymerase iiassociated factor complex protein parafibromin and was recently reported to potentiate notch signaling by binding to the nicd.36 we also found an ep300 mutation in the validation cohort that could abrogate signaling of the notch transcriptional complex . of note , notch signaling was reported to have a tumor suppressor function in gist by downregulating kit mrna expression.37 this prior study also demonstrated that patients with gists with low hes1 expression had shorter rfs times than those with high hes1 expression . whether loss - of - function mutations in notch pathway genes that lead to decreased hes1 expression contribute to gist tumorigenesis remains to be determined . 
nf1 regulates mek / smad3 / jagged1 / hes1dependent glial and neuronal differentiation.38 notch has also been reported to mediate transformation of benign plexiform neurofibromas into mpnsts in the setting of nf - 1 , 39 which suggests an alternate oncogenic role of notch in nf - 1 . 
more studies are required to elucidate the biologic significance of notch pathway mutations in gist , including nf - 1associated gist . finally , the mechanism by which nf1 mutations lead to gists that specifically arise at the djf remains unclear . 
summary of duodenal - jejunal flexure genomic alterations . tumor size , mitotic index ( mi ) , and genomic alterations for patients with duodenal - jejunal flexure gi stromal tumors ( gists ) from both cohorts are presented and organized by known driver genes and notch pathway alterations . somatic and germline mutations are annotated by color for mutation type . snp , single nucleotide polymorphism . cdc73 ep300 notch2 maml2 asxl1 men1 erbb4 tsc2 bcor ( per 5 mm2 ) high unknown genomic alteration nonsense frameshift missense in - frame indel deletion splicing ( somatic ) ( germline ) ( somatic ) braf ( somatic ) arid1a ( somatic ) notch pathway other pacemaker activity.42 one could postulate that this region of chromatin on 17q is open and transcriptionally active at the djf . 
belinsky no relationship to disclose margaret von mehren consulting or advisory role : cytrx , blueprint medicines , janssen pharmaceuticals , deciphera pharmaceuticals research funding : arqule travel , accommodations , expenses : janssen pharmaceuticals , blueprint medicines , novartis , arog pharmaceuticals other relationship : national comprehensive cancer network john a . 
thorson no relationship to disclose lisa madlensky employment : janssen pharmaceuticals ( i ) patents , royalties , other intellectual property : husband has patents through his employment with janssen pharmaceuticals ; these are methods patents and not related to any therapeutics ( i ) timothy bowler employment : regenxbio ( i ) stock and other ownership interests : regenxbio ( i ) francesco dangelo no relationship to disclose dwayne g . 
sicklick , university of california , san diego , la jolla , ca ; martina de siena and francesco dangelo , sapienza e universit `a di roma , rome , italy ; benjamin medina , timothy bowler , and ronald p . 
ma gl , murphy jd , martinez me , et al : epidemiology of gastrointestinal stromal tumors in the era of histology codes : results of a population - based study . 
hechtman jf , zehir a , mitchell t , et al : novel oncogene and tumor suppressor mutations in kit and pdgfra wild type gastrointestinal stromal tumors revealed by next generation sequencing . 
burgoyne am , somaiah n , sicklick jk : gastrointestinal stromal tumors in the setting of multiple tumor syndromes . curr opin oncol 26 : 408 - 414 , 2014 11 . 
belinsky mg , rink l , cai kq , et al : somatic loss of function mutations in neurofibromin 1 and myc associated factor x genes identified by exome - wide sequencing in a wild - type gist case . 
miettinen m , fetsch jf , sobin lh , et al : gastrointestinal stromal tumors in patients with neurofibromatosis 1 : a clinicopathologic and molecular genetic study of 45 cases . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
messiaen l , vogt j , bengesser k , et al : mosaic type - 1 nf1 microdeletions as a cause of both generalized and segmental neurofibromatosis type - 1 ( nf1 )  . 
dematteo rp , ballman kv , antonescu cr , et al : adjuvant imatinib mesylate after resection of localised , primary gastrointestinal stromal tumour : a randomised , double - blind , placebo - controlled trial . 
gastrointestinal stromal tumor meta - analysis group ( metagist ) : comparison of two doses of imatinib for the treatment of unresectable or metastatic gastrointestinal stromal tumors : a meta - analysis of 1 , 640 patients . 
pietrantonio f , cremolini c , petrelli f , et al : first - line anti - egfr monoclonal antibodies in panras wild - type metastatic colorectal cancer : a systematic review and meta - analysis . 
sorich mj , wiese md , rowland a , et al : extended ras mutations and anti - egfr monoclonal antibody survival benefit in metastatic colorectal cancer : a meta - analysis of randomized , controlled trials . 
hansen , md , phd1 , 2 ; lars henrik jensen , md , phd1 , 2 ; and anders jakobsen , dmsc1 , 2 purpose analysis of circulating tumor dna ( ctdna ) is a potential improvement in precision medicine . 
plasma samples represented healthy donors and localized and metastatic crcs . results the level of neuropeptide ymethylated dna in the tissue cohorts differed between nonmalignant and malignant / premalignant tissues with minimal overlap . 
furthermore , meth - ctdna was detected in plasma from 100% of patients with metastatic disease , compared with 67% of those with localized disease and 8% of healthy donors . 
2019 by american society of clinical oncology introduction circulating tumor - specic dna ( ctdna ) is an attractive biomarker for obvious reasons , such as easily repeated access , better reection of overall tumor biology , and timely correct tumor mutational status . 
consequently , the era is marked by enthusiasm , but it should be noted that its clinical utility , with few exceptions , remains to be proven.1 ctdna can be determined by the presence of tumorspecic genetic or epigenetic alterations . 
ngs allows identication of mutations of interest and is a relatively broad approach applicable in the identication of mutational patterns and quantication of specic mutations , but this method is time and resource consuming . 
different platforms may lead to contradictive results , as shown in a recent report , with only 60% agreement between analyses of the same mutations.2 the second method relies on pcr with analysis of specic mutations . 
compared with ngs , pcr , especially digital pcr , has a faster data turnaround time3 and lower costs , but direct pcr is only applicable in the fraction of patients with known specic mutations . in colorectal cancer ( crc ) , ras / raf mutations are of interest , because their presence in tumor tissue contraindicates treatment with monoclonal antibodies cetuximab and panitumumab . 
multiplex technology allows for screening of 27 mutations in the same analysis , 4 but even so , only approximately 60% of patients present with a ras / raf - mutated tumor . 
 thomsen et al context key objective can circulating tumor - specic methylated dna serve as a universal biomarker in colorectal cancer ? knowledge generated methylated dna was tumor specic in different cohorts including tissue as well as plasma . 
high correlation was found between mutated and methylated dna in both localized and metastatic disease . relevance circulating tumor - specic methylated dna holds promise as a universal colorectal cancer marker that can be analyzed in a simple , highly reproducible approach . most of these patients , the same tumor - specic mutation is detectable in ctdna ( mut - ctdna )  . 
dna was eluted in 50 ml of nuclease - free water and further diluted with nuclease - free water , if necessary . aberrant methylation is an early event in carcinogenesis and occurs in almost all malignant tumors as methylation of promoter regions causing inactivation of genes . 
this , along with the stability of methylation changes , makes it a relevant biomarker for early diagnosis.5 analysis of abnormally methylated dna in plasma ( meth - ctdna ) has been proposed for screening , 6 and a test for crc has been approved by the us food and drug administration.7 however , because of insufcient sensitivity and specicity , it has not been generally accepted . 
the neuropeptide y gene ( npy ) has been correlated with invasion and proliferation.8 , 9 exogenous npy has been shown to inuence the growth of malignant cholangiocarcinoma cells in vitro and inhibit invasion of crc cells . 
in this disease , hypermethylation of the npy promoter region has been correlated with inactivation of gene expression and therefore carcinogenesis , and currently , hypermethylation of the npy gene is suggested as a blood - based biomarker.10 - 12 the purpose of our study was to compare mutated dna with methylated dna in malignant and nonmalignant colorectal tissue and plasma with the aim of determining a method applicable in all patients with crc . plasma sampling and purication in edta tubes , 9 - ml blood samples were collected from donors and different cohorts of patients . 
plasma was centrifuged again at 10 , 000 g for 10 minutes before purication , and cysteine - rich polycomb - like protein 1 ( cpp1 ) dna fragments were added as exogenous internal control.13 dna from patients was puried from 2 2 or 2 4 ml of plasma on the qiasymphony sp instrument using the qiasymphony circulating nucleic acid kit ( qiagen , hilden , germany ) according to manufacturer instructions . dna from healthy donors was puried from 1 4 ml of plasma . dna was eluted in 60 ml of the supplied buffer and water added to 400 ml . 
three 15 - mm slices of ffpe tissue were subjected to 180 ml of incubation buffer and 20 ml of protein kinase k overnight at 70c ; 400 ml of lysis buffer adenomas and tumor tissue samples were screened for ras / raf mutations as previously described.14 samples from adjacent tissue and plasma were analyzed for the specic mutation by droplet digital pcr in two wells . positive controls for each mutation ( gblocks ; idt , carolville , ia ) or mutated fragments generated according to spindler et al , 15 genomic donor dna , and water were included in the analyses as controls . 
patients with mcrc had plasma analyzed both at baseline and after the rst treatment cycle . tissue methylation in 25 nonneoplastic specimens , all but one were nonmethylated ( 4% were npy positive )  . 
figure 1 shows the results of the tissue npy methylation analysis , with methylated npy presented as actual clearly shows that the level of npy methylation was different in malignant and nonmalignant tissues . 
the range of npy fractions . mutated and methylated dna in colorectal cancer methylation analysis tumor - specic methylated dna was dened as dna with methylation of the npy gene.12 before droplet digital pcr methylation analysis , bisulte conversion of circulating and ffpe - isolated dna was performed as recommended by the manufacturer ( zymo research , irvine , ca )  . 
water and a pool of lymphocyte dna from cancer - free individuals were included in each round of analyses as negative controls ; universal human methylated control dna ( zymo research ) and epitech control dna ( qiagen ) were included as positive controls . 
the upper limit of the 95% ci was 1.8% , and therefore , the cutoff value was set to 2% . limit of blank in plasma limit of blank ( lob ) in the mutation analysis was determined by donor controls . 
because fewer than two fampositive droplets were observed in all analyses of the genomic donor dna controls and plasma dna from healthy individuals as previously described , 16 plasma samples with more than two fam - positive droplets in two wells were classied as positive . lob in the methylation analysis was dened as the quantity of npy droplets counted in control dna samples from a test cohort of healthy individuals ( n = 50 )  . 
correlations are also illustrated in the scatter plots shown in figures 3 , 4a , and 4b , which represent correlation between meth - ctdna and mut - ctdna in the localized and metastatic settings at baseline and in the metastatic setting after the rst treatment cycle , respectively . discussion our study on tissue and plasma samples from donors and different stages of crc indicates that meth - ctdna correlated with mut - ctdna across the cohorts and that methctdna was detectable in cases without activating dna mutations . 
therefore , meth - ctdna may be a universal biomarker in crc from the perspectives of detection and monitoring . key challenges in cancer management are early diagnosis and close monitoring during treatment and follow - up with minimal harm to the patients . 
in the landscape of tumor markers , ctdna represents a relatively new approach with the potential for progress in cancer management , because it contains the same molecular alterations as the corresponding tumor and potential metastases . 
even so , clinical utility has been limited . recently , presence of ctdna was conrmed in wt patients using epigenetic modications such as hypermethylation.17 - 19 meth - ctdna seems to represent tumor - specic dna and is found in nearly all colorectal adenocarcinomas , 10 as opposed to ctdna based on ras / raf mutations . 
three recent studies have suggested that hypermethylation of the npy gene may serve as a biomarker for monitoring the treatment of crc , 10 , 11 , 20 but a detailed comparison of mutated and methylated dna motivated our study . the level of methylation varied among different cohorts with malignant and nonmalignant tissues , the latter in general being negative . 
fifty healthy donors were only analyzed for methylated ctdna ( methctdna ) , and 20 healthy donors were analyzed for kras g12d mutated ctdna ( mut - ctdna )  . 
plasma was drawn from patients with localized ras / raf - mutated tumors ( n = 27 ) and metastatic ras / rafmutated tumors ( n = 20 ) and analyzed for the same specic mutations and methylation . methylation in tumors did not overlap that of nonmalignant npy methylation , except in three cases ( 3% )  . plasma methylation figure 2 shows the plasma analysis of different cohorts . samples from healthy donors showed that four ( 8% ) of 50 had detectable meth - ctdna above the lob . 
furthermore , the analysis showed that 67% ( 18 of 27 ) of patients with localized crc had mut - ctdna , compared with 52% ( 14 of 27 ) with meth - ctdna . 
scatter plot showing correlation of methylated circulating tumor dna ( ctdna ) and mutated ctdna ( mut - ctdna ) in localized disease . almost complete separation of malignant and nonmalignant tissues based on the statistical variation of the observed frequencies in healthy individuals . 
adjacent tissue showed npy hypermethylation in one third of the samples , although they were just positive ; none were mutated , despite ras / raf mutations in the nearby tumor . 
scatter plots showing correlation of methylated circulating tumor dna ( ctdna ) and mutated ctdna ( mut - ctdna ) in metastatic colorectal cancer ( a ) at baseline and ( b ) after one cycle of treatment . furthermore methylation is an early event in carcinogenesis , 22 which may represent a potential universal biomarker.23 all tumor tissue samples were hypermethylated irrespective of mutational status and to the same extent as ras / raf - mutated adenomas . 
this indicates that aberrant methylation takes place as an early part of cancer progression , which can predispose to additional genetic alterations.24 possibly , these epigenetic changes give an important and early window for therapeutic interventions in patients with crc . plasma analyses showed that the frequency of patients with ctdna varied depending on disease stage . 
lob of two positive droplets per reaction determined in a test cohort of 50 healthy donors seems in good agreement with previous results reporting an lob of 3.30 on the basis of 17 control dna samples without hypermethylated npy.11 the two self - reported healthy donor cohorts with 4% ( two of 50 ) and 8% ( four of 50 ) of patients having more than two positive droplets per reaction indicate good agreement . 
therefore , occurrence of aberrant methylation with increasing age does not seem to be a major problethe false - positive rate may be acceptable for patients with advanced disease , and therefore , clinical application of meth - ctdna in metastatic disease seems encouraging . 
in localized tumors , two thirds of patients harbored mut - ctdna and meth - ctdna , which is in accordance with current literature.10 in the metastatic setting , all indicating its potential as patients had meth - ctdna , a general biomarker . 
they found that 77% had detectable ctdna at baseline , assessed by mut - ctdna and meth - ctdna . boeckx et al11 reported that 87.5% of all baseline plasma samples were positive for npy methylation in 24 patients with mcrc . 
varying methods of analysis , which lack standardization throughout the eld of research in ctdna , are one obvious reason for the lower frequency of patients with detectable ctdna compared with our ndings . correlation between mut - ctdna and meth - ctdna was investigated to compare the two potential biomarkers . 
this is in agreement with previous studies concluding that methylated markers in ctdna could replace tumor - specic mutations in the blood.10 this conclusion was based on a correlation coefcient of r = 0.94 between mut - ctdna ( kras , braf , and tp53 ) and meth - ctdna ( npy )  . 
we did not investigate the potential role of meth - ctdna in monitoring wt patients , but we found a strong correlation between mut - ctdna and meth - ctdna after one cycle of treatment , which emphasizes the role of meth - ctdna in treatment monitoring . 
taken together , the current literature indicates high agreement between mut - ctdna and meth - ctdna , 10 , 11 , 20 further supported by our results . a minor disadvantage in the methylation assays is the need for bisulte conversion , which can degrade dna and therefore requires additional material . 
however , the need for prior establishment of mutation status , as with mutctdna , is bypassed , and therefore , meth - ctdna is applicable in nearly all patients with crc . 
it should be noted that meth - ctdna is not relevant in the selection of patients for targeted treatment with epidermal growth factor receptor inhibitors . in conclusion , to our knowledge , our study represents the rst detailed comparison of methylated and mutated dna in normal tissue as well as in localized and metastatic tumors . 
this indicates that hypermethylated npy in plasma is applicable as a universal biomarker in all patients with mcrc , but further investigation of clinical utility is warranted . affiliations 1danish colorectal cancer center south , vejle hospital , vejle , denmark 2university of southern denmark , odense , denmark relationships are self - held unless noted . 
all relationships are considered compensated . lars henrik jensen research funding : merck sharp & dohme ( inst ) travel , accommodations , expenses : bayer healthcare pharmaceuticals no other potential conicts of interest were reported . acknowledgment we thank the study participants and the staff at vejle hospital who collected samples for this study . 
merker jd , oxnard gr , compton c , et al : circulating tumor dna analysis in patients with cancer : american society of clinical oncology and college of american pathologists joint review . 
jama oncol 4 : 868 - 870 , 2018 thierry ar , el messaoudi s , mollevi c , et al : clinical utility of circulating dna analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti - egfr treatment . 
front mol biosci 2 : 13 , 2015 church tr , wandell m , lofton - day c , et al : prospective evaluation of methylated sept9 in plasma for detection of asymptomatic colorectal cancer . 
boeckx n , op de beeck k , beyens m , et al : mutation and methylation analysis of circulating tumor dna can be used for the follow - up of metastatic colorectal cancer patients . 
roperch jp , incitti r , forbin s , et al : aberrant methylation of npy , penk , and wif1 as a promising marker for blood - based diagnosis of colorectal cancer . 
spindler kl , pallisgaard n , vogelius i , et al : quantitative cell - free dna , kras , and braf mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan . 
thomsen cb , hansen tf , andersen rf , et al : monitoring the effect of rst line treatment in ras / raf mutated metastatic colorectal cancer by serial analysis of tumor specic dna in plasma . 
garlan f , laurent - puig p , sefrioui d , et al : early evaluation of circulating tumor dna as marker of therapeutic efcacy in metastatic colorectal cancer patients ( placol study )  . 
step two was 44 cycles at 95c for 15 seconds and 56c for 1 minute , with a 1.5c per second ramp rate . step three was at 98c for 10 minutes . 
 c subgrouping of unfavorable histology neuroblastomas with immunohistochemistry toward precision prognosis and therapy stratication naohiko ikegaki , phd1 and hiroyuki shimada , md , phd2 , for the international neuroblastoma pathology committee introduction neuroblastoma , as well dened by willis , 1 is an embryonal tumor of neural crest origtumors of the neuroblastoma group include neuroblastoma , ganglioneuroblastoma , and ganglioneuroma . 
we believe that all ganglioneuromas were once neuroblastomas in the early stage of tumor development.1 , 2 they are collectively called peripheral neuroblastic tumors ( pnts ) and are known to present with a wide range of clinical behavior , from spontaneous regression and tumor maturation to aggressive progression that is refractory to intensive treatment . 
these observations have led us to seek additional immunohistochemical biomarkers that are tightly associated with aggressive behaviorsthat is , unresponsiveness or resistance to the current intensive multimodal therapyof certain uh tumors . 
to address this problem , we and others have sought and identied the expression of potentially drug - targetable proteins that are responsible for their aggressive progression in the uh group . 
when we seek such biomarkers , they are preferably actionable / druggable by existing pharmaceutical agents , us food and drug administration approved , or currently tested in human clinical trials . 
of note , these three types of highly aggressive neuroblastomasthat is , myc - driven neuroblastoma , alt phenotype neuroblastoma because of atrx loss , and tert - overexpressing neuroblastoma with gene rearrangementare mutually exclusive and seem to comprise the majority of therapy - resistant or refractory uh neuroblastomas . these three markersthat is , myc family proteins , tert , and atrx can be the subjects of immunohistochemical assay and potentially incorporated in future inpc . it should be noted that other than the above gene and expression alterations found in aggressive uh neuroblastomas , alk mutations and amplication are found in approximately 10% of sporadic neuroblastoma cases.20 , 21 it was also found that alk gene amplication and f1174 mutations are associated with mycn amplication.22 moreover , alk overexpression as a result of alk amplication / mutations seems to be responsible for its oncogenic activity23 ; however , the prognostic signicance of alk mutations / overexpression has been controversial , as some reports suggest an association of alk mutations / overexpression with fatal outcomes of the disease , whereas others suggest otherwise.22 , 23 in particular , regairaz et al24 showed immunohistochemically that expression of alk and its active form palk was observed in many neuroblastomas independent of alk mutation / amplication . 
in this regard , myc / mycn and tert overexpression26 - 29 has been linked to the stemness of tumor cells , and direct and indirect therapeutic targeting of these alterations suppresses the stemness of aggressive and therapy - resistant uh neuroblastomas . 
similarly , alt is linked to tumor stem cells.30 , 31 these observations allow us to incorporate the information into the inpc toward the goal of precision prognosis and therapy stratication . our plan is to rene the uh neuroblastoma stratication using immunohistopathologic analysis with antipan - myc antibody ( or anti - mycn and anti - myc ) , anti - tert antibody , and anti - atrx antibody . 
it is expected that the nonresponder to current high - risk therapymyc , tert , and atl subgroups could be separated from the possible respondernull subgroupwith a high probability before the initiation of therapy : myc subgroup : myc - driven neuroblastoma shows augmented expression of mycn protein and / or myc proteas myc family protein overexpression stimulates rrna synthesis and protein translation , neuroblastoma cells in this subgroup often exhibit prominent nucleolar formationnucleolar hypertrophyand hypertrophic cell morphology.13 , 14 because of the presence of one to a few prominent nucleoli , myc - driven neuroblastoma is cytologically distinguishable from the conventional small blue - cell neuroblastoma with salt - and - pepper nuclei . myc - driven neuroblastomas , which comprise more than 50% of high - risk neuroblastomas , includes rare and unique tumor , named large - cell neuroblastoma , characterized by the bulls eye appearance of enlarged and uniquely open euchromatin - rich nuclei that contain highly conspicuous nucleoli.33 most large - cell neuroblastomas overexpress higher levels of mycn or myc protein than other myc - driven neuroblastomas . 
their euchromatin - rich open nuclei suggest the stem celllike nature of the tumor cells.29 a considerable number of myc - driven neuroblastomas are also associated with tert overexpression , as tert is a direct transcriptional target of myc family proteins . 
tert can also stabilize myc protein via its noncanonical enzymatic function , creating a positive feedback loop between mycn / myc and tert expression.34 , 35 tert subgroup : tert is the protein component of telomerase , which also includes terc , the telomerase rna component . 
immunohistochemical grading of tert is performed in essentially the same way as that of myc family proteins . estimated survival of each subgroup is based on wang et al12 and valentijn et al.18 to exclude the possibility of overlooking alt cases with atrx point mutations with no atrx loss and / or rare daxx mutations , patients older than age 5 years in the null group can be subjected to additional screening by either rdna32 or telomeric uorescence in situ hybridization assay.32a likely a result of long - range genomic rearrangements but not to promoter mutations in neuroblastoma.36 tert promoter hypermethylation may also be involved in its activating mechanism.37 , 38 alt subgroup : atrx loss results in the alt phenotype . atrx mutation is rarely observed in the myc and tert subgroups , and loss of atrx is exclusively observed in patients with neuroblastoma older than age 5 years.17 because a normal atrx function is to insert the variant histone h3 , namely h3.3 , in concert with daxx , into chromatin to maintain transcriptionally active euchromatin , 39 atrx loss may result in a more transcriptionally inactive heterochromatic state . 
thus , histologic / cytologic appearance of the alt subgroup could be the conventional type with salt - and - pepper nuclei . null subgroup : there are still uh neuroblastomas without myc family protein overexpression , tert overexpression , and atrx loss . 
strategies under consideration include , but are not limited to , transcriptional repression of myc and mycn genes by g - quadruplex ( g4 ) stabilizers , 42 , 43 bromodomain and extraterminal protein inhibitors , 44 , 45 and inhibitors of a transcriptional cyclin - dependent kinase 746 , 47 ; destabilization of mycn by aurora kinase a inhibitors48 and of myc and mycn proteins by ras signaling pathway inhibitors49 - 51 ; and translational blockade of myc mrna by eif4f inhibition and stabilization of rna g4 by rna g4 ligands.52 , 53 we recognize that uh tumors with high - level myc family protein expression tend to exhibit hypertrophic nucleoli , 13 , 14 which indicates that they are highly active in rrna synthesis and protein translation . 
small - molecule inhibitors , including cx - 5461 , an rna polymerase i inhibitor , and halofuginone , a protein translation inhibitor , could therefore effectively target these pathophysiologic features . as we have shown , these inhibitors in fact downregulate myc family protein expression in neuroblastoma cells.13 for those patients with tert - overexpressing neuroblastoma , telomerase inhibitors can be considered . 
if so , is there any other way by which the alt phenotype can be suppressed ? to address this question , we need to understand the mechanism of how atrx loss leads to alt . 
it has become evident that the acquisition of the alt phenotype uses the dna replication stress response , 30 which involves a cascade of events , including the obligatory activation of atr ( ataxia telangiectasia and rad3 related ) kinase . azd6738 is a novel potent and selective inhibitor of atr kinase with ic50 values of less than 1 mm in cell - based assays58 and would effectively target alt tumor cells . conclusion prognosis and therapy stratication of neuroblastomas have steadily improved since the introduction of the evans staging system59 with age used as a prognostic factor60 ; however , 5 - year survival of patients with high - risk neuroblastoma , dened by the international neuroblastoma risk group system ( inrg ) , remains at approximately 40% or lower.61 , 62 the same is true for the uh neuroblastoma group , which essentially overlaps high - risk neuroblastomas.8 , 63 unfortunately , no additional renement for the inrg has been proposed since 2009.61 the proposed histologic subgrouping of uh neuroblastomas represents a practical and immediately implementable approach with which to create the category leading to the most devastating clinical outcome , namely extremely unfavorable histology ( euh ) neuroblastoma . 
we expect that conventional uh neuroblastoma tumorsthat is , the null subgroupwould , we hope , respond to the current multimodal high - intensity therapy designed for high - risk neuroblastoma , whereas euh tumorsthat is , those of the myc , tert , and alt subgroupswould not . 
for those euh tumors , appropriate therapy protocols will have to be designed and tested . on the basis of our ndings , 12 myc family protein immunohistochemistry is already incorporated in the childrens oncology group pathology analysis as an integrated biomarker of neuroblastoma . 
similar attempts are now underway for tert and atrx immunohistochemistry for precision prognosis and the stratication of neuroblastoma . furthermore , we are planning to perform correlative studies between tert protein expression and tert gene rearrangements and between atrx protein loss and atrx gene mutations / deletions . 
for more information about ascos conict of interest policy , please refer to or po.ascopubs.org / site / ifc . no potential conicts of interest were reported . references willis r : neuroblastoma and ganglioneuroma . 
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 o graft - versus - leukemia effect of allogeneic stem - cell transplantation and minimal residual disease in patients with acute myeloid leukemia in first complete remission purpose the detection of minimal residual disease ( mrd ) in patients with acute myeloid leukemia ( aml ) may improve future risk - adapted treatment strategies . 
we assessed whether mrd - positive and mrd - negative patients with aml benefit differently from the graft - versusleukemia effect of allogeneic hematopoietic stem - cell transplantation ( allohsct )  . methods a total of 1 , 511 patients were treated in subsequent dutch - belgian hemato - oncology cooperative group and the swiss group for clinical cancer research aml trials , of whom 547 obtained a first complete remission , received postremission treatment ( prt ) , and had available flow cytometric mrd before prt . 
prt consisted of allohsct ( n = 282 ) , conventional prt by a third cycle of chemotherapy ( n = 160 ) , or autologous hematopoietic stem - cell transplantation ( n = 105 )  . results mrd was positive in 129 patients ( 24% ) after induction chemotherapy before proceeding to prt . 
2017 by american society of clinical oncology introduction acute myeloid leukemia ( aml ) is a heterogeneous malignancy characterized by a variety of underlying cytogenetic and molecular aberrations , which are associated with distinct prognostic features.1 although current treatment approaches induce high percentages of hematologic remission , relapse rates are high and vary according to the underlying risk profile.2 recently , the european leukemianet ( eln ) developed an updated classification on the basis of cytogenetic and molecular aberrancies distinguishing patients with a favorable , intermediate , jurjen versluis burak kalin wendelien zeijlemaker jakob passweg carlos graux markus g . 
therefore , we evaluated whether and to what extent allohsct quantitatively reduces relapse compared with conventional prt in upfront - treated patients with mrd - positive or mrd - negative aml in first cr ( cr1 )  . assessed for eligibility hovon - sakk aml42a hovon - sakk aml92 hovon - sakk aml102 ( n = 1 , 511 ) ( n = 511 ) ( n = 142 ) ( n = 858 ) excluded obtained no cr received no prt no available mrd ( n = 964 ) ( n = 255 ) ( n = 234 ) ( n = 475 ) patients eligible for analysis ( n = 547 ) mrd negative ct auto allo ( n = 418 ) ( n = 118 ) ( n = 89 ) ( n = 211 ) mrd positive ct auto allo ( n = 129 ) ( n = 42 ) ( n = 16 ) ( n = 71 ) methods patients patients participated in three prospective , consecutive dutch - belgian hemato - oncology cooperative group and the swiss group for clinical cancer research ( hovon - sakk ) collaborative group trials ( aml42 , aml92 , and aml102 ; the netherlands trial register numbers ntr230 , ntr1446 , and nrt2187 , respectively ) , for whom assessment of mrd after induction therapy and before prt by allohsct , chemotherapy , or autologous hsct ( autohsct ) was performed.25 , 26 the results of the aml92 trial have not yet been published , but trial information is available in the netherlands trial register ( ntr1446 )  . 
patients were excluded if cr1 did not occur after two induction cycles of chemotherapy ( n = 255 ; 17% ) or the patient did not receive prt after obtaining cr1 ( n = 234 ; 15% )  . 
in addition , a total of 475 patients ( 31% ) received prt in cr1 ; however , their mrd status was not available within a time window of 4 months before prt . 
patients were classified by aml prognostic risk on the basis of the cytogenetic and molecular profile of the underlying aml , according to the eln2017 risk classification.3 molecular analysis was available for the majority of patients , specifically for npm1 ( 93% ) ; fms - like tyrosine kinase 3 internal tandem duplication ( flt3 - itd ; 91% ) , including the flt3 - itd mutant to wild - type ratio ( 86% ) ; evi1 ( 79% ) ; asxl1 ( 83% ) ; runx1 ( 47% ) ; and tp53 ( 47% )  . 
patient characteristics mrd negative ( n = 418 ) mrd positive ( n = 129 ) wbc count at diagnosis , 109 / l cytogenetics of aml cytogenetic abnormalities monosomal karyotype characteristic male female age , years median range < 100  . 
prt included a third cycle of chemotherapy with mitoxantrone and etoposide , high - dose chemotherapy with busulfan and cyclophosphamide followed by autohsct , or allohsct after either myeloablative conditioning or reduced intensity conditioning ( ric )  . 
the myeloablative conditioning regimen contained high - dose cyclophosphamide with at least 8 gy total body irradiation ( tbi ) in 83 patients ( 76% ) ; the remainder of the patients received busulfan with cyclophosphamide . 
although ric regimens varied , the majority contained low - dose ( 2 or 4 gy ) tbi preceded by fludarabine ( n = 126 ; 77% ) , whereas 23% of the patients received fludarabine with busulfan . 
these different prt modalities were applied according to a riskadapted strategy : patients with aml classified as favorable risk , according to cytogenetic and molecular analysis , were planned for a third cycle of chemotherapy ; intermediate - risk patients were preferentially treated with allohsct using an hla - matched sibling donor or a fully hlamatched unrelated donor , if available ; patients with adverse - risk aml proceeded to allohsct using a sibling donor , an unrelated donor , or cord blood grafts ; and patients alternatively received autohsct or a third cycle of chemotherapy if no suitable donor was available.25 - 28 mrd detection and sample selection mrd flow cytometric analysis was performed in a two - step procedure , as previously described.18 in summary , the immunophenotype was determined on blasts defined by cd45 expression with a low sideward scatter . 
mrd percentage was defined as the percentage of laip cells within the wbc compartment multiplied by the correction factor ( 100% divided by the percentage of laippositive blasts at diagnosis )  . 
patient characteristics ( continued ) characteristic year of prt median range mrd negative ( n = 418 ) mrd positive ( n = 129 ) p = .11 2011 2006 - 2014 2010 2006 - 2014 abbreviations : aml , acute myeloid leukemia ; cn - x - y , cytogenetically normal or only loss of x or y chromosome ; cr , complete remission ; eln2017 , european leukemianet 2017 ; hsct , hematopoietic stem - cell transplantation ; iqr , interquartile range ; flt3 - itd , fms - like tyrosine kinase 3 internal tandem duplication ; mrd , minimal residual disease ; npm1 , nucleophosmin - 1 ; prt , postremission treatment . * the cutoff of the flt3 - itd ratio is defined as 0.50. number of induction courses ( ie , i or ii ) was performed to allow the baseline hazard to differ between these two patient groups . 
all analyses were performed with stata software ( release 13.1 ; stata corporation , college station , tx )  . results patient characteristics a total of 547 patients with aml in cr1 and available mrd status proceeded to prt with either allohsct ( n = 282 ) , chemotherapy ( n = 160 ) , or autohsct ( n = 105 )  . 
patients with mutated npm1 were more frequently mrd negative , whereas mrd - positive patients more frequently tended to obtain a late cr1 ( ie , after induction cycle 2 )  . 
the eln2017 risk classification was similarly distributed among the mrd - negative and mrd - positive patients . interestingly , the time interval from cr1 to prt for patients with mrd - positive aml was shorter compared with their negative counterparts , which was mainly apparent in favorablerisk patients with aml . 
no other differences with regard to donor source , conditioning , cytomegalovirus serostatus , and european group for blood and marrow transplantation ( ebmt ) risk score were apparent between mrd - negative and mrdpositive patients . 
patients with a high risk of nrm according to the ebmt risk score32 ( > 3 points ) represented 45% of the patients who underwent transplantation . treatment outcome os and rfs were significantly better in patients without mrd before prt compared with mrdpositive patients ( 65% 6 2% v 50% 6 5% at 4 years ; p = .002 ; and 58% 6 3% v 38% 6 4% ; p , .001 , respectively ; figs 2a and 2b )  . 
the sample with the shortest time interval between prt and the date of collection was selected for analysis . end points the primary end point of the study was the cumulative incidence of relapse . 
the cumulative risks of relapse and nrm over time were calculated as competing risks with actuarial methods , where patients alive in continuing cr1 were censored at the date of last contact . statistical methods a time - dependent analysis of prt was performed as described previously28 , 29 by applying multivariable cox regression with allohsct as the timedependent covariable . 
in both the multivariable analysis and the estimation of the survival curves , these patients were counted as at risk in the chemotherapy group from the start of prt until allohsct and after that as at risk in the allohsct group . 
more detailed outcome estimates according to mrd status , type of prt , and risk of nrm on the basis of the ebmt risk score are presented in the data supplement . the cumulative incidence of relapse was significantly lower in patients without mrd receiving allohsct compared with chemotherapy or autohsct ( 26% 6 3% v 38% 6 3% at 4 years ; p = .027 , respectively ; fig 3a )  . 
the cumulative incidence of relapse in mrd - positive patients is estimated to be 45% 6 6% compared with 66% 6 6% at 4 years ( p = .058 ) for recipients of allohsct compared with recipients of chemotherapy or autohsct , respectively ( fig 3b )  . 
rfs after allohsct proved similar compared with prt with chemotherapy or autohsct in patients without mrd before prt ( 58% 6 4% v 58% 6 4% at 4 years ; p = .99 , respectively ; fig 3c )  . 
rfs after allohsct in mrd - positive patients before prt was 44% 6 6% compared with 31% 6 6% at 4 years ( p = .20 ) after chemotherapy or autohsct , respectively ( fig 3d )  . 
only 46 of relapsing patients ( 22% ) proceeded to allohsct after obtaining a second cr . multivariable analysis the following variables significantly predicted for relapse in the univariable analysis : mrd status , type of prt , age , wbc category , flt3 - itd category , year of prt , time from diagnosis to cr , time from cr to prt , cytogenetics , number of cycles to cr , eln2017 risk classification , npm1 mutation , evi1 overexpression , and cebpa double mutation . 
after forward selection , the multivariable analysis was performed , stratified by the total number of induction courses with adjustment for mrd status , type of prt , age , wbc category at diagnosis , flt3 - itd , eln2017 risk classification , number of cycles to cr , and year of prt ( table 3 )  . 
different variables in the multivariable model were significantly associated with relapse , with the eln2017 risk classification , flt3 - itd mutant to wild - type ratio , number of cycles to reach cr , and type of prt being the most important variables . discussion the development of treatment approaches in patients with aml is increasingly personalized by using genetic and molecular leukemia characteristics at diagnosis and individual treatment response.3 - 6 response and especially mrd , detected by either multiparametric flow cytometry or quantitative polymerase chain reaction , has become an important parameter in a more precise treatment approach for patients with aml.10 - 24 currently , it is unknown whether and how the presence or absence of mrd should guide the application of allohsct as prt . 
recently , the quantitative detection of mutated npm1 has been shown to have high predictive value , and recommendations to tailor the application of allohsct by mrd were made.10 - 12 balsat et al10 suggested refraining from allohsct in npm1 mrdnegative patients and to selectively proceed to allohsct as prt in cr1 in mrd - positive patients or adverse - risk patients on the basis of karyotype or the presence of flt3 - itd . 
kaplan - meier estimates of the cumulative incidence ( ci ) of relapse in ( a ) minimal residual disease ( mrd ) negative patients ; ( b ) ci of relapse in mrdpositive patients ; ( c ) relapse - free survival ( rfs ) in mrd - negative patients ; and ( d ) rfs in mrdpositive patients by type of prt in patients with acute myeloid leukemia in first complete remission from start of prt . 
nevertheless , the advantages of personalized treatment are obvious and continuously being refined by updated and better methods of risk assessment . also , new technologies to better define mrd , such as quantification of leukemic stem - cell content , standardized protocols and antibody panels , and novel software possibilities , are emerging.45 - 47 a personalized approach including mrd identifies patients with a high risk of relapse who qualify for allohsct , but who might benefit from attempts to induce mrd negativity before transplantation . 
previously , a number of investigators reported that patients in cr1 with persistence of mrd before allohsct have worse survival compared with recipients of allohsct with an mrd - negative cr1.17 , 19 - 24 although allohsct is clearly indicated in mrd - positive patients , it is important to study the value of approaches intended to induce mrd negativity before allohsct . 
it has been suggested that continued chemotherapy with one or two consolidation cycles may not be the preferred strategy to obtain an mrd - negative cr before allohsct , 48 but several new drugs are currently being developed and evaluated for aml.49 possible other strategies may include efforts to improve allogeneic immunotherapy by early tapering of immunosuppression and / or preemptive donor lymphocyte infusions , which also could be guided by mrd . 
of events / patients allo ct / auto hr & 95% ci ( ct / auto : allo ) reduction ( sd ) mrd status ( cut - off 0.1 ) negative positive 93 / 207 40 / 58 90 / 211 40 / 71 total 133 / 265 ( 50% ) 130 / 282 ( 46% ) allo better ct / auto better 45% ( 8 ) reduction 2p < .001 collectively , our study shows that the gvl effect was strikingly similar in mrd - positive and mrdnegative patients . 
additional prospective studies are needed to evaluate whether the conversion of mrd positivity into an mrd - negative remission before allohsct further optimizes outcome , and how the gvl effect after allohsct can be optimized . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . jurjen versluis no relationship to disclose burak kalin no relationship to disclose wendelien zeijlemaker patents , royalties , other intellectual property : i have a patent for a laboratory test to identify specific leukemia cells that can be used in patients with acute myeloid leukemia . 
manz consulting or advisory role : amgen , novartis , roche , pfizer marie - christiane vekemans consulting or advisory role : amgen ( inst ) , celgene ( inst ) , bristol - myers squibb ( inst ) , janssen ( inst ) , takeda ( inst ) travel , accommodations , expenses : amgen , bristol - myers squibb , teva , janssen bart j . 
bargetzi no relationship to disclose juergen kuball employment : gadeta stock and other ownership interests : gadeta consulting or advisory role : gadeta , novartis ( inst ) speakers bureau : gadeta research funding : gadeta , miltenyi biotec , novartis patents , royalties , other intellectual property : multiple patents on gamma - delta t cell receptor sequences isolation strategies and targets in combination with gamma - delta t cell receptor sequences travel , accommodations , expenses : gadeta , novartis , miltenyi biotec harry c . 
van der velden consulting or advisory role : celgene ( inst ) research funding : bd biosciences ( inst ) patents , royalties , other intellectual property : patent for euroflow mrd antibody tubes . 
janssen honoraria : bristol - myers squibb netherlands , pfizer europe consulting or advisory role : pfizer europe , novartis research funding : novartis travel , accommodations , expenses : novartis thomas pabst no relationship to disclose bob lowenberg consulting or advisory role : astex pharmaceuticals , clear creek bio , agio pharmaceuticals , celgene mojca jongen - lavrencic no relationship to disclose gerrit jan schuurhuis research funding : becton dickinson ( inst ) gert ossenkoppele honoraria : roche consulting or advisory role : celgene , sunesis pharmaceuticals , janssen , novartis , cti , amgen , pfizer , roche research funding : amgen ( inst ) , janssen ( inst ) , celgene ( inst ) travel , accommodations , expenses : roche jan j . 
cornelissen no relationship to disclose acknowledgment following dutch - belgian hemato - oncology cooperative group and the swiss group for clinical cancer research ( hovon - sakk ) publication rules , coauthorship was offered to centers contributing the highest number of patients . nevertheless , the authors highly appreciate the contribution of many physicians and data managers throughout hovonsakk , who made this analysis possible . affiliations jurjen versluis , burak kalin , bob lowenberg , mojca jongen - lavrencic , and jan j . 
van der velden , erasmus university medical center , rotterdam ; wendelien zeijlemaker , jeroen j.w.m. janssen , gerrit jan schuurhuis , and gert ossenkoppele , vu university medical center ; bart j . 
wijermans , haga hospital , the hague ; mels hoogendoorn , medical center leeuwarden , leeuwarden ; juergen kuball , university medical center utrecht , utrecht ; harry c . schouten , maastricht university medical center , maastricht , the netherlands ; jakob passweg , university hospital basel , basel ; markus g . 
grimwade d , hills rk , moorman av , et al : refinement of cytogenetic classification in acute myeloid leukemia : determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the united kingdom medical research council trials . 
d ohner h , estey eh , amadori s , et al : diagnosis and management of acute myeloid leukemia in adults : recommendations from an international expert panel , on behalf of the european leukemianet . 
cornelissen jj , gratwohl a , schlenk rf , et al : the european leukemianet aml working party consensus statement on allogeneic hsct for patients with aml in remission : an integrated - risk adapted approach . 
ossenkoppele g , schuurhuis gj : mrd in aml : does it already guide therapy decision - making ? hematology ( am soc hematol educ program ) 2016 : 356 - 365 , 2016 8 . 
grimwade d , freeman sd : defining minimal residual disease in acute myeloid leukemia : which platforms are ready for prime time ? blood 124 : 3345 - 3355 , 2014 9 . 
buccisano f , walter rb : should patients with acute myeloid leukemia and measurable residual disease be transplanted in first complete remission ? curr opin hematol 24 : 132 - 138 , 2017 10 . 
balsat m , renneville a , thomas x , et al : postinduction minimal residual disease predicts outcome and benefit from allogeneic stem cell transplantation in acute myeloid leukemia with npm1 mutation : a study by the acute leukemia french association group . 
kr onke j , schlenk rf , jensen ko , et al : monitoring of minimal residual disease in npm1 - mutated acute myeloid leukemia : a study from the german - austrian acute myeloid leukemia study group . 
freeman sd , virgo p , couzens s , et al : prognostic relevance of treatment response measured by flow cytometric residual disease detection in older patients with acute myeloid leukemia . 
schnittger s , kern w , tschulik c , et al : minimal residual disease levels assessed by npm1 mutation - specific rq - pcr provide important prognostic information in aml . 
shayegi n , kramer m , bornhauser m , et al : the level of residual disease based on mutant npm1 is an independent prognostic factor for relapse and survival in aml . 
jourdan e , boissel n , chevret s , et al : prospective evaluation of gene mutations and minimal residual disease in patients with core binding factor acute myeloid leukemia . 
araki d , wood bl , othus m , et al : allogeneic hematopoietic cell transplantation for acute myeloid leukemia : time to move toward a minimal residual disease - based definition of complete remission ? j clin oncol 34 : 329 - 336 , 2016 18 . 
bastos - oreiro m , perez - corral a , martnez - laperche c , et al : prognostic impact of minimal residual disease analysis by flow cytometry in patients with acute myeloid leukemia before and after allogeneic hemopoietic stem cell transplantation . 
walter rb , buckley sa , pagel jm , et al : significance of minimal residual disease before myeloablative allogeneic hematopoietic cell transplantation for aml in first and second complete remission . 
walter rb , gooley ta , wood bl , et al : impact of pretransplantation minimal residual disease , as detected by multiparametric flow cytometry , on outcome of myeloablative hematopoietic cell transplantation for acute myeloid leukemia . 
zhou y , othus m , araki d , et al : preand post - transplant quantification of measurable ( minimal ) residual disease via multiparameter flow cytometry in adult acute myeloid leukemia . 
l owenberg b , pabst t , maertens j , et al : therapeutic value of clofarabine in younger and middle - aged ( 18 - 65 years ) adults with newly diagnosed aml . 
cornelissen jj , versluis j , passweg jr , et al : comparative therapeutic value of post - remission approaches in patients with acute myeloid leukemia aged 40 - 60 years . 
versluis j , hazenberg cl , passweg jr , et al : post - remission treatment with allogeneic stem cell transplantation in patients aged 60 years and older with acute myeloid leukaemia : a time - dependent analysis . 
buccisano f , maurillo l , piciocchi a , et al : pre - transplant persistence of minimal residual disease does not contraindicate allogeneic stem cell transplantation for adult patients with acute myeloid leukemia . 
cornelissen jj , breems d , van putten wl , et al : comparative analysis of the value of allogeneic hematopoietic stemcell transplantation in acute myeloid leukemia with monosomal karyotype versus other cytogenetic risk categories . j clin oncol 30 : 2140 - 2146 , 2012 35 . 
lamers ch , wijers r , van bergen ca , et al : cd4 + t - cell alloreactivity toward mismatched hla class ii alleles early after double umbilical cord blood transplantation . 
norde wj , overes im , maas f , et al : myeloid leukemic progenitor cells can be specifically targeted by minor histocompatibility antigen lrh - 1 - reactive cytotoxic t cells . 
terwey th , hemmati pg , martus p , et al : a modified ebmt risk score and the hematopoietic cell transplantationspecific comorbidity index for pre - transplant risk assessment in adult acute lymphoblastic leukemia . 
sorror ml , maris mb , storb r , et al : hematopoietic cell transplantation ( hct ) - specific comorbidity index : a new tool for risk assessment before allogeneic hct . 
sorror ml , sandmaier bm , storer be , et al : comorbidity and disease status based risk stratification of outcomes among patients with acute myeloid leukemia or myelodysplasia receiving allogeneic hematopoietic cell transplantation . 
versluis j , labopin m , niederwieser d , et al : prediction of non - relapse mortality in recipients of reduced intensity conditioning allogeneic stem cell transplantation with aml in first complete remission . 
shouval r , labopin m , bondi o , et al : prediction of allogeneic hematopoietic stem - cell transplantation mortality 100 days after transplantation using a machine learning algorithm : a european group for blood and marrow transplantation acute leukemia working party retrospective data mining study . 
zeijlemaker w , kelder a , oussoren - brockhoff yj , et al : a simple one - tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia . 
flores - montero j , sanoja - flores l , paiva b , et al : next generation flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma . 
cornelissen jj , van norden y , van gelder m , et al : early post - transplant epigenetic therapy by panobinostat and decitabine followed by donor lymphocyte infusion ( dli ) : interim results of the hovon - 116 phase i / ii feasibility study in poor - risk aml recipients of allogeneic stem cell transplantation ( allohsct )  . 
bug g , burchert a , wagner e , et al : phase i / ii study of the deacetylase inhibitor panobinostat as maintenance therapy after an allogeneic stem cell transplantation in patients with high - risk mds or aml : the panobest - trial . 
stone rm , mandrekar s , sanford bl , et al : the multi - kinase inhibitor midostaurin prolongs survival compared with placebo in combination with daunorubicin / cytarabine induction , high - dose c consolidation , and as maintenance therapy in newly diagnosed acute myeloid leukemia ( aml ) patients age 18 - 60 with flt3 mutations : an international prospective randomized - controlled double - blind trial ( calgb 10603 / ratify [ alliance ] )  . 
dinardo c , de botton s , pollyea da , et al : molecular profiling and relationship with clinical response in patients with idh1 mutation - positive hematologic malignancies receiving ag - 120 , a first - in - class potent inhibitor of mutant idh1 , in addition to data from the completed dose escalation portion of the phase 1 study . 
 plasma dna - based molecular diagnosis , prognostication , and monitoring of patients with ewsr1 fusion - positive sarcomas purpose ewing sarcoma ( es ) and desmoplastic small round cell tumors ( dsrcts ) are aggressive sarcomas molecularly characterized by ewsr1 gene fusions . 
as pathognomonic genomic events in these respective tumor types , ewsr1 fusions represent robust potential biomarkers for disease monitoring . methods to investigate the feasibility of identifying ewsr1 fusions in plasma - derived cell - free dna ( cfdna ) from patients with es and dsrct , we evaluated two complementary approaches in samples from 17 patients with radiographic evidence of disease . 
the first approach involved identification of patient - specific genomic ewsr1 fusion breakpoints in formalin - fixed , paraffinembedded tumor dna using a broad , hybridization capture - based next - generation sequencing ( ngs ) panel , followed by design of patient - specific droplet digital polymerase chain reaction ( ddpcr ) assays for plasma cfdna interrogation . 
2017 by american society of clinical oncology introduction ewing sarcoma ( es ) and desmoplastic small round cell tumor ( dsrcts ) are aggressive sarcomas with peak incidences in adolescence and young adulthood.1 both are characterized by fusions involving the ewsr1 gene on chromosome 22q12.2 , 3 the fusions in es involve ewsr1 and a member of the ets family . 
most commonly , the partner gene is ets family member fli1 , with alternative genes such as erg , etv1 , etv4 , and fev less commonly observed.4 in dsrcts , the fusion involves ewsr1 and the wilms tumor gene wt1 . 
given their exquisite specificity for es and dsrct , respectively , the detection of these fusions in a tumor biopsy has become a standard part of the diagnostic assessment of patients with these sarcomas . over the past several decades , numerous trials for es have been conducted , leading to current therapeutic standards of care that result in long - term cure for approximately 75% of patients who present with localized disease but only approximately 25% in patients who present with metastatic disease.5 , 6 dsrct was only recognized as a distinct malignancy 25 years ago , with the characterization of its pathognomonic oncogenic fusion following several years later.3 , 7 despite intensive regimens evaluating combinations of chemotherapy , aggressive debulking surgical approaches , neerav n . 
 radiation therapy , and autologous stem cell rescue , outcomes for dsrct remain poor , with locoregional recurrences representing the most common type of relapse.8 the establishment of clinically valid prognostic and predictive biomarkers has been challenging in es and largely unstudied in dsrct.9 specifically , numerous studies have been conducted in an effort to establish platforms to identify and follow subclinical levels of disease burden.10 - 14 the two strategies most commonly used have been reverse transcription polymerase chain reaction ( rtpcr ) evaluation for the ewsr1 fusion transcript from cellular rna extracted from peripheral blood and / or bone marrow , or , for es , flow cytometric evaluation of peripheral blood and / or bone marrow cells on the basis of a cd99 + gating strategy . 
these studies have had varying results and , to date , have not satisfied criteria as suitable clinical biomarkers . more recently , studies in various cancer types have demonstrated the potential use of identifying and following tumor - specific mutations in cell - free dna ( cfdna ) isolated from plasma as a marker for subclinical disease.15 - 17 a study of various different cancer types by bettegowda et al18 demonstrated highly variable levels of mutations in baseline cfdna in patients with different tumor types , suggesting that the use of cfdna as a clinically relevant signal for subclinical disease will be partly based on the cancer type itself . 
these studies all rely on the ability to identify tumorspecific aberrations as a marker of disease burden . tumors such as es and dsrcts have low mutational burdens , because it is believed that the pathognomonic ewsr1 fusions are the primary driving oncogenic lesions in these tumor types , with few recurrent secondary alterations.19 therefore , the identification of tumor - specific ewsr1 fusions in cfdna as a marker for active disease is an especially attractive option , given that these tumors are defined by these translocations . 
moreover , issues such as clonal evolution , which can affect the detection of certain mutations in cfdna in other tumor types , are less pertinent , because these sarcomas maintain these specific fusions through an individuals disease course . 
importantly , even identical ewsr1 fusion transcripts are the result of genomic breakpoints in the corresponding introns that are unique to each patient.20 although a genomic dna - based clinical assay to monitor these translocations in plasma cfdna should be more robust than rna - based assays in this context , the heterogeneity of intronic breakpoint regions between tumors from different patients had previously presented a major challenge for the design and routine application of such a dna - based assay , a hurdle now overcome by the application of more powerful sequencing approaches . in this study , we directly compared two strategies to identify baseline ewsr1 genomic rearrangements in cfdna of patients with es and dsrct . the first approach uses droplet digital pcr ( ddpcr ) of cfdna to detect fusions identified by targeted next - generation sequencing of formalinfixed paraffin - embedded tumor biopsy material.21 this approach has been successfully used in numerous studies of other tumor types . 
the second approach uses next - generation sequencing ( ngs ) via a modified hybridization capture approach to identify ewsr1 fusions as well as potentially prognostic mutations in cfdna samples . 
the primary aim of this study was to evaluate each platform , ddpcr and ngs , on paired cfdna samples from patients with es and dsrct with confirmed radiographic evidence of disease . methods patient population between august 2014 and january 2016 , patients with a confirmed diagnosis of es or dsrct and formalin - fixed paraffin - embedded clinical tumor material available for memorial sloan kettering integrated mutation profiling of actionable cancer targets ( msk - impact ) tumor genotyping were eligible to enroll on this study . 
for each patient , at least one baseline 10 - ml blood sample was collected in streck blood collection tubes ( bcts ; streck , la vista , ne )  . msk - impact tumor ngs genomic dna from tumor tissue and patientmatched normal blood were subjected to targeted sequencing using msk - impact , a custom , deepcoverage targeted sequencing assay approved by the new york state department of health as a clinical test.21 , 22 tumors were sequenced to an average coverage depth of 7663 ( range , 4053 to 1 , 2513 ) in the clinical laboratories of the memorial sloan kettering cancer center molecular diagnostics service . 
 cancer - associated genes using bioinformatics pipelines previously described.21 gene fusions involving ewsr1 were identified in all patients included in this study using delly.23 , 24 plasma cfdna extraction and analyses whole blood collected in 10 - ml cell - free dna bcts ( streck ) was centrifuged in two steps to separate plasma from cells . 
in step 2 , separated plasma was further centrifuged in a high - speed microcentrifuge at 18 , 000 3 g for 10 minutes ( ambient temperature )  . cell - free plasma was aliquoted and frozen at 280c until ready to extract . 
extraction of cfdna was performed using a fully automated qiagen platform , qiasymphony sp , and qiasymphony dsp virus / pathogen midi kit ( catalog #937055 ; qiagen , valencia , ca )  . 
quality and quantity of cfdna was evaluated with automated electrophoresis using either tapestation with high sensitivity d1000 screentape and reagents ( agilent technologies , santa clara , ca ) or fragment analyzer with high sensitivity genomic dna analysis kit ( advanced analytical , ankeny , ia )  . breakpoint - specific ddpcr for each sample , a fusion - specific assay was designed using primer3plus and ordered through biorad ( hercules , ca )  . 
pcr reactions contained fusion - specific primers and probes , biorad validated copy number control primers and probes , and digital pcr supermix for probes ( no 2 / - deoxyuridine 59 - triphosphate ) and circulating free dna . 
emulsified reactions were amplified on a 96 - well thermal cycler using cycling conditions identified during the optimization step ( 95c 109 ; 40 cycles of 94c 3099 58c 19 , 98c 109 , 4c hold )  . 
plates were read and analyzed with the quantasoft software to assess the number of droplets positive for mutant dna , wild - type dna , both , or neither . the assay threshold sensitivity was set at two mutant droplets . targeted plasma ngs cell - free dna ( cfdna ) from plasma was extracted using the qiasymphony sp system ( qiagen )  . sequencing libraries were prepared according to the kapa hyper protocol ( kapa biosystems , wilmington , ma ) with the ligation of illumina sequence adaptors followed by pcr amplification and clean - up . 
custom dna probes targeting all coding exons of stag2 and tp53 and selected introns of ewsr1 ( intron 7 to 13 ) were synthesized by idt ( integrated dna technologies , coralville , ia )  . 
an equal amount of each dna library ( 200 ng per sample ) was pooled for hybridization capture using a customized double - capture protocol modified from the nimblegen seqcap target enrichment system ( roche sequencing solutions , pleasanton , ca )  . 
after pcr clean - up , the captured target library was processed by a secondary capture ( using the same custom dna probes ) incubated at 65c for 4 hours and followed by postcapture washes and three to five cycles of pcr amplification . 
the pooled , cleaned - up libraries containing captured dna fragments were sequenced on the illumina hiseq system with paired end reads ( 2 3 100 bp )  . analysis was performed using the same bioinformatics pipeline as for msk - impact tissue sequencing . 
sequence alignment and mutation calling were performed on reads from all tumor samples , normals , and cfdna samples simultaneously to ensure consistent realignment around indels and consistent annotation of shared mutations . the scatterplot for concordance between ngs and ddpcr measurement of fusion molecules was generated using the r package ggplot2 . 
clinical features of patients with es in the study patient id sex age at dx ( years ) disease type fusion partner gene disease status primary tumor location metastatic site ( s ) at diagnosis site of recurrence es - 3 right chest wall n / a left lung fli1 fli1 fli1 fli1 fli1 fli1 fli1 fli1 newly diagnosed , right fibula newly diagnosed , right scapula multiple bones , bone multiple bones marrow newly diagnosed , left femur multiply recurrent , right chest wall multiple bones lungs , multiple newly diagnosed , left chest wall lungs multiply recurrent , pelvis vertebrae , lungs lungs bones lungs lungs pelvis lungs left ankle lungs left tibia lungs , multiple bones , multiple bones bone marrow left chest wall pleura , abdomen localized metastatic recurrent , metastatic localized metastatic metastatic metastatic recurrent , metastatic recurrent , metastatic recurrent , metastatic recurrent , metastatic abbreviations : dx , diagnosis ; es , ewing sarcoma ; id , identification ; n / a , not applicable . disease were analyzed by both ddpcr and custom capture ngs ( tables 1 and 2 )  . 
all patients with dsrct had abdominal / pelvic disease involvement , being the characteristic clinical presentation of this sarcoma , and four out of six patients also had metastatic disease involving the mediastinum . tumor - specific ewsr1 fusions were successfully identified by msk - impact in all 17 tumor biopsyderived dna samples ( table 3 ; data supplement )  . 
no false positives were detected in 165 , 000 wild - type copies . we identified tumor - specific fusions as evidence of circulating tumor dna ( ctdna ) in all 11 baseline plasma samples from patients with es ( table 3 )  . 
we also identified ewsr1 - wt1 fusions in five out of six dsrct baseline plasma samples . using our custom capture ngs assay involving ewsr1 , tp53 , and stag2 , we sequenced parallel cfdna samples from the cohort of 17 patients to a mean total depth of coverage of 147 , 5493 ( mean unique coverage , 3 , 7543 after removing inferred pcr duplicates )  . 
by achieving such deep coverage , we achieved high sensitivity for detecting different ewsr1 fusions in a single universal assay , regardless of the precise location of the genomic breakpoint in ewsr1 . fusions were detected in 14 out of 17 patients ( table 3 ) , including 10 of 11 patients with ewing sarcoma and four of six patients with dscrt . 
as a complementary approach , cfdna samples were also directly profiled using a custom capturetargeted ngs assay designed to identify alterations in ewsr1 , tp53 , stag2 , and cdkn2a . dna extraction msk - impact breakpoint identication tumor blood cfdna extraction personalized ddpcr primers ddpcr assay targeted ngs prior knowledge from sequencing the tumor tissue ( data supplement )  . 
this is the first report to our knowledge to directly compare two complementary methodologies , ddpcr and hybrid - capture ngs , to evaluate tumor - specific ewsr1 fusions in cfdna from patients with es and dsrct . tumor - specific fusions are particularly attractive target substrates for mutation - based biomarker studies of ewsr1 translocation - associated sarcomas . 
although tumor cfdna studies typically target oncogene point mutations , we previously demonstrated the relative scarcity of such recurrent somatic mutations in fusion - associated sarcomas.19 specifically , in 75 es and 24 dsrct samples screened for 275 recurrent point mutations in 29 oncogenes frequently mutated across different cancer types , mutations were identified in only 4% of es samples , and none of the dsrct samples . 
recent comprehensive wholeexome and whole - genome sequencing studies of es tumor specimens have validated this finding.26 - 28 conversely , ewsr1 fusions are defining molecular features of es and dsrcts and have never been reported to undergo modification or clonal evolution through a disease course . previous studies using rt - pcr to identify ewsr1 fusion transcripts in peripheral blood or bone marrow samples from patients with es report successful identification in , 50% of patients.10 , 11 , 13 it is perhaps not surprising that the cfdna - based approach used in our study yielded more sensitive results , because previous studies attempted to identify rare occult tumor cells through the identification of cellular ewsr1 fusion transcripts . 
recent studies have established the increased sensitivity of plasma - derived cfdna assays as compared with circulating tumor cell assays for blood - based identification of disease.18 a similar sensitivity advantage of cfdna over cell - free rna is also likely . 
mu , mutant droplets ; wt , wild - type droplets . ddpcr ( mu / ( mu + wt ) ) rt - pcr methodologies used rna as a substrate , which undergoes rapid degradation leading to potential false - negative results if samples are not processed immediately . 
finally , previous attempts at using rt - pcr used primers for only the most common transcripts seen in es tumors among the many reported ewsr1 - fli1 fusion types.20 therefore , a subset of patients whose tumors harbored less common ewsr1 - fli1 fusion types or other ewsr1 fusion partners was uncaptured , reducing the clinical sensitivity of the assay . in this study , both platforms were highly sensitive at detecting ewsr1 fusions in baseline cfdna samples , although ddpcr demonstrated increased sensitivity and was able to detect levels < 0.1%. 
the characterization of tp53 alterations as an adverse prognostic finding was proposed on the basis of several retrospective studies29 - 32 but was not confirmed in a recent prospective study.33 however , another recent large - scale genomic profiling study of es identified tumors with comutation of stag2 and tp53 as defining a particularly high - risk subset of patients.28 it is therefore important future biomarker studies continue to collect these mutational data in an effort to establish more definitively their clinical use . that a significant advantage of the ngs approach is that it can be performed directly on cfdna without a priori knowledge of tumor - specific mutations . 
this is a particularly attractive aspect of this methodology , because reliable acquisition of baseline tumor tissue has proven difficult in north american cooperative group es banking studies.34 in this study , we demonstrated the ability to identify fusion breakpoint sequences in the majority of plasma cfdna samples , along with potentially prognostic alterations in tp53 and stag2 . 
although ngs was successful at identifying fusions in the majority of baseline cfdna samples , there were several examples in which ddpcr was able to identify a low level of fusion positivity that was not identified by ngs . 
a similar ngs - based approach was recently described in patients with advanced lung cancer , with 88% and 100% sensitivity and specificity , respectively , for detecting previously charat 0.1% allele acterized mutations present frequency or higher.35 the higher sensitivity of a ddpcr approach is potentially clinically important for identifying early evidence of relapse in followup samples during and after therapy . our study highlights the advantages of both platforms . 
furthermore , ngs provides the ability to capture the heterogeneity of a patients disease more completely than ddpcr , as reflected in the patient cfdna sample ( es - 2 ) with multiple tp53 mutations . 
nonetheless , these results provide important data for the establishment of an optimal strategy for future cooperative group prospective biomarker studies of ewsr1 - defined sarcomas . given these findings , we propose a hybrid approach for future clinical trials ( fig 3 )  . 
this will also allow for identification of the precise fusion breakpoint as well as additional tp53 and stag2 mutational status on the majority of patients , without the need to profile tumor material . 
proposed schema for prospective ewsr1 fusion - based cell - free dna ( cfdna ) studies . the depicted proposed schema for future prospective studies evaluating the use of cfdna as a marker for subclinical disease in ewing sarcoma or desmoplastic small round cell tumors incorporates the advantages of each methodology ( nextgeneration sequencing [ ngs ] and downstream droplet digital polymerase chain reaction [ ddpcr ] )  . breakpoint determination , as well as quantification of ewsr1 fusion , and identification of additional mutation data are identifiable on the majority of baseline plasma cfdna samples using a targeted custom - capture ngs approach . 
 ( * ) for cases where ngs fails to identify genomic fusion sequences , tissue samples will need to be collected for breakpoint determination through tumor profiling . baseline cfdna extraction follow - up blood cfdna extraction targeted ngs breakpoint determination * quantication of fusion additional mutation detection ddpcr assay biomarker development in rare cancer types , and certain characteristics are imperative for the successful development of predictive markers in this clinical context.9 our proposed approach is especially compelling given these criteria . 
first , our approach should capture almost 100% of patients with es and dsrct , because the platforms are designed to capture any ewsr1 fusion type , including those with rare variant ewsr1 translocations . 
cell - free specific bcts contain stabilizing reagents , which have been shown to minimize both ctdna degradation and sample contamination with blood cellderived genomic dna.36 , 37 this is particularly important for potential biomarkers in rare diseases , where cooperative group studies require long - distance sample deliveries , necessitating strategies to eliminate the issue of substrate degradation . 
ddpcr has been established as an attractive platform for circulating nucleic acids on the basis of the ease of quantitation of mutant fragments as compared with realtime pcr.38 furthermore , broad , hybrid - capture ngs platforms are becoming increasingly used for clinical use , as exemplified by the msk - impact sequencing platform , with well over 10 , 000 patient tumor samples studied to date , 39 and even greater numbers studied by similar ngs - based assays at commercial reference laboratories . our primary aim in this study was to establish a robust methodology suitable for the unique and stringent requirements for biomarker development in prospective studies of rare diseases . 
 heather magnan no relationship to disclose ahmet zehir no relationship to disclose daoqi you no relationship to disclose jiabin tang no relationship to disclose fanli meng no relationship to disclose aliaksandra samoila no relationship to disclose emily k . 
fletcher c , bridge j , hogendoorn p , et al ( eds ) : world health organization classification of tumours : pathology and genetics of tumours of soft tissue and bone ( ed 4 )  . 
lessnick sl , ladanyi m : molecular pathogenesis of ewing sarcoma : new therapeutic and transcriptional targets . 2837 - 2840 , 1994 annu rev pathol 7 : 145 - 159 , 2012 5 . 
womer rb , west dc , krailo md , et al : randomized controlled trial of interval - compressed chemotherapy for the treatment of localized ewing sarcoma : a report from the childrens oncology group . 
kolb ea , kushner bh , gorlick r , et al : long - term event - free survival after intensive chemotherapy for ewings family of tumors in children and young adults . 
desai nb , stein nf , laquaglia mp , et al : reduced toxicity with intensity modulated radiation therapy ( imrt ) for desmoplastic small round cell tumor ( dsrct ) : an update on the whole abdominopelvic radiation therapy ( wap - rt ) experience . 
zoubek a , ladenstein r , windhager r , et al : predictive potential of testing for bone marrow involvement in ewing tumor patients by rt - pcr : a preliminary evaluation . 
schleiermacher g , peter m , oberlin o , et al : increased risk of systemic relapses associated with bone marrow micrometastasis and circulating tumor cells in localized ewing tumor . 
ash s , luria d , cohen ij , et al : excellent prognosis in a subset of patients with ewing sarcoma identified at diagnosis by cd56 using flow cytometry . 
roschewski m , dunleavy k , pittaluga s , et al : circulating tumour dna and ct monitoring in patients with untreated diffuse large b - cell lymphoma : a correlative biomarker study . 
hyman dm , diamond el , vibat cr , et al : prospective blinded study of brafv600e mutation detection in cell - free dna of patients with systemic histiocytic disorders . 
dawson sj , tsui dw , murtaza m , et al : analysis of circulating tumor dna to monitor metastatic breast cancer . n engl j med 368 : 1199 - 1209 , 2013 18 . 
shukla n , ameur n , yilmaz i , et al : oncogene mutation profiling of pediatric solid tumors reveals significant subsets of embryonal rhabdomyosarcoma and neuroblastoma with mutated genes in growth signaling pathways . 
plougastel b , zucman j , peter m , et al : genomic structure of the ews gene and its relationship to ewsr1 , a site of tumor - associated chromosome translocation . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
huang hy , illei pb , zhao z , et al : ewing sarcomas with p53 mutation or p16 / p14arf homozygous deletion : a highly lethal subset associated with poor chemoresponse . 
tsuchiya t , sekine k , hinohara s , et al : analysis of the p16ink4 , p14arf , p15 , tp53 , and mdm2 genes and their prognostic implications in osteosarcoma and ewing sarcoma . 
lerman dm , monument mj , mcilvaine e , et al : tumoral tp53 and / or cdkn2a alterations are not reliable prognostic biomarkers in patients with localized ewing sarcoma : a report from the childrens oncology group . pediatr blood cancer 62 : 759 - 765 , 2015 34 . 
integrative genomics viewer screenshot of es - 2 bam file showing tp53 r280k ( right ; frequency , 0.04 ; identified in plasma ) and e285k ( left ; frequency , 0.44 ; identified in both plasma and tissue ) mutations . 
paweletz cp , sacher ag , raymond ck , et al : bias - corrected targeted next - generation sequencing for rapid , multiplexed detection of actionable alterations in cell - free dna from advanced lung cancer patients . 
denis mg , knol ac , theoleyre s , et al : efficient detection of braf mutation in plasma of patients after long - term storage of blood in cell - free dna blood collection tubes . 
 detection of circulating tumor dna in patients with leiomyosarcoma with progressive disease purpose leiomyosarcoma ( lms ) is a soft - tissue sarcoma characterized by multiple copy number alterations ( cnas ) and without common recurrent single - nucleotide variants . 
 we evaluated the feasibility of detecting circulating tumor dna ( ctdna ) with next generation sequencing in a cohort of patients with lms whose tumor burden ranged from no evidence of disease to metastatic progressive disease . patients and methods we evaluated cell - free dna in plasma samples and paired genomic dna from resected tumors from patients with lms by ultra - low passage whole genome sequencing . 
sequencing reads were aligned to the human genome and cnas that were identified in cell - free dna and tumor dna by ichorcna software to determine the presence of ctdna . 
clinical data were reviewed to assess disease burden and clinicopathologic features . results we identified lms ctdna in 11 ( 69% ) of 16 patients with disease progression and total tumor burden greater than 5 csixteen patients with stable disease or low disease burden at the time of blood draw were found to have no detectable ctdna . 
ctdna levels declined after resection of progressive disease in one case and became detectable upon disease relapse in another individual patient . conclusion these results suggest that ctdna , assayed by a widely available sequencing approach , may be useful as a biomarker for a subset of patients with uterine and extrauterine lms . 
2018 by american society of clinical oncology introduction leiomyosarcoma ( lms ) is a malignant neoplasm derived from smooth muscle that represents one of the most common subtypes of soft - tissue sarcoma.1 , 2 although the majority of lms cases are sporadic , predisposing factors include li fraumeni syndrome , hereditary retinoblastoma , and radiation exposure.3 , 4 there are no oncogenic single - nucleotide variants ( snvs ) that characterize lms , although loss of tumor suppressors , including tp53 , rb1 , and pten , are commonly observed , as are multiple copy number alterations ( cnas ) .5 - 8 lms is frequently a clinically aggressive disease , and patients are at high risk for local and metastatic relapse after initial complete resection.9 , 10 efforts to improve outcomes for patients would benefit from more reliable indicators of high - risk disease and biomarkers of response to therapy . detection of circulating tumor dna ( ctdna ) has emerged as a new approach for identifying oncogenic mutations , measuring disease burden , clinical prognostication , and assessing tumor response to therapy.11 , 12 most ctdna assays matthew l . 
 have been developed to detect snvs that are highly recurrent in many types of carcinomas.13 although the lack of recurrent snvs in lms limits efforts at targeted sequencing , the numerous cnas that are characteristic of this disease represent an ideal target for detection . 
 a next - generation sequencing approach using ultra - low passage whole - genome sequencing ( ulp - wgs ) can detect cnas in cell - free dna , which indicates the tumor fraction of ctdna present in blood.14 , 15 previous studies have shown that ctdna can be detected in cell - free dna samples that are sequenced at a minimum coverage of 0.1 across the whole genome.14 sequencing for ulp - wgs uses the standard illumina next - generation sequencing platform without modifications or special adaptions ( illumina , san diego , ca )  . 
the ichorcna algorithm is used to detect megabase - scale cnas from ulp - wgs data in which ctdna comprise as little as 3% of the total cell - free dna extracted from a plasma sample.14 in the current study , we evaluated plasma from patients with uterine and extrauterine lms for the presence of ctdna using ulp - wgs . 
paired resected tumors from each patient were also sequenced when available ( 29 of 30 patients ) , which enabled the identification and comparison of cnas between primary tumors and ctdna . 
high - molecular - weight dna contamination of cell - free dna was determined by bioanalyzer ( agilent technologies , santa clara , ca ) and size selection was performed if necessary ( ampure xp beads ; beckman coulter , brea , ca )  . 
up to 40 ng of cell - free dna , 100 ng of dna from fresh frozen tissue , and 200 ng of dna from ffpe tissue were used for kapa hyper library preparation ( kapa biosystems , wilmington , ma )  . 
 after bioruptor sonication ( diagenode ) of formaldehyde - fixed fresh frozen sample , with library preparation using a thruplex dna sequencing kit ( rubicon ) and sequencing on an illumina nextseq 500 . 
this higher sequencing coverage did not alter downstream analyses or data interpretation . sequencing results were demultiplexed , aligned , and processed using picard , burrows - wheeler alignment tool , 17 and genome analysis toolkit.18 , 19 to assess for cnas in tumor and cellfree dna and determine tumor fraction or percentage of ctdna in cell - free dna , we used ichorcna software14 with manual curation of results as necessary to confirm tumor percentages . 
previous studies have demonstrated that this technique can be used to identify and quantitate ctdna that constitutes as little as 3% of the cell - free dna in a sample.14 copy number analysis copy number segments and estimates of tumor fraction and ploidy were generated by ichorcna . 
this analysis was performed separately on tumor resection samples , including the eight samples for which plasma was not available , and on plasma samples that were positive for detectable levels of ctdna . statistical analysis group comparisons between patients with lms with active or indolent disease at the time of plasma collection was performed by nonparametric mann - whitney test , and we performed pearson correlation coefficient between tumor fraction or cell - free dna and tumor burden using graphpad prism ( version 7.0 ; graphpad software , la jolla , ca )  . 
one was added to integer values before log2 transformation to generate non - negative values for data representation . results detection of lms ctdna we retrospectively identified 30 patients who were diagnosed with metastatic lms who had plasma banking performed at variable time points in their treatment history between january 2007 and november 2017 . 
median age at diagnosis was 51 years , most patients were female , the most common primary tumor location was the uterus ( n = 16 ) followed by the retroperitoneum ( n = 8 ) , and most tumors were originally of intermediate or high grade ( table 1 )  . 
twenty - nine of 30 patients had a tumor resection sample available for comparison with cell - free dna . in review of clinical data at the time of plasma banking , the cases could be divided into active disease or indolent disease groups on the basis of tumor volume and evidence of disease progression . 
the active disease group consisted of 16 patients and was defined by having a total tumor burden greater than 5 cm in greatest diameter and progressive disease at the time of blood draw on the basis of imaging or clinical determination . 
in contrast , 16 patients in the indolent disease group had stable disease at the time of blood draw and / or tumor burden less than 5 camong the active disease group , 11 ( 69% ) of 16 patients had detectible ctdna , including patients with both uterine and extrauterine primary tumors . 
none of the samples obtained from the indolent disease group had detectible ctdna ( fig 1a )  . when comparing the amount of ctdna measured as a fraction of total cell - free dna in the plasma sample to the volume of tumor burden reported by computed tomography scan , there was a significant association between higher ctdna levels and increasing tumor burden ( fig 1b )  . 
 abbreviations : a , abdomen ; b , bone ; c , chest ; ctdna , circulating tumor dna ; f , female ; hpf , high - power field ; l , liver ; m , male ; na , not available ( data not reported during pathology review ) ; p , pelvis ; st , soft tissue , including subcutaneous , muscle , and paraspinal locations . cna concordance between lms tumors and ctdna in the 11 plasma samples with detectible levels of ctdna , there was a high concordance of cnas between the tumor sample and ctdna ( figs 2a - 2d ) , with blood collection occurring less than 2 years apart from resection of the matched tumor specimen . 
the active disease group consists of patients with tumors > 5 cm in size and with progressive disease at the time of blood draw as indicated by computed tomography ( ct ) scan or clinical report . 
labels indicate the active disease subgroup ( red ) with tumor size > 5 cm and progressive disease ( n = 16 ) , or indolent disease subgroups ( gray ) with tumor size > 5 cm and stable disease ( n = 2 , diamond ) , tumor size < 5 cm and progressive disease ( n = 4 ; triangle ) , and tumor size < 5 cm and stable disease ( n = 10 ; circle )  . 
overall , recurrent cnas from tumors in this cohort are remarkably similar to those found in prior studies.5 , 8 the most significantly amplified genomic region was found on chromosome 17p , which includes the transcriptional regulator myocd ( copy gained in 20 of 37 tumors )  . 
this gene has previously been reported as significantly amplified in lms and is associated with smooth muscle differentiation.22 additional putative oncogenic and lms associated genes that were recurrently amplified include hdgf in 20 , myc in 14 , cthrc1 in 17 , tnfrsf19 in 10 , and myh2 in 16 of 37 tumors ( fig 3a )  . 
copy gains in these genes were also observed in a portion of cell - free dna samples with detectable ctdna ( myocd in four , hdgf in six , myc in eight , cthrc1 in nine , tnfrsf19 in three , and myh2 in four of 11 ctdna - positive samples ) ; however , the number of evaluable samples was too small for any copy gains of these genes to reach statistical significance ( fig 3b )  . 
from tumor samples , recurrent deletions in tumor suppressors were found , which are characteristic of lms , 5 , 8 , 23 including pten deletions in 27 , rb1 in 27 , and tp53 in 11 of 37 tumors profiled ( fig 3c )  . 
deletions that involved these genes were also detected in ctdna ( pten in eight , rb1 in six , and tp53 in two of 11 ctdna - positive samples ) , but sample size was too small for many of these deletions to reach significance ( fig 3d )  . 
 thus , ulp - wgs is capable of detecting recurrent cnas characteristic of lms , and these methods may be helpful in identifying genelevel cnas in ctdna . ctdna longitudinally correlates with disease status to determine whether ctdna levels change in agreement with longitudinal disease status , we evaluated serial plasma samples in two patients after either resection of localized lms or with disease recurrence after surgery . 
in contrast , in the patient with disease recurrence 32 months after surgical resection , cell - free dna tumor fraction increased from undetectable postoperatively to detectable at the time of disease recurrence ( fig 4b )  . 
 ( a - e ) copy number plots generated from ultra - low passage whole - genome sequencing of five representative tumor surgical ( left panels ) and plasma ( right panels ) sample pairs . 
tumor fraction is indicated for each plot . discussion in the current study , we analyzed cell - free dna from the plasma of patients with lms for evidence of ctdna . 
a significant association between tumor burden and the amount of ctdna was identified , which suggests that larger and actively growing tumors are more likely to release tumor dna into circulation . 
 ( a - d ) gistic2.0 analysis identifying recurrent focal amplified ( a and b ) and deleted ( c and d ) regions in lms tumors ( n = 37 ; left panels ) and cell - free dna samples with detectable circulating tumor dna ( n = 11 ; right panels )  . 
the green line indicates an fdr of 0.25. tumor cell - free dna amplifications tnfrsf19 myocd myh2 amplifications cthrc1 6 7 8 9 10 chromosome 12 14 16 18 20 x 12 14 16 18 20 6 7 8 9 10 chromosome deletions hdlbp deletions cdkn2aip brca2 tp53 pten tsc1 ypel3 12 14 16 18 20 x 12 14 16 18 20 x 6 7 8 9 10 chromosome 6 7 8 9 10 chromosome 10 - 1 0.25 10 - 2 10 - 1 0.25 less than 5 cm in size , regardless of evidence of disease progression , which may represent a technologic sensitivity threshold . 
we further found that ctdna levels decline with resection of disease and increase with disease recurrence in individual patients , which indicates that these methods may be useful for supporting a diagnosis of disease recurrence or response to therapy . sequencing of snvs in cell - free dna has proven clinical utility in detecting resistance mutations to targeted therapies and directing treatment strategies.24 emerging use of whole - genome sequencing to identify and quantify cnas from tumor - derived dna circulating in patients with cancer has been evaluated in a number of cancer types.14 , 25 - 27 this noninvasive approach can be useful in the genomic characterization of malignancy and provide diagnostic and prognostic information relevant for clinical care.15 , 21 , 28 in a recent study of patients with lms , a highly customized lms - specific assay ( cancer personalized profiling by deep sequencing , or cappseq ) was designed to detect selected snvs and combined with cna analysis to identify and quantify ctdna in patients with progressive disease.29 in this study , the investigators found that approximately 20% of patients ( two of nine ) had tumors that did not have somatic events that were detectable by the assay . 
in contrast , we found that tumors from 100% of cases had cnas that were detectable by ulp - wgs , which suggests that ulp - wgs may be more broadly applicable for patients with lms . 
 the interval between surgical date and cell - free dna collection is indicated . in six of seven baseline samples of patients who were eligible for study ( 86% ) and had a sensitivity of 68% among all samples tested from their cohort . 
although these numbers are small , this suggests that capp - seq may have a higher sensitivity for ctdna , which might be expected given the deep sequencing coverage used in this assay . 
for example , our recent study in osteosarcoma demonstrated that pretreatment ctdna levels detected by ulpwgs are prognostic , 28 whereas a more sensitive assay , such as capp - seq , may be advantageous in the setting of disease surveillance.29 there are several potential clinical uses of ctdna in the diagnosis and management of patients with lms . 
crompton strategies for uterine tumors that could potentially harbor lms.32 second , there is significant clinical uncertainty regarding which patients with lms derive benefit from adjuvant chemotherapy and radiation.10 , 33 , 34 should ctdna levels bear prognostic significance for tumors that are at highest risk of recurrence or identify the presence of residual disease , their measurement may help guide clinical decisions regarding adjuvant therapies . 
finally , ctdna levels may be a useful indicator of response to systemic therapy and provide an early indication for switching or intensifying treatment regimens used in this disease.3 , 35 together with technologic improvements in sensitivity and throughput , these initial reports that identify ctdna in lms may quickly evolve to transform clinical practice . 
andersen no relationship to discloses edwin thai no relationship to discloses suzanne george stock and other ownership interests : abbott laboratories , abbvie ( i ) , allergan ( i ) consulting or advisory role : blueprint medicines , deciphera , astrazeneca , blueprint medicines , bayer , eli lilly research funding : pfizer ( inst ) , novartis ( inst ) , bayer ( inst ) , ariad pharmaceuticals ( inst ) , blueprint medicines ( inst ) , deciphera ( inst ) patents , royalties , other intellectual property : uptodate expert testimony : bayer other relationship : research to practice brian d . 
crompton , broad institute of massachusetts institute of technology and harvard , cambridge , ma ; and gavin ha , fred hutchinson cancer research center , seattle , wa . supported by the american society of oncology conquer cancer foundation young investigator award ( m.l.h. ) , national institutes of health grant no . 
toro jr , travis lb , wu hj , et al : incidence patterns of soft tissue sarcomas , regardless of primary site , in the surveillance , epidemiology and end results program , 1978 - 2001 : an analysis of 26 , 758 cases . 
ducimetire f , lurkin a , ranchre - vince d , et al : incidence of sarcoma histotypes and molecular subtypes in a prospective epidemiological study with central pathology review and molecular testing . 
hensley ml , ishill n , soslow r , et al : adjuvant gemcitabine plus docetaxel for completely resected stages i - iv high grade uterine leiomyosarcoma : results of a prospective study . 
oxnard gr , paweletz cp , kuang y , et al : noninvasive detection of response and resistance in egfr - mutant lung cancer using quantitative next - generation genotyping of cell - free plasma dna . 
klega k , imamovic - tuco a , ha g , et al : detection of somatic structural variants enables quantification and characterization of circulating tumor dna in children with solid tumors . 
mermel ch , schumacher se , hill b , et al : gistic2.0 facilitates sensitive and confident localization of the targets of focal somatic copy - number alteration in human cancers . 
stover dg , parsons ha , ha g , et al : association of cell - free dna tumor fraction and somatic copy number alterations with survival in metastatic triple - negative breast cancer . 
oxnard gr , thress ks , alden rs , et al : association between plasma genotyping and outcomes of treatment with osimertinib ( azd9291 ) in advanced nonsmall - cell lung cancer . 
van roy n , van der linden m , menten b , et al : shallow whole genome sequencing on circulating cell - free dna allows reliable noninvasive copy - number profiling in neuroblastoma patients . 
heitzer e , ulz p , belic j , et al : tumor - associated copy number changes in the circulation of patients with prostate cancer identified through whole - genome sequencing . 
shulman ds , klega k , imamovic - tuco a , et al : detection of circulating tumour dna is associated with inferior outcomes in ewing sarcoma and osteosarcoma : a report from the childrens oncology group . 
hensley ml , wathen jk , maki rg , et al : adjuvant therapy for high - grade , uterus - limited leiomyosarcoma : results of a phase 2 trial ( sarc 005 )  . 
reed ns , mangioni c , malmstrm h , et al : phase iii randomised study to evaluate the role of adjuvant pelvic radiotherapy in the treatment of uterine sarcomas stages i and ii : an european organisation for research and treatment of cancer gynaecological cancer group study ( protocol 55874 )  . 
 translating in vivo metabolomic analysis of succinate dehydrogenasedeficient tumors into clinical utility purpose mutations in the mitochondrial enzyme succinate dehydrogenase ( sdh ) subunit genes are associated with a wide spectrum of tumors , including pheochromocytomas and paragangliomas , gi stromal tumors , renal cell carcinomas , and pituitary adenomas . 
our aim was to investigate the potential clinical applications of proton - 1 magnetic resonance spectroscopy ( 1h - mrs ) in a range of suspected sdh related tumors . patients and methods fifteen patients were recruited to this study . 
sequential imaging in a patient with a metastatic abdominal paraganglioma demonstrated loss of the succinate peak after four cycles of [ 177lu ] dotatate , with a corresponding biochemical response in normetanephrine . conclusion this study has demonstrated the translation into clinical practice of in vivo metabolomic analysis using 1h - mrs in patients with sdh - deficient tumors . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction the succinate dehydrogenase ( sdh ) enzyme is composed of four subunits ( a to d ) and plays a key role in the krebs cycle and oxidative phos phorylation.1 in the past two decades , germ line mutations in the genes encoding the four sdh subunits ( sdha , sdhb , sdhc , and sdhd ) , collectively known as sdhx , have emerged as an important cause of human neoplasia and a par adigm for the role of disordered cellular meta bolism in oncogenesis.27 sdhx mutations were described initially in association with head and neck paragangliomas ( pgls ; derived from para sympathetic ganglia ) and in pheochromocytomas and pgls ( ppgls ; derived from sympathetic ganglia and often secreting catecholamines ) .2 , 3 it is now recognized that approximately 40% of patients with ppgls harbor germ line muta tions in inherited ppgl genes , and sdhx mutations comprise the most common cause of ppgl predisposition.8 in addition , germ line sdhb mutations are associated with a high risk of malignancy in ppgls.9 other tumor types associated with sdhx mutations include gi stromal tumors ( gists ) and renal cell car cinomas ( rccs ) .1013 gists are mesenchymal tumors of the gi tract and in adults are usually associated with somatic activating mutations in ruth t . 
 the kit or pdgfra gene.4 , 11 however , gists without kit or pdgfra gene mutations , 4 known as wildtype gists ( wtgists ) , account for 15% of adult and 85% of pediatric gists , and recent studies suggest that up to 88% of wtgists are sdh deficient.11 a wtgist with sdh deficiency may harbor a germ line sdhx mutation ( 75% of cases ) or an sdhc gene epi mutation with hypermethylation of the pro moter region.11 only approximately one third of patients with sdhdeficient wtgists achieve disease stabilization with imatinib therapy , 12 and the risk of metastatic disease is higher with sdhdeficient gists compared with conven tional gists.11 , 12 sdhx - associated rcc may present in patients with a personal or family his tory of ppgl or may present with an rcconly phenotype.13 finally , germ line sdhx mutations have been described in rare patients with pitu itary adenomas.10 despite recent advances in the understanding of the sdhx genes , there are many areas of unmet clinical need , including a lack of robust biomarkers to predict aggressive biologic behavior and inform clinical surveil lance and management.14 succinate has been shown to be elevated by 100fold in sdhxmutated ppgls ex vivo compared with nonsdhxmutated ppgls.15 recently , in vivo detection of succinate by mag netic resonance spectroscopy ( mrs ) was reported in two patient cohorts with sdhdeficient ppgls.16 , 17 similarly , the noninvasive detec tion of 2hydroxyglutarate with proton1 mrs ( 1hmrs ) has been demonstrated within glio mas in patients with gainoffunction mutations in another citric acid cycle enzyme , isocitrate dehydrogenase 1.18 the ability to measure succi nate in vivo has a number of important potential clinical applications , including early identifica tion of sdh deficiency , which could enable tai lored patient surveillance and management . 
suitable patients were identi fied based on sdhx germ line status , suspicious clinical phenotype ( metastatic ppgl , pgl , or wtgist ) , and / or immunohistochemistry of tumor tissue showing absent sdhb immuno staining . 
all participants provided written informed consent , and the study was approved by the cambridge south research ethics committee . mrs analysis both sage ( ge healthcare , waukesha , wi ) and lcmodel ( sprovencher.com ) 19 spectros copy analysis programs were used to recon struct , analyze , and display spectra . 
 ( c ) axial fused [ 18f ] fluorodeoxyglucose ( [ 18f ] fdg ) positron emission tomography ( pet ) / computed tomography ( ct ) image demonstrating an [ 18f ] fdgavid carotid body paraganglioma after sdh deficiency was demonstrated on proton1 mr spectroscopy ( 1hmrs )  . 
three patients had multicentric primary tumors , including patient 5 , who presented with a metastatic wtgist and was subsequently diagnosed with a 1.9cm carotid body pgl ( fig 1d ) ; patient 9 , with an abdominal pgl and a small leftsided 1.5cm carotid pgl ( fig 2b ) ; and patient 8 , with a large leftsided glomus pgl and a 2cm prolactin secreting pituitary adenoma ( data supplement )  . 
 genotype a germ line mutation in an sdhx gene was iden tified in nine ( 60% ) of 15 patients : five in sdhb ( four missense variants and one truncating vari ant ) and four in sdha ( one missense and three truncating )  . 
two additional patients were found to have somatic sdhc epimutations ( table 1 )  . 1h - mrs succinate analysis the 1hmrs characteristics of the 15 patients are listed in the data supplement . 
the liver was the most common site to be assessed ( n = 6 ) , but goodquality spectra were also obtained from a pituitary tumor ( n = 1 ) and ppgls ( n = 5 )  . 
patient 4 had a met astatic wtgist with no detectable germ line sdhx mutation and preserved sdhb protein expression in the tumor tissue ; choline was con fidently fitted on lcmodel , but no succinate was seen . 
patient 6 demonstrated a goodquality spectrum from the remnant pituitary adenoma ; choline was detected on lcmodel and sage processing , but no succinate was detected , and this finding was consistent with the preserva tion of sdhb protein expression in the pitu itary tumor by immunohistochemistry ( fig 4 )  . 
patient 12 demonstrated no reliable detection of succinate or choline because of motion artifact and a low signaltonoise ratio ( snr ) , which probably resulted from inconsistent breathing , because the voxel was at the edge of the liver . 
a metastasis on the edge of the liver was imaged in patient 14 , where again incon sistent respiration probably led to displace ment of the voxel into adjacent adipose tissue . 
 ( a ) t2weighted magnetic resonance image from patient 1 and ( b ) t1weighted image from patient 3 demonstrating liver metastases from which spectra were acquired in the locations indicated by the white arrows . 
after four cycles of treatment , a repeat 1hmrs examina tion on the same pelvic nodal metastases revealed a choline peak but no succinate peak ( fig 5c )  . 
 although the magnetic resonance imaging fea tures of the metastatic lesions were unchanged pre and posttreatment , the loss of a succi nate peak was correlated with a reduction in plasma normetanephrine levels ( from 1 , 861 to 1 , 193 pmol / l ) and tumor avidity on [ 18f ] fluo rodeoxyglucose ( [ 18f ] fdg ) positron emission tomography ( pet ) / computed tomography ( ct ; standardized uptake value : pretreatment , 16.1 ; posttreatment , 9.3 ; figs 5d to 5f )  . 
the detec tion of choline on the acquired spectra both before and after treatment indicates that tumor necrosis is unlikely to account for the absent suc cinate peak posttreatment . a sequential 1hmrs study was performed for patient 5 because of evidence of progressive disease on surveillance ct , despite treatment with a multikinase inhibitor , regorafenib . 
the results for scr were almost identical in these two patients , suggesting good test reproducibility ( data supplement )  . discussion this proofofprinciple study demonstrates that detection of a succinate peak and an increased scr was specific for a variety of sdhdeficient tumor types . 
all six tumors with a positive suc cinate peak and elevated scr were associated with a germ line sdhx mutation ( n = 4 ) or an sdhc epimutation ( n = 2 )  . 
 ( c ) succinate dehydrogenase b ( sdhb ) immunohistochemistry demonstrating preserva tion of the sdhb protein performed on a section of tumor tissue debulked from the pituitary tumor . adequate 1hmrs demonstrated preservation of sdhb expression in the tumors analyzed . 
our findings are complementary to a previous study in which 1hmrs was applied in nine patients with pgls , and succinate peaks were detected in all five with sdhx mutations but not in the four patients without mutations.16 we demonstrate for the first time to our knowledge that 1hmrs can also be used to determine the sdh status of gists and pituitary adenomas and that succinate peaks can be detected in sdhdeficient tumors with epigenetic inactivation of sdhc . 
potential diagnostic applications of this new approach include : assessing the pathoge nicity of patients with germ line sdhx variants of uncertain significance and potentially sdh related tumors ; investigating possible metastatic lesions ( eg , in the liver ) in patients with germ line sdhx mutations and primary sdhdeficient tumors ; assessing patients with multiple pri mary tumors to determine if all are sdh defi cient ; identifying patients without detectable germ line sdhx mutations who might benefit from specialist genetic investigations , such as sdhc promoter methylation status ; and assess ing sdh tumor status preoperatively , particu larly for patients with possible wtgists , because standard adjuvant treatment with imatinib has proven to be less effective in patients with sdh deficient disease.12 notably , here we use the presence of a choline signal as an internal control for viable tissue to discriminate technical failures from a negative finding . 
 this trend is the opposite of what would be expected if necrosis were artificially lowering the overall succinate levels in large tumors and therefore suggests that the method is measuring real differences in succinate , which are indepen dent of tumor size . 
 ( b ) spectra acquired before treatment illustrating succi nate accumulation at 2.4 pp ( c ) spectra ac quired after four cycles of [ 177lu ] dotatate with no detectable succinate peak at 2.4 pp ( d ) plasma metanephrine and methoxytyramine levels before and after treatment with [ 177lu ] dotatate . 
as a consequence , sdhdeficient tumors demonstrate epigenetic abnormalities and activated hypoxic gene responses , and more recently , evidence has suggested that succinate may have a paracrine effect on stromal tis sue.2022 understanding the molecular mech anisms of sdhrelated tumorigenesis provides a rationale for novel therapeutic interventions such as reversing the epigenetic abnormalities or exploiting metabolic vulnerabilities , similar to the recent discovery that tumoral 2hydroxyglutarate accumulation may increase responsiveness to olapa rib , a poly ( adpribose ) polymerase inhibitor.23 the availability of sensitive noninvasive bio markers would greatly facilitate precision medicinebased clinical trials . 
1hmrs is highly specific and allows in vivo detection of individual metabolites without the use of ionizing radiation ; however , 1hmrs is significantly less sensitive than pet , which could limit the detection of low levels of succinate , and it can be challenging to differen tiate intracellular from extracellular metabolites . 
 in the future , 1hmrs may be complemented by other techniques , such as hyperpolarized car bon13 mrs , which can increase 13mrs snr by several orders of magnitude , allowing assess ment of enzyme flux in vivo.26 we show that 1hmrs could be a valuable tool for the assessment of tumor response in the con text of radionuclide and other therapies , because alterations in succinate levels were detected despite stable appearance of the tumor diam eters . 
this important application of 1hmrs could be expanded to include other tumors with specific metabolic defects , including fuma rate hydratasedeficient tumors , 27 idh1mutant tumors , 18 and the recently identified malate dehydrogenase 2deficient tumors.28 however , important limitations of in vivo metabolomic analysis using 1hmrs were also revealed by our study ; for example , spectral quality was poor in close proximity to metal dental work , in areas adjacent to air spaces such as the lung , and in bone metastases and was susceptible to motion artifact . 
in this study , the technical failure rate was 26% , which is similar to the failure rate reported in previous studies using 1hmrs.16 importantly , no patient cases were excluded from this prospective study , with the intention that this would inform the translation of this imaging modality into clinical practice . 
on the basis of the evidence from this exploratory study , we recommend that tumors be selected for 1hmrs analysis based on the following : ideally the largest tumor deposit but at least > 2 cm in size ; tumors located close to bone or lung should be avoided ; tumors with significant necrosis or hemorrhage should be avoided ; superficial tumor deposits should be selected preferentially ; and respiratorytriggered acquisition should be used for tumors in the upper abdomen , such as hepatic metastases . although the use of 1hmrs as a diagnos tic tool is likely to be limited to specialist cen ters , the number of scan averages in our study during spectral acquisition was less than half those reported in a previous study16 ( 200 v 512 ) , without demonstrating a reduction in sensi tivity . 
furthermore , this imaging modal ity could be used to investigate other metaboli cally driven tumors . in conclusion , this study is the largest to date to our knowledge to evaluate 1hmrs in patients with sdh deficiency . 
it reveals that 1hmrs has the potential to be used as a noninvasive biomarker in the precision management of sdhdeficient disease and could have a role as a biomarker of successful treatment response . 
mclean stock and other ownership interests : veryan medical patents , royalties , other intellectual property : patent for stent design for arterial bypass graft basetti madhu no relationship to disclose benjamin g . 
challis no relationship to disclose rogier ten hoopen no relationship to disclose thomas roberts no relationship to disclose consulting or advisory role : bayer healthcare pharmaceuticals , ariad pharmaceuticals speakers bureau : pfizer travel , accommodations , expenses : pierre fabre , teva pharmaceuticals europe helen l . 
bulusu honoraria : pierre fabre kieran allinson no relationship to disclose lisa happerfield no relationship to disclose soo - mi park no relationship to disclose alison marker no relationship to disclose olivier giger stock and other ownership interests : cambridge pathology eamonn r . 
xekouki p , stratakis ca : succinate dehydrogenase ( sdhx ) mutations in pituitary tumors : could this be a new role for mitochondrial complex ii and / or krebs cycle defects ? endocr relat cancer 19 : c33c40 , 2012 7 . 
boikos sa , pappo as , killian jk , et al : molecular subtypes of kit / pdgfra wildtype gastrointestinal stromal tumors : a report from the national institutes of health gastrointestinal stromal tumor clinic . 
holdsworth ch , badawi rd , manola jb , et al : ct and pet : early prognostic indicators of response to imatinib mesylate in patients with gastrointestinal stromal tumor . 
 o comparative analysis of durable responses on immune checkpoint inhibitors versus other systemic therapies : a pooled analysis of phase iii trials elvire pons - tostivint , md1 , 2 ; aur elien latouche , phd3 ; pauline vaard , md1 ; francesco ricci , md , phd1 ; delphine loirat , md , phd1 ; s egol `ene hescot , md , phd1 ; marie - paule sablin , md1 ; roman rouzier , md , phd3 , 4 ; maud kamal , phd1 ; claire morel , msc1 ; charlotte lecerf , msc1 ; vincent servois , md1 ; xavier paoletti , phd5 ; and christophe le tourneau , md , phd1 , 3 , 6 purpose immune checkpoint inhibitors ( icis ) have been demonstrated to improve overall survival ( os ) in several tumor types . 
the proportion of patients who experienced an os that exceeded two times the median os of the whole patient population also was estimated . results nineteen studies involving 11 , 640 patients treated in 42 treatment arms ( 26 ici and 16 non - ici arms ) were included . 
the mean proportion of patients who experienced a durable response was 2.3 times higher in those treated with an ici compared with those treated in the control arms ( 25% v 11% )  . 
durable responses were more frequent in patients treated with antipd - 1 / pd - l1 agents than in patients treated with antictla - 4 agents ( 28% v 18% )  . 
the mean proportion of patients who had an os that exceeded two times the median os was also higher in those treated with icis than in those treated in the control arms ( 30% v 23% )  . 
2019 by american society of clinical oncology introduction immune checkpoint inhibitors ( icis ) that target cytotoxic t - lymphocyte protein - 4 ( ctla - 4 ) and programmed cell death protein 1 ( pd - 1 ) and its ligand pd - l1 have been demonstrated to improve overall survival ( os ) in several tumor types.1 - 12 in 2011 , ipilimumab , a fully human immunoglobulin g1 monoclonal antibody that targets ctla - 4 , was the rst ici to be approved by the food and drug administration for the treatment of patients with previously treated advanced melanoma because of a signicant improvement in os in two randomized phase iii clinical trials.8 , 13 antipd - 1 icis , including nivolumab and pembrolizumab , and antipd - l1 icis , including atezolizumab , avelumab , and durvalumab , were subsequently approved by the food and drug administration not only in recurrent melanoma but also in several other tumor types , including nonsmall - cell lung cancer ( nsclc ) , renal cell carcinoma , head and neck squamous cell carcinoma , urothelial carcinoma , merkel cell carcinoma , and hodgkin lymphoma . patients with cancer with recurrent and / or metastatic disease usually have been considered as being incurable . 
when a duplicate publication of the same trial was found , the study with the most complete and updated report was included . selection criteria studies meeting all of the following criteria were selected : prospective randomized phase iii trials of icis in patients with cancer treated in the recurrent and / or metastatic setting , trials with os and / or progression - free survival ( pfs ) as a primary end point , and availability of the os and pfs kaplan - meier survival curves in the publications . 
finally , trials were excluded if the follow - up was insufcient to estimate the proportion of patients who experienced a durable response ( see statistical analyses )  . data extraction data extracted from eligible trials were tumor type , class of drugs in each treatment arm , number of patients in each treatment arm , number of previous lines of treatment in the recurrent and / or metastatic setting , median pfs , median os , the proportion of patients with a pfs exceeding three times the median pfs in each arm , and the proportion of patients with an os exceeding two times the median os in each arm ( fig 1 )  . statistical analyses because no denition of durable response existed , we arbitrarily dened as a durable response for a specic patient a pfs that exceeded three times the median pfs of all patients treated with the same drugs in the same trial . 
we also estimated the proportion of patients who experienced an os that exceeded two times the median os of all patients treated with the same drugs in the same trial . 
using digitizelt software ( digitzeit , braunschweig , germany ) , proportions of patients who experienced a pfs / os that exceeded three / two times the median pfs / os in each arm were measured directly from the extracted kaplan - meier curves.17 , 18 proportions were compared using the mannwhitney u test and represented using graphpad ( graphpad software , la jolla , ca )  . 
twelve of these were excluded for the following reasons : absence of a monotherapy ici arm ( n = 3 ) , adjuvant setting ( n = 3 ) , duplicate trials ( n = 5 ) , and insufcient follow - up ( n = 1 )  . 
the 19 remaining phase iii trials involved 11 , 640 patients treated in 42 different treatment arms ( table 1 )  . tumor types included melanoma ( seven trials ) , 8 - 12 , 23 , 24 nsclc ( ve trials ) , 1 - 3 , 19 urothelial carcinoma ( two trials ) , 6 , 20 prostate cancer ( two trials ) , 21 , 22 head and neck squamous cell carcinoma ( one trial ) , 4 gastric cancer ( one trial ) , 5 and renal cancer ( one trial ) .7 a total of 7 , 769 patients were randomly assigned to an ici treatment in 26 arms . 
the remaining 3 , 871 patients were treated with chemotherapy ( 11 arms ) , a molecularly targeted agent ( one arm ) , a vaccine ( one arm ) , and a placebo ( three arms )  . 
twelve ( 63% ) of the 19 trials were conducted in patients having received at least one prior line of treatment in the recurrent and / or metastatic setting , whereas patients were treated in the rst - line recurrent and / or metastatic setting in ve trials ( 26% ) and without restriction in terms of prior treatments in the two remaining trials ( 11% )  . 
crossover from the control arm to the ici arm was allowed in ve trials ( 26% )  . proportion of durable responses the mean of the median follow - ups was 15.7 months ( range , 5 to 38 months )  . 
the mean proportion of patients who experienced an os that exceeded two times the median os was 30% ( 95% ci , 28% to 32% ) in the ici arms versus 23% ( 95% ci , 21% to 26% ) in the other arms ( p , .001 , mann - whitney u test ; fig 3b ; table 2 )  . predictors of durable responses because durable responses were well reported in patients with melanoma treated with icis , 15 we compared the proportion of durable responses in patients with and without melanoma . 
estimated proportion of patients with a survival probability that exceeded two times the median survival in each treatment arm . articles that did not match the selection criteria ( n = 322 ) phase iii randomized trials that involved at least one immune checkpoint inhibitor arm ( n = 31 ) combination trials adjuvant setting duplicate insufficient follow - up ( n = 3 ) ( n = 3 ) ( n = 5 ) ( n = 1 ) subgroup of patients with nsclc , given the high number of trials in this latter tumor type . 
nonmelanoma trials involved 6 , 991 patients treated in 13 ici arms and 12 non - ici arms ( table 1 )  . the mean proportion of patients in the ici arms who had a pfs that exceeded three times the median pfs was 28% ( 95% ci , 22% to 34% ) in the melanoma trials versus 23% ( 95% ci , 20% to 26% ) in the nonmelanoma trials ( p not signicant , mann - whitney u test ; fig 4a ; table 2 )  . 
the mean proportion of patients in the ici arms who experienced an os that exceeded two times the median os was 30% ( 95% ci , 26% to 33% ) in the melanoma trials versus 30% ( 95% ci , 26% to 33% ) in the nonmelanoma trials ( p not signicant ; mann - whitney u test )  . 
results were similar when the subgroup of nonmelanoma trials was restricted to nsclc trials ( table 2 )  . we then evaluated whether the number of previous lines of treatment in the recurrent and / or metastatic setting affected the results . 
trials beyond the rst - line recurrent and / or metastatic setting involved 6 , 991 patients treated in 14 ici arms and 12 non - ici arms ( table 1 )  . 
the mean proportion of patients in the ici arms who had a pfs that exceeded three times the median pfs was 30% ( 95% ci , 20% to 40% ) in the rst - line trials versus 23% ( 95% ci , 19% to 26% ) in the beyond - the - rst - line trials ( p not signicant , mann - whitney u test ; fig 4b ; table 2 )  . 
the mean proportion of patients who had an os that exceeded two times the median os in the rst - line setting could not be calculated because of a too - short follow - up . we nally evaluated whether the proportion of durable responses depended on the target of the icis ( pd - 1 / pd - l1 v ctla - 4 )  . 
the mean proportion of patients treated with an antipd - 1 / pd - l1 agent who had a pfs that exceeded three times the median pfs was 28% ( 95% ci , 24% to 31% ) versus 18% ( 95% ci , 15% to 21% ) for patients treated with an antictla - 4 agent ( p , .001 , mann - whitney u test ; fig 4c ; appendix table a1 )  . 
the mean proportion of patients who had an os that exceeded two times the median os was 31% ( 95% ci , 28% to 34% ) in patients treated with an antipd - 1 / pd - l1 agent compared with 29% ( 95% ci , 26% to 32% ) in patients treated with an antictla - 4 agent ( p not signicant , mann - whitney u test ; appendix table a1 )  . the effect of in multivariable analysis , treatment with antipd - 1 / pd - l1 agents ( p , .001 ) and the effect of rstline treatment ( p = .02 ) were statistically associated with a higher mean proportion of durable responses ( appendix table a2 )  . discussion our study conrms that a higher proportion of patients experience durable responses to icis than to other drug classes ( 25% v 11% )  . 
proportion of patients who had ( a ) a progression - free survival ( pfs ) that exceeded three times the median pfs and ( b ) an overall survival ( os ) that exceeded two times the median os according to drug classes . 
these results are of primary importance because it cannot be excluded that some patients who experience a durable response might be cured of their recurrent and / or metastatic cancer , which represents the overarching goal of cancer research . 
estimation of the proportion of patients with a pfs that exceeds three times the median pfs of the whole cohort of patients treated with the same drugs instead of arbitrarily dening a minimum pfs is an elegant way not to be biased by heterogeneity in terms of the natural history of the disease , clinical setting , and drugs used . 
we selected a threshold of at least two times the median for os because the followup was insufcient in most of the trials to get the information for three times the median os . 
using durable response as we dened it provides additional information that is not given by any other classic end point , such as duration of response or pfs because it is calculated on the basis of the median pfs of the whole treatment arit gives an insight into the proportion of patients who experience durable responses and takes into account the natural history of the disease and the overall activity of the treatment . 
whether this measure correlates with os , which can be evaluated only with individual data , remains to be determined . twenty - ve percent of patients treated with icis in the trials retrieved for our study had a durable response according to our denition . 
although the proportion of durable responses is high and conrms one of the specicities of icis , 11% of patients treated with other drug classes , including chemotherapy and targeted therapy , also experienced durable responses , which means that this phenomenon is not unique to immunotherapy . 
how the results of our study would have been affected by such patient populations remains to be determined . the proportion of patients who had an os that exceeded two times the median os was also higher in patients treated with an ici compared with those treated with other drug classes , although to a lesser extent ( 30% v 23% )  . 
this difference might be explained by two factors . first , the threshold chosen of two times the median was lower for os than for pfs ( for the reasons explained at the beginning of the discussion )  . 
proportion of patients who had a progression - free survival ( pfs ) that exceeded three times the median pfs according to ( a ) tumor type ( melanoma v nonmelanoma ) , ( b ) clinical setting ( rst v second line or greater of treatment ) , and ( c ) drug classes ( antiprogrammed cell death 1 [ pd - 1 ] / programmed death - ligand 1 [ pd - l1 ] v anticytotoxic t - cell lymphocyte - 4 [ ctla - 4 ] agents )  . 
for example , 58% of patients crossed over in the nivolumab versus chemotherapy nsclc trial.19 reporting of long - term os data will be key to estimate the real effect on os . one of the limitations of our study is the small number of non - ici arms , which hampered the separate evaluation of the proportion of durable responses in patients treated in the various control arms . 
we attempted as well to include in our study phase iii trials that did not involve ici arms to have a better representation of patients treated with chemotherapy and targeted therapy . 
however , the lower proportion of patients who experienced durable responses to other drugs than icis in our study conrms the usual short - lived responses reported with chemotherapy and targeted therapy ( mainly those related to the occurrence of resistance mutations in this latter case )  . 
finally , we restricted our analysis to trials with sufcient follow - up to estimate the proportion of patients who experienced a durable response , as dened in patients and methods under statistical analyses , which means that the use of our end point cannot be generalized to all trials . in conclusion , our pooled analysis of randomized phase iii trials objectively conrms that durable responses are more frequent with icis than with other drug classes , especially with icis that target pd - 1 and pd - l1 . 
our study also shows that durable responses according to our denition are not specic to icis because this phenomenon also exists with targeted therapies and chemotherapy , although to a lower extent . 
the identication of predictive biomarkers of efcacy , and especially durable response , is of upmost importance to select patients who will need only a single - agent ici and patients for whom a drug combination will be necessary . 
horn l , spigel dr , vokes ee , et al : nivolumab versus docetaxel in previously treated patients with advanced nonsmall - cell lung cancer : two - year outcomes from two randomized , open - label , phase iii trials ( checkmate 017 and checkmate 057 )  . 
herbst rs , baas p , kim d - w , et al : pembrolizumab versus docetaxel for previously treated , pd - l1 - positive , advanced non - small - cell lung cancer ( keynote010 ) : a randomised controlled trial . 
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lancet 389 : 255 - 265 , 2017 ferris rl , blumenschein g jr , fayette j , et al : nivolumab for recurrent squamous - cell carcinoma of the head and neck . 
n engl j med 375 : 1856 - 1867 , 2016 kang y - k , boku n , satoh t , et al : nivolumab in patients with advanced gastric or gastro - oesophageal junction cancer refractory to , or intolerant of , at least two previous chemotherapy regimens ( ono - 4538 - 12 , attraction - 2 ) : a randomised , double - blind , placebo - controlled , phase 3 trial . 
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larkin j , minor d , dangelo s , et al : overall survival in patients with advanced melanoma who received nivolumab versus investigators choice chemotherapy in checkmate 037 : a randomized , controlled , open - label phase iii trial . 
lee ck , davies l , wu y - l , et al : getinib or erlotinib vs chemotherapy for egfr mutation - positive lung cancer : individual patient data meta - analysis of overall survival . 
in patients with stage iii or iv melanoma , early changes in ctdna within 1 month after initiation of treatment correctly predicted recist response categories in 19 of 20 patients . 
 braune et al context key objective we evaluated ctdna in stage iii and iv melanoma to detect active disease and to predict response to treatment . knowledge generated ctdna had superior sensitivity to ldh and s100 in detecting active disease in stage iv melanoma . 
written informed consent was obtained from all patients . isolation and quantitation of ctdna from plasma blood samples were collected in edta tubes ( sarstedt , n umbrecht , germany ) and centrifuged twice within the 2 hours after collection . 
the supernatant was stored at 80c . cell - free ctdna was extracted from 2 ml using the qiasymphony circulating dna kit ( qiagen , hilden , germany ) according to the manufacturers instructions and was eluted in 60 l of elution buffer . 
the limit of blank , limit of detection , and lowest detectable copy ratio were determined for each assay ( data supplement )  . imaging analysis imaging and quantitative analyses were performed by a single radiologist blinded to results of the ctdna analyses . 
workows for ct and magnetic resonance imaging are capable of automated segmentation and volume measurements . pet imaging and quantication 18f - uorodeoxyglucose scans were performed with a 64 - slice gemini tf positron emission tomography ( pet ) / ct scanner or a 16 - slice gemini tf big bore pet / ct scanner ( philips healthcare for both ) .17 a contrast - enhanced diagnostic ct ( 120 kvp , 100 , 400 mas , dose modulation applied ) or a low - dose ct ( 120 kvp , 25 mas ) scan was performed for attenuation correction and spatial allocation . 
for each pet study , a quantitative analysis was performed using rover v2.1.12 ( abx , radeberg , germany ) .18 , 19 a threshold of 40% of maximum standardized uptake value was chosen for the determination of the metabolic tumor volume ( mtv ) .20 statistical analysis data analysis was performed separately by stage for all eligible patients . 
correlations were calculated by using a log scale , and ldh and s100 measurements below the standard value were set to the standard value . within - patient correlation was accounted for in the correlation analysis and in the estimation of the progression - free survival ( pfs ) hazard ratio ( hr ) in stage iv patients . 
patient characteristics ( continued ) characteristic 66 ( 28 - 92 ) tissue mutation status braf exon 15 v600e v600k v600r k601n d594g l597s w604c nras q61 q61k q61r total patients female male age , years median ( range ) 60  . 
60 stage stage iii newly diagnosed previous treatment treatment after inclusion surgery surgery radiation systemic treatment relapsed previous treatment of relapse treatment of relapse after inclusion surgery radiation systemic treatment no . 
of previous lines of therapy stage iv mean ( range ) treatment at inclusion braf / mek inhibitor immunotherapy treatment after inclusion immunotherapy braf / mek inhibitor radiotherapy surgery interferon vaccination ( continued in next column ) 1.7 ( 0 - 7 ) * systemic treatment after progression to stage iv . three patients received systemic treatment for unresectable stage iii melanoma , and three patients received systemic treatment after progression to stage iv melanoma . total number of tissue mutations is 64 . 
patient 62 was tissue positive for nras q61r and braf w604c , patient 55 had resection of two different melanomas before inclusion in the study ( one was positive for braf v600e , and the other was positive for nras q61k )  . 
the mean number of samples was 6.8 ( range , 1 to 15 samples ) per patient with stage iii disease , and the mean was 5.8 ( range , 1 to 27 samples ) for stage iv patients . in patients with stage iii disease , 10 ( 83.3% ) of 12 were positive for ctdna at least once ; for patients with stage iv disease , 44 ( 88% ) of 50 patients were positive for ctdna at least once ( table 2 )  . 
s100 and ldh are indicated as being increased when level is above the normal range of , 0.105 mg / l for s100 and 135 to 225 u / l for ldh . 
we identied 10 stage iii and 30 stage iv patients with 56 samples obtained up to 30 days before surgery , initiation of systemic treatment , or change in systemic treatment . 
even when they were combined , ldh and s100 detected measurable disease in only 34 ( 60.7% ) of 56 samples before initiation of treatment ( table 3 ; fig 1c )  . 
thus , ctdna detected a higher proportion of patients with active disease . amount of mutated ctdna correlates with ldh and s100 to compare ctdna with the established biomarkers ldh and s100 , we studied measurement pairs obtained at the same time points . 
ctdna strongly correlated with s100 ( r = 0.64 based on 278 measurement pairs from 62 patients ) and ldh ( r = 0.50 based on 288 measurement pairs from 61 patients )  . ctdna reects tumor volume and metabolic activity we next asked whether mutant ctdna levels correlate with tumor burden . 
analysis was performed according to change in the sum of the diameters of target tumor lesions from baseline to rst ( fig 2a ) and subsequent ( fig 2b ) follow - up imaging scans according to recist 1.1. 
 ( a ) percentage of positive ( ctdna ) or increased ( lactate dehydrogenase [ ldh ] , s100 ) samples from 274 time points with matched ctdna , ldh , and s100 samples for stage iii and stage iv patients , respectively . 
red lines indicate the mean value for ctdna , ldh , or s100 . dotted lines indicate the thresholds of ldh ( 225 u / l ) and s100 ( 0.105 ug / l ) above which values are stated as being increased . 
 ( c ) sensitivity of ctdna , ldh , and s100 to detect disease within 30 days before surgery , initiation of treatment , or change in treatment ( see table 3 for details )  . 
of note , one patient who displayed a new lesion despite favorable ctdna course had an additional braf w604c mutation detectable at baseline that converted from negative at day 43 to positive at day 91 . 
conversely , all nine patients with partial response according to recist had a positive - to - negative conversion , had a decrease in ctdna , or remained ctdna negative . thus , early ctdna dynamics correctly predicted progressive disease or partial response according to recist in 21 of 24 patients . 
the remaining patient had a ctdna - positive sample available 1 day after imaging ( patient 60 ; fig 2c )  . ctdna predicts outcome we analyzed stage iii patients with a ctdna measurement within 30 days after surgery or at initiation of systemic therapy . 
four patients did not meet these criteria because they had no samples within 30 days after surgery ( n = 3 ) or at the initiation of systemic treatment ( n = 1 )  . 
thus , the higher levels of ctdna that appeared early after initiation of treatment or change in treatment indicated inferior pfs . dynamic ctdna proles of individual patients results of biomarker assessment in individual patients demonstrate that ctdna analysis over time is informative with respect to tumor dynamics as shown in the data supplement . 
ctdna demonstrated a superior dynamic signal range compared with ldh and s100 , and ctdna detected progression whereas ldh ( data supplement ) or ldh and s100 ( fig 1c , g ) failed to detect progression . 
of note , the combination of all three biomarkers provides an 8.9% increase in sensitivity to ctdna alone . thus , ctdna might be used together with ldh and s100 in a combined biomarker score . 
we observed a signicant association of ctdna at baseline with ldh , s100 , tumor size , and mtv ( r2 = 0.87 ) , the latter displaying the strongest correlation . 
this is in agreement with two other studies demonstrating a strong correlation between the number of ctdna copies and mtv in melanoma ( r = 0.63 , 23 and r2 = 0.6949 , 24 respectively )  . 
 ( d ) correlation between ctdna and ( continued on following page ) recist 1.1 , ( e ) tumor volume at baseline assessed by radiologic analysis , and ( f ) tumor metabolic activity , assessed by positron emission tomography / computed tomography ( pet / ct ) scan and expressed as the mean total lesion glycolysis ( tlg ) in [ g / ml x ccm ]  . 
the tlg in the equation [ g / ml x ccm ] was calculated by multiplying the lesion volume with the mean standardized uptake value in the volume of interest . 
eleven patients with stage iii and 22 with stage iv disease with a baseline ctdna sample , radiographic imaging before intervention , and a ctdna sample taken within 30 days after initiation of treatment or change in treatment were analyzed . 
 ( a ) waterfall plot showing the change in sum of diameter ( sod ) of target tumor lesions from baseline to rst follow - up imaging according to recist 1.1 in relation to early changes in ctdna . 
t0 denotes baseline ctdna sample , and t1denotes rst sample after baseline at day 8 to 28 after initiation of or change in treatment with ctdna detectable ( + ) or not detectable ( )  . 
dotted lines represent 20% increase in sod , which indicated progressive disease ( pd ) , or 30% decrease , which indicated partial response ( pr ) according to recist 1.1. 
 ( b ) spider plot showing changes in sod from baseline to rst and subsequent follow - up imaging according to early ctdna dynamic changes from baseline to rst sample after baseline taken within 28 days after institution of or change in treatment . 
cr , complete response ; sd , stable disease . importantly , early changes in ctdna levels were superior to ldh and s100 for early assessment of treatment response . of all the patients who achieved partial response , 76.9% had favorable ctdna dynamics as early as 8 to 28 days after initiation of or change in treatment . 
in line with these observations , detectable ctdna as early as within 30 days after initiation of or change to therapy predicted inferior pfs for patients with stage iii or iv disease . 
in stage iv disease , ctdna with more than 10 copies per ml within 30 days after initiation of or change in treatment indicated a population with a particularly short pfs of only 4 months . 
we included patients who were alive and free of progression 1 month after surgery or at the start of systemic therapy and who had a ctdna measurement within 1 month . 
pfs was dened as the time from initiation of treatment to rst reported progression or to the most recent visit . progression events included progression as dened by recist 1.1 , clinical progression , or melanoma - specic death . 
 ( a ) pfs in eight patients with stage iii disease alive and free of progression after 1 month with a ctdna measurement within 1 month after surgery or start of systemic treatment . 
 ( b - c ) pfs in 36 patients with stage iv disease with 46 pfs sections , alive and free of progression at 1 month by the rst ctdna value taken within 30 patients with days ( median , day 22 ; range , days 1 to 30 ) after start of or change in systemic treatment with a ctdna measurement within 1 month , by ( b ) positive v negative , and ( c ) negative or 10 or fewer copies per ml v more than 10 copies per ml . 
our data suggest that ctdna dynamics allow prediction of response and pfs within in the rst month after implementing braf / mek inhibitors or immunotherapy . in contrast to previous reports , 27 - 30 ctdna at baseline was not a prognostic marker for pfs in patients with stage iv disease in our cohort , whereas the level of ctdna at rst measurement after baseline was clearly predictive for pfs , most likely as an expression of response to treatment . notably in the aforementioned studies , most patients were treated with braf / mek inhibitors whereas the majority of our patients received immunotherapy . 
in two other studies of patients with or without melanoma who were receiving pd - 1 antibodies , there was no association between baseline ctdna and pfs.25 , 31 however , subgroup analyses did not reveal signicant differences because this study was not designed to examine the signicance of baseline ctdna in patients treated with braf / mek inhibitors versus immunotherapy . 
 braune et al nras mutations.27 , 32 this is a possible explanation for the increasing copies of nras - mutated ctdna detected in patient 53 after changing the treatment to dabrafenib and trametinib , which suggests selection of a subclonal mutation that mediates resistance to treatment with braf / mek inhibitors . 
nevertheless , previous studies have demonstrated tumor interand intraheterogeneity involving additional genetic events that lead to mapk or pi3k / pten / akt reactivation when disease progresses during treatment with braf inhibitors.33 , 34 few studies address the use of ctdna as a biomarker in stage iii melanoma . 
but in one recent study , ctdna positivity within 12 weeks after surgery indicated adverse disease - free survival and os.35 in our cohort , ctdna positivity after resection of the primary tumor and within 1 month after denitive locoregional treatment or start of systemic therapy predicted a poor pfs . 
with adjuvant therapies showing benet for high - risk stage iii melanoma , 9 , 10 ctdna positivity might help to stratify patients for adjuvant treatment . one limitation of this study is the small sample size . 
validation in a larger prospective trial is required , especially for detection of minimal residual disease in stage iii and to examine whether changes of ctdna early after the start of treatment constitute an independent marker for pfs and os for braf / mek inhibitor treatment versus immunotherapy . 
although mutationspecic assays might be most suitable for monitoring response and minimal residual disease , a broader sequencing approach using next - generation sequencing might be used in previously treated patients to track subclonal mutations to understand treatment resistance and to guide further therapy.34 in addition , including tert promoter mutations present in 70% of melanomas36 could increase coverage when added to brafand nrasspecic ctdna assays.37 in conclusion , our results demonstrate that ctdna is superior to ldh and s100 for detecting active disease and suggest that changes in ctdna early after initiation of treatment predict response to treatment and pfs . 
 ctdna as a biomarker in malignant melanoma dagmar von bubnoff honoraria : roche , iomedico travel , accommodations , expenses : bristol - myers squibb frank meiss honoraria : bristol - myers squibb , novartis , pierre fabre consulting or advisory role : bristol - myers squibb , novartis , pierre fabre , roche pharma ag speakers bureau : bristol - myers squibb travel , accommodations , expenses : novartis , bristol - myers squibb , pierre fabre nikolas von bubnoff honoraria : astrazeneca , amgen , bristol - myers squibb , novartis consulting or advisory role : novartis ( inst ) research funding : novartis ( inst ) no other potential conicts of interest were reported . acknowledgments the authors thank renata koch and aurelia winter for sample collection , patient management , and administrative support . references cancer genome atlas network : genomic classication of cutaneous melanoma . 
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 precision oncology strategy in trastuzumab - resistant human epidermal growth factor receptor 2positive colon cancer : case report of durable response to ado - trastuzumab emtansine introduction colorectal cancer is the third most common cancer and the fourth leading cause of cancer death worldwide.1 metastatic disease is ultimately identified in the majority of patients with colorectal cancer and , with rare exceptions , is considered incurable . 
the treatment of metastatic colorectal cancer ( mcrc ) has historically included the use of fluorouracil in combination with other chemotherapeutic agents , including oxaliplatin or irinotecan . the addition of the antivascular endothelial growth factor antibody , bevacizumab , to standard chemotherapy regimens has improved the progression - free and overall survival of patients with mcrc and has signaled a shift toward the addition of biologic and targeted agents in the treatment of advanced cancer.2 , 3 the transition toward molecularly targeted therapies has gained additional momentum with the us food and drug administration approval of the antiepidermal growth factor receptor agents cetuximab and panitumumab for the treatment of kras wild - type colorectal cancers.4 , 5 unfortunately , many patients with mcrc progress through these therapies , or are not candidates to receive them on the basis of the molecular profile of their particular tumor , and are left with few treatment options . 
 the tumor exhibited normal expression of the mismatch repair genes mlh1 , msh2 , msh6 , and pms2 . shortly after diagnosis , the patient began palliative chemotherapy with capecitabine and oxaliplatin every 3 weeks . 
however , follow - up imaging 6 months after treatment initiation identified progressive disease in the liver and lung , as well as a new adrenal gland metastasis . the patients treatment was then changed to capecitabine plus irinotecan and , ultimately , capecitabine , irinotecan , plus cetuximab , but his tumor continued to progress through each of these regimens , as determined by an increasing size of multiple hepatic metastases ( table 1 )  . 
we used a standard set of her2 - specific taqman primers and probes ( thermo fisher scientific , foster city , ca ) compared with standard references using an ultraconserved region on chromosome 1 . 
briefly , taqman pcr reaction mixtures were assembled using 2x ddpcr supermix for probes , 20x assays ( 18 mm primers and 5 mm probe ) and restriction digested dna samples ( bio - rad )  . 
this standard reference assay used the following primers : forward primer : 59 - tgagggattcggcagatgttg - 39 ; reverse primer : 59 - ctgaaaggctggacttgacaga - 39 ; and probe : 59 - vic - actgtgtgctggacctmgb - 39 . 
all assay primers were ordered from integrated dna technologies ( coraville , ia )  . thermal cycling conditions were 95c for 10 minutes ( 1 cycle ) ; 94c for 30 seconds and 60c for 60 seconds ( 40 cycles ) ; 98c for 10 minutes ( 1 cycle ) , and a 12c hold . 
her2 copy number per cell was estimated as the ratio of the her2 and rna polymerase 30 ( rpp30 ) concentrations multiplied by two to account for the two copies of rpp30 that are expected per diploid genome . 
quadruplicate ddpcr wells were analyzed for each sample . results in the absence of additional treatment options , and given the patients good performance status , another liver biopsy was performed and nextgeneration sequencing was completed in a certified laboratory , which identified several tumor - specific somatic mutations , including apc r232 * , tp53 splice site 97_1g.a , and her2 amplification . the patients case and tumor genomics were then presented to a multi - institutional molecular tumor board for interpretation . the identification of a mutation in the adenomatous polyposis coli ( apc ) gene was expected and consistent with previous reports in which up to 85% of spontaneous colorectal cancers harbor somatic mutations in the classic tumor suppressor.16 a splice site mutation in the tp53 gene was equally unsurprising , given the relative frequency of tp53 mutations in human cancers.17 , 18 the molecular tumor board considered neither the apc nor tp53 mutations clinically actionable . treatment duration , months best outcome table 1 . 
cross - reference with the cancer genome atlas confirmed that approximately 4% to 5% of spontaneous colorectal cancers harbor high - level her2 amplifications , 19 and preclinical data predicted that anti - her2 therapy might be clinically effective , particularly when used in a combinatorial approach ( table 2 ) .20 , 21 gene alteration r232 * tp53 splice site 97_1g > a erbb2 amplification fig 1 . 
tumor - specific somatic alterations and quantification of the her2 copy number alteration . ( a ) next - generation sequencing identified three validated alterations in the patients hepatic metastatic colon cancer . 
 ( b ) using a droplet digital polymerase chain reaction assay , the total copies of her2 per genome in the patients metastatic tumor ( blue dot ) were measured and compared with the total copies of her2 in the patients germline ( peripheral blood , gold dot )  . 
error bars represent standard error of the mean . peripheral blood tumor to further characterize the her2 amplification and to validate the finding using an orthogonal method , a ddpcr assay was used . 
in contrast , a control gene , rpp30 , had the expected diploid copy number of two ( fig 1 )  . given the genomic findings and the patients clinical circumstances , treatment with trastuzumab was initiated . 
follow - up ct imaging , compared with baseline evaluation , revealed disease progression in the liver , with the largest lesion measuring 5.8 cm ( figs 2a and 2b )  . trastuzumab treatments were discontinued and the patient then received trastuzumab - emtansine ( t - dm1 ) infusions every 21 days for three total treatments . 
follow - up imaging revealed the largest hepatic metastasis measured 4.6 cm , compared with 5.3 cm at the initiation of t - dm1 ( fig 2c )  . three additional infusions at 21 - day intervals with subsequent imaging yielded ongoing disease response , with the largest hepatic mass measuring 3.2 cm ( fig 2d )  . 
the patient ultimately received t - dm1 infusions every 21 days for 15 months without any discernible negative clinical effect . the largest hepatic metastasis decreased to 2.1 cm ( fig 2e ) and two other metastases responded completely . 
the use of next - generation sequencing panels to identify actionable mutations and guide treatment has become increasingly scrutinized as a potentially viable approach for improving outcomes in a variety of cancer subtypes , including lung cancer and melanoma . 
these findings are bolstered by an interim analysis report from the mypathway study2 investigating the use of the combination of trastuzumab plus pertuzumab in various solid tumors , including mcrc . the case reported here suggests that genomicsbased analysis of refractory colorectal cancer can lead to effective targeted treatments . 
the expected progression - free survival and overall survival of a patient with mcrc with refractory disease is approximately 1.7 and 5 months , respectively , 25 in contrast to the 15 - month progression - free survival observed in this case . additional data and outcomes are required to confirm that precision oncology routinely yields superior outcomes ; however , this report illustrates an additional treatment option in patients with refractory disease who have exhausted all of the commonly used therapeutics for their respective disease and yet retain an acceptable performance status and desire additional treatmenta common clinical scenario . 
kamangar f , dores gm , anderson wf : patterns of cancer incidence , mortality , and prevalence across five continents : defining priorities to reduce cancer disparities in different geographic regions of the world . 
saltz lb , clarke s , daz - rubio e , et al : bevacizumab in combination with oxaliplatin - based chemotherapy as first - line therapy in metastatic colorectal cancer : a randomized phase iii study . 
douillard jy , siena s , cassidy j , et al : randomized , phase iii trial of panitumumab with infusional fluorouracil , leucovorin , and oxaliplatin ( folfox4 ) versus folfox4 alone as first - line treatment in patients with previously untreated metastatic colorectal cancer : the prime study . 
slamon dj , clark gm , wong sg , et al : human breast cancer : correlation of relapse and survival with amplification of the her - 2 / neu oncogene . 
sartore - bianchi a , trusolino l , martino c , et al : dual - targeted therapy with trastuzumab and lapatinib in treatmentrefractory , kras codon 12 / 13 wild - type , her2 - positive metastatic colorectal cancer ( heracles ) : a proof - ofconcept , multicentre , open - label , phase 2 trial . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors based on molecular profiles : early results from mypathway , an open - label , phase iia umbrella basket study . 
richman sd , southward k , chambers p , et al : her2 overexpression and amplification as a potential therapeutic target in colorectal cancer : analysis of 3256 patients enrolled in the quasar , focus and piccolo colorectal cancer trials . 
sartore - bianchi a , ardini e , bosotti r , et al : sensitivity to entrectinib associated with a novel lmna - ntrk1 gene fusion in metastatic colorectal cancer . 
bertotti a , migliardi g , galimi f , et al : a molecularly annotated platform of patient - derived xenografts ( xenopatients ) identifies her2 as an effective therapeutic target in cetuximab - resistant colorectal cancer . 
parikh a , atreya c , korn wm , et al : prolonged response to her2 - directed therapy in a patient with her2amplified , rapidly progressive metastatic colorectal cancer . 
bensch f , van rooijen jm , schr oder cp , et al : a 21 - year - old patient with a her2 - positive colorectal cancer . discov 5 : 832 - 841 , 2015 gastroenterology 148 : 20 - 21 , 2015 cancer . 
grothey a , van cutsem e , sobrero a , et al : regorafenib monotherapy for previously treated metastatic colorectal cancer ( correct ) : an international , multicentre , randomised , placebo - controlled , phase 3 trial . 
 impact of variable rna - sequencing depth on gene expression signatures and target compound robustness : case study examining brain tumor ( glioma ) disease progression alexey stupnikov paul g . 
mcart , phd , msc , bioinformatics group , health sciences ( continued ) purpose gene expression profiling can uncover biologic mechanisms underlying disease and is important in drug development . 
the impact of sequencing depth alteration on rna - seqbased downstream analyses such as gene expression connectivity mapping is not known , where this method is used to identify potential therapeutic compounds for repurposing . methods in this study , published rna - seq profiles from patients with brain tumor ( glioma ) were assembled into two disease progression gene signature contrasts for astrocytoma . 
gene signatures were subsampled to simulate sequencing alterations and analyzed in connectivity mapping to investigate target compound robustness . results data loss to gene signatures led to the loss , gain , and consistent identification of significant connections . 
 building , centre for cancer research and cell biology , queens university belfast , 97 lisburn rd , belfast , bt9 7bl , united kingdom ; twitter : @drdmcart ; e - mail : d.mcart@qub.ac.uk. measurement is rna sequencing ( rna - seq ) of cdna transcripts in a high - throughput manner . 
rna - seq has wider analytical capabilities , including single nucleotide variants , insertion - deletions , gene splice variants , post - transcriptional modifications , and gene fusion detection , but remains costly.6 , 7 experimental techniques developed to minimize sequencing costs include sample multiplexing . 
 multiplexing involves labeling each sample library with a barcode identifier , allowing multiple libraries to be pooled and sequenced simultaneously , reducing costs.7 - 10 smaller volumes of rna are analyzed for multiplexed samples ; thus , the downside to multiplexing is reduced sequencing depth for this library type . accurate assessment of transcripts depends on length , abundance , and mappability to the reference and sufficient sequencing depth , particularly for genes with low transcript abundance.11 , 12 sequencing depth alterations can affect the detection of differentially expressed genes ( degs ) and potentially the accuracy of rna - seqbased downstream analysis . 
few studies have assessed the impact of sequencing depth alterations on rna - seq downstream applications.13 more studies are required , particularly to assess applications that rely on precise gene signatures , informative in classifying cancer subtypes and improved prognostic and predictive outcomes.14 , 15 a gene signature is summarized by degs that collectively represent the most prominent features of a cancer subtype or disease progression phenotype . 
this could be particularly problematic for connectivity mapping that examines a gene expression signature contrast with the aim of predicting potentially therapeutic us food and drugapproved target compounds for repurposing.16 there is urgent need for new targeted therapies for gliomas , which are the most common form of primary brain tumor . 
gliomas can be classified from grade i to iv on the basis of histologic and molecular information.17 depending on the cell of origin , each neoplasm is classified as an astrocytoma , oligodendroglioma , or ependymoma . 
herein , reference gene signatures were compiled from publically available sequenced tumors for astrocytoma disease progression.2 subsampling was applied to simulate sequencing depth alterations of gene signatures , and the performance of connectivity mapping was assessed . 
results reveal that information loss to gene signatures significantly affects target compound robustness . methods published whole transcriptome sequencing data of brain tumor biopsy specimens from adults ( accession : gse48865 ; bao et al2 ) was downloaded from the sequence read archive.25 on average , samples had 50 million reads each . 
reads were quality controlled using trimmomatic software26 and aligned using bowtie2 , 27 allowing one mismatch against the human genome version hg38.28 aligned reads were mapped to genes from the grch38.81 annotation29 using samexplorer software.30 , 31 to benchmark a diverse range in performance of the rna - seq analysis , mapped reads were subsampled to simulate samples with a range of lower cdna library sequencing depths using a bioinformatics pipeline32 ( appendix fig a1 ; data supplement )  . 
the impact of information loss to gene signatures for degs , gene ontology ( go ) terms , and target compound detection was assessed using differential expression , go , and gene expression connectivity mapping analysis , respectively , with deseq2 , goseq , and the qub accelerated drug and transcriptomic connectivity ( quadratic ) software33 - 35 ( data supplement )  . 
the reproducibility of significant connections to the library of integrated cellular signatures identified for all cell lines and neuronal specific cell lines ( data supplement ) by quadratic was investigated16 , 36 - 38 ( data supplement )  . 
results and associated false discovery rates ( fdrs ) were visualized using the r packages venndiagram and ggplot2.39 , 40 results assessment of the l - h and h - h gene expression signatures l - h ( dataset_i ) and h - h ( dataset_ii ) gene signature contrasts comprised 47 and 33 patients , respectively ( data supplement )  . 
 when data input was reduced , the fdr for the number of degs detected increased linearly and by approximately the same amount for both data sets ( appendix fig a2 )  . 
dataset_i gene signature therefore demonstrated better resilience to data loss for deg detection compared with dataset_ii . for the full data set ( f = 1.0 ) , > 200 go terms described the functions of the degs identified for dataset_i ( appendix fig a3a )  . 
given this low number , which reduced to zero on f = 0.5 , go results for subsampled dataset_ii are not depicted . impact of information loss to gene signatures used in gene expression connectivity mapping for the full data set , a greater number of significant reverse ( rev ) and progress ( prog ) connections were identified for dataset_i compared with dataset_ii ( fig 2a )  . 
effect of decreased cdna library sequencing depth on the number of differentially expressed genes ( degs ) detected from ( a ) dataset_i , and ( b ) dataset_ii gene signatures ( data supplement )  . 
visualization of the global stratification ability of ( c ) dataset_i , low to high ( l - h ) , and ( d ) dataset_ii , high to high ( h - h ) gene signatures . 
heatmap was generated using unsupervised hierarchical clustering with the full rna - seq data ( f = 1 ) and depicts the gene expressional patterns of the top 100 differentially expressed genes identified between the gene signature contrast groups . 
the who disease grades of samples as determined by bao et al2 are overlaid . target compound identification , dataset_i was therefore more resilient to alterations in cdna sequencing depth compared with dataset_ii . the impact of data loss to gene signatures and the reproducibility of connectivity mapping is presented in figures 3 - 5 . 
for example , 3 , 135 rev connections were identified for dataset_i using f = 1.0 ; this increased to approximately 5 , 000 when subsampled to f = 0.01 , but approximately 60% of compounds were infrequently detected ( figs 3b and 3c )  . 
for dataset_i , when 50% of reads were removed , approximately 62.5% rev ( approximately 2 , 500 of 4 , 000 ) and approximately 50% prog significant connections ( approximately 1 , 500 of 3 , 000 ) were identified with every iteration ( fig 3 )  . 
for dataset_ii , when 50% of reads were removed , just approximately 13% rev ( approximately 400 of 3 , 000 ) and 9% prog significant connections ( approximately 180 of 2 , 000 ) were identified with every iteration ( fig 4 )  . 
 significant connections that potentially could progress ( prog ) or reverse ( rev ) the disease phenotype and fdrs are plotted against the data fraction included in the analysis ( f )  . 
when affected by data loss , connectivity mapping results were more robust for dataset_i compared with the dataset_ii gene signature . reducing data to the gene signature led to the loss , gain , and consistent identification of significant connections to target compounds ( fig 5 )  . 
for dataset_i , a large proportion of the significant connections across all cell lines ( 69% ; 2 , 195 of 3 , 135 ) and neuronal - specific cell lines ( 70% ; 144 of 205 ) were detected with all data fractions . 
similarly for dataset_ii , a proportion of the significant connections across all cell lines ( 7% ; 100 of 1 , 339 ) and neuronal specific cell lines ( 5% ; nine of 172 ) were detected with all data fractions . 
for example , 350 and 105 compounds were detected across all cell lines for dataset_i , f = 0.01 , that were not identified by the full data set ( fig 5a )  . 
when 50% of the reads were removed , nine and 27 target compounds identified for neuronal specific cell lines were lost , respectively , for dataset_i and ii ( figs 5c and 5d ; table 1 )  . 
thus , for dataset_ii , more target compounds identified by the full gene signature were lost , and some of these included compounds of potential clinical utility for glioma , such as suramin and dasatinib ( table 1 )  . 
a comparison of the rate of impact of data loss on go terms and significant connections detection for dataset_i can be seen by comparing figures a3 and 2a . discussion understanding molecular pathways and regulatory networks driving cancer is essential for the development of new therapies . 
gene expression profiling using rna - seq has led to the development of clinically relevant gene signatures that are informative for cancer subtypes.14 , 15 rnaseq experimental approaches such as sample multiplexing reduce cdna sequencing depth and potentially affect gene signature accuracy . 
significant connections to target compounds identified from the neuronal derived cell lines using ( c ) dataset_i and ( d ) dataset_ii across gene signatures are also compared . more patient gene expression profiles matched their who grade classifications . 
results support the subjective nature of tumor classification , which has interobserver variability.41 gene signatures provided a framework to assess connectivity mapping output for a well - performing accurate versus a poorer - performing less accurate contrast . characterization of the disease progression gene signatures revealed they differed in biologic complexity . 
thus , the l - h gene signature was more resilient to data loss for deg detection and had greater test sensitivity compared with the h - h gene signature . 
overall , the number of significant connections detected for the l - h gene signature was greater , most likely explained by the heterogeneous biologic mechanisms involved in lowto high - grade astrocytoma transition . 
with data loss , this fdr threshold was reached by the l - h and h - h gene signatures , respectively , when 70% and just 1% of reads were removed . 
thus , the l - h gene signature was more resilient to data loss for the detection of significant connections to target compounds using connectivity mapping . subsampling of gene signatures for connectivity mapping revealed that the suite of significant connections to target compounds became modified with data loss . 
compounds lost by the h - h gene signature ( who grade iii to iv ) included suramin , lopinavir , dasatinib , and vincristine , which have already been considered as glioma treatments . 
suramin , an anticancer agent , inhibits the binding of growth factors understood to play a role in glioma progression , angiogenesis , and radioresistance and has been used to treat newly diagnosed gbms.42 , 43 lopinavir , a protease inhibitor , has reached phase ii clinical trials for the treatment of high - grade glioma.44 dasatinib , a kinase inhibitor that acts on members of the src family of kinases , is well studied in glioma and has shown preclinical promise.45 vincristine , a spindle poison , is used in combination with procarbazine and lomustine to treat high - grade glioma and has also been successful in a phase iii trial for the treatment of low - grade gliomas.22 , 46 , 47 reductions in transcript abundance probably led to the loss of low - abundance genes from the full gene signature and altered the degs detected , leading to the loss of these connectivity mapping connections . 
fewer significant connections identified by the full data sets were lost by the l - h gene signature compared with the h - h gene signature , suggesting it was more resilient to data loss . 
similarly , in another subsampling rna - seq study of healthy organisms from multiple taxa , highly expressed genes regulating metabolism and pathogenesis of disease were consistently identified even when downsampling rna - seq reads to only 1 million reads , 13 thereby corroborating our findings from diseased tumors . results highlight the need for determining the optimal cdna sequencing depth for accurately identifying degs when compiling gene signatures . 
in the future , rna standard and spike - in controls may be useful to inform rna - seq best practices.48 the accuracy of a gene signature was particularly important when carrying out additional downstream analyses , such as connectivity mapping . 
given the instability of gene expression , perhaps using ontology types or ontotypes49 to characterize contrast phenotypes may be a more reliable approach compared with gene lists in connectivity mapping . 
other target compounds sensitive to data loss are being tested for their biologic efficacy against glioma stem cells using clonogenic cell survival assays and western blot analyses in ongoing studies by this research group . 
roddy no relationship to disclose manuel salto - tellez consulting or advisory role : philips healthcare , visiopharm , bristol - myers squibb , merck patents , royalties , other intellectual property : co - inventor of qupath ( open - source system for digital pathology analysis ) darragh g . 
mcart , queens university belfast ; tom flannery , estelle healy , and manuel salto - tellez , belfast health and social care trust , belfast , united kingdom ; alexey stupnikov , johns hopkins university , baltimore , md ; hayley p . 
 kurian , brain tumour research centre , university of bristol , bristol , united kingdom ; and frank emmert - streib , tampere university of technology , tampere , finland . supported by funding from the brainwaves northern ireland charity ( registered charity number : nic103464 )  . 
bao zs , chen hm , yang my , et al : rna - seq of 272 gliomas revealed a novel , recurrent ptprz1 - met fusion transcript in secondary glioblastomas . 
verhaak rg , hoadley ka , purdom e , et al : integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in pdgfra , idh1 , egfr , and nf1 . 
wang l , si y , dedow lk , et al : a low - cost library construction protocol and data analysis pipeline for illumina - based strand - specific multiplex rna - seq . 
liu y , zhou j , white kp : rna - seq differential expression studies : more sequence or more replication ? bioinformatics 30 : 301 - 304 , 2014 13 . 
turkington rc , hill la , mcmanus d , et al : association of a dna damage response deficiency ( ddrd ) assay and prognosis in early - stage esophageal adenocarcinoma . 
lamb j , crawford ed , peck d , et al : the connectivity map : using gene - expression signatures to connect small molecules , genes , and disease . 
okamoto y , di patre pl , burkhard c , et al : population - based study on incidence , survival rates , and genetic alterations of low - grade diffuse astrocytomas and oligodendrogliomas . 
ziu m , kalkanis sn , gilbert m , et al : the role of initial chemotherapy for the treatment of adults with diffuse low grade glioma : a systematic review and evidence - based clinical practice guideline . 
stupp r , hegi me , mason wp , et al : effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase iii study : 5 - year analysis of the eortc - ncic trial . 
takano s , gately s , engelhard h , et al : suramin inhibits glioma cell proliferation in vitro and in the braj neurooncol 21 : 189 - 201 , 1994 44 . 
yu mk , kramer m , dutkowski j , et al : translation of genotype to phenotype by a hierarchy of nat rev genet 18 : 473 - 484 , 2017 cell subsystems . 
effect of decreased cdna library sequencing depth on the number of gene ontology ( go ) terms identified for ( a ) dataset_i and ( b ) the false discovery rate ( fdr ) for the number of go terms identified . 
 combined targeted therapy for braf - mutant , treatment - related acute myeloid leukemia a 44 - year - old woman with a prior history of myxoid liposarcoma who was previously treated with radiation , chemotherapy , and resection , was admitted with syncope and pancytopenia . she was diagnosed with a high - grade therapy - related myelodysplastic syndrome and treated with decitabine . 
she was treated with cytotoxic chemotherapy including cytarabine and topotecan , but she was not a candidate for further anthracycline exposure given her prior treatment for sarcoma and concern for cardiotoxicity . 
her aml was refractory to two sequential induction regimens . given that targeted genomic sequencing had revealed a braf v600e mutation with a high allelic fraction , she was then placed on combined targeted braf / mek therapy with dabrafenib and trametinib for her refractory disease . 
this resulted in a dramatic response with clearance of circulating myeloblasts , restoration of normal hematopoiesis , a significant decrease in marrow leukemic burden , and a concordant decrease in the braf v600e allelic burden . 
it emerges spontaneously or from antecedent myeloid neoplasms , such as myelodysplastic syndromes ( mdss ) .1 mds and aml can be related to prior chemotherapy or radiotherapy , in which case they have a worse prognosis . 
substantial molecular heterogeneity underlies myeloid malignancies , 2 - 5 and the presence of certain mutations has an impact on prognosis and response to treatment.6 for aml , npm1 , cebpa , and flt3 mutations are incorporated into clinical practice as important prognostic characteristics . such alterations reflect only a subset of the genomic heterogeneity underlying aml , and additional mutational abnormalities may soon influence treatment selection and prognostication.7 across malignancies , focus has shifted toward a genomics - driven framework promoting therapies that target specific molecular alterations . 
this personalized approach contrasts with traditional treatment with indiscriminately cytotoxic agents.8 therapies targeting the braf pathway are pervasive in melanoma , in which activating mutations are common.9 most alterations involve a substitution of glutamic acid for valine at position 600 ( v600e ) within the activation loop , causing constitutive activation and signaling through the mek / erk pathway . 
in metastatic melanoma , combinatorial regimens targeting braf and mek have demonstrated superiority to braf inhibitors alone in the phase iii setting and are approved by the us food and drug administration.10 braf mutations have also been noted in colorectal and nonsmall - cell lung cancers , gastric carcinomas , 11 and in virtually all patients with hairy cell leukemia in whom therapeutic responses are noted with braf inhibition.12 - 14 braf mutations are much less common in myeloid malignancies . 
in a study of more than 1 , 500 patients with aml , braf mutations were rare ( nine individuals [ , 1% ] ) , but their presence was associated with worse prognosis.15 in another series of 140 patients with therapy - related myeloid neoplasms , three braf mutations were po.ascopubs.org po precision oncology seth a . 
she had received neoadjuvant chemotherapy and radiation therapy 20 months earlier as treatment for sarcoma , including three cycles of doxorubicin , ifosfamide , and mesna ( aim )  . 
resection was followed by three additional cycles of aim . one year later , after presenting with the aforementioned symptoms , laboratories revealed pancytopenia , with white blood cell count ( wbc ) of 1 , 620 / ml , neutrophil count of 370 / ml , hemoglobin of 4.9 g / dl , and platelet count of 21 , 000 / ml . 
two weeks later , another marrow sample revealed persistent disease , with myeloblast involvement above 80% . her disease thereafter progressed , with wbc and circulating blast percentage reaching peaks of 9 , 530 / ml and 69% , respectively . 
although no pathogenic genomic variants were reported at initial diagnosis of therapy - related mds , a braf v600e mutation was identified ( at an allelic fraction of approximately 43% ; fig 1 ) upon leukemic progression . 
 therapy - related mds sample revealed that this braf variant had indeed been present , but at a variant allelic frequency ( vaf ) below detection threshold ( four reads , , 2% vaf ; fig 1 ; table 1 )  . no additional pathogenic mutations were present according to our institutions sequencing platform . because of rapid and refractory aml progression and the unique genomic data noted above , she was initiated on combined targeted braf / mek inhibition . 
she received dabrafenib ( 150 mg twice per day ) and trametinib ( 2 mg once per day ) , per established regimens for braf - mutant melanoma.17 two weeks later , her wbc had decreased to 500 / ml , and circulating blasts had disappeared . 
retrospective identification of a small population of braf - mutant cells in the original mds sample likely reflects the presence of an early subclone that expanded under the selective pressure of conventional treatment . because of limited therapeutic options and a wealth of clinical experience with braf / mek blockade in melanoma , she was treated with dabrafenib and trametinib . 
a dramatic clinical response was noted , with clearance of circulating myeloblasts , restoration of hematopoiesis , decrease in marrow leukemic burden , and a concordant decrease in the braf v600e vaf . 
of note , the braf v600e vaf rose at the time of progression because the acquisition of novel kras subclones ( all of which also harbored the original braf v600e mutation ) drove resistance and outgrowth . mechanisms governing resistance to braf / mek inhibition have been well characterized in cell culture models , xenografts , and patients.18 primary resistance to braf blockade in colorectal and papillary thyroid cancers has been attributed to loss of erk - dependent negative feedback on other oncogenic signaling pathways , table 1 . 
 ( c ) hematoxylin and eosin staining on continued braf / mek - directed therapy demonstrating hypocellular marrow with regenerating erythroid elements and several blasts . ( d ) terminal deoxynucleotidyl transferase dutp nick - end labeling staining of ( top ) pretreatment bone marrow sample showing no staining of the leukemic blasts and ( bottom ) first posttreatment bone marrow sample showing scattered positive apoptotic cells . including those involving egfr and her2 / 3 receptor tyrosine kinases.19 - 21 acquired resistance to braf blockade , also described in translational studies , does not seem to be the result of gatekeeper mutations in the braf kinase , as seen with other kinase inhibitors.22 instead , bypass mechanisms activate the map kinase pathway through mutations that activate key enzymes , such as nras , mek1 , or mek2.23 - 25 upregulation of pi3k / akt or her2 pathways have also been implicated as mechanisms of acquired resistance , 26 , 27 as have amplification of braf v600e , novel mek2 variants , and activating nras mutations via study of therapy - resistant tissue samples.28 - 30 in the presented case , four kras mutations were identified at disease progression . 
resistance to vemurafenib exposure co - occurred with spontaneous acquisition of a kras g12d activating mutation in papillary thyroid carcinoma cells in vitro.31 in recent clinical studies of vemurafenib in hairy cell leukemia , a patient with resistant disease acquired two subclonal kras mutations ( g12d and k117n ) .32 there have also been reports of rasdependent tumor outgrowth after treatment with braf inhibitors . 
additional work will be necessary to validate this assumption in cellular models . braf mutations are rare in mds and aml . nevertheless , it is intriguing that all braf - mutant cases among the previously published cohort of patients with therapy - related disease , like our case , harbored a concurrent t ( 9 ; 11 ) ( p22 ; q23 ) mll rearrangement.16 in a large cohort of patients with mll - rearranged aml , ras signaling alterations were identified in 50% of patients ( 59 of 118 ) , three of whom harbored braf mutations.35 the presence of a braf or ras mutation may interact with the mll - rearranged protein to drive leukemic genesis and evolution in this population and could contribute to decreased therapeutic responsiveness and poor prognosis . 
such a potential interaction requires further study . therapy in mds or aml leading to an impressive antileukemic response , as well as the first description of convergent kras mutations in aml at the time of therapeutic resistance . although braf and mek inhibitors hold tremendous promise in various malignancies , divergent mechanisms of resistance have been described , many of which rely upon constitutive activation of ras kinases and related pathways . 
hasserjian stock and other ownership interests : cerner , gilead pharmaceuticals , halyard health , healthcare services group , johnson & johnson , medtronic , novo nordisk , perrigo consulting or advisory role : amgen , celgene , promedior kwadwo oduro no relationship to disclose krzysztof glomski no relationship to disclose valentina nardi no relationship to disclose gregory m . 
cote research funding : pharmamar ( inst ) , epizyme ( inst ) , boston biomedical ( inst ) , agios pharmaceuticals ( inst ) , macrogenics ( inst ) timothy a . 
graubert stock and other ownership interests : biogen idec ( i ) , vertex ( i ) , bristol - myers squibb , express scripts consulting or advisory role : biogen idec ( i ) , novartis ( i ) , agios pharmaceuticals , juno therapeutics andrew m . 
chen consulting or advisory role : insys therapeutics , incyte , takeda pharmaceuticals , regimmune , seattle genetics research funding : seattle genetics , novartis , bayer healthcare pharmaceuticals , celgene amir t . 
mardis er , ding l , dooling dj , et al : recurring mutations found by sequencing an acute myeloid leukemia genome . n engl j med 361 : 1058 - 1066 , 2009 3 . 
papaemmanuil e , gerstung m , bullinger l , et al : genomic classification and prognosis in acute myeloid leukemia . 2847 - 2855 , 2016 n engl j med 374 : 2209 - 2221 , 2016 16 . 
christiansen dh , andersen mk , desta f , et al : mutations of genes in the receptor tyrosine kinase ( rtk ) / ras - braf signal transduction pathway in therapy - related myelodysplasia and acute myeloid leukemia . 
long gv , weber js , infante jr , et al : overall survival and durable responses in patients with braf v600 - mutant metastatic melanoma receiving dabrafenib combined with trametinib . 
corcoran rb , ebi h , turke ab , et al : egfr - mediated re - activation of mapk signaling contributes to insensitivity of braf mutant colorectal cancers to raf inhibition with vemurafenib . 
montero - conde c , ruiz - llorente s , dominguez jm , et al : relief of feedback inhibition of her3 transcription by raf and mek inhibitors attenuates their antitumor effects in braf - mutant thyroid carcinomas . 
nazarian r , shi h , wang q , et al : melanomas acquire resistance to b - raf ( v600e ) inhibition by rtk or n - ras x274 - x279 , 2006 upregulation . 
emery cm , vijayendran kg , zipser mc , et al : mek1 mutations confer resistance to mek and b - raf inhibition . proc natl acad sci usa 106 : 20411 - 20416 , 2009 25 . 
villanueva j , vultur a , lee jt , et al : acquired resistance to braf inhibitors mediated by a raf kinase switch in melanoma can be overcome by cotargeting mek and igf - 1r / pi3k . 
wagle n , van allen em , treacy dj , et al : map kinase pathway alterations in braf - mutant melanoma patients with acquired resistance to combined raf / mek inhibition . 
long gv , fung c , menzies am , et al : increased mapk reactivation in early resistance to dabrafenib / trametinib combination therapy of braf - mutant metastatic melanoma . 
danysh bp , rieger ey , sinha dk , et al : long - term vemurafenib treatment drives inhibitor resistance through a spontaneous kras g12d mutation in a braf v600e papillary thyroid carcinoma model . 
andrews mc , behren a , chionh f , et al : braf inhibitor - driven tumor proliferation in a kras - mutated colon carcinoma is not overcome by mek1 / 2 inhibition . 
callahan mk , rampal r , harding jj , et al : progression of ras - mutant leukemia during raf inhibitor treatment . n engl j med 367 : 2316 - 2321 , 2012 35 . 
staining was performed by using an anti - braf v600e mouse monoclonal antibody ( clone ve1 , ventana medical systems , tucson , az , catalog #790 - 4855 )  . 
terminal deoxynucleotidyl transferasemediated dutp nick - end labeling detection was completed manually by using millipore apoptag in situ detection kit ( emd millipore , darmstadt , germany , s7100 ) and visualized with diaminobenzidine substrate ( dako agilent pathology solutions , santa clara , ca , k4000 )  . this next - generation sequencing assay ( snapshot ) , using a multiplex polymerase chain reaction technology called anchored multiplex pcr ( amp ; zheng z , et al : nat med 20 : 1479 - 1484 , 2014 ) for single nucleotide variant ( snv ) and insertion / deletion ( indel ) detection , was used . 
briefly , genomic dna was isolated from blood or marrow aspirate . genomic dna was sheared with the covaris m220 instrument , followed by end - repair , adenylation , and ligation with an adapter . 
illumina misequation 2 3 147 base - paired end - sequencing results were aligned to the hg19 human genome reference using bwa - mem ( li h and durbin r : bioinformatics 25 : 1754 - 1760 , 2009 )  . 
mutect ( cibulskis k , et al : nat biotechnol 31 : 213 - 219 , 2013 ) and a laboratory - developed indel analysis algorithm were used for snv and indel variant detection , respectively . 
 acquired alk and ret gene fusions as mechanisms of resistance to osimertinib in egfr - mutant lung cancers introduction approximately 20% of patients with metastatic lung adenocarcinoma have somatic activating mutations in the epidermal growth factor receptor ( egfr ) gene , egfr . 
1 patients with egfr - mutant lung adenocarcinomas have a 70% response rate to first - line egfrtyrosine kinase inhibitor ( tki ) therapy ( ie , erlotinib , gefitinib , or afatinib ) .2 egfr t790m is the dominant resistance mechanism to earlier - generation egfr - tkis.3 osimertinib is approved by the us food and drug administration for the treatment of egfr - mutant lung cancers that have an acquired egfr t790m after failure of a previous egfr - tki4 and now is approved in the first - line setting as well.5 response to osimertinib eventually is followed by progression with known resistance mechanisms , including small - cell transformation3 , 6 ; acquired egfr mutations , including g796 / c797 , l792 and l718 / g7197 ; and non - egfr mediated resistance , including alterations / amplification in met , her2 , braf , mek , kras , and pik3ca . 
ngs with archerdx and msk - impact confirmed an acquired eml4 - alk fusion ( eml4 exons 1 through 6 fused with alk exons 20 through 29 ; c.667 + 516 : eml4_c.3173 - 415 : alkinv ) in addition to the egfr exon19del and t790m mutations . 
the patient started combination treatment with osimertinib 80 mg daily and crizotinib 200 mg twice daily , remained on treatment with stable disease ( by recist version 1.1 ) , and continued to receive clinical benefit from treatment , with no report of toxicity . 
the preosimertinib biopsy was a fine - needle aspiration ( fna ) and was partially fragmented on staining , whereas the acquired resistance ( ar ) sample was a core - needle biopsy . 
the pretreatment egfr exon19del staining was performed with epidermal growth factor receptor ( egfr ) e746 ; the post - treatment ihc stained more diffusely for egfr exon19del , likely related to the egfr amplification ( fold change : 3.0 ) noted on next - generation sequencing with msk - impact . 
 the ihc data suggest the presence of the alk rearrangement and egfr mutation within the same cell ( however , this cannot be confirmed by targeted next - generation sequencing because of technical limitations )  . 
 case 2 a 68 - year - old woman , who was never a smoker , presented with a 9 - cm right apical lung mass , right hilar lymphadenopathy , and a left temporal lobe mass ( staging t4n1m1b )  . 
 ( of note , no alk rearrangement was detected on ngs or fish . ) the patient started treatment with osimertinib and had an initial disease response , but oligoprogression occurred in the lung after 6 months . 
 ( a ) osimertinib and crizotinib in patient case 1 after one cycle ( 28 days ) of treatment ; image shows overall stable disease ( 0% best response )  . 
 ( b ) osimertinib and alectinib in patient case 2 after one cycle ( 28 days ) of treatment ; image shows regression of the dominant right upper lobe mass ( 25% reduction )  . precombination targeted therapy postcombination targeted therapy precombination targeted therapy postcombination targeted therapy l858r and t790m mutations as well as the new eml4 - alk rearrangement ( eml4 exons 1 through 2 fused to alk exons 20 through 29 ; c.208 + 4890 : eml4_c.3173 - 373 : alkinv ; fig 3b ; appendix table a2 )  . 
the patient started treatment with alectinib 300 mg twice daily and osimertinib 80 mg daily ; the first interval scan showed a decrease in the dominant right lung mass ( 25% reduction by recist version 1.1 ) as well as ongoing clinical benefit and no report of toxicity at the last follow - up . 
a right lung biopsy showed the known egfr l747s and l858r mutations as well as a new ncoa4 - ret fusion on msk - impact ; the fusion was confirmed by archerdx . 
 ( * ) treatment ongoing as of april 1 , 2018 . which showed that the fusion partner did not influence ret activity in cell lines.19 , 20 each condition was assayed in triplicate in at least two independent experiments . 
the pc9 cells were used to assess the effect of ret fusions on sensitivity of growth and apoptosis ( caspase 3 / 7 activity ) in the presence of osimertinib and cabozantinib . 
however , erk1 / 2 phosphorylation was not sensitive to osimertinib treatment in pc9 - pcx4.1ccdc6 - ret cells but was diminished with cabozantinib treatment ( fig 4b )  . 
these results suggest that ret rearrangements can cause bypass activation of growth - promoting pathways in cells that express oncogenic egfr . growth of pc9 cells that stably expressed ccdc6 - ret or kif5b - ret rearrangements was at least 10 - fold less sensitive to osimertinib than pc9 - pcx4.1empty cells were ( 50% inhibitory concentrations described in fig 4e )  . 
expression of ret fusions in pc9 cells prevented osimertinib - induced activation of caspase 3 / 7 , similar to the effect on growth rate ( fig 4f [ left panel ] )  . 
treatment of control - group pc9 cells or pc9 - ret cells with cabozantinib did not lead to activation of caspase 3 / 7 ( fig 4f [ left panel ] )  . 
these results suggest that ret rearrangements can induce resistance to osimertinib in cells that have egfr mutations and that response to osimertinib is restored when it is used in combination with cabozantinib . in conclusion , osimertinib is now a first - line treatment for metastatic egfr - mutant lung cancers.5 although potential resistance mechanisms to osimertinib have been identified , these have primarily been observed after later - line osimertinib in the setting of egfr t790m mutations and have focused on ctdna.9 - 11 many alterations identified as potential resistance mechanisms are seen concurrently with egfr in pretreatment samples , including amplifications of met and her2 as well as pik3ca mutations , which makes the comparison with pretreatment tissue critical to identify truly acquired alterations.13 as ngs becomes standard of care , it is feasible and fruitful to molecularly profile tumors broadly before treatment and at progression to identify acquired alterations that mediate resistance . it remains unclear whether mechanisms of resistance to first - line osimertinib will differ from mechanisms of resistance to later - line osimertinib or earlier - generation egfr - tkis . 
osimertinib is a potent mutant - egfr inhibitor that inhibits egfr t790m , so there is a potential to see novel on - target21 , 22 and off - target23 - 25 resistance mechanisms . 
this is corroborated by the low frequency ( 15% to 25% ) of egfr c797s and other acquired egfr alterations after osimertinib use compared with 60% frequency of acquired egfr t790m after use of earlier generation egfr - tkis.9 , 13 , 22 we may see new acquired alterations not previously seen or seen rarely with earlier - generation egfr - tkis . 
 moreover , although cabozantinib can inhibit growth of egfrand ret - mutant / rearranged cell lines , a combination of osimertinib and cabozantinib induced apoptosis . at our institution , among 174 patients with egfr - mutant lung cancer in whom ngs was performed on tumor tissue after progression developed during treatment with erlotinib or afatinib , we found no alk or ret fusions . 
it is unknown whether this potential enrichment of acquired fusions is related to the more potent egfr inhibition of osimertinib or to the later - line setting after multiple lines of egfr inhibition . 
pc9 cells were infected with lentivirus that harbored an empty plasmid ( pcx4.1 - empty ) or pcx4.1 with either ccdc6 - ret or kif5b - ret complementary dna ( cdna ) , and cells that expressed the plasmids were selected to generate stable cell lines . 
 ( a ) cell extracts were immunoblotted ( indicated by ib ) for ( left panel ) total ret ( left panel ) or ( right panel ) phosphorylated ret . 
 ( b ) cell lines were treated with either osimertinib 0.05 m or cabozantinib or 0.25 m for 1 h ; cell extracts were prepared and then immunoblotted for the indicated proteins . 
 ( c ) cells were treated with increasing concentrations ( as indicated on the figure ) of ( left panel ) osimertinib or ( right panel ) cabozantinib for 96 h , and then growth was determined . 
 ( f ) cells were treated with the indicated concentrations of ( left panel ) osimertinib or ( right panel ) cabozantinib for 48 hours , and then caspase 3 / 7 enzymatic activity determined . 
 ( g ) pc9 cells that expressed either pcx4.1 - empty vector or pcx4.1ccdc6 - ret were treated with the indicated concentrations of cabozantinib in the presence of osimertinib 0.1 m for 48 h , and then caspase 3 / 7 enzymatic activity was determined . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . michael offin no relationship to disclose romel somwar research funding : helsinn healthcare travel , accommodations , expenses : helsinn healthcare natasha rekhtman no relationship to disclose ryma benayed no relationship to disclose jason c . 
li consulting or advisory role : roche , biosceptre international , thermofisher scientific , mersana , guardant health research funding : roche ( inst ) , genentech ( inst ) , illumina ( inst ) , biomed valley discoveries ( inst ) , astrazeneca ( inst ) , grail ( inst ) gregory j . 
riely research funding : novartis ( inst ) , roche ( inst ) , genentech ( inst ) , millenium ( inst ) , glaxosmithkline ( inst ) , pfizer ( inst ) , infinity pharmaceuticals ( inst ) , ariad ( inst ) patents , royalties , other intellectual property : patent application submitted covering pulsatile use of erlotinib to treat or prevent brain metastases ( inst ) travel , accommodations , expenses : merck sharp & dohme charles m . 
kris consulting or advisory role : astrazeneca , regeneron william travis speakers ' bureau : genentech alexander drilon consulting or advisory role : ignyta , loxo , tp therapeutics , astrazeneca , pfizer , blueprint medicines , genentech , roche , takeda , helsinn therapeutics , beigene maria e . 
arcila consulting or advisory role : astrazeneca travel , accommodations , expenses : astrazeneca , invivoscribe , raindance technologies marc ladanyi honoraria : merck ( i ) consulting or advisory role : national comprehensive cancer network , boehringer ingelheim , astrazeneca ( tagrisso rfp advisory committee ) research funding : loxo ( inst ) helena a . 
jui , the chinese university of hong kong , sha tin new town , hong kong special administrative region , china . supported in part by the national cancer institute of the national institutes of health grants no . 
jnne pa , wang xf , socinski j , et al : randomized phase ii trial of erlotinib ( e ) alone or in combination with carboplatin / paclitaxel ( cp ) in never or light former smokers with advanced lung adnocarcinoma : calgb 30604 trial . 
yang z , yang n , ou q , et al : investigating novel resistance mechanisms to third - generation egfr tyrosine kinase inhibitor osimertinib in nonsmall - cell lung cancer patients . 
yang jc , ahn mj , kim dw , et al : osimertinib in pretreated t790m - positive advanced non small - cell lung cancer : aura study phase ii extension component . 
yu ha , suzawa k , jordan e , et al : concurrent alterations in egfr - mutant lung cancers associated with resistance to egfr kinase inhibitors and characterization of mtor as a mediator of resistance . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
jackman d , pao w , riely gj , et al : clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in nonsmall - cell lung cancer . 
national cancer institute : osimertinib and necitumumab in treating patients with egfr - mutant stage iv or recurrent nonsmall - cell lung cancer who have progressed on a previous egfr tyrosine kinase inhibitor ( nct02496663 )  . 
okamoto k , kodama k , takase k , et al : antitumor activities of the targeted multi - tyrosine kinase inhibitor lenvatinib ( e7080 ) against ret gene fusiondriven tumor models . 
arai s , kita k , tanimoto a , et al : in vitro and in vivo anti - tumor activity of alectinib in tumor cells with ncoa4 - ret . 
klempner sj , mehta p , schrock ab , et al : cis - oriented solvent - front egfr g796s mutation in tissue and ctdna in a patient progressing on osimertinib : a case report and review of the literature . 
ou si , horn l , cruz m , et al : emergence of fgfr3 - tacc3 fusions as a potential bypass resistance mechanism to egfr tyrosine kinase inhibitors in egfr - mutated nsclc patients . 
detailed molecular and immunohistochemical characterization of patient case 1 presented timing of molecular test diagnosis type of molecular test result tissue pcra impactb 15base pair egfr exon 19 deletion detected egfr exon19 p.e746_a750del ; tp53 exon10 p.r337l ; atr exon41 p.d2331y detected erlotinib progression cfdnac egfr t790m detected ( 0.79% ) d tissue pcrc egfr t790m detected ( 15.15% ) d alk : negative ; egfr exon 19 deletion : positive osimertinib + necitumumab ihce alk : positive ; egfr exon 19 deletion : positive progression impactb ihce impactb archerdxf fishg egfr exon19 p.e746_a750del ; egfr exon20 p.t790m ; kmt2c exon38 p.d2690n ; rad51b exon7 p.l209v detected no alk fusion detected no evidence of alk rearrangement . 
the single base extension product is detected by tandem mass - spectrometry on a sequenom massarray spectrometer ( sequenom , san diego , ca )  . bmsk - impact ( integrated mutation profiling of actionable cancer targets ) was used to identify specific mutations in 468 genes . cdigital pcr of cfdna and / or tissue amplification of part of egfr exon 20 in the presence of fluorescent probes specific to the wild - type and mutant alleles . dratio of mutant allele / ( mutant + wild - type allele )  . eimmunohistochemistry ( ihc ) for pertinent proteins performed with the following clones : egfr - exon19del ( egfr - e746 ) : clone 6b6 ; alk : clone d5f3 . farcher fusionplex custom solid panel ( archer fusionplex , boulder , co ) uses the anchored multiplex pcr used to detect gene fusions in tumor samples consisting of 62 cancer - related genes previously reported to be involved in chromosomal rearrangements . 
the single base extension product is detected by tandem mass - spectrometry on a sequenom massarray spectrometer . carcher fusionplex custom solid panel uses the anchored multiplex pcr used to detect gene fusions in tumor samples consisting of 62 cancer - related genes previously reported to be involved in chromosomal rearrangements . 
 mithat g onen lipika goyal stacy gray patrick grohar william grubb joshua gruber michael hall balazs halmos heather hampel sigurdis haraldsdottir derrick haslem aaron hata robert hayashi matthew hellmann andrew hendifar brian henick daniel hertz j . 
kevin hicks cinta hierro masahiro hiratsuka kim hirshfield eran hodis david hong david hsu eric huang hatim husain david hyman david ilson steven isakoff haruka itakura gopa iyer katherine janeway fabrice jardin russell jenkins lingyun ji chris jones goran jonsson mark kelley jonathan killian won kim hyunjae kim kevin kim randall kimple eric klein samuel klempner todd knepper andrew ko rachel koff wendy kohlmann tomonobu koizumi jill kolesar carl koschmann vessela kristensen rajiv kumar shaji kumar shivaani kummar pamela kunz allison kurian kwong kwok wong norman lacayo joseph lasky ulrik lassen josh lauring christophe le tourneau sarah leary adrian lee j . 
jack lee jochen lennerz mark lewis jessica lin carol liu charles lopez patricia lorusso christine lovly ying lu cynthia ma anant madabhushi subha madhavan michael maitland xavier maldonado david malka diana mandelker aaron mansfield richard marais laetitia marisa renato martins daniela matei joaquin mateo jes sloth mathiesen ignacio matos caroline mccoach rohit mehra diana miao james miser irvin modlin todd morgan alessandro morotti jean mulcahy levy peter m uller arno mundt katherine nathanson j.t. 
raymond , ms3 ; eduardo vilar , md , phd1 ; michael overman , md1 ; bryan kee , md1 ; cathy eng , md1 ; kanwal raghav , md1 ; and scott kopetz , md , phd1 purpose atypical , non - v600 braf ( abraf ) mutations represent a rare molecular subtype of metastatic colorectal cancer ( mcrc )  . 
preclinical data are used to categorize abraf mutations into class ii ( intermediate to high levels of kinase activity , ras independent ) and iii ( low kinase activity level , ras dependent )  . 
among patients with abraf ras wild - type mcrc who received anti - egfr antibodies ( monotherapy , n = 1 ; combination therapy , n = 10 ) , no responses to anti - egfr therapy were reported , and six patients ( four with class iii abraf mutations , one with class ii , and one unclassied ) achieved stable disease as best response . 
in the ctdna cohort , there was an increased prevalence of abraf mutations and subclonal abraf mutations ( p , .001 for both ) among predicted anti - egfr exposed compared with nonexposed patients . conclusion efcacy of anti - egfr therapy is limited in class ii and iii abraf mcrc . 
2019 by american society of clinical oncology introduction brafv600e missense mutations are present in 6% to 10% of patients with metastatic colorectal cancer ( mcrc ) .1 , 2 within the braf kinase domain , substitution of a valine to glutamic acid at position 600 manifests as constitutive activation and oncogenic signaling along the mitogenactivated protein kinase ( mapk ) pathway.1 brafv600e mcrc represents an aggressive molecular subtype of colorectal cancer inherently refractory to standard chemotherapy ; thus , tremendous research focus has been directed toward novel therapeutic development.2 because of increased use of next - generation sequencing ( ngs ) in the management of mcrc , various mutational hotspots of clinical signicance have emerged within interest , such as expanded ras testing . genes of however , with such broad testing including circulating tumor dna ( ctdna ) , alterations of evolving signicance without clear predictive , prognostic , or therapeutic implications have also been identied . 
atypical , non - v600 braf ( abraf ) mutations that occur outside of codon 600 are one example of an emerging molecular subtype in colorectal cancer . abraf mutations were retrospectively studied at two large centers comprehensively describing the clinical , pathologic , and survival implications of these mutations in patients with mcrc.3 a total of 9 , 643 patients with mcrc underwent ngs testing and 208 patients with abraf mutations were identied , representing 2.2% of all patients tested . 
 johnson et al context key objective to determine if atypical , non - v600 braf ( abraf ) mutations , stratied by class , affect outcomes with anti - egfr therapy and , secondarily , if acquisition of abraf mutations imparts resistance to egfr inhibition . knowledge generated clinical outcomes on the basis of functional class and their effect on anti - egfr efcacy for abraf metastatic colorectal cancer ( mcrc ) have not been dened . 
in patients with abraf wild - type ras mcrc who received anti - egfr antibodies , there were no responses among class ii or iii abraf mcrc , with stable disease as best response in our internal cohort . evaluation of a large external cohort of patients with circulating tumor dna interrogated by an anti - egfr exposure score revealed increased prevalence of abraf and subclonal abraf mutations among predicted anti - egfr exposed compared with nonexposed patients , suggesting an acquired mechanism of resistance to egfr inhibition . relevance anti - egfr therapy is limited in abraf mcrc , with class ii abraf mutations emerging as a negative predictive biomarker . detection of abraf mutations in ctdna may represent a novel mechanism of resistance warranting additional investigation . can be used sequentially throughout the course of the disease . although brafv600e mcrc is known to be predictive of poor response to anti - egfr therapy , the clinical utility of egfr inhibition in abraf mcrc remains unclear.4 , 5 of note , previous retrospective work investigating a cohort of 150 patients with refractory mcrc identied seven patients with abraf mutations and reported poor progression - free survival when they were exposed to anti - egfr therapy in comparison with a cohort with ras / raf wild - type ( ras / rafwt ) mcrc.6 preclinical work has highlighted unique biology among traditional brafv600e mutations ( class i ) and abraf mutations ( classes ii and iii ) .7 , 8 brafv600e mutations , designated as class i , result in feedback inhibition of gtp - bound ras , are ras independent , and signal as active monomers . 
in contrast , class ii abraf mutations have intermediate to high levels of kinase activity , are ras independent , signal as constitutive dimers , and are resistant to vemurafenib , whereas class iii abraf mutations have low or absent kinase activity and are primarily ras dependent and sensitive to erk - dependent feedback of ras.7 , 8 these tumors bind more tightly to ras - gtp than do wild - type braf tumors , and binding to wild - type craf is enhanced , resulting in increased erk signaling.8 these stark differences in underlying tumor biology may explain the varying response to therapy , specically the potential limitations to anti - egfr inhibition in abraf mcrc . 
cohort 1 included patients with stage iv colorectal cancer who were seen at mdacc between march 1 , 2012 , and june 27 , 2017 , and underwent targeted exome sequencing . 
all clinical and outcomes data were derived from this cohort . cohort 2 included patients with mcrc who underwent a targeted ngs ctdna assay ( guardant360 ; guardant health , redwood city , ca ) as part of their care between june 1 , 2014 , and december 26 , 2017 , at multiple institutions . 
 abraf mutations as a mechanism of resistance to egfr inhibition frequencies for both cohorts of abraf mutations : predicted anti - egfr exposed and nonexposed . statistical analysis clinicopathologic features were compared by 2 test or fishers exact test for categorical variables and assessment of odds ratios for associations as appropriate , as well as by t test to compare continuous variables . 
matched analysis adjusted by age and sidedness was undertaken to compare os among all molecular classes . 2 or fishers exact tests , as well as t test , were used to evaluate the adequacy of the matching . 
statistical analyses were performed using graphpad prism , version 6.07 ( graphpad software , la jolla , ca ) , and spss , version 23.0 ( ibm , armonk , ny )  . 
among 36 patients with abraf mcrc , 19 ( 53% ) had left - sided primary tumors and 24 ( 67% ) had microsatellite stable ( mss ) disease and were a median age of 56 years ( table 1 )  . 
of note , in contrast to brafv600e mcrc , which is mutually exclusive with ras mutations , 12 patients with abraf mcrc had ras mutations ( class iii , n = 7 of 21 [ 33% ] ; class ii , n = 5 of 10 [ 50% ] )  . 
among these , one patient with class ii abraf mcrc also had microsatellite instabilityhigh ( msi - h ) disease . for a more accurate comparison of survival outcomes among respective molecular classes , we performed a matched analysis by age and sidedness among the 36 patients with abraf mcrc compared with 36 patients with brafv600e mcrc and 36 with ras / rafwt disease ( fig 3 ; appendix table a1 )  . 
 ( % ) unless otherwise indicated . abbreviations : , not applicable ; abraf , atypical , non - v600 braf ; msi - h , microsatellite instabilityhigh ; mss , microsatellite stable ; wt , wild exposed to anti - egfr therapy in their treatment course . among patients with abraf raswt mcrc , 11 ( 50% ) received anti - egfr monoclonal antibodies ( mabs ; monotherapy , n = 1 , combination therapy , n = 10 ; class iii , n = 7 of 14 [ 50% ] , class ii , n = 3 of 5 [ 60% ] , unclassied , n = 1 )  . there were no responses reported , and six patients ( class iii , n = 4 ; class ii , n = 1 ; unclassied , n = 1 ) achieved stable abraf ( n = 36 ) brafv600e ( n = 221 ) wild - type braf ( n = 438 ) time ( months ) fig 2 . 
in line with previous reports , patients with class i or brafv600e mcrc had worse survival outcomes when exposed to egfr inhibition ( fig 4 )  . abraf mutations as a potential mechanism of resistance to anti - egfr therapy to understand our institutional ndings of limited efcacy of anti - egfr therapy in abraf mcrc , we analyzed an external ctdna cohort of 5 , 257 samples ( cohort 2 ) from 4 , 465 patients with mcrc . 
 abraf mutations as a mechanism of resistance to egfr inhibition abraf ( n = 36 ) brafv600e ( n = 36 ) wild - type braf ( n = 36 ) time ( months ) fig 3 . 
abraf , atypical , nonv600 braf . cohort , after excluding ras and brafv600e mutations , we applied a previously validated anti - egfr exposure score to divide patients into predicted prior exposure to anti - egfr ( n = 644 ) versus predicted nonexposed ( n = 2 , 880 ) .10 this revealed that the prevalence of abraf mutations increased from 3.99% among patients predicted to have no prior antiegfr exposure to 8.38% among those with predicted antiegfr exposure ( p , .001 ; fig 5a )  . as a sensitivity analysis , we evaluated the prevalence of abraf mutations among patients with egfr ectodomain mutations , compared with those without , because egfr ectodomain mutations are unequivocally present only after exposure to egfr inhibition . 
class ii activating g469a mutations ( p , .001 ) and class iii d594n ( p , .001 ) were the most common alterations among patients with predicted antiegfr exposure ( fig 5c )  . 
agents such as vemurafenib are not active , because the drug is designed to target the shape of the v600e - mutated protein and is more than 10 - fold less active against nonmutated forms . 
 ( a ) ctdna prevalence of abraf ( 3.99% ) among non - exposed patients ( n = 2 , 880 ) v ctdna prevalence of abraf ( 8.38% ) among predicted anti - egfr exposed ( n = 644 )  . 
schematic gure of the conserved region3 ( cr3 ) of the braf protein , also known as the braf kinase domain ( amino acids 457 to 717 ) , and the location of the class ii and iii hotspot mutations . 
the kinase domain contains the p - loop ( amino acids 464 - 471 ) , the c helix ( amino acids 492 - 504 ) , the dimerization interface ( dif , amino acids 504 - 511 ) , the catalytic loop ( cl , amino acids 574 - 581 ) , the dfg motif ( amino acids 594 - 596 ) , and the activation segment ( as , amino acids 594 - 623 )  . 
class ii , activating g469a mutations ( p , .001 ) and class iii , d594n ( p , .001 ) were the most common , respectively , among predicted anti - egfr exposed patients . 
although previous reports have suggested class iii abraf mcrc may achieve responses with antiegfr therapy owing to ras dependency , our cohort , albeit small , only revealed stable disease as best response.6 , 8 , 12 - 15 , 19 these results support the ndings of the breac study , which revealed no responses in six patients with abraf mcrc ( n = 2 each with class ii , class iii , and unclassied ) .6 of note , the nding of concomitant ras mutations with abraf mcrc in tissue , conrmed in our external ctdna cohort , clearly precludes the use of egfr inhibition for all patients , highlighting the need for novel combination strategies that consider this distinct signaling biology.20 , 21 in our internal and external cohorts , we did not nd a difference between patients with class ii and class iii abraf mcrc in regard to the presence of concurrent ras mutations . 
 abraf mutations as a mechanism of resistance to egfr inhibition in our mdacc internal cohort , four patients had unclassied disease ( y656d , a415v , k752r , f109i ) and had poor survival outcomes . 
in the external ctdna cohort , we found numerous unclassied abraf mutations that are yet to be functionally described ; most were commonly comutated with ras , pik3ca , and egfr . development of acquired ras and subclonal egfr ectodomain mutations are known mechanisms of resistance to anti - egfr therapy in raswt mcrc.22 - 26 considering the limitation of egfr inhibition noted in our internal cohort , the emergence of abraf potentially reects whether a novel mechanism of acquired resistance to egfr inhibition was investigated . 
analysis of our ctdna cohort showed statistically signicant increases in the prevalence of abraf mutations , subclonal abraf mutations , and multiple abraf mutations in individual ctdna samples among patients predicted to be exposed to egfr inhibition versus those predicted to be nonexposed . 
it is important to note that the rate of abraf mutation in our ctdna cohort was higher than in our internal cohort , as well as higher than what has been published in tissue - based testing.3 these ndings highlight the potential of abraf mutations as a novel acquired resistance mechanism to egfr inhibition and warrant additional prospective investigation . of note , in patients with egfr - mutant nonsmall - cell lung cancer , abraf mutations have been reported as a mechanism of acquired resistance to egfr tyrosine kinase inhibition.27 our results underscore the need for innovative combinatorial targeted approaches for abraf mutations exploiting class biology . 
data suggest that downstream signaling with novel inhibitors of mek or erk may be useful , because the pathway is still active.20 furthermore , because both class ii and class iii mutations signal through braf and / or craf target didimers , next - generation raf inhibitors that merization is of interest in combination with these agents as a rationale next step for therapeutic intervention.21 of note , regimen using safety lead - in data for a novel triplet encorafenib , binimetinib , and cetuximab for refractory brafv600e mcrc have been published showing impressive response rates and signicant in survival outcomes.28 subsequent studies will need to investigate whether this novel triplet combination strategy can also enhance optimal mapk inhibition and provide durable benet in patients with abraf mcrc . improvement there are limitations to this study . 
second , considering the rarity of abraf mcrc , we were only able to identify 36 patients in our internal cohort , of whom 30% received egfr inhibition therapy . third , detailed clinical annotation is not available for the external ctdna cohort , and we were unable to conrm that these patients had received prior anti - egfr therapy . therefore , we applied a previously validated and published anti - egfr exposure score to identify patients predicted to have anti - egfr exposure versus patients predicted to be nonexposed to conduct our ctdna analysis . our results suggest selection criteria for the use of antiegfr therapy in abraf - mutated mcrc should be based not only on raswt status but also the functional class of the atypical variant . 
in our cohort , although limited by sample size , ndings continue to generate more hypotheses in that class ii abraf mutations may represent a negative predictive biomarker of egfr inhibition , and exposure to anti - egfr mabs may adversely affect survival outcomes , whereas efcacy of egfr inhibition was limited in patients with class iii abraf mcrc . 
increased subclonal abraf mutations and prevalence of class ii and class iii variants in ctdna among patients with predicted anti - egfr exposure suggest a novel mechanism of resistance warranting additional prospective investigation . also , our data support the clinical utility of comprehensive genomic proling ( both tissue and ctdna ) in the management of advanced mcrc , because standard hotspot polymerase chain reaction testing would not capture emerging clinically signicant alterations . 
loree , kanwal raghav , scott kopetz provision of study material or patients : all authors collection and assembly of data : all authors data analysis and interpretation : benny johnson , alexandre a . 
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br j cancer 117 : 1450 - 1458 , 2017 yao z , torres nm , tao a , et al : braf mutants evade erk - dependent feedback by different mechanisms that determine their sensitivity to pharmacologic inhibition . 
cancer cell 28 : 370 - 383 , 2015 yao z , yaeger r , rodrik - outmezguine vs , et al : tumours with class 3 braf mutants are sensitive to the inhibition of activated ras . 
nature 548 : 234 - 238 , 2017 strickler jh , loree jm , ahronian lg , et al : genomic landscape of cell - free dna in patients with colorectal cancer . 
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parseghian cm , loree jm , morris vk , et al : anti - egfr resistant clones decay exponentially after progression : implications for anti - egfr re - challenge . 
cremolini c , di bartolomeo m , amatu a , et al : braf codons 594 and 596 mutations identify a new molecular subtype of metastatic colorectal cancer at favorable prognosis . 
sullivan rj , infante jr , janku f , et al : first - in - class erk1 / 2 inhibitor ulixertinib ( bvd - 523 ) in patients with mapk mutant advanced solid tumors : results of a phase i dose - escalation and expansion study . 
rankin a , klempner sj , erlich r , et al : broad detection of alterations predicted to confer lack of benet from egfr antibodies or sensitivity to targeted therapy in 19 . 
schirripa m , biason p , cortiula f , et al : clinico - pathological and molecular characterization of braf mutant metastatic colorectal cancer ( mcrc ) : are all advanced colorectal cancer . 
yu ha , suzawa k , jordan e , et al : concurrent alterations in egfr - mutant lung cancers associated with resistance to egfr kinase inhibitors and characterization of mtor as a mediator of resistance . 
distribution of abraf ( atypical , non - v600 braf ) mutations in cohort 1 ( n = 36 )  . abraf mutation class appendix johnson et al table a1 . 
faden , md1 , 2 ; roberto gomez - casal , ms3 ; diego alvarado , phd4 ; and umamaheswar duvvuri , md , phd3 introduction the epidermal growth factor receptor / human epidermal growth factor receptor ( her ) family plays a critical role in the growth of numerous human cancers . multiple therapeutics targeted to members of the her family have been developed , including the epidermal growth factor receptortargeting monoclonal antibody cetuximab , which is fda approved for treatment of head and neck squamous cell carcinoma ( hnscc )  . her3 / erbb3 is expressed in most solid tumors , including hnscc , and has been shown to correlate with a poor prognosis.1 , 2 phosphorylation of erbb3 ( perbb3 ) affects numerous pathways , including activation of the pi3k / akt pathway , driving cell survival and growth . 
for this reason , blocking erbb3 activity could be an effective strategy to decrease tumor growth and resistance to other tyrosine kinase inhibitors.3 - 5 cdx - 3379 is a human immunoglobulin g1 lambda monoclonal antibody that binds erbb3 and inhibits its activity by multiple mechanisms . 
after pathologic conrmation by biopsy , informed consent was obtained , the patient was enrolled in nct02473731 and given two 1 , 000 mg intravenous doses of cdx - 3379 . 
a signiresponse was noted within the rst cant clinical 48 hours after administration of the rst dose , with concomitant reduction in pain from 8 of 10 to 2 of 10 . on examination before surgical extirpation , the primary tumor was found to have a nearly complete response , demonstrating a 92% reduction in greatest dimension . 
tumor and germline dna underwent whole exome sequencing ( wes ) library construction with the illumina truseq kit ( san diego , ca ) , with exonic regions captured using agilent sureselect v4 ( santa clara , ca )  . 
rna underwent library preparation with the illumina truseq rna access kit followed by pairedend ( 2 76 ) sequencing on an illumina nextseq 500 , with average coverage of 45 million reads per sample . primary processing of sequence data was performed using illumina casava software v1.8. 
somatic mutations and copy number variants ( cnvs ) were identied by variantdx as previously described.10 all nonsynonymous coding variants , splice site variants , indels , and cnvs above or below 2.0 were considered potentially deleterious . 
after adapter trimming with cutadapt v1.5 , reads were mapped to the human reference genome grch37 / 38 using hisat2 v2.0.4 , and gene expression was quantitated using htseq v0.9. 
differential gene expression was performed using edger v3.20.9. quantitative real - time polymerase chain reaction total rna was isolated from human hnscc cell lines ( te6 , kyse510 , te9 , fadu , pecapj34 , cal33 , scc4 , bhy , osc19 , scc25 ) using the qiagen rneasy kit . 
 faden et al real - time polymerase chain reaction ( pcr ) was performed using the bio - rad iq sybr green supermix and applied biosystems 7900ht fast real - time pcr systethe following primers were used to detect fzd3 mrna : 5 ( cid : 2 ) - gcttccacagtgacacaagg - 3 ( cid : 2 ) and 5 ( cid : 2 ) - accatacactgccagccata - 3 ( cid : 2 )  . to detect erbb3 mrna , the following primers were used : 5 ( cid : 2 ) - actctccatatcccttcctctc - 3 ( cid : 2 ) and 5 ( cid : 2 ) - gtttcctccctttcctctct - 3 ( cid : 2 )  . pcr amplications were run in triplicate for each cell line . comparative quantication of gene expression was performed using the ct method , and values were referenced to gapdh mrna expression . erbb3 phosphorylation erbb3 phosphorylation was measured by veratag assay ( san francisco , ca ) on ffpe tissue.6 colony - formation assays five thousand cells per well were seeded in six - well plates and allowed to attach overnight . 
the area covered by stained cells was measured using imagej . statistical analysis was performed using graphpad prism 7 . perbb3 was assessed by veratag assay to examine changes in activated erbb3 pretreatment to post - treatment , revealing a 69% decrease from 0.45 to less than 0.14 ( lower limit of quantitation of the assay ) , demonstrating successful blockage of erbb3 activation by cdx - 3379 . 
we hypothesized that loss of somatic alterations from pretreatment to post - treatment , in combination with a signicant reduction in clinical tumor volume , could represent selective killing of clones more sensitive to cdx - 3379 and thus give insight terrogating the list of alterations that were present in the tumor pretreatment but not post - treatment ( on the basis of known mechanisms of action of each gene ) identied amplication of fzd3 ( frizzled class receptor 3 ) , a receptor in the wnt signaling pathway , as a possible candidate conferring sensitivity to cdx - 3379 ( table 1 )  . 
rna - seq demonstrated a 2.5 - foldhigher expression of fzd3 pretreatment versus post - treatment ( p , 4.5e - 13 ) , consistent with the 3.0 amplication identied on wes in the pre ( fig 1a )  . 
six percent ( 28 of 496 ) of hnsccs in tcga possess an fzd3 alteration that could result in an increase in downstream wnt signaling , consistent with the case reported here ( one amplication and 27 mrna upregulations )  . 
patients with hnsccs from tcga that possess an fzd3 alteration have poorer overall survival , suggesting that alterations in this gene may be important in the biology of a subset of hnsccs ( fig 1b )  . tumor to explore the possibility that fzd3 plays a role in sensitivity to erbb3 inhibition , we examined fzd3 mrna expression lines , by quantitative real - time pcr in 10 hnscc cell nding high variability among cell lines ( fig 1c )  . 
to test the hypothesis that increased fzd3 expression could render cells more sensitive to cdx - 3379 , akin to the pretreatment tumor , we performed colony - formation assays with table 1 . 
the role of wnt signaling pathways in cancer is well established.11 fzd upregulation has been identied in numerous cancers , correlates with poor outcomes , and is an active area of investigation for targeted therapeutics.11 , 12 fzd3 , in particular , is believed to play a role in both the canonical and noncanonical wnt pathways and has been found to be upregulated in lung , esophageal , and colon cancers.13 - 15 here , we report fzd3 gene amplication and overexpression in pretreatment but not post - treatment tumor from an exceptional responder to cdx - 3379 . 
review of fzd3 in hnsccs from tcga supports the potential biologic importance of alterations in this gene . we further demonstrate that fzd3 expression levels correlate with sensitivity to cdx - 3379 and that activation of fzd3 by its ligand wnt3a further potentiates the effects of cdx - 3379 . 
the mechanism by which fzd3 confers sensitivity to erbb3 inhibition is unclear ; however , the wnt and erbb pathways have been reported to closely interact and collude in tumorigenesis.16 , 17 it remains to be seen whether this is by convergence on a common downstream molecule , such as - catenin , 18 or via effects on different target genes . 
in breast cancer , wnt overexpression activates signaling via erbb.19 , 20 interestingly , we identied a tight correlation between erbb3 and fzd3 mrna expression levels in hnscc cell lines , suggesting a similar relationship and highlighting the interconnected nature of these singling pathways . 
images of a representative colony formation ( top ) with quantication of stained area represented in bar graph ( middle ) and culture additives ( bottom ) p , .001 for all comparisons by one - way analysis of variance . 
semin cell dev biol 21 : 944 - 950 , 2010 takikita m , xie r , chung jy , et al : membranous expression of her3 is associated with a decreased survival in head and neck squamous cell carcinoma . 
j transl med 9 : 126 , 2011 lee - hoeich st , crocker l , yao e , et al : a central role for her3 in her2 - amplied breast cancer : implications for targeted therapy . 
cancer res 68 : 5878 - 5887 , 2008 sergina nv , rausch m , wang d , et al : escape from her - family tyrosine kinase inhibitor therapy by the kinase - inactive her3 . 
nature 445 : 437 - 441 , 2007 brand tm , hartmann s , bhola ne , et al : cross - talk signaling between her3 and hpv16 e6 and e7 mediates resistance to pi3k inhibitors in head and neck cancer . 
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j clin oncol 34 , 2016 ( suppl ; abstr 2501 ) duvvuri u , george j , kim s , et al : molecular and clinical activity of cdx - 3379 , an anti - erbb3 monoclonal antibody , in head and neck squamous cell carcinoma : a preoperative window of opportunity study . 
schroeder ja , adriance mc , mcconnell ej , et al : erbb - beta - catenin complexes are associated with human inltrating ductal breast and murine mammary tumor virus ( mmtv ) - wnt - 1 and mmtv - c - neu transgenic carcinomas . 
faivre ej , lange ca : progesterone receptors upregulate wnt - 1 to induce epidermal growth factor receptor transactivation and c - src - dependent sustained activation of erk1 / 2 mitogen - activated protein kinase in breast cancer cells . 
these preclinical observations suggest a role for the targeted inhibitors of the bcr pathway in the treatment of dlbcl.1 dlbcl is most commonly present as malignant inltration of lymph nodes . 
the use of btk inhibitors is now incorporated into standard therapy for these conditions , and the genetic basis of acquired resistance in cll , dominated by acquired mutation of btk or plcg2 , has been well - studied.6 , 7 by contrast , the role of bcr inhibition in dlbcl remains less clear and little is known about the genetics of acquired resistance . a 75 - year - old male presented with rapidly enlarging cutaneous nodules . 
clinical examination revealed cutaneous nodules up to 25 mm in diameter affecting the feet , legs , arms , and abdomen . a repeat biopsy showed features identical to his original diagnostic biopsy ( fig 1a ) , and computed tomography scan conrmed exclusively cutaneous disease . 
the patient was managed with palliative radiotherapy . the rapid clinical response and prolonged remission , followed by later re - emergence of tumor , suggested the acquisition of new genetic alterations driving resistance to bcr inhibition . 
this study was approved by the east of england cambridge south research ethics committee ( approval reference number 07 / mre05 / 44 )  . full variant and copy number data are presented in the data supplement . 
 ( a ) histology and immunohistochemistry from tumor biopsy showing a diffuse inltration by large atypical cells with predominantly centroblast - like morphology expressing cd20 , mum1 , bcl2 , weak bcl6 , no cd10 , and mib1 proliferation fraction 90% . 
 ( b ) images of selected lesions on the forearm , hand , popliteal fossa , and abdomen taken prior to therapy ( top row ) , at 28 days ( middle row ) , and at 6 months ( bottom row ) of b cell receptortargeted therapy . 
 ( a ) summary of whole - exome sequencing of samples taken from three different timepoints ; diagnosis , prior to bcr - targeted therapy , and at the time of relapse . 
gray indicates no mutation or copy number change identied . ( b ) a simplied schematic showing the critical signaling components of the bcr pathway that converge onto activation of canonical nf - b . components targeted pharmacologically in this patient are indicated . 
we manually examined the loci of plcg2 and btk , mutations that are commonly acquired in btki - resistant cll , and conrmed both genes to be wild type at all timepoints . 
 case report in the bcr pathway to activate canonical nf - b signaling.1 activating mutation in the coiled - coil domain of card11 was previously proposed as a mechanism of resistance to bcr inhibition in mantle cell lymphoma8 and primary resistance in dlbcl.9 the acquired k215t mutation in our patient is located in the coiled - coil domain and has been previously shown to activate nf - b.10 we conrmed the near - clonal presence of the card11 k215t mutation at relapse ( variable allele frequency 40% ) but zero mutant reads at this position in either of the two pretreatment biopsies ( read depth , 180 and 210 , appendix fig a3 )  . 
taken together , these data strongly suggest that card11 mutation was the genetic driver of the acquired resistance in this case . the limited information about acquired resistance to bcr inhibition in dlbcl suggests potential differences compared with that of cll . 
the latter is predominantly associated with mutation of btk and plcg2.6 , 7 card11 identied in 10% - 15% of dlbcl at mutation is presentation11 but is rarely identied ( , 1% ) in cll.12 this suggests that the bcr signal in cll and dlbcl may be qualitatively different and therefore that genetic mechanisms of resistance to bcr inhibition may also differ . however , establishing the genetic basis of resistance to bcr inhibition in dlbcl presents specic challenges that contrast with the situation in cll . 
first , patients with dlbcl receiving bcr inhibitors are typically treated simultaneously with multiagent immunochemotherapy regimens , making it hard to establish which mutations provide resistance to which agent , or indeed if sensitivity to the bcr inhibitor ever existed in the rst place . 
combined with the biopsy - accessible cutaneous location of disease , this provided a rare opportunity to study genetic mechanisms of resistance to bcr inhibition in abc dlbcl . indeed , we have found only one other case describing the genetic basis of acquired resistance to bcr inhibition in dlbcl . 
this also involved a case of pcdlbcl - lt and also reported acquisition of card11 mutation ( interesting also k215 mutant ) in a patient who relapsed following an initial response to a btk inhibitor.13 however , the concurrent nding of nfkbie mutation and igh - irf8 translocation reported in that study might also have contributed to resistance , leaving the role of the card11 mutation uncertain . our ndings of acquired activating card11 mutation , the absence of any likely alternative genetic explanation , and the demonstrated ability of the k215t mutation to affect btki resistance in vitro strongly suggest that card11 mutation is the dominant driver of bcr independence in our case . as we move toward the introduction of precision medicine in dlbcl and the real - time monitoring of clonal evolution , it will become increasingly important to understand genetic mechanisms of drug resistance . 
our study uses the unique features of this case of cutaneous dlbcl to highlight the importance of card11 mutation as a driver of acquired resistance to bcr inhibition in dlbcl . 
hodson provision of study materials or patients : nimish shah collection and assembly of data : rebecca caeser , ieuan walker , jie gao , nimish shah , livia rasso - barnett , jose - ezequiel martin , daniel j . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . rebecca caeser consulting or advisory role : karus therapeutics nimish shah consulting or advisory role : abbvie speakers bureau : roche , janssen travel , accommodations , expenses : abbvie livia rasso - barnett leadership : cambridge pathology stock and other ownership interests : cambridge pathology honoraria : cambridge pathology consulting or advisory role : cambridge pathology daniel j . 
semin hematol 52 : 77 - 85 , 2015 swerdlow sh , campo e , pileri sa , et al : the 2016 revision of the world health organization classication of lymphoid neoplasms . 
blood 127 : 2375 - 2390 , 2016 grange f , beylot - barry m , courville p , et al : primary cutaneous diffuse large b - cell lymphoma , leg type : clinicopathologic features and prognostic analysis in 60 cases . 
mareschal s , pham - ledard a , viailly pj , et al : identication of somatic mutations in primary cutaneous diffuse large b - cell lymphoma , leg type by massive parallel sequencing . 
j invest dermatol 137 : 1984 - 1994 , 2017 pham - ledard a , prochazkova - carlotti m , andrique l , et al : multiple genetic alterations in primary cutaneous large b - cell lymphoma , leg type support a common lymphomagenesis with activated b - cell - like diffuse large b - cell lymphoma . 
j clin oncol 35 : 1437 - 1443 , 2017 ahn ie , underbayev c , albitar a , et al : clonal evolution leading to ibrutinib resistance in chronic lymphocytic leukemia . 
fox lc , yannakou ck , ryland g , et al : molecular mechanisms of disease progression in primary cutaneous diffuse large b - cell lymphoma , leg type during ibrutinib therapy . 
nagel d , spranger s , vincendeau m , et al : pharmacologic inhibition of malt1 protease by phenothiazines as a therapeutic approach for the treatment of aggressive abc - dlbcl . 
 case report chr1 chr3 chr5 chr11 chr13 chr15 chr17 chr19 chr21 chrx prdm1 tnfaip3 chr7 cdkn2a chr9 rb1 gna13 chr2 chr4 chr6 chr8 chr10 chr12 chr14 chr16 chr18 chr20 chr22 chry mir17 - 92 bcl2 malt1 tcf4 cd79a spib mir17 - 92 bcl2 malt1 tcf4 cd79a spib chr1 chr3 chr5 chr7 chr9 chr11 chr15 chr17 chr19 chr21 chrx chr2 chr4 chr6 chr8 chr10 chr12 chr14 chr16 chr18 chr20 chr22 chry prdm1 tnfaip3 rb1 chr13 gna13 appendix relapse pre - treatment diagnosis chr1 chr3 chr5 chr7 chr9 chr11 chr13 chr15 chr17 chr19 chr21 chrx chr2 chr4 chr6 chr8 chr10 chr12 chr14 chr16 chr18 chr20 chr22 chry fig a1 . 
 role of community - based genomics programs see accompanying article doi : increasingly , genomic changes in patients tumors are used to inform individualized management , although the optimal approach and impact of tumor profiling on cancer care , especially in the community setting , remain important research questions.1 , 2 challenges in applying precision medicine approaches include cost and access to genomic testing and to indicated therapies , lack of actionable gene alterations , education of all stakeholders , patient expectations , complexity of interpretation of data , access to big data solutions , and expansion of physicians comfort zones . 
arguably , the latter represents the most formidable challenge . in the article accompanying this editorial , burkard et al3 address many of these challenges in their report , implementation and clinical utility of an integrated academic - community regional molecular tumor board . 
the implementation of a novel wisconsin state - wide model , including a precision - medicine molecular tumor board ( pmmtb ) , an institutional review boardapproved registry protocol , and a journal club , allows for addressing many of the challenges of precision medicine . 
the authors point out that in the united states , much of precision medicine is implemented in an academic - center context , often in conjunction with robust clinical trial platforms , to include big data analytic informatics solutions . conversely , most patients are cared for outside of such academic systems.4 the challenge is to provide patients access , through their community - based providers , to the potential benefits of precision medicine . 
the described model is an academic and community partnership that allows for nextgeneration sequencing ( ngs ) through any clinical laboratory improvement amendmentscertified laboratory , and uses as its cornerstone a twiceweekly web - based pmmtb teleconference , provided as a free service . 
as with most molecular tumor boards ( mtbs ) , the focus is on prioritizing labeled and off - label therapies , as well as clinical trial options , on the basis of biologic profiling of the patients tumor . 
the model addresses the increasingly dynamic nature of the molecular pathology report with its interpretation of a specific genotype and its clinical importance often becoming outdated in the short term . the presence of an institutional review board approved registration trial assists in observing the clinical course of patients to include relevant outcomes . 
patients may have molecular profiling performed early in their clinical course ; thus , long - term follow - up allows for evaluating the downstream impact of the original molecular profiling or that of any subsequent repeat molecular profiling . this type registry is especially important given the uncertainties regarding the value proposition of precision medicine in daily practice . 
the construct of this tripartite model seeks to address the challenges of precision medicine , though the results reported by burkard et al3 emphasize the remaining barriers to successful implementation of precision medicine platforms in community settings and beyond . over approximately 1 year , a modest number of patients with metastatic / incurable cancer ( n 5 74 ) were presented at the pmmtb ; only 38 evaluable patients were enrolled in the registration trial , and some of these were enrolled retrospectively . 
 actionable target ( ie , molecularly targeted clinical trial and / or off - trial treatment ) was identified in 32 patients , with treatment accepted in only nine patients ( 28% )  . 
only one patient was enrolled in an instate clinical trial , although a total of 14 patients had clinical trial options within the state , and no patients were enrolled in out - of - state clinical trials . 
during much of the reported period in the registry trial , the national cancer institute molecular analysis for therapy choice ( match ) basket trial was on hold , and there was no access to the asco targeted agent and profiling utilization registry ( tapur ) basket trial . 
clinical benefit was seen in three of eight ( 38% ) evaluable patients , with a response evaluation criteria in solid tumors ( recist ) - defined response in one patient . this novel precision - medicine model aims at networking academic and community providers to deliver genomically driven care to patients closest to where they live and under the care of their community physicians . 
what role does cost play with regard to accessing ngs testing and targeted drugs ? given the wide range of ngs testing platforms , how is quality assured ? this is especially important because clinical laboratory improvement amendments certification does not address many quality dimensions inherent to ngs testing . in this report , the mean number of prior therapies for patients was two . 
when is the most effective time to perform ngs testing during a patients clinical course : earlier or later ? similarly , a range of ngs panel sizes was used , with a not - surprising suggestion that larger comprehensive panels are more frequently associated with treatment options . where does cost effectiveness guide us in selecting ngs panel size ? with regard to actionable gene alterations , it is remarkable that by recent analysis , the us food and drug administration has only approved 22 gene alterations with associated biomarkers.7 , 8 the key to defining labeled indications for targeted therapies is participation in clinical trials such as match and tapur . additional clinical trial approaches include prospective registration - type trials that contain detailed data elements , including demographic data , clinical outcomes measures , and quality - of - life and economic metrics that allow for long - term follow - up and planned statistical analyses . one aspect not referenced in the burkard et al3 report is the underlying informatics platforthe scalability of such a platform , as well as its interfacing capabilities , along with its analytic functions , are key , especially across a multisite network . 
in particular , a clinical - trials matching function that allows for prioritizing variables such as geographic distance and relevance to an individual patients unique characteristics , and those of their tumor , will facilitate identification of the most promising clinical trials for a given patient . 
in addition , engaging busy clinicians is always a challenge , and one presumed solution is to have approaches and platforms that align with their daily work flow , including integration into existing informatics platforms , most especially the electronic medical record , along with automatic extraction of data elements from existing electronic databases , which limits the need for form completion , whether electronic or manual . 
for example , maximal use of high quality databases , such as tumor registries or the seer program database , can help overcome the challenges of relying on semistructured data within the electronic medical record . the demonstration of the value proposition for precision medicine is a work in progress . 
it will require clinical trial experience with genomically driven therapies across a broad population of patients with varied tumor types , cared for by a range of providers , and in varied settings , to include the broad variety of community practices . 
the newer formulation of clinical trial design , whether the basket or umbrella type design or that of sophisticated registry trials , such as those described by burkard et al , will substantially facilitate the access to targeted therapy trials in the community setting.9 , 10 these trials provide guidance for community physicians in identifying gene alterations or biomarkers with appropriate targeted therapies and provide access to off - label targeted therapies . 
brown td , tittel pd , gold pj , et al : impact of a personalized medicine research program ( pmrp ) , using targeted tumor profiling and a cloud based clinical trials matching platform , on clinical decision - making . 
 effects of molecular heterogeneity on survival of patients with brafv600 - mutated melanoma treated with vemurafenib with or without cobimetinib in the cobrim study purpose the treatment of advanced brafv600 - mutated melanomas with braf inhibitors ( brafi ) has improved survival , but the efficacy of brafi varies among individuals and the development of acquired resistance to brafi through reactivation of mitogen activated protein kinase ( mapk ) signaling is common . 
we performed an exploratory , retrospective analysis to investigate the effects of brafv600 allelic balance , coexisting oncogene mutations , cell proliferation signaling levels , and loss of pten expression on progression - free survival ( pfs ) in patients in the phase iii cobrim study , which compared the combination of the mek inhibitor cobimetinib with the brafi vemurafenib versus vemurafenib as monotherapy . methods baseline tumor samples from the intention - to - treat population were analyzed by targeted deep sequencing at a median coverage of 3 , 600 and by immunohistochemistry for cell proliferation markers , brafv600e , and pten . 
gene expression in relation to loss of pten was profiled by rna sequencing in 205 patient samples and 42 brafv600 - mutated melanoma cell lines . results neither brafv600 allelic balance nor coexisting mutations in the ras / raf / rtk pathway affected pfs in either treatment group . 
loss of function of the tumor suppressor pten ( via both genetic alterations and other mechanisms such as epigenetic silencing ) occurs in > 20% of patients with brafv600 - mutated melanoma.15 - 17 loss of functional pten has been associated with a shorter pfs response to braf inhibition , 17 , 18 reduced overall survival , and a shorter time to the development of brain metastases.15 , 19 pten / akt signaling is known to interact with mapk signaling . 
for example , meki have been shown to enhance epidermal growth factorinduced akt activation , and mutations that activate pi3k / akt signaling reduce mapk signaling.20 to understand the impact of baseline tumor heterogeneity on the efficacy of treating advanced melanoma with meki and brafi , we systematically examined the effect of braf allelic balance , coexisting oncogene mutations , and cell proliferation signaling levels on pfs in all evaluable cobrim study participants . 
here , we report the results of this retrospective , exploratory analysis . methods analysis population samples for biomarker analysis were collected from patients with unresectable locally advanced or metastatic brafv600 - mutated melanoma in the cobrim study of cobimetinib combined with vemurafenib versus vemurafenib monotherapy , 6 with the aim of exploring intrinsic mechanisms of resistance to mek and braf inhibition . 
brafv600 allelic balance , coexisting oncogene mutations , cell proliferation biomarkers , and loss of pten expression were analyzed in samples from the cobrim intention - to - treat ( itt ) population . 
most analyses could not be performed on the complete itt population ( n = 495 ) because of insufficient tissue availability or assay failure . genetic marker analysis archival formalin - fixed paraffin - embedded ( ffpe ) melanoma tumor samples , biopsy specimens of accessible tumor lesions , and blood samples were obtained from consenting patients at study entry ( baseline )  . 
rna was extracted using the high pure rna paraffin isolation kit ( roche diagnostics , indianapolis , in )  . from the 495 patients in the cobrim itt population , 400 pretreatment tumor samples were evaluable by targeted next - generation sequencing . 
 ( a ) distribution of variant allele frequency ( vaf ; ratio of mutant to wild type [ wt ] ) in brafv600 in 400 baseline tumor samples from cobrim study patients . 
 ( b ) impact on progression - free survival ( pfs ) indicated by kaplan - meier plots of pfs in patients grouped by vaf above or below the median for each treatment ar ( c ) immunohistochemistry of brafv600e protein levels in tumor samples with a range of vafs ; increased vaf was associated with greater staining intensity . 
cobi , cobimetinib ; pbo , placebo ; vem , vemurafenib . mutations ( appendix table a1 ) was performed for a focused panel of 17 oncogenes ( suraseq targeted next - generation sequencing assay ; asuragen , austin , tx ) 21 at a median coverage of 3 , 600 . 
different cutoff points were explored earlier in the analysis and gave results similar to those shown here using the median vaf as the high and low cutoff ( appendix fig a1 )  . 
the median was chosen to be representative of the overall range of cutoff points . analysis of pten copy number and methylation status in 42 brafv600 - mutant melanoma cell lines was performed as described by haverty et al.22 analysis of methylation status was performed in bisulfite - treated genomic dna using the illumina 450k array ( infinium humanmethylation450 beadchip kit ; illumina , san diego , ca )  . 
methylation data were analyzed using the methyanalysis package for r.23 gene expression in relation to loss of pten was profiled by rna sequencing in 205 patient samples and in 42 braf v600 - mutant melanoma cell lines . 
rna was prepared for high throug hput sequencing with the truseq rna access library prep kit ( illumina ) , and 40 million 2 100 - bp reads were sequenced per sample using illumina hiseq . 
coexisting oncogene mutations and impact on progression - free survival ( pfs ) in patients treated with cobimetinib ( cobi ) combined with vemurafenib ( vem ) or vem monotherapy . 
 ( b ) pfs in patients with or without ras / raf / rtk mutations ( both treatment arms were combined to give sufficient numbers of patients with ras / raf / rtk mutations for statistical analysis )  . 
 ( c ) patients with coexisting ras / raf / rtk mutations had elevated levels of phosphorylated extracellular regulated kinase ( perk ) , measured by immunohistochemistry , compared with patients with wild - type ( wt ) ras / raf / rtk , indicating greater mitogen - activated protein kinase signaling pathway activity . 
 defined by the molecular signatures database emt hallmark collection , 24 was compared by z - score ( sum of log2 reads per kilobase of transcript per million mapped reads for all genes on the emt hallmark collection ) between samples with functional and nonfunctional pten . and immunohistochemical markers and a pfs data cutoff date of january 16 , 2015 . 
 expression levels were quantified by histo - score ( h - score ) , and samples were grouped as high or low , depending on whether the h - score was above or below the median , respectively . 
in a subset of samples , brafv600e expression was also evaluated by ihc ( antibrafv600e [ ve1 ] mouse monoclonal primary antibody ; ventana [ n = 39 ] )  . in vitro studies the effect of pten loss on drug sensitivity was evaluated in 42 brafv600 - mutant melanoma cell lines by analyzing cell viability after vemurafenib treatment . 
 in all samples , > 95% of tumor cells stained positive for brafv600e mutation , suggesting that differences in vaf reflect cells across the tumor rather than indicate a subclonal mutation , and that increased vaf was associated with greater staining intensity ( fig 1c )  . 
similar results were seen in a set of four melanoma cell lines ( appendix fig a2 )  . association between oncogene mutations and pfs to explore potential drivers of brafi resistance affecting survival , in addition to previously identified mechanisms such as mek mutation , 27 coexisting mutations in a panel of 17 oncogenes21 ( appendix table a1 ) were assayed in baseline braf - mutated tumor tissue from 400 of 495 patients in the cobrim itt population and were identified in 43 patients ( 11% ; fig 2a )  . 
h - score , histo - score ; mut , mutation ; pbo , placebo ; pd , progressive disease ; pr , partial response ; sd , stable disease . 
cell proliferation biomarkers and impact on progression - free survival ( pfs ) in patients treated with cobimetinib ( cobi ) combined with vemurafenib ( vem ) or vem monotherapy . 
 ( a ) kaplan - meier plots of pfs in patients grouped by high ( above the population median histo - score [ h - score ] of 40 ) or low ( below the population median ) phosphorylated extracellular regulated kinase ( perk ) levels . 
 ( b ) kaplan - meier plots of pfs in patients grouped by high or low baseline proliferation index , measured by the protein encoded by themki67gene ( ki - 67 ) immunohistochemistry ( population median ki - 67 level is 20% ) or ( c ) phosphorylated s6 immunohistochemistry ( ps6 ; population median h - score is 71 )  . 
 ( c ) rna sequencing profiling of melanoma samples from cobrim comparing the epithelial - mesenchymal transition ( emt ) gene expression signatures in tumors with pten loss versus tumors with normal pten levels . 
patient characteristics were similar between patients with and without loss of pten expression in the cobimetinib combined with vemurafenib and the vemurafenib monotherapy cohorts , with the exception that more patients with loss of pten expression had elevated lactate dehydrogenase in the cobimetinib combined with vemurafenib arm ( appendix table a3 )  . 
 ( b ) rna sequencing profiling of braf - mutant melanoma cell lines with or without pten loss comparing the epithelial - mesenchymal transition ( emt ) gene expression signatures . 
 loss of pten expression was associated with upregulation of the emt gene expression signature in these melanoma cell lines ( fig 5b ) , similar to that seen in the baseline tumor samples . 
when tested in an in vitro wound healing assay , in the absence of vemurafenib or cobimetinib , cell lines with loss of pten showed a higher capacity for migration ( fig 5c ) , consistent with observed clinical manifestations and previous preclinical findings.28 , 29 attempts to analyze whether vemurafenib or cobimetinib combined with vemurafenib affected the increased cell migration behavior associated with loss of pten were confounded by their effect on cell viability . discussion to our knowledge , this retrospective biomarker analysis of brafv600 - mutated melanoma samples from the cobrim study is the largest such study to date . 
although there was an association between signaling pathway activity and pfs in patients treated with vemurafenib monotherapy , increased mapk signaling and cell proliferation did not affect pfs in patients treated with a combination of cobimetinib and vemurafenib . 
 the combination of mek and braf inhibition thus seems to override the poor prognostic effect of increased mapk pathway activation . loss of pten expression is linked to reduced survival in advanced melanoma , most likely as a result of suppression of apoptosis and enhanced cell survival mediated by hyperactivation of pi3k / akt signaling.17 , 18 our analysis suggests that combined mek and braf inhibition can overcome the effect of loss of pten on pfs . 
 loss of pten function in addition to brafv600 mutation has been linked to the rapid development of brain metastases in advanced melanoma , which may also contribute to reduced pfs.15 , 26 our analysis confirms that brafv600 - mutated melanoma cells with loss of pten function have a higher expression of genes associated with emt , as well as enhanced migration . 
functional pten inhibits cell migration through dephosphorylation of focal adhesion kinase ( fak ) ; loss of pten relieves this inhibition.29 in prostate cancer models , ras / mapk activation cooperated with pten loss to promote emt and metastasis , and mek inhibition was able to reduce metastatic progression in these models.30 mek can also activate fak to promote cell migration , and mek inhibition could therefore downregulate fak - mediated migration , 31 addition to reducing the cell survival resulting from ras / mapk activation in brafv600 - mutated melanoma . 
this suggests a potential mechanism by which cobimetinib combined with vemurafenib could contribute to a survival benefit in patients with braf v600 - mutated melanoma and loss of pten . the large patient cohort available for analysis was a significant strength of this study ; however , the analysis was limited by the inability to evaluate baseline tumor samples for approximately 20% of patients included in the cobrim study . additional investigation of the mechanisms underlying intrinsic and acquired resistance is warranted . 
 the molecular mechanisms at work in patients with prolonged responses to combination therapy with cobimetinib and vemurafenib are still under investigation but may involve regulation of the host antitumour immune response.33 the overall findings of this study suggest that deeper inhibition of the mapk pathway through combined inhibition of both mek and braf overrides the effects of molecular heterogeneity to provide a survival benefit to all patients . 
 this both supports and provides a rationale for the greater efficacy of combination therapy with braf and meki , which has become the standard of care , compared with braf inhibition alone . 
ascierto , brigitte drno , anna maria di giacomo , claus garbe , ilsung chang , jessie hsu , hartmut koeppen , james larkin , yibing yan , grant a . 
wongchenko employment : genentech / roche stock and other ownership interests : genentech / roche , ariad pharmaceuticals antoni ribas stock and other ownership interests : compugen , flx bio , cytomx therapeutics , five prime therapeutics , advaxis , arcus biosciences , tango therapeutics , pact pharma consulting or advisory role : merck , amgen , genentech / roche , novartis paolo a . 
 james larkin honoraria : eisai , bristol - myers squibb , msd , glaxosmithkline , pfizer , novartis , genentech / roche , sectra , pierre fabre , eusa pharma , consulting or advisory role : eisai , bristol - myers squibb , msd , glaxosmithkline , pfizer , novartis , genentech / roche , sectra , pierre fabre , eusa pharma research funding : pfizer ( inst ) , novartis ( inst ) , msd ( inst ) , bristol - myers squibb ( inst ) travel , accommodations , expenses : bristol - myers squibb , pfizer , novartis , genentech / roche yibing yan employment : genentech / roche stock and other ownership interests : genentech / roche patents , royalties , other intellectual property : genentech / roche grant a . 
wongchenko , ilsung chang , jessie hsu , isabelle rooney , william lu , hartmut koeppen , and yibing yan , genentech , south san francisco ; antoni ribas , jonsson comprehensive cancer center at the university of california , los angeles , los angeles , ca ; paolo a . 
pascale , naples ; anna maria di giacomo , azienda ospedaliera universitaria senese , siena , italy ; brigitte drno , nantes university , nantes , france ; claus garbe , university of tbingen , tbingen , germany ; james larkin , the royal marsden hospital , london , united kingdom ; grant a . 
ascierto pa , mcarthur ga , drno b , et al : cobimetinib combined with vemurafenib in advanced braf ( v600 ) - mutant melanoma ( cobrim ) : updated efficacy results from a randomised , doubleblind , phase 3 trial . 
hauschild a , grob j - j , demidov lv , et al : dabrafenib in braf - mutated metastatic melanoma : a multicentre , open - label , phase 3 randomised controlled trial . 
wilmott js , menzies am , haydu le , et al : braf ( v600e ) protein expression and outcome from braf inhibitor treatment in braf ( v600e ) metastatic melanoma . 
wagle n , van allen em , treacy dj , et al : map kinase pathway alterations in braf - mutant melanoma patients with acquired resistance to combined raf / mek inhibition . 
bucheit ad , chen g , siroy a , et al : complete loss of pten protein expression correlates with shorter time to brain metastasis and survival in stage iiib / c melanoma patients with brafv600 mutations . 
xing f , persaud y , pratilas ca , et al : concurrent loss of the pten and rb1 tumor suppressors attenuates raf dependence in melanomas harboring ( v600e ) braf . 
nathanson kl , martin a - m , wubbenhorst b , et al : tumor genetic analyses of patients with metastatic melanoma treated with the braf inhibitor dabrafenib ( gsk2118436 )  . 
latham g , hough j , sah s , et al : suraseq targeted ngs assays : integrated , cross - platform reagents that enable accurate detection of cancer gene mutations in residual clinical ffpe , fna , and biofluid biopsies . 
mcarthur ga , larkin j , ascierto pa , et al : impact of baseline allelic frequencies of brafv600 mutation and coexisting oncogenic mutations on progression - free survival from the cobrim phase 3 study evaluating cobimetinib + vemurafenib in advanced brafv600 - mutated melanoma . 
larkin jmg , yan y , mcarthur ga , et al : update of progression - free survival ( pfs ) and correlative biomarker analysis from cobrim : phase iii study of cobimetinib ( cobi ) plus vemurafenib ( vem ) in advanced braf - mutated melanoma . 
tamura m , gu j , takino t , et al : tumor suppressor pten inhibition of cell invasion , migration , and growth : differential involvement of focal adhesion kinase and p130cas . 
mulholland dj , kobayashi n , ruscetti m , et al : pten loss and ras / mapk activation cooperate to promote emt and metastasis initiated from prostate cancer stem / progenitor cells . 
wongchenko mj , mcarthur ga , drno b , et al : gene expression profiling in braf - mutated melanoma reveals patient subgroups with poor outcomes to vemurafenib that may be overcome by cobimetinib plus vemurafenib . 
exploration of the impact of variant allele frequency ( vaf ) on progression - free survival ( pfs ) indicated by kaplan - meier plots of pfs in patients grouped by vaf above or below cutoff points of ( a ) 25% and ( b ) 50% for each treatment arfor the analysis , the median was chosen to be representative of the overall range of cutoff points . 
cell viability of 42 brafv600 - mutant melanoma cell lines treated with vemurafenib , stratified by expression versus loss of expression of pten . pten + ( n = 20 ) pten loss ( n = 22 ) table a1 . 
leach , ms , lgc1 ; jessica marquard , ms , lgc1 ; manmeet ahluwalia , md1 ; hetty carraway , md , mba1 ; petros grivas , md , phd2 ; davendra p.s. 
recent data sets indicate that the incidence of hereditary cancer may be as high as 17.5% in patients with cancer , and a notable subset is missed if screening is solely by family history and current syndrome - based testing guidelines . identication of germline variants has implications for both patients and their families . 
we aimed to summarize genes linked to hereditary cancer and the somatic and germline panels that include such genes . methods germline predisposition genes were chosen if commercially available for testing . 
penetrance was dened as low , moderate , or high according to whether the gene conferred a 0% to 20% , 20% to 50% , or 50% to 100% lifetime risk of developing the cancer or , when percentages were not available , was estimated on the basis of existing literature descriptions . results we identied a total of 89 genes linked to hereditary cancer predisposition , and we summarized these genes alphabetically and by organ systewe considered four germline and six somatic commercially available panel tests and quantied the coverage of germline genes across thecomparison between the number of genes that had germline importance and the number of genes included in somatic testing showed that many but not all germline genes are tested by frequently used somatic panels . conclusion the inclusion of cancer - predisposing genes in somatic variant testing panels makes incidental germline ndings likely . 
2019 by american society of clinical oncology introduction cancer is a genetically driven disease triggered by the accumulation of variants in genes responsible for regulation of cell growth , invasion , survival , and migration . 
most cancers are sporadic and result from acquired genetic changes known as somatic variants , which are caused by stochastic events that accumulate over time as well as by specic environmental factors , such as smoking and ultraviolet radiation , among others.1 precision oncology currently consists of an effort to understand the acquired genetic variants that underlie each patients cancer , which thus allows the opportunity to tailor therapy to a patients individual tumor biology.2 although tumor testing previously involved sequencing of individual genes or short hotspot fragments of dna , recent advances in next - generation sequencing ( ngs ) technology have enabled much more frequent and precise identication of pathogenic variants.2 , 3 such sequencing platforms are rapidly becoming faster and cheaper , which makes them as available and as popular as many other clinical testing tools.4 , 5 germline variants occur in germ cells , which are responsible for reproduction . 
although somatic variants occur in a single cell and are passed down only to that cells offspring , germline variants exist in all somatic cells and can inuence cancer susceptibility by affecting multiple cellular processes ( eg , cancer clone growth , somatic variant acquisition rates , and carcinogen metabolism ) .1 discovery and understanding of germline variants are of major clinical importance to the patient diagnosed with a hereditary cancer ; the identication of an inherited genetic variant often has therapeutic and prognostic value for a patient undergoing therapy . 
 akras et al showing promise in the context of both somatic and germline variants in brca and other homologous repair genes and represent a breakthrough niche in precision oncology.7 therapeutic consequences of other germline ndings are also expanding ; this is best highlighted by the recent nding that lynch syndromerelated cancers are exquisitely sensitive to programmed cell death - 1 inhibition.8 finally , a germline nding is clinically important in that members of the patients broader family who also carry inherited pathogenic variants will be at an increased risk of cancer as well.9 targeted surveillance is important for all carriers of germline pathogenic variants , whether they are cancer survivors or asymptomatic relatives ; the vast majority of inherited cancer predisposition syndromes increase the risk of multiple cancers , and targeted surveillance for the discrete subset of cancers associated with that particular gene should be undertaken . 
in addition , data support that gene - specic , directed cancer surveillance programs can be life - saving for carriers.10 , 11 thus , identication of pathogenic variants in the germline is critical for the improvement of health outcomes in cancer treatment and prevention . previously , it had been estimated that a small minority of cancers ( 5% to 10% ) are a result of inherited germline pathogenic variants.9 emerging data sets in the post - ngs era indicate that the incidence of hereditary cancer may be as high as 17.5% in patients who carry a diagnosis of cancer and even higher in specic histologic types , such as ovarian cancer and urothelial carcinoma.12 - 14 multiple studies have shown that somatic tumor dna sequencing may lead to incidental ndings of hereditary risk.15 , 16 this realization magnies the potential impact of somatic testing and prompts the need for a re - evaluation of companion germline diagnostic methodology . 
in this review , we provide a summary of genes related to hereditary cancer and assess the comprehensiveness of somatic and germline panels that test them . genes with germline implications table 1 catalogs cancer predisposition genes organized by tumor type ; each row lists all associated germline genes for a given tumor type . 
as an alternate reference , table 2 lists cancer predisposition genes in alphabetical order ; each row lists associated cancers and the estimated penetrance of these cancers for that particular gene . 
the dual table organization allows an oncologist to use either the clinical presentation or the results of somatic genetic testing as a screening tool to understand if a patients family history or somatic genetic testing result , respectively , may represent a germline variant . reviewed genes were chosen on the basis of their identication as cancer - susceptibility genes for which commercial germline testing is available from the four major germline testingfocused companies in the united states . 
for each cancer predisposition gene in table 2 , associated cancers are broadly categorized by the estimated penetrance categories ( high , medium , and low penetrance ) for that gene . 
penetrance was dened as low , moderate , or high , according to a 0% to 20% , 20% to 50% , or 50% to 100% lifetime chance of developing that particular cancer or , when percentages were unavailable , an estimate based on descriptions in the existing literature.17 it is important to note that different studies may declare varying penetrance rates for a gene , so we aimed to reect the ndings of the literature at large . as seen in table 2 , cancer - associated genes have greatly varying penetrance rates for different phenotypes ( ie , different types of cancer )  . 
for example , pathogenic variants in lynch syndrome genes ( ie , epcam , mlh1 , msh2 , msh6 , pms2 ) are not highly penetrant for pancreatic cancer but are for colorectal cancer . 
these differences in penetrance also underscore the importance of gene - based , rather than syndromic or histology - based , interpretations , given the unique physiology that underlies specic variants . in addition , several germline variants confer an increased risk only if they are biallelic ( ie , both alleles of the gene carry pathogenic variants )  . 
if genes are not marked as such , they can be assumed to confer risk when monoallelic ( ie , only one allele is altered )  . in the appropriate scenario , these tables can be used to guide genetic testing and counseling ; however , they should the current clinical be used with an understanding of limitations of such data . 
cancer subtypes and associated hereditary genes that confer risk ( continued ) cancer type associated germline genes skin ( scc ) small bowel ( ac ) thyroid acd , blm , cdkn2a , fancc , recql4 , terc , tert , tp53 apc , bmpr1a , epcam , mlh1 , msh2 , msh6 , mutyh , pms2 , smad4 , stk11 apc ( ptc ) , hras , mutyh , pten ( ftc , ptc ) , ret ( mtc ) , wrn ( ftc , ptc ) abbreviations : ac , adenocarcinoma ; acc , adrenocorticocarcinoma ; all , acute lympocytic leukemia ; aml , acute myeloid leukemia ; bcc , basal cell carcinoma ; cel , chronic eosinophilic leukemia ; cll , chronic lymphocytic leukemia ; cmml , chronic myelomonocytic leukemia ; eoc , epithelial ovarian cancer ; erms , embryonal rhabdomyosarcoma ; ftc , follicular thyroid cancer ; gbm , glioblastoma multiforme ; gist , gi stromal tumor ; hb , hepatoblastoma ; hcc , hepatocellular carcinoma ; lam , lymphangioleiomyomatosis ; mds , myelodysplastic syndrome ; mel , melanoma ; mtc , medullary thyroid cancer ; nb , nephroblastoma ; nsclc , nonsmall - cell lung cancer ; pnet , pancreatic neuroendocrine tumors ; ppb , pleuropulmonary blastoma ; ptc , papillary thyroid cancer ; rcc , renal cell carcinoma ; scc , squamous cell carcinoma ; sccoht , small - cell carcinoma of the ovary , hypercalcemic type ; scst , sex cord stromal tumors ; sctat , sex cord tumors with annular tubules ; seb , sebaceous gland carcinoma ; sega , supependymal giant cell astrocytoma ; tcc , transitional cell carcinoma ; t - pll , t - cell prolymphocytic leukemia . clinical benet to the patient . 
testing relatives can help identify those at an increased risk of developing similar cancers ; however , a challenge to counseling still exists , because these variants lack the cohesive screening guidelines of their high - penetrance counterparts , and they may require a more individualized plan . 
differences between the number of genes currently tested in widely available germline panels , even compared with the number of genes linked to cancer predisposition as listed in table 2 , underscore the rapidly shifting understanding of clinically pertinent germline genes and the wide differences in opinion about what constitutes a clinically useful gene list . variants noted as rare ( labeled - r in table 2 ) are included for a more comprehensive guide to known germline - related malignancies . 
given the young age at which cancers develop in these patients or the other profound organ dysfunction associated with the respective syndromes , germline variants in such genes are unlikely to be carried through generations . furthermore , these syndromes often have clearly associated features that readily identify them clinically , and genetic testing only conrms a suspected diagnosis . 
one such example is wrn , the gene implicated in werner syndrome ; individuals with this disease display severe growth retardation , lipodystrophy , and progeria that would be obvious on examination . 
although familial inheritance of these genes has on occasion been described , such cases should be regarded as the rare exception rather than the rule . currently available somatic variant panels somatic variant panels test many more genes than their germline counterparts do . 
some prominent somatic tests are caris molecular intelligence ( caris life sciences , irving , tx ) , foundationone cdx ( foundation medicine , cambridge , ma ) , genpath onkosight ( genpath oncology , elmwood park , nj ) , msk - impact ( memorial sloan kettering cancer center , new york city , ny ) , omniseq comprehensive ( omniseq corporation , buffalo , ny ) , and tempus xt ( tempus labs , chicago , il )  . 
table 4 lists the number of genes in these somatic panels and the number of genes in each panel with germline implications ( according to table 2 ) at the time of publication . 
in general , a notable number of genes with germline implications are sequenced within common somatic panels . importantly , however , no somatic panel covers all known genes of germline importance , and sizeable numbers of cancer - susceptibility genes are excluded . currently available germline variant panels discussion several companies offer panels that test genes linked to hereditary cancer for various body systems . 
well - established companies with longer commercial track records include ambry genetics ( aliso viejo , ca ) , genedx ( gaithersburg , md ) , invitae ( san francisco , ca ) , and myriad genetics ( salt lake city , ut )  . 
criteria for inclusion of a company in this summary were a primary focus in commercial clinical laboratory improvement amendmentscertied germline testing and exclusion of direct - to - consumer testing or testing several cancer - related genes hold germline implications , and these genes are not uniformly included in currently available germline panel tests ( table 3 )  . 
given the state of current germline testing guidelines , which are syndrome based , and available commercial germline panels , which increasingly are organ - system based , a degree of disconnect between what is clinically relevant and what is offered with germline panels is to be expected . 
our group found that tumor sequencing had a notable number of potential germline variants and that referrals based on somatic variant sequencing could reveal otherwise unknown germline variants.22 within the 315 - gene panel used for the initial 222 - patient cohort , 41 genes ( 13% ) were classied as genes known to cause cancer susceptibility when mutated in the germline . 
coverage of genes within comprehensive or body systemspecic panel offerings from each major germline testing company ambry cancer type genedx invitae myriad * breast / gynecologic colorectal melanoma pancreatic prostate renal frequently somatically mutated in tumors , including tp53 , apc , and cdkn2a , were excluded , 91 patients ( 41% ) had a potential germline variant . 
twenty - three patients ( 10% ) were evaluated by genetics professionals ; three genetics referrals resulted directly from somatic testing results , which ultimately yielded one previously unknown germline variant . 
with formal integration of medical genetics into somatic testing review by the genomics tumor board at our institution , the overall percentage of referrals to genetics increased from 15% to 33% , and 41% of those referrals were the direct result of a somatic test review.23 in another study , speare et al24 reviewed 74 germline tests that followed identication of somatic variants in the tumor specimens of 54 patients and found that 10 variants ( 13.5% ) were conrmed in the germline . 
last , schrader et al25 surveyed germline variants in 187 genes that overlapped with a concurrent 341 - gene tumor somatic panel and identied pathogenic germline variants in 246 ( 15.7% ) of 1 , 566 patients . 
moreover , of the 198 individuals with variants in genes specically associated with increased cancer risk , only 81 of these germline changes ( 40.9% ) were concordant with the tumor type . 
that is , more than half of the patients who had pathogenic germline variants likely would not have had a recommendation for germline testing of that particular gene on the basis of their cancer type alone . 
in our own data , a high proportion of patients who were discovered by somatic tumor testing alone to carry a germline pathogenic variant ( ve of seven patients , or 71.4% ) did not meet any guidelinebased genetic testing criteria.23 together , these data demonstrate that somatic variant sequencing can inform germline testing and act as a screening tool for inherited germline carrier status , which suggests an additional clinical purpose for somatic variant testing separate from the intended goal of screening for possible targeted therapies . * myriad myrisk simultaneously tests 28 genes linked to hereditary cancers in various body systems . 
although some of these tests ask for blood or saliva samples and can test germline variants , they are primarily advertised as panels used to test tumor samples for somatic variants . abbreviation : mskcc , memorial sloan kettering cancer center . in a 1 , 000 - patient cohort with tumor and normal dna sequenced with a 202 - gene panel , meric - bernstam et al20 reported 43 patients ( 4.3% ) with potentially germline variants . 
more than half of these germline variantpositive individuals ( 101 patients ) would not have met guideline - based criteria for clinical germline testing . these studies add credence to the idea that the identication of germline variants from somatic testing can benet patients who would not otherwise have been identied by means of striking personal or family histories and would likely identify inherited cancer susceptibility in additional patients that targeted germline panel or syndrome testing alone may miss . although somatic variant testing can unearth germline variants that traditional means of screening have not , it is important to point out that some common types of germline variants are not picked up with the current technology used for somatic sequencing . 
large rearrangements , inversions , promoter variants , and pseudogene differentiation are not consistently addressed with current somatic sequencing . thus , a germline variant that involves a large rearrangement of brca or is within pms2 , which is notoriously difcult to distinguish from its pseudogene , may be missed these genes or misinterpreted despite the inclusion of within the somatic test panel.26 , 27 despite technical differences between germline and somatic testing , somatic sequencing still has promise as a clinically useful , but not independently adequate , tool for the screening of inherited cancer predisposition . 
somatic tumor sequencing may be considered additive to a proper family history and of particular utility in screening the subset of patients whose family or tumor histology would not otherwise meet traditional germline genetic testing criteria . tables 3 and 4 demonstrate the complexity of current offerings in germline and somatic variant testing . 
even within the category of commercial germline testing , the composition of clinically relevant genes within panels designed for the same organ ( eg , breast ) varies widely from company to company . 
these tables underscore the difculty of interpreting exactly which genes are clinically relevant , whether in a germline or somatic setting , and in some ways reect the issues of offering tests in a eld with a rapidly expanding knowledge base . 
correspondingly , one of the largest challenges in the writing of this review was the expansive and rapidly changing nature of both genes and tumors , which require multidisciplinary expertise and multiple revisions to interpret emerging data . although these tables are designed as a foundational primer specically for the oncologist and should serve well as a quick reference in clinic , the involvement of genetic counselors whenever available in the interpretation of germline implications of somatic testing and the orders for follow - up germline testing is recommended . in conclusion , although family history remains the most reliable screening tool for inherited cancer predisposition syndromes , several studies suggest that a clinically notable subset of germline carriers would be missed by family history alone . because limited family history or an incomplete gene penetrance can result in ambiguity , family historybased germline testing alone may be inadequate to completely assess a patients , and the familys , hereditary cancer risk . 
the plethora of cancer susceptibility genes tested in widespread somatic testing tumor panels makes incidental germline ndings likely , and numerous studies have conrmed that somatic testing can be a useful , but not fully adequate , screen for germline variants . 
sohal , pauline funchain collection and assembly of data : zade akras , brandon bungo , brandie h . leach , jessica marquard , manmeet ahluwalia , hetty carraway , davendra p.s. 
leach consulting or advisory role : invitae speakers ' bureau : myriad genetics references manmeet ahluwalia stock and other ownership interests : mimivax honoraria : prime oncology , elsevier , itamar medical ( i ) consulting or advisory role : monteris medical , astrazeneca , bristol - myers squibb , abbvie , cbt pharmaceuticals , kadmon , vbi vaccines research funding : novartis , tracon pharma , novocure , spectrum pharmaceuticals , lilly , imclone , boehringer ingelheim , astrazeneca hetty carraway consulting or advisory role : amgen , agios , myriad genetics , celgene speakers ' bureau : baxalta , celgene , novartis , jazz pharmaceuticals research funding : celgene travel , accommodations , expenses : celgene , agios , jazz pharmaceuticals , novartis , baxalta petros grivas consulting or advisory role : genentech , merck , bristol - myers squibb , astrazeneca , biocept , clovis oncology , emd serono , seattle genetics , foundation medicine , driver , pzer , qed therapeutics , heron speakers ' bureau : genentech , bristol - myers squibb research funding : mirati therapeutics ( inst ) , genentech ( inst ) , roche ( inst ) , merck ( inst ) , oncogenex ( inst ) , bayer ( inst ) , pzer ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) davendra p.s. 
sohal honoraria : foundation medicine consulting or advisory role : perthera research funding : novartis ( inst ) , celgene ( inst ) , oncomed ( inst ) , bayer ( inst ) , genentech ( inst ) , bristol - myers squibb ( inst ) , agios ( inst ) , incyte ( inst ) travel , accommodations , expenses : foundation medicine pauline funchain consulting or advisory role : eisai no other potential conicts of interest were reported stratton mr : exploring the genomes of cancer cells : progress and promise . 
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carlo mi , zhang l , mandelker d , et al : cancer predisposing germline mutations in patients ( pts ) with urothelial cancer ( uc ) of the renal pelvis ( r - p ) , ureter ( u ) and bladder ( b )  . 
catenacci dvt , amico al , nielsen sm , et al : tumor genome analysis includes germline genome : are we ready for surprises ? int j cancer 136 : 1559 - 1567 , 2015 16 . 
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klek s , heald b , milinovich a , et al : genetic counseling ( gc ) and germline ( gl ) testing rates after adoption of an integrated clinical cancer genetics ( ccg ) approach to genomics tumor board ( gtb )  . 
 e introducing the jco precision oncology molecular tumor board case discussion series see accompanying article doi : as the idea and application of precision oncology gains momentum among cancer practitioners , the need for evidence - based guidelines and expert opinions is essential . 
many challenges exist for implementing precision oncology , but knowledge gaps in the interpretation of cancer variants and off - label access to approved agents or experimental drugs in clinical trials are major barriers faced by medical oncologists . 
a cancer genome interpreter must deal with the huge amount of unstructured information on effectiveness of therapies matched to driver alterations that is being generated constantly by clinical trials and preclinical experiments . 
moreover , potential and confirmed germline genomic events identified during tumor and / or normal dna sequencing may pose significant challenges for oncologists who are not prepared to handle incidental findings that have therapeutic effect on the individual patient with cancer and clinical implications for at - risk family members . mtbs bridge this gap by facilitating the exchange of information among a wide range of experts ( eg , medical oncologists , cancer geneticists and biologists , molecular pathologists , genetic counselors , bioinformaticians ) who review patients medical histories and cancer genomic profiles to guide treatment options . 
ford research funding : myriad genetics ( inst ) , natera ( inst ) author affiliations and support information ( if applicable ) appear at the end of this article . corresponding author : james m . 
ford md , fasco , stanford university school of medicine , 1115 ccsr , 269 campus dr , stanford , ca 94305 ; e - mail : jmf@ stanford.edu. the following represents disclosure information provided by authors of this manuscript . 
others have an intermediate phenotype and are classified as glomus tumors of uncertain malignant potential ( gt - ump ) .3 herein , we present a case of an 18 - year - old patient with two glomus tumors involving the lateral and posterior cords of the right brachial plexus . 
physical examination demonstrated no neurologic deficit . imaging mr imaging of the right upper extremity demonstrated two distinct brachial plexus lesions , one emanating from the distal aspect of the lateral cord and the other from the posterior cord ( fig 1 )  . 
after extensive discussion with the neurosurgeon , the patient opted for surgical exploration with open biopsy and possible removal of the infraclavicular tumors to provide a histologic diagnosis and guidance on further management . intraoperative findings open exploration of the right infraclavicular brachial plexus was performed . 
these were gently mobilized but did not stimulate and were divided , allowing the lesion to be removed en bloc and sent for histopathological analysis . in the posterior cord region , the axillary nerve was clearly enlarged with tumor . 
 ( a ) on coronal fluid - sensitive magnetic resonance sequence through the chest , the right brachial plexus demonstrates asymmetric hyperintensity and thickening of the c5 , c6 , c7 , and t1 nerve roots and upper trunk ( not shown ) in addition to two discrete masses arising from the distal brachial plexus involving the posterior ( short arrow ) and lateral ( long arrow ) cords . 
 ( c ) on diffusion weighted imaging with apparent diffusion coefficient mapping , there is qualitative and quantitative restricted diffusion ( apparent diffusion coefficient [ adc ] values range from 0.6 to 1 103 mm2 / s in the larger mass and 0.9 to 1 103 mm2 / s in the smaller mass )  . 
on frozen section , this tumor appeared relatively benign , but given its widespread involvement with the axillary nerve , further aggressive dissection of the remaining tumor was deferred to avoid morbid neurologic deficits . histopathology and molecular analysis histopathologic analysis revealed a well circumscribed proliferation of relatively monomorphic cells with round nuclei and abundant eosinophilic cytoplasm ( figs 2a and 2b )  . 
mart1 was faint , caldesmon was focal , cd34 highlighted blood vessels , ini - 1 expression was intact , and ki - 67 proliferation index was mild to moderate . 
these findings together led to the diagnosis of malignant glomus tumor per current criteria.3 molecular genetic analysis using polymerase chain reactionbased sequencing of braf exon 15 revealed the braf v600e mutation at a mutant allele frequency of 40% . 
 to support this finding , we performed immunohistochemical analysis using antibodies directed against phosphoextracellular signal - regulated kinase ( erk ) ; this revealed variable immunostaining , with both cytoplasmic and nuclear immunoreactivity . 
in addition to isolated braf testing , a conventional cytogenetic analysis of the tumor revealed a normal 46 xx karyotype in all cells examined . therapeutic management management of this patients malignant tumor was discussed at the johns hopkins hospital multidisciplinary sarcoma tumor board . 
 ( a ) low - power view of glomus tumor associated with peripheral nerve fascicles , hematoxylin and eosin sta ( b ) tumor cells had ample eosinophilic cytoplasm and evident mitotic activity . 
mr imaging and 18f - fluorodeoxy - d - glucose positron emission tomographycomputed tomography obtained 2 weeks after the addition of trametinib demonstrated interval decrease in tumor size ( table 1 )  . 
 mr imaging at 3 and 6 months after initiating raf inhibitor therapy showed continued decrease in tumor size ( figs 3 and 4 ) and at 9 months showed no further change in size or imaging characteristics relative to most recent prior imaging . 
she experiences no pain , and the extremity function is normal . discussion the ras - raf - mek - erk signaling pathway is a frequent site of oncogenic mutations , with approximately 30% of human cancer harboring at least one mutation that results in its dysregulation . 
activating braf mutations are found in approximately 7% of cancers , roughly half of malignant melanomas , 5 and in other tumors , including colorectal , ovarian , papillary thyroid , and nonsmall - cell lung cancers , gliomas , and histiocytic disorders.6 - 12 the most common mutation , v600e , results in constitutive activation of the erk pathway . small molecule inhibitors of raf kinase were first studied in clinical trials of patients with melanoma . 
to determine whether the benefit of targeted raf inhibition would extend to nonmelanoma tumors with braf v600e , a phase ii basket trial explored the use of vemurafenib in patients selected for this mutation . 
this study demonstrated modest antitumor activity , although not all tumors with braf mutation responded , thus enforcing that a single driver oncogene may be associated with a nonuniform response to targeted therapy.16 braf mutations also occur rarely in a wide spectrum of cancers , including 1.4% of soft tissue sarcomas.17 a single patient with braf - mutated clear cell sarcoma was treated in the vemurafenib basket trial and sustained a partial response.16 the role of raf inhibitors in other sarcomas , however , has not been explored . glomus tumors are mesenchymal neoplasms that are rare and generally benign.18 they can occur as solitary or multiple soft tissue tumors and are most commonly located on the digits , supporting early hypotheses about their origin in the temperature - sensing glomus body . 
in a targeted sequencing analysis , three of 28 glomus tumors were found to harbor braf v600e mutations and a single tumor mutation in kras.19 a single case of a glomus tumor of the median nerve was found by next - generation sequencing to harbor a braf v600e mutation.20 this patient , and those in the retrospective analysis of 28 cases , was treated surgically without consideration for systemic therapy . 
finally , in a collection of 102 glomus tumors ( 56% benign , 15% gt - ump , and 29% malignant ) braf v600e mutation was detected in six tumors ( 6% ) , all of which were classified as either malignant glomus tumor or gt - ump ( and 0 of 57 benign glomus tumors harbored braf mutations ) .21 these data , although reflective of an overall small sample size , are suggestive of braf mutation as a predictor of more aggressive clinical behavior . 
 interestingly , glomus tumor has previously been associated with neurofibromatosis type 1 , 22 suggesting a shared terminal event in activation of erk signaling in these tumors . our patients response to targeted braf inhibition is an extraordinary one and highlights promise for future use in sarcomas with this mutation . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center  . andrea cuviello no relationship to disclose aviad zick research funding : merck ( inst ) , eli lilly ( inst ) shivani ahlawat no relationship to disclose fausto j . 
folpe al , fanburg - smith jc , miettinen m , et al : atypical and malignant glomus tumors : analysis of 52 cases , with a proposal for the reclassification of glomus tumors . 
kimura et , nikiforova mn , zhu z , et al : high prevalence of braf mutations in thyroid cancer : genetic evidence for constitutive activation of the ret / ptc - ras - braf signaling pathway in papillary thyroid carcinoma . 
mcarthur ga , chapman pb , robert c , et al : safety and efficacy of vemurafenib in braf ( v600e ) and braf ( v600k ) mutation - positive melanoma ( brim - 3 ) : extended follow - up of a phase 3 , randomised , open - label study . 
hauschild a , grob jj , demidov lv , et al : dabrafenib in braf - mutated metastatic melanoma : a multicentre , open - label , phase 3 randomised controlled trial . 
dahlin lb , scherman p , besjakov j , et al : intraneural glomus tumor of uncertain malignant potential and with braf mutation in the median nerve an unusual case . 
karamzadeh dashti n , bahrami a , lee sj , et al : braf v600e mutations occur in a subset of glomus tumors , and are associated with malignant histologic characteristics . 
dahlin lb , mller g , anagnostaki l , et al : glomus tumours in the long finger and in the thumb of a young patient with neurofibromatosis - 1 ( nf - 1 )  . 
i believe there are two other important aspects of the us food and drug administration ( fda ) approval not mentioned in her editorial . first , haraldsdottir described this as fda approval , although in fact this was an fda accelerated approval . 
as a result , the fda requires the sponsor to do additional studies involving patients with different tumor types.2 the different tumor types studied should be tumors derived from different tissues , and studies should include dmmr / msi - h tumors derived from the same tissue of origin to determine whether additional molecular markers might predict which patients with dmmr / msi - h tumors are among the roughly 40% of patients whose tumors do respond or the 60% of patients whose dmmr / msi - h tumors do not respond to pembrolizumab . 
seventy - eight percent of the roughly 40% of patients whose tumors responded to pembrolizumab in the key study were still responding or had stable disease for at least 6 months from the initiation of pembrolizumab.2 the likelihood of durable responses among responders to checkpoint inhibitors does seem to distinguish the benefit of these drugs from that typically seen in patients responding to chemotherapy . 
other molecular markers harbored in dmmr / msi - h tumors might further distinguish which patients with dmmr / msi - h tumors are more likely to have durable responses . biomarkers have long been used as predictors of response to specific therapies . 
 targeted therapy for colorectal cancers with non - v600 braf mutations : perspectives for precision oncology braf mutations are found in up to 10% of colorectal cancers ( crc )  . 
it has been established that braf v600e mutations in crc predict unresponsiveness to epidermal growth factor receptor ( egfr ) inhibitioncetuximab and / or panitumumabas a result of the constitutive activation of the mitogen - activated protein kinase pathway downstream of egfr signaling . 
in many instances , clinicians may be hesitant to use egfr inhibitors for these patients , as it is largely assumed that tumors with non - v600 braf mutations activate the mitogen activated protein kinase pathway in a similar manner to ras or braf v600e mutations and would therefore be equally refractory to egfr inhibition ; however , the evidence that currently exists to substantiate this claim is mixed and incomplete . 
preclinical data and several case reports suggest that a subset of braf non - v600 mutations that impair the protein 's kinase activity may in fact confer heightened sensitivity to egfr inhibition because of dependency on upstream receptor tyrosine kinase signaling . 
 ( a ) in braf wild - type ( wt ) tumors , braf signals downstream from receptor tyrosine kinases ( rtks ) and ras as a dimer with braf or craf . 
it signals downstream to mitogen - activated protein kinase kinase 1 / 2 ( mek1 / 2 ) and extracellular regulated kinase 1 / 2 ( erk1 / 2 ) , sustaining cell survival and proliferation . 
for this reason , targeting them with egfr and mek inhibitors is the optimal combination of approved agents , but may be improved upon with the development of next - generation pan - raf and erk inhibitors . 
jones et al2 identified an impressive cohort of 208 patients with braf non - v600 mutant crc , which accounted for 2.2% of patients in their crc cohort and 22% of patients with braf mutations . 
among non - v600 mutants , class ii mutations were found in eight patients ( 22% of non - v600 mutants ) , class iii mutations in 17 patients ( 46% ) and unknown braf mutations in 12 patients ( 32% )  . 
it is likely that the prevalence of non - v600 mutants compared with v600e braf mutants is overestimated in this population because braf v600e is associated with microsatellite instability high ( msi - h ) tumor status.4 together , these data imply that non - v600 braf mutations are present in 2% to 3% of crc , which makes them more prevalent than was previously speculated.29 in addition to the prevalence of non - v600 mutations in crc , these studies also revealed important insights into the characteristics of patients with braf mutant crc . 
braf v600 mutant tumors are commonly right sided , of high clinical grade , msi - h , and experience inferior survival compared with both braf wild - type and non - v600 mutants.2 , 4 , 30 meanwhile , braf non - v600 mutant tumors tend to be found in younger patients with left - sided disease who experience a more indolent disease course than patients with either braf v600e or braf wild - type mutant tumors.2 , 30 these data clearly segregate braf v600 and non - v600 mutant tumors as distinct clinical entities . 
while jones et al2 did not observe differences between class ii and class iii mutants in these characteristics , results from yaeger et al4 suggest that class ii mutants may cluster more closely with class i mutants than class iii mutants as a result of their common right - sided frequency and mutual exclusivity from complimentary genetic lesions in the mapk pathway . 
 this result was underwhelming compared with the response rates observed in patients with melanoma and later with nonsmall - cell lung cancer.37 , 39 a subsequent study evaluated combined inhibition of brafvemurafeniband egfr panitumumabfor the treatment of patients with braf v600e mutant crc . 
the results of this trial showed modest efficacy for a small number of patients , with responses seen in two of 15.40 similar response rates were observed in braf plus egfr inhibited arms of more recent trials . 
encorafenib and cetuximab elicited responses in 19% of patients , 41 whereas 10% of dabrafeniband panitumumab - treated patients experienced a response.42 given that braf v600e is a strong predictor of nonresponsiveness to egfr inhibition , 10 , 43 - 45 these studies are successful in demonstrating that braf inhibition can sensitize braf v600e crc to egfr inhibitors . 
however , it is clear that this strategy is still insufficient at effectively inhibiting the mapk pathway and achieving responses in a majority of patients . safety lead - in data from 30 patients in an ongoing clinical trial using encorafenib ( braf inhibitor ) , binimetinib ( mek inhibitor ) , and cetuximab ( egfr inhibitor ) indicate the strongest results with targeted therapy for braf v600e crc thus far , achieving response rates of 48%.46 this is in contrast with a completed study by corcoran et al42 that used dabrafenib ( braf inhibitor ) , trametinib ( mek inhibitor ) , and panitumumab ( egfr inhibitor ) which only achieved a response rate of 21% . 
whereas it is difficult to make cross - study comparisons , both studies had similar patient characteristics , which makes it possible to speculate that this large discrepancy between trials comes from differences between the drugs themselves . 
panitumumab and cetuximab are considered similarly effective agents , and different mek inhibitors have been shown to be highly similar mechanistically.47 , 48 for this reason , the differences between encorafenib and dabrafenib may account for the improved outcomes in the beacon safety lead - in data . 
in braf class i mutant metastatic melanoma , treatment with encorafenib plus binimetinib has produced higher response rates and progression - free survival compared with what has been previously reported with dabrafenib plus trametinib.49 this suggests that these new agents may be superior in their ability to sustain the inhibition of the mapk pathway in braf v600e mutant cells . 
 this approach is currently under exploration in melanoma.64 , 65 given that combinations of targeted therapies are beginning to elicit encouraging responses in braf v600 mutant microsatellite stable crc , additional studies that explore novel combinations are expected to continue . 
additional preclinical studies that explore patient - derived crc cell lines and xenografts may help to determine which , if any , of the nonv600 braf mutations confer increased sensitivity to egfr inhibition . 
 important areas of preclinical research include understanding the oncogenic potential of these mutations to assign them to class ii , class iii , or as passenger mutations of no significance . as the development of novel targeted agents , such as erk inhibitors and next - generation raf inhibitors , reach clinical trials , it will be important to assess their potential for patients with crc with non - v600 braf mutations . 
furthermore , preclinical research in understanding the mechanisms that underlie co - occurrence of non - v600 braf mutations with other lesions in the mapk pathway may provide important insights into the pathogenesis and vulnerabilities of these tumors . 
as the field refines targeted therapies for braf v600e crc , it is likely that more effective treatment strategies will become understood and available for crc with non - v600 braf mutations . 
seligmann jf , fisher d , smith cg , et al : investigating the poor outcomes of braf - mutant advanced colorectal cancer : analysis from 2530 patients in randomised clinical trials . 
shitara k , yonesaka k , denda t , et al : randomized study of folfiri plus either panitumumab or bevacizumab for wild - type kras colorectal cancer - wjog 6210g . 
de roock w , claes b , bernasconi d , et al : effects of kras , braf , nras , and pik3ca mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy - refractory metastatic colorectal cancer : a retrospective consortium analysis . 
shinozaki e , yoshino t , yamazaki k , et al : clinical significance of braf non - v600e mutations on the therapeutic effects of anti - egfr monoclonal antibody treatment in patients with pretreated metastatic colorectal cancer : the biomarker research for anti - egfr monoclonal antibodies by comprehensive cancer genomics ( breac ) study . 
shen y , wang j , han x , et al : effectors of epidermal growth factor receptor pathway : the genetic profiling of kras , braf , pik3ca , nras mutations in colorectal cancer characteristics and personalized medicine . 
corcoran rb , ebi h , turke ab , et al : egfr - mediated re - activation of mapk signaling contributes to insensitivity of braf mutant colorectal cancers to raf inhibition with vemurafenib . 
herr r , halbach s , heizmann m , et al : braf inhibition upregulates a variety of receptor tyrosine kinases and their downstream effector gab2 in colorectal cancer cell lines . 
planchard d , kim tm , mazieres j , et al : dabrafenib in patients with braf ( v600e ) - positive advanced nonsmall - cell lung cancer : a single - arm , multicentre , open - label , phase 2 trial . 
cremolini c , di bartolomeo m , amatu a , et al : braf codons 594 and 596 mutations identify a new molecular subtype of metastatic colorectal cancer at favorable prognosis . 
schirripa m , biason p , cortiula f , et al : clinico - pathological and molecular characterisation of braf mutant metastatic colorectal cancer ( mcrc ) : are all mutations created equal ? j clin oncol 36 : 3590 , 2018 ( suppl ) 32 . 
planchard d , besse b , groen hjm , et al : dabrafenib plus trametinib in patients with previously treated braf ( v600e ) - mutant metastatic nonsmall - cell lung cancer : an open - label , multicentre phase 2 trial . 
van geel rmjm , tabernero j , elez e , et al : a phase ib dose - escalation study of encorafenib and cetuximab with or without alpelisib in metastatic braf - mutant colorectal cancer . 
guren tk , thomsen m , kure eh , et al : cetuximab in treatment of metastatic colorectal cancer : final survival analyses and extended ras data from the nordic - vii study . 
price tj , peeters m , kim tw , et al : panitumumab versus cetuximab in patients with chemotherapy - refractory wild - type kras exon 2 metastatic colorectal cancer ( aspecct ) : a randomised , multicentre , open - label , non - inferiority phase 3 study . 
dummer r , ascierto pa , gogas hj , et al : encorafenib plus binimetinib versus vemurafenib or encorafenib in patients with braf - mutant melanoma ( columbus ) : a multicentre , openlabel , randomised phase 3 trial . 
niessner h , sinnberg t , kosnopfel c , et al : braf inhibitors amplify the pro - apoptotic activity of mek inhibitors by inducing er stress in nras - mutant melanoma . 
pietrantonio f , oddo d , gloghini a , et al : met - driven resistance to dual egfr and braf blockade may be overcome by switching from egfr to met inhibition in braf - mutated colorectal cancer . 
ahronian lg , sennott em , van allen em , et al : clinical acquired resistance to raf inhibitor combinations in braf - mutant colorectal cancer through mapk pathway alterations . 
sinicrope fa , mahoney mr , smyrk tc , et al : prognostic impact of deficient dna mismatch repair in patients with stage iii colon cancer from a randomized trial of folfox - based adjuvant chemotherapy . 
tran b , kopetz s , tie j , et al : impact of braf mutation and microsatellite instability on the pattern of metastatic spread and prognosis in metastatic colorectal cancer . 
overman mj , mcdermott r , leach jl , et al : nivolumab in patients with metastatic dna mismatch repair - deficient or microsatellite instability - high colorectal cancer ( checkmate 142 ) : an open - label , multicentre , phase 2 study . 
donia m , fagone p , nicoletti f , et al : braf inhibition improves tumor recognition by the immune system : potential implications for combinatorial therapies against melanoma involving adoptive t - cell transfer . 
hu - lieskovan s , mok s , homet moreno b , et al : improved antitumor activity of immunotherapy with braf and mek inhibitors in braf ( v600e ) melanoma . 
sullivan rj , gonzalez r , lewis kd , et al : atezolizumab ( a ) + cobimetinib ( c ) + vemurafenib ( v ) in brafv600 - mutant metastatic melanoma ( mel ) : updated safety and clinical activity . 
miller wh , kim tm , lee cb , et al : atezolizumab ( a ) + cobimetinib ( c ) in metastatic melanoma ( mel ) : updated safety and clinical activity . 
bahadoran p , allegra m , le duff f , et al : major clinical response to a braf inhibitor in a patient with a braf l597r - mutated melanoma . 
noeparast a , teugels e , giron p , et al : non - v600 braf mutations recurrently found in lung cancer predict sensitivity to the combination of trametinib and dabrafenib . 
sullivan rj , infante jr , janku f , et al : first - in - class erk1 / 2 inhibitor ulixertinib ( bvd - 523 ) in patients with mapk mutant advanced solid tumors : results of a phase i dose - escalation and expansion study . 
de roock w , de vriendt v , normanno n , et al : kras , braf , pik3ca , and pten mutations : implications for targeted therapies in metastatic colorectal cancer . 
hampson , phd1 ; bjoern bornkamp , phd1 ; antonella maniero , msc2 ; frank bretz , phd1 ; and emmanuel zuber , phd1 the diversity of patient journeys can raise fundamental questions regarding the evaluation of drug effects in clinical trials to inform clinical practice . 
when dening the treatment effect of interest in a trial , the researcher needs to account for events occurring after treatment initiation , such as the start of a new therapy , before observing the end point . 
this estimand framework provides a structured approach to discuss how to account for intercurrent events that occur after random assignment and may affect the assessment or interpretation of the treatment effect . 
on the basis of the patients clinical and pathologic ndings , he is offered entry into a randomized trial of a new drug plus standard - of - care therapy ( soc ) versus soc alone . 
the examples provided earlier include discontinuation of treatment as a result of an adverse reaction , start of a new anticancer regimen before observing the end point of interest ( eg , progression - free survival [ pfs ] or overall survival [ os ] ) , or death of a patient while on treatment . some of these events may reect a desired or undesired activity of the treatment , which is not directly captured by the end point ; some may prevent observation of the end point ; and still others may complicate interpretation of the end point . 
 degtyarev et al context key objective a clinical researcher is confronted with unique challenges when dening the treatment effect of interest in oncology clinical trials as a result of the diversity of patient journeys . 
in this article , the relationship between patient journeys and treatment effects is highlighted , and it is illustrated how the newly introduced estimand framework can structure discussions among interdisciplinary teams regarding the design and interpretation of oncology trials . knowledge generated the estimand framework facilitates a precise denition of the scientic question of interest accounting for different patient journeys . 
all parties involved in the design of clinical trials need to consider how to dene the treatment effect of interest in the study population , along with the relevant end point and data analysis , to adequately address the trial objective in the presence of intercurrent events . understanding the treatment effect measured in a clinical trial will be important for oncologists so that they can make informed treatment decisions . the international council for harmonization of technical requirements for pharmaceuticals for human use ( ich ) aims to align the development of new medicinal products in europe , japan , and the united states . 
the ich e9 note for guidance focuses on the design , conduct , analysis , and reporting of conrmatory clinical trials . however , there is a growing awareness that an imprecise denition of the scientic question of the trial may create a misalignment between its objectives and analysis and confuse its interpretation . 
in response to this , in 2017 , the ich released a draft addendum1 to its e9 guideline.2 this proposes a structured framework for clinical researchers to better align trial objectives , design , analysis , and interpretation , in view of the diversity of patient journeys and intercurrent events . 
the addendum introduces the concept of an estimand to precisely describe the treatment effect of interest in terms of the following four attributes : the target population ; the end point ( or variable ) to be obtained for each patient ; a specication of how to account for intercurrent events ; and a population - level summary for the variable , such as the hazard ratio for pfs . these four attributes are always interlinked . 
for those three patients , events occurring after random assignment either prevent the observation of the end point ( patient 2 : how long would the pfs be otherwise ? ) or raise important questions on its interpretation ( patient 1 : is the observed pfs a result of the effect of the investigational treatment or of the new therapy ? patient 2 : is the withdrawal of consent indicative of any treatment effect ? patient 3 : is death a random event , or is it indirectly related to the disease or treatment ? )  . 
eot , end of treatment ; soc , standard of care . a change in anticancer treatment reect the reality of clinical practice , including the possible impact of the investigated treatment on the choice or activity of subsequent therapies . 
for example , estimand 3 ( table 1 ) targets the effect of the studied treatment on pfs as if no alternative therapy existed ( ie , excluding its possible impact )  . 
this has implications for how we dene the end point , as well as how we collect and analyze the data . for example , for estimands 2 and 3 , collection of tumor assessments after the start of new therapy is not required , whereas this is necessary for estimand 1 . 
the choice of the estimand will be informed by discussions between all parties involved in the clinical trial , the disease cluding sponsor and regulators , about setting , the anticipated mode of action of the treatment , and the relevance of intercurrent events . example with os table 1 lists the four attributes ( population , end point , intercurrent events , and summary ) of several estimands for the lung cancer trial , each of which is dened with a pfs end point . 
it is interesting to further illustrate how the estimand framework can accommodate a patients journey when os ( ie , time to all - cause mortality ) is the end point of interest . 
how the treatment effect denition drives the choice of end point and handling of an intercurrent event in the estimand framework for the lung cancer trial example attributes of the estimand description per ich e9 addendum estimand 3 estimand 2 estimand 1 population variable ( end point ) intercurrent event : start new therapy population - level summary prolonging time to progression or death prolonging time to progression , death , or new therapy prolonging time to progression or death if new therapy is not initiated because none is available population according to targeted indication pfs based on progression death as event new time to event end point considering progression , death , or new therapy as event pfs based on progression or death as event treatment policy composite hypothetical hazard ratio note . 
the intercurrent event addressed here is the start of a new anticancer therapy before the assessment of tumor progression ( as illustrated by patient 1 in fig 1 )  . 
the treatment policy strategy ignores the event , and the end point is expected to be assessed and analyzed regardless of its occurrence ; traditionally , this was addressed in the analysis by not censoring observations at the start of new therapy . 
the estimand framework allows clarication of how different choices of strategy to account for intercurrent events affect the end point denition , assessment , and analysis , and consequently , the interpretation of trial results with respect to the treatment effect of interest . 
note that in all three estimands , the intercurrent event of death ( as for patient 3 in fig 1 ) is incorporated in the variable using a composite strategy . abbreviations : ich , international council for harmonization of technical requirements for pharmaceuticals for human use ; pfs , progression - free survival . if one assumes that patient journeys observed in the trial after treatment discontinuation reect real - world clinical practice , the treatment effect of interest can be dened as follows : does the investigational treatment prolong survival compared with soc , regardless of treatment discontinuation ? this reects a treatment policy strategy for handling intercurrent events such as the start of new therapy . 
this denes the estimand typically used with os in oncology trials . however , it is debatable whether this estimand with os always yields a clinically meaningful comparison of treatments , in particular if the assumption that subsequent new therapies reect that clinical practice is violated . 
even if it does become available ( eg , after regulatory approval based on pfs results ) , the investigational treatment would likely be used in lieu of soc , rather than after disease progression on soc . 
a similar situation arises when patients randomly assigned to soc have access , upon tumor progression , to other therapies outside the trial with the same mechanism of action ( moa ) as the investigational treatment . 
 estimands and the patient journey example in the adjuvant treatment setting a 70 - year - old woman with a 2.0 - cm adenocarcinoma of the breast and no involved lymph nodes comes to her oncologist for treatment . 
in her seventh year after random assignment , the patient dies from complications after a myocardial infarction . the scientic question of interest in this adjuvant trial is whether the new treatment prolongs patients disease - free survival time . 
should the appearance of a contralateral breast cancer be considered as disease recurrence ? and what about another primary cancer or death not related to breast cancer ? such discussions may further rene the scientic question . 
for example , the researcher may choose between the following three questions to dene the objective of this trial : does the drug delay relapse , secondary malignancy , or death from any cause ? does the drug delay relapse or death from any cause ? does the drug delay relapse or disease - related death ? the choice of scientic question should be informed by a treatments moa and the specic disease setting . 
for instance , published data suggest that adjuvant hormone therapies for breast cancer may effectively reduce the risk of contralateral breast cancer but increase the risk of other primary malignancies.14 - 16 therefore , in this setting , the effect of treatment on the risk of developing these secondary malignancies seems to be relevant . 
consequently , the composite strategy could be applied to include these secondary malignancies in the denition of the end point . the researcher would select the rst question ( does the drug delay relapse , secondary malignancy , or death from any cause ? ) as the primary treatment effect of interest for the study . such discussions are not new in oncology , and efforts have been undertaken in the adjuvant setting to standardize end points and handle intercurrent events without using the ich e9 terminology . 
hudis et al8 ( p2127 ) noted that standardized denitions of breast cancer clinical trial end points must be adopted to permit the consistent interpretation and analysis of breast cancer clinical trials and to facilitate cross - trial comparisons and meta - analyses . 
recommendations for breast cancer were provided as part of the denition for the assessment of time - to - event end points in cancer trials initiative , further distinguishing between different types of relapses and secondary malignancies.17 the authors also recognized that handling intercurrent events differently may require new end points . 
alternatively , she could receive an investigational personalized therapy , a chimeric antigen receptor t - cell therapy ( car - t ) , currently approved to treat patients with diffuse large b - cell lymphoma in a later setting.18 , 19 the oncologist anticipates the different journeys this patient could encounter on each of the treatment options . if the patient is to receive soc , she will initially receive salvage chemotherapy . 
the best soc therapy after rituximab plus cyclophosphamide , doxorubicin , vincristine , and prednisone is high - dose chemotherapy followed by asct for patients who achieve a deep remission on salvage chemotherapy . 
if a deep remission is not obtained after two to three cycles of a rst salvage chemotherapy ( approximately 6 weeks ) , the patient will receive a new salvage therapy ( second salvage ) , still with the goal to achieve a deep remission and subsequently receive transplantation . 
in this situation , the car - t therapy could be offered because it is approved in this later setting . alternatively , the patient may receive car - t immediately , as an experimental option in this earlier setting . 
she might then require chemotherapy to control the disease and prevent rapid progression ( bridging chemotherapy ) during the approximate 4 to 6 weeks required to manufacture and deliver the car - t therapy to the clinic . 
however , in general and in contrast with the administration of asct in the soc option , tumor response or progression before cart infusion is not relevant for the administration of car - t and , at this stage , would not constitute a nal outcome for the patient . 
if there is no improvement of disease after approximately 12 weeks into the journey , then the strategy of car - t administration would be deemed to have failed , and other treatment options would need to be considered . adopting now the perspective of the researcher who wants to compare both options in a randomized clinical study , a review of the different patient journeys just described raises a number of challenges to identify the adequate research question and the treatment effect of interest for this trial . 
those challenges that need to be addressed to design a study enabling a scientically sound and interpretable comparison are as follows . first , the therapies to be compared represent two treatment strategies , not simply two different treatments . 
during the manufacturing period , patients may take bridging therapy . their tumor may progress or respond to the bridging therapy , but the tumor response status before car - t infusion is not relevant for the initiation of car - t . 
therefore , the treatment effect of the car - t strategy relative to the soc strategy is not expected to manifest fully during the waiting period . finally , car - t may be administered after failure of the soc treatment strategy . 
for patients receiving the soc treatment strategy , once two lines of salvage therapy have failed to be effective , patients become eligible to receive the car - t therapy , which has already been approved in that setting . 
this means that the patient outcome observed afterward in the soc treatment group may not reect the effect of the soc strategy alone , but also the effect of this crossover . the estimand framework helps to address the challenges raised by the complexity of patient journeys on both arms . 
the researcher will be able to give a more precise description of the treatment effect of interest using the language offered by the estimand framework , starting with the two treatment strategies to be a description of compared . treatment strategies to be compared the study could evaluate the effect of car - t against asct administration only , focusing on the possibly curative component of each treatment strategy . 
this would ideally be studied by randomly assigning patients only when they would be ready for both the administration of car - t ( ie , after successful car - t manufacturing ) and asct ( ie , after reaching a sufcient remission from salvage therapy )  . 
in this case , patients would be randomly assigned to receive either car - t or asct , with all necessary preparatory steps in their therapeutic journey being part of a common run - in phase before random assignment . however , the population for the comparison would then constitute only a subset of the population initially considered eligible to receive the car - t or soc treatment strategies . 
this would not address the following scientic question of interest : what is the relative clinical benet across the entire patient journey once a car - t or soc treatment strategy is prescribed ? therefore , it seems of interest to compare the entirety of both treatment strategies instead , including all possible paths as dened in the patient journeys . 
this also corresponds to clinical practice , where not all patients will receive all components of the treatment . population aligned with the denition of the treatment strategies described earlier , the population of interest includes all patients eligible to receive either car - t or soc treatment strategies , to be further specied with adequate inclusion and exclusion criteria . variable typically in this disease setting , the primary variable , or end point , is event - free survival ( efs ) , representing the time from random assignment to failure of the treatment strategy . 
intercurrent events affecting the comparison of the treatment strategies , with their respective handling strategy intercurrent event handling strategy justication manufacturing failure in car - t arm or failing treatment policy intrinsic to treatment strategy : those events to receive asct in soc arm reect the clinical reality of the treatment strategy new cancer therapy before observing efs event hypothetical not part of treatment strategy , and affecting the sd / pd at week 6 treatment policy observation and interpretation of further assessments does not reect an outcome of treatment strategy ; only used for treatment decision in soc arm ; not used for car - t arm abbreviations : asct , autologous stem - cell transplantation ; car - t , chimeric antigen receptor t - cell therapy ; efs , event - free survival ; pd , progressive disease ; sd , stable disease ; soc , standard of care . constitute a failure of a treatment strategy if observed at or after the week 12 assessment . 
however , death represents the most informative and denitive outcome of either treatment strategy , which can be observed regardless of when it occurs . new cancer therapy started before observing efs event . 
if a patient takes an unplanned anticancer therapy before his or her assigned treatment strategy reaches failure , it will be difcult to interpret the outcome as reecting the effect of that treatment strategy . 
this intercurrent event can be handled using the hypothetical strategy ( ie , to evaluate the effect had patients not been offered such an alternative treatment )  . insufcient remission ( stable disease or progressive disease ) at week 6 . 
they are ignored , following the treatment policy strategy , as reected in the variable denition . intercurrent events table 2 lists the intercurrent events affecting the comparison of the two treatment strategies and the estimand strategy used to handle each of them . summary measure manufacturing failure in the car - t arm or failing to receive asct in the soc arthe patient may not receive the nal treatment ( car - t infusion or asct ) as a result of the nature of both treatment strategies . 
following the treatment policy strategy , such events should be ignored , and patients should still be observed until the nal event of interest . the summary measure often used to quantify the relative treatment effect on efs in a randomized study is the hazard ratio . 
in addition , both car - t and asct are population variable ( cid : 129 ) patients with dlbcl who have experienced relapse or are refractory after first - line therapy ( cid : 129 ) event - free survival with sd / pd on or after week 12 or death at any time as event treatment strategies ( cid : 129 ) soc : salvage therapy followed by asct ( cid : 129 ) car - t : optional bridging therapy and lymphodepleting therapy followed by car - t infusion ( cid : 129 ) hazard ratio summary measure ( cid : 129 ) failure / delay to receive final treatment ( cid : 129 ) sd / pd at week 6 ( cid : 129 ) new therapy intercurrent events fig 2 . 
in the soc arm , patients receive up to two salvage chemotherapies , with the aim to receive autologous stem - cell transplantation ( asct ) when disease responds to chemotherapy . 
if no sufcient response to the rst round of chemotherapy is achieved in soc patients , car - t is manufactured in parallel to the patient receiving a new round of chemotherapy . 
birc , blinded independent review committee ; cr , complete response ; lymphoma ; pd , progressive disease ; pet , positron ct , computed tomography ; dlbcl , diffuse large b - cell emission tomography ; pr , partial response ; r , random assignment ; sd , stable disease . potentially curative therapies , and hence , a proportion of patients may have a similar outcome when they are cured . 
alternative summary measures to quantify treatment effects in the presence of nonproportional hazards are currently being investigated.20 - 22 having dened the treatment strategies to be compared , together with the four other attributes of the primary estimand , as summarized in figure 2 , the study design presented in figure 3 could be proposed . conclusion patients with cancer experience different journeys , and researchers need to account for this diversity when dening the treatment effect of interest in a clinical trial . 
various events occurring after random assignment may affect the assessment and interpretation of the treatment effect of interest . the estimand framework provides a structured approach to transparently discuss and account for different patient journeys . 
identifying an estimand leads to a precise denition of the scientic question of interest and to tailored study designs and data analyses.23 , 24 experience with the estimand framework in clinical research is still limited , 25 and its use in oncology is being explored in dedicated regulatory and industry forums and working groups . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . evgeny degtyarev employment : novartis stock and other ownership interests : novartis yiyun zhang employment : novartis kapildeb sen employment : novartis stock and other ownership interests : novartis david lebwohl employment : novartis , semma therapeutics stock and other ownership interests : novartis , semma therapeutics expert testimony : novartis mouna akacha employment : novartis stock and other ownership interests : novartis lisa v . 
hampson employment : novartis , astrazeneca stock and other ownership interests : novartis bjoern bornkamp employment : novartis stock and other ownership interests : novartis , alcon travel , accommodations , expenses : novartis antonella maniero employment : novartis , bristol - myers squibb stock and other ownership interests : novartis , bristol - myers squibb travel , accommodations , expenses : novartis frank bretz employment : novartis stock and other ownership interests : novartis travel , accommodations , expenses : novartis emmanuel zuber employment : novartis , novartis ( i ) stock and other ownership interests : novartis , alcon , air liquide , novartis ( i ) , alcon ( i ) travel , accommodations , expenses : novartis , novartis ( i ) no other potential conicts of interest were reported . acknowledgment we thank aby buchbinder for his enlightening critical review of the rst version of this article . 
we are indebted to ying lu , md , and jack lee , md , for inviting us and gratefully appreciate the opportunity to contribute this review article . references international council for harmonization of technical requirements for pharmaceuticals for human use : e9 ( r1 ) draft guideline : estimands and sensitivity analysis in clinical trials . 
international council for harmonization of technical requirements for pharmaceuticals for human use : e9 guideline : statistical principles in clinical trials . rubach k : treatment effect quantication for time - to - event endpoints : estimands , analysis strategies , and beyond . 
pharm stat 18 : 145 - 165 , 2019 bengoudifa b - r , weber h , gathmann i , et al : estimand framework in aml based on a randomized , placebo - controlled , phase 3 study investigating the effect of adding midostaurin to standard chemotherapy in patients with newly diagnosed aml without a flt3 mutation . 
j clin oncol 27 : 2874 - 2880 , 2009 korn el , freidlin b , abrams js : overall survival as the outcome for randomized clinical trials with effective subsequent therapies . 
j clin oncol 29 : 2439 - 2442 , 2011 saad ed , buyse m : overall survival : patient outcome , therapeutic objective , clinical trial end point , or public health measure ? j clin oncol 30 : 1750 - 1754 , 2012 8 . 
hudis ca , barlow we , costantino jp , et al : proposal for standardized denitions for efcacy end points in adjuvant breast cancer trials : the steep systej clin oncol 25 : 2127 - 2132 , 2007 larkin j , minor d , dangelo s , et al : overall survival in patients with advanced melanoma who received nivolumab versus investigators choice chemotherapy in checkmate 037 : a randomized , controlled , open - label phase iii trial . 
rittmeyer a , barlesi f , waterkamp d , et al : atezolizumab versus docetaxel in patients with previously treated non - small - cell lung cancer ( oak ) : a phase 3 , open - label , multicentre randomised controlled trial . 
reck m , rodr guez - abreu d , robinson ag , et al : treatment switchingadjusted overall survival ( os ) in keynote - 024 : first - line pembrolizumab versus chemotherapy in patients with advanced non - small cell lung cancer ( nsclc )  . 
sternberg cn , hawkins re , wagstaff j , et al : a randomised , double - blind phase iii study of pazopanib in patients with advanced and / or metastatic renal cell carcinoma : final overall survival results and safety update . 
motzer rj , escudier b , oudard s , et al : phase 3 trial of everolimus for metastatic renal cell carcinoma : final results and analysis of prognostic factors . 
goss pe , ingle jn , martino s , et al : a randomized trial of letrozole in postmenopausal women after ve years of tamoxifen therapy for early - stage breast cancer . 116 : 4256 - 4265 , 2010 n engl j med 349 : 1793 - 1802 , 2003 15 . 
gourgou - bourgade s , cameron d , poortmans p , et al : guidelines for time - to - event end point denitions in breast cancer trials : results of the datecan initiative ( denition for the assessment of time - to - event endpoints in cancer trials )  . 
saad ed , zalcberg jr , p eron j , et al : understanding and communicating measures of treatment effect on survival : can we do better ? j natl cancer inst 21 . 
royston p , parmar mk : restricted mean survival time : an alternative to the hazard ratio for the design and analysis of randomized trials with a time - to - event outcome . 
 functional rna studies are a useful tool in variant classication but must be used with caution : a case study of one brca2 variant paola nix , phd1 ; erin mundt , ms1 ; susan manley , ms / cgc , mba1 ; bradford coffee , phd1 ; and benjamin roa , phd1 the use of genetic testing to evaluate hereditary cancer risk is increasing , because it can inform clinical management and cancer treatment decisions . 
a germline pathogenic variant in one of these genes is associated with an increased risk of cancer and therefore merits altered including increased surveilmedical management , lance and the option of risk - reducing surgeries.1 in addition , the identication of a pathogenic brca1 or brca2 variant may affect treatment decisions and clinician recommendations for women with breast and / or ovarian cancer.1 - 4 given these clinical implications , laboratories accurately that classify variants as part of hereditary cancer genetic testing . is essential although established guidelines from the american college of medical genetics ( acmg ) and association for molecular pathology ( amp ) provide a framework to help with variant classication , 5 there may be challenges interpreting the available evidence for some variants.6 , 7 this includes some sequence variants that have the potential to affect mrna splicing . 
a notable fraction of disease - causing variants in cancer predisposition genes affect splicing.8 - 10 many of these occur at the 5 ( cid : 1 ) and 3 ( cid : 1 ) exon - intron boundaries , and the potential impact on splicing is well established , based on the expected sequence at the canonical splice donor and acceptor ( 6 1 , 2 splice sites ) .5 however , sequence variants located beyond these canonical splice sites may also alter mrna processing . 
pathogenicity for these variants is more difcult to assess , and additional functional evidence is often required to determine the actual impact of the variants on splicing . clinical laboratories commonly use functional rna studies to aid in the classication of variants that may affect splicing . 
although these studies can provide useful evidence about the pathogenicity of such variants , there are important caveats to consider , and care must be taken in both how the studies are performed and how the data are interpreted.11 , 12 one important consideration when using rna analysis to classify variants that may affect splicing is that the observation of a splice defect alone may not be sufcient evidence for classication . 
aberrant splicing may result either in no normal transcript produced by the variant allele ( complete splice defect ) or in some residual normal transcript produced by the variant allele.13 these partial , or leaky , splice defects may result in enough functional transcript being produced to support normal protein function . 
therefore , it is essential to quantify any aberrant splice product and determine whether the variant allele produces any normal transcript when using rna analysis in variant classication . functional rna studies often involve reverse transcription polymerase chain reaction ( rt - pcr ) analysis of rna extracted from a patient blood sample or lymphoblastoid cell line . 
one method to distinguish variant - specic transcripts is to use informative single nucleotide in the same pcr polymorphisms ( snps ) present amplicon as the putative spliceogenic variant being tested ( ie , tag snps )  . 
in the case of a heterozygous variant carrier , these tag snps can differentiate the transcripts produced by the normal allele versus the variant allele , especially when the variant allele is located in the intron and not present in the processed mrna . 
rt - pcr products representing these transcripts can be cloned into a vector system or used directly as templates for sequencing analysis . laboratories that do not require rna analysis to include allele - specic transcript quantication for variant classication , either as part of their own studies or from published studies , may misclassify splice - related variants and potentially report false - positive results . 
in addition , care must be taken in assigning pathogenicity if a variant allele has been shown to produce functional transcript , because it may be unclear whether the amount transcript could support normal protein function . 
 commentary splice variants causing brca2 exon 3 skipping , a variant allele may express up to 20% aberrant product yet not be associated with even a moderate risk of cancer . case study of brca2 c.426 - 12_426 - 8del one example of a splice variant with a complex interpretation is brca2 c.426 - 12_426 - 8delan intronic variant resulting in the deletion of ve nucleotides from the dna sequence adjacent to the splice acceptor site of exon 5 . 
published functional studies support this prediction , with studies by zhang et al in 200916 and sanz et al in 201017 demonstrating that this variant causes skipping of exon 5 , which is out of frame . 
however , both studies also noted that the variant allele produces full - length transcript , indicating a partial splice defect . the variant recently published rna analyses from one clinical testing laboratory showed that brca2 c.426 - 12_426 - 8del caused aberrant splicing , resulting in the deletion of exon 5.18 , 19 there was no allele - specic quantication of however , normal transcripts to determine how much , if any , was produced from the variant allele . 
still , the authors concluded that this variant could be classied as likely pathogenic in accordance with acmg / amp guidelines5 on the basis of rna studies demonstrating abnormal splicing from ( evidence category ps3 ) , absence of population controls ( evidence category pm2 ) , and in silico splicing models predicting a weakening of the native site ( evidence category pp3 ) .18 conversely , our laboratory initially classied brca2 c.42612_426 - 8del as a vus on the basis of the evidence available at the time ( fig 1 )  . 
this evidence included the 2 previously published functional studies showing that the variant allele produced some normal transcript.16 , 17 these functional data alone are not sufcient to determine whether the variant is associated with increased cancer risk , because there is uncertainty in how much normal transcript must be expressed by the variant allele to support normal brca2 function . 
although population databases and in silico modeling can provide useful evidence for classication , these tools also must be considered carefully . benign variants also can be rare ( eg , absent from population controls ) , and in silico splicing models cannot discriminate between a full or partial splice defect . 
as a result , this body of evidence was not sufcient for our laboratory to classify brca2 c.42612_426 - 8del as likely pathogenic . our laboratory was later able to reclassify brca2 c.42612_426 - 8del from vus to benign using additional clinical evidence . 
this included evidence from a validated history weighting algorithm ( hwa ) that scores a variant on the basis of the personal and family histories of multiple individuals who carry the specic variant of interest.20 the variant - specic score is compared with matched pathogenic controls ( individuals with known pathogenic variants in brca2 ) and matched benign controls ( individuals with only benign variants in brca2 and other breast and ovarian cancer - risk genes ; appendix fig a1 )  . 
in this case , the hwa called brca2 c.426 - 12_426 - 8del benign , indicating that carriers did not have a personal or family in brca2 history consistent with a pathogenic variant ( appendix fig a1 )  . in addition , co - occurrence of a vus with known pathogenic variants in the same gene can provide evidence that a variant is benign on the basis of associated clinical phenotypes . 
this provides additional evidence that time of brca2 c.426 - 12_426 - 8del hwa and co - occurrence data are consistent with a benign classication and suggest the variant allele does produce enough functional transcript to support normal brca2 function . 
the recent uptake in laboratories performing their own functional rna studies for variant classication can lead to denitive , actionable ndings for more patients ; however , it is critical that rna analysis be held to a high standard to ensure that variant classications also retain a high level of accuracy . to this end , there are several important considerations in the classication of variants in cancer predisposition genes that may affect splicing . 
snp , single - nucleotide polymorphism . some functional transcript , additional clinical evidence may be needed to determine whether the variant is pathogenic . the importance of this additional clinical evidence is exemplied by brca2 c.426 - 12_426 - 8del . 
karam et al18 came to the conclusion that the variant was likely pathogenic on the basis of rna analysis that identied aberrant splicing , in combination with population frequency and in silico modeling data ; however , the rna analysis performed in that study did not elucidate whether the transcript . 
the variant allele produced any functional previously published rna analyses were considered insufcient for a pathogenic classication by our laboratory because of evidence that the variant allele does produce some normal transcript . 
previous studies have highlighted the complex situation that discordant classications create for patients and providers regarding patient management and the potential for discordant test results for family members tested at different laboratories.6 , 7 second , classications made without careful consideration of rna analysis performed by the laboratory or from published studies may result in a false - positive test result . 
professional societies provide recommendations for the medical management of patients with a pathogenic variant in brca2 , including consideration of risk - reducing surgical interventions and eligibility for cancer drug therapies.1 - 4 the example of brca2 c.426 - 12_426 - 8del highlights the need for caution when interpreting functional evidence from rna analysis for variants that may affect splicing to avoid potential mismanagement and inappropriate medical intervention . in this case , variant classication made without full the partial splice defect consideration of resulted in a classication of likely pathogenic for a variant that our laboratory classied as benign when assessed with additional clinical evidence . acmg / amp guidelines provide useful guidance for variant classication5 but do not inform on every caveat or complexity that may be encountered during variant classication . given the severity of the potential clinical consequences of incorrectly classifying a benign variant as pathogenic , additional guidance for interpreting rna sequencing data may be necessary to enhance accurate classication of splicerelated variants . 
this could involve a stepwise process until sufcient data ( from functional rna studies and other sources ) are obtained to accurately perform variant classication ( fig 1 )  . 
for example , the rna transcripts observed in a patient who carries a sequence variant should be compared with the range of full - length and alternative transcripts that naturally occur in normal controls . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . erin mundt employment : myriad genetics stock and other ownership interests : myriad genetics susan manley employment : myriad genetics stock and other ownership interests : myriad genetics bradford coffee employment : myriad genetic laboratories stock and other ownership interests : myriad genetics benjamin roa employment : myriad genetics leadership : myriad genetics stock and other ownership interests : myriad genetics research funding : myriad genetics patents , royalties , other intellectual property : intellectual property held by employer myriad genetics ( inst ) travel , accommodations , expenses : myriad genetics no other potential conicts of interest were reported . acknowledgment we thank krystal brown , phd , and stephanie meek , phd , for their assistance in preparing the manuscript . references daly mb , pilarski r , berry mp et al : nccn clinical practice guidelines in oncology : genetic / familial high - risk assessment : breast , ovarian , and pancreatic ( version 1.2020 ) , nccn clinical practice guidelines in oncology , 2020 . 
 nix et al armstrong d , plaxe s , alvarex r , et al : nccn clinical practice guidelines in oncology : ovarian cancer including fallopian tube cancer and primary peritoneal cancer , version 4.2017. 
curr oncol 25 : s151 - s160 , 2018 richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
genet med 17 : 405 - 424 , 2015 gradishar w , johnson k , brown k , et al : clinical variant classication : a comparison of public databases and a commercial testing laboratory . 
oncologist 22 : 797 - 803 , 2017 balmaa j , digiovanni l , gaddam p , et al : conicting interpretation of genetic variants and cancer risk by commercial laboratories as assessed by the prospective registry of multiplex testing . 
j clin oncol 34 : 4071 - 4078 , 2016 rhine cl , cygan kj , soemedi r , et al : hereditary cancer genes are highly susceptible to splicing mutations . 
whiley pj , de la hoya m , thomassen m , et al : comparison of mrna splicing assay protocols across multiple laboratories : recommendations for best practice in 12 . 
brnich se , abou tayoun an , couch fj , et al : recommendations for application of the functional evidence ps3 / bs3 criterion using the acmg / amp sequence standardized clinical testing . 
gelli e , colombo m , pinto am , et al : usefulness and limitations of comprehensive characterization of mrna splicing proles in the denition of the clinical relevance of brca1 / 2 variants of uncertain signicance . 
sanz dj , acedo a , infante m , et al : a high proportion of dna variants ofbrca1 and brca2 is associated with aberrant splicing in breast / ovarian cancer patients . 
pruss d , morris b , hughes e , et al : development and validation of a new algorithm for the reclassication of genetic variants identied in the brca1 and brca2 genes . 
 prospective feasibility study for using cell - free circulating tumor dnaguided therapy in refractory metastatic solid cancers : an interim analysis purpose retrospective studies have demonstrated that cell - free circulating tumor dna ( ctdna ) hotspot testing predicts matched therapy response to firstand second - line therapies in patients with advanced nonsmall - cell lung cancer ( nsclc )  . 
however , no prospective outcomes studies have evaluated ctdna - guided matched therapy decision making on the basis of comprehensive plasma genomic testing including all four major classes of alterations . 
here , we report the clinical utility of this approach in advanced solid tumor cancers . patients and methods we conducted a multiple parallel cohort , open - label , clinical trial using ctdna - guided matched therapy when tissue was insufficient or unobtainable for next - generation sequencing . 
patients with prespecified targetable alterations in metastatic nsclc , gastric cancer ( gc ) , and other cancers were matched to several independent targeted agent trials at a tertiary academic center . results somatic alterations were detected in 59 patients with gc ( 78% ) , and 25 patients ( 33% ) had targetable alterations ( erbb2 , n = 11 ; met , n = 5 ; fgfr2 , n = 3 ; pik3ca , n = 6 )  . 
2017 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction the national comprehensive cancer network guidelines recommend genotyping in seven solid tumor cancers ( nonsmall - cell lung cancer [ nsclc ] , breast , gastric , esophageal , colorectal , melanoma , and gi stromal tumors ) for 11 genomic targets ( gts ) to inform targeted therapy selection.1 - 6 however , biopsy specimens can be inadequate for comprehensive profiling in 25% to 50% of patients , 7 - 10 leading to incomplete genotyping or repeat invasive biopsy to obtain more tissue . 
repeat biopsy is also recommended at progression in patients with breast cancer and nsclc to capture targetable genomic changes such as erbb2 ( human epidermal growth factor receptor 2 [ her2 ] ) copy number amplification ( cna ) or egfr and alk resistance mutations , respectively.4 , 5 , 11 comprehensive ctdna testing covering point mutations , insertions / deletions ( indels ) , fusions , seung tae kim kimberly c . 
 and cna may obviate the need for repeat invasive biopsies for genotyping when tissue is of insufficient quantity or unobtainable at initial diagnosis or at progression.12 , 13 in general , next - generation sequencing ( ngs ) seems to detect more actionable variants in target genes than non - ngs methods ( hotspot testing ) such as polymerase chain reaction ( pcr ) , immunohistochemistry ( ihc ) , or fluorescence in situ hybridization.14 - 18 beyond the benefits of invasive biopsy avoidance and higher sensitivity compared with nonngs methods , comprehensive ctdna ngs may provide a global summary of multiple lesions , whereas tissue genotyping of small biopsies may fail to capture intraand intertumor heterogeneity.19 - 21 retrospective studies in nsclc using ctdna genotyping for egfr mutations in the firstline ( egfrl858r / exon19del ) 22 and second - line ( egfrt790m ) 23 , 24 settings have produced response rates similar to studies of therapies directed by tissue - based genotyping . 
the study was conducted in accordance with the current ethical principles outlined in the declaration of helsinki and good clinical practice guidelines . patients eligible patients were older than age 20 years with histologically confirmed metastatic cancer , who had sufficient tumor tissue to test cancer - specific biomarkers but not to undergo comprehensive genomic profiling ( ngs )  . 
rr and disease control rate ( dcr = rr + stable disease ) were centrally adjudicated in accordance with response evaluation criteria in solid tumors ( recist ) version 1.1.28 statistical analysis descriptive statistics were calculated for demographics , ctdna alteration detection rate , and substudy matching . 
after double ultracentrifugation , 5 to 30 ng of cfdna was isolated for digital sequencing as previously described.12 , 26 , 29 all exons in 30 genes and critical exons ( those known to harbor somatic mutations ) of 40 genes were completely sequenced . 
sequencing data were analyzed using a custom bioinformatics pipeline to identify single nucleotide variants ( snvs ) in 70 genes ( 150 - kb panel footprint ) , cnas in 18 genes , indels in three genes ( egfr and erbb2 exons 19 and 20 ; met exon 14 ) , and alk , ret , ros1 , ntrk1 , fgfr2 , and fgfr3 fusions ( appendix fig a2 )  . 
tumor - derived dna shed into the bloodstream increases this value but , as a result of the relative proportions of tumorderived versus leukocyte - derived cfdna , typically a minor contributor . 
90th percentiles , respectively , of all cna calls in the guardant360 database . results patient enrollment and demographics from august 2014 to february 2016 , informed consent was obtained from 210 consecutive patients with metastatic cancer whose tissue was available for cancer - specific biomarker testing , but insufficient for ngs , at initial diagnosis or at progression . 
sixteen patients were lost to follow - up or withdrew consent , leaving 194 patients molecularly profiled by ctdna ngs ( appendix fig a3 )  . median age was 60 years ( range , 28 to 78 years ) for nsclc and 57 years ( range , 23 to 82 years ) for gc , melanoma , and other cancers ; 43% , 58% , 56% , and 89% of patients with these cancers were male , respectively ( table 1 )  . 
newly diagnosed ( firstline ) patients composed 29% of patients with nsclc , 37% of those with gc , 68% of those with melanoma , and 11% of those with other table 1 . 
erbb2 ( her2 ) cna was identified in two patients at progression ( one co - occurring with egfrt790m and one with the erbb2 g778_p780dup ) , and met was amplified in four patients at progression ( one with erbb2 insertion and three with egfrt790m )  . 
however , cnas in nsclc were not prespecified gts . on the basis of rolling substudy availability , inclusion criteria , and patient comorbidities , 10 ( 40% ) of the 25 patients with gc ( erbb2 , n = 6 ; met , n = 1 ; fgfr2 , n = 1 ; and pik3ca , n = 2 ) and 17 ( 50% ) of the 34 patients with nsclc ( egfr , n = 7 ; egfrt790m , n = 7 ; and alk , n = 1 ) with prespecified gts were matched to a molecularly targeted therapy ( tables 2 and 3 )  . 
one patient with gc and two patients with nsclc were lost to follow - up , leaving nine patients ( 90% ) and 15 patients ( 88% ) evaluable for response , respectively . ctdna - guidable targeted therapies and response by cancer type in gc , cnas in erbb2 ( n = 5 ) , fgfr2 ( n = 1 ) , and met ( n = 1 ) and snvs in pik3ca ( n = 2 ) were targeted with one patient achieving complete response ( cr ) , five partial response ( pr ) , and three stable disease ( sd ) for an rr of 67% ( 95% ci , 31% to 91% ) and dcr of 100% ( 95% ci , 63% to 100% ; table 4 , fig 2a )  . 
of patients ctdna alterations detected patients with prespecified second line of therapy third or greater line of therapy treated with matched therapy evaluable for response treated with matched therapy evaluable for response treated with matched therapy evaluable for response treated with matched therapy evaluable for response abbreviations : ctdna , circulating tumor dna ; gt , genomic target . erbb2 amplification amplification fgfr2 amplification pik3ca mutation no . 
one patient with ctdna - detected erbb2 ( her2 ) amplification ( + + + ) achieved complete remission after six cycles of capecitabine , oxaliplatin , and lapatinib ( fig 2b )  . in nsclc , egfrexon19del ( n = 5 ) , egfrl858r ( n = 2 ) , egfrt790m ( n = 7 ) , and alk fusion ( n = 1 ) were targeted , with 13 patients achieving pr and two sd for a rr of 87% ( 95% ci , 58% to 98% ) and a dcr of 100% ( 95% ci , 75% to 100% ; table 4 , fig 2c )  . 
of the seven patients receiving first - line epidermal growth factor receptor inhibitors ( egfri ) , six achieved pr on afatinib , erlotinib , or gefitinib , whereas the one patient with sd received rociletinib . 
similarly , six egfrt790m patients achieving pr were treated with osimertinib or olmutinib , whereas the patient with sd was treated with afatinib plus insulin - like growth factor ligand monoclonal antibody . 
% clinical status at time of ctdna collection new diagnosis treated with matched therapy evaluable for response second line of therapy treated with matched therapy evaluable for response treated with matched therapy evaluable for response treated with matched therapy evaluable for response abbreviations : ctdna , circulating tumor dna ; gt , genomic target . third or greater line of therapy all patients discussion to our knowledge , this is the first prospective ctdna - guided molecular testing program with objective response evaluated in solid tumors . 
this program guided patients in whom biopsy was not readily available or in whom tumor material was not sufficient for comprehensive sequencing to genomically matched therapies available in practice or clinical trials . 
of 194 patients , 30 ( 15.5% ) were successfully enrolled onto one of the ongoing matched therapy clinical trials , a rate comparable to tumor sequencing - based trials . responses to ctdna - guided matched therapy in gc and nsclc were similar to those published in tissue - based matched therapy studies , although the sample sizes here are modest . in gc , cnas were found in erbb2 ( her2 ) , met , and fgfr2 in 31% of our patients , split evenly between newly diagnosed and pretreated patients , consistent with previous primary tumor estimates of these cnas at 20% to 22%.30 , 31 significantly , four ( 80% ) of five patients with erbb2 ( her2 ) - amplified gc responded , including one cr ( fig 2a ) , with all achieving clinical benefit ( cr , pr , or sd )  . 
the one patient with gc with erbb2 cna without pr ( but with sd ) was on lapatinib monotherapy , raising the question of whether chemotherapy produced most of the benefit here . 
however , addition of lapatinib to chemotherapy did produce a significant overall survival benefit in asian patients in the lapatinib optimization study in her2 - positive gastric cancer ( logic ) study.32 in addition , a patient with refractory colon cancer with erbb2 cna achieved sd as best response ( table 4 )  . 
matched therapy response and disease control rate by cancer cohort response gc ( n = 78 ) nsclc ( n = 73 ) melanoma ( n = 34 ) other ( n = 9 ) no . 
thus , an advantage of ctdna over tissue genotyping is that quantitation of the relative vafs can provide an indication of the subclonality and potentially predict treatment response , in contrast to a binary positive or negative result . 
however , a ratio , 10% may be misleading if there is focal amplification of the egfr driver mutation and not egfrt790m.42 beyond t790m , recent reports suggest that comprehensive profiling at progression may be important in nsclc given the multiple other resistance mechanisms after egfri therapy.43 these include non - egfrt790m on - target point mutations , as well as bypass mutations in braf , kras , mek , and pik3ca ; cnas in met and erbb2 ; fusions in alk ; or rb1 inactivation heralding epithelial to mesenchymal cell transition.34 , 44 - 47 because egfrt790m is the resistance mechanism in only half of patients experiencing progression on firstline egfri , a comprehensive ctdna ngs test covering all major types of genomic alterations is particularly relevant . small sample sizes for targeted therapy in melanoma and colon cancer limit the conclusions that can be drawn in those cohorts ; however , the rr cis in gc and nsclc are consistent with tissue - guided matched therapy rrs . 
single - arm objective rrs exceeding 30% have led to us food and drug administration regulatory approval of matched therapies.48 , 49 the rrs to ctdna - detected alterations in this interim analysis ( 67% [ 95% ci , 31% to 91% ] for gc and 87% [ 95% ci , 58% to 98% ] for nsclc ) support clinical utility for guardant360 in patients with advanced nsclc and gc in whom tissue is insufficient or inaccessible and build upon previous validation studies of the diagnostic test used herein.12 , 26 because this study was not randomized , its primary limitation is the potential for selection bias to enroll patients more likely to benefit . 
not all patients with targetable alterations could receive matched therapy because of the various requirements of the multiple parallel matched therapy substudy protocols , performance status , or loss to follow - up . 
future studies should examine ctdnaguided matched therapy outcomes in more racially diverse cohorts . to our knowledge , this is the first prospective study to examine the clinical utility of comprehensive ctdna genomic testing to guide matched therapy selection . 
lanman , amirali talasaz , keunchil park , jeeyun lee financial support : young suk park , amirali talasaz administrative support : young suk park , amirali talasaz provision of study materials or patients : seung tae kim , se hoon park , joon oh park , young suk park , ho yeong lim , won ki kang , amirali talasaz , keunchil park , jeeyun lee collection and assembly of data : seung tae kim , kimberly c . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . ho yeong lim no relationship to disclose won ki kang no relationship to disclose seung tae kim no relationship to disclose kimberly c . 
banks employment : guardant health stock and other ownership interests : guardant health se - hoon lee honoraria : astrazeneca / medimmune , roche , bristol - myers squibb , merck consulting or advisory role : pfizer , novartis , astrazeneca , bristol - myers squibb travel , accommodations , expenses : novartis kyung kim no relationship to disclose joon oh park honoraria : celgene consulting or advisory role : celgene speakers bureau : celgene research funding : celgene , astrazeneca se hoon park no relationship to disclose young suk park no relationship to disclose richard b . 
lanman employment : guardant health , veracyte leadership : guardant health stock and other ownership interests : guardant health , veracyte research funding : guardant health amirali talasaz employment : guardant health leadership : guardant health stock and other ownership interests : guardant health research funding : guardant health patents , royalties , other intellectual property : guardant health travel , accommodations , expenses : guardant health keunchil park consulting or advisory role : astellas pharma , astrazeneca , boehringer ingelheim , clovis oncology , eli lilly , hanmi , kyowa hakko kirin , novartis , ono pharmaceutical , roche speakers bureau : boehringer ingelheim research funding : astrazeneca jeeyun lee no relationship to disclose affiliations support seung tae kim , se hoon park , kyung kim , joon oh park , se - hoon lee , young suk park , ho yeong lim , won ki kang , keunchil park , and jeeyun lee , samsung medical center , sungkyunkwan university school of medicine , seoul , korea ; and kimberly c . 
j natl compr canc netw 13 : 448 - 475 , 2015 von mehren m , randall rl , benjamin rs , et al : gastrointestinal stromal tumors , version 2.2014. 
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lim sm , kim ey , kim hr , et al : genomic profiling of lung adenocarcinoma patients reveals therapeutic targets and confers clinical benefit when standard molecular testing is negative . 
schrock ab , frampton gm , herndon d , et al : comprehensive genomic profiling identifies frequent drug - sensitive egfr exon 19 deletions in nsclc not identified by prior molecular testing . 
ali sm , hensing t , schrock ab , et al : comprehensive genomic profiling identifies a subset of crizotinib - responsive alkrearranged non - small cell lung cancer not detected by fluorescence in situ hybridization . 
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kim st , lee w - s , lanman rb , et al : prospective blinded study of somatic mutation detection in cell - free dna utilizing a targeted 54 - gene next generation sequencing panel in metastatic solid tumor patients . 
zill oa , mortimer sa , banks kc , et al : somatic genomic landscape of over 15 , 000 patients with advanced - stage cancer from clinical next - generation sequencing analysis of circulating tumor dna . 
zill oa , greene c , sebisanovic d , et al : cell - free dna next - generation sequencing in pancreatobiliary carcinomas . cancer discov 5 : 1040 - 1048 , 2015 30 . 
deng n , goh lk , wang h , et al : a comprehensive survey of genomic alterations in gastric cancer reveals systematic patterns of molecular exclusivity and co - occurrence among distinct therapeutic targets . 
hecht jr , bang y - j , qin sk , et al : lapatinib in combination with capecitabine plus oxaliplatin in human epidermal growth factor receptor 2 - positive advanced or metastatic gastric , esophageal , or gastroesophageal adenocarcinoma : trio - 013 / logica randomized phase iii trial . 
arrieta o , cardona af , martn c , et al : updated frequency of egfr and kras mutations in nonsmall - cell lung cancer in latin america : the latin - american consortium for the investigation of lung cancer ( clicap )  . 
rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as first - line treatment for european patients with advanced egfr mutation - positive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
sequist lv , yang jc - h , yamamoto n , et al : phase iii study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with egfr mutations . 
vallee a , audigier - valette c , herbreteau g , et al : rapid clearance of circulating tumor dna during treatment with azd9291 of a lung cancer patient presenting the resistance egfr t790m mutation . 
marchetti a , palma jf , felicioni l , et al : early prediction of response to tyrosine kinase inhibitors by quantification of egfr mutations in plasma of nsclc patients . 
piotrowska z , niederst mj , karlovich ca , et al : heterogeneity underlies the emergence of egfrt790 wild - type clones following treatment of t790m - positive cancers with a third - generation egfr inhibitor . 
liang w , he q , chen y , et al : metastatic eml4 - alk fusion detected by circulating dna genotyping in an egfrmutated nsclc patient and successful management by adding alk inhibitors : a case report . 
stewart el , tan sz , liu g , et al : known and putative mechanisms of resistance to egfr targeted therapies in nsclc patients with egfr mutations : a review . 
most prostate cancers that develop resistance to abiraterone or enzalutamide have active ar signaling , and amplication of the ar gene or its enhancer is thought to be a common resistance mechanism.2 - 4 in the jayaram et al study , plasma was collected before starting treatment from one of four clinical cohorts that used either abiraterone plus low - dose steroid or enzalutamide for treatment of metastatic castration - resistant prostate cancer ( mcrpc ) ( pcr2023 cohort , clinicaltrials.gov identier : nct01867710 ; premier cohort , clinicaltrials.gov identier : nct02288936 ; british columbia cohort , clinicaltrials.gov identier : nct02125357 ; and cohort a , which was on biospecimen collection protocols )  . 
the investigators used droplet digital polymerase chain reaction and / or targeted next - generation sequencing to determine cn gain of the ar gene within the cell - free dna ( cfdna ) in plasma . 
their study adds support for a noninvasive prognostic factor to improve the management of mcrpc . not surprisingly , the rate of ar gain ( 16.5% ) identied in the jayaram et al1 study and in other evaluations of plasma ar status5 , 6 is signicantly lower than what has been found in tissue ( 70% ) , 3 which is even higher when including gain of the ar enhancer ( 81% )  . 
this suggests that the prognostic value of detecting ar gain may be as much in its indication of high ctdna fractionlikely a reection of tumor burdenas in the identication of a putative resistance mechanisindeed , plasma ar gain has been associated with worse os in patients receiving docetaxel8 and worse os and pfs in patients receiving cabazitaxel.9 concentration of cfdna alone is predictive and prognostic in the setting of taxane therapy.10 the authors should be commended for acknowledging a prognostic bias of tumor burden . 
vanderweele research funding : prostate cancer foundation , department of defense - prostate cancer research program , astrazeneca ( inst ) , fortis ( inst ) travel , accommodations , expenses : genzyme no other potential conicts of interest were reported . references jayaram a , wingate a , wetterskog d , et al : plasma androgen receptor copy number status at emergence of metastatic castrationresistant prostate cancer : a pooled multicohort analysis . 
j transl med 12 : 313 , 2014 salvi s , casadio v , conteduca v , et al : circulating cell - free ar and cyp17a1 copy number variations may associate with outcome of metastatic castration - resistant prostate cancer patients treated with abiraterone . br j cancer 112 : 1717 - 1724 , 2015 conteduca v , wetterskog d , sharabiani mta , et al : androgen receptor gene status in plasma dna associates with worse outcome on enzalutamide or abiraterone for castration - resistant prostate cancer : a multiinstitution correlative biomarker study . 
ann oncol 28 : 1508 - 1516 , 2017 annala m , vandekerkhove g , khalaf d , et al : circulating tumor dna genomics correlate with resistance to abiraterone and enzalutamide in prostate cancer . 
cancer discov 8 : 444 - 457 , 2018 conteduca v , jayaram a , romero - laorden n , et al : plasma androgen receptor and docetaxel for metastatic castration - resistant prostate cancer . eur urol 75 : 368 - 373 , 2019 conteduca v , castro e , wetterskog d , et al : plasma ar status and cabazitaxel in heavily treated metastatic castration - resistant prostate cancer . eur j cancer 116 : 158 - 168 , 2019 10 . 
these results highlight the importance of screening for ntrk fusions as part of the tumor genomic proling for patients with pediatric cancer . jco precis oncol 5 : 204 - 214 . 
2021 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction rearrangements involving the neurotrophic tyrosine receptor kinase ( ntrk ) genes ntrk1 , ntrk2 , and ntrk3 encode fusion proteins containing the intact ntrk kinase domain that are oncogenic drivers of a histologically diverse group of tumors . 
 ntrk fusions in pediatric tumors : frequency , partner , and outcome content key objective ntrk fusions are tumor - agnostic or age - independent biomarkers that identify patients suitable for treatment with the us food and drug administrationapproved trk inhibitors . 
this single institutional study examined the frequency , fusion partner , and clinical outcome of ntrk fusions in a large cohort of unselected patients with pediatric cancer . knowledge generated our study demonstrated that ntrk fusions are more frequent in pediatric tumors and involve a broader panel of fusion partners and a wider range of tumors those than previously recognized and highlights pediatric cancers in which ntrk fusions are more or less likely to occur . relevance this study provided important ndings regarding ntrk fusionpositive pediatric cancers and cancers where ntrk fusions are rarely seen . 
immunohistochemistry was performed in a subset of cases using pan - trk antibody ( abcam , cambridge , uk , 1 : 100 dilution ) ( table 1 )  . fusion gene detection was performed using the chop cancer fusion panel as previously described.15 briey , targetspecic primers covering 673 exons were custom designed to identify known fusion genes and potential novel fusion genes associated with 110 cancer genes using anchored multiplex polymerase chain reaction ( pcr ) technology ( archerdx , inc , boulder , co )  . 
all fusions , when identied in our laboratory for the rst time , were conrmed by nested pcr followed by sanger sequencing . mutations ( single nucleotide variants [ snvs ] and small insertion / deletions [ indels ] ) and copy number alterations ( cnas ) were evaluated by using the chop hematological malignancy panel ( chmp ) or solid tumor panel ( cstp ) as described previously.16 briey , genomic dna was extracted from the tumor samples . 
libraries were prepared using probes targeting 118 genes ( chmp ) or 238 genes ( cstp ) , respectively , and sequenced on illumina hiseq platform using 150 bp paired - end sequencing . 
sequence data were analyzed using the home brew software concords v2 ( for snvs and indels ) and nextgene v2 next - generation sequencing analysis software ( for cnas ; softgenetics , llc , state college , pa )  . 
clinically signicant variants including indels , and cnas were conrmed by sanger sesnvs , quencing , multiplex ligation - dependent probe amplication ( mlpa ) , real - time pcr , or droplet digital pcr ( ddpcr ) when necessary . 
by contrast , different ntrk fusions were present in tumors with the same histological diagnosis , such as kctd16 - ntrk2 and trim24 - ntrk2 in two male patients of similar age with ganglioglioma , who grade i ( table 1 )  . 
in ptc , tumorigenesis is associated with constitutive activation of the mapk and pi3k signaling pathways secondary to somatic point mutations in braf , pten , dicer1 , and ras as well as fusions involving ret , alk , and ntrk.3 , 23 gene fusions have been reported in both sporadic and radiation - induced ptcs and are more common in pediatric ( 50% - 60% ) compared with adult tumors ( approximately 15% ) .2 , 23 ret and ntrk fusions are the most common , reported in 25%30% and approximately 10% ( range , 0% - 26% ) of pediatric ptcs , respectively.2 , 23 ntrk3 fusions are usually observed more commonly in ptcs than ntrk1 fusions , 2 , 23 which is similar to what we observed in our patient cohort . 
all the ptcs in our cohort were sporadic , none associated with ntrk1 ntrk2 ntrk3 snvs / indels cnas 10 - 15 16 - 20 gender female male snvs and indels missense frameshift inframe cnas gain loss gain and loss cnloh fig 1 . 
case 17 demonstrates a low - grade tumor with strn3 - ntrk3 showing moderately cellular spindle to round cell proliferation with prominent vascular proliferation ( c , h&e , 200 )  . 
case 21 shows widely invasive follicular variant of papillary thyroid carcinoma with etv6 - ntrk3 demonstrating extrathyroidal extension and angioinvasion ( d , h&e , 50 ) and nuclear features of papillary thyroid carcinoma ( inset , 400 )  . 
two cases demonstrate the diffuse sclerosing variant of papillary thyroid carcinoma showing solid and papillary groups inltrating the thyroid parenchyma with numerous psammomatous calcications ( e , h&e , 100 )  . 
the data are limited but suggest that the presence of an ntrk fusion may have diagnostic , prognostic , and therapeutic signicance in ptcs and may hold clinical utility for stratifying surgical and medical care . 
standard therapy for advanced ptcs in children involves surgical resection of gross disease followed by radioactive - iodide ( rai ) therapy . in a recent multicenter , open - label phase i or ii study of larotrectinib for the treatment of pediatric patients with solid tumors , two children with advanced ptcs were treated for  . 
7 months and remained progression - free , although , unfortunately , the objective response to treatment was not reported.25 ntrk fusions in pediatric cns tumors all the cns tumors identied with ntrk fusions were either gliomas or mixed neuronal glial tumors . 
four of the six ntrk2 fusions were novel fusions at the time of discovery with different 5 ( cid : 1 ) - partner genes including kank1 , c2orf4 , kctd16 , and specc1l . 
ntrk fusion genes have been described in pediatric lowand highgrade gliomas at a low prevalence , 26 although one study reported a nding of 40% of nonbrainstem high - grade gliomas in children younger than 3 years old containing an ntrk fusion gene ( n = four of the 10 samples ) .27 six of the 7 ntrk fusionpositive cns tumors were low - grade gliomas ( lgg ) , which may be partially due to the higher frequency of lgg in our unselected pediatric cohort ( approximately 45% of all cns tumors )  . 
although the duration of follow - up in our study is limited , the majority of patients underwent standard of care for their cns tumor subtype with resection of the primary tumor without recurrence . 
larotrectinib and entrectinib have shown antitumor effect for both primary brain tumors and solid tumors with brain metastases with systemic administration suggesting adequate penetration of the blood - brain barrier.11 in a study of nine patients with primary cns tumors treated with ntrk inhibitors , disease control was observed in all evaluable patients with stable disease in seven patients . 
some tumors exhibited typical age , morphology , and etv6 - ntrk3 fusion compatible with if , showing densely cellular fascicular growth of primitive ovoid cells and inltration of surrounding tissue ( cases 13 and 14 )  . 
clinical classication and treatment based on histology only , particularly in soft - tissue tumors with spindle morphology , have proven to be challenging in some cases because of variable histologic features and immunohistochemical patterns.30 as more tumors are being studied for fusions , the morphologic spectrum is expanding , such that the fifth edition of the soft tissue and bone tumors who classication31 now includes an emerging entity titled ntrk - rearranged spindle cell neoplasm to encompass spindle cell soft - tissue tumors with ntrk gene rearrangements ( other than if )  . 
it remains to be seen if soft - tissue histologic classication , molecular classication , or some combination of both will provide clinicians with the most accurate information for personalized treatment . 
magnetic resonance imaging at diagnosis ( a ) , 1 month post - targeted therapy ( b ) , and 5 months post - therapy ( c ) showing marked tumor shrinkage . 
at diagnosis , the tumor contained densely cellular areas with spindled to round cells and increased mitosis ( d , h&e 400 ) with cytoplasmic and membranous pan - trk immunohistochemistry ( inset )  . 
ccd , coiled - coil domain ; chd , calponin homology domain ; ecd - lb , extracellular ligand binding domain ; h&e , hematoxylin and eosin staining ; pkt , protein tyr kinase ; tm , transmembrane domain ; ss , signal sequence . ntrk fusions facilitate precision diagnosis , prognosis , and therapy follow - up information is available for all patients , and follow - up times ranged from 6 to 46 months . 
in almost all cases , the detection of an ntrk fusion conrmed the morphologic diagnosis , and in ve cases , the nal tumor diagnosis was largely based on the discovery of an ntrk fusion ( excluding other differential diagnostic considerations )  . 
one exception was the case of secretory carcinoma , which was initially diagnosed as mucoepidermoid carcinoma , but later changed to secretory carcinoma following the detection of the etv6 - ntrk3 and additional immunohistochemical evaluation ( case 10 )  . 
collectively , we analyzed 261 common embryonal solid tumors ( 79 neuroblastomas , 29 medulloblastoteratoid / rhabdoid mas , 28 wilms tumors , 13 atypical tumors , and 11 hepatoblastomas ) , bone tumors ( 27 osteosarcomas and 24 ewing sarcomas ) , and skeletal muscle tumors ( 50 rhabdomyosarcomas ) , and none had ntrk fusions . 
thus , although it is difcult to exclude the possibility that ntrk fusions might occur in individual tumors of these subtypes , which accounted for about one third of all solid tumors in this cohort , they are likely to be rare . case 15 was a 6 - month - old male infant with a left upper extremity mass . 
prospectively , the rest of the patients who survived with the standard therapy may also benet from trk inhibitor therapy if their tumors progress or recur . received neoadjuvant we assessed the clinical outcome of all ntrk fusionpositive patients for up to 46 months ( median 21 months )  . 
the median follow - up time for patients with cns tumors was 21 months . the majority of ntrk - positive lgg demonstrated superb outcome with gross total resection without additional therapy . one patient with congenital glioblastoma and an etv6ntrk3 fusion died 3 months after birth , and the tumor specimen was obtained via autopsy . 
our review of 1 , 217 patients showed that ntrk fusions are more frequently seen in pediatric tumors than in adult tumors and involve a broader panel of fusion partners and a wider range of pediatric tumors than previously recognized . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / cci / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . elizabeth fox other relationship : helsinn therapeutics gerald b . 
wertheim employment : johnson & johnson stock and other ownership interests : johnson & johnson vinodh pillai consulting or advisory role : foundation medicine travel , accommodations , expenses : foundation medicine jennifer e . 
genes ( basel ) 10 : 723 , 2019 prasad ml , vyas m , horne mj , et al : ntrk fusion oncogenes in pediatric papillary thyroid carcinoma in northeast united states . 
hsiao sj , zehir a , sireci an , et al : detection of tumor ntrk gene fusions to identify patients who may benet from tyrosine kinase ( trk ) inhibitor therapy . 
nat rev clin oncol 15 : 731 - 747 , 2018 suurmeijer aj , dickson bc , swanson d , et al : the histologic spectrum of soft tissue spindle cell tumors with ntrk3 gene rearrangements . 
papadopoulos kp , gandhi l , janne pa , et al : first - in - human study of ds - 6051b in patients ( pts ) with advanced solid tumors ( ast ) conducted in the us . 
drilon a , ou si , cho bc , et al : repotrectinib ( tpx - 0005 ) is a next - generation ros1 / trk / alk inhibitor that potently inhibits ros1 / trk / alk solventfront mutations . 
lopez gy , perry a , harding b , et al : cdkn2a / b loss is associated with anaplastic transformation in a case of ntrk2 fusion - positive pilocytic astrocytoma . neuropathol appl neurobiol 45 : 174 - 178 , 2019 19 . 
surrey lf , jain p , zhang b , et al : genomic analysis of dysembryoplastic neuroepithelial tumor spectrum reveals a diversity of molecular alterations dysregulating the mapk and pi3k / mtor pathways . 
tuttle rm , haugen b , perrier nd : updated american joint committee on cancer / tumor - node - metastasis staging system for differentiated and anaplastic thyroid cancer ( eighth edition ) : what changed and why ? thyroid 27 : 751 - 756 , 2017 25 . 
laetsch tw , dubois sg , mascarenhas l , et al : larotrectinib for paediatric solid tumours harbouring ntrk gene fusions : phase 1 results from a multicentre , 26 . 
okamura r , boichard a , kato s , et al : analysis of ntrk alterations in pan - cancer adult and pediatric malignancies : implications for ntrk - targeted theropen - label , phase 1 / 2 study . 
nat genet jones ka , bossler ad , bellizzi am , et al : bcr - ntrk2 fusion in a low - grade glioma with distinctive morphology and unexpected aggressive behavior . 
robinson gw , gajjar aj , gauvain km , et al : phase 1 / 1b trial to assess the activity of entrectinib in children and adolescents with recurrent or refractory solid tumors including central nervous system ( cns ) tumors . 
 o persistent severe hyperlactatemia and metabolic derangement in lethal sdhb - mutated metastatic kidney cancer : clinical challenges and examples of extreme warburg effect see accompanying editorial doi : purpose to describe the unique clinical features , determine the genomics , and investigate the metabolic derangement of an extremely rare form of a hereditary lethal kidney cancer syndrome . patients and methods three patients with lethal kidney cancer ( age 19 , 20 , and 37 years ) exhibiting persistent ( 1 to 3 months ) extremely high levels of blood lactate ( > 5 mm ) despite normal oxygen perfusion , highly avid tumors on [ 18f ] fluorodeoxyglucose positron emission tomography ( pet ) , and pleomorphic histopathologic features were identified and treated in a single institute . 
integrated studies including whole - genome sequencing ( wgs ) , targeted sequencing , immunohistochemistry , cell - based assays , and 18f - glutamine pet imaging were performed to investigate this rare kidney cancer syndrome . results all three patients with kidney cancer were initially given various diagnoses as a result of diverse tumor histopathology and atypical clinical presentations . 
genomic studies of the blood and tumors of these patients identified three different kinds of germline lossof - function mutations in the sdhb gene and the common loss of heterozygosity in the remaining sdhb allele thorough somatic chromosome 1p deletion . 
in one patient , wgs revealed that a germline mutation of sdhb coupled with loss of heterozygosity was the sole genetic event . cancer evolution analysis of sdhb tumors based on wgs demonstrated that sdhb in kidney epithelium fulfills the knudson two - hit criteria as a major tumor suppressor gene . 
right panels are corresponding blood lactate levels over the monitoring period for patients 1 , 2 , and 3 . mutated gene in sdh - rcc is sdhb ( 83% ) .18 - 20 as a result of its rarity and recent designation , clinical and molecular features associated with aggressive sdhb - deficient rcc are unknown . the aerobic glycolysis warburg effect in cancer was recognized by otto warburg , 21 , 22 who first described a phenomenon where glucose undergoes lactic acid fermentation in lieu of aerobic respiration despite normal oxygen tension , providing adaptive advantages for proliferating cancer cells.23 - 26 direct investigation of the warburg effect in human cancer remains a challenge.27 , 28 the increased activity detected by [ 18f ] fluorodeoxyglucose ( fdg ) positron emission tomography ( pet ) reflects glucose uptake and thereby renders indirect evidence of aerobic glycolysis . 
cb - 839 was provided by calithera biosciences ( south san francisco , ca )  . germline dna analysis germline dna was purified from blood and subjected to sequencing on all coding exons of the sdhb , sdhc , sdhd , and af2 genes by genedx ( elmwood park , nj ) .45 histopathology and immunohistochemistry immunohistochemistry for sdhb was performed using anti - shdb antibody ( ab14714 , clone 21a11 ; abcam , cambridge , united kingdom ) .20 normal pair samples were prepared from the primary kidney tumor of patient 1 and the primary tumor and metastases of patient 3 . 
paired - end 2 3 100 base pair sequence reads were performed using illumina hisequation 2500 instruments ( illumina , san diego , ca ) , mapped using the burrows - wheeler aligner , and processed with the genome analysis toolkit . 
target - specific probes for hybrid selection were designed to capture all protein - coding exons of 341 cancer genes.48 metabolic assessment of patient - derived sdhb - deficient cancer cells short - term primary cell cultures were established from patient 1s tumor ( sdhb2 / 2 tumor ) , patient 1s tumor - adjacent tissue ( sdhb + / 2 cells ) , and an unrelated patients tumor - adjacent normal kidney tissues ( normal kidney )  . 
metabolites were extracted by 80% methanol , and count values were normalized to cell number . whole - genome sequencing 18f - glutamine pet whole - genome sequencing ( wgs ) was performed at the new york genome center ( new york , ny )  . 
 [ 18f ] fdg - pet confirmed extensive metastatic lesions and noted additional involvement of the parietal lobe and cranium ( maximum suv , 44.1 ; fig 1 )  . 
his hospital course was complicated by multiple unexplainable hemolytic and thrombotic events , including pulmonary emboli , numerous cerebral vascular events , and infarction of the superior mesenteric artery necessitating exploratory laparotomies for small bowel and colon resections . 
the increasing blood lactate was associated with increasing tumor burden . patient 3 had persistently elevated lactate levels with a baseline of 4.4 mm / l and a peak of 7.5 mm / l ( fig 1 )  . 
as a result of the high blood lactate level and suspicion of sdhbdeficient rcc based on fdg - pet images , an arterial blood gas was obtained ( ph , 7.43 ; partial pressure of carbon dioxide , 28.6 mm hg ; bicarbonate , 19.3 meq / l ; and oxygen saturation , 98% ; fig 1 )  . diverse histopathology of sdhb - deficient the histopathologic features varied significantly among our patients with sdhb - deficient cancer , making the initial diagnosis challenging . 
 1 7 3 4 4 0 0 0 17352000 17360000 17368000 sdhb exons 4 - 5 patient 1 260 substitutions 72 deletions and insertions 7 rearrangements complex insertion deletion other deletion repeat deletion m - homology t . 
common histology described for sdhbdeficient renal tumors , such as bubbly eosinophilic cytoplasm and cytoplasmic inclusions , 56 was detected only in patient 1s primary tumor but not in the tumors of patients 2 and 3 . loss of sdhb staining in tumors as a result of unusual clinical and histopathologic presentations , sdhb - deficient rcc was suspected in these patients . 
 the two innermost tracks depict the copy number changes ( red , increase ; blue , decrease ; purple , allelic imbalance , one allele amplified or deleted ) in the tumor when compared with the normal for patient 1 . the outermost track shows the intermutation distance for substitutions each plotted according to the type of nucleotide change . the middle track shows the genomic positions of the small insertion ( green ) and deletion ( red ) along the genome . 
immunohistochemistry stain for sdhb protein was subsequently established at memorial sloan kettering cancer center , demonstrating the loss of sdhb staining in cancer but not adjacent non - neoplastic cells ( fig 2a )  . 
as a control , oncocytoma , a kidney tumor with wild - type sdhb , showed strong sdhb staining ( fig 2a )  . patient 1s family pedigree the young age of patient 1 and the unusual histopathologic features of his tumor were concerning for a hereditary rcc . 
accordingly , germline dna analysis was performed by direct sequencing of the sdhb , sdhc , sdhd , and af2 genes , 45 which revealed a germline exon 45 deletion within the sdhb gene in patient 1 ( fig 2b )  . 
germline duplication in sdhb and somatic loss of 1p , where sdhb resides ( 1p36 ) , resulted in complete genetic loss of sdhb in patient 2s tumor ( fig 2b )  . wgs of patient 1s primary tumor to determine genetic events contributing to the pathogenesis of sdhb - deficient rcc , wgs of patient 1s tumor and matched normal tissue was performed to mean haploid coverage of approximately 903 with greater than 96% of the genome at > 303 . 
wgs identified only a single nonsynonymous somatic substitution ( srebf1 p.d329y ) with no significant impact based on functional prediction.57 copy number the tumor revealed few genomic analysis of alterations including loss of the short arm of chromosome 1 where sdhb resides ( fig 3 )  . 
furthermore , close examination of the sdhb locus in the retained allele found that both tumor and normal tissue have the same deletion spanning exons 4 and 5 of the sdhb coding sequence ( chr1 : 17 , 353 , 986 to 17 , 359 , 321 ; fig 3 ) , resulting in a frameshift mutation . 
altogether , a germline intragenic deletion in sdhb and a somatic loss of 1p resulted in complete loss of sdhb in the tumor ( fig 2b )  . wgs and clonal evolution of patient 3s primary and metastatic tumors wgs was performed on patient 3s primary kidney tumor as well as his metastatic liver and scapula bone lesions ( fig 4 and data supplement )  . 
of note , the same sdhb mutation has been reported in patients with gi stromal tumor and paraganglioma.58 detailed characterization of the subclonal structure using the substitution data established a long trunk with 5 , 198 of 8 , 047 substitutions in 100% of cells across tumor sites . 
the second largest subclone supported by 820 substitutions was present in approximately 55% and 100% of cells in the liver and bone metastases , respectively , likely reflecting tumor heterogeneity.46 overall , subclonal relationship among the three tumor sites suggests relatively late metastasis during clonal evolution . disruption of mitochondrial architecture in sdhb - deficient cancer cells because sdhb encodes a tca cycle protein , we examined the mitochondria morphology of patient 1s sdhb - deficient primary tumor cells under electron microscopy . 
 primary tumor 5 , 809 substitutions liver metastasis 0 10 20 30 40 50 60 5 , 916 substitutions 394 deletions and insertions 50 100 150 200 ( most recent common ancestor ) 81 rearrangements patient 3 mrca complex insertion deletion other deletion repeat deletion m - homology t . 
duplication deletion inversion translocation 50 100 150 99 rearrangements 0 10 20 30 40 50 60 70 8 , 817 substitutions 679 deletions and insertions complex insertion deletion other deletion repeat deletion m - homology t . 
whole - genome sequencing and clonal evolution analysis of patient 3s paired primarymetastatic kidney tumors . mrca denotes the most recent common ancestor cell identified during the cancer evolution of individual tumors.46 loh , loss of heterozygosity ; mtor , mammalian target of rapamycin . electron transport chain ( etc ) complex v , was used to inhibit mitochondrial oxygen consumption , thereby activating glycolysis , and carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ( fccp ) , an uncoupler of the mitochondrial proton gradient , was used to induce maximum mitochondrial oxygen consumption . 
 ( d ) relative succinate levels . intracellular succinate was quantified by gas chromatographymass spectrometry and then normalized to cell number . error bars indicate standard deviation ( n = 3 )  . fccp , carbonyl cyanide4 - ( trifluoromethoxy ) phenylhydrazone . primary cell production at baseline as a result of the preexisting defect in oxidative respiration . 
measurement of intracellular metabolites showed that sdhb2 / 2 kidney cancer cells had increased succinate compared with sdhb + / 2 cells and rcc4 and a498 ( fig 5d ) , suggesting that loss of both copies of sdhb resulted in succinate accumulation . glutamine uptake in the sdhb - deficient kidney cancer to determine whether the use of other carbon sources was altered in sdhb - deficient kidney cancer , an 18f - glutamine pet was used to measure glutamine uptake in patient 2 ( fig 6a )  . patient 2s tumors exhibited elevated 18fglutamine uptake , implicating increased dependence on glutamine metabolisusing primary sdhb2 / 2 tumor cells from patient 1 , glutamine deprivation resulted in decreases in glutamate and succinate , which are downstream catabolites of glutamine ( fig 6b )  . 
similarly , lactate downstream of glucose metabolism was unaffected by glutamine deprivation ( fig 6b )  . furthermore , prolonged glutamine deprivation in sdhb2 / 2 tumor cells resulted in increased cell death ( fig 6c )  . discussion here we report three patients with sdhbdeficient lethal rcc . 
annexin v staining of apoptotic cells was determined in sdhb2 / 2 culture after 72 hours of glutamine deprivation . error bars indicate standard deviation ( n = 3 )  . ( d ) model depicts the increased glucose flux and lactate production in sdhb - deficient cancer cells . 
all three individuals carry distinct germline mutations in one copy of the sdhb gene , and through chromosome 1p loss , the remaining copy of sdhb is lost in their tumors , consistent with the knudson two - hit hypothesis.59 mitochondrion generates atp and provides biosynthetic intermediates via two interlinked processes ( ie , etc and tca cycle )  . 
unlike the other dedicated tca cycle enzymes , the sdh enzyme also functions as complex ii in etc , coupling oxidation of succinate to fumarate in the tca cycle with electron transfer to ubiquinone in the etc . 
sdh enzyme is highly conserved and consists of a , b , c , and d subunits , with sdha and sdhb as the catalytic subunits extending into the mitochondrial matrix and anchored to its inner membrane with sdhc and sdhd ( fig 6d )  . 
of note , homozygous mutations in sdha cause severe neurologic defects in infants , known as leigh syndrome , characterized by subacute necrotizing encephalomyelopathy , failure to thrive , ataxia , seizures , and severe lactic acidosis.37 to our knowledge , the association between chronic hyperlactatemia and overt metastatic sdhb kidney cancer has never been reported . metabolic assessment of patient 1s sdhb2 / 2 tumor cells demonstrated the uncoupling between etc and tca , manifested by the lack of oxidative phosphorylation , the overproduction of lactate , and the dependence on aerobic glycolysis , exemplifying the extreme warburg effect in human cancer ( fig 6d )  . 
the mechanisms by which sdhb loss might contribute to tumorigenesis have been investigated using cell - based assays and mouse paraganglioma models.60 - 63 these studies highlighted the role of accumulated succinate as an oncometabolite that inhibits a - ketoglutaratedependent dioxygenases , resulting in metabolic and epigenetic changes.60 - 63 in fact , our genomic study of these patients demonstrated that the complete loss of sdhb constitutes the first event in the pathogenesis of sdhbdeficient kidney cancer . from 2011 to 2015 , 4 , 328 patients were diagnosed with rcc at memorial sloan kettering cancer center , among whom eight ( 0.18% ) have sdhb - deficient rcc . 
limited clinical data presented in our small series suggested varied treatment benefit with vascular endothelial growth factor and possibly mammalian target of rapamycin inhibitors , and the use of glutaminase inhibitor in this rare disease deserves further investigation . 
hsieh honoraria : novartis , pfizer , chugai , eisai , cancer genomics consulting or advisory role : novartis , pfizer , chugai , eisai , cancer genomics research funding : novartis , pfizer , chugai , eisai , cancer genomics acknowledgment we dedicate this article to the patients and their families and are forever grateful for their inspirational courage instilled upon us when fighting against this deadly disease . ying - bei chen no relationship to disclose justin r . 
cheng no relationship to disclose ramaprasad srinivasan no relationship to disclose dayna oschwald no relationship to disclose affiliations support chung - han lee , gunes gundem , william lee , ying - bei chen , justin r . 
moch h , cubilla al , humphrey pa , et al : the 2016 who classification of tumours of the urinary system and male genital organspart a : renal , penile , and testicular tumours . 
hsieh jj , manley bj , khan n , et al : overcome tumor heterogeneity - imposed therapeutic barriers through convergent genomic biomarker discovery : a braided cancer river model of kidney cancer . 
ricketts cj , forman jr , rattenberry e , et al : tumor risks and genotype - phenotype - proteotype analysis in 358 patients with germline mutations in sdhb and sdhd . 
williamson sr , eble jn , amin mb , et al : succinate dehydrogenase - deficient renal cell carcinoma : detailed characterization of 11 tumors defining a unique subtype of renal cell carcinoma . 
lu j , tan m , cai q : the warburg effect in tumor progression : mitochondrial oxidative metabolism as an antimetastasis mechaniscancer lett 356 : 156 - 164 , 2015 27 . 
fraley ds , adler s , bruns fj , et al : stimulation of lactate production by administration of bicarbonate in a patient with a solid neoplasm and lactic acidosis . 
friedenberg as , brandoff de , schiffman fj : type b lactic acidosis as a severe metabolic complication in lymphoma and leukemia : a case series from a single institution and literature review . 
martinez - outschoorn ue , whitaker - menezes d , valsecchi m , et al : reverse warburg effect in a patient with aggressive b - cell lymphoma : is lactic acidosis a paraneoplastic syndrome ? semin oncol 40 : 403 - 418 , 2013 36 . 
xu j , pham cg , albanese sk , et al : mechanistically distinct cancer - associated mtor activation clusters predict sensitivity to rapamycj clin invest 126 : 3526 - 3540 , 2016 45 . 
miyakita h , tokunaga m , onda h , et al : significance of 18f - fluorodeoxyglucose positron emission tomography ( fdg - pet ) for detection of renal cell carcinoma and immunohistochemical glucose transporter 1 ( glut - 1 ) expression in the cancer . 
ho cl , chen s , ho km , et al : dual - tracer pet / ct in renal angiomyolipoma and subtypes of renal cell carcinoma . clin nucl med 37 : 1075 - 1082 , 2012 53 . 
voss mh , molina am , chen yb , et al : phase ii trial and correlative genomic analysis of everolimus plus bevacizumab in advanced non - clear cell renal cell carcinoma . 
collins n , dietzek a : contiguous bilateral head and neck paragangliomas in a carrier of the sdhb germline mutation . nucleic acids res 39 : e118 , 2011 j vasc surg 55 : 216 - 219 , 2012 59 . 
letouze e , martinelli c , loriot c , et al : sdh mutations establish a hypermethylator phenotype in paraganglioma . cancer cell 23 : 739 - 752 , 2013 61 . 
xiao m , yang h , xu w , et al : inhibition of a - kg - dependent histone and dna demethylases by fumarate and succinate that are accumulated in mutations of fh and sdh tumor suppressors . 
craig lockhart , md17 ; cristiana sessa , md18 ; thomas zander , md19 ; matthew ng , md , phd20 ; giuseppe curigliano , md , phd21 , 22 ; jennifer bendiske , mba23 ; xueying chen , phd23 ; somesh choudhury , phd23 ; diana graus - porta , phd24 ; nancy lewis , md23 ; jose manuel perez garcia , md , phd25 ; and mara jos e de miguel - luken , md , phd26 purpose concurrent pik3ca mutations and broblast growth factor receptor ( fgfr ) alterations occur in multiple cancer types , including estrogen receptorpositive breast cancer , bladder cancer , and endometrial cancer . 
archival tumor samples were sequenced to explore genomic correlates of response . results in combination , both agents were escalated to full , single - agent recommended doses ( alpelisib , 300 mg per day continuously ; ingratinib , 125 mg per day 3 weeks on followed by 1 week off )  . 
the toxicity prole of the combination was consistent with the established safety prole of each agent , although 71% of all patients required at least one treatment interruption or dose reduction . 
molecularly selected dose expansions in breast cancer and other solid tumors harboring pik3ca mutations , alone or in combination with fgfr alterations , identied sporadic responses , predominately in tumor types and genotypes previously dened to have sensitivity to these agents . conclusion the combination of alpelisib and ingratinib can be administered at full single - agent doses , although the high rate of dose interruption or reduction suggests long - term tolerability may be challenging . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction genome - driven oncology has been focused primarily , to date , on the targeting of individual genomic alterations present in each tumor.1 although this approach has led to the development of many highly effective therapies , 2 - 6 we now understand that most oncogenes exist within a complex genomic landscape characterized by additional alterations that may , themselves , be targetable or modify sensitivity to therapy.7 even in instances in which the paradigm of targeting an individual genomic alteration has been successful , adaptation of the tumor eventually leading to acquired resistance ultimately limits effectiveness . 
 hyman et al context key objective to determine if the - elective pi3k inhibitor alpelisib can be combined safely with the selective broblast growth factor receptor ( fgfr ) inhibitor ingratinib . knowledge generated alpelisib and ingratinib were successfully escalated in combination to full single - agent doses ; however , high rates of treatment interruption and dose reduction suggest long - term tolerability may be challenging . 
in exploratory signal - seeking cohorts of patients harboring dual pik3ca and fgfr1 - 3 alterations , no clear evidence of synergistic activity was observed . relevance additional studies are necessary to determine the potential role of combined pi3k and fgfr inhibition in treatment of various advanced solid tumors with alterations in pik3ca and / or fgfr1 - 3 . suggested that the combined inhibition of these oncogenes might be benecial.14 moreover , in breast cancer , pik3ca mutations often co - occur with fgfr1 gene amplications , indicating pik3ca and fgfr1 could cooperate as potential dual oncogenic drivers in this tumor type.12 , 15 similarly , concurrent activating pik3ca and fgfr2 mutations have been observed in endometrial carcinomas , with 90% or more fgfr2 - mutant endometrial cancers also exhibiting genomic activation of the pi3k pathway.16 - 18 taken together , these data provided a rationale for evaluating the feasibility and preliminary activity of combined fgfr and pik3ca inhibition in the clinic . to evaluate this therapeutic strategy , we studied the combination of alpelisib ( byl719 ) and ingratinib ( bgj398 )  . 
alpelisib is a selective class i pi3k inhibitor that recently demonstrated a statistically signicant and clinically relevant improvement in progression - free survival in a phase iii study ( solar - 1 ) of patients with pik3camutant , er + metastatic breast cancer.19 ingratinib is an orally bioavailable , selective pan - fgfr kinase inhibitor that has demonstrated single - agent activity in fgfr2 / 3altered bladder cancers and fgfr2 fusion - positive cholangiocarcinomas.20 , 21 we hypothesized that the combination of a selective fgfr and pik3ca inhibitors would be safe and would synergize in tumors with concurrent alteration of these oncogenes . 
dose escalation was guided by a bayesian logistic regression model with escalation with overdose control.22 in total , three arms in the expansion cohort were planned : dual pik3camutant and fgfr - altered breast ( arm 1 ) and nonbreast ( arm 2 ) cancers and pik3ca - mutant solid tumors ( arm 3 )  . because of the slow rate of accrual and overall safety prole , the study was closed before the target enrollment was completed . the primary objective was to determine the maximum tolerated dose and / or recommended dose for expansion of the combination of ingratinib and alpelisib . 
the secondary objectives included assessment of safety and tolerability of the combination , characterization of pharmacokinetic ( pk ) proles , assessment of preliminary antitumor activity as measured by overall response rate ( orr ) , and progressionfree survival ( pfs ) , per recist , version 1.1. study drug administration eligibility patients were at least 18 years of age and had advanced solid tumors with pik3ca mutation with or without fgfr genetic alteration in which standard therapy was unsuccessful . 
patients remained in the study until disease progression , unacceptable adverse investigator events ( aes ) , or withdrawal at patient or discretion . study assessments tumor responses were measured by recist , version 1.1 ( investigator assessed ) , using computed tomography or magnetic resonance imaging.23 assessments were performed at screening and every 8 weeks after rst treatment until disease progression . 
safety was monitored throughout institute the study according to the national cancer common terminology criteria for adverse events , version 4.03.24 ecgs were assessed and independently reviewed by a central laboratory . pharmacokinetic analysis during dose escalation , blood samples were collected to measure alpelisib and ingratinib plasma concentrations . samples were collected predose and 0.5 , 1 , 2 , 3 , 4 , 6 , 8 , and 24 hours postdose on cycle 1 day 1 , cycle 1 day 15 , and cycle 2 day 1 . 
drug plasma concentrations ( and active metabolites ) were measured using validated liquid chromatographytandem mass spectrometry with a lower limit of quantication of 1 ng / ml . genomic sequencing genomic alterations potentially associated with fgfr and pik3ca signaling were assessed from archival tumor specimens of fresh , pretreatment tumor biopsy specimens . for samples with sufcient tissue material ( n = 41 ) , nextgeneration sequencing ( ngs ) analysis was performed in a clinical laboratory improvement amendmentscertied laboratory ( foundation medicine , cambridge , ma ) using previously published methods.25 statistical analysis the data cutoff for all statistical analyses was august 23 , 2016 . 
the sample size for each expansion cohort was selected primarily for continued safety evaluation and was not powered for any specic efcacy end point . results patient demographics a total of 62 patients were enrolled in the study between october 2013 and august 2016 across 22 clinical centers in 12 countries . 
the median age of patients at the time of study entry was 60 ( range , 30 to 78 ) years , and all patients had an eastern cooperative oncology group performance status of 1 or less . 
patients were heavily pretreated , having received a median of three prior lines of systemic therapy . safety and tolerability dose - limiting toxicities were reported in three patients ( 10.7% ) who were included in the dose - escalation cohort ( n = 38 ) during the rst 28 days of treatment . 
dose - limiting toxicities included grade 4 hyperglycemia in one patient receiving alpelisib 300 mg and ingratinib 40 mg ; and grade 3 stomatitis in two patients ( one receiving alpelisib 300 mg and ingratinib 75 mg ; and the other receiving alpelisib 300 mg and ingratinib 100 mg )  . 
the recommended dose for expansion was determined to be alpelisib 300 mg once per day ( administered continuously ) and ingratinib 125 mg once per day ( administered on a schedule of 3 weeks on followed by 1 week off )  . overall , the safety prole of alpelisib and ingratinib was similar to the known safety proles of each agent ; no new major aes were noted ( table 2 )  . 
serious aes suspected to be drug related were reported in 12.9% of patients ( n = 8 of 62 ) overall and 16.1% ( n = 5 of 31 ) treated at the recommended dose for expansion . 
a total of 13 deaths ( 21% ) were reported during the study , including three ( 4.8% ) deaths resulting from disease progression while the patient was receiving study treatment . 
similarly , hyperglycemia , which is a biomarker of on - target pi3k inhibition , was also observed in 38.7% of all patients treated with alpelisib 200 mg or 300 mg , consistent with prior experience with this agent.27 for ingratinib , 37 patients ( 59.7% ) reported at least one dose interruption and 23 patients ( 37.1% ) reported at least one dose reduction . 
there was no or negligible pk interaction between ingratinib and alpelisib . antitumor efcacy in total , 52 patients were evaluable for response and 10 ( n = 6 in the dose escalation study ; n = 4 in the dose expansion study ) were nonevaluable on the basis of discontinuing therapy before the rst disease assessment ( table 3 )  . 
among 60 patients with a locally annotated pik3ca mutation reported , sequencing with high condence coverage was obtained centrally in 35 samples and the locally reported pik3ca variant conrmed in 80% patients ( n = 28 of 35 )  . 
 ( % ) , except where n = 0 . abbreviations : cr , complete response ; pd , progressive disease . * for patients who only had nontarget lesions at baseline and did not experience either a cr or pd , response is reported as non - cr / non - pd . patient with unknown best overall response due to ( 1 ) no valid postbaseline assessment , or ( 2 ) stable disease occurred too early ( , 6 weeks ) dened as patients who have best overall response cr , partial response , or stable disease , in which stable disease needs to occur at least 6 weeks after the rst dosing . squamous anal cancer with pik3ca e545k mutation . 
interestingly , in the patient with melanoma who had a pr , the only genomic activation of either pathway identied by central sequencing was an fgfr2 m722i mutation , an alteration not known to be recurrent or of biologic signicance.27 we also explored whether genetic alterations of the mapk pathway co - occurred with mutations in the fgfr and / or pi3k pathways and may account for lack of response , at least partially , in those settings . 
overall , ngs studies showed no clear association of alterations in the fgfr , pi3k , or mapk pathways with response . discussion to our knowledge , this is the rst study to evaluate the safety and preliminary efcacy of combined selective pi3k and fgfr inhibition in patients . 
we dened a recommended dose for expansion of alpelisib 300 mg once per day and ingratinib 125 mg once per day , 3 weeks on followed by 1 week off , which is the full single - agent dose and schedule of each drug . 
although this combination did not raise new specic safety concerns , 71% of patients required a dose interruption of alpelisib and 60% of patients reported at least one dose interruption of ingratinib during the course of treatment . 
of note , dose interruptions in the range of 50% to 70% have previously been reported with each agent when used individually , again suggesting that their combination may not be associated with signicant additive toxicity over the component parts but that long - term tolerance each drug alone can exhibit issues.20 , 28 , 29 no drug - drug interactions were reported between ingratinib and alpelisib . this study does have some important limitations . 
the relatively small number of patients treated with relevant concurrent alterations , as well as the heterogeneity of the population , do not permit a denitive assessment of synergistic efcacy . as such , any efcacy data presented from these expansions should be considered hypothesis generating only . second , we note that in the subset of 58% of patients ( n = 35 of 60 ) with tumor tissue in which successful central sequencing was completed , the pik3ca mutations were centrally conrmed in 80% of patients . 
as a result , some patients may have been enrolled on the basis of pik3ca mutations characterized by subclonality , spatial or temporal heterogeneity , or even test failures , as has been observed in prior genome - driven studies.7 finally , enrollment in the expansion cohorts for patients harboring concurrent pik3ca and fgfr1 - 3 alterations was not restricted to variants functionally characterized as activating but to regions of the affected genes that are recurrently the target of alteration . 
efcacy by ( a ) a waterfall plot depicting best percent change in the target tumor burden from baseline according to cohort and ( b ) a swimmer plot depicting progression - free survival by patients according to cohort . 
integrated genomic analysis of efcacy according to mutations in the fgfr , pi3k , and mapk pathways by central next - generation sequencing . ( * ) missing response data , as a result of discontinuation before rst response assessment . 
indel , insertion and / or deletion . fgfr amplication is partially dependent on the degree of amplication.30 both the inclusion of variants of unknown signicance as well as copy number amplications with a specic fold - change requirement may have adversely affected the observed orr by enrolling tumors without true pathway dependency on fgfr . despite these important limitations , a relatively low orr of only 9.7% across the entire study suggests that these two classes of agents may not provide synergistic activity . moreover , responses were generally observed in tumors arising from anatomic sites and harboring genetic alterations previously described as sensitizing to either alpelisib or ingratinib . 
it is also noteworthy that a signicant proportion of patients did not respond , or had stable disease as their best response , despite harboring pik3ca mutations alone or in combination with fgfr alterations . 
 hyman et al implementation has been identication of targeted therapy combinations have often been cited as a means of overcoming both intrinsic and acquired resistance , but a number of scientic , technical , and logistic impediments have made successful implementation challenging.31 , 32 perhaps the most difcult barrier to successful right drug combinations . 
enrichment for concurrent alterations of two oncogenes in one or multiple cancer types has served as one basis for selecting potential drug combinations and formed the basis of the hypothesis evaluated in this clinical study . 
recently , ndings were reported from two precision medicine studies , i - predict and winther , suggesting that a higher degree of molecular matching between somatic alterations detected at baseline and the therapy administered was associated with improved outcomes.33 , 34 although these results imply that targeting multiple oncogenic alterations with polytherapy may be a worthwhile strategy in select circumstances , the data presented here demonstrate that more rigorous evaluation of specic biomarker and drug combinations is required before the approach is more widely embraced . in conclusion , the combination of pik3ca and fgfr inhibition with alpelisib and ingratinib was feasible , although overall tolerability was limited by each agent . 
schellens , mario campone , cristiana sessa , giuseppe curigliano , jennifer bendiske , xueying chen , somesh choudhury , nancy lewis , jose manuel perez garcia , mara jos e de miguel - luken provision of study material or patients : luis paz - ares , jan h . 
craig lockhart , cristiana sessa , thomas zander , matthew ng , giuseppe curigliano , jose manuel perez garcia , mara jos e de miguel - luken collection and assembly of data : david m . 
craig lockhart , cristiana sessa , thomas zander , giuseppe curigliano , xueying chen , somesh choudhury , nancy lewis , jose manuel perez garcia , mara jos e de miguel - luken data analysis and interpretation : david m . 
schellens employment : modra pharmaceuticals stock and other ownership interests : modra pharmaceuticals honoraria : debiopharm group consulting or advisory role : debiopharm group research funding : dutch cancer society ( inst ) patents , royalties , other intellectual property : patent on oral taxanes philippe l . 
bedard research funding : bristol - myers squibb ( inst ) , sano ( inst ) , astrazeneca ( inst ) , roche ( inst ) , servier ( inst ) , glaxosmithkline ( inst ) , novartis ( inst ) , signalchem ( inst ) , ptc therapeutics ( inst ) , nektar ( inst ) , merck ( inst ) , seattle genetics ( inst ) , mersana ( inst ) , immunomedics ( inst ) , eli lilly ( inst ) mario campone honoraria : novartis , eli lilly consulting or advisory role : novartis ( inst ) , servier ( inst ) , menarini , sano ( inst ) , eli lilly ( inst ) , pzer ( inst ) , astrazeneca / medimmune ( inst ) , abbvie ( inst ) , pierre fabre ( inst ) , accord ( inst ) , sandoz - novartis ( inst ) speakers bureau : novartis , amgen research funding : novartis ( inst ) travel , accommodations , expenses : novartis , astra zeneca , pzer other relationship : roche philippe a . 
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all four patients received selpercatinib ( capsule or liquid formulation ) orally in continuous 28 - day cycles at a starting dose of 90 mg / m2 twice per day . this dose was intended to deliver exposure equivalent to the recommended adult phase ii dose of 160 mg twice per day . 
one patient was enrolled to the 80 - mg cohort of trial ( clinicaltrials.gov identier : nct03157128 ) and underwent intrapatient dose escalation to 160 mg per protocol . the ongoing selpercatinib phase i / ii pharmacokinetic analysis serial blood samples were collected for pharmacokinetic analyses . 
molecular analysis showed that the tumor harbored an ret exon 6 deletion ( d378_ g685.e ) , and the patient was treated sequentially with four multitargeted kinase inhibitors : vandetanib , sunitinib , cabozantinib , and lenvatinib , which were each discontinued because of either adverse events or lack of efcacy . 
a 7 - year - old boy with neonatal hypotonia , abdominal pain , hollow feet , and unexplained laryngeal spasms was referred for diagnostic exome sequencing of genomic dna ( sureselect xt clinical research exome , agilent , santa clara , ca )  . 
this revealed a constitutional de novo ret m918t mutation , a pathogenic variant associated with men2b that confers the highest risk of early onset mtc.16 the patient was subsequently diagnosed with an mtc and underwent thyroidectomy with tracheostomy followed by vandetanib therapy , which was discontinued because of the onset of grade 3 colitis . selpercatinib was subsequently initiated at 90 mg twice per day . 
magnetic resonance imaging demonstrated new lung nodules indicative of metastatic disease . an rna - based next - generation sequencing ( ngs ) fusion assay ( solid fusion assay v2 ; archerdx , boulder , co ) indicated that the tumor harbored an myh10 - ret gene fusion . 
the patient was started on vandetanib and achieved a partial tumor response by response evaluation criteria in solid tumors ( recist ) 1.1.22 however , after 4 months of treatment , she developed disease progression at the primary site . 
repeat imaging after 6 months of treatment showed complete resolution of the paraspinal mass and a 50% decrease in the size of the retroperitoneal , pelvic , and lumbosacral mass ( fig 2a - d )  . 
during repair of congenital bilateral inguinal hernias , a 2 - month - old girl was found to have a right renal mass , and she underwent a right nephrectomy . 
patient 1 : magnetic resonance imaging scans of ( a ) right lateral and ( b ) left anterior liver metastatic lesions at baseline and after 22 months of treatment with selpercatinib of ( c ) right lateral and ( d ) left anterior in a heavily pretreated patient with ret - mutated medullary thyroid cancer . 
patient 3 : ( a , c ) computed tomography ( ct ) scans of the abdomen at baseline and ( b , d ) after 6 months of treatment with selpercatinib , revealing multiple paraspinal retroperitoneal and pelvic lesions in a patient with infantile myobroma / hemangiopericytoma harboring an myh10 - ret fusion . 
a partial response was observed after one cycle of selpercatinib ; after six cycles , the paraspinal lesion had completely resolved , and the patient regained lower extremity neurologic function . 
patient 4 : ct scans at baseline of ( e ) the lungs and ( g ) brain and after 8 months of treatment with selpercatinib of ( f ) the lungs and ( h ) brain in a patient with an specc1l - ret fusionpositive congenital mesoblastic nephroma and infantile brosarcoma . 
patient 5 : ct scans ( i , k ) at baseline and ( j , l ) after 2 months of treatment with selpercatinib of the left foot in a patient with an ncoa4 - ret fusionpositive lipobromatosis . 
abdominal ultrasound and chest x - ray identied a mass in the left kidney and a large left lower lung mass ; computed tomography imaging conrmed these lesions and identied additional , multiple small lung lesions . 
biopsy of a lung mass revealed high - grade spindle cell sarcoma consistent with an infantile brosarcoma ; pathology review of the original right nephrectomy specimen conrmed the initial diagnosis of mesoblastic nephroma . 
the patient received additional chemotherapy with cyclophosphamide and topotecan , but treatment was complicated by febrile neutropenia and enterococcus faecalis ventriculitis . dna - based ngs ( using memorial sloan kettering integrated mutation proling of actionable cancer targets [ msk - impact ] ) of the tumor from the renal and lung masses identied the same specc1l - ret gene fusion , and the patient initiated treatment with selpercatinib . after two cycles , a partial response was observed with a 41% tumor reduction , ( fig 2e - h )  . 
a selpercatinib concentration of 4.5 ng / ml was achieved in cerebrospinal uid at a dose level of 48 mg twice per day ( 90 mg / m2 per dose )  . upon detection and resection of an isolated metastasis at the right posterior temporal occipital junction , the dose of selpercatinib was increased to 94 mg ( 180 mg / m2 ) twice per day , raising the concentration of the drug to 16 ng / ml in the cerebrospinal uid . 
 selpercatinib in pediatric ret - altered cancer libretto - 001 ( 160 mg , day 8 ) patient 3 patient 4 patient 5 6 , 000 4 , 000 2 , 000 time ( hours ) fig 3 . 
plasma samples from three patients revealed that adequate plasma concentrations of selpercatinib were achieved ( greater than ret wild - type concentration that inhibits 90% [ ic90 ] ) , which were within the range seen in adult patients treated with the recommended dose of 160 mg in the libretto - 001 trial . 
the gray area represents the 95% cis for the median plasma concentrations observed in patients treated in the libretto - 001 trial ( red circles )  . fusion , which was conrmed by whole genome and transcriptome analysis . 
selpercatinib was initiated , and imaging after 2 months revealed a partial response by recist 1.1 , with a 59% reduction in tumor volume and resolution of tumor inltration of the metatarsals ( fig 2i - l )  . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . sandya govinda raju employment : loxo oncology dahlia henry employment : loxo oncology stock and other ownership interests : loxo oncology , allergan , axovant sciences , palatin technologies steve smith consulting or advisory role : various , loxo oncology patents , royalties , other intellectual property : various patents and applications travel , accommodations , expenses : various s . 
cox employment : bayer , loxo oncology , merck kgaa , amgen , day one biopharmaceuticals stock and other ownership interests : loxo oncology , bayer , merck kgaa , amgen , day one biopharmaceuticals patents , royalties , other intellectual property : us patent 62 / 318 , 041 issued to loxo oncology ( inst ) julia glade bender consulting or advisory role : abbvie ( inst ) research funding : celgene ( inst ) , merck ( inst ) , pzer ( inst ) , amgen ( inst ) , ignyta ( inst ) , bristol - myers squibb ( inst ) , eisai ( inst ) , novartis ( inst ) , eli lilly ( inst ) , loxo oncology ( inst ) , genentech ( inst ) travel , accommodations , expenses : novartis , amgen , merck , bayer , genentech uncompensated relationships : springworks therapeutics , bristol - myers squibb a . 
lindsay frazier stock and other ownership interests : decibel therapeutics consulting or advisory role : decibel therapeutics peter anderson stock and other ownership interests : healios consulting or advisory role : enlivity patents , royalties , other intellectual property : patent for glutamine and trehalose compositions ; priority date 13 september 2013 ; app 14 / 470 , 545 , led august 27 , 2014 ; patent 61 / 878 , 084 issued december 26 , 2017 other relationship : enlivity alberto s . 
pappo honoraria : bayer , roche consulting or advisory role : merck , loxo oncology / bayer , eusa pharma no other potential conicts of interest were reported . acknowledgments we thank the patients and their families and contributing clinical staff across all sites . 
medical writing services were provided by jim heighway of cancer communications and consultancy , knutsford , united kingdom , and were funded by loxo oncology , stamford , ct . references kato s , subbiah v , marchlik e , et al : ret aberrations in diverse cancers : next - generation sequencing of 4 , 871 patients . 
clin cancer res 23 : 1988 - 1997 , 2017 ceccherini i , pasini b , pacini f , et al : somatic in frame deletions not involving juxtamembranous cysteine residues strongly activate the ret proto - oncogene . oncogene 14 : 2609 - 2612 , 1997 prescott jd , zeiger ma : the ret oncogene in papillary thyroid carcinoma . 
nat commun 9 : 4821 , 2018 ferrara r , auger n , auclin e , et al : clinical and translational implications of ret rearrangements in non - small cell lung cancer . 
j thorac oncol 13 : 27 - 45 , 2018 cordioli mi , moraes l , bastos au , et al : fusion oncogenes are the main genetic events found in sporadic papillary thyroid carcinomas from children . 
thyroid 27 : 182 - 188 , 2017 fenton cl , lukes y , nicholson d , et al : the ret / ptc mutations are common in sporadic papillary thyroid carcinoma of children and young adults . 
j clin endocrinol metab 85 : 1170 - 1175 , 2000 prasad ml , vyas m , horne mj , et al : ntrk fusion oncogenes in pediatric papillary thyroid carcinoma in northeast united states . 
cancer 122 : 1097 - 1107 , 2016 vanden borre p , schrock ab , anderson pm , et al : pediatric , adolescent , and young adult thyroid carcinoma harbors frequent and diverse targetable genomic alterations , including kinase fusions . 
carvalho d , mackay a , bjerke l , et al : the prognostic role of intragenic copy number breakpoints and identication of novel fusion genes in paediatric high 11 . 
al - ibraheemi a , folpe al , perez - atayde ar , et al : aberrant receptor tyrosine kinase signaling in lipobromatosis : a clinicopathological and molecular genetic grade glioma . 
antonescu cr , suurmeijer aj , zhang l , et al : molecular characterization of inammatory myobroblastic tumors with frequent alk and ros1 gene fusions and rare novel ret rearrangement . 
elisei r , cosci b , romei c , et al : prognostic signicance of somatic ret oncogene mutations in sporadic medullary thyroid cancer : a 10 - year follow - up study . j clin endocrinol metab 93 : 682 - 687 , 2008 16 . 
latteyer s , klein - hitpass l , khandanpour c , et al : a 6 - base pair in frame germline deletion in exon 7 of ret leads to increased ret phosphorylation , erk activation , and men2a . 
wirth lj , kohno t , udagawa h , et al : emergence and targeting of acquired and hereditary resistance to multikinase ret inhibition in patients with ret - altered 20 . 
drilon ae , subbiah v , oxnard gr , et al : a phase 1 study of loxo - 292 , a potent and highly selective ret inhibitor , in patients with ret - altered cancers . 
antonescu cr , dickson bc , swanson d , et al : spindle cell tumors with ret gene fusions exhibit a morphologic spectrum akin to tumors with ntrk gene 26 . 
wells sa jr , robinson bg , gagel rf , et al : vandetanib in patients with locally advanced or metastatic medullary thyroid cancer : a randomized , double - blind 28 . 
schlumberger m , jarzab b , cabanillas me , et al : a phase ii trial of the multitargeted tyrosine kinase inhibitor lenvatinib ( e7080 ) in advanced medullary thyroid 30 . 
gautschi o , milia j , filleron t , et al : targeting ret in patients with ret - rearranged lung cancers : results from the global , multicenter ret registry . 
yoh k , seto t , satouchi m , et al : vandetanib in patients with previously treated ret - rearranged advanced non - small - cell lung cancer ( luret ) : an open - label , multicentre phase 2 trial . 
 emerging role for precision therapy through next - generation sequencing for sarcomas see accompanying article doi : sarcomas are rare malignancies , collectively accounting for only 1% of adult and 15% of pediatric cancers . 
there are at least 100 different histologic subtypes of bone and soft tissue cancers , and significant genetic and biologic heterogeneity exists within the more common subtypes , such as high - grade pleomorphic sarcomas , liposarcomas , and leiomyosarcomas.1 as a result , development of novel therapies for sarcoma has been slow , because of the difficulty in conducting large clinical trials for rare and heterogeneous populations . 
particularly in smaller phase ii trials , signals of activity can be diluted by the inclusion of many different sarcoma histologies , with limited power to detect predictive biomarkers for patients likely to respond . 
 although cytotoxic chemotherapies have greatly improved cure rates for localized pediatric sarcomas , such as ewing sarcoma , rhabdomyosarcoma , and osteosarcoma , metastatic sarcomas remain incurable in most cases , with median overall survival of 12 to 18 months.2 thus , novel therapies are desperately needed for this challenging group of cancers . recently , wu et al3 presented a pediatric patient with an aggressive , poorly differentiated retroperitoneal sarcoma found to harbor a fusion of ntrk2 . 
shortly thereafter , the patient was enrolled in a clinical trial using larotrectinib , which subsequently led to a dramatic and prolonged clinical improvement of her cancer symptoms , with a near - complete response that has lasted more than 1 year . 
she exhibited an early and dramatic clinical tumor response . the tropomyosin - related kinase ( trk ) family of proteins are receptor tyrosine kinases encoded by the neurotrophic receptor tyrosine kinase 1 , 2 , and 3 ( ntrk1 , 2 , and 3 ) that are activated by their ligands : nerve growth factor , brain - derived growth factor , and neurotrophin 3 / 4.5 there have been many different fusion partners that seem to drive the development of cancers , with several drugs currently in development that target these fusions , including larotrectinib and entrectenib.6 , 7 these drugs have had astonishing efficacy and , importantly , seem to have activity regardless of cancer subtype . 
for example , larotrectinib showed a 75% response rate and 13% complete response rate in ntrk - associated tumors , with median durations of response and median progression - free survival not reached in this study.6 although incidence of these fusions in most cancers is low , the importance of finding these alterations cannot be overemphasized . in the last 20 years , cancer therapy has dramatically evolved by advances in genetics and molecular biology . 
 facilitated diagnostic capabilities in cases where immunohistochemistry and pathology review have been indeterminate . in the article that accompanies this editorial , boddu et al8 present their data on a single institution experience using ngs in patients with sarcoma . 
from a total population of 114 patients , they report a median of three ( range , zero to 19 ) driver variants per tumor tested , with the most common variants being found in tp53 , cdkn2a / b , cdk4 , mdm2 , atrx , and rb1 . 
 perhaps more importantly , five patients ( 4.4% ) ended up having a change in their pathology on the basis of sequencing findings ( including a new diagnosis of a desmoid tumor , synovial sarcoma , and several more specific subtypes of small round blue cell tumors )  . gounder et al9 recently reported a retrospective analysis of more than 5 , 700 patients who underwent ngs with the foundation medicine genomic profiling platforthe results of genomic profiling changed or refined the pathologic diagnosis in 8% of patients . 
in addition , 8% of patients were found to have genetic mutations that were already biomarkers for approved drugs for sarcoma , whereas an additional 9% of patients had genetic biomarkers associated with approved drugs for other cancers . 
overall , for the majority of patients , genetic profiling identified potential therapeutic options either as standard of care or clinical trial opportunities . in some sarcoma subtypes , targeted therapies have shown remarkable efficacy and have become the standard - of - care approach over traditional chemotherapy ( table 1 )  . 
in 2000 , gastrointestinal stromal tumors ( gists ) were first identified to carry activating mutations in kit , and clinical trials of imatinib produced remarkable and durable responses in these sarcomas that were completely resistant to cytotoxic chemotherapy . 
however , although 70% of gists will carry exon 11 mutations , up to 10% of gists have now been shown to be driven from other genetic mutations , many of which are resistant to imatinib.10 to avoid futile therapy at diagnosis , the national comprehensive cancer network guidelines recommend mutation testing for all patients who are planned to be treated with imatinib . 
novel kit inhibitors are being rationally designed to target these mutations ; thus , biopsy at relapse may allow more strategic enrollment in clinical trials , with agents likely to target the specific alterations identified . despite the advances for these subtypes , the majority of mutations and abnormalities identified in metastatic sarcomas are still lacking effective therapies . 
in a recently published analysis from the cancer genome atlas network , multiplatform genomic characterization of 206 soft tissue sarcomas ( leiomyosarcomas , pleomorphic sarcomas , dedifferentiated liposarcomas , myxofibrosarcomas , synovial sarcoma , and malignant peripheral nerve sheath tumors ) revealed that the majority of tumors are characterized predominantly by copy - number changes , with low mutational loads and only a few conserved recurrent genetic mutations across subtypes , including tp53 , atrx , and rb1.11 however , in some subsets , clustering was able to identify genetic signatures associated with prognosis and potentially allow for better stratification of patients for future clinical trials . 
for example , in dedifferentiated liposarcomas , somatic copy - number alteration analysis and dna methylation data revealed three distinct clusters : k1 ( jun amplified ) , k2 ( tert amplified and chromosomally unstable ) , and k3 ( 6q25.1 amplified with fewer unbalanced segments )  . 
 efforts to develop validation trials on the basis of these and other identified genetic subsets will be critical to better customize therapy and improve outcomes . in summary , better understanding of key oncogenic driver mutations and fusions along with development of potent , specific inhibitors is creating meaningful treatment options for patients who do not respond to traditional therapies . 
selected examples of targeted therapy options for sarcomas and other bone / soft tissue tumors sarcoma histologic subtype genetic driver therapies gi stromal tumor c - kit exon 11 ( 70% ) exon 9 ( 10% ) exon 13 / 14 / 17 ( < 1% ) pdgfr ( 10% ) exon 12 exon 18 d842v sdh deficient raf v600e nf - 1 pi3k igf - 1r overexpression ntrk fusions imatinib 400 mg imatinib 800 mg imatinib resistant panotinib * imatinib imatinib resistant crenolanib or avapritinib * sunitinib or regorafenib raf inhibitor raf inhibitor * raf / mek inhibitors * pi3k inhibitors igf - 1r inhibitors * larotrectinib * dermatofibrosarcoma protuberans t ( 17 ; 22 ) ( fusion col1a1 / pdgfb ) > pdgf - b imatinib alveolar soft part sarcoma cediranib , sunitinib , pazopanib t ( 17 ; x ) ( fusion asps1 / tfe3 ) > vegf cofactors tsc1 / tsc2 mutations > mtorch1 activation perivascular epithelial cell tumor ( pecoma ) sirolimus , temsirolimus desmoid fibromatosis giant cell tumor of bone notch signaling gamma secretase inhibitors * rank / rank ligand overexpression denosumab pigmented villonodular synovitis t ( 1 ; 2 ) translocation ( collagen / ) csf1 > csf1 csf1r inhibitors * inflammatory myofibroblastic tumor alk rearrangements dedifferentiated liposarcomas cdk4 / mdm2 amplification solitary fibrous tumor epithelioid sarcoma chondrosarcomas ewing sarcoma ini1 loss idh1 / idh2 mutations ews - fli1 translocation crizotinib palbociclib ezh2 inhibitors idh inhibitors * nab2 - stat6 translocation > vegf temozolomide / bevacizumab abbreviations : csf1r , colony stimulating factor 1 receptor ; igfr1 , insulin growth factor receptor 1 ; vegf , vascular endothelial growth factor . * being explored in ongoing clinical trials . tk - 216 ( disrupts binding of fusion protein to rna helicase a ) * alterations such as ntrk fusions in sarcomas remains relatively low , the potential benefit for this subset of patients is without question . 
wu lw , pavlock t , patterson a , et al : durable clinical response to larotrectinib in an adolescent patient with an undifferentiated sarcoma harboring an strn - ntrk2 fusion . 
doebele rc , davis le , vaishnavi a , et al : an oncogenic ntrk fusion in a soft tissue sarcoma patient with response to the tropomyosin - related kinase ( trk ) inhibitor loxo - 101 . 
drilon a , siena s , ou s - hi , et al : safety and antitumor activity of the multi - targeted pan - trk , ros1 , and alk inhibitor entrectinib ( rxdx - 101 ) : combined results from two phase 1 trials ( alka - 372 - 001 and startrk - 1 )  . 
gounder mm , ali sm , robinson v , et al : impact of next - generation sequencing ( ngs ) on diagnostic and therapeutic options in soft - tissue and bone sarcoma . 
blay jy , le cesne a , cassier pa , et al : gastrointestinal stromal tumors ( gist ) : a rare entity , a tumor model for personalized therapy , and yet ten different molecular subtypes . 
donahue , md , memorial sloan kettering cancer center , department of surgery , 353 east 68th st , new york , ny 10065 ; e - mail : donahuet@mskcc.org. purpose patients with lynch syndrome ( ls ) have a significantly increased risk of developing upper - tract urothelial carcinoma ( utuc )  . 
here , we sought to identify differences in the patterns of mutational changes in ls - associated versus sporadic utucs . patients and methods we performed targeted sequencing of 17 utucs from patients with documented ls - associated germline mutations ( ls - utucs ) using the memorial sloan kettering integrated molecular profiling of actionable cancer targets targeted exon capture assay and compared the results with those from a recently characterized cohort of 82 patients with sporadic utuc . results patients with ls - utuc were significantly younger , had had less exposure to tobacco , and more often presented with a ureteral primary site compared with patients with sporadic utuc . 
although fgfr3 mutations were identified in both cohorts , the r248c hotspot mutation was highly enriched in ls - utuc . conclusion lsand sporadic utucs have overlapping but distinct genetic signatures . 
2018 by american society of clinical oncology introduction lynch syndrome ( ls ) is an autosomal dominant cancer predisposition syndrome caused by germline mutations in the mismatch repair ( mmr ) genes mlh1 , msh2 , msh6 , or pms2 . 
 patients with ls have an increased risk of developing a variety of tumors , particularly those arising in the colon , but also extracolonic cancers , including urothelial carcinomas ( ucs ) .1 - 3 uc is the third most frequent malignancy in ls , occurring in approximately 5% of patients.3 , 4 patients with ls have up to a 22 - fold greater risk of developing upper - tract urothelial carcinoma ( ls - utuc ) over the general population and a median age of onset 10 to 15 years earlier than patients with sporadic utuc.2 - 5 although both uc of the bladder ( ucb ) and utuc are believed to arise from a common precursor cell population within the urothelium , increasing evidence suggests that these two malignancies represent different disease entities from both clinicopathologic and genetic perspectives.6 - 10 in support of this concept , tumor genomic sequencing of ucb and utuc has identified distinct mutational profiles between these two urothelial malignancies.11 , 12 here , we sought to determine whether patients with utuc with known germline mutations uniformly harbored loss of heterozygosity of the wild - type allele of the gene that was the basis of their ls diagnosis and whether their tumors exhibited a pattern of hypermutation consistent with mmr deficiency . 
the ccfr activities are conducted across six multipleprincipal investigator sites and six subaward sites : university of melbourne ( melbourne , victoria , australia ) , fred hutchinson cancer research center ( seattle , wa ) , sinai health system ( comprising mount sinai hospital and lunenfeld tanenbaum research institute [ toronto , ontario , canada ] ) , mayo clinic ( mayo clinic arizona [ scottsdale , az ] with a subaward to mayo clinic minnesota [ rochester , mn ] ) , cedars - sinai consortium ( comprising cedars - sinai medical center [ los angeles , ca ] with subawards to cleveland clinic [ cleveland , oh ] , university of minnesota [ minneapolis , mn ] , dartmouth college [ hanover , nh ] , and university of virginia [ charlottesville , va ] ) , and university of hawaii ( university of hawaii medical center [ honolulu , hi ] with a subaward to cancer prevention institute of california [ fremont , ca ] )  . 
a previously characterized cohort of 82 patients with presumed sporadic utuc treated with radical nephroureterectomy was used as a comparison group.11 germline dna was extracted from peripheral blood lymphocytes provided by the ccfr for each patient . 
when not provided by the ccfr , dna was extracted from paraffin sections as previously described.12 clinical and demographic information were obtained from the prospectively maintained registry of the ccfr . targeted sequencing all protein - coding exons of 341 cancer - associated genes were sequenced using the memorial sloan kettering integrated molecular profiling of actionable cancer targets ( msk - impact ) assay as previously described ( data supplement ) .14 all candidate mutations and indels were reviewed manually . 
the quasi p value for a particular signature was calculated as the fraction of samples with a signature proportion greater than a precalculated noise threshold ( 1 105 )  . 
all analyses were conducted using r software ( version 2.13.1 ; r foundation , vienna , austria )  . results genomic characterization of ls - utuc of the 21 tumors analyzed , four were reclassified as renal cell carcinoma ( rcc ) on central pathologic review . 
genomic dna from all 21 sample tumors ( ls - utuc , n = 17 ; rcc , n = 4 ) and matched normal peripheral blood lymphocytes were analyzed for alterations in 341 cancer - associated genes . 
 thirteen ( 76% ) of 17 patients had loss of heterozygosity in the dna repair genes that corresponded to the germline alterations identified in their germline dna ( fig 1a ; appendix fig a1 )  . a total of 1 , 834 somatic coding alterations were identified across all patients with ls - utuc , involving 300 different genes ( data supplement )  . 
characteristic of mmr - deficient neoplasms , the mean msisensor score was high ( median , 25.1 ; range , 6 to 37.7 ) , with an excess of frameshift mutations ( 142 [ 8% ] in 1 , 834 ) , whereas copy - number alterations were uncommon.19 using mutational decomposition analysis in samples with 10 somatic mutations , we identified evidence of msi / mmr signatures in seven tumors , whereas the mitotic clock / aging signature was predominant in nine ( fig 1b )  . 
notably , the four rccs found to arise in the patients with ls had low mutation rates ( median , two ; range , one to nine ) , low msisensor scores ( median , 4.7 ; range , 3 to 11.6 ) , and no evidence of loss of heterozygosity of their ls associated germline mutation , suggesting that the pathogenesis of these tumors was biologically independent of their ls diagnosis . the most frequently mutated genes in the 17 ls - utuc tumors ( present in > 10 patients ) in decreasing frequency were kmt2d , crebbp , arid1a , smarca4 , cic , fat1 , fgfr3 , foxp1 , kmt2c , notch1 , and notch3 ( appendix fig a2 )  . 
with the exception of cic , foxp1 , and kmt2c , these genes were also those with the highest mutation rate ( > 0.002 mutations per nucleotide ; data supplement )  . 
 tp53 was mutated in a smaller subset of carcinomas ( five [ 29% ] of 17 ) , and in four of these , the tp53 mutation co - occurred with fgfr3 mutations . 
moreover , cdkn1b amplification showed a tendency toward mutual exclusivity with both fgfr3 and tp53 mutations . comparison of genomic profiles of ls - utuc and sporadic utuc to identify potential differences in the mutational landscape between ls - utuc and sporadic utuc , we compared the results of the ls - utuc cohort with those from a previously reported analysis by our group of 82 patients with sporadic utuc . 
one patient in this prior cohort had an ultramutated phenotype ( 422 mutations and no focal copy - number alterations ) attributable to a hotspot mutation in pole.11 patient demographic and clinicopathologic characteristics of the two groups are listed in table 1 . 
 this revealed that , although the mutational landscapes overlapped , some genes were targets of somatic alteration in both cohorts ; however , the frequency of alteration was significantly higher in ls - utuc ( ie , kmt2d , crebbp , arid1a , and smarca4 )  . 
when adjusting for grade , most of the differences between the two cohorts remained significant , although the frequency of gene alterations was different between lowand high - grade tumors ( table 2 )  . finally , we noted that both cohorts had similar frequencies of fgfr3 mutations ( ls - utuc , 11 [ 65% ] of 17 v sporadic utuc , 41 [ 50% ] of 82 ; p = .269 ; appendix fig a3 )  . 
 however , whereas the most prevalent fgfr3 hotspot mutation in sporadic utuc was s249c ( 26 [ 63% ] of 41 mutations ; 13 of 22 and 13 of 19 for lowand high - grade tumors , respectively ) , in ls - utuc , the fgfr3 mutations noted were predominantly r248c ( nine [ 82% ] of 11 mutations ; four of four and five of seven for lowand high - grade tumors , respectively ) and to a lesser extent g380r ( four [ 36% ] of 11 mutations ; two of four and two of seven for lowand high - grade tumors , respectively ; fig 2a )  . given this unexpected finding , we rereviewed all 41 fgfr3 mutations in our sporadic cohort of 82 utucs and identified two carcinomas with an fgfr3 r248c mutation . 
however , their msisensor scores were low ( 0 and 0.11 ) , and the mutational decomposition analysis revealed an aid / apobec signature . validation of fgfr3 r248c as marker of ls - utuc to determine the extent to which the presence of an fgfr3 r248c mutation may be associated with ls or an ls - like hypermutator phenotype , we queried 14 , 800 tumors prospectively sequenced as part of clinical care at memorial sloan kettering cancer center using the msk - impact assay . 
twenty - three patients had an fgfr3 r248c mutation , corresponding to 11 utucs , nine ucbs , and three tumors with other primary sites ( one squamous cell carcinoma of the lung , one lung metastasis from breast carcinoma , and one squamous cell carcinoma of the tongue ; fig 3 )  . 
 of the nine evaluable patients with presumed sporadic utuc and an fgfr3 r248c hotspot mutation , seven displayed germline evidence of ls ( six germline mutations in msh2 and one in msh6 )  . 
upwards of 80% of patients with ls will develop a malignancy , with colorectal cancer being the most common.20 uc is the third most common site of extracolonic malignancy in the ls spectrum and occurs in approximately 5% of patients . 
few studies to date have sought to define the somatic mutational profile of mmr - deficient urothelial tumors , in part because of the perception that the high mutational load would limit the ability to identify true driver events.19 in this study , we performed targeted sequencing of utuc from 17 patients with known germline mutations in an ls - associated gene to characterize the genomic landscape of these tumors and compared the results with those from a cohort of 82 patients with clinically presumed sporadic utuc . next - generation sequencing ( ngs ) can be used to discover novel , targetable , or pathogenic somatic alterations and identify patients with mutations for which therapies already exist . 
 a fgfr3 ls - utuc ( n = 17 ) presumed sporadic utuc ( n = 81 ) sporadic utuc with pole mutation ( n = 1 ) fgfr3 r248c fgfr3 s249c fgfr3 other mutation fgfr3 wild type p < .001 patients r248c s249c mutation fig 2 . 
 ( a ) number of somatic alterations in lynch syndromeassociated upper - tract urothelial carcinoma ( ls - utuc ) and sporadic utuc tumors and their association with fgfr3 mutation status . 
 ( b ) total mutation count in patients with somatic fgfr3 r248c and fgfr3 s249c mutations . the ls and sporadic utuc cohorts tended to be similar ( fgfr3 , kdm6a , pic3ca , and tp53 ) , differences in the genomic profiles were observed . 
of note , however , hypermutation or loss of heterozygosity of the ls germline mutation was not noted in the four rcc samples collected from patients with ls , suggesting that the pathogenesis of these tumors was unrelated to the ls diagnosis . although we were able to identify somatic alterations in most patients driving loss of heterozygosity in the germline ls genes relevant to each corresponding patient , four patients with ls had germline mutations without evidence of loss of heterozygosity as detectable by tumor dna sequencing . 
flow chart of identification of the patients with fgfr3 r248c mutation in the clinical memorial sloan kettering integrated molecular profiling of actionable cancer targets ( msk - impact ) cohort . 
 signatures consistent with msi , suggesting that these patients may have had transcriptional silencing by promoter hypermethylation , as previously reported.21 regarding the mutational decomposition analysis , only seven ls - utuc tumors had an mmr / msi signature . 
this mutational decomposition analysis only uses single - nucleotide polymorphisms , and these are not the most prevalent type of mutation present in msi / mmr cases.19 this may partially explain why the decomposition analysis did not identify a strong msi / mmr signature in some patient cases , including many with high msisensor scores . among the most striking findings in our study was the identification of an r248c hotspot mutation in fgfr3 that may serve as a potential biomarker for ls in patients with utuc . 
activating mutations of fgfr3 are particularly common in low - grade ucs.22 , 23 fgfr3 r248c leads to increased fgfr3 dimer stability and constitutive receptor activation in the absence of ligand , resulting in activation of the phosphatidylinositol 3 - kinaseakt and mitogen - activated protein kinase signaling pathways.24 , 25 this mutation has already been described in ucb , 26 but its presence in utuc was highly associated with a significantly increased median number of somatic mutations . some hereditary cancers are misclassified as sporadic , although their identification might have consequences for both patients and their family members , because this information would prompt germline testing and , if positive , screening for ls - related malignancies.27 our data suggest that the finding of an fgfr3 r248c somatic mutation is a potential biomarker for ls and its identification in a patient should prompt germline testing for ls . 
lindor tumors with mmr deficiency are more responsive to immune checkpoint blockade than mmr proficient tumors.28 it is possible that patients with ls - utuc may derive greater benefit from anti pd - 1 therapy , suggesting an immediate clinical implication for screening and potential personalization of therapy in these patients . one potential limitation of our study was the use of a targeted sequencing approach focusing on a panel of 341 cancer - related genes . 
recently , the analysis of 28 samples of sporadic utuc identified four rna expression profiles with different clinical characteristics , and it would be biologically important to study how ls - utucs cluster within this classification.29 additionally , although ours is the largest series to date to our knowledge profiling ls - utuc using ngs , the sample size remains small , and a larger study may have revealed other significant patterns of comutated genes . 
consequently , there is a need for a larger cohort with external validation . in conclusion , we performed targeted ngs of 17 utucs that arose in patients with ls to compare the genetic characteristics of such tumors with those of sporadic utuc tumors . 
although the spectrums of mutated genes showed some similarities , ls - utucs were characterized by a unique genetic signature , consisting of hypermutation and frequent hotspot mutations at fgfr3 r248c . 
 for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . christophe rosty no relationship to disclose daniel d . 
sfakianos consulting or advisory role : emd serono speakers bureau : astellas medivation dahlia sperling employment : next generation stock and other ownership interests : pharma ( i ) consulting or advisory role : next generation travel , accommodations , expenses : next generation hikmat al - ahmadie consulting or advisory role : bristol - myers squibb , emd serono mark clendenning no relationship to disclose zsofia k . 
stadler consulting or advisory role : allergan ( i ) , genentech / roche ( i ) , regeneron ( i ) , optos , ( i ) , adverum ( i ) gopa iyer no relationship to disclose barry taylor no relationship to disclose jonathan coleman no relationship to disclose noralane m . 
lindor consulting or advisory role : uptodate patents , royalties , other intellectual property : royalties as editor for section in uptodate ; royalties as editor for section in uptodate ( i ) travel , accommodations , expenses : intercept pharmaceuticals ( i ) david b . 
lynch ht , smyrk tc , watson p , et al : genetics , natural history , tumor spectrum , and pathology of hereditary nonpolyposis colorectal cancer : an updated review . 
rink m , ehdaie b , cha ek , et al : stage - specific impact of tumor location on oncologic outcomes in patients with upper and lower tract urothelial carcinoma following radical surgery . 
wagle n , berger mf , davis mj , et al : high - throughput detection of actionable genomic alterations in clinical tumor samples by targeted , massively parallel sequencing . 
duperret ek , oh sj , mcneal a , et al : activating fgfr3 mutations cause mild hyperplasia in human skin , but are insufficient to drive benign or malignant skin tumors . 
juanpere n , agell l , lorenzo m , et al : mutations in fgfr3 and pik3ca , singly or combined with ras and akt1 , are associated with akt but not with mapk pathway activation in urothelial bladder cancer . 
al - ahmadie ha , iyer g , janakiraman m , et al : somatic mutation of fibroblast growth factor receptor - 3 ( fgfr3 ) defines a distinct morphological subtype of high - grade urothelial carcinoma . 
audenet f , colin p , yates dr , et al : a proportion of hereditary upper urinary tract urothelial carcinomas are misclassified as sporadic according to a multi - institutional database analysis : proposal of patient - specific risk identification tool . 
moss tj , qi y , xi l , et al : comprehensive genomic characterization of upper tract urothelial engl j med 372 : 2509 - 2520 , 2015 carcinoma . 
oncoprint of the most frequent genes altered in the lynch cohort . kmt2d crebbp arid1a smarca4 fat1 fgfr3 foxp1 kmt2c notch1 notch3 kdm6a pik3ca cdkn1b tp53 tsc1 genetic alterations truncating mutation inframe mutation missense mutation ( putative driver ) missense mutation ( putative passenger ) amplification i - set pkinase_tyr lynch 806 aa fgfr3 fgfr3 r248c g380r s249c i - set i - set fig a3 . 
 frequent molecular subtype switching and gene expression alterations in lung and pleural metastasis from luminal atype breast cancer max klebe , ms1 ; carlo fremd , md2 ; mark kriegsmann , md1 ; katharina kriegsmann , md3 ; thomas albrecht , md1 ; verena thewes , phd2 , 6 ; martina kirchner , phd1 ; pornpimol charoentong , phd2 , 6 ; nadine volk , phd1 , 2 ; johannes haag , md4 ; ralph wirtz , phd5 ; thordur oskarsson , phd6 ; alexandra schulz , ms1 ; j org heil , md7 ; andreas schneeweiss , md8 ; hauke winter , md4 ; and peter sinn , md1 purpose conversion of tumor subtype frequently occurs in the course of metastatic breast cancer but is a poorly understood phenomenon . 
in luma cancers , 62 genes were identied with differential expression in metastatic versus primary disease , compared with only 10 differentially expressed genes in lumb , human epidermal growth factor receptor 2 ( her2 ) enriched , and basal subtypes combined . gene expression changes in luma cancers affected not only the repression of the estrogen receptor pathway and cell cyclerelated genes but also the wnt pathway , proteinases ( mme , mmp11 ) , and motility - associated cytoskeletal proteins ( ck5 , ck14 , ck17 )  . 
this involved 83 notable gene expression changes . conclusion our results indicate that gene expression changes and subsequent subtype conversion occur on a large scale in metastatic luminal atype breast cancer compared with other molecular subtypes . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction visceral metastases often occur as late events in the course of metastatic breast cancer and are generally followed by a rapidly fatal outcome . 
we examined changes in pam50 gene signatures in a matched - pair series of 57 breast cancer primaries and corresponding lung or pleural metastases to determine the nature and extent of tumor subtype conversion . knowledge generated subtype conversion to pam50 luminal b subtype was observed in the majority of luminal atype breast cancers . 
only few cases of primary luminal b , human epidermal growth factor receptor 2enriched , and basal - like subtypes converted to another pam50 category in metastasis . relevance pam50 - based subtype classication of breast cancer lung and pleural metastases may be different than that seen in the primary tumor , especially in primary luminal a cancers . 
this change in pam50 classication between primary and metastatic disease may affect response to systemic therapy and must be investigated . other three subtypes by tissue microarray analysis , 7 and basal subtypes in particular were more frequent than expected.9 in this study , we included patients with invasive breast cancer of all molecular subtypes and metachronous breast cancer with lung or pleural metastasis ( bclpm ) to provide insights into the upand downregulation of genes during the course of the disease . 
therefore , we retrospectively examined gene expression proles in a wellcharacterized , prospectively assembled group of patients with breast cancer and metachronous lung or pleural metastasis ( data supplement 2 , fig s1 )  . 
exclusion criteria included no availability or insufciency of tumor tissue from pbc or bclpm tumor tissue ( n = 11 ) , primary lung cancer after review of patient records ( n = 2 ) , and low rna content or not meeting quality control criteria of rna data ( n = 11 )  . gene expression analysis for the selection of tumor tissue for rna extraction , ffpe tissue blocks from the pbc and metastatic lesions were selected after reviewing all original tissue slides and were recut for hematoxylin & eosin sections , to be used for reference and to determine tumor cell content ( tumor surface area )  . 
microdissection was performed in most cases to avoid normal breast tissue contamination . a minimum of approximately 50 ng of total rna was used . hybridization time per cartridge was 16 hours before measurement . rna expression analysis was performed by measuring a custom panel of 269 breast cancerrelated genes and 11 housekeeping genes ( data supplement 2 , table s2 ) , using the ncounter platform ( nanostring technologies , seattle ca ) according to the manufacturers instructions . 
the gene panel cludes 25 published gene signatures with prognostic or predictive properties in luminal - type breast cancer with the aim to determine their role in metastatic disease ( data supplement 2 , table s3 )  . 
the selection of gene signatures covered by this list of genes includes nine proliferationrelated signatures , four estrogen receptorrelated signatures , one immune - related signature , and 11 signatures related to cancer pathways . 
for gene function , david bioinformatics resources were used ( version 6.8 ) , 17 , 18 and functional annotation analysis in the biological process category and kegg pathway enrichment were computed . 
all statistical calculations , except gene function analysis , were done using r , version 3.6.1.20 additional information is available in data supplement 1 . results patient characteristics a total of 57 patients with bclpm were included in this study . 
pam50 molecular subtype conversion occurred in the majority of luminal atype pbc ( n = 16 ; 57.1% ) , evolving mostly into luminal btype or her2 - type metastasis ( table 1 ; fig 1b )  . 
gene expression clustering of bclpm was clearly different from pbc in the low - risk category but not with cancers of high - risk subtypes ( figs 2a - 2b )  . this indicates a similar tumor biology of bclpm and pbc for tumors of high - risk subtypes but a different biology in the low - risk ( luminal a ) category . 
by tnm classication luma lumb her2 basal ( range ) initial metastatic site before the treatment of metastatic breast cancer lung or pleural metastasis endocrine therapy at time of metastatic other unknown biopsy yes , tamoxifen yes , aromatase inhibitor yes , gnrha no . 
similarly , heatmap clustering conrmed separate luminal a clusters for primary tumors and metastases ( fig 3 )  . pgr was signicantly downregulated ( p = .0015 , wilcoxon rank - sum test ) , and mki67 was not signicantly increased ( data supplement 2 , fig s5 )  . differential gene expression analysis to characterize which genes are involved in the process of bclpm , differential gene expression analysis was performed . 
these included 3 cytoskeletal proteins ( krt14 , krt17 , krt5 ) , 2 matrix metalloproteinases ( mmp11 , mme ) , and ankrd30a . the latter is a breast differentiation gene that is frequently expressed in the breast and in receptor - positive breast cancer.22 other molecular changes in metastases were linked to proliferation ( cdc6 , ccnb1 , mki67 , top2a , aurkb , pttg1 ) , cell cycle checkpoints ( brca2 , bub1 , bub1b , chek1 ) , and epithelial - mesenchymal transition ( emt ) genes . 
among the genes most relevant for therapy in breast cancer ( esr1 , pgr , her2 , mki67 ) , the expressions of esr1 and her2 were unaffected in bclpm , but subtype - specic changes the pam50 subtypes of pbc showed marked variation with regard to the extent of differential gene expression of this gene panel in bclpm . 
the greatest number of differentially expressed genes was seen in luminal atype breast cancer , with 47 and 11 downand upregulated genes , respectively , compared with fewer expression changes in metastatic high - risk tumors ( luminal b , her2 - enriched , basal - like ) , with six and four downand upregulated genes , respectively ( figs 4a - 4b )  . 
only 1 gene was downregulated in bclpm that was her2 enriched , and no genes reached signicance after adjustment for multiple testing in the basal - like subtype , which in part may be attributed to the comparably small sample sizes of 6 and 7 , respectively ( data supplement 2 , table s6 )  . 
of the genes repressed in luminal a metastases , several are associated with emt . four of these ( twist2 , twist1 , zeb1 , tgfb3 ) are key regulators of emt . 
multidimensional scaling ( umap ) of gene expression , showing ( a ) gene expression in primary versus metastatic cancers , and ( b ) pam50 subtypes of the primary and metastatic tumors . 
for luminal a ( luma ) subtype , two subtype clusters can be distinguished , ( bottom ) one for primary breast cancers , and ( top ) the smaller one representing lung metastases . 
lumb , luminal b ; her2 , human epidermal growth factor rector 2enriched ; basal , basal - like ; normal , normal - like subtype . with bclpm ( figs 2a - 2b ) , and the magnitude and type of gene expression changes in lung metastasis ( table 1 )  . 
thirty - four and 49 genes were signicantly upand downregulated , respectively , in bclpm from luminal a cancers with subtype switch ( adjusted p , .05 ) compared with only 7 genes downregulated and none upregulated in the nonsubtype switch group ( figs 4c - 4d )  . 
in luminal a cancers , subtype switch affected cell cycle and cell proliferation genes ( mitotic cell cycle checkpoint , sister chromatid cohesion , mitotic nuclear division , cell division ) and the p53 - signaling as well as progesterone - signaling pathways ( data supplement 2 , figs 4a and 4b )  . clustering of gene expression in luminal atype cancers to better characterize the phenomenon of subtype conversion in luminal a cancers , we set up a logistic model for the assessment of differential gene expression with and without subtype conversion . 
in the group of 28 luminal asubtype tumors , a subtype conversion was more likely to occur when genes involved in certain growth factors , nuclear proteins , and signaling molecules were upregulated ( gnaz , hoxb13 , vegfa ) and some genes involved in immune response and inammation ( foxp3 , cd8a , cd68 , csf1r ) were downregulated ( fig 5a )  . 
this included 10 genes of various functions with downregulation in metastases and 52 genes , also of diverse functions , that were upregulated after subtype switch ( fig 5b )  . 
this upregulation of a substantial number of genes suggests a common mechanism of gene regulation that is involved in subtype switching . comparison of gene signatures in luminal a and btype cancers for additional insight into the classes of genes involved in subtype switching , a couple of well - characterized gene signatures for estrogen receptor signaling , tumor proliferation , and hallmarks of cancer were analyzed in bclpm ( data supplement 2 , figs s6a and s6b )  . 
interestingly , and despite the different genes involved in these signatures , all 4 tumor proliferation signatures tested showed similar patterns of gene expression in metastases derived from luminal atype tumors with and without subtype switch . 
 klebe et al subtype luminal a luminal b basal - like her2 - enriched normal - like site breast primary lung metastasis expression subtype site brca2 diaph3 chek2 ccne2 aurka bub1 mki67 rrm2 cenpa exo1 mybl2 cep55 plk1 pttg1 cdc6 top2a tyms hif1a flvcr2 cd44 rragd uchl1 scrg1 qsox2 oxct1 krt7 cd24 fgfr4 phgdh cdca7 adgrg6 prkcb pim2 ptgds zeb2 tp53 il17rb rbbp8 krt5 krt14 krt17 tspyl5 elf5 vgll1 prom1 sfrp1 mmp12 mmp9 igfbp5 cxcl14 abl1 zeb1 twist2 twist1 snai2 aldh1a1 slc2a3 csnk1d fam213b fzr1 bbc3 rassf7 plau mmp11 tgfb3 ankrd30a tmem45b pik3ca col4a2 cdc42bpa gstm1 esm1 csnk1e znf703 ccnd1 ebf4 esr1 thsd4 tbc1d9 gata3 cxxc5 ca12 tff1 tff3 foxa1 spdef krt18 pak1 blvra cdk7 stk32b bcl2 stc2 greb1 mapt scube2 igf1r slc39a6 siah2 krt19 cldn4 ddr1 azgp1 fig 3 . 
tumors are discriminated according to their molecular subtype , with separation of metastatic luminal a tumors from primary luminal a cancers ( shown as blue bars across top )  . 
different patterns of gene expression changes are observed , with ( a ) signicantly more changes in the luminal a subtype compared with ( b ) high - risk tumors ( lumb , human epidermal growth factor receptor 2 ( her2 ) enriched , basal - like )  . 
within the luminal a subtype , gene expression changes were mostly conned to ( c ) tumors with subtype switch in lung metastasis compared with ( d ) nonswitched metastases . intrinsic tumor subtype as subtype conversion or subtype switch.26 our results indicate that evolution within cancer cell populations during metastasis leads to more transcriptomic and phenotypic changes than might be expected from the acquisition of genetic events . 
 molecular subtype switching in luminal atype breast cancer the pam50 subtype , subtype conversion only partly reects the changes occurring in tumor evolution , and our data indicate that , even the molecular without change of phenotype may still be different in the metastasis , especially in low - grade tumors . 
in multidimensional scaling , tumors of luminal a subtype in bclpm form a different cluster than luminal a subtype in the primary ( fig 2 ) , and , in the differential expression heatmap , they cluster with more aggressive subtypes ( fig 3 )  . 
along that line , relevant gene expression changes in brain metastases were found without change of pam50 subtype.28 when pam50 subtype conversion occurs , it can be regarded as an indicator of genetic remodeling in metastasis and as a risk factor of additional disease progression . 
of note , although estrogen receptor and her2 statuses as predictive clinical markers remained unchanged in this group , the proliferation rate greatly increased , and breast hormone receptor signaling declined . 
as such , although therapeutically relevant markers were unaltered upon subtype switch , remarkable changes occurred that are generally associated with a worsened malignant behavior and prognostic impact . the metastatic tumor cell the gene expression changes that were evident across all subtypes included evidence for increased mobility and invasive behavior of through downregulation of cytokeratins ( krt5 / 14 ) 29 ; tumor activation through downregulation of mmp1130 , 31 ; and deactivation of estrogen receptordependent pathways , as indicated by the downregulation of the progesterone receptor and the ankyrin repeat - domain gene ( ankrd30a )  . conversely , genes involved in emt and growth signaling were upregulated in all molecular subtypes ( data supplement 2 , fig s3 )  . 
as far as datasets are comparable , most changes found in bclpm have been reported to occur also in other metastatic sites , 27 , 32 with the exception of her2 , which was demonstrated to be upregulated in the brain28 ; however , it was not in our dataset . luminal a cancers undergoing a subtype switch were characterized by a much higher variability in gene expression in bclpm compared with high - risk carcinomas . this variability affected various molecular pathways . 
from gene expression data alone , it appears not possible to name a common denominator for the changes in metastasis of luminal a cancers that affected several dozens of genes . 
when trying to dissect the alterations that occur in subtype conversion , we found that primary tumors with low expression of inammation - related genes but increased expression of growth signalingassociated genes were more likely to undergo subtype conversion . 
in the metastatic tumor cell , the subtype switch was characterized by quite extensive gene expression changes of various molecular pathways , similar to the nding of important gene expression changes in  . 
100 genes in brain metastasis.29 no hypothesis about the root cause of changes for subtype conversion can be put forward , but the manifold changes of expression status in bclpm from luminal a cancers do indicate a major reprogramming of the tumor cell . limitations of this study include the use of a curated gene list . 
last but not least , although the discrimination of patients with breast cancer into specic cancer subtypes is a widely applied clinical concept with profound prognostic implications , luminal a and b cancers may much more represent a continuum rather than true distinct subtypes from a biologic point of view . in conclusion , we have shown that luminal atype breast cancer is most affected by subtype conversion and differential gene expression in matched metastases to lung or pleura involving but not limited to estrogen receptor signaling ( downregulation ) and tumor proliferation ( upregulation )  . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . carlo fremd honoraria : roche , pzer , celgene , astrazeneca , amgen consulting or advisory role : roche , pzer travel , accommodations , expenses : celgene , roche ralph wirtz employment : stratifyer molecular pathology gmbh stock and other ownership interests : stratifyer molecular pathology gmbh consulting or advisory role : janssen oncology ( inst ) , biontech ag ( inst ) research funding : janssen research & development ( inst ) patents , royalties , other intellectual property : royalties from biontech ( inst ) ; royalties from qiagen ( inst ) thordur oskarsson stock and other ownership interests : neovasc , cyclacel pharmaceuticals , windtree therapeutics , enzon pharmaceuticals patents , royalties , other intellectual property : migrastatins and uses thereof . 
description : the present invention provides compounds , pharmaceutically acceptable compositions thereof , and methods of using the same . other relationship : genentech j org heil honoraria : roche , siemens healthcare diagnostics , bard consulting or advisory role : roche , siemens healthcare diagnostics research funding : bard travel , accommodations , expenses : celgene andreas schneeweiss honoraria : roche pharma ag , celgene , astrazeneca , pzer , novartis , msd oncology , lilly , tesaro research funding : roche pharma ag ( inst ) , celgene ( inst ) , abbvie , molecular partners expert testimony : roche , astrazeneca travel , accommodations , expenses : roche , celgene hauke winter expert testimony : astrazeneca travel , accommodations , expenses : astrazeneca peter sinn honoraria : nanostring technologies , roche pharma ag research funding : roche pharma ag travel , accommodations , expenses : nanostring technologies no other potential conicts of interest were reported . references vanharanta s , massagu e j : origins of metastatic traits . 
j surg oncol 23 : 175 - 180 , 1983 van den hurk cj , eckel r , van de poll - franse lv , et al : unfavourable pattern of metastases in m0 breast cancer patients during 1978 - 2008 : a population - based analysis of the munich cancer registry . 
smyth gk : limma : linear models for microarray data , in gentleman r , carey v , dudoit s , et al ( eds ) : bioinformatics and computational biology solutions using r and bioconductor . 
aomatsu e , takahashi n , sawada s , et al : novel scrg1 / bst1 axis regulates self - renewal , migration , and osteogenic differentiation potential in mesenchymal stem cells . 
thompson am , jordan lb , quinlan p , et al : prospective comparison of switches in biomarker status between primary and recurrent breast cancer : the breast recurrence in tissues study ( brits )  . 
this , in turn , leads to a profound defect in homology - mediated dna repair and inappropriate use of error - prone repair pathways , which result in gross genomic instability that contributes to tumorigenesis.1 , 2 this dna - repair defect in brca1 / 2 - mutant cancers renders them exquisitely vulnerable to certain kinds of dna damage , including those caused by poly ( adp - ribose ) polymerase ( parp ) inhibitors and certain classic chemotherapy agents , including platinum.3 the vulnerabilities of brca1 / 2 - mutant cancers to these agents have been translated successfully into treatment approaches . 
parp inhibitors are now approved by the us food and drug administration for treatment of brca1 / 2 mutant ovarian cancers , 4 and data are accumulating that suggest that parp inhibitors are likely active in brca1 / 2 - mutant cancer regardless of the tissue of origparp inhibitors are thought to be toxic to brca1 / 2 - mutant cancers not only because of their catalytic inhibition of parp but by their ability to trap parp - 1 enzyme on dna.5 , 6 the most potent parp inhibitors that induce cell death in brca1 / 2 - mutant cells are those that most efficiently trap parp protein on dna , creating a bulky protein dna adduct.7 in this sense , parp inhibitors work similarly to topoisomerase inhibitors , which trap topoisomerases to create a bulky protein dna adduct . 
other dna - damaging agents including platinum , mitomycin c , and topoisomerase inhibitors , that also induce dna adducts , are also effective in the treatment of brca1 / 2 - mutant cancers in animal models and clinically . given the widespread use of germline and tumor sequencing , and recent us food and drug administration approval of parp inhibitors , more patients who have tumors with pathogenic brca1 / 2 mutations are being treated with parp inhibitors and / or platinum agents . 
one important mechanism of acquired resistance is reversion mutations in brca1 or brca2 that partly restore wild - type gene function.8 , 9 the three reports10 - 12 of reversion mutations in brca2 in breast and prostate cancers that accompany this editorial highlight the importance and increasing awareness of this mechanism of resistance . 
the presence of reversion mutations in brca1 / 2 also reveals some insights about the role of brca1 / 2 function in tumorigenesis and chemosensitivity . most pathogenic mutations in brca1 and brca2 are small insertion / deletions that result in a frameshia frameshift will introduce a premature stop codon , which leads to a truncated , nonfunctional protein product . 
often , frameshift mutations lead to effective null mutations , because rna transcripts harboring premature stop codons can be recognized and degraded by the nonsense - mediated decay pathway.13 reversion mutations are secondary mutations , often small deletions , in a mutant brca1 / 2 allele that convert the initial frameshift mutation into an in - frame internal deletion that still produces a partly functional protein product . 
 complex rearrangements or abnormal use of alternative start sites that bypass the frameshift mutation may also occur.14 rarely , there is full reversion of the pathogenic mutation with restoration of the full wild - type sequence , as seen in the article by banda et al.10 reversion mutations can occur in the setting of either germline or somatic brca1 / 2 mutations , as demonstrated by carneiro et al , 12 and can lead to acquired resistance not only to parp inhibitors but to other classes of dna - damaging agents , such as platinum11 . intriguingly , the mechanism underlying many reversion mutations is inappropriate use of the nonhomologous end - joining pathway , resulting in small deletions , as seen by presence of microhomology at junction sequences.15 longer deletions associated with single - strand annealing may also be present but more difficult to detect . 
it is possible that reversion mutations may already be present in a small population of cells , especially if there is high tumor burden as seen , for example , in ovarian cancer , and are simply selected with ongoing treatment . reversion mutations can be difficult to detect by standard sequencing methods . 
large deletions may be completely missed by short - read sequencing , and even small deletions have to be carefully curated to determine in which allele they originate , and the effect on the final reading frame . 
some full reversions to wild - type sequence may only be detected by careful analysis of tumor purity and mutant allele frequencies in serial samples over time , as demonstrated by banda et al.10 heterogeneity of reversion mutations may also hinder detection . 
analysis of circulating - cell free dna , such as reported by cheng et al11 and carneiro et al , 12 can detect multiple reversion mutations simultaneously at the time of clinical resistance to parp inhibitors or platinum , each restoring the reading frame and arising from a different small tumor - cell population.16 it is possible that only a fraction of reversion mutations in brca1 / 2 are being identified by current sequencing methods . 
new technologies , such as long - read sequencing , and higher depth sequencing may allow more robust detection of reversion mutations and better define their frequency . the selection for reversion mutations in brca1 / 2 under certain treatments does give us some insights into the biology of brca1 . 
 the induction of reversion mutations by platinum is clear genetic evidence that this agent acts on the dna - repair defect associated with brca1 / 2 loss and exerts direct selection pressure to restore brca1 / 2 function . 
this finding also suggests that perhaps , in general , dna - damaging chemotherapies function not as gross metabolic poisons but as targeted therapies for cancers with underlying defects in dna repair and / or checkpoint control . 
if this is true , we may need to reconsider how to optimally dose and schedule platinum and other chemotherapy agents , especially in the setting of cancers with underlying dna - repair defects . the presence of reversion mutations demonstrates that loss of brca1 or brca2 function and the associated dna - repair defect is only required for initiation of tumorigenesis and is not required for maintenance of the cancer phenotype . 
 this feature can be labeled tumor - suppressor tolerance to place it in contrast to oncogene addiction . tumor - suppressor tolerance may operate in cancers that have underlying mutations in genes critical for genomic stability . 
loss of tumor - suppressor function of these genes may only be required for initial tumorigenesis ; once the tumor is established , there may be selection pressure to restore the tumor - suppressor function and reestablish dna - repair function . 
in brca1 - mutant cancers , resistance to parp inhibitors can occur not only by reversion mutations that directly restore brca1 function but also by compensating mutations in other genes such as 53bp1 and its downstream factors such as rif1 , ptip and rev7 , which also can restore homology mediated repair pathways independent of functional brca1.21 - 25 similarly , loss of ptip and chd4 may allow brca2 - mutant cells to reestablish replication fork stability and become resistant to cisplatin and parp inhibitors.26 these findings suggest that to better predict sensitivity to parp inhibitors and platinum , we will need to develop assays capable of distinguishing a cancer with ongoing genomic instability from a cancer with just a history of genomic instability followed by functional reversion of a dna - repair defect . with increasing use of parp inhibitors and platinum for targeted therapy of brca1 / 2 - mutant cancers , we will likely see increased incidence of acquired resistance that exploits tumor - suppressor tolerance and restores brca1 / 2 function . 
it is possible that some hypomorphic alleles of brca1 and brca2 that arise by reversion mutation may still have some targetable vulnerability.27 alternatively , parp inhibitors , or other dna - damaging agents such as platinum , will need to be combined with other drugs that target a different vulnerability in these cancers . 
carneiro b , et al : acquired resistance to the parp inhibitor olaparib in brca2 - associated prostate cancer due to biallelic brca2 reversion mutations restoring both germline and somatic loss of function mutations . 
perrin - vidoz l , sinilnikova om , stoppa - lyonnet d , et al : the nonsense - mediated mrna decay pathway triggers degradation of most brca1 mrnas bearing premature termination codons . 
quigley d , alumkal jj , wyatt aw , et al : analysis of circulating cell - free dna identifies multiclonal heterogeneity of brca2 reversion mutations associated with resistance to parp inhibitors . 
kondrashova o , nguyen m , shield - artin k , et al : secondary somatic mutations restoring rad51c and rad51d associated with acquired resistance to the parp inhibitor rucaparib in high - grade ovarian carcinoma . 
escribano - daz c , orthwein a , fradet - turcotte a , et al : a cell cycle - dependent regulatory circuit composed of 53bp1 - rif1 and brca1 - ctip controls dna repair pathway choice . 
bouwman p , aly a , escandell jm , et al : 53bp1 loss rescues brca1 deficiency and is associated with triple - negative and brca - mutated breast cancers . 
chapman jr , barral p , vannier jb , et al : rif1 is essential for 53bp1 - dependent nonhomologous end joining and suppression of dna double - strand break resection . 
 o genomic characterization of erbb2 - driven biliary cancer and a case of response to ado - trastuzumab emtansine sebastian mondaca , md1 ; pedram razavi , md , phd1 , 2 ; chongrui xu , md , phd1 ; michael ofn , md1 ; mackenzie myers , ms1 ; maurizio scaltriti , phd1 ; jaclyn f . 
abou - alfa , md , mba1 , 2 purpose biliary tract cancers ( btcs ) , which include intrahepatic cholangiocarcinoma ( icc ) , extrahepatic cholangiocarcinoma ( ehc ) , and gallbladder cancer ( gbc ) , have limited treatment options . 
twenty - eight patients ( 5.4% ) had erbb2 alterations , including 2.7% with erbb2 gene amplication , 2.3% with erbb2 mutation , and 0.4% with concurrent amplication and mutation . 
frequent co - altered genes in this cohort were tp53 ( 54% ) , pik3ca ( 21% ) , and cdkn2a ( 18% ) ; kras amplication / mutation was found in 7% of patients . 
2019 by american society of clinical oncology introduction biliary tract cancer ( btc ) comprises three clinically and molecularly distinct entities , intrahepatic cholangiocarcinoma ( icc ) , extrahepatic cholangiocarcinoma ( ehc ) , and gallbladder cancer ( gbc )  . 
genomic proling of btcs with next - generation sequencing ( ngs ) platforms has shown that the three clinical subtypes have signicant molecular differences.5 whereas all three have a high prevalence of tp53 and cdkn2a mutations , genomic alterations in fgfr2 and idh1 are mostly limited to icc ( approximately 20% each )  . 
 mondaca et al context key objective to describe the landscape of erbb2 genomic alterations in biliary tract cancer and illustrate a case of a patient who responded to antihuman epidermal growth factor receptor 2 ( her2 ) targeted therapy . knowledge generated erbb2 amplication or mutations were identied in 28 ( 5.4% ) of 517 patients with biliary tract cancer . 
erbb2 - driven biliary tract tumors had higher concurrent alterations of tp53 and pik3ca , while kras alterations seemed to be less common . one patient with erbb2 - amplied extrahepatic cholangiocarcinoma had a partial response to the her2 - targeted antibodydrug conjugate ado - trastuzumab emtansine . relevance potentially targetable erbb2 amplication and mutation are present in a signicant group of biliary tract tumors , particularly in gallbladder cancer . 
this study supports prospective testing for erbb2 alterations to develop novel anti - her2 strategies in this population . rst - line chemotherapy compared with patients with erbb2 wild type . on the basis of demonstrated improved os in pivotal phase iii trials , drugs that target her2 are standard treatment in her2 - overexpressed breast and gastric cancers.9 , 10 given the low incidence of btcs , the clinical characterization of relevant molecular subgroups , such as erbb2 - amplied or erbb2 - mutant btcs , has been limited , and the clinical experience with anti - her2 agents in this group has been largely anecdotal . 
a recent basket trial showed objective response in four ( 36% ) of 11 patients with erbb2amplied or erbb2 - mutant btc treated with the combination of the anti - her2 antibodies trastuzumab and pertuzumab.11 the aim of the current study was to comprehensively describe the clinical and molecular characteristics of erbb2 - amplied or erbb2 - mutant btcs identied within the context of a prospective tumor proling initiative at memorial sloan kettering ( msk ) cancer center . we also report a durable response to the her2 - targeted antibody - drug conjugate ado - trastuzumab emtansine ( t - dm1 ) in a patient with erbb2 - amplied btc enrolled in a basket trial ( clinicaltrials.gov identier : nct02675829 )  . methods patients demographic data and treatment histories were collected for all patients with erbb2 - altered btc who consented for prospective tumor genomic proling using the us food and drug administrationauthorized msk - impact assay between february 2014 and june 2018 . 
informed consent for tumor proling and clinical data collection was obtained under the genomic proling in cancer patients protocol ( clinicaltrials.gov identier : nct01775072 ) , which was approved by the msk institutional review board . data collection clinical data were extracted from electronic medical records . variables collected were demographic characteristics ( age , sex , race ) , eastern cooperative oncology group performance status , tumor type ( icc , ehc , gbc ) , metastatic sites , systemic treatments received , and clinical outcomes . 
all tumors were prospectively reviewed to conrm histology and to estimate tumor purity . molecular proling and genomic analysis memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) was performed as previously described in a clinical laboratory improvement amendmentscertied laboratory.12 , 13 msk - impact sequences at high coverage all exons and selected intronic and noncoding regions of up to 468 cancer - associated genes . 
baseline characteristics characteristic illustrative case one patient with metastatic erbb2 - amplied ehc was treated with t - dm1 in a basket trial at msk for patients with her2 - amplied or her2 - mutant cancers in the cohort of other solid tumors . 
methods and partial results of this trial have been presented previously , 15 , 16 but to our knowledge , this would be the rst publication about the patient with btc from the basket trial . 
this study was approved by the msk institutional review board , and all patients signed informed consent in accordance with the precepts of the declaration of helsinki . results patients and molecular prole during the period considered in this study , 517 patients with btc underwent msk - impact testing . 
in the subset of patients with btc with erbb2 - amplied or erbb2 - mutant tumors , the median age at diagnosis was 64 years ( range , 27 - 82 years ) , and the most frequent stage of presentation was stage iv ( 50% )  . 
the most frequently mutated domain of her2 was the extracellular domain ( 32% ) , and all mutations in this region arose at the s310 codon ( fig 1 )  . missense mutations represented the most common alteration in the mutated group ( 94% )  . 
of patients age , years median ( range ) male female race white asian black unknown primary tumor stage at diagnosis genomic alteration erbb2 amplication erbb2 mutation erbb2 amplication / mutation first sites of metastasis liver lung bone peritoneum other btc , no . 
the patient then received adjuvant gemcitabine , but within 3 months of initiation of systemic chemotherapy , imaging showed progression of disease in the posterior mediastinum , liver , and peritoneua biopsy specimen of the table 3 . 
aa , amino acid ; furin - like , furin - like cysteine - rich region ; gf_recept - iv , growth factor receptor domain iv ; pkinase_tyr , protein tyrosine kinase domain ; recep_l , receptor l domain . mediastinal metastasis conrmed recurrence ; immunohistochemistry revealed 3 + her2 protein overexpression . the patient was then treated with t - dm1 3.6 mg / kg once every 21 days in a basket trial and achieved a conrmed partial response after 12 weeks of treatment ( fig 3 )  . 
despite ongoing treatment with t - dm1 , the patients disease progressed after 8.6 months with the development of a new 2.2 - cm ( short - axis ) retrocaval lymph node . 
cancer antigen 19 - 9 was 10 , 700 u / ml before starting t - dm1 and decreased to 73 u / ml after achieving a conrmed partial response , with cancer antigen 19 - 9 remaining stable at the time of disease progression ( 62 u / ml )  . 
however , with the development of a new site of disease , t - dm1 was discontinued . discussion this study reports that potentially targetable erbb2 amplication or mutation was present in 5.4% of a cohort of 517 patients with btc who underwent prospective tumor molecular characterization at our institution . 
a similar gradient was described in a previous study that reported 16% , 11% , and 3% of erbb2 amplication in gbc , ehc , and icc , respectively.5 we also found that msi - high tumors were more frequent in the erbb2 - altered cohort compared with tumors without these alterations . 
similar ndings have been described in erbb2 - mutant colon cancer17 ; however , it is not clear whether this represents a biologically meaningful association or passenger erbb2 mutations are more frequent in tumors with an increased tumor mutational burden . 
erbb2activating mutations have been previously described to be present in 1% to 8% of btcs ; the potential therapeutic signicance of these mutations is an active area of research.5 as an example , 20 patients with her2 - mutant btc were enrolled in a basket trial of the pan - her tyrosine kinase inhibitor neratinib ( clinicaltrials.gov identier : nct01953926 ) , with two ( 10% ) achieving a partial response.18 this trial the clinical activity of found that neratinib varied as a function of tumor site , with the most promising clinical activity observed in her2 - mutant breast cancer ( 32% ) but no recist responses in several other cancer types , including bladder and colon cancers.19 the likelihood of clinical response to neratinib also varied as a function of mutation type . 
in another basket study , the response rate of patients with her2 - mutant nonsmall - cell lung cancer to t - dm1 was 44%.15 it remains unknown whether her2targeted antibody drug conjugates would have similar activity in her2 - mutant btc . we report here a patient with erbb2 - amplied btc who achieved a partial response to the her2 - targeted antibodydrug conjugate t - dm1 . 
 mutation count mutation spectrum msi type cancer type sample type erbb2 egfr erbb3 erbb4 fgfr2 pik3ca akt1 tsc2 tp53 mdm2 smad4 arid1a arid1b arid2 kmt2c kras cdkn2a idh1 bap1 mondaca et al stable indeterminate instable primary metastatic female male 100% genomic alterations missense mutation ( putative driver ) missense mutation ( unknown significance ) truncating mutation in - frame mutation fusion amplification deletion no alterations fig 2 . 
ehc , extrahepatic cholangiocarcinoma ; gbc , gallbladder cancer ; icc , intrahepatic cholangiocarcinoma ; msi , microsatellite instability . basket trials , conduct of dedicated trials that include an erbb2 - altered btc cohort has been more challenging . currently , a phase ii trial in china is evaluating the combination of gemcitabine plus oxaliplatin and trastuzumab in erbb2 - amplied btc ( clinicaltrials.gov identier : nct02836847 )  . 
her2 overexpression by immunohistochemistry and uorescence in situ hybridization has been pivotal for the approval of her2 - targeted therapies in breast and gastric cancers.9 , 10 however , tissuebased ngs analyses can identify both mutation and amplication along with other potential actionable targets . given the good correlation for amplication among these methods14 and the increasing clinical use of ngs , we believe that this approach represents an appropriate inclusion criterion in clinical trials . 
first , the cohort represents a selected population treated at a single academic cancer center that favors clinical trial enrollment ; hence , the patients with btc studied may have had different characteristics compared with those being treated in a community setting . 
 erbb2 amplication and mutation in biliary tract cancer baseline 3 months 8.6 months 30% present 80% present new retrocaval ln target lesions ( liver ) nontarget lesions new lesions 10 , 000 8 , 000 6 , 000 4 , 000 2 , 000 months fig 3 . 
ca , cancer antigen ; ln , lymph node . reported treatment is greater than 1.3 times the pfs on the prior line of therapy , the reported treatment can be considered benecial.26 in the case presented here , the patient progressed within 3 months after starting adjuvant chemotherapy and had a pfs of 8.6 months on rst - line t - dm1 , which supports a clinically meaningful benet . 
abou - alfa , md , memorial sloan kettering cancer center , 300 e 66th st , new york , ny 10065 ; twitter : @gaboualfa , @sloan_ kettering ; e - mail : abou - alg@mskcc.org. support supported by national institutes of health memorial sloan kettering cancer center core grant no . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . sebastian mondaca consulting or advisory role : foundation medicine pedram razavi consulting or advisory role : novartis research funding : grail ( inst ) , illumina ( inst ) chongrui xu honoraria : astrazeneca , boehringer ingelheim , eli lilly , msd , novartis , pzer , roche consulting or advisory role : boehringer ingelheim , pzer travel , accommodations , expenses : astrazeneca , boehringer ingelheim , eli lilly , novartis , pzer , roche , bristol - myers squibb , msd michael ofn consulting or advisory role : pharmamar , novartis , targeted oncology travel , accommodations , expenses : bristol - myers squibb , merck sharp & dohme maurizio scaltriti stock and other ownership interests : loxo oncology honoraria : menarini , adc therapeutics consulting or advisory role : menarini research funding : menarini ( inst ) , immunomedics ( inst ) , targimmune therapeutics ( inst ) , puma biotechnology ( inst ) , daiichi sankyo ( inst ) patents , royalties , other intellectual property : pi3k inhibitors and uses thereof , us provisional application no . 
62 / 742 , 163 , inventors : maurizio scaltriti , dan heller , jos e baselga , yosef shamay , carles monterrubio , amaia arruabarrena ( inst ) ; methods for detecting her2 dimerization in patients with her2 mutant lung cancers and predicting responsiveness to ado - trastuzumab emtansine therapy , sk2018038 - 01 , inventors : maurizio scaltriti , bob t . 
solit stock and other ownership interests : loxo oncology consulting or advisory role : pzer , loxo oncology , illumina , intezyne technologies , vivideon therapeutics , eli lilly travel , accommodations , expenses : merck kgaa bob t . 
li consulting or advisory role : roche , biosceptre international , thermo fisher scientic , mersana , guardant health , hengrui therapeutics research funding : roche ( inst ) , genentech ( inst ) , illumina ( inst ) , biomed valley discoveries ( inst ) , astrazeneca ( inst ) , grail ( inst ) , daiichi sankyo ( inst ) , hengrui therapeutics ( inst ) , guardant health ( inst ) , amgen ( inst ) ghassan k . 
n engl j med 362 : 1273 - 1281 , 2010 okusaka t , nakachi k , fukutomi a , et al : gemcitabine alone or in combination with cisplatin in patients with biliary tract cancer : a comparative multicentre study in japan . 
br j cancer 103 : 469 - 474 , 2010 lamarca a , palmer d , wasan h , et al : abc - 06 | a randomised phase iii , multi - centre , open - label study of active symptom control ( asc ) alone or asc with oxaliplatin / 5 - fu chemotherapy ( asc + mfolfox ) for patients ( pts ) with locally advanced / metastatic biliary tract cancers ( abc ) previously - treated with cisplatin / gemcitabine ( cisgem ) chemotherapy . 
j clin oncol 37 , 2019 ( suppl ; abstr 4003 ) javle m , bekaii - saab t , jain a , et al : biliary cancer : utility of next - generation sequencing for clinical management . 
cancer 122 : 3838 - 3847 , 2016 roa i , de toro g , schalper k , et al : overexpression of the her2 / neu gene : a new therapeutic possibility for patients with advanced gallbladder cancer . gastrointest cancer res 7 : 42 - 48 , 2014 galdy s , lamarca a , mcnamara mg , et al : her2 / her3 pathway in biliary tract malignancies ; systematic review and meta - analysis : a potential therapeutic target ? cancer metastasis rev 36 : 141 - 157 , 2017 lowery ma , ptashkin r , jordan e , et al : comprehensive molecular proling of intrahepatic and extrahepatic cholangiocarcinomas : potential targets for intervention . 
clin cancer res 24 : 4154 - 4161 , 2018 slamon dj , leyland - jones b , shak s , et al : use of chemotherapy plus a monoclonal antibody against her2 for metastatic breast cancer that overexpresses her2 . 
bang yj , van cutsem e , feyereislova a , et al : trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of her2 - positive advanced gastric or gastro - oesophageal junction cancer ( toga ) : a phase 3 , open - label , randomised controlled trial . 
lancet 376 : 687 - 697 , 2010 javle mm , hainsworth jd , swanton c , et al : pertuzumab + trastuzumab for her2 - positive metastatic biliary cancer : preliminary data from mypathway . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
ross ds , zehir a , cheng dt , et al : next - generation assessment of human epidermal growth factor receptor 2 ( erbb2 ) amplication status : clinical validation in the context of a hybrid capture - based , comprehensive solid tumor genomic proling assay . 
li bt , shen r , buonocore d , et al : ado - trastuzumab emtansine for patients with her2 - mutant lung cancers : results from a phase ii basket trial . 
ross js , fakih m , ali sm , et al : targeting her2 in colorectal cancer : the landscape of amplication and short variant mutations in erbb2 and erbb3 . 
harding j , cleary j , shapiro g , et al : o - 005 : treating her2 - mutant advanced biliary tract cancer with neratinib : benets of her2 - directed targeted therapy in the phase 2 summit basket trial . 
nature 554 : 189 - 194 , 2018 [ erratum : nature 566 : e11 - e12 , 2019 ] janjigian yy , sanchez - vega f , jonsson p , et al : genetic predictors of response to systemic therapy in esophagogastric cancer . 
cancer discov 8 : 49 - 58 , 2018 iwata h , tamura k , doi t , et al : trastuzumab deruxtecan ( ds - 8201a ) in subjects with her2 - expressing solid tumors : long - term results of a large phase 1 study with multiple expansion cohorts . 
park h , doi t , modi s , et al : oa02.07 : updated results of phase 1 study of ds - 8201a in her2 - expressing or mutated advanced non - small - cell lung cancer . 
meric - bernstam f , beeram m , mayordomo ji , et al : single agent activity of zw25 , a her2 - targeted bispecic antibody , in heavily pretreated her2 - expressing 25 . 
von hoff dd , stephenson jj jr , rosen p , et al : pilot study using molecular proling of patients tumors to nd potential targets and select treatments for their thorac oncol 13 : s324 , 2018 cancer res 77 , 2017 ( suppl ; abstr 31 ) cancers . 
rod rassekh , md , mhsc5 ; rebecca deyell , md5 ; jennifer rauw , md1 ; meg knowling , md2 ; kong khoo , md6 ; ursula lee , md6 ; krista noonan , md6 ; jason hart , md1 ; r . 
taylor2 ; simon chan , msc2 ; karen mungall2 ; eric chuah2 ; yongjun zhao , dvm2 ; andrew mungall , phd2 ; richard moore , phd2 ; howard lim , phd2 ; daniel j . 
jones , phd2 ; marco marra , phd2 ; and janessa laskin , md2 purpose this study investigated therapeutic potential of integrated genome and transcriptome proling of metastatic sarcoma , a rare but extremely heterogeneous group of aggressive mesenchymal malignancies with few systemic therapeutic options . methods forty - three adult patients with advanced or metastatic non - gi stromal tumor sarcomas of various histology subtypes who were enrolled in the personalized oncogenomics program at bc cancer were included in this study . 
fresh tumor tissues along with blood samples underwent whole - genome and transcriptome sequencing . results the most frequent genomic alterations in this cohort are large - scale structural variation and somatic copy number variation . 
outlier rna expression as well as somatic copy number variations , structural variations , and small mutations together suggest the presence of one or more potential therapeutic targets in the majority of patients in our cohort . 
point mutations or deletions in known targetable cancer genes are rare ; for example , tuberous sclerosis complex 2 provides a rationale for targeting the mammalian target of rapamycin pathway , resulting in a few patients with exceptional clinical benet from everolimus . 
2019 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction adult sarcoma is one of the most difcult cancer types to study in the clinical trial setting , owing to its rarity along with complexity and heterogeneity in histology , tissue origin , and molecular subtypes . 
although improved survival is seen over time in metastatic sarcoma , the prognosis continues to be poor , with a median overall survival of approximately 18 months.1 the genomic prole of adult sarcomas was recently unveiled by a comprehensive genomic study published by the cancer genome atlas ( tcga ) consortium.2 unlike other cancer types , adult sarcomas are characterized by a predominance of copy number variations ( cnvs ) , with relatively few single - nucleotide variants ( snvs ) .2 however , these data were limited to localized primary disease in only six major sarcoma subtypes and did not explore any direct correlation with potential targeted therapies . targeted oncological therapeutics based on genomic proling ( ie , personalized cancer medicine ) , despite many controversies , seem to improve clinical outcome of patients with multiple cancer types . 
 feng et al context key objective using whole - genome and transcriptome sequencing technology , we attempt a systemic analysis of comprehensive proling on a cohort of 43 patients with metastatic non - gi stromal tumor sarcomas to identify therapeutic implication . knowledge generated our data showed large - scale structural variant aberration is the primary mechanism of mutagenesis in metastatic sarcoma . 
in addition , we reported cases of clinical outcomes of personalized oncogenomicsdirected therapy and demonstrated the clinical utility of genomic proling in metastatic sarcomas . furthermore , we reported several novel mutational signatures in metastatic sarcomas , one of which is homologous recombination deciency signature that may associate with superior sensitivity to double - strand dnadamaging agents in several sarcoma subtypes . relevance our results support the use of genomic data in precision medicine and clinical trial design to improve care for patients with metastatic sarcoma . 19 distinct types of advanced metastatic sarcoma as part of the personalized oncogenomics ( pog ) project ( clinicaltrials.gov identier : nct02155621 ) at bc cancer in canada . 
these represent cases refractory to standard therapies that were genomically analyzed with the aim to identify molecular aberrations amenable to a clinically approved targeted therapy or a theoretical targeted therapy under investigation in clinical trials . 
clinical benet of pogdirected therapy was determined by radiographic response on the basis of response evaluation criteria in solid tumors ( recist ; version 1.1 ) and / or progression - free survival of 6 months or more . methods patient samples as previously described , 5 , 6 candidate patients enrolled in pog underwent metastatic tumor biopsies and peripheral blood collection for comparison with detected somatic alterations . 
genome libraries from tumors and blood samples ( normal control ) as well as transcriptome libraries from tumors were constructed using protocols previously described.7 , 8 all patients provided written informed consent for tumor biopsies , blood sample collection , genomic proling , data analysis , and publication of results as part of pog of british columbia approved by the university of british columbia research ethics committee ( reb# h12 - 00137 ) and registered ( clinicaltrials.gov identier : nct02155621 )  . 
raw sequence data and downstream analytics were maintained within a secure computing environment . whole - genome and transcriptome sequencing and bioinformatics paired - end reads from all libraries were generated on illumina hiseq2000 or hiseq2500 sequencer ( illumina , san diego , ca )  . 
determination of somatic snvs , structural variants ( svs ) , cnvs , and gene expression was performed as previously described.5 , 6 transcript outlier expression was determined either by a greater than or a less than two - fold change against tumor illumina bodymap ( arrayexpress id : e - mtab - 513 ) or percentile less than 10% or greater than 90% ( tumor v compendium of tcga ; nih.gov / tcga / )  . 
for gene expression clustering , data were t using the deseq2 package , and hierarchical clustering of log - transformed reads per kilobase of transcript per million mapped reads was performed for genes signicant at an adjusted p value threshold of .01 ( euclidean distance , complete linkage clustering )  . 
the common histologies in this cohort were leiomyosarcoma ( lms ; nongynecologic : n = 7 [ 16% ] and gynecologic : n = 3 [ 7% ] ) , osteosarcoma ( osa ; n = 5 [ 12% ] ) , undifferentiated pleomorphic sarcoma ( ups ; n = 5 [ 12% ] ) , dedifferentiated liposarcoma ( ddlps ; n = 4 [ 9% ] ) , and ewing sarcoma ( n = 3 [ 7% ] ; table 1 )  . 
there were 29 male patients ( 67% ) and 14 female patients ( 33% ) , with a median age at time of metastatic diagnosis of 55 years ( range , 18 to 79 years )  . 
in this cohort , 28 of 43 patients ( 65% ) had received one standard systemic treatment of advanced metastatic disease at the time of sequencing biopsy , and 15 of 43 patients ( 35% ) had received multiple lines ( range , 2 to 7 )  . 
details of standard systemic treatments received in this cohort are described in appendix table a1 . at the time of data analysis , 26 of 43 patients ( 60% ) were deceased , and 17 of 43 ( 40% ) were still alive . potentially targetable alterations in a heterogeneous sarcoma cohort we sought to systematically characterize the genomic landscapes of adult metastatic sarcomas in this heterogeneous cohort and explore how these landscapes relate to therapeutic approaches . 
however , this level of information can potentially be used to inform treatment options for therapies currently available . overall clinical outcome of pog - directed therapy of 42 patients with potentially targetable alterations , 18 ( 43% ) patients attempted pog - directed therapies either in the clinical trial setting or through a compassionate access these , eight of 18 patients ( 44% ) program ( cap )  . 
of derived clinical benet from pog - directed therapy ( table 2 ) and are described in case series of patients who derived clinical benet from pog - directed therapy . response could not be assessed in four cases ( 22% ) owing to short duration ( 2 weeks ) on pog - directed toxicities and / or deterioration of therapies because of performance status ( appendix table a2 )  . 
there were six patients ( 33% ) considered nonresponders to pogdirected therapies , including tyrosine kinase inhibitors , histone deacetylase inhibitors , and immunotherapies ( appendix table a2 )  . 
this result is comparable to a recent study using genomic proling in the clinical setting.15 case series of patients who derived clinical benet from pog - directed therapy infrequent tsc2 mutation / deletion has cases 1 and 2 : therapeutic implications for mammalian target of rapamycin inhibitors . 
case 1 is a 62 - year - old male who presented with recurrent metastatic renal epithelioid angiomyolipoma with lung , liver , peritoneum , and bone metastases ( table 2 )  . 
pog identied a tsc2 pathologic truncating mutation , a relatively well - known event.18 everolimus was given through cap , yielding prolonged partial response ( pr ; appendix fig a3 ) as predicted and continued to show response at 15 months of therapy at the time of analysis . case 2 is a 57 - year - old female who presented with a large right upper lobe mass with mediastinal / hilar lymphadenopathy and pleural effusion and was diagnosed with undifferentiated round cell sarcoma . 
however , the above two cases suggest that the landscape of small mutations and deletions , although rare , has therapeutic relevance in some sarcoma subtypes . our systematic analysis of somatic events based on wholegenome sequencing reveals that the most frequent recurrent alterations in this cohort of adult metastatic sarcomas occur through large - scale structural changes in the genome ( fig 2a ) , rather than through point mutations or small deletions that are frequent in many other cancer types , 20 with a few exceptions of known tumor suppressor genes including tp53 ( approximately 30% ; 13 of 43 ) , atrx ( approximately 12% ; ve of 43 ) , and rb1 ( approximately 7% ; three of 43 ; fig 2b )  . 
 personalized oncogenomics of adult metastatic sarcoma case 3 : targeting common oncogenic amplications such as mouse double minute 2 homolog and cyclin - dependent kinase 4 may have therapeutic implications . 
she subsequently had a pr to a combination of mdm2 and cdk4 inhibitors in a phase ib / ii clinical trial ( clinicaltrials.gov identier : nct02343172 ) and continued to receive therapy for approximately 1 year before death as a result of disease progression . 
although the synergistic effect of mdm2 and cdk4 inhibitors remains controversial22 , 23 pending ongoing clinical trials , the case of this exceptional responder provides rationale for investigating and targeting these genomic abnormalities in this tumor and possibly other sarcoma types . recurrent amplications have been described as a common feature in genomically complex sarcoma subtypes.2 , 24 the canonical 12q13 - 15 amplications targeting mdm2 / cdk4 were observed in ddlps , with an additional case of highgrade osa also harboring this event , suggestive of a dedifferentiated osa arising from a low - grade osa with mdm2 amplication.25 amplication of the oncogene myc on 8q24 was also observed in three of 43 sarcomas . 
the most common region of amplication across this adult sarcoma cohort is 17p11 - 12 altered in 42% ( 18 of 43 ) of cases ( figs 3a and 3b )  . 
strikingly , when we examined the frequency of our recurrent amplication events across tcga cancer types , the amplication on 17p11 - 12 was found to be highly sarcoma specic , which is rarely observed in other tumor types ( fig 3c )  . 
furthermore , the 17p11 - 12 amplicon has been previously described only in osa26 , 27 and lms.28 in our study , this amplicon is observed in a variety of sarcoma subtypes , including ddlps , pleomorphic liposarcoma , osa , lms , chordoma , ups , and undifferentiated endometrial sarcoma ( ues ) , suggesting it harbors an oncogene that is important for sarcoma tumorigenesis . myocd , pmp22 , and cops3 are believed to be potential oncogenic targets within this amplicon in lms and osa.26 - 28 we examined transcriptome expression data for potential oncogenic targets in this region ( fig 3d )  . 
myocd and pmp22 are both within the region most frequently amplied in this cohort , with myocd showing particularly higher expression in amplied samples and pmp22 showing higher expression overall and compared with many other in addition , adora2b was noted as having tumors . amplicon - associated expression ; this gene has not previously been examined as a potential target for this amplicon , but it has been reported to have growth - promoting properties in oral and breast cancers.29 , 30 this analysis suggests that additional functional analysis of genes within this amplicon is required to clearly dene the oncogenic target ( s )  . there are few therapies available that to date , target common amplications in metastatic sarcomas . 
the future development of therapies targeting recurrent amplications such as 17p11 - 12 may be warranted , potentially improving therapeutic options in this disease . case 4 , 5 , and 6 : mutation signatures and homologous recombination deciency may delineate sarcoma subtypes and indicate sensitivity to certain dna - damaging agents . mutation signatures are characteristic patterns of mutation , which may result from specic cellular or environmental pressures and may have implications for therapy.12 the most comprehensive validated set of mutation signatures described to date is reported in cosmic ( sanger.ac.uk / cosmic / signatures )  . 
on the basis of singlenucleotide alterations , we performed mutation signature detection in our sarcoma cohort and identied eight signatures through this de novo process , denoted v1 to v8 ( appendix fig a6 )  . 
comparison of discovered sarcoma signatures to the 30 previously described mutation signatures reported in cosmic31 revealed that sarcoma signatures v1 , v3 , v4 , v5 , v6 , and v8 matched signatures 30 , 5 , 2 , 1 , 8 , and 9 , respectively ( fig 4 )  . 
for example , ddlps possessed similar rearrangement proles , with elevated signature r1 and high mutation burden . signatures v6 and v7 matched signatures 3 and 8 , both of which are associated with hrd signature , 31 also known as brcaness , which has been well described in breast , fig 2 . 
genomic structural complexity is calculated as the average of the proportion of copy segments that are nondiploid and the proportion of the genome represented in nondiploid copy segments , similar to that per the cancer genome atlas research network.2 ( b ) oncoprint of recurrent somatic dna alterations in cancer - related genes observed in sarcomas , grouped by sarcoma subtype . 
types of gene alterations include amplication ( red ) , homozygous deletion ( blue ) , gene fusion ( purple ) , truncating mutation ( black ) , or missense mutation ( green )  . 
 personalized oncogenomics of adult metastatic sarcoma ovarian , and other relatively common cancer types32 including osa.33 we further computed hrd indices and found that in addition to osa , high hrd scores were also seen in lms , ues , and a few other sarcoma subtypes ( fig 4 )  . 
the brcaness phenotype in lms that we observed is consistent with a recent report.34 interestingly , we did not observe deleterious genomic or transcriptomic aberrations of genes such as brca1 and brca2 , which have been described as accounting for the majority of hrd signatures in other tumor types . 
we do observe losses , svs , or downregulated expression of various components implicated in the homologous recombination repair pathway , such as pten , atm , and rad51 ( data not shown ) , suggesting potential alternative contributions to the hrd signature . 
a clear - cut relationship between genetic aberrations and hrd signature is also not observed in other studies.12 , 31 case 4 is a 44 - year - old woman who presented with metastatic ues with lung metastasis . 
the response to ifosfamide was not long - lasting , like all other chemotherapies , and both patients eventually experienced progression , did not respond to any additional treatment , and died as a result of disease progression . 
the hrd signature or brcaness phenotype is associated with therapeutic response to dna - damaging agents , including platinum - based chemotherapies.12 although platinums are not generally used in metastatic soft tissue sarcomas , the dna damaging chemotherapeutic agent ifosfamide has similar , though less - pronounced , dna crosslinking effects . although ifosfamide is an active drug in various sarcoma subtypes , with an overall response rate between 10% and 40% depending on dosing schedule , single - agent response is generally considered low ( , 10% ) , especially in the third - line setting or beyond for sarcomas aside from synovial sarcoma.35 we speculate that the response may relate to partial dna cross - linking property of ifosfamide alkylating agent , which may have therapeutic advantage in hrd tumors . case 6 is 53 - year - old man who presented with recurrent metastatic splenic vein lms with liver and lung metastasis . his disease progressed through docetaxel plus gemcitabine treatment . 
because of a similar hrd signature identied through pog study , he was enrolled in a phase i clinical trial ( clinicaltrials.gov identier : nct02873975 ) investigating an experimental drug ( prexasertib ) targeting hrd tumors . 
these cases support the clinical utility of genomic analysis of hrd and mutation signatures across sarcoma subtypes . case 7 and 8 : potentially targetable aberrations identied solely through transcriptome / expression analysis . 
case 7 is a 77 - year - old male presenting with recurrent multiple intraabdominal ups , which progressed through two cycles of doxorubichis tumors exhibited high rna expression of various tyrosine kinase receptors , such as plateletderived growth factor receptors ; therefore , sunitinib was attempted through cap . 
he then received pog - directed therapy in the form of sunitinib on the basis of his tumor exhibiting high rna expression of fms - like tyrosine kinase 3 and colony - stimulating factor 1 receptor . 
expression in amplicon refers to log ( reads per kilobase of transcript per million mapped reads ) ; increase in amplied samples is measured as expression in amplied samples divided by expression in nonamplied samples in this cohort ; expression percentile is determined relative to all tcga tumors ( see methods )  . 
 ( a ) structural variant ( sv ) mutation signatures ( r1 to r4 ) and single - nucleotide variant ( snv ) mutation signatures ( v1 to v8 ) were deciphered de novo from metastatic sarcomas . 
three dedifferentiated liposarcomas demonstrated high sv and snv burden , with similar r1 rearrangement signature and signatures v4 , v5 , and v6 . signature v4 matched catalogue of somatic mutations in cancer ( cosmic ) signature 2 and is known to be associated with apobec deamination . 
 ( b ) three sarcoma cases ( labeled in red ) with elevated homologous recombination deciency ( hrd ) score and / or the hrd - associated signature 3 demonstrated response to double - strand dnadamaging chemotherapy . 
 ( c ) mutation signatures deciphered de novo from sarcomas were compared against 30 canonical signatures from the cosmic using the cosine similarity metric . signatures that map most closely to a cosmic signature are highlighted . 
 personalized oncogenomics of adult metastatic sarcoma selection who had these two histology subtypes.36 , 37 taken together , these results support the notion that drug efcacy may be dictated by biology measurable through gene expression biomarkers in addition to histology subtype . furthermore , these data support additional research on these putative biomarkers in the context of a complex signaling network , which may help advance the drug development of various tyrosine kinase inhibitors in future sarcoma clinical trials . immune landscape of sarcoma subtypes despite being regarded as a poorly immunogenic tumor because of relatively low mutation burden compared with other cancer types , 2 checkpoint inhibitor immunotherapies have produced promising durable response in a few sarcoma histology subtypes such as ups and alveolar soft part sarcoma thus far.38 , 39 pd - l1 expression has been correlated with efcacy of immunotherapy in some , but not all , cancer types40 and reported as a prognostic marker in soft tissue and bone sarcoma , 41 , 42 but it has not yet been shown to correlate with immunotherapy sensitivity in sarcoma.43 the tumor immune microenvironment plays a key role in determining immunotherapy sensitivity , and emerging data demonstrated a positive correlation between clinical response and preexisting tumor immune microenvironment.44 we examined immune inltration on the basis of gene expression proles ( see methods ) and observed that a subset of tumors show high iiss ( fig 5 ; appendix fig a9 )  . 
we did not observe any correlation between high level of pd - l1 expression and immunotherapy response in a small number of cases ( n = 4 ) within our cohort . interestingly , of the four patients in our cohort treated with immunotherapies , the only patient ( case 8 , lms ) who exhibited some response to immunotherapy also demonstrated an unusually high iis ( fig 5 ; table 2 )  . 
two other cases with high iis , both ups , were not treated with immunotherapy , and two other sarcomas that were treated with immunotherapy , an lms and an osa , had low iis and did not respond . 
overall , our experience suggests that although iis alone may not be sufcient as a predictive biomarker for immunotherapy in investigation , all sarcoma types , particularly in lms . it warrants additional cases 9 and 10 : detection of fusion genes assists in sarcoma diagnosis and therapeutic intervention . 
t - cell inltration score is the sum of all t - cell subtypes excluding regulatory t cells , which are negative modulators . immune inltration score is the sum of scores for all 22 immune cell types proled . 
arms , alveolar rhabdomyosarcoma ; asps , alveolar soft part sarcoma ; ccs , clear cell sarcoma ; chsa , chondrosarcoma ; ddlps , dedifferentiated liposarcoma ; eaml , epithelioid angiomyolipoma ; ews , ewing sarcoma ; mlps , myxoid liposarcoma ; mpnst , malignant peripheral nerve sheath tumor ; msft , malignant solitary brous tumor ; osa , osteosarcoma ; other , groups urs , plps , mlps , ccs , mpnst , eaml , arms , ss , chsa , asps , and sef ; plps , pleomorphic liposarcoma ; sef , sclerosing epithelioid brosarcoma ; ss , synovial sarcoma ; stlms , nongynecologic leiomyosarcoma ; ues , undifferentiated endometrial sarcoma ; ulms , gynecologic leiomyosarcoma ; ups , undifferentiated pleomorphic sarcoma ; urs , undifferentiated round cell sarcoma . sarcoma and change to standard alternating multi - agent chemotherapies with substantial pr . 
her systemic disease also responded to second - line and third - line standard chemotherapies for ewing sarcoma , but she eventually developed brain metastasis and died as a result of disease progression . case 10 is a 68 - year - old man presenting with recurrent metastatic osa ( initial pathologic diagnosis ) of the right femur with lung metastasis . 
 feng et al rapid deterioration shortly after this diagnosis was made from genomic analysis and died as a result of disease progression . within our cohort , in all cases where a fusion would be expected based on pathologic features , we observed a fusion consistent with the sarcoma type ( appendix table a3 )  . the exceptional above two cases , where we identied fusions that had not initially been expected based on pathology , support the utility of genomic proling in rening sarcoma diagnosis and therapeutic intervention , echoing the results of a large retrospective study.17 conclusion our data lend additional support to the current literature data showing that sv and cnv are the primary mechanisms of mutagenesis in sarcomas , as opposed to single - nucleotide alteration . 
we provide anecdotal evidence showing the potential of genomic alterations , such as loss of tsc2 and amplication of mdm2 / cdk4 , to predict exceptional response to targeted treatments . 
overall , we were able to identify potentially actionable genomic alterations in the majority of cases , largely on the basis of outlier rna expression and cnv and sv aberrations . 
renouf , karen gelmon , stephen yip , marco marra , janessa laskin data analysis and interpretation : xiaolan feng , erin pleasance , eric y . zhao , tony ng , jasleen k . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . xiaolan feng honoraria : astrazeneca , nanostring / innomar strategies consulting or advisory role : astrazeneca , ipsen , genomic health , nanostring / innomar strategies , roche tony ng honoraria : nanostring technologies travel , accommodations , expenses : nanostring technologies sara k . 
 personalized oncogenomics of adult metastatic sarcoma krista noonan consulting or advisory role : merck , sano canada , bristol - myers squibb , janssen , pzer , astrazeneca speakers bureau : merck stephen yip consulting or advisory role : bayer , pzer , roche research funding : pzer , bayer , roche , foundation medicine travel , accommodations , expenses : bayer , roche , pzer jason hart honoraria : novartis martin jones employment : qiagen , thermo fisher scientic ( i ) howard lim honoraria : eli lilly / imclone systems , ipsen , merck , roche canada , eisai , taiho pharmaceutical , bristol - myers squibb canada travel , accommodations , expenses : eisai , taiho daniel j . 
renouf honoraria : celgene , servier , taiho pharmaceutical , ipsen , shire research funding : bayer ( inst ) , astrazeneca ( inst ) karen gelmon honoraria : astrazeneca , merck sharp & dohme consulting or advisory role : pzer , novartis , astrazeneca , merck , eli lilly , bristol - myers squibb , nanostring technologies , genomic health , janssen oncology , roche , mylan research funding : pzer ( inst ) , bristol - myers squibb ( inst ) , astrazeneca ( inst ) , roche ( inst ) expert testimony : genentech marco marra travel , accommodations , expenses : roche canada janessa laskin honoraria : boehringer ingelheim , roche canada , astrazeneca , pzer research funding : astrazeneca ( inst ) , roche canada ( inst ) no other potential conicts of interest were reported . acknowledgment we thank katherine mui , jessica nelson , and lindsay zibrik from the personalized oncogenomics ( pog ) program for coordinating our study . this work would not be possible without the participation of our patients and families , the entire pog team , and the generous support of the bc cancer foundation and genome british columbia ( project b20pog )  . 
cell 171 : 950 - 965.e28 , 2017 schwaederle m , zhao m , lee jj , et al : impact of precision medicine in diverse cancers : a meta - analysis of phase ii clinical trials . 
j clin oncol 33 : 3817 - 3825 , 2015 schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
jama oncol 2 : 1452 - 1459 , 2016 jones mr , lim h , shen y , et al : successful targeting of the nrg1 pathway indicates novel treatment strategy for metastatic cancer . 
ann oncol 28 : 3092 - 3097 , 2017 laskin j , jones s , aparicio s , et al : lessons learned from the application of whole - genome analysis to the treatment of patients with advanced cancers . 
cold spring harb mol case stud 1 : a000570 , 2015 shefeld bs , tinker av , shen y , et al : personalized oncogenomics : clinical experience with malignant peritoneal mesothelioma using whole genome sequencing . 
plos one 10 : e0119689 , 2015 jamshidi f , pleasance e , li y , et al : diagnostic value of next - generation sequencing in an unusual sphenoid tumor . 
oncologist 19 : 623 - 630 , 2014 gao j , aksoy ba , dogrusoz u , et al : integrative analysis of complex cancer genomics and clinical proles using the cbioportal . 
timms km , abkevich v , hughes e , et al : association of brca1 / 2 defects with genomic scores predictive of dna damage repair deciency among breast cancer 15 . 
breast cancer res 16 : 475 , 2014 oncotarget 8 : 39254 - 39267 , 2017 italiano a , khalifa e , laizet y , et al : genetic landscape of soft - tissue sarcomas : moving toward personalized medicine . 
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cytogenet genome res 150 : 52 - 59 , 2016 van dartel m , redeker s , bras j , et al : overexpression through amplication of genes in chromosome region 17p11.2 approximately p12 in high - grade osteosarcoma . 
lee sh , chang mh , baek kk , et al : high - dose ifosfamide as secondor third - line chemotherapy in refractory bone and soft tissue sarcoma patients . 
mahmood st , agresta s , vigil ce , et al : phase ii study of sunitinib malate , a multitargeted tyrosine kinase inhibitor in patients with relapsed or refractory soft tissue sarcomas . 
tawbi ha , burgess m , bolejack v , et al : pembrolizumab in advanced soft - tissue sarcoma and bone sarcoma ( sarc028 ) : a multicentre , two - cohort , single - arm , 39 . 
kim jr , moon yj , kwon ks , et al : tumor inltrating pd1 - positive lymphocytes and the expression of pd - l1 predict poor prognosis of soft tissue sarcomas . 
wei s , shreiner ab , takeshita n , et al : tumor - induced immune suppression of in vivo effector t - cell priming is mediated by the b7 - h1 / pd - 1 axis and transforming growth factor beta . 
the number of potentially targetable alterations was determined by comparing observed somatic mutations and gene expression alterations to a database of events that have clinical or preclinical evidence of potential therapeutic targeting , on the basis of therapeutics currently available or in development . mutation copy number expression only samples with potentially targetable alterations ( % ) fig a2 . 
samples with potentially targetable alterations were determined by comparing observed somatic mutations and gene expression alterations to a database of events that have clinical or preclinical evidence of potential for therapeutic targeting , on the basis of therapeutics currently available or in development . 
 feng et al baseline before standard chemotherapy ( april 2016 ) progression after standard chemotherapy ( august 2016 ) response to pog - directed therapy ( july 2017 ) fig a3 . 
 personalized oncogenomics of adult metastatic sarcoma baseline before standard chemotherapy ( january 2015 ) progression after standard chemotherapy ( may 2015 ) response to pog - directed therapy ( december 2015 ) fig a4 . 
annotation tracks show exposure values calculated from mutation analysis ( structural variant [ sv ] , single - nucleotide variant [ snv ] burden ) , clinical covariates , and histologic properties of the sarcoma cohort . 
 feng et al baseline before standard chemotherapy ( august 2015 ) progression after standard chemotherapy ( july 2016 ) response to pog - directed therapy ( october 2016 ) fig a7 . 
 personalized oncogenomics of adult metastatic sarcoma baseline ( feb 2015 ) progression after standard chemotherapy ( april 2015 ) response to pog - directed therapy ( october 2015 ) fig a8 . 
other than undifferentiated pleomorphic sarcoma ( ups ) , some leiomyosarcoma ( lms ) and dedifferentiated liposarcoma ( ddlps ) and one case of synovial sarcoma ( ss ) seem to have relatively high iis compared with others . 
 precision oncology strategy in trastuzumab - resistant human epidermal growth factor receptor 2positive colon cancer : case report of durable response to ado - trastuzumab emtansine introduction colorectal cancer is the third most common cancer and the fourth leading cause of cancer death worldwide.1 metastatic disease is ultimately identified in the majority of patients with colorectal cancer and , with rare exceptions , is considered incurable . 
the treatment of metastatic colorectal cancer ( mcrc ) has historically included the use of fluorouracil in combination with other chemotherapeutic agents , including oxaliplatin or irinotecan . the addition of the antivascular endothelial growth factor antibody , bevacizumab , to standard chemotherapy regimens has improved the progression - free and overall survival of patients with mcrc and has signaled a shift toward the addition of biologic and targeted agents in the treatment of advanced cancer.2 , 3 the transition toward molecularly targeted therapies has gained additional momentum with the us food and drug administration approval of the antiepidermal growth factor receptor agents cetuximab and panitumumab for the treatment of kras wild - type colorectal cancers.4 , 5 unfortunately , many patients with mcrc progress through these therapies , or are not candidates to receive them on the basis of the molecular profile of their particular tumor , and are left with few treatment options . 
 the tumor exhibited normal expression of the mismatch repair genes mlh1 , msh2 , msh6 , and pms2 . shortly after diagnosis , the patient began palliative chemotherapy with capecitabine and oxaliplatin every 3 weeks . 
however , follow - up imaging 6 months after treatment initiation identified progressive disease in the liver and lung , as well as a new adrenal gland metastasis . the patients treatment was then changed to capecitabine plus irinotecan and , ultimately , capecitabine , irinotecan , plus cetuximab , but his tumor continued to progress through each of these regimens , as determined by an increasing size of multiple hepatic metastases ( table 1 )  . 
we used a standard set of her2 - specific taqman primers and probes ( thermo fisher scientific , foster city , ca ) compared with standard references using an ultraconserved region on chromosome 1 . 
briefly , taqman pcr reaction mixtures were assembled using 2x ddpcr supermix for probes , 20x assays ( 18 mm primers and 5 mm probe ) and restriction digested dna samples ( bio - rad )  . 
this standard reference assay used the following primers : forward primer : 59 - tgagggattcggcagatgttg - 39 ; reverse primer : 59 - ctgaaaggctggacttgacaga - 39 ; and probe : 59 - vic - actgtgtgctggacctmgb - 39 . 
all assay primers were ordered from integrated dna technologies ( coraville , ia )  . thermal cycling conditions were 95c for 10 minutes ( 1 cycle ) ; 94c for 30 seconds and 60c for 60 seconds ( 40 cycles ) ; 98c for 10 minutes ( 1 cycle ) , and a 12c hold . 
her2 copy number per cell was estimated as the ratio of the her2 and rna polymerase 30 ( rpp30 ) concentrations multiplied by two to account for the two copies of rpp30 that are expected per diploid genome . 
quadruplicate ddpcr wells were analyzed for each sample . results in the absence of additional treatment options , and given the patients good performance status , another liver biopsy was performed and nextgeneration sequencing was completed in a certified laboratory , which identified several tumor - specific somatic mutations , including apc r232 * , tp53 splice site 97_1g.a , and her2 amplification . the patients case and tumor genomics were then presented to a multi - institutional molecular tumor board for interpretation . the identification of a mutation in the adenomatous polyposis coli ( apc ) gene was expected and consistent with previous reports in which up to 85% of spontaneous colorectal cancers harbor somatic mutations in the classic tumor suppressor.16 a splice site mutation in the tp53 gene was equally unsurprising , given the relative frequency of tp53 mutations in human cancers.17 , 18 the molecular tumor board considered neither the apc nor tp53 mutations clinically actionable . treatment duration , months best outcome table 1 . 
cross - reference with the cancer genome atlas confirmed that approximately 4% to 5% of spontaneous colorectal cancers harbor high - level her2 amplifications , 19 and preclinical data predicted that anti - her2 therapy might be clinically effective , particularly when used in a combinatorial approach ( table 2 ) .20 , 21 gene alteration r232 * tp53 splice site 97_1g > a erbb2 amplification fig 1 . 
tumor - specific somatic alterations and quantification of the her2 copy number alteration . ( a ) next - generation sequencing identified three validated alterations in the patients hepatic metastatic colon cancer . 
 ( b ) using a droplet digital polymerase chain reaction assay , the total copies of her2 per genome in the patients metastatic tumor ( blue dot ) were measured and compared with the total copies of her2 in the patients germline ( peripheral blood , gold dot )  . 
error bars represent standard error of the mean . peripheral blood tumor to further characterize the her2 amplification and to validate the finding using an orthogonal method , a ddpcr assay was used . 
in contrast , a control gene , rpp30 , had the expected diploid copy number of two ( fig 1 )  . given the genomic findings and the patients clinical circumstances , treatment with trastuzumab was initiated . 
follow - up ct imaging , compared with baseline evaluation , revealed disease progression in the liver , with the largest lesion measuring 5.8 cm ( figs 2a and 2b )  . trastuzumab treatments were discontinued and the patient then received trastuzumab - emtansine ( t - dm1 ) infusions every 21 days for three total treatments . 
follow - up imaging revealed the largest hepatic metastasis measured 4.6 cm , compared with 5.3 cm at the initiation of t - dm1 ( fig 2c )  . three additional infusions at 21 - day intervals with subsequent imaging yielded ongoing disease response , with the largest hepatic mass measuring 3.2 cm ( fig 2d )  . 
the patient ultimately received t - dm1 infusions every 21 days for 15 months without any discernible negative clinical effect . the largest hepatic metastasis decreased to 2.1 cm ( fig 2e ) and two other metastases responded completely . 
the use of next - generation sequencing panels to identify actionable mutations and guide treatment has become increasingly scrutinized as a potentially viable approach for improving outcomes in a variety of cancer subtypes , including lung cancer and melanoma . 
these findings are bolstered by an interim analysis report from the mypathway study2 investigating the use of the combination of trastuzumab plus pertuzumab in various solid tumors , including mcrc . the case reported here suggests that genomicsbased analysis of refractory colorectal cancer can lead to effective targeted treatments . 
the expected progression - free survival and overall survival of a patient with mcrc with refractory disease is approximately 1.7 and 5 months , respectively , 25 in contrast to the 15 - month progression - free survival observed in this case . additional data and outcomes are required to confirm that precision oncology routinely yields superior outcomes ; however , this report illustrates an additional treatment option in patients with refractory disease who have exhausted all of the commonly used therapeutics for their respective disease and yet retain an acceptable performance status and desire additional treatmenta common clinical scenario . 
kamangar f , dores gm , anderson wf : patterns of cancer incidence , mortality , and prevalence across five continents : defining priorities to reduce cancer disparities in different geographic regions of the world . 
saltz lb , clarke s , daz - rubio e , et al : bevacizumab in combination with oxaliplatin - based chemotherapy as first - line therapy in metastatic colorectal cancer : a randomized phase iii study . 
douillard jy , siena s , cassidy j , et al : randomized , phase iii trial of panitumumab with infusional fluorouracil , leucovorin , and oxaliplatin ( folfox4 ) versus folfox4 alone as first - line treatment in patients with previously untreated metastatic colorectal cancer : the prime study . 
slamon dj , clark gm , wong sg , et al : human breast cancer : correlation of relapse and survival with amplification of the her - 2 / neu oncogene . 
sartore - bianchi a , trusolino l , martino c , et al : dual - targeted therapy with trastuzumab and lapatinib in treatmentrefractory , kras codon 12 / 13 wild - type , her2 - positive metastatic colorectal cancer ( heracles ) : a proof - ofconcept , multicentre , open - label , phase 2 trial . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors based on molecular profiles : early results from mypathway , an open - label , phase iia umbrella basket study . 
richman sd , southward k , chambers p , et al : her2 overexpression and amplification as a potential therapeutic target in colorectal cancer : analysis of 3256 patients enrolled in the quasar , focus and piccolo colorectal cancer trials . 
sartore - bianchi a , ardini e , bosotti r , et al : sensitivity to entrectinib associated with a novel lmna - ntrk1 gene fusion in metastatic colorectal cancer . 
bertotti a , migliardi g , galimi f , et al : a molecularly annotated platform of patient - derived xenografts ( xenopatients ) identifies her2 as an effective therapeutic target in cetuximab - resistant colorectal cancer . 
parikh a , atreya c , korn wm , et al : prolonged response to her2 - directed therapy in a patient with her2amplified , rapidly progressive metastatic colorectal cancer . 
bensch f , van rooijen jm , schr oder cp , et al : a 21 - year - old patient with a her2 - positive colorectal cancer . discov 5 : 832 - 841 , 2015 gastroenterology 148 : 20 - 21 , 2015 cancer . 
grothey a , van cutsem e , sobrero a , et al : regorafenib monotherapy for previously treated metastatic colorectal cancer ( correct ) : an international , multicentre , randomised , placebo - controlled , phase 3 trial . 
johnson , phd1 purpose given regulatory approval of immune checkpoint inhibitors in patients with mismatch repairdecient ( mmr - d ) cancers agnostic to tumor type , it has become important to characterize occurrence of mmr - d and develop cost - effective screening approaches . 
using a next - generation sequencing ( ngs ) panel ( oncopanel ) , we developed an algorithm to identify mmr - d frequency in tumor samples and applied it in a clinical setting with pathologist review . methods to predict mmr - d , we adapted methods described previously for use in ngs panels , which assess patterns of single base - pair insertion or deletion events occurring in homopolymer regions . 
for tumors tested after june 2017 , sequencing results were presented to pathologists in real time for clinical mmr determination , in the context of tumor mutation burden , other mutational signatures , and clinical data . results of 20 , 301 tumors sequenced , 2.7% ( 553 ) were retrospectively classied as mmr - d by the algorithm . of 4 , 404 samples with pathologist sign - out of mmr status , the algorithm classied 147 ( 3.3% ) as mmr - d : in 116 cases , mmr - d was conrmed by a pathologist , ve cases were overruled by the pathologist , and 26 were assessed as indeterminate . 
overall , the highest frequencies of oncopanel - inferred mmr - d were in endometrial ( 21% ; 152 / 723 ) , colorectal ( 9.7% ; 169 / 1 , 744 ) , and small bowel ( 9.3% ; 9 / 97 ) cancers . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction as regulatory approvals for immunotherapy agents expand across tumor types , efforts to identify predictive biomarkers of response or resistance to treatment are similarly expanding . 
comprehensive analysis of colorectal cancers has shown that tumors with defects in mismatch repair ( mmr ) , resulting in microsatellite instability ( msi ) , have elevated lymphocytic inltration.1 in addition , tumors with msi demonstrate upregulated expression of inhibitory immune checkpoints , including pd - l1 , on tumorinltrating lymphocytes and myeloid cells . 
overall , the algorithm classied 2.7% of samples as mmr - d . relevance we show the value of using a routine ngs panel to identify new , clinically relevant biomarkers that might otherwise not be assessed because of low prevalence in certain tumor types and to identify potential candidates for immunotherapy . testing using ihc or pcr is not routinely pursued in clinical practice in other tumor types , despite national comprehensive cancer network testing recommendations for the rectal , endometrial , following cancers : cervical , colon , esophageal , gastroesophageal junction , gastric carcinoma , gallbladder , intrahepatic cholangiocarcinoma , extrahepatic cholangiocarcinoma , neuroendocrine , ovarian , pancreatic , prostate , testicular , and vulvar . 
broad implementation of a targeted , tumor - only next - generation sequencing ( ngs ) assay ( oncopanel ) for patients at dana - farber cancer institute ( dfci ) , brigham and womens hospital ( bwh ) , and boston childrens hospital ( bch ) allowed us to assess whether bioinformatic analysis of oncopanel7 data could characterize the prevalence of mmr - d / msi - h ( hereafter mmr - d ) across tumors and increase the number of patients who could be considered for immunotherapy . methods such as msings , 8 msi - sensor9 and cell - free dna10 have previously been reported for detection of mmr - d . 
however , given the wide implementation and relatively low cost of tumor - only ngs , it is important to develop approaches suited for this paradigm and understand potential differences in results . 
it is also critical to better characterize the frequency of mmr - d across tumors to inform selective use of ihc , pcr , or ngs assays in clinical settings where ngs panels and mmr testing are used less frequently . 
this study expands on previous work to develop oncopanel for clinical assessment of mmr - d using ihc and pcr for validation11 and to characterize mmr status in 645 upper gi tract cancers12 and 304 sarcomas.13 here we describe a bioinformatics algorithm that infers mmr status using an institution - wide ngs panel and report the prevalence of mmr - d across  . 
of these tumors , 4 , 488 were sequenced after the implementation on june 27 , 2017 of a process for pathologist sign - out of ngsbased , mmr - d assessment , described in more detail below . 
a total of 4 , 404 of these 4 , 488 tumors met criteria for mmr assessment via the oncopanel algorithm , which measures insertion or deletion ( indel ) events within homopolymeric microsatellite loci to infer mmr status , and were presented for nal interpretation by a molecular pathologist . 
signed - out oncopanel reports , including mmr - d calls , were returned to physicians for clinical use . patients from the aforementioned study of 304 sarcomas13 are also included in the summary data here , although with different subclassications . 
here , we used the original pathologists diagnoses as recorded at oncopanel sign - out , rather than more detailed subclassication of sarcomas using chart review . sequencing from august 2013 to july 2014 , patient samples were tested with oncopanel version 1 , which covers 0.753334 mb and the full coding regions of 275 genes , plus selected intronic regions of 30 genes for rearrangement detection . 
at the time of clinical pathology assessment for mmr deciency ( on or after june 27 , 2017 ) , all samples were being sequenced using oncopanel version 3.14 variants observed in public genome databases at a frequency of  . 
finally , variants were tiered and reported based on clinical significance by a molecular pathologist using methods previously described.16 all testing was conducted in a clinical laboratory improvement amendmentscertied environment . mmr algorithm on the basis of methods described previously for identifying mmr - d in colorectal cancer using oncopanel versions 1 - 2 , 11 an internally developed algorithm was adjusted to predict mmr status across all tumor types . 
only single base - pair patterns of single base - pair indels in homopolymers were considered ( with a minimum threshold of 1.5 / mb ) , and tumor mutational burden ( tmb ) was ignored . possible values resulting from the oncopanel algorithm are procient ( mmr - p ) , decient ( mmr - d ) , and cannot assess ( for tumors with , 12 variants ) , although a pathologist may override algorithmic calls or mark a tumor indeterminate . for the 4 , 488 samples where mmr results were communicated to the ordering physician , a pathologist manually reviewed the algorithmic mmr status to assign nal status . 
pathologists interpreted the algorithmic mmr determination in the context of tmb , other mutational signatures validated on the platform ( uv [ ultraviolet ] , tobacco , apobec [ apolipoprotein b mrna editing enzyme , catalytic polypeptide - like ] , temozolomide , and pole [ dna polymerase epsilon catalytic subunit ] ) , and existing clinical data such as mmr protein expression , when available . 
recognizing that the positive and negative predictive values of the mmr algorithm are lower near the dened cutoff , and that patterns of mmr - d are not well dened for tumors outside of colon and endometrium , the pathologist could elect to override the algorithmic classication . 
possible reasons for overriding the classication included homopolymer indel rates near the cutoff , low tmb for that tumor type , or no mmr gene alterations in the sequencing data . the treating clinician was also encouraged to seek conrmatory testing with ihc or pcr in this setting when clinically relevant . tmb was calculated by including all nonsynonymous mutations in coding regions of the genome covered by oncopanel . 
we reported three tmb metrics for each tumor : mutations per megabase , percentile of tmb across all tumors sequenced in the current oncopanel version , and percentile of tmb across tumors of each cancer type . orthogonal testing twelve of the 26 tumor samples with discrepant calls between algorithm and pathologist , specically those with algorithm - predicted mmr - d , a pathologist call of indeterminate , and sufcient leftover dna , underwent orthogonal , tumor - only pcr testing . 
overall , among 3 , 779 noncolorectal , nonendometrial samples for which oncopanel results were returned to clinicians , 43 ( 1.14% ) tumors were detected as msi - h / mmr - d , and results were reported to the patients treating clinicians . without another indication for clinical msi / mmr testing , these 43 patients are unlikely to have been tested outside of the oncopanel program . across the 816 pediatric tumors proled , eight were predicted as mmr - d algorithmically . 
pcr - based orthogonal validation was performed on the small bowel case to conrm msi - h status , and msi was detected for three of four microsatellite markers . we identied two adult appendiceal tumors with predicted mmr - d : one predicted by the algorithm in the historical cohort and the other identied by a pathologist during routine clinical sign - out . 
17 - 465 all represents the overall oncopanel cohort using algorithm prediction , and 17 - 465 pathologist assessed represents the subset with pathology sign - out . abbreviations : mmr - d , mismatch repair deciency ; n / a , tumor type not assessed or specied . mmr - d has not been reported previously in appendiceal cancer . 
in addition , we found one thymic mmr - d tumor among ve cases ( 20% ) , indicating that thymic cancers too can be mmr - d , corroborating a recent report.20 the cancer genome atlas ( tcga ) and therapeutically applicable research to generate effective treatments ( target ) projects20 reported msi - h status in 3.8% of tumors , using paired tumor - normal sequencing across cancer types and the algorithm mantis , 40 and identied in 27 cancer types ( see discussion )  . 
one factor among many that could play a role in discordance between algorithmic prediction and pathologist assessment ( table 5 ) is tmb , which was relatively high in concordant mmr - d cases ( mean tmb , 55.8 mut / mb )  . 
for the 4 , 404 we found 2.72% of 20 , 301 tumor samples to be mmr - d across a wide range of adult and pediatric malignancies using a bioinformatics algorithm to process tumor - only ngs data . 
mapping of tumor types from sources original tumor type mapped tumor type colon adenocarcinoma rectal adenocarcinoma colorectal adenocarcinoma barret esophagus adenocarcinoma esophageal carcinoma gastric adenocarcinoma stomach or gastric adenocarcinoma esophagogastric junction carcinoma pancreatic adenocarcinoma uterine carcinosarcoma kidney chromophobe kidney renal clear cell carcinoma kidney renal papillary cell carcinoma kidney cancer sarcoma glioblastoma multiforme lower - grade or low - grade glioma merkel cell carcinoma colorectal cancer colorectal cancer colorectal cancer esophagogastric carcinoma esophagogastric carcinoma esophagogastric carcinoma esophagogastric carcinoma esophagogastric carcinoma pancreatic cancer uterine sarcoma renal cell carcinoma renal cell carcinoma renal cell carcinoma renal cell carcinoma soft tissue sarcoma glioma glioma skin cancer , nonmelanoma small intestinal malignancies small bowel cancer extrahepatic bile duct adenocarcinoma biliary cancer malignant solitary brous tumor of the pleura soft tissue sarcoma ovarian surface epithelial carcinoma nonepithelial ovarian cancer ovarian cancer ovarian cancer cervical squamous cell carcinoma / endocervical cervical cancer adenocarcinoma pediatric acute myeloid leukemia acute myeloid leukemia pediatric high - risk wilms tumor wilms tumor poorly differentiated carcinoma , nos cancer of unknown combined small - cell lung carcinoma lung bronchioloalveolar carcinoma thyroid carcinoma thymic carcinoma head and neck squamous carcinoma nut midline carcinoma of the head and neck prostatic adenocarcinoma primary small - cell lung cancer nonsmall - cell lung cancer thyroid cancer thymic tumor head and neck carcinoma head and neck carcinoma prostate cancer abbreviations : nos , not otherwise specied ; nut , nuclear protein in testis . agreement between purely algorithmic calls and formal pathologist classication . 
it should be noted that these two measures are not independent , because the pathologists included the algorithmic calls as a part of their assessment , along with tmb , results of other mutational signatures , and existing clinical data . 
orthogonal laboratory validation of tumors predicted to be mmr - d but signed out as indeterminate demonstrated the value of manual pathologist review as a nal step in the mmr - d classication process and suggests that inclusion of other data types could improve future algorithms . conrming prior clinical observations , frequencies of predicted mmr - d were in endometrial and colorectal cancers , but mmr - d was also observed in many different cancer types not commonly assessed for mmr - d in routine clinical practice , including appendiceal cancer and nonmelanoma skin cancer . the highest further manual review of predicted mmr - d tumors signed out as indeterminate highlighted the tendency for tumors with a pattern of indels in homopolymer regions but low tmb to be called indeterminate . 
our results suggest conrmatory laboratory testing should continue to be used , especially when borderline ngs results are obtained or mmr - d is predicted in a tumor with low mmr - d prevalence . 
correlation between high tmb and msi - h has been reported in other ngs studies , including in prostate cancer , where 71% of 111 tumors with high tmb ( 20 mut / mb ) were msi - h , 41 and in adrenocortical carcinoma ( n = 92 ) and cervical squamous cell and cervical adenocarcinoma ( n = 305 ) .20 in a larger ngs panel study ( n = 11 , 000 ) , only 27% of tumors with high tmb ( dened as 17 mut / mb ) were msi - h ( 46 altered microsatellite loci / mb ) , whereas 70% of msi - h cases had high tmb.21 as exemplied in these studies , however , there is signicant variability in the denitions of high tmb and the methodologies used to measure it.42 recent fda approval of pembrolizumab for high - tmb tumors dened high tmb as 10 mut / mb , with a targeted gene panel as a companion diagnostic . 
 clinical assessment of mmr deciency using targeted ngs measure names oncopanel pathologist oncopanel all bonneville middha vanderwalde dudley colorectal cancer endometrial cancer esophagogastric carcinoma head and neck carcinoma hepatocellular carcinoma prostate cancer renal cell carcinoma soft tissue sarcoma thyroid cancer fig 1 . 
across all studies described in table 3 , 18 - 39 the frequency of mmr - d or msi - h was reported across all six cohorts in nine tumor types : colorectal , endometrial , esophagogastric , head and neck , hepatocellular , prostate , renal cell carcinoma , thyroid cancer , and soft tissue sarcoma . 
hepatocellular carcinoma and thyroid cancer had considerably higher msi - h rates reported in the review by dudley et al.19 it is encouraging , however , that mmr - d prevalence in most tumor types across independent studies was generally consistent ( fig 1 ) , especially for other ngs - based table 4 . 
mean tmb across 4 , 404 oncopanel tumors , subcategorized by algorithm - predicted mmr status and pathologist - assessed mmr status pathologist assessment algorithm mmr - d algorithm mmr - p pathologist mmr - d pathologist mmr - p pathologist indeterminate 54.96 ( 116 ) 17.34 ( 5 ) 17.61 ( 26 ) 47.36 ( 147 ) mean tmb 72.24 ( 6 ) 7.58 ( 4 , 213 ) 22.71 ( 38 ) 8.40 ( 4 , 257 ) 55.81 ( 122 ) 7.59 ( 4 , 218 ) 20.64 ( 64 ) 8.99 ( 4 , 404 ) note . 
across tumor types , it remains unclear which methodology to determine mmr status provides greater accuracy , and ngs computational algorithms may improve identication of these tumors compared with tmb , pcr , or ihc testing . 
it will also be important for future studies to measure the impact of mmr - d assessment on treatment decisions and patient outcomes . given the low overall frequency of mmr - d across most noncolorectal , nonendometrial tumors , we believe our algorithm provides a cost - effective initial screening tool to identify patients for pcror ihc - based testing . 
from a clinical implementation standpoint , rather than testing all patients with cancer with pcr or ihc to identify the , 3% of patients who may be candidates for immune checkpoint inhibitors on the basis of mmr - d , implementing the algorithm in the context of an existing , multipurpose ngs tumor - only panel , can provide an efcient initial screen and allow clinicians to select only those tumors most likely to be mmr - d for further testing . 
biomarker studies in patients with mmr - d tumors treated with immune checkpoint inhibition will provide further insight . in the near future , combined testing of many biomarkers across a large panel of genes as described here may be more efcient in cost , time , and sample material than sequential or more selective biomarker testing , and it also offers the ability to quickly incorporate new biomarker discoveries into clinical practice and bring effective treatments to patients who would otherwise not benet from these discoveries . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / authors / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . mayur amonkar employment : merck stock and other ownership interests : merck alanna j . 
church honoraria : biorad laboratories , jackson laboratories consulting or advisory role : alphasights , samba scientic , perceptive advisors , jackson laboratories , bayer travel , accommodations , expenses : jackson laboratories , bayer adem albayrak employment : health catalyst stock and other ownership interests : health catalyst honoraria : bc platforms research funding : merck ( inst ) travel , accommodations , expenses : health catalyst other relationship : guidepoint marios giannakis research funding : bristol myers squibb , merck renato umeton consulting or advisory role : health catalyst patents , royalties , other intellectual property : patent : portable medical device and method for quantitative retinal image analysis through a smartphone ; patent : epstein barr virus genotypic variants and uses thereof as risk predictors , biomarkers , and therapeutic targets of multiple sclerosis elizabeth h . 
johnson consulting or advisory role : novartis , foundation medicine , hengrui therapeutics , daiichi sankyo , chugai pharmaceuticals , lilly , checkpoint therapeutics research funding : toshiba ( inst ) , novartis ( inst ) , novartis patents , royalties , other intellectual property : dana - farber cancer institute kai - li liaw employment : merck sharp & dohme stock and other ownership interests : merck sharp & dohme research funding : merck sharp & dohme travel , accommodations , expenses : merck sharp & dohme katherine a . 
nowak patents , royalties , other intellectual property : provisional patent application involving novel methods for characterizing immune cell distributions in solid tumors . lynette sholl honoraria : astrazeneca consulting or advisory role : loxo oncology , emd serono research funding : roche / genentech nancy u . 
lin consulting or advisory role : seattle genetics , puma biotechnology , daiichi sankyo , california institute for regenerative medicine , denali therapeutics research funding : genentech , pzer , seattle genetics , merck patents , royalties , other intellectual property : royalties for chapter in upto - date regarding management of breast cancer brain metastases ; royalties , jones & bartlett jason m . 
johnson consulting or advisory role : ucb , patientslikeme research funding : merck travel , accommodations , expenses : ucb no other potential conicts of interest were reported . acknowledgment we thank kaitlyn t . 
bifolck , ba , for her editorial support . references llosa nj , cruise m , tam a , et al : the vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter - inhibitory checkpoints . 
newsroom / pressannouncements / ucm560167.htm. le dt , durham jn , smith kn , et al : mismatch repair deciency predicts response of solid tumors to pd - 1 blockade . 
murphy km , zhang s , geiger t , et al : comparison of the microsatellite instability analysis system and the bethesda panel for the determination of microsatellite instability in colorectal cancers . 
am j cancer res 8 : 1977 - 1988 , 2018 salipante sj , scroggins sm , hampel hl , et al : microsatellite instability detection by next generation sequencing . 
nowak ja , yurgelun mb , bruce jl , et al : detection of mismatch repair deciency and microsatellite instability in colorectal adenocarcinoma by targeted nextres 25 : 7035 - 7045 , 2019 generation sequencing . 
christakis ag , papke dj , nowak ja , et al : targeted cancer next - generation sequencing as a primary screening tool for microsatellite instability and lynch syndrome in upper gastrointestinal tract cancers . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
vanderwalde a , spetzler d , xiao n , et al : microsatellite instability status determined by next - generation sequencing and compared with pd - l1 and tumor mutational burden in 11 , 348 patients . 
glavac d , volavsek m , potocnik u , et al : low microsatellite instability and high loss of heterozygosity rates indicate dominant role of the suppressor pathway in squamous cell carcinoma of head and neck and loss of heterozygosity of 11q14.3 correlates with tumor grade . 
chiappini f , gross - goupil m , saffroy r , et al : microsatellite instability mutator phenotype in hepatocellular carcinoma in non - alcoholic and non - virally infected normal livers . 
murphy ma , wentzensen n : frequency of mismatch repair deciency in ovarian cancer : a systematic review this article is a us government work and , as such , is in the public domain of the united states of america . 
hammerschmied c , stoehr r , walter b , et al : the role of elevated microsatellite alterations at selected tetranucleotides ( emast ) in renal cell carcinoma ( rcc )  . cancer res 67 , 2007 ( abstr 1934 ) 35 . 
palmieri g , ascierto pa , cossu a , et al : assessment of genetic instability in melanocytic skin lesions through microsatellite analysis of benign naevi , dysplastic naevi , and primary melanomas and their metastases . 
cesinaro am , ubiali a , sighinol p , et al : mismatch repair proteins expression and microsatellite instability in skin lesions with sebaceous differentiation : a study in different clinical subgroups with and without extracutaneous cancer . 
reuschenbach m , sommerer c , hartschuh w , et al : absence of mismatch repair deciency - related microsatellite instability in non - melanoma skin cancer . j invest dermatol 132 : 491 - 493 , 2012 39 . 
vokes ni , liu d , ricciuti b , et al : harmonization of tumor mutational burden quantication and association with response to immune checkpoint blockade in non - small - cell lung cancer . 
boland cr , thibodeau sn , hamilton sr , et al : a national cancer institute workshop on microsatellite instability for cancer detection and familial predisposition : development of international criteria for the determination of microsatellite instability in colorectal cancer . 
comparison of oncopanel mmr algorithm with historical clinical msi / mmr testing laboratory assessment algorithm mmr - d algorithm mmr - p total laboratory msi - h laboratory mss total note . 
 clinical use of precision oncology decision support purpose precision oncology is hindered by the lack of decision support for determining the functional and therapeutic significance of genomic alterations in tumors and relevant clinically available options . 
to bridge this knowledge gap , we established a precision oncology decision support team that provides annotations at the alteration level and subsequently determined whether clinical decision making was influenced . methods genomic alterations were annotated to determine actionability on the basis of a variants known or potential functional and / or therapeutic significance . 
a webbased survey was implemented to capture the reasons genotype - matched therapies were not pursued . results the precision oncology decision support team processed 1 , 669 requests for annotation of 4 , 084 alterations ( 2 , 254 unique ) across 49 tumor types for 1 , 197 patients . 
sixty - six percent of patients had at least one actionable / potentially actionable alteration , and 20.6% of patients ( 110 of 535 ) annotated enrolled in a genotype - matched trial . 
clinicians cited a variety of reasons that patients with actionable alterations did not enroll in trials . conclusion over half of alterations annotated were of unknown significance or nonactionable . physicians were more likely to enroll a patient in a genotype - matched trial when an annotation supported actionability . 
 research indicates that clinicians are unlikely to use sources that take longer than 30 seconds to retrieve data10 and that information presented in graphical summaries enhances interpretation , improving health care quality.11 , 12 second , limited information may be exposed because these web sites are public facing . 
third , novel alterations continue to be discovered , and information is rapidly evolving , generally outpacing the rate of updates . thus , there is a need for an on - demand , real - time clinical interpretation service that annotates all requested alterations , beyond those available within accessible knowledge bases , to determine their actionability . the precision oncology decision support system ( pods ) was established at the university of texas md anderson cancer center in recognition of these needs.9 herein , we report our experience with large - scale , institution - wide decision support and its initial clinical utility . 
all requestors indicating the same physician as the contact for correspondence were considered a teastatistical significance was evaluated by a x2 test . annotation process and return of reports pods scientists receive requests , access our knowledge base of variant annotations , and review and update content as applicable.9 , 13 reports consist of a data summary with references and alteration frequency from external ( cbioportal , cosmic [ catalogue of somatic mutations in cancer ] ) and internal databases . 
in 2015 , reports evolved to routinely contain a functional significance , gene , and alteration - level actionability call ( table 1 ) .7 , 12 all reports are reviewed by the medical director and returned via e - mail . 
starting in august 2015 , reports referencing clia - validated panels were deposited within md andersons electronic health record ( ehr )  . manual review of clinical data medical records of 539 patients annotated in 2015 through clinician - initiated requests or for treatment planning conferences were reviewed for the presence of a clinical trial entrance note in the patients ehr . 
potential matches between the annotated variants and targeted therapy used within the trial were manually recorded . clinical decision follow - up surveys a web - based survey was initiated in 2015 , and 236 surveys were sent to annotation requestors with a 1 - month postannotation delivery date to ascertain whether the patient matched to genomically guided therapy . 
for patients not enrolled to receive therapy on the basis of the annotation provided , the survey inquired as to the reasons why . results patient annotation requests the pods team processed 1 , 669 requests for 1 , 197 patients , most in 2014 and 2015 ( data supplement )  . 
multiple requests for the same patient may have been due to additional alterations requested for annotation , different clinicians requesting annotation of the same molecular report , or the same molecular report requested for different venues in which the patient was evaluated ( eg , clinic visit v treatment planning conference )  . 
clinician - initiated requests originated from treating physicians and team members for point - of - care decisions on the basis of molecular testing or from clinical trial teams screening for patients to enroll in genotype - matched trials . fifty - six physician teams initiated requests for 724 patients and ranged from one to 176 per team , with two requests being the median ( data supplement )  . 
because not all aspects of protein function can possibly be determined , the functional significance is likely benign there is peer - reviewed published literature showing that the alteration has an inactivating or inferred inactivating effect on an oncogene . there is peer - reviewed published literature showing that the alteration has an activating or inferred activating effect on a tumor suppressor that enhances its function as a tumor suppressor overall , 4 , 084 variant - level annotations were provided for 2 , 254 unique alterations . 
genes most commonly annotated as part of a full panel request included tp53 , kras , and pik3ca , representing the three most commonly mutated genes in the genome ( fig 1c )  . 
the percentage of requests is shown for ( a ) each indicated tumor type ( top 25 ) and ( b ) annotation type ( full panel or select alterations )  . 
genes with 20 or more annotations are shown . actionable genes and alterations a gene is actionable if there are clinically available therapies that directly or indirectly targe alterations in the gene and / or there are clinical trials selecting for alterations in the gene . of the 4 , 084 annotations , 3 , 166 ( 78% ) were within a gene defined as actionable at the time of annotation ( fig 2a ; data supplement )  . 
in 2015 , annotation reports evolved to contain an actionable variant call describing the alterations functional and therapeutic significance , with classification into four broad categories actionable ( further subcategorized by source : literature based , inferred , or functional genomics ) , potentially actionable , unknown , and nonactionable ( table 1 ) 9which was provided with 2 , 444 annotations delivered for 669 patients . 
a total of 794 ( 32.5% ) annotations were actionable , 230 ( 9.4% ) were potentially actionable , 725 ( 29.7% ) were unknown , and 697 ( 28.4% ) were not actionable ( fig 2b )  . 
the total number of annotations in 2015 and the subset that was actionable / potentially actionable are shown per the patients tumor type ( fig 2e and data supplement , respectively )  . 
the actionability of kras has been controversial , particularly in colorectal cancer.14 - 16 when excluding kras as actionable , most actionable annotations were provided for breast cancer patients ( n = 90 ; data supplement )  . clinical utility of annotations we then asked whether physicians acted more often on alterations with evidence of actionability ( data supplement )  . 
we manually recorded clinical follow - up data by accessing the ehrs of 539 patients requested for annotation by clinicians or for treatment planning conferences . four patients were excluded : two had mutations that were actionable only for resistance to therapy , and two because of insufficient follow - up since physician notification . 
for the remaining 535 patients , variant annotation data were filtered , such that each patient in the data set is represented by a single alteration ( fig 3a )  . 
the fraction of patients represented by the highest variant call ( yellow column ) and the corresponding fraction of patients with that call enrolled in a trial ( gray column )  . 
the fraction of patients represented by the highest variant call ( yellow column ) and the corresponding fraction of patients with that call enrolled in a trial ( gray column )  . 
of patients with variant call enrolled in a trial ( % ) yes : literature based 214 ( 40.0 ) 49 ( 22.9 ) yes : inferred 54 ( 10.1 ) 26 ( 48.1 ) potentially 65 ( 12.1 ) 17 ( 26.2 ) unknown 136 ( 25.4 ) 16 ( 11.8 ) no ( nonactionable ) 66 ( 12.3 ) 2 ( 3 ) p < .001 actionable / potentially actionable alterations unknown alterations nonactionable alterations actionable variant classification total no . 
alterations of 214 patients ( 40% ) were classified into the yes : literature based category ; 54 ( 10.1% ) were classified into the yes : inferred category ; 65 ( 12.1% ) were classified into the potentially category ; 136 ( 25.4% ) were classified into the unknown category ; and 66 ( 12.3% ) were classified into the no ( nonactionable ) category ( fig 3a , yellow column )  . 
patients with actionable alterations in pten ( n = 20 ) , pik3ca ( n = 11 ) , and erbb2 ( n = 10 ) most frequently enrolled in a trial ( fig 4 ) , paralleling the data showing a large number of actionable / potentially actionable annotations delivered for pik3ca and pten ( fig 2d ; data supplement )  . to understand why physicians may not have acted on potentially actionable alterations , we introduced a web - based survey in 2015 to accompany physician - initiated requests . 
a combination of survey responses and manual review revealed that 26.8% of annotations ( 59 of 221 ) led to a genotypematched trial ( fig 3c , gray column )  . 
the latter had another well - characterized oncogenic variant ( idh1 r132c , akt1 e17k , or fgf3 and fgf4 amplifications ) or an alteration clearly inferable as actionable ( early truncating ptch1 mutation ) , where either decision support was not needed or was previously supplied outside the survey group . finally , three patients ( 3.3% ) elected to be treated elsewhere . among 161 patients with variants classified as yes : literature based and yes : inferred , there were fig 4 . 
reasons patients did not enroll in genotype - matched trials after precision oncology decision support annotations , as stated by the survey respondents patients ( n = 161 ) patients with yes : literature based and yes : inferred variants ( n = 42 ) patients with variants annotated as potentially actionable ( n = 29 ) patients with variants annotated as unknown and no for actionability ( n = 90 ) 71 ( 44.1 ) 5 ( 11.9 ) 9 ( 31.0 ) 57 ( 63.3 ) reasons patient did not enroll in trial physician stated annotation does not support trial enrollment , no . 
reasons included another alteration pursued ( two patients ) , the patient was responding to current therapy ( six patients ) , and the patient elected treatment elsewhere ( nine patients )  . 
other reasons included nongenotype - matched treatment options pursued ( five patients ) and ineligibility ( 11 patients )  . in five instances , physicians indicated that the annotation did not support trial enrollment , which we investigated further . 
 toxicity - related deaths in patients treated with targeted therapies.17 - 20 however , few biomarkers have an indication for treatment with a food and drug administrationapproved drug specific to the patients tumor type , 21 - 27 and a limited number of patients with potentially actionable alterations receive genotype - matched therapies in experimental contexts.3 - 7 one contributing factor may be a lack of decision support , as suggested by the cancer genome evaluation committee , which found that physicians are often overwhelmed by data of uncertain significance and that sound guidelines are essential for determining clinical action.28 moreover , we previously reported modest differences in trial enrollment between patients with or without potentially actionable alterations.3 assessing a new population of patients where decision support was provided , we observed that physicians acted more often when the function of the alteration was known . the necessity for real - time decision support is clear from the large volume of requests received from numerous physicians ( data supplement ) treating patients with diverse tumor types for alterations in a range of genes . 
however , bias likely exists toward physicians leading targeted therapy trials and for genes targeted by those therapies . the pods team is frequently asked to provide an annotation for sequencing reports from commercial vendors that produce end - to - end reports , some already containing alteration - level annotations . distinguishing factors of pods reports are a clear call of functional effect9 ( eg , activating , inactivating , unknown ) , a range of variant - level actionability categories on the basis of experimental evidence , and inclusion of all md anderson genotype - matched clinical trials in current reports . previous studies reported high rates of alterations in actionable genes , 4 , 29 - 33 and we provided an annotation in at least one actionable gene for 97% of patients in our cohort . 
using the target ( tumor alterations relevant for genomics - driven therapy ) database and phial ( precision heuristics for interpreting the alteration landscape ) algorithm to rank alterations followed by manual annotation for only selected patients , a study showed that 90% of patients have clinically relevant alterations.34 conversely , we found that 66% of patients annotated have at least one potentially actionable alteration on the basis of manual curation of all aberrations . 
potential differences between the studies include ( 1 ) the inclusion of potentially actionable diagnostic or prognostic alterations by van allen et al34 and not in our study ; ( 2 ) we provided annotations for only requested alterations , and few annotation requests were obtained for well - established alterations ( eg , braf v600e ) ; and ( 3 ) the difference in the number of patients assessed by manual curation in the two studies . among all annotations with an actionable variant call , only 32.5% were classified as actionable , with another 9.4% classified as potentially actionable . even within genes that are considered actionable , 47.4% of the annotations either had no evidence to support actionability or were not actionable . moreover , 58% of the genomic alterations annotated and evaluable for frequency have not been reported in the cosmic database ( data supplement )  . 
these data highlight the need to define actionability at the variant , rather than gene , level . to determine whether physicians acted according to the evidence presented in the pods reports , we followed 535 patients . 
thus , physicians more often act on alterations when sufficient evidence is provided that they may be driver events . the most often - cited reason that patients did not enroll in genotype - matched trials was that the annotation did not support trial enrollment . 
mills shaw , john mendelsohn , funda meric - bernstam provision of study materials or patients : sarina piha - paul , vivek subbiah , david hong , kenna r . 
mills shaw , funda meric - bernstam data analysis and interpretation : amber johnson , yekaterinab . khotskaya , lauren brusco , jia zeng , vijakumar holla , ann m . bailey , md abu shufean , russell broaddus , gordon b . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . jia zeng stock and other ownership interests : amgen , mckesson , mylan , adamas pharmaceuticals amber johnson no relationship to disclose yekaterina b . 
s anchez no relationship to disclose md abu shufean no relationship to disclose sarina piha - paul consulting or advisory role : genentech research funding : glaxosmithkline , xuanzhu , puma biotechnology , novartis , merck sharp & dohme , curis , principa biopharma , biomarin , helix biopharma , bayer , abbvie , incyte , five prime therapeutics , cerulean pharma , medimmune , medivation vivek subbiah research funding : novartis , glaxosmithkline , nanocarrier , northwest biotherapeutics , roche / genentech , bergpharma , bayer , incyte , fujifilm , pharmamar , d3 , pfizer , amgen , abbvie , multivir , bluprint medicines travel , accommodations , expenses : novartis , pharmamar , fujifilm david hong stock and other ownership interests : molecularmatch , oncorena honoraria : adaptimmune , baxter , merrimack , bayer consulting or advisory role : baxter , bayer research funding : novartis , genentech , eisai , astrazeneca , pfizer , mirna therapeutics , amgen , daiichi sankyo , merck , mirati therapeutics , eli lilly travel , accommodations , expenses : loxo , mirna therapeutics other relationship : oncorena mark routbort no relationship to disclose russell broaddus no relationship to disclose kenna r . 
stockley tl , oza am , berman hk , et al : molecular profiling of advanced solid tumors and patient outcomes with genotype - matched clinical trials : the princess margaret impact / compact trial . 
j pers med 3 : 306 - 325 , 2013 johnson a , zeng j , bailey am , et al : the right drugs at the right time for the right patient : the md anderson precision oncology decision support platfordrug discov today 20 : 1433 - 1438 , 2015 10 . 
forsman j , anani n , eghdam a , et al : integrated information visualization to support decision making for use of antibiotics in intensive care : design and usability evaluation . 
rinehart j , adjei aa , lorusso pm , et al : multicenter phase ii study of the oral mek inhibitor , ci - 1040 , in patients with advanced non - small - cell lung , breast , colon , and pancreatic cancer . 
bennouna j , lang i , valladares - ayerbes m , et al : a phase ii , open - label , randomised study to assess the efficacy and safety of the mek1 / 2 inhibitor azd6244 ( arry - 142886 ) versus capecitabine monotherapy in patients with colorectal cancer who have failed one or two prior chemotherapeutic regimens . 
schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
jardim dl , schwaederle m , wei c , et al : impact of a biomarker - based strategy on oncology drug development : a meta - analysis of clinical trials leading to fda approval . 
haslem ds , van norman sb , fulde g , et al : a retrospective analysis of precision medicine outcomes in patients with advanced cancer reveals improved progression - free survival without increased health care costs . 
van allen em , wagle n , stojanov p , et al : whole - exome sequencing and clinical interpretation of formalin - fixed , paraffin - embedded tumor samples to guide precision cancer medicine . 
this , in turn , leads to a profound defect in homology - mediated dna repair and inappropriate use of error - prone repair pathways , which result in gross genomic instability that contributes to tumorigenesis.1 , 2 this dna - repair defect in brca1 / 2 - mutant cancers renders them exquisitely vulnerable to certain kinds of dna damage , including those caused by poly ( adp - ribose ) polymerase ( parp ) inhibitors and certain classic chemotherapy agents , including platinum.3 the vulnerabilities of brca1 / 2 - mutant cancers to these agents have been translated successfully into treatment approaches . 
parp inhibitors are now approved by the us food and drug administration for treatment of brca1 / 2 mutant ovarian cancers , 4 and data are accumulating that suggest that parp inhibitors are likely active in brca1 / 2 - mutant cancer regardless of the tissue of origparp inhibitors are thought to be toxic to brca1 / 2 - mutant cancers not only because of their catalytic inhibition of parp but by their ability to trap parp - 1 enzyme on dna.5 , 6 the most potent parp inhibitors that induce cell death in brca1 / 2 - mutant cells are those that most efficiently trap parp protein on dna , creating a bulky protein dna adduct.7 in this sense , parp inhibitors work similarly to topoisomerase inhibitors , which trap topoisomerases to create a bulky protein dna adduct . 
other dna - damaging agents including platinum , mitomycin c , and topoisomerase inhibitors , that also induce dna adducts , are also effective in the treatment of brca1 / 2 - mutant cancers in animal models and clinically . given the widespread use of germline and tumor sequencing , and recent us food and drug administration approval of parp inhibitors , more patients who have tumors with pathogenic brca1 / 2 mutations are being treated with parp inhibitors and / or platinum agents . 
one important mechanism of acquired resistance is reversion mutations in brca1 or brca2 that partly restore wild - type gene function.8 , 9 the three reports10 - 12 of reversion mutations in brca2 in breast and prostate cancers that accompany this editorial highlight the importance and increasing awareness of this mechanism of resistance . 
the presence of reversion mutations in brca1 / 2 also reveals some insights about the role of brca1 / 2 function in tumorigenesis and chemosensitivity . most pathogenic mutations in brca1 and brca2 are small insertion / deletions that result in a frameshia frameshift will introduce a premature stop codon , which leads to a truncated , nonfunctional protein product . 
often , frameshift mutations lead to effective null mutations , because rna transcripts harboring premature stop codons can be recognized and degraded by the nonsense - mediated decay pathway.13 reversion mutations are secondary mutations , often small deletions , in a mutant brca1 / 2 allele that convert the initial frameshift mutation into an in - frame internal deletion that still produces a partly functional protein product . 
 complex rearrangements or abnormal use of alternative start sites that bypass the frameshift mutation may also occur.14 rarely , there is full reversion of the pathogenic mutation with restoration of the full wild - type sequence , as seen in the article by banda et al.10 reversion mutations can occur in the setting of either germline or somatic brca1 / 2 mutations , as demonstrated by carneiro et al , 12 and can lead to acquired resistance not only to parp inhibitors but to other classes of dna - damaging agents , such as platinum11 . intriguingly , the mechanism underlying many reversion mutations is inappropriate use of the nonhomologous end - joining pathway , resulting in small deletions , as seen by presence of microhomology at junction sequences.15 longer deletions associated with single - strand annealing may also be present but more difficult to detect . 
it is possible that reversion mutations may already be present in a small population of cells , especially if there is high tumor burden as seen , for example , in ovarian cancer , and are simply selected with ongoing treatment . reversion mutations can be difficult to detect by standard sequencing methods . 
large deletions may be completely missed by short - read sequencing , and even small deletions have to be carefully curated to determine in which allele they originate , and the effect on the final reading frame . 
some full reversions to wild - type sequence may only be detected by careful analysis of tumor purity and mutant allele frequencies in serial samples over time , as demonstrated by banda et al.10 heterogeneity of reversion mutations may also hinder detection . 
analysis of circulating - cell free dna , such as reported by cheng et al11 and carneiro et al , 12 can detect multiple reversion mutations simultaneously at the time of clinical resistance to parp inhibitors or platinum , each restoring the reading frame and arising from a different small tumor - cell population.16 it is possible that only a fraction of reversion mutations in brca1 / 2 are being identified by current sequencing methods . 
new technologies , such as long - read sequencing , and higher depth sequencing may allow more robust detection of reversion mutations and better define their frequency . the selection for reversion mutations in brca1 / 2 under certain treatments does give us some insights into the biology of brca1 . 
 the induction of reversion mutations by platinum is clear genetic evidence that this agent acts on the dna - repair defect associated with brca1 / 2 loss and exerts direct selection pressure to restore brca1 / 2 function . 
this finding also suggests that perhaps , in general , dna - damaging chemotherapies function not as gross metabolic poisons but as targeted therapies for cancers with underlying defects in dna repair and / or checkpoint control . 
if this is true , we may need to reconsider how to optimally dose and schedule platinum and other chemotherapy agents , especially in the setting of cancers with underlying dna - repair defects . the presence of reversion mutations demonstrates that loss of brca1 or brca2 function and the associated dna - repair defect is only required for initiation of tumorigenesis and is not required for maintenance of the cancer phenotype . 
 this feature can be labeled tumor - suppressor tolerance to place it in contrast to oncogene addiction . tumor - suppressor tolerance may operate in cancers that have underlying mutations in genes critical for genomic stability . 
loss of tumor - suppressor function of these genes may only be required for initial tumorigenesis ; once the tumor is established , there may be selection pressure to restore the tumor - suppressor function and reestablish dna - repair function . 
in brca1 - mutant cancers , resistance to parp inhibitors can occur not only by reversion mutations that directly restore brca1 function but also by compensating mutations in other genes such as 53bp1 and its downstream factors such as rif1 , ptip and rev7 , which also can restore homology mediated repair pathways independent of functional brca1.21 - 25 similarly , loss of ptip and chd4 may allow brca2 - mutant cells to reestablish replication fork stability and become resistant to cisplatin and parp inhibitors.26 these findings suggest that to better predict sensitivity to parp inhibitors and platinum , we will need to develop assays capable of distinguishing a cancer with ongoing genomic instability from a cancer with just a history of genomic instability followed by functional reversion of a dna - repair defect . with increasing use of parp inhibitors and platinum for targeted therapy of brca1 / 2 - mutant cancers , we will likely see increased incidence of acquired resistance that exploits tumor - suppressor tolerance and restores brca1 / 2 function . 
it is possible that some hypomorphic alleles of brca1 and brca2 that arise by reversion mutation may still have some targetable vulnerability.27 alternatively , parp inhibitors , or other dna - damaging agents such as platinum , will need to be combined with other drugs that target a different vulnerability in these cancers . 
carneiro b , et al : acquired resistance to the parp inhibitor olaparib in brca2 - associated prostate cancer due to biallelic brca2 reversion mutations restoring both germline and somatic loss of function mutations . 
perrin - vidoz l , sinilnikova om , stoppa - lyonnet d , et al : the nonsense - mediated mrna decay pathway triggers degradation of most brca1 mrnas bearing premature termination codons . 
quigley d , alumkal jj , wyatt aw , et al : analysis of circulating cell - free dna identifies multiclonal heterogeneity of brca2 reversion mutations associated with resistance to parp inhibitors . 
kondrashova o , nguyen m , shield - artin k , et al : secondary somatic mutations restoring rad51c and rad51d associated with acquired resistance to the parp inhibitor rucaparib in high - grade ovarian carcinoma . 
escribano - daz c , orthwein a , fradet - turcotte a , et al : a cell cycle - dependent regulatory circuit composed of 53bp1 - rif1 and brca1 - ctip controls dna repair pathway choice . 
bouwman p , aly a , escandell jm , et al : 53bp1 loss rescues brca1 deficiency and is associated with triple - negative and brca - mutated breast cancers . 
chapman jr , barral p , vannier jb , et al : rif1 is essential for 53bp1 - dependent nonhomologous end joining and suppression of dna double - strand break resection . 
 duality of purpose : participant and parent understanding of the purpose of genomic tumor profiling research among children and young adults with solid tumors purpose increasing use of genomic tumor profiling may blur the line between research and clinical care . 
we aimed to describe perspectives of research participants about the purpose of genomic tumor profiling research in pediatric oncology . methods we surveyed 45 participants ( response rate , 85% ) in a pilot study of genomic profiling in pediatric solid tumors at four academic cancer centers after the return of sequencing results . 
we defined understanding according to a one - item ( basic ) definition ( recognition that the primary purpose was not to improve the patients treatment ) and a four - item ( comprehensive ) definition ( primary purpose was not to improve patients treatment ; primary purpose was to improve treatment of future patients ; there may not be direct medical benefit ; most likely result of participation was not increased likelihood of cure )  . results sixty - eight percent of respondents ( 30 of 44 respondents ) demonstrated basic understanding of the study purpose ; 55% ( 24 of 44 respondents ) demonstrated comprehensive understanding . 
ninety - three percent of respondents who believed the primary purpose was to improve the patients care simultaneously stated that the research also aimed to benefit future patients . conclusion most participants in pediatric tumor profiling research understand that the primary goal of this research is to improve care for future patients , but many express dual goals when they participate in sequencing research . 
2019 by american society of clinical oncology introduction parents of children with cancer1 , 2 and adults with cancer3 - 5 often fail to understand the purpose of clinical trials in which they participate . 
understanding the distinction between the goals of research and clinical care is of particular importance in early - phase oncology trials , in which response rates approximate 10%.6 , 7 up to 60% of research participants demonstrate evidence of therapeutic misconception , 3 , 4 , 8 , 9 the belief that the primary purpose of research is therapeutic in nature rather than acquisition of generalizable knowledge.10 , 11 the precision medicine era invites new exploration of these findings . 
 among adults16 , 17 and children.17 - 20 although advances in targeted therapeutics generate great excitement , they may also blur the line between research and clinical care.21 , 22 young adult patients and parents of children with cancer have high hopes / expectations for tumor sequencing , 23 , 24 though only a minority experience clinical benefit.25 - 29 this mirrors findings among adult patients with cancer30 - 33 and highlights the need for a deeper understanding of the tumor profiling consent process . 
furthermore , many participants simultaneously identified dual purposes for genomic tumor profiling research in pediatric oncology . relevance consenting clinicians should query and explore participant goals during presequencing counseling to identify both ( 1 ) those who do not recognize that the primary purpose of research sequencing is to generate knowledge to help future patients and ( 2 ) those who report dual purposes for this research . 
additional work is necessary to better understand the perspectives and motivations of those who express this duality and to develop and test interventions to improve equitable understanding of the purpose of genomic tumor profiling research in pediatric oncology . methods we surveyed consenting participants in the icat ( individualized cancer therapy ) pilot study of genomic profiling in children with relapsed , recurrent , and high - risk solid tumors ( clinicaltrials.gov identifier : nct01853345 ) .25 participants were approached at dana - farber / boston childrens cancer and blood disorders center ( boston , ma ) , university of california at san francisco ( san francisco , ca ) , columbia university medical center ( new york , ny ) , and childrens national medical center ( washington , dc )  . 
the study was approved by the institutional review board of all participating institutions . individualized cancer therapy study icat study procedures have been reported previously.25 all patients receiving care at participating institutions were eligible for enrollment if they were age 30 years or younger at enrollment and had a recurrent , refractory , or high - risk ( expected likelihood of cure < 25% ) extracranial solid tumor with sufficient tumor for submission . 
the study consent document described the purpose of the study as follows : to determine how often the panel of experts can [ use tumor sequencing results to ] make an individual treatment recommendation and to use this information to help future patients with cancer . 
 consent discussions were not standardized , nor were data collected on the content of these discussions . enrolled participants underwent tumor profiling via targeted next - generation sequencing and copy number assessment or a sequenom assay . 
survey items for assessment of participant understanding of the purpose of genomic profiling research in pediatric oncology question stem answer choice agree unsure * disagree * the main reason this study was done was to improve the treatment of myself / my child . the main reason this study was done was to improve the treatment of future patients with cancer . there may not have been direct medical benefit to me / my child from participation . what of the following did you think was most likely to happen because of your participation in this research study ? agree * unsure * disagree agree * unsure * disagree i / my child would have a better chance of being cured . doing this testing would give me peace of mind . * doctors would be better able to find cures for future patients . * doctors would be able to learn more about my / my childs cancer . * i / my child would have a greater number of treatment options . * nothing was likely to happen as a result of this research . * i would learn about my / my childs genes . * i would learn about my familys genes . * other * alterations for which a matched targeted therapy was available via clinical trial or us food and drug administrationapproved medication ; the recommendation described actionable alteration ( s ) found and strength of evidence for each treatment recommendation . study population icat participants were offered a self - administered written survey after return of study results to the patients oncologist . 
surveys were offered in english to the consenting individual : the patient if he / she was age 18 years or older at enrollment , or the patients parent / guardian if the patient was younger than age 18 years at enrollment . 
 surveys were not offered if the patient died between the time of enrollment and approach by the study team ( n = 41 ) ; the patient / parent did not understand english sufficiently to complete the survey ( n = 3 ) ; the patient / parent declined additional contact from study investigators after enrollment ( n = 0 ) ; and / or the oncologist did not permit approach by the study team ( n = 4 )  . survey methods survey procedures have been reported previously.24 surveys consisted of 103 items and included scales that addressed participant understanding of the purpose of clinical research , 34 genetic knowledge , 35 and the short form - 36 general health perceptions question . 
secondary outcomes were participant - level predictors of understanding ( demographic characteristics , genetic knowledge , experience with genetics , clinical status , receipt of icat recommendation / targeted therapy )  . 
participants were enrolled between september 2012 and november 2013 ; surveys were administered between september 2014 and july 2015 . participant understanding of the purpose of research sequencing we assessed participant understanding with four independent items ( table 1 )  . 
three items were adapted from the quality of informed consent measurea validated measure to assess understanding of the purpose of oncology clinical trials , 34 by adult patients with cancer and further validated in parents of children with cancer1 ; answer choices were agree , unsure , and disagree.34 the fourth item offered respondents multiple choices about their perceived most likely result of study participation . participants were asked how well they understood conversation ( s ) they had with the doctor about the icat study and the testing involved in it , and responses were collected on a 5 - point likert scale ( responses of extremely well , well , moderately , poorly , extremely poorly )  . 
 to measure patient knowledge about genetics / genomics.24 , 36 , 37 respondents were asked if they had regular exposure to genetics and / or genetic information through their job and if they had ever attended any classes / lectures on genes / genetics . statistical methods understanding of the purpose of the study was defined in two ways . 
 comprehensive understanding was defined as an understanding of all four of the following : ( 1 ) the primary purpose was not to improve their / their childs treatment ; ( 2 ) the primary purpose was to improve treatment of future patients with cancer ; ( 3 ) there may not have been direct medical benefit to them / their child ; and ( 4 ) the most likely result of participation was not an increased likelihood of cure for themselves / their child . 
for example , if a participant identified that the primary purpose of the study was not to improve her childs treatment , he or she demonstrated basic understanding of the studys purpose . 
to be as inclusive as possible , and because of the complexity and uncertainty inherent in tumor profiling research , responses of unsure to any of the first three items were coded as consistent with understanding . 
for the fourth item , only responses that the most likely result of participation in the study was cure were coded as inconsistent with understanding ; all other responses , including answers of other , were coded as understanding . 
missing responses to any of the four understanding items were excluded from analysis of comprehensive understanding ; only those that were missing the first item were excluded from analysis of basic understanding . self - report of degree of understanding of the consent conversation ( s ) was dichotomized as well or extremely well ( coded as good self reported understanding ) versus all others . 
those who answered extremely true or very true to the item about how helpful participation was to them / their child were coded as feeling the study to have been helpful , and those who answered with the other answer choices were coded as feeling it was not . experience with genetics was defined as an affirmative response to questions of regular exposure to genetics or experience with genetics / genetic information and / or enrollment in any classes / lectures on genes or genetics . 
high genetic knowledge was defined as correct answers of all four items from the gki.35 those who answered fewer than four gki items correctly were coded as having low genetic knowledge . respondent demographics and clinical characteristics and understanding of results and the purpose of testing were evaluated using descriptive statistics . 
item nonresponse was less than 10% , and participants with nonresponse to an item were excluded from analyses of that ite all analyses were performed using stata , version 13.1 ( statacorp , college station , tx )  . results respondent characteristics of 101 participants who underwent profiling on the icat study , 53 were eligible for survey administration . 
characteristics of survey respondents are listed in table 2 for the overall cohort and are subdivided into patient ( n = 11 ; 24% ) and parent / guardian ( n = 34 ; 76% ) respondents . 
participant and patient demographics , overall and separately , according to whether the survey was completed by the patients parent / guardian or by the patient himself or herself overall ( n = 45 ) patient respondents ( n = 11 ) no . 
participant and patient demographics , overall and separately , according to whether the survey was completed by the patients parent / guardian or by the patient himself or herself ( continued ) overall ( n = 45 ) patient respondents ( n = 11 ) no . 
sixty - eight percent of respondents ( 30 of 44 respondents ) recognized that the primary reason the study was performed was not to improve the treatment of them / their child , which met our definition of basic understanding of the purpose of the study . 
no significant differences were seen according to respondent age , sex , or race / ethnicity ; according to self - reported health status or likelihood of cure , receipt of an icat treatment recommendation or matched targeted therapy ; or according to participant - identified understanding of what they were told about the study . 
 results were similar when responses of unsure were excluded from analysis ( appendix fig a1 )  . similar results were seen with understanding defined by the composite four - item scale . 
comprehensive understanding of the purpose of genomic profiling research was seen with statistically greater frequency among those with at least a college education ( 73% v 28% ; p = .01 ) and with higher genetic knowledge ( 71% v 23% ; p = .01 ) and among white / non - hispanic respondents ( 68% v 37% ; p = .07 ) , though the comparison by race / ethnicity was not statistically significant . 
similarly , no statistical difference in understanding was seen according to receipt of an icat treatment recommendation or matched targeted therapy or according to whether the respondent reported a good understanding of what they were told about the study / testing . 
among those who mistakenly identified the primary purpose as improvement of their / their childs treatment , 93% ( 13 of 14 respondents ) simultaneously recognized that it aimed to benefit future patients . 
twenty - eight percent ( 12 of 43 respondents ) of those who identified that the primary purpose was to benefit future patients also reported that the primary purpose was to improve their / their childs treatment . discussion in this multi - institutional study that examined the role of molecular profiling of pediatric solid tumors , nearly all participants recognized that the primary purpose was to benefit future patients . 
 however , approximately one third of respondents believed that the primary purpose of the trial was to improve their / their childs treatment , and nearly one fifth expected participation to impart a greater chance of cure . although these responses raise concerns about the quality of informed consent for tumor fig 1 . 
relationship between participant demographics and understanding of purpose of research tumor profiling ( continued ) helpfulness of participating in the study not helpful to myself / my child helpful note . 
 ( % ) 17 ( 71 ) 7 ( 35 ) sequencing , they must be considered in context of a complex technology with an evolving role in clinical care . 
this duality is echoed by the american society of clinical oncology , which states that early - phase clinical trials in oncology simultaneously generate new knowledge and provide participants the opportunity for psychological and clinical benefit.22 , 38 oncologists often balance dual goals for patients by recommending enrollment in a phase i trial while hoping for patient benefit or by simultaneously providing palliative and cancer - directed ( ie , blended ) care.39 in the era of precision cancer medicine , it is reasonable that patients / families might perceive such dualities as well . this duality has important clinical implications . 
however , an initial approach could be to discuss the primary goal of the study as gaining new knowledge to help future patients , followed by an acknowledgment , tempered with realistic expectations , that many patients / parentsand many clinicianshold hope that the child will also benefit from participation . 
in the case of next - generation sequencing , for example , it is important to note that the number of patients who experience direct benefit via receipt of a targeted therapy is quite low , likely in the range of 3% to 19%.25 - 29 our results also underscore the importance of hope among patients and parents of children with cancer in clinical and research settings.40 - 42 hopeful thinking may partially explain why participants who felt the study had helped them / their child and those who were receiving cancer directed therapy at the time of survey completion less frequently demonstrated understanding of the purpose of research tumor sequencing . in this cohort , understanding was observed more frequently in those with at least a college education and those with good genetic knowledge . 
 this finding , also reported elsewhere , 23 is not surprising , given the complexity of these concepts and the expected link between understanding and health literacy / numeracy.43 understanding also varied according to race / ethnicity , consistent with similar work in the pediatric oncology phase i literature , 2 although the difference did not reach statistical significance in this pilot study . 
these disparities underscore the importance of attention to the needs of vulnerable populations when they are counseled about genomic results ; however , the optimal mechanism for such counseling remains unclear.44 prior work in pediatric oncology has identified that refinement of the consent process may improve understanding , 45 but optimal strategies to adequately convey the complexities of tumor sequencing and support fully informed consent for participation in sequencing research are not yet known . 
a follow - up study is in development to examine the benefit of such an intervention for those who demonstrate less than comprehensive understanding , as defined in this cohort . 
patients / parents may have better understood the purpose of profiling closer to the time of consent , but understanding did not vary statistically with time to survey completion in this cohort . 
many study participants died before surveys could be administered ; however , demographic and clinical characteristics of respondents mirrored those of the overall cohort.25 finally , participants were enrolled at four large academic centers , so results may not be generalizable to those from smaller and / or community centers . 
this could , for example , explain the unexpectedly high genetic knowledge and experience seen in this cohort . although some participants misidentified the primary goal of tumor profiling research as therapeutic in nature , participant views are nuanced . 
dubois honoraria : loxo consulting or advisory role : loxo research funding : millennium ( inst ) , merck ( inst ) , novartis ( inst ) , roche ( inst ) , genentech ( inst ) , lilly ( inst ) , curis ( inst ) , loxo ( inst ) travel , accommodations , expenses : loxo , roche , genentech julia glade - bender research funding : bristol - myers squibb ( inst ) , pfizer ( inst ) , novartis ( inst ) , ignyta ( inst ) , amgen ( inst ) , celgene ( inst ) , eisai ( inst ) , lilly ( inst ) , merck ( inst ) travel , accommodations , expenses : novartis , amgen , merck aerang kim no relationship to disclose brian d . 
weinfurt kp , seils dm , lin l , et al : research participants high expectations of benefit in earlyphase oncology trials : are we asking the right question ? j clin oncol 30 : 4396 - 4400 , 2012 6 . 
italiano a , massard c , bahleda r , et al : treatment outcome and survival in participants of phase i oncology trials carried out from 2003 to 2006 at institut gustave roussy . 
druker bj , sawyers cl , kantarjian h , et al : activity of a specific inhibitor of the bcr - abl tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the philadelphia chromosome . 
kantarjian hm , shah np , cortes je , et al : dasatinib or imatinib in newly diagnosed chronicphase chronic myeloid leukemia : 2 - year follow - up from a randomized phase 3 trial ( dasision )  . 
wagle n , grabiner bc , van allen em , et al : activating mtor mutations in a patient with an extraordinary response on a phase i trial of everolimus and pazopanib . 
kushner bh , cheung nv , modak s , et al : a phase i / ib trial targeting the pi3k / akt pathway using perifosine : long - term progression - free survival of patients with resistant neuroblastoma . 
laetsch tw , dubois sg , mascarenhas l , et al : larotrectinib for paediatric solid tumours harbouring ntrk gene fusions : phase 1 results from a multicentre , open - label , phase 1 / 2 study . 
weber js , levit la , adamson pc , et al : reaffirming and clarifying the american society of clinical oncologys policy statement on the critical role of phase i trials in cancer research and treatment . 
oberg ja , ruiz j , ali - shaw t , et al : whole - genome and whole - exome sequencing in pediatric oncology : an assessment of parent and young adult patient knowledge , attitudes , and expectations . 
marron jm , dubois sg , glade bender j , et al : patient / parent perspectives on genomic tumor profiling of pediatric solid tumors : the individualized cancer therapy ( icat ) experience . 
harris mh , dubois sg , glade bender jl , et al : multicenter feasibility study of tumor molecular profiling to inform therapeutic decisions in advanced pediatric solid tumors : the individualized cancer therapy ( icat ) study . 
chang w , brohl as , patidar r , et al : multidimensional clinomics for precision therapy of children and adolescent young adults with relapsed and refractory cancer : a report from the center for cancer research . 
harttrampf ac , lacroix l , deloger m , et al : molecular screening for cancer treatment optimization ( moscato - 01 ) in pediatric patients : a single - institutional prospective molecular stratification trial . 
miller fa , hayeems rz , bytautas jp , et al : testing personalized medicine : patient and physician expectations of next - generation genomic sequencing in late - stage cancer care . 
gray sw , park er , najita j , et al : oncologists and cancer patients views on whole - exome sequencing and incidental findings : results from the canseq study . 
carere da , couper mp , crawford sd , et al : design , methods , and participant characteristics of the impact of personal genomics ( pgen ) study , a prospective cohort study of direct - to - consumer personal genomic testing customers . 
weber js , levit la , adamson pc , et al : american society of clinical oncology policy statement update : the critical role of phase i trials in cancer research and treatment . 
ferrell br , temel js , temin s , et al : integration of palliative care into standard oncology care : american society of clinical oncology clinical practice guideline update . 
sensitivity analyses for the relationship between participant demographics and understanding of the purpose of research tumor profiling basic understanding ( n = 33 ) comprehensive understanding ( n = 26 ) variable survey respondent characteristic no . 
 ( % ) respondent attitudes about icat study understanding of icat information poor self - reported understanding good self - reported understanding helpfulness of participating in icat study not helpful to myself / my child helpful basic understanding ( n = 33 ) comprehensive understanding ( n = 26 ) 10 ( 77 ) 9 ( 45 ) 11 ( 69 ) 8 ( 47 ) 8 ( 80 ) 8 ( 50 ) 10 ( 77 ) 6 ( 46 ) note . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction as our knowledge of hereditary cancer risk has testing of cancer preevolved , multigene panel disposition genes has become an important component of patient care . 
the national comprehensive cancer network provides gene - specic recommendations for changes in medical management on the basis of the identication of pathogenic variants.1 to this end , accurate and denitive variant classication is a critical component of clinical genetic testing . 
the american college of medical genetics and genomics ( acmg ) and the association for molecular pathology ( amp ) provide guidelines for clinical variant classication2 ; however , there is often insufcient evidence to support a denitive classication of pathogenic or benign when a variant is rst detected . 
as a result , testing laboratories may vary in their clinical terpretation of acmg / amp guidelines for hereditary cancer genetic testing and , ultimately , in their variant classications.3 - 6 with the increased use of multigene panels , number of variants of uncertain signicance ( vuss ) detected has also increased.7 , 8 in addition to a larger number of genes , many cancer predisposition genes have been more recently incorporated into clinical testing , and there is less evidence available to support variant classication . 
 evolution of cancer gene variant reclassication context key objective with the increasing use of hereditary cancer genetic testing and introduction of multigene panels , robust and accurate methods for variant re / classication of cancer predisposition genes are of utmost importance . 
laboratory - developed methods were the most common lines of evidence used in variant reclassication , with pheno analysis ( a clinical history weighting algorithm ) being used in approximately 40% of cases . relevance many tools traditionally used for variant re / classication are not informative for rare variants ; however , rare variants collectively affect millions of patients . 
this was followed by testing for lynch syndrome ( mlh1 , msh2 , msh6 , and pms2 ) and melanoma - pancreatic cancer syndrome ( cdkn2a ) in 2002 and for familial adenomatous polyposis ( fap ) / attenuated fap / mutyh - associated polyposis ( apc and mutyh ) in 2003 . 
in 2013 , a hereditary cancer multigene panel was introduced , which was analytically validated and described in detail previously.9 the panel included 25 genes : apc , atm , bard1 , bmpr1a , brca1 , brca2 , brip1 , cdh1 , cdk4 , cdkn2a ( p16ink4a and p14arf ) , chek2 , epcam , mlh1 , msh2 , msh6 , mutyh , nbn , palb2 , pms2 , pten , rad51c , rad51d , smad4 , stk11 , and tp53 . 
individuals who had single - site or founder mutation testing were reclassication program the reclassication program used by this testing laboratory included a multidisciplinary committee of laboratory directors , genetic counselors , phd - level scientists , and supporting variant specialists . 
variant classication and reclassication followed a stringent , welldocumented protocol as previously described.13 on the basis of acmg / amp guidelines , variants were classied into ve categories of increasing clinical severity : benign ( b ) , likely benign ( lb ) , vus , likely pathogenic ( lp ) , and pathogenic ( p ) .2 , 14 , 15 automated processes proactively monitored and gathered all available evidence to expedite reclassication . 
multiple classication tools could have been used to evaluate the pathogenicity of a variant at the time of original classication and / or if a variant was reclassied , an reclassication ( fig 1 )  . amended report was sent to health care providers for all patients who were carriers of the variant . 
 esterling et al pheno ( n = 56 , 596 ) clinical history weighting algorithm to assess phenotype severity for variant carriers v carriers of pathogenic and benign variants pt / ha ( n = 36 , 314 ) phase testing or haplotype analysis for genes associated with embryonic lethality or severe clinical phenotypes because of homozygosity or compound heterozygosity homozygosity confirmed continuous evidence evaluation threshold reached population frequency ( n = 17 , 664 ) threshold reached allele frequency from exac and gnomad functional or clinical evidence literature ( n = 4 , 509 ) expert review of automated search results and curated repository structure disrupted structural analysis ( n = 850 ) using validated protein structures mco ( n = 12 , 753 ) likelihood of co - occuring pathogenic variants in the same gene or in genes associated with the same hereditary cancer syndrome threshold reached cosegregation in multiple families segregation analysis ( n = 8 , 255 ) published pedigrees and provider communications rna analysis ( n = 610 ) in - house comprehensive rna analysis to directly assess rna splicing effects splicing disrupted splicing analysis ( n = 3 , 148 ) functional data and expert review other ( n = 4 , 598 ) knowledge of similar variants , species conservation in conjunction with structural analysis , additional statistical analysis , and classification / reclassification protocol refinements evidence queuing for review based on variant type and new evidence multiple expert reviews evidence insufficient wait for additional evidence committee discussion variant reclassified amended reports fig 1 . 
phase testing / homozygosity analysis ( pt / ha ) was only applied to genes associated with autosomal dominant cancer risks that may also be associated with embryonic lethality or severe clinical phenotypes in individuals carrying pathogenic variants in the affected gene on different parental chromosomes as a result of either homozygosity or compound heterozygosity . 
common single nucleotide polymorphisms ( snps ) observed in the patient population were used to develop gene - specic haplotypes , which were automatically assigned to determine the variants phase ( ie , in cis or in trans ) , whenever possible . 
mutation co - occurrence ( mco ) analysis was based on an assumption of a low likelihood that an individual will carry two autosomal dominant pathogenic variants that cause an increased risk for the same or a similar cancers.18 mco analysis measured the statistical signicance of a variant co - occurring multiple times with one or more pathogenic variants in the same patient ( in the same gene or in a different gene for the same syndrome )  . 
99.5%. rna splicing analysis and protein structural analysis . assessment of in silico rna splicing predictions was used to initially evaluate the potential effects of a variant on rna splicing.19 published reports of biochemical splicing analysis , which used high - quality data and appropriate controls , were considered for variant classication . 
in some cases where evidence was unavailable or of insufcient quality or detail to be conclusive , quantitative rna splicing analyses were performed in house , using informative snps , to assess potential rna splicing effects resulting from the variant of interest.20 expert protein structural analysis was also used to assess the impact of variants predicted to affect protein structure at the amino acid level . 
jack lee , phd2 precision oncology exploits genomic , immunologic , and other biomarkers to drive discovery , drug development , and clinical care for patients with cancer . precision oncology helps devise individualized treatment strategies and therapies on the basis of a patients specic characteristics , such as genetic information , health history , environmental exposure , and needs and preferences , to guide discovery of effective drugs and treatment interventions . 
because precision oncology research raises so many interesting and challenging clinical and statistical questions that require new approaches to statistical thinking , design , and analysis , it is critical that multidisciplinary teams include clinicians , basic scientists , and statisticians who can collaborate on study design , analysis , and interpretation . statistical methods play an important role in discovering signals and ltering out noises embedded in the rich sources of high - dimensional - omics data , but they must also contend with the complexity of cancer biology and medical conditions . 
for example , the umbrella trial design was used in battle1 ( clinicaltrials.gov identier : nct00409968 ) and i - spy 2 ( clinicaltrials.gov identier : nct01042379 ) 2 trials , and it greatly improved efciency in these phase ii cancer trials , which screened drugs for efcacy signals . 
a basket trial design was used successfully in a trial of vermurafenib3 in multiple non - melanoma cancers with braf v600 mutations ( clinicaltrials.gov identier : nct01524978 )  . master protocols have been developed for the lungmap ( clinicaltrials.gov identier : nct02154490 ) trial4 and nci - match ( clinicaltrials.gov identier : nct02465060 ) trial , among others.5 , 6 in recent years , statisticians have worked closely with oncologists to develop new statistical methods that will provide scientically rigorous answers to research questions and substantially improve the efciency of the drug development process and the feasibility of doing the research . 
for this reason , jco precision oncology has organized this special series of tutorial and review articles on statistical methods for precision oncology that will serve as a bridge between the clinical researcher and statistician communities , will help facilitate the application of new methods , and will stimulate the development of more and better methods to address various needs in precision oncology research . the journal solicited 13 papers from well - known statisticians who are developers of novel statistical methods for precision oncology or are experts in their application . 
some articles introduce and review various methods for developing precision drugs in oncology , and some provide expert opinions on discoveries , precision oncology care , and decision making . cluded here are articles on drug discoveries , earlyphase dose determination , phase ii biomarker - driven trials , phase iii trials , companion and predictive diagnoses , master protocols for basket trials and umbrella trials , selection of estimands for clinical trials , and clinical trials for pediatric cancer . 
the following are brief descriptions of highlights from each article . high - dimensional data have much promise in advancing precision oncology , whereas data on individual biomarkers contribute relatively small amounts of information on complex diseases . 
newly developed statistical tools allow researchers to aggregate pathwaylevel information from multiple relatively weak signals . zhao , chang , and long7 provide a comprehensive review of knowledge - guided statistical supervised and unsupervised learning methods for - omics data . 
the review of recent developments in risk assessment models of clinical outcomes by halabi , li , and luo8 covers a topic that is identifying new prognostic factors and critical building new risk assessment prognostic models for managing patients and decision making . 
in precision oncology , evaluating treatment effects in populations dened by their histology , choosing biomarker - driven clinical trial designs , and planning analyses require additional considerations , depending on the nature and utility of the biomarkers . simon9 gives a historical account of the evolution of trial design in precision oncology and provides a review of new biomarker - driven clinical trial designs that can be used for various purposes , including adaptive validation of companion diagnostic tests for targeted treatments . polley and cheung10 have reviewed the concepts and methodology of early - phase cancer trial designs with a focus on dose nding and treatment screening . 
because most cancer trial designs have been developed for cytotoxic targeted cancer therapies , the authors discussed the relevance of these time - tested design principles in the context of platform trials for targeted cancer therapies . 
yuan , lee , and hilsenbeck11 introduce and review a new class of model - assisted designs to combine the transparency and simplicity of conventional algorithm - based designs with the superiority and rigorousness of novel model - based designs . thall12 reviews two clinical trial designs that help researchers make subgroup - specic decisions and compare each of them to a simpler design that ignores patient heterogeneity . 
these trial designs illustrate the benets of prospectively accounting for treatment - subgroup interactions and show how utilities may be used to quantify riskbenet trade - offs . several articles also discuss newly developed methods that can be used in later - phase trials of drug efcacy and can also be used to identify subgroups dened by predictive biomarkers . 
they then discuss the adaptive enrichment design , which uses subgroup analysis to inform random assignments to various treatments . progress in the areas of genomics , disease pathways , and in clinical and drug discovery is beginning to be felt trials have been translational cancer research . 
the article by kaizer et al14 provides a comprehensive review of basket trial designs in oncology settings and discusses several issues that arise with their design and methods of analysis . hu and dignam15 provide a comprehensive overview of the design elements , types , features , and practical considerations of master protocols for multiagent and / or multihistology master protocol trials . 
polley , korn , and freidlin provide an updated review of commonly used biomarkerbased randomized clinical trial design for a single agent and subpopulation for simultaneous inferences , with a particular focus on design efciency.16 ou et al17 provide case studies of ve types of trial designs for selecting patruly benet from a target therapy and tients who will discuss the advantages and disadvantages of each design . renfro et al18 discuss the unique challenges often encountered in any rare tumor setting and the best practices used by the childrens oncology group to address these challenges . 
now that clinical trials can use precision oncology to dene molecular subtypes of cancers and specic targeted treatments , those trials increasingly encounter the same challenges as those faced by the childrens oncology group . 
thus , their solutions to these challenges are highly relevant beyond pediatric oncology trials . one of the challenges in evaluating precision oncology agents is the diversity of each patients journey in clinical practice . 
degtyarev et al19 address the estimand framework , which provides a structured approach for discussing how to account for intercurrent events that occur after random assignment and may affect the assessment or interpretation of the treatment effect . 
they illustrate this framework by using a trial for chimeric antigen receptor t - cell therapy . the main purpose of this collection of tutorial and review articles is to inform clinical researchers about statistical concerns that should be considered in their research projects and to encourage professional statisticians to use cutting - edge methods in their analyses . 
because the authors of this collection are leading developers of these methods , it is also a great resource for statisticians who are interested in learning about oncology research by providing a succinct overview of the literature . although there are many novel statistical methods currently being developed to address the challenges of precision oncology , they are published primarily in statistical it is important to increase awareness of these methods among clinical researchers so that theory can be brought into practice . 
we hope this series will serve as a bridge between clinical researchers and statisticians and will help disseminate information and transfer knowledge so these powerful designs and methods can be used to advance precision oncology . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . ying lu consulting or advisory role : paxvax , nektar therapeutics , zap surgical systems , abeona therapeutics ( inst ) research funding : merck ( inst ) j . 
jack lee consulting or advisory role : abbvie no other potential conicts of interest were reported . references liu s , lee jj : an overview of the design and conduct of the battle trials . 
n engl j med 375 : 83 - 84 , 2016 diamond el , subbiah v , lockhart ac , et al : vemurafenib for braf v600 - mutant erdheim - chester disease and langerhans cell histiocytosis : analysis of data from the histology - independent , phase 2 , open - label ve - basket study . 
herbst rs , gandara dr , hirsch fr , et al : lung master protocol ( lung - map ) : a biomarker - driven protocol for accelerating development of therapies for squamous cell lung cancerswog s1400 . 
n engl j med 377 : 62 - 70 , 2017 chen ap , eljanne m , harris l , et al : national cancer institute basket / umbrella clinical trials : match , lungmap , and beyond . 
cancer j 25 : 272 - 281 , 2019 zhao y , chang c , long q : knowledge - guided statistical learning methods for analysis of high - dimensional - omics data in precision oncology . 
renfro la , ji l , piao j , et al : trial design challenges and approaches for precision oncology in rare tumors : experiences of the childrens oncology group . 
 prevalence of homologous recombinationrelated gene mutations across multiple cancer types purpose the prevalence of homologous recombination dna damage repair ( hr - ddr ) deficiencies among all tumor lineages is not well characterized . 
therapy directed toward homologous recombination ddr deficiency ( hrd ) is now approved in ovarian and breast cancer , and there may be additional opportunities for benefit for patients with other cancers . 
comprehensive evaluations for hrd are limited in part by the lack of a uniform , cost - effective method for testing and defining hrd . methods molecular profiles of 52 , 426 tumors were reviewed to identify pathogenic mutations in the hr - ddr genes arid1a , atm , atrx , bap1 , bard1 , blm , brca1 / 2 , brip1 , chek1 / 2 , fanca / c / d2 / e / f / g / l , mre11a , nbn , palb2 , rad50 , rad51 , rad51b , or wrn . 
 a total of 17 , 566 tumors were sequenced with ngs600 ( n = 592 genes ) , and 34 , 860 tumors underwent hotspot illumina miseq platform testing ( n = 47 genes )  . results of the tumors that underwent ngs600 testing , the overall frequency of hrddr mutations detected was 17.4% , and the most commonly mutated lineages were endometrial ( 34.4% ; n = 1 , 475 ) , biliary tract ( 28.9% ; n = 343 ) , bladder ( 23.9% ; n = 201 ) , hepatocellular ( 20.9% ; n = 115 ) , gastroesophageal ( 20.8% ; n = 619 ) , and ovarian ( 20.0% ; n = 2 , 489 )  . 
germline mutations in one or both of these genes place patients at heightened risk for development of breast , 1 - 6 ovarian , 1 - 6 prostate , 7 - 9 melanoma , 7 , 10 and pancreatic cancers7 , 10 - 12 during their lifetime . 
it has become apparent that brca interacts with a number of other dna repair proteins to form a complex system for ddr , including atm , rad51 , palb2 , mre11 , rad50 , nbn , and the fanconi anemia proteins.13 , 14 recent evidence suggests mutations in palb2 , atm , and the genes responsible for the mrn complex , rad50 , mre11 , and nbn , play a role in hereditary cancers.15 , 16 for example , palb2 mutation carriers have a lifetime risk of breast cancer development of approximately 50% , 17 , 18 and atm mutation carriers are at higher risk for development of breast , 19 , 20 pancreatic , 21 , 22 and prostate cancers.23 , 24 arielle l . 
in germline brca1 / 2 mutation carriers , exposure to platinum chemotherapy led to improved objective response rates in advanced triple - negative breast cancer versus taxanes ( 68% v 33% ) , 25 and overall survival in pancreatic cancer versus other nonplatinum chemotherapy ( 22 months v 9 months ) .26 mychoice hr - ddr deficiency ( hrd ) score - high triple - negative breast cancer responded better to platinum - based neoadjuvant therapy , with pathologic complete response ( cr ) rates of 27.5% versus 0% in the hr - ddrproficient cohort.27 the mychoice hrd score is frequently used to identify patients with hrd . 
it is a proprietary diagnostic test to assess a hrd phenotype , including an evaluation of loss of heterozygosity , telomeric allelic imbalance , and large - scale state transitions . on exposure to another class of dna - damaging agents , poly - adp ribose polymerase ( parp ) inhibitors , patients with germline or somatic deleterious mutations in the hr - ddr pathway have also achieved favorable responses . 
in this study , three patients had germline brca2 mutations , three patients had germline atm mutations , and the remaining responders had tumor expression of a deleterious mutation ( including palb2 , brca2 , brca1 , chek2 , fanca , and atm ) .35 all germline mutation carriers except for one patient with atm mutation responded to therapy . despite the exciting therapeutic potential of dna - damaging agents in patients with broader evidence of hrd , the prevalence of hrd among all tumors is largely unknown . 
the aim of our study was to determine the prevalence of hr - ddr pathogenic or presumed pathogenic mutations detected on tumor next - generation sequencing ( ngs ) testing across multiple cancer lineages , using commercially available dna sequencing ( ngs or sanger sequencing panel testing , multiplatform profiling ; caris life sciences [ caris ] , irving , tx ) to better define the proportion of patients who may benefit from such therapy . methods study design approval for this study was obtained from the georgetown university institutional review board . 
we defined hrd on tumor ngs testing as a mutation in the following genes , each of which has some activity within the hr - ddr pathway36 - 45 and has been included previously in hr - ddr biomarker clinical trials : arid1a , atm , atrx , bap1 , bard1 , blm , brca1 / 2 , brip1 , chek1 / 2 , fanca / c / d2 / e / f / g / l , mre11a , nbn , palb2 , rad50 , rad51 , rad51b , or wrn . 
frequencies of each mutation were determined for the total cohort , as well as for each cancer lineage ( biliary tract , bladder , breast , cervix , colorectal [ crc ] , endometrial , gastro esophageal [ ge ] , gastrointestinal stromal [ gist ] , glioma , head and neck , hepatocellular [ hcc ] , melanoma , neuroendocrine / small cell lung , nonsmall - cell lung [ nsclc ] , ovarian , pancreas , prostate , renal , sarcoma , thyroid , and unknown primary )  . ngs testing platforms hr - ddr mutation analysis from solid tumor biopsy specimens was determined by ngs at caris , a clinical laboratory improvement amendmentscertified laboratory . 
while 17 , 566 tumors were sequenced with ngs600 , 34 , 860 tumors underwent hotspot illumina miseq platform testing ( illumina truseq amplicon cancer hotspot panel , evaluating 47 genes including the hr - ddr genes atm , brca1 , and brca2 ; illumina , san diego , ca )  . 
tumor enrichment was achieved by harvesting targeted tissue by manual microdissection performed on all cases before molecular testing . the pathogenicity of gene variants identified were interpreted by board - certified molecular geneticists and categorized as pathogenic , presumed pathogenic , variant of unknown significance , presumed benign , or benign , according to american college of medical genetics and genomics standards on the basis of the level of evidence of published studies on the identified variants.46 , 47 only pathogenic or presumed pathogenic mutations were considered deleterious ; variants of unknown significance and variants that have not been previously reported in individuals affected by cancer in the literature were excluded . 
 deleterious mutations reported included frameshift mutations , premature stop codons , mutations shown to disrupt natural splicing , as well as point mutations ; deleterious mutations included those that have and have not been reported as causal for hereditary cancers . statistical analysis the proportion of pathogenic or presumed pathogenic mutations identified from all tumor specimens tested for each specific mutation were calculated for the total cohort and for each cancer lineage investigated . 
the total frequency of hr - ddr mutations in the complete cohort and per cancer lineage was calculated by dividing the number of tumors carrying at least one mutation by the total number of tumors tested , to avoid counting tumors carrying more than one hr - ddr mutation multiple times . 
the 95% cis were computed using the pearson - klopper exact method using r ( org / ) , and the graphics were generated by spss statistics , version 24 ( ibm , armonk , ny )  . results we evaluated 52 , 426 solid tumor pathologic specimens that underwent extended ngs for hrd . 
molecular profiling was performed on the primary tumor in 47.9% of cases ( n = 25 , 102 ) , and on a metastatic site of disease in 41.9% ( n = 21 , 956 )  . 
 within the tumor lineages , the frequencies of mutations varied ( tables 2 and 3 ; fig 2 )  . hr gene mutation frequency overall , pathogenic mutations within the homologous recombination pathway were seen in 17.4% of the 17 , 566 tumors tested with ngs600 , and 8.3% of the 52 , 426 solid tumors overall ( including 34 , 860 tumors that were evaluated for atm , brca1 , and brca2 mutations only on the hotspot panel )  . 
 brca1 / 2 mutations were seen predominately in ovarian and breast cancers , though pathogenic brca2 mutations were seen in high frequencies among gi and nonovarian genitourinary malignancies , as well . 
although palb2 mutations were less common overall and appreciated in only 0.6% of tumors tested , a significant proportion of palb2 mutations was found in bladder , breast , and gi malignancies . 
no pathogenic mutations were identified in bard1 , chek1 , fanca / d2 / e / f / g / l , rad51 , or rad51b ( table 4 )  . hr - ddr gene mutations were appreciated in both primary and metastatic lesions , though with different patterns . 
the remaining mutated hr - ddr genes ( blm , brip1 , chek2 , fancc , mre11a , nbn , palb2 , rad50 , and wrn ) had similar frequencies among primary and metastatic tissue evaluations . tumors with multiple hr gene mutations of the 17 , 566 tumors that underwent extended molecular profiling with the ngs600 platform , 362 were found to carry more than one hr - ddr pathway mutation , including 112 endometrial ( 7.6% ; n = 1 , 475 ) , 75 crc ( 3.1% ; n = 2 , 454 ) , 34 ovarian ( 1.4% ; n = 2 , 489 ) , 29 nsclc ( 0.9% ; n = 3 , 245 ) , 23 ge ( 3.7% ; n = 619 ) , 20 breast ( 1.2% ; n = 1 , 625 ) , and 15 biliary tract ( 4.4% ; n = 343 ) tumors . 
total bar height represents the overall frequency of hr - ddr deficient tumors within each lineage ; colored bar length represents the relative mutation frequency of individual genes in each cancer type . 
summary of homologous recombination dna damage repair mutations identified * overall , % ngs600 , % hotspot , % mutation arid1a brca2 brca1 atrx chek2 bap1 palb2 brip1 fancc rad50 mre11a abbreviation : n / a , not applicable . * no mutations identified : bard1 , chek1 , fanca / d2 / e / f / g / l , rad51 , rad51b . in this large - scale study of ngs molecular profiling of solid tumor samples across multiple cancer lineages , we confirmed hrd is common and was observed in 17.4% of the solid tumors evaluated by the ngs600 platform ( spanning 21 cancer lineages )  . 
in a recent evaluation of brca1 / 2 mutational patterns , for example , a model to detect brca1 / 2 deficiency failed to correlate brca1 / 2 mutational patterns with pathogenic mutations in several additional hr - ddr pathway genes , including atm , atr , chek2 , the fanc group of genes , palb2 , pten rad50 , and rad51c.48 the unique genetic signatures across the breadth of hr - ddr pathway genes are not well characterized , and , as such , it is unknown if the lack of a brca - like mutational pattern is associated with altered responses to parp inhibitors and dna - damaging chemotherapy . our reported frequencies of hrd are similar to previously published work , though a range of frequencies is appreciated ( table 5 )  . 
because there is not yet an established way to measure hrd , and previous studies have measured hrd by an hrd assay assessing large - scale transition scores and telomere allelic imbalances , germline mutations , somatic mutations , brca - like genetic signatures , or a combination of these methods , the differences in hrd prevalence may be related to nonuniformity of assessment . 
in addition , significant variation will occur between whole - exome sequencing versus use of hotspot panels , with the identification of hrd more common among studies that used whole - exome sequencing . 
within our study , 17 , 566 tumors ( 33.5% of the total 52 , 426 tumors tested ) underwent sequencing with the targeted whole - exome sequencing platform ngs600 , which evaluated tumors for 592 genes , assuming dna was sufficient . 
consequently , studies including an evaluation for pten mutations are expected to report higher frequencies of hrd . it is also possible frequencies of hrd may differ across studies as a result of differences in patient population . 
patients with tumors harboring hr - ddr pathway mutations may be more sensitive to dna - damaging chemotherapy and therefore less likely to recur after initial treatment of localized disease . 
although the clinical stage of patients included in our analysis at the time of tissue acquisition ( and if tissue was obtained at presentation ) was unknown , it is possible our population was enriched with patients with metastatic disease without hrd who were less likely to respond favorably to frontline treatment and , therefore , recurred . looking forward , classifying tumors as hr - ddr deficient will likely become increasingly important , as we now appreciate that hrd is common and has been associated with improved outcomes in some patients treated with dna - damaging therapies . 
several clinical trials are assessing in part the role of hrd in response to anticancer therapies and outcomes , including treatment with parp inhibitor therapy alone , but also in combinations with chemotherapy , radiation therapy , or immunotherapy to enhance antitumor efficacy . 
in tandem , it will be important to generate a consensus regarding uniform testing to identify appropriate patients for these tailored therapies . there are some limitations of this study to note . 
given the nature of the study , we evaluated patients tumor tissue for the presence of hr - ddr mutations and thus are unable to distinguish whether a given mutation was a somatic or germline mutation . 
we were also unable to assess tumors for epigenetic modifications such as dna methylation , which could also lead to significant and clinically relevant alterations in gene expression affecting functions of the hr pathway.57 , 58 in addition , hrd is defined by currently available literature regarding the suspected pathogenicity of each mutation . 
it is certainly possible that mutations labeled as a variant of unknown significance may prove important in the future , although the majority are reclassified as benign polymorphisms.59 - 61 we also recognize our results are influenced by the overrepresentation of certain cancer lineages , including ovarian ( n = 9 , 630 ) , nsclc ( n = 8 , 119 ) , crc ( n = 6 , 650 ) , breast ( n = 5 , 910 ) , and endometrial ( n = 5 , 540 ) cancers in the caris database . 
baker employment : caris life sciences , roche travel , accommodations , expenses : caris life sciences , roche honoraria : roche , amgen , bayer / onyx , taiho pharmaceutical , caris life sciences , celgene consulting or advisory role : roche , amgen , bayer / onyx , taiho pharmaceutical , caris life sciences , celgene speakers ' bureau : roche , amgen , bayer / onyx , celgene , taiho pharmaceutical research funding : bayer / onyx ( inst ) , roche ( inst ) , pfizer ( inst ) , amgen ( inst ) , boehringer ingelheim ( inst ) , medimmune ( inst ) claudine isaacs honoraria : roche , astrazeneca , pfizer , novartis , syndax , nanostring technologies consulting or advisory role : pfizer , roche , novartis , astrazeneca , medivation , nanostring technologies , syndax speakers ' bureau : genentech , pfizer , astrazeneca research funding : novartis ( inst ) , pfizer ( inst ) , genentech ( inst ) , tesaro patents , royalties , other intellectual property : uptodate , mcgraw hill publishing affiliations arielle l . 
antoniou a , pharoah pd , narod s , et al : average risks of breast and ovarian cancer associated with brca1 or brca2 mutations detected in case series unselected for family history : a combined analysis of 22 studies . 
van der kolk dm , de bock gh , leegte bk , et al : penetrance of breast cancer , ovarian cancer and contralateral breast cancer in brca1 and brca2 families : high cancer incidence at older age . 
kote - jarai z , leongamornlert d , saunders e , et al : brca2 is a moderate penetrance gene contributing to young - onset prostate cancer : implications for genetic testing in prostate cancer patients . 
casadei s , norquist bm . , walsh t , et al : contribution to familial breast cancer of inherited mutations in the brca2 - interacting protein palb2 to familial breast cancer . 
telli ml , timms km , reid j , et al : homologous recombination deficiency ( hrd ) score predicts response to platinum - containing neoadjuvant chemotherapy in patients with triple - negative breast cancer . 
swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
oza am , tinker av , oaknin a , et al : antitumor activity and safety of the parp inhibitor rucaparib in patients with high - grade ovarian carcinoma and a germline or somatic brca1 or brca2 mutation : integrated analysis of data from study 10 and ariel2 . 
mccabe n , turner nc , lord cj , et al : deficiency in the repair of dna damage by homologous recombination and sensitivity to poly ( adp - ribose ) polymerase inhibition . 
aarts m , bajrami i , herrera - abreu mt , et al : functional genetic screen identifies increased sensitivity to wee1 inhibition in cells with defects in fanconi anemia and hr pathways . 
pennington kp , walsh t , harrell mi , et al : germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian , fallopian tube , and peritoneal carcinomas . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
yap kl , kiyotani k , tamura k , et al : whole - exome sequencing of muscle - invasive bladder cancer identifies recurrent mutations of unc5c and prognostic importance of dna repair gene mutations on survival . 
stjepanovic n , kim rh , wilson m , et al : clinical outcome of patients with advanced triple negative breast cancer with germline and somatic variants in homologous recombination gene . 
shahda s , timms k , ibrahim a , et al : homologous recombination deficiency ( hrd ) in patients with pancreatic cancer ( ps ) and response to chemotherapy . 
bang yj , im sa , lee kw , et al : randomized , double - blind phase ii trial with prospective classification by atm protein level to evaluate the efficacy and tolerability of olaparib plus paclitaxel in patients with recurrent or metastatic gastric cancer . 
easton df , deffenbaugh am , pruss d , et al : a systematic genetic assessment of 1 , 433 sequence variants of unknown clinical significance in the brca1 and brca2 breast cancer - predisposition genes . 
dabrafenib , a braf inhibitor , and pazopanib , a multikinase inhibitor that targets vascular endothelial growth factor and platelet - derived growth factor , have not been combined previously . 
this phase i study was designed to evaluate the safety , pharmacokinetics , and pharmacodynamics of the combination . patients and methods patients with any advanced braf mutated malignancy with adequate organ function were eligible . 
dosages started at dabrafenib 50 mg twice a day and pazopanib 400 mg daily on dose level ( dl ) 1 , with maximum dosages of 150 mg twice a day and 800 mg daily on dl5 . 
pharmacokinetics and braf v600e plasma clone were measured , and efficacy was evaluated by imaging and tumor markers every 8 weeks . results twenty - three patients with 11 different tumor histologies were enrolled in five dls . 
common drug - related adverse events included nausea ( 52% ) , skin papules ( 43% ) , diarrhea ( 39% ) , hand - foot syndrome ( 30% ) , anemia ( 26% ) , rash ( 22% ) , vomiting ( 22% ) , hypophosphatemia ( 22% ) , and transaminitis ( 22% )  . 
five patients ( 22% ) experienced a partial response , including low - grade ovarian serous carcinoma , thyroid cancer , and glioblastoma multiforme , and two patients ( appendiceal and thyroid cancer ) had stable disease > 6 months . 
2018 by american society of clinical oncology introduction considered driver mutations in some tumor types , braf mutations are found in several types of cancer , 1 including melanoma , papillary thyroid cancer , 2 colorectal cancer , 3 and low - grade serous ovarian cancer ( lgsoc ) .4 braf inhibitor therapy is associated with an impressive , albeit shortlived , response rate in the treatment of malignant melanoma.5 in other malignancies , the activity of single - agent braf inhibitor is mixed . 
for example , limited efficacy was observed in colorectal cancer , 6 but favorable activity was shown in non small - cell lung cancer.7 mechanisms of resistance differ by tumor type . 
in melanoma , upregulation of the mitogen - activated protein kinase ( mapk ) pathway via mutations in alternate splicing of nras , mek , and braf leads to primary resistance.8 - 10 in addition , upregulation of platelet - derived growth factorin melanoma tumors from patients and in melanoma cell lines has been observed.11 in colorectal cancer , primary resistance seems to be via upregulation of the epidermal growth factor receptor and the mapk pathway , 12 , 13 although increased platelet - derived growth factor ( pdgf - b ) and signal transducer and activator of transcription 3 ( stat - 3 ) phosphorylation have also been sigurdis haraldsdottir filip janku ming poi cynthia timmers susan geyer larry j . 
an example of a fruitful attempt is the combination of dabrafenib , a braf inhibitor , with trametinib , a mek 1 / 2 inhibitor , in melanoma.16 collectively , it is compelling to combine therapies that target the aforementioned pathways with a braf inhibitor to synergize the effects of the agents and enhance the durability of response by abating drug resistance . we conducted an investigator - initiated multicenter phase i clinical trial that combined dabrafenib and pazopanib in the treatment of advanced braf mutated malignancies . 
eligibility criteria included any advanced malignant tumor carrying a mutation in the braf gene for which no standard curative or palliative measures existed , no treatment within 2 weeks of study enrollment , eastern cooperative oncology group performance status of 2 , life expectancy > 12 weeks , age 18 years , no prior use of dabrafenib or pazopanib , no more than three prior multikinase inhibitor regimens , normal organ function defined by an absolute neutrophil count of 1 , 500 / ml , hemoglobin 9 g / dl , and a platelet count of 100 , 000 / ml , as well as normal renal function ( creatinine 1.5 upper limit of normal and urine protein to creatinine ratio < 1 ) , hepatic function ( alt / ast 2.5 upper limit of normal and bilirubin 1.5 upper limit of normal ) , and cardiac function ( left ventricular ejection fraction 50% )  . 
patients with symptomatic , untreated brain metastases , hiv , hepatitis b or c , poorly controlled hypertension ( defined as systolic blood pressure 140 mm hg and / or diastolic blood pressure 90 mm hg measured on more than one occasion and not responsive to antihypertensives ) , qt interval > 480 ms , or who received excluded medications ( warfarin , antiepileptics , and / or antifungals , among others ) were excluded . 
braf mutation testing for study entry must have been performed at a clinical laboratory improvement amendmentscertified laboratory . dose escalation and administration dose escalation was performed on the basis of a standard cohorts - of - 3 designdose level 1 ( dl1 ) was dabrafenib 50 mg twice a day and pazopanib 400 mg daily ; dl2 was dabrafenib 50 mg twice a day and pazopanib 600 mg daily ; dl3 was dabrafenib 100 mg twice a day and pazopanib 600 mg daily ; dl4 was dabrafenib 100 mg twice a day and pazopanib 800 mg daily ; and dl5 was dabrafenib 150 mg twice a day and pazopanib 800 mg daily , administered in 28 - day cycles without interruption . 
all patients who were treated within a dl had to complete 28 days of therapy before escalation to the next dl and had to receive 80% of the doses of each drug ( or else they were replaced with another enrolled study patient )  . safety and efficacy assessments after drug initiation , patients were assessed on days 8 , 15 , 22 , 29 , and 43 , then every 4 weeks for adverse events , vital signs , hematology , chemistries , prothrombin time / partial thromboplastin time , urinalysis , and spot urine protein - to creatinine ratio . 
 dlts included grade 3 hematologic toxicities ( except grade 3 that resolved 7 days of stopping drugs or grade 3 lymphopenia ) ; fever 38.5c associated with dehydration , hypotension , or renal insufficiency ( two or more occasions ) ; grade 2 proteinuria ( unresolved 2 weeks of holding drugs ) ; grade 2 valvular toxicities ; asymptomatic decrease in left ventricular ejection fraction 10% that resulted in left ventricular ejection fraction at or below the institutional lower limit of normal ; symptomatic hypertension or recurring 160 / 100 mm hg despite antihypertensive medication ; and any grade 3 nonhematologic toxicities , excluding nausea , vomiting , diarrhea , electrolyte disturbance , fatigue , rash , or acute kidney injury that resolved 7 days of holding drugs . radiologic assessment was performed by computed tomography or magnetic resonance imaging ( as long as the same consistent measure was used serially ) every 8 weeks , and responses were measured according to recist 1.1. 
tumor markers , if applicable , were measured every 8 weeks . pharmacokinetic studies a single dose of dabrafenib was administered on cycle 1 , day 1 ( c1d1 ) , and a single dose of pazopanib on c1d2 . 
blood samples for dabrafenib and pazopanib pks were obtained predose , at 0.5 , 1 , 2 , 3 , 4 , 6 , 8 0.5 hours , and at 24 1 hours after drug administration on c1d1 , c1d2 , c1d3 , and c2d1 . 
 noncompartmental analysis of concentration time data were performed using phoenix winnonlin ( version 6.3 ; pharsight corporation , mountain view , ca )  . pharmacodynamic studies correlative studies included the quantification of the copy number of the braf v600e mutant allele in cell - free dna that was purified from plasma . 
we performed cell - free dna analysis on plasma samples that were collected at c1 to c6 and every other cycle beyond c6 using an allele - specific digital polymerase chain reaction assay on the qx200 droplet digital polymerase chain reaction system ( bio - rad laboratories , hercules , ca )  . 
archival formalin - fixed , paraffin embedded tumor tissue was collected , and dna was extracted to sequence the coding exons of 409 tumor suppressor and oncogenes using the ion torrent comprehensive cancer panel ( thermo fisher scientific , waltham , ma )  . 
maximum grade , frequency , and perceived attribution of adverse events were summarized across and within dls per national cancer institute common terminology criteria for adverse events criteria ( version 4 )  . 
per these criteria , toxicity was defined as an adverse event that was at least possibly related to treatment . in an exploratory manner , clinical outcomes were summarized , including overall response , time to progression ( ttp ) , and duration of response , and were based on recist criteria . 
of note , two patients on dl5 ( patients 5b and 5f ) received < 80% of drug doses and were replacedone patient experienced rapid disease progression with new brain metastasis , the other developed radiation recall after 3 days of therapy . 
two dlts were observed , which included grade 3 bowel perforation on dl3 , which was attributed to pazopanib , and grade 3 arthralgias on dl5 , which was attributed to dabrafenib . 
therapy was discontinued in three patients because of adverse events that included grade 3 bowel perforation that developed 4 days after therapy initiation , grade 2 radiation recall that developed 3 days after therapy initiation , 17 and grade 2 uveitis that developed 10 months after therapy initiation . 
serious adverse events possibly related to study drugs that led to hospital admission included a grade 3 to 4 pulmonary emboli , grade 3 abdominal pain , grade 2 intracranial hemorrhage , and grade 2 fever . response rate and ttp five ( 22% ; 95% ci , 7% to 44% ) of 23 patients achieved partial responses after 2 to 4 months of therapy ( fig 1 )  . 
 ( % ) , unless otherwise noted . abbreviations : ecog , eastern cooperative oncology group ; q , quartile . * nineteen patients with available data . the study and enrolled them in five dls ( table 1 )  . 
 grade grade grade toxicity ophthalmologic floaters photosensitivity uveitis skin skin papule or tag palmar - plantar erythrodysesthesia syndrome rash maculopapular dry skin actinic keratosis pruritus radiation recall reaction rash acneiform skin hyperpigmentation skin induration treatment - related secondary malignancy ( melanoma ) miscellaneous acute kidney injury dysmenorrhea flank pain menorrhagia oral pain thromboembolic event total events 1 ( 4 ) 1 ( 4 ) 1 ( 4 ) 10 ( 43 ) 7 ( 30 ) 4 ( 17 ) 3 ( 13 ) 2 ( 9 ) 1 ( 4 ) 1 ( 4 ) 1 ( 4 ) 1 ( 4 ) 1 ( 4 ) 1 ( 4 ) 1 ( 4 ) 1 ( 4 ) 1 ( 4 ) 1 ( 4 ) 1 ( 4 ) 1 ( 4 ) abbreviation : dl , dose level . * expanded to six patients ( two patients on dl5 had < 80% of doses and were replaced )  . grade 4 or 5 events were not observed . pk parameters of first - dose and steady - state pazopanib and dabrafenib are listed in tables 3 and 4 , respectively . 
appendix figures a1 and a2 ( online only ) depict mean plasma concentration time profiles stratified by dl for first - dose ( monotherapy ) and steady - state ( combination ) pazopanib and dabrafenib , respectively . 
 papillary thyroid cancer ( follicular variant ) ovarian cancer ( low grade , serous ) papillary thyroid cancer ae , papillary thyroid cancer papillary thyroid cancer appendiceal adenocarcinoma ovarian cancer ( low grade , serous ) glioblastoma multiforme * hrthle cell thyroid cancer cholangiocarcinoma nonsmall - cell lung cancer * parotid gland carcinoma melanoma melanoma bladder cancer melanoma colon cancer nonsmall - cell lung cancer papillary thyroid cancer melanoma ae , neuroendocrine carcinoma of colon * patient withdrawal , colon cancer ae , nonsmall - cell lung cancer time on treatment ( months ) time to response time to off treatment fig 1 . 
 ( * ) found to have discordance in braf mutation testing . concentrationtime profiles stratified by dl on c1d2 , whereas appendix figure a4b shows the predicted mean steady - state on the basis of firstdose pazopanib monotherapy pk data in the absence of drug interaction . 
only 39% and 20% of patients had a trough pazopanib concentration of > 17.5 g / ml on c1d15 and at steady state ( c2d1 ) , and only 13% of patients had a trough pazopanib concentration of > 20.5 g / ml at steady state ( c2d1 )  . 
samples from 16 patients were collected for both dabrafenib and pazopanib 24 - hour profile pharmacokinetics analysis on c2d1 . abbreviations : auc ( 0 - ) , observed area under the curve from 0 to infinity ; cmax , maximum observed concentration ; cssavg , average steady - state concentration ; cl / f , clearance ; fd , first dose ; ss , steady state ; t1 / 2 , terminal phase elimination half - life ; v / f , apparent volume of distribution on the basis of terminal phase . * the accumulation of dabrafenib is insignificant ; therefore , auc0is presented here for first dose and steady state for direct comparison . pharmacodynamics / braf v600e plasma measurements fig 2 . 
log ratio of braf v600e / braf wild type is depicted before therapy , lowest value on therapy , and value upon progression for patients who remained on therapy for more than two cycles . we measured braf v600e copies in circulating tumor dna in 19 patients . 
changes in copies along with tumor measurements and / or tumor markers in patients in the study for more than two cycles are shown in appendix figure a5 ( online only )  . 
the most common adverse events included nausea , vomiting , diarrhea , transaminitis , arthralgia , and skin toxicities , but the combination did not increase toxicities compared with the administration of either agent alone.18 , 19 two dltsa grade 3 bowel perforation on dl3 and grade 3 arthralgia on dl5were observed . 
 long - term toxicities were not prohibitive , as six patients were on therapy for > 6 months . ideally , time - dependent pks would have been measured with a run - in , with each drug measuring single dose and steady state , but given the refractory study population , a single drug run - in was not considered feasible . 
dabrafenib mean terminal half - lives grew shorter with repeated dosing at steady state , and mean auc ratios of c2d1 : c1d1 were < 1.0 , which suggests autoinduction of the drug.20 this correlates with previous reports in monotherapy studies.21 , 22 pk parameters for first single - dose pazopanib were overall consistent with previously published data from pazopanib monotherapy studies23 - 25 ; however , the steady - state concentration of pazopanib was lower than expected . 
in preclinical models , a steady - state concentration of > 17.5 g / ml was required for in vivo activity of pazopanib , 26 and a target pazopanib trough concentration of > 20.5 g / ml is associated with improved efficacy in patients with renal cell carcinoma.21 only 20% of patients in this study reached trough pazopanib concentrations of > 17.5 g / ml at steady state ( c2d1 ) , and only 13% of the patients achieved a trough pazopanib concentration of > 20.5 g / ml at steady state ( c2d1 )  . 
 the efficacy of single - agent multikinase inhibitors in thyroid cancer is well known , 30 - 32 with two agentssorafenib and lenvatinibapproved by the us food and drug administration . 
dabrafenib demonstrated partial responses in 33% of patients with braf mutated papillary thyroid carcinoma.33 , 34 braf mutations are found in 36% of patients with lgsoc tumors.4 two case reports of the administration of a braf inhibitor in heavily pretreated patients with unresectable lgsoc demonstrated a long lasting response.35 , 36 both patients with lgsoc in this trial were heavily pretreated and one had received an mek inhibitor as well as sorafenib / vemurafenib combination therapy . 
approximately 2% of glioblastomas have braf mutations , 37 and a response to vemurafenib was observed in a basket trial that included glioblastoma.36 we did not observe any activity in four patients with melanoma who had previously experienced progression on vemurafenib . 
it is possible that the combination does not target resistance mechanisms that promote progression on prior braf inhibitor therapy . measurements of the plasma braf v600e clone were positive in 58% of patients at baseline . 
this suggests that this type of monitoring could be used to determine clinical efficacy but should be undertaken in a larger trial . tumor heterogeneity was observed in three nonresponding patients with discordant outside braf testing that was obtained after coming off the trial . 
furthermore , we allowed tumor types with non - v600 braf mutations in this study , as dabrafenib does not exclusively inhibit braf v600 mutations , but we did not observe any activity in these patients . 
tumor sequencing of 409 tumor suppressor and oncogenes did not reveal any other significant findings . strengths of our study include the robust pk data that were obtained as well as assessment of braf v600e plasma clone levels during therapy and upon progression . 
therefore , a randomized phase ii trial is warranted in certain tumor types to determine whether the combination is superior to single - agent therapy or combination braf / mek inhibitor therapy . 
shah ming poi stock and other ownership interests : immunomedics , cti biopharma , juno therapeutics , propanc biopharm , pfizer , merck consulting or advisory role : salveo health communications , sandoz - novartis financial support : manisha h . 
shah collection and assembly of data : sigurdis haraldsdottir , filip janku , cynthia timmers , jennifer sexton , lai wei , jennifer thurmond , vivianne velez - bravo , erin m . 
schaaf , lai wei , kari kendra , amir mortazavi , vivek subbiah , mitch phelps manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors cynthia timmers no relationship to disclose susan geyer no relationship to disclose larry j . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . stock and other ownership interests : abbott nutrition research funding : medimmune ( inst ) , pfizer ( inst ) , bristol - myers squibb ( inst ) , ignyta ( inst ) patents , royalties , other intellectual property : us and foreign patents travel , accommodations , expenses : medimmune sigurdis haraldsdottir no relationship to disclose filip janku stock and other ownership interests : trovagene consulting or advisory role : deciphera , trovagene , novartis , sequenom , foundation medicine , guardant health , immunome research funding : novartis , biomed valley discoveries , roche , agios , astellas pharma , deciphera , symphony evolution , plexxikon , piqur , fujifilm other relationship : bio - rad vivianne velez - bravo no relationship to disclose vanda m . 
shah consulting or advisory role : eisai , loxo , novartis research funding : eisai , merck , loxo amir mortazavi honoraria : motive medical intelligence consulting or advisory role : genentech research funding : acerta pharma ( inst ) , genentech ( inst ) , merck ( inst ) , novartis ( inst ) , seattle genetics ( inst ) vivek subbiah consulting or advisory role : medimmune research funding : novartis ( inst ) , glaxosmithkline ( inst ) , nanocarrier ( inst ) , northwest biotherapeutics ( inst ) , genentech ( inst ) , berg pharma ( inst ) , bayer ( inst ) , incyte ( inst ) , fujifilm ( inst ) , pharmamar ( inst ) , d3 oncology solutions ( inst ) , pfizer ( inst ) , amgen ( inst ) , abbvie ( inst ) , multivir ( inst ) , blueprint medicines ( inst ) , loxo ( inst ) , vegenics ( inst ) , takeda ( inst ) , alfasigma ( inst ) , agensys ( inst ) , idera ( inst ) , boston biomedical ( inst ) travel , accommodations , expenses : pharmamar , bayer affiliations sigurdis haraldsdottir , ming poi , cynthia timmers , susan geyer , larry j . 
shah , ohio state university medical center , columbus , oh ; sigurdis haraldsdottir , stanford university , stanford , ca ; filip janku , vivianne velez - bravo , vanda m . 
stepanek , and vivek subbiah , university of texas md anderson cancer center , houston , tx ; and susan geyer , university of south florida , tampa , fl . funded by national comprehensive cancer network oncology research program from general research support provided by novartis , the sidney kimmel foundation for cancer research , a 2015 university of texas brain seed grant , and the glioblastoma moonshot translational research on brain tumors grant . presented at the asco annual meeting , chicago , il , may 30 - june 3 , 2014 , and the 26th european organisation for research and treatment of cancer national cancer instituteamerican association for cancer research symposium on molecular targets and cancer therapeutics , barcelona , spain , november 18 - 21 , 2014 . support prior presentation references 417 : 949 - 954 , 2002 1 . 
nikiforova mn , kimura et , gandhi m , et al : braf mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas . 
sieben nl , macropoulos p , roemen gm , et al : in ovarian neoplasms , braf , but not kras , mutations are restricted to low - grade serous tumours . 
planchard d , kim tm , mazieres j , et al : dabrafenib in patients with braf ( v600e ) - positive advanced nonsmall - cell lung cancer : a single - arm , multicentre , open - label , phase 2 trial . 
nazarian r , shi h , wang q , et al : melanomas acquire resistance to b - raf ( v600e ) inhibition by rtk or n - ras upregulation . 
wagle n , van allen em , treacy dj , et al : map kinase pathway alterations in braf - mutant melanoma patients with acquired resistance to combined raf / mek inhibition . 
ahronian lg , sennott em , van allen em , et al : clinical acquired resistance to raf inhibitor combinations in braf - mutant colorectal cancer through mapk pathway alterations . 
long gv , stroyakovskiy d , gogas h , et al : dabrafenib and trametinib versus dabrafenib and placebo for val600 braf - mutant melanoma : a multicentre , double - blind , phase 3 randomised controlled trial . 
hauschild a , grob jj , demidov lv , et al : dabrafenib in braf - mutated metastatic melanoma : a multicentre , open - label , phase 3 randomised controlled trial . 
ouellet d , gibiansky e , leonowens c , et al : population pharmacokinetics of dabrafenib , a braf inhibitor : effect of dose , time , covariates , and relationship with its metabolites . 
shibata si , chung v , synold tw , et al : phase i study of pazopanib in patients with advanced solid tumors and hepatic dysfunction : a national cancer institute organ dysfunction working group study . 
kumar r , knick vb , rudolph sk , et al : pharmacokinetic - pharmacodynamic correlation from mouse to human with pazopanib , a multikinase angiogenesis inhibitor with potent antitumor and antiangiogenic activity . 
lawrence sk , nguyen d , bowen c , et al : the metabolic drug - drug interaction profile of dabrafenib : in vitro investigations and quantitative extrapolation of the p450 - mediated ddi risk . 
goh bc , reddy nj , dandamudi ub , et al : an evaluation of the drug interaction potential of pazopanib , an oral vascular endothelial growth factor receptor tyrosine kinase inhibitor , using a modified cooperstown 5 + 1 cocktail in patients with advanced solid tumors . 
brose ms , nutting cm , jarzab b , et al : sorafenib in radioactive iodine - refractory , locally advanced or metastatic differentiated thyroid cancer : a randomised , double - blind , phase 3 trial . 
brose ms , cabanillas me , cohen ee , et al : vemurafenib in patients with braf ( v600e ) positive metastatic or unresectable papillary thyroid cancer refractory to radioactive iodine : a non - randomised , multicentre , open - label , phase 2 trial . 
combe p , chauvenet l , lefrre - belda ma , et al : sustained response to vemurafenib in a low grade serous ovarian cancer with a braf v600e mutation . 
 ( a through g ) patients who were on study for more than two cycles with measurable braf v600e plasma clone levels ( gold ) are depicted along with recist measurements ( sum of target lesions in centimeters depicted as blue columns , if disease was measurable ) and tumor marker ( gray , if measurable )  . 
in three patients with a partial response ( 3c , 4c , and 5e ) , plasma copies decreased , and in two patients ( 3c and 4c ) they became undetectable . 
two patients with stable disease > 6 months ( 2c and 5h ) had a decrease in copies ( one to undetectable levels , rising again upon progression )  . 
patient and tumor characteristics , tumor shrinkage , and tumor mutations in patients with partial responses and stable disease > 6 months patient id cancer type and time since diagnosis of metastatic disease until trial entry ( months ) appendiceal adenocarcinoma ( 44 ) lgsoc ( 6 ) lgsoc ( 105 ) ptc ( 119 ) ptc ( 24 ) gbm ( 45 ) follicular variant ptc ( 3 ) no . 
of prior therapies * 5 ( mek inhibitor , sorafenib + vemurafenib ) none none 1 ( sunitinib ) 3 ( vemurafenib ) shrinkage , sd ( nm ) tumor mutations ( deleterious ) braf v600e , ep400 , braf v600e sd ( nm ) braf v600e braf v600e , ddb2 braf v600e braf v600e time since acquiring tissue for testing until trial entry , months abbreviations : gbm , glioblastoma multiforme ; lgsoc , low - grade serous ovarian cancer ; mek , mitogen - activated pathway kinase kinase ; nm , nonmeasurable ; nt , not tested ; ptc , papillary thyroid cancer ; sd , stable disease . 
one year later , endometrial adenocarcinoma , endometrioid type international federation of gynecology and obstetrics grade 2 ( figo ; stage ii1 ) was diagnosed and treated with total abdominal hysterectomy and bilateral salpingo oophorectomy , followed by postoperative external beam radiotherapy and high - dose vaginal brachytherapy . 
immunohistologic studies for dna mismatch repair proteins on the endometrial adenocarcinoma revealed loss of expression of mlh1 and pms2 , and mlh1 promoter hypermethylation analysis was positive . another year later , positron emission tomography computed tomography scans indicated bilateral lung metastases suggestive of metastatic endometrial adenocarcinoma and a left axillary hypermetabolic lymph node suggestive of metastatic breast adenocarcinoma ( estrogen receptor negative , progesterone - receptor negative [ 1 + , 10% ] , and her2 negative )  . 
a biopsy specimen was taken of the chest wall lesion , which was found to be consistent with metastatic carcinoma most likely of breast origin , based on immunohistochemical and clinical findings . 
 follow - up magnetic resonance imaging testing showed metastases to bone and bra because of systemic progression , tumor molecular profiling for one of the chest wall lesions was performed using the stanford actionable mutation panel ( stamp ) assay , which is a 198 gene , hybrid - capturebased , solid - tumor , next generation sequencing panel . 
the case was discussed by the molecular tumor board ; the molecular profiling was considered to be suggestive of endometrial origin rather than breast orig given that the primary endometrial adenocarcinoma had been microsatellite unstable , microsatellite instability ( msi ) testing was recommended on the metastasis to enable the potential use of immune - checkpoint inhibitors , if positive . 
msi testing by polymerase chain reaction demonstrated that the tumor was msi high . after liposomal doxorubicin chemotherapy , stereotactic radiotherapy for the brain metastases , and palliative radiation therapy to a painful right acetabulum metastasis , the patient started treatment with pembrolizumab on a compassionate use basis because she did not qualify for current open trials ( having two active cancers ) , and this was before food and drug administration approval of immunotherapies for msi tumors . 
hence , although nonspecific , the - catenin ( ctnnb1 ) somatic mutation ( as opposed to an amplification ) is virtually never seen in ductal breast carcinoma and thus was a unique finding suggesting that the molecular signature of the biopsy specimen was more consistent with an endometrial adenocarcinoma origin than that of a breast adenocarcinoma . to more convincingly distinguish the tissue of origin of the patients metastatic disease , the molecular tumor board members recommended testing of mismatch repair protein status by polymerase chain reaction and agreed that if the tumor demonstrated microsatellite instability , the patient would be eligible to receive pembrolizumab as a participant in a local clinical trial or off label ( this was before the food and drug administration approval of pembrolizumab for msi - high tumors )  . discussion of possible alternatives to immune checkpoint inhibitors included therapeutic routes focused on targeting the akt - mtor pathway , because of pten and pik3ca mutations . 
 which has been demonstrated to respond to immunotherapy.6 published online on ascopubs.org / journal / po on september 19 , 2018 . rochelle reyes no relationship to disclose meredith mills research funding : myriad genetics ( inst ) recipient : natera ( inst ) james l . 
zehnder no relationship to disclose george sledge leadership : syndax stock and other ownership interests : syndax honoraria : symphony evolution consulting or advisory role : symphony evolution , coherus biosciences , radius health , peregrine pharmaceuticals , taiho pharmaceutical research funding : roche ( inst ) travel , accommodations , expenses : radius health , taiho pharmaceutical christina curtis consulting or advisory role : grail james m . 
 validation of her2 amplification as a predictive biomarker for anti epidermal growth factor receptor antibody therapy in metastatic colorectal cancer purpose her2 amplification has been implicated in resistance to therapy with anti epidermal growth factor receptor antibodies ( anti - egfrabs ) in metastatic colorectal cancer ( mcrc )  . 
the purpose of the study was to validate the predictive impact of her2 amplification in mcrc . patients and methods we analyzed patients with ras / braf wild - type mcrc across two distinct cohorts . 
 cohort 2 ( n = 70 ) included 16 patients with her2 amplification and 54 her2 nonamplified controls identified by next - generation sequencing ( her2 amplification : four or more copies ) who had received prior anti - egfrabs . 
2019 by american society of clinical oncology introduction antiepidermal growth factor receptor ( egfr ) antibodies ( anti - egfrabs ) , cetuximab and panitumumab , are routinely used , either alone or in combination with chemotherapy , in the treatment of metastatic colorectal cancer ( mcrc )  . 
 activating mutations in ras ( kras and nras ) genes are the only validated predictive biomarker of resistance to these agents.1 therefore , current practice guidelines recommend restricting anti - egfrab use to patients with ras wild - type tumors , which compose 45% to 50% of the population with mcrc.2 even among this kanwal raghav jonathan m . 
 select group , many patients do not derive clinical benefit from treatment with anti - egfrabs as a result of diverse inherent and acquired resistance mechanisms.3 amplification in the her2 ( human epidermal growth factor receptor 2 ) gene ( also known as erbb2 ) is one such mechanisher2 amplification has been implicated in therapeutic resistance to anti - egfrab therapy in preclinical studies in mcrc.4 , 5 aberrant her2 signaling results in bypass activation of the ras - mek - erk signaling pathway , thereby blunting the effect of egfr blockade.4 although the clinical significance and therapeutic potential of her2 amplification is well established in breast and gastric cancer , its characterization in mcrc remains deficient . 
her2 amplification is seen in a small subset of mcrc , predominantly in kras wildtype tumors , for which anti - egfrabs are used as targeted therapies.6 , 7 limited retrospective evidence shows that her2 amplification is associated with poor survival outcomes with anti - egfrabbased regimens.4 , 5 , 8 given the preliminary nature of these studies , these findings need additional clinical corroboration . accordingly , we systematically analyzed two distinct cohorts of patients with mcrc to validate the impact of her2 amplification on anti egfrabbased therapy . 
our in situ hybridization ( ish ) cohort ( cohort 1 ) included 98 sequential patients with ras / braf wild - type mcrc who were enrolled on a molecular screening program at the university of texas md anderson cancer center ( mdacc ; data supplement ) between august 2010 and june 2015 ( fig 1 ; data supplement ) .1 , 9 in this cohort , we identified 13 her2 - amplified and 85 her2 - nonamplified patients . 
among these patients , the 12 her2 amplified patients and 65 her2 - nonamplified patients who had received anti - egfrab in the secondor third - line setting after treatment on a nonanti - egfr regimen in the first - line setting were used for primary analyses ( fig 1 )  . 
 our next - generation sequencing ( ngs ) cohort ( cohort 2 ) comprised 16 patients with ras / braf wild - type mcrc with her2 amplification and 54 patients with ras / braf wild - type mcrc without her2 amplification who were identified by an ngs platform at mdacc and city of hope cancer center and had received prior treatment with anti - egfrabs in a second or third - line setting after treatment on a non anti - egfr regimen in the first - line setting , similar to cohort 1 ( fig 1 ; data supplement )  . 
 data on patient and tumor characteristics , treatment course , and vital status were collected from a prospectively maintained database and electronic medical records ( data supplement )  . the primary objective was to compare the efficacy of anti - egfrab therapy in the secondor third - line setting after treatment on prior therapy without anti - egfrab between patients with her2 - amplified and her2 - nonamplified mcrc . 
the choice of this specific line of therapy was based on practice patterns of treating mcrc ; anti - egfrab therapy is most commonly used as secondor third - line therapy.10 her2 amplification testing and interpretation pretreatment formalin - fixed paraffin - embedded archival tumor tissues were used for her2 testing . 
focal her2 amplification was defined as an her2 / cep17 ratio of 2.0 or greater , as per asco / college of american pathologists clinical practice guidelines for her2 testing in breast and gastric cancer ( data supplement ) .11 , 12 her2 amplification status in cohort 2 was determined with ngs . 
 a cohort 1 ( ish cohort ) cohort 2 ( ngs cohort ) patients with mcrc enrolled in attacc molecular prescreening protocol ( n = 747 ) systematic search of patients with mcrc who had ngs under clia excluded patients kras mutant nras mutant ras unknown braf mutant ( n = 489 ) ( n = 368 [ 49% ] ) ( n = 28 [ 4% ] ) ( n = 41 [ 6% ] ) ( n = 52 [ 7% ] ) patients excluded as a result of absence of necessary tissue for her2 dual ish evaluation ( n = 160 ) patients eligible for her2amp testing ( n = 258 ) ras / braf wild - type patients with successful her2amp testing ( n = 98 ) step 1 identification of patients step 2 identification of controls patients satisfying all of following criteria : her2 amp detected no ras mutations no braf mutation patients satisfying all of following criteria : no her2 amp detected no ras mutations no braf mutation patients were excluded if patients either had : not received any anti - egfr therapy received anti - egfr therapy in first - line setting her2 - amplified mcrc ( n = 13 ) her2 - nonamplified mcrc ( n = 85 ) patients : her2 - amplified mcrc ( n = 16 ) controls : her2 - nonamplified mcrc ( n = 54 ) anti - egfrbased therapy used in second or third - line setting * ( n = 12 ) ( n = 65 ) fig 1 . 
the analyses to determine the impact of her2 amplification ( her2amp ) on progression - free survival with antiepidermal growth factor receptor ( egfr ) therapy was tested in two independent cohorts of patients with metastatic colorectal cancer ( mcrc )  . 
 ( a ) cohort 1 consisted of patients with ras / braf wild - type mcrc with adequate tissue available for her2 testing using dual in situ hybridization ( ish )  . 
 ( b ) cohort 2 consisted of patients with ras / braf wild - type mcrc with her2amp detected on next - generation sequencing ( ngs ) and matched controls with her2 - nonamplified tumors who underwent similar sequencing and were treated with an anti - egfr therapy regimen in a similar setting . 
at time of database lock , 21 patients had received either no anti - egfr therapy ( n = 20 ) or anti - egfrbased therapy in the first - line setting ( n = 1 )  . 
attacc , assessment of targeted therapies against colorectal cancer ; clia , clinical laboratory improvement amendments ; pep , primary end point . using the hybrid capturebased foundationone assay ( data supplement ) .14 all testing was performed in clinical laboratory improvement amendmentscertified laboratories . statistical analysis the primary end point was progression - free survival ( pfs ) in patients treated with anti egfrabbased therapy in the secondor thirdline setting . 
to ascertain the predictive value of her2 amplification , we then compared the pfs of these patients on a regimen devoid of anti egfrabs ( nonanti - egfrabbased regimen in first - line setting )  . 
we also performed additional analyses to ascertain whether the reduction in pfs in her2 - amplified patients is exclusively a result of rapid progression on treatment with anti - egfrabs or whether these patients are rapid progressors irrespective of type of therapy by using the pfs ratio ( ratio of pfs on antiegfrbased therapy in secondor third - line setting to pfs on nonanti - egfr regimen in first - line setting )  . 
key secondary end points were overall survival and time to treatment failure ( defined as time from start of treatment to discontinuation of treatment for any reason or death )  . 
baseline characteristics and treatment variables were well balanced between cohorts 1 and 2 ( data supplement ) and between her2 - amplified and nonamplified subsets in both cohorts ( table 1 )  . 
in cohort 1 , cetuximab was the most commonly administered anti - egfr agent ( 88% of patients ) , and irinotecan was the most common cytotoxic chemotherapy backbone ( 88% ) used with anti - egfrabs . 
likewise , in cohort 2 , cetuximab and an irinotecan - based regimen were used in 79% and 87% of patients , respectively . her2 amplification and pfs on treatment with anti - egfrabbased therapy to evaluate the impact of her2 amplification on efficacy of anti - egfrabs , we first compared pfs of her2 - amplified and her2 - nonamplified patients receiving these agents in the secondor third - line setting . 
in both ( a ) cohort 1 and ( b ) cohort 2 , her2amp was associated with shorter median pfs ( mpfs ) on anti epidermal growth factor receptor ( egfr ) antibody ( anti - egfrab ) therapy compared with the her2 - nonamplified ( her2na ) group . 
in contrast , there was no difference in mpfs on therapy devoid of anti - egfrabs between her2amp and her2na groups in both ( c ) cohort 1 and ( d ) cohort 2 . 
 ( e and f ) in addition , the pfs ratio ( ratio of pfs on anti - egfrab therapy [ pfs2 ] to pfs on nonanti - egfrab therapy [ pfs1 ] ) for individual patients is lower for her2amp patients compared with her2na patients . 
the direction and magnitude of differences between her2 - amplified and nonamplified patients on anti - egfrab and non anti - egfrab therapy were comparable to the previously presented results in both cohorts ( data supplement )  . 
 is a pivotal step in refining the risk - benefit profile of anti - egfrabs and toward the eventual goal of true personalized therapy in mcrc . this clinical analysis was performed to determine the role of her2 amplification as a predictive biomarker . 
using two distinct cohorts , we showed that when patients with ras wild - type mcrc are treated with anti - egfrabbased regimens , the pfs of her2 - amplified patients is significantly shorter ( less than 3 months ) compared with the pfs of her2 - nonamplified patients ( 8 to 9 months )  . in addition to corroborating our findings in two independent cohorts , our complementary analysis helped to assess the predictive nature of this finding in the absence of an observation or placebo arm.15 the precept underlying this approach was that if the poor pfs seen with anti egfrabbased regimen is a result of poor prognostic impact of her2 amplification , then we would expect a similar lower pfs in these her2 amplified patients irrespective of type of therapy.16 however , we found that the pfs of these patients on therapy devoid of anti - egfrabs was similar to that of her2 - nonamplified patients . 
 we also found that the ratio of pfs on antiegfr therapy to pfs on nonanti - egfr therapy in her2 - amplified patients was lower , indicating that her2 - amplified patients had relatively better pfs on nonanti - egfr therapy . 
thus , we demonstrated that poorer survival found in her2 - amplified patients compared with her2 - nonamplified patients was uniquely associated with anti - egfr therapy ( data supplement ) .1 our findings are comparable to and confirm other smaller series reported in literature . 
our study included two independent groups , and in addition , our analysis uniquely explored the predictive value of her2 amplification by comparing the effect of her2 amplification on nonanti - egfrab therapy . 
preclinical data have shown that activating genetic aberrations in her2 overcome efficacy of anti - egfrabs by either bypassing egfr signaling and activating pi3k / akt / mtor and ras / raf / mapk cascade or by heterodimerization with egfr.18 using a large xenograft cohort derived from patients with mcrc ( n = 85 ) , bertotti et al5 showed that her2 amplifications were enriched in a subset of cetuximab - resistant , kras / nras / braf / pik3ca wild - type patients and caused resistance to anti egfrab therapy . 
together , these robust preclinical and clinical data strengthen the hypothesis that her2 amplifications are negative predictors of response to anti - egfr therapy.4 , 5 the prevalence of her2 amplifications and the appropriate setting and optimal methodology for testing are critical concerns moving forward . 
we performed her2 amplification analyses in cohort 1 using dual ish , as was done in the her2 amplification for colorectal cancer enhanced stratification ( heracles ) study ( fig 3 ) .22 in cohort 2 , ngs was used for her2 amplification determination , as was done in the my pathway study.21 our analysis shows comparable impact of her2 amplification on pfs with anti - egfr therapy , irrespective of the methodology used . 
representative photomicrographs of colorectal cancer ( crc ) samples with ( a ) her2 protein expression using immunohistochemistry ( ihc ; scores of 0 , 1 + , 2 + , and 3 + ) and ( b ) her2amp using dual in situ hybridization ( ish ; gene status expressed as her2 / cep17 ratio )  . 
 additional studies in colon cancer are needed for cross - validation of these methodologies to assess concordance between ish and ngs and to determine the optimal platform ; however , both platforms supported similar findings of predictive impact in our study . the discovery of her2 amplification in a patient with mcrc has implications beyond identification of a negative predictive factor . 
 unlike patients with ras - mutant tumors where we struggle from the lack of therapies that target the ras pathway , myriad agents ( eg , trastuzumab , lapatinib , pertuzumab , trastuzumab emtansine , neratinib ) that target her2 exist . 
 these have shown favorable clinical activity in her2 - amplified tumor types such as breast cancer and gastric cancer.24 - 27 her2 - amplified patient - derived xenografts resistant to cetuximab show rapid and dramatic tumor shrinkage with dual anti - her2 inhibition by combination therapy with pertuzumab and lapatinib.5 in line with these preclinical findings , two distinct proof - of - concept , single - arm , phase ii studies using dual anti - her2 targeting in patients with her2 - amplified refractory mcrc have shown promising preliminary results . 
however , given the lack of major differences between the her2 - amplified and nonamplified patients , the relatively large size of this cohort of a rare subset of mcrc , the strength of the observed effect , and the clear imitation in a second independent data set , this study indicates strong evidence for this predictive effect . 
additional study of her2 amplifications in tissue specimens from randomized prospective cohorts such as the swog 80405 trial may provide a higher level of validation , although the low prevalence of this biomarker makes this challenging . 
despite these limitations , we consider these findings to be representative of this subset of patients with mcrc , and although not definitive , the findings strongly indicate that her2 amplification is a negative determinant of response to anti - egfrabbased therapy in mcrc . 
we propose that although the level of evidence is insufficient to completely forego therapy with anti - egfrabs in these patients , the limited benefit with these agents in this population argues in favor of using her2 testing for facilitating anti - egfr treatment decision making and for clinical trial actionability . her2 amplification is not the only resistance mechanism to anti - egfr therapy . 
 loree , david menter , shalini singh , riham katkhuda , kandavel shanmugam , scott kopetz administrative support : david menter provision of study materials or patients : michael j . 
shaw , riham katkhuda , dipen maru , marwan fakih , scott kopetz studies have implicated other alterations , such as her2 mutations and met amplification , in this resistance.29 , 30 emerging data have also shown that acquired resistance as a result of her2 amplifications occurs in a small subset of patients ( three [ 14% ] of 22 patients ) .31 follow - up studies are necessary to assess the role of these acquired her2 amplifications in resistance to anti - egfrabs . 
activation as measured by phosphoher2 and downstream effector pik3 / akt pathway in amplified tumors and in tumors where it occurs independent of amplification ( data supplement ) may predict response to her2 - targeted therapies , as is the case with breast cancer.32 , 33 in conclusion , her2 amplifications predict a lack of response to anti - egfrab therapy in patients with mcrc . 
early integration of her2 testing for patients with ras / braf wild - type mcrc can allow for a more informed discussion with patients regarding risks and benefit of anti - egfrabs , adding a layer of personalization to therapy in this subset of patients with mcrc . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . kanwal raghav consulting or advisory role : genentech ( inst ) , bayer ( inst ) travel , accommodations , expenses : tracon pharma jonathan m . 
overman consulting or advisory role : bristol - myers squibb , genentech , gritstone oncology , medimmune research funding : bristol - myers squibb , merck , roche , medimmune ruoxi yu no relationship to disclose funda meric - bernstam honoraria : sumitomo group , dialectica consulting or advisory role : genentech , inflection biosciences , pieris pharmaceuticals , clearlight diagnostics , darwin health , samsung bioepis , spectrum pharmaceuticals , aduro biotech , origimed , xencor , debiopharm group research funding : novartis , astrazeneca , taiho pharmaceutical , genentech , calithera biosciences , debiopharm group , bayer , aileron therapeutics , puma biotechnology , cytomx therapeutics , jounce therapeutics , zymeworks , curis , pfizer , effector therapeutics , abbvie , boehringer ingelheim ( i ) , guardant health ( inst ) david menter no relationship to disclose krittiya korphaisarn no relationship to disclose brian kee stock and other ownership interests : medtronic stock and other ownership interests : roche patents , royalties , other intellectual property : roche travel , accommodations , expenses : roche shalini singh employment : ventana medical systems stock and other ownership interests : ventana medical systems patents , royalties , other intellectual property : method of identifying treatment - responsive nonsmall - cell lung cancer using alk as a marker ( inst ) mark routbort no relationship to disclose ken chen no relationship to disclose kenna r.m. 
shaw no relationship to disclose riham katkhuda no relationship to disclose kandavel shanmugam employment : ventana medical systems stock and other ownership interests : roche patents , royalties , other intellectual property : patents ( inst ) travel , accommodations , expenses : ventana medical systems dipen maru no relationship to disclose marwan fakih consulting or advisory role : amgen , array biopharma , genentech speakers ' bureau : amgen , taiho pharmaceutical research funding : novartis ( inst ) , amgen ( inst ) , astrazeneca ( inst ) scott kopetz stock and other ownership interests : molecularmatch , navire pharma consulting or advisory role : roche , genentech , emd serono , merck , karyopharm therapeutics , amal therapeutics , navire pharma , symphogen , holy stone , biocartis , amal therapeutics , amgen , novartis research funding : amgen ( inst ) , sanofi ( inst ) , biocartis ( inst ) , guardant health ( inst ) , array biopharma ( inst ) , genentech ( inst ) , emd serono ( inst ) , medimmune ( inst ) , novartis ( inst ) affiliations kanwal raghav , jonathan m . 
allegra cj , rumble rb , hamilton sr , et al : extended ras gene mutation testing in metastatic colorectal carcinoma to predict response to anti - epidermal growth factor receptor monoclonal antibody therapy : american society of clinical oncology provisional clinical opinion update 2015 . 
misale s , di nicolantonio f , sartore - bianchi a , et al : resistance to anti - egfr therapy in colorectal cancer : from heterogeneity to convergent evolution . 
bertotti a , migliardi g , galimi f , et al : a molecularly annotated platform of patient - derived xenografts ( xenopatients ) identifies her2 as an effective therapeutic target in cetuximabresistant colorectal cancer . 
richman sd , southward k , chambers p , et al : her2 overexpression and amplification as a potential therapeutic target in colorectal cancer : analysis of 3256 patients enrolled in the quasar , focus and piccolo colorectal cancer trials . 
martin v , landi l , molinari f , et al : her2 gene copy number status may influence clinical efficacy to anti - egfr monoclonal antibodies in metastatic colorectal cancer patients . 
overman mj , morris v , kee b , et al : utility of a molecular prescreening program in advanced colorectal cancer for enrollment on biomarker - selected clinical trials . 
wolff ac , hammond me , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
bartley an , washington mk , ventura cb , et al : her2 testing and clinical decision making in gastroesophageal adenocarcinoma : guideline from the college of american pathologists , american society for clinical pathology , and american society of clinical oncology . 
jeong jh , kim j , hong ys , et al : her2 amplification and cetuximab efficacy in patients with metastatic colorectal cancer harboring wild - type ras and braf . 
hurwitz h , raghav kps , burris ha , et al : pertuzumab + trastuzumab for her2 - amplified / overexpressed metastatic colorectal cancer ( mcrc ) : interim data from mypathway . 
ross ds , zehir a , cheng dt , et al : next - generation assessment of erbb2 ( human epidermal growth factor receptor 2 ) amplification status : clinical validation in the context of a hybrid capture - based , comprehensive solid tumor genomic profiling assay . 
bang yj , van cutsem e , feyereislova a , et al : trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of her2 - positive advanced gastric or gastrooesophageal junction cancer ( toga ) : a phase 3 , open - label , randomised controlled trial . 
sartore - bianchi a , trusolino l , martino c , et al : dual - targeted therapy with trastuzumab and lapatinib in treatment - refractory , kras codon 12 / 13 wild - type , her2 - positive metastatic colorectal cancer ( heracles ) : a proof - of - concept , multicentre , open - label , phase 2 trial . 
raghav k , morris v , tang c , et al : met amplification in metastatic colorectal cancer : an acquired response to egfr inhibition , not a de novo phenomenon . 
wulfkuhle jd , yau c , wolf dm , et al : evaluation of the her / pi3k / akt family signaling network as a predictive biomarker of pathologic complete response for patients with breast cancer treated with neratinib in the i - spy 2 trial . 
pathologic examination showed a 2.9 - cm collecting duct carcinoma ( cdc ) with metastases in eight of 15 resected hilar , suprahilar , and periaortic lymph nodes . after surgery , the patient was initiated on therapy with cisplatin and gemcitabine on a dosing schedule of once every 3 weeks . 
cytologic examination of pleural fluid retrieved during thoracentesis confirmed malignant pleural effusions . given the absence of any standard second - line treatment of cdc , comprehensive genomic profiling ( cgp ) was performed using a commercially available assay in a clinical laboratory improvement amendmentscertified , college of american pathologistsaccredited laboratory ( foundation medicine ; cambridge , ma )  . 
formalin - fixed paraffin - embedded tumor derived from the original nephrectomy specimen was analyzed by hybridization capture of all coding exons from 315 cancerrelated genes and select introns of 28 genes commonly rearranged in cancer . 
sequencing and bioinformatics methods for characterization of genomic alterations ( ga ) have previously been described in detail.1 the sole ga observed in this case was homozygous deletion of cdkn2a / b . 
multiple variants of unknown significance were identiincluding bard1 ( p358_s364del ) , brip1 fied , ( r264w ) , crlf2 ( e213k ) , dicer1 ( v594i ) , fgf6 ( n113k ) , igf1r ( p190s ) , mll2 ( r1292c ) , notch1 ( v1232m ) , pdgfrb ( s408c ) , ros1 ( e401k ) , socs1 ( a44_v45insvp ) , and tet2 ( e1405q )  . 
cdkn2a alteration has been suggested to be a potential predictive biomarker for cdk4 / 6 inhibitors.2 , 3 in light of loss of cdkn2a / b , the patient was offered therapy with the cdk4 / 6 inhibitor palbociclib . 
chung , foundation medicine , cambridge ; and toni k . choueiri , dana - farber cancer institute / brigham and womens cancer center , boston , ma corresponding author : sumanta k . 
the genomic landscape of cdc was defined in a previous report from our group.11 potential biomarkers for response to cdk4 / 6 inhibitors such as palbociclib include alterations in genes involved in the regulation of cdk4 / 6cyclin d kinase complexes , 2 , 3 such as cdk4 amplification , and palbociclib has demonstrated encouraging activity in phase 2 studies of patients with liposarcoma who harbor this driver.12 , 13 although preclinical studies of cell lines from multiple cancer types , including rcc , have reported that cdkn2a / p16ink4a loss of function is associated with sensitivity to cdk4 / 6 inhibitors , 14 - 18 there is limited clinical evidence supporting cdkn2a as a predictive biomarker , with one case study reporting that a patient with uterine leiomyosarcoma harboring cdkn2a homozygous deletion and a concurrent nf2 mutation experienced 4 months of stable disease on palbociclib treatment . in contrast , for patients with advanced breast cancer , for which palbociclib is approved with either letrozole or fulvestrant , cdkn2a loss ( assessed by fluorescent in situ hybridization ) was not associated with further benefit from palbociclib in combination with letrozole , in the palbociclib ongoing trials in the management of breast cancer ( paloma ) - 1 trial for patients with er + / her22 breast cancer.19 similarly , in a basket trial of the cdk4 / 6 inhibitor ribociclib , no responses were observed among the 73 patients with cancer whose tumor harbored a cdkn2a ga , as assessed by cgp.20 in this context and that of limited single - agent efficacy of cdk4 / 6 inhibitors in unselected populations , 19 , 20 the partial response to palbociclib experienced here by a patient with metastatic refractory cdc harboring cdkn2a / b homozygous deletion is unusual . 
in a series of 17 patients with cdc assessed by cgp , the most frequently identified gas were in nf2 ( 29% ) , setd2 ( 24% ) , smarcb1 ( 18% ) , and cdkn2a / b ( 12% , including homozygous deletion and mutation )  . 
characteristics for cases with cdkn2a or cdkn2a / b homozygous deletion characteristic total cases , no . total cases with cdkn2a or cdkn2a / b homozygous deletion , no . cases with cdkn2a or cdkn2a / b homozygous deletion and no co - occurring alteration , no . characteristics of patients with cdkn2a or cdkn2a / b homozygous deletion and no co - occurring alteration female male age , years , median ( range ) median tumor mutational burden , no . 
cdkn2a ( or cdkn2a / b ) homozygous deletion was identified as the sole driver alteration in 1.2% ( 16 of 1 , 322 ) rcc cases and also in other disease types , including 15.2% of salivary gland acinic cell tumors , 14.3% of bone giant - cell tumors , and 11.3% of bone chordomas ( table 1 )  . 
it is conceivable , however , that some of these cases with cdkn2a homozygous deletion as the sole known ga may harbor concurrent driver alterations that have not yet been discovered . 
a larger subset of cases ( 16% ) had homozygous deletion of this locus as well as other gas in other genes ( table 1 ) , and additional cases had loss of heterozygosity of cdkn2a with the remaining allele mutated . 
more investigation is also required to understand how responses to cdk4 / 6 inhibitors for patients with cdkn2a alteration may be modulated by the presence or absence of different driver gene alterations . for patients with cdc , additional genomic profiling studies are needed , given the response to palbociclib observed in this study , which suggests that cdk4 / 6 inhibitors should be further explored for patients with cdkn2a - altered cdc . 
more broadly , this study identifies cdkn2a homozygous deletion as the sole known ga in a small subset ( 0.25% ) of advanced cancer cases and at higher frequency in specific tumor types ( table 1 )  . 
 patents , royalties , other intellectual property : patents via foundation medicine , seres health on microbiome research in non - neoplastic disease ( inst ) genentech , eisai , foundation medicine , cerulean pharma , astrazeneca , peloton therapeutics , exelixis , prometheus , alligent jeffrey ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine honoraria : pfizer , emd merck serono research funding : foundation medicine toni k . 
choueiri honoraria : national comprehensive cancer network , uptodate consulting or advisory role : pfizer , bayer , novartis , glaxosmithkline , merck , bristol - myers squibb , roche / research funding : pfizer ( inst ) , novartis ( inst ) , merck ( inst ) , exelixis ( inst ) , tracon pharma ( inst ) , glaxosmithkline ( inst ) , bristol - myers squibb ( inst ) , astrazeneca ( inst ) , peloton therapeutics ( inst ) , roche / genentech ( inst ) , celldex ( inst ) , agensys ( inst ) travel , accommodations , expenses : advisory boards / consultancy jon h . 
curr oncol 20 : e223 - e232 , 2013 von der maase h , hansen sw , roberts jt , et al : gemcitabine and cisplatin versus methotrexate , vinblastine , doxorubicin , and cisplatin in advanced or metastatic bladder cancer : results of a large , randomized , multinational , multicenter , phase iii study . 
miyake h , haraguchi t , takenaka a , et al : metastatic collecting duct carcinoma of the kidney responded to sunitinib . int j clin oncol 16 : 153 - 155 , 2011 10 . 
dickson ma , schwartz gk , keohan ml , et al : progression - free survival among patients with well - differentiated or dedifferentiated liposarcoma treated with cdk4 inhibitor palbociclib : a phase 2 clinical trial . 
dickson ma , tap wd , keohan ml , et al : phase ii trial of the cdk4 inhibitor pd0332991 in patients with advanced cdk4 - amplified well - differentiated or dedifferentiated liposarcoma . 
logan je , mostofizadeh n , desai aj , et al : pd - 0332991 , a potent and selective inhibitor of cyclin - dependent kinase 4 / 6 , demonstrates inhibition of proliferation in renal cell carcinoma at nanomolar concentrations and molecular markers predict for sensitivity . 
young rj , waldeck k , martin c , et al : loss of cdkn2a expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the cdk4 / 6 inhibitor pd0332991 in melanoma cell lines . 
finn rs , crown jp , lang i , et al : the cyclin - dependent kinase 4 / 6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first - line treatment of oestrogen receptor - positive , her2 - negative , advanced breast cancer ( paloma - 1 / trio - 18 ) : a randomised phase 2 study . 
peguero ja , oneil bh , sohal d , et al : genomic mutation profiling ( gmp ) and clinical outcome in patients ( pts ) treated with ribociclib ( cdk4 / 6 inhibitor ) in the signature prograj clin oncol 34 , 2016 ( suppl ; abstr 2528 ) 21 . 
wang j , papanicolau - sengos a , chintala s , et al : collecting duct carcinoma of the kidney is associated with cdkn2a deletion and slc family gene up - regulation . 
 translating gene signatures into a pathologic feature : tumor necrosis predicts disease relapse in operable and stage i lung adenocarcinoma purpose the high 5 - year disease relapse rate in patients with stage i lung adenocarcinoma indicates the need for additional risk stratification parameters . 
this study assessed whether gene signatures translate into a pathologic feature for better disease stratification . materials and methods the mutual interdependence and risk stratification power of three gene signatures , cell cycle , hypoxia , and mammalian target of rapamycin ( mtor ) , were investigated in nine cohorts of patients with lung adenocarcinoma and five cohorts of patients with lung squamous cell carcinoma . 
the translation of signature score to pathway activity was further investigated by integrative analyses using the cancer genome atlas and the cancer protein atlas lung adenocarcinoma data sets . results the results showed that the three gene signatures were mutually interdependent in lung adenocarcinoma but not in lung squamous cell carcinoma . 
2018 by american society of clinical oncology introduction nonsmall - cell lung cancers ( nsclcs ) account for 85% of lung cancer cases and are the leading cause of cancer - related death worldwide.1 - 4 although a high proportion of nsclcs are diagnosed at advanced stages , the percentage of patients with early stage disease is increasing with the implementation of lung cancer screening programs.5 nevertheless , although surgical resection may increase the potential cure for patients with early stage disease , tumor recurrence is an almost inevitable outcome in reality , including those with stage i tumors . 
the 5 - year survival rate after potential curative resection is approximately 73% for stage ia and 58% for stage ib disease.6 current evidence shows that adjuvant chemotherapy is detrimental to patients with stage ia disease and is beneficial to patients with stage ib disease only among those with primary tumor size > 4 cm.7 - 9 despite numerous studies in the field of prognostic biomarkers for better patient stratification during the past decade , t stage remains the only available parameter . 
 the new - generation adjuvant trials are evaluating drugs beyond chemotherapy.10 advances in cancer genomics have led to the generation of several prognostic gene expression signatures.11 , 12 although these signature - based biomarkers represent a new milestone in lung cancer biomarker research , the sophisticated processing procedures limit their feasibility in clinical use . 
however , several retrospective clinicopathologic studies suggested that tumor necrosis serves as an additional stratification factor for stage i disease.13 - 16 nevertheless , somewhat discrepant results from these studies precluded the consensus for adopting tumor necrosis as an additional parameter . from the biologic aspect , tumor necrosis is the end result of dysregulated cell proliferation that occurs in a hypoxic tumor microenvironment poor with nutrient supply.17 depletion of intracellular atp levels switches apoptotic cell death to necrotic cell death.18 poly ( adp - ribose ) polymerase - 1 interacts with hypoxia - inducible factors , and its overactivation leads to a massive intracellular atp depletion that results in necrotic cell death.19 , 20 the mammalian target of rapamycin ( mtor ) kinase integrates signals from nutrients , growth factors , energy , and stress to regulate cell growth.18 autophagy is a cellular machinery that antagonizes necrotic cell death , and the mtor signaling pathway has been shown to promote necrotic cell death via suppression of autophagy.21 balance among apoptosis , necrosis , and autophagy determines the ultimate destiny of the cell.18 this study investigated the association between tumor necrosis and clinical prognosis of patients with lung adenocarcinoma as well as the association between tumor necrosis and three prognostic gene expression signatures in lung adenocarcinoma . 
the mutual interdependence of three gene signatures , representing cell cycle activity , tissue hypoxia , and nutrient balance , was assessed , as was the ability of these signatures to predict risk in patients with lung adenocarcinomas . 
 lung squamous cell carcinoma gene signatures were analyzed in four clinical cohorts : gse8894 , gse11969 , gse4573 , and tcga lung squamous cell carcinoma data sets ( data supplement )  . 
the tcga data set was downloaded from the tcga web site , and other data sets were downloaded from the gene expression omnibus database.22 the reverse phase protein array data of lung adenocarcinoma data sets were downloaded from the cancer protein atlas ( tcpa ) web site.23 determination of gene signatures three gene signatures , the cell - cycle , hypoxia , and mtor signatures , were used in this study . 
 the cell - cycle signature is composed of genes whose expression levels were altered during synchronized cell cycle phase.24 the hypoxia signature consists of hif targeted genes identified as core responders to hypoxia.25 the mtor signature consists of genes downregulated after rapamycin treatment of human bjab lymphoma cells.26 , 27 the genes involved in these three signatures are listed in the data supplement . 
the r values derived then served as the null distribution for evaluating the significance of our observation . statistical analysis differences in signature scores between two groups , such as normal and tumor samples , were estimated by t tests . 
the results were considered statistically significant when two - tailed p values were < .05. results three mutually interdependent gene signatures active in operable lung adenocarcinomas bearing areas of necrosis assessment of the gse11969 lung adenocarcinoma cohort showed that overall survival was significantly shorter in necrosis - positive than necrosis - negative tumors ( p = .04 ; fig 1a )  . 
three gene signatures , cell cycle , hypoxia , and mtor , were selected to estimate the activities of three corresponding processes in tumor biology : cell proliferation , tissue hypoxia , and nutrient balance , respectively . 
evaluation of the gse11969 cohort showed that each gene signature possessed significantly higher activity in necrosis - positive than in necrosis - negative tumors ( p < .001 each ; fig 1a ) and was predictive of the presence of necrosis ( area under the curve > 0.7 each ; data supplement )  . 
each of these three gene signatures also showed higher activities in the sample containing tumor cells than in adjacent normal areas ( p < .001 each ; data supplement )  . to determine whether these three gene signatures were mutually interdependent , they were evaluated in six clinical cohorts : the shedden cohort , gse8894 , gse31210 , gse20853 , gse13213 , and gse11969 . 
these results showed that the three gene signatures were mutually interdependent and possessed higher activities in necrosis - positive than necrosis - negative operable lung adenocarcinomas . three gene signatures stratify patients with operable lung adenocarcinoma into different risk groups we next investigated whether the three interdependent gene signatures correlated with overall or relapse - free survival in patients with operable lung adenocarcinoma . 
five data sets , the shedden cohort of michigan , gse8894 of korea , gse11969 , gse31210 , and gse13213 of japan , were included in univariable cox regression analysis . 
for every gene signature selected , the signature activity was positively associated with shorter overall ( the shedden cohort , gse11969 , and gse13213 ) or relapse - free ( gse8894 and gse31210 ) survival in almost all study cohorts ( data supplement )  . 
these cohorts , the shedden cohort ( n = 442 ) and the gse11969 , gse31210 , and gse13213 cohorts ( total n = 453 ) , included a total of 895 patients from both western and asian populations . 
the only exception was the hypoxia signature , with which the association did not reach statistical significance when it was applied to the gse11969 ( p = .31 ) and gse13213 ( p = .09 ) cohorts ( table 1 )  . 
 however , the unit - weighted composite score derived from the hypoxic and mtor signatures predicted overall and relapse - free survival ( all p < .05 ) in all cohorts ( data supplement )  . in addition , the predictive power of these three gene signatures was independent of patient age and tumor stage , with gene signature tending to be a stronger predictor of overall survival than clinical stage ( table 1 )  . 
in the shedden cohort , for example , the three gene signatures stratified adenocarcinoma patients into two groups with different overall survival times on the basis of kaplan - meier survival analyses ( fig 2a )  . 
high signature activity was defined when the signature score was weighted in the upper half of the distribution , whereas low signature activity was defined when the signature score was weighted in the lower half of the distribution . 
stratification of stage ii and stage iii patients by signature activities showed that the survival curve of patients with stage ii and high activity almost overlapped with that of patients with stage iii and low activity . 
taken together , the results showed that the three gene signatures were significantly associated with overall and relapse - free survival in patients with operable lung adenocarcinoma , with predictive powers independent of and somewhat stronger than clinical stage . three gene signatures predict disease relapse in patients with stage i lung adenocarcinoma to determine whether the three gene signatures were associated with overall and relapsefree survival in patients with stage i disease , patients with stage i disease in the gse13213 cohort were examined . 
to further determine whether these gene signatures predicted relapse - free survival , the okayama cohort ( gse31210 ) , which includes 162 patients with stage i adenocarcinoma and information on clinical stage , age , and relapse - free survival , was evaluated by multivariable cox regression analysis . 
significant associations between signature scores and relapse - free survival time were observed , with similar results observed with all three gene signatures ( data supplement ; fig 3a )  . three gene signatures are indicative of pathologic tumor necrosis , which predicts disease relapse in patients with stage i lung adenocarcinoma a gene signature is composed of gene sets that react to biologic stimulation and is more representative of a biologic function than a single gene . 
the results showed that the signature score correlated with the expression level of the representative protein ( cell cycle versus cyclin b1 , hypoxia versus pai1 , and mtor versus mtor - ps2448 ) , which implicated the activity of each pathway ( data supplement ) .28 , 29 the biologic meanings of these signatures were found to converge into one pathologic feature , tumor necrosis . 
in the japanese gse11969 cohort , tumor necrosis was associated with shorter overall survival and higher signature activities . these findings were independently validated using the tcga lung adenocarcinoma data set . 
the three gene signatures were mutually interdependent and associated with overall and relapse - free survival in patients with operable and stage i lung adenocarcinomas ( data supplement )  . 
 ( a ) kaplan - meier survival analysis showing that tumor necrosis was associated with poor overall survival in the gse11969 cohort ( log - rank p = .04 , right upper panel )  . 
 ( b ) kaplan - meier analysis showing that stratification of patients with stage ii and stage iii disease by signature activities resulted in the survival curve of patients with stage ii disease with high activity almost overlapping with that of patients with stage iii disease with low activity . 
mtor , mammalian target of rapamycin . between mtor signature activity and relapsefree survival in patients with stage i disease , in which the differences did not reach statistical significance ( data supplement )  . compatible with what was observed in the gse11969 cohort , tumor necrosis was associated with higher signature activities ( p < .001 ; fig 3b ) and shorter overall as well as relapsefree survival ( p < .05 ; fig 3c )  . 
although signature activities were associated with the presence of necrosis , the signatures remained prognostic , even in patients with necrosis - negative tumors , indicating the prognostic value of the gene signatures by themselves ( data supplement )  . 
in addition , because the three gene signatures were interdependent and associated with the presence of necrosis , we further included a proliferative marker , the cyclin b1 , as a surrogate for ki - 67 in our multivariable cox regression analyses with the tcga and tcpa data sets . 
the independent validation results supported the role of tumor necrosis as the pathologic sum of the three gene signatures combined . three gene signatures are not interdependent or predictive of risk in lung squamous cell carcinoma adenocarcinoma and squamous cell carcinoma are both categorized as nsclcs and share the same tnm classification criteria . 
 ( c ) tumor necrosis predicts overall and relapse - free survival in patients with operable and stage i lung adenocarcinoma of tcga lung adenocarcinoma cohort . study showed that tumor size matters differently in these two cancer types.30 we therefore evaluated whether the three gene signatures play a role in squamous cell carcinoma similar to that in adenocarcinoma . 
assessment of four cohorts , the gse11969 , gse4573 , gse8894 , and tcga data sets , showed that the three gene signatures were no longer interdependent , with the only correlation between the cell - cycle and mtor signatures ( fig 4a )  . 
 ( c ) inability of the three gene signatures to predict overall survival in patients with lung squamous cell carcinoma . the three gene signatures were unable to stratify patients with squamous cell carcinoma into different risk groups in regard to overall survival ( fig 4c )  . 
these findings provide further evidence that lung adenocarcinoma and squamous cell carcinoma are two distinct disease entities with different biologic behavior . discussion proper risk stratification in patients with stage i lung adenocarcinoma is important for postoperative treatment strategy decisions , because it may influence the long - term outcome of patients . 
 in patients with stage ib disease with tumors > 4 cm.8 on the basis of these two studies , t stage seems to be the only available risk stratification parameter for stage i disease . ironically , approximately 30% to 50% of patients with stage i disease died as a result of disease relapse within 5 years after potential curative surgery.6 , 32 , 33 this fact indicates that a substantial percentage of stage i tumors are more aggressive than thought , which is compatible with the results obtained from the uracil - tegafur and calgb 9633 trials.8 , 31 the issue of how we can identify these high - risk individuals , for whom postoperative adjuvant therapy will do good more than bad , remains . 
genomically , the cell - cycle , hypoxia , and mtor signatures were found to be mutually interdependent , more active in necrosis - positive than necrosis negative tumors , and associated with overall and relapse - free survival independent of clinical stage . 
in contrast , the three gene signatures were no longer mutually interdependent and lost their risk stratification power in patients with lung squamous cell carcinoma . although genome - wide approaches have been used for risk stratification of biomarkers , the comprehensive process of gene signature derivation limits their clinical application . 
therefore , translation from sophisticated cancer genomics to corresponding pathologic features that can be applied in routine clinical practice would be especially important . although limited by its retrospective nature , this study showed that gene signatures could be translated into a pathologic feature . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . konan peck no relationship to disclose takashi takahashi no relationship to disclose pan - chyr yang honoraria : astrazeneca , novartis , merck , ono pharmaceutical speakers ' bureau : astrazeneca , roche , novartis , merck , ono pharmaceutical acknowledgment the authors thank the laboratory of takashi takahashi , md , phd , for preparing the information needed in analyzing data sets gse11969 and gse13213 . 
lozano r , naghavi m , foreman k , et al : global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010 : a systematic analysis for the global burden of disease study 2010 . 
morgensztern d , ng sh , gao f , et al : trends in stage distribution for patients with non - small cell lung cancer : a national cancer database survey . 
travis wd , brambilla e , noguchi m , et al : international association for the study of lung cancer / american thoracic society / european respiratory society international multidisciplinary classification of lung adenocarcinoma . 
goldstraw p , chansky k , crowley j , et al : the iaslc lung cancer staging project : proposals for revision of the tnm stage groupings in the forthcoming ( eighth ) edition of the tnm classification for lung cancer . 
pisters km , evans wk , azzoli cg , et al : cancer care ontario and american society of clinical oncology adjuvant chemotherapy and adjuvant radiation therapy for stages i - iiia resectable non small - cell lung cancer guideline . 
crin l , weder w , van meerbeeck j , et al : early stage and locally advanced ( non - metastatic ) non - small - cell lung cancer : esmo clinical practice guidelines for diagnosis , treatment and follow - up . 
shedden k , taylor jm , enkemann sa , et al : gene expression - based survival prediction in lung adenocarcinoma : a multi - site , blinded validation study . 
yoshizawa a , motoi n , riely gj , et al : impact of proposed iaslc / ats / ers classification of lung adenocarcinoma : prognostic subgroups and implications for further revision of staging based on analysis of 514 stage i cases . 
duhig ee , dettrick a , godbolt db , et al : mitosis trumps t stage and proposed international association for the study of lung cancer / american thoracic / european respiratory society classification for prognostic value in resected stage 1 lung adenocarcinoma . 
wu yt , tan hl , huang q , et al : activation of the pi3k - akt - mtor signaling pathway promotes necrotic cell death via suppression of autophagy . 
benita y , kikuchi h , smith ad , et al : an integrative genomics approach identifies hypoxia inducible factor - 1 ( hif - 1 ) - target genes that form the core response to hypoxia . 
ebi h , tomida s , takeuchi t , et al : relationship of deregulated signaling converging onto mtor with prognosis and classification of lung adenocarcinoma shown by two independent in silico analyses . 
liao h , hyman mc , lawrence da , et al : molecular regulation of the pai - 1 gene by hypoxia : contributions of egr - 1 , hif - 1alpha , and c / ebpalpha . 
sawabata n , asamura h , goya t , et al : japanese lung cancer registry study : first prospective enrollment of a large number of surgical and nonsurgical cases in 2002 . 
 multigene hereditary cancer panels reveal high - risk pancreatic cancer susceptibility genes purpose the relevance of inherited pathogenic mutations in cancer predisposition genes in pancreatic cancer is not well understood . 
we aimed to assess the characteristics of patients with pancreatic cancer referred for hereditary cancer genetic testing and to estimate the risk of pancreatic cancer associated with mutations in panel - based cancer predisposition genes in this high - risk population . methods patients with pancreatic cancer ( n = 1 , 652 ) were identified from a 140 , 000 patient cohort undergoing multigene panel testing of predisposition genes between march 2012 and june 2016 . 
gene - level mutation frequencies relative to exome aggregation consortium and genome aggregation database reference controls were assessed . results the frequency of germline cancer predisposition gene mutations among patients with pancreatic cancer was 20.73%. 
mutations in atm , brca2 , cdkn2a , msh2 , msh6 , palb2 , and tp53 were associated with high pancreatic cancer risk ( odds ratio , > 5 ) , and mutations in brca1 were associated with moderate risk ( odds ratio , > 2 )  . 
in a logistic regression model adjusted for age at diagnosis and family history of cancer , atm and brca2 mutations were associated with personal history of breast or pancreatic cancer , whereas palb2 mutations were associated with family history of breast or pancreatic cancer . conclusion these findings provide insight into the spectrum of mutations expected in patients with pancreatic cancer referred for cancer predisposition testing . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction pancreatic cancer ( pc ) is the fourth most common cause of death resulting from cancer in the united states.1 epidemiologic studies have suggested that 10% to 20% of pcs are associated with an inherited component , with familial pc , defined as kindreds containing at least two affected first - degree relatives , as an established entity of inherited disease.2 pc is a component of hereditary breast - ovarian cancer syndrome , 3 , 4 lynch syndrome , 5 , 6 familial adenomatous polyposis , 7 familial atypical multiple mole melanoma syndrome , 8 hereditary pancreatitis , 9 peutz - jeghers syndrome , 10 and li - fraumeni syndrome.11 recent studies involving familial pc kindreds have further characterized the role of brca1 / 2 , cdkn2a , atm , and palb2 in pc susceptibility.12 - 14 until recently , germline studies of pcs have focused on single cancer predisposition genes.15 , 16 the first panel - based study of 13 cancer predisposition genes among patients with pc identified 11 mutations ( 3.8% ) in atm , brca1 / 2 , mlh1 , msh2 , msh6 , and tp53 , 17 whereas a 22 - gene panelbased study identified atm , brca1 / 2 , chek2 , and palb2 mutations in 13% of 96 sequentially collected pcs.18 a majority of these mutations were identified in pcs with a family history of pancreatic , breast , ovarian , or colorectal cancer , suggesting that a better understanding of pc risk will depend on evaluation of families with broad constellations of tumors.18 more recently , panel - based approaches identified germline chunling hu holly laduca hermela shimelis eric c . 
 mutations in 4% ( 33 of 854 ) of patients with apparently sporadic pc19 and in 25% ( 44 of 176 ) of patients with advanced pc.20 here , we report results from panel - based clinical testing of 1 , 652 patients with pc from a large cohort of > 140 , 000 patients evaluated by a single diagnostic laboratory and calculate gene - specific risks of pc by comparison with exome aggregation consortium ( exac ) and genome aggregation database ( gnomad ) reference controls.21 , 22 methods study population patients with pc ( n = 1 , 819 ) were identified from a large cohort of > 140 , 000 patients undergoing multigene panel testing of seven to 49 cancer predisposition genes between march 2012 and june 2016 at ambry genetics23 ( aliso viejo , ca ; appendix table a1 )  . 
exclusion criteria , including the presence of neuroendocrine tumors or intraductal papillary mucinous neoplasms , reduced the number of patients for analysis ( n = 1 , 652 ; appendix )  . 
 the study was approved by the western institutional review board . multigene panel testing mutation testing was performed by sequencing of targeted custom capture products from several multigene panels and targeted chromosomal microarray analysis , as previously described.24 genomic dna was isolated from each patients blood or saliva specimen using a standardized methodology ( qiagen , valencia , ca )  . 
sequence enrichment was performed by incorporating the genomic dna into microfluidics chip or microdroplets along with primer pairs or by a bait - capture methodology using long biotinylated oligonucleotide probes ( raindance technologies , billerica , ma ; integrated dna technologies , san diego , ca ) , followed by polymerase chain reaction and then next - generation sequencing analysis ( illumina , san diego , ca ) of all coding exons plus at least five bases into the 5 and 3 ends of all the introns and untranslated regions . 
all mutations identified by ambry genetics are submitted to the clinvar public database . statistical methods the observed frequency of all pathogenic mutations within each gene in white patients with pc was compared with the frequency of pathogenic mutations in the exac non - finnish european ( nfe ) nonthe cancer genome atlas ( tcga ) reference control after data cleaning and filtering ( appendix ) as previously described.23 copy number variants in all genes and mutations in pseudogene homology regions ( pms2 exons 9 and 11 to 15 ) were excluded from cases and controls for risk estimation , because these alterations were not individually validated in exac or gnomad controls . 
although these gnomad controls partially overlap with exac nfe non - tcga controls , the substantially increased number along with updated variant calling algorithms identified gnomad as an independent reference control data set . 
 ( % ) patients of all ethnicities ( n = 1 , 652 ) white patients ( n = 1 , 256 ) age at pc diagnosis were evaluated using the kolmogorov - smirnov test . 
 compared with a median age at pc diagnosis of 70 years in surveillance , epidemiology , and end results registries between 2010 and 2014 , 26 the median age at diagnosis was 63 years among patients with pc . 
among white patients with pc , 38.1% had a firstor second - degree relative with pc , and 48.8% had a family history of breast cancer ( table 1 )  . 
similar frequencies were observed for patients with pc of all races and ethnicities . pathogenic mutations among patients with pc the combined frequency of mutations in genes from all hereditary cancer testing panels was 20.73% for patients with pc of any race or ethnicity and 21.12% for white patients ( appendix table a2 )  . 
brca2 was the most frequently mutated predisposition gene ( 4.64% ) among patients diagnosed at age 63 years , and atm was most frequently mutated ( 4.03% ) in patients with pc diagnosed at age > 63 years ( appendix table a4 )  . 
association analyses using gnomad reference controls confirmed all significant associations , and gene - specific risk estimates were highly similar , except for slightly attenuated risk for palb2 mutations and increased risk for tp53 ( appendix table a6 )  . the same genes were associated with increased pc risk when considering patients of all races and ethnicities compared with exac all race and ethnicity controls ( appendix table a7 ) and after excluding those who had previously tested negative for brca1 / 2 mutations before panel testing ( appendix table a8 )  . 
risk estimates for most genes were slightly diminished when including only those patients with pc for whom pc was the first cancer diagnosis , although msh2 and tp53 mutations were no longer significantly associated with moderate risk of pc because of the decreased number of mutations in patients with pc , and the modest or associated with chek2 was marginally significant ( appendix table a9 )  . 
in contrast , analyses using only exac nfe non - tcga variants in the high - quality pass category marginally increased the ors for each gene ( appendix table a10 )  . 
mutations in atm , brca2 , and palb2 were also more frequent in patients with pc with a family history of breast cancer ( first or second - degree relative ; table 3 )  . 
in contrast , only palb2 and msh2 displayed a substantial increase in mutation frequency among patients with a family history of pc , and only chek2 , msh2 , and tp53 had increased frequencies of mutation among patients with pc with a family history of colorectal cancer ( table 3 )  . 
 family history of pc ( p = .029 ) or breast cancer ( p = .0056 ) and the association of chek2 mutations with family history of colorectal cancer ( p = .014 ; table 4 )  . performance of genetic testing criteria among mutation carriers consensus clinical genetic testing guidelines include pc as a component tumor for seven of the confirmed pc genes in this study ( brca1 / 2 , msh2 , msh6 , atm , palb2 , and cdkn2a ) .27 - 29 clinical histories of patients with mutations in these genes were evaluated to determine whether the respective genetic testing criteria were met ( table 5 )  . 
although a majority of brca1 / 2 and all msh2 mutation carriers displayed histories consistent with testing criteria , 50.0% of atm , cdkn2a , palb2 , and msh6 carriers met criteria . 
in addition , no cdkn2a families met diagnostic criteria for familial atypical multiple mole melanoma syndrome , 30 and 38.9% ( seven of 18 ) did not report any personal or family history of melanoma . discussion here we report a study of cancer predisposition gene mutations among patients with pc on the basis of a cohort of individuals undergoing hereditary cancer multigene panel testing from a single clinical laboratory . 
results from case control studies of the pc cases and exac reference controls identified six genes associated with high risk ( or , > 5 ) of pc ( atm , brca2 , cdkn2a , msh6 , palb2 , and tp53 ) , consistent with previous smaller studies and segregation studies from pc families . 
msh2 was also associated with a high risk of pc ; however , additional studies are needed to confirm these findings , because this association was based on a limited number of mutations detected among pc cases . 
 here we show that brca1 mutations are associated with a moderate risk ( or , > 2 ) of pc , even in a series of sensitivity analyses accounting for potential modifying effects of other cancers . 
 chek2 mutations were also associated with a moderate risk of pc ; however , this association was either diminished ( or , < 2 ) or nonsignificant in several sensitivity analyses . 
one likely explanation is that stk11 mutations are unlikely to occur in the absence of pathognomonic clinical characteristics associated with peutz - jeghers syndrome , and therefore , patients with suspected peutz - jeghers syndrome may be referred for single - gene testing more often than multigene testing . 
pathogenic mutations in other panel genes were still sufficiently uncommon to allow assessment of associations with risk ( eg , apc , mlh1 )  . the risk estimates for pc associated with each of these established predisposition genes will help improve clinical pc risk assessment . 
for some genes , these results offer more precise estimates than previously reported , whereas for others , such as palb2 and atm , we are the first to characterize the level of risk , to our knowledge . 
 it should be noted that the interpretation of the risks reported here is specific to patients referred for hereditary cancer genetic testing based on a personal or family history of cancer ( at least one diagnosis of pc in the family ) , and thus , these data may not be applicable to the general population or unselected pc cohorts . 
despite the enrichment for cases with personal or family history of cancer , these risks are derived from a broader clinical cancer testing cohort compared with previous studies selected for classic syndromic phenotypes such as fammm and therefore demonstrate that pc risk from syndromic genes remains high across a range of clinical histories . 
furthermore , this enrichment presented an opportunity to explore predictors of germline mutations . in total , 13% of patients had mutations in genes significantly associated with increased risk for pc across a range of sensitivity analyses ( atm , brca1 , brca2 , cdkn2a , msh6 , palb2 , and tp53 )  . 
family history of breast , pancreatic , or colorectal cancer was a significant predictor of positive results , suggesting that histories of these cancers should specifically be considered as genetic testing guidelines evolve for pc . 
the remaining 9% ( 15 of 173 ) of mutations were found in the approximately 65% of patients with pc without a family history of these cancers , suggesting a mutation rate of only 2.1% in white patients with pc without a family history of cancer ( 15 mutations in 698 ) in the clinically tested cohort . 
additional studies of population - based series of patients with pc are needed to determine whether clinical panel testing should be considered for patients with pc unselected for family history . in practice , patients with pc may not benefit directly from genetic testing because of the high mortality rate for this cancer . 
however , knowledge of mutation status for genes such as brca1 / 2 and palb2 with respect to clinical trial eligibility for targeted agents such as poly ( adp - ribose ) polymerase inhibitors may make genetic testing more appealing . 
in addition , mutation - positive family members can significantly benefit from knowledge of increased risk for a variety of cancers , including pc , and mutation - negative family members can also adjust their cancer screening protocols accordingly . 
in addition , the international cancer of the pancreas screening consortium and the american college of gastroenterology 29 , 31 recommend that pc surveillance , including annual endoscopic ultrasound and / or magnetic resonance imaging , be considered for individuals with > 5% lifetime or relative risk for pc . 
in addition , although brca1 mutation carriers with a firstor second - degree relative with pc are included in the list of patients for whom pc screening should be considered , the moderate pc risk categorization for brca1 in this study suggests this may not be clinically indicated . exac nfe non - tcga controls were used in this study because of the lack of a large series of matched controls . 
although the use of large reference data sets is not ideal , the large sample size allows precise estimation of the frequency of mutations in individuals without cancer and is likely reflective of the general population . 
in addition , we applied many data cleaning steps and used consistent criteria for selection of mutations in the clinical cohort of patients with pc and the exac controls to ensure that the data sets were adequately normalized for case - control association analyses . 
in a recent assessment of the quality of clinical history information for patients undergoing hereditary cancer panel testing , pedigrees and / or clinic notes were provided for 46% of randomly selected patient cases ( unpublished data )  . 
 when compared with pedigrees and clinic notes , a vast majority of proband cancers were reported completely ( 95% ) and accurately ( > 99% ) on test requisition forms . 
among family members , 76% of melanomas and > 80% of breast , ovarian , colorectal , endometrial , and pancreatic cancers were reported with 98% accuracy on test requisition forms . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . stock and other ownership interests : ambry genetics chunling hu no relationship to disclose holly laduca employment : ambry genetics hermela shimelis no relationship to disclose eric c . 
lee no relationship to disclose brigette tippin davis employment : ambry genetics stock and other ownership interests : ambry genetics research funding : ambry genetics ( inst ) travel , accommodations , expenses : ambry genetics mary helen black employment : ambry genetics research funding : ambry genetics travel , accommodations , expenses : ambry genetics tina pesaran employment : ambry genetics stock and other ownership interests : ambry genetics travel , accommodations , expenses : ambry genetics david e . 
rahib l , smith bd , aizenberg r , et al : projecting cancer incidence and deaths to 2030 : the unexpected burden of thyroid , liver , and pancreas cancers in the united states . 
risch ha , mclaughlin jr , cole de , et al : population brca1 and brca2 mutation frequencies and cancer penetrances : a kin - cohort study in ontario , canada . 
ruijs mw , verhoef s , rookus ma , et al : tp53 germline mutation testing in 180 families suspected of li - fraumeni syndrome : mutation detection rate and relative frequency of cancers in different familial phenotypes . 
mandelker d , zhang l , kemel y , et al : mutation detection in patients with advanced cancer by universal sequencing of cancer - related genes in tumor and normal dna vs guideline - based germline testing . 
laduca h , stuenkel aj , dolinsky js , et al : utilization of multigene panels in hereditary cancer predisposition testing : analysis of more than 2 , 000 patients . 
canto mi , harinck f , hruban rh , et al : international cancer of the pancreas screening ( caps ) consortium summit on the management of patients with increased risk for familial pancreatic cancer . 
 appendix patient cases of pancreatic cancer a total of 1 , 819 patients with pancreatic cancer were identified in a cohort of 140 , 449 individuals undergoing clinical germline cancer panel testing between march 2012 and june 2016 at a clinical testing laboratory ( ambry genetics , aliso viejo , ca )  . 
all variants classified by ambry were submitted to clinvar . exome aggregation consortium reference controls the exome aggregation consortium ( exac ) contains exome sequence data from 60 , 706 unrelated individuals sequenced as part of various disease - specific and population genetic studies . 
exclusion of sequence data from these patient cases yielded exac non - nfe non - tcga reference controls . genome aggregation database reference controls the genome aggregation database ( gnomad ) contains sequencing data of 123 , 136 exomes and 15 , 496 genomes from unrelated individuals sequenced as part of various disease - specific and population genetic studies . 
 gnomad data cleaning and filtering restricted to gnomad nfe exome data combined with gnomad ashkenazi jewish exome data . genomics viewer and by frequency in control data from dbsnp . pathogenic variant classification rules : same as in exac rules 1 to 8 . review variants with allele count 15 by integrative allele number was calculated as average of all variants within the coding region of a gene of interest . 
we previously reported our prospective experience of precision oncology in solid tumors , and here we report our longitudinal experience , focusing on therapeutic impact . patients and methods we conducted a retrospective review of 600 consecutive patients seen at cleveland clinic from 2013 to 2016 for treatment of incurable solid tumor malignancies for whom tumor genomic profiling was ordered using foundationone ( cambridge , ma )  . 
 after results , 313 patients ( 52.2% ) started any subsequent therapy ; of these , 95 ( 30% ; 15.8% overall ) received genomics - driven therapy ( g ) , and 218 ( 70% ) received non genomics - driven treatment ( ng )  . 
for the g versus ng group , the on - label , off - label , and clinical trial therapy breakdowns were 23% versus 88% , 47% versus 3% , and 30% versus 9% , respectively . 
more patients received treatment via clinical trials in the g cohort , and although not statistically significant , there was a trend toward increased os in the g ( v ng ) group . 
2018 by american society of clinical oncology introduction the sequencing of the human genome in the early 21st century ushered in the era of precision oncology , an era of tailoring anticancer treatments to individual patients by targeting the specific molecular alterations in their tumors.1 , 2 notable examples include pembrolizumab for microsatellite instability ( msi ) high tumors , vemurafenib for braf v600e alterations in melanoma , and olaparib for brca - positive ovarian cancer . 
however , clinical data supporting precision oncology in its broadest sense ( ie , applying these methods across a variety of solid tumors in large patient cohorts ) remain sparse . 
obstacles to generating real - world applications in precision oncology include small study populations , unclear distribution of biomarkers in various subsets , ethical considerations or treatment crossover , cost and availability of resources , and limitations in external validity . 
delays in accessing new treatments for our patients have also been a dilemma , frequently leading to off - label prescribing or use on compassionate grounds , which feed into the lack of available data . 
the study did not receive any external funding . patients and procedures all consecutive adult patients with a diagnosis of an incurable solid tumor malignancy who were seen at the cleveland clinic between january 1 , 2013 , and december 31 , 2016 , and underwent tumor genomic profiling were eligible for inclusion in this study . 
 briefly , this assay is capable of detecting any of the four classes of alterations in > 300 genes , including > 25 introns that are commonly altered in solid tumor malignancies . 
the assay has > 99% specificity , and results include msi and tumor mutational burden.4 all patients undergoing such testing are presented at our biweekly , multidisciplinary genomics tumor board ( gtb ) , composed of clinical oncologists , genetic counselors , pathologists , and patient care coordinators . 
next - generation sequencing ( ngs ) reports for each patient were reviewed comprehensively , and therapeutic treatment recommendations , made on the basis of reviewed literature , abstract presentations , and clinical trial availability , were discussed . 
all recommendations were documented in the electronic medical record system and were communicated directly to the treating oncologist . criteria for actionability of genomic alterations gtb considered an alteration to be actionable if one of the following criteria was met : ( 1 ) the target had a food and drug administration ( fda ) approved drug available for the specific tumor type ( on - label use ) , ( 2 ) the target had a drug approved by the fda for a different tumor type ( off - label use ) , or ( 3 ) the target had a drug available via clinical trial ( available at either the home institution or other institutions )  . 
when outside clinical trials were recommended , they were generally within approximately 200 miles of the patients primary residence . germline mutations predisposing patients to hereditary cancer syndromes and standardof - care alterations , such as her2 amplification , braf , alk fusions , or egfr mutations identified previously through routine testing for currently approved indications , were not considered to be actionable . 
however , if standard - of - care alterations had not been tested previously or the results of such testing performed at outside institutions were not available , then the gtb made appropriate on - label recommendations for such findings . study measurements clinical and pathologic information was acquired from electronic medical record review . 
demographics ( age , sex , ethnicity ) , date of diagnosis , location of primary malignancy , treatment history , gtb recommendations made on the basis of ngs results , treatment received after gtb recommendations were made , reason for non genomics - based treatment ( if known ) , date of last contact , and date of death ( if applicable ) were all collected . 
all data were entered into a secure electronic database for analysis . statistical analysis study data were collected and managed using redcap ( research electronic data capture ) tools hosted at cleveland clinic . 
overall survival ( os ) was calculated as time from date of gtb discussion of the case to either date of death or date of last contact ( the latter cases were censored for os calculation )  . 
 ( % ) unless indicated otherwise . 79% of patients had received two or more prior lines of therapy . on the basis of sequencing results , targeted therapy was recommended in 310 patients ( 51.7% ) , 95 of whom received more than one recommendation . 
of all gtb recommendations ( n = 405 ) , 32 ( 7.9% ) were for on - label treatment , 42 ( 10.4% ) were for off - label treatment , and 331 ( 81.7% ) were for clinical trials . 
for genomics - driven versus nongenomics - driven groups , the distributions of on - label , off - label , and clinical trial therapy received were 23% versus 88% , 47% versus 3% , and 30% versus 9% , respectively . 
the use of genomics - driven treat ment increased from 13% ( 29 of 223 ) in the first cohort ( reported earlier ) 3 to 18% ( 66 of 377 ) in the subsequent cohort . 
of note , of the 33 patients with cancer of unknown primary , the gtb was able to determine the primary origin in eight of these cases : three lung , two cholangiocarcinoma , and one each of colorectal , gastric , and pancreas . discussion this study represents one of the largest cohorts of patients with multiple types of incurable solid - tumor malignancies who had ngs performed to determine therapeutic options . 
 awareness and use of precision medicine ; and ( 5 ) although not statistically significant , there was a trend toward longer os in the genomics - driven versus nongenomics - driven treatment group ; however , this finding may also be related to unmeasured confounders . a review of the current literature indicated that a few other studies at various institutions have analyzed actionability and the percentage of patients who receive genomics - driven therapy . 
one such study was conducted at johns hopkins with 155 patients ; 85% of these patients received a treatment recommendation , and 24 ( 15% ) went on to receive genomics - driven therapy.5 although , in comparison , 51.7% of our cohort received recommendations , it is important to keep in mind our exclusion , in many cases , of germline mutations and alterations known to exist from standard - of - care testing in approved indications ( such as her2 in breast cancer )  . 
as in our findings , the researchers at johns hopkins commented on poor performance status being a limiting factor to clinical trial enrollment in addition to the unavailability of local trials . 
the md anderson cancer center reported on 2 , 000 patients who underwent ngs , of whom 789 ( 39% ) were found to have an actionable alteration and 83 ( 4.1% ) enrolled in genomics - driven trials.6 another study , of 1 , 932 patients with ngs testing , found that management was altered in 21% of patients ( 331 of 1 , 593 ) in need of a treatment change , as reported by the treating physicians.7 although our cohort was smaller , we were still able to find an increased percentage of patients who were provided with gtb recommendations and a similar , if not also increased , percentage of those receiving genomics - driven treatment . it is also important to note that the benchmark of precision oncology ( actionability ) is a fluid concept itself.8 it depends on the availability of clinical trials , locally , regionally , and nationally . 
 data on molecular markers and targeted therapy emerge rapidly , requiring up - to - date searches and algorithms to keep up with the literature.9 as more evidence accumulates and drugs receive regulatory approval , off - label use may shift to on label . 
for example , our data set predates the blanket approval of pembrolizumab for msi high or mismatch repair deficient tumors ; msi results on ngs are now increasingly being used to determine the use of immune checkpoint inhibitors , which is likely to increase the proportion of patients receiving genomics - driven therapy . 
 similarly , after additional evidence , other agents such as braf and alk inhibitors may move from the off - label to the on - label arena in additional tumor types over time . 
although the reasons are beyond the scope of this discussion , it leads to more on - label recommendations and has contributed somewhat to increased actionability over time in our cohort as well . an interesting point to note is the large cohort of patients not receiving genomics - driven therapy because of physician preference . 
of the cases found to have actionable alterations by expert curators , physicians were able to identify actionable alterations in 81% ( 362 of 445 ) .7 this finding showed that clinicians have substantial awareness of actionability . 
 the os benefit of genomics - driven therapy has been evaluated only in a limited number of studies , the largest being the impact ( initiative for molecular profiling and advanced cancer therapy ) study.12 this retrospective analysis included a cohort of 1 , 436 patients , of whom 637 ( 44.4% ) were found to have one or more clinically actionable alterations . 
the impact trial researchers used their institutions in - house ngs ; however , recommendations for treatment were not reviewed individually at a multidisciplinary gtb , as they were in our case , making the definition we used for a clinically actionable alteration more stringent . 
 although the impact trial compared survival among patients who were all determined to have clinically actionable alterations , we compared survival between those who received genomics driven treatment and those who did not ( 54.1% of whom did not have a clinically actionable target or receive a gtb recommendation ) ; our approach was to evaluate the overall impact of precision oncology in all comers . 
the shiva trial was an open - label randomized controlled phase ii trial that spanned eight french hospitals to produce a cohort of 195 patients ; 99 were randomly assigned to the experimental group ( who received molecularly targeted therapy ) and 96 were randomly assigned to the control group ( who received treatment at physicians choice )  . 
of these , 22 patients received genomics - driven therapy and the other half received standard chemotherapy ( n = 17 ) or best supportive care ( n = 5 )  . 
although the treatment of every patient with cancer is certainly individualized , it is evident from the data that most patients get such testing performed only after they have progressed on multiple lines of treatment . 
in addition , given that trial ineligibility secondary to poor performance status is a deterrent to patients receiving genomics - driven therapy as recommended by the gtb , it could be suggested that patients would benefit from having ngs performed earlier in the course of their disease . it is notable that a number of different ngs platforms exist using diverse assays and gene panels , introducing a degree of variability in the field ; we report results that are based on a specific assay , which is now fda approved . 
relevant additional parameters to take into account and discuss with patients when ordering ngs testing include turnaround time , cost , and the possibility of identifying an incidental germline mutation . 
because the implications of the latter can be dire , pretest awareness and the need for genetic counseling for the patient and more broadly , for the family , before and after testing , is crucial.15 given that this was a retrospective study , we were reliant on medical record review to determine the reason behind treatment decisions ; this approach has inherent putative selection and confounding biases . 
although real - world analyses of the benefit of precision oncology are required , they are obviously limited by heterogeneity , with a mixture of histologies , prior therapies , various performance statuses , and a slew of different tumor - specific prognostic features . 
last but not least , the vast majority of patients in our cohort were white , highlighting the need to generate more data that include other racial and ethnic populations , which is actually aligned with the priorities of the national cancer institute regarding equity in cancer care delivery and health care disparities . 
in that regard , our group has launched a prospective study evaluating ngs testing in underserved populations . tumor genomic profiling influenced treatment in 15.8% of patients in this cohort , and its use has increased over time . 
more patients received treatment via clinical trials in the genomics driven treatment cohort , and although not statistically significant , there was a trend toward longer os in the genomics - driven versus the nongenomics - driven treatment group . 
 sohal amy pritchard no relationship to disclose administrative support : amy pritchard provision of study material or patients : all authors collection and assembly of data : all authors data analysis and interpretation : meena sadaps , davendra p.s. 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . meena sadaps no relationship to disclose pauline funchain consulting or advisory role : eisai haider mahdi no relationship to disclose petros grivas consulting or advisory role : genentech , dendreon , exelixis , merck , bristol - myers squibb , astrazeneca , biocept , clovis oncology , emd serono , seattle genetics , foundation medicine , driver speakers ' bureau : genentech , bristol - myers squibb research funding : mirati therapeutics ( inst ) , genentech / roche ( inst ) , merck ( inst ) , oncogenex ( inst ) , bayer ag ( inst ) , pfizer ( inst ) , astrazeneca ( inst ) stefan klek no relationship to disclose bassam estfan no relationship to disclose jame abraham honoraria : pfizer , genentech / roche speakers ' bureau : pfizer g . 
thomas budd research funding : genentech / roche ( inst ) , eisai ( inst ) , cytrx corporation ( inst ) , tracon pharma ( inst ) , threshold pharmaceuticals ( inst ) james p . 
pennell consulting or advisory role : astrazeneca , eli lilly , regeneron , cota healthcare research funding : genentech ( inst ) , celgene ( inst ) , astrazeneca ( inst ) , pfizer ( inst ) , merck alok a . 
khorana honoraria : janssen pharmaceuticals , halozyme , pfizer , bayer ag , angiodynamics , pharmacyte biotech consulting or advisory role : sanofi , janssen , halozyme , bayer ag , pfizer , pharmacyte biotech research funding : amgen ( inst ) travel , accommodations , expenses : janssen , pfizer , bayer ag brian j . 
sohal honoraria : foundation medicine consulting or advisory role : perthera research funding : novartis ( inst ) , celgene ( inst ) , oncomed ( inst ) , bayer ag ( inst ) , genentech ( inst ) , bristolmyers squibb ( inst ) travel , accommodations , expenses : foundation medicine affiliations meena sadaps , pauline funchain , haider mahdi , amy pritchard , stefan klek , bassam estfan , jame abraham , g . 
tsimberidou a - m , hong ds , ye y , et al : initiative for molecular profiling and advanced cancer therapy ( impact ) : an md anderson precision medicine study . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , openlabel , proof - of - concept , randomised , controlled phase 2 trial . 
genomics - driven therapy received after gtb recommendations ( n = 95 ) ( continued ) histology drug therapy type target os ( months ) pancreatobiliary idh1 inhibitor clinical trial idh1 prostate prostate prostate prostate prostate renal sarcoma sarcoma temsirolimus olaparib everolimus + bicalutamide trastuzumab olaparib + bicalutamide cabozantinib palbociclib sunitinib unknown primary lenvatinib unknown primary trastuzumab + folfox unknown primary idh1 inhibitor unknown primary trastuzumab + pertuzumab unknown primary everolimus unknown primary trastuzumab abbreviations : gtb , genomics tumor board ; os , overall survival . 
the latest innovations in clinical trials have followed with master protocols , which are dened by inclusive eligibility criteria and devised to interrogate multiple therapies for a given tumor histology and / or multiple histologies for a given therapy under one protocol . 
conventionally conducted within the phase ii setting , basket designs have become popular as drug developers seek to effectively evaluate and identify preliminary efcacy signals among clinical indications identied as promising in preclinical study . 
the latest innovations in clinical trials have followed with master protocols , which are dened by inclusive eligibility criteria and devised to interrogate multiple therapies for a given tumor histology and / or multiple histologies for a given therapy under one protocol.1 , 2 the use of master protocols for oncology has become more common with the desire to improve the efciency of clinical research and accelerate overall drug development . 
the lung - map study , for example , used a master protocol for a phase ii / iii comparative trial designed to evaluate biomarkermatched therapies in patients with previously treated advanced squamous nonsmall - cell lung cancer.3 shortly after initiation , the us food and drug administration approved a new immunotherapy for the same population . 
the designs exibility facilitated subsequent modication of the standard of care , which preserved the relevance of the study.1 a second example includes the create study master protocol , which evaluated the efcacy of crizotinib in patients with alk or met mutations among six different tumor histologies , where each histology constituted a subtrial of alk / met - positive or alk / met - negative tumors.2 emerging cancer therapies may target relatively rare molecular subtypes , which poses practical challenges to completing a clinical trial devised to enroll a single clinical indication . 
second , biomarkerguided treatment selection supersedes traditional clinical indicators for the studied populations such that the presence of a molecular marker predicts response to a targeted therapy irrespective of tumor histology.2 , 4 , 5 thus , basket trials are designed to enroll multiple clinical subpopulations in whom it is assumed that the therapy in question offers benecial efcacy in the presence of the targeted molecular prole . 
most often conducted within the phase ii setting , basket designs have become popular as drug developers seek to effectively evaluate and identify preliminary efcacy signals among clinical indications identied as promising in preclinical study.6 - 8 in certain circumstances , however , evidence acquired from a basket trial has served as the basis for regulatory approval.9 , 10 the framework also poses new challenges to statistical analysis . 
 kaizer et al context key objective describe recent developments in the design of basket trials for oncology studies and elucidate several issues that pertain to their statistical analysis and design . knowledge generated basket trials have evolved from their initial use in exploratory contexts to conrmatory , randomized , and platform trials . 
the context of each trial requires consideration of statistical properties for trial design ( strength and type of type i error control and implications for power ) and methods for analysis ( to pool clinical indications with a common target or accommodate the potential for heterogeneous activity among tumor histologies )  . relevance advances in genomics , disease pathways , and drug discovery have progressed into clinical cancer research . 
innovations in trial design with basket trials facilitate interrogations of multiple therapies for a given tumor histology and / or multiple histologies for a given therapy - target combination that bridges aspects of translational and clinical research paradigms . 
molecularly targeted treatment strategies may not offer acceptable efcacy to all putatively promising clinical indications . proliferation rates of tumor cells are known to vary within and between tumor types.11 moreover , contemporary sequencing methodologies have revealed extensive genetic variation among different types of cancer . 
as drug developers pursue drug approvals with broad labels ( eg , pembrolizumab for which indication was expanded in 2017 to include any unresectable or metastatic solid tumors with microsatellite instability , 12 larotrectinib in 2018 for adults and children who harbor ntrk gene fusion without a known acquired resistance mutation13 ) , trials will evaluate multiple disease indications for agnostic effects . 
for these trials , subpopulation analysis becomes central to the study design and should be considered in the evaluation of trial operating characteristics . trials , discusses this article reviews designs of basket several issues that arise with their implementation , and examines the statistical criteria considered in the development of such trials . 
this section discusses examples of exploratory trials designed as basket trials and introduces recent extensions that convey the evolving landscape of basket trial design in oncology . exploratory designs most basket trials have been conducted within exploratory settings to evaluate agent - specic estimates of tumor response . 
cunanan et al5 described three studies implemented in oncology settings that extend the basic formulation of a basket trial to multiple targets and / or agent combinations . the nci - match trial used a master protocol to enroll participants into drugand mutation - specic baskets while also incorporating genetic screening . 
a basket trial approach was selected to accommodate the rare disease setting , which is limited by traditional large prospective trials.14 the custom trial was a phase ii study with a two - stage design that aimed to identify molecular biomarkers and determine their clinical relevance in patients expected to benet from multiple targeted therapies.15 these trials facilitated the study of less common types of malignancies . 
in addition , these studies were devised to identify potential baskets in which the respective therapies demonstrated promising efcacy and , thus , were exploratory . extensions to randomized , conrmatory designs the methodology for basket trials has been extended to accommodate a wide variety of potential motivations beyond exploratory studies . 
one such extension includes randomized design such that participants are randomly assigned to either a standard - of - care arm without regard to specic genetic mutations or to an arm where a specic mutation is hypothesized to be susceptible to targeted treatment.16 , 17 these designs test the hypothesis that biomarker - guided selection demonstrates benecial treatment efcacy . 
 basket designs : statistical considerations for oncology trials fusion of basket and platform trials may accommodate exploratory and conrmatory study.18 one example of this approach is the signature program , which included a series of eight agent - specic , open - label , phase ii basket trials under a single overarching master protocol.21 the signature program was tissue agnostic and driven by molecular indications with the intention to demonstrate the feasibility of rapid signal nding across multiple tumors . 
molecularly targeted treatment strategies , however , may not offer acceptable efcacy to all putatively promising clinical trials pose challenges to the traditional paradigm for trial design and analysis , which assumes that individual patients who enroll in the same clinical study represent exchangeable units that can be averaged . 
imatinib , for example , was shown to be efcacious in treating malignancies , such as philadelphia chromosomepositive chronic myelogenous leukemia , through the dysregulation of imatinib - sensitive protein tyrosine kinases.23 to determine whether other malignancies with similar kinases respond to treatment , a single - arm , open - label , phase ii study was conducted with inclusive enrollment across 40 different pathologic diagnoses.24 neither the number of indications was constrained nor the design devised to examine subpopulation - specic effects . 
the study represents an example of the challenges posed by indication heterogeneity with conventional study design ( eg , see lacombe et al25 ) , the prediction of which may not be feasible in advance of a studys implementation . the opposite end of the spectrum is dened by studies that evaluate the effectiveness of a treatment strategy for a given subpopulation independent of the evidence acquired from patients in differing patient subsets . 
subpopulation - specic inference also fails to delineate which patient subtypes should be considered nonexchangeable on the basis of the observed data . for example , upon establishing that melanomas that exhibit braf v600 mutations respond to vemurafenib , 26 a basket trial was designed to evaluate the effectiveness of vemurafenib in patients without melanoma , with baskets dened by primary tumor origthe study was devised to interrogate the effectiveness of vemurafenib independently among patients who harbor braf v600 mutations within several prespecied tumor organ sites.5 , 9 despite having designed the study with success criteria that were invariant by tumor indication , the studys analysis plan evaluated evidentiary measures of effectiveness independently for each basket . 
with some baskets having observed low accrual and others yielding one or no response , overall conclusions are difcult to ascertain.27 the polarities of independence and pooling require strong a priori assumptions , which yield poor statistical designs when violated . 
these include multistage designs that merge subtypes at interim analyses28 or bayesian adaptive designs with hierarchical modeling strategies.29 hobbs and landin30 adapted the multisource exchangeability model ( mem ) , 31 which facilitates bayesian inference with respect to all possible pairwise exchangeability relationships among the studied subpopulations . 
the design enables the identication of disjointed subpopulations that comprise meta - subtypes or singleton subtypes on the basis of accumulating evidence during the course of study . design criteria and operating characteristics a basket designs operating characteristics should be determined by the trials intention and phase of development . control of falsely concluding that an inferior experimental therapy offers benet to any clinical indication should be emphasized for conrmatory trials that enroll multiple indications . 
statistical designs of clinical studies are conventionally devised in consideration of distributions of test statistics that measure the extent of evidence acquired against a prespecied null hypothesis upon repeated sampling of the trial data for a given decision rule . 
for hypotheses of superiority , null hypotheses dene value ( s ) of statistical parameters for which the underlying true state of nature is characterized by the absence of treatment effectiveness . 
for one - sample the null often is represented by a xed , prestudies , specied value that represents an expected response rate considered inadequately low to justify additional investigation . 
the decision rule determines the extent to which the trials conclusions could be explained as a result of chance . in this context , a type i error occurs when the null hypothesis is true but the trial yields data for which the decision rule rejects the null hypothesis in favor of a positive nding . 
for basket trials designed to examine multiple indications , the possibility of committing a type i error may occur for tests of each subpopulation , yielding two types of false conclusions : marginal type i error rates estimate the type i error rate for each indication separately , whereas family - wise type i error rates consider the entire trial violation if a single indication falsely rejects the null hyrepresents stronger pothesis . 
when selecting a statistical design devised to enroll and evaluate multiple indications , trialists must synthesize the trials operating characteristics with respect to a diverse array of possible trial outcomes , which we denote hereafter as scenarios . 
the strongest control is therefore conferred by the scenario with only one null indication . decisions that pertain to statistical approaches for analysis of multiple subpopulations , expected enrollment distribution , type i error method , and the strength of type i error control have implications for a designs statistical power . 
in the absence of heterogeneity among indications , the pooling of data acquired from all indications has the potential to increase statistical power ( identify smaller effect sizes )  . 
in addition , increasing imbalance among subpopulation sample sizes may reduce power and / or inate bias under strategies that enable data pooling . hierarchical models devised to share information across indications in relation to estimates of statistical exchangeability have been proposed by various authors18 , 35 - 38 with the intention of increasing statistical power while controlling bias . 
hierarchical models were calibrated for strong control of type i error such that marginal type i error was controlled under scenarios for which only one ( of ve or 10 ) subpopulation was truly ineffective . 
it should be noted , however , that under scenarios with multiple null subpopulations , the designs that resulted from hierarchical models demonstrated both commensurate power and reduced type i error when compared with subpopulation - specic analyses . 
in some cases , drastic reductions in type i error were observed for hierarchical models , such as the global null scenario , where strong borrowing reduced the type i error rate from a target of 10% to approximately 1% when considering 10 indications.39 moreover , the authors failed to adjust frequentist tests for multiple comparisons , which would have attenuated their power . 
in the presence of limited or imbalanced enrollment , however , one may wish to combine subpopulations or use statistical methods to facilitate information sharing among patient subpopulations.5 , 30 guidelines for evaluating the operating characteristics of subpopulation analyses are needed for both conrmatory and nonconrmatory settings.33 , 34 to provide an illustration of the existing strategies and their trade - offs , a simulation study was completed for a xedsample basket trial with ve indications each enrolling 25 patients . 
type i error , however , inates quickly to unacceptable levels in the presence of basket heterogeneity . the independent analysis approach , which adjusts for multiplicity , maintains family - wise error rates , 5% , but it is at the expense of power ( only 0.66 probability of identifying any active basket as efcacious )  . 
finally , the analysis of each basket independently and failure to adjust for multiple comparisons yielded high power ( 0.91 ) with large familywise error rates for all scenarios except the one with four active baskets ( ie , one null basket )  . the method for calibrating basket results for the bayesian model are provided for three posterior probability decision thresholds to illustrate the exibility of trials of various types . 
calibration 1 maintains a marginal type i error at 10% with no active baskets as well as increased probability for identifying effective baskets relative to independent approaches with two or more active baskets . family - wise error and marginal type i error rates are inated with fewer active baskets , however . 
reported is the basket - specic probability of being declared efcacious for null ( nonbold ) and active ( bold ) baskets for multisource exchangeability models ( mems ) with different pp threshold calibrations . 
this stands in contrast multiplicity ( low power of 0.66 ) or do not adjust for multiplicity ( higher power at the expense of higher family - wise error rates of 40% )  . 
the three calibrations presented were selected to demonstrate acceptable designs for different types of basket trials , with calibrations 1 and 2 formulated for exploratory settings and calibration 3 appropriate for conrmatory trials . discussion subpopulation heterogeneity is intrinsic to evaluations of cancer therapies that emerge within the evolving oncology landscape . 
basket trials provide a natural framework for studying molecularly targeted treatment strategies in several potentially promising clinical subpopulations while acknowledging the possibility of heterogeneous results . initiated in exploratory settings , basket designs have evolved to include randomization , enrichment of the enrolled population , and incorporation to platform trials and even acquire conrmatory evidence . 
further innovations and renements may make it possible to use the framework across the full spectrum of clinical translation with seamless design.19 as our simulation study demonstrates , basket trials should require analysis strategies and designs that acknowledge the potential for subpopulation effects . 
several aspects of basket trials , including the recommendations for weak versus strong control of type i error and adjustment for multiplicity , require additional consideration and guidance from academic and regulatory leaders to promote best practices for each stage of the research process.33 as basket trials evolve from simple exploratory trialists trials to increasingly complex master protocols , should avoid simple replication of designs from prior study and instead adapt the trial to its context with appropriate assumptions for the phase of inquiry . our simulation study demonstrates that nave pooling of all subpopulations results in unacceptable type i error , whereas independent examination of each basket without correction for multiplicity may result in inated type i error or , conversely , decreased power to detect efcacious baskets when correcting for multiplicity . 
the mem was evaluated with three different posterior probability threshold calibrations , each demonstrating appropriate operating characteristics for basket trials devised for exploratory and conrmatory settings . further improving their accessibility is the availability of the basket package in r to implement the symmetric mem approach.41 these results stand in contrast to challenges and limitations to information sharing previously reported.34 , 39 one should note a few limitations , however , that remain unresolved by the basket trial framework . 
patients described by a common subpopulation may exhibit sources of heterogeneity on the basis of other important considerations , such as immune prole , prior treatment histology , informatics that redemographics , or additional clinical quire patient - level data analysis . 
for a single tumor histology , this can yield insufcient efcacy not because the drug is necessarily ineffective for the indication but because the subtype in the trial happened to enroll a disproportionate number of patients with refractory disease.27 in settings where indication heterogeneity is not only known but also well understood , an effective means for addressing this type of heterogeneity is to construct baskets derived from information contained in clinical and auxiliary data sources . 
given sufcient enrollment , an indication can be partitioned into prognostic subpopulations and thereby provide new avenues for delineating drug versus prognostic effects at the subpopulation level without randomization . basket trials , like many master protocols , require tissue analysis and molecular proling before registration , which often limits their scope to a small number of leading academic institutions . 
molecular match rates for trials of targeted therapies have been reported to be as low as 4%.42 a single - institution study of the msk - impact assay evaluated 1 , 932 patients and a span of 49 cancer types in the real - world setting and in a matched found that 13% of patients were enroll study.43 by facilitating a centralized platform for acquiring and processing genomic data in conjunction with their functional and therapeutic implications , basket trials may overcome such deciencies . 
this has prompted interrogations of electronic health recordderived measures of real - world progression as alternatives to the response evaluation criteria in solid tumors ( recist ) .44 with the emergence of data science and machine learning , pathologic and radiologic data may offer new avenues for rapid , noninvasive patient proling . 
innovations in radiomics , for example , have effectively yielded prognostic signatures from informatics acquired from scanning technologies used routinely in clinical settings.45 - 48 to address the potential for heterogeneity within a clinical indication , innovations that facilitate use of noninvasive biomedical informatics and algorithms should be pursued with the aim of developing prognostic models in concert with the trials design . 
koopmeiners patents , royalties , other intellectual property : listed as an inventor for us patent 9 , 858 , 665 , which describes a computer - aided design system for predicting prostate cancer using magnetic resonance imaging satrajit roychoudhury employment : pzer leadership : pzer stock and other ownership interests : pzer , novartis david s . 
hong stock and other ownership interests : molecularmatch , oncorena , presagia honoraria : adaptimmune , baxter , merrimack , bayer ag consulting or advisory role : baxter , bayer ag , guidepoint global , janssen pharmaceuticals , genentech , eisai , glg , alpha insights , axiom biotechnologies , adaptimmune , grouph , merrimack , medscape , numab , pzer , seattle genetics , takeda pharmaceuticals , trieza therapeutics research funding : novartis , genentech , eisai , astrazeneca , pzer , mirna therapeutics , amgen , daiichi sankyo , merck , mirati therapeutics , eli lilly , adaptimmune , abbvie , bayer ag , bristol - myers squibb , genmab , ignyta , innity pharmaceuticals , kite pharma , kyowa hakko kirin , loxo oncology , medimmune , molecular templates , takeda pharmaceuticals , seattle genetics , amgen , fate therapeutics , mologen , nci - ctep travel , accommodations , expenses : loxo oncology , mirna therapeutics , genmab brian p . 
n engl j med 377 : 62 - 70 , 2017 renfro l , sargent d : statistical controversies in clinical research : basket trials , umbrella trials , and other master protocols : a review and examples . 
ann oncol 28 : 34 - 43 , 2017 steuer ce , papadimitrakopoulou v , herbst rs , et al : innovative clinical trials : the lung - map study . 
clin pharmacol ther 97 : 488 - 491 , 2015 redig aj , j anne pa : basket trials and the evolution of clinical trial design in an era of genomic medicine . 
j clin oncol 33 : 975 - 977 , 2015 cunanan km , gonen m , shen r , et al : basket trials in oncology : a trade - off between complexity and efciency . 
nakamura y , komatsu y , kato k , et al : btmb - high basket trial : a multicenter phase ii trial of nivolumab monotherapy in patients with advanced gastrointestinal cancers with high blood tumor mutational burden ( btmb )  . 
j clin oncol 37 , 2019 ( suppl ; abstr tps179 ) diamond el , subbiah v , lockhart ac , et al : vemurafenib for braf v600 - mutant erdheim - chester disease and langerhans cell histiocytosis : analysis of data from the histology - independent , phase 2 , open - label ve - basket study . 
sch offski p , wozniak a , escudier b , et al : crizotinib achieves long - lasting disease control in advanced papillary renal - cell carcinoma type 1 patients with met mutations or amplication . 
lopez - chavez a , thomas a , rajan a , et al : molecular proling and targeted therapy for advanced thoracic malignancies : a biomarker - derived , multiarm , multihistology phase ii basket trial . 
chen c , li x , yuan s , et al : statistical design and considerations of a phase 3 basket trial for simultaneous investigation of multiple tumor types in one study . 
heinrich mc , joensuu h , demetri gd , et al : phase ii , open - label study evaluating the activity of imatinib in treating life - threatening malignancies known to be associated with imatinib - sensitive tyrosine kinases . 
freidlin b , korn el : borrowing information across subgroups in phase ii trials : is it useful ? clin cancer res 19 : 1326 - 1334 , 2013 40 . 
grifth sd , tucker m , bowser b , et al : generating real - world tumor burden endpoints from electronic health record data : comparison of recist , radiologyanchored , and clinician - anchored approaches for abstracting real - world progression in non - small cell lung cancer . 
yu w , tang c , hobbs bp , et al : development and validation of a predictive radiomics model for clinical outcomes in stage i non - small cell lung cancer . 
lacking multivariable specication of relationships among subpopulations , conventional analysis strategies represent the extremes of either pooling all indications or analyzing each indication independently of the evidence acquired for other indications . 
more specically , pooling in this context refers to an analysis strategy devised to evaluate evidence of effectiveness for all indications through statistical inference of a single response rate that is estimated using data from all indications . 
by relying on only one test , marginal and family - wise type i error rates are identical for the pooled approach . at the other extreme , independent analyses infer a total of ve response rates estimated from the data observed within specic indications . 
our simulation study considers the constrained empirical bayes ( ceb ) priors for symmetric mems described by hobbs and landin.30 briey , unconstrained empirical bayes approach to prior specication identies and conditions the analysis with respect to the mem that maximizes the marginal data likelihood . 
while acknowledging the uncertainty about identifying the true mem from the marginal likelihood , the ceb prior constrains shrinkage using an upper bound ( ub ) on the prior probability of inclusion for each source . 
for the basket trial with ve indications , there are six possible scenarios that range from all indications being null ( scenario 1 ) to all indications being alternative ( scenario 6 ) and four intermediate combinations ( scenarios 2 to 5 )  . 
for the bayesian mem approach with ub = 0.10 , posterior probability thresholds were identied that maintained the basket - specic ( marginal ) type i error rate under the global null scenario , the type i error rate for the scenario with one null basket , and the family - wise type i error rate for the global null scenario . 
the other two articles are case reports of breast and gastroesophageal cancer with mismatch repair deficiency ( dmmr ) where immunotherapy resulted in remarkable responses . these articles are highly relevant because the us food and drug administration ( fda ) approved pembrolizumab in may 2017 for the treatment of all unresectable or metastatic cancer with msi / dmmr irrespective of tissue origit was the first time that any therapy had been approved for cancers that share a specific genetic feature irrespective of site of origfurthermore , in late july , nivolumab was approved for msi / dmmr metastatic colorectal cancer . 
the fda approvals are based on studies showing disease control rates of 70% to 90% in patients with colorectal cancer and noncolorectal msi / dmmr cancers , most of whom with disease that was refractory to standard chemotherapy upon entering these trials.1 - 3 these trials had not reached median progression - free survival1 , 2 or overall survival1 - 3 at the time of publication , indicating that the responses are durable . msi is defined as a difference in length of microsatellites ( dna nucleotide repeats ) when comparing tumor dna with normal dna from the same individual . 
deficiency in the mmr system , a dna repair system in charge of correcting errors during dna replication , causes a distinct accumulation of errors in microsatellites , as well as a hypermutated phenotype . 
the mmr system is most commonly inactivated in tumors by either somatic inactivation via methylation of the mlh1 gene , 4 germline mutations in the mmr genes ( mlh1 , msh2 , msh6 , and pms2 [ lynch syndrome ] ) , 5 or , more rarely , somatic mmr mutations.6 , 7 dmmr can be detected by two widely implemented methods : immunohistochemistry ( tumor is stained for the four mmr proteins ) and msi testing via polymerase chain reaction ( pcr )  . 
these two methods have a high concordance.8 traditionally , msi testing has been done via pcr focusing on five predefined microsatellites ( monoor dinucleotide microsatellites ) , which were initially chosen during a meeting in bethesda in 1997.9 , 10 with the advent of ngs with targeted gene sequencing or whole exome / genome sequencing , myriad microsatellites can now be investigated simultaneously with computational tools . 
at least four different computational tools have been developed : msings , 11 msisensor , 12 mantis , 13 and mosaic.14 so how common is msi / dmmr in cancer ? it is well known that the highest msi / dmmr incidences are observed in endometrial cancer ( 20% to 30% ) , 15 - 17 colorectal cancer ( 12% to 15% ) , 18 , 19 and gastric cancer ( 15% to 20% ) , 20 although rates may be lower when focusing on metastatic disease ( 4% to 5% in colorectal cancer in populationbased studies ) .21 , 22 bonneville et al23 examined msi with mantis software in 11 , 139 tumors ( 39 cancer types ) , mostly derived from the cancer genome atlas , using whole exome sequencing data ( including 2 , 530 microsatellites )  . 
the study by middha et al24 describes lower rates of msi compared with bonneville et al , 23 but it is probably as a result of the more advanced stages examined in the study by middha et al . 
both studies looked at mutational burden and found that msi tumors have a significantly higher mutational burden compared with microsatellite stable ( mss ) tumors . do msi cases found via msi - ngs always signify tumors with dmmr ? or could they have other etiologies causing msi ? mantis had previously been validated against samples with msi by pcr in colorectal cancer , endometrial cancer , gastric cancer , esophageal cancer , and prostate cancer , as well as uterine carcinomasarcoma.13 in the study by bonneville et al , 23 the investigators examined ngs data of the mmr genes and found mmr mutations in some cases , but do not have data on mlh1 hypermethylation , which is the most common reason for msi . 
in the study by middha et al , 24 the msi status was validated in 138 colorectal and endometrial cancer cases by performing mmr immunohistochemistry and conventional msi by pcr . 
they found a high concordance between the methods ( 99.4% ) , and msi by ngs seems slightly more sensitive than msi by pcr because they discovered three msi cases by msisensor that scored as msi - low or mss on pcr but were dmmr on immunohistochemistry . 
furthermore , they investigated 456 other cancer cases that scored low ( 3 to 10 ) on msisensor and found that only 3.5% were discordant ( msi by pcr ) , reflecting the fact that most cases with low msi are not truly dmmr.10 ideally , validation would be done for every tumor type to ensure that msi is detected across a range of different cancers and truly represents dmmr . 
in the study by middha et al , 24 one case with a pole mutation was msh6 deficient and msi ; all other pole - mutated cases were mss by msisensor and had normal mmr immunohistochemistry , suggesting that msi in pole - mutated cases is secondary to mutations in mmr genes . other studies have described similar findings.6 , 25 the case reports are great examples of how tumors that are not typically associated with msi status can respond dramatically to immunotherapy . 
in the study by kok et al , 26 a 69 - year - old patient with metastatic breast cancer responded to nivolumab , with complete resolution of a malignant gastric ulcer after three cycles and an ongoing response after 12 cycles of nivolumab . 
in the study by klempner et al , 27 an 85 - yearold patient with stage iii gastroesophageal adenocarcinoma , deemed not a surgical candidate , had a complete response to pembrolizumab , with an ongoing response for 8 months . 
because mlh1 - hypermethylated tumors are more commonly observed in older patients , 16 , 20 , 28 a patients age should not deter physicians from investigating msi / dmmr . with the fdas recent approval of immunotherapy in msi / dmmr advanced cancers , and the finding of this molecular signature across a broad range of tumor types , screening all metastatic cancer cases for msi should become standard practice . 
companies and / or institutions who offer multiple gene sequencing panels should strongly consider integrating msi analysis into their existing ngs protocols and include this as part of their reporting . 
some panels are already reporting this , such as the foundation one platform . it is unclear how many of the msi cases found in the studies by bonneville et al23 and middha et al24 were caused by lynch syndrome . 
in prior studies , 10% to 15% of msi / dmmr colorectal cancer cases and 10% of msi / dmmr endometrial cancer cases were caused by lynch syndrome.21 , 22 , 29 lynch syndrome can increase the risk of many of the malignancies found to be msi in the studies presented in jco precision oncology ; thus it is important for physicians to refer patients with msi / dmmr cancers to the genetics clinic ( the exception being mlh1 / pms2 - absent cancers that test positive for mlh1 hypermethylation , because most of these cases are somatic )  . 
overman mj , mcdermott r , leach jl , et al : nivolumab in patients with metastatic dna mismatch repair - deficient or microsatellite instability - high colorectal cancer ( checkmate 142 ) : an open - label , multicentre , phase 2 study . 
kane mf , loda m , gaida gm , et al : methylation of the hmlh1 promoter correlates with lack of expression of hmlh1 in sporadic colon tumors and mismatch repair - defective human tumor cell lines . 
haraldsdottir s , hampel h , tomsic j , et al : colon and endometrial cancers with mismatch repair deficiency can arise from somatic , rather than germline , mutations . 
bartley an , luthra r , saraiya ds , et al : identification of cancer patients with lynch syndrome : clinically significant discordances and problems in tissue - based mismatch repair testing . 
boland cr , thibodeau sn , hamilton sr , et al : a national cancer institute workshop on microsatellite instability for cancer detection and familial predisposition : development of international criteria for the determination of microsatellite instability in colorectal cancer . 
billingsley cc , cohn de , mutch dg , et al : polymerase e ( pole ) mutations in endometrial cancer : clinical outcomes and implications for lynch syndrome testing . 
 o comparative analysis of public knowledge bases for precision oncology steffen pallarz , phd1 ; manuela benary , phd1 , 2 ; mario lamping , md2 ; damian rieke , md2 , 3 ; johannes starlinger , md2 ; christine sers , phd2 , 4 ; david luis wiegandt1 ; marc seibert1 ; jurica seva , phd1 ; reinhold sch afer , phd2 , 4 ; ulrich keilholz , md2 ; and ulf leser , phd1 purpose precision oncology depends on the availability of up - to - date , comprehensive , and accurate information about associations between genetic variants and therapeutic options . 
we performed a quantitative and qualitative comparison of clinical interpretations of variants in cancer , oncokb , cancer gene census , database of curated mutations , cgi biomarkers ( the cancer genome interpreter biomarker database ) , tumor alterations relevant for genomics - driven therapy , and the precision medicine knowledge base . methods we downloaded each kb and restructured their content to describe variants , genes , drugs , and genedrug associations in a common format . 
for the analysis of clinically relevant gene - drug associations , we obtained lists of genes affected by genetic alterations and putative drug therapies for 113 patients with cancer whose cases were presented at the molecular tumor board ( mtb ) of the charit e comprehensive cancer center . results our analysis revealed that the kbs are largely overlapping but also that each source harbors a notable amount of unique information . 
the relative importance of a kb in terms of cancer genes was assessed in more detail by logistic regression , which revealed that all but one source had a notable impact on result quality . 
we conrmed these ndings using a second data set obtained from an independent mtb . conclusion to date , none of the existing publicly available kbs on gene - drug associations in precision oncology fully subsumes the others , but all of them exhibit specic strengths and weaknesses . 
2019 by american society of clinical oncology introduction precision oncology ( po ) is based on the molecular characterization of tumors and the integration of these data into clinical decision making.1 molecular characterizations typically are based on the identication of genomic variants by genomic sequencing , which results in patient - specic variant proles of single nucleotide variants , small insertions or deletions , copy number variations , and gene fusion load of the events . 
this bottleneck of clinical interpretation in po currently is based mostly on manual work and still lacks standardization.2 clinical variant interpretation must be based on published information.3 however , such information currently is dispersed across a number of databases , repositories , and web sites . 
 pallarz et al context key objective several knowledge bases that support the clinical evaluation of genetic variants in patients with cancer have emerged during the past years , but their mutual overlaps and extents are unknown . knowledge generated we compared seven knowledge bases and found that they share information only to a moderate extent and that they altogether lack important data . 
also , none of the knowledge bases is clearly more comprehensive than the others . relevance the creation of structured and easy - to - use knowledge bases to support precision oncology has made notable progress in the past years , yet the current systems remain incomplete and inhomogeneous . 
our results underline the necessity that , to obtain comprehensive information , oncologists must interrogate multiple knowledge bases and search relevant literature directly . we compared these kbs by measuring their mutual overlap of genes , drugs , and gene - drug associations and , if possible , by comparing each kbs content to actual treatment recommendations for 113 patients with cancer whose cases were discussed at the molecular tumor board ( mtb ) of the charit e comprehensive cancer center . 
results were conrmed using a second , independent set of recommendations for 10 patients with cancer . methods we performed pubmed searches and expert interviews to obtain a list of seven publicly available knowledge bases that aim at supporting medical decision making in precision oncology ( po - kb ) by providing structured information regarding the relationship between genes , variants , cancer entities , and therapies . 
specically , we used ( 1 ) civic ( clinical interpretations of variants in cancer ) , ( 2 ) oncokb , ( 3 ) cgc ( cancer gene census ) , ( 4 ) docm ( database of curated mutations ) , ( 5 ) cgi biomarkers ( the cancer genome interpreter biomarker database ) , ( 6 ) target ( tumor alterations relevant for genomics - driven therapy ) , and ( 7 ) pmkb ( the precision medicine knowledge base )  . 
the technical data integration procedure and normalization methods we developed for genes , variants , and drugs can be found in the data supplement . overlap comparison for a quantitative comparison , we computed the mutual overlap of all sources with regard to the set of genes , variants , drugs , and gene - drug associations they contain and visualized them using upset diagrams.12 for clustering of po - kbs , we rst computed pairwise distances using jaccards formula for set similarity and then applied hierarchical clustering ( data supplement )  . comparison of clinically relevant gene - drug associations we compared the content of each po - kb alone and in combinations versus recommendations of the charit e comprehensive cancer centers mtb ( n = 113 patient cases ) , at which results of comprehensive molecular table 1 . 
diagrams show the overlap between different precision oncology knowledge bases ( po - kbs ; identied by colors ) the matrices in the lower panels indicate po - kbs involved in an intersection , and the bar plots show the number of elements in this intersection . 
cgi , cgi biomarkers ( the cancer genome interpreter biomarker database ) ; civic , clinical interpretations of variants in cancer ; db , database ; docm , database of curated mutations ; pmkb , precision medicine knowledge base ; target , tumor alterations relevant for genomics - driven therapy . analysis ( whole - exome or panel sequencing , rna sequencing , immunohistochemical validations ) from patients with advanced cancer are discussed on a weekly basis . clinical interpretation of molecular alterations in the mtb is performed by a trained physician3 , 13 ; the data supplement contains details on this process . 
we measured the overlap between this reference set and different combinations of po - kbs for each patient using the metrics precision ( percentage of associations of a [ set of ] po - kb that is also contained in the reference ) , recall ( percentage of associations in the reference that is also contained in a [ set of ] po - kb ) and f1 measure ( harmonic mean between precision and recall )  . 
these measures are visually displayed later in the results , and more details can be found in the data supplement . to assess the predictive power of the different po - kbs , we proceeded as follows : for each association present in any of the po - kbs , a vector representation was constructed to encode its coverage by civic , cgi biomarkers , target , and oncokb , respectively . 
every gene - drug association that also was listed in the mtb associations was considered positive ( association relevant ; n = 345 cases ) , and all others were considered negative ( association not relevant ; n = 808 cases )  . 
 ( a ) percentage of patients for which any gene - drug associations were found when their presence was required in one to four po - kbs ( y - axis )  . 
each dot represents one patient , and only patients for whom at least one gene - drug association had been found were used . a 10 - fold cross - validation scheme to compute the out - ofsample error . 
eventually , a new model was trained on the entire training set to take this error into account ( using r package caret ) ; we report the results of the second model when applied on the test set . to validate our ndings , we used a second data set , which described treatment recommendations for patients with cancer , as derived at the molecularly aided stratication for tumor eradication ( masters ) 14 program at the national center for tumor diseases in heidelberg ( n = 10 ) and obtained from perera - bel et al.15 the normalization of gene and drug names for both data sets is described in the data supplement . 
to make results comparable to our rst data set , we removed all affected genes for which we did not have a single association in any of the po - kbs or for which no drug recommendation had been provided . 
to compensate for the positive bias in this set , we then added twice the number of genes chosen randomly from those we removed before . results overlap of studied kbs we downloaded and normalized seven publicly available databases that aim to support clinical decision making in po . we found that all databases overlap to a certain degree in the information they provide but also that each provided unique information . 
civic ( n = 89 genes ) and oncokb ( n = 86 genes ) have the second largest number of unique genes not mentioned in any other po - kb ; 49 genes were contained in all seven databases . 
the data supplement shows a hierarchical clustering of po - kbs on the basis of their overlaps in genes , variants , and gene - drug associations . although civic , oncokb , and pmkb share a highly similar goal and creation process and also obtain their data from the same origin ( scientic publications ) , each of them contains a substantial amount of unique genes . 
the most probable reason for this observation is that all po - kbs are small compared with the huge amounts of published data . the data supplement lists all genes contained in one or more of the databases . 
in addition , the four sources that contain drug information provide both shared and unique information ( fig 1b ) : 17 drugs are mentioned in all sources , and civic has the largest number of unique drugs ( n = 223 )  . 
similarly , civic shows the largest number of unique gene - drug associations ( n = 698 ; fig 1c )  . comparison of clinically relevant gene - drug associations we next compared the content of each po - kb and combinations thereof versus expert considerations for the 113 patients discussed at the charit e mtb at the level of genedrug associations . 
the number of drugs is high , because , for the analysis , drug classes ( eg , mammalian target of rapamycin inhibitors ) were expanded to all drugs of the respective class to match the granularity of information in different data sets . 
we analyzed which patients for whom the associations presented to the mtb also were contained in different po - kbs ( fig 2 ) and which fractions of gene - drug associations presented in the mtbs also would be found by specic combinations of po - kbs ( fig 3 )  . overall , the mtb discussed 203 different genes and 245 different drugs , of which 152 and 176 , respectively , were found in at least one po - kb . 
this implies that 25% of genes and 28% of drugs discussed in the mtb could not be found by our software in any of the po - kbs we considered . 
next , we computed the set of prioritized genes per patient and checked for how many of those had associations with drugs in at least one , two , three , or four po - kbs . 
 comparison of knowledge bases for precision oncology precision true positives ( tp ) false negatives ( fn ) recall false positives ( tn ) true negatives ( tn ) in po - kbs precision recall no . 
 ( b ) median precision ( blue ) , recall ( red ) and f1 ( harmonic mean between precision and recall ) score ( gray ) when considering an increasing number of precision oncology knowledge bases ( po - kbs ) to support gene - drug associations discussed by the molecular tumor board ( mtb ) experts . 
 ( c ) precision and ( d ) recall obtained by using different combination of po - kbs as boxplots ( median , 25th and 75th percentiles ; whiskers extend to the maximum values within 1.5 the interquartile range ; dots represent outliers )  . 
cgi , cgi biomarkers ( the cancer genome interpreter biomarker database ) ; civic , clinical interpretations of variants in cancer ; pr , precision and recall ; target , tumor alterations relevant for genomics - driven therapy . gene - drug association , the number of patients decreases to 42 . 
figure 2b shows that the number of potential suggestions per patient drops sharply with the required number of supporting po - kbs : when support by any two po - kbs was required , we obtained a median of ve associations per patient . 
the median increased to 12 if only one po - kb was required and decreased to two for three po - kbs and zero for four po - kbs . next , we considered the mtb associations as reference and the contents of the four po - kbs that contained genedrug associations as predictors . 
when the mtb reference gene - drug association was required to be contained in only one po - kbsthat is , the union of all po - kbs was used as predictorthe median recall for all patients reached approximately 46% ( fig 3b , blue line ) , whereas the median precision was low because of the large number of associations found in the po - kbs that were not discussed in the mtb ( fig 3b , orange line ; data supplement contains evaluation metrics )  . 
intuitively , this means that searching in more than one po - kb in our evaluation always led to nding more relevant data but at the cost of also nding more data that are irrelevant for the concrete patient according to the mtb . finally , we trained a logistic regression classier on evidence from all po - kbs using mtb associations as ground truth . 
on our withheld test set ( n = 288 gene - drug associations ) , this model reached an f1 score of 43% ( precision , 64% ; recall , 33% )  . 
on the nct data set , this model reached a precision of 100% at a recall of 38% , which resulted in a higher f1 score of 56% . the data supplement contains an interactive html page with the complete list of gene - drug associations of the berlin and the heidelberg data sets together with the information about which of the po - kbs contains which reference associations . discussion we performed a quantitative and qualitative comparison of the current content ( february 2019 ) of seven knowledge bases specialized in po . 
our results indicate that the pokbs have partly overlapping and partly unique results . compared with associations discussed for 113 patients in an mtb of a comprehensive cancer center at a university clinic , we found that po - kbs contain relevant information not present in any of the others . 
however , we also want to point out some limitations of our work that warrant additional studies . even after careful normalization of gene and drug names in all po - kbs ( data supplement ) , 809 of the 1 , 154 gene - drug associations of the mtb data set were not found in any of the po - kbs . 
possible reasons for their absence are that these genes are simply not yet curated , are grossly misspelled , or designate gene families rather than individual genes and so could not be matched by our normalization procedure . 
similarly , 157 associations with 69 unique drugs were not found because these drugs are not contained in any of the kbs ; 383 associations were missing because none of the po - kbs reported this particular association , although both the genes and the drugs were found in principle . our analysis showed that civic has the highest recall and f1 score compared with mtb associations when data from only one po - kb are considered ( ie , no combinations were allowed )  . 
these submissions could be the reason for the higher recall of po - kb combinations contained in civic ( fig 3d ) ; however , civic also simply contains the highest number of gene - drug associations among all po - kbs considered , which also positively inuences recall . 
separation of these two effects is difcult , but we note that regression results on the heidelberg data set , for which no bias toward civic exists , are even better than for the mtb data set . 
on both data sets , removal of civic from the training data left the precision of the learned model unchanged but induced a notable decrease in recall ( mtb , 33% decreased to 19% ; heidelberg , 38% decreased to 21% ) and , thus , in the f1 score ( mtb , 43% decreased to 29% ; heidelberg , 56% decreased to 35% )  . 
the fact that the decrease in recall was comparable in both data sets is an argument against a strong bias of the charit e mtb analysis toward civic ; the overall strong decrease originates from the large number of gene - drug associations only contained in civic . as reported , we trained a classier on combinations of the different po - kbs using the mtb associations as ground truth . 
however , we note that the reported results in terms of prediction accuracy cannot be considered as the ability to predict treatments in a clinical decision support setting for multiple reasons . 
second , we considered all associations in the mtb data as relevant without taking into account the nal decision of the mtb , which could be the recommendation of a single drug , could be a combination of drugs , or could disregard all discussed associations . 
fifth , the patient cases presented in the mtb represent a large variety of different tumor types , such as breast cancer , pancreatic cancer , leiomyosarcoma , ovarian cancer , liposarcoma , and neuroendocrine tumors ( data supplement )  . our analysis ignored the information on cancer types , because this level of detail frequently is missing in the po - kbs and , when present , uses a highly idiosyncratic nomenclature . 
note that analysis of recommendations derived from genomic information across different cancer types is commonplace in current studies about po.16 , 17 in conclusion , we here report a quantitative and qualitative comparison of precision oncology knowledge databases . our analysis shows that each of the databases contains , besides a rather small common core , a relevant amount of unique information . 
when we compared po - kb contents with gene - drug associations discussed in two mtbs , we found that a considerable fraction of information indeed can be found in some po - kbs but also that many presumably clinically relevant associations are not yet contained in any of them and that relevant information often is dispersed across different po - kbs . 
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perera - bel j , hutter b , heining c , et al : from somatic variants towards precision oncology : evidence - driven reporting of treatment options in molecular tumor boards . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular proling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
massard c , michiels s , fert e c , et al : high - throughput genomics and clinical outcome in hard - to - treat advanced cancers : results of the moscato 01 trial . cancer discov 7 : 586 - 595 , 2017 18 . 
chinnaiyan , md , phd4 , 5 case description in january 2018 , a 79 - year - old man was referred to our genitourinary medical oncology clinic for management of his prostate adenocarcinoma metastatic to multiple bones . 
his prostate cancer spread to additional bony sites , which were likewise treated with radiation , including radiation to the thoracic spine in july 2017 and the proximal humerus in august 2017 , rather than any systemic in december 2017 , he had prophylactic therapy . nipple irradiation to prevent gynecomastia in preparation for noncastrating medical therapy with singleagent bicalutamide . 
on meeting the patient for the rst time in january 2018 , we elected to continue with the plan for single - agent bicalutamide 50 mg per day , given the patients preference and the uncertain prognosis from his metastatic melanoma . 
his prostatespecic antigen ( psa ) was 30.8 ng / ml at the time of treatment initiation and responded rapidly to bicalutamide , reaching a nadir of 1.1 ng / ml in september 2018 ( fig 1a )  . his melanoma was diagnosed from a shave biopsy of the right superior lateral lower back in august 2015 as localized ulcerated malignant melanoma , unclassied , with nevoid features . 
he subsequently had a microstaging excision and a right back excision with a negative sentinel lymph node in the right groin 2017 , the patient noticed a new lump on his right lower back scar in 2017 . 
in november 2017 , excisional biopsies of his right lower back and right groin both showed metastatic melanoma , as conrmed by immunohistochemistry ( ihc ) showing positive staining for sox - 10 and mart - 1 . 
a 7 - mm lesion was also detected on his posterior scalp and was also visible on magnetic resonance imaging of his brain and metabolically active on positron emission tomography ( pet ) / computed tomography ( ct ) ; hence , it was diagnosed as stage iv melanoma . 
he began treatment of his metastatic melanoma with pembrolizumab 200 mg intravenous every 3 weeks and received four treatments before the pembrolizumab was stopped because of the development of pneumonitis . 
his pneumonitis was treated with a taper of prednisone , which was reduced to physiologic dosing by august 2018 . however , despite evidence of response in his melanoma by physical examination , there was concern for progression on pet / ct in june 2018 , with fdg update in the descending colon / small bowel wall , perisplenic region , a mildly fdg - avid left internal iliac lymph node , and a right external iliac nodal conglomerate encasing the right ureter , 4.1 cm in diameter and with an suv max of 11.7. 
 case report systemic therapy : gem - carbo docetaxel bicalutamide pembro bicalutamide leuprolide 1.1 ureter stent and pelvic mass biopsy abdominal wall excisional biopsy psa : ( ng / ml ) 30.8 event : date : 1 / 2018 6 / 2018 1 / 2019 6 / 2019 1 / 2020 6 / 2020 before gem - carbo after gem - carbo before docetaxel after docetaxel fig 1 . 
psa , prostate - specic antigen . pelvic mass was judged to be atypical for either prostate cancer or melanoma , it was biopsied by pelvic laparoscopy in november 2018 and was found on surgical pathology assessment to be consistent with a high - grade carcinoma with squamous features ( fig 2a )  . 
we performed exome sequencing using the university of michigan oncoseq panel ( mi - oncoseq ) and whole transcriptome analysis on formalin - xed parafn - embedded tissue from this biopsy . 
all patients enrolled in the mi - oncoseq study provided written informed consent approved by the university of michigan institutional review board . consent is inclusive of publishing information and / or images from participants ( or their designate )  . 
because the pelvic mass was causing pain and urinary obstruction , we elected to treat it with a platinum doublet active in urothelial carcinoma and carcinoma of unknown primary , despite not knowing the tissue of origin january 2019 , we began carboplatin area under the curve 5 mg / ml / minute on day 1 and gemcitabine 1 , 000 mg / m2 on days 1 and 8 of 21 - day cycles , and we completed six cycles before stopping because of progressive fatigue . 
 ( d ) ihc for erg of the primary prostate tumor showing strong labeling . ( e ) h&e histology of the abd wall nodule biopsy specimen used for molecular analysis . 
 ( f ) ihc for erg of the abd wall nodule showing much weaker labeling . in june 2019 , while being treated only with bicalutamide , the patient was noted on ct scan as having a 1.4 - cm nodule in the supercial anterior abdominal wall , which subsequently became easily palpable and caused skin together with smaller adjacent erythema . 
this nodule , nodules , was removed by excisional biopsy in july 2019 , where surgical pathology assessment demonstrated tumor features consistent with metastatic squamous cell carcinoma ( fig 2b )  . 
a fresh portion of this specimen was sent for analysis using the mi - oncoseq platforthis specimen showed a much higher tumor content of 54% , allowing the discovery of additional molecular alterations . precision medicine tumor board discussion results were discussed at the university of michigan precision medicine tumor board in september 2019 . somatic aberrations detected in the previous sample were detected in the new biopsy specimen , suggesting clonal relatedness , and additional alterations consistent with the increased tumor content were noted as well ( table 2 )  . 
for prostate adenocarcinoma , the addition of medical castration , likely in addition to either abiraterone and prednisone or a nonsteroidal second - generation antiandrogen , would have been a reasonable next line of therapy . 
however , the transcriptomics data of the mi - oncoseq platform showed low expression of the androgen receptor and androgen responsive genes klk2 , klk3 ( psa ) , tmprss2 , acpp , and slc45a3 ( fig 3b )  . 
therefore , we did not plan chemotherapy with a regimen such as carboplatin and etoposide , which is active against small - cell neuroendocrine carcinomas . we examined the remainder of the molecular results in an effort to nd alternative , clinically actionable molecular targets . 
however , overall , the results for mtor inhibitors in prostate cancer have been disappointing.3 some randomized data support the use of pi3k inhibitors in metastatic castration - resistant prostate cancer.4 however , this was in combination with abiraterone , which made pi3k inhibitors less attractive in this case , given the patients near total lack of androgen signaling . 
however , we decided against this option because cdk4 / 6 inhibitors have thus far shown disappointing results in prostate cancer.5 therefore , we elected to treat with docetaxel , the most common cytotoxic chemotherapy drug for castration - resistant prostate cancer . at this point , the patients lung nodules , which were previously indeterminate , had enlarged greatly ( fig 1d )  . 
we started treatment with docetaxel at 75 mg / m2 for one cycle , then decreased the dose to 60 mg / m2 because of fatigue , and completed three more cycles . 
ct completed after three cycles showed an interval decrease in the size of multiple bilateral lung nodules , and a decrease in size of his right pelvic mass , compatible with a therapeutic response ( fig 1e )  . discussion in this case , we used next - generation sequencing to determine that the patients squamous cell carcinoma was actually of prostate origin , because of the presence of a gene fusion between tmprss2 and erg . 
 case report prostate cancer subtypes has become more common since the development of abiraterone and nonsteroidal secondgeneration antiandrogens , 6 possibly as a means to escape continual selective pressure against androgen signaling , a phenomenon that had been described rarely in the past.9 some of these double - negative prostate cancer ( dnpc ) tumors seem to be driven by broblast growth factor ( fgf ) alterations , and trials of fgf inhibitors have recently begun in advanced prostate cancer.10 our patient did not have an fgf abnormality . 
in the current case , despite having squamous differentiation , this patient had only stable disease in response to platinum - based chemotherapy . after using whole exome sequencing and rna sequencing to identify this tumor as a squamous neoplasm of prostate origin , we elected to treat him with an agent approved for prostate cancer ( docetaxel ) , an agent we would not have elected to use without knowing the tissue of origwe elected not to pursue the therapies that could target molecular alterations in his tumor because of the known survival benet of docetaxel in men with advanced prostate cancer , but therapies targeting his tumors molecular alterations remain options down the road if his disease progresses . 
alumkal consulting or advisory role : merck sharp & dohme , dendreon research funding : aragon pharmaceuticals ( inst ) , astellas pharma ( inst ) , zenith epigenetics ( inst ) , gilead sciences ( inst ) travel , accommodations , expenses : astellas pharma , merck sharp & dohme other relationship : astellas pharma arul chinnaiyan stock and other ownership interests : oncopia , tempus , esanik , oncofusion therapeutics , medsyn consulting or advisory role : tempus patents , royalties , other intellectual property : co - inventor on a patent on the detection of ets gene fusions in prostate cancer issued to the university of michigan . no other potential conicts of interest were reported . acknowledgment we thank the mi - oncoseq clinical sequencing team , leslie fecher , md , jeff montgomery , md , and michael sabel , md , for collaborative clinical care and discussion ; alex hopkins for assistance with gures ; and jyoti athanikar for assistance with scientic editing and manuscript preparation . 
cell 161 : 1215 - 1228 , 2015 [ erratum : cell 162 : 454 , 2015 ] statz cm , patterson se , mockus sm : mtor inhibitors in castration - resistant prostate cancer : a systematic review . 
target oncol 12 : 47 - 59 , 2017 de bono js , de giorgi u , rodrigues dn , et al : randomized phase ii study evaluating akt blockade with ipatasertib , in combination with abiraterone , in patients with metastatic prostate cancer with and without pten loss . 
clin cancer res 25 : 928 - 936 , 2019 clinicaltrials.gov : a phase ii study of androgen deprivation therapy with or without palbociclib in rb - positive metastatic prostate cancer . 
gov / ct2 / show / nct02059213 bluemn eg , coleman im , lucas jm , et al : androgen receptor pathway - independent prostate cancer is sustained through fgf signaling . 
cancer cell 32 : 474 - 489.e6 , 2017 labrecque mp , coleman im , brown lg , et al : molecular proling straties diverse phenotypes of treatment - refractory metastatic castration - resistant prostate cancer . 
j clin invest 129 : 4492 - 4505 , 2019 parwani av , kronz jd , genega em , et al : prostate carcinoma with squamous differentiation : an analysis of 33 cases . 
weindorf sc , taylor as , kumar - sinha c , et al : metastatic castration resistant prostate cancer with squamous cell , small cell , and sarcomatoid elementsa clinicopathologic and genomic sequencing - based discussion . 
with the recent increase in integrative clinical sequencing for pediatric patients with cancer , our understanding of the dicer1 syndrome continues to evolve , as new and rare pathogenic variants are reported . 
as the frequency of integrative clinical sequencing increases , discussions regarding challenges encountered in the interpretation of sequencing results are essential to continue to advance the eld of cancer predisposition . 
the purpose of this work was to identify patients with somatic and / or germline dicer1 variants in our patient population and to discuss sequencing interpretation and the clinical recommendations that result from the integrative clinical sequencing results . methods patients were enrolled in the peds - mioncoseq study . 
2019 by american society of clinical oncology introduction the dicer1 gene is located on chromosome 14 ( 14q32.13 ) and encodes the dicer protedicer is an rnase iii enzyme functioning in micro - rna ( mirna ) processing . 
mirnas are single - stranded , noncoding rnas that target mrnas by binding to complementary mrna sequences , increasing their degradation and suppressing translation.1 through its ability to regulate mirna processing , dicer1 plays a critical role in cellular mrna expression and in early development.1 , 2 with respect to human disease , more than 130 germline and 95 somatic dicer1 mutations have been reported.2 the majority of somatic dicer1 mutations are hotspot mutations in the rnase iiib domain ; however , somatic mutations are reported to be elsewhere , including the domain of unknown function.2 germline dicer1 mutations have been reported in all of the 10 major domains and spanning regions.3 dicer1 syndrome , caused by pathogenic variants in dicer1 , is an inherited condition associated with an increased risk of developing pleuropulmonary blastoma ( ppb ) , multinodular goiter , cystic nephroma , sertoli - leydig cell tumors of the ovary , and other rare tumors . 
 bailey et al context the goal of this work was to describe sequencing interpretation challenges and clinical recommendations in patients with dicer1 somatic and / or germline variants identied on integrative sequencing . this work suggests that postzygotic mosaicism may play a signicant role in the development of dicer1 - associated tumors . furthermore , we report a patient with a dicer1 variant of uncertain signicance whose clinical course and response to treatment was highly unusual , suggesting this variant could be clinically meaningful . the use of integrative clinical sequencing has signicantly expanded in pediatric oncology in the past 5 years . 
reports also include a dicer1 syndrome family with both somatic and germline pathogenic variants found in family members with different types of dicer1 - related cancers.15 to our knowledge , our group reported the rst real - time , integrative clinical sequencing ( ics ) study in the pediatric oncology population in 2015.16 since that time , the role of real - time ics of tumors and germline samples in the pediatric oncology population has continued to expand.17 - 19 however , the interpretation of genetic variants and their translation into the clinical setting remain challenging . when sequencing results identify germline dicer1 variants in pediatric oncology patients , counseling and screening implications for family members are affected . 
the purpose of this work was to highlight ( 1 ) somatic and / or germline dicer1 pathogenic variants identied in our pediatric oncology sequencing cohort , ( 2 ) challenges encountered with sequencing result interpretation with respect to dicer1 , and ( 3 ) current patient and family screening recommendations . methods patients the pediatric patients described were treated at c.s. 
patients were enrolled in a prospective , institutional review boardapproved ics trial ( peds - mioncoseq ) 16 and underwent paired tumor / normal whole - exome sequencing and tumor transcriptome sequencing . 
peds - mioncoseq is an ongoing precision oncology cohort and focused on enrolling highrisk , relapsed / refractory , and rare pediatric and young adult ( younger than 25 years of age ) patients with cancer . integrative clinical sequencing specics of the sequencing procedure and bioinformatics analyses have been previously described.16 briey , germline sequence and copy number variants , as well as somatic mutations , copy number alterations / variants , insertions and deletions , gene fusions , and gene expression , were identied.20 , 21 pathogenicity of germline variants was determined through a review of the published literature and publically available databases ( ie , clinvar )  . clinical relevance of somatic variants was investigated using an integrated approach incorporating technical considerations ( eg , recurrence , variant allele fraction , expression levels , and predictive algorithms for pathogenicity ) , variant - specic information ( ie , clinvar , published literature , and curated gene - specic resources ) , and published correlations of drug and variant sensitivity proles . results four patients with somatic and / or germline dicer1 pathogenic variants were identied among the rst 300 patients enrolled in peds - mioncoseq ( table 1 )  . 
we detail the patient presentations , diagnoses , variant terpretation , and clinical actions taken on the basis of these results . patient 1 : a patient with a hotspot somatic variant and a germline pathogenic dicer1 variant patient 1 presented at 3 years of age with a history of left arm and abdominal paan abdominal x - ray was performed , and a left - sided pleural effusion was incidentally noted . 
surveillance for dicer1 - related tumors was initiated for the patients sister ( 6 years old at time of genetic testing ) and included baseline chest ct ( to screen for ppb ) , with a chest x - ray at 8 years of age , annual ultrasound scans of the abdomen and neck ( to screen for ovarian and kidney tumors and thyroid tumors , spectively ) , and annual serum markers to screen for ovarian tumors . 
the father received an annual ultrasound scan of the thyroid gland only , because dicer1 - associated cancers affecting males primarily present in childhood . the bony metastatic lesions completely responded to chemotherapy , and the patient underwent primary mass resection followed by two more cycles of chemotherapy . the resected mass showed mostly viable ppb , with malignant cartilaginous nodules overrun by an anaplastic prostate cancer multinodular goiter s / p thyroidectomy prostate cancer fig 1 . 
patient 1 continued to have disease progression despite chemotherapy , and the decision was made to transition to comfort care . patient 2 : a patient with a germline dicer1 variant of unknown signicance patient 2 initially presented at 3 years of age with a left lower quadrant / pelvic mass . 
remarkably , the patient had an excellent response to therapy and was deemed disease free at age 5 years . at 19 years of age , patient 2 presented to our medical center with severe abdominal and back paimaging revealed concern for new / recurrent widely metastatic disease , because a pelvic mass was noted along with multiple spine and liver lesions . 
palliative radiation was initiated for pain control , and the patient enrolled in the peds - mioncoseq study . sequencing results revealed a germline dicer1 variant of uncertain signicance ( vus ) ( table 1 )  . 
sequencing of the original tumor ( from 3 years of age ) was attempted for comparison / to determine whether this was a metachronous tumor ; however , only sparse , poor - quality material was available , and sequencing could not be performed . 
although vuss are not disclosed per the pedsmioncoseq protocol , at times , a recommendation for clinical germline genetic testing may be made for patients with striking personal / family histories . 
the majority of vuss will be reclassied as likely benign / benign , 22 but for those that are reclassied as likely pathogenic / pathogenic , there can be signicant implications for patients and their relatives . 
patient 2 died at 20 years of age as a result of metastatic disease . patient 3 : a patient with low - level dicer1 mosaicism patient 3 initially presented at 22 months of age with a leftsided pneumonia and pneumothorax . 
a persistent air - lled cystic cavity was present in the left chest and observed with imaging for approximately 3 months before a diserial agnosis of ppb was made after resection of the left lower lobe of the lung . 
regardless , clinical germline genetic testing of dicer1 was recommended but not pursued because of the out - of - pocket cost . approximately 6 months later , patient 3 was enrolled in the peds - mioncoseq study . 
 ( a ) he image ( 40 ) of the original tumor showing mixed morphology with alveolar pattern in the top half and more abundant rhabdomyoblastic differentiation in the lower half . ( b ) he image ( 400 ) of the late recurrence tumor showing spindle - shaped and round cell tumor cells with focal rhabdomyoblastic differentiation . 
as such , no signicant somatic copy number variations , point mutations , indels , or gene fusions were able to be called . no germline variants were considered reportable at this time . this patient also enrolled in the national institutes of healths ( nihs ) pleuropulmonary blastoma dicer1 syndrome study , which includes germline and somatic sequencing , approximately 1 year after initial diagnosis . germline sequencing identied a mosaic germline dicer1 pathogenic variant ( table 1 ; fig 3 ) in 2% of the allelic reads in the patients blood sample . 
both parents enrolled in the study , and their germline testing was negative for the dicer1 pathogenic variant , consistent with the low - level mosaicism detected in the patients blood sample . 
this somatic variant is commonly seen in dicer1 syndromeassociated tumors and involves the rnase iiib domain of dicer1 . analysis of the raw data available from the peds - mioncoseq the germline dicer1 pathogenic study indicates that variant identied by the nih was present in 2% of germline variant reads in our study as well . 
this patient continues to receive annual chest ct scans with no evidence of disease and undergoes annual kidney ultrasound scans , thyroid examinations , and targeted physical examinations as screening for other potential tumors . patient 4 : a patient with a rare dicer1 - associated tumor patient 4 presented at 3 years of age with headaches , photophobia , and hydrocephalus . 
the patient received ve cycles of chemotherapy , consisting of vincristine , etoposide , cyclophosphamide , and cisplatin , with tumor resection performed after the second cycle because of an increase in tumor size . 
the patient also underwent autologous bone marrow transplantation . a year later , an mri scan revealed an enhancing mass overlying the left middle cerebellar peduncle , suggestive of recurrent disease . 
in a previous study , 25 low - level mosaicism in four children with multiple dicer1 - associated tumors in whom no germline dicer1 variant had been identied by conventional sequencing techniques was observed . 
patient 4 contributes to the literature on dicer1 syndrome and illustrates how ics can be useful in identifying germline variants in individuals with rare cancers . from a cancer treatment perspective , all four of these challenging patients revealed dicer1 variants ; however , to date , there are no there are no clinically available pediatric therapies targeting specic mirnas . 
the possibility of targeting mirnas and dicer function in the future is currently being explored.26 from a genetic counseling standpoint , once a germline dicer1 pathogenic variant is identied , germline genetic testing is recommended for rst - degree relatives . 
approximately 13% to 20% of individuals with detectable germline dicer1 pathogenic variants have de novo variants , whereas 80% to 87% are inherited.9 , 27 germline genetic testing can provide tumor risk information to probands and their relatives and provide information regarding recurrence risk for future pregnancies . 
in families where the dicer1 variant is inherited , reports indicate it is uncommon to have multiple individuals diagnosed with ppb ; however , there may be higher penetrance of other manifestations , including goiters and benign lung cysts . 
family members lacking the familial dicer1 mutation do not need surveillance , whereas those who have inherited this variant should be observed according to current surveillance recommendations . screening recommendations at our institution evolved over time and are based on literature review , expert opinion , and patient preferences . 
beyond this age , for female adolescent and adult patients , we discuss symptoms and signs of hormone excess related to sex cordstromal tumors , such as hirsutism , virilization , and menstrual abnormalities . 
for all other manifestations , we recommended a detailed physical examination . recently , consensus guidelines for surveillance were published by the international ppb registry , which are largely in accordance with our institutions age - specic recommendations . 
these guidelines were developed at the rst international dicer1 symposium in may 2016.7 recommendations for surveillance begin in the prenatal period , with a third - trimester ultrasound for detection of large lung cysts that require intervention at birth . 
these differences reect the natural uncertainty in screening approaches for a recently described hereditary tumor syndrome , which aim to integrate the morbidity and the unknown mortality of syndrome - associated tumors , incidence in the target population , and presumed cost effectiveness and patient burden . least 10 years of age and do not tumor and germline sequencing in the pediatric oncology patient population has rapidly expanded in recent years . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . no potential conicts of interest were reported . references peng y , croce cm : the role of micrornas in human cancer . 
nat rev cancer 14 : 662 - 672 , 2014 kurzynska - kokorniak a , koralewska n , pokornowska m , et al : the many faces of dicer : the complexity of the mechanisms regulating dicer gene expression and enzyme activities . 
nucleic acids res 43 : 4365 - 4380 , 2015 kim j , field a , schultz kap , et al : the prevalence of dicer1 pathogenic variation in population databases . 
int j cancer 141 : 2030 - 2036 , 2017 slade i , bacchelli c , davies h , et al : dicer1 syndrome : clarifying the diagnosis , clinical features and management implications of a pleiotropic tumour predisposition syndrome . 
hum mutat 32 : 1381 - 1384 , 2011 schultz kap , williams gm , kamihara j , et al : dicer1 and associated conditions : identication of at - risk individuals and recommended surveillance strategies . clin cancer res 24 : 2251 - 2261 , 2018 8 . 
science 325 : 965 , 2009 brenneman m , field a , yang j , et al : temporal order of rnase iiib and loss - of - function mutations during development determines phenotype in pleuropulmonary blastoma / dicer1 syndrome : a unique variant of the two - hit tumor suppression model . 
schultz ka , pacheco mc , yang j , et al : ovarian sex cord - stromal tumors , pleuropulmonary blastoma and dicer1 mutations : a report from the international pleuropulmonary blastoma registry . 
schultz ka , yang j , doros l , et al : dicer1 - pleuropulmonary blastoma familial tumor predisposition syndrome : a unique constellation of neoplastic conditions . dev pathol 18 : 504 - 511 , 2015 pediatr blood cancer 61 : 1695 - 1697 , 2014 pathol case rev 19 : 90 - 100 , 2014 22 : 564 - 567 , 2014 14 . 
fern andez - martnez l , villegas ja , santamara i , et al : identication of somatic and germ - line dicer1 mutations in pleuropulmonary blastoma , cystic nephroma and rhabdomyosarcoma tumors within a dicer1 syndrome pedigree . 
kline cn , joseph nm , grenert jp , et al : targeted next - generation sequencing of pediatric neuro - oncology patients improves diagnosis , identies pathogenic germline mutations , and directs targeted therapy . 
marks lj , oberg ja , pendrick d , et al : precision medicine in children and young adults with hematologic malignancies and blood disorders : the columbia university experience . 
messinger yh , stewart dr , priest jr , et al : pleuropulmonary blastoma : a report on 350 central pathology - conrmed pleuropulmonary blastoma cases by the international pleuropulmonary blastoma registry . 
therefore , less is known about the prevalence and extent of msi among other types of cancer . methods using our recently published msi - calling software , mantis , we analyzed wholeexome data from 11 , 139 tumor - normal pairs from the cancer genome atlas and therapeutically applicable research to generate effective treatments projects and external data sources across 39 cancer types . 
within a subset of these cancer types , we assessed mutation burden , mutational signatures , and somatic variants associated with msi . results we identified msi in 3.8% of all cancers assessedpresent in 27 of tumor typesmost notably adrenocortical carcinoma ( acc ) , cervical cancer ( cesc ) , and mesothelioma , in which msi has not yet been well described . 
in addition , msi - high acc and cesc tumors were observed to have a higher average mutational burden than microsatellite - stable acc and cesc tumors . conclusion we provide evidence of as - yet - unappreciated msi in several types of cancer . 
2017 by american society of clinical oncology introduction large - scale sequencing projects of cancer genomes have opened the door to studies that have identified putative biomarkers with potential clinical and therapeutic value , among them the presence or absence of microsatellite instability ( msi )  . 
microsatellites are defined as 10 to 60 base pair regions that contain multiple repeats of 1 to 5 base pair motifs.1 microsatellites occur at microsatellite loci , which are widely dispersed throughout the human genome . 
in normal cells , repeat count of microsatellites is verified and maintained during cell division by the mismatch repair ( mmr ) system , 2 , 3 one of many cellular dna repair mechanisms . 
after multiple cycles of cell division , cells with an impaired mmr system will develop varying lengths in their microsatellite sequences . mismatch repair deficiency is known to occur in some tumors , 2 either by somatic hypermutation of mmr genes , most commonly , mlh14 , 5 ; an inherited germline mmr pathway mutation , such as in lynch syndrome6 , 7 ; or double somatic mutations in mmr genes . 
 durable responses and a statistically significant improvement in overall survival.12 msi polymerase chain reaction ( pcr ) and immunohistochemistry are two molecular biology based methods that are in routine use for clinical msi testing . 
msi - pcr analyzes the distribution of microsatellite lengths at five standardized loci ( bethesda panel ) , 14 and immunohistochemistry detects the presence or absence of four proteins that are involved in the mmr pathway ( msh2 , msh6 , mlh1 , and pms2 )  . 
examples of such software include msings , 15 msisensor , 16 and mantis.17 a recent study by our group17 demonstrated that mantis achieves high sensitivity ( 97% ) and specificity ( 99% ) across six cancer typestested using samples with known msi status by msipcrand provides stable performance with varying numbers of microsatellite loci . 
because of this , mantis is particularly well suited for application to a wider variety of cancer types . as clinical msi testing is routinely performed only on colorectal and endometrial tumors , 18 the prevalence of msi in many other cancer types has been less well described . 
in addition , evidence exists that msi - pcr may be less accurate in other cancer types.19 a recent study by hause et al20 developed and applied the msi detection tool , mosaic , to perform a detailed survey of msi across 18 cancer types ( n = 5 , 930 cases ) ; however , many other cancer types have yet to be analyzed for msi . 
the ability to detect msi in novel cancer types would permit the investigation of immuneenhancing therapies in these cancers , with the potential to benefit previously unknown subsets of patients with cancer with msi . to perform a more comprehensive assessment of msi across many additional cancer types than those analyzed by hause et al , our study determined the prevalence of msi in 39 distinct cancer types ( n = 11 , 139 tumors from 11 , 080 patients ) by using our previously published msi - calling tool , mantis . methods data preprocessingthe cancer genome atlas and therapeutically applicable research to generate effective treatments for analysis , 10 , 701 cases of paired tumor - normal whole - exome sequencing data were obtained from the cancer genome atlas ( tcga ) 21 - 44 and therapeutically applicable research to generate effective treatments ( target ) 45 , 46 projects . 
data from all of these cases , with the exception of diffuse large b - cell lymphoma ( dlbcl ) were processed via our in - house automated pipeline , l - map ( landscape microsatellite analysis pathway )  . 
l - map is implemented in python and mysql and was run on the oakley supercomputer at the ohio supercomputing center.47 first , the metadata for all dna whole - exome bam files were downloaded from the genomic data commons ( gdc ) 48 and were converted to sql database entries . 
premarked duplicate reads were removed as above . data preprocessingother sources four hundred thirty cases of paired tumor - normal whole - exome sequencing data were obtained from the sequence read archive51 : 338 chronic lymphocytic leukemia cases from 279 patients from landau et al , 52 32 cutaneous t - cell lymphoma cases from choi et al , 53 51 nasopharyngeal carcinoma cases from zheng h et al , 54 and 8 cholangiocarcinoma cases from ong et al.55 fifteen additional cholangiocarcinoma cases were obtained from the european nucleotide archive56 from chan - on et al.57 all sample identifiers used are available in the data supplement . these cases were processed via l - map . 
coordinates for 2 , 539 microsatellite loci within or near the exomeoriginally introduced by salipante et al15 and used by later studies17 were converted from hg19 to hg38 by using liftover.61 nine unlifted loci were discarded , which left 2 , 530 regions that were used for analysis with mantis in all cohorts , with the exception of dlbcl ( data supplement )  . 
mantis was run with authorrecommended settings for whole - exome data minimum read quality , 20 ; minimum locus quality , 25 ; minimum locus coverage , 20 ; minimum repeat reads , one ; all other settings left at defaults . eight samples were observed to have fewer than 10 loci sufficiently covered and were dropped . 
variant annotation was performed by using annovar ( version 201602 - 01 ) 63 and gnu parallel.64 somatic mutations in the repair genes msh2 , msh6 , mlh1 , pms2 , exo1 , pold1 , and pole were determined by filtering variants with a dann68 , 69 pathogenicity score greater than 0.96 ( included in annovar )  . this threshold for dann was chosen as it was previously shown to provide optimal sensitivity and specificity.69 mutational signature calling was performed by using the tool deconstructsigs70 with the nature 2013 signatures set , which contains 27 signatures , 71 and the exome2genome normalization method . 
of 2 , 530 loci , we identified 22 loci that , within at least five cohorts , had an msi - h versus mss difference score greater than 0.75 and were sufficiently covered by at least 50% of samples in the cohort ( appendix table a2 )  . 
note that for chronic lymphocytic leukemia ( cll ) , the listed msi prevalence in panel a is out of 279 patients , and all 338 tumors are shown in panel b . 
acc , adrenocortical carcinoma ; aml , pediatric acute myeloid leukemia ( target ) ; blca , bladder carcinoma ; brca , breast carcinoma ; cesc , cervical squamous cell carcinoma and endocervical adenocarcinoma ; chol , cholangiocarcinoma ; coad , colon adenocarcinoma ; ctcl , cutaneous t - cell lymphoma ; dlbc , diffuse large b - cell lymphoma ; esca , esophageal carcinoma ; gbm , glioblastoma multiforme ; hnsc , head and neck squamous cell carcinoma ; kich , kidney chromophobe ; kirc , kidney renal clear cell carcinoma ; kirp , kidney renal papillary cell carcinoma ; laml , acute myeloid leukemia ( tcga ) ; lgg , lower - grade glioma ; lihc , liver hepatocellular carcinoma ; luad , lung adenocarcinoma ; lusc , lung squamous cell carcinoma ; meso , mesothelioma ; nbl , pediatric neuroblastoma ; npc , nasopharyngeal carcinoma ; ov , ovarian serous cystadenocarcinoma ; paad , pancreatic adenocarcinoma ; pcpg , pheochromocytoma and paraganglioma ; prad , prostate adenocarcinoma ; read , rectal adenocarcinoma ; sarc , sarcoma ; skcm , skin cutaneous melanoma ; stad , stomach adenocarcinoma ; tcgt , testicular germ cell tumor ; thca , thyroid carcinoma ; thym , thymoma ; ucec , uterine corpus endometrial carcinoma ; ucs , uterine carcinosarcoma ; uvm , uveal melanoma ; wt , wilms tumor . were within the set of 22 top - performing loci . these results indicate a striking heterogeneity of msi patterns across various types of cancer . all four disease types with the highest rates of msi prevalence were lynch syndromeassociated tumor types that have been previously known to exhibit msi : endometrial carcinoma , colon adenocarcinoma , gastric adenocarcinoma , and rectal adenocarcinoma . 
somatic variant calling was performed on whole - exome samples from these four cancer types , and the mean absolute number of somatic mutationsboth nonsynonymous and synonymouswas found to be increased among msi - h versus mss tumors within their own cohorts ( fig 3 )  . 
p values were calculated by using two - sided fishers exact test ( using signature presence or absence ) , with benjamini correction for multiple hypotheses.78 mmr pathway alterations msi - h lynch syndromeassociated tumors are known to lack the expression or function of at least one mmr protein ; therefore , we analyzed somatic mutations that were predicted to be deleterious ( by dann68 ) in the mmr genes msh2 , msh6 , mlh1 , pms2 , and exo1 , and the proofreading dna polymerases pold1 and pole , among msi - h and mss samples within acc , cesc , and meso ( appendix table a3 ; data supplement )  . 
although pold and pole are not considered mmr proteins , mutations in these genes have been shown to lead to somatic hypermutation.22 , 79 within these cohorts , 64% of msi - h cases and 7% of mss cases were found to contain at least one predicted deleterious somatic mutation in at least one of these genes ; however , given that these samples were sequenced with potentially different exome captures , together with the increased mutational burden of msi - h tumors , we could not determine the statistical significance of this finding . discussion in this study , we have performed , to our knowledge , the largest analysis of msi in human cancer exomes to date , including 11 , 139 whole - exome tumor - normal pairs from 39 types of cancer . compared with a study by hause et al , 20 we mantis score mantis score mantis score ascopubs.org / journal / po jco precision oncology 5 fig 2 . 
kernel density plots of mantis scores within ( a ) adrenocortical carcinoma ( acc ) , ( b ) cervical squamous cell carcinoma and endocervical adenocarcinoma ( cesc ) , and ( c ) mesothelioma ( meso )  . 
somatic mutational burden correlates with microsatellite instability high ( msi - h ) status within adrenocortical carcinoma ( acc ) and cervical squamous cell carcinoma and endocervical adenocarcinoma ( cesc )  . mutational burden is listed for ( a ) acc , ( b ) cesc , and ( c ) mesothelioma ( meso )  . 
previous studies of msi in acc have implicated lynch syndrome as a risk factor for familial acc80 , 81 ; however , to our knowledge , ngs - based msi analysis has not yet been applied to acc . msi - h colorectal tumors have been previously shown to be exceptionally sensitive to therapy with pd - 1 immune checkpoint inhibitors.12 identification of msi in novel tumor types may lead to an expanded role for immunotherapy and a broader scope of clinical msi testing.82 in addition , msi is known to be prognostic within colorectal cancer , 83 which may apply in other cancer types as well . 
clinical trials of immune checkpoint inhibitors are beginning or are underway in acc ( clinicaltrials.gov identifier : nct02673333 ) , cesc ( clinicaltrials.gov identifier : nct02635360 ) , and meso ( clinicaltrials.gov identifiers : nct02784171 , nct02991482 , nct02707666 , and nct02399371 ) , and a previous study of dendritic cell immunotherapy in acc84 demonstrated tumor marker but not clinical response . these studies may benefit from the retrospective evaluation of msi - h as a biomarker . 
prospective expansion of clinical msi testing to other cancer types may enlighten the prognostic and predictive value of msi - h for noncolorectal cancers . mmr deficiency is well recognized as the predominant cause of msi within colorectal , endometrial , and gastric cancers . 
if future studies indicate that msi in acc , cesc , and / or meso is indeed a result of mmr deficiency , the findings of this study may implicate previously unappreciated cancer types as being part of lynch syndrome . 
compared with germline alterations in mmr genes , somatic events are most often a result of hypermethylation of cpg islands in the promoter region of mlh1.4 additional investigation is needed to elucidate other molecular mechanisms that can lead to msi , as well as the downstream effects of msi on tumor - specific biology . 
in addition , of 9 , 569 tumors assessed in this study not within colorectal , endometrial , or gastric cancer , 77 ( 0.8% ) were msi - h . 
only 14 of these were within acc , cesc , or meso , which compromised the statistical power of our mutational signature analysis . a larger cohort of msi - h tumors would permit more comprehensive studies , including correlation with clinical data . in summary , we have detected msi in multiple cancer types , including acc , cesc , and meso , which indicates that msi may affect nonlynch syndrome tumor types . 
kautto no relationship to disclose affiliations all authors : the ohio state university , columbus , oh . support sameek roychowdhury stock and other ownership interests : johnson & johnson ( i ) research funding : takeda , ignyta acknowledgment we thank current and past members of the roychowdhury laboratory for their helpful insight and discussion . 
the chronic lymphocytic leukemia sequencing data ( dbgap : phs000922.v1.p1 ) used in this work was supported by national human genome research institute large - scale sequencing program grant no . 
kane mf , loda m , gaida gm , et al : methylation of the hmlh1 promoter correlates with lack of expression of hmlh1 in sporadic colon tumors and mismatch repair - defective human tumor cell lines . 
study of two large midwestern kindreds . 812 - 816 , 1993 arch intern med 117 : 206 - 212 , 1966 imai k , yamamoto h : carcinogenesis and microsatellite instability : the interrelationship between genetics and epigenetics . 
boland cr , thibodeau sn , hamilton sr , et al : a national cancer institute workshop on microsatellite instability for cancer detection and familial predisposition : development of international criteria for the determination of microsatellite instability in colorectal cancer . 
niu b , ye k , zhang q , et al : msisensor : microsatellite instability detection using paired tumor - normal sequence data . chem 60 : 1192 - 1199 , 2014 bioinformatics 30 : 1015 - 1016 , 2014 17 . 
giardiello fm , allen ji , axilbund je , et al : guidelines on genetic evaluation and management of lynch syndrome : a consensus statement by the us multi - society task force on colorectal cancer . 
faulkner rd , seedhouse ch , das - gupta ep , et al : bat - 25 and bat - 26 , two mononucleotide microsatellites , are not sensitive markers of microsatellite instability in acute myeloid leukaemia . 
cancer genome atlas research network : comprehensive genomic characterization of squamous cell lung cancers . nature 489 : 519 - 525 , 2012 [ erratum : nature 491 : 288 , 2012 ] 24 . 
hoadley ka , yau c , wolf dm , et al : multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origcell 158 : 929 - 944 , 2014 33 . 
zheng s , cherniack ad , dewal n , et al : comprehensive pan - genomic characterization of adrenocortical carcinoma . cancer cell 29 : 723 - 736 , 2016 [ erratum : cancer cell 30 : 363 , 2016 ] 42 . 
zheng h , dai w , cheung akl , et al : whole - exome sequencing identifies multiple loss - of - function mutations of nf - kb pathway regulators in nasopharyngeal carcinoma . 
chan - on w , nairismagi m - l , ong ck , et al : exome sequencing identifies distinct mutational patterns in liver flukerelated and non - infection - related bile duct cancers . 
oleary na , wright mw , brister jr , et al : reference sequence ( refseq ) database at ncbi : current status , taxonomic expansion , and functional annotation . 
quang d , chen y , xie x : dann : a deep learning approach for annotating the pathogenicity of genetic variants . bioinformatics 31 : 761 - 763 , 2015 69 . 
bacher jw , flanagan la , smalley rl , et al : development of a fluorescent multiplex assay for detection of msi - high 1113 - 1120 , 2013 tumors . 
gatalica z , vranic s , xiu j , et al : high microsatellite instability ( msi - h ) colorectal carcinoma : a brief review of predictive biomarkers in the era of personalized medicine . 
challis bg , kandasamy n , powlson as , et al : familial adrenocortical carcinoma in association with lynch syndrome . j clin endocrinol metab 101 : 2269 - 2272 , 2016 81 . 
patterns of mutational signatures ( s ) across microsatellite instability cancers : ( a ) adrenocortical carcinoma ( acc ) , ( b ) cervical squamous cell carcinoma and endocervical adenocarcinoma ( cesc ) , and ( c ) mesothelioma ( meso )  . 
listed are the number of samples ( mss or msi - h ) with at least one predicted deleterious mutation in msh2 , msh6 , mlh1 , pms2 , exo1 , pold1 , and pole . mutations were called by using mutect ( variant calling in methods ) and included in this table if the dann pathogenicity score was  . 
velez - velez1 ; horacio asbun , md1 ; and gerardo colon - otero , md1 introduction solid pseudopapillary neoplasms ( spns ) of the pancreas are rare , accounting for between 1% and 2% of all pancreatic tumors.1 - 5 these tumors are primarily seen in young women between the ages of 20 and 40 years who usually present with large pancreatic masses.1 , 2 surgical resection is highly effective , with 5 - year survival rates of 95% in patients able to have a complete resection.3 - 5 a recent retrospective review of spn cases treated surgically in the united states from 2004 to 2013 as part of the national cancer database identied 369 cases , with a 9.6% incidence of positive surgical margins and a 95.5% 5 - year overall survival.6 an english medical literature review up to december 2003 found a total of 718 well - documented published cases of spn of the pancreas.5 among 497 cases with adequate data , 97 ( 19.5% ) had evidence of either metastasis or local invasion.5 the most common sites of invasion or metastasis were the liver ( n = 27 ) , portal vein ( n = 26 ) , and spleen ( n = 17 ) .5 radiation is frequently used when margins of surgical resection are positive . 
surgical resection of metastasis is recommended whenever feasible , given the frequently indolent nature of the disease , with long - term diseasefree survival noted.6 - 8 given the rarity of this disease and the highly favorable outcomes with complete surgical resection , there is no standard of care for the systemic management of unresectable recurrent or metastatic disease.7 hao et al9 recently reported a meta - analysis of all aggressive spn cases , including locally invasive , recurrent , or metastatic tumors , reported in the english medical literature.9 a total of 59 patients were included in this review , most of whom ( 91.5% ) were able to undergo a curative resection . 
a 5 - year disease - free survival rate of 26.8% with a 5 - year overall survival of 71.1% was noted in this series.9 the presence of unresectable disease at presentation and recurrence within 3 years of initial diagnosis correlated with worse outcomes.9 we report a case of a patient with unresectable spn , treated with neoadjuvant chemotherapy followed by attempt at surgery and radiation therapy , who developed rapidly progressing biopsy - proven liver metastasis . 
an ultrasound - guided biopsy of the mass was positive for neoplasm and showed cells positive for cd10 and - catenin and negative for cytokeratin 7 , cam5.2 , p16 , pax - 8 , chromogranin , and synaptophysin , thus conrming the diagnosis of spn . involvement of blood vessels , the tumor was considered unresectable . 
there was some mild reduction in the tumor , which was followed by a pyloricpreserving total pancreatectomy with vascular construction of the superior mesenteric vein ( smv ) and the portal veat the time of surgery , the pancreatic mass was readily evident and was noted to involve the transverse colon mesentery , which was subsequently resected . 
 jorgensen et al context key objective is everolimus of potential benet in the treatment of patients with unresectable metastatic solid pseudopapillary neoplasms ( spns ) of the pancreas ? knowledge generated a patient with aggressive metastatic spn of the pancreas to the liver with ctnnb1 and pten mutations had a sustained signicant response to everolimus treatment , with excellent tolerability . 
a review of the literature shows no data on the frequency of pten mutations in spn tumor of the pancreas and no previous reported cases of everolimus treatment of spns . 
there are limited data on the effectiveness of systemic treatments in this rare tumor . relevance given the lack of standard treatments for metastatic unresectable spn of the pancreas , this case illustrates the potential role for genomic testing in this rare cancer . 
vascular reconstruction of the smv and the portal vein was performed using an 8 - mm ringed polytetrauoroethylene graft . mri of the abdomen and pelvis taken 6 weeks postoperatively showed recurrent tumor in the midabdomen with encasement of the sma and both replaced hepatic arteries ( fig 2 )  . 
a computed tomography scan performed 9 months postoperatively was consistent with liver metastases , which were conrmed on biopsy ( fig 3a )  . comprehensive genomic proling was performed on the primary tumor specimen using foundationone ( foundation medicine , cambridge , ma ) that revealed the following genomic alterations : ctnnb1 d32y and pten g165fs * 2 , y16fs * 1 pathogenic mutations . 
figure 4 summarizes the evaluation and treatment timeline . discussion spns are uncommon pancreatic neoplasms that typically present with localized disease , with excellent long - term prognosis after complete surgical resection . 
 ( c ) ct scan taken 18 months postoperatively showing continued response . current literature providing specic details on systemic treatments for unresectable , recurrent and / or metastatic spns of the pancreas is mostly limited to case reports ( table 1 )  . 
responses in single - case reports have been described with a combination chemotherapy regimen of ifosfamide , cisplatin , and etoposide , 11 with hyperthermic intraperitoneal chemotherapy with oxaliplatin and irinotecan , and with infusional uorouracil , leucovorin , and oxaliplatin ( folfox ) .12 , 13 selective intrahepatic radiation therapy was successful in controlling hepatic metastasis in a patient with recurrent disease.14 tamoxifen has been associated with stable disease in one case report.15 isolated case reports of different combination chemotherapy regimens listed in table 1 had failed to show signicant responses.13 , 16 a patient with metastatic disease responded to folfox chemotherapy.13 neoadjuvant treatment of unresectable spns with singleagent gemcitabine , with a combination of cisplatin , ifosfamide , etoposide , and vincristine , and with concomitant uorouracil and radiation therapy , have been shown to be effective in single - case reports.11 , 18 , 19 these treatment approaches resulted in signicant tumor size reductions , making surgical resection possible . 
in another report of a patient with unresectable hepatic metastases , treatment with intrahepatic perfusions with melphalan resulted in stable disease for 10 months.21 another report of a patient with multiple liver metastases after surgical resection of spn treated with intra - arterial chemotherapy ( cisplatin , doxorubicin , and uorouracil ) and systemic therapy with gemcitabine and erlotinib failed to provide long - lasting responses.2 a liver transplantation was ultimately performed in this patient , with no recurrence on follow - up 1 year after transplantation.2 in the case reported here , comprehensive genomic prole of the tumor revealed ctnnb1 and pten pathogenic mutations . 
 jorgensen et al ( 83% ) , and 14 of 14 ( 100% ) spn tumors analyzed , for an average of 90.3% ; therefore , ctnnb1 alterations have been implicated in the pathogenesis of spns.24 - 26 besides the wnt / - catenin pathway , whole - genome mrna expression analyses of 14 spn tumors by kim et al6 showed that genes involved in activation of the hedgehog and androgen receptor signaling pathways are also differentially up - regulated in spns compared with pancreatic adenocarcinomas , neuroendocrine tumors , and non - neoplastic pancreatic tissues . 
a proteomic analysis demonstrated upregulation of proteins that directly interact with the key proteins of notch , hedgehog , and androgen receptor signaling pathways.27 these ndings have paved the way to the development of many preclinical and clinical studies exploring cancer therapies that aim to exploit loss of pten function and investigating the role of pten loss in tumorigenesis and resistance to cancer therapy.25 there are limited data available on the frequency of pten mutations in spn tumors . 
pten was not listed as one of the genes that was down regulated in spn in the studies reported by kim et al6 and cavard et al.28 although inhibitors of the mtor pathway , such as everolimus , have been effective in decreasing growth of pten - decient tumors in preclinical studies , clinical studies in several tumor subtypes have yielded mixed results.29 the phosphatase and tension homolog ( pten ) tumor suppressor gene is a potent regulator of the phosphatidylinositol 3 - kinase / akt / mammalian target of rapamycin ( mtor ) pathway , which stimulates cell growth , proliferation , and survival . 
deciencies in pten due to mutations or homozygous loss , as in the case reported here , tumor subtypes . are frequently found across different everolimus ( rad - 001 [ 40 - o - ( 2 - hyrdoxyethyl ) - rapamycin ] ) is an orally administered rapamycin analog with various indications approved by the us food and drug administration , including advanced renal cell carcinoma , progressive pancreatic neuroendocrine tumors , renal angiomyolipoma , and subependymal giant cell astrocytoma associated with tuberous sclerosis.30 - 32 everolimus table 1 . 
 everolimus in spn pancreas adverse effects include stomatitis , rash , fatigue , infection , pulmonary toxicities , hyperglycemia , anemia , and thrombocytopenia.31 because genetic alterations or loss of pten have been proposed as predictors of sensitivity to everolimus independently of advanced tumor type , we hypothesized that the spn recurrence in our patient might be responsive to everolimus.30 , 32 the effective use of everolimus on the basis of low level of pten expression has also been reported in a patient with metastatic renal medullary carcinoma , another rare type of tumor without well - dened management.33 moreover , a study using a mouse model with a gain - of - function mutation in the - catenin gene and homozygous deletion of pten in cells of the mouse ovary to study ovarian endometrioid adenocarcinoma showed that dysregulation of wnt and pten signaling pathways resulted in increased mtor activity and more aggressive tumors.34 the same group showed that mtor inhibition with rapamycin reduced tumor growth in these mice . these data and our patients ndings suggest that the pten mutation may have contributed to the aggressiveness of her tumor . 
recently , results from a phase ii trial provided encouraging evidence that treatment with the mtor inhibitor temsirolimus in combination with chemotherapy improves progression - free survival in patients with advanced endometrial cancer with identied tsc2 somatic mutations.35 similar to oncogenic alterations in pten , tsc2 mutations are also known to cause aberrant activation of the phosphatidylinositol 3 - kinase / akt / mtor pathway . 
these data suggest that the combination of bevacizumab and everolimus might be of added benet in tumors with ctnnb1 and pten mutations like our patients tumor . in summary , this unique patients experience suggests that metastatic spn tumors with pathogenic mutations of ctnnb1 and pten may have signicant long - term benet from treatment with everolimus . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . gerardo colon - otero research funding : novartis ( inst ) no other potential conicts of interest were reported . references reddy s , cameron jl , scudiere j , et al : surgical management of solid - pseudopapillary neoplasms of the pancreas ( franz or hamoudi tumors ) : a large singleinstitutional series . 
j am coll surg 208 : 950 - 957 , discussion 957 - 959 , 2009 dovigo ag , daz mb , g utierrez mg , et al : liver transplantation as treatment in a massive metastasis from gruber - frantz pancreatic tumor : a case report . transplant proc 43 : 2272 - 2273 , 2011 vollmer cm , jr . , dixon e , grant dr : management of a solid pseudopapillary tumor of the pancreas with liver metastases . 
hpb 5 : 264 - 267 , 2003 casanova m , collini p , ferrari a , et al : solid - pseudopapillary tumor of the pancreas ( frantz tumor ) in children . 
med pediatr oncol 41 : 74 - 76 , 2003 papavramidis t , papavramidis s : solid pseudopapillary tumors of the pancreas : review of 718 patients reported in english literature . 
j am coll surg 200 : 965 - 972 , 2005 leraas hj , kim j , sun z , et al : solid pseudopapillary neoplasm of the pancreas in children and adults : a national study of 369 patients . 
j pediatr hematol oncol 40 : e233 - e236 , 2018 kim cw , han dj , kim j , et al : solid pseudopapillary tumor of the pancreas : can malignancy be predicted ? surgery 149 : 625 - 634 , 2011 lubezky n , papoulas m , lessing y , et al : solid pseudopapillary neoplasm of the pancreas : management and long - term outcome . 
hao eu , hwang hk , yoon d - s , et al : aggressiveness of solid pseudopapillary neoplasm of the pancreas : a literature review and meta - analysis . 
gao h , gao y , yin l , et al : risk factors of the recurrences of pancreatic solid pseudopapillary tumors : a systematic review and meta - analysis . 
rebhandl w , felberbauer fx , puig s , et al : solid - pseudopapillary tumor of the pancreas ( frantz tumor ) in children : report of four cases and review of the 12 . 
krug s , bartsch dk , schober m , et al : successful selective internal radiotherapy ( sirt ) in a patient with a malignant solid pseudopapillary pancreatic neoplasm 15 . 
hofmann h , von haken r , werner j , et al : unresectable isolated hepatic metastases from solid pseudopapillary neoplasm of the pancreas : a case report of chemosaturation with high - dose melphalan . 
abraham sc , klimstra ds , wilentz re , et al : solid - pseudopapillary tumors of the pancreas are genetically distinct from pancreatic ductal adenocarcinomas and almost always harbor beta - catenin mutations . 
lee l , ito t , jensen rt : everolimus in the treatment of neuroendocrine tumors : efcacy , side - effects , resistance , and factors affecting its place in the treatment sequence . 
lipkin js , rizvi sm , gatalica z , et al : therapeutic approach guided by genetic alteration : use of mtor inhibitor in renal medullary carcinoma with loss of pten 8 : 55582 - 55592 , 2017 expression . 
tanwar ps , zhang l , kaneko - tarui t , et al : mammalian target of rapamycin is a therapeutic target for murine ovarian endometrioid adenocarcinomas with dysregulated wnt / - catenin and pten . 
these results highlight the importance of screening for ntrk fusions as part of the tumor genomic proling for patients with pediatric cancer . jco precis oncol 5 : 204 - 214 . 
2021 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction rearrangements involving the neurotrophic tyrosine receptor kinase ( ntrk ) genes ntrk1 , ntrk2 , and ntrk3 encode fusion proteins containing the intact ntrk kinase domain that are oncogenic drivers of a histologically diverse group of tumors . 
 ntrk fusions in pediatric tumors : frequency , partner , and outcome content key objective ntrk fusions are tumor - agnostic or age - independent biomarkers that identify patients suitable for treatment with the us food and drug administrationapproved trk inhibitors . 
this single institutional study examined the frequency , fusion partner , and clinical outcome of ntrk fusions in a large cohort of unselected patients with pediatric cancer . knowledge generated our study demonstrated that ntrk fusions are more frequent in pediatric tumors and involve a broader panel of fusion partners and a wider range of tumors those than previously recognized and highlights pediatric cancers in which ntrk fusions are more or less likely to occur . relevance this study provided important ndings regarding ntrk fusionpositive pediatric cancers and cancers where ntrk fusions are rarely seen . 
immunohistochemistry was performed in a subset of cases using pan - trk antibody ( abcam , cambridge , uk , 1 : 100 dilution ) ( table 1 )  . fusion gene detection was performed using the chop cancer fusion panel as previously described.15 briey , targetspecic primers covering 673 exons were custom designed to identify known fusion genes and potential novel fusion genes associated with 110 cancer genes using anchored multiplex polymerase chain reaction ( pcr ) technology ( archerdx , inc , boulder , co )  . 
all fusions , when identied in our laboratory for the rst time , were conrmed by nested pcr followed by sanger sequencing . mutations ( single nucleotide variants [ snvs ] and small insertion / deletions [ indels ] ) and copy number alterations ( cnas ) were evaluated by using the chop hematological malignancy panel ( chmp ) or solid tumor panel ( cstp ) as described previously.16 briey , genomic dna was extracted from the tumor samples . 
libraries were prepared using probes targeting 118 genes ( chmp ) or 238 genes ( cstp ) , respectively , and sequenced on illumina hiseq platform using 150 bp paired - end sequencing . 
sequence data were analyzed using the home brew software concords v2 ( for snvs and indels ) and nextgene v2 next - generation sequencing analysis software ( for cnas ; softgenetics , llc , state college , pa )  . 
clinically signicant variants including indels , and cnas were conrmed by sanger sesnvs , quencing , multiplex ligation - dependent probe amplication ( mlpa ) , real - time pcr , or droplet digital pcr ( ddpcr ) when necessary . 
by contrast , different ntrk fusions were present in tumors with the same histological diagnosis , such as kctd16 - ntrk2 and trim24 - ntrk2 in two male patients of similar age with ganglioglioma , who grade i ( table 1 )  . 
in ptc , tumorigenesis is associated with constitutive activation of the mapk and pi3k signaling pathways secondary to somatic point mutations in braf , pten , dicer1 , and ras as well as fusions involving ret , alk , and ntrk.3 , 23 gene fusions have been reported in both sporadic and radiation - induced ptcs and are more common in pediatric ( 50% - 60% ) compared with adult tumors ( approximately 15% ) .2 , 23 ret and ntrk fusions are the most common , reported in 25%30% and approximately 10% ( range , 0% - 26% ) of pediatric ptcs , respectively.2 , 23 ntrk3 fusions are usually observed more commonly in ptcs than ntrk1 fusions , 2 , 23 which is similar to what we observed in our patient cohort . 
all the ptcs in our cohort were sporadic , none associated with ntrk1 ntrk2 ntrk3 snvs / indels cnas 10 - 15 16 - 20 gender female male snvs and indels missense frameshift inframe cnas gain loss gain and loss cnloh fig 1 . 
case 17 demonstrates a low - grade tumor with strn3 - ntrk3 showing moderately cellular spindle to round cell proliferation with prominent vascular proliferation ( c , h&e , 200 )  . 
case 21 shows widely invasive follicular variant of papillary thyroid carcinoma with etv6 - ntrk3 demonstrating extrathyroidal extension and angioinvasion ( d , h&e , 50 ) and nuclear features of papillary thyroid carcinoma ( inset , 400 )  . 
two cases demonstrate the diffuse sclerosing variant of papillary thyroid carcinoma showing solid and papillary groups inltrating the thyroid parenchyma with numerous psammomatous calcications ( e , h&e , 100 )  . 
the data are limited but suggest that the presence of an ntrk fusion may have diagnostic , prognostic , and therapeutic signicance in ptcs and may hold clinical utility for stratifying surgical and medical care . 
standard therapy for advanced ptcs in children involves surgical resection of gross disease followed by radioactive - iodide ( rai ) therapy . in a recent multicenter , open - label phase i or ii study of larotrectinib for the treatment of pediatric patients with solid tumors , two children with advanced ptcs were treated for  . 
7 months and remained progression - free , although , unfortunately , the objective response to treatment was not reported.25 ntrk fusions in pediatric cns tumors all the cns tumors identied with ntrk fusions were either gliomas or mixed neuronal glial tumors . 
four of the six ntrk2 fusions were novel fusions at the time of discovery with different 5 ( cid : 1 ) - partner genes including kank1 , c2orf4 , kctd16 , and specc1l . 
ntrk fusion genes have been described in pediatric lowand highgrade gliomas at a low prevalence , 26 although one study reported a nding of 40% of nonbrainstem high - grade gliomas in children younger than 3 years old containing an ntrk fusion gene ( n = four of the 10 samples ) .27 six of the 7 ntrk fusionpositive cns tumors were low - grade gliomas ( lgg ) , which may be partially due to the higher frequency of lgg in our unselected pediatric cohort ( approximately 45% of all cns tumors )  . 
although the duration of follow - up in our study is limited , the majority of patients underwent standard of care for their cns tumor subtype with resection of the primary tumor without recurrence . 
larotrectinib and entrectinib have shown antitumor effect for both primary brain tumors and solid tumors with brain metastases with systemic administration suggesting adequate penetration of the blood - brain barrier.11 in a study of nine patients with primary cns tumors treated with ntrk inhibitors , disease control was observed in all evaluable patients with stable disease in seven patients . 
some tumors exhibited typical age , morphology , and etv6 - ntrk3 fusion compatible with if , showing densely cellular fascicular growth of primitive ovoid cells and inltration of surrounding tissue ( cases 13 and 14 )  . 
clinical classication and treatment based on histology only , particularly in soft - tissue tumors with spindle morphology , have proven to be challenging in some cases because of variable histologic features and immunohistochemical patterns.30 as more tumors are being studied for fusions , the morphologic spectrum is expanding , such that the fifth edition of the soft tissue and bone tumors who classication31 now includes an emerging entity titled ntrk - rearranged spindle cell neoplasm to encompass spindle cell soft - tissue tumors with ntrk gene rearrangements ( other than if )  . 
it remains to be seen if soft - tissue histologic classication , molecular classication , or some combination of both will provide clinicians with the most accurate information for personalized treatment . 
magnetic resonance imaging at diagnosis ( a ) , 1 month post - targeted therapy ( b ) , and 5 months post - therapy ( c ) showing marked tumor shrinkage . 
at diagnosis , the tumor contained densely cellular areas with spindled to round cells and increased mitosis ( d , h&e 400 ) with cytoplasmic and membranous pan - trk immunohistochemistry ( inset )  . 
ccd , coiled - coil domain ; chd , calponin homology domain ; ecd - lb , extracellular ligand binding domain ; h&e , hematoxylin and eosin staining ; pkt , protein tyr kinase ; tm , transmembrane domain ; ss , signal sequence . ntrk fusions facilitate precision diagnosis , prognosis , and therapy follow - up information is available for all patients , and follow - up times ranged from 6 to 46 months . 
in almost all cases , the detection of an ntrk fusion conrmed the morphologic diagnosis , and in ve cases , the nal tumor diagnosis was largely based on the discovery of an ntrk fusion ( excluding other differential diagnostic considerations )  . 
one exception was the case of secretory carcinoma , which was initially diagnosed as mucoepidermoid carcinoma , but later changed to secretory carcinoma following the detection of the etv6 - ntrk3 and additional immunohistochemical evaluation ( case 10 )  . 
collectively , we analyzed 261 common embryonal solid tumors ( 79 neuroblastomas , 29 medulloblastoteratoid / rhabdoid mas , 28 wilms tumors , 13 atypical tumors , and 11 hepatoblastomas ) , bone tumors ( 27 osteosarcomas and 24 ewing sarcomas ) , and skeletal muscle tumors ( 50 rhabdomyosarcomas ) , and none had ntrk fusions . 
thus , although it is difcult to exclude the possibility that ntrk fusions might occur in individual tumors of these subtypes , which accounted for about one third of all solid tumors in this cohort , they are likely to be rare . case 15 was a 6 - month - old male infant with a left upper extremity mass . 
prospectively , the rest of the patients who survived with the standard therapy may also benet from trk inhibitor therapy if their tumors progress or recur . received neoadjuvant we assessed the clinical outcome of all ntrk fusionpositive patients for up to 46 months ( median 21 months )  . 
the median follow - up time for patients with cns tumors was 21 months . the majority of ntrk - positive lgg demonstrated superb outcome with gross total resection without additional therapy . one patient with congenital glioblastoma and an etv6ntrk3 fusion died 3 months after birth , and the tumor specimen was obtained via autopsy . 
our review of 1 , 217 patients showed that ntrk fusions are more frequently seen in pediatric tumors than in adult tumors and involve a broader panel of fusion partners and a wider range of pediatric tumors than previously recognized . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / cci / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . elizabeth fox other relationship : helsinn therapeutics gerald b . 
wertheim employment : johnson & johnson stock and other ownership interests : johnson & johnson vinodh pillai consulting or advisory role : foundation medicine travel , accommodations , expenses : foundation medicine jennifer e . 
genes ( basel ) 10 : 723 , 2019 prasad ml , vyas m , horne mj , et al : ntrk fusion oncogenes in pediatric papillary thyroid carcinoma in northeast united states . 
hsiao sj , zehir a , sireci an , et al : detection of tumor ntrk gene fusions to identify patients who may benet from tyrosine kinase ( trk ) inhibitor therapy . 
nat rev clin oncol 15 : 731 - 747 , 2018 suurmeijer aj , dickson bc , swanson d , et al : the histologic spectrum of soft tissue spindle cell tumors with ntrk3 gene rearrangements . 
papadopoulos kp , gandhi l , janne pa , et al : first - in - human study of ds - 6051b in patients ( pts ) with advanced solid tumors ( ast ) conducted in the us . 
drilon a , ou si , cho bc , et al : repotrectinib ( tpx - 0005 ) is a next - generation ros1 / trk / alk inhibitor that potently inhibits ros1 / trk / alk solventfront mutations . 
lopez gy , perry a , harding b , et al : cdkn2a / b loss is associated with anaplastic transformation in a case of ntrk2 fusion - positive pilocytic astrocytoma . neuropathol appl neurobiol 45 : 174 - 178 , 2019 19 . 
surrey lf , jain p , zhang b , et al : genomic analysis of dysembryoplastic neuroepithelial tumor spectrum reveals a diversity of molecular alterations dysregulating the mapk and pi3k / mtor pathways . 
tuttle rm , haugen b , perrier nd : updated american joint committee on cancer / tumor - node - metastasis staging system for differentiated and anaplastic thyroid cancer ( eighth edition ) : what changed and why ? thyroid 27 : 751 - 756 , 2017 25 . 
laetsch tw , dubois sg , mascarenhas l , et al : larotrectinib for paediatric solid tumours harbouring ntrk gene fusions : phase 1 results from a multicentre , 26 . 
okamura r , boichard a , kato s , et al : analysis of ntrk alterations in pan - cancer adult and pediatric malignancies : implications for ntrk - targeted theropen - label , phase 1 / 2 study . 
nat genet jones ka , bossler ad , bellizzi am , et al : bcr - ntrk2 fusion in a low - grade glioma with distinctive morphology and unexpected aggressive behavior . 
robinson gw , gajjar aj , gauvain km , et al : phase 1 / 1b trial to assess the activity of entrectinib in children and adolescents with recurrent or refractory solid tumors including central nervous system ( cns ) tumors . 
mandrekar , phd1 with the launch of the national cancer institutes precision medicine initiative in 2015 , there has been a shift to trial designs that tailor health care solutions to individual patients by using a screening platform and by moving away from the one - trial / one - biomarker - at - a - time approach . 
2019 by american society of clinical oncology introduction precision medicine takes into account the variability of an individual patients genes , environment , and lifestyle when deciding the best approach for disease prevention and treatment.1 since the rst publication of the human genome in february 2001 , 2 scientists have made great progress in understanding inherited differences in genes , making precision medicine a tangible reality . 
even though precision medicine is a relatively new term , tailoring intervention on the basis of a patients specic features is not a new idea . many currently available us food and drug administration ( fda ) approved therapies are based on specic patient subgroups . 
for example , rituximab , the rst monoclonal antibody treatment for cancer , was approved by the fda in 1997 for patients with relapsed or refractory cd20 + , b - cell , low - grade , or follicular non - hodgkin lymphoma . 
the addition of cetuximab to rst - line chemotherapy in patients with kras wild - type metastatic colorectal cancer has been shown to signicantly improve treatment outcomes compared with chemotherapy alone in two randomized clinical trials , crystal ( clinicaltrials.gov identier : nct00154102 ; cetuximab combined with irinotecan in first - line therapy for metastatic colorectal cancer ) 3 and opus ( clinicaltrials.gov identier : nct00125034 ; oxaliplatin and cetuximab in first - line treatment of metastatic colorectal cancer [ mcrc ] ) .4 erlotinib and crizotinib are fda - approved therapies for patients with advanced lung cancer who test positive for an epidermal growth factor receptor ( egfr ) mutation and the alk rearrangement , respectively . 
it was later found that patients with higher programmed death - ligand 1 expression treated with pembrolizumab had longer progression - free survival ( pfs ) and overall survival ( os ) .5 most recently , on may 23 , 2017 , the fda granted accelerated approval to pembrolizumab as the treatment for adult and pediatric patients with unresectable or metastatic microsatellite instability - high or mismatch repair decient solid tumors , 6 which is the rst time that the fda has approved a cancer treatment for an indication on the basis of a common biomarker rather than the primary site or origin ( ie , tissue agnostic ) .7 there are continuing efforts in drug development to move away from the one - size - ts - all approach and toward precision medicine . in this article , we aim to introduce novel trial designs by using case studies as examples . 
 ou et al context key objective to discuss types of clinical trial designs for accelerating development of new drugs in the era of precision medicine . knowledge generated clinical trial designs ( such as umbrella , basket , and subgroup ) that evaluate multiple hypotheses are efcient precision medicine initiatives . 
other designs include enrichment designs for validating the efcacy of an agent in biomarker - driven subgroups and window of opportunity designs to help understand the antitumor activity of an agent in a treatment - naive patient population . relevance the clinical trial designs discussed in this article are practical and relevant for precision medicine trials . 
several case studies are provided to demonstrate the utility of these clinical trial designs . the results of tumor gene sequencing or other testing techniques , such as polymerase chain reaction , ow cytometry , uorescence in situ hybridization ( fish ) , chromogenic in situ hybridization , and immunohistochemistry . 
specic to genotype - driven trials , patients will be matched to trials that are developed on the basis of prior understanding of certain molecular aberrations that may determine sensitivity to certain therapies . 
if a patient is eligible for more than one genotype - driven trial , the trial will be selected by the treating physician on the basis of the totality of information , such as the patients preferences and status , expected benets , and molecular characteristics . 
enrichment designs test selective therapeutic options for matching patients that can result in better patient outcomes.9 the genotype - driven trial mentioned in the case study is one type of enrichment design ; other types of enrichment , such as that based on the epigenome , is also appropriate . 
enrichment trial design is best suited when the rationale for the trial strongly suggests that the agent would be effective in a specic biomarkerdened patients with a specific tumor type with biomarker status unknown may be part of the trial protocol biomarker profiling with targeted biomarker trial enrollment with targeted therapy without targeted biomarker standard of care fig 1 . 
it is particularly advantageous if the biomarker used to dene the patient population is considered soc ; if so , the trial design does not need to take into account the molecular testing because the information would be available for all patients . 
highly specic screening tests are required to identify the low - prevalence molecular prole and to avoid assigning patients who tested false positive to treatment options that may not be efcacious . 
finally , certain molecular proles may have low prevalence and may result in a large proportion of screened patients receiving the soc , which can be inefcient ( eg , if the prevalence of the molecular prole of interest is only 5% , 500 patients will need to be proled to identify 25 patients who are potentially eligible )  . umbrella design umbrella trials include patients with a single tumor type or disease and for which several biomarkers have been identied and used to dene disease subtypes.10 patients are screened for a panel of biochemical , genetic , and / or immunologic markers associated with their disease and , on the basis of the markers detected , assigned to a biomarkerdriven treatment strategy or targeted therapy that is most likely to result in favorable outcomes . 
thus , an umbrella trial consists of multiple substudies , each with independent subgroups of patients receiving different therapies and with the option of assuming different statistical parameters for independent designs . 
the substudies , however , exist under an overarching master protocol that uses a common infrastructure for screening and treatment assignment to reduce the cost and time associated with enrollment to unrelated and often sequential biomarker - informed studies . 
lung - map ( clinicaltrials.gov identier : nct03851445 ; a master screening protocol previously - treated non - small cell lung cancer ) is an example of a phase ii / iii umbrella study in solid tumors.11 lung - map opened to accrual in 2014 for patients with advanced squamous cell carcinoma ( scc ) of the lung . similar to aml , scc of the lung is a biologically complex disease . 
roughly 60% of patients with lung scc harbor genetic abnormalities that might be targeted with therapy.14 lung - map provided a common infrastructure to test patient samples for multiple biomarkers , which informed therapy assignment within 10 to 14 days . 
patients were assigned to a substudy according to a predened algorithm that used specic genetic abnormalities detected via ngs in patients with a specific tumor type biomarker profiling targeted biomarker 1 targeted biomarker 2 targeted biomarker 3 targeted biomarker n no actionable biomarker regimen a regimen b regimen c regimen n standard of care or non - targeted regimen fig 2 . 
a substudy of new therapy versus soc remained open for patients without an actionable biomarker . lung - map was designed to test new therapies against soc for a particular subgroup using multiple , randomized phase ii trials with pfs as the primary end point . 
within a substudy , if a new therapy met criteria that suggested large the substudy was expanded to improvements in pfs , a phase iii study with pfs and os as co - primary end points . patients who were part of the phase ii interim analysis were included in the phase iii assessment . 
with a well - established screening platform in place , an umbrella trial design can be quite efcient in directing patients into different treatment regimens and accelerating the drug development process . 
the concept of umbrella trials can be applied in any disease setting , but it is most appropriate when there is a strong scientic rationale informing which therapies might work best in biomarker - dened subgroups . 
although not used in beat aml or lung - map , adaptive randomization can be applied within a substudy that has multiple arms to assign patients to a therapy that shows the most favorable outcome , given the data already collected.1 , 15 , 16 bayesian posterior probabilities that compare efcacy of experimental therapy with soc can be used in decision making . umbrella trials are exible and have the ability to open or close new substudies with different targeted therapies or statistical designs if appropriate . basket design basket trials are an efcient way of screening experimental therapeutics across patients with a variety of tumor types and / or histology under one master trial . 
basket trials involve several biomarker proles whereby patients are assigned to treatments based on the molecular alterations their tumors contain , regardless of the histologic type of the tumor.17 , 18 the basic premise for this trial design is that the average response to one or more experimental treatments is expected to vary across these different biomarker proles . like umbrella trials , there may be a common genetic screening platform , especially if the cohorts dened by the study are histology - independent and dened only by the presence of a single molecular aberration . 
figure 3 provides a generic representation of a basket trial design schema . basket trial designs may be appropriate when there is reason to believe that one or more experimental treatments may be effective across a range of primary tumor sites or histologies , and in particular , when patients with specic biomarker proles are expected to respond well to a particular treatment , regardless of their specic disease or histology . 
one such design is the bayesian basket design.20 in this design , a bayesian approach is used to model the response probabilities for the various histologic strata , and two hypotheses are considered : ( 1 ) the response probabilities for a particular targeted agent are equal across the corresponding histologic strata , and ( 2 ) the activity of the drug is independent across these strata . 
a hierarchical bayesian design has recently been proposed for randomized phase ii trials with multiple groups ( or baskets ) , allowing for information sharing across baskets while at the same time having the ability to help with decision making for each individual trial or basket.21 within the hierarchical bayesian design framework , the estimate of the treatment effect for each phase ii trial basket within this master protocol shrunk toward the overall mean treatment effect , which alleviates concerns regarding randomness across the trials or baskets.21 , 22 this approach is most powerful when the treatment effects are heterogeneous across the different baskets and inates the type i error in the inferior treatment effect groups or baskets in the case of heterogeneous treatment effects across baskets . 
it has been noted that the results of a basket trial can be heavily tilted toward positive conclusions , 23 even with specication of weak priors in a bayesian setting . 
the goal of this study , as noted in the publication , was to identify molecular biomarkers and determine their frequency and clinical relevance in patients with advanced nonsmall - cell lung cancer , and thymic malignancies and to evaluate the efcacy of multiple targeted therapies in specic molecular subsets of patients.27 ( p1001 ) a limitation of this trial was the lack of the lung cancer , small - cell adaptive trial feature , because seamless addition of new treatment arms for molecular targets or replacing treatments for a particular molecular subtype was not possible . another issue was the timeliness of the availability of some of the core molecular proling results , which had an impact on enrollment to the trial.27 acs e and tapur case studies . 
the french national cancer institutes acs e initiative ( secured access to innovative therapies ) 28 and ascos tapur trial ( clinicaltrials.gov identier : nct02693535 ; testing the use of food and drug administration [ fda ] approved drugs that target a specic abnormality in a tumor gene in people with advanced stage cancer ) 29 are examples of large - scale trials with basket - type design that evaluate the safety and efcacy prole of approved agents in other indications ( ie , tumor types )  . 
the goals of these trials are to provide patients with advanced diseases ( without promising treatment options ) off - label access to approved agents and to evaluate the safety and efcacy of these agents outside their approved indications . 
the acs e - crizotinib trial ( clinicaltrials.gov identier : nct02034981 ; phase 2 study assessing efcacy and safety of crizotinib in patients harboring an alteration on alk , met or ros1 ) is the rst trial coordinated by the acs e prograit includes patients with various solid and hematologic malignancies . 
basket designs test a single agent in different histologic subtypes , which can be efcient if the goal is to screen for antitumor activity of the agent in different disease settings . 
for example , only two ( nsclc with egfr mutations and nsclc with kras or braf mutations ) of the 15 substudies in the custom trial completed accruing ; accrual was unsuccessful for the other 13 rare histologic subtypes . 
 ou et al experimental therapy in the subgroup of patients positive for the biomarker as well as in either the subgroup of patients negative for the biomarker or in all patients . 
with co - primary objectives , the signicance level ( ) is allocated or split between the two objectives to maintain an acceptable overall type i family - wise error rate using a conservative bonferroni correction or a less conservative correction that considers the correlation between the two tests.31 in the case with co - primary objectives dened for the biomarker - positive subgroup and all patients , the design can be subgroup focused or all - population focused.32 a subgroup - focused design is most appropriate when there is evidence that the experimental therapy will be most effective in patients with the biomarker of interest , but it could also have a broad impact in the general disease population . 
a design with an all - population focus is most appropriate when there is less evidence that the experimental therapy will be most effective in patients with the biomarker of interest but could be effective in the general disease population . 
with an accrual goal of 618 patients egfr - positive by fish , there was 92% power to detect a hazard ratio of 0.75 for pfs in the cetuximab arm relative to the control arm with a type i error rate ( ) of 2% . 
with an accrual goal of 1 , 546 total patients , there was 86% power to detect a hazard ratio of 0.83 for os in the cetuximab arm relative to the control arm with a one - sided of 1.5%. 
because of slow accrual and a lower - than - expected percentage of patients who were egfr positive , the study was amended to 400 egfrpositive patients to detect planned differences in pfs with 80% power . 
 case studies for innovative trial designs multiple hypotheses in a subgroup design allows formal testing of efcacy for a biomarker - dened subgroup of patients and the overall patient population . 
by testing multiple hypotheses , the power for testing a specied effect in the overall population is reduced.32 furthermore , in a subgroup - focused design , as the prevalence of a biomarker decreases , the total sample size increases.32 costs also increase because tissue procurement and biomarker testing is required for all patients to reach the target number of subgroup patients.33 these additional resources can be offset by testing for larger treatment effects in the biomarker - dened subgroup.32 window of opportunity design the primary goal of window of opportunity ( woo ) design , is to unalso known as window or phase 0 design , derstand the antitumor activity of an agent in a disease state that is not disrupted by previous or simultaneous treatments . 
these trials occur between patient diagnosis and initiation of standard treatment and typically have a clinical end point such as tumor response or pfs at a predened early time point , often assessed by radiographic imaging ( figure 5 )  . 
woo designs are distinct from neoadjuvant trial settings , in which an investigational agent is commonly given preoperatively along with cytotoxic chemotherapy or hormonal therapy for a longer period of time than in a woo trial . 
woo trials have recently gained attention , specically in the context of evaluating molecular agents or in early advanced or metastatic disease in which the tumor may not be resistant to selective inhibition of a novel cancer target.38 evaluating molecular end points can be hindered by difculties in procuring tumor tissue before and after drug administration and by heterogeneity in previous exposure to cancer therapies . 
woo trials help to overcome these difculties by enrolling treatment - nave patients , including a translational research component , and occurring in a short time frame before the initiation of the standard treatment of the disease . 
the ultimate hope of woo trials is that they can expedite the drug development process by improving understanding of an agents biologic effect early in its development , validating markers that may predict subsets of patients who will benet , and targeting select short courses of experimental treatment diagnosis of disease standard of care ( eg , surgery ) standard treatment option fig 5 . 
it is a design best suited for trials in which the treatment cycles are short , and a short - term outcome ( eg , biomarker change ) is the end point of interest . 
in addition , although it is not yet documented , there is the theoretical possibility that the investigational agent may in fact induce resistance to subsequent therapies . logistically , woo trials require careful coordination among members of a multidisciplinary study team because diagnosis , workup , and study treatment must all be arranged in a short time frame . 
furthermore , tumor heterogeneity can be an issue , and thus it is essential to have the infrastructure capable of performing a centralized and high - quality review of all biomarkers.40 woo trials were initially used to inform a go / no - go decision in development of anticancer agents , to expose a small number of patients to a limited duration and dose of a drug and subsequently proceed to phase i / ii trials on the basis of the results.41 ( p2574 ) woo trials may not be advantageous in settings in which an agent has already been well - studied in other disease setfor which the toxicity prole is already welltings , understood , or even for which little safety data are available regarding the risk of delaying surgery.40 despite these concerns and considerations , woo trials have proved benecial in some settings . 
 ou et al active in untreated patients.42 , 43 woo trials should therefore be considered as a promising alternative trial design . discussion with the ability to simultaneously evaluate the effects of therapies on multiple biomarkers and diseases , umbrella and basket trial designs can help accelerate the discovery of targeted therapy . 
because each substudy of the umbrella and basket trials is essentially an enrichment trial design , we would expect a larger effect size , given the available prior knowledge of certain genotype proles and their sensitivity to therapy . 
woo trials allow investigators to understand the antitumor activity of an agent in the de novo disease setting . trial designs in the precision medicine era require a platform to carry out the biomarker proling . 
given the advances in liquid biopsies , cell - free dna from plasma may provide a minimally invasive alternative to standard tumor biopsies.44 even though we have more options for proling patients , the proling is not without challenge . 
at the same time , a screening cut point will need to be established and validated to ensure the cut point value ( ie , the high and low levels of the biomarker value to predict the response to treatment ) is appropriate.45 in an early work , mandrekar et al46 outlined the criteria for choice of phase ii designs for initial validation of a predictive marker in a review article that considered assay performance , turnaround time , preliminary evidence , and marker prevalence . with the advances in targeted therapy , we now have many therapeutic options for specic targeted biomarkers ; however , mutations that have high prevalence in a certain disease may not be common in another ( for example , human epidermal growth factor receptor 2 overexpression has been reported in approximately 15%47 of patients with breast cancer but only approximately 2% of patients with colorectal cancer ) .48 designing a trial with a biomarker target with extremely low prevalence is a challenge because it is difcult to obtain a sufcient number of patients to properly power the study . 
a statistical trial design that uses small sample sizes is needed to tackle this issue , as well as potentially international partnerships . in this article , we have focused our discussion on trial designs that are better suited for phase ii and iii studies . 
seamless phase i / ii designs , which blur the distinction between dose selection and efcacy evaluation , are one such design . hobbs et al49 provided an overview of the use of seamless designs in rst - in - human studies based on abstracts submitted to asco annual meetings from 2010 to 2017 and provided guidance on the design and conduct of such trials . 
to validate biomarker associations identied from such studies , sufcient statistical rigor is still required , such as clear denition of the primary end point , a prespecied interim and nal analysis plan , and well - dened safety monitoring rules . with immunotherapy gaining traction in oncology , it uncertain whether the current designs can adequately address the needs for these agents . 
if the primary objective is to show efcacy in a biomarker - dened , tumor - specic patient population , then both enrichment design and umbrella design ( ie , as one of the substudies ) are applicable . 
as mentioned in the introduction , pembrolizumab received the rst tissue - agnostic fda approval , and there may be several factors that could have contributed to successfully ling for approval of pembrolizumab.50 first , the tissue collection in the initial randomized pembrolizumab trials allowed investigators to retrospectively test the tissue - agnostic hypothesis . 
second , immunotherapies are designed with a strong foundation of preclinical data . third , multiple prospective clinical trials have veried the hypotheses generated by the retrospective data that contributed to its approval . 
in the case of pembrolizumab , approval was based on the data from ve different trials in which patients were retrospectively identied from two studies and prospectively enrolled in three studies.7 because this design is the rst of its kind , it is unclear which design discussed in this article will be best suited for future agents targeting tissue - agnostic indications . 
nature 409 : 860 - 921 , 2001 van cutsem e , k ohne ch , hitre e , et al : cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer . 
n engl j med 360 : 1408 - 1417 , 2009 bokemeyer c , bondarenko i , makhson a , et al : fluorouracil , leucovorin , and oxaliplatin with and without cetuximab in the rst - line treatment of metastatic colorectal cancer . 
j clin oncol 27 : 663 - 671 , 2009 daud ai , wolchok jd , robert c , et al : programmed death - ligand 1 expression and response to the anti - programmed death 1 antibody pembrolizumab in melanoma . 
clin cancer res [ epub ahead of print on february 20 , 2019 ] zardavas d , maetens m , irrthum a , et al : the aurora initiative for metastatic breast cancer . 
br j cancer 111 : 1881 - 1887 , 2014 tsimberidou am , wen s , hong ds , et al : personalized medicine for patients with advanced cancer in the phase i program at md anderson : validation and landmark analyses . 
herbst rs , gandara dr , hirsch fr , et al : lung master protocol ( lung - map ) : a biomarker - driven protocol for accelerating development of therapies for squamous cell lung cancerswog s1400 . 
burd a , levine rl , shoben a , et al : initial report of the beat aml umbrella study for previously untreated aml : evidence of feasibility and early success in molecularly driven phase 1 and 2 studies . 
lopez - chavez a , thomas a , rajan a , et al : molecular proling and targeted therapy for advanced thoracic malignancies : a biomarker - derived , multiarm , multihistology phase ii basket trial . 
herbst rs , redman mw , kim es , et al : cetuximab plus carboplatin and paclitaxel with or without bevacizumab versus carboplatin and paclitaxel with or without bevacizumab in advanced nsclc ( swog s0819 ) : a randomised , phase 3 study . 
herbst rs , kelly k , chansky k , et al : phase ii selection design trial of concurrent chemotherapy and cetuximab versus chemotherapy followed by cetuximab in advanced - stage non - small - cell lung cancer : southwest oncology group study s0342 . 
kim es , moon j , herbst rs , et al : phase ii trial of carboplatin , paclitaxel , cetuximab , and bevacizumab followed by cetuximab and bevacizumab in advanced nonsquamous non - small - cell lung cancer : swog s0536 . 
lynch tj , patel t , dreisbach l , et al : cetuximab and rst - line taxane / carboplatin chemotherapy in advanced nonsmall - cell lung cancer : results of the randomized multicenter phase iii trial bms099 . 
pirker r , pereira jr , von pawel j , et al : egfr expression as a predictor of survival for rst - line chemotherapy plus cetuximab in patients with advanced nonsmall - cell lung cancer : analysis of data from the phase 3 flex study . 
machiels jp , bossi p , menis j , et al : activity and safety of afatinib in a window preoperative eortc study in patients with squamous cell carcinoma of the head 40 . 
schmitz s , caballero c , locati ld : perspectives on window of opportunity trials in head and neck cancer : lessons from the eortc 90111 - 24111 - noci - hncg and neck ( scchn )  . 
clark pi , slevin ml , joel sp , et al : a randomized trial of two etoposide schedules in small - cell lung cancer : the inuence of pharmacokinetics on efcacy and 44 . 
our real - time tumor sequencing program , which makes precision treatment decisions for patients with cancer , produces matched germline information , providing a unique opportunity to efficiently implement pharmacogenetics and benefit patients . methods the germline genetic database from the michigan oncology sequencing ( mi - oncoseq ) program was searched for 21 clinically actionable polymorphisms in five cancer - relevant genes : tpmt , dpyd , cyp2c19 , cyp3a5 , and ugt1a1 . 
the medical records of mi - oncoseq patients with actionable phenotypes were searched for receipt of relevant drugs and to determine whether having genetic information at the time of treatment would have led to a treatment recommendation . results all nine variants in tpmt , dpyd , and cyp2c19 that were detected in mi - oncoseq were confirmed by external genotyping . 
on the basis of retrospective assessment of 115 adult and pediatric patient records , 4.3% ( n = 5 ) had a potentially clinically actionable phenotype for tpmt , dpyd , or cyp2c19 and received a relevant medication . 
 after accounting for differences in adult and pediatric recommendations , three of these patients could have received a treatment recommendation at the time of prescribing . conclusion germline genotype determinations for tpmt , dpyd , and cyp2c19 can be used to make evidence - based treatment recommendations in mi - oncoseq patients . 
 although the proportion of patients for whom recommendations can be made is small , this added value to mi - oncoseq and patient care comes at no additional genotyping cost . 
2018 by american society of clinical oncology introduction the genomes of patients ( germline ) and their tumors ( somatic ) each provide useful information to guide precision medicine in oncology . 
vali dated pharmacogenetic associations exist for several germline polymorphisms with com monly used cancer treatments , including thiopu rines ( tpmt ) , 1 fluorouracil ( fu ) / capecitabine ( dpyd ) , 2 irinotecan ( ugt1a1 ) , 3 and tacrolimus ( cyp3a5 ) .4 additionally , there are several known associations with supportive care agents com monly used in patients during treatment , such fungal prophylaxis with voriconazole ( cyp2c19 ) 5 and antiemetic treatment with ondansetron ( cyp2d6 ) .6 despite their established clinical validity , few of these associations have been implemented into clinical practice . 
st jude childrens research hospital has led the way , implementing pre emptive pharmacogenetic testing to guide per sonalized treatment of several genedrug pairs.7 the experiences of early adopters has identified formidable challenges to implementing phar macogenetics into clinical practice , 8 , 9 which require substantial investment and expertise.10 , 11 daniel l . 
mody arul chinnaiyan author affiliations and support information ( if applicable ) appear at the end of this article . the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health . corresponding author : daniel l . 
although there is debate about the necessity and feasibility of demon strating clinical utility for pharmacogenetic implementation , 12 , 13 the lack of uptake indicates that health systems are not willing to incur the costs of pharmacogenetic implementation for unproven clinical benefit . 
however , available germline genetic information should be con sidered when making treatment decisions.14 in anticipation of a time when genetic informa tion is available for many patients , perhaps as a result of the proliferation of directtoconsumer genotyping , 15 the pharmacogenetic community has developed evidencebased pharmacogenetic treatment guidelines.3 , 16 in oncology , there has been a tremendous expan sion in the availability of genetic information as a result of the proliferation of tumor sequenc ing programs that use somatic genetic informa tion to personalize selection of targeted cancer treatments.17 although some programs analyze only the somatic genome , others have found that matched germline analysis improves quality control18 and enables simultaneous assessment of familial predisposition to cancer.19 this cre ates a unique situation in which germline genetic information for clinically relevant pharmaco genes is freely available.20 however , the oppor tunity to use these data in clinical practice has not yet been capitalized upon . the michigan oncology sequencing ( mi oncoseq ) program at the university of michi gan comprehensive cancer center ( umccc ) performs targeted sequencing of somatic and matched germline dna , in addition to somatic wholetranscriptome analysis . 
21 the targeted sequencing panel includes approximately 100 pharmacogenes that could be used to provide evidencebased pharmacogenetic treatment rec ommendations , if the accuracy of the germline genetic determinations are verified . 
detailed study information including inclusion and exclusion criteria , data analysis and processing , and return of results has been pre viously published.2123 briefly , patients treated at umccc with refractory tumors are invited to participate in a research protocol in which they provide tumor and blood samples for matched genetic sequencing , among other somatic anal yses . 
 since the initiation of mioncoseq , the dna sequencing platform has transitioned from wholeexome sequencing to a targeted exon sequencing panel , and sequencing methods have been previously described in detail.21 , 23 this tar geted panel sequences primarily exonic regions of approximately 1 , 700 genes , including approx imately 100 pharmacogenes selected based on curation within pharmgkb ( appendix table a1 )  . 
these five genes were selected based on their relevance to cancer treatment or sup portive care , likelihood of clinician interest in prospective implementation of the genedrug pairs , and existence of evidencebased treatment guidelines from the clinical pharmacogenet ics implementation consortium ( cpic ) 16 or the dutch pharmacogenetics working group ( dpwg ) .3 the polymorphisms in each gene were selected based on the validated variant lists within each guideline . 
note that ugt1a1 * 28 ( rs8175347 ) and * 80 ( rs887829 ) were both included to represent the ugt1a1 * 28 geno type , because they are highly linked , and * 80 is often substituted for * 28 as a result of its relative ease of genotyping.24 the mioncoseq germ line genetic database was screened to identify all variant calls at any of these polymorphisms for all patients sequenced on onco1700_v4 , and variant calls were compiled in a single data set for further analysis . confirmatory genotyping genotype determinations from onco1700 were manually screened for potentially unreliable calls by assessing standard sequencing quality con trol parameters , including read depth . 
in the second stage , polymor phisms with > five but < 30 occurrences were selected , by prioritizing patients who also car ried a variant at one of the common snp posi tions . 
finally , as many samples as necessary were selected that carried these common variants so that each variant was represented in five samples . selected samples were sent for college of american pathologistsaccredited , clinical lab oratory improvement amendmentsapproved genotyping at genelex laboratories ( seat tle , wa )  . 
genotypes were obtained using a laboratorydeveloped , multiplex polymerase chain reactionbased test followed by single base primer extension for variant detection by mass spectrometry ( massarray analyzer 4 system ; agena bioscience , san diego , ca )  . 
percent concordance for each snp was calculated as the number of concordant genotypes divided by the total number of samples compared . retrospective analysis of clinically actionable phenotypes patient genotypes were translated to activity phenotypes based on the appropriate cpic26 or dpwg3 guidelines ( appendix table a2 )  . 
genotype data for ugt1a1 * 28 ( * 80 ) and cyp3a5 * 3 was only available for the 25 samples sent for confirma tory genotyping ; therefore , these patients and genotypes were included in assessments of clin ical usefulness . 
medical record screening was performed using an automated screening tool ( emerse27 ) that searches the text of notes in michart , the version of epic used at mich igan medicine . 
text used to screen the medical records included all generic and brand drug names and commonly used acronyms ( eg , fu for fluourouracil )  . a pharmacy student , in consultation with a phar macist with oncology pharmacogenomic expertise , manually reviewed the electronic medical record for each patient who had an actionable pheno type and received the relevant drug to determine whether a treatment modification would have been recommended had this genetic information been available to the clinician before treatment initia tion . 
these patients represent the diversity of the mioncoseq cohort , including adult ( n = 82 ; 71% ) and pediatric patients ( n = 33 ; 29% ) who were evenly divided between male ( n = 58 ; 50% ) and female sex ( n = 57 ; 50% ) , were primarily white ( n = 97 ; 84% ) , and had a variety of solid ( n = 80 ; 70% ) and liquid tumor types ( n = 35 ; 30% )  . no genotype determinations for cyp3a5 * 3 , ugt1a1 * 28 , or ugt1a1 * 80 were made by onco1700 because of low read depth ( cyp3a5 * 3 and ugt1a1 * 80 ) or sequence repeat misalign ment ( ugt1a1 * 28 ) ; therefore , these polymor phisms were excluded from genotype concordance analyses . 
across the 18 remaining snps , a total of 139 variant calls were made in these 115 samples , and nine unique snps were detected in at least one patient ( table 1 )  . 
the frequency of actionable phenotypes was highest for cyp2c19 ( 43.5% ) , followed by cyp3a5 ( 20% ) , ugt1a1 ( 20% ) , and tpmt ( 13% ) , and lowest for dpyd ( 3.5% ) , as expected . the electronic medical record for each patient carrying an actionable phenotype was screened for relevant drugs . 
one patient with cyp2c19 rapid metabolizer phenotype never received voriconazole ; two patients with cyp2c19 rapid metabolizer phenotype received voriconazole treatment , but they were pediatric patients , so no dose adjustment is recommended per cpic guidelines5 ; and one patient with ugt1a1 poor metabolizer phenotype treated with irinotecan received a standard pediatric dose ( 49 mg / m2 ) , which is below the recommended threshold for dose adjustment based on dpwg guidelines.3 three patients with actionable phenotypes who received the relevant drug , in whom a treatment recommendation could have been made , were identified . 
one patients dose was held after 5 days because of neutro penia ( absolute neutrophil count [ anc ] , 200 ) , and the second patients dose was reduced to 70% of the standard dose after 7 days because of neutropenia ( anc , 500 )  . 
 are many challenges to pharmacogenetic imple mentation , 10 but perhaps the primary challenge is the current lack of evidence of clinical util ity to justify the upfront cost of establishing a pharmacogenetic service.31 , 32 in anticipation of a future in which genomic information is more readily available , cpic and other groups have published evidencebased treatment recommen dations for patients with known genotypes.3 , 16 there is a unique opportunity to integrate evidencebased pharmacogenetic treatment into tumor sequencing programs that analyze germline genetic information , 20 such as the mioncoseq program at umccc.21 the objective of this analysis was to confirm the accuracy of germ line genotype determinations produced during mioncoseq sequencing and then to retrospec tively assess the clinical usefulness of integrating pharmacogenetics into mioncoseq . attempted confirmatory genotyping of 21 clin ically actionable snps in five cancerrelevant pharmacogenes confirmed genotyping accu racy for common and uncommon variants in three genes ( tpmt , dpyd , and cyp2c19 ) but revealed an inability to genotype common vari ants in cyp3a5 and ugt1a1 . 
this finding is easily explained by the targeted exonic coverage of onco1700 and the location of these polymor phisms at a splice site ( cyyp3a5 * 3 , rs776746 ) and in the promoter region ( ugt1a1 * 28 , rs8175347 )  . 
other variants that were not detected are extremely rare , and several have not been found in white patients . prior studies have estimated that > 90% of the population has an actionable phenotype of at least one candidate gene33 , 34 ; however , this is only relevant if the patient is treated with the drug of interest and the guidelines apply to the patient . 
 in our cohort , 4% ( five of 115 ) of patients had a potentially actionable phenotype in cyp2c19 , tpmt , or dpyd and received the relevant drug , and in 2.6% ( three of 115 ) of patients , a guideline based treatment recommendation could have been made . manual review of these three patients identified several interesting findings . 
despite tpmt gen otype information available at the time of treat ment , two patients with heterozygous genotypes initiated treatment at standard mercaptopurine doses , per the childrens oncology group protocols on which they were enrolled . 
cpic recommends a preemptive 30% to 70% dose decrease with enhanced monitoring and titration based on tolerability.1 although germline tpmt determination from mioncoseq would not have changed these patients treatment in any way , it would have prevented external genetic testing , resulting in cost savings to the health systethe third patient carried a dpyd geno type that confers risk of severe toxicity with fu , which was not known at the time of treatment . 
 cpic guidelines recommend a preemptive dose reduction of 50% with monitoring and titra tion.2 although it is impossible to attribute tox icity to any single factor , it is interesting that two patients experienced toxicity requiring a reactive dose reduction , which may have been prevented if care had been based on cpic guidelines . the 2% to 4% absolute increase in the propor tion of patients with clinically actionable findings from germline pharmacogenetics represents a minimal estimate , because the emerse screen ing tool does not automatically screen prescrib ing data , and there is some chance that a patient with an actionable phenotype received a relevant medication that was never mentioned in a clini cal note . 
regardless , this represents a meaning ful increase in the clinically actionable findings from our tumor sequencing program.35 in addi tion to its usefulness for quality control , 18 , 36 matched germline analysis identifies validated cancer predisposition variants in an estimated 15% of patients.19 several tumor sequencing programs , including mioncoseq , 21 use their matched germline dna for this purpose.37 , 38 our results represent a critical first step toward integration of germline pharmacogenetics into mioncoseq . although several programs have reported that pharmacogenetics is considered in their deci sion making , 39 , 40 we are not aware of any detailed reports of the integration of pharmacogenetics into these programs . 
 used to inform irinotecan treatment decisions3 or how the incidental finding of gilberts syn drome would be conveyed to the patient.42 , 43 generalization of our estimate of the proportion of patients who could benefit from pharmacog enetic implementation in other tumor sequenc ing programs is challenging for several reasons . 
 first , there are differences in the frequencies of clinically actionable alleles or phenotypes among racial cohorts.44 additionally , institutional differ ences in the distribution of tumors that are treated and sent for sequencing could dramatically affect this estimate . 
 pharmacogenetic integration would be most ben eficial in programs that sequence tumors early in treatment , particularly at institutions that treat many pediatric patients with all . the proportion of patients who would benefit from pharmacogenetic implementation could be increased in several ways . 
mody , arul chinnaiyan substantial upfront investment to build and maintain clinical decision support within the electronic health record , hire or train individu als with pharmacogenetic expertise , and provide clinician and patient education . 
pharmacoge netic implementation within tumor sequencing programs that analyze matched germline dna is particularly efficient because there is no geno typing cost and the bioinformatic workflow to detect actionable germline phenotypes can be integrated into the existing infrastructure . 
additional work is necessary to develop infrastructure to embed active clini cal decision support into the electronic health record so that actionable phenotypes can be stored and used indefinitely.7 , 11 in conclusion , onco1700 produces reliable germline pharmacogenetic information for three clinically relevant pharmacogenes ( tpmt , dpyd , and cyp2c19 )  . 
elliott ls , henderson jc , neradilek mb , et al : clinical impact of pharmacogenetic profiling with a clinical decision support tool in polypharmacy home health patients : a prospective pilot randomized controlled trial . 
caudle ke , dunnenberger hm , freimuth rr , et al : standardizing terms for clinical pharmacogenetic test results : consensus terms from the clinical pharmacogenetics implementation consortium ( cpic )  . 
hanauer da , mei q , law j , et al : supporting information retrieval from electronic health records : a report of university of michigans nineyear experience in developing and using the electronic medical record search engine ( emerse )  . 
luzum ja , pakyz re , elsey ar , et al : the pharmacogenomics research network translational pharmacogenetics program : outcomes and metrics of pharmacogenetic implementations across diverse healthcare systems . 
ji y , skierka jm , blommel jh , et al : preemptive pharmacogenomic testing for precision medicine : a comprehensive analysis of five actionable pharmacogenomic genes using nextgeneration dna sequencing and a customized cyp2d6 genotyping cascade . 
stockley tl , oza am , berman hk , et al : molecular profiling of advanced solid tumors and patient outcomes with genotypematched clinical trials : the princess margaret impact / compact trial . 
crews kr , gaedigk a , dunnenberger hm , et al : clinical pharmacogenetics implementation consortium ( cpic ) guidelines for codeine therapy in the context of cytochrome p450 2d6 ( cyp2d6 ) genotype . 
hicks jk , bishop jr , sangkuhl k , et al : clinical pharmacogenetics implementation consortium ( cpic ) guideline for cyp2d6 and cyp2c19 genotypes and dosing of selective serotonin reuptake inhibitors . 
elvin , md , phd1 purpose vulvar squamous cell carcinoma ( vscc ) encompasses two predominant variants : one associated with detectable high - risk strains of human papillomavirus ( hrhpv ) and a second form often occurring in the context of chronic dermatitis in postmenopausal women . 
sixty - one percent of hpv + vsccs had a pathogenic alteration in the pi3k / mtor pathway , whereas hpv vsccs showed alterations in tp53 , tertp , cdkn2a , ccnd1 , and egfr , and biomarkers associated with responsiveness to immunotherapy . jco precis oncol 4 : 647 - 661 . 
90% of vulvar cancers and nearly 5% of all gynecologic cancers.1 , 2 radical excision imposes high morbidity , and one third of patients have been shown to experience recurrence after primary treatment.3 recent reports have shown durable responses with denitive or neoadjuvant chemoradiation for unresectable cancers.4 , 5 for patients with recurrence or distant metastasis , prognosis is poor , with an overall 2 - year survival rate of less than 15%.6 there is a critical need to improve our understanding of the molecular pathogenesis of vscc to provide insights that may guide more effective therapies . vscc develops through two distinct oncogenic pathways . 
this subgroup often associates with usual - type vulvar intraepithelial neoplasia ( vin ) , also known as high - grade squamous intraepithelial lesion.7 although other anogenital squamous cell neoplasms have  . 
 williams et al context key objective previous analyses have suggested genetic differences in vulvar squamous cell carcinoma based on human papillomavirus ( hpv ) status , but restricted sample volume and testing platforms have limited comprehensive identication of statistically signicant differences . 
in a large - scale comparative genomic study , what undiscovered genomic alterations distinguish high - risk hpv - driven versus dystrophic / inammatory - associated vulvar squamous cell carcinoma ? knowledge generated we identify signicantly different molecular proles based on hpv status . 
for genomic analyses , 60 ng of dna was extracted from 40 - m sections of 255 , 008 tumor samples , including 280 vscc specimens and 1 , 031 cervical squamous cell carcinoma ( cscc ) , each from a different patient , in formalin - xed parafnembedded ( ffpe ) tissue blocks . 
hpv genome sequences were detected by de novo assembly of nonhuman sequencing reads and nucleotide basic local alignment search tool ( blastn ) comparison against all viral nucleotide sequences in the national center for biotechnology information refseq database . 
hpv types identied in this study were stratied according to the hpv classication described by muoz et al , 8 with hpv 16 , 18 , 31 , 33 , and 58 labeled hrhpv + and hpv 6 labeled low risk . 
sequencing was performed on the primary tumor in 200 patients and on metastases in 80 ( 57 regional lymph nodes and 23 distant sites )  . hpv status and typing were determined on all 280 patient samples ; 102 / 280 vsccs ( 36% ) contained hrhpv sequences , predominantly hpv 16 ( 88% ; table 1 )  . 
for the 177 hpv vsccs , 137 were sequenced using the original primary tumors and 40 from metastatic site biopsies ( 10 distant , including seven lung , one pleural , one abdominal , and one to bone )  . comprehensive genomic proling figure 1 displays the distribution of gas by hpv status . 
the only specic point mutation with a signicant difference between hpv + and hpv was pik3ca e545k , an activating mutation that was signicantly enriched in hpv + vsccs ( table 2 )  . 
of vsccs cell - free circulating tumor dna ( ctdna ) was evaluated from blood specimens collected from 10 patients with vscc ( liquid biopsy ) using the hybrid capture - based illumina hi - seq ( illumina , san diego , ca ) technology . 
maximum somatic allele frequency was used to estimate the fraction of ctdna per methods previously described.30 , 31 mutational signatures mutational signatures were assessed for all tumor samples with at least 20 nondriver somatic missense alterations . signatures were given by analysis of the trinucleotide context and proled using the sanger cosmic signatures of mutational processes in human cancer.32 a positive signature required a sample to have at least a 40% t to a characterized mutational process , including apobec overexpression , exposure to ultraviolet light , hypofunction of the brca tumor suppressor , and defects in mismatch repair.32 immunohistochemistry programmed death - ligand 1 ( pd - l1 ) immunohistochemistry ( ihc ) was performed regularly in tandem with cgp to guide patient selection for immunotherapy . 
pd - l1 protein expression was assessed by ihc on 5 - micron ffpe tissue sections using the dako pd - l1 ihc22c3 pharmdx assay ( agilent ; santa clara , ca ; n = 52 vsccs ) or the ventana ( oro valley , az ) pd - l1 ( sp142 ) assay ( n = 21 vsccs ) , following each manufacturers instructions . 
dako pd - l1 expression was reported as a tumor proportion score , and ventana pd - l1 was reported as percent tumor area covered by positively staining tumor cells and immune cells . less than 1% staining was dened as negative , 1% - 49% was dened as low positive , and 50% was dened as high positive . clinicopathologic analysis of the vscc cohort a total of 280 vsccs were assayed with cgp ( foundation medicine ) , using material sent from treating institutions , from 2014 to 2019 . 
human investigations were performed after approval by a local human investigations committee and in accordance with an assurance led with and approved by the department of health and human services , where appropriate . 
amp , amplication ; mb , megabase ; msi , microsatellite instability ; msi - h , microsatellite instability - high ; mss , microsatellite stable ; mut , mutation ; pd - l1 , programmed death - ligand 1 ; tmb , tumor mutation burden . of each ga in the hpv and hpv + cohorts is included in appendix figures a2a and a2b , respectively . in the entire cohort : an hpv 16 ( + ) vscc with an mlh1 splice site mutation . frequencies of specic biomarkers associated with responsiveness to immunotherapy differed between the vscc subgroups ( fig 2 )  . 
all other gas , as well as age and pd - l1 ihc staining , showed no signicant differences between primary versus metastatic samples controlled for hpv status . comparison of hpv 16 with other hrhpv subtypes revealed no signicant differences in demographics , tmb , or sequenced site . 
thirty - three ( 12.6% ) were identied with an apobec ( apolipoprotein b mrna - editing enzyme , catalytic polypeptide - like ) signature ( 12 hpv + and 21 hpv ) , two with brca signature ( one hpv + , one hpv ) , seven with mismatch repair ( two hpv + , ve hpv ) , and a single tumor with ultraviolet signature ( hpv )  . 
three of four liquid biopsies showed at least one pathogenic ga present in the associated tissue biopsy ( appendix table a2 )  . separate from our 280 patients in the vscc cohort , ctdna was evaluated on six patients with known vscc but without tissue biopsy sequencing data . 
gas were detected in ve of six of these patients ( appendix table a2 )  . hpv + cscc ( n = 864 ) showed gas that were largely similar to what we found in hpv + vscc ( n = 103 ; appendix table a3 )  . 
although low in frequency , gas in kdm6a , ar , and cdk12 were signicantly higher in vscc versus cscc ( appendix table a3 )  . discussion in this study , hybrid capture - based dna sequencing was applied to a large series of patient tumors to better characterize the genomic landscape of vscc and to identify important genetic differences between hpv + and hpv disease . 
consistent with prior studies , a high rate of mutation was identied overall , with 98% of tumors in the analysis containing one or more known oncogenic mutations.17 , 18 mutational proles sharply differentiated hpv + and hpv disease . 
hpv + vscc showed mutations in the pi3k / mtor pathway , with 61% of tumors containing gas in the pathway , with the majority of gas showing signicant association with hpv + status ( table 2 )  . 
choschzick et al22 specically examined ccnd1 copy number changes in 183 vsccs and identied amplications in 22% , with a signicant association with hpv tumors.23 growdon pd - l1 ihc negative low positive high positive genomic alterations rearrangement short variant deletion not performed amplification fig 2 . 
of the 73 vulvar squamous cell carcinoma for which pd - l1 ihc was performed , 33% of hpv - negative and 9% of hpv - positive were pd - l1 tumor high - positive ; p = .04. 
histopathology of vulvar squamous cell carcinoma ranged from ( a ) well differentiated with abundant keratin to ( b ) poorly differentiated ( hematoxylin and eosin stains , 400 )  . 
 ( c ) programmed death - ligand 1 ( pd - l1 ) staining of human papillomavirus ( hpv ) negative vsccs showed signicantly higher frequency of high - positive tumors , whereas ( d ) hpvpositive disease was largely negative for pd - l1 stain ( pd - l1 immunohistochemistries , 400 )  . et al24 evaluated egfr amplication in 51 vsccs , and identied amplication in 12% of tumors , with signicant association with poor prognosis and hpv status . 
zie ba et al19 performed sequencing of 81 vsccs with a 50 - gene panel , and the results differed from other studies , most strikingly in the absence of clear genomic differences between hpv + and hpv disease . 
the authors reported tp53 and cdkn2a mutations in both hpv + and hpv vscc , whereas mutations in pik3ca , fbxw7 , hras , fgfr3 , stk11 , akt1 , smad4 , and pten were found at low frequencies in both types of vscc.19 zie ba et al19 noted , however , that the 2 hpv tests that they used gave highly inconsistent results and that those hpv tests had not been developed for analyzing tissue - derived dna.19 these difculties in identifying hpv + and hpv disease may account for the divergence of their results from several prior studies.16 - 18 , 20 in our study , hpv status clearly divided our large cohort into two signicantly different genomic - dened diseases . pi3k / mtor pathway mutations , including stk11 , a negative regulator of mtor signaling , have been described in a wide range of hpv - driven cancers.33 , 34 in our cohort , a signicantly higher rate of stk11 gas was observed in hpv + tumors sequenced from metastases , compared with hpv + tumors sequenced from the primary site . stk11 has been previously correlated with poor response to antiprogrammed death - 1 therapy in kras - mutant lung adenocarcinoma.35 it is conceivable that a similar role could exist in hpv + vscc as a putative tumor immuneescape mechanism , but additional studies are needed . a minority of vsccs showed distinctive mutational signatures . 
this signature reects apobec cytidine deaminase dna - editing activity36 , 37 and has been noted to be important in development of thoracic cancers , with possible implications for predicting response to immunotherapy.38 , 39 several of the gas observed to be signicantly enriched in the hpv cohort are in pathways functionally relevant to hpv pathogenesis . 
tp53 , tert , and cdkn2a are deregulated by hpv e6 and / or e7 , whereas egfr recycling is altered by hpv e5.40 - 42 beyond specic gas , key differences were identied in tmb and pd - l1 ihc staining patterns . 
hpv induces genomic instability , which may account for the increased tmb in the primary hpv + cohort.43 in addition , hpv infection reduces the cellular immune response by decreasing the interferon antiviral response.44 cgp may reveal opportunities for targeted therapies to be tested in clinical trials . 
kmt2d , an epigenetic modier , and transcription factor sox2 can activate and interact with the pi3k pathway.49 - 51 gas in both are enriched in hpv + vscc ( table 2 )  . 
tumors with gas in kmt2d may be sensitive to aurora kinase inhibition.52 in light of the many recent successes of immune checkpoint inhibitors , a careful approach to patient selection for clinical trials of these agents may be valuable . 
in our cohort , hpv vsccs showed a signicantly higher rate of pd - l1 ihc highpositive tumor staining , a higher rate of pdl1 amplication , and signicantly lower rates of stk11 alterations . 
vsccs in this category may benet from novel targeted therapeutics.53 other identied potential therapeutic targets include gas in receptor tyrosine kinases , cell cycle regulation , and the mapk pathway . 
early work in gas that affect epigenetic regulation indicates ezh2 inhibitors may be a viable therapeutic strategy.54 , 55 our study also provides a proof of concept that liquid biopsy detects ctdna in vscc , with three of four demonstrating at least one pathogenic ga detected in the tissue biopsy from the same patient . 
liquid biopsy may be a valuable method in vscc , and additional investigation is warranted . limitations in the study include the distinct patient population . tumor samples undergoing cgp are usually sent by clinicians seeking targeted therapy for patients with advanced disease . an additional limitation is the inadequate data on treatment history of the patients before tumor sequencing ; controls for tmb and resistance gas that may have arisen from local radiation or systemic treatment were not available . 
future work is needed to correlate genetic ndings with treatment exposure and follow - up data , which are not included in this study . in this study , we provided evidence that hpv + and hpv vscc are two distinct diseases , each with a characteristic molecular prole . 
 genomics of hpv + versus hpv vulvar squamous cell carcinoma rachel erlich employment : foundation medicine stock and other ownership interests : foundation medicine kevin jon williams stock and other ownership interests : hygieia stock and other ownership interests : gemphire therapeutics consulting or advisory role : gemphire therapeutics , inc . research funding : novo nordisk jeff m . 
venstrom employment : genentech , foundation medicine leadership : genentech stock and other ownership interests : genentech research funding : genentech , foundation medicine travel , accommodations , expenses : genentech brian m . 
alexander employment : foundation medicine leadership : foundation medicine stock and other ownership interests : roche research funding : eli lilly ( inst ) , puma ( inst ) , celgene ( inst ) open payments link : 854258 / summary nikunj shah employment : foundation medicine natalie danziger employment : foundation medicine eric a . 
severson employment : foundation medicine , partners healthcare stock and other ownership interests : foundation medicine jonathan keith killian employment : foundation medicine stock and other ownership interests : foundation medicine douglas i . 
ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine consulting or advisory role : celsius therapeutics research funding : foundation medicine julie y . 
hemmerich employment : foundation medicine stock and other ownership interests : foundation medicine ( inst ) acknowledgment we thank our colleagues for their helpful comments during the preparation of this article . references judson pl , habermann eb , baxter nn , et al : trends in the incidence of invasive and in situ vulvar carcinoma . 
moore dh , ali s , koh wj , et al : a phase ii trial of radiation therapy and weekly cisplatin chemotherapy for the treatment of locally - advanced squamous cell carcinoma of the vulva : a gynecologic oncology group study . 
n engl j med 348 : 518 - 527 , 2003 alemany l , saunier m , alvarado - cabrero i , et al : human papillomavirus dna prevalence and type distribution in anal carcinomas worldwide . 
adv anat pathol 24 : 201 - 214 , 2017 van der avoort ia , shirango h , hoevenaars bm , et al : vulvar squamous cell carcinoma is a multifactorial disease following two separate and independent pathways . 
virgili a , borghi a , toni g , et al : prospective clinical and epidemiologic study of vulvar lichen sclerosus : analysis of prevalence and severity of clinical features , together with historical and demographic associations . 
weberpals ji , lo b , duciaume mm , et al : vulvar squamous cell carcinoma ( vscc ) as two diseases : hpv status identies distinct mutational proles including oncogenic broblast growth factor receptor 3 . 
trietsch md , nooij ls , gaarenstroom kn , et al : genetic and epigenetic changes in vulvar squamous cell carcinoma and its precursor lesions : a review of the current literature . 
swarts dra , voorham qjm , van splunter ap , et al : molecular heterogeneity in human papillomavirus - dependent and - independent vulvar carcinogenesis . cancer med 7 : 4542 - 4553 , 2018 24 . 
halec g , alemany l , lloveras b , et al : pathogenic role of the eight probably / possibly carcinogenic hpv types 26 , 53 , 66 , 67 , 68 , 70 , 73 and 82 in cervical 9 : 34 , 2017 cancer . 
sakamoto j , kamiura s , okayama k , et al : single type infection of human papillomavirus as a cause for high - grade cervical intraepithelial neoplasia and invasive cancer in japan . 
clark ta , chung jh , kennedy m , et al : analytical validation of a hybrid capturebased next - generation sequencing clinical assay for genomic proling of cell - free circulating tumor dna . 
gregg jp , li g , pavlick d , et al : comprehensive genomic proling of ctdna in patients with colon cancer and its delity to the genomics of the tumor biopsy . j clin oncol 36 : 569 , 2018 ( 4 ; suppl ) 32 . 
wang s , jia m , he z , et al : apobec3b and apobec mutational signature as potential predictive markers for immunotherapy response in non - small cell lung cancer . 
gameiro sf , kolendowski b , zhang a , et al : human papillomavirus dysregulates the cellular apparatus controlling the methylation status of h3k27 in different human cancers to consistently alter gene expression regardless of tissue of origoncotarget 8 : 72564 - 72576 , 2017 42 . 
zhang b , srirangam a , potter da , et al : hpv16 e5 protein disrupts the c - cbl - egfr interaction and egfr ubiquitination in human foreskin keratinocytes . oncogene 24 : 2585 - 2588 , 2005 43 . 
song d , li h , li h , et al : effect of human papillomavirus infection on the immune system and its role in the course of cervical cancer . 
tinker av , ellard s , welch s , et al : phase ii study of temsirolimus ( cci - 779 ) in women with recurrent , unresectable , locally advanced or metastatic carcinoma of the cervix . 
oncol lett 12 : 4107 - 4116 , jung k , kang h , mehra r : targeting phosphoinositide 3 - kinase ( pi3k ) in head and neck squamous cell carcinoma ( hnscc )  . 
pi3k pathway regulates er - dependent transcription in breast cancer through the epigenetic regulator kmt2d . oncogene 37 : 1354 - 1368 , 2018 science 355 : 1324 - 1330 , 2017 51 . 
kalu nn , mazumdar t , peng s , et al : comprehensive pharmacogenomic proling of human papillomavirus - positive and - negative squamous cell carcinoma identies sensitivity to aurora kinase inhibition in kmt2d mutants . 
strauss j , heery cr , schlom j , et al : phase i trial of m7824 ( msb0011359c ) , a bifunctional fusion protein targeting pd - l1 and tgfb , in advanced solid tumors . clin cancer res 24 : 1287 - 1295 , 2018 idris s , lindsay c , kostiuk m , et al : investigation of ezh2 pathways for novel epigenetic treatment strategies in oropharyngeal cancer . 
lindsay cd , kostiuk ma , harris j , et al : efcacy of ezh2 inhibitory drugs in human papillomavirus - positive and human papillomavirus - negative oropharyngeal squamous cell carcinomas . 
 hpv negative ( n = 177 ) hpv positive ( n = 103 ) rearrangement short variant deletion amplification multiple rearrangement short variant deletion amplification multiple williams et al fig a2 . 
30 years ago with the identication of the reciprocal translocation , t ( 11 ; 22 ) ( q24 ; q12 ) , otherwise known as ews - fl1.1 , 2 in the time since , multiple other fusion partners with ews have been identied that t a similar ewing sarcoma phenotype.3 , 4 when ews fusions are not identied , tumors with histologic features of ewing sarcoma have been labeled as primitive neuroectodermal tumors . 
in 2012 , pierron et al5 identied a subset of ewing - like tumors harboring paracentric inversion on the short arm of chromosome x , resulting in the fusion of the bcor and ccnb3 genes.5 since that discovery , several small case series have further elucidated the clinical , morphologic , and genomic differences that make this diagnosis distinct from other round cell sarcomas , most notably ewing sarcoma.6 - 8 though distinct from ewing sarcoma , most bcorccnb3fused sarcomas ( bcs ) are treated with upfront compressed chemotherapy with vincristine , doxorubicin , cyclophosphamide , ifosfamide , and etoposide plus local control with surgery and / or radiation . bcs shares similar event - free and overall survival rates with the standard ews - fli1fused ewing sarcoma using this treatment strategy.6 - 8 despite the growing knowledge base related to bcs , little is known about potential drug targets related to this disease entity , especially with regard to treatment of disease recurrence . 
we highlight the treatment of a young patient who had multiply - relapsed disease with the us food and drug administrationapproved cyclindependent kinase 4 / 6 ( cdk4 / 6 ) inhibitor palbociclib ; the tumor harbored a bcor - ccnb3 fusion and a germline variant in cdkn2b , and treatment resulted in a complete response and no evidence of disease 25 months into therapy . case history our male patient initially presented in 2010 at 1 year of age with a xed mass on his back . 
a core needle biopsy was performed , which revealed a malignant , small , round , blue cell tumor along with small amounts of benign brofatty tissue and skeletal muscle . 
immunohistochemical stains were positive for cd99 , fli1 , and vimentin and were negative for nse , synaptophysin , myf4 , gaf , cd45rb , and tdt consistent with a primitive neuroectodermal tumor . 
the patient started chemotherapy per childrens oncology group protocol aews0031 , regimen b2 , with ifosfamide , etoposide , vincristine , doxorubicin , and cyclophosphamide . gross total resection was not feasible at the time per neurosurgery , and the patient received 57.6 gy of proton beam radiation in october 2010 . 
2 years but then developed multiple local recurrences without metastases from 2013 to 2017 and underwent numerous surgeries , along with multiple different early - phase childrens oncology group therapeutic studies , as outlined in the timeline in figure 1a . 
after the most recent recurrence in october 2016 , the patient was referred to our pediatric cancer precision genomics prograbecause of the ndings outlined here in the results , we chose to start palbociclib in february 2017 . 
 diagnosis radiation debulking surgeries ( n = 4 ) case report molecular tumor analysis debulking surgery debulking surgery april 2010 2010 july 2013 2014 2015 2016 june 2016 2016 2016 2017 march 2019 initial therapy aews0031 arm b : vincristine , doxorubicin , cyclophosphamide , ifosfamide , etoposide cog phase i advl1212 : crizotinib , cyclophosphamide , topotecan phase ii advl1322 : pazopanib phase i advl1315 : axitinib metronomic therapy molecular - guided therapy palbociclib initial diagnosis before palbociclib 2 cycles of palbociclib 3 cycles of palbociclib 2 years of palbociclib fig 1 . 
there was concern for progression after 2 cycles of palbociclib , but there was a 2 - month lag between the before palbociclib scan and actually starting drug , so interval progression likely occurred in this timeframe . 
ned , no evidence of disease . results whole - genome sequencing , rna sequencing ( rna - seq ) analysis , germline exome sequencing , and protein evaluation were performed at the clinical laboratory improvement amendment ( clia ) approved laboratory , nantomics ( culver city , ca )  . 
the mutational burden of the tumor was relatively low at 75 , 046 somatic mutations , with only 88 somatic mutations mapping to protein coding regions ( circos plot in fig 2a )  . 
additionally , an undescribed somatic mutation in the smo gene ( smo n476s ) was identied in the tumor , and germline sequencing revealed a cdkn2b n41d missense variant , which was heterozygous in both the germline and tumor genomes of this patient . 
rna - seq was also performed by nantomics , and mrna transcripts were ranked by abundance , which could be associated with increased pathway activity and sensitivity to a targeted drug . 
with the exception of the destruction box in ccnb3 , all functional domains from each encoded protein remain intact in the bcor - ccnb3 fusion prote ( c ) the cdk4 / 6 pathway is a gene regulatory program controlled by multiple tiers of protein kinases and transcriptional regulators . increases in cyclin - d or cdk4 or 6 protein can lead to phosphorylation of the rb1 - e2f tumor suppressor complex . 
upon phosphorylation of rb1 , the e2f1 - 3 transcription factors are released from the complex and are able to bind to the promoters of target genes , driving activation of transcription . 
in the several case series describing bcs , the median age of diagnosis is in the teenage years , with the youngest patient recorded at age 2.5 - 8 , 11 again , because of the age of presentation , one could be concerned about an inherited cancer syndrome . 
this congenital tumor also harbored a smarcb1 / ini1 gene deletion common to malignant rhabdoid tumor , epithelioid sarcomas , and epithelioid malignant peripheral nerve sheath tumor that also , table 1 . 
overexpression of relevant tumor - promoting pathways gene status gene function ccnd1 ccnd2 cdk4 e2f1 e2f2 e2f3 cdc25a cdc25c cdc25b top2a bub1 bub1b overexpressed activating cyclin for cdk4 and cdk6 overexpressed activating cyclin for cdk4 and cdk6 overexpressed rb1 protein kinase overexpressed rb1 - regulated transcription factor overexpressed rb1 - regulated transcription factor overexpressed rb1 - regulated transcription factor overexpressed e2f - regulated gene overexpressed e2f - regulated gene overexpressed e2f - regulated gene overexpressed e2f - regulated gene overexpressed e2f - regulated gene overexpressed e2f - regulated gene note . 
the expression status for a gene was classied as overexpressed if its tpm exceeded the genes upper 5th percentile.30 abbreviations : cdk , cyclin - dependent kinase ; e2f , e2 transcription factor ; rb1 , retinoblastoma gene ; tpms , transcripts per million . when found germline , is known to cause rhabdoid tumor predisposition syndrome.12 - 16 in the case reported by alfaro - cervello et al , 12 ini1 germline analysis was not performed . 
our patient also harbored a germline heterozygous missense variant , cdkn2b n41d . is unclear what role this germline cdkn2b n41d variant could play in sarcomagenesis , as cancer risks associated with cdkn2a / b gene variants include melanoma , pancreatic cancer , and astrocytomas.17 , 18 there is a recent short report from jouenne et al19 that found an increased risk of soft tissue sarcoma development with germline loss of cdkn2a , though no data exist conrming this risk with cdkn2b variants . 
sunita et al20 showed that the specic cdkn2b n41d variant , which encodes p15 ( ink4b ) , is unable to bind to the cdk6 protein , leading to loss of function of cdkn2b , which could lead to dysregulated control of s - phase entry . 
though this variants contribution to tumorigenesis is intriguing , ckdn2b was normally expressed in our patients tumor , and there are no data suggesting that this impaired binding to cdk6 leads to mrna overexpression along multiple levels of the cdk4 / 6 pathway . despite discovering alterations of several key regulators of the cdk4 / 6 pathway in this tumor , none have been proven to serve as clinical biomarker for sensitivity to cdk4 / 6 inhibitors.21 in a preclinical ewing sarcoma orthotopic xenograft model with cdkn2a deletion , palbociclib was able to greatly suppress growth despite doxorubicin resistance of this model.22 in other sarcoma subtypes , palbociclib reduced tumor burden in murine preclinical models.23 - 25 clinically , there is phase ii evidence of palbociclibs efcacy in adults with liposarcoma26 , 27 and leiomyosarcoma.28 despite growing evidence in these sarcomas , there are no published data testing cdk4 / 6 inhibitors in bcs . 
for our patient with bcs , the expression status for a gene was classied as overexpressed if its tpm exceeded the genes upper 5th percentile of per - gene rna - seq by expectation - maximization ( rsem ) transcript - per - million ( tpm ) values for a collection of rna sequencing ( rna - seq ) datasets from the cancer genome atlas normal samples . 
in the study by pierron et al , 5 10 bcs tumors were analyzed for certain mrna expression ; overexpression was determined as described in pierron manuscript methods . abbreviations : bcs , bcor - ccnb3fused sarcomas ; nd , not disclosed . pathway , and ( 3 ) the presence of a germline cdkn2b variant . 
ferguson , md , ms , 705 riley hospital dr , ri 4340 , indianapolis , in 46202 ; twitter : @rileychildrens ; e - mail : micjferg@iu.edu. support supported by grant no . 
cancer genet cytogenet 21 : 185 - 208 , 1986 turc - carel c , aurias a , mugneret f , et al : chromosomes in ewings sarcoma : i . 
an evaluation of 85 cases of remarkable consistency of t ( 11 ; 22 ) ( q24 ; q12 )  . cancer genet cytogenet 32 : 229 - 238 , 1988 ginsberg jp , de alava e , ladanyi m , et al : ews - fli1 and ews - erg gene fusions are associated with similar clinical phenotypes in ewings sarcoma . 
j clin oncol 17 : 1809 - 1814 , 1999 shing dc , mcmullan dj , roberts p , et al : fus / erg gene fusions in ewings tumors . 
cancer res 63 : 4568 - 4576 , 2003 pierron g , tirode f , lucchesi c , et al : a new subtype of bone sarcoma dened by bcor - ccnb3 gene fusion . 
nat genet 44 : 461 - 466 , 2012 peters tl , kumar v , polikepahad s , et al : bcor - ccnb3 fusions are frequent in undifferentiated sarcomas of male children . 
mod pathol 28 : 575 - 586 , 2015 puls f , niblett a , marland g , et al : bcor - ccnb3 ( ewing - like ) sarcoma : a clinicopathologic analysis of 10 cases , in comparison with conventional ewing sarcoma . 
am j surg pathol 38 : 1307 - 1318 , 2014 kao , yc , owosho aa , sung ys , et al : bcor - ccnb3 fusionpositive sarcomas : a clinicopathologic and molecular analysis of 36 cases with comparison to morphologic spectrum and clinical behavior of other round cell sarcomas . 
riggi n , suv `a ml , suv `a d , et al : ews - fli - 1 expression triggers a ewings sarcoma initiation program in primary human mesenchymal stem cells . 
matsuyama a , shiba e , umekita y , et al : clinicopathologic diversity of undifferentiated sarcoma with bcor - ccnb3 fusion : analysis of 11 cases with a reappraisal of the utility of immunohistochemistry for bcor and ccnb3 . 
chan ak , han sj , choy w , et al : familial melanoma - astrocytoma syndrome : synchronous diffuse astrocytoma and pleomorphic xanthoastrocytoma in a patient with germline cdkn2a / b deletion and a signicant family history . 
campa d , pastore m , gentiluomo m , et al : functional single nucleotide polymorphisms within the cyclin - dependent kinase inhibitor 2a / 2b region affect pancreatic cancer risk . 
oncotarget 7 : 57011 - 57020 , 2016 jouenne f , chauvot de beauchene i , bollaert e , et al : germline cdkn2a / p16ink4a mutations contribute to genetic determinism of sarcoma . 
agarwal sk , mateo cm , marx sj : rare germline mutations in cyclin - dependent kinase inhibitor genes in multiple endocrine neoplasia type 1 and related states . j clin endocrinol metab 94 : 1826 - 1834 , 2009 21 . 
murakami t , singh as , kiyuna t , et al : effective molecular targeting of cdk4 / 6 and igf - 1r in a rare fus - erg fusion cdkn2a - deletion doxorubicin - resistant ewings sarcoma patient - derived orthotopic xenograft ( pdox ) nude - mouse model . 
perez m , muoz - galv an s , jim enez - garca mp , et al : efcacy of cdk4 inhibition against sarcomas depends on their levels of cdk4 and p16ink4 mrna . oncotarget 6 : 40557 - 40574 , 2015 24 . 
vlenterie m , hillebrandt - roeffen mh , schaars ew , et al : targeting cyclin - dependent kinases in synovial sarcoma : palbociclib as a potential treatment for synovial sarcoma patients . 
b ohm mj , marienfeld r , j ager d , et al : analysis of the cdk4 / 6 cell cycle pathway in leiomyosarcomas as a potential target for inhibition by palbociclib . 
dickson ma , tap wd , keohan ml , et al : phase ii trial of the cdk4 inhibitor pd0332991 in patients with advanced cdk4 - amplied well - differentiated or dedifferentiated liposarcoma . 
dickson ma , schwartz gk , keohan ml , et al : progression - free survival among patients with well - differentiated or dedifferentiated liposarcoma treated with cdk4 inhibitor palbociclib : a phase 2 clinical trial . 
elvin ja , gay lm , ort r , et al : clinical benet in response to palbociclib treatment in refractory uterine leiomyosarcomas with a common cdkn2a alteration . oncologist 22 : 416 - 421 , 2017 1072 - 1074 , 2015 29 . 
 development and validation of a clinical polygenic risk score to predict breast cancer risk elisha hughes , phd1 ; placede tshiaba , ms1 ; shannon gallagher , mph1 ; susanne wagner , phd1 ; thaddeus judkins , ms1 ; benjamin roa , phd1 ; eric rosenthal , phd , ms1 ; susan domchek , md2 ; judy garber , md , mph3 ; johnathan lancaster , md , phd1 ; jeffrey weitzel , md4 ; allison w . 
lanchbury , phd1 ; alexander gutin , phd1 ; and mark robson , md6 purpose women with a family history of breast cancer are frequently referred for hereditary cancer genetic testing , yet , 10% are found to have pathogenic variants in known breast cancer susceptibility genes . 
largescale genotyping studies have identied common variants ( primarily single - nucleotide polymorphisms [ snps ] ) with individually modest breast cancer risk that , in aggregate , account for considerable breast cancer susceptibility . 
candidate polygenic risk scores ( prss ) as predictors of personal breast cancer history were developed through multivariable logistic regression models adjusted for age , cancer history , and ancestry . 
an optimized prs was validated in 2 independent cohorts ( n = 13 , 174 ; n = 141 , 160 )  . results within the training cohort ( n = 24 , 259 ) , 4 , 291 women ( 18% ) had a personal history of breast cancer and 8 , 725 women ( 36% ) reported breast cancer in a rst - degree relative . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction although breast cancer ranks among the cancer types with the highest heritability , 1 characterization of the incomplete . 
pathounderlying genetic causes is still genic variants in high - penetrance genes ( eg , brca1 , brca2 , palb2 ) associated with substantial increases in risk are individually rare in the general population.2 - 4 all known highand moderate - penetrance breast cancer susceptibility genes are estimated to account for a combined 20% of familial relative risk.5 as a result , most women who are referred for hereditary cancer genetic testing are negative for a pathogenic variant and without a clear understanding of the magnitude of their risk.6 genome - wide association studies ( gwas ) have identied several hundred , common , single - nucleotide polymorphisms ( snps ) as breast cancer susceptibility variants.7 - 11 although their individual contribution to risk is minor , polygenic risk scores ( prss ) of such variants can dene variant combinations with potentially actionable clinical risk.9 , 11 together , pathogenic variants in highand moderate - risk genes and panels of snps are estimated to explain up to 40% of the familial risk for developing breast cancer.11 the rst well - characterized prs combined 77 snps ( prs77 ) and was evaluated in  . 
this prs was highly predictive of breast cancer in two independent validation cohorts . relevance prss that aggregate genotypes from common variants have emerged as a new approach to improve breast cancer risk assessment . 
we developed and validated an 86 - snp score that was adjusted for family breast cancer history and was highly predictive of breast cancer risk , making it appropriate for clinical use to identify women at increased risk of developing breast cancer . recently , a polygenic score of 313 snps was deduced from the same gwas and then validated in an independent population cohort.11 the same study also supported previous work indicating snp - based risk scores may have to be optimized depending on breast cancer estrogenreceptor ( er ) status . 
composition and individual contribution of variants of the ideal snp - based breast cancer risk score remain to be dened . family history ( fh ) of breast cancer , a combination of biologic , social , and environmental factors , includes a genetic component that is expected to overlap with snpbased risk.9 , 11 to avoid double counting of risk due to correlation between genetic components of fh and prs , joint modeling of both genetic factors is required . 
here , we describe the development and validation of a prs in which we co - estimate snp - based risk and familial risk to obtain effect sizes independent of fh . 
additional details are provided in the data supplement . snp genotyping amplicons for 94 snp markers published at the time of this study were incorporated into a next - generation sequencing panel.8 , 9 two markers failed design due to their location within repetitive elements , leaving 92 markers for evaluation . 
clinical information from provider - completed test request forms included ancestry , personal and family cancer history , cancer type ( s ) , and age ( s ) at diagnosis . 
 clinical polygenic risk score for breast cancer previous work supports a multiplicative model as the best method for capturing the combined snp effects.9 , 11 we centered the multiplicative model according to generalpopulation allele frequencies so the or for an individual patient represents the fold - change in risk relative to the general population . 
patient clinical characteristics validation study tested the association of the prs with invasive breast cancer after adjusting for age , personal and family cancer history , and ancestry . in exploratory analyses , we assessed the predictive ability of the optimized prs compared with a previously described prs77 by adding both to a multivariable model . 
the observed versus theoretical effect of the prs on breast cancer risk under the multiplicative polygenic model was assessed by comparing ors from the continuous prs with those obtained from analysis of patients binned in categories according to prs percentiles . 
heterogeneity of the prs effect size according to age , ancestry , and fh of breast cancer was evaluated by tting additional interaction terms and by repeating the primary analysis restricted to subcohorts stratied by age , ancestry , and fh severity . 
overall , 8 , 725 patients ( 36% ) reported a breast cancer diagnosis in 1 rst - degree relative and 13 , 230 ( 55% ) had no rst - degree relatives with breast or ovarian cancer . effect sizes and population frequencies were used to rank 92 susceptibility variants by their informativeness and to construct a polygenic score by successive addition of the most informative variant . 
distribution of relative risks , or fold changes , in breast cancer risks due to the 86 - snp score in unaffected women was similar between the rst and second validations ( data supplement figures s1 and s2 )  . 
ors for developing breast cancer by 86 - snp score percentile in each of the two validation cohorts are given in table 2 . consistently , women in the top 95th percentile of the 86snp score distribution had a more than 2 - fold higher risk of breast cancer development than women with an average 80 86 snps ranked by informativeness * fig 1 . 
effect sizes of the 86 - snp score were consistent across subcohorts stratied by age ( table 3 ; data supplement figure s4 ) , ancestry ( data supplement table s3 ) , and fh ( table 4 ; data supplement figure s5 ) in both validation cohorts . 
these results indicate the simple multiplicative model remains the best method for capturing the risk conferred by the combined effects of snps after accounting for family cancer history . discussion prss aggregating genotypes from common variants offer new ways to improve breast cancer risk assessment and can contribute to better understanding of breast cancer risk in all women . 
modication of risk estimates by prs - based stratication has been shown in a variety of clinical settings , including women who carry a pathogenic variant , high - risk women , and population cohorts.11 , 15 - 17 these novel tools , however , are of particular interest to women who present for genetic risk assessment with increased risk due to a fh of breast cancer . 
fh and susceptibility variants contribute independently to risk prediction , yet the approximately 13% reduction in the or for fh when adjusted for the prs77 ( or prs313 ) indicates an overlapping contribution to risk.9 , 11 , 18 adjustment for this correlation between snps and fh can be achieved by co - estimating ors from multivariable models . 
the reported interaction was only signicant in er - positive disease , a subanalysis not available in our data set.11 since the inception of this study , additional breast cancer risk variants have been described , and expanded breast cancer prss have been evaluated and validated in independent data sets.10 , 11 the enlarged prss are estimated to account for a larger fraction of familial risk and promise observed theoretical observed theoretical > 1 - 5 5 - 10 10 - 20 20 - 40 40 - 60 60 - 80 80 - 90 90 - 95 95 - 99 > 99 > 1 - 5 5 - 10 10 - 20 20 - 40 40 - 60 60 - 80 80 - 90 90 - 95 95 - 99 > 99 percentiles of the 86 - snp score percentiles of the 86 - snp score fig 2 . 
the approach described here of co - estimation in multivariate models could be applied to any emerging prs , irrespective of number of variants or ancestral origin . among the strengths of this study are the use of large , contemporary , and fully independent cohorts for development and 2 validation studies . 
thus , to our knowledge , the 86 - snp score is the rst prs with an snp component adjusted for fh and , as such , would be an appropriate addition to conventional clinical models . limitations of the study include the inability to apply the 86snp score to patients of non - european ancestry because changes in genetic structure and linkage disequilibrium may affect the predictive ability of individual risk variants when present in a different ancestral background . 
although association studies with equivalent explanatory power in other ancestries have yet to be described , they are the subject of ongoing research and the expansion of breast cancer prs to other ancestries is a high priority . 
however , previous studies have demonstrated that unbiased risk estimates can be obtained from analysis of a clinical testing population through multivariable adjustment for the factors related to ascertainment.14 , 19 although the 86 - snp score described here adjusted for fh , genetic factors are not the sole contributors to breast cancer risk , even in cohorts with exceptional fh . 
validated clinical risk models incorporate variables such as body mass index , age at menarche , parity , age at rst birth and hormone replacement therapy use , as well as details of fh.20 - 23 other important risk factors , such as mammographic density and diet , are not taken into account here.24 - 26 more studies will be needed to accommodate these additional variables and to determine their dependent contributions . 
lanchbury data analysis and interpretation : elisha hughes , placede tshiaba , shannon gallagher , susanne wagner , benjamin roa , eric rosenthal , susan domchek , judy garber , jeffrey weitzel , allison w . 
komen for the cure ( i ) , american association for cancer research , diane helis henry medical foundation , james p . wilmot foundation ( i ) , adrienne helis malvin medical research foundation ( i ) , breast cancer research foundation , facing our risk of cancer empowered johnathan lancaster employment : myriad genetics , regeneron stock and other ownership interests : myriad genetics , regeneron jeffrey weitzel speakers bureau : astrazeneca allison w . 
jama 315 : 68 - 76 , 2016 peto j , collins n , barfoot r , et al : prevalence of brca1 and brca2 gene mutations in patients with early - onset breast cancer . 
j natl cancer inst 91 : 943 - 949 , 1999 anglian breast cancer study group : prevalence and penetrance of brca1 and brca2 mutations in a population - based series of breast cancer cases . 
 hughes et al collaborative group on hormonal factors in breast cancer : familial breast cancer : collaborative reanalysis of individual data from 52 epidemiological studies including 58 , 209 women with breast cancer and 101 , 986 women without the disease . 
j mammary gland biol neoplasia 9 : 221 - 236 , 2004 desmond a , kurian aw , gabree m , et al : clinical actionability of multigene panel testing for hereditary breast and ovarian cancer risk assessment . 
michailidou k , beesley j , lindstrom s , et al : genome - wide association analysis of more than 120 , 000 individuals identies 15 new susceptibility loci for breast cancer . 
am j hum genet 104 : 21 - 34 , 2019 judkins t , leclair b , bowles k , et al : development and analytical validation of a 25 - gene next generation sequencing panel that includes the brca1 and brca2 genes to assess hereditary cancer risk . 
li h , feng b , miron a , et al : breast cancer risk prediction using a polygenic risk score in the familial setting : a prospective study from the breast cancer family 17 . 
n aslund - koch c , nordestgaard bg , bojesen se : common breast cancer risk alleles and risk assessment : a study on 35 441 individuals from the danish registry and kconfab . 
cuzick j , brentnall ar , segal c , et al : impact of a panel of 88 single nucleotide polymorphisms on the risk of breast cancer in high - risk women : results from two randomized tamoxifen prevention trials . 
lindstr om s , thompson dj , paterson ad , et al : genome - wide association study identies multiple loci associated with both mammographic density and breast cancer risk . 
van zogchel , md1 , 3 ; jalenka van wijk , msc1 , 2 ; ilse timmerman , phd1 , 3 ; ngoc - kim vo1 ; lily zappeij - kannegieter1 ; boris decarolis , md4 ; thorsten simon , md4 ; max m . 
studies have found that neuroblastoma tissue contains adrenergic ( adrn ) and mesenchymal ( mes ) cells ; the latter express low levels of commonly used markers for minimal residual disease ( mrd )  . 
after selecting a panel of markers , serial bone marrow , peripheral blood , and peripheral blood stem cell samples were obtained from patients with high - risk neuroblastoma and tested for marker expression ; survival analyses were also performed . results prrx1 , postn , and fmo3 mrnas were used as a panel for specically detecting mes mrna in patient samples . 
mes mrna was detected only rarely in peripheral blood ; moreover , the presence of mes mrna in peripheral blood stem cell samples was associated with low event - free survival and overall survival . 
 van wezel et al context key objective the aim of this study was to identify a panel of markers to detect mesenchymal ( mes ) neuroblastoma mrna and to study these markers dynamics in patients with high - risk neuroblastoma . knowledge generated we identied a panel of mrna markers ( prrx1 , postn , and fmo3 ) to specically detect mes mrna in peripheral blood , bone marrow ( bm ) , and peripheral blood stem cell samples . 
in contrast to the frequently used adrenergic ( adrn ) mrna markers in real - time quantitative polymerase chain reaction studies , mes mrna increased during therapy in bm , which supports the idea that mes neuroblastoma cells have a different response to therapy than adrn neuroblastoma cells . furthermore , mes mrna was more frequently detected in bm and peripheral blood stem cells of patients with adverse outcomes . relevance neuroblastoma cells that undergo epithelial to mes transition are believed to become more chemoresistant and give rise to relapse . 
clinical samples were collected in edta tubes , processed within 24 hours , transferred to paxgene blood rna tubes ( qiagen , venlo , the netherlands ) , and stored at 20 c . 
dna was isolated from mononuclear cells stored in 10% dimethylsulfoxide at 180 c , and hypermethylated rassf1a rt - qpcr was performed as described previously.34 control samples and isolation of nonpathologic cell subsets to assess expression patterns of candidate markers in control tissue , pediatric bm samples of patients with leuleast 3 months after kemia in molecular remission ( at therapy ; n = 48 ) , pb samples from healthy volunteers ( n = 104 ) , and pbsc samples from children treated for a disease other than neuroblastoma ( n = 29 ) were tested as described previously.28 different pb populations were isolated as described previously.10 cultured mscs35 were provided by carlijn voermans , phd , at the department of hematopoiesis , sanquin blood supply foundation ( amsterdam , the netherlands )  . 
all samples were obtained with informed consent . rna extraction and rt - qpcr rna was isolated from clinical and control samples using the paxgene blood rna kit ( qiagen )  . 
expression was normalized to gusb expression using the following equation : normalized threshold cycle ( dct ) = ( ctgusb ctmarker )  . data analysis for the newly identied mes markers , a threshold for positivity in control bm , pb , and pbsc samples was determined on the basis of their expression in each sample ( see results )  . in the pbsc cohort , survival was analyzed using the kaplanmeier method , and signicant differences between the estimated survival curves were analyzed using the log - rank test . fishers exact test was used to analyze the differences in mes marker positivity between the relapse and survivor groups . 
subsequently , we ltered for genes with a minimal expression level in pb data sets , which resulted in an initial list of 14 candidate genes ( fig 2a )  . 
although prrx1 was not included in this initial list because its expression in 700 - mes cells was less than 400 , we included this gene in our additional analyses because prrx1 was reported as an immunohistochemistry marker for mes neuroblastoma.24 validation of candidate markers using rt - qpcr with sybr green i dye ( applied biosystems , foster city , ca ) , we examined the expression of all 15 candidate markers in mes cells ( 691 - mes and shep2 ) , adrn cells ( 691 - adrn , sh - sy5y , and imr - 32 ) , and a control pb sample ( data supplement )  . 
our analysis revealed three genes with high expression in mes cells , a signicant difference in expression between the mes and adrn cell lines , and either low or no measurable expression in the control pb these three genes were postn , prrx1 , and sample ; col3a1 , which encode the proteins periostin , paired related homeobox 1 , and collagen type iii 1 , respectively . rt - qpcr using taqman probes validated expression of these markers , which demonstrated high postn , prrx1 , and col3a1 expression in 691 - mes and sh - ep2 and low expression in 691 - adrn , sh - sy5y , and imr - 32 ( fig 3a )  . col3a1 was then excluded because it did not adequately discriminate between adrn and mes cell lines and was detected in the control pb samples . next , to set thresholds for dening positivity , we measured the expression of postn and prrx1 in normal hematologic cells : control bm samples ( n = 48 ) , pb samples ( n = 104 ) , and pbscs ( n = 29 )  . 
however , because both postn and prrx1 were expressed at variable levels in the control bm and pb samples ( fig 3b ) , we examined their expression levels in several subsets of hematologic cells and in mscs ( fig 3c )  . 
blue circles , sh - ep2 ; blue squares , 691 - mes ; red squares , 691 - adrn ; red circles , sh - sy5y ; red diamonds , imr - 32 . robustly expressed in mscs and , to a lesser extent , in t cells ; in contrast , postn was expressed at extremely low levels in b cells . 
taken together , these results suggest that the presence of both postn and prrx1 in control bm and pb samples is likely the result of their expression in mscs . identication of an msc - discriminating marker next , we searched for a marker that can be used to discriminate between mscs and mes mrna . 
microarray analysis identied 14 candidate genes that were highly expressed in mscs but poorly expressed both in mes cell lines and in hematologic cells ( not containing mscs ; fig 4a ; data supplement )  . 
the specicity of these markers was then tested using mscs obtained from healthy individuals ( n = 2 ) , neuroblastoma cell lines ( n = 5 ) , and control pb samples ( n = 2 ) using sybr green i dye ( data supplement )  . our analysis revealed that the fmo3 gene ( encoding avincontaining monooxygenase 3 ) was the most promising marker for discriminating between mscs and mes cells . testing using an fmo3 - specic taqman subsequent probe conrmed high expression in mscs ( comparable to the expression levels of prrx1 and postn ) , low expression ( or no expression ) in all neuroblastoma cell lines ( fig 4b ) , and no expression in hematologic subsets that lacked mscs . threshold for positivity in bm , pb , and pbscs because mscs , and to a lesser extent , b cells and t cells , express low levels of postn and prrx1 , we established a threshold for positivity for mes mrna detection in control bm , pb , and pbsc samples . 
 ( b ) heat map that shows the expression proles of the indicated 35 genes with high expression ( red ) in mes cell lines ( right ) and low expression ( green ) in adrenergic ( adrn ) cell lines ( left ) ; shown underneath are genes that are commonly used for mrd testing in neuroblastoma . 
 ( a ) the indicated genes were measured in mes cell lines ( shown in blue ) and adrenergic ( adrn ) cell lines ( shown in red )  . 
 ( b ) normalized expression ( dct = ctgusb ctmarker ) of postn and prrx1 measured in control bone marrow ( bm ) , peripheral blood ( pb ) , and peripheral blood stem cell ( pbsc ) samples . 
on the basis of these results , the positivity threshold for postn and prrx1 was set to a dct value greater than 9 between postn / prrx1 and gusb and a dct value greater than 3 between postn / prrx1 and fmo3 ( fig 4c )  . 
the threshold for positivity for pb was determined using 104 control pb samples ( data supplement ) and was set to a dct value greater than 12 for prrx1 and a dct value greater than 10 for postn , with no measurable expression of fmo3 . 
because the background expression of both postn and prrx1 was extremely low in control pbsc samples , each pbsc sample was scored as r2 microarray mscs neuroblastoma mes cell lines 74 genes r2 and haematlas no or low expression in whole blood hematologic subgroups 14 genes candidate msc marker negative fmo3 postn prrx1 phox2b postn prrx1 ctgusb ctpostn > 9 and ctfmo3 ctpostn > 3 ctgusb ctpostn > 10 and fmo3 negative ctgusb ctprrx1 > 9 and ctfmo3 ctpostn > 3 ctgusb ctprrx1 > 12 and fmo3 negative pbsc quantifiable range and fmo3 negative quantifiable range and fmo3 negative fig 4 . 
 van wezel et al positive for mes mrna if the ct value was within the quantitative range , 38 with no measurable expression of fmo3 ( fig 4c )  . 
phox2b mrna was detected in all diagnostic samples ; this expression decreased to undetectable levels in most patients during induction chemotherapy ( ic ) and was detected again at relapse . 
in contrast , mes mrna was detected in only six samples ; ve of these samples were pbscs , and of note , these pbsc samples were negative for phox2b mrna . next , we examined pbsc samples obtained from 53 patients with high - risk neuroblastoma who were previously studied using adrn mrna39 ( data supplement )  . 
mes mrna and adrn mrna were detected in 15 ( 28% ) and six ( 11% ) of 53 samples , respectively , with little overlap ( both mes and adrn mrna were detected in only one sample )  . 
we previously reported that adrn mrna positivity is not correlated with outcome.39 however , here we found that the presence of postn and / or prrx1 mrna in pbsc samples was signicantly associated with low eventfree survival ( p = .045 ) and low overall survival ( p = .047 ; fig 5 ; data supplement )  . 
specically , we compared phox2b mrna with mes mrna measured in 95 serial bm samples collected at diagnosis , during therapy , during follow - up , and ( where applicable ) during relapse ( fig 6 ; data supplement )  . 
phox2b mrna was detected in all 27 diagnostic bm samples ; this expression decreased during ic treatment and was undetectable by the end of ic in most patients ( nine of 10 patients with complete remission and 10 of 15 patients who experienced relapse )  . 
in the patients with recurrent disease , phox2b mrna was detected again in 75% ( nine of 12 patients ) with systemic relapse ( three of the 15 patients with recurrent disease had a local relapse )  . 
 a negative mesenchymal minimal residual disease panel in neuroblastoma n716 asct relapse g04 survivor asct time since diagnosis ( days ) time since diagnosis ( days ) negative negative negative diagnosis mid - induction end - induction after asct relapse n = 13 n = 14 n = 10 n = 16 n = 10 n = 15 n = 12 both adrn negative s urviv ors r ela pse s urviv ors r ela pse s urviv ors r ela pse s urviv ors r ela pse r ela pse negative fig 6 . 
 ( c ) summary of the results obtained from all serial samples obtained from patients who ultimately experienced relapsed and patients who remained in complete remission ( survivors )  . 
 ( d ) a peripheral blood stem cell sample obtained from patient 774 and two bm samples obtained from patients 576 and 690 were measured for phox2b ( squares ) , postn ( lled circles ) , prrx1 ( triangles ) , and hypermethylated rassf1a ( open circles )  . 
we found that gd2s is expressed at high levels in both mes and adrn cell lines ; however , specicity is limited because of its relatively high expression in normal hematologic cells.10 to study the expression of mes - specic markers at the time of diagnosis as well as the dynamic expression pattern during follow - up , we identied a panel of markers that includes postn and prrx1 , which have been linked to emt in several cancer types.24 , 40 - 44 an mrd marker ideally should be expressed at extremely low levels in normal hematologic cells . 
however , a possible limitation of this approach is that dependent on the platform used , the actual gene expression levels can be underestimated , for example , as in the case of prrx1 expression in one cell line used in our study . 
of note , although we ascribed the expression of postn and prrx1 in control bm samples to mscs , we cannot rule out the possibility that other cell types , such as osteoblasts , may also contribute to this expression . 
nevertheless , because we established strict thresholds for dening positivity , we believe that we avoided detection of normal stromal cells as well as other cell types . because mes mrna was rarely detected in pb samples obtained from patients with high - risk neuroblastoma , its clinical relevance remains unclear , and these results need to be conrmed using a larger cohort . 
in contrast , mes mrna was detected in 28% of the pbsc samples obtained from 53 patients and was signicantly associated with low event - free survival and overall survival . 
of note , we previously reported a relatively low prevalence ( 9% ) of adrn mrna in pbscs obtained from this cohort , and the presence of adrn mrna was not associated with either low event - free survival or low overall survival.39 therefore , we speculate that the mes cells that reside in the bm circulate during stem - cell mobilization . by focusing on serial bm samples in high - risk patients , we found that mes mrna and adrn mrna have distinct temporal dynamics . 
specically , adrn mrna levels were high at diagnosis and during relapse but decreased during treatment , whereas mes mrna levels increased during treatment and were associated with patients who ultimately had a relapse . 
this nding suggests that mes cells may respond differently to therapy compared with adrn neuroblastoma cells and may play an important role in disease progression and / or recurrence ; this notion is consistent with reports that demonstrated the importance of emt in disease progression and treatment resistance.24 , 45 moreover , our nding that mes mrna was detected in only 14 of 27 patients at diagnosis ( and only rarely at relapse ) is consistent with the hypothesis that metastatic cells can undergo an mes - to - epithelial conversion and thus revert to an adrn phenotype.46 the relatively small size of our patient cohorts precluded extensive multivariable analyses of survival ; however , these exploratory ndings suggest that detection of mes markers in the bm and pbscs during treatment may have prognostic value . dna markers for mrd ( and recently , circulating cell - free dna markers ) have been shown to provide added value when combined with rna - based methods for monitoring mrd and for measuring tumor - derived genetic aberrations.34 , 47 - 49 on the other hand , we previously reported discrepancies between rna - based and dna - based markers using either methylated rassf1a or patientspecic dna markers.34 , 47 we hypothesize that these discrepancies reect mes neuroblastoma cells that express reduced levels of adrn markers but can still be detected using dna markers . in conclusion , we report that postn , prrx1 , and fmo3 mrna can be used to detect mes neuroblastoma cells in bm and pbscs in patients with high - risk neuroblastoma . 
moreover , although the mes markers are more prevalent in the bm of patients who will ultimately experience relapse , they are rarely present at the actual time of relapse . 
van zogchel , jalenka van wijk , ngoc - kim vo , lily zappeij - kannegieter , boris decarolis , thorsten simon , rogier versteeg , jan koster , johan van nes data analysis and interpretation : esther m . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . thorsten simon consulting or advisory role : eusa pharma ( inst ) consulting or advisory role : merck kgaa ( inst ) huib n . 
caron employment : roche stock and other ownership interests : roche travel , accommodations , expenses : roche no other potential conicts of interest were reported . references laprie a , michon j , hartmann o , et al : high - dose chemotherapy followed by locoregional irradiation improves the outcome of patients with international neuroblastoma staging system stage ii and iii neuroblastoma with mycn amplication . 
matthay kk , villablanca jg , seeger rc , et al : treatment of high - risk neuroblastoma with intensive chemotherapy , radiotherapy , autologous bone marrow transplantation , and 13 - cis - retinoic acid . 
n engl j med 341 : 1165 - 1173 , 1999 yu al , gilman al , ozkaynak mf , et al : anti - gd2 antibody with gm - csf , interleukin - 2 , and isotretinoin for neuroblastoma . 
n engl j med 363 : 1324 - 1334 , 2010 seeger rc , reynolds cp , gallego r , et al : quantitative tumor cell content of bone marrow and blood as a predictor of outcome in stage iv neuroblastoma : a childrens cancer group study . 
j clin oncol 18 : 4067 - 4076 , 2000 beiske k , burchill sa , cheung iy , et al : consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow , blood and stem cell preparations by immunocytology and qrt - pcr : recommendations by the international neuroblastoma risk group task force . 
stutterheim j , gerritsen a , zappeij - kannegieter l , et al : detecting minimal residual disease in neuroblastoma : the superiority of a panel of real - time quantitative 11 . 
stutterheim j , zappeij - kannegieter l , versteeg r , et al : the prognostic value of fast molecular response of marrow disease in patients aged over 1 year with pcr markers . 
burchill sa , lewis ij , abrams kr , et al : circulating neuroblastoma cells detected by reverse transcriptase polymerase chain reaction for tyrosine hydroxylase mrna are an independent poor prognostic indicator in stage 4 neuroblastoma in children over 1 year . 
marachelian a , villablanca jg , liu cw , et al : expression of ve neuroblastoma genes in bone marrow or blood of patients with relapsed / refractory neuroblastoma provides a new biomarker for disease and prognosis . 
hartomo tb , kozaki a , hasegawa d , et al : minimal residual disease monitoring in neuroblastoma patients based on the expression of a set of real - time rt - pcr markers in tumor - initiating cells . 
hirase s , saitoh a , hartomo tb , et al : early detection of tumor relapse / regrowth by consecutive minimal residual disease monitoring in high - risk neuroblastoma patients . 
viprey vf , gregory wm , corrias mv , et al : neuroblastoma mrnas predict outcome in children with stage 4 neuroblastoma : a european hr - nbl1 / siopen 19 . 
burchill sa , beiske k , shimada h , et al : recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the international neuroblastoma response criteria bone marrow working group . 
debruyne dn , bhatnagar n , sharma b , et al : alk inhibitor resistance in alk ( f1174l ) - driven neuroblastoma is associated with axl activation and induction of emt . 
piskareva o , harvey h , nolan j , et al : the development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro . cancer lett 364 : 142 - 155 , 2015 [ erratum : cancer lett 369 : 428 , 2015 ] 28 . 
burchill sa , bradbury fm , smith b , et al : neuroblastoma cell detection by reverse transcriptase - polymerase chain reaction ( rt - pcr ) for tyrosine hydroxylase oncol 26 : 5443 - 5449 , 2008 mrna . 
bate - eya lt , ebus me , koster j , et al : newly - derived neuroblastoma cell lines propagated in serum - free media recapitulate the genotype and phenotype of primary neuroblastoma tumours . 
eur j cancer 50 : 628 - 637 , 2014 van nes j , chan a , van groningen t , et al : a notch3 transcriptional module induces cell motility in neuroblastoma . 
kraal kc , bleeker gm , van eck - smit bl , et al : feasibility , toxicity and response of upfront metaiodobenzylguanidine therapy therapy followed by german pediatric oncology group neuroblastoma 2004 protocol in newly diagnosed stage 4 neuroblastoma patients . 
maijenburg mw , noort wa , kleijer m , et al : cell cycle and tissue of origin contribute to the migratory behaviour of human fetal and adult mesenchymal stromal cancer res 18 : 808 - 814 , 2012 cells . 
cheung iy , lo piccolo ms , kushner bh , et al : quantitation of gd2 synthase mrna by real - time reverse transcriptase polymerase chain reaction : clinical utility in evaluating adjuvant therapy in neuroblastoma . 
viprey vf , lastowska ma , corrias mv , et al : minimal disease monitoring by qrt - pcr : guidelines for identication and systematic validation of molecular markers prior to evaluation in prospective clinical trials . 
gabert j , beillard e , van der velden vh , et al : standardization and quality control studies of real - time quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemiaa europe against cancer prograleukemia 17 : 2318 - 2357 , 2003 van wezel em , stutterheim j , vree f , et al : minimal residual disease detection in autologous stem cell grafts from patients with high risk neuroblastoma . 
wu sq , lv ye , lin bh , et al : silencing of periostin inhibits nicotine - mediated tumor cell growth and epithelial - mesenchymal transition in lung cancer cells . 
j clin invest 119 : 1420 - 1428 , 2009 van wezel em , zwijnenburg d , zappeij - kannegieter l , et al : whole - genome sequencing identies patient - specic dna minimal residual disease markers in neuroblastoma . 
chicard m , colmet - daage l , clement n , et al : whole - exome sequencing of cell - free dna reveals temporo - spatial heterogeneity and identies treatmentresistant clones in neuroblastoma . 
 mesenchymal minimal residual disease panel in neuroblastoma appendix microarray analysis mesenchymal ( mes ) - specic candidate markers were identied by comparing gene expression proles ( human genome u133 plus 2.0 array ; affymetrix , santa clara , ca ) among the following isogenic pairs of adrenergic ( adrn ) and mes cell lines : 691 - mes / 691b - adrn , sh - ep2 ( mes ) / sh - sy5y ( adrn ) , 700 - mes / 700 - adrn.24 specically , we screened for genes with a 1 log - fold or more difference in expression ( within each isogenic pair ) and an expression level greater than 400 units , thereby selecting only highly expressed marker genes . 
to identify discriminating markers for normal mesenchymal stromal cells ( mscs ) , the top 300 genes with the highest expression were used ( gene expression omnibus : gse68374 ) ; the genes with the highest fold difference between mscs and mes neuroblastoma cell lines were then selected . 
for each analysis , each genes expression was compared with its corresponding expression level in reference blood samples obtained from ve different data sets ( gse13159 , gse17186 , gse10715 , gse8121 , and gse6575 ) ; only genes that are not expressed in hematologic cell types ( using haematlas ; watkins et al : blood 113 : e1 - e9 , 2009 ) were selected . 
the reverse transcription was then heat inactivated , and the reaction volume was increased to 100 to 150 l . primers and probes were designed using primer express version 1.5 software ( applied biosystems , foster city , ca ) or oligo 6 ( molecular biology insights , colorado springs , co ) and synthesized by eurogentec ( li `ege , belgium ; data supplement )  . 
the primer / probe combinations for glucuronidase ( gusb ) , - 1 , 4 - n - acetylgalactosaminyltransferase 1 ( b4galnt1 , also known as gd2s ) , paired - like homeobox 2b ( phox2b ) , tyrosine hydroxylase ( th ) , dopa decarboxylase ( ddc ) , growth - associated protein 43 ( gap43 ) , cholinergic receptor nicotinic 3 ( chrna3 ) , and dopamine - hydroxylase ( dbh ) have been published previously10 , 36 , 37 ( beillard et al : leukemia 17 : 2474 - 2486 , 2003 )  . 
the initial screening for candidate molecular markers was performed using sybr green i dye ( applied biosystems ) combined with a melting curve analysis followed by specic taqman probes ( eurogentec )  . 
 effects of molecular heterogeneity on survival of patients with brafv600 - mutated melanoma treated with vemurafenib with or without cobimetinib in the cobrim study purpose the treatment of advanced brafv600 - mutated melanomas with braf inhibitors ( brafi ) has improved survival , but the efficacy of brafi varies among individuals and the development of acquired resistance to brafi through reactivation of mitogen activated protein kinase ( mapk ) signaling is common . 
we performed an exploratory , retrospective analysis to investigate the effects of brafv600 allelic balance , coexisting oncogene mutations , cell proliferation signaling levels , and loss of pten expression on progression - free survival ( pfs ) in patients in the phase iii cobrim study , which compared the combination of the mek inhibitor cobimetinib with the brafi vemurafenib versus vemurafenib as monotherapy . methods baseline tumor samples from the intention - to - treat population were analyzed by targeted deep sequencing at a median coverage of 3 , 600 and by immunohistochemistry for cell proliferation markers , brafv600e , and pten . 
gene expression in relation to loss of pten was profiled by rna sequencing in 205 patient samples and 42 brafv600 - mutated melanoma cell lines . results neither brafv600 allelic balance nor coexisting mutations in the ras / raf / rtk pathway affected pfs in either treatment group . 
loss of function of the tumor suppressor pten ( via both genetic alterations and other mechanisms such as epigenetic silencing ) occurs in > 20% of patients with brafv600 - mutated melanoma.15 - 17 loss of functional pten has been associated with a shorter pfs response to braf inhibition , 17 , 18 reduced overall survival , and a shorter time to the development of brain metastases.15 , 19 pten / akt signaling is known to interact with mapk signaling . 
for example , meki have been shown to enhance epidermal growth factorinduced akt activation , and mutations that activate pi3k / akt signaling reduce mapk signaling.20 to understand the impact of baseline tumor heterogeneity on the efficacy of treating advanced melanoma with meki and brafi , we systematically examined the effect of braf allelic balance , coexisting oncogene mutations , and cell proliferation signaling levels on pfs in all evaluable cobrim study participants . 
here , we report the results of this retrospective , exploratory analysis . methods analysis population samples for biomarker analysis were collected from patients with unresectable locally advanced or metastatic brafv600 - mutated melanoma in the cobrim study of cobimetinib combined with vemurafenib versus vemurafenib monotherapy , 6 with the aim of exploring intrinsic mechanisms of resistance to mek and braf inhibition . 
brafv600 allelic balance , coexisting oncogene mutations , cell proliferation biomarkers , and loss of pten expression were analyzed in samples from the cobrim intention - to - treat ( itt ) population . 
most analyses could not be performed on the complete itt population ( n = 495 ) because of insufficient tissue availability or assay failure . genetic marker analysis archival formalin - fixed paraffin - embedded ( ffpe ) melanoma tumor samples , biopsy specimens of accessible tumor lesions , and blood samples were obtained from consenting patients at study entry ( baseline )  . 
rna was extracted using the high pure rna paraffin isolation kit ( roche diagnostics , indianapolis , in )  . from the 495 patients in the cobrim itt population , 400 pretreatment tumor samples were evaluable by targeted next - generation sequencing . 
 ( a ) distribution of variant allele frequency ( vaf ; ratio of mutant to wild type [ wt ] ) in brafv600 in 400 baseline tumor samples from cobrim study patients . 
 ( b ) impact on progression - free survival ( pfs ) indicated by kaplan - meier plots of pfs in patients grouped by vaf above or below the median for each treatment ar ( c ) immunohistochemistry of brafv600e protein levels in tumor samples with a range of vafs ; increased vaf was associated with greater staining intensity . 
cobi , cobimetinib ; pbo , placebo ; vem , vemurafenib . mutations ( appendix table a1 ) was performed for a focused panel of 17 oncogenes ( suraseq targeted next - generation sequencing assay ; asuragen , austin , tx ) 21 at a median coverage of 3 , 600 . 
different cutoff points were explored earlier in the analysis and gave results similar to those shown here using the median vaf as the high and low cutoff ( appendix fig a1 )  . 
the median was chosen to be representative of the overall range of cutoff points . analysis of pten copy number and methylation status in 42 brafv600 - mutant melanoma cell lines was performed as described by haverty et al.22 analysis of methylation status was performed in bisulfite - treated genomic dna using the illumina 450k array ( infinium humanmethylation450 beadchip kit ; illumina , san diego , ca )  . 
methylation data were analyzed using the methyanalysis package for r.23 gene expression in relation to loss of pten was profiled by rna sequencing in 205 patient samples and in 42 braf v600 - mutant melanoma cell lines . 
rna was prepared for high throug hput sequencing with the truseq rna access library prep kit ( illumina ) , and 40 million 2 100 - bp reads were sequenced per sample using illumina hiseq . 
coexisting oncogene mutations and impact on progression - free survival ( pfs ) in patients treated with cobimetinib ( cobi ) combined with vemurafenib ( vem ) or vem monotherapy . 
 ( b ) pfs in patients with or without ras / raf / rtk mutations ( both treatment arms were combined to give sufficient numbers of patients with ras / raf / rtk mutations for statistical analysis )  . 
 ( c ) patients with coexisting ras / raf / rtk mutations had elevated levels of phosphorylated extracellular regulated kinase ( perk ) , measured by immunohistochemistry , compared with patients with wild - type ( wt ) ras / raf / rtk , indicating greater mitogen - activated protein kinase signaling pathway activity . 
 defined by the molecular signatures database emt hallmark collection , 24 was compared by z - score ( sum of log2 reads per kilobase of transcript per million mapped reads for all genes on the emt hallmark collection ) between samples with functional and nonfunctional pten . and immunohistochemical markers and a pfs data cutoff date of january 16 , 2015 . 
 expression levels were quantified by histo - score ( h - score ) , and samples were grouped as high or low , depending on whether the h - score was above or below the median , respectively . 
in a subset of samples , brafv600e expression was also evaluated by ihc ( antibrafv600e [ ve1 ] mouse monoclonal primary antibody ; ventana [ n = 39 ] )  . in vitro studies the effect of pten loss on drug sensitivity was evaluated in 42 brafv600 - mutant melanoma cell lines by analyzing cell viability after vemurafenib treatment . 
 in all samples , > 95% of tumor cells stained positive for brafv600e mutation , suggesting that differences in vaf reflect cells across the tumor rather than indicate a subclonal mutation , and that increased vaf was associated with greater staining intensity ( fig 1c )  . 
similar results were seen in a set of four melanoma cell lines ( appendix fig a2 )  . association between oncogene mutations and pfs to explore potential drivers of brafi resistance affecting survival , in addition to previously identified mechanisms such as mek mutation , 27 coexisting mutations in a panel of 17 oncogenes21 ( appendix table a1 ) were assayed in baseline braf - mutated tumor tissue from 400 of 495 patients in the cobrim itt population and were identified in 43 patients ( 11% ; fig 2a )  . 
h - score , histo - score ; mut , mutation ; pbo , placebo ; pd , progressive disease ; pr , partial response ; sd , stable disease . 
cell proliferation biomarkers and impact on progression - free survival ( pfs ) in patients treated with cobimetinib ( cobi ) combined with vemurafenib ( vem ) or vem monotherapy . 
 ( a ) kaplan - meier plots of pfs in patients grouped by high ( above the population median histo - score [ h - score ] of 40 ) or low ( below the population median ) phosphorylated extracellular regulated kinase ( perk ) levels . 
 ( b ) kaplan - meier plots of pfs in patients grouped by high or low baseline proliferation index , measured by the protein encoded by themki67gene ( ki - 67 ) immunohistochemistry ( population median ki - 67 level is 20% ) or ( c ) phosphorylated s6 immunohistochemistry ( ps6 ; population median h - score is 71 )  . 
 ( c ) rna sequencing profiling of melanoma samples from cobrim comparing the epithelial - mesenchymal transition ( emt ) gene expression signatures in tumors with pten loss versus tumors with normal pten levels . 
patient characteristics were similar between patients with and without loss of pten expression in the cobimetinib combined with vemurafenib and the vemurafenib monotherapy cohorts , with the exception that more patients with loss of pten expression had elevated lactate dehydrogenase in the cobimetinib combined with vemurafenib arm ( appendix table a3 )  . 
 ( b ) rna sequencing profiling of braf - mutant melanoma cell lines with or without pten loss comparing the epithelial - mesenchymal transition ( emt ) gene expression signatures . 
 loss of pten expression was associated with upregulation of the emt gene expression signature in these melanoma cell lines ( fig 5b ) , similar to that seen in the baseline tumor samples . 
when tested in an in vitro wound healing assay , in the absence of vemurafenib or cobimetinib , cell lines with loss of pten showed a higher capacity for migration ( fig 5c ) , consistent with observed clinical manifestations and previous preclinical findings.28 , 29 attempts to analyze whether vemurafenib or cobimetinib combined with vemurafenib affected the increased cell migration behavior associated with loss of pten were confounded by their effect on cell viability . discussion to our knowledge , this retrospective biomarker analysis of brafv600 - mutated melanoma samples from the cobrim study is the largest such study to date . 
although there was an association between signaling pathway activity and pfs in patients treated with vemurafenib monotherapy , increased mapk signaling and cell proliferation did not affect pfs in patients treated with a combination of cobimetinib and vemurafenib . 
 the combination of mek and braf inhibition thus seems to override the poor prognostic effect of increased mapk pathway activation . loss of pten expression is linked to reduced survival in advanced melanoma , most likely as a result of suppression of apoptosis and enhanced cell survival mediated by hyperactivation of pi3k / akt signaling.17 , 18 our analysis suggests that combined mek and braf inhibition can overcome the effect of loss of pten on pfs . 
 loss of pten function in addition to brafv600 mutation has been linked to the rapid development of brain metastases in advanced melanoma , which may also contribute to reduced pfs.15 , 26 our analysis confirms that brafv600 - mutated melanoma cells with loss of pten function have a higher expression of genes associated with emt , as well as enhanced migration . 
functional pten inhibits cell migration through dephosphorylation of focal adhesion kinase ( fak ) ; loss of pten relieves this inhibition.29 in prostate cancer models , ras / mapk activation cooperated with pten loss to promote emt and metastasis , and mek inhibition was able to reduce metastatic progression in these models.30 mek can also activate fak to promote cell migration , and mek inhibition could therefore downregulate fak - mediated migration , 31 addition to reducing the cell survival resulting from ras / mapk activation in brafv600 - mutated melanoma . 
this suggests a potential mechanism by which cobimetinib combined with vemurafenib could contribute to a survival benefit in patients with braf v600 - mutated melanoma and loss of pten . the large patient cohort available for analysis was a significant strength of this study ; however , the analysis was limited by the inability to evaluate baseline tumor samples for approximately 20% of patients included in the cobrim study . additional investigation of the mechanisms underlying intrinsic and acquired resistance is warranted . 
 the molecular mechanisms at work in patients with prolonged responses to combination therapy with cobimetinib and vemurafenib are still under investigation but may involve regulation of the host antitumour immune response.33 the overall findings of this study suggest that deeper inhibition of the mapk pathway through combined inhibition of both mek and braf overrides the effects of molecular heterogeneity to provide a survival benefit to all patients . 
 this both supports and provides a rationale for the greater efficacy of combination therapy with braf and meki , which has become the standard of care , compared with braf inhibition alone . 
ascierto , brigitte drno , anna maria di giacomo , claus garbe , ilsung chang , jessie hsu , hartmut koeppen , james larkin , yibing yan , grant a . 
wongchenko employment : genentech / roche stock and other ownership interests : genentech / roche , ariad pharmaceuticals antoni ribas stock and other ownership interests : compugen , flx bio , cytomx therapeutics , five prime therapeutics , advaxis , arcus biosciences , tango therapeutics , pact pharma consulting or advisory role : merck , amgen , genentech / roche , novartis paolo a . 
 james larkin honoraria : eisai , bristol - myers squibb , msd , glaxosmithkline , pfizer , novartis , genentech / roche , sectra , pierre fabre , eusa pharma , consulting or advisory role : eisai , bristol - myers squibb , msd , glaxosmithkline , pfizer , novartis , genentech / roche , sectra , pierre fabre , eusa pharma research funding : pfizer ( inst ) , novartis ( inst ) , msd ( inst ) , bristol - myers squibb ( inst ) travel , accommodations , expenses : bristol - myers squibb , pfizer , novartis , genentech / roche yibing yan employment : genentech / roche stock and other ownership interests : genentech / roche patents , royalties , other intellectual property : genentech / roche grant a . 
wongchenko , ilsung chang , jessie hsu , isabelle rooney , william lu , hartmut koeppen , and yibing yan , genentech , south san francisco ; antoni ribas , jonsson comprehensive cancer center at the university of california , los angeles , los angeles , ca ; paolo a . 
pascale , naples ; anna maria di giacomo , azienda ospedaliera universitaria senese , siena , italy ; brigitte drno , nantes university , nantes , france ; claus garbe , university of tbingen , tbingen , germany ; james larkin , the royal marsden hospital , london , united kingdom ; grant a . 
ascierto pa , mcarthur ga , drno b , et al : cobimetinib combined with vemurafenib in advanced braf ( v600 ) - mutant melanoma ( cobrim ) : updated efficacy results from a randomised , doubleblind , phase 3 trial . 
hauschild a , grob j - j , demidov lv , et al : dabrafenib in braf - mutated metastatic melanoma : a multicentre , open - label , phase 3 randomised controlled trial . 
wilmott js , menzies am , haydu le , et al : braf ( v600e ) protein expression and outcome from braf inhibitor treatment in braf ( v600e ) metastatic melanoma . 
wagle n , van allen em , treacy dj , et al : map kinase pathway alterations in braf - mutant melanoma patients with acquired resistance to combined raf / mek inhibition . 
bucheit ad , chen g , siroy a , et al : complete loss of pten protein expression correlates with shorter time to brain metastasis and survival in stage iiib / c melanoma patients with brafv600 mutations . 
xing f , persaud y , pratilas ca , et al : concurrent loss of the pten and rb1 tumor suppressors attenuates raf dependence in melanomas harboring ( v600e ) braf . 
nathanson kl , martin a - m , wubbenhorst b , et al : tumor genetic analyses of patients with metastatic melanoma treated with the braf inhibitor dabrafenib ( gsk2118436 )  . 
latham g , hough j , sah s , et al : suraseq targeted ngs assays : integrated , cross - platform reagents that enable accurate detection of cancer gene mutations in residual clinical ffpe , fna , and biofluid biopsies . 
mcarthur ga , larkin j , ascierto pa , et al : impact of baseline allelic frequencies of brafv600 mutation and coexisting oncogenic mutations on progression - free survival from the cobrim phase 3 study evaluating cobimetinib + vemurafenib in advanced brafv600 - mutated melanoma . 
larkin jmg , yan y , mcarthur ga , et al : update of progression - free survival ( pfs ) and correlative biomarker analysis from cobrim : phase iii study of cobimetinib ( cobi ) plus vemurafenib ( vem ) in advanced braf - mutated melanoma . 
tamura m , gu j , takino t , et al : tumor suppressor pten inhibition of cell invasion , migration , and growth : differential involvement of focal adhesion kinase and p130cas . 
mulholland dj , kobayashi n , ruscetti m , et al : pten loss and ras / mapk activation cooperate to promote emt and metastasis initiated from prostate cancer stem / progenitor cells . 
wongchenko mj , mcarthur ga , drno b , et al : gene expression profiling in braf - mutated melanoma reveals patient subgroups with poor outcomes to vemurafenib that may be overcome by cobimetinib plus vemurafenib . 
exploration of the impact of variant allele frequency ( vaf ) on progression - free survival ( pfs ) indicated by kaplan - meier plots of pfs in patients grouped by vaf above or below cutoff points of ( a ) 25% and ( b ) 50% for each treatment arfor the analysis , the median was chosen to be representative of the overall range of cutoff points . 
cell viability of 42 brafv600 - mutant melanoma cell lines treated with vemurafenib , stratified by expression versus loss of expression of pten . pten + ( n = 20 ) pten loss ( n = 22 ) table a1 . 
konat e , phd1 ; biswajit das , phd3 ; chris karlovich , phd3 ; chih - jian lih , phd3 ; eric polley , phd9 ; richard simon , dsc1 ; ming - chung li , phd1 ; richard piekarz , md , phd1 ; and james h . 
although patients and physicians were blinded to the sequencing and random assignment results , a higher pretreatment dropout rate was observed in the control arm ( 22% ) compared with the experimental arm ( 6% ; p = .038 ) , suggesting that some patients may have had prior tumor mutation proling performed that led to a lack of participation in the control arm . conclusion further investigation , better annotation of predictive biomarkers , and the development of more effective agents are necessary to inform treatment decisions in an era of precision cancer medicine . 
increasing prevalence of tumor mutation proling and preference for targeted therapy make it difcult to use a randomized phase ii design to evaluate targeted therapy efcacy in an advanced disease setting . jco precis oncol 5 : 133 - 144 . 
to explore this , we conducted a randomized , histology - agnostic clinical to examine whether patients with advanced , refractory cancer who had a tumor mutation in a gene in one of three signaling pathways ( dna repair , pi3k , or ras / raf / mek ) were more likely to derive clinical benet if treated with regimens targeting that pathway ( the experimental arm ) than if they were treated with regimens that did not ( the control arm )  . 
 chen et al context key objective a molecular aberration in a patients tumor is expected to render the tumor susceptible to a drug targeting that aberration , but randomized , controlled , blinded phase ii trials conrming the efcacy of this precision medicine approach are both sparse and challenging to design . knowledge generated efcacy in patients with study - dened actionable mutations in the dna repair , ras / raf / mek , or akt / pi3k / mtor pathways either did not achieve the target objective response rate ( trametinib or adavosertib with carboplatin ) or indicated futility despite accrual challenges ( everolimus or veliparib with temozolomide )  . 
patients randomly assigned to the nontargeted control arm had a higher pretreatment dropout rate than the experimental arm , suggesting a preference for targeted therapy based on prestudy genetic proling . relevance the prevalence of genetic data makes it challenging to randomly assign patients to a nontargeted control arbetter geneand variant - specic biomarkers that predict response to drugs are needed for patients with cancer . patients were required to have measurable disease , be willing to undergo tumor biopsy to establish presence of a study - dened actionable mutation of interest ( amoi ) , and have tumor amenable to interventional radiologyguided percutaneous biopsy with a 16to 18 - gauge neeindicated and dle ; excisional biopsy was allowed if evaluable . 
patients who had prior treatment with any of the investigational agents were eligible to participate but were not assigned that same agent . agent - specic eligibility criteria are included in the data supplement . this trial was conducted under a national cancer institute ( nci ) - sponsored investigational new drug application with institutional review board approval . 
protocol design and conduct followed all applicable regulations , guidances , and local policies ( clinicaltrials.gov identier : nct01827384 )  . the investigators obtained informed consent from each participant . trial design this was a multihistology , multicenter , randomized phase ii study of four investigational drug regimens demonstrated to inhibit the dna repair pathway , ras / raf / mek pathway , or akt / pi3k / mtor pathway . 
eligible patients with an amoi detected were randomly assigned 2 : 1 to receive the recommended phase ii dose of either ( 1 ) a predened targeted regimen based on mutation status ( experimental arm ) or ( 2 ) a regimen , chosen from the four study regimens , that did not target their amois ( control arm )  . 
the specic study amois ( data supplement ) were selected on the basis of published functional evidence or implications for protein translation and pathway function ; they were detected in patient samples via a clinical laboratory improvement amendments ( clia ) sequencing assay developed and validated by the molecular characterization laboratory at the frederick national laboratory for cancer research , as previously described.5 patient eligibility determination and randomized treatment assignment were performed with the genemed informatics system as described.6 if more than one amoi was detected for a patient randomly assigned to the experimental arm , the targeted treatment was based on the selection of higher allele frequency ; if allele frequencies were within 15% , the patient was assigned to the targeted treatment cohort with the fewest patients enrolled ( with the option to receive the other treatment regimen upon disease progression )  . 
if a patient randomly assigned to the control arm had multiple nontargeted treatment options , their regimen was chosen based on the proportions of treatment assignments on the experimental arm , so as to balance the two arms with respect to proportions receiving each of the four regimens . for the purpose of assessing the primary end point of this trial , only response to the rst regimen was used . 
adverse events ( aes ) were graded according to nci common toxicity criteria version 4.0 until march 31 , 2018 , when version 5.0 was implemented ; all aes were mapped to version 5.0 before the nal analysis . 
 random assignment and clinical outcome on the nci - mpact trial study agents veliparib ( abt - 888 ; nsc 737664 ) , adavosertib ( azd1775 ; nsc 751084 ) , trametinib ( nsc 763093 ) , and everolimus ( nsc 733504 ) were supplied by the division of cancer treatment and diagnosis , nci , under collaborative research and development agreements with abbvie ( north chicago , il ) , astrazeneca ( cambridge , united kingdom ) , glaxosmithkline ( brentford , united kingdom ) , and novartis ( basel , switzerland ) , respectively . 
study drugs were administered at the recommended phase ii doses and schedules ( data supplement )  . statistical study design the accrual ceiling for each regimen cohort of the experimental arm was set at 30 patients to discriminate between tumor response rates of 20% versus 5% . 
the design included n = 198 enrolled n = 5 did not have biopsy n = 193 had biopsy collected n = 13 insufficient tumor or dna n = 1 declined to participate detceted ioma on dah 17 = n detceted ioma na dah 801 = n n = 7 had no targeted regimen n = 5 had no control regimen but were offered treatment n = 64 randomly assigned to targeted arm n = 32 randomly assigned to control arm n = 10 assigned everolimus n = 25 assigned trametinib n = 3 assigned veliparib plus tmz n = 10 assigned everolimus n = 10 assigned trametinib n = 2 assigned veliparib plus tmz n = 26 assigned adavosertib plus carboplatin n = 10 assigned adavosertib plus carboplatin n = 7 clinically excluded n = 4 died n = 4 declined to participate n = 7 clinically excluded n = 1 died n = 7 declined to participate n = 6 received adavosertib plus carboplatin n = 8 received everolimus n = 20 received trametinib n = 3 received veliparib plus tmz n = 3 received everolimus n = 6 received trametinib n = 2 received veliparib plus tmz n = 8 off treatment : n = 2 death ( pd ) n = 4 pd n = 1 toxicity n = 1 withdrew n = 20 off treatment : n = 19 pd n = 1 withdrew n = 3 off treatment : n = 3 pd n = 3 off treatment : n = 3 pd n = 6 off treatment : n = 5 pd n = 1 withdrew n = 2 off treatment : n = 1 pd n = 1 withdrew n = 6 off treatment : n = 5 pd n = 1 toxicity n = 18 received adavosertib plus carboplatin n = 18 off treatment : n = 13 pd n = 1 intercurrent illness n = 3 toxicity n = 1 withdrew fig 1 . 
seven patients had an amoi but could not be randomly assigned because they were ineligible for the targeted treatment ( six patients : pancreatic cancer with an ras mutation ; one patient : unknown reason )  . 
 chen et al an interim futility analysis ; if no objective responses were observed among the initial 12 patients in each experimental cohort , the cohort was to be terminated early ( with 54% likelihood under the null hypothesis ) , with 93% condence that the response rate would be lower than the target 20% rate . 
this design yields at least 84% power to detect a true objective response rate of at least 20% and at least 0.94 probability of a negative result if the true objective response rate was no more than 5% . 
four - month progression - free survival ( pfs ) , dened as the time from random assignment to progression or death from any cause ( whichever comes rst ) , was evaluated as a secondary end point using kaplan - meier estimates and cis calculated using greenwoods formula . 
twelve or more instances of 4 - month pfs ( at least 40% ) among the 30 patients in an experimental cohort was to be considered promising ; this would occur with 90% likelihood if the true 4 - month pfs rate is 50% ( median pfs of 4 months ) and with 5% likelihood if the true 4 - month pfs rate is 25% ( median pfs of 2 months )  . results enrollment and treatment assignment one hundred ninety - eight patients who met study eligibility criteria were enrolled from january 2014 to april 2018 ( fig 1 , table 1 , data supplement ) , 108 ( 55% ) of whom underwent a tumor biopsy procedure and ultimately had a study - actionable mutation detected ( fig 2a )  . 
patient characteristics characteristic patients enrolled median age , years ( range ) sex ( female / male ) median number of prior lines of therapy ( range ) a patients with karnofsky performance status ( % ) dna repair pi3k ras / raf / mek patients with an amoi in a targeted pathwayb number 60 ( 23 - 83 ) 109 / 89 4 ( 1 - 12 ) abbreviations : amoi , actionable mutation of interest . areported for patients who received at least one dose of study agent on nci - mpact . breported for all patients with an amoi detected on nci - mpact . 
patients with an amoi in more than one pathway are counted here for each affected pathway . 136 2021 by american society of clinical oncology the genes with highest frequency of study amois were tp53 and kras ( 56% and 46% of patients with an amoi , respectively ) ( fig 2c , data supplement )  . 
all patients assigned to the two dna repair pathway cohorts of the experimental arm had a tp53 mutation and 75% of patients assigned to the trametinib experimental cohort had a kras mutation . 
the majority of amois were nonsynonymous single - nucleotide variants ( data supplement )  . toxicity thirty ( 45% ) of the 66 patients who received at least one dose of study agent ( s ) experienced an ae of grade 3 or greater that was considered at least possibly related to study treatment ( data supplement )  . 
seven patients died within 30 days of their last dose of study drugs but none of the on - study deaths were considered related or likely related to the study treatments . treatment compliance interim analysis revealed unanticipated differences in compliance for patients who were assigned to targeted versus nontargeted therapy ( fig 3a )  . 
the overall percentage of patients assigned to the control arm who never initiated treatment was 47% ( 15 / 32 ) , signicantly higher than the rate for patients assigned to the experimental arm ( 23% ; 15 / 64 ; p = .034 , two - sided , by fishers exact test )  . 
evaluation of the reasons why patients came off study , as documented in the clinical database , reveals that a signicantly higher percentage of the patients randomly assigned to the control arm chose not to start treatment ( 22% [ 7 / 32 ] ) compared with the percentage of patients in the experimental arm who chose not to start treatment ( 6% [ 4 / 64 ] ; p = .038 , two - sided , by fishers exact test )  . interim futility analysis accrual to the veliparib plus tmz and everolimus cohorts was slow such that accrual did not reach the 12 - patient threshold for interim analysis of the primary end point , objective response rate ; no responses were measured on either regimen ( fig 3b )  . 
slow accrual to the veliparib plus tmz arm was because of the fact that every dna repair pathway amoi detected on study was within tp53 ; on the basis of preclinical evidence that loss - of - function mutations in tp53 affect regulation of cell cycle progression , patients who were randomly assigned to the experimental arm with these mutations were assigned adavosertib plus carboplatin as their rst - line study treatment if eligible , not veliparib plus tmz . 
pfs events were experienced by 12 / 17 randomly assigned and treated patients in the control arm ; among the patients randomly assigned to the experimental cohorts , pfs events were documented in 6 of 8 treated with everolimus , 20 of 20 treated with trametinib , 13 of 18 treated with adavosertib plus carboplatin , and 3 of 3 treated with veliparib plus tmz . 
caution must be used in comparing the individual experimental cohorts to the control arm as the restriction of an experimental cohort to a particular target may be prognostic of better ( or worse ) pfs . 
 ( a ) nci - mpact treatment compliance rates : the percent of randomly assigned patients who initiated their assigned study treatment or did not start treatment because of death , clinical exclusion that developed after enrollment , or patient choice . 
 ( b ) the proportions of randomly assigned and treated patients who experienced objective response ( complete response or conrmed partial response ) are presented by arm or targeted treatment cohort . 
none of the three patients in the experimental veliparib plus tmz cohort were progressionfree at 4 months ( ci not provided because of small sample size )  . two patients experienced sustained stable disease ( sd ) for a noteworthy 24 cycles of targeted treatment ( fig 5 )  . one patient with endometrial cancer and pik3ca h1047l and pten r130 * amois received everolimus and experienced sd for 24 cycles before progressing . 
the other patient , a 52 - year - old woman with low - grade ovarian cancer with an nras q61r amoi , experienced sd for 27 cycles of trametinib treatment before her disease progressed . 
in the experimental adavosertib plus carboplatin cohort , one patient with endometrial carcinoma and tp53 r213 * and pik3ca c420r amois had an unconrmed pr before coming off treatment because of toxicity . 
none of the patients who crossed over at disease progression from the control arm to a targeted treatment or from one targeted treatment to another experienced clinical benet on the crossover regimen ( data supplement )  . discussion highly effective , tailored therapy targeting specic genetic aberrations is a primary goal of precision medicine.1 , 2 reports of exceptional responders , retrospective studies , and several nonrandomized trials indicate clinical benet for genome - driven treatments , prompting optimism and discussion about the appropriate evidence framework for precision oncology.3 , 8 - 24 the results of our study , which was important considerdesigned in 2012 , highlight several ations for randomized precision oncology studies in the advanced disease setting that have evolved since then.25 - 28 with the greater prevalence of genetic tests and emphasis on precision medicine , it may be very challenging to randomly assign patients to a nontargeted control arour data suggest that some patients and physicians may have had prior tumor mutation prole knowledge and , when randomly assigned to the control arm , appeared to show a bias favoring the presumed precision medicine approach , declining to participate in the study . 
the low dropout rate on the similarly designed shiva trial may be explained by having the patients randomly assigned to a physicians choice control arm ; results from the shiva trial also did not demonstrate the superiority of molecularly targeted agents over the control treatment.25 the results of the winther and coppo trials , two nonrandomized studies , describe clinical benet for a small number of patients with personalized treatment but the pfs - based end point was not met in either study.29 , 30 our results are similar in that the two statistically evaluable experimental cohorts indicate that neither trametinib nor adavosertib plus carboplatin was more effective than standard therapy at achieving objective response when assigned to target nci - mpact - dened amois in ras / raf / mek or dna repair pathways ( eg , kras gain of function or tp53 loss of function mutations , respectively )  . 
 random assignment and clinical outcome on the nci - mpact trial experimental arm control arm 120 - day rate median 120 - day rate median 100 150 200 250 300 350 100 150 200 250 300 350 days since random assignment days since random assignment number at risk number at risk everolimus experimental cohort trametinib experimental cohort 120 - day rate median 120 - day rate median 100 150 200 250 300 350 days since random assignment number at risk 100 150 200 250 300 350 days since random assignmentt number at risk ( b ) control arm , fig 4 . 
 chen et al x age at dx prior disease category cycles best response werdhtiw yticixot withdrew toxicity illness toxicity werdhtiw female genitourinary genitourinary lower gi breast lower gi female genitourinary gender female male female male female female female male female female male male female female female female male female male male female female male male female female female female female male female male male female female female female female male male male female female male female female female male male male female male female male male male male female female female female elamef genitourinary head and neck genitourinary sarcoma genitourinary lower gi lower gi lower gi lung upper gi genitourinary neuroendocrine lower gi genitourinary lower gi lower gi lower gi lower gi lower gi breast sarcoma lower gi genitourinary gi upper head and neck breast lower gi melanoma lower gi breast sarcoma lower gi lower gi genitourinary genitourinary lower gi lung lower gi lower gi lower gi lower gi lower gi breast lower gi lower gi lung lower gi lower gi lower gi lower gi lower gi lower gi female genitourinary lower gi melanoma melanoma lung genitourinary female genitourinary fig 5 . 
each patients detected nci - mpact amois are listed and color - coded to indicate the level of evidence that the mutation is susceptible to the assigned nci - mpact treatment ( based on the information in the oncokb and civic precision oncology knowledge bases at the time of writing )  . 
where available , the results of whole exome sequencing are presented as the number of genetic alterations detected that are annotated in oncokb as either oncogenic ( # oncogenic mutations ) or as oncogenic and actionable with available therapeutic agents ( # oncokb mutations )  . 
 random assignment and clinical outcome on the nci - mpact trial medicine approach , the lack of signicant response on the experimental arm argues for need of better therapies and further validation . 
extensive genetic sequencing and clinical trials evaluating broader aspects of pathway alterations may reveal additional information about actionable sensitivity markers . the targeted treatment assignments in nci - mpact were made based on molecular aberrations that were expected to confer therapeutic sensitivity at the level of a signaling pathway . 
since the trials initiation , the cancer research community has been engaged in an ongoing effort identify geneand variant - specic biomarkers that predict response to drugs.31 - 37 retrospective review of two precision oncology knowledge bases , oncokb and civic , suggests that there is little curated evidence to support many of the amoi - drug associations targeted in the ncimpact trial , which is consistent with the lack of clinical activity . 
for example , tp53 mutations , which were detected in every nci - mpact patient randomly assigned to a targeted dna repair inhibitor cohort , are understood to be oncogenic but have remained largely undruggable.38 the experimental trametinib cohort is the exception in that for each ras / mek / erk pathway amoi that was detected , there is evidence curated in oncokb or civic suggesting effective inhibition by trametinib . 
notably , all three patients who were treated with trametinib to target an nras q61r mutation experienced clinical benet , in line with published clinical evidence of mek inhibitor activity in patients carrying an nras q61 mutation.39 most of the patients treated in the experimental trametinib arm carried an amoi in kras . 
although there is preclinical support for treating kras amois with an mek inhibitor , the results of that approach were modest in this study , perhaps because of reported mechanisms of kras mutant resistance to atpnoncompetitive mek inhibitors.40 - 42 the presence of a therapy - targeted amoi furthermore , in a patient tumor does not indicate whether the amoi is a driver or passenger mutation.43 , 44 in nci - mpact , the most frequently detected amoisthose in tp53 and kraswere detected in all four treatment cohorts and might have contributed to disease progression in cases where treatment was assigned to target an amoi in a different pathway ( eg , akt / pi3k / mtor pathway )  . 
treatment assignment in such cases was made based on the amoi with the highest allele frequency but there is precedent for subclonal molecular alterations driving disease progression.45 - 48 two of the three patients who tolerated everolimus treatment and had amois conned to only the pi3k pathway experienced prolonged sd ( 10 cycles )  . it is important to note that we were able to achieve a successful biopsy collection rate of  . 
90%.49 even with a turnaround time of 10 days for sequencing results , 5 19 biopsied patients ( 10% ) progressed to the point of ineligibility or death before treatment initiation , reecting the advanced disease status of this patient population . 
biopsy sampling could not have identied spatially or temporally isolated tumor subclones that may have been driving tumor growth or conferring resistance in these patients , 43 , 44 , 50 , 51 challenges that can be explored further in preclinical studies as can the presence of putative driver mutations in other signaling pathways . we are completing the analysis of a preclinical study performed in parallel with the nci - mpact trial using xenograft models derived from the tumors of cancer patients to overcome the hurdles inherent to clinical investigations of molecularly targeted treatment response ( manuscript in preparation )  . 
our preliminary data suggest that functional in vivo evidence of activity in molecularly dened models should be the basis on which to evaluate clinical benet of targeted agents in specied patient subgroups . 
chen , md , national cancer institute , 31 center drive , room 3a44 , bethesda , md 20814 ; twitter : @ncitreatment ; e - mail : chenali@ mail.nih.gov. support this project has been funded in whole or in part with federal funds from the national cancer institute , national institutes of health , under contract no . 
mickey williams , richard piekarz , james h . doroshow provision of study materials or patients : shivaani kummar , geraldine osullivan coyne , funda meric - bernstam , stephen leong , chris karlovich collection and assembly of data : alice p . 
konat e , biswajit das , chris karlovich , chih - jian lih , eric polley , richard simon , ming - chung li , richard piekarz , james h . 
mickey williams research funding : illumina patents , royalties , other intellectual property : i was a co - inventor of the dlbcl cell of origin patent recently led by the nih kanwal p . 
trends mol med 23 : 874 - 898 , 2017 tate jg , bamford s , jubb hc , et al : cosmic : the catalogue of somatic mutations in cancer . 
nucleic acids res 47 : d941 - d947 , 2019 lih c - j , sims dj , harrington rd , et al : analytical validation and application of a targeted next - generation sequencing mutation - detection assay for use in treatment assignment in the nci - mpact trial . 
j mol diagn 18 : 51 - 67 , 2016 zhao y , polley ec , li m - c , et al : genemed : an informatics hub for the coordination of next - generation sequencing studies that support precision oncology clinical trials . 
eur j cancer 45 : 228 - 247 , 2009 ottmann og , druker bj , sawyers cl , et al : a phase 2 study of imatinib in patients with relapsed or refractory philadelphia chromosome - positive acute lymphoid leukemias . 
blood 100 : 1965 - 1971 , 2002 sawyers cl , hochhaus a , feldman e , et al : imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis : results of a phase ii study . 
obrien sg , guilhot f , larson ra , et al : imatinib compared with interferon and low - dose cytarabine for newly diagnosed chronic - phase chronic myeloid leukemia . 
fukuoka m , wu yl , thongprasert s , et al : biomarker analyses and nal overall survival results from a phase iii , randomized , open - label , rst - line study of getinib versus carboplatin / paclitaxel in clinically selected patients with advanced non - small - cell lung cancer in asia ( ipass )  . 
rosell r , carcereny e , gervais r , et al : erlotinib versus standard chemotherapy as rst - line treatment for european patients with advanced egfr mutationpositive non - small - cell lung cancer ( eurtac ) : a multicentre , open - label , randomised phase 3 trial . 
hauschild a , grob jj , demidov lv , et al : dabrafenib in braf - mutated metastatic melanoma : a multicentre , open - label , phase 3 randomised controlled trial . lancet 380 : 358 - 365 , 2012 16 . 
haslem ds , van norman sb , fulde g , et al : a retrospective analysis of precision medicine outcomes in patients with advanced cancer reveals improved progression - free survival without increased health care costs . 
massard c , michiels s , ferte c , et al : high - throughput genomics and clinical outcome in hard - to - treat advanced cancers : results of the moscato 01 trial . cancer discov 7 : 586 - 595 , 2017 19 . 
hainsworth jd , meric - bernstam f , swanton c , et al : targeted therapy for advanced solid tumors on the basis of molecular proles : results from mypathway , an open - label , phase iia multiple basket study . 
freidlin b , allegra cj , korn el : moving molecular proling to routine clinical practice : a way forward ? j natl cancer inst 112 : 773 - 778 , 2019 25 . 
le tourneau c , delord j - p , gonalves a , et al : molecularly targeted therapy based on tumour molecular proling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
herbst rs , gandara dr , hirsch fr , et al : lung master protocol ( lung - map ) - a biomarker - driven protocol for accelerating development of therapies for squamous cell lung cancer : swog s1400 . 
goncalves a , bachelot t , lusque a , et al : high - throughput genome analysis and therapeutic decision for patients with her2 - negative metastatic breast cancer : first feasibility and molecular results of the randomized phase ii study safir02 breast ( ucbg - 0105 / 1304 )  . 
tuxen iv , rohrberg ks , oestrup o , et al : copenhagen prospective personalized oncology ( coppo ) clinical utility of using molecular proling to select patients to phase i trials . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
dummer r , schadendorf d , ascierto pa , et al : binimetinib versus dacarbazine in patients with advanced nras - mutant melanoma ( nemo ) : a multicentre , open - label , randomised , phase 3 trial . 
ferry - galow kv , datta v , makhlouf hr , et al : what can be done to improve research biopsy quality in oncology clinical trials ? j oncol pract 14 : e722 - e728 , 2018 50 . 
the aim of our study was to investigate whether ctdna can be used for response monitoring in neuroblastoma . methods one hundred forty - nine plasma samples from 56 patients were analyzed by quantitative polymerase chain reaction ( qpcr ) for total cell free dna ( cfdna ; albumin and - actin ) and ctdna ( hypermethylated rassf1a )  . 
ctdna results were compared with mrna - based minimal residual disease ( qpcr ) in bone marrow ( bm ) and blood and clinical patient characteristics . results ctdna was detected at diagnosis in all patients with high - risk and stage m neuroblastoma and in 3 of 7 patients with localized disease . 
the discrepancies indicated either low - level bm inltration ( ctdna negative / mrna positive ) or primary tumor / soft tissue lesions with no bm involvement ( ctdna positive / mrna negative )  . conclusion ctdna can be used for monitoring disease in patients with neuroblastoma . 
it is likely that ctdna can originate from both primary tumor and metastases and may be of special interest for disease monitoring in patients who experience relapse in other organs than bm . jco precis oncol 4 : 291 - 306 . 
2020 by american society of clinical oncology creative commons attribution non - commercial no derivatives 4.0 license introduction neuroblastoma is the most common extracranial solid tumor of childhood . in approximately 50% , patients present with high - risk ( hr ) disease and are treated with intensive multimodality treatment protocols that encompass induction therapy , primary tumor surgery , myeloablative chemotherapy with autologous stem - cell rescue , local irradiation , and anti - gd2based immunotherapy1 ( appendix fig a1 )  . 
despite this intensive therapy , in approximately one half of hr patients , the tumor will relapse and result in a fatal outcome.2 assessment of treatment response is based on the international neuroblastoma response criteria . 
meta - iodobenzylguanidine ( mibg ) scintigraphy , imaging ( magnetic resonance imaging / positron emission tomography scans ) , and bone marrow ( bm ) examinations by histology or ( immuno ) cytology are combined to assess the extent of disease.3 because the median age at diagnosis is 18.8 months , 4 response evaluation that is based on imaging and bm testing often must be performed under general anesthesia . 
 van zogchel et al context key objective can hypermethylated rassf1a be used as a circulating tumor dna ( ctdna ) marker for minimal residual disease detection in neuroblastoma ? knowledge generated when testing cell free dna , we were able to detect tumor - derived hypermethylated rassf1a in all patients with stage m disease at diagnosis . 
comparison between ctdna and bloodor bone marrow ( bm ) derived mrna revealed that discrepancies were found when bm inltration was low or when there were primary tumor lesions without bm involvement . relevance ctdna is an interesting source for monitoring disease in patients with neuroblastoma . 
our data indicate that testing of both ctdna and mrna increases the sensitivity of molecular disease monitoring . several studies have shown the feasibility of detecting mutations or tumor - specic translocations in the ctdna by high - depth targeted sequencing or mutation - specic pcr to monitor disease in various types of adult cancer.13 - 19 however , neuroblastoma tumors , like many pediatric tumors , lack recurrent mutations and translocations . 
broader analysis , such as whole - exome sequencing ( wes ) and shallow whole - genome sequencing ( swgs ) , to detect tumor - specic mutations or copy number alterations have been performed successfully using cfdna of patients with neuroblastoma at diagnosis and relapse.20 - 26 nevertheless , these techniques are only informative when the ctdna content is approximately 10%27 and are , therefore , less suited for the detection of mrd , when the tumor burden is low . in contrast to the copy number alterations and tumorspecic mutations , a methylation - specic qpcr assay could potentially be a more general and sensitive ctdna marker . 
hypermethylated rassf1a ( rassf1am ) can be detected in bm with a similar sensitivity as mrna and has shown added value in mrna - negative bm.28 in addition , rassf1am already has been described as a prognostic ctdna marker at diagnosis.29 the aim of this study was to investigate the feasibility of using ctdna ( rassf1am in plasma ) to monitor treatment response in patients with hr / stage m neuroblastoma . 
we retrospectively performed qpcr for rassf1am on cfdna from stored remains of previously collected plasma samples of patients with localized or metastatic neuroblastoma at diagnosis and for patients with hr / stage m neuroblastoma during treatment and at relapse . 
to test the additional value of ctdna monitoring , we compared it with other techniques for disease monitoring : mibg scans , urinary catecholamines , immunocytology , and rt - qpcr rna - based mrd detection in bm and peripheral blood ( pb )  . methods between 2013 and 2016 , from all consecutively diagnosed patients who were included in this study ( n = 56 ) , 149 pb samples for mrna and cfdna and 105 bm samples for mrna were tested . 
pediatric pb control samples were collected from the amsterdam umc ( appendix table a1 )  . sample collection , dna isolation , bisulte conversion , and real - time qpcr methods for sample collection , dna isolation , bisulte conversion , and real - time qpcr for rassf1am28 and mrna markers31 can be found in the appendix and appendix table a2 . data analysis total cfdna was quantied by qpcr for albumin ( alb ) or - actin ( actb )  . 
all statistical analyses were performed with spss version 23 ( ibm corporation , chicago , il ) or graphpad prism 8 ( graphpad software , la jolla , ca ) software . results patients and samples from 48 patients with hr and / or stage m and 8 patients with non - hr neuroblastoma , 149 samples were tested in this study ( fig 1 )  . 
six of the 149 patient samples and 12 of 73 healthy control samples were not included for rassf1am qpcr because too much dna had been lost during bisulte conversion ( fig 1 )  . 
flowchart of samples tested for total cell free dna ( cfdna ) , number of samples excluded after too much dna had been lost after bisulte conversion , and number of samples tested for circulating tumor dna ( ctdna ) by hypermethylated rassf1a ( rassf1am )  . 
the percentage of ctdna of total cfdna , calculated with the equation [ rassf1am / ( rassf1am + unmethylated rassf1a ) 100 ] , was 94% ( range , 82% - 98% ) in the 14 diagnostic samples from patients with stage m disease . 
compared with the individual mrna markers , rassf1am was more often positive , but the combined mrna markers identied the same positive samples as rassf1am ( appendix table a4 )  . in 93 matched bm mrna and ctdna ( pb ) samples , double - negative or double - positive results were found in 77% ( fig 3b )  . 
in contrast to pb , the bm mrna panel identied more positive samples than ctdna , and the individual markers phox2b and th correlated best with rassf1am ( appendix table a4 )  . discrepant findings between ctdna and pb or bm mrna mrd discrepant results between ctdna and mrna were detected in 27 pb and 21 bm samples , respectively , and listed in table 3 and appendix table a4 . 
from 3 of 5 bm mrna - negative / ctdnapositive samples , cryopreserved bm cells were available and tested all negative for rassf1ain some patients ( n850 , n865 , n732 ) , the high levels of ctdna probably correlated with the large primary or local relapse tumors , and these patients had no or very little bm inltration . in the ctdna - negative samples , in general , the cfdna levels were lower , with a median of 6.1 ng / ml for the bm mrna - positive / ctdna - negative and 1.52 ng / ml for the pb mrna - positive / ctdna - negative samples . 
from 15 of negative samples , cryopreserved cells were available and tested for rassf1am ; of the 5 positive samples , 4 were not in the quantitative range , which indicated low levels of bm inltration . in the samples from patients n777 , n798 , n2011 , n2014 , and n802 , no ctdna was detected . 
begin induction indicates until 2 courses of induction therapy ; mid - induction indicates after 3 - 5 courses of induction chemotherapy , unless additional courses were given after 6 courses , and samples before last course were also included at this time point ; end induction indicates at the end of induction therapy ; surveillance indicates during follow - up or at relapse suspicion ; and event indicates relapse or progression . 
of samples diagnosis samples localized and stage ms stage m follow - up samples beginning of induction therapy mid - induction therapy end of induction therapy postconsolidation surveillance progression relapse / refractory disease relapse therapy note . 
positive indicates that rassf1am circulating tumor dna was detected and quantied , and negative indicates that no rassf1am circulating tumor dna was detected . abbreviations : pnq , positive not quantiable ; rafssf1am , hypermethylated rafssf1a . complete remission at that time . 
patient n802 was treated for an isolated cns relapse . in the samples from patients n2012 , n2013 , n2016 , n2024 , n2029 , and n2031 , no ctdna was detected , while low amounts of mrna were detected in the bm . in the case of restricted , minimal bm disease , mrna detection was more sensitive than ctdna ( appendix table a4 )  . 
however , in some patients , a primary tumor was still present ( median , 50 mm ) while ctdna was negative ( table 3 )  . discussion ctdna in plasma is a powerful source for the detection of tumor - derived aberrations in a minimally invasive setting . many ctdna studies in adults for the detection of mrd are based on detection of tumor - specic mutations by targeted sequencing or digital droplet pcr ( ddpcr ) .12 , 16 , 17 because recurrent mutations are not common in neuroblastoma , 32 tumor - specic aberrations need to be characterized before they can be used as an mrd marker . 
however , temporospatial heterogeneity has been reported in neuroblastoma by several studies , 20 , 26 which raises the question of whether we should only use the small part of the tumor that is derived from the biopsy to design tumor - specic mrd markers . 
first , it is a sensitive marker , with a sensitivity of 1 tumor cell in 105 mononuclear cells.28 second , rassf1am qpcr can be used in all patients with stage m neuroblastoma because it has been shown that rassf1a is hypermethylated in all previously tested stage m neuroblastoma tumors.28 third , detection of rassf1am is less costly compared with wes and even swgs ( approximately 40and 10 - fold less expensive , respectively )  . 
hypermethylation of rassf1a has been described in several types of cancer and in physiologic circumstances in placental cells.33 rassf1a is not methylated in normal hematologic cells.28 , 33 , 34 however , in 2 of 61 samples from healthy individuals , we detected very low , nonquantiable levels of rassf1ain diagnosis or relapse other diagnosis or relapse other n = 11 n = 12 n = 23 n = 16 n = 12 n = 124 positive negative positive negative n = 66 n = 12 n = 4 n = 36 n = 4 n = 1 negative ctdna ( rassf1am ) positive negative ctdna ( rassf1am ) positive fig 3 . 
 van zogchel et al addition , in other studies , infrequent detection of rassf1am has been observed in plasma samples from healthy control participants.35 , 36 therefore , when detecting verylow levels of rassf1am in patients with neuroblastoma ( indicated as pnq range ) , results should be analyzed with caution . it has been shown that neuroblastoma tumors shed high amounts of ctdna in the plasma.25 , 26 , 37 in the current study , we found a median cfdna concentration of 73.1 ng / ml at diagnosis for patients with stage m disease . 
however , the levels we found are lower compared with previously published studies.25 , 26 , 37 this inconsistency may be due to differences in isolation of cfdna because we did not use a circulating nucleic acid kit or to differences in quantication methods . 
we found the majority of the cfdna ( 94% at diagnosis ) to be tumor derived in patients with stage m or hr disease , which is also supported by previous research.26 , 37 we tested 143 samples from 54 patients with neuroblastoma and detected ctdna in 57 samples . 
ctdna was detected at diagnosis in all 14 patients with stage m and 4 of 8 patients with localized and stage ms neuroblastoma . misawa et al29 described detection of rassf1am at diagnosis in the serum of 17 of 68 patients ( all stages ) and in 11 of 18 patients with stage m disease . 
there are two likely causes for the increased ctdna detection in our study . first , we used plasma , whereas misawa et al tested serum , which is known to be more contaminated by genomic dna originated from leukocytes during ex vivo clotting.38 second , misawa et al used conventional pcr , which is less sensitive than qpcr . 
our group has previously described that hypermethylation of rassf1a is variable in tumors of patients with stage ms ( median , 65% ) and localized ( median , 30% ) disease28 ; therefore , the level of ctdna can be slightly underestimated in these patients when using rassf1am as marker . we compared the performance of ctdna with pb and bm mrna in 128 and 93 samples , respectively . 
most patients in whom we detected relatively high levels of ctdna compared with pb or bm mrnas still had considerable tumor volumes or negative or low mibg scores ( data not shown ) ; therefore , it is likely that the ctdna in these patients originated from the primary tumor . 
two of these patients were in complete remission but in the other 15 patients , considerable tumor volumes were detected on imaging or urine catecholamines were still positive , which indicate the need to optimize pre - analytic sample handling and prospective study of cfdna kinetics in well - characterized patient cohorts with available paired ( nuclear ) imaging and bm assessment . while the detection of ctdna is very promising for future mrd studies , the current study has some limitations . stored remains were used , which resulted in missing samples and paired clinical data . 
for detection and quantication of low levels of ctdna in the plasma , dna extraction methods can be optimized with an isolation method specic for cfdna , and ddpcr39 may be a more suited technique compared with qpcr.40 moreover , large amounts of cfdna ( up to 96% ) could be destroyed during bisulte conversion41 ; therefore , we are investigating alternative methylation - specic ddpcr methods . 
previous studies showed that rassf1a was the most frequent hypermethylated tumor suppressor gene in neuroblastoma as well as identied other hypermethylated tumor suppressor genes , and inclusion of these genes as mrd markers might increase the sensitivity.42 , 43 in this study , we used rassf1am as a ctdna marker . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . no potential conicts of interest were reported . references pinto nr , applebaum ma , volchenboum sl , et al : advances in risk classication and treatment strategies for neuroblastoma . 
j clin oncol 33 : 3008 - 3017 , 2015 park jr , kreissman sg , london wb , et al : effect of tandem autologous stem cell transplant vs single transplant on event - free survival in patients with high - risk neuroblastoma : a randomized clinical trial . 
jama 322 : 746 - 755 , 2019 park jr , bagatell r , cohn sl , et al : revisions to the international neuroblastoma response criteria : a consensus statement from the national cancer institute clinical trials planning meeting . 
j clin oncol 35 : 2580 - 2587 , 2017 london wb , castleberry rp , matthay kk , et al : evidence for an age cutoff greater than 365 days for neuroblastoma risk group stratication in the childrens oncology group . 
j clin oncol 23 : 6459 - 6465 , 2005 viprey vf , gregory wm , corrias mv , et al : neuroblastoma mrnas predict outcome in children with stage 4 neuroblastoma : a european hr - nbl1 / siopen study . 
j clin oncol 32 : 1074 - 1083 , 2014 cheung nk , ostrovnaya i , kuk d , et al : bone marrow minimal residual disease was an early response marker and a consistent independent predictor of survival after anti - gd2 immunotherapy . 
j clin oncol 33 : 755 - 763 , 2015 kreissman sg , seeger rc , matthay kk , et al : purged versus non - purged peripheral blood stem - cell transplantation for high - risk neuroblastoma ( cog a3973 ) : a randomised phase 3 trial . 
lancet oncol 14 : 999 - 1008 , 2013 burchill sa , beiske k , shimada h , et al : recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the international neuroblastoma response criteria bone marrow working group . 
cancer 123 : 1095 - 1105 , 2017 stutterheim j , zappeij - kannegieter l , versteeg r , et al : the prognostic value of fast molecular response of marrow disease in patients aged over 1 year with stage 4 neuroblastoma . 
gormally e , caboux e , vineis p , et al : circulating free dna in plasma or serum as biomarker of carcinogenesis : practical aspects and biological signicance . mutat res 635 : 105 - 117 , 2007 2017 12 . 
mcbride dj , orpana ak , sotiriou c , et al : use of cancer - specic genomic rearrangements to quantify disease burden in plasma from patients with solid tumors . genes chromosomes cancer 49 : 1062 - 1069 , 2010 16 . 
tsao sc , weiss j , hudson c , et al : monitoring response to therapy in melanoma by quantifying circulating tumour dna with droplet digital pcr for braf and 18 . 
van roy n , van der linden m , menten b , et al : shallow whole genome sequencing on circulating cell - free dna allows reliable noninvasive copy - number proling in neuroblastoma patients . 
chicard m , colmet - daage l , clement n , et al : whole - exome sequencing of cell - free dna reveals temporo - spatial heterogeneity and identies treatmentresistant clones in neuroblastoma . 
yanik ga , parisi mt , shulkin bl , et al : semiquantitative mibg scoring as a prognostic indicator in patients with stage 4 neuroblastoma : a report from the 31 . 
stutterheim j , gerritsen a , zappeij - kannegieter l , et al : detecting minimal residual disease in neuroblastoma : the superiority of a panel of real - time quantitative 32 . 
chan kc , ding c , gerovassili a , et al : hypermethylated rassf1a in maternal plasma : a universal fetal dna marker that improves the reliability of noninvasive prenatal diagnosis . 
scheffer pg , de haas m , van der schoot ce : the controversy about controls for fetal blood group genotyping by cell - free fetal dna in maternal plasma . 
white he , dent cl , hall vj , et al : evaluation of a novel assay for detection of the fetal marker rassf1a : facilitating improved diagnostic reliability of noninvasive opin hematol 18 : 467 - 473 , 2011 prenatal diagnosis . 
wong fc , sun k , jiang p , et al : cell - free dna in maternal plasma and serum : a comparison of quantity , quality and tissue origin using genomic and epigenomic approaches . 
 circulating tumor dna in neuroblastoma appendix sample collection clinical samples were collected in edta tubes and processed within 24 hours , and 2 ml peripheral blood ( pb ) or 0.5 ml bone marrow ( bm ) was transferred to paxgene blood rna tubes ( qiagen , venlo , the netherlands ) and stored at 20c . 
mononuclear cells were isolated from the remaining bm sample by ficoll density centrifugation and cryopreserved in 10% dimethyl sulfoxide . dna isolation and bisulte conversion dependent on the available plasma volume , dna was extracted by using the qiaamp dna mini blood kit ( qiagen ) for 200 l plasma , or the magna pure 96 isolation robot ( roche , basel , switzerland ) for 500 - 1 , 000 l plasma and eluted in h2o . 
converted dna samples were used directly or stored at 20c . real - time quantitative polymerase chain reaction real - time quantitative polymerase chain reaction ( qpcr ) was performed as previously described ( van wezel em , et al : j mol diagn 17 : 43 - 52 , 2015 )  . 
primer and probe sequences have been described previously28 ( scheffer pg , et al : bjog 118 : 1340 - 1348 , 2011 ) and are listed in appendix table a2 . 
qpcr for alb , actb , and unmethylated rassf1a was performed in duplicate ; hypermethylated rassf1a was tested in triplicate . pb and bm mrna reverse transcriptase qpcr total rna from whole blood and bm samples was extracted using the paxgene blood rna kit ( qiagen ) according to the manufacturers instructions . 
cdna was synthesized and reverse transcriptase ( rt ) qpcr , with a maximum of 50 cycles was performed for - glucuronidase ( gusb ) , paired ctdna in neuroblastoma like homeobox 2b ( phox2b ) , tyrosine hydroxylase ( th ) , dopa decarboxylase ( ddc ) , growth - associated protein 43 ( gap43 ) , cholinergic receptor nicotinic - 3 ( chrna3 ) , and dopamine - hydroxylase ( dbh ) , as has been described previously.31 expression was normalized to gusb expression using the following equation : [ normalized threshold cycle ( ct ) = ( ctgusb ctmarker ) ]  . 
samples were scored for positivity according to previously published thresholds9 , 31 ( stutterheim j , et al : j clin oncol 26 : 5443 - 5449 , 2008 )  . 
we examined the frequency and characteristics of braf - mutated lgsc and described the response to treatment with braf inhibitors . patients and methods mutations were assessed in lgsc ( n = 65 ) by using targeted , exome , and whole - genome sequencing . 
braf inhibitors were trialed in two patients with a somatic braf v600e mutation : one patient received dabrafenib monotherapy and was monitored clinically , biochemically ( cancer antigen [ ca ] - 125 levels ) , and with positron emission tomography ( pet ) imaging . 
expression of the braf v600e protein in this patient was assessed by immunohistochemistry . results among patients with lgsc , nine ( 13.8% ) of 65 had a somatic braf mutation . 
two patients with braf v600e mutation received braf inhibitors at relapse and both achieved durable responses . conclusion braf mutations are not uncommon in patients with lgsc and should be routinely tested , because braf inhibitors can be an effective treatment for these patients . 
2018 by american society of clinical oncology introduction epithelial ovarian cancer ( eoc ) is a heterogeneous disease composed of several histologic and molecular subtypes , and emerging molecular analyses are challenging longstanding clinical treatment paradigms . 
eoc subtypes are characterized by different gene expression and somatic mutation patterns and by varying degrees of sensitivity to current standard carboplatin - paclitaxel combination chemotherapy.1 - 3 the predominant type of eoc is serous carcinoma , which accounts for approximately 80% of occurrences . 
 serous carcinoma is then classified into two main subtypes : the more common and better characterized high - grade serous carcinoma , and the less common low - grade serous carcinoma ( lgsc )  . 
lgsc generally is not responsive to standard platinum - based chemotherapy , 4 , 5 and the outcome is poor in women who have residual disease after debulking surgery , 6 - 8 which underscores the need for alternative therapeutic options . lgsc is molecularly distinct from high - grade serous carcinoma and is characterized by activating mutations of the ras sarcomamitogen - activated protein kinase ( ras - mapk ) pathway . 
 the mapk pathway and can be found in several cancer types , most often in melanoma.13 the reported frequency of braf mutations in lgsc varies from 2% to 33%11 , 14 - 16 and these mutations also are found in up to 46% of serous borderline tumors.17 , 18 clinical trials have shown impressive response rates to braf inhibitors in braf - mutant melanoma , and braf inhibitor use in metastatic melanoma is now considered standard of care.19 , 20 responses in other tumor types , including gastrointestinal stromal tumor , thyroid papillary cancer , hairy cell leukemia , and high - grade colorectal neuroendocrine tumors , also have been reported.21 - 26 however , some cancer types , such as colorectal adenocarcinoma , have much lower response rates to braf inhibitors despite the presence of the same somatic braf mutations.27 - 29 thus , although oncogenic braf represents a potential therapeutic target , responses vary according to tumor type , and clinical benefit is not always achieved . 
 we incorporated the shift from a three - tier grading system to a two - tier grading system , as recommended in the 2014 who classification of ovarian tumors.31 patient cases of lgsc were identified from review of diagnostic pathology reports and independent review of hematoxylin and eosinstained diagnostic slides by expert gynepathologists . 
in addition , grade 2 occurrences were screened for tp53 mutations , and only those that were wild type , consistent with a molecular classification of lgsc , were included in the lgsc cohort . clinical definitions progression - free survival was calculated as the time interval from date of histologic or cytologic diagnosis to the date of first progression based on gynecologic cancer intergroup ( gcig ) criteria.32 overall survival was calculated from date of diagnosis to date of death as a result of any cause . 
treatment response was assigned according to gcig cancer antigen ( ca ) 125 criteria.32 briefly , 50% or greater reduction in serum ca - 125 from an elevated pretreatment level , confirmed and maintained for at least 28 days , was considered a response . 
the aocs was approved by the human research ethics committee at the peter maccallum cancer centre , queensland institute of medical research , westmead hospital , and at all other participating hospitals . 
the gynbiobank and this study were approved by the western sydney local health district human research ethics committee . written informed consent was obtained from the patient treated with dabrafenib to include clinical information and imaging . 
this process also was approved by the western sydney local health district human research ethics committee . sequencing and immunohistochemistry frozen or fixed tumor samples were sectioned , and hematoxylin and eosin stains were used to assess tumor content before and after serial sectioning for nucleic acid extraction . 
for samples that contained less than 70% tumor , needle dissection of tumor cells was performed on sections of up to 50 10 extractions were performed using the dneasy blood and tissue kit or qiaamp dna mini kit ( qiagen , hilden , germany )  . 
mutations in exon 15 of braf , exon 2 of kras , exon 20 of erbb2 , and exons 2 to 11 of tp53 were screened by high - resolution melt analysis and validated by direct sequencing , as previously described.8 , 9 a subset of cases was screened by next - generation sequencing . 
target enrichment of the dna samples was performed according to the manufacturers protocol.34 the polymerase chain reactions for each sample were pooled , and target enriched dna samples were purified with agencourt ampure xp beads ( beckman coulter , indianapolis , in )  . 
the dna libraries were prepared according to qiagen protocols.35 dna libraries ( > 4 nm ) were sequenced on a miseq ( illumina , san diego , ca ) at the australian genome research facility ( melbourne , victoria , australia ) , with a read length of 2 150 base pairs . 
samples of the ovarian tumor from the primary surgery specimen and in biopsies at disease relapse were stained for mutated braf v600e with a mutation - specific monoclonal antibody ( mouse antihuman braf v600e monoclonal antibody , clone ve1 ; spring biosciences , pleasanton , ca ) by using the ventana benchmark ultra immunohistochemistry staining module ( ventana medical systems , tucson , az ) with a 1 - in - 200 antibody dilution . 
most ( eight [ 89% ] of nine ) were braf v600e mutations , a shared hot - spot mutation locus found in diverse cancer types ; one was a braf l597r mutation , an uncommon missense variant ( table 1 )  . in three braf - mutant tumors , additional variants identified by wes and wgs predicted alteration in the protein sequence ( range , 13 to 30 variations per patient case ; table 2 ) at lower allele frequencies than the braf mutations , which suggests subclonal events.8 , 37 there was no evidence of additional driver mutations , and no genes with deleterious mutationsother than brafwere common to the three patient cases . clinical features of braf mutation positive lgsc occurrences among patients with braf - mutation positive lgsc , age at diagnosis ranged from 22 to 77 years ( median , 51 years ; table 1 )  . 
the majority had international federation of gynecology and obstetrics ( figo ) stage iii or iv disease at diagnosis ( seven [ 78% ] of nine patients )  . all patients with braf - mutated lgsc had surgery as part of primary treatment , and most tumors ( seven [ 78% ] of nine ) were optimally debulked to no macroscopic residual disease . 
 the median follow - up time was 61.5 months , and five ( 56% ) of the nine patients remained progression free ( table 1 )  . treatment and clinical course in patients with braf mutationpositive lgsc after disease progression four patients experienced progression , and all four had relatively short progression - free survival ( table 1 ; fig 2 )  . 
sections ( 4 m ) from formalin - fixed , paraffin - embedded tumor tissue blocks were stained with hematoxylin and eos ( a ) patient case 1 , publishing id 65928 ; ( b ) patient case 2 , publishing id 11368 ; ( c ) patient case 3 , publishing id 5711 ; ( d ) patient case 4 , publishing id 65917 ; ( e ) patient case 5 , publishing id 65854 ; ( f ) patient case 6 , publishing id 10693 ; ( g ) patient case 7 , publishing id 9125 ; ( h ) patient case 8 , publishing id 11014 ; and ( i ) patient case 9 , publishing id 66198 . progression and died 2 months after the completion of treatment . 
overall survival was less than 2 years from her initial diagnosis . patient case 8 was diagnosed with stage iv ovarian cancer at age 31 years ( fig 2b )  . 
at additional disease progression , the patient entered a phase i trial of the braf inhibitor dabrafenib21 ( glaxosmithkline , sydney , australia ) and received 100 mg twice daily . 
the serum cancer antigen ( ca ) - 125 levels and treatment throughout the clinical course of patients : ( a ) patient case 6 , publishing id 10693 ; ( b ) patient case 8 , publishing id 11014 ; ( c ) patient case 4 , publishing id 65917 ; and ( d ) patient case 9 , publishing id 66198 . which promptly decreased after resumption of dabrafenib . 
expression of braf v600e in patient case 4 : immunohistochemical staining that used an antibody specific for braf v600e in formalin - fixed , paraffin - embedded tumor tissue sections of ( a ) left ovarian tumor from the primary surgical specimen , ( b ) left supraclavicular fossa lymph node core biopsy before second dabrafenib treatment at 78 months , and ( c ) left para - aortic lymph node core biopsy before second dabrafenib treatment at 78 months . 
the bar indicates a 50 - m scale . during treatment with dabrafenib 150 mg twice daily ( 50% higher than her previous dose ) , there was an impressive clinical response , which included reduced analgesic requirements , improved well - being , and marked reduction in the palpable supraclavicular lymph node . 
a positron emission tomography / computed tomography scan confirmed a significant partial radiologic response to treatment ( 63% decrease in sum of diameters of measured lesions ) and significantly fewer metabolically active lesions throughout almost all nodal regions and pulmonary nodules ( fig 4 )  . at the time of censoring , the patient continued to receive dabrafenib , and her ca125 had remained within normal range for 4 months . 
with disease progression , she was enrolled into a braf inhibitor basket trial ( desai et al , manuscript in preparation ) and has received treatment for more than 12 months . 
this patient experienced a partial radiologic response ( bo gao , personal communication , december 2017 ) and normalization of her ca - 125 level with treatment with a braf inhibitor ( fig 2d )  . discussion here , we report clinical outcomes in , to our knowledge , one of the largest series of patients with braf mutationpositive lgsc reported to date . 
we also report the first , to our knowledge , response to the braf inhibitor dabrafenib in a patient who has lgsc with a somatic braf v600e mutation . lgsc typically is tp53 wild type and commonly harbors ras and raf pathway mutations.9 , 14 , 18 this finding has led to clinical trials to evaluate mapk / erk kinase ( mek ) inhibitor activity . 
 a phase ii trial to evaluate the response to the mek inhibitor selumetinib in lgsc reported a promising response rate of 15%.39 response was not associated with known braf / kras mutation status , although this may be due to the small number of cases with known mutations in the trial . 
it is unclear whether all mutation subtypes of lgsc respond similarly to mek inhibition . we found braf mutations in 13.8% ( nine of 65 ) of patients with lgsc , which falls within the broad range reported previously ( 2% to 33% ) .11 , 14 - 16 , 40 this was similar to the 17.9% rate ( 10 of 56 patients ) reported by xing et al40 in a similarly sized cohort and was slightly higher than rate in the american association for cancer research genie project database , for which the frequency of patients with braf mutations in lgsc was four ( 7% ) of 56 patients.41 the genie project contains genomic records generated in clia / isocertified laboratories obtained at multiple tertiary referral centers41 and may be enriched for patients with late - stage disease who seek biomarker - driven clinical trials , whereas patients enrolled in this study were prospectively recruited from clinics . some studies have suggested that braf v600e mutations in lgsc are rare and are associated with early - stage disease and improved prognosis.15 , 16 , 42 however , we found that most women with a braf mutationpositive carcinoma were diagnosed at an advanced stage . 
 [ 18f ] - fluorodeoxyglucose ( 18f - fdg ) pet / ct ( siemens biograph mct , knoxville , tn ) images ( a , b , and c ) before and ( d , e , and f ) after 7 months of second dabrafenib treatment that indicated response to treatment with decrease in size and metabolic activity of lesions . 
the right common iliac node ( c ) before and ( f ) after 7 months of second treatment with dabrafenib . before dabrafenib 7 months on dabrafenib to no residual disease , which is associated with improved prognosis . 
 two others received braf inhibitors , and both achieved sustained response . patient case 4 initially received dabrafenib as part of a dose - finding phase i trial , 21 and , although some response was observed with deceased serum ca - 125 levels , a much more profound response was seen when the patient was retreated with dabrafenib 46 months later . 
 the explanation for the improved response with subsequent dabrafenib treatment is potentially multifactorial and may include the increased dosage , in line with current melanoma dosing guidelines , which suggests that her original dose was subtherapeutic.43 the patient also had a 25 - kg weight loss between dabrafenib treatment periods , although there is no evidence to date that the dabrafenib dose requires adjustment for weight.43 there also is emerging evidence that braf inhibitors may act in part via an effect on host immunity , 44 , 45 and it is possible that an uncharacterized immunologic component contributed to the response in this patient . consistent with our findings , responses to another braf inhibitor , vemurafenib , have been reported in lgsc . 
 the only patient with lgsc in a basket trial of a novel braf inhibitor ( desai et al , manuscript in preparation ) ; her disease has shown a sustained response , which provides additional evidence that braf v600e mutationpositive tumors in lgsc are broadly responsive to braf inhibition . in conclusion , recurrent lgsc is relatively chemotherapy resistant , and targeted treatment may play an important role in improvement of patient outcomes . 
our results are consistent with recent reports of response to braf inhibition in at least two other studies30 , 46 and suggest that braf inhibitors may be an effective option in patients with relapsed , braf mutationpositive lgsc . 
wain , russell hogg , sian fereday , nadia traficante , alexander dobrovic , anna defazio data analysis and interpretation : tania moujaber , dariush etemadmoghadam , yoke - eng chiew , rosemary l . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center  . tania moujaber no relationship to disclose dariush etemadmoghadam no relationship to disclose catherine saunders employment : macquarie medical imaging yoke - eng chiew no relationship to disclose rosemary l . 
wain no relationship to disclose bo gao no relationship to disclose russell hogg no relationship to disclose sivatharsny sirangan no relationship to disclose casina kan no relationship to disclose sian fereday research funding : astrazeneca ( inst ) nadia traficante research funding : astrazeneca ( inst ) ann - marie patch no relationship to disclose john v . 
 alexander dobrovic honoraria : astrazeneca , amgen consulting or advisory role : astrazeneca , amgen research funding : astrazeneca ( inst ) patents , royalties , other intellectual property : hold a patent related to a novel treatment target in ovarian cancer david d.l. 
we also thank georgina long , bsc , phd , mbbs , fracp , a primary investigator on the phase i dabrafenib trial ; glaxosmithkline and novartis oncology for providing the treatment drug , dabrafenib , on a compassionate use basis ; the american association for cancer research ( aacr ) for its financial and material support in the development of the aacr project genie registry ; and members of the consortium for their commitment to data sharing . 
bowtell , dariush etemadmoghadam , and alexander dobrovic , university of melbourne ; alexander dobrovic , olivia newton john cancer research institute , heidelberg ( melbourne ) , and la trobe university , bundoora , victoria ; ann - marie patch , john v . 
12 / rig / 1 - 17 and 15 / rig / 1 - 16 and is a member bank of the australasian biospecimens network - oncology , funded by nhmrc nos . 
damd17 - 01 - 1 - 0729 , the cancer council victoria , queensland cancer fund , the cancer council new south wales , the cancer council south australia , the cancer foundation of western australia , the cancer council tasmania , and the nhmrc nos . 
seidman jd , horkayne - szakaly i , haiba m , et al : the histologic type and stage distribution of ovarian carcinomas of surface epithelial origint j gynecol pathol 23 : 41 - 44 , 2004 2 . 
schmeler km , sun cc , bodurka dc , et al : neoadjuvant chemotherapy for low - grade serous carcinoma of the ovary or peritoneugynecol oncol 108 : 510 - 514 , 2008 5 . 
grabowski jp , harter p , heitz f , et al : operability and chemotherapy responsiveness in advanced low - grade serous ovarian cancer : an analysis of the ago study group metadatabase . 
emmanuel c , chiew ye , george j , et al : genomic classification of serous ovarian cancer with adjacent borderline differentiates ras pathway and tp53 - mutant tumors and identifies nras as an oncogenic driver . 
singer g , sthr r , cope l , et al : patterns of p53 mutations separate ovarian serous borderline tumors and lowand high - grade carcinomas and provide support for a new model of ovarian carcinogenesis : a mutational analysis with immunohistochemical correlation . 
grisham rn , iyer g , garg k , et al : braf mutation is associated with early stage disease and improved outcome in patients with low - grade serous ovarian cancer . 
anglesio ms , arnold jm , george j , et al : mutation of erbb2 provides a novel alternative mechanism for the ubiquitous activation of ras - mapk in ovarian serous low malignant potential tumors . 
sieben nl , macropoulos p , roemen gm , et al : in ovarian neoplasms , braf , but not kras , mutations are restricted to low - grade serous tumours . 
hauschild a , grob j - j , demidov lv , et al : dabrafenib in braf - mutated metastatic melanoma : a multicentre , open - label , phase 3 randomised controlled trial . 
falchook gs , long gv , kurzrock r , et al : dabrafenib in patients with melanoma , untreated brain metastases , and other solid tumours : a phase 1 dose - escalation trial . 
falchook gs , trent jc , heinrich mc , et al : braf mutant gastrointestinal stromal tumor : first report of regression with braf inhibitor dabrafenib ( gsk2118436 ) and whole exomic sequencing for analysis of acquired resistance . 
klempner sj , gershenhorn b , tran p , et al : brafv600e mutations in high - grade colorectal neuroendocrine tumors may predict responsiveness to braf - mek combination therapy . 
mao m , tian f , mariadason jm , et al : resistance to braf inhibition in braf - mutant colon cancer can be overcome with pi3k inhibition or demethylating agents . 
rustin gj , vergote i , eisenhauer e , et al : definitions for response and progression in ovarian cancer clinical trials incorporating recist 1.1 and ca 125 agreed by the gynecological cancer intergroup ( gcig )  . 
farley j , brady we , vathipadiekal v , et al : selumetinib in women with recurrent low - grade serous carcinoma of the ovary or peritoneum : an open - label , single - arm , phase 2 study . 
gershenson dm , sun cc , wong kk : impact of mutational status on survival in low - grade serous carcinoma of the ovary or peritoneubr j cancer 113 : 1254 - 1258 , 2015 43 . 
castelli c , rivoltini l , rodolfo m , et al : modulation of the myeloid compartment of the immune system by angiogenicand kinase inhibitor - targeted anti - cancer therapies . 
combe p , chauvenet l , lefrre - belda ma , et al : sustained response to vemurafenib in a low grade serous ovarian cancer with a braf v600e mutation . 
bowtell , peter maccallum cancer centre , east melbourne and university of melbourne , parkville , victoria , australia ; and imperial college london , london , england , united kingdom ; g . 
 treatment of patients with lobular breast cancer harboring human epidermal growth factor receptor 2 mutation with her2 - directed therapy introduction human epidermal growth factor receptor 2 ( her2 ) is a tyrosine kinase encoded by the erbb2 gene . 
mutations in cdh1 have been reported in 62% to 100% of cases of invasive lobular carcinoma , and many investigators consider a mutation in cdh1 to represent a genomic alteration characteristic of lobular carcinoma.5 mutations in erbb2 have been reported in six of 22 ( 27% ) cdh1 - mutated lobular carcinomas.5 the presence of activating somatic mutations in erbb2 could provide a rationale for her2 targeted therapy , 4 , 6 and there have now been several case reports of successful treatment of patients with such mutations with her2 targeted therapy.7 - 10 we initially identified a patient with progressive metastatic lobular carcinoma resistant to chemotherapy and hormonal regimens , whose liver metastasis harbored both cdh1 and erbb2 mutations . 
on the basis of the presence of the erbb2 mutation , we elected to treat the patient with her2 - targeted therapy , and she has achieved significant and durable responses to three consecutive her2 - targeted therapies during a 2.5year period of time , despite the lack of erbb2 amplification . after our experience with this patient , we then reviewed all of our patients with metastatic lobular carcinoma of the breast for whom next generation sequencing had been obtained . 
on the basis of reports that erbb2 exon 20 insertion has been shown to confer sensitivity to trastuzumab and tyrosine kinase inhibitors , we elected to treat the patient with dual her2 blockade with the combination of trastuzumab and pertuzumab.2 , 3 treatment was continued for 8 months , with major subjective clinical improvement , which included better appetite and resolution of abdominal pa clinical response was confirmed by ct scan on april 4 , 2016 showing a significant decrease in hepatic lesions , which has been maintained ( fig 2 )  . 
by july 2017 , the carcinoembryonic antigen , ca19 - 9 , and alkaline phosphatase increased aga ado - trastuzumab emtansine was discontinued , and she was started on third - line anti - her2directed therapy with lapatinib and capecitabine in july 2017 . she once again demonstrated significant response in all four tumor markers as well as her liver enzymes ( figs 3 and 4 )  . 
 only one of the five was also her2 amplified and had received prior her2 - directed therapy . all five patients have received her2 - targeted therapy for their metastatic disease , and four have achieved clinical responses as defined by response evaluation criteria in solid tumors ( recist ; tables 1 and 2 )  . 
liver biopsy showing infiltrating cords and nests of tumor cells ( july 2011 )  . in december 2010 , tumor markers were noted to be elevated , and a magnetic resonance imaging scan of the abdomen demonstrated hepatic metastases . 
 subsequent positron emission tomography / computed tomography ( ct ) scan showed progression of her liver lesions , and an ultrasound guided biopsy of the liver in july 2011 confirmed metastatic lobular carcinoma ( fig 1 )  . 
as with the primary tumor , the tumor specimen from liver biopsy was estrogen receptor and progesterone receptor positive and her2 negative by ihc . during the next 2 years she was treated with several chemotherapy regimens , including carboplatin and gemcitabine , capecitabine , and eribul she experienced progression on these regimens after initial responses of approximately 6 months , and progression of liver metastases was seen in august of 2014 . eribulin was then discontinued and she was started on tamoxifen , followed by exemestane and everolimus . 
her disease did not respond to these treatments , and she presented to cancer treatment centers of america at midwestern regional medical center for a second opinion in march of 2015 with extensive hepatic and bone metastases . 
comprehensive metabolic panel : alkaline phosphatase , ast , and alt . has received only two cycles of treatment , and it is too early to evaluate for radiologic response , although her tumor markers have fallen significantly . discussion we have identified erbb2 mutations in five of 16 patients ( 31% ) with cdh1 - mutated metastatic lobular carcinoma , an incidence similar to that previously reported.5 our experience suggests that such patients may well respond favorably to her2 - targeted therapy despite the absence trastuzumab and pertuzumab of her2 amplification . 
if the prevalence of erbb2 mutations is confirmed in larger studies , it would indicate that a significant number of patients with metastatic lobular carcinoma are likely to benefit from her2 - targeted therapy . 
this , together with the fact that one patient ( patient 3 ) had received no systemic treatment before biopsy , suggests that erbb2 mutation may be an intrinsic characteristic of lobular carcinoma of the breast and does not necessarily arise from prior systemic therapy . there is currently considerable interest in the possibility that patients with her2 - mutated breast cancer can be successfully treated with drugs already approved by the us food and drug administration for her2 - amplified disease . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . speakers ' bureau : astrazeneca , pfizer , r - pharm us , agendia bradford a . 
ali employment : foundation medicine leadership : incyte stock and other ownership interests : exelixis , blueprint medicines , agios , genocea biosciences patents , royalties , other intellectual property : patents via foundation medicine , patents via seres health on microbiome in non - neoplastic disease ( i ) ankur r . 
parikh consulting or advisory role : foundation medicine maurie markman employment : cancer treatment centers of america consulting or advisory role : amgen , celgene , crititech , pfizer , myriad genetics , merck , clovis oncology dennis l . 
 expert testimony : actavis , acorn research , apotex , emcure pharmaceuticals , dr reddy 's laboratories , fresenius kabi , hikma pharmaceuticals , hospira , pharma international , sagent pharmaceuticals , strides arcolab , sun pharma , wockhardt jeffrey s . 
ross employment : foundation medicine leadership : foundation medicine references stock and other ownership interests : foundation medicine research funding : foundation medicine arun kadamkulam syriac no relationship to disclose sara j . 
wolff ac , hammond me , hicks dg , et al : recommendations for human epidermal growth factor receptor 2 testing in breast cancer : american society of clinical oncology / college of american pathologists clinical practice guideline update . 
ross js , slodkowska ea , symmans wf , et al : the her - 2 receptor and breast cancer : ten years of targeted anti - her - 2 therapy and personalized medicine . 
ross js , wang k , sheehan ce , et al : relapsed classic e - cadherin ( cdh1 ) - mutated invasive lobular breast cancer shows a high frequency of her2 ( erbb2 ) gene mutations . 
ali sm , alpaugh rk , downing sr , et al : response of an erbb2 - mutated inflammatory breast carcinoma to human epidermal growth factor receptor 2 - targeted therapy . 
chumsri s , weidler j , ali s , et al : prolonged response to trastuzumab in a patient with her2nonamplified breast cancer with elevated her2 dimerization harboring an erbb2 s310f mutation . 
ben - baruch ne , bose r , kavuri sm , et al : her2 - mutated breast cancer responds to treatment with single - agent neratinib , a second - generation her2 / egfr tyrosine kinase inhibitor . 
shih j , bashir b , gustafson ks , et al : cancer signature investigation : erbb2 ( her2 ) - activating mutation and amplification - positive breast carcinoma mimicking lung primary . 
hyman dm , piha - paul sa , rodon j , et al : neratinib in her2 or her3 mutant solid tumors : summit , a global , multi - histology , open - label , phase 2 basket study . 
 loss of g2032r resistance mutation upon chemotherapy treatment enables successful crizotinib rechallenge in a patient with ros1 - rearranged nsclc sebastian michels matthias scheffler svenja wagener dennis plenker andreas scheel lucia nogov anne schultheis rieke n . 
sos reinhard bttner jrgen wolf author affiliations and support information ( if applicable ) appear at the end of this article . licensed under the creative commons attribution 4.0 license correspondence author : jrgen wolf , md , lung cancer group cologne , department i for internal medicine , center for integrated oncology , university hospital of cologne ; kerpener str . 
lacking targeted treatment options at that time , we treated him for approximately 3 months within a clinical phase i trial testing a combination of nintedanib and everolimus , without radiographic response of the disease . while on treatment , an extended molecular profiling was performed of a tumor sample collected 4 years after first diagnosis . 
a break - apart fluorescence in - situ hybridization assay ( zytovision , bremerhaven , germany ) revealed a rearrangement of ros1 in 100% of the tumor cells ( baseline ) .10 massively parallel sequencing ( mps ) and further alk fluorescence in - situ hybridization assays revealed no co - occurring genetic aberration ( fig 2 )  . 
disease progression in january 2015 most notably became apparent by a new ascites ( t1 ) , of which a sample was collected for molecular analyses and for the establishment of a patient - derived tumor model using the conditionally reprogramming of cells ( crc ) protocol established by liu et al.11 , 12 the culture was positive for malignant cells by immunohistochemical analysis , and ros1 fish confirmed the high level of rearrangement in the native ascites as well as the crc model ( fig 2 )  . 
because our mps panel did not cover the ros1 gene , sanger sequencing of ros1 exons 34 to 41 ( exon 38 forward primer sequence : 5 - actccataaagaccctcggc - 3 ; reverse primer sequence : 5 - gttttaccactgcagcctattaa - 3 ) was performed on an eight - capillary 3500 genetic analyzer ( thermo fisher scientific ) and showed a g2032r substitution in exon 38 in the ascites and in the derived cells ( fig 2 ) .7 to assess whether the g2032r mutation was acquired during treatment with crizotinib , we additionally sequenced the pretreatment biopsy specimen , which did not harbor any point mutation in ros1 . 
treatment of the crc with crizotinib and cabozantinib showed a shift of the half maximal growth - inhibitory dose ( gi50 ; cabozantinib , 0.3 m ; crizotinib , 1.2 m ) for crizotinib toward higher concentrations , as compared with cabozantinib in the viability assay ( fig 2d )  . 
 ( a ) table listing the results of the molecular pathological analyses before first crizotinib treatment ( baseline ) , at resistance to first crizotinib treatment ( t1 ) , and at progression to chemotherapy , before the initiation of the second crizotinib treatment ( t2 ) period . 
 ( b ) ros1 mutation status as determined by sanger sequencing of exons 34 to 41 at baseline , t1 , and t2 , revealing the substitution c.6094g > a / p.g2032r. 
 ( c ) magnifications of hematoxylin and eosin ( h&e ) staining , ttf1 and calretinin immunohistochemistry ( ihc ) , and ros1 break - apart fluorescence in situ hybridization ( fish ) of the patients conditionally reprogramming of cells ( crc ) tissue collected at t1 . 
the first 18f - labeled fluorodeoxyglucose positron emission tomographycomputed tomography ( ct ) scan at week 4 of treatment showed a dramatic metabolic and morphologic response with a reduction of the largest dimension of target lesions by 41.2% and a reduction of maximum standardized uptake value of the single hottest lesion by 45.9%. 
another ct scan 4 weeks later confirmed the partial response and , at time of data cutoff for this report ( december 2017 ) , the patient has continued treatment of almost 1 year . discussion this case is a proof of concept that rechallenge with crizotinib after intervening chemotherapy is feasible in ros1 - rearranged nsclc with acquired resistance . 
 it is commonly thought that darwinian selection underlies the emergence of resistant clones during targeted therapy , 4 such as those that carry the ros1 p.g2032r resistance mutation , like in our case . 
a similar phenomenon has been described in vitro in egfr p.t790mpositive cells that grew slower than did cells with sensitizing egfr mutations only.14 otherwise , crizotinib - resistance mutations in alk - rearranged cell - line models seem to induce an increased proliferation rate.15 also , studies proving a higher sensitivity toward chemotherapy of ros1 p.g2032positive cells have not been reported . 
thus , our finding needs to be interpreted with caution , and comprehensive studies are required to explain this observation . apart from the clinical findings , this case also illustrates the feasibility and the viability of crc establishment for individual patients to guide treatment decisions . 
sos , reinhard bttner , jrgen wolf administrative support : sebastian michels , reinhard bttner provision of study material or patients : sebastian michels , matthias scheffler , lucia nogov , wolfgang baus , sabine merkelbach - bruse , reinhard bttner , jrgen wolf collection and assembly of data : sebastian michels , matthias scheffler , svenja wagener , dennis plenker , andreas scheel , lucia nogov , rieke n . 
 abdulla , carsten kobe , wolfgang baus , sabine merkelbachbruse , reinhard bttner , jrgen wolf sensitive to treatment with cabozantinib , which is consistent with the clinical loss of crizotinib efficacy . 
tpx0005 , a next - generation ros1 / alk inhibitor , has promising activity in the presence of these mutations , including g2032r and is in early clinical development.17 our theoretical understanding of the evolutionary dynamics in cancer has impressively improved , and it seems that clonal evolution under therapy follows deterministic and stochastic forces . 
 in particular , in the patient in this case report who has been treated sequentially with targeted and nontargeted therapy , a precise allocation to one of these mechanisms may not be possible . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . sebastian michels honoraria : novartis , pfizer , astrazeneca , boehringer ingelheim , roche pharma consulting or advisory role : boehringer ingelheim , pfizer , roche pharma research funding : pfizer ( inst ) , novartis ( inst ) , bristolmyers squibb ( inst ) travel , accommodations , expenses : novartis matthias scheffler honoraria : healthcare consulting cologne , boehringer ingelheim , takeda consulting or advisory role : boehringer ingelheim , takeda travel , accommodations , expenses : boehringer ingelheim svenja wagener no relationship to disclose dennis plenker stock and other ownership interests : roche , foundation medicine patents , royalties , other intellectual property : a patent of nrg1 fusions has been filed . andreas scheel honoraria : msd , bristol - myers squibb , roche , dako / agilent technologies consulting or advisory role : msd , bristol - myers squibb , roche , dako / agilent technologies lucia nogov honoraria : pfizer , celgene , pfizer , novartis consulting or advisory role : roche , novartis , boehringer ingelheim , bristol - myers squibb research funding : pfizer ( inst ) , bristol - myers squibb ( inst ) , novartis ( inst ) , msd ( inst ) travel , accommodations , expenses : novartis , pfizer , celgene , boehringer ingelheim anne schultheis consulting or advisory role : novartis research funding : roche research funding : bristol - myers squibb ( inst ) , msd ( inst ) diana s.y. 
abdulla honoraria : boehringer ingelheim , roche pharma , bristolmyers squibb , merck consulting or advisory role : boehringer ingelheim , roche pharma , novartis research funding : bristol - myers squibb ( inst ) , pfizer ( inst ) , novartis ( inst ) travel , accommodations , expenses : bristol - myers squibb , roche pharma , boehringer ingelheim , novartis , loxo , merck richard riedel consulting or advisory role : boehringer ingelheim , novartis , eli lilly travel , accommodations , expenses : loxo anne bunck no relationship to disclose carsten kobe no relationship to disclose wolfgang baus travel , accommodations , expenses : novartis ( i ) sabine merkelbach - bruse honoraria : astrazeneca , bristol - myers squibb , novartis consulting or advisory role : bristol - myers squibb , novartis martin l . 
sos research funding : novartis reinhard bttner stock and other ownership interests : cofounder and chief scientific officer for targos molecular pathology ( kassel , germany ) and tamp ( atlanta , ga ) honoraria : astrazeneca , abbvie , bayer , bristol - myers squibb , boehringer ingelheim , merck serono , msd , novartis , qiagen , pfizer , roche research funding : roche ( inst ) jrgen wolf honoraria : abbvie , astrazeneca , bristol - myers squibb , boehringer ingelheim , msd , novartis , roche consulting or advisory role : abbvie , astrazeneca , bristol - myers squibb , boehringer ingelheim , chugai pharma , ignyta , eli lilly , msd oncology , novartis , pfizer , roche research funding : bristol - myers squibb , novartis , pfizer affiliations all authors : university hospital of cologne ; martin l . 
gainor jf , tseng d , yoda s , et al : patterns of metastatic spread and mechanisms of resistance to crizotinib in ros1 - positive nonsmall - cell lung cancer . 
facchinetti f , loriot y , kuo ms , et al : crizotinib - resistant ros1 mutations reveal a predictive kinase inhibitor sensitivity model for ros1and alk - rearranged lung cancers . 
shaw at , felip e , bauer tm , et al : lorlatinib in non - small - cell lung cancer with alk or ros1 rearrangement : an international , multicentre , open - label , single - arm first - in - man phase 1 trial . 
cui jj , zhai d , deng w et al : abstract b185 : tpx - 0005 , a supreme ros1 inhibitor , overcomes crizotinib - resistant ros1 mutations including solvent front mutation g2032r and gatekeeper mutation l2026m . 
 tumor - infiltrating lymphocyte function predicts response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer purpose the presence of tumor - infiltrating lymphocytes ( tils ) in tumors is superior to conventional pathologic staging in predicting patient outcome . 
here we developed an assay that tests til cytotoxicity in patients with locally advanced rectal cancer before definitive treatment , identifying those who will obtain a pathologic complete response ( pcr )  . 
we also used the assay to demonstrate the rescue of til function after checkpoint inhibition blockade ( cib )  . patients and methods thirty - four consecutive patients were identified initially , with successful completion of the assay before surgery in those 17 patients who underwent full treatment . 
cib ( antiprogrammed cell death protein 1 [ antipd - 1 ] antibody ) response was also assessed in a subset of patient specimens . results six of the 17 patients achieved an objective pcr on final evaluation of the resected specimen after neoadjuvant chemoradiotherapy . 
assessment of the effectiveness of cib revealed partial restoration of cytotoxicity in tils with increased pd - 1 expression with antipd - 1 antibody exposure . conclusion evaluating til function can be undertaken within weeks of the diagnostic biopsy , affording the potential to alter patient management decisions and refine selection for a watch - and - wait protocol . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license introduction the patients immune system plays a vital role in tumor eradication.1 this has been demonstrated by gene expression studies of colorectal cancer ( crc ) , identifying tumor - infiltrating lymphocytes ( tils ) , associated cytokines such as interferon gamma ( ifn - ) , and cytolytic granules ( granzyme b ) , correlating with a good prognosis.2 subsequent validation by immunohistochemistry ( ihc ) defined cd8 + ( cytotoxic ) tils to be a superior predictive marker to tnm staging.3 similarly , the immune landscape in rectal cancer before neoadjuvant chemoradiotherapy ( nacrt ) is well characterized , with high densities of cd8 + tils seeming to be an enticing arbiter of good tumor regression grade.4 - 6 although the location of tils correlates with outcome , this is not to an extent whereby management can be confidently altered . 
inclusion criteria were patients with t3 to t4 and / or n1 to n2 rectal cancer who were scheduled for long - course nacrt , had no or solitary hepatic metastasis amenable to surgery , and had subsequent total mesorectal excision surgery with curative intent performed at least 10 to 12 weeks from the last crt dose . 
patients were excluded if they were younger than 18 years of age or if they had received short - course , or failed to complete long - course , crt . 
the study was approved by the peter maccallum cancer centre human research ethics committee , and written consent was obtained from each patient according to national health and medical research council guidelines . clinical assessment each patient was treated by a specialist lower gi cancer multidisciplinary board consisting of a nuclear medicine physician , two radiologists , two medical oncologists , two radiation oncologists , two pathologists , and four colorectal trained surgeons and research scientists . 
radiotherapy took place during a period of 5 weeks , for a total of 45 gy in 25 fractions to the pelvis , followed by a 5.4 gy boost in three fractions . histology was examined by two gi oncology specialist - pathologists . 
 if no tumor was identified macroscopically , the whole tumor bed was examined . pretreatment immune cytotoxicity measurement collection of biopsy specimens to derive tumoroids and tils was performed by one of the five scientists , after 3 months of training . 
experimental assessors were blinded to clinical outcomes . tumoroid and til expansion tumoroids are an established in vitro model.20 , 27 fresh tumor biopsy specimens were collected at the time of endoscopy , with a proportion of the specimens processed into tumoroids.27 their origin was confirmed by short tandem repeat analysis by the australian genomics research foundation , melbourne , and by staining for cdx2 and ck20 ( data supplement )  . 
 a portion of the endoscopic biopsy specimen was used for the expansion of tils , using a modified protocol.28 tils were analyzed for t - cell subsets ( cd3 + , cd4 + , cd8 + , foxp3 + , and cd56 + cells ) using a fluorescence - activated cell sorting ( facs ) platform ( bd lsrfortessa x - 20 , north ryde , australia ) at days 0 and 14 ( data supplement )  . multiplex ihc , imaging , spectral unmixing , and phenotyping multiplex ihc staining was performed using the perkinelmer opal 6 - color kit ( perkinelmer , waltham , ma ) as per manufacturers instructions . 
representative images , detailed methodology , and a list of antibodies can be found in the data supplement . immune cytotoxicity assay analysis a comprehensive protocol is reported in the data supplement . 
briefly , tumoroids were expanded over two passages and were plated onto an ibidi 96 - well microscopy - grade plate in their third passage and allowed to grow for 5 to 10 days to a size of > 50 each well contained 10 to 500 tumoroids with the appropriate media included during their coculture with matched tils . 
two fluorescent dyes were added to the wells : an apoptotic marker ( thermo fisher , scoresby , australia cellevent caspase - 3 / 7 - green readyprobes reagent ) and a cell death marker ( themo fisher propidium iodide [ pi ] ) activated by nucleic acid binding . 
 tumoroids death was quantified by measuring the mean fluorescence intensity ( mfi ) of pi only , given the stability of the dye . four wells , consisting of two negative controls with tumoroids alone and two test wells , cocultured with matched tils , were used for each experiment . 
tumoroids at the matrigel periphery ( < 100 m from the edge ) were viewed at random , with at least 10 tumoroids imaged every 2 hours for 48 hours . 
the images were then analyzed by measuring the maximum mfi for each tumoroid ( overall fluorescence intensity emitted / surface area of tumoroid ) to obtain a mean for the 10 + tumoroids . 
morphologic killing was examined by scanning electron microscopy ( scem ; jcm - 6000 , jeol , frenchs forest , australia ; at 15 kv under high vacuum )  . 
cultivation of tils in tumoroid media had no effect on the viability or the capacity to respond to activation stimuli ( data supplement )  . results study participants thirty - four consecutive patients consented , with completion of the cytotoxic assay before surgery in 17 . 
after additional optimization , we eliminated in vitro culture infection and , with sufficient tumor tissue , achieved a culture success rate of 87% ( 19 of 22 patients )  . 
an additional four had disease progression and thus were not amenable to surgery , two patients had immediate surgery , and one patient was excluded because of poor tolerance to nacrt and proceeded to surgery . 
the mean number of sections examined for patients with pcr was 6.8 , and the mean number of lymph nodes inspected was 19.5 ( table 1 )  . functional immune cytotoxicity assay we compared tumor sections and matched embedded tumoroids by costaining with cdx2 and ck20 antibodies ( fig 1a )  . 
to show that tils were capable of interaction with tumoroids , we used scem to visualize the morphologic integrity of the tumoroids with and without their matched tils ( fig 1c )  . 
patient - specific tils of those who eventually achieved a pcr , initiated tumor cell killing ( apoptosis by activated caspase3 / 7green fluorescence [ data supplement ] ) and subsequent tumoroid death ( pi red fluorescence ) as shown in figs 1d - 1f . 
in those patients who did not respond to nacrt , their tils did not induce substantive tumoroid death , whereas some tumoroids are intrinsically resistant to immune attack and / or the tils lack cytotoxic activity ( figs 1g - 1i ; data supplement )  . implicit to the concept of til - mediated cytotoxicity is that by increasing the ratio of effector cells to target cells , it would be expected that the kinetics of til killing would accelerate . 
by monitoring the pi fluorescence of 10 to 30 tumoroids over time , it was apparent that there was a range of til - induced tumoroid deaths across patient samples ( fig 2b )  . prospective patient cohort and matched primary and metastasis assays the prospective recruitment of patients with rectal cancer afforded an opportunity to explore the predictive power of our assay without modifying the patient treatment course . 
when the results are presented as a box plot of for maximum killing ( fig 3c ) , an unambiguous difference ( p < .001 ) between pcr and incomplete response at 48 hours is evident ( mean pcr mfi of 27 , 982 [ 95% ci , 25 , 340 to 30 , 625 ] compared with 12 , 428 [ 95% ci , 9 , 434 to 15 , 423 ] ) for incomplete response . 
 we also evaluated the percentage of tumoroids killed by exposure to tils in the pcr group ( mean sem , 85% 5% ) versus incomplete response ( 32% 7% ) on the basis of morphologic disruption ( bright field microscopy ) as well as pi uptake ( fluorescence microscopy ) , finding a highly significant difference by this measure ( p < .0001 , two - tailed t test )  . 
 in parallel , we showed that the primary tumor tils kill primary tumorderived tumoroids , whereas the liver tumoroids are not killed readily by their matched tils ( data supplement )  . 
 in one patient , we propagated sufficient tils to add primary tils to metastasis tumoroids to find that these have a slightly greater capacity to kill than do metastasis tils . 
on antipd - 1 antibody treatment , the increase in cytokine production tracks with improved cytotoxicity of the increased pd - 1expressing tils in one patient ( figs 5d , 5g ) partial improvement in another ( figs 5e , 5h ) , and no improvement in a third ( figs 5f , 5i )  . 
facs separation of til subsets cd8 + , cd4 + , cd56 + / cd8 ( natural killer cells ) , and cd8 + / cd56 + ( natural killer t ) was performed to distinguish the cytotoxic effect of each in the assay ( data supplement )  . 
visualization of the killing capacity of cd8 + / cd56 tils compared with the other subpopulations is documented in fig 4d , whereas production of ifnor tumor necrosis factor ( tnf - ) did not discriminate between groups ( data supplement )  . data from this prospective study advance the concept that it is possible to assess til cytotoxicity and that measuring the function of patient specific tils accurately predicts pcr in patients with locally advanced rectal cancer undegoing nacrt . 
this study has demonstrated the functional characterization of individual patient - specific tils against matched primary or metastatic tumor cells . our study ensured that all eligible patients received standardized treatment without variation , including an interval time to surgery of at least 10 weeks ( from the last radiation dose )  . 
 ( b ) tumoroids ( approximately 500 cells ) embedded in matrigel are readily generated ex vivo from surgical biopsy specimens and can be combined with patient - matched tils to allow measurement of patient - specific cytotoxicity . 
the addition of tils ( rendered pink on the basis of characteristic cell size ; pink arrows ) to patient - matched tumoroids leads to morphologic irregularities on the tumoroid surface and progressive structural disruption ( images at 8 hours )  . 
 ( d ) tumoroids ( black arrow ) at time 0 show no uptake of propidium iodide ( pi ) as indicated by red fluorescence , or activation of caspase - 3 / 7 ( apoptosis ) as indicated by green fluorescence in the absence of tils . 
 ( gi ) not all tumoroids ( white arrow ) are subject to til - mediated killing , as demonstrated in this series of images , suggesting that intrinsic resistance to immune - mediated killing is a characteristic in some patients with rectal cancer . 
 assessment was performed by two independent , blinded pathologists across the entire irradiated tumor bed to ensure reporting consistency . the usefulness of tumoroid models lies in their resemblance to the original tumor in terms of tissue architecture , mutations , and heterogeneity.30 the restoration of tils in this model activated caspase 3 / 7 target : effector ratios 12 h 20 h 48 h 12 h 20 h 12 h killing 1 : 10 1 : 10 30 , 000 25 , 000 20 , 000 15 , 000 10 , 000 5 , 000 8 12 16 20 24 28 32 36 40 44 48 time ( hours ) fig 2 . 
 ( a ) target effector ratios of tumoroid cells ( stained blue with hoechst 33258 ) to matched tils measured by activated caspase 3 / 7 fluorescence ( green ) demonstrates that til - mediated killing is effector cell density dependent . 
 ( b ) cytotoxic assays measured in six patient samples by propidium iodide uptake ( mean fluorescence intensity [ mfi ] ) in rectal tumoroids and patient - matched tils at a 1 : 10 ratio demonstrate patient - to - patient heterogeneity in terms of kinetics and extent of killing of tumoroids ( mfi of 30 to 100 tumoroids / patient tracked over time ; six patient samples ; means depicted )  . partially recapitulates the tumor microenvironment . 
of greatest importance , the rapid establishment of tumoroids and tils within a clinically relevant timeline ( 3 to 4 weeks of patient biopsy ) lends this assay to multiple applications . phenotypic characterization of tils identified cd8 + tils as a key arbiter of good tumor regression grade.4 - 6 , 31 - 34 however , quantification of tils is limited by an inability to predict response accurately and thereby allows treatment decisions to be altered.5 , 6 our study advances the phenotypic characterization to include functional evaluations , reaffirming that cd8 + tils are the predominant t cells responsible for cytotoxicity . 
importantly , the assay uses the same ratio of tils to target cells across samples , further emphasizing that til density alone does not predict response , rather , their functional quality . to determine whether tils have the ability to migrate toward and interact with patientmatched tumoroids , we performed scem of mixed cultures of matched tils and tumoroids . 
by staining tils with nucleic acid binding fluor - syto11 , we tracked the migration of patient - matched peripheral blood mononuclear cells through matrigel ( data supplement ) and identified tumoroids that are intrinsically resistant to immune attack ( data supplement )  . 
 ( b ) by measuring the cytotoxicity of tils against matched tumoroids ( that contain approximately 500 cells ) over a 48 - hour period with an effector - to - target ratio of 10 : 1 , distinct killing profiles were ascertained . 
 ( d ) pretreatment biopsy tissue blocks were processed for immune makers by multiplex immunohistochemistry ( ihc ) and digitally evaluated for immune cells subsets within the tumor and the treg / cd8 + cell ratio ( e ) for the cohort of patients achieving a pcr ( four ) and incomplete responses ( six ) whereby no significant differences were observed . 
 the addition of antipd - 1 antibody into this setting partially rescued killing activity for two of three patient samples , despite increases in ifn and tnfrelease in all three . 
this uncoupling has been reported by others , who showed that there can be target engagement by cytotoxic t cells but failure to kill , which in turn leads to chronic cytokine production.41 interestingly , despite having a greater proportion of cd8 + tils , our crossover study in one patient with synchronous primary and metastatic liver tumors showed a decreased killing efficacy of met - tils compared with primary tils . 
however , met - cd8 + tils also displayed a higher proportion of pd - 1expressing cells and showed the same , but no greater , killing efficacy as primary tils on addition of antipd - 1 antibody . 
 ( ac ) tils were fluorescence - activated cell sortingenriched to generate different subsets ( cd4 + / cd56 , cd8 + / cd56 , cd8 / cd56 + , and cd8 + / cd56 + ) to identify the cell types responsible for the killing of patient - matched rectal tumoroids . 
mfi , mean fluorescence intensity . including receptor and ligand expression by ihc , have had low yield in predicting response to immunotherapy.26 in conclusion , others have recently explored the usefulness of examining patient - derived organotypic tumor spheroids or tumor organoidlike cultures in combination with other microenvironment components in the setting of cib treatments , 42 as well as peripheral blood - derived cells from the same patient.43 the relative merits of our assay platform are tabulated in the data supplement . 
it is instructive that this assay is performed before the effects of the tumor cell damage induced by nacrt are presented to the immune milieu and that measuring de novo immune competency appears to predict response to treatment . 
 ( ac ) pd - 1 ( programmed cell death protein 1 ) expression of cultured tils is induced after 2 days of exposure to anti - cd3 / cd28 dynabeads ( blue bar ) compared with unstimulated ( gray bar ) ; shown for three patient samples . 
mfi , mean fluorescence intensity . thus , in some patients , the immune microenvironment is poised to take advantage of the effects of nacrt to achieve a pcr , and this implies , but does not prove , that the quality of the tils affects the multiple actions of nacrt ; these are issues for future evaluation . 
this pretreatment biomarker has the potential to influence the management of patients with rectal cancer by improving the accuracy of patient selection for a watch - and - wait protocol . 
 alexander graham heriot no relationship to disclose robert george ramsay honoraria : merck serono research funding : merck serono , targovax , fisher and paykel travel , accommodations , expenses : fisher and paykel acknowledgment we thank rebecca abbot for her assistance in tissue culture , pasquale petrone and heloise halse for their assistance in multiplex immunohistochemistry data acquisition and initial til analysis respectively , and chad johnson , jill dane , and sarah ellis for their guidance and assistance in the usage of high - resolution fluorescence microscopy instruments . affiliations joseph cherng huei kong , glen robert guerra , rosemary magdalena millen , sara roth , huiling xu , paul joseph neeson , phillip kevin darcy , michael henry kershaw , shienny sampurno , jordane malaterre , david shi hao liu , toan duc pham , vignesh narasimhan , minyu wang , yu - kuan huang , jacob mccormick , andrew craig lynch , satish warrier , michael michael , jayesh desai , william murray , catherine mitchell , samuel ngan , wayne allen phillips , alexander graham heriot , and robert george ramsay , peter maccallum cancer centre , university of melbourne , melbourne ; and kumar visvanathan and rosemary magdalena millen , st vincents hospital , east melbourne , australia . supported by the colorectal surgical society of australia , the new zealand foundation , the royal australasian college of surgeons foundation for surgery , the national health and medical research council of australia , and the victorian cancer agency . presented in part at the american society for clinical oncology annual meeting , chicago , il , june 2 - 6 , 2017 . support prior presentation references 1 . 
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dalton rs , velineni r , osborne me , et al : a single - centre experience of chemoradiotherapy for rectal cancer : is there potential for nonoperative management ? colorectal dis 14 : 567 - 571 , 2012 39 . 
maas m , lambregts dm , nelemans pj , et al : assessment of clinical complete response after chemoradiation for rectal cancer with digital rectal examination , endoscopy , and mri : selection for organ - saving treatment . 
perez ro , habr - gama a , gama - rodrigues j , et al : accuracy of positron emission tomography / computed tomography and clinical assessment in the detection of complete rectal tumor regression after neoadjuvant chemoradiation : long - term results of a prospective trial ( national clinical trial 00254683 )  . 
jenkins mr , rudd - schmidt ja , lopez ja , et al : failed ctl / nk cell killing and cytokine hypersecretion are directly linked through prolonged synapse time . 
tsai s , mcolash l , palen k , et al : development of primary human pancreatic cancer organoids , matched stromal and immune cells and 3d tumor microenvironment models . 
 drug - related pneumonitis in the era of precision cancer therapy drug - related pneumonitis as a result of novel cancer therapy provides new challenges for providers of cancer care in the era of precision medicine . 
here , we provide a detailed review of drug - related pneumonitis that develops during precision cancer therapies using immune - checkpoint inhibitors and molecular targeting agents , and we summarize the emerging data that have been obtained by recent investigations to provide a state - of - the - art overview for clinicians involved in cancer care . we focus on immune - checkpoint inhibitorrelated pneumonitis , which is an immune - related adverse event of growing interest and increasing clinical significance in current oncology practice that has rapidly expanding access to these agents . 
clinical characteristics , radiographic spectrum , and risk factors and outcome of pneumonitis are described for each class of agents , and current treatment guidelines and monitoring recommendations are discussed . 
with recent advances in precision cancer therapy using molecular targeting agents and immune - checkpoint inhibitors ( icis ) , pneumonitis in patients who are treated with novel anticancer agents is increasingly recognized as a significant clinical challenge . 
in addition , rapidly expanding access to icis , such as programmed death 1 ( pd - 1 ) and programmed death - ligand 1 ( pd - l1 ) inhibitors has brought new and emerging challenges . 
recent studies of pneumonitis in patients who are treated with these novel agents have provided important new knowledge and insight that are relevant to cancer care providers across disciplines . this review focuses on drug - related pneumonitis during precision cancer therapies , including icis and molecular targeting agents , and provides an overview for clinicians who are involved in the care of patients with cancer . 
the spectrum of radiographic manifestations of pneumonitis is presented in the context of patient management . we also provide up - to - date guidelines and recommendations for the treatment and monitoring of pneumonitis during these therapies . 
 been made to characterize the radiographic patterns of pneumonitis caused by various therapeutic agents.1 , 2 several recent reports have indicated that the radiographic patterns of drug - related pneumonitis may be categorized according to patterns that correspond to american thoracic society / european respiratory society ( ats / ers ) classifications of idiopathic interstitial pneumonias and related disorders , 3 which are based on ct findings and their extent and distributions on imaging.2 , 4 - 8 in these studies of pneumonitis that developed during treatment with molecular targeting agents and icis , the commonly noted radiographic patterns of pneumonitis included cryptogenic organizing pneumonia ( cop ) pattern , nonspecific interstitial pneumonia ( nsip ) pattern , hypersensitivity pneumonitis ( hp ) pattern , and acute interstitial pneumonia ( aip ) / acute respiratory distress syndrome ( ards ) pattern2 , 4 - 10 ( table 1 )  . 
radiographic patterns of pneumonitis have been shown to correlate with the toxicity grades assessed by the common terminology criteria for adverse events.5 recent advances in precision cancer therapy have brought the new and emerging challenges of pneumonitis as a result of treatment with novel agents . 
regulatory approvals for these agents have recently been granted , including nivolumab for melanoma , nonsmall - cell lung cancer ( nsclc ) , renal cell carcinoma ( rcc ) , and hodgkin lymphoma ; pembrolizumab for melanoma , nsclc , hodgkin lymphoma , and squamous cell head and neck cancer ; and atezolizumab for urothelial carcinoma and nsclc . 
combination therapy of nivolumab and ipilimumab , a cytotoxic t - lymphocyte - associated protein 4 inhibitor , has also been approved for treatment of advanced melanoma and is actively investigated in nsclc and other malignancies . as a result , these agents are rapidly expanding their treatment roles in clinical oncology practice , and more agents are in the drug development and testing pipeline . immune - checkpoint inhibition of these agents is associated with a unique set of toxicities that are known as immune - related adverseevents ( iraes ) .11 - 14 among a variety of iraes that can affect organs from head to toe , ici - related pneumonitis is a clinically significant and potentially life - threatening irae and is therefore recognized as an event of special interest . phase i trials of pd - 1 inhibitors have resulted in pneumonitis - related deaths in patients with advanced solid tumors , including nsclc , melanoma , and colorectal cancer.15 - 18 in a report of long - term safety in a nsclc cohort that was treated in a phase i trial , pneumonitis occurred in 7% ( 9 of 129 patients ) , with three pneumonitisrelated deaths.15 in a phase ii trial of nivolumab in squamous nsclc , pneumonitis was one of the most common iraes , occurring in 5% of patients ( 6 of 117 patients ) , including four patients with grade 3 pneumonitis.19 more recent data suggest that pneumonitis can be an even more significant issue in patients who are treated with combination therapies . 
radiographic pattern was associated with clinical severity of pneumonitis represented by toxicity grades , where aip / ards pattern had the highest grade , followed by cop pattern , whereas nsip pattern and hp pattern had lower grade ( median grade : 3 , 2 , 1 , 1 ) .5 results indicate that the radiographic patternbased approach , in addition to early and accurate diagnosis , may help guide patient management and follow - up in the setting of pneumonitis . 
another recent study of pd - 1 / pd - l1 inhibitor - related pneumonitis has also reported the diverse radiographic and pathologic features of the entity , 25 further indicating the importance of a standardized common language to describe the findings of ici - related pneumonitis.10 treatment , clinical course , and management guidelines treatment of ici - related pneumonitis consists mainly of holding back on the use icis and administering corticosteroids ; however , some cases may be refractory to corticosteroids and require additional treatment . 
 ( d ) aip / ards pattern is characterized by diffuse or multifocal ggos or consolidations , along with lung volume loss and traction bronchiectasis . ( n = 12 ) or as intravenous therapy ( n = 5 )  . 
although the cause of death was solely attributed to pneumonitis in one patient alone , the serious outcome in 12% ( 5 of 43 ) of patients again emphasizes the clinical significance of the entity.25 twelve patients were retreated with icis after complete clinical resolution of initial pneumonitis , and three patients experienced recurrent pneumonitis , which again resolved by drug holding ( n = 1 ) or oral corticosteroids ( n = 2 ) as in the course of their initial pneumonitis treatment.25 a recently published guideline of pneumonitis management is in agreement with the observations in these reports.31 the guideline recommends oral corticosteroid treatment , including prednisone 1 to 2 mg / kg / d or methylprednisolone 0.5 to 1 mg / kg / d in mild to moderate cases . 
in mild to moderate cases , bronchoscopy is recommended to exclude infectious etiologies before starting immunosuppression.31 in severe cases , hospitalization is necessary , with treatment to include high - dose corticosteroids , such as methylprednisolone 2 to 4 mg / kg / d , and additional immunosuppression , including mycophenolate mofetil , cyclophosphamide ; infliximab can be administered if necessary.31 although limited to a small percentage of patients , an additional unique phenomenon of pneumonitis flare has been reported where ici - related pneumonitis recurs after the termination of corticosteroid taper and in the absence of ici retreatment after the initial episode of pneumonitis has been successfully treated ( fig 2 )  . 
this was first reported in a patient with nsclc who was treated with commercial nivolumab and who experienced pneumonitis.6 the patient was successfully treated with corticosteroids for the initial pneumonitis ; however , the patient experienced another episode of pneumonitis after completing corticosteroid taper without resuming pd - 1 inhibitor therapy or starting any other therapy , indicating a pneumonitis flare.6 although similar to the initial presentation both clinically and radiographically , flare pneumonitis tends to be more severe and extensive than the initial episode . 
such a phenomenon has not been described in the setting of pneumonitis related to other anticancer agents and this that indicates the clinical course of icirelated pneumonitis may be more complex than other drug - related pneumonitis in some patients . in a series of 20 patients with ici - related pneumonitis , one patient with lymphoma experienced two episodes of pneumonitis flare in which pneumonitis came back with similar but more extensive radiographic presentations compared with the initial episode after completion of corticosteroid taper without pd - 1 retreatment5 ( fig 2 )  . 
this observation further confirms the phenomenon , which is likely unique to iraes and may involve autoimmune mechanisms . unmet clinical needs that require additional investigations although several recent reports provide important knowledge and observations of ici - related pneumonitis , significant knowledge gaps still exist for this emerging entity . 
because early and accurate diagnosis is key for this mostly treatable conditionwith good response to corticosteroids in the majority of the casesadditional studies are needed to identify risk factors and early markers for pneumonitis to improve diagnostic accuracy . treatment and clinical management guidelines need to be further optimized as the immuneoncology community accumulates the experience of this entity . 
pneumonitis with a cryptogenic organizing pneumonia ( cop ) pattern in a 33 - year - old female with hodgkin lymphoma who was treated with nivolumab and ipilimumab combination therapy , with a recurrence during retreatment ( 2a ; ah ) and two episodes of pneumonitis flare after completion of corticosteroid taper ( 2b ; ah )  . 
reprinted with permission from nishino et al.5 2a : ( a and b ) computed tomography ( ct ) scan of the chest at 1.4 months of therapy demonstrated ground - glass and reticular opacities and consolidations with multifocal distribution , which are indicative of a cop pattern of pneumonitis ( arrowheads )  . 
 ( 2b : a and b ) the patient completed 2 months of corticosteroid taper , and after 1 month , experienced another episode of pneumonitis with a similar radiographic pattern , without nivolumab retreatment or other systemic therapy , which indicated a pneumonitis flare . 
 ( 2b : e and f ) the 2.7 - month course of corticosteroid taper was completed , and after 2 weeks , the patient again developed a pneumonitis flare with a similar radiographic pattern as prior episodes . 
findings included lymphocyte - predominant interstitial pneumonitis ( arrowhead , 2b [ g ] , hematoxylin and eosin stain , 3200 ) with rare eosinophils ( arrow , 2b [ g ] ) , and areas of organizing pneumonia with fibroblast plugs and foamy macrophages filling the airspaces ( asterisks , 2b [ h ] , hematoxylin and eosin stain , 3200 )  . 
the patient started another course of prednisone taper with subsequent clinical improvement and is schedule for a follow - up ct scan . in addition , awareness of pneumonitis related to icis other than pd - 1 / pd - l1 inhibitors is becoming increasingly important . 
everolimus is approved for the treatment of several cancers , including advanced rcc , advanced pancreatic neuroendocrine tumors ( nets ) , subependymal giant cell astrocytomas , and advanced hormone receptorpositive , human epireceptor 2negative dermal growth factor breast cancer ( in combination with exemestane )  . 
temsirolimus is approved for the treatment of advanced rcc . drug - related pneumonitis is a recognized class effect toxicity of mtor inhibitors and has been described in detail in the context of advanced rcc treatment . 
in a study by maroto et al , 34 among 178 patients with rcc who were treated with singleagent temsirolimus , 52 ( 29% ) had radiographically identified drug - related pneumonitis . 
of these , 16 patients ( 31% ) had respiratory symptoms at the time of the onset of radiographically detected pneumonitis , whereas others ( 36 of 52 ; 69% ) were asymptomatic and thus may not be clinically diagnosed for pneumonitis . 
the estimated cumulative probability of radiologically identified drug - related pneumonitis was 21% ( 95% ci , 15% to 29% ) at 8 weeks , 31% ( 95% ci , 24% to 40% ) at 16 weeks , and 45% ( 95% ci , 36% to 57% ) at 13 months.34 another study of 46 patients with rcc who were treated with temsirolimus or everolimus reported that 14 patients ( 30% ) developed drug - related pneumonitis.35 median time of the onset of radiologic manifestations of pneumonitis was 56 days ( range , 31 to 214 days )  . 
these 14 patients more frequently achieved stable disease than did others , which indicates a possibility of pneumonitis as a treatment benefit marker.35 reports of mtor inhibitorrelated pneumonitis in tumors other than rcc indicate a somewhat different incidence of pneumonitis according to tumor types . 
radiographic patterns of mtor inhibitorrelated pneumonitis have been characterized in patients with wm and in those with advanced net.7 , 8 in both cohorts , the most common findings were bilateral ground - glass opacities ( ggos ) and reticular opacities , sometimes with consolidations , in a peripheral and lower distribution . 
cop pattern was the leading pattern observed in the majority of cases ( 70% in wm and 57% in net ) , followed by nsip pattern ( 30% in wm and 36% in net ) , whereas one patient in the net cohort demonstrated the hp pattern.7 , 8 similar imaging manifestations are noted in other cohorts with mtor inhibitor related pneumonitis.34 , 35 recognition of the characteristic radiographic patterns of pneumonitis during mtor inhibitor therapy helps with the early detection and accurate diagnosis of this entity . clinical management of mtor inhibitorrelated pneumonitis mostly depends on the severity of clinical symptoms . 
egfr - tki therapy should be discontinued , and systemic corticosteroids are usually administered.49 although egfr - tkirelated pneumonitis has mostly been a specific concern among the japanese population and has often had a low clinical impact in other populations , a recent observation from a trial of a novel ici and third - generation egfr - tki , osimertinib , raises a new concern in the setting of combination therapy . 
 agents may limit , in part , the destructive remodeling of the lung and impair the lungs ability to respond to injury.37 , 49 , 52 these possible mechanisms are also likely modified by various host and environmental factors , which further complicates the issue.52 likewise , risk factors for pneumonitis need to be defined , especially for ici - related pneumonitis , to allow for optimal patient selection with regard to treatment safety . 
the risk of recurrent pneumonitis after reintroduction of therapy is another important debate for these agents , as they may be the only available or effective therapy for patients with advanced cancers.5 , 37 the impact of radiotherapy on pneumonitis is a major topic , given a number of ongoing combination therapy trials that use ici and radiotherapy.53 in some studies of patients with advanced nsclc who were treated with egfr - tkis , prior thoracic irradiation and concomitant radiotherapy have been noted as risk factors for pneumonitis.49 , 54 although the exact effect of radiotherapy on ici - related pneumonitis awaits additional data , prior chest radiation has influenced the radiographic appearance of pd - 1 pneumonitis in patients with lymphoma , which indicates a need for caution in treatment monitoring.30 prior reports have often focused on radiation to the chest ; however , radiotherapy in the extrathoracic sites may require more attention given the concept of abscopal effects , which are increasingly discussed in the context of ici and radiation treatment.53 in the clinical setting , drug - related pneumonitis is ultimately a diagnosis of clinical correlation and exclusion , and there is no specific test for the entity.55 among various lung conditions that can affect patients with advanced cancer who undergo systemic therapy , infection is an important differential diagnosis that should be excluded before starting corticosteroid therapy . 
fiberoptic bronchoscopy is helpful for this purpose as it allows for targeted and deep sampling of specimens for microbiology , cytology , and histology via bronchoalveolar lavage and transbronchial biopsy.55 a minority of patients may present with respiratory distress in the absence of radiographic confirmation , thereby leading to delays in appropriate management . 
of note , some of the findings of pneumonitis , such as ggos , are not adequately evaluated on chest radiographs , and thus diagnostic chest ct scans are needed to sensitively detect and fully characterize the lung abnormalities in the setting of suspected pneumonitis . in conclusion , knowledge of drug - related pneumonitis has increasingly emerged in recent investigations , providing important clues for accurate diagnosis , patient management , and monitoring . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . mizuki nishino honoraria : bayer yakuhin consulting or advisory role : bristol - myers squibb , worldcare clinical , toshiba medical systems research funding : merck ( inst ) drug - related pneumonitis in the era of precision cancer therapy the following represents disclosure information provided by authors of this manuscript . 
stephen hodi employment : dana - farber cancer institute consulting or advisory role : merck sharp & dohme , novartis , genentech , amgen , emd serono research funding : bristol - myers squibb ( inst ) , merck sharp & dohme ( inst ) , genentech ( inst ) , novartis ( inst ) patents , royalties , other intellectual property : patent pending as per institutional policy , patent pending royalties received on mica - related disorders application to institution per institutional policy travel , accommodations , expenses : novartis , bristol - myers squibb other relationship : bristol - myers squibb , genentech nikhil h . 
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ghobrial im , gertz m , laplant b , et al : phase ii trial of the oral mammalian target of rapamycin inhibitor everolimus in relapsed or refractory waldenstr om macroglobulinemia . 
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min jh , lee hy , lim h , et al : drug - induced interstitial lung disease in tyrosine kinase inhibitor therapy for nonsmall cell lung cancer : a review on current insight . 
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 o inference of germline mutational status and evaluation of loss of heterozygosity in high - depth , tumor - only sequencing data purpose inherited germline defects are implicated in up to 10% of human tumors , with particularly well - known roles in breast and ovarian cancers that harbor brca1 / 2 mutated genes . 
however , because of the frequent lack of patient matched control normal dna and / or low tumor purity , there is limited ability to determine the genomic status of these alterations ( germline v somatic ) and to assess the presence of loss of heterozygosity ( loh )  . 
these analyses , especially when applied to genes such as brca1 / 2 , can have significant clinical implications for patient care . materials and methods lohgic ( loh - germline inference calculator ) is a statistical model selection method to determine somatic versus germline status and predict loh for mutations identified via clinical grade , high - depth , hybrid capture , tumor - only sequencing . 
lohgic incorporates statistical uncertainties inherent to high - throughput sequencing as well as specimen biases in tumor purity estimates , which we used to assess brca1 / 2 mutations in 1 , 636 specimens sequenced at rutgers cancer institute of new jersey . results evaluation of lohgic with available germline sequencing from brca1 / 2 testing demonstrates 93% accuracy , 100% precision , and 96% recall . 
2018 by american society of clinical oncology introduction inherited germline genomic alterations contribute to the pathogenesis of 5% to 10% of cancers.1 , 2 hereditary breast and ovarian cancer ( hboc ) is one of the most common familial cancer syndromes , with a prevalence of one in 400 to 500 people in the general population2 and a higher ocurrence in specific ethnic and racial groups.3 women harboring germline mutations in the brca1 and brca2 genes have a significantly increased lifetime risk of developing hboc.4 , 5 mutations in these genes increase the risk of other cancers , including fallopian tube and peritoneal cancers in women , 6 prostate and breast cancers in men , 7 , 8 and pancreatic cancer in both sexes9 ; they are also associated with subtypes of fanconi anemia.10 , 11 brca1 and brca2 are tumor - suppressor genes that encode two proteins involved in homologous recombination ( hr ) mediated dna repair . 
tumors that arise in patients with germline brca1 / 2 mutations often lose the wildtype allele through additional somatic mutations or copy number changes that result in loss of hossein khiabanian kim m . 
us food and drug administration approval of olaparib is only for germline brca1 / 2 mutations , because response to both chemotherapy and targeted therapy is affected by their presence.16 second is continued cancer risk . 
relatives of patients with germline brca1 / 2 mutations may benefit from genetic testing to assess risk.17 for patients with cancer who may possess inherited brca1 / 2 alterations , the standard approach to investigate mutational status has been sequenc ing normal tissue , such as peripheral blood lymphocytes or buccal swabs.18 , 19 with the availability of high - quality assays , tumoronly sequencing is being performed on more patients , 20 , 21 and pathogenic mutations in genes associated with hereditary cancer syndromes , such as brca1 / 2 , can be unexpectedly detected.22 the identification of brca1 / 2 variants in tumor - only data raises two important questions . 
 is the variant acquired somatically in the tumor , or is it in the germline ? and is loh affecting brca1 / 2 loci , suggesting a role for targeted therapy ? the latter question , specifically , cannot be answered by routine germline testing . 
 for example , if a male patient with a known pathogenic germline brca2 mutation develops a tumor not typically associated with the brca2 tumor syndrome ( eg , lung cancer ) , standard approaches cannot determine whether the cancer cells have undergone loh at the brca2 locus and thus may be susceptible to treatment with parp inhibitors . 
similarly , in the setting of a somatic pathogenic brca1 / 2 mutation , determining the loh status can resolve whether the alteration is a heterozygous passenger mutation or whether it has led to a true loss of brca1 / 2 function and , hence , susceptibility to parp inhibitors . 
because loh is the most common mechanism for biallelic inactivation , 23 , 24 its identification in a tumor may also help classify variants of unknown significance . analysis of variant allele frequencies ( vafs ) coupled with robust estimates of copy number changes and tumor purity can be used to infer loh status . 
in this study , we address this problem and introduce lohgic ( loh - germline inference calculator ) to assess germline versus somatic status of mutations and predict presence of loh in tumor - only deep - sequencing data . 
all classes of genomic alterations , including substitutions , small indels , rearrangements , and copy number changes , were determined for each sample , as previously described.26 each tumor sample was analyzed alongside a process - matched normal control ( an internally validated mixture of 10 heterozygous diploid hapmap samples ) , which allowed normalizing sequence coverage distribution across baited targets.26 purity and base ploidy were derived using empirical bayesian sampling methodologies . 
 ( a ) a tumor specimen consists of both cancer cells ( brown ) , normal stromal cells ( gray ) , and possibly normal lymphocytes ( light red )  . 
observed variant allele frequency ( vaf ) of a mutation in hybrid capture dna sequencing depends on various factors including depth of sequencing and chromosomal copy number ( cn ; ploidy ) at its locus , as well as specimen purity ( see materials and methods )  . 
 the brca1 / 2 mutations were classified as candidate pathogenic when they were reported in clinvar or other brca1 / 2 mutation classification databases , 29 , 30 were listed as verified mutations in the catalogue of somatic mutations in cancer database , 31 or were truncating indels or nonsense substitutions in functional protein domains of the genes . lohgics mutational status modeling assuming tumor purity of p and a mutations chromosomal copy number of two in normal cells and ploidy of y in tumor cells , the expected vaf for a somatic variant is given by vaf = c mut p _____________ 2 ( 1 p ) + y p , where c mut is the variant alleles unknown copy number . 
akaike information criterion ( aic ) can then be used to compare these likelihoods.32 because there is one fitted parameter in a binomial distribution , aic ( f , d ) = 2 log l ( f , d ) + 2  . 
to select the most consistent model for each variant , w for a c mut estimate and mutational status were computed across purity and vaf cis and were then summed . 
the ratio of w or sums of ws for a set of models indicates their relative strength of evidence.33 thus , aggregate weights , wgerm and wsom , were calculated by summing all germline and all somatic models , respectively . 
similarly , aggregate weights for loh status in germline and somatic models were calculated independently ; wloh > 0.5 was considered as significant presence of loh in tumors . results modeling mutational status of substitution and small indels observed vaf of a mutation in hybrid capture dna sequencing depends on the relative number of cancer cells compared with normal cells ( tumor purity ) in a specimen ( fig 1a )  . 
 however , for germline models , the mutant allele is present in both normal and tumor cells ( fig 1c )  . lohgic is developed on a model - selection scheme using aic weighting.33 lohgic infers the most consistent model describing the germline versus somatic mutational status with the corresponding number of mutated copies for each mutation . 
loh events , especially at the brca1 / 2 loci , can occur by either deletion of the wild - type allele ( ie , reduction to copy number one ) or duplication of mutant allele with loss of the wild - type under copy - neutral loh ( gene conversion or isodisomy ) .34 in our approach , models for all these mechanisms are evaluated . 
 biallelic mutations are considered if more than one pathogenic mutation is detected ; however , lohgic cannot infer rare rearrangements in the second allele or focal deletions aic weights can be interpreted as likelihoods for mutational status and presence of loh and strongly rely on the statistical confidence in assessing vaf and tumor purity . 
appendix figures a1 , a2 , and a3 illustrate the changes in aic weights at higher ploidy and across possible models , indicating how some combinations of vaf and tumor purity will not permit the selection of one model out of several , yielding ambiguous calls . 
moreover , because accuracy in vaf estimates depends on sequencing depth , inferring loh is statistically robust at depths that provide sufficient confidence in measuring vaf within 1% to 5% . lohgic is available at the khiabanian lab web site ( html )  . 
given confidences in these measurements , lohgic reports and visualizes aic weights for all possible mutational models . mutational status of brca1 / 2 alterations we identified 64 patients with cancer with candidate pathogenic brca1 / 2 mutations , detected by hybrid capture , tumor - only sequencing , in a set of 1 , 636 sequenced specimens at the rutgers cancer institute ( data supplement )  . 
in a sample with purity of 60% , sequenced at 1 , 000 , akaike information criterion ( aic ) weights ( w ) are shown across the variant allele frequencys ( vaf ) 99% ci . 
 used lohgic and predicted germline versus somatic mutational status of the brca1 / 2 mutant alleles with high confidence in 88% of patients ( 56 of 64 patients ) , 66% of whom ( 37 of 56 patients ) had germline aic weights > 0.7 and 34% of whom ( 19 of 56 patients ) had somatic aic weights > 0.7. 
our predictions significantly corroborated these data , showing 93% accuracy , 100% positive predictive value ( precision ) , 96% true - positive rate ( recall ) , 75% negative predictive value , and 25% false omission rate ( table 1 )  . 
 in only one patient , lohgic could not correctly genotype a previously validated germline brca2 mutation ; this single nucleotide deletion had a vaf of 35% ( depth , 844 ; purity , 29% )  . 
 further inspection of raw sequencing reads at the position of the deletions revealed the presence of a somatic reversion mutation ( insertion of a guanine ) that restored the reading frame and thus affected the vaf of the germline thymine deletion ( appendix fig a4 )  . six patients had data from multiple sequenced specimens . 
in 33% of these patients ( two of six patients ) , lohgics predictions agreed with genetic testing for one specimen while remaining ambiguous for the other ( table 2 )  . evaluating the loss of wild - type allele and we predicted the presence of loh when the sum of aic weights for germline or somatic models with loh was > 0.5. 
as expected for pathogenic brca1 / 2 mutations , we found loh to be prevalent in our cohort ; loh was present in 76% of patients ( 28 of 37 patients ) inferred to have a germline mutation and in 63% of patients ( 12 of 19 patients ) inferred to have a table 1 . 
in two patients , we observed two brca2 mutations ; one patient ( patient 31 ) harbored both a somatic and a germline ( without loh ) mutation , and the other patient ( patient 60 ) harbored one somatic mutation , whereas the inference of his second mutation was ambiguous ( data supplement )  . prevalence and sex specificity of brca1 / 2 mutations germline brca1 mutations were exclusively found in tumors arising in women , 87% of whom ( 13 of 15 patients ) were diagnosed with hboc or fallopian tube cancers . 
germline brca2 mutations were observed equally in cancers occurring in men and women , each at 50% ( 11 of 22 patients ) , with a wide range of tumor types . 
graphs represent the relationship between the expected vaf and purity for each model ( see materials and methods ) ; the shaded gray area represents vaf and purity cis , within which w is calculated . 
further inspection of the data from the second breast tumor indicated low purity of the sequenced specimen , which prohibited robust assessment of loh status . discussion inherited mutations in the brca1 / 2 genes contribute to 1% to 5% of breast cancers . 
these tumors almost invariably undergo loh with the loss of the wild - type allele and are shown to be sensitive to parp inhibitor and platinum treatment.13 - 15 parp inhibitors and platinum are also selectively effective in other cancers harboring mutations in brca1 / 2.35 therefore , early detection of these mutations and precise determination of their germline versus somatic status and loh is extremely important to therapeutic approaches.36 germline genetic testing men with malignancies in the prostate ( three patients ) , pancreas ( two patients ) , lung ( one patient ) , and breast ( one patient )  . 
in the setting of somatic brca2 mutation , presence of loh was inferred in 67% of patients ( 10 of 15 patients ) , uncorrelated with sex . co - mutations in patients with brca1 / 2 mutations the most common genomic alterations in brca1 / 2 - mutated tumors were tp53 mutations ( 67% ; 43 of 64 patients ) , myc amplifications ( 17% ; 11 of 64 patients ) , and cdkn2a / b deletions ( 17% ; 11 of 64 patients )  . 
tp53 mutations were present in 87% of patients ( 13 of 15 patients ) with germline brca1 mutations , co - occurring in 100% of patients ( 11 of 11 patients ) with inferred loh . 
 cnv , copy number variant ; f , female ; gist , gi stromal tumor ; m , male ; snv , single nucleotide variant ; na , not available ( lack of testing results )  . primary tumor tissue somatic brca1 heterozygous somatic brca1 with loh germline brca1 heterozygous germline brca1 with loh ambiguous brca1 somatic brca2 heterozygous somatic brca2 with loh germline brca2 heterozygous germline brca2 with loh ambiguous brca2 genetic testing patients sex tp53 mutation myc amplification cdkn2a / b deletion no . 
however , various ethical complications , uninformative family histories , and / or miscommunication between patients , families , and health care providers may result in missed opportunities to incorporate brca1 / 2 mutational status , and that of related genes , in treatment and prevention of future cancers.37 with the advent of precision oncology and implementation of high - depth sequencing in the clinic , it is now possible to partially resolve the genetic profile of tumors and their prognostic mutations soon after disease diagnosis . 
in the absence of normal dna controls , computational analysis of tumor - only genetic data are essential for identifying patients who may harbor germline brca1 / 2 mutations and prioritizing them for germline testing.38 here , we presented lohgic , which statistically infers mutational status and predicts the loss of wild - type allele under loh from deep - sequencing data . 
lohgic predictions are strongly concordant with independent germline genetic testing results and provide additional information on the likely presence of loh at brca1 / 2 in tumor cells ; patient - matched control dna genotyping is required to further support lohgics results . this analysis highlights differences in the tumor spectrum associated with the brca1 / 2 mutations . 
tumors arising in the setting of germline brca1 mutations with loh were all found in women and were exclusively breast cancer or of mllerian origmoreover , they universally harbored pathogenic tp53 mutations . 
in contrast , tumors showing germline brca2 mutation with loh had a much wider spectrum of cancers , were seen in both sexes , and did not frequently harbor tp53 mutations . 
tumors with evidence of germline brca1 / 2 mutation , but without loh , may represent sporadic tumors that arose in mutation carriers ; however , careful molecular analysis is required to rule out the presence of reversion mutations or other mechanisms including epigenetic silencing of the wild - type allele.24 , 39 haploinsufficiency may also play a role in tumorigenesis in some settings.40 in the case of somatic brca1 / 2 alterations , most were in the setting of a relatively high mutation burden , suggesting their heterozygous passenger status that may not confer defects in hr and thus sensitivity to parp inhibitors or platinum until biallelic inactivation occurs . 
although these tumors are predicted not to harbor genomic signatures of brca1 / 2 loss , deficiency in hr cannot be completely ruled out.23 , 24 our analysis may be confounded by low tumor purity and insufficient sequencing data . 
the presence of reversion mutations in brca1 / 2 , which occur under acquired resistance to platinum and parp inhibitors , 41 can also lead to incorrect or ambiguous inference . 
in our data set , a somatic reversion mutation in one patient and low tumor purity in another led to discrepant results , further illustrating the importance of incorporating statistical variations in vaf , depth , and purity , especially considering inherent biases in high - throughput sequencing . 
of note , the requirement for robust vaf determination limits this approach to hybrid capture based sequencing , because interpreting vaf in amplicon - based assays can be confounded by polymerase chain reaction efficiency . in summary , lohgic , which is freely available for academic use , will facilitate investigating the molecular epidemiology of brca1 / 2 and hr - associated mutations . 
 hirshfield , shridar ganesan deborah toppmeyer employment : novartis ( i ) leadership : novartis ( i ) financial support : hossein khiabanian , shridar ganesan stock and other ownership interests : novartis ( i ) serena wong no relationship to disclose nancy chan no relationship to disclose kalyani dhar no relationship to disclose jinesh gheeya no relationship to disclose hetal vig no relationship to disclose mohammad hadigol no relationship to disclose dean pavlick no relationship to disclose sepand ansari no relationship to disclose administrative support : lorna rodriguez - rodriguez , shridar ganesan provision of study material or patients : kim m . 
 hirshfield , mendel goldfinger , mark stein , joseph aisner , deborah toppmeyer , serena wong , nancy chan , kalyani dhar , jinesh gheeya , hetal vig , lorna rodriguez rodriguez , shridar ganesan collection and assembly of data : hossein khiabanian , kim m . 
hirshfield , mendel goldfinger , mark stein , joseph aisner , deborah toppmeyer , serena wong , nancy chan , kalyani dhar , jinesh gheeya , hetal vig , dean pavlick , siraj ali , lorna rodriguez - rodriguez , shridar ganesan data analysis and interpretation : hossein khiabanian , kim m . 
 for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . siraj ali employment : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine ; patents via seres health on microbiome stuff in non - neoplastic disease ( i ) bing xia no relationship to disclose lorna rodriguez - rodriguez no relationship to disclose shridar ganesan employment : merck ( i ) stock and other ownership interests : ibris , inspirata , merck ( i ) consulting or advisory role : inspirata , novartis patents , royalties , other intellectual property : i hold two patents for digital imaging that may be licensed to ibris and inspirata travel , accommodations , expenses : inspirata hossein khiabanian no relationship to disclose kim m . 
hirshfield , mendel goldfinger , simon bird , mark stein , joseph aisner , deborah toppmeyer , serena wong , nancy chan , kalyani dhar , jinesh gheeya , hetal vig , mohammad hadigol , sepand ansari , bing xia , lorna rodriguez - rodriguez , and shridar ganesan , rutgers cancer institute of new jersey , rutgers university ; hossein khiabanian , kim m . 
samimi g , bernardini mq , brody lc , et al : traceback : a proposed framework to increase identification and genetic counseling of brca1 and brca2 mutation carriers through family - based outreach . 
finch a , beiner m , lubinski j , et al : salpingo - oophorectomy and the risk of ovarian , fallopian tube , and peritoneal cancers in women with a brca1 or brca2 mutation . 
giri vn , obeid e , gross l , et al : inherited mutations in men undergoing multigene panel testing for prostate cancer : emerging implications for personalized prostate cancer genetic evaluation . 
de bono j , ramanathan rk , mina l , et al : phase i , dose - escalation , two - part trial of the parp inhibitor talazoparib in patients with advanced germline brca1 / 2 mutations and selected sporadic cancers . 
wagle n , berger mf , davis mj , et al : high - throughput detection of actionable genomic alterations in clinical tumor samples by targeted , massively parallel sequencing . 
alexandrova em , mirza sa , xu s , et al : p53 loss - of - heterozygosity is a necessary prerequisite for mutant p53 stabilization and gain - of - function in vivo . 
christie el , fereday s , doig k , et al : reversion of brca1 / 2 germline mutations detected in circulating tumor dna from patients with high - grade serous ovarian cancer . 
 ploidy 1 or 2 ploidy 3 somatic loh cnmut = 1 germline loh cnmut = 1 somatic loh cnmut = 1 germline loh cnmut = 1 somatic , cnmut = 1 germline , cnmut = 1 somatic , cnmut = 1 germline , cnmut = 1 somatic loh , cnmut = 2 germline loh , cnmut = 2 somatic loh , cnmut = 2 germline loh , cnmut = 2 somatic loh , cnmut = 3 germline loh , cnmut = 3 somatic loh , cnmut = 3 germline loh , cnmut = 3 depth : 1000 purity 0.60 depth : 1000 purity 0.60 somatic loh cnmut = 1 germline loh cnmut = 1 somatic loh cnmut = 1 germline loh cnmut = 1 somatic , cnmut = 1 germline , cnmut = 1 somatic , cnmut = 1 germline , cnmut = 1 somatic loh , cnmut = 2 germline loh , cnmut = 2 somatic loh , cnmut = 2 germline loh , cnmut = 2 somatic loh , cnmut = 3 germline loh , cnmut = 3 somatic loh , cnmut = 3 germline loh , cnmut = 3 fig a2 . 
similar to the models shown in figure 1 , here , the sum of aic weights across vaf and purity cis ( shown in parentheses ) larger than 0.7 indicates unambiguous prediction of a model . 
expected variant allele frequency ( vaf ) versus specimen purity for all possible models is shown at mutational ploidy of ( a ) one and two and ( b ) three . 
akaike information criterion weights for a sample with purity of 60% , sequenced at 1 , 000 are shown at ploidy of ( c ) one and two and ( d ) three . 
overlapping regions show how some combinations of tumor purity , ploidy , and sequencing depth may not permit the selection of one model out of several , resulting in ambiguous results . 
pathology demonstrated metastatic colloid / mucinous adenocarcinoma ( stage pt4a , pn2b , pm1c )  . immunohistologic testing for dna mismatch repair ( mmr ) proteins on tissue biopsy specimen showed preservation of muts protein homolog 2 ( msh2 ) and muts homolog 6 ( msh6 ) , but loss of mutl homolog 1 ( mlh1 ) and mismatch repair endonuclease pms2 ( pms2 ) , consistent with mmr deciency . 
initially , the patient was treated with four doses of uorouracil , leucovorin , oxaliplatin , and irinotecan ( folfoxiri ) until computed tomography scanning revealed an increase in peritoneal masses and liver metastases . 
he then was treated with pembrolizumab on the basis of mmr deciency ; however , this was discontinued after three doses when scans revealed progressive disease , and a doubling of carcinoembryonic antigen since initiation was noted ( 401 ng / ml to 848 ng / ml )  . 
all patient information was curated under an institutional review board protocol ( mcc 19161 ) , which was formally reviewed and granted approval by moftt cancer center ( mcc )  . 
this institutional review board protocol includes a waiver of consent , because no identifying information is reported in the case . a foundationone cdx test ( foundation medicine , cambridge , ma ) was ordered on the diagnosed omental tissue with subsequent review by the personalized medicine clinical service ( pmcs ) at mcc for guidance regarding future therapeutic options ( table 1 )  . 
the molecular tumor board ( mtb ) discussion focused on approaches for targeted therapeutic considerations in a patient with high tumor mutation burden ( tmb ) after disease progression on an immune checkpoint inhibitor . the patient had 93 reported alterations ; the foundationone cdx report provided interpretive content for 26 of them for correlated therapies with clinical benet , clinical trials , or therapies with a lack of response . 
in a patient with high tmb status and microsatellite instability ( msi ) , the likelihood of an individual alteration primarily driving the cancer is difcult to ascertathe pmcs categorized alterations into common repair / signaling pathways , associating the alterations with possible therapies and ranking therapeutic options on the available evidence ( table 1 )  . 
figure 1 and appendix table a1 illustrate the workow taken by the pmcs to identify and prioritize treatment options . of the 26 alterations for which foundationone cdx provided interpretive content , impaired homologous repair ( hr ) was a commonly altered pathway . 
highlight alterations with targeted therapies tmb : high ( 105 muts / mb ) microsatellite status : unstable kras a59t atm r2227c brca2 n319fs * 8 nf1 i679fs * 21 ptch1 r1308fs * 64 akt1 e17k msh6 f1088fs * 2 men1 r612h mtor a469t apc h298fs * 28 apc r1114 * apc v2194fs * 5 arid1a g889fs * 2 asxl1 g646fs * 12 bcorl1 p1681fs * 20 ctcf t204fs * 18 keap1 f478fs * 2 mll2 p2354fs * 30 notch3 a1701v notch3 g2035fs * 60 notch3 p1317fs * 103 pbrm1 w992fs * 23 ptpn11 s502p qki k134fs * 14 setd2 f1650v sox9 v306fs * 77 pold1 r17q flt1 s733del 66 vus 2 . 
categorize by mechanism tmb - high msh6 arid1a brca2 akt1 kras * nf1 * mmr deficiency mmr deficiency mmr deficiency hr deficiency hr deficiency hr deficiency akt activation ras activation ras activation 3 . 
correlate mechanisms to therapies parp or atr inhibitor second line of immunotherapy ( combination therapy or novel agent on trial ) pi3k - akt - mtor inhibitor arid1a brca2 tmb - high msh6 arid1a brca2 akt1 4 . 
 ( 3 ) correlating grouped repair / signaling mechanisms from step 2 with possible targeted therapy options assists collecting and correlating supporting evidence to be considered by a molecular tumor board ( mtb )  . 
 ( 4 ) the mtb reviewed the evidence and prioritized therapy options according to the patients current disease state , current failed line of therapy , patient preference , strength of evidence associated with an alteration and therapy option , and available clinical trials . 
nct03188965 ( first - in - human study of atr inhibitor bay1895344 in patients with advanced solid tumors and lymphomas ) was recruiting patients with atm alterations and was further supported by clinical , in vivo , and in vitro data . 
 ( * ) the kras a59t and nf1 i679fs * 21 alterations produced conicting likelihoods of response to epidermal growth factor receptor ( egfr ) inhibitors and were based on minimal case report data . 
 nelson et al ( parp ) inhibitors or immunotherapy.2 , 3 parp inhibition in the context of brca alterations has demonstrated clinical benet in breast , ovarian , and prostate cancer and has produced discussion of expansion to other ddr deciencyrelated alterations.4 atm encodes a protein kinase that plays a key role in dna repair , cell cycle checkpoints , and apoptosis in response to dna damage.5 the atm r2227c alteration is clinically relevant , because it produces a missense alteration in the frap - atm - trrap ( fat ) domain , has been categorized as deleterious in breast cancer and adenoid cystic carcinoma , and is classied as pathogenic by clinvar ( clinvar / )  . 
in a phase ii trial of patients with ddr - defective metastatic castrate - resistant prostate cancer receiving olaparib , patients with germline or somatic alterations in genes involved in ddr showed signicantly increased activity , with responses occurring in 14 out of 16 patients ( 88% ) .6 , 7 the at - rich interactive domain - containing protein 1a ( arid1a ) g889fs * 2 alteration is likely inactivating ( catalogue of somatic mutations in cancer mutation id : cosm6911235 )  . arid1a is a subunit of the chromatin remodeling complex swi / snf that is involved in facilitating access of proteins to dna , and inactivating alterations are common across various cancer types.8 pathways involved in ddr have been shown to interact with arid1a , including msh2 . 
during dna replication , it has been demonstrated that arid1a recruits msh2 to chromatin and promotes mmr.8 thus , inactivation of arid1a has been shown to impair mmr . arid1a deciency has demonstrated sensitization to parp inhibitors in vitro and in vivo.9 atr , another important genetic component of ddr , has been the target of novel investigational drugs that inhibit atr and arid1a signaling in vitro and in vivo.5 , 10 collectively , arid1a , atm , and brca2 produce a common theme of ddr / hr deciency , which leads to atr or parp inhibition alone or in combination with platinum or another dna - damaging chemotherapy agent in an off - label or ( more preferred ) clinical trial setting were recommended as viable options . akt1 e17k has been reported as a missense alteration that occurs in the pleckstrin homology domain of rac - alpha serine / threonine - protein kinase ( akt1 ) and activates akt signaling leading to inhibition of cell apoptosis and promotion of cellular proliferation.11 this alteration is therefore considered oncogenic and is well described in the literature . 
in a phase i , dose - escalation trial of the pan - akt inhibitor azd5363 , 58 patients with solid tumor found to have somatic akt1 e17k alterations were enrolled . 
the median progression - free survival was 5.5 months for patients with estrogen - positive breast cancer , 6.6 months for patients with gynecologic disease , and 4.2 months for patients with other solid tumors , including two patients with colorectal cancer ( crc ) .12 however , in a patient with a high tmb and msi , the value of this akt1 e17k mutation as a clinically relevant driver is unclear . 
in case the clinical team decided to target akt , clinical trials , including nct02465060 ( nci - match - arm z1k ) , nct03310541 , or nct02761694 , could be considered . finally , the kras a59t alteration is less common than other known activating kras alterations but has been reported as resulting in increased activation of the map kinase and pi3k pathways in vivo.13 interestingly , this alteration may demonstrate an exception to the observation that patients with kras - mutated metastatic crc do not receptor respond to epidermal growth factor ( egfr ) targeted monoclonal antibodies cetuximab or panitumumab . 
there is one case report of a patient with metastatic crc and the kras a59t alteration who was treated with panitumumab plus uorouracil , leucovorin , and irinotecan and experienced a partial radiographic response ( 36% decrease per response evaluation criteria in solid tumors 1.1 ) , stable carcinoembryonic antigen biomarker , and decreased size of metastases for an 8 - month duration.14 the patient showed no other alterations in exons 2 and 3 of kras , nras , and hras genes ; exon 15 of braf ; and exons 2 , 5 , 9 , 10 , 20 , and 21 of pik3ca . this seems to be the rst observed documentation of the a59t allele not producing resistance to egfr - targeting monoclonal antibodies and may provide a potential future consideration for our patient.14 however , there is limited evidence that the nf1 frame shift may predict poorer response to cetuximab or panitumumab therapy on the basis of a small case series of four chinese patients with metastatic crc who were kras g12 and g13 wt but found to have inactivating nf1 alterations . 
the alterations involved in hr deciency ( brca2 , atm , and arid1a inactivation ) provided the most robust combined evidence of predicted response to future therapy options . the akt1 e17k alteration is known to be activating , but its importance in a highly mutated , unstable tumor was less well dened . 
 therapy option prioritization in crc with high mutation burden follow - up of the patients case , unfortunately , showed that after three treatments of pembrolizumab , there was no sign of response , which led the clinical team to refer back to the above options to consider treatment beyond immunotherapy . 
the clinical team discussed the predominant grouping of alterations ( arid1a , atm , and brca2 ) with the common theme of ddr / hr deciency and ultimately decided to enroll the patient in a clinical trial ( clinicaltrials.gov identier : nct03188965 ) investigating the atr inhibitor bay1895344 in patients with solid tumor with atm loss . the lesson learned with this patient was one of categorizing a large list of alterations and prioritizing each according to therapeutic targets on the basis of dominant signaling / repair pathways and strength of available evidence . 
in doing so , the pmcs was able to provide the treating physician with a succinct , prioritized grouping of alterations validating the initial line of therapy and considerations for future therapeutic options , which led to the patient being enrolled in a clinical trial with the greatest anticipated therapeutic benet . 
walko , pharmd , 12902 magnolia dr , tampa , fl 33612 ; twitter : @mofttnews ; e - mail : christine.walko@moftt.org. support supported in part by national cancer institute comprehensive cancer center support grant no . 
mcleod leadership : cancer genetics stock and other ownership interests : cancer genetics , interpares biomedicine honoraria : genentech , illumina consulting or advisory role : gentris , cancer genetics , saladax biomedical , national institutes of health / national cancer institute , admera health , evicore healthcare , pharmazam speakers bureau : genentech christine m . 
walko employment : mission healthcare stock and other ownership interests : alexion pharmaceuticals ( i ) consulting or advisory role : foundation medicine , jackson laboratory for genomic medicine , intermountain precision genomics other relationship : mission hospital no other potential conicts of interest were reported . references cerniglia m , xiu j , grothey a , et al : association of dna damage response and repair genes ( ddr ) mutations and microsatellite instability ( msi ) , pd - l1 expression , tumor mutational burden ( tmb ) in gastroesophageal cancers . 
j clin oncol 37 , 2019 ( suppl 4 ; abstr 60 ) overman mj , mcdermott r , leach jl , et al : nivolumab in patients with metastatic dna mismatch repair - decient or microsatellite instability - high colorectal cancer ( checkmate 142 ) : an open - label , multicentre , phase 2 study . 
lancet oncol 18 : 1182 - 1191 , 2017 ghiringhelli f , richard c , chevrier s , et al : efciency of olaparib in colorectal cancer patients with an alteration of the homologous repair proteworld j gastroenterol 22 : 10680 - 10686 , 2016 pili e pg , gay cm , byers la , et al : parp inhibitors : extending benet beyond brca - mutant cancers . 
clin cancer res 25 : 3759 - 3771 , 2019 shiloh y , ziv y : the atm protein kinase : regulating the cellular response to genotoxic stress , and more . 
n engl j med 373 : 1697 - 1708 , 2015 shen j , ju z , zhao w , et al : arid1a deciency promotes mutability and potentiates therapeutic antitumor immunity unleashed by immune checkpoint blockade . 
nat med 24 : 556 - 562 , 2018 shen j , peng y , wei l , et al : arid1a deciency impairs the dna damage checkpoint and sensitizes cells to parp inhibitors . 
banerji u , dean ej , p erez - fidalgo ja , et al : a phase i open - label study to identify a dosing regimen of the pan - akt inhibitor azd5363 for evaluation in solid tumors and in pik3ca - mutated breast and gynecologic cancers . 
mei z , shao yw , lin p , et al : smad4 and nf1 mutations as potential biomarkers for poor prognosis to cetuximab - based therapy in chinese metastatic colorectal cancer patients . 
ninety - three alterations were reported on the foundationone cdx report ( including variants of known and unknown signicance )  . twenty - six of the alterations had interpretive content for correlated therapies with clinical benet , clinical trials , or therapies with a lack of response . 
twenty - seven alterations had potentially relevant information ( maf , listed in exac , protein domain location , in oncokb and others ) as reported in the initial screening of ndings . 
 enrichment of fgfr3 - tacc3 fusions in patients with bladder cancer who are young , asian , or have never smoked purpose fgfr3 - tacc3 ( fibroblast growth factor receptor 3transforming acidic coiled coil - containing protein 3 ) fusions have recently been identified as driver mutations that lead to the activation of fgfr3 in bladder cancer and other tumor types and are associated with sensitivity to tyrosine kinase inhibitors . 
we examined the clinical and molecular characteristics of patients with fgfr3 - tacc3 fusions and hypothesized that they are enriched in a subset of patients with bladder cancer . materials and methods we correlated somatic fgfr3 - tacc3 fusions with clinical and molecular features in two cohorts of patients with bladder cancer . 
the second cohort consisted of patients with mibc or high - grade nonmibc at the dana - farber cancer institute that had targeted capture sequencing of a selected panel of cancer genes ( n = 356 )  . 
the clinical response of one patient with fgfr3 - tacc3 bladder cancer to an fgfr3 inhibitor was investigated . results overall , 751 patients with high - grade bladder cancer without fgfr3 - tacc3 fusions and 17 with fgfr3 - tacc3 fusions were identified in the pooled analysis of the data sets from the cancer genome atlas and the dana - farber cancer institute . 
 fgfr3 - tacc3 fusions were enriched in patients age 50 years versus age 51 to 65 years versus those older than 65 years ( pooled , p = .002 ) , and were observed in four ( 12% ) of 33 patients age 50 years in the pooled analysis . 
in 2017 , 79 , 030 new cases of bladder cancer are expected to be diagnosed , and approximately 16 , 870 deaths are predicted to occur from the disease in the united states.1 , 2 compared with other cancer subtypes , advances in the management of bladder cancer have been limited in the past three decades , and there is an unmet need to develop novel therapeutic agents that target potentially actionable alterations.3 , 4 genomic alterations in fibroblast growth factor receptors ( fgfrs ) are among the most frequent events during bladder cancer development . 
fgfrs are receptor tyrosine kinases that orchestrate various cellular processes , including cell proliferation , differentiation , and survival.5 fgfr mutations lead to developmental syndromes when present in the germline , and contribute to cancer growth when acquired somatically.6 fgfr fusions with an intact kinase domain have been identified in several cancer amin h . 
consort diagram for 850 patients with bladder cancer . clinical cohort ( n = 850 ) dana - farber cancer institute ( n = 438 ) the cancer genome atlas ( n = 412 ) whole - exome sequencing ( n = 412 ) paired tumor - normal muscle - invasive bladder cancer rna sequencing for structural variant calls assessment of clinical and molecular characteristics in fgfr3 - tacc3positive versus negative bladder tumors institutional targeted next - generation sequencing assay oncopanel ( n = 438 ) computational workflow ( n = 365 ) mutational calling ( mutect ) copy number variations ( viscap - cancer ) breakmer algorithm ( structural variants ) exclusions low - grade nonmuscle invasive histology ( n = 73 ) exclusions low tumor purity ( < 20% ; n = 9 ) final analysis ( n = 356 ) assessment of clinical and molecular characteristics in fgfr3 - tacc3positive versus negative bladder tumors types , including cervical cancer , bladder carcinoma , glioblastoma multiforme , squamous lung carcinoma , and head and neck cancer.7 - 15 fgfr3 , a member of this family , has been reported to be involved in fusions with several genes in bladder carcinoma , including tacc3 ( transforming acidic coiled coil - containing protein 3 )  . 
tacc3 normally is thought to mediate the stabilization and organization of the mitotic spindle during mitosis.14 in the cancer genome atlas ( tcga ) muscle invasive bladder cancer ( mibc ) cohort , in - frame activating fgfr3 - tacc3 fusionsobserved in 10 ( 2.4% ) of 412 patientswere the most common gene fusions identified.7 fgfr3 - tacc3 fusion proteins consist of the immunoglobulin , transmembrane , and tyrosine kinase domains of fgfr3 , fused to the coiled - coil domain of tacc3 . 
through the promotion of dimerization , these fusions lead to a constitutively active fgfr3 kinase protein that has been demonstrated to promote cell proliferation in vivo and in vitro.7 - 9 , 13 phase i and ii trials of fgfr inhibitors have reported promising antitumor activity in patients with fgfr genetic alterations , especially bladder cancer.16 certain genetic alterations , particularly gene fusion events , are enriched in clinical subsets of patients with cancer . 
 patients with mibc and high - grade non - mibc were pooled together in the dfci cohort as there is substantial evidence that the two subtypes are biologically and genomically similar.21 - 23 overall , seven patients with fgfr3 - tacc3 fusions were identified in the dfci cohort using an institutional targeted next - generation sequencing assay24 ( oncopanel )  . 
figure 1 shows the sample inclusion and exclusion criteria and workflow . tissue collection and dna extraction tumor specimens and clinicopathologic information were collected with institutional review board approval at dfci . 
tumor areas that contained at least 20% of tumor cells ( mean tumor purity , 58% ; range , 20% to 100% ) were isolated from normal tissue and chosen for dna extraction . 
dna was quantified by nanodrop and pico - green assays . targeted sequencing two hundred nanograms of genomic dna from each sample was subjected to targeted exon capture and sequencing using oncopanel_v1 to v3 cancer gene panels at brigham and womens hospital ( boston , ma )  . 
the oncopanel gene panel includes capture probes for 275 to 560 cancer - associated genes , as well as intronic portions of 60 genes for rearrangement detection , including fgfr3.24 sample dna was captured using oncopanel_v1 to v3 bait sets using a solution - phase agilent sureselect hybrid capture kit ( agilent technologies , santa clara , ca )  . 
single - nucleotide variants and small indels were analyzed using mutect version 1 0.27200 ( display / cgatools / mutect ; accessed may 2013 ) and annotated by oncotator ( broadinstitute.org / oncotator ; accessed may 2013 )  . 
 copy number alterations were analyzed using a custom r - based tool26 , 27 ( viscap - cancer )  . mean depth of read coverage for the targeted genes was 283 . 
mean , median , and range of percentage of target bases with read depth > 30 was 98% , 99% , and 78% to 99% , respectively . identification of rearrangements and analysis of genomic breakpoints fgfr3 fusion sequences were identified using the breakmer algorithm28 and were manually reviewed using integrated genomic viewer29 to exclude sequencing or alignment artifacts . 
all analyses of sequencing data and mutation and fusion calls were performed blinded to clinical data . clinical response to anti - fgfr3 therapy one patient with fgfr3 - tacc3 mibc received anti - fgfr3 therapy along with docetaxel and the clinical response was monitored . statistical analysis we used fisher 's exact test for categorical data and the wilcoxon rank - sum test for quantitative data . 
 ( % ) , unless otherwise noted . abbreviations : dfci , dana - farber cancer institute ; fgfr3 , fibroblast growth factor receptor 3 ; hg , high grade ; nd , not determined ; nmibc , nonmuscle invasive bladder cancer ; tacc3 , transforming acidic coiled - coil - containing protein 3 ; tcga , the cancer genome atlas . fusion partner fgfr3 - tacc3 fgfr3 - tacc3 fgfr3 - tacc3 fgfr3 - tacc3 fgfr3 - tacc3 fgfr3 - tacc3 fgfr3 - tacc3 fgfr3 - lmnb2 fgfr3 - tnip2 fgfr3 - fam184b fgfr3 - jakmip1 fgfr3 4 : 1808826 ; 4 : 1737404 4 : 1808771 ; 4 : 1740268 4 : 1808744 ; 4 : 1740297 4 : 1808806 ; 4 : 1741100 4 : 1808737 ; 4 : 1741297 4 : 1808690 ; 4 : 1741336 4 : 1808937 ; 4 : 1732863 4 : 1808556 ; 19 : 2436887 4 : 1808750 ; 4 : 2752213 4 : 1809003 ; 4 : 17691454 4 : 1808936 ; 4 : 6091989 fig 2 . 
schematic representation of the genomic rearrangements observed in 11 tumor samples that harbor fibroblast growth factor receptor 3 ( fgfr3 ) fusion variants identified using the oncopanel assay in the dana - farber cancer institute cohort . 
fam184b , family with sequence similarity 184 member b ; lmnb2 , lamin b2 ; jakmip1 , janus kinase and microtubule interacting protein 1 ; tacc3 , transforming acidic coiled - coil - containing protein 3 ; tnip2 , tnfaip3 interacting protein 2 . results formalin - fixed , paraffin - embedded tumor specimens were obtained from 438 patients at dfci . 
 we excluded 82 tumors from the analysis because they were of low - grade nonmuscle invasive histology ( n = 73 ) or had low ( < 20% ) tumor purity ( n = 9 )  . 
we mapped the genomic breakpoints of fgfr3 and its corresponding fusion partners that were identified in the dfci cohort , which included four non - tacc3 fusions ( fig 2 )  . 
 ( % ) , unless otherwise noted . abbreviations : dfci , dana - farber cancer institute ; fgfr3 , fibroblast growth factor receptor 3 ; tacc3 , transforming acidic coiled - coil - containing protein 3 ; tcga , the cancer genome atlas . patients age 50 harboring a fusion ( p = .03 ; table 2 )  . 
fgfr3 - tacc3 fusions in tcga were also more frequent in asians ( six [ 14% ] of 44 patients ) compared with other races ( p < .001 ) , as well as in never smokers ( eight [ 7.2% ] of 111 patients ) compared with ever smokers ( p < .001 ; table 2 )  . 
similarly , fgfr3 - tacc3 fusions were more common in dfci patients age 50 years ( one [ 12% ] of eight patients ) compared with other age groups ( p = .001 ; table 2 )  . 
eleven ( 65% ) of 17 patients with fgfr3tacc3 fusions were associated with least one of these three clinical characteristics , and three ( 18% ) of the 17 patients were asian never smokers age 50 years . we next examined whether tumors with fgfr3 - tacc3 fusions had molecular features that distinguished them from other tumors . 
as the oncopanel analysis was performed on tumor samples only , we excluded variants that were observed at any frequency in the exome aggregation consortium database , 30 as they were considered likely germline variants . 
the 17 patients whose tumors harbored fgfr3 - tacc3 fusions were enriched for cdkn1a mutations ( 5 [ 29% ] of 17 v 76 [ 10% ] of 751 ; p = .03 ; table 3 )  . 
conversely , fgfr3 - tacc3 fusion - positive tumors had significantly fewer tp53 mutations ( p = .02 ) , and none had rb1 mutations ( p = .054 ; table 3 )  . 
 somatic copy number alterations were also analyzed in both cohorts using criteria for loss , deletion , gain , and amplification that were developed and applied independently in the two cohorts ( table 3 )  . 
dfci cohort : amplification : log2 ( copy ratio ) > 1.8 , gain : 1.1 log2 ( copy ratio ) < 1.8 ; deletion : log2 ( copy ratio ) < 2 , loss : 2 log2 ( copy ratio ) < 1 . abbreviations : cnv , copy number variation ; dfci , dana - farber cancer institute ; fgfr3 , fibroblast growth factor receptor 3 ; snv , single nucleotide variant ; tacc3 , transforming acidic coiled - coil - containing protein 3 ; mdm2 , murine double minute 2 ; tcga , the cancer genome atlas . mdm2 ( murine double minute 2 ) gain ( p = .04 ) , deletion of pten ( p = .02 ) , and deletion of cdk2na ( p = .0033 ; table 3 )  . as a result of differences in the extent of genome sequencing in the tcga and dfci cohorts , we analyzed the overall mutational burden in each cohort separately . 
in the tcga cohort , the nonsynonymous somatic mutation rate across 18 , 862 genes was significantly higher in patients without fgfr3 - tacc3 fusions compared with those with fusions ( median 224 v 128 ; p = 0.04 ; table 3 )  . 
 in addition , there were no significant differences in the frequency of somatic copy number alterations in either the tcga or dfci cohorts ( table 3 )  . one patient who harbored the fgfr3 - tacc3 fusion in mibc in the dfci cohort was treated with an fgfr3 inhibitor and docetaxel and experienced complete remission for approximately 10 months . discussion our results demonstrate that patients with bladder cancer with fgfr3 - tacc3 fusions have distinct clinical and molecular features compared with the general population of patients with bladder cancer . 
in addition , fgfr3tacc3 fusions were associated with a low frequency of tp53 and rb1 mutations and a higher frequency of cdkn1a mutations , fgfr3 and mdm2 amplifications , and pten deletions . 
 because fgfr3 - tacc3 fusion - positive tumors can be sensitive to fgfr inhibitors , 9 , 31 , 32 these observations suggest that molecular testing to detect fgfr3 - tacc3 fusions in bladder cancer should be prioritized for patients who are young ( age 50 years ) , of asian race , and / or who have never smoked . 
most strikingly , we observed that all patients with bladder cancer who were asian never smokers younger than age 50 years ( n = 3 ) had fgfr3 - tacc3 fusions . we emphasize that our study has significant limitations as a result of the small number of patients with fgfr3 - tacc3 fusions included ( n = 17 ) , which reflects that this is a relatively rare molecular subset of bladder cancer . 
most importantly , we strongly advocate additional studies of this association to extend and confirm these findings . in conclusion , fgfr3 - tacc3 fusion - positive bladder cancer is highly enriched in asians , never smokers , and those age 50 years . 
nassar no relationship to disclose kevin lundgren no relationship to disclose mark pomerantz honoraria : bayer eliezer van allen stock and other ownership interests : synapse , tango therapeutics , genome medical consulting or advisory role : synapse , roche , third rock ventures , takeda , novartis , genome medical , invitae speakers ' bureau : illumina research funding : bristol - myers squibb , novartis lauren harshman consulting or advisory role : medivation , astellas pharma , pfizer , genentech , theragene , kew , corvus pharmaceuticals , merck , exelixis , bayer research funding : medivation , astellas pharma ( inst ) , bayer ( inst ) , sotio ( inst ) , genentech ( inst ) , dendreon ( inst ) , bristol - myers squibb ( inst ) , takeda ( inst ) , merck ( inst ) , janssen oncology ( inst ) , pfizer ( inst ) travel , accommodations , expenses : bayer atish d . 
sonpavde honoraria : uptodate consulting or advisory role : bayer , genentech , sanofi , merck , novartis , pfizer , argos therapeutics , agensys , eisai , astrazeneca , janssen pharmaceuticals , amgen , bristolmyers squibb , exelixis speakers ' bureau : clinical care options , national comprehensive cancer network , physician education resource , onclive , research to practice research funding : onyx pharmaceuticals ( inst ) , bayer ( inst ) , boehringer ingelheim ( inst ) , celgene ( inst ) , merck ( inst ) , pfizer ( inst ) other relationship : boehringer ingelheim , astrazeneca affiliations amin h . 
1p01ca120964 : molecular pathogenesis of the hamartoma syndromes . a directly related abstract was accepted as a poster presentation in the 2018 genitourinary cancer symposium of the american society of oncology , san francisco , ca , february 8 - 10 , 2018 . support prior presentation references 1 . 
acquaviva j , he s , zhang c , et al : fgfr3 translocations in bladder cancer : differential sensitivity to hsp90 inhibition based on drug metabolis mol cancer res 12 : 1042 - 1054 , 2014 4 . 
nogova l , sequist lv , perez garcia jm , et al : evaluation of bgj398 , a fibroblast growth factor receptor 1 - 3 kinase inhibitor , in patients with advanced solid tumors harboring genetic alterations in fibroblast growth factor receptors : results of a global phase i , dose - escalation and dose - expansion study . 
shaw at , yeap by , mino - kenudson m , et al : clinical features and outcome of patients with nonsmall - cell lung cancer who harbor eml4 - alk . 
humphrey pa , moch h , cubilla al , et al : the 2016 who classification of tumours of the urinary system and male genital organs - part b : prostate and bladder tumours . 
balbs - martnez c , sagrera a , carrillo - de - santa - pau e , et al : recurrent inactivation of stag2 in bladder cancer is not associated with aneuploidy . 
lindgren d , frigyesi a , gudjonsson s , et al : combined gene expression and genomic profiling define two intrinsic molecular subtypes of urothelial carcinoma and gene signatures for molecular grading and outcome . 
garcia ep , minkovsky a , jia y , et al : validation of oncopanel : a targeted next - generation sequencing assay for the detection of somatic variants in cancer . 
tabernero j , bahleda r , dienstmann r , et al : phase i dose - escalation study of jnj - 42756493 , an oral pan - fibroblast growth factor receptor inhibitor , in patients with advanced solid tumors . 
 use of a targeted exome next - generation sequencing panel offers therapeutic opportunity and clinical benet in a subset of patients with advanced cancers scott kopetz , md , phd1 ; kenna r . 
jack lee , md1 ; jiexin zhang , ms1 ; beate litzenburger , phd1 ; vijaykumar holla , phd1 ; walter kinyua , ms1 ; emily broaddus1 ; molly s . 
broaddus , md , phd1 purpose smaller hotspot - based next - generation sequencing ( ngs ) panels have emerged to support standard of care therapy for patients with cancer . 
survival and the impact of matched therapy use were determined by kaplan - meier estimate , log - rank test , and cox proportional hazards regression . results the larger ngs panel identied at least one alteration in an actionable gene not previously identied in the smaller sequencing panel in 214 ( 41% ) of 521 of enrolled patients . 
with current clinical molecular diagnostics , sequencing is faster , uses less tumor dna , and costs less when genes are multiplexed into small next - generation sequencing ( ngs ) panels . 
historically , the focus was often on the identication of mutations found in hotspotsareas of the genome where known drivers are frequently mutated , many of which can be mapped to therapeutic intervention . 
these panels typically emphasize genes that are recommended by cancer treatment guidelines . an emerging utility of ngs is the screening of other types of cancers for mutations in clinically actionable genes that can be targeted therapeutically . 
such treatment approaches are associated with improved in retrospective analyses screening , 1 prosurvival spective trials , 2 - 4 and meta - analyses , 5 - 7 but this approach is not universally embraced.8 , 9 an important issue in these studies is the low number of patients who benet from this approach . 
 kopetz et al context key objective can additional actionable information be identied by expanding sequencing coverage to the full exome of hundreds of genes beyond hotspot analysis and , secondarily , does that additional information affect patient outcomes ? knowledge generated only a small number of genes have alterations that are specically listed in a us food and drug administrationapproved indication that would match a patient with cancer to a specic actionable agentfor example , braf v600e to vemurafenib . 
limited access to targeted therapy trials is an issue , as some trials enroll only certain tumor types or are only active in a few geographic sites . in patients with advanced cancer that is refractory to standard treatment , any benet gained from the detection of mutations in clinically actionable genes beyond those detected using focused hotspot sequencing panels remains unknown . 
hotspot - focused ngs panels fail to robustly identify copy number alterations or mutations that are found outside commonly altered loci , which would represent an advantage of larger ngs initiative was to panels . 
enrollment criteria included the following : adult patient with pathologic documentation of a single solid tumor malignancy ; completed frontline treatment and any standard treatments that extended life by at least 3 months ; eastern cooperative oncology group performance status of 0 or 1 ; no active brain metastases ; and completed tumor testing using a smaller sequencing panel and either disease progression on a matched therapy that targeted a previously identied actionable nding or no clinically actionable mutations detected . molecular assays and decision support that consisted of hotspot mutation testing was primarily performed on archival formalin - xed , parafn - embedded primary and metastatic cancers acquired during the course of routine clinical care using a 46or 50 - gene ngs panel described previously10 ( actionable genes summarized in the data supplement )  . 
subsequent testing used a larger ngs the entire coding regions of panel 409 cancer - related genes with methodologies previously detailed11 ( actionable genes and details of larger ngs panel provided in the data supplement )  . 
the protocol did not provide for image - guided biopsies of the most recent metastasis for testing ; median time since tissue acquisition to ngs was 467 days ( appendix fig a1 )  . relevant germline ndings were reported to the referring oncologist.12 for comparisons with protocol patients , the cancer genome atlas ( tcga ) datagene mutations and gene amplicationsfor the same 409 genes from 316 ovarian high - grade serous carcinomas , 212 colorectal adenocarcinomas , and 230 lung adenocarcinomas were extracted using cbioportal.org. 
for patients with colorectal cancer , mismatch repair was assessed as part of routine clinical care.13 somatic mutations and amplications that were reported for each patient were reviewed by the precision oncology decision support team.14 , 15 this involved annotation for function and potential clinical trial matching for actionability . 
characteristics for patients enrolled and tested in the prospective protocol characteristic female male performance status missing race asian black white hispanic other unknown tumor type bladder breast colorectal endometrial hawaiian / pacic islander gastric / gastroesophageal junction germ cell tumor head and neck lung neuroendocrine others ovarian prostate renal sarcoma thyroid age , years 20 20 - 30 30 - 40 40 - 50 50 - 60 60 - 70  . 
for the smaller 46and 50 - gene ngs panel , 34 and 38 genes , respectively , were considered actionable , whereas for the larger 409 - gene ngs panel , 96 genes were actionable ( data supplement )  . statistical analysis study sample size was dictated by the capacity of the clinical laboratory improvement amendmentscertied clinical molecular diagnostics laboratory that performed the 409 - gene ngs panel , estimated to be 25 cases per week . with a target of 600 patients , it was determined a priori that the rate of identication of alterations in actionable genes could be estimated with a standard error of no more than 0.02. 
we used fishers exact test to assess whether the gene mutation frequency was signicantly different between protocol patients with colorectal adenocarcinoma , lung adenocarcinoma , or ovarian high - grade serous carcinoma and corresponding tcga data sets . 
the probability of overall survival was determined by kaplan - meier estimate , and we used a log - rank test to assess the statistical signicance of the difference between patient groups . 
to evaluate the effect of clinical variables on overall survival , we performed multivariable survival analysis using a cox proportional hazards regression model that included variables that were signicant ( p , .05 ) in a univariable analysis . results characteristics of the patient population enrolled in the protocol a total of 675 patients were enrolled , with large - panel testing completed in 569 patients . 
five hundred twenty - one patients who received at least 6 months of follow - up or who died within 6 months were included in the analysis ( appendix fig a2 )  . colorectal cancer , sarcoma , head and neck cancer , ovarian cancer , and breast cancer were the most frequently represented cancers in the cohort ( table 1 ) , but the protocol enrolled patients with a broad spectrum of cancer types . sequencing results once material was received by the molecular diagnostics laboratory , a median of 13 days passed before the molecular reports were nalized and entered into the electronic medical record ( appendix fig a3 )  . 
of the 521 patients who were tested with the 409 - gene ngs assay , 214 ( 41% ) had at least one tumor alteration in an actionable gene that was not identied in a prior assay using a smaller ngs panel ( figs 1a and 1b )  . full 409 - gene ngs assay results are summarized for all patients in the data supplement . 
not counting the sequencing results from the smaller panel , the mean number of alterations detected in actionable genes in the 409 - gene panel was 1.0 ( range , 0 to 66 ; fig 1c )  . 
after the application of decision support , 201 ( 41% ) of 495 alterations in actionable genes were considered to affect the function of the gene and were deemed actionable ( mean per patient , 0.4 ; range , 0 to 5 ; fig 1d )  . 
the rate of detection of an alteration in an actionable gene with additional testing was dependent on tumor type , with low rates observed in thyroid and ovarian cancers ( fig 2a )  . 
alterations in a variety of actionable genes from the 409 - gene panel were identied , with the most common mutations in genes nf1 , mdm2 , atm , kdr , notch2 , erbb2 , mtor , and pten ( fig 2b )  . matching tested patients to targeted therapy of the 214 patients who had alterations in actionable genes in the large ngs platform that were not detected in the previous small - panel ngs testing , 40 ( 19% ) were matched gender race male female unknown other hispanic hawaiian / pacific islander white black asian disease site others thyroid sarcoma renal ovarian lung colorectal breast age , years > q3 : 65 - 85 q2 - q3 : 56 - 65 q1 - q2 : 46 - 56 < q1 : 18 - 46 fig 1 . 
on average , only one alteration in an actionable gene per tumor was detected . ( d ) on average , only 0.4 actionable alterationsmutation or amplication in an actionable gene that is known or has inferred biologic functionwere detected per tumor . 
 ( a ) stacked bar chart shows the percent of cases for each broad class of tumor type listed with 0 , 1 , 2 , or 3 or more alterations in actionable genes not detected in the smaller testing panel . data are shown for tumor types in which at least 10 patient tumors were sequenced . 
these patients had mutations in actionable genes that were classied as actionable , potentially actionable , or a variant of unknown signicance but the oncologist chose a therapy that was not a match to the gene alterations treating with matched therapy found . 
reasons for not protocols were similar to those from a previously reported patient population18 and included declining performance status , a desire to seek treatment closer to home , or an oncologist who did not consider the gene mutation clinically actionable or the targeted therapy sufciently active in the given tumor type ( data supplement )  . decision support decision support was critical as alterations were detected in a number of actionable genes that were less familiar to oncologists , and the availability of matching clinical trials varied during the study period . 
given the hypermutated nature of this patients tumor , it was felt that none of the mutations would act as a driver or represented a good target for matched therapy ; thus , they were not annotated ( fig 3a )  . 
not counting the sequencing results from the smaller panel , nearly 70% of tumor alterations were identied in patients with tumors that were found to be in the top quartile of overall tumor mutation burden . 
 kopetz et al ( n = 201 ; 41% ) yes : inferred ( n = 35 ; 17% ) potentially ( n = 81 ; 40% ) yes : literature based ( n = 85 ; 42% ) ( n = 66 ; 13% ) ( n = 11 ; 2% ) unknown ( n = 21 ; 44% ) all gene alterations ( n = 2 , 673 ) bottom quartile 3rd quartile 2nd quartile top quartile alterations in actionable genes ( n = 495 ) actionable alterations ( n = 201 ) bottom quartile 3rd quartile bottom quartile 2nd quartile top quartile 3rd quartile top quartile 2nd quartile fig 3 . 
forty - four percent of the alterations found in actionable genes were variants of unknown signicance ( unknown ) , and 13% were not annotated because they were all from one hypermutated patient ( na )  . 
of the alterations with known function , 2% are known to be benign or not actionable ( no ) , with the remainder having some annotated function on the basis of the existing literature . 
presence of clinically actionable alterations was not related to high overall tumor mutation burden . application of decision support , the number of actionable alterations was poorly correlated with mutation burden , as less than 50% of actionable alterations in actionable genes are in the top quartile of mutation load ( fig 3b )  . 
thus , tumor mutation burden was not a good predictor of the presence of a clinically actionable alteration . patient outcomes neither the number of overall gene alterations in the tumor , nor the presence of alterations in actionable genes signicantly inuenced patient survival ( figs 4a and 4b )  . 
survival was assessed by manual curation of the medical record along with the date when a patient was last known to be alive as documented by a physician note or date of death provided by obituary , tumor registry , or reported by a family member . 
this study is limited by the lack of randomization ; however , comparison of matched and unmatched patients helps to minimize concerns related to comorbidities or declining performance status as an explanation for the differences in outcomes observed in figures 4c and 4d , as all patients in this analysis received another line of therapy , including participation in clinical trials . two patients were found to have deleterious germline mutations that were not previously known to the patient or oncologistpatient management was not altered in either case . 
 ( a - d ) kaplan - meier plots for overall survival by ( a ) total number of alterations ( actionable and nonactionable ) detected , ( b ) whether the patient had a tumor with an alteration in an actionable gene , ( c ) type of therapy and absence of alterations in actionable genes , and ( d ) whether the patient had a tumor with an alteration in an actionable gene on the 409 - genes next - generation sequencing panel received matched treatment . 
only treatment with matched targeted therapy ( c and d ) was associated with signicantly improved survival ( p = .017 compared with patients treated with nonmatched therapy )  . 
tcga did not select for patients with advanced cancers ; therefore , we compared mutation frequencies in our cohort with those of the tcga for colorectal adenocarcinoma , lung adenocarcinoma , and ovarian high - grade serous carcinoma.19 - 21 these three cancers were examined because of their relatively homogeneous histology and sufcient representation in the study . 
for colorectal cancer , kras mutation frequency was higher in our patient population , which was consistent with their resistance to epidermal growth factor receptordirected therapy22 ( data supplement )  . 
protocol patients also had a signicantly higher incidence of tp53 mutations ( 72% v 52% ; p = .004 ; data not shown )  . only 3% of protocol colorectal cancers were microsatellite instabilityhigh compared with 13% in tcga . 
at risk : wild type 507 439 315 197 112 508 440 318 198 113 508 439 319 199 114 time ( months ) time ( months ) time ( months ) mutation mutation mutation no . 
 ( a - j ) kaplan - meier plots for overall survival for nine actionable genes commonly altered in clinical protocol patients ( a - i ) and for the most common cancer types represented in the protocol ( j )  . 
only two clinically actionable genescdkn2a and cdkn2bhad lower frequencies of alteration in protocol patients with lung adenocarcinoma ( data supplement )  . discussion survival for the overall cohort was poor in this institutionwide prospective protocol that enrolled patients with advanced solid tumor malignancies refractory to treatment . for 41% of these patients , we identied an alteration in a clinically actionable gene that was not detected by previous small - panel testing . 
the overall utility of singleas opposed to sequential smallpanel , then large - panel testinglarge - panel ngs testing combined with decision support can be estimated by combining the 11% enrollment in clinical trials from our prior 46and 50 - gene panel study18 with the 8% derived from the incremental addition of the large - panel testing in this study . 
as metastases can accumulate mutations over time , this represents another potential reason for limited patient benet . with increasing gene targets and matched drugs , it is becoming increasingly difcult for an individual physician to reasonably interpret the molecular ndings from larger ngs panels . 
the importance of decision support a comprehensive precision medicine strategy is highlighted by the fact that less than one half of actionable alterations in actionable genes had been reported previously in the literature . 
over time , at least some of these variants of undetermined signicance may be found to be activating mutations after they are examined in functional assays . examples of variants that have been reclassied as activating mutations in a functional genomics platform26 include pdgfra k385m , pik3ca e110del , and ret d627n . 
the reliability of quality decision support is crucial as patients with advanced cancers have a nite lifespan . patients with advanced cancers that are refractory to standard therapy may have mutational frequencies of individual genes that differ from published frequencies derived from unselected patients . 
this was apparent in patients with colorectal cancer and ovarian high - grade serous carcinoma in whom frequencies likely reected a bias toward more clinically aggressive or treatment - resistant tumor biology . frontline treatment of ovarian cancer is for example , platinum - based chemotherapy ; is possible that , by selecting for patients with platinum - resistance , the protocol also selected for patients with ovarian cancers with therapydriven alterations in the molecular landscape . 
the phenomenon is supported by the comparison of survival populations from stage - matched patients in tcga and clinical trials , where patient survival from clinical trials was substantially lower.27 patients with advanced cancers may have higher incidences of mutations associated with other forms of treatment resistance , such as egfr t790m and esr1 mutations.28 the potentially unique molecular features in patients with advanced , chemotherapy - resistant cancers may represent one factor that contributes to the the matched targeted therapy low success rate of approach . treatment directed against actionable genes remains suboptimal . 
pathways that were previously thought to be universally targetable have proven to be difcult to treat , such as the phosphatidylinositol 3 - kinase / akt pathway , 29 or subject to variation in results by tumor type , such as braf30 or her2 / 3 mutations.31 , 32 in protocol patients , we were unable to ascertain any strong individual signals of activity for particular alterations in specic tumor types in part because of insufcient numbers of patients . in conclusion , these results demonstrate the utility of largepanel ngs testing when combined with decision support . the derived benet is realized in only a small subset of patients . 
future efforts should emphasize high - quality and timely decision support and minimize barriers to patient enrollment , with the ultimate goal of broadly delivering precision medicine to patients with solid tumor malignancies . 
broaddus , md , phd , department of pathology , unit 85 , university of texas md anderson cancer center , 1515 holcombe blvd , houston , tx 77030 ; twitter : @mdandersonnews ; e - mail : rbroaddus@ mdanderson.org. support supported , in part , by undesignated donor funds from the md anderson annual funds program provided by the provosts ofce , khalifa bin zayed al nahyan foundation , cancer prevention research institute of texas grant no . 
for more information about asco 's conict of interest policy , please refer to or po.ascopubs.org / site / ifc . scott kopetz stock and other ownership interests : molecularmatch , navire consulting or advisory role : roche , genentech , emd serono , merck , karyopharm therapeutics , amal therapeutics , navire pharma , symphogen , holy stone , biocartis , amgen , novartis research funding : amgen ( inst ) , sano ( inst ) , biocartis ( inst ) , guardant health ( inst ) , array biopharma ( inst ) , genentech ( inst ) , emd serono ( inst ) , medimmune ( inst ) , novartis ( inst ) j . 
jack lee consulting or advisory role : abbvie kopetz et al beate litzenburger employment : qiagen funda meric - bernstam honoraria : sumitomo group , dialectica consulting or advisory role : genentech , inection biosciences , pieris pharmaceuticals , clearlight diagnostics , darwin health , samsung bioepis , spectrum pharmaceuticals , aduro biotech , origimed , xencor , debiopharm group research funding : novartis , astrazeneca , taiho pharmaceutical , genentech , calithera biosciences , debiopharm group , bayer , aileron therapeutics , puma biotechnology , cytomx therapeutics , jounce therapeutics , zymeworks , curis , pzer , effector therapeutics , abbvie , boehringer ingelheim ( i ) , guardant health ( inst ) no other potential conicts of interest were reported . references tsimberidou am , iskander ng , hong ds , et al : personalized medicine in a phase i clinical trials program : the md anderson cancer center initiative . 
massard c , michiels s , fert e c , et al : high - throughput genomics and clinical outcome in hard - to - treat advanced cancers : results of the moscato 01 trial . 
cancer discov 7 : 586 - 595 , 2017 radovich m , kiel pj , nance sm , et al : clinical benet of a precision medicine - based approach for guiding treatment of refractory cancers . 
cancer res 76 : 3690 - 3701 , 2016 jardim dl , schwaederle m , wei c , et al : impact of a biomarker - based strategy on oncology drug development : a meta - analysis of clinical trials leading to fda approval . 
j natl cancer inst 107 : djv253 , 2015 schwaederle m , zhao m , lee jj , et al : impact of precision medicine in diverse cancers : a meta - analysis of phase ii clinical trials . 
j clin oncol 33 : 3817 - 3825 , 2015 schwaederle m , zhao m , lee jj , et al : association of biomarker - based treatment strategies with response rates and progression - free survival in refractory malignant neoplasms : a meta - analysis . 
singh rr , patel kp , routbort mj , et al : clinical validation of a next - generation sequencing screen for mutational hotspots in 46 cancer - related genes . 
singh rr , patel kp , routbort mj , et al : clinical massively parallel next - generation sequencing analysis of 409 cancer - related genes for mutations and copy number variations in solid tumours . 
bartley an , luthra r , saraiya ds , et al : identication of cancer patients with lynch syndrome : clinically signicant discordances and problems in tissue - based mismatch repair testing . 
cancer prev res ( phila ) 5 : 320 - 327 , 2012 johnson a , zeng j , bailey am , et al : the right drugs at the right time for the right patient : the md anderson precision oncology decision support platfordrug discov today 20 : 1433 - 1438 , 2015 15 . 
liang h , cheung lw , li j , et al : whole - exome sequencing combined with functional genomics reveals novel candidate driver cancer genes in endometrial cancer . genome res 22 : 2120 - 2129 , 2012 33 : 2753 - 2762 , 2015 18 . 
andr e f , bachelot t , commo f , et al : comparative genomic hybridisation array and dna sequencing to direct treatment of metastatic breast cancer : a multicentre , prospective trial ( safir01 / unicancer )  . 
hyman dm , piha - paul sa , rodon j , et al : neratinib for erbb2 mutant , her2 non - amplied , metastatic breast cancer : preliminary analysis from a multicenter , open - label , multi - histology phase ii basket trial . 
 t clinical benet of larger ngs panels assessed for eligibility ( n = 675 ) excluded ( n = 101 ) died before testing declined to return to the institution for treatment insufficient tissue for testing ngs testing ( n = 574 ) final analysis ( n = 521 ) excluded ( n = 53 ) no death within 6 months follow - up < 6 months cms46 / 50 testing ( n = 507 ) without cms46 / 50 ( n = 14 ) no actionable gene found ( n = 371 ) actionable gene found , then progression ( n = 78 ) actionable gene found , no progression ( n = 58 ) cms400 testing ( n = 507 ) no actionable genes in cms400 that are not identified in cms46 / 50 ( n = 301 ) no actionable gene identified in cms400 ( n = 6 ) cms400 testing ( n = 14 ) 1 actionable gene in cms400 not identified in cms46 / 50 ( n = 206 ) no additional actionable gene identified in cms400 ( n = 307 ) 1 actionable gene identified in cms400 ( n = 8 ) 1 actionable gene in cms400 not in previous panel ( n = 214 ) matched therapy ( n = 40 ) nonmatched therapy ( n = 108 ) no therapy ( n = 66 ) fig a2 . 
summary of the turnaround time ( in days ) for large panel next - generation sequencing testing to be nalized once tissue was received by the clinical molecular diagnostics lab . 
we then used polymerase chain reaction to quantify gene expression in diagnostic biopsy tissue across a prospectively designed archival cohort of 754 consecutive thinand intermediate - thickness primary cutaneous melanomas . 
a penalized maximum likelihood estimation algorithm was used to train logistic regression models in a repeated cross - validation scheme to predict the presence of sln metastasis from molecular , clinical , and histologic variables . results expression of genes with roles in epithelial - to - mesenchymal transition ( glia - derived nexin , growth differentiation factor 15 , integrin - 3 , interleukin 8 , lysyl oxidase homolog 4 , transforming growth factorreceptor type 1 , and tissue - type plasminogen activator ) and melanosome function ( melanoma antigen recognized by t cells 1 ) were associated with sln metastasis . 
per current guidelines ( table 1 ) , slnb is not recommended if the risk of nodal metastasis is , 5% , as in melanoma with a breslow thickness of , 0.8 mm and no adverse features . 
the key objective of this study was to identify primary melanoma clinicopathologic ( cp ) variables and a gene expression prole ( gep ) that associate with a low risk of sln metastasis . knowledge generated cp variables in combination with an eight - gene gep tied to epithelial - to - mesenchymal transition as a biologic process inherent to metastasis effectively stratied melanoma according to its likelihood of sln metastasis . relevance our cp - gep model promises to work as an sln biopsy reduction tool . 
10% risk of shortand long - term complications , including bleeding , infection , lymphocele , lymphatic stula , pain , neuropathy , and lymphedema , 7 as well as an up to 5% risk of hospital readmission within 30 days because of postsurgical complications.8 better methods are needed to identify patients whose risk of nodal metastasis is so low that they may safely forgo slnb . 
here , we report the design of a model that combines established clinicopathologic ( cp ) variables with a gene expression prole ( gep ) to identify patients who have , on average , a risk of nodal metastasis of , 5% . 
of the 754 patients in this cohort , 373 were included in a previously published cohort.10 all specimens were analyzed by quantitative polymerase chain reaction ( pcr ) between february 2018 and october 2018 . eligibility was determined on the basis of histopathology data derived from patient medical records and established by two or more board - certied mayo clinic dermatopathologists . inclusion was determined by the ajcc 7th edition on the basis of institutional practice guidelines of the mayo clinic for recommending slnb , which were based on breslow thickness , ulceration , mitoses , and age . 
 molecular model to assess sentinel lymph node metastasis risk exclusion criteria were m1 disease within 90 days of primary diagnosis ; insufcient primary tumor diagnostic biopsy tissue ; inadequate rna harvested ; and , for minnesota , denial of access to medical records for research purposes ( per minnesota state law )  . 
because there is an ongoing debate about the relevance of , 0.1 mm metastasis in sln ( ie , isolated tumor cells [ itcs ] and cell clusters , 0.1 mm in diameter ) , patients with , 0.1 mm metastasis were excluded from model development . 
isolated benign melanocytes and histiocytic melanophages can be present elsewhere in the sln and mimic itcs.13 some authors cautioned against hidden tumor burden in , 0.1 mm metastatic sln and highlighted the need for enhanced pathology assessment protocols.14 , 15 others found that , 0.1 mm metastasis has no impact on prognosis compared with negative slns.16 , 17 enrollment of patients and inclusion and exclusion criteria are summarized in appendix figure a1 . 
this study was approved by the mayo clinic institutional review board . gene expression by quantitative pcr see the appendix for details . statistical methods logistic regression and least absolute shrinkage and selection operator . 
features with a tolerance 0.15 were removed from the input data set ( the tolerance represents the fraction of variance in the kth feature that cannot be accounted for by other features )  . 
the output of logistic regression models estimated the probability of sln metastasis and was converted into binary results : samples with a probability of metastasis greater than the cutoff were classied as positive , whereas samples with a probability lower than the cutoff were classied as negative . 
the performance metrics of the classiers are listed in appendix table a2 and are cutoff specic , except the area under the receiver operating characteristic curve ( auc )  . double - loop cross - validation . 
however , splitting the available data just once into a training set and a test set may be viewed as inefcient.19 a better solution is to estimate the average performance of the model by repeated cross - validation or bootstrapping . 
here , we opted for a repeated crossvalidation scheme ( ie , double - loop cross - validation [ dlcv ] ) .20 the key idea of dlcv is to get a reliable estimate of the outof - sample performance of a classier by averaging the performance of multiple classiers trained in cross - validation a number of times ( appendix fig a2 )  . 
see the appendix for details . memorial sloan kettering cancer center nomograsee the appendix for details . results epithelial - to - mesenchymal transition in high - risk melanoma to identify candidate genes tied to biologic processes inherent to metastasis and differentially expressed between metastatic and nonmetastatic melanoma , we rst reviewed rna sequencing data obtained previously.10 genes with a false discovery rate of , 0.01 in a comparison of either benign nevi and cutaneous melanoma or cutaneous melanoma with and without sln metastasis were selected for further qualication . 
a total of 194 candidate biomarkers and 3 control genes were screened for performance in breslow thickness and age - matched case - control studies by quantitative pcr ( appendix table a3 )  . 
we noted that genes predictive of nodal metastasis had been associated with epithelial - to - mesenchymal transition ( emt ) , a biologic process known to promote metastasis in primary cutaneous melanoma.21 our prospectively designed archival cohort22 comprised 754 patients with thinand intermediate - thickness primary cutaneous melanoma who underwent an slnb within 90 days of diagnosis ( table 2 )  . 
of 754 patients , 128 ( 17% ) were sln positive , in agreement with the typical prevalence in an slnb - eligible population.3 our approach was to develop models of the likelihood of sln metastasis on the basis of either cp variables ( cp models ) or geps of the primary tumor ( gep models ) and then to assess the performance of a combined model of cp and gep factors ( cpgep models )  . 
 ( % ) characteristic other mixed not documented negative ( n = 626 ) positive ( n = 128 ) 4 of 626 ( 0.6 ) 6 of 626 ( 1.0 ) 31 of 626 ( 5.0 ) 2 of 128 ( 1.6 ) 6 of 128 ( 4.7 ) abbreviations : sd , standard deviation ; slnb , sentinel lymph node biopsy . acomparisons of patients with slnb - negative and - positive outcomes were performed using the 2 test for categorical variables , the twosample t test for patient age , and the wilcoxon rank sum test for all other variables . using gene expression to predict sln metastasis dlcv and lasso were used to identify a gep dened from 11 genes that differentiated the patients with and without nodal metastasis detected by slnb within 90 days of primary diagnosis : adam metallopeptidase domain 12 ( adam12 ) , interleukin 8 ( cxcl8 ) , growth differentiation factor 15 ( gdf15 ) , integrin - 3 ( itgb3 ) , galectin 1 ( lgals1 ) , lysyl oxidase like 4 ( loxl4 ) , melanoma antigen recognized by t cells 1 ( mlana ) , tissue - type plasminogen activator ( plat ) , protein kinase c ( prkcb ) , glia - derived nexin ( serpine2 ) , and transforming growth factor ( tgf ) receptor 1 ( tgfbr1 )  . 
finally , logistic regression modeling was used to develop a novel model combining cp factors ( ie , breslow thickness and patient age ) and a gep . the combined cp - gep model was based on the expression of mlana , a melanosome marker , 23 and seven genes functionally linked to emt and with specic roles in angiogenesis / hypoxia and coagulation : gdf15 , 24 - 26 cxcl8 , 27 , 28 loxl4 , 29 , 30 tgfbr1 , 31 , 32 itgb3 , 33 - 36 plat , 37 , 38 and serpine239 , 40 ( table 3 )  . 
the cp - gep model , therefore , promised to work as an slnb reduction tool : patients with a negative test may forgo slnb because their risk of nodal metastasis is , on average , , 5% , a reduction from the pre - test probability41 ( table 1 )  . for a predictor of sln status to be clinically relevant , it must change the pretest probability within each t category of melanoma . 
the high slnb reduction rate for t1b melanoma is particularly meaningful in light of the increasing incidence of thinner melanoma , 42 for which cp variables are less predictive.5 to further dene the clinical relevance of the cp - gep model , we compared cp - gep performance to the wellknown memorial sloan kettering cancer center ( mskcc ) nomogram for predicting sln metastasis . 
shown are the models that are based on the mskcc nomogram , clinicopathologic variables ( cp model ) , and gene expression prole ( gep ) and cp variables combined ( cp - gep model )  . 
curves are averages over 100 repeats obtained by concatenating the threefold crossvalidation test results . that the mskcc nomogram performed similarly to the cp model but was outperformed by the cp - gep model in auc ( appendix fig a5 ) and slnb reduction rate ( fig 2 )  . discussion while completion lymphadenectomy for which slnb was a key determinant has fallen out of favor , 43 , 44 slnb continues to determine patient eligibility for adjuvant therapy . unfortunately , the majority of slnb procedures performed today are negative , which conrms only the low - risk nature of the primary tumor without inuencing decision making toward adjuvant therapy . 
here , we present a model that considers gene expression and cp variables ( ie , breslow thickness and patient age ) to assess the likelihood of sln metastasis in patients diagnosed with thinand intermediate - thickness primary cutaneous melanoma . 
the ability to characterize melanoma at the molecular level reduces the need for slnb , a surgical procedure that carries a risk of complications.7 our approach of combining cp factors with molecular proling better identies patients who may forgo the slnb procedure because of their low risk of metastasis . for melanoma with a 5% - 10% chance of sln metastasis ( breslow thickness , 0.8 - 1 mm ) , slnb is optional but should be discussed with the patient.45 even though slnb in this risk group is optional ,  . 
50% of affected patients in the united states undergo slnb.46 the majority of these patients have negative slnb ndings , which highlights our current dilemma with melanoma risk stratication and the limitations of histopathology alone as a predictor of regional metastasis . 
multivariable models have used breslow thickness , tumor ulceration , and patient age to predict sln status , with age being a negative predictor and breslow thickness as well as tumor ulceration being strong positive predictors.47 - 50 angiolymphatic invasion was also found to positively correlate with sln metastasis in some models.5 , 51 the most ambitious cp models , such as those developed from a large bi - institutional series , achieved slnb reduction rates of 18% - 30% , with a negative predictive value table 4 . 
in comparison , the cp plus molecular model developed here showed an slnb reduction rate of 42% at a negative predictive value of 96% ( appendix table a4 )  . 
there seems to be a clear limit in the ability of cp factors to predict sln metastasis . to improve the performance of predictive models , we developed a gep from primary diagnostic biopsy tissue . gep has been used successfully in breast cancer to individualize therapy.52 previous research on gene expression in invasive breast cancer , 53 prostate cancer , 54 colon cancer , 55 melanoma , 10 and other solid cancers56 has consistently demonstrated the upregulation of adhesion receptors and secreted factors that remodel the tumor microenvironment and are involved in emt.10 , 53 - 57 here , we have conrmed this upregulation and found genes involved in emt with specic roles in angiogenesis ( growth differentiation factor 15 , 25 interleukin 8 , 28 lysyl oxidase homolog 4 , 58 tgfreceptor type 1 , 32 and integrin 334 ) and coagulation ( tissue - type plasminogen activator , 38 and gliaderived nexin40 ) as well as the melanosome biogenesis marker melanoma antigen recognized by t cells 123 to be associated with sln metastasis ( table 3 )  . 
the functional roles of these genes have been demonstrated by genetic approaches32 , 34 , 38 , 59 and pharmacologic efcacy studies where the inhibition of integrin - 3 by cyclic peptide , 60 tgfreceptor type 1 by kinase inhibitor , 61 and interleukin 8 by neutralizing antibody62 reduced tumor angiogenesis , tumor growth , and metastasis . 
tumor vascularity in melanoma diagnostic biopsy tissue is well known to associate with nodal and distant metastasis but has been difcult quantify in the past.63 likewise , constitutive brinolytic activity in tumor tissue has been described as early as 191164 and attributed largely to plasminogen activators38 and other serine proteases , such as glia - derived nexin , 65 which promote metastasis , 66 disseminated intravascular coagulation , and bleeding in patients with metastatic cancer.67 a drawback of the simultaneous selection of cp variables and genes by our feature selection algorithm is the absence of established variables easily recognizable by clinicians , such as ulceration , in the cp - gep model . 
moreover , we excluded t4 lesions ( ie , melanoma with a breslow thickness , 4 mm ) because the pretest probability of regional metastasis for these patients is very high . 
for example , 21 ( 70% ) of 30 patients with t4 lesions in our cohort presented with regional metastasis , which is well above the recommend threshold for recommending slnb . 
finally , eligibility of patients with t1 melanoma was determined by the mayo clinic institutional practice guidelines for recommending slnb , which select for higher - risk patients , such as those , 40 years of age and with t1b melanoma . 
however , slnb to slnb metastasis risk was only , 5% if cohorts were enriched for a priori low - risk cases , such as by including t1a melanoma or restricting the analysis to patients  . 
previous attempts to develop molecular risk factors have been limited by small cohort sizes , incomplete tnm staging data , or limited clinical utility of the resulting models.49 , 69 , 70 our approach of combining cp factors and gene expression variables improved the performance of cp factors alone by outperforming current clinical practice and important benchmarks , such as the mskcc nomograthe combined cp - gep model maintained an average negative predictive value of  . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . domenico bellomo employment : skylinedx stock and other ownership interests : synlogic , skylinedx patents , royalties , other intellectual property : gene signatures for predicting metastasis of melanoma julia s . 
van vliet employment : skylinedx stock and other ownership interests : skylinedx patents , royalties , other intellectual property : named inventor on patents in the multiple myeloma eld jvalini dwarkasing employment : skylinedx stock and other ownership interests : skylinedx alexander meves research funding : skylinedx patents , royalties , other intellectual property : mayo clinic has led several patent applications on which i am listed as an inventor ; technologies are in the area of wound healing and skin cancer ( inst ) open payments link : 478139 no other potential conicts of interest were reported . acknowledgment we thank vera j . 
simon , and the mayo clinic gene expression core facility for excellent technical assistance . references saranga - perry v , ambe c , zager js , et al : recent developments in the medical and surgical treatment of melanoma . 
ca cancer j clin 64 : 171 - 185 , 2014 chang jm , kosiorek he , dueck ac , et al : stratifying sln incidence in intermediate thickness melanoma patients . 
n engl j med 370 : 599 - 609 , 2014 gershenwald je , scolyer ra , hess kr , et al : melanoma staging : evidence - based changes in the american joint committee on cancer eighth edition cancer staging manual . 
hieken tj , grotz te , comfere ni , et al : the effect of the ajcc 7th edition change in t1 melanoma substaging on national utilization and outcomes of sentinel lymph node biopsy for thin melanoma . 
eur j surg oncol 43 : 270 - 277 , 2017 ascha m , ascha ms , gastman b : identication of risk factors in lymphatic surgeries for melanoma : a national surgical quality improvement program review . 
ann plast surg 79 : 509 - 515 , 2017 oude ophuis cmc , verhoef c , rutkowski p , et al : the interval between primary melanoma excision and sentinel node biopsy is not associated with survival in sentinel node positive patients an eortc melanoma group study . 
fahy as , grotz te , keeney gl , et al : frozen section analysis of slns in trunk and extremity melanoma has a high false negative rate but can spare some patients 13 . 
mahmood mn , lee mw , linden md , et al : diagnostic value of hmb - 45 and anti - melan a staining of sentinel lymph nodes with isolated positive cells . 
murali r , desilva c , mccarthy sw , et al : sentinel lymph nodes containing very small ( , 0.1 mm ) deposits of metastatic melanoma cannot be safely regarded as 33 : 2509 - 2515 , 2015 121 : 1628 - 1636 , 2015 a second operation . 
ann surg oncol 15 : 1547 - 1548 , 2008 van akkooi ac , de wilt jh , verhoef c , et al : clinical relevance of melanoma micrometastases ( , 0.1 mm ) in sentinel nodes : are these nodes to be considered negative ? ann oncol 17 : 1578 - 1585 , 2006 18 . 
alonso sr , tracey l , ortiz p , et al : a high - throughput study in melanoma identies epithelial - mesenchymal transition as a major determinant of metastasis . epidemiol 54 : 774 - 781 , 2001 21 : 3755 - 3762 , 2005 cancer res 67 : 3450 - 3460 , 2007 22 . 
subramanian j , simon r : what should physicians look for in evaluating prognostic gene - expression signatures ? nat rev clin oncol 7 : 327 - 334 , 2010 23 . 
hoashi t , watabe h , muller j , et al : mart - 1 is required for the function of the melanosomal matrix protein pmel17 / gp100 and the maturation of melanosomes . 
mullican se , lin - schmidt x , chin cn , et al : gfral is the receptor for gdf15 and the ligand promotes weight loss in mice and nonhuman primates . 
fernando ri , castillo md , litzinger m , et al : il - 8 signaling plays a critical role in the epithelial - mesenchymal transition of human carcinoma cells . 
yang j , shultz rw , mars wm , et al : disruption of tissue - type plasminogen activator gene in mice reduces renal interstitial brosis in obstructive nephropathy . j clin invest 110 : 1525 - 1538 , 2002 38 . 
mocellin s , thompson jf , pasquali s , et al : sentinel node status prediction by four statistical models : results from a large bi - institutional series ( n = 1132 )  . 
wong sl , faries mb , kennedy eb , et al : sentinel lymph node biopsy and management of regional lymph nodes in melanoma : american society of clinical oncology and society of surgical oncology clinical practice guideline update . 
hayek sa , munoz a , dove jt , et al : hospital - based study of compliance with nccn guidelines and predictive factors of sentinel lymph node biopsy in the setting of thin melanoma using the national cancer database . 
kather jn , charoentong p , zoernig i , et al : prognostic value of histopathological tumor - stroma ratio and a stromal gene expression signature in human solid tumors . 
nat rev cancer 18 : 533 - 548 , 2018 [ erratum : nat rev cancer 19 : 179 , 2019 ] jiang wp , sima zh , wang hc , et al : identication of the involvement of loxl4 in generation of keratocystic odontogenic tumors by rna - seq analysis . 
zhang m , kleber s , r ohrich m , et al : blockade of tgfsignaling by the tgfr - i kinase inhibitor ly2109761 enhances radiation response and prolongs clin cancer res 11 : 6270 - 6279 , 2005 survival in glioblastoma . 
huang s , mills l , mian b , et al : fully humanized neutralizing antibodies to interleukin - 8 ( abx - il8 ) inhibit angiogenesis , tumor growth , and metastasis of human 63 . 
boulaftali y , ho - tin - noe b , pena a , et al : platelet protease nexin - 1 , a serpin that strongly inuences brinolysis and thrombolysis . 
 molecular model to assess sentinel lymph node metastasis risk appendix methods quantitative polymerase chain reaction ( pcr ) was performed as previously described.10 rna purication was from formalin - xed parafn - embedded tissue ( qiagen , hilden , germany )  . 
gene expression was corrected by the mean of housekeeping genes ( rlp0 , rlp8 , and - actin ) using the ct method . statistical methods double - loop cross - validation . 
in the inner loop ( 10 - fold cross - validation ) , we optimized the parameter by determining the number of features ( ie , the weight of the least absolute shrinkage and selection operator penalty term ) , and in the outer loop ( threefold cross - validation ) , we designed a classier on the training set ( two of the three folds )  . 
next , we assessed the performance of the trained classier on the remaining fold ( test set ) , with the parameter xed to the value estimated in the training set . 
the nal classiers were trained on the entire data set using the average ? parameter over 300 runs . memorial sloan kettering cancer center nomograthe majority of online tools for melanoma provide prognostic information47 ( zabor et al : ann surg oncol 25 : 2172 - 2177 , 2018 )  . 
the memorial sloan kettering cancer center ( mskcc ) nomogram , in contrast , is a tool specically designed to predict the probability of primary cutaneous melanoma metastasis to sln.23 the nomogram corresponds to a logistic regression model that is based on ve clinicopathologic variables : age ( range , 20 - 95 years ) , breslow thickness ( range , 0.1 - 10 mm ) , clark level ( ii , iii , iv , or v ) , biopsy location ( trunk , extremity , or head and neck ) , and tumor ulceration ( yes or no )  . 
while attempting to apply the nomogram to our cohort , we could not calculate the probability of sln metastasis for 16 patients because of missing values or because the values were outside the allowable range for the nomograsix patients were , 20 years of age , seven had a missing clark level ( one of whom was also , 20 years of age ) , and four did not have ulceration status available . 
study ow diagrathe gure depicts the enrollment of patients , inclusion and exclusion criteria , and other potential sources of retrospective bias such as the absence of research consent , the unavailability of tissue for molecular analysis , or failed gene expression proling ( gep )  . 
 molecular model to assess sentinel lymph node metastasis risk x 10 cvs x 100 repeats x 3 cvs train train test train test train average test performance test train train select optimal for lasso penalty fig a2 . 
the dlcv consists of two nested cross - validation loops : in the inner loop ( tenfold cross - validation ) , we estimate the optimal parameter , namely , the weight of the lasso penalty term ( ie , optimal feature selection ) ; in the outer loop ( threefold cross - validation ) , we assess the performance of the classier on each test set , with the parameter as determined in the training set . 
moreover , in each training set of the outer loop , we choose and x an operating point on the receiver operating characteristic curve , and we assess the performance of the classier at that operating point in the corresponding test set . 
the cross - validation procedure has been repeated 100 times , and unless otherwise stated , we reported the average performance over 300 test sets ( three test sets per outer loop , repeated 100 times )  . 
lasso , least absolute shrinkage and selection operator . cp1 ; auc , 0.78 cp2 ; auc , 0.78 cp3 ; auc , 0.78 cp4 ; auc , 0.77 cp5 ; auc , 0.77 cp6 ; auc , 0.77 false - positive rate fig a3 . 
 bellomo et al cp model ; auc , 0.78 gep model ; auc , 0.78 combined cp - gep model ; auc , 0.82 false - positive rate fig a4 . 
blue line , model for predicting sentinel based on cp variables ( cp model ) ; red line , model based on gene expression prole ( gep model ) ; teal line , model based on combined gep and cp variables ( cp - gep model )  . 
curves are averages over 300 double loop crossvalidationgenerated test sets . mskcc nomogram ; auc , 0.77 cp model ; auc , 0.78 combined cp - gep model ; auc , 0.82 false - positive rate fig a5 . 
orange line , model based on mskcc nomogram ; blue line , model based on cp variables ( cp model ) ; teal line , model based on gene expression and cp variables combined ( cp - gep model )  . 
clinicopathologic variables available for statistical modeling clinicopathologic variable age categorical biopsy location biopsy location mskcc breslow thickness breslow thickness categorical mitotic rate categorical clark level ulceration regression tumor - inltrating lymphocytes microsatellitosis angiolymphatic invasion histology type abbreviation : mskcc , memorial sloan kettering cancer center . head and neck , trunk , upper extremity , lower extremity , acral value female , male years 15 - 39 years , 40 - 59 years , 60 years head and neck , trunk , extremity millimeters , 1 mm , 1 - 2 mm , 2 - 4 mm 0 / mm2 , 1 - 6 / mm2 ,  . 
6 / mm2 , not determined ii , iii , iv , v yes , no , not determined yes , no , not determined absent , nonbrisk , brisk , not determined yes , no , not determined yes , no , not determined supercial spreading , nodular , desmoplastic , lentigo maligna , acral lentiginous , spindled , dermal , spitzoid , nevoid , unclassied , other , mixed , not determined table a2 . 
average performance of the cp , gep , and combined cp - gep models were trained in double - loop cross - validation 300 times . performance is shown for the 754 - patient cohort assembled on the basis of inclusion criteria and devoid of patients with equivocal sln metastasis ( ie , patients with , 0.1 mm metastatic disease )  . 
 year 1 in the molecular era of pediatric brain tumor diagnosis : application of universal clinical targeted sequencing in an unselected cohort of children purpose next - generation sequencing is gaining acceptance as a clinical tool to aid diagnosis and guide treatment of pediatric cancer . 
here , we report an unselected prospective cohort study to evaluate the clinical use of universal targeted sequencing in pediatric patients with brain tumors . methods we applied a universal sequencing protocol for all tumors of the cns undergoing diagnostic workup at seattle childrens hospital during the study period of november 2015 to november 2016 . 
all tumors were sequenced using the uw - oncoplex platform , which is a multiplexed targeted deep gene sequencing panel that detects genetic alterations in 262 cancer - related genes performed in a college of american pathologists accredited clinical laboratory improvements amendmentscertified laboratory . results eighty - eight patients underwent diagnostic evaluation during the study period , of which 85 tumors ( 95% ) yielded sufficient dna for sequencing , including 59 newly diagnosed and 26 relapsed . 
of these , 57 ( 67% ) had disease - defining or disease - modifying mutations , 44 ( 52% ) had potentially targetable mutations , and 31 ( 36% ) had mutations requiring germline follow - up . 
2018 by american society of clinical oncology introduction high - throughput genomic and epigenomic technologies have recently been used to thoroughly describe the genomic landscape of pediatric brain tumors.1 - 3 this work has transformed our understanding of the molecular basis of these diseases , 4 which are the leading cause of pediatric cancer death.5 the current challenge for the field of pediatric neuro - oncology is to translate this knowledge into clinical practice . the 2016 revision of the who classification of cns tumors has begun to incorporate molecular findings into tumor diagnosis in addition to traditional histopathology.6 however , there is currently no consensus regarding best - practice diagnostics for routine clinical practice . 
recently , a few institutions have reported the use of clinically validated next generation sequencing ( ngs ) panels to identify molecular alterations in selected subsets of pediatric patients with brain tumors.7 - 10 these studies demonstrated that molecular profiling is bonnie l . 
atrt , atypical teratoid rhabdoid tumor ; dnet , dysembryoplastic neuroepithelial tumor ; etmr , embryonal tumor with multilayer rosettes ; gg , ganglioglioma ; nos , not otherwise specified ; pxa , pleomorphic xanthoastrocytoma . feasible and that clinically relevant genetic alterations may be identified . methods instability the uw - oncoplex version 5 test is a multiplex gene sequencing panel used to detect alterations in 262 cancer - related genes , including single nucleotide variants , small insertions or deletions , amplifications , selected translocations , and microsatellite ( washington.edu / tests / genetics / uw - oncoplex )  . 
in a retrospective pilot study using this test , sequencing identified a large proportion of targetable mutations in high - grade supratentorial tumors.11 we hypothesized that the use of a selected or convenience cohort in our retrospective pilot as well as all prior studies may have identified a higher proportion of actionable findings that would not necessarily be applicable to the wider population of all patients undergoing pathologic diagnosis of pediatric brain tumor.7 - 12 we designed this follow - up , prospective , unselected cohort study to evaluate the feasibility and impact of the universal application of molecular tumor testing on routine pediatric brain tumor diagnosis and to also assess how molecular results may alter medical management . all tumors of the cns undergoing diagnostic evaluation at seattle childrens hospital from november 2015 to november 2016 were included in this study . 
details of the laboratory workflow and methods are included in the data supplement . results demographics and sample characteristics we identified 88 patients who met the clinical and diagnostic inclusion criteria ( fig 1 )  . 
 of these , 97% ( 85 of 88 ) had sufficient dna obtained for sequencing , and only three patients did not have adequate tissue in formalin - fixed paraffin - embedded ( ffpe ) blocks to perform ngs . 
of patients ( % ) * targeted agent braf inhibitor mek inhibitor fgfr inhibitor braf v600e , brafpa598t599insv ganglioglioma , pleomorphic xanthoastrocytoma , pilocytic astrocytoma , desmoplastic infantile ganglioglioma / astrocytoma kiaa1549 - braf fusion , nf1 mutations and deletions , ptpn11 mutation pilocytic astrocytoma , anaplastic astrocytoma , pleomorphic xanthoastocytoma fgfr1 mutations and duplication , fgfr3 fusions pilocytic astrocytoma , dysembryoplastic neuroepithelial tumor , diffuse astrocytoma , ganglioglioma mtor pathway inhibitor mtor , fbxw7 diffuse midline glioma , medulloblastoma smo inhibitor ptch1 mutations medulloblastoma pd1 / pdl1 inhibitor biallelic msh2 loss , pms2 mutation with loh anaplastic astrocytoma , medulloblastoma pdgfra inhibitor pdgfra amplification diffuse midline glioma met inhibitor kit inhibitor met amplification kit amplification diffuse midline glioma diffuse midline glioma cdk4 / 6 inhibitor cdk4 amplification glioblastoma ezh2 inhibitor loss of smarcb1 atypical teratoid rhabdoid tumor * ten patients had more than one targetable mutation in their tumors . 10 ( 12 ) 18 ( 21 ) 10 ( 12 ) 3 ( 4 ) 3 ( 4 ) 2 ( 2 ) 2 ( 2 ) 2 ( 2 ) 2 ( 2 ) 1 ( 1 ) 1 ( 1 ) were sequenced , including 59 newly diagnosed tumors and 15 tumors from patients with a radiologic relapse during the time of this study . 
 eleven additional patients had sequencing of newly resected recurrent tumors . patients were a median age of 10 years ( range , 3 months to 22 years ) and 49% female ( 42 of 85 )  . 
medical history included only three patients with previously known cancer predisposition syndrome ; two patients had neurofibromatosis type 1 and one had neurofibromatosis type 2 . genetic results the average depth of sequencing coverage was 441 ( range , 102 to 1 , 112 )  . 
five reports with turnaround times of > 60 days were from patients with minimal tissue that required cutting additional ffpe sections with subsequent repeat dna extraction . genetic result details are shown in data supplement 1 , and criteria for tiers of alterations are listed in appendix table a1 . 
genetic alterations were found to be disease defining or modifying in 67% ( n = 57 ) , targetable with an available therapeutic drug in 52% ( n = 44 ; table 1 ) , and potentially germline associated with cancer predisposition in 36% ( n = 31 )  . 
as of the last follow - up , seven patients ( 8% of total , 16% of those with an identified molecular target ) had been prescribed targeted agents ; molecularly targeted agents were obtained through insurance - approved off - label use in five patients and through enrollment in a clinical trial in two patients . analysis of tumor subtypes tumors were analyzed separately as high - grade glioma ( hgg ) , low - grade glioma ( lgg ) , medulloblastoma , and other rare pediatric cns tumors . 
 medulloblastoma ngs results were correlated with traditional histology , immunohistochemistry , and fluorescence in situ hybridization studies.6 , 13 all 20 medulloblastomas were able to be classified into subgroups as defined by the who , 6 , 14 including a wingless ( wnt ; n = 1 ) , sonic hedgehog ( shh ; n = 7 ) , or non - wnt / non - shh group ( n = 12 ; fig 3 )  . 
for example , one patient with a wnt pathway medulloblastoma had only weak nuclear beta catenin staining ; in this patient , the finding of a ctnnb1 mutation and copy loss consistent with monosomy 6 by ngs helped place this tumor firmly in the wnt - activated subgroup . 
in another instance , an shh group medulloblastoma that was gab1 immunopositive displayed ambiguous p53 immunohistochemical staining of approximately 40% of tumor cell nuclei ; ngs not only confirmed the shh subgroup by finding sufu mutations , but also confirmed that this tumor contained a tp53 mutation . 
as of the last follow - up , two patients in the relapsed shh group with ptch1 mutations had been prescribed smo inhibitors , one of whom experienced a partial response to treatment . forty - four lggs were analyzed ( fig 4 )  . 
 a , high - grade astrocytoma ; aa , anaplastic astrocytoma ; apa , anaplastic pilocytic astrocytoma ; dipg , diffuse intrinsic pontine glioma ; gbm , glioblastoma ; hgg , high - grade glioma , not otherwise specified . pik3ca pten smarcb1 msh2 idh1 pdgfra h3f3a atrx cdk6 cdkn2a tp53 ptpn11 v600 mutations and 10 braf - kiaa translocations . 
 twenty - nine patients with lgg had targetable mutations , and at the last follow - up , three patients had been prescribed targeted therapy with a braf inhibitor for braf v600e mutation , two at the time of recurrence and one as initial therapy for unresectable brainstem tumor . 
 three patients with lgg were confirmed to have germline mutations , including the two known patients with nf1 and one patient with a recurrent pleomorphic xanthoastrocytoma who was confirmed to have a germline tp53 mutation in addition to a braf v600e mutation in the tumor ; the patient was treated with a braf inhibitor instead of radiation for recurrent tumor and continues without disease progression with > 12 months of receiving targeted therapy . 
molecular profiling did not alter treatment of the 17 patients in the lgg group with completely resected primary tumors . eleven other uncommon tumors were evaluated ( fig 5 ) , including six nonmedulloblastoma embryonal tumors , choroid plexus carcinoma , choroid plexus papilloma , vestibular schwannoma , rhabdoid meningioma , and chordoma . 
one patient had a known nf2 mutation , and the other two patients tested negative for smarcb1 ( atypical teratoid rhabdoid tumor ) and tp53 ( choroid plexus carcinoma )  . analysis of primary versus recurrent tumors considering newly diagnosed patients , 85% ( n = 50 of 59 ) had clinically relevant mutations , including disease - defining or disease - modifying mutations in 76% of tumors ( n = 45 ) , targetable mutations in 46% of tumors ( n = 27 ) , and potentially germline mutations in 41% of tumors ( n = 24 )  . of the 15 primary tumors sequenced at the time of radiologic relapse , 10 ( 67% ) were found to have clinically significant molecular alterations considered disease defining , and nine ( 60% ) identified molecular targets for therapy . 
 of 11 tumors sequenced using newly resected tumor obtained at the time of recurrence , clinically significant alterations were identified in eight ( 73% )  . discussion germline sequencing germline dna was not collected at the time of somatic sequencing in this study . 
however , somatic sequencing identified mutations considered to be potentially germline in 31 of 85 of tumor samples ( 36% ) on the basis of variant interpretation , which led to initiation of clinical follow - up and genetic counseling to recommend germline evaluation . 
in this population , germline sequencing has been performed on 13 patients , and germline mutations were confirmed in nine patients ( 75% of those tested ; 10.6% of the study population ) as of the last follow - up . 
 one patient had ctnnb1 mutation with monosomy of chromosome six diagnostic of wingless ( wnt ) subgroup , seven had either ptch1 or sufu mutations characteristic of sonic hedgehog ( shh ) subgroup , and the other 12 were non - shh / nonwnt group ; three of our seven shh group tumors were tp53 mutant . 
in the group of newly diagnosed patients , alterations were identified in 85% of patients ( n = 50 of 59 ) , a finding that was contrary to our hypothesis that selected cohorts previously reported by our group and others may have been limited by a selection bias increasing the likelihood of clinically relevant findings.7 , 8 , 11 , 12 targetable mutations were identified in 44 tumors ( 52% ) , and seven of these patients ( 8% ) have been prescribed targeted therapeutic agents to date . 
this number likely underestimates the longer - term use of targeted therapy because the study has a relatively short follow - up period and there are effective standard treatment regimens of cytotoxic therapy for most diagnoses . 
if successful , the routine use of targeted agents will greatly increase in patients with pediatric brain tumors . the main aims of cancer genomic profiling tests are to identify somatic mutations . 
it is crucial to include genetic counseling in oncology care , ideally with a geneticist or genetic counselor embedded into the pediatric oncology clinic.15 in this study , we identified 31 patients ( 36% ) with potential germline mutations . 
 be difficult to overstate in a patient for whom radiation therapy is being considered , because the long - term second malignancy rate in these patients who undergo therapeutic irradiation approaches 100%.16 molecular profiling is important for the diagnosis and management of pediatric patients with brain tumors , 6 although at present , there are several competing platforms and currently no diagnostic gold standard . 
whatever platform is used must be clinically validated and should balance cost , turnaround time , and ability to identify clinically relevant mutations in small samples , which is no small task . 
 alternatively , many research studies have argued the advantages of potentially subclassifying pediatric brain tumors primarily by rna - based gene expression17 or methylation analyses.18 however , these types of platforms are difficult to establish fig 4 . 
the majority of low - grade gliomas in our series had dysregulated braf signaling , which included 10 ( 26% ) with braf fusions and 10 ( 26% ) with codon 600 mutations ; these alterations were considered both targetable and either disease defining or modifying . 
ag , angiocentric glioma ; da , diffuse astrocytoma ; dig / dia , desmoplastic infantile ganglioglioma or desmoplastic infantile astrocytoma ; dnet , dysembryoplastic neuroepithelial tumor ; ep , ependymoma ; gg , ganglioglioma ; lgg , low - grade glioma , not otherwise specified ; o , oligodendroglioma ; pa , pilocytic astrocytoma ; pma , pilomyxoid astrocytoma ; pxa , pleomorphic xanthoastrocytoma . atrx cdk6 cdkn2a tp53 ptpn11 fgfr3 fgfr1 braf with the degree of reliability that clinical testing requires for use on a single - patient basis , and even if molecular classification was established , expression or methylation analysis might not identify candidates for targeted therapy . 
as an example , although shh pathway medulloblastoma may be reliably identified with immunohistochemistry , 13 nanostring , 17 or methylation profiling , 18 none of these platforms is specific for the site of mutation , which is required for consideration of therapy with a smoothened inhibitor that would be predicted to be effective in ptch1 mutant tumors but unlikely to be effective in the case of downstream alterations , such as gli1 amplification . 
 similarly , braf inhibitor therapy is indicated in braf v600 mutant tumors but contraindicated in braf fusion.19 the detection of fusions such as braf using a dna - based platform such as uw - oncoplex requires the specific inclusion of intronic regions in sequencing . 
alternatively , rna - based sequencing analysis may have an advantage in identifying fusions and splice variants that would be missed on a dnabased platform that does not include intronic sequencing . although 80% of our patients had clinically relevant mutations identified , this means that 20% of tumors sequenced did not have clinically relevant mutations . 
one limitation of this study and other similar studies is that not all known clinically relevant genetic alterations are yet able to be identified on any one clinically validated panel . 
for example , the angiocentric glioma would be expected to have an myb fusion , 20 embryonal tumor with multilayer rosettes would be expected to have mir c19 cluster amplification , 21 and a subset of midline gliomas would be expected to have acvr1 alterations , which are not yet included in this version of the test.22 one advantage of using a targeted sequencing panel is the ability to add and validate targets rapidly with minimal increase in cost . 
however , turnaround time can be shortened with experience , because it is most dependent on clinical interpretation and data generation may be relatively short . another question that our study did not address was the cost comparison between sequential single - gene sequencing in specific diagnostic entities . 
although single - gene testing for the most common variations costs less than a panel , it is not clear that the overall cost of sequential sequencing would be decreased for the population . 
if even a small minority of patients required three - gene testing or subsequent biopsy if tissue were depleted , then there may be a benefit to the population from an initial multiplexed approach . molecular profiling may be particularly useful in patients in whom the histologic diagnosis is unclear . 
 mutations in smarcb1 ( p.p374rfs * 100 ) , bap1 ( p.q392 * ) , pten ( p.g36e ) , nf2 ( p.q319 * ) , and tp53 ( p.r175h ) were all considered both disease defining and potentially germline . 
because only two of our h3f3a k27m mutant diffuse midline gliomas were classic radiologic diffuse intrinsic pontine gliomas , it is important to evaluate for this diagnostic entity whenever a diffuse midline glial neoplasm is encountered . this study provides rigorous evidence regarding the use of routine molecular profiling of an unselected cohort of pediatric brain tumors . 
 on the basis of the analysis presented here , we recommend incorporation of routine molecular profiling into standard clinical practice for all newly diagnosed pediatric hggs , medulloblastomas , and incompletely resected lggs . 
this may be especially important in patients who have undergone stereotactic biopsy in which tumor tissue is severely limited . there was one subgroup in which sequencing did not affect medical care . 
lockwood travel , accommodations , expenses : cambridge healthtech institute shannon stasi no relationship to disclose jeffrey stevens stock and other ownership interests : seattle genetics amy lee no relationship to disclose jeffrey g . 
leary no relationship to disclose acknowledgment we extend our deepest thanks to michael astion , md , medical director of seattle childrens hospital department of laboratories , for his tireless support of this project . 
kool m , korshunov a , remke m , et al : molecular subgroups of medulloblastoma : an international meta - analysis of transcriptome , genetic aberrations , and clinical data of wnt , shh , group 3 , and group 4 medulloblastomas . 
kline cn , joseph nm , grenert jp , et al : targeted next - generation sequencing of pediatric neuro - oncology patients improves diagnosis , identifies pathogenic germline mutations , and directs targeted therapy . 
ramkissoon sh , bandopadhayay p , hwang j , et al : clinical targeted exome - based sequencing in combination with genome - wide copy number profiling : precision medicine analysis of 203 pediatric brain tumors . 
bandopadhayay p , ramkissoon la , jain p , et al : myb - qki rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanisnat genet 48 : 273 - 282 , 2016 21 . 
patients with medulloblastoma were subgrouped on the basis of both histology and standard immunohistochemistry methods for beta - catenin ( dako 1 : 200 ) , gab1 ( santa cruz 1 : 100 ) , and p53 ( protein tech 1 : 50 ) .13 the study pathologist estimated the percentage of tumor and selected appropriate formalin - fixed paraffin - embedded ( ffpe ) blocks for testing . 
it was collected at a separate encounter only if a possible deleterious germline mutation was detected that needed to be investigated further . template preparation , gene capture , massively parallel sequencing , and bioinformatics . sequencing was preformed using a clinically validated method performed in a college of american pathologistsaccredited clinical laboratory improvements amendmentscertified laboratory as previously reported ( pritchard et al , j mol diagn 16 : 56 - 67 , 2014 )  . 
sequencing libraries were prepared from dna samples and hybridized to a custom set of complementary rna biotinylated oligonucleotides targeting the exons of 262 genes and select intronic regions , for a total of > 1.4 mb of targeted dna sequenced ( agilent sureselect ; agilent technologies , santa clara , ca )  . 
uw - oncoplex assay version 5 is a targeted , massively parallel gene sequencing assay that detects mutations in 262 cancer - related genes ( washington.edu / uw - oncoplex )  . 
the test uses next - generation deep sequencing to detect mutations , including single nucleotide variants ( snvs ) , insertions and deletions ( indels ) , copy number changes including gene amplifications , selected structural rearrangements such as gene fusions , and microsatellite instability . 
uw - oncoplex was performed on samples from the seattle childrens hospital cohort , as previously described ( pritchard et al , j mol diagn 16 : 56 - 67 , 2014 ; salipante et al , clin chem 60 : 1192 - 1199 , 2014 )  . dna was extracted from ffpe solid tumor tissue samples using the gentra puregene dna isolation kit ( catalog #158489 ; qiagen , valencia , ca )  . 
hematoxylin and eosinstained slides were reviewed before dna extraction for all ffpe samples , and when feasible , macrodissection of tumor - containing regions was performed to enrich tumor cellularity . 
sequencing libraries were constructed from a minimum of 200 ng of dna using kapa hyper prep kits ( kapa biosystems , wilmington , ma ) , and hybridization was performed with custom agilent sureselect probes ( agilent technologies , santa clara , ca )  . 
dna sequencing was performed on a hiseq2500 sequencing system ( illumina , san diego , ca ) with 2 101bp , paired - end reads , and on a nextsequation 500 ( illumina ) with 2 150bp , paired - end reads according to the manufacturers instructions . 
for copy number variation ( cnv ) analysis , copy number states for individual probes were initially called using contra version 2.0.5 ( files ) with reference to a cnv control comprising reads from two independent rounds of library preparation and sequencing of the hapmap individual na12878 . 
adjacent exons were merged into larger segments if the read depths of their component baits were not significantly different ( p > .001 ) by students t test , and read - depth statistics were recalculated for the larger segments . 
filters are applied to exclude variants extremely unlikely to be clinically significant , including variants in intergenic dna , and variants are removed that are present at > 1% frequency in general population databases ( as reported by the exome variant server , 1000 genomes , and the exome aggregation consortium )  . 
variants of interest are subsequently evaluated in conjunction with a combination of multiple external databases ( cosmic , iarc , insight , clinvar ) , literature review ( for newly described mutations ) , and algorithms ( splice prediction tools )  . three separate expert molecular oncology reviewers independently cull data to a list of potentially reportable variants and review quality of data for variant calls by examining genotype quality score metrics , evaluating base call quality , assessing mapping quality , and evaluating other quality assessments as indicated for specific alterations . 
 once the molecular oncology experts agree on a final list of reportable variants , one laboratory director composes a fully customized , formal report that is available in the electronic health record . 
the shh subgroup was initially identified by mutations ( snvs , indels , structural alterations , and copy number alterations ) in ptch1 , smo , sufu , and copy loss for genes on the long arm of chromosome 10 . 
each month , all new patients were systematically reviewed in a presentation format consisting of clinical features and magnetic resonance imaging presented by a neuro - oncologist ( s.e.s.l. ) , histologic findings and photomicrographs by a pediatric pathologist ( b.l.c. ) , molecular results by a molecular pathologist ( c.m.l. ) followed by group discussion . 
the molecular tumor board welcomed rotating trainees from all disciplines and included attendance of trainees from oncology , neurosurgery , pathology , laboratory medicine , medical genetics , and medical students . 
when applicable , a new final integrated diagnosis was reported in the pathology addendum . genetic alterations were considered to be clinically significant as defined by meeting criteria for at least one of the following three categories : ( 1 ) alterations that define a diagnosis or molecular subtype , for example , an activating beta - catenin mutation in wnt - subtype medulloblastoma ; ( 2 ) alterations that identify a proven therapeutic target , such as braf v600e ; and / or ( 3 ) alterations that identify possible deleterious germline mutation associated with cancer predisposition syndrome , such as tp53 . 
with consideration of the variant allele fraction as well as previously described genes associated with cancer predisposition , additional constitutional follow - up testing was recommended for the patients with potential germline mutations , which was coordinated by a genetic counselor . 
 o personalized clinical decision making through implementation of a molecular tumor board : a german single - center experience rouven hoefflin anna - lena geiler ralph fritsch rainer claus julius wehrle patrick metzger meike reiser leman mehmed lisa fauth dieter henrik heiland thalia erbes friedrich stock agnes csanadi cornelius miething britta weddeling frank meiss dagmar von bubnoff christine dierks isabell ge volker brass steffen heeg henning schfer martin boeker justyna rawluk gian kayser simone hettmer hauke busch christoph peters martin werner justus duyster tilman brummer melanie boerries ( continued ) purpose dramatic advances in our understanding of the molecular pathophysiology of cancer , along with a rapidly expanding portfolio of molecular targeted drugs , have led to a paradigm shift toward personalized , biomarker - driven cancer treatment . 
here , we report the 2 - year experience of the comprehensive cancer center freiburg molecular tumor board ( mtb ) , one of the first interdisciplinary molecular tumor conferences established in europe . 
the role of the mtb is to recommend personalized therapy for patients with cancer beyond standard - of - care treatment . methods this retrospective case series includes 198 patients discussed from march 2015 through february 2017 . 
the mtb guided individual molecular diagnostics , assessed evidence of actionability of molecular alterations , and provided therapy recommendations , including approved and off - label treatments as well as available matched clinical trials . results the majority of patients had metastatic solid tumors ( 73.7% ) , mostly progressive ( 77.3% ) after a mean of 2.0 lines of standard treatment . 
 treatment recommendations were implemented in 33 of 104 patients ( 31.7% ) , of whom 19 ( 57.6% ) showed stable disease or partial response , including 14 patients ( 7.1% of the entire population ) receiving off - label treatments . conclusion personalized extended molecular - guided patient care is effective for a small but clinically meaningful proportion of patients in challenging clinical situations . 
2018 by american society of clinical oncology licensed under the creative commons attribution 4.0 license personalized cancer medicine uses molecular biomarkers for standard - of - care treatment stratification , such as activating braf mutations for the treatment of melanoma with braf inhibitors.1 in parallel , it has become evident that therapeutic strategies with targeted drugs are no longer specific for the treatment of distinct entities but rather for particular molecular profiles across different cancers.2 - 4 thus , testing for single - drug targets can provide therapeutic information , but its predictive value may vary between entities . 
although an activating braf v600e mutation will predict response to braf inhibitors in melanoma , 1 it may not do so in colorectal cancers because of epidermal growth factor receptor ( egfr ) feedback activation with requirement of additional egfr targeting.5 , 6 moreover , non - v600 braf mutations might not be responsive to braf inhibition at all.7 thus , one - mutationone - drug approaches may be ineffective , especially in heavily pretreated patients with cancer . 
examples include the selection of ras mutant clones in colorectal cancer treated with egfr antibodies , such as cetuximab or panitumumab , 11 or the acquisition of a secondary egfr t790m kinase domain mutation mediating resistance to egfr kinase inhibitors , such as gefitinib or erlotinib in non small - cell lung cancer.12 , 13 this increasing amount of complexity requires tools to translate individual information into personalized treatment concepts . 
the initial discussion includes a clinical case presentation , review of the pathology data and the tumor - specific genetic landscape , known molecular predictive or prognostic markers , active clinical trials , and potential inand off - label molecular targeted treatments . 
 diagnostic results are presented to the mtb by the molecular pathology and / or the computational biologist teaafter discussion , treatment recommendations are given and are supported by levels of evidence ( data supplement )  . 
these are based on published molecular biomarker recommendations.14 patients and patient informed consent all patients discussed ( n = 198 ) were included in this retrospective single - center case series . 
patients with individual or family history indicative of germline disease - causing mutations were referred to the institute of human genetics for counseling and possibly germline genetic analyses . diagnostic molecular pathology appropriate tissues were subjected to molecular analyses as recommended by the mtb ( fig 1 )  . 
 a molecular diagnostic tests 36 16 immunohistochemistry per case 15 6 ddseq ( exon / hotspot ) hpv testing tngs rna - seq ( total = 867 ) ( total = 492 ) 7 - 8 antibodies ( total = 139 ) type of tngs 48 - gene panel 8 - gene panel brca1 / brca2 54 - gene myeloid panel 1 antibody 2 antibodies 3 antibodies 4 antibodies 5 antibodies 6 antibodies not annotated cosmic ( no hotspot ) hotspot ( not actionable ) actionable ( sensitizing ) actionable ( resistence ) tumor entities brain colorectal melanoma thyroid other gastrointestinal fig 1 . 
 ( a ) the panels depict the type of molecular diagnostic testing performed ( left panel ) and specify the number of immunohistochemical stains ( one to eight antibodies ) per case ( middle panel ) as well as the type of targeted next - generation sequencing ( tngs ) library sequenced ( right panel )  . 
tngs was performed either by a custom panel ( eight - gene panel ) , a 48 - gene panel ( truseq amplicon cancer panel , illumina , san diego , ca ) , a 54 - gene myeloid panel ( trusight myeloid sequencing panel , illumina ) or a custom brca1 / 2 consortium panel . 
differentially expressed genes were identified using the limma voom package from r / bioconductor.34 , 35 results from march 2015 through february 2017 , 49 mtb meetings were attended by a median of 16 physicians and scientists , ensuring continuous interdisciplinary data interpretation and discussions with diagnostic and therapeutic decision making . 
the workflow of the mtb included a case and literature review , molecular diagnostic recommendations , and follow - up discussions of the molecular diagnostic results , including treatment recommendations ( appendix fig a1 )  . 
these included 305 diagnostic and 104 treatment recommendations . patient characteristics the average patient age at the time of the initial mtb presentation was 58 years ( range , 1 to 85 years )  . 
the majority of patients ( n = 146 ; 73.7% ) suffered from metastatic disease , and 77.3% ( n = 153 ) showed disease progression while receiving the standard treatment ( table 2 )  . 
of all diagnostic recommendations , 234 ( 76.7% ) were implemented , resulting in 867 single diagnostic tests ( mean , five per patient ) , including 815 routine molecular tests and 52 extended genetic analyses ( fig 1a , left panel )  . routine molecular diagnostics included immunohistochemical ( ihc ) staining for biomarkers ( n = 492 ; fig 1a , middle panel ) , such as programmed death - ligand 1 ( pd - l1 ) and mismatch repair proteins , in situ hybridizations ( ish ) for gene copy number analyses ( n = 92 ) , and testing for microsatellite instability and / or gene hotspot variations ( n = 89 ) and tngs ( n = 139 ; fig 1a )  . 
the most frequent cosmic annotated sequence variants detected by tngs occurred in tp53 , brca1 , kdr , kit , kras , pik3ca , brca2 , and braf ( fig 1b ; data supplement )  . 
therapeutically relevant mutations in hotspot regions were identified in 41 of 139 patients ( 29.5% ) , including drug - sensitizing variants in braf , pik3ca , idh1 , egfr , and kit , as well as drug resistance variants in kras and nras . extended genetic analyses including exome and transcriptome assays were performed for 36 patients ( 18.2% ; wes and rna - seq : n = 35 ; rna - seq only : n = 1 )  . 
patient characteristics characteristic total , no . female male ( range ) tumor type soft tissue unknown primary site colorectal urogenital thyroid breast lung skin hepatobiliary upper gi tract hematologic neuroendocrine pediatric head and neck others complete remission localized disease metastatic disease 2 to 3 rare tumor others solid tumors ( n = 189 ) : stage at presentation no . 
ninety of 104 treatment recommendations ( 86.5% ) were either off - label therapies ( n = 77 ) or trial inclusions ( n = 13 )  . the implementation rate of treatment recommendations was 31.7% ( 33 of 104 )  . 
in - label recommendations were pursued in nine of 14 cases ( 64.3% ) , whereas off - label recommendations and trial inclusions were implemented in only 28.6% ( 22 of 77 ) and 15.4% ( two of 13 ) of the cases , respectively . 
intended trial inclusion in 11 patients failed because of poor performance status or patient death ( n = 5 ) , closed trial arm ( n = 4 ) , or patient will ( n = 2 )  . 
main reasons for nonimplementation of treatment recommendations included loss to follow - up ( 22.5% ) , recommendation in the future ( 19.7% ) , patient death ( 16.9% ) , patient will ( 14.1% ) , and medical reasons ( 14.1% ; data supplement )  . 
of note , evidence level of individual off - label recommendations did not affect implementation rates ( data not shown )  . clinical outcome dgi and target databases , respectively ( data supplement )  . 
 the disease impact of non - hotspot mutations is more difficult to evaluate ; however , it can lead to in 33 patients with implemented treatment recommendations , partial remissions ( pr ) and stable diseases ( sd ) were seen in 11 ( 33.3% ) and eight patients ( 24.2% ; table 1 ) , respectively . 
of 14 responders receiving off - label therapies , eight ( 57.1% ) showed a progression - free survival ( pfs ) ratio ( pfs2 / pfs1 ; pfsr ) > 1.3 , supporting the impact of the recommended therapies.36 three patients had a pfsr < 1.3 with ongoing responses , meaning that their pfsr is still increasing . 
to assess whether implementation of treatment recommendations affected overall survival from first mtb discussion , we analyzed all patients with stage iv malignancies according to three subgroups ( n = 148 ; fig 3 )  . 
the median survival was not reached for patients with implemented treatment recommendations ( n = 33 recommendations pursued ; 95% ci , 9 months to not reached ) , 8 months for patients for whom treatment recommendations were not implemented ( n = 43 recommendations not pursued ; 95% ci , 3 to 10 months ) , and 10 months for patients who did not receive a treatment recommendation ( n = 72 no recommendations ; 95% ci , 7 to 17 months )  . 
predictive biomarkers for individualized immunotherapies are emerging and changing rapidly , with strong differences between entities.44 here , we used ihc for programmed cell death protein 1 ( pd - 1 ) / pd - l1 , tumor - infiltrating lymphocytes , microsatellite instability testing , and mutational burden assessment as predictive biomarkers . 
in the near future , identifying individual cancer neoantigens might allow a more precise prediction of responses to immunotherapies.45 this highlights the importance of an interdisciplinary mtb team that analyzes and interprets biomarkers to identify patients who might benefit from off label immuno - oncology treatments . in an mtb workflow , the portfolio of molecular diagnostic tests , as well as criteria to match and prioritize targeted therapies to molecular biomarkers , affects the probability to identify patients with actionable targets . 
our approach shares similarities with memorial sloan kettering integrated mutation profiling of actionable cancer targets ( msk - impact ) , focusing on therapeutically targetable biomarkers for fast clinical decision making and referral of patients to available clinical trials.51 targeted drug combinations might offer better dcr over single - agent therapies.52 - 55 in part , this is due to crosstalk between signaling pathways as well as spatial and temporal clonal heterogeneity , especially in patients with advanced cancer who failed standard - of - care treatment.56 , 57 most current programs for precision oncology use prespecified , genetically matched , single - agent treatments ( nci - match , clinicaltrials.gov identifier : nct02465060 ; or targeted agent and profiling utilization registry [ tapur ] , clinicaltrials.gov identifier : nct02693535 )  . 
these patients did not suffer from grade 3 to 4 adverse effects , although treatment combinations may bear a higher risk of toxicity.58 earlier referral to an mtb ( eg , after failure of first - line treatment ) might prevent the institution of ineffective treatments , improve the implementation rate , and increase the likelihood of success of molecular biomarkermatched treatments . 
 because of the low sample size and the heterogeneous composition of patients in the cohorts , the validity of this survival analysis is limited . access to molecular biomarkermatched , off label agents for cancer treatment is limited . 
in a recent single - center study , only 5% of molecular biomarkermatched treatment recommendations were implemented , mainly because of limited access to clinical trials or to restricted use of drugs outside their marketed label.59 thus , it is crucial to build up platforms for patients and treating physicians to link individual molecular information of the tumor to appropriate nonapproved drugs and available clinical trials . 
in rare entities , and especially in the setting of treatment - refractory cancers , precision oncology networks should allow hypothesis - driven in vitro studies and validation in small sets of individuals . 
thus , within the concept of patient - centric , biomarker - driven trial designs , 60 an mtb might constitute a critical tool to identify informative patients for clinical trials of targeted therapies in rare molecular subgroups . in summary , this mtb experience illustrates that patient management , on the basis of individual molecular biomarker profiling and analysis , is feasible in patients beyond standard - of - care treatment . 
 hauke busch no relationship to disclose christoph peters leadership : university medical center freiburg honoraria : university medical center freiburg travel , accommodations , expenses : novartis martin werner honoraria : diakovere consulting or advisory role : roche , novartis , johnson & johnson research funding : agilent ( inst ) , novartis ( inst ) , roche ( inst ) justus duyster honoraria : novartis , genentech , pfizer consulting or advisory role : novartis , roche travel , accommodations , expenses : novartis tilman brummer no relationship to disclose melanie boerries no relationship to disclose silke lassmann honoraria : novartis , astrazeneca travel , accommodations , expenses : novartis , astrazeneca nikolas von bubnoff honoraria : astrazeneca , amgen , bristol - myers squibb consulting or advisory role : novartis research funding : novartis travel , accommodations , expenses : novartis acknowledgment we thank the team of the genomics and proteomics core facility , german cancer research center / dkfz , heidelberg , germany , for their sequencing service . affiliations all authors : university of freiburg , freiburg ; ralph fritsch , julius wehrle , cornelius miething , christoph peters , martin werner , justus duyster , tilman brummer , melanie boerries , silke lassmann , and nikolas von bubnoff , german cancer consortium , partner site freiburg , and german cancer research center , heidelberg ; rainer claus , augsburg medical center , augsburg ; and hauke busch , university of lbeck , lbeck , germany . supported by comprehensive cancer center freiburg and by deutsches konsortium fr translationale krebsforschung grant no . 
hoadley ka , yau c , wolf dm , et al : multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origcell 158 : 929 - 944 , 2014 3 . 
hong ds , morris vk , el osta b , et al : phase ib study of vemurafenib in combination with irinotecan and cetuximab in patients with metastatic colorectal cancer with brafv600e mutation . 
goss g , tsai cm , shepherd fa , et al : osimertinib for pretreated egfr thr790met - positive advanced non - small - cell lung cancer ( aura2 ) : a multicentre , open - label , single - arm , phase 2 study . 
li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
kovaleva v , geissler al , lutz l , et al : spatio - temporal mutation profiles of case - matched colorectal carcinomas and their metastases reveal unique de novo mutations in metachronous lung metastases by targeted next generation sequencing . 
kumar p , henikoff s , ng pc : predicting the effects of coding non - synonymous variants on protein function using the sift algorithnat protoc 4 : 1073 - 1081 , 2009 32 . 
von hoff dd , stephenson jj jr , rosen p , et al : pilot study using molecular profiling of patients tumors to find potential targets and select treatments for their refractory cancers . 
johnson db , dahlman kh , knol j , et al : enabling a genetically informed approach to cancer medicine : a retrospective evaluation of the impact of comprehensive tumor profiling using a targeted next - generation sequencing panel . 
massard c , michiels s , fert c , et al : high - throughput genomics and clinical outcome in hardto - treat advanced cancers : results of the moscato 01 trial . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
tos , tumorboard online system . online registration ( tos ) > 5 days prior to mtb treating physician analysis : adherence to recommendation entity expert review literature check trial availability molecular pathology review pathology review biomarkers treatment decision patient analysis : outcome 1 . 
 genomic and immune proling of a patient with triple - negative breast cancer that progressed during neoadjuvant chemotherapy plus pd - l1 blockade david casadevall , md1 , 2 ; xiaotong li , phd1 ; ryan l . 
wali , phd1 ; natalia buza , md1 ; vasiliki pelekanou , md , phd1 ; arjun dhawan , md1 ; julia foldi , md , phd1 ; borbala szekely , md , phd1 , 3 ; francesc lopez - giraldez , phd4 ; christos hatzis , phd1 ; and lajos pusztai , md , dphil1 introduction preliminary results from neoadjuvant trials combining immune checkpoint blockade ( icb ) with standardof - care chemotherapy suggest high pathologic complete response rates that range between 50% and 80% in triple - negative breast cancer ( tnbc ) .1 - 3 adding atezolizumab to nab - paclitaxel for rst - line treatment of metastatic tnbc signicantly improved response rate and progression - free survival compared with nabpaclitaxel alone , suggesting synergy between icb and chemotherapy.4 however , not all patients respond to icb , and a minority exhibit rapid progression of their disease.5 patients who experience exceptionally favorable or unfavorable responses provide unique opportunities for studying disease biology and for identifying response markers . 
progression during neoadjuvant chemotherapy is a rare event in tnbc . here , we report results from the molecular analysis of a tnbc that rapidly progressed during neoadjuvant chemotherapy plus programmed death - ligand 1 ( pdl1 ) blockade in a clinical trial neoadjuvant medi4736 concomitant with weekly nab - paclitaxel and dosedense ac for stage i - iii triple negative breast cancer ( clinicaltrials.gov identier : nct02489448 )  . 
baseline tumor - inltrating lymphocyte ( til ) count was 10% ( cd4 , 5% ; cd8 , 5% ; and cd20 , 1% ) , macrophage ( cd68 ) was 1% , and tumor cellularity was 50% . 
the patient agreed to participate in a neoadjuvant phase i / ii clinical trial that combined durvalumab ( 10 mg / kg once every 2 weeks ) with once - per - week nabpaclitaxel ( 100 mg / m2 ) for 12 cycles and dosedense doxorubicin plus cyclophosphamide ( ddac ) for 4 cycles . 
after 8 weeks of nab - paclitaxel plus durvalumab , physical examination showed increased tumor size and new skin edema conrmed by repeat mammogram and ultrasonograrepeat cnb showed 60% tumor cellularity but also an increase in til count to 20% ( cd4 , 10% to 15% ; cd8 , 5% ; and cd20 , 0% ) and an increase in macrophages ( cd68 , 20% )  . 
nab - paclitaxel was stopped but because of the increased immune inltration , durvalumab was continued , and the patient was administered ddac , which led to disease stabilization after four courses of therapy . 
because of the apparent clinical benet , she received two additional courses of ddac without durvalumab off protocol and underwent right skin - sparing mastectomy and lymph node dissection . pathology showed extensive multifocal disease ( largest focus , 2.4 cm ; tumor cellularity , 40% ) with lymphovascular invasion in the breast and more than 10 positive axillary lymph nodes ( ypt2 , ypn3 )  . 
immune cell proportions in the mastectomy were tils , 20% ; cd4 , 10% ; cd8 , 10% ; cd20 , 1% ; and cd68 , 10% . dna and rna were extracted from formalin - xed parafn - embedded sections of tumor samples obtained at baseline , at week 8 , and from the mastectomy . 
til counts and immune cell subtypes were determined by routine pathology and immunohistochemistry . when we compared our patients baseline expression of 26 immune cell types and 100 immune function metagenes with those in the reference cohort , her neutrophil metagene expression was below the 2.5th percentile , whereas the cell cycle and immunosuppression metagenes were above the 97.5th percentile of the reference distribution ( fig 1a ) , indicating a highly proliferative tumor with an immunosuppressed microenvironment . 
the tumor also showed low expression of two previously validated immunotherapy predictive gene signatures , 8 , 9 indicating low probability of response to icb ( fig 1b )  . 
single - gene level analysis revealed signicantly low expression of 27 genes including pdcd1 , cd8a , and klrc2 ( fig 1c ) , suggesting impaired t - cell and natural killer ( nk ) cell activity . 
in addition , we found high exincluding tgfb , hla - g , cd63 , pression of 33 genes , ccl28 , and cxcl16 ( fig 1d ) , which are associated with an immune evasive phenotype , increased cell motility , and invasion . 
overall , greater changes were observed between week 8 and mastectomy ( ddac treatment ) than between baseline and week 8 ( nab - paclitaxel treatment )  . we observed upregulation of fos , abcb1 , kir3dl3 , gzma , and gzmk in the mastectomy ( single - gene and metagene expression changes are provided in the data supplement )  . 
to study this further , we next analyzed t - cell exclusion and dysfunction features using the tumor immune dysfunction and exclusion ( tide ) method ( data supplement ) .10 in concordance with our previous ndings , cytotoxic t - cell inltration increased during the second period . 
however , this was accompanied by an increase in t - cell dysfunction score , indicating an ineffective immune response ( fig 2b )  . we also performed whole - exome sequencing of baseline and mastectomy specimens . 
other mutations with the highest variant allele frequency at baseline included znf385c , cacna1e , nxpe1 , and dync1h1 , which have poorly understood functions in cancer ( fig 2c ; data supplement )  . 
we also detected amplications in fgfr1 , fgf2 , fgf3 , myc , mcl1 , ccnd1 , and tgfb2 and deletions in cdkn2a and cdkn2b ( data supplement )  . 
by using sciclone ( waltham , ma ) 11 and clonevol , 12 we identied four distinct tumor clones , all present at baseline and in the mastectomy specimen , with little evidence for clonal selection during therapy ( figs 2c - d )  . 
compound heterozygous inactivating mutations cause severe combined immune deciency.14 discussion this cancer showed primary resistance to nab - paclitaxel and ddac chemotherapy concurrent with an anti - pd - l1 agent . 
the tumor harbored several poor prognostic genomic alterations at diagnosis , including a p53 mutation and coamplication of myc and mcl1 , both of which are implicated in chemotherapy resistance.15 , 16 the expression of permeability glycoprotein ( p - glycoprotein ; abcb1 ) , a drugefux transporter that mediates taxane and anthracycline resistance in vitro , also increased during treatment.17 we observed a measurable increase in intratumor ammatory response by the end of the treatment ( data supplement ) , but unfortunately this did not translate into clinical antitumor activity . 
tgf activation in cancers has been linked to primary chemotherapy resistance and also to immune evasion and resistance to pd - l1 blockade in multiple experimental systems.18 - 21 furthermore , r175h p53 mutation that this cancer harbored has been shown to render cancers insensitive to the growth inhibitory effects of tgf.22 on the basis of these ndings , we hypothesize that the high expression of tgfb2 by this tumor may have contributed to its immune escape and its resistance to chemotherapy . 
another notable feature of this cancer was the low level of expression of programmed cell death protein 1 ( pd1 ) at the time of diagnosis and the high level of expression of hla - g . 
categories in which our patients score is above the 97.5th percentile ( red dots ) or below the 2.5th percentile ( teal dots ) are highlighted , are shown as bold numbers , and are described in the legend key . 
finally , genes for which our patient had expression values below the 2.5th ( panel c , teal squares ) or above the 97.5th ( panel d , red squares ) bootstrap distribution percentiles are shown . 
legend boxes and bold numbers show categories for which we observed an increase or decrease of 0.5 log2 fold change between either baseline and 8 weeks ( rst period ) and / or 8 weeks and mastectomy ( second period )  . 
 ( b ) tumor immune dysfunction and exclusion ( tide ) - based ( data supplement ) t - cell dysfunction and exclusion shown together with correlation coefcients of the gene expression values for our patient with cancer - associated broblast ( caf ) , myeloid - derived suppressor cell ( mdsc ) , and m2 tumor - associated macrophages ( tam m2 ) signatures . 
 ( c ) variant allele frequency ( vaf ) plot ( left ) and shplot ( right ) illustrating the clonal architecture and evolution ( based on sciclone [ waltham , ma ] and clonevol tools ; data supplement ) of the tumor between baseline and mastectomy time points . 
ifng , interferon gamma signature ; math , mutant allele tumor heterogeneity ; til , tumor - inltrating lymphocyte ; tlr , toll - like receptor . contributed to the poor outcome . 
however , the ndings pose at least one testable therapeutic hypothesis : tgf - targeting therapies ( eg , galunisertib , m7824 , tew7197 , ly - 3200882 , fresolimumab , and nis793 ) may have improved the efcacy of pd - l1 blockade in this particular individual . in summary , this case demonstrates the complexity of chemotherapy and immunotherapy resistance mechanisms and suggests that multiple different biologic processes may contribute to disease progression during treatment . we nd it reassuring that previously published icb response signatures predicted low sensitivity to pd1 / pd - l1 blockade for this patient . 
for more information about asco 's conict of interest lajos pusztai honoraria : merck , astrazeneca / medimmune , pzer , syndax pharmaceuticals , almac diagnostics , pieris pharmaceuticals , genentech , immunomedics , eisai , seattle genetics / astellas pharma , biotheranostics consulting or advisory role : h3 biomedicine , merck , novartis , pieriandx , seattle genetics , syndax pharmaceuticals , athenex research funding : merck , genentech , seattle genetics , astrazeneca no other potential conicts of interest were reported . acknowledgment we thank erika esteve , md , for her invaluable support throughout the study period ; xavier monzonis , md , and santiago balseiro , md , for their insightful suggestions ; tao qing and michal marczyk for their frequent inputs regarding methodologic and technical aspects of the manuscript ; and jeremia walah ( developer of structural variation and indel analysis by assembly ) and peng jiang ( developer of tumor immune dysfunction and exclusion score ) for their feedback regarding the use and interpretation of their tools . references pusztai l , hofstatter ew , chung gg , et al : durvalumab ( medi4736 ) concurrent with nab - paclitaxel and dose dense doxorubicin cyclophosphamide ( ddac ) as neoadjuvant therapy for triple negative breast cancer ( tnbc )  . 
j clin oncol 36 , 2018 ( suppl ; abstr 586 ) loibl s , untch m , burchardi n , et al : randomized phase ii neoadjuvant study ( geparnuevo ) to investigate the addition of durvalumab to a taxane - anthracycline containing chemotherapy in triple negative breast cancer ( tnbc )  . 
nanda r , liu mc , yau c , et al : pembrolizumab plus standard neoadjuvant therapy for high - risk breast cancer ( bc ) : results from i - spy 2 . 
j clin oncol 35 , 2017 ( suppl ; abstr 506 ) schmid p , adams s , rugo hs , et al : atezolizumab and nab - paclitaxel in advanced triple - negative breast cancer . 
n engl j med 379 : 2108 - 2121 , 2018 ferrara r , mezquita l , texier m , et al : hyperprogressive disease in patients with advanced non - small cell lung cancer treated with pd - 1 / pd - l1 inhibitors or with single - agent chemotherapy . 
ann oncol 29 : 2232 - 2239 , 2018 shi w , jiang t , nuciforo p , et al : pathway level alterations rather than mutations in single genes predict response to her2 - targeted therapies in the neo - altto trial . 
ann oncol 28 : 128 - 135 , 2017 ayers m , lunceford j , nebozhyn m , et al : ifn - - related mrna prole predicts clinical response to pd - 1 blockade . 
j clin invest 127 : 2930 - 2940 , 2017 fehrenbacher l , spira a , ballinger m , et al : atezolizumab versus docetaxel for patients with previously treated non - small - cell lung cancer ( poplar ) : a multicentre , open - label , phase 2 randomised controlled trial . 
lancet 387 : 1837 - 1846 , 2016 jiang p , gu s , pan d , et al : signatures of t cell dysfunction and exclusion predict cancer immunotherapy response . 
ann oncol 28 : 3076 - 3082 , 2017 imamura t , takase m , nishihara a , et al : smad6 inhibits signalling by the tgf - beta superfamily . 
lee k , giltnane jm , balko jm , et al : myc and mcl1 cooperatively promote chemotherapy - resistant breast cancer stem cells via regulation of mitochondrial oxidative phosphorylation . 
noonan , mbbchbao , msc1 - 3 ; sagar sardesai , md1 - 3 ; jeffrey vandeusen , md , phd1 - 3 ; robert wesolowski , md1 - 3 ; nicole williams , md1 - 3 ; clara n . 
we evaluated three prespecied questions to assess patients perceptions of genomic testing . results in all , 100 patients underwent genomic testing , with a median of ve mutations ( range , 0 to 13 mutations ) detected per patient . 
genomic testing revealed one or more potential therapies in 98% of patients ( 98 of 100 ) , and 60% of patients ( 60 of 100 ) had one or more recommended treatments with level i / ii evidence for actionability . 
we did not detect a statistically signicant difference in time - to - treatment failure ( log - rank p = .87 ) or overall survival ( p = .71 ) among patients who had treatment change supported by genomic testing versus those who had no treatment change . 
this study provides a prospective analysis of physician decision making and outcomes of patients receiving tumor sequencing as part of mbc clinical care . knowledge generated genomic testing revealed that most patients ( 60% ) had at least one recommended treatment with level i / ii evidence for actionability ; however , only a subset of patients were given a recommendation for a change in treatment , and even fewer actually changed therapy on the basis of genomic testing results . 
patients whose therapy did not change on the basis of genomic testing had a decrease in condence of treatment success . relevance even though standard of care is improving , relatively few patients with mbc changed therapy on the basis of genomic testing in this prospective study . 
this suggests a need to better understand barriers to implementation of mutation - directed therapy as well as a need for improved understanding of patient perception of somatic genomic testing . ( ia ) , pik3ca hotspot - activating missense mutations ( ia ) , microsatellite instability ( ic ) , ntrk fusions ( ic ) , esr1 hotspot - activating missense mutations ( iia ) , pten loss ( iia ) , akt1 mutations ( iib ) , and erbb2 hotspot - activating missense mutations ( iib ) .16 although genomic testing is promising for improving treatment efcacy , it is unclear how frequently it changes treatment.17 - 19 complex interactions between choice of genomic testing and patient perception of care make it imperative to understand the impact of genetic sequencing in clinical oncology.17 , 20 , 21 to date , we have little understanding of how patients perceive the value or accuracy of genomic testing , and whether changes in treatment recommendation based on genomic testing affect patients perceptions of care . 
there are discrepancies between cancer patient and physician expectations of therapy efcacy ; patients demonstrate greater optimism about therapy efcacy.22 - 25 expectations of patients with mbc and their motivations for undergoing somatic genetic testing have not yet been explored . clinical tumor genomic analyses typically rely on targeted next - generation sequencing ( ngs ) of a panel of specic , typically actionable cancer - related genes to analyze somatic gene alterations from biopsy specimens . 
most commercial targeted panel sequencing approaches , including foundationone cdx ( foundation medicine , cambridge , ma ) in this study , provide treatment suggestions that are based on literature.26 we genomic alterations and available clinical hypothesized that prospective implementation of foundationone cdx testing into clinical care for patients with mbc will identify patient - specic approaches that offer improved patient outcomes and perceptions of care . 
in this study , patients with mbc received foundationone cdx testing when a new treatment was initiated and genomic testing results released to the provider at the next progression event . 
the study was approved by the ohio state university institutional review board and informed consent was obtained from all patients . testing was performed by using foundationone cdx , formalin - xed parafnwhich used ngs on patients embedded tumor tissue . 
the foundationone cdx results were then released to the provider . survey measures physicians received a ve - item questionnaire after the foundationone cdx report was released to assess whether their treatment recommendation should be changed on the basis of those results . 
the questionnaire asked how the physician used the foundationone cdx test ( multiple choice ) and what else they would like to see in the report ( multiple choice plus an open - ended other option )  . 
attitudes toward foundationone cdx testing were assessed with three items : ( 1 ) by having the test , i will feel more condent of my treatments success , ( 2 ) i will trust the test results , and ( 3 ) i think the test results will be accurate . 
patient motivation and expectations for study participation were assessed with 15 questions using a ve - point likert scale that ranged from strongly disagree to strongly agree ( data supplement )  . because of small numbers in each group , these questions were assessed as a three - level outcome ( agree v neutral v disagree ) and as a binary outcome ( agree v neutral and disagree )  . statistical and survival analyses mcnemars test of agreement ( 2 level or 3 level ) was used to compare preand post - test survey responses and preversus post - test survey agreement by foundationone cdxsupported treatment change results from the physician questionnaire . 
the offstudy date was dened as the date of the patients second progression ( rst while on study ) , second treatment change ( rst while on study ) , death , or loss to follow - up . 
patients still alive on december 18 , 2018 , were censored as of this date . all statistical analyses were performed using sas version 9.4 ( sas institute , cary , nc ) and r version 3.4.1 , and survival curves were created using the packhv package.27 results study population a total of 142 patients with mbc provided consent and were assessed for eligibility ( fig 1 )  . 
patients were excluded from analysis if there was no available tissue , insufcient tissue , or poor dna quality ( n = 21 ) , died before the foundationone cdx report was released ( n = 13 ) , did not receive foundationone cdx testing within 10 weeks of starting their current line of therapy ( n = 4 ) , were lost to follow - up ( n = 2 ) , withdrew consent ( n = 1 ) , or had a biopsy that revealed no metastatic disease ( n = 1 )  . 
of these samples , 71% ( 71 of 100 ) were metastatic biopsies and 29% ( 29 of 100 ) were primary tumors ( breast or axillary lymph node )  . 
the study population was predominantly age 55 years or older , white , and collegeeducated with a high income ( table 1 )  . foundationone cdx genomic testing results among the 100 patients with successful foundationone cdx ngs testing , the number of mutations identied per patient ranged from 0 to 13 with a median of ve mutations ( data supplement )  . 
as anticipated , the most common mutations were missense or frameshift mutations in tp53 ( n = 49 ) and pik3ca ( n = 40 ) or amplication of myc ( n = 20 ; fig 2a )  . 
among er - positive / her2 - negative patients , 10 ( 16.7% ) of 63 harbored an esr1 mutation . in terms of therapy recommendations , 98 ( 98.0% ) of 100 patients had at least one potential therapy identied on foundationone cdx testing , with a median of nine potential treatments ( range , 0 to 35 potential treatment ; fig 2b )  . 
the most commonly recommended therapy based on a genomic alteration was an mtor inhibitor ( everolimus or temsirolimus ) for pik3ca mutations ( data supplement )  . among all patients , 60 ( 60% ) of 100 had at least one recommended treatment with level i / ii evidence ( not including erbb2 amplications ) , 16 with a median of one potential treatment with level i / ii evidence ( range , 0 to 2 potential treatments ; fig 2b )  . potential germline alterations in somatic tumor testing a known challenge in somatic tumor sequencing is potential identication of germline alterations.28 - 32 among the 100 total patients , 14 had alterations identied in brca1 ( n = 4 ) , brca2 ( n = 8 ) , or palb2 ( n = 2 )  . 
of these six patients with no prior knowledge of germline mutations , two had germline alterations conrmed ( one brca1 and one brca2 ) and one underwent germline testing in which no alterations were identied ( presumed somatic brca2 mutation )  . 
the six patients whose foundationone cdx reports were not therapy without proreleased remained on their initial gression as of september 1 , 2018 ; ve of these patients had er - positive / her2 - negative and one had er - positive / her2 - positive breast cancer . 
across the study population , 10 ( 10.0% ) of 100 total patients or 10 ( 11.5% ) of 87 patients with physician questionnaire data experienced a treatment change supported by foundationone cdx data . 
physicians described not changing treatment because the patient could not be enrolled on a clinical trial ( ie , the recommended trial closed , the patient did not want to enroll , or the patient was ineligible ; n = 10 ) , patient hospitalization ( n = 3 ) , insurance denied treatment charges ( n = 2 ) , patient transferred care to other provider ( n = 1 ) , or patient pursued a nonrecommended trial ( n = 1 ; data supplement )  . genomic alterations and survival among 76 patients evaluable for ttf analysis , there was no signicant difference in ttf ( log - rank p = .87 ) by foundationone cdx - supported treatment change status ( fig 3a )  . 
despite a high frequency of mutation , there was no signicant difference in os by pik3ca mutation status among er - positive / her2 - negative patients ( p = .18 ) or tnbc patients ( p = .66 ) , or when evaluating alterations that could activate the pi3k pathway ( pik3ca mutation , pten loss , or akt1 mutation ) among er - positive / her2 - negative patients ( p = .60 ) or tnbc ( p = .81 ) patients ( data supplement )  . 
in the msk - impact study ( clinicaltrials.gov identier : nct01775072 ) , hrpositive / her2 - negative patients with detected tumor mutations in esr1 , map kinase pathway , or myc or other transcription factors genes had a worse response to aromatase inhibitor these alterations.15 we evaluated the association of these alterations with os and found no signicant difference among the four groups ( fig 3d )  . therapy than patients without patients perceptions of genetic testing a total of 58 ( 58.0% ) of the 100 evaluable patients completed at least a portion of the survey at enrollment ( pretest ) , and 40 ( 40.0% ) completed at least a portion of the survey at study conclusion ( post - test )  . 
in the pretest survey , most patients strongly agreed or somewhat agreed that by having the genetic testing they would feel more condent in their treatments success ( 36 [ 65.5% ] of 55 ) , whereas at study completion , a minority of patients ( 12 [ 30.8% ] of 39 ) felt more condent in their treatments success by having the genetic testing . 
there was no signicant difference in patients trust of test results or thinking the results were accurate ( mcnemars p = .06 for both questions )  . foundationone cdx - supported treatment change status ( fig 3b )  . 
by subtype , there were signicant differences in os ( log - rank p = .01 ) ; patients who had tnbc had worse os than er - positive / her2 - negative patients , as anticipated ( data supplement )  . among genomic alterations , we evaluated several mutations known to be prognostic ( esr1 mutations ) or potentially predictive of outcomes ( pik3ca )  . 
in msk - impact ( a targeted tumor - sequencing assay developed at memorial sloan kettering cancer center [ msk ] termed integrated mutation proling of actionable cancer targets [ impact ] ) , there was evidence that patient response to aromatase inhibitor therapy was signicantly worse for patients whose tumors harbored one of several alterations.15 among we then assessed whether pretest and post - test survey results were associated with treatment change on the basis of foundationone cdx results in patients who completed both the pretest and post - test survey questions of interest . for treatment condence , we found that patients who did not have a treatment change supported by foundationone cdx data were signicantly more likely to change their response from agree to neutral or disagree ( mcnemars test of agreement p = .001 ; fig 4 )  . 
of potential therapies with level i / ii evidence 50 40 30 20 10 0 % mutant tp53 pik3ca cdh1 znf703 esr1 ccnd1 fgfr1 fgf4 fgf19 gata3 fgf3 map2k4 znf217 pten mcl1 brca2 erbb2 map3k1 arid1a smad4 myst mdm2 mutations or potential therapies for each patient fig 2 . 
comut plot created by using the genvisr package.47 ( b ) for each patient ( n = 100 ) , the graph shows number of detectable mutations , number of potential therapies identied , and number of potential therapies with level i / ii evidence for actionability in breast cancer based on condorelli , et al.16 ( * ) indicates patients whose treatment was changed based on genomic testing results . treatment ( data supplement )  . 
 ( a ) kaplan - meier plot of time to treatment failure stratied by patients with treatment change based on genomic testing ( red line ) v no treatment change . 
time to treatment failure dened as time from release of foundationone cdx results to the off - study date , dened as date of second progression or treatment change ( rst during the study period ) , death , or loss to follow - up . 
 ( c ) kaplan - meier plot of overall survival for patients with estrogen receptor ( er ) positive / human epidermal growth factor receptor 2 ( her2 ) negative metastatic breast cancer , stratied by presence or absence of mutation in esr1 . 
we prospectively evaluated the impact of ngs on mbc treatment decisions and potential associations with patient outcomes and their perceptions of genomic testing . in this study , more than 60% of patients had a genomic alteration associated with level i or ii evidence for actionability ( not including her2 amplication ) based on a recent consensus panel16an impressive number that emphasizes the potential for widespread use of ngs . 
a total of 37 patients answered the question both before ( pre - genomic test survey ) and after ( post - genomic test survey ) genomic test results were shared with physicians . 
this is consistent with a previous study that identied barriers such as trial ineligibility , distance to trial , and physical / emotional exhaustion.18 treatment change was not signicantly associated with ttf or os ; however , the small number of patients experiencing treatment change limits the conclusions that can be drawn from these analyses . this is the rst study to our knowledge to prospectively evaluate the inuence of somatic ngs on patient perception of care and it provides important initial data , but the lower patient survey response rate limits denitive conclusions . 
when comparing study surveys at admission to surveys at conclusion , patients seemed less condent in their treatment , particularly those whose treatment was not changed by foundationone cdx results . 
this is consistent with other studies that suggest genetic testing may increase negative emotions in patients with metastatic cancer , 18 although most studies evaluated effects from germline testing.36 - 40 our data preliminarily suggests that there may be ramications of somatic genomic testing for patients despite its known utility in selecting therapy for mbc . because negative emotions are associated with decreased quality of life and potentially with survival , 41 - 44 the possible negative implications of broadly integrating ngs into clinical practice should be considered . furthermore , pretest survey data suggest that patients have misconceptions about what somatic mutation testing can inforwe found that patients had overly optimistic expectations for somatic tumor ngs : almost half believed the test would tell them which medication to take whereas only 11.5% of the total patients switched therapy on the basis of foundationone cdx reports . 
this is similar to previous studies that demonstrate that patients have greater optimism regarding treatment efcacy than physicians do , 24 possibly because physicians provide patients with more optimistic information than their true assessment.45 in addition , most patients incorrectly believed the test would predict future genetic mutations , assess childrens disease risk , or estimate treatment success , despite the informed consent stating that participants would rarely benet personally or therapeutically from this study . 
therefore , this supports further exploration of the readability of consent forms , an area currently under investigation.46 we hypothesize that patients limited knowledge of genetics may explain their decreased condence after genomic testing , 17 - 19 and we also hypothesize that improved decision support for genomic testing for patients will improve condence in their treatment , particularly in the context of increases in available targeted treatments for mbc . there are known limitations to our study that prevent us from making denitive conclusions . 
furthermore , unlike the methodologies in the existing literature , we used a unique prospective and longitudinal methodology to evaluate patient perceptions . future studies may benet by including patient interviews to assess the experience of somatic ngs . 
furthermore , the potential treatments identied by foundationone cdx test results reect drugs approved for any indication , not necessarily for mbc , and therapies available at the time of the test . 
for example , both everolimus and temsirolimus were reported as potential therapies for patients with alterations in nf1 , pten , ak1 , pik3r1 , rptor , akt3 , fbxw7 , kit , pdgfra , stk11 , and vhl ; however , only everolimus is approved by the us food and drug administration for mbc . 
in addition , since this study was completed , multiple new therapies approved by the us food and drug administration have not been considered , suggesting that clinical implications of ngs should be assessed regularly . in conclusion , our ndings suggest that ngs by tools such as foundationone cdx may provide valuable insight for physicians and patients when determining the best treatment option for patients with mbc upon disease progression . 
there are clear clinical challenges involved with integrating ngs into the framework of mbc care , including barriers to preferred treatment options and complex interactions between genomic testing and patient perceptions , which warrant further study . affiliations 1the ohio state university college of medicine , columbus , oh 2the ohio state university comprehensive cancer center , columbus , oh 3stefanie spielman comprehensive breast center , columbus , oh 4the ohio state university , columbus , oh 5foundation medicine , cambridge , ma 6the ohio state university college of public health , columbus , oh 7mt . 
chen consulting or advisory role : novartis , immune design , syapse speakers bureau : novartis , foundation medicine research funding : eisai patents , royalties , other intellectual property : matchtx , a genomics software package that helps researchers , oncologists , and clinical trial managers identify the full set of biomarkers that collectively predict the outcome of patients with cancer to treatment siraj mahamed ali employment : foundation medicine leadership : incysus stock and other ownership interests : exelixis , blueprint medicines , agios , genocea biosciences consulting or advisory role : revolution medicines , azitra ( i ) , princeps tx ( i ) patents , royalties , other intellectual property : patents via foundation medicine and via seres health on microbiome in non - neoplastic disease ( i ) anne m . 
noonan consulting or advisory role : helsinn healthcare , qed therapeutics sagar sardesai consulting or advisory role : novartis speakers bureau : immunomedics travel , accommodations , expenses : novartis jeffrey vandeusen stock and other ownership interests : immunomedics robert wesolowski consulting or advisory role : pzer ( inst ) , puma biotechnology research funding : acerta pharma , astrazeneca travel , accommodations , expenses : pzer , puma biotechnologybhuvaneswari ramaswamy consulting or advisory role : pzer maryam b . 
lustberg consulting or advisory role : tempus , pledpharma other relationship : hologic / cynosure no other potential conicts of interest were reported . references oshaughnessy j : extending survival with chemotherapy in metastatic breast cancer . 
condorelli r , mosele f , verret b , et al : genomic alterations in breast cancer : level of evidence for actionability according to esmo scale for clinical actionability 17 . 
gray sw , park er , najita j , et al : oncologists and cancer patients views on whole - exome sequencing and incidental ndings : results from the canseq study . of molecular targets ( escat )  . 
gollust se , gray sw , carere da , et al : consumer perspectives on access to direct - to - consumer genetic testing : role of demographic factors and the testing 21 . 
meropol nj , weinfurt kp , burnett cb , et al : perceptions of patients and physicians regarding phase i cancer clinical trials : implications for physician - patient communication . 
lux mp , bayer cm , loehberg cr , et al : shared decision - making in metastatic breast cancer : discrepancy between the expected prolongation of life and treatment efcacy between patients and physicians , and inuencing factors . 
parsons dw , roy a , plon se , et al : clinical tumor sequencing : an incidental casualty of the american college of medical genetics and genomics recommendations for reporting of incidental ndings . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation proling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
tanioka m , fan c , parker js , et al : integrated analysis of rna and dna from the phase iii trial calgb 40601 identies predictors of response to trastuzumabbased neoadjuvant chemotherapy in her2 - positive breast cancer . 
bakos ad , hutson sp , loud jt , et al : brca mutation - negative women from hereditary breast and ovarian cancer families : a qualitative study of the brca37 . 
hay jl , meischke hw , bowen dj , et al : anticipating dissemination of cancer genomics in public health : a theoretical approach to psychosocial and behavioral negative experience . 
collins vr , meiser b , ukoumunne oc , et al : the impact of predictive genetic testing for hereditary nonpolyposis colorectal cancer : three years after testing . genet med 9 : 290 - 297 , 2007 40 . 
hendriks ks , hendriks mm , birnie e , et al : familial disease with a risk of sudden death : a longitudinal study of the psychological consequences of predictive testing for long qt syndrome . 
chida y , hamer m , wardle j , et al : do stress - related psychosocial factors contribute to cancer incidence and survival ? nat clin pract oncol 5 : 466 - 475 , 2008 42 . 
giese - davis j , collie k , rancourt km , et al : decrease in depression symptoms is associated with longer survival in patients with metastatic breast cancer : a secondary analysis . 
perni s , rooney mk , horowitz dp , et al : assessment of use , specicity , and readability of written clinical informed consent forms for patients with cancer undergoing radiotherapy . 
 o implementation and clinical utility of an integrated academiccommunity regional molecular tumor board see accompanying editorial doi : purpose precision oncology develops and implements evidence - based personalized therapies that are based on specific genetic targets within each tumor . 
however , a major challenge that remains is the provision of a standardized , up - to - date , and evidenced - based precision medicine initiative across a geographic region . materials and methods we developed a statewide molecular tumor board that integrates academic and community oncology practices . 
the precision medicine molecular tumor board ( pmmtb ) has three components : a biweekly web - based teleconference tumor board meeting provided as a free clinical service , an observational research registry , and a monthly journal club to establish and revise evidence - based guidelines for off - label therapies . 
the pmmtb allows for flexible and rapid implementation of treatment , uniformity in practice , and the ability to track outcomes . results we describe the implementation of the pmmtb and its first year of activity . 
nine subjects ( 28% ) received recommended therapy with a response rate of 17% ( one of six ) and a clinical benefit rate ( partial response + stable disease ) of 38% ( three of eight )  . 
today , next - generation sequencing assays are widely available and considered to be part of the standard of care in specific clinical situations.1 , 2 at the same time , precision medicine promises to match patients to gene - targeted treatments.3 however , many challenges exist in implementing precision medicine , including knowledge gaps , privacy , systems barriers , and reimbursement issues.4 , 5 the knowledge gaps arise from the extensive breadth and depth of knowledge required to effectively use genomic information , which is compounded by tumor complexity and the wide variation of genomic landscapes in tumors.6 , 7 to meet this challenge , knowledge in genomic technologies , cancer biology , human pathology , pharmacology , clinical patient care , and often detailed knowledge of gene structure and function are required . molecular tumor boards ( mtbs ) address gaps in knowledge and clinical utility by providing a forum for individuals with wide - ranging expertise to review patient medical histories and mutation profiles to guide patient - specific treatment options.8 - 12 in contrast to organ - specific tumor boards , mtbs consider patients with diverse mark e . 
rehrauer jill kolesar author affiliations appear at the end of this article . supported by national cancer institute grant no . p30 ca014520 to the university of wisconsin carbone cancer center . corresponding author : mark e . 
although mtbs have been established at academic institutions , they are largely unavailable at nonacademic sites . to expand access to mtbs , we have partnered with community practitioners at other health networks to develop an integrated mtb available to any provider in our geographic region , the precision medicine molecular tumor board ( pmmtb )  . 
in addition to its regional scope , the pmmtb is novel in its tripartite structure by separating clinical service , a registry protocol , and a journal club to establish parameters for evidencebased use of off - label therapies . 
moreover , some patients are prospectively consented to the registry at the time that molecular testing and biopsy are performed ; these patient cases are later presented at the tumor board . the third part of the structure is a monthly journal club meeting . 
at the conclusion of the journal club , the tumor board decides whether to adopt or reject the proposed drug - biomarker pair and to update the guidance document that specifies when offlabel therapies will be recommended . 
in addition , periodic review of cases in the tumor registry ineffective treatment approaches , may reveal which in turn will trigger a journal club to seek to retract a previously issued criterion in the guidance document . tumor board the first part of the pmmtb structure consists of a twice - monthly tumor board convened through web - based teleconference . 
participants are experts in diverse fields that span basic cancer biology , medical / clinical genetics , molecular pathology , surgical pathology / cytopathology , pharmacology , medical oncology , and radiation oncology . 
molecular test results from a clinical laboratory improvement amendmentscertified laboratory are included with the case submission information . up to six cases are discussed per 1 - hour meeting ; when additional cases are submitted , review is either postponed or performed administratively by the pmmtb chairs . 
the molecular pathology team reviews alterations and assesses their analytic validity , that is , whether a particular mutation is likely to alter gene function on the basis of site and allele frequency or whether an amplicon encompasses many reported genes . 
 are systematically discussed in the context of cancer databases ( cancer gene census / cosmic [ catalogue of somatic mutations in cancer ] ) , the cancer genome atlas , and human polymorphism databases ( dbsnp , 1000 genomes project , esp6500 ) along with knowledge tools ( cbioportal , my cancer genome , targeted cancer care , personalized cancer therapy ) .13 - 18 the board identifies and discusses possible clinical trials and off - label targeted therapies . 
final recommendations are provided in the form of a formal letter to the submitting physician , with acceptance at the discretion of the treating clinician ( data supplement )  . journal club the pmmtb considers targeted food and drug administrationapproved drugs that are not labeled for use in that particular patients tumor type according to a guidance document . 
the guidance document includes , for example , the targeting of oncogenes with known activating mutations by using drugs that directly inhibit their activity , combination therapies for braf mutations , and specific criteria for using genomic decisions for immune checkpoint inhibitors . 
during the meeting , up to three studies , including human and preclinical evidence , are provided and the pmmtb decide reviewed ; members of whether to amend the guidance document , to provisionally amend it ( if human data are extremely limited ) , or to reject it . 
in this manner , all decisions to recommend off - label therapies are based on careful , but timely review of evidence . registry patients are enrolled in a registry to allow for the tracking of outcomes . 
the registry protocol was approved by our institutional review board ( university of wisconsin [ uw ] hs - irb approval #2015 - 1370 ) and was conducted in accordance with the declaration of helsinki . 
all clinical laboratory improvement amendments certified genomic tumor profiling assays are allowed , including commercial tests , tests performed at outside academic facilities , and tests performed at the uw collaborative genomics core . subjects registered and presented at the pmmtb were followed for acceptance of recommendations , treatment , and disease response or progression . 
the primary objective of this study was to assess the the pmmtb , defined as clinical utility of the frequency of recommendation acceptance . descriptive summaries of response to therapy , disease status , and demographic information are provided . 
for nonmeasurable tumors , stable disease versus partial response was reported on the basis of radiologic follow - up and clinical assessment of the treating physician at the follow - up study performed 2 to 4 months after baseline . results participant and disease characteristics because registry consent was not a prerequisite to pmmtb presentation , overlap was incomplete between patient cases presented and subjects enrolled . 
seventy - seven patient cases were submitted to the tumor board between september 17 , 2015 , and september 1 , 2016 : 74 were presented to the board , and three were reviewed administratively . 
four registered patients who had planned genomic testing were not presented at the tumor board because molecular analysis of their tumor was not complete ( eg , insufficient tissue )  . 
of the 44 patient cases presented , 38 were deemed to have adequate followup by september 1 , 2016 , to report results here . table 1 includes demographics and other baseline characteristics for these 38 patients . 
the most common genomic test used was the foundationone ( foundation medicine , cambridge , ma ) assay , followed by the uw collaborative genomics core 50 - gene panel . pmmtb recommendations and acceptance figure 3 depicts recommendations and acceptance of the 38 patient cases . 
an actionable target was defined as one that allowed identification of a molecular - targeted clinical trial or off - trial treatment that is based on the pmmtb guidance document . 
the area of the circle is proportional to the number of patient cases presented from that health system . uwccc , university of wisconsin carbone cancer center . actionable target was found in 32 ( 84% )  . 
treatment was accepted for nine ( 28% ) of these 32 and not accepted for 11 ( 34% ) , and the remainder of patients continued on standard - of - care therapy , pending progression . 
in general , we found that patients were able to obtain these drugs when recommended by the pmmtb , which highlights the access that physicians and patients have to off - label therapies . 
the pmmtb may have facilitated this access by establishing and implementing a uniform set of criteria and recommendations provided as an official letter ( data supplement )  . although clinical trials were preferentially recommended , they were rarely accepted . 
for clinical trials available outside the state , no patients enrolled . the major reason was that patients with metastatic cancer often were unable or unwilling to travel long distances for investigational therapy . within the state , restrictive and often unanticipated eligibility criteria prohibited enrollment . for example , one patient was ineligible because of a recent amendment that precluded any history of drug allergy . 
molecular - targeted basket studies were largely unavailable in the region partly because of a national hold placed on the national cancer institute molecular analysis for therapy choice ( nci - match ) trial during most of the period covered . 
these findings highlight poor access to clinical trials of precision medicines and the need for local availability of clinical trials with minimal eligibility restrictions and fast startup . in the first year of the pmmtb , journal clubs primarily were used to expand the guidance document ( data supplement )  . 
in this diagram , some samples were tested for large gene sets ( foundationone , including gray boxes for nonaltered genes ) and others for limited gene sets through the uw collaborative genomics core 50 - gene panel or subset ; genes not tested are depicted as white areas . 
additional genes identified in these subjects are listed in the data supplement . the pmmtb identified treatment options that targeted 18 distinct genes in these patients ( red boxes )  . 
more treatment options were identified for tumors tested with comprehensive panels . with the inclusion of both trial and nontrial treatments , we identified a mean of 1.7 treatment options with the 405 - gene panel versus 1.1 with a more - restrictive panel of < 50 genes . 
of the remaining eight patients who received the recommended therapy , the clinical benefit rate was 38% ( three of eight ) and the response rate was 17% ( one of six ) by central review . 
the pmmtb recommended genetic counseling for three patients , and two received this counseling . discussion olaparib for brca1 / 2 mutant cancers and pembrolizumab for tumors with mismatch repair deficiency confirmed by immunohistochemistry or microsatellite testing . 
for example , vandetanib was adopted provisionally for the treatment of tumors with fumarate hydratase loss - of - function mutations , which led to an updated recommendation issued at the next pmmtb meeting . 
to bridge this gap , mtbs that comprise experts with a diverse knowledge base have been established at academic institutions8 - 11 but remain largely inaccessible to community oncologists and their patients . 
 patients ( n = 38 ) no treatment identified ( n = 6 ) treatment recommendation ( n = 32 ) accepted ( n = 9 ) not accepted ( n = 11 ) waiting for progression ( n = 12 ) condition deteriorated physician discretion unable to travel trial unavailable ( n = 6 ) ( n = 3 ) ( n = 1 ) ( n = 1 ) fig 3 . 
identification and acceptance of precision medicine molecular tumor board recommendations . to develop and implement the tripartite pmmtb . by separating the clinical service from the registry , we were able to rapidly launch and provide service across the region , although this method partially limited the number of reportable patients . 
moreover , the pmmtb used a separate decisionmaking process ( journal club ) for standardizing recommendations for off - label therapy , which allowed the board to focus on clinical cases rather than on time - consuming excursions to the clinical / preclinical literature . 
moreover , the monthly journal club allowed for a timely and careful review of conditions for which off - label therapies are reasonably expected to be effective . most previously described mtbs appear to be focused on a single institution or a single precision medicine clinic within the institution.8 - 12 such centralization can standardize clinical practice and facilitate access to clinical trials . 
despite this limitation , the decentralized approach is expected to be superior in fostering regional collaboration and access of patients to precision medicine . we found actionable mutations in 86% of the selected cohort . 
although this number appears high , we note that the number of patients with actionable mutations depends on the particular definition of actionable as well as on the clinical trials and approved off - label drugs available at that time . 
in addition , this statistic can be biased because physicians are expected to select for submission patient cases with actionable alterations . moreover , although we were able to identify molecular - based clinical trials for many patients , a number of these were inaccessible to patients unable to travel . 
if we limit the data by excluding out - ofstate trials , the rate of actionable mutations becomes 63% , which is similar to the 71% reported by a another tumor board.9 an important finding of this study is that clinical trials often are unavailable for patients . 
although this registry study was launched contemporaneously with nci - match , the expected competition for the two approaches was not realized partly because of the relative complexity of match and the clinical hold on accruals . 
nevertheless , our experience and that of others suggest that off - trial therapies will remain an important component of an mtb.3 thus , the tracking of patient outcomes is important to provide data on treatment outcomes by molecular profile . on the basis of our experience and that reported by others , 3 , 8 - 10 continued identification of food and drug administrationapproved therapies and maintenance of a registry that tracks patient outcomes will be important . 
 # genes osimertinib everolimus enzalutamide pd dabrafenib dabrafenib + trametinib everolimus everolimus enzalutamide everolimus key : tumor type key : genomic profile lung mutation amplification recommendation breast other deletion no alteration treated fig 4 . 
the genes with identified alterations are displayed as columns , with rows displaying the tumor profile for a given subject . areas shown in white organize genomic data and matched clinical outcomes will facilitate national efforts , such as cancerlinq , 19 , 20 the american association for cancer research genie ( genomics evidence information exchange ) project , 21 neoplasia orien ( oncology research information exchange network ) , commercial databases , and anticipated nci / moonshot data banks . few trials have compared molecular - targeted therapies versus standard of care to determine the benefit of genome sequencing . 
cancer type by color code is shown on the right , as are the number of clinical trials and nontrial treatments recommended by the precision medicine molecular tumor board , the response to therapy , and the treatment given . the chart on the bottom displays the frequency of alterations in each gene shown at the top by tumor type . 
a , adenoid cystic ; m , melanoma ; o , papillary serous ovary ; p , prostate ; s , alveolar rhabdomyosarcoma ; u , carcinoma of unknown primary . expression of hormone receptors , assigned treatments on the basis of amplifications , and used single - agent targeted therapies . 
by contrast , other studies have reported improved outcomes and enhanced cost effectiveness through a precision medicine approach.12 , 23 similarly , clinical benefit of adaptable use of nonstandard molecular - targeted therapy has been seen in lung cancer24 , 25 and by other mtbs.9 , 10 , 12 we observed a 13% objective response rate by standardized criteria and a clinical benefit rate of 38% ( partial response plus stable disease )  . 
although modest with a small sample , the observed rate compares favorably with the 5% response reported for unmatched phase i trials and the 4% to 16% response rate reported for secondline chemotherapy for colorectal cancer.26 , 27 similarly , a retrospective analysis has found improved progression - free survival without increased cost when patients are treated with a precision medicine approach.23 thus , our initial findings are consistent with these observations and can be improved by experience . this work has some limitations . 
the study was observational with a small sample size and patient responses were assessed by their treating physicians as part of standard - of - care evaluations . in addition , the majority of patients presented were of advanced - stage disease and had limited standard - of - care treatment options , which suggests that these results may not be generalizable to patients earlier in the course of their disease . 
local access to molecularly targeted therapies is the most pressing need for our patients with advanced cancer . in conclusion , we have developed and implemented a regional mtb that integrates clinical service , a registry , and a journal club . 
the principal weaknesses are that the registry is observational , largely retrospective , and does not include all patient cases presented at the tumor board . our experience demonstrates that we can identify actionable mutations for a high proportion of patients and identify off - trial targeted therapies . 
kruglyak km , lin e , ong fs : next - generation sequencing in precision oncology : challenges and opportunities . expert rev mol diagn 14 : 635 - 637 , 2014 2 . 
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tafe lj , gorlov ip , de abreu fb , et al : implementation of a molecular tumor board : the impact on treatment decisions for 35 patients evaluated at dartmouth - hitchcock medical center . 
ortiz mv , kobos r , walsh m , et al : integrating genomics into clinical pediatric oncology using the molecular tumor board at the memorial sloan kettering cancer center . 
johnson a , zeng j , bailey am , et al : the right drugs at the right time for the right patient : the md anderson precision oncology decision support platfordrug discov today 20 : 1433 - 1438 , 2015 17 . 
shah a , stewart ak , kolacevski a , et al : building a rapid learning health care system for oncology : why cancerlinq collects identifiable health information to achieve its vision . 
le tourneau c , delord jp , gonalves a , et al : molecularly targeted therapy based on tumour molecular profiling versus conventional therapy for advanced cancer ( shiva ) : a multicentre , open - label , proof - of - concept , randomised , controlled phase 2 trial . 
haslem ds , van norman sb , fulde g , et al : a retrospective analysis of precision medicine outcomes in patients with advanced cancer reveals improved progression - free survival without increased health care costs . 
kaderbhai cg , boidot r , beltjens f , et al : use of dedicated gene panel sequencing using next generation sequencing to improve the personalized care of lung cancer . 
johnson be , kris mg , berry ld , et al : a multicenter effort to identify driver mutations and employ targeted therapy in patients with lung adenocarcinomas : the lung cancer mutation consortium ( lcmc )  . 
tsimberidou a - m , iskander ng , hong ds , et al : personalized medicine in a phase i clinical trials program : the md anderson cancer center initiative . 
 genomic profiling to expand management options for locally advanced esophagogastric cancers : a proof of principle case introduction the management of locally advanced esophageal and gastric cancer is an evolving field , but despite improvement in response rates and surgical techniques , recurrence rates for stage iii disease remain high.1 , 2 surgical resection is potentially curative and associated with established morbidity and mortality risk , limiting surgery to medically fit patients . 
emerging data suggest a role for positron emission tomography ( pet ) adaptive treatment strategies and possibly incorporation of trastuzumab in neoadjuvant therapy for patients with human epidermal growth factor receptor 2 ( her2 ) positive disease.3 , 4 however , additional tumor biologic features are not incorporated routinely in the management of locally advanced disease . 
large collaborative molecular characterization efforts , including the cancer genome atlas , the asian cancer research group , and commercial sequencing databases , have highlighted genomically defined subgroups.5 - 9 within gastric and esophageal cancers , 4% to 22% are reportedly microsatellite instable ( msi - h ) , a subgroup that has demonstrated significant responsiveness to immune checkpoint inhibitors ( icpis ) in small metastatic data sets.10 , 11 the potential to incorporate this approach in earlier - stage disease is understudied , and , to our knowledge , we report the first case describing a complete response to pembrolizumab in an msi - h stage iii gastroesophageal junction ( gej ) adenocarcinoma with persistent disease after definitive chemoradiotherapy ( crt )  . 
staging petcomputed tomography ( ct ) demonstrated a large and intensely avid mass at the gej ( standardized uptake value of 20.3 ) with no evidence of distant disease ( fig 2 )  . 
pet - ct scanning was repeated 10 weeks after crt completion , demonstrating partial response with residual pet - avid mass at the gej ( standardized uptake value of 7.1 ) , confirmed by endoscopic evidence of persistent disease and symptomatic dysphagia ( fig 2 )  . 
to explore additional therapeutic options , the original gej biopsy was submitted for comprehensive genomic profiling ( foundationone ) , which revealed msi - h , a high tumor mutational burden ( tmb ) of 36 mutations per dna megabase , an absence of erbb2 ( her2 ) alterations , and several additional genomic alterations ( appendix table a1 )  . 
orthogonal immunohistochemical mismatch repair testing confirmed loss of mlh1 and pms2 , and mlh1 promoter polymerase chain reaction testing confirmed mlh1 promoter hypermethylation , all consistent with a sporadic msi - h tumor ( fig 1 )  . 
 ( a ) scattered luminal necrosis as well as punctate single cell apoptoses and frequent mitoses are visible . mismatch repair immunohistochemistry demonstrates loss of ( b ) mlh1 and ( c ) pms2 , with intact ( d ) msh6 and ( e ) msh2 . 
 ( a ) scattered tumor infiltrating lymphocytes were present , at a density of approximately one lymphocyte per five to 10 tumor cells . response with resolution of pet - avid gej mass ( fig 2 )  . 
at the time of submission , he continues on pembrolizumab and remains clinically asymptomatic with ongoing complete response now over 8 months . discussion the ability to identify the optimal therapy for each patient is the hallmark of the precision medicine paradigm but has largely remained elusive in esophagogastric cancers to date . 
in the nonmetastatic setting , attempts to incorporate biologic agents , including the antiepidermal growth factor receptor monoclonal antibodies and antiangiogenesis agents , have not improved outcomes.12 , 13 the fully accrued radiation therapy oncology group ( rtog ) - 1010 trial examining a role for trastuzumab in combination with crt in locoregional esophageal / gej adenocarcinoma may ultimately be the first to identify a molecularly selected group to derive benefit from a biologic agent during neoadjuvant therapy for resectable disease . 
however , at the present time , patients with persistent disease after definitive crt who are unfit for resection represent a difficult clinical scenario with limited established approaches and 3 - year survival less than 10% in some series.14 , 15 although only a single case is presented herein , we suggest that in locally advanced unresectable esophagogastric cancer there may be clinical utility to molecular profiling to expand potential treatment options . 
the cancer genome atlas data sets for gastric and esophageal cancer are almost exclusively derived from nonmetastatic surgical samples and report rates of msi - h of 22% and 0% , respectively ( 0% for confirmed esophageal adenocarcinoma , although three of 36 among gej tumors not clearly of esophageal origin ) .5 , 6 this differs somewhat from the 6% rate of msi - h reported from the capecitabine and oxaliplatin adjuvant study in stomach cancer ( classic ) trial analysis of resected stage ii to iii gastric cancer and the 6.7% rate of msi - h identified in the european medical research council adjuvant gastric infusional chemotherapy ( magic ) trial of resectable gastroesophageal cancer.16 in both data sets , msi - h appears to confer a favorable prognosis and unclear benefit from adjuvant or perioperative chemotherapy , analogous to stage ii colon cancer.17 - 19 although analyses from ongoing metastatic and locoregional trials will refine the incidence of msi - h tumors , a portion will be msi - h and thus will be optimal candidates for immunotherapy consideration as supported by our case . 
complete resolution of [ 18f ] fluorodeoxyglucose activity was observed after three doses of pembrolizumab , and imaging is shown in coronal , sagittal , and axial planes for reference . pretreatment 10 weeks postchemoradiotherapy 10 weeks postpembrolizumab start strong biologic rationale , including in nonmetastatic locally advanced unresectable patients who have progressed on prior therapy.20 the ongoing checkmate - 577 trial ( nct02743494 ) will be important to more clearly establish the role of icpis in the adjuvant / locoregional setting after concurrent crt and surgery with persistent disease , and analyses for msi testing as well as tmb will be important.21 importantly , there is scientific rationale for moving these agents into the true neoadjuvant setting when the primary tumor remains.22 , 23 notably , our patient was not deemed a surgical candidate and would not be eligible for this trial . additional small studies incorporating programmed death - 1 inhibitors into concurrent crt will likely add more details , and there is strong scientific rationale for this approach ( nct03044613 ) .22 more broadly , predictive biomarkers for icpis are critical to identify patients who are most likely ( or least likely ) to benefit from icpi incorporation . not surprisingly , our patient harbored an elevated tmb at 36 mutations / dna megabase . 
however , a fraction of microsatellite stable gi cancers have also been shown to have high tmb.24 next - generation sequencingderived tmb has emerged as a robust biomarker for icpi benefit across several tumor types.25 - 27 in a small retrospective series of esophagogastric cancers , it was suggested that msi - h or tmb of > 14 mutations / megabase was associated with icpi benefit , although larger series will be critical to refine tmb in esophago - gastric cancers ( note that tmb determination was slightly different in this study ) .28 overall , our case highlights the potential role for genomic profiling in this nonmetastatic clinical situation and suggests a possible treatment approach . 
klempner consulting or advisory role : eli lilly , boston biomedical , celgene , astellas pharma speakers bureau : foundation medicine travel , accommodations , expenses : eli lilly the following represents disclosure information provided by authors of this manuscript . 
schrock employment : foundation medicine stock and other ownership interests : foundation medicine joseph chao consulting or advisory role : eli lilly , bayer , five prime therapeutics , boston biomedical , merck research funding : merck ( inst ) , novonco therapeutics ( inst ) siraj m . 
ali employment : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine , patents via seres health on microbiome stuff in non - neoplastic disease ( i ) affiliations samuel j . 
klempner , cedars - sinai medical center ; winnie wu , providence saint johns hospital , los angeles ; joseph chao , city of hope comprehensive cancer center , duarte , ca ; and alexa b . 
oppedijk v , van der gaast a , van lanschot jj , et al : patterns of recurrence after surgery alone versus preoperative chemoradiotherapy and surgery in the cross trials . 
smyth ec , fassan m , cunningham d , et al : effect of pathologic tumor response and nodal status on survival in the medical research council adjuvant gastric infusional chemotherapy trial . 
lordick f , ott k , krause bj , et al : pet to assess early metabolic response and to guide treatment of adenocarcinoma of the oesophagogastric junction : the municon phase ii trial . 
goodman ka niedzwiecki d , hall n , et al : initial results of calgb 80803 ( alliance ) : a randomized phase ii trial of pet scan - directed combined modality therapy for esophageal cancer . 
ali sm , sanford em , klempner sj , et al : prospective comprehensive genomic profiling of advanced gastric carcinoma cases reveals frequent clinically relevant genomic alterations and new routes for targeted therapies . 
gainor jf , tan ds , de pas t , et al : progression - free and overall survival in alk - positive nsclc patients treated with sequential crizotinib and ceritinib . 
le dt , durham jn , smith kn , et al : mismatch - repair deficiency predicts response of solid tumors to pd - 1 blockade . science 357 : 409 - 414 , 2017 12 . 
cunningham d , stenning sp , smyth ec , et al : peri - operative chemotherapy with or without bevacizumab in operable oesophagogastric adenocarcinoma ( uk medical research council st03 ) : primary analysis results of a multicentre , open - label , randomised phase 2 - 3 trial . 
intergroup phase iii trial of neo - adjuvant chemotherapy , followed by chemoradiation and surgery with and without cetuximab in locally advanced esophageal carcinoma : first results from the sakk 75 / 08 trial . 
smyth ec , wotherspoon a , peckitt c , et al : mismatch repair deficiency , microsatellite instability , and survival : an exploratory analysis of the medical research council adjuvant gastric infusional chemotherapy ( magic ) trial . jama oncol 3 : 1197 - 1203 , 2017 17 . 
bang yj , kim yw , yang hk , et al : adjuvant capecitabine and oxaliplatin for gastric cancer after d2 gastrectomy ( classic ) : a phase 3 open - label , randomised controlled trial . 
kelly rj , lockhart ac , jonker dj , et al : checkmate 577 : a randomized , double - blind , phase 3 study of adjuvant nivolumab ( nivo ) or placebo in pts with resected esophageal ( e ) or gastroesophageal junction ( gej ) cancer . 
rosenberg je , hoffman - censits j , powles t , et al : atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum - based chemotherapy : a single - arm , multicentre , phase 2 trial . 
ku gy , sanchez - vega f , chatila w , et al : correlation of benefit from immune checkpoint inhibitors with next gen sequencing ( ngs ) profiles in esophagogastric cancer ( egc ) patients . 
 clinical utility of real - time targeted molecular proling in the clinical management of ovarian cancer : the allocate study olga kondrashova , phd1 , 2 , 3 ; gwo - yaw ho , mbchb2 , 4 , 5 ; george au - yeung , mbbs , phd1 , 5 ; leakhena leas1 ; tiffany boughtwood , mba1 ; kathryn alsop , phd5 ; giada zapparoli , msc6 , 7 ; alexander dobrovic , phd1 , 6 , 7 ; yi - an ko , msc5 ; arthur l . 
wakeeld , phd1 , 2 ; orla mcnally , mbbao , mbbch1 , 4 ; michael quinn , mbchb , mgo4 ; sumitra ananda , mbbs1 , 2 , 4 , 5 ; deborah neesham , mbbs4 ; anne hamilton , mbbs1 , 4 , 5 ; marisa grossi , mbbs5 ; alison freimund , mbbs1 , 5 ; yada kanjanapan , mbbs5 ; danny rischin , md5 ; nadia tracante5 ; david bowtell , phd1 , 5 ; clare l . 
targeted molecular and methylation proling of fresh biopsy and archived tumor samples were performed by screening for mutations or copynumber variations in 44 genes and for promoter methylation of brca1 and rad51c . results somatic genomic or methylation events were identied in 85% of all patient cases , with potentially actionable events with dened targeted therapies ( including four resistance events ) detected in 60% of all patient cases . 
on the basis of these ndings , six patients received molecularly guided therapy , three patients had unsuspected germline cancerassociated brca1 / 2 mutations and were referred for genetic counseling , and two intermediate differentiated ( grade 2 ) serous ovarian carcinomas were reclassied as low grade , leading to changes in clinical management . 
additionally , secondary reversion mutations in brca1 / 2 were identied in fresh biopsy samples of two patients , consistent with clinical platinum / poly ( adp - ribose ) polymerase inhibitor resistance . 
these include histopathologic features , gene expression patterns , driver mutations , copy - number variations ( cnvs ) , and mutational signatures.1 - 4 the survival rates for ovarian cancer have remained largely unchanged during the past three decades , despite increasing knowledge of the molecular and genetic mechanisms underlying most subtypes of ovarian cancer.5 , 6 there is , therefore , an urgent need to rapidly translate this knowledge into improved clinical outcomes . molecular proling of cancer has become increasingly helpful not only in diagnosis , disease monitoring , and prognostication but also in identication of patients who may benet from molecularly guided treatments.7 the feasibility of molecular proling in routine clinical practice has now been evaluated in unselected cancer cohorts8 and in a number of cancer types , such as lung , brain , and pancreatic cancers9 - 11 as well as ovarian cancer.12 , 13 the allocate ( australian ovarian cancer assortment trial evaluation ) study was designed to assess the feasibility of real - time multimodal molecular proling of recurrent and advanced ovarian cancers in routine clinical practice . 
 kondrashova et al context key objective this study was designed as a pilot to assess the feasibility and potential clinical utility of multimodal real - time molecular proling in the clinical management of ovarian cancer . knowledge generated we showed that molecular proling was technically possible and acceptable to patients and has a potential place in clinical management of ovarian cancer . 
we also showed that factors like timely reporting of results , availability of matched clinical trials , and early referral for patients with platinum - resistant cancers are important in achieving the maximum clinical benets of molecular proling . relevance real - time molecular proling should play a role in the clinical management of ovarian and multiple other cancer types . 
eligibility criteria included recurrent ovarian , fallopian tube , or primary peritoneal cancer of serous , mucinous , or clear cell histology , eastern cooperative oncology group performance status of 2 or less , life expectancy of 3 months or longer , suitability for enrollment in clinical trials , and residency in the state of victoria . 
informed consent was obtained from all patients , and all analyses were performed according to the national health and medical research council of australia human ethics guidelines . sample collection matched tumor ( either snap - frozen biopsies , ascites , or formalin - xed parafn - embedded [ ffpe ] samarchival ples ) and peripheral blood samples were obtained from all patients . 
a pathologist examined a hematoxylin and eosin section from the same tumor fragment to assess sample suitability and determine the proportion of neoplastic cells . unsuitable patient cases were excluded from the study ( appendix fig a1 )  . clinical data collection clinical history and follow - up data were collected where possible ( 88 of 99 patients ) from medical records and an internal hospital database . 
dna quality of ffpe samples was assessed by genomic dna fragment size analysis ( labchip ; perkinelmer , waltham , ma ) or by pothe gene gapdh lymerase chain reaction ( pcr ) of ( appendix fig a2 )  . 
custom - designed truseq amplicon ( version 1.0 ) or dual - stranded truseq amplicon low input ( version 1.5 ; illumina , san diego , ca ) ovarian cancer panels were used to assess somatic and germline mutations in 29 and 38 genes , respectively ( appendix table a1 )  . both panels underwent thorough analytic validation and accreditation to conrm that they were suitable for diagnostic purposes . 
variant effect predictor was used for variant annotation and ltering.16 analysis was restricted to variants within coding exons with 20 bp of anking intronic sequences within the genes of interest . 
droplet digital pcr was performed on the bio - rad qx - 200 system ( bio - rad , hercules , ca )  . results patient demographics between december 2013 and october 2016 , 113 patients with advanced or recurrent ovarian cancer were recruited to the allocate study from two tertiary hospitals : rwh and pmcc ( appendix fig a1 )  . 
a total of 14 patient cases ( 12.4% ) were excluded from the study , mostly because of insufcient neoplastic cellularity or poor dna quality in the tumor samples available for analysis ( appendix fig a1 )  . multiple histopathologic subtypes were analyzed in the study , which included 69 cases of hgsoc ( grade 2 to 3 ) and eight cases of lgsoc ( grade 1 ) ; the remaining subtypes included mucinous , clear cell , carcinosarcoma , transitional cell carcinoma , mixed histology , and unknown primary peritoneal carcinoma ( table 1 )  . clinical history and poststudy follow - up data were collected for 88 patients , with the data cutoff date set to october 1 , 2018 . 
on average , patients had received between one and two lines of treatment prior to their enrollment in the study ( fig 1b )  . median time from obtaining the patient samples to the reporting of mutation assay results was 87 days ( range , 11 to 357 days )  . 
tumor biopsies were attempted on 36 of the total 113 study patients ( 31.9% ) where the patient had consented and where it was possible to perform the biopsy safely . 
of these , 28 biopsies ( 77.8% ) obtained sufcient tissue for successful molecular proling , and eight failed because of insufcient neoplastic cellularity of less than 10% , as estimated by the pathologist . for the archival ffpe samples ( n = 69 ) , the level of dna fragmentation was assessed by genomic dna fragment size analysis or by pcr of the gene gapdh ( appendix fig a2 )  . 
nonconventional therapies ( eg , naturopathic therapies ) are not shown as lines of treatment . poor quality ( highly fragmented ) and were excluded from the mutation analysis . molecular and methylation proling one or more genomic variants were detected in 86.9% of all screened patient cases by mutation and cnv proling , with 85% of these being somatic . 
not all samples were tested with all three assays ( mutations , copy - number variations [ cnvs ] , and methylation ) because of dna fragmentation or limited amounts . the assays used for each particular patient case are marked with triangles above each testing panel . 
germline and somatic likely pathogenic ( 4 ) and pathogenic ( 5 ) variants , as well as somatic variants of uncertain signicance ( vus ; 3 ) , are shown . 
brca1 and brca2 class 4 or 5 mutations were detected in nine ( 9.6% ) and eight ( 8.5% ) of all screened cases ( n = 94 ) , respectively ; 12 of these were germline , and ve were somatic . 
an additional seven patients had somatic class 3 variants of uncertain signicance ( including one somatic heterozygous brca2 exon 2 to 27 deletion )  . variants in other homologous recombinationrelated genes , including palb2 , chek2 , atr , fanca , and rad51 , were identied in 10 patient cases . 
after adjusting the methylation proportions by percentage of neoplastic cells , two of the brca1 - methylated patient cases were predicted to have heterozygous methylation19 and ve were predicted to have homozygous methylation ; the rest could not be determined accurately ( appendix table a3 )  . potential clinical impact we assessed the potential clinical utility of the allocate study by annotating the detected mutations , cnvs , or methylation events using the oncokb database.22 a total of 78 potentially actionable events and four resistance events were detected in 60% ( 60 of 99 ) of the patients ( table 2 )  . the indicated targeted therapies included poly ( adpribose ) polymerase ( parp ) inhibitors for 31 detected events , including brca1 / 2 mutations ( evidence level 1 ) , brca1 or rad51c methylation ( level 3a ) , and other homologous recombination pathway events ( level 4 ) ; phosphatidylinositol 3 - kinase / mammalian target of rapamycin / akt inhibitors for four pik3ca hotspot mutations ( level 3b ) and 12 pten or arid1a mutations ( level 4 ) ; cdk2 / wee1 inhibitors for 11 ccne1 amplications ( level 4 ) ; human epidermal growth factor receptor 2 inhibitors for six erbb2 amplications ( level 4 ) ; mek inhibitors for eight kras or nf1 mutations ( level 4 ) ; ttk inhibitors for three ctnnb1 mutations ( level 4 ) ; braf inhibitors for two braf v600e mutations ( level 4 ) ; and alk / met inhibitors for one met amplication ( level 4 ; table 2 )  . improving selection of patients to determine which patients were most or least likely to benet from molecular proling , we assessed a number of including the number of potentially actionable criteria , events detected and the overall median survival in the patient cohort . 
likewise , stratication of patients by the histopathologic subtypes of their tumors did not greatly affect the number of potentially actionable events detected by the allocate testing ( fig 3c )  . clinical utility the primary aim of this study was to assess the feasibility and potential clinical utility of the molecular proling of ovarian cancer and not to determine the direct clinical benet that any patient or the cohort may derive , because the study was not directly linked to clinical trials for targeted therapies . 
nevertheless , a number of patients enrolled in the study went on to receive molecularly matched treatments on the basis of the results of the molecular proling of their tumors . as a result of molecular proling and subsequent pathology two of eight grade 2 serous carcinomas were review , reclassied from hgsoc to lgsoc , because in both cases a somatic kras mutation was detected with no tp53 mutation . 
one of these patients was subsequently enrolled in a phase ii clinical trial of anastrozole , an aromatase inhibitor , and achieved a partial response ( fig 4a )  . 
an additional six patients received matched therapy on the basis of the allocate results : one patient with high - level erbb2 amplication was treated with trastuzumab but did not respond ; one patient with lgsoc with a braf mutation was enrolled in a braf and epidermal growth factor receptor inhibitor combination clinical trial and achieved a partial response ( fig 4b ) ; and four patients with somatic ( n = 3 ) or germline ( n = 1 ) brca1 / 2 mutations were treated with parp inhibitors ( appendix table a4 )  . 
furthermore , a germline brca1 / 2 mutation was detected in three patients with hgsoc who had not previously undergone the genetic testing that has become routine for all patients with newly diagnosed hgsoc . 
these patients were subsequently referred to a familial cancer clinic for further management and cascade testing . in two of nine patients with brca1 / 2 mutations , where a recurrent fresh sample was tested , secondary mutations in brca1 / 2 were identied ( figs 4c to 4d ; appendix table a5 )  . 
 ( a ) kaplan - meier overall survival analysis of the patients from the time of referral to the allocate study stratied by duration of the primary platinum - free interval ( pfi ; n = 84 ) , where pfi could be determined . 
 ( b ) percentage of patient cases with potentially actionable events , as dened by oncokb , detected in the patients stratied by primary pfi ( n = 84 )  . 
 ( c ) percentage of patient cases with potentially actionable events identied by the allocate testing stratied by histologic subtype ( n = 99 )  . brca1 / 2 function and thereby caused platinum and parp inhibitor resistance , as has been previously reported by multiple studies.24 , 27 , 28 discussion the allocate study aimed to assess the feasibility and potential clinical utility of real - time molecular proling in the clinical management of advanced or recurrent ovarian cancer . 
we showed that events detected using multimodal targeted molecular proling panels , ( designed to identify point mutations , insertions , deletions , duplications , copynumber alterations , and methylation changes ) reected known common clinically relevant molecular events in different ovarian cancer subtypes.12 , 13 , 21 furthermore , we showed that molecular proling was clinically acceptable and feasible and had the potential to inuence clinical management of more than half of all screened patients , despite no formal linkage to open clinical trials . we showed that molecular proling has clinical utility by matching patients with targetable alterations to the corresponding targeted therapies , identifying clinically unsuspected germline familial cancerassociated mutations , reclassifying intermediate - grade serous tumors , and identifying secondary resistance mutations . we also assessed the acceptability , feasibility , and value of obtaining contemporary fresh biopsies for molecular proling . 
 ( a ) diagnosis - adjusted treatment ( aromatase inhibitor ) of one of two patient cases reclassied from high - grade to low - grade serous on the basis of the mutational ndings . 
 ( b ) recist ( version 1.1 ) measurements of the monitored tumor lesion in the patient treated with braf inhibitor ( combined with epidermal growth factor receptor inhibitor ) on the basis of the allocate ndings ( somatic braf v600e mutation ; treatment currently ongoing )  . 
the additional somatic variant is located 15 bases upstream from exon 8 and is predicted to create a new acceptor splice site ( hsf3 analysis ) , extending the length of exon 8 by 13 bases . 
taking into consideration both mutations , the reading frame of the protein is likely to be restored , with the predicted protein having an 11amino acid substitution and fouramino acid insertion . 
the study had to rely heavily on analysis of archival ffpe samples , which often pose challenges for molecular proling because of dna fragmentation and formalin - induced dna artifacts.29 , 30 although there have been a number of technologic improvements in recent years to allow generation of reliable sequencing results from ffpe samples , analysis of archival tissue may not provide up - to - date information about the current genomic status of the cancer . 
 kondrashova et al invasive way of obtaining an up - to - date snapshot of cancer molecular status.31 however , comprehensive molecular proling of liquid biopsies has technologic challenges resulting from the often low proportion of tumor - derived dna and is considerably more expensive compared with traditional biopsy proling , 32 and its feasibility still needs to be assessed for ovarian cancer . although molecular proling of cancer is becoming more acceptable in routine clinical practice , there is still a need to identify those patients who will derive the most clinical benet . 
optimized patient selection for molecular proling would maintain cost effectiveness , help reduce the potential harm of repeat biopsies , and avoid psychological stress and false hope where such testing is unlikely to alter patients management plans . 
we showed that the number of treatment lines received before study enrollment did not inuence the survival of patients and was deemed an unsuitable standalone indicator for whether the patient should undergo molecular proling of her cancer . 
it is especially important to include rare ovarian cancer subtypes in the molecular proling initiatives because of the limited treatment options available to these patients and the often - aggressive nature of such tumors.33 because molecular proling was often performed at cancer recurrence , the short survival limits the window of opportunity for patient enrollment in targeted therapy trials . 
importantly , patients with platinum - resistant cancers and rare subtypes are in most need of molecular proling as early as possible to help conrm the diagnosis and determine alternative treatment options . we showed that multimodal molecular proling was ethically acceptable , technically feasible , and logistically possible and has a potential place in the clinical management of ovarian cancer ; however , it also needs to match available drug targets to have clinical utility . 
waring provision of study material or patients : gwo - yaw ho , george au - yeung , orla mcnally , deborah neesham , marisa grossi , danny rischin , linda mileshkin collection and assembly of data : olga kondrashova , gwo - yaw ho , george au - yeung , leakhena leas , tiffany boughtwood , kathryn alsop , alexander dobrovic , yi - an ko , clare j . 
wakeeld , orla mcnally , michael quinn , sumitra ananda , deborah neesham , anne hamilton , marisa grossi , yada kanjanapan , danny rischin , nadia tracante , david bowtell , graham r . 
 allocate : molecular proling of ovarian cancer data analysis and interpretation : olga kondrashova , gwo - yaw ho , george au - yeung , giada zapparoli , arthur l . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . george au - yeung research funding : roche / genentech ( inst ) kathryn alsop research funding : astrazeneca ( inst ) alexander dobrovic honoraria : astrazeneca , amgen consulting or advisory role : astrazeneca , amgen patents , royalties , other intellectual property : provisional patent travel , accommodations , expenses : bio - rad matthew j . 
wakeeld travel , accommodations , expenses : merck sharp & dohme ( i ) michael quinn employment : tesaro anne hamilton research funding : glaxosmithkline ( inst ) , janssen ( inst ) , amgen ( inst ) , eli lilly ( inst ) , abbvie ( inst ) , clovis oncology ( inst ) alison freimund patents , royalties , other intellectual property : certied principal investigator of the clinical trial precise , which is receiving study drug and partial study funding from beigene pharmaceuticals travel , accommodations , expenses : roche yada kanjanapan travel , accommodations , expenses : merck sharp & dohme , astrazeneca / medimmune danny rischin research funding : genentech / roche , merck , amgen , regeneron , bristolmyers squibb , glaxosmithkline travel , accommodations , expenses : merck nadia tracante research funding : astrazeneca ( inst ) david bowtell honoraria : astrazeneca consulting or advisory role : astrazeneca research funding : roche / genentech , astrazeneca , beigene patents , royalties , other intellectual property : astrazeneca , genentech , roche clare l . 
scott research funding : roche / genentech , clovis oncology , sierra oncology , eisai patents , royalties , other intellectual property : royalty agreement for venetoclax ( inst ) expert testimony : astrazeneca travel , accommodations , expenses : astrazeneca other relationship : clovis oncology linda mileshkin travel , accommodations , expenses : beigene paul m . 
waring employment : dorevitch pathology , australian clinical laboratories , roche honoraria : illumina , roche , novartis , pzer , astrazeneca , merck consulting or advisory role : pillar biosciences , xing technologies travel , accommodations , expenses : pillar biosciences , dorevitch pathology no other potential conicts of interest were reported . acknowledgment we thank all of the women who participated in these research programs . the australian ovarian cancer study group ( aocs ) also acknowledges the cooperation of the participating institutions in australia and acknowledges the contribution of the study nurses , research assistants , and all clinical and scientic collaborators to the study . 
nat genet 50 : 1262 - 1270 , 2018 tothill rw , tinker av , george j , et al : novel molecular subtypes of serous and endometrioid ovarian cancer linked to clinical outcome . 
clin cancer res 14 : 5198 - 5208 , 2008 alexandrov lb , nik - zainal s , wedge dc , et al : signatures of mutational processes in human cancer . 
nature 500 : 415 - 421 , 2013 [ erratum : nature 502 : 258 , 2013 ] romero i , bast rc jr : minireview : human ovarian cancerbiology , current management , and paths to personalizing therapy . 
lancet 384 : 1376 - 1388 , 2014 goff b : measuring ovarian cancer care : why are we still failing ? gynecol oncol 136 : 1 - 2 , 2015 syn nl - x , yong w - p , goh b - c , et al : evolving landscape of tumor molecular proling for personalized cancer therapy : a comprehensive review . 
expert opin drug metab toxicol 12 : 911 - 922 , 2016 sholl lm , do k , shivdasani p , et al : institutional implementation of clinical tumor proling on an unselected cancer population . 
jci insight 1 : e87062 , 2016 barlesi f , mazieres j , merlio j - p , et al : routine molecular proling of patients with advanced non - small - cell lung cancer : results of a 1 - year nationwide programme of the french cooperative thoracic intergroup ( ifct )  . 
pfaff e , kessler t , balasubramanian gp , et al : feasibility of real - time molecular proling for patients with newly diagnosed glioblastoma without mgmt promoter hypermethylationthe nct neuro master match ( n2m2 ) pilot study . 
ross js , ali sm , wang k , et al : comprehensive genomic proling of epithelial ovarian cancer by next generation sequencing - based diagnostic assay reveals new routes to targeted therapies . 
richards s , aziz n , bale s , et al : standards and guidelines for the interpretation of sequence variants : a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology . 
kondrashova o , love cj , lunke s , et al : high - throughput amplicon - based copy number detection of 11 genes in formalin - xed parafn - embedded ovarian tumour samples by mlpa - seq . 
swisher em , lin kk , oza am , et al : rucaparib in relapsed , platinum - sensitive high - grade ovarian carcinoma ( ariel2 part 1 ) : an international , multicentre , open - label , phase 2 trial . 
aziz d , etemadmoghadam d , caldon ce , et al : 19q12 amplied and non - amplied subsets of high grade serous ovarian cancer with overexpression of cyclin e1 differ in their molecular drivers and clinical outcomes . 
 allocate : molecular proling of ovarian cancer appendix patients recruited ( n = 113 ) ( n = 59 ) pmcc ( n = 54 ) excluded insufficient neoplastic ( n = 8 ) cellularity or dna quantity poor - quality dna ( n = 3 ) no sample available ( n = 2 ) died before sample collection ( n = 1 ) patient samples analyzed ( n = 99 ) ( n = 51 ) pmcc ( n = 48 ) mutation panel version 1 , 29 genes ( n = 56 ) version 1.5 , 38 genes ( n = 38 ) panel , 11 genes no . 
patients were recruited from two tertiary hospitals in melbourne : royal womens hospital ( rwh ) and peter maccallum cancer centre ( pmcc )  . biopsy or archival tumor samples were screened by mutation and copy - number variation ( cnv ) panels where possible . 
dna fragmentation levels were assessed by a qc multiplex pcr assay targeting the gapdh gene , which should generate amplied products of 100 , 200 , 300 , and 400 bp in an unfragmented dna sample . 
each sample was scored on the basis of the number of visible amplied products : 100 - bp band corresponds to dna score of 1 ; 100 - bp and weak 200 - bp bands correspond to dna score of 1.5 ; 100 - bp and 200 - bp bands correspond to dna score of 2 ; 100 - bp , 200 - bp , and weak 300 - bp bands correspond to dna score of 2.5 ; 100 - bp , 200 - bp , and 300 - bp bands correspond to dna score of 3 ; 100 - bp , 200 - bp , 300 - bp , and weak 400 - bp bands correspond to dna score of 3.5 ; and 100 - bp , 200 - bp , 300 - bp , and 400 - bp bands correspond to dna score of 4 . the mutation panel required dna with little to no fragmentation ; therefore , very poor - quality dna samples had to be excluded from the analysis , and poor - quality dna samples had to be repeated in duplicate to reduce the number of dropout target regions . kondrashova et al very poor poor moderate good primary pfi < 12 months ( n = 37 ) 12 months ( n = 47 ) time ( years ) no . 
 ( a ) kaplan - meier overall survival analysis of the patients from the time of diagnosis stratied by the duration of the primary platinumfree interval ( pfi ; n = 84 ) where pfi could be determined . 
neoplastic cellularity on the basis of pathologist review of a representative tumor section stained with hematoxylin and eosin . pathogenicity classication : score of 3 represents variant of uncertain signicance ; score of 5 , pathogenic . 
 response of a metastatic breast carcinoma with a previously uncharacterized erbb2 g776v mutation to human epidermal growth factor receptor 2targeted therapy introduction we present a case of a patient with advanced estrogen receptor / progesterone receptorpositive , her2 - negative ( by fluorescent in situ hybridization ) breast cancer , refractory to estrogen receptortargeted therapy . 
in vitro experiments showed that erbb2 g776v is an oncogenic driver , and further largescale analysis of genomic profiles of breast carcinomas suggested the existence of a distinct subtype of her2 - negative breast cancer driven by a combination of activating erbb2 kinase domain mutations and inactivating map3k1 alterations . 
these observations suggest that patients with breast cancer who have relapsed / refractory disease can benefit from cancer genomic profiling to identify unanticipated opportunities for targeted therapy , including the use of combinatorial therapies with anti - her2 agents for those cases with erbb2 mutations . management of estrogen receptor ( er ) / progesterone receptor ( pr ) - positive breast cancer refractory to endocrine therapy presents a clinical challenge , although recent studies demonstrating the acquisition of esr1 mutations1 - 5 or alterations in the pi3k / mtor pathway6 - 8 have identified mechanisms of such resistance . 
in addition , unlike the breast cancer described in this case report , where the tumor was her2 negative , a small subset of er - positive breast cancers are also her2 positive and have been dubbed triple - positive cases ; in the absence of her2 - targeted therapy , these have a poor prognosis and worse response to endocrine therapy relative to er - positive breast cancers in general , 9 - 11 but they have been reported to respond to anti - her2 agents.11 , 12 recently , several lines of investigation have demonstrated that mutations in the her2 gene ( erbb2 ) are oncogenic drivers in breast carcinoma , as evidenced by patient responses to her2 - targeted therapy . 
erbb2 mutations such as s310f , l755s , v777l , and l869q have been shown to be strong drivers associated with clinical sensitivity to her2 - targeted agents.13 - 16 in addition , erbb2 mutations have been reported to be prevalent in invasive lobular breast carcinoma , which is typically er / pr positive , her2 negative , and cdh1 mutant.17 - 19 we report on a patient with advanced breast cancer harboring a previously uncharacterized erbb2 g776v variant , who derived clinical benefit from treatment with trastuzumab plus pertuzumab ( tp ) regimens . 
furthermore , breast carcinomas with erbb2 kinase domain ( kd ) mutations are marked by cosegregation with map3k1 alterations , and the majority of breast cancer cases with the g776v mutation are er positive , which are not general features of her2 - positive or erbb2 - mutant breast cancer and , thus , may denote a distinct class of breast carcinoma driven by erbb2 kd alterations . methods comprehensive genomic profiling dna was extracted from 40 microns of formalinfixed paraffin - embedded sections , and comprehensive yakov chudnovsky runjun d . 
 genomic profiling was performed in a clinical laboratory improvement amendments certified , college of american pathologistsaccredited reference laboratory ( foundation medicine ) on hybridization - captured , adaptor ligationbased libraries to a mean coverage depth of  . 
the construct was then shuttled into the pcfg5 retroviral vector ( which includes a zeocyn resistance marker and ires - gfp sequence ) using the in - fusion hd cloning system kit ( clonetech laboratories , mountain view , ca ) , and verified using sanger sequencing . retroviral particles were produced using fnx amphotrophic packaging cells . 
cells were selected under 10 mg / ml zeocin for 3 weeks . agar colony - formation assays , wells were seeded with 7 , 000 cells and grown for 2 weeks before imaging . 
codons 23652 were defined as the extracellular domain ( ecd ) , and codons 720987 were defined as the kd.22 the p value was calculated using logistic regression , and p values were corrected for multiple testing using the falsediscovery rate23 method to generate q values . the odds ratio ( or ) for each variant type is calculated from the logistic regression coefficient ; an or  . 
1.0 indicates more frequent co - occurrence than expected by chance , whereas an pr , 1.0 indicates less frequent co - occurrence than expected by chance . results case description for western blots , cells were serum starved for 6 hours before lysate harvesting . 
lysates were probed using the following antibodies : her2 ( monoclonal , ab - 17 ) from thermo fisher scientific ( waltham , ma ) , phospho - her2 ( py1248 ) from emd millipore ( billerica , ma ) , p44 / 42 mapk and phospho p44 / 42 mapk from cell signaling technologies ( danvers , ma )  . 
in july 2003 , the patient presented with a t2 n0 m0 , er - positive , pr - positive , her2negative ( nonamplified by fluorescent in situ hybridization , 2 + by immunohistochemistry ) invasive ductal carcinoma of the left breast . 
in december 2005 , she developed metastasis to the sternum and lung , and was initially treated with ovarian suppression and zoledronate . she received radiation therapy to the sternal lesions in december 2008 and again in may 2012 . 
additional antiestrogen therapies used included letrozole ( january 2009 to january 2011 and again from february 2012 to may 2013 ) and fulvestrant ( february 2011 to january 2012 )  . in early 2013 , positron emission tomographycomputed tomography ( pet - ct ) scans showed increasing activity in the lung lesions , and the patient underwent a video - assisted thoracoscopic surgery procedure in may 2013 . 
the ca15 - 3 tumor marker level decreased with capecitabine ( down from 391 to 85 u / ml , but then rising to 213 u / ml ) and also with estradiol ( decreasing further to 74 u / ml , but then rising to 132 u / ml ; fig 1 )  . 
trastuzumab , pertuzumab , and exemestane were started in october to november 2014 , and the patient responded well to this , with marked improvement on pet - ct scan and decrease in ca15 - 3 level to 43 u / ml . 
however , by february 2015 , the ca15 - 3 level rose to 58 u / ml and the patients treatment was changed to nabpaclitaxel , trastuzumab , and pertuzumab . 
the ca15 - 3 level decreased to a nadir of 31 u / ml ( normal range for ca15 - 3 is 0 to 31 u / ml ) , the pet - ct scan showed no areas of increased uptake , and the patient remained on this regimen for a total of seven cycles . 
in december 2015 , pet - ct showed new uptake in her lung nodules , and the patient enrolled in an unrelated clinical trial . preclinical characterization of her2 ( erbb2 ) g776v the her2 g776v mutant was stably overexpressed in nih 3t3 cells by retroviral transduction . capecitabine estradiol trastuzumab pertuzumab exemestane nabpaclitaxel trastuzumab pertuzumab maintanence trastuzumab date ascopubs.org / journal / po jco precision oncology 3 fig 1 . 
immunoblotting showed that these cell lines expressed elevated levels of active phosphorylated mapk ( similar to cells expressing her2 v777l ) , but nonelevated levels of phosphorylated her2 , which may be an artifact of the overexpression system ( fig 2a )  . 
we also assayed these cell lines for colony formation in soft agar . we found that cells expressing her2 g776v formed colonies at a rate similar to that of the her2 v777l mutant ( figs 2b and 2c )  . 
together , these results suggest that g776v is an activating mutation , causing increased signaling through the her2 / mapk pathway and allowing for anchorage - independent growth of nih 3t3 cells . genomic co - occurrence of map3k1 alterations with erbb2 alterations among the 10 , 790 breast carcinoma cases that have undergone cgp in our laboratory , 1 , 329 ( 12.3% ) have been found to harbor alterations in erbb2 , including 1 , 041 cases ( 9.6% ) with erbb2 amplification , 229 cases ( 2.1% ) with erbb2 short variants ( substitutions or inframe insertions or deletions ) , and 59 ( 0.5% ) with both ( table 1 )  . 
to identify additional genomic variants that may cooperate with erbb2 mutations in the pathogenesis of her2 - negative breast cancer , we calculated the co - occurrence of alterations in each of 345 genes profiled at foundation medicine with various alterations in erbb2 ( data supplement )  . 
as discussed , cdh1 alterations have been reported to co - occur with erbb2 mutations in the context of invasive lobular breast carcinoma.17 - 19 tbx3 has also been reported to be enriched for mutations in lobular breast carcinoma , 25 as well in neuroendocrine breast cancer , 26 but its role in breast cancer is not entirely clear : it has been suggested to be a tumor suppressor27 , 28 and an oncogene29 , 30 in different studies . 
we chose to further examine the tumor suppressor gene map3k1 , which was mutated in the patient described in this case report . alterations in map3k1 co - occur with erbb2 short variants significantly more frequently than expected by chance in breast cancer cases profiled in our laboratory , but erbb2 alterations , in general , are not significantly enriched for cooccurrence with map3k1 variants ( table 1 )  . furthermore , although activating mutations in erbb2 are found in both the ecd and the kd , 21 only kd mutations , and not ecd mutations , are significantly enriched for co - occurrence with map3k1 alterations ( table 1 )  . 
map3k1 alterations have been identified in 513 ( 4.8% ) of the 10 , 790 breast carcinoma cases profiled at foundation medicine and at a similar frequency range of 0% to 8% of breast cancers in several large - scale sequencing studies.27 , 31 - 33 map3k1 inactivation via mutation or copy number loss has been reported to be particularly prevalent in the luminal a subtype of breast cancer , which is distinct from the subtype that is characterized by erbb2 amplification.33 , 34 consistent with this , among the breast carcinoma cases profiled using our hybrid capture based assay , erbb2 copy number alterations co - occur with map3k1 variants significantly less frequently than expected by chance ( table 1 )  . 
notably , although subtype data were not available for the breast cancer cases profiled in our laboratory , logistic regression g776va v777l her2 wt parental ascopubs.org / journal / po jco precision oncology fig 2 . 
 ( a ) western blot showing expression of active phosphorylated and total her2 and mapk in untransduced ( parental ) nih 3t3 cells and those transduced with wt human her2 or the indicated mutants . 
g776va and g776vb are duplicate cell lines that were transduced independently . ( b ) representative images and ( c ) quantification of soft - agar colony formation ( anchorage - independent growth ) by untransduced ( parental ) nih 3t3 cells and those transduced with wt human her2 or the indicated mutants . 
 analysis to control for alteration status of the genes reported to be most frequently mutated in luminal a breast cancer ( ie , tp53 , pik3ca , gata3 , and cdh1 ) 33 retained significant enrichment of cooccurrence of map3k1 alterations with all erbb2 short variants and with erbb2 kd short variants ( table 2 )  . 
this indicates that the co - occurrence of erbb2 and map3k1 variants in a subset of breast carcinoma cases is independent of the status of luminal aassociated genes and cannot be attributed solely to these cases belonging to the luminal a subtype . 
these findings suggest that there may be a particular subtype of her2 - negative , predominantly er - positive breast cancer driven by a combination of activating erbb2 kinase domain mutations and inactivating map3k1 alterations . discussion this is the first demonstration of clinical response to tp regimens ( tp plus exemestane followed by tp plus nab - paclitaxel ) in a patient with a her2 kinase domain mutation ( erbb2 g776v ) and expands our knowledge of treatment options for patients with metastatic breast cancer containing erbb2 mutations . 
although it cannot be excluded that the clinical benefit was due to the exemestane or nab - paclitaxel , these findings are consistent with four previous studies that have reported responses of patients with erbb2 - mutant ( s310f , l755s , v777l , l869q ) breast cancer to antiher2 agents including antibodies and tyrosine kinase inhibitors , with a cytotoxic chemotherapy backbone in three of the four cases.13 - 16 in addition , studies in cultured cells and animal xenograft models have shown that multiple erbb2 mutations associated with erbb2 amplification - negative breast cancer are activating , oncogenic , and sensitive to the irreversible pan - her inhibitor neratinib and , in some cases , to the reversible pan - her inhibitor lapatinib and the anti - her2 antibody trastuzumab.21 the effects of the g776v mutation on her2 functions had not previously been characterized in published studies , although one study reported that this alteration is untargetable.35 however , including the substitution similar alterations , g776s36 and the compound substitution and insertion g776vinsc , 37 - 39 have been shown to be activating . 
 her2 - targeted agents have shown high rates of clinical benefit in her2 - positive breast cancers and are approved for both er - negative and erpositive patients.11 , 12 the clinical data for her2negative breast carcinoma cases driven by erbb2 mutations are considerably more limited . 
a recent large - scale study of nonamplification genomic alterations in erbb2 in breast cancer18 reported three patients who responded to anti - her2 therapy , two with er - positive tumors and one with triple - negative cancer.13 - 15 , 18 consistent with this , the patient we report here had er - positive cancer , and additional analysis showed that 80% ( four of five ) of breast cancers profiled at foundation medicine and found to carry the erbb2 g776v mutation were er positive . 
the previous study reported that several genes are frequently coaltered with erbb2 in erbb2 - nonamplified breast cancers , particularly the tumor suppressor tp53 and the oncogene pik3ca.18 here , we show that erbb2 - nonamplified breast carcinomas driven by erbb2 kinase domain mutations specifically are significantly enriched for co - alteration with the tumor suppressor map3k1 . 
although frequent alteration of map3k1 in the luminal / erpositive breast cancer subtype has been reported , 33 the current study provides evidence of association between erbb2 and map3k1 mutations independent of subtype and suggests that activating mutations in the erbb2 kinase domain may cooperate with inactivation of map3k1 to promote cancer progression . 
additional work will also be needed to elucidate the effects of er status on the clinical efficacy of her2 - targeted agents in erbb2 - mutated breast cancer . in this patients case , the clinical decision to use tp - based regimens was based on prior reports of similar erbb2 mutations in breast cancer or nsclc.36 - 42 , 46 , 48 the preclinical experiments reported here confirmed that erbb2 g776v is an activating mutation , and the patient had a good clinical outcome on tp - based therapy . 
this study adds to a body of evidence supporting the use of her2 - targeted therapy in patients with metastatic breast cancer with erbb2 mutations , although given that the tp - based regimens in this case included exemestane or nab - paclitaxel that may have significantly contributed to the clinical benefit , caution must be exercised in applying these findings to the treatment of other patients . 
for more information about ascos conflict of interest policy , please refer to or po.ascopubs.org / site / ifc . yakov chudnovsky employment : foundation medicine stock and other ownership interests : foundation medicine travel , accommodations , expenses : foundation medicine runjun d . 
schrock employment : foundation medicine stock and other ownership interests : foundation medicine caitlin connelly employment : foundation medicine kyle gowen employment : foundation medicine stock and other ownership interests : foundation medicine travel , accommodations , expenses : foundation medicine garrett m . 
miller employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine patents , royalties , other intellectual property : receive periodic royalties related to t790m patent awarded to memorial sloan - kettering cancer center jeffrey s . 
ross employment : foundation medicine leadership : foundation medicine stock and other ownership interests : foundation medicine honoraria : pfizer , emd merck serono research funding : foundation medicine siraj m . 
ali employment : foundation medicine stock and other ownership interests : exelixis , blueprint medicines , agios patents , royalties , other intellectual property : patents via foundation medicine , patents via seres health related to microbiome in non - neoplastic disease ( inst ) ron bose honoraria : genentech , novartis consulting or advisory role : genentech affiliations yakov chudnovsky , alexa b . 
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tokunaga e , kimura y , oki e , et al : akt is frequently activated in her2 / neu - positive breast cancers and associated with poor prognosis among hormone - treated patients . 
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schwab rb , koehler m , ali sm , et al : genomic profiling and treatment of her2 + , er + , pgr + triple positive breast cancer : a case report and literature review . 
ross js , slodkowska ea , symmans wf , et al : the her - 2 receptor and breast cancer : ten years of targeted antiher - 2 therapy and personalized medicine . 
ali sm , alpaugh rk , downing sr , et al : response of an erbb2 - mutated inflammatory breast carcinoma to human epidermal growth factor receptor 2 - targeted therapy . 
ben - baruch ne , bose r , kavuri sm , et al : her2 - mutated breast cancer responds to treatment with single - agent neratinib , a second - generation her2 / egfr tyrosine kinase inhibitor . 
chumsri s , weidler j , ali s , et al : prolonged response to trastuzumab in a patient with her2 - nonamplified breast cancer with elevated her2 dimerization harboring an erbb2 s310f mutation . 
grellety t , soubeyran i , robert j , et al : a clinical case of invasive lobular breast carcinoma with erbb2 and cdh1 mutations presenting a dramatic response to anti - her2 - directed therapy . 
ross js , wang k , sheehan ce , et al : relapsed classic e - cadherin ( cdh1 ) - mutated invasive lobular breast cancer shows a high frequency of her2 ( erbb2 ) gene mutations . 
ross js , gay lm , wang k , et al : nonamplification erbb2 genomic alterations in 5605 cases of recurrent and metastatic breast cancer : an emerging opportunity for anti - her2 targeted therapies . 
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shimamura t , ji h , minami y , et al : non - small - cell lung cancer and ba / f3 transformed cells harboring the erbb2 g776insv_g / c mutation are sensitive to the dual - specific epidermal growth factor receptor and erbb2 inhibitor hki - 272 . 
suzuki t , fujii a , ohya j , et al : antitumor activity of a dual epidermal growth factor receptor and erbb2 kinase inhibitor mp - 412 ( av - 412 ) in mouse xenograft models . 
wang se , narasanna a , perez - torres m , et al : her2 kinase domain mutation results in constitutive phosphorylation and activation of her2 and egfr and resistance to egfr tyrosine kinase inhibitors . 
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kris mg , camidge dr , giaccone g , et al : targeting her2 aberrations as actionable drivers in lung cancers : phase ii trial of the pan - her tyrosine kinase inhibitor dacomitinib in patients with her2 - mutant or amplified tumors . 
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sohal , md , mph1 a 53 - year - old woman with no signicant medical history presented to her primary care physician with fatigue and anorexia in may 2016 . 
she subsequently underwent a liver biopsy in september 2016 , and the pathology was consistent with adenocarcinoma ( strongly and diffusely positive for ck7 , with weak , patchy expression for ck20 ; programmed death ligand 1 , 2% )  . 
she was diagnosed with cancer of unknown primary , despite extensive workup revealing caudal - type homeobox 2 , thyroid transcription factor 1 , napsin a , gata - 3 , arginase , receptor 2 , and human epidermal growth factor mammaglobin negativity on immunohistochemical staining and no lesions on esophagogastroduodenoscopy or repeat colonoscopy ; however , the pathology was thought to be suggestive of a biliary origin . she received three doses of uorouracil and oxaliplathowever , she deteriorated clinically . 
genomic sequencing performed on her initial biopsied tissue ( foundationone ; foundation medicine , cambridge , ma ) was notable for mutations in braf v600e , apc , and cdkn2a ( table 1 )  . 
her case was reviewed at our genomics tumor board in december 2016 , and the recommendation was made to trial ( nci - match [ national pursue either a clinical cancer institute molecular analysis for therapy choice ; clinicaltrials.gov identier : nct02465060 ] or mypathway [ clinicaltrials.gov identier : nct02091141 ] ; each trial , the vemurafenib arm was open and available at the time ) or off - label treatment with dabrafenib and trametinib based on the asco 2016 update.1 at our institution , if two outside experts agree with the scientic data presented , off - label therapy can be approved on a case - by - case basis . given that she was not a clinical trial candidate because of the high bilirubin , off - label treatment with dabrafenib and trametinib was started in december 2016 . 
it is important to note that hepatic dysfunction , although closely monitored , is not a contraindication to initiating treatment with dabrafenib and trametinib . although serum alt elevations can occur in approximately 35% to 42% of patients receiving combination therapy , elevations greater than ve times the upper limit of normal occur in only 4% of such patients . 
in addition , there have been no instances of clinically apparent acute liver injury or hepatic failure in prelicensure studies , and there have been no published reports of clinically apparent hepatotoxicity with either drug.2 our patient was noted to experience clear clinical benet , with her total bilirubin and ast normalizing within just 2.5 months of treatment initiation and the remainder of her liver function enzymes continuing to downtrend . 
she had remarkable disease response , with radiographic ( fig 1 ) and clinical improvement until may 2018 ( a duration of almost 18 months ) , at which point there was concern about radiographic progression . 
cisplatin and gemcitabine were considered but not agreed to by the patient and family because of prior potential failure of oxaliplatin . her other option was to pursue a phase i clinical trial at an outside institution . 
her case was again discussed at the genomics tumor board in july 2018 , at which time there was deemed to be no targetable therapeutic option beyond the braf alteration , for which she had already been treated . 
interestingly , her second biopsy did identify some additional , albeit nontherapeutic , alterations not seen on the rst : rictor amplication and mtap loss of exons 6 to 8 . 
in a study analyzing these alterations in melanoma cell lines , it was found that v600e - mutant braf inhibits mammalian target of rapamycin complex 2induced phosphorylation of akt ser473 in the presence of pten . 
therapeutic implications of genomic sequencing , november 2016 sadaps and sohal fda - approved therapies genomic alterations detected * patients tumor type another tumor type potential clinical trials braf v600e none cobimetinib dabrafenib regorafenib nct01827384 nct01531361 ( phase i , targeting braf ) nct02034110 ( phase ii , targeting braf , mek ) trametinib nct02465060 ( phase ii , nci - match ) vemurafenib nct02693535 ( phase ii , tapur study ) apc r856c cdkn2a / b : cdkn2a loss exon 1 and cdkn2b loss none none none none none none abbreviations : fda , us food and drug administration ; nci - match , national cancer institute molecular analysis for therapy choice ; tapur , targeted agent and proling utilization registry . * variants of unknown signicance , 10 ; microsatellite stable ; low tumor mutational burden , 4 . discovered rictor amplication contributed to the development of braf inhibitor resistance . cleveland clinic to allow publication of this case have been obtained . per the request of the patient and her family and preliminary data extrapolated from the keynote 022 phase ii trial ( clinicaltrials.gov identier : nct02130466 ) , pembrolizumab ( via compassionate use ) was added to her regimen of dabrafenib plus trametinib starting in august 2018 . 
although high tumor mutational burden ( tmb ) has been demonstrated to be a useful biomarker in predicting immunotherapy responsiveness , 4 , 5 most studies ( eg , mypathway ) have used much higher tmb cutoffs , generally of at least 16 or more mutations per megabase , which far exceeds the tmb score obtained for our patient in either of her next - generation sequencing reports . 
she died in january 2019 , secondary to septic shock with multiorgan failure . because the patient died , consent could not be obtained ; however , no identifying personal health information has been disclosed . 
patients who receive standard - of - care gemcitabine and cisplatin chemotherapy have an overall survival ( os ) benet of 3.6 months when compared with gemcitabine monotherapy.6 even then , most patients develop treatment resistance within a few months , and median os remains less than 1 year . 
outcomes for second - line therapies have not been promising , with median progression - free survival of only a few months.7 - 13 recent advances in precision medicine have allowed for the identication of potentially targetable mutations in the effort to improve therapeutic outcomes in this population . promising targets identied in cholangiocarcinoma thus far include idh 1 / 2 , fgfr , her2 , and , the focus of our patient case , braf mutations.14 braf encodes protein kinases that function downstream of ras as part of the mitogenactivated protein kinase signaling cascade , which promotes cell proliferation , survival , and transformation . 
therapeutic implications of repeat genomic sequencing , june 2018 fda - approved therapies genomic alterations detected * patients tumor type another tumor type potential clinical trials braf v600e none cobimetinib dabrafenib regorafenib trametinib vemurafenib nct02091141 ( phase ii , mypathway ) nct02070549 ( phase i , targeting mek ) nct01989585 ( phase i / ii ) nct02428712 ( phase i / ii , targeting braf / craf ) nct02693535 ( phase ii , tapur study ) nct02034110 ( phase ii , targeting braf , mek ) apc r856c cdkn2a / b : cdkn2a loss exon 1 and cdkn2b loss riktor amplication none none none none none none none none nct02795156 ( phase ii ) nct02466802 ( phase i ) nct02097225 ( phase i ) nct01531361 ( phase i ) nct02576444 ( phase ii ) nct02142803 ( phase i ) nct03065062 ( phase i ) nct02159989 ( phase i ) nct02719691 ( phase i ) nct02583542 ( phase i / ii ) mtap loss of exons 6 to 8 none none none abbreviations : fda , us food and drug administration ; tapur , targeted agent and proling utilization registry . * variants of unknown signicance , 10 ; microsatellite stable ; low tumor mutational burden , 1 . and hyperactivate its downstream signaling.15 braf mutations are reported in up to 22% of biliary tract cancers , among which it is most commonly seen in intrahepatic cholangiocarcinoma . 
braf mutations have typically been associated with poor prognosis in patients with cholangiocarcinoma , including worse os , higher tumor grade , and chemotherapy resistance.16 there have been a few case reports on the use of dabrafenib and trametinib in cholangiocarcinoma , describing remarkable clinical and radiographic responses in patients who experienced progression during standard - of - care chemotherapy and who continued to do well 5 to 9 months out.17 , 18 most recently , a phase ii international clinical trial of dabrafenib plus trametinib in braf - mutated rare gi cancers was reported as a late - breaking presentation at the 30th european organisation for research and treatment of cancernational cancer instituteamerican association for cancer research symposium on molecular targets and cancer therapeutics . 
sohal honoraria : foundation medicine consulting or advisory role : perthera research funding : novartis ( inst ) , celgene ( inst ) , oncomed ( inst ) , bayer healthcare pharmaceuticals ( inst ) , genentech ( inst ) , bristol - myers squibb ( inst ) , agios ( inst ) , incyte ( inst ) , loxo ( inst ) travel , accommodations , expenses : foundation medicine no other potential conicts of interest were reported . references flaherty k , davies ma , grob jj , et al : genomic analysis and 3 - y efcacy and safety update of combi - d : a phase iii study of dabrafenib ( d ) + trametinib ( t ) vs d monotherapy in patients with unresectable or metastatic braf v600e / k - mutant cutaneous melanoma . 
plos one 7 : e42598 , 2012 chan ta , yarchoan m , jaffee e , et al : development of tumor mutation burden as an immunotherapy biomarker : utility for the oncology clinic . 
ann oncol 30 : 44 - 56 , 2019 legrand f , gandara d , mariathasan s , et al : association of high tissue tmb and atezolizumab efcacy across multiple tumor types . 
j clin oncol 36 , 2018 ( suppl ; abstr 12000 ) valle j , wasan h , palmer dh , et al : cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer . 
croitoru a , gramaticu i , dinu i , et al : fluoropyrimidines plus cisplatin versus gemcitabine / gemcitabine plus cisplatin in locally advanced and metastatic biliary tract carcinomaa retrospective study . 
fornaro l , vivaldi c , cereda s , et al : second - line chemotherapy in advanced biliary cancer progressed to rst - line platinum - gemcitabine combination : a multicenter survey and pooled analysis with published data . 
brugnara s , sicher m , bonandini em , et al : treatment with combined dabrafenib and trametinib in brafv600e - mutated metastatic malignant melanoma : a case of long - term complete response after treatment cessation . 
loaiza - bonilla a , clayton e , furth e , et al : dramatic response to dabrafenib and trametinib combination in a braf v600e - mutated cholangiocarcinoma : implementation of a molecular tumour board and next - generation sequencing for personalized medicine . 
presented at the 30th eortc - nci - aacr symposium on molecular targets and cancer therapeutics , dublin , ireland , november 13 - 16 , 2018 ( abstr 2 ) 20 . 
george , md1 ; and susan halabi , phd8 purpose androgen receptor splice variant 7 ( ar - v7 ) detection in circulating tumor cells ( ctcs ) is associated with a low probability of response and short progression - free ( pfs ) and overall survival ( os ) in men with metastatic castration - resistant prostate cancer ( mcrpc ) treated with enzalutamide or abiraterone . 
os , conrmed prostate - specic antigen ( psa ) , and objective radiologic responses were secondary end points . results we enrolled 118 men with mcrpc treated with abiraterone or enzalutamide , 51 of whom received subsequent docetaxel or cabazitaxel . 
all patients provided written informed consent under stitutional review board approval at all participating centers within the department of defensefunded prostate cancer clinical trial consortium.16 study design and assessments prophecy is a prospective multicenter study evaluating the ability of baseline ( pretreatment ) ar - v7 status in ctcs to predict treatment outcomes with abiraterone or enzalutamide as well as subsequent taxane chemotherapy upon disease progression . 
cellsearch ( menarini silicon biosystems , huntington valley , pa ) ctc enumeration was performed at these time points for all patients and processed in a college of american physicians / clinical laboratory improvement amendmentsapproved central laboratory at memorial sloan kettering cancer center.17 , 18 treatment selection was at the discretion of the treating physician without knowledge of ar - v7 status . 
all data sets were separately sent to the study statistician ( s.h. ) , who unblinded the data after database lock . analysis of ctcs ctcs were analyzed in two central laboratories , each blinded to the results of the other . 
baseline pretaxane characteristics of patients enrolled according to epic sciences ar - v7 status based on ar - v7 detection after abiraterone or enzalutamide and before taxane epic sciences ar - v7 status ( % ) positive ( n = 8 ) negative ( n = 42 ) 62 - 79 45 - 87 6 - 881 96.5 1 - 850 0 - 325 characteristic age , years median range race white black other gleason sum 8 - 10 kps 90 poor - risk feature psa , ng / ml median range taxane used docetaxel cabazitaxel months median range hemoglobin , 12 g / dl elevated alp elevated baseline serum ldh presence of liver metastasis presence of clinically signicant pain requiring cellsearch ctcs , cells per 7.5 ml opiates range median , % ( n = 32 ) psadt , 3 months prior docetaxel for mhspc  . 
20 bone metastases time from ar - v7 sample to chemotherapy start , 0 - 17 0 - 33 abbreviations : alp , alkaline phosphatase ; ar - v7 , androgen receptor splice variant 7 ; ctc , circulating tumor cell ; kps , karnofsky performance score ; ldh , lactate dehydrogenase ; mhspc , metastatic hormone - sensitive prostate cancer ; psa , prostate - specic antigen ; psadt , prostate - specic antigen doubling time . registration to clinical or radiographic progression or death , whichever occurred rst . 
radiographic progression was assessed at each center using pcwg3 - modied recist ( version 1.1 ) soft tissue and bone scan criteria.22 clinical progression was dened as a composite end point including death , escalating pain or other symptomatic progression , initiation of new systemic therapy , or a skeletal - related event . secondary clinical end points included conrmed 50% prostate - specic antigen ( psa ) decline , radiographic response per recist ( verson 1.1 ) , 23 and os , and these same outcomes for subsequent taxane chemotherapy . data analysis the primary objective of prophecy was to validate that patients with pretreatment ar - v7negative ctcs have prolonged pfs with abiraterone or enzalutamide compared with ar - v7positive patients . 
with longer follow - up , analyses of the time - toevent end points ( pfs and os ) were also updated , and patients who received taxane chemotherapy were observed for response , radiographic progression , and death . 
details regarding study design have been published elsewhere.9 patients with no evaluable ctcs were considered ar - v7 negative , and all patients with sufcient blood collection were analyzed regardless of their evaluable ctcs . 
in secondary analyses , the proportional hazards model was used for assessing the prognostic value of ar - v7 status for pfs and os after adjusting for validated prognostic factors ( risk score ) .14 , 15 , 24 for taxane outcomes , pfs was dened from the date of starting chemotherapy to clinical or radiographic progression or death , whichever occurred rst . 
no power or sample size analysis is provided for this secondary descriptive analysis , because prophecy was powered around the primary objective and previously reported . between may 2015 and january 2017 , we prospectively enrolled 118 men with high - risk mcrpc who were initiating treatment with abiraterone , enzalutamide , or both at one of ve academic medical centers . 
baseline characteristics of the cohort were previously published and included 36 men who had received prior enzalutamide or abiraterone therapy.9 of these 118 men treated with subsequent ar inhibitor therapy , 51 experienced progression and were treated with taxane chemotherapy , including docetaxel ( n = 42 ) or cabazitaxel ( n = 9 ; consort diagram provided in appendix fig a1 )  . 
baseline characteristics of these patients at the time of taxane chemotherapy are listed in table 1 based on epic sciences ar - v7 status and appendix table a1 based on johns hopkins ar - v7 status . 
as of the nal database lock on october 15 , 2019 , median follow - up times from study registration ( before abiraterone or enzalutamide treatment ) and taxane initiation were 35 and 23 months , respectively . 
table 2 and figure 1 summarize the results of pfs and os by baseline ar - v7 status for each ctc assay . conrmed 50% psa declines were observed in 11% and 30% of patients with ar - v7positive and ar - v7negative disease by johns hopkins assay , respectively , and radiographic responses ( complete or partial responses by recist [ version 1.1 ] criteria ) were seen in 7% and 9% of patients , respectively . 
results were unchanged when two patients with psa - only progression were considered censored ( appendix table a3 )  . os did not differ from the start of taxane chemotherapy by ar - v7 status . 
table 3 and figure 2 summarize the results of pfs and os by baseline ar - v7 status for each ctc assay . conrmed 50% psa responses with taxane therapy were observed in 36% and 32% of patients with ar - v7positive versus ar - v7negative disease by johns hopkins assay , respectively , and radiographic responses ( complete or partial responses by recist [ version 1.1 ] ) were observed in 10% and 20% of patients , respectively . 
conrmed 50% psa responses were observed in 40% and 33% of those with ar - v7positive versus ar - v7negative disease by epic sciences nuclear assay , respectively , and radiographic responses ( complete or partial responses by recist [ version 1.1 ] ) were observed in 13% and 17% of patients , respectively ( appendix fig a2 )  . 
 a pre - abi / enza at progression on abi / enza at progression on taxane nuclear localized ar - v7 ctc total ctc nuclear localized ar - v7 ctc nuclear localized ar - v7 ctc total ctc total ctc 123456789 2345789 * * * * * * * * patient id patient id patient id armstrong et al 123456789 2345789 patient id patient id patient id 123456789 2345789 patient id patient id patient id * * * * * * * fig 3 . 
bar plot demonstrating heterogeneity of androgen receptor splice variant 7 ( ar - v7 ) expression in circulating tumor cells ( ctcs ) by either the johns hopkins ( jhu ) messenger rna assay or the epic sciences nuclear protein assay collected from men in the prophecy study at one of three time points : ( a ) before abiraterone ( abi ) or enzalutamide ( enza ) therapy , ( b ) at progression during abiraterone or enzalutamide treatment , and ( c ) at progression during subsequent taxane chemotherapy . 
for the epic sciences nuclear protein assay , approximately 1 ml of blood is analyzed , and total and nuclear - localized ar - v7positive ctcs are expressed per 1 ml of blood . 
 ( * ) no data . disease pretreatment who received abiraterone or enzalutamide and pretaxane therapy . finally , we examined how ar - v7 changes during the treatment course from before abiraterone or enzalutamide to progression and then after progression during taxane chemotherapy . 
at baseline , ar - v7 positivity was 10% versus 24% by epic sciences and johns hopkins assays , respectively , with a majority of men with ar - v7positive disease by epic sciences ( nine of 11 ; 82% positive agreement ) also testing positive by johns hopkins assay . johns hopkinspositive , epic sciencesnegative results were observed , particularly in those men with low epic sciences ctcs or nonnuclear ar - v7 protein expression . although a majority of ctcs in men with mcrpc were ar - v7 negative , even in those with ar - v7positive disease , the proportion of ar - v7positive cells ranged from 1% to 100% ( median , 20% ; fig 3 )  . 
at progression during taxane chemotherapy , 33% ( four of 12 ) and 48% ( 12 of 25 ) of evaluable men had ar - v7 detected by epic sciences and johns hopkins criteria , respectively ( appendix table a4 )  . 
these results conrm previously published data showing ar - v7positive disease remains sensitive to taxane chemotherapy.11 - 13 in the secondor third - line mcrpc setting , cabazitaxel improves os and should be considered a reasonable third - line treatment option , 5 and docetaxel or cabazitaxel are effective treatments after progression with abiraterone or enzalutamide , with pfs estimates of 4 to 6 months in these poor - risk men.14 , 24 , 26 , 27 the results are particularly important with regard to current practice , given the recent proven benet and use of these same ar inhibitors in the metastatic hormone - sensitive pc and nonmetastatic crpc settings , where ar therapy cross - resistance concerns remain when a patient experiences progression to mcrpc during therapy.28 - 32 a majority of men with ar - v7positive disease by epic sciences assay ( nine [ 82% ] of 11 ) were also positive by johns hopkins assay ; however , 17 ( 60% ) of 28 patients with positive disease by johns hopkins assay were negative by epic sciences assay at baseline . 
these differences largely reect the differential sensitivities of the assays for ar - v7 detection in ctc - based rna expression versus protein and the distinction between nuclear versus cytoplasmic localization . 
some patients developed ar - v7positive disease at progression during abiraterone or enzalutamide treatment , despite being ar - v7 negative at baseline , whereas some men with ar - v7positive disease converted to arv7negative status at progression , illustrating the need to assess ctc biomarker status at each management decision point ( fig 3 )  . 
a limitation of our study includes the lack of random assignment to taxane or ar therapy based on arv7 test results , and as such , our results show a prognostic rather than predictive utility . 
 armstrong et al support supported by a grant from the prostate cancer foundation and movember ; by the department of defense prostate cancer clinical trials consortium , which provided infrastructural support ; by the national institutes of health and grant 1r01ca233585 - 01 , duke cancer institute ( dci ) grant no . 
p30 ca014236 , and dci shared resources for biostatistics , ow cytometry , and sequencing and genomic technologies ( a.j.a. ) ; in part by department of defense grants no . 
george , susan halabi manuscript writing : all authors final approval of manuscript : all authors accountable for all aspects of the work : all authors consulting or advisory role : bayer , sano , dendreon , medivation , janssen biotech , pzer , astellas scientic and medical affairs , clovis oncology , astrazeneca speakers bureau : dendreon , bayer research funding : dendreon ( inst ) , sano ( inst ) , bayer ( inst ) , pzer ( inst ) , novartis ( inst ) , janssen oncology ( inst ) , medivation ( inst ) , astellas pharma ( inst ) , gilead sciences ( inst ) , roche / genentech ( inst ) , active biotech ( inst ) , bristol myers squibb ( inst ) , constellation pharmaceuticals ( inst ) , merck ( inst ) patents , royalties , other intellectual property : circulating tumor cell novel capture technology ( inst ) travel , accommodations , expenses : dendreon , janssen biotech , bayer , astellas scientic and medical affairs jun luo consulting or advisory role : sun pharma , janssen oncology , tolero pharmaceuticals research funding : sano ( inst ) , orion pharma ( inst ) , mirati therapeutics ( inst ) , gilead sciences ( inst ) , astellas pharma ( inst ) , constellation pharmaceuticals ( inst ) , calibr ( inst ) , cardiff oncology ( inst ) patents , royalties , other intellectual property : coinventor of a technology assigned to johns hopkins university , which licensed to tokai pharmaceuticals ( inst ) ; coinventor of a technology licensed to qiagen ( inst ) ; coinventor of a technology licensed to a&g pharmaceuticals ( inst ) david m . 
nanus consulting or advisory role : roche / genentech research funding : novartis ( inst ) , boehringer ingelheim ( inst ) , zenith epigenetics ( inst ) , astrazeneca ( inst ) , immumedics ( inst ) , janssen ( inst ) , clovis oncology ( inst ) , pzer ( inst ) paraskevi giannakakou employment : novartis ( i ) stock and other ownership interests : novartis ( i ) patents , royalties , other intellectual property : coinventor on international patent application docket no . 
danila honoraria : angle , bayer , screencell , janssen oncology , pzer , aximmune , pzer , clovis oncology , astellas pharma consulting or advisory role : angle , bayer , sanador , aximmune , pzer , clovis oncology , astellas pharma , janssen scientic affairs research funding : prostate cancer foundation , genentech , janssen research & development ( inst ) patents , royalties , other intellectual property : gene expression prole associated with prostate cancer travel , accommodations , expenses : cambridge healthtech institute , prostate cancer foundation , screencell , stopcancer , american austrian open medical institute , janssen biotech , genzyme , pzer , janssen scientic affairs , astellas pharma andrew j . 
berry honoraria : pzer , merck , genomic health , janssen oncology consulting or advisory role : merck , pzer , genomic health , janssen oncology research funding : merck ( inst ) travel , accommodations , expenses : merck , pzer , genomic health , janssen oncology tian zhang leadership : capio biosciences ( i ) , archimmune therapeutics ( i ) stock and other ownership interests : capio biosciences ( i ) , archimmune therapeutics ( i ) , nanorobotics ( i ) honoraria : exelixis , genentech / roche , mjh life sciences , pacic genuity consulting or advisory role : janssen , genentech / roche , sano , exelixis , astrazeneca , pzer , bristol myers squibb , foundation medicine , pharmacyclics , amgen , merck , seattle genetics speakers bureau : exelixis , genentech / roche , genomic health , sano research funding : janssen ( inst ) , acerta pharma ( inst ) , pzer ( inst ) , merrimack ( inst ) , stem centrx ( inst ) , novartis ( inst ) , omniseq ( inst ) , personal genome diagnostics ( inst ) , regeneron ( inst ) , merck ( inst ) , mirati therapeutics ( inst ) , astellas pharma patents , royalties , other intellectual property : circulating tumor cell novel capture by c - met technology ( inst ) ; prochelators as targeted prodrugs for prostate cancer ( inst ) travel , accommodations , expenses : acerta pharma , genomic health , astrazeneca michael r . 
harrison consulting or advisory role : bayer , sano , exelixis , genentech , argos therapeutics , fujilm , janssen oncology , astrazeneca , pzer , bristol myers squibb speakers bureau : genentech , exelixis research funding : argos therapeutics ( inst ) , bristol myers squibb ( inst ) , genentech ( inst ) , pzer ( inst ) , medivation / astellas pharma ( inst ) , merck ( inst ) , clovis oncology ( inst ) , acerta pharma ( inst ) , astrazeneca ( inst ) changxue lu patents , royalties , other intellectual property : inventor of the patent that was licensed to qiagen and received royalty joseph d . 
scher leadership : asterias biotherapeutics stock and other ownership interests : asterias biotherapeutics honoraria : research to practice consulting or advisory role : janssen biotech , amgen , janssen research & development , menarini silicon biosystems , wirb - copernicus group , essa , sano , ambry genetics , konica minolta , pzer , bayer research funding : janssen ( inst ) , illumina ( inst ) , epic sciences ( inst ) , menarini silicon biosystems ( inst ) , thermosher scientic biomarkers ( inst ) travel , accommodations , expenses : asterias biotherapeutics , menarini silicon biosystems , amgen , wirb - copernicus group , konica minolta , essa , prostate cancer foundation , sano , bayer , phosplatin therapeutics richard wenstrup employment : epic sciences leadership : epic sciences stock and other ownership interests : epic sciences consulting or advisory role : blueprint genetics , resolys patents , royalties , other intellectual property : patent royalties from assurexhealth travel , accommodations , expenses : epic systems scott t . 
tagawa consulting or advisory role : medivation , astellas pharma , dendreon , janssen , bayer , genentech , endocyte , immunomedics , karyopharm therapeutics , abbvie , tolmar , qed , amgen , sano , pzer , clovis oncology , novartis , genomic health , point biopharma research funding : lilly ( inst ) , sano ( inst ) , janssen ( inst ) , astellas pharma ( inst ) , progenics ( inst ) , millennium pharmaceuticals ( inst ) , amgen ( inst ) , bristol myers squibb ( inst ) , dendreon ( inst ) , rexahn pharmaceuticals ( inst ) , bayer ( inst ) , genentech ( inst ) , newlink genetics ( inst ) , inovio pharmaceuticals ( inst ) , astrazeneca ( inst ) , immunomedics ( inst ) , novartis ( inst ) , aveo ( inst ) , boehringer ingelheim ( inst ) , merck ( inst ) , stem centrx ( inst ) , karyopharm therapeutics ( inst ) , abbvie ( inst ) , medivation ( inst ) , endocyte ( inst ) , exelixis ( inst ) , clovis oncology ( inst ) travel , accommodations , expenses : sano , immunomedics , amgen uncompensated relationships : telix pharmaceuticals , atlab pharma , phosplatin therapeutics emmanuel s . 
antonarakis honoraria : sano , dendreon , medivation , janssen biotech , essa , astellas pharma , merck , astrazeneca , clovis oncology consulting or advisory role : sano , dendreon , janssen biotech , essa , merck , astrazeneca , clovis oncology , lilly , bayer research funding : janssen biotech ( inst ) , johnson & johnson ( inst ) , sano ( inst ) , dendreon ( inst ) , aragon pharmaceuticals ( inst ) , exelixis ( inst ) , millennium pharmaceuticals ( inst ) , genentech ( inst ) , novartis ( inst ) , astellas pharma ( inst ) , tokai pharmaceuticals ( inst ) , merck ( inst ) , astrazeneca ( inst ) , clovis oncology ( inst ) , constellation pharmaceuticals ( inst ) patents , royalties , other intellectual property : coinventor of a biomarker technology that has been licensed to qiagen travel , accommodations , expenses : sano , dendreon , medivation daniel j . 
george leadership : capio biosciences honoraria : sano , bayer , exelixis , emd serono , onclive , pzer , urotoday , acceleron pharma , american association for cancer research , axess oncology , janssen oncology , millennium medical publishing consulting or advisory role : bayer , exelixis , pzer , sano , astellas pharma , innocrin pharma , bristol myers squibb , genentech , janssen , merck sharp & dohme , myovant sciences , astrazeneca , michael j . 
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armstrong aj , szmulewitz rz , petrylak dp , et al : arches : a randomized , phase iii study of androgen deprivation therapy with enzalutamide or placebo in men with metastatic hormone - sensitive prostate cancer . 
aggarwal r , huang j , alumkal jj , et al : clinical and genomic characterization of treatment - emergent small - cell neuroendocrine prostate cancer : a multiinstitutional prospective study . 
de laere b , van dam pj , whitington t , et al : comprehensive proling of the androgen receptor in liquid biopsies from castration - resistant prostate cancer reveals novel intra - ar structural variation and splice variant expression patterns . 
kohli m , ho y , hillman dw , et al : androgen receptor variant ar - v9 is coexpressed with ar - v7 in prostate cancer metastases and predicts abiraterone commun 7 : 13668 , 2016 resistance . 
de laere b , oeyen s , mayrhofer m , et al : tp53 outperforms other androgen receptor biomarkers to predict abiraterone or enzalutamide outcome in metastatic castration - resistant prostate cancer . 
 armstrong et al appendix assessed for eligibility ( n = 131 ) did not meet inclusion criteria ( n = 13 ) received hormone therapy abiraterone enzalutamide both ( n = 118 ) ( n = 55 ) ( n = 58 ) ( n = 5 ) progression during treatment death during treatment ( n = 105 ) ( n = 4 ) received subsequent taxane therapy docetaxel cabazitaxel ( n = 51 ) ( n = 42 ) ( n = 9 ) progression during treatment death during treatment ( n = 46 ) ( n = 3 ) fig a1 . 
waterfall plots of best overall prostate - specic antigen ( psa ) decline from pretaxane baseline during therapy with docetaxel or cabazitaxel according to ( a ) epic sciences and ( b ) johns hopkins androgen receptor splice variant 7 ( ar - v7 ) status . 
baseline pretaxane characteristics of patients enrolled according to johns hopkins ar - v7 status johns hopkins ar - v7 status ( % ) positive ( n = 20 ) 48 - 82 negative ( n = 31 ) 45 - 87 elevated baseline serum ldh presence of liver metastasis presence of clinically signicant pain requiring opiates cellsearch ctcs , cells per 7.5 ml baseline pretaxane characteristic age , years median range race white black other gleason sum 8 - 10 poor - risk feature hemoglobin , 12 g / dl elevated alp range median , % ( n = 37 ) psadt , 3 months prior docetaxel for mhspc  . 
 successful targeting of an atg7 - raf1 gene fusion in anaplastic pleomorphic xanthoastrocytoma with leptomeningeal dissemination mehdi touat , md1 ; nadia younan , md1 ; philipp euskirchen , md1 , 2 , 3 , 4 , 5 ; maxime fontanilles , md1 ; karima mokhtari , md1 ; caroline dehais , md1 ; patrick tilleul , pharmd6 ; amithys rahimian - aghda , msc1 ; adam resnick , phd7 ; anne - paule gimenez - roqueplo , md , phd8 , 9 , 10 ; h el `ene blons , pharmd , phd8 , 9 , 11 ; kh e hoang - xuan , md , phd1 ; jean - yves delattre , md , phd1 ; ahmed idbaih , md , phd1 ; pierre laurent - puig , md , phd8 , 9 , 11 ; and marc sanson , md , phd1 introduction pleomorphic xanthoastrocytoma ( pxa ) is an uncommon primary brain tumor occurring primarily in children and young adults.1 although pxa is typically considered a relatively indolent entity , some tumors are accompanied by anaplastic features associated with high rates of recurrence after local treatment and unfavorable outcome.2 accordingly , anaplastic pxa ( who grade 3 ) has been added to the 2016 who classication of cns tumors as a distinct entity.1 there is no standard management for patients with anaplastic pxa that recurs after surgery ; neither chemotherapy nor radiotherapy have demonstrated clinical benet in this disease.2 , 3 the identication of activating in recent years , brafv600e mutations in 50% of patients with pxa has rened our understanding of these disorders as malignancies driven by aberrant activation of the mitogenactivated protein kinase ( mapk ) signaling pathway.1 , 3 - 5 this was further supported by the observation of both preclinical and clinical activity of vemurafenib , a selective brafv600e inhibitor , in brafv600e - mutant gliomas.6 , 7 however , patients with braf wild - type pxa are not candidates for treatment with selective brafv600e inhibitors , and little is known about the spectrum of molecular alterations occurring in this population . 
recent studies showed that a subset of braf wild - type pxa might harbor rare braf or raf1 fusions , 8 , 9 which have been associated with exquisite sensitivity to mapk signaling inhibition in preclinical models.10 it remains unknown whether patients who have glioma with braf or raf1 gene fusions might respond to therapies targeting mapk signaling . 
histopathologic analysis showed a tumor composed of pleomorphic glial cells with the presence of mitoses , necrosis , and microvascular proliferation , consistent with who grade 3 anaplastic pxa ( fig 2 )  . 
although rhabdoid morphology was observed in some sectors , both epithelial membrane antigen and p53 immunostainings were negative , and we did not observe loss of ini1 , features that were previously associated with rhabdoid glioblastomas and cns atypical teratoid / rhabdoid tumors , respectively . 
furthermore , targeted sequencing of the tumor did not nd mutations in braf ( exons 11 and 15 ) , idh1 , idh2 , h3f3a , hist1h3b , or smarcb1 . 
array comparative genomic hybridization revealed a 9p deletion involving cdkn2a and cdkn2b , which have been previously associated with poor outcome in brafv600e - mutant gliomas.3 , 11 , 12 given the unfavorable prognosis , she was rst treated with adjuvant chemoradiotherapy ( 60 gy in 30 fractions over 6 weeks along with once - per - day temozolomide 75 mg / m2 ) followed by temozolomide ( 200 mg / m2 ) for 6 cycles until november 2015 ( fig 1a )  . in february 2016 , routine surveillance mri scans showed increased contrast enhancement in the right occipital lobe . 
 touat et al context key objective to report the successful treatment of a patient with refractory braf - wild - type anaplastic pxas harboring an in - frame atg7raf1 fusion with the mek inhibitor cobimetinib . knowledge generated we report for the rst time , to our knowledge , that raf1 gene fusions are actionable molecular events in high - grade gliomas . this report highlights the oncogenic role of mapk - activating alterations including raf1 rearrangements and brafv600e mutations in a subset of pediatric and adult gliomas . relevance cobimetinib can achieve therapeutic exposure within the cns , including the cerebrospinal uid in patients with raf1translocated high - grade gliomas . showed leptomeningeal enhancement in the posterior cerebral fossa ( fig 1b )  . 
neoplastic meningitis was conrmed by lumbar puncture showing 42 white blood cells per l ( 84% lymphocytes ) , hyperproteinorachia ( 0.55 g / l ) , and the presence of tumor cells in the cerebrospinal uid ( csf ; fig 1b )  . 
intravenous thiotepa ( 45 mg / m2 ) was begun , the treatment was discontinued after one infusion because of grade 4 thrombopenia and febrile neutropenia with infectious pneumonia , which required intravenous antibiotics . the patient was enrolled in the precision medicine program exorare for tumor molecular characterization using rna sequencing and matched tumor or normal whole - exome sequencing . 
reverse transcriptase - polymerase chain reaction and sanger sequencing conrmed the expression of an in - frame atg7raf1 fusion transcript in the recurrent tumor ( second surgery , 2016 ; fig 3b )  . 
interestingly , no additional molecular alteration was found by whole - exome among 475 genes previously associated with cancer , which suggested that the atg7 - raf1 fusion was the main oncogenic driver . moreover , previous reports showed that fusions involving exon 8 of raf1 are recurrent events in cancer including pxa8 , 9 , 13 and can result in constitutive activation of the tyrosine kinase domain and downstream mapk signaling through the loss of the n - terminal autoinhibitory domain of raf1.14 treatment with sorafenib ( 200 mg twice per day ) was then started in october 2016 through off - label use after informed consent . 
unfortunately , her clinical condition rapidly deteriorated with partial seizures , confusion , cerebellar ataxia , diplopia , hypoacousia , headaches , and diffuse pain in the lower limbs , for which treatment with intravenous morphine , steroids , and midazolam was begun . on the basis of preclinical data suggesting that mek inhibition in astrocytomas harboring raf1 fusions has superior efcacy , 10 sorafenib was discontinued and treatment with off - label cobimetinib ( 60 mg per day , 3 weeks on and 1 week off ) was started in november 2016 , after family members provided informed consent ( fig 1a )  . 
the patient dramatically improved after a few days of treatment , with complete regression of the confusion and headaches and partial regression of the cerebellar ataxia and pain in the lower limbs . 
subsequent lumbar puncture showed complete cytologic response in the csf ( fig 1b ) with regression of both pleocytosis ( 1 white blood cell per l ) and hyperproteinorachia ( 0.15 g / l ; fig 1b )  . 
 ( b ) top : serial contrast - enhanced t1 - weighted images of brain magnetic resonance imaging ( mri ) and cerebrospinal uid ( csf ; may - gr unwald - giemsa stain ) cytology at ( left ) initial presentation , ( middle ) pretreatment , and ( right ) during treatment with cobimetinib showing the right occipital lesion , the leptomeningeal enhancement on the brain mri ( white arrows ) , and the presence of neoplastic cells in the csf ( black arrowheads ) before treatment with cobimetinib was started . 
the observation of clinical activity with both braf and mek inhibitors suggest that brafand raf1 - driven gliomas , even in patients who have been heavily pretreated , display a high level of mapk pathway dependency and possibly less molecular heterogeneity than the majority of gliomas driven by other alterations such as egfr variants.20 - 22 despite previous reports indicating activity of sorafenib in treating patients with cancer who harbor mutations of braf or araf ( both paralogs of raf1 ) , 23 , 24 our patient did not respond to treatment with sorafenib . 
 ( a ) hematoxylin - eosin and ( b ) reticulin colorations showing a pleomorphic tumor proliferation , with the presence of xanthomatous cells with nuclear atypia , reticulin bers , microvascular proliferation , and necrosis . 
 ( c - f ) immunohistochemical stains showing tumor cell immunoreactivity for ( c ) glial brillary acidic protein , ( d ) cd34 , and ( e ) oligodendrocyte transcription factor 2 . 
 ( f ) high mitotic activity was noted on mib - 1 staining . alterations is currently ongoing ( nct02639546 ; safety and pharmacokinetics of cobimetinib in pediatric and young adult participants with previously treated solid tumors [ imatrixcobi ] ) , although adult patients are not yet eligible for this study . 
furthermore , novel mek inhibitors optimized for achieving higher brain exposure might have a role in these diseases.28 in conclusion , our report provides evidence for the oncoincluding raf1 reargenic role of mapk alterations , in pediatric and adult brain tumors and rangements , suggests that raf1 fusions may represent potential therapeutic targets in a subset of patients with braf wild - type high - grade gliomas . 
a recent report of a patient with melanoma with an ano10raf1 fusion reported long - lasting clinical improvement after treatment with the mek inhibitor trametinib , 29 suggesting that patients with raf1 fusiondriven tumors might respond to mek inhibition regardless of histology . 
for more information about asco 's conict of interest policy , please refer to or ascopubs.org / po / authorcenter . mehdi touat consulting or advisory role : agios pharmaceutical , taiho oncology travel , accommodations , expenses : merck sharp & dohme maxime fontanilles travel , accommodations , expenses : roche patrick tilleul honoraria : takeda , astellas pharma , roche , biogen , evidera , baxter , gr unenthal group , chiesi , bristol - myers squibb , msd , sandoz - novartis , pzer , astrazeneca h el `ene blons honoraria : astrazeneca , bristol - myers squibb , msd oncology kh e hoang - xhuan travel , accommodations , expenses : abbvie ahmed idbaih travel , accommodations , expenses : carthera pierre laurent - puig honoraria : amgen , astrazeneca , boehringer ingelheim , merck serono , merck , roche , sano consulting or advisory role : merck , bristol - myers squibb , boehringer ingelheim patents , royalties , other intellectual property : inventor of mir31 - 3p ( license to integragen ) travel , accommodations , expenses : roche , merck marc sanson consulting or advisory role : abbvie , genenta travel , accommodations , expenses : abbvie no other potential conicts of interest were reported . acknowledgment the authors thank ines detrait and delphine le corre for tissue preparation and acid nucleic extractions , and dr yvan bieche and integragen for the genomic analyses performed in this patient . references louis dn , ohgaki h , wiestler od , et al : world health organization histological classication of tumours of the central nervous system , 4th edition revised . lyon , france , international agency for research on cancer , 2016 ida cm , rodriguez fj , burger pc , et al : pleomorphic xanthoastrocytoma : natural history and long - term follow - up . 
brain pathol 25 : 575 - 586 , 2015 lassaletta a , zapotocky m , mistry m , et al : therapeutic and prognostic implications of braf v600e in pediatric low - grade gliomas . 
j clin oncol 35 : 2934 - 2941 , 2017 dias - santagata d , lam q , vernovsky k , et al : braf v600e mutations are common in pleomorphic xanthoastrocytoma : diagnostic and therapeutic implications . plos one 6 : e17948 , 2011 schindler g , capper d , meyer j , et al : analysis of braf v600e mutation in 1 , 320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma , ganglioglioma and extra - cerebellar pilocytic astrocytoma . 
clin cancer res 17 : 7595 - 7604 , 2011 kaley t , touat m , subbiah v , et al : braf inhibition in brafv600 - mutant gliomas : results from the ve - basket study . 
brain pathol 29 : 85 - 96 , 2018 jain p , fierst tm , han hj , et al : craf gene fusions in pediatric low - grade gliomas dene a distinct drug response based on dimerization proles . 
huillard e , hashizume r , phillips jj , et al : cooperative interactions of brafv600e kinase and cdkn2a locus deciency in pediatric malignant astrocytoma as a basis for rational therapy . 
 cobimetinib in anaplastic pleomorphic xanthoastrocytoma with raf1 fusion jones dt , hutter b , j ager n , et al : recurrent somatic alterations of fgfr1 and ntrk2 in pilocytic astrocytoma . 
nat genet 45 : 927 - 932 , 2013 johnson a , severson e , gay l , et al : comprehensive genomic proling of 282 pediatric lowand high - grade gliomas reveals genomic drivers , tumor mutational burden , and hypermutation signatures . 
reinhardt a , stichel d , schrimpf d , et al : anaplastic astrocytoma with piloid features , a novel molecular class of idh wildtype glioma with recurrent mapk pathway , cdkn2a / b and atrx alterations . 
szerlip nj , pedraza a , chakravarty d , et al : intratumoral heterogeneity of receptor tyrosine kinases egfr and pdgfra amplication in glioblastoma denes subpopulations with distinct growth factor response . 
cancer discov 4 : 956 - 971 , 2014 imielinski m , greulich h , kaplan b , et al : oncogenic and sorafenib - sensitive araf mutations in lung adenocarcinoma . 
casadei gardini a , chiadini e , faloppi l , et al : efcacy of sorafenib in braf - mutated non - small - cell lung cancer ( nsclc ) and no response in synchronous braf wild type - hepatocellular carcinoma : a case report . 
choo ef , ly j , chan j , et al : role of p - glycoprotein on the brain penetration and brain pharmacodynamic activity of the mek inhibitor cobimetinib . 
gampa g , kim m , cook - rostie n , et al : brain distribution of a novel mek inhibitor e6201 : implications in the treatment of melanoma brain metastases . 
blumenthal , md1 ; and reena philip , phd1 purpose next - generation sequencing ( ngs ) oncology panels are becoming integral in hospital and academic settings to guide patient treatment and enrollment in clinical trials . 
although ngs technologies have revolutionized decision - making for cancer therapeutics , physicians may face many challenges in parsing and prioritizing ngs - based test results to determine the best course of treatment for individual patients . 
here , we discuss the presentations and discussion highlights across the four sessions of the workshop . methods the goal of the public workshop was to engage stakeholders and solicit input from experts in precision oncology to discuss the integration of complex ngs data into patient management and regulatory innovation within the precision oncology community . 
the us food and drug administration gathered representatives from academia , industry , patient advocacy , government , and professional organizations for a series of presentations followed by panel discussions . 
after the workshop , the transcript and speaker presentation slides were reviewed and summarized for manuscript preparation . results speakers and panelists provided diverse perspectives on the integration of ngs technology into patient care for oncology and on the complexities that surround data interpretation and sharing . 
2019 by american society of clinical oncology introduction the goal of precision oncology is to use the genetic informationgermline or somaticfrom a patient with cancer to help determine which drug ( s ) might be most effective in treating his or her disease . 
next - generation sequencing ( ngs ) is increasingly used in precision oncology because of its ability to identify many mutations simultaneously , thereby maximizing the amount of information obtained from a single test.1 the global endeavor to identify somatic mutations within a patients tumor genome has led to an unprecedented amount of genomic information , with varying degrees of associated clinical evidence . 
ngs can report a vast number of mutations which may lead to clinician uncertainty in the interpretation and prioritization of mutations with respect to the clinical signicance and optimal course of treatment.2 in january 2017 , the association for molecular pathology ( amp ) , asco , and the college of american pathologists ( cap ) published a joint consensus recommendation for standards and guidelines for the interpretation and reporting of sequence variants in cancer3 ; however , stakeholders have not consistently implemented these recommendations . 
 horne et al context key objective to provide an overview of a public workshop hosted by the us food and drug administration that discussed key concepts and considerations for the classication and interpretation of genetic variants in precision oncology . knowledge generated key highlights and ndings from the discussion include the conclusion that as the use of next - generation sequencing oncopanels in the clinical setting increases , it is critical to develop a framework that ensures accurate and consistent interpretation of genetic variants . 
in addition , transparency in data sourcing and reporting methodologies , as well as frequent updating of variant interpretations , are necessary to advance science and appropriately care for patients . relevance as precision medicine advances , clear communication among stakeholders in oncology will be key to creating an environment that facilitates generating and sharing data that have value to patients . treatement options , in addition to providing tumor proling data.6 , 7 with fda approval and authorization of these two ngs - based pan - tumor oncology panels ( oncopanels ) , the agency was in a unique position to foster a dialogue among key stakeholders about this innovative technology . input on january 29 , 2018 , the fda held a public workshop to engage stakeholders and solicit from experts in precision oncology and patient representatives to discuss how genetic sequencing data could be best implemented in patient management . 
to set the stage for the workshop discussion , dr philip outlined the fdas new regulatory pathway for ngs tumor - proling tests , as summarized in the fdas center for devices and radiologic health fact sheet published online on november 15 , 2017.8 as described by dr philip , the agencys three - tiered approach for reporting biomarkers in tumor - proling ngs tests includes the following : level 1 , companion diagnostic claims ; level 2 , cancer mutations with evidence of clinical signicance ; and level 3 , cancer mutations with potential clinical signicance . 
tumor - proling ngs tests may include companion diagnostic claims ( level 1 ) that are prescriptive for a specic therapy and are supported by the clinical validity of the test for each biomarker reported . 
bowles , phd , myriad genetic laboratories best practices for the use of clinical databases for variant classication and facmg interpretation in clinical oncology session 4 : future directions for data sharing , standardization , and establishing consistency in precision oncology dane dickson , md cureone us food and drug administration public workshop : variant classication dennis dean , phd seven bridges genomics creating a community view : standards , analyses , and data to advance robert grossman , phd university of chicago the important role of data commons for sharing variant classication and consistency in precision oncology interpretation data in precision oncology len lichtenfeld , md american cancer society cancer action panelist network * afliations noted in the table are based on speaker afliations at the time of the public workshop and may not reect the current afliation . validity has not been demonstrated either in professional guidelines or with the test , but is suggestive on the basis of clinical / biologic evidence . session 1 : overview of the state of science for sequence variant classication in oncology and its practical use in treating patients use of an individuals mutation prole for clinical decision making is driving the ever - expanding number of biomarkerbased clinical trials and clinical regimens in precision oncology . 
 horne et al vincent miller , md , indicated that fmi lters out germline mutations but further noted that when reporting germline mutations , laboratories need to have an operation in place , or at least have an understanding with the clinicians , so that the information can be followed up with genetic counseling . comprehensive variant annotation is necessary to inform treatment selection and match patients to clinical trials . 
the number of patients who are matched to either clinical trials or personalized treatments on the basis of their genomic data were reported by panelists to be between 10% and 37% . 
to compile and interpret the evidence , scientists and pathologists continually gather new information from guidelines , published literature , public databases , clinical trials , fdaapproved therapeutic labeling , and other sources . 
this comprehensive information is then used to sort biomarkers , either manually or in combination with bioinformatics software algorithms , on the basis of levels of evidence . panelists concluded that a key to the success of somatic genotyping will be developing statistical tools and bioinformatics infrastructure to identify clinically meaningful mutations . 
dr berger noted that as more sequencing data accumulates , integrated analyses of big data will allow for the identication of novel hotspot mutations occurring at frequencies greater than would be expected by chance . this approach would allow for these novel mutations to be directly tested in targeted clinical trials to assess drug sensitivity . 
panelists noted that the eld will continue to evolve and become more challenging with the increase in tissue - agnostic trials in which patients are enrolled on the basis of biomarker status , irrespective of the tumor type , which could , in turn , lead to an increase in tissue - agnostic drug indications . 
john deeken , md , also highlighted some of the unique challenges faced when running an in - house 50 - gene oncopanel at a community - based hospital system . these challenges included the cost of developing and supporting an oncopanel test and the lack of reimbursement for the majority of ngs testing performed . 
donna roscoe , phd , noted that the fda is striving to ensure that there are validated tests on the market that will allow for dynamic use within clinical settings to enable clinicians to optimize patient decisions in oncology and other therapeutic areas . session 2 : levels of evidence required for reporting variants and guiding patient treatment widespread implementation of ngs data in clinical settings has been accompanied by challenges in standardization , interpretation , and reporting of genetic tests . 
shashikant kulkarni , phd , provided an overview of the proposed guidelines published by a multidisciplinary working group convened by amp , asco , and cap for variant interpretation and reporting.3 the four - tiered system to categorize somatic sequence variations on the basis of their levels of evidence are as follows : tier i , variants with strong clinical signicance ; tier ii , variants with potential clinical signicance ; tier iii , variants of unknown clinical signicance ; and tier iv , variants deemed benign or likely benign.3 guidelines were developed to provide a general framework , on the basis of an evidence - based variant classication approach , for the standardized interpretation and reporting of somatic variants in cancer . 
for example , variants with strong clinical evidence include those with level a evidence ( fda - approved therapies or included in professional guidelines ) or level b evidence ( well - powered studies with consensus from experts in the eld )  . 
variants with potential clinical evidence are those with level c evidence ( fda - approved therapies for different tumor types or investigational therapies of multiple small published studies with some consensus ) or level d evidence ( preclinical trials or found in a few case reports without consensus )  . in general , it is widely accepted that tests should be optimized to ensure coverage across targets and high sensitivity for detecting low - frequency mutations ; however , as noted in the consensus manuscript , 3 the eld is split on the use of variant allele frequency ( vaf ) in reporting and clinical practice . 
one perspective , provided from a workshop attendee , was that if frequencies are too low , the detected mutations could represent subclonal events and may not necessarily be appropriate therapeutic targets . 
on the contrary , apostolia - maria tsimberidou , md , phd , noted that , as a clinical investigator , there is utility in having allele fraction present in the patient report as the data could be used to interpret clinical outcomes , particularly because patients have multiple molecular alterations . 
a consensus was not reached by workshop panelists or attendees regarding the clinical use or validity of reporting vaf in precision oncology . standardizing the annotation and reporting of somatic mutations from ngs data remains a challenge . 
john pfeifer , md , phd , pointed out that there is a gray zone between where the technical portion of the ngs test ends and the practice of medicine begins . 
although many of the speakers and panelists used the amp / asco / cap guidelines , several groups used a slightly modied tiering system . panelists generally agreed that treatment options for tier ii and below , as noted in the amp / asco / cap guidelines , should be considered only once tier i treatment options have been exhausted . 
moreover , panelists expressed the need to create a databasethat is , a knowledge basethat can assist in resolving discordances in variant interpretation and allow for the generation of community standards . session 3 : best practices for use of public / private databases for variant classication and interpretation in oncology to positively affect patient care , mutations detected using ngs technology must be appropriately interpreted and classied . 
the panel unanimously agreed that a known drawback to using publicly available databases for variant classication is that interpretation is not always consistent . heidi rehm , phd , noted that the quality of the data rests in the hands of those who deposit the information into the database and that amassing sufcient information to provide high - quality classication data requires global sharing . kenna shaw , phd , afrmed that standards and rules are needed to not only interpret but reinterpret variant classication , and she summarized the efforts being conducted at her institution to amass a personalized cancer therapy knowledge base . 
dr shaw provided an overview of aacr project genie ( genomics evidence neoplasia information exchange ) , which presently includes 17 institutions across the world that are working together to develop a regulatorygrade registry that aggregates and links cancer genomic data with clinical outcomes from tens of thousands of cancer patients . 
project genie will allow for sharing of the accumulated data with the global research community through cbioportal and the synapse platform with the noted caveat that the underlying functional evidence int the knowledge base will continue to change over time . panelists agreed that databases should be transparent about data sources , methods , and rules used for reporting mutation - associated clinical claims . 
karla bowles , phd , noted that subjective tools , such as literature reviews , population data , and structural analysis , should be used with caution , as interpretations from these data are often sources of discrepancies . 
the number of session 4 : future directions for data sharing , standardization , and establishing consistency in precision oncology many mutations identied by genomic tests for oncology are novel , rare , or otherwise lack conclusive evidence to support a clinical these mutations will only increase as additional genes are added to oncopanels and the costs of sequencing decrease . understanding the clinical utility of these mutations requires addressing barriers that prevent accurate classication . 
dane dickson , md , cited improved data sharing as a critical need and described the utility of high - quality databases that include both diagnostic results and clinical outcomes . dennis dean , phd , described the need for interoperability of complex systems and tools to enable analysis of large data sets . interoperability requires standards and languages to support reproducibility . 
dean discussed rabix , a suite of open - source development tools for creating and running computation workows , as a development environment that uses common workow language to promote the analysis of combined large data sets . 
robert grossman , phd , emphasized the need for data commonsthat is , a unied data system that promotes sharingsuch as the national cancer institute that use data models and genomic data commons , metadata services to harmonize and share cancer genomic datasets . 
grossman discussed the concept of leveraging funders , journal editors , and payers to incentivize the data sharing required to build a data commons . as a patient advocate , len lichtenfeld , md , discussed the challenges of translating the information generated by large - scale projects to communities that lack the resources and infrastructure to access the latest information necessary to improve care . 
 horne et al in conclusion , precision oncology is a rapidly evolving eld . with more oncopanels being developed , and as more genomic and patient outcome data accumulate , physicians are faced with new challenges in interpreting ngs - based test results to determine the best treatment course for each patient . 
in addition , there are multiple public and private databases available that aid in variant classication and interpretation ; however , methods used to classify variants and how these databases align remain unclear . 
to that end , the fda recently nalized a guidance document entitled , use of public human genetic variant databases to support clinical validity for genetic and genomic - based in vitro diagnostics . 
david litwack employment : prevail therapeutics travel , accommodations , expenses : prevail therapeutics no other potential conicts of interest were reported . acknowledgment the authors thank all event staff at the us food and drug administration for helping to execute a seamless public workshop that was benecial to both in - person and online attendees . 
the authors thank the workshop presenters who graciously provided their permission to publish details about their titles and summaries in the manuscript table and text . references strom sp : current practices and guidelines for clinical next - generation sequencing oncology testing . 
n engl j med 375 : 711 - 713 , 2016 li mm , datto m , duncavage ej , et al : standards and guidelines for the interpretation and reporting of sequence variants in cancer : a joint consensus recommendation of the association for molecular pathology , american society of clinical oncology , and college of american pathologists . 
us food and drug administration : use of public human genetic variant databases to support clinical validity for genetic and genomic - based in vitro diagnostics : guidance for stakholders and food and drug administration staff . 
us food and drug administration : public workshop - - weighing the evidence : variant classication and interpretation in precision oncology , january 29 , 2018 , workshops and conferences ( medical devices )  . 
 c long - term complete response of an androgen receptorpositive triple - negative metastatic breast cancer to abiraterone acetate introduction several gene expression array studies have identified a new breast cancer subtype characterized by the expression of the androgen receptor ( ar ) , absence of the estrogen receptor ( er ) , and paradoxical expression of many genes that are typically expressed in er - positive luminal tumors.1 - 3 two thirds of this tumor subtype are human epidermal growth factor receptor 2 ( her2 ) positive . 
these ar - positive and triple - negative tumors have been identified as a stable subtype in two series of tnbcs.4 , 5 of note , ar is also expressed in approximately 75% of er - positive / her2 - negative breast cancers.6 however , clinical and preclinical data suggest that ars act as a tumor suppressor gene in er - positive tumors and as an oncogene in er - negative tumors.7 in both retrospective series8 and prospective clinical trials , 9 - 11 the following immunohistochemistry ( ihc ) definition is classically used to identify this breast cancer subtype : ar positive and er , progesterone receptor ( pr ) negative , and her2 negative . 
there is no universal consensus on defining ar positivity to date , although the accepted definition of ar positivity is 10% . preclinical in vitro2 , 12 - 14 and in vivo14 , 15 data suggest that androgens are involved in the development and growth of the mda - mb - 453 cell line , which is used as an in vitro model for the ar - positive tnbc subtype.2 consequently , in three prospective clinical trials , either competitive antagonists of the ar ( bicalutamide or enzalutamide ) or androgen synthesis suppressor , cyp17 inhibitor ( abiraterone acetate , aa ) , were evaluated in ar - positive tnbcs . 
 she received neoadjuvant chemotherapy ( three cycles of fluorouracil , epirubicin , and cyclophosphamide followed by three cycles of docetaxel ) followed by mastectomy and axillary lymph node dissection ( level 1 and 2 )  . 
 the arrows show the index masses with changes with therapies . multiple sclerosis and administered interferon at a dose of 30 g per week . in november 2007 , the patient was diagnosed with an ipsilateral axillary lymph node relapse without distant metastasis . 
no further treatment was proposed . in july 2013 , she presented with a 4 - cm left axillary mass with skin involvement ( fig 1a ) and two supraclavicular 1 - cm lymph nodes ; a core biopsy of the axillary mass confirmed tnbc , with a proliferation index ( ki - 67 ) of 15% . 
on the computed tomography scan ( figs 1b and 1c ) , in addition to the axillary mass , several chest soft tissue masses were observed ; no visceral or bone metastasis was observed . 
the patient was included in the ucbg 2012 - 1 trial and received experimental treatment with 1 , 000 mg of aa and 10 mg of prednisone administered orally , daily . 
ten months after the treatment initiation , clinical and computed tomography scan evaluations showed that the patient achieved a cr ( on the basis of the response evaluation criteria in solid tumors criteria )  . 
after 4 years , the patient is still receiving the treatment with aa and low - dose prednisone , with a prolonged cr ( figs 1d - 1f )  . 
concomitantly , she has been receiving interferon treatment for multiple sclerosis . further ihc analyses later performed on the 2006 mastectomy and the 2007 axillary relapse showed that 100% of cells were ar positive . 
 furthermore , ihc analyses on additional markers in the 2013 axillary relapse confirmed a pronounced apocrine feature with marked forkhead box a1 and gross cystic disease fluid protein 15 expression . 
ongoing clinical trials on ar - positive triple negative breast cancer in metastatic setting single agent combination antiandrogen ar antagonist enzalutamide ( phase iii , clinicaltrials.gov identifier : nct02929576 , withdrawn ) bicalutamide v chemotherapy ( phase iii / first line , clinicaltrials . gov identifier : nct03055312 , recruiting ) darolutamide v chemotherapy ( randomized phase ii , first and second line , clinicaltrials.gov identifier : nct03383679 , recruiting ) bicalutamide plus cdk4 / 6 inhibitor ribociclib ( phase i / ii , clinicaltrials.gov identifier : nct03090165 , recruiting ) bicalutamide plus cdk4 / 6 inhibitor palbociclib ( phase i / ii , clinicaltrials.gov identifier : nct02605486 , recruiting ) enzalutamide plus pi3k inhibitor alpelisib ( phase i , clinicaltrials.gov identifier : nct03207529 , not yet recruiting ) or plus pi3k inhibitor taselisib ( phase i / ii , clinicaltrials.gov identifier : nct02457910 , recruiting ) cyp17a1 inhibitors orteronel ( tak - 700 ; phase ii , clinicaltrials.gov identifier : nct01990209 , recruiting ) seviteronel ( phase i / ii , clinicaltrials.gov identifier : nct02580448 , recruiting ) selective ar modulator cr1447 ( phase ii , clinicaltrials.gov identifier : nct02067741 , recruiting ) enobosarm plus pembrolizumab ( phase ii , clinicaltrials.gov identifier : nct02971761 , recruiting ) enobosarm ( phase ii , clinicaltrials.gov identifier : nct02368691 , terminated ) abbreviations : ar , androgen receptor ; cdk , cyclin - dependent kinase ; her2 , human epidermal growth factor receptor 2 ; pi3k , phosphatidylinositol 3 - kinase . from a clinicians perspective , the cbrs at 6 months ( 19% to 29% ) observed in this breast cancer subtype in the three prospective trials testing ar antagonist or androgen synthesis inhibitor are modest but clinically relevant . 
patients with ar - positive tnbcs seem to be older compared with the whole tnbc population.9 - 11 , 17 they also tend to relapse after a long interval after the primary tumor treatment . 
in our opinion , in ar - positive tnbcs , treatment choice between antiandrogens and chemotherapy could take into account different factors , including disease - free interval , tumor burden , and the need for rapid disease / symptom control , similar to luminal erand / or pr - positive tumors . 
of note , it is important to remember that progression - free survival for first - line chemotherapy is merely 4 to 6 months.18 , 19 from a researchers perspective , cbrs at 6 months with antiandrogens in this tumor subtype are much lower than those observed with anti - aromatase inhibitors used as single agents in luminal erand / or pr - positive cancers . 
in the latter group , the historical cbrs range from 49% to 59% for first - line and from 24% to 37% for second - line treatments , respectively.20 hence , there is a need to improve these results in ar - positive tnbcs , and we briefly outline four key areas that could be improved . first , ihc is probably not ideal to identify this breast cancer subtype . 
of note , in the enzalutamide trial , 10 patients with low ar ( 1% to 9% ) were included in an exploratory cohort , and some of these patients responded . 
in two large series of tnbcs analyzed by gene expression array , 11% and 17% of tumors were ar positive.4 , 5 in a tnbc series analyzed by ihc , 22% to 35% of patients were ar positive.8 it is difficult to explain this difference . 
hence , there is a need to better identify this subtype in clinical practice , for example , by using rna signatures with a selected number of transcripts.5 second , predictors of antiandrogen treatment sensitivity in this breast cancer subtype should be developed . 
new preclinical models are needed ; the reliability of the mda - mb - 453 cell line has been in question because of genomic alterations , such as ar mutation.22 , 23 this ar q865h mutation in its ligand - binding domain is reported to decrease sensitivity to dihydrotestosterone , limiting its use to test antiandrogen therapy.22 a new patient - derived xenograft model of molecular apocrine tumors , directly derived from patients , could be a solution.24 finally , we need better drugs and combinations . 
 however , both enzalutamide and abiraterone have shown to improve the sensitivity of tnbc cell lines toward immune - mediated lysis , providing potential rationale for combination of immunotherapy with antiandrogen therapy.25 in conclusion , we believe that this patients case is important for clinicians . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center  . thomas grellety consulting or advisory role : novartis herv bonnefoi consulting or advisory role : abbvie , astellas pharma , bayer healthcare pharmaceuticals , innocrin pharmaceuticals , puma biotechnology research funding : bayer ( inst ) travel , accommodations , expenses : roche , pfizer acknowledgment we thank ravi nookala , phd , of institut bergoni for the medical writing service . affiliations thomas grellety and herv bonnefoi , institut bergoni unicancer , universit bordeaux , institut national de la sant et de la recherche mdicale u1218 , institut national de la sant et de la recherche mdicale cic1401 ; and thomas grellety , gaetan macgrogan , camille chakiba , michele kind , and herv bonnefoi , institut bergoni , bordeaux , france . support supported by the fondation pour la lutte contre le cancer pour les recherches medico - biologiques , site de recherche intgre sur le cancer - bordeaux recherche intgre oncologie , and groupement interrgional de recherche clinique et dinnovation sud - ouest outre - mer grant no . 
vera - badillo fe , templeton aj , de gouveia p , et al : androgen receptor expression and outcomes in early breast cancer : a systematic review and meta - analysis . 
hickey te , robinson jl , carroll js , et al : minireview : the androgen receptor in breast tissues : growth inhibitor , tumor suppressor , oncogene ? mol endocrinol 26 : 1252 - 1267 , 2012 8 . 
gucalp a , tolaney s , isakoff sj , et al : phase ii trial of bicalutamide in patients with androgen receptor - positive , estrogen receptor - negative metastatic breast cancer . 
traina ta , miller k , yardley da , et al : results from a phase 2 study of enzalutamide ( enza ) , an androgen receptor ( ar ) inhibitor , in advanced ar + triple - negative breast cancer . 
bonnefoi h , grellety t , tredan o , et al : a phase ii trial of abiraterone acetate plus prednisone in patients with triple - negative androgen receptor positive locally advanced or metastatic breast cancer ( ucbg 12 - 1 )  . 
lehmann bd , bauer ja , schafer jm , et al : pik3ca mutations in androgen receptor - positive triple negative breast cancer confer sensitivity to the combination of pi3k and androgen receptor inhibitors . 
arce - salinas c , riesco - martinez mc , hanna w , et al : complete response of metastatic androgen receptor - positive breast cancer to bicalutamide : case report and review of the literature . 
oshaughnessy j , schwartzberg l , danso ma , et al : phase iii study of iniparib plus gemcitabine and carboplatin versus gemcitabine and carboplatin in patients with metastatic triple - negative breast cancer . 
miles dw , diras v , corts j , et al : first - line bevacizumab in combination with chemotherapy for her2 - negative metastatic breast cancer : pooled and subgroup analyses of data from 2447 patients . 
moore nl , buchanan g , harris jm , et al : an androgen receptor mutation in the mda - mb - 453 cell line model of molecular apocrine breast cancer compromises receptor activity . 
vranic s , gatalica z , wang zy : update on the molecular profile of the mda - mb - 453 cell line as a model for apocrine breast carcinoma studies . 
 response of nf1 - mutated melanoma to an mek inhibitor introduction molecular classification of melanoma has established three subtypes of braf wild - type tumors , 1 nf1 mutant ( 10% to 15% ) , ras mutant ( 28% ) , and triple - negative ( 10% ) , for which treatment options are limited . 
his sister had been diagnosed with acute leukemia but family history was otherwise noncontributory . after wide local excision , histopathologic examination revealed an ulcerated nodular melanoma , breslow 4.8 mm , clark v , and 2 mitoses / mm2 ( stage pt4b , american joint committee on cancer , 7th edition )  . 
the sentinel lymph node biopsy was positive , motivating a parotidectomy and left cervical lymphadenectomy , which removed 33 lymph nodes ( seven positive , one with extracapsular extension )  . 
sequencing of the sentinel biopsy with 52 - gene hotspot panel ( appendix ) revealed no mutations in braf , nras , or kit . in accordance with recommendations , 2 adjuvant radiotherapy ( 48 gy ) was administered . 
the patient participated in a clinical trial of adjuvant pembrolizumab versus placebo ( nct02362594 ) and was randomly assigned to the pembrolizumab ar treatment was interrupted after four doses because of the appearance of liver , bone , cutaneous , and subcutaneous metastases . excision of a subcutaneous lesion confirmed metastatic relapse with no programmed death ligand 1 ( pd - l1 ) expression , and the patient received ipilimumab plus nivolumab for 2 months , followed by nivolumab for 3 months.3 the disease progressed with new metastases in the skin , liver , pancreas , spleen , lungs , adrenal glands , and lymph nodes . 
chemotherapy with cisplatin , vinblastine , dacarbazine , and interferon alpha was administered for two cycles , until additional disease progression.4 additional sequencing was performed using a panel covering 409 cancer - related genes ( appendix ) , and 43 somatic mutations ( appendix table a1 ) , corresponding to 29.5 nonsynonymous mutations / mb , were identified . 
the mutational profile ( fig 1a ) was typical of cutaneous melanoma , 5 with the wide spectrum of allelic frequencies ( af ) being consistent with the progressive accumulation of mutations over time ( fig 1b )  . 
ptpn11 contained two mutations ( fig 1d ) : one resulting in an early stop codon ( trp423 * , af = 26% ) and another , a single amino acid change ( thr468pro , af = 31% ) , both in the protein - phosphatase domain , which autoinhibit ras activation of shp26 and are likely to induce gain - of - function . on the basis of these findings , from july 2017 , we administered trametinib , a mek inhibitor , and , 6 weeks later , clinically evaluable cutaneous and subcutaneous lesions had visibly regressed . 
this was confirmed by positron emission tomography / computed tomography ( fig 2 ) , in which no new metastases were observed , no measurable lesions had increased , and most lesions had cline py yann christinat mario kreutzfeldt thomas a . 
whole - brain radiotherapy was initiated , followed by pembrolizumab , but the patient died 11 weeks after the end of trametinib therapy . two biopsy specimens were obtained during and after trametinib treatment and were analyzed with the 409 - gene panel . 
the three samples sequenced shared 39 mutations ( appendix table a1 ) , and the pretrametinib and post - trametinib samples had four additional nonshared mutations each ( table 1 )  . the pretrametinib sample showed diffuse nuclear expression of phosphorylated extracellular regulated kinase ( erk ) , consistent with mitogen - activated protein kinase ( mapk ) activation , in contrast to the other samples ( fig 3a )  . 
samples obtained before and after trametinib did not express pd - l1 and had negligible infiltration by cd8 lymphocytes , but during treatment , both pd - l1 expression and cd8 + density increased significantly ( fig 3b )  . discussion herein , we document , to our knowledge , the first response to trametinib in nf1 - mutated melanoma . 
radiologic evaluation with [ 18f ] fluorodeoxyglucose ( [ 18f ] fdg ) positron emission tomography ( pet ) / computed tomography ( ct ) before and during treatment with trametinib . 
 ( a ) baseline image showed multiple supra and infra - diaphragmatic lesions in the skin , liver , pancreas , spleen , lungs , bone , adrenal glands , and lymph nodes . 
 ( c ) ct scan and fdgpet / ct showed a morpho - metabolic decrease of the cervical lesion . in a phase i trial of trametinib , four of 39 patients with braf wild - type tumor had a partial response , 22 but none carried an nf1 mutation . 
with the exception of the nf1 splice mutation in the initial sample , none of the remaining unique mutations is predicted to be pathogenic or known to be a cancer hotspot mutation . 
a patient with nf1 - associated optic glioma treated with trametinib had rapid improvement that persisted for 4 months , 24 and trametinib treatment of one patient with a refractory ependymoma harboring mutations in both nf1 and ptpn11 resulted in disease stabilization for 10 weeks.25 nf1 - mutated tumors , as in this case , have a higher mutation burden than do other melanoma subtypes10 , and the accumulation of protein - coding mutations favors response to immune checkpoint inhibitors by generating mutated peptides that are recognized as neoantigens.30 nevertheless , this tumor did not have significant cd8 + infiltration , did not express pd - l1 before treatment with trametinib , and did not respond to pembrolizumab or the association of ipilimumab and nivolumab . in our patient , disease progression occurred after 5 months of trametinib , and none of the mutations found exclusively in the midor post - treatment biopsy specimens are known to induce resistance to mek inhibitors . 
although crebbp loss may activate the ras pathway , it does not modify sensitivity to mek inhibition in vitro26 and would not be expected to induce resistance to trametinib in our patient . 
genomic screens suggest that resistance to trametinib may be acquired through changes in the atxn1lcic - ets transcription factor axis , downstream of mek.29 our analyses would not capture atxn1l or etv upregulation , but would detect cic mutations , which were not present . 
 these events could recapitulate the effects of erk activation without nuclear phosphorylated erk localization , which we saw before , but not during and after trametinib . the increase in cd8 + infiltration and pd - l1 expression during trametinib treatment ( fig 3 ) is in accordance with the findings of previous studies . 
a mouse model treated with an raf inhibitor resulted in a notable increase in cd8 + infiltration that might have contributed to response , 31 and the same pattern has been observed in melanomas treated with braf or braf plus mek inhibitrs.32 the increase in cd8 + infiltrate may be explained by the effect of mek inhibition , which can protect t cells from t - cell receptordriven apoptosis.33 alternatively , major histocompatibility complex class i internalization driven by mapk activation34 could also be reversed by mek inhibition ( not observed in this case )  . 
 ( b ) cd8 t - cell infiltrate , programmed deathligand 1 ( pd - l1 ) , and hla class i expression before , during , and after treatment with trametinib . 
pd - l1 expression in the treatment sample is estimated at approximately 2% . might be required to achieve a robust immune response.35 - 37 changes that occurred under treatment might be enhanced by combined immunotherapy . this report supports the exploration of mek inhibitors for nf1 - mutated melanoma in clinical trials , with attention to the presence of concomitant ptpn11 mutations . 
mckee speakers ' bureau : novartis travel , accommodations , expenses : roche pierre - yves dietrich research funding : novartis ( inst ) petros tsantoulis honoraria : roche , pfizer travel , accommodations , expenses : amgen financial support : thomas a . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . affiliations all authors : university hospital of geneva , geneva , switzerland . references 161 : 1681 - 1696 , 2015 1 . 
atkins mb , hsu j , lee s , et al : phase iii trial comparing concurrent biochemotherapy with cisplatin , vinblastine , dacarbazine , interleukin - 2 , and interferon alfa - 2b with cisplatin , vinblastine , and dacarbazine alone in patients with metastatic malignant melanoma ( e3695 ) : a trial coordinated by the eastern cooperative oncology group . 
martinelli s , torreri p , tinti m , et al : diverse driving forces underlie the invariant occurrence of the t42a , e139d , i282v and t468m shp2 amino acid substitutions causing noonan and leopard syndromes . 
falchook gs , lewis kd , infante jr , et al : activity of the oral mek inhibitor trametinib in patients with advanced melanoma : a phase 1 dose - escalation trial . 
tafe lj , gorlov ip , de abreu fb , et al : implementation of a molecular tumor board : the impact on treatment decisions for 35 patients evaluated at dartmouth - hitchcock medical center . 
dixon za , nicholson l , zeppetzauer m , et al : crebbp knockdown enhances ras / raf / mek / erk signaling in ras pathway mutated acute lymphoblastic leukemia but does not modulate chemotherapeutic response . 
frederick dt , piris a , cogdill ap , et al : braf inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma . 
ebert pjr , cheung j , yang y , et al : map kinase inhibition promotes t cell and anti - tumor activity in combination with pd - l1 checkpoint blockade . 
bradley sd , chen z , melendez b , et al : brafv600e co - opts a conserved mhc class i internalization pathway to diminish antigen presentation and cd8 + t - cell recognition of melanoma . 
bendell jc , kim tw , boon cg , et al : clinical activity and safety of cobimetinib ( cobi ) and atezolizumab in colorectal cancer ( crc )  . 
desai j , hong ys , kim je , et al : efficacy and safety of cobimetinib ( cobi ) and atezolizumab ( atezo ) in an expanded phase 1b study of microsatellite - stable ( mss ) metastatic colorectal cancer ( mcrc )  . 
sullivan r , gonzalez r , lewis kd , et al : atezolizumab ( a ) + cobimetinib ( c ) + vemurafenib ( v ) in brafv600 - mutant metastatic melanoma ( mel ) : updated safety and clinical activity . 
 clinical utility of clia - grade ar - v7 testing in patients with metastatic castration - resistant prostate cancer purpose a splice variant of the androgen receptor , ar - v7 , confers resistance to ar - targeted therapies ( atts ) but not taxane chemotherapies in patients with metastatic castrationresistant prostate cancer . 
since august 2015 , a clinical - grade assay to detect ar - v7 messenger rna expression in circulating tumors cells ( ctcs ) has been available to providers through a clinical laboratory improvement amendmentscertified laboratory at johns hopkins university . methods we contacted ordering providers of the first 150 consecutive tests by using a questionnaire - based survey to determine how the results of ar - v7 testing were used to influence clinical practice . results in all , 142 ( 95% ) of 150 questionnaires were completed by 38 providers from 29 sites across the united states and canada . 
prevalence of ar - v7 detection increased with prior exposure to atts ( abiraterone and enzalutamide nave , 22% ; after abiraterone or enzalutamide , 35% ; after abiraterone and enzalutamide , 43% )  . 
2017 by american society of clinical oncology introduction metastatic castration - resistant prostate cancer ( mcrpc ) remains dependent on androgen receptor ( ar ) signaling for survival in the presence low testosterone levels.1 two novel artargeted therapies ( atts ) , abiraterone and enzalutamide , induced high objective response rates and improved overall survival in patients with mcrpc.2 , 3 treatment with abiraterone or enzalutamide in the prechemotherapy setting led to a 50% decline in prostate - specific antigen ( psa50 ) response in 62% and 78% of these patients , respectively.2 , 3 however , sequential use of abiraterone and enzalutamide has resulted in much lower psa response rates with the use of the second att agent ( ie , abiraterone after enzalutamide , 10% to 15% ; enzalutamide after abiraterone , 20% to 30% ) .4 - 6 these data underscore the importance of identifying a predictive biomarker of resistance to att to prevent the use of subsequent futile therapy . potential mechanisms of resistance to att include ar amplification and mutation , as well as the expression of arsplicevariants.7a well - characterized mark c . 
eisenberger jun luo emmanuel s . antonarakis author affiliations and support information ( if applicable ) appear at the end of this article . the content is solely the responsibility of the authors and does not necessarily represent the official views of the national cancer institute or the national institutes of health . corresponding author : emmanuel s . 
 splice variant , ar - v7 , is a truncated form of fulllength ar ( ar - fl ) that lacks the ligand - binding domain but retains both the transactivation and dna - binding domains , allowing for constitutive ar signaling in the absence of androgen.8 - 10 in patients with mcrpc , detection of ar - v7 in circulating tumors cells ( ctcs ) was shown to predict resistance to novel atts.11 - 13 moreover , ar - v7 + patients had a significantly shorter overall survival , suggesting a prognostic value of ar - v7 in addition to its use as a predictive biomarker.11 , 13 chemotherapy is an alternative to att for arv7 + patients with mcrpc . 
detection of ar - v7 has been shown not to preclude response to taxanebased chemotherapy.14 , 15 further prospective investigation found a significant survival benefit with the use of taxanes versus atts in patients with ar - v7 + disease.16 interestingly , the presence of this splice variant is a dynamic feature with possible conversion from ar - v7 + to ar - v7 status after chemotherapy with taxanes.14 , 17 these data suggest that serial arv7 testing may guide the clinical treatment of patients with mcrpc . 
those patients with ar - v7 prostate cancer may continue to benefit from att , whereas chemotherapy may be more effective in patients with detectable ar - v7 transcript.18 if the clinical utility of ar - v7 testing can be confirmed , it may also have an economic benefit . in a recent study , we modeled the cost of treating all patients with mcrpc with abiraterone or enzalutamide versus using ar - v7 testing to direct treatment.19 by using a clinical scenario in which ar - v7 + patients were changed from treatment with abiraterone and / or enzalutamide to chemotherapy thus avoiding the cost of futile att therapy , ar - v7 testing resulted in a theoretical cost savings to the health care system of $150 million per year . 
to this end , since august 2015 , clinicalgrade ar - v7 testing performed in a clinical laboratory improvement amendments ( clia ) certified laboratory at johns hopkins university has been available to health care providers for clinical use.20 however , despite the commercial availability of this ar - v7 test , its clinical utility is unknown . 
here , we have retrospectively compiled questionnaire - based data on how ordering providers are applying the results of ar - v7 testing in their clinical practice to influence decision making . methods the analytical validation and test characteristics of our clia - grade ar - v7 assay have been described previously.20 our molecular pathology database was queried for all ar - v7 tests ordered by internal and external providers for clinical purposes . 
clinical results of this test were reported as ctc , ctc + / ar - v7 , or ctc + / ar - v7 + , because each of these categories is associated with different outcomes.12 a clinical utility questionnaire ( data supplement ) was generated for each ar - v7 test ordered and was mailed or e - mailed to each ordering provider . we defined a biomarker - based change in treatment as a confirmation of treatment choice or a change from one therapy to another after ar - v7 testing . 
the institutional review board at johns hopkins university approved this study and granted a waiver of consent to contact the provider of each ar - v7 test ordered because that was considered a clinical audit . 
the ordering provider was then contacted for participation and asked to complete a questionnaire pertaining to treatment decisions that were made on the basis of results of that specific ar - v7 test . 
if a provider did not wish to participate or the questionnaire was not returned after two attempts to contact the provider , data for that patient were not included in the analysis . one hundred fifty consecutive ar - v7 clinical test results were obtained between august 31 , 2015 , and august 31 , 2016 , representing the first 150 tests ordered . 
from these , 142 questionnaires ( 95% ) were completed and returned by 38 providers across 29 sites ( 28 in the united states [ in 22 states ] and one in canada )  . 
the significance level was set at p , .05 , and corrections were not performed for multiple comparisons . results information from 142 of 150 questionnaires sent ( 95% participant response rate ) were included in this analysis . 
the number of lines of additional systemic therapies for mcrpc among these patients was reported as follows : 24% ( n = 33 ) had received no other lines , 27% ( n = 39 ) had received one line , 27% ( n = 39 ) had received two lines , 13% ( n = 18 ) had received three lines , and 9% ( n = 13 ) had received four or more lines of systemic therapy before testing . 
of tests abiraterone enzalutamide abiraterone or enzalutamide abiraterone enzalutamide nave test result ctc 40 / 142 28.17 ctc + / ar - v7 42 / 142 29.58 8 / 40 20.0 11 / 40 27.5 21 / 40 52.5 8 / 42 19.0 20 / 42 47.62 14 / 42 33.33 ctc + / ar - v7 + 60 / 142 42.25 26 / 60 43.33 21 / 60 35.0 13 / 60 21.66 abbreviations : ar - v7 , androgen receptor splice variant 7 ; att , ar - targeted therapy ; ctc , circulating tumor cell ; n / n , number of patients in that category divided by total number of patients . overall , the prevalence of a ctc result was 28% , the prevalence of a ctc + / ar - v7 result was 30% , and the prevalence of a ctc + / ar - v7 + result was 42% . 
patients who were treated with abiraterone and / or enzalutamide resulted in a higher prevalence of ar - v7 detection compared with patients who were not treated with an att : 22% of treatment - nave patients were ar - v7 + ; after treatment with abiraterone or enzalutamide , 35% of patients were ar - v7 + ; and after treatment with abiraterone and enzalutamide , 43% of patients were ar - v7 +  . to assess the clinical utility of ar - v7 testing , providers were asked whether the ar - v7 status influenced their decision making . 
the majority of ar - v7 tests ( ctc or ctc + / ar - v7 ) did not change the clinical practice of the providers ( table 2 )  . 
however , almost two thirds ( 62% ) of ar - v7 + tests resulted in a change in management . in patients for whom treatment was changed , providers were then asked to specify the type of therapy selected on the basis of the test result . 
patients with an ar - v7 result ( ctc or ctc + / ar - v7 ) were preferentially treated with an att agent ( confirmed ar treatment , or changed from taxane to ar therapy )  . 
conversely , after an ar - v7 + result , most patients were changed from an att agent to taxane chemotherapy ( 43% ) or were enrolled in a clinical trial ( 43% )  . 
psa50 rr data were missing from 16% ( n = 12 ) and 22% ( n = 15 ) of questionnaires in which management was changed or not changed , respectively . we also investigated which systemic therapy was used in patients with mcrpc after progression on both abiraterone and enzalutamide , according to ar - v7 status ( table 5 )  . 
by contrast , ar - v7 + patients who had already received abiraterone and enzalutamide were more often treated on a clinical trial ( 54% ) compared with treatment using chemotherapy ( 19% )  . 
in the patients who were treated with docetaxel , those who were ar - v7 were commonly treated with standard chemotherapy ( ie , cabazitaxel ; 67% [ six of nine ] ) whereas ar - v7 + patients were more frequently placed on a clinical trial ( 58% [ 11 of 19 ] )  . finally , we examined provider treatment preferences for ar - v7 + patients irrespective of the prior therapies received ( table 6 )  . 
a minority of patients ( 7% ) received either enzalutamide or abiraterone despite an ar - v7 + result , whereas all ar - v7 + patients managed with observation ( 10% ) enrolled in hospice shortly thereafter . to summarize the data compiled from providers real - world experience with ar - v7 testing , we propose a hypothetical treatment algorithm for making decisions regarding patients with mcrpc using ar - v7 as a potential treatment - selection biomarker ( fig 1 )  . 
after first - line systemic therapy with abiraterone or enzalutamide , ar - v7 + patients would preferentially cross over to taxane - based therapy , whereas those who are ar - v7 may continue on a second att . 
because of occasional conversions from ar - v7 + to ar - v7 status , men progressing on taxane treatment can be retested and could potentially consider treatment with an att if the ar - v7 status reverts to negative . 
finally , even patients progressing after treatment with abiraterone and enzalutamide may be considered for ar - v7 testing if an ar - v7directed clinical trial is available . discussion the optimal sequencing of therapeutic agents in patients with mcrpc is unknown and remains a major challenge . 
to this end , the national comprehensive cancer network prostate cancer guidelines now suggest that ar - v7 testing can be considered and may play a role in guiding therapy selection in mcrpc , but at this time , these guidelines have not gone as far as recommending testing to determine treatment choice . 
in another recent consensus report , the majority of prostate cancer specialists polled ( 59% ) stated that ar - v7 testing would be useful for some ( majority or minority of ) patients with mcrpc.21 although further prospective validation of the predictive ability of ar - v7 is currently ongoing , here we investigated the clinical utility of ar - v7 testing in a real - world setting . we asked providers whether the result of the arv7 test influenced their clinical practice for that specific patient . 
although our findings are retrospective , they suggest that ar - v7 testing may possibly lead to improved clinical responses to treatment , at least in terms of psa50 rr . 
we did not assess for radiographic progression - free or overall survival , which may not have correlated with psa50 rr . the statistically significant psa50 rr difference between the change and no - change groups is largely driven by the ar - v7 + subgroup ( described in appendix table a3 )  . 
ar - v7 + patients for whom management did not change had a psa50 rr of 5% ( v 39% in ar - v71 patients for whom treatment was changed )  . 
we hypothesize that ar - v7 + patients in the change group were more commonly treated with chemotherapy compared with the no - change group , resulting in improved outcomes . 
we also acknowledge a high psa50 rr in ar - v7 + patients treated with chemotherapy and ar - v7 patients treated with att ( both in the change group )  . 
nonetheless , these data suggest that further prospective investigation is warranted to study biomarker - driven clinical outcomes . by using the treatment history captured by our questionnaires , we were able to observe an increasing prevalence of ar - v7 detection with prior exposure to atts , as expected . 
this finding is corroborated by the recent biomarker data from the armor3 - sv ( a study of galeterone compared with enzalutamide in men expressing androgen receptor splice variant - 7 mrna [ ar - v7 ] metastatic crpc ) trial , in which first - line patients with mcrpc who had previously received docetaxel for metastatic hormone - sensitive disease had a higher prevalence of ar - v7 detection compared with chemotherapy - nave patients.22 moreover , 79% of men with newly developed mcrpc had prior exposure to bicalutamide . 
the higher trend of ar - v7 + tests after treatment with docetaxel or bicalutamide , in the absence of a novel att , may suggest that total number of therapies ( ie , more advanced disease ) contributes to ar - v7 expression in addition to the known relationship with prior att exposure . 
another provocative hypothesis might be that docetaxel works as an ar - modulating therapy in prostate cancer , inhibiting microtubule - dependent nuclear transport of wild - type ar but not ar - v7 , thereby selecting for the emergence of ar - v7expressing clones during or after chemotherapy treatment . this study had some additional limitations . 
first , the prevalence of ar - v7 was probably overestimated compared with earlier reports because providers were more likely to order a test if the clinical scenario suggested that the test might be positive . other significant weaknesses of this analysis were its retrospective nature and reliance on selfreporting by providers . 
an audit of 14 randomly selected questionnaires determined that providers gave accurate responses to the questionnaire in most instances ( see appendix ) , suggesting that our data represent the true clinical course for each patient . 
finally , we concede that many patients will receive all atts approved by the us food and drug administration , regardless of ar - v7 status , because clinical responses to atts are sometimes observed in arv7 + patients.13 however , the clinical utility of arv7 testing may potentially be clearest in ar - v7 + patients who have additional atts still available for treatment . 
by identifying high - risk patients ( ie , arv7 + ) who have adequate performance status earlier in their mcrpc treatment , this would allow them to be treated with taxane therapy before the chemotherapy window closes . 
potential decision algorithm based on serial androgen receptor splice variant 7 ( ar - v7 ) testing across the castration - resistant prostate cancer landscape . after each line of therapy , we propose an algorithm for consideration of ar - v7 testing . 
after abiraterone and enzalutamide treatment , patients should be subject to ar - v7 testing only if an ar - v7directed clinical trial is available . ( * ) indicates limited clinical data supporting the use of atts in ar - v7 + patients that subsequently convert to ar - v7 . 
 ( ) denotes a clinical trial that does not require a positive ar - v7 test for ar - v7 patients , whereas ar - v7 + patients should consider an arv7directed trial , if available . first - line therapy clinical ar - v7 testing ? second - line therapy clinical ar - v7 testing ? third - line therapy abiraterone enzalutamide consider ar - v7directed clinical trial / taxane docetaxel taxane / trial enzalutamide abiraterone consider ar - v7directed clinical trial / taxane receive either chemotherapy or atts at their physicians discretion . we propose a decision algorithm using serial arv7 testing across the mcrpc landscape . 
this algorithm is based largely on published data suggesting that the presence of ar - v7 confers resistance to atts but does not influence response to taxane chemotherapy.11 , 13 , 14 , 16 we note that at this time , there is no clinical trial evidence to support the use of atts in ar - v7 + patients who convert to ar - v7 status after chemotherapy . 
in our study , 17 of 22 ar - v7 + patients were enrolled on a clinical trial that mandated ar - v7 detection as an entry criterion , suggesting that many of the tests in this study were ordered for the purpose of screening for a clinical trial . 
finally , there is ongoing debate in the prostate cancer community about whether ar - v7 detection is merely a proxy for ar amplification or overexpression and not an independent predictor of response to therapy or clinical outcomes.21 although several studies have shown that ar - v7 detection is indeed correlated with ar - fl expression , ar - v7 still remains independently prognostic in multivariable analysis after controlling for ar - fl levels.11 , 13 , 23 in conclusion , to our knowledge , this is the first study to examine the preliminary real - world clinical utility of clia - grade ar - v7 testing in patients with mcrpc . 
we show that ar - v7 testing influenced clinical decision making overall ( regardless of test results ) but that its utility was greatest in the setting of ar - v7 + results . 
 jun luo honoraria : gilead sciences , sanofi consulting or advisory role : tokai pharmaceuticals , sun pharmaceutical industries , janssen oncology research funding : sanofi ( inst ) , orion pharma ( inst ) , mirati therapeutics ( inst ) , gilead sciences ( inst ) , astellas pharma ( inst ) patents , royalties , other intellectual property : co - inventor of an ar - v7 biomarker technology assigned to johns hopkins university who licensed to tokai pharmaceuticals ; co - inventor of a technology licensed to qiagen emmanuel s . 
antonarakis honoraria : sanofi , dendreon , medivation , janssen biotech , essa , astellas pharma consulting or advisory role : sanofi , dendreon , medivation , janssen biotech , essa , astellas pharma research funding : janssen biotech ( inst ) , johnson & johnson ( inst ) , sanofi ( inst ) , dendreon ( inst ) , aragon pharmaceuticals ( inst ) , exelixis ( inst ) , millennium pharmaceuticals ( inst ) , genentech ( inst ) , novartis ( inst ) , astellas pharma ( inst ) , tokai pharmaceuticals ( inst ) travel , accommodations , expenses : sanofi , dendreon , medivation mark c . 
w81xwh - 15 - 2 - 0050 and w81xwh - 12 - 1 - 0605 ( j.l. ) , johns hopkins prostate specialized programs of research excellence grant p50 ca058236 ( j.l. ) , the patrick c . 
beer tm , armstrong aj , rathkopf de , et al : enzalutamide in metastatic prostate cancer before chemotherapy . n engl j med 368 : 138 - 148 , 2013 n engl j med 371 : 424 - 433 , 2014 4 . 
maughan bl , luber b , nadal r , et al : comparing sequencing of abiraterone and enzalutamide in men with metastatic castration - resistant prostate cancer : a retrospective study . 
dehm sm , schmidt lj , heemers hv , et al : splicing of a novel androgen receptor exon generates a constitutively active androgen receptor that mediates prostate cancer therapy resistance . 
hu r , dunn ta , wei s , et al : ligand - independent androgen receptor variants derived from splicing of cryptic exons signify hormone - refractory prostate cancer . 
antonarakis es , lu c , wang h , et al : ar - v7 and resistance to enzalutamide and abiraterone in prostate cancer . n engl j med 371 : 1028 - 1038 , 2014 12 . 
antonarakis es , lu c , luber b , et al : clinical significance of androgen receptor splice variant - 7 mrna detection in circulating tumor cells of men with metastatic castration - resistant prostate cancer treated with firstand second - line abiraterone and enzalutamide . 
antonarakis es , lu c , luber b , et al : androgen receptor splice variant 7 and efficacy of taxane chemotherapy in patients with metastatic castration - resistant prostate cancer . 
onstenk w , sieuwerts am , kraan j , et al : efficacy of cabazitaxel in castration - resistant prostate cancer is independent of the presence of ar - v7 in circulating tumor cells . 
scher hi , lu d , schreiber na , et al : association of ar - v7 on circulating tumor cells as a treatment - specific biomarker with outcomes and survival in castration - resistant prostate cancer . 
sprenger c , uo t , plymate s : androgen receptor splice variant v7 ( ar - v7 ) in circulating tumor cells : a coming of age for ar splice variants ? ann oncol 26 : 1805 - 1807 , 2015 19 . 
markowski mc , frick kd , eshleman jr , et al : cost - savings analysis of ar - v7 testing in patients with metastatic castration - resistant prostate cancer eligible for treatment with abiraterone or enzalutamide . 
lokhandwala pm , riel sl , haley l , et al : analytical validation of androgen receptor splice variant 7 detection in a clinical laboratory improvement amendments ( clia ) laboratory setting . 
gillessen s , attard g , beer tm , et al : management of patients with advanced prostate cancer : the report of the advanced prostate cancer consensus conference apccc 2017 . 
taplin me , antonarakis es , ferrante kj , et al : clinical factors associated with ar - v7 detection in armor3 - sv , a randomized trial of galeterone ( gal ) vs enzalutamide ( enz ) in men with ar - v7 + metastatic castration - resistant prostate cancer ( mcrpc )  . 
silberstein j , luber b , wang h , et al : clinical significance of ar mrna quantification from circulating tumor cells ( ctcs ) in men with metastatic castration - resistant prostate cancer ( mcrpc ) treated with abiraterone ( abi ) or enzalutamide ( enza )  . 
j clin oncol 35 , 2017 ( suppl ; abstr 132 ) we further queried our database to investigate the difference in the 50% decline in prostate - specific antigen ( psa50 ) response rate ( rr ) between the change and no - change groups on the basis of androgen receptor splice variant 7 ( ar - v7 ) status . 
for patients with ar - v7 + disease , the psa50 rr in those who did not change therapy was 5.3% ( one of 19 ) compared with 38.7% ( 12 of 31 ) in the change group ( table a3 )  . 
in the ar - v72 patients , the psa50 rr was 45.45% ( 15 of 33 ) in the no - change group versus 68.88% ( 22 of 32 ) in the change group . 
within both the ar - v7 + and ar - v7 groups , there was no discernible difference in number of lines of therapy between the change and no - change group . 
we also examined psa50 rr by treatment choice in those patients who had a change in treatment ( these data were not solicited for patients who did not have a treatment change in the questionnaire )  . 
in the 14 questionnaires audited , we found two minor discrepancies : ( 1 ) bicalutamide was listed in error as a prior therapy ( one time ) , and ( 2 ) the correct clinical trial was misidentified for a patient ( one time )  . subjective responses ( ie , clinical helpfulness and change in management ) were not assessed . 
dinardo , md1 introduction germline predispositions to hematologic malignancies in adult patients presenting with a diagnosis of myelodysplastic syndrome ( mds ) or acute myeloid leukemia ( aml ) are commonly underestimated . 
through next - generation sequencing ( ngs ) , many genes involved in hereditary mds or aml are now evaluated , including runx1 , cebpa , gata2 , tp53 , etv6 , and ddx41.1 , 2 germline gata2 mutations are a common cause for predisposition to mds or aml.3 patients with gata2 deciency can have manifestations ranging from mild cytopenias to life - threatening infections and myeloid neoplasia . 
the incidence of gata2 deciency is still unknown ; however , gata2 mutations may account for approximately 75% of adolescent patients with monosomy 7 mds.4 approximately 20% to 30% of gata2 mutations are inherited in an autosomal dominant fashion , whereas others are sporadic or de novo mutations.5 because molecular testing for mds and aml has become more commonplace , we recognize that some clinicians may incorrectly equate a negative ngs panel as ruling out any potential inherited malignancy predisposition . 
in addition , promoter mutations ( ie , ankrd26 ) and intronic mutations ( ie gata2 ) may often be missed if the ngs panel is restricted to exonic sequences . 
specic testing for fanconi anemia and dyskeratosis congenita when clinically indicated should be performed , for example , diepoxybutane or mitomycin cinduced chromosomal breakage assay and telomere length owuorescence in situ hybridization analysis , respectively . 
publically available registries and genomic data sets such as clinvar and gnomad are increasingly useful to provide insight into clinical signicance of identied variants . here , we report on two patients with a history of immunodeciency and myeloid neoplasia . 
1 is a 24 - year - old hispanic man who presented for evaluation of pancytopenia , mainly leukopenia associated with recurrent extensive warts on bilateral hands and recurrent panniculitis . 
his blood counts were as follows : white blood cell count , 3.2 109 / l ; hemoglobin , 14.1 g / dl ; and platelet count , 186 109 / l . 
concurrent ow cytometry analysis demonstrated a small population of aberrant myeloblasts , lack of hematogones , virtual absence of monocytes , and markedly reduced natural killer ( nk ) cells . 
a peripheral blood sample was obtained for ow cytometry study that showed no monocytes , nk cell lymphopenia ( 0.4% of lymphocyte gate ; fig 1b ) , and decreased b cells ( 3.8% of lymphocyte gate )  . 
a targeted exome sequence analysis of the following genes was performed on cultured skin broblasts : ankrd26 , cebpa , ddx41 , etv6 , gata2 , runx1 , srp72 , terc , tert , and tp53 . 
her blood counts showed a white blood cell count of 2.9 109 / l , with 75% neutrophils , 21% lymphocytes , 1% monocytes , hemoglobin of 10.1 g / dl , and platelet count of 164 109 / l . 
her past medical history was unremarkable , and her family history was negative for any immunologic or hematologic diseases . because of persistent ebv viremia , she received autologous ebv - specic cytotoxic t - lymphocyte cells . 
she received intensive cytarabine - based chemotherapy , and her induction course was complicated by an infection with mycobacterium fortuituflow cytometry analysis showed that she achieved complete remission with negative minimal residual disease . 
before proceeding with allogeneic hematopoietic stem - cell transplantation ( hsct ) from her sibling , the patient was referred to our hereditary hematologic malignancy clinic.6 a skin biopsy was performed for cultured skin broblasts , and genetic testing demonstrated a heterozygous deletion that included at least exon 2 of the gata2 gene . 
top : prehsct , the bone marrow shows a near absence of monocytes on ( left ) cd45 / ssc and ( middle ) cd14 ( thick arrows ) ; ( right ) no stage i or stage ii hematogones or mature b cells ( thin arrows )  . 
this variant has been previously reported in at least 29 other patients.4 , 10 - 14 the gata2 mutation detected in patient no . 2 was a large deletion involving at least exon 2 . 
to our knowledge , this is the rst time this specic deletion has been reported , although other in - frame or full gene gata2 deletions have been described.4 , 15 gata2 deciency can have various clinical presentations.11 , 16 patient no.1 presented initially with dermatologic problems , including extragenital warts and recurrent severe panniculitis ; mild cytopenias and hypocellular mds occurred later in his disease course . 
laboratory ndings include monocytopenia , dendritic cell cytopenias , b and nk lymphocytopenia , relative increase in t cells with an inverted cd4 : cd8 ratio , lack of hematogones , and various degrees of dysplasia.13 bm cellularity may vary from hypocellular marrow with severe cytopenias to normoor hypercellular marrow . 
 abou dalle et al stem cell exhaustion.3 the median age at diagnosis of 380 younger patients was approximately 20 years ( range , 12 to 35 years ) , with a high risk of progression to aml in 75% of carriers . 
complex cytogenetics and del ( 5q ) were not usually observed.5 patients with immunodeciency and recurrent infections along with a new diagnosis of mds at a younger age should be screened for an underlying germline mutation . 
genetic testing for germline mutations should sequence the most common genes responsible for familial predisposition to mds or aml , including ankrd26 , cebpa , ddx41 , etv6 , gata2 , runx1 , srp72 , samd9 , samd9l , terc , tert , and tp53 using nonhematopoietic tissue , usually cultured skin broblasts . gata2 sequencing should involve deletion and duplication analysis and intron 5 of the gata2 gene to detect all possible pathogenic variants . 
dinardo , md , department of leukemia , the university of texas md anderson cancer center , 1515 holcombe blvd , unit 0428 , houston , tx 77030 ; e - mail : cdinardo@mdanderson.org. support supported in part by specialized programs of research excellence grant no . 
cortes consulting or advisory role : bristol - myers squibb , biolinerx , novartis , pzer , amphivena therapeutics , daiichi sankyo , bio - path holdings , astellas pharma , takeda pharmaceuticals research funding : bristol - myers squibb ( inst ) , novartis ( inst ) , pzer ( inst ) , celgene ( inst ) , arog pharmaceuticals ( inst ) , astellas pharma ( inst ) , ambit biosciences ( inst ) , celator pharmaceuticals ( inst ) , immunogen ( inst ) , sun pharmaceutical industries ( inst ) , takeda pharmaceuticals ( inst ) alessandra ferrajoli research funding : celgene , genentech , acerta pharma dimitrios p . 
kontoyiannis employment : md anderson cancer center honoraria : gilead sciences , astellas pharma , t2 biosystems , mylan consulting or advisory role : merck , astellas pharma , fast track to growth , pzer , astellas pharma sa a . 
blood 129 : 424 - 447 , 2017 arber da , orazi a , hasserjian r , et al : the 2016 revision to the world health organization classication of myeloid neoplasms and acute leukemia . 
semin hematol 54 : 81 - 86 , 2017 dinardo cd , bannon sa , routbort m , et al : evaluation of patients and families with concern for predispositions to hematologic malignancies within the hereditary hematologic malignancy clinic ( hhmc )  . 
blood 80 : 575 - 581 , 1992 vicente c , conchillo a , garca - s anchez ma , et al : the role of the gata2 transcription factor in normal and malignant hematopoiesis . 
crit rev oncol hematol 82 : 1 - 17 , 2012 tsai fy , orkin sh : transcription factor gata - 2 is required for proliferation / survival of early hematopoietic cells and mast cell formation , but not for erythroid and myeloid terminal differentiation . 
hsu ap , johnson kd , falcone el , et al : gata2 haploinsufciency caused by mutations in a conserved intronic element leads to monomac syndrome . blood21 : 3830 - 3837 , 2013 11 . 
kazenwadel j , secker ga , liu yj , et al : loss - of - function germline gata2 mutations in patients with mds / aml or monomac syndrome and primary lymphedema reveal a key role for gata2 in the lymphatic vasculature . 
 c mechanistic learning for combinatorial strategies with immuno - oncology drugs : can model - informed designs help investigators ? joseph ciccolini , pharmd , phd1 ; dominique barbolosi , phd1 ; nicolas andr e , md , phd1 , 2 ; fabrice barlesi , md , phd3 ; and s ebastien benzekry , phd4 the past couple of years have seen an unprecedented number of failures of clinical trials investigating combinatorial strategies with immuno - oncology drugs ( iods )  . beyond the highly publicized crashes of the mystic study1 ( antipd - l1 plus anti - ctla4 in nonsmall - cell lung cancer [ nsclc ] ) or the echo - 301 trial2 ( antipd - 1 and anti - ido in melanoma ) , many other attempts to combine immunotherapy with radiation therapy ( rt ) , 3 chemotherapy , 4 metronomic chemotherapy , 5 or antiangiogenics6 have similarly led to disappointing results . 
because successful immunotherapy is restricted today to a limited number of patients in a limited number of cancers ( melanoma , lung cancer , head and neck cancer , and kidney cancer , with 5 - year survival rates , 40% ) , developing appropriate strategies to stretch efcacy remains critical ( eg , by turning cold tumors [ noninammatory with lack of inltrating t cells ] into hot ones [ inltrated by t cells ] )  . 
several studies successfully associated conventional treatments with iods in comparative phase iii trials.7 , 8 however , these trials should not hide the high attrition rate of too many other studies . 
one of the common characteristics of the trials is the lack of computational pharmacology support , plus the lack of prior knowledge regarding the pharmacokinetics ( pk ) / pharmacodynamics ( pd ) relationships of the combined treatments . 
actually , comes from the fact that , sometimes , some combinatorial clinical trials manage to be successful . author affiliations and support information ( if applicable ) appear at the end of this article . accepted on march 20 , 2020 and published at ascopubs.org / journal / po on may 8 , 2020 : doi 1200 / po.19.00381 to improve the design of such combinatorial trials beyond trial - and - error methods , we propose an innovative strategy termed mechanistic learning ( fig 1 )  . we dene it as the combination of mechanistic modelingsimulation of the kinetics of pathophysiologic processesand statistical ( machine ) learning . using data generated from previous clinical trials and preclinical experiments , it consists in building computational models able to simulate and predict the toxicity and efcacy outcomes of candidate regimens . the optimal scheduling is then selected for clinical trial testing . 
limited options when performing combinatorial trials are therefore a major caveat , possibly explaining many failures , or at least limiting the conclusions regarding the real intrinsic potential of a given combination . 
for instance , in the checkmate032 study , the antipd - 1 nivolumab and the antictla4 ipilimumab were administered to patients with nsclc using only 2 dosing modes ( ie , 1 and 3 mg / kg and vice versa )  . 
these doses were chosen because they were already combined in a previous phase i study.9 in dose - nding trials , up to 5 dose levels ranging from 0.1 to 20 mg / kg have been tested for nivolumab and ipilimumab used as single agents without reaching dose - limiting toxicities.10 , 11 consequently , at least 25 different combinations in dosing could have been explored in checkmate - 032 , not to mention the countless variations in sequencing and scheduling . 
in the subsequent checkmate - 143 study , the same strategy failed to improve survival in patients with glioblastoma.12 another example of a poorly designed study is the modul umbrella trial , which tested a triple combination of uoropyrimidines , the antiangiogenic bevacizumab , and the antipd - l1 atezolizumab in patients with metastatic colorectal cancer ( mcrc )  . 
baseline data can be composed of demographic , clinical , pathologic ( eg , histologic type ) , molecular ( eg , genetic mutations ) , or biologic ( eg , blood counts ) variables . 
survival data ( eg , progression - free or overall survival ) can also be modeled with a mechanistic basis ( instead of biologically agnostic survival analysis based on , eg , cox regression ) , using adapted , survival learning statistical methods . 
ct1 , rst chemotherapy ; ct2 , second chemotherapy ; mtki , maintenance tyrosine kinase inhibitor ; ps , performance status ; tgi , tumor growth inhibition ; tki , tyrosine kinase inhibitor . surprisingly , in this study , all drugs were administered concomitantly at xed dosing . 
this combination failed to exhibit signicant efcacy in terms of progression - free survival , and thus , the conclusion was that adding atezolizumab to standard of care for maintenance in mcrc showed no benet.13 actually , this conclusion may sound peremptory because it is not possible to know whether or not different dosing or scheduling with exactly the same drugs would have performed better . 
for instance , trial of nivolumab , 23 the impact of dosing ( ie , 0.1 - 10 mg / kg ) on pd - 1 receptor occupancy was investigated . 
similar target engagement ( ie , 64% - 70% ) was achieved regardless of the dosing , thus prompting several observers to conclude that pk / pd relationships were at with iods and , therefore , that interpatient variability was not an issue . 
however , in this seminal study , pd - 1 inhibition was measured only on circulating t lymphocytes extracted from peripheral blood and not on inltrated t lymphocytes in the tumor microenvironment . 
importantly , the pharmacokinetics of most therapeutic monoclonal antibodies is characterized by large interindividual variability and reduced ability to diffuse the vascular space and reach solid tumors.24 out of therefore , to what extent differences in nivolumab dosing could affect or not target engagement at the tumor levels , and not only in circulating t cells , remains to be fully investigated . 
critically , it would also be highly informative to analyze the data generated using the initial model to close the mechanistic learning loop . using similar principles for tumor heterogeneity , others have proposed the concept of adaptive therapy , suggesting individuals be treated only upon disease progression.38 this was further successfully translated at bedside in patients with metastatic castration - resistant prostate cancer.39 such a strategy could help to personalize combinations of iods and aromatase inhibitors in the neoadjuvant treatment of breast cancer.40 despite their scarcity , these studies highlight how application of mathematical modeling in clinical trials in oncology is now feasible and can rationalize study design . these innovative methods are yet to be extended to iod combinations . 
modeling immunotherapy has recently been applied to gain insights on optimal modes for combinations with rt.41 - 43 for instance , kosinsky et al42 were able to simulate multiple sequences of antipd - 1 treatment in combination with rt and validated their results using preclinical data . 
 commentary the general modeling methodology that could be undertakenmechanistic learningis a combination of mechanistic modeling and statistical learning , either from machine learning , mixed - effects learning , 45 or survival learning , 46 for integration of the multimodal data arising from clinical trials or routine management ( fig 1 )  . 
as output , it would provide informative simulations of the effect of various doses and schedules to aid decisions in early clinical studies . these would consist of predicted probabilities of graded toxicities , tumor growth kinetics in response to treatment , and survival outcomes . 
of note , there is a major trend by health regulatory agencies such as the us food and drug administration to call for incorporating extensive modeling and simulation into clinical research.47 in this respect , and regarding the current challenges with iods , 48 we believe that mechanistic learning could be a valuable tool for decision making when setting up combinatorial strategies . conclusion after a rst phase of enthusiasm , success stories with immunotherapy seem to have reached a glass ceiling because many studies now fail to further stretch either response rates or survival.49 consequently , combining immune checkpoint inhibitors with other treatments likely to boost tumor immunity is a rising strategy in clinical oncology . 
for more information about ascos conict of interest policy , please refer to or ascopubs . org / po / author - center . open payments is a public database containing information reported by companies about payments made to us - licensed physicians ( open payments )  . joseph ciccolini honoraria : pierre fabre , pzer , roche , novartis research funding : roche , merck serono travel , accommodations , expenses : astrazeneca nicolas andr e research funding : bristol - myers squibb ( inst ) travel , accommodations , expenses : bristol - myers squibb fabrice barlesi honoraria : genentech , pzer , pierre fabre , astrazeneca , bristol - myers squibb , boehringer ingelheim , eli lilly , novartis , merck serono , msd oncology , takeda , bayer consulting or advisory role : genentech , pzer , novartis , pierre fabre , bristol - myers squibb , astrazeneca / medimmune , boehringer ingelheim , eli lilly , merck serono , msd oncology , takeda , bayer research funding : genentech ( inst ) , astrazeneca / medimmune ( inst ) , bristol - myers squibb ( inst ) , pierre fabre ( inst ) , abbvie ( inst ) , amgen ( inst ) , bayer ( inst ) , boehringer ingelheim ( inst ) , eisai ( inst ) , eli lilly ( inst ) , ipsen ( inst ) , innate pharma ( inst ) , novartis ( inst ) , merck serono ( inst ) , msd oncology ( inst ) , pzer ( inst ) , sano - aventis ( inst ) , takeda ( inst ) travel , accommodations , expenses : genentech , bristol - myers squibb , astrazeneca / medimmune , msd oncology no other potential conicts of interest were reported . acknowledgment we thank elena ivanchenko for graphic assistance . 
in memory of marie - christine masini . references 41 : 41 - 48 , 2019 rizvi na , chul cho b , reinmuth n , et al : durvalumab with or without tremelimumab vs platinum - based chemotherapy as rst - line treatment for metastatic nonsmall cell lung cancer : mystic . 
mcbride sm , sherman ej , tsai cj , et al : a phase ii randomized trial of nivolumab with stereotactic body radiotherapy ( sbrt ) versus nivolumab alone in metastatic ( m1 ) head and neck squamous cell carcinoma . 
 ciccolini et al rothschild s , zippelius a , savic s , et al : sakk 16 / 14 : anti - pd - l1 antibody durvalumab ( medi4736 ) in addition to neoadjuvant chemotherapy in patients with stage iiia ( n2 ) non - small cell lung cancer ( nsclc ) a multicenter single - arm phase ii trial . 
j clin oncol 36 , 2018 ( suppl 15 ; abstr tps8584 ) 1200 / jco.2018.36.15_suppl.tps8584 toulmonde m , penel n , adam j , et al : use of pd - 1 targeting , macrophage inltration , and ido pathway activation in sarcomas : a phase 2 clinical trial . 
jama oncol 4 : 93 - 97 , 2018 gao j , karam ja , tannir nm , et al : a pilot randomized study evaluating nivolumab ( nivo ) or nivo + bevacizumab ( bev ) or nivo + ipilimumab ( ipi ) in patients with metastatic renal cell carcinoma ( mrcc ) eligible for cytoreductive nephrectomy ( cn ) , metastasectomy ( ms ) or post - treatment biopsy ( bx )  . 
j clin oncol 36 , 2018 ( suppl 15 ; abstr 4520 ) socinski ma , jotte rm , cappuzzo f , et al : atezolizumab for rst - line treatment of metastatic nonsquamous nsclc . 
hellmann md , rizvi na , goldman jw , et al : nivolumab plus ipilimumab as rst - line treatment for advanced non - small - cell lung cancer ( checkmate 012 ) : results of an open - label , phase 1 , multicohort study . 
ribas a , camacho lh , lopez - berestein g , et al : antitumor activity in melanoma and anti - self responses in a phase i trial with the anti - cytotoxic t lymphocyteassociated antigen 4 monoclonal antibody cp - 675 , 206 . 
grothey a , tabernero j , arnold d , et al : fluoropyrimidine and bevacizumab plus or minus atezolizumab as rst - line treatment for braf wild type metastatic colorectal cancer : findings from the modul trial of biomarker - driven maintenance . 
schaer da , beckmann rp , dempsey ja , et al : the cdk4 / 6 inhibitor abemaciclib induces a t cell inamed tumor microenvironment and enhances the efcacy of pd - l1 checkpoint blockade . 
heery cr , osullivan - coyne g , madan ra , et al : avelumab for metastatic or locally advanced previously treated solid tumours ( javelin solid tumor ) : a phase 1a , multicohort , dose - escalation trial . 
lindauer a , valiathan cr , mehta k , et al : translational pharmacokinetic / pharmacodynamic modeling of tumor growth inhibition supports dose - range selection of the antipd - 1 antibody pembrolizumab . 
long gv , tykodi ss , schneider jg , et al : assessment of nivolumab exposure and clinical safety of 480 mg every 4 weeks at - dosing schedule in patients with cancer . 
h enin e , meille c , barbolosi d , et al : revisiting dosing regimen using pk / pd modeling : the model1 phase i / ii trial of docetaxel plus epirubicin in metastatic breast cancer patients . 
barlesi f , imbs d - c , tomasini p , et al : mathematical modeling for phase i cancer trials : a study of metronomic vinorelbine for advanced non - small cell lung cancer ( nsclc ) and mesothelioma patients . 
yu ha , sima c , feldman d , et al : phase 1 study of twice weekly pulse dose and daily low - dose erlotinib as initial treatment for patients with egfr - mutant lung cancers . 
poleszczuk jt , luddy ka , prokopiou s , et al : abscopal benets of localized radiotherapy depend on activated t cell trafcking and distribution between j immunother cancer 6 : 17 , 2018 metastatic lesions . 
claret l , girard p , hoff pm , et al : model - based prediction of phase iii overall survival in colorectal cancer on the basis of phase ii tumor dynamics . 
coosemans a , vankerckhoven a , baert t , et al : combining conventional therapy with immunotherapy : a risky business ? eur j cancer 113 : 41 - 44 , 2019 49 . 
murray leslie samuel paolo nuciforo jose jimenez guillem argiles rodrigo dienstmann josef tabernero lucia picariello luca messerini stefania nobili enrico mini kieran sheahan elizabeth ryan ( continued ) purpose transcriptomic profiling of colorectal cancer ( crc ) has led to the identification of four consensus molecular subtypes ( cms1 to 4 ) that have prognostic value in stage ii and iii disease . 
2018 by american society of clinical oncology introduction colorectal cancer ( crc ) has the third highest worldwide incidence.1 although 75% of patients present with operable diseasemainly stages ii and iiiapproximately 40% experience disease recurrence.2 compared with surgery alone , adjuvant chemotherapy improves survival in only approximately 3% of patients with stage ii disease , rising to 15% to 20% for those with stage iii disease . 
 contributed equally to this work . corresponding author : daniel longley , phd , centre for cancer research and cell biology , queens university belfast , 97 lisburn rd , belfast , bt9 7bl united kingdom ; e - mail : d.longley@qub.ac.uk. licensed under the creative commons attribution 4.0 license significant advances have been made in the molecular stratification of crc , leading to the identification of four consensus molecular subtypes ( cms1 to 4 ) .3 cms1 is enriched for microsatellite instability , is immune rich , and correlates with good prognosis , and cms4 is stromal rich , with high levels of cancer - associated fibroblasts , and has a relatively poor prognosis . 
 cms3 is defined by the activation of multiple metabolic pathways , potentially as a result of its enrichment for kras mutations.4 the epithelial - rich cms2 is the largest group , accounting for approximately 40% of all tumors . 
of these , 188 samples with > 50% tumor content passed quality control and were subjected to rna and dna analysis ( appendix )  . transcriptomics high - quality transcriptomics data were obtained for 156 of the 188 samples ( almac xcel array ; almac diagnostics , craigavon , united kingdom )  . 
residual technical batch effects were corrected using the combat method ( sva package ) , and data were deposited in the national center for biotechnology information gene expression omnibus repository ( gse103479 )  . 
the clinical pathologic details of this cohort are provided in table 1 . data analysis gse3958210 and gse1433311 crc data sets were downloaded from the national center for biotechnology information gene expression omnibus repository and their respective cel files uploaded into r . 
similar results were obtained in the larger gse39582 cohort for stage ii disease ( log - rank test p = .071 ) , whereas in stage iii disease , this correlation reached significance ( p = .001 ; data supplement )  . 
when we combined the taxonomy and gse39582 cohorts to increase statistical power , the benefit from adjuvant chemotherapy for cms2 was significant in both stage ii disease ( log - rank test p = .02 ; hazard ratio [ hr ] , 0.21 [ wald test p = 3.52 102 ] ; fig 1a ) and stage iii disease ( log - rank test p < .001 ; hr , 0.22 [ wald test p = 1.48 104 ] ; fig 1b )  . 
of note , no significant benefit from adjuvant chemotherapy was observed in cms1 or cms4 , although a nonsignificant trend was observed in cms4 stage iii disease ( log - rank test p = .089 ; data supplement )  . these results suggest that the more epithelial cms2 and cms3 subgroups benefit from adjuvant chemotherapy . 
in support of this , in a combined analysis of cms2 and cms3 , benefit from adjuvant chemotherapy was significant in both stage ii disease ( log - rank test p = .0042 ; hr , 0.16 [ wald test p = .012 ] ; fig 1c ) and stage iii disease ( log - rank test p < .001 ; hr , 0.20 [ wald test p = 1.14 106 ] ; fig 1d )  . 
in contrast , in a combined analysis of the cms1 and cms4 subgroups , no benefit from adjuvant chemotherapy was observed ( data supplement )  . when cms2 subgroup results for stage ii disease were adjusted for t stage ( t4 v t3 ) , age , and sex using cox proportional hazards regression analysis , the significance of the benefit from chemotherapy was lost ( hr , 0.31 ; wald test p = .121 ; log likelihood ratio , 7.0 105 ; fig 1e ) ; however , in stage iii disease , adjusting for t stage ( t4 or n2 v t1 to t3 / n1 ) , age , and sex , the significance of benefit from chemotherapy in cms2 was maintained ( hr , 0.27 ; wald test p = .007 ; log likelihood p = 3.25 105 ; fig 1f )  . 
in cox proportional hazards regression analyses of the combined cms2 and cms3 subgroups , the significance of benefit from chemotherapy was maintained in stage ii disease ( hr , 0.23 ; wald test p = .049 ; log likelihood ratio , 2.72 105 ; fig 1g ) and stage iii disease ( hr , 0.21 ; wald test p < .001 ; log likelihood p = 3.19 107 ; fig 1h )  . clinical implications of tumor - intrinsic stratification in cms2 as previously reported , 6 there are limited associations between cms and cris classifications for example , cms4 is distributed relatively evenly between the five cris subtypes ( data supplement ) ; however , some clear patterns were observed , with cms2 almost exclusively distributed between cris - c , - d , and - e ( fig 2a )  . 
 subsequently , we investigated whether substratification of cms2 into cris - c , - d , or - e could identify a more specific subset of patients with stage ii and iii disease who derive benefit from adjuvant chemotherapy . 
these results were confirmed in an additional independent cohort , gse14333 ( logrank test p = .02 ; hr , 0.12 [ wald test p = .05 ] ; data supplement )  . 
in the other 2 cris subgroups ( cris - a and - b ) , no significant benefit from adjuvant chemotherapy was observed in either stage ii or iii disease , although a nonsignificant trend was observed in cris - a for stage iii disease ( p = .057 ; data supplement ) , which is consistent with this subgroup being enriched for cms3 , where a significant benefit was observed ( data supplement )  . 
when cris - c results were adjusted for t stage , age , and sex using cox proportional hazards regression analyses , the benefit from chemotherapy maintained significance in stage ii disease ( hr , 0.12 ; wald test p = .045 ; log likelihood p = .0054 ; fig 2d ) and stage iii disease ( hr , 0.27 ; wald test p = .02 ; log likelihood p = 1.11 103 ; fig 2e ) ; furthermore , for combined stage ii and iii disease , adjusted hr was 0.20 ( wald test p < .001 ; log likelihood p = 7.5 106 ; data supplement )  . 
 ( a and b ) kaplan - meier plots of 5 - year overall survival ( os ) for consensus molecular subtype 2 ( cms2 ) patients who received adjuvant fluorouracil ( fu ) - based treatment ( gold ) and those who did not receive treatment ( surgery alone , blue ) , in the ( a ) stage ii combined ( taxonomy and gse39582 ) cohort and the ( b ) stage iii combined ( taxonomy and gse39582 ) cohort . 
 ( c and d ) kaplan - meier plots for 5 - year os for combined cms2 and cms3 patients in the ( c ) stage ii combined ( taxonomy and gse39582 ) cohort and the ( d ) stage iii combined ( taxonomy and gse39582 ) cohort . 
to account for potential intratumoral heterogeneity of putative biomarkers in specific tumor regions , tmas were generated from the taxonomy cohort to incorporate three cores each from ct , if , and tumor - adjacent sr regions . 
cd8 and cd3 levels were defined using immunohistochemistry , and patients were stratified into high and low groups using the median as cutoff ( representative cd8 and cd3 images ; figs 3a and 3b )  . 
we further assessed cd8 + lymphocytes in histologically normal tissue adjacent to the tumor and found no difference in survival between high and low cd8 levels ( log - rank test p = .72 ; data supplement )  . 
of note , no correlations were found between cd3 + lymphocyte levels and prognosis ( if log - rank test p = .55 ; sr log - rank test p = .75 ; ct log - rank test p = .8 ; normal log - rank test p = .82 ; data supplement )  . for each patient , cd8 scores in each tumor subregion correlated closely with one another ( p < .001 ; data supplement )  . 
 as expected on the basis of correlations for individual regions ( data supplement ) , applying this tumor average score to the combined stage ii and iii taxonomy cohort revealed that high levels of cd8 + lymphocytes identified cris - c patients with good prognosis ( log - rank p = .0031 ; hr , 12.18 [ wald test p = .0191 ] ; fig 4a )  . 
in contrast , average cd3 scores , which again did not correlate closely with average cd8 scores ( fig 3c ) , were not prognostic ( fig 4b )  . 
of note , cd8 mrna expression was unable to distinguish good and poor prognosis patients ( log - rank p = .83 ; data supplement ) , which indicates the need for immunohistochemistry in combination with transcriptomic profiling for effective prognostication . 
 ( e - h ) forest plots show the results from the adjusted cox proportional hazards regression analysis for the ( e ) stage ii cms2 combined cohort , the ( f ) stage iii cms2 combined cohort , the ( g ) stage ii cms2 and cms3 combined cohort , and the ( h ) stage iii cms2 and cms3 combined cohort . 
for adjusted analyses , data are stratified by treatment and adjusted for t stage , sex , and age in stage ii , and for age and sex in stage iii . 
 ( a ) caleydo plots display mapping of patient samples from the consensus molecular subtype 2 to the colorectal cancer intrinsic subtypes ( cris ) in the combined ( taxonomy and gse39582 ) data sets . 
 ( a and b ) representative images of samples display high and low ( a ) cd8 and ( b ) cd3 expression in each of the three tumor regions sampled : invasive front ( if ) , stromal rich ( sr ) , and central tumor ( ct )  . 
 ( a - c ) kaplan meier plots of patients with ( a ) cd8 - high versus cd8 - low stage ii and iii disease in the colorectal cancer intrinsic subtype ( cris ) - c surgery - only , taxonomy cohort ; ( b ) cd3 - high versus cd3 - low stage ii and iii disease in the cris - c surgery - only , taxonomy cohort ; and ( c ) cd8 - high versus cd8low stage ii disease in the cris - c surgery - only , taxonomy cohort . 
therapeutic benefit from adjuvant chemotherapy is modest for this group as a whole , with an absolute improvement in survival of approximately 3%.18 - 22 currently , additional pathologic characteristics , such as obstruction , perforation , extramural venous invasion , and t stage ( t4 ) , are used to identify poor prognostic stage ii disease and guide the decision of whether to start chemotherapy treatment.23 additional methods of assessing the risk of recurrence in the adjuvant disease setting have been the focus of many studies in recent years , 24 - 29 which has led to the development of the 12 - gene oncotype dx assay.30 this algorithm has been extensively clinically tested31 - 33 and demonstrated to identify patients with stage ii disease who are at higher risk of recurrence and patients with stage iii disease who are at lower risk of recurrence.34 , 35 additional signatures have been proposed , including the 18 - gene prognostic classifier , known as coloprint , 36 and the 634 - gene prognostic classifier , known as coldx.27 the current study indicates that the transcriptionally definable cms2 / cris - c patient subgroup may be the cohort of patients within stage ii disease that benefits from standard adjuvant fu - based chemotherapy . 
these data correlate well with our previous study on the prognostic significance of immune - derived programmed death ligand 1 mrna expression in crc , in which we postulated that patients with low immune infiltrates would significantly benefit from adjuvant fu - based chemotherapy after surgery.9 , 37 meta - analysis of the idea ( international duration evaluation of adjuvant chemotherapy ) collaboration examined whether a 3 - month duration of oxaliplatin containing adjuvant chemotherapyfolfox4 , modified folfox6 , or xeloxis as effective as a 6 - month schedule in patients with stage iii crc . 
this study found that the 3 - month treatment was almost as effective as the 6 - month treatment and reduced the risk of treatment associated toxicity , thus concluding that a 3 - month treatment would be more beneficial for patients with low - risk ( t1 to 3 / n1 tumors ) stage iii disease.38 our study suggests that levels of cd8 + lymphocytes could also be used to identify such low - risk patients , at least in the cris - c subgroup . 
of note , cris - c is enriched for mutant tp53 and wild - type kras tumors , 6 but neither of these established molecular markers provided additional information with regard to disease outcome within the cris - c subgroup . collectively , these results provide the first evidence of the predictive value of the now well established cms and more recently described cris transcription - based classification systems . 
 our results also emphasize the utility of combining cms and cris subtyping in a substratification strategy to maximize clinical benefit from adjuvant fu - based chemotherapy in patients with stage ii and iii crc . 
murray , leslie samuel , jose jimenez , guillem argiles , lucia picariello , luca messerini , stefania nobili , enrico mini , elizabeth ryan , sandra van schaeybroeck , daniel b . 
longley tumor - infiltrating lymphocytes , would potentially enable the prospective identification of the cris - c / cd8 - low stage ii patients who significantly benefit from adjuvant fu - based chemotherapy . 
however , we recognize that there are a number of limitations in the current study , which was conducted on a relatively small number of retrospective samples that were collected outside of clinical trials , and we realize that this hypothesis - generating study now requires validation in either larger patient cohorts or stratified trial cohorts enriched for the cris - c patient subtype . 
nonetheless , this study suggests that transcription - based classification systems , such as cms and cris , have the potential to be developed into patient stratification tools and , when used alone or alongside other molecular pathology approaches , such as immunohistochemistry , could enable the selection of patients with crc who are most likely to benefit from adjuvant chemotherapy , while at the same time sparing nonresponders the potentially harmful treatment related adverse events and sequelae of chemotherapy . 
of importance , the cris subtyping method uses gene expression from tumor epithelial cells only and is independent of stromal - derived signals ; therefore , the cris subgroups can be detected irrespective of the profiling technology used or the tissue source.15 such robustness and reproducibility are critical for clinical translation . 
murray , leslie samuel , paolo nuciforo , jose jimenez , guillem argiles , josef tabernero , lucia picariello , luca messerini , stefania nobili , enrico mini , kieran sheahan , elizabeth ryan , sandra van schaeybroeck , mark lawler , daniel b . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . richard wilson consulting or advisory role : merck serono , sirtex medical , amgen , servier , clovis oncology , halozyme , bristol - myers squibb research funding : almac group ( inst ) travel , accommodations , expenses : merck serono , amgen vicky m . 
murray research funding : vertebrate antibodies ( inst ) leslie samuel consulting or advisory role : mundipharma research funding : mologen ( inst ) , roche ( inst ) , merck ( inst ) , eli lilly ( inst ) , taiho pharmaceutical ( inst ) travel , accommodations , expenses : mologen wendy l . 
longley stock and other ownership interests : fusion antibodies consulting or advisory role : astex pharmaceuticals sandra van schaeybroeck no relationship to disclose mark lawler honoraria : pfizer research funding : astex pharmaceuticals patents , royalties , other intellectual property : inhibitors of the antiapoptotic protein flip travel , accommodations , expenses : astex pharmaceuticals affiliations wendy l . 
murray and leslie samuel , national health service grampian , aberdeen , united kingdom ; najeeb ashraf syed and puthen veettil jithesh , sidra medical and research center , qatar ; paolo nuciforo , jose jimenez , guillem argiles , rodrigo dienstmann , and josef tabernero , university hospital vall dhebron , barcelona , spain ; lucia picariello , luca messerini , stefania nobili , and enrico mini , university of florence , florence , italy ; and kieran sheahan and elizabeth ryan , university college dublin , dublin , ireland . funded by cancer research uk grants no . 
lenz h - j , ou f - s , venook ap , et al : impact of consensus molecular subtyping ( cms ) on overall survival ( os ) and progression free survival ( pfs ) in patients ( pts ) with metastatic colorectal cancer ( mcrc ) : analysis of calgb / swog 80405 ( alliance )  . 
dunne pd , mcart dg , bradley ca , et al : challenging the cancer molecular stratification dogma : intratumoral heterogeneity undermines consensus molecular subtypes and potential diagnostic value in colorectal cancer . 
marisa l , de reynis a , duval a , et al : gene expression classification of colon cancer into molecular subtypes : characterization , validation , and prognostic value . 
jorissen rn , gibbs p , christie m , et al : metastasis - associated gene expression changes predict poor outcomes in patients with dukes stage b and c colorectal cancer . 
galon j , costes a , sanchez - cabo f , et al : type , density , and location of immune cells within human colorectal tumors predict clinical outcome . 
hutchins gga , treanor d , wright a , et al : intratumoral stromal morphometry predicts disease recurrence but not response to 5 - fluorouracil : results from the quasar trial of colorectal cancer . 
roepman p , schlicker a , tabernero j , et al : colorectal cancer intrinsic subtypes predict chemotherapy benefit , deficient mismatch repair and epithelial - to - mesenchymal transition . 
figueredo a , charette ml , maroun j , et al : adjuvant therapy for stage ii colon cancer : a systematic review from the cancer care ontario program in evidence - based cares gastrointestinal cancer disease site group . 
kelley rk , venook ap : prognostic and predictive markers in stage ii colon cancer : is there a role for gene expression profiling ? clin colorectal cancer 10 : 73 - 80 , 2011 26 . 
kennedy rd , bylesjo m , kerr p , et al : development and independent validation of a prognostic assay for stage ii colon cancer using formalin - fixed paraffin - embedded tissue . 
tabernero j , salazar r , roepman r , et al : additional validation of a genomic signature ( coloprint ) for the risk stratification of stage ii colon cancer patients . 
oconnell mj , lavery i , yothers g , et al : relationship between tumor gene expression and recurrence in four independent studies of patients with stage ii / iii colon cancer treated with surgery alone or surgery plus adjuvant fluorouracil plus leucovorj clin oncol 28 : 3937 - 3944 , 2010 31 . 
gray rg , quirke p , handley k , et al : validation study of a quantitative multigene reverse transcriptase - polymerase chain reaction assay for assessment of recurrence risk in patients with stage ii colon cancer . 
venook ap , niedzwiecki d , lopatin m , et al : biologic determinants of tumor recurrence in stage ii colon cancer : validation study of the 12 - gene recurrence score in cancer and leukemia group b ( calgb ) 9581 . 
cartwright t , chao c , lee m , et al : effect of the 12 - gene colon cancer assay results on adjuvant treatment recommendations in patients with stage ii colon cancer . 
srivastava g , renfro la , behrens rj , et al : prospective multicenter study of the impact of oncotype dx colon cancer assay results on treatment recommendations in stage ii colon cancer patients . 
dunne pd , mcart dg , oreilly pg , et al : immune - derived pd - l1 gene expression defines a subgroup of stage ii / iii colorectal cancer patients with favorable prognosis who may be harmed by sdjuvant chemotherapy . 
shi q , sobrero af , shields af , et al : prospective pooled analysis of six phase iii trials investigating duration of adjuvant ( adjuv ) oxaliplatin - based therapy ( 3 vs 6 months ) for patients ( pts ) with stage iii colon cancer ( cc ) : the idea ( international duration evaluation of adjuvant chemotherapy ) collaboration . 
 appendix development of a multiomics patient cohort the taxonomy cohort was assembled from an initial cohort of 363 patients with stage ii and iii disease from four european centers ( vall dhebron institute of oncology , barcelona , spain ; st vincents university hospital , dublin , ireland ; university of florence , florence , italy ; and university of aberdeen , aberdeen , united kingdom )  . 
this work was approved by the medicine , dentistry , and biomedical sciences school ethics committee ( ref : 12 / 12v4 )  . samples from 194 patients with 50% or more tumor content as assessed by hematoxylin and eosin staining were subjected to rna extraction . 
 data for samples were preprocessed and aligned according to best practices ( broad institute : tute.org / gatk / discussion / 3060 / how - should - i - pre - process - data - from - multiplexed - sequencing - and - multi - library - designs ) using burrows - wheeler aligner for alignment and gatk 3.4 for realignment and recalibration . 
cd8 was detected using the anti - cd8 antibody c8 / 144b ( dako , carpinteria , ca ) using the leica bond max staining platform ( leica microsystems , wetzlar , germany )  . 
for each tumor core , cd3 + and cd8 + t - cell populations were scored using the open access image analysis software qupath ( bankhead p , et al : sci rep 7 : 16878 , 2017 )  . 
measurements of tissue area and positive cell counts for each core provided cell density measurements , which are expressed as the number of positive cells per square millimeter of tissue . 
high and low cd3 / cd8 levels were calculated using the median level as cutoff . statistical analyses for kaplan - meier survival analysis and cox proportional hazards regression analysis , significance was assessed using log - rank and wald tests , respectively . 
cox proportional hazards regression analysis was used to assess overall survival at 5 years for adjuvant chemotherapy before and after adjustment for age , t stage , and sex ( stage ii and iii ) and age , sex , and stage ( stage ii and iii combined )  . 
 palbociclib in patients with pancreatic and biliary cancer with cdkn2a alterations : results from the targeted agent and proling utilization registry study tareq al baghdadi , md1 ; susan halabi , phd2 ; elizabeth garrett - mayer , phd3 ; pam k . 
schilsky , md3 purpose the targeted agent and proling utilization registry ( tapur ) study identies signals of antitumor activity of commercially available targeted agents in patients with advanced cancers that harbor genomic alterations known as drug targets . 
in this article , data from two cohorts of patients with pancreatic and biliary cancers with cdkn2a loss or mutation treated with palbociclib are reported . methods eligible patients age 12 years and older with advanced measurable or evaluable solid tumors are provided treatment according to protocol - specied genomic matching rules . 
secondary end points include safety , progression - free survival ( pfs ) , and overall survival ( os )  . results between july 2016 and november 2017 , 12 and 10 patients with pancreatic and biliary cancer , respectively , with cdkn2a loss or mutation were treated with palbociclib . 
despite recent advances , outcomes remain poor with a median overall survival ( os ) of less than 12 months in stage iv biliary and pancreatic cancers , 1 - 4 and new therapies are urgently needed . cyclin - dependent kinase ( cdk ) inhibitor 2a ( cdkn2a ) encodes p16ink4a , which plays an important role in cell - cycle regulation through inhibition of cdk4 / 6 . cdkn2a loss or mutation is found in a wide array of malignancies and may lead to increased cdk activity.5 in a report of the mutational landscape of advanced pancreatic cancer , 46.5% of tumors harbored alterations in cdkn2a.6 palbociclib is an orally available selective cdk inhibitor approved for the treatment of hormone receptorpositive , human epidermal growth factor receptor 2 ( her2 ) negative breast cancer in combination with endocrine therapy . the targeted agent and proling utilization registry ( tapur ) study is a prospective , phase ii , pragmatic basket trial designed to identify signals of antitumor activity of commercially available targeted agents in patients with advanced cancers that harbor genomic alterations known to be drug targets . 
this study examined whether patients with advanced biliary and pancreatic cancer with cdkn2a loss or mutation would be responsive to palbociclib , an oral cyclin - dependent kinase inhibitor . knowledge generated the results demonstrated that palbociclib monotherapy has no meaningful clinical activity in patients with cdkn2a mutated or deleted advanced pancreatic or biliary adenocarcinoma . relevance off - label prescribing of palbociclib for the treatment of patients with pancreatic and biliary tract cancers with cdkn2a loss or mutation is not recommended . 
these patients should be offered participation in clinical trials of new treatment approaches . methods trial design the rationale and study design have been reported previously in detail.7 patients with advanced cancer and no standard treatment options are eligible if they have a tumor that harbors a genomic alteration known to be a target of or to predict sensitivity to one of the available study drugs . the primary study end point is antitumor activity , dened as objective response ( or ) at 8 weeks or later or stable disease ( sd ) at 16 weeks or later from the time of enrollment . secondary end points include progression - free survival ( pfs ) , os , and toxicity . 
patients are matched to one of according to prespecied protocol matching rules on the basis of genomic testing performed by laboratories selected by clinical sites that have certication under the clinical laboratory improvement amendments and accreditation from the college of american pathologists . 
treating physicians may choose to consult with the tapur molecular tumor board for appropriate targeted treatment options where none or multiple treatment matches surface within the tapur matching rules or if the treating physician would like to propose a treatment match outside the matching rules . 
radiographic assessment and clinical evaluation for response evaluation are performed at 8 and 16 weeks after initiation of treatment ; then every 12 weeks while the patient remains in the study ; and then at the end of study treatment , where possible . 
patients are placed in multiple parallel cohorts dened by study drug , genomic alteration , and tumor type . each cohort has the same optimal simon two - stage design , with a total possible cohort size of 28 patients . 
the design requires 10 patients to be enrolled in a cohort in stage i and if fewer than two patients have or or sd of at least 16 weeks duration , the cohort is permanently closed . 
if at least seven patients in the total cohort have a response of at least 16 weeks duration , the null hypothesis is rejected , and it is concluded that a signal of activity has been identied . patients eligible patients are required to meet both protocoland drugspecic inclusion and exclusion criteria . 
protocol - specic eligibility criteria include advanced measurable or evaluable solid tumors per recist version 1.1 ; eastern cooperative oncology group ( ecog ) performance status ( ps ) of 0 , 1 , or 2 ; and a protocol - specied genomic target identied by a test performed in a laboratory that has clinical laboratory improvement amendments certication and college of american pathologists accreditation . 
 palbociclib in pancreatic / biliary cancer with cdkn2a alterations addition , to match to palbociclib , patients must have a cancer type other than breast and not have received food or drugs within 7 days before the start of study treatment that are known to be cyp3a4 inhibitors or inducers . 
detailed inclusion and exclusion criteria for tapur have been previously reported.7 patients treated with palbociclib received standard doses ( 125 mg orally daily for 21 days followed by 7 days off treatment to complete a 28 - day cycle ) until disease progression . 
all patients were tested for alterations using next - generation sequencing ( ngs ) platforms , with more than half the patients determined to have cdkn2a loss ( table 1 )  . patients in both cohorts continued treatment with palbociclib until disease progression , and no patients withdrew from the study . trial oversight the tapur study protocol was reviewed and approved by a central institutional review board and , in some cases , by the local institutional review board at participating sites . patients provide written consent before any screening activities or data collection . 
the tapur study was designed by asco staff with input from asco volunteer members and the participating pharmaceutical companies . the tapur data and safety monitoring board ( dsmb ) is an independent board appointed by asco and meets biannually to monitor the study and review the safety and efcacy ndings . 
in the case of the cohorts reported herein , the dsmb reviewed the safety and efcacy data and determined that both cohorts should permanently close at the end of stage i enrollment and that the ndings be released . study end points the primary end point is or , dened as complete or partial response at or before 16 weeks , or sd at 16 weeks or later as reported per recist version 1.1. 
 al baghdadi et al results patients with pancreatic cancer twelve patients with pancreatic cancer and cdkn2a loss or mutation treated with palbociclib were enrolled in the study across seven sites from july 25 , 2016 , to april 28 , 2017 . 
the median age was 62 years ( range , 52 to 70 years ) ; 67% of patients were male ; and 10 patients were white , one black , and one other . 
other common genomic alterations noted in this cohort were mutations in braf , fgfr1 , frs2 , kras , and tp53 , with kras mutations being the most commonly reported in 10 of 12 patients . two patients who were enrolled and received treatment were subsequently found to be ineligible because they had not met the eligibility requirement for a hemoglobin level of 9.0 g / dl or greater at the time of enrollment . 
of the 10 eligible patients , nine experienced progression at or before 8 weeks ; the remaining patient had tumor progression at 16 weeks . all patients were included in the analysis for safety , pfs , and os . 
no other grade 3 or higher adverse event or serious adverse event was observed as at least possibly related to palbociclib . patients with biliary cancer ten patients with biliary cancer with cdkn2a loss or mutation treated with palbociclib were enrolled in the study across six sites from august 9 , 2016 , to november 11 , 2017 . 
other genomic alterations noted in this cohort included arid1a , atm , braf , fgfr2 , fh , kras , nras , pik3ca , and vhl . all 10 patients experienced progression at or before 10 weeks . 
four patients experienced grade 3 or 4 adverse events of thrombocytopenia at least possibly related to palbociclib . discussion this phase ii study in patients with advanced pancreatic or biliary cancers with cdkn2a loss or mutation treated with single - agent palbociclib demonstrated no clinical activity . the toxicity is similar to previous reports of monotherapy with palbociclib . cdkn2a is a tumor suppressor gene frequently altered by mutations , deletions , and epigenetic silencing . 
of patients loss mutation loss cdkn2a / 2b homozygous loss p16ink4a a4_p11del , p16ink4a l16fs * 9 , loss exons 2 - 3 splice site 151 - 2a.g p16ink4a r58 * and p14arf p72l p16ink4a a36fs * 17 , p16ink4a n71fs * 49 and p14arf q85fs * 50 + splice site 151 - 1g.c truncation exon 2 foundationone mi - oncoseq foundationone foundationone foundationone foundationone foundationone foundationone abbreviations : mi - oncoseq , michigan oncology sequencing project ; ngs , next - generation sequencing . cdkn2a mutations / deletions occur frequently in pancreatic adenocarcinoma.11 moreover , alterations that abrogate the rb / p16 tumor suppressor pathway are seen in virtually all pancreatic carcinomas.12 cdkn2a mutations occur in a minority of cholangiocarcinomas and are associated with a poor prognosis.13 , 14 prior studies of multiple rb - positive tumors have shown that most are sensitive to some degree to cdk4 / 6 inhibition.15 - 18 preclinical studies assessing the impact of cdk4 / 6 inhibition on pancreatic cancer cell lines and xenograft models showed conicting data . 
for example , one study showed that palbociclib inhibited the growth of pancreatic cancer cell lines but resulted in upregulation of the expression of genes that promote invasion and metastasis.19 another showed that established pancreatic cell lines displayed a relatively weak response to palbociclib , but palbociclib had potent activity in patient - derived xenografts.20 palbociclib is approved for the treatment of patients with advanced hormone receptorpositive , her2 - negative breast cancer in combination with an aromatase inhibitor or fulvestrant.21 , 22 palbociclib showed promising activity in patients with liposarcoma23 and cdkn2a - mutated nonsmallcell lung cancer.24 however , palbociclib was not effective in lung cancer25 or unselected patients with squamous cell urothelial cancer.26 prior studies have suggested several mechanisms of resistance to cdk4 / 6 inhibitors in human cancer cell lines and xenograft models , including cdk4 amplication , rb loss , and cyclin e1 amplication.27 - 31 the potential for prior exposure to systemic chemotherapy to induce resistance to palbociclib through these or other mechanisms has not been explored but might have contributed to the lack of efcacy of palbociclib in this trial . 
it is conceivable , therefore , that the use of palbociclib alone or in combination earlier in the treatment course could be benecial . the association of cdkn2a mutations / deletions with clinical activity of cdk4 / 6 inhibitors is undetermined . 
in patients with hormone receptorpositive , her2 - negative breast cancer , cdkn2a mutations do not predict activity of either palbociclib or ribociclib.32 , 33 a report revealed that cdkn2a mutant cancers show only intermediate sensitivity to cdk4 / 6 inhibition with abemaciclib , whereas genetic events that activate d - type cyclins were associated with high sensitivity.30 the effects of combining other targeted agents with palbociclib in pancreatic adenocarcinoma cell lines have been explored . 
the addition of a mek inhibitor31 and a mammalian target of rapamycin inhibitor30 , 34 was synergistic . the latter may relate to metabolic reprogramming of pancreatic cancer cells by cdk4 / 6 inhibition , which results in increased oxidative phosphorylation , increased consumption of glucose and glutamine , and activation of mammalian target of rapamycin pathway.34 these and other novel combinations hopefully will allow successful treatment of pancreatic adenocarcinoma and other malignancies on the basis of a sound understanding of mechanisms that underlie cancer cell resistance . 
for more information about ascos conict of interest policy , please refer to or ascopubs.org / po / authorcenter . tareq al baghdadi stock and other ownership interests : array biopharma , astrazeneca , biogin , bristol - myers squibb , merck , portola pharmaceuticals , spectrum pharmaceuticals , tracon pharma , celgene , seattle genetics , sunesis pharmaceuticals honoraria : cardinal health , medscape consulting or advisory role : celgene , bristol - myers squibb , heron therapeutics travel , accommodations , expenses : cardinal health , celgene , bristolmyers squibb , heron therapeutics susan halabi consulting or advisory role : eisai , ferring pharmaceuticals vaibhav sahai consulting or advisory role : celgene , halozyme , newlink genetics , ipsen , incyte research funding : celgene ( inst ) , bristol - myers squibb ( inst ) , agios ( inst ) , incyte ( inst ) , clovis oncology ( inst ) , debiopharm group ( inst ) , fibrogen ( inst ) , halozyme ( inst ) , medimmune ( inst ) , rafael pharmaceuticals ( inst ) , ipsen ( inst ) ricardo h . 
alvarez employment : cancer treatment centers of america leadership : cancer treatment centers of america consulting or advisory role : eisai , puma biotechnology , r - pharma , pzer speakers bureau : eisai , pzer other relationship : eisai , puma biotechnology , pzer edward s . 
kim honoraria : astrazeneca , boehringer ingelheim , pzer , merck , takeda pharmaceuticals , roche , genentech consulting or advisory role : astrazeneca , boehringer ingelheim , pzer , merck , takeda pharmaceuticals , roche , genentech research funding : boehringer ingelheim , merck , ignyta , genentech , roche travel , accommodations , expenses : astrazeneca , boehringer ingelheim , takeda pharmaceuticals , genentech , roche , pzer , merck kathleen j . 
schilsky research funding : astrazeneca ( inst ) , bayer ag ( inst ) , bristol - myers squibb ( inst ) , genentech ( inst ) , roche ( inst ) , eli lilly ( inst ) , merck ( inst ) , pzer ( inst ) , boehringer ingelheim ( inst ) , varian ( inst ) no other potential conicts of interest were reported . references conroy t , desseigne f , ychou m , et al : folfirinox versus gemcitabine for metastatic pancreatic cancer . 
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n engl j med 362 : 1273 - 1281 , 2010 sahai v , catalano pj , zalupski mm , et al : nab - paclitaxel and gemcitabine as rst - line treatment of advanced or metastatic cholangiocarcinoma : a phase 2 clinical trial . 
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finn rs , dering j , conklin d , et al : pd 0332991 , a selective cyclin d kinase 4 / 6 inhibitor , preferentially inhibits proliferation of luminal estrogen receptor - positive human breast cancer cell lines in vitro . 
baughn lb , di liberto m , wu k , et al : a novel orally active small molecule potently induces g1 arrest in primary myeloma cells and prevents tumor growth by specic inhibition of cyclin - dependent kinase 4 / 6 . 
young rj , waldeck k , martin c , et al : loss of cdkn2a expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the cdk4 / 6 inhibitor pd0332991 in melanoma cell lines . 
cristofanilli m , turner nc , bondarenko i , et al : fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone - receptor - positive , her2negative metastatic breast cancer that progressed on previous endocrine therapy ( paloma - 3 ) : final analysis of the multicentre , double - blind , phase 3 randomised controlled trial . 
dickson ma , schwartz gk , keohan ml , et al : progression - free survival among patients with well - differentiated or dedifferentiated liposarcoma treated with cdk4 inhibitor palbociclib : a phase 2 clinical trial . 
gopalan pk , pinder mc , chiappori a , et al : a phase ii clinical trial of the cdk 4 / 6 inhibitor palbociclib ( pd 0332991 ) in previously treated , advanced non - small cell lung cancer ( nsclc ) patients with inactivated cdkn2a . 
edelman mj , redman mw , albain ks , et al : a phase ii study of palbociclib ( p ) for previously treated cell cycle gene alteration positive patients ( pts ) with stage iv squamous cell lung cancer ( scc ) : lung - map sub - study swog s1400c . 
rose tl , chism dd , alva as , et al : phase ii trial of palbociclib in patients with metastatic urothelial cancer after failure of rst - line chemotherapy . 
herrera abreu mt , asghar u , elliot r , et al : pi3 kinase / mtor inhibition increases sensitivity of er positive breast cancers to cdk4 / 6 inhibition by blocking cell cycle re - entry driven by cyclind1 and inducing apoptosis . 
gong x , litcheld lm , webster y , et al : genomic aberrations that activate d - type cyclins are associated with enhanced sensitivity to the cdk4 and cdk6 31 . 
finn rs , crown jp , lang i , et al : the cyclin - dependent kinase 4 / 6 inhibitor palbociclib in combination with letrozole versus letrozole alone as rst - line treatment of oestrogen receptor - positive , her2 - negative , advanced breast cancer ( paloma - 1 / trio - 18 ) : a randomised phase 2 study . 
andre f , stemmer sm , campone m , et al : ribociclib + letrozole for rst - line treatment of hormone receptor - positive ( hr + ) , human epidermal growth factor receptor 2 - negative ( her2 - ) advanced breast cancer ( abc ) : efcacy by baseline tumor markers . 
 entrectinib in two pediatric patients with inflammatory myofibroblastic tumors harboring ros1 or alk gene fusions introduction inflammatory myofibroblastic tumor ( imt ) is a rare , indolent spindle cell tumor that typically affects children and young adults.1 , 2 imts occur predominantly in visceral soft tissue and are categorized as an intermediate malignancy because they have a potential for local recurrence but rarely progress to distant metastases.2 , 3 approximately 50% of imts harbor alk rearrangements , although gene fusions involving ros1 , ntrk3 , and pdgfr have also been identified.2 , 4 - 6 previously , the presence of alk gene fusions was typically identified using immunohistochemistry7 ; however , newer techniques such as hybridization capture - based next - generation sequencing ( ngs ) and ngs - based anchored multiplex polymerase chain reaction ( pcr ) testing can now be used to detect gene fusions.8 - 10 identifying patients with actionable gene fusions is important , given the availability of promising therapeutic strategies.2 , 4 surgical resection is the standard of care for patients with a solitary imt , 11 but patients with unresectable or advanced disease have limited treatment options . 
the current national comprehensive cancer network guidelines for soft tissue sarcoma recommend the use of alk inhibitors for patients with alk gene fusions12 ; however , there are no targeted agents approved specifically for use in fusion - positive imts . 
 entrectinib is an investigational , cns - active , potent , and selective inhibitor of trk , ros1 , and alk.8 , 13 , 14 in phase i trials , entrectinib demonstrated clinical efficacy in patients with different tumor types harboring ntrk , ros1 , or alk fusions , regardless of the fusion partner gene.8 a majority of the responses to entrectinib occurred in a rapid and durable manner , and some patients remained on study because they received clinical benefit , even after experiencing disease progression.8 here , we present two patients with fusion positive imts ( dctn1 - alk and tfg - ros1 ) who were enrolled in the ongoing startrk - ng phase i / ib trial ( nct02650401 : study of rxdx 101 in children with recurrent or refractory solid tumors and primary cns tumors , with or without trk , ros1 , or alk fusions ) and treated with entrectinib . 
further molecular testing using a hybrid capture - based targeted exome sequencing assay15 revealed a dctn1 - alk fusion ( fig 1a ) , which determined eligibility to enroll in the startrk - ng trial . 
 ( a ) the dctn1alk rearrangement identified in patient 1 is a deletion that results in the fusion of dctn1 exons 1 - 26 with alk exons 20 - 29 . 
 ( d ) axial t1 postcontrast image shows complete response of the tumor to entrectinib treatment ( no measurable tumor present , red arrow )  . the patient provided informed consent , and entrectinib was started in may 2017 at a dose of 550 mg / m2 taken orally once per day ( 1 cycle was 28 days )  . 
 at the time of this report , she continues daily entrectinib and has no limitations in academic or athletic activities . patient 2 is a 10 - year - old girl who presented with daily high - grade fevers , cough , weight loss , and changes in physical activity over a 2 - month period . 
hybrid capture - based targeted exome sequencing and anchored multiplex pcr as described above15 , 16 identified a tfg - ros1 gene fusion ( fig 2a ) , and she was subsequently enrolled in the startrk - ng trial . the patient provided informed consent and entrectinib was started in may 2017 at a dose of 550 mg / m2 orally once per day . 
in addition , by month 4 of treatment , the residual mass was primarily nonenhancing on contrast - enhancing magnetic resonance imaging , suggesting relative lack of activity ( fig 2b - 2c )  . 
while on treatment , she experienced constipation , mild bloating , nausea , and weight gashe also experienced nighttime urinary incontinence that was potentially related to treatment and improved with simple changes in her nighttime routine . 
both patients exhibited clinically significant responses to the investigational agent entrectinib , which was well tolerated , and the observed adverse effects were consistent with the reported safety profile of entrectinib.8 these cases are in agreement with previous reports demonstrating the clinical efficacy and tolerability of entrectinib in patients with solid tumors harboring ntrk , ros1 , or alk gene fusions , regardless of fusion partner.8 , 17 - 20 entrectinib seems to be well tolerated , as demonstrated in 203 patients treated at the recommended phase ii dose of 600 mg once per day.21 the most common treatment - related adverse events ( traes ) of any grade were dysgeusia ( 38% ) , fatigue ( 29% ) , constipation ( 23% ) , and dizziness ( 23% )  . 
in preclinical studies , entrectinib was a potent inhibitor of trk - driven neuroblastoma , 22 and it has demonstrated clinical activity in a 20 - month - old patient with infantile fibrosarcoma harboring an ntrk fusion.23 the benefit observed in the two patients reported here along with published data on the benefit of entrectinib in trk fusion - positive infantile fibrosarcoma highlight the potential of entrectinib in pediatric tumors harboring trk , ros1 , or alk fusions . 
the startrk - ng trial is currently enrolling pediatric patients with cancer , including primary cns tumors , neuroblastoma , and other non - neuroblastoma extracranial solid tumors harboring gene fusions in ntrk , ros1 , or alk , or harboring alk molecular alterations.23 these cases add to the growing body of evidence that demonstrates the diversity of gene fusion drivers in the formation of imt and underscore the importance of screening patients with imts for ntrk , ros1 , and alk gene fusions by using the best available diagnostic techniques . 
 the gene fusions present in these patients were identified by using a workflow that sequentially uses two different diagnostic platforms : a hybrid capture - based targeted exome sequencing assay ( msk - impact ) and anchored multiplex pcr ( archer fusionplex )  . 
 these cases also highlight the importance of treating patients with imts who harbor trk , ros1 , or alk fusions with drugs , such as entrectinib , that bind and inhibit the kinase domains of these fusion proteins . 
imt is often characterized by the presence of alk rearrangements ; however , additional gene fusions have also been identified in this rare cancer , including ros1 , ntrk3 , and pdgfr , 2 , 4 , 5 including a patient with etv6 - ntrk3 fusion - positive imt who is being considered for treatment with entrectinib.27 in patients with alk fusion positive imt , there have been varying efficacy results reported when patients are treated with alk inhibitors , including crizotinib , ceritinib , and alectinib.28 - 32 in addition , a recent report noted that a patient with imt harboring a tfg - ros1 fusion exhibited a poor response to crizotinib.6 there remains an unmet need in this patient population for safe and effective therapies that can treat patients across a wide range of gene fusion drivers . 
for more information about asco 's conflict of interest policy , please refer to or ascopubs.org / po / author - center . the potential benefit of entrectinib in that it inhibits select targeted kinases with high potency , allowing for the treatment of multiple distinct patient populations with one therapy . we have demonstrated clinically significant improvement in two patients with imts who harbor actionable gene fusions treated with entrectinib in the startrk - ng trial . 
basu no relationship to disclose acknowledgments the authors thank the patients who granted us consent to present their cases for the benefit of the scientific , medical , and patient communities worldwide and nick cianciola , of the lockwood group , for providing medical writing support funded by ignyta . accountable for all aspects of the work : all authors stock and other ownership interests : ignyta affiliations srikanth r . 
basu , memorial sloan kettering cancer center , new york , ny ; and edna chow - maneval , ignyta , san diego , ca . support supported by ignyta ; funded by national institutes of health national cancer institute grant no . 
antonescu cr , suurmeijer aj , zhang l , et al : molecular characterization of inflammatory myofibroblastic tumors with frequent alk and ros1 gene fusions and rare novel ret rearrangement . 
drilon a , siena s , ou si , et al : safety and antitumor activity of the multitargeted pan - trk , ros1 , and alk inhibitor entrectinib : combined results from two phase i trials ( alka - 372 - 001 and startrk - 1 )  . 
loke bn , lee vkm , sudhanshi j , et al : novel exon - exon breakpoint in cic - dux4 fusion sarcoma identified by anchored multiplex pcr ( archer fusionplex sarcoma panel )  . 
menichincheri m , ardini e , magnaghi p , et al : discovery of entrectinib : a new 3 - aminoindazole as a potent anaplastic lymphoma kinase ( alk ) , c - ros oncogene 1 kinase ( ros1 ) , and pantropomyosin receptor kinases ( pan - trks ) inhibitor . 
ardini e , menichincheri m , banfi p , et al : entrectinib , a pan - trk , ros1 , and alk inhibitor with activity in multiple molecularly defined cancer indications . 
cheng dt , mitchell tn , zehir a , et al : memorial sloan kettering - integrated mutation profiling of actionable cancer targets ( msk - impact ) : a hybridization capture - based next - generation sequencing clinical assay for solid tumor molecular oncology . 
green dc , deharvengt sj , de abreu fb , et al : use of anchored multiplex pcr enrichment for detection of gene fusions in solid tumors by next generation sequencing . 
drilon a , li g , dogan s , et al : what hides behind the masc : clinical response and acquired resistance to entrectinib after etv6 - ntrk3 identification in a mammary analogue secretory carcinoma ( masc )  . 
sartore - bianchi a , ardini e , bosotti r , et al : sensitivity to entrectinib associated with a novel lmna - ntrk1 gene fusion in metastatic colorectal cancer . 
ahn mj , cho bc , siena s , et al : entrectinib in patients with locally advanced or metastatic ros1 fusion - positive non - small cell lung cancer ( nsclc )  . 
rangaraju s , li g , christiansen j , et al : pediatric phase 1 / 1b study of entrectinib in patients with primary brain tumors , neuroblastoma , and ntrk , ros1 , or alk fusions . 
vendrell ja , taviaux s , bganton b , et al : detection of known and novel alk fusion transcripts in lung cancer patients using next - generation sequencing approaches . 
subbiah v , mcmahon c , patel s , et al : stump unstumped : anti - tumor response to anaplastic lymphoma kinase ( alk ) inhibitor based targeted therapy in uterine inflammatory myofibroblastic tumor with myxoid features harboring dctn1 - alk fusion . 
ono a , murakami h , serizawa m , et al : drastic initial response and subsequent response to two alk inhibitors in a patient with a highly aggressive alk - rearranged inflammatory myofibroblastic tumor arising in the pleural cavity . 
mansfield as , murphy sj , harris fr , et al : chromoplectic tpm3 - alk rearrangement in a patient with inflammatory myofibroblastic tumor who responded to ceritinib after progression on crizotinib . 
 la radiologia medica ( 2019 ) 124 : 408413 neuroradiology singlecenter experience using the3max or4max reperfusion catheter forthetreatment ofacute ischemic stroke withdistal arterial occlusions inpatients noteligible forintravenous fibrinolysis danielegiusepperomano1 ignaziomariavallone1 paolagennari1 maurizioacampa2 giuseppemartini2 sandrabracco1 tommasocasseri1 saraleonini1 rossanatassi2 samuelecioni1 received : 10 september 2018 / accepted : 4 december 2018 / published online : 13 december 2018 italian society of medical radiology 2018 abstract background in acute stroke , distal cerebrovascular occlusions can be linked to severe clinical symptoms , and treatment by mechanical thrombectomy may have an important clinical impact . 
when intravenous fibrinolytic therapy is firmly contraindicated , it remains the only treatment option . methods a total of 42 patients with isolated distal arterial occlusions and absolute contraindication for intravenous fibrinolytic therapy were retrospectively included . 
in a cohort of patients with absolute contraindication for intravenous fibrinolytic therapy , a significant percentage achieved good revascularization . keywords acute stroke distal occlusions mechanical thrombectomy introduction in 2015 , five randomized trials investigated the efficacy of modern endovascular therapies for stroke [ 15 ]  . 
they provided strong evidence to support the use of mechanical thrombectomy when initiated within 6h from stroke onset , prompting worldwide changes in the guidelines for management of acute stroke due to large - vessel occlusion . * daniele giuseppe romano dromano80@gmail.com 1 unit ofneuroimaging andneurointervention ( nint ) , department ofneurological andsensorineural sciences , azienda ospedaliera universitaria senese , policlinico santa maria alle scotte , viale m . 
bracci 2 , siena , italy 2 stroke unit , department ofneurological andsensorineural sciences , azienda ospedaliera universitaria senese , policlinico santa maria alle scotte , siena , italy these trials used a variety of treatment approaches , including the mandatory use of intravenous thrombolysis before the initiation of endovascular therapy . consequently , american heart association ( aha ) / american stroke association ( asa ) published its scientific rationale for the inclusion and exclusion criteria for intravenous alteplase in acute ischemic stroke [ 6 ]  . 
even if some of the original exclusion criteria have proven to be unnecessarily restrictive in clinical practice , absolute contraindications remain . the occlusion of smaller arteries , such as the m2 or m3 segments of the middle cerebral artery ( mca ) , a2 segments of the anterior cerebral artery ( aca ) or the posterior cerebral artery ( pca ) , sometimes causes ischemic strokes with pronounced clinical impact on the patient [ 7 ]  . the updated aha 2018 stroke guidelines [ 8 ] specifically talk about distal occlusions . 
 the number of attempts was guided by tici score after aspiration . before treatment , informed consent was obtained from technical success was defined as recanalization of the the patient if conscious or a legal representative . target vessel according to tici score 2b . for each patient , we considered age , sex , risk factors , baseline mrs , nihss and aspect score ( or early signs of ischemia in vascular territory of a2 or p1p2 ) , time from symptoms onset to groin puncture , time from groin puncture to revascularization , type of endovascular procedure ( adapt alone vs stent retriever ) and device used , tici score at the end of the procedure , kind of anesthesia , aspect score at 24h or signs of ischemia in vascular territory of a2 or p1 , complications , nihss at 24h and 30days , rankin score ( mrs ) at 90 - days follow - up . all patients underwent unenhanced ct to exclude hemorrhage and ct angiography to detect cerebrovascular occlusions . 
in case of uncertain onset of the symptoms ( i.e. , wake - up stroke ) , patients were considered eligible for treatment when salvageable brain parenchyma was depicted by perfusion ct mismatch . 
all patients were evaluated by a stroke - dedicated neurologist . two blinded neuroradiologists with 8 and 23years of experience in interventional neurovascular procedures ( dgr and sb ) reviewed all imaging data and assessed aspect score from the baseline ct scan and the tici scores after intervention . 
they also reviewed the imaging data for complications of emboli to new territory , vessel dissection , vasospasm , vessel perforation and ich . statistical analysis continuous parameters were compared using the students t test for normally distributed data or the mannwhitney u test for not normally or ordinally distributed data . 
a p value of 0.05% or lower was considered to be statistically significant . results we retrospectively included 42 consecutive patients with acute ischemic stroke from distal arterial occlusions and absolute contraindication to fibrinolytic therapy for the following reasons : current use of anticoagulant with inr > 1.7 or pt > 15s ( n = 13 ) , platelet count < 100 000 / mm3 [ 1 ] , after 4.5h time window ( n = 28 )  . patients with wake - up stroke were 11 / 42 . all patients were treated in our hospital with thromboaspiration as first intention using the penumbra reperfusion catheter . target vessel occlusions were : m2 ( n = 46 ) , p1 ( n = 4 ) , a2 ( n = 2 )  . 1 3 la radiologia medica ( 2019 ) 124 : 408413 410 fig . 
1 male patient , 72 y.o with right m2 parietal branch occlusion ( a , b ) , ace 64 reperfusion catheter was advanced over a 3max aspiration catheter into the right mca to the site of occlusion and left at the face of the thrombus ( c , d ) , aspiration was applied resulting in successful revascularization 4h after symptom onset ( e , f ) 57.1% were female ( n = 24 )  . 
 [ 18 ] achieved substantially the same clinical outcome in the treatment of m2 occlusions using stent retriever versus thromboaspiration , with slightly less complications in the last . notably aspiration catheters offer an alternative strategy for achieving thrombus removal by applying suction at the proximal portion of the occlusion and drawing it into the catheter lumen . 
the smaller percentage of successful outcome was explained by the inclusion criteria ( patients with absolute contraindication for intravenous fibrinolytic therapy ) and clinical characteristics of our population ( moderate to severe stroke with a mean nihss score of 18 )  . the duration of the endovascular treatment ( time from groin puncture to reperfusion ) was significantly lower in patients who underwent adapt alone because a stent retriever was used in case of failure of adapt . 
nonetheless , switching to other techniques might provide better angiographic outcomes in cases with unsuccessful reperfusion after a few attempts with the adapt technique . general anesthesia ( used in 71.4% of cases ) , keeping the patient motionless , allows a safer procedure when dealing with a small intracranial vessel occlusion and permits the operator to concentrate on the procedure . procedural or anesthetic complications did not occur in the present study . the risk profile for symptomatic bleeding was similar to that in recently reported controlled clinical trials [ 15 ]  . main limitations of this paper are the lack of randomization and the fact that it is a single - center , retrospective and observational study over a long period . conclusions in a cohort of patients with absolute contraindication for intravenous fibrinolytic therapy , a significant percentage achieved good revascularization . direct thromboaspiration appears a safe technique in acute isolated distal arterial occlusions . 
fifty - two patients ( study group ) underwent 256 - mdct low - dose ccta ( 80kv ; automated - mas ; 60ml of cm , 350 mgl / ml ) with prospective ecg - triggering acquisition and imr . 
a control group of 46 patients underwent 256 - mdct standard prospective ecg - gated protocol ( 100kv ; automated - mas ; 70ml of cm , 400 mgl / ml ; idose4 )  . 
radiation dose exposure was quantified as dlp , ctdivol and ed . results mean values of mas were significantly lower for imr - ccta ( 167 62 mas ) compared to idose - ccta ( 278 55 mas ) , p < 0.001. 
therefore , according to alara principle , low - dose coronary ct angiography has recently been performed in several studies [ 11 , 12 ] , thanks to the use of wide detector panels [ 13 , 14 ] and due to a reduction of tube - current and / or tube - voltage setting . 
for this reason , hybrid - iterative reconstruction ( hir ) algorithm for ct , such as asir ( ge healthcare ) , aidr 3d ( toshiba medical ) and idose ( philips healthcare ) , was introduced vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 350359 in clinical practice in the last years , in order to reduce the image noise due to the use of low - dose protocol with filtered back projection ( fbp ) reconstruction technique . 
unfortunately , some artifacts and a certain amount of image noise was still present in hir images , so the new model - based iterative reconstruction algorithm ( i.e. , imr , philips healthcare ; veo , ge healthcare ; admire , siemens ; first , toshiba medical , canon ) was introduced in clinical practice , trying to solve these problems . 
this new reconstruction technique can effectively control the image noise while maximizing the spatial resolution with a significant radiation dose sparing than those obtained with fbp and hybrid - iterative reconstruction algorithm [ 17 ]  . one of the concerns of the model - based iterative reconstruction algorithm is the time spend to generate the final images ; but , thanks to new faster computing hardware and algorithmic optimization , nowadays the reconstruction time for imr is of about 2min for cardiac ct ( about 200 images / minute ) [ 17 ] , making imr applicable also in clinical practice . moreover , this reconstruction technique combined to low - dose protocol leads to lower contrast medium volume use thanks to higher attenuation levels of iodinated contrast medium at low kv - setting due to greater photoelectric effect and decreased compton scattering [ 12 , 1820 ]  . 
in order to obtain an optimal intra - luminal contrast enhancement , the start of scanning was individually obtained for each patient by using bolustracking technique ( bt ) , with a trigger level of 120 hu and a delay time between 5 and 8s . 
the trigger area was manually placed at the proximal ascending aorta , in order to reduce the time needed for the ct bed to reach the starting level of acquisition . 
in all 52 patients of this group , low volume ( 60ml ) of intermediate iodine concentration contrast agent ( xenetix 350 , 350 mgl / ml , guerbet aulnay , france ) was administered , with a flow rate of 4.5ml / s followed by saline flushing ( volume 35ml ; flow rate 4.5ml / s ) , for a total iodine amount of 21g . all the raw data were reconstructed with the new modelbased iterative reconstruction algorithm ( imr level 1 ) and standard filter cardiac routine , as suggested from the vendor . control group protocol control group patients were evaluated on a different 256mdct scanner ( brilliance ict , philips medical systems , best , the netherlands ) , with a standard 100kv protocol and automated tube - current modulation ( range of 200800 mas )  . 
the other scan parameters were : collimation 128 0.625 ; rotation time 0.33s ; thickness 1mm ; increment 1mm ; fov 350mm ; matrix 512 512 ( table1 )  . in all patients of the control group , we used a total of 70ml of a high iodine concentration cm ( iomeron 400 , 400 mgl / ml , bracco imaging , italy ) at a flow rate of 4.5ml / s , followed by saline flushing ( 50ml ; flow rate 4.5ml / s ) , for a total iodine amount of 28g . the dual - scout image was used to optimize the acquisition volume and the same prospective ecg - gating protocol of the study group ( step & shoot ) was used to acquire the cardiac examination . the raw data were reconstructed with hybrid - iterative reconstruction algorithm ( idose4 , level 4 ) and standard filter cardiac routine was applied . radiation dose quantification to analyze the radiation dose exposure , the ct dose - length product ( dlp , mgy cm ) and the ct dose index ( ctdivol , mgy ) were recorded for all scans . 
we also calculated the effective radiation dose ( ed ) of coronary cta , using the known formula ed = k dlp , where dlp ( mgy m ) is the dose - length product and k is the region - specific normalized effective dose ( msv / mgy1cm1 ) , derived from the paper by deak etal . 
 [ 28 ] ; the region - specific conversion coefficients of k = 0.0146msv / mgy1cm1 were used for both the 80 - kv and 100 - kv protocols [ 28 ]  . quantitative image evaluation images were anonymized and then processed on a dedicated workstation ( intellispace portal , philips ) in order to produce multiplanar reconstructions , maximum intensity projections ( mip ) and volume rendering ( vr ) images . 
final images were analyzed by two radiologists in consensus for the quantitative evaluation . vessel contrast enhancement ( mean attenuation value , hu ) was measured in the axial native images , by manually placing a circular region of interest ( roi ) in the center of vascular lumen in the left ventricle , ascending aorta , in the proximal and distal segment of left anterior descendant ( lad ) , left circumflex ( lcx ) and right coronary artery ( rca )  . 
the rois size was as large as possible depending on artery size , avoiding wall calcifications and atherosclerotic plaque , and in order not to be affected by pixel variability . we also compared the image noise of two groups , determined as the standard deviation ( sd ) of the attenuation value in each proximal coronary segment . 
moreover , we evaluated the contrast resolution by calculating the contrastto - noise ratio ( cnr ) using the formula : cnr = huahub where hua is the mean attenuation of the proximal tract of rca , hub and sdb are the mean attenuation and standard deviation of the adjacent adipose tissue , as reported in the paper by park etal . 
for subjective evaluation , the overall diagnostic quality of the images of ascending aorta and the previously described coronary arteries ( lad , lcx and rca ) were evaluated using a 4 - points subjective scale [ 30 ] : 4 as excellent ( minimal image noise and artifacts with excellent visualization of boundary of each coronary segment ) ; 3 as good ( some image noise , good visualization of the boundary , minor artifacts especially in small middle and distal coronary segments and good visualization of proximal tract ) ; 2 as acceptable ( delineation of the boundary equivocal but within an acceptable range , minor artifacts and sufficient visualization of proximal and middle tract ) ; 1 as low ( major artifacts , unsatisfactory delineation of the boundary or poor visualization of coronary arteries at any levels )  . moreover , subjective image noise , image texture and image sharpness were evaluated by the two radiologists using a similar 4 - point scale . 
for the evaluation of the image texture and noise : 4 was excellent ( noise and unnatural texture not present , with high diagnostic images ) ; 3 was good ( image noise and unnatural texture present but do not interfere with depiction of adjacent structures ) ; 2 was acceptable ( image noise and unnatural image texture that interfere with depiction of adjacent structures ) ; 1 was low ( image noise and unnatural texture unacceptable ; low diagnostic images )  . 
image sharpness was graded by evaluating ascending aortic wall : 4 excellent , the best sharp image ; 3 good , better than average ; 2 as acceptable but poorer than average ; 1 as low because of a blurry image [ 7 ]  . statistical analysis all continuous variables were expressed as mean standard deviation ( sd ) and compared with t - student test and mannwhitney u test , as appropriate . 
categorical variables are expressed as number and percentage and compared with 2 test , using bonferroni correction . the agreement between the two observers in the subjective image evaluation was measured with the kappa statistic , where a value of 0 = no agreement ; > 0 and 0.20 = poor ; 0.210.40 = fair ; 0.410.60 = moderate ; 0.610.80 = substantial ; and 0.810.99 = almost perfect agreement . all statistical analyses were performed with commercially available software ( med calc , med calc software 14.8.1 , mariakerke , belgium )  . 
a value of p < 0.05 was considered to be a statistically significant difference . table 2 comparison of patients characteristics of the study and control groups data / protocol study group control group p value no . 
in the last 10years , the patients radiation burden concerns led to the optimization of ccta protocol with the introduction of new techniques , such as the step and shoot acquisition , and by modifying acquisition parameters settings , as lower tubecurrent and mas modulation . 
c , d curved and straight reconstruction of right coronary artery with a good visualization of artery wall due to high snr and cnr leading to high spatial resolution 1 3 356 la radiologia medica ( 2019 ) 124 : 350359 fig . 
a , b shows the overall image quality between the idose4 image ( a ) and imr ( b ) , with lower image noise and higher spatial resolution in the image on the right . 
on the contrary , the attenuation value of the three coronary arteries was better in the imr studies compared to idose4 ( p < 0.05 ) , obtaining a mean attenuation value in coronary lumen measured in all patients above to the minimum value of 300 hu , as suggested in literature [ 32 , 33 ]  . 
the overall subjective image quality was better for imr - group compared to idose one for both the readers , without any statistical differences ( p = 0.23 and p = 0.47 for reader 1 and reader 2 , respectively )  . 
also for the other qualitative evaluations , the scores were better for study group compared to control one , without any difference ( p > 0.05 ) except for the texture evaluation of reader 1 , who assigned higher rate to imr - images , as shown in table5 . these results demonstrated that low - dose ccta with imr reconstruction provides comparable diagnostic images compared to standard dose examination . 
moreover , the use of reduced kv - setting , increasing the image noise in the raw data , avoids the plastic appearance of mbir into the final images , previously described in literature by barras etal . 
moreover , we used only the first level ( imr1 ) of the model - based iterative reconstruction ( imr 13 ) , suggested from the vendor and selected as standard by our institution . 
thirdly , the patients in our series had a bmi 30kg / m2 , and therefore , further studies are needed to test the lowkv protocol combined to imr in patients with higher bmi . 
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park ch , lee j , oh c , han kh , kim th ( 2015 ) the feasibility of sub - millisievert coronary ct angiography with low tube voltage , prospective ecg gating , and a knowledge - based iterative model reconstruction algorithint j cardiovasc imaging 31 ( 2 ) : 197203 . 
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therefore , dce - mri parameters can predict tumor aggressiveness and crt efficacy , playing a role as imaging biomarkers in patients with larc . keywords locally advanced rectal cancer magnetic resonance imaging imaging biomarkers neoadjuvant therapy patient outcome * andrea laghi andrea.laghi@uniroma1.it introduction 1 department ofsurgical andmedical sciences andtranslational medicine , sapienza university ofrome , radiology unit santandrea university hospital , via di grottarossa , 1035 - 1039 , 00189rome , italy 2 department ofradiological sciences , oncology andpathology , uoc radiologia , policlinico umberto i hospital , sapienza university ofrome , viale regina elena , 324 , 00161rome , italy 3 department ofradiological sciences , oncology andpathology , uoc radioterapia , policlinico umberto i hospital , sapienza university ofrome , viale regina elena , 324 , 00161rome , italy 4 department ofradiological sciences , oncology andpathology , uoc anatomia - patologica , policlinico umberto i hospital , sapienza university ofrome , viale regina elena , 324 , 00161rome , italy neoadjuvant chemoradiotherapy ( crt ) is the treatment of choice in patients with locally advanced rectal cancer ( larc ) [ 13 ]  . 
thus , a reliable diagnostic method able to stratify larc patients could be beneficial to reduce crt - related toxicity and select the proper treatment strategy for each patient . magnetic resonance imaging ( mri ) represents the noninvasive reference standard in the diagnosis of larc due to its excellent contrast resolution [ 5 , 6 ]  . 
however , conventional mri only relies on morphological parameters such as changes in tumor size , volume , and signal intensity [ 7 ] , resulting inadequate in predicting response to crt [ 8 ]  . 
due to their correlation with tumor aggressiveness [ 11 ] , dce - mri parameters quantification could represent an appealing strategy in management of patients with larc , helping in the selection of the most appropriate treatment for each patient and eventually resulting in a better prognosis [ 12 ]  . in the last decades , dce - mri parameters for larc have been widely investigated , with particular interest in the assessment of tumor response after crt and patient prognosis prediction [ 1318 ]  . 
however , inconsistent results have been returned among different investigations , reporting either good efficacy [ 15 , 19 ] of dce - mri in predicting response to crt or opposite results [ 14 , 17 ]  . hence , the aim of our study was to correlate dce - mri parameters to tumor grading and to assess their reliability in predicting pathological complete response ( pcr ) before neoadjuvant crt in patients with larc . materials andmethods patients characteristics this retrospective study was approved by the institutional review board of ( blinded ) with a waiver for informed consent . 
the study included data from consecutive patients with histologically proven larc at stage ii ( ct34 , n0 , m0 ) or stage iii ( ct1 - 4 , n + , m0 ) who had undergone clinical assessment by dce - mri before and after crt between august 2015 and february 2016 . 
a routine clinical imaging protocol wasacquired , which included high - resolution t2 - weighted fast recovery fast - spin echo ( 2d frfse ) sequencewith the following parameters : tr : 20864172ms ; te : 11.4122.3ms ; acceleration factor ( nex ) : 2 ; slice thickness : 4mm ; matrix : 512 512 . 
dedicated axial oblique and coronal oblique planes wereobtained , respectively , orthogonal and parallel to the long axis of the rectuaxial dwi images were obtained using a single - shot echo - planar imaging sequence with spectral adiabatic inversion recovery fat saturation technique ( tr : 4400ms ; te : 81.4ms ; nex , 2 ; slice thickness : 4mm ; matrix : 256 256 ; b values : 0 , 200 , 800s / mm2 )  . for the specific purpose of our study , dce - mri was added to the standard rectal cancer mri clinical protocol . 
 dce images wereobtained with a 3d gradient recalled echo ( gre ; tr : 14.05 ms ; te : 3.66 ms ; nex , 1 ; slice thickness : 3 mm ; matrix : 320 320 ) sequence which started simultaneous to the intravenous administration of gadolinium chelate ( gadobenate dimeglumine , multihance ; bracco imaging ; 0.1mmol / kg ) at 2ml / s followed by 15ml of saline flush at the same flow rate . 
the entire volume was acquired in 1s , and the acquisition was repeated over a scan time of 60s using a thin collimation ( 2mm ) in order to obtain an accurate evaluation of the contrast media kinetics in the tumor tissue during all vascular phases . neoadjuvant therapy chemoradiotherapy was administered following the standard of care in our institution [ 20 ]  . 
an additional dose of 5.49gy was administered to the tumor volume in 35days ( 615 mv energy photons )  . chemotherapy was administered through a central venous access as follows : 5 or 6 cycles of oxaliplatin ( 2 - h infusion 50mg / m2 ) on the first day of each week of radiotherapy followed by five daily continuous infusions of 5 - fu 200mg / m2 / die . 
toxicity was evaluated according to nci - ctc version 3.0dsds. table 1 dce - mri exclusion criteria incomplete dce - mri acquisition or histopathological analysis contraindication to the use of neoadjuvant therapy or surgical treatment suspension of neoadjuvant combination chemotherapy and radiation treatment prior to surgery treatment with concurrent and experimental drugs , or participation in another clinical trial technical problems in post - processing of dce - mri images diagnosis of mucinous rectal adenocarcinoma 1 3 la radiologia medica ( 2019 ) 124 : 331338 surgical technique during a time frame of 6 to 8weeks after the completion of crt , all patients underwent total mesorectal excision [ 21 ] performed by experienced colorectal surgeons , each of them with at least 10years of experience in this technique . histopathological assessment a single gastrointestinal pathologist with 10years of experience in rectal cancer performed all of the histopathology analyses , which were carried out as assessments of the basic histopathology of the primary tumor ( type and grade of the tumor ) samples obtained as a biopsy specimen taken before the crt treatment . 
all analyses comprised evaluation of surgical margins and other histological features , including t stage and n stage according to the 7th edition of the american joint committee on cancer [ 22 ]  . for the specific purpose of our study , patients were divided into two groups : complete responders ( cr ) and noncomplete responder ( ncr )  . 
cr group includedpatients who showed downstaging after crt and whose surgical intervention was classified as pathological complete response ( ypt0n0 ) , hereafter indicated as pcr . perfusion imaging mri_1 and mri_2 images were processed with a dedicated software ( olea sphere , v2.3 ; olea medical , la ciotat , france ) and were analyzed using the extended tofts model [ 23 ] to evaluate quantitative perfusion parameters . 
two board certified radiologists with 8 and 5years of experience in rectal mridrew in consensus the region - of - interests ( roi ) on perfusion maps around the central tumor ( carefully avoiding necrosis or cystic areas ) and the external iliac artery . 
other additional rois were placed in the gluteus maximus and in the healthy intestinal wall to normalize signal intensities as reported in other studies to overcome the issue of high range of absolute perfusion values [ 2426 ]  . 
for each roi , the software provided values for ktrans ( plasma - toextravascular volume transfer ) , kep ( extravascular - to - plasma volume transfer constant ) , ve ( extravascular extracellular volume fraction per unit of tissue volume ) , vp ( plasma volume ) , wash - in ( velocity of enhancement ) , wash - out ( velocity of enhancement loss ) , curve - washout ( wash - out slope ) , peak enhancement , peak ( maximal concentration of contrast agent ) , and tme ( time - to - maximal enhancement )  . statistical analysis test was used to assess normality of data distribution . 
tumor roi values were normalized to the healthy intestinal wall and maximum gluteus muscle . spearmans test was used to assess correlation between dce - mri parameters and tumor grading , and tumor pathological status related to crt . 
comparison between mri_1 values and mri_2 values , cr patients and ncr patients , and g3 , g2 , and g1 tumors was carried out using either mannwhitney u test ( if data presented a nonparametric distribution ) or unpaired t test ( if data presented a parametric distribution )  . 
discriminatory power of each parameter to predict tumor grading or cr was assessed by receiver operating characteristic ( roc ) curve analysis and the calculated area under the curve and the corresponding p value . 
p value of < 0.05 was considered statistically significant . results patient characteristics from an initial cohort of 51 patients , 11 were excluded due to incomplete mri examination ( n = 1 ) , suspension of crt prior to surgery ( n = 5 ) , and participation in other clinical trials ( n = 5 )  . 
 wash - out returned the largest area under the roc curve ( = 0.801 ; [ 95% ic 0.7630.851 ] ) ; a wash - out value of 0.79 provided a sensitivity of 80% and a specificity of 70% in differentiating g3g2 from g1 larc . 
in particular , 17 patients ( 68% ) showed partial response , 5 patients ( 20% ) showed stable disease , and 3 patients ( 15% ) were classified as non - responders . 
these perfusion parameters showed a moderate discriminatory power ( area under the curve : 0.709 [ 95% ic 0.6720.737 ] for ktrans ; 0.71 [ 95% ic 0.6850.749 ] for tme ; and 0.68 [ 95% ic 0.6500.716 ] for auc , respectively )  . 
we documented the correlation of some dce - mri parameters with tumor grading , their changes during crt and their potential to distinguish , at baseline examination , patients with larc that will achieve pcr from those non - responsive to crt . it has been reported that the degree of angiogenesis in a tumor correlates with tumor grading and aggressiveness , which in turn are related to prognosis and response to treatment [ 27 , 28 ]  . 
more mature vessels can permit a higher exchange between the blood bed and the interstitial space , with more effective passage and permeability of different substances , including mri contrast media . 
on the contrary , poorly differentiated tumors consist of disorganized and leaky vessels , with ineffective exchange and inefficient delivery of substances , such as oxygen which may contribute to tumor hypoxia [ 29 ]  . we demonstrated that wash - out and kep are two perfusion parameters significantly correlated with tumor grading . 
in detail , our results indicate that wash - out values are positively correlated with tumor grade resulting higher in g3 - tumors , while kep values show a negative correlation with cancer grading resulting higher in g1 - tumors . 
kep , defined as the ratio between ktrans and ve , is the rate constant of contrast agent escaped from the extracellular extravascular space into plasma compartment [ 30 ]  . 
higher kep in well - differentiated tumors means that there is an increased return flow from the tumor interstitial space to neo - vessels and this reflects a more permeable neo - vasculature allowing better exchange between interstitial space and vessels . 
on the contrary , poorly differentiated tumors presented a lower kep , reflecting a lower vessel permeability and a lower capability of the tumor to exchange substances with the vascular network . an inverse correlation between kep and tumor grade has also been found by yeo etal . 
 [ 11 ] , which investigated quantitative perfusion parameters in 46 patients with rectal cancer , reporting higher kep values in well - differentiated tumors . the analysis of perfusion parameters changes between baseline and post - crt examination showed a significant ve increase . 
 [ 32 ] documented higher ve associated with tumor regression in patients with cervical cancer during crt , and with liver metastases shrinkage from rectal cancer during chemotherapy , respectively . 
6 receiver operating characteristic ( roc ) curves of tme , auc , and ktrans patient management by the implementation of wait - andwatchstrategy [ 35 ] , ultimately reducing the number ofsurgical interventions and their related risks . 
our results indicate ktrans as a reliable indicator of complete response to crt : at the baseline mri examination , we demonstrated higher ktrans in those patients lately identified as pcr . 
ktrans has been widely investigated as a new tumor biomarker , and our finding of a higher ktrans in cr group is in accordance with other investigations [ 4 , 19 , 34 ]  . 
 first , our sample size was relatively small and further investigations with larger sample sizes are advisable to confirm our results and toobtain robust data on overall survival and disease - free survival . 
third , despite a consensus session with two expert readers , the manual roi placement is less reproducible than an automated placement , which would be more appropriate to guarantee standardization of measurements . conclusion mri - derived ktrans , tme , and auc measured before crt are reliable in discriminating cr and ncr patients with larc , while kep and wash - out significantly correlate with tumor grading . 
we evaluated the relationship between maximum and mean standardized uptake values ( suvmax and suvmean , respectively ) , metabolic tumor volume and total lesion glycolysis ( tlg ) of primary tumor and cervical lymph nodes with disease - free survival ( dfs ) and overall survival ( os )  . 
the corresponding planning target volume consisting of gtvs plus asymmetric margins of 0.51.5 cm to account for microscopic diseases and setup uncertainties was planned to receive a total dose of 70gy with conventional or moderately accelerated fractionation ( 22.12gy per fraction , 5 fractions per week )  . 
radiotherapy ( rt ) was delivered by relying on a conventional static fields technique ( conventional imrt ) or volumetric modulated arc therapy ( vmat ) with sequential or simultaneous integrated boost approaches . chemotherapy patients staged i and iia received exclusive rt , whereas patients with stage iibiiiiv received concomitant platinum - based cht . 
a boundary box to autocontour and 1 3 416 la radiologia medica ( 2019 ) 124 : 414421 segment the region of interest was placed over the image , reviewed and adjusted to ensure that the entire lesion was included in all three sectional pet images ( axial , coronal , sagittal )  . 
these vois were segmented automatically using an iterative adaptive algorithm to detect the threshold level that separated the target volume from the background tissue by weighting the maximum and mean standardized uptake value ( suvmax and suvmean ) within the target volume with a weighting factor . 
this weighting factor was automatically set at 0.5 [ 16 , 17 ]  . using the generated voi , suvmax , suvmean and metabolic tumor volume ( mtv ) were automatically recorded . 
 total lesions glycolysis ( tlg ) was calculated multiplying suvmean by mtv . in patients with multiple hypermetabolic lymph nodes , the metabolic parameters of each node were summed . followup after rt completion , patients received follow - up examinations including clinical exams and mri at predefined intervals , typically every 36months for the first 3years and annually thereafter . 
pearson correlation coefficients ( ) were determined to test the correlation between pet and clinical and biomarker features . overall survival ( os ) and disease - free survival ( dfs ) were evaluated starting from the first day of treatment ( cht or rt , whichever came first ) to the day of death or recurrence . 
maximal chi - square method was used to determine the optimal cutoff values for pet parameters and for the following factors : age , t stage , n stage , overall stage and gtvs . 
additionally , tlg - t had the highest hr for os . 1 3 la radiologia medica ( 2019 ) 124 : 414421 table 1 general characteristics of population ( n = 49pts ) table 2 distributions for pet and clinical parameters characteristics no . 
although limited to a small retrospective cohort of patients , the present study is the most comprehensive to date performed in a low - incidence area . at baseline , no correlation was discovered between pet and clinical , biological and therapeutic parameters . 
specifically , the correlation between pet indicators and clinical stage is a controversial issue in literature [ 1820 ]  . although some differences can be found in methods of t and n staging among ours and other series , we can bolster the idea that tnm staging and pet could detect different information . 
moreover , the fact we did not find any correlation between pet parameters and gtv strengthens the 1 3 418 la radiologia medica ( 2019 ) 124 : 414421 fig . 
ned no evidence of disease , dod dead of disease supposition that tumor volume cannot be related to its metabolic activity in npc . metabolic information provided by pet / ct imaging was investigated into target volume delineation by many authors . 
we 1 3 420 la radiologia medica ( 2019 ) 124 : 414421 think that additional ad hoc studies are necessary to clarify the use of pet / ct in this setting . the identification of prognostic factors plays an important role for cancer treatment stratification and improvement of clinical outcome in npc patients . two recent meta - analyses on the prognostic role of 18ffdg pet / ct in npc patients found that suvmax , tlg and mtv of primary tumor predict the risk of failure or death , notwithstanding the differences in method adopted in the literature taken into consideration [ 26 , 27 ]  . in our study , we showed that suvmax , suvmean and tlg of primary tumor were significant predictors of os in non - endemic ebv - related npc patients treated with imrt with or without systemic treatments ; tlg was demonstrated to have the highest hr for os . 
in particular , in the largest series including 371 patients , those showing a suvmax lower than 9 , 3 significantly had better 5 - year os compared to patients with higher values [ 9 ]  . 
 [ 19 ] in a study on 179 patients concluded that suvmax > 10.22 predicted worse moreover , in our analysis , a prognostic role of primary tumor suvmean was identified . 
similarly , patients having a tlg > 65 showed lower os and dfs compared to patients who with tlg < 65cc [ 11 ]  . on the other side , we did not find any association between pet parameters and dfs . although a crucial reason may be a low number of patients for the study , it is also true that a factor that can predict the outcome of loco - regional / distant metastasis survival and dfs may not always yield the same results for os . 
 xiao suggested that high fdg accumulation may primarily reflect the proliferative activity of npc and thus tumor aggressiveness [ 19 ]  . we can state that suv - t and tlg - t may refer to very aggressive tumors unsuited to salvage treatment . 
standard lumbar mri protocol of sagittal , and axial t1 weighted images ( wi ) and t2 wi and coronal short tau inversion recovery ( stir ) t2 wi were obtained . 
the level and above the level of lstv were evaluated for the lumbar disc space and facet degeneration based on grading methods which compares subtype groups with each other . results prevalence of lstv was 32% ( 600 of 1875 )  . 
but the groups with higher grade of disc degeneration were type iv and iii . conclusion in young male patients with lbp , lstv was found to be high in frequency and mostly occurred to be subtype i . 
coronal t2 stir images are useful in showing lumbosacral region anomalies and variants , and should be included in the routine lumbar mri protocol . keywords lowback pain lstv ( lumbocral transitional vertebra ) magnetic resonance imaging male maging in the lumbosacral transitional vertebra , the extreme elongation in the transverse process of the last lumbar vertebra fuses with the first sacral segment in different shapes in different subtypes called sacralization [ 1 ]  . 
in previous studies , lstv was studied in both sexes in different age groups and very variable results were obtained [ 4 ]  . the subgroups and types of lstv in regarding to disc and facet degenerations in young male patients with low vol . : ( 0123456789 ) 1 3 376 la radiologia medica ( 2019 ) 124 : 375381 back pain were investigated retrospectively by using castellvis lstv classification [ 8 ]  . materials andmethods the research was approved by the institutional review board at the local hospital . 
between march 2012 and january 2017 , 1875 consecutive lumbar spine mri studies on adult male patients younger than 40years of age with lower back pain , lumbar radiculopathy , or both at least four weeks were reviewed . 
both t1 - weighted spin - echo images ( tr / te , 637 / 12 ; repetition time msec / echo time msec ) and t2 - weighted fast spin - echo images ( 3073 / 85 ) were obtained through the lumbosacral spine in the sagittal plane and t2 - weighted fast spin - echo images in the axial plane ( 4.400 / 85 ) of the lumbar spine with slice thickness of 4mm were performed . 
also coronal t2 short tau inversion recovery ( stir ) images were added ( 2580 / 35 ) for exact evaluation of lumbosacral area and measurements of transverse process according to the literature with slice thickness of 4.5mm , sectioned parallel to the cauda equina at l3 to l5 vertebral body levels , was obtained [ 9 , 10 ]  . 
the field of view was 200 200mm for axial scans and 280 280mm for sagittal scans , with a matrix of 256 256 , slice thickness of 4.3mm , and a 1mm gap for both axial and sagittal imaging . lstvs were classified into incomplete or complete as well as uni or bilateral origin according to the castellvis classification [ 8 ]  . 
the disc degeneration and facet degeneration of the lstv level and above were recorded and correlated among the lstv subtypes i - iv . statistical analyses were performed using spss statistical software . 
1 coronal stir t2wi demonstrate the castellvi classification of lstv : a type ia : dysplastic unilaterally enlarged transverse process ( thick arrow ) , bone changes in iliac bone ( thin arrow ) ; b type ib : dysplastic bilaterally enlarged transvers process ( thick arrow ) , bone changes in sacrum ( thin arrow ) ; c type iia unilaterally pseudoarticulation of the transverse process with the sacrum with increased sclerosis ( arrow ) ; d type iib bilateral pseudoarticulation of the transverse process with the sacrum with increased sclerosis ( arrows ) 1 3 la radiologia medica ( 2019 ) 124 : 375381 fig . 
2 coronal stir t2 wi demonstrate the castellvi classification of lstv : a type iiia : unilaterally fusion with the sacrum ( arrows ) ; b type iiib : bilaterally fusion with the sacrum ( arrows ) ; c type iv : lstv type ii ( long arrow ) with type iii on the contralateral side ( short arrow ) using bonferroni - corrected mannwhitney u test to compare disc degeneraton , facet arthrosis and the t test for continuous variables , and chi square test . 
for comparisons , continuous variables were compared by using analysis of variance or ranked analyzes of variance as required , and categorical variables were compared by using chi square test . 
these variations can be detected easily with coronal mr images especially in type 1 lstv , otherwise axial and sagittal images are not enough to detect type i ( fig.3 a , b ) [ 13 ]  . 
these results correlated well with the previous reports [ 4 ]  . facet arthrosis , disc degeneration at the lstv level and above level were evaluated and compared among the lstv subtypes . 
the bonferroni - corrected mannwhitney u test analysis was done and facet degeneration has been found to be most affected in type i to the type ii and type iii on both sides ( p < 0.0083 ) on above level of the transition . 
 short tau inversion recovery ( stir ) coronal mri sequences which produces fat supression and yields high tissue contrast especially for the diagnosis of bone and soft tissue lesions were obtained for detailed anatomy ( fig.2 ) [ 12 ]  . more lstvs detected than most of the studies in the study ( 32% )  . 
the high prevalence of lstv may be explained by the superiority of the mr protocol used to diagnose lstv and the selection of young male patients with lbp for the study . patients with lstv can show age - related lumbar degeneration over time , which may mask early degenerative disease associated with lstv [ 4 , 8 , 18 ]  . 
for these reasons , this study was conducted in young male population to prevent age or sex - related outcomes for degeneration . the reason of pain in lstv is also a debate . 
 the articulation anomaly , contralateral facet ( in one sided anomaly ) degeneration , early degeneration and instability of the above level of lstv and nerve compression from bone hypertrophy are the main causes of low back pain in lstv [ 14 ]  . 
transitional level degeneration among subgroups of the lstv is most common in patients with lstv type ii in their study [ 14 ]  . the anatomy and variations is always complicated in nature . 
so , the pain is found to be much resistant to the therapy in lstv [ 21 ]  . the lstv prevalence was reported 4.635.9% in patients with low back pain and has been suggested to be investigated in the differential diagnosis especially in young population [ 15 , 17 , 21 , 22 ]  . 
all of the sagittal , axial and coronal mri images should be obtained for the correct identification of the transitional lumbosacral component [ 11 , 14 , 15 ]  . 
secondly , due to the large number of cases , we have not been able to assess the professions and physical activities of our patients , their body mass index as well . despite these limitations , to the best of our knowledge , this study was the first that was performed with mri in a young male group . 
in order to distinguish this variant from age - related degenerative changes and different age - related anatomic findings , it has been planned and studied in young male patients for the standardization of the study . conclusion lstv were found in 32% of selected young male population . 
it was known to be disc and facet degeneration were seen widely by the age which can mask lstv related degenerative findings alone [ 4 , 8 , 18 ]  . 
for this type of study formal consent is not required . la radiologia medica ( 2019 ) 124 : 422431 radiotherapy highdose intensitymodulated radiation therapy asprimary treatment ofprostate cancer : genitourinary / gastrointestinal toxicity andoutcomes , asingleinstitution experience beatricedetti1 muhammedbaki1 carlottabecherini1 calogerosaieva2 danielescartoni1 irenegiacomelli1 lauratrombetta1 cinziaciabatti1 giuliocarta1 lindapoggesi1 camilladellipaoli1 francescaterziani1 robertagrassi1 lorenzolivi1 cristinamuntoni1 emanuelaolmetto1 giuliofrancolini1 anaturkaj1 julianatopulli1 received : 7 july 2017 / accepted : 5 december 2018 / published online : 3 january 2019 italian society of medical radiology 2019 abstract purpose prostatectomy , radiotherapy and watchful waiting are the main therapeutic options available for local stage of prostate cancer ( pca )  . 
we report our experience on 394 patients affected by prostate cancer primarily treated with high - dose , image - guided , imrt , focusing on gastrointestinal , genitourinary toxicities and biochemical control . methods from july 2003 to august 2014 , 394 patients were treated with radical high - dose radiotherapy ( hdrt ) for prostate cancer ; the mean total radiation dose was 79gy in standard fractions . 
hormonal therapy ( ht ) was administered to 7.6% of low - risk patients , to 20.3% of intermediate - risk patients and to 72% of high - risk patients . 
patients were evaluated for biochemical failure , local recurrence ( lr ) and metastases . results ninety - seven patients ( 26.65% ) developed acute gu toxicity at the medium dose of 25.4gy , grade 1 ( g1 ) or grade 2 ( g2 ) in 94 cases . 
moreover , continued technologic advancements , as image - guided radiotherapy , could lead to further reduction in toxicity , thus increasing the therapeutic index . keywords prostate cancer intensity - modulated radiation therapy ( imrt ) genitourinary toxicity gastrointestinal toxicity introduction prostate cancer is the second most common cancer and the sixth leading cause of death among men [ 1 ]  . 
 radical prostatectomy ( rp ) , radiotherapy ( rt ) and watchful waiting / active surveillance in selected cases are the main available therapeutic options for early - stage disease . 
biochemical control and survival rates obtained with either treatment are comparable , at least in localized prostate cancer [ 3 ]  . randomized trials supported the indication of higher doses of rt in the treatment of prostate cancer [ 4 , 5 ] , associated with better results and higher biochemical disease control [ 69 ]  . 
in fact , the proximity of the rectum and bladder has been a limiting factor in safe dose escalation both in 2d and in 3d treatment planning era [ 6 , 11 ]  . 
 nevertheless , the technological evolution and improvements led to spare organs at risk ( oars ) and to limit their exposure : intensity - modulated radiotherapy ( imrt ) in association with image - guided radiation therapy ( igrt ) allowed to lower rates of severe toxicity , as well as a better quality of life [ 12 ] , often respecting normal tissue - sparing goals since igrt allows to correct target and oars movement immediately . 
in particular , imrt is associated with a significant reduction in acute g2 + gastrointestinal ( gi ) toxicity with a trend for a decrease in late g2 + gi toxicity [ 13 ]  . 
we , herein , report our institutional experience in 394 patients with localized prostate cancer treated with definitive high - dose , image - guided - intensity - modulated radiotherapy ( ig - imrt )  . 
we have also reported disease outcomes , in terms of loco - regional recurrence ( lr ) and distant metastasis occurrence ( dm )  . materials andmethods from our institutional database , we retrospectively reviewed the clinical data of 394 patients , treated between july 2003 and august 2014 with radical high - dose rt for localized prostate cancer . 
the main eligibility criteria were added which include : untreated histologically confirmed adenocarcinoma of the prostate and stage ct1c - t4 n0 m0 according to the sixth edition american joint committee on cancer staging systeprognostic risk groups stratification was made based on damico criteria . 
 data that were extracted from our medical records included age and tumor characteristics like gleason score and pretreatment psa . staging magnetic resonance imaging ( mri ) to evaluate for extraprostatic extension of disease . 
the planning ct scan was performed with 3 - mm slices in the supine position with leg immobilization system ( combifix - sinmed , civco , kalona , ia , usa )  . 
the clinical target volume ( ctv ) was limited to the prostate in low - risk patients , while in high - risk patients the entire seminal vesicles were included in the ctv . the planning treatment volume ( ptv ) was generated by adding an 8 - mm isotropic expansion to the ctv excepting 6mm posteriorly . 
for treatment planning , the dosevolume constraints for the bladder were v65 < 50% and a maximum dose < 65 gy ; for the small bowel v15 < 120 ccs and v45 195 ccs ; for the rectum : v50 gy 50% , v60gy 35% , and v70gy 20% . 
dose constraints for the organs at risk ( oar ) were selected based upon quantitative analyses of normal tissue effects in the clinic ( quantec ) data [ 17 ]  . 
ht was administered to 7.6% of low - risk patients ( subgroup treated in the earlier period ) , 20.3% of intermediate - risk patients and 72% of high - risk patients . 
 two hundred and forty - three patients received neoadjuvant , concurrent and adjuvant ht for a total of 6 - month duration in intermediate risk and for a total of 2years in high - risk disease . 
during rt , patients were visited at least once weekly by a physician . followup andtoxicity evaluation staging of pelvic lymph node and bone metastases was performed by computed tomography ( ct ) scan and bone scan , as indicated , and these were negative for all examined patients . 
radiological assessments were performed only as indicated by symptomatology or in case of biochemical failure according to astro criteria [ 21 ]  . statistical analysis loco - regional recurrence disease - free survival ( lr - dfs ) was defined as the time from the end of radiation therapy to the date of the first event , between loco - regional recurrence and biochemical failure . 
loco - regional recurrence was defined as the appearance of a new lesion in the prostate bed or in the pelvis lymph nodes detected by ct scan or by choline - pet . 
distant metastasis disease - free survival ( dm - dfs ) was defined as the time from the end of radiation therapy to the date of the first event of appearance of distant lesions , detected by ct scan , choline - pet or bone scintigraphy . 
in 38 cases ( 9.6% of the entire cohort , 54.3% of all deaths ) , death was deemed to be cancer related . the treatment was overall well tolerated , and all patients completed the prescribed course . 
 moreover , at mannwhitney statistical test no significant correlations were recorded between the dvh parameters and the toxicities developed by the patients ( see table3 )  . furthermore , we evaluated the comparison between available individual parameters of patients and toxicity by applying an appropriate statistical test according to the variables under consideration ( mannwhitney or chisquare test ) ( see table4 )  . 
4 dm - dfs according to administration of hormonal therapy ( ht ) before radiotherapy ( rt ) discussion comparison between radiotherapy and radical prostatectomy ( rp ) for localized prostate cancer is still a controversial issue . 
randomized clinical studies are necessary to better understand the benefits and risks of each therapeutic approach . we know that higher doses administered are associated with more probability of local control in prostate cancer . 
however , despite the better local control rates and outcomes that can be achieved with higher doses , we know that it can be translated into a big cost in terms of toxicities , especially to the rectum and bladder . 
the rates of acute and late toxicities after definitive rt vary according to the published studies ; generally grade 2 / 3 side effects can reach 40% of cases [ 3133 ]  . 
literature helps us to estimate them ; in fact , urinary incontinence and chronic proctitis are common and well - described side effects of radiotherapy , and sometimes they can develop into complications that can compromise the quality of life , some of which may even necessitate hospital admission or surgical intervention , namely posttreatment urinary or rectal bleeding , infections in the urinary tract or lower gastrointestinal tract and recto - urethral fistulae [ 30 ]  . 
the availability of three - dimensional conformal radiation therapy ( 3d - crt ) in addition to intensity - modulated radiation therapy ( imrt ) has prompted several investigators in the last 20years to explore the use of these technologies to escalate the radiation dose in prostate cancer avoiding worsening of the possible side effects . 
3d - crt , not long ago , had allowed a better sparing of the adjacent oars and a resultant decrease in rectal and bladder toxicity when compared to earlier techniques [ 25 , 28 , 29 ]  . 
in recent years , this advantage has even become greater with the development of imrt that allows a sharper dose falloff gradient , concave dose distributions and narrower margins with a more conformal delivery of isodose lines to ptv . 
most of the trials involving imrt use for prostate cancer therapy have demonstrated both safety and efficacy of dose escalation supported by the results shown in the literature [ 34 , 35 ]  . 
based on the growing body of literature in this regard , imrt has become the standard of care and most commonly employed technique . in our study , despite the use of higher radiation doses , the use of ig - imrt resulted in a low incidence of severe toxicities . 
 [ 13 ] published their results from the radiation therapy oncology group 0126 prostate cancer trial and demonstrated a significant reduction in acute gi / gu toxicity > g2 and a trend for a clinically meaningful reduction in late gi toxicity > g2 in patients treated with imrt compared as 3d - crt , while vora etal . 
with regard to disease outcomes , our results are in line with other published experiences in demonstrating that definitive ig - imrt in combination with adt ( as indicated ) can achieve long - lasting disease control in low - / intermediaterisk patients . we also showed favorable outcomes for patients with high - risk prostate cancer . 
however , the high precision of this technique requires precise delivery methods in order to reduce to the maximum the possibility of geographical miss that may result in increased dosevolume effects on the oars and in increased side effects , such as proctitis or cystitis [ 42 ]  . 
such image guidance for dose - escalated imrt should be the standard of care . several studies have analyzed both interand intrafractional displacements of the prostate , which ranged from 0.2 to 21mm depending primarily on rectal and bladder filling . 
 the magnitude and effect of organ motion of the prostate can be reduced significantly by standardized rectal and bladder filling and an accurate imaging [ 19 ] so their measures were used in our treatment . in our study , ht pre - rt was associated with a worse survival and a higher risk of distant metastasis . 
 [ 43 ] demonstrated that the use of long - course ht , from 3months before high - dose rt to 2years after , is associated with a better survival and a lower percentage of biochemical failure , without increasing radiotherapy toxicity . 
they showed that neoadjuvant ht , before radical prostatectomy , has no effect on survival , while neoadjuvant and adjuvant ht in combination with radiotherapy result in an increasing disease - specific and overall survival , although the duration of ht remains under debate . 
there appears to be an increased risk of cardiovascular morbidity and mortality associated with luteinizing hormone - releasing hormone agonists , particularly in men with preexisting cardiovascular disease , but the relevance of this in the adjuvant / neoadjuvant setting is currently unclear . 
a potential explanation for our results could be the heterogeneity of our population on the correlation of ht use with higher - risk disease based on damico classification and consequently with a worse prognosis . 
a particular strength of this analysis is that all of the patients included were treated at the same institution with the same positioning technique , identical delivery and immobilization devices , and a similar protocol for drinking and evacuation before treatment . 
this led to more standardization accounting for less interand intrafractional organ motion than in the previous reports . conclusions our data confirm the efficacy and limited toxicity of doseescalated ig - imrt for prostate cancer . 
further improvements in treatment delivery and image guidance should be the focus of future investigation to further improve the therapeutic ratio . compliance with ethical standards conflict of interest we declare that the absence of conflict of interest and financial relationships relevant to the content of this article have been disclosed by all authors . 
we had full access to all data in this study , and we take complete responsibility for the integrity of the data and the accuracy of the data analysis . research involving human participants all procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and its later amendments or comparable ethical standards . 
for our retrospective study , formal consent is not required . informed consent all authors declare that informed consent was obtained from all individual participants included in the study and to have used adequate strategies for protecting anonymity . 
our objective is to compare outcomes of conventional laparoscopic ligation ( ll ) and occluding balloon embolization ( obe ) a percutaneous interventional procedure . materials and methods we treated retrospectively a total of 98 patients , divided in two cohorts ; arm a with 48 and arm b with 50 patients . 
we performed a comparative analysis ( fishers test ) of intra - operative time , hospitalization and patients postoperative recovery time . results and limitations outcomes have been in line with the recent literature ones , allowing the occluding balloon embolization a small advantage for quicker operative average time , hospitalization needed and full recovery to normal activities for the patient . 
86 , campusul universitar vasile goldis , arad , romania 4 department ofradiology andradiotherapy , policlinico universitario gaspare rodolico , azienda ospedaliera universitaria policlinico vittorio emanuele , via santa sofia n.29 , 95100catania , italy vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 438443 background varicocele is a relatively complex pathology that affects the pampiniform plexus of veins in the spermatic cord . 
 according to the modern literature [ 1 , 2 ] , this condition affects 2025% of the normal adult male population and is well known as the most common treatable cause of male infertility ; percentages are higher in studies applied to infertile population ( up to 40% )  . 
a gross , first sight of well - developed va is the enlargement of the testicle , and at this level veins can be easily seen through patients skin , aspect named in the past and modern literature as a bag of worms . objective this paper aims to compare the conventional laparoscopic ligation ( ll ) with a specific approach among other methods used to treat this condition in interventional radiology ( ir ) , which is not yet known as standard one , named occluding balloon embolization ( obe )  . 
 a great concern from patients perspective is the potential benefit of treating an established va in order to enhance chances of natural conception ; for its characteristics , management of this pathology has indeed a sensitive nature . 
this original research will be focused on patients advantages and outcomes of these techniques . methodology the study plan is a retrospective comparative analysis , and the design is a parallel one with a total of 98 adult patients divided in two cohorts ; arm a with 48 and arm b with 50 patients ( with observational purposes ) , from two european institutions . 
hence , all patients have been asked to respect 3 and 6months follow - up ; clinical examination and ultrasonography were performed . we treated group - a patients with ll , two operators for the entire cohort with more than 200 cases experience . 
the cohort is made of 48 patients , 22 with symptomatology and 24 with spermiographic abnormalities ; age range varies from 16 to 45years old , with average of 29years . 
for surgical methodology , our team has followed tu and glassberg [ 5 ] surgical steps . patients from group b underwent obe with the technique explained in the literature [ 6 ] by a single operator with more than 200 cases . 
1 shows a lsv phlebography , the white arrow specifies position of the catheter tip ( mid - portion of the vein ) , contrast reflux ensure the presence of va 1 3 440 la radiologia medica ( 2019 ) 124 : 438443 4 . 
2 a shows the balloon in position and inflated before testicles wrapping and sclerosant agent injection ( white arrow ) , b post - embolization check - up shows lsv occlusion ( black arrow ) fig . 
 a correct position of the ob catheter ( proximal barrage ) , b rubber band / tourniquet ( distal barrage ) , c : valved introducer , d pampiniform plexus within the scrotum considering complications , three patients encountered minor allergic reactions ( no medical therapy required ) , just one case with major allergic reaction and subsequent injection of betamethasone . 
the retrospective study did not require ethical approval by local commission . patients involved in this study have been selected from june 2014 up to april 2016 ; in cases of partial occlusion of the lsv , embolization has been finalized with coils . inclusion andexclusion criteria main criterion of acceptance is va grade iii according to the sarteschis classification [ 7 ] , thus diagnosed by cdus ; hence clinically recognized as grade iii , left sided only . 
both persistent pain and spermiogram anomalies have been recognized as criteria of inclusion . exclusions criteria , instead , are : previous therapeutic attempts , coagulopathies and age below 14years old and above 42years of age . 
no other comorbidities have been accepted such as diabetes , hypertension and other existing diseases ; particular attention has been devoted to further possible scrotal conditions . results andlimitations both techniques showed a good amenability in terms of results ; moreover , outcomes of this research have been in line with the recent literature ones [ 811 ] , allowing the obe a small advantage for quicker operative average time , hospitalization needed and full recovery to normal activities for the patient ( fig4 )  . 
medical therapy suggested after treatment is ibuprofen ( brufen 600mgabbot ) if required by symptomatology . for laparoscopic procedure , the operative time average is of 45min ( door to door ) , and is practiced in day - hospital regime ( 24h ) due to patients preparation and post - anesthesiology care . 
analgesic prescribed post - operatory outcome measurements andstatistical analysis our method concerning outcome measurements has been focused on patients benefits , such as : operative time , hospital stay and full recovery . 
fisher test has been used for statistical analysis , creating a 2 2 contingency table and applying a two - tailed p value ; table1 shows rate of complications and technical success / recurrence . operative time ( minutes ) hospital stay ( hours ) full recovery ( days ) fig . 
the single intra - operative dose of antibiotic therapy is considered sufficient . we have decided not to run a cost - effectiveness analysis due to lack of time and possible biases driven by price differences of materials used among different countries . 
nevertheless , some publications [ 8 ] assess similar costs between percutaneous embolization and ll , while others [ 12 ] claim microsurgery repair to be cheaper than these two approaches analyzed in this paper . 
being this work a retrospective one with a limited number of cases evaluated , the common decision has been to avoid semen analysis ; results would eventually be not statistically relevant in this particular effort . discussion treating varicocele improves semen parameters and testicle dimension , providing support to further therapies of reproductive medicine / biology such as icsi ( intra - cytoplasmic sperm injection ) and tese ( testicular sperm extraction ) , hence is the only strategy at our disposition in the case of painful symptomatology of the scrotum related to va . 
the technique proposed in this research is already known as a useful maneuver in some situation of spermatic embolization [ 13 ] , but it has been suggested as standard technique for va only in one paper [ 6 ] ; up - to - date reviews such as the one proposed by halpern etal . 
laparoscopic varicocelectomy has been previously compared to percutaneous embolization [ 8 ] ; nonetheless , more in detail , for a certain period and probably even nowadays coils usage is preferred by many operators . 
underline the effectiveness of percutaneous sclerotherapy as first line of treatment , this group has used mainly the aethoxysclerol as agent of choice , similarly to our experience ; hence , they do mention the use of the occluding balloon in cases of severe renal vein reflux [ 10 ]  . 
this original research , despite a limited casuistry , has provided more glances of viable approaches and techniques ; the obe has not been compared to surgical procedures up until now . 
the advantage of injecting sclerosant agent through an occluding balloon is mainly the enhanced control of the injection , accompanied by maximum concentration of aethoxysclerol at static flow and without dilution ( occurring with free flow technique )  . 
additionally , in the hands of an experienced operator , obe technique could be performed even in the cases of right - sided va , adapting opportunely to eventual anatomical variants [ 15 ]  . 
our results prove , in line with the modern literature [ 14 ] , comparable outcomes of these interventions ; conceding obe a slight advantage in terms of hospital stay and full recovery to normal activity of patients , minding the use of radiologic equipment and irradiation at acceptable levels . 
further studies among differences of these and other approaches such as microsurgery could uncover the best option suitable in terms of cost - effectiveness , patients safety and satisfaction of the procedure and resulting complication rates . 
last but not least , we believe this topic needs further efforts in order to improve quality of data available , besides randomized controlled trials including live birth and pregnancy rates as suggested by the latest cochrane review [ 14 ]  . conclusion in consonance with the modern literature , these two techniques have not showed a significant statistical difference . 
in ll , a longer recovery period is needed as well , though this time may vary slightly from patient to patient . one of the disadvantages produced by radiological procedures are the effects of radiation to patients body and gonad , and these have been statistically analyzed and discussed before attempting such intervention . 
color and pw - doppler analysis showed a reduction in renal blood flow in kidneys during the hypoperfusion with a progressive and significant change from baseline values of resistive index ( ri )  . 
at the histological evaluation , 60min of hypoperfusion resulted in ischemic changes in the kidneys . conclusions the results of this experimental study encourage the use of the described model to study acute renal ischemia trough severe hypoperfusion . 
cardarelli hospital , via cardarelli 9 , 80131naples , italy 3 division ofpathology , department ofpublic , clinic andpreventive medicine , second university ofnaples , piazza miraglia 2 , 80138naples , italy 4 department ofradiology , university ofmolise , via francesco de sanctis , 1 , 86100campobasso , italy introduction acute ischemia is the most common cause of acute renal failure [ 1 ]  . 
the acute kidney injury following the hemorrhagic shock remains a crucial problem : it occurs in approximately 30% of patients admitted in intensive care units and is commonly associated with multiple organ dysfunction syndrome ( mods ) [ 2 ]  . 
ultrasound ( us ) is the first choice for its non - invasiveness , thus permitting repeated measurements invivo [ 4 ] , as already demonstrated in previous studies [ 5 , 6 ] , and renal blood flow can be evaluated using us color and pulsed - wave ( pw ) doppler [ 4 ]  . 
in the literature , many studies use animal models reproducing ischemia blocking locally renal vessels , with a clamp or with a surgical ligation [ 79 ]  . none of them evaluate acute kidney injury caused by systemic hypoperfusion , assessable recreating hemorrhagic shock , and explored by micro - us . 
 other models are not severe enough [ 11 , 14 ] or the ischemic injury is followed by reperfusion , or the histological damage caused by ischemia is not analyzed [ 15 , 16 ]  . 
furthermore , it is not clear whether they are suitable for a radiological evaluation , thus permitting to systematically study the imaging findings . the aim of this work is to create an animal model of acute renal ischemia induced by systemic hypoperfusion , which may result controllable and reproducible , to study , in real time , hemorrhagic shock in rats with micro - us . 
this model allows not only to define the morphological changes that occur in renal parenchyma and in other organs after hemorrhagic shock , but also to test diagnostic markers and drugs , which is becoming crucial in the success and timeliness of research and is allowing a more efficient approach in investigating small living animal models longitudinally studied by noninvasive imaging tools [ 17 , 18 ]  . 
the progression of renal damage was monitored by a high resolution micro - us system , dedicated for small animals . materials andmethods animal preparation all procedures performed on animals were in accordance with the ethical standards of the institution and were approved by the institutional animal care and use committee . 
mean arterial blood pressure ( map ) was continuously measured using a catheter placed in the right femoral artery and connected to a pressure transducer , displaying on a monitor the map values . appendages were secured to electrocardiogram pads to allow constant monitoring of the heart and the respiratory rate . surgical procedures andcatheter placement after a bilateral inguinal incision , polyethylene catheters pe50 ( inner diameter , 0.58 ; outer diameter , 0.96mm ; clay adams , becton dickinson & co , parsippany , nj , usa ) were placed in the femoral arteries and fixed by polyglycolic acid suture ( vicryl 3 / 0 , ethicon inc , summerville , nj )  . 
the catheter inserted in the right femoral artery was connected to a pressure transducer ( apt300 , harvard apparatus , holliston , massachusetts , usa ) calibrated to the atmospheric pressure . 
it was achieved in 2530mfor the next 60min , the map was maintained at a constant pressure value , between 25 and 30mmhg , by automatic pump infusion and withdrawal . us imaging rats were placed in a supine position on the imaging platform ( vevo rat platform , visualsonics , toronto , canada )  . 
 depilatory cream was used to remove fur from the abdominal skin , and medical ultrasound acoustic gel ( aquasonic 100 ; parker laboratories , inc , fairfield , nj ) was used as a coupling fluid between the real - time micro - visualization scanhead and the skus imaging was performed using 1 3 la radiologia medica ( 2019 ) 124 : 323330 fig . 
1 a vevo 2100 us system ; b inguinal incision with isolated femoral artery ( yellow clip ) and positioning of the catheter ; c view of the bilateral inguinal incisions with catheters placed in the femoral arteries ; d syringe pump ; e pressure transducer the vevo 2100 system with 30 micron resolution ( visualsonics , toronto , canada ) using the ms250 transducer ( 1324 mhz ) , positioned and fixed by a dedicated arm ( visualsonics vevo integrated rail system ii )  . 
renal size , morphology and echogenicity were evaluated in b - mode ; it was used to acquire two dimensional images of the kidneys . the probe was positioned on the abdominal skin with a thin layer of ultrasound acoustic gel . 
the main renal arteries arise laterally from the aorta and were visualized using a transverse midline approach . renal blood flow was evaluated using color - doppler us , in a transversal scan of each kidney , and the area of interest was positioned including the kidney vessels to examine the arterial and venous flow . 
the sample volume was placed within the renal artery at the hilum , and the system generates a pw - doppler spectrum which provides the velocity information with the software . 
the same pw - doppler analysis was done for intrarenal arteries , the arcuate arteries ( at the cortico - medullary junction ) or interlobar arteries ( adjacent to the medullary pyramids )  . 
the arcuate arteries are relatively small and tortuous , and determination of the angle of doppler insonation relative to the vessel , required for blood flow velocity calculation , cannot be accurately achieved . 
therefore , analysis of arcuate artery flow spectra is mainlybased on ri evaluation , where doppler insonation angle is irrelevant [ 20 , 21 ]  . interobserver variability assessment the evaluation of renal echogenicity , before and after the hypoperfusion , was assessed independently by two observers ( fi and ts ) , who have graded the kidney echogenicity in comparison with the image acquired at t0 . the kidney echogenicity was graded and recorded using the following scale : grade 1 hypoechoic ; grade 0 : equally echoic ; grade 1 : slight hyperechoic ; grade 2 : moderately hyperechoic ; grade 3 : markedly hyperechoic . interobserver agreement among the two readers was assessed using cohen statistic . 
reliability of categorical data is commonly quantified by the kappa coefficient , which indicates the beyond chance agreement and ranges 1 3 326 la radiologia medica ( 2019 ) 124 : 323330 from 0 ( indicating no agreement beyond chance ) to 1 ( when agreement is perfect )  . to calculate the interobserver agreement by cohen statistic , the grade 2 of kidney echogenicity was considered as positive ( + ) and the grade 1 as negative ( ) ( table1 ) , and p < 0.05 was considered to indicate statistical significance . 
a colordoppler us scan in axial plane showing the physiological blood flow in the renal artery ( red arrow ) and in the renal vein ( white arrow ) ; b color - doppler us scan in axial plane showing a marked reduction in renal blood flow after 60min of renal hypoperfusion ; c pulsed - wave doppler us image showing the renal blood velocity at the renal artery before the induction of hypoperfusion ; d pulsed - wave doppler us image showing reduced blood flow velocity in the left renal artery after 60min of hypoperfusion fig . 
4 renal blood flow at both the renal and intrarenal arteries showing the increase in vascular resistance at different time : a pwdoppler analysis at the renal artery after 60 min of hypoperfusion , showing a high peak systolic frequency shift with a ri > 0.70 ; b renal blood flow at intrarenal arteries after 30min of ischemia . 
histology revealed anatomopathological alterations in both cortex and medulla , such as dilatation and congestion of glomerular arterioles , edema of the connective tissue with some polymorphonuclear cells ( pmn ) , intraparenchymal hemorrhage , red blood cell extravasation , cellular debris , flaked cells and amorphous material in tubular lumens . 
in models with a map of 3035mmhg [ 22 , 23 ] , regulatory mechanism are still effective , making it difficult to maintain a severe shock requiring repeated withdrawal of blood , negatively affecting reproducibility . 
models of severe shock protracted for 60min have been realized , but none of them evaluates the suitability to study organs with imaging techniques in comparison with histological findings [ 15 , 16 ]  . 
5 results of the two observers evaluation of the kidney echogenicity , graded and recorded using the following scale : grade 1 hypoechoic ; 0 , equally echoic ; grade 1 : slight hyperechoic ; grade 2 : moderately hyperechoic ; grade 3 : definitely hyperechoic fig . 
 a cortex of the left kidney 40 : dilatation of bowmans space with red blood cell extravasation ( black arrow ) , glomerular congestion ( blue arrow ) and edema of the connective tissue with rare pmn ( red arrow )  . 
b cortico - medullary junction of left kidney 40 : cellular debris , flaked cells ( blue arrow ) and amorphous material in tubular lumens ( black arrow ) 1 3 la radiologia medica ( 2019 ) 124 : 323330 ischemia , but most of them are focused on chronic injury , studied on models of localized ischemia obtained by surgical ligation or clamping of the renal artery [ 9 , 24 ]  . 
the model we here describe is a systemic model that allows to study with micro - us the physiopathology of acute ischemia in the kidneys , by a pure model of hypoperfusion , which is the most common cause of renal failure . 
for its realization , several issues have been evaluated . in rats with a weight equal to or less than 320g , the reduced caliber of the vessel caused a difficult catheterization of the femoral arteries . 
on the other side , in animals with a weight exceeding 420g , the acquisition of ultrasound images is hampered by the interposition of the intestinal loops , considering the forced decubitus . 
consequently , the choice of the weight has to range from 400 to 410 g , preferably with the aid of a few drops of papaverine that locally dilate the artery , facilitating the access of the catheter . the supine position is obliged by the presence of the catheters , but is also ideal for the acquisition of ultrasound images of the kidney and other abdominal and thoracic organs during the state of hypoperfusion . another issue to be considered in the model planning was the choice of the blood pressure value to achieve during the shock period : from data literature , a value equal to or less than 20mmhg results in the death of rats in about 20min [ 15 ]  . 
achieving a map between 21 and 24mmhg , there is a high variability in terms of survival [ 15 ] , while achieving a value of map higher than 30mmhg the compensation mechanisms are still too effective [ 15 ]  . 
this means that the most severe model of hypoperfusion reproducible must maintain a map between 25 and 30mmhg . the length established for the shock period is 60min as it is the optimal ischemic duration for depiction of renal ischemia on us [ 9 ]  . 
the state of suffering is evident as altered respiratory rate and sudden drop in blood pressure , immediately after the 60th min , which cannot be compensated by the syringe pump . micro - us with pw and color - doppler allows to examine and to monitor in real - time vessels blood flow and organs during hypoperfusion in the animal model , being a useful and versatile tool in the study of physiopathological mechanisms and in the evaluation of the efficacy of new experimental therapy . 
in intrarenal ( segmental and arcuate ) arteries , ri slightly increased during the first 30mafter 30min of hypoperfusion ri decreased remaining , however , below the threshold of 0.70 , this is probably due to the intervention of compensatory mechanisms . the rat model used in this study has several limitations : blood withdrawal was slow , thus not causing a significant increase in the heart rate , while in clinical practice bleeding can be more rapid leading to a compensatory tachycardia . 
 severe hemorrhagic shock cannot be prolonged for more than 60min , as evidenced by the decrease in the respiratory rate and by the sudden fall of the map value at the end of the first hour of hypoperfusion . 
our study has no correlation with laboratoristic data ; however , the kidney injury was histologically confirmed . conclusions this study protocol reproduces a severe hemorrhagic shock in a rat model , with a reasonable survival rate of the animals for the experimental period . 
the analytic data set included the interpreting radiologists years of experience , patient age , patient gender , radiologist gender , ordering service and clinical question to be answered as collected from the radiology request forms . results of the 1996 us and ct examinations performed between october and december 2016 in the inpatient setting , 34% ( 683 examinations ) had a radiologists rai . 
indeed , the growth rate of imaging reflects expanded applications that have occurred over the years in high - tech imaging services such as computed tomography ( ct ) , magnetic resonance ( mr ) imaging and ctpositron emission tomography ( pet - ct )  . 
some publications have suggested that as many as 2050% of high - tech imaging procedures may fail to provide information that improves patient welfare and therefore may represent , at least in part , unnecessary imaging services [ 3 ]  . 
aside from referring physicians , it has been reported that radiologists too make recommendations for further imaging tests in their vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 432437 interpretative reports [ 4 ]  . 
the aim of our study was to measure the proportion of radiologists additional recommended imaging examinations ( rai ) at a hospitalbased inpatient setting and to estimate the influence on rai of radiologist gender and radiologistsyears of experience , of patient gender , patient age , ordering service and clinical question to be answered as collected from the radiology request forms . methods inpatient ct and us examinations interpreted by fifteen radiologists between october and december 2016 at the fondazione ptv policlinico tor vergata were evaluated by two radiologists . 
reports that recommended correlate with clinical information or other suggestions to glean missing clinical information were excluded . the influence on rai of radiologist gender and radiologistsyears of experience and of patient gender , patient age and ordering service were evaluated . 
also the clinical question to be answered was collected from the radiology request forfor each of the clinical question to be answered collected , the number and percentage of examinations with at least one rai were enumerated . 
then the effect size ( odds ratios ) and significance ( confidence intervals ) of each clinical question to be answered on the likelihood of rai was calculated . this retrospective study was approved and waived for patients consents by the institutional review board ( irb )  . statistical analysis data were analyzed with the nonparametric mannwhitney u test . 
 odds ratio with the respective confidence intervals was calculated using medcalc software . results analysis ofct andus imaging reports of the 1996 us and ct examinations performed between october and december 2016 in the inpatient setting at the fondazione ptv policlinico tor vergata , 34% ( 683 examinations ) had a radiologists recommendation for additional ct , pet - ct or mri . 
the largest proportion of imaging examinations prompted by a radiologists recommendation was for chest ct ( n = 260 , 38% ) , followed by pet - ct ( n = 198 , 29% ) , abdominal ct ( n = 164 , 24% ) and abdominal mri ( n = 61 , 9% )  . 
chest ct was most often prompted by a recommendation in chest ct ( 58% ) and whole body ct reports ( 42% ) ; pet - ct , in chest ct ( 52% ) and in whole body ct ( 48% ) ; abdominal ct , in abdominal us ( 93% ) and in whole body ct ( 7% ) reports ; abdominal mri , in abdominal ct ( 56% ) and in abdominal us ( 44% ) reports . 
 also , inconclusive radiology examinations were another relatively common reason for additional imaging recommendation ( 11% )  . pulmonary solid or subsolid nodules < 6mm were the most common findings that prompted a rai ( 31% ) , followed by adenopathy ( 23% )  . 
the other findings leading to additional imaging were renal lesions ( 14% ) , liver lesions ( 12% ) and pulmonary parenchyma abnormalities other than nodules ( 9% )  . correlation withclinical variables in our cohort , there were 438 patients ( 172 males and 266 females , mean age 52 14years ) , 203 were hospitalized in the clinical medicine department , 132 in the surgery department and 103 in the neuroscience department . 
the authors found that the individual radiologist influenced the frequency of recommendations for additional imaging , the percentages of recommendations for additional imaging ranged from 12% to 45% . in our study , chest ct was the most represented rai . 
we hypothesize that the higher proportion of recommendations for follow - up chest ct of small nodules < mm 6 resulted from our radiologists not adherence to these recognized guidelines [ 8 ]  . pet - ct was the second most frequently rai . 
most of the published studies have shown high pet - ct sensitivity and specificity when maximum standardized uptake value of 2.5 was used as the cutoff to differentiate benign from malignant conditions [ 9 ]  . 
 however , no clear guidelines have been established for the employment of pet - ct in the evaluation of enlarged lymph nodes [ 9 ]  . liver and renal lesions were the other findings that often generated radiologist - recommended mri or ct evaluation in our cohort . 
6 scatterplot shows distribution of rai percentages by use of the two discriminant functions obtained for linear discriminant analysis : y - axis , radiologists years of experience ; x - axis , radiologist gender . 
blaivas and lyon [ 5 ] and baumgarten and nelson [ 6 ] found that in abdominal computed tomographic scans the rate of radiologists recommendations for additional imaging in the outpatients setting was , respectively , 31% and 19% , which is comparable to what we found in our inpatient setting ( 34% )  . 
 table 1 rai stratified according to clinical question to be answered as collected from the radiology request forms 1 3 436 la radiologia medica ( 2019 ) 124 : 432437 evaluation by which they can be characterized as benign versus malignant . 
although it is known that most incidental findings often have little or no clinical significance , the drive to evaluate them is often prompt by radiologist and patient unwillingness to accept uncertainty , even given the rare possibility of an important diagnosis . 
by the inappropriate or suboptimal selection of the initial imaging modality . indeed , the excessive ordering of diagnostic tests is one of the most frequently encountered forms of defensive medicine ( a ) , which is defined as a deviation from sound medical practice induced mainly by the fear of liability [ 13 ]  . 
radiologists aware that malpractice liability can make clinical errors more costly , resort to diagnostic tests that , they hope , will reduce the probability of diagnostic error . however , defensive medicine typically operates together with several other variables to motivate clinical practice decisions [ 14 ]  . 
some of these variables may be clinical such as patient symptoms , seriousness of the suspected disease , degree of certainty about diagnosis and accuracy of the available diagnostic tests . 
others are nonclinical , such as availability of technology , years of training and communication skills . with respect to the radiologist gender , we found that male radiologists were more prone to recommend a supplement examination . 
thus , it maybe not surprising that in our cohort female radiologists recommend for less supplement imaging than male radiologists . on the other side , a communication gap between the radiologist and the referring clinician could result in ( b . ) inappropriate or suboptimal selection of the initial imaging modality [ 16 ]  . 
a radiologistclinician consultation would be advisable for a better and more complete understanding of the clinical scenario , especially for complicated patients with multi - morbidities such as those hospitalized in the clinical medicine department . 
however , whether or not these systems will lead to changes in radiologists recommended clinical imaging also remains to be seen . notably , in our cohort of radiologists there was a negative correlation between the radiologists years of experience and rai meaning that there is a tendency in younger participants to recommend supplement imaging . 
we hypothesize that radiologists improve their accuracy and confidence as they build their experience base and gain more knowledge through continuing education . when rai was stratified according to the clinical question to be answered , pneumonia showed the highest rate of rai due to follow - up of lung nodules . 
 it is possible that recommendations for further imaging are not acted upon by the referring physician [ 18 ]  . in conclusion , a high percentage of rai resulted from ct and us radiologists reports in our inpatient setting . 
chest ct follow - up was the most frequently rai driven by the presence of pulmonary nodule in prior ct imaging , followed by pet - ct driven by adenopathy documented in prior ct . 
also , follow - up studies are warranted to assess the number of rai that are actually acted upon by the referring physicians . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical standards this study was approved and waived for patients consents by the institutional review board ( irb )  . 
these methods seem to have similar efficacies for the evaluation of vessel stenosis , legitimizing the use of the developed phantom as a versatile and reproducible instrument that could be used during quality controls programs . keywords carotid atherosclerosis diagnosis ultrasound magnetic resonance imaging quality controls introduction atherosclerosis is a chronic disease that affects medium and large arteries . 
the resultant plaque can obstruct lumen or disseminate material into blood stream and could be a cause of myocardial infarction , stroke and peripheral vascular disease [ 1 ]  . in particular , carotid atherosclerosis is very important in the pathogenesis of cerebral ischemia [ 2 ]  . 
although advances in the understanding and treatment of these lesions * giuseppe acri gacri@unime.it italy 1 department ofbiomorf , university ofmessina , messina , irccs centro neurolesi bonino - pulejo , messina , italy 3 biomedical department ofinternal andspecialistic medicine , university ofpalermo , palermo , italy 4 casa di cura cristo re , messina , italy 5 high school campo calabro ( rc ) , campocalabro , italy have been performed , thrombotic complications of atherosclerosis remain one of the most important causes of morbidity and mortality in western society [ 3 ]  . it should be important to have a noninvasive imaging technique capable to identify , not only the presence , but also the stage of intra - plaque hemorrhage . 
currently , the predominant noninvasive imaging modalities investigated for this purpose are ( a ) ultrasonography ( us ) and ( b ) magnetic resonance imaging ( mri ) techniques [ 4 ]  . in particular , us and mri are highly sophisticated imaging modalities for an accurate and timely diagnosis [ 2 , 5 , 6 ]  . 
mri utilizes powerful static magnetic fields to align the magnetic spins of the protons in water molecules and to drive the larmor precession which provides the signal used to construct the image [ 7 ] , whereas the principle of ecography is similar to that of sonar or radar ; in essence , following an us pulse transmission , echoes from the medium being interrogated are detected and used to form an image [ 8 ]  . these us waves are transmitted from probe to body , and the signal is reflected wherever there is an interface between two tissues with different acoustic impedances . 
it should be possible to differentiate soft and unstable plaques with liquid constituents from solid , hard , and stable plaques . these diagnostic modalities are based on acquisition and correct evaluation of images . 
underhill [ 13 ] , crowe [ 14 ] and mani [ 15 ] were the first who investigated the relation between carotid mri and us . in any case , it is important to guarantee the maintenance of the consistent image quality of the radiological equipments . 
in this context , quality controls ( qc ) have an important role because qc enables a complete evaluation of system status and image quality [ 1618 ] and permit the identification of image quality degradation and source of possible equipment malfunction [ 5 , 19 ]  . 
in fact , the purpose of qc testing is to detect changes that may result in a clinically significant degradation in image quality [ 20 ] which reduces the ability to detect and correctly interpret abnormal findings . for this reason , the optimization of image reproducibility motivates the calibration of acquisition protocols that use phantoms with defined dimensions , inserts etc . phantoms are usually used in order to guarantee the maintenance of consistent image quality over lifetime of the diagnostic equipment . in this context , to geometrically characterize carotid atherosclerotic lesions , diameter stenosis percentage and area stenosis percentage have to be determined . 
therefore , accuracy of stenosis size represents a significant parameter that should be estimated during qc , to correctly evaluate area stenosis percentage . however , most us and mri scanners adopt specific automated procedures that require the use of dedicated phantoms . 
1 upper and lateral view of the phantom modalities and require specific protocols depending also on specific diagnostic device [ 2123 ]  . recently , we realized a single polymethyl methacrylate ( pmma ) phantom that was suitable to perform quality inspection both on ct and on mri that may be used to provide a complete quality inspection of a ct and mri equipment [ 24 ]  . 
in this work , we performed a us and mri examination , by using the novel phantom , for a qualitative and quantitative stenosis evaluation . the reliability of this study was evaluated by a detailed statistical analysis . 
the parameters that characterize the blood vessel are : diameter 5mm and thickness 1min order to mimic the us and mri characteristics of a stenosis , we inserted a thin semirigid plastic wall . 
the choice of pmma and semirigid plastic tubing was related to the necessity of using no ferromagnetic elements in order to perform geometric stenosis measurements both on an mri scanner and on an us device . 
in previous works , stenosis models with different diameter and / or area reductions have been manufactured in order to study the effect of the degree of stenosis and flow rate in large vessels in ct , mri and us [ 2123 ] , but no comparison among different 1 3 370 la radiologia medica ( 2019 ) 124 : 368374 table 1 physical and acoustic properties of blood mimicking fluid compared with the human blood properties human blood ( 37c ) blood mimicking characteristics ( e.g. , viscosity , acoustic properties ) of human blood , as indicated in table1 . 
in particular , models which mimicked area stenosis in large vessels ranging from 18% up to 90% were made . the pmma box was filled with distillate water , while the hollow cylinder was filled with a liquid solution , mimicking human blood , and containing : water , glycerol , orgasol , detergens , citric acid and acnibio ocs ( dansk fantom service )  . 
 orgasol , characterized by nylon particles 5m in diameter , has been used in blood mimicking fluids when natural buoyancy of the particles has been achieved , whereas glycerol avoids refraction [ 2527 ]  . 
2 us images of the novel dedicated phantoa longitudinal cross section ; b longitudinal cross section and measurements performed us andmri data acquisition the common carotid artery ( cca ) us data were obtained as longitudinal cross sections using two different us devices : a philips iu22 us and a philips ie33 systein both us systems , a broadband linear l9 - 3 probe , operating in 39mhz frequency range , was used . 
in both cases , the sequences we considered were a t1 - weighted turbo spin echo ( t1w tse ) sequence and a t2 - weighted turbo spin echo ( t2w tse ) one . 
in addition , a t2 spectral presaturation with inversion recovery ( t2 spir ) sequence was also considered in order to suppress fat and reconstruct carotid artery wall [ 2830 ]  . 
 the concordance correlation coefficient combines measures of precision and accuracy , to determine how the observed data deviate from the line of perfect concordance ( line at 45 on a square scatter plot )  . 
intra - rater analysis was used to determine test rest - reliability , comparing the different scores marked by the same rater on the same phantom with different methods . finally , inter - rater agreement was assessed between the various measurements obtained with the different techniques in order to test the performance stability with different raters . 
statistical significance was set at p < 0.05. results accuracy measurements in tables4 and 5 , the results of the measurements , respectively , conducted on us and mri devices are reported . 
in this study , we assess the diagnostic ability of us and mri techniques to identify and measure carotid stenosis . in order to choose a method for investigation , different parameters should be considered : availability of modality , level of optimization and the capability of patient and operator . 
on the other hand , by using mri it is possible to acquire tridimensional images with the high contrast resolution . other authors have conducted studies about the use of dedicated phantoms to evaluate stenosis parameter [ 2123 ] , but no comparison between diagnostic devices was performed . 
this is a first study that reports a comparison among two different us modalities and two different mri strength fields ( 1.5 and 3 t ) with a novel phantom approach . in this context , we developed a novel phantom , simulating a vessel stenosis that can be used both on mri and on us devices . 
its preparation requires only the filling of the hollow cylinder and the pmma box , using , respectively , a solution , simulating human blood and distillate water . the accuracy obtained using the 3 t device is significantly higher than that obtained using the other diagnostic scanners . however , statistical analysis showed that no significant difference exist between us and mri techniques . 
in fact , us and mri have shown similar diagnostic efficacies for the evaluation of vessel stenosis , using the novel phanto the obtained results showed that there was concordance between the two different diagnostic techniques ; however , we found significant results in inter - rater agreement . 
this could be due to the fact that the 3.0 t magnet has the capability to provide a better image quality as the base for improved diagnostic performance , because doubling the field strength ( almost ) doubles signal - to - noise ratio , that is , the quantity of signal made available from the patient in order to build mri images . the proposed method employs a new universal phantom , which can be used on any us and mri device , in a quick manner , to evaluate stenosis vessel . 
mean adc values ( b50 + b400 + b800 / 3 ) of parathyroid lesions were compared with that of normal appearing thyroid parenchyma ( tp ) , sternocleidomastoid muscle ( scm ) and jugulodigastric lymph nodes ( jdln )  . results of lesions , 4 were parathyroid hyperplasia , 13 parathyroid adenoma and 3 parathyroid adenocarcinoma . 
by increasing strength ( b value ) of diffusion tensor on dwi , solid parathyroid lesions still kept their brightness comparing other soft tissue structures of head and neck region because of their high t2 properties . conclusion solid parathyroid lesions had higher diffusion properties comparing other soft tissues structures of head and neck region . 
 preoperative evaluation of parathyroid pathology includes studies such as usg , scintigraphy , ct , spect - ct fusion scan ( 4d ct ) and mri [ 2 ]  . 
 accurate localization of parathyroid adenoma is valuable to * seyma yildiz drseymayildiz@gmail.com 1 department ofradiology , bezmialem vakif university , istanbul , turkey limit the extent of surgery with the goal of a unilateral small incision with minimal distortion of the normal anatomy , and fewer structures placed at risk . mri has been widely used in the evaluation of parathyroid disease . 
recently , diffusion - weighted mri has gained a widespread usage for diagnostic purposes due to its ability to provide a new contrast in evaluation of head and neck masses . 
the apparent diffusion vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 360367 coefficient ( adc ) , a measure of diffusion , is valuable quantitative metric , and helpful in distinguishing malignancy from benign lesions [ 5 ]  . 
the aim of this study was to evaluate the mri findings of solid parathyroid lesions and to elaborate on a possible improvement of mri detection of parathyroid lesions by the use of additional dwi . materials andmethods patients the surgeons in our institution prefer at least two imaging techniques to localize the parathyroid lesions before surgery to minimize the extent of surgery . 
twenty patients ( 4 men , 16 women , mean age 56 , 7years , range 3487years ) with hpt who underwent conventional mri for parathyroid pathology between august 2011 and january 2014 were enrolled . 
our institutional review board approved the study . mri examination all mri examinations were performed using a 1.5 t wholebody superconducting mri machine ( siemens , avanto , erlangen , germany ) with a head and neck coil . 
mean adc values ( b50 + b400 + b800 / 3 ) of the lesions were calculated automatically on images with b factors of 50 , 400 and 800s / mm2 , and adc values were expressed in square millimeters per second . image analysis one neuroradiologist ( aa ) reviewed the routine mri images only visually . 
at the same time , mean adc measurements were taken from normal appearing thyroid parenchyma ( tp ) , sternocleidomastoid muscle ( scm ) , jugulodigastric lymph nodes ( jdln ) and thyroid gland lesions ( tl ) for comparison with that of parathyroid lesions . 
the size of the rois was big enough to cover the interested area excluding outer borders of regions under evaluation . results of total 20 solid parathyroid lesion , 4 were parathyroid hyperplasia , 13 parathyroid adenoma and 3 parathyroid adenocarcinoma . 
however , similar to parathyroid adenoma and hyperplasia , parathyroid carcinomas had high diffusion properties compared to other soft tissue structures of head and neck region ( table2 )  . 
by increasing strength of diffusion tensor on dwi , all three types of parathyroid lesions still kept their brightness comparing other soft tissue structures of head and neck region because of their high t2 properties . 
however , scintigraphy has also imaging pitfalls since coexistent thyroid lesions , including hyperplastic thyroid nodules , chronic thyroiditis , hurthle cell lesions and adenomas , can retain radiotracer [ 13 ]  . recently , contrast - enhanced thin - collimation ct has been used for preoperative evaluation of patients with hpt . 
 [ 16 ] suggested multiphase ct as an ideal imaging method for preoperative evaluation hyperfunctioning parathyroid tissue because of its higher sensitivity than usg and scintigraphy ( 88% for ct , 21% for us , 54% for scintigraphy )  . 
1 af coronal fat - saturated t2 ( a ) , axial t2 ( b ) , fat - saturated t2 ( c ) , t1 ( d ) and postcontrast t1w ( e ) images demonstrate small , well - defined , homogenous , t2 bright and avidly enhancing nodular lesion within the posterior aspect of the thyroid gland on the right , suggesting parathyroid adenoma or hyperplasia . 
on dwi views , comparing to the nearby structures , parathyroid lesion keeps its brightness as the strength of tensor increases on dwi images due to its high t2 content . 
spectct is more informative by giving the exact anatomical localization of the lesion in major ectopic lesions and distorted neck anatomy [ 17 ]  . although less commonly used for preoperative evaluation of patients with hpt than other modalities , the 1 3 364 la radiologia medica ( 2019 ) 124 : 360367 fig . 
2 af coronal fat - saturated t2 ( a ) , axial t2 ( b ) , fat - saturated t2 ( c ) , t1 ( d ) and postcontrast t1w ( e ) images demonstrate large , illdefined , heterogeneous , infiltrative , t2 bright and enhancing lesion in the posterior and inferior aspect of the thyroid gland on the left , suggesting parathyroid adenocarcinoma . 
on dwi views , comparing to the nearby structures , parathyroid lesion keeps its brightness as the strength of tensor increases on dwi images due to its high t2 content . 
surgical result was reported as parathyroid adenocarcinoma reported sensitivity of mri , including both patients who have undergone surgery and patients who have not , is 4288% [ 6 , 9 , 11 , 18 , 19 ]  . 
also multiparametric mr perfusion can distinguish ptas from adjacent thyroid tissue or lymph nodes with a highly diagnostic accuracy . images of the neck are generally obtained from the skull base to the sternal notch . 
the imaging characteristics of parathyroid adenomas are most commonly hypo - to - isointense on t1w and high intensity on t2w conventional imaging [ 3 , 4 , 21 ]  . 
another potential pitfall in the detection of the parathyroid adenomas is the presence of associated thyroid disease which may be present approximately in % 30 of patients with hpt [ 23 ]  . 
however , false - negative results are most commonly associated with adenoma that is isointense on t1 and t2w sequences ; the addition of contrast - enhanced images can increase the sensitivity in these cases [ 18 ]  . parathyroid carcinoma is a rare malignancy developed from the parenchymal cells of the parathyroid glands . 
the mri characteristics of parathyroid adenocarcinoma have not been described well in the literature . in our study group , small sized , well - defined borders , t2 brightness , increased diffusion and homogenous appearance on t1 , dwi , t2 and postcontrast t1w images were properties of parathyroid hyperplasia and parathyroid adenoma . 
large sized , ill - defined borders , t2 brightness and heterogeneity on all sequences including t1 , t2 , dwi and postcontrast t1w images were properties of parathyroid adenocarcinoma . 
when evaluating dwi with increasing diffusion strength ( b50 , 400 , 800 ) , parathyroid lesion keeps their brightness longer on images when comparing with other soft tissues structures ( tp , jdln , tl ) of head and neck region . 
our results were in concordance with the literature . routine conventional mri is useful examination for parathyroid pathologies and helpful in differentiation of benign lesions from malignant ones , but it has disadvantages of high cost and longer examination . 
diffusion properties of parathyroid hyperplasia , parathyroid adenoma and parathyroid adenocarcinoma are higher than that of nearby structures ; this feature makes them easily differentiate from adjacent structures but the overlap of measured adc values of all three parathyroid lesions is too wide to enable differentiation from each other . in conclusion , mri is very successful in demonstration of parathyroid lesions . 
 solid parathyroid lesions , whether being parathyroid hyperplasia , parathyroid adenoma or parathyroid adenocarcinoma , have higher diffusion properties comparing the other soft tissues structures of head and neck region . 
this feature enables us to easily differentiate solid parathyroid lesions from nearby structures on fat - saturated t2w and dwi . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . informed consent our studies is retrospective . 
the results of imaging evaluation were compared with surgical findings , considered as reference standard . results during the study period , 124 patients operated on for anal fistulas underwent complete preoperative imaging assessment . 
the fistulas were classified as simple in 68 / 126 ( 53.9% ) and complex in 58 / 126 ( 46.03% ) cases , according to fistulas parks classification and the most recent american guidelines . 
in both simple and complex anal fistulas , 3d - eaus did not show a significantly higher accuracy in the evaluation of internal openings , if compared with mr ( p = 0.47 ; mcnemars chi - square test )  . 
in the complex anal fistulas , mr showed a significantly higher accuracy in the evaluation of secondary extensions if compared with 3d - eaus ( p = 0.041 ; mcnemars chi - square test ) , whereas in the simple anal fistulas , no significant difference was found . conclusion in the preoperative work - up of patients with anorectal fistulas , 3d - eaus may represent the first - line diagnostic tool . 
vanvitelli , piazza miraglia 2 , 80138naples , italy 3 department ofsurgery , villa esther hospital , via due principati 169 , 83100avellino , italy 4 department ofsurgery , pellegrini hospital , asl na1 centro , via portamedina alla pignasecca 41 , 80134naples , italy 5 department ofsurgery , university ofcampania l . 
exclusion criteria included age less than 18 , refusal to participate in the study , absolute contraindication to surgery or to mr and anal stenosis precluding the execution of 3d - eaus . all patients who met these criteria were preoperatively assessed by anamnestic evaluation , clinical examination , 3d - eaus and mr . clinical examination and 3d - eaus were performed , respectively , by a colorectal surgeon and a radiologist together with the same surgeon . mr examinations were evaluated in consensus by two radiologists ( fi and ar ) with 4 and 11years of experience in the pelvic imaging . surgery was performed by another colorectal surgeon blinded to 3d - eaus and mr results . the local ethical committee approved the study protocol . 
all patients gave written informed consent . clinical examination clinical examination included anamnesis , combined inspection and palpation of perineal area and anorectal digital examination , to evaluate the swellings and tenderness of perineal area and the suspected external and internal openings . 
the injection of the h2o2 through the external orifice allows a better depiction of the fistula tract that appears hyperechoic ( arrows ) : a axial plane , b sagittal plane fig . 
after an initial localizer in the three different planes , the study protocol starts with the following sequences including the small pelvis , the perineum and the gluteal regions ( table1 ) : axial tse t1 - w ; axial tse t2 - w ; and sagittal tse t2 - w . 
the use of fat saturation avoids the use of intravenous contrast medium as active fistula tracts appear spontaneously hyperintense , thus allowing a detailed evaluation of the fistula anatomy [ 9 , 25 ]  . 
 examination underanesthesia ( eua ) surgery was performed under subarachnoid anesthesia with patients in the lithotomy position . in the presence of suspected external orifices , lockhartmummery fistula probes and h2o2 were used to search for the fistula tracks and identify the internal openings . 
all the abscesses were incised and evacuated . the site and number of external openings , the site of internal openings and the anatomic relationship between fistulas or abscesses and anorectal musculature were examined and recorded . based on the parks classification [ 24 ] , and on the ascrs ( american society of colorectal surgeons ) guidelines [ 3 ] , the anal fistulas with intersphincteric and low transsphincteric primary tracks ( crossing < 30% of the external sphincter ) were classified as simple , whereas the anorectal fistulas with high transsphincteric ( crossing < 30% of the external sphincter ) , suprasphincteric and extrasphincteric primary tracks were considered complex . the anterior fistulas in women , the horseshoe fistulas and those associated with malignancy , ibd and radiation were also classified as complex disease . statistical analysis values are expressed , according to distribution , as mean standard deviation ( sd ) or medians and range . 
 prevalence data were compared between groups using the fishers exact test , whereas continuous data were compared between each group using the student t test or the mannwhitney u test , when indicated . the concordance between 3d - eaus and eua in defining the anorectal fistulas severity was evaluated through the simple kappa coefficient ( k )  . 
the diagnostic value in the identification of primary tracks did not show any difference . in table3 the 3d - eaus and mr sensitivity and specificity in the detection of simple fistulas internal openings , primary tracks and secondary tracks are compared . 
 although 3d - eaus showed higher sensitivity in the detection of internal openings and mr exhibited higher sensitivity in the detection of secondary extensions , no significant differences were found . table4 shows the 3d - eaus and mr sensitivity and specificity in the detection of complex fistulas internal openings , primary tracks and secondary extensions . 
 discussion although 3d - eaus and mr are generally considered useful tools in the preoperative evaluation of anorectal fistulas , the specific role of each imaging technique is still not well clarified and their weight in the different anorectal sepsis severity grades is still a matter of debate . in this study we evaluated the role of both diagnostic imaging techniques in the simple and complex perianal sepsis . first of all , our results suggest an excellent agreement between 3d - eaus and surgery in defining the anorectal sepsis severity . 
according to previous studies [ 1923 ] , this finding seems to encourage the routine use of 3d - eaus in the preoperative evaluation of patients with anorectal fistulas , with the aim to support the surgeon in planning the more appropriate therapeutic strategy . overall , our data show high and comparable sensitivity and specificity of 3d - eaus and mr in the evaluation of fistulas primary tracks , internal openings and secondary extensions . 
however , the diagnostic value of the two imaging techniques showed some differences depending on the sepsis severity grade . particularly , in the cases of fistulas classified as simple on the basis of assessment with 3d - eaus , the accuracy of mr in the detection of internal openings , primary tracks 1 3 la radiologia medica ( 2019 ) 124 : 339349 fig . 
therefore , in the simple fistulas , the additional mr evaluation did not provide substantial advantages , over ultrasound study , in the definition of fistulas anatomy . however , in the cases of fistulas graded as complex by 3d - eaus , mr showed a significantly higher accuracy in the evaluation of secondary extensions if compared with ultrasound , proving a definite advantage , over 3d - eaus alone , in the study of fistulas complete morphology ( figs.4 , 5 , 6 )  . although the reasons for this different accuracy cannot be inferred from the reported data , it is plausible that the secondary extensions of complex fistulas tend to be more numerous and distant from the anus and rectum 1 3 346 la radiologia medica ( 2019 ) 124 : 339349 fig . 
williams jg , farrands pa , williams ab , taylor ba , lunniss pj , sagar pm , varma js , george bd ( 2007 ) the treatment of anal fistula : acpgbi position statement . 
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reginelli a , mandato y , cavaliere c , pizza nl , russo a , cappabianca s , brunese l , rotondo a , grassi r ( 2012 ) threedimensional anal endosonography in depicting anal - canal anatomy . 
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siddiqui mr , ashrafian h , tozer p , daulatzai n , burling d , hart a etal ( 2012 ) a diagnostic accuracy meta - analysis of endoanal ultrasound and mri for perianal fistula assessment . 
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ognibene nm , basile m , di maurizio m , petrillo g , de filippi c ( 2016 ) features and perspectives of mr enterography for pediatric crohn disease assessment . 
foti pv , farina r , coronella m , palmucci s , ognibene n , milone p , conti bellocchi c , samperi l , inserra g , laghi a , ettorre gc ( 2015 ) crohns disease of the small bowel : evaluation of ileal inflammation by diffusion - weighted mr imaging and correlation with the harveybradshaw index . 
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buchanan gn , halligan s , bartram ci , williams ab , tarroni d , cohen cr ( 2004 ) clinical examination , endosonography , and mr imaging in preoperative assessment of fistula in ano : comparison with outcome - based reference standard . 
these patients underwent planning ct ( pct ) for radiation therapy ( rt ) and contrast - enhanced three - dimensional fast - spoiled gradientecho image ( 3d fspgr ) to assist tumor delineation . 
hypofractionated stereotactic radiotherapy ( hsrt ) with 3 or 5 fractions is the preferred mode of radiotherapy treatments for non - removable vs , where the gross tumor volume is bigger and / or of irregular shape [ 1 , 2 ]  . 
in mr - only planning , the image registration between ct and mr scans is eliminated , and at the same time , one can get rid of the radiation exposure as a result of a ct scan . radiation therapy planning ( rtp ) depends on threedimensional ( 3d ) computed tomography ( ct ) for dose calculation . 
significant development in treatment planning systems for past two decades has led to the generation of 3d dose color wash overlapped on the patient ct images at all three planes . 
many countries still depend on ct - based planning , and mri primarily assists in delineation of targets and critical structures , where appropriate mr sequence is registered to the planning ct ( pct ) images . 
ct scans are assumed to be geometrically accurate , but in case of mri , there might be geometric imperfections due to main and gradient magnetic fields and / or due to distribution of magnetic susceptibility values with biological tissues [ 11 ]  . there are quite a few studies that have employed mr images for radiation planning . 
 autosegmentation of structures from one or more mr sequence ( s ) was developed where 3d - pseudo - ct is generated and used for subsequent radiation planning [ 1315 ]  . 
 this study is one of a kind where bulk densities are assigned mainly to three structures ( bone , air , and soft tissue ) , derived directly from mr images . 
our aim is to compare the dose calculation accuracy of plans done on planning ct image and ct - value - assigned mr image . materials andmethods patient selection the scientific and ethics board has approved this work . 
ultra - fast - spoiled gradient - echo image sequences use a small flip angle and very short repetition time and optimized k - space filling to reduce the overall acquisition time . 
fspgr image is registered with the ct image using rigid registration algorithm version 11.0 ( varian medical systems , palo alto , usa )  . automatic registration is done using 3d coordinate system and it is followed by manual correction and verification by the oncologists for valid image fusion . 
treatment planning was performed for 6 mv beams on clinac - ix linear accelerator equipped with 120 leaves millennium multi - leaf collimator and with eclipse treatment planning software ( varian medical systems , palo alto , usa ) by senior clinical physicists . 
the dose calculation grid size greater than 2mm gives a dose difference of 2.3% of the prescribed dose for imrt treatments as compared with 1.5mm especially in high - dose gradient areas [ 16 ]  . 
as the image registration is already performed , the structures , the treatment plan along with optimization parameters , and priorities are copied from pct and pasted on fspgr image set . 
in this study , the fspgr image set is assigned hounsfield units ( hu ) values for three structures created viz : bone = + 1000 , air = 1000 , and body ( soft tissue ) = 25 . 
the corresponding mass densities recommended by icru 46 [ 17 ] are 1.61g / cm3 for bone , 0.001g / cm3 for air cavities and 1.025g / cm3 for average soft tissue . 
figure1 shows the comparison of 50% isodose coverage of plans generated on ( a ) planning ct ( b ) mrct without optimization and ( c ) mrct with optimization . evaluation parameters parameters such as conformity indices ( ci ) , max dose ( dmax ) to brainstem ( bs ) , and dose received by 0.5cc of brainstem ( d0.5cc ) were chosen for evaluation of the quality of plans generated . 
gamma agreement index ( gai ) and correlation coefficient ( r ) are used to predict the agreement of plans generated on mrct versus pct using omnipro imrt quality assurance ( qa ) software ( iba dosimetry , gmbh , germany )  . 
the extent of correlation coefficient varies between minus one and plus one , i.e. , 1 0 1 where r = 1 indicates perfect inverse correlation , while r = + 1 indicates perfect direct linear correlation between two variables [ 19 ]  . 
detailed descriptions of characteristics of parameters selected for evaluation are found in table2 with respect to hsrt treatments . single - factor analysis of variance ( anova ) test is carried out to find the statistically significant difference between the plans generated on ctplan , ddc and opt_dc . 
if p value < 0.05 , we reject the null hypothesis and conclude that there is a significant difference between the parameters generated from plans . results the metrics mentioned in table2 are applied to the extracted data and the results of deviation of ddc and opt_dc of mrct from original plan done on pct are obtained . 
the range and mean deviation values of dmax and d0.5cc of bs for ddc versus ctplan and 1 3 la radiologia medica ( 2019 ) 124 : 400407 table 2 evaluation parameters parameter conformity of prescribed dosea dose to the brainstem ( bs ) ( 5fx hsrt ) ( endpointcranial neuropathy ) b gamma agreement index ( gai ) correlation coefficient ( r ) a rtog 0915 b aapm task group 101 description / dose constraint 1 . 
 ci1 - ratio of the prescription isodose volume to the planning target volume ; ctplanplans done on planning ct ; ddcplans dose on mrct without optimization ; opt_dcplans done on mrct with optimization opt_dc versus ctplan are given in table4 . 
the dosimetric verification of mr versus ct - based planning for prostate patients using intensity modulated radiation therapy has been validated by chen etal [ 8 ]  . in this study , the delivered fluences of ddc and opt_ dc were not acquired / analyzed and compared only with approved plans ( calculated fluences ) done using ctplan . 
this criterion is sufficient since the treated plans done using pct passes the routine qa procedures of hsrt treatments.1 the mean values of ci2 are not relevant and not discussed here since the passing criterion of ci2 value falls under a range of value and are different for different ptv volumes2 . 
it is inferred from fig.1 that the plans done on mrct with and without optimization are qualitatively similar to those plans done on pct and statistically proved with anova test . 
the differences in dose volume patients considered under this study , bs lies in close proximity to the ptv and in addition hsrt involves delivering unusually high dose per fraction of 5gy ( steep dose fall off )  . 
4 dose color washes in coronal plane in omnipro imrt software for a original ct b ddc and c opt_dc deviation may also because of mild / negligible peripheral distortions from mr image set as the soft tissue structure ( body ) is created using mr image set . 
this is also evident from the anova tests where p value is > 0.05. the location of tumors in this study is in the region of hetero - density area ( cp angle )  . 
also generation 1 3 406 la radiologia medica ( 2019 ) 124 : 400407 compliance with ethical standards conflict of interest all authors contributed to this study declare that they have no conflict of interest with respect to the manuscript . ethical standards institutional scientific and ethics board has approved this study . 
comparing with hydrocephalus , the width of prepontine cistern ( ppc ) / the width of aqueductus sylvii ( as ) was significantly higher and other csf metrics with standardized systolic and sum of systolic and diastolic flow durations were significantly lower . 
compared with normal group , ppc / as and reverse / forward flow duration were significantly lower and other csf metrics were significantly higher . conclusion in hydrocephalus , significant increase in asv and peak velocities were noted . 
communicating hydrocephalus could be with obstruction to csf absorption secondary to subarachnoid bleeding , leptomeningeal carcinomatosis or infective meningitis or could be without obstruction to csf absorption such as in normal pressure hydrocephalus , vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 382391 hydrocephalus ex - vacuo or colpocephaly secondary to parenchymal volume loss or in choroid plexus papillomas due to overproduction of csf . 
most common imaging findings of hydrocephalus are : ventriculomegaly ( evans index > 0.3 ) , enlargement of the third ventricular recesses and lateral ventricular horns , decreased mamillopontine distance and frontal horn angle , thinning and elevation of the corpus callosum , normal or narrowed cortical sulci , periventricular white matter hyperintensities due to interstitial edema and aqueductal flow void phenomenon in t2w images . 
in csf flow studies , dilated aqueductus sylvii and increased asv may be seen [ 1 , pseudotumor cerebri or iih is a disorder of unknown etiology that is characterized by elevated intracranial pressure , usually occurs in obese women in the childbearing years [ 35 ]  . 
there is no evidence of deformity or obstruction of the ventricular system , and neurodiagnostic studies are otherwise normal except for increased cerebrospinal fluid pressure ( greater than 200mm of water in the non - obese and probably greater than 250mm of water in the obese patient )  . 
mri findings of iih are widening of perioptic subarachnoid space , optic nerve tortuosity , posterior scleral flattening , preliminary enhancement of optic nerves , empty sella , tonsillar herniation , perimesencephalic cistern obliteration , shrinkage of ventricles , enlargement of meckels cave and bilateral transverse sinus compression [ 6 ]  . 
in the literature , only one study was present detailing csf flow metric in iih suggesting statistically decreased peak flow velocity in untreated iih patients [ 4 ]  . with recent advances on mri , mri is not only beneficial in the diagnosis of csf - related diseases , but also helps clinicians in planning the treatment and follow - up of patients . 
new mri sequences including phase - contrast magnetic resonance imaging ( pc - mri ) , 3d - heavily t2w ( 3d constructive interference in steady state ) ( 3d - ciss ) and 3d sampling perfection with application - optimized contrasts using different flip angle evolutions ( 3d - space ) enable evaluation of csf - related pathologies with higher sensitivity and specificity . 
this study is retrospective casecontrol observational study , and our purpose is to evaluate csf dynamics of iih and communicating hydrocephalus and search for any correlation between mri findings , csf metrics and csf opening pressure in iih . materials andmethods subjects twenty - nine patients ( average age 37.28 , m / f 2 / 27 ) ( age range 1857years ) with a diagnosis of iih and 43 patients ( average age 29.12 , m / f 23 / 20 ) ( age range 1178years ) with a diagnosis of high - pressure communicating hydrocephalus based on standard clinical and laboratory criteria , referred from our neurology department , were included . 
 cases with normal pressure hydrocephalus , hydrocephalus ex - vacuo or colpocephaly secondary to parenchymal volume loss or hydrocephalus due to overproduction such as in choroid plexus papilloma were all excluded . 
control group included 30 healthy volunteers ( average age 36.57 , m / f 25 / 5 ) ( age range 2065years ) , with normal neurological examination , clinical and laboratory findings . 
for all patients in this study , a detailed history , neurological examination and laboratory findings were retrospectively evaluated in our hospital via computer - based archive systehealthy volunteers also underwent csf flow study . 
all subjects were fully informed and gave their written informed consent . imaging technique mri was performed on a 1.5 tesla system ( avanto ; siemens medical solution , erlangen , germany ) using head coil . 
mean modulus , magnitude of complex difference and directional phase difference images perpendicular to the cerebral aqueduct in the sagittal plane were then obtained at the semi - axial plane . 
1 axial images acquired perpendicular to ampulla la radiologia medica ( 2019 ) 124 : 382391 1430 cardiac phase sections according to heart rate were : tr : 31.25ms , te : 8.06ms , slice thickness : 3mm , nsa : 1 , fov : 16 10cm , matrix 128 256 and flip angle , 10 . 
flow in the caudocranial direction ( diastolic or reverse flow ) was identified as negative , and flow in the craniocaudal direction ( systolic or forward flow ) was identified as positive . 
 parameters used for 3d - t2 ciss were : slice thickness : 1mm , fov : 200mm , matrix 290 320 , tr : 6.06ms , tr : 2.61ms and flip angle 70 . 
parameters used for 3d - t2 space were : slice thickness : 1 mm , fov : 240 mm , matrix 231 256 , tr : 2500 and tr : 501 . mri analysis the images obtained from all subjects ( subjects with iih , subjects with hydrocephalus and healthy subjects ) were evaluated by two separate radiologists ( aa and tfy ) using the siemens user console ( argus software , siemens , erlangen , germany )  . 
on midline sagittal t2 ciss images , at the midpontine level , width of prepontine cistern ( ppc ) and width of cerebral aqueductus sylvii ( as ) were measured and ppc / as ratio was calculated . 
velocity versus time , peak flow velocity versus time ( fig.3 ) , flow versus time and net flow versus time curves of the flow passing through the as during one cardiac cycle were obtained . 
systolic ( arrow on a ) and diastolic durations ( arrow on b ) are shown volume ( l ) , net forward flow volume ( l ) , asv ( aqueductal stroke volume ) ( l ) , as area ( mm2 ) , ppc / as ratio , the average flow volume in one cardiac cycle ( ml / min ) , systolic and diastolic flow times ( ms ) , standardized ( by heart rate - 60 beats / min ) systolic and diastolic flow times ( ms ) , the ratio of diastolic and systolic time , the sum and difference of systolic and diastolic flow times ( ms ) and the sum and difference of standardized systolic and diastolic flow times ( ms ) were calculated . 
the average volume flow in one cardiac cycle was calculated by the formula : forward volume + reverse volume / range ( 0680ms ) 60ml / mthe asv was calculated by the formula : forward volume + reverse volume / 2 . 
 positive values of peak flow velocity were recorded . in iih patients , accompanying conventional mri and mr venography findings including empty sella , optic nerve sheath edema , posterior scleral flattening , perimesencephalic cistern obliteration , shrinkage of ventricles , enlargement of meckels cave , tonsillar herniation and transverse sinus compression were also noted . statistical analysis all statistical analyses were performed using spss for windows version 23.0 software package ( ibm corp . , new york , ny ; formerly spss inc . , chicago , il )  . 
subjects were evaluated in three separate groups : group with communicating hydrocephalus ( n = 43 ) , group with iih ( n = 29 ) and group with healthy volunteers ( n = 30 )  . 
for each three groups , mean standard deviation of frequently used csf flow metrics ( asv , average volume flow in one cardiac cycle , and peak flow velocities ) was calculated . first , distribution of variables from three groups was evaluated by using shapirowilk normality test . 
when sum of standardized systolic and diastolic flow time was evaluated , a significant difference between healthy subjects and hydrocephalus was not present ( p = 0.382 ) , [ median values in healthy group , 636ms ( minimum 374ms , maximum 821ms ) / hydrocephalus , 618ms ( minimum 324ms , maximum 807ms ) ]  . 
a statistically significant difference was not determined in other csf flow metrics between two groups ( table2 )  . in patients with iih , there was no correlation of empty sella , optic nerve sheath edema , posterior scleral flattening , perimesencephalic cistern obliteration , shrinkage of ventricles , enlargement of meckels cave , tonsillar herniation , transverse sinus compression and ppc / as ratio with asv values . 
among conventional mr and mr venography findings including ppc / as ratio and asv , there was only statistically significant difference in csf opening pressure between iih patients with transverse sinus compression and iih patients without transverse sinus compression . 
aqueductal stroke volume ( asv ) , a flow metric , is the average of forward flow volume during systole and reverse flow volume during diastole in each cardiac cycle . 
however , location of axial views of pc - mri in our study was through ampulla of as , which is widest and middle - third area of as . normal csf flow at the level of as and foramen magnum is biphasic due to pump mechanissystolic expansion of intracranial vessels and brain parenchyma results in csf movement from ventricular system toward the spinal canal ( craniocaudal flow , systolic csf flow , forward flow ) , while diastolic contraction of intracranial vessels and brain parenchyma results in reduction in intracranial pressure and secondary csf movement from spinal region to subarachnoid space and ventricular system within the brain ( caudocranial flow , diastolic flow , reverse flow )  . in this study , we measured diastolic flow time in iih and healthy subjects . 
in iih comparing with healthy subjects , standardized diastolic flow time ( median value in iih , 395ms / healthy subjects , 484ms ) and difference between standardized diastolic and systolic flow time ( median value in iih , 206ms / healthy subjects , 289ms ) and sum of standardized systolic and diastolic flow times ( median value in iih 542ms / healthy subjects 636ms ) were found statistically significantly short . 
comparing the sum of standardized systolic and diastolic flow times among three groups , it was statistically shortest in iih ( median value in healthy subjects 636ms , hydrocephalus 618ms , iih 542ms ) , but not statistically significant difference noted between hydrocephalus and healthy subjects . 
in iih , we also observed abnormal csf flow pulsatile resulting from attempt to flow against the increased pressure . ct and mri findings in patients with iih are dilated optic nerve sheath , empty sella , posterior scleral flattening , prelaminar optic nerve enhancement , vertical tortuous appearance of orbital optic nerves , enlargement of meckel cave and rarely tonsillar herniation . 
 in a study of diyvata etal . , mri findings were found similar in both iih and secondary intracranial hypertension [ 15 ]  . they reported that optic nerve head protrusion and posterior scleral flattening were significantly associated with iih with no statistically significant difference found in the occurrence of rest of the findings . 
they suggested that clinical features , not the degree of stenosis , should be used to determine management in iih . in our study , we compared csf opening pressure and csf flow metrics with cranial mri and mr venography findings . 
in our study , in comparison with as area and ppc / as ratio , there was no significant difference between patients with iih and healthy subjects but a statistically significant difference was found between hydrocephalus and other two groups . 
this led to a statistically significant decrease in ppc / as ratio in hydrocephalus 1 3 390 la radiologia medica ( 2019 ) 124 : 382391 compared to two other groups . 
in the present study , we found statistically significant increase in asv , peak flow velocity , forward flow volume , reverse flow volume , net forward flow volume , forward flow velocity , reverse flow velocity and average flow volume in one cardiac cycle in hydrocephalus , compared to healthy subjects . high asv values in hydrocephalus are due to increased csf volume and increased as area . 
however , only statistically significant difference was found between patients with hydrocephalus and iih . in comparison with asv values of all three groups , a significant increase in asv was detected in hydrocephalus , whereas no significant difference between iih and healthy subjects ( median asv 180l in hydrocephalus , 15l in iih and 21.2l in healthy subjects )  . 
the average flow volume in one cardiac cycle showed similar ranking ( median value 31.76ml / min in hydrocephalus , 2.6ml / min in iih , and 3.7ml / min in healthy subjects )  . 
asv shows the volume of csf passing through as , and it has a positive correlation with area of as and volume of csf within the lateral and third ventricles . 
we thought that these findings were expected effects of decreased intraventricular csf volume as well as increased intracranial pressure . conclusion csf flow dynamic is so complex , and pathways of csf production and absorption are still speculative . 
this mainly comes from shortening in diastolic flow duration that is probably effects of increased impedance of csf flow against increased intracranial pressure and unchanged or even decreased intraventricular csf volume . 
in conclusion , csf flow dynamic is not fully understood ; further , more detailed , larger - scale studies should be performed to reveal the role and variability of csf flow in diseases . authors contribution tfy and ht contributed to data collection , data archiving , manuscript writing ; aa was involved in project development , data collection , data archiving , manuscript writing ; em contributed to data collection , data archiving ; gk , neurologist , took care of the patients ; sk and aa were involved in project development ; mok contributed to biostatistical analysis . compliance with ethical standards conflict of interest the authors have declared no conflict of interest . ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 declaration of helsinki and its later amendments or comparable ethical standards . 
restoring the normal alignment and articular congruity after a distal radius fracture is important in order to obtain good functional results [ 2 , 3 ] , in addition to preventing the development of osteoarthritis [ 4 ]  . 
open * alessandro stecco a.stecco@libero.it 1 radiology institute , services diagnosis andtherapies department , maggiore della carit hospital , university ofeastern piedmont upo university , corso mazzini 18 , 28100novara , italy 2 orthopaedics andtraumatology department , maggiore della carit hospital , university ofeastern piedmont , novara , italy 3 medical physics department , maggiore della carit hospital , university ofeastern piedmont , novara , italy reduction and internal fixation ( orif ) with plates and screws has been demonstrated to be effective in the treatment of unstable distal radius and ulnar fractures [ 5 ]  . magnetic resonance imaging ( mri ) is a useful tool to study the most common complications associated with plating . 
however , metallic implants can cause significant image distortion in mri and are known to interfere with the accurate identification of fractures , infections and recurrent tumours surrounding an implant , or even loosening of the implant [ 5 ]  . two mri techniques have been implemented in order to reduce metal - induced artefacts : the view angle tilting ( vat ) technique and the higher readout bandwidth ( hibw ) technique [ 611 ]  . 
in our department , we have a dedicated low - field mri scanner with metal artefact reduction ( mar ) sequences , which was applied in the study of distal radius and ulna fractures treated with volar plating . the purpose of this study was to compare these two optimised mar sequences in their efficiency in reducing vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 392399 metallic artefacts , using a low - field mri scanner . 
to our knowledge , this is the first study to evaluate the role of mar sequences in wrists after plating using a dedicated low - field mri scanner . materials andmethods patients we evaluated 21 patients who underwent mri from november 2014 to march 2016 at our institution . 
ten patients underwent an mri due to pain , one due to a dorsal wrist tumefaction ( suspicion of a ganglion cyst on ultrasound ) and five due to a slight decrease in range of motion . one patient had extensor digitorum communis tendon rupture , four had a partial scapholunate ligament tear , one had a luno - triquetral ligament tear , one had an extensor digitorum communis tenosynovitis , one patient had an extensor carpi ulnaris tendon ganglion cyst , one had a trapeziometacarpal osteoarthritis , one had carpometacarpal osteoarthritis , and two patients had a triangular fibrocartilage complex ( tfcc ) lesion ( one patient with a 1a and one with a 1d lesion , according to the palmer classification [ 13 ] )  . 
the patients had been treated with a 2.4mm variable angle locking compression plate ( lcp ) two - column volar distal radius plate ( depuy synthes , johnson & johnson , west chester , pa , usa )  . 
patients gave their consent prior to mri , and as the study was retrospective in nature , approval from the local ethics committee was not sought . magnetic resonance imaging sequences all patients were examined using a dedicated 0.3t scanner ( o - scan , esaote , italy )  . 
coronal fse t2 vat sequence ( 4460 / 96 ; 3mm section thickness , 0.3 gap , 512 512 field of view , 12ms echo spacing , 9 echo train length , 15 sections , 5min 21s acquisition time )  . our protocol usually also contains standard axial fse t2w , axial se ( spin echo ) t1w , coronal stir ( short - tau inversion recovery ) and coronal gradient - echo ( gre ) t1 sequences ; however , these were not analysed in this study . 
the artefacts volumes were then , after rois manual tracing , automatically calculated using the measure stack plugin ( available at av.com / measu resta ck.htm ) , which performed measurements on a series of distinct regions of interest ( roi ) in the slices of a stack . 
after entering the slice spacing ( section thickness , thus 3mm ) , the results were summed to determine the total volume of the artefact . the two components of teams reader 1 and reader 2 collaborated in consensus to perform and validate , in a doublereading fashion , the measurements of cumulative areas of the signal voids and artefacts on standard and mar images . this type of work does not require approval by ethics committee , because of the retrospective nature of the study , 1 3 394 la radiologia medica ( 2019 ) 124 : 392399 but before all diagnostic exams patients received and signed informed consent . statistical analyses cohens k value . 
quantitative statistics were used to compare the signal void ( black artefact ) and artefact distortion ( bright artefact )  . a preliminary testretest statistical evaluation has been carried out by the two equipes of raters ( reader 1 and reader 2 ) , who performed independently multiple measurements ( 10 ) on the same predefined area of artefact , in the same randomly selected data set among the case series . 
1 standard coronal fast spin echo ( fse ) t2 , b coronal fse t2 optimised with high bandwidth ( fse - hibw ) and c coronal fse t2 view angle tilting ( vat ) sequences . 
single manually drawn regions of interest ( roi ) were then summed , and the total volume was calculated 1 3 la radiologia medica ( 2019 ) 124 : 392399 fig . 
2 standard coronal fast spin echo ( fse ) t2 , b coronal fse t2 optimised with high bandwidth ( fse - hibw ) and c coronal fse t2 view angle tilting ( vat ) sequences . 
3 standard coronal fast spin echo ( fse ) t2 , b coronal fse t2 optimised with high bandwidth ( fse - hibw ) and c coronal fse t2 view angle tilting ( vat ) sequences . 
 open reduction and internal fixation ( orif ) with plates and screws is considered to be the most effective treatment for displaced and unstable fractures.. the most common complications associated with volar or dorsal plating are a loss of reduction after plate fixation , irritation of the median nerve [ 5 ] and lesions or irritation of the extensor or flexor tendons . 
many of these complications are related to the features of the device ( thickness , shape , screws or material ) or the surgical approach and technique . rupture or irritation of the extensor tendons is the most frequent problem associated with the dorsal approach . 
carpal tunnel syndrome has also been associated with volar plating [ 18 , 22 , 23 ] ; however , this may occur in distal radius fractures regardless of the treatment method used . 
the inhomogeneity correction arises from the additional tilting gradient in the slice - select direction , so that the slice is effectively viewed at a tan1 ( gz / gx ) angle . 
 when viewed at this angle , shifts in the slice - selection plane compensate for shifts during readout , such that plane shifts are re - registered in the images . 
however , vat images are prone to blurring in the frequency - encoding direction , which has hindered the widespread use of this technique [ 7 ]  . the hibw technique increases the resolution in the frequency - encoding direction [ 8 , 9 ]  . 
as the bandwidth ( pixels ) increases , the effects from the geometric distortion and intravoxel phase dispersion form a smaller proportion of the total signal , thereby reducing image distortion . 
 alternatively , the field of view ( fov ) could be decreased for the same image matrix , but this is not routinely performed because the anatomical region of interest would not be completely visible . 
the disadvantage of using stronger gradients for these measurements is a decrease in the signal - to - noise ratio ( snr ) [ 10 , 11 ]  . dedicated mri scanners are increasingly used to study extremities as they have many advantages over whole - body scanners . 
the main advantages of extremity scanners over whole - body high - field scannersare : decreased purchase and maintenance costs , lower weight , easier installation in a small space and faster patient diagnosis ( in the office setting )  . 
the installation of low - field mr scanners is also beneficial for interventional and intraoperative use . in our study , we observed a significant decrease in both the black and bright artefacts for the hibw and vat sequences . 
 [ 21 ] described seven patients who had lost fixation of a volar lunate facet fragment , with subsequent carpal displacement 1 3 398 la radiologia medica ( 2019 ) 124 : 392399 blurred which reduced the overall quality of the images . 
the poorer quality of images obtained by the vat sequence may make the evaluation of subtle alterations in small anatomical structures ( ligaments or tendons ) more difficult when compared to standard fse t2 and hibw sequences . on the other hand , bone is easily evaluated ; therefore , vat sequences could be used to obtain a more detailed evaluation of the bone surrounding the screws or metallic implants . 
in our opinion , hibw sequences represent a good compromise , both quantitatively and qualitatively , as it provides images of acceptable quality in addition to significant reduction in artefacts . this study has several limitations . 
another limitation of this retrospective study was that the patients were randomly chosen , and because of this , there was a wide range in the time between surgery and mri among the patients . 
 we were also unable to qualitatively assess the artefacts , even though our primary goal was to perform a quantitative analysis . more studies are required to verify the clinical impacts of these different sequences . 
due to the use of the drape , radiation exposure of primary operators abdominal area , genitals , thyroid and eye lenses was reduced by an average of 59% , 60% , 65% and 59% , respectively . 
however , dose area product ( dap ) and peak skin dose ( psd ) were increased by 20% when part of the drape was placed into the x - ray field . conclusion during evar and pta procedures , primary operators organs are exposed to considerable radiation doses . 
box2208 , 71003heraklion , crete , greece interventional radiology unit , department ofradiology , university hospital ofheraklion , medical school , university ofcrete , crete , greece 3 vascular surgery unit , department ofcardiothoracic andvascular surgery , university hospital ofheraklion , medical school , university ofcrete , crete , greece 4 department ofmedical physics , university hospital ofheraklion , medical school , faculty ofmedicine , university ofcrete , p.o. 
box2208 , 71003iraklion , crete , greece introduction atherosclerosis of aortoiliac arteries , abdominal aorta and below the knee arteries can cause intermittent claudication , gangrene , limb ischemia and loss of a limb [ 13 ]  . 
pta is the most common fluoroscopically guided intervention for the treatment of peripheral artery disease [ 4 ]  . the most frequent form of aortic aneurysm is the abdominal aortic aneurysm ( aaa ) [ 5 ]  . 
the procedures were performed by three experienced interventionalists . the following data were recorded for all patients : total fluoroscopy time , dap , psd and the number of digital subtraction angiography ( dsa ) series . 
positioning of the c - arm in left / right ( lao / rao ) direction was between 0 and 45 for both procedures . occupational doses in both procedures were determined using thermoluminescence dosimetry ( tld ) chips ( tld200 , hashaw , solon , oh )  . 
at the beginning of each procedure , tld chips were placed on the left side of the head , on the protective eyewear , over and underneath the thyroid collar , on chest level over and underneath the apron and on the middle finger of both hands of the primary operator . 
estimation of the effective dose with this algorithm is independent of aprons thickness . to evaluate the efficacy of a radioprotective drape , experiments were performed using two physical anthropomorphic phantoms ( rando - alderson research labs , ca , usa ) and a 0.25mm pb equivalent drape ( ecolab , saint paul , minnesota , usa )  . 
total fluoroscopy time and the number of dsa runs were the same for all simulations . statistical analysis statistical analysis was performed using prism software ( graphpad , ca , usa ) , and office excel ( microsoft , wa , usa )  . 
2 positioning of the drape in the first experiment ( left ) , second experiment ( middle ) , third experiment ( right ) results there was a statistically significant difference in fluoroscopy time ( p = 0.0008 ) and dap ( p < 0.0001 ) , between pta and evar procedures ( table1 )  . table 2 presents occupational doses of the primary operator , for evar and pta procedures , respectively . 
figures3 and 4 show the correlations between chest dose over the apron , right - hand dose , thyroid dose over the collar , eye lens dose and fluoroscopy time for the primary operator in pta and evar procedures . table3 presents the maximum number of evar or pta procedures an interventionalist can perform annually . 
the calculated maximum workloads were based on the annual effective dose limit and equivalent dose to the lens of the eye limit recommended by icrp as well as on median ed and eye lens doses presented in table2 . the results of this study show that the drapes significantly contribute to the operators radioprotection . 
specifically , radiation exposure of the abdominal area , genitals and thyroid , was reduced by an average of 59% , 60% , 65% , respectively , due to the use of the drape . 
on the contrary , differences up to 20% were noted when half of the drape was placed into the field ( table5 )  . discussion dap and fluoroscopy time are directly related with the radiation exposure of the interventionalists . 
4 correlations between different body parts doses and fluoroscopy time in evar procedures table 3 maximum permissible annual workload based on ed and eye lens doses procedure number of procedures ( based on ed ) number of procedures ( based on eye lens dose ) evar 4255 4545 1626 tabulated data ( table2 ) show that radiation exposure of the primary operator was higher in evar procedures than in pta procedures . 
based on the literature , hand doses of the interventionalists during pta procedures range from 21 to 190 sv [ 1315 , 23 , 24 ]  . eye lens doses during pta procedures have been evaluated by a number of studies [ 1315 , 2325 ]  . 
the wide range of these values indicates that several parameters including complexity of the procedures as well as the experience of the operator affect the radiation exposure of the interventionalists . 
further , a mean reduction up to 29% , 60% , and 60% , of the radiation exposure , was recorded for the right side of the head , the forehead and the left side of the head , respectively . 
these results are in agreement with the corresponding results presented in the literature [ 1517 ]  . the efficacy of the drape has been evaluated by a number of studies . 
specifically , dap and psd were 1 3 la radiologia medica ( 2019 ) 124 : 539545 increased by 20% when part of the drape was placed into the x - ray field . 
radiation dose increases when a drape is in the x - ray field because aec increases the exposure in an attempt to maintain image quality . our study has some limitations that should be considered . 
a patient study is needed to confirm the effect of the drapes positioning on aec . conclusion during evar and pta procedures , primary operators organs are exposed to considerable radiation doses . 
the non - contrast - enhanced scan sequences were performed with noise indexes ( nis ) of 11 , 14 , and 16 for the 0 to < 1 , 1 to < 3 , and 3 to < 6 year age groups , respectively . 
enhanced scan sequence : the ni default was 9 , and the data were subjected to the filtered back projection reconstruction algorithall other scanning parameters were the same as those used in the non - contrast - enhanced scan sequence . results in the 0 to < 1year group , the image qualities were scored as 3 or above with asir weights of 50% for the lung window and 40% for the mediastinal window ; in the 1 to < 3year group , the image qualities were scored as 3 or above with asir weights of 60% for the lung window and 50% for the mediastinal window ; in the 3 to < 6year group , the image qualities were scored as 3 or above with asir weights of 70% for the lung window and 60% for the mediastinal window . conclusion for low - dose chest ct scans of preschool - age children , application of the asir technique significantly improved image quality and reduced image noise . 
however , the reduction in tube potential increases image noise , decreases the signal - to - noise ratio ( snr ) , and influences the detection of subtle lesions . 
 as reported [ 8 ] , under the conditions of assured quality of ct reconstruction images and not influencing the diagnosis by the radiologist , asir allows 3265% reductions in x - ray radiation dosage compared with the traditional fbp . 
however , the use of an asir reconstruction technique with over - reduction in ct scan conditions may cause over - smoothing of images and mask some image information , which can affect the clinical diagnosis . 
to optimize the ct scan dose , it is required to explore the best asir ratios for different groups of people and in different scanning areas while practicing ct scans . currently , there are several reports on the study of applying asir on adult chest ct examinations , but there are fewer reports on the application of asir in childrens chest ct scans . 
this study aims to evaluate the combined application of asir and ni on the chest ct scans of preschool - age children . information andmethod this is a prospective study , this study was approved by the independent medical ethics committee of our hospital , and the family members of all children provided informed consent before the study . general information clinical pretest children who underwent ct non - contrast - enhanced and enhanced scans at our department due to clinical needs from june 2015 to june 2016 were included . 
slice thickness was 5mm with 5 - mm intervals , the field of view ( fov ) was small body , the pitch was 1.375 : 1 , the rotation time was 0.6s , and the matrix was 512 512 . 
for enhanced scans , the ni was set at 9 for all examinations , and all other scanning parameters were the same as in normal scan . interest ( roi ) was circular in shape . 
image snrs were calculated as follows : left ventricle snr = left ventricle ct value / erector spinae sd value , whereas the erector spinae snr = erector spinae ct value / erector spinae sd value . 
 all other scan parameters were the same as those used in the clinical pretest . data measurement data measurements were performed on the non - contrastenhanced chest ct images of the children . 
the ct and sd values of the left ventricle and erector spinae were measured from the slice with the maximum left ventricle area and the two slices above and below . 
the region of the ct dose index volume ( ctdivol ) and dose length product ( dlp ) of non - contrast - enhanced and enhanced ct scans for each group were recorded . 
 the k values were as follows : 0.0039msvmgy1cm1 for the 0 to < 1year age group , 0.0026msvmgy1cm1 for the 1 to < 3year age group , and 0.0018msvmgy1cm1 for the 3 to < 6year age group [ 9 ]  . 
the average of each group was calculated . subjective evaluation ofimage quality the 4 - point scale used in the chest ct image evaluation by the quality control team of the laboratory of the researchers was adapted . 
this scale was developed by the members of the quality control team through referencing high - impact studies in the literature and evaluation standards implemented in other pediatric hospitals with consideration of the conditions of our equipment [ 1015 ]  . 
the evaluation team was composed of an associate chief physician in the department of radiology and two radiology physicians with more than 5years of work experience . the images of the mediastinal window ( w280 , l70 ) and lung window ( w1300 , l - 500 ) taken from children in each group were evaluated by the two senior physicians using a 4 - point image quality evaluation scale with scan parameters concealed . 
b measurement of the anteroposterior and horizontal diameters on the slice with maximum area in the left ventricle 1 3 470 la radiologia medica ( 2019 ) 124 : 467477 points indicated good image quality with clear lesion and anatomical details ; 3 points indicated good image quality with clear illustration of most of the lesion and anatomical details except for a few images , which did not affect the diagnosis ; 2 points indicated poor image quality that could not satisfy the diagnostic requirements ; and 1 point indicated poor image quality that did not allow diagnosis . 
 the quantified parameters are expressed as x s . clinical pretest comparisons of the baseline data ( age , anteroposterior thoracic diameter , and axial thoracic diameter ) , subjective evaluation of image quality , scan radiation doses , image snr , and image noise of each patient group under different ni conditions were conducted with one - way analysis of variance ( anova )  . 
if the results were statistically significant , multiple comparisons were performed using the bonferroni method ; a value of p < 0.05 was considered statistically significant . clinical test : the subjective evaluation results of image quality , image snr , and image noise of the children using asir with different weights were examined using anova for repeated measurements . 
such a phenomenon was more apparent at the edges of the images ( table9 )  . for images of the group aged 0 to < 1year , the image qualities were scored as 3 and above with asir weights of 50% for the lung window and 40% for the mediastinal window . 
2 a six - month - old boy , lung window , asir weights of 40% , b six - month - old boy , mediastinal window , asir weights of 30% , c six - monthold boy , lung window , asir weights of 50% , d six - monthold boy , mediastinal window , asir weights of 40% , e six - month - old boy , lung window , asir weights of 60% , f six - month - old boy , mediastinal window , asir weights of 50% 1 3 la radiologia medica ( 2019 ) 124 : 467477 474 fig . 
3 a two - year - old girl , lung window , asir weights of 50% , b two - year - old girl , mediastinal window , asir weights of 40% , c two - year - old girl , lung window , asir weights of 60% , d two - year - old girl , mediastinal window , asir weights of 50% , e two - year - old girl , lung window , asir weights of 70% , f two - year - old girl , mediastinal window , asir weights of 60% for images of the group aged 1 to < 3years , the image qualities were scored as 3 and above with asir weights of 60% for the lung window and 50% for the mediastinal window . 
to reduce the effects of these differences , we divided preschool - age children into groups of 0 to < 1 , 1 to < 3 , and 3 to < 6years . 
stringent requirements were set for inclusion and exclusion of patients to assure the universality of the research results . in non - contrast - enhanced scan sequences , it is unknown whether the ct images obtained after adjusting the scan parameter ni could satisfy the diagnostic requirements . 
4 a four - year - old boy , lung window , asir weights of 60% , b four - year - old boy , mediastinal window , asir weights of 50% , c four - year - old boy , lung window , asir weights of 70% , d four - year - old boy , mediastinal window , asir weights of 60% , e four - year - old boy , lung window , asir weights of 80% , f four - year - old boy , mediastinal window , asir weights of 70% scan images were from children who also needed enhanced scans . 
in other words , if the quality levels of the non - contrast - enhanced ct images were reduced due to the increase in ni and the diagnosis was affected , the use of regular parameters in their enhanced scan sequence ensured a satisfactory image quality for diagnosis . 
with this method , the disadvantage of poor image quality obtained in a nonenhanced scan was overcome , and repetitive scans on children , which increase the radiation dose , could be avoided . in our study , dlp , ctdivol , and ed were considered as the indexes to evaluate the radiation dose in the ct examinations of children . 
the image reconstruction method implemented in our study was asir , which has been reported and demonstrated various advantages , such as effectively reducing image noise and increasing image quality [ 2228 ]  . the clinical pretest results showed that the choice of ni is crucial and directly affects the radiation dose and ct image quality of the children subjected to ct scans . 
the optimal ni value increased with the age of the child and thus varied for different age groups as follows : 11 for the 0 to < 1year group , 14 for the 1 to < 3year group , and 16 for the 3 to < 6year group . 
so , for the clinical tests in our research , the nis were set at 11 , 14 , and 16 for the 0 to < 1 , 1 to < 3 , and 3 to < 6year age groups , respectively . the clinical pretest results can be used to guide clinical practice and provide the optimal ni values for ct scans for preschool children of all ages , which can be directly applied to the ct equipment models used in this study while providing references for ct equipment of other models . 
we also 1 3 476 la radiologia medica ( 2019 ) 124 : 467477 found that the weight of asir directly affects the image quality , and its impact on children and adults is different . 
 in the case of adults , except for some body types , the asir weight is essentially the same ; however , the asir weights for children of different ages differ significantly , and different age groups have their respective optimal weights . the clinical test results showed that the asir technique can significantly reduce the effective radiation dose by 42% for children aged 0 to < 1year , by 63% for children aged 1 to < 3years , and by 72% for children aged 3 to < 6years , without reducing image quality . 
when the children in each age group were subjected to a scan with the optimal ni value , the image quality was closely correlated with the weight of asir , and the optimal asir weights of different age groups were as follows : 50% for the lung window and 40% for the mediastinal window for the 0 to < 1year group , 60% for the lung window and 50% for the mediastinal window for the 1 to < 3year group , and 70% for the lung window and 60% for the mediastinal window for the 3 to < 6year group . 
this result provides guidance for the application of the asir technique in children , and this method can also be extended to other age groups so that their optimal asir weights can be determined . 
this result can also guide the choice of parameters for other similar iterative reconstruction techniques , in which the optimal iterative reconstruction parameters of children of different age groups can be explored . a limitation of our study is the small sample size . 
further studies are needed to refine the application of asir in pediatric chest ct scans . in summary , for low - dose chest ct scans of preschoolage children , application of the asir technique significantly improved image quality and reduced image noise . 
contrast - enhanced magnetic resonance angiography ( ce - mra ) and digital subtraction angiography ( dsa ) were performed at 3months post - procedure and correlated . results primary cp was used in 13 patients , while sac was used in three patients . 
no sac reperfusion was seen in aneurysms with packing densities 29% , irrespective of either embolization method . conclusion favorable midterm results for coil packing of saas seem to depend on the coil packing density with a coil volume approximately a quarter of the aneurysm volume being most effective . 
follow - up should involve the use of ce - mra as this modality has been shown to be superior over dsa in detecting aneurysm reperfusion and coil compaction . level of evidence level iv , therapeutic study . keywords splenic artery aneurysm visceral aneurysm aneurysm embolization selective coiling stent - assisted coiling packing density mra follow - up protocol * mikoaj wojtaszek nwojtaszek@gmail.com 2nd department ofclinical radiology , medical university ofwarsaw , banacha 1a street , 02 - 097warsaw , poland 2 vascular unit , kent & canterbury hospital , east kent hospital university nhs trust , canterbury , kentct13ng , 3 department ofgeneral andendocrine surgery , medical university ofwarsaw , banacha 1a street , 02 - 097warsaw , poland introduction true splenic artery aneurysms ( saas ) are the most common visceral artery aneurysms . the acc / aha guidelines recommend treating saa when symptomatic or larger than 2cm and in women of childbearing age and patients undergoing liver transplantation , regardless of size [ 1 ]  . based on autopsy studies , the prevalence of visceral artery aneurysms has been estimated to be up to 10% , with up to 25% complicated by rupture and a mortality rate of up to 70% [ 24 ]  . 
1 a final angiogram of a treated 17 mm cp splenic aneurysm , b cta shows an infarct of the posterior aspect of the spleen and a coil packed splenic aneurysm , c selective angiogram performed just after the cta shows translocation of the coils from the aneurysm sac , occlusion of a major dorsal branch of the middle segment of the splenic artery and class ii reperfusion of the aneurysm sac , d a self - expandable xpert stent ( abbott medical ) is implanted across the aneurysm neck to secure the translocated coils , e a follow - up 3month dsa shows patency of the splenic artery without aneurysm sac reperfusion , f no sac reperfusion and splenic artery patency is further confirmed by ce - mra coils and preserving the parent artery has been reported with diligent follow - up imaging necessary to detect aneurysmal re - growth and plan intervention , but the optimal method and timing of follow - up has not yet been established . 
aneurysms with a wide neck received stent - assisted coil exclusion ( sac ) of the aneurysm sac with the parent artery supported with a self - expandable stent ( xpert self - expandable stent , abbott vascular , abbott park , il , usa )  . 
all patients received single - agent antiplatelet therapy ( acetylsalicylic acid , 75mg ) post - procedure for the following 3months . patient population followup methods , techniques andparameters all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . 
this retrospective review evaluated 16 consecutive patients ( 4 men ; mean age 46.7 , range 2879 ) , who underwent selective true saa embolization between june 2012 and march 2016 using detachable fibered embolization coils ( concerto , medtronic , minneapolis , mn , usa )  . 
next , coronal breath - hold fatsaturated spoiled gradient - recalled echo sequences ( fast lowangle shotflash 3d ) were performed before and after iv bolus administration of paramagnetic contrast material . 
after contrast administration , four measurements were performed with pauses at 0s , 5s , 5s and a total acquisition time of 1 3 la radiologia medica ( 2019 ) 124 : 450459 1 3 454 la radiologia medica ( 2019 ) 124 : 450459 fig . 
2 a initial selective angiogram of a 26mm splenic artery aneurysm in the mid - distal segment of the splenic artery , b final angiogram after cp of the splenic aneurysm , c initial , 3 month ce - mra of the cp splenic aneurysm ( coils and thrombus depicted by white arrowheads ) together with class iii reperfusion ( depicted by black arrows ) of the aneurysm sac , d selective angiogram before reembolization ( also 3 months ) showing coil compaction and class iii reperfusion of the aneurysm sac , e completion angiogram after reembolization showing complete occlusion of the aneurysm , f a 3month control ce - mra showing persistent reperfusion ( class ii ) of the aneurysm sac ( depicted by black arrows ) 46s . 
as mra is not a validated diagnostic method for the assessment of recanalization in coil embolized visceral aneurysms , these results were correlated with a dsa study performed on the same day . 
the follow - up period was defined as the time from treatment to the last ce - mra study . image analysis andpacking density ce - mr angiography image analysis was performed on a 3d workstation ( syngovia , siemens , erlangen , germany ) using subtracted source images to obtain multiplanar reformatted , maximum - intensity projection . 
aneurysm occlusion was classified as follows : class 1 , complete occlusion ; class 2 , residual neck ; class 3 , residual aneurysm using a neuroradiology assessment scheme as proposed by roy etal . 
results were compared using the fisher and student t tests . results sixteen patients with saa treated with fibered detachable coils had complete datasets available ( ce - mra and dsa )  . 
one patient presented with acute left flank pain 23days after the initial procedure , and cta showed an infarction of approximately 20% of the spleen and an occluded dorsal splenic branch . 
there was a significant difference in the incidence of aneurysm sac reperfusion on cemra study in between coil packing and stent - assisted coiling 1 3 la radiologia medica ( 2019 ) 124 : 450459 1 3 456 la radiologia medica ( 2019 ) 124 : 450459 fig . 
3 a initial selective angiogram of a 23 - mm splenic artery aneurysm in the mid - distal segment of the splenic artery , b a self - expandable xpert stent ( abbott medical ) is placed and expanded across the splenic artery aneurysm neck , c final angiogram after sac of the mid - segment of the splenic artery with complete aneurysm occlusion , d a 3 - month control dsa shows occlusion of the aneurysm and supporting stent with a large network of collaterals between the major and dorsal pancreatic arteries supplying the distal segment of the splenic artery , e ce - mra also showing complete occlusion of the aneurysm and underlying segment of the splenic artery . 
in the sac group , no statistical differences could be determined between the different aneurysm size groups in relation to aneurysm coil packing ( table2 )  . no sac reperfusion or coil compaction was seen in aneurysms in which a packing density of 29% was achieved , irrespective of either embolization method . 
 the thick line denotes a cutoff value of 29% discussion endovascular techniques , including embolization and stent graft placement , are now considered alternatives to conventional surgery in the treatment of splenic aneurysms [ 8 , 9 ]  . 
several authors have reported follow - up intervals at 1 and 6months after coil embolization using cta imaging , but it is now believed that this modality is hampered with excessive beam hardening artifacts making assessment of aneurysm reperfusion unreliable [ 10 , 11 ]  . 
as dsa missed 1 / 3 of the sac reperfusions in this study , and considering the low rate of rupture for visceral aneurysms , we suggest a simplified follow - up protocol which comprises a cemra performed at 3months . 
if reperfusion or compaction is present , a follow - up dsa should be performed with the intent to repack the residual aneurysa satisfactory scan at 3months should be followed up by ce - mra at 1year . 
if residual neck ( class ii ) is observed , a consensus decision should be reached , in which the risk of rupture is assessed in relation to the potential technical challenge of reembolization . 
a detailed follow - up flowchart is presented in fig.6. the relationship between packing density , coil compaction and sac reperfusion in visceral aneurysms has been previously studied by yasumoto etal . 
in this paper , a packing density of above 24% has been shown to be sufficient in preventing reperfusion in long - term follow - up while in our study a packing density of 29% was enough to prevent aneurysm reperfusion . 
 this technique is much more demanding and often impossible and will worsen splenic artery patency , as we have shown . one of the limitations of the present study is that it was conducted retrospectively in a single center and had a small series size . 
the small population group hampers adequate statistical analysis of the influence of the size of the aneurysm , but also the location on the packing density threshold , making comparison of cp and sac groups difficult statistically . 
we were unable to study the effects of anticoagulation therapy and blood pressure control in this study for similar reasons . conclusions in conclusion , favorable midterm results for coil packing of saas seem to depend on the coil packing density with a coil volume approximately a quarter of the aneurysm volume being most effective . 
 ( * technical and clinical risk for reembolization should be stratified before undertaking the procedure , especially in class ii reperfusions ) a single stage procedure , accurate follow - up of these patients is essential , as some will require further interventions . 
follow - up should involve the use of ce - mra at regular intervals as this modality has been shown to be superior over dsa in detecting aneurysm reperfusion and coil compaction . declaration and its later amendments or comparable ethical standards . 
to find the superior plan , h - vmat with three different arc designs including , two partial arcs ( 2a ) , four partial arcs ( 4a ) and four tangential arcs ( ta ) were created for each study case by combining 3dcrt and vmat with 75% 3dcrt / 25% vmat dose proportion of prescription dose . results all h - vmat plans achieved the expected target coverage . 
further , 2a h - vmat delivers less mu and beam - on time compared to 4a h - vmat . keywords breast cancer whole breast vmat hybrid - vmat introduction breast cancer is among the most common cancers in women worldwide [ 1 ]  . 
as an adjunct to surgery and chemotherapy , rt in early - stage breast cancer patients halves the rate of disease recurrence and reduces the breast cancer mortality by sixth [ 2 ]  . 
with advancement in rt techniques , the 5 - year , 10 - year and 15 - year overall survival of the breast cancer patients has improved to 89% , 83% and 78% , respectively [ 3 ]  . 
to avoid long - term complications , sparing organs at risk ( oar ) such as ipsilateral lung ( il ) , contralateral lung ( cl ) , heart and contralateral breast ( cb ) at different dose volume levels has been suggested in numerous clinical studies [ 514 ]  . 
further , reducing the dose inhomogeneity and skin dose helps in minimizing the rt - induced toxicities such as fibrosis , erythema and moist desquamation [ 15 , 16 ]  . 
the most common rt techniques available for whole breast irradiation in a linear accelerator setup are bi - tangential 3 - dimensional conformal radiotherapy ( 3dcrt ) , field - in - field ( finf ) , intensity modulated radiotherapy ( imrt ) and volumetric modulated arc therapy ( vmat )  . 
in contrast , imrt and vmat techniques provide improved dose conformity , homogeneity and sparing of high dose irradiation , however , vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 546554 at the expense of increased low dose spread to normal tissue ( nt )  . though these advanced treatment techniques generate clinically acceptable plans , innovative methods are needed to get a well - adjusted result between ptv and oar dose parameters . 
 several researchers have reported on the benefit of hybrid techniques compared to other techniques in various disease sites ( whole breast [ 1722 ] , bilateral breast [ 23 ] , chest wall [ 24 ] , oral cavity [ 25 ] , lung and esophagus [ 26 ] , prostate [ 27 ] )  . 
in previous hybrid studies on breast rt , commonly 3dcrt had been kept as a base dose plan and imrt or vmat as a hybrid component [ 1721 ]  . 
 [ 22 ] kept imrt as a base dose plan and vmat as a hybrid plan . most of the published data have discussed the benefit of h - imrt for whole breast rt , whereas studies using hybridvmat ( h - vmat ) are sparse [ 1724 ]  . 
 further , in non - hybrid dosimetric studies , the vmat - only plans with different arc designs have shown to be superior compared to other plans for whole breast rt [ 2830 ]  . 
the objective of the present study is to evaluate different vmat designs in hybrid setting combined with 3dcrt as a base dose plan for left - sided whole breast rt . methods andmaterials patient selection andcontouring this study included twenty - six patients , median age of 50years ( range 2370years ) , with left - sided early breast cancer without nodal involvement . 
all scans were exported to eclipse ( version 13.6 , varian medical systems , usa ) treatment planning system ( tps ) for target and oar delineation and treatment planning . 
also , normal tissue ( nt : external body volume minus ptv ) and skin defined as a 4mm of tissue between the ptv and the body surface in the region encompassing 2cm around the ptv were contoured . 
for high dose control in nt , a virtual ring structure of 1cm thickness around the ptv was contoured . treatment planning to find the optimal plan , h - vmat plans were generated by summation of the 3dcrt plus vmat plans with three different arc arrangements comprising : two partial arcs ( 2a ) , four partial arcs ( 4a ) and four tangential arcs ( ta ) in the eclipse tps as described below . 
a single isocenter was placed at the center of ptv in the cranio - caudal direction and in the axial slice it was kept at lungptv interface to avoid beam divergence to il and heart . 
dose calculations were done using analytical anisotropic algorithm ( aaa ) with 2.5 - mm dose grid matrix and normalized to ptv mean dose equal to the prescribed dose [ 31 ]  . 
the dose objectives used for ptv and oar are described in table1 . the 3dcrt plan consisted of two coplanar open tangential fields with gantry angles based on the ptv contour . 
in order to accommodate the ptv shape changes as well as breathing effect during treatment , 2cm air gap was given from the body surface . the 2a vmat plan consisted of two coplanar partial arcs : arc1 rotated clockwise from 300 to 50 ( arc length 110 ) with collimator angle 15 , and arc2 rotated clockwise from 50 to 160 ( arc length 110 ) with collimator angle 345 . 
 for each patient , dose to oar was minimized as low as possibly achievable without compromising the ptv coverage ; however , same objective functions were used for all three vmat plans for a particular patient . 
 similarly , arc3 rotated clockwise from 110 to 160 and arc4 dosimetric evaluation to evaluate the plan quality , quantitative analysis was performed by using dose volume histograms ( dvh )  . 
the paddick [ 32 ] ci was calculated , as ci = v_ptvref v_ptvref v_ref v_ptv where v_ptvref is the volume of reference isodose ( 95% ) inside the ptv ; and v_ptv , v_ref are the volume of ptv and reference isodose ( 95% ) , respectively . 
the hi was calculated , as hi = d2% d98% where d2% and d98% are the doses received by 2% , 98% of the ptv and dp is the prescription dose [ 31 ]  . 
the gi was calculated , as gi = v50% v_ptv 1 3 la radiologia medica ( 2019 ) 124 : 546554 where v50% is the volume of 50% isodose and v_ptv is the volume of ptv . 
treatment delivery parameters such as mu and beam - on time ( bot : time taken to deliver first mu to last mu ) were also recorded . additionally , the chest wall separation ( cws ) , maximum heart distance ( mhd ) and central lung distance ( cld ) were measured as illustrated in fig.2 and defined by das etal . 
the correlation coefficients ( r ) were calculated between ptv coverage , ci , hi versus cws ; heart dose parameters versus mhd ; il dose parameters versus cld . statistical analysis the dosimetric results of three h - vmat plans were statistically analyzed using the mannwhitney u test for nonparametric paired group comparison . 
the beam - on time was 46s less in the 2a h - vmat plan ( p < 0.00001 ) compared to other plans . the cws , mhd , cld and average volumes of the ptv , heart , il are summarized in table3 . 
table4 summarizes the correlation coefficients ( r ) between ptv coverage , ci , hi versus cws and ptv volume ; heart dose parameters versus mhd ; il dose parameters versus cld for all plans . 
similarly , intermediate correlations ( r ~ 0.8 ) were noticed between cld and il mean , v20gy , v40gy for all plans . cws = chest wall separation ; mhd = maximum heart distance ; cld = central lung distance fig . 
the second cancer risk was lower in 3dcrt [ 34 ] than vmat , and the cosmetic outcomes were improved in imrt / vmat compared to 3dcrt [ 16 ]  . 
 h - vmat technique efficiently limits the setup and breathing effect by delivering most of the dose using open 3dcrt beams with widened skin flash margin . in several non - hybrid dosimetric studies , different vmat designs were used for whole breast rt . 
 the present study utilized two split arcs and tangential arcs design in h - vmat plans . to get optimal beam geometry , several plans with different arc lengths were generated prior to this study . 
these plans included a 190 arc length between the two tangential fields used in 3dcrt ( 300130 ) , a 220 arc length between 300 and 160 and a 240 arc length between 300 and 180 . 
therefore , in this study , average dose proportion ( 75% dose weighting for 3dcrt and 25% dose weighting for vmat plans ) was chosen by considering the balance between the risks of low and high dose levels . the main aim of hybrid plan is to improve the ptv coverage , ci , hi and reduce low and high dose volumes to heart , il , cl and cb , thereby reducing the risk of longterm complications such as heart disease , pneumonitis and radiation induced secondary cancers . 
robust clinical studies with strong dosimetric correlation have made a set of doseconstraint recommendations to limit the dose of radiation received by specific volumes of oar in order to prevent long - term adverse outcomes [ 514 ]  . 
moreover , planning technique , beam geometry , optimization , dose calculation algorithms used and planners skill play a significant role in generating optimal plans . the main uncertainties in whole breast rt are setup and breathing effect . 
deep inspiration breath - hold ( dibh ) and gated techniques have been 1 3 la radiologia medica ( 2019 ) 124 : 546554 shown to reduce high dose to heart and il . 
to decrease setup uncertainties , appropriate ptv margin and daily image guidance should be considered . the key factor to get an optimal h - vmat plan would be its ability to reduce high dose as well as low dose radiation to oar without compromising the ptv parameters . 
2a h - vmat provided statistically similar results compared to 4a h - vmat , in addition reduced mu ( ~ 23 mu ) as well as beam - on time ( ~ 46s )  . 
however , in whole breast rt with simultaneous integrated boost or nodal volumes or hypo fractionated dose settings , 4a h - vmat might provide favorable results by increased beam modulation . conclusion in the present dosimetric study , 2a and 4a h - vmat techniques were effective in reducing oar doses as well as improving ptv dosimetric parameters . 
prospective clinical trials with long - term follow - up in patients would provide insight into actual benefits of such innovative techniques . compliance with ethical standards conflict of interest all authors who contributed to this study declare that they have no conflict of interest with respect to the manuscript . ethical approval institutional scientific and ethics board has approved this study . 
this article does not contain any studies with human participants performed by any of the authors . informed consent the informed consent has been waived off by the ethics board of the institute considering this as a retrospective study with no human involved . 
we measured the correlations using the spearman correlation coefficients ( rs ) for all combinations of the bivariate pair , performed pairwise comparisons of the rs values , and calculated the coefficients of determination . 
all analyses were conducted with the jmp pro software . results the stronger bivariate correlations were represented by the estotal cites rs = 0.968 , p < 0.001 , r2 = 0.937 ; and the citescoresjr rs = 0.911 , p < 0.001 , r2 = 0.829. 
from 105 possible combinations of pairwise comparisons , 38 depicted a p value > 0.050 which would suggest interchangeability among bivariate correlations . conclusions our findings support our hypothesis that the if does not show the best correlation between other metrics . 
cuauhtemoc , 06726mexicocity , mexico 2 escuela de medicina , universidad de sonora , hermosillo , mexico 3 departamento de metodologia de la investigacion , instituto nacional de pediatria , mexicocity , mexico 4 department ofradiology , i.m. 
2 , 119992moscow , russia introduction with the emergence of new databases offering competitive algorithms to participate in the bibliometric boosting game , new metrics are around , each with their strengths and weaknesses ; the bibliometric analysis of these metrics attempts to measure the impact of a journals published material that can reinforce their prestige [ 1 ]  . 
some questions remain unanswered in this field : which index metric should i use to select the journal to submit for publication ? in which journal will my research have the most extensive circulation ? several alternative metrics are available for the journals editor in the last years , and many editors have decided to publish them parallel to the impact factor ( if ) : the scimago journal rank ( sjr ) , the source normalized impact per paper ( snip ) , the eigenfactor score ( es ) , and vol . : ( 0123456789 ) 1 3 496 la radiologia medica ( 2019 ) 124 : 495504 the citescore ( citescore )  . 
of articles [ published ] , cited half - life , es , and article influence score ] , the es was the best predictors of 2 - years - ahead citations . 
this evidence was proved for the journals in the gastroenterology and hepatology [ 3 ] ; neurosciences [ 4 ] ; and radiology , nuclear medicine & medical imaging [ 5 ] categories . several journals have published comparisons of the if with sjr , snip , and es in some specialties such as otolaryngology [ 6 ] , nuclear medicine [ 7 ] , pediatric neurology [ 8 ] , occupational therapy [ 9 ] , psychiatry and psychology [ 10 ] , nephrology [ 11 ] , obstetrics and gynecology [ 12 ] , clinical medicine and infectology [ 13 ]  . 
furthermore , the use of citescore has been reported for journals in behavioral neuroscience [ 14 ] , dermatology [ 15 ] , pharmaceuticals science and molecular medicine [ 16 ] , trade journals [ 17 ] , structural chemistry [ 18 ] , and nursing [ 19 ]  . 
studies about bibliometrics continue being a trending topic , as many editors and authors in this category comment on several aspects of the if and its association with concurrent bibliometrics [ 2024 ]  . we aimed to obtain correlation values between the if and the metrics mentioned above and to assess significant differences between bivariate correlations of if with sjr , snip , es , and citescore in the radiology , nuclear medicine & medical imaging category . 
our findings might help authors in this category to understand which indices offer the best association with total cites or if ; then , authors could use this information to decide which bibliometric they use to rank their target journals before submission . table 1 basic bibliometric concepts [ 26 ] bibliometric index concept total cites snip the total number of times that a journal has been cited by all journals included in the database in the jcr year . 
citations to journals listed in jcr have compiled annually from the jcr years combined database , regardless of which jcr edition lists the journal and regardless of what kind of article was cited or when the cited article was published . 
each unique article - to - article link is counted as a citation all citations to the journal in the current jcr year to items published in the previous 2years , divided by the total number of scholarly items ( these comprise articles , reviews , and proceedings papers ) published in the journal over the last 2years . 
though not a strict mathematical average , the journal impact factor provides a functional approximation of the mean citation rate per citable item it is a measurement factor that establishes the quality of scientific publications based on the count of citations obtained by each publication . 
it is performed on the calculation of citations received by journals in 3years , giving higher weight to citations from prestigious journals ( those with high citation rates and low self - citations ) using the algorithm of google pagerank it measures contextual citation impact by weighting citations based on the total number of citations in a subject field . 
unlike the well - known journal impact factor , snip corrects for differences in citation practices between scientific fields , thereby allowing for more accurate between - field comparisons of citation impact . 
cwts journal indicators also provide stability intervals that indicate the reliability of the snip value of a journal it measures the number of times the articles from the journal published in the past 5years have been cited in the journal citation reports ( jcr ) year . 
every reference from one article in a journal to another article from the same journal is discounted , and weights each reference according to a stochastic measure of the number of time researchers spend reading the journal citescore citescore is a metric that measures the list of citations per article published . 
it provides added value to compare and evaluate scientific citescore calculates the citations of all the documents of a specific year in all the papers published in the previous 3years . 
that number is divided by the number of papers indexed in scopus published in those same years 1 3 la radiologia medica ( 2019 ) 124 : 495504 materials andmethods study design retrospective study . 
if , total cites , and es of journals in the radiology , nuclear medicine & medical imaging category from the web of knowledge [ 2 ] were recorded from the journal citation reports ( jcr ) [ 25 ] available for 2017 . 
the impact factor ( if ) is a measure of the frequency with which the average article in a journal has been cited in a particular year which can be consulted in the platform web of knowledge every year . 
the context within which the snip is evaluated is determined by the reference practices of the journals within the thematic field in question , as well as the extent to which the database covers this field . 
the citescore is the youngest metric , was proposed in december 2016 [ 29 ] , and is one of the three major indices included in scopus by elsevier to rank publication sources [ 17 ] ; the definition can be explained by its formula [ 17 ]  . 
citescore for year n ( citescore n ) sums the citations received in year n to documents published in years n 1 , n 2 , and n 3 and divides this by the number of documents published in the three consecutive years n 1 , n 2 , and n 3 . citescore n = citation count in n documents documents ( n 3 ) ( n 1 ) for instance , citescore n = citation count in 2019 documents 2016 2018 according to scopus , the 3 - year citescore time window was chosen as the best fit for all subject areas [ 17 ]  . we only chose journals in the radiology , nuclear medicine & medical imaging that coincidentally depicted values for if , es , sjr , snip , and citescore reported for the year 2017 . 
we additionally recorded the number of total cites as it is the standard variable considered for these metrics , and we also wanted to know the correlation values for this variable . 
a total of 122 journals were selected ; see table a in the supplementary online - only file . sample size calculation we used a sample size formula based on correlation coefficients in a cross - sectional study [ 30 ]  . 
the pairs of bivariate correlations were calculated using the formula for combinations c ( n , r ) = p ( n , r ) / r ! ; then , we use n = 6 for the number of bibliometrics and r = 2 for the number of pairs , which gave us 15 possible pairs . 
the previous three examples revealed that the average difference in r values of 0.146 is enough to warrant significant differences when you compare correlation values ; this difference represents a coefficient of determination of 0.021 ; it means a 2.1% of the variance in r values would suggest not interchangeability between bivariate correlations . 
table4 shows the pairwise comparisons with a p value < 0.050 , indicating not interchangeability among bivariate correlations of bibliometric indices ( table5 )  . comparison ofcorrelation coefficients withearlier studies we found only three previous studies in the literature that included some of the bivariate correlations evaluated in this studyour findings showed very strong correlation values for the citescoreif and sjrif were very similar to the values reported in the category of occupational therapy [ 9 ]  . 
figure2 shows the correlation matrix for all selected variables . pairwise comparison ofcorrelation coefficients from 105 possible combinations of pairwise comparisons , 38 depicted a p value > 0.050 which would indicate interchangeability among bivariate correlations . 
among those pairs with p values > 0.700 , sjr and citescore were not significant when each one was compared with the total cites ; similar observations were found for citescorees and esif . 
table4 shows the the measure of academic performance using bibliometrics usually applies to the impact of the scientific work of a journal or an author ; or the eligibility of a researcher to compete for a national scientific qualification , to receive funds or to become professor [ 18 ]  . 
the assessment of the scientific impact of journals evaluated by bibliometrics is a complex , multi - dimensional construct , and therefore the use of a single bibliometric index is considered inappropriate to rank , evaluate , and value journals [ 33 ]  . 
then , the use of multiple newer metrics provides a more comprehensive view of journals and their relative placements in their fields [ 34 ] ; this approach let referees go beyond the if limits . in this study , we were able to calculate correlations coefficients and pairwise comparisons between six selected bibliometrics : total cites , if , sjr , snip , es , and citescore in the radiology , nuclear medicine & medical imaging 1 3 500 la radiologia medica ( 2019 ) 124 : 495504 fig . 
it is evident the non - normal distribution of the majority of selected indices category in his study , we performed comprehensive pairwise comparisons among bibliometrics from the leader databases ( web of science , scopus , and scimago )  . the citescore was proposed in december 2016 by elsevier ; we only found one study that compared ranking positions of nuclear medicine journals using the sjr , if , and es from 2010 , but the authors did not present correlation coefficients [ 7 ]  . 
independently of the limitations based in the years that each index considered in their calculations , we were surprised to discover that the index that reached the highest correlation with the total cites in our chosen category was total citeses . 
this pair had an rs = 0.967 , other rs correlations were represented by total citesif rs = 0.729 , total cites snip and total citessjr rs = 0.686 , and the lower correlation was observed for total citescitescore with rs = 0.665 ; we had believed that citescore would depict the highest relationship as it is the more contemporary algorithour findings agree with a previous study in the neurology category that showed a moderate association between the citescore ; however , this study did not include pairwise comparisons of the r coefficients [ 8 ]  . 
one of the reasons for this finding could be related to the fact that citescore covers twice as many journals22 , 000 to the ifs 11 , 000and that its formula counts the total number of documents in the previous 3years [ 17 ]  . although if became the leader metric for consideration of tenure and promotion , and budget and resource planning in most of the universities , research institutions , and colleges [ 35 ] , contradictory opinions have stated that the if is not a perfect metric and has severe limitations [ 24 , 3638 ]  . a previous study [ 5 ] revealed the es was bibliometric that captured the best prediction of citations of a journal . 
our findings reveal that correlation values show the same behavior as the if : each medical speciality depicts different r threshold for significance [ 35 ]  . because es is a per - journal measure that represents each journal size , based on its total citations [ 39 ] , we considered the total cites as an additional variable in our analyses and graphs . 
however , we considered this interpretation misleading because each medical speciality should be analyzed independently and with a clear understanding of the correlation values among metrics for that selected category . 
the same study reported for the sjrif a negative r = 0.35 ; we found for the same variables an r = 0.884 , but the studies were performed in different categories of medical journals [ 6 ]  . 
 we did not include altmetrics in our comparisons , because they were out of the scope of this article . additional factors , such as longer time frames , the number of articles published in each issue , circulation of each journal , host of factors impacting citation ( self - citation , semi - mandatory , and mandatory citation ) might all influence citation calculations . 
we did not include these possible confounding factors , as the web of knowledge , scj , and scopus do not consider thealso , some factors that may affect author where to submit their work were outside the scope of this study : topic , affiliation with society , geography , rejection by earlier submission , familiarity with the submission and revision process , turnaround time , and invitation by editors . 
readers should be aware that the jcr contains approximately 171 categories in the sciences and 54 in the social sciences ; then , publication of future studies validating our model in other specialities would be desirable . in conclusion , bivariate correlations of bibliometric indices report a wide range of values from very strong to weak and even negative associations ; but these results vary depending on the selected categories . 
there is a growing trend to report on bibliometric analyses in particular disciplines looking for a better assessment of the significance and importance of scientific radiologists and other medical specialities ( nuclear medicine , interventional radiologists , and related fields ) should have the reference values of correlations among the essential bibliometric indices in their area ; these values may become a new standard of the quality of research . acknowledgements moises villaseor - almaraz , m.d. , was a research fellow at directorate of research , hospital general de mexico eduardo liceaga , during 2018 . 
univariate radiomic analysis was performed to find highly correlated radiomic features with imrt response , and a paired t test was used to find significant features between responders and non - responders . 
to find high predictive radiomic models , tenfold cross - validation as the criterion for feature selection and classification was applied on the pre - , postand delta imrt radiomic features , and area under the curve ( auc ) of receiver operating characteristics was calculated as model performance value . results of 33 patients , 15 patients ( 45% ) were found as responders . 
studies have indicated that intensity - modulated radiation therapy ( imrt ) has several benefits for prostate cancer patients in terms of both tumor control and normal tissue toxicity [ 1 ]  . 
 for example , the common marker , prostate - specific antigen ( psa ) , has some limitations including substantial differences in its accuracy , sensitivity , specificity and positive predictive value [ 3 ]  . 
on the other hand , prostate cancer diagnosis , staging and grading using some current methods suffer from several limitations such as invasiveness , low sensitivity and chance of patient infections [ 4 ] , and also , treatment effectiveness assessment using imaging is mostly based on morphological parameters and contrast media kinetics which are not best predictors of short - term response [ 5 ]  . as an advanced image quantification issue , radiomics may potentially serve as a new promising approach to reach precise personalized therapy in prostate cancer [ 6 , 7 ]  . 
 some previous studies have indicated that radiomic features extracted from different prostate mri scans including t2weighted ( t2w ) , dw , dynamic contrast - enhanced ( dce ) and adc could be the feasible biomarkers for prostate cancer detection / diagnosis [ 8 , 9 ] , aggressiveness [ 10 ] , staging and therapy response prediction [ 11 ]  . 
previous prostate radiomic studies also have revealed that there are good correlations among mr image features and gleason scores ( gs ) of prostate cancer zones , biochemical recurrence [ 12 ] and gene expression [ 13 ]  . regarding prostate cancer , on the other hand , a wealth of data has been published on the radiation therapy ( rt ) response evaluation using diffusion - weighted imaging ( dwi ) and identified that apparent diffusing coefficient ( adc ) obtained from dw images is a feasible noninvasive approach to assess rt response in different cancers [ 1417 ]  . 
 for prostate cancer , studies have found that adc is a favorable indicator to predict and evaluate response to different rt approaches including intensity - modulated radiation therapy ( imrt ) [ 18 ] , three - dimensional conformal rt ( 3dcrt ) [ 19 ] and ion therapy [ 20 ]  . in recent years , researchers have investigated a variety of machine learning ( ml ) approaches to build high accuracy , reliability and efficiency predictive and prognostic radiomic models in different cancers [ 2123 ]  . 
hence , some studies have suggested that it is essential to compare different ml models for radiomics - based clinical decision making [ 21 , 24 ]  . to the best of our knowledge , there is no report on prostate cancer imrt response prediction using radiomic approaches . 
also , we tested several ml methods for gleason scores and prostate cancer stage prediction . materials andmethods the general framework of this study is depicted in fig.1. this wok was conducted as a prospective research study , and all ethical issues relating to the patients are approved by the ethical committee . 
all procedures performed in studies involving human participants were in accordance with the 1964 helsinki declaration and its later amendments . patients andtherapy a total of 33 prostate cancer patients who were diagnosed and treated from april 2015 to june 2017 were included in this study . 
all imrt plans were generated using varian eclipse system . imaging all patients underwent two mri scans before and after treatment called pre - imrt and post - imrt scans , respectively . 
transverse t1 - weighted images were acquired as follows : repetition time / echo time ( msec ) , 563 / 14 ; section thickness , 3mm ; intersection gap , 2mm ; field of view , 2240cm ; and matrix , 256 224 . 
transverse , coronal and sagittal t2 - weighted were acquired as follows : repetition time / echo time ( msec ) 3000 / 101 ; section thickness , 3mm ; intersection gap , 2mm ; field of view , 1424cm ; and matrix , 265 205 . 
transverse dw sequences were acquired with a single - shot spin - echo echo - planar imaging sequence with single b value ( 1200s / mm2 ) ; section thickness , 3.5mm ; intersection gap , 2mm ; field of view , 1424cm ; matrix , 150 150 ; and with the same orientation and location used to acquire transverse 1 3 la radiologia medica ( 2019 ) 124 : 555567 data acquisition t2 weighted adc map image preprocessing 3 , 64 gray level image segmentation & response evaluation feature extraction classification model evaluation fig . 
1 flowchart of the present study intensity shape texture ( glrlm , glcm , glszm , ngldm , gldm ) feature selection select from model ( sm ) select k best ( sb ) select percentile ( sp ) variance threshold ( vt ) adaptive boost ( ab ) decision tree ( dt ) linear support vector machine ( lsvm ) gaussian nave bayesian ( gnb ) k - nearest neighborhood ( knn ) gaussian nave bayesian ( gnb ) k - nearest neighborhood ( knn ) logistic regression ( lreg ) bernoulli nave bayesian ( bnb ) t2 - weighted images . 
preand post - imrt scan protocols including t1w , t2w and dw images for all patients were same . response evaluation in the present study , prostate cancer response was assessed based on the changes of adc values before and after imrt . 
in this situation , roi was drawn in the same area as that initially used in the pre - therapy , based on the anatomical markers and was also described by previous studies [ 25 , 26 ]  . 
the percentage changes ( pc ) of adc values 1 3 558 la radiologia medica ( 2019 ) 124 : 555567 pre - post - imrt were considered as imrt response ( ( adcpost - imrtadcpre - imrt / adcpre - imrt ) 100 )  . 
this therapy response evaluation was obtained from previous studies [ 27 , 28 ]  . preprocessing before feature extraction and in order to normalize the intensities across all the patients , to reduce noise and to increase sensitivity , image intensities were normalized between 3 , where is the mean value of gray levels inside the region of interest ( roi ) and is the standard deviation . 
in this issue , 3 limits image intensities in the range [ 3 , + 3 ] and removes the dependency on the shift of the mean value and on multiplicative change in the image intensity and also minimizes intraand interscanner effects in mri texture analysis . 
also , all images were quantized to 64 gray - level intensities for noise reduction and image uniformity . tumor segmentation in this study , t2w and adc images were used for radiomic analysis . 
we also obtained delta imrt features by relative changes of pre - / post - imrt features : ( features post - imrtfeatures pre - imrt / features pre - imrt )  . feature selection because many of features extracted from images are simply noise or highly correlated with each other , they could enhance the chance of overfitting , decreasing prediction accuracy , maximizing the computational cost . 
in this regard , feature selection is a critical issue in radiomic analysis . in this study , we used four feature selection methods : selectkbest_chi2 ( sk ) , variance threshold ( vt ) , select from model ( sm ) and select percentile ( sp )  . 
in sp method , an anova test is used with percentile of 20 , 40 , 60 and 80% . univariate analysis in this study , our main focus was on the imrt response prediction . 
our univariate analysis has two main steps : ( 1 ) highly correlated radiomic features with imrt response were selected among four groups of feature selection and ( 2 ) a paired t - test was performed to find significant features among responder and non - responders . 
 box plots were made to compare selected features among responder and non - responder groups . classification the objective of this study was to stratify patients into two labeled classes using different ml classifiers . 
this issue also was repeated to classify patients with stages t2 and t3 ( t2 patients were labeled as 1 , and t3 patients were labeled as 0 ) and patients with gs 7 and gs > 7 ( t 2 patients with gs 7 were labeled as 1 , and patients with gs > 7 were labeled as 0 )  . a total of nine classification methods based on ml approaches were used in the analysis : linear support vector machine ( lsvm ) , logistic regression ( lreg ) , bernoulli naive bayes ( benb ) , stochastic gradient descent ( sgd ) , k - nearest neighbors ( knn ) , decision tree ( dt ) , random forest ( rf ) , adaptive boosting ( adbo ) and gaussian naive bayes ( ganb )  . 
in balance method , we have equal data from each class ( binary classification ) , whereas in unbalance , there are no equal data and data are selected randomly . all information regarding all ml algorithms for feature selection and classification is summarized in supplementary table2 . statistical analysis all analyses regarding the feature selection and classification were performed using in house - developed python codes . 
 1 3 la radiologia medica ( 2019 ) 124 : 555567 in order to find best predictive models for these classifications , we tested different combinations of feature selection and classification methods . 
classifiers were trained using the tenfold cross - validation method , and their predictive performance was then evaluated using the area under the receiver operator characteristic ( roc ) curve ( auc )  . 
the predictive performance of all models was compared based on the mean aucs . results patients andimrt response thirty - three prostate cancer patients with median age of 73years ( 5182 ) were studied . 
the box plots comparing selected radiomic features are shown in fig.2 ( a for t2w and b for adc )  . imrt response prediction models for imrt response prediction , several radiomic models based on the image features , classification and feature selection methods were obtained . 
the models performance ( auc ) as cross - combination of feature selection and classification approaches for pre - imrt t2 w and adc image features is depicted in fig.3 ( a for t2w and b for adc )  . 
in comparison with other methods , e.g. , psa , quantified radiomic features may be more feasible , noninvasive and easy to use . in our predictive modeling , in addition to pre - treatment radiomic features , we also analyzed features extracted from post - treatment mr images for response predicting in prostate cancer patients . 
because the main aim of the radiomic analysis is to assess intra - tumor heterogeneity , post - treatment radiomic analysis will show the impact of therapy on this heterogeneity . 
on the other hand , after the treatment , the tumor status underwent several structural changes and therefore radiomic feature changes , which will be interesting to understand how a therapy modality acts . 
because response monitoring in prostate cancer patients is based on the several months to years follow - up , our radiomic models may be useful for tailor the therapy and individualized radiotherapy . 
several studies have applied radiomic analysis on post - treatment images and indicated that these issues have the potentials for several clinical decision makings such as adaptive radiotherapy , new therapy prescription and late response prediction [ 3032 ]  . 
they found that most of the post - rt features were significant with the pet response , whereas clinical stage was not associated with the response . for delta radiomics , to our knowledge , no published reports have investigated the possibility of using delta radiomic features extracted from mr images for predicting response in prostate cancer patients . 
 several investigations have mentioned the utility of delta radiomics for prognosis predicting in cancers including liver metastases in colorectal cancer [ 34 ] , metastatic renal cell cancer [ 35 ] , esophageal cancer which would develop radiation pneumonitis [ 36 ] and non - small cell lung cancer [ 37 ]  . 
in some previous studies , delta radiomic model had found more performance which differs from our results . in this study , we observed that several pre - treatment radiomic features are highly correlated with imrt response . 
2 ( continued ) cluster shade are good measures of tumor homogeneity , heterogeneity , smoothness , randomness and symmetry which finally determine how a specific tumor respond to a specific therapy . 
our results are in concordance with some previous studies [ 3840 ]  . as was discussed by some previous studies , for the successful realization of predictive or prognostic radiomic modeling , different feature selection and classification methods have to be evaluated and compared . 
there are few studies which compared different machine learning approaches as feature selection and classification methods for developing radiomic models in non - small cell lung cancer ( survival prediction ) [ 41 ] , lung cancer ( survival prediction ) [ 21 ] and head and neck squamous cell carcinoma ( survival prediction ) [ 42 ]  . 
3 a heatmap depicting the predictive performance ( auc ) of feature selection ( in rows ) and classification ( in columns ) methods of pre - imrt t2w models . 
4 box plot comparing of the predictive performance ( mean auc ) of all the radiomic models 1 3 la radiologia medica ( 2019 ) 124 : 555567 564 fig . 
5 a heatmap depicting the predictive performance ( auc ) of classification ( in rows ) and all the image sets ( in columns ) methods of pre - imrt t2w models . 
in regard to correlation between gleason scores and radiomic features , although it is not new and have been reported by some previous studies , we performed this investigation by using new machine learning approaches , and for classification of gs 7 and gs > 7 , fehr etal . 
they used generalized linear regression and generalized estimating equations methods to differentiate between gleason scores of 3 + 3 = 6 versus ( 3 + 4 = 7 or 4 + 3 = 7 ) , 3 + 3 = 6 ver sus > 7 and 3 + 4 versus > 3 + 4 using mri - derived texture parameters [ 44 ]  . 
it could be a new noninvasive , simple and easy method for cancer staging without any patient discomforts . although our results are significant , this study has several limitations : first , small number of patients . 
whole liver dose , tumor dose and healthy injected liver dose , lung dose and if applicable the gallbladder dose were all calculated by using the medical internal radiation dose ( mird ) formula from spectct images . 
there were no detected grade 2 or 3 adverse events . conclusion tare is safely performed without cystic artery embolization when its origin is close to the treatment area . keywords cystic artery embolization transarterial radioembolization y90 introduction transarterial radioembolization ( tare ) with yttrium - 90 ( y90 ) , also known as selective internal radiotherapy ( sirt ) , has been increasingly used as a locoregional therapy option for primary and secondary malignant hepatic tumors [ 14 ]  . 
the most feared complication is nontarget delivery of y90 - labeled microspheres into the surrounding tissues through the hepatico - enteric arteries ( cystic artery , right gastric artery , gastroduodenal artery and hepatic falciform artery ) resulting in gastrointestinal ulceration or radiation cholecystitis [ 68 ]  . 
radiation cholecystitis resulting from the nontarget delivery of microspheres through the cystic artery is generally a subclinical , self - limiting process and rarely requires surgery [ 9 , 10 ]  . 
however , gallbladder involvement after the pretreatment angiography , with technetium - 99m - labeled macroaggregated albumin ( 99mtc - maa ) , is not rare and could be detected in 812% of the patients [ 1113 ]  . 
however , pre - treatment coil embolization does not always preclude nontarget embolization due vol . : ( 0123456789 ) 1 3 576 la radiologia medica ( 2019 ) 124 : 575580 to recanalization of the occluded artery and / or development of collaterals [ 1417 ]  . 
in addition , coil embolization of the cystic artery itself , although occasionally observed , can cause ischemic cholecystitis which results in even more serious complications [ 10 ]  . 
some other groups do not routinely perform coil embolization [ 17 ] and position the microcatheter distal to the hepatico - enteric artery origin when possible or alternatively they may induce vasospasm or temporary embolization during the y90 treatment when distal positioning is not possible [ 18 ]  . 
the debates about how to manage the cystic artery during tare are continuing and have not yet arrived at a definitive conclusion [ 5 , 18 ]  . in the present study , we aimed to assess radiation - induced cholecystitis in cases of cystic artery origin nearby the treatment zone for tare treatment . materials andmethods patient population local institutional review board approved this retrospective single - center study . 
all of the hepatic angiograms of the patients with primary or secondary malignant liver tumors treated with tare were extracted from the radiology database between january 2014 and january 2018 , and patients in whom cystic artery origin was located in the surrounding area of the treatment zone on 99mtc - maa angiograms were included in this study . ct angiography of the liver and blood tests including liver functions were obtained from all patients before the procedure . 
all of the patients in this cohort were deemed suitable for tare by the multidisciplinary tumor board of the hospital . pre - treatment evaluation hepatic angiography obtained with delivery of 99mtc - maa into the target hepatic arteries was performed in all patients , and single - photon emission computed tomography ( spectct ) or fusion of spect and ct images was obtained . patients with cholecystectomy , non - enhanced or embolized cystic arteries or microcatheter position distal to the cystic artery on pre - treatment evaluation angiograms with 99mtc - maa were excluded . assessment ofgallbladder withspectct the gallbladder was assessed for 99mtc - maa involvement both qualitatively by visual evaluation and quantitatively by calculating the radiation doses from the spectct images . 
whole liver dose , tumor dose and healthy injected liver dose , lung dose and if applicable the gallbladder dose were all calculated by using the medical internal radiation dose ( mird ) formula ( 20 )  . 
only the wall of the gallbladder was used for dose calculation , and the gallbladder lumen was excluded . procedure all procedures were performed via standard right femoral approach with a 4f or 5f short vascular sheath under local anesthesia . 
microcatheters with a distal luminal size of 2.8f ( progreat , terumo , somerset , new jersey , usa , or embocath , biosphere medical , rockland , ma ) were used for right hepatic artery selective catheterization . 
in all patients , the cystic artery was visualized , and when feasible , the cystic artery origin was bypassed and the microcatheter was placed distal to the cystic artery origifbypass was not technically possible , 99mtc - maa infusion was performed from the placed microcatheter and a decision to proceed with y90 treatment was made based on spectct findings . 
if it was decided to proceed with y90 treatment , on the day of the tare ( 715days after the pre - treatment session ) , all of the angiographic devices were selected same as with the pretreatment 99mtc - maa angiography in order to imitate the same conditions . 
temporary or permanent coil embolization of the cystic artery was not felt necessary in this cohort , neither was preferred an anti - reflux infusion system , nor provocation of vasospasm within the cystic artery . 
all procedures were performed by two interventional radiologists with > 10years and 5years of individual experience in embolization procedures . followup clinical assessment with laboratory workup ( including liver function tests ) was repeated on the first day and at 1month after the treatment . 
spectct images show radioactivity in whole gallbladder wall ( arrows ) which was scored as moderatesevere involvement failure ( increasing ascites , icterus and elevated laboratory test results ) , liver abscess , intrahepatic bilomas and liver infarction were also noted . with ultrasound ( us ) , dynamic computed tomography ( ct ) and / or position emission tomography ( pet ) and / or dynamic liver magnetic resonance imaging ( mri ) at 13months following treatment and at 36 - month intervals thereafter . 
the observed adverse events regarding both gallbladder problems ( cholecystitis , right upper quadrant pain , etc . ) and others were classified according to the national cancer institutes common terminology criteria for adverse events ( ctcae v5.0 ) [ 20 ]  . 
a total of 34 tare procedures from 29 patients ( 18 men and 11 women ) , with a mean age of 65 13.3years meeting the inclusion criteria , were involved in the current study . 
increasing ascites , icterus , elevated laboratory test results , liver abscess , intrahepatic bilomas or liver infarction was not encountered during follow - up visits . discussion the current study demonstrated that without taking a precaution for the cystic artery , y90 - labeled microspheres might be safely delivered into the target hepatic artery when cystic artery origin was located in the close proximity ( less than 8 - 10mm ) of the tare treatment zone . 
however , administration of y90 microspheres into the cystic artery has always been avoided whenever possible in our unit , and the cystic artery origin was always bypassed with the microcatheter which was placed distal to the cystic artery orighowever , it became complicated when the bypass or la radiologia medica ( 2019 ) 124 : 575580 coil embolization was technically impossible , and the cystic artery was retained in the treatment zone . 
in those group of patients , if there is no moderatesevere maa uptake ( none or mild ) on spectct , we proceed with tare , whereas if there is moderatesevere maa uptake on spectct , tare is postponed until cholecystectomy . 
nevertheless , as a result of not very uncommon anatomical variations and technical infeasibility of super - selective catheterization of the cystic artery , in some cases it might be impossible to refrain from the delivery of some radioactive - labeled spheres into the cystic artery [ 21 , 22 ]  . 
we know from previous studies that the proximal microcatheter position might not always cause radiation - induced cholecystitis ( ric ) , and pathological evaluation of some hepatectomy cases showed multiple microspheres within the gallbladder wall in totally asymptomatic patients [ 23 ]  . 
 [ 18 ] that the microcatheter could be repositioned to change the flow and diminish the gallbladder perfusion , and therefore , cystic artery might be safely included [ 18 ]  . 
 [ 18 ] also reported different options to cope with cystic artery to prevent ric , despite the fact that they noted proximal microcatheter position did not always cause ric . 
moreover , although the cystic artery was located within the treatment zone , we observed even no radioactivity in the gallbladder wall on spectct images in 23 procedures in this series . 
 [ 24 ] reported three patients with detected radioactivity in the gallbladder wall without any signs or symptoms of ric during the follow - up and they did not use any precaution for the cystic artery before the procedure . 
within the quality improvement guidelines , it was also noted that radioactivity detected in the gallbladder wall on spectct is not uncommon and not always resulted in ric clinically or radiologically , and if any ric is diagnosed , it does not always need surgical treatment [ 25 ]  . in a comparison study of cystic artery embolization , powerski etal . 
 at our institution , we prefer distal microcatheter position whenever possible or feasible , or to leave the cystic artery within the tare treatment zone because both embolization and delivery of y90 - labeled microspheres may result in cholecystitis [ 10 , 19 ]  . 
pre - treatment coil embolization does not always preclude nontarget embolization due to 1 3 la radiologia medica ( 2019 ) 124 : 575580 recanalization of the occluded artery and / or development of collaterals [ 1417 , 27 ]  . 
if there is a significant gallbladder radioactivity on spectct images , we perform a proximal coil embolization of the cystic artery before tare . spectct is useful for gallbladder assessment before tare , and a risk of cholecystitis could be assigned by measuring the radioactivity in the wall of the gallbladder [ 25 ]  . 
however , the detected radioactivity in the gallbladder wall may not always mean an increased risk of ric because it was not the radioactivity itself , but the concentration or amount of it in the gallbladder wall on spectct , which enabled us to make a reliable sirt without a complication of ric . 
and , nine out of 11 patients were totally symptoms free or negative for any changes in laboratory tests . several alternatives were described to eliminate the risk of ric during hepatic tare procedure : ( 1 ) permanent or temporary embolization of the cystic artery with coils or gelfoam [ 26 , 27 ] , ( 2 ) positioning the microcatheter distal to the cystic artery [ 6 , 11 ] , ( 3 ) causing vasospasm with a guidewire [ 18 ] or ( 4 ) utilization of an anti - reflux infusion system [ 30 ] , ( 5 ) antibiotic usage [ 24 ] and repositioning of the microcatheter to change the flow direction [ 18 , 28 ] or ( 6 ) delivery of separate doses for right anterior and right posterior hepatic arteries [ 19 ] were all previously reported . our findings might only hold true for tare with glass particles . 
it is well known that because of the mildly larger size and larger number of particles , resin microspheres have more embolic effect over glass microspheres and this critical point may create a tendency to cholecystitis [ 31 ]  . 
in one case , there was a left kidney in left iliac fossa . conclusion although rare and probably overlooked , a plras can be encountered in patients with situs viscerum inversus or presenting a left - sided ivc with hemiazygos continuation . 
these vessels can cause technical problems during surgery at the left renal hilum and should be specifically searched for in patients with vascular anomalies . keywords renal vessels anomalies ivc hemiazygos continuation of ivc left - sided precaval renal artery * lorenzo e . 
gemelli , rome , italy 4 diagnostica perimmagini ospedaliera , ospedale evangelico internazionale , presidio ospedaliero di voltri , genoa , italy 5 department ofradiology , univ med pharm iuliu hatieganu , cluj - napoca , romania 6 department ofradiology , faculty ofmedicine , dokuz eylul university , izmir , turkey 7 department ofhealth sciences ( dissal ) , radiology section , university ofgenoa , radiologia durgenza , ospedale policlinico san martino ist , largo r . 
benzi , 10 , 16132genoa , italy introduction knowledge of the number , course and relationships of the renal arteries is of great importance for correct planning of surgical procedures to kidney , aorta and retroperitoneal region . 
 both anatomical and radiologic studies report the presence of vascular anomalies in about 30% of examined subjects [ 1 ]  . the right renal artery takes origin from the aorta and crosses the midline passing posteriorly to the inferior vena cava ( ivc )  . 
anomalous right renal arteries with precaval course ( prras ) can be encountered , and their presence has been associated with renal rotational anomalies and pelvic dilatation [ 2 , 3 ]  . to the best of our knowledge , there are no reports of left renal arteries with a precaval course ( plras )  . 
only one case with a complex vascular anomaly ( common renal arterial trunk originating from the aorta with a recurrent precaval and preaortic left renal artery ) has been described in vol . : ( 0123456789 ) 1 3 la radiologia medica ( 2019 ) 124 : 445449 446 fig . 
there is a left ivc ( open arrow ) with hemiazygos continuation which passes behind the left diaphragmatic crus ( c , d ) , gives a retroaortic branch to the azygos vein ( e ) and continues cranially as a small accessory hemiazygos ( curved arrow in f )  . 
b oblique reconstruction along the course of the left and lower right renal arteries shows the left ( open arrow ) and right ( arrowhead ) ivcs , as well as the two ( right and left ) renal arteries , both with a precaval course . 
we describe herein the imaging findings observed in eight patients in whom a plra was observed . materials andmethods we reviewed the teaching files of six radiologic departments for patients with lpras . 
 this study was approved , and the need for informed consent was waived by the ethics committee of the institution of the corresponding author . results all patients have been examined for problems not related to the vascular anomalies that are the object of the present study . 
 two patients had undergone evaluation of the chest ( one with unenhanced ct and one with mr ) , and the left - sided ivc and the plra were visible in the most caudal parts of the studies ; only selected images were available for review in these as well as in three other patients . 
 ( the patient had undergone unenhanced ct of the chest only . ) the remaining patient had situs viscerum inversus ; he had absent infrarenal right ivc , and the renal vein from the right kidney continued cranially into a vein interpreted as a right - sided hemiazygos . 
the hemiazygos passed between the left diaphragmatic crus and the vertebral bodies , gave a branch to the azygos vein , passing posteriorly to the aorta and then 1 3 case # ivc right renal artery left renal artery renal veins right kidney left kidney table 1 summary of imaging findings hemiazygos continuation of left ivc la radiologia medica ( 2019 ) 124 : 445449 non - visible normal normal plra plra plra non - visible normal normal normal normal normal retroaortic right normal normal renal vein main from iliac normal normal main artery normal ; accessory prra to lower pole artery ; accessory plra ectopic , left lower quadrant , with anterior malrotation normal normal normal plra plra plra normal normal normal normal normal normal normal left normal right to azygos left normal right to rightsided hemiazygos 448 non - visible double , right smaller left only double , right interrupted , continuing into hepatic collaterals . 
however , most left - sided inferior ivcs , at the level of the renal hilum , do not continue cranially , but flow into the left renal vein , which crosses the midline and then ascends as a right - sided ivc with a normal course to the right of the aorta . 
the same happens also in most patients with double ivc : the left one , again , flows into the left renal vein to join into a single ivc on the right side [ 5 ]  . 
embryologically , this is considered as due to persistence of the left supracardinal vein these cases , in fact , the left ivc turns backwards , passes between the left diaphragmatic crus and the lateral aspect of the vertebral bodies and ascends into the thorax . 
then , the left renal artery seems to run in a normal way , and it is the ivc that takes a course posterior to the artery . the findings observed in the patient with an ectopic left kidney and in the one with situs viscerum inversus are somewhat different and , possibly , the developmental anomalies causing the plra are also different . as regards the first case , it is known that ectopic kidneys have a higher number of associated vascular anomalies and that it is the final position of the kidney to determine the position and number of renal arteries . 
 we did not encounter a patient with combined presence of a left ivc without hemiazygos continuation and an ectopic kidney in the left lower inferior abdominal quadrant ; however , we believe that a plra could be possible also in this case . as regards the second one , it must be remembered that prras are considered the result of a persistent caudal vessel arising ventrally from the aorta after formation of the normal ivc [ 3 ]  . 
the same mechanism , but in the presence of a left - sided ivc due to situs viscerum inversus , could possibly explain the anomalous relationship between the two vessels . to the best of our knowledge , no attention has been paid to the relationship of the left renal artery with the ivc in the reported patients with a left - sided ivc and hemiazygos continuation [ 610 ]  . 
however , in one of the illustrations in a paper on multidetector ct of the ivc by sheith and fishman [ 11 ] , this anomaly seems present , but has not been described either in the caption or in the text . 
 [ 12 ] reported on a patient with left - sided ivc with hemiazygos continuation who underwent surgery for a nutcracker phenomenon affecting the right renal vethe paper shows a drawing illustrating the relationships among the vessels at the renal hilum in which the left renal artery is shown posteriorly to the ivc , but in the description of the surgical intervention , no special mention to this relationship was given . there are some limitations to our study . 
although a plra was recognized in all of our cases , we cannot exclude other relationships between the renal vessels and the left - sided ivc with hemiazygos continuation or other associated anomalies in a larger series of patients . to conclude , although rare and probably overlooked until now , a plra can be encountered in patients with situs viscerum inversus or presenting a left - sided ivc with hemiazygos continuation . 
it can be an additional cause of technical problems during retroperitoneal surgery at the left renal hilum and should be specifically searched for in patients with these vascular anomalies . funding this study has received no funding . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical standards this article does not contain any studies with human participants or animals performed by any of the authors . informed consent this study was approved , and the need for informed consent was waived by the ethics committee of the institution of the corresponding author ( 335reg2014 )  . la radiologia medica ( 2019 ) 124 : 568574 ultrasonography clinical value ofhighresolution ( 517mhz ) echocolor doppler ( ecd ) foridentifying filling materials andassessment ofdamage orcomplications inaesthetic medicine / surgery mariascottodisantolo1 candidamassimo1 giovannitortora2 valeriaromeo1 micheleamitrano1 arturobrunetti1 massimoimbriaco1 received : 4 august 2017 / accepted : 5 december 2018 / published online : 5 january 2019 italian society of medical radiology 2019 abstract purpose the purpose of this retrospective study is to evaluate the role of echo - color - doppler ( ecd ) imaging in identifying a series of characteristics pursuant to aesthetic filling material such as their degree of absorbability and their potential complications which include their propensity to stimulate the formation of encapsulated foreign - body granulomas . 
in the latter case , ecd can be of aid by giving indication for surgical therapy . materials and methods over a 4 - year period , we studied 180 patients ( 60 ) who underwent an aesthetic medical / surgical treatment . 
we used ecd to evaluate the implant material , its thickness , the injection site , the integrity of dermal layers and the presence of any associated complications . results in 97% ( 174 / 180 ) of our patients , we were able to identify the type of material used ; furthermore , 57% of patients had a hyaluronic acid implant , 14% a lipofilling and 29% a non - absorbable filler ( with 10% of silicone )  . 
in 6 / 180 ( 3% ) , we could not recognize the material used ; 89% ( 161 / 180 ) of our patients presented post - injection complications ; moreover , 67% showed peri - implant dermalhypodermal thickening areas with adjacent lymphostasis , 6% displayed an abnormal implant site , and 17% showed inflammation with encapsulated foreign - body granulomas that required subsequent surgical excision . 
 biopsy samples were obtained from 37 / 180 patients ( 21% ) ; among these , 31 patients had an ecd evidence of granuloma and on 6 patients we were not able to define the injected material . 
bianchi , naples , italy vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 568574 introduction high - resolution ( 517mhz ) color - doppler ultrasound ( ecd ) is widely used in the evaluation of normal and pathological skin [ 19 ] ( figs.1a , b , 2 ) ; it plays an important role in aesthetic medicine / surgery , in the filling technique , in identifying filling materials and in the assessment of subsequent damage or complications [ 1020 ] ( table1 )  . 
 in the study of superficial organs and in particular in the dermatological field , the depth of the region to be investigated is 12cm and pathological skin lesions are often less then 1mm thick ; as a result , the use of high - frequency / high - resolution / reduced scan depth probes is crucial [ 1 , 2 ]  . 
the lesion is hyper vascular with regular margins there are several types of fillers : absorbable , non - absorbable , synthetic and autologous adipose tissue in the lipofilling technique . among the absorbable fillers , the most used is hyaluronic acid ( pure or mixed with lidocaine ) which is a glycosaminoglycan component of the dermis consisting of long , unbranched chains of disaccharide units . 
the mixed formulation ( hyaluronic acid and lidocaine ) appears as a circumscribed hypoechoic areolae with regular margins localized in the dermis or subcutaneous adipose tissue or as pseudocysts with inner echoes ( debris ) and septa [ 11 , 13 , 1517 ] ( table1 )  . 
polymethylmethacrylate appears as hyperechoic dots with a small comet tail artifact , and calcium hydroxyapatite appears as hyperechoic bands with a posterior acoustic shadowing artifact ; the latter artifact is the one classically described in calcified structures [ 1113 , 1517 ] ( table1 )  . polyacrylamide gel , another synthetic filler , appears as mostly oval anechoic pseudocysts with increased echogenicity of the surrounding subcutaneous tissue [ 1113 , 1517 ] ( table 1 ) ; its deposits have been reported to not change in size at least for 1year [ 1117 ] ( table1 )  . the most well - known non - absorbable filler is silicone ( pure or in oily formulations ) whose medical aesthetic use was forbidden in the usa in 1991 and in italy in 1992 because of the high incidence of complications [ 11 , 13 , 15 , 16 ] ( table1 )  . 
silicone echo - genicity depends on the formulation type ; pure silicone is anechoic , similar to intact breast implants , while silicone oil is hyperechoic , with posterior reverberation / scattering artifact ( snow - storm appearance ) , similar to ruptured silicone implants when pure silicone is fig . 
1 a longitudinal scan of healthy skin on the dorsal region of the arm in a 36 - year - old male with mild photo aging ; sonographic representation of all the layers of the dermis , epidermis and subcutaneous adipose tissue . 
b longitudinal scan of the healthy nail bed in a 30 - year - old woman patient 1 3 570 la radiologia medica ( 2019 ) 124 : 568574 table 1 filler materials characteristics observed on ecd hyaluronic acid glycosaminoglycan component of the dermis consisting anechoic pseudocysts that decrease in size over 3 to of long , unbranched chains of disaccharide units 6months in pure formulation pseudocysts with inner echoes ( debris ) and septa in mixed formulation hyaluronic acid and lidocaine collagen bovine glycosaminoglycan component of the dermis consisting anechoic pseudocysts that decrease in size over 3 to of long , unbranched chains of disaccharide units 6months poly - l - lactic acid synthetic , biodegradable , biocompatible , immunologianechoic pseudocysts that decrease in size over 3 to cally inert peptide polymer 6months silicone highly purified long - chain polydimethylsiloxane pure silicone implant appeared anechoic , similar to echotrimethylsiloxy - terminated silicone oil genicity described in intact breast implants calcium hydroxyapatite synthetic filler , semi - permanent inorganic material hyperechoic bands with a posterior acoustic shadowing polymethylmethacrylate synthetic filler , semi - permanent inorganic material lipofilling grafting autologous adipose tissue silicone oil was hyperechoic , showing a posterior reverberation or scattering artifact , similar to the one seen in ruptured silicone implants when the pure silicone is mixed with the fatty tissue of the breast artifact hyperechoic dots with a mini comet tail artifact area of hyper echo - genicity with regular margins corresponding to the implanted adipose tissue mixed with the fatty breast tissue [ 1113 ] ( table1 )  . 
finally , the lipofilling technique consists in grafting autologous adipose tissue to correct soft tissue volumetric defects secondary to trauma , cancer excisions , congenital defects and aging [ 2 , 12 ] ( table1 )  . 
among the most frequent complications is the foreign - body reaction ( initial inflammation and subsequent granuloma formation ) , whose recognition is crucial to direct the patient to surgery . 
less common adverse reactions include fistulous tracts of fluid collections , skin necrosis due to intravascular filler injection and secondary capillary or larger - vessel thrombosis [ 1820 ]  . 
the aim of this study is to evaluate the role of ecd in identifying different types of filling material , their possible complications and the accuracy of ecd in diagnosing and indicating treatment for possible subsequent foreign - body granulomas . materials andmethods our retrospective study was performed on material and data available in our department in accordance to the 1964 declaration of helsinki ethical standards ( and its subsequent amendments )  . 
exclusion criteria were : pregnancy or breast - feeding , general contraindication to surgical or aesthetical procedures and psychological problems . over a 4 - year period ( january 2012september 2015 ) , 180 patients ( 60 ) aged between 20 and 76years were evaluated with ecd after the aesthetic treatment to identify filler materials , to evaluate the possible resulting damage and post - op complications , especially the onset of persistent ecchymosis and nodules . 
ecd was performed by a 16 - year experienced operator ( msds ) on the affected skin region using a high - frequency transducer ( 517mhz ) and a philips iu22 ultrasound device ( best , the netherlands )  . 
the echostructure and echo - genicity of the dysmorphic region was studied in order to evaluate the implant type ( table1 ) , its thickness , the site of implantation , dermal layer integrity , the presence of a newly formed granuloma ( capsulated or not ) , vascularization and inflammation ( using slow - flows ecd settings )  . 107 / 180 patients showed the presence of filler within the nasolabial folds , 21 / 180 patients showed filler lips , 50 / 180 cheekbones filler , 1 patient showed filler at the level of a sternal scar and 1 patient into the arms ( table2 )  . 
ecd foreign - body granuloma diagnosis was based on the visualization of an inhomogeneous hypoechoic collection with some internal echoes with or without a hyperechoic peripheral rim ( capsule ) exhibiting a rich vascular supply on color - doppler 1 3 la radiologia medica ( 2019 ) 124 : 568574 table 2 filler materials characteristics observed on ecd , in 180 patients studied after filler treatment , injection sites number of patients filling material identified sonographically injection sites hyaluronic acid lipofilling calcium hydroxyapatite polymethylmethacrylate silicone material unidentified clearly nasolabial folds ( 75 ) lips ( 14 ) cheekbones ( 10 ) nose ( 1 ) nasolabial folds ( 19 ) cheekbones ( 3 ) lips ( 1 ) sternum ( 1 ) cheekbones ( 25 ) nasolabial folds ( 6 ) nasolabial folds ( 2 ) cheekbones ( 12 ) lips ( 5 ) nasolabial folds ( 5 ) lips ( 1 ) fig . 
4 ultrasound examination obtained in the transverse view at the level of skin region affected by dimorphism ( sternum ) and at the adjacent region , showing measurement of the autologous fat implant fig . 
3 longitudinal scan of nasolabial groove in a 40 - year - old woman shows hyaluronic acid implant 10 days after treatment , well located and without ultrasonographic evidence of complications examinations . 
 the vascular signal was evaluated at the injection site using colorand power - doppler functions set to slow speed blood flow ( color doppler : prf 500hz , wall filter 27hz , gain 75% , persistent media ; power doppler : prf 350hz , wall filter 45hz , gain 85% , low optimization ) [ 1 , 2 ] , therefore providing a flow qualitative analysis as well as the peak systolic velocity [ 1 , 2 ]  . results ecd was able to identify the type of filler material used in medical treatments and cosmetic surgery according to data already present in the literature ( table1 )  . 
5 longitudinal scan of biceps in a 43 - year - old man shows ultrasonographic evidence of diffuse and large area of ultrasound beam attenuation as traditional silicone implant patients ( 97% ) , we identified the type of material used ( table2 )  . 
 50 / 174 ( 29% ) patients had a non - absorbable filler implant ( tables1 and 2 ) primarily calcium hydroxyapatite ( 36 / 50 , 72% ) which appeared as hyperechoic bands with a posterior acoustic shadowing artifact ( table1 and 2 )  . 
14 / 50 ( 28% ) patients had another type of non - absorbable filler , polymethylmethacrylate , which appeared as hyperechoic dots with a mini comet tail artifact ( table1 and 2 )  . 
in 20 / 180 ( 11% ) patients , ecd showed previous undeclared implants , 5 ( 3% ) 1 3 572 table 3 complications observed on ecd in 161 / 180 patients studied after filler treatment la radiologia medica ( 2019 ) 124 : 568574 number of complications type of complications locations lymphostasis abnormal implant site hyaluronic acid granuloma polymethylmethacrylate granuloma hydroxyapatite granuloma nasolabial folds ( 108 ) lips ( 5 ) cheekbones ( 7 ) suprafascial cheekbones ( 10 ) nasolabial folds ( 11 ) lips ( 2 ) nasolabial folds ( 6 ) nasolabial folds ( 5 ) cheekbones ( 2 ) previous undeclared silicone implant granuloma lips ( 5 ) fig . 
transverse view showing the inflammatory reaction after filler treatment of these were silicone implants with a foreign - body granuloma allowing to avoid a second aesthetic treatment and preventing possible medical legal issues ( fig.5 ; tables2 and 3 )  . 
in 6 / 180 patients ( 3 , 3% ) , we could not recognize the material used because of the intense adjacent inflammation and because of the evidence of repeated different absorbable filler treatments ( table3 )  . 
6 a longitudinal scan of zygomatic region in a 42 - year - old woman who complained of pain and swelling , 1month after the aesthetic treatment ; abnormal implant site ( suprafascial cheekbones )  . 
biopsy samples were obtained from 37 patients , 31 ( 17% ) with us evidence of encapsulated foreign - body granulomas and 6 in which ecd was not able to successfully define the injected material . 
 among these , 5 specimens that were identified sonographically as silicone granulomas showed empty vacuoles surrounded by fibrous tissue , giant cells with cytoplasmic phagocytosed silicone particles and inflammatory cells . 
as for the 6 patients in which ecd could not define filler material , histology determined the presence of intense chronic inflammation with macrophages , giant cells ( fig.7 ) ; 3 / 6 ( 50% ) also showed cystic granulomas including hyaluronic acid . 
 a comparison between histological results and ultrasound diagnosis is shown in table4 . discussion the results of this study show that ecd can identify different filling materials and possible post - surgical complications ; in addition , when performed by an expert sonographer and with the state of the art technique , ecd shows a 78% diagnostic accuracy in identifying the presence of foreign - body encapsulated granulomas , directing the patient to the most appropriate surgical procedure . 
the use of us in the dermatological field is strongly tied to a series of innovations : the availability of high - frequency transducers ( > 15mhz ) , the introduction of techniques that reduce artifacts while optimizing resolution power and the possibility of studying both the vascularization of expansive skin lesions and angiogenesis with the color - doppler function [ 1 , 2 , 4 , 5 , 9 , 11 , 1317 ]  . 
the massive recourse to medicine and cosmetic surgery in a perpetual pursuit of beauty has increased the number of legal medical practices and , as a consequence , also the number of medical errors and complications related to the type of equipment used . 
a skin ultrasound , performed by doctors with sonography and cosmetic medicine experience , allows for the recognition the type of material used , the exclusion of the presence of silicone , of great importance for subsequent medical legal issues and the evaluation of possible complications in time . 
this will provide both qualitative and quantitative data ( volumetric follow up of implant material dimension through repeated us ) regarding the successful resolution of a potential problem [ 11 , 13 , 1520 ]  . 
sonographically , the granuloma is not easy recognized ; it can either have a thick hypoechoic rim or , if fibrotic phenomena prevail , a hyperechoic rim that shows vascular signal at color and power doppler especially if there is associated inflammation . 
in our study , us was also helpful in patients with foreign - body granulomas caused by post multidistrict infiltration of acidlidocaine ; in this case us targeted the infiltration of hyaluronidase which allowed for resolution . 
our study shows the importance of us in recognizing different types of filling materials used in medicine and cosmetic surgery procedures as well as their complications ; it can also help to identify previous implants that were not declared by the patient . 
this noninvasive , precise and detailed diagnostic methodology is of great value especially to those patients who usually have a high cosmetic expectancy and try to avoid invasive procedures such as biopsies [ 1120 ]  . conclusion ecd is a low - cost widespread noninvasive diagnostic tool that allows the identification of filling materials used as well as the evaluation of damage and complications from aesthetic medicine / surgical treatments [ 47 , 917 ]  . 
diagnostic accuracy of the dect maps ( qualitative assessment ) and of the ct numbers ( quantitative assessment ) , interobserver and intraobserver agreements were calculated . results mri revealed 61 edematous vertebrae and 52 collapsed non - edematous vertebrae . 
 dect numbers were significantly different between positive ( mean 23 hu , range 189 , 29 hu ) and negative cases ( mean 126 hu , range 321 , 66 hu ) with p < 0.001. 
sempreboni 10 , 37024negrar , vr , italy 2 department oforthopaedic surgery , irccs sacro cuore don calabria hospital , negrar , italy 3 department ofanesthesia andanalgesic therapy , irccs sacro cuore don calabria hospital , negrar , italy introduction vertebral compression fractures represent a common clinical problem , especially in the elderly population , and are associated with a decreased quality of life and increased mortality [ 1 , 2 ]  . 
common causes of vertebral compression fractures include postmenopausal osteoporosis , where skeletal mass and bone strength diminish with the aging process , trauma , malignancies and secondary osteoporosis [ 3 ]  . 
for example , percutaneous vertebroplasty stabilizes the fracture and strengthens the vertebrae immediately after the procedure [ 5 , 6 ]  . imaging procedures such as magnetic resonance ( mr ) imaging , bone scintigraphy and computed tomography ( ct ) help to determine the treatment choice and relative location [ 7 ]  . 
however , mri cannot be routinely vol . : ( 0123456789 ) 1 3 488 la radiologia medica ( 2019 ) 124 : 487494 used in all patients in the acute settings owing to reduced availability , contraindications and / or metal artefacts . 
also , ct is commonly used in trauma to rule out thoracic and abdominal traumatic lesions [ 14 ]  . with the advancements in dual - energy ct ( dect ) technology , material decomposition and the elimination of misregistration artefacts are possible [ 15 ]  . 
 also dect have been successfully used to evaluate abdominal and pelvic trauma [ 20 ]  . the possibility to demonstrate acute vertebral fractures by using dect have been shown in previous studies using second generation scanners [ 4 , 8 ]  . 
 [ 22 ] focused on the performance of 5 readers and used both a second and a third - generation dual - source scanner , although the number of patients studied with the newer dual - source ct was relatively limited . the purpose of our study was to investigate the reproducibility of dect in the evaluation of vertebral fractures and bone marrow edema with a 384 - slice third - generation systethe diagnostic accuracy values of dect were calculated by using mri as the standard of reference . materials andmethods patient population this prospective study was approved by the institutional review board , and all patients provided informed consent . 
five patients were excluded because of non - mri - compatible pacemaker ( n = 3 ) , incomplete mri protocol ( n = 1 ) and claustrophobia ( n = 1 )  . 
one pathologic fracture due to tumor infiltration and three vertebral bodies with subtotal vertebral collapse were excluded from this study . dect protocol the dect examinations were performed with a third - generation 384 - slice dual - source ct scanner ( somatom definition force , siemens healthcare , forchheim , germany )  . 
the predefined tube currenttime product was set at a ratio of 1.6 : 1 ( tube a , 220 quality reference mas ; tube b , 138 quality reference mas )  . 
 soft - tissue kernel ( qr32 ) 80 - kvp and 150 - kvp set images were transferred to an offline workstation ( syngovia vb20 ; siemens , erlangen , germany )  . the software uses a three - material decomposition resulting in a virtual non - calcium image . 
the information was color - coded with a color lookup table which codes bone marrow and edema in shades of green - yellow to orange - red ( parallely to the progressive increase of density ) ; 3d volume rendering maps coding bone marrow edema in shades of green and normal bone in blue were also available . 
the de - specific information was fused with the conventional grayscale morphologic images ( thickness , 1mm ; increment , 1mm )  . mri protocol mr imaging was performed with a commercially available 1.5 - t unit ( magnetom avanto fit ; siemens healthcare , erlangen , germany )  . 
standard t1 - weighted spin echo , t2 - weighted turbo spin echo , and turbo inversion - recovery magnitude ( tirm ) sequences were performed in the sagittal orientation . 
mr imaging parameters are summarized in table1 . image analysis on all images datasets available , each vertebral body was evaluated for the presence of bone marrow edema using a binary classification system ( 1 = presence of fracture / bone marrow edema and 0 = absence of acute findings )  . as the standard of reference , all mr images were evaluated in random order , by an experienced radiologist ( 20years of experience ) blinded to ct findings . 
the vertebral bodies were graded as normal ( grade 0 ) , minimal fracture ( grade 1 with 2025% decrease in anterior , middle , and / or posterior height ) , moderate fracture ( grade 2 with 2540% decrease in height ) , and severe fracture ( grade 3 with > 40% decrease in height )  . standard axial , coronal and sagittal reconstructed ct and dect images ( 3d and 2d color - coded maps ) were analyzed by two independent radiologists ( 35 and 15years of experience in radiology , respectively ) in a random order , blinded to clinical and mri findings on a dedicated syngovia workstation . 
to determine the intraobserver agreement , the dect images were re - evaluated randomly after 30days . quantitative analysis of dect numbers was performed by achieving two circular regions of interest ( roi ) for each vertebral body , placed on the suspected area , and using mean value for further analysis [ 8 ]  . 1 3 490 statistical analysis the statistical analyses were performed with statacorp . 
mri images served as the standard of reference . table 2 clinical data of patients enrolled la radiologia medica ( 2019 ) 124 : 487494 the receiver operating characteristic ( roc ) curve analysis and calculations of the area under the roc curve ( auc ) were used to evaluate the attenuation values from dect scans . 
the sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and accuracy of dect for the diagnosis of vertebral fracture were calculated on a per - vertebra basis both as regards qualitative and quantitative assessment . 
2 sixty - two - year old man with traumatic acute dorso - lumbar pathe mri study ( 3a ) shows a grade 1 acute fracture of l1 vertebral body , with mild hyperintensity on the sagittal tirm image ( yellow arrow )  . 
 on the sagittal reconstructed dect color - coded image ( 3b ) the bone marrow edema is confirmed by the presence of shades of green ( yellow arrow )  . 
 on the parasagittal 3d dect map ( 3c ) , the acute fracture can be easily recognized appearing green ( yellow arrow ) , while the normal vertebral bodies are coded in blue 1 3 la radiologia medica ( 2019 ) 124 : 487494 fig . 
3 sixty - four - year old woman with atraumatic acute lumbar pathe mri study ( 4a ) shows a grade 1 acute fracture of l4 vertebral body , with mild hyperintensity on the sagittal tirm image ( yellow arrow )  . 
 on the sagittal reconstructed dect color - coded image ( 4b ) and on the parasagittal 3d dect map ( 4c ) , the bone marrow edema is confirmed by the presence of shades of green ( yellow arrow )  . 
in line with previous studies [ 8 , 15 , 21 , 22 ] , our results demonstrated that dect represents a reliable imaging technique for the identification of fresh fracture and to rule out the presence of bone marrow edema in the case of non - edematous collapsed vertebral bodies . 
used a cutoff value of - 80 hu for the ct numbers , obtaining a sensitivity of 96.3% , a specificity of 98.2% , and an overall accuracy of 97.6% in the subgroup of vertebral bodies with less than 50% sclerosis and / or air . 
although we did not perform a sub - analysis of sclerotic vertebral bodies , we obtained a relatively high value of overall accuracy that could be explained by the advancement in vnc post processing technique . 
 by using a cutoff of 47 hu ( similar to our 50 hu threshold ) , the authors obtained a sensitivity of 92.0% , specificity of 82.6% , accuracy of 84.0% , positive predictive value of 48.9% , and negative predictive value of 98.3% for the differentiation of edematous vertebral bodies . in the study of kaup etal . 
the multiplanar reformatted 2d color - coded maps , created and superimposed to the bone window grayscale dataset , were the most adequate for the evaluation of bone marrow edema . 
 the level of superimposed map can be modified from a level showing only the colors of the map to a level showing only the standard grayscale ct images : this allowed us to evaluate traditional ct images and color - coded maps together , reducing reading time . 
 in this case , the sclerosis caused an abnormal subtraction process , covering the bone marrow edema on color - coded maps with subsequent misdiagnosis . our study population was relatively younger compared with the populations in the studies by wang etal . 
 however , as previously stated , our findings confirmed that the diagnostic accuracy of dect in depicting bone marrow edema tends to increase in patients with osteoporotic bone structure because of the prevalence of fatty instead 1 3 la radiologia medica ( 2019 ) 124 : 487494 of red bone marrow and reduced trabecular bone structures [ 8 , 21 ]  . 
however , as previously described , the mean age of patients enrolled in our study was inferior when compared to the populations of similar studies [ 8 , 22 ]  . 
moreover , a comparative analysis of qualitative versus quantitative analysis was not performed ; as previously discussed , we believe that the dect numbers and dect maps are strictly correlated , so that the quantitative analysis could represent an additional tool that helps radiologists to discriminate subtle imaging findings . 
any abnormal middle ear attenuation on high - resolution ct images ( hrct ) or dect color - coded maps , and any abnormal signal on mri images was evaluated by four experienced radiologists . 
ct numbers were significantly different between positive ( mean 57.6hu , range 65 , 112hu ) and negative cases ( mean 5.4hu , range 100 , 66hu ) with p < 0.001. 
the disease is treated surgically often followed by a second - look approach to check for residual tissue or recurrence [ 4 ]  . high - resolution computed tomography ( hrct ) of the temporal bone is widely accepted to detect cholesteatoma and to assess the extension and possible complications of the disease [ 5 ]  . 
however , ct opacity is nonspecific for vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 478486 cholesteatoma , and the diagnosis is based on the high specificity of lesion location and patterns of bone erosion [ 6 ]  . 
 also , ct is not reliable in case of atypical locations , especially to distinguish recurrence from postoperative changes [ 68 ]  . because of its high keratin content , cholesteatoma has specific signal - intensity characteristics on magnetic resonance imaging with very high signal intensity on diffusionweighted imaging ( dwi ) at high b value ( 8001000s / mm2 ) [ 911 ]  . 
however , mri cannot be routinely used in all patients with suspected cholesteatoma owing to reduced availability , contraindications and / or metal artifacts . with the advancements in dual - energy ct technology ( dect ) , material decomposition and the elimination of misregistration artifacts are possible [ 15 ]  . 
materials , particularly those with high atomic weight numbers ( e.g. , iodine and calcium ) , display different attenuation measurements at different kilovoltage peak levels [ 16 , 17 ]  . 
dect has been proposed to demonstrate the presence of bone marrow edema in vertebral compression fractures , to evaluate brain hemorrhage and iodine extravasation in the brain [ 1823 ]  . 
 however , dect has not yet been proposed for the evaluation of cholesteatoma . in the setting of a filled hypodense post - surgical cavity with suspect of local recurrence or residual disease , dect materials decomposition capabilities could be potentially used to differentiate cholesteatoma from other non - cholesteatomatous tissues ( i.e. , trapped fluids , granulation tissue )  . thus , the purpose of our study was to prospectively evaluate the diagnostic accuracy of dect and mri to identify residualrecurrent cholesteatoma using the second - look surgery as the reference standard . materials andmethods patient population this prospective study was approved by the institutional review board , and all patients provided informed consent . 
six patients were excluded because of non - mri - compatible pacemaker ( n = 2 ) , incomplete mri protocol ( n = 1 ) , claustrophobia ( n = 1 ) , and absence of surgical correlation ( n = 2 )  . 
1 flowchart with inclusion and exclusion criteria of patients enrolled in previously surgically treated ears ( fetid discharge , pain , hear deterioration ) or presence of soft tissue density mass in the surgical cavity at standard ct . dect protocol the dect examinations were performed with a third - generation 384 - slice dual - source ct scanner ( somatom definition force , siemens healthcare , forchheim , germany ) [ 15 ]  . 
 soft tissue kernel ( qr32 ) 80 - kvp and 150 - kvp set images were transferred to an offline workstation ( syngovia vb20 ; siemens , erlangen , germany )  . 
a customized postprocessing based on the de liver virtual non - contrast application [ 1619 ] was used in order to enhance subtle differences between tissues with similar , intermediate density . 
the images were evaluated by two independent radiologists ( xx and xx with 35 and 15years of experience in radiology , respectively ) in a random order , blinded to clinical and mri findings . 
the maps were available on three planes ; each reader was free to modify window settings and imaging parameters including slice thickness , magnification , and level of superimposition between hrct images and color - coded maps . 
moreover , standard hrct images were always available for review on a dedicated window in order to correlate the maps with the presence of subtle erosions and for anatomical reference . 
in particular , by using the default settings of the fat map , a tissue appearing in blueviolet on the look - up table was coded cholesteatoma , whereas the tissues coded in gray / yellow were characterized as scar and trapped inflammatory fluid , respectively . for any disagreement on presence of cholesteatoma , a consensus reading was appended , and the consensual results were used for further analysis . 
quantitative analysis of dect numbers was performed on the blended images on syngovia software by achieving two circular regions of interest ( roi ) for each ear , covering half to 2 / 3 of the suspected soft tissue mass ( identified on the dect maps or on standard reconstruction according to the reader choice ) , and using mean value for further analysis . the mr examinations were analyzed in a separate session , in random order , by two blinded radiologists ( xx and xx , 25 and 5years of experience in radiology , respectively )  . 
surgery served as the standard of reference . the sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and accuracy of the dect color - coded maps ( qualitative analysis ) and of the ct numbers ( quantitative analysis ) , and of the mri images for the diagnosis of residual or recurrent cholesteatoma were calculated on a per - ear basis . 
interobserver and intraobserver agreement for the qualitative analysis were calculated with weighted k statistics . for the quantitative analysis , the receiver operating characteristic ( roc ) curve analysis and calculations of the area under the roc curve ( auc ) were used to evaluate the attenuation values from dect scans . 
the ear - based results and relative diagnostic performance values of the imaging methods employed are shown in tables2 and 3 , respectively . surgical andpathological results at surgery , the diagnosis of cholesteatoma was obtained in 16 / 24 ears ( 66.6% recurrence rate ) because of the presence 1 3 la radiologia medica ( 2019 ) 124 : 478486 table 1 clinical data in 19 patents / 24 ears with 16 residual / recurrent cholesteatoma parameter value aage ( years ) aage of male ( years ) aage of female ( years ) no of male no of female treated on one side only treated on both sides canal wall up technique canal wall down technique total cholesteatomas single cholesteatomas bilateral cholesteatomas right lesions left lesions 62 . 
on the coronal reconstructed highresolution ct image of the left ear ( a ) , intermediate density tissue can be depicted within the surgical cavity , without significant erosions ( thin arrow )  . 
by using an algorithm intended for the evaluation of liver fat content to create color - coded maps , an overall accuracy of 87.5% in determining the presence versus the absence of residual or recurrent cholesteatoma was achieved , without any significant difference with respect to dwi images . 
an additional explanation is that the structural properties of cholesteatoma , rich of keratin proteins , could have led to its appearance on dect . in line with the results of previous studies [ 26 , 27 ] , conventional imaging signs on hrct images alone allowed us to depict cholesteatoma in 4 of 16 ears following mastoid 1 3 la radiologia medica ( 2019 ) 124 : 478486 fig . 
on the coronal reconstructed high - resolution ct image of the right ear ( a ) , a thickened hypodense fold ( thin arrow ) can be depicted without erosive changes of the adjacent neo - cavity wall and without signal changes on coronal dwi image ( b )  . 
the high overall accuracy of mri could be also explained by the absence of cholesterinic granulomas or other confounding lesions in our series . sensitivity , specificity , ppv , npv and accuracy of dect maps were 87.5%. 
an additional peripherally located lesion , missed at mri as well , was misdiagnosed as a fn at dect maps both from reader 1 and reader 2 . one false positive shared by mri , and dect ( for both readers ) was represented by a squamous cell carcinoma : the lesion was misdiagnosed at mri because of its dwi hyperintensity but lack of significant contrast enhancement . 
at dect , an intermediate density lesions were depicted , coded in blueviolet on the maps , which was consistent with the incorrect diagnosis of cholesteatoma . at dect , an additional fp for reader 2 was due to the presence of an earwax plug left from previous surgery . 
in particular , cholesteatomas represented high density tissue on the maps and were coded as blueviolet , whereas fluid and scars represented medium and low density areas and were coded as yellow and gray , respectively . 
conversely , inflammatory fluid and cholesteatoma were relatively spared from this subtraction process , showing medium and high density values ( with dect numbers similar to those expected on conventional ct images )  . 
moreover , when considering the intended use of the color - coded maps employed , we may argue that inflammatory fluid and cholesteatoma were coded as yellow and blueviolet areas on the color - coded maps , respectively , as expected for fatty and non - fatty areas of the liver . this study had some limitations . 
first , few patients were enrolled and larger series are need to confirm our preliminary results ; also , a possible selection bias may depend on the enrollment of patients with high clinical suspicion of residual / recurrent cholesteatoma . 
although the methods described in this paper represent a reproducible approach , new dedicated decomposition algorithms would be desiderable and could lead to more reliable results in the future . in conclusion , by using surgical and pathological data as the standard of reference , both mri and dect provided an accurate demonstration of residual / recurrent cholesteatoma . 
 1 3 la radiologia medica ( 2019 ) 124 : 478486 while mri can be considered the most accurate imaging method , dect may represent a promising imaging tool in patients with contraindications for mri . 
sarmento kma jr , sampaio all , santos tgt etal ( 2017 ) highfrequency conductive hearing loss as diagnostic test for incomplete ossicular discontinuity in non - cholesteatomatous chronic suppurative otitis media . 
ms - estells f , mateos - fernndez m , carrascosa - bisquert b etal ( 2012 ) contemporary non - echo - planar diffusion - weighted imaging of middle ear cholesteatomas . 
alvo a , garrido c , salas etal ( 2014 ) use of non - echo - planar diffusion - weighted mr imaging for the detection of cholesteatomas in high - risk tympanic retraction pockets . 
dremmen mh , hofman pa , hof jr etal ( 2012 ) the diagnostic accuracy of non - echo - planar diffusion - weighted imaging in the detection of residual and / or recurrent cholesteatoma of the temporal bone . 
chen s , ikawa f , kurisu k etal ( 2001 ) quantitative mr evaluation of intracranial epidermoid tumors by fast fluid - attenuated inversion recovery imaging and echo - planar diffusion - weighted imaging . 
aikele p , kittner t , offergeld c etal ( 2003 ) diffusion - weighted mr imaging of cholesteatoma in pediatric and adult patients who have undergone middle ear surgery . 
palmer we , simeone fj ( 2018 ) can dual - energy ct challenge mr imaging in the diagnosis of focal infiltrative bone marrow lesions ? radiology 286 ( 1 ) : 214216 . 
petritsch b , kosmala a , weng am etal ( 2017 ) vertebral compression fractures : third - generation dual - energy ct for detection of bone marrow edema at visual and quantitative analyses . 
mallinson pi , coupal tm , mclaughlin pd et al ( 2016 ) dual - energy ct for the musculoskeletal syste radiology 281 ( 3 ) : 690707 ( review ) 22 . 
bodanapally uk , shanmuganathan k , issa g etal ( 2018 ) dualenergy ct in hemorrhagic progression of cerebral contusion : overestimation of hematoma volumes on standard 120 - kv images and rectification with virtual high - energy monochromatic images after contrast - enhanced whole - body imaging . 
bonatti m , lombardo f , zamboni ga etal ( 2018 ) iodine extravasation quantification on dual - energy ct of the brain performed after mechanical thrombectomy for acute ischemic stroke can predict hemorrhagic complications . 
magarelli n , de santis v , marziali g etal ( 2018 ) application and advantages of monoenergetic reconstruction images for the reduction of metallic artifacts using dual - energy ct in knee and hip prostheses . 
hu r , daftari besheli l , young j etal ( 2016 ) dual - energy head ct enables accurate distinction of intraparenchymal hemorrhage from calcification in emergency department patients . 
osman nm , rahman aa , ali mt ( 2017 ) the accuracy and sensitivity of diffusion - weighted magnetic resonance imaging with apparent diffusion coefficients in diagnosis of recurrent cholesteatoma . 
when biliary communication is clearly shown on t2w haste sequences , it should be reported as cysto - biliary communication even if there is no leakage of gadoxetic acid into the cyst on ce - mrcp . keywords biliary system gadoxetic acid hydatid cyst magnetic resonance cholangiopancreatography introduction hydatid disease is a widespread zoonosis and more commonly seen in the mediterranean area [ 1 , 2 ]  . 
no : 1 , umraniye , 34764istanbul , turkey 2 general surgery department , university ofhealth sciences umraniye training andresearch hospital , istanbul , turkey there are some major complications of hydatid cyst . 
 the rupture of the cyst into peritoneal cavity which leads to peritonitis , transdiaphragmatic rupture into thorax , or rupture into biliary system and abscess formation are lifethreatening complications [ 2 , 4 ]  . 
if there is any suspicion of biliary communication in hydatid cyst , surgical procedures such as cystectomy and unroofing with primary closure of biliary communication can be preferred [ 3 , 5 ]  . 
therefore , preoperative diagnosis of cysto - biliary communication is vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 460466 necessary for selecting the most appropriate treatment option , guiding surgical planning , and decreasing the risk of mortality of the patient . magnetic resonance imaging ( mri ) is usually performed for the detection of complications . 
there are two kinds of hepatobiliary - specific contrast agent : gadobenate dimeglumine ( gd - bopta , multihance ; bracco imaging , milan , italy ) and gadoxetic acid ( gd - eob - dtpa , primovist ; bayer pharma ag , berlin , germany )  . 
contrast - enhanced magnetic resonance cholangiopancreatography ( cemrcp ) is performed by administration of gadoxetic acid because of its hepatobiliary specificity and high biliary excretion ( 50% ) [ 8 ]  . the aim of our study was to evaluate the effectiveness of ce - mrcp and t2 - weighted half - fourier acquisition singleshot turbo spin - echo ( t2w haste ) sequences for diagnosis of cysto - biliary communication in hydatid cysts compared to surgical results . 
we aimed to present the results and our experiences about ce - mrcps of hepatic hydatid cysts with biliary communication . materials andmethods patients the institutional review board approved this retrospective study , and informed consent was waived . 
between october 2014 and october 2016 , at a single institution , 52 consecutive patients who underwent surgery because of hepatic hydatid cysts were reviewed from our hospitals picture archiving and communications systewe excluded a total of seven patients : six without preoperative mri examinations and one patient with suboptimal image due to patient motion and susceptibility artifacts . 
a final cohort of 45 patients [ 21 ( 46.70% ) men and 24 ( 53.30% ) women with a mean age of 45 15 ( sd ) years ( range 2067years ) ] were included in our study and preoperative abdominal contrastenhanced mri examinations were reviewed . 
there were no patients with > 3mg / dl serum bilirubin level which causes insufficient visualization of the biliary system in ce - mrcp due to diminished biliary excretion . magnetic resonance imaging andimage evaluation all mris were performed on a 1.5 tesla system ( magnetom avanto , siemens healthineers , erlangen , germany )  . 
the parameters of routine abdominal mri sequences were as follows : coronal t2w haste [ tr / te : 2000 / 130 , field of view ( fov ) : 420mm , slice thickness 7mm ] , axial t2 - weighted haste ( tr / te : 2000 / 162 , fov : 440mm , flip angle 90 , slice thickness 6mm ) with and without fat saturation , spoiled dual - gradient echo t1 - weighted inand opposed - phase mr imaging ( tr / te : 209209 / 2.34.8 , slice thickness 7mm ) , axial precontrast and postcontrast dynamic t1 3d vibe ( tr / te : 800 / 22 , fov : 440mm , slice thickness 5mm ) sequences . 
 in 27 patients , 0.1ml / kg ( 0.25mmol / kg ) gadoxetic acid was intravenously administered as a contrast agent at a rate of 2ml / s , followed by a bolus of 20ml saline . 
on t2w haste sequences , extension or contact of an intrahepatic bile duct with the contour of the cyst and also focal dilatation of a bile duct near the contour defect of the cyst were accepted as positive findings for cysto - biliary communication . 
 these roi measurements inside the cyst were performed on non - contrast t1 - weighted sequence , hepatobiliaryspecific delayed phase , at 1 - h , 2 - h , and 24 - h delayed t1 - weighted sequences after contrast injection . 
the degree of observer agreement , as indicated by kappa coefficients ( k ) , was interpreted as follows : 00.20 , slight agreement ; 0.210.40 , fair agreement ; 0.410.60 , moderate agreement ; 0.610.80 , substantial agreement ; and 0.811.00 , almost perfect agreement [ 10 ]  . results a total of 45 patients with 45 hepatic hydatid cysts were enrolled in our study . 
the excretion of contrast agent into the intrahepatic biliary system and the common bile duct was observed in all patients with ce - mrcps ( n = 27 )  . 
the duration between preoperative mri and surgery ranged 130days with the mean duration of 12days . the radiological findings were divided into two groups as t2w haste sequences ( n = 45 ) and ce - mrcps ( n = 27 ) findings . 
of 45 hydatid cysts , 23 cysts were 10cm and 22 were < 10cwhen we compared the findings of < 10cm cysts with 10cm cysts , we also found that larger cysts ( 10 cm ) increased the specificity , ppv , and accuracy of findings on t2w haste sequences . 
1 ce - mrcp at hepatobiliary phase shows contrast agent inside the gall bladder ( * ) , the common bile duct ( white arrow ) and intrahepatic bile duct ( black arrow ) ( a , b )  . 
the contour defect ( small black arrow ) of hydatid cyst with extension of a dilated biliary duct ( white arrow ) through defect was demonstrated on t2w - haste sequence ( c )  . 
 among interpretations of ce - mrcps , there was also perfect interobserver agreement ( k = 1 )  . discussion the complications of hydatid cysts can increase morbidity and mortality . 
2 ce - mrcp shows contrast inside the intrahepatic bile ducts ( white arrows ) : gall bladder ( black arrow ) ( a ) and the common bile duct ( white arrow ) ( b )  . 
there was no difference between roi measurements inside the surgically proven cyst with biliary communication at non - contrast ( c ) and at hepatobiliary phase ( d ) axial t1 - weighted images . 
 the contour defect of cyst with extension of a bile duct ( white arrow ) through defect ( small black arrow ) was illustrated on axial t2w haste sequence ( e , f ) the surgery is required and the type of the surgery [ 1114 ]  . 
the visualization of the biliary system and the bile leakage can be shown by excretion of contrast agent within the biliary systethus , the presence of contrast agent inside the hydatid cyst indicates cysto - biliary communication . in a previous study , kantarci etal . 
among hydatid cysts with biliary communication , there was one false - negative result in < 5cm cysts ( n = 6 ) , one in cysts between 5 and 10cm ( n = 15 ) , and two false - negative results in > 10cm cysts ( n = 14 ) on ce - mrcps [ 8 ]  . 
although 51% of patients had 10cm cysts ( n = 23 ) in our study , no leakage of contrast agent inside the cyst with biliary communication was observed . a few previous studies were published on the role of cemrcp for diagnosis of the postoperative biliary leakage [ 1821 ]  . 
 [ 18 ] detected five false - negative results in traumatic group ( n = 20 ) , six false - negative results in postoperative group ( n = 49 ) , and four false - negative results in hydatid cyst group ( n = 30 ) after combined analysis with mrcps and ce - mrcps [ 18 ]  . 
 [ 19 ] found the sensitivity of 96.4% , specificity of 100% , and accuracy of 97.1% for detection of an active bile leakage in ce - mrcps ( n = 34 ) at 2025min , 6090min , and 150180min delayed images . 
in contrast to some previous studies [ 18 , 20 ] , the sensitivity ratio in ce - mrcp at 2025min images ( 42.9% ) was lower , and there was one false - negative result [ 19 ]  . 
they concluded that although the visualization of the biliary system was sufficient at hepatobiliary phase and liver function tests are normal , the bile leakage may not be demonstrated on ce - mrcp and prolonged delayed phase images are required [ 21 ]  . 
moreover , in two patients with documented cysto - biliary communication after interventional procedures , no leakage of gadoxetic acid into the cyst was noted at prolonged delayed phase images [ 21 ]  . 
 on the contrary to previous studies [ 8 , 1821 ] , no contrast agent inside the cyst was detected in our study , although all cysto - biliary communications were clearly shown on t2w haste sequences . 
in our study , we investigated the relationship between hydatid cyst and the biliary system by comparing signs on routine axial and coronal t2w haste sequences , instead of mrcps , with findings of ce - mrcps . 
to decrease the bias of reader , two radiologists evaluated mris without knowledge of surgical findings . the reasons for non - visualization of gadoxetic acid in the cysts with biliary communications may be related to the size of biliary communication between the cyst and the biliary system , small volume leakage and high pressure inside the cyst [ 12 , 21 , 22 ]  . 
 we hypothesized that the increased pressure inside the cyst and the compression of bile ducts could be the reasons for no leakage of contrast agent inside the cyst . several studies were conducted to investigate the effect of the size of the hydatid cysts on biliary communication and the size of the cyst was found as an important predictor . 
all cysts 10cm had biliary communication in our study . we noticed a mismatch of findings in diagnosis of biliary communication in the cyst on routine abdominal t2whaste sequences and ce - mrcps . 
we recommend that if biliary communication of a cyst is seen on t2w haste sequences but no leakage of gadoxetic acid inside the cyst is shown on delayed phase of ce - mrcp , it should still be reported as cysto - biliary communication . 
the contour defect of a hydatid cyst with extension of a dilated bile duct through the defect is an important clue for cysto - biliary communication on t2w haste sequences . 
 so , on t2w haste sequences , the existence of marginal defect of cyst is an essential finding . the limitations of our study were as follows : retrospectively collected data were analyzed and the sample size was small . 
in a prospective study , subtracted images could be more valuable to demonstrate the leakage of contrast agent . in conclusion , ce - mrcp is an important noninvasive modality for depicting detailed anatomy and the visualization of the biliary system preoperatively . 
on the basis of our experience , we believe that when the contour defect of hydatid cyst with extension of a dilated bile duct through the defect is clearly shown on axial or coronal t2w haste sequences , it should be reported as a cyst with biliary communication . 
di diagnostica perimmagini ed ecografia interventistica , ospedale evangelico internazionale , genova , italy 7 dipartimento di diagnostica perimmagini , pineta grande hospital , castelvolturno ( ce ) , italy 8 unit di diagnostica perimmagini , casa di cure fornaca , torino , italy 9 universit degli studi di varese , bustoarsizio , italy 10 radiologia pediatrica e specialistica , azienda ospedaliera universitaria ospedali riuniti , universit politecnica delle marche , ancona , italy 3 department ofapplied clinical science andbiotechnology , 11 dipartimento di scienze biomediche perla salute , universit university oflaquila , laquila , italy degli studi di milano , milan , italy 4 unit operativa di radiologia diagnostica ed interventistica , irccs istituto ortopedico galeazzi , via riccardo galeazzi 4 , 20161milano , italy 12 u.o. 
they are sequences with long time of repetition ( tr ) and intermediate time of echo ( te ) halfway between pd - weighted sequences ( te = 1020ms ) and t2 - weighted sequences ( te = 80100ms )  . 
the advantage of these sequences is that te is short enough to maintain optimal anatomic detail ( as pd - weighted sequences ) and high sensitivity to fluid ( as t2 - weighted sequences )  . 
in this setting , strict adherence to standardized protocol is crucial to increase diagnostic performance and minimize variability among different diagnostic centres and readers [ 2 ]  . the aim of this paper is to provide standardized technical recommendations for musculoskeletal mri scans proposed by the italian college of musculoskeletal radiology . 
these recommendations are designed to give a uniform application of mri protocols over the national territory , to increase reproducibility and improve diagnostic performance . these technical recommendations can be applied to mri equipment of any manufacturer with different technical features . 
orientation of scan planes for shoulder examination : a axial plane ; b coronal oblique plane ; c sagittal oblique plane ; d aber 1 3 524 la radiologia medica ( 2019 ) 124 : 522538 in case of musculoskeletal tumours , the mri protocol often depends on tumour location . 
orientation of scan planes for elbow examination : a axial plane ; b coronal plane ; c sagittal plane macro - instability and micro - instability pathologies axial fat - saturated pd - weighted in extrarotation of the axial fat - saturated pd - weighted in intra - rotation of the humerus humerus elbow patients position patient does not tolerate the prone position , alternatively he can be positioned supine with elbow extended along the body in wide gantry scanners . elbow flexion at 6090 ( for the bicipital tendon , coracobrachialis tendon and tricipital tendon pathologies ) , consider the humerus as a reference for the planes positioning . flexed , abducted and supinated ( fabs ) elbow position for distal insertion of the biceps brachii tendon pathologies ( optional )  . scan planes extended elbow , patient in prone position with extended elbow and abducted limb ( superman position )  . 
orientation of scan planes for wrist examination : a axial plane ; b coronal plane ; c sagittal plane 1 3 la radiologia medica ( 2019 ) 124 : 522538 sagittal fat - saturated pd - / int - weighted mra examination saline solutionbased contrast agent indications sequences axial fat - saturated pd - / int - weighted coronal t2 - weighted or stir coronal fat - saturated pd - / int - weighted sagittal fat - saturated pd - / int - weighted wrist patients position pronated wrist in superman position . if the patient is not able to maintain the prone position for his clinical conditions , it is possible to consider the supine position with limb along the body in wide gantry scanners . scan planes axial parallel to the radiocarpal articular plane . 
orientation of scan planes for thumb examination : a axial plane ; b coronal plane ; c sagittal plane la radiologia medica ( 2019 ) 124 : 522538 fov maximum 120mm ; maximum thickness 3mm . fingers standard mri examination sequences axial fat - saturated pd - weighted axial t1 - weighted coronal fat - saturated pd - weighted sagittal fat - saturated pd - / int - weighted sagittal t2 - weighted this examination should be preferably performed on highfield scanners ( 1.5 or 3 t )  . patients position the superman position 1 3 la radiologia medica ( 2019 ) 124 : 522538 fig . 
alberto bellelli , roberto caudana , carlo faletti , eugenio genovese , carlo masciocchi and vincenzo spina are past chairs of the italian college of musculoskeletal radiology . compliance with ethical standards conflicts of interests the authors declare that they have no conflict of interest . ethical approval this article does not contain any studies with human participants or animals performed by any of the authors . 
itt can present as a thyroid nodule , and be confused with malignancy with its hyperechoic pattern ; this might cause unnecessary fine - needle aspiration biopsies and / or surgical interventions . 
we also aim to describe the most sensitive and most specific diagnostic parameters of itt . methods we have evaluated us examination reports of 56 children for whom differential diagnosis included itt between february 2015 and august 2018 . 
we have recorded sonographic characteristics of the lesions , cdus data , and thyroid hormone levels . results study population consists of 56 patients ( 22 itt , 34 other diagnoses )  . 
the most valuable criteria to predict itt presence were the fusiform shape and the longest diameter of the lesion . conclusions fusiform shape and a maximum diameter of 9mm are the most selective criteria to predict itt diagnosis . keywords intrathyroidal ectopic thymus tissue ultrasound diagnosis introduction the prevalence of thyroid nodules in pediatric population is 0.22% , which is lower than adults [ 1 ]  . 
when located in cervical region , it is generally seen between the * sonay aydin sonaydin89@hotmail.com 1 department ofradiology , sami ulus training andresearch hospital , 06340altnda , ankara , turkey 2 department ofradiology , erzincan university , erzincan , turkey 3 department ofradiology , ankara training andresearch hospital , ankara , turkey angle of the mandible and the manubrium [ 3 ]  . 
the most ectopic thymic pathways are located laterally according to thyroid gland [ 4 ] ; however , occasionally , ectopic thymus tissue can be found intrathyroidically , too . ultrasonography ( us ) is the modality of choice for evaluating thyroid gland pathologies in children as being noninvasive and not containing ionizing radiation . 
itt can present as a thyroid nodule , and be confused with malignancy with its hyperechoic pattern [ 5 ] ; this might cause unnecessary fine - needle aspiration biopsies and / or surgical interventions . sonographic detection of itt can be easier for the ones involved in pediatric radiology , but it is still a relatively unknown and difficult diagnosis . 
us examinations were performed by a 7 - mhz linear transducer ( toshiba , xario )  . we have recorded sonographic characteristics of the lesions , cdus data , and thyroid hormone levels . 
youdens index was used to define predictive values about lesion diameters . a two - tailed value of p < 0.05 was considered statistically significant . results study population consists of 56 patients ( 22 itt , 34 other diagnoses )  . 
it is also fusiform in shape ; however , its maximum diameter is measured 15mm , and typical punctate and thin linear internal echoes are not present table 1 pathological diagnosis other than itt diagnosis n ( % ) thyroiditis papillary cancer benign cytology atypia 13 ( 38.2% ) 2 ( 5.9% ) 15 ( 44.1% ) 4 ( 11.8% ) table 2 distribution of parameters according to pathological diagnovariables other diagnoses n = 34 itt n = 22 9.5 ( 516 ) 10 ( 417 ) 0.680 between itt and other diagnosis groups . 
also , a 1mm decrease in the longest diameter of the lesion increases the possibility of itt diagnosis 1.32 times ( or = 0.76 ; p = 0.006 ) ( table3 )  . discussion the development of thymus begins in the sixth week of gestation from ventral sacculation of the third pharyngeal pouch and minor portions of the fourth pharyngeal pouch . 
the bilateral primordial thymus fuses at midline in the eighth week to form the bilobed thymus , which will later descend into the superior mediastinuthe routes of descent of the thymus and of the thyroid are alike because of the proximity of the thyroid diverticulum to the third branchial pouch . 
in the current study , we mainly aim to define the most sensitive and most specific diagnostic parameters to predict itt diagnosis in order to prevent unnecessary invasive procedures and follow - up examinations . typical us appearance of itt is defined as a hypoechoic ( more hypoechoic than the surrounding strap muscles ) lesion with punctate and thin linear internal echoes [ 9 ]  . 
it is stated in the literature that the echogenicities in itt can correspond to hassalls corpuscles rather than microcalcifications [ 10 ]  . in the literature , itt can generally be presented as a fusiform lesion [ 3 , 5 , 9 ]  . 
our results are consistent with the literature ; fusiform shape is the most common itt shape , as well as the rate of being fusiform shape is higher in itt group than the other diagnoses . 
the information contributes to the literature , as far as we know this is the first study to evaluate the diagnostic power of the sonographic parameters . itt was defined to be hypovascular or isovascular compared to the adjacent thyroid parenchyma [ 5 , 11 ]  . 
in other studies [ 5 , 12 ] , it is emphasized that itt lesions are generally subcentimeter , but they did not define any cutoff values , or do not use the diameter of the lesion as a diagnostic criterion like fusiform shape , and typical us appearance . 
different from the literature , we showed that the diameter of the lesion is one of the most powerful criteria to predict itt diagnosis . most of the itt cases were euthyroid in our study . 
we believe that abnormal thyroid hormone levels seen in itt cases are caused by concurrent , independent pathologies , such as thyroiditis . 1 3 la radiologia medica ( 2019 ) 124 : 505509 36 ( 4 ) : 299308 . 
hernandez - cassis c , poniecka a , vogel ck , mckenzie jm ( 2008 ) a six - year - old boy with a suspicious thyroid nodule : intrathyroidal thymic tissue . 
vlachopapadopoulou ea , vakaki m , karachaliou fe , kaloumenou i , kalogerakou k , gali c , michalacos s ( 2016 ) ectopic intrathyroidal thymus in childhood : a sonographic finding leading to misdiagnosis . 
avula s , daneman a , navarro om , moineddin r , urbach s , daneman d ( 2010 ) incidental thyroid abnormalities identified on neck us for non - thyroid disorders . 
yildiz ae , ceyhan k , siklar z , bilir p , yagmurlu ea , berberoglu m , fitoz s ( 2015 ) intrathyroidal ectopic thymus in children : retrospective analysis of grayscale and doppler sonographic features . 
first , unfortunately , some of the patients did not have follow - up us examinations ; hence , the study can offer limited information about followup change of itts . 
lastly , we did not examine the prevalence of itt according to age subgroups , because we did not have enough itt cases for such an evaluation . in the literature , the articles about itt are generally case reports or case series . 
there are not so many original articles , and the present articles are not inclusive of our study ; such as some of them concentrate on only gray - scale us findings , while some of them contain localization data mainly . 
also , our study contributes to the growing literature by defining the diagnostic power of examined parameters . to conclude , itt must be kept in mind in the differential diagnosis of thyroid lesions in pediatric population in order to prevent unnecessary interventional procedures and follow - up examinations . 
simple clinic data and imaging findings were considered : ( a ) location ( craniocaudal and axial ) , ( b ) size , ( c ) morphology , ( d ) dural contact , ( e ) signal characteristics , ( f ) enhancement degree and patterns . 
the surgical treatment of schwannomas requires incision of both the dura mater and arachnoid membrane , vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 510521 because the tumors reside in the subarachnoid space . 
the attachment site of the meningioma to the dura mater must be resected with the tumor to remove any residual tumor cells ; thus , preservation of the arachnoid membrane helps prevent postoperative csf leakage . 
to solve this issue , we conducted this retrospective study to develop the usefulness of mr imaging for the differential diagnosis of these two entities . materials andmethods population data this retrospective study was approved by our institutional review board , and the requirement for informed consent was waived . 
the following inclusion criteria were : ( a ) pathology - proven meningiomas or schwannomas , ( b ) patients with multiparametric mr imaging including t1wi , t2wi , and enhanced t1wi sequences , and ( c ) patients with surgical resection . of these , 27 patients were excluded for the following reasons : ( a ) patient with operation before ( n = 4 ) , ( b ) neurofibromatosis ( n = 5 ) , ( c ) unsatisfactory imaging quality for review ( n = 6 ) , ( d ) main part of lesion - located paravertebral region ( n = 4 ) , or ( e ) without enhancement mr images ( n = 8 )  . 
therefore , a final cohort of 157 patients was included . we reviewed each case with respect to age , gender , the nature of preoperative symptoms , duration of the symptoms , location of the tumor and mri findings . imaging data all mr examinations in all 157 patients were performed on 1.5 - tesla mr scanner ( 65 cases , magnetom aera ; siemens healthcare , erlangen , germany ) or 3.0 - tesla mr scanner ( 92 cases , siemens magnetom trio tim , erlangen , germany ) , and surface coils were used for all patients . including t1wi spin - echo ( se ) sagittal ( for 1.5t , tr / te = 550 / 11ms , nex = 4 , for 3.0t tr / te = 500 / 9.5ms , nex = 4 ) , and fast spin - echo ( fse ) t2 - weighted sequences ( tr / te = 3000 / 500ms for 1.5t ; tr / te = 2500 / 100ms for 3.0t ) sequences t2wi axial ( for 1.5t , tr / te = 500 / 12ms , nex = 4 ; for 3.0t , tr / te = 470 / 11ms , nex = 4 ) , and followed by enhanced fat - suppressed t1wi in the axial plane , the sagittal and the coronal plane . 
both of them were blinded to age , gender , clinical history , symptoms and histopathologic results at the time of interpretation . following items were evaluated onmr images its location ( a ) craniocaudal : cervical region , cervicothoracic region , thoracic region , thoracolumbar region , lumbar region , lumbosacral region or sacral region ; and ( b ) axial location : anterolateral , posterolateral or lateral , and ventral ( midline anterior ) or dorsal ( midline posterior )  . size as the average of long and short axes more accurately reflects three - dimensional tumor volume [ 7 ]  . 
the greatest diameter of each tumor was obtained on the contrast - enhanced images . morphologic characteristics ( 1 ) oval or round shape ( mainly depending on sagittal / coronal images ) , ( 2 ) dumbbell type , ( 3 ) intervertebral foramen widening , ( 4 ) the ginkgo leaf sign . the ginkgo leaf sign : the shape of the deformed cord and the streak resembled a ginkgo leaf on contrast - enhanced axial mr images [ 8 ]  . dural contact the dural tail sign was considered according to the following criteria [ 9 ] : ( 1 ) the tail should be identified at least on two successive sections through the tumor , ( 2 ) the tail should taper smoothly away from the tumor , and ( 3 ) the tail has an 1 3 512 la radiologia medica ( 2019 ) 124 : 510521 enhancement greater than that of the tumor itself , with bone signal abnormalities occasionally . signal characteristics on t1wi ( precontrast ) and t2wi the signal intensity of the lesions was compared with that of the spinal cord . 
 ( 1 ) hypointense was lower than spinal cord , not equal to csf , ( 2 ) isointense was equal to spinal cord , ( 3 ) hyperintense was higher than spinal cord and ( 4 ) miscellaneous signal was formed by of different signs but not classified as any of the aforementioned , and ( 5 ) cystic degeneration ( for t2wi ) as indicated by signal intensity equal to that of the cerebrospinal fluid , exhibiting low intensity on t1wi and high intensity on t2wi . enhancement degree andpatterns on t1wi 1 . 
and a multivariate logistic regression analysis was performed to assess which factors affected the diagnosis results between schwannomas and meningiomas based on the performance of mr image findings and simple clinic data . 
diagnostic parameterssensitivity ( se ) , specificity ( sp ) , positive predictive value ( ppv ) and negative predictive value ( npv ) were calculated on a lesion and a patient basis for mr imaging . interobserver agreement analysis with k statistic was performed to determine consistency between the two radiologists for detection of tumor for the different mr imaging findings . 
the k values were interpreted as follows : 0.000.20 , slight agreement ; 0.210.40 , fair agreement ; 0.410.60 , moderate agreement ; 0.610.80 , substantial agreement ; and 0.811.00 , almost perfect agreement ( 28 )  . 
most of the patients had progressive transverse spinal cord lesions , such as progressive paraparesis , nerve palsy , shoulder and back pain , sciatica , impotence , and bowel dysfunctions . 
among the meningioma group , there was a predominance in the female group ( 40 cases , 75.5% ) , and there were 43 ( 81.1% ) patients with the age of 6187years . 
concerning schwannomas , 9 cases ( 8.6% ) were ventral or anterolateral ( one case , 0.96% ; 8 cases , 7.6% , right - anterolateral / left - anterolateral , 3 : 1 ) , 42 cases ( 40.4% , right - posterolateral / left - posterolateral , 4 : 3 ) were posterolateral , while there was no case identified in the dorsal region , and 43 ( 41.4% ) schwannomas were lateral ( right / left , 22 : 21 )  . 
for the schwannomas , there were 13 ( 12.5% ) cases in the vertebral junction regions ( cervical - thoracic , thoracic - lumbar and lumbosacral : 3 , 6 and 4 cases , respectively ) , and multiple schwannomas ( 10 cases , 9.6% ) occurred in anywhere of intraspinal canal ( cervical , thoracic , thoracic - lumbar and lumbosacral : 5 , 3 , 1 and 1 cases , respectively ) , of which features were not manifested in the meningiomas group . statistical analysis demonstrated difference between the two groups : meningiomas seldom occurred in the vertebral junction regions . 
oval or round shape was observed in 63 cases of schwannomas , and a dumbbell shape with intervertebral foramen widening was manifested in 31 cases ( multiple lesions not involved )  . 
thirty - two ( 60.4% ) meningiomas demonstrated the dural tail sign , and only 6 ( 6.8% ) schwannomas did the dural tail sign on contrast - enhanced t1wi . 
 the dural tail sign was highly significant difference between the two groups ( 2 = 51.55 , p < 0.001 ) 1 3 la radiologia medica ( 2019 ) 124 : 510521 fig . 
there was no the dural tail sign in multiple schwannomas in our work . signal intensity on t1wi ( precontrast ) and t2wi on precontrast t1wis , 3 ( 5.7% ) meningiomas were hypointense , and 50 ( 94.3% ) cases were isointense ( hetero - / homogenous , 15 / 35 )  . 
in contrast to meningiomas , 23 ( 22.1% ) schwannomas demonstrated isointense signal on t2wis ( hetero - / homogenous , 1 3 516 la radiologia medica ( 2019 ) 124 : 510521 12 / 11 ) , 31 ( 29.8% ) schwannomas demonstrated hyperintense signal ( hetero - / homogenous , 18 / 13 ) , and 31 schwannomas ( 29.8% ) demonstrated fluid signal intensity ( cyst degeneration )  . 
thirty - four ( 32.7% ) schwannomas demonstrated rim enhancement , and 5 ( 4.8% ) schwannomas did focal enhancement , of which moderate and marked enhancement were as follows : diffuse : 0 / 65 , rim : 1 / 33 , focal : 1 / 4 , respectively . 
 ( table2 ) multivariate logistic regression forty - six ( 86.8% ) meningiomas demonstrated diffuse enhancement and 7 ( 13.2% ) meningiomas demonstrated rim enhancement , of which moderate and marked enhancement were manifested on contrast - enhanced mr images ( moderate / marked 2 / 44 , 1 / 6 , respectively )  . 
meningiomas were almost hypointense ( 50 , 94.3% ) , while schwannomas ( multiple lesions involved , the maximum lesion was analyzed ) , 56 ( 53.8% ) cases were hypointense , 40 ( 38.5% ) cases homogenous isointense , 2 cases hyperintense and 6 cases miscellaneous signal . 
schwannomas usually demonstrated rim enhancement , while meningiomas did diffuse enhancement ( 2 = 10.58 , p = 0.005 ) 1 3 la radiologia medica ( 2019 ) 124 : 510521 fig . 
a miscellaneous signal oval lesion with marked heterogeneity ( rim with focal enhancement ) was manifested in the left - posterolateral area ( white arrow ) , and it extended over several vertebra along the spinal canal ( white arrow ) , there was a fluidfluid level inside it ( b and e , black arrowhead )  . 
the four most common intradural spinal tumors are the following : schwannoma ( 30% ) , which is the most common intradural extramedullary spinal tumor , occurring at a rate of approximately 0.30.4 per 100 , 000 persons per year . 
intradural intramedullary of which concerning 90% is glial tumors ; ependymoma ( 60% ) is the most common spinal cord tumor in adults , and astrocytoma ( 30% ) is the second most common spinal cord tumor in adults [ 1 , 10 ]  . mri is considered the best preoperative imaging technique to diagnose spinal tumors . 
studying the statistical weight of these modalities on basis of multivariate logistic regression , six statistically significant findings ( age , size , dural tail sign , morphology , sign of t2wi and axial location ) were screened to facilitate the differential diagnosis . 
the capability of logistic equation analysis is 87.1% , indicating that these imaging findings may be useful for differential diagnosis of the two lesions . based on a certain finding only , such as sex , craniocaudal distribution , gave excellent results when evaluated with our series , but they were not included in the multivariate logistic equation . 
an oval lesion manifested hypointense on t1wi ( white arrow ) , marked rim enhancement in the right lateral area ( white arrowhead ) , and the adjacent spinal cord was compressed to the dorsal area . 
an isointense round lesion ( white arrow ) with marked homogeneity enhancement was demonstrated in the right lateral area ( white arrowhead ) , the adjacent spinal cord was compressed to the lea 41 - year - old man with two intraspinal schwannomas in the lumbar . 
the big lesion manifested fluid signal intensity on t2wi ( white arrow ) with marked rim enhancement and septal enhancement ( black arrowhead ) and the round small lesion manifested isointense signal with marked homogenous enhancement ( white arrowhead )  . 
an oval lesion manifested hypointense on t1wi ( white arrow ) , marked rim enhancement with focal mural nodule enhancement in the right - anterolateral area ( white arrowhead ) it gets the most appropriate regression model ; moreover , the multicollinearity does not exist anymore . more than 95% of meningiomas were benign tumors and occurred most frequently in older women [ 4 ]  . 
the female preponderance in the adult population was even stronger than that associated with intracranial meningiomas , and it was thought to be due to the effect of estrogen [ 11 , 12 ]  . 
most spinal meningiomas ( 80% ) arise in the thoracic region , with less common 1 3 la radiologia medica ( 2019 ) 124 : 510521 involvement of the cervical ( 15% ) or lumbar ( 5% ) regions [ 10 ]  . 
intramedullary schwannomas are overwhelmingly rare [ 13 ]  . the typical spinal meningioma is a small , single , discrete , round or oval intradural tumor , but occasionally it may present multiple lesion ( 2% )  . 
in general , schwannomas are solitary , well - circumscribed and encapsulated tumors , commonly arising from the dorsal sensory roots of the cervical and lumbar spine with less frequent involvement of the thoracic region and rarely occur in the lumbosacral region [ 12 , 14 ]  . 
consequently , we suggest that coronal images ( for lateral tumors ) and sagittal images ( for ventral or dorsal tumors ) can be used to study the relation between the tumor and the dura , and to search for the dural tail sign . 
but our results do not conform to these findings , 50 ( 94.3% ) meningiomas manifested isointensity , while 56 ( 53.8% ) schwannomas cases did hypointensity , 6 cases hyperintense and 2 cases miscellaneous signal on t1wi . 
 we speculate that focal areas of even greater hyperintensity on t2wi often corresponds to cystic portions , whereas hypointensity may represent hemorrhage , dense cellularity or collagen deposition [ 12 , 25 ]  . 
schwannomas tendency to cyst formation and hemorrhage were greater in the spine than the intracranial examples and other extramedullary spinal tumors , such as meningiomas [ 26 ]  . 1 3 520 la radiologia medica ( 2019 ) 124 : 510521 in our series , all schwannomas and meningiomas were easily detected on contrast - enhanced mri , and none of schwannomas or meningiomas were not enhanced . 
 the first and foremost , the study was retrospective , and the reviewers were all specialized in differentiating schwannomas from meningiomas , not include any cases of other intradural extramedullary tumors such as cysts , capillary hemangiomas , or ependymoma , which might possess the influenced sensitivity of mr imaging for differentiating the two lesion types . 
finally , because the cases were retrospectively collected over 8years from two different types of mr machines , protocols were inevitably not standardized . conclusions to sum up , simple clinical data associated with mri findings can be used to an accurate diagnosis in the vast majority spinal meningiomas and schwannomas , if various imaging features are analyzed carefully and discreetly . a thoracic vertebra canal tumor with diffuse homogenous enhancement and the dural tail sign in females proved statistically significant as predictors of meningioma , while a tumor with widening of neural foramen , fluid signal intensity on t2wi , rim enhancement proved statistically significant as predictors of schwannoma , especially occurred in vertebral junction regions or lumbar locations . 
in the future , diffusion - weighted imaging and susceptibility weighted imaging may increase the accuracy of imaging findings in the differentiation of spinal schwannomas and meningiomas . compliance with ethical standards conflict of interest no conflict of interest exists in the submission of this manuscript , and manuscript is approved by all authors for publication . 
i would like to declare on behalf of my co - authors that the work described was original research that has not been published previously , and not under consideration for publication elsewhere , in whole or in part . 
all the authors listed have approved the manuscript that is enclosed . ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . informed consent this retrospective study was approved by our institutional review board , and the requirement for informed consent was waived . la radiologia medica ( 2019 ) 124 : 662670 radiotherapy concomitant radiotherapy andtki inmetastatic egfr oralkmutated nonsmall cell lung cancer : amulticentric analysis onbehalf ofairo lung cancer study group paoloborghetti1 marcolorenzobon1 rachelegiubbolini2 niccologiajlevra3 rosariomazzola3 marcoperna4 lucavisani4 fiammettameacci4 mariataraborrelli5 lucatriggiani1 davidefranceschini6 carlogreco7 alessiobruni2 stefanomariamagrini1 vieriscotti4 received : 25 july 2018 / accepted : 29 january 2019 / published online : 15 february 2019 italian society of medical radiology 2019 abstract purpose to investigate the role of radiotherapy ( rt ) in the management of egfror alk - mutated metastatic non - small cell lung cancer ( nsclc ) treated with tki . materials and methods clinical data of 106 patients ( pts ) from five institutions treated with rt concomitant to tki were retrospectively revised . 
patients with four or less metastasis were defined as oligometastatic / oligoprogressive ( om / op ) ; sites of rt were brain , bone , lung or others in 46% , 27% , 14% and 13% , respectively . 
multivariate analysis confirmed srt ( hr 0.355 , ci 95% 0.2120.595 ; p < 0.001 ) and median duration of tki > 14months ( hr 0.17 , 95% ci 0.100.30 ; p < 0.001 ) as independent factors related to better os . 
therefore , platinum - based chemotherapy has been replaced by anti - egfr ( gefitinib , erlotinib , afatinib , osimertinib ) or anti - alk ( crizotinib , ceritinib , alectinib ) tkis in the first - line treatment of this subset of patients [ 13 ]  . median progression - free survival ( pfs ) and overall survival ( os ) of patients treated with tkis range between 813 * marco lorenzo bon marcolorenzo89.mlb@gmail.com extended author information available on the last page of the article and 1825months , respectively . 
however , it is becoming clearer that patients with stage iv egfror alk - mutated nsclc represent a heterogeneous group in terms of disease characteristics and , therefore , survival outcomes . 
clinical presentation can occur with low or high burden of metastatic disease , and it can be affected by the number , dimension and / or location of the metastatic lesions [ 46 ]  . oligometastatic status represents a condition with a low disease burden at presentation ( commonly 46 metastatic lesions )  . 
according to these inclusion criteria , patients were divided in three groups : those who underwent rt no more than 30days before the beginning of the drug ( group a ) , those who underwent rt no more than 30days after the definitive suspension of the tki ( group b ) and those who underwent rt during the administration of the drug ( group c )  . in terms of disease burden at rt presentation , the patients were classified in oligometastatic or oligoprogressive ( om / op ) state , defined as four or less metastatic lesions , and polymetastatic or polyprogressive ( pm / pp ) state , defined as more than four lesions , according to the institutional policy and supported by the literature [ 12 ]  . all the dose fractionation schedules were considered ; the term hypofractionated rt ( hrt ) includes treatments with a palliative aim , as 30gy in 10 fractions , 20gy in 5 fractions or 8gy in 1 fraction . 
the term stereotactic rt ( srt ) means an ablative treatment able to deliver bed over 60gy in a few fractions ( mean 80gy , range 60178gy , considering an alpha / beta for the tumor equal to 10 )  . mutation analysis was conducted by extracting dna and identifying egfr exon 19 deletion and exon 21 l858r mutations by standard sequencing and fragment analysis , while to detect alk gene translocations fluorescence insitu testing ( fish ) has been used . 
in order to analyze the efficacy of the combined treatment , overall survival ( os ) , defined as time from the beginning of drug treatment to death ( any cause ) or until the last follow - up , was estimated with kaplanmeier curves . 
the same variables reported above and potentially affecting os were investigated at univariate analysis with the logrank test ; multivariate analysis was performed with the cox regression model , including all variables resulted statistically significant at univariate analysis . 
acute toxicities , according to rt schedule , were defined as adverse events occurring at the site of irradiation within 90days , whereas late toxicities are those becoming evident after 90days . 
the majority of the patients ( 65 , 61% ) were nave to previous chemotherapy ; 33 ( 31% ) were treated with a first chemotherapy line , while eight patients ( 8% ) received two or more chemotherapy lines before tki . 
fifty - nine patients were treated with gefitinib , 19 with erlotinib , 18 with crizotinib and 10 with other tkis ; four patients were on a secondline tki ( osimertinib and ceritinib )  . 
only two cases of g2 toxicity have been recorded in the srt group , while 5 , 7 and 2 cases with grade 1 , 2 and 3 toxicities , respectively , were registered in the hypofractionated group . 
srt stereotactic radiotherapy , hrt hypofractionated radiotherapy , om / op oligometastatic / oligoprogressive disease , pm / pp polymetastatic / polyprogressive disease discussion preclinical studies demonstrate the possible synergistic effect of tkis given concurrently with radiation at several levels , including effects on cell cycle kinetics , apoptosis induction and the targeting of accelerated cellular repopulation . 
a potential relationship between egfr signaling and dna damage repair has been recently supported by data regarding the inhibition of rad51 expression , a protein involved in the cell double - strand break repair systewhen combined with radiation , erlotinib enhances the induction of apoptosis , inhibits egfr autophosphorylation and rad51 expression following radiation exposure , promoting an increase in radiosensitivity [ 16 , 17 ]  . available data suggest that the om / op condition in nsclc is characterized by better outcomes , also in the absence of ablative treatments [ 7 ]  . 
furthermore , some authors suggest that oligometastatic patients more frequently progress with a further oligoprogressive pattern in comparison with polymetastatic patients [ 18 ]  . recent retrospective and randomized prospective studies show that rt given at progression or on residual disease after partial response on oligometastatic foci fairly improves pfs and may allow longer disease - free intervals [ 9 ]  . 
it is still unclear if ablative treatment of om / op disease may change the natural history of the disease and contribute to a real survival benefit [ 1215 ]  . accordingly , recent retrospective and some prospective studies tried to identify which patients subgroup can achieve a clinical advantage from local treatments . 
nevertheless , some authors remark that the site of metastasis , a longer disease - free interval and the presence of driver mutations characterize patients who can benefit more from locally ablative treatment . 
probably , these parameters indirectly reflect a more favorable tumor biology , in which srt can sterilize metastatic foci , ablate lesion in sanctuary locations , overcome drug resistance and promote drug holidays [ 19 , 20 ]  . our study corroborates these hypotheses , finding that srt schedule is a factor independently associated with better os at multivariate analysis . 
the authors find out that the ablation of all the metastatic foci in op patients treated with crizotinib allowed an extended duration of drug exposure and was associated with longer overall survival [ 21 ]  . these retrospective findings have been recently confirmed by the preliminary results of a prospective randomized trial [ 22 ]  . in our series , the majority of patients treated with concomitant tkis did not interrupt the drug administration . 
our data are consistent with the available evidence identifying patients with a good performance status , om / op disease and the presence of a driver mutation as the best candidates for srt . 
srt can be safely administered also concurrently with tkis . increasing evidences suggest that the ablative treatment of all sites of disease in patients with a limited number of metastases improves clinical outcomes . 
a multi - disciplinary discussion each time this condition occurs is strongly encouraged . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical standards the study was performed in accordance with the declaration of helsinki . 
the questionnaire was developed by the board of the italian college of chest radiology of the italian society of medical and interventional radiology ( sirm ) and by an experienced group of italian academic chest radiologists . 
the link to the online electronic survey was submitted by email to all the sirm members . results a total of 767 radiologists , corresponding to 7.5% of all the sirm members , participated in the online survey . 
the majority of participants ( 92% ) routinely describe the attenuation of pulmonary nodules in the report , and 84.1% recommend the further follow - up , with 92.7% of respondents taking ct nodule morphological features into consideration . 
the majority of sirm members express a need for an update on this topic . keywords pulmonary nodule computed tomography management italian survey chest radiology introduction the continuous technical development and widespread use of computed tomography ( ct ) have led to an increasing number of pulmonary nodules detected , which represents a considerable diagnostic and management challenge [ 1 ]  . the proper management of lung nodules needs an appropriate evaluation , in terms of adequate ct technique chosen , correct identification and interpretation of imaging findings , and optimal follow - up recommendation according to the nodules characteristics . to date , several guidelines from the major medical societies are available for the management of pulmonary nodule * cristiano rampinelli cristiano.rampinelli@ieo.it extended author information available on the last page of the article incidentally identified on ct [ 24 ] , with distinct algorithms for solid and subsolid nodules ( namely , part - solid and ground - glass nodules )  . 
however , in the current clinical practice it is unclear whether radiologists agree with such guidelines , since a variety of strategies can be adopted for nodule detection and management . 
the final version of the survey , which consisted in 23 multiple - choice questions , was created as an online electronic survey using the surveymonkey webbased tool [ 5 ]  . 
the first four questions of the survey were aimed to collect generic information on survey participants , namely the years of experience in radiology , the italian area of belonging , the site of work ( university / research hospital , public hospital , private hospital ) and the daily time committed to thoracic imaging . the following five questions were related to technical aspects and reading tools that are routinely used in chest ct analysis , while ten questions were aimed to collect information on nodule characteristics , management and diagnostic algorithms . the final section included four questions on the role of trans - thoracic ct - guided biopsy , on the multidisciplinary approach to lung nodule and the need of congresses and refresher courses focused on pulmonary nodule . multiple answers were allowed only for two questions . 
the mean time to complete the questionnaire was 620 . the experience in radiology of respondents was quite homogeneously distributed with a faintly higher prevalence of experienced radiologists ( 20.1% with less than 5 - year experience , 16.7% with 5to 9 - year experience , 28.3% with 10to 19 - year experience and 34.9% with more than 20 - year experience ) ( q1 )  . 
slightly more than half of participants ( 53.7% ) were employed in a public hospital while one - fourth ( 25.6% ) in a university / research hospital ( q3 )  . 
significant differences in the distribution of the answers among the two groups ( p < 0.05 ) have been observed for some questions , and results are summarized in table2 . 
particularly , radiologists working at university / research hospitals use two - reconstruction ct protocol for evaluating pulmonary nodules and dedicated software for volumetric analysis when nodule diameter assessment is inconclusive , more frequently than the other respondents ( q5 , q9 )  . 
lung nodule density is always reported by the majority of radiologists in both groups , even though with a higher rate among university / research radiologists ( q10 ) , who also prefer to measure part - solid nodule by reporting the maximum diameter of both the solid component and the entire nodule ( q11 )  . 
with regard to nodule management , university / research radiologists rely more on the fleischner society guidelines and consider pet - ct not indicated for the characterization of a solid nodule < 8mm compared to the rest of respondents ( q15 , q18 )  . 
ct - guided lung biopsies are performed in the majority of university / research hospitals , with a higher rate than the other institutions , mostly carried out by radiologists ( q21 )  . 
finally , university / research radiologists attend a weekly lung nodule multidisciplinary meeting in the 45.9% of cases as compared to the 21.6% of the rest of participants ( q22 )  . discussion the present study evaluated the approach of the sirm members to the complex topic of pulmonary nodule in ct . 
the questionnaire was aimed to identify potential critical issues in regard to ct technique , image reading and interpretation as well as management of incidental pulmonary nodules . as expected , we observed a strong consensus among respondents on some questions . 
particularly , the vast majority of participants ( 92% ) routinely described the attenuation appearance ( solid , part - solid , ground - glass ) of pulmonary nodules identified on ct scan in the radiological report , and 84% recommended the further work - up . 
1 the graph shows results of the sub - analysis conducted for q15 on the basis of the years of experience in radiology ( < 5years ; 59years ; 1019years ; 20years ) ( q1 )  . 
2 the graph shows results of the sub - analysis conducted for q20 on the basis of the years of experience in radiology ( < 5years ; 59years ; 1019years ; 20years ) ( q1 )  . 
 radiologists with experience 20years tend to suggest a biopsy for the characterization of a ground - glass nodule with a diameter > 10mm more often than the other groups 1 3 la radiologia medica ( 2019 ) 124 : 602612 606 fig . 
3 the graph shows results of the sub - analysis conducted for q18 on the basis of the years of experience in radiology ( < 5years ; 59years ; 1019years ; 20years ) ( q1 )  . 
 a significantly higher number of radiologists with experience < 5years does not indicate a pet - ct for characterizing a solid pulmonary nodule with a diameter < 8mm than the other groups features of pulmonary nodules to indicate the subsequent management , demonstrating that morphology is still considered a significant determinant by radiologists , regardless of the nodule size and density . 
these results denoted the awareness of the different strategies for managing pulmonary nodules , indicating an active role of radiologists in suggesting the optimal work - up strategy . on the other hand , we noted a variety of practices regarding the ct technique and reading tools routinely used in the chest examinations . 
for example , while 91.5% of the respondents use thin section thickness for lung nodules evaluation , as suggested by the fleischner society [ 6 ] , only half of themslightly more if we consider the setting of university / research radiologistsregularly perform two reconstructions , respectively , with sharp and standard kernel ( q5 )  . 
 the former reconstruction is suitable for the evaluation of nodule borders , attenuation and measurements , while the latter is suggested for the assessment of the nodules internal content ( calcifications , fat tissue )  . we also found a variability on the use of contrast medium for pulmonary nodule characterization . 
 actually , the dynamic assessment of lung nodule enhancement has not been widely used in the clinical practice over the years , probably because of the relatively high radiation dose and its variability in diagnostic performance in characterizing nodules according to the technique used , mainly in terms of specificity ( 5893% ) [ 7 ]  . concerning radiation exposure , only 44.1% of participants always use low - radiation dose ct protocol for the follow - up of pulmonary nodules ( q7 )  . 
several dose reduction strategies are available in the current generation of ct scanners , including lowering ma and kvp , and using high - pitch technique , automatic tube current modulation and iterative reconstructions [ 811 ]  . 
particularly for follow - up ct scans , further efforts are therefore required to expand the adoption of optimized ct protocols to minimize patient radiation exposure . among italian radiologists , mpr and mip were the tools of choice for pulmonary nodule evaluation ( q8 , q9 )  . 
this could be explained by the convenience and user - friendliness of these widely integrated reporting tools , compared to the limited availability of cad systems and software for volumetric analysis , which are rarely incorporated into the pacs system and often time - consuming . 
volumetric analysis software is more frequently used , even if not extensively , by radiologists working at university / research hospitals , due to a greater availability / integration of these systems in these institutions , as demonstrated by the results of the specific sub - analysis ( q9 )  . from the questions focused on pulmonary nodule management , we observed again a variety of approaches , which reflect the challenge of this topic . 
in particular , we found that the 57.7% of participants preferred the fleischner society guidelines for the management of pulmonary nodules detected incidentally , specifically in the subgroups of radiologists less experienced and employed in university / research hospitals ( q15 )  . 
however , in our survey the adherence to the fleischner society guidelines is not comprehensive , as can be observed from the response concerning the follow - up of solid nodules < 6mm in adult patients without 1 3 la radiologia medica ( 2019 ) 124 : 602612 1 3 608 la radiologia medica ( 2019 ) 124 : 602612 risk factors . 
although no follow - up is suggested for such nodules in the fleischner guidelines [ 2 ] , only 32.3% of participants follow this indication , while about 57% ask for 3 to 6months follow - up and 11% send the subject to the referring physician or specialist ( q15 )  . 
similarly , in the case of a ground - glass nodule stable at annual follow - up , only 11.2% of the participants followed the fleischner societys recommendation of a further 2 - year follow - up , while favoring instead a shorter follow - up ( 75% ) ( q16 )  . 
therefore , even if the fleischner society guidelines are the primary choice , the respondents tend to adopt a more cautious approach with a shorter follow - up for both solid and ground - glass nodules . 
 in fact , the pulmonary nodule guidelines , that also take into account several aspects , including the patients will , provide recommendations and not mandatory rules . with regard to interventional lung procedures ( q21 ) , even though in a substantial rate of institutions ct - guided biopsies are not performed at all ( 35.8% ) , our analysis demonstrated that where biopsies are carried out , radiologists are mostly involved . since the management of pulmonary nodules is challenging , the multidisciplinary team plays an important role [ 13 ]  . 
 unfortunately , more than half of respondents reported that there are no multidisciplinary meetings in their workplace ( q22 ) , especially among those who do not work in university / research hospitals . 
this observation underlines the need for congresses or refresher courses to continue education in the field of nodule work - up ( q23 )  . pet - ct plays an important role in the characterization of pulmonary nodule due to its capacity of assessing nodule metabolism by evaluating the 18f - fluorodeoxyglucose ( 18f - fdg ) uptake . 
these data underline that radiologists need a more in - depth knowledge of the advantages and limitations of the application of nuclear medicine in this field . the present study has some limitations . 
with almost 800 respondents , this is one of the largest surveys among italian radiologists ever reported , but it corresponds to a rather low survey response rate of 7.5%. 
when compared to other surveys , our response rate is , however , in the range of two recent surveys among sirm members , conducted on teleradiology and musculoskeletal interventional procedures , which achieved a response rate of 17% and 5.4% , respectively [ 15 , 16 ]  . 
the reason of the low response rate was possibly related to the specialist topic of the survey ( pulmonary nodule ) proposed to a general radiology audience ( all sirm members )  . 
moreover , physicians are a professional group with low survey response rates in general [ 17 , 18 ]  . another limitation was that the questionnaire was sent to the sirm members 4days prior to the online publication of the recommendations on pulmonary nodule measurement from the fleischner society ( 26 june 2017 ) [ 6 ]  . 
moreover , also in presence of specific guidelines the adherence of radiologists may be not complete . in conclusion , the results of this national survey allowed us to obtain a comprehensive view of the attitudes of the italian radiologists toward the complex and debated topic of pulmonary nodule management . 
this retrospective study investigated safety and efficacy of embolization agents with small - particle embolization treating patients with massive hemoptysis due to ipvs . methods patients with massive hemoptysis ( n = 207 ) underwent computed tomography angiography of bronchial artery . 
only four patients experienced mild hemoptysis during the 24 - month follow - up with the efficiency of 75.0%. conclusions intercostal artery embolization with 300500m alone or combined with microcoils is a safe and effective procedure in patients with ipvs - induced bronchial hemoptysis . keywords catheter intercostal pulmonary venous shunt massive hemoptysis interventional therapy introduction massive hemoptysis , defined as the coughing up of blood or bloodstained mucus , is a common life - threatening sign of respiratory disease [ 1 ]  . 
systemic arterial ( bronchial or nonbronchial ) pulmonary circulation shunt , known as systemic - to - pulmonary venous shunt , is anatomically abnormal that induces recurrent and life - threatening * xiaolin zhang sxdxfzg@sina.com 1 first college ofclinical medical science , china three gorges university , yichang443003 , china 2 department ofradiology , yichang central peoples hospital , 183 yiling road , yichang443003 , china hemoptysis [ 25 ]  . 
 ipvs is characterized by earlier development of thickened interatrial ( ia ) shunts , hyperplastic blood vessels , and pulmonary veins observed in digital subtraction angiography ( dsa )  . 
however , ipvs has not been reported as an independent disease . current therapeutic methods for bronchial massive hemoptysis include hemostatics , surgery , and bronchial artery embolization [ 16 ]  . 
treatments using small - particle embolization agents ( i.e. , smallparticle embolization ) , such as 300500m gel foam or polyvinyl alcohol ( pva ) particles , have been recently investigated . 
these agents greatly reduce the extent of embolism and improve hemostatic efficacy . nonbronchial artery pulmonary circulation shunt , especially intercostal arterypulmonary artery shunt , is usually accompanied by massive hemoptysis when pulmonary lesions spread to the pleura [ 12 , 13 ]  . 
this may be due to the low number of clinical reports about ipvs , which is usually ignored during treatment because the incidence rate is low [ 14 ]  . this retrospective study used dsa to investigate the safety and efficacy of small - particle embolization in patients with massive hemoptysis and ipvs . methods patients from february 2009 through september 2015 , 207 patients underwent bae , for the management of massive hemoptysis [ 111 ]  . 
twenty - four of these patients received a diagnosis of ipvs determined by dsa and were included for the final analysis . of the 24 ipvs patients in the study population ( 7 women , 17 men ; aged 3272years ) , 15 and 9 , respectively , had first - time and recurrent hemoptysis . 
these patients did not achieve effective hemostasis by the traditional hemostatic medications and were subjected to examination with dsa , computed tomography angiography ( cta ) of the bronchial artery , and then interventional embolization . 
in addition , based on the location of lung lesions and associated pleura determined by ct , the following were explored : bronchial artery dominated by the vagus nerve ; ias ; branches of the subclavian artery ; esophageal inherent artery ; and phrenic arteries . vagus nervedominated bronchial artery intravascular embolization based on the angiographic findings for the bronchial artery and nonbronchial systemic arteries , embolization was performed with a 3f superselective microcatheter ( terumo , tokyo , japan )  . 
embolization agents included pva particles ( 300500m ) and microcoils ( mwce - 35 - 3 - 3 , mwce35 - 3 - 4 , mwce - 35 - 3 - 5 ) , both supplied by cook medical ( bloomington , usa )  . for ia embolization , pva was used when the volume of ipvs was 4ml . 
when the vascular angiography was performed , a dose of contrast agent ( adequately displayed the shunting phenomenon and did not flow back ) greater than 4ml was larger flow volume , and less than 4ml was small flow volume . clinically successful embolization was determined by disappearance of the ipvs and abnormal ia network , or retention of contrast agents in the ia . 
the bronchial artery was embolized in the same way as described above . postoperative treatment dsa was performed using the standard angiography suites innova 3100iq ( ge , fairfield , usa ) and fd20 ( philips amsterdam , netherlands ) , with odixanol isotonic non - ionic contrast agent ( visipaque , 320mgi / ml ; ge , carrigtohill , ireland )  . 
the flow rate of contrast agent was manually adjusted to guarantee clear visualization all patients were required to lie and relax in bed , without extensive right leg movement , for 24h . 
 percussion on the back was performed if it was difficult for the patient to discharge sputum or hemoptysis . 1 3 590 la radiologia medica ( 2019 ) 124 : 588594 patients were followed for 148months , with an average of 30months . 
the ct results of all patients suggested involvement of the pleura ( figs.1a and 2a ) , with clearly thickened pleura in some cases ( table1 )  . dsa images ofculprit arteries a total of 51 culprit bronchial arteries ( diameter 2mm ) , two left internal thoracic arteries , and two left phrenic arteries were identified by dsa . 
contrast agents entered the branches of the pulmonary vein through the ia in a spray pattern , and flowed back to the left atriuthe main pulmonary vein was straight and clear in dsa images . 
c the right ia embolism and pulmonary vein shunt disappeared after embolization with microcoils combined with 300500m pva particles 1 3 la radiologia medica ( 2019 ) 124 : 588594 fig . 
a ct scan showed a shadow on the right upper lobe of the lung and involved pleura , in which thickened blood vessels could be observed ( arrow )  . 
however , the main pulmonary vein was light , due to the small shunt volume . in this study , we identified nine large volume ipvss and 30 small - volume ipvss . 
the dsa results of all patients corresponded well with the results of the cta of the bronchial artery , except for one culprit left phrenic artery that was identified during the operation . interventional embolization the superselective microcatheter was inserted into the ia , without disturbing the individual spinal artery . 
for embolization , pva particles ( 300500m ) were used in 7 smallvolume ipvss , and pva particles combined with microcoils ( pva + mc ) in 30 small - volume ipvss . embolization was not performed for one ia , since the mouth of this ia was too narrow to form an acute angle with the main artery and the cobra catheter could not be applied . 
 embolization could not be accomplished for another ia , because the wall of the artery was damaged by the catheter during operation . artery dissection was found after 2min when the microwire began to smoke during repeat micro - wire operation . 
the complications improved within 1week after proper treatment . 1 3 592 la radiologia medica ( 2019 ) 124 : 588594 the short - term follow - up found that hemoptysis recurred in two patients , with blood volume < 300ml ( the hemoptysis volume before embolization ) ( table3 )  . 
massive hemoptysis was cured after treatment with bae . during the mid - term follow - up , hemoptysis reoccurred in one patient 6months after the embolization ( table3 )  . 
the long - term effective rate was 75.0%. discussion hemoptysis with intercostal artery involvement is often accompanied by body - pulmonary circulation shunt , with thickened pleura and the involved lung tissue close to pleura . 
 the pathogenesis of ipvs indicates an increased intercostal artery flow due to the pleural or subpleural inflammatory disease , following by the formation of a shunt with the adjacent pulmonary venous system . in this study , we found that 11.6% ( 24 / 207 ) of patients with massive hemoptysis had ipvs , which is a lower occurrence rate than intercostal arterypulmonary artery shunt . 
since patients in this study mainly had bronchiectasis , thus the overall incidence of body - pulmonary circulation shunt is relatively low . patients with intercostal arterial - pulmonary vein shunt showed thickened intercostal artery , with early development during intercostal arteriography . 
the larger area or the thicker pleura indicated greater abnormal proliferation of the vascular network and a bigger shunt volume . in our study , dsa with hand - push was carried out to avoid blood vessel damage caused by the high - pressure syringe , and the proper flow rate according to the shunt volume was selected to guarantee clear development of the pulmonary vein without reflux . 
the direction of blood flow in the pulmonary artery was away from heart . for patients with diabetes or hyperlipidemia , the placement of the catheter should be gentle enough to avoid the artery wall , spinal artery damage , artery dissection , or vagal reflex . 
for example , in our study , one phrenic artery was not found by bronchial artery cta , but was identified by further examination during the operation . in our study , all microcatheters were inserted into the main intercostal artery with care to avoid the spinal artery during the operation . 
the small pva particles could more closely approach the shunt to reduce the possibility of re - communication between blood vessels and the opening of collateral circulation , achieving better hemostasis . 
the lesson we learned from these cases was that the catheter insertion 1 3 la radiologia medica ( 2019 ) 124 : 588594 must be as gentle as possible to avoid damage to the artery wall and angiorrhexis . 
for example , rlg or yashiro catheter could be used to replace the cobra catheter if the cobra catheter insertion failed . the intercostal artery has an important role in blood supply to the thoracic cage , including the spinal cord , thoracodorsal muscle , and thoracodorsal skit usually does not supply the lung . 
this indicated that main artery embolization alone cannot successfully stop hemoptysis . embolization particles > 350 m or nbca ( n - butyl cyanoacrylate ) agents are safe for bps and do not induce serious complications [ 16 ]  . 
thus , we suggest that coils can be applied first to slow the blood flow before performing the embolization . it has been reported that simultaneous embolization of the bronchial , intercostal , thoracic , and phrenic arteries can cause respiratory muscle ischemia , or even dyspnea . 
we should be cautious to avoid embolization of the intercostal artery , especially embolizing multiple intercostal arteries , for patients with poor respiratory function . taken together , we think that blocking the main intercostal artery and shunt can correct abnormal blood flow , reduce the pressure in branches of the pulmonary vein to stop bleeding , and decrease the possibility of bacterial spread and ectopic embolization formed by a microthrombus . in summary , 300500m pva particles alone or combined with microcoils was safe for intercostal artery embolization . 
together with excellent postoperative prevention and management of lung infection [ 19 ] , our embolization achieved good efficacy . the limitation of this manuscript is that this is a retrospective study and monocentric review , and prospective studies include multiple centers with more patients should be conducted in the future to verify our findings . intercostal artery embolization with 300500m alone or combined with microcoils is a safe and effective procedure in patients with ipvs - induced bronchial hemoptysis . compliance with ethical standards conflict of interest yonghui liang declares that he has no conflict of interest . 
written informed consent was obtained from each included patient . informed consent informed consent was obtained from all individual participants included in the study . la radiologia medica ( 2019 ) 124 : 671681 radiotherapy trends incombined radiochemotherapy forlocally advanced rectal cancer : asurvey amongradiation oncology centers ofsicily region onbehalf ofairo giuseppeprivitera1 robertomilazzotto1 , 2 alessandratocco1 , 2 graziaacquaviva3 corradospatola1 francescomarletta3 lorenzamarino4 alfiodigrazia4 rosalbasalvo5 giovannicartia5 angeloplatania6 lauramolino2 , 7 annasantacaterina6 marilenamattaceraso8 pasqualefrosina8 robertoianni9 michelebono9 lucaliardo10 salvatorebonanno10 leonardalapaglia11 manuelafederico11 ivanfazio11 gianlucamortellaro12 giuseppeferrera12 antonellatripoli12 giovannaevangelista12 antoninodaidone13 giampaolobiti13 marcobadalamenti13 luciaognibene13 albertocacciola2 , 7 silvanaparisi2 , 7 stefanopergolizzi2 , 7 received : 20 november 2018 / accepted : 11 february 2019 / published online : 26 february 2019 italian society of medical radiology 2019 abstract aim to conduct a survey among sicilian centers of radiation oncology belonging to associazione italiana di radioterapia ed oncologia clinica ( airo ) , to record the different methods of integration of radio - chemotherapy both in neoadjuvant and adjuvant settings , to evaluate surgical procedures in relation to the sphincter preservation and to report the different toxicity profiles of the treatment strategies . methods a questionnaire was sent at the end of 2017 to all the radiation oncology centers of sicily region in order to collect the data from individual centers and the treatment characteristics retrospectively over the previous 5years , from 2012 to 2016 . 
the requested data concerned the type of integrated treatment ( neoadjuvant vs adjuvant vs radical ) , combination with chemotherapy ( induction , concomitant , adjuvant ) , type of surgical intervention ( sphincter - saving vs abdomino - perineal resection ) , disease stage , schedule and radiotherapy technique adopted , as well as toxicity detected over the treatment period . results a total of 784 pts ( m / f : 509 / 275 ) were treated between 2012 and 2016 , with a median age of 67years ( range 2592 )  . 
 the majority of patients was treated in the neoadjuvant phase ( 62% of the total ) compared to the adjuvant phase ( 31% ) and to those treated radically ( 7% )  . 
the use of chemotherapy alone before concomitant treatment is more common for patients treated in the adjuvant phase ( 64% of this subgroup ) , while 14% of patients treated in the neoadjuvant phase received induction chemotherapy before the concomitant phase ; in both cases of chemotherapy alone , the majority of patients ( 91% ) received oxaliplatin - based protocols ( folfox / xelox / capox )  . 
the overall rate of sphincter - saving surgery ( anterior resection ) was 72% , but the contribution of neoadjuvant treatment allowed to reach a rate of 83% in this subgroup ( against 65% found in the subgroup of patients treated in adjuvant phase )  . 
traditional radiotherapy schedule ( 4550gy in 2528 fractions ) was used in 90% of patients , of which an intensified treatment in neoadjuvant phase ( 45gy + boost of 910gy ) was used in 11% of patients . 
interestingly , the toxicity rates were significantly higher in the adjuvant group compared to the neoadjuvant ( gi : 58% vs 31% , gu : 21% vs 10% )  . extended author information available on the last page of the article vol . : ( 0123456789 ) 1 3 672 la radiologia medica ( 2019 ) 124 : 671681 conclusion the present survey shows that in the sicily region integrated therapies for rectal cancer have allowed a neoadjuvant approach in the majority of patients , thus resulting in a greater use of sphincter conservative surgery . 
the toxicity has also been reported to be significantly less in this treatment setting . keywords rectal cancer survey airo neoadjuvant treatment radio - chemotherapy introduction from an epidemiological point of view , cancer of the rectum is usually associated with colon cancer , as the majority of studies does not consider the two pathologies separately ; generally , in clinical trials we often refer to colorectal cancer . 
in italy , about 53.000 new cases are diagnosed per year [ 2 ] , representing the second most incident cancer in men ( after prostate cancer ) and in women ( after breast cancer )  . 
of these , around 30% arises from the rectucolorectal cancer prognosis is considered favorable , with survival rates for rectal cancer at 5 and 10years of 62% and 58% , respectively . 
the overall mortality in the world for colorectal cancer is about half of the incidence : in particular , mortality is decreasing in both males and females due to early diagnosis and admission to better surgical treatments and adjuvant therapies . 
in italy , compared to northern regions , the southern regions register lower survival rates of about 68% in males and 56% in females , with a trend toward improvement in recent years . 
in sicily , the incidence and prevalence of colorectal cancer is lower than other italian regions ; on average , based on the regional cancer registries report of years 20032011 , 1623 , 4 new cases are estimated per year in male and 1410 , 8 new cases in female . 
the 5 - year survival rate is 60% for both sexes [ 2 , 3 ]  . management of rectal cancer is a matter of debate , in particular regarding the timing of single therapeutic approaches . 
 surgery still represents the main treatment for rectal cancer , and the implementation of total mesorectal excision has dramatically reduced the local recurrence rates for locally advanced tumors [ 4 ]  . 
radiotherapy maintains a relevant role in increasing local control rates , both in preoperative and postoperative setting compared to surgery alone [ 5 ]  . in locally advanced rectal carcinoma ( larc ) of the middle - lower rectum ( localized to < 12cm from the anal verge ) , preoperative long - course radiotherapy in combination with chemotherapy with 5 - fluorouracil or capecitabine is the standard treatment , statistically reducing the incidence of local recurrences and prolonging overall survival [ 69 ]  . 
adjuvant radio - chemotherapy should be considered the standard care for patients with pt1 - 3 n1 - 2 m0 cancers , while management of pt3 n0 m0 patients is still a matter of debate . herein , we report the results of a survey conducted among sicilian centers of radiation oncology in order to assess the trend in the management of locally advanced rectal cancer , in light of the changes emerging over time from clinical trials and from the new technological developments . 
the current survey would represent the starting point to improve the integration between different therapeutic modalities and to optimize and standardize the therapeutic strategy . materials andmethods the survey was organized in radiation oncology centers of sicily region belonging to associazione italiana di radioterapia ed oncologia clinica ( airo ) by sending a questionnaire at the end of 2017 to collect the data from individual centers regarding clinical and technical aspects of radiotherapy treatments for patients affected by locally advanced rectal cancer . it was decided to collect data over a period of 5years , from january 2012 to december 2016 , in order to have a sufficient follow - up time for reporting the results and toxicity of the treatments . 
the data collected refer , in relation to the single catchment areas , to about 85% of the sicilian population that was , according to the last surveys of istituto nazionale di statistica ( istat ) , of 5 , 026 , 989 people . 
three other centers did not return the questionnaire within 6months from the date of submission and have been therefore excluded . patient data a total of 784 pts ( male / female : 509 / 275 ) were treated with radiation therapy for larc between 2012 and 2016 , so they were included in the survey and all clinical and technical data were collected . 
the distribution of patients by gender and age reflects what was shown at national level by the aforementioned aiom - airtum report , with the exception of a significant higher incidence in the male sex compared to the female , that did not correspond to the epidemiological data from the sicilian regional cancer registries [ 2 , 3 ]  . 
the incidence of the disease in the sicilian regional population appears to be smaller than the national average , and it is worth to note that about one - third of the incident cases have been treated with radiotherapy at sicilian centers . 
this would probably be related , at least in part , to interregional health migration , to a missed indication to radiotherapy or to the patients difficulties to reach radiotherapy departments . staging procedures atdiagnosis andtemporal distribution ofpatients all patients received an endoscopic procedure in order to perform a biopsy for histological confirmation of the disease before to be treated . 
an eus staging was performed in a minority of patients ( 28% ) , while a higher percentage ( 52% , 497 pts ) received a pelvic mr for local staging . 
a small amount ( 14% ) of pts received a pet or pet / ct as a part of initial staging of disease . histology was adenocarcinoma in almost all cases , with only 2% of other histologies ( i.e. , squamous cell cancer ) and 6% of carcinoma nos . 
tumor regression grade ( trg , according to dworak ) was evaluated in patients undergoing neoadjuvant radiotherapy in almost all cases . the temporal distribution of patients recruitment at radiotherapy centers is specified below : year 2012 : 137 pts year 2013 : 166 pts year 2014 : 159 pts year 2015 : 140 pts year 2016 : 182 pts treatment strategy the differences reported across the years do not reach the statistical significance . radiation therapy was prescribed with curative intent in 729 patients ( 93% ) , before or after surgery . 
the treatment had instead a radical purpose in 55 patients ( 7% ) , for whom 1 3 674 la radiologia medica ( 2019 ) 124 : 671681 no surgical indication was given . 
patients with local recurrence of carcinoma of the rectum were excluded from the survey . the spread of the neoadjuvant preoperative strategy has been significant , and indeed this therapeutic approach has been implemented in 486 patients ( 62% ) , while the adjuvant postoperative strategy has been applied in 243 patients ( 31% )  . 
oral capecitabine was preferred in 429 patients ( 73% ) , and continuous infusion of 5 - fluorouracil was used in 159 patients ( 27% )  . treatment strategy also included a systemic chemotherapy before concomitant radio - chemotherapy phase in 224 patients : this strategy has been more frequently used in patients treated in the adjuvant phase ( 64% of the subgroup ) , compared to those treated in the neoadjuvant phase ( 14% of the subgroup )  . 
the induction chemotherapy strategy , before concomitant rt - ct , was in the majority of cases oxaliplatin - based , with folfox , xelox or capox protocols . twenty - five percent of patients ( n = 196 ) did not receive combined chemotherapy , mainly for cardiovascular diseases . the therapeutic strategy is obviously dependent on tumor staging at the time of recruitment ( table2 )  . 
consequently , considering that the diagnosis of larc is conducted differently in the different centers , we classified the patients recruited as follows : ct2 - 3nx 186 pts , ct2 - 3n + 253 pts , ct4nx / + 47 pts . 
the patients treated in the postoperative adjuvant phase had the following distribution by stage : pt2n1 - 2 18 pts , pt3n0 74 pts , pt3n1 - 2 123 pts , pt4n0 12 pts , pt4n1 - 2 16 pts . 
patients treated without surgery had the following distribution by stage : ct3nx / + 19 pts , ct4nx / + 36 pts . table 2 patients distribution for treatment strategy , systemic therapy and stage at recruitment treatment strategy curative ( with surgery ) neoadjuvant treatment adjuvant treatment radical or palliative systemic chemotherapy concomitant capecitabine concomitant 5fu induction oxa - based chemotherapy before rt - ct adjuvant chemotherapy after rt - ct no chemotherapy stage distribution in neoadjuvant setting ct2 - 3nx ct2 - 3n + ct4nx / + stage distribution in adjuvant setting pt2n1 - 2 pt3n0 pt3n1 - 2 pt4n0 pt4n1 - 2 stage distribution in radical / palliative setting ct3nx / + ct4nx / + number ( percentage ) 729 ( 93 ) 486 ( 62 ) 243 ( 31 ) 55 ( 7 ) 588 ( 75 ) 429 ( 73 ) 159 ( 27 ) adjuvant strategy : 156 ( 64 ) neoadjuvant strategy ( intensification regimens ) : 68 ( 14 ) 18 ( 3 ) 196 ( 25 ) 486 ( 62 ) 186 ( 38 ) 253 ( 52 ) 47 ( 10 ) 243 ( 31 ) 18 ( 7 ) 74 ( 30 ) 123 ( 51 ) 12 ( 5 ) 16 ( 7 ) 55 ( 7 ) 19 ( 35 ) 36 ( 65 ) 5fu 5 - fluorouracil , oxa oxaliplatin , rt - ct radio - chemotherapy , rt radiotherapy 1 3 la radiologia medica ( 2019 ) 124 : 671681 radiotherapy schedule anddata fromsurgery radiotherapy was performed in most cases with a longcourse schedule ( 706 pts , 90% ) , with a total dose ranging from 45 to 60gy , both in neoadjuvant and adjuvant settings . 
 in neoadjuvant setting , a portion of the sample of patients ( 88 pts , 11% of total , but 18% of neoadjuvant group ) was treated with an intensified schedule . 
a small sample of patients ( 35 pts , 4.5% ) was treated with long - course regimen to a radical dose of 60gy , because they were judged inoperable , because of medical reasons or technically unresectable tumor . 
figure1a , b shows the details of the regimens used in the radiotherapy treatments . conven ( cid : 31 ) onal frac ( cid : 31 ) onated rt : 706 pts ( 90% ) hypofrac ( cid : 31 ) onated rt : 78 pts ( 10% ) toxicity 45 gy wp ( neoadjuvant / adjuvant ) 50 gy wp ( neoadjuvant / adjuvant ) 45 gy wp + 9 gy boost ( intensied neoadjuvant ) 50 gy wp + 10 gy boost ( radical rt ) 25 gy in 5 frac ( cid : 31 ) ons ( neoadjuvant short - course ) 30 - 39 gy in 10 - 13 frac ( cid : 31 ) ons ( pallia ( cid : 31 ) ve ) legend : wp : whole - pelvis radiotherapy fig . 
1 radiotherapy schedule used in conventional fractionated rt ( a ) and in hypofractionated rt ( b ) three - dimensional conformal technique , performed using a 3 - field technique with the patient in the prone position or with a box technique with the patient in the supine position , was preferred in the majority of centers and it was employed in 572 patients ( 73% )  . 
an intensity - modulated technique , with static step - and - shoot technique or volumetric arc - therapy , was employed in 212 patients ( 27% ) , in order to reduce dose to small bowel and bladder and increase treatment tolerability , especially for patients treated in the postoperative adjuvant phase . the survey questionnaire did not explicitly require the target volumes to be specified , which will probably be the subject of evaluation of a subsequent multicentric survey . 
 nevertheless , most of the centers participating in the survey ( 10 / 13 ) reported to have followed the airo guidelines on gastrointestinal tumors during the treatment planning procedures . surgery was performed in 729 patients ( 93% ) , and information on surgical treatment received is available for 690 patients ( 88% )  . 
data from surgery are available and complete for patients treated in adjuvant phase , while those for patients treated preoperatively are partially complete , as sometimes the follow - up is carried out by other specialists . 
the rate of sphincter - saving surgery reaches 83% in the subgroup treated with neoadjuvant combined radio - chemotherapy , versus the rate of 65% of pts treated in adjuvant phase . adjuvant radiotherapy was performed after a median time interval from surgery of 7 weeks , with a range of 512weeks . 
in the subgroup of neoadjuvant radiotherapy , the conventional interval time to surgery of 1week , in case of short - course rt , and 69weeks , in case of long - course rt , was respected in the majority of patients ( 85% )  . data on toxicity were complete for what concerns acute and subacute toxicity , since the visits during radiotherapy and in the immediate post - treatment are conventionally carried out every week in all the centers . 
as specified before , the common terminology criteria for adverse events ( ctcae v5.0 ) criteria were utilized commonly . overall , most patients reported grade i and ii toxicities , both gastrointestinal ( gi , 39% ) , cutaneous ( 23% ) and genitourinary ( gu , 14% ) , without any need to stop treatment . 
 grade iii diarrhea was developed by 31 patients ( 4% ) , of whom 20 were treated in the adjuvant phase , nine in the neoadjuvant phase and two treated radically ; a treatment interruption of 613days was then necessary in these cases , with a definitive suspension of chemotherapy . 
2 acute / subacute toxicity report in patients treated with neoadjuvant , adjuvant or radical strategy la radiologia medica ( 2019 ) 124 : 671681 toxicity report neoadjuvant treatment adjuvant treatment radical / pallia ( cid : 15 ) ve treatment gi grade i - ii tox gu grade iii tox gi grade iii tox gu grade i - ii tox skin grade i - ii tox skin grade iii tox hematologic grade i - ii tox hematologic grade iii tox legend : gi : gastro - intes ( cid : 15 ) nal . 
overall , 56% of patients performed follow - up at the radiotherapy centers , so late toxicity data are only partially available , with 83 patients ( 10% ) reporting symptoms of anal sphincter discomfort , rectal tenesmus , fecal incontinence or , on the contrary , evacuative difficulties . 
 cases of chronic post - actinic enteritis with subocclusive crises were not common ( 2% )  . discussion the management of locally advanced rectal cancer has undergone remarkable changes in recent years . 
the development of total mesorectal excision surgery and the evidences from randomized clinical trials about the use of radiotherapy and , especially , the addition of fluorouracil to radiotherapy have definitively confirmed their role in the management of this pathology in order to reduce local recurrences and to improve survival . 
randomized clinical trials have also shown that radiotherapybefore surgery further improves local control [ 8 , 1416 ]  . nevertheless , there are still many inconsistencies in the management of these patients , both during the diagnostic and staging phase , both for the treatment and the timing of the various interventions . 
the development of multidisciplinary teams can aid in increasing the use of preoperative radiotherapy and the rates of sphincter - saving procedures , as reported in the literature [ 17 ]  . the present survey was conducted in order to assess the trend in the management of larc in sicily , to record the different methods of staging and treat such disease , through the integration of radio - chemotherapy both in neoadjuvant and adjuvant settings , with the aim to improve , optimize and standardize its management . the epidemiological data from sicilian cancer registries , updated to 2016 but related to the period 20032011 , show a regional median incidence of 5509 new cases per year of rectal cancer in male and 4062 new cases per year in female . 
 according to the regional cancer registries , the male / female ratio is 58% / 42% ; our report , on the other hand , shows a higher prevalence of male sex , with a ratio of 65% / 35% . 
the overall number of new patients in the regional population is , moreover , quite higher than the number of patients treated with radiotherapy at the regional centers . the difference is probably due to a series of motivations . 
moreover , the latter are related to all tumors of the rectum , including initial stages and local recurrences , while the survey refers only to cases of locally advanced cancer . 
in this regard , the difficulty of patients to reach radiotherapy centers and the lack of treatment indication , especially for > 75yearold patients , may play a significant role . 
finally , it must be remembered that the survey was completed by 13 out of 17 sicilian radiotherapy centers , which overall treat a catchment area of about 85% of the regional population . 
the relatively low absolute number of rectal cancer patients treated at sicilian radiotherapy centers can be explained by all these motivations , as well as by the controversies related to the 1 3 la radiologia medica ( 2019 ) 124 : 671681 indication to the radiation treatment for patients suffering from pt3n0 disease . the survey highlighted significant differences between the various centers in relation to the pre - treatment staging procedures , especially for patients undergoing neoadjuvant therapy . 
regarding local staging for patients undergoing neoadjuvant radiotherapy , airo guidelines [ 18 ] , while not giving clear indications , confirm the role of transrectal echoendoscopy and pelvic mri in assessing the depth of parietal invasion and lymph nodes involvement . 
 the literature data [ 1113 , 19 , 20 ] show that mri has a greater accuracy than echoendoscopy on the definition of t ( 94% vs 90% ) and above all of n ( 85% vs 7383% )  . 
our survey showed a considerable diffusion of the use of mri in local staging , used in 52% of patients , while the use of eus appears to be reduced ( 28% of patients )  . 
regarding systemic staging , the use of chest and abdomen ct was superior than that of pet / ct scan ( 55% vs 14% )  . the main purpose of the survey was to effectively evaluate the diffusion of the organ preservation strategies at the sicilian regional radiotherapy centers , and in particular of the radio - chemotherapy neoadjuvant treatment , as within the regional airo sicilia group there was feeling of reduced use in clinical practice of this therapeutic strategy . 
surprisingly , however , we could point out that around two - thirds of the treatments carried out followed a neoadjuvant treatment strategy ( 62% ) , whose main objective is to reduce the percentage of local recurrences and to increase the resectability . 
 patients treated with radical intent to a dose of 60gy ( 4.5% ) should be added to the sample of patients treated in neoadjuvant : they were not candidates for surgery for various reasons , but nevertheless they maintained an organ preservation . 
unfortunately , the proportion of patients treated postoperatively is quite high ( 31% ) , and it deserves a further reduction , but this is due to the persistent resistance of some surgeons and the slow diffusion of multidisciplinary groups , especially in peripheral hospital centers . trials published over the last decades have indisputably demonstrated the superiority of neoadjuvant compared to adjuvant approach [ 14 , 16 ]  . 
from a careful analysis of the collected data , it can be pointed out that this practice remains more widespread in radiotherapy centers that do not have a direct collaboration with the surgeons , generally due to logistic problems . 
this involves often a unilateral therapeutic management by the surgeons at the time of recruitment , with consequent less indications to the neoadjuvant strategy . adjuvant treatment has the advantage to allow a better selection of patients , according to the pathological stage , but at the expenses of an increased acute and late toxicity , mainly gi . 
there is no difference between adjuvant and neoadjuvant approaches in terms of distant metastases [ 6 ]  . concurrent 5 - fluorouracil chemotherapy in combination with radiotherapy is considered the standard of care for larc [ 7 , 8 ]  . 
oral capecitabine has demonstrated the non - inferiority to 5 - fu [ 9 ] , so today the two drugs are considered equivalent , and capecitabine is often preferred for a better clinical compliance , with reduced issues related to the central venous access . 
the survey has demonstrated a broad adherence in the regional territory to this therapeutic standard : concomitant capecitabine was preferred in 73% of the sample , while continuous infusion of 5 - fu was administered in 27% of patients . 
regarding the use of other compounds , mainly oxaliplatin , 224 patients ( 64% of the adjuvant group and 14% of the neoadjuvant group ) were treated with folfox or capox or xelox protocol , for a median of 36 cycles , before concomitant radio - chemotherapy . 
the combination of 5fu and oxaliplatin is widely employed in adjuvant setting , both in colon and in rectal cancer , due to the evidence of a significant improvement in disease - free and overall survival [ 21 ] , but there is yet no clear evidence for the efficacy of adding oxaliplatin to the neoadjuvant multimodal treatment of patients with locally advanced rectal cancer . 
therefore , the data of currently available randomized trials using oxaliplatin - based chemotherapy in neoadjuvant setting did not allow a diffusion in clinical practice , so their use remains experimental [ 15 , 22 , 23 ]  . 
chemotherapy was not administered in 25% of the sample , mainly due to cardiovascular diseases . long - course radiotherapy is considered the standard of care both in adjuvant and neoadjuvant settings , and our data are in accordance with this clinical practice : 706 patients ( 90% of the total ) were treated with conventional fractionation to 4550 , 4gy , 125 of which treated up to 5460gy . 
 this regimen is widely considered the most appropriate when the treatment goal is to maximize tumor downsizing , increase resectability rate of initially bulky tumors and to ameliorate the rate of sphincter - saving surgery in patients initially candidate to abdomino - perineal resection . as previously reported , a fair number of patients in the survey ( 88 pts , 11% of the sample ) were treated with an intensified preoperative regimen with a concomitant boost to a total dose of 54gy . 
according to the standard of care of 4550gy , a median of 15% of pathological complete response ( pcr ) is reported in randomized trials [ 8 , 1416 ]  . 
 it is believed that an increased dose to the site of primitive tumor may allow to improve the rate of tumor regression and 1 3 678 la radiologia medica ( 2019 ) 124 : 671681 of pathologic complete response [ 24 ] ; recent trials reported a pcr rate up to 30% when an intensification regimen was adopted , generally using a boost to the macroscopic disease attempting to increase the rate of organ preservation [ 25 ]  . 
it is worth to note that , to date , there is no consensus about prescription dose , target volume definition and combination chemotherapy , so further confirmations from randomized trials are needed to allow the intensified treatment to be disseminated in clinical practice . short - course neoadjuvant protocol , 25gy in 5 fractions in a week , has been used in a few patients ( 58 pts , 7% )  . 
however , the lack of combination chemotherapy and of tumor downsizing make it not useful when patients are not candidate initially to a sphincter preservation and when mesorectal fascia is infiltrated or close to the tumor . it has been demonstrated that the rate of local control is similar between long - course and short - course radiotherapy [ 26 ]  . 
other hypofractionated regimens were adopted for 20 patients ( 2.5% ) , mainly with palliative intent for inoperable patients with low performance status , in order to reduce local symptoms ( pain or bleeding ) or the risk of occlusion . the survey reports information on the main technical aspects of radiotherapy treatment : a three - dimensional conformal technique was largely preferred toward intensity - modulated ones ( 73% vs 27% )  . 
the rationale for using imrt or vmat is to reduce dose to the small bowel and bladder and to reduce the incidence of gastrointestinal and genitourinary toxicity , especially for patients treated in postoperative adjuvant phase or when clinically or technically required . 
almost all centers participating in the survey followed the above - mentioned airo guidelines to define setup procedures , volumes of interest definition , planning , dose reporting and delivery , according to icru 62 and quality assurance procedures [ 18 , 2729 ]  . organ preservation is considered an additional goal of larc treatment . 
in recent years , it has been playing an increasing role and this has fostered novel targeted trials of intensification of treatment , both for chemotherapy and for radiotherapy [ 15 , 25 , 30 , 31 ]  . 
however , it should be noted that often the information related to the surgical treatment received by patients treated with neoadjuvant strategy is incomplete , as patients are lost to follow - up , which is often conducted by other specialists . 
in our case series , about 15% of patients have no data on the surgical treatment received after neoadjuvant radio - chemotherapy , while the surgical data of patients treated in the adjuvant phase are comprehensive . 
 taken together this data suggest that greater diffusion and integration of multidisciplinary groups is desirable in order to better understand and integrate the surgical information and the follow - up data . the increased rate of sphincter - saving surgery reported in our case series in the group treated with neoadjuvant approach does not allow definitive conclusions to be drawn , as the survey was not aimed at evaluating this parameter . 
 however , the data is in line with previously reported studies , according to which the use of the neoadjuvant approach should be considered in all patients with low rectal cancer that are not candidates for organ preservation at diagnosis . as regard toxicity , the literature clearly shows that combined radio - chemotherapy increased the rate of gastrointestinal toxicity [ 69 ] compared with the use of radiation only . 
 protocols of chemotherapy and / or radiotherapy intensification [ 15 , 21 , 22 , 25 , 31 ] have reported a further increased toxicity compared with conventional radio - chemotherapy . 
as previously pointed out , most patients reported grade iii side effects ( ctcae v5.0 ) , while grade iii gastrointestinal toxicity was reported in only 4% of sample , most of which were treated in the adjuvant phase [ 32 , 33 ]  . the tolerance of combined radio - chemotherapy is greater with the neoadjuvant approach compared to the adjuvant one . 
in the postoperative phase , in fact , as a consequence of the surgical amputation and the poor use of the surgical devices supporting the intestinal loops , the latter have greater exposure to irradiation , especially with the use of 3d - conformal radiotherapy technique . 
reports and images were evaluated by neurologists who assessed : disease - specific muscular involvement pattern ; presence of sufficient information to order the appropriate genetic / diagnostic tests ; presence of sufficient information to make therapeutic decision / perform biopsy and necessity to review mri images . 
mannwhitney and fishers exact tests were used to compare the number of key features for nsr and sr and neurologists answers for reports produced by neuroradiologists with different experience . results thirty - one srs and 101 nsrs were reviewed . 
when reports produced by expert neuroradiologists were evaluated , no significant difference in neurologists answers was observed . conclusion sr of ind - mri contained more often clinically relevant information considered important for disease management than nsr . 
radiologists expertise affects completeness of nsr reports . keywords magnetic resonance imaging neuromuscular diseases limb - girdle muscular dystrophies sarcoglycanopathies structured reporting * francesco alessandrino falessandrino@bwh.harvard.edu 1 neuroradiology department , irccs c . 
gemelli , rome , italy 3 radiology unit , istituto dermopatico dellimmacolata - irccs - flmm , rome , italy 4 neuroradiology unit , fondazione irccs ca granda ospedale maggiore policlinico , milan , italy 5 unit ofneuroradiology , irccs san raffaele scientific institute , milan , italy 6 neuroradiology unit , department ofimaging , irccs bambino ges childrens hospital , rome , italy 7 neuromuscular andrare diseases unit , department ofneuroscience , fondazione irccs ca granda ospedale maggiore policlinico , milan , italy 8 pediatric neurology andnemo clinical centre , universit cattolica del sacro cuore , fondazione policlinico universitario a . 
mondino foundation , pavia , italy 10 department ofbrain andbehavioural neuroscience , university ofpavia , pavia , italy 11 department ofradiology , harvard medical school , brigham andwomens hospital , 75 francis street , boston , ma02115 , usa vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 628635 introduction over the last few years , substantial progress has been made in the genetic diagnosis of inherited neuromuscular disorders ( ind ) with the identification of over 400 genetically distinct forms . 
this complex genetic heterogeneity makes the diagnosis extremely challenging since the most commonly used diagnostic tests , including muscle enzymes , electrophysiological studies , and muscle biopsy , are not always specific [ 1 ]  . 
it is well known that mutations in the same gene can originate different phenotypes and that the same phenotype and even the same muscle alteration detected by electron microscope can be the consequence of different genetic defects [ 2 , 3 ]  . there has been increasing evidence that muscle mri can be an important additional tool in diagnosis and follow - up of patients with ind , providing valuable information on muscle bulk , shape , volume , dystrophic , and inflammatory changes [ 48 ]  . 
mri often allows recognition of specific patterns of muscle involvement and has proved to be helpful in narrowing the differential diagnosis , aiding in the selection of the appropriate genetic and biochemical diagnostic investigations , as well as identifying which muscle to target for pathological studies [ 48 ]  . the advent of new next - generation sequencing and other molecular tools has significantly facilitated the diagnostic pathway in ind ; nonetheless , muscle mri still plays a relevant role in solving diagnostic dilemmas and in the interpretation of the results obtained from molecular panels . 
despite the bulk of evidence published in the last two decades , the use of muscle mri is still relatively limited to research settings or to a limited number of tertiary care centers . 
in the past decades , to overcome variability and increase completeness , consistency and readability of radiologic reports , structured report ( sr ) templates have been adopted in many fields of radiology , from oncologic imaging to neuroradiology [ 912 ]  . various studies analyzed the impact of clinician experience in evaluating sr in different fields of radiology , showing that sr more often contains adequate information for patient care with decreased variability compared to non - structured report ( nsr ) , in most cases [ 916 ]  . 
thus far , since muscle mri of ind has been mainly limited to tertiary care centers in which clinicians are often involved in the interpretation of the mri , and consequently , little has been reported about the possible use of sr in neuromuscular disorders and how srs are perceived by the referring clinician . 
the difficulties in this field are related to the accuracy of the reports but also to the fact that their interpretation should be based on the knowledge of the patterns of muscle involvement reported in the literature . the aim of this study was to establish whether sr of mri in patients with ind provides more clinically relevant information for disease management compared with nsr and whether the completeness of mri reports is affected by neuroradiologists level of expertise . materials andmethods study population institutional review board approval at all the institutions was obtained for this hipaa compliant study . 
from each institution , the radiology information system ( ris ) at the radiology departments of the various institution was queried for reports of mri of the lower limbs with indication of suspected / known ind generated 3years before ( from november 1 , 2013 , to october 31 , 2016 ) and 1year after implementation of a sr template ( from november 1 , 2016 , to october 1 , 2017 )  . 
only reports produced by a randomly selected neuroradiologist for each institution were included . in order to establish whether the level of expertise might have affected the accuracy of the sr and nsr , it was noted whether the mri reports were produced by neuroradiologists with more than 15years of expertise ( experienced neuroradiologists in reading mri for suspected / known ind ) or by less experienced neuroradiologists ( less than 15years of expertise ) in the corresponding referral clinical center for ind . 
all neuroradiologists reported a minimum of 50 mri reports for suspected / known ind per year . mri protocol given the retrospective nature and multi - institutional nature of the study , technical mri parameters varied among the different institutions . 
at a minimum , sequential , non - contrastenhanced , axial turbo spin echo ( tse ) t1 - weighted ( t1w ) and short - time inversion recovery ( stir ) sequences were used to study the lower limb muscles including the pelvic girdle , the thighs , and lower legs bilaterally . subjects lay in the scanner in the feet - first supine position , with the lower limbs lying in a comfortable position on the scanner bed . 
a fabricated thermoplastic splint and sandbags were used to stabilize the limbs and to minimize motion , 1 3 630 la radiologia medica ( 2019 ) 124 : 628635 when necessary . 
the patients did not receive any sedation and the total examination time was approximately 30min . mentioned in the report , regardless if the finding was positive or negative , and absent if not mentioned . turbo spin echo , t1w , and stir sequences were acquired on axial plane selected with respect to the long axis of the femoral shaft for the thighs and with respect to the long axis of the tibia and fibula for the lower legs . 
each muscle was evaluated throughout its length . scan parameters were as follows : tse t1 > tr 600ms , te 20ms , fov 1638cm ( selected according to patients size ) , pixel 1 1 , slice thickness 5 mm , flip angle 90 , interslice gap 0.5mstir > tr 4500ms , te 100ms , ti 150ms , fov 1638cm ( selected according to patient size ) , pixel 1 1 , slice thickness 5mm , interslice gap 0.5mm. descriptive analysis was used to identify the muscles that were more frequently affected in the different segments . 
each feature was considered present if the key features , in part based on a previous paper describing the pattern of muscular involvement in ind , were : ( 1 ) type of sequences acquired ( mention of type of sequence and part of body imaged ) ; ( 2 ) entity of the subcutaneous tissue relative to underlying muscular bulk ( increased , similar , or reduced ) ; ( 3 ) fat replacement of single muscle bundles ( mention of specific muscle ) ; ( 4 ) selective muscular involvement pattern , defined according to literature data ; ( 5 ) specific muscular involvement at the pelvic girdle ( mention of specific muscles ) ; ( 6 ) specific muscular involvement at the thigh ( mention of specific muscles ) ; ( 7 ) specific muscular involvement at the lower leg ( mention of specific muscles ) ; ( 8 ) specific muscle bundles hyperintense on t2 - stir sequences ; ( 9 ) overall impression [ 19 ]  . 
figure1 shows three examples of the key features evaluated on lower limb mri . evaluation ofreports andmri byclinicians after sr and nsr were selected for neuroradiologists evaluation of key features , a sample of de - identified randomly selected sample of sr and nsr was provided to clinicians for their evaluation . 
one neurologist for each institute with more than 15years of experience in ind management evaluated independently the reports and mri images . firstly , clinicians evaluated the reports : for each evaluation , clinicians were asked the following questions : 1 . 
case 1 : 16 - year - old male patient with suspected facioscapulohumeral muscular dystrophy ( fshd ) presenting with lagophthalmos , horizontal smile , winging scapulae , horizontal clavicula , limited upper limbs abduction and mild steppage gait . 
a t1 - weighted image acquired at the level of the thigh shows mild hyperintensity of the right long head of the biceps femoris muscle ( arrows ) and of both semimembranosus muscles , consistent with fat infiltration . 
b t2 / stir image acquired at the same level of the thigh showing hyperintensity of the left long head of the biceps femoris muscle and mildly of the semimembranosus of the same side , as a radiological sign of intramuscular edema ( arrows )  . 
c t1 - weighted image acquired at the level of the pelvic girdle shows fat infiltration of the bilateral gluteus maximus and gluteus medius muscles ( black arrowheads )  . 
case 3 : 11 - year - old male child with suspected limb - girdle muscular dystrophy presenting with positive gowers sign , waddling gait , prominent calf , winging scapulae and hyperlordosis . 
reports were initially screened for the presence of artifacts , 1 3 632 la radiologia medica ( 2019 ) 124 : 628635 completeness , and the presence of positive findings . 
mri reports mentioning the presence of artifacts ( n = 2 ) , interruption of the mri examination ( n = 1 ) , and mri reports with the absence of any positive findings ( n = 14 ) were excluded . 
 a total of 132 reports of lower limbs mri with indication of suspected / known ind created by a total of 6 neuroradiologists were included ; of these 101 were nsrs , 31 were srs . 
 sixty - eight mri reports were produced by neuroradiologists with more than 15years of experience in ind diagnosis ( 48 nsr and 20 sr ) , and 64 mri reports by less experienced neuroradiologists ( 53 nsrs and 11 srs )  . 
 regarding single key features , entity of the subcutaneous tissue relative to underlying muscular bulk ( p value : < 0.0001 ) , fat replacement of single muscle bundles ( p value : < 0.0001 ) , the specific muscular involvement at the thigh ( p value : < 0.0001 ) , at the lower leg ( p value : 0.0003 ) , and the presence of specific muscle bundles hyperintense on stir sequences ( p < 0.0001 ) were significantly more often reported in sr than nsr . 
the application of sr in various fields of radiology , from mammography to neuroradiology to have shown that this system has been useful to improve completeness and clarity of reports , facilitate data mining , and improve communications of results to the referring physician [ 11 , 21 , 22 ]  . our study showed that sr of mri in patients with ind contained more clinically relevant and important information for disease management compared with nsr . 
specifically , the entity of the subcutaneous tissue relative to underlying muscular bulk , the fat replacement of single muscle bundles , the specific muscular involvement at the thigh and lower leg and the t2 - stir hyperintensity of specific muscle bundles , were significantly more often reported in sr than in nsr . 
even though these findings are considered crucial for mri diagnosis of ind , our multi - institutional study shows these are not always mentioned in the mri reports , and the clinical practice varies widely according to neuroradiologists experience . 
a potential bias of this study is that the clinicians were often involved in the multidisciplinary discussion of the mri findings and that the reports may not always reflect the multidisciplinary discussion . 
nevertheless , since radiologic reports are formal documents that should be made available for patients , clinicians and other radiologists , our findings suggest that more attention should be paid to make sure that clinically relevant information is consistently present in the report . 1 3 634 la radiologia medica ( 2019 ) 124 : 628635 neurologists understood disease - specific pattern of muscular involvement and had enough information for making adequate clinical decision or to perform a biopsy more often when reading sr rather than nsr . 
furthermore , neurologists needed to evaluate images significantly less often to make decisions in patient care , when reading sr rather than nsr . these findings are in agreement with previous studies reporting that sr conveys adequate clinical information on imaging studies more often than nsr in other areas of neuroradiology and musculoskeletal radiology [ 11 , 12 , 2325 ]  . 
our study confirmed that also for mri reports of suspected / known ind , sr more often contains more often a significantly higher number of key features affecting management of this condition . similarly , regarding neurologist evaluation of reports , a previous study of our group showed that when evaluating brain mri reports of patients with known / suspected multiple sclerosis , experienced neurologists found that sr had more often sufficient information for clinical decision - making than nsr and needed to evaluate images to make clinical decision significantly more often with nsr [ 12 ]  . this study has some limitations , including its retrospective nature . 
we divided radiologists in only two categories based on their experience , more than 15years or less than 15 years of experience , clustering together novices and radiologists with moderate experience in reading mri for ind . 
more recent nsr might have been more informative than nsr performed years before , both for increased radiologist experience , and for the potential improvement related to peerreview of the cases . 
in this study , we did not evaluate this aspect , as we focused in comparing nsr with sr , although it is reasonable to predict that more recent nsr , might have been more complete than the nsr performed years before . our results clearly showed that the mri reports produced by neuroradiologists with more than 15years of experience in ind diagnosis , gave neurologists adequate information for patient care both when sr as when nsr , whereas when the report was produced by a radiologist with less than 15years of experience , only the sr gave the clinician sufficient information to understand the pattern of muscular involvement to make clinical decisions / to perform a biopsy . 
this does not suggest that muscle mri should be performed or interpreted only by very experienced neuroradiologists but rather that less experienced neuroradiologists should use sr as it will provide a structured approach to fill the mri report with all the relevant information . 
further studies are needed to establish the compliance to the use of sr within the same institution or across different institutions . in conclusion , our study suggests that sr can facilitate communication of findings and support neurologists in medical or therapeutic decisions and provide more complete information in patients with ind especially when reported by neuroradiologists with lower level of expertise . funding this research did not receive any specific grant from funding agencies in the public , commercial , or not - for - profit sectors . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . informed consent for this type of study formal consent is not required . la radiologia medica ( 2019 ) 124 : 682692 radiotherapy interobserver variability ofclinical target volume delineation indefinitive radiotherapy ofneck lymph node metastases fromunknown primary . 
acooperative study oftheitalian association ofradiotherapy andclinical oncology ( airo ) head andneck group mariannatrignani1 esterorlandi6 domenicogenovesi1 almalinabacigalupo7 angelaargenone2 saidedibiase1 danielamusio3 annamerlotti4 stefanoursino5 received : 31 october 2018 / accepted : 11 february 2019 / published online : 9 march 2019 italian society of medical radiology 2019 abstract background this study , promoted by italian association of radiotherapy and clinical oncology ( airo ) head and neck group , aimed to assess the current national practice of target volume delineation on a case of neck lymph node metastases from unknown primary evaluating inter - observer variability , in a setting of primary radiotherapy . materials and methods a case of metastatic neck lymph node from occult primary was proposed to 17 radiation oncologists . 
a comparison between following parameters of the ctvs was performed : centroids distances , dice similarity index ( dsi ) , jaccard index and mean distance to agreement ( mda )  . 
accurate target volume definition is thus an important goal and standard of quality to attain radiation oncology , in order to avoid geographic miss , reduced local control and poor survival , excessive irradiation of surrounding normal tissues and increased radiotherapy toxicity [ 16 ]  . 
highly conformal radiotherapy * marianna trignani marianna.trignani@unich.it extended author information available on the last page of the article techniques such as intensity - modulated radiotherapy ( imrt ) can increase the effects of contouring variability and inaccuracy , particularly in head and neck ( h&n ) cancer treatment where target definition is challenging and requires a detailed knowledge of h&n anatomy and pathways of tumor spread . 
several guidelines and contouring atlases have been proposed in an effort to standardize the h&n target delineation process defining both neck nodal levels for radiation oncologist practice and site - specific treatment recommendations regarding nodal station coverage [ 79 ]  . for h&n district , several authors studied the impact of inter - observer variation in organs at risk delineation , such as parotid glands , but few studies studied inter - observer vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 682692 variability in relation to h&n target volumes [ 10 , 11 ] and particularly in the setting of occult primary tumors . uptake value ( suv ) of 3.6. 
no other areas of abnormal accumulation were found . neck lymph node metastases from unknown primary represent a rare and heterogeneous malignant disease , accounting for 28% of the overall malignancies and 35% of all solid tumors [ 1216 ]  . 
prospective randomized studies are not available ; thus , treatment strategies differ widely and are based on retrospective studies , clinical experience and institutional policy [ 12 ]  . the present study , promoted by italian association of radiotherapy and clinical oncology ( airo ) head and neck group , was carried out with the aim of assessing the current national practice of h&n contouring with regard to a case of neck lymph node metastases from unknown primary and to evaluate inter - observer variability of target volume definition in a setting of primary radiotherapy . material andmethod this was a multi - institutional contouring dummy run in the context of a contouring course promoted by the airo head and neck cooperative group for a case of a patient affected by metastatic neck lymph nodes squamous cell carcinoma with an unknown primary site . case description in 2014 , a non - smoking 53 - year - old man found , by a selfexamination , a solid nodule formation located on the left site of the neck . 
at the neck and thyroid ultrasound procedure , two hypo - echoic thyroid nodules , measuring 4mm ( right lobe ) and 6mm ( left lobe ) , not vascularized and without suspect elastosonography characteristics , were identified ; a solid formation of oval shape , with inhomogeneous echogenicity ( maximum diameter of 25mm ) , adjacent to the left submandibular glands was also found . 
furthermore , it was required to define the anatomical node levels and mucosal site encompassed in the clinical target volume ( ctv ) ; considering three different dose levels corresponding to the risk of microscopic disease , ctvs were distinguished as follows : ctv high dose , ctv intermediate dose and ctv low dose [ 1719 ]  . 
finally , it was requested to indicate whether any ctv changes would be felt necessary in case of hpv positivity . contouring section a national reference rt center was identified on the basis of per - year head and neck cancer ( hnc ) - treated patients ( more than 30 ) and of hnc expertise according to scientific publications . 
contouring data from the latter were considered as the benchmark in the comparison analysis . methodology participant centers to the contouring dummy run promoted by the airo h&n group were requested to delineate target volumes by their clinical experience without specific instructions in terms of guidelines or recommendations and tools of contouring ( i.e. , atlas of reference )  . 1 3 684 la radiologia medica ( 2019 ) 124 : 682692 the planning ct scan was obtained with a standard acquisition protocol ( supine position , thermoplastic mask immobilizing both head and shoulders ; slice thickness of 2mm and reconstruction interval of 2mm )  . 
 a comparison between these parameters of the ctvs was performed [ 2022 ]  . descriptive statistics ( minimum , maximum , mean , standard deviation , median , 25th and 75th percentile and coefficient of variation [ cv ] ) was calculated for each parameter . statistical analysis was performed using spss advanced statistica 11.0 software ( spss inc , chicago , illinois , usa ) and r open source software . dice similarity index ( dsi ) , jaccard index , mean distance toagreement andhausdorff distance dice similarity index ( dsi ) , jaccard index , mean distance to agreement and hausdorff distance were computed to compare target volume of participating centers in respect of reference center . dsi is expression of contours overlap . 
it was used as a statistical validation metric to evaluate the spatial overlap accuracy of the different delineations of ctvs [ 2325 ] and compared with the contouring of the reference contour considered as the benchmark . given two observers contouring the volumes a and b , dsi is defined as : dsi = 2|a b| |a| + |b| the value of a dsi is a scalar coefficient ranging from 0 to 1 , with 0 indicating no spatial overlap between two sets of binary segmentation results and 1 complete overlap . in addition to dsi , a closely related similarity metric called jaccard index was also calculated [ 26 , 27 ]  . the jaccard index measures similarity between finite sample sets and is defined as the size of the intersection divided by the size of the union of the sample sets : j ( a , b ) = |a b| |a b| |a b| ( |a| + |b| |a b| ) 2d and d = 2j ( if a and b are both empty , we define j ( a , b ) = 1 ) .0 j ( a , b ) 1 . 
mda is a geometrical parameter that measures the per voxel shortest distance from the surface of one structure to another , ideally when two structures overlap the mda results in 0mm [ 28 , 29 ]  . the hausdorff distance is a shape comparison method based on a distance measure of the edge maps of two objects . 
diagnostic and therapeutic approach for this disease is well codified , even if some controversies exist [ 35 , 36 ]  . in the context of treatment management , surgery represents the first therapeutic step , while radiotherapy is reserved in case of macroscopic tumor residual , pn > 1 , extra - capsular extension ( ece ) , neck violation or in patient not amenable to surgery . 
the center of reference is represented in white in the intermediate dose volume ; node stations at low risk of microscopic disease should be included in the low dose volume [ 17 , 3741 ]  . 
it seems easy , but discrepancies can affect the putative primary tumor site definition , the inclusion or not of the oral cavity , the extension of the pharynx to be irradiated [ 38 ] , such as neck irradiation extension ( i.e. , bilateral vs unilateral neck , extensive irradiation )  . in this scenario , inter - observer variability must be further considered . 
target variability delineation is a well - known source of errors in radiotherapy that has been well investigated in several tumors sites but in hn district , particularly in the setting of neck nodes metastases of occult primary . the present airo head and neck group study aim was to assess current national practice of h&n contouring with regard to a case of neck lymph node metastases from unknown primary , evaluating inter - observer variability of target volume definition in a setting of primary radiotherapy . 1 3 688 la radiologia medica ( 2019 ) 124 : 682692 fig . 
considering the uncertainty regarding the occult primary management as well as the anatomical complexity of head and neck district , a dsi > 0.60 could be considered acceptable , and in this study , a dsi near 0.60 was obtained in five ctvs hd and three ctvs ld [ 24 , 46 ]  . to the best of our knowledge , this is the first experience evaluating dsi values for the inter - observer variability in delineation of h&n district in relation to nodes metastases from occult primary rt . although dsi value is often applied in studies of interobserver variability , it could not be fully representative of the variability between two volumes . 
 these parameters contributed to define spatial differences of ctvs and confirmed variability shown by dsi . in the hpv era , the viral infection represents an additional factor to consider prescribing radiotherapy volumes in nodes metastases from occult primary and it could contribute to increase inter - observer variability . 
this translates into the qualitative analyses of our study ; in fact , when it was requested to indicate any ctvs 1 3 690 la radiologia medica ( 2019 ) 124 : 682692 changes taking into account hpv status , 59% of participating ros suggested a de - intensification of the prescription . conclusion in this study , a notable heterogeneity of global radiotherapy management in nodes metastases from occult primary was detected . 
four patients presented a peak systolic velocity ( psv ) between 130 and 150cm / s , six a psv between 150 and 180cm / s , and three a psv > 180cm / s . 
further studies with longer - term outcome are necessary to confirm our results . keywords carotid artery stent in - stent restenosis dual - layer stent introduction carotid endarterectomy ( cea ) is the gold standard for treating symptomatic carotid stenosis [ 1 ]  . 
although cas is no more effective than cea in preventing subsequent stroke , [ 2 ] , its less invasiveness , decreased patient discomfort and * gianpaolo carrafiello gcarraf@gmail.com 1 diagnostic andinterventional radiology department , asst santi paolo e carlo , san paolo hospital , university ofmilan , via a di rudin 8 , 20142milan , italy 2 vascular surgery unit , surgical sciences department , asst santi paolo e carlo , san paolo hospital , university ofmilan , milan , italy 3 department ofradiology , cto hospital , azienda dei colli , naples , italy shorter hospitalization make it a good alternative in selected patients . in the last years , cases with cas have been increasing , while those with cea have been decreasing [ 3 ]  . two concerns are related to cas : the intraand periprocedural risk of vascular events and the risk of in - stent restenosis ( isr ) during follow - up [ 3 ]  . 
the use of embolic protection devices limited the risk of periprocedural stroke to 6% , which is comparable to that in patients with cea [ 4 ]  . new dual - layer stents were designed with the aim of increasing the plaque coverage and decreasing the risk of dislodging through the stent strut [ 5 , 6 ]  . 
 [ 6 ] showed good safety and efficacy of duallayer stent in the treatment of carotid stenosis , with a low vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 704709 table 1 characteristics of patients and devices used during carotid stenting with roadsaver stents incidence of delayed embolic events and new ipsilateral ischemic brain lesions . the prevalence of plaque prolapse after cas , as assessed by oct ( optical coherence tomography ) , has been estimated between 29% for closed - cell design and 69% for open - cell design [ 7 ]  . in patients with lipid - rich plaques , a higher incidence of in - stent restenosis is registered . 
plaque prolapse , as assessed by oct , is relatively low with this novel device , as revealed in a multicenter italian study [ 5 ]  . the present study is a retrospective single - center study . 
 our aim was to evaluate the incidence of in - stent restenosis at 12 - month follow - up , in patients treated with new duallayer roadsaver stent . materials andmethods we selected all patients who underwent cas and received a roadsaver ( terumo corp . , tokyo , japan ) stent between february 2017 and july 2018 ; in a total of 21 patients , 13 ( 8 men and 5 women , mean age 75.8years old ; range 6584years old ) presented a follow - up of 12months ( table1 )  . principal inclusion criteria were age > 18years and documented carotid artery stenosis . 
symptomatic patients had to have a stenosis 50% evaluated by duplex ultrasound ( dus ) according to the nascet criteria [ 8 ] ; for asymptomatic patients , the stenosis had to be 70% and suitable for treatment according to the vascular and neurological specialists . principal exclusion criteria were carotid obstruction , previous stenting at the same site , acute stroke within the last 30days , myocardial infarction within 72h and intracranial hemorrhage in the previous 12months . the clinical evaluation included a neurological examination performed by an independent neurologist using the national institute of health stroke scale ( nihss ) [ 9 ]  . plaque morphological characteristics were determined by dus and defined as echolucent ( lipidic plaque ) , echogenic ( fibrotic plaque ) and heterogeneous texture ( heterogeneous plaque )  . carotid stenosis was revealed by dus and multidetector ct ( mdct ) scan . 
the degree of stenosis determined at grayscale and dus , considering internal carotid artery ( ica ) peak systolic velocity ( psv ) , should be stratified into the categories of normal ( no stenosis ) , < 50% stenosis ( psv < 130 cm / s ) , 5069% stenosis ( 130 < psv < 150 cm / s ) , 7079% severe stenosis ( 150 < psv < 180cm / s ) , 80% near occlusion to total occlusion ( psv > 180cm / s ) [ 10 ]  . 
 angiography revealed the extension and the morphology of the plaque . 1 3 706 la radiologia medica ( 2019 ) 124 : 704709 all patients have been evaluated by a team including an expert on stroke as suggested by the iccs - spread joint committee [ 11 ] ; in particular on the basis of their risk factors and / or comorbidities , asymptomatic patients were considered susceptible to cas . all patients had at least one coexisting condition on specific anatomic and comorbid clinical criteria that potentially increased the risk posed by cea , according to sapphire criteria [ 12 ]  . 
neurological complications were quantified by the national institutes of health stroke scale ( nihss ) , and recovery was evaluated by median rankin scale ( mrs )  . characteristics of patients and their lesions are summarized in table1 . the study was conformed to the declaration of helsinki , and patients gave written informed consent for the procedure . procedure each patient was on dual antiplatelet therapy ( aspirin 100mg and clopidogrel 75mg daily for at least 3days before the procedure or loading doses of 300mg of aspirin and clopidogrel the day before the procedure )  . 
moreover , in those patients a mdct was performed to confirm findings of dus . results technical success was achieved in all cases with a residual stenosis < 30% at postprocedural angiographic examination . 
in both patients ceus and mdct denied the presence of significant stenosis ( fig.2ah ) ( table1 )  . discussion restenosis has been one of the most important remote complications of both cas and cea , although more frequently associated with the cas [ 14 ]  . 
 based on dus criterions , crest reported a cumulative isr incidence of 6.0% in 2years after cas [ 16 ]  . three forms of isr were described [ 15 ]  . 
the commonest is in - stent restenosis , which probably is due to neointimal hyperplasia , caused by a symmetrical or asymmetrical 1 3 la radiologia medica ( 2019 ) 124 : 704709 fig . 
1 ct image reveals ica stenosis and its morphology ( a ) ; angiogram performed before stenting ( b ) ; angiogram performed at the end of the procedure with a minimal residual stenosis of ica ( c ) narrowing of the lumen within the stent after an initial good result . 
the end - of - stent restenosis is similar to the recurrent stenosis after carotid patch thromboendarterectomy , which has been ascribed to local factors or technical errors . the less frequent type is tandem restenosis that may represent the most benign type regarding treatment durability [ 15 ]  . several factors seem to be implicated with the risk of isr : female gender and advanced age and concomitant conditions like diabetes mellitus , hypertension and dislipidemia may be more frequently associated with isr ; moreover , patients who received cas for stenosis related to radiotherapy or post - cea and the use of multiple stents , the use of open - cell stents and patients with post - cas residual stenosis are more prone to isr [ 3 , 6 , 1720 ]  . technological advances in endoluminal equipment made more frequent the indication for cas even in patients not strictly at risk of cea [ 21 ]  . the increase in indications for cas will require not only demonstration of feasibility and immediate clinical success but also durability in long - term studies . in a published single - center experience with 1000 carotid wallstent implantations , restenosis occurred in 3.2% ( 32 ) stented lesions ; among the retreated lesions , 1% [ 9 ] were those who had > 30% residual stenosis on the control angiogram ; all had calcified plaque [ 17 ]  . other authors analyzed the clinical impact and predictors of carotid artery isr [ 19 ] ; during the long - term followup period , the combined rate of ipsilateral stroke and death was 33.3% in the group with an isr > 70% ( 2 strokes and 2 deaths )  . 
they did not indicate the stent used , but the clinical impact of isr is important and they invited to a closer surveillance in the high - risk subgroup . double - layer stents are one of the recently introduced new stent designs to treat carotid stenoses as well . 
to date , we have literature data about feasibility , clinical success and safety about the use of roadsaver double - layer stent [ 6 , 2224 ]  . no data are published about the incidence of isr using this new generation of stent ; unfortunately , the too small population represents the biggest weakness of our study . 
no meaningful statistical analysis was possible to obtafirst of all , our preliminary results need confirmation with more patients . in one patient with suspected residual intra - stent stenosis at the final angiogram , a minimal intimal hyperplasia was revealed at 6 - month follow - up , not increased at 12 - month examinations . 
the only first large series published [ 24 ] revealed a restenosis rate which does not seem different 1 3 708 la radiologia medica ( 2019 ) 124 : 704709 fig . 
2 ct image reveals ica stenosis and its morphology ( a ) ; angiogram performed before stenting ( b ) ; angiogram performed at the end of the procedure with a minimal residual stenosis of ica ( c ) ; b - mode us image ( d ) and dus image ( e ) at 12 - month follow - up : the latter shows a minimal intra - stent intimal hyperplasia ( white arrow ) ; psv of about 80cm / s reveals a nonsignificant intra - stent stenosis ( f ) ; ct scan confirms minimal nonsignificant intra - stent intimal hyperplasia ( g , h ) from other stents , although randomized studies are still lacking . 
the same authors hypothesized a novel pattern of restenosis , with plaque growth between the two layers , probably associated with a lower embolic risk . our data should be considered as supportive to those just published . 
a second objective was to assess whether there were other histopathologic features that could affect mean adc value . methods in this 4 - year retrospective study were included 125 patients who underwent radical or modified mastectomy for monofocal bc . 
adc value was shown to be significantly related to tils level ( p < 0.0001 ) and cancer histotype ( p = 0.0006 ) , with a lower mean adc value correlated to absentlow til level and ductal histotype . conclusion bcs with absentlow til showed a statistically significant lower mean adc value than those with mediumhigh til . 
the latter is the treatment of choice for patients with locally advanced breast cancer , and those who show pathologic complete response ( pcr ) to nac may experience prolonged disease - free survival . 
 [ 16 ] have shown that higher til level is associated with her2 + dcis . in this study , we hypothesized that adc value could predict til level , which could help to identify bcs with a better response to nac and patients with prolonged disease - free survival . 
a second objective was to assess whether there were other histopathologic features that could affect mean adc value . materials andmethods study population the ethics committee of our institution approved this retrospective study . 
exclusion criteria were : preoperative breast mri in another institution ( n = 5 ) ; mri performed without dwi sequence ( n = 3 ) ; and history of neoadjuvant treatment for bc ( n = 28 )  . 
according to these criteria , the final cohort consisted of 125 patients . mri technique all mri examinations were performed with 1.5 - t machine ( achieva , philips healthcare , andover , ma ) and a 7 - channel dedicated breast coil ( sense breast 7 ch ) with patients in prone position . 
in order to assess adc value , a region of interest ( roi ) was manually drawn in each slice in which the lesion was detectable on the b = 800smm 2 dw images . 
only mean adc value , which corresponds to the area - weighted average of the adc values found within each lesion slice , was used in the analysis . pathologic analysis all patients underwent radical or modified unilateral mastectomy for monofocal bc . 
the surgical piece was fixed in formalin and included in paraffthin sections were evaluated by the same pathologist with more than 10 - year experience in breast pathology , and the presence of til was assessed . 
 [ 15 ] , based on til level , we divided bcs into two groups : tumors with til level 10% were classified as absentlow til and those with til level > 10% were classified as mediumhigh til . 
 tumor size , histotype , estrogen and progesterone hormone receptor expression ( er , pr ) , human epidermal growth factor receptor 2 ( her2 ) expression , proliferation defined by the ki - 67 labeling index , histologic subtype , grade and tumor cellularity were also recorded . 
her2 positivity was defined as 3 + score by ihc in > 30% of invasive 1 3 la radiologia medica ( 2019 ) 124 : 581587 tumor cells using the hercept test ( dako , glostrup , denmark )  . 
equivocal cases at ihc ( 2 + score or 3 + in 30% of invasive tumor cells ) were subjected to fluorescence insitu hybridization ( fish ) analysis [ 17 ]  . 
tumor cellularity was defined as percentages in 10% increments and was calculated from five arbitrarily selected high - power fields in each specimen using a computer prograaccording to tumor cellularity , we divided bcs into two groups : tumors with low cellularity ( < 60% ) and tumors with high cellularity ( 60% )  . 
adc threshold was chosen by the area under roc curve ( auc ) and youden index . in order to assess histotype , grade and ki - 67 expression differences between absentlow til group and mediumhigh til group , mannwhitney u test was used . 
moreover , tils were shown to be the most important factor on which mean adc value depends . within the tumor , there are different cellular and stromal components , on which the average adc value of the neoplastic lesion may depend . 
moreover , adc is also dependent on histotype , and this finding is probably attributable to the lower cellular density of invasive lobular cancer compared to ductal ones . dwi is an mri technique that does not require contrast medium , with a short acquisition time and high specificity in bc detection [ 2022 ]  . 
1 a 34 - year - old woman with a 15 - mm invasive ductal cancer in her left breast with absentlow til that appears hyperintense on dwis obtained at b = 0 ( a ) and b = 800 ( b ) and hypointense on adc map ( 0.891 * 103mm2s1 ) ( c ) including histotype and proliferative index [ 8 , 9 , 23 ]  . 
 [ 24 ] demonstrated that adc value was significantly higher in er - negative than in er - positive cancer , but we did not find this . to our knowledge , there are no previously published works in scientific literature comparable to this one . 
 [ 25 ] obtained a non - statistically significant difference in mean adc value between bc with low and high til levels , but they only considered triple - negative bcs and used a til threshold value of 50% to distinguish the two groups . 
 [ 26 ] reported that only tumor cellularity and ki - 67 were independently associated with mean adc value , and not tils . in the present study , the choice to stratify bcs into two til groups , as reported by asamo etal . 
 [ 15 ] , was performed because most of the previous studies that evaluated til as a predictor of nac response used 10% as a cutoff value [ 11 ]  . 
however , til level assessment , to date , presents a poor intra - observer reproducibility , and it is a subject of debate in the current scientific literature [ 2729 ]  . 1 3 la radiologia medica ( 2019 ) 124 : 581587 fig . 
2 a 46 - year - old woman with a 12 - mm invasive ductal cancer in her right breast with mediumhigh til that appears hyperintense on dwis obtained at b = 0 ( a ) and b = 800 ( b ) and hypointense on adc map ( 1.023 * 103mm2s1 ) ( c ) fig . 
3 a 39 - year - old woman with a 18 - mm invasive lobular cancer in her right breast with absentlow til that appears faintly hyperintense on dwis obtained at b = 0 ( a ) and b = 800 ( b ) and hypointense on adc map ( 1.208 * 103mm2s1 ) ( c ) fig . 
4 a 45 - year - old woman with a 20 - mm invasive lobular cancer in her left breast with mediumhigh til that appears faintly hyperintense on dwis obtained at b = 0 ( a ) and b = 800 ( b ) and hypointense on adc map ( 1.354 * 103mm2s1 ) ( c ) til level in bc has recently taken an important oncological role [ 30 , 31 ]  . 
 in some studies , it showed a positive predictive value for response to nac , and patients that show a pcr to nac may experience prolonged disease - free survival . 
we review the various surgical techniques used in pe , discuss optimal ct protocols for postsurgical evaluation and describe cross - sectional imaging appearances of normal postoperative anatomic changes as well as early and late complications . 
gemelli 8 , 00168rome , italy introduction pelvic exenteration ( pe ) is a radical en bloc resection of multiple endoand exopelvic organs affected by cancer , followed by reconstructive surgical procedures to restore compromised urinary , intestinal and sexual functions [ 1 ]  . 
early and late postoperative complications after pe in patients treated for advanced , persistent or recurrent gynecologic cancers are vol . : ( 0123456789 ) 1 3 694 la radiologia medica ( 2019 ) 124 : 693703 the focus of this review . 
we describe the types of pe and the expected ct imaging findings after surgery , showing the ct appearances of most common complications including bleeding , wound infection , urinary injury ( anastomotic leak , stricture , fistula ) and gastrointestinal injury ( anastomotic leak , bowel occlusion , fistula )  . 
lastly , the imaging pattern of local tumor recurrence after pe is discussed . surgical techniques different types of pe can be performed depending on the tumor location and extent to other pelvic structures . 
supralevatoric pe is a resection above levator ani muscle , with spare of anal sphincter and urogenital diaphragtranslevatoric pe is an excision below pelvic floor muscles , with partial or complete removal of levator ani muscle , anal sphincter and urogenital diaphragmore extended exenteration is required when tumor affects the pelvic sidewall [ 8 ]  . 
the laterally extended endopelvic resection ( leer ) includes resection of all pelvic viscera with any of the endopelvic parietal structures , such as paravisceral fat pad , internal iliac vessels , obturator internus and levator ani muscles [ 9 ]  . 
extended pelvic resection ( epr ) is characterized by an extensive pelvic excision including pelvic sidewall muscles , bones and major neural and / or vascular structures as common and / or external iliac vessels [ 10 ]  . 
b sagittal ct scan , acquired 7days after total pe , shows the absence of bladder and the presence of bilateral ureteral stent ( curved arrow ) due to urinary diversion . 
 b sagittal ct scan illustrates the pelvic cavity after anterior pe with the absence of lower urinary tract organs ( urinary bladder and urethra ) 1 3 la radiologia medica ( 2019 ) 124 : 693703 fig . 
 the imaging protocol usually includes abdomen and pelvis ct scan ( with or without chest ) before and after intravenous ( iv ) administration of contrast material [ 13 ]  . 
enhanced arterial and portal phases ( 3040s and 7080s delayed acquisition after injection of contrast media ) are mandatory to detect active bleeding , in case of suspected hemorrhage [ 14 ]  . 
after scanning , the radiologist can use post - processing techniques as multiplanar reformatting ( mpr ) , maximum intensity projection ( mip ) and volume rendering ( vr )  . 
therefore , the familiarity with the type of pe performed helps the radiologists to interpret imaging and differentiate between normal findings and postsurgical complications . after anterior pe , ct allows recognition of the absence of bladder / urethra and the presence of a urinary reconstruction , also known as urinary diversion . 
mpr and mip postprocessed images may be useful to accurately detect ureters and the integrity of ureteroenteric anastomosis [ 17 ]  . when vaginectomy is performed during anterior pe , the reconstruction of functioning vagina is indicated . 
coronal reformatted mip image , from a ct scan acquired at excretory phase ( 15 min delayed after contrast media injection ) , shows the ileal conduit ( white arrow ) located in the right lower abdomen and the ureters anastomosed at its proximal end . 
 the black arrow indicates the ureteroenteric anastomosis such as the absence of rectus abdominis muscle in its normal site and the evidence of vram flap vascular pedicle represented by the deep inferior epigastric artery [ 22 ]  . postoperative pelvic imaging afterposterior pe posterior pe consists of two main surgery procedures : translevatoric pe with creation of end colostomy and supralevatoric pe with colorectal / coloanal anastomosis . during translevatoric pe , rectum and anus are resected , and a permanent colostomy is usually created in the left lower abdominal quadrant . 
combined positron emission tomography ( pet ) / ct might be considered a complementary method if tumor 1 3 la radiologia medica ( 2019 ) 124 : 693703 recurrence is suspected , because of its higher sensitivity and specificity compared to ct [ 25 ]  . the linea alba and the sacrum [ 23 ]  . 
ct images show the bowel anastomosis line evidenced by surgical clips and the enlargement of the presacral space with anterior displacement of the rectua marked ( 35cm ) dislocation of rectum from the sacrum associated with persistent ( > 6months ) fluid or air in the presacral space is suspicious for anastomotic leak , fistula or tumor recurrence [ 24 , 26 ]  . pelvic soft tissue reconstruction after posterior pe is based on the use of autologous vascularized pedicle flaps . 
due to its angiogenetic property , omental flap helps the wound healing and reduces postsurgical complications , like abscesses and adhesions between pelvic floor and organs [ 22 ]  . 
axial enhanced ct image shows the omental pedicle flap with fat density ( star ) in the presacral space and the small bowel dislocated in the anterior compartment of the pelvic cavity postoperative pelvic imaging aftertotal pe total pe is a complete exenteration of pelvic organs that consists of both anterior and posterior pe . 
pelvic reconstruction is also required to restore sexual functioning and to provide an acceptable body image . postsurgical complications postoperative complications after pe can be classified as early ( < 30days ) and late ( > 30days ) according to the time interval from surgery [ 27 ]  . 
common postoperative complications include hemorrhage , pelvic abscess , wound infection , urinary injury ( leakage , obstruction , fistula ) and gastrointestinal injury ( anastomotic leak , bowel occlusion , fistula )  . 
ct with the use of multiplanar reformatted images offers an accurate identification of complications by providing a support for the surgeon to decide the best treatment approach for each patient . general complications hemorrhage andhematoma hemorrhage is an intra - operative and early postoperative major complication with an incidence rate of 39.4% and a potential fatal prognosis if not diagnosed on time [ 29 ]  . 
bleeding commonly originates in the presacral plexus or in the internal table 2 type of pe and their most common related complications anterior pe posterior pe total pe hemorrhage infection / abscess wound dehiscence urinary tract injury gastrointestinal tract injury + pe pelvic exenteration + , low risk ; + + , high risk 1 3 698 la radiologia medica ( 2019 ) 124 : 693703 fig . 
bilateral deep thrombosis of external iliac veins ( arrowheads , c ) is also present iliac vein , or it can be locally confined to wound site [ 29 ]  . 
bleeding may also develop as pelvic hematoma , which typically shows higher density ( 7090 hu ) at unenhanced ct scan [ 13 ]  . infection andpelvic abscess infection remains one of the most common complications of surgical procedure in gynecology [ 30 ]  . 
the management is various , and if no surgical revision is required , ct - guided drainage in combination with antibiotics is the preferred treatment approach [ 30 ]  . wound infection anddehiscence about 214% of patients who underwent pe develop wound infection , most commonly involving the laparotomy site [ 27 ]  . 
sagittal enhanced ct scan shows a fluid collection containing a large amount of gas in the presacral space ( line arrow ) associated with a widening of the presacral space ( > 35 cm )  . 
urography ct is useful to study enterourinary fistula ( fig.9 ) , while ct with rectal administration of water - soluble iodinate contrast is indicated in case of low enterogenital fistula [ 16 , 17 ]  . 
as recurrent tumor may cause fistulas , accurate evaluation of any soft tissue mass or other signs of malignancy should always be done . complications afteranterior pe urinary leak urinary leak from a newly formed conduit is a major , early postoperative complication after anterior pe . 
the urine leak rate in the pe surgery is reported in excess of 15% , while the urine leak rate after conduit formation for a primary urological malignancy is < 5% [ 3537 ]  . 
the diagnosis is related to the presence of creatinine - rich fluid from abdominal drains or wound sites , or evidence of sites of iodinated urine extravasation on imaging [ 35 ]  . 
depending on the site , there are two types of leak : ( a ) anastomotic leak , when urine extravasation originates from ureteroenteric anastomosis , and ( b ) conduit leak , when urine extravasation originates anywhere else along the conduit . 
urography ct may show iodinated urine outside the injured ureteral / conduit segment and the shedding into the retroperitoneum or less commonly into 1 3 700 la radiologia medica ( 2019 ) 124 : 693703 fig . 
9 malignant enterourinary fistula between the ileal conduit and the cecum in a 52 - year - old woman who underwent total pe for recurrence of cervical cancer ( 10 months earlier )  . 
a coronal and b axial images , from a ct scan acquired after administration of water - soluble iodinated contrast through the ileal stoma , show the opacification of the cecum and the presence of a fistulous tract ( line arrows ) that connects the ileal conduit ( dashed line arrows ) to the cecu the presence of solid enhancing masses ( arrowhead , b ) in the right side of the pelvis , adjacent to the ileal conduit , represents recurrence of tumor the peritoneal cavity . 
the management is conservative if the urine leak is small and asymptomatic ; otherwise , it needs surgical revision or radiological intervention ( percutaneous nephrostomies or percutaneous drain insertion ) [ 37 ]  . urinoma urinoma is a urine collection located commonly in perirenal space or in the retroperitoneum along psoas muscle . 
ct scan , during excretory phase , shows the progressive increase of attenuation inside the urinoma due to iodinated urine entering into the collection [ 15 ]  . urinary stricture urinary stricture generally involves the ureteroenteric anastomosis and occurs lately within 12 years after surgery . 
ureteral strictures are classified as benign , in case of ureteral fibrosis secondary to ischemic lesion or improperly fashioned anastomosis , and malignant when tumor recurrence grows close to ureteroenteric anastomosis . 
b axial excretory phase ct image , acquired 10min later , shows an influx of contrast material into the collection ( arrow ) 1 3 la radiologia medica ( 2019 ) 124 : 693703 fig . 
c coronal excretory phase ct scan confirms the soft tissue lesion ( arrow ) located close to the right ureteroenteric anastomosis associated with dilation of the urinary tract above on the right side ( black star ) complications afterposterior pe bowel anastomotic leak bowel anastomotic leak is a major , early complication after posterior pe associated with a high morbidity . 
the extravasation of rectal contrast medium is the most predictable sign for an anastomotic leak [ 39 , 40 ]  . bowel occlusion bowel occlusion is a late complication that occurs in 4.415.4% of cases after posterior pe and involves more frequently the small bowel [ 27 ]  . 
closed - loop obstruction is a type of small bowel occlusion where a bowel segment is obstructed at two adjacent points along its course ( fig.12 ) ; when the closed - loop rotates on its vascular axis , it forms small intestinal volvulus . 
radiologist should exclude bowel strangulation , perforation and peritonitis that are potential fatal sequelae of bowel occlusion . adynamic ileus is a common early cause of functional bowel occlusion due to the paralysis of intestinal motility in the immediate postsurgical timing . 
generally , this condition does not last more than 48h after surgery and can be conservatively managed . complications aftertotal pe total pe is a more radical exenteration surgery than partial pe with higher rate of postoperative complications . 
urinary and gastrointestinal tract complications are both documented after this surgical operation ( table2 )  . 1 3 702 la radiologia medica ( 2019 ) 124 : 693703 conclusion pe remains the most challenging surgical operation in patients with advanced , persistent or recurrent gynecological cancers . 
12 small bowel obstruction due to adhesions in a 48 - year - old woman who underwent posterior pe for recurrence of cervical cancer ( 4 months earlier )  . 
an inter - observer reliability analysis was performed . results the prevalence of emissary veins in mri was found in the right mastoid emissary vein ( mev ) 82.7% and left mev 81.4%. 
there was a statistically significant difference between the left mev and oev and transverse sinus anatomic variations . conclusion mr imaging is a noninvasive and irradiating imaging method for detecting posterior fossa major emissary veins , and we recommend using mr imaging for preoperative evaluation of posterior fossa major emissary veins and related dural venous sinuses . keywords magnetic resonance cranial fossa cerebral veins mastoid introduction emissary veins are venous structures without valves and are located between the venous sinuses and the extracranial venous circulation . 
the blood flow in the emissary veins is usually from external * pinar gulmez cakmak pinarcakmak20@gmail.com 1 department ofradiology , faculty ofmedicine , pamukkale university , kinikli kampusu , 20100denizli , turkey to internal . 
in some conditions , such as intracranial vascular malformations or internal jugular venous hypoplasia of craniosynostosis , venous drainage of the brain is provided by emissary veins [ 5 ]  . 
anatomic variations of the major venous sinuses appear as hypoplasia or aplasia in cross - sectional imaging [ 69 ]  . preoperative detection of emissary veins , particularly in temporal bone surgery , within mastoid bone or in close proximity , may prevent complications such as bleeding or venous thrombosis . 
digital subtraction angiography , computerized tomography angiography and magnetic resonance venography ( mrv ) are used in the radiological evaluation of the brain venous systemrv can be achieved by using either non - enhanced ( two - dimensional time of flight mrv , phase contrast mrv ) or contrast - enhanced vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 620627 ( gadolinium enhanced 3 - dimensional mrv , auto - triggered 3d gd - enhanced mrv ) methods . 
contrast - enhanced mrv has the advantage of showing the detail of intracranial venous anatomy and has relatively faster acquisition time compared to non - enhanced mrv [ 1113 ]  . 
there are studies about transverse sinus and sigmoid sinus anatomic variations in the literature [ 79 ]  . we found no other radiological study investigating the relationship between the presence of emissary veins and the anatomic variations of venous sinuses . 
the aim of this study was to find the prevalence of emissary veins in the posterior cranial fossa and to compare the presence of these emissary veins with the anatomic variations of the dural venous sinuses detected in mr venography . materials andmethods study population the study protocol was approved by the local ethics committee of our university . 
posterior fossa emissary veins and dural venous sinus anatomic variations in 247 patients who underwent mr venography ( mrv ) examinations were evaluated retrospectively between january 2015 and september 2017 . 
1 axial mr images show that mastoid emissary vein ( mev ) diameter measurements ( close to the medial wall of the cranial bone ) mri was performed using two 1.5t superconducting magnets ( ge signa excite hd ; ge medical systems , usa , ingenia ; philips medical systems , best , the netherlands ) with an 8 - channel neurovascular coil . 
subsequently , the statistical analysis presence of the emissary veins was compared with the dural venous sinus anatomic variations . data analysis was performed on a personal computer using statistical software ( spss 16 for windows , chicago , il )  . 
the absence of left mev ( p = 0.007 for r1 , p = 0.011 for r2 ) and the presence of oev ( p = 0.005 for r1 , p = 0.004 for r2 ) and right pcev ( p = 0.038 for r1 , p = 0.024 for r2 ) were found to be statistically significant in women for both radiologists . 
no correlation was found between the other variations of transverse and sigmoid sinus with gender . there was not any statistically significant difference between transverse and sigmoid venous sinus anatomic variations and the presence of posterior condylar emissary veins . 
however , a statistically significant difference was found between the anatomic variations of the left mev and oev and transverse sinus ( table4 )  . discussion various sizes of emissary vein have been described in previous studies [ 1420 ]  . 
 other studies investigating pcev prevalence have been performed with smaller numbers of patients or cadavers according to our study , and different prevalence values have been reported [ 24 , 14 , 18 ]  . 
in our study , the largest mastoid emissary vein diameter was 4.9mm and posterior condylar emissary vein diameter was 6.0mm. the comparison of the frequency of mev , pcev and oev in the present study and the previous studies is presented in table5 . there are some differences in the prevalence of mev among the studies performed in the literature using cadaver and imaging modalities . 
 [ 19 ] reported that the prevalence of occipital foramen ( of ) in over 200 specimens was 7% on the right and 4% on the lein the literature , there is no imaging study about the prevalence of oev . 
 [ 23 ] reported that emissary veins serve to cool the venous blood table 5 the comparison of the frequency of the emissary veins study type mev prevalence ( % ) pcev prevalence ( % ) oev prevalence ( % ) ruiz etal . 
 bleeding is common , and tactics for achieving hemostasis of the mev include bipolar coagulation , vessel ligation , and packing the vein with hemostatic material [ 10 ]  . 
a case of dural arteriovenous malformation embolized via enlarged mev has been reported [ 29 ]  . in the literature , there are ct and cadaver studies in which the emissary canal is evaluated [ 3 , 1416 , 30 ]  . 
in addition , the ultrashort tr and te enable has extremely short acquisition times ( shorter than fse ) , and the images can be post - processed using mip , volume rendering or 3d techniques . 
mr venography is frequently used in dural venous sinus imaging , except for patients with contraindications to mr ( e.g. , pacemakers , cochlear implants , non - mr compatible cerebral aneurysm clips )  . 
magnetic resonance venography have made it possible to visualize the dural sinuses and cerebral veins without the use of invasive procedures or ionizing radiation [ 1113 ]  . dural venous sinuses are the major blood drainage system in the craniuthe sinuses in the superior group are superior sagittal ( sss ) , inferior sagittal ( iss ) , straight ( ss ) , occipital ( os ) , transverse ( ts ) and sigmoid sinuses . 
reported that 21 ( 20% ) patients had aplasia of the left transverse sinus , 41 ( 39% ) patients hypoplasia of the left transverse sinus , 33 ( 31% ) patients symmetric sinuses , 6 ( 6% ) patients hypoplasia of the right transverse sinus , and 4 ( 4% ) patients aplasia of the right transverse sinus [ 8 ]  . 
in our study , 119 ( 54.1% ) of the symmetric transverse sinus and 67 ( 30.4% ) of the left hypoplastic / aplastic transverse sinus , 165 ( 75% ) of the symmetric sigmoid sinuses and 35 ( 15.9% ) of the left hypoplastic sigmoid sinus were found . 
we did not find any significant difference between the rest of anatomic variations of the venous sinus and the gender . embryologically , emissary veins were identified from the third month of gestation and the development of the emissary veins has been reported to develop after transverse sinus ballooning [ 21 ]  . 
these findings in our study demonstrate a strong association between the presence of the emissary veins and 1 3 626 la radiologia medica ( 2019 ) 124 : 620627 the dural venous sinus variations starting with ballooning of the sinuses in embryological development . our study has several limitations such as being retrospective . 
in our study , brain mr and mrv were noninvasive and irradiating imaging methods and no comparison was made with other imaging modalities ( ct angiography , digital subtraction angiography )  . 
finally , the number of patients is limited and to increase the statistical significance of the relationship between dural venous sinus variations and the presence of emissary veins could be done with increasing the number of patients . conclusion mr imaging with a balanced gre sequence is a successful method for detecting posterior fossa major emissary veins . 
 the prevalence of emissary veins in mri was found at right mev to be 82.7% , left mev 81.4% , right pcev 77.3% , left pcev 75% and oev 28.6%. 
from a radiological perspective , this correlation proves that the posterior fossa emissary veins were formed by a ballooning from the transverse sinus . authors contributions pgc was involved in protocol development ; data collection and management ; data analysis ; and manuscript writing and editing . 
fracture lines and comminution zones were identified by reduced three - dimensional computed tomography reconstructions and then graphically superimposed onto a standard template to create two - dimensional fracture maps , followed by the conversion into heating maps . 
based on qualitative descriptive fracture mapping analyses , the patterns of intra - articular distal radius fractures were determined . results it was observed that the highest fracture line intensity was located as an inverted t shape zone in the dorsal aspect of the joint with high incidence of fractures and the prominently intense color in heat mapping . 
in this study , a new surgical approach was attempted , but due to the lack of evidence - based evidence , long - term postoperative complications and hand function should be further evaluated . conclusion this study redefines a new method for the classification of intra - articular fractures of the distal radius , which allows doctors to have a clearer understanding of the characteristics of distal radius fractures . 
 although the surgical management for drfs is variable , the principle is to restore the bone anatomic structures and to recover the function of the wrist joint after rigid internal fixation as soon as possible . 
recently , with the development of volar locking palmar plates , which have been demonstrated to have a low - profile design , a capacity of neutralizing the load across the fracture sites and the non - requirement of vol . : ( 0123456789 ) 1 3 614 la radiologia medica ( 2019 ) 124 : 613619 good bone quality , volar locking plate fixation has been widely used in the treatment of distal radius intra - articular fractures . 
in particular , due to the unexposed joint capsule through the volar locking approach , the location and dependence of internal fixation for fractures of the distal radius articular surface is principally relied on x - ray examinations and surgeons experience , requiring precise and effective methods . 
therefore , it has become a strong interest for surgeons to know how to achieve the articular surface fracture fixation after restoring the articular surface and whether to add other screws when volar locking plate distal screws are used to fix important fracture fragments , especially for the elderly patients with osteoporosis [ 2 ]  . 
 recently wrist arthroscopy may be an effective method to observe the fracture line of distal fractures but not popular because of technique and expense [ 3 ]  . the current study aimed to explore the distribution characteristics of the fracture lines in drfs by building fracture maps reconstructed using axial computed tomography ( ct ) scan images of patients with intra - articular fractures of distal radius . 
as fracture mapping can contribute to the better understanding and accurate evaluation on the fracture patterns of drfs ( e.g. , location and frequency of the injury of the articular surface ) , through fracture mapping , surgeons hence could further anticipate the fracture patterns through superimposing fracture lines , zones of comminution and articular involvement , which provide novel mechanistic insights and develop therapeutic strategies of drfs [ 4 ]  . materials andmethods patients consecutive image sampling was conducted for the patients with distal radial fractures at department of orthopedics of tongji hospital ( tongji university , shanghai , china ) between july 2014 and january 2016 by using the international classification of diseases , ninth revision , clinical modification ( icd - 9 - cm ) codes . 
in total , 40 of 265 patients with distal radial fractures were screened for entry into the current study . fracture mapping three - dimensional computed tomography ( q3dct ) analyses were performed to visualize the distributions and locations of fracture lines . 
 the selected ct images were then reloaded into software mimics 17.0 ( mimics , boston , ma ) , and bone structures were marked manually in axial , sagittal and coronal planes to create 3d polygon mesh reconstructions . 
all the articular images were subsequently superimposed by software photoshop cc ( version 2015.5 ; adobe systems incorporated , san jose , ca ) to build a frequency diagram as determined by the density of fracture lines . heat mapping anddata analysis as a mean to obtain a more intuitive understanding of the 2d fracture mapping , the initial diagrams were also converted into fracture heat maps by using photoshop cc ( adobe systems incorporated )  . 
the heat maps were created based on the fracture lines and represented the relative frequency of fracture lines that have been graphically displayed as colors . the analysis of the fracture maps was descriptive . 
in the heat mapping , the distribution frequency of the fracture on the articular surface is distinguished by the change in appearance of the intensity . results there were 23 ( 57.5% ) female and 17 ( 42.5% ) male cases for intra - articular fractures of the distal radius eligible in the current study . 
according to the association for osteosynthesis / association for the study of internal fixation ( ao / asif ) fracture classification system , there were 3 cases ( 7.5% ) of type b1 , 2 cases ( 5% ) of 1 3 la radiologia medica ( 2019 ) 124 : 613619 fig . 
a free bone fragments marked with different colors ; b a 3d reconstructed model ; c a 3d image manually reset ; d a 3d image with the fracture lines drawn fig . 
a in the process of 3d reconstruction , the backbone of radius was in translucent mode and the position of the backbone was then adjusted to form the ellipse through overlapping the margin of the bony cortex . 
by adjusting the parameters in the diagram ( width and depth ) , the center of the ellipse was adjusted at the centroid of the articular surface ( i.e. , the intersection of dashed red lines ) to form a standard articular surface . 
b illustration of the articular view and some important anatomy landmarks such as flexor pollicis longus ( fpl ) , the dangerous zone ( fpl would be threatened in this zone during the volar plate implanting ) and the safe zone . 
on the volar aspect of the joint , the fracture lines were shown to be less distributed and dispersed , which are accompanied with the lower color intensity in heat mapping . 
as the functions of the wrist joint mostly depend on the integrity and axial direction of the distal radius , precise reconstruction of the articular surface and rigid fixation of main fracture fragments are the key to reduce the risk of traumatic osteoarthritis and achieve satisfactory function recovery . 
in this fixation technique , to be able to insert the distal locking screws into the proper locations and to the accurate depth is well known 1 3 616 la radiologia medica ( 2019 ) 124 : 613619 fig . 
b the intensity of the color represents the frequency of the fracture occurrence , and fracture line intensity is illustrated by heat mapping analyto be the foundation to achieve adequate fixed capacity and the compression of debris . 
however , the proper treatment of intra - articular fracture of distal radius requires great understanding of the fracture pattern and optimal preoperative planning for surgeons ; and thus , there is an urgent requirement of the surgeons in predictably defining the fracture patterns in the articular surface of distal radius before operation . 
although fracture mapping techniques have obtained the substantial advancements and have been widely used in clinical practice to further understand the injury patterns [ 5 ]  . in the current study , intra - articular distal radius fractures were mapped by 3d reconstruction of articular surface with the data transferred from preoperative ct examination , which is currently widely used in assessing the distal radius fractures [ 6 , 7 ]  . 
thus , articular surface details such as the ankle , fossa and protrusion of the articular surface were clearly displayed . in addition , by analyzing fracture line distribution , the location of the fragments could be inferred , and the area of the metaphyseal collapse ( effective bone loss ) companied with potential instability also could be estimated . 
two of the fracture lines traverse the articular surface and intersect . as the most common type , type is observed to be with more bone loss and less lunate and scaphoid fossa surface . 
a lunate fossa and scaphoid fossa : two independent stress distribution areas in the radial articular surface , which could form the radiocarpal joint with the lunar and scaphoid ; sigmoid notch : an important anatomical structure of the radius , which forms the distal radioulnar joint with ulnar head and serves as the pivot of forearm pronation and postpronation . 
b the keystone area : the intermediate column ( ic ) in the three - column biomechanical construction and the main conduction area of the energy load which is often used as a key point for screw placement 1 3 la radiologia medica ( 2019 ) 124 : 613619 fig . 
dorsal approach is thus recommended in surgery . in addition , there was also acceptable bone loss found in type , which could be treated by volar approach surgery , so that bone grafting may not be performed intraoperatively . 
 the fracture lines at the lunate fossa or scaphoid fossa were characterized to be intensively distributed at the edge of the fossa and directly through the center of the fossa sparsely . 
 previous studies have shown that the reconstruction of the articular surface of the lunate fossa and scaphoid fossa is critical to facilitate the recovery of the wrist function and to reduce postoperative complications [ 9 ]  . since type fractures involved sigmoid notch , aside from reconstruction of the distal radioulnar joint surface and exploring the damage of surrounding soft tissue ( tfcc ) , the volar approach cold also be achieved . 
as the triangular fibrocartilage complex ( tfcc ) was considered as the major ligamentous stabilizer of the distal radioulnar joint [ 10 ] , more attention should be paid for restoring the stability of tfcc , which is dependent on the distribution of the fracture lines at the sigmoid notch , during the fracture reduction and fixation . for type fractures , the focus of reduction is on the styloid process of the radius . 
these findings indicated that the keystone area is not an isolated articular surface of the distal radius but an important area with dense trabecular bone structure in the dorsal aspect of the distal radius , strongly supporting the rationality and safety of the keystone area screwing . 
apart from the keystone area , the radial styloid process was shown to be the area with the lowest fracture rate , which suggests it could be served as another screw inserting point . 
however , due to the lack of a precise intraoperative instrument to assess the positions ( orientation and depth ) of the screws , it still relies on the surgeons experience and skill level to reach the proper position and decrease iatrogenic complications ( e.g. , the extensor tendon problems ) in the clinic [ 11 , 12 ]  . by retrospectively analyzing the typical x - rays of each type before surgery , it was found that for the and type fractures , the preoperative anteroposterior x - ray has 1 3 618 la radiologia medica ( 2019 ) 124 : 613619 observation . 
both the anteroposterior x - ray and the lateral x - ray can achieve effective in conclusion , mapping analyses of the number and distribution of the fracture lines could reveal the classification of distal radial intra - articular fractures . 
the results from the current study will be useful for the further understanding of the fracture patterns , providing the basis for the preoperative use of diagnostic imaging methods as well as for further developments in surgical approaches and fixation techniques for specific fracture types . 
a , c , e , g , i the anteroposterior x - ray film ; b , d , f , h , j represents the lateral x - ray fil although fracture lines can be seen in ( a , c ) , it is difficult to distinguish the distribution of fracture lines on the dorsal and dorsal sides . 
separation and displacement of the volar and dorsal fractures of the distal radius can be observed in ( j ) 1 3 la radiologia medica ( 2019 ) 124 : 613619 size and the assignment of all fracture fragments to one of the quadrants . 
the study group used 100kvp and reduced cm concentration of 270mgi / ml , and the images in this group were reconstructed with 50% adaptive statistical iterative reconstruction ( asir ) and mbir . 
the subjective quality included overall image noise , enhancement degree , lesion ( including mediastinum mass , pulmonary space - occupying lesions , and parenchymal infiltrative lesions ) conspicuity , and beam - hardening artifacts . 
56 , nanlishi road , xicheng district , beijing100045 , china 2 department ofradiology , tokyo womens medical university & medical center east , tokyo116 - 8567 , japan 3 respiratory department , beijing childrens hospital , capital medical university , no . 
56 , nanlishi road , xicheng district , beijing100045 , china vol . : ( 0123456789 ) 1 3 596 introduction low - dose computed tomography ( ct ) is being rapidly adopted in clinical practice [ 1 ] , especially in pediatric population . 
a large number of studies have been focused on using low - dose ct in children [ 2 , 3 ] , and on using iterative reconstruction ( ir ) algorithms to maintain image quality under low - dose conditions in imaging various body parts such as chest , abdomen , and blood vessels [ 48 ]  . 
related researches have shown that full model - based iterative reconstruction ( mbir ) is more effective than adaptive statistical iterative reconstruction ( asir ) , resulting in more significant reduction in image noise [ 912 ]  . 
studies have shown that the use of low tube voltage in ct scanning can improve contrast - enhancing effects [ 13 ] , which can be utilized to reduce the amount of iodine in contrast mediuin our study , we performed low - radiation - dose contrast - enhanced chest ct in a group of children using reduced contrast medium concentration ( 270mgi / ml ) to lower the total contrast dose and used the low voltage of 100kvp to offset the contrast dose reduction . 
our objective was to determine the effects of the full model - based iterative reconstruction ( mbir ) algorithm in improving the image quality under low - dose conditions , using the state - of - the - art adaptive statistical iterative reconstruction ( asir ) as the reference of standard . materials andmethods general information this study was approved by the ethics committee of our hospital . 
a total of 56 children who underwent chest ct examination from august 1 to october 31 , 2015 , were enrolled in the study group , and the projections of the contrast - enhanced phase in this group were reconstructed with both asir ( default reconstruction ) and mbir . 
clinically , all patients la radiologia medica ( 2019 ) 124 : 595601 underwent the contrast - enhanced ct scans to evaluate the parenchymal lesions and their correlation with surrounding tissues . instruments imaging data were collected using a ct scanner with a gemstone detector ( discovery hdct 750 , ge , usa )  . 
both patient groups shared the following same scan and contrast injection protocol : helical pitch value of 1.375 and rotation speed of 0.4s ; automatic tube current modulation ( atcm ) in the range of 10700ma during the scan to obtain agebased image noise index ( ni ) settings : ni = 11 hu for children with age of 012months , ni = 13 hu for 12years old , and ni = 15 hu for 314years old ; total contrast agent volume based on body weight at ratios of 1.21.8ml / kg ( 1.8ml / kg for child with weight of 35kg , 1.6ml / kg for 510kg , 1.4ml / kg for 1015kg , and 1.2ml / kg for 1535kg )  . 
the contrast agent was administrated through intravenous injection using a single - tube high - pressure syringe at the rate of 0.43.0ml / s and total injection time of 15s . 
for the study group , the tube voltage was set at 100kv , and the 270 mgi / ml concentration contrast medium ( iodixanol , ge healthcare , america ) was used . 
the original data obtained were reconstructed with mbir ( study group 1 ) and 50% asir ( study group 2 ) at image slice thickness of 0.625m for the control group , tube voltage was set at 120kv and the 320 mgi / ml concentration contrast medium ( iodixanol ) was injected . 
images in the control group were reconstructed with 50% asir only at slice thickness of 0.625mduring the ct scans , the children were lying on their back with arms raised overhead without holding breath . 
oral sedative medicine ( 10% chloral hydrate , 0.5ml / kg ) was given to children who could not cooperate , and scan was performed after subjects falling to sleep . 
additionally , the normal 5 - mm slice thickness images were reconstructed for the routine clinical diagnostic purpose . subjective image quality evaluation the three sets of images were transmitted to a ge aw4.6 ct workstation for analysis and measurement . 
two physicians , one with 12years of experiences ( 3years in adult imaging and 8years in pediatric imaging ) and the other with 17years of experiences in pediatric imaging , examined the images arranged in random order . 
during the evaluation process , the doctors were allowed to adjust the width of display window 1 3 la radiologia medica ( 2019 ) 124 : 595601 according to personal habits and use the multi - plane recombination tool equipped on the workstation to observe the images at multiple perspectives . quantitative evaluation of image quality was performed in refer to relevant studies [ 4 , 14 ]  . 
for overall image noise : 4 indicated that the image was exquisite with very little noise ; 3 meant that the image was fine with acceptable noise ; 2 referred to an image with some noise , but the overall quality still met the diagnostic requirement ; while 1 was defined as that the image quality was difficult to accept . 
for evaluation of the degree of enhancement , 4 referred to that images of the soft tissue and the mediastinum structure were obviously enhanced with the boundary between the edge and the surrounding clearly defined ; 3 indicated that the degree of enhancement was obvious , with the slightly blurred edge with clear resolution ; 2 meant that image was somewhat enhanced with the surrounding tissue distinguishable , but the edge was not clear , still meeting the diagnostic requirement ; and 1 was defined as poor structural enhancement with marginal boundary unclear and could not meet the diagnostic requirement . 
lesion display ability was in concern with the display of the ranges and details of lesions , including mediastinum mass , pulmonary spaceoccupying lesions , and parenchymal infiltrative lesions , as well as the display of small blood vessels in or around the lesion : 4 stood for a clear lesion structure display with good contrast and good detail ; 3 indicated decent display of the lesion and detailed structure with clear boundary ; 2 meant low clarity of the lesion and detailed structure and poor contrast , but still met the basic diagnostic requirement ; while 1 referred to image with lesion and surrounding tissue difficult to be distinguished . 
for evaluation of beam - hardening artifacts : 4 indicated that the image was even and delicate with no obvious beam - hardening artifact ; 3 stood for a small amount of observable beam - hardening artifacts but without impact on the diagnosis ; 2 referred to images with more apparent beam - hardening artifacts but the diagnosis was not affected ; while 1 meant that image was with more beamhardening artifacts that affected the diagnosis . 
a unified score was given after discussion according to the scoring standard , and the original scores from the two reviewers were tested for consistency . objective image quality measurement after the subjective image quality evaluation was completed , the two doctors performed the objective quality evaluation of the image together by measuring the image noise . 
 the sizes of rois for ct value and sd measurement in the back muscle were set to be half of the cross - sectional area of the descending aorta at the same plane . 
the signal - to - noise ratio ( snr ) was calculated as the ratio between ct value and the noise value in each tissue , and the contrast - to - noise ratio ( cnr ) of the left ventricle was calculated using the following formula : cnr = ( ct value ( left ventricular ) ct value measurement ofradiation dose andcontrast dose ( muscle ) ) sd ( muscle ) the radiation dose , volumetric ct dose index ( ctdivol ) , dose length product ( dlp ) , and the total volume of contrast agent used were recorded in both groups . 
the amount of iodine used was then calculated according to the concentration of the contrast agent . statistical analysis measurement data including subjective score and objective noise measurement were represented as x s . 
the analysis of variance ( anova ) was used to compare the differences in the subjective score and objective measurement among the three image groups , and kappa test was used to evaluate the consistency of the subjective scores by the two radiologists . 
all statistical analysis was performed using spss version 17.0 ( ibm , usa ) with p < 0.05 considered as statistically significant . results a total of 56 children ( 36 males and 20 females , with a median age of 4 years , ranging 4 months14 years ) were enrolled in the observation group . 
the noise of mbir image was reduced by 42.80% ( in left ventricle ) and 59.36% ( in muscle ) compared to those of the asir image in the control group . 
however , the snr and cnr of mbir images were significantly increased , with cnr of mbir image in the study group increased by 165.70% compared to the asir image of the control group . 
evaluation of asir images from the study group and the control group showed that there was no statistical difference in the overall image noise scores , both lower than the diagnostic requirements . 
although the thickness of layer in a was 0.625mm , the noise was significantly lower than that of c and e , close to that of b and d , indicating that mbir could significantly reduce image noise . 
the edge of the low - density component ( white arrow ) in the lesion of c was unclear , while it could be observed relatively more clearly in mbir images with both the thick and thin layers . 
similar changes could be observed in control group images of d and e ; horizontal beam - hardening artifact ( black arrow ) could be observed in c , resulted from reduced electron penetration in the low - voltage scanning , which was not obvious in e with the conventional voltage , and in a in which mbir eliminated the artifact ; the mediastinal small blood vessel ( white arrow ) in c was blurred and not continuous , although it could not be clearly displayed in b due to the layer thickness ; mbir images could clearly show small lesions due to low noise of solid tissues or organs , it is still recommended to use a 100kv setting [ 18 ]  . 
 according to our previous experience , children before the age of 14 who did not enter puberty are usually with a relatively constant body size significantly smaller than adults , therefore suitable for low - voltage ct scans . 
in our study , we have limited the use of 100 kvp for patients with age less than 14years of age . scanning at low voltage can significantly improve the contrast of the image , especially the ct value of the contrast agent [ 20 , 21 ] , which facilitates observation of the lesion . 
on the other side , low - voltage scanning increases the beam - hardening artifact of the image , which is not conducive to image observation [ 19 ] , especially the observation of detailed structures . 
while the increased beam - hardening artifacts at 100kv scanning had a certain impact on the image quality , especially at a layer thickness of 0.625mm which usually with high noise level , the detail display capability was reduced . 
compared to the asir images of the control group , cnr of the mbir images was increased by 165.70% , which was similar to the previous research results [ 14 ]  . 
in these settings , when only asir images from the study group and the control group were evaluated , it was found that both the subjective quality and objective quality of the images from the two groups were with no significant difference . 
therefore , the 100kv low - voltage scan can reduce the amount of contrast agent by about 20% compared with the conventional 120kv scan under the premise of ensuring image quality , similar to the previous findings in the heart scanning [ 23 ]  . there are several end goals when designing the research projects with iterative reconstruction algorithms . 
the most common one is to reduce dose ( either radiation and / or contrast medium dose ) while maintaining similar image quality in terms of enhancement and image noise by using more advanced iterative reconstruction algorithms . 
in our research , the main object was to demonstrate the ability of mbir algorithm to significantly improve image quality compared with the stateof - the - art asir algorithm at similar radiation dose . 
we assigned same noise index for both the study and control groups in our study , and the ct system adjusted tube current to achieve the target noise setting , which resulted in similar radiation dosage for the two groups . according to previous research , mbir could significantly reduce the noise of images , with the quality of 0.625 - mm mbir image similar to that of the 5 - mm fbp images [ 10 ]  . 
considering that chest - enhanced ct is mainly to observe the mediastinal occupancy or vascular condition , the 0.625 - mm image allows observation of more details in the chest lesion . 
the results of the current study have demonstrated the potential of the full model - based iterative reconstruction in imaging solid tissues or organs with the 0.625 - mm thin slice thickness rather than the standard 5 - mm images : mbir could reduce image noise more effectively than asir algorithm , improve image quality , and facilitate the observation of fine structures of lesions under thinner images . 
however , due to the limited noise reduction in asir , the microstructure observation was not satisfactory for small enhanced lesions and small blood vessels , and the fine structures of lesions could not be clearly observed . 
the mbir significantly reduced the noise and eliminated excessive beam - hardening artifacts , which not only highlighted the high degree of enhancement brought about by 100kv , but also further improved the observation of fine structures . 1 3 la radiologia medica ( 2019 ) 124 : 595601 there are still some limitations in the current study : first , skewed distributed prevented age stratification or weight stratification . 
osteochondral defects were most commonly detected at the medial talar dome : in 29 of 37 patients ( 78.3% ) in the ocd without loose bodies group and in 16 of 29 ( 55.2% ) patients in the ocd with loose bodies group . 
 ocds of the distal tibial plafond are not rare in the ankle joint and are often associated with loose bodies . keywords ankle joint mr arthrography anterolateral impingement osteochondritis dissecans loose body introduction traditionally , the majority of osteochondral lesions of the tibiotalar joint have been described as occurring in the posteromedial and anterolateral talar dome [ 13 ]  . 
our * hayri ogul drhogul@gmail.com 1 department ofradiology , medical faculty , ataturk university , erzurum , turkey 2 department ofradiology , erzincan university mengcek gazi training andresearch hospital , erzincan , turkey 3 department oforthopedic , medical faculty , ataturk university , erzurum , turkey 4 niversite mah . , niversite lojmanlar kme evleri lojman sitesi 45 blok no : 50 kap no : 3 , yakutiye / erzurum , turkey experience with ankle magnetic resonance ( mr ) arthrography suggests that an osteochondral lesion of the tibial and fibular plafond is not rare in patients with osteochondritis dissecans ( ocd )  . 
for an optimal treatment approach , it is also crucial to determine the stage of the osteochondral lesion . computed tomography ( ct ) and mr arthrography examinations are very sensitive imaging methods for the evaluation of loose bodies and ocds . 
in patients with loose bodies , intra - articular - injected contrast media might leak into adjacent anatomic compartments , and this situation shows the possible migration pathways of loose bodies . 
the purpose of our study was to use mr arthrography to determine the relationship between joint capsule thickness and the stage of osteochondral lesions of the tibiotalar joint and to evaluate their clinical results . 
additionally , this arthrographic study demonstrates the unusual locations of osteochondral lesions of the tibiotalar joint . materials andmethods patients this retrospective study included 82 consecutive patients referred to ataturk university hospital for ankle mr arthrography between march 2015 and february 2017 . 
 diluted contrast medium ( 0.5mmol / l gadopentetate dimeglumine , magnevist , bayer schering pharma , germany ) at a concentration of 1 : 200 was injected ( 0.1ml contrast medium diluted in 20 ml normal saline )  . 
our mr arthrography protocol includes turbo spin - echo ( se ) t1 - weighted ( tr / te , 650 / 15ms echo train length , 8 section thickness , 3mm spacing , 0.3mm field of view , 130200mm matrix , 256 256 three signals acquired ) and fat - suppressed turbo se t1 - weighted images . 
two staff radiologists with 11 and 4years of experience in musculoskeletal imaging , respectively , who were blinded to the diagnosis of the patients independently analyzed the mr arthrography images . 
all mr arthrography images were evaluated in a randomized fashion followed by evaluation of the same mr arthrography images4weeks later in a different randomized order for assessment of intraand interobserver agreement . 
volumetric measurements of extra - articular contrast material leakage ( into adjacent synovial compartments ) and tibiotalar joint capacity were taken with a 3d volume measurement workstation ( myriad pro , intrasense , france )  . osteochondral lesions of the tibiotalar joint on the basis of ct and mr arthrography were divided into four subtypes . 
a p value was calculated for each comparison , and values 0.05 were considered to indicate statistical significance . we evaluated other features of anterolateral impingement ( e.g. , scar tissue of the synovium , meniscoid lesions , and synovial plicae / bands )  . results arthroscopy andct arthrography the time interval between ct or mr arthrography and arthroscopy was between 2weeks and 2months . 
all ct arthrography examinations were performed using a second - generation somatom definition flash 256 - slice dual - source multidetector ct scanner ( siemens healthcare , forchheim , germany )  . 
the mannwhitney u test was used to compare the relationship between anteromedial , anterolateral , and posterior joint capsule thickness for both the ocd without loose bodies and ocd with loose bodies groups . 
four of the remaining six patients ( 23.5% ) had grade ii anterolateral impingement syndrome ( fig.3 ) , and two of the remaining six patients ( 11.8% ) had grade iii anterolateral impingement syndrome . 
locations of the ocd , loose bodies , and extravasation are summarized in table3 . we detected a moderate interobserver agreement in the detection of the osteochondral defects and loose body localizations in the ocd with loose bodies group . 
anterolateral capsular thickening in the ocd patients with loose bodies clinically was associated with anterolateral impingement syndrome . intra - articular loose bodies might associate with impingement syndrome in the ankle joint [ 10 , 11 ]  . 
c axial t1 - weighted fat - suppressed conventional spin - echo mr image shows thickening of the inferior portion of the anterior tibiofibular ligament ( grade ii thickening of the inferior anterior tibiofibular ligament )  . 
f axial t1 - weighted fat - suppressed conventional spin - echo mr image shows a smooth contour of the anterolateral joint capsule 1 3 658 la radiologia medica ( 2019 ) 124 : 653661 it has been alleged that traumatic occasions account for a substantial percentage of ocd etiologies , although this has not been definitively established [ 12 , 13 ]  . 
it is known that inversion injury of the ankle joint is an important precursor for the development of osteochondral damage at the medial anterior tibial plafond and the medial talar dome [ 14 ]  . 
current study results advance the trauma theory for the development of anterolateral impingement syndrome , as lesions originating from medial talar dome and medial tibial plafond corresponded with 86.3% of the ocd patients with loose bodies . prior to the last two decades , when osteochondral lesion of the ankle joint was diagnosed it exclusively referred to ocd of the talar dome . 
furthermore , it has also been reported that these lesions were frequently encountered , contrary to popular belief , when examinations were re - checked , and the low prevalence was most likely due to overlooking of the anatomical location [ 5 , 8 , 14 ]  . 
 for the last 2 years , we have detected nine lesions in our database , for which the annual encounter rate was similar to those authors . in a biomechanical study , athanasiou etal . 
 in contrast to our findings , most of these tibial and fibular lesions were kissing lesions , and furthermore , none of these lesions at the distal tibia and fibula was reported as stage 4 ocd . 
the sensitivity of conventional mr imaging is somewhat lower compared to mr arthrography in the staging of ocd due to the insufficient delineation of loose bodies [ 17 ]  . 
d axial t1 - weighted non - suppressed conventional spin - echo mr image shows diffuse irregular thickening of the anterolateral capsular tissues and obliterating of the anterolateral ankle recess ( grade iv irregular nodular soft tissue thickening ) radiological evidence representing varying degrees of anterolateral impingement that we found in all patients with loose bodies supports the above idea . 
in our study , all of the detected ocds of the tibial plafond were grade 4 , and all those lesions were associated with the clinical and arthrographic findings of high - grade ( grade 34 ) anterolateral soft tissue impingement syndrome . 
one of the reasons for the absence of the above - mentioned lesions might be healing of stage 1 or 2 ocds of the talar dome . in ankle joint ocds , free fragments of cartilage or bone might stay within the joint space or migrate to adjacent synovial compartments [ 17 ]  . 
ct and mr examinations performed after the injection of contrast material in the joint space are the most sensitive imaging methods for the evaluation of loose bodies and ocds [ 13 , 17 ]  . 
for instance , a loose body located adjacent to the tendon of the flexor hallucis longus can be a cause of chronic irritation of this tendon and is thus one of the reasons underlying constant and recurrent pain of the heel and posterior foot . 
in our series , eleven cases ( 38% of all loose bodies ) had loose bodies located in adjacent anatomic compartments ( posterior talocalcaneal joint and synovium of the tendon of the flexor hallucis longus )  . 
we think that these rates were considerable amounts and that intra - articular - injected contrast material that leaked into adjacent compartments eased our work for the detection of loose bodies . no age restrictions in patient populations should be recognized as a limitation in this study . 
in these patients , especially in those with loose bodies , fractures of osteophytic fragments might complicate the situation . concerning ocds of the ankle joint , this mr arthrography study showed that clinic and mr arthrographic findings of anterolateral impingement syndrome often accompany grade 4 ocds . 
routine head mri and three - dimensional pseudo - continuous arterial spin labeling were performed using a 3.0 - t system within 7days prior to operations , and at 4 consecutive time - points ( 24 , 48 , 72 , and 96h ) after operations . 
comparisons within groups were made using paired t test , and comparisons between groups were made using independent - sample t test . results the cbf values markedly increased at 24h after cas and cea ( p < 0.05 ) compared with baseline . 
most patients showed peak cbf values on the ipsilateral side at 72h ( 13 / 19 , 68% ) after cas and at 48h ( 10 / 13 , 77% ) after cea , which then declined . 
previous studies have only revealed an improvement in cbf in the ipsilateral side by monitoring 1day after cas [ 912 ] and cea [ 13 , 14 ]  . a number of methodologies , including quantitative magnetic resonance angiography ( qmra ) [ 9 ] , dynamic susceptibility contrast perfusion mr ( dsc ) [ 10 ] , xenon - 133 single - photon emission computed tomography ( spect ) [ 11 ] , continuous arterial spin labeling ( casl ) [ 13 ] , and perfusion computed tomography ( pct ) [ 14 ] , have been applied to investigate hemodynamic changes after cas and cea . 
 recently , three - dimensional ( 3d ) pseudo - casl ( pcasl ) was shown to be a promising technology with high signalto - noise ratio ( snr ) and spatial resolution and was able to vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 636642 quantify absolute cbf invivo [ 15 , 16 ]  . 
the highly precise 3d - pcasl , which does not need a gadolinium - based tracer , can be used repeatedly to evaluate cbf changes over time and has favorable intraand inter - scanner reliability and reproducibility [ 17 ]  . 
all study protocols were approved by the institutional review board of our hospital , and written informed consents were obtained from all patients . all the patients were diagnosed using conventional angiography . 
to ensure patient safety , blood pressure was monitored in accordance with the patients specific condition by two experienced clinicians , and a systolic blood pressure value of < 120mm hg / diastolic blood pressure value of < 80mm hg was maintained during the 2496 - h period after cas and cea to reduce the influence on postoperative cbf changes . 
patients who satisfied the following criteria were enrolled : ( 1 ) symptomatic severe unilateral intracranial ica stenosis ( 7099% stenosis ) ; ( 2 ) no obvious abnormality in the rest of the cerebral artery ; and ( 3 ) ica stenosis caused by atherosclerosis . 
first , fast - spoiled gradient - recalled echo was normalized to montreal neurological institute template space using spm8 ( statistical parametric mapping , university college of london , available at softw are / spm8 ) on the matlab platform ( r2013b ; math works , natick , ma , usa )  . 
subsequently , cbf map was coregistered to the normalized t1 - weighted template and spatially smoothed with a 6 - mm full - width - at - half - maximum gaussian kernel . 
according to the time of data acquisition , cbf values of ipsilateral ica territory of cas ( g1 ) , contralateral ica territory of cas ( g2 ) , ipsilateral ica territory of cea ( g3 ) , and contralateral ica territory of cea ( g4 ) were divided into five groups : within 7days before operation 1 3 638 la radiologia medica ( 2019 ) 124 : 636642 fig . 
mean cbf value of ipsilateral and contralateral sides was measured by using voi ( t0 ) , and at 4 consecutive time - points post - treatment : 24h ( t1 ) , 48h ( t2 ) , 72h ( t3 ) , and 96h ( t4 )  . 
comparisons between t0 and t1t4 post - treatment cbf values for the treated side and those for the contralateral side were made separately for cea and cas groups using a paired t test . 
meanwhile , demographics including blood pressure , age , degree of stenosis , sex , hypertension , hyperlipidemia , diabetes mellitus , obesity , and cigarette smoking were compared between patients who underwent cea and cas . 
data are presented as mean sem . results the baseline characteristics of patients treated with cea and cas , including age , sex , blood pressure , degree of stenosis , and vascular risk factors , are summarized in table1 . 
in the later time - points , most patients showed peak cbf values on the ipsilateral side 72h ( 13 / 19 , 68% ) after cas , which then declined at 96h ; most patients showed peak cbf values on the ipsilateral side 48h ( 10 / 13 , 77% ) after cea , which descended 72 h and remained relatively stable at 96h . 
however , the major challenge of the revascularization of ica stenosis following cea or cas is the postoperative management including blood pressure control and cerebral hyperperfusion syndrome ( chs ) [ 7 , 8 ] , which are all associated with cerebral hemodynamic changes due to a state of maximal vasodilation of the cerebral arterioles and poor cerebrovascular pressure autoregulation as a result of prolonged perfusion reduction caused by the stenosis [ 46 ]  . 
 in the present study , we had found that the cbf values markedly increased at 24h after cas and cea compared with baseline , but the peak cbf values on the ipsilateral side were presented at 72h ( 13 / 19 , 68% ) after cas and at 48h ( 10 / 13 , 77% ) after cea , which then declined . 
what is more , the cbf values for the ipsilateral ica territory of cea group were significantly higher than those of cas group whether at 24 , 48 , 72 , or 96h . 
to the best of our knowledge , this is the first study to investigate the pattern of cbf changes after cea or cas and compare the differences of postoperative effect between cea and cas by using 3d - pcasl . as revealed by the linear graph , we postulated that postoperative 72 h after cas and 48 h after cea might be focused to prevent subsequent severe complications such as chs which is associated with the fluctuation of cbf changes , and an early discharge within the time frame should also be avoided . 
 from these data , it can be inferred that the cbf values between the ipsilateral and contralateral sides are close after the treatments , which is consistent with our results . 
the conclusion may suggest the importance of first achieving collateral circulation to relieve the hypoperfusion effects of the ipsilateral side and providing metabolic compensation before treatment ; these actions additionally moderate the high perfusion pressure after cea and cas . the prevalence of chs between cas and cea for patients with severe ica stenosis has long remained a contentious medical issue . 
we found that cea resulted in higher cbf values of ipsilateral ica territory than cas at the 24 - h , 48 - h , 72 - h , and 96 - h time - points . 
however , we believe that the results might imply both types of explanation for the individual differences : for patients with risk factors for chs , such as bilateral carotid artery stenosis , previous stroke , and peri - procedural hypertension [ 5 ] , a higher cbf might be harmful after cea , and for patients without risk factors for chs , a higher cbf might be beneficial or neutral fig . 
2 absolute cbf quantification in ica territory of ipsilateral and contralateral sides on 7 days before cas and cea and consecutive scan once per 24 h after cas and cea . 
t0 , before operation ; t1 , 24 h after operation ; t2 , 48 h after operation ; t3 , 72 h after operation ; t4 , 96h after operation . 
data are mean sem we also found that the cbf values of the contralateral and ipsilateral ica territories showed similar variations , but the increase in cbf on the contralateral side was smaller than that on the ipsilateral side . 
moreover , previous studies have shown that cbf values increased not only in the ipsilateral side , but also in the contralateral side after cea and cas ; for example , yun etal . 
secondly , only a single pld of 2.0s as recommended in asl white paper was used in this study [ 33 ] , which might induce error since the flow arrival time might be shifted for the sake of blood velocity changes after cea and cas . 
considering the multiple parametric evaluation including arterial arrival time map and delay - corrected cbf map and considering the superiority to visualize slow flow , the multi - delay asl protocol might be useful to solve the problehowever , the multi - delay asl required long scanning time . 
this distends the spinoglenoid notch and can be enlarged in cases of suprascapular neuropathy which is evident on mri . keywords suprascapular neuropathy magnetic resonance imaging arthroscopic suprascapular nerve decompression suprascapular vein spinoglenoid notch introduction suprascapular neuropathy ( ssn ) is caused by entrapment of the suprascapular nerve under the transverse scapular ligament and can result in chronic shoulder pain [ 1 , 2 ]  . 
multiple etiologies for ssn exist including cysts originating from the glenoid labrum , tumor , ossification of the transverse scapular ligament , and traction from either overhead activity or retraction of the supraspinatus in a rotator cuff tear [ 1 , 36 ]  . 
 the diagnosis of ssn is difficult and is typically made on * yoshihiro katsuura yoshikatsuura@gmail.com 1 department oforthopaedic surgery , university oftennessee college ofmedicine , chattanooga , 975 east third st . , hospital box260 , chattanooga , tn37403 , usa 2 department ofradiology , erlanger university hospital , chattanooga , usa 3 royal colleges ofsurgeons inireland , dublin , ireland the basis of clinical examinationnotably pain and rotator cuff weakness [ 1 , 7 , 8 ]  . 
magnetic resonance imaging ( mri ) may be used to detect signs of denervation in the supraspinatus and infraspinatus , identify mass lesions , and rule out other causes of shoulder pain such as rotator cuff tears [ 7 , 12 , 13 ]  . 
currently , there are no simple and definedradiographic markers of suprascapular neuropathy . the suprascapular vein passes under the transverse scapular ligament along with the surpascapular nerve as it courses toward the jugular system where it empties [ 14 ]  . 
the healthy controls had a varicosity size on average of 2.3mm , whereas the ssn patients vol . : ( 0123456789 ) 1 3 644 la radiologia medica ( 2019 ) 124 : 643652 had an average of 8.4mthe second was by van meir etal . 
we suggest that examining the spinoglenoid notch for distension ( an indirect marker of venous engorgement ) on magnetic resonance imaging ( mri ) can aid in the diagnosis of ssn . 
the goal of this study was to compare spinoglenoid notch distension size on mri between patients with electrodiagnostic confirmed ssn treated with arthroscopic release to normal shoulder controls . institutional review board approval was granted for this study in february 2018 , which was compliant with the health insurance portability and accountability act ( hipaa )  . 
a waiver for informed consent was obtained . methods patients a retrospective review of prospectively collected data was performed of patients who had failed conservative therapy and undergone arthroscopic suprascapular nerve release by the senior author between november 2013 and november 2014 . 
patients were included if they had the following : a clinical diagnosis of ssn based on examination by the senior author , an electrodiagnostic confirmation of ssn , and an available shoulder mri . 
data collected included age , sex , body mass index ( bmi ) , diabetic status , smoking status , and workers compensation status . sixtyssn patients were compared to 47 normal shoulders of patients ages 1830years who were randomly selected based on our population of shoulder mris on the pacs systepatients in this cohort were included if they had no significant derangement of the shoulder per the radiology report . 
patients of this age - group were selected because of the highest likelihood of having a normal shoulder free of degenerative change . image protocol andanalysis due to the retrospective nature of this study , performed over several years across two institutions , mr imaging protocol and parameters varied . 
all non - arthrogram examinations included an axial t2 fast spin echo ( fse ) sequence with fat saturation or proton density ( pd ) sequence with fat saturation ( tr = 20003920 , te = 3048 ) and all arthrogram examinations included either an axial t2 fse with fat saturation or axial t1 fat saturated ( fs ) sequence ( tr = 339.0900.0 , te = 1123 ) from which measurements were obtained . the spinoglenoid notch distension was evaluated by two independent observers who did not refer to each others findings . 
to ensure reproducibility of the measurements and calculate the intra - rater reliability , a second set of blinded measurements were made by each observer 6weeks after the first set . 
moreover , muscle quality was rated according to the goutallier classification where grade 0 was no fatty infiltration , grade 1 was some fatty streaking , grade 2 was fat < muscle , grade 3 was muscle = fat and grade 4 was fat > muscle [ 18 ]  . 1 3 la radiologia medica ( 2019 ) 124 : 643652 fig . 
the coronal section ( red line ) is scrolled progressively posterior until at the base of the spinoglenoid notch is reached ( green line ) while still within the bone of the glenoid . 
sensitivity and specificity were tabulated using cross - tabs function . statistics were performed using the statistical package for the social sciences software ( ibm , version 24.0 , chicago , usa )  . 
a dependent t - test was used to compare means within the same group ( i.e. , vas improvement ) , and an independent t test was used to compare the means of the varicosity size and glenoid neck width as well results patients the average age of the ssn group ( n = 60 , female = 29 ) was 52years ( range 2172 )  . 
with the image set on the correct coronal section ( fig.1 ) , the axial section ( red line ) is scrolled progressively inferior until at the base of the suprascapular notch within the bone of the glenoid ( green line ) current or former smokers , and 16 were workers - comp patients . 
the average age of the control group ( n = 47 , female = 21 ) was 24years ( range 1830years )  . reliability andreproducibility analysis the interclass correlation coefficient for the intra - rater reliability for was 0.89 ( p < 0.001 ) for spinoglenoid notch distension and 0.87 ( p < 0.0001 ) for the glenoid neck 1 3 la radiologia medica ( 2019 ) 124 : 643652 fig . 
the interclass correlation coefficient for the intra - rater reliability for was 0.95 ( p < 0.0001 ) for spinoglenoid notch distension and 0.98 ( p < 0.0001 ) for the glenoid neck measurement again indicating excellent agreement . 
 the mean diameter of the transverse scapular ligament was 7.54mm ( sd = 2.03 ) in the ssn group and 4.49 mm 1 3 648 la radiologia medica ( 2019 ) 124 : 643652 fig . 
e axial proton density view of the same patient shows distension of the spinoglenoid notch measuring 7mm sensitivity andspecificity discussion using a spinoglenoid notch distension value of 7mm as a cutoff for a screening test , the sensitivity for suprascapular neuropathy was 74% and the specificity was 78% . 
the nerve then courses with the suprascapular vein under the spinoglenoid ligament in the spinoglenoid notch on its way to innervate the infraspinatus [ 14 , 19 , 20 ]  . 
despite being a relatively small caliber 1 3 la radiologia medica ( 2019 ) 124 : 643652 1 3 650 la radiologia medica ( 2019 ) 124 : 643652 fig . 
7 bar chart comparing glenoid width averages between suprascapular neuropathy ( ssn ) patients and controls nerve , it supplies the significant amount of sensory and motor innervation to the shoulder [ 14 , 2022 ]  . 
as the nerve is tethered to the scapula at this location , it is sensitive to compression from mass effect caused by cysts , varicosities , or hypertrophy of the transverse scapular ligament [ 46 , 23 ]  . the diagnosis of suprascapular neuropathy is often elusive . 
part of the reason for this is that it is often difficult to make the diagnosis asthere are no quantitative imaging methods to aid in the clinical work up [ 1 ]  . 
 additional tools to aid in the diagnosis of ssn should be developed . on examining the images of normal shoulder mris from the control group , we noted that the infraspinatus invests into the spinoglenoid notch . 
we found that the distance between the glenoid and the infraspinatus at the spinoglenoid notch ( spinoglenoid notch distension ) to be increased in patients with suprascapular neruropathy ( m = 8.36 mm ) , which was almost identical to the vein caliber measured by carroll etal . 
as the suprascapular vein runs underneath the transverse scapular ligament with the suprascapular nerve [ 14 ] , we postulate that the anatomical basis for the distension of the spinoglenoid notch is obstruction of the suprascapular veins caused by hypertrophy of the transverse scapular ligament . 
additionally , in cases of overhead use of the arm , the veins may suffer intimal damage via 1 3 la radiologia medica ( 2019 ) 124 : 643652 compression by the transverse scapular ligament accelerating formation of varicosities . 
while previous studies have implicated varicose veins as a cause of ssn , we suggest instead that varicose veins are the result of impeded flow back toward the jugular systespinoglenoid notch distension may be used as an adjunct diagnostic sign to aid the clinician in patients with refractory shoulder pawe suggest that a varicosity notch size of > 7mm is a good cutoff to support the diagnosis of ssnin conjunction with clinical and electrophysiologic findings as this value had the best combined sensitivity and specificity of a range of values we tested . 
however , a simpler test is examining the spinoglenoid notch on an axial view to see whether the infraspinatus is pushed away from the groove . the treatment of suprascapular neuropathy is a trial of conservative therapy followed by open or arthroscopic release which has been shown to be effective in a number of studies [ 2629 ]  . there were several weaknesses with this study . 
we also performed a sub - analysis examining the difference in notch size between non - contrast and arthrogram studies in the control group and were unable to find a significant difference . 
to account for the age difference between the two groups , we specifically examined the agematched subset of patients in the ssn group and the difference in varicosity size remained significant from controls . 
 we hope this study will help lead to future prospective studies comparing well - shoulder to ill - shoulder with bilateral mri scans as well as comparing mris in patients with negative emg / ncs studies to patient with confirmed ssn . in conclusion , the varicosity size is a simple measurement which can aid in the diagnosis of ssn and should be examined in suspected cases . compliance with ethical standards conflict of interest disclosure of potential conflicts of interest : no financial support was utilized for this study . 
all authors declare that they have no conflicts of interests . research involving human participants and / or animals all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 declaration of helsinki and its later amendments or comparable ethical standards . informed consent institutional review board approval was obtained from ut - college of medicine institutional review board , study number 18 - 021 . 
prospective studies are needed to further assess its role among locoregional treatment options for liver metastases from crc . keywords liver metastases stereotactic body radiotherapy colorectal cancer oligometastases introduction colorectal cancer ( crc ) is estimated to be the second most frequently diagnosed cancer in europe , accounting for 447 , 000 new cases in 2012 , and one of the most relevant causes of cancer mortality worldwide with 694 , 000 deaths in the same year [ 1 , 2 ]  . over the last two decades , the clinical outcome of patients with metastatic crc ( mcrc ) has improved due to many factors , such as local ablative treatments ( lats ) [ 3 ]  . 
potentially , lats can have a curative intent for oligometastatic patients [ 4 ] and could play a role in the management of an oligoprogressive disease , in which only a solitary or few ( 5 ) metastatic localizations progress , while all other sites of disease are stable or responding to the current regimen [ 5 ]  . 
over the past two decades , extensive clinical experience demonstrated sbrt to be a safe and high - precision technology method to deliver ablative treatments for different metastatic sites [ 8 ] , including liver metastases [ 9 ]  . since 2012 , robotic sbrt has been offered in our institution as a possible alternative to surgery or other lats for liver oligometastases in colorectal cancer patients unsuitable for surgery . the aim of our retrospective study was to report the clinical outcome of robotic sbrt in this setting . 
during treatment planning , the following organs at risk ( oars ) were delineated : left and right lung , esophagus , heart , thoracic wall or ribs , left and right kidneys , small and large bowel , duodenum , stomach , spinal canal , whole liver , great vessels . 
for most patients , the intended prescription dose was 37.5gy in three fractions , prescribed to the 70% isodose line to cover 95% of the ptv [ 10 ]  . 
 patients were prescribed proton pump inhibitors , serotonin 5 - hydroxytryptamine antagonists , benzodiazepines and antacids as clinically indicated . followup andtoxicity evaluation after the last application , a clinical examination was performed and treatment toxicity was scored , if present , according to the common terminology criteria for adverse events ( ctc - ae ) scale , v 4.0.3. 
treatment response was 1 3 872 la radiologia medica ( 2019 ) 124 : 870876 evaluated by serial contrast - enhanced spiral ct or mri scan 2months after end of sbrt . 
complete response ( cr ) was defined as disappearance of all target lesions ; partial response ( pr ) as at least a 30% decrease in the sum of diameters of target lesions ; progression disease ( pd ) as at least a 20% increase in the sum of diameters of target lesions . 
local control ( lc ) was defined as the time of absence of progression disease at the site of sbrt from the end of the treatment . statistical analysis local control , local and distant progression - free survival ( pfs ) and overall survival ( os ) curves were evaluated by the kaplanmeier method . 
for cb , additional investigation included treatment planning related parameters such as number of fractions , physical and biological effective dose ( bed10 , considering / = 10 ) coverages . 
in order to comply with dose constraints to organs at risk , a risk - adapted approach was followed for the treatment of nine lesions , which were irradiated in five fractions instead of three . 
 then , we compared the bed10 mean dose covering the ptvs between lf and lc , and we found that its median value was smaller in the lf group ( 89.26gy ) than in lc fig . 
actuarial 1 3 874 la radiologia medica ( 2019 ) 124 : 870876 although surgical resection is presently accepted as the first choice among strategies for ablation , for many reasons only a small percentage of patients qualify for surgical procedures [ 14 ]  . up to now , ct - guided percutaneous radiofrequency ablation ( rfa ) is the most common local treatment option to be offered to such patients [ 15 ]  . 
in recent years , sbrt has become the current standard in medically inoperable early lung cancer patient [ 16 ] and , potentially , it could be the same for unresectable liver metastases treatment [ 17 ]  . our analysis counted one of the most numerous series addressing the use of high doses of radiation by means of robotic sbrt in the treatment of liver metastases exclusively from crc . 
moreover , our study has other limitations : its retrospective nature , the heterogeneity of the prescription doses , the differences in the timing of sbrt with respect to other treatments . our results demonstrated that it is possible to have good outcomes on response rate and survival analysis , keeping an excellent safety profile . 
 [ 18 ] in their subgroup analysis on liver metastasis from adenocarcinoma , confirming that colorectal adenocarcinoma has a statistically significant worst lc than other histologies [ 19 , 20 ] , which is perhaps related to the fact that these patients are generally heavily pretreated and surgically unresectable [ 21 ]  . moreover , as chang etal . 
 [ 22 ] had already concluded in their meta - analysis , a dose of 4652gy in three fractions or higher is required to achieve 90% lc at 1year for the treatment of hepatic cr metastases . although a good performance in terms of lc , in our analysis , pfs ( both hepatic and distant ) was poor , if compared with the results obtained by berkovic etal . 
swaminath and dawson [ 23 ] had already noticed in their analysis that , although excellent local control rates were achieved , many patients developed out - of - field progression probably because of the relatively high number of patients with extrahepatic disease and / or more than three metastatic lesions , suggesting that a closer attention should be reserved to patients who really belong to a relatively good prognostic group with longer survival expectancy and offering a strong rationale for combining sbrt with systemic treatments , as hypothesized by scorsetti etal . 
in fact , comparing survival curves of patients with or without extrahepatic disease before sbrt , there is a clear advantage for the second ones , both in os and in pfs ( supplementary materials , figures1 and 2 )  . 
previous extrahepatic disease and number of metastatic lesions > 3 showed a statistically significant correlation at univariate analysis ; this result was confirmed at multivariate analysis only for the presence of previous extrahepatic disease , as in the pfs analysis . toxicity no complications were observed during or after fiducials placement . 
as referred by hellman and weichselbaum , the theory of oligometastases considers that there could be a subgroup of patients with metastatic disease that is intermediate between completely absent and widely metastatic [ 12 ]  . 
on the other hand , their cohort was less homogeneous than ours , since also patients treated for extrahepatic localizations ( lung , lymph nodes , suprarenal gland ) were included in the analysis . 
 [ 29 ] performed a phase 1 / 2 study enrolling about 70% of patients with liver metastases from non - colorectal origin ( lung , breast , ovary , esophagus and others )  . 
likewise , in scorsettis study [ 9 ] , more than 50% of patients had metastases from non - colorectal primary tumor , and more than 80% of patients received a prescription dose of 75gy in three fractions ( mean dose to ptv )  . 
as expected , very limited treatment - related toxicity was observed in our cohort ; however , it has to be acknowledged that our local control rate is at the lower range of published data . in particular , the rate of actuarial lc at 2years in these experiences ranges between 64 and 92% ( supplementary table2 )  . 
as a matter of fact , the net effect of sbrt on the irradiated lesion is critically dependent on the delivered physical total dose and fractionation , the applied bed , the size of the gtv , the prescription modality and the intrinsic biological radiosensitivity . 
overall , patients selection refinement and prospective trials based on strict inclusion criteria in the setting of oligometastatic crc should be warranted . conclusions survival and toxicity results of our retrospective analysis support the hypothesis that sbrt for liver metastases from crc is a feasible treatment , combining an excellent safety profile with acceptable efficacy results . 
in addition , only controlled studies could allow us to define how to best select oligometastatic patients potentially profiting from a locoregional treatment with the aim to defer further systemic therapies . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical standards informed consent was obtained from all individual participants included in the study . 
to detect urine leakage , we initially performed conventional cystography after retrograde distention of the bladder with dilute iodinated contrast material , followed by ultra - low - dose ct cystography . 
the diagnostic accuracy of these two modalities was compared , and the technical characteristics of ultra - low - dose ct cystography were examined . results all 31 referred patients were included in this study . 
of the 31 patients , 27 ( 87.1% ) underwent bladder repair after radical prostatectomy , 3 ( 9.7% ) after radical cystectomy , and 1 ( 3.2% ) after bladder diverticulectomy . 
based on these findings , we were able to establish a proper treatment plan . conclusions ultra - low - dose ct cystography is an accurate method for evaluating urine leakage after bladder repair , and this technique may help determine the most appropriate treatment strategy for patients with urine leakage after bladder repair . keywords anastomotic leakage radiation dosage computed tomography fluoroscopy introduction recently , awareness and concern about radiation exposure have increased . 
in the republic of korea , the number of radiation - generating devices has rapidly increased * seong hoon choi 0733280@uuh.ulsan.kr 1 department ofurology , ulsan university hospital , university ofulsan college ofmedicine , ulsan , korea 2 department ofradiology , ulsan university hospital , university ofulsan college ofmedicine , 877 bangeojinsunwando - ro , dong - gu , ulsan44033 , korea from 59 , 739 in 2008 to 72 , 626 in 2012 [ 2 ]  . 
in a study involving conventional non - contrast ct , the reported exposure dose ( ed ) for the abdomen and pelvis was between 10 and 20msv [ 6 ]  . 
therefore , if dose - reduced ct can be shown to accurately diagnose bladder injury , low - dose and ultra - lowdose ct could be applied to a wide variety of cases . one of the advantages of ct compared with conventional cystography is the ability to detect anatomical relationships . 
 a major concern associated with dose - reduced ct is higher noise , which results in lower image quality that may reduce the accuracy of this technique for detecting anatomical relationships and facilitating differential diagnoses . 
moreover , while low - dose and ultra - low - dose ct are effective for detecting urolithiasis [ 7 , 11 ] , it remains uncertain whether low - dose ct cystography can detect bladder injury or urine leakage . 
therefore , we performed a prospective cohort study to compare the accuracy and technical characteristics of ultra - low - dose ct cystography with those of conventional retrograde cystography . materials andmethods study participants from november 2015 to october 2017 , we prospectively examined 31 consecutive patients referred for cystography after bladder repair . 
 to diagnose urine leakage , we initially performed conventional cystography after retrograde distention of the bladder with dilute iodinated contrast material , followed by ultralow - dose ct cystography ( table1 )  . imaging technique andinterpretation conventional cystography was performed in the following manner : with the patient supine on the examination table , the urine bag was disconnected from the foley catheter and the foley catheter was suctioned using a 50 - ml syringe until the bladder was completely emptied . 
additional pelvic ap radiographs were obtained after the initial infusion of approximately 100ml of contrast material and after either infusion of another 150ml of contrast material or occurrence of one of the following endpoints : ( 1 ) 300ml of contrast material was completely infused into the bladder , ( 2 ) urinary leakage occurred due to continuous bladder detrusor activity with infusion less than 300ml , or ( 3 ) the patient complained of pain in the abdomen and pelvis . 
oblique radiographs were also obtained , followed by an ap radiograph after bladder emptying . ct cystography was performed in the following manner : with the patient supine on the examination table , the foley catheter was suctioned using a 50 - ml syringe , as described above . 
ultra - low - dose ct images were reconstructed using idose level 5 , which is one of the iterative reconstruction ( ir ) algorithms [ 12 , 13 ]  . 
the ct parameters were as follows : a pitch of 0.915 , collimation of 0.625mm , matrix size of 512 512 pixels , sectional thickness / reconstruction interval of 2mm , collimation of 2 64 0.6mm , pitch of 1.2 , and gantry rotation time of 0.5s. 
the reductions in radiation dose were compared between the two modalities and also with regard to the patients bmi [ 14 ]  . image findings were interpreted by an expert radiologist in the genitourinary division of the radiology department of our institution . 
the following conventional cystography and ct cystography findings were recorded : ( 1 ) presence or absence of leakage , ( 2 ) location of leakage , and ( 3 ) amount of leakage . 
the amount of leakage analyzed by conventional cystography was scored according to a previous report as follows : grade i , extraperitoneal leakage within 6cm of the vesicourethral anastomosis ( vua ) ; grade 2 , extraperitoneal leakage extending beyond 6cm from the vua ; and grade 3 , intraperitoneal leakage [ 15 ]  . 
informed consent was obtained from all subjects when they were enrolled . results the mean conventional cystography dose area product , ct cystography dose length product values , and the effective radiation dose are summarized in table2 . 
the estimated dose reduction using ultra - low - dose ct compared with the dose in conventional cystography was 60.7%. four of the 31 patients were diagnosed with urine leakage by conventional cystography . 
in all of the patients who were diagnosed with urine leakage by conventional cystography , leakage was observed at the posterior aspect of the vua ; moreover , all had grade 1 leakage ( leakage within 6cm of the vua )  . 
the amount of leakage in these patients was not large ( average , 1.31cm2 ; range , 0.581.74cm2 ) ( table3 )  . by performing ct cystography , we were able to track the precise locations and amounts of urine leakage . 
all nine patients who had urinary leakage , including the two with longer retention of the foley catheter , experienced no further complications and were discharged . discussion conventional cystography has been accepted as the standard for the assessment of bladder trauma [ 8 ]  . 
our study also showed that conventional cystography detected leakage at the posterior aspect of the vua in all patients who were diagnosed with urine leakage ; however , it is thought that the contrast collects in the dependent position by gravity . 
in particular , according to ultra - low - dose ct cystography , patient i had urine leakage at the 3 oclock position of vua , whereas 1 3 la radiologia medica ( 2019 ) 124 : 812818 fig . 
ct cystography identified the location of leakage at the 4 oclock position of vua , and the amount of leakage was estimated to be 7.08cm2 ( black arrow )  . 
c ct cystography showed patient i to have urine leakage at the 3 oclock position of the vua ( black arrow ) , but conventional cystography ( white arrow ) detected leakage at the posterior aspect of the vua according to conventional cystography , there was leakage at the posterior aspect of vua . lately , iterative reconstruction ( ir ) algorithms have received attention as good new methods for reducing radiation dose during ct examination [ 17 ]  . 
this 4th generation iterative reconstruction technique in preventing photon starvation artifacts ( streaks , bias ) prior to image creation and in maintaining image texture to overcome the artificial or plastic look of images that have been frequently reported when using previous iterative reconstruction techniques . 
evidence from rigorous clinical evaluations demonstrates the potential of idose to improve image quality and / or lower radiation dose levels beyond those previously achievable with conventional , routine - dose acquisitions , fbp reconstructions [ 12 , 13 ]  . 
for the ultra - lowdose protocols , the assumed effective dose was 0.44msv , which was very low and comparable to the dose used for conventional single plain radiography of the kidneys , ureter , and bladder [ 19 ]  . 
thus , low - dose or ultra - low - dose protocols could potentially contribute toward reducing the lifetime cumulative radiation dose as well as cancer risk [ 2 , 20 ]  . urinary leakage is a challenging , short - term complication that can occur regardless of the surgical approach used and may reduce patients quality of life [ 21 ]  . 
however , although most urine leakages are self - limited , urine leakage - induced complications include infection , metabolic abnormality as a result of intraperitoneal urine reabsorption , and urinoma formation [ 23 , 24 ]  . 
although there have been no previous reports providing definitive criteria for persistent or massive vua urine leakage , some studies have defined anastomotic urine leakage as a persistent daily amount of urine ( over 1500ml ) in the suction drain for more than 6days and for which an invasive interventional procedure had to be performed [ 27 , 28 ]  . 
our findings suggest that the use of ultra - low - dose ct may help to guide treatment and avoid unnecessary procedures in addition to reducing the risks associated with radiation exposure . our study has several limitations . 
moreover , although ultra - low - dose ct cystography was the more accurate modality for determining urine leakage compared to conventional cystography , we cannot rule out the possibility that extravasations outside the urethra or bladder lumen detected by ultra - low - dose ct cystography were not urine leakages . 
nonetheless , to our knowledge , this study is the first to evaluate the accuracy and technical characteristics of ultra - low - dose ct cystography as an optimal modality to track the precise location and amount of urine leakage and limit the radiation dose . 
 these results establish a foundation for future studies and treatments . conclusions ultra - low - dose ct cystography enables accurate evaluation of urine leakage after bladder repair despite the small radiation dose . 
the survey consisted of 11 multiple - choice questions about participants demographics , current local modalities of pic acquisition and storage , perceived advantages and disadvantages of pic dematerialisation over conventional paper - based pic , and overall opinion about pic dematerialisation . results a total of 1791 radiologists ( amounting to 17.4% of active sirm members for the year 2016 ) joined the survey . 
conversely , the need to create dedicated areas for pic acquisition inside each radiological unit ( 64.0% ) and to gain preliminary approval for the use of advanced digital signature tools from patients ( 51.8% ) were seen as potential disadvantages . 
however , concerns were raised that its practical implementation might face hurdles due to its complexity in current real life working conditions . keywords informed consent dematerialisation online survey paperless * lorenzo faggioni lfaggioni@sirm.org 1 department ofexperimental , diagnostic andspecialty medicine , s . 
orsola malpighi university hospital , bologna , italy 2 diagnostic andinterventional radiology , university hospital ofpisa , via paradisa 2 , 56100pisa , italy 3 department ofradiology , university ofcampania l . 
vanvitelli , naples , italy 4 snr foundation , rome , italy 5 uoc radiodiagnostica , aou policlinico vittorio emanuele , catania , italy 6 department ofimaging , asl 3 genovese villa scassi hospital , genoa , italy 7 department ofradiology , usl n . 
bosco hospital , turin , italy 9 department ofinterventional anddiagnostic radiology , arcispedale santanna , ferrara , italy 10 radiology department , universit politecnica delle marche , ancona , italy 11 department ofradiology , candiolo cancer institute , fpo - irccs , candiolo , turin , italy vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 846853 introduction dematerialisation is a complex process involving the development of a digital document flow with full legal value aimed to support and , in perspective , replace conventional document production and archiving for public or private activities [ 112 ]  . 
the advantages of digitisation over paperand film - based workflow in radiology have been widely demonstrated [ 1317 ] , and attempts have since been made to extend the process to administrative key areas of healthcare data management , including , on a national level , the introduction of the digital medical recipe in most italian regions [ 312 ]  . 
however , to our knowledge , no systematic initiatives have been taken so far towards dematerialisation of patients informed consent ( pic ) for diagnostic and interventional radiology procedures , despite incentives from international [ 1 , 2 ] and national government agencies and healthcare institutions to promote digitisation in the public administration as well as in the healthcare system [ 312 ]  . 
such delay is due to the need for dematerialised pic to be formally equivalent to its conventional , paper - based version , implying for it to fully conform to current laws and privacy regulations , ensure proper patients understanding of medical procedures for which written informed consent is required , and provide seamless integration with existing ris / pacs infrastructure at radiological centres [ 18 , 19 ]  . 
while the development of a fully functional programme for pic dematerialisation may pose significant legal and technical challenges , evidence exists in the literature that pic dematerialisation with the aid of portable devices is feasible and as effective or superior to conventional methods in several aspects [ 2024 ]  . the italian society of medical radiology ( sirm ) has promoted the development of guidelines for pic dematerialisation and has put the imaging informatics chapter and informed consent commission of sirm in charge for its implementation . 
in this context , we sought to gain insight about the current modalities of pic acquisition for radiological procedures in italy and the opinion of italian radiologists about pic dematerialisation via an online survey . materials andmethods the online survey was launched as part of an initiative by the imaging informatics chapter of sirm aimed to promote dematerialisation of pic for radiological procedures ( both diagnostic and interventional ) , in cooperation with a number of it firms . 
the survey was devised following suggestions from a multidisciplinary expert panel of sirm and consisted of 11 questions , of which 9 were single choice and 2 multiple choice ( appendix )  . 
a free text field was left at the bottom of each question for additional comments . based upon a similar approach to two previous sirm surveys on teleradiology and radiological structured reporting [ 25 , 26 ] , every single sirm member received a personal email invitation to join the survey via a direct web link from the president of the sirm imaging informatics working group . 
b multiple answers allowed medical directors [ 23.7% ( 423 / 1780 ) ] or consultants [ 16.9% ( 301 / 1780 ) ] , respectively ( question #4 )  . the majority of responders deemed their it skills good [ 67.9% ( 1207 / 1778 ) ] or excellent [ 12.6% ( 224 / 1778 ) ] , whereas a minority declared to have barely sufficient [ 18.7% ( 332 / 1778 ) ] or poor [ 0.8% ( 15 / 1778 ) ] it skills , respectively ( question #5 )  . pic forms were mostly developed locally at each survey participants institution [ 77.0% ( 1311 / 1702 ) ] or , less frequently , by sirm [ 38.5% ( 655 / 1702 ) ] or the health department of each region [ 10.5% ( 180 / 1702 ) ] ( question #6 )  . pic could be revoked by the patient anytime before the examination according to 62.4% ( 1105 / 1772 ) of survey respondents ( question #7 ) , and pic forms were mainly stored in the hospital archive [ 82.7% ( 1428 / 1726 ) ] or , much less frequently , outside of the hospital [ 6.9% ( 119 / 1726 ) ] or were digitally archived into the ris [ 6.8% ( 117 / 1726 ) ] ( question #8 )  . the main advantages of dematerialised pic over conventional , paper - based pic as perceived by the survey respondents ( question #9 ) were its easier and faster recovery [ 96.5% ( 1669 / 1729 ) ] , safer storage and conservation [ 94.5% ( 1643 / 1738 ) ] , reduced cost due to the workflow going paperless [ 90.7% ( 1573 / 1735 ) ] , higher degree of protection in case of medico - legal litigation [ 83.8% ( 1439 / 1717 ) ] , and simplified patient identification and registration [ 74.3% 1 3 la radiologia medica ( 2019 ) 124 : 846853 ( 1274 / 1714 ) ] ( table2 )  . 
this latter circumstance may be due to different factors , including the current lack or inadequacy of technical infrastructures for digital pic collection ( including tablet devices running ad hoc software and connected to dedicated hospital wireless networks ) and / or of integration with existing ris / pacs environments , in spite of substantial investments into dematerialisation by government agencies . 
besides , some features of conventional , paper - based pic acquisition have emerged from the survey that should be incorporated in a dematerialised , digital pic version , including the possibility for patients to revoke their consent anytime before radiological procedures ( question #7 )  . 
in other terms , such basic aspects of conventional pic should be taken into account and preserved in the design and practical implementation of dematerialised pic solutions in order to maintain their legal validity , so that the transition from conventional to dematerialised pic should not substantially alter workflow . 
 furthermore , the current , more frequent usage of pic forms developed locally ( 77.0% of respondents to question #6 ) rather than by regional health departments ( 10.5% ) or , better , national scientific societies such as sirm ( 38.5% ) could be 1 3 la radiologia medica ( 2019 ) 124 : 846853 a potential , yet surmountable hurdle to standardisation and large - scale setup of dematerialised pic tools . 
these latter would actually benefit from the adoption of a single , predefined validated pic model for each radiological procedure , which could also be advantageous for data mining purposes and eventually in case of medico - legal issues [ 2731 ]  . overall , the majority of survey respondents were favourable to pic dematerialisation and acknowledged several advantages of digital over conventional pic , the main ones being easier and faster data retrieval , safer data storage , and reduced running costs . 
in this perspective , tablet - based methods for pic collection have shown to be preferred over conventional pic by both patients and medical staff and has proven more effective at ensuring adequate patients understanding of medical procedures and research trials [ 2024 , 32 , 33 ]  . 
interestingly , our finding of a rate of pic dematerialisation supporters consistently higher than 90% across all age classes of survey participants ( independent of and greater than their self - assessed it skills ) may suggest that radiologists are confident about the usability of digital pic and / or believe that its advantages would outweigh any potential difficulties related to its use as a routine working tool . on the other hand , the main potential drawbacks of pic dematerialisation as pointed out in the survey were related to technical difficulties in carrying out the process in real working environments . 
actually , tablet - based pic acquisition resulted to be more time consuming than paper - based pic collection [ 21 , 24 ] , and earlier work by haller etal . 
 [ 34 ] cautioned against the use of palmtops for electronic data collection in clinical research due to a significant increase in data entry time and risk of typing errors and missing data in comparison with paper - based questionnaires . 
 [ 21 ] found a positive correlation between the duration ofelectronic ipad - based briefings and patient age , paralleled by a negative correlation between patient age and computer skills , but nonetheless the majority of patients would prefer ipad briefings to conventional written informed consent forms in the future . 
even more specifically , it has been suggested that the higher degree of understanding and overall satisfaction provided by tabletbased pic may improve adhesion to clinical trials [ 23 ]  . a limitation of our study is the relatively small number of sirm members joining the survey ( 1791 / 10304 , corresponding to 17.4% of all active sirm members in full standing for the year 2016 ) , which could restrict the validity of our findings to a minority of all members . 
however , the participation rate to our survey was slightly higher than two recent online surveys involving sirm members on teleradiology [ 16.5% ( 1599 / 9662 ) ] [ 25 ] and radiological structured reporting [ 12.1% ( 1159 / 9560 ) ] [ 26 ] , and much higher than a similar european online survey on teleradiology ( 368 radiology professionals from 35 european countries ) [ 35 ] , confirming that pic dematerialisation has gained significant interest within the radiological community . conclusions our findings show that the majority of radiologist members of sirm involved in the survey were favourable to pic dematerialisation . 
though most of them were aware of the potential long - term advantages of pic dematerialisation over conventional , paper - based pic ( mainly related to greater ease , speed , and safety of data collection , storage and retrieval ) , concerns emerged about potential technical and organisational hurdles that its practical implementation may pose , including the need for extra space and time for digital pic collection ( which would add to regular radiologists workload ) , authorisation to use patients advanced digital signature , and to cater for patients who wish to remain anonymous . 
cchf is a life - threatening disease observed endemically over a wide geographical regions in the world , and there is limited information about pulmonary findings in cchf patients . purpose we aimed to investigate the pulmonary findings belonging to a large cchf patient cohort and to determine if there is any relationship between laboratory findings and disease severity . materials and methods a total of 165 patients who were diagnosed with cchf and examined through chest x - ray ( cxr ) due to respiratory symptoms at their first examination and / or during their hospitalization were included in this study . 
the general non - specific symptoms of cchf are fever , malaise , * fatma akta fatmakokcu79@hotmail.com 1 department ofradiology , faculty ofmedicine , gaziosmanpaa university school ofmedicine , 60100tokat , turkey 2 pulmonary diseases department , versa hospital , nevehir , turkey anorexia , nausea / vomiting , and myalgia , which are observed after the contact . 
in addition , intracranial bleeding , gastrointestinal bleeding , and alveolar hemorrhage that are life - threatening and often responsible for mortality can be observed as well [ 14 ]  . 
however , the number of studies related to pulmonary imaging in cchf is very rare and limited despite the increasing number of cases in the world and in our country [ 14 ]  . 
 we may propose simple pulmonary imaging such as cxr to be used within the following 5days after the first examination as an effective tool to evaluate clinical course and severity of the disease . the aim of this study was to investigate the chest x - ray findings of cchf patients with respiratory findings and to determine if there is any relationship between the laboratory findings and severity of the disease . materials andmethods a total of 464 patients with confirmed cchf were admitted to emergency department and department of infectious diseases and clinical microbiology between january 2011 and december 2016 . 
 patients using medicines that may cause pulmonary toxicity and having acute or chronic lung diseases , malignancies , heart diseases , and blood diseases , and a history of thoracic radiotherapy or surgery in addition to cchf were excluded from the study . 
additionally , patients who were clinically diagnosed with pulmonary infection ( such as cough sputum ) and who were diagnosed with other microorganisms in their cultures were not included in the study as well . ethical approval was obtained from ethics committee of the university . 
in addition to demographical findings of the patients such as age , gender , occupation , place of residence , tick exposure , clinical findings , and medical history , laboratory findings and radiological images of lungs were evaluated . 
the cchf diagnosis was made based on the detection of virus genome by using enzyme - linked immunosorbent assay ( elisa ) or polymerase chain reaction ( pcr )  . statistical analysis data are expressed as mean standard deviation . 
 out of 40 patients with comorbidity , 23 were found to have hypertension , three of them had diabetes mellitus , 13 were diagnosed with both hypertension and diabetes mellitus , and one of them had hypothyroids . 
the frequency of general symptoms of the patients observed during the first examination is presented in table2 . based on the laboratory findings , the mean values of liver - specific enzymes such as alkaline phosphatase ( alp ) , alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , gamma - glutamyltransferase ( ggt ) , creatine kinase ( ck ) , and lactate dehydrogenase ( ldh ) were seen to be high . single and / or multiple abnormal imaging findings were detected in 93 patients ( 56.4% ) as a result of cxr examination during the first examination . 
in a similar vein , four patients , claimed to have ground glass densities on cxr , and one patient , claimed to have pleural effusion , were not found to have these findings according to the results of the thorax ct . 
the comparison of survival and cxr findings of patients is shown in table5 . discussion cchf is a life - threatening zoonotic viral disease that can be seen endemically in many regions in the world . 
the most frequent clinical symptoms are fatigue ( 92.3% ) , fever ( 89.4% ) , myalgia ( 69.7% ) , headache ( 68.1% ) , and nausea ( 64.7% ) ; 23% of patients had bleeding ( petechiae , epistaxis , gingival bleeding , vaginal bleeding , and bleeding in internal organs ) [ 10 ]  . 
this situation might be related to the possibility that males work in occupations with high risk of being in contact with ticks . as in many other viral diseases , cchf may cause symptoms of pulmonary involvement and may lead to death . 
the results of the thorax ct revealed that 20 of these patients ( 86% ) had pleural effusion , 12 of them ( 52% ) had consolidation , nine ( 39% ) had atelectasis , and 14 ( 60% ) had 1 3 la radiologia medica ( 2019 ) 124 : 826832 fig . 
however , hemoptysis was detected in a total of nine out of 27 patients with hemorrhage ( six of those patients died )  . there have been a very limited number of studies on pulmonary radiological findings in cchf in the literature . 
however , it has been stated that it can share similarities with mechanism of pulmonary pathologies such as pleural effusion , pneumonitis , hemoptysis , and alveolar hemorrhage in other febrile viral hemorrhagic diseases [ 15 , 16 ]  . 
in a study performed on dengue hemorrhagic fever , a total of 468 cxr taken from 363 patients were examined and parenchymal infiltration and pleural effusion were observed in more than half of the patients on the third day of follow - up . 
 moreover , these radiological findings were found to be closely related to laboratory findings of leukocyte count , platelet count , aptt , alt , and albumin levels in the same study [ 18 ]  . 
 in conclusion , pleural effusion and other findings related to pulmonary involvement can be seen in cchf due to endothelial damage caused by viral load in the circulation , increased capillary permeability , and hemorrhages due to low platelet . in the study by akta etal . 
the results of the study revealed that there was no statistically significant difference between the two examination techniques in terms of detecting parenchymal infiltration , alveolar infiltration , and pleural effusion . 
when these patients were compared , it was found that there was consolidation in cxr of the four patients and pleural effusion in one patient while they were not observed in thorax ct . 
on the other hand , based on thorax ct results , ground glass density was detected in four patients and atelectasis in two patients , and these were not observed in cxr . 
therefore , there was a possibility of response to the treatment and spontaneous regression or progression in the findings . the mortality rate of cchf generally ranges from 2 to 80% . 
this result can be associated with the fact that cchf is endemic in this region ; therefore , medical staff and people are informed about the disease , and thus , they are alert . 
in addition , the experience in the clinical follow - up might also contribute to the decrease in mortality rate besides improvements in medical care conditions in cchf and increase in awareness of the seriousness of the disease . it is known that mortality and some laboratory parameters prolonged a ptt , increased inr , high alt , ast and ldh , low platelet , and increased leukocyte are related to each other [ 23 , 24 ]  . 
for this reason , all of the cchf patients who were found to have pathologic cxr result should be considered at high risk . our choice to conduct a retrospective study is the first limitation ; on the other hand , medical information of the patients and their radiological images has been recorded in a proper way . 
the other limitation was the inability to confirm the cxr findings of the patients as not all of these patients had thorax ct . in conclusion , cchf is a serious public health problem that can be fatal . 
according to the results of our study , we suggest that 1 3 832 la radiologia medica ( 2019 ) 124 : 826832 radiological imaging of pulmonary system should be performed primarily with cxr . 
pulmonary involvement ( pleural effusion , consolidation , and ground glass opacity ) affects the survival in cchf negatively , and further examination should be performed with thorax ct in case of necessity . 
the association between findings was assessed using fishers exact test , while correlation at ct scan was evaluated with the spearman analysis . results bronchiectasis / bronchioloectasis ( 89.8% ) , nodule ( s ) ( 81.6% ) , tree - in - bud ( tib ) , and consolidation ( 79.6% each ) figured among the most common parenchymal findings . 
2 , university offlorence , azienda ospedaliero - universitaria careggi , largo brambilla 3 , 50134florence , italy 2 rheumatology unit , department ofexperimental andclinical medicine , university offlorence , azienda ospedaliero - universitaria careggi , 50134florence , italy infectious diseases unit , department ofexperimental andclinical medicine , university offlorence , 50134florence , italy infectious andtropical diseases unit , azienda ospedaliero - universitaria careggi , 50134florence , italy 5 department ofhealth science , university offlorence , viale g.b. 
morgagni 48 , 50134florence , italy vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 838845 introduction tuberculosis ( tb ) continues to be a public health concern in many european countries , despite the remarkable progress made in the last decade with respect to diagnosis and treatment availability . 
in many non - european countries , the prevalence of tb is still high , even in the multi - drug - resistant foreuropean countries , such as italy , have a high number of cases , especially among groups of vulnerable populations , such as immigrants and people co - infected with hiv [ 1 , 2 ]  . 
 the presence of immigrants from high - endemic areas is considered by many authors as one of the main reasons for the recent spurt in the number of tb patients in italy . 
to avoid misunderstanding the description of the epidemiological picture , it needs to be specified that a foreign - born population does not contribute significantly to the transmission of infection to the native population [ 6 ]  . tuberculosis is often diagnosed late , many times after the onset of the symptoms , and can involve any organ , with variable manifestations . 
although laboratory tests are essential , a radiological analysis can aid clinicians in the diagnosis , allowing the early start of infection control measures ( airborne precautions ) , that can be discontinued only when ( 1 ) another diagnosis explains the clinical signs and / or tb disease is considered unlikely ; ( 2 ) patient has three negative afb sputum smear results [ 7 ]  . while symptoms and physical findings can be nonspecific , specific signs of tb can be identified using radiological techniques . 
a computed tomography ( ct ) of the chest increases the specificity of the diagnosis , better showing distinct findings such as ground glass opacity ( ggo ) and tree - in - bud ( tib ) , and may be helpful , particularly when the x - ray does not show typical findings associated with tb ( cavities , consolidations , calcifications , etc . ) [ 8 , 9 ]  . against this backdrop , after observing a series of pulmonary tb patients until their healing , we propose this retrospective study to identify the most frequent radiological findings , to determine those with the highest degree of association , and to identify the most suggestive radiological findings for acid - fast bacilli ( afb ) positive disease . materials andmethods patients andstudy design we retrospectively reviewed the medical records of patients admitted to the infectious and tropical diseases unit at azienda ospedaliero - universitaria ( aou ) careggi ( florence , italy ) between 2014 and 2016 . 
on 28 may 2018 , the study obtained the approval of the local ethics committee ( protocol #12819 )  . all patients had provided their written informed consent for submission of their chest ct and , if necessary , of an iodinated contrast agent , according to the principles of the declaration of helsinki . 
tuberculosis ; hospitalization at other hospitals ; diagnosis of disease with primary or secondary pulmonary involvement due to other comorbidities ( cystic fibrosis , primary ciliary dyskinesia , autoimmune diseases ) ; diagnosis of respiratory tb in the absence of pulmonary involvement ; chest ct performed at a radiological service other than aou careggi . from january 2014 to december 2016 , 191 patients were admitted with a generic diagnosis of mycobacterial infection to the infectious and tropical diseases unit of aou careggi , and 127 of these patients were infected with m . 
only 109 showed pulmonary involvement , 102 of them didnt show comorbidities ( 5 had previously received a diagnosis of autoimmune disease and 2 had previously received a diagnosis of cystic fibrosis ) , 54 of whom executed a chest ct during diagnosis at one of the aou careggi radiological services . 
five patients were excluded from the study because of incomplete clinical data ( 2 ) , ct imaging of low quality ( 2 ) , and absence of dicom files of ct scans ( 1 )  . 
medical records provided the following data : ( 1 ) demographic data ( age , sex , nationality , time of permanence in italy ) ; ( 2 ) signs and symptoms before hospitalization ( asthenia , chest pain , cough , dyspnea , fever , haemoptysis , hyporexia , night sweating , weight loss ) ; ( 3 ) laboratory data ( leucocyte count , neutrophils percentage , c - reactive protein ) ; ( 4 ) microbiological data ( afb stain , culture methods , gene amplification test performed on sputum and , if present , on bronchoalveolar lavage )  . 1 3 840 la radiologia medica ( 2019 ) 124 : 838845 all ct scans in dicom were evaluated in consensus by two expert radiologists , vs and lm ( 5 and 7years of experience , respectively )  . 
a third evaluation was performed by sc ( 30years of experience ) , when the opinions differed . the observers checked for the presence or the absence of 10 parenchymal ct findings , as reported in the third paragraph : ( 1 ) bronchiectasis and bronchioloectasis ; ( 2 ) bulla ; ( 3 ) calcification ( s ) ; ( 4 ) cavity ; ( 5 ) consolidation ; ( 6 ) ggo ; ( 7 ) micronodule ( s ) ; ( 8 ) nodule ( s ) ; ( 9 ) serous reaction ; and ( 10 ) tib . 
they also looked for the presence or the absence of four mediastinal lymph node findings , as reported later : ( 1 ) calcification ( s ) ; ( 2 ) cavity ; ( 3 ) colliquation ; and ( 4 ) lymphadenopathy . hence , we defined the association between various findings as a pattern and calculated the degree of correlation among parenchymal findings . we then studied the prevalence of each finding in the entire sample and in the afb stain - positive subgroup , looking for findings that strongly correlated with positivity , to identify patterns that could predict afb positivity , and vice versa . it should be underlined that in the last years , the radiological difference between primary and post - primary tb has been questioned . 
many evidences show that the radiographic appearance of pulmonary tb does not depend on the time since infection , and other factors , like the immunocompetence of the host , can condition it [ 10 ]  . 
so , we avoided to differentiate patients in the two categories primary and post - primary tb . imaging studies helical multiphasic ct was performed on 49 patients using somatom plus volume zoom 4 ( siemens medical solutions ) unit with 37 - mm contiguous sections . 
the routine ct protocol comprised volumetric end - inspiratory and end - expiratory low - dose scans in a 16or 64 - row scanner ( somatom sensation , siemens , erlangen , germany ) , using the following parameters : 120kv for patients 35kg , 100kv for patients < 35kg , mas 1540 , 0 , 6mm collimation , slice thickness 2mm , reconstruction increment 0.8mm , pitch 1 , kernel b30 . 
ct scans were evaluated for the presence of the following 10 signs , as defined by the fleischner society [ 11 ] : bronchiectasis this refers to irreversible localized or diffuse bronchial dilatation . 
morphological criteria on thinsection ct scans include bronchial dilatation with regard to the accompanying pulmonary artery , lack of tapering of bronchi , and the identification of bronchi within 1cm of the pleural surface . bronchioloectasis it is the dilatation of bronchioles . 
 when dilated bronchioles are filled with exudate and are thick - walled , they are visible as a tib or as centrilobular nodules . bulla an airspace measuring more than 1cm in diameter sharply demarcated by a thin wall no greater than 1mm in thickness . 
it appears as a rounded focal lucency or an area of decreased attenuation , 1cm or more in diameter , bounded by a thin wall . calcification this refers to a zone of lung parenchyma with a very high attenuation value . cavity it is a gas - filled space , seen as a lucency or lowattenuation area , within pulmonary consolidation , a mass , or a nodule . consolidation it refers to an exudate or other product of a disease that replaces alveolar air , rendering the lung solid . 
it appears as a homogeneous increase in pulmonary parenchymal attenuation , which obscures the margins of vessels and airway walls . ggo it appears as hazy increased opacity , with preservation of bronchial and vascular margins . lymphadenopathy it is a lymph node with a short axis greater than 1cm . micronodule it refers to a discrete , small , round , focal opacity , with a diameter less than 3mm . nodule it is a rounded or irregular opacity , well or poorly defined , measuring up to 3cm in diameter . serous reaction the term serous reaction refers to all the findings that prove an involvement of serous membranes : pachypleuritis , pericardial effusion , pleural effusion , pleural empyema , and pleural thickening . tib it refers to multiple areas of centri - lobular nodules with a linear branching pattern that resembles a budding tree . 
it reflects a spectrum of endoand peribronchiolar disorders , including mucoid impaction , inflammation , and / or fibrosis . statistics in our study , data were presented in percentage terms . 
the association between suggestive findings for a transmissible disease ( scored positive or negative at afb stain ) and each ct scan finding ( yes or no ) was assessed using fishers exact test . 
the variables with significant association with afb positivity were entered into a multivariate logistic regression model , considering , as dependent variable , the afb positivity ( 1 for positive results , 0 for negative results )  . 
 among the 39 foreign patients ( 7 , 8 , 10 , and 14 , from africa , asia , south america , and eastern europe , in that order ) , only five ( 12.8% ) had been in italy for less than 1year , eight ( 20.5% ) from 1 to 5years , and 26 ( 66.7% ) for more than 5years . 
tuberculosis ( n = 64 ) excluded : absence of pulmonary involvement ( n = 18 ) excluded : presence of comorbidities ( n = 7 ) excluded : ct scan not performed ( n = 48 ) excluded : lack of data ( n = 5 ) patients infected by mycobacterium tuberculosis ( n = 127 ) patients with pulmonary involvement ( n = 109 ) patients without comorbitidies ( n = 102 ) patients that have performed a ct scan at diagnosis ( n = 54 ) enrolled patients ( n = 49 ) 1 3 842 la radiologia medica ( 2019 ) 124 : 838845 bulla serous reaction calcification ground glass opacity micronodule cavity tree - in - bud consolidation nodule bronchi ( olo ) ectasis cavity colliquation calcification limphadenopathy fig . 
2 frequency of parenchymal and nodal finding ( s ) nodule ( s ) and ggo ( r = 0.488 ; p < 0.05 ) bronchi ( olo ) ectasis and cavity ( r = 0.465 ; p < 0.05 ) tib and micronodule ( s ) ( r = 0.464 ; p < 0.05 ) tib and ggo ( r = 0.428 ; p < 0.05 ) nodule ( s ) and micronodule ( s ) ( r = 0.400 ; p < 0.05 ) high correlation involved only the seven most frequent parenchymal findings ( see fig.2 ) ; no nodal finding showed high correlation . 
no patient showed all the 10 parenchymal findings . radiological findings inafbpositive patients we found that only three findings ( consolidation , tib , cavity , p < 0.05 ) have a positive predictive value for afb stain , as shown in table1 . 
consolidation , tib , and cavity can be seen as a pattern , the only one with a positive predictive value of 100% if all three were detected together ( 25 , 60 and 77.8% if zero , one or two of them were present , respectively )  . consolidation , tib , cavity and serous reaction were included in the first multivariate logistic regression model as independent variables . 
serous reactions might indicate afb stain negativity bold values represent a significantvalue discussion as far as we know , our series is the only recent one that examined the radiological features of pulmonary tb in a low - incidence area . 
however , there are some findings which are more frequent , such as bronchi ( olo ) ectasis , nodule ( s ) , tib , and consolidation ( fig.3 ) , found in 44 , 40 , 39 , and 39 patients , respectively . 
other associations showed a contradictory positive predictive value , as that one among the four findings bronchi ( olo ) ectasis , nodule , micronodule and ggo , which demonstrated a positive predictive value of 95.7% if all four findings were present , but also conflicting values of 75% versus 71.4% if one or two of them were present , respectively . 
however , we believe this is a statistical result with no clinical meaning , since 65% of the subjects that do not have a serous reaction are afb positive as well . 
ct scans show scattered bronchiectasis ( arrows ) and tib in both lungs ( a ) and in the rear portion of both lungs ( b ) 1 3 844 la radiologia medica ( 2019 ) 124 : 838845 which is reported in 61% of our patients . 
in the literature , however , this frequency ranges from high values ( 7695% ) [ 1416 , 18 ] to low values ( 312% ) [ 2024 ]  . 
 for other frequent findingsnodule , consolidation , and tibour percentages do not significantly differ from the majority of data present in the literature , which shows , for example , the frequency of consolidation ranging from 54.7 to 96% [ 1519 , 2127 ]  . from an epidemiological point of view , our study population is mainly represented by foreign patients ( 39 of 49 ) , most of whom lived in italy for more than 5years . 
this confirmed that , despite the reduction in italian native population , the presence of migrants from high - endemic areas is one of the main causes of a stable incidence rate of tb cases ( approximately six per 100 , 000 inhabitants ) [ 4 ] , even if it does not contribute to infect the native population [ 6 ]  . the present study is not without limitations . 
nevertheless , the aim of the present study was to identify the most frequent findings with a view to examining them in a future prospective study . third , our report lacks inter - observer correlation index , because we adopted consensus evaluation . 
the application of an inter - observer correlation index in only 49 patients should have been not so confident between the two readers . finally , information about chronic obstructive pulmonary disease and smoking habit is not reported because the data is discontinuously acquired . 
however , since the average age is 40years , the induced risk is not so high . in conclusion , our data confirm ( 1 ) extreme heterogeneity of pulmonary tb while also revealing that no finding or pattern is clearly prevalent over others or unique to the disease . 
 ( 2 ) there are couples of findings ( tib and cavity ; tib and bronchi ( olo ) ectasis ; tib and consolidation ; nodule ( s ) and ggo ; bronchi ( olo ) ectasis and cavity ) with high correlation , which can help us to make the right diagnosis . 
 ( 3 ) although we identify a triad ( tib , cavity , and consolidation ) that strongly predicts afb positivity , we have not found any predictive sign of afb negativity . 
 ct scan shows in the left lower lobe a cavity of cm 4.20 ( asterisk ) surrounded by tib , and in the left upper lobe a consolidation ( arrowhead )  . 
moreover , in the right lung ct shows scattered bronchiectasis ( arrow ) for afb , but also 82% of patients without calcification ( s ) were positive for afb . 
this confirms that microbiological examination continues to be crucial for tb diagnosis . in our series , bronchi ( olo ) ectasis was the most represented finding , which is clearly not of the case with pulmonary tb . 
however , after having excluded the presence of other primary causes of bronchiectasis ( cystic fibrosis , primary ciliary dyskinesia , and autoimmune diseases ) , the correlation we found between bronchi ( olo ) ectasis and cavity ( r = 0.465 ; p < 0.05 ) could have a new meaning . 
in fact , these strong correlations and those between tib - cavity ( r = 0.577 ; p < 0.05 ) , tib - bronchi ( olo ) ectasis ( r = 0.498 ; p < 0.05 ) , and tib - consolidation ( r = 0.497 ; p < 0.05 ) suggest a unique pathogenetic path that was previously reported in the literature [ 12 ]  . 
accordingly , tib indicates mainly an endobronchial diffusion of the disease , but is recognized in a large number of conditions as diffuse panbronchiolitis and cystic fibrosis ; bronchiectasis is an expression of both bronchial wall malacia and chronic disease [ 11 , 13 ]  . 
the latter leads to parenchymal inflammation , consolidation , and ultimately , to cavity . some of the frequency values detected by us ( see the results section ) lack a precise confirmation in the literature . 
in the literature , the frequency values of bronchiectasis sensu stricto in tb patients range from 64% [ 18 ] and 89.3% [ 19 ] ( high frequency ) to 5% 14.3% [ 20 ] , 21% [ 21 ] , and 29% [ 22 ] ( low frequency )  . 
we can notice a similar trend with ggo , 1 3 la radiologia medica ( 2019 ) 124 : 838845 compliance with ethical standards conflict of interest all authors declare that they do not have conflict of interest . ethical standards this article does not contain any studies with animals performed by any of the authors . 
all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . 
on 28 may 2018 , the study obtained the approval of the local ethics committee ( protocol #12819 )  . informed consent informed consent was obtained from all individual participants included in the study . 
all patients had provided their written informed consent for submission of their chest ct and , if necessary , of an iodinated contrast agent , according to the principles of the declaration of helsinki . 
cisplatin concomitant chemotherapy was administered during imrt . results fourteen patients with cervical cancer were prospectively recruited between august 2014 and june 2017 , 13 ( 93% ) had a lacc , one ( 7% ) patient was not evaluable because 18fdg - pet / ct evidenced metastases to the liver undetected by previous ct / mri . 
imrt median dose to the pelvis was 48.6gy in 27 fractions , sib median dose 54gy in 27 fractions , hdr - bt boost median dose 21gy in 3 fractions . 
there were no grade 4 acute and / or late toxicities . conclusions the 18fdg - pet / ct influenced stage assessment and rt treatment planning due to its high specificity in distant metastases and nodal involvement detection . 
maria hospital , terni , italy vol . : ( 0123456789 ) 1 3 820 introduction cervical cancer remains the third most common malignancy worldwide and up to half of patients present with locally advance disease even if screening and prevention are improved [ 1 ]  . 
since 1999 exclusive concomitant radiotherapy ( rt ) plus chemotherapy is the standard treatment for locally advance cervical cancer ( lacc ) , and 5 - year overall survival ( os ) rate is near 70% [ 2 ]  . 
literature data show that 18fdg - pet / ct has a higher sensitivity than ct and magnetic resonance imaging ( mri ) , not only in detecting occult metastasis but also nodal involvement , while mri remains the best imaging technique to evaluate local tumor extension [ 4 , 5 ]  . 
therefore , 18fdg - pet / ct can influence the staging of cervical cancer and resulting therapeutic decision , particularly for rt field extension and dose to administer to involved lymph nodes [ 5 ]  . 
few experiences in literature evaluated outcome of 18fdgpet / ct based treatment plan and the use of simultaneous integrated boost ( sib ) for treatment of lacc [ 3 , 69 ]  . in clinical practice , a sib associated to conventional fractionation rt , is used with the aim to increase rt total dose to the tumor without increasing total treatment time . 
 moreover , overall treatment time of rt is crucial for the results because it has been shown that lc and os decrease by about 1% per day when time to rt exceeds 8weeks [ 1012 ]  . 
the influence of 18fdg - pet / ct for diagnosis and treatment decision were also discussed . materials andmethods patients with lacc diagnosed with 18fdg - pet / ct and enrolled between august 2014 and june 2017 were prospectively evaluated after definitive chemo - radiotherapy . 
 a concomitant weekly cisplatin ( 40mg / sq ) and subsequent three - dimensional - high - dose - rate brachytherapy la radiologia medica ( 2019 ) 124 : 819825 ( hdr - bt ) boost to the primary tumor were administered . 
the imrt was delivered with a ct - based planning system of the 18fdg - pet / ct , and it was done in supine position with empty rectum and filled bladder . 
 low dose ct was used for attenuation correction . the clinical target volume ( ctv ) included the entire uterus , vagina based on disease extension , lower common iliac , external and internal iliac , obturator and presacral nodes . 
for imrt , ctv was defined by a 7mm expansion of nodal regions with subtraction of the pelvic bones , femoral heads and vertebral bodies , then a margin of generally 5mm in all directions was added to ctv to obtain planning treatment volume ( ptv )  . 
based on the institutional set - up verification protocols , for the first 3days of treatment , patients position was verify by kv cone beam ct system integrated in the machine . 
then any variation of set - up was checked once a week . for hdr - bt , a ct - based planning was performed using a single uterine fletchers applicator and two vaginal ovoids , then a ct was done each fraction to verify appropriateness of placement . 
the brachytherapy ctv included disease volume based on clinical and mri evaluation done at the end of imrt and on viswanathan guidelines [ 14 ] ; prescription and optimization were performed on ctv based on uterine size , configuration and disease location . 
treatment plans were 1 3 la radiologia medica ( 2019 ) 124 : 819825 generated on the plato brachytherapy planning system , version 14.2.6 ( nucletron ) or oncentra brachytherapy planning system , version 4.1.2. the hdr - bt was administered 3 times a week , fractionation schemes were selected taking into account equivalent dose , quantec organs at risk ( oar ) constrains , patient compliance and previous imrt dose [ 15 , 16 ]  . 
treatment planning parameters adopted for hdr - bt were those of the american brachytherapy society [ 15 ]  . for combined treatment , cumulative dose ( i.e. , imrt plus hdr - bt ) was calculated with an / ratio of 10 for ctv ; for tolerance dose concerning late effects , an / ratio of 3 was used in the calculation of the equivalent dose in 2 - gy fraction to oar [ 17 ]  . for each patient , an informed consent was obtained both for imrt and hdr - bt according to the rules of our institution . response was assessed 12 months after the end of treatment with physical and mri exams . 
further follow - up was done using clinical and mri exams every 34months for first 2years , then every 6months ; ct was performed every 6months and 18fdg - pet / ct only when there was a suspicious of progression or relapse . in accord with the response evaluation criteria in solid tumors [ 18 ] , complete response ( cr ) was defined as complete resolution of enhancing lesion , partial response ( pr ) , 30% decrease in size of the lesion , progression of disease ( pd ) , > 20% increase in size of the lesion , and stable disease ( sd ) , no change in dimension of the lesion or does not meet criteria for cr , pr or pd . 
local relapse was defined as failure inside the rt fields in patients with local control and distant relapse as a failure outside rt fields . common terminology criteria for adverse events version 4.03 was used to grade toxicity . 
statistical analysis was performed using a software package ( medcalc 11.1 broekstraat 52 , b - 9030 mariakerke belgium ) and the kaplanmeier product - limit method [ 19 ]  . 
a value of p < 0.05 ( two tailed ) was considered statistically significant . results fourteen patients with cervical cancer treated at two italian centres ( terni and ancona , 8 and 6 cases , respectively ) were prospectively assessed . 
there were 13 of 14 patients evaluable for all study endpoints ; one ( 7% ) patient was not evaluable because 18fdg - pet / ct evidenced metastases to the liver undetected by previous ct / mri . 
apart from this case , 18fdg - pet / ct changed the stage derived from ct / mri findings , in other 7 ( 50% ) patients in which a more extensive disease due to nodal involvement was evidenced . 
 so , after 18fdg - pet / ct we modified treatment volume in these 7 patients . with respect to ct / mri findings , 18fdg - pet / ct changed local tumor extension in no case . 
clinical stage was as follows : apart from patient with liver metastasis , the other 13 patients were in figo stage iia , iib , iiib , iva and ivb in 1 , 6 , 2 , 1 and 3 cases , respectively . 
it is worth of note that 11 patients had nodal involvement , pelvic and / or lumbo - aortic in 8 and 3 cases , respectively . all patients underwent imrt to the primary tumor and pelvic draining lymph nodes , 3 of 13 ( 23% ) patients were treated also on para - aortic region . 
median total treatment time was 7weeks ( range , 616weeks ) , 2 patients temporary interrupted imrt for haematological grade ( g ) 3 and gastrointestinal g2 toxicity , respectively . after a median follow - up of 30months ( range , 641 ) , all patients were alive , 9 ( 69% ) without disease , 3 ( 23% ) with metastatic disease but no local relapse , and 1 ( 8% ) patient maintained her pr . 
at first control after treatment , 12 ( 92% ) of 13 patients obtained clinical and radiological cr , and one 1 3 822 la radiologia medica ( 2019 ) 124 : 819825 ( 8% ) a pr . 
2 distant control probability 1 3 la radiologia medica ( 2019 ) 124 : 819825 such as age , histology , stage and dose were assessed and had no prognostic impact . regarding acute toxicity , g1 - 2 nausea was registered in 3 ( 23% ) cases , g2 dysuria in 2 ( 16% ) , g2 proctitis in 3 ( 23% ) , g1 - 2 diarrhea in 3 ( 23% ) , g3 neutropenia in 3 ( 231% ) and g3 thrombocytopenia in 1 ( 8% ) patient ( table1 )  . generally , chemo - radiotherapy resulted feasible : 10 of 13 ( 77% ) patients completed treatment as planned , 3 ( 23% ) had iatrogenic toxicity . 
in 1 patient with acute grade 3 neutropenia and g2 diarrhea , chemotherapy was stopped after the fourth cycle and rt temporary interrupted ; in another case , chemo - radiotherapy was temporary interrupted for g3 neutropenia and thrombocytopenia ; 1 patient with grade 2 diarrhea interrupted treatment for 5days ( table1 )  . 
regarding late toxicity , g1 vaginal stenosis was found in 1 ( 8% ) case , g1 hematuria and dyspareunia in 1 ( 8% ) , and g3 rectal bleeding in 1 ( 8% ) patient . 
no g4 toxicity was registered ( table1 )  . discussion recently 18fdg - pet / ct has become an interesting modality for staging , re - staging and assessing response to therapy in lacc management . 
this exam , resulting from a combination of metabolic pet information and anatomic ct images , can be more accurate than ct or mri in detecting regional lymph node involvement and extra - pelvic disease [ 5 ]  . 
lymph node status , though not included in the figo system , is an important prognostic factor which should be considered in the therapeutic strategy [ 3 , 5 , 6 ]  . 
although mri remains the best imaging technique to evaluate local tumor extension , several data have shown that 18fdg - pet / ct has a higher sensitivity with respect to ct and mri , not only in detecting occult metastases ( 95% ) but also nodal involvement ( 100% ) [ 4 , 5 , 9 ]  . 
in clinical practice , 18fdg - pet / ct staging altered lacc management in a population variable from 18% to 44% by changing treatment strategy from surgery to chemo - radiotherapy or palliation . 
moreover , rt planning can be influenced by 18fdg - pet / ct imaging both in defining field extension and in prescribing higher doses to involved lymph nodes [ 5 , 9 , 20 ]  . our series confirmed these indications . 
in fact , 18fdgpet / ct changed the stage derived from ct / mri findings , in 8 of 14 ( 57% ) patients in which a more extensive disease due to nodal involvement ( 7 cases ) or metastatic disease ( 1 case ) was evidenced , and did not change local tumor extension assessed by mri . 
so , in the 7 patients with an evidence of nodal disease unidentified at ct / mri , ctv was enlarged to encompass nodal metastases which received also a higher dose using the sib technique . in our experience this discrepancy between 18fdg - pet / ct and ct / mri is higher with respect to literature data [ 5 , 9 , 20 ]  . 
a possible explication of this discrepancy is the prevalence of advanced figo stage in our patients and the consequent higher probability of nodal and / or metastatic involvement . in clinical practice the planning with 18fdg - pet / ct is uncommon . 
however , patients have a long follow - up ( median 30months ) and pet / ct did change figo stage and treatment approach in 57% ( 8 of 14 ) of cases . 
so , our study , although suboptimal for the paucity and inhomogeneity of patients recruited , can result of some interest considering the role of 18fdg - pet / ct in changing not only the stage and therapeutic strategy , but also in better defining imrt targets in lacc . our patients underwent 18fdg - pet / ct in rt treatment position to have a more precise sib - ctv contour and to reduce set - up error probability . 
as in other published similar series of 18fdg - pet / ct simulation , for sib - ctv a margin of only 5mm was done to obtain sib - ptv [ 21 ]  . 
 when 18fdg - pet / ct was available only for staging and not for rt simulation , margins between ctv and ptv were generally > 5mm [ 3 , 8 , 9 ]  . 
this difference in size of expansion is very important because can condition the table 1 acute and late toxicities registered with common terminology criteria for adverse events version 4.03 scale entity upper gastro - enteric ( nausea / diarrhea ) lower gastro - enteric ( proctitis / rectal bleeding ) genito - urinary ( dysuria / vaginal stenosis / dyspareunia ) hematologic ( neutropenia / thrombocytopenia ) acute ( % ) late ( % ) acute ( % ) late ( % ) acute ( % ) late ( % ) acute ( % ) late ( % ) grade 1 grade 2 grade 3 grade 4 1 3 824 la radiologia medica ( 2019 ) 124 : 819825 appearance of iatrogenic toxicity . 
in fact , the tighter is tumor margin the lesser is expected toxicity to surrounding healthy tissues . the administration of a boost dose to positive lymph nodes by sib is a modern technique which allows to increase total dose without increasing total treatment time and toxicity [ 13 , 22 , 23 ]  . 
with 30months of median follow - up , our 86% of lc rate at 3years was similar to vargos experience in which lc and regional control rates at 3years were 76% and 94% , respectively [ 3 ]  . 
in another paper , local and pelvic control rates were > 90% but number of patients was limited and median follow - up of only 9months [ 13 ]  . the 67% of dc probability at 3years was quite inferior to literature data where it results between 77 and 90% [ 3 , 6 ]  . 
 nevertheless , only 3 ( 23% ) patients had local or distant progression , compliance to the treatment was very high , and toxicity acceptable considering that all patients completed scheduled treatment . although our prospective study had recruited a relatively small number of patients , follow up is quite long and standardized 18fdg - pet / ct was used for rt simulation and imrt / sib administration according to the best modern concept of diagnosis and treatment of lacc . 
the aim of this study is to calculate the accuracy of chest computed tomography to detect surgically resectable blebs or bullae in patients with primary spontaneous pneumothorax . methods this is a retrospective study includes all patients with primary spontaneous pneumothorax who underwent chest computed tomography evaluation for their disease over the period from january 2005 to december 2015 . 
patients who underwent surgical exploration were sub - grouped to calculate the sensitivity and the specificity of the chest computed tomography to detect surgically resectable pulmonary blebs or bullae . results a total of 143 patients were included in the study . 
therefore , the routine use of chest computed tomography scan before the surgical exploration in patients with primary spontaneous pneumothorax should depend on the clinical judgment . keywords blebs bullae computed tomography primary spontaneous pneumothorax introduction spontaneous pneumothorax is usually classified into primary and secondary types . 
while the primary type occurs without the presence of underlying lung disorder , the secondary one occurs when the lung has a disorder leading to the formation of cystic cavities [ 1 ]  . although the patients with primary spontaneous pneumothorax ( psp ) have no obvious lung disease , abnormalities in the visceral pleura such as blebs or bullae are usually present . 
the intuitional review board approves this study on february 9 , 2017 , with the number irb - 2017 - 01 - 017 . the study includes all patients with psp who underwent chest ct evaluation during their admission . 
the chest ct in this study was a highresolution non - contrast - enhanced ct of the chest with 64 - row mdct ( somatom definition , siemens healthcare sector ; forchheim , germany ) using a standardized chest ct protocol with the following parameters : collimation : 32 2 0.6mm ; weight - adapted selection of the tube voltage for both tubes ( ranging between 100 and 120kv ) with adapted tube current setting ( ranging between 90 and 100 effective mas ) ; 4d dose modulation ( care dose 4d ) ; pitch : 3 ; rotation time : 0.28s. 
all patients underwent ct scan examinations in the supine position and were required to hold their breath at full inspiration . ct images of 1 - mm slice thickness for 2 - mm intervals in the lung parenchyma in axial and coronal [ window level : 600 hu ; window width : 1500 hu ] were used for analysis . all images were displayed using a picture archiving and communication system ( pacs ) workstation ( syngo.plaza. siemens healthcare sector , erlangen , germany )  . the diagnosis of psp was confirmed by reviewing the patients initial chest x - ray and charts . 
all chest ct la radiologia medica ( 2019 ) 124 : 833837 examinations of the patients were reviewed by one expert radiologist looking for the presence of ipsilateral and contralateral blebs or bullae and the presence of other abnormalities in the lung or pleura . 
 the surgical management includes intercostal chest tube drainage of the pneumothorax that may be followed by surgical exploration of the pleural cavity depending on the patient response to the drainage procedure . 
the recurrence rate of psp is lower in the operated patients whether they were operated after the first or the second episode of psp as the surgical intervention reduces the risk of recurrence [ 5 ]  . 
moreover , the surgical intervention reduces the length of hospital stay as the operated patients usually require three hospitalstay days , while it requires about five to seven days for the conservative management patients . 
1 high - resolution computed tomography of the chest , coronal ( a ) and axial cuts ( b , c ) : left pneumothorax , red arrows ( b , c )  . 
2 an algorithm of the study sample 174 patients 143 patients 31 patients excluded as they dont have chest ct evaluation 23 patients underwent only drainage procedure 120 patients underwent surgical exploration taken as a subgroup to determine the sensitivity if chest ct scan in detecting surgically resectable blebs or bullae table 1 chest computed tomography finding of 143 patients with primary spontaneous pneumothorax number of patients ( % ) table 2 2 2 table correlates the chest computed tomography finding to the surgical exploration finding of 120 patients with primary spontaneous pneumothorax chest computed tomography findings of pulmonary blebs or bullae surgical findings of resectable pulmonary blebs or bullae total finding pneumothorax side right left pulmonary blebs ipsilateral contralateral pulmonary bullae ipsilateral contralateral 70 ( 49.0% ) 73 ( 51.0% ) 119 ( 83.2% ) 103 ( 72.0% ) 32 ( 22.4% ) 21 ( 14.7% ) total surgery looking for pulmonary blebs or bullae for resection with or without the use of pleurodesis . 
the patient who underwent surgical exploration was taken as a sample to determine the sensitivity and the specificity of chest ct scan in detecting surgically resectable blebs or bullae . statistical analysis of the sample was carried using statistical package for the social sciences version 21 ( ibm corp . , armonk , ny , usa )  . 
out of the 94 patients who found to surgically resectable pulmonary blebs or bullae , the chest ct scan could detect the pulmonary blebs or bullae in 90 patients of them with a calculated sensitivity of 95.7%. 
on the other hand , 11 patients have a negative chest ct scan among the 26 patients who did not found to have surgically resectable pulmonary blebs or bullae with a calculated specificity of 42.3%. 
most of the samples are males ( n = 141 , 98.6% ) with a mean age of 24 6.2years. through evaluation of the chest ct scan of the patients , 119 patients ( 83.2% ) have ipsilateral blebs and 32 patients ( 22.3% ) have ipsilateral bullae . 
contralateral blebs were present in 103 patients ( 72.0% ) and contralateral bullae were present in 21 patients ( 14.7% ) ( table1 )  . psp occurs usually in males who are smokers , tall , and thin [ 4 ]  . 
chest x - ray can also detect abnormalities in the pleura that may have an effect on the recurrence rate [ 7 ]  . chest ct scan is an evolving method that is commonly used for psp to detect the presence of abnormalities in the lung or pleural cavity , mainly the presence of pulmonary 1 3 836 la radiologia medica ( 2019 ) 124 : 833837 blebs or bullae [ 8 ]  . 
the sensitivity of the chest ct scan to detect those changes is increased when the ct scan that is used is high resolution with thin slice and reviewing both the axial and coronal view [ 9 , 10 ]  . 
 moreover , chest ct scan can show abnormalities in the pattern of the thorax that is commonly found in patients with psp as they tend to be taller , wider transversely , and flatter anteroposteriorly [ 1214 ]  . 
unexpected lesions can also be detected by the use of chest ct scan in patients with psp especially in children and females [ 15 , 16 ]  . the ipsilateral recurrence rate of psp is increased with the presence of ipsilateral blebs and bullae in the chest ct scan if no surgical exploration offered [ 17 ]  . 
this is a very crucial element in the discussion with the patients in our culture as the recurrence on the contralateral side can occur even with doing a surgery on the ipsilateral side . 
on the other hand , an absence of pulmonary blebs or bullae on the chest ct scan fails to predict the recurrence rate psp [ 16 , 21 , 22 ]  . even though chest ct scan has those benefits , the current guidelines limit the use of chest ct scan to the complicated cases of psp [ 23 , 24 ]  . 
in fact , some studies advocate the use of surgical exploration even in the absence of abnormalities in the chest ct scan [ 21 , 25 ]  . the study was limited as it was a retrospective study involving only one center with specific geographic distribution . 
a more prospective study is needed to study the recurrence rate of psp in relation to chest ct scan findings with consideration of the histopathological evaluation of the resected specimen . conclusion the sensitivity of the chest ct scan is high in detecting surgically resectable pulmonary blebs or bullae . 
the aim of our work was to evaluate the potentialities of carotid computed tomography angiography ( ccta ) in assessing composition of atherosclerotic plaque . materials and methods we retrospectively evaluated 29 patients ( 7 women and 22 men , age range 5481 ; mean age 69 ) who underwent carotid endarterectomy . 
mcnemars test was used for comparison of dichotomous variables . results a significant correlation between histology and ccta was found with respect to the areas corresponding to adipose , fibrotic and calcified plaques . 
carotid atherosclerotic disease is a common condition preferably affecting the elderly population , considering that a carotid artery stenosis has been reported in up to 75% of men and 62% of women aged 65years [ 13 ]  . 
these components associated with endothelial erosion may lead to plaque instability and contribute to severe disease and clinical risk [ 810 ]  . on the basis of their degree , carotid stenoses have been classified as moderate ( 5069% of vessel lumen ) or severe ( 70% of lumen )  . 
since cerebrovascular events causing acute occlusion of a cerebral vessel are the consequence of embolisms typically arising from a carotid bifurcation plaque , several recent studies suggest investigating other processes , besides the degree of stenosis , as possible causes of the atherosclerotic plaque . 
vulnerable plaques are associated with crucial morphologic findings : rupture of the fibrous cap , signs of intra - plaque hemorrhage , the presence of adipose large core and irregularities ( ulcerations ) on the plaque surface [ 1214 ]  . 
among the different diagnostic imaging techniques currently available , multidetector ct angiography ( mdct ) is considered the most effective thanks to its availability , velocity of execution , high spatial resolution and multi - planar reconstructions . 
as suggested by many authors [ 15 , 16 ] , mdct plays an important role in predicting vulnerability of atherosclerotic plaque as the inner density of the plaque is inversely proportional to the risk of rupture and consequent cerebral embolisbased on these considerations , we retrospectively analyzed atherosclerotic carotid plaque composition using mdct with the aim to assess quantitatively adipose , fibrous and calcified intra - plaque tissue and compare the imaging data with the histological features obtained from carotid endarterectomy ( cea ) samples . 
the institutional review board of our university approved this retrospective study protocol . materials andmethods from january 2016 to february 2017 , we selected 29 patients ( 7 women and 22 men , age range 5481 ; mean age 69 ) who underwent carotid endarterectomy ( cea ) due to the presence of hemodynamic internal carotid artery stenosis 70% with or without neurological symptoms . 
all patients received 60ml of contrast medium 400mg / ml ( iomeprolo , iomeron ; bracco imaging italia ) followed by 40ml saline bolus chaser , both with an injection rate of 4ml / s . 
poor - quality ct images were excluded from our study . image analysis ct images were sent to a stand - alone workstation ( vitrea workstation , toshiba medical systems europe ) operating with dedicated post - processing 3d analysis software sureplaquetm . 
in our experience , range 100 to 49 ( hu ) was displayed in red ( adipose plaque component ) , range 50149 ( hu ) in blue ( fibrotic component ) , and range 1501300 ( hu ) in yellow ( calcific component )  . 
to assess the atherosclerotic plaque composition , the vessel was studied at the level of maximum stenosis and 3mm above and below the slice selected as the one showing the largest plaque area . 
 the percentage of the three different components of the 1 3 la radiologia medica ( 2019 ) 124 : 795803 atherosclerotic plaque ( adipose , fibrous and calcified tissue ) was assessed using pre - specified hounsfield unit ( hu ) densitometric values . 
according to our pre - defined protocol , the plaque component presenting a hu value between 100 and 49 was classified as adipose tissue , between 50 and 150 fibrous tissue , and higher than 150 calcified tissue . 
to avoid rupture or fragmentation of the plaque the specimen wasobtainedbyerodingthe outer media and adventitia . of the different areas occupied by each of the components , by calculating it on the total surface of the plaque . statistical analysis continuous variables were summarized as means with standard deviation , and categorical variables were reported as absolute or relative frequencies . 
vessel and plaque parameters were compared between ccta and histology using the pearson correlation coefficient ( r ) with two - sided p values and with 95% confidence intervals ( ci ) for r . 
 similarly to ccta image acquisition and analysis , the arteries were sectioned serially 5mm above and below the level of major stenosis , dehydrated in graded ethanol and embedded in low - temperature - fusion paraffin for histological studies . 
serial 3 - m - thick sections were cut in order to reach the maximum plaque thickness ; the sections located above and below the maximum plaque extension were discarded as they did not show a complete view of the lesion . 
the whole image was therefore employed to select the corresponding calcified , fibrotic and adipose areas within the plaque . these evaluations were independently performed by two pathologists ( av and rs ) in a double - blinded fashion . finally , through an image analysis system ( ias , delta system , rome , italy ) it was possible to obtain the percentage considering the differences in thickness between ccta slices ( 0.5mm ) and histological sections ( 3m ) , the matching between ccta and histology was considered with great care to avoid , as much as possible , misregistration . 
the table on the top shows percentages of the different components of the plaque showing very good correlation with histological values obtained from the specimen using massons trichrome staining protocol . 
2 maximum intensity projection ( mip ) ct ( left ) and volume rendering ( right ) reconstructions of a mainly fibrotic carotid bifurcation atherosclerotic plaque , presenting a relatively high adipose component . 
3 maximum intensity projection ( mip ) ct ( left ) and volume rendering ( right ) reconstructions of a carotid bifurcation atherosclerotic plaque presenting a prevalence of calcified tissue . 
in the center of the figure , residual vessel lumen is shown in green 1 3 la radiologia medica ( 2019 ) 124 : 795803 discussion the results of our study confirm that mdct can be a reliable diagnostic imaging tool to assess different components of the atherosclerotic carotid plaque , useful to identify vulnerability criteria . 
on the other hand , a relatively moderate correlation with histology regarding adipose and fibrotic tissue was reported in exvivo studies [ 16 ] , where the authors found a wide range of overlapping values for differentiation of non - calcified from mixed plaques . 
they explained the obtained data with the frequent coexistence of calcium and the consequent blooming effect ; in fact , the correlation for adipose tissue was particularly inaccurate in severely calcified plaques . 
the blooming effect , caused by calcifications , leads to overestimation , on ct images , of the calcified component and often does not allow adequate evaluation of the non - calcified parts . 
 [ 20 ] who reported sensitivity and specificity values of 93% , however , excluding from the analysis all the slices presenting a cluster of calciusaremi and achenbach [ 21 ] reported that differentiation of soft and lipid - rich plaque from more stable fibrous ones is still difficult and significant overlap in attenuation values exists . on current clinical computed tomography scanner , thin fibrous cap detection is not possible , and the vulnerable plaques may be misdiagnosed or overlooked [ 22 ]  . 
nevertheless , a low attenuation core surrounded by a rim - like area of higher attenuation also known as napkin sign may be suggestive of plaque at high risk of cerebrovascular events . when performing mdct , it is important to consider that hyperdense areas projecting outside vessel lumen could be referable to plaque ulcerations but also to focal calcifications ; it is thus recommended to acquire unenhanced scans in order to assess the correct diagnosis , since a true ulceration will appear hypodense on unenhanced scans . the vulnerable plaque is reportedly 60 hu in density [ 23 , 24 ]  . 
on the coronary plane , a low - density plaque is considered as vulnerable , and the boundary hu between lipid - rich and non - lipid - rich plaque is established at 5070 [ 25 , 26 ]  . our data aided by these considerations suggest that limitations still persist in the characterization of plaques even using a wide - detector apparatus with thin 0.5 - mm detectors [ 27 ] and considering lipid - rich plaque with 50 hu in density . 
we also observed a moderate correlation between histology and ccta in adipose tissue ( r = 0.55 ; p < 0.012 ) with respect to fibrotic ( r = 0.90 ; p < 0.0001 ) and calcified ( r = 0.79 ; p < 0.0001 ) tissue within the plaques . 
overestimation of the calcified component may be the consequence of blooming artefacts also present in our experience . according to our experience , in heavily calcified plaques , a slight modification of the aforementioned thresholds when analyzing different components of the plaque might be appropriate in order to overcome limitations depending on blooming effects ; this concept , however , has still to be demonstrated in larger case series . the moderate correlation of the adipose component with histology may be more reliably related to the coexistence of the calcified component hindering an adequate estimation of the adipose one . this affirmation may be supported by the evidence that the adipose area is underestimated at ccta if compared to histology considering that it was excluded that discrepancies could be related to the manipulations of the plaque . 
for all these reasons , our experience confirms the good potentialities of ccta in plaque characterization that , combined with an accurate stenosis assessment , may suggest an interesting role in the one - shot evaluation . currently , other diagnostic imaging techniques provide several fundamental information regarding plaque morphology and clinical relevance . 
ultrasonography with doppler examination is considered a fast and first - line imaging technique able to estimate the degree of stenosis and useful to identify echo - lucent plaques more prone to rupture due to their high content in fat tissue ; nevertheless , its reproducibility is not always adequate [ 2830 ]  . some studies , however , showed the superiority of contrast - enhanced ultrasound ( ceus ) to conventional us , particularly regarding the possibility to identify ulcerated plaques in terms of sensitivity and inter - observer agreement [ 31 ]  . mri is considered the best imaging technique for plaque characterization based on the evidence and status of the fibrous cap ( thick , thin or fissured ) usually appearing as a juxta - luminal low - signal band on time - of - flight ( tof ) 1 3 802 la radiologia medica ( 2019 ) 124 : 795803 sequences ; lack of the hypointense zone in tof sequences between vessel lumen and plaque represents a sign of ulceration . 
thanks to the different imaging patterns of blood products on t1 - weighted , t2 - weighted and tof sequences , it is also possible to age the areas of hemorrhage . moreover , mri , performed with contrast enhancement in dynamic technique , is a valuable tool to identify possible intra - plaque enhancing areas , referrable to signs of inflammation or hemorrhage and to assess the possible presence of neovascularization , which is considered a sign of vulnerability since these vessels are highly prone to rupture . these patients would be better candidates for carotid endarterectomy than for carotid stenting , considering that the latter has been demonstrated to present a higher risk of intra - procedural complications [ 15 ] due to possible thromboembolic complications . however , mri has some limitations including poor spatial resolution , scarce ability to demonstrate the calcified components and quite long acquisition times [ 3234 ]  . 
on the other hand , mri may prove useful in selected patients showing unfavorable morphologic features of the plaque on ct imaging and its use may prove of support during plaque analysis in the attempt of evaluating the individual risk of developing cerebral stroke [ 35 , 36 ]  . limitations our study presents some limitations . 
first , histology is considered as the reference standard in quantitative assessment of carotid plaque composition using mdct ; nevertheless , carotid plaque specimens may be damaged during transportation between operating rooms and histological examinations ; these phenomena are likely to affect mostly the softest component of the plaque , represented by fat . 
this was necessary to have a correlation technique represented by histological analysis , but on the other hand , this could limit the clinical application of our data , since many cerebral strokes occur in patients presenting a lower degree of stenosis [ 37 ] and correlation between ct imaging and histopathological findings should be investigated . 
finally , our data were obtained from a relatively small cohort , while a larger sample size may have provided more reproducible results . conclusions our data suggest that ccta has adequate characteristics to identify correctly the different components of carotid atherosclerotic plaques , with some limitations in plaques with a relevant adipose component . 
postnatal imaging , postoperative report , histology , autopsy , and clinical outcomes were the reference standard to calculate sensitivity , specificity , accuracy , positive predictive value ( ppv ) , and negative predictive value ( npv ) of prenatal mri in detecting airway patency . 
we also assessed mri performance in the diagnosis of the mass nature . results we obtained data about postnatal airway status in 19 of 23 patients ; prenatal mri demonstrated a sensitivity of 9 / 9 [ 100% , 95% confidence interval ( ci ) 66100% ] , specificity 8 / 10 ( 80% , 4498% ) , accuracy 17 / 19 ( 89% , 6799% ) , ppv 9 / 11 ( 82% , 4898% ) , and npv 8 / 8 ( 100% , 63100% ) ; the interobserver agreement was perfect . 
buzzi childrens hospital asst fatebenefratelli - sacco , via castelvetro 32 , 20154milan , italy 4 radiology unit , irccs policlinico san donato , via morandi 30 , 20097sandonatomilanese , milan , italy 5 department ofbiomedical sciences forhealth , universit degli studi di milano , via morandi 30 , 20097sandonatomilanese , milan , italy introduction congenital neck masses are rare and usually benign [ 1 ] ; however , they may compress and distort foetal airway resulting in hypoxic injury and neonatal death [ 25 ]  . 
the antenatal detection of a neck mass and its effect with the foetal airway is of paramount importance as it may change the therapeutic strategy and management of the delivery [ 2 , 3 ]  . in order to prevent respiratory distress , at birth is available the ex utero intrapartum therapy ( exit ) procedure . 
in fact , the progress of imaging techniques allows antenatal selection of those foetuses with congenital abnormalities , who need special care at birth in highly specialized hospital [ 5 , 6 ]  . 
in clinical practice , ultrasound ( us ) is the first line imaging modality to evaluate pregnancies , thanks to its wide availability , low cost , and absence of radiation exposure [ 10 , 11 ]  . 
despite its limitations , in most cases us is sufficient to characterize foetal malformations , but sometime , it does not provide complete information for a proper treatment planning , as in case of neck mass , where it can only identify indirect signs of airway obstruction , such as polyhydramnios , tongue protrusion , and absence of swallowing [ 2 , 5 , 10 , 12 , 13 ]  . 
magnetic resonance imaging ( mri ) is now a complementary tool for evaluating foetuses with foetal airway and neck mass anomalies at prenatal us [ 3 , 11 , 13 ]  . 
as previously published in case reports and small case series , mri is actually considered the best imaging modality to directly visualize the foetal airway , thanks to its intrinsic high softtissue contrast , high spatial resolution , and application of new ultrafast sequences [ 11 , 1316 ]  . the aim of our study was to investigate the diagnostic performance of prenatal mri in neck mass characterization , with special focus on the foetal airway status . materials andmethods patient selection we retrospectively reviewed all prenatal mri examinations performed in our institution from may 2001 to february 2016 , performed after a second - level ultrasound suspicious for neck mass carried out at most 7days before . 
all mothers signed a written informed consent prior to prenatal mri and a disclaimer regarding data collection about clinical and imaging follow - up . prenatal mri protocol prenatal mri examinations were performed on a 1.5 - t system ( philips achieva , the netherlands )  . 
they were blinded to the patients name , indication to mr , and final diagnosis ; instead , they were aware of the gestational age at the time of the examination . foetal airway the readers evaluated the involvement of foetal airway on t2 - weighted images , where the airway was better identifiable because of the brightness of its fluid content . 
foetal airway was considered involved if the lumen was : obstructed : not visible at all at the level of the mass narrowed : any narrowing of the airway diameter at the level of the mass respect to the airway diameter above or under the mass . displacement of the airway alone was not sufficient for considering airway threatened . mass characteristics we divided neck in anterior , posterior , and laterocervical regions . anterior region : limited posteriorly by the transverse processes of the cervical vertebrae , including infrahyoid and suprahyoid regions and the median visceral space of the neck posterior region : located behind the transverse processes of the cervical vertebrae , including cervical spine and 1 3 la radiologia medica ( 2019 ) 124 : 917925 the posteriorly surrounding soft tissues together with the nuchal region laterocervical region : including the neurovascular bundle region , the sternocleidomastoid muscle , and the supraclavicular region . we also assessed the development of the mass into the mediastinum . the structure of the masses was assessed considering the characteristics of the signal on t1 and t2 weighted sequences ; hence , we classified each mass as cystic ( high signal on t2w and low signal on t1w ) , solid ( low signal on t2w and low signal on t1w ) , or mixed ( cystic - solid signal )  . to estimate the size of the mass , we considered the maximum diameter in any plane . for each mass , a hypothesis of diagnosis of nature was given by the two readers , distinguishing among lymphatic vascular malformation , teratoma , or other lesion . 
the prenatal us diagnosis is also reported in table2 . reference standard the need for respiratory care in neonatal period , determined by clinical outcomes , postnatal imaging , surgery , histology , or autopsy , was considered the reference standard for determination of airways involvement . 
respiratory care included exit procedure and intubation . the final diagnosis was confirmed by histopathology in case of resection or biopsy of the mass or by autopsy in case of non - delivered foetuses or neonatal death . 
unequivocal clinical course was used for final diagnosis of some lymphatic vascular malformations thatwere followed up with us , mri , and surgical visitand which showed regression , spontaneously or after sclerotherapy . statistical analysis for each foetus , the following variables were recorded : the gestational age and diagnosis at the time of us , the gestational and maternal age at the time of prenatal mri . 
distributions were given as counts and percentages for categorical variables and as median and interquartile range ( iqr ) for continuous variables . we calculated the diagnostic performance of prenatal mri as sensitivity , specificity , overall accuracy , positive predictive value ( ppv ) , and negative predictive value ( npv ) with their 95% confidence intervals for the involvement of the airway by the neck mass . 
the interobserver agreement in evaluating airway status , as well as in the diagnosis of mass nature , was estimated by using the cohen statistics . differences between lymphatic vascular malformations and teratomas were assessed in terms of location , structure , maximum diameter , gestational , and maternal age at the time of the prenatal mri . 
 significance level was set at 0.05. results postnatal follow - up was available in 23 of the 26 foetuses , 14 females and 9 males ; three patients were lost to postnatal follow - up . 
of these 23 , 19 women carried the pregnancy to term , three voluntarily terminated the pregnancy , and a woman experienced intrauterine foetal demise . at the time of the us , the median gestational age was 25weeks ( iqr 1934 )  . 
at the time of the prenatal mri , the median gestational age was 28weeks ( iqr 2037 ) and the median maternal age was 32years ( iqr 2149 ) ; see table1 for more detail . among the 19 delivered neonates , 17 ( 90% ) survived through neonatal period and were alive after a follow - up of at least 1year ( range 113years )  . airway involvement postnatal confirmation about airway involvement was available only for the 19 delivered foetuses . eight of 19 babies did not need any neonatal respiratory assistance at birth ; nine of 19 babies needed neonatal respiratory care : six exit procedures and three endotracheal intubations were performed . 
exit procedure was performed in three babies affected by a lymphatic malformation , two by a teratoma and one by a venous vascular malformation ; all newborns had long - term survival but one with a huge teratoma extending into the mediastinum , died on the third day due to respiratory insufficiency . 
among babies undergone intubation : one had a thyroid goitre , one a lymphatic malformation , and one a branchial cyst ; the newborn with the goitre died shortly after the intubation for multi - organ failure . 
in 4 exit procedures , airway was secured by intubation ; in 2 exit procedures , tracheotomy was necessary . in diagnosing the foetal airway involvement by the neck mass , prenatal mri demonstrated a sensitivity of 9 / 9 ( 100% , 66100% ) , a specificity of 8 / 10 ( 80% , 4498% ) , an accuracy of 17 / 19 ( 89% , 6799% ) , a ppv of 9 / 11 ( 82% , 4898% ) , and a npv of 8 / 8 ( 100% , 63100% )  . 
 only one lymphatic vascular malformation and one teratoma developed into the mediastinum ; in both cases , exit procedure was successfully performed . prenatal mri correctly identified 21 / 23 ( 91% ) cervical masses , while prenatal us correctly identified 17 / 23 ( 74% )  . there were two wrong mri diagnoses : a branchial cyst ( fig.3 ) misdiagnosed as lymphatic vascular malformation and a lymphatic vascular malformation misdiagnosed as teratoma . 
the two cases of disagreement were the venous vascular malformation and the neurocristic cutaneous hamartoma , both correctly diagnosed only by the more experienced reader . at the time of prenatal mri , teratomas had a median maximum diameter ( 68mm , iqr 6178 ) greater ( p = 0.032 ) than that of lymphatic vascular malformations ( 52 mm , iqr 3762 )  . 
instead , there was no significant difference between lymphatic vascular malformations and teratomas in terms of median gestational age ( 28 weeks for both , p = 0.750 , mannwhitney u test ) , median maternal age ( 32 vs 31years , respectively ; p = 0.385 , mannwhitney u test ) , 1 3 la radiologia medica ( 2019 ) 124 : 917925 fig . 
ss - tse t2 - weighted axial mr image ( a ) shows a high signal multiseptated left neck mass ( * ) , deviating and compressing the oropharynx ( arrow ) ; ss - tse t2 - weighted sagittal image ( b ) demonstrates the mass ( * ) compressing the oropharynx ( arrow ) , while larynx and trachea are patent ( arrowheads ) ; sense btfe coronal image ( c ) shows the mass ( * ) compressing the airway at the level of oropharynx ( arrow ) ; trachea is patent ( arrowhead ) fig . 
sstse t2 - weighted sagittal ( a ) , coronal ( b ) , and axial ( c ) images show an exophytic mass ( * ) with mixed solid and cystic structure , obstructing oropharynx , hypopharynx , and larynx ( arrow ) with hyperextension of the foetal neck ; nasopharynx and trachea are patent ( arrowheads ) and gestational age at the discovery of the disease ( 23 vs 26weeks , respectively ; p = 0.892 , mannwhitney u test )  . among foetuses affected with teratomas , three had hyperextension of the neck and two had polyhydramnios . details are reported in table3 . discussion foetal neck masses , although uncommon and usually benign , may become life - threatening and have clinical relevance if they compromise the airway , leading to the risk of severe respiratory distress at birth . 
if a foetal neck mass is detected , parents and clinicians are forced to take critical decisions about time and mode of delivery and management of the newborn . lymphatic vascular malformations and teratomas are the most common foetal cervical masses [ 2 , 3 , 17 , 20 ] ; about 30% of childhood and 25% of all neonatal teratomas occur in the neck [ 21 , 22 ]  . 
congenital lymphatic malformations are slow - flow vascular malformations due to an abnormal development of the lymphatic system , presenting in microand / or macrocystic form [ 3 , 23 , 24 ]  . 
 ss - tse t2 - weighted sagittal ( a ) , axial ( b ) , and coronal ( c ) mr images show a hypersignal cystic mass ( * ) slightly compressing the oropharynx ( arrow ) ; coronal image ( c ) shows patent trachea ( arrowhead )  . 
the misdiagnosis of lymphatic malformation was made in this foetus table 2 prenatal ultrasound diagnoses , prenatal magnetic resonance imaging diagnoses , and postnatal confirmation case us diagnosis r1 mr diagnosis r2 mr diagnosis postnatal diagnosis other other teratoma other teratoma teratoma teratoma teratoma teratoma teratoma teratoma teratoma other other other other teratoma teratoma teratoma teratoma teratoma teratoma teratoma other teratoma other teratoma teratoma teratoma teratoma teratoma teratoma teratoma goitre neurocristic cutaneous hamartoma venous malformation rich branchial cyst teratoma teratoma teratoma teratoma teratoma teratoma us ultrasound , mr magnetic resonance , r1 12years experience reader , r2 2years experience reader , lm lymphatic malformation , rich rapidly involuting congenital hemangioma teratomas are germ cell tumour originating from the midline of the body , composed of all embryonal layers : endoderm , ectoderm , mesoderm [ 3 , 17 ]  . our data , about location and structure of lymphatic vascular malformations and teratomas , confirmed data of the literature . 
in fact , lymphatic vascular malformations 1 3 la radiologia medica ( 2019 ) 124 : 917925 table 3 mass features observed at prenatal magnetic resonance imagcase site structure postnatal diagnosis max diameter ( mm ) solid mixed solid solid cystic mixed cystic mixed cystic cystic cystic cystic cystic cystic cystic cystic cystic mixed mixed mixed mixed mixed mixed goitre neurocristic cutaneous hamartoma venous malformation rich branchial cyst teratoma teratoma teratoma teratoma teratoma teratoma a anterior , p posterior , lc laterocervical , lm lymphatic malformation , rich rapidly involuting congenital hemangioma are more often located posteriorly or posterolaterally in the neck , with cystic texture ( in our series 83% , 10 / 12 ) , and teratomas are more commonly anteriorly located , with mixed cystic - solid structure ( in our series 100% , 6 / 6 ) [ 3 , 11 , 19 , 25 , 26 ] ; two cases of lymphatic vascular malformations with mixed appearance were probably due to the coexistence of microcystic and macrocystic pattern . we agree with hedrick [ 7 ] , when he affirms that the involvement of the airway depends on the location rather than the mass size . 
in fact , in our experience , 5 of 6 anteriorly located masses required respiratory care at birth , while none of the posteriorly located needed it , and less than half of the lesions ( 4 / 9 ) located in the laterocervical space underwent neonatal respiratory assistance . 
this could be explicated by the proximity of the anteriorly located mass to the airway . the extension of the masses into the mediastinum is an additional risk factor for compromising the airway , as in one of our cases , where a huge teratoma completely obstructed the foetal trachea , requiring exit procedure at birth . we also found that teratomas had median maximum diameter greater than lymphatic vascular malformations at the time of the prenatal mri . 
teratomas are often huge when diagnosed , determining sometimes hyperextension of the foetal neck , as we saw in three foetuses , polyhydramnios , as we saw in two foetuses , and dystocia [ 19 ]  . the fact that no significant difference was found between lymphatic vascular malformations and teratoma , in term of median gestational age at the time of the prenatal mri ( 28weeks ) , could be explained based on our obstetric surveillance system , which establishes a period to perform us to detect morphological anomalies . in the presence of a foetal neck mass , in addition to lymphatic vascular malformation and teratoma , must be considered other rare expansive lesions , such as thyroid goitre , hemangioma , neuroblastoma , branchial cyst , laryngocele , and soft - tissue tumours [ 17 , 26 ]  . 
in most cases , us is sufficient to identify foetal body anomalies and choose the proper therapy , but in some cases , sonographic features are unclear and the abnormality is not completely characterized [ 10 ]  . mri begun to be used in pregnant women in 1983 [ 12 , 27 ]  . 
currently , thanks to its absence of radiation exposure , large field of view , intrinsic high soft - tissue contrast , high spatial resolution , multiplanar capability , and application of new ultrafast sequences , it allows to obtain high detail about extent , location , and structure of the mass , making clinicians more confident in the diagnosis [ 6 , 10 , 13 ]  . in our experience , mri demonstrated substantial accuracy in prenatal diagnosing of mass nature , having correctly identified 21 of 23 masses . 
the different level in interpreting prenatal mri could explain this difference , allowing the more expert reader to understand when a mass was other than teratoma or lymphatic vascular malformation , which have specific features . 
we did not calculate the agreement between prenatal us and postnatal diagnosis about mass nature , because the us examinations were all performed in second - level centres but by different operators , so without any standardization . correct diagnosis of mass nature and details about mass characteristics are certainly necessary for paediatric surgeons , anaesthesiologists , gynaecologists , and other members of the multidisciplinary team to plan the best treatment ; however , the most important issue to consider 1 3 924 la radiologia medica ( 2019 ) 124 : 917925 is the status of the foetal airway [ 7 ] , because airway obstruction could bring newborn to death . despite us has the advantage to be a real time examination , which allows to evaluate the presence of foetal movements , it can only identify indirect signs of airway obstruction , such as polyhydramnios , tongue protrusion , or absence of swallowing [ 2 , 5 ]  . 
conversely , visualization of the foetal anatomy is possible with high - resolution mr images , beingwhite foetal airway easily identifiable on t2 - weighted images thanks to the high signal of the amniotic fluid content , which enables accurate diagnosis [ 2 , 11 , 15 , 19 , 25 ]  . from our study emerges that prenatal mri has a sensitivity of 100% and a positive predictive value of 82% in evaluating the involvement of foetal airway in case of neck mass , with an acceptable rate of wrong positive ( 18% )  . in fact , only two babies with mri examination positive for airway impairment were misdiagnosed ; one was intubated at birth and extubated some hours later , after a postnatal mri study revealing spontaneous regression of the malformation , by now , far from the airway ; the other baby was delivered by planned caesarean section and could breathe spontaneously . 
we think that to obtain updated information , it might have been better to repeat mri closer to the date set for the childbirth . moreover , there was another case of overtreatment : prenatal mri at 21weeks of gestation correctly showed patent airway , very close to a large lymphatic malformation . 
thus , we can state that a negative mri examination , in consideration of its very high sensitivity and npv , is a reliable tool to exclude significant airway impairment and avoid invasive maternal foetal procedures . regarding the status of the airway , the two readers agreed in all cases . 
indeed , the bright foetal airway is relatively simple to identify on t2 - weighted images , so if it is not visible or thinned at the level of the mass , its involvement can be reasonably supposed . 
hence , the performance of prenatal mr in the assessment of foetal airway seems not influenced by the readers experience . conclusion prenatal us remains the first line tool in the evaluation of foetal neck anatomy . 
in case of potential airway obstruction , we believe in the need for serial foetal mri , repeating it as close as possible to the planned delivery date . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . 
the authors did not receive any funding for the present study . ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . informed consent for this type of study , formal consent is not required . 
strohmenger. diagnostic imaging in dental anomalies diagnostic imaging in dental traumatology table of recommendations glossary introduction technological improvement in diagnostic imaging techniques and equipment has recently led to a great qualitative and quantitative improvement . dental imaging has dramatically changed over the last fifteen years , in particular in the field of oral surgery and paediatric dentistry . 
in both these specialties , scientific evidence has increased , highlighting the need for guidelines concerning radiation dose justification and optimisation in dental imaging . as a matter of fact , several scientific articles and communications on dose issues in dental imaging increased , with particular focus on applied physics in medicine . 
in ministry of health secretariat general office 2 national guidelines for dental diagnostic imaging in the developmental age november 2017 introduction work group bibliographical research current regulations considerations on radiological risks and containment strategies in x - ray examinations diagnostic imaging for caries and periodontal disease diagnostic imaging in orthodontics and gnathology diagnostic imaging in cranio - maxillofacial malformations this sense , interest also arose in the international scientific community , leading to publication of guidelines by several institutions and societies ( 1 , 2 , 3 , 4 , 5 , 6 )  . among the campaigns for oral health promotion , in 2016 the national department of health decided to prepare guidelines on dental diagnostic imaging procedures in children and adolescents . therefore , a dedicated workgroup was created under the coordination of laura strohmenger , member of the w.h.o. 
collaborating centre for epidemiology and community dentistry . 1 3 la radiologia medica ( 2019 ) 124 : 887916 this document aims to support the dental professional in choosing the adequate diagnostic technique , minimising the radiation dose in observance of the as low as reasonably achievable ( alara ) principle ( 7 )  . 
medical subject headings used for bibliographical research radiological field patient field dental radiology dental radiography dental cone beam dental cbct intraoral panoramic cephalometer * child * adolescent paediatric from the identified publications , 185 articles and 7 web pages were selected , whose references are reported in this document . for evaluation of the gathered documents , we used prisma ( preferred reporting items for systematic reviews and meta - analyses ) , one of the tools suggested by the methodological manual of the 2011 national guidelines system how to produce , disseminate and update public health guidelines . bibliography guidelines for the safe use of dental and maxillofacial cbct : a review with recommendations for south africa . 
4 principle of optimisation introduces the diagnostic reference levels ( drl ) with the aim of providing a working tool to guarantee an optimisation process standardisation for the most common radiological practices . 
these are easily measureable dose - level thresholds that should never be exceeded in normal examination of average build patient groups , and for well - defined appliances and techniques . 
in the field of dental radiological imaging , there are currently no defined drls in italy , whereas some drl values defined in other countries will be illustrated in a dedicated section of this document . specific aspects regarding apparatuses are featured in art . 
8 , including the obligation to define quality assurance programmes , to make acceptance and performance tests , and for the head of radiological equipment to state their clinical adequacy . 
so , within the context of these guidelines purpose , it is highlighted that all dental radiological practices involving exposure of paediatric patients must be considered as special practices , in accordance with the above - mentioned art . 
all provisions of the latter article must therefore be adhered to , including periodic dosimetric assessments by a medical physics expert , based on which the head of radiological equipment shall judge the technical service quality and the diagnostic procedure . generally , in the current regulatory framework and in particular with regard to the implementation modes , the european guidelines on radiation protection must be taken into consideration . 
among these , we point out : european guidelines on radiation protection in dental radiologyno 136 [ 1 ] 1 3 la radiologia medica ( 2019 ) 124 : 887916 criteria for acceptability of medical radiological equipment used in diagnostic radiology , nuclear medicine and radiotherapyno 162 [ 2 ] cone beam ct for dental and maxillofacial radiology no 172 [ 3 ] moreover , the reference technical regulations defined by the italian electrotechnical committee are of fundamental importance in verifying the equipment technical features . 
all these methods use x - rays and thus ionising radiation that may interact at a cellular level . when ionising radiation passes through a cell , there may be ionisations of atoms in the molecules that constitute the cell itself . 
damage to the dna may be entirely repaired by means of complex mechanisms ; it may be irreparable , with consequent cellular death ; or it may be partially repairable with the consequent appearance of a genetic mutation . 
 this mutation could be the primum movens of a complex series of events potentially resulting in carcinogenesis or , in the case of the involvement of germ cells , causing hereditary diseases in the radiated persons progeny . the biological effects of ionising radiation may be grouped in deterministic effects and stochastic effects . deterministic effects are caused by exposure to ionising radiation doses that are far higher than those used in radiological diagnostics , occurring only if specific threshold values are exceeded and with a gravity that increases with an increase in the absorbed dose . 
they are caused by cellular death and may manifest themselves in pancytopenia , alopecia , sterility , burns , pneumonia and serious gastrointestinal disturbances , with possible death of the radiated person . stochastic effects are caused by radiation doses lower than those required for the manifestation of deterministic effects and are due to non - lethal and unrepaired cells damage that may become manifest with a variable latency , even of several years . for the purposes of radiation protection , the accepted assumption is that there is a linear relationship between risk and radiation dose , and that there is no threshold value below which risk is zero . 
in the absence of certainty in this regard , a prudential approach is preferred to ensure that the dose of radiation employed for radiological diagnostics is the minimum indispensable for producing an image of adequate diagnostic quality . the absorbed dose for ionising radiation may be defined as the quantity of energy deposited in the exposed tissues or organs . 
 the gray is a very large unit of measurement in diagnostic imaging so it is therefore more practical to use the milligray as unit of measurement , corresponding to one thousandth of a gray . 1 3 892 la radiologia medica ( 2019 ) 124 : 887916 the risks due to exposure to different types of ionising radiation may be compared in terms of equivalent dose . 
moreover , the effective dose allows comparison of doses resulting from different techniques employed for the same medical examination and / or comparison of doses resulting from similar procedures carried out in several institutions . 
it follows that the risk must always be kept in mind , especially in the case of using radiological procedures that expose patients to high doses of radiation , this being justifiable only when there is a clear benefit to the patient deriving from execution of the procedure itself . 
for radiological procedures in the dental field , the scientific reference literature consistently reports the values of effective dose and equivalent dose to be at levels of microsieverts ( sv ) , where one sv is equal to one thousandth of an msv . 
in the context of cbct procedures in dentistry during the developmental years , ludlows recent review [ 1 ] provides a metanalysis of assessments of effective doses with paediatric protocols and phantoms , derived from the use of 10 different apparatuses . 
the assessments are subdivided into those deriving involving the use of a medium or large field of view ( height greater than 10cm ) or small field of view ( height less than 10cm ) for maxillary bone and for mandible . 
ludlow underscores that the effective dose assessed using paediatric simulation phantoms with cbct apparatuses is around 36% greater than that found with adult phantoms , an increase to be correlated mainly with the greater proximity of the thyroid to the inferior margin of the jaw in children , with the consequence of a greater irradiation , both direct and as scatter from the maxillary structures . 
median values of effective dose , thyroid and salivary glands doses for a range of cbct equipments and protocols employed on paediatric simulation phantoms [ 1 ] effective dose dose to thyroid dose to salivary small maxillary bone small jaw medium or large ( 16177 ) ( 24331 ) ( 39430 ) ( 53575 ) ( 3273382 ) 1003 ( 3844265 ) glands 1930 ( 4384974 ) 1654 ( 4045937 ) 2045 ( 5316622 ) the values in brackets are the minimummaximum range . 1 3 la radiologia medica ( 2019 ) 124 : 887916 examination carried out , dose indicators for the patient have been defined , specific to the different radiological methodologies . 
the availability of such dosimetric indicators makes it feasible to carry out multicentric data gathering that , with appropriate statistical analyses , can be used to define diagnostic reference levels . the obligation to have this dose indication available is prescribed by the italian regulations 187 / 00 in article 8 , subparagraph 8 : in the case of utilisation of a newly installed radiodiagnostic equipment , this must be equipped , if feasible , with a device that informs the specialist about the quantity of ionised radiation produced by the device during the radiological procedure . 
the latest european directive on the subject of protection from ionising radiation ( 2013 / 59 / euratom ) , currently being integrated into the national regulations , confirms this obligation and , moreover , prescribes the recording of the pertinent parameters for assessment of the dose to the patient for each examination . 
indicators of doses used in the various apparatuses for dental radiology magnitude apparatus on which used definition unit of measurement normally used air kerma ( kair ) entrance skin air kerma ( esak ) entrance skin dose ( esd ) mgy cm dose area product ( dap ) kerma area product ( kap ) computed tomography dose index ( ctdi ) intraoral air kerma value measured in correspondence to the entrance of the beam into patient cephalometry unit intraoral , orthopantomograph , cephalometry unit , cbct dose value resulting from product of air kerma and backscatter factor measured in correspondence to entrance of beam into patient product of the area of a radiological beam section and the average air kerma on that section multislice ct computed tomography dose index . 
see [ 2 ] for a more complete definition 1 3 894 la radiologia medica ( 2019 ) 124 : 887916 kerma ( kinetic energy released per unit mass ) means the kinetic energy transferred by the x photons to the charged particles of the irradiated material : for the x - ray energies used in radiodiagnostics , its air value coincides with the value of the dose , and therefore , the two terms may be used in an equivalent manner . 
the esak ( entrance skin air kerma ) for an intraoral apparatus therefore corresponds to the air dose assessed on the axis of the beam at the exit of the cylinder and is a quantity directly proportional to the duration of exposure and to the intensity of the anodic current . 
the dap ( dose area product ) takes into account of both the intensity of the beam and its dimensions , and its assessment does not depend on the distance from the source for which it is evaluated . 
where these data are not available on already installed devices , the medical physics expert in charge of dosimetry must measure and tabulate them in such a way that the clinician can take account of these indicators in the choice of the protocols to use . 
in the case of availability of these data , the medical physics expert must check their accuracy , with a maximum acceptable tolerance of 50% . what are thetypical dose indicator values givenintheliterature forthevarious examinations ? in the absence of diagnostic reference levels defined at european or national level , the dosimetric indicators presented in the previous paragraph and available with newly installed radiological equipment may be compared with the values published in the literature . 
values of dose indicators in the literature for cephalometry units used with paediatric patients reference data typology of datum magnitude utilised kim 2014 [ 10 ] 121.3mgycm2 diagnostic reference holroyd 2011 [ 13 ] dap 25mgycm2 diagnostic reference reference level resulting from data analysis of 50 centres in germany ence level resulting from data analysis of 62 centres in greece level resulting from data analysis of 20 centres in korea level resulting from data analysis of 27 centres in great britain for cbct equipment , to date there are no published studies with multicentric data collection of dosimetric indicators . 
values of dose indicators in the literature for cbct with protocols for paediatric patients reference magnitude utilised data ludlow 2015 typology of datum average values deriving from the setting of paediatric protocols on 10 different apparatuses fov large and medium 529mgycm2 fov small maxillary 121mgycm2 fov small jaw 153mgycm2 what characteristics must theacquisition protocols andparameters have forradiodiagnostic dental examinations inthedevelopmental years ? in general , studies published in recent years underline the need to define acquisition protocols with parameters adapted to the reduced dimensions and the diagnostic requirements specific for the paediatric patient , with the published multicentric assessments often highlighting the use of inadequate parameters . an anglo - saxon study of 2013 [ 15 ] of intraoral examinations showed the national diagnostic reference level to be exceeded in 35% of the operational modes used for adult patients and in no less than 61% of those used for paediatric patients . 
the same study highlights how , notwithstanding the fact that the technological evolution of digital detectors and collimation systems offers the possibility of significant dose reductions , in many cases the technical parameters used lead to patient exposures greater than those required to obtain the correct diagnostic information . a finnish work of 2015 [ 16 ] presents an investigation carried out on 241 orthopantomographs and 118 cephalometric units used on paediatric patients : the panoramic images turned out to be wider than necessary in 70% of the cases and of greater length in 96% . 
thyroid protection was used in roughly 71% of cases . image gently is an awareness - raising and educational campaign concerning proper radiological risk management for paediatric patients , promoted by american scientific associations in the paediatric and radiology field . 
they have summarised in the following six steps the key points for minimising dose in dental radiology practices in the developmental years [ 17 ] : selection of radiological images in conformity with the patients specific needs ; the use of detectors with maximum sensitivity ( high speed in the case of film or equivalent digital systems ) ; collimation of the beam on the area of interest ; use of thyroid collar for all intraoral examinations , and for extraoral examinations when it does not interfere with the area under examination ; suitable reduction in exposure parameters ( time , intensity of anodic currents , etc . ) in comparison with parameters employed for adult patients ; use of cbct only when necessary . 1 3 896 la radiologia medica ( 2019 ) 124 : 887916 among the most common technical errors that might cause artifacts in the images , one must consider : carrying out examinations in the presence of hairclips , metal jewellery , food in the mouth ( sweets , chewing gum ) , removable dental prostheses ; incorrect positioning of the patient ; incorrect positioning of the tongue ; errors in limiting patient movement during image acquisition . increase the exposure times which , coupled with the method of positioning the apparatus , augments the risk of movement artefacts . 
so special attention must be paid to justification of the use of these devices and , while awaiting further evidence from the literature , it is expected that their use will remain limited to very particular cases where it is not effectively possible to employ a fixed intraoral unit . in drawing up these guidelines , comparative evaluation of the recent reference bibliography has allowed the formulation of the following recommendations . the main actions to be adopted in order to reduce the frequency of these errors are : recommendation 1 programme suitable child - friendly time schedules for preparing and carrying out the examination , longer than those necessary for adults ; train staff in suitable communications skills with kids and their families ; calm the patient : in the case of excessive anxiety and restlessness , do not insist ; if necessary , postpone the session ; check also with the parents that jewellery , hairclips and dental prostheses have been removed , as well as sweets or gum from the childs mouth ; pay great attention to correct positioning of the patient ( neck , back , shoulders ) ; without rush or fuss , show the patient the correct positioning of the tongue ( in contact with the palate ) ; for teleradiography of the cranium : in the case of roll , pitch or rotation , always consider carefully the real need to repeat the examination ; most errors influence assessment of the main angles [ 18 ]  . the 2012 document radiation protection 162 [ 9 ] defines new acceptability criteria for technical and functional parameters of all radiological apparatuses , including those used in the dental sector . 
in the same document , it is underlined that this kind of equipment generally works with lower voltage and ma at conventional intraoral examinations , involving the need to strength of recommendation : a degree of evidence : iv recent dosimetric studies [ 22 , 23 ] show that the use of an adult protocol on a paediatric patient may involve unjustified exposure doses . 
limiting the fov in line with the other parameters and compatibly with diagnostic requirements is in general a rule to follow in order to minimise the dose to the paediatric patient . 
there are , however , studies 1 3 la radiologia medica ( 2019 ) 124 : 887916 showing that with some equipment a reduction in fov may be associated with an increase in other exposure parameters , as the beam intensity , such that the dose may even be greater [ 24 ]  . 
it is , therefore , necessary to pay attention to all the parameters associated with the different equipment settings , and in order to compare them from a dose viewpoint , it is appropriate to refer to the dap indication supplied . a recent study by pauwels [ 25 ] evaluated image quality indices on variation in the anatomical dimension concerned . 
the results show the possibility of reducing the dose in the paediatric field by maintaining the kv value relatively high ( 90kv on the apparatus examined ) and reducing the value of the mas prescribed for an adult by 40% for paediatric patients of around 3years old and by 20% for those around 10years old . recommendation 4 the lead thyroid collar contributes significantly toreducing thedose tothethyroid forall radiodiagnostic dental examinations inthedevelopmental years . 
in thecontext ofcbct , it isparticularly recommended forextended fields , exceptincases where , atthetime ofpositioning thepatient , thetechnician , theradiologist orthespecialist ascertains there tobe arisk ofartifacts orpossible superimpositions withregard totheanatomical structures ofinterest . 
as forcephalometry , theuse isrecommended wherethere isnoneed tovisualise bony structures belowthesecond cervical vertebra strength ofrecommendation : adegree ofevidence : iv the assessments made by hidalgo [ 26 ] on a cbct apparatus with fov diameter 17cm and height 12cm , and with various types of thyroid collar , show dose reductions to the thyroid of between approximately 20 and 40% . numerous recent studies [ 27 , 28 , 29 ] highlight the compatibility of thyroid protection in carrying out cephalometry on paediatric patients , which involves a significant reduction in the dose at the thyroid , with the limitation that the bone structures represented start from the second cervical vertebra . bibliography ludlow jb , timothy r , walker c , hunter r , benavides e , samuelson db , scheske mj . 
 2013 ; 64 ( 6 ) : 7559 . european commission radiation protection no 162 criteria for acceptability of medical radiological equipment used in diagnostic radiology , nuclear medicine and radiotherapy directorate - general for energy directorate dnuclear energy unit d4 radiation protection 2012 . berkhout we , suomalainen a , brullmann d , jacobs r , horner k , stamatakis hc justification and good practice in using handheld portable dental x - ray equipment : a position paper prepared by the european academy of dentomaxillofacial radiology ( eadmfr )  . 
2011 ; 40 : 471475 . diagnostic imaging forcaries andperiodontal disease numerous studies ( 110 ) agree that visual examination , especially in primary dentition , is a key factor in assessing the presence or absence of dental caries . 
in fact , for the former type , it seems that radiographic analysis is not necessary to confirm diagnosis . regarding non - invasive diagnostic systems ( 13 ) , the aid of fibre - optic transillumination ( foti ) may significantly favour the diagnostic process ( 14 , 15 )  . ex ante caries risk assessment of a patient ( 1620 , b ) appears to be another discriminating factor in the diagnostic process of carious lesions in primary dentition . 
further radiographic examinations are notjustified strength of recommendation : a degree of evidence : i assessment of carious processes in permanent teeth appears to be sufficiently supported by inspective clinical examinations , to be performed according to well - established criteria ( 1720 ) and backedparticularly for high caries risk patientsin case there is clinical suspicion by a bitewing x - ray and , if necessary , by periapical images to be taken using image receptor holders and beam aiming appliances . 1 3 la radiologia medica ( 2019 ) 124 : 887916 taking an x - ray , using the paralleling technique , of teeth with suspected caries lesions that may have jeopardised pulpal health is particularly indicated for high caries risk patients ( 21 )  . 
it should be stressed that , in certain areas of the mouth , especially the upper and lower posterior areas , the superimposition of other bony structures might make it difficult to examine and outline the periapical image . 
 examinations such ascbct are notindicated inthis diagnostic phase strength ofrecommendation : adegree ofevidence : iii if a fistula is present in the vestibular mucosa , close to the apex of either primary or permanent teeth , it should always raise suspicion of a septic lesion of the dental pulp also affecting the periapical tissues . intraoral x - ray , with the aid of a beam aiming device , is diagnostic investigation of choice . 
5 xray examination ofapermanent tooth showing infection signs onthemarginal gingiva seems tobe indicated , especially forpatients withconfirmed high risk ofperiodontal disease strength ofrecommendation : b degree ofevidence : iii procedures such as direct pulp capping , apexogenesis , apecification and also complete root canal treatment require taking a preoperative x - ray with a paralleling syste this may give sufficient clues regarding the degree of maturity of the root apex and the estimated working length ( 35 )  . clinicians are advised to be in possession of this image already in the diagnostic phase . 
it is worth remembering that , where cleaning and shaping of the endodontium are necessary in immature teeth , electronic apex locators are not reliable ( 36 , 37 )  . 
the followup images should be taken three months , six months , one year , sincetheend oftherapy andthen annually forthenextthree years strength ofrecommendation : adegree ofevidence : ii based on findings from the literature , radiographic treatment longterm follow - up of primary teeth is deemed justifiable ( 38 )  . in case of septic or traumatic pathological processes leading to eradication or conservative ( partial or complete ) treatment of the pulp , a careful observation phase allows accurate assessment of positive or negative signs of the health conditions of the periapical tissues ( 38 , a , b , c , d , e , f )  . 
 on this subject , there are no studies indicating exact timing to mandatorily comply with for following checkups ( 34 ) ; there are , however , longitudinal studies about the assessment of healing processes of edodontically originated periapical lesions , in which patients were checked over time spans varying between six and twelve months , for the initial phases of treatment , and then once a year for a period from three to five years ( 35 )  . 
radiographic followup isrecommended afterthree andsix months andthen annually forthenextthree years , depending ontheclinical situation strength ofrecommendation : adegree ofevidence : ii on the other hand , indications do emerge in checkup schedule to comply with , for assessing radiographic appearance of the pulp chamber and root canalas well as of the apex of permanent teeth which , having undergone conservative pulp treatment ( direct or indirect pulp capping / pulpotomy ) , must be regularly checked to avoid complications , such as obliteration of the root canal , or the onset of endodontically originated periapical lesions , in case partial or complete necrosis of the pulp occurs ( 3436 , a )  . clinicians should also keep track of problems that may arise in case the pulp disease justifies an endodontic treatment , although the tooth has not completed formation of the root apex , by performing an apexogenesis or apecification procedure which , based on scientific evidence , should be then supervised for a six months to three years time span ( 33 )  . bibliography pitts nb , ekstrand kr , foundation i . 
international caries detection and assessment system ( icdas ) and its international caries classification and management system ( iccms ) methods for staging of the caries process and enabling dentists to manage caries . 
community dentistry and oral epidemiology 2013 ; 41 ( 1 ) : e4152 . gimenez t , piovesan c , braga mm , raggio dp , deery c , ricketts dn , etal . 
journal of dental research 2015 ; 94 ( 7 ) : 895904 . gimenez t , piovesan c , braga mm , raggio dp , deery c , ricketts dn , etal . 
 caries research 2015 ; 49 ( 5 ) : 523530 . lino jr , ramos - jorge j , coelho vs , ramos - jorge ml , moyses mr , ribeiro jc . 
international dental journal 2015 ; 65 ( 4 ) : 178181 . ferraz eg , silva lr , sarmento va , de jesus campos e , de oliveira tf , magalhaes jc , etal . 
can we trust visual methods alone for detecting caries in teeth ? evidence - based dentistry 2016 ; 17 ( 2 ) : 4142 . melo m , pascual a , camps i , del campo a , ata - ali j . 
jbr - btr : organe de la societe royale belge de radiologie 2011 ; 94 ( 5 ) : 254265 . castilho ls , cotta fv , bueno ac , moreira an , ferreira ef , magalhaes cs . 
european archives of paediatric dentistry : official journal of the european academy of paediatric dentistry 2016 ; 17 ( 1 ) : 1325 . bussaneli dg , restrepo m , boldieri t , pretel h , mancini mw , santos - pinto l , etal . 
lasers in medical science 2015 ; 30 ( 7 ) : 18731879 . kuhnisch j , sochtig f , pitchika v , laubender r , neuhaus kw , lussi a , etal . 
evidence on existing caries risk assessment systems : are they predictive of future caries ? community dentistry and oral epidemiology 2013 ; 41 ( 1 ) : 6778 . gauba k , goyal a , mittal n . 
the journal of clinical pediatric dentistry 2016 ; 40 ( 1 ) : 3643 . goolsby sp , young da , chiang hk , carrico ck , jackson lv , rechmann p . 
 european archives of paediatric dentistry : official journal of the european academy of paediatric dentistry 2010 ; 11 ( 4 ) : 166174 . mejare ia , axelsson s , davidson t , frisk f , hakeberg m , kvist t , etal . 
international endodontic journal 2012 ; 45 ( 7 ) : 597613 . american academy on pediatric dentistry ad hoc committee on pedodontic r , american academy on pediatric dentistry council on clinical a . 
diagnostic accuracy of periapical radiography and 1 3 902 la radiologia medica ( 2019 ) 124 : 887916 cone beam computed tomography in detecting apical periodontitis using histopathological findings as a reference standard . 
international endodontic journal 2013 ; 46 ( 6 ) : 483491 . kuhnisch j , ekstrand kr , pretty i , twetman s , van loveren c , gizani s , etal . 
since every patient is unique , the need for dental x - rays can only be determined upon the analysis of his medical and dental history , and of his clinical condition and after assessing exposure to environmental risk factors that could jeopardise oral health . x - ray should only be used as a diagnostic examination when considered useful for the patients health , and when it may provide additional information helpful for treatment , complying with the principle of justification . 
given the importance and the current relevance of this issue , numerous guidelines have been published.17 these studies unanimously agree that prescribing x - ray examinations is solely advisable as necessary . 
in compliance with all the international guidelines , 17 this section will describe indications and levels of evidence for each type of radiographic examination suitable for identifying disease , also concerning the costbenefit ratio . for the orthodontic patient , radiographic examination generally consists of panoramic radiography , i.e. 
orthopantomography ( opt ) , and latero - lateral teleradiography of the head , which may be supplemented , in the most complex cases , by more specific examinations . opt allows assessing the presence or agenesis of permanent teeth , the position of teeth yet to erupt , and any supernumerary teeth.8 it is not , however , advisable for evaluating caries in patients suffering from dental decay about to undergo orthodontic treatment . 
in such cases , in fact , a bitewing image is to be preferred.9 teleradiography of the head and cephalometric analysis are indicated for orthodontic diagnosis , treatment planning and the monitoring of results following therapy on the maxillo - mandibular bony structure . 
a useful aid in choosing the appropriate age to prescribe this radiographic examination is the index of orthodontic treatment need ( iotn ) .10 this index allows objective distinction between severe cases requiring early treatment ( iotn 4 and iotn 5 ) and cases which treatment may begin by the age of 1011.11 the use of teleradiography for pa projection for facial asymmetries must be carried out with great care , considering the difficulty in locating the cephalometric points in this projection.12 , 13 moreover , it should be kept in mind that even slight rotations of the head in the cephalostat might mask the presence of facial asymmetry.12 , 14 cone beam computed tomography ( cbct ) guarantees the volumetric graphic 3d representation of all anatomical bony structures , as well as overcoming the limitations of ordinary 2d radiographic imagesi.e. 
geometric distortion and superimposition of bony and dental structures , and improper positioning of the patients head in the cephalostat15since usual practice continues to favour 2d , even though 3d16 cephalometric analyses do exist . 1 3 la radiologia medica ( 2019 ) 124 : 887916 routine prescription of this examination for young patients is considerably limited , by both the literature and all the existing guidelines , 15 , 17 upon radiation dose , to individual clinical cases . 
it should be highlighted , though , that it is current common practice among orthodontists to convert 3d images into 2d , for cephalometric analysis.15 , 17 in all cases , choosing the fov is crucial when prescribing this examination , in order not to expose the patient to a non - optimised radiation dose with an acquisition field too extensive for the target zone . 
in orthodontics , small and medium fovs are indicated for the assessment of impacted teeth , root resorption caused by impacted teeth , and the designated area for mini - screw insertion in patients with malocclusion.14 , 1820 moreover , successful application of mini - implants is influenced by cbct14 evaluation of bone density , as it reduces the risk of screw detachment . 
in all these cases , with one single exposure , despite the higher radiation dose being used , the orthodontist and the oral and maxillofacial surgeons get access all the required information . the use of large fovs for a 3d cephalometric examination in simple clinical cases is not validated by the literature , nor by existing guidelines.17 temporomandibular disorders ( tmd ) affect the temporomandibular joint ( tmj ) , the masticatory muscles and associated structures . 
in a recent update , the american academy of orofacial pain divided tmds into two large categories : tmj disorders and masticatory muscle disorders.24 the prevalence of tmd in newborns , children and teenagers is highly variable in the literature , inasmuch as data between 6 and 68% are reported . 
 radiological examination is only indicated when insufficient information is obtained from the anamnesticclinical examinations.24 , 34 different radiological techniques are employed in study of the tmj , with different sensitivities , specificity and appropriateness in relation to the diagnostic question . 
 magnetic resonance ( mr ) and computed tomography ( ct ) and / or cbct scanning should be preferred over conventional radiography . ct is held to be the gold standard imaging technique for bone lesion assessment , while mri is considered the gold standard for ligament and articular capsule structure analysis.24 , 34 recommendation no . 
1 for acorrect orthodontic diagnosis andtreatment planning , apanoramic radiography andateleradiography are necessary strength ofrecommendation : adegree ofevidence : i teleradiography of the skull in norma lateralis , panoramic radiography and radiography of the non - dominant hand and wrist should be prescribed based on the degree of malocclusion ( iotn ) 10 and the patients age.17 frequent radiographic checkups to monitor progress of therapy are advised against and inappropriate , unless there is a precise clinical indication . in case of serious craniofacial dysmorphoses or of skeletal iii class , orthodontic treatment will begin early , getting radiographic records of the patient is advisable at an early stage . 
2 limit prescription ofcbct and , preferably , use small andmedium fovs strength ofrecommendation : adegree ofevidence : i in the past decade , a considerable increase in the use of cbct in dentistry , especially in orthodontics , was witnessed . 
nonetheless , numerous recent guidelines and studies have drastically limited prescription of cbct to selected clinical cases . cbct is therefore indicated for diagnosis and treatment planning for severe dysmorphosescraniofacial syndromes , impacted teeth , bone anomalies , serious facial asymmetries and condylar aplasia or hypoplasia.17 1 3 904 recommendation no . 
4 posterioranterior teleradiography , withrelated cephalometric analysis , requires ahighly experienced clinician strength of recommendation : a degree of evidence : i cephalometric analysis of the posterioranterior teleradiograph , compared to the latero - lateral analysis , requires the clinician to have extensive experience , as localisation of the cephalometric points is far more complex due to bone superimpositions . 
they are insufficient for assessment of la radiologia medica ( 2019 ) 124 : 887916 the bony structures ( condyle and glenoid cavity ) and may reproduce the condyleglenoid cavity relationships unreliably , due to projection errors bound to the imaging technique.24 , 34 bibliography k . 
diagnostic criteria for temporomandibular disorders ( dc / tmd ) for clinical and research applications : recommendations of the international rdc / tmd consortium network * and orofacial pain special interest groupdagger . 
clinical classification of 1416 - year - old danish children according to functional status of the masticatory systecommunity dent oral epidemiol 1988 ; 16 : 4751 . pahkala r , laine t , narhi m , ettala - ylitalo um . 
pediatric dentistry sept - oct 2015 ; 37 ( 5 ) : 7884 . guarnieri r , cavallini c , vernucci r , vichi m , leonardi r , barbato e . 
med oral patol oral cir bucal 2016 ; 21 : e743 - e750 . 1 3 906 la radiologia medica ( 2019 ) 124 : 887916 diagnostic imaging incraniomaxillofacial malformations recommendation no . 
craniosynostoses and craniofacial stenoses , as well as orofacial clefts and branchial arch syndromes , are an example . since we are dealing with deformations that are often present at birth , radiographic investigations should be proportionate to the diagnostic and therapeutic objectives of the single case.6 , 13 the golden rule is that radiographic investigations should be carried out only when they are essential to diagnosis and treatment planning.6 , 13 , 14 by way of example , the diagnosis of skull malformations and of cranial suture anomalies is purely clinical.3 , 13 , 14 , 15 direct x - ray imaging is not indicated as a routine examination , since the same information may be obtained by ultrasound imaging.2 , 5 , 15 , 24 multislice computer tomography ( msct ) or cbct is indicated in selected cases , when necessary for treatment planning.15 , 22 msct and cbct are not indicated as a routine examination for cleft lip and palate , since their diagnosis is clinical . 
2 diagnosis ofcraniostenosis isstrictly clinical , 4 , 14 , 15 , 18 , 21 butct may be of assistance23 strength ofrecommendation : adegree ofevidence : ii for treatment planning , a ct examination is indicated.5 , 14 , 23 for suspected anomalies of the cranial sutures and the anterior fontanelle , an ultrasound examination is often sufficient.2 , 5 , 15 , 24 msct orcbct may be indicated foranalysis ofi andii branchial arch syndromes craniofacial defects andwiththepurpose oftreatment planning.1 , 7 , 8 , 14 , 15 , 17 , 18 , 20 strength of recommendation : a degree of evidence : ii in cranio - maxillofacial defects , cbct may be indicated as an investigation alternative to msct , 2 , 24 since the radiation dose , based on scientific evidence , is lower where evaluation of the soft tissues is not required.3 , 6 , 9 , 19 , 23 bibliography adam m . 
australian dental journal 2014 ; 59 ( 174185 )  . boeddinghaus r , whyte a / european journal of radiology 66 ( 2008 ) 396418 . cabrera - martos i etal . 
2011 jan ; 22 ( 1 ) : 47 kuijpers mar , chiu yt , nada rm , carels cel , fudalej ps ( 2014 ) three - dimensional imaging methods for quantitative analysis of facial soft tissues and skeletal morphology in patients with orofacial clefts : a systematic review . 
2015 oct ; 37 ( 8 ) : 989 diagnostic imaging indental anomalies tooth impaction . an impacted tooth is one that , somehow , fails to erupt in the oral cavity within the expected developmental window.1 an impacted tooth may also be defined as a tooth , which is partially or not erupted , showing clinical , anatomical and radiological signs that suggest its correct eruption is unlikely.2 dental impaction is a common problem ( 2550% of the world population ) , which prevalence is strongly related to age , gender , ethnicity and anatomical localisation ( quadrants ) , 3 , 4 while its incidence appears to be increasing.5 the teeth of normal permanent dentition that are most often impacted are , in order of frequency , third molars , upper canines , premolars , and upper central incisors.6 in europe , impaction of third molars is observed in more than 70% of young adults , 7 and extraction of third molars is the most frequent procedure in oral surgery . possible causes of dental impaction are lack of space , position anomalies , supernumerary teeth , persistence of primary teeth , odontogenic cysts / tumours , trauma and systemic diseases . 
1 on suspicion ofdental impaction , level i radiological examination must notbe carried outbeforetheage of6 strength of recommendation a degree of evidence i at 6years of age , permanent tooth buds , except those of the iii molars , are visible in the panoramic image , a level i radiographic investigation . 
2 la radiologia medica ( 2019 ) 124 : 887916 impacted tooth and its relationship with adjacent anatomical structures , especially with the maxillary sinus floor.19 , 21 on suspicion ofdental impaction , orthopantomography should be thefirst diagnostic examination recommendation no . 
this image provides information on the position of the impacted tooth , its relationship with the alveolar ridge , its projective relationships with the adjacent teeth and structures and the presence of any associated lesions ( radiotransparent and / or radio - opaque periradicular lesions ) .12 however , 2d imaging techniques turn out to be less accurate in assessing dental impaction than 3d cbct . 
3 level i radiographic investigations are anefficient tool indefining therisk ofinjuring thealveolar nerve and , innoncritical cases , may be considered sufficient intreatment planning fortheextraction ofaiii lower molar strength ofrecommendation adegree ofevidence iv with reference to impaction of the third lower molars , orthopantomography , and , in some cases , intraoral radiography , is an efficient imaging technique for estimating the risk of injuring the lower alveolar nerve.14 , 15 so , if level i radiographic investigations exclude any chance contact between the iii lower molar and the mandibular canals , 2d imaging may be held to be sufficient and 3d cbct imaging unnecessary.16 , 18 recommendation no . 
in particular , identifying and evaluating the extent of an external root resorption processsometimes observed in dental impaction , especially in the upper caninesrequire a 3d evaluation.22 , 24 so , on suspicion of external root resorption associated with dental impaction , a 3d cbct imaging technique is indicated.22 , 25 recommendation no . 
6 periapical intraoral imaging may be used fordental impaction oftheupper incisorcanine region strength ofrecommendation : b degree ofevidence : iv periapical radiography is an imaging technique generally used in dentistry to evaluate periapical lesions , root morphology ( when planning extraction / endodontic treatment ) and post - implant surgery checkup . 
this anomaly , only found in permanent dentition , causes an alteration of the normal sequence in the dental arch.28 , 29 tooth transposition may be found in two forms , complete ( true / complete transposition ) and incomplete ( pseudo / incomplete transposition ) .30 in its complete form , transposition involves the whole permanent tooth ( crown and root ) , while , in the incomplete form , only the crown is involved . transposition is a relatively rare , usually unilateral , anomaly , which prevalence in the population is around 0.3%.28 it is more frequently found in female patients ( female / male ratio 3 : 1 ) and in the upper arch , 29 and it is often associated with other dental anomalies.31 in most cases , tooth transposition occurs between the canine and the neighbouring teeth , in particular between the upper canine and first premolar , followed by transposition between the upper canine and lateral incisor ( 28 , 29 )  . the aetiology of tooth transposition remains unknown to date . 
1 on suspicion oftooth transposition , level i radiological investigation should notbe carried outearlier thantheage ofsix strength of recommendation : a degree of evidence : i at age 6 , permanent tooth buds , third molars excepted , are visible on the panoramic image . 
2 on suspicion oftooth transposition , theindicated level i radiological investigation isorthopantomography , which may be sufficient fortreatment planning strength ofrecommendation : adegree ofevidence : i level i radiographic investigations are the first step in the diagnostic approach to tooth transposition , and the panoramic image is indicated in order to confirm diagnostic suspicion . 
the main advantages , compared to those of intraoral images , are it shows both jaws in one single two - dimensional image , and it allows reducing exposure to radiation ( 10 times less than radiographic status ) .11 there is no clinical evidence supporting the use of cbct as an initial diagnostic examination for tooth transposition . 
4 intraoral periapical imaging may be used intooth transpositions strength ofrecommendation : b degree ofevidence : v periapical radiography is an imaging technique generally used in dentistry to evaluate periapical lesions , root morphology ( when planning extraction / endodontic treatment ) and post - implant surgery checkup . 
this radiographic image provides information on the number and position of supernumerary teeth , their relationship with the alveolar ridge , the projective relationships with teeth and adjacent structures and the presence of any associated lesions ( radio - transparent lesions and / or periradicular radio - opaque lesions ) .12 however , in the assessment of a supernumerary tooth that has caused dental impaction , 2d imaging techniques turn out to be less accurate than 3d cbct . 
in particular , it should be considered that 2d imaging is significantly less effective than 3d imaging in establishing correct positioning of the supernumerary teeth with regard to adjacent structures ( teeth and critical structures such as the mandibular canal , the nasal fossa and the maxillary sinus ) 13 and also in supplying necessary information for correct surgical planning . 
it may occur in a sporadic form or associated with hypodontia , within the picture of genetic syndromes . the term dilaceration describes the presence of a sharp curvature of the root with regard to the crown . 
according to some authors , for a tooth to be defined as dilacerated , the angle formed by the axis along the root must be 90 , while others say 20.33 the aetiology of dilaceration is not fully known yet . 
the most supported explanation is that this shape anomaly results from a traumatic event , occurring to the corresponding deciduous tooth in early childhood , even though idiopathic forms are described , probably consequent upon ectopic development of the tooth germ.33 dens invaginatus ( dens in dente ) is an anomaly mainly involving the upper lateral incisors and originates from infolding and consequent development of part of a tooth within another tooth . the fusion and germination of two neighbouring permanent germs determine the formation of teeth with separate pulp chambers and completely fused , either at the level of 1 3 la radiologia medica ( 2019 ) 124 : 887916 the crown in the former case or with a single pulp chamber in the latter . 
 2012 ; 54 ( 2 ) : 197203 . celikoglu m , miloglu o , oztek o ( 2010 ) investigation of tooth transposition in a non - syndromic turkish anatolian population : characteristic features and associated dental anomalies . 
2015 aug 15 ; 8 ( 8 ) : 118905 . diagnostic imaging indental traumatology for dento - alveolar traumatology , please refer to guidelines for prevention and clinical management of dental trauma in children and adolescents ( 2012 ) ( 1 , 11 , a )  . diagnostic imaging in dental trauma is used to evaluate the degree of root formation of the tooth affected by trauma and to assess tooth displacement or possible root fracture . 
2 periapical intraoral xray isnecessary inthediagnostic investigation ofdentoalveolar trauma inpaediatric patients strength ofrecommendation : adegree ofevidence : iv periapical intraoral x - ray performed with an adequate paralleling system is the imaging technique of choice , which finds indication in identifying dento - alveolar modifications subsequent to localised trauma . 
dental radiographic examinations : recommendations for patient selection and limiting radiation exposure ; 2012 . govindarajan m , reddy vn , ramalingam k , durai ks , rao pa , prabhu a . 
dental trauma guide : a source 1 3 914 la radiologia medica ( 2019 ) 124 : 887916 a e.gov.it / imgs / c_17_pubbl icazi diagnostic imaging for caries and periodontal disease . strength of recommendation : a degree of evidence : i of evidence - based treatment guidelines for dental trauma . 
in all patients , when carious lesions are suspected in permanent teeth , the favoured radiographic image of choice is the bitewing . this film size is large enough to evaluate the whole crown of a permanent tooth . 
intraoral x - ray is indicated for assessment , over time , of the periapical and pulp health conditions of permanent teeth that have undergone conservative treatment of the dental pulp . 
intraoral x - ray is indicated for periapical health condition periodical assessment of mature or immature teeth that have undergone provisional endodontic treatment ( apecification ) , pulp regeneration or definitive rct . 
msct or cbct may be indicated for analysis of i and ii branchial arch syndrome craniofacial defects with the purpose of treatment planning . strength of recommendation : a degree of evidence : i strength of recommendation : a degree of evidence : ii strength of recommendation : a degree of evidence : ii strength of recommendation : a degree of evidence : i strength of recommendation : a degree of evidence : iv strength of recommendation : a degree of evidence : iv strength of recommendation : b degree of evidence : iv strength of recommendation : a degree of evidence : iv strength of recommendation : b degree of evidence : iv strength of recommendation : a degree of evidence : iv strength of recommendation : a degree of evidence : i strength of recommendation : a degree of evidence : i strength of recommendation : a degree of evidence : v strength of recommendation : b degree of evidence : v strength of recommendation : a degree of evidence : iv 1 . 
cbct is the level ii examination indicated for selected cases in which level i investigations ( periapical intraoral radiography ) do not supply sufficient diagnostic information for suitable therapeutic planning . glossary strength of recommendation : a degree of evidence : iv strength of recommendation : a degree of evidence : iv strength of recommendation : a degree of evidence : iv strength of recommendation : a degree of evidence : iv diagnostic imaging in dental traumatology . alara as low as reasonably achievable : each exposure to radiation must be kept as low as it is reasonably achievable base on both economic and social considerations . 
under these circumstances , it is reasonable to minimise a risk that may be presumed to exist also at levels lower than the recommended limits , considering that what fovfield of view : the field of investigation prescribed in order to avoid subjecting the patient to useless radiation with areas of acquisition that are over - extensive with regard to the district under examination . 
availability of different fovs ( small , medium , large ) depends on the device used . iotnindex of orthodontic treatment need : devised to rate the need for an orthodontic treatment based on dental position alterations that may be associated with selected functional alterations . 
besides being used for epidemiological research , it is also used as a tool by the national health service to assess childrens orthodontic treatment priority and eligibility for free care . 
the iotn index has 5 classes for orthodontic condition grading . drlsdiagnostic reference levels : radiation dose levels for each diagnostic imaging system or , in nuclear medicine diagnostics , levels of activity , for each type of examination , performed with standard appliances , for groups of patients of standard body size or for standard dummies . 
for six selected radiologists , the number and types of reported examinations as well as the related radiology report turnaround times ( rtats ) were analyzed in detail and compared between the two 1 - year periods . results overall , there was a significant increase of 10.3% in the total number of examinations performed in the whole department in 2016 compared with 2014 . 
subspecialized reporting allows the individual radiologist to focus on a special field of professional competence but can result in longer overall rtat . keywords subspecialization reporting time professional competence radiologists * martin h . 
15 , 81377munich , germany vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 860869 introduction a high degree of subspecialization among referring physicians from different clinical specialties has implications for radiologists in that it requires increasingly specialized diagnostic skills tailored to the specific needs of referrers [ 1 , 2 ]  . 
referring physicians expect radiologic examinations to adhere to high technical standards and expect high - quality radiology reports to be available within a short time [ 3 ]  . in this era of lower health care spending and reimbursement models mostly based on the payment of fixed amounts per service , efficient organization of examination and reporting workflow becomes essential for both hospital - based and officebased radiologists [ 46 ]  . 
in europe , radiology departments and practices organize reporting workflow by imaging modality such as computed tomography ( ct ) , magnetic resonance imaging ( mri ) , mammography , and conventional radiography . an alternative to general reporting is subspecialized reporting . 
with this option , reporting is organized by organ system such as abdomen , chest , musculoskeletal , and vascular system , and radiologists form teams focusing on the examination of one organ system with any imaging modality [ 7 , 8 ]  . 
using subspecialized reporting , radiologists can develop in - depth skills and experience in a specific area , allowing them to better meet the needs of subspecialized referring physicians and ensuring consistently high reporting quality [ 9 , 10 ]  . 
in addition to improving quality , subspecialized reporting has the potential to also enhance reporting efficiency . based on these considerations , the radiologic department of a swiss university hospital introduced an internal subspecialized examination and reporting system in mid - 2015 . 
the subspecialties defined were thoracic and cardiovascular imaging , abdominal and urogenital imaging ( including mammography ) , musculoskeletal imaging , and emergency imaging . in this study , we analyzed the effects of switching from general to subspecialized reporting on reporting workflow in a representative group of radiologists from our department who worked under both systems during the study period . 
specifically , we investigated how the implementation of subspecialized reporting altered the range of types of examinations reported by individual radiologists and reporting efficiency in terms of reporting volumes and radiology report turnaround time ( rtat )  . materials andmethods we conducted an internal analysis in the radiology department of a swiss university hospital to compare workflow before and after implementation of subspecialized reporting . 
subspecialized reporting was introduced on may 1 , 2015 . before the introduction of subspecialized reporting in 2015 , the radiologists selected for this analysis worked by imaging modality , i.e. , ct , mri , conventional radiography , and mammography . 
rtat was defined as the interval from the end of the examination to finalization of the report . for detailed analysis , six radiologists who worked in the department throughout the two 1 - year periods under investigation were selected . 
the preliminary report is then discussed with the staff or senior radiologist responsible for the respective modality and then finalized by the latter after any necessary editing was done to minimize effects of outliers or of imaging material only stored in the picture archiving and communication system ( pacs ) for documentation purposes on the calculation of average rtats . all data for the analysis were extracted from the radiology information system ( ris ; centricity ris - i 4.2 plus , ge medical systems , milwaukee , wi , usa )  . 
the following data were recorded : type of examination , date of examination , end of examination , beginning and end of reporting time , and finalizer . for each report , rtat was calculated in minutes . 
for comparison of rtats before and after introduction of subspecialized reporting , the following classification was used : more than 2h shorter with subspecialized reporting [ ] , 12h shorter with subspecialized reporting [ ] , no more than 1h longer or shorter with subspecialized reporting [ 0 ] , 15h longer with subspecialized reporting [ + ] , more than 5h longer with subspecialized reporting [ + + ]  . statistical analysis the cochranmantelhaenszel test was used to test for significant differences in the number of different types of examinations following the introduction of subspecialized reporting for all six radiologists taken together . 
the chisquare test was applied to analyze possible differences in the comparison of individual radiologists . changes in the range of examinations reported by each radiologist in terms of a shift toward a higher share of subspecialized imaging studies were assessed using a one - sided cochranmantelhaenszel test . 
the impact of subspecialized reporting on the range of types of imaging studies reported by each radiologist was assessed by the chi - square test . whether subspecialized reporting had a significant effect on rtat was tested for each radiologist using a two - tailed mannwhitneywilcoxon test and was based on an analysis of the subset of the 10 most common types of imaging studies reported by that radiologist in 2016 . 1 3 la radiologia medica ( 2019 ) 124 : 860869 fig . 
with the introduction of the subspecialized reporting system , teams for different organ systems or body regions were formed , who worked independently of the imaging modality used for the examination . 
the subspecialties defined were thoracic and cardiovascular imaging , abdominal and urogenital imaging ( including ent imaging and breast imaging ) , musculoskeletal imaging , emergency imaging , and pediatric imaging . 
statistical calculations were done using the r software ( r foundation for statistical computing , vienna , austria )  . results we included a total of 67 , 585 reports from 2014 and 72 , 224 reports from 2016 . 
 the proportion of reports finalized within the evaluated 72 - h period was 97.8% in the full year before and 95.2% in the full year after implementation of a subspecialized reporting . for each of the six radiologists , introduction of the subspecialized system led to an approximately 25% reduction in the range of examination types reported . 
the reduction was statistically significant for five of the six radiologists ( p < 0.05 , see table2 )  . this reduction was associated with an increase in the share of reports pertaining to the respective subspecialty for four of the six radiologists . 
following introduction of subspecialized reporting , this radiologists 10 most commonly reported types of examinations accounted for 78.1% of his / her overall reporting volume in his / her specialized area . 
on the contrary , rtats were in part even markedly longer after switching to subspecialized reporting . these findings must be interpreted in light of an overall increase of approximately 7% in the departments total reporting volume , and this increase in workload also included the six radiologists selected for this study . 
the increase in imaging studies performed by our department is in agreement with general national and international data indicating a steady increase in radiologic examinations [ 11 , 12 ]  . as expected , the introduction of subspecialized reporting in 2016 led to a concentration on examinations of the respective subspecialties among the six radiologists included in our analysis in that the range of different examination types reported became smaller for all of them compared with 2014 ( see table2 )  . 
reporting of a narrower range of examinations was especially conspicuous for radiologist a ( who became head of the abdominal imaging team with the introduction of subspecialized reporting , 30% decrease ) and radiologist c ( head of the thoracic and vascular imaging team , 34% decrease )  . as expected after switching to the new system , subspecialized reports accounted for a higher proportion of the total reporting volume ( for examinations performed during standard weekday working hours ) in 2016 compared with 2014 . 
this high increase is attributable to the fact that , with his / her extensive expertise and experience , radiologist a covered nearly the complete range of imaging studies in his / her reporting activities under the earlier general reporting system ; hence , the impact of subspecialization was especially marked . 
in contrast , the range of examination types reported by radiologist e ( head of pediatric imaging before and after introduction of the new system ) was nearly unchanged after the switch of systems . 
this is not surprising since pediatric radiology with its focus on examinations tailored to the specific needs of young patients already was a largely autonomous unit before the introduction of subspecialized reporting . while a concentration on the defined subspecialties was expected , it was surprising that subspecialized reporting , rather than making reporting workflow more efficient , even resulted in longer rtats for many types of examinations . 
a possible explanation is that a large proportion of residents , as in our department , which is one of the largest teaching institutions for radiology residents in switzerland , results in a more complex and time - consuming reporting workflow . 
 [ 13 ] show that , median rtats can be dramatically shortened , from approximately 17h to approximately 3h and 30min , when fully trained radiologists directly report imaging studies under a subspecialized system . another , specific factor contributing to longer rtats for radiologist a in our analysis may be that , as head of the abdominal imaging team , he / she was also responsible for all complex reports and imaging examinations revealing rare findings . 
an effect of this policy is that radiologist a tends to get a large share of difficult abdominal imaging cases for a second opinion . conversely , for radiologist b ( head of emergency imaging ) , focusing on a specialized range of examinations to be reported ( 28% decrease , from 87 to 63 types of examinations , in 2016 , see table 2 ) resulted in consistently shorter or nearly unchanged rtats compared to 2014 ( see table3 radiologist b )  . 
moreover , the spatial arrangement , with the supervising radiologist being based directly next to the emergency ct scanner during standard weekday working hours , facilitates workflow and finalization of preliminary reports generated by the residents of the team . overall , our results show that subspecialized reporting has not only benefits but also disadvantages and risks . 
greater in - depth skills in a well - defined area of their field allow radiologists to solve more complex problems , which is likely to have a positive mental effect . 
 in addition , the growing complexity of a specialty like radiology makes it more and more difficult for an individual radiologist to master all its many facets with the required depth of expertise [ 17 , 18 ]  . subspecialization can also have a positive impact on residents who rotate through different fields of radiology during their training . 
clearly defined rotations allow radiologists to focus on a specialized area for a predefined time [ 19 ] rather than having to constantly switch between many different types of examinations . 
various studies have so far shown that subspecialization in radiology can lead to a higher quality and precision in radiologic reports in the individual sub - disciplines [ 20 , 21 ]  . 
we could also recognize this effect in our clinic after the implementation of a subspecialized reporting systealthough it was not the aim to quantify changes in the quality of reports after introducing a subspecialized system , we nevertheless achieved an apparently significantly higher perceived quality among our referrals with multiple positive feedbacks . on the other hand , subspecialization can have negative effects [ 22 ] , because repetition can be boring and less motivating . 
monotony can lead to greater fluctuation , making staff to leave and seek employment where they work on a broader , more generalized range of imaging studies if they prefer such an approach to focus on a narrow subspecialized area . 
from our perspective , there are different possible reasons for this : ( 1 ) the number of reports that are being sent between different senior radiologists to get a second opinion had significantly increased after the implementation of the subspecialized systein a radiology information system ( ris ) database research , we found that there were only about 2% of the reports signed by two senior radiologists in 2014 , while this percentage increased to over 10% in the year 2016 . 
a possible reason for this especially in the group of abdominal imaging was that reporting on liver imaging was dedicated to three specific radiologists with the best experience in this field . 
obviously , this not only leads to a higher quality of the reports , but also causes longer reporting time ; ( 2 ) difficult findings and reports are intentionally withheld by individual specialized radiologists in order to first discuss these with the referring colleagues in a weekly tumor board . 
the reports then become more focused and there are fewer misunderstandings in difficult situations , but the reporting time increases ; ( 3 ) nonurgent reports were not finally completed before weekends or public holidays . according to the directive of the european union as of february 2018 , it is mandatory for the member states to make sure that for radiologic examinations which are based on ionizing radiation , the dose is being documented afterward in the radiologic report ( article 58 , b ) [ 23 ]  . 
this is due to the fact that we wished to include radiologists from each of the four subspecialized areas introduced with the new systemoreover , all radiologists selected had to be present throughout the two study periods , i.e. , throughout 2014 and 2016 , without longer leaves of absence . 
 the involvement of residents in reporting workflow in all areas including complex and highly specialized imaging studies further contributed to a much greater complexity and hence longer rtats in our study . 
from our perspective , ultrasound is different from all the other diagnostic methods like ct , mri or conventional radiography as the reporting time as a part of the overall procedure time is less important than for all the other procedures . 
cchf is a life - threatening disease observed endemically over a wide geographical regions in the world , and there is limited information about pulmonary findings in cchf patients . purpose we aimed to investigate the pulmonary findings belonging to a large cchf patient cohort and to determine if there is any relationship between laboratory findings and disease severity . materials and methods a total of 165 patients who were diagnosed with cchf and examined through chest x - ray ( cxr ) due to respiratory symptoms at their first examination and / or during their hospitalization were included in this study . 
the general non - specific symptoms of cchf are fever , malaise , * fatma akta fatmakokcu79@hotmail.com 1 department ofradiology , faculty ofmedicine , gaziosmanpaa university school ofmedicine , 60100tokat , turkey 2 pulmonary diseases department , versa hospital , nevehir , turkey anorexia , nausea / vomiting , and myalgia , which are observed after the contact . 
in addition , intracranial bleeding , gastrointestinal bleeding , and alveolar hemorrhage that are life - threatening and often responsible for mortality can be observed as well [ 14 ]  . 
however , the number of studies related to pulmonary imaging in cchf is very rare and limited despite the increasing number of cases in the world and in our country [ 14 ]  . 
 we may propose simple pulmonary imaging such as cxr to be used within the following 5days after the first examination as an effective tool to evaluate clinical course and severity of the disease . the aim of this study was to investigate the chest x - ray findings of cchf patients with respiratory findings and to determine if there is any relationship between the laboratory findings and severity of the disease . materials andmethods a total of 464 patients with confirmed cchf were admitted to emergency department and department of infectious diseases and clinical microbiology between january 2011 and december 2016 . 
 patients using medicines that may cause pulmonary toxicity and having acute or chronic lung diseases , malignancies , heart diseases , and blood diseases , and a history of thoracic radiotherapy or surgery in addition to cchf were excluded from the study . 
additionally , patients who were clinically diagnosed with pulmonary infection ( such as cough sputum ) and who were diagnosed with other microorganisms in their cultures were not included in the study as well . ethical approval was obtained from ethics committee of the university . 
in addition to demographical findings of the patients such as age , gender , occupation , place of residence , tick exposure , clinical findings , and medical history , laboratory findings and radiological images of lungs were evaluated . 
the cchf diagnosis was made based on the detection of virus genome by using enzyme - linked immunosorbent assay ( elisa ) or polymerase chain reaction ( pcr )  . statistical analysis data are expressed as mean standard deviation . 
 out of 40 patients with comorbidity , 23 were found to have hypertension , three of them had diabetes mellitus , 13 were diagnosed with both hypertension and diabetes mellitus , and one of them had hypothyroids . 
the frequency of general symptoms of the patients observed during the first examination is presented in table2 . based on the laboratory findings , the mean values of liver - specific enzymes such as alkaline phosphatase ( alp ) , alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , gamma - glutamyltransferase ( ggt ) , creatine kinase ( ck ) , and lactate dehydrogenase ( ldh ) were seen to be high . single and / or multiple abnormal imaging findings were detected in 93 patients ( 56.4% ) as a result of cxr examination during the first examination . 
in a similar vein , four patients , claimed to have ground glass densities on cxr , and one patient , claimed to have pleural effusion , were not found to have these findings according to the results of the thorax ct . 
the comparison of survival and cxr findings of patients is shown in table5 . discussion cchf is a life - threatening zoonotic viral disease that can be seen endemically in many regions in the world . 
the most frequent clinical symptoms are fatigue ( 92.3% ) , fever ( 89.4% ) , myalgia ( 69.7% ) , headache ( 68.1% ) , and nausea ( 64.7% ) ; 23% of patients had bleeding ( petechiae , epistaxis , gingival bleeding , vaginal bleeding , and bleeding in internal organs ) [ 10 ]  . 
this situation might be related to the possibility that males work in occupations with high risk of being in contact with ticks . as in many other viral diseases , cchf may cause symptoms of pulmonary involvement and may lead to death . 
the results of the thorax ct revealed that 20 of these patients ( 86% ) had pleural effusion , 12 of them ( 52% ) had consolidation , nine ( 39% ) had atelectasis , and 14 ( 60% ) had 1 3 la radiologia medica ( 2019 ) 124 : 826832 fig . 
however , hemoptysis was detected in a total of nine out of 27 patients with hemorrhage ( six of those patients died )  . there have been a very limited number of studies on pulmonary radiological findings in cchf in the literature . 
however , it has been stated that it can share similarities with mechanism of pulmonary pathologies such as pleural effusion , pneumonitis , hemoptysis , and alveolar hemorrhage in other febrile viral hemorrhagic diseases [ 15 , 16 ]  . 
in a study performed on dengue hemorrhagic fever , a total of 468 cxr taken from 363 patients were examined and parenchymal infiltration and pleural effusion were observed in more than half of the patients on the third day of follow - up . 
 moreover , these radiological findings were found to be closely related to laboratory findings of leukocyte count , platelet count , aptt , alt , and albumin levels in the same study [ 18 ]  . 
 in conclusion , pleural effusion and other findings related to pulmonary involvement can be seen in cchf due to endothelial damage caused by viral load in the circulation , increased capillary permeability , and hemorrhages due to low platelet . in the study by akta etal . 
the results of the study revealed that there was no statistically significant difference between the two examination techniques in terms of detecting parenchymal infiltration , alveolar infiltration , and pleural effusion . 
when these patients were compared , it was found that there was consolidation in cxr of the four patients and pleural effusion in one patient while they were not observed in thorax ct . 
on the other hand , based on thorax ct results , ground glass density was detected in four patients and atelectasis in two patients , and these were not observed in cxr . 
therefore , there was a possibility of response to the treatment and spontaneous regression or progression in the findings . the mortality rate of cchf generally ranges from 2 to 80% . 
this result can be associated with the fact that cchf is endemic in this region ; therefore , medical staff and people are informed about the disease , and thus , they are alert . 
in addition , the experience in the clinical follow - up might also contribute to the decrease in mortality rate besides improvements in medical care conditions in cchf and increase in awareness of the seriousness of the disease . it is known that mortality and some laboratory parameters prolonged a ptt , increased inr , high alt , ast and ldh , low platelet , and increased leukocyte are related to each other [ 23 , 24 ]  . 
for this reason , all of the cchf patients who were found to have pathologic cxr result should be considered at high risk . our choice to conduct a retrospective study is the first limitation ; on the other hand , medical information of the patients and their radiological images has been recorded in a proper way . 
the other limitation was the inability to confirm the cxr findings of the patients as not all of these patients had thorax ct . in conclusion , cchf is a serious public health problem that can be fatal . 
according to the results of our study , we suggest that 1 3 832 la radiologia medica ( 2019 ) 124 : 826832 radiological imaging of pulmonary system should be performed primarily with cxr . 
pulmonary involvement ( pleural effusion , consolidation , and ground glass opacity ) affects the survival in cchf negatively , and further examination should be performed with thorax ct in case of necessity . 
to detect urine leakage , we initially performed conventional cystography after retrograde distention of the bladder with dilute iodinated contrast material , followed by ultra - low - dose ct cystography . 
the diagnostic accuracy of these two modalities was compared , and the technical characteristics of ultra - low - dose ct cystography were examined . results all 31 referred patients were included in this study . 
of the 31 patients , 27 ( 87.1% ) underwent bladder repair after radical prostatectomy , 3 ( 9.7% ) after radical cystectomy , and 1 ( 3.2% ) after bladder diverticulectomy . 
based on these findings , we were able to establish a proper treatment plan . conclusions ultra - low - dose ct cystography is an accurate method for evaluating urine leakage after bladder repair , and this technique may help determine the most appropriate treatment strategy for patients with urine leakage after bladder repair . keywords anastomotic leakage radiation dosage computed tomography fluoroscopy introduction recently , awareness and concern about radiation exposure have increased . 
in the republic of korea , the number of radiation - generating devices has rapidly increased * seong hoon choi 0733280@uuh.ulsan.kr 1 department ofurology , ulsan university hospital , university ofulsan college ofmedicine , ulsan , korea 2 department ofradiology , ulsan university hospital , university ofulsan college ofmedicine , 877 bangeojinsunwando - ro , dong - gu , ulsan44033 , korea from 59 , 739 in 2008 to 72 , 626 in 2012 [ 2 ]  . 
in a study involving conventional non - contrast ct , the reported exposure dose ( ed ) for the abdomen and pelvis was between 10 and 20msv [ 6 ]  . 
therefore , if dose - reduced ct can be shown to accurately diagnose bladder injury , low - dose and ultra - lowdose ct could be applied to a wide variety of cases . one of the advantages of ct compared with conventional cystography is the ability to detect anatomical relationships . 
 a major concern associated with dose - reduced ct is higher noise , which results in lower image quality that may reduce the accuracy of this technique for detecting anatomical relationships and facilitating differential diagnoses . 
moreover , while low - dose and ultra - low - dose ct are effective for detecting urolithiasis [ 7 , 11 ] , it remains uncertain whether low - dose ct cystography can detect bladder injury or urine leakage . 
therefore , we performed a prospective cohort study to compare the accuracy and technical characteristics of ultra - low - dose ct cystography with those of conventional retrograde cystography . materials andmethods study participants from november 2015 to october 2017 , we prospectively examined 31 consecutive patients referred for cystography after bladder repair . 
 to diagnose urine leakage , we initially performed conventional cystography after retrograde distention of the bladder with dilute iodinated contrast material , followed by ultralow - dose ct cystography ( table1 )  . imaging technique andinterpretation conventional cystography was performed in the following manner : with the patient supine on the examination table , the urine bag was disconnected from the foley catheter and the foley catheter was suctioned using a 50 - ml syringe until the bladder was completely emptied . 
additional pelvic ap radiographs were obtained after the initial infusion of approximately 100ml of contrast material and after either infusion of another 150ml of contrast material or occurrence of one of the following endpoints : ( 1 ) 300ml of contrast material was completely infused into the bladder , ( 2 ) urinary leakage occurred due to continuous bladder detrusor activity with infusion less than 300ml , or ( 3 ) the patient complained of pain in the abdomen and pelvis . 
oblique radiographs were also obtained , followed by an ap radiograph after bladder emptying . ct cystography was performed in the following manner : with the patient supine on the examination table , the foley catheter was suctioned using a 50 - ml syringe , as described above . 
ultra - low - dose ct images were reconstructed using idose level 5 , which is one of the iterative reconstruction ( ir ) algorithms [ 12 , 13 ]  . 
the ct parameters were as follows : a pitch of 0.915 , collimation of 0.625mm , matrix size of 512 512 pixels , sectional thickness / reconstruction interval of 2mm , collimation of 2 64 0.6mm , pitch of 1.2 , and gantry rotation time of 0.5s. 
the reductions in radiation dose were compared between the two modalities and also with regard to the patients bmi [ 14 ]  . image findings were interpreted by an expert radiologist in the genitourinary division of the radiology department of our institution . 
the following conventional cystography and ct cystography findings were recorded : ( 1 ) presence or absence of leakage , ( 2 ) location of leakage , and ( 3 ) amount of leakage . 
the amount of leakage analyzed by conventional cystography was scored according to a previous report as follows : grade i , extraperitoneal leakage within 6cm of the vesicourethral anastomosis ( vua ) ; grade 2 , extraperitoneal leakage extending beyond 6cm from the vua ; and grade 3 , intraperitoneal leakage [ 15 ]  . 
informed consent was obtained from all subjects when they were enrolled . results the mean conventional cystography dose area product , ct cystography dose length product values , and the effective radiation dose are summarized in table2 . 
the estimated dose reduction using ultra - low - dose ct compared with the dose in conventional cystography was 60.7%. four of the 31 patients were diagnosed with urine leakage by conventional cystography . 
in all of the patients who were diagnosed with urine leakage by conventional cystography , leakage was observed at the posterior aspect of the vua ; moreover , all had grade 1 leakage ( leakage within 6cm of the vua )  . 
the amount of leakage in these patients was not large ( average , 1.31cm2 ; range , 0.581.74cm2 ) ( table3 )  . by performing ct cystography , we were able to track the precise locations and amounts of urine leakage . 
all nine patients who had urinary leakage , including the two with longer retention of the foley catheter , experienced no further complications and were discharged . discussion conventional cystography has been accepted as the standard for the assessment of bladder trauma [ 8 ]  . 
our study also showed that conventional cystography detected leakage at the posterior aspect of the vua in all patients who were diagnosed with urine leakage ; however , it is thought that the contrast collects in the dependent position by gravity . 
in particular , according to ultra - low - dose ct cystography , patient i had urine leakage at the 3 oclock position of vua , whereas 1 3 la radiologia medica ( 2019 ) 124 : 812818 fig . 
ct cystography identified the location of leakage at the 4 oclock position of vua , and the amount of leakage was estimated to be 7.08cm2 ( black arrow )  . 
c ct cystography showed patient i to have urine leakage at the 3 oclock position of the vua ( black arrow ) , but conventional cystography ( white arrow ) detected leakage at the posterior aspect of the vua according to conventional cystography , there was leakage at the posterior aspect of vua . lately , iterative reconstruction ( ir ) algorithms have received attention as good new methods for reducing radiation dose during ct examination [ 17 ]  . 
this 4th generation iterative reconstruction technique in preventing photon starvation artifacts ( streaks , bias ) prior to image creation and in maintaining image texture to overcome the artificial or plastic look of images that have been frequently reported when using previous iterative reconstruction techniques . 
evidence from rigorous clinical evaluations demonstrates the potential of idose to improve image quality and / or lower radiation dose levels beyond those previously achievable with conventional , routine - dose acquisitions , fbp reconstructions [ 12 , 13 ]  . 
for the ultra - lowdose protocols , the assumed effective dose was 0.44msv , which was very low and comparable to the dose used for conventional single plain radiography of the kidneys , ureter , and bladder [ 19 ]  . 
thus , low - dose or ultra - low - dose protocols could potentially contribute toward reducing the lifetime cumulative radiation dose as well as cancer risk [ 2 , 20 ]  . urinary leakage is a challenging , short - term complication that can occur regardless of the surgical approach used and may reduce patients quality of life [ 21 ]  . 
however , although most urine leakages are self - limited , urine leakage - induced complications include infection , metabolic abnormality as a result of intraperitoneal urine reabsorption , and urinoma formation [ 23 , 24 ]  . 
although there have been no previous reports providing definitive criteria for persistent or massive vua urine leakage , some studies have defined anastomotic urine leakage as a persistent daily amount of urine ( over 1500ml ) in the suction drain for more than 6days and for which an invasive interventional procedure had to be performed [ 27 , 28 ]  . 
our findings suggest that the use of ultra - low - dose ct may help to guide treatment and avoid unnecessary procedures in addition to reducing the risks associated with radiation exposure . our study has several limitations . 
moreover , although ultra - low - dose ct cystography was the more accurate modality for determining urine leakage compared to conventional cystography , we cannot rule out the possibility that extravasations outside the urethra or bladder lumen detected by ultra - low - dose ct cystography were not urine leakages . 
nonetheless , to our knowledge , this study is the first to evaluate the accuracy and technical characteristics of ultra - low - dose ct cystography as an optimal modality to track the precise location and amount of urine leakage and limit the radiation dose . 
 these results establish a foundation for future studies and treatments . conclusions ultra - low - dose ct cystography enables accurate evaluation of urine leakage after bladder repair despite the small radiation dose . 
prospective studies are needed to further assess its role among locoregional treatment options for liver metastases from crc . keywords liver metastases stereotactic body radiotherapy colorectal cancer oligometastases introduction colorectal cancer ( crc ) is estimated to be the second most frequently diagnosed cancer in europe , accounting for 447 , 000 new cases in 2012 , and one of the most relevant causes of cancer mortality worldwide with 694 , 000 deaths in the same year [ 1 , 2 ]  . over the last two decades , the clinical outcome of patients with metastatic crc ( mcrc ) has improved due to many factors , such as local ablative treatments ( lats ) [ 3 ]  . 
potentially , lats can have a curative intent for oligometastatic patients [ 4 ] and could play a role in the management of an oligoprogressive disease , in which only a solitary or few ( 5 ) metastatic localizations progress , while all other sites of disease are stable or responding to the current regimen [ 5 ]  . 
over the past two decades , extensive clinical experience demonstrated sbrt to be a safe and high - precision technology method to deliver ablative treatments for different metastatic sites [ 8 ] , including liver metastases [ 9 ]  . since 2012 , robotic sbrt has been offered in our institution as a possible alternative to surgery or other lats for liver oligometastases in colorectal cancer patients unsuitable for surgery . the aim of our retrospective study was to report the clinical outcome of robotic sbrt in this setting . 
during treatment planning , the following organs at risk ( oars ) were delineated : left and right lung , esophagus , heart , thoracic wall or ribs , left and right kidneys , small and large bowel , duodenum , stomach , spinal canal , whole liver , great vessels . 
for most patients , the intended prescription dose was 37.5gy in three fractions , prescribed to the 70% isodose line to cover 95% of the ptv [ 10 ]  . 
 patients were prescribed proton pump inhibitors , serotonin 5 - hydroxytryptamine antagonists , benzodiazepines and antacids as clinically indicated . followup andtoxicity evaluation after the last application , a clinical examination was performed and treatment toxicity was scored , if present , according to the common terminology criteria for adverse events ( ctc - ae ) scale , v 4.0.3. 
treatment response was 1 3 872 la radiologia medica ( 2019 ) 124 : 870876 evaluated by serial contrast - enhanced spiral ct or mri scan 2months after end of sbrt . 
complete response ( cr ) was defined as disappearance of all target lesions ; partial response ( pr ) as at least a 30% decrease in the sum of diameters of target lesions ; progression disease ( pd ) as at least a 20% increase in the sum of diameters of target lesions . 
local control ( lc ) was defined as the time of absence of progression disease at the site of sbrt from the end of the treatment . statistical analysis local control , local and distant progression - free survival ( pfs ) and overall survival ( os ) curves were evaluated by the kaplanmeier method . 
for cb , additional investigation included treatment planning related parameters such as number of fractions , physical and biological effective dose ( bed10 , considering / = 10 ) coverages . 
in order to comply with dose constraints to organs at risk , a risk - adapted approach was followed for the treatment of nine lesions , which were irradiated in five fractions instead of three . 
 then , we compared the bed10 mean dose covering the ptvs between lf and lc , and we found that its median value was smaller in the lf group ( 89.26gy ) than in lc fig . 
actuarial 1 3 874 la radiologia medica ( 2019 ) 124 : 870876 although surgical resection is presently accepted as the first choice among strategies for ablation , for many reasons only a small percentage of patients qualify for surgical procedures [ 14 ]  . up to now , ct - guided percutaneous radiofrequency ablation ( rfa ) is the most common local treatment option to be offered to such patients [ 15 ]  . 
in recent years , sbrt has become the current standard in medically inoperable early lung cancer patient [ 16 ] and , potentially , it could be the same for unresectable liver metastases treatment [ 17 ]  . our analysis counted one of the most numerous series addressing the use of high doses of radiation by means of robotic sbrt in the treatment of liver metastases exclusively from crc . 
moreover , our study has other limitations : its retrospective nature , the heterogeneity of the prescription doses , the differences in the timing of sbrt with respect to other treatments . our results demonstrated that it is possible to have good outcomes on response rate and survival analysis , keeping an excellent safety profile . 
 [ 18 ] in their subgroup analysis on liver metastasis from adenocarcinoma , confirming that colorectal adenocarcinoma has a statistically significant worst lc than other histologies [ 19 , 20 ] , which is perhaps related to the fact that these patients are generally heavily pretreated and surgically unresectable [ 21 ]  . moreover , as chang etal . 
 [ 22 ] had already concluded in their meta - analysis , a dose of 4652gy in three fractions or higher is required to achieve 90% lc at 1year for the treatment of hepatic cr metastases . although a good performance in terms of lc , in our analysis , pfs ( both hepatic and distant ) was poor , if compared with the results obtained by berkovic etal . 
swaminath and dawson [ 23 ] had already noticed in their analysis that , although excellent local control rates were achieved , many patients developed out - of - field progression probably because of the relatively high number of patients with extrahepatic disease and / or more than three metastatic lesions , suggesting that a closer attention should be reserved to patients who really belong to a relatively good prognostic group with longer survival expectancy and offering a strong rationale for combining sbrt with systemic treatments , as hypothesized by scorsetti etal . 
in fact , comparing survival curves of patients with or without extrahepatic disease before sbrt , there is a clear advantage for the second ones , both in os and in pfs ( supplementary materials , figures1 and 2 )  . 
previous extrahepatic disease and number of metastatic lesions > 3 showed a statistically significant correlation at univariate analysis ; this result was confirmed at multivariate analysis only for the presence of previous extrahepatic disease , as in the pfs analysis . toxicity no complications were observed during or after fiducials placement . 
as referred by hellman and weichselbaum , the theory of oligometastases considers that there could be a subgroup of patients with metastatic disease that is intermediate between completely absent and widely metastatic [ 12 ]  . 
on the other hand , their cohort was less homogeneous than ours , since also patients treated for extrahepatic localizations ( lung , lymph nodes , suprarenal gland ) were included in the analysis . 
 [ 29 ] performed a phase 1 / 2 study enrolling about 70% of patients with liver metastases from non - colorectal origin ( lung , breast , ovary , esophagus and others )  . 
likewise , in scorsettis study [ 9 ] , more than 50% of patients had metastases from non - colorectal primary tumor , and more than 80% of patients received a prescription dose of 75gy in three fractions ( mean dose to ptv )  . 
as expected , very limited treatment - related toxicity was observed in our cohort ; however , it has to be acknowledged that our local control rate is at the lower range of published data . in particular , the rate of actuarial lc at 2years in these experiences ranges between 64 and 92% ( supplementary table2 )  . 
as a matter of fact , the net effect of sbrt on the irradiated lesion is critically dependent on the delivered physical total dose and fractionation , the applied bed , the size of the gtv , the prescription modality and the intrinsic biological radiosensitivity . 
overall , patients selection refinement and prospective trials based on strict inclusion criteria in the setting of oligometastatic crc should be warranted . conclusions survival and toxicity results of our retrospective analysis support the hypothesis that sbrt for liver metastases from crc is a feasible treatment , combining an excellent safety profile with acceptable efficacy results . 
in addition , only controlled studies could allow us to define how to best select oligometastatic patients potentially profiting from a locoregional treatment with the aim to defer further systemic therapies . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical standards informed consent was obtained from all individual participants included in the study . 
the survey consisted of 11 multiple - choice questions about participants demographics , current local modalities of pic acquisition and storage , perceived advantages and disadvantages of pic dematerialisation over conventional paper - based pic , and overall opinion about pic dematerialisation . results a total of 1791 radiologists ( amounting to 17.4% of active sirm members for the year 2016 ) joined the survey . 
conversely , the need to create dedicated areas for pic acquisition inside each radiological unit ( 64.0% ) and to gain preliminary approval for the use of advanced digital signature tools from patients ( 51.8% ) were seen as potential disadvantages . 
however , concerns were raised that its practical implementation might face hurdles due to its complexity in current real life working conditions . keywords informed consent dematerialisation online survey paperless * lorenzo faggioni lfaggioni@sirm.org 1 department ofexperimental , diagnostic andspecialty medicine , s . 
orsola malpighi university hospital , bologna , italy 2 diagnostic andinterventional radiology , university hospital ofpisa , via paradisa 2 , 56100pisa , italy 3 department ofradiology , university ofcampania l . 
vanvitelli , naples , italy 4 snr foundation , rome , italy 5 uoc radiodiagnostica , aou policlinico vittorio emanuele , catania , italy 6 department ofimaging , asl 3 genovese villa scassi hospital , genoa , italy 7 department ofradiology , usl n . 
bosco hospital , turin , italy 9 department ofinterventional anddiagnostic radiology , arcispedale santanna , ferrara , italy 10 radiology department , universit politecnica delle marche , ancona , italy 11 department ofradiology , candiolo cancer institute , fpo - irccs , candiolo , turin , italy vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 846853 introduction dematerialisation is a complex process involving the development of a digital document flow with full legal value aimed to support and , in perspective , replace conventional document production and archiving for public or private activities [ 112 ]  . 
the advantages of digitisation over paperand film - based workflow in radiology have been widely demonstrated [ 1317 ] , and attempts have since been made to extend the process to administrative key areas of healthcare data management , including , on a national level , the introduction of the digital medical recipe in most italian regions [ 312 ]  . 
however , to our knowledge , no systematic initiatives have been taken so far towards dematerialisation of patients informed consent ( pic ) for diagnostic and interventional radiology procedures , despite incentives from international [ 1 , 2 ] and national government agencies and healthcare institutions to promote digitisation in the public administration as well as in the healthcare system [ 312 ]  . 
such delay is due to the need for dematerialised pic to be formally equivalent to its conventional , paper - based version , implying for it to fully conform to current laws and privacy regulations , ensure proper patients understanding of medical procedures for which written informed consent is required , and provide seamless integration with existing ris / pacs infrastructure at radiological centres [ 18 , 19 ]  . 
while the development of a fully functional programme for pic dematerialisation may pose significant legal and technical challenges , evidence exists in the literature that pic dematerialisation with the aid of portable devices is feasible and as effective or superior to conventional methods in several aspects [ 2024 ]  . the italian society of medical radiology ( sirm ) has promoted the development of guidelines for pic dematerialisation and has put the imaging informatics chapter and informed consent commission of sirm in charge for its implementation . 
in this context , we sought to gain insight about the current modalities of pic acquisition for radiological procedures in italy and the opinion of italian radiologists about pic dematerialisation via an online survey . materials andmethods the online survey was launched as part of an initiative by the imaging informatics chapter of sirm aimed to promote dematerialisation of pic for radiological procedures ( both diagnostic and interventional ) , in cooperation with a number of it firms . 
the survey was devised following suggestions from a multidisciplinary expert panel of sirm and consisted of 11 questions , of which 9 were single choice and 2 multiple choice ( appendix )  . 
a free text field was left at the bottom of each question for additional comments . based upon a similar approach to two previous sirm surveys on teleradiology and radiological structured reporting [ 25 , 26 ] , every single sirm member received a personal email invitation to join the survey via a direct web link from the president of the sirm imaging informatics working group . 
b multiple answers allowed medical directors [ 23.7% ( 423 / 1780 ) ] or consultants [ 16.9% ( 301 / 1780 ) ] , respectively ( question #4 )  . the majority of responders deemed their it skills good [ 67.9% ( 1207 / 1778 ) ] or excellent [ 12.6% ( 224 / 1778 ) ] , whereas a minority declared to have barely sufficient [ 18.7% ( 332 / 1778 ) ] or poor [ 0.8% ( 15 / 1778 ) ] it skills , respectively ( question #5 )  . pic forms were mostly developed locally at each survey participants institution [ 77.0% ( 1311 / 1702 ) ] or , less frequently , by sirm [ 38.5% ( 655 / 1702 ) ] or the health department of each region [ 10.5% ( 180 / 1702 ) ] ( question #6 )  . pic could be revoked by the patient anytime before the examination according to 62.4% ( 1105 / 1772 ) of survey respondents ( question #7 ) , and pic forms were mainly stored in the hospital archive [ 82.7% ( 1428 / 1726 ) ] or , much less frequently , outside of the hospital [ 6.9% ( 119 / 1726 ) ] or were digitally archived into the ris [ 6.8% ( 117 / 1726 ) ] ( question #8 )  . the main advantages of dematerialised pic over conventional , paper - based pic as perceived by the survey respondents ( question #9 ) were its easier and faster recovery [ 96.5% ( 1669 / 1729 ) ] , safer storage and conservation [ 94.5% ( 1643 / 1738 ) ] , reduced cost due to the workflow going paperless [ 90.7% ( 1573 / 1735 ) ] , higher degree of protection in case of medico - legal litigation [ 83.8% ( 1439 / 1717 ) ] , and simplified patient identification and registration [ 74.3% 1 3 la radiologia medica ( 2019 ) 124 : 846853 ( 1274 / 1714 ) ] ( table2 )  . 
this latter circumstance may be due to different factors , including the current lack or inadequacy of technical infrastructures for digital pic collection ( including tablet devices running ad hoc software and connected to dedicated hospital wireless networks ) and / or of integration with existing ris / pacs environments , in spite of substantial investments into dematerialisation by government agencies . 
besides , some features of conventional , paper - based pic acquisition have emerged from the survey that should be incorporated in a dematerialised , digital pic version , including the possibility for patients to revoke their consent anytime before radiological procedures ( question #7 )  . 
in other terms , such basic aspects of conventional pic should be taken into account and preserved in the design and practical implementation of dematerialised pic solutions in order to maintain their legal validity , so that the transition from conventional to dematerialised pic should not substantially alter workflow . 
 furthermore , the current , more frequent usage of pic forms developed locally ( 77.0% of respondents to question #6 ) rather than by regional health departments ( 10.5% ) or , better , national scientific societies such as sirm ( 38.5% ) could be 1 3 la radiologia medica ( 2019 ) 124 : 846853 a potential , yet surmountable hurdle to standardisation and large - scale setup of dematerialised pic tools . 
these latter would actually benefit from the adoption of a single , predefined validated pic model for each radiological procedure , which could also be advantageous for data mining purposes and eventually in case of medico - legal issues [ 2731 ]  . overall , the majority of survey respondents were favourable to pic dematerialisation and acknowledged several advantages of digital over conventional pic , the main ones being easier and faster data retrieval , safer data storage , and reduced running costs . 
in this perspective , tablet - based methods for pic collection have shown to be preferred over conventional pic by both patients and medical staff and has proven more effective at ensuring adequate patients understanding of medical procedures and research trials [ 2024 , 32 , 33 ]  . 
interestingly , our finding of a rate of pic dematerialisation supporters consistently higher than 90% across all age classes of survey participants ( independent of and greater than their self - assessed it skills ) may suggest that radiologists are confident about the usability of digital pic and / or believe that its advantages would outweigh any potential difficulties related to its use as a routine working tool . on the other hand , the main potential drawbacks of pic dematerialisation as pointed out in the survey were related to technical difficulties in carrying out the process in real working environments . 
actually , tablet - based pic acquisition resulted to be more time consuming than paper - based pic collection [ 21 , 24 ] , and earlier work by haller etal . 
 [ 34 ] cautioned against the use of palmtops for electronic data collection in clinical research due to a significant increase in data entry time and risk of typing errors and missing data in comparison with paper - based questionnaires . 
 [ 21 ] found a positive correlation between the duration ofelectronic ipad - based briefings and patient age , paralleled by a negative correlation between patient age and computer skills , but nonetheless the majority of patients would prefer ipad briefings to conventional written informed consent forms in the future . 
even more specifically , it has been suggested that the higher degree of understanding and overall satisfaction provided by tabletbased pic may improve adhesion to clinical trials [ 23 ]  . a limitation of our study is the relatively small number of sirm members joining the survey ( 1791 / 10304 , corresponding to 17.4% of all active sirm members in full standing for the year 2016 ) , which could restrict the validity of our findings to a minority of all members . 
however , the participation rate to our survey was slightly higher than two recent online surveys involving sirm members on teleradiology [ 16.5% ( 1599 / 9662 ) ] [ 25 ] and radiological structured reporting [ 12.1% ( 1159 / 9560 ) ] [ 26 ] , and much higher than a similar european online survey on teleradiology ( 368 radiology professionals from 35 european countries ) [ 35 ] , confirming that pic dematerialisation has gained significant interest within the radiological community . conclusions our findings show that the majority of radiologist members of sirm involved in the survey were favourable to pic dematerialisation . 
though most of them were aware of the potential long - term advantages of pic dematerialisation over conventional , paper - based pic ( mainly related to greater ease , speed , and safety of data collection , storage and retrieval ) , concerns emerged about potential technical and organisational hurdles that its practical implementation may pose , including the need for extra space and time for digital pic collection ( which would add to regular radiologists workload ) , authorisation to use patients advanced digital signature , and to cater for patients who wish to remain anonymous . 
considering the overall issues involving your professional activity , do you believe this process would be needed ? la radiologia medica ( 2019 ) 124 : 854859 radiotherapy extrapleural pneumonectomy intheera ofimageguided intensitymodulated radiotherapy marcotrovo1 davidefranceschini2 carlofurlan3 francescapietrobon3 stefanovagge4 eleonorafarina5 albertorevelant5 lucavisani6 virginiamaragna6 giuseppeparisi1 vieriscotti6 received : 18 july 2018 / accepted : 14 march 2019 / published online : 8 april 2019 italian society of medical radiology 2019 abstract purpose to assess the outcome of malignant pleural mesothelioma patients treated with extra - pleural pneumonectomy ( epp ) and adjuvant radiotherapy ( rt ) , using the most advanced radiotherapeutic techniques , namely image - guided intensitymodulated rt ( ig - imrt )  . methods and materials fifty - four patients were analyzed . 
the study endpoints included loco - regional control ( lrc ) , distant metastases free survival ( dmfs ) , and overall survival ( os ) , as well as radiation - related toxicity . results major patients and treatment characteristics were the following : median age 62years , epithelioid histology in 51 ( 94% ) cases , locally advanced disease in 41 ( 90% ) cases , and metastatic mediastinal lymph nodes in 27 patients ( 50% )  . 
the predominant pattern of failure was distant : 34 patients ( 62.9% ) developed some component of distant failure , and only 5 patients ( 9.2% ) developed an isolated loco - regional recurrence . 
other major toxicities included : grade 2 and 3 pneumonitis in 1 and 2 cases , respectively , 1 case of bronchial fistula , pleural empyema , and grade 3 esophagitis , respectively . conclusions although executed in the era of high - technology radiotherapy ( ig - imrt ) , epp should not be routinely performed . keywords malignant pleural mesothelioma image - guided imrt extra - pleural pneumonectomy introduction malignant pleural mesothelioma ( mpm ) is a rare cancer , strictly correlated with asbestos exposure . 
its prognosis is usually poor , with a median survival of 68months in untreated patients [ 1 ]  . extra - pleural pneumonectomy ( epp ) represents an aggressive surgical approach that has been shown to increase survival as part of a trimodal therapeutic strategy including * giuseppe parisi giuseppe.parisi@asuiud.sanita.fvg.it extended author information available on the last page of the article adjuvant chemotherapy and radiotherapy . 
 reported a 5 - year survival of 46% in selected patients , including who had an epithelial histology , with negative resection margins and without metastatic extra - pleural nodes [ 2 ]  . 
due to these results , in recent years such surgical approach fell out of favor among the scientific community , mainly due to the severe perioperative stress , the noticeable complication rate , and the long - term detrimental anatomical and functional effects [ 3 ]  . 
a less invasive approach , as lungsparing surgery , found place particularly after the publication of the mars trial , a feasibility study in which patients were randomized to receive epp or not [ 4 ]  . 
in a meta - analysis comparing epp with lung - sparing surgery , it was documented that pleurectomy / decortication might be performed vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 854859 with lower morbidity and mortality than epp while resulting in comparable long - term survival [ 5 ]  . 
however , the comparison of both procedures has several limitations and the choice for a specific therapy is still highly individual based on the extension of the disease , the patient comorbidities , and the center experience . also , the role of adjuvant radiotherapy ( rt ) was questioned after the recent publication of the sakk trial , a randomized trial that failed to show a clinical advantage of adjuvant rt following epp [ 6 ]  . 
it must be underlined that in this study different fractionation schemes and rt techniques , including 3d conformal rt , were adopted . considering the uncertainties of the published literature , we conducted the present study to assess the outcome of mpm patients treated with epp and adjuvant rt , using the most advanced radiotherapeutic techniques , namely imageguided intensity - modulated radiation therapy ( ig - imrt ) , in major academic centers in our country . loco - regional ( lrr ) and distant relapses were assessed using chestabdomen ct , which was performed every 46months ; fdg - pet / ct was added at the workflow in case of concerns of disease progression . 
lrr was defined as any clinical or radiographic recurrence in the chest wall ( local ) or in the regional lymph nodes ( hilar , mediastinal , or internal mammary lymph nodes )  . the study endpoints , including loco - regional control ( lrc ) , distant metastases free survival ( dmfs ) , and overall survival ( os ) , were estimated using the kaplanmeier method , starting from the date of completion of rt until death or the last available follow . 
the hazard ratios for potential risk factors included age , sex , performance materials andmethods table 1 patient , tumor , and treatment characteristics this is a retrospective studies including patients data collected in prospective databases in four academic centers in italy . 
each patient signed an informed consent form that was approved by each institutional review board before entry into the registry . in the study period , 66 patients were treated with epp and adjuvant ig - imrt for mpm . 
ten patients were excluded from the study : 7 were lost at follow - up immediately after the completion of rt ( were followed in other facilities ) , and 5 were treated with palliative radiation doses . 
planning target volume ( ptv ) was generated adding an isotropic margin of 0.5cm to the clinical target volume ( ctv ) , which encompassed the entire hemithorax , including the ipsilateral mediastinum if interested by disease , the pericardium and diaphragm , as well as the thoracotomy scars and any drain sites , according to the technique proposed by the researchers from the md anderson cancer center [ 7 ]  . 
imrt was delivered with tomotherapy in 17 patients [ 8 ] , using a volumetric imrt technique in 27 patients [ 9 ] , and static 7 - field sliding window imrt in 10 patients [ 10 ]  . 
cone beam ct or megavoltage ct scans were performed daily for each patient , in order to properly guide the radiation treatment . patients were followed at regular intervals to determine tumor status and presence of symptoms . 
1 kaplan - mayer estimates of overall survival of patients treated with extrapleural pneumonectomy and imageguided intensity modulated radiation therapy table 2 pattern of failure failure pattern local local only local and nodal local and distant local , nodal , and distant nodal nodal only nodal and distant distant distant only month n ( % ) 13 ( 24.0% ) 4 ( 7.4% ) 1 ( 1.8% ) 5 ( 9.2% ) 3 ( 5.5% ) 9 ( 16.6% ) 5 ( 9.2% ) 34 ( 62.9% ) 21 ( 38.8% ) chemotherapy , 4 patients ( 7% ) received adjuvant chemotherapy , and 3 patients ( 5% ) received both neoadjuvant and adjuvant chemotherapy . 
the median number of chemotherapy cycles , consisting of cisplatin / carboplatin and pemetrexed , was 4 ( range 37 cycles )  . all patients underwent postoperative imrt , with a total radiation dose ranging between 50 and 60gy in 2530 fractions . 
the predominant pattern of failure was distant : 34 patients ( 62.9% ) developed some component of distant failure , and only 5 patients ( 9.2% ) developed an isolated loco - regional recurrence . 
we did not find any risk factor correlating with overall survival at the univariate analysis . three fatal ( grade 5 ) pneumonitis were documented within 6months from the completion of radiotherapy . 
other major status , histologic subtype , stage of disease , gross residual disease after surgery , and chemotherapy administration . results patients , tumor , and treatment characteristics are shown in table1 . 
2 kaplan - mayer estimates of loco - regional control ( a ) and distant metastasis - free survival ( b ) of patients treated with extrapleural pneumonectomy and imageguided intensity modulated radiation therapy months months toxicities included : grade 2 and 3 pneumonitis in 1 and 2 cases , respectively , 1 case of bronchial fistula , pleural empyema , and grade 3 esophagitis , respectively . 
nine patients reported severe fatigue . discussion in the present paper , we wished to report the clinical outcome of mpm patients treated with epp , followed by the 1 3 858 la radiologia medica ( 2019 ) 124 : 854859 table 3 trimodality with extra - pleural pneumonectomy and intensity - modulated radiation therapy for malignant pleural mesothelioma author year no . 
of patients who completed trimodality therapy key results : median overall survival ( months ) key results : 2 - year overall survival ( % ) krug [ 13 ] patel [ 14 ] gomez [ 15 ] thieke [ 16 ] present study 2009 2011 2013 2015 2018 predominant pattern of failure distant ( 65% ) distant ( 60% ) distant ( 75% ) not reported distant ( 63% ) most advanced radiotherapeutic treatment , namely imageguided imrt , with the hypothesis that the adoption of such technology could lead to better clinical results . 
these results are particularly unsatisfying if considering that the patient population was selected positively : only patients that successfully underwent epp were included in the analysis . it has been reported that almost 50% of patients who are suitable candidates for trimodality therapy actually complete the proposed treatment . 
authors from the european institute of oncology reported that of the 83 patients candidate to chemotherapy , epp , and rt , only 37 ( 45% ) completed the planned trimodality treatment [ 11 ]  . 
reported that only 30 out of 60 patients completed high - dose hemithoracic rt after epp ; the median os for all patients intended to undergo trimodality therapy was 14months [ 12 ]  . 
similarly , a multicentric study conducted in usa on neoadjuvant chemotherapy followed by epp and radiation reported that only 57% of the enrolled patients started radiation therapy [ 13 ]  . 
the author documented a median os of 17months and 29months in the intention to treat population and in the population that completed hemithoracic radiotherapy , respectively . our results are comparable with those published in the literature by the researchers from major academic centers in north america . 
the principal findings of these studies are summarized in table3 . in our study , only 5 ( 9.2% ) patients experienced an isolated loco - regional failure , and predominant pattern of failure was distant : 62.9% of patients failed distantly . 
 these data suggest that the high control rate obtained with extensive surgery and high - dose ig - imrt did not translate into a survival advantage . according to this , it has been showed that less aggressive therapeutic approaches , such as lung - sparing surgery followed by hemithoracic rt , might be suitable to manage a systemic disease such as mpm while guaranteeing an adequate rate of loco - regional control [ 17 , 18 ]  . the main limit of our study is a possible patient selection bias , as previously discussed . 
it is implicit that surgery - related toxicities and deaths were not considered in the present analysis , and patients who progressed immediately after surgery or chemotherapy were not enrolled in the study . 
on the other hand , these features highlight the poor results obtained in our study . taken together , our findings , including survival and pattern of failure , imply that epp , although executed in the era of high - technology radiotherapy ( ig - imrt ) , should not be routinely performed . 
probably , lung - sparing surgery represents a valid therapeutic option for mpm patients . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical standards all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 declaration of helsinki and its later amendments or comparable ethical standards . 
 intravascular attenuation ( iva ) , signal - to - noise ratio ( snr ) , contrast - to - noise ratio ( cnr ) and image noise were compared . 
computed tomography angiography ( cta ) is less invasive , safe and with diagnostic performance comparable to dsa and is often used as * marco fogante marco.fogante89@gmail.com 1 department ofradiology , azienda ospedalierouniversitaria , ospedali riuniti di ancona , via conca 71 , 60126torrette , ancona , italy 2 department ofradiology - school ofradiology , universit politecnica delle marche , ancona , italy preoperative examination in pad [ 7 , 8 ]  . 
but , with this technique there is the risk to overrun contrast bolus , especially with vascular pathologies , such as stenosis and aneurysmal dilatations , which delay the arrival of contrast vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 184190 medium at the peripheral arteries with inadequate filling of small distal arteries at acquisition time [ 1518 ]  . modulation , care dose 4d ( siemens , medical solution ) ; and matrix , 350 350mm . dual - source ct ( dsct ) , with two x - ray tubes placed at 95 to each other , has allowed , compared to single - source ct ( ssct ) , greater spatial and temporal resolution , by the use of flash imaging characterized by high pitch and halving rotation time [ 1921 ]  . our hypothesis is to experiment a new protocol , with dsct flash caudo - cranial acquisition , rather than craniocaudal , to reduce radiation dose and obtain a better contrastographic peripheral arteries attenuation . 
the aim of this study is to compare radiation dose and image quality of lower extremity cta between ssct cranio - caudal acquisition and dsct flash caudo - cranial acquisition . materials andmethods patients selection this prospective study received institutional review board approval . 
exclusion criteria were : allergy to iodinated contrast medium , renal insufficiency ( estimated glomerular filtration rate < 60ml / min / 1.73m2 , according to european society of urogenital radiology ) and history of stents or bypass in lower limbs arteries . 
based on a random - number table , all patients were assigned into a control group , group a ( n = 30 ) , submitted to protocol 1 ( p1 ) , with ssct cranio - caudal acquisition and an experimental group , group b ( n = 30 ) , subjected to protocol ( p2 ) , with dsct flash caudo - cranial acquisition . ct scan protocol all cta examinations were performed using 192 2 - sections dsct scanner ( somatom force , siemens medical solutions , forchheim , germany ) with patients in supine position and feet toward the gantry . 
a 20 - gauge cannula was inserted into superficial vein of the right antecubital fossa connected to a two - way injector with 1.0ml / kg infusion of contrast medium ( iopamidol , 370mgi / ml ) at flow rate of 4.0ml / s , followed by 60ml of saline at the same flow rate . in both protocols , was obtained anteriorposterior scout from the xyphoid process to the feet . 
the acquisition parameters were : tube voltage , 80kvp ; automatic tube current control group a underwent p1 with ssct cranio - caudal acquisition , bolus tracking technique and monitoring region positioned at the abdominal aorta bifurcation level . 
the different acquisition parameters used in two protocols are summarized in table1 . image analysis the ct images were reconstructed with slice thickness of 1mm and interval thickness of 0.7mm with advanced modeled iterative reconstruction ( admire , siemens healthcare ) , strength 3 and body vascular 36 ( bv36 ) algorith all datasets were transferred to a dedicated workstation ( syngo.via , siemens medical solution , forchheim , germany )  . 
maximum intensity projection ( mip ) , curved planar reformation ( cpr ) and volume rendering ( vr ) images were obtained for each patient . images quality evaluation to evaluate objective image quality , all measurements were taken by a radiologist with 10years of cta experience . 
 intravascular attenuation ( iva ) was measured , by positioning a region of interest ( roi ) in axial images in five locations : aortic artery bifurcation , common iliac arteries , common femoral arteries , middle third of superficial femoral artery and middle third of popliteal artery . 
before image evaluation , the observers were instructed to divide the vascular structures into three segments ( aortoiliac , femoropopliteal and the below the knee ) and then score the image quality in these segments using a 4 - point scale according to the degree of noise artefacts , the presence of streak artefacts and graininess of the images . 
 the scoring criteria were as follows [ 22 ] : poor ( grade 1 ) , graininess or streak artefacts were significant and did not provide sufficient information for the diagnosis ; adequate ( grade 2 ) , the examination provided acceptable information but unsatisfactory image quality ; good ( grade 3 ) , image quality was satisfactory enough to provide the information necessary to make an adequate radiological diagnosis ; excellent ( grade 4 ) , image quality provided optimal information for a radiological diagnosis . 
in the event of observer disagreement , another reading session was convoked to reach an agreement . radiation dose estimation ctdivol and dlp were used to estimate radiation dose of each patient and were recorded in the dose report . statistical analysis statistical analysis was performed using version 19 of the spss software . 
 the analysis of variance was used to compare mean value of iva , snr , cnr and in . kappa analysis was used to assess the inter - observer agreement of the subjective image quality rating . 
a k value less than 0.20 indicates poor agreement ; 0.210.40 , fair agreement ; 0.410.60 , moderate agreement ; 0.610.80 , good agreement ; and 0.811.00 , very good agreement . 
there were no statistically significant differences in age , gender , weight and height between the two groups ( table2 )  . image quality table3 summarizes mean values of iva , snr , cnr and in of two protocols . 
the use of low - voltage , automated modulation systems of mas and iterative reconstruction techniques achieves , with the same radiation dose , better iva , snr and cnr [ 1114 ]  . 
 to obtain an appropriate acquisition time of angiographic phase , bolus tracking is the technique that guarantees , in most cases , better results [ 1518 , 28 , 29 ]  . 
dsct with flash protocol has allowed to improve spatial and temporal resolution , compared to ssct , using high pitch and half rotation time [ 19 , 20 ]  . 
our hypothesis is to experiment , with dsct a flash caudo - cranial acquisition , rather than craniocaudal , to reduce radiation dose and obtain a better contrastographic peripheral arteries attenuation . 
possible explanation is that the use of high pitch and high rapid scan provides less radiant exposure . mean values of iva , snr and cnr were higher in p2 than in p1 with better image quality for small arteries below the knee . 
possible explanation of these results is that the caudo - cranial acquisitions were started when the radiologist saw contrast medium in the popliteal fossa , avoiding the risk of overrunning contrast bolus , especially with vascular pathologies , such as stenosis and aneurysmal dilatations , which delay the arrival of contrast medium with little filling of the small distal arteries at the time of scanning . 
moreover , the rapid acquisition with high pitch assured , even if starting from the ankles , an excellent opacification up to cranial portions of the abdominal aorta . to date , this is the first study in scientific literature to experiment a flash acquisition protocol with caudocranial scan . 
in this work , group a was submitted to p1 with 70kvp , cranio - caudal acquisition in ssct and were obtained mean iva , snr , cnr and in values comparable to those of qi etal . 
for an accurate evaluation of the effective radiation dose , direct organ measurements should be taken . in conclusion , with dsct and flash caudo - cranial acquisition we obtained a dose reduction administered to the 1 3 la radiologia medica ( 2019 ) 124 : 184190 patient of 40.9% in ctdivol and of 42.8% in dlp with better mean values of iva , snr and cnr compared to a ssct with 70 kvp and cranio - caudal acquisition protocol . 
this new protocol may contribute to reduce radiation dose and to obtain better image quality with correct acquisition time of angiographic phase in the lower extremity cta examination . compliance with ethical standards conflict of interest the authors declared no potential conflicts of interests associated with this study . ethical standards all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 declaration of helsinki and its later amendments or comparable ethical standards . 
ceus might be used instead of contrast - enhanced computed tomography ( cect ) during spleen injury follow - up in order to reduce radiation exposure . objective assess diagnostic comparability between ceus and cect in the evaluation of dsvi and dae during spleen injury follow - up . subjects and methods a total of 139 trauma patients ( 101 males , 38 females ; mean age 48.6years ) with cect diagnosed spleen injury were prospectively evaluated . 
twelve patients performed digital subtraction angiography ( dsa ) during follow - up , and the diagnostic performance comparability between ceus and dsa was evaluated . results ceus showed 17 delayed spleen injury complications , and in 122 patients no complication was suspected . 
compared to dsa , ceus showed a sensitivity of 100% and a positive predictive value of 91.7%. conclusions ceus can be used during spleen injury follow - up instead of cect . 
positive ceus examinations could perform cect and , when necessary , dsa in order to confirm and treat spleen injury complications . keywords contrast - enhanced ultrasound ( ceus ) delayed splenic vascular injury delayed active extravasation blunt splenic trauma non - operative management introduction the spleen is the second most frequent organ involved in blunt abdominal trauma ( 13% ) after the liver ( 16% ) [ 1 ]  . 
 delayed splenic vascular injury ( dsvi ) and delayed active extravasation ( dae ) are reported to occur , respectively , in * corrado tagliati corrado.tagliati@gmail.com 1 school ofradiology , universit politecnica delle marche , ancona , italy 2 sod clinica di radiologia , durgenza e dellarea oncologica , azienda ospedaliero universitaria ospedali riuniti , ancona , italy 3 department ofradiological sciences , azienda ospedaliero universitaria ospedali riuniti , universit politecnica delle marche , ancona , italy 323% and 1.5% of blunt splenic injuries ( bsi ) [ 25 ]  . 
nonoperative management ( nom ) of blunt splenic trauma is the treatment of choice for hemodynamically stable patients in an optimal setting in which monitoring and frequent clinical evaluations are possible , and an operating room is available for urgent laparotomy . 
nom allows the preservation of spleen role in immune system , reducing the risk of future infections , the most serious of which are part of the overwhelming post - splenectomy infection ( opsi ) syndrome . 
 delayed splenic rupture is a life - threatening injury complication , and it is thought that it could be related to the rupture of a pseudoaneurysm ( psa ) , clot disruption or expansion of vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 170175 a subcapsular hematoma [ 4 , 8 , 9 ]  . 
there are different classifications of spleen trauma injury grade ; the american association for the surgery of trauma ( aast ) spleen injury scale ( sis ) is the most used [ 10 ] ( table1 )  . 
according to the western trauma association ( wta ) algorithm , in all patients with aast - sis grade ii or higher - grade injuries a repeat imaging may be considered before hospital discharge to rule out psa formation [ 11 ]  . conventional ultrasound ( us ) , contrast - enhanced ultrasound ( ceus ) and ct are used to assess patients with blunt abdominal trauma . 
conventional us is generally used in the early assessment of polytrauma patients in order to assess intra - abdominal free fluid , a technique known as focused assessment with sonography for trauma ( fast )  . 
in adult patients , ct is the most used imaging modality in spleen injury follow - up , but ceus is a promising alternative imaging modality in this field [ 13 ]  . 
according to efsumb guidelines , ceus can be used in the follow - up of conservatively managed abdominal trauma to reduce the number of ct examinations , particularly in children [ 14 ]  . 
according to the world society of emergency surgery ( wses ) guidelines , ct scan repetition during the admission should be considered in adult patients with an initial diagnosis of moderate lesions ( hemodynamically stable aast - sis grade iii - v lesions ) as well as when there is lowering hematocrit levels , vascular anomalies , underlying splenic pathology or coagulopathy , and in neurologically impaired patients . 
according to the same guidelines , ceus follow - up seems reasonable to minimize the risk of life - threatening hemorrhage and associated complications in children [ 15 ]  . 
dsvi are splenic - contained table 1 aast spleen injury scale ( 1994 revision ) [ 10 ] grade injury type description of injury vascular injuries , and consist of pseudoaneurysm ( psa ) and post - traumatic arteriovenous fistula ( avf ) : these two entities are usually indistinguishable by means of ceus or ct [ 1921 ]  . 
another study demonstrated a high sensitivity ( 83% ) and specificity ( 92% ) of ceus at detection of post - traumatic liver and splenic psa in the pediatric population [ 23 ]  . to our knowledge , no previous study evaluated ceus diagnostic performance in diagnosing dsvi and dae in adult patients with serial ceus examinations during follow - up . the study aim was to assess diagnostic comparability between ceus and cect in the evaluation of dsvi and dae during spleen injury follow - up . subjects andmethods patient population during the 2years period 20162017 , 824 hemodynamically stable trauma patients without diffuse peritonitis underwent contrast - enhanced ct ( cect ) in our emergency department . 
the study population consisted , therefore , of 139 adult patients ( 101 males ; 38 females ; m : f 2.7 : 1 ; mean age 48.6years , range 1886 ) who underwent ceus and cect follow - up between january 2016 and december 2017 . hematoma laceration hematoma laceration hematoma laceration laceration laceration vascular subcapsular , < 10% surface area capsular tear , < 1cm parenchymal depth subcapsular , 1050% surface area intraparenchymal , < 5cm in diameter capsular tear , 13cm parenchymal depth that does not involve a trabecular vessel subcapsular , > 50% surface area or expanding ; ruptured subcapsular or parenchymal hematoma ; intraparenchymal hematoma > 5cm or expanding > 3cm parenchymal depth involving trabecular vessels laceration involving segmental or hilar vessels producing major devascularization ( > 25% of spleen ) completely shattered spleen hilar vascular injury with devascularized spleen 1 3 172 la radiologia medica ( 2019 ) 124 : 170175 the study was approved by the institutional review board and ethics committee of our institution . 
informed consent was obtained from all individual participants included in the study . statistical analysis ceus technique ceus follow - up consisted of one - to - eight ( mean 4.7 ) serial examinations at 13715306090180days ( 1day ) after trauma until spleen injury became no more visible . 
ceus were performed with continuous image acquisition , starting immediately after contrast injection and lasting 5min . ct technique all the 139 patients underwent a cect examination between 3 and 7days after trauma . 
cect examination was performed using a 16 - slice ct ( philips brilliance ) , with arterious , venous and delayed post - contrast images after intravenous injection of 90120ml iopamidol 370mg i / ml ( iopamiro 370 , bracco , milan , italy ) with an electronic power injector ( stellant ; medrad ) at a flow rate of 3ml / s . image assessment two radiologists , with more than 10years experience in ceus and cect abdominal imaging , evaluated in consensus ceus and cect images in order to detect a dsvi or a dae . 
 dsvi was diagnosed by ct when a hyperdense round or oval area of contrast enhancement with distinct margins in arterial phase did not increase in size in portal venous phase . 
 in the thirteen patients eligible for interventional procedures , dsa images , considered as the standard of reference , were reviewed by an experienced interventional radiologist ( with 10years of experience in interventional radiology ) , who was unaware of the ceus and cect findings . 
dsa diagnosis was compared with that made using ceus to assess the two modalities comparability . in the 139 patients included in the study , ceus diagnosis was compared with cect one , and diagnostic performance comparability was assessed . 
cect diagnosed 16 delayed spleen injury complications in these 17 patients ( 12 dsvi and 4 dae ) ; cect diagnosed a small dsvi ( 2mm of maximum diameter ) in another patient . 
previous studies showed that ceus can detect active bleeding [ 16 , 22 ] or demonstrated the usefulness of performing one or two ceus during follow - up [ 24 , 25 ]  . 
some other studies reported the usefulness of performing a follow - up ct scan 48 or 72h after admission in patients with grade ii in order to diagnose a possible psa development [ 4 , 25 , 26 ]  . 1 3 la radiologia medica ( 2019 ) 124 : 170175 fig . 
3 63 - year - old man with active bleeding diagnosed by ceus ( a ) 3days after blunt abdominal trauma and confirmed by subsequent ct scan ( b ) in the present study , all delayed splenic injury complications were diagnosed in the first 7days after trauma , and none occurred in aast - sis grade i spleen injuries . 
this study demonstrate that ceus is a valuable imaging modality that could be performed during splenic injury followup ; in fact , splenic injury complications could be promptly diagnosed and embolized before a splenectomy would be necessary . 
this is particularly useful during the first 7days after trauma in patients with aast - sis grade ii , when complications are more probably to occur [ 9 ]  . 
4 50 - year - old man with a delayed splenic vascular injury showed by ceus ( a ) and ct scan ( b ) , and confirmed by subsequent dsa ( c ) la radiologia medica ( 2019 ) 124 : 170175 lower grade blunt splenic injuries in order to avoid a lot of unuseful ct scans [ 13 ]  . 
ceus allows to perform serial spleen injury evaluation at short time intervals , and our study results strongly support the use of ceus as a follow - up imaging modality of splenic injury . 
in fact ceus is safe , relatively cheap , and can be performed at the patients bedside in critical - ill patients , such as in intensive care unit ( icu ) ones . 
therefore , we could affirm that ceus could fill a gap in diagnostic imaging , and that it could represent the reference imaging modality in splenic injury follow - up . 
we do not have the haughtiness to think that the study cohort could be the representative of all the patient cohorts in other hospitals , but we think that the use of an imaging modality like ceus could be used at least in the first seven days after trauma in patients with aast - sis grade ii at pre - established intervals ( e.g. , 137days after trauma ) in order to diagnose dsvi and dae , and to reduce ct scans . this study has some limitations : it is a single - institute analysis , and the study population is small . 
however , to our knowledge , this is the first study which evaluated ceus efficacy in diagnosing dsvi and dae with respect to ct and dsa performing many serial ceus examinations . compliance with ethical standards conflict of interest the authors declared no potential conflicts of interests associated with this study . ethical standard all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 declaration of helsinki and its later amendments or comparable ethical standards . 
the morphometry of the pituitary stalk with 1.5t mr images was useful in diagnosing the infundibular lesion and to assess the efficacy of treatment given by chemotherapy for many pathological conditions . 
there are no data regarding the morphometry of the pituitary stalk in the adult population . methods one hundred and fifty normal brain mr images ( 75 males and 75 females ) were analyzed for the evaluation of stalk and other parameters . 
these normative data shall be of help in the evaluation of stalk in various neurosurgical and endocrinological pathologies and lead the radiologist and endocrinologist in the early diagnosis and management of hypothalamo - pituitary axis diseases . keywords fendersons funnel infundibulum morphometry mri pituitary stalk introduction pituitary stalk or infundibular stalk is an axonal tract of magnocellular neurons of supraoptic and paraventricular nuclei which connects the hypothalamic nuclei with * amirthalingam umamageswari dr_amirthauma@yahoo.co.in 1 department ofanatomy , jawaharlal institute ofpostgraduate medical education andresearch [ jipmer ] , dhanvantri nagar , pondicherry605006 , india 2 department ofradiodiagnosis , jawaharlal institute ofpostgraduate medical education andresearch [ jipmer ] , dhanvantri nagar , pondicherry605006 , india 3 department ofradiodiagnosis , sri manakulavinayagar medical college andhospital , kalitheerthalkuppam , puducherry605107 , india neurohypophysis . 
the normal morphometry of pituitary stalk and infundibular recess , in relation to adjacent floor of the third ventricle , helps to diagnose the pituitary infundibular lesion in an early stage which can help to avoid delay in diagnosis of pathological conditions like inflammation , infections , neoplasm , lymphocytic infundibuloneurohypophysitis , and neurosarcoidosis [ 37 ]  . 
high - resolution heavily t2 - weighted 3d sequence space ( sampling perfection with application optimized contrasts using different flip - angle evolution ) was used to acquire thin 1 - mm sections . 
true sagittal sections were confirmed by the presence of stalk , infundibular recess , cerebral aqueduct , and anteriorposterior commissural line in the same visualized midsagittal plane image . one hundred and fifty mr images ( 75 males and 75 females ) that were reported as normal by a neuroradiologist were included in the study . 
the ratio shows a gradual increase in the age - groups below 50years , and then , it starts decreasing in the elderly populations . 1 3 208 la radiologia medica ( 2019 ) 124 : 206210 fig . 
being a midline structure , the position and displacement of stalk give vital clues in the evaluation and differentiation of pituitary and hypothalamic lesions . in many diseases , stalk itself may be involved in the form of either stalk interruption as in congenital pituitary insufficiency , or enlargement in various inflammatory and neoplastic causes , or elongation in empty sella syndrome which has varied clinical presentations [ 8 ]  . 
there is a need for normative data in the evaluation of stalk to compare its normal morphologysize ( length ) , thickness ( diameter ) , and enhancementand position including stretch into the sellar compartment . mri is considered as the investigation of choice to diagnose the pituitary and infundibular lesions [ 9 ]  . 
there was a gradual increase in the length of the pituitary stalk from 18 to 60years ( table2 )  . the length and the position of the pituitary stalk can be influenced by the surrounding structure like tuberculum sellae , dorsum sellae , and diaphragm sellae . 
 the relation between the type of chiasm and the pituitary stalk was more often ( p < 0.05 ) 90 or greater for prefixed chiasmata and acute angles for normal or postfixed chiasmata . 
a prefixed chiasm will displace the pituitary stalk more anteriorly , which is prone to injury during anterior approaches to the midline skull base structures [ 12 ]  . the thickness of the stalk was considered larger in girls than boys in the adolescent age - groups in both mri and ct study [ 10 , 13 ]  . 
this correlates well with the present study . the thickness is important in diagnosing the hypothalamic lesions including glioma and neuroepithelial tumors like dermoid , teratoma , dysgerminoma , and hamartoma [ 13 ]  . 
isolated thickness can be seen in diabetes insipidus , but the presence of both diabetes insipidus and stalk thickening may be seen in germinomas [ 14 ]  . the stalk thickness is an important factor in follow - up of chemotherapy in case of diabetes and gh deficiency [ 15 ]  . 
the present study shows 5.24m there is a gradual increase in the depth from younger to elder individuals ( table2 )  . the recess may be elongated or enlarged with the apparent lengthening of the stalk in cases of empty sellathe extension of suprasellar csf space into the bony sellar compartment . 
in extreme cases , the suprasellar structure of optic chiasma itself may be dragged into the sella causing serious table 3 comparison of measurements of pituitary stalk with the previous study study study number of subjects diameter of the stalk ( mm ) length of the stalk ( mm ) depth of infundibular recess ( mm ) ratio of length of the stalk to the depth of the recess satogami etal . 
in the literature , the ratio between the pituitary stalk and the basilar artery was considered as a screening tool to evaluate the tumor involving the pituitary stalk [ 10 , 13 ]  . 
since the basilar artery localization is difficult in routine mri , this ratio can be considered as an alternative tool for the pituitary stalk - to - basilar artery ratio . 
an extensive study is needed to evaluate the sensitivity of the ratio in the clinical settings and its implications . conclusion the morphometry of the pituitary stalk is equally important as the pituitary gland . 
it can also be used in planning the appropriate management for the infundibular lesions based on the thickness and the modularity including metastatic disease in the follow - up neuroimaging . 
ceus spleen injury diagnostic sensitivity was 96.9% and , according to the american association for the surgery of trauma ( aast ) spleen injury scale ( sis ) , ceus - ct concordance was 95.8%. conclusions spleen injury healing time in blunt abdominal trauma nonoperatively managed is significantly related to aastsis grade , sch presence and grade , and spleen infarct development , and ceus can be used in order to evaluate spleen injury grade . keywords contrast - enhanced ultrasound ( ceus ) abdominal imaging spleen nonoperative management introduction the spleen is one of the most frequent splanchnic organs involved in blunt abdominal trauma , frequently reported as the second one after liver [ 1 , 2 ]  . 
nonoperative management ( nom ) is currently considered the best management option in stable patients in an optimal setting in which monitoring and frequent clinical evaluations are possible , and an * corrado tagliati corrado.tagliati@gmail.com 1 school ofradiology , universit politecnica delle marche , ancona , italy 2 sod clinica di radiologia , durgenza e dellarea oncologica , azienda ospedaliero universitaria ospedali riuniti , universit politecnica delle marche , ancona , italy 3 sod radiologia pediatrica e specialistica , azienda ospedaliero universitaria ospedali riuniti , universit politecnica delle marche , ancona , italy operating room is available for urgent laparotomy . 
nom allows the preservation of the important immunological spleen role , reducing the risk of future infections , the most serious of which are part of the overwhelming post - splenectomy infection ( opsi ) syndrome . 
moreover , avoidance of surgery - related complications , a shorter hospitalization period and a concomitant reduction in costs have been reported [ 3 ]  . some recent articles and guidelines define the timing of contact restriction and the conditions that require to repeat imaging after a splenic injury managed nonoperatively [ 46 ] , and a few previous studies assessed spleen injury healing time in adult patients nonoperatively managed [ 79 ]  . ct is considered the reference standard in order to detect and assess spleen injury grade , but a high ceus performance in these fields was reported in some previous studies and guidelines [ 1015 ]  . 
clinical follow - up lasted at least 6months ( range 612months )  . as serial ceus examinations were performed at preestablished intervals , spleen injury healing time was evaluated considering the number of ceus performed during follow - up . all patients underwent an abdomen - pelvis ct examination ( 16 slices ct philips brilliance ; philips medical systems , best , netherlands ) with iv administration of 90120 of iopamidol ( 370mg i / ml of iopamiro 370 ) using an electronic power injector ( ct injector ; medrad ) at a flow rate of 3ml / s . 
all patients with a ct - diagnosed spleen injury underwent at least one ceus study and no more than 8 ones during 6months period at pre - established intervals ( 1 , 3 , 8 , 15 , 30 , 60 , 90 and 180days after trauma ) , until spleen injury became no more identifiable . 
all scans were reported by a group of six radiologists with more than 10 - years experience in abdominal imaging ( ct , us and ceus ) and reviewed by two of theeach ct and ceus report stated the injury presence and its location and size . 
ct performed in the ed and the first ceus were evaluated in order to classify spleen injury grade using american association for the surgery of trauma ( aast ) spleen injury scale ( sis ) [ 17 ] ( table1 )  . 
the patients ( 5 out of 101 ) that developed a spleen infarct after splenic artery embolization ( ae ) were excluded from ceus sensitivity analysis and from the comparison of ct and ceus spleen injury grade evaluation , as the first ceus was done after ae and spleen infarct prevented a correct evaluation of injury grade . the study was approved by the institutional review board and the ethics committee of our institution . 
15.8 ( ostend , bel )  . table 1 association for the surgery of trauma ( aast ) spleen injury scale ( sis ) ( 1994 revision ) [ 10 ] grade injury type description of injury hematoma laceration hematoma laceration hematoma laceration laceration laceration vascular subcapsular , < 10% surface area capsular tear , < 1cm parenchymal depth subcapsular , 1050% surface area intraparenchymal , < 5cm in diameter capsular tear , 13cm parenchymal depth that does not involve a trabecular vessel subcapsular , > 50% surface area or expanding ; ruptured subcapsular or parenchymal hematoma ; intraparenchymal hematoma > 5cm or expanding > 3cm parenchymal depth involving trabecular vessels laceration involving segmental or hilar vessels producing major devascularization ( > 25% of spleen ) completely shattered spleen hilar vascular injury with devascularized spleen 1 3 la radiologia medica ( 2019 ) 124 : 163169 results table 3 mean ceus follow - up examinations and spleen injury healing time patients characteristics are summarized in table2 . mean ceus follow - up examinations and spleen injury healing time are reported in table 3 . 
the first follow - up ceus examination identified no - one spleen injury in 3 patients ( 3.1% ) , and all these patients were diagnosed as grade i by ct . 
 spleen injury healing time could be evaluated by ceus , and it is significantly related to aast - sis grade , sch presence and grade , and spleen infarct development . 
moreover , ceus is able to detect and correctly grade spleen injury severity . blunt abdominal trauma could cause hematoma or laceration that are showed by ct and ceus as hypodense or hypoechoic areas after contrast media injection . 
aast - sis , association for the surgery of traumaspleen injury scale ; sch , subcapsular hematoma ; ae , spleen artery angioembolization 1 3 166 la radiologia medica ( 2019 ) 124 : 163169 fig . 
1 78 - year - old man with aast - sis grade i showed by ct performed at the emergency department ( a ) and by ceus examination one day later ( b )  . 
two months later the lesion was no more detectable by ceus follow - up examination ( c ) spleen injuries progressively heal and , at least , become no more visible . western trauma association algorithm recommends contact restrictions for at least 4weeks for patients with aast - sis grade i injuries , 8weeks for patients with grade ii and grade iii injuries , and 12weeks for grade iv and grade v splenic injuries managed nonoperatively [ 4 ]  . 
the same algorithm reports that a routine post - discharge imaging study to confirm complete healing in asymptomatic patients has typically not been recommended except in patients with professions involving a high risk for contact [ 4 ]  . 
according to the world society of emergency surgery ( wses ) guidelines , activity restriction may be suggested for 46weeks in minor injuries ( aast - sis grade iii ) and up to 24months in moderate and severe injuries ( grade iiiv ) [ 5 ]  . 
in the outpatient setting , repeat imaging may also be helpful when a patient desires to return to a vigorous activity , such as contact sports [ 6 ]  . some previous studies assessed spleen injury healing time using us or ceus in pediatric population , and using us , ceus or ct in adult one [ 79 , 1820 ]  . 
in adult patients , ct is the most used imaging modality in spleen injury follow - up , but ceus is a promising alternative imaging modality in this field [ 21 ]  . 
to the best of our knowledge , only a previous study used ceus in order to evaluate spleen and liver injury healing time in adult patients [ 9 ]  . 
in particular , ceus was performed in the emergency room and repeated at 12 , 24 , 48h after the trauma ; later , ceus follow - up was performed at 30days and , in the event of persistent structural parenchymal alterations related to the trauma , again at 90days [ 9 ]  . 
therefore , our study is the first one that evaluated spleen injury healing time using ceus even in adult patients with aast - sis 1 3 la radiologia medica ( 2019 ) 124 : 163169 fig . 
no - one injury was showed by ceus examination in 3 patients ; therefore , they were considered false negative cases pts number of patients , aast - sis association for the surgery of traumaspleen injury scale grade iv and in which ceus examination was repeated up to 180days when necessary . 
our data confirmed previously reported direct correlation between aast - sis spleen trauma grade detected in the first ct carried out in the emergency department ( ed ) and the time required for radiographic healing [ 7 , 8 ]  . 
in the present study was found shorter healing time period than those previously reported by two studies in which spleen injury follow - up was performed in adult patients , respectively , with conventional us and ct [ 7 , 8 ]  . 
 previous studies assessed ceus performance and proved 1 3 168 la radiologia medica ( 2019 ) 124 : 163169 its accuracy in spleen injury detection and grade evaluation , even after angioembolization , and the present study is in line with them [ 10 , 16 , 22 ]  . although in recent years some publications have shown that there are some advantages in the use of the new grading system of splenic injury proposed by marmery etal . 
 [ 2326 ] particularly for predicting which patients with blunt splenic trauma need arteriography or splenic interventionaast - sis still continues to be the most widely used splenic injury grading system [ 3 , 2729 ] , and therefore , we have decided to use the latter . 
furthermore , using aast - sis in our study it was possible to compare spleen injury grade between ct examinations performed in the ed and ceus ones carried out the following day . 
this would not have been possible using the new classification system , as the patients who would have been classified as grades iva or ivb for ctdiagnosed active bleeding would have been classified as grades iiii by ceus examination , because the latter has always been performed after ae in these patients . this study could be useful in clinical practice as it allows to know spleen injury healing time evaluated by ceus in adult patients ; it also recognized some factors that are significantly related to healing time , such as aast - sis grade , sch presence and grade , and spleen infarct development . 
therefore , many factors related to spleen injury , and not only spleen injury grade , need to be taken into account in order to recommend activity limitations and contact restriction , particularly if follow - up us , ceus , or ct is not performed to demonstrate spleen injury healing . 
since ceus and ct scans were interpreted by two examiners consensually , inter - observer variability was not considered . compliance with ethical standards conflict of interest the authors declared no potential conflicts of interests associated with this study . ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . 
we assessed qualitative ( presence and extent of wall thickening , degree of contrast enhancement of the thickened wall on cect images , and signal intensity of the thickened wall on t2 - weighted images ) and quantitative ( maximum thickness of the thickened wall ) imaging findings . results eccentric and smooth wall thickening was observed in 11 / 11 ( 100% ) patients . 
the signal intensity of the thickened wall on t2 - weighted images was isointense relative to that of normal lymph nodes in 7 / 8 ( 88% ) and mildly hyperintense in 1 / 8 ( 12% ) patient . 
they demonstrate * hiroki kato hkato@gifu - u.ac.jp 1 department ofradiology , gifu university school ofmedicine , 1 - 1 yanagido , gifu501 - 1194 , japan 2 department ofotolaryngology , gifu university school ofmedicine , gifu , japan 3 department oftumor pathology , gifu university school ofmedicine , gifu , japan bimodal patient age peaks at < 5years ( accounting for 20% of all branchial cleft anomalies ) and 2040years ( accounting for 75% of all branchial cleft anomalies ) without sex predilection . sbccs usually present as solitary , painless , fluctuant , and slow - growing masses in the lateral neck . 
sbccs are usually located at the anteromedial border of the sternocleidomastoid muscle , lateral to the carotid space , behind the angle of the mandible , and generally posterior to the submandibular gland . 
although atypical imaging features may be observed when the lesions are accompanied with infection , hemorrhage , or carcinomatous transformation [ 6 ] , to the best of our knowledge , no previous study has reported on imaging findings focused on wall thickening of infection - free and benign sbccs . 
hence , this study was performed with the aim of assessing the ct and mr imaging findings of infection - free and benign sbccs with an emphasis on qualitative and quantitative characteristics of wall thickening . materials andmethods patients the study was approved by the human research committee of the institutional review board , and complied with the guidelines of the health insurance portability and accountability act . 
we searched electronic medical records of gifu university hospital for patients histopathologically diagnosed with benign sbccs treated by complete excision between april 2004 and may 2018 and identified consecutive 18 patients with benign sbccs . 
among them , 5 patients with infected sbccs and 2 patients who underwent only unenhanced ct as preoperative radiological examination were excluded . in total , 11 patients ( 6 males and 5 females ; age range 1459years ; mean age 40years ) met the inclusion criteria ; these patients underwent preoperative contrast - enhanced ct ( cect ) and / or mr imaging . 
ten patients presented with painless neck swelling , and the remaining one patient presented with neck swelling with mild papatients characteristics are summarized in table1 . ct imaging all 11 patients were examined using multidetector - row ct . 
ct imaging was performed using a 8 - slice ct scanner ( lightspeed ultra ; ge healthcare , milwaukee , wi , usa ) , a 16 - slice ct scanner ( lightspeed 16 ; ge healthcare , milwaukee , wi , usa ) , or a 64 - slice ct scanner ( brilliance table 1 patient characteristics number of patients age ( year ) mean range gender male female location right left clinical symptom painless neck swelling neck swelling with mild pain examination ct ( contrast - enhanced ct ) mri ( contrast - enhanced mri ) 1459 11 ( 7 ) 8 ( 5 ) ct 64 ; philips medical systems , best , the netherlands )  . 
 unenhanced ct images were obtained in all patients , and contrast - enhanced ct images were obtained in 7 patients 45s after initiating intravenous bolus injection of 100ml of nonionic iodine contrast material containing 240 or 300 - mg iodine per ml . 
all mr images were obtained using the parallel imaging technique at 4 - mm section thickness with 1 - mm intersection gap with axial field of view ( fov ) of 20cm or coronal fov of 26caxial non - fat - suppressed t2 - weighted fast spin - echo ( tr / te , 41024269 / 90 ) , axial non - fat - suppressed t1 - weighted spin - echo ( tr / te , 679827 / 915 ) , coronal fat - suppressed t2 - weighted fast spin - echo ( tr / te , 39835283 / 8090 ) , and coronal non - fatsuppressed t1 - weighted spin - echo ( tr / te , 400713 / 1014 ) images were obtained in all patients . 
axial and coronal gadolinium - enhanced fat - suppressed t1 - weighted spin - echo ( tr / te , 623841 / 912 ) images were obtained in 5 patients after the intravenous injection of 0.1mmol / kg of gadopentetate dimeglumine ( magnevist , bayer healthcare , leverkusen , germany ) or gadobutrol ( gadavist , bayer healthcare , leverkusen , germany )  . 1 3 la radiologia medica ( 2019 ) 124 : 199205 image assessment two radiologists with 19 and 5years of post - training experience of head and neck imaging , respectively , reviewed the ct and mr images , and any disagreements among the radiologists were resolved by consensus . first , sbccs were classified into the following four subtypes on the basis of the bailey classification : i , the most superficial subtype , which reaches as deep as the platysma surface and lies along the anterior surface of the sternomastoid muscle ; ii , the most common subtype , identified along the surface of the sternomastoid muscle and posterior to the submandibular gland ; iii , extends medially at the bifurcation of the internal and external carotid arteries to the lateral pharyngeal wall ; and iv , arises in the pharyngeal mucosal space [ 7 ]  . second , we assessed qualitative imaging findings ( presence and extent of wall thickening , degree of contrast enhancement of the thickened wall on cect images , signal intensity of the thickened wall on t2 - weighted images , and signal intensity of intracavitary fluid on t2and t1 - weighted images )  . 
the extent of wall thickening ( percentage of the thickened wall to the circumference of the wall ) was qualitatively graded using the following fourpoint scale : 1 , small ( 1%25% ) ; 2 , moderate ( 26%50% ) ; 3 , extensive ( 51%75% ) ; and 4 diffuse ( 76%100% )  . 
the signal intensity of the thickened wall on t2 - weighted images was compared with that of normal lymph nodes , and the signal intensity of the intracavitary fluid on t2and t1 - weighted images was compared with that of the spinal cord . third , we assessed quantitative imaging findings ( maximum and minimum diameter of sbcc , maximum thickness of the thickened wall , and ct value of the intracavitary fluid )  . 
the maximum thickness of the thickened wall was measured on cect or mr images , whereas ct value of the intracavitary fluid was measured on unenhanced ct images . results table2 summarizes the imaging findings of sbccs . 
the maximum diameter was always consistent with the superior - to - inferior axis . with regard to the cyst wall , eccentric and smooth wall thickening was observed in 11 / 11 ( 100% ) patients , with the absence of mural irregularity and mural nodules ( figs.1 , 2 , 3 )  . 
the signal intensity of the thickened wall on t2 - weighted mr images was isointense relative to that of normal lymph nodes in 7 / 8 ( 88% ) and mildly hyperintense in 1 / 8 ( 12% ) patients . 
the signal intensity of the intracavitary fluid on t2 - weighted images was homogeneously table 2 ct and mr imaging findings of infection - free and benign sbcc patient no . / age / sex bailey classification maximum diameter ( mm ) eccentric and smooth wall thickening extent ( percentage ) of thickened wall maximum thickness of thickened wall ( mm ) ce of thickened wall on si of thickened wall on t2wi 1 / 44 / m 2 / 48 / f 3 / 49 / m 4 / 40 / m 5 / 20 / f 6 / 32 / m 7 / 50 / f 8 / 39 / f 9 / 14 / f 10 / 48 / m 11 / 59 / m extensive ( 51%75% ) small ( 1%25% ) moderate ( 26%50% ) small ( 1%25% ) moderate ( 26%50% ) moderate ( 26%50% ) moderate ( 26%50% ) moderate ( 26%50% ) small ( 1%25% ) moderate ( 26%50% ) small ( 1%25% ) mild , homo mild , homo mild , homo mild , homo mild hyper mild , homo mild , homo mild , homo sbcc = second branchial cleft cyst , ce = contrast enhancement , homo = homogeneous , na = not available , si = signal t2wi = t2 - weighted image , iso = isointensity , hyper = hyperintensity intensity , si of thickened wall on t2wi is compared with normal lymph nodes 1 3 202 la radiologia medica ( 2019 ) 124 : 199205 fig . 
coronal gadoliniumenhanced fat - suppressed t1 - weighted image ( tr / te , 650 / 12 ms ) shows a unilocular cystic lesion ( arrow ) with mildly enhanced eccentric and smooth wall thickening ( arrow head ) fig . 
 b axial t2 - weighted image ( tr / te , 4102 / 90ms ) shows a unilocular cystic lesion ( arrow ) with isointense wall thickening relative to normal lymph nodes ( arrow head )  . 
axial gadolinium - enhanced fatsuppressed t1 - weighted image ( tr / te , 841 / 9ms ) shows a unilocular cystic lesion ( arrow ) with mildly enhanced eccentric and smooth wall thickening ( arrow head ) 1 3 la radiologia medica ( 2019 ) 124 : 199205 fig . 
 b axial t2 - weighted image ( tr / te , 4415 / 100ms ) shows a unilocular cystic lesion ( arrow ) with slightly hyperintense wall thickening relative to normal lymph nodes ( arrow heads )  . 
 axial gadolinium - enhanced fatsuppressed t1 - weighted image ( tr / te , 607 / 10ms ) shows a unilocular cystic lesion ( arrow ) with mildly enhanced eccentric and smooth wall thickening ( arrow heads )  . 
hematoxylin and eosin ( h&e ) stain shows focal proliferation of lymphoid tissue ( arrows ) adjacent to the epithelium within thin cyst wall low , isointense , and hyperintense relative to those of the spinal cord in 1 / 8 ( 13% ) , 1 / 8 ( 13% ) , and 6 / 8 ( 75% ) patients , respectively . 
the signal intensity of intracavitary fluid on t1 - weighted images was homogeneously low , isointense , and hyperintense relative to those of the spinal cord in 1 / 8 ( 13% ) , 3 / 8 ( 38% ) , and 4 / 8 ( 50% ) patients , respectively . discussion sbccs are unilocular and usually between 2 and 6cm in diameter . 
histopathologically , sbccs are most commonly lined by stratified squamous epithelium ( 90% ) and less commonly by columnar respiratory epithelium ( 8% ) , with occasional goblet cells and transitional areas of both epithelia . 
because one of the characteristics of pathologic findings of sbccs is a lymphoid tissue with reactive germinal centers and without true lymph node architecture beneath the epithelium within the cyst wall , sbccs are also referred to as lymphoepithelial cysts . 
 although branchiogenic carcinoma , a squamous cell carcinoma ( scc ) arising in a branchial cyst , has been reported previously , it is currently considered to be an exceptional diagnosis , with < 40 patients fulfilling the strict diagnostic criteria [ 8 ]  . typical ct findings of sbccs include well - circumscribed and homogeneously hypodense cystic masses surrounded by a uniformly thin , smooth wall [ 2 , 5 ]  . 
the mural thickness is attributed to the response of the lymphoid tissue , and the degree of mural thickness varies depending on the presence and severity of any associated inflammatory processes [ 2 , 5 ]  . in the present study , infection - free and benign sbccs were always accompanied by eccentric and smooth wall thickening , which did not exceed 4mm in total thickness . 
conversely , infection of sbccs leads to necrosis of the epithelial lining , with variable degrees of acute and chronic inflammation and 1 3 204 la radiologia medica ( 2019 ) 124 : 199205 granulation of the tissue . 
because circumferential wall thickening is expected in infected sbccs , eccentric wall thickening may be a characteristic imaging feature of infection - free and benign sbccs . although the intracavitary fluid of sbccs typically showed low - to - intermediate signal intensity on t1 - weighted images , the proteinaceous content of sbcc or its chronic infection can also cause hyperintensity on t1 - weighted images [ 2 , 4 ]  . 
 although hyperintensity has been observed on t2 - weighted images because of the presence of intracystic fluid [ 4 ] , hypointensity on t2 - weighted images has been observed , albeit rarely , because of solidification of the cystic fluid [ 6 ]  . differential diagnoses of sbccs include benign and malignant conditions with cystic lesions in the lateral neck . 
 among patients with clinically diagnosed lateral cervical cysts , postoperative histopathological examination revealed malignancies in 9.2%14.4% of the patients ; the most common malignancy was cystic nodal metastasis from scc , followed by that from papillary thyroid carcinoma ( ptc ) [ 911 ]  . 
conversely , differential diagnoses of benign cystic lesion in the lateral neck include lymphangioma , venous malformation , diving ranula , abscess , and tuberculous / suppurative lymphadenitis [ 4 ]  . 
cystic schwannoma should also be included as a differential diagnosis of sbccs [ 12 ]  . the differential diagnosis between sbccs and cystic nodal metastases from scc can be challenging . 
this phenomenon is thought to be caused by the eccentric and smooth wall thickening in sbccs . cystic changes within nodal metastases occur in 26%70% of patients with nodal metastases from ptcs [ 16 , 17 ]  . 
however , because characteristic findings of nodal metastases from ptcs include a location in the mid or lower jugular chain , internal nodules , internal septum [ 18 ] , and a relatively small size ( mean 12mm ) [ 16 ] , these imaging findings could be the key for differentiating ptcs from sbccs . 
as for the signal intensity of cystic components on t1 - weighted images , although hyperintensity compared with fat was observed in 27% of nodal metastases from ptcs [ 16 ] , hyperintensity related to sbccs was observed inconsistently . this study had several limitations . 
however , because patients who underwent cect and / or mr imaging were included , we believe that assessment of cyst wall thickening could be performed accurately . conclusions in conclusion , eccentric and smooth wall thickening was common on cect and mr imaging findings in infectionfree and benign sbccs . 
histopathologically , the thickened walls of sbccs were mainly composed of lymphoid tissue adjacent to the epitheliuthese characteristic imaging findings of the walls of sbccs may be useful for differentiating them from other cystic lesions in the lateral neck . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 declaration of helsinki and its later amendments or comparable ethical standards . ethical standards this article does not contain any studies with human participants performed by any of the authors . informed consent for this type of study , formal consent is not required . 
a three - month retention time is reasonable for airway stents . keywords post - tracheotomy tracheal stenosis post - intubation tracheal stenosis airway stent fluoroscopy complications introduction tracheal intubation and tracheotomy with mechanical ventilation may bring a variety of potentially life - threatening complications , such as post - tracheotomy tracheal stenosis ( ptts ) and post - intubation tracheal stenosis ( pits ) , with an incidence ranges from 0.3 to 80% [ 1 ]  . 
surgical resection and end - to - end anastomosis are the classic techniques for dreamweaver08@126.com * xinwei han * gang wu wuganghenan2004@126.com 1 department ofinterventional radiology , the first affiliated hospital ofzhengzhou university , no.1 , east jian she road , zhengzhou450052 , china the treatment of tracheal stenosis [ 2 ]  . 
unfortunately , surgery is not suitable for patients with long segmental stenosis or complicated underlying diseases , and those patients may not be willing to undergo open surgery under general anesthesia [ 3 ]  . 
nowadays , a variety of minimally invasive interventional treatments , such as balloon dilation and tracheal stent implantation , is suggested as an alternative to surgery for patients with medical contraindications or recurrence restenosis [ 4 , 5 ]  . since first adoption of metallic airway stents to treat tracheal stenosis in 1989 [ 6 ] , airway stents have been widely used in clinic [ 710 ]  . 
although airway stents are well tolerated , safe and effective [ 1114 ] , this treatment is still debatable and even abandoned in clinical practice for benign tracheal stenosis due to high rate of symptomatic vol . : ( 0123456789 ) 1 3 192 la radiologia medica ( 2019 ) 124 : 191198 recurrence and complications [ 1517 ]  . 
covered stents should be removed within a certain time to reduce longterm complications for benign stenosis [ 18 , 19 ] , which was usually performed by flexible bronchoscopy under general anesthesia or local anesthesia [ 11 , 12 , 16 , 17 , 2032 ]  . 
we reported our clinical results in stent removal for ptts and pits under fluoroscopic guidance and local anesthesia . materials andmethods patients this study was approved by the ethics committee and medical records management section of our university ( permit number : keyan - 2017 - 023 )  . 
all patients were indicated surgical resection ; however , they were unable to tolerate anesthesia due to severe dyspnea and had to implant stents first to alleviate the symptoms , to avoid unexpected accidents or even death . 
a 5f vertebral artery catheter ( cook corporation , bloomington , ind , usa ) was advanced along a 0.035 inch stiff hydrophilic guide wire ( cook corporation )  . 
routine removal defines as stent removal in patients with no obvious symptom or sign , although complications such as excessive granulation tissue or stent migration may able to be found during stent removal or by endoscopy . 
a severe pits was shown by ct before stenting ( a ) , which was dilated after stenting and 2months after stent removal ( b ) 1 3 la radiologia medica ( 2019 ) 124 : 191198 fig . 
one piece of strut residue was successfully removed endoscopically ( c ) patency defines as patent airway condition after stent removal without restenosis , tracheal intubation , tracheotomy , surgical resection or death . 
statistical significance was considered when p < 0.05. results the patients characteristics thirty - eight airway stents were successfully inserted in 32 consecutive patients over a five - year period . 
two patients showed dyspnea immediately after removal and required mechanical ventilation in ptts ( table3 )  . second stenting afterremoval intolerance of stenting , stent migration and replacement of bare stent . 
following doctors advice , more than half of patients were admitted to hospital for stents removal to avoid long - term complications , although they complained no obvious symptom or sign . 
 restenosis caused by tissue hyperplasia was the most common indication for later stent removal . complications ofstent insertion andremoval insufficient expansion of stents was most common complication immediately after stent insertion ; balloon tracheoplasty was performed for those with severe tracheal stenosis . 
besides , three patients showed stent migration and one patient complained of intolerance of stenting in ptts . except strut residue in three stent removal , 35 of 38 airway stents were successfully removed fluoroscopically , with a technical success rate of 92.1%. 
there were six complications of second stenting was performed in six cases ; included four stents were inserted again after removal due to restenosis in pits , and two stents were inserted after removal due to stent migration in ptts . 
coronal ct showed that the stent was in good position immediately after stent placement ( a ) , and the stent was obviously migrated downwards more than 2months later ( b )  . 
airway stents have been widely used in clinic [ 710 ] , which are reported as well tolerated , safe and effective for management of benign tracheal stenosis [ 1114 ]  . 
for benign stenosis , airway stents should be removed within a certain time to reduce complications caused by long - term stent retention , because a limited retention is capable of relieving dyspnea and minimizing recurrence of stenosis by routine stent removal [ 18 , 19 ]  . stent insertion and extraction was usually performed by flexible bronchoscopy under general anesthesia or conscious sedation [ 11 , 12 , 16 , 2024 ]  . 
our data indicated that fluoroscopic insertion and removal of airway stent is safe and effective for pits and ptts . the application of airway stent is becoming more and more widespread in benign tracheal stenosis . 
the incidence of complications was similar as that previously reported in rigid bronchoscopy [ 27 , 28 , 32 , 35 ]  . major complications ( such as stent migration and stent rupture ) are generally considered indications of stent removal [ 14 ]  . 
in our opinion , the shorter the stent retention time ( 3months ) the better once achieves the purpose of treatment to avoid long - term complications . in conclusion , fluoroscopic insertion and removal of airway stent is safe and effective for pits and ptts . 
indications for treatment were the risk of rupture / hemorrhage due to size greater than 4cm and symptomatology ; in one case , a previous hemorrhage was the indication for treatment . 
technical and clinical success , safety , and quality of life ( qol ) were evaluated in a mean follow - up of 9months ( range 312 )  . results technical success was obtained in all cases . 
clinical success was obtained in all cases ; the volume of the ablated amls was not related with symptoms relief ; all patients referred a significant improvement in their qol , with a regularization of daily activities . 
post - ablation syndrome was registered in 5 cases and was self - limited in all cases . conclusions symptoms relief , lower risk of hemorrhage and a normalized qol were obtained in all patients with a safe and mini - invasive procedure . keywords angiomyolipoma tuberous sclerosis quality of life microwaves introduction tuberous sclerosis complex ( tsc ) is a genetic disorder characterized by the growth of hamartomas in multiple organs . 
 up to 80% of patients with tsc have at least 1 angiomyolipoma ( aml ) in their lifetime [ 1 ]  . their growth can be rapid and unpredictable and may cause problems including hemorrhage , renal dysfunction , and rarely venous thrombosis [ 2 ]  . 
it has been shown that renal amls > 4cm are at increased risk of further growth and eventual hemorrhage [ 3 ]  . nephron preservation is a key treatment consideration , especially in patients with multiple , bilateral , and often very large amls , like those suffering from tsc [ 4 ]  . 
for many years , first - line treatment of these lesions was surgical excision ; nephron sparing surgery permits to preserve renal function , but in particular in patients with tsc , the * gianpaolo carrafiello gcarraf@gmail.com 1 diagnostic andinterventional radiology department , asst santi paolo e carlo , san paolo hospital , university ofmilan , via a di rudin 8 , 20142milan , italy 2 radiology department , uninsubria , varese , italy 3 nephrology department , san paolo hospital , university ofmilan , via a di rudin 8 , 20142milan , italy 4 nuclear medicine department , irccs istituto nazionale dei tumori , milan , italy vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 176183 propensity for recurrences is high so these patients have to be undergone several surgical treatments for multiple amls [ 5 ]  . advances in minimally invasive techniques allow for most of these tumors to be successfully treated [ 6 ]  . 
transarterial embolization has an established role in the modern management of renal lesions amenable to nephron sparing options , especially in larger aml and in acutely bleeding lesions [ 5 ]  . a number of investigators have explored the use of percutaneous ablative techniques such as radiofrequency ( rf ) ablation , cryoablation , and microwave ( mw ) ablation for the treatment of amls [ 5 ]  . compared to rf , mw energy produces larger and hotter ablation zones in less time [ 7 ] and more effectively penetrates macroscopic fat [ 8 ] , which is the major component of most amls . thanks to its known less intra - procedural pain , safety , and repeatabilities , percutaneous mwa is generally well tolerated by all patients treated [ 9 ]  . the aim of the present study was to evaluate safety , efficacy , and quality of the life of the patients with tsc treated with percutaneous mwa . materials andmethods patients this retrospective study was compliant with the health insurance portability and accountability act ( hipaa )  . 
 clinical and radiological data were retrospectively reviewed for all patients who had tcs associated with amls . our series includes nine patients ( 7 females and 2 males ; mean age 27.6years , range 2334 ) , with 10 renal amls ( all tcs associated ) with a mean size 42.8cm ( range 2470 ) ( table1 ) treated with image - guided percutaneous mw ablation . 
the primary indication for treatment was risk of rupture / hemorrhage due to size greater than 4cm ; moreover , in all patients a persistent growth was observed and all patients were symptomatic , flank pain and pressure except that one ; in the latter case , a previous hemorrhage was the indication for treatment ( table1 )  . the procedures were performed between july 2016 and december 2017 . 
patients were referred to our interventional radiology department by nephrologists of our hospital where patients with tcs refer . each patients case was discussed in the contest of a multidisciplinary meeting , during which the indication for thermal ablation was given . 
indications , benefits , and risks of the procedure were explained and discussed with every patient , and informed consent was obtained before treatment . three of the patients treated presented neurological involvement , well controlled with medications ; all patients presented cutaneous angiofibromas . coagulation blood tests resulted within the reference values in all patients [ 10 ]  . a first - generation cephalosporin ( cefazolin 2 gr b.i.d. ; pfizer srl , milan , italy ) was administered at the beginning of every procedure as antibiotic prophylaxis . procedure a 10 ml solution of lidocaine was injected to obtain local anesthesia in correspondence with the entrance site of the antenna . 
vital parameters ( heart rate , respiratory rate , and blood pressure ) , together with oxygen saturation and electrocardiographic tracing , were continuously monitored during the procedure . the antenna was positioned under us guidance ( arietta v70 ( hitachi aloka medical , tokyo , japan ) ) in 2 cases . 
after the guided placement of the antenna into the target tumor , the ablative procedure itself consisted in the delivery of thermal energy to the tissue , produced by maintenance of a power of 100w for a total time between 5 and 9min , as specified by the manufacturer , obtaining the desired necrosis volume . outcomes technical success consisted in a correct position of the antenna into the target lesion , as planned before treatment . 
 time of ablation was registered in all patients . clinical success was defined as the improvement or disappearance of the symptomatology and the normal performance for daily activities . as mentioned above , all patients had a pre - procedural mri . 
1 ct scan performed during the procedure to assess the correct position of the antenna ( a , b ) ; dynamic mri acquisition after iv administration of contrast medium performed 4weeks after the procedure confirmed the ablated area within the aml ( c , d ) fig . 
moreover , they were asked about the tolerance of the procedure ( post - ablation syndrome ( fever , malaise , pain , myalgia , nausea and vomiting ) usually resolving within 4days post - procedure ) [ 12 ] and post - procedural paa single trained interviewer registered prospectively the data about the qol before treatment , and then , the information obtained by a phone interview about symptomatology and daily activities after 4 weeks , 3 , 6 , and 12months after the procedure . 
sf - 36 includes one multiitem scale that assesses eight health concepts : ( 1 ) limitations in physical activities because of health problems ; ( 2 ) limitations in social activities because of physical or emotional problems ; ( 3 ) limitations in usual role activities because of physical health problems ; ( 4 ) bodily pain ; ( 5 ) general mental health ( psychological distress and wellbeing ) ; ( 6 ) limitations in usual role activities because of emotional problems ; ( 7 ) vitality ( energy and fatigue ) ; and ( 8 ) general health perceptions . in table3 , we indicated the tolerance of the procedure , post - procedural pain , and the scored qol of our patients before the procedure , and then 4weeks , 3 , 6 and 12months after the procedure . complications were classified according to common terminology criteria for adverse events [ 13 , 14 ]  . safety was evaluated on the basis of the complications recorded immediately after the procedure and during the follow - up . 
a complication was defined as immediate when table 3 quality - of - life questionnaire included : procedure tolerance ; health concepts ( physical or emotional problems concerning health ) procedure tolerance ( pa sdr ) ; post - procedural pain pre - procedure sf - 36 health status 4w post - procedure sf - 36 health status 3m post - procedure sf - 36 health status 6m post - procedure sf - 36 health status 12m post - procedure sf - 36 health status m , 28 yo 3 d mild fever ; f , 25 yo no pain 2 d fever , myalgia ; no pain no ; no f , 34 yo no ; no f , 25 yo 3 d fever , myalgia , nausea ; 2 d myalgia ; f , 23 yo f , 31 yo no ; no f , 26 yo no ; no m , 30 yo no ; no f , 27 yo 3 d myalgia , nausea ; no ( dancer ) ( runner ) m male , f female , yo years old , pa sdr post - ablation syndrome , w weeks , mmonths , d days 1 3 la radiologia medica ( 2019 ) 124 : 176183 it occurred up to 24h following the procedure , as periprocedural if occurred within 30days , and as delayed if occurred more than 30days after the procedure [ 14 ]  . 
major complications were defined as events that , if untreated , could leave to life - threatening , or events that led to substantial morbidity and disability , or could be cause of hospital readmission or substantially lengthened the patients hospital stay [ 14 ]  . 
minor complications included characteristic symptoms of the post - ablation syndrome ( fever , pain , nausea , and vomiting ) if lasting more than 4days after the ablation procedure . was not significantly related with the improvement in the symptoms . no patients have experienced renal hemorrhage , and all patients are asymptomatic at 12 - month follow - up . 
 one patient developed a small post - procedural subcapsular hematoma which was self - limited and clinically occult . post - ablation syndrome was registered in 5 cases and was statistic analysis spss v25.0 ( chicago , il ) was used for all statistical analyses . 
mean ablation time was 5min and 3s ( range 39min )  . technical success was obtained in all cases : antenna was correctly positioned within the lesion . post - ablation imaging demonstrated the area of ablation , compared with pre - procedural mri . 
the volume of ablation renal manifestations are the second most common findings associated with tsc , with amls occurring in 80% and renal cystic disease in 50% of patients , but only 3% of these patients present polycystic kidney disease ( pkd ) [ 15 ]  . typically children with tsc are born with normal kidneys , but develop cystic disease and angiomyolipomas as they age . 
3 scatter plot of distribution of health status ( assessed by sf - 36 questionnaire ) per time 1 3 182 la radiologia medica ( 2019 ) 124 : 176183 ( ckd ) , affecting approximately one million patients with tsc worldwide [ 15 ]  . ckd represents the most common causes of death in adult patients , with increasing morbidity and mortality [ 16 ]  . a cross - sectional study of tsc patients revealed an increase in amls during childhood and adolescence that then stabilized throughout adulthood [ 17 ]  . 
angiomyolipomas significantly affect the lives of patients because these lesions are at high risk of hemorrhage when a diameter is larger than 4cm and can invade adjacent normal renal parenchyma leading to ckd with development , sometimes , of uremia . given the frequent association of tsc and amls , the most logical intervention is to treat amls with diameter greater than 4cm to reduce risk of hemorrhage , and not to actually remove the aml . 
due to the bilateral nature of the renal lesions in tsc , and in order to preserve functional renal mass , nephrectomy should not be undertaken without a very careful risk - benefit analysis . the indications for the treatment of amls are different : to avoid hemorrhage , that is one of the most important aims , to destroy the mesenchymal tumor elements ; these results must be obtained by thermal coagulation . like in other districts ( uterus , liver , etc . ) , if technically possible , we preferred thermo - ablation over embolization , because in most cases post - procedural course is uneventful and post - ablation syndrome , when present , is milder than post - embolization symptomatology . the results of preliminary studies suggest that mw ablation of renal amls is technically feasible and appears to be safe and effective at early follow - up [ 8 ]  . thermal ablation is widely accepted as second - line therapy for medically or surgically inoperable patients with small renal cell carcinomas ( rcc ) [ 18 , 19 ]  . in the literature , the use of other percutaneous ablative techniques such as radiofrequency ( rf ) ablation and cryoablation was described . 
as for radiofrequency , recently published series include small amls ( 1.52.5cm ) [ 6 ]  . although rfa and cryoablation appear to be safe in smalland medium - sized renal amls , long - term efficacy data are lacking [ 6 ]  . the indications for the treatment of aml are different ; avoiding hemorrhage is one of the most important aims whereby immediate vascular thrombosis and destroying the mesenchymal tumor elements by thermal coagulation must be obtained . 
tissues with low electrical conductivity ( such as fat ) force current to higher conductivity pathways in the electrical circuit , thereby reducing the amount of heat generated in the low - conductivity fat , resulting in less effective tissue heating [ 20 ]  . 
in 2 cases , patients started to play sports again and in all cases an improvement in symptoms was registered ; moreover , a lower risk of hemorrhage was immediately obtained , because mri images performed during follow - up revealed the volume of ablation and in particular the necrosis of the most vascularized areas of the aml ( compared to pre - procedural images )  . 
our patients with amls are all affected by tcs ; clinicians consider quality of life an important parameter in this class of patients , because tcs may lead to neurological symptoms , pulmonary involvement as well as renal disease discussed [ 15 ]  . 
moreover the vogts triad ( facial angiofibromas , mental retardation and intractable epilepsy ) may characterize these patients , even if less than 40% of them have all three features [ 23 ]  . 
in our series , all well - controlled patients presented extra - renal disease ; thermal ablation of amls gave them a restitutio to normal daily activities with an estimated increase in health status of 0.683 per week ( data from standardized regression coefficient )  . 
symptoms relief , lower risk of hemorrhage and a normalized quality of life were obtained in all patients . pearson analysis could not demonstrate a statistically significant correlation between preand post - procedural health status and aml volume or treated area volume . in conclusion , a longer follow - up and most numerous samples are necessary to confirm our preliminary results . 
 moreover , a randomized study to compare mwa and embolization should be planned to evaluate safety , effectiveness , and compliance of the patient . 1 3 la radiologia medica ( 2019 ) 124 : 176183 compliance with ethical standards conflict of interest all authors declare that they have no conflict of interest . ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . informed consent informed consent was obtained from all individual participants included in the study . la radiologia medica ( 2019 ) 124 : 234240 radiotherapy evaluation ofitalian radiotherapy research from1985 to2005 : preliminary analysis albafiorentino1 rosariomazzola2 valentinalancellotta3 simonettasaldi3 sarachierchini3 annaritaalitto4 paoloborghetti5 fabianagregucci1 michelefiore6 isaccodesideri7 lorenzamarino8 danielagreto7 giovannidomenicotebala9 airo giovani italian association of radiation oncology - young members working group received : 13 september 2018 / accepted : 2 november 2018 / published online : 14 november 2018 italian society of medical radiology 2018 abstract aim the difficulty in conducting meaningful clinical research is a multifactorial issue , involving political , financial and cultural problems , which can lead to unexpected negative long - term consequences , in terms of knowledge advancement and impact on patient care . 
the aims of the present review were to evaluate the publications of italian radiotherapy ( rt ) groups during a 20 - year period and to verify whether research is still appealing to young radiation oncologists ( ros ) in italy . methods pubmed database was searched for english - language articles published by italian groups from january 1985 to december 2005 . 
analyzed variables were : publication / year , kind of study , geographical area and age of the first author . results the systematic review identified 3291 articles : 1207 papers fulfilled the inclusion criteria . 
the age of the first / second author was evaluated on 716 papers : in more than 50% of cases , the first author was younger than 40 . conclusion despite a general gradual improvement , rt clinical research suffers in italy ( as elsewhere ) from insufficient funding , with a negative impact on evidence production . 
it is worth noting that clinical research is still appealing and accessible to junior italian ro . keywords radiotherapy italian research * alba fiorentino albafiorentino@hotmail.it 1 radiotherapy oncology department , general regional hospital f . 
127km 4 , 70021acquavivadellefonti , bari , italy 2 radiotherapy oncology department , sacro cuore don calabria hospital , negrar , verona , italy 3 radiation oncology section , university ofperugia , perugia , italy 4 radiotherapy oncology department , fondazione policlinico universitario a . 
gemelli irccs , uoc radioterapia , rome , italy 5 radiation oncology department university andspedali civili , brescia , italy 6 radiation oncology , campus bio - medico university , rome , italy 7 department ofbiomedical , experimental andclinical sciences mario serio , section ofradiation oncology , university offlorence , florence , italy 8 radiotherapy oncology department , rem , viagrande , catania , italy 9 surgical department , east kent hospitals university nhs foundation trust , ashford , uk vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 234240 introduction for the oncological community , as well as for the whole medical community , clinical research is crucial to determine the safety and effectiveness of medications , devices , diagnostic procedures and treatment regimens for cancer patients . 
the so - called pyramid of levels of evidence provides a way to evaluate the quality of the available evidence ( level of evidence , loe ) and the reliability of consequent recommendations ( grade of recommendation , gor )  . 
it is therefore important to understand , both from a public policy perspective and from clinicians , including universities and national association of radiation oncology , point of view , the evolution of radiation therapy research , to identify strengths , weaknesses and opportunities . the aim of the present research was to perform a critical appraisal of the italian scientific amount in the field of radiation oncology in a 30 - year period ( 19852015 ) , to define its trend and to emphasize its strength and weaknesses . 
moreover , we aimed at evaluating the impact of junior radiation oncologists ( ros ) on the whole scientific production and to speculate whether research is still accessible and appealing to young specialists . this preliminary analysis reports an evaluation of the first 20years ( from 1985 to 2005 ) , just to get an early idea of the big picture , in order to be able to plan the subsequent analysis on the correct indicators . materials andmethods literature search andselection ofstudies pubmed database was searched by two independent ros , for articles published in english from january 1985 to december 2015 using as keywords radiotherapy combined with italy [ ( radiotherapy ) and ( italy ) ]  . 
studies eligible for inclusion in this analysis were : case reports , radiobiological studies , clinical and dosimetric retrospective or prospective studies ( phase iiiiii ) in which rt was used , systematic or not reviews and meta - analyses . 
 exclusion criteria were : study not conducted in italy , study in which rt was not used or was not investigated , study in which no italian ro was in the authors list . statistical analysis the abstracts of the selected papers were analyzed , and the following variables were considered : ( 1 ) year of publication ; ( 2 ) type of trial ( retrospective , prospective , including phase iiiiii trials , reviews , metanalysis , case reports , dosimetric study , translational and radiobiological research ) ; ( 3 ) geographical area of rt institution ( northern , central and southern italy ) ; ( 4 ) age of the first / second author ( only in the papers whose authors were a ro )  . 
to summarize the most relevant findings of the analyzed variables , descriptive statistics and linear regression analysis were performed on medcalc ver 18.2. results the systematic pubmed search ( 19852015 ) identified 9729 records . 
the present study is not a systematic review aimed at the statistical analysis of pooled data , but just a gross evaluation of the scientific production in a single country and in a specific time frame . 
the peak of number of papers in 1994 could be explained due to the improvements of tc simulation in the early 1990s ; in fact , most of papers are retrospective analysis analyzing tc simulation . based on the type of study , retrospective analysis , prospective phase iii trials and literature reviews were 44 , 20 and 14.5% of all published data , respectively . 
since 2000s , the regression analysis showed that randomized trials quantitatively increased with a linear trend ( r2 = 0.78 ) , although their absolute number remains low ( 48 / 1207 , 4% of all the publications )  . as regards the geographical area where the research was conducted , despite a general increase in the research production in all the areas ( fig.4 ) , northern italy provided the bulk of italian research in rt ( 58.7% ) , whereas southern italy published only 7.2% of all the studies here analyzed . after further review , the articles whose first / second author was not a ro were excluded , and in the remaining papers ( 716 / 1207 , 59.3% ) , the age of the first / second author was recorded . 
5 rate of young ( < 40yo ) first authors per year authored by junior ro increased , the percentage of studies where the first / second author was under 40 showed a progressive reduction . [ 4 , 5 ] , but we must not forget that the continuous widening of the spectrum of indications to rt takes the steps from the findings of clinical research . discussion during the natural history of oncologic diseases , approximately 60% of patients receive rt as part of their therapeutic course . 
in the last few years , the scientific community saw a worldwide improved access to rt , to deliver a comprehensive cancer treatment in 2012 , the european society for radiotherapy and oncology ( estro ) published a document on the future development and growth of rt and clinical oncology both within europe and at a global level in the forthcoming decades , where the society confirmed the importance of optimal individualized patient care that could be reached by integrating new clinical and preclinical evidence from biology , molecular / functional and anatomic imaging and the use of new systemic agents together with the delivery of high precision radiation therapy [ 7 ]  . 
although the investments in rt are often heterogeneous , the implementation of 1 3 238 la radiologia medica ( 2019 ) 124 : 234240 high - quality rt needed technical advances , such as imageguided rt , intensity - modulated rt , stereotactic body rt and radiosurgery , and heavy particles therapy . 
in fact , the increasing amount of robust evidence about new rt approaches allowed an expansion of the population for whom rt will be part of evidence - based cancer [ 8 , 9 ]  . 
what is the part played by italian scholars in rt within the big worldwide picture ? in other terms , what is the impact of italian scientific production , specific to rt , on the worldwide knowledge ? as junior members of the italian association of radiation oncologist ( airo ) , we aimed to perform a critical appraisal of the scientific production in rt coming from italian centers in quite a longtime span ( 30years )  . 
 therefore , we decided to carry on a preliminary analysis on the first two decades ( 19852005 ) to have a rough idea of the trend during the years , in terms of number of publications , geographical area of production of the study and age of the first / second author . 
in consideration that new techniques and technologies ( e.g. , intensity - modulated rt or igrt ) have been implemented in the first years of 2000 , the idea that from 2005 to nowadays the italian research was more complex than the past was the background for splitting the analysis of entire period . 
the subsequent evaluation ( 19852015 ) will be based on the findings of the present preliminary results . to our knowledge , this is the first study aimed at performing critical analysis of the amount and type of research on rt in italy with the intent to identify straight and weakness for the future planning of rt research , investments for rt centers and universities . 
about the type of publications , retrospective studies represented more than 40% of the whole scientific production , because collection and analysis of data are easier , the absence of implications such as randomization , informed consent and ethical and legal issues and the much lower costs . 
however , these studies provide evidence of a lower level ( loe - 2b ) so they usually cannot be used to draw sensible recommendations ( gor - 2 )  . 
moreover , in terms of phase iii trial , usa , japan , italy , republic of korea and spain had a relatively low ratio of phase iii / phase ii trials ; conversely , uk , netherlands , sweden and poland published more phase iii studies [ 10 ]  . 
this discrepancy is not clear but , probably , could be related to economic and cultural factors [ 10 ]  . at the same time , the number of reviews and meta - analyses is increasing , meaning that probably a growing number of authors decided to devote themselves to cheaper , timeefficient and office - based studies , which can be published on high - rank journals and attract funding and career improvements , instead of embarking in difficult , time - consuming and expensive rcts [ 2 ]  . 
several authors pointed out how , at least in the last decade , there has been a worldwide linear increase in rcts and an exponential increase in systematic reviews and meta - analyses [ 2 , 1214 ]  . 
the authors showed that only 5.3% of the oncological studies were rt trials and that sponsorship or funding for rt trials was significantly less than that for other oncological studies [ 15 ]  . 
these data support the hypothesis that , to date in oncology , research funding seems to be mainly focused to improve the armamentarium of new drugs , and unfortunately , private and governmental support for research in radiation oncology is significantly lower than for medical oncology [ 14 ]  . 
we wonder if there is a direct causative relation between the paucity of funding and the absence of well - structured changing practice rcts in rt . about the geographical area of research , northern italy produced the very most of italian scientific papers . 
probably , the northsouth gap can be explained by several aspects : ( 1 ) the number of rt centers , ( 2 ) the number of research centers , including universities , ( 3 ) the unequal distribution of funds and ( 4 ) the differences in terms of technologies . 
in fact , the numbers of rt centers increased during the analyzed period ( 77 in the 1989 , 96 in 1995 and 138 in 2002 ) , but about 50% of the centers were in the northern italy , despite similar population density . 
on the other hand , in the next years , the italian universities will be called to improve the young specialists formation in terms of methodological scientific education and we are sure that this will represent a great boost for research in italy , in particular in the analyzed field . 
this too entails the need for adequate financing . finally , the present analysis was the first report about the role of the contribution of young ro to rt research publication . 
thus , an increasing number of young specialists result as first / second author of papers in the 1990s , probably due to the relatively young age of radiation oncology as a single specialty in medicine . 
however , the trend of the rate of junior authors on the total number of papers published per year showed that in the same period , the percentage of papers whose first author is under 40 is gradually reducing . 
 the growing clinical commitment of junior ro and trainees , and the modifications of their timetables after the introduction of the european working time directive represent two factors which might have diverted time from research . 
scientific societies , including airo , and universities should support trainees and junior ro in getting a good training in research methodology , either during or after the specialist training , and to have access to research grants and facilities . 
 italian ministries for health and for education , university and research recently promoted research programs explicitly reserved for younger researchers , and more than 1300 university research positions for younger people have been financed for the current year . 
however , considering the costs of clinical research and randomized trials , especially if no profit , this financial effort should be increased to obtain a significant impact on the research systeit should be also noted that trials in radiation oncology are often less costly than trials in other clinical disciplines . 
other assets that can be exploited to facilitate an increased involvement of young specialists in radiation oncology research are the estro school of radiotherapy and oncology that aims to improve , professionalize and standardize knowledge and practice in radiation oncology and the young associations , including estro and airo young [ 1620 ]  . 
these associations can establish a solid network of connections and promote educational and research projects , contributing to an increasing role of young specialists in clinical research in radiation oncology . conclusion in conclusion , despite the limitation of the present data ( analysis of published papers in which major pitfall is to balance methodological and scientific issues with political ones ) showed that italian research in rt in the period 19852005 has been generally good , in terms of numbers of publications , and its quality , in terms of number of changing practice studies , is slowly but steadily improving . 
the simple parameters we have evaluated represent sufficiently reliable indicators to get a gross picture of rt research in italy , in the period considered , allowing to study its evolution in the subsequent periods analyzing bibliometrics parameters to evaluate also the quality of research . compliance with ethical standards conflict of interest alba fiorentino declares that she has no conflict of interest . 
it was requested that only one physician per facility completed the survey , and that he / she was dedicated to pc management in his / her daily clinical practice . 
more than 80% of respondents would perform radiotherapy ( rt ) to both the prostate and all metastases sites , but mostly up to 23 metastases ; furthermore , > 80% of physicians would perform rt on both nodal and bone secondary lesions . 
most respondents deem a cholineor prostate - specific membrane antigen ( psma ) - positron emission tomography ( pet ) mandatory before considering a patient affected by ompc for non - palliative rt . 
more than 90% of respondents confirmed to be interested in participating in a multicentre study regarding this subject . conclusions this survey investigated the current opinion of italian radiation oncologists and confirmed their interest in ompc management . keywords prostate cancer oligometastatic radiotherapy survey introduction local control of primary tumour in the presence of metastatic disease has been associated with improved outcome in several malignancies . 
nevertheless , the recent development of imaging techniques , such as cholineand prostate - specific membrane antigen ( psma ) - positron emission tomography ( pet ) / computed tomography ( ct ) , allows an earlier detection of metastatic disease in an intermediate state of tumour spread with better prognosis . 
in this subset of patients , a more aggressive approach could improve tumour control , postpone biochemical and clinical disease progression and delay the start of more toxic systemic treatments [ 4 ]  . 
limited retrospective studies suggested that interventions , including local and / or metastasis - directed therapy using surgery and rt , can improve survival outcomes with minimal risk of adverse effects [ 5 ]  . 
 since there are no guidelines to address this kind of treatment approach and studies providing rt + adt in this cohort are ongoing , the aim of this survey was to evaluate the current management of abinitio oligometastatic prostate cancer ( ompc ) patients in the italian radiotherapy facilities and to investigate the interest in participating in a multicentre study . table 1 list of questions included in the survey methods the study was conducted on behalf of the uro - oncologic study group of associazione italiana di radioterapia e oncologia clinica ( airo )  . 
it was requested that only one physician per facility completed the survey and that he / she was dedicated to pc management in his / her daily clinical practice . the questionnaire consisted of 15 questions concerning the management of abinitio ompc . 
the rate of completion after beginning the questionnaire was 100% , and the standard completion time was 4min . at question 1 , 27% of respondents answered they have been working as radiation oncologists for less than 5years , 15% for 510years , 30% for 1020years and 28% for more for how long have you been working as a radiation oncologist ? are you specifically involved in the treatment of pc ? in what kind of facility are you currently working ? where is your department geographically located ? is a regular prostate multidisciplinary team meeting performed in your facility ? how many patients affected with abinitio ompc are annually referred to your department for non - palliative rt ? which criteria do you adopt to evaluate if a patient affected with abinitio ompc is a candidate to non - palliative rt ? in a fit non - symptomatic patient affected with abinitio ompc what treatment would you recommend ? what imaging technique do you consider mandatory to evaluate this subset of patients ? which secondary lesions would you irradiate with non - palliative intent ? what volumes would you treat ? what technique and fractionation would you adopt ? how would you associate adt ? how would you perform the follow - up of these patients after treatment ? would you be interested in participating in a multicentre study of rt associated with adt in patients affected with abinitio ompc ? 1 3 la radiologia medica ( 2019 ) 124 : 211217 than 20 years . 
the majority of respondents work in a public non - academic facility ( 50% ) , while 31% work in a public academic or research institute and 19% in a private centre . 
 regarding geographic location of the departments , in northern , central and southern ( and isles ) italy work 41% , 34% and 23% of respondents , respectively , while 2% of italian radiation oncologist who completed the survey work abroad . 
seven percentage of respondents do not evaluate this subset of patients because they are not referred for rt evaluation . management andtreatment regarding selection criteria for treatment of prostate and / or metastatic sites , 26% of respondents would consider age and performance status , 19% biological features of the disease ( psa , gleason score , etc . ) , 28% the extent of the disease ( number and volume of the lesions ) , 13% technical feasibility and 76% all previous alternatives . 
physicians could choose more than one answer . in a fit asymptomatic patient affected with ompc , 9% of responders would offer rt to all secondary lesions , 1% to the prostate alone and 81% to both the prostate and all metastases , while 9% would not perform any rt , only adt . clinicians were asked which imaging technique they would consider mandatory before treating these patients : results are listed in table2 ( multiple answers were allowed )  . the sites the responders would most likely irradiate with non - palliative intent were : lymph nodes only , only bone metastases , both lymph nodal and bone lesions and none in 13% , 1% , 81% and 5% , respectively . 
regarding the number and site of lesions the responders would treat , results are summarized in table3 . a stereotactic body radiotherapy ( sbrt ) would be performed to both the prostate and metastases by 18% of clinicians ; 48% would offer sbrt to metastatic sites and standard or moderately hypofractionated schedule to the prostate ; 33% would choose a standard or moderately hypofractionated schedule for both ; no radiation treatment would be performed by 1% . the association of rt with adt was the topic of 13th question : 46 months of adt were chosen by 8% of respondents , 1236months of adt by 53% , continuous adt until biochemical progression by 24% ; 11% would not add any systemic treatment to rt ; 4% declared they do not deal with adt in their daily practice . the proposed follow - up scheme after treatment is shown in table4 ( multiple answers were allowed )  . 
numerous recent studies clearly show that metastatic pc is an extremely heterogeneous condition ranging from abinitio castration - naive low - volume disease to metachronous castration - resistant high - volume cancer . 
one hundred and eleven radiation oncologists filled in the questionnaire ; since we recommended to complete only one questionnaire per each centre , and 195 centres are active in italy , the response rate is higher than usually achieved when online surveys are conducted in our country . 
 we think actually that younger specialists may be probably more interested in this area of developing treatments . more than half of the respondents work , as expected , in a non - academic public facility ; over 30% work in an academic one , instead . 
it is possible that physicians working in an academic context are more likely to consider and perform innovative treatments , which also may require adequate technological equipment . not surprisingly , the percentage of the respondents according to geographic areas roughly follows the distribution of the radiation oncology centers in the country : 42% of the respondents come from the northern italy ( where 44% of the centres are located ) , 34% from the central italy ( 24% of the centres ) and 24% from southern italy ( 32% of the centres )  . almost two - thirds of the respondents work in a centre where regular mdt meetings are performed . 
it has been recently pointed out that the mdt meetings had an impact upon patient assessment and management practices in 445% of cases ; moreover , patients discussed at mdt meetings are more likely to receive more accurate and complete pre - operative staging and neo - adjuvant / adjuvant la radiologia medica ( 2019 ) 124 : 211217 treatments [ 8 ]  . 
another study showed that an uro - oncology mdt modifies management plans in about one quarter of cases , especially in metastatic patients , also improving cross - referral and consideration for clinical trials [ 9 ]  . 
the percentage of physicians who evaluate these patients for a non - palliative treatment appeared higher than expected : 27% see more than 20 of these patients in a year . 
the actual rate of abinitio oligometastatic patients is not clear , but with the development of new diagnostic tools , as whole - body mri and psma - pet / ct , the number is expected to increase . selection criteria for treatment were quite homogeneous , with 76% of respondents considering all listed criteria ( age and performance status , biological features of the disease , extent of the disease and technical feasibility ) useful in assessing the indication for a more aggressive treatment . 
 according to nccn guidelines , there are no specific criteria regarding age or comorbidities with regard to the indication to radical pc treatment ; however , life expectancy of at least 5years for high or regional risk patients is recommended . 
in elderly patients with a poor performance status , a palliative - only rt should be offered . the diagnostic topic is an interesting one , both in the initial / staging and in follow - up phase , as in case of progression ( re - staging )  . 
the standard work - up of staging for high - risk and locally advanced or metastatic pc includes abdominal ct or mri and bone scan , to assess distant disease . 
in recent years , since the widespread use of new systemic agents for upfront ( oligo ) metastatic patients and metastases - directed local treatments , the interest in a precise diagnosis of number and site of secondary lesions is growing . 
comparative reviews of whole - body ( wb ) mri and pet / ct have already demonstrated superior accuracy in metastasis detection in advanced - stage prostate cancer than ct and bone scans . 
 a recent study investigated the role of wb - mri to assess oligometastatic disease ( defined as 3 synchronous lesions ) , 1 3 la radiologia medica ( 2019 ) 124 : 211217 finding out that only about one fourth of the patients with hormone - naive prostate cancer had disease that could be classified as truly oligometastatic [ 13 ]  . 
the high sensitivity and specificity ( 80% and 97% , respectively ) of psma - pet / ct also may allow discriminating the true oligometastatic patients from those who have actually a widespread disease that has not been detected by ct or bone scan [ 14 ]  . 
over 90% of respondents would indeed perform a cholineor psma - pet / ct before evaluating a patient for aggressive treatment ; a local staging with mpmri would be recommended by 38% of respondents : this would be useful in locally advanced pc , as ct3 tumours , to avoid target missing in prostate rt . 
another relevant aspect is the appropriateness of employing such an expensive and time - consuming examination in a metastatic scenario , whereas other patients waiting for a radical treatment would probably benefit the most of it . it has been suggested that oligometastatic disease may represent an intermediate state of tumour spread with limited metastatic capacity , with a better prognosis compared to a more extensive metastatic disease [ 15 ]  . 
it would be crucial to identify patients whose disease is really less aggressive , so that the treatment of both primary and all metastatic would actually impact on local control , time to progression , castration resistance and even survival . 
it might be reasonable to consider as prognostic factors psa value , psa - doubling time , number of sites and pattern of metastatic spread , in order to select patients more suitable for an aggressive treatment , which can actually impact on patients outcome [ 16 ]  . 
the site , number and volume of secondary lesions also have to be considered carefully , since it would be complicated ( other than pointless ) to irradiate several or very large metastasis at a high , biologically effective dose , even with modern techniques [ 17 ]  . technical feasibility is a crucial issue . 
it does not mean that being able to do something implies that we should , but the irradiation of small isolated lymph nodes , for example , would be more complicated without advanced diagnostic tools ( as cholineor psma - pet / ct ) and high precision up to date rt equipment . 
if an extreme hypofractionated / stereotactic approach is considered for the treatment of the primary tumour , it should be performed in centres with adequate clinical experience and with the technological tools needed for both delivery and image guidance [ 18 ]  . over 80% of responders would irradiate both the prostate and all metastatic sites , if technically feasible and clinically indicated . 
the rationale of treating this subset of patients is indeed to achieve long - term local control , and therefore longer time to progression , so the irradiation of only a quote of disease would be probably insufficient . 
if there is growing evidence in treating metastatic sites in castration - sensitive pc , studies evaluating the role of prostate irradiation in this subset of patients are ongoing , and few retrospective experiences have been published [ 17 , 1922 ]  . 
nevertheless , 9% of the respondents would not perform any radiation treatment , but only adt as suggested by nccn guidelines [ 11 ]  . defining the oligometastatic state in prostate cancer is controversial and has varied across published and ongoing trials : most studies consider the limit up to 35 metastases [ 19 ]  . 
over twothirds of respondents would irradiate both the prostate and up to 23 secondary lesions : the treatment of the prostate and 45 metastases would be probably considered a technical challenge and clinically less indicated for most of the regarding the type of metastases to be irradiated , more than 80% of respondents would treat both nodal and bone lesions , if technically feasible . 
most studies have been performed delivering extreme hypofractionated treatments ( 1850gy in 110 fractions ) on secondary lesions , due to their excellent local control results as well as convenience [ 17 , 23 ]  . 
although the majority of respondents would offer a sbrt approach to metastatic sites , and only 18% also to the prostate , if technically feasible , about one third would perform a conventional or moderately hypofractionated treatment on both primary and secondary lesions [ 24 ]  . 
this can be understood in the case of nodal metastasis , or with bone lesions within the pelvic area , so that a single comprehensive treatment plan can be performed . 
it might be more difficult to justify a large number of fractions in a patient affected with more than one extra - pelvic lesion . the use of high doses delivered in a limited number of fractions , as we do with sbrt , is not only a convenient approach for the patient and the facility : due to its peculiar radiobiological features , characterized by a low / value , pc should be the ideal candidate for extreme hypofractionation [ 24 ]  . regarding systemic treatment , more than half of respondents would prescribe adt for 1236months , as current 1 3 216 la radiologia medica ( 2019 ) 124 : 211217 guidelines recommend for high - risk or locally advanced pc . 
even though metastases - directed sbrt in hormone sensitive ompc already treated with rt or surgery may be performed to delay the onset of adt ( and therefore its side effects ) , in this subset of abinitio oligometastatic patients the primary tumour is not controlled , and it is in fact a potential source for further metastatic spread . 
moreover , since long - term adt associated with rt is considered a standard approach in high - risk and locally advanced / node - positive pc , it should be mandatory in metastatic patients , who have been so far treated with continuous or intermittent adt alone [ 11 ]  . 
indeed , 24% of respondents suggested the use of adt until progression , which usually occurs within 2years from adt onset in this subset of patients . a small number of respondents ( 4% ) asserted that they do not deal with adt in their daily practice : radiation oncologists in italy are authorized to prescribe hormonal therapies , so the decision not to do it might be either a personal or a facility policy . cholineand psma - pet / ct also represent high sensitivity and specificity techniques for the detection of locoregional and distant metastases in pc patients with recurrence of disease [ 25 ] and have been proposed for re - staging for disease progression by 59% of respondents . 
although a standard follow - up scheme in this subset of patients has not been defined yet , it has been proposed to perform clinical examination , psa value and testosterone levels ( systematically or in case of psa rise ) every 36months by 92% of respondents . a number of prospective studies regarding the treatment of abinitio ompc are being developed , and the interest in this topic is growing ; 91% of respondents confirmed to be interested in participating in a multicentre study of non - palliative rt in this subset of patients . 
one of the aims of the uro - oncologic study group of airo is to elaborate shared guidelines and to promote multicentric trials regarding cutting - edge topics of our clinical practice . 
the development of a study protocol regarding the combination of adt and rt to the prostate and secondary lesions in abinitio ompc is indeed under investigation . strength points of our survey are represented by the relatively high response rate and by good heterogeneity for experience , geographic origin and centre type . 
many interesting sparks about assessment , management and treatment of abinitio ompc emerged , as outlined above . the major limitation of the survey is that , despite our request , about 20% of respondents declared not to be pc specialists : maybe a quote of them work in a facility where radiation oncologists are not divided into pathology - directed groups , so these colleagues are dealing also but not mainly with pc in their daily practice . 
in oncology , the role of wb - mri has expanded to the point of being recommended in international guidelines for the assessment of several cancer histotypes ( multiple myeloma , melanoma , prostate cancer ) and cancer - prone syndromes ( lifraumeni and hereditary paragangliomapheochromocytoma syndromes )  . 
the literature shows growing use of wb - mri for the staging and follow - up of other cancer histotypes and cancer - related syndromes ( including breast cancer , lymphoma , neurofibromatosis , and von hippellindau syndromes )  . 
the main aim of this review is to examine the current scientific evidence for the use of wb - mri in oncology . keywords magnetic resonance imaging diffusion - weighted imaging oncology whole - body mri cancer screening cancer - related syndromes * paul e . 
2 , university offlorence azienda ospedaliero - universitaria careggi , florence , italy 8 department ofradiology , ospedali riuniti , universit politecnica delle marche , ancona , italy introduction in 1905 , albert einstein published an article in annalen der physik describing the random motions of small particles suspended in a fluid that provided the first quantitative theory for the natural phenomenon widely known as the brownian motion . 
essentially , einsteins theory allows one to relate the diffusion constant to physical quantities , such as the mean squared displacement of a particle in a given interval of time [ 1 ]  . 
denis le bihan published the first article displaying diffusion - weighted magnetic resonance images of the central nervous system in 1985 [ 2 ] , some 80years after einsteins theory on water diffusion . 
since then , diffusion - weighted imaging ( dwi ) has evolved as a clinical magnetic resonance imaging ( mri ) technique ; it first entered practice in neuroradiology for the assessment of cerebral ischemia [ 3 ] and subsequently has extended into oncological applications [ 4 , 5 ]  . in 2004 , taro takahara started applying dwi in a single , whole - body ( wb ) examination , thus giving birth to the wbmri examination as we know it today : used mostly in oncological applications to provide a combination of morphologic vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 218233 and diffusion - weighted images from head to mid - thigh . 
taking steps to ensure good background body suppression of the signal , this first whole - body mri ( wb - mri ) with dwi examinations took almost an hour to acquire and required off - line image concatenation to produce an unified stack of slices for visualization [ 6 ]  . 
as highlighted in recent meta - analyses [ 7 , 8 ] , the growing evidence regarding the use of wb - mri in oncology has come to support recommendations of whole - body mri in international guidelines [ 911 ] and widening clinical adoption for different cancer histotypes [ 12 , 13 ]  . our aim is to review the current scientific evidence for the use of wb - mri in oncology . imaging acquisition protocol where f and w are , respectively , the fat and water images produced by the dixon technique . t2 - weighted images are acquired in the axial plane without fat suppression using turbo spin - echo ( tse ) sequences , with half - fourier acquisition single - shot turbo spin - echo ( haste ) being preferred . 
notably , some centers do not include t2 - weighted images in the wb - mri protocol in order to limit the acquisition time as similar information is provided by low b - value dwi images , but at a lower spatial resolution . t1 - weighted images and t2 - weighted images with fat suppression via short tau inversion recovery ( stir ) , acquired the spine in the sagittal plane , are usually warranted for investigation of suspected skeletal metastasis . the uptake of wb - mri as a radiological technique is closely tied to technical developments that have contributed to achieving a good spatial resolution with good signal - to - noise ratio throughout the body , including continuous moving - table acquisitions , multi - channel surface receiver coils , and parallel imaging acquisition . 
these developments have dramatically reduced the scanning time for wb - mri , such that dwi can now be performed along with supporting morphologic t1and t2 - weighted images of the whole body in a reasonably short acquisition time ( 3045min )  . the anatomical coverage of a wb - mri examination is usually from the skull base to mid - thigh analogous to positron emission tomography ( pet ) and computed tomography ( ct ) scans , though in specific clinical contexts it may be extended from vertex to feet . 
imaging is usually performed with a large field of view ( 4045cm ) [ 14 ]  . morphologic images due to their short acquisition times , t1 - weighted images of the chest and abdomen are usually performed within breath - holds as this prevents misinterpretation due to motion artifacts . 
gradient echo ( ge ) and dixon techniques are increasingly used for the t1 - weighted images due to their capability to derive multiple images ( including in - phase , opposite phase , water , and fat images ) in a single acquisition . 
intravenous contrast agent administration with subsequent post - contrast t1 image acquisition is performed only for specific clinical requests , such as the detection of brain metastases or characterization of liver masses . post - processing of the dixon images and specifically derivation of a fat fraction ( f% ) map that describes fat distribution are recommended [ 15 ]  . 
a fat fraction map can be computed on most of the mri post - processing consoles as : 100 f ( f + w ) diffusionweighted images dwi is usually performed using relatively thick sections ( from 5 to 7mm ) in an axial orientation during free breathing using a single - shot spin - echo planar imaging ( ssh - epi ) acquisition , to reduce both acquisition time and image distortion . 
fat suppression with the stir is strongly recommended in order to provide homogeneous fat signal suppression at larger fields of view . in the interest of limiting examination duration , two b - values are usually sufficient but three or more b - values are likely to more precisely quantify the apparent diffusion coefficient ( adc ) value , as requested for the tumor response assessment . 
the lowest b - value is usually in the range from 50 to 100s / mm2 to minimize perfusionrelated signal , while the highest is typically between 800 and 1000s / mm2 to allow good detection of hyper - cellular lesions with a good signal - to - noise ratio . 
depending on the mr scanner homogeneity and patient size , between four and six stacks of contiguous slices ( corresponding to different bed positions ) are usually necessary to perform wb - mri from skull base to mid - thigh . post - processing of the dwi data is mandatory and should consist of : unification of the high b - value images into a single series ( consecutive from superior to inferior ) , generation of maximum intensity projections ( mips ) of the unified high b - value series at small angular increments ( typically 3 ) rotating around the cranialcaudal axis , generation of coronal multi - planar reconstructions ( mprs ) of the unified high b - value series , if dedicated t2 - weighted images were not acquired , unify the low b - value images into a single series , unification of the adc maps into a single series ( consecutive from superior to inferior )  . 1 3 220 la radiologia medica ( 2019 ) 124 : 218233 mip reconstructions are usually displayed with an inverted gray scale , to provide a panoramic view similar to pet examinations . 
a complete image acquisition protocol is summarized in table1 , and a typical example of wb - mri imaging , including dwi , is displayed in fig.1. other possible wbmri protocols other wb - mri protocols for oncology have been used in the studies published over the last 15years . 
their differences can be described in terms of the following four concepts : anatomical coverage , imaging planes , mr sequences and reconstructions , and contrast administration . the anatomical coverage can be extended from the vertex to the feet ( including upper limbs ) when evaluating bone involvement in diseases that frequently involve the long bones , such as multiple myeloma ( mm ) [ 16 , 17 ] , when evaluating soft tissues in melanoma patients [ 18 , 19 ] , as well as for cancer screening in patients with lifraumeni syndrome [ 20 ]  . 
extending the anatomical coverage implies longer acquisition times . as regards imaging planes , many studies have made use of the coronal plane for morphologic whole - body images , alone or in combination with axial imaging [ 16 , 2129 ]  . 
in some cases , this choice may allow a better trade - off between scan time and image coverage , but is also a matter of scanner performance , radiologist preference , local practices , and considerations of readability , for example , the axial skeleton [ 30 ]  . 
the addition of dedicated single body part imaging is worth considering for specific assessment of brain , liver [ 31 , 32 ] , and chest [ 32 ]  . 
sagittal acquisition of the whole spine is included in the majority of wb - mri published protocols [ 2125 , 27 , 3134 ] , as it allows rapid acquisition and efficient evaluation of vertebral lesions , spinal cord compression , and vertebral fractures [ 26 ]  . the choice of the mr sequences and reconstructions used in wb - mri protocols has seen considerable evolution , and while not all include dwi [ 2125 , 32 ] , the majority of studies without dwi were published between 2002 and 2013 , when dwi of the whole body was quite time - consuming and quality inconsistent . 
mr technology developments in the last few years have significantly improved the quality and time efficiency of dwi in large volumes , allowing its wider and easier application in wb - mri protocols . 
similarly , the use of f% maps that we suggest in this review for bone assessment is a recent introduction to wb - mri and is only described in two studies published in 2017 , using wbmri for multiple myeloma [ 17 ] and breast cancer [ 34 , 35 ] patients . 
that earlier studies did not make use of f% maps is probably due to progressive acceleration of the dixon acquisition and prior lack of experience with this application in wb - mri . contrast administration is reported primarily in studies where wb - mri was used for patients with tumors requiring brain assessment , such as melanoma [ 18 , 31 ] and lung adenocarcinoma [ 27 ]  . 
 however , dwi has largely replaced the administration of contrast agents in many studies performed in patients with osteotropic tumor histotypes [ 26 , 3436 ] and lymphoma [ 29 , 37 ]  . clinical applications inoncology multiple myeloma multiple myeloma ( mm ) is a hematological disorder characterized by the accumulation of neoplastic plasma cells in the bone marrow . 
axial scans include multiple t1 - weighted images from a single dixon acquisition ( namely in - phase , out of phase , water , and fat images ) and t2weighted images performed without fat suppression . 
two different diffusion weightings ( with b - values of 50s / mm2 and 900s / mm2 , respectively , in this example ) are used to calculate the adc map ( third column )  . 
for this reason , the international myeloma working group ( imwg ) has affirmed that even the presence of asymptomatic bone disease on conventional radiography should be considered a criterion of symptomatic mm , requiring treatment [ 38 ]  . 
involving 611 mm patients , wb - mri detected more focal lesions than conventional whole - body x - rays in three of the most common metastatic sites for mm , including spine ( 78% versus 16% ; p value 0.001 ) , pelvis ( 64% versus 28% ; p value 0.001 ) , and sternum ( 24% versus 3% ; p value 0.001 ) [ 39 ]  . 
similar results were observed in a prospective cohort study conducted by baurmelnyk on 41 newly diagnosed mm patients , in which wb - mri showed diagnostic performance superior to conventional wholebody computed tomography ( ct ) in the detection of skeletal lesions ( ct understaged 11 / 41 patients compared to mri , p < 0.001 ) [ 40 ]  . 
finally , wb - mri is also recommended for the staging of solitary bone plasmacytoma ( sbp ) , an early - stage malignancy with a clinical course between monoclonal gammopathy of undetermined significance ( mgus ) and mm [ 38 ]  . melanoma although newly discovered immunological treatments for advanced melanoma have significantly increased overall survival , most patients with stage iiiiv melanoma will still die of the disease [ 42 ]  . 
in a later study involving 71 scans , wb - mri with dwi but without contrast - enhanced scans and wb - dwi without dwi but with contrast agent were seen to have an equivalent diagnostic performance in the detection of extracranial metastases from advanced melanoma [ 31 ]  . 
moreover , wb - mri is recommended for the follow - up of melanoma patients staged from iic to iv [ 9 , 44 ]  . prostate cancer mortality rates for prostate cancer are low , despite it being the most common cancer among males in europe [ 45 ]  . 
furthermore , prostate - specific membrane antigen ( psma ) pet / ct scans may fail to provide information on tumor viability during androgen receptor inhibition [ 48 ]  . 
2 contrast - enhanced ( gadolinium - ethoxybenzyl - diethylenetriamine pentaacetic acid , gd - eob - dtpa ) wb - mri with dwi performed on a patient with a stage iii melanoma . 
the same lesion is clearly detectable in the high b - value ( 900s / mm2 ) diffusion - weighted image performed in the same session ( b ) cancer ( eortc ) considers wb - mri as a one - size - fitall solution for evaluating treatment efficacy in advanced prostate cancer patients [ 50 ]  . the advanced prostate cancer consensus conference ( apccc ) has confirmed that psa alone is not reliable for monitoring disease activity in mcrpc , suggesting the use of a robust imaging technique before deciding to start a new line of treatment . 
in this respect , their guidelines recognize the superior diagnostic performance of wb - mri relative to ct and bs in the detection and assessment of skeletal metastasis [ 51 ] , though they note limited availability of the wb - mri technique . lymphoma fluorodeoxyglucose ( fdg ) - pet / ct is the imaging technique recommended for the most common lymphomas , fig . 
consistent with this , the national comprehensive cancer network ( nccn ) recommends serial ct or pet / ct examinations for both the staging and follow - up of lymphoma patients [ 53 ]  . 
the sensitivity of wb - mri with dwi to hyper - cellular lesions , independent of glucose metabolism , allows a reliable radiological evaluation of these lymphoma subtypes [ 54 ]  . in a prospective study conducted by mayerhoefer etal . 
 [ 13 ] on 140 patients , wb - mri with dwi demonstrated better sensitivity ( 94.4% ) than fdg - pet / ct ( 60.9% ) and contrast - enhanced ct ( 70.7% ) in staging patients with lymphoma subtypes of variable fdg avidity . 
 ( the majority were malt lymphomas . ) further , the same group found wbmri with dwi to have a diagnostic performance similar to fdg - pet / ct and ct in fdg - avid lymphomas [ 55 ]  . a growing application of wb - mri in lymphoma involves young patients irrespective of histotype . 
in a 1 3 224 la radiologia medica ( 2019 ) 124 : 218233 breast cancer in the last two decades , conservative treatments and early detection have substantially improved the prognosis for patients with low - stage breast cancer ( bc ) [ 66 ]  . 
nevertheless , according to the american cancer society , 5 - year overall survival for patients with advanced ( stage iv ) bc remains unfavorably low at just 22% [ 67 ]  . 
notably , bone metastases were present at the first staging procedure in 49.7% of invasive ductal cancer ( idc ) and in 61.7% of invasive lobular cancer ( ilc ) subtypes , respectively [ 68 ]  . 
 the widely accepted response evaluation criteria in solid tumors ( recist ) are not suited to a proper assessment of bone metastases and in fact consider them to not be measurable . 
according to the new criteria , bone metastases are considered measurable only once they have spread to the surrounding soft tissue with an extent larger than 10mm in diameter , which rarely occurs in clinical practice [ 69 ]  . 
this situation points to a significant unmet clinical need for means to evaluate bony metastases , with implications beyond breast cancer . in a 2011 meta - analysis by yang etal . 
 [ 70 ] covering 145 studies with 15 , 221 metastatic cancer patients , wb - mri showed diagnostic performance comparable to pet and superior to ct and bs in the detection of skeletal metastases ( table3 )  . 
 observed that out of the 46 treatment changes made due to progressive disease ( pd ) detected at imaging , in 34.7% of the cases ( 16 paired examinations ) , pd was visible only on wb - mri and not on the ct - cap examinations [ 12 ]  . 
similarly among 40 cases of progressive disease findings in a group of 58 patients who had both wb - mri and ct - cap or an 18f - fdg - pet / ct within 8weeks , zugni etal . 
 [ 71 ] found that all 40 ( 100% ) have been identified on wb - mri but only 23 ( 58% ) were also identified by ct - cap or 18f - fdg - pet / wb - mri has also shown promising diagnostic performance and established itself as a safe and accurate diagnostic imaging method for the systemic staging of pregnant bc patients ; some 40% of whom receive a diagnosis when bc is already at an advanced stage [ 72 ]  . 
4 a patient admitted with an initial diagnosis of prostate adenocarcinoma ( gs 5 + 4 , biopsy positive in 8 / 8 cores ) , psa = 29 ng / ml , positive digital rectal examination ( ct2c )  . 
the systemic staging , previously assessed with contrast - enhanced ct examinations of chest , abdomen , and pelvis ( ct - cap ) and bs , was negative for metastatic disease . 
subsequent systemic staging with wb - mri with dwi showed two metastases , one in the anterior arch of a left rib ( arrow ) and the other at level of retroperitoneal lymph node ( arrowhead )  . 
a multiparametric mri examination performed in the same session showed a pi - rads five lesion ( dashed line ) , visible on t2 - weighted axial images , high b - value diffusion - weighted images , and adc map recent study by brenner etal . , the overall 10 - year survival for lymphoma patients younger than 35years of age was seen to be above 90% ( 94.7% for patients < 24years and 89.4% for patients comprised between 25 and 35years , respectively ) [ 56 ]  . 
despite this long survival , the nccn guidelines still recommend pet / ct or ct for the staging and follow - up of lymphoma patients [ 57 ] , with 69 examinations to be performed in the first two years after diagnosis . 
after multiple lines of systemic treatments fdg - pet / ct and wb - mri were performed before and 6 months after initiating chemotherapy under the eshap scheme and peripheral blood stem cell transplantation . 
it has been reported that patients who have symptoms at onset have a poorer prognosis , and this is mainly related to the fact that vision reestablishment starts earlier compared with patients with mild symptoms , but restoration of visual function is still possible [ 6 , 7 ]  . 
corticosteroid treatment hastens recovery following on , but this does not improve final visual function . magnetic resonance imaging ( mri ) may help on diagnosis as it allows detecting optic nerve inflammation , to rule out differential diagnoses , and to predict the risk for developing ms [ 1 , 4 ]  . the application of mri in the assessment of optic nerve damage in a single episode of acute on has been rarely performed [ 4 ] , and its role in predicting the prognosis of visual impairment and visual outcome after on is not clear [ 4 , 610 ]  . optical coherence tomography ( oct ) is a noninvasive imaging technique used in ophthalmology to visualize the layers of the retina . 
it allows the depiction of unmyelinated central nervous system axons within the retina , the so - called retinal nerve fiber layer ( rnfl ) , and provides a measurement of its thickness ( micron - scale )  . 
the rnfl thickness is used as a noninvasive biomarker of neurodegeneration and axonal loss in ms [ 11 , 12 ]  . our aim was to assess brain and orbital mri findings of patients with a first attack of acute on , and their correlation with visual acuity at presentation , visual recovery , ms development and a possible correlation between orbits mri pattern and rnfl on oct . patients we retrospectively revised the ophthalmological , neurological and imaging data of patients who were admitted to our emergency department ( ed ) with the first episode of acute on from january 2015 to january 2017 ( n = 85 )  . the exclusion criteria were : patients aged < 18years ; previous episodes of on ; history of ms or optic neuromyelitis ( nmo ) ; patients affected by known ophthalmological diseases ; patients affected by immunological / infective disorders ; patients with previous neurological events ; patients with family history of leber hereditary optic neuropathy ; lack of complete ophthalmological , neurological , imaging data ; time between symptoms onset and presentation > 14days ; time between ed arrival and mri execution > 14days ; mri executed in other institutions ; lack of follow - up data ; clinical follow - up < 1year . we therefore included in our study 37 patients ( age range 2157years ; mean age 33years ) , 26 females and 11 males . 
twenty had right on , 13 had on in the left eye , and 4 had bilateral on , for a total of 41 affected eyes . after the arrival , each patient underwent a complete ophthalmological examination , including fundoscopic examination , visual acuity assessment , visual field and oct , brain and orbits unenhanced computed tomography , according to the protocol of our emergency department , to exclude intracranial and orbital abnormalities , blood tests to exclude infective / autoimmune causes of on and to test aqp4 antibodies myelin oligodendrocyte glycoprotein ( mog ) igg and cerebrospinal fluid ( csf ) collection for detection of the oligoclonal bands , as previously described [ 13 ]  . following the neuro - ophthalmological confirmation of the diagnosis of on , steroid therapy was administered intravenously within 24h from the patient access ( methylprednisolone 1g / day for 5days , followed by a low - dose steroid oral regimen for 15days )  . methods andmaterials this retrospective study has been approved by our institutional review board ( institutional review board area 1 milan ; reference number : 2018 / st / 116 )  . 
all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 helsinki declaration and its later amendments . 
the number of brain and spinal cord lesions was recorded . optic nerves mris were reported as ( 1 ) normal , ( 2 ) stiralteration without contrast enhancement ( ce ) , ( 3 ) stir signal abnormalities + ce . 
optic nerves pathologic findings were localized into three sites [ intra - orbital ( io ) , canalicular ( ca ) and chiasmal ( ch ) ] , and the length of altered signal or ce was measured in cm . visual acuity visual acuity was measured at patients arrival , according to snellen chart ( decimal )  . 
light perception and motus manus ( mm ) were assigned a decimal visual acuity of 0 . the degree of visual acuity was further divided into three groups : severe deficit ( mm4 / 10 ) , intermediate deficit ( 57 / 10 ) , slight deficit ( 8 / 10 , 9 / 10 )  . 
each patient was followed - up for at least 12months ( mean follow time : 16 4months )  . oct test was acquired using the same machine ( spectralis version 6.3.2 , eye explorer software 1.6.1.0 , heidelberg engineering , heidelberg , germany ) , after mydriasis induced with 1% tropicamide . oct produces a cross - sectional image of the peripapillary retina which is color - coded and shows the temporal , superior , nasal , inferior and temporal sections on the same image . the software automatically recognizes the retinal nerve fiber layer ( rnfl ) layer , measures the rnfl thickness along the scan and reports the results on a graphic display showing the rnfl thickness ( in m )  . 
pearson chi - square test for categorical variables was used to assess the correlation between brain mri patterns and visual acuity , visual outcome and diagnosis , and between orbits mri patterns and visual acuity , visual outcome and diagnosis . 
logistic regression model was applied to variables with p < 0.05. we then performed mannwhitney u test to assess the correlation between the lesions number and the diagnosis , applied the logistic regression model and finally performed roc curve analysis , and to evaluate the correlation between orbital mri pattern and extension of the altered signal of the optic nerve . kruskalwallis test was performed to assess possible correlation between orbits mri pattern and rnfl thickness at oct . results brain mris were classified as follows : ( 1 ) normal in 9 patients out of 37 patients ; ( 2 ) millimetric hyperintensities in t2 and flair with location and morphology not typical for demyelinating lesions in 9 out of 37 patients ; ( 3 ) lesions with ms - like shape , localized in typical ms sites with dissemination in space without contrast enhancement in 13 out of 37 patients and ( 4 ) lesions with ms - like shape , localized in typical ms sites with dissemination in space and coexistence of gadolinium - enhancing and non - enhancing lesions in 6 out of 37 patients . ms - like shape lesions number range was : 025 , with a mean number of 8 2 . ophthalmological evaluation including visual acuity assessment was executed at 6months to establish the visual outcome . 
optic nerves pathologic findings were localized into the io segment in 9 out of 27 eyes with signal abnormalities ( 33.3% ) ; into the ca segment in 16 out of 27 eyes ( 59.2% ) ; io + ca in 2 out of 27 eyes ( 7.4% ) , whereas no localization was observed in the ch segment . 
mean visual acuity at follow - up was 7 1 / 10 . twenty - five patients ( 67.5% ) received a diagnosis of ms ; 12 patients ( 32.4% ) received a diagnosis of cis ; in our group no patients received a diagnosis of nmo . 
3 roc curve shows the correlation between the lesions number and the diagnosis of ms 1 3 la radiologia medica ( 2019 ) 124 : 12961303 1301 discussion different disorders determine the inflammation of the optic nerve , but the form caused by ms related demyelination is the most common [ 2 ]  . 
demyelinating on represents the first manifestation of ms in about 20% of patients [ 19 ] and a diagnosis that brings great concerns to patients who are often young and previously healthy . the execution of mri , soon after the onset of symptoms , has important implications : first , mri helps clinicians to confirm the diagnosis of on , but it can also provide with useful information for patient management and treatment planning . in our case series , most optic nerve lesions were in the intra - orbital segment , and this is in line with the evidence of another study by soelberg etal . 
in a study on 37 adult patients with a recent or past attack of on who executed obits mri with stir sequence [ 20 ] , highsignal abnormalities of the optic nerves were found in 84% of symptomatic subjects and poor visual outcome was observed in patients with more extensive lesions , or with lesions located in the optic canal . 
our study shows that visual evoked potentials were more sensitive than mri in detecting lesions and in diagnosing demyelinating disease of the optic nerve ; however , we must consider that this study was conducted in 1998 and the mr equipment was certainly less efficient than the currently available scanners . stir sequence showed abnormalities in 88% of acute on in another retrospective study [ 21 ]  . we observed a lower rate of optic nerves altered signal ; however , differences in rates of mri abnormalities detected at optic nerves can be explained by different timings of mri execution after symptoms onset and by the beginning of the steroid treatment in our patients : berg etal . , for example , performed the mri within 24h form the patients arrival , before the corticosteroid therapy . in accordance with the literature , in our group of patients , all optic nerve abnormalities were in the io or ca segment without any involvement of the ch portion , which is instead considered typical for neuromyelitis optica spectrum disorder [ 22 , 23 ]  . in our study , we did not demonstrate a significant correlation between orbits mri pattern and visual outcome at follow - up . in a prospective study by hickman et al . 
 [ 6 ] in 33 patients , 15 of whom performed serial gadoliniumenhanced orbits mri until enhancement ceased , short acute lesion on triple - dose gadolinium - enhanced imaging are associated with initial improvement in vision ( p < 0.01 ) , but recovery was not related to the duration of enhancement . 
 [ 24 ] analyzed a group of 107 patients and observed that optic nerves with gadolinium enhancement in the optic canal had poorer color vision , compared with optic nerves with abnormalities in other segments . 
 they also report that that when there was complete involvement of all segments of the nerves the threshold perimetry and color vision were mostly impaired , and that optic nerves with enhancing segments longer than 17mm had poorer baseline visual acuity , threshold perimetry and color vision . 
 however , visual recovery was similar regardless of location or length of abnormal enhancement at baseline , and this fact is in line with our results . the extension of optic nerve abnormalities has been proposed as predictor of visual outcome in a study on 22 patients [ 25 ] , where complete visual recovery was observed in patients with lesions extension less than 17.5 mm , whereas lesions greater than 17.5mm and / or lesions with intracanalicular location were associated with incomplete or partial recovery . the intracanalicular location was identified as a risk factor for poor outcome also in a retrospective study of 50 patients acute on , who executed mri including stir sequence of the optic nerve [ 21 ] , together with initial low visual acuity ( less than 2 / 10 ) and the absence of orbital pain . in the attempt to predict visual recovery after on , zhang etal . 
 [ 26 ] proposed the use of texture analysis in a study on 25 patients with acute optic neuritis : open source software imagej was applied for the assessment of texture heterogeneity calculated on the coronal stir image showing the largest cross - sectional area of the lesion in the optic nerve . 
they observed that only baseline lesion texture independently correlated with visual recovery at 6 and 12months , whereas no other mri variables at baseline , as altered signal length and gadolinium enhancement length , nor ophthalmic variables , as rnfl , correlated with visual outcome . 
the absence of a correlation between signal abnormalities in stir and gadolinium enhancement of the optic nerves and visual recovery is in line with our results . we confirmed that mri brain lesions at presentation with optic neuritis ( on ) increase the risk of developing clinically definite multiple sclerosis . 
the evidence that the presence of brain lesions at the onset of on is related to the diagnosis of ms is in accordance with the literature : previous studies have reported that patients with no brain lesions at the onset of on have a 1522% risk of ms compared to a 5688% risk 1 3 1302 la radiologia medica ( 2019 ) 124 : 12961303 in patients with one or more brain lesions [ 14 , 27 , 28 ]  . 
this evidence has important clinical implications , since these patients may be later at a greater risk of disability [ 4 ]  . all patients with spinal lesions received a diagnosis of ms , and this agrees with other studies [ 4 , 29 ]  . another option for the assessment of patients affected by on is represented by oct . 
data in the literature demonstrated the relationships between rnfl thickness and visual acuity , visual field and color vision , and previous results affirmed that oct can be used as a noninvasive biomarker of neurodegeneration and axonal loss in ms [ 30 ] and of optic nerve atrophy [ 31 , 32 ]  . 
moreover , a strong correlation between rnfl thickness and visual outcome has been observed in ms patients [ 3335 ] , suggesting that a lower rnfl thinning in early ms is related to a better visual outcome [ 33 ] .we use this ophthalmological technique in combination with mri examination . limitations our study has some limitations , first of all the 1 - year clinical follow - up : it is indeed possible for the patient to develop ms at a greater distance of time ; however , the same followup period was also considered in other studies in patients with on [ 4 , 8 , 20 , 24 ]  . 
this is a retrospective study ; however , we considered only the patients with complete clinical , ophthalmological and imaging documentation , eliminating from our case series all patients with partial or incomplete data ; the patient population is not huge , but comparable to that proposed in other similar studies . 
another limitation is the execution of mri after the beginning of the steroid treatment , since the administration of steroids reduces active lesions contrast enhancement of the optic nerves , brain and spinal cord . conclusions in conclusion , the correlation between orbits mri pattern at baseline and visual recovery remains unclear , whereas the observation of brain lesions and their number at the first episode of on can help predict the development of ms . 
the identification of those patients who may convert to ms is important , as they may later be at greater risk of disability [ 30 ]  . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical standards as stated in materials and methods , this retrospective study has been approved by our institutional review board ( institutional review board area 1 milan ; reference number : 2018 / st / 116 )  . 
therefore , we want to evaluate the diagnostic performance of preoperative mri with a dedicated axillary sequence for axillary lymph node ( aln ) metastasis in patients with early ductal breast cancer and determine potential predictors of axillary nodal positivity . materials and methods we retrospectively reviewed the mri findings for 74 consecutive patients diagnosed with invasive breast cancer . 
peritumoral high signal intensity on t2 - weighted images ( p = 0.015 ) of the primary tumor was significantly associated with aln metastasis . conclusion our findings suggest that preoperative axial + reconstructed coronal mr images exhibit good diagnostic performance for aln metastasis in patients with early ductal breast cancer . 
in addition , peritumoral high signal intensity on t2 - weighted images of the primary tumor can be used as a predictor of aln metastasis in these patients . keywords axilla breast cancer lymph node magnetic resonance imaging metastasis introduction axillary staging of primary breast cancer is important to determine the treatment and prognosis [ 1 ]  . 
axillary lymph node ( aln ) dissection ( alnd ) used to be a conventional * mijung jang mjjang74@gmail.com 1 department ofradiology , chung - ang university hospital , chung - ang university college ofmedicine , 102 heukseok - ro , dongjak - gu , seoul06973 , republicofkorea 2 department ofradiology , seoul national university bundang hospital , gumi - ro , bundang - gu , seongnam - si , gyeonggi - do13620 , republicofkorea 3 department ofradiology , chungbuk national university hospital , 776 1sunhwan - ro , seowon - gu , cheongju , cungcheongbuk28644 , republicofkorea staging method ; however , the performance of this invasive procedure may result in overtreatment for patients without lymph node metastasis . 
therefore , alnd has largely been replaced by sentinel lymph node biopsy ( slnb ) for surgical axillary staging , and alnd is performed for patients with positive slnb findings in recent use [ 2 , 3 ]  . ultrasound ( us ) is considered the best imaging tool for breast cancer diagnosis , which can have high sensitivity and high specificity for axillary nodal staging [ 4 ]  . 
in addition , us - guided fine needle aspiration biopsy ( fnab ) exhibited high specificity and a high positive predictive value ( ppv ) for axillary staging in previous studies [ 46 ]  . 
however , following the reporting of the acosog z0011 trial findings [ 7 ] , the usefulness of us - guided fnab for aln staging in patients with breast cancer is needed to be revisited [ 8 , 9 ]  . 
the results of the acosog z0011 trial suggested that only patients with extensive nodal involvement ( more than vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 12201228 1221 three axillary nodes ) should undergo alnd in the setting of early breast cancer . 
considering that the identification of metastases on us images commits patients to alnd , the use of us and us - guided fnab , particularly for patients with early breast cancer and relatively few axillary nodal metastases , was challenged after publication of the acosog z0011 trial findings [ 11 ]  . 
these changes have spurred a reappraisal of the role of many other noninvasive imaging modalities for aln staging . magnetic resonance imaging ( mri ) is widely used for the preoperative evaluation of breast cancer . 
staging of aln metastasis using mri is generally based on the size and additional morphological characteristics such as irregular margins , an inhomogeneous cortex , perifocal edema , and asymmetry [ 12 ]  . 
many advanced techniques such as ultrasound particles of iron oxide ( uspio ) - enhanced t2 * imaging , diffusion - weighted imaging ( dwi ) , and dedicated high - spatial resolution axillary mri have been attempted to improve the accuracy of aln metastasis detection [ 13 , 14 ]  . 
 however , these techniques are very difficult to use in clinical practice , considering they are time - consuming because of the addition of experimental sequences or a dedicated axillary coil . 
 [ 14 ] demonstrated that the sensitivity and specificity of any advanced mri techniques were not better than those of snlb for the detection of aln metastasis in patients with early breast cancer . 
therefore , the aims of the present study were to evaluate the diagnostic performance of preoperative mri using a dedicated axillary sequence on a 3 - tesla magnet with the addition of coronal multiplanar reconstruction images for aln metastasis in patients with early ductal breast cancer . 
in addition , we also determined the clinicopathological and imaging factors predicting axillary nodal positivity in early ductal breast cancer patients . materials andmethods study population this study was a retrospective review of the medical records of patients with early breast cancer diagnosed and treated at our institution between october 2015 and december 2015 . 
 the protocol of this observational study was approved by the institutional review board of our hospital , a tertiary referral center , which waived the requirements for informed written consent for use of these data ( irb no . : b - 1907 - 550 - 109 )  . 
of these , 103 were excluded for the following reasons : no invasive component on final pathology [ ductal carcinoma insitu only ( n = 36 ) ] , advanced disease requiring preoperative systemic therapy ( n = 27 ) , bilateral cancer ( n = 14 ) , other breast cancers such as invasive lobular carcinoma ( n = 11 ) , unavailable mri data or mri performed elsewhere ( n = 8 ) , unknown primary tumor with axillary metastasis ( n = 3 ) , performance of excisional biopsy instead of breast conserving surgery ( n = 3 ) , and recurrent breast cancer ( n = 1 )  . 
finally , a total of 74 consecutive patients were included in this study . mri protocol mri was performed using a 3.0 - tesla ( t ) mri device ( achieva , philips healthcare , best , the netherlands ) with a 16 - channel dedicated breast surface coil . 
coronal reconstruction was performed on the basis of the axial images using a commercial computeraided diagnosis ( cad ) workstation ( cadstream version 4.1.3 ; confirma , inc , kirkland , wa )  . 
the cad system was configured to compare the pixel intensity values of the precontrast and immediate post - contrast image series using a 50% enhancement threshold . axillary mr image analysis two radiologists with experience in breast mr image interpretation retrospectively reviewed the mri images , respectively . 
the reviewers were informed 1 3 1222 la radiologia medica ( 2019 ) 124 : 12201228 about the primary cancer site only for the purpose of comparisons with the contralateral axilla as a control . 
clinical aln positivity was recorded when one or more positive findings were noted by both reviewers on axial or axial + coronal mr images . positive mri findings indicating lymph node metastasis included the following : a short - axis diameter of > 0.5cm , a maximal cortical thickness of > 0.3cm , eccentric cortical thickening , and loss or compression of the fatty hilu both axillae were evaluated at the same time , and the aln ipsilateral to the breast cancer was compared with the contralateral node . 
if there were no differences in number , size , or shape between the ipsilateral and contralateral alns , we recorded nodal negativity in accordance with the concept of asymmetry described by baltzer etal . 
we additionally included a cn0 stage , which represented negative mri findings , in the present study . breast mr image analysis breast mr images were evaluated for the primary tumor morphology and size and the presence of peritumoral edema by one of three radiologists with experience in breast mr image interpretation . 
the lesion morphology was evaluated as mass or non - mass enhancement ( nme ) , and peritumoral edema was evaluated on t2 - weighted image as absent or present . 
the maximal diameter of the tumor was recorded as the radiological tumor size . reference standard ( pathological nodal stages ) andstatistical analysis the final histopathological findings in either alnd or slnb specimens were used as the reference standard . 
in cases of n0 ( i + ) , i.e. , when very small deposits of isolated tumor cells were found in a lymph node ( less than 0.2mm or 200 cells ) , the nodal stage was still designated as n0 . 
in other words , the n0 ( i + ) stage represented negative findings in the present study . the clinical nodal status was correlated with the corresponding pathological nodal status , and the diagnostic performance of mri for aln metastasis was assessed on the basis of these correlations . 
in addition , these values , along with the area under the receiver operating characteristic curve ( auc ) value , were calculated for axial and axial + coronal images and compared . 
the patient and primary tumor characteristics , including age ; family history ; clinical symptoms ; the primary tumor size ( pathological ) , multiplicity , and molecular subtype ; and the mri findings for the primary tumor ( morphology , size , and presence of peritumoral edema ) , were correlated with the pathologic nodal stage . 
the primary malignancies included invasive ductal carcinoma in 71 patients , microinvasive ductal carcinoma in two patients , and mixed invasive ductal and lobular carcinoma in one patient . diagnostic performance ofmri according to axial scan analysis , there were 50 patients with negative findings and 24 patients with positive findings . 
final pathological analysis revealed positive findings for eight ( 33.3% ) of the 24 patients with positive mri findings ; the remaining 16 ( 66.7% ) patients showed negative pathological findings . 
with regard to the 50 patients showing negative mri findings , final pathological analysis confirmed negative findings for 41 ( 82% ) and positive findings for nine ( 18% ; table1 )  . 
six ( 40% ) of the 15 patients with positive mri findings showed positive findings in the final pathological analysis ; the remaining nine ( 60% ) showed negative findings . 
1 receiver operating characteristic curve ( roc ) to assess the diagnostic performance of axial alone and axial + reconstructed coronal magnetic resonance images for axillary lymph node metastasis in patients with early ductal breast cancer correlation ofclinicopathological andmri findings fortheprimary tumor withthepresence ofaln metastasis table3 shows the clinicopathological and mri findings for the primary breast lesions in the aln positive and negative groups . 
the tumor stage was significantly higher ( p < 0.001 ) , and the pathological tumor size was significantly greater ( p = 0.008 ) in the positive group than in the negative group . 
the dedicated axillary sequence used in this study can eliminate pulsation artifact due to use of phase encoding direction and can be easily added the routine dce - mri protocol because it uses the same breast coil for the primary breast cancer instead of a separate coil for the axilla . 
3 preoperative breast magnetic resonance images of a 43 - yearold woman who underwent total mastectomy for left breast cancer a contrast - enhanced axial t1 - weighted image shows a proven malignant mass in the outer quadrant of the left breast . 
these findings suggest that postprocessing using cad software can improve the diagnostic performance of axillary mri for aln metastasis without the need for supplemental advanced sequences such as uspio - enhanced t2 * sequences , dwi , and dedicated high - spatial resolution axillary sequences . 
we also compared various clinicopathological and mri features of the primary tumor between the node - positive and node - negative groups and found that venous / lymphatic invasion , the pathological stage , and the pathological size , which are widely known prognostic factors for breast cancer , showed significant differences between the two groups . 
the presence of peritumoral 1 3 1226 la radiologia medica ( 2019 ) 124 : 12201228 edema on t2 - weighted images was also associated with aln metastasis . in a recent article evaluating the practical approach to breast mr image interpretation and reporting , the authors described that the use of image postprocessing , such as subtraction , maximum intensity projections ( mips ) , threedimensional reconstruction , region of interest analysis , and automatic color mapping using cad software , should also be included in the breast mri report [ 16 ]  . 
although image postprocessing is widely used in practice , to the best of our knowledge , this is the first study comparing axial images with axial + reconstructed coronal images for axillary staging . 
the authors obtained two additional sequences , including a t2 - weighted haste and a t1 - weighted , contrast - enhanced , fat - saturated sequence with coronal orientation and demonstrated a clinically feasible fast protocol for local and locoregional staging of breast cancer . 
 [ 14 ] demonstrated that uspioenhanced t2 * sequences showed superior sensitivity ( 98% ) and inferior specificity ( 96% ) for aln metastasis in patients with early breast cancer . 
therefore , this technique could be a useful addition to current diagnostic techniques , although it cannot compete with slnb , which exhibits high sensitivity ( 9395% ) and specificity ( 100% ) [ 17 , 18 ]  . 
 while the difference in the diagnostic performance between our study and previous studies is not remarkable , our result is significant with regard to clinical practice , because reconstructed coronal images were obtained by postprocessing after routine mri . 
that is , the diagnostic performance for axillary staging in early breast cancer was significantly improved without additional experimental mri sequences or a dedicated axillary coil . the aln disease status is the most significant prognostic factor for patients with early - stage breast cancer . 
in the present study , venous / lymphatic invasion by the primary tumor cells and the pathological stage and size of the primary tumor were found to be potential predictors of aln metastasis . 
on the basis of these findings , we can assume that venous / lymphatic invasion by the primary tumor and the pathological stage and size of the primary tumor is associated with peritumoral edema on t2 - weighted images . 
in particular , peritumoral edema on t2 - weighted images or rim enhancement of the primary breast lesion on dynamic contrast - enhanced images was significantly correlated with the disease prognosis . 
 [ 28 ] reported that preoperative breast mri findings of rim enhancement and peritumoral edema may be used as prognostic biomarkers for predicting distant metastasis in patients with breast cancer . 
although multivariate analysis for the association between peritumoral edema and other possible correlated factors such as molecular subtypes ( luminal a / b , human epidermal growth factor - positive , and triple - negative ) was not performed because of the small total sample size , peritumoral edema by itself was confirmed to be a significant predictor of aln metastasis in our study . our study was limited by the small sample size and selection bias due to the retrospective design . 
to minimize discordant results , the sizes of the 1 3 la radiologia medica ( 2019 ) 124 : 12201228 1227 most suspicious alns on mri were correlated with those of pathologically proven metastatic lymph nodes ; however , we could not confidently confirm any correlation . 
third , we used reconstructed coronal images created by postprocessing techniques , but this protocol may be unsuitable for screening postoperative patients because of the possibility of metal artifacts . in conclusion , preoperative breast mri showed a relatively high specificity value and npv for the detection of aln metastasis in patients with early ductal breast cancer . 
ishmael pastore roberta perri marco pesapane filippo petralia giuseppe petrillo antonella petrillo mario piacentino filippo pintari robert pinto antonio pinto fabio pinzi valentina pirtoli luigi pofi enrico pozzi mucelli fabio pradella silvia quaia emilio la radiologia medica ( 2019 ) 124 : 13331335 quattrocchi carlo cosimo rampado osvaldo regge daniele renzulli padiglione 2 radiologia golfieri righini andrea romanini laura romano andrea rosi giovanni rossi andrea rossi michele saba luca sabatini umberto ahin ertan salerno sergio salvolini ugo samarakoon rushani sartori paolo scevola germano sconfienza luca maria secinaro aurelio seitun sara sessa barbara shikhare sumer sibilla luisella somma francesco spampinato sofia sperandeo marco splendiani alessandra stabile ianora amato antonio stasolla alessandro stecco alessandro stramare roberto sverzellati nicola tagliafico alberto tartaglione tommaso tedeschi enrico teke memik tofani alessandro toma paolo tonerini michele tonolini massimo torricelli pietro tortora fabio triulzi fabio trovato guglielmo turgut bekir ugga lorenzo ukmar maja 1 3 la radiologia medica ( 2019 ) 124 : 13331335 1335 valeri gianluca valeriani maurizio vanzulli angelo varrassi marco verrone giovanni volterrani luca woniak krzysztof xiaoyun su zappia marcello zeng jing zuiani chiara zampa virna zanirato rambaldi giuseppe a . 
the role of whole - body computed tomography ( wb - ct ) in self - referred and asymptomatic patients has been debated . aim to determine frequency and spectrum of wb - ct findings in average - risk subjects derived from a medical - check - upunit , to evaluate recommendations reported and distribution according to sex and age - groups . materials and methods we retrospectively reviewed 6516 subjects who underwent wb - ct ( june 2004 / february 2015 )  . 
de tarazona , k3 , 31500tudela , navarra , spain 4 present address : complejo hospitalario de navarra , calle de irunlarrea 3 , 31008pamplona , navarra , spain vol . : ( 0123456789 ) 1 3 1200 introduction since the development of the first x - ray scanner in 1971 , computed tomography ( ct ) has emerged as a potential tool for diagnosis in symptomatic patients [ 1 ]  . 
in the recent years , the role of this technique has also been advocated to allow early detection of certain diseases in specific asymptomatic individuals , such as screening for lung cancer [ 25 ] or coronary calcium screening as a robust predictor of cardiovascular outcomes [ 68 ]  . 
furthermore , institutions as the american college of radiology ( acr ) have developed the acr appropriateness criteria for performing screening for colorectal cancer with ct colonography in specific clinical conditions [ 911 ]  . in the current health care environment , there is a growing awareness that prevention and early diagnosis may reduce the high mortality associated with cancer , cardiovascular and other severe diseases . 
this fact , together with the high accuracy , wide availability and fast image acquisition of ct [ 2 ] , may allow the use of this technology as an effective whole - body diagnostic strategy in patients derived from a check - up - unit . 
whole - body ct ( wb - ct ) can be defined as the integration of calcium scoring and cross - sectional chest and abdominopelvic imaging for the recognition of any abnormality . 
the role of wb - ct in self - referred and asymptomatic patients has been continuously debated since the publication of first article in 2002 , as a way to provide early diagnosis of cancer as well as cardiac and other diseases [ 1215 ]  . 
however , there are no more recent publications available that assess the usefulness of wb - ct and its findings . the aims of our study were to determine the frequency and spectrum of wb - ct findings in average - risk subjects derived from a medical - check - up - unit , to establish the recommendations reported by radiologists in these examinations and to evaluate the distribution of wb - ct findings according to sex and age - groups . materials andmethods patient population we retrospectively reviewed 6516 consecutive subjects who underwent wb - ct from june 2004 to february 2015 . 
a physician specialized in internal medicine from a specific la radiologia medica ( 2019 ) 124 : 11991211 medical - check - up - unit composed of three physicians ( with 2532years of experience ) who evaluated every patient . 
they determined the indication of performing a ct based on a table of risk factors previously defined by a committee of experts in radiology , internal medicine , cardiology and vascular pathology . 
all subjects were older than 40years ( mean age 58.4years ) , non - symptomatic , with no history of malignancy , and with one or more risk factors , including arterial hypertension , diabetes , history of cigarette smoking , obesity or hypercholesterolemia . written informed consent for study protocol was obtained from all patients before ct examinations . wholebody ct protocol all examinations were performed using a sixty - four - row multidetector ct ( 64 - mdct ) ( somatom definition and somatom sensation - 64 , siemens healthcare , forchheim , germany ) , with tube voltage and reference tube current time product variable depending on region included . 
all images were stored in pacs . the protocol of wb - ct included : low - dose chest ct ( 120kv and 40ma / s ) without contrast material . coronary artery calcium measurement ( 120 kv and 138ma / s ) in cases where coronary calcification was identified in low - dose chest ct . abdominopelvic ct ( 120kv and 180ma / s ) performed after intravenous injection of 120 - ml iodinated contrast medium ( omnipaquetm 300 ( iohexol ) , 300mgi / ml , ge healthcare bio - sciences , madrid , spain ) , at 2ml / s . 
sometimes , a delayed phase was necessary to confirm a diagnosis . additionally , osteo - ct ( 80kv and 250ma / s ) was performed in women to quantify bone density . 
for the analysis , bone density values of three consecutive vertebrae were compared with a reference phantototal trabecular , cortical and final bone mineral density according to age were obtained . findings anddatabase all ct images were reviewed at a computer workstation by eight expert radiologists with 1317years of experience . 
radiologists completed a specific questionnaire with three modules : chest ct , abdominopelvic ct and bone ct 1 3 la radiologia medica ( 2019 ) 124 : 11991211 1201 findings ( including calcium score and bone mass measurement )  . 
radiological findings : it is categorized according to location and classified as normal / abnormal for ( i ) every organ , ( ii ) every module , and ( iii ) every examination . 
we multiplied the recorded dose - length product ( dlp ) by a weighting factor of 0.014 msv / mgy ( for chest and calcium score ) and 0.017msv / mgy ( for abdomen and spine ) [ 19 ]  . statistical analysis descriptive statistics to analyze the number of imaging findings and recommendations were used . 
a chi - square test and a linear - by - linear association test were used to evaluate the distribution of the most important radiological findings detected in the chest , abdomen , pelvis and bones , in relation to gender and age - group , respectively . all p values < 0.05 were considered statistically significant . 
the data analysis was performed using spss 20.0 software ( armonk , ny : ibm corp )  . results subjects data a total of 6516 subjects ( 4499 men ( 69% , mean age of 58.1years ) and 2017 women ( 31% , mean age of 59.1years ) , overall mean age of 58.4years and mean body - mass - index of 27.69 ) underwent wb - ct . according to age , we classified them into four groups : group 1 ( 4049years , 324 women and 826 men ) , group 2 ( 5059 years , 776 women and 1806 men ) , group 3 ( 6069 years , 589 women and 1294 men ) and group 4 ( > 69years , 328 women and 573 men )  . radiological findings global ct results only 1.60% ( 104 / 6516 ) of individuals had a completely normal wb - ct . 
a physician from a specific medical check - up - unit evaluated every patient and determined the indication of performing a ct based on a table of risk factors previously defined by a multidisciplinary committee of experts . 
all these findings are important since they would encourage for risk factor modification , specific treatments and appropriate follow - up . the abdominal findings we observed were more numerous than those described with other non - ionizing examinations , such as ultrasound , performed in asymptomatic patients . 
therefore , wb - ct allows detecting a greater number and type of significant positive abdominal findings compared to ultrasound . 1 3 la radiologia medica ( 2019 ) 124 : 11991211 1209 as described by furtado etal . , in our cohort , we observed male predominance and we did not observe statistically significant differences in the main radiological findings between both sex groups , except for emphysema , bronchiectasis , bronchial disease and coronary calcium . in line with previous studies , the proportion of patients with significant radiological findings increased with age , except for emphysema and fatty liver , in which the distribution between the four age - groups was similar . in our cohort , a total of 1179 recommendations were performed . 
 [ 20 ] described abdominal recommendations more frequently ( 289 of 1192 patients )  . since ct is a technique available but expensive and uses ionizing radiation , it is controversial to be considered at the moment in a screening context [ 12 ]  . 
we do not intend to defend this exploration as a screening method , but we believe it can be considered as a useful tool in specific patients to identify important abdominal pathologies , when they are still asymptomatic , especially in a context of medical check - up - unit , where a physician evaluates each patient and determines the need to indicate or not a ct . one of the main discussible problems of wb - ct is the radiation dose . 
although some articles support that no dose of radiation is safe , the number of predicted radiation - induced cancers is tiny compared to the very large cancer incidence rate in humans , making statistical limitations for evaluating the impact of radiation on cancer rate [ 1 , 2429 ]  . 
that is the reason why we consider a physician should provide the indication for a wb - ct . some authors have debated the ethical problem of overimaging using x - ray such as salerno etal . 
this equipment employs an automated dose management that adapts scanning parameters to any patient , allowing to use high - quality ultra - low - dose imaging with a tin filter , providing significantly optimized dose efficiency . 
 however , radiologists adherence to these guidelines is not universal [ 3336 ]  . it is important to note that wb - ct in all patients of this study was indicated by a physician specialized in internal medicine . 
this had been already defended in recent articles , such as mazzei and volterrani [ 37 ] who defended that both clinicians and radiologists should be involved in choosing the most appropriate diagnostic imaging for each patient and each pathology . besides , the majority of patients who come to a check - up unit are people intrinsically concerned about their health status . 
in general , they feel more anxious about not to know how they are at that moment , than for the possible nonsignificant findings that may be detected in the wb - ct . incidental findings in radiologic explorations are becoming more frequent due to the improvement of the different radiological techniques , which implies greater sensitivity in the detection of pathological and non - pathological findings . 
 hence , the importance of radiologists , who should transmit in their reports the need to perform determined follow - up or other complementary techniques , depending on the incidental finding . 
this information will be also very helpful for the physician , who has requested the ct , to understand the relevance of a particular findings and , consequently , to adequately explain 1 3 1210 la radiologia medica ( 2019 ) 124 : 11991211 to the patient the importance of thethis fact highlights the importance of a fluent radiologistphysician and physicianpatient communication [ 38 ]  . thing is that many of these findings were detected at an early stage , allowing less aggressive and sometimes curative treatment . the last controversy of this technique is the cost - effectiveness [ 31 , 39 ]  . 
some previous works have found low prevalence of cancer in the evaluated populations and high rate of falsepositive findings that contribute to increased cost , individuals anxiety and over diagnosis , leading to unnecessary follow - ups and treatments . we think there is not enough information to use this exploration as a screening strategy . 
but on the basis of our results , although further research is needed to optimize wb - ct programs , we think this technique may be useful in a multidisciplinary well - planned check - up unit where radiologists and internal physicians maintain a close relationship in order to obtain the maximum benefit of diagnostic tests requested and to improve the patients health . 
with a standardized reporting system , a medical control about recommendations and a wellmanaged follow - up system , wb - ct may have an important role in providing real benefits to patients from early diagnosis of disease and changes in lifestyle to improve global health . the main limitation of this study is that our findings were based on a retrospective review of radiologist reports . 
also , the explorations were interpreted by different expert radiologists , and in most of relevant findings , the radiologists used the same consensus guidelines , such as the elcap protocol for lung nodules or the agatston classification for coronary calciu however , other non - tumoral findings were classified as mild , moderate or severe , according to a qualitative method , being more suggestive of undergoing inter - observer variations . 
 however , these variations were probably not very significant , taking into account that they were analyzed by expert radiologists in a specific area ( thorax or abdomen )  . a second limitation is that although the majority of findings ( mainly tumors ) were confirmed , few patients did not come back to our hospital and we could not achieve the final diagnosis . 
in these years , various changes , mainly in relation to radiation dose optimization and recommendations , were improved . finally , we did not evaluate the development of other diseases in short - term or long - term follow - up . 
therefore , we only have a complete assessment of the health of a specific individual at a particular time point , and we cannot predict the development of a future pathology . conclusion this examination allows identifying a number of non - depreciable findings that significantly increasing with age , highlighting different types of tumors . 
and the most important however , it should not be forgotten that most of findings are benign , so individuals should be aware of the high number of benign positive results that are diagnosed with this test and the unknown value of a negative test . the development of new radiological equipment in the recent years has increased the quality of the image with a significant decrease in the dose . 
given the lack of available literature in this line of research and our recent publication [ 2 ] discussed by ghaffari , we would like the opportunity to comment and provide our respective opinion . 
we have been utilizing rectal spacer implants in our practice over the last 7years [ 36 ] and have not had any major adverse events to report and , therefore , have recommended it as a safe and effective adjunct treatment for post - prostatectomy radiotherapy . 
ghaffari commented on multiple aspects of our work that we would like to address . first , conducting a randomized two - arm study is of potential value ; however , given ours is a large community cancer clinic ( and not an academic institution ) , we had interest in providing this treatment to all patients electing to receive hydrogel spacer implantation . 
additionally , given our radiation therapy protocols had changed prior to the use of our hydrogel rectal spacer implant practice in post - prostatectomy patients , we did not find value in comparing the outcomes against our own historical controls . 
this is a valid methodology in clinical practice medicine . second , we agree that rectal dosimetry planning preand post - implant would be of value to the field and plan to complete this study in the near future as an adjunct to the original presentation of our work . 
however , hydrogel rectal spacer implants are now covered by the majority of insurance companies in the usa as an authorized procedure with little to no cost to the patient . 
we would be keener to suggest that the use of a rectal retractor may be more uncomfortable to the patient , especially with a fractionated regimen for post - prostatectomy radiotherapy [ 7 ]  . third , in regard to ctv volume reporting , we did not find it necessary to report this given we had reported the ptv volume and mean dose in our original manuscript . 
additionally , in the manuscript we had defined for the first 25 fractions , the ctv as the residual prostatic bed , plus internal / external iliac nodes , and for the subsequent 15 fractions the ctv was defined as the residual prostatic bed plus margin [ 2 ]  . lastly , we welcome ghaffaris suggestion for the consideration of alternative technologies for rectal displacement from radiotherapy targets , namely his rectal retractor . 
the field of rectal spacer implants for prostate radiotherapy has evolved over the years , and many technologies have been utilized , including hyaluronic acid [ 3 , 4 ] , collagen [ 8 ] , and blood patch [ 9 ]  . 
the hydrogel spacer implant has withstood robustclinical investigations over the last decade and continues to be used as a valid technique for prostate spacing [ 5 , 10 ]  . 
1 , east jian she road , zhengzhou450052 , china 2 department ofhistology andembryology , college ofbasic medicine , zhengzhou university , zhengzhou , china although rare , pus in the mediastinum or pleural cavity may corrode vessels and result in an even higher mortality [ 7 ]  . 
over the past two decades , various conservative management protocols have been reported , such as endoscopic transluminal drainage or clipping , metallic stent placement and the application of biodegradable fistulae plugs or fibrin glue [ 4 , 810 ]  . 
despite these therapeutic modalities , treating an esophagogastric anastomotic leakage remains challenging and the optimal treatment has not yet been determined [ 2 , 4 , 11 ]  . considering the patients pathophysiologic changes and anatomic characteristics , we designed a protocol consisting of three - tube method ( abscess drainage tube , gastrointestinal decompression tube and jejunal feeding tube ) with or without temporary covered esophageal stent placement . 
this study enrolled all patients with esophagomediastinal and esophagobronchial fistulas after esophagogastro - anastomosis , who received three - tube method ( jejunal feeding tube , and thoracic drainage tube , gastrointestinal decompression tube and jejunal feeding tube placement ) with or without temporary esophageal stent placement in our department between january 2013 and february 2018 . 
a esophagography showing the site of fistula in the upper esophagus and irregular abscess in the mediastinub esophagography shows that the abscess cavity disappeared 1.7months after transnasal drainage tube placement . 
c , d at 1.7months after three - tube treatment , a chest ct scan in the mediastinal window shows disappearance of mediastinal abscess , re - expansion of the lungs with no pleural effusion 1 3 la radiologia medica ( 2019 ) 124 : 12531261 1255 fig . 
c , d at 1.6months after three - tube treatment and stent placement , a chest ct scan in the mediastinal window shows disappearance of esophagopleural fistula and reexpansion of the left lungs chest ( figs.2a , b ; 3a , b )  . 
esophageal covered stent placement is used for patients with enough length ( > 100mm ) of esophagus to effectively fix stents to avoid stent migration or failure of stent placement . 
the catheter tip was delivered into the distal end of the abscess cavity and then exchanged with a 5 - f pigtail catheter ( cook medical , inc . , bloomington , in , usa ) or a 5 - f straight catheter ( cook medical , inc . , bloomington , in , usa )  . 
a jejunal feeding tube was inserted into the proximal jejunusimilarly , gastrointestinal decompression tube was introduced into the gastral cavity . esophageal stent placement eight patients were treated with fluoroscopic placement of retrievable covered esophageal stent ( nanjing micro - tech medical company , nanjing , china )  . 
the diameter of the stents ranges from 18 to 20mthe length of the stents ranges from 7 to 14cthe stent was chosen based on the leakage location , and adequate stent coverage is allowed on either side of the leakage to ensure complete occlusion , with 3 to 5cm longer than the leakage . 
d after removal of stent and drainage tube , esophagography shows that the contrast agent flows though the esophagus without any leakage saline was injected and irrigated through the drainage tube twice a day . 
the covered stent and drainage tube was removed when chest ct showed complete disappearance of abscess cavity , full re - expansion of the lungs , with no pleural effusion ( figs.2c , d ; 3c , d )  . results general information this study involved a total of 23 patients with esophagogastric anastomotic leakage , including 19 men and 4 women ( table1 )  . 
 there were 17 cases of esophagopleural fistula , and six patients showed esophagomediastinal fistula . intervention andcomplications three - tube method was used for all patients , of which , eight patients received covered stent placement . 
a total of 10 covered esophageal stents were placed , with a median diameter of 20mm , median length of 120mall patients underwent abscess drainage tube placement or exchange of chest tube . 
no massive hemorrhage , esophageal 1 3 la radiologia medica ( 2019 ) 124 : 12531261 1257 1 3 1258 la radiologia medica ( 2019 ) 124 : 12531261 1 3 la radiologia medica ( 2019 ) 124 : 12531261 1259 rupture or other complications occurred during procedures . 
no grade 4 , grade 5 or grade 6 complications were observed . postoperative management all patients showed a reducing amount of pus daily , and the drainage fluid gradually became clear . 
chest ct , esophagography and radiographic examination showed that the abscess cavity had markedly decreased in seven patients or disappeared in 16 cases , indicating that continuous negative pressure suction via the drainage tube was effective . 
thirteen patients were able to return to their normal living conditions without any symptoone patient showed slight reflux with no need of medicine , and two patients showed mild choking feeling due to anastomotic restenosis . 
various 1 3 1260 la radiologia medica ( 2019 ) 124 : 12531261 conservative management protocols have been reported , such as endoscopic transluminal drainage or clipping , metallic stent placement and the application of biodegradable fistulae plugs or fibrin glue [ 4 , 810 ]  . 
esophageal stent placement is an alternative approach , which was initially used as palliative treatment for dysphagia in patients with esophageal cancer . recently , applications of esophageal stent have expanded to treat benign disease [ 14 , 15 ]  . 
owning to the ability to be removed easier with less damage , plastic stents are commonly used for the treatment for leaks and strictures of benign disease [ 1618 ]  . 
however , metallic stents also have been used for temporary treatment for benign diseases in recent years ; the appropriate type of stent for anastomotic leakage remains controversial [ 14 ]  . successful management of an esophagogastric anastomotic leakage requires prompt elimination of ongoing contamination by insertion of the covered stent and by adequate therapy of the associated mediastinal and thoracic infection . 
there was no perioperative death in this study , which is substantially lower than reported in other series [ 16 , 17 , 21 ] or conventional surgical therapy [ 6 ]  . 
in our series , all patients received continue chest drain , with a median duration of 2.5months. the duration between surgery and diagnosis is essential for the clinical outcomes [ 13 ]  . 
only eight patients received stent placement , which prohibit drawing a clear conclusion on the use of the covered stent . in conclusion , interventional treatment for esophagogastric anastomotic leakage can be considered a safe and effective alternative to operative treatment . 
examination protocol includes coronal and axial fisp , t2 - w half - fourier rare and dwi sequences , a baseline coronal t1 - w fat - saturated ultrafast ( gre ) sequence followed by contrast 3d t1 - w gre . 
all images were assessed by two radiologists who graded each of bowel segments for the presence of inflammation on a four - point confidence scale on the basis of wall thickening and wall signal on dwi and adc maps and comparing their results with post - contrast images . 
one false positive case corresponded to the absence of inflammatory histopathology changes at the level of the terminal ileum in a 15 - year - old male , and one false negative case was in a 10 - year - old female with only jejunal lesion . 
 these results emphasize the utility to include the dwi / adc in standard mr enterography protocols and suggest that dwi could replace t1 - weighted post - contrast sequences . keywords crohn disease pediatric mr enterography diffusion small bowel * gabriele masselli gabriele.masselli@uniroma1.it 1 radiology department , umberto i hospital , sapienza university , viale del policlinico 155 , 00161rome , italy 2 pediatric department , umberto i hospital , sapienza university , viale del policlinico 155 , 00161rome , italy 3 radiology department , careggi university hospital , university offlorence , largo g.a. 
vanvitelli , piazza miraglia 2 , 80138naples , italy introduction crohns disease ( cd ) is a chronic inflammatory and remitting disorder of the gastrointestinal tract ; in 25% of cases , it is diagnosed in childhood with an increasing incidence in children . 
one recent study showed that the incidence of pediatric cd has doubled in the usa over the last 12years [ 1 ]  . magnetic resonance enterography ( mre ) is an increasingly important pediatric imaging modality that is most often used to evaluate inflammatory bowel disease ( ibd ) , sparing children and adolescents from potential risks of ionizing radiation exposure [ 26 ]  . 
conventional mre requires vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 13061314 1307 intravenous administration of gadolinium - based contrast agents ( ca ) ; the most current concern about ca in children has been the accumulation of gd in the basal ganglia , which is of uncertain clinical significance but should be considered in patients with ibd , who undergo multiple imaging examinations over time to evaluate symptom recurrence [ 7 , 8 ]  . a non - contrast mre does not need glucagon injection and would improve patient comfort and compliance , reducing scanning time and in some cases avoiding the need for pediatric sedation ; moreover , it would reduce sanitary costs ( in the usa , contrast adds up to $1000 to the mri price for out - of - pocket paying patients ) [ 9 ]  . in this regard , diffusion - weighted imaging ( dwi ) has been increasingly used for abdominal and pelvic examinations due to its ability to measure molecular water diffusion into the extracellular space and to detect microscopic changes of inflammatory processes . 
its values decrease with increased tissue cellularity or cell density and may help in the quantitative analysis of disease activity [ 1012 ]  . dwi appears well adapted for children thanks to rapid acquisition time , a free breathing mode that reduced motion artifacts , high tissue contrast and finally because it obviates the need for contrast enhancement . 
currently , dwi is considered an additional sequence in the mre and only a few studies have been done in order to evaluate its potential and diagnostic accuracy alone and in comparison with standard mri sequences [ 13 , 14 ]  . the aim of this study was to determine whether mre performed with dwi sequences , without administration of intravenous gd - ca , is comparable to contrast - enhanced mre ( ce - mre ) in the detection of small - bowel inflammation in pediatric patients with cd . materials andmethods patients andmethods this prospective study was approved by the institutional review board of our university . 
parent or guardian informed consent and subject assent were obtained at the time of study enrollment . sixty - eight consecutive patients ( 38 females and 30 males ; mean age 10.3years ; range , 616years ) treated at the ibd pediatric unit of our university hospital were consecutively and prospectively included between april 2015 and june 2018 . inclusion criterion was the diagnosis of cd according to lennardjones criteria [ 6 ]  . 
coronal and axial fisp images were obtained first , and if distension was adequate , t2 - w images were obtained in the coronal and axial planes with a singleshot half - fourier rare sequences ( i.e. , haste half - fourier acquisition single - shot turbo spin echo )  . coronal dwi and axial dwi were obtained by using a single - shot spin - echo - type echo - planar imaging ( epi ) sequence with fat suppression and parallel technique ( reduction factor 2 )  . 
imaging parameters for dwi were as follows : tr / te 2500 ms / 80 ms ; matrix 128 128 ; field of view 280 400mm ; slice thickness / gap 6mm / 1mm ; 6 averages ; bandwidth 1930hz / pixel . 
the time required to acquire the dwi set was 3mb values of 50 , 400 and 800s / mm2 , as used in other abdominal applications [ 13 ] , were employed ( table1 )  . 
isotropic adc maps were generated with a commercially available software workstation system ( leonardo ; siemens , erlangen , germany ) using all b values and taking an average value of the three directions of diffusion sensitization . 
the bowel wall was considered to be thickened when it was > 3mon dwi , grading scores of 0 and 1 were regarded as indicating normal bowel wall and scores of 2 and 3 bowel wall inflammation . on t2 - w images , we analyzed the following signs : wall thickness ( setting a grading score of 1 : 3mm , 2 : 46mm and 3 : > 6 mm ) , submucosal edema , aphthous ulcers , lymphadenopathy . on t1 - w post - contrast images , we analyzed the following signs : comb sign ( defined as mural stratification with engorged vasa recta that penetrate the bowel wall perpendicular to the bowel lumen ) , mesenteric hyperemia and stratified contrast enhancement of the mucosa . to minimize recall bias , two reading sessions were performed 4weeks apart from each other and mr images ( t2 , contrast t1 and diffusion - weighted images ) were randomly analyzed during the different sessions . patient information was removed from all images . 
during each imaging analysis , the readers were asked to determine the presence and location of small - bowel inflammation . moreover , after 3weeks , during a third interpretation , the two readers ( 1 ) resolved discrepancies in consensus and determined the diagnoses that were compared with the reference standard diagnoses ; and ( 2 ) evaluated image quality of t1 - weighted images post - contrast and dwi images by using a point scale : a score of 0 indicated non - diagnostic images ; 1 , diagnostic images with numerous artifacts ; 2 , diagnostic images with a few artifacts ; and 3 , diagnostic images without artifacts ; ( 3 ) objective measurements of adcs signal intensity were calculated using the standard tools of the workstation . 
regions of interest ( rois ) on the adc maps were drawn by an mr imaging physicist ( s.s. , 10years of experience in abdominal mr imaging ) and the two radiologists in consensus by using visual correlation of the adc maps with the anatomic information derived from the corresponding t2 - weighted images . 
three values for each zone were recorded , and the final adc value was the average . 1 3 la radiologia medica ( 2019 ) 124 : 13061314 1309 the largest possible polygonal - shaped regions of interest ( rois ) , ranging from 8 to 18mm2 , were placed at the level of the small - bowel wall . inflammation ( eais = 0 ) and those with histopathological inflammation ( ais score > 1 ) using the mannwhitney u test . reference standards the standard of reference for the presence of active lesion of ibd was histopathological findings ( obtained by biopsy during endoscopic examination in 63 cases and by surgical specimens in 5 cases : 1 lesion of the jejunum and 4 lesions of the ileum )  . the mr findings were correlated with the corresponding histological references for each segment by a coordinator not involved in the studies . histopathology activity score the intestinal biopsies were stained with hematoxylineosin and retrospectively reviewed by an experienced pathologist > 10years of experience , who was unaware of clinical information or mri findings . 
the histopathologist applied an endoscopic biopsy acute inflammatory score ( eais : table2 ) based on the typical morphological features of crohns disease described in guidelines published by the european crohns and colitis organization . at least three samples of biopsy were collected for each patient , and the highest score for each was used for that patient , in accordance with the standard procedure in our institution . statistical analysis quantitative variables are given as means and standard deviation ( s.d. ) or as medians in the case of an abnormal distribution . 
proportions are expressed as percentages and 95% confident intervals ( cis )  . differences in quantitative measures were tested by students test or kruskalwallis test if the conditions of students test were not met . 
qualitative variables were compared by exact fishers test or chi - squared test . using the histopathological standard of reference , adc values were compared between patients with no histological table 2 histopathology grading for acute inflammation score ( ais ) histological variable erosion or ulceration polymorphs in the lamina propria cryptitis crypt abscess formation inflammatory exudates granulomas grade 0 = no , 1 = yes 0 = no , 1 = yes 0 = no , 1 = yes 0 = no , 1 = yes 0 = no , 1 = yes 0 = no , 1 = yes eais scores were then compared across qualitative dwi grades using kruskalwallis test with post hoc correction . the sensitivity and specificity of dwi grades 2 and 3 for active disease ( ais > 1 ) were calculated . 
a threshold was determined by calculating receiver operating characteristic ( roc ) curves . all correlations were studied using spearmans nonparametric correlation coefficients ( r and p value , respectively )  . 
calculations were done with stata v10 ( stata corp . , college station , tx , usa )  . the strength of agreement was considered poor for values less than 0.20 , fair for values of 0.210.40 , moderate for values of 0.410.60 , good for values of 0.610.80 and very good for values of 0.811.00. results the mean time between mre and endoscopy with biopsy was 5.5days ( range 121days )  . based on the histopathology , findings were classified 48 / 68 ( 70% ) patients in acute inflammatory phase and 20 / 68 ( 30% ) patients in no - acute inflammatory phase , respectively . baseline patient characteristics are shown in table3 . 
particularly , this last characteristic could avoid the venous access , the risk of adverse reactions to the contrast medium and minimize patient discomfort particularly important in the pediatric population that can preclude their cooperation at this examination . there are several advantages in using dwi instead of ce t1 - w images : a shorter examination duration ( if postgadolinium mr imaging is not performed ) and no need for catheter placement and for glucagon injection . in our study , images obtained by dwi displayed better image quality than ce t1 - w images . 
a coronal steady - state true fisp image showed thickening of the wall of the terminal ileum ( arrows ) with evidence of increased layered enhancement on b coronal post - contrast t1 - weighted image . 
e axial t2 - weighted single - shot fast spin - echo image with fat saturation shows thickness of the wall of the terminal ileum ( short arrow ) with a hyperintense fluid collection ( arrow )  . 
in the future , further studies investigating dwi findings using different b values may determine their role in the detection of bowel inflammation . finally , comparison of imaging results with histological activity as gold standard is debatable since inflammation in crohns disease is discontinuous , leading to sampling error for histological assessment . 
transmural inflammation in crohns disease may also mean that histological mucosal healing does not represent quiescent disease , since deeper inflammation , although invariably in the form of lymphoid hyperplasia , and therefore reflecting chronic inflammation rather than acute inflammation , may persist [ 34 , 35 ]  . in conclusion , our study has shown that dwi sequences with the corresponding adc maps have a high accuracy in detecting the bowel segment affected by cd and that , associated with the standard t2 - weighted images , allows to increase the diagnostic accuracy . 1 3 la radiologia medica ( 2019 ) 124 : 13061314 1313 fig . 
a coronal t2 - weighted haste image showed mild thickening of jejunal small - bowel loop ( arrows ) with no evidence of increased enhancement on b coronal post - contrast t1 - weighted image . 
based on the restriction of the diffusion cd in the active phase was diagnosed that was confirmed by endoscopy this evidence suggests the utility to include the dwi / adc in the standard protocols of mr enterography . 
dwi could replace t1 - weighted post - contrast sequences for detecting small - bowel acute inflammation in patients with cd , avoiding injection of contrast medium and reducing the examination time making the examination better tolerated by pediatric patients . compliance with ethical standards conflict of interest the authors declare that there is no conflict of interest regarding the publication of this paper . ethical standards the present study was conducted in accordance with the institutional ethical committee of sapienza university of rome . 1 3 1314 la radiologia medica ( 2019 ) 124 : 13061314 informed consent parent or guardian informed consent was obtained at the time of study enrollment . la radiologia medica ( 2019 ) 124 : 13241332 radiotherapy locally advanced inoperable primary orrecurrent nonsmall cell lung cancer treated with4week hypofractionated radiation therapy ( 3gy / fraction ) mauriziovaleriani1 mattiafalchettoosti1 lucamarinelli1 lucanicosia1 chiarareverberi1 vitalianadesanctis1 davidemollo1 received : 7 february 2019 / accepted : 7 july 2019 / published online : 17 july 2019 italian society of medical radiology 2019 abstract background the prognosis of locally advanced non - small cell lung cancer ( nsclc ) treated with conventional radiotherapy remains poor . 
hypofractionation reduces overall treatment time increasing biological effect in patients not suitable for concurrent chemo - radiotherapy . method from january 2009 to october 2016 , 76 inoperable locally advanced primary or recurrent nsclc patients were treated with 60gy in 20 fractions of 3gy / each for 4weeks as exclusive or post - chemotherapy treatment . 
univariate and multivariate analyses demonstrated that patients with complete response presented better outcomes , whereas no statistically relevant difference was evidenced in terms of previous chemotherapy , recurrent vs primary disease , volume and stage . 
complete local response was a predictor of better outcomes , and any efforts will be made to perform prospective clinical trials to further evaluate hypofractionated regimens with increased lesional bed . keywords locally advanced nsclc radiation therapy 3d - rt hypofractionation introduction lung cancer is the leading cause of death related to cancer [ 1 ]  . 
nevertheless , the prognosis remains poor , with a 5 - year * maurizio valeriani mauval1@libero.it 1 department ofradiation oncology , sant andrea hospital , sapienza university , via di grottarossa 1035 - 1039 , 00189rome , italy overall survival ( os ) rate ranging from 5 to 10% [ 5 ] and a median survival time approximately of 16 to 17months after concurrent treatment ; intensification of regional heed has been attempted to improve local control and survival rates [ 6 ]  . 
treatment intensification may be obtained also by shortening the overall treatment time with the purpose to reduce accelerated repopulation of tumor cells that could start 46weeks after the beginning of treatment [ 8 ]  . 
these studies demonstrated good local control with acceptable acute and late toxicity rates after hyport based on three - dimensional conformal radiation therapy ( 3d - crt ) modern planning system [ 10 , 11 ]  . based on these postulations on 2009 , we started to treat patients affected by inoperable advanced - stage nsclc not suitable to concurrent chemo - radiotherapy for comorbidities . 
 response , local control , toxicity rates and survivals were evaluated . methods andmaterials patients we retrospectively collected data regarding 76 histologically confirmed inoperable locally advanced primary or recurrent nsclc patients treated with curative hypofractionated radiotherapy in our department between january 2009 and october 2016 . 
all patients had performance status ( eastern cooperative oncology group criteria ) 2 . pre - treatment evaluation consisted of contrast - enhanced total body computed tomography ( ct - tb ) scan , bronchoscopy with histological examination , respiratory functional tests , 18 - fluorodeoxyglucose positron - emission tomography ( fdg - pet / ct ) scan . 
all patients signed written informed consent . treatment all patients underwent pre - treatment ct planning in the supine position , merged with diagnostic ct and pet - ct for the target volume delineations ( gross tumor volumegtv , clinical target volumectv )  . 
the gtv1 and the gtv2 included the primary tumor and the clinically positive lymph nodes with a short - axis diameter 1cm and / or positive at pet - ct scan . 
during treatment , all patients underwent medical examination and blood test examinations . followup andstatistics the first follow - up was planned 2months after rt completion with contrast - enhanced ct - tb and blood test examinations ; then , follow - up period was about 3months for the first 2years after rt completion and every 6months afterward . 
 fdg / pet - ct was performed until 6months after rt or for suspected radiological progression . toxicities were evaluated using the common terminology criteria for adverse events ( ctcae ) version 4.0 and were classified as acute ( within 3months from rt ) or late ( after 3months from rt )  . the response evaluation criteria in solid tumors ( recist 1.1 ) measurement was used to determine response rates and progression of disease [ 13 ]  . 
patients , who underwent local or distant progression , were treated with rt or local therapy when possible and / or systemic therapy . kaplanmeier method was used for univariate analysis and cox regression model for multivariate analysis . 
progression - free survival ( pfs ) and the loco - regional pfs ( lr - pfs ) were defined as the time to loco - regional / systemic progression or death and the time to loco - regional progression , respectively . 
subgroup analysis was performed by stage ( iiia vs iiibiiic vs iva ) , chemotherapy ( cht ) prior to rt ( yes vs no ) , type of disease ( primary vs recurrent disease ) , responses ( cr vs pr vs nc - pd ) and volume ( < 250cc vs 250cc ) ( table1 )  . statistical analysis was performed using the spss statistical software package version 24.0 ( spss , inc . , chicago , il , usa )  . 
previous cht ( p = 0.518 ) and volume ( p = 0.173 ) were not statistically relevant . fifty - eight patients ( 76.4% ) progressed : 12 ( 15.8% ) only loco - regionally , 23 ( 30.3% ) only at distance and 23 ( 30.3% ) both loco - regionally and at distance . 
previous cht ( p = 0.303 ) , type of disease ( p = 0.342 ) , volume ( p = 0.620 ) and stage ( p = 0.148 ) were not statistically relevant . forty - six patients ( 60.5% ) developed distant metastasis in one or multiple sites ; contralateral lung was involved in 17 cases , brain in 14 , bone in 16 and liver in 7 . 
 no correlation between dosimetric parameters and grade 2 or 3 lung toxicity was found , whereas grade 2 or 3 acute esophageal toxicity was correlated with mean dose ( > 13.5 gy ) and v42 ( > 20% )  . 
data are summarized in table5 . discussion up to date , there is an increasingly interest to hypofractionation for the treatment of locally advanced nsclc with the aim to intensify the treatment and to shortening the total treatment time . 
in the past , the use of hypofractionation was limited by concerns about high rate of late toxicities until improvement of planning and delivery techniques was empowered . therefore , a significant percentage of patients develop an advanced - stage nsclc presenting comorbidities , advanced age , poor performance status and potentially poor tolerance of standard concurrent chemo - radiotherapy ( crt )  . 
then , many different hypofractionated schedules were used with doses ranging from 45gy in 15 fractions to 84gy in 35 fractions [ 1519 ]  . minding all the above points , we analyzed in our study a cohort of inoperable locally advanced primary or recurrent nsclc patients , not suitable to concurrent chemoradiotherapy for comorbidities , with exclusive or postchemotherapy hypofractionated radiotherapy . 
a randomized eortc study [ 25 ] reported a median survival similar to our ( 17months ) in patients treated with 2 cycles of induction 1 3 la radiologia medica ( 2019 ) 124 : 13241332 1331 cisplatin and gemcitabine or concurrent low dose daily cisplatin and 66gy in 24 fractions . 
six out of 75 patients ( 8% ) presented grade 45 toxicities ( for damage of proximal bronchial tree and surrounding vasculature ) , and most of them received a total dose of at least 75gy . hypofractionation was generally well tolerated with three studies reporting any significant differences in terms of toxicities with respect to conventional fractionation . 
our study presented some limitations : retrospective evaluation , limited number of patients and wide - range interval in recruiting study population . conclusion outcomes in locally advanced nsclc remain non - satisfactory , and concurrent chemo - radiotherapy represents , actually , the standard treatment . 
cdi should be suspected in case of unexplained diarrhea and abdominal pain in patients with a recent history of antibiotic use and healthcare exposures ; diagnosis is based on a combination of clinical and laboratory findings with demonstration of c . 
considerable overlap exists between the cect findings of cdi and those of colitis of other origins , such as typhlitis , ischemic colitis , graft - versus - host disease , radiation colitis and inflammatory bowel diseases ; however , some features may help distinguish between these conditions . 
this paper provides a comprehensive overview of the imaging features of clostridium difficile colitis and its mimics , with a view to assist the radiologist in reaching the correct diagnosis . keywords clostridium infections pseudomembranous enterocolitis differential diagnosis contrast - enhanced computed tomography abbreviations c . 
difficile can colonize and proliferate in the gut of individuals with altered microbiota , producing two major exotoxins ( toxin a and toxin b ) able to damage the human colonic epithelium and to stimulate the inflammatory response ; histologically , the consequence of mucosal necrosis is the formation of pseudomembranes , an inflammatory exudate overlaying the denuded mucosa [ 1 , 2 ]  . 
the incidence of clostridium difficile infection ( cdi ) has dramatically increased in the last decades , and estimates predict a continuing increase in the coming years [ 3 ] ; today cdi is a leading cause of hospital - associated infections in europe ( about seven cases for every 10 , 000 hospitalized patients ) ; incidence is similar in the usa , where cdi causes 14 , 000 deaths each year [ 1 ]  . 
a considerable rise in mortality was observed since the worldwide emergence of new vol . : ( 0123456789 ) 1 3 1186 la radiologia medica ( 2019 ) 124 : 11851198 hypervirulent strains , such as c . 
clinicians should consider the possibility of cdi in patients with unexplained diarrhea associated or not with abdominal pain , in patients with a recent history of antibiotic use and healthcare exposures [ 6 , 7 ]  . 
however , severe cases of cdi have been reported in otherwise healthy patients , without traditional risk factors [ 8 ]  . the diagnosis of cdi is based on the combination of clinical and laboratory findings and requires the demonstration of c . 
although it is not routinely part of the evaluation of cdi , colonoscopy with biopsies may help to confirm the diagnosis of cdi ruling out other colonic etiologies , particularly when results from stool testing are negative or still pending [ 9 , 10 ] ; however , colonoscopy is an invasive procedure with an associated risk of bleeding and perforation . contrast - enhanced computed tomography ( cect ) plays an important role in the management of patients with suspected colonic disease thanks to its ability to evaluate both the colonic wall and the adjacent soft tissues ; cect also helps in determining the extent and severity of the disease and in detecting potential complications , such as toxic megacolon and perforation [ 11 , 12 ]  . 
however , a significant number of patients shows no abnormalities on abdominal cect scan and often cect findings do not allow a reliable distinction to be made between cdi and other colonic diseases ; the list of possible differential diagnoses is long and includes , among others , typhlitis , ischemic colitis , graft - versus - host disease ( gvhd ) , radiation colitis and inflammatory bowel disease ( ibd ) [ 13 , 14 ]  . 
the aim of this paper is to review the cect findings that can help to identify and differentiate cdi from other inflammatory conditions of the colon . imaging features ofclostridium difficile colitis since clinical presentation can be unclear or misleading and due to the in - depthness of the differential diagnosis , computed tomography plays a crucial role in evaluating the patient , especially in case of severe abdominal paeven if the examination protocol is not fixed and should be adapted to each situation , in patients with abdominal pain with suspicion of a colonic origin , a triple - phase ct protocol ( unenhanced , late arterial and portal venous phase ) of the abdomen and pelvis is indicated . 
the standard intravenous contrast material is a nonionic , low - osmolar iodinated one with a concentration between 350 and 400mgi / ml , administered using a power injector at a rate of 34ml / s with a bolus tracking technique . 
a delayed arterial phase ( 15s after peak aortic enhancement ) is optimal to depict the enhancement pattern of colonic wall and is required for the assessment of mesenteric arterial supply ; portal phase ( 7080s ) allows to exclude other causes of acute abdominal pain and to outline the venous drainage . at our institution , oral contrast material is not routinely administered because it is time - consuming ( at least 6090min for adequate opacification of the entire colon )  . 
 however , oral contrast agents can be used to improve image quality ; in this case , low - density oral contrast agents are preferable because they do not obscure mural enhancement . intestinal ct findings intestinal cect findings suggestive of cdi include colonic wall thickening and colon wall nodularity . colonic wall thickening represents the most common finding in cdi ; it can be circumferential or eccentric and it is generally more prominent in comparison with other inflammatory colonic diseases with the exception of crohns disease [ 14 ]  . 
in the majority of the cases , cdi presents as a pancolitis with a marked thickening that can be seen throughout the whole colon ( fig.1 ) ; in up to 3040% of the cases , there is a limited involvement of the right colon , and in other cases , the disease starts in the rectum and extends proximally to the left colon [ 14 , 18 ]  . 
some cases of small bowel involvement have been reported in the literature , mainly as isolated case reports , particularly in patients with surgically altered small bowel anatomy [ 1922 ]  . the administration of intravenous contrast material provides several important advantages in assessing wall abnormalities . 
wall thickening is typically irregular and often asymmetric with a shaggy internal outline , probably due to mucosal edema , and cect can better depict the presence of nodular swelling projections ( wall nodularity ) protruding into the lumen ( the ct equivalent to thumbprinting classically seen in barium enema ) [ 15 ]  . 
 ( 64 patients who underwent abdominal ct scan within 3days of stool sample for cytotoxin assay ) , the presence of wall nodularity was statistically associated with a more severe clinical disease , regardless of the degree of colonic involvement [ 23 ]  . in association with thickened colonic wall and wall nodularity , other cect findings can be a clue for diagnosis . 
 mucosal hyperemia and submucosal inflammation can give rise to an abnormal pattern of wall enhancement , with a 1 3 la radiologia medica ( 2019 ) 124 : 11851198 1187 fig . 
1 clostridium difficile colitis : axial ( a , b ) and coronal ( c ) cect images show marked and irregular wall thickening ( maximum thickness of 23 mm ) , with intense enhancement of the mucosa ( white arrows )  . 
mild inflammatory changes are present in the pericolic fatty tissue ( dashed arrow ) stratified multilayered appearance of colonic wall ( target sign ) [ 14 ]  . even if it is not always possible in urgent cases , oral contrast material can be helpful in order to distend the colon and to better define its luminal surface . 
2 clostridium difficile colitis : axial cect scan demonstrates low - attenuation wall thickening involving the entire length of the colon with intense mucosal enhancement ( white arrow , a ) ; the enhancing mucosa is stretched over the edematous haustral folds , resembling an accordion even in the absence of intra - luminal contrast ( black arrow , b )  . 
a large amount of ascites is also present ( dashed arrow , c ) 1 3 1188 la radiologia medica ( 2019 ) 124 : 11851198 extraintestinal ct findings pericolic stranding and ascites are the most common extraintestinal findings . 
in some series , ascites has been reported in a high number of patients with cdi and can serve as a useful adjunct in the differential diagnosis with crohns disease [ 13 , 14 , 16 , 25 ]  . 
however , ascites is not specific since it can be seen in a large variety of other inflammatory and infectious diseases and also in case of coexisting medical conditions ( e.g. , portal hypertension , congestive heart failure and metastatic cancer ) [ 26 ]  . as mentioned above , a considerable rise in mortality was observed following the spread of hypervirulent strains of cd . 
to the best of our knowledge , only one study analyzed the ct features of cdi after the outbreak of epidemic hypervirulent strains , trying to outline the existence of differences in ct presentation with the classic form of cdi . 
retrospectively reviewed the abdominal ct of 165 patients with confirmed cdi ( ct performed within 72h from positive stool sample examination ) ; patients were stratified in two groups depending on the year of diagnosis ( 19982002 , 43 patients ; 20032006 , 122 patients ) , which corresponds , respectively , toa low or high likelihood of 027 hypervirulent strain infection . 
the following ct features were reviewed : increased colonic wall thickness , diffuse colon involvement ( pancolitis ) , target sign and the presence of pleural effusion , ascites and subcutaneous edema . 
difficile , and the radiological presentation of patients with severe cdi was similar either before or during the epidemic period ( 20032006 ) [ 27 ]  . differential diagnosis due to the poor specificity and the overlap of imaging findings between different colonic diseases , the radiologist should be aware of the extensive differential diagnosis and analyze the diagnostic clues and their distribution carefully in order to reach a correct diagnosis of cdi . typhlitis typhlitis , also known as neutropenic colitis , is a life - threatening disease that primarily affects immunocompromised patients [ 28 , 29 ]  . 
in its classic form , typhlitis affects the cecum , which maybe due to its limited blood supply ; the ascending colon and the terminal ileum can also be involved . 
 clinical and laboratory findings are often non - specific : patients typically present with fever , diarrhea abdominal tenderness and abdominal pain predominantly in the right lower quadrant ; intestinal bleeding is usually less common [ 28 , 30 ]  . on cect , findings suggestive of typhilitis include cecal distension and circumferential wall thickening ; the affected colonic wall may show an intramural pattern of low attenuation due to submucosal edema [ 31 ]  . 
 while typhlitis is usually limited to the cecum ( even if any segment of the small and large bowel can be involved ) , cdi is more likely to cause a pancolitis ; however , some of these cases of limited involvement of cecum and right - side colon have been described , making it more difficult to distinguish among the two conditions [ 13 ]  . ischemic colitis ischemic colitis ( ic ) is the most common form of intestinal ischemia , representing about the 70% of the cases [ 33 ]  . 
 ic results from a reduced vascular flow to the colon either for arterial and venous occlusion ( i.e. , occlusive forms ) and low flow states due to hypotension , congestive heart failure , cardiac arrhythmias and hemorrhagic or septic shock ( nonocclusive mesenteric ischemia : nomi ) ; ischemia is usually limited to the mucosa or submucosa , but in severe and advanced cases transmural necrosis may develop [ 34 ]  . 
early colonoscopy performed within 48h of presentation is the gold standard for the diagnosis of ic , but this is an invasive procedure with an associated risk of complications such as perforation ; according to the american college of gastroenterology ( acg ) , the diagnosis of ic can be suggested relying on cect findings ; in case of high clinical suspicions , ct should be the first - line imaging to assess the distribution and the phase of ic [ 36 ]  . cect findings vary in relation to the stage and the extent of the disease , depending on the underlying pathogenesis . 
in vascular occlusive forms , the ischemic injury is followed by reperfusion from riolans arcades collateral vessels : in the acute phase , the colonic wall is thickened and has a stratified appearance due to edema and / or hemorrhage with a subsequent narrowing of the lumen [ 37 ]  . 
the involved tract can show a low - attenuation pattern due to edema ( with a possible target sign on cect ) or , in case of intramural hemorrhage , a hyperdense colonic wall on non - contrast - enhanced ct . 
3 typhlitis : axial ( a , b ) cect images show moderate wall thickening of the cecum and terminal ileum ( white arrows ) in a man with hodgkin lymphoma who has recently completed chemotherapy . 
 coronal cect image ( c ) demonstrates an ill - defined , infiltrating soft tissue mass ( dashed arrow ) in the mesentery that displaces the small intestine and abdominal vessels be present [ 14 ]  . 
if riolans arcade provides an adequate blood supply , the colonic wall returns to normal thickness and pericolic and free fluids gradually decrease , if not the loss of muscular tone and the absence of blood supply lead to the thinning of the bowel wall with a paper - thin appearance [ 37 ]  . 
pneumatosis with or without air in the mesenteric vessels or portal vein is detected in severe cases of ic when transmural necrosis occurs and indicates a poor prognosis [ 37 ]  . 
in chronic stages , weeks or months later or with long - standing arthrosclerosis , fibrotic changes lead to a mild and irregular circumferential thickening of the colonic wall ; gaping lumen and disappearance of the haustral folds are common associated findings [ 38 ]  . since the above - mentioned cect findings are not pathognomonic , emphasis should be placed on geographic distribution of the abnormalities and on the presence of extra - intestinal findings to reach the correct diagnosis . in the occlusive form of ic , the pattern of bowel involvement in ic is related to vascular anatomy , showing a clear demarcation with the non - ischemic tract [ 38 ]  . 
ic is more likely to occur in certain weak points , known as watershed areas , at borders of territories supplied by different vascular systems ; these include the splenic flexure ( griffiths point ) , the retto - sigmoid junction ( sudecks point ) and also the ilealcecal region because of the scarcity of marginal vessels [ 39 ]  . 
the presence of arterial filling defects , aortic dissection with the involvement of mesenteric arteries , small arterial aneurism and occlusion ( in the setting of vasculitides ) and venous thrombosis often with engorged mesenteric veins due to outflow obstruction is typically observed ; the presence of signs of ischemic damage involving other visceral organs ( i.e. , the liver , kidneys , spleen and pancreas ) may help the radiologist , suggesting the diagnosis ( fig.4 ) [ 40 ]  . nomi , which accounts ~ 2030% of all cases of acute mesenteric ischemia , represents a real diagnostic challenge for the radiologist [ 41 ]  . 
as a result of this , the affected colon usually appears dilated with a classic paper - thin wall due to the marked decrease in muscular tone and vascular volume loss ; the geographic distribution simulates an obstruction of both the superior and inferior mesenteric arteries . 
 this presentation may mimic the appearance of normal distended colon and the presence of pericolic fluid often 1 3 1190 la radiologia medica ( 2019 ) 124 : 11851198 fig . 
4 ischemic colitis : axial ( a , b ) and coronal ( c ) cect images show circumferential , symmetric wall thickening and abnormal enhancement of the left colon with a stratified appearance due to edema ( white arrows )  . 
the left kidney is reduced in size due to vascular - based suffering ( e ; open arrow ) and multiple ischemic lesions of the spleen are present ( f ; open arrow ) representing the only other associated finding [ 37 , 38 ]  . 
the reduction in contrast enhancement of other visceral organs , along with the patients clinical history , is an important diagnostic clue [ 38 ]  . graftversushost disease the gastrointestinal tract is one of the principal target organs in acute gvhd ( beyond the first month after allogeneic bone marrow transplantation ) ; the colon can be affected in isolation or as a part of a pan - intestinal involvement [ 42 ]  . 
the development of intestinal gvhd is due to mucosal cell and intestinal stem cells disruption as a consequence of immune dysregulation ; alteration of gut microbiota also plays an important role [ 43 ]  . 
presenting symptoms include diarrhea , with or without hematochezia , and crampy abdominal pain ; these symptoms should raise the suspicion for gvhd or infection . the gastrointestinal tract is one of the principal target organs in acute gvhd . 
the pattern of bowel involvement shows a discontinuous distribution with normal portions of bowels separating the involved tract , and frequently , the small bowel is also affected ; the length of bowel involvement in gvhd is generally less extensive in comparison with radiation - induced colitis [ 24 ]  . 
wall thickening is less marked than in typhlitis and cdi , and although a certain overlap of wall thickness ranges can be seen , a measurement > 7mm should lead to exclude gvhd as a possible diagnosis ( fig.5 ) [ 16 ]  . radiation colitis radiation therapy to the pelvis is often used as adjuvant or neoadjuvant therapy in many gynecologic , urologic and rectal cancers . 
one of the major adverse effects is the development of radiation - induced injuries to the surrounding healthy tissues , such as small bowel and colon [ 46 ]  . 
5 graft - versus - host disease : circumferential wall thickening , with intense enhancement of the mucosa involving the entire length of the colon ( a , b ; white arrows )  . 
this appearance and the extent of colon involvement can mimic that of clostridium difficile colitis , but the thickness of colonic wall is inferior ( maximum thickness of 7mm ) and the small bowel is also affected ( c ; open arrow )  . 
note also the marked engorgement of the vasa recta and fat stranding ( dashed arrows ) acute radiation colitis usually occurs within 24weeks after the treatment ; the clinical presentation is non - specific with diarrhea , abdominal pain and tenesmus , which are usually self - limited [ 46 ]  . 
clinical history is fundamental to suggest the diagnosis , and cect is generally not necessary ; if performed , cect in the acute phase demonstrates wall thickening of the affected tract with adjacent inflammatory stranding ( fig.6 ) [ 14 ]  . 
difficile , a large variety of bacterial , viral and parasitic pathogens are responsible for infectious colitis ; in developed countries , bacteria are the most common cause of infectious colitis and include escherichia coli , shigella , salmonella , yersinia , campylobacter , staphylococcus and chlamydia trachomatis [ 14 ] ; cytomegalovirus is a common cause of colitis in immunosuppressed patients [ 24 ]  . generally , the diagnosis of infectious colitis is based on clinical symptoms and confirmed on the basis of stool analysis and / or colonoscopy [ 48 ]  . cect is generally not required for the diagnosis of infectious colitis , but may be performed incidentally or in cases with equivocal clinical presentation . 
in the acute phase , all these types of infectious colitis manifest with wall thickening ( which usually enhances homogeneously ) , inflammation of the pericolic fat and various degrees of ascites ( fig.7 ) [ 49 ]  . 
colonic distension and multiple air - fluid levels may be seen due to increased fluid production [ 14 ]  . inflammatory colitis ibd include crohns disease ( cd ) and ulcerative colitis ( uc ) , two long - term , relapsingremitting disorders characterized by inflammation of the gastrointestinal tract [ 50 ]  . 
uc typically begins in the rectum and extends proximally ; the inflammation is confined to the mucosa and uniform in distribution with a preponderance of left - sided colon [ 51 ]  . 
cd can affect any segment of the gastrointestinal tract and shows a discontinuous pattern of inflammation with uninvolved areas separated by areas of transmural inflammation ( skip lesions ) ; over 60% of patients show colonic involvement [ 52 ]  . 
6 radiation colitis : axial ( a , b ) and sagittal ( c ) cect images demonstrate smooth wall thickening in the rectum and sigmoid colon ( white arrows )  . 
7 infectious colitis : moderate wall thickening of the transverse colon with homogeneous enhancement ( a ; white arrow ) in a young man with severe diarrhea or abdominal pathe small bowel is also affected ( b ; dashed arrow )  . 
stool cultures were positive for e.coli 1 3 la radiologia medica ( 2019 ) 124 : 11851198 1193 wall thickening is associated with mural stratification ; in the acute phase , the water halo sign is frequently encountered on cect , even if non - specific [ 24 ]  . 
submucosal edema and pericolonic soft tissue changes with dilatation and engorgement of the vasa recta ( comb sign ) are other important additional findings ( fig.8 ) [ 53 ]  . 
fatty proliferation of the mesentery and the presence of pericolic lymphadenopathy are very suggestive of cd [ 24 , 54 ] ; on the other side , proliferation of perirectal fat with enlargement of pre - sacral space is more often seen in uc , even if non - specific [ 14 , 53 ]  . 
radiographs show a total or segmental distension of the colon ( > 6cm in the transverse colon ) with a loss of haustral folds [ 56 , 58 ]  . clinical presentation in association with family history can suggest the diagnosis . 
even in acute onset , the presence of antecedent rectal bleeding is an important clue for the diagnosis [ 11 , 54 ]  . evolution ofclostridium difficile infection with successful therapy , symptomatic improvement is generally observed after 12days , while diarrhea takes more time to resolve ( usually 36days ) [ 59 ]  . 
in case of complicated cdi , ct scan of the abdomen and the pelvis should be performed to determine whether perforation or toxic megacolon is present [ 60 ]  . 
progression to toxic megacolon should be suspected in case of rapid abdominal distension and lessening of diarrhea due to paralytic ileus ; the diagnosis requires radiological proof of colonic dilatation ( diameter of transverse colon > 6cm ) in the presence of signs of severe systemic inflammatory response [ 62 , 63 ]  . today cdi is essentially a medical disease , and surgical intervention is generally not required [ 60 , 63 ]  . 
8 inflammatory colitis : circumferential wall thickening in a patient with cd in the acute phase ( coronal image a , sagittal image b and coronal with zoom image c ; white arrows ) ; associate findings such as engorgement of the vasa recta ( i.e. , comb sign ) ( b , c ; dashed arrows ) and the presence of mesenteric lymphadenopathy ( coronal with zoom image d ; black arrow ) are often important clues for the diagnosis 1 3 1194 la radiologia medica ( 2019 ) 124 : 11851198 fig . 
9 evolution of clostridium difficile infection : colonic wall thickness ( white arrows ) and pericolic free fluid ( dashed arrows ) at the time of diagnosis of cdi ( a , b ) and 7days after the start of medical therapy ( c , d ) ; the decrease in wall thickness and pericolic and free fluids is evident intervention . 
the following parameters were recorded : wall thickness , extent of colon abnormalities , caliber of the largest portion of the colon , presence / absence of the accordion sign and target sign , the degree of pericolonic stranding and ascites , presence / absence of pleural effusion and subcutaneous edema . 
 none of the parameters evaluated significantly differs in comparison with nonsurgical control group , and cect findings are not necessarily correlated with the severity of the disease [ 64 ]  . 
no statistical correlation was found between cect findings and clinical parameters and none of the cect parameters evaluated was useful to predict the need for surgical intervention [ 65 ]  . 
 in the series by boland etal . , nodular haustral thickening ( including the accordion pattern ) was the only imaging finding that correlated with the severity of the disease , being significantly more frequent in patients with elevated wbc ( > 11.000 / mm3 ) regardless of the extent of colonic involvement [ 23 ]  . 
 reported that increased colonic wall thickness ( 11mm ) and the presence of pleural effusion were independently associated with an increased likelihood of developing complicated cdi [ 27 ]  . conclusion although the confirmation of the diagnosis is based on the demonstration of toxigenic c . 
difficile toxins by stool test , cect is a useful examination to raise the suspicion of 1 3 la radiologia medica ( 2019 ) 124 : 11851198 1195 1 3 1196 la radiologia medica ( 2019 ) 124 : 11851198 clostridium difficile colitis and to evaluate the extension of the disease as well as of possible complications . 
although colitis of other origins can occasionally mimic the cect appearance of cdi , the degree of wall thickening , the extension and location of colonic involvement and the presence of associated extracolonic findings , especially in patients with a recent history of antibiotic use and healthcare exposures , are important points of differential diagnosis . 
inclusion criteria were : preoperative mr including diffusion - weighted imaging ( dwi ) and perfusion dynamic contrast - enhanced ( dce ) sequence ; cytoreductive surgery performed within a month from mr ; and minimum follow - up of 12months . 
dwi parameters included apparent diffusion coefficient ( adc ) of the largest ovarian mass ( o - adc ) and normalized ovarian adc as a ratio between ovarian adc and muscle adc ( m - adc )  . 
none of the other functional parameters showed either difference between r0 and r1 patients or association with pfs in the first 12months . conclusions this preliminary study demonstrated a slightly significant association between normalized ovarian adc and the presence of residual tumor at surgery . 
 in the usa , oc ranks as the second most common female * stefania rizzo stefania.rizzo@eoc.ch 1 postgraduate school ofradiodiagnostics , universit degli studi di milano , via festa del perdono 7 , 20122milan , italy 2 department ofradiology , aslvc , corso mario abbiate 21 , 13100vercelli , italy 3 division ofepidemiology andbiostatistics , ieo european institute ofoncology irccs , milan , italy 4 department ofgynecology , ieo european institute ofoncology irccs , via ripamonti 435 , 20141milan , italy genital cancer , and the first cause of death from gynecological malignancies , with estimated new cases and deaths for 2019 of 22530 and 13980 , respectively [ 2 ]  . 
standard treatment for oc patients consists of primary cytoreductive surgery followed by six cycles of platinum - based chemotherapy 5 department ofoncology andhemato - oncology , universit degli studi di milano , milan , italy 6 department ofradiotherapy , ieo european institute ofoncology irccs , via ripamonti 435 , 20141milan , italy 7 department ofradiology , ieo european institute ofoncology irccs , via ripamonti 435 , 20141milan , italy 8 clinica di radiologia eocistituto imaging della svizzera italiana , via tesserete 47 , 6900lugano , switzerland vol . : ( 0123456789 ) 1 3 1316 la radiologia medica ( 2019 ) 124 : 13151323 [ 3 ]  . 
no residual tumor at surgery is still the most important prognostic factor because of a demonstrated inverse relationship between the extent of residual disease and patient survival [ 4 ]  . in some cases , tumor spread may not allow the complete resection of disease , or it may require a very aggressive approach that might trigger important postoperative complications . 
these cases are usually sent to three or more cycles of neoadjuvant chemotherapy and subsequent interval debulking surgery followed by adjuvant chemotherapy [ 4 ]  . according to the european society of urogenital radiology guidelines , preoperative staging of oc is based on computed tomography ( ct ) [ 5 ] , whereas magnetic resonance ( mr ) is reserved to patients that may not undergo a contrast - enhanced ct . 
nevertheless , recent studies have demonstrated that mr including diffusion - weighted imaging ( dwi ) may predict the extent of disease better than ct in oc patients , thus offering a better prediction of the outcome of cytoreductive surgery [ 6 ]  . 
moreover , several recent studies have focused on functional quantitative parameters derived from mri , such as dwi and dynamic contrastenhanced ( dce ) perfusion [ 710 ] , suggesting that a combination of functional and morphologic information may represent a powerful tool for improving preoperative staging as well as for response assessment [ 1113 ]  . dwi depicts molecular diffusion , mainly measuring the cellularity within a tissue based on the movements of water protons in the extracellular space . 
dce perfusion parameters assess the microvascular environment within a lesion , taking into account the permeability of vessels and giving the opportunity to quantify the extravascular space , the intravascular space and the movements of blood between each of thesome authors have investigated the use of mri perfusion in gynecological malignancies , such as for the detection of uterine cervical cancer , for staging of endometrial cancer and for characterization of adnexal masses [ 9 ]  . to the best of our knowledge , no previous study has investigated the role of mr functional imaging techniques for prediction of outcome of oc patients . therefore , the first purpose of this study was to evaluate whether the dwi and dce quantitative parameters derived from staging mr in oc patient may be associated with the presence of residual tumor at surgery ; secondary purpose was to assess whether the above - mentioned functional sequences may predict progression free survival ( pfs ) in the first 12months after surgery . materials andmethods patients selection this prospective study includes patients affected by oc who underwent cytoreductive surgery after a whole - body mr for preoperative staging at our institution between april 2016 and july 2017 . 
 inclusion criteria consisted of surgery performed at our institution within 1month from mri , high - grade serous oc histological type , preoperative mr including dwi and dce quantitative perfusion sequences and minimum follow - up of 12months . 
exclusion criteria were : lack of a proper follow - up ; mr performed after neoadjuvant chemotherapy ; and mr examination performed on a different mr machine . the following patients characteristics were retrieved : age at surgery ; body mass index ; surgical staging according to the international federation of gynecology and obstetrics ( figo ) ; presence of residual tumor ( rt ) at surgery , indicated as r0 = no rt or rt smaller than 10mm ; r1 = rt higher than 10mm ; date of last visit ; and status ( relapse or not )  . 
afterward , dce imaging sequence was acquired before , during and after injection of a gadolinium chelate ( gadoteric acid ) , at a dose of 0.2ml / kg and rate of 3ml / s . 
images were obtained sequentially at 15 - s intervals for 315s . dwi measurements the adc measurement was taken on a four - view display , by uploading the t2 images of the pelvis as morphologic images , the b = 0 dw images that most closely reflect the t2 images , thus allowing an evaluation of the entire tumor diffusion , and the adc maps . 
a radiologist with 15years of experience in gynecological radiology traced freehand twodimensional ( 2d ) regions of interest ( rois ) on the largest ovarian mass including the most large solid component ( excluding cystic components , if present ) on the b = 0 diffusion - weighted images , and then applied to the corresponding image in the adc map . 
subsequent adc of ovarian masses ( o - adc ) and adc of ovarian mass normalized to the muscle ( m - adc ) were calculated . dce measurements a radiologist ( sr ) with 15years of experience in gynecological radiology placed a volume of interest ( voi ) on the solid part of the largest ovarian mass ; each voi was elaborated by a specific tool ( geniq , ge healthcare ) , which is a software application running on aw ( advantage workstation tm ) on the volumeshare platform , where quantitative functional data about the tissue flow , permeability and contrast leakage from the vascular space into the extravascular extracellular space ( ees ) were collected . 
specifically , the following parameters , whose definitions are shown in table1 , were table 1 definitions and units of geniq map parameters geniq map definition included for analysis : bolus arrival time ( bat ) area ; contrastenhancement ratio ( cer ) area ; initial area under the gadolinium concentration time curve ( iaugc ) ; rate transfer constant of gadolinium from the extravascular to the vascular space ( kep ) ; volume transfer constant from vascular space to extravascular and extracellular space ( ktrans ) ; maximum slope of increase area ( msl ) ; volume of extracellular and extravascular space ( ve ) ; and intracellular space ( int )  . statistical analysis distribution of o - adc and normalized m - adc was described using mean and standard deviation . 
difference in o - adc and normalized m - adc between groups of patients with different characteristics was assessed with the student t test and also using the nonparametric median test . 
all p values were two - sided , and those < 0.05 were considered statistically significant . results this study included 49 patients with high - grade serous oc ( mean age at diagnosis 58years , range 4178 )  . 
rt after surgery was present in 8 / 49 patients ( 16% ) ( table2 )  . an example of the extraction of dce quantitative parameters by the geniq tool used in this study is shown in fig.1. the analysis evaluating o - adc and normalized m - adc according to patients characteristics did not demonstrate significant association , as shown in table2 . pfs curve for 40 patients with r0 disease and available follow - up is shown in fig.2. 
1 an example of the extraction of dce quantitative parameters by the geniq tool used in this study 1 3 1320 la radiologia medica ( 2019 ) 124 : 13151323 our results show that the value of normalized ovarian adc imaging at preoperative mri may be associated with the risk of residual tumor at surgery . 
the association of adc values with clinical outcome is concordant with previous studies , demonstrating that adc values increased the precision of diagnosis and helped to predict the prognosis as a therapeutic response [ 22 ]  . 
a recent study has also shown that the adc value was associated with histological severity of oc and predicted the clinical outcome : namely , low adc values were associated with traditional histopathological prognostic markers , such as poorly differentiated tumors , high ki - 67 expression and vegf receptor expression [ 23 ]  . many studies are in support of the use of perfusion dce sequences , especially in the field of oncology . 
indeed , dcemri enables noninvasive characterization of tissue vasculature , blood volume and permeability , thereby providing information about tumor angiogenesis , which is essential in the development and metastatic dissemination of malignant tumors [ 24 ]  . 
dce analysis may include qualitative semiquantitative and quantitative measures [ 24 , 25 ]  . in gynecological malignancies , semiquantitative analysis , referring to an analysis of kinetics of the curve of contrast enhancement , has been suggested in cervical cancer as an useful tool to predict percentage tumor regression after nonsurgical therapy [ 7 ] ; in endometrial cancer for the detection of small tumors , and in combination with t2w images and dwi to better evaluate deep myometrial invasion [ 13 , 26 ] ; and in the evaluation of complex adnexal masses undetermined at ultrasound as an adjunctive sequence to dwi for characterization [ 27 ]  . on the other hand , quantitative measures are complex but most generalizable and standardized [ 25 , 28 ]  . 
these measures refer to a two - compartment model where the extracellularextravascular space ( ees , tissue ) and the capillary vascular plasma can be considered two compartments that are individually well mixed . 
dce - mri can generate quantitative parameters such as the volume transfer coefficient , ktrans between blood plasma and ees , the volume of the ees , ve , and the rate constant between ees and blood plasma , kep . 
therefore , many previous papers have hypothesized scoring systems or other methods to predict the outcome of cytoreductive surgery [ 1519 ]  . for the evaluation of ovarian masses , mr is considered helpful in evaluating the location , size and nature of the ovarian lesion , as well as in determining the response to treatment [ 20 ]  . 
besides the usual morphologic sequences , functional sequences such as dwi and dce , which are able to offer semiquantitative or quantitative information , have been introduced for use in different clinical settings and / or research settings [ 8 , 11 ]  . dwi displays information about water mobility , tissue cellularity and the integrity of the cellular membranes . 
the quantitative parameter adc is derived from the exponential attenuation of signal intensity between at least two b values and represents the gradient of the signal intensity logarithm line produced between two b values . 
the adc value describes microscopic water diffusibility and is decreased in the presence of factors that restrict water diffusion , such as the cell membrane and the viscosity of the fluid [ 10 , 12 ]  . 
despite its recognized value in diagnosing and monitoring of disease , there is some controversy in the literature regarding the limited reproducibility of adc estimates across different imaging platforms and imaging sites . 
the lack of significance of the dce quantitative parameters may be related to the small group of patients evaluated as well as to the inclusion of a single histological type ( high grade serous oc ) that may show similar behavior in terms of angiogenesis and vasculature . this study has several limitations . 
however , the patients included in this study are part of an undergoing prospective clinical trial , aiming to assess whether wholebody mr may perform preoperative evaluation of oc patients better than ct . 
we have decided to perform this ad interim analysis to assess the overall performance of quantitative dce imaging on the ovarian mass , in order to decide whether this sequence was worthy of an adjunctive time , especially for those patients uncomfortable with the duration of the mr examination . 
 however , we included only patients undergoing surgery at our institution , which is a third referral center for oc treatment ; therefore , we can assume that surgeons with comparable technical skills and experience operated all patients . 
 moreover , the percentage of patients with rt at surgery may appear low to evaluate data trend ; however , since the percentage of rt in this study ( 16% ) is comparable to the one reported in the literature , an increase in the patients number would have not changed this proportion . 
 however , the relapse within the first 12months is an important clinical endpoint for oc patients because it is the basis to define the platinum resistance of patients . in conclusion , in this study we demonstrated a slightly significant association between m - adc and the presence of residual tumor at surgery , whereas dwi - related parameters and dce perfusion quantitative parameters did not show association with residual tumor at surgery and pfs in the first 12months . 
these results may suggest that dce perfusion parameters in oc patients undergoing mr for staging are not needed for the prediction of outcome . acknowledgements we hereby thank antonello vidiri for sharing his knowledge about the use of the software tool for quantitative dce analysis . 
the recent developments in ct technology with the introduction of the third - generation dual - source ( ds ) dual - energy ( de ) ct scanners well suited to respond to these challenges . 
 the aim of this article is to describe the state - of - the - art in dsct protocol with de technology in pediatric chd patients , providing some case examples of our experience over an 18 - month period . keywords dual - source ct dual - energy ct congenital heart disease pediatric patients state - of - the - art introduction congenital heart disease ( chd ) is the most common inborn pathology with an incidence of 1% and a prevalence of 810 per 1000 live births . 
 table1 shows the main chds . imaging assessment of pediatric chd patients is challenging for the wide spectrum of disease and complex congenital defects , high heart rates , motion artifacts from cardiac and respiration movements and limited patient cooperation . 
imaging modalities for evaluating chd in pediatric patients include echocardiography , conventional invasive angiography , magnetic resonance imaging ( mri ) and computed tomography ( ct ) [ 2 , 3 ]  . 
despite these advantages , the high heart rate , the use of ionizing radiation and the limited patient cooperation are limiting factors and ct is not the modality of choice for imaging in pediatric patients [ 5 ]  . 
however , the development in ct technology with the introduction of the third - generation dual - source ( ds ) dualenergy ( de ) ct scanners well suited to respond to these challenges [ 6 ]  . the aim of this article is to describe the state - of - the - art in dsct protocol with de technology in pediatric chd patients to obtain high - quality images with low radiation dose , providing some case examples of our experience over an 18 - month period . technical parameters * marco fogante marco.fogante89@gmail.com 1 radiology department , azienda ospedaliero universitaria ospedali riuniti , 60126ancona , italy third - generation dsct scanner uses two x - ray tubes working at the same energy ( single - energy ) or at different energy ( de )  . 
two x - ray tubes are placed approximately at 95 to each other and can work at high ( from 120 to vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 12381252 1239 table 1 clinical classification of congenital heart diseases table 2 technical parameters of third - generation dual - source dualenergy ct scanner acyanotic heart disease increased pulmonary venous flow ( pulmonary edema ) hypoplastic left heart aortic coarctation interrupted aortic arch congestive heart failure neonatal sepsis increased pulmonary arterial flow ( shunt vascularity ) atrial septal defect ventricular septal defect patent ductus arteriosus atrio - ventricular canal defect cyanotic heart disease decreased pulmonary vascularity with cardiomegaly ebstein anomaly decreased pulmonary vascularity without cardiomegaly tetralogy of fallot usually increased pulmonary vascularity without cardiomegaly transposition of great arteries truncus arteriosus tricuspid atresia partial / total anomalous pulmonary venous return single ventricle 150kv ) and at low ( from 70 to 100kv ) energies [ 79 ]  . 
 third - generation ds systems have a tin filter in front of the high - energy tube , that attenuates lower energy photons , thereby increasing the difference of spectral energy . 
the fov of the low - energy tube is 50cm , whereas the fov of the highenergy tube is 35cthe potential disadvantage of the fov of the smaller tube is the evaluation of the structures located peripherally in larger patient . 
third - generation ds - dect allows a submillimeter spatial resolution of the heart and coronary arteries in one or a few heartbeats , a high temporal resolution , a radiation dose reduction with high - pitch values and low tube voltage , new algorithms of iterative reconstruction , and provides additional clinical information with de technology . 
thirdgeneration dsct makes possible to scan non - cooperative pediatric patients with minimal sedation , free breathing and without - blockage of heart rate , even to heart rate around 140beats / mthe highest temporal resolution should be used to minimize motion artifacts . 
nevertheless , beta - blockers are safe in pediatric patients without contraindications and should be considered if high - definition imaging is required because image quality remains heart - pulse - dependent [ 21 ]  . contrast injection third - generation dsct allows contrast volume saving , because using a low tube voltage ( 70kv ) x - rays generated have an average energy close to the k - edge of iodine ( 33.2kev ) , improving contrast enhancement . 
moreover , the fast scan protocol provides precise shaping and timing 1 3 1240 la radiologia medica ( 2019 ) 124 : 12381252 of the contrast peak enhancement for optimal intravascular attenuation and allows reducing contrast volume [ 22 ]  . 
for these reasons , keeping the iodine delivery rate constant , contrast medium ( cm ) with low iodine concentration ( 300mgi / ml ) showed a higher vascular enhancement than cm with high iodine concentration ( 400mgi / ml ) , allowing iodine load reduction . 
intravenous line size ranging from 24 - gauge in neonates to 18 - gauce in older children , with flow rates between 0.5 and 5ml / s and pressure settings of 50300lb per square inch ( psi )  . 
central venous catheter might be used in critically pediatric patient , but it allows for adequate injection in children with age less than 1 - year because maximum flow rate is 0.41.2ml / s and maximum pressure value is 2550 psi . 
the triphasic approach consists of a two - phase contrast administration ( about half at the regular rate plus the remainder either at a slower rate ) followed by saline with slower flow rate . 
 this method should be used for the assessment of complex cardiovascular disease to obtain simultaneous evaluation of right and left - side structures [ 24 , 25 ]  . scan timing scan time can be decided with three different techniques : fixed - time , bolus - test and bolus - tracking . 
the site of cannulation and scan protocol must be considered ; an extra delay of 24s should be added when injecting from the leg and when using shorter scan methods . 
nevertheless , this technique is prone to inaccuracy and it is challenging because it is limited reproducibility from the different time of contrast transit for anatomical , functional and technical reasons [ 26 ]  . bolus - test technique consists of calculating vascular peak enhancement time of a small contrast volume by monitoring cm transit on a selected slice . 
this method involves additional cm and radiation and it is not routinely applicate in children [ 27 ]  . bolus - tracking technique is the most useful in pediatric patients . 
when is necessary the visualization of the cardiac chambers and the descending aorta in the same plane , the region of interest could be placed within the left ventricle and the trigger threshold should be set 100 hu . 
 visual start should be used with restless patients because with automatic approach , the movements of the patient during the bolus measurement can cause to start too early or too late . 
to reduce radiation exposure , monitoring should start at the end of contrast injection and should be reduced in frequency ( one each second ) , and tube voltage should be reduced ( 70kv ) [ 28 ]  . scan protocol the scan range should include the heart , the thoracic aorta and the pulmonary arteries . the scan acquisition can be non - electrocardiogram ( ecg ) - synchronized or ecg - synchronized . nonecgsynchronized acquisition non - ecg - synchronized acquisition should be used when the diagnostic purpose concerns extra - cardiac anatomy . 
 the high temporal resolution allows for good quality imaging of extra - cardiac structures because they are less sensitive to heart motion than the heart itself [ 29 ]  . 
 moreover , respiratory artifacts are responsible for imaging degradation and non - synchronized acquisition is obtained more quickly than synchronized acquisition , and thus is responsible for fewer respiratory artifacts [ 30 ]  . 
finally , synchronized acquisition requires a much higher radiation dose than non - synchronized acquisition because the exposure time is longer due to low pitch acquisitions [ 31 ]  . 1 3 la radiologia medica ( 2019 ) 124 : 12381252 1241 fig . 
the main acquisition modes include : prospective ecg - triggered high - pitch spiral , prospective ecg - gated sequential , and retrospective ecg - gated spiral [ 33 ]  . prospective ecg - triggered high - pitch spiral mode should be used in uncooperative patients because it reduces respiratory and cardiac motion artifacts , as well as the need for sedation and general anesthesia . 
this mode uses two x - ray tubes to achieve both maximum temporal resolution and z - axis scan speed , resulting in rapid and large gapless non - overlapping imaging in a single heartbeat . 
the length of the rr interval in pediatric patients is around 500ms at a heart rate of around 120 beats / man ecg - triggered high - pitch scan with a 12cm scan length takes up to 130ms . 
mostly , only information on the anatomy is required and not the rule out of coronary artery disease as in adults , providing sufficient diagnostic image quality [ 34 ]  . prospective ecg - gated sequential mode should be used in cooperative patients with irregular heart rate . 
this mode allows to increase the percentage of the cardiac cycle phase acquired ( from 30 to 60% of the diastolic phase ) , to select the ecg phase without motion artifacts , and avoid nondiagnostic scans [ 35 ]  . 
an issue of prospective ecg - gated sequential mode is the possibility of a stack artifact when scanning a larger length in the z - axis than the collimation width . 
in children , because the length of the z - axis is not elevated and the focus is mainly on anatomy assessment , the stack artifacts can be less problematic [ 36 ]  . retrospective ecg - gated spiral mode should be use with very irregular heart rate where the need for ecg editing is expected , or when the functional cardiac information could be necessary . 
this technique loses efficiency at higher heart rate because of less efficient slope - up time and slope - down time of the ecg - based tube current modulation [ 37 ]  . de clinical applications de clinical applications in pediatric chd patients include better evaluation of vascular structures and shunts , assessment of the myocardial and lung perfusion and reduction in metal artifact . 
the post - processing techniques that best exploit the advantages of de in chds are material decomposition and virtual mono - energetic [ 38 ]  . material decomposition allows discriminating structures based on attenuation characteristics at different energy levels . 
the system uses three - material decomposition algorithm in the image - space domathe three materials used for material differentiation in pediatric chd are soft tissue , calcium , and iodine . 
type a interrupted aortic arch , after the left subclavian artery origin , with a gap between the ascending and descending thoracic aorta , in sagittal ( a ) and coronal ( b ) views ( yellow arrows )  . 
iodine overlay images allow quantitative evaluation of iodine content , a reference region is placed in an area of maximum contrast enhancement and another region of interest in placed in the target area of a contrast materialenhanced image . 
because absolute threshold values for increased or decreased iodine content have not been defined and iodine measurements may vary among vendors , the quantitative analysis is best performed by assessing relative contrast enhancement values [ 40 ]  . 
another application is the evaluation of myocardial perfusion , which provides the myocardial iodine distribution during the first pass arterial enhancement and can detect myocardial blood supply [ 41 ]  . 
medium smooth convolution kernel should be used with a low - intermediate strength [ 49 ]  . dsct , electrocardiogram ; bv , body vascular ; ctdivol , volume computed tomography dose index ; dlp , dose length product ; ssde , sizespecific dose estimates postprocessing application allows assessing qualitative and quantitative lung perfusion . 
dect may be an efficient method to assess perfused lung blood volume in chds , such as pulmonary atresia , arteriovenous malformations and tetralogy of fallot [ 42 ]  . 
 these perfusion defects correlate with increased right - toleft shunt and combined with the morphologic analysis can improve detectability of the cyanotic chd [ 43 ]  . virtual mono - energetic allows reconstruction of virtual images that simulate the attenuation values of an image acquired by using a single - energy value . 
these values reduce metal and beam - hardening artifacts from injected cm in the subclavian vein , axillary vein , or superior vena cava [ 45 , 46 ]  . for extra - cardiac structures evaluation are necessary multi planar reformations in sagittal and coronal planes . 
volume rendering reconstructions are necessary for a tridimensional overview of cardiac and great vessel anatomy [ 50 , 51 ]  . radiation dose radiation dose reduction in pediatric patient is important because organ sensitivity to radiation is much higher than in adults , and a risk of developing cancer in the future cannot be totally ruled out . 
using this protocol , radiation exposure is estimated to be less than 1msv , which is equivalent to the dose delivered by natural radiation over a 6 - month period [ 54 ]  . 
retrospective ecg - gated scan mode provides from 2 to 4msv [ 55 , 56 ]  . concerning dect in the pediatric population , in the few prior publications , the radiation exposure level from dect is equivalent to or even less than that of a comparable single - energy examination [ 57 ]  . 
moreover , dect scanners can generate virtual non - enhanced images , which have the potential to reduce radiation exposure by eliminating the need for a true nonenhanced acquisition [ 58 , 59 ]  . case examples clinical indications for cardiac ct in the pediatric population with chds cover a wide spectrusome case examples are provided , all acquired on a dsct scanner with la radiologia medica ( 2019 ) 124 : 12381252 de technology ( somatom force , siemens heathineers , forchleim , germany )  . hypoplastic left heart syndrome hypoplastic left heart syndrome ( hlhs ) comprises a wide spectrum of cardiac malformations , including hypoplasia or atresia of the aortic and mitral valves and hypoplasia of the left ventricle and ascending aorta . 
hlhs is the fourth most common cardiac malformation to manifest in the first year of life behind ventricular septal defect , transposition of the great arteries , and tetralogy of fallot . 
however , the recent evolution of palliative surgical procedures has increased the survival rate in children with these malformations [ 60 ]  . ct has become a valuable modality in evaluating the complex anatomic findings associated with hlhs . 
ct provides information for surgical planning , include location and size of the hypoplastic ascending aorta , and post - surgical assessment , such as the modified fontan procedure , a lateral tunnel created within or near the right atrium to direct blood from the inferior vena cava to the right pulmonary artery to reduce patient cyanosis [ 61 ]  . 
ct protocol and radiation dose are summarized in table3 . interrupted aortic arch withpatent ductus arteriosus interrupted aortic arch ( iaa ) accounts for approximately 1.5% of all chds and 15% of iaa patients have digeorge syndrome . 
 according to the site of interruption , three different types are described : type a ( 30% ) , the interruption is distal to the origin of left subclavian artery ; type b ( 43% ) , the interruption is between the left carotid and left subclavian arteries and digeorge syndrome is reported in about 50% of patients ; type c ( 17% ) , the interruption is between the innominate and left carotid arteries . 
bicuspid aortic valve occurs in 60% of all cases , sub - aortic stenosis occurs in about 20% , and truncus arteriosus in about 10% [ 62 ]  . ct has become a valuable modality in evaluating the complex anatomic findings associated with iaa . 
ct can be used to evaluate the type and the level of iaa , the pda , the aortic valve and potential post - surgical complications [ 63 ]  . 
large ventricular septum defect ( a ) , anterior shift of the aorta over the ventricular septum defect ( b ) , right ventricular outflow tract obstruction with pulmonary artery atresia and dextro - position of the aortic arch ( c )  . 
ct protocol and radiation dose are summarized in table4 . ebstein anomaly ebstein anomaly ( ea ) accounts for < 1% of all chds with an equal distribution between male and female . 
as a consequence of this apical and anterior displacement of the tv functional orifice , functionally rv is variably hypoplastic ; the myocardium above the orifice becomes atrialized and , thus , thin and dysfunctional ; the myocardium below the orifice typically possesses a more normal ventricular wall thickness but is still dysfunctional . 
the rv impairment and the tv regurgitation decrease forward flow across the pulmonary valve reducing systemic cardiac output and increase right atrial dimensions and pressure thus favoring a right - to - left shunt through the interatrial communication ; cyanosis depends upon the rightto - left shunting [ 64 ]  . ct has become a valuable modality in evaluating the complex anatomic findings associated with ea . 
ct protocol and radiation dose are summarized in table5 . tetralogy offallot tetralogy of fallot ( tof ) occurs in 10% of all chds and is the most common cyanotic defect seen in children beyond infancy . 
it involves the following four anatomic abnormalities of the heart : large ventricle septal defect ( vsd ) , anterior shift of the aorta over the vsd ( overriding aorta ) , rv outflow tract ( ot ) obstruction , and rv hypertrophy . 
tof and pulmonary atresia with vsd occurs in approximately 1520% of all tetralogy cases and the pulmonary supply is most commonly mediated through a pda ( 70% ) and less commonly through major aorto - pulmonary collateral arteries ( mapcas ) ( 30% ) [ 66 ]  . ct has become a valuable modality in evaluating the complex anatomic findings associated with both unrepaired and repaired tof patients . 
ct can be used to evaluate the rvot obstruction , the aortic root anatomy and the overriding aorta , the vsd , the pda , the mapcas , the patency of surgical palliative shunt placement and for long - term sequelae and complications . 
ct protocol and radiation dose are summarized in table6 . transposition ofthegreat arteries transposition of great arteries ( tga ) accounts for 25% of all chds with a prevalence of about 0.20.3 of 1000 births ; it is more common in males than in females with a ratio of 23 : 1 . 
it derives from truncal ridge and infundibulum normal spiraling rotation failure during fetal life which results in discordant ventriculo - arterial connection : aorta arises from the morphologically right ventricle ( rv ) and is located anteriorly and to the right of pulmonary artery ( pa ) ( d - transposition ) , whereas the pa arises from morphologically left ventricle ( lv )  . 
during neonatal period , patent ductus arteriosus ( pda ) and mainly patent foramen ovale ( pfo ) usually maintain an adequate mixing because they ensure the effective systemic / pulmonary blood flow going , respectively , into the aorta and the pa ; as the pda starts to close and pfo by itself is restrictive in size , infant develops severe cyanosis [ 69 ]  . 1 3 la radiologia medica ( 2019 ) 124 : 12381252 1249 fig . 
 volume rendering reconstructions in superior view with ( e ) and without aorta ( f ) ct has become a valuable modality in evaluating the complex anatomic findings associated tga . 
ct can be used to evaluate the origin and course of the great arteries , the presence and size of the pda and / or pfo , and any associated anomalies such as lvot obstruction and aortic coarctation [ 70 ]  . 
ct protocol and radiation dose are summarized in table7 . conclusion dsct with de technology is an important low - invasive diagnostic tool for the evaluation of chd in pediatric patients , providing relevant information for optimal surgical management . 
then , we selected retrospectively , among the 1500 patients underwent to cedm at the breast diagnostics department of the careggi university hospital of florence and the national cancer institute of milan from september 2016 to november 2018 , 31 women ( mean age 57.1 aa ; range 4178 aa ) with a definitive histological diagnosis of ilc . 
specificity in the characterization of additional lesions was 66.7% , and the diagnosis of the extension of disease was correct in 77.4% of cases : nme also led to a decrease in diagnostic accuracy in the evaluation of disease extension up to 40% versus 85% for masses and 80% for masses associated with nme ( m / nme )  . 
moreover , in 12 / 31 ( 38.7% ) , cedm allowed to correctly identify lesions not shown by mammography + ultrasonography + tomosynthesis : in the half of these ( 6 / 12 ) , there was a multicentricity , thus allowing an adequate surgical planning change . 
cedm was also very accurate in analyzing the maximum diameter of the masses , while it was much less reliable in the case of the m / nme and pure nme . 
 in conclusion , cedm is a new promising imaging technique in the loco - regional preoperative staging and in the evaluation of disease extension for ilc , especially in case of mass enhancement lesions . keywords breast contrast - enhanced digital mammography contrast - enhanced spectral mammography invasive lobular breast cancer breast cancer staging * giulia bicchierai giulia.bicchierai@gmail.com 1 department ofradiology , university ofpalermo , palermo , italy 2 diagnostic senology unit , azienda ospedaliero - universitaria careggi , largo g . 
brambilla 3 , 50134florence , italy 3 general management staff , azienda ospedaliero - universitaria careggi , florence , italy 4 breast imaging unit , fondazione irccs istituto nazionale 5 department ofradiology , azienda ospedaliero - universitaria dei tumori , milan , italy careggi , florence , italy introduction invasive lobular carcinoma ( ilc ) is the second most frequent invasive breast cancer ( 515% ) after ductal histotype ( idc ) ( 7090% ) , compared to which is more difficult to diagnose by mammography and ultrasonography and has a greater tendency to multifocality , multicentricity or bilaterality [ 14 ]  . 
then , magnetic resonance imaging ( mri ) is recommended from international guidelines in the preoperative work - up for all ilc patients , thanks to its high sensitivity ( 9598% ) , also in detecting of adjunctive lesions in the ipsilateral or contralateral breast , not previously identified with mammography or us , not even with the most recent advances in these diagnostic techniques [ 514 ]  . 
recently , contrast - enhanced digital mammography ( cedm ) has shown a similar sensitivity and an even greater specificity vol . : ( 0123456789 ) 1 3 1230 la radiologia medica ( 2019 ) 124 : 12291237 compared to mri , in the detection of breast lesions , both for index lesions and for possible additional lesions , with lower costs , greater rapidity , greater patient compliance , especially in the claustrophobic , greater availability , and with the possibility to verify if microcalcifications have contrast enhancement , thanks to the perfect correspondence between full - field digital mammography ( ffdm ) and cedm images [ 1521 ]  . 
the aim of our study was to assess the performance of cedm in the preoperative loco - regional staging of ilc patients , about the valuation of the extension of disease and in measurement of lesions . 
we also calculated the distribution of frequencies of various histological parameters ( i.e. , positivity to the receptor for estrogens , positivity to the receptor for the progesterone , ki67 , her - 2 positivity ) in our sample . 
 cedm technique cedm was performed using a selenia dimensions mammography system ( hologic , marlborough , ma ) capable of performing full - field 2d digital mammography , 3d tomosynthesis and cedm ( high and low energy )  . 
an intravenous injection of 1.5ml / kg body weight of an iodinebased contrast agent ( ultravist 370 , bayer healthcare llc , whippany , nj ) was administered with an automated bolus injection with a flow of 3ml / s , followed by 20ml of saline solution . 
1 flow chart of patient enrollment cedm image , 2 acquisitions were performed at 2631kvp with rhodium and silver filters ( rh and ag ) for low - energy acquisition , and at 4549kvp with a copper filter for high acquisition power . 
a recombination algorithm was used to subtract the non - enhanced breast tissue , and then to provide a subtracted image in which only the areas of post - contrastographic enhancement were highlighted : this will allow to evaluate the neoangiogenesis of the tumor , as in mri . 
 acquisitions in both standard projections ( cc and mlo ) , carried out also 8min after the administration of the contrast medium , also make it possible to carry out a qualitative assessment of the enhancements kinetics . 
 [ 22 , 23 ] imaging interpretation andhistological parameters two different radiologists , with more than 30years of experience in breast imaging in the two different hospitals , performed ultrasonography and analyzed mammograms and tomosynthesis of the 31 patients of our study ( of which they knew the medical history ) , according to bi - rads criteria established by the american college of radiology ( acr )  . 
as in mri , cedm classifies all 31 index lesions detected in the subtracted images into three main groups : focus , mass and non - mass enhancement ( nme ) ; we also included a further category , called m / nme , when the index lesion is composed by a mass closely associated with a nme component . 
according to the bi - rads , they analyzed morphological and kinetics findings of the enhancement of lesions and valuated the number of lesions and then the extension of the disease . 
the two radiologists measured the maximum diameter of index lesions at cedm : in case of m / nme , they measure the maximum diameter of the sum of the two components . 
 statistical analysis we calculated cedm sensitivity and specificity in detecting of adjunctive lesions and the accuracy in assessing the local extent of disease first in the total of the sample and then for the various subgroups , divided for type of contrast enhancement ( masses , nme and m / nme )  . 
then , we have analyzed if and how cedm sensitivity , specificity and accuracy in assessing of the local extent of disease were influenced by the different types of contrast enhancement . 
regarding the dimensional analysis of index lesions , mean , median , standard deviation and the ranges of their measurements on the entire sample and in the subgroups were calculated . 
 the blandaltman plot was used to explore the agreement between cedm index lesion measurements and histology ( gold standard ) , and the intraclass correlation coefficient ( icc ) was calculated [ 31 , 32 ]  . 
freemanhalton extension of fishers exact probability test was used to verify any significant differences between the masses , nme , m / nme , in the distribution of lesion histological parameters . 
the index lesion occurred in 20 / 31 ( 64.5% ) of the cases like a mass , in 6 / 31 ( 19.3% ) like nme , in 5 / 31 ( 16.1% ) t 1 3 la radiologia medica ( 2019 ) 124 : 12291237 1233 like a m / nme , in 0 / 31 ( 0% ) like focus , and the distribution of their enhancement features is described in tab.1. cedm , additional lesions , extension ofdisease andhistological parameters in 12 / 31 ( 38.7% ) , cedm allowed to correctly identify lesions not shown by mammography + ultrasonography + tomosynthesis : in the half of these ( 6 / 12 ) , there was a multicentricity , thus allowing an adequate surgical planning change [ 3338 ]  . 
other histological features are shown in tab.3 : they were correlated with the three main categories ( masses , nme , m / nme ) , and no significant variations were found between the various groups [ 39 ]  . 
nme is associated , although without statistical significance ( p : 0.20 ) , with a higher risk of a progesterone receptor negativity ( 3 / 6 , 50.0% ) , and with statistical significance ( p : 0.03 ) to a moderate ( 2 + ) or high ( 3 + ) her2 positivity : this significance is further greater ( p : 0.021 ) if we consider the sum of nme and m / nme rather than masses only . 
fish analysis in moderate ( 2 + ) her2 positivity cases showed no gene amplification for all , and then the real and relevant positivity to her2 was detected in only 1 / 31 ( mass - like lesion )  . 
then , we have calculated the intraclass correlation coefficient ( icc ) for the entire sample and its result ( 0.858 ; 95% ic 0.7060.932 ) suggests a good performance of the cedm in the valuation of the maximum diameter of the lesion . 
then , a correct loco - regional staging with an evaluation of the extent of the disease is fundamental , even more than in other histological variants , to allow the most appropriate treatment and avoid any complications [ 4548 ]  . 
3 blandaltman plots 1 3 la radiologia medica ( 2019 ) 124 : 12291237 1235 cedm , which is more recent than mri [ 11 , 35 , 39 ]  . 
furthermore , in our study , unlike what was done in previous ones , we analyzed the differences in the various categories of enhancements such as m , nme and m / nme : the presence of a nme led to a lowering of cedm sensitivity in detecting any additional lesions up to 25% , versus 100% demonstrated in presence of a mass - like enhancement or a m / nme . 
nme led to a decrease in diagnostic accuracy in the evaluation of disease extension up to 40% , unlike what happens for masses ( 85% ) and for m / nme ( 80% ) : for the latter categories , data are absolutely similar to the performance of the mri reported in the literature [ 35 ]  . 
etal , cedm proves to be accurate in assessing the maximum diameter of the index lesion , being in agreement with its dimensions at the definitive histology [ 41 ]  . 
even if the sample size is very small , and therefore requires a multicenter study with a significantly larger sample , the performance seems to be better in case of masses or nme , and worse in case of masses closely associated with a non - mass enhancement . 
as well as in patels study and in other studies , the literature about ilc is described that almost all of lesions present expression of the estrogen receptor , in the ours the 100% of lesions shows a positivity to er . 
nme is associated , although not significantly , with a higher risk of a progesterone receptor negativity , while is correlated significantly with an increased expression of her2 ( 2 + or 3 + ) , compared to the presence of a mass enhancement : the necessary additional study with fish technique on moderate positivity to her2 , however , showed that all were not amplified , and then the real positivity was present only in 1 / 31 ( mass - like lesion )  . 
because this is a bi - centric study , the evaluation of the images was performed by different radiologists , as were different the surgeons who managed the patients , planning and performing surgical interventions and the pathologists . 
being a relatively recent diagnostic technique , the lack of experience of radiologists may have been a limitation for the study , especially in the early stages of it , even if as in case of introduction of each new diagnostic technique , it is to be considered a learning curve of the operators ; we believe that the experience of over 30years in breast imaging of our two radiologists of the study has reduced , although only partially , the influence of this limitation . 
the assessment of the intensity of the enhancement of lesions moreover was qualitative , in the absence , to date , of a validated quantitative system for its measurement in cedm , and thus also the evaluation of its kinetics . 
furthermore , the sample size is small , making definitive results more difficult ; therefore , a larger prospective study is needed to confirm our conclusions . conclusions cedm has proved to be a new promising imaging technique in case of loco - regional preoperative staging for ilc , especially in case of mass enhancement lesions . 
imaging assessment included : pre - procedural mdct , intra - procedural dp - cbct performed before first and second dsm - taces and 1 - month follow - up mdct . 
in particular , degradable starch microsphere trans - arterial chemo - embolization ( dsm - tace ) combines the advantages of loco - regional approach ( maximization of local drug delivering whilst reducing the systemic toxicity ) with the ones of a resorbable carrier that significantly increases the safety profile of the procedure by reducing the ischaemia time and vessel occlusion rate [ 2 ]  . 
in fact , a patient with bilobar spread will complete treatment within 2months ( four cycles ) and will be reassessed for lesion response with mdct only after 1month from the last dsm - tace . during the last few years , attention has been focused on the optimum modality for guidance of these treatments . 
the introduction of cone - beam computer tomography ( cbct ) to the field of liver catheter - based procedures has contributed to significant improvement in clinical outcomes , versus the use of standard digital subtraction angiography ( dsa ) guidance alone [ 3 ]  . 
in particular , cbct has been able to depict a significant rate of occult lesions missed at pre - procedural mdct [ 4 ] and the presence of extra - hepatic feeders [ 3 , 5 , 6 ] and provide a roadmap of the tumour vascular supply to facilitate successful and complete embolization [ 79 ]  . 
 recently , dual - phase cbct , consisting in introducing an additional delayed acquisition , has shown to facilitate hypovascular lesion visualization [ 10 ]  . despite these advantages in aiding the embolization procedure , only a few studies have focused on this technique ability to intra - procedurally predict the treatment outcome , with controversial results [ 11 , 12 ]  . 
for these reasons the aim of this study is to evaluate the potential prognostic value of sequential dual - phase cbct imaging performed during dsm - tace session in predicting the nodules response to treatment . materials andmethods this study was approved by the ethical institutional review board . 
all enrolled hccs were of typical vascular behaviour ( exhibiting washin in the arterial phase and washout on the delayed venous phase ) diagnosed on the basis of mdct only being greater than 2cm in diameter . 
the scan delay before the initiation of late arterial phase imaging was determined by means of bolus tracking with automated scan triggering ( care bolus ct ; siemens medical system )  . 
digital subtraction angiography ( dsa ) and cbct were acquired after having positioned the 4 - fr catheter in the common hepatic artery coupled with a power injector ( mark v provis ; medrad , beek , the netherlands )  . 
power injector parameters were 3.5ml / s , 900 psi , intra - arterial injection lasted 15s and first cbct acquisition started with 8 - s delay from the injection start . 
c - arm rotates 200 around the patient in 8 - s acquiring images every 0.5 at 60 frame / second for a total of 419 images with a 512x512x387 matrix and isotropic resolution of 0.49mm resulting in a 25 25 19cm fov . 
the data sets were automatically transferred to a workstation ( x - workplace , siemens healthineers , erlangen , germany ) for reconstruction and analysis . dsmtace protocol the protocol stipulated two sessions of dsm - tace on each involved lobe with 28 days between the two procedures . 
second row ( yellow box ) , management of monolobar disease 1 3 la radiologia medica ( 2019 ) 124 : 12121219 1215 imaging timing imaging assessment included a pre - procedural mdct performed less than a month before treatment , a first dual - phase cbct performed before the beginning of the first dsmtace session , a second dual - phase cbct performed before the beginning of the second dsm - tace session and a follow - up mdct performed after 1month after completion of treatment . 
attenuation values are expressed in hounsfield unit ( hu ) for mdct acquisitions , and pseudo - attenuation values are expressed in arbitrary unit in all intra - procedural cbct acquisition . to assess the predictive value of dp - cbct regarding the dsm - tace protocol per lesion , the modification over time of the lesions attenuation was correlated with the post - procedural modify response evaluation criteria in solid tumours ( mrecist )  . 
in detail , the modification of pseudoattenuation and attenuation were calculated as the difference of : first cbct and second cbct values ( arterial and venous acquisitions ) and pre - procedural mdct and post - procedural mdct values ( arterial and venous acquisitions )  . followup evaluation follow - up mdct was performed to assess treatment outcome , according to mrecist criteria [ 13 ] for hcc lesions . 
response to treatment was categorized as : complete response ( cr ) , partial response ( pr ) , stable disease ( sd ) and progressive disease ( pd )  . statistical analysis the normality of each continuous variable group was tested using the kolmogorovsmirnov z test . 
digital subtracted angiography ( a ) performed from the common hepatic artery showing multiple hypervascular hcc distributed in the both lobes ; the arrow shows the hcc of the vi segment detailed in the following images . 
in particular , it is possible to demonstrate the reduction in attenuation arterial ( b , e ) and venous ( f , i ) phases and pseudo - attenuation arterial ( c , d ) and venous ( g , h ) phases in a complete response nodule 1 3 1216 la radiologia medica ( 2019 ) 124 : 12121219 positive and negative cases = 0.33 for cr / pr + sd + pr , 1 for or / sd + pd and 3 for dc / pd . 
roc curve analysis was also performed and the auc were calculated for the difference of attenuation between first and second cbcts and preand post - mdct ( both arterial and venous phases )  . 
roc curve analysis ( fig.3 ) showed that the higher the attenuation ( hu and hu * ) difference between preand postprocedural mdct and first and second dp - cbcts was , the better the clinical outcome will be . 
the complete analysis is shown in table3 and in fig.4. discussion a strong association was found between the change in lesion attenuation assessed intra - procedurally over a set of multiple dsm - tace sessions and the prediction of treatment outcome evaluated at post - procedural mdct . 
they focused their studies on dp - cbct imaging before single - session tace procedures , failing to identify a statistically significant association ( univariate and multivariate analysis ) between lesions attenuation and post - procedural mrecist criteria . 
in fact , no standard treatment scheme was defined in the literature regarding the eventual readministration of tace ( conventional tace , deb - tace or dsm - tace ) protocol and several factors should be taken 1 3 1218 la radiologia medica ( 2019 ) 124 : 12121219 fig . 
the first column shows , for arterial ct phase ( first row ) and arterial cbct phase ( second row ) the sensitivity / specificity trend in relation to cut - off change , respectively , for complete response ( a ) , objective response ( b ) and disease control ( c )  . 
the second column highlighted the optimal cut - off with the corresponding sensitivity and specificity for complete response ( a ) , objective response ( b ) and disease control ( c ) into account , including : procedure timeline and embolization drug choice [ 17 , 18 ]  . 
on - demand scheme , over the scheduled one , is usually considered safer being re - treatment proposed only in case of progression of disease [ 19 , 20 ]  . 
 [ 22 ] demonstrated that switching from epirubicin to cisplatin in patients with refractory response to epirubicin - loaded deb - tace is a safe , well - tolerated and efficacious treatment strategy . 
however , unlike liver function , the effect of the dsm - tace could be assessed only at the end of the pre - defined administrative protocol ( 2 sessions per lobe ) by ct or mri . 
therefore , the possibility to predict intra - procedurally , with a predetermined cut - off , the effectiveness of the ongoing dsm - tace treatment introduces another parameter that impacts the management of the patient , avoiding unnecessary drug administration that could potentially impair the limited liver functional reserve . 
 for instance , the execution of dp - cbct by demonstrating lesion attenuation modification by early prediction of treatment failure could permit either drug switch , drug combination or dosage adjustment . our study presents several potential limitations . 
firstly , sample size is small ( 24 patients ) , although the usage of a per - nodule analysis ( 96 nodules ) in a subset of advanced disease patients supplies this limitation according to our sample size analysis . 
despite these limitations that can be 1 3 la radiologia medica ( 2019 ) 124 : 12121219 1219 addressed by increasing the sample size , we strongly believe that the lesion attenuation modification measurement is a simple tool that could lead to a significant clinical benefit . conclusion dp - cbct can predict intra - procedurally , by assessing lesion pseudo - attenuation modification , the dsm - tace 1 - month treatment outcome . 
 specific attention will be focused on the interaction of x - rays with matter , on the principles of attenuation of x - rays in ct toward the intrinsic limits of conventional ct , on the material decomposition algorithms ( twoand three - basis - material decomposition algorithms ) and on effective rho - z methods . 
the progresses in material decomposition algorithms , in computational power of computers and in ct hardware , lead to the development of different technological solutions for dect in clinical practice . 
this was the first empirical observation of material decomposition in computed tomography ( ct ) opening a new research field : material decomposition in dual - energy computed tomography ( dect ) [ 2 , 3 ]  . 
in the following years , several technologies have been developed and the applications of dect are more widely spread in the clinical routine . this review will provide a brief overview on theoretical principles behind dect ( better understood after reviewing the interactions of x - rays with matter ) , the available technologies and the main clinical applications . xrays : interaction withmatter the polychromatic spectrum ( photons with different wavelengths and energies ) of x - rays produced by the tube interacts and exchange energy with the biological matter with a combination of several phenomena : rayleigh scattering , photoelectric absorption , compton scattering , and pair production . 
the probability of each of them , expressed as cross section ( ) , is related to the energy of the incident photon and properties of the material [ 5 ]  . 
the rayleigh scattering ( r ) and the pair production give minor contribution to x - ray attenuation and are considered of minor relevance at energies used in ct [ 5 ]  . the photoelectric absorption ( ) involves the electrons of the inner shells ( k or l )  . 
the energy is completely transferred from the photon to the electron which is ejected ; the vacancy is filled by an electron from outer shells with emission of characteristic radiation or an auger electron . 
the probability of photoelectric interaction as linear attenuation coefficient ( , see the next section ) follows the rule : = k a ( h ) 3 z4e3 la radiologia medica ( 2019 ) 124 : 12811295 where k is a constant depending on the electron shell , is the density , z is the atomic number of the target , a the atomic weight , h is the plancks constant , is the speed of photon , and e is the energy of the incident photon . 
when the photon energy reaches the binding energy of the electron , there is a peak in the probability of photoelectric absorption ( absorption edge , k - edge if an electron in the k - shell is involved )  . 
for energies above the absorption edge , the probability of interactions rapidly decreases ( 1 / e3 ) [ 5 , 6 ]  . the compton scattering ( c ) involves the electrons in the outer shell . 
the incident photon exchanges part of the kinetic energy with the electron ; the electron is ejected , and the scattered photon continues the travel with a lower energy and a scatter angle . 
the probability of compton scattering is proportional to the electron density ( number of electrons per gram , ne ) , which is quite constant for most materials except for hydrogen . 
the probability of compton scattering , described as linear attenuation coefficient ( c ) , can be approximated as proportional to the electron density and inversely proportional to the x - ray photon energy [ 7 ] : c nee1 limits ofcomputed tomography ( ct ) andphysical principles ofdualenergy ct ( dect ) experimentally , when a monochromatic ( one specific energy ) x - ray beam with an energy e and intensity i0 is attenuated by a homogeneous material , the residual intensity after at a thickness x ( ix ) follows the lambertbeers law : ix = i0ex = i0e ( , z , e ) x where e is th eulers or napiers constant and the linear attenuation coefficient ( the fractional attenuation of the x - ray beam per unit thickness of the attenuating material ) is a function of the energy of the x - ray beam ( e ) , the atomic number ( z ) and the physical density ( ) of the material [ 8 ]  . 
 in ct , the linear attenuation coefficient of each scanned voxel ( r , the considered unit volume ) can be derived from the ct number ( ct# ) in the hounsfields equation [ 1 , 9 ] : ct# = ( r ) water water 1000 the linear attenuation coefficient is related to the physical density , and its application in the attenuation of 1 3 la radiologia medica ( 2019 ) 124 : 12811295 1283 biological tissues can be less convenient . 
the physical density of the attenuating material is influenced by the physical state of the matter ; however , the energies involved in photonelectron interactions are much greater than the energies involved in molecular bindings ; thus , the linear attenuation coefficient can be considered approximately proportional the physical density and eq.3 can be written as : ix = i0e ( z , e ) where / can is the mass attenuation coefficient [ 8 ]  . 
in the case of a mixture of materials , such as biological tissues , the mass attenuation coefficient can be approximated as the sum of the mass attenuation coefficients of each compound : where wi is the weight fraction of each compound of the mixture [ 8 ]  . 
this is the mathematical expression of the intrinsic limits of ct : considering the attenuation of a mixture with two known materials at unknown proportions , eq.6 has no solution because it is an equation in two unknowns ( w1 and w2 ) and one solution ( , or ct# ) [ 8 , 9 ]  . 
the plot shows the linear attenuation coefficient ( total ) of iodine at different densities ( = 0.1 g / cm3 , light blue ; = 0.01 g / cm3 , dark blue ) and calcium ( = 0.1 g / cm3 , red ) as function of energy of the incident x - ray photon . 
approximating the x - ray beam in ct to monochromatic , the colored curves are a representation of the ct numbers ( total ) of a given material at a given density when irradiated with a given energy . 
the attenuation curve is characteristic of the material and models the compton and photoelectric interactions : the blue curves of iodine at two different densities are equal with the same absorption edges ; the value of linear attenuation coefficient ( total ) is function of energy ( the shape of the curve ) and of density ( light and dark blue curves of iodine )  . 
if the same materials at the same densities are scanned at a different energy levels ( e.g. , 10kev , black dashed line ) , the linear attenuation coefficients and the ct numbers are clearly different ( black arrowheads )  . 
by knowing the attenuation curves , a couple of materials can be characterized and quantified if scanned at two energy levels ( i.e. , material decomposition in dualenergy ct )  . 
national institute of standards and technology ( : / / www.nist.gov / ; accessed on october 8 , 2019 ) 1 3 1284 la radiologia medica ( 2019 ) 124 : 12811295 fig . 
in the mixed images , the ct numbers in each pixel pixels are a weighted sum of the correspondent ct numbers in the lowand highenergy reconstructed images and can be considered as similar to conventional ct . 
the material decomposition evaluates the different attenuation curves of the basis materials , allowing for material labeling ( iodine : blue map and arrowheads ; calcium : red map and arrowheads ) ( e ) = ( e ) + ( e ) + ( e ) ( e ) + two - basis - material decomposition in the projection domain ( raw data ) ( fig.3 ) [ 2 , 3 ]  . this principle is at the basis of the first two - basis - material decomposition algorithm developed by alvarez and macovski . 
this demonstrates that when using a conventional x - ray beam , the effective attenuation coefficient can be calculated from the contribution of compton and photoelectric interactions modeled on the effective eff ( density ) and zeff ( atomic number ) of the material : ( e ) e ( e ) e e = e + fkn ( e ) ( e ) ( e ) + ( e ) + where and are constants , d 3 4 , g 3 3.5 and fkn ( e ) is the kleinnishina function . 
the right term of the third part of eq.8 is the linear combination , respectively , of the ] and compton [ fkn ( e ) ] interphotoelectric [ actions . 
 the work of alvarez and macovski is at the basis of the rho - z ( z ) algorithms for material decomposition and the eeg starting from the work of alvarez and macovski , kalender etal . 
defined the mass attenuation coefficient of a given mixture , as function of energy [ ( / ) ( e ) ] , with a linear combination of the mass attenuation coefficients of two hypothetical known basis materials ( 1 and 2 ) present at different mass densities ( 1 and 2 , see also the weight fractions wi in eq.6 ) at the spatial location r : ( r , e ) = 1 ( r ) ( e ) + 2 ( r ) the linear attenuation coefficient at a given spatial location ( r , e ) ( i.e. , the measured attenuation in ct ) is the sum of two known materials with known mass attenuation coefficients [ ( / ) 1 , 2 ] present at unknown mass densities within the voxel ( unknown 1 , 2 ) ; the equation has no solution when a single x - ray spectrum is used [ 13 ]  . if we measure the attenuation coefficients after irradiation with two x - ray energies ( high and low , h and l , ih and il ) , eq.9 can be solved as a system of two equations with two solutions ( ( r , e ) h , l ) and two unknowns ( 1 , 2 )  . 
vmi at low kev are useful for highlighting focal lesions in condition of low contrast ( e.g. , in liver or pancreas ) ( fig.4 ) or for improvement ofthe contrast - to - noise ratio and reduction of the administeredcontrastmaterial [ 14 ]  . 
conversely , vmi at high kev are useful for reduction of proton - starving and beam - hardening artifacts in the presence of metallic objects at the expense of contrast ( fig.5 ) [ 15 ]  . 
a drawback of some vmi algorithms is the increasing noise at lower energies ; this problem has been addressed with the introduction of noise - corrected vmi ( monoplus , monoenergetic plus , siemens healthineers ) [ 16 ]  . 
the theoretical advantage of the approach in the projection domain is the avoidance of beam - hardening artifacts : the linear attenuation coefficient is obtained from the attenuation coefficient of the basis materials at the calculated mass densities [ 13 ]  . 
 a critical issue of the projection domain approach is the large amount of data processed and the high computational power required : the post - processing of dual - energy data could be easier on reconstructed images ( fig.3 ) [ 13 ]  . 
equation8 can be defined as the linear combination of the mass attenuation coefficients of two basis materials present in the mixture with fractional masses of m1 + m2 = 1 ( being m2 = 1m1 )  . 
also , the effective linear attenuation coefficient ( eff ) can be calculated from the ct numbers ( ct# ) as in eq.4 : e = water = e + fkn ( e ) 1000 = e 1 m1 ( 12 ) 1 3 1286 la radiologia medica ( 2019 ) 124 : 12811295 fig . 
despite the strong weighting of the low voltage on image a , the monochromatic plus reconstruction allows for better visualization of a higher number of liver metastases ( empty arrowheads ) fig . 
 a , b dual - energy acquisitions 100 / 150kvp , mixed 0.8 , venous phase , soft - tissue window ( a ) and bone window ( b )  . 
marked beam - hardening and photonstarving artifacts are present close to the prosthesis ( arrowheads ) limiting the evaluation of the soft tissues ( a ) and the cortical bone ( b )  . 
the artifacts are reduced , the visualization of pelvic soft tissues ( c ) and the cortical bone ( d ) are improved ( empty arrowheads ) at the expense of contrast ( * ) the effective linear attenuation coefficient is calculated from the ct number ( ct# ) , it is a function of photoelectric and compton interaction of a mixture with an atomic number zeff but can also be defined as a function of two unknowns : the effective mass density ( eff ) and the fractional mass of one basis material ( m1 )  . 
the ct acquisition with two different x - ray energy spectra ( h and l ) provides two datasets of reconstructed images that can be processed to obtain the two unknowns : ct#h 1000 + 1 ct#l 1000 + 1 water = e water = e 1 m1 1 m1 ( 13 ) this is the concept behind the material decomposition on image domain this case , since the reconstructed images are used , the beam - hardening artifacts are not automatically removed andneed to be corrected [ 17 ]  . 
proposed a hybrid algorithm with a pre - processing in the projection domain to reduce beam - hardening artifacts [ 18 ]  . 1 3 la radiologia medica ( 2019 ) 124 : 12811295 1287 an interesting point is that any material with different atomic number than the chosen basis material variably contributes ( and is characterized as ) to the attenuation of each of the basis material in all of these algorithms [ 13 ]  . 
a classical case was the calcium quantification in osteoporosis with dual - energy acquisitions of vertebral bones , with a mixture of calcium , fat ( yellow marrow ) and water or soft tissue ( red marrow )  . 
the authors started from the hypothesis that the effective linear attenuation coefficient in function of x - ray energy eff ( e ) comes from the sum of the mass attenuation coefficients of each basis material ( i ) multiplied by their concentrations ( the concentration ci is defined as the product between the fractional volume vi and mass density i ) , similarly to what expressed in eqs.6 , 9 , 10 , 12 , and 13 : ( 15 ) ct ( h ) = v1ct#1 ( h ) + v2ct#2 ( h ) + v3ct#3 ( h ) ct ( l ) = v1ct#1 ( l ) + v2ct#2 ( l ) + v3ct#3 ( l ) v1 + v2 + v3 = 1 where ct#i ( e ) are the ct numbers of pure basis materials at the given x - ray energy , previously obtained ( e.g. , calcium hydroxyapatite as a model for bone )  . 
this model allows for calculation , in the image domain , of relative quantities ( fractional volumes ) of three basis materials , by two measurements ( acquisitions at two different energies ) with a third condition ( the conservation of volume )  . 
the model cannot be generalized because the conservation of volume is not always respected ( e.g. , salt in water or iron in fat or soft tissue )  . a more generalized model was proposed by liu etal . 
in b , the blue dot over the separation line is the pixel labeled as iodine , while the red dot is the pixel labeled as calcium ( see also fig.2 ) 1 3 1288 la radiologia medica ( 2019 ) 124 : 12811295 fig . 
a dual source : two tubes working at different voltages and two asymmetrical detectors are mounted with an offset of nearly 90 , increased in the latest generation to improve the fov of system b up to 35.5cthe tin filtration for better spectral separation is possible with these scanners . 
the main purpose of this architecture was of achieving a temporal resolution acceptable for cardiac studies ( < 100ms : 6585ms ) with both tubes working ( flash or turboflash acquisition , siemens healthineers ) ; the dual - energy mode with the two tubes at different kvp is a natural corollary in these scanners [ 4 , 23 ]  . due to engineering issues ( balance and space in the gantry ) , the two detectors are asymmetrical with the smaller detector b limited to a field of view ( fov ) of 266mm in the first generation ( thus limiting the fov for material decomposition )  . 
the straton tube in the first generation was capable of 20kv steps between 80 and 140kv in the first generation , allowing for the combination of 80 / 140kv in dual - energy acquisitions . 
in the second generation , a tin filter ( 0.4mm ) was added to improve the spectral separation ( the robustness of material decomposition algorithms is proportional to separation of the two x - ray spectra ) allowing for two combinations in dual - energy acquisitions ( 80 / 140kv ; 100 / 140sn kv ) [ 24 ]  . 
in the third generation , the vectron tube was introduced : it is capable of 10kv steps between 70kv and 1 3 la radiologia medica ( 2019 ) 124 : 12811295 1289 150kv with a maximum current of 1300ma at 70kv . 
this technology allows for ( 1 ) a more frequent use of low voltages ( contrast and dose reduction ) and ( 2 ) better spectral separation ( 70100 kv / 140150 sn kv )  . 
moreover , the higher power allows for a more aggressive filtration at high voltages ( 0.6mm tin ) with further improvement of spectral separation ( fig.7 ) [ 25 ]  . with the newer generations , the rotation speed was improved ( up to 0.25s ) with no limitations for dual energy , together with the z - axis coverage and the introduction of integrated stellar detectors ( the lower noise in the stellar detectors improved the material decomposition )  . 
iterative reconstructions can be used with both tubes [ 25 ]  . this technical solution requires the material decomposition to be performed on image domain ( due to ~ 90 offset of the projections ) , and adequate algorithms are implemented to reduce the cross - scattered radiation between the two systems . 
besides the material decomposition , virtual monochromatic and rho - z methods , the scanner provides the blended ( mixed ) images for reading and reporting , in which the pixels are a weighted sum of the ct numbers from the highand low - energy datasets , without any material decomposition performed ( fig.3 ) [ 23 ]  . fast kvp switching in the fast kvp switching scanners ( discovery ct 750 hd and revolution , ge healthcare , milwaukee , wi ) , the x - ray tube switches between 80 and 140 kvp in less than 0.2ms , thus acquiring highand low - energy projections within the same rotation with almost no - temporal mismatch and at a full fov of 50cm . regarding the x - ray tube , the spectral separation ( and the material decomposition ) is affected by the ramp - up and ramp - down of the tube voltages while switching . 
at low voltage , the photon output is lower and the noise increases : higher current and longer exposure time are necessary at low kv ( typical exposure ratio : 80 / 140 kvp = 60% / 40% ) ; the automatic current modulation is not feasible in this configuration , and the dose reduction is implemented only with the iterative reconstructions . 
other challenges in detector design are the position of the photodiodes and the necessary presence of an antiscatter grid reducing the optical sensitivity when comparedto conventional detectors with photodiodes below the scintillator . 
conventional ct images are reconstructed by weighted summation of the highand low - energy datasets ; iterative reconstructions can be used for dose reduction [ 22 ]  . the split filter the split filter in the somatom edge twinbeam ( siemens healthineers , forchheim , germany ) is composed of two different metals : tin and gold , which , respectively , filter the low and high components to split a polychromatic x - ray beathe dual - energy datasets are acquired with each half of the detector receiving the split beathis allows for dualenergy information at full fov of 50cm with no limitations in rotation speed . 
potential drawbacks are the short pitch values ( 0.30.5 ) , the necessary high power of the tube because of filtration , with potential limitations in larger patients , and the limited spectral separation ( fig.7 ) [ 27 ]  . consecutive acquisitions this method does not require a dedicated hardware . 
dose reduction techniques , such as automatic current modulation , can be used ( fig.7 ) [ 28 ]  . 1 3 1290 la radiologia medica ( 2019 ) 124 : 12811295 1 3 la radiologia medica ( 2019 ) 124 : 12811295 1291 fig . 
 b iodine map ( red ) showing a perfusion defect ( * ) in the right lower lobe close to the pulmonary embolus ( arrowhead ) ; dual - energy acquisition 90 / 150sn , iodine overlayct dualenergy ct : postprocessing andclinical applications the applications of dual - energy can be divided in materialselective and energy - selective . 
the former includes material decomposition with material labeling , quantification ( distribution maps ) and subtraction ( vnc ) ; the latter includes the monoenergetic imaging , effective atomic number and effective electron density ( rho - z maps )  . 
table1 provides an overview of the algorithms and main clinical application dual - energy techniques . materialselective images : material subtraction , quantification andlabeling with material - selective images , a given basis material is detected , labeled and subtracted : iodine , calcium or other known materials ( e.g. , monosodium urate ) are frequently used as basis material . in neuroradiology , the main application of material decomposition is the material labeling and quantification of iodine in the presence of hemorrhagic material . 
the evaluation of contrast enhancement of a hemorrhagic brain lesion may be challenging in both ct and in magnetic resonance ( mri ) , while dect is able to distinguish the different spectral characteristics of iodine and iron . 
 [ 29 ] demonstrated a significantly higher sensitivity of iodine maps fused with virtual unenhanced images for detection of enhancing hemorrhagic brain lesions . in thoracic imaging , iodine maps allow for the evaluation of perfusion defects in pulmonary embolism , beyond the ct angiography , in acute and chronic setting ( fig.8 ) [ 30 , 31 ]  . 
 in cardiovascular imaging , the iodine maps are useful for the evaluation of myocardial perfusion while the selective subtraction of iodine in virtual non - contrast ( vnc ) images provided good results for the calcium score [ 3436 ]  . 
calcium labeling and subtraction with dual energy is useful for bone removal and plaque removal , improving the evaluation of vascular studies and the evaluation of vascular lumen [ 35 , 36 ]  . 
recorded an improved diagnostic accuracy with dect in characterization lipidic , necrotic , fibrous components and calcium in atherosclerotic plaques [ 37 ]  . an application of material decomposition in abdominal and oncological studies is the vnc reconstructions with avoidance of basal acquisitions and dose reduction , particularly relevant in selected populations ( e.g. , pediatric population ) [ 3842 ]  . 
it has to be pointed that small , often unimportant , differences in attenuation values need to be accounted when using virtual non - contrast images in abdomen ( fig.9 ) [ 43 , 44 ]  . 
the evaluation of iodine maps allows for accurate discrimination between enhancing and unenhancing lesions : this may be relevant in complicated cystic lesions of the kidney [ 45 ]  . 
regarding treatment response , the iodine quantification showed promising results : changes in iodine uptake are a more accurate indicator of treatment response to target or antiangiogenic agents than morphological criteria [ 4749 ]  . 
the evaluation of iodine maps has demonstrated their utility in the evaluation of the gastrointestinal tract , improving the detection of inflammatory , vascular ( ischemic ) and neoplastic lesions [ 50 ]  . 
b dual - energy acquisition , venous phase ( 90150sn kvp ) , 3 - basis - material decomposition ( liver vnc , siemens healthineers ) , virtual non - contrast . 
virtual non - contrast acquisitions are of good quality , may be useful for avoiding basal acquisitions , but sometimes present variable differences in attenuation when compared to basal acquisitions fig . 
material decomposition allows for labeling of monosodium urate crystals in green on coronal ( b ) and volume rendering ( vr ) reconstructions ( c ) ( arrowheads ) with volumetric estimation in liver imaging , the iron quantification with dect is an important tool when mri is contraindicated [ 53 ]  . in musculoskeletal radiology , material labeling is useful for the quantification of monosodium urate crystals in gout and differentiation with other crystal arthropathies ( fig.10 ) [ 54 , 55 ]  . 
the evaluation of bone marrow disorders , typically evaluated with mri , is an expanding application of material decomposition : dect is useful for depiction of bone marrow edema and bone lesions ( three - material decomposition and virtual non - calcium ) [ 57 , 58 ]  . 
vnc and iodine subtraction are also useful in the case of intravenous contrast injection or in ct arthrography [ 59 , 60 ]  . energyselective applications : virtual monoenergetic images andrhoz methods vmi are produced with the linear attenuation coefficients of basis materials obtained from the material decomposition , with the possibility of reconstruction of simulated images in a wide range of energy levels , within or out of the ranges effectively used . 
the vmi reconstructions have demonstrated promising results for the evaluation of pancreatic adenocarcinomas , focal liver lesions ( fig.4 ) or for the extension of endometrial adenocarcinomas [ 6163 ]  . 
another field of applications is the optimization of contrast , similarly to acquisitions at low voltages ( not always possible ) , with potential reduction of the dose of contrast material , in particular in cardiovascular imaging , and in the evaluation of pulmonary embolism [ 14 , 6466 ]  . 
vmi at high kev simulate the use of high - energy x - ray beams and are useful for attenuation of beam - hardening artifacts at the expense of contrast ( fig.5 ) [ 15 ]  . a material or a mixture with unknown attenuation coefficients can be semiquantitatively assessed on the spectral curve and highlighted ; this is the case of biliary noncalcific stones or foreign bodies , such as in drug mules [ 6769 ]  . finally , the rho - z methods still have more limited implementation : interesting applications in tissue characterization , mainly in cartilages and tendons [ 59 ]  . conclusion in this review , we have provided a brief overview on physical and theoretical principles , and clinical applications of dual - energy ct , which is a growing technique with expanding applications . funding no funding was received for this paper . compliance with ethical standards conflict of interest aa is a speaker for siemens healthineers . 
all the staging ct scans were evaluated by three experienced radiologists dedicated to thoracic disease in order to radiologically define the predominant pattern of presentation . results the following parenchymal patterns were observed : 11 patients with single / multiple nodules , five with masses / mass - like consolidations , 14 with consolidations with air bronchogram , 16 with ground - glass opacity , ten with angiogram sign , 22 with perilymphatic and / or peribronchovascular spread , 15 with associated lymphadenopathies , and 13 with pleural / chest wall involvement . 
the main characteristics of ppls were the presence of consolidations and ground - glass opacities , with perilymphatic and / or bronchovascular spread . conclusion all the characteristics of the work should alert the radiologist to consider lymphoma among the possible differential diagnoses , always correlating the results of the ct examination with appropriate clinical laboratory evaluations . keywords lung neoplasm lymphoma malt high - resolution computed tomography diagnosis differential diagnosis introduction lymphoproliferative disorders of the lung can be divided into primary pulmonary lymphomas ( ppls ) and secondary pulmonary involvement by lymphomas arising from adjacent nodal sites ( such as mediastinal or hilar lymph nodes and thymus ) or by haematogenous dissemination from an extrapulmonary site of involvement . 
ppl is defined as a malignant monoclonal lymphoid proliferation within the lung parenchyma , in a patient without any detectable extrapulmonary involvement at least 3months after initial * vittorio miele vmiele@sirm.org 1 department ofemergency radiology , university hospital careggi , largo brambilla 3 , 50123florence , italy 2 haematology unit department ofoncology , university hospital careggi , florence , italy 3 haematology unit andbone marrow transplant unit , san camillo forlanini hospital , rome , italy diagnosis [ 1 ]  . 
it is a rare disease , representing approximately 34% of extranodal lymphomas ( less than 1% of all nhl cases ) and 0.51% of all primary malignancies involving the lung [ 26 ]  . 
ppls can be malt lymphoma ( maltomalow - grade marginal zone b - cell lymphoma of mucosa - associated lymphoid tissue ) , other non - hodgkin lymphomas or hodgkin lymphoma . 
 malt lymphoma is the most common type , representing 5890% of all the ppls ; it is more common in females , often associated with autoimmune disease [ 7 ]  . 
others rare types of ppls include mantle - cell lymphoma ( mcl ) , follicular lymphoma ( fl ) or lymphoplasmacytic lymphoma ( ll ) [ 3 , 4 , 8 ]  . 
in such case , parenchymal involvement usually occurs by direct extension vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 12621269 1263 of mediastinal pathology or it is associated with disseminating disease [ 5 , 810 ]  . 
clinical findings are often unspecific or absent ; pulmonary symptoms such as cough , dyspnoea , haemoptysis , or chest pain may occur , as well as systemic symptoms like fever , weight loss , and fatigue . 
this study aimed to describe the predominant ct patterns of plls evidenced in our population of patients , focusing on the differential diagnosis to address the problematic role of the radiologist in making a correct diagnosis and to help the clinicians to set the right therapy . materials andmethods inclusion criteria our institutional board review approved this retrospective study . 
inclusion criteria were patients age between 18 and 90years , acquisition of informed consent , histological diagnosis of ppl , pre - treatment chest ct with contrast medium utilization . reference standard the diagnosis was histologically confirmed by lung biopsy in all cases . 
clinical staging was referred to as ann - arbor staging , also assessing the presence of systemic symptoms ( fever , night sweats , weight loss of > 10% of body weight over 6months ) [ 11 ]  . imaging protocol because of the length of the considered period ( 16years ) , images were acquired by the use of different ct scanners and technique protocols ( scanners : lightspeed vct 64 , ge ; sensation 16 , siemens ; sensation 64 , siemens ; optima 64 , ge ; ict 128 , philips ; somatom definition flash , siemens )  . 
the acquisitions were all in inspiration phases , and high - resolution images were contiguously reconstructed at 11.5mm using a smooth kernel for mediastinal structures and a sharp one for parenchymal evaluation . imaging review anddata analysis all images were stored in a picture archiving and communication system ( pacs )  . 
an independent and retrospective review of chest imaging for each patient was performed by two radiologists to define the predominant pattern of ppl presentation ; in case of discordance , a consensual agreement was reached . 
ct scans were assessed for the presence of single or multiple pulmonary nodules ( diameter < 2.5cm ) , monolateral or bilateral , masses ( diameter > 2.5cm ) or mass - like consolidations , with or without the coexistence of a positive angiogram sign , ground - glass opacities , parenchymal distribution ( bronchovascular or perilymphatic ) , the presence of hilarmediastinal lymph nodes ( long axis > 1.5cm ) and pleura / chest wall involvement . 
all thoracic images were also assessed for evidence of other associated pulmonary pathology ( such as bronchiectasis / bronchiolectasis , emphysema , and fibrosis )  . results patients were 14 males and 16 females , aged 5686years at diagnosis ( mean age 72years )  . 
angiogram sign and pleural involvement were similar in both malt and dlbcl . discussion the lung is frequently involved in metastatic localization of hodgkin and non - hodgkin lymphomas ( up to 38% in hl and up to 24% un nhl ) [ 7 , 9 , 12 ]  . 
b one - year follow - up ct after chemotherapy shows multiple bronchiectasis in the medium lobe that remains stable for many years 1 3 1266 la radiologia medica ( 2019 ) 124 : 12621269 fig . 
b 8 - month follow - up ct after treatment lymphoid tumours are rare ; as discussed before , malt lymphoma is more common than dlbcl ( 5890% and 1020% respectively )  . 
although dlbcl can occur as a primary proliferation , it can also arise from the transformation of a malt lymphoma into a more aggressive dlbcl [ 6 , 13 ]  . 
imaging manifestations of ppls may overlap with other ct features of completely different diseases and usually make a radiological diagnosis is difficult . in our case series , the most prevalent ct findings in both malt and dlbcl are the presence of masses and consolidations , together with diffuse / focal ground - glass opacities and with perilymphatic and peribronchovascular spread of these lesions . 
on imaging , the ct features of malt and dlbcl can overlap ; in both cases , patients can present nodules or consolidations , although cavitation and necrosis of the lesion are more frequent in dlbcl than in malt lymphoma itself [ 2 , 9 ]  . 
in a majority of patients , a followup ct scan shows any growth of the lesion , which can be stable for many years [ 20 , 21 ]  . 
however , a similar pattern can be seen in other entities and the differential diagnosis , in this case , includes organizing pneumonia , eosinophilic or lipoid pneumonia , lymphoid interstitial pneumonia ( lip ) or lung adenocarcinoma with lepidic growth [ 13 , 22 ]  . 
chest radiograph in a shows a mass - like consolidation in the lower right lobe , confirmed at ct study in b crazy paving appearance was associated with the lesion in the medium lobe . 
b one - year follow - up ct after therapy patient , but it is unlikely that there is a transformation of lip into a malignant process [ 9 ]  . ( transplant , autoimmune disease , clinical indolent behaviour , hiv infection , smoke ) [ 19 , 23 , 26 ]  . usually , air bronchogram is common in ppls because the bronchi and bronchioles tend to be unaffected from the proliferation of the tumour . 
angiogram sign , that is an enhancing pulmonary vessel within a homogeneous area of consolidation , is a feature of ppl ; it is a usual sign but non - specific . 
it could be present in other conditions such as mucinous adenocarcinoma , post - obstructive consolidation , and also in all the lymphoproliferative diseases [ 19 , 2325 ]  . 
 in general , a consolidation with homogeneous attenuation , air bronchograms , and angiogram signs are typical features of ppls but non - specific ; radiologist should keep high suspicion in patients with these ct features and high - risk factors the typical perilymphatic spread of ppls lesions could simulate other diseases , in particular , sarcoidosis and lymphangitic carcinomatosis . 
all these three entities are characterized by abnormalities of the bronchovascular bundles , subpleural interstitium , and interlobular septa ; the thickened interlobular septa and alterations of the subpleural interstitia are significantly greater in lymphangitic carcinomatosis than in the other two diseases [ 27 , 28 ]  . 
 lymphangitic carcinomatous often involves the peripheral interstitium at first , and then progresses towards the central area ( retrograde spread ) ; instead , lung lymphoma and sarcoidosis tend to progress from the lymph nodes 1 3 1268 la radiologia medica ( 2019 ) 124 : 12621269 to the peripheral lymphatics ( anterograde spread )  . 
mediastinal hilar lymphadenopathies and pleural involvement are not frequent in our case series as well as in the other most recent clinical studies [ 23 , 30 , 31 ]  . 
pleural involvement can be shown as a focal thickening , a secondary effusion or a pyothorax , especially in patients with epsteinbarr viruspositive dlbcl [ 32 , 33 ]  . differential diagnosis of ppls includes also two other clinical situations : lymphomatoid granulomatosis ( lg ) and post - transplant lymphoproliferative disorders ( ptld )  . 
radiologically lg can be indistinguishable with ppls but , when both cavitations and ground - glass halos are present , the disease appears similar to granulomatosis with polyangiitis ( wegener granulomatosis , especially in 30% cases here are necrotic coalescing and cavitated nodules ) [ 35 ]  . 
ptld collects a group of lymphoproliferative disorders occurring in the post - solid organ or post - stem cell transplant , varying from benign polyclonal conditions to malignant monoclonal diseases [ 3 , 5 , 9 ]  . 
imaging manifestations are similar to dlbcl , including solid masses - nodules , consolidations , ground - glass opacities ; solid masses in ptld tend not to cavitate [ 19 ]  . 
 both lg and ptld lesions are avidly hypermetabolic at fdg - pet [ 3638 ]  . despite everything , nowadays lung biopsy is still necessary to achieve a definitive diagnosis , essentially in all cases of ppls . 
percutaneous ct - guided or bronchoscopic biopsy may be sufficient for solid lesions ; in the case of groundglass opacity , consolidation , and interstitial involvement , an open surgical biopsy may be required [ 3 , 15 , 30 ]  . this study has some limitations . 
second , the statistical analysis did not reveal any significant correlation between our data , may be because of the various heterogeneity of ppls at ct imaging and the small number of patients . 
however , to the best of our knowledge in the most recent literature , this work contains one of the numerically most significant case series of ppl [ 23 , 30 , 31 , 39 , 40 ]  . in conclusion , we have described the imaging findings in a series of 30 ppls , confirmed by lung biopsy . 
radiologists have to be aware of the various radiological patterns of penile traumatic lesions , in order to establish a prompt and correct diagnosis . keywords penile trauma fracture priapism ultrasonography magnetic resonance retrograde urethrography introduction acute penile traumas are uncommon , with an incidence of 1 / 175.000 in emergency department [ 1 ]  . 
the primary and clinical distinction is between penetrating and non - penetrating traumas : furthermore , it should also be evaluated if the trauma occurred when the penis was flaccid or erect , because the resulting injury is different [ 2 ]  . 
radiological approach is the first choice in cases of blunt trauma , while clinical evaluation , before surgical exploration , is usually sufficient in patients with penetrating traumas [ 3 ]  . 
ultrasonography ( us ) is the first - choice imaging modality in evaluating patients with penile trauma : in particular in expert hands , using high - resolution and high - frequency probes , with color and spectral doppler investigation , us can be really useful and can guide the management of the patient [ 46 ]  . 
magnetic resonance imaging ( mri ) is the best diagnostic tool in evaluating the integrity of tunica albuginea , even in patient with severe injury , because of its panoramic and multiplanar capabilities and excellent tissue contrast [ 79 ]  . 
in emergency department , us is the first ( and often unique ) imaging technique and mri is used only in particular situations in the clinical practice , for example when it is necessary to identify a small tear of ta in a suspect penile fracture . 
the cc are composed of sinusoidal spaces with smooth muscles and endothelium ( erectile tissue ) : the inter - cavernosal septum divides these two structures , and it is porous , allowing the passage of blood from one side to the other . 
in mri , the cc and cs have variable signal intensity depending on the rate of blood flow within the cavernous spaces : usually , they have an intermediate t1w and t2w signal . 
 the thin dartos fascia lies superficial to bucks fascia ; it is not always seen in mri and separates the connective subcutaneous tissue from relatively t2 - hyperintense skin epidermis . 
the vascular anatomy of penis is very variable [ 10 ] , but there are three main paired sources of arterial inflow in the penis : the dorsal artery , which is lateral to the deep dorsal vein and supplies the glans , penis and skin ; the cavernosal artery , a terminal branch of the internal pudendal artery located in the center of each cc , which provides arterial inflow to the cc during erection ; the bulbourethral artery which supplies the urethral bulb and posterior cs . 
 the venous drainage is through the dorsal veins : the superficial dorsal vein , which is superficial to the bucks fascia , and the deep dorsal vein , which is deep to the bucks fascia . 
normal t2 sequence mri ( b ) and sonographic ( c ) anatomy of the penis 1 3 1272 la radiologia medica ( 2019 ) 124 : 12701280 superficial one drains the external envelopes of the penis . 
 the two systems , although separated from the bucks fascia , communicate widely with each other and can , therefore , mutually substitute each other . because of its superficial location , the penis is ideally suited for us imaging . 
sonographic evaluation of the penis is performed with patient in a supine position , and the penis should be in the anatomic position , lying superiorly against the anterior abdominal wall . 
mr imaging of the penis is facilitated by appropriate positioning of the patient : in supine position , the penis is dorsiflexed against the lower abdominal wall in the midline . 
imaging protocols are customized to each patient , but , in general , an axial t1w spin - echo and axial , sagittal , coronal fast spinecho t2w images are obtained . 
both in us and mr imaging , if there is the necessity to have imaging of erect penis , prostaglandin e1 is injected into one corpus cavernosum . penetrating trauma the severity of penetrating traumas to the penis is variable , depending on the involvement of the skin , urethra and erectile tissue , and most of these are caused by selfinflicted mutilation in psychiatric patients or inflicted by a second part [ 2 ]  . 
one of the main reasons of penetrating injury is self - insertion of foreign bodies into urethra on purpose of sexual eroticism : in these cases , retrograde urethrography can be used to assess any possible urethral lesions [ 15 , 16 ]  . 
us and color doppler investigations have a role in identifying hematomas and any possible foreign bodies retained within the penis : in particular , color doppler interrogation of cavernosal arteries allows evaluation of associated vascular injuries . 
 us imaging of penile hematoma from penetrating injuries , as well as hematomas from other causes , depends on the moment of onset : hyperechoic lesion in acute phase , with time it appears as hypoechoic area also with cystic alterations and septated wall . 
cavernosal hematomas can evolve with complete restoration or with a fibrotic scar [ 17 ]  . table 1 basic protocol for post - trauma penile mri basic protocol for post - trauma penile mri baseline standard sequences axial , coronal , sagittal t2w tse ( slice thickness 35mm ) axial t1w tse ( slice thickness 3mm ) additional sequences for trauma axial t1w gre ( slice thickness 35mm ) sagittal , axial t2w stir ( slice thickness 35mm ) anatomic evaluation of cc and cs integrity of ta characterizing fluid collections anatomic evaluation of cc and cs integrity of ta characterizing fluid collections characterizing blood collections characterizing fluid collections and edema after gadolinium administration standard sequences axial , sagittal post - contrast t1w fat suppressed gre ( slice thickness 35mm ) tissues evaluating the enhancement of penile soft tse turbo spin - echo , gre gradient - echo , stir short - inversion time - inversion recovery , cc corpus cavernosum , cs corpus spongiosum , ta tunica albuginea 1 3 la radiologia medica ( 2019 ) 124 : 12701280 nonpenetrating trauma erect penis most of traumas to the erect penis happen during sexual activities and often result in penile fracture , urethral lesions and extratunical hematomas . 
 generally , a tear occurs only in one of the cc ; however , cs fracture can occur and urethral lesions are associated in 1020% of cases [ 9 , 18 , 19 ]  . 
erect penis has an increased risk of fracture because the ta stretches and gets thinner during erection : it has to be remembered that ta is one of the strongest human fasciae and its thickness decreases from 2 to 0.50.25mm during erection [ 11 , 21 ]  . 
penile fractures are rare urologic emergencies : patients hear a popping or cracking sound associated with pain and rapid detumescence , swelling and penile deformity [ 19 , 22 ]  . 
most authors are for an immediate surgical repair of the fracture and making an imaging diagnosis before surgery is recommended : in fact , preoperative imaging is helpful in confirming the correct clinical diagnosis and can also differentiate true penile fracture from false / pseudofracture ( a false fracture is , for example , a hematoma without any ta defect ) [ 3 , 6 , 23 ]  . 
early surgical intervention can prevent late complications such as fibrosis , scarring and angulation of the organ ; surgery is generally recommended in case of suspected urethral injury and ta tear . 
the us detection rate in identifying ta tear is between 88 and 100% [ 6 , 25 ] , but us was unable to detect any ta tear in 12% of patients in the study of mehrjardi etal . 
however , sometimes us evaluation may be nondiagnostic , especially in the detection of small tears or in the presence of a thrombus that closes the ta defect . mri can be used in diagnosing penile fracture because of its panoramic view and multiplanar reconstructions ; however , it is often not performed in acute setting [ 3 , 8 ]  . 
the mri detection rate in identifying tear of ta was between 98 and 100% [ 6 , 28 ]  . in the literature , some authors have found that an accurate delineation of the site of rupture can enable surgeon to use a small focal incision , rather than a more invasive approach used in the past [ 3133 ]  . 
evaluation of the urethra with us is limited : us may be able to show edema or hematoma of corpus spongiosum , but a small isolated injury can be misdiagnosed [ 35 ]  . 
in the proximal third of the penile shaft of the right cc , there is an inhomogeneous oval hypoechoic area , referable to hematoma with a maximum axial diameter of 11.4mm ( arrow , figure a ) ; the tunica albuginea appears intact ( arrowhead )  . 
b , c sagittal t1w pre - contrast and post - contrast sagittal reconstructions that help in recognizing the focal interruption of the tunica albuginea ( arrows ) presence of air in the cavernosa corpora is suggestive of urethral injury , and it should be evaluated . 
a better visualization of the urethra is possible through sonourethrography : this is a retrograde technique in which a saline solution or a lubricant anesthetic jelly is inserted into the urethra via either a foley catheter or a syringe placed directly in the meatus [ 11 ]  . 
torn veins usually collapse and are not visible with us imaging , but sometimes they may undergo a post - traumatic thrombosis ( enlarged vessels with echogenic content )  . 
usually , aspiration of the hematoma is recommended to prevent septal fibrosis and its symptoms ( rigidity and penile shortening )  . 1 3 la radiologia medica ( 2019 ) 124 : 12701280 1275 fig . 
in this patient , the penis crushed against the pelvic bone ( asterisks ) after a blunt trauma : there is neither evidence of hematoma nor a ta laceration fig . 
catheter is placed in the urethra in the corpus spongiosum ( asterisk ) flaccid penis non - penetrating traumas to flaccid penis are usually caused by blunt perineal injuries , where the basis of the penile shaft is crushed against the pelvic bone [ 2 ]  . 
injury to the deep venous plexus or to the smooth muscle trabeculae can lead to intracavernosal hematoma : ta is usually intact in these patients [ 2 , 11 ]  . 
intracavernosal hematoma is usually bilateral , and its us appearance varies depending on the moment of bleeding : hematomas appear as a hyperechoic mass in the acute phase and then become cystic , often with septations . 
priapism is defined as a prolonged ( > 3h ) and sometimes painful penile erection without sexual desire , and it is classified as high - flow and low - flow priapism [ 3 , 5 , 41 ]  . 
patients usually come to the emergency department because of a painless persistent erection : the disease is occasionally recognized several days or month 1 3 la radiologia medica ( 2019 ) 124 : 12701280 1276 fig . 
9 isolated disruption of penile septuaxial ( a ) and sagittal ( b ) t2w images show a round well - circumscribed hematoma near the septum , with a hypointense wall ( arrow ) in subacute phase fig . 
a we can see a tear of the right cc with a large hypoechoic area ( with diameter equal to the diameter of the cc ) ( arrow )  . 
the tear shows a connection with the cavernous artery and has an arterial color doppler signal ( b ) : this sign indicates the presence of a fistula in high - flow priapis tunica albuginea and left cc are intact after the trauma , because of its symptoms that are not dramatic in low - flow priapism [ 43 , 44 ]  . 
nowadays , color doppler us is the imaging modality of choice because of its sensitivity and non - invasiveness and wide availability in radiology emergency department [ 42 , 44 ]  . 
11 artero - cavernosum fistula and high - flow priapisa a large hypoechoic area within the right corpus cavernosum , which shows a connection with the cavernous artery and has an arterial color doppler signal ( b )  . 
below the laceration , both corpora cavernosa are erected ( c ) and the doppler signal is suggestive of the presence of high - flow priapism ( d )  . 
any post - traumatic morphostructural alterations of the left corpus cavernosus are appreciated hypoechoic area , with well - circumscribed margins in the cc : in this area , color doppler interrogation demonstrates a characteristic arterial color blush ( extravasation of blood from the torn artery ) with high velocity and turbulent flow in the site of fistulization . 
12 arteriography pre ( a ) and post ( b ) insertion of spirals , as therapy for high - flow priapisin figure a , it is appreciable the flushing of contrast media from the cavernosal right artery ( arrow ) 1 3 1278 la radiologia medica ( 2019 ) 124 : 12701280 penis is rigid and painful . 
low - flow priapism is usually not caused by a direct penile trauma , but it can follow a central nervous system trauma or it can be secondary to medications , thrombophilic state , malignancy and neurological disorders [ 47 ]  . 
in low - flow priapism , there is a malfunction in penile vascular outflow : obstruction to the physiological venous outflow leads to a high intracavernosal pressure , irreversible ischemic alterations and permanent erectile disease . 
usually , priapism is well evaluated clinically : sometimes the analysis of cavernosal blood gas level may help , with a low oxygen tension in case of low - flow priapis the main aim of the therapy is to have a rapid reduction of the ischemic state , and in emergency , there is a limited role for imaging . 
the area ( arrows ) is hypointense both in t1w ( a ) and t2w ( b , c ) sequences , thus indicating a fibrotic area that could be mistaken for a ta tear , especially in sagittal reconstruction ( c ) 1 3 la radiologia medica ( 2019 ) 124 : 12701280 1279 fig . 
 axial t1w ( a ) and t2w ( b ) , sagittal t2w ( c ) images show diffuse hypointense fibrotic areas in both cc and the crura with thickening of the ta ( asterisks )  . 
it is possible to appreciate widespread fibrotic alterations of both cc in the entire length of the penis , except only for the peripheral portion of the cc themselves ( arrow )  . 
 after gadolinium administration ( d , e ) , the fibrotic areas do not enhance post - traumatic scarring and fibrosis may involve the ta , the corpora cavernosa and the surrounding tissues [ 35 ]  . 
usually , a diffuse penile fibrosis is a consequence of permanent low - flow priapism ; rarely , it can follow highflow priapism as well , as it has been described by savoca etal . 
radiologists must be aware that they have the key role to optimize clinical management of patients with penile trauma , escaping from errors and vain therapies , in order to avoid the potential irreversible sequelae . author contributions cozzi d , verrone gb and pradella s contributed to manuscript writing / editing ; damico g and agostini s contributed to manuscript writing / editing , data collection and management ; bartolini m and miele v contributed to protocol / project development . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest related to the publication of this article . ethical standard this article does not contain any studies with human participants or animals performed by any of the authors . la radiologia medica ( 2019 ) 124 : 10371042 ultrasonography receiver operating characteristic analysis ofultrasound joint inflammation inrelation tostructural damage anddisease activity inrheumatoid arthritis huihuali1 johncarsonallenjr.2 julianthumboo3 , 4 , 5 yorkkiattan3 , 4 , 5 received : 23 april 2019 / accepted : 24 june 2019 / published online : 3 july 2019 italian society of medical radiology 2019 abstract objective to investigate whether ultrasound greyscale ( gs ) and power doppler ( pd ) joint inflammation may be useful in identifying rheumatoid arthritis ( ra ) patients in different states of structural damage and disease activity . methods in this cross - sectional study utilizing 36 - joint ultrasonography , bone erosion was scored dichotomously ( 1 = yes / 0 = no ) while gs and pd joint inflammations were graded semi - quantitatively ( 03 ) at each joint recess . 
ultrasound power doppler ( pd ) imaging provides valuable real - time information on synovial vascularity , while ultrasound greyscale ( gs ) imaging provides essential structural information at the joints such as extent of synovium thickening and erosive changes in bone [ 2 ]  . 
because pd and gs imaging are both core components of ra ultrasound joint assessment , the increased focus on standardization and clinical validation work over the past decade is not surprising [ 3 ]  . 
 both the european league against rheumatism ( eular ) and the american college of rheumatology ( acr ) have vol . : ( 0123456789 ) 1 3 1038 la radiologia medica ( 2019 ) 124 : 10371042 published recommendations on ultrasonography use in the clinical management of patients with ra [ 4 ] and rheumatology clinical practice [ 5 ]  . 
despite efforts to define the role of musculoskeletal ultrasound in ra clinical management , important practical issues remain to be addressed , one being a determination of the accuracy of ultrasound - diagnosed joint inflammation as a clinical marker for identifying ra patients in different states of disease activity and structural damage . 
in the recent literature , only a single recent study was found that utilized roc analysis to determine optimal cut - off values of ultrasound pd and gs joint inflammation metrics to identify ra patients in remission based the 28 - joint disease activity score ( das28 ) [ 8 ] , and none investigating optimal cut - off values for identifying patients with high bone erosion burden detected on ultrasonography . 
by applying roc curve analysis , our study aims to fill a current knowledge gap by investigating whether ultrasound gs and pd joint inflammation scores may be useful for identifying ra patients in different states of structural damage and disease activity . methods ra patients with at least one swollen and / or tender joint were recruited into this cross - sectional study from march 2016 till january 2017 . 
 [ 10 ] as follows : a semi - quantitative score of grade 0 refers to no colour signal ; a score of grade 1 refers to up to either ( a ) 2 single and 1 confluent signal or ( b ) up to three colour signals ; a score of grade 2 refers to greater than grade 1 to less than 50% filled with colour signals ; a score of grade 3 refers to greater or more than 50% filled with colour signals . 
ultrasound gs scoring relied on a previously published ultrasonographic atlas [ 11 ] which utilized representative ultrasound images for b - mode ( combined score for synovitis and joint fluid ) semi - quantitative ( 03 ) severity scoring . 
score of 0 = absence ; score of 1 3 la radiologia medica ( 2019 ) 124 : 10371042 1039 1 = mild ( describing the presence of a small hypoechoeic / anechoic line which is beneath the joint capsule ) ; score of 2 = moderate ( describing the joint capsule being elevated parallel to the joint area ) ; score of 3 = severe ( where there is a strong distension of the joint capsule )  . 
dichotomously scoring of ultrasound - detected bone erosion was utilized , as there has been no general consensus on which other more sophisticated methods of scoring bone erosion to use ( such as using a semi - quantitative scoring scale )  . 
das28 is a widely accepted composite disease activity marker for ra in the routine clinical practice and the threshold criteria of ( a ) 2.6 , ( b ) 3.2 and ( c ) 5.1 were used in our study as these are the generally accepted threshold score levels for das28 to distinguish patients in ( a ) remission versus low disease activity , ( b ) low versus moderate disease activity and ( c ) moderate versus high disease activity in ra respectively . 
 [ 8 ] previously looked at optimal cut - off values for ultrasound inflammatory scores to identify ra patients in disease remission , although a similar analysis using ultrasound joint inflammation scores was not performed for ultrasound - detected bone erosion . 
from the auc results of both our study and that by leng etal . , it appears that ultrasound pd inflammation is more useful than ultrasound gs inflammation in identifying ra patients in different states of disease activity . 
this may be explained in part by the inability of ultrasound pd vascularity to reflect the overall cumulative inflammatory burden at the joints over time when performed as a single time - point measurement . 
there is evidence to suggest that measuring joint inflammation over time ( time - integration ) can provide better prognostic information on disease outcomes compared to single time - point measurements as demonstrated by the two studies [ 13 , 14 ]  . 
although mri synovitis and bone marrow oedema at the dominant wrist were independent predictors of 3 - year radiographic progression , in this study , the 1 - year cumulative inflammatory measures of mri synovitis and bone marrow oedema were shown to be more informative in explaining variation in radiographic changes compared to baseline mri values . ultrasound gs joint inflammation has been shown to be associated with ra structural joint damage by various imaging modalities , including ultrasound - detected bone erosion [ 15 ] , radiographic [ 16 ] and mri structural damage progression [ 17 ] , thereby showing evidence of construct validity . 
to the best of our knowledge , the present study is the first to use extended 36 - joint ultrasonography ( with 60 joint recesses scanned per patient ) in an ra cohort with overall longstanding and moderate disease activity in conjunction with roc analysis to determine an optimal gs ultrasound joint inflammation score cut - off to identify patients with high ( ultrasound - detected ) bone erosion burden . 
we arbitrarily utilized the median score in the definition of high ultrasound - detected bone erosion burden as there is presently no general consensus on what constitutes a high or low erosive burden on ultrasonography at the patient level . 
 [ 18 ] has previously shown that improvement in ultrasound gs synovial hypertrophy can occur as early as 2weeks in a cohort of 89 ra patients treated with abatacept followed up longitudinally for 24weeks . 
future well - designed longitudinal studies on ultrasound gs joint inflammation in large ra cohorts will be required to shed light on this aspect of dmards therapy . one limitation of our study is the relatively small sample size , although we have gone to great lengths to study ultrasound joint inflammation and structural damage by using extended 36 - joint ultrasonography on each patient . 
 we believe that including a greater number of joints in the assessment will give a more comprehensive evaluation of 1 3 1042 la radiologia medica ( 2019 ) 124 : 10371042 how gs and pd joint inflammation relate to ultrasounddetected bone erosion . 
notwithstanding the claims of greater feasibility , better representation and use of logistic regression methods in joint selection by some ra ultrasound researchers advocating for using fewer numbers of joints in ultrasonography [ 19 ] , there is presently no formal consensus on a minimum joint set to scan . 
because our study cohort also has an overall longstanding disease duration and moderate disease activity , with the majority of the patients on conventional dmardsand none on biologics , our derived cut - off values for ultrasound gs and pd joint inflammation scores for identifying patients with high bone erosion burden and high disease activity state will require further validation in other independent ra cohorts with different clinical and treatment profiles . in summary , via the application of an extended 36 joints ultrasonography , we performed roc analysis to obtain sensitivity , specificity and other diagnostic parameters to determine whether ultrasound gs and pd joint inflammation scores may be helpful in identifying ra patients in different states of structural damage and disease activity . 
early diagnosis and treatment of lvv are paramount to reduce the risk of ischemic complications such as visual loss and strokes , vascular stenosis and occlusion , and aortic aneurysm formation . 
use of imaging modalities [ ultrasound ( us ) , magnetic resonance imaging ( mri ) , computed tomography ( ct ) and [ 18f ] - fluorodeoxyglucose positron emission tomography ( pet ) ] has steadily increased to enable assessment of cranial and extracranial arteries , as well as the aorta . 
however , these various imaging tools are not yet uniformly used in routine clinical practice and controversy exists as to which imaging modality best provides meaningful assessments of disease activity and damage in lvv . 
early diagnosis and treatment of lvv are paramount to reduce the risk of ischemic complications such as visual loss and strokes , vascular stenosis and occlusion , and aortic aneurysm formation [ 3 , 4 ]  . the first temporal artery biopsy ( tab ) was performed at the mayo clinic in 1932 and was held as the gold standard test to confirm the diagnosis of gca until recently [ 5 ]  . 
thus , use of imaging modalities [ ultrasound ( us ) , magnetic resonance imaging ( mri ) , computed tomography ( ct ) and [ 18f ] - fluorodeoxyglucose positron emission tomography ( pet ) ] has steadily increased to enable assessment of cranial and extracranial arteries , as well as the aorta [ 10 , 11 ]  . 
further , extracranial arteries are not accessible for histological assessment , and in lv - gca , temporal arteries are spared in 40% of patients [ 12 , 13 ]  . 
ultrasound - guided fast track approach in assessing gca patients has led to a reduction in irreversible vision loss and has introduced the concept of gca with and without cranial involvement , or gca with or without large vessel involvement ( lv - gca ) [ 1416 ]  . in tak , temporal arteries are frequently spared , and biopsies of the large vessels are generally not feasible . 
due to strong evidence and high taskforce agreement , emphasis is placed on early confirmation or exclusion of gca to prevent disease complications of luminal changes caused by vasculitis ( such as stenosis , aneurysm or occlusion ) but cannot identify vessel wall pathology . 
these advances have created controversy regarding which imaging modality best provides meaningful assessments of disease activity and damage [ 4 ]  . aortitis is a common finding in gca and tak and can occur secondary to infections , other forms of vasculitis , but rarely occurs as an isolated idiopathic disease found incidentally on radiologically or histological assessments from resected aortic aneurysms [ 18 , 19 ]  . several studies have evaluated the use of us , mri , ct and pet in lvv over the last two decades . 
the aim of this review is to summarize the current evidence of imaging in patients with or suspected of having lvv , and to highlight the clinical implications of the eular recommendations . eular recommendations the eular recommendations were informed by a systematic literature review ( slr ) by duftner etal . 
 [ 21 ] that assessed 43 prospective studies , published until march 2017 , with sample sizes of at least 20 patients with suspected and / or established primary lvv . 
of note , some of the articles assessed overlap in domains and imaging modalities . when reviewing the guidelines , ct and mri also refer to ct angiography ( cta ) and mr angiography ( mra ) , fig . 
the halo sign typically resolves within 24 weeks of glucocorticoid therapy [ 23 , 24 , 2835 ] and has not been prospectively studied as a marker to assess disease activity . majority of the us studies in the slr ( n = 16 / 17 studies ) in gca tested for the halo sign . 
analysis of 8 studies ( 605 patients ) comparing the halo sign to a clinical diagnosis of gca revealed a positive likelihood ratio ( lr + ) of 19 , negative likelihood ratio ( lr ) of 0.2 with a pooled sensitivity ( sn ) of 77% and specificity ( sp ) of 96% . 
when the halo sign was compared with tab as the reference standard , a comparably good diagnostic performance was obtained ( n = 7 studies , 289 patients )  . 
the inclusion of vessel stenosis or occlusion on us did not provide further diagnostic accuracy versus halo alone [ 21 ] , since vascular stenosis or occlusion occurs due to inflammatory wall swelling , which is a correlate of the halo sign [ 9 , 23 , 32 , 36 , 37 ]  . 
imaging should ideally occur within 1week of glucocorticoid therapy initiation , as treatment rapidly reduces the sensitivity of imaging beyond this time frame [ 9 , 2325 ]  . the working group highly agreed based on expert opinion that imaging examinations must be done by trained specialist using appropriate equipment , operational procedures and settings . 
 development of training programmes , as well as national and international courses for imaging in lvv ( particularly for us ) is needed to facilitate implementation of these recommendations . cranial giant cell arteritis role ofultrasound incranial giant cell arteritis ultrasound should be the primary imaging test in patients with cranial symptoms due to a high level of evidence , good test performance , rapid access , lack of radiation or need of contrast material , safety , with relative low costs compared with other modalities . 1 3 968 la radiologia medica ( 2019 ) 124 : 965972 role ofmagnetic resonance imaging incranial giant cell arteritis high - resolution mri refers to the use of 1.5 to preferably 3 tesla mri to visualize vessel wall thickening and contrast enhancement as signs of vasculitis in the superficial temporal and occipital arteries . 
 thus , based on quality evidence and high agreement , mri of the cranial arteries can be used as an alternative to diagnose gca if us is not available or inconclusive . when mri was compared to tab , mri yielded a sn of 93% and a sp of 81% ( n = 6 studies , 443 patients )  . 
it is tempting to compare these results to that of us , and to consider better diagnostic accuracy in mri ; however , these data must be interpreted with caution . 
for example , in most mri studies , tab was only performed in cases with high a suspicion of gca , while in us studies , most patients underwent a tab [ 21 ]  . 
mri allows for the detection of concurrent structural lesions such as vessel wall thickening , luminal stenosis , occlusion , as well as arterial wall enhancement [ 21 ]  . 
specific sequences ( laid out in the eular guidelines ) are required to image both the arterial wall and lumen . advantages of mri include standardization of data acquisition , assessment of multiple cranial and extracranial arteries , including the aorta . 
this remains an area that requires further study [ 48 ]  . challenges with full mra include specific technical settings , multiple coils and long acquisition time in the scanner . 
since patients with suspected gca are treated with high dose glucocorticoids prior to completing investigations , mri must be performed urgently within 4days of treatment initiation to avoid false negatives . role ofpositron emission tomography andct scan incranial giant cell arteritis utilization of pet . 
practically , most centers do not have rapid access to pet which limits its use in routine practice . large vessel giant cell arteritis lv - gca can present with the absence of typical cranial symptoms and non - specific complaints . 
if gca cannot be confirmed or excluded based on clinical , laboratory and imaging results , use of tab and / or additional imaging is warranted , as it may be a case of lv - gca . 
based on expert consensus , the working group agreed that mural inflammation and / or luminal changes in extracranial arteries present on either us , mri , ct and / or pet support the diagnosis of lv - gca . 
notably , tab has a lower sensitivity in patients with predominant lv - gca as compared with cranial gca [ 12 , 13 , 50 ]  . role ofultrasound inlarge vessel giant cell arteritis in patients with a high clinical suspicion of gca with negative or inconclusive temporal artery scans , assessment of axillary and other cranial and / or extracranial arteries is valuable . 
despite this , based on expert consensus , the working group endorses assessment of axillary arteries . currently , no study has answered whether us is useful to assess relapse or monitoring of disease activity [ 29 ]  . 
also , new large vessel vasculitis lesions have been reported in up to 10% of gca patients followed by serial us despite treatment [ 34 ]  . role ofmagnetic resonance imaging inlarge vessel giant cell arteritis no study was identified in the slr addressing the role of mri for evaluating extracranial lv - gca . 
mri can be used to monitor inflammation and / or aortic dilatation . role ofpositron emission tomography andct scan inlarge vessel giant cell arteritis the working group agreed that ct and pet could not be recommended in diagnosing cranial gca . 
this is pertinent 1 3 la radiologia medica ( 2019 ) 124 : 965972 in elderly patients with constitutional symptoms without specific clinical features of gca and / or polymyalgia rheumatica ( pmr )  . 
this example highlights how the performance of a diagnostic test is influenced by the reference standard , and that a reliable standard is needed in extracranial lvv to facilitate future studies . in the same study by blockmans etal . 
two - thirds of patients in full clinical remission still had a positive pet at both follow - up visits , and pet scores did not significantly differ between remission and relapse . 
whether ongoing tracer uptake in patients in full clinical remission is caused by low - grade inflammation or remodeling , and whether targeting treatment to modify these changes has any impact on future outcomes requires further studies [ 21 ]  . disadvantages of pet include high costs , lower availability , radiation exposure , rapid reduction of sn to 36% after 10days of glucocorticoid treatment and misinterpretation of atherosclerosis as lvv with inexperienced readers [ 53 , 54 ]  . ct may be useful to detect structural lesions and vessel wall inflammation while enabling a higher resolution with shorter procedural time compared to mri . 
 there is only one small study ( 24 patients ) that reported a sn of 73% and sp of 78% using cta compared to a clinical diagnosis of gca [ 48 ]  . takayasus arteritis the taskforce agreed based on expert consensus , that us , pet and / or ct may be used as alternative imaging modalities in patients with suspected of tak . 
note , us is of limited value for assessment of the thoracic aorta . role ofultrasound intakayasus arteritis no studies have evaluated us in the diagnosis of tak , but it is of value in the assessment of patients with upper and lower limb claudication . 
us can be used to screen for structural damage in patients with signs or symptoms of stenosis , occlusion , aneurysm and / or in those with recurrent / persistent large vessel inflammation . 
however , us has not been able to demonstrate the capability to differentiate active disease from remission in tak [ 55 ]  . role ofmagnetic resonance imaging intakayasus arteritis based on expert consensus and a high level of agreement , patients with suspected tak should be investigated with mri assuming prompt availability and expertise . 
the most significant limitation of mri is availability . role ofpositron emission tomography andct scan intakayasus arteritis no studies have evaluated pet in tak ; it may be useful in patients suspected to have infection or malignancy . 
one small study ( 25 patients ) using cta compared to conventional angiography for diagnosing tak revealed a sn and sp of 100% [ 58 ]  . role ofimaging infollowup ofgiant cell arteritis andtakayasus arteritis in patients with lvv in full clinical and biochemical remission , the utility of additional imaging to determine disease activity is unknown . 
involved extracranial arteries may have residual inflammatory changes that remain for months , 1 3 970 la radiologia medica ( 2019 ) 124 : 965972 making them valuable targets to assess for disease remission [ 28 , 30 , 31 , 34 , 35 ]  . cranial and lv - gca . 
studies to clarify the role of imaging in disease monitoring are also required . mra , cta and / or us may be used for long - term monitoring of structural damage to detect stenosis , occlusion , dilatation and / or aneurysm formation . 
risk factors for aortic dilatation include baseline aortic inflammation , male sex , hypertension and smoking history [ 52 , 60 ]  . angiography conventional angiography was not evaluated in the slr though it was the gold standard in diagnosing lvv . 
based on expert opinion , the task force was in agreement that due to the invasiveness , high resource utilization , procedural risk and lack of vessel wall morphology evaluation , angiography should be superseded by modern imaging modalities . 
the main indication for conventional angiography in lvv is for interventions such as angioplasty or stenting [ 61 ]  . conclusions prompt recognition of lvv is vital to prevent complications and harms of unnecessary treatment . 
as these imaging modalities have reshaped our understanding of lvv , they have led to new unexplored questions . mri and pet / ct will have an expanding role in the diagnosis and monitoring of lvv as us is operator dependent and lacks the ability to assess the thoracic aorta . 
us in comparison has good test performance , rapid access , lack of radiation or need for contrast material , along with a relative low cost . going forward , there is a need for prospective studies comparing the diagnostic value of us to mri and pet / ct . 
studies to clarify whether a standardized hierarchical approach to assess different vascular territories ( e.g. , temporal , carotid , subclavian and axillary arteries ) with us , or other imaging modalities improve the diagnostic certainty of as novel treatments are explored in lvv , the need for better classification criteria with a greater emphasis on imaging is required . 
by means of t2 - wi , ece was evaluated in a qualitative manner , according to pi - rads v.2 ( two groups with low and high risk of ece ) ; sensitivity and specificity were calculated for both groups . 
we performed a quantitative analysis on two tractographic parameters , fractional anisotropy ( fa ) and apparent diffusion coefficient ( adc ) and computed the ratio between the lesion quadrant and its contralateral ( l / h ratio )  . 
these cut - off values were used in association with t2 - wi to reassess patients and to evaluate whether specificity and sensitivity of ece detection change . results t2 - wi showed a sensitivity of 80% and a specificity of 71% in detection of ece . 
the simultaneous use of t2 - wi and tractography revealed high sensitivity ( 100% ) on patients with low suspect of ece ( on t2 - wi ) and high specificity ( 83% ) on patients with high suspect of ece ( on t2 - wi )  . conclusion the morphologic component of t2 - weighted imaging and functional aspect of dti should be interpreted together to more successfully assess the presence of ece . keywords prostate cancer extra - capsular extension dti tractography staging introduction radical prostatectomy is a commonly used treatment modality for localized prostate cancer that has been shown to present higher cancer - specific survival than conservative management in high - risk patients [ 1 , 2 ]  . radical retropubic prostatectomy ( rrp ) is one of the most commonly used surgical techniques and provides excellent long - term cancer control in patients with clinically localized prostate cancer ( pca )  . 
erectile dysfunction ( ed ) is one of the complications after rrp , and the recovery of * matteo catania id634iff@studenti.univr.it 1 ospedale policlinico gb rossi , verona , italy erectile function is quantitatively related to the preservation of the neurovascular bundles ( nvbs ) [ 3 ]  . 
however , extra - capsular extension ( ece ) is found in a significant number of patients with clinically localized prostate cancer treated with radical prostatectomy [ 5 ]  . 
therefore , it would be valuable to assess the risk of ece before surgery for correct therapeutic planning [ 1 ]  . several studies have investigated the role of preoperative mri in patients with prostate cancer to evaluate ece [ 1 ]  . 
the method is based on the sensitivity to anisotropic diffusion of the water protons in the biological tissues with a strictly orientated texture , such as nervous central and peripheral fibres . 
in this type of tissues , the spread of water protons is not accidental or brownian , but oriented along a specific axis , thus being referred to as anisotropic [ 10 ]  . 
 through the measurement of fractional anisotropy ( fa ) for each single voxel in at least six non - collinear and noncoplanar directions , dti can quantify the phenomenon of anisotropic diffusion . 
by integrating the fa values of all voxels , it becomes possible to represent the direction of the nerve fibres in all three dimensions of the space , guaranteeing both quantitative and qualitative information [ 11 ] .this technology has also recently been used also to map peri - prostatic nerve fibres ( pnf ) because it offers optimal representation of the peri - prostatic plexus , and could be used as a potential biomarker in predicting ece in patients with pca [ 3 , 12 ]  . 
however , the assessment of ece using t2 - wi is based on a strictly morphologic viewpoint , whereas the dti is a functional marker and cannot be directly compared with t2 - weighted imaging findings due to the low spatial resolution of the dti sequence . 
we believe that instead of comparing the accuracies of t2 - wi and dti with tractography in determining ece , these two sequences should be used together . purpose the aim of this study was to evaluate the role of dti ( with fibretracking ) and t2 - wi used together for predicting extra - capsular extension in patients with localized prostate cancer , using radical prostatectomy as the reference standard . method andmaterials patient selection this prospective study was approved by our institutional review board and included male patients with biopsy - proven clinically significant prostate cancer ( gleason > 6 ) in the peripheral zone , candidate to radical prostatectomy , who gave their written informed consent to mri examination before surgery . 
after standard anatomical tse and dwi sequences , the diffusion tensor imaging was acquired in 32 directions ( tr / te 1449 / 88ms , thickness 3mm , b - value = 0.800s / mm 2 ) , with a scan duration for dti sequence of 3min and 14s . 
at last , the dynamic imaging was performed during gadolinium chelate injection ( 0.1mmol per kilogram of body weight gadolinium chelates , gadovist , schering , berlin , germany ) with a power injector ( medrad spectris solaris ; medrad , pittsburgh , pa , usa ) at a rate of 2ml / s . 
the dynamic imaging consisted of the repetition of 14 t1 dixon acquisitions after contrast injection . image analysis two types of analysis had been conducted in this study : a qualitative analysis and a quantitative one . qualitative analysis : morphologic assessment ont2weighted imaging two radiologists with 20 and 7years of experience in prostate imaging ( readers 1 and 2 , respectively ) independently reviewed the images at a pacs system ; just in case there was no consensus between the two radiologists , then it was tried to reach a consensus together . 
in this way , we tried to overcome the bias that could arise from the difference experience of the two radiologists . ece was evaluated according to pi - rads second version on t2 - w images . 
we wanted to keep reader 1 in both analyses as he has more experience ( 20years ) , while we replaced reader 2 with reader 3 in the quantitative analysis so that it was blind to the qualitative analysis . 
the reader 1 instead performed the qualitative and quantitative analyses in two different periods at time , so as not to correlate the two analyses . the reconstruction was performed by using the fiber tracking software provided by philips ( version 4.1 ) , which consisted of a deterministic tracking algorithm based on the criterion of linear propagation using the following parameters : angle threshold of 45 , fa threshold of 0.15 and minimum length threshold of 5mm , analogously to other published studies [ 11 ]  . in the dti post - processing phase , the dti images were synchronized with the axial t2 - weighted images to correct eventual movement artefacts of the patients ; subsequently , the axial t2 - w images were fused with the synchronized dti images , obtaining a t2 - w / dti hybrid images . 
b voluminous lesion of the left posterior quadrant with extracapsular invasion ( arrows ) and involvement of the seminal vesicle and peri - prostatic adipose tissue 1 3 la radiologia medica ( 2019 ) 124 : 946954 fig . 
2 a positioning of the rois in a patient with lesion involving the capsule in the posterior right quadrant : on an axial plane passing through the lesion , two orthogonal planes are tracked passing through the centre of the gland , dividing it into four quadrants and placing a roi on the side of the lesion ( red ) and in the opposite side ( green ) , obtaining the tractographic reconstructions shown in b after software elaboration the number , the fa and adc values and the length in millimetres were calculated for each reconstructed tractography at the tumour level , both on the lesion quadrant and on the opposite healthy side . pathologic data collection surgical specimens were assessed by the dedicated genitourinary pathologist ( 19years of experience ) and processed according to the stanford protocol [ 13 ]  . 
reader 3 reviewed the records in the pathology database to evaluate the status of extra - prostatic extension , which is the preferred term for the presence of tumour beyond the confines of the prostate gland ( note h in the stanford protocol ) , and descriptors of extra - prostatic extension ( focal versus non - focal ) were used [ 13 ]  . the patient population was clustered into two groups according to the extra - prostatic extension status : the first group with intra - prostatic tumour ( controls group ) and the second group with histological extra - prostatic extension , both focal and non - focal ( cases group )  . statistical analysis in the qualitative analysis , in both groups ( a and b ) based on t2 - wi , sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) were calculated for assessment of ece . in the quantitative analysis , only in the group of patients with extra - capsular infiltration ( cases group ) , the ratio was calculated for each of the five output parameters of the tractographic reconstruction between the lesion quadrant and the contralateral one ( t3 / health ratio )  . 
these analyses were applied to the fa and adc values . results pathological assessment ofece based on radical prostatectomy , ece was found in 15 of 36 ( 42.7% ) patients ; in this group ( cases group , with histological evidence of ece ) , the lesion infiltrating the capsule was localized : in the right anterior quadrant in 2 / 15 ( 13% ) , in the right posterior quadrant in 6 / 15 ( 40% ) , in the left posterior in 4 / 15 ( 27% ) and finally in the left anterior quadrant in 3 / 15 ( 20% ) ; thus , the cases group was formed by 15 patients , while the controls group was composed by 21 patients . qualitative analysis on the t2 - wi , the morphologic evaluation of ece was found in 18 patients ( group a ) , while in the other 18 patients there was no evidence of ece ( group b ) ( table2 )  . 
thus , the ece assessment on t2 - wi showed values of sensibility , specificity , ppv and ppn , respectively , of 80% , 71% , 66% and 83% ( table2 )  . table 2 a representation of the two groups ( low and high risk of ece ) based on the presence or absence of ece on the t2 - w images , b calculation of the sensitivity , specificity , npv , ppv , fn and fp of t2 - w images t2 - wi histological ece ( cases ) histological no ece ( controls ) total t2 - w risk ece t2 - w risk ece sensitivity specificity n = 3 n = 6 quantitative analysis the t3 / health ratio of the five tractographic parameters showed a univocal trend only for the fa and adc parameters ; for fa , all the ratios were < 1 ; for adc , they were all > 1 ( table3 )  . 
 these cut - off values are potentially applicable as an aid in the reporting of multiparametric mri of the prostate in preoperative local staging to improve surgical planning and treatment decisions . given the low spatial resolution of dti , trying to identify direct evidence of ece on dti maps will be limited and nobody can think to use only dti in the ece evaluation . 
there is suspect of ece on t2 - w images : this is confirmed with dti and tractographic reconstructions ( fa = 0.68 [ cut - off < 0.87 ] and adc = 1.41 [ cut - off > 1.23 ] ) 1 3 la radiologia medica ( 2019 ) 124 : 946954 fig . 
6 blandaltman analysis related to interobserver concordance of the adc and fa parameters fundamentally morphologic , dti is known to provide functional information . of the values on different scanners , and we are already applying in this sense . therefore , the key of our analytic approach was to comprehensively incorporate both sequences in determining ece of prostate cancer rather than to directly compare the accuracies of the two sequences . a further important result of our study was represented by the high interobserver agreement values , demonstrating that dti of the nvbs is reliable technique . 
moreover , neither the bias nor limits of agreement between dti output metrics across repeated assessments were not clinically important for either observer , suggesting the potential repeatability of the technique [ 11 ]  . 
however , repeated dti acquisitions under the same conditions were not performed . with regard to the feasibility in clinical practice , we are convinced that it is absolutely feasible as the sequence does not last long ( 3min and 14s ) and the post - processing analysis lasts about 2 min ( launch the application , draw two rois and calculate the ratio between two numbers )  . 
regarding reproducibility on different machines , we have no data regarding the reproducibility of the same sequence on different scanners in prostate imaging as we have used only one scanner . 
having said that , we know from the literature that the quantitative analysis of adc and fa values obtained in the dti sequence provides different values depending on the scanner used in neuroimaging . 
in fact , we have tried to limit this variability by normalizing the values of fa and adc with the healthy contralateral side so as to obtain an absolute value and not a measurement . 
this is in fact a preliminary study , and surely other studies will be needed in this area , both to implement the number of patients studied , and to assess the reproducibility in the study we did not use the endorectal coil ; even if panebianco etal . 
we aligned ourselves with the di paola method [ 11 ]  . the main limitations of the present study were the potential miscount of nerve fibres because of the presence of linear non - nerve structures ( fibromuscular tissue , arteries and veins ) and the relatively small number of patients included in the study . 
analyzed variables included patients sex , age , cirrhosis etiology , childpugh status , size of the lesion , liver segment , subcapsular or central liver site , type of imaging used for fusion ( mr / ct ) , and the presence of surrounding anatomical landmarks ( sal ) < 3cm from the index lesion . results the primary efficacy was 59.4% ( 22 / 37 nodules ) ; nine lesions ( 24.3% ) were partially ablated ( pa ) , six lesions ( 16.7% ) were mistargeted ( ma )  . 
the outcome of the procedure is heavily affected by the presence of sal . keywords hepatocellular carcinoma fusion imaging thermal ablation * marco calandri marco.calandri@unito.it 1 radiology unit , department ofoncology , san luigi gonzaga hospital , orbassano ( torino ) , university oftorino , turin , italy 2 department ofsurgical sciences , radiology institute , citt della salute e della scienza , torino , university oftorino , turin , italy 3 gastro - hepatology unit , department ofmedical sciences , aou citt della salute e della scienza , turin , italy 4 liver transplant center , a.o.u. 
citt della salute e della scienza di torino , university ofturin , turin , italy introduction image - guided percutaneous thermal ablation is a well - established curative treatment for the very early and early stage of hepatocellular carcinoma ( hcc ) [ 1 , 2 ] that remains the sixth most common cancer worldwide and the third leading cause of cancer - related deaths in the world [ 3 ]  . nowadays , ultrasound ( us ) guidance , especially in europe and eastern countries , remains the most used guiding modality because of its several advantages , such as real - time control , accessibility , relatively low cost without ionizing radiation exposure [ 1 , 4 ]  . nevertheless , due to the great recent developments in the ct and mri fields , interventional radiologists are asked to reach and treat percutaneously smaller nodules , not clearly detectable on conventional ultrasound imaging [ 5 , vol . : ( 0123456789 ) 1 3 1044 la radiologia medica ( 2019 ) 124 : 10431048 6 ]  . 
indeed , according to the literature data , approximately 14.633.1% of the hcc nodules are inconspicuous on conventional b - mode evaluation and ablation could not be performed under the sole us guidance [ 4 , 79 ]  . contrast - enhanced us ( ceus ) has been reported as a valuable aid in these cases to target properly the nodules . 
 however , because of poor conspicuity , location or low arterial phase wash - in , there are some cases ( up to 45% in the literature series ) in which even ceus fails to clearly detect the nodule [ 10 ]  . in recent years , fusion imaging ( fi ) has been proposed as a solution to overcome these limitations of us guidance , enabling a real - time display of mr or ct images on the same screen during ablation [ 1115 ]  . despite the presence in the literature of several reports on this topic , factors affecting fi guidance during ablation of us inconspicuous hcc nodules have never been fully investigated , especially if performed under conscious sedation , when patients can breathe spontaneously , potentially affecting the synchronization process of fi . the aim of our study is to evaluate , in this specific setting , the most relevant factors affecting the outcome of thermal ablation of completely inconspicuous hcc nodules with fi guidance . undetectable with conventional b - mode ultrasound only . 
 among these , 21 nodules were visible with good conspicuity at ceus examination by injecting 2.4ml of sonovue ( bracco , milan , italy ) ; 37 nodules in 26 patients were completely undetectable at b - mode or ceus examination and fi guidance was performed . 
variables extracted from the database or updated by review of electronic medical records for each patient and procedure included sex , age , cirrhosis etiology , childpugh status , size of the lesion , liver segment , subcapsular or central liver site , type of imaging used for fusion ( mri sequences or ct phases ) , and the presence of surrounding anatomical landmarks ( sal ) ; sal were defined as any anatomical structure ( such as vessels larger than 3mm , cysts or ligaments ) no more distant than 3cm on axial ct or mr images from the index lesion . ablations were done under conscious sedation by interventional radiologists using a 17 - gauge rfa electrode multi - tined needle ( med - italia , medolla , italy ) with a radiofrequency generator ( rf3000 , 200w capacity , radiofrequency ablation system , boston scientific )  . 
before the needle insertion , 10ml of 2% lidocaine hydrochloride was injected at the puncture site . materials andmethods study design this study was approved by the institutional ethics review board . 
the prospectively compiled interventional radiology liver ablation registry of our institution was retrospectively evaluated and updated by review of the electronic medical records to identify consecutive patients who underwent percutaneous liver ablation for the treatment of hcc nodules from january 2016 through may 2018 . patient selection , ablation eligibility criteria andtechnique patients were considered for percutaneous ablation in case of no more than three hcc nodules , measuring 3cm each . 
 hcc diagnosis was performed with cross - sectional imaging ( ct or mri ) according to easl criteria . indication for ablation was discussed at the institutions multidisciplinary hcc tumor board . 
with the plane registration , the physician found the same plane on real - time us and uploaded ct or mr images ( any plane can be used but , for simplicity , the umbilicus axial plane was considered the first choice ) [ 16 ]  . 
other available functions , such as rotate or drag , were useful to optimize the real - time us - cect / cemri matching [ 17 ]  . assessment oftumor ablation results standardized terminology and reporting criteria for tumor ablation were utilized to determine ablation endpoints [ 18 ]  . 
residual unablated tumor ( partial ablation pa ) was defined as the presence of peripheral or nodular enhancement within 1cm of the ablated area at the first imaging follow - up ( triple - phase contrast - enhanced computed tomography [ ct ] or magnetic resonance [ mr ] )  . 
we defined missed ablation ( ma ) as the condition of complete 1 3 la radiologia medica ( 2019 ) 124 : 10431048 1045 table 1 patients and nodules characteristics mistargeting of the nodule at the first ablation attempt , when the distance between ablated volume center and ideal target point preoperatively established was greater than 5mm [ 19 ]  . 
local tumor progression ( ltp ) was defined as the appearance of tumor foci at the edge of the ablation zone or within 1cm after at least one cross - sectional imaging had demonstrated complete ablation . statistical analysis the normality of distribution for continuous numeric data was assessed with saphirowilk test . 
thus , according to the defined groups , data were reported as mean , standard deviation ( sd ) , and ranges for normally distributed variables , while median , ranges , and interquartile ranges were used for not normally distributed variables . 
 statistical analysis was performed using graphpad stats software ( 2018 )  . results from january 2016 to may 2018 , a total of 37 hcc nodules in 26 patients ( see table1 for nodule and patient characteristics ) underwent image - guided ablation relying only on fi guidance in consideration of their undetectability at conventional b - mode or ceus imaging . 
 ( see table1 for patients characteristics . ) according to claviendindo classification , only two minor complications requiring standard medical therapy ( grade i ) were observed after two ablation sessions . 
according to cirse classification of complications , only two nonsevere complications occurred ( grade ii )  . complete ablation ( primary efficacy ) was obtained in 22 / 37 nodules ( 59.4% ) ; nine lesions ( 24.3% ) were partially ablated ( pa ) , whereas six lesions ( 16.7% ) were mistargeted ( ma )  . 
among the seven remaining nodules , in four cases ( two patients ) , intrahepatic progression was observed and tace was performed ; in two cases , patients underwent liver transplantation , and in one case , patient was lost to followup . 
 discussion our study investigated the role of fusion imaging guidance in treating hcc nodules completely undetectable at conventional b - mode us or ceus evaluation . the main findings of our study were : the primary efficacy of fi - guided treatments is 59.4% ; the secondary efficacy reaches the 81.1% with a ltp rates at 1year f.u. 
 [ 19 ] analyzed a series of fi - guided ablation of completely inconspicuous liver metastases and they demonstrated a similar primary success rate ( 56.7% 17 / 30 patients , in the pet - ct group )  . 
in our series , however , even in case of missed ablation at the first ablation , the ablation scar was used as reliable internal anatomical landmark to repeat the fi targeting and to perform ablation . 
indeed , we reached a 100% ca as a second attempt with an overall ltp rate ( 6.7% ) in line with the other literature reports about poorly visible or visible nodules [ 13 , 22 , 26 ]  . as for the anatomical landmarks , they are well known among fi operators for their role in the second part of the plane and point registration [ 17 ]  . 
our study suggests for them a role of paramount importance in case of ablation of completely inconspicuous nodules in procedures performed in critical technical conditions , such as under conscious sedation , spontaneous breathing and with a pre - acquired ct . 
 in fact , with endotracheal general anesthesia , the breath of the patient can be well controlled using a breathing machine for as long as 2min , greatly facilitating co - registration and subsequent ablation . furthermore , our series was characterized by the usage of pre - acquired cross - sectional imaging ( ct or mr )  . 
in other series , the availability of ct in the operatory room allowed for acquisition of real - time ct images with the patient already under general anesthesia and in the desired decubitus , so the correspondence between ct and us images is increased [ 27 ]  . however , these facilities are rarely available in most of the ir services worldwide and physicians are usually asked to merge with real - time us a pre - acquired ct or mri in supine position . 
therefore , we believe our study gives pertinent information on the relevance of anatomical landmarks in a specific but widespread clinical setting of fusion imaging . as for other possible predicting factors that may affect the outcome of the ablation , our study did not find any significant statistical correlation . 
the ct scans were reviewed by three different radiologists , who evaluated the findings potentially associated with acute rejection such as air trapping , tree - in - bud , consolidations , crazy paving , ground - glass opacity , bronchiectasis , thickening of intralobular or interlobular septa and presence of pleural effusion . 
the association between a tissue diagnosis of acute rejection and the above - mentioned ct findings was assessed using a multivariate model of logistic regression . results based on our results , none of the ct findings included in the study , alone or in combination , showed significant statistical association with the diagnosis of acute rejection . conclusion ct is a very useful technique for the assessment of lung transplant recipients although it has limited accuracy for the assessment of acute rejection . 
 oneand five - year survival rates are approximately 75% and 50% [ 1 , 2 ]  . postoperative graft surveillance includes laboratory data , pulmonary function tests , bronchoscopy with broncho - alveolar lavage ( bal ) and biopsies , and imaging . 
many studies have examined the role of hrct in the diagnosis of chronic rejection , but only a few attempted to assess the accuracy of hrct for the diagnosis of acute rejection [ 3 ]  . 
the aim of this study is to evaluate the sensitivity , specificity and predictive values of hrct for the detection of acute rejection ( ar ) and to determine which computed tomography findings are associated with histopathologically proven acute rejection . materials andmethods study population one hundred sixty lung transplants have been performed at our institute between 2005 and 2018 . 
our final population included 78 patients ; 37 patients with no histopathologic evidence of pulmonary abnormalities ( control group ) ; and 41 patients with a diagnosis of acute rejection . 
 our study was approved by the ethical committee of our hospital . histopathology sampling in our institution , symptomatic and asymptomatic lung transplant recipients are followed up with a regular schedule of blood work , respiratory function tests and clinical assessment . 
a bronchoscopy and ct scan are performed at 1month , 6months and then yearly or when required clinically . bronchoscopy includes a broncho - alveolar lavage and multiple trans - bronchial biopsies ( tbb ) ; a minimum of 6 samples is usually procured from all lobes of one lung . 
the degree of acute rejection is evaluated according to the conventional criteria established for pulmonary rejection : degree a0 , no rejection ; degree a1 , minimal rejection ; degree a2 , mild rejection ; degree a3 , moderate rejection ; degree a4 , serious rejection [ 4 ]  . 
bal samples are also assessed for standard and opportunistic infections , including bacteria , fungal , and viral species . ct scan evaluation ct scans were acquired by lightspeed 16 slice scanner ( ge healthcare , usa ) or lightspeed vct 64 slice scanner ( ge healthcare , usa ) systems . 
when used , the contrast medium was administered by a mechanical injector ( medrad double head ct injector stellant siemens , indianola , pa , usa ) ; 30ml of normal saline solution was administered at the end of the intravenous contrast medium infusion . 
the arterial phase , conventionally acquired in the caudocranial direction , started 6s after reaching the peak of the contrast medium concentration by smart prep technique , with a region of interest on the common trunk of the pulmonary artery . 
the late phase was acquired 50s after the contrast medium administration . images were later reconstructed in the post - processing phase , by dedicated consoles ( advantage windows 4.6 , ge ) , to obtain reconstructions by multi - planar images ( mpr , multi - planar reconstruction ) , images that enhance the contrast density ( mip , maximum - intensity projection ) or highlight hypodensity on a hyperdense background ( minip , minimum intensity projection ) , and volumetric images ( vr , volume rendering ) , to evaluate both vascular and bronchial structures . 
images were then retrospectively analyzed by image archiving and communication systems ( pacs , ge medical systems ) , with two high - definition screens barco ( 5 megapixel ) as visualization support . 
ct scans were assessed by visualization of a window for both soft tissues ( level , 40 uh ; width , 440 uh ) and lung ( level , 700 uh , 1 3 1002 la radiologia medica ( 2019 ) 124 : 10001005 width , 10001500 uh )  . 
a single thoracic radiologist ( ad ) selected the ct scans included in the study . three independent thoracic radiologists ( ad , sc and gm ) reviewed all the ct scans ; they revised and evaluated the single ct tests in a consensus . 
the following ct parameters were evaluated in a non - quantitative fashion ( absent or present ) in order to express a judgment on the presence of acute rejection : air trapping , tree - in - bud , consolidations , crazy paving , ground - glass opacity , bronchiectasis , thickening of intralobular or interlobular septa and pleural effusion . statistical analysis descriptive statistics of the population are presented using absolute and relative frequencies , as appropriate . 
multivariate analysis was performed with the logistic regression model . results among the 41 patients with ar , the degree of acute rejection was a1 and a2 except for 7 patients ( 17% ) with a3 rejection . 
three years after lung transplantation she has a recurrence of acute rejection characterized by peribronchial wall thickening ( yellow arrows ) and tree - in - bud opacities ( red circle )  . 
according to fishers exact test , none of the ct findings included in the study , alone or in combination , showed any significant statistical association with the diagnosis of acute rejection , as showed by p values > 0.05.figures1 , 2 , 3 , 4 , 5 show some cases with the above mentioned ct findings . 
it may occur early ( days or weeks ) or late ( months or years ) after transplantation and require treatment adjustments in case of recurrence [ 6 ]  . 
 recurrent episodes of acute rejection may increase the chance of developing chronic rejection , which is the main cause of late morbidity and mortality in lung transplant recipients [ 7 ]  . 
tbb is the gold standard for the diagnosis 1 3 1004 la radiologia medica ( 2019 ) 124 : 10001005 with the diagnosis of acute rejection [ 3 ]  . 
their population with acute rejection consisted of 34 patients . our results are consistent with the findings of gotway and colleagues , although there are some methodological differences between the two studies . 
as showed in other studies , the casual tbb procurement is a significant bias for the assessment of acute rejection , especially in its mild - moderate phases [ 8 , 9 ]  . 
a limitation of both studies is the small number of cases of severe rejection ( 7 patients with a3 and no patient with a4 in our study ) included in the analysis , a finding that could possibly increase the sensitivity of single ct results , as demonstrated by other authors [ 11 ]  . in 6 cases , despite evidence of acute rejection on histopathology , the ct was considered normal , without any ct abnormality , probably because of the early stage of ar . 
 similarly , in the study by gotway , 5 patients with negative ct scans showed histopathologic diagnosis of acute rejection . on the other hand , in 29 of 37 patients ( 78% ) with no histopathologic evidence of acute rejection ( control group ) , ct scans identified at least one of the radiological abnormalities suggested as possible indicators of acute rejection . 
however , their population consisted of only 8 patients diagnosed with acute rejection by pathology , of which only 5 had significant ct findings , mostly bilateral . unlike the studies by loubeyre etal . 
 [ 12 ] , in our research we did not take into account repeated findings in the same patient , but only those corresponding to the first biopsy - proven episode of acute rejection . 
two - thirds of the centers reporting to the international society for heart and lung transplantation ( ishl ) registry include a tbb in their surveillance protocol within the first year post - transplantation ; nevertheless , the role of tbb in completely asymptomatic patients is still controversial . 
surveillance with tbb showed the presence of acute rejection in up to 73% of clinically and physically stable patients , highlighting that a timely treatment of these silent episodes could reduce the development of chronic rejection [ 8 ]  . it is a matter of debate whether episodes of grade a1 ar without recurrence or progression to higher grades may be a significant independent predictor of obliterative bronchiolitis . 
none of the ct findings included in the study , alone or in combination , showed any significant statistical association 1 3 la radiologia medica ( 2019 ) 124 : 10001005 1005 of acute rejection may influence the likelihood and course of subsequent episodes . as demonstrated in our study , hrct has limited accuracy for the assessment of acute rejection , and none of the findings considered in our study is significantly associated with acute rejection . 
it has proven value for the characterization of other postoperative complications [ 13 ] such as bronchial stricture and dehiscence , infections , vascular complications , malignancies and chronic rejection . 
 moreover , ct scan may facilitate trans - bronchial biopsies by guiding the sampling of the most affected areas of the lung . in conclusion , hrct is a very useful technique and may support clinicians in the follow - up of lung transplant recipients . 
in adult populations , 9398% of all scalp lesions are benign , with the most common diagnosis being trichilemmal cysts ( 41% of all scalp lesions ) , followed by epidermal cysts , lipoma , nevi , and sebaceous cysts [ 24 ]  . 
in pediatric populations , 9799% of all scalp lesions are benign , with the most common diagnosis being nevus sebaceous ( 60% of all scalp lesions ) , followed by infantile hemangioma , melanocytic nevi , and juvenile xanthogranuloma [ 5 , 6 ]  . 
a clear understanding of the scalps anatomy is essential for the topographic characterization of the lesion as the first step in the differential * hiroki kato hkato@gifu - u.ac.jp 1 department ofradiology , gifu university school ofmedicine , 1 - 1 yanagido , gifu501 - 1194 , japan diagnosis ( table1 ) , as the majority ( 82% ) of scalp lesions in adults originate from the skin layer [ 2 ]  . the rate of correct preoperative diagnosis for scalp lesions ranged from 13 to 27% among neurosurgery , plastic surgery , or general surgery departments [ 2 ]  . 
although radiological investigations of some scalp lesions have been reported [ 810 ] , to the best of our knowledge , there have been no review articles with emphasis on ct and mri features of scalp lesions . 
therefore , the aim of this review article is to describe a variety of cutaneous and subcutaneous lesions of the scalp in terms of the anatomic origin with emphasis on ct and mri features . anatomy the soft - tissue envelope of the cranial vault is named the scalp , extending from the external occipital protuberance and superior nuchal lines to the supraorbital margins and vol . : ( 0123456789 ) 1 3 la radiologia medica ( 2019 ) 124 : 10491061 1050 origin table 1 classification of scalp lesions melanocytic origin appendageal origin follicular differentiation benign malignant keratinocytic origin epidermoid cyst / dermoid cyst / teratoid cyst , keratoasquamous cell carcinoma , basal cell carcicanthoma , seborrheic keratosis melanocytic nevus , blue nevi noma , bowen disease malignant melanoma pilomatricoma , trichilemmal cyst / proliferating pilomatrical carcinoma , malignant prolifertricholemmal tumor ating tricholemmal tumor sebaceous differentiation apocrine and eccrine differentiation sebaceoma / sebaceous adenoma eccrine poroma , hidrocystoma , syringoma sebaceous carcinoma adenocarcinoma , porocarcinoma , apocrine carcinoma hematolymphoid origin langerhans cell histiocytosis , rosaidorfman disease , malignant lymphoma juvenile xanthogranuloma soft tissue origin adipocytic vascular neural origin others lipoma , lipoma variant infantile hemangioma , venous malformation / cavernatypical lipomatous tumor , liposarcoma angiosarcoma ous hemangioma lymphangioma lymphatic smooth and skeletal muscle leiomyoma fibrous , fibrohistiocytic and histiocytic dermatofibroma , keloid / hypertrophic scar , infantile myofibromatosis neurofibroma , schwannoma leiomyosarcoma dermatofibroma protuberance , fibrosarcoma merkel cell carcinoma , malignant peripheral nerve sheath tumor dermoid / teratoid cyst metastasis anchored laterally by each ear . 
the scalp consists of five histologic layers : skin , subcutaneous tissue ( superficial fascia ) , galea aponeurotica ( deep fascia ) , loose areolar connective tissue , and pericranium ( fig.1 ) [ 1 , 3 , 7 ]  . 
this single unit can move along the loose areolar tissue over the pericranium adherent to the calvaria . the skin is the outermost region of the scalp , which is composed of thin external epidermis and thick inner dermis , which both decrease in thickness with age . 
the epidermis is composed of stratified squamous epithelium , with a thickness of 0.040.4mthe dermis is usually thinnest over the forehead and thickest over the occipital region [ 1 , 3 ] with a thickness of 0.52.5mthe skin also contains closely arranged adnexa ( consisting of sebaceous glands , hair follicles , and eccrine and apocrine glands ) surrounded by dense networks of blood vessels and lymphatics [ 3 ]  . the subcutaneous tissue , also known as the subcutis or superficial fascia , is a fibrofatty layer that connects the skin to the underlying galea aponeurotica of the occipitofrontalis muscle and provides a passageway for blood vessels , nerves , and lymphatic tissue . 
at the vertex , the subcutaneous tissue has a thickness of 47mm . the galea aponeurotica , also called the deep fascia , is a strong , thin , tendinous sheath that descends laterally into the temporal fossa as the superficial temporal fascia . 
the galea aponeurotica has a thickness of 12mm . the loose connective tissue , which is also referred to as the areolar tissue , comprises the subgaleal layer between the galea aponeurotica and the pericraniuthis layer contains the most widely distributed connective tissues and is composed of a central dense collagenous layer surrounded by vascularized areolar tissue . 
this layer has several functions , including support and binding , fluid retention , defense , and nutrient storage . the periosteum covering the outer surface of the skull is known as the pericraniuthe pericranium is a fibrous membrane that is tightly attached to the outer table of the skull . 
laterally , it terminates at the origin of the temporalis muscle along the superior temporal line . imaging the skin layer presents on ct as a soft - tissue density and on mri as isointense to muscle . 
upon encountering cutaneous lesions arising from the skin layer , the depth of the lesion 1 3 la radiologia medica ( 2019 ) 124 : 10491061 1051 and the invasion of nearby subcutaneous tissue should be assessed by radiologists on the basis of ct and mri . the subcutaneous tissue layer appears as a hypodense area on ct and as an area with hyperintensity on t1and t2 - weighted mr images because of its fat content . 
on fat - suppressed contrast - enhanced t1 - weighted images , the enhancement of subcutaneous tissue can be observed because of its rich vascular network . the galea aponeurotica layer is shown on mri as an area that is isointense to muscles . 
in contrast , the loose connective tissue and the pericranium cannot normally be visualized by either ct or mri and can only be detected pathologically [ 7 ]  . the depth of tumor invasion is an important prognostic factor for local recurrence and distant metastasis after surgery . 
if cutaneous lesions invade the subcutaneous tissue , the subcutaneous tissue appears as an area of hyperintensity on fat - suppressed t2 - weighted images and as an area of hypointensity on t1 - weighted images . 
the tumor extends down to the cranial bone ; full - depth resection is required , including the dura if necessary . benign lesions trichilemmal cysts , proliferating trichilemmal tumors trichilemmal cysts , otherwise known as pilar cysts or isthmus - catagen cysts , are the most common form of intradermal or subcutaneous cysts of the scalp , accounting for 510% of the population . 
histologically , these cysts are lined by squamous epithelium , containing a granular layer ( trichilemmal keratinization ) that is filled with homogenous keratcalcifications are common features in trichilemmal cysts . 
the presence of calcification causes inhomogeneity on t2 - weighted images ; the lack of intralesional enhancement can be observed on contrast - enhanced images ( fig.2 ) [ 12 ]  . 
as the underlying skull vault and skin surface are not affected by trichilemmal cysts , enhanced soft - tissue components with irregular margins , invasion into surrounding structures , and erosion of adjacent bony tissue are indicative signs of malignant transformation . sebaceoma sebaceous neoplasms are uncommon cutaneous tumors arising in the sebaceous glands , which are responsible for secreting lipids and other substances into the hair follicle infundibulum [ 14 ]  . 
the scalp consists of five histologic layers : skin , subcutaneous tissue ( superficial fascia ) , galea aponeurotica ( deep fascia ) , loose connective tissue , and periosteum ( pericranium ) 1 3 1052 la radiologia medica ( 2019 ) 124 : 10491061 fig . 
the cystic wall is lined by stratified squamous epithelium , with sebaceous lobules present in , or adjacent to , the cystic wall . sebaceomas are located in the dermis and frequently expand downward to the subcutaneous tissue layer [ 16 ]  . 
in contrast , if the cystic cavities are filled with large amounts of keratin and hair , they appear as areas with hypoto isointensity on t1 - weighted images and hyperintensity on t2 - weighed images . epidermoid / dermoid / teratoid cysts epidermoid , dermoid , and teratoid cysts are common , related , cutaneous cysts lined by squamous epithelium , classified according to whether they are lined by simple squamous epithelium ( epidermoid cysts ) , the presence of skin adnexa in the cystic wall ( dermoid cysts ) , or the presence of other tissues , such as muscle , cartilage , or bone ( teratoid cysts )  . 
these types 1 3 la radiologia medica ( 2019 ) 124 : 10491061 1053 of cysts occur in the head and neck region , generally occurring in young to middle - aged adults with a male - to - female ratio of 3 : 1 . 
dermoid and teratoid cysts are usually found as congenital masses in pediatric patients [ 17 ]  . on ct and mri , these types of cysts appear as welldemarcated , oval - shaped , subcutaneous cystic masses adjacent to the overlying skepidermoid cysts appear as areas with isoto hyperintensity on t1 - weighted images and hyperintensity with variable hypointense foci , including internal linear dark debris , on t2 - weighted images ( fig.5 ) [ 10 ]  . 
although peripheral , thin - rim enhancement without central enhancement is usually observed , the rupture of epidermoid cysts results in the enhancement of internal septa and a thickened , irregular rim , with fuzzy enhancement in surrounding subcutaneous tissues [ 18 ]  . 
dermoid cysts typically appear as areas with hyperintensity due to their fat content on t1 - weighted images and variable intensities on t2 - weighted images [ 19 ]  . 
teratoid cysts show as the presence of calcification on ct , reflecting the formation of cartilage or bone . keratoacanthoma keratoacanthoma is a self - limiting , epidermal tumor that is cryptic because of its nosological position at the border between benignity and malignancy [ 20 ]  . 
 lesions show an intermediate intensity on t1 - weighted images and a mixture of intermediate and high signal intensities on t2 - weighted images with peripheral thin - rim enhancement [ 22 ]  . pilomatricoma pilomatricoma , which is also known as calcifying epithelioma , is a rare , benign tumor of the dermis or subcutaneous tissue , which originates from pluripotent cells that normally differentiate into hair matrix cells [ 8 ]  . 
a t1 - weighted image shows a exophytic , cup - shaped , well - demarcated cutaneous lesion ( arrow ) with central recesses of keratotic horn ( arrow head )  . 
approximately , half of the tumors occur in the head and neck region , followed by the upper extremities , trunk , and lower extremities ; the sites that are most commonly affected by pilomatricoma are the cheek , neck , eyebrows , and scalp [ 23 ]  . 
reticular and ring - like hyperintensities on fat - suppressed t2 - weighted and fat - suppressed contrast - enhanced t1 - weighted images are frequently observed , corresponding to intratumoral stroma and tumor capsule with various degrees of inflammatory cell infiltration and vascular proliferation , respectively ( fig.7 ) [ 8 ]  . 
 melanocytic nevi melanocytic nevi , which is also called moles , are common skin lesions that are characterized by benign clonal proliferation of cells expressing a melanocytic phenotype , with heterogeneous clinical and molecular characteristics [ 24 ]  . 
 melanocytic nevi are formed by nests of melanocytes in epidermal junctions ( junctional nevi ) , dermis ( intradermal nevi ) , or both compartments ( compound nevi )  . 
as the risk of malignant transformation of melanocytic nevi strongly depends on size , giant congenital melanocytic nevi are associated with an increased risk of malignant melanoma . melanocytic nevi present as flat patch lesions on the sk hyperdensity relative to soft tissue has been observed on ct owing to the melanin content of melanocytic nevi . 
although melanocytic nevi rarely invade the underlying fascia , muscle , or deeper structures , giant congenital melanocytic nevi may invade the subcutaneous tissue or beyond with ill - defined margins . neurofibroma neurofibroma is a benign nerve sheath tumor with a neuroectodermal origin , accounting for 5% of all benign soft - tissue tumors [ 25 ]  . 
at least 10% of diffuse neurofibromas are associated with neurofibromatosis type 1 . diffuse neurofibroma involves both the skin and the subcutaneous tissue , also frequently extending to the fascia over muscle , commonly with plaque - like or infiltrative growth patterns . 
despite being mildly or markedly hyperintense to muscle in t2 - weighted images and isointense or mildly hyperintense to muscle in t1 - weighted images , mr signal intensities of neurofibroma are typically nonspecific [ 25 ]  . vascular anomalies / infantile hemangioma vascular anomalies can be divided into two main categories : vascular tumors and malformations . 
infantile hemangiomas are the most common childhood tumors , with an incidence of 1223% among low - birth - weight , preterm infants and with a female - to - male ratio of 3 : 1 [ 26 ]  . 
c fat - suppressed contrast - enhanced t1 - weighted image shows reticular and ring - like enhancement ( arrow ) with perilesional fat stranding ( arrow heads ) 1 3 1056 la radiologia medica ( 2019 ) 124 : 10491061 fig . 
t2 - weighted image shows a heterogeneously hyperintense lesion with plaque - like growth ( arrow ) lipoma lipoma is the most common type of benign mesenchymal tumor , which is composed of mature fat cells . 
surgical approaches and planning differ between superficial and deep lipomas ; therefore , accurate assessment of localization is necessary for radiologists . on ct , lipomas appear as round or ovoid , well - delineated homogeneous masses associated with fat attenuation . 
the findings of a large tumor size , thick septa with moderate or marked enhancement , and nodular or patchy nonadipose components are indicative of well - differentiated liposarcomas [ 31 ]  . malignant tumors basal cell carcinoma ( bcc ) andsquamous cell carcinoma ( scc ) bcc and scc are the most common histologic subtypes of malignant cutaneous tumors of the scalp , accounting for 41% and 17% of all cases , respectively [ 32 ]  . 
b t1 - weighted image shows heterogeneous isoto hyperintensity ( arrow ) frequently occur in the head and neck region ( 60% of the cases ) , typically presenting two to 6weeks after birth as small lesions and rapidly growing in the first 1218months ( proliferating phase )  . 
subsequently , half of all the cases are completely resolved by 5years of age ( involuting phase )  . infantile hemangiomas typically appear as well - defined , lobulated , noninfiltrating masses , with hypointensity on t1 - weighted images and hyperintensity on t2 - weighted images ( fig.10 ) [ 27 , 28 ]  . 
as bccs and sccs both show nonspecific hypointensity on t1 - weighted images and isoto hyperintensity on t2 - weighted images ( fig.11 ) [ 33 ] , bccs and sccs are indistinguishable from one another with mri . 
sccs are more likely to be invasive than bccs ; however , peritumoral or deeper soft - tissue enhancement may be seen more often in cases of sccs than in those of bccs on contrast - enhanced t1 - weighted images . malignant melanoma malignant melanoma is a malignancy of pigment - producing cells ( melanocytes ) , which are located predominantly in the skhead and neck melanomas comprise 18% of all melanomas , and 7% of all melanomas arise on the scalp [ 34 ]  . 
 melanomas of the scalp frequently occur in the sixth decade , with a male predilection ( male - to - female ratio = 4 : 1 ) , which is significantly greater than for other sites [ 34 ]  . 
the tumor thickness of melanomas is greater in the scalp ( 2.6mm ) than in other sites ( 1.71.9mm ) , correlating positively with the risk of lymph node and distant metastasis . 
the location of melanoma on the scalp is , therefore , an independent adverse prognostic factor [ 34 ]  . stable - free radicals within melanin pigments are paramagnetic and induce shortening of t1 and t2 relaxation times ; therefore , the expected signal pattern for melanotic melanoma is hyperintensity on t1 - weighted images and hypointensity on t2 - weighted images ( fig.12 ) [ 33 ]  . 
the signal intensity varies depending on several factors , and intralesional hemorrhage also has a significant effect on mri . merkel cell carcinoma ( mcc ) mcc is a rare , aggressive , primary cutaneous neuroendocrine carcinoma . 
as sun exposure is considered a significant risk factor for mcc ( 81% ) , cases frequently occur in the head and neck region , accounting for 2948% of all primary mccs [ 35 ]  . 
b fat - suppressed contrast - enhanced t1 - weighted image shows homogeneously intense enhancement ( arrow ) of bccs and 38% of sccs are located on the scalp [ 3 ]  . 
 bccs and sccs of the scalp usually occur in patients in the sixth to seventh decades , with a female - to - male ratio of 1.1 : 1 and 1.3 : 1 for bccs and sccs , respectively [ 32 ]  . 
bccs and sccs of the scalp have a greater tendency to ulcerate , compared with other locations on the skas sccs of the scalp are often diagnosed at an advanced stage , this location may be considered an independent risk factor for poor prognosis [ 3 ]  . 
for sccs and bccs of the scalp , the maximum diameter greater than 10mm , the poorly defined borders , the recurrent tumor , and the history of immunosuppression and prior radiation therapy are clinical manifestations indicative of high - risk tumors . 1 3 1058 la radiologia medica ( 2019 ) 124 : 10491061 fig . 
 t2 - weighted image shows an ill - demarcated , heterogeneously isointense , cutaneous lesion ( arrow ) lymph node enlargement is often observed , alongside fine , compressed , retained fatty tissue [ 9 ]  . angiosarcoma angiosarcoma is a malignant tumor of vascular endothelial cells , often associated with local recurrence and metastasis , resulting in a poor prognosis . 
the characteristic findings for angiosarcoma include the presence of high - flow serpentine vessels , whereas low - flow vessels may show hyperintensity on t2 - weighted images [ 37 ]  . 
on contrast - enhanced images , solid components are intensely enhanced , whereas tumor necrosis is depicted as nonenhancing areas [ 37 ]  . metastasis cutaneous metastasis from primary visceral malignancy is clinically relatively uncommon , with a reported incidence fig . 
b t1 - weighted image shows heterogeneous hyperintensity ( arrow ) ranging from 0.22 to 10% , whereas 40% of the cases with cutaneous metastases also present with internal metastases [ 38 ]  . 
on ct and mri , cutaneous and subcutaneous metastases commonly manifest as multiple , variable - sized , poorly defined nodules , or as infiltrative softtissue masses with homogeneous or heterogeneous contrast enhancement . 1 3 la radiologia medica ( 2019 ) 124 : 10491061 1059 common subtype , which occurs on the scalp [ 3 ]  . 
if lymphomas present with subcutaneous bulky masses of the scalp with extensive cranial vault involvement , both bone lymphoma with scalp invasion and cutaneous lymphoma with bone invasion should be considered as differential diagnoses . 
 although ct and mri play important roles in tissue characterization and evaluating the depth of invasion of scalp lesions , it is challenging for radiologists to differentiate between scalp tumors because the imaging findings are typically similar and nonspecific . 
unenhanced ct image shows a homogeneous soft - tissue lesion ( arrow ) conclusions malignant lymphoma malignant lymphoma accounts for 0.64.8% of all malignant scalp tumors [ 2 , 32 ]  . 
the most common b - cell lymphomas of the scalp are primary cutaneous follicle center lymphoma and primary cutaneous marginal zone lymphoma , both of which have excellent prognoses [ 3 ]  . 
t1 - weighted image shows a hypointense subcutaneous nodule ( arrow ) 1 3 1060 la radiologia medica ( 2019 ) 124 : 10491061 utility of fine - needle aspiration in the diagnosis of primary scalp lesions . 
yang cc , chen ya , tsai yl , shih ih , chen w ( 2014 ) neoplastic skin lesions of the scalp in children : a retrospective study of 265 cases in taiwan . 
kato h , kanematsu m , watanabe h , nagano a , shu e , seishima m , miyazaki t ( 2016 ) mr imaging findings of pilomatricomas : a radiological - pathological correlation . 
anderson se , beer kt , banic a , steinbach ls , martin m , friedrich ee , stauffer e , vock p , greiner rh ( 2005 ) mri of merkel cell carcinoma : histologic correlation and review of the literature . 
kim hk , kim sm , lee sh , racadio jm , shin mj ( 2011 ) subcutaneous epidermal inclusion cysts : ultrasound ( us ) and mr imaging findings . 
prior a , anania p , pacetti m , secci f , ravegnani m , pavanello m , piatelli g , cama a , consales a ( 2018 ) dermoid and epidermoid cysts of scalp : case series of 234 consecutive patients . 
hong sh , chung hw , choi jy , koh yh , choi ja , kang hs ( 2006 ) mri findings of subcutaneous epidermal cysts : emphasis on the presence of rupture . 
sorenson ep , powel je , rozzelle cj , tubbs rs , loukas m ( 2013 ) scalp dermoids : a review of their anatomy , diagnosis , and treatment . 
t2 - weighted image shows a homogeneously isointense subcutaneous lesions ( arrows ) compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . 
 for this type of study , formal consent is not required . human and animal rights this article does not contain any studies with human participants performed by any of the authors . informed consent for this type of study , formal consent is not required . 
angulated , sharply defined borders occurred in 82.7% of itts in our series and accompanied both fusiform - shaped and bizarre - shaped itts ( fig.1 ) [ 2 ]  . 
this distinction is important since nodules in children , even when < 1cm , should undergo fine - needle aspiration ( fna ) when ultrasonography indicates malignancy [ 3 ]  . 
consequently , geometric / geographic instead of irregular better describes itts various shapes [ 2 ]  . itts reportedly exhibit a typical hypoechoic pattern with echogenic linear and punctate foci [ 15 ]  . 
in our study , itts internal echogenic dots either evenly or unevenly distributed showed a variable thin or thick configuration and were routinely compared with and found identical to those of the same patients thymic speckles ( fig.2 ) [ 2 ]  . 
however , itts tend to occur at the posterior aspect of the middle and lower thirds of the thyroid lobes , reflecting the embryological relationship between thymus and thyroid gland [ 2 , 4 , 5 ]  . 
thymic tongues should be differentiated from extrathyroid extension of an aggressive lesion . sparse or no vascularity in itts has been repeatedly reported [ 2 , 4 , 5 ]  . 
consequently , a strong aspect of this original article is the radiologic - pathologic correlation which provides solid proof for the diagnosis . conclusively , small - sized fusiform thyroid lesions could suggest the diagnosis of itt provided that attention is paid to additional details . 
further studies that will compare the occurrence of features like identical pattern of speckles with mediastinal thymus , low - lying and posterior location of lesion , well demarcated , angulated borders and sparse or no internal vascularity with / without extrathyroidal extension in both itts and non - itt thyroidal lesions , are necessary vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 10641065 1065 fig . 
the lesion is small , sharply demarcated , is located at the posterior aspect of the thyroid lobe , exhibits four angulations ( arrows ) and contains thin speckles which are identical to the speckles of the neighbouring cervical thymus ( * ) fig . 
we underline how important it is to determine whether a vascular anomaly has a regional vascular origin , or if there are other entities , ranging from benign to malignant lesions , which have flow - signal or blood degradation products . 
even though clinical examination and patients history are the first and indispensable steps in the initial diagnosis , the role of imaging is crucial , not only to determine whether a mass represents a true tumour / pseudo - tumour , but also to achieve a more correct diagnosis and determine the extension of the tumour / pseudo - tumour and its relation with the nearby anatomic structures . keywords soft tissue tumour pseudo - tumour vascular malformation vascular tumour children paediatric introduction the purpose of this article is to provide an up - to - date overview on imaging of paediatric vascular soft tissue masses , including neoplastic and non - neoplastic lesions , also considering non - vascular differential diagnosis . advanced clinical evaluation should be the first step in framing the diagnosis ( age , site , texture , colour , etc . ) , but soft tissue tumours and pseudo - tumours are more and * paolo tom paolo.toma@opbg.net 1 department ofimaging , bambino ges childrens hospital irccs , piazza santonofrio , 4 , 00165rome , italy 2 radiology department , santobono - pausilipon children 3 department ofradiology , g . 
gaslini institute , irccs , hospital , naples , italy genova , italy interventional radiology unit , department ofimaging , bambino ges childrens hospital irccs , rome , italy 5 department ofbiopathology andmedical biotechnologies ( di.bi.med ) , policlinico university ofpalermo , palermo , italy 6 department ofadvanced biomedical sciences , university federico ii , naples , italy more frequently referred to primary care sonography ( us ) as a contemporary first - line approach . 
 the first approach to vascular soft tissue masses in children is often challenging , due to their heterogeneous origin , which leads to a different treatment and prognosis [ 14 ]  . malignant lesions are very rare in paediatric age group : benign soft tissue tumours outstrip their malignant equivalents by about 100 to 1 , but most cutaneous and subcutaneous masses are very small and are often excised without imaging studies [ 5 ]  . 
however , 4% of solid malignancies in children are in the soft tissues , and this is why clinical and radiological approach is crucial for patient management [ 68 ]  . the diagnostic approach to paediatric soft tissue masses should follow an organic process , which consists in clinical evaluation , imaging examination , and eventual biopsy . age as systematically reported by brisse et al . 
for example , vascular tumours , infantile myofibromatosis , infantile fibrosarcoma , and subcutaneous metastases from neuroblastoma are more commonly reported in neonates and in early infancy , whilst others , such as rms , lipoblastomas or infantile aggressive fibromatosis , arise before the 5th year of age . 
indeed , lesions bigger than 5cm associated with pain and deeper involvement are more commonly associated with malignancy than to a benign lesion [ 711 ]  . imaging examination after clinical examination , the second crucial step is imaging evaluation of the tumour , not only to determine whether a mass represents a true tumour / pseudo - tumour , but also to achieve a more correct diagnosis and determine its extension and relation with the nearby anatomic structures [ 14 ]  . biopsy or fine - needle aspiration when both imaging and clinical examination have not achieved definitive proofs of 1 3 la radiologia medica ( 2019 ) 124 : 935945 table 1 sonographic pattern of soft tissue masses . 
in all the other cases , when the benign origin of the mass has been determined , patient management will only include simple observation , medical treatment or surgery [ 7 ]  . the aim of this review is to propose a rational diagnostic approach for soft tissue masses in children , which may mimic vascular anomalies . imaging evaluation the first choice examination is ultrasonography . ultrasound ( us ) with grey scale and colour doppler , with its wide availability , low costs , lack of ionizing radiation and no need for children sedation , are considered as the technique of choice for soft tissue lesion assessment , especially when dealing with small and superficial lesions [ 14 ]  . during examination , the practitioner should assess the following elements : anatomic location , depth of the lesion ( dermis , subcutaneous tissues , fascia , muscle , bone ) , its shape and margins , its echo texture and internal features , and , last but not least , its vascularity . moreover , when performing soft tissue ultrasound , a crucial and diriment tip is always to look at the symmetry of the lesions . 
 its appearance is hypoechoic with vascular density patternboth arterial and venous flow , in the proliferative stage , becoming smaller , more echogenic and less vascular , infantile haemangioma venous malformations lipoblastoma plexiform schwannoma / neurofibroma fibrous hamartoma of infancy clear cell sarcoma rhabdomyosarcoma spiradenoma cellulitis venous malformations plexiform schwannoma / neurofibroma infantile myofibromatosis infantile haemangioma congenital haemangiomas kaposiform haemangioendothetufted angioma arteriovenous malformation lioma ( avm ) infantile myofibromatosis pilomatrixoma lymph nodes ( metastases ) nasal glioma rhabdomyosarcoma phleboliths congenital haemangiomas kaposiform haemangioendothelioma pilomatrixoma myositis ossificans infantile myofibromatosis bone tumours sarcomas lympho - adenomegalies lymphatic malformations benign cysts popliteal , dermoid dacriocistocele necrotic areas of malignant tumours pilomatrixoma target pattern hyper - vascular pattern calcifications cystic lesions hyperechoic pattern infantile haemangioma lipoma / lipoblastoma fibrous hamartoma of infancy cellulitis subcutaneous fat necrosis bone fracture ( muscles ) echoic inhomogeneous pattern venous malformations cellulitis nodular fasciitis subcutaneous granuloma annular calme ( childhood asymmetrical labium majus enlargement ) axillary breast tissue in bold the proper vascular anomalies , in standard mimickers of vascular anomalies 1 3 938 fig . 
 hypoechoic lobular pattern of the intra - parotid mass ( white arrows ) , showing , on doppler us , high vessel density , high flow velocity , low resistance index , broadening of the spectrum ( a , b ) la radiologia medica ( 2019 ) 124 : 935945 fig . 
2 infantile haemangioma in the forearm of a 40 - day - old female : colour doppler examination shows afferent vessels due to fibro - fatty proliferation in involution stage ( fig.1 , 2 ) [ 1 , 4 , 14 , 15 ]  . 
differently from infantile haemangioma , vm presents with phleboliths , which , although not common , are very helpful in differential diagnosis ( dd ) , because they are rare in other paediatric soft tissue masses ( fig.3 ) [ 1 , 4 , 15 ]  . 
moreover , under the clinical point of view , they may expand with valsalva manoeuvre , after compression , and with gravity ( in the dependent limb ) [ 4 , 15 ]  . mr shows , in both cases , hypo / iso - density on t1 ( signal increases during involution phase due to fat deposition of theih ) compared with the surrounding muscles , hyper - density on t2 , and homogeneous enhancement after contrast media administrationwhich instead appears less prominent in case of involutions [ 1 , 4 ]  . vm differs from ih for the absence of flow in gradientecho ( ge ) imaging and shows spare signal void foci in all sequences , due to phleboliths and foci without enhancement in case of thrombi or a target sign ( described in the dedicated section ) [ 1 , 4 ]  . 
c t2w fat sat sequence shows multiple venous lakes with coagula inside ( target pattern ) mimickers of vascular anomalies : lipoblastoma may enter in dd due to its myxocollagenous stroma . 
however , the reported flow voids are normally less prominent in schwannomas and neurofibromas than in haemangioma due to the lower vascularity , as well as the entity of contrast enhancement ( and the entity of colour signal on colour doppler us ) , which is greater in haemangioma than in neurogenic tumours [ 2 , 17 , 18 ]  . target signs ( included in the dedicated section ) can be presented as well , both on us , due to the visualization of 1 3 la radiologia medica ( 2019 ) 124 : 935945 the entering and exiting nerve , and on mr , as a central zone of tightly packed fibbers , consisting of collagen or a highly cellular component , and a peripheral zone of non - fibrillary stromal or myxoid material , exhibiting various enhancements ( fig.4 ) [ 2 , 14 , 15 ]  . 
they differ from ce to ih since they show rapid infiltrative growth pattern , endothelium spindling , micro - thrombi , hemosiderin deposits ( depicted on mr ) , and often lymphatic aberrations [ 1 , 4 , 27 , 28 ]  . 
7 rapidly involuting congenital haemangioma ( rich ) of the neck in a newborn , colour doppler shows hyper - vascularization , but venous flow is arterialized ( a )  . 
blood supply and drainage anomalies can lead to asymmetric overgrowth or the involved body part , trophic changes may be present due to ischemia from arterial steal , haemorrhage , and highoutput heart failure , when capillaries are massive in size [ 1 , 4 , 2932 ]  . precautions should be taken when scanning these lesions , because they can be extremely fragile and a large amount of blood can be quickly lost . 
 suggest preparing emergency plans , including a haemostasis kit [ 4 ]  . on sonography , they appear as hypoechoic conglomerates of tortuous vessels , with or without a discrete soft tissue mass , associated with hyper - echogenic fibro - fatty proliferation in the surrounding tissues [ 1 , 4 ]  . on colour doppler interrogation , avm show high flows and high vascular density with a multidirectional flow [ 4 , 27 , 28 ]  . 
although it can show vascular patterns and calcifications , these features are never present at the same time , since the first tends to appear in the early stages due to inflammation , whilst the second only appears in the late phase . 
in dd , mr plays a pivotal role , since it demonstrates an intramuscular soft tissue mass deep into the fascia , with iso - hyper - signal intensity on t1 - weighted imagescompared to the surrounding musclesand a very high signal intensity on t2 - weighted images [ 2 , 38 ]  . also synovial sarcomas show , in 30% of cases , calcifications with a non - specific us appearance ( that can vary widely on the basis of the malignant potential of the tumour : from hypoechoic mass with lobulated margins to inhomogeneous hypo - echogenity with irregular margins , with potential vascularization in the areas of viable malignancy ) ( fig.11 ) [ 2 , 39 ]  . 
it appears as a firm , red to bluish skin - covered mass , which , nevertheless , does not exhibit any pulsations or increasing in size with the valsalva manoeuvres or compression of the ipsilateral jugular vein . us and doppler flow differentiate these entities , depicting the different cystic or solid nature of the masses and the characteristic low arterial flow velocity during the enddiastolic phase , unlike haemangioma , which shows a high arterial doppler flow velocity [ 12 , 35 , 36 ]  . 
nevertheless , mr is the imaging modality of choice , and can display nasal gliomas being often isointense compared to normal brain at mr imaging [ 12 ]  . calcifications proper vascular anomalies and some of the mimickers of vascular anomalies have been discussed in the previous paragraphs . 1 3 la radiologia medica ( 2019 ) 124 : 935945 fig . 
lymphatic vesicles can be detected on the skin or the mucosae , and they represent the most useful sign for clinical diagnosis [ 1 , 4 , 2932 , 40 , 41 ]  . imaging on us macro - cystic lymphatic malformations do not collapse after compression ( unlike venous malformations ) and appear as soft tissues composed of multiple cystic spaces ( anechoic or having varying degrees of echogenicity , or presenting with fluidfluid levels , especially when haemorrhage occurs ) and thin septa . 
the multiple cystic spaces show no inner flow on doppler investigation , however , it can be detected in the septa ( fig.12 ) [ 1 , 4 , 14 ]  . in case of micro cystic lymphatic malformations , us appearance is different as they show an increased echogenicity , which is attributed to the multiple interfaces caused by tiny cysts that are beyond the resolution of the us equipment [ 1 , 4 , 14 , 40 , 41 ]  . on mri they appear as multi septate cystic masses that can infiltrate surrounding tissues , which are typically hypointense on t1 - weighted images and hyperintense on t2 - weighted images , and do not show enhancementwhich is an important feature in differential diagnosis from venous malformations . 
nevertheless , inner septa may show various degrees of post - contrast media administration enhancement , since they are vascularized [ 1 , 4 , 14 , 40 , 41 ]  . cysts with protein content or prior haemorrhage can show a heterogeneous intensity [ 1 , 4 , 14 ]  . mimickers of vascular anomalies : dacriocystocele may enter in dd with vascular anomalies and nasal gliomas since , under the clinical point of view , it appears as a grey - blue cystic swelling below the medial canthus . 
laffan ee , ngan by , navarro om ( 2009 ) pediatric soft - tissue tumors and pseudotumors : mr imaging features with pathologic correlation : part 2 tumors of fibroblastic / myofibroblastic , socalled fibrohistiocytic , muscular , lymphomatous , neurogenic , hair matrix , and uncertain origradiographics 29 ( 4 ) : e36 3 . 
morn fe , morriss mc , jones jj , hunter jv ( 2004 ) lumps and bumps on the head in children : use of ct and mr imaging in solving the clinical diagnostic dilemma . 
arioni c , bellini c , oddone m , risso fm , scopesi f , nozza p , serra g , tom p ( 2006 ) congenital fibrous hamartoma of the knee . 
de beuckeleer lh , de schepper am , vandevenne je , bloem jl , davies am , oudkerk m , hauben e , van marck e , somville j , vanel d , steinbach ls ( 2000 ) mr imaging of clear cell sarcoma ( malignant melanoma of the soft parts ) : a multicenter correlative mri - pathology study of 21 cases and literature review . 
jin w , kim gy , lew bl , yang dm , kim hc , ryu jk , park js , ryu kn ( 2008 ) sonographic findings of an eccrine spiradenoma : case report and literature review . 
wortsman x , wortsman j , arellano j , oroz j , giugliano c , benavides mi , bordon c ( 2010 ) pilomatrixomas presenting as vascular tumors on color doppler ultrasound . 
abate m , salini v , rimondi e , errani c , alberghini m , mercuri m , pelotti p ( 2011 ) post traumatic myositis ossificans : sonographic findings . 
pang lm , roebuck dj , griffith jf , kumta sm , metreweli c ( 2001 ) alveolar soft part sarcoma : a rare soft - tissue malignancy with distinctive clinical and radiological features . 
murphey md , gibson ms , jennings bt , crespo - rodrguez am , fanburg - smith j , gajewski da ( 2006 ) imaging of synovial sarcoma with radiologic - pathologic correlation . 
khuu a , yablon cm , jacobson ja , inyang a , lucas dr , biermann js ( 2014 ) nodular fasciitis : characteristic imaging features on sonography and magnetic resonance imaging . 
pham h , fessell dp , femino je , sharp s , jacobson ja , hayes cw ( 2003 ) sonography and mr imaging of selected benign masses in the ankle and foot . 
gokli a , neuman j , lukse r , koshy j , kong f , laor t ( 2016 ) childhood asymmetrical labium majus enlargement sonographic and mr imaging appearances . 
a pivotal prospective randomized controlled trial has shown that hydrogel spacer has a great role in reducing the rectal wall doses and improving rectal toxicities during definitive prostate radiotherapy [ 1 ]  . 
 [ 3 ] have reported gastrointestinal ( gi ) and genitourinary ( gu ) toxicities in salvage and / or adjuvant post - prostatectomy radiotherapy with hydrogel spacer implant , and i would like to thank the authors for sharing wonderful work . 
their study did not have control group to compare observed results , and even they did not perform a comparative planning study between the preand post - implantation of hydrogel to elucidate the effect of a hydrogel spacer implant on rectal wall doses . 
 initially , it is important to know the impact of a hydrogel * hamed ghaffari hamedghaffari@yahoo.com 1 department ofmedical physics , school ofmedicine , iran university ofmedical sciences , tehran , iran spacer on rectal dosimetry during post - prostatectomy radiotherapy because hydrogel injection has an expensive and invasive procedure . 
 [ 2 ] have previously expressed the prerequisites for hydrogel spacer application in the post - prostatectomy setting , including single and macroscopically visible tumor recurrence located in the proximity of the rectal wall and a very high level of safety that the lesion is the only site of recurrence . 
it should be noted that the entire prostatic fossa ( from the pubic bone to the rectal wall in the anteriorposterior axis , as well as potential microscopic tumor spread ) is defined as the ctv in the standard treatment [ 4 ]  . 
a strategy that can be applied to reduce the potential risk of the remaining tumor cells at the anterior rectal wall is to include the prostatic fossa and hydrogel spacer within the ctv ; however , the improvement in rectal dosimetry will be reduced . 
 [ 3 ] have confidently expressed that the application of a hydrogel spacer does not compromise biochemical control rates and maintains and / or reduces grades of gu and gi toxicities in comparison with previously published reports without the use of a hydrogel spacer injection . 
as stated above , reliability of these results is difficult . in summary , i think that a two - arm study with large sample size and well - defined inclusion and exclusion criteria is required to elucidate the role of a hydrogel spacer in reducing rectal wall doses and rectal toxicities during post - prostatectomy radiotherapy . 
as stated above , reducing gi and gu toxicities in salvage and / or adjuvant post - prostatectomy radiotherapy with hydrogel spacer implant in a single - arm study is not reliable . 
meanwhile , based on my experience on the application of a rectal retractor in definitive prostate radiotherapy [ 5 , 6 ] , the rectal retractor can be used in the postprostatectomy radiotherapy setting with promising results as an alternative method to a hydrogel spacer . 
where are we now ? marwingutierrez1 , 2 antoniogarca4 chiarabertolazzi1 marikatardella3 luisrodriguez1 jaimemendoza1 deniseclavijocornejo1 received : 4 april 2019 / accepted : 18 june 2019 / published online : 2 july 2019 italian society of medical radiology 2019 abstract lung ultrasound ( lus ) achieved an intriguing role in the management of pulmonary involvement in patients affected by connective tissues diseases ( ctds )  . 
few studies have been performed to support its usefulness in the evaluation of the presence and the severity of interstitial lung disease ( ild ) , relating it to the information obtained with chest high - resolution computed tomography ( hrct )  . 
nevertheless , ild is the second cause of death in systemic sclerosis ( ssc ) and rheumatoid arthritis ( ra ) [ 3 ]  . in daily clinical practice , conventional chest radiography is the first imaging tool used to evaluate the presence of ild , but it is of limited use due to its low sensitivity , particularly in early stage of disease . 
chest high - resolution computed tomography ( hrct ) is considered the gold standard in the diagnosis of ild and in the ability to identify lung pattern of different interstitial pneumonia [ 4 ]  . in the last 20years , many authors demonstrated the utility of ultrasound ( us ) assessment in the evaluation of lung and pleural diseases , offering new tools for the management of acute and chronic pulmonary conditions [ 512 ]  . 
the elementary findings detectable are vol . : ( 0123456789 ) 1 3 990 la radiologia medica ( 2019 ) 124 : 989999 artifacts generated from the thickened interlobular septa at lung surface level . 
in the evaluation of methodological quality of the included studies , we applied the newcastle - ottawa scale ( nos ) , which is a tool developed to assess quality of nonrandomized observational studies [ 20 ]  . methods we reviewed all relevant scientific articles regarding ild in ctds published in the last 18years , according to the preferred reporting items for systematic review and meta - analysis ( prisma ) guidelines [ 19 ]  . 
we performed a systemic research on the electronic databases ( pubmed and embase ) using the following search terms in all possible combinations : ultrasound , ultrasonography , sonography , interstitial lung disease , pulmonary fibrosis , interstitial pulmonary fibrosis , interstitial fibrosis , systemic sclerosis , rheumatoid arthritis , sjgrens syndrome and systemic lupus erythematosus . 
two independent rheumatologists ( mg , mt ) screened all the titles , abstracts and full reports of articles identified , and in case of disagreement , a third investigator ( cb ) was consulted , obtaining a consensus . 
in table1 are summarized demographic data , number of patients enrolled and type of diseases included . the largest number of articles concerns the ssc , since the incidence of ild in this disease is higher than in other ctds . 
the first study was conducted by an italian group in 2009 [ 13 ] ; they subjected 33 consecutive ssc patients to a lus assessment , using a 2.53.5mhz cardiac transducer , in the anterior , middle and posterior chest . 
sixty - two pulmonary intercostal spaces ( lis ) were included with an average lus examination time of 10mlus data were correlated with hrct data , using the score proposed by warrick etal . 
they obtained a linear correlation between the lus and hrct data and a significant correlation between the number of b lines and the diffusion capacity values of carbon monoxide ( dlco )  . the same group of authors [ 22 ] conducted a study on 25 patients to compare the evaluation of lus performed with two different probes : a 2.53.53.5mhz heart probe and a 612mhz linear probe used at 6mhz , and with hrct chest data , using the warrick score . 
lus evaluations were performed on 50 lis in anterior , middle and posterior chest , with a 27mhz convex broadband multifrequency transducer , using an average time of 23min for each patient . 
they found a positive correlation between the lus data and the dlco values and , in addition , a significant linear correlation between the semiquantitative lus score and the warrick score . 
among the ssc patients , eight were classified as limited ssc , five as diffuse ssc and three as ssc without scleroderma dssc ( 24 ) / lssc ( 15 ) lssc ( 33 ) , dssc ( 15 ) , sjgrens syndrome lssc ( 42 ) , dssc ( 21 ) , sine scleroderma ( 4 ) sd standard deviation ; n / a not applicable ; ssc systemic sclerosis ; ctd connective tissue disease ; ra rheumatoid arthritis ; ss sjgren syndrome ; dssc diffuse systemic sclerosis ; lssc limited systemic sclerosis ; sle systemic lupus erythematosus ; ild interstitial lung disease were observed in the lis at the level of the lower posterior regions of the chest , which were proposed by the authors as the first lis to be evaluated in the initial phase of the ild . the same group of researchers [ 17 ] proposed a simplified evaluation of the lus , performing a post hoc analysis . 
they included 36 ctd patients ( 28 ssc , 2 sjgrens syndrome , one undifferentiated ctd , two antisynthetase syndrome , two dermatomyositis , one mixed ctd ) , performing the evaluation of lus with a 27mhz broadband multifrequency convex transducer . 
they found a significant correlation between the complete and simplified lus score ( p = 0.0001 ) and a positive correlation between the simplified lus score and the hrct score ( p = 0.0006 ) , both in the quantification and extension of the ild . 
they correlated the lus data with chest hrct and proposed the definition of pleural irregularities when the thickening of the pleural line was greater than 2.8mm and a pleural score : 0 = without areas of irregularity , 1 = 15 areas of pleural irregularity and 2 = > 5 areas of pleural irregularity . 
 they performed lus evaluation in 62 lis of cw , using a 2.53.5mhz cardiac transducer with a length of 2.5cm , and correlated ultrasound data with hrct and pulmonary function tests . 
they found 100% sensitivity and 59% specificity when the total number of b lines was > 5 , and an agreement of 83% between hrct and lus for ild detection . 
the evaluation of lus included the evaluation of ten lis : for the anterior chest : the fourth lis along the midclavicular line ; for the lateral chest : the fourth lis along the anterior and middle - axillary axillary lines ; and for the posterior chest : the eighth lis along the subscapular and posterior axillary lines . 
the purpose of the study was to evaluate the ability of the lus to detect pleural line thickness ( usually < 3.0mm ) for the study of subclinical ild in patients with ssc to plan hrct evaluation . 
pleural line thickening ( 3.05.0mm ) was found in 97 patients , subpleural nodules in 32 patients and major pleural line thickening ( > 5.0mm ) in 35 patients , while normal pleural line thickening was found in 26 patients without ild . 
all lus data showed good agreement with the hrct score , classified as extended pulmonary fibrosis ( pf ) ( definitely involving the mediumhigh lung ) , limited or baseline pf ( only involving the posterior lower - base lung ) or absent pf ( no apparent sign )  . in accordance with the previous study , buda etal . 
 [ 26 ] proposed a new method to describe the results of the lus for the ild criteria : pleural line irregularity , pleural line narrowing , the fragmentary nature of the pleural line , pleural line blurring , pleural line thickening , b - line artifacts 3 and subpleural consolidations < 5mthey performed a study at a single center to correlate lus results with hrct results , using warricks score , in a cohort of 52 ild patients compared to 50 healthy subjects . 
the most frequent result was a thickening of the pleural line ( thickness 2mm ) , mainly in the lower lung fields in patients with ssc , while in severe cases of ild a blurred pleural line was detected , which is detected in patients with honeycombing in the hrtc scan . 
 [ 27 ] conducted a transversal study with the aim of correlating the results of lus , hrct thoracic and pfts in 39 patients with ssc , including the modified rodnan skin score ( mrss ) as a clinical variable . 
 [ 29 ] in 2018 designed a cross - sectional study to determine a cutoff point of the number of b lines to detect the presence of significant ild in 40 consecutive ssc patients in relation to the warrick hrct score . 
the authors adopted the previous lus score of 14 lis [ 17 ] for the evaluation of each patient and showed that a value of 10 b lines is highly predictive for the significant presence of ssc - ild in hcrt , using as external criterion a warrick score of 7 . 
 they also found a strong correlation between the total lus 1 3 la radiologia medica ( 2019 ) 124 : 989999 score and the dlco and a moderate correlation between the total lus score and quality of life measures . also in 2018 , hassan etal . 
in 29 patients with abnormal hrct ( warrick score > 7 ) and lus , two had a low score ( 615 b lines ) and 27 had moderate or severe scores ( 16 b lines )  . 
a total of 29 patients were studied with a ps - usd , whose sensitivity and specificity with respect to chest hrct were 89% and 50% , respectively . an interesting element was introduced by hasan etal . 
 [ 33 ] who studied the accuracy of lus in ild diagnosis by comparing it with hrct chest data ( including ground glass , reticular , nodular or honey combing ) and with pft . 
they divided the chest into four areas and considered a positive region when they found three or more b lines in a longitudinal plane between two ribs and defined an examination as positive when there were two or more positive regions bilaterally . 
 [ 34 ] proposed a transverse study to estimate the value of lus as a diagnostic screening tool in patients with ra who did not show clinical signs or symptoms of ild . 
on the other hand , lus showed sporadic abnormalities in 7% of healthy controls . recently , the same authors [ 14 ] conducted a new study with the aim of determining the diagnostic value of lus in the diagnosis of ild in patients with ctds [ ra , ssc and systemic lupus erythematosus ( sle ) ] and , as a second aim , to determine the possible correlation between the frequency of pathological results of lus and the underlying disease . 
lus was correlated with chest hrct showing a sensitivity of 1 ( 95% ci 0.3981.0 ) , a specificity of 0.89 ( 95% ci 0.5180.997 ) and a positive probability of 9.00 ( 95% ci 7.111.3 ) to detect ild . 
 lus achieved an excellent correlation with hrct data in sjgrens syndrome with ild . table2 shows the feasibility , reliability , sensitivity and specificity of lus , while the technical characteristics of lus and the type of score used in all studies involved in the review are represented in table3 . discussion identification and quantification of early manifestations of ild in ctds are an important objective to improve the quality of life of patients affected by ctds and for prognosis [ 1 , 3741 ]  . 
in this regard , lus has recently demonstrated a high sensitivity in the detection of signs indicative of ild , even in the early stages of the disease and , especially , a high negative predictive value . 
hrct remains the gold standard in ild identification ; however , lus , due to the absence of ionizing radiation and its simplicity of execution , even in the patients bed , can be considered as an excellent screening tool . 
lus has demonstrated encouraging validity , reliability and feasibility , and currently , it could be considered as an excellent methodology to establish the correct timing of hrct in ild assessment . how to behave in the preclinical stages of disease , however , remains a topic of debate [ 30 ]  . 
interesting the cutoff point of ten b lines to detect the presence of sscild proposed as a reference on the basis of which to send patients to perform a hrct [ 29 ]  . despite the progress of studies in the literature , there is still much to be done , and some crucial points need to be discussed . 
currently , the number of lis used in the studies is very variable , from 10 to 72 on the other hand , new possibilities of interpretation of pathological findings are emerging , in particular with regard to pleural abnormalities . 
pleural abnormalities appear to be adequately related to specific hrct findings such as ground - glass opacity and extensive fibrosis and could therefore be considered an ild imaging biomarker [ 15 ]  . 
 moreover , in the future , there will be an increasing interest in pocket - sized ultrasound machines , which can be used to evaluate lung diseases without the need for top of the range machines to acquire reliable information . in conclusion , this review showed how lus can become an important technique for assessing lung disease in rheumatic diseases with suspected lung involvement . 
applications of this imaging method are still growing , and further opportunities are likely to arise in the light of the vibrant field of research . funding the authors have not declared a specific grant for this research from any funding agency in the public , commercial or not - for - profit sectors . compliance with ethical standards conflict of interest the authors would like to make the following statements with regard to their conflicts of interest / financial disclosures : mg has attended advisory board meetings and scientific consultancies and has obtained speaking fees for : abbvie , novartis , ucb , esaote spa , janssen , bristol - myers squibb , merck sharp & dohme , pfizer , sanofi aventis . 
in this scenario , the radiologist plays a key role in both diagnostic and therapeutic workups of pavms : chest x - ray , computed tomography and magnetic resonance are effective tools for pavms identification and confirmation of the suspected diagnosis . 
furthermore , imaging modalities provide most of the elements for pavms classification according to their angioarchitecture ( simple and complex ) and help the clinicians in establishing which lesion requires prompt treatment and which one will benefit of imaging follow - up alone . 
endovascular management of pavms has grown up as the first - line treatment in respect of surgery during last decades , showing lower risk of intraand post - procedural complications and offering a wide number of treatment options and materials , ensuring effective management in virtually any clinical situation ; interventional treatment aims to exclude pavms from pulmonary circulation , and specific technique and embolic agents should be selected according to pre - treatment imaging , in order to obtain the best procedural outcome . 
this paper proposes a review of the clinical and radiological features that a radiologist needs to know for pavms diagnosis and proper management , also showing an overview of the most common endovascular treatment strategies and embolization materials . keywords pulmonary arteriovenous malformation pulmonary arteriovenous fistula hereditary hemorrhagic telangiectasia oslerweberrendu disease ct imaging endovascular treatment introduction pulmonary arteriovenous malformations ( pavms ) or fistulas are a rare condition , firstly described by churton etal . 
 in this scenario , the radiologist plays a key role in both the diagnostic and therapeutic workup of pavms : chest x - ray ( cxr ) , computed tomography ( ct ) and magnetic resonance ( mr ) are essential tools for diagnosis , treatment planning and follow - up of pavms , providing the majority of the elements required for a proper management ; on the other hand , endovascular management of pavms is vol . : ( 0123456789 ) 1 3 974 la radiologia medica ( 2019 ) 124 : 973988 carving out a role as first - line treatment option for these lesions , showing high safety and effectiveness . the article focuses on the clinical and radiological features of pulmonary arteriovenous malformations according to what the radiologist needs to know for a correct patient management ; etiology and classification are also described , and an overview of the current endovascular treatment techniques and materials available is proposed . etiology congenital pavms most of pavms are congenital ( 80% ) , and a tight association with hereditary hemorrhagic telangiectasia ( hht ) is described in the literature ( 4790% ) [ 38 ]  . 
hht is an autosomal dominant disorder associated with the mutation of one of the following proteins : endoglin ( eng gene , hht1 subtype ) or activin a receptor type ii - like 1 ( akl1 gene , hht2 subtype ) [ 9 , 10 ] ; among hht subtypes , hht1 has the strongest association with pavms [ 11 ] , probably because endoglin is part of the cellular membrane receptor for tgfthat is a key factor in the regulation of the angiogenetic process [ 12 ]  . 
in a small percentage of cases ( ~ 2% ) , a mutation in smad4 [ 13 ] is observed , coexisting in these patients the juvenile polyposis [ 14 ]  . on the other hand , only 1050% of patients with hht will develop pavms during their lives [ 4 , 15 ] , and there is lack of evidence about possible genetic locus , other than eng , akl1 and smad4 , associated with pavms development [ 16 , 17 ] , suggesting that congenital pavms conceal genetic basis that are still widely unclear . pavms inpregnancy pregnancy is associated with the growth of preexisting pavms [ 21 ] , especially in hht patients : probably , the underlying cause is the increased blood flow / volume and cardiac outputtypical hemodynamic effects observed in pregnancyassociated with the complex changes promoted by progesterone on the pulmonary venous bed . 
the consequence is an increased risk of major clinical events associated with pavms rupture / embolization , such as hemoptysis , hemothorax and cerebrovascular disorders [ 21 , 22 ]  . classification the classification of pavms currently used was proposed by white [ 23 ] : pavms are classified as simple and complex , on the basis of their angioarchitecture . 
two particular subtypes of complex pavms are diffuse and telangiectatic ones : diffuse subtype is characterized by the involvement of a large part of parenchyma , sometimes even an entire lung , by a tangle of malformed vessels ; telangiectatic pavms involve only part of pulmonary lobe , and their vascular network is usually microscopic , beyond the spatial resolution power of ct [ 24 ]  . pavms are also classified on the basis of the feeding arteries size : if a feeding artery has a diameter of 3mm or greater , pavm is classified as macroscopic , otherwise as microscopic ( fig.1 ) [ 25 ]  . acquired pavms clinical features pavms are acquired in 20% of cases and associated with pathological conditions such as hepatic cirrhosis , infectious diseases , traumas , mitral valve stenotic disease , fanconis syndrome and metastatic cancer [ 2 ]  . 
among these , chest traumas , cardiothoracic surgery , hepatic cirrhosis , metastatic cancer , mitral stenosis , infections and amyloidosis are described most frequently [ 3 ] ; sporadic cases of pavms in constrictive pericarditis [ 18 ] , lung parenchymal cyst [ 19 ] and chronic thromboembolic disease [ 20 ] have also been reported . in our opinion , this great variability of conditions associated with acquired pavms subtends a common underlying developmental mechanism , probably triggered by a chronic angiogenetic impulse to the lung vasculature . clinical presentation of pavms is extremely heterogeneous : some patients are completely asymptomatic , and some others present from vague to even life - threatening respiratory symptoms such as dyspnea and hypoxemia at rest or during exertion . 
platypnea ( orthostatic dyspnea ) and orthodeoxia ( orthostatic hypoxemia ) are typical features and usually affect patients with pavms located in the lower lobes , probably as the consequence of a blood flow redistribution to the site of pavms in the upright position [ 26 ] ; less frequently , pavms manifest as a clinical emergency with hemoptysis [ 27 ] and / or hemothorax ( table1 ) [ 7 ]  . hht patients have a major risk of complications , severe symptomatology , multi - organ disease and rapid progression of pavms [ 3 ]  . 
simple pavms are characterized by the presence of a single segmental feeding artery ; their angioarchitecture may consist of a direct communication between the segmental artery and a usually aneurysmatic draining vein ( a ) or of multiple sub - segmental arteries merging in a tangle of malformed vessels draining in one ( b ) or multiple ( c ) veins . 
complex pavms receive blood supply from two or more segmental arteries ( d ) ; diffuse subtype shows a wide parenchymal involvement with a mess of malformed vessels of similar size ( e ) that in the telangiectatic subtype are too small to be discerned with imaging ( f ) or assessed with certainty , hht should be promptly confirmed by the presence of curacao criteria ( table2 ) [ 28 ] : the presence of three or four criteria confirms the diagnosis of hht , and the presence of two criteria makes the diagnosis possible or suspected , whereas one or less criteria make the diagnosis unlikely . imaging diagnostic workup the radiologist is like a metronome in the pavms decision - making process ; cxr , ct and mr provide essential elements for both treatment and careful observation of 1 3 976 la radiologia medica ( 2019 ) 124 : 973988 table 1 clinical features of pavms symptoms signs complications absence of sympanemia brain abscess toms chest pain cough dizziness dyspnea dyspnea on exertion clubbing cyanosis hypoxemia polycythemia pulmonary hypertencerebrovascular stroke endocarditis epistaxis hemoptysis hemothorax sion pulmonary rumors telangiectasia heart failure splanchnic abscess splanchnic embolization attack transient ischemic fatigue migraine orthodeoxia palpitation platypnea seizures syncope pavms pulmonary arteriovenous malformation table 2 curacao criteria recurrent and spontaneous epistaxis ( > 1 episode ) first - degree relative with hht specific anatomical locations of telangiectasia ( face , fingers , mouth , oral and nasal mucosa ) arteriovenous malformations at visceral sites ( lung , liver and brain ) hht hereditary hemorrhagic telangiectasia diagnosed pavms . chest xray respiratory symptoms are usually the first clinical event among patients with pavms [ 29 , 30 ]  . 
in this scenario , cxr is the ideal first imaging tool , due to the mini - invasiveness and diffuse availability , providing good sensitivity ( 70% ) and excellent specificity ( 98% ) for diagnosis of pavms , when nodular opacities are evident [ 31 ]  . 
these characteristics allowed cxr to be proposed in screening programs for pavms in patients with hht , in association with transthoracic contrast echocardiography ( ttce ) [ 31 , 32 ]  . according to the european guidelines on quality criteria for diagnostic radiographic images [ 33 ] , the best diagnostic results are obtained when cxr is performed in the upright position and both postero - anterior ( pa ) and lateral projections are performed . 
a correct acquisition of cxr is essential , in order to avoid misdiagnosis of small pavms or fistulas located behind normal structures such as heart or diaphragm [ 34 ]  . 
cxr appearance of pavms is chameleonic : some patients show no evidence of parenchymal involvement , due to microvascular telangiectasia ; some others may have a heavy impairment of pulmonary parenchyma with multiple and diffuse pavms in both lungs [ 2 , 34 ] , but usually are less than 5cm in the largest diameter [ 2 , 10 , 24 ]  . 
frontal chest x - ray radiogram shows two well - defined homogeneous mass - like opacities with lobulated shapes ( circle ) in the sub - pleural parenchyma of the left lung ( a ) confirmed in the lateral radiogram ( b ) that better shows also serpiginous opacities directed from the lesions to the hilum , referable to feeding vessels ( arrowheads ) 1 3 la radiologia medica ( 2019 ) 124 : 973988 from the pulmonary hilum and reach the fistula , whereas draining veins arise from pavm to reach the left atrium [ 2 , 24 ]  . 
when visible , feeding vessels appear as tubular opacities that connect the suspected fistula to the mediastinum and veins are usually larger than arteries ; the diameter of vessels is usually > 4mm but < 7mm , even if vessels with diameter > 20mm have been previously reported [ 36 ]  . 
 in some cases , parenchymal hemorrhage , resulting from the rupture of the lesion , or atelectasis , as a consequence of ab estrinseco bronchial compression , is observed [ 2 ]  . computed tomography ct is the gold standard among noninvasive diagnostic tools for pavms [ 24 , 36 ]  . 
there is lack of evidence about ct acquisition protocols for dose reduction , and a wide inter - study and intra - study variability is observed in the literature , probably as the consequence of different patients populations ( screening , follow - up , etc . ) and ct scanners ( singleto multi - slice )  . 
some authors suggest a low - dose unenhanced ct acquisition of the thorax [ 41 , 42 ] , in order to identify pavms and exclude other pathologies such as calcified granulomas [ 35 ]  . 
maximum intensity projection images confirm the presence of a macroscopic pavm characterized by the presence of a nidus ( circle ) receiving an arterial feeder ( arrow ) from the apical segmental pulmonary artery and draining in a large venous collector ( arrowhead ) ( b )  . 
complex pavms are also recognized on ct scans with simplicity and are distinguished from simple type for the presence of more than one feeding artery , originating from multiple segmental arterial branches ( fig.3 ) [ 24 ]  . 
the recognition of diffuse and telangiectatic subtypes may be more laborious : in the diffuse subtype , a tangle of pathological feeding arteries and veins in association with a homogeneous involvement of a whole segment , lobe or fig . 
c the presence of multiple simple pavms ( arrows ) in the lower lobes strengthened the suspected diagnosis 1 3 la radiologia medica ( 2019 ) 124 : 973988 lung is evident . 
telangiectatic pavms appear as a groundglass area , with possible solid nodular component inside , and both feeding artery and vein are frequently absent ; this type of lesions is usually diagnosed in children , and their diagnosis may be challenging , but multiple pavms with typical appearance usually coexist in both lungs , suggesting the nature of the lesions ( fig.4 ) [ 24 ]  . whatever the nature of pavm , the identification of at least a feeding artery and vein is mandatory for establishing the diagnosis on ct images [ 35 ]  . 
the identification of arterial feeders is also necessary to assess the vessel diameter ; this measurement should be performed as near as possible to the nidus of the pavm , however , beyond non - afferent parenchymal vessels , because an arterial diameter of 3mm or larger is the current cutoff to indicate endovascular treatment [ 25 ] , in the face of an increased risk of complications [ 23 ]  . 
their study reported that the dynamic contrast - enhanced mra with the improved temporal and spatial resolution can be used in children and infants for the assessment of congenital vascular disease . in the study of schneider etal . , the contrast - enhanced mra was reported to be an effective screening tool for the detection of pavms in patients with hereditary hemorrhagic telangiectasia . 
contrast - enhanced mra is also a superior technique to global or selective pulmonary angiography , permitting the detection of significantly more pavms both over all ( p = 0.0003 ) and when compared solely with the initial global or selective pulmonary angiography ( p < 0.0001 ) [ 46 ]  . time - resolved mr angiography was recently reported to be a more useful option than ct for assessing the reperfusion of pavms after coil embolization because of its high sensitivity in detecting blood flow and the absence of ionizing radiation , and it offers a noninvasive examination with high resolution [ 47 , 48 ]  . unfortunately , most of the series enrolled few patients and the level of evidence remains basically low . in general , 3d contrast - enhanced mr angiography has shown good identification rate for pavms > 5cm , with an acceptable demonstration of number and size of feeding vessels ; however , the diagnostic accuracy seems to be significantly lower for lesions < 5cm , limiting the use of this technique in setting indication to treatment [ 49 ]  . interventional management endovascular treatment of pavms has carved out a significant role over the years , proving to be superior to surgery in terms of complications rate and hospitalization length [ 50 ]  . 
5 pavms treatment complications : ct axial image of a 54 - year - old man presenting with left thoracic pain , 7 days after endovascular occlusion of an inferior left lobe pavm with coiling ( arrow )  . 
a well - demarcated triangular sub - pleural consolidation area is evident , distally to the treated lesion ( circle ) ; the ct appearance and the clinical onset suggest the nature of the lesion as a post - procedural pulmonary infarction 1 3 980 la radiologia medica ( 2019 ) 124 : 973988 this goal is achieved by closing the feeder vessels of the pavm as near as possible to the nidus , in order to preserve normal vessels , lung parenchyma and preventing collateral reperfusion . 
coil selection is a delicate step : the diameter should not be smaller than that of the feeder vessel , because the risk of paradoxical embolization may increase [ 52 ] ; on the other hand , coils with a too large diameter may unfit the vessel , causing local complications , such as vessel rupture or migration in nontarget adjacent vessels ; a 2030% coil oversizing , with respect to vessel diameter , has been described as a good compromise to prevent complications [ 52 ]  . 
also , coil length may affect deployment : a short length can lead to the use of many coils , with a rising in procedural costs and increasing the risk of paradoxical embolization or retrograde migration ; moreover , an excessive length makes difficult the coil folding process in the target vessel . 
a 30 - year - old woman presenting with dyspnea : chest radiogram ( a ) shows the presence of a round bilobated opacity in the right middle lobe ( arrow ) , with two tubular opacities converging to the pulmonary hilum ( arrowheads ) , suspected for pavm . 
contrast - enhanced ct ( b ) confirms the presence and the nature of the lesion , revealing a well - defined nidus ( arrow ) , a feeding artery ( white dot ) and an efferent vein ( white star ) ; a little collateral feeding artery originating from the same segmental artery is also evident , defining the lesion as simple pavm . 
the occlusion of the main ( c ) and collateral ( d ) arterial feeders to the pavm by means of metallic coils , allowed pavm complete vascular exclusion ( e ) 1 3 982 la radiologia medica ( 2019 ) 124 : 973988 fig . 
7 anchor technique : ( a ) selective angiography of an artery suppling an inferior right lobe malformation ( star ) showing the presence of multiple collateral vessels near the lesion ( arrow )  . 
a catheter is placed into one of the collaterals by mean of coaxial technique ( b ) and a long metallic coil is deployed starting from the collateral vessel ( c ) into the lumen of feeding artery of the vascular malformation ( d )  . 
b the small size of the feeders and their collaterals suggested the operator to choice a scaffold technique placing an oversized , rigid coil in each feeding vessel ( arrowheads ) , in order to create a supportive system for the deployment c of multiple filling coils . 
d final control showing the complete vascular exclusion of the fistulas 1 3 la radiologia medica ( 2019 ) 124 : 973988 whatever the embolization technique , advantages of using coils consist of a relative ease of use , the possibility of deployment control and excellent adaptability to the vascular lumen . 
on the other hand , the major limit lies in the necessity to reach the target vessel and the need for sufficient lumen space for the deployment . there is no evidence in the literature about changes in procedural outcome for different coils type [ 56 ] , and vascular recanalization is observed in up to 19% of cases [ 26 ]  . onyx vascular plugs amplatzer vascular plugs are dense nitinol mesh devices that reduce blood flow in the target vessel and promote thrombosis once deployed . 
each device has a relatively rigid delivery system which carries the plug , through the catheter , till the site of embolization ; the plug is anchored to the delivery system by a screw mechanism which , once the target vessel is reached , is manually turned counterclockwise , with subsequent plug deployment [ 57 ]  . 
a new generation of plugs composed by microvascular plugs ( medtronic , dublin , ireland ) and endoluminal occlusion system ( artventive , carlsbad , california ) has been recently described for pavms treatment [ 5860 ] ; they differ from classical vascular plug for the presence of a polytetrafluoroethylene membrane that induces vessels occlusion and allows the reduction in both profile and size of the device in respect of amplatzer vascular plugs . 
once in contact with blood , dmso rapidly dissolves and ethylene vinyl alcohol begins the polymerization process [ 52 ]  . the great advantage of onyx resides in the injection technique : the non - adhesive nature of the mixture , and the relatively long time of polymerization , ensures in - vessel navigation by onyx , favoring the embolization of distant lesions ; in addition , onyx progressive polymerization helps to prevent nontarget embolization and reflux , making the embolization process safer with respect to other liquid embolic agents . 
onyx disadvantages consist of the necessity of dmso - compatible catheters , higher procedural costs and a good experience in onyx injection / manipulation , in order to prevent nontarget or distal embolization . venous sac embolization ( vse ) vse has been also proposed as an alternative technique to feeding artery embolization for pavms endovascular management . 
maximum intensity projection coronal reconstructions show vascular plugs ( arrowheads ) in the lumen of the feeding arteries of two pavms ( arrows ) in the right lower lobe ( a ) ; to notice that the site of deployment is distant from the nidus for both vascular plugs , increasing the risk of vascular collateralization to the lesions . 
on the other hand , coils produce more metallic artifacts in respect of vascular plugs ( b ) that can make posttreatment assessment more difficult 1 3 984 la radiologia medica ( 2019 ) 124 : 973988 fig . 
10 digital angiography ( a ) in a 28 - year - old woman with a pulmonary fistula of the left lung characterized by unique feeding artery ( dot ) and a large aneurysmatic vein ( star )  . 
the deployment of a plug ( thin arrow ) proximal to the shunt was preferred over the coil due to the high risk of systemic migration ; marked flow reduction in the aneurysmatic vein was obtained ( c )  . 
embolization is completed by the deployment of two coils ( thick arrow ) behind the plug ( d ) , with no evidence of fistula reperfusion at the final angiographic control ( e ) embolization is the complete savings of the arterial pulmonary circulation , with a reduced risk of nontarget embolization and complications , such as pulmonary infarction . 
in this sense , the procedure may be useful when feeder arteries are too short to allow safe treatment or when the target of embolization is a high - flow pavm , with an increased risk of paradoxical embolization [ 62 , 63 ]  . 
given the increased risk of coil migration on the venous side of the pavm , due to the presence of larger vessels , some authors suggest the use of a detachable coil as first , in order to obtain a safe coil deployment and reduce complication during the filling phase of the procedure [ 62 , 63 ]  . there is poor literature evidence and no consensus about which is the best way among venous and arterial approach for venous sac embolization ; in essence , both techniques seem to be effective [ 64 , 65 ]  . followup andposttreatment evaluation follow - up of patients with pavms is also important : according to the international guidelines for diagnosis and management of hht [ 25 ] , patients with small pavms not requiring embolotherapy , or patients with suspected pavms , should undergo ct examination after case - by - case analysis , every 15years ; for those undergoing embolotherapy , first ct examination should be performed at 612months and then every 3years ; this reduced follow - up time interval is recommended because up to 20% of patients with treated pavms will experience lesion recanalization as the consequence of pavm reperfusion at the site of embolization , new vessels recruitment , preexisting feeding vessels hypertrophy or as the consequence of an incomplete exclusion during the first 1 3 la radiologia medica ( 2019 ) 124 : 973988 fig . 
contrast - enhanced ct is considered the standard technique for follow - up of these patients ; good treatment outcome is confirmed by the reduction in size of the nidus and the absence of enhancement in the treated feeding arteries ; draining veins may preserve contrast enhancement , but this sign is not specific because a retrograde perfusion of the lesion is possible [ 26 ]  . 
 [ 68 ] , authors suggest that an absolute diameter of 2.5mm or more of the draining 1 3 986 la radiologia medica ( 2019 ) 124 : 973988 vein at follow - up unenhanced ct was strongly associated with pavm patency after endovascular treatment . time - resolved mr may offer advantages in the follow - up of treated pavms , especially for those treated with coils , because the induced metallic artefacts , that limit ct diagnostic performance , have less impact on mr images evaluation [ 69 ]  . 
a diagnostic and therapeutic flowchart for patients with suspected pavm is shown in fig.11. conclusion pavms persist to be a medical challenge in the face of their complex anatomy , their vague and variable clinical appearance and the particular population ( hht ) involved by this disease . 
the radiologist plays a key role in the diagnostic workup of pavms , but there is still much to be done to improve both ct and mr protocols , in order to modulate dose and personalize the examination . endovascular treatment is now identified as the treatment of choice for pavms , offering lower complication rates and less hospitalization time , compared to surgery . 
however , chemotherapy and radiation therapy comprehend a complex scenario due to the wide choice of strategies including neoadjuvant vol . : ( 0123456789 ) 1 3 956 la radiologia medica ( 2019 ) 124 : 955964 chemotherapy ( nact ) , followed by surgery [ 4 ]  . 
the choice of the most adequate treatment usually depends on certain prognostic factors , such as tumor volume , parametrial and lymphovascular space invasion revealed through biopsy and locoregional lymph node status [ 5 , 6 ]  . 
mr imaging is the gold standard for accurate preoperative assessment of tumor volume and parametrial invasion as well as detection of lymphadenopathy [ 4 ] ; indeed , great importance has been given to nodal disease in the last figo staging guidelines of 2018 , being considered one major prognostic factor . on the other hand , histopathological analysis is the gold standard for establishing tumor heterogeneity , but in many cases it is difficult to assess structural , phenotypic or genetic intratumoral features on the basis of random sampling or biopsy [ 7 ]  . 
however , identification of some phenotypic or genetic features can be useful to predict response to neoadjuvant therapy in order to make therapeutic decisions and to avoid over - toxicity in connection with ineffective treatment . differentiation of histological features using conventional mr imaging is still challenging because tumors may be very similar in appearance on conventional t2 - weighted images despite histological diversity [ 8 , 9 ]  . 
in this contest , texture analysis ( ta ) applied to imaging has proved to be an imaging prognostic marker for poor survival in several tumor types ( colorectal primary tumor and liver metastases , lymphoma , lung , head and neck , esophageal renal and prostatic cancer ) correlating with glucose metabolism , stage and unfavorable genetic mutation [ 9 , 1221 ]  . in particular , magnetic resonance ta has proved to be an imaging biomarker of tumor response to neoadjuvant therapy in breast cancer ( using t2 and t1 contrast - subtracted sequences ) [ 22 ] and to neoadjuvant chemoradiotherapy in rectal cancer ( using t2 sequences ) [ 23 ]  . 
another recent study has demonstrated that texture analysis applied to mrdynamic contrast - enhanced images is useful for outcome prediction [ 24 ]  . regarding cervical cancer , few preliminary studies evaluated the efficacy of texture to identify node involvement or to predict local recurrence after chemiotherapy , with promising results . 
the aim of this study was to investigate any correlation between tumor features ( grading and histology ) and ta parameters or conventional parameters ( maximum diameter and adc value ) to evaluate which of the conventional parameters and ta parameters better predict the response to nact , and if there was any possible correlation between each other . materials andmethods study population from november 2015 to april 2016 , 28 patients ( age range 3565years ) with lacc were retrospectively evaluated applying texture analysis to mr images obtained prior to nact . 
diagnosis of cervical cancer was previously confirmed in all patients through diagnostic hysteroscopy and biopsy , and histopathological examination had established histological type and tumor grading . all patients were staged according to the figo criteria and all underwent vaginal and rectal examination under anesthesia as well as a standard chest radiograph . 
patients were excluded from the study if : ( a ) mr imaging acquisition was incomplete ; and ( b ) nact was discontinued before surgery . mr examination mr imaging was carried out using a 3t scanner ( discovery mr750 ; general electrics , milwaukee , wi ) equipped with high - performance gradients using an 8 - channel - phased array coil with the patient in the supine position . 
mr protocol included high - resolution morphological imaging of the pelvis using turbo - spin echo ( tse , repetition time , 20864172ms ; echo time , 11.4122.3ms ; nex , 2 ; slice thickness , 4mm ; matrix , 512 512 ) oriented in the sagittal plane , axial oblique plane ( perpendicular to the major axis of the cervix ) and coronal oblique plane ( parallel to the major axis of the cervix )  . neoadjuvant therapy andsurgical technique patients affected by lacc were scheduled to receive three 21 - day cycles of platinum - based nact followed by radical surgery as already described in the literature [ 25 ]  . 
in patients whose lesions remained unresectable also after nact , tumor response was assessed through clinical 1 3 la radiologia medica ( 2019 ) 124 : 955964 evaluation and assessment of tumor volume / disease extension revealed at the most recent mr examination after nact . texture analysis heterogeneity of the cervical tumor was assessed with texrad , a proprietary software algorithm ( texrad ltd , somerset , england , uk )  . 
a region of interest ( roi ) was delineated around the cervical lesion area on a single - slice t2 - weighted mr image by a radiologist with 6years experience in abdominal mr imaging . 
texture analysis of image within the roi was obtained using the filtration - histogram method that comprises an initial filtration step that highlights image features of a specified size , followed by histogram analysis of the filtered image [ 9 ]  . the filter basically extracts and enhances intensity - variation features of different sizes corresponding to the spatial scale of the filter itself ( ssf in radius ) : fine ( ssf = 1mm ) , medium ( ssf = 1.51.8mm ) and coarse ( ssf = 2mm )  . 
quantification of histograms ( with and without filtration ) was based on the calculation of the following parameters : mean : average value of pixel intensity ; mean positive pixels ( mpp ) : average value of positive standard deviation ( sd ) : variation or dispersion from the ( hyperintense ) pixels ; mean value ; skewness : a measure of the asymmetry of the histograhyperintense features within the image increase the mean value and result in positive skewness , whereas hypointense features within the image decrease the mean value and result in negative skewness [ 8 , 9 ] ; entropy : the disorder and randomness of gray - level distribution in terms of pixel orientation within the image ; kurtosis : a measure of the peakedness of the histograa positive kurtosis indicates a histogram that is more peaked and consequently that the collected data are distributed mainly around their mean . 
this indicates that there is more variability and heterogeneity in the dataset [ 8 , 9 ]  . conventional evaluation radiologists collected for every case the maximum diameter expressed in millimiters and evaluated on t2 oblique plane . to calculate adc parameters , radiologists draw a region of interest ( roi ) on the peripheral portion of the lesion on the adc map ( mean size 3 mm2 ; range 25 mm2 ) , with careful exclusion of the necrotic or cystic portions inside the tumor . statistical analysis descriptive statistics are provided as mean standard deviation for continuous variables . 
unpaired t test was used to evaluate whether the obtained data were statistically different comparing squamous cell carcinoma to adenocarcinoma , g3 to g1g2 tumors , and responders to non - responders . 
ta showed that adenocarcinoma exhibited more bright pixels on mr images than squamous cell carcinoma , thereby presenting high values , both mean pixel values ( mean ) and mean of positive ( hyperintense ) pixels ( mpp )  . 
 the mean area under the curve ( auc ) used to discriminate between adenocarcinoma and squamous cell carcinoma was 0.91 using coarse filtration ( ssf 2 ) with a significant discriminatory power ( p = 0.002 ) : the optimal cutoff for identifying adenocarcinoma was 29 . 
 kurtosis showed a tendency to be lower in patients with adenocarcinoma , but there was no statistically significant difference between the two histological groups . ta andtumor grading dividing the lesions into three categories : well - differentiated ( g1 ) , moderately differentiated ( g2 ) and poorly differentiated ( g3 ) , histopathological examination classified one patient in g1 , 15 in g2 and 12 in g3 . 
 a total of 15 patients responded to nact : 3 presented complete response ( cr ) and 12 presented partial response ( pr ) ; 13 patients were non - responders : 6 had disease progression ( pd ) and 7 had stable disease ( sd )  . ta andtumor response tonact ta parameters related to response to nact are listed in table 3 . 
this filtration seemed to yield the lowest p values ( p = 0.002 ) when unpaired t test was applied to compare the difference between responders and non - responders . 
the optimal cutoff found for kurtosis to identify non - responders was 3.7 yielding a sensitivity of 92% and a specificity of 75% conventional parameters did not show any significative correlation to the tumor response to nact . 
few studies reported that lower value of adc correlated with poorer prognosis [ 26 , 27 ] even in our sample we had a lower mean value , however , not statistically relevant . 
in contrast , adenocarcinoma presents an alternation of solid and fluid components ( glandular elements and contents of the glandular duct , respectively ) which may explain a more heterogeneous distribution of pixels in the histogram with both alternating positive pixels ( the vast majority ) and negative fig . 
 the greater structural homogeneity seems to reflect also the better response rates and complete response to nact which are usually obtained in cases of squamous cell carcinoma as reported in the literature [ 2932 ]  . 
the structural heterogeneity of adenocarcinoma expressed the tendency of this tumor type to be more aggressive and less sensitive to nact . texture analysis andhistological grading well - differentiated tumors have a better prognosis than poorly differentiated tumors [ 32 ] , but in this study none of the texture parameters yielded a statistically significant correlation with histological grading . 
however , it may be interesting to mention that , even if not statistically significant , kurtosis was slightly lower in patients with high - grade lesions ( g3 ) than in patients with intermediate - grade lesions ( g1g2 ) , thus reflecting a greater dispersion of pixel distribution in g3 tumors linked to the higher heterogeneity of tumor structure . texture analysis andtumor response tonact at ta , responders presented a higher degree of kurtosis expressing a more homogeneous distribution of pixels within the lesions , whereas non - responders presented greater heterogeneity with lower kurtosis . 
this variability may reflect a more disorganized histological structure with great alteration of tumor cell morphology , nuclear enlargement and an ineffective vascular net causing a reduced efficacy of chemotherapeutics . 
in line with the more heterogeneous histological structure and to the more aggressive biological behavior , adenocarcinoma showed a lower peak of the histogram ( arrow ) than squamous cell carcinoma and a lower kurtosis 1 3 962 la radiologia medica ( 2019 ) 124 : 955964 fig . 
responders showed a more homogenous pixels intensity , with a less dispersion of pixel distribution and a higher peak of the histogram ( red arrow ) and a consequent higher kurtosis compared to non - responders . 
from a clinical point of view , being able to early detect responders patients would allow to avoid ineffective treatments , the collateral effects directly associated , and at last start alternative therapies . 
even if the use of a 3t mr system can reduce the impact of image noise on biological heterogeneity , which can affect texture analysis , especially using fine texture scale [ 16 ] , we should consider that mri can introduce a higher variability in data acquisition than ct can , related mainly to the complexity of the technique and the higher number of parameters that can be modified . 
this variability could , in theory , affect reproducibility of the results . certainly it would have been better to normalize the whole image before applying texture analysis , but we decided to use the original non - normalized images to demonstrate the feasibility of texture analysis using t2w mr images . 
moreover , other authors in literature have successfully used mrta in non - normalized t2 - weighted images in rectal and breast cancer [ 23 , 33 ]  . finally , our considerations are based solely on ta values correlated to histological tumor type , grading and response to treatment but there is no correlation with markers of tumor angiogenesis , glucose metabolism and hypoxia which may influence tumor aggressiveness according to previous studies . 
it would be interesting to compare pre - treatment texture data with post - nact texture analysis and with patient survival . conclusion in conclusion , ta applied to t2 - weighted mr sequences of lacc seems to be a promising tool for describing tumor heterogeneity having demonstrated a strong potential for differentiating adenocarcinoma from squamous cell carcinoma and predicting response to nact . 
 therefore , ta may add information to patient prognosis , 1 3 la radiologia medica ( 2019 ) 124 : 955964 especially when data from histological specimens are limited and insufficient . 
diagnostic accuracy values for diagnosing bme were calculated for dect maps ( qualitative assessment ) and for ct numbers ( quantitative assessment ) by using receiver operator curves and area under the curve ( auc ) , using mri as the gold standard . 
sempreboni 10 , 37024negrar , vr , italy 2 department oforthopedic surgery , irccs sacro cuore hospital , negrar , italy introduction bone marrow edema ( bme ) of the ankle represents a common clinical problem in adult population [ 13 ]  . 
it may be diagnosed following acute traumas , or in a chronic clinical scenario without recent traumas , as , for example , in the bone marrow edema syndrome or in post - traumatic arthritis [ 46 ]  . 
early preventive measures , including restriction or cessation of activity after the diagnosis , may be crucial in preventing further deterioration of articular lesions [ 8 , 9 ]  . 
a recently published meta - analysis confirmed that the diagnostic performance of dect for the evaluation of the spine ( sensitivity , 0.84 ; specificity , 0.98 ) and of the appendicular skeleton ( sensitivity , 0.84 ; specificity , 0.93 ) was excellent [ 20 ]  . 
nevertheless , the majority of these studies focused on patients in the acute settings , and there no papers available describing the role of dect in diagnosing bme of the ankle in patients without recent trauma . 
moreover , dect has been used for the evaluation of urate deposition in patients with gouty arthritis and for the assessment of achilles tendinopathy [ 2427 ]  . the purpose of our study was to investigate the diagnostic accuracy values of a third - generation dect scanner in detecting bme in the ankle , by using mri as the standard of reference . materials andmethod patient population this prospective study was approved by the institutional review board , and all patients provided informed consent . 
eligibility criteria were represented by acute pain following sprained ankle ( n = 13 ) , or chronic pain ( n = 27 ) in patients with negative conventional radiographs . 
the ct examinations were used for the evaluation of ankle anatomy and to rule out subtle meshed fracture , while mri was used to assess the presence of bme and to evaluate ankle ligaments and tendons . four patients were excluded because of non - mri - compatible pacemaker ( n = 1 ) , incomplete mri protocol ( n = 1 ) , metal artifacts from previous surgery ( n = 1 ) , and claustrophobia ( n = 1 )  . 
1 diagram showing flowchart of patients enrolled 1 3 1030 la radiologia medica ( 2019 ) 124 : 10281036 product was set at a ratio of 1.6 : 1 ( tube a , 220 quality reference mas ; tube b , 138 quality reference mas )  . 
soft tissue kernel ( qr32 ) 80 - kvp and 150kvp set images were transferred to an offline workstation ( syngovia vb20 ; siemens , erlangen , germany )  . the software uses a three - material decomposition resulting in a virtual non - calcium image ( vnca )  . 
the information was color - coded with a color lookup table which codes bone marrow and edema in shades of green - yellow to orange - red ( parallelly to the progressive increase in density ) ; 3d volume rendering maps coding bone marrow edema in shades of green and normal bone in blue were also available . 
the de - specific information was fused with the conventional grayscale morphologic images ( thickness , 1mm ; increment , 1mm )  . mri protocol mr imaging was performed with a commercially available 1.5 - t unit ( magnetom avanto fit ; siemens healthcare , erlangen , germany )  . 
standard t1 - weighted spin - echo , t2 - weighted turbo spin - echo , and turbo inversion - recovery magnitude ( tirm ) sequences were performed in the axial and sagittal orientation . 
 image analysis on all images datasets available , each ankle was evaluated for the presence of bone marrow edema using a binary classification system ( 1 = presence of bme and 0 = absence of bme )  . 
at mri , the diagnosis of bme was based on signal intensity increase at tirm imaging with a signal decay at t1 - weighted imaging . in the second reading session , dect images were analyzed by two independent radiologists ( reader 1 and reader 2 , with 15 and 35years of experience in radiology , respectively )  . 
standard axial , coronal , and sagittal reconstructed ct and dect images ( 3d and 2d color - coded maps ) were analyzed in a random order , blinded to clinical and mri findings on a dedicated syngovia workstation , in order to calculate diagnostic accuracy values . 
the diagnosis of bme was based on the presence of shades of green - yellow to orange - red on 2d maps and on the presence of shades of green on 3d maps . 
for any disagreement on the presence of bme , a consensus reading was appended , and the consensual results were used for further analysis . in the third reading session , the dect images were reevaluated randomly after 30days by reader 2 and reader 3 in order to determine the intraobserver agreement . finally , the quantitative analysis of dect numbers was performed by the same two readers on dect maps or standard ct images according to radiologist choice by achieving two circular regions of interest ( roi ) for each involved area , and using mean value for further analysis [ 13 ]  . 
in positive cases , normal adjacent bone in the spared segment was used for comparison . the site of bme ( involved bones ) and presence of associated imaging findings including ocl of the talus , meshed fractures , or stress fractures were recorded by the readers 1 3 la radiologia medica ( 2019 ) 124 : 10281036 1031 on mri and dect images with mri images available , we placed the rois at the location with highest density changes on dect maps according to the radiologist choice . table 2 clinical data of patients enrolled parameter value statistical analysis the statistical analyses were performed with statacorp . 
the receiver operating characteristic ( roc ) curve analysis and calculations of the area under the roc curve ( auc ) were used to evaluate the attenuation values from dect scans . 
the optimal cutoff value in hu showing the highest accuracy for distinguishing presence versus absence of bone marrow edema was determined by using youdens index and roc curves and used for calculation of diagnostic accuracy values . 
the sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) , and accuracy of dect for the diagnosis of bme were calculated on a per - patient basis as regards both qualitative and quantitative assessment . 
interobserver and intraobserver agreements for the qualitative analysis were calculated with weighted k - statistics as follows : poor agreement = less than 0.20 ; fair agreement = 0.200.40 ; moderate agreement = 0.400.60 ; good agreement = 0.600.80 ; very good agreement = 0.801.00. results clinical data of patients enrolled are summarized in table2 . 
bme was depicted in patients with recent trauma in 9 / 13 cases ( 69.2% ) and in 16 / 27 patients ( 59.2% ) suffering from chronic pain . the diagnostic accuracy values of dect on a perpatient analysis are summarized in table3 . 
on the sagittal reconstructed 1 - mm 2d dect color - coded image ( b ) , the bme areas are characterized by shades of green to yellow ( white arrows )  . 
on the coronal t1 - weighted image ( b ) , the ocl ( white arrow ) is hyperintense , whereas the bme appears slightly hypointense ( arrowheads )  . 
on the coronal 1 - mm reconstructed 2d dect color - coded map ( c ) , the ocl is imaged as a red area ( white arrow ) with surrounding bme appearing as shades of green ( arrowheads )  . 
on the coronal 3d dect image ( d ) , the bme is coded in green ( arrowheads ) , whereas the ocl , not directly recognized , is characterized by a subtle transparence ( white arrow ) fig . 
on the sagittal 1 - mm reconstructed 2d dect map ( c ) , the stress fractures ( white arrows ) can be recognized on the posterior aspect of the heel , associated with diffuse bme . 
 while our results confirmed similar values as regards the sensitivity ( 90% and 87.5% , respectively ) , we obtained higher values of specificity ( 86.6% for both readers )  . 
moreover , the qualitative assessment of 2d and 3d maps was superior to the attenuation measurements in the detection of bone bruises around the ankle joint ( 90% accuracy for reader 1 versus 87.5% accuracy of quantitative analysis )  . 
achieved different cutoff values , namely 80hu , 70hu , and 39hu , for ankle mortise , talar dome , and talar body / head , respectively [ 21 ]  . 
 the difference of ct numbers and of the relative cutoff with respect to previous studies [ 16 , 21 ] could be partly due to the use of different parameters ( 80kv150kv with a tin filter versus 80140kv ) and software employed . 
conversely , ct numbers may become less accurate going distally in the foot , because of the relative small size of mid - foot bones , with relative prevalence of cortical bone . 
however , in our experience , the analysis of ct numbers may help in selected cases to characterize subtle or equivocal imaging findings at the visual assessment . bone sclerosis represents a pitfall for vnca imaging in the ankle . 
bone sclerosis could cover a mild area of bme , especially in the subchondral regions ; an abnormal subtraction process in these cases would lead to the presence of normal ct numbers at the quantitative assessment . 
conversely , a mild sclerotic area can generate a local increase in ct numbers in a bme spared area , leading to a fp result in both the dect maps and on the quantitative assessment of ct numbers . 
the patient was experiencing pain relief at the time of the 3 - month imaging follow - up , showing the reduction in the bme of the talus and of the conspicuity of the ocl . in our study , we had a prevalence of patients suffering from chronic pain of the ankle with respect to patients with acute traumatic paalso , all the two cases of meshed fractures not diagnosed on x - ray examinations and the three stress fractures were correctly pointed out at dect . 
in these cases , the standard high - resolution ct images represent an additional tool for the assessment of cortical bone involvement [ 29 , 30 ]  . the ocls of the talus were correctly diagnosed in 4 of 5 cases , with good correlation with mri images . 
in this case , the possibility to evaluate the high - resolution ct images on multiple planes allowed us to rule out the presence of migrated fragments and to confirm the integrity of cortical bone . 
first , a relatively limited number of patients were included and larger studies are 1 3 la radiologia medica ( 2019 ) 124 : 10281036 1035 needed to corroborate our results . 
second , a comparative analysis of qualitative versus quantitative analysis was not performed ; as previously discussed , we believe that the quantitative analysis could represent an additional tool that helps radiologists to discriminate subtle imaging findings . 
diagnostic performance parameters and receiver operating characteristic ( roc ) curves of cesm were calculated and compared to those of ffdm or ffdm + bus ( mcnemars test )  . 
good agreement between tumor diameters measured using cesm and histopathology was observed ( spearmans rank correlation , r = 0.891 , p < 0.0001 ) , although this technique tended to produce an overestimation of the size ( + 7mm )  . conclusions cesm has high diagnostic accuracy and can be considered as a useful technique for the assessment of breast lesions . keywords contrast - enhanced dual - energy spectral mammography ( cesm ) digital mammography breast cancer iodinated contrast medium breast ultrasound * octavio prez - luzardo octavio.perez@ulpgc.es 1 breast imaging section , department ofradiology , grupo hospitalario san roque , c / dolores de la rocha , 35001laspalmasdegrancanaria , spain 2 breast surgery service , insular andmaternal andchild hospitals , plaza pasteur s / n , 35016laspalmasdegrancanaria , spain 3 research institute ofbiomedical andhealth sciences ( iuibs ) , university oflas palmas de gran canaria , paseo blas cabrera felipe s / n , 35016laspalmasdegrancanaria , spain 4 spanish biomedical research centre , physiopathology ofobesity andnutrition ( ciberobn ) , madrid , spain introduction an estimated 1.7 million new breast cancer cases are diagnosed each year , and it is also the cause of the highest number of cancer deaths among women ( around 570 , 000 deaths per year ) [ 1 ]  . 
the implementation of screening programs based on the performance of mammograms on the general population , together with the development of neoadjuvant chemotherapy and anti - estrogenic therapy , has contributed to decreasing mortality due to this disease [ 2 ]  . 
this allows ffdm to use a dual - energy subtraction algorithm to depict contrast - enhanced cancers that would otherwise be occult on standard unenhanced mammography [ 59 ]  . 
this makes cesm a very promising technique , since it combines the relative simplicity , low cost , and practicality of the mammography with the high sensitivity of the mri [ 912 ]  . cesm was launched in 2010 ( senobright cesm , general electric healthcare , usa ) , but it is still available only in a relatively small number of breast imaging centers , and it is estimated that only around 200 , 000 cesm examinations have been performed to date in both research and clinical settings [ 13 ]  . 
in fact , the available studies in which the diagnostic performance of this technique is evaluated include about 3000 patients in total ( average 160 patients / study ) [ 1420 ] , and a recent review has indicated that further studies with larger series of patients are needed to validate the initial literature [ 4 ]  . since cesm implementation in our hospital in 2013 , it has been employed routinely and a retrospective analysis of our imaging bank has been made . 
we have included 644 breast lesions studied by cesm ( 465 patients ) , and we have compared its performance to that of ffdm , alone or combined with breast ultrasound ( bus )  . patients andmethods patients andstudy design in our hospital , women without contraindications for the intravenous administration of an iodine - based contrast agent are eligible for cesm if : ( a ) a clarification of already identified abnormalities by mammography and / or bus is needed , namely mass lesions , areas of parenchyma distortion , focal asymmetries , or suspicious microcalcifications , or ( b ) they need further evaluation of heterogeneous dense breast parenchyma . 
since cesm implementation in our hospital , all patients have been informed that this is a novel technique and that their radiological data could be used in future scientific studies . 
only those patients from whom written informed consent was obtained , according to a model previously approved by the ethics committee of our institution , were included in this study . 
exclusion criteria included pregnancy , breast feeding , age less than 18 , known brca1 / brca2 carriers ( in whom mri is the only cost - effective technique for supplemental screening [ 21 ] ) , history of renal disease , and allergy to iodine . we collected and recorded the following information from each patient : age , personal and familial history of breast cancer , clinical symptoms ( nipple discharge , palpable mass , skin retraction , inflammatory breast ) , indication of cesm , and the presence of additional malignant lesions . 
all the imaging techniques were performed within a time window of maximum 8days . the senobright application allows the production of low and high energy image for each mammography view which in turn gives a recombined image that shows the enhanced structures . 
two minutes after injecting the iv contrast media , low and high energy images are acquired following this order : a cranialcaudal view of non - suspicious breast ; b mediallateral oblique view of non - suspicious breast ; c mediallateral oblique view of suspicious breast ; and d cranialcaudal view of suspicious breast . 
they did not even have the information about which breast was suspicious of malignancy / pathology , since the workstation always presents the images in the same way , independently of the order in which they were obtained . 
the radiologists were provided with a template in which they could categorize the information related to each lesion observed by cesm according to the qualitative morphological descriptors previously described for this technique , based on the bi - radsmri [ 23 , 24 ]  . 
subsequently , they assigned the lesion a 15 classification as proposed previously for cesm ( 1negative ; 2benign findings ; 3probably benign ; 4suspicious abnormality ; and 5highly suspicious of malignancy ) [ 23 , 24 ]  . 
breast density was categorized according to the recommendations of the american college of radiology [ 25 ] as class a : fatty ; class b : scattered fibroglandular density ; class c : heterogeneously dense ; or class d : extremely dense . in the second stage , two other radiologists with more than 10years of experience ( > 12 , 000 mammograms / year ) reviewed all the discrepant cases ( 9.8% of cases ) that is , when a given descriptor was scored differently by the three radiologists [ 23 , 24 ] and provided a consensus opinion . 
 finally , the principal investigator correlated all data between both techniques and was also responsible for relating the imaging results to the gold standard employed as reference . reference standard procedures used toaddress true disease status to assess the current disease status of each patient disease status of each patient , different strategies were followed , depending on the type of lesion [ 3 , 26 , 27 ]  . when available , histopathological result , from surgery or biopsy , was used as the reference standard . 
in the case of cysts , an additional ultrasound was performed in which an aspiration of the contents of the cyst was done , in order to prove its non - solid nature . 
when the result of the histopathological analysis of this aspirate revealed the presence of atypical cells , the lesion was similarly biopsied , and the histopathological result was considered the gold standard . 
 in the cases in which superimposed fibroglandular tissue gave rise to a suspicious lesion , an additional localized mammography projection of the area and also a supplementary bus study oriented to that area were performed . 
in these recalls , if the images remained unchanged , the patient was discharged and in the other eventuality a biopsy of the suspicious area was performed , and the histopathological result obtained was considered as the gold standard . 1 3 la radiologia medica ( 2019 ) 124 : 10061017 1009 the biopsy was usually performed immediately after cesm examination . 
however , when the patients were referred for the cesm imaging for cancer staging ( 84 patients , 118 lesions ) , they had previously undergone the biopsy before cesm examination . 
however , as described above , the radiologists participating in this study did not have this information when they retrospectively reviewed the images . for evaluation of the diagnostic performance of ffdm , ffdm + bus , and cesm , lesion images classified 13 were considered as benign and 45 as malignant . all histopathological diagnostics were performed by the same certified pathologist with 20years of experience in breast cancer . 
immunohistochemical determination of estrogen receptor ( er ) and progesterone receptor ( pr ) was carried out , and her - 2 / neu status , tumor grade , and nodal involvement in malignant cases were also assessed . statistical analysis all the statistical analyses were done using graphpad prism v6.0 ( graphpad software , ca , usa )  . 
using the benignmalignant cutoff value , sensitivity , specificity , positive predictive value ( ppv ) , and negative predictive value ( npv ) were calculated for the three imaging modalities . 
the mean difference between the image and the histological results was calculated and related to the interval in which 95% of the calculated differences ( limits of agreement , loa ) were found . 
the differences between the imaging techniques and histological measurements did not follow a normal distribution ( kolmogorovsmirnov test ) , and therefore , nonparametric statistical tests were used in the subsequent analyses involving this variable ( mannwhitney u test or the kruskalwallis test [ for comparisons of more than two groups ] ) [ 9 ]  . 
 a total of 283 patients ( 367 lesions ) had a previous ffdm study , and 182 patients ( 277 lesions ) had a previous complementary ffdm + bus study . 
therefore , 367 lesions were seen by ffdm and cesm , and 277 lesions were seen by ffdm + bus and cesm . we employed the histopathological diagnosis of the surgical piece as the gold standard for the 426 malignant lesions obtained from the patients who underwent surgery . 
of these non - enhancing malignant lesions , three belonged to patients bearing breast prostheses ( 1 infiltrating ductal carcinoma , 1 ductal carcinoma insitu , and 1 invasive lobular carcinoma ) , and the remaining lesions were : infiltrative intraductal carcinoma ( 6 lesions ) ; intraductal carcinoma insitu ( 3 lesions ) ; angiosarcoma ( 2 lesions )  . 
seven of the 14 non - enhancing lesions were located in breasts that were classified as heterogeneously dense ( n = 3 , category c ) or extremely dense ( n = 4 , category d )  . 
for the 10 remaining benign lesions that were enhanced by the contrast medium , there did not seem to be any particular characteristic that explained the reason for said uptake . 
however , cesm did not improve the negative predictive value with respect to any of the other two diagnostic imaging methods . a similar trend was observed for the roc curves . 
the comparison among the auc values was statistically significant in all the cases : p < 0.001 for the comparison between ffdm and ffdm + bus ; p < 0.001 for the comparison between ffdm and cesm ; and p < 0.05 for the comparison between ffdm + bus and cesm . table3 shows only 63 lesions were misclassified by cesm ( 9.7% of the total series )  . 
compared to previous studies with fewer numbers of cases [ 3 , 1518 , 20 , 23 , 2830 ] , it practically quadruples the average number of lesions reported to date ( a range of 49261 lesions , an average of 158 lesions per study )  . several authors have demonstrated the similarity of the performance of cesm and contrast - enhanced breast mri [ 5 , 15 , 29 , 31 , 32 ]  . 
due to the similarities between the two techniques , it has been proposed that the bi - rads - mri could be used to characterize the cesm image of the breast lesions [ 23 , 24 ]  . 
the underlying causes of this misdiagnosis were related , in most cases , either to characteristics of the patients or to lesions , or to diagnostic limitations of digital mammography in general and of cesm in particular ( such as bearing breast prostheses )  . 
in our series , 87% of the misdiagnosed cases were included in one of the following groups : ( a ) lesions in high density breasts , which are more difficult to diagnose either by ffdm or cesm [ 17 ] ; ( b ) lesions in small breasts of thin , middle - aged women [ 9 , 35 , 36 ] ; ( c ) lesions in patients bearing breast prostheses [ 6 , 8 ] ; ( d ) belonged to special benign pathological conditions that have been reported to simulate malignant pathology , such as in the case of diabetic mastopathy [ 37 ] ; or ( e ) corresponded to microcalcifications without associated mass [ 28 , 38 ]  . 
4 prediction of tumor size by mammography ( left column ) , mammography plus breast ultrasound ( center column ) and cesm ( right column ) : ( upper panels ) box plots illustrating the median size difference between the imaging technique ( s ) and histopathology , with the corresponding interquartile range and whiskers from the 5th to the 95th percentile ; ( middle panels ) scatterplots and spearmans correlation coefficient of maximum tumor diameter measurements between the imaging technique ( s ) and histopathology ( lower panels ) ; bland altman plots illustrating the size difference between the imaging technique ( s ) and histology compared to the histological size . 
the recombined image shows the contrast enhancement of the two mass lesions without an enhancement of the microcalcification area ( which was finally part of a moderately differentiated infiltrative ductal carcinoma ) fig . 
the recombined image of cesm ( b ) does not clarify anything regarding the low energy image ( a ) , since the mammary parenchyma diffusely uptakes contrast media bilaterally , without highlighting the lesion in a specific way , and without a clear cause that may explain this behavior . 
it allowed detection of even some very difficult lesions , such as a carcinoma hidden behind a fibroadenoma , which has already been published by our group as an interesting case report [ 39 ]  . 
 this means that our results support the previous studies , indicating that cesm has high diagnostic accuracy , even higher than the combination of two complementary imaging techniques such as ffdm + bus . 
in any case , our results indicate that this technique can be considered as a useful imaging technique for the initial assessment of breast lesions , at least in the clinical setting . 
in our experience , cesm would have allowed us to rule out malignancy directly in up to 13.8% of cases ( 89 benign lesions out of a total series of 644 lesions were correctly classified as benign by cesm ) , avoiding delays in diagnosis ( and the associated stress ) , as well as additional costs . additionally , our results also indicate that tumor measurement with cesm has good correlation rates with the histopathologically determined size . 
 both the correlation indexes and the concordance percentages were similar or even higher than those described for the mri [ 4246 ] and , despite the overestimation of the size , definitely superior to that of the ffdm and ffdm + bus , as we have previously reported [ 9 ]  . 
preoperative accurate estimation of tumor size is very necessary for adequate surgical planning , and several authors have proposed that the most accurate measurement is obtained by mri [ 42 , 43 , 47 ]  . 
 according to the results of this study , cesm presented the best prediction rates of the actual size of the tumor piece , so it can also be considered a suitable imaging technique for this purpose . this study has several limitations . 
in addition , when analyzing the results , we found that there was a low representation of clusters of microcalcifications , as well as very few cases of carcinomas insitu . 
additionally , it should be also taken into account that certain patients , particularly those with benign lesions , were asked to participate voluntarily in the study in order to also observe the behavior of the lesions with low suspicion of malignancy , which might introduce a new case selection bias . 
it is also noteworthy that in a retrospective study , like the one presented here , it is not possible to directly compare the diagnostic performance of cesm with traditional imaging tools , such as magnified views , digital tomosynthesis of the breast , or targeted ultrasound . 
this may lead to bias in the results , although there are still no data to indicate the extent to which the menstrual cycle influences the uptake of contrast in cesm . conclusions our data allow the conclusion that the diagnostic performance of cesm is higher than that of ffdm or ffdm + bus . 
all the bodies were pmct - scanned prior to autopsy , and internal putrefactive state was determined using the radiological alteration index ( rai ) by a radiologist with expertise in forensic radiology and a forensic pathologist trained in forensic imaging . 
after pmct scans , grade of external putrefaction ( gep ) was assigned during the external examination and the complete autopsy was performed by forensic pathologists . results the pmct images evaluation revealed that the rai index was > 61 in all bodies , corresponding to a moderatemassive presence of putrefactive gas . 
the gas grade was > ii in correspondence of the major vessels , heart cavities , liver parenchyma , vertebra l3 and subcutaneous pectoral tissues , and varied from i to iii in correspondence of the kidney . 
cadaveric external examination revealed the presence of advanced transformative phenomena , with a gep3 and gep4 in most of the cases , with body swelling , eyes and tongue protrusion , body fluids expulsion and fat liquefaction . conclusion radiological imaging by pmct as an adjunct to autopsy in advanced decomposed bodies represents a useful tool in detecting post - mortem gas , even in very small amounts . 
following the most recent advances , ct technology is today frequently requested as a complement before conventional autopsy , or in some cases as a supplement , for non - invasive post - mortem diagnosis [ 24 ]  . 
 pmct offers a detailed visualization of anatomical structures , injury patterns or localization of foreign bodies prior to dissection [ 5 ] , allowing the study of body parts or areas that are not routinely dissected during a standard autopsy , such as the viscerocranium , craniocervical junction , larynx , shoulder girdle , pelvis , extremities and soft tissue of the back [ 69 ]  . 
furthermore , while the traditional autopsy determines a destruction of the residual findings , pmct allows preserving images for re - evaluation and reinterpretation over vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 10181027 1019 time , also with the aid of post - processing 3d reconstructions . 
pmct represents a useful procedure in many forensic scenarios that are often very difficult to interpret only with conventional autopsy [ 1014 ] , as in the case of decomposed bodies , because putrefaction processes can impressively alter the appearance of corps and consequently add more difficulty in determining the cause of death . 
in fact , body decomposition is determined by a combination of fermentation processes by gastrointestinal flora , aggression of external bacteria and other micro - organisms , and autolytic digestion of body tissues by endogenous enzymes . 
the rate and extent of post - mortem changes and decomposition processes are quite variable and are dependent on the cause of death and the external environment [ 15 ]  . 
as a consequence , examination of a putrefied body represents a remarkable challenge for the forensic pathologists [ 16 , 17 ]  . gas formation is a hallmark feature of putrefaction , since gaseous bloating in advanced decomposed bodies is determined by vascular and intra - parenchymatous gases . 
pmct allows to detect exact localization of post - mortem gas in the whole body , even in very small amounts , and to distinguish between normal decomposition processes and pathologic gas collections that may have contributed to causing the death , such as air embolism , pneumothorax or pneumoperitoneum [ 1829 ]  . the aim of the present study is to report radiological findings and features in advanced decomposed bodies obtained by pmct , even if possible with autopsy correlation . 
an overview of the radiological cadaveric modifications in different anatomical sites , due to natural decomposition processes that can be mistaken for pathologic processes , is also offered . materials andmethods subjects this retrospective descriptive multicentric study was conducted between may 2013 and november 2016 , including forensic cases selected among those admitted to the medicolegal centres of rome , foggia and ferrara ( italy ) by the local enquiring authorities to ascertain the cause of death . 
eleven bodies ( cases 111 ) were exhumed on the order of a judge , while 30 bodies ( cases 1241 ) were found at the crime scenes ( table1 )  . 
cases 111 consisted of subjects deceased in a hospital with an ascertained pmi as obtained from clinical records , while cases 1241 were found dead after a presumptive pmi ( sd ) , reconstructed on the base of testimonies and forensic considerations . 
 pmct scans were performed on a 64 - section ct system ( somatom sensation cardiac 64 - slice scanner ; siemens , forchheim , germany ) according to standardized scanning protocol : tube voltage 120kv ; tube current 250mas ; section thickness 0.6 mm ; reconstruction interval 0.5 mm ; gantry rotation time 0.5 s ; and pitch 0.9. 
pmct data were transferred to a workstation for post - processing images reconstruction , performed with a slice thickness of 1.25mm in increments of 0.7mm , using soft tissue deconvolution algorithm ( b20 kernel ) and bone - weighted deconvolution filter ( b46 kernel )  . 
images were finally analysed using a viewing software ( osirix v.5.8.2 32 - bit ; pixmeo , geneva , switzerland ) , calculating two - dimensional sagittal and coronal reformations and volume rendering ( vr ) reconstructions . the present study was conducted in accordance with the helsinki declaration . 
as all the examinations were parts of a complete forensic examination ordered by the local enquiring authorities , no specific consensus by the relatives was necessary prior to the realization of all pmct ; after the examination , all results were analysed anonymously . image analysis images viewing and assessment was performed by 2 radiologists with expertise in forensic radiology ( 2 and 30years of experience , respectively ) and 2 forensic pathologists trained in forensic imaging ( 3 and 35years of experience , respectively )  . 
to promote consistent assessments , a concordance was preliminarily tested between the investigators . the internal putrefactive state was determined by the aforementioned observers using the radiological alteration index ( rai )  . 
rai was determined by pmct in 7 selected sites , including the major vessels ( left innominate vena and abdominal aorta ) , selected bones ( vertebra l3 ) , selected organs ( heart cavities , liver parenchyma and vessels , and kidney parenchyma ) and subcutaneous tissues and muscles ( subcutaneous pectoral tissues ) , according to the standardized protocol by egger etal . 
in particular , the grade of gas presence ( 0 , i , ii or iii ) was determined for the major vessels and selected bones ( one gas bubble to completely filled with gas ) , and for the selected organs , subcutaneous 1 3 1020 table 1 description of the main characteristics of 41 advanced decomposed bodies including sex , age , cause of death and post - mortem interval ( pmi ) cases age ( years ) cause of death pmi ( days ) la radiologia medica ( 2019 ) 124 : 10181027 haemorrhagic shock after femur fracture cardiogenic syncope heart failure by bleeding and anaemia acute bleeding in chronic subdural haematoma heart failure by anaemia haemorrhagic shock after femur fracture bleeding in non - hodgkins lymphoma haemorrhagic shock cardiogenic pulmonary oedema multi - organ failure septic shock ischaemic cardiomyopathy heart failure in alcohol intoxication traumatic brain injury fatal firearm injury fatal firearm injury asphyxia heart failure fatal firearm injury heart failure in chronic alcohol abuse heart failure arrhythmia in cocaine abuse dismemberment after fatal strangling accidental train falling injury drowning fatal firearm injury traumatic brain injury cardiogenic pulmonary oedema cardiogenic pulmonary oedema asphyxia drowning drowning drowning drowning drowning asphyxia slaughtering cardiogenic pulmonary oedema hanging frostbite cardiogenic pulmonary oedema 634 89 7 1 5 1 90 5 4 1 14 5 33 1 120 10 30 5 5 1 4 1 4 1 4 1 180 90 30 5 4 1 12 2 6 1 12 2 25 5 25 5 150 30 15 5 13 2 13 2 35 5 7 1 6 1 25 5 4 1 pmi post - mortem interval . 
cases 111 : ascertained pmi as obtained from clinical records ; cases 1241 : presumptive pmi ( sd ) reconstructed on the base of testimonies and forensic considerations tissues and muscles ( one gas bubble to extensive emphysema )  . 
once a grade was assigned for each site , according to the corresponding scores , the rai was calculated . conventional autopsy after pmct scans , the complete conventional autopsy of each body ( examination of the cranial , thoracic and abdominal cavities ) was performed by forensic pathologists . 1 3 la radiologia medica ( 2019 ) 124 : 10181027 1021 the grade of external putrefaction ( gep ) was assigned by the forensic pathologist during the external examination of the bodies , according to the standardized classification of maujean etal . 
 [ 31 ] , based on the putrefaction signs described in the forensic literature : sites heart cavities table 2 gas grade and score determined for 7 selected sites gas grades scores number of cases gep1 ( beginning ) : green skin discoloration was preliver parenchyma and vessent in the abdomen area ; gep2 ( moderate ) : detachment of the skin and / or blisters containing reddish purplish serous liquid occurred at the extremities and in the sloping regions ; gep3 ( advanced ) : putrefactive gases produced body swelling , eyes and tongue protrusion and body fluids expulsion ; gep4 ( major ) : fat liquefaction and / or darkening colouring of the integument and drying of the body extremities were observed ; gep5 ( mummified ) : drying of the whole body with sels left innominate vena abdominal aorta kidney parenchyma leather - like skin was evident . vertebra l3 a complete autopsy report was provided in all the cases , and causes of death were subsequently investigated by histological and toxicological examinations . 
in particular , the correlation between gep and pmi , and rai and pmi has been studied ; the correlation between gep and rai has also been investigated . results the pmct images evaluation and the analysis of putrefactive internal state revealed that , in all the examined cases , gas grade was > ii in correspondence of the major vessels ( left innominate vena and abdominal aorta ) , heart cavities , liver parenchyma , vertebra l3 and subcutaneous pectoral tissues ; in correspondence of the kidney , the gas grade varied from i to iii ( table2 )  . in the selected 7 sites , the rai index was > 61 in all bodies ( interval 61100 , median 85 ) ( table3 )  . 
in particular , intracranial gas accumulation was observed in all cases , distributed on the top of the calvarium and in the vascular structures of the posterior cranial fossa ; in 22 / 41 ( 54% ) cases , gas accumulation filled more than 50% of the whole intracranial space . 
small gaseous collections were ubiquitously distributed within the brain parenchyma , with a swiss - cheese aspect ; brain volume was reduced in all cases , placed in the sloping region of the calvariuin all cases , loss of definition of the greywhite matter junction and effacement of the sulci and ventricles were detectable . 
 as a consequence of the degenerative processes , adipose degeneration and fat liquefaction associated with multiple gaseous collections were observed in all examined bodies , with a marked fluid collection in the pleural and peritoneal space . 
soft and connective tissues were collapsed and showed gaseous degeneration , notably in correspondence of periskeletal muscular structures or in elective areas , i.e. , gaseous ballooning of the scrotuparenchymatous organs were not completely recognizable in shape and appearance , liquefied and characterized by the collapse of the residual parenchymas . 
grade of external putrefaction ( gep ) assigned in all the examined bodies resulted gep3 ( 11 / 41 , 27% ) and gep4 ( 18 / 41 , 42% ) in most fig . 
five out of 41 ( 12% ) bodies presented widespread phenomena of aggression by the fungal 1 3 1024 la radiologia medica ( 2019 ) 124 : 10181027 greyish pink colour , colliquated , homogenized and with pultaceous consistency . 
in the thoracic cavity , the pleural space showed accumulation of serosanguinous putrefactive fluid or resulted covered with a mixture of brownish - blackish putrefactive liquid and fine yellowish granular residue ; the lungs appeared collapsed towards the mediastinum , with pleura raised in bubbles , soft and pasty at palpation . 
8 autopsy findings : dura mater detached from the endocranium ( a ) ; brain colliquated , homogenized and with pultaceous consistency ( b ) ; heart flattened and empty , strongly reduced in form and consistency ( c ) ; intra - abdominal putrefactive phenomena ( d )  . 
furthermore , in advanced putrefaction , the texture can be completely dissolved , leading to liquefaction of entire organs , as shown in the images analysis carried out in the present study [ 9 , 16 , 19 ]  . 
these alterations often lead to misinterpretation of the radiological findings and to difficulties in determining the cause and manner of death . the knowledge of the standard pmct findings following the post - mortem changes and the decomposition status is important when interpreting pmct images , because decomposition has a great impact on the imaging features . 
the aetiology of post - mortem gas detected on pmct , in fact , can be multifactorial and not necessarily related to putrefaction [ 6 , 9 , 13 , 1621 ]  . decomposition gas normally occurs within 2448 - h postmortem and is typically produced by intestinal flora [ 20 ]  . 
 gas can be also detected in many other conditions such as sepsis , ischaemia and tissue damage , which is associated with rapid decomposition and putrefaction [ 32 ]  . 
intravascular gas could also be produced during cardiopulmonary resuscitation ( cpr ) : chest compression causes the vaporization of dissolved gas in blood and the rupture of pulmonary vessels , allowing the air to enter the pulmonary vein and reach the systemic circulation . 
furthermore , in hospitalized subjects , the bronchovenous fistulas caused by endotracheal intubation and the alveolar rupture caused by positive - pressure ventilation barotrauma may lead to intravascular gas entry [ 3335 ]  . on the other hand , putrefactive gas should not be mistaken for pathologic gas collections that may have contributed or caused the death , such as air embolism , pneumothorax or pneumoperitoneum [ 2226 ]  . in all the above - mentioned cases , asymmetric or focal gas collections should be considered suspicious and related to pathological or traumatic causes . 
in our study , an asymmetric soft tissue gas distribution was detected in perforating gunshot wounds through the chest in 2 cases , but these findings were clearly related to the underlying traumatic injury . 
in fact , gas presence was reported in many cases of penetrating trauma with associated lacero - contusive foci and air bubble at the entry site [ 19 , 36 ]  . radiological imaging by pmct , as an adjunct to conventional autopsy in advanced decomposed bodies , has been proven to be a useful tool in detecting post - mortem gas localization , even in very small amounts . 
in all the examined cases , a symmetrical ubiquitous gas accumulation has been detected within the heart cavities , vascular system , body cavities , parenchymatous organs , soft tissues and bones , as shown in results . our results have demonstrated in all cases ubiquitously moderate - diffuse presence of gas ( grade ii to iii ) , particularly in correspondence of soft subcutaneous tissues , vertebral body l3 and cardiovascular structures such as heart cavities , abdominal aorta and left innominate veinterestingly , parenchymatous organs have shown a different distribution pattern , as in correspondence of liver ( grade ii to iii ) and kidney ( grade i to iii ) a different gaseous amount has been found . 
this partial exception could be explained by the fact that the perirenal capsule protects more the renal structure from the putrefactive phenomena , coinciding with the autoptic data confirming that kidney moderately resists putrefaction . furthermore , according to pmct standardized imaging assessment and the subsequent statistical analysis conducted on gep and rai values , a strong positive correlation between the external and internal grade of putrefaction has been established . 
therefore , from the present study , the combined use of gep and rai resulted useful in the assessment of the putrefactive state in advanced decomposed bodies , providing reliable data prior to autoptic investigation and supporting the forensic pathologist in the evaluation of cadaveric post - mortem changes [ 37 ]  . conclusion according to this standardized approach , an objective description of the post - mortem transformative phenomena was offered . 
assessment of external ( gep ) and internal ( rai ) grade of putrefaction is even more important , considering its decisive role as an aid in discriminating the various causes of death , particularly confused by the overlap of putrefactive phenomena . 
scientific organizations supporting clinical radiologists and medical physicists have published guidelines reporting parameters useful to quantify the radiation output and to assess patient dose . conclusions our research revealed that there is not a shared interpretation of patient exposure information to be included in radiological report . 
in this respect , this directive should emphasize the need for justification of medical exposure , including the exposure of asymptomatic individuals , and should strengthen the requirements concerning information to be provided to patients , the recording and reporting of doses from medical procedures , the use of diagnostic reference levels and the availability of dose - indicating devices . 
after all , iaea basic safety standards also stressed the issue that patients have to be informed about benefits and radiation risks associated with radiological procedures [ 2 ]  . secondly , because of the increasing number of radiological examinations , such as interventional radiology ( ir ) procedures , or ct scansas reported , for example , by the organization for economic co - operation and development ( oecd ) [ 3 ] ( fig.1 ) , the knowledge about patient doses in medical procedures has become an important topic for population dose assessment [ 46 ]  . information included in radiological report should make aware the patient about radiological risk index associated with an increased probability of cancer over the remaining lifespan , arising from a medical exposure . 
the doses involved in diagnostic radiology , indeed , are below the minimum threshold dose for deterministic effects ( 100msv ) , but a potential dose for stochastic effects , i.e. , cancer induction and genetic changes . 
this radiation risk index is critically dependent on the age and sex distribution of the exposed population . without any other specification relative to art.58 point b , its implementation in european member states could lead to choice among four types of information : patient exposure , understood as a pure physical quantity related to x - ray tube output and thus a machine parameter ; absorbed dose , a physical quantity more complex than exposure , requiring a professional evaluation from a medical physics expert ( mpe ) starting from machine parameters ; effective dose , a radiological protection quantity providing a single number proportional to the radiobiological fig . 
 ( source : : / / data.oecd.org / healt heqt / compu tedtomog raphy - ct - scann ers.htm ) 1 3 la radiologia medica ( 2019 ) 124 : 783793 detriment from a particular type of radiation exposure [ 7 ]  . relative radiological risk index expressed as an average effective dose range , but identified using symbols or comparative scales [ 810 ]  . especially regarding the most known dosimetric quantity effective dose , scientific community knows that it results inappropriate for the estimation of individual risk , as it does not consider health status , age and sex [ 11 ] , and it is calculated using uniformly irradiated phantoms ( a different setting respect to routine radiological examinations )  . 
moreover , icrp 103 stated that the effective dose is calculated for a reference person and not for an individual ; for individual retrospective dose and risk assessment , individual parameters and uncertainties have to be taken into account , with the direct involvement of an mpe [ 7 ]  . the aim of this study was to properly define the information regarding patient exposure to ionizing radiations in the radiological report , according to the european directive 2013 / 59 / euratom ( eu 2013 / 59 art.58 ( b ) )  . 
to better understand the meaning of quantities analyzed later in the paper , a brief description is given in the following paragraphs . radiation dose metrics it is clear to scientific community that patient exposure does not mean patient dose , but it suggests some useful information to evaluate dose released to patient . using laymans term , exposure can be thought as how much is it raining ? , absorbed dose as how much did you get wet ? and estimated dose as what are the chances of getting a cold due to getting wet in the rain ? [ 12 ]  . patient exposure the information on exposure is a machine - related parameter representing the device output measured using specific tube settings ( tube voltage , tube current , exposure time , field of view , sourcesurface distance and so on )  . 
x - ray tube output is used to calculate incident air kerma ( ki ) , i.e. , the energy absorbed in air per mass unit on beam axis ( definition valid in diagnostic radiology energy range )  . 
a dose index representing incident air kerma can be easily defined for each radiological modality , using technical devices added to the equipment . this index is not the patient absorbed dose , but it could be correlated with patients features ( the thickness ) when , for example , an automatic exposure control system is activated ; anyway , it is related to patient dose . 
moreover , the dose index has to be previously validated by the mpe involved in radiological procedures optimization process and in quality assurance program . patient absorbed dose energy transported by radiation and then released to patient is the absorbed dose , i.e. , the energy imparted to matter ( the patient ) per mass unit . 
the biological effect of radiation is enclosed in the quantity equivalent dose , defined in a tissue t , given a physical absorbed dose dt released from different types of radiation . patient effective dose in order to take into account the contribution to the overall detriment derived from different organs radiosensitivity , icrp [ 13 ] introduces the effective dose ( expressed in millisievert , msv ) as the sum of products of tissue weighting factors and absorbed dose inside tissues and organs of the body . 
tissue weighting coefficients wt are derived from epidemiologic studies on population exposed to high dose of radiation and represent reference values for humans averaged over both sexes and all ages , thus not relating to characteristics of each individual [ 7 ]  . 
 the european report rp 154 [ 11 ] also confirms that effective dose should be used for comparison purpose only in order to assess , for example , doses from different diagnostic procedures performed in different hospitals and countries . 
 furthermore , rp 154 affirms that effective dose is not recommended for epidemiological evaluations . different softwares are available allowing to calculate effective or equivalent patient dose in medical exposure using different human phantoms and calculation models ( pcxmc [ 14 ] , ct - expo [ 15 ] , virtualdose [ 16 ] )  . 1 3 786 la radiologia medica ( 2019 ) 124 : 783793 relative radiological risk index in order to make aware patients about differences between radiological procedures and their associated risk , a relative radiation risk index could be included in clinical report . 
effective dose ranges are based on reviews of the current literature , us and european diagnostic reference level publications , and experience of medical physicists and radiologists . two examples of relative radiological risk index employed by radiologists as appropriateness criteria are described : relative radiation level ( rrl ) comparative scale [ 8 ] developed by the american college of radiology ( acr ) appropriateness criteria and dose classes system as reported by italian reference guidelines on diagnostic imaging [ 9 ]  . 
recently the european society of radiology provided an iguide reporting the rrls scale for common diagnostic procedures [ 10 ]  . acr suggested a comparative scale assigning a symbol for each radiation level associated with an effective dose range ( see table1 )  . 
a similar scale is given for pediatric patients [ 8 ]  . the italian association of medical radiology ( sirm ) [ 9 ] together with national health institutions also published a dose classification system for radiological procedures ( see table2 )  . a good approach for communicating medical exposure risk to patients in a radiological examinations , using effective dose or related levels , could be to quantify risk as the time needed to receive the same effective dose from natural background . 
table3 summarizes this comparison , assuming a background exposure of 3msv / year as reported by peck and samei [ 17 ]  . another way to convey the risk is to consider a chest radiography as a reference and then comparing a radiological procedure with the equivalent number of chest radiographies [ 9 ]  . international legislative background currently not all european member states have transposed the eu directive 2013 / 59 yet . in may 2016 , the european community approved a tender project to monitor the transposition of the basic safety standards ( bss ) directive ( eu 2013 / 59 ) into member states national legislation and to support its implementation in the medical area . 
 ( adapted from [ 8 ] ) relative radiation level adult effective dose estimate range ( msv ) example examination ultrasound ; mri limbs and chest radiography pelvis radiography and mammography abdomen ct and nuclear medicine bone scan abdomen ct with or without contrast and whole - body pet total body ct with contrast ; stent or shunt placement ; and interventionl radiology procedures table 2 risk classification of radiological procedures as provided by italian guidelines . 
one of the projects objectives was to prepare and perform a survey collecting information on member states strategies and plans for bss transposition ( medical area )  . regarding the effort of countries to transpose the art . 
 no more specification about which country and dose metrics definition was reported . in october 2014 , the board of heads of european radiological protection competent authorities ( herca ) approved an action plan [ 19 ] in relation to the transposition and implementation of eu 2013 / 59 . 
at the time of writing , the action 4 is still going on . since before the entering into force of new directive , some european countries provided in their legislation the inclusion of dosimetric information in radiological report , also specifying which dose unit should be indicated . the french legislation [ 20 ] , for example , established in 2006 that useful information for assessing dose received by patient ( [ ] les informations utiles lestimation de la dose recue par le patient [ ] ) has to be included in the radiological report ( more details in table4 )  . 
 french nuclear safety authority ( asn ) [ 22 ] is working with government on production of ministerial orders and on implementation texts of eu 2013 / 59 . in england , the department of health and social care has published in june 2018 the guidance to the ionising radiation ( medical exposure ) regulations 2017 [ 23 ]  . 
the sociedad espanola de fisica medica ( sefm ) during the vii national meeting on radiation protection in medical field ( april 2018 ) debated about eu 2013 / 59 transposition outcomes . 
in particular , sefm highlighted that the patient dose management , strongly hoped by the new directive , has stoked the business on dose recording systems [ 26 ]  . in italy , national law on radiation protection ( medical exposures ) [ 27 ] orders that the responsible practitioner and the employer , according to their own authority , provide for registration of every single radiological procedure although in a synthetic way ( article 12 )  . 
in particular , aifm contributed to insert in a regional health plan the population dose monitoring from medical exposures through radiological information system ( ris ) , as well as the faculty to inform patients about dose received by radiological examination . furthermore , because of the increasing spread of dose monitoring systems , aifm instituted in 2014 a working group with the aim of establishing reference guidelines on acceptance test of dose monitoring systems , claiming the need for a coherent , complete and uncorrupted migration of information from radiology modality to the software . 
the group published at the end of 2016 a complete report on general aspects of dose monitoring systems and on essential dose parameters to be collected for each modality [ 29 ]  . 
 the french decree [ 21 ] indicates that information relating to patient exposure , executed procedures and all information useful to assess dose received by patient , shall be included in radiological report . 
french legislation system was so sensitive to this issue maybe because of the great ct number per 1000 inhabitants in 2016 ( the greatest among the five countries considered ) [ 3 ]  . english regulations [ 23 ] in addition to what provided by france specify that such information might enable the estimation of the effective dose to the individual to be made at a later date , if necessary . german x - ray ordinance [ 24 ] only refers to dosimetric information recoding for each radiological procedure , without specifying dose metrics . 
article 58 ( b ) is not yet embodied in national law as well as in spain and italy . our perception is that the transposition process is recently started in europe . 
it is important that manufacturers specify the maximum uncertainty of dosimetric index . step 3 the third step is to transfer dosimetric parameter related to each radiological procedure to physicians report . 
 any diagnostic modality can provide exposure data in a different way : dose screen image ( fig.3 ) : a report produced by the device , containing exposure parameters ( kv , mas , etc . ) and a dosimetric index ( ctdi , dlp , dap , etc . ) if available ; image header dicom ( fig.4 ) : the file associated with image , containing metadata related to exposition ( for example , in a ct series it collects a cumulative dose information )  . 
data are stored in image header as attributes of standardized dicom tags . modality performed procedure step ( mpps ) ( fig.5 ) : a notification message sent from the modality to a dicom server in order to trace the procedures status . radiation dose structured report ( rdsr ) ( fig.6 ) : the document - like report able to collect both dosimetric and non - dosimetric ( geometry , patient name , patient id , etc . ) data for each exposure event . 
efomp also recommends that medical equipment used for interventional radiology equipment used for computed tomography not mandatory for equipment installed before february , 6 , 2018 new medical radiodiagnostic equipment producing ionizing radiation mandatory for all machines mandatory for all machines mandatory capability to transfer relevant not mandatory for equipment not mandatory for equipment mandatory installed before february 6 , 2018 installed before february 6 , 2018 table 5 extract summary of art.60 [ 1 ] feature requested a device or a feature informing of quantity of radiation produced by the equipment during the procedure a device or a feature informing at the end of the procedure , of relevant parameters for assessing the patient dose parameters for assessing patient dose to the record of the examination 1 3 la radiologia medica ( 2019 ) 124 : 783793 790 table 6 efomp recommended parameters for eu directive transposition of art.60 ( adapted from [ 32 ] ) modality metrics computed tomography dose index ctdivol [ mgy ] or ctdiw [ mgy ] indicating phantom size size - specific dose estimatesssde [ mgy ] dose length productdlp [ mgycm ] interventional radiology air kermaarea product kap [ gycm2 ] general fluoroscopy planar x - ray mammography cbct * dental radiography ( panoramic projection ) * * reference air kerma [ mgy ] air kermaarea product kap [ gycm2 ] air kermaarea product kap [ gycm2 ] entrance surface air kermaesak [ mgy ] incident air kermaiak [ mgy ] mean glandular dosemgd [ mgy ] air kermaarea product kap [ gycm2 ] * incident air kermaiak air kerma length product * metrics recommended by aifmsirm etal . 
3 an example of dose screen image , from ct modality conclusion the literal transposition of art.58 ( b ) indicates that patient exposure information and not patient dose information shall be added in radiological report . 
on the other hand , the mpe can estimate individual patient dose finalized to provide the radiological risk starting from machine parameters . anyway , in this review we considered four possible scenarios of art.58 ( b ) implementation : patient exposure data on the report . different organizations suggested the recommended machine parameters / exposure data for each radiological modality ; this information will provide a record for retrospective calculation of patient doses when needed . 
if requested by patient , mpe can assess the patient absorbed dose . patient absorbed dose on the report . in this scenario , the mpe should always be involved and , as responsible of patient dose estimation , he has to sign the radiological report . 
absorbed dose calculation is based on complex process that needs deep knowledge of software , phantoms and calculation models . effective dose and relative radiological risk index on the also allow : report . these values , expressed in units of millisievert ( msv ) , are based on effective doses and give a very approximate ment : assessment of radiological risk without taking into account patient specificity and then individual risk . 
in spite of this , we have to acknowledge that the use of relative radiological risk index is advantageous because it makes easy patient communication and understanding of risk : patients prefer symbols and scales to indicate risk [ 37 ]  . as stated and analyzed so far , the authors wish that italian implementation of art.58 ( b ) will provide exposure information on clinical report . the machine parameters are the most simple solution to implement art.58 ( b ) in radiological practice . 
they indeed , derived directly from equipment , are objective quantities that do not require further evaluation and may be easily transfer to physicians report , as described before in this paper . 
in order to assure accuracy and quality of these steps and to previously validate these data , a mpe has to be always involved . the use of appropriate optimized exam protocols for every type of medical radiological procedure is relevant to give meaningful patient exposure information . 
moreover , a team working among radiographers , physicians and medical physicists is desirable . dosimetric data entry on the radiological report will radiological individual risk estimates in terms of detri1 3 792 la radiologia medica ( 2019 ) 124 : 783793 a better implementation of art.64 eu 2013 / 59 regarding dose estimates of population from medical exposure , in terms of collective effective dose ; the dose management should be used as an optimization mean to decrease overall base dose ( from medical exposures ) ; an easier and more effective checking and review of diagnostic reference levels ( drl ) , in order to optimize radiological practice and then to support international audits as , for example , the eurosafe initiative promoted by european society of radiology [ 38 ]  . it is also important to highlight that dosimetric information included in patient radiological report will assume a legal value . 
the patient , indeed , could have one more tool for taking legal actions against the facility where he has been exposed ; in a future bad scenario , it would be possible that a patient undergoing to radiological examination uses his dosimetric information to compare doses from similar examinations executed in different facilities . 
that is why dosimetric information must be certified and it cannot disregard from a good optimization policy inside the radiology department . the national institution involved to drl collection should provide to a more frequently drl monitoring in order to make dosimetric data from medical exposures as uniform as possible in the majority of facilities . 
the european directive 2013 / 59 / euratom takes into account the new recommendations on reduction in the dose limit for the lens of the eye for planned occupational exposures released in 2012 by the international commission on radiological protection ( icrp 118 )  . materials and methods different dose - monitoring procedures and devices were considered . 
 the current status of eye lens radiation protection and the main methods for dose reduction were investigated . results the analysis showed that the workers , potentially exceeding the new limit , are clinical staff performing interventional procedures with a relatively high x - ray dose . 
regarding radiological protection issues , the considered literature reports that the proper use of personal protective equipment may reduce the eye lens absorbed dose . conclusion the evaluation of the occupational eye lens dose is essential to establish which method of personal dose monitoring should be preferred . 
furthermore , education and training about the right use of personal protective equipment are important for medical staff working with ionizing radiation . keywords eye lens dose radiation protection occupational dose dose limit dose reduction european directive 2013 / 59 introduction the new european directive 2013 / 59 / euratom ( eu 2013 / 59 ) introduces several changes to the protection of workers against the dangers arising from exposure to ionizing radiation [ 1 ]  . 
during these procedures , indeed , the upper body is not usually protected as trunk , which is systematically shielded by lead aprons . as a direct consequence of article 9 , eu 2013 / 59 article 40 updates the criterion for classification of workers by specifying that workers who are liable to receive an equivalent loe dose greater of 15msv / year shall be classified as category a . 
this more restrictive criterion , which equals the equivalent dose for public , is likely to result in an increase in workers classified in category a . a second aspect to be considered is the detection dose modality for individual monitoring of workers most at risk , likely to exceed the dose limit of 20msv / year for the eye lens . the aim of this article is to understand whether and how the new loe dose limit will affect the daily routine of clinical staff working with ionization radiation . 
we considered only x - ray exposures in interventional cardiology and radiology or surgery procedures using x - ray imaging such as ct - guided interventions [ 4 ] , fluoroscopically guided procedures , electrophysiology procedures , etc . 
the role of the radiation protection equipment in reducing the dose absorbed by the lens of the eye is also discussed . 2017 approved code of practice introduced a flexibility for 5 - year averaging for dose limit to lens of the eye , subject to conditions specified by hse [ 6 ]  . 
belgium , the netherlands , slovenia , and france actual legislation [ 7 ] provide the effective dose limit of 20msv each year rather than 20msv / year on average over 5years and mandatory personal dosimetry for category b workers . the new french decree on workers radiation protection [ 8 , 9 ] provides that the classification in category a is for exposed workers likely to receive an annual effective dose greater than 6msv or an equivalent dose greater than 150msv for skin and extremities ; the classification in category b is addressed to workers likely to exceed an effective dose of 1msv and an equivalent loe dose exceeding 15msv or 50msv for skin and extremities . 
in italy , current legislation has not transposed the new directive yet : italian exposed workers are included in category a if they are likely to overcome 6msv of effective dose for whole body , or equivalent dose of 45msv for eye lens or 150msv for extremities [ 10 ]  . 
with the new loe dose limit ( 20msv ) , an increase in classified a workers and the contextual decrease in classified b workers are expected . in other european union member states , the expressions category a and category b workers are often used . 
 the category a worker is equivalent to a classified worker , category b to a non - classified worker [ 11 ]  . materials andmethods lens dosemonitoring devices exposed workers classification we have already mentioned that lowering the loe dose limit will increase the number of workers classified in category a . 
it should be kept in mind that a non - negligible contribution to individual doses derives from radiation scattered from the patient . looking at international state of the art , the england transposition of eu 2013 / 59 , for example , reports that classified workers are those likely to receive an effective dose greater than 6msv / year or an equivalent loe dose greater than 15msv / year or an equivalent dose of 150msv for skin and extremities [ 5 ]  . 
regarding the risk of exceeding the loe dose limit of 20msv , the english radiation regulations for individual monitoring of occupational exposures to external radiation , the operational quantity recommended by icrp is the personal dose equivalent ( hp ( d ) ) whose unit of measure is the sievert ( sv ) [ 12 ]  . 
icrp proposed a depth of 3mm ( d = 3mm ) for the assessment of the loe dose because it corresponds to the depth at which the part of the eye sensitive to ionizing radiation is located . in the case of homogeneous irradiation conditions , it is also possible to obtain the hp ( 3 ) value from hp ( 0.07 ) and hp ( 10 ) quantities using appropriate correction factors [ 13 ]  . 
hp ( 3 ) values come from dosimeters positioned close to the eyes . among different technologies , thermoluminescent dosimeters ( tlds ) are the most used for medical workers monitoring . 
there are different types on market such as the eyed dosimeter ( fig.1a ) which can be worn on a narrow headband adjacent to the eye [ 14 ] , the dosiris dosimeter 1 3 730 la radiologia medica ( 2019 ) 124 : 728735 fig . 
in these devices , the stimulated light emission during the reading procedure is produced by a laser pulse rather than heat [ 17 ]  . tlds and oslds can only provide retrospective information ( passive dosimeters )  . 
 another example of active dosimeter is the doseaware system ( fig.2b ) [ 19 ]  . a more general approach to choose the appropriate dosimeter for workers is provided by the international atomic energy agency ( iaea ) and the international radiation protection association ( irpa ) [ 20 , 21 ]  . 
they divided exposed workers into three categories : those exposed to a relatively uniform whole - body radiation field for which the whole - body dosimeter will provide a good estimate of the loe dose ; those exposed to highly non - uniform radiation field in which the loe may be preferentially exposed . 
in this case , estimation of potential eye lens dose is this case is required ; those exposed to weakly penetrating radiation , such as beta particles or photons of low energies , significantly contributing to the loe dose but not to the effective dose . 
2 different active dosimeters : a the unfors edd 30 dosimeter [ 18 ] , b the doseaware system [ 19 ] dosemonitoring procedure the physical surveillance of people working with ionizing radiations , one of the issues ascribed on radiation protection experts ( rpe ) includes the pivotal theme of dose - monitoring procedures . before starting with individual regulatory monitoring , it is necessary to identify the workers most at risk of exceeding the eye lens dose limit for the population ( 15msv )  . according to the new directive art . 
besides , an adequate system for dose monitoring when [ ] category a workers are liable to receive significant internal exposure or significant exposure of the lens of the eye or extremities it is required . for an adequate system , it is intended a system able to measure a dose value as closer as possible to the one really absorbed by the tissue considered . it is therefore essential to establish what level of exposure should be considered significant and consequently to decide whether and which dosimeter to provide to the worker . irpa guidance on implementation of eye dose monitoring of workers [ 21 ] suggests dose - monitoring procedures based on potential absorbed doses . 
it considers hp ( 3 ) value and identifies two cases : ( 1 ) annual dose range from 1msv to 6msv and ( 2 ) annual dose exceeding 6msv . 
table 1 summarizes all the abovedescribed approaches . reduction inoccupational eye lens dose the new dose limit introduced in eu 2013 / 59 also requires a more careful evaluation of all possible solutions to reduce the loe dose . 
the main exposure source for an operator is the scattered radiation leaving the patient , so reducing the patient exposure will directly yield a lower exposure of the medical staff [ 24 ]  . factors contributing to eye lens dose include : the distance between operator and patient , the incident angle of radiation source , the position of the display unit , which determines the operators head orientation with regard to the scattered field , the type and position of the protection equipment . 
 ( university of glasgow ) 16 610 > 10 initial monitoring with collar or head dosimeter to establish dose levels regular monitoring is recommended regular monitoring with collar or head dosimeter is mandatory routine monitoring routine monitoring regular monitoring with collar dosimeter is recommended regular monitoring with head dosimeter is recommended regular monitoring with head dosimeter is required in interventional radiology , in order to optimize patients and workers radiation protection . regarding the occupational dose reduction in interventional cardiology procedures , a practical guide by european heart rhythm association ( ehra ) [ 24 ] described how each technical parameter can affect the dose for patients and staff ; this guide proposed practical ways to reduce radiation exposure . secondly , radiation protection devices ( shielding ) and personal protective equipment must be evaluated , based on exposure source parameters ( energy and position )  . eye lens exposure can be reduced by wearing protective eyewear with leadglass lens , adapted to face geometry . 
 it should be positioned as close as possible to patient and closer to image detector to decrease scattered radiation ; a dose reduction factor ( drf ) of 5 is typically applied for ceiling - suspended shields [ 26 ]  . 
in some situations , a lead protective cabinet protecting the operator can be recommended , thought it could physically obstruct the simultaneous presence of other protective devices . irpa proposes guidance on the use of protection device for the eye [ 21 ]  . 
it suggests the use of ceiling - suspended screens or protective eyewear when the annual unprotected dose is in the range from 3 to 6msv ; the use of both of them when annual unprotected dose exceeds 6msv . 
usually , the use of collective protection devices ( i.e. , ceilingsuspended shields ) is preferred to the use of personal protective equipment [ 27 ]  . a new device recently available on the market is a disposable radiation absorbing pad that results in a mean primary operator reduction dose of about 40% in interventional radiology [ 28 ]  . 
3 different ceiling - suspended eye shields 1 3 la radiologia medica ( 2019 ) 124 : 728735 protection topics to guarantee the effective employment of protective devices described above . table 3 equivalent eye lens doses per procedure ( sv ) for different categories of clinical staff adapted from [ 32 ] overview oftheestimated occupational equivalent dose the transposition of eu 2013 / 59 and consequently the possible adoption of the new loe dose limit put the attention on the current values absorbed by workers exposed to ionizing radiations . 
table2 shows a literature summary of occupational equivalent loe doses in interventional procedure [ 4 , 2933 ]  . the wide range of doses reported in table2 is mainly due to the presence of protective devices ( not always specified in the study ) as well as the different x - ray tube setting during procedures . 
the study of karolinska university hospital [ 34 ] highlighted the importance of the position of workers during interventional procedures in order to distinguish staff categories ( primary physician , scrub nurse and / or circulating nurse ) more at risk of exceeding the dose limit of 20msv / year . 
as predictable , primary physicians resulted the most exposed workers [ more details in table3 ] ; radiologists and cardiologist have the highest occupational equivalent eye dose . interventional procedures radiology ( sv ) cardiology ( sv ) neuroradiology ( sv ) primary physician scrub nurse circulating nurse results anddiscussion the analyzed studies demonstrate that exposed workers involved in interventional procedures using x - rays could potentially exceed the eye lens equivalent dose of 20msv / year if a radiation protection environment is not properly structured . 
the reported loe occupational doses are not homogeneous because they are not always normalized per time exposure and the work pattern is not indicated , so these studies cannot be considered as representative of state of the art . the retrospective assessment of occupational eye lens dose could be conducted throughout chest or neck dose measurements from personal dosimeter [ 29 , 31 , 34 ]  . as far as occupational eye lens dose reduction is concerned , the three well - known factors time , distance and shielding must be kept in mind . 
besides , optimizing the patient exposure , directly related to the worker exposure , is essential . among protection devices mentioned , ceiling - suspended systems provide the highest drf , and their use is more functional than glasses . 
 ( 2017 ) not specified ceiling - suspended screen osl on glasses hp ( 3 ) no eyewear edd30 near the left eye active dosimeter clipped onto eyewear sciahbasi a . 
 ( 2017 ) a ct - guided interventions mobile leaded glass suspended from the ceiling hp ( 3 ) uncorrected use of shields tld hp ( 3 ) category average dose / procedure ( sv ) interventional radiologya interventional cardiology neuroangiography interventional radiology and cardiology interventional cardiology interventional cardiology ~ 169 ~ 34 ~ 114 1 3 734 study table 4 the maximum procedures number ( mpn ) calculated for each study average dose / procedure ( sv ) vano e . 
 ( 2017 ) ~ 169 ~ 34 ~ 114 ~ 118 ~ 589 ~ 175 radio - induced cataract is not a stochastic effect with a probability of occurring related to absorbed dose , but it is a tissue reaction related to a threshold value . 
thus , the optimization process has to be balanced on potential exceeding of eye lens dose limit of 20msv . in order to prevent the exposed workers to exceed the dose limit , we suggest to previously estimate the maximum number of procedures ( mpn ) that each worker should perform and to consider the type and the duration of these procedures . 
the estimates of mpn should be performed in each interventional radiology facility , based on averaged equivalent eye lens dose values per procedure obtained from individual monitoring and for each category ( primary physician , scrub nurse , and circulating nurse )  . 
similarly , each procedure should be performed according to a pre - established written protocol . regarding workers classification and dose limits , all countries that have already transposed the new directive , confirmed the loe dose limit of 20msv / year . 
some member states does not distinguish exposed workers in two categories : england is an example of this , and it defines an equivalent loe dose greater than 15msv / year as exposed workers classification criterion . conclusion the new loe dose limit ( 20msv / year ) and the new threshold value for the equivalent loe dose for exposed workers a ( 15msv / year ) suggested by the european directive 2013 / 59 / euratom are significantly lower than the previous ones ( 150msv / year and 45msv / year )  . 
strategies for la radiologia medica ( 2019 ) 124 : 728735 effective reduction , protection , monitoring , and dosimetry must be implemented . the first topic to address is classification of workers : many workers , currently classified as category b , could be classified as category a because possibly exceeding the new limit value of 15msv / year , the same of population limit . 
consequently , the number of workers belonging to category a and then management costs could increase . in this regard , a monitoring loe dose campaign could be necessary for each exposed worker ; a first retrospective assessment could be done from whole body dosimeters placed above lead apron . 
whole - body personal dosimeter for workers belonging to category b is recommended for a transition period . for regulatory monitoring , a personal eye - dedicated dosimeter is recommended and the best solution is an active dosimeter providing real - time information about radiation exposures as well as alarms at for particular dose levels . optimization of procedures ( e.g. , source and operator position , beam collimation , exposure time , etc . ) and of workloads ( in terms of number of procedures performed by each operator ) is a relevant aspect for the reduction in the absorbed loe dose . 
an adequate distribution of workloads , using the mpn calculation , could prevent the removal of that exposed workers who are most at risk of exceeding the loe dose limit . education and training on the effective and appropriate use of collective and personal protection equipment are essential elements to achieve good protection and acceptableeyelens doses . the results reported in this paper show that loe doses can be significantly reduced if lead glasses and ceiling mounted shields are properly used . 
a close cooperation and collaboration across all the physicians responsible for patient care in requiring imaging examination is also important , balancing possible ionizing radiation disadvantages and patient benefits in terms of care . keywords overimaging overdiagnosis ethics radiation protection * sergio salerno sergio.salerno@unipa.it 1 department ofdiagnostic radiology , university ofpalermo , policlinico via del vespro 127 , 90127palermo , italy 2 department ofsurgical andmedical sciences andtranslational medicine , santandrea university hospital , sapienza - university ofrome , via di grottarossa 1035 , 00189rome , italy 3 department ofbiomedical , surgical anddental sciences , university ofmilan , via pascal 36 , 20133milan , italy 4 department ofradiology , ss giovanni e paolo hospital , castello 6777 , 30122venice , italy 5 department ofradiology , cto hospital , azienda ospedaliera dei colli , naples , italy 6 department ofdiagnostic radiology , hopital europen georges pompidou paris aphp , universit paris - descartes , paris , france introduction the field of biomedical imaging , in particular the area of radiology , has expanded intensely over the last decade . 
the contemporary role of the radiologist is being challenged as the use of images by clinicians is continuously increasing . the development of medical imaging over the past two decades has produced undisputable benefits to patients in terms of life expectancy and quality of life [ 1 ]  . 
this evolution reflects the utilization of complex ionizing and nonionizing radiation technologies such as multidetector computed tomography ( mdct ) , positron emission tomography ( pet ) , and magnetic resonance imaging ( mri )  . 
part of such growth , however , can be attributed to the overuse of imaging services . overuse can be defined as the application of imaging procedures where conditions clearly show that they are unlikely to increase the patient outcome . key factors influencing imaging overuse include : the practice behaviour of referring physicians , self - referral ( including referral for additional radiological examinations ) , vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 714720 duplicate imaging studies , defensive medicine , missed educational opportunities when inappropriate procedures are requested , patient demand , payment mechanisms , and financial incentives ( as in the us healthcare system ) [ 2 ]  . self - referral was known since 1920s , well before the advent of high technology in radiology , but observers generally agree its volume has increased in the last 2030years [ 3 ]  . 
self - referral can be manifested in two main ways forms : a physician who is not an imaging specialist ( or a non - physician provider ) referring patients to his or her own on - site imaging services represents the first form of self - referral ; the second form of self - referral consists of physicians referring their patients to outside facilities in which they has personal a financial interest . 
self - referral denotes a conflict of interest for the referring physician when the financial profit from conducting the procedure may prevail , the medical necessity of the procedure . defensive medicine has been defined as diagnostic or therapeutic actions applied mainly to safeguard against potential claims of malpractice rather than to benefit the patient . 
too often these studies are expensive and provide doubtful benefits to individuals undergoing the moreover , physicians should encourage patients to report imaging examinations they have undergone previously in order to avoid duplicate studies . 
 duplicate studies contribute to overuse of medical imaging [ 6 ]  . recent research shows that approximately one - third of healthcare spending is duplicative , is unhelpful , or makes patients worse [ 7 ]  . 
unnecessary imaging studies rarely reveal the cause of patients complaint , yet may disclose incidental findings , which necessitate further imaging or interventional procedures to be clarified [ 8 ]  . 
therefore , imaging information must not be considered absolute , but rather must be understood inside a specific clinical context including the patient history , previous radiological examinations , and other clinical data to form a combined picture that reasonably either confirms or excludes a given diagnosis [ 9 ]  . because of the possible selfinterest by policy makers , payers , physicians , imaging industry , and patients , it clearly emerges the ethical imperatives of trust and right conduct . 
it is clear that a serious part of the increasing healthcare costs and fragmentation of medical care resides in cooperation and collaboration across many sectors , including radiologists , industry , referring clinicians , healthcare service payers , and public interest groups . the bioethical principle of distributive justice holds that healthcare resources should be distributed as equitably as possible . 
given finite resources for health services , it is unethical to provide expensive non - essential health services to one sector of the society when another sector lacks of essential services [ 11 ]  . 
despite persistent inequity in the distribution of healthcare resources in the usa , we should ask about new medical interventions , does this test or treatment undermine the fair distribution of health resources ? given the importance of this subject and the magnitude of its potential economic impact , it is important to restore objectivity . speaking more in deep about radiological protection , we daily deal not only with useless or inappropriate examinations , but with imaging modalities which may themselves lead to detrimental effects on patients , especially when using contrast media administration and radiation exposure . 
with respect to other planned exposure situations , the justification lies more often with the profession , rather than with government or regulatory authorities , and the final responsibility for the justification , in the use of a particular procedure , lies with the relevant physician , who should be aware of risks and benefits of the involved procedures [ 12 , 13 ]  . 
in case of radiological examinations exceeding the needs of good medical practice , the only result may be an 1 3 716 la radiologia medica ( 2019 ) 124 : 714720 unjustifiable possible risk for patients , not providing any real benefit , with the result of inconvenience application of the main principles of radiological protection and medical ethics [ 14 ]  . 
the potential risks and the uncertainties regarding the risk associated with radiation exposures have a significant role in the decision to undertake a procedure , and also the complexity , in communicating the risk , has to be taken into consideration . 
in fact , informed consent is more than simply getting a patient to sign a written consent form , it is a communication process , which requires additional time for a real and comprehensive informed consent , and this is also related to the critical question of having the proper time to dedicate to the individual patients . 
value judgements on appropriateness of a procedure require knowledge on the implications of the act and about the ethical and societal values on which to base the decisions for that act . 
the system of radiological protection developed by the international commission on radiological protection ( icrp ) has evolved and continues to evolve on the basis of three recognized pillars : science of radiological protection , a set of ethical values , and experience accumulated from the daily practice of radiological protection by professionals [ 15 , 16 ]  . 
it is evident that quantifying benefits and harms is often problematic and the threshold between an appropriate and an inappropriate act can vary among patients and patient groups [ 17 ]  . 
it is also evident that ethics alone cannot offer a definitive solution to questions and dilemmas , but it can certainly provide useful insights into the principles and philosophy of radiological protection . 
in such way , it can be of help in the dialogue among experts , and among experts , patients , and people , to highlight values and preferences in view of a positive balance between potential benefits and harms . regarding the ethical dimension of radiological protection , it is worthwhile to mention the contribution by giovanni silini , who reviewed the ethical foundation of the radiological protection system in his sievert lecture , in 1992 [ 18 ] , by emphasizing that the system has been rationally developed and , at the same time , with the desire to act reasonably . 
in radiological protection , the core ethical values , as addressed in the recent icrp publication on ethical foundations of the system of radiological protection [ 15 ] , support the aims of the radiological protection system and its three fundamental principles : justification , optimization , and individual dose limitation . 
 as well known , the origin of medical ethics lies with hippocratic oath and more recent with the approach by beauchamp and childress [ 19 , 20 ] given through the four principles of biomedical ethics : autonomy , the right of patients to make their own choice ; beneficence , to act taking in mind the best interest for the patient ; non - maleficence , to consider not to harm ; justice , to be fair . 
the core ethical values recognized in the system of radiological protection are clearly consistent with the principles of biomedical ethics , when considering beneficence and non - maleficence in one single concept ; the autonomy replaced by dignity ; and the addition of prudence . overimaging as applications of imaging procedures where circumstances indicate that they are unlikely to provide additional positive outcomes for the patient may increase the average dose to the population resulting from medical exposures . 
given the moral obligation of healthcare providers , first , do not harm , the ethical indication is to lower the risk , with benefit outweighing the risk , by considering procedures appropriately prescribed and appropriately performed ( justification and optimization ) [ 21 ]  . 
at the same time , there is an ethical relevance related to benefit to the society , which is not achieved and quite disregarded , if an unbalance between health outcome and corresponding costs is created [ 2224 ]  . supply and demand are considered the main mechanisms at the base of overimaging . 
expanded availability of technological medical procedures and increased demand from patients / individuals , together with referring clinicians for assurance , can create the feeling and perception of imaging as a mean to comfort patients and clinicians , where the benefits may easily be overestimated , while risks and costs are somewhat neglected [ 25 ]  . 
individual health assessment which is addressed in the last european directive on radiation protection ( bss ) could also play a role in increasing unnecessary examinations . the obligation to benefit the patient must be balanced against the obligation not to cause harm , with the purpose of ensuring that benefits will outweigh , harms ( beneficence , non - maleficence ) [ 26 ] and the attention to these ethical values may become a difficult task if the risks are uncertain , as for the low doses . 
overestimation of risks might result in not performing an imaging procedure that could be of benefit and underestimation of risk might result in an increase in risk for patient and society without any advantage for the patient [ 27 , 28 ]  . 
prudence should not be taken as synonymous of conservatism or never taking risk , but it defines and sustains the way in which decisions are made , and it does not refer only to the outcome of those decisions . 
we can say that prudence represents the ability to take an informed and well - considered decision under uncertainty , without having a full knowledge of the consequences of the undertaken action . 
a proper imaging examination , at the right time , with attention to justification and optimization , 1 3 la radiologia medica ( 2019 ) 124 : 714720 can have significant value for the patient and the society , while overutilization of imaging brings inappropriate use of resources that could be used for other medical purposes , thus violating the justice in the distribution of advantages and disadvantages . 
justice relates our sense of fairness , and we can say that considering radiation risk a particular attention has to be given to avoid overimaging children in view of their higher risk of adverse effects of radiation , compared with adult [ 28 ]  . this is particularly true when considering the exponential increase in radiological examinations during the last decades , and the consequent increased number of imaging findings which are not related to the original diagnostic dilemma . 
indeed , market research studies have shown that in the usa the number of computed tomography ( ct ) examinations increased from 3 million / year in 1980 to 6 million / year in 2006 ; the actual estimated increased rate is between 8 and 10% / year [ 2931 ]  . it also results from the medicare program ( reimbursing diagnostic imaging services in the usa ) that during this period ct scans of the abdomen tripled and those of the thorax quintupled . 
furthermore , the number of magnetic resonance imaging ( mri ) studies is constantly increasing , with brain examinations rising by fourfold , spinal examinations by sixfold , and knee examinations by tenfold [ 32 ]  . 
a recent study reported that in these tests , in 68% of the cases , abnormalities causing no symptoms , called incidentalomas , were detected [ 32 , 33 ]  . 
of course , a finding in a non - symptomatic patient causes negative psychological effects on the patient . in this regard , for example , it is reported that the risk of death for a liver biopsy performed for the purpose of investigating an incidentaloma ( from about 12 per 1000 to 4 ) is of the same order of magnitude as the probability that incidentaloma is a deadly tumour [ 35 ]  . also , in the field of ct , an important aspect concerns the detection of anomalies affecting venous circulation and their possible consequences at the pulmonary level . 
in fact , the prevalence of pulmonary embolism is significantly influenced by the diagnostic tests performed , while from a clinical point of view , few patients with venous thrombosis of the lower limbs have respiratory disorders [ 36 ]  . currently in most cases , nuclear medicine was replaced by the spiral ct . 
this investigation , in patients with thrombosis of the peripheral veins of the lower limbs , identifies 34% more patients with micro pulmonary embolisms compared to the nuclear medicine test [ 37 ]  . 
therefore , although ct increases the micropulmonary embolisms detection rate [ 38 ] , it also entails the risk of contrast media injection , radiation exposure , and increased costs [ 39 ]  . ultrasound investigations can also be a source of overdiagnosis . 
occasional finding of thyroid nodules almost always leads to the request of at least one subsequent follow - up test , if not directly to the direct execution of a fine needle aspiration . 
the diffusion of transrectal ultrasound ( trus ) and trus - guided biopsy has led to a clear increase in the diagnosis , without , however , significant effects on reduction in mortality rate . 
one study showed that performing six samples in symptomatic patients , no neoplasm was found , but referring the same patients to saturation biopsy with 3238 samples , prostate cancer was detected in 14% [ 42 ]  . the american cancer society has recently stated that prostate cancer has a slow growth and no tests should be done in men who have a life expectancy of less than 10years . 
a recent us study even states that screening programs would increase prostate cancer mortality by 13% , as a result of post - surgical and therapeutic complications in patients who would otherwise have not undergone any treatment [ 43 ]  . professor albin , the discoverer of the psa , recently published in the new york times an article entitled the great error of the prostate stating that unfortunately psa test fails to distinguish between two types of prostate cancer : the one that can develop up to be mortal and the other one which remains silent [ 44 ]  . 
ulterior source of overdiagnosis is mr examinations , especially of the musculoskeletal syste approximately 40% of people undergoing mri of the knee , despite being substantially asymptomatic , may have meniscal injuries [ 45 , 46 ]  . 
approximately 50% of people undergoing mri of the lumbar spine , although without significant symptoms in the back , may have disc protrusions [ 47 ]  . the matter of the costs of overdiagnosis is then strongly correlated with that of inappropriateness . 
in italy , a sector study has shown a rate of inappropriateness in outpatient radiology requests of 44% [ 48 ]  . the chairman of the italian society of medical and interventional radiology ( sirm ) , in a recent article , confirmed that in the country one - third of the radiological investigations performed is inappropriate [ 49 ]  . 
calculating that in italy about 100 million radiological examinations per year 1 3 718 la radiologia medica ( 2019 ) 124 : 714720 are performed and that the average cost of radiological services , taking into account the various types , is about 80 , it is likely to estimate the economic value of the inappropriate tests in about 3 billion eur / year . but now , we must consider the most human and important factor of overdiagnosis and overusing of medical radiological examinations : the inappropriate and potentially useless radiation exposure of the population , without any improvement of cost / benefit ratio . the overuse of medical imaging , when involves methods delivering ionizing radiation , increases the radiation exposure and thus the associated risks such as cancer induction [ 50 , 51 ]  . 
in particular , overdiagnosis is associated with ductal carcinoma insitu , a rare tumour ( less than 5% of the annual incidence of breast cancers ) which progresses to invasive disease in a wide time frame ranging between 5 and 15years according to the grade . 
it has been evaluated that the mean glandular dose for screening mammography ranges between 3 and 10mgy and it is mainly influenced by the number of views acquired , the technology used and by patient - related factors [ 57 , 58 ]  . 
 in this setting , the number of false positive is also crucial since it has been demonstrated that 9.251% of positive controls at the first round turns to be a false positive which become 21% at the second round and 33% at the third round determining 2.3% of minor and 2.73% of major invasive procedures [ 63 , 64 ]  . 
since the effect of screening on lung cancer mortality has been estimated to be about 5% , the risk of radiation - induced cancer can be considered acceptable [ 60 , 62 , 66 ]  . the last major setting where overdiagnosis increases overuse and thus radiation exposure is the field of emergency . 
in particular , the use of computed tomography has increased fourfold in the last 10years for any clinical condition such as trauma , pulmonary infections , abdominal pain , and pulmonary embolism [ 53 , 68 ]  . 
the indiscriminate use of computed tomography determines an increase in incidental findings leading to a costly and potentially harmful diagnostic , therapeutic , or interventional cascade [ 68 ]  . 
in a recent survey , over 85% of emergency physicians interviewed felt that 22% of advanced imaging studies ordered were not clinically justified . conclusion while the radiation exposure risks related to screening imaging seem justified by the benefit of a greater mortality reduction , despite the negative influence of not negligible overdiagnosis , in the setting of emergency imaging this phenomenon as well as overuse negatively influences the radiation exposure of patients without the compensation of clinical benefits . in practice , searching for reasonableness and tolerability is a permanent effort directed to act wisely , based on accumulated knowledge , ethical values , and experiences . 
a single - source 64 - mdct ( lightspeed vct , ge ) scan was performed in 705 patients from january to august 2017 ( 207 coronary examinations and 498 vascular examinations ) and 753 patients underwent thirdgeneration 192 2 - dsct ( somatom force , siemens ) scan from january to august 2018 ( 302 coronary examinations and 451 vascular examinations )  . 
volume ct dose index ( ctdivol ) , dose length product ( dlp ) , effective dose ( ed ) , tube voltage ( tv ) and exposure time ( et ) , pitch factor ( pf ) were registered for each patient . 
 cta has been widely used in the diagnostic evaluation of many vascular diseases , and serves as first - line modality in the early diagnosis of abdominal aortic aneurysm or aortic dissection and to follow - up patients treated with endovascular stents and stent grafts , with the aim of determining their patency or potential complications [ 310 ]  . 
 indeed , an effective dose of 5msv adds only a small , negligible additional risk to lifetime cancer risk , but the diagnostic information and the clinical consequences resulting from a coronary cta may outweigh this very small theoretical additional cancer risk [ 24 ]  . related to this , the introduction of dual - source ct ( dsct ) scanners provided a series of improvements , such as fast gantry rotation speed ( from 280 to 250ms ) , increased longitudinal detector coverage ( from 38mm for 128 - slice dsct to 58mm for 192 - slice dsct ) and more powerful roentgen tube . 
this causes reduced gantry rotation time and high - pitch values , which combined with the possibility to reach higher mas and consequently to reduce kv , should improve image quality and reduce radiation dose , compared with the other ct scanner . 
moreover , the dcst scanners equipped of two x - ray sources at 95 to each other [ 25 ] provided a new scan protocol , defined turboflash ( tfp ) , suitable for patient with regular heart rate lower than 65 beats per minute ( bpm ) that allows minimal radiation exposure and concurrent reduction in any motion artifacts from moving structures , such as heart , valves or pulsating aortic root [ 2631 ]  . 
exclusion criteria were : groups of cv type examinations with size smaller than 30 patients in 64 - mdct or in dsct ( superior limbs artery examination , n = 2 with ssct and n = 15 with dsct ; coronary examinations performed with pp in 64 - mdct n = 7 )  . 
we chose to collect patients from the same period of two different years to obtain two cohorts as homogenous as possible . the final population was made up of 1458 patients . 
a 20 - gauge cannula was inserted into superficial vein of the right antecubital fossa , connected to a two - way injector : one with contrast medium ( cm ) ( iopamidol , 370mg i / ml , bracco ) and the other with saline solution . 64mdct ( lightspeed vct , ge ) coronary protocol retrospective ecg trigger was used . 
cm volume was weight - based ( 1.5 ml / kg ) with flow rate of 5 ml / s followed by 50ml of saline solution at the same flow rate . 
scan started , automatically , 10 s after that a region of interest ( roi ) enhancement reached 150 hounsfield unit ( hu )  . table2 summarized scan parameters for cv protocols with 64 - mdct . 192 2dsct ( somatom force , siemens ) coronary protocol ecg - triggered scan was performed with tfp in patients with rhythmic heart rate lesser than 65bpm , with pp in patients with rhythmic heart rate between 66 and 80bpm and with rp in patients with heart rate greater than 80bpm or in case of arrhythmia . bolus test technique was used : 4ml of cm at flow rate of 5ml / s followed by 35ml of saline solution at the same flow rate was used to evaluate peak time ( pt ) in ascending aorta . 
scan started , automatically , 6s after that roi enhancement reached 250 hounsfield unit ( hu )  . table3 summarized scan parameters for cv protocols with 192 2 - dsct . radiation dose volume ct dose index ( ctdivol ) and dose length product ( dlp ) were registered for each patient . 
effective dose ( ed ) , an useful parameter to optimize rd , was calculated by multiplying dlp value by k factor ( k = 0.014 for thoracic examinations and k = 0.015 for abdominal or thoracicabdominal examinations ) , according to guidelines from the american association of physicists in medicine [ 35 ]  . 
for all comparisons , p value less than 0.05 was considered statistically significant . results coronary study : radiation dose andscanning parameters in table4 are summarized and compared ctdivol , dlp , ed , tv , et and pf between dsct and mdct with rp . 
figure1 compares ed between mdct and dsct in each coronary protocol . vascular study : radiation dose andscanning parameters in table5 are summarized and compared ctdivol , dlp , ed , kvp , et and pf between tfp , pp and rp in dsct . 
in all three protocols , mean tv was between 80 and 90kv , but it was significantly lower with pp compared to tfp and rp , which instead showed the greater tv ( 87.8kv ) ; this can partly justify the higher dose delivered compared with the other protocols . 
in fact , the most relevant difference between the three protocols is related with et , significantly lower in rp compared , respectively , with pp ( 60.0% ) and rp ( 74.8% ) , which conceivably has to be considered the largest cause of radiation dose reduction . concerning vascular examinations , the greater radiation dose reduction , with dsct , was found in inferior limb artery study , with 73.1% dlp and ed decrease . 
in particular , the greatest et reduction occurred in thoracic aorta examinations where at the same time , there was the greater pf increase , compared with the other vascular examinations . 
on the contrary , due to the need of leaving time to contrast media to arrive at limb extremities , the lowest et reduction was found in the examinations of lower limb arteries associated with the smaller pf increase , compared to the other vascular exam types . 
another remarkable aspect to underline is that in thoracic aorta and thoracicabdominal aorta , et and kv were significantly lower with dsct than with 64 - mdct , because the prospective ecg triggering sometimes used in the latter one was very burdensome in terms of radiation exposure , due to smaller tube coverage and lower pf , which results in higher et . to the best of our knowledge , no work has compared radiation dose and scanning parameters between 192dstc and 64 - ssct . 
however , they used 70kv for all examinations and disabled ecg - controlled tube current modulation , as a standard protocol , instead of our experience in which we prefer to use an automatic modulation of tube voltage ( care kv , siemens , medical solution ) and tube current ( care dose 4d , siemens )  . 
first , the study is based on a historical comparison , with no guarantee that the populations are entirely comparable , even if patients were selected from the database of the same hospital . 
intra - individual comparisons with repeated examinations using different protocols would strengthen our claims , but this is not possible for obvious ethical reasons . in conclusion , in cv examinations , ctdi , dlp and ed considerably decrease with 192 - dsct in comparison with conventional 64 - mdct , and we can hypothesize that the reduction is mainly associated with higher pf and tv used . funding this research did not receive any specific grant from funding agencies in the public , commercial or not - for - profit sectors . 1 3 760 la radiologia medica ( 2019 ) 124 : 753761 compliance with ethical standards conflict of interest the authors declared no potential conflict of interest associated with this study . ethical standards all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . informed consent informed consent was obtained from all individual participants included in the study . la radiologia medica ( 2019 ) 124 : 711713 editorial editorial fromguest editors current euratom legislation ( de 59 / 2013 ) : new patient management inradiation protection giuseppeguglielmi1 antoniopinto2 sergiosalerno3 received : 29 may 2019 / accepted : 6 august 2019 / published online : 29 august 2019 italian society of medical radiology 2019 keywords euratom legislation radiation protection radiation dose it is our great pleasure to introduce this issue of la radiologia medica focussed in the current euratom legislation on radiation protection . 
 furthermore , the new european directive replacing and integrating in some cases the previous directives 89 / 618 / euratom , 90 / 641 / euratom , 96 / 29 / euratom , 97 / 43 / euratom and 2003 / 122 / euratom , is expected to have a significant and constructive impact on european radiodiagnostic and radiotherapeutic procedures . 
one of the main goals of the directive , born based on the continuous increase of radiation exposure of general population for medical reason , is the information and communication to the patients . 
new scientific data on tissue reactions call for the optimization principle to be applied to equivalent doses as well , where appropriate , in order to keep doses as low as reasonably achievable , alara principle . 
this new directive should also follow new international commission on radiation protection ( icrp ) guidance on the limit for equivalent dose for the lens of the eye in occupational exposure . 
it should be noted also that according to the world health organization the concept of health is understood to cover the physical , mental and social well - being of an individual and not merely the absence of disease or infirmity . in the opening article , salerno etal . 
 [ 2 ] analysed the key factors that influence imaging overuse with x - ray such as self - referral , defensive medicine ( diagnostic or therapeutic actions applied mainly to safeguard against potential claims of malpractice rather than to benefit the patient ) and duplicate imaging studies . 
 [ 3 ] described the wide spectrum of changes introduced by council directive 2013 / 59 / euratorelevant changes include new definitions , a new dose limit for the eye lens , non - medical imaging exposures , procedures in asymptomatic individuals , the use and regular review of diagnostic reference levels ( including interventional procedures ) , dosimetric information in imaging systems and its transfer to the examination report , new requirements on responsibilities , the registry and analysis of accidental or unintended exposure and population dose evaluation . 
 [ 7 ] compared radiation exposure associated with daily practice cardiovascular examinations performed on two different multidetector computed tomography ( mdct ) scanners : a conventional 64 - mdct and a third - generation dual - source ( ds ) mdct . 
 [ 8 ] analysed the main human factors that may increase patient dose as inaccurate scan over - length and patients positioning in ct colonography ( ctc ) and their effect in radiation exposure : radiographers and radiologists need to be aware of dose variation and noise effects on vertical positioning and over - scanning . 
 [ 10 ] described several contemporary and emerging concerns related to radioprotection in radiation therapy , including quality and safety in external beam radiotherapy and brachytherapy , foetal dose , secondary malignancies and the safety issues related to the new techniques and treatment strategies . in the last closing article , cornacchia etal . 
 [ 11 ] reported the information regarding patient exposure to ionizing radiations in the radiological report , according to the european directive 2013 / 59 / euratom ( eu 2013 / 59 art.58 ( b ) ) : for such purpose , results from other member states eu 2013 / 59 transpositions and from guidelines recommendation published by international organizations involved in diagnostic radiology have been evaluated . 
each of these two groups were further divided in two age groups ( or > 10years old ) including patients scanned in the same period of two consecutive years , in 2017 with a 16 - row lightspeed ct ( ge healthcare ) or in 2018 with a somatom force dsct ( siemens healthineers )  . 
in low - dose chest ct , the frequent use of tin filter required higher tube current ; a total dlp 3 times lower was achieved with dsct in patients 10years old . 
the growing number of pediatric ct examinations and radiation exposure raised the concern of a hypothetical increased risk of radiation - induced cancer in adult age [ 24 ]  . 
even though these evidences still need to be fully understood and validated , they had a major impact on global media and a positive influence on scientific research [ 5 ]  . recent technological advances in mdct for pediatric applications focused on dose reduction and faster acquisition for reduction in artifacts in children [ 6 , 7 ]  . 
in particular , the dual - source ct technology ( dsct ) , conceived for cardiovascular imaging , is equipped with two independent x - ray tubedetector systems ( somatom definition , flash and force , siemens healthineers , forchheim , germany ) [ 8 ]  . 
when the tubes work at the same voltage ( kvp ) , an extremely fast acquisition with low rotation times and high pitch is feasible ; vol . : ( 0123456789 ) 1 3 746 la radiologia medica ( 2019 ) 124 : 745752 this is worthy in pediatric population reducing the need of sedation [ 9 ]  . 
the obtained virtual non - contrast images may represent an acceptable solution for avoiding the pre - contrast acquisition with considerable dose reduction [ 12 ]  . going beyond the dect and ultrafast acquisitions already mentioned , several technological solutions contribute to significant dose reduction . 
on the opposite side , the implementation of efficient detector technologies and iterative reconstructions also cooperates to noise and then dose reduction resulting in better image quality [ 1719 ]  . the new - generation ct scanners , with dual - energy technology , efficient dose reduction and ultrafast acquisitions , will realistically expand pediatric ct applications with diagnostic advantages and good image quality . in the present study , we will provide an overview in terms of dose reduction and image quality in a pediatric population after installation of a third - generation dsct in our department . 
examination protocols from two different scanners ( a single - source ct , ssct and dsct ) will be compared in the same period of two consecutive years . materials andmethods inclusion criteria andpatient population we included pediatric patients ( < 20years old ) who underwent to ct examinations at division of special and pediatric radiology of university hospital ospedali riuniti umberto i - gm lancisi - g salesi . 
we included patients scanned in the first 7months of 2017 ( for ssct ) or in the first 7months of 2018 ( for dsct )  . the exam protocols included were studies of neck , thorax and abdomen in oncological patients , dividing the patient population in two groups group a ( 010years old ) and group b ( 1120years old )  . 
pediatric patients undergoing low - dose chest ct for lung diseases ( lung malformations , pneumonia and cystic fibrosis ) were also divided in group c ( 010years old ) and group d ( 1120years old )  . 
emergency ct , neuroradiological and musculoskeletal ct examinations were excluded . image acquisition in the first 7months of 2017 , all pediatric patients were scanned with a ssct , a 16 - row lightspeed scanner ( ge healthcare , milwaukee , wi )  . 
patients less than 6years old required sedation . after installation of a third - generation dsct in january 2018 , oncological and thoracic examinations in pediatric patients were performed with a somatom force ( siemens healthineers , forchheim , germany ) when possible . 
the somatom force scanner is equipped with two tubes delivering high power at voltages between 70kv and 150kv in steps of 10kv with automatic modulation of kv and ma , tin filters for spectral shaping and with high - efficiency stellar detectors ( siemens healthineers , forchheim , germany )  . 
the low - dose chest protocols included highpitch ultrafast acquisitions ( turboflash , siemens healthineers , forchheim , germany ) , dual - energy acquisitions and spectral shaping with tin filter , tailored on patients age and body weight . 
 protocols with dsct did not require sedation . data retrieval data were acquired by a radiation dose index monitoring ( rdim ) system ( dosewatch , ge healthcare , buc , france ) , collecting all the information present on the dicom header and on the dicom radiation dose structured report ( rdsr ) , in order to get information on the whole exam and on the singles scans . patients age , body mass index ( bmi ) , kvp , x - ray tube current per rotation ( mas ) , total exposure time ( ms ) , slice thickness ( st ) and spacing ( sp ) , number of series ( excluding localizers , bolus tracking or bolus test ) and the number of images per series were retrieved . 
when dect was performed , the low kvp was reported ; the high kvp was always 150 sn kv . regarding the dose parameters , we extracted the mean ctdivol ( mgy ) , the total dlp ( mgy * cm ) and the calculated ssde ( mgy ; dosewatch , ge healthcare , buc , france )  . 1 3 la radiologia medica ( 2019 ) 124 : 745752 image quality , data analysis andstatistics image quality , technical parameters anddoses the image quality was evaluated in consensus by two radiologists ( cl , aa ) in consensus on a semiquantitative fivepoint scale as follows : 1 . 
statistical analysis was performed with medcalc v12.5 ( ostend , belgium )  . results patient population table1 summarizes the patient demographics of each group of the population included in the study . 
dect dual - energy ct , hp ultrafast acquisition at high pitch ( siemens healthineers ) , ssh spectral shaping with tin filter 1 3 748 la radiologia medica ( 2019 ) 124 : 745752 1 3 la radiologia medica ( 2019 ) 124 : 745752 1 3 750 la radiologia medica ( 2019 ) 124 : 745752 fig . 
80 kvp ; pitch 0.9375 ; slice thickness 2.5 mm ; kernel standard ; asir 50% ; ctdivol 6.147.56 mgy ; total dlp : 320.29 mgy * c dg follow - up with dsct ( somatom force , siemens healthineers ) and dual - energy acquisition . 
the image quality was significantly higher with the dsct . discussion the therapeutic successes in the managements of clinical conditions such as infantile cancers or lung diseases lead to a significant improvement of the survival of these patients with long follow - up periods of pathologies and their complications [ 1 , 20 ]  . 
the longer survival has led to an increase in ct examinations in pediatric patients with concerns about potential risks in terms of induced cancers after radiation exposure [ 24 ]  . 
the debate on benefits and potential harms of ct in pediatric patients pushed the scientific progress ; fast acquisitions matched with dose reduction strategies are of great value in this setting [ 5 ]  . in our study , we provided an overview in terms of image quality and dose reduction in pediatric patients in two different settings : oncological staging and low - dose chest ct . 
however , considering the same period of the year , a lower number of patients have been evaluated on dsct within groups a , b ( the oncological patients ) and c ( patients with lung diseases , less than 10 years old )  . 
it has to be explicated that the pediatric department , the pediatric oncology and the ssct scanner 1 3 la radiologia medica ( 2019 ) 124 : 745752 ( lightspeed 16 , ge healthcare ) on one side , and the dsct on the other side is in two separated facilities . 
this may have had influence in patients transportation and logistics in particular for younger patients with worse clinical conditions . in our oncological population ( tables2 , 3 ) , we achieved significantly lower delivered dose in both patients groups . 
a better image quality , beyond the implementation of iterative reconstructions present on both scanners , has in part to be related to reduction in motion artifacts in faster acquisitions with dsct . the significant reduction in motion artifacts plays a main role in chest examinations where lower rotation times and acquisitions at ultra - high pitch possible with dsct significantly increased image quality ( tables4 , 5 ) [ 9 , 25 ]  . 
in younger and uncooperative patients , the acquisitions at ultrahigh pitch mode no longer required sedation on dsct , while with the ssct sedation was necessary for patients younger than 6years . as expected , with low - dose chest ct examinations ( tables4 , 5 ) , we achieved lower ctdi values in all patients groups . 
the higher median tube current with dsct in these groups of patients has to be related to the frequent use of spectral shaping with tin filter , which is compensated by the tube current . 
even though the evidences in terms of dose reduction with the spectral shaping are less robust for the pediatric population , in particular when considering smaller children , in our pediatric population , we recorded a lower dose also in terms of total dlp in smaller patients ( table4 ) , while in older patients was comparable ( table5 ) [ 14 , 15 ]  . 
 moreover , in chest ct with dsct , sequential acquisitions planned on ssct were avoided with only volumetric acquisitions with more images but also more information and better anatomical coverage . this study has some limitations . 
even if there are no strictly defined limits for patient radiation exposure , it is recommended to try to keep doses as low as reasonably achievable ( the alara principle )  . 
finally , the risk - benefit analysis of each examination , and careful communication of this risk to the patient , is emphasized . keywords pediatric radiology radiation protection computed tomography radiation - induced cancer child pregnancy introduction the use of medical imaging in children has increased during the past few decades , raising concerns about risks associated with radiation . 
new techniques are now employed in medical fields where justification , optimization , quality assurance and training may need careful evaluation , such as * paolo tom paolo.toma@opbg.net 1 dipartimento diagnostica perimmagini , ospedale pediatrico bambino ges , irccs , rome , italy 2 dipartimento di diagnostica perimmagini , policlinico universit degli studi di palermo , via del vespro 127 , cap90127palermo , italy 3 uoc radiologia ospedale pediatrico giannina gaslini , genoa , italy 4 servizio prevenzione e protezione / fisica sanitaria , ospedale pediatrico bambino ges , irccs , rome , italy 5 department ofclinical radiology , great ormond street hospital forchildren , nhs foundation trust , london , uk use of cone - beam ct in dentistry . 
10% or more of ct examinations worldwide are performed for patients under the age of 18 . managing exposure to radiation from medical imaging is a complex challenge ; efforts that aim to reduce exposure in childhood include improvements in pediatric protocols , technological advances in equipment and implementation of diagnostic reference levels ( drls )  . european directive framework the new european directive 2013 / 59 / euratom sets out basic safety standards for protection against the dangers arising from exposure to ionizing radiation and highlights the need for justification of medical exposure , requirements concerning patient information , quality assurance programs ( recording and reporting doses from radiological procedures ) , the use of drls , the availability of dose - indicating devices and vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 736744 the improved role and support of the medical physics experts in imaging . damage ( greater in middle age ) , which may vary according to cancer type [ 7 ]  . article 61 of the european directive 2013 / 59 / euratom [ 1 ] is focused on children , emphasizing that special attention will be paid to quality assurance programs and to the evaluation of the dose or verification of the activity administered for the practices involving medical exposure of children and to high doses procedures , which may be the case in interventional radiology , nuclear medicine , computed tomography or radiotherapy . article 62 of the european directive 2013 / 59 / euratom states that if pregnancy cannot be excluded , depending on the medical radiological procedure , special attention shall be given to the justification and optimization , taking into account both the pregnant mother and the unborn child . factors thatinfluence therisks ofradiation the biochemical and physiologic damage produced by radiation generally occurs within hours or days , but the impact of these changes , such as the induction of cancer , can take decades to manifest . 
the third step is tumorigenicity [ 2 ]  . radiation exposures , like the medical ones , induce a cellular genomic instability that is transmitted to progeny , which was little described as a persistent enhancement in the rate of which genetic changes arise in the descendants of the irradiated cells after many generations of replication has been termed a non - targeted effect of radiation , as genomic damage occurs in the cells that receive no direct radiation exposure [ 3 ]  . 
most childhood tumors occur sporadically , but in 1015% of the cases , a strong family association and genetic basis for radiation sensitivity are present , such as li - fraumeni syndrome or neurofibromatosis type 1 , although the exact mechanism for this is unclear . the effects of radiation are the greatest on rapidly developing tissues and organsin fetuses , infants and young children [ 4 ]  . 
in pregnancy , the major biological effects of fetal demise , growth restriction , organ malformations and cognitive deficits are seen only with doses in excess of routine diagnostic imaging [ 5 ]  . 
recently noted that cancer induction risk ( greater at younger ages ) must be balanced with the radiation - induced promotion of premalignant evolution ofknowledge estimates of the carcinogenic risk of radiation are derived from epidemiological studies of large populations , such as the atomic bomb survivor cohort and nuclear industry workers and also from dose - response models , such as the linear no threshold ( lnt ) [ 8 ]  . 
diagnostic imaging uses lowlevel radiation that is defined , for the purposes of radiation risk , as < 100150msv ( i.e. , < 20msv for a single - phase pediatric ct examinations )  . 
 [ 11 ] , the lnt model is commonly adopted because it ensures a conservative approach ( i.e. , the model may overestimate the risk of cancer induction at low doses )  . the radiation - induced clustering of dna double - strand breaks into repair centers shows a nonlinear doseresponse in human cells and is much higher at smaller doses , implying that lnt extrapolation could lead to overestimation of cancer risk in the low dose range [ 12 ]  . 
the life span study ( lss ) cohort of about 120 000 subjects included atomic bomb survivors and residents of hiroshima and nagasaki who were not in either city at the time of the bombing . 
the fukushima health management survey will then contribute to future epidemiological research on nodular thyroid diseases in children and adolescents [ 15 ]  . the risk associated with low doses is estimated to be small , such that it cannot be quantified accurately , as even very large studies would lack adequate statistical power to demonstrate small differences . 
published the preliminary results of the epi - ct study ( epidemiological study to quantify risks for pediatric computerized tomography and to optimize doses ) that recruited a total of about 950 000 patients who had undergone ct at least once before the age of 22years [ 16 ]  . 
when considering time since the exposure greater than 5years , the cancer standardized mortality ratios ( smrs , i.e. , the ratio between observed and expected number of deaths based on national reference rates ) decreased to the level of the general population while the non - cancer smrs remained significantly increased . 
these authors conclude that the study population was less healthy than the general population , and reverse causation bias should be considered for cancer risk analyses . the american association of physicists in medicine ( aapm ) supports the position that when such exposures are medically appropriate , the anticipated benefits to the patient are highly likely to outweigh any small potential risks . 
in the position statement pp 25 - c , aapm declares that at the present time , epidemiological evidence supporting increased cancer incidence or mortality from radiation doses below 100msv is inconclusive.and that any predictions of hypothetical cancer incidence and mortality from the use of diagnostic imaging are highly speculative this may lead some patients to fear or refuse safe and appropriate medical imaging , to the detriment of the patient [ 17 ]  . 
it may be hypothesized that low - dose radiation could enhance the immune system response and thus reduce cancer incidence overall . imaging dose optimization there are no absolute defined limits for patient radiation exposure . 
two international campaigns aim to reduce exposure of children : ( a ) image gently [ 24 ] sponsored by the alliance for radiation safety in pediatric imaging in the us and in 70 other countries around the world ; ( b ) eurosafe imaging [ 25 ] sponsored by the european society of radiology ( esr )  . gonadal shielding the use of gonadal shielding is debated . 
 [ 27 ] determined the incidence of missing or misplaced gonadal shields in pediatric orthopedic practice and determined the frequency with which visualization of bony landmarks is compromised by pelvic shielding . 
further , they reported that , because of the identified errors in misplacement of pelvic shielding and potential repeated imaging , pelvic shielding does not reduce the risk of radiation exposure for boys and potentially increases the risk of radiation exposure for girls . furthermore , slovis and strauss [ 29 ] highlighted how a significant fraction of the gonadal dose in both genders is from internal scatter , which is not attenuated by a properly placed gonadal shield . 
assuming that with proper collimation , added filtration and technique selection , the gonadal 1 3 la radiologia medica ( 2019 ) 124 : 736744 dose without lead shielding from the examination should be 2550gy for boys and 1325gy for girls , the estimated increased risk from omitting gonad shielding is relatively small . 
these authors conclude that in order to lower the radiation dose , reducing the number of radiographs and teaching the proper collimation of images can have a greater effect than placing gonadal shields . radiation protection influoroscopy radiation reduction is not always appropriate because some examinations require multiple and additional projections , greater fluoroscopy time or magnification , or lower image noise to answer specific clinical questions . 
planning includes clarifying with requesting physicians what the clinical question is , i.e. , whether the radiation imaging test is justified , and also preoperative planning for efficiency can minimize dose during fluoroscopic and angiographic examinations . a number of dose management strategies exist for fluoroscopy and interventional radiology , including those by the international atomic energy agency and alliance for radiation safety in pediatric imaging [ 30 ]  . 
radiation exposure in fluoroscopy is widely variable across and even within different institutions , [ 31 ] hindered by lack of technical standardization . the only fluoroscopic examination included in the pidrl report was the micturating cystourethrography , and no national pediatric drls have been set for interventional radiology procedures in any eu country to date [ 22 ]  . 
drls for non - standard or newer life - saving interventional radiology procedures may be neither possible nor useful for more complex procedures . icrp 121 reports some key concepts to reduce dose during fluoroscopic examinations [ 32 ]  . 
still images acquired using last - image hold should be used to review findings instead of live fluoroscopy ( repeated exposure )  . the radiation field adjustments should be done with the light beam and not with the fluoroscopy function ( x - ray beam )  . 
collimation of the x - ray beam is essential to reduce the exposed area and to keep radiosensitive areas ( breast , eyes , thyroid and gonads ) away from the x - ray beam , when possible . 
finally , patient dose needs to be recorded and reviewed . interventional radiology procedures account for only 1% of x - ray procedures , but they are responsible of 10 - 15% of cumulative x - ray exposure . 
suggested values for the trigger level for a deterministic effect are a skin dose of 3gy , a kerma - area product of 500gycm2 , or an air kerma at the patient entrance reference point of 5gy [ 34 ]  . when the patients radiation dose from the procedure exceeds the institutions trigger level , clinical follow - up should be performed for early detection and management of skin injuries . optimization ofpediatric ct protocols relative to other imaging modalities , ct can provide a comparatively large dose of ionizing radiation . 
technical innovations allowing faster examinations and better quality imaging have spread the use of ct in children in the last two decades [ 37 ]  . clearly , imaging modalities that do not depend on ionizing radiation , such as ultrasound or mri , are realistic alternatives . 
several authors emphasize that attention shall be given not to reduce the ct dose too much : the real risk to the patient of an inadequate examination , either not yielding the correct diagnosis or needing to be repeated because it was non - diagnostic , is far greater than a small risk of latent malignancy [ 39 ]  . 
mdct scanners are now installed with default settings providing 1 3 740 la radiologia medica ( 2019 ) 124 : 736744 a perfect standard image and deliver the corresponding standard dose . optimization is a process by which a substantial portion of the standard dose is eliminated with a consequent increase in noise , but without loss in diagnostic performance and / or confidence . 
a number of factors are involved in the optimization of ct protocols . tube current : a reduction in tube current ( ma ) is directly related to an increase in image noise and the opposite . 
however , very small children generally have less adipose tissue between organs and tissue planes and this can result in an excessive image noise related to tube current reduction and may need a proportional increase of the product of tube current and tube rotation time ( s )  . 
weight - based or girthbased protocols should be used when imaging pediatric patients , rather than age - based protocols . optimization includes consideration of the following factors : peak kilovoltage : increasing the kilovoltage ( kvp ) increases the energy of spectrums photons , which results in a more penetrating x - ray beakvp has an exponential relationship with dose , and a decrease of 20kvp will decrease the dose by about 3540% . 
a lower kvp generally improves bone detail and substantially increases contrast for ct angiography , while soft tissue studies without the use of a contrast agent are typically improved by increases in the kvp with appropriate reductions in the ma to result in reasonable patient doses [ 34 ]  . pitch : a pitch of approximately 1.31.4 and a short rotation time ( ~ 0.5s ) to minimize total scanning time are generally recommended for pediatric body ct examinations . 
cardiac studies require a faster rotation time and a lower pitch , thus resulting in increased patient dose . automatic exposure control ( aec ) : this system is available in most modern scanners . 
the improper use of aec can result in increased patient dose or non - diagnostic examination [ 42 ]  . patient positioning : centering the patients body in the isocenter of the ct gantry reduces the radiation dose to the patient . 
if the patient is not correctly positioned , the bowtie filters produce an inappropriate compensation of the x - ray beam resulting in more x - rays penetrating thinner peripheral portions of the patients body and less x - rays penetrating in the central , thicker portion of the patient [ 43 ]  . iterative reconstructions : this method processes several passes over the raw data ( obtained using low - dose techniques ) to produce more accurate model of images and to reduce the amount of noise [ 44 ]  . 
implementation requires time , careful adjustment of many acquisition parameters and diagnostic image acceptance . clinical indication - based ct : acceptable levels of image noise are not the same for all ct examinations . 
this changes according to the body site ( i.e. , imaging of the chest or skeletal system , can tolerate relatively increased noise , thus requiring a smaller dose , compared to brain , liver and other solid abdominal organs ) , and the clinical indications , including cumulative patient exposure . 
for example , a low - dose ct technique may be used to detect renal stones in children without compromising detection [ 46 ]  . single - phase scan : a single - phase scan is generally all that is needed in pediatric imaging [ 41 ]  . 
in order to reduce the dose , the length of the scout and the scan length should be limited to the clinical area of interest [ 47 ]  . radiation protection inpregnancy thousands of pregnant women are exposed to diagnostic radiation examinations each year . 
a misinformation on the radiological risks related to diagnostic examinations in pregnant or potentially pregnant women always causes anxiety in radiologists and patients and can lead to voluntary interruption of pregnancy . several well - recognized published documents provide guidance regarding imaging of pregnant women . 
the ircp 84 [ 48 ] states that prenatal doses from most properly done diagnostic procedures present no measurably increased risk of prenatal death , malformation , or impairment of mental development over the background incidence of these entities . 
this evidence is based on atomic bombs survivors and survivors of childhood cancers treated with radiotherapy . 1 3 la radiologia medica ( 2019 ) 124 : 736744 the american college of radiology established that fetal doses below 100mgy should not be considered a reason for terminating a pregnancy [ 49 ]  . 
the american college of obstetricians and gynecologists [ 50 ] published the following policy statement : women should be counseled that x - ray exposure from a single diagnostic procedure does not result in harmful fetal effects . 
specifically , exposure to < 5rad ( 50mgy ) has not been associated with an increase in fetal anomalies or pregnancy loss . according to these documents , the risk to the unborn baby from radiation doses of < 50mgy is negligible . 
doubling that dose ( i.e. , 100mgy ) , the increase over background incidence for organ malformation and the development of childhood cancer combined results in about 1% . according to the international commission on radiological protection , prenatal doses from most correctly performed diagnostic procedures present no measurably increased risk of prenatal or postnatal death , developmental damage including malformation , or impairment of mental development over the background incidence of these entities ; lifetime cancer risk following in utero exposure is assumed to be similar to that following irradiation in early childhood [ 51 ]  . 
however , there is no safe level : the alara principle requires that we use diagnostic methods without ionizing radiation whenever they are equivalent in reaching the diagnosis to those with radiation . 
the risks to the mother require justification , such as the proliferating breast gland that is more sensitive to radiation , or the metabolic adaptations to as well as anatomical changes of the pregnancy that may predispose to certain diseases , modifying indirectly the risks or benefits of a diagnostic imaging procedure . 
effects may be either stochastic or deterministic , with a rather high threshold [ 52 ]  . while the natural risk for malformations at birth is 4% , 100mgy of conceptus dose will only slightly reduce the proportion of children without a malformation from 96 to 95.8% [ 5 ] , and similarly , the natural rate of 99.3% of children without a cancer during childhood will just marginally decrease to 99.07%. in summary , after implantation of the conceptus in utero exposure by less than 100mgy has no proven deterministic effects , but the stochastic effects of cancer induction , although small , are estimated to exist and to increase in proportion to the dose [ 53 ]  . 
deterministic effects exhibit a threshold of around 100mgy even during the most sensitive phase of organogenesis . the alara principle means that ultrasonography and magnetic resonance imaging and any non - imaging diagnostic examinations should be considered before x - ray imaging or nuclear medicine techniques are used . 
when ionizing radiation is appropriate , lower exposure is preferred to higher exposure as long as imaging quality is adequate to answer the clinical question . the anatomical area exposed to direct radiation is the most important factor predicting the uterine dose . in general , when performing a radiological examination in a pregnant woman , the clinical risk of not performing the examination must be evaluated . 
if the fetus is in the direct beam , the procedure should be tailored to reduce the fetus dose ( i.e. , for radiographic examinations : collimate the beam , increase kvp , remove the anti - scatter grid ; for ct : collimate the beam and reduce the scan to the very specific area of interest )  . 
fluoroscopic time must always be limited to the minimum [ 52 ]  . any department offering imaging services , beyond complying with general quality standards , has a number of duties regarding radiation exposure of pregnant patients . duties ofadepartment ofradiology , communication anddecision duties of a department of imaging include information , screening for pregnancy , counseling , documentation and the decision for the best justified examination . 
the stronger the arguments for a critical situation of one of them are , the easier is the justification ; in contrast , a vague suspicion would not justify an important exposure . 
while policies in different departments may differ slightly , it is important that each department formally states its rules in written form and follows them consistently . imaging thenonpregnant woman every adolescent girl and woman of childbearing age has to be considered as potentially pregnant and should be asked whether she is pregnant or thinks she could be [ 52 , 54 ]  . 
as the hormone level does not increase before implantation ( with individual variation ) , it is suggested not to perform the test earlier than 10days after ovulation ; blood tests are more sensitive and , thus , become positive around 2days earlier [ 56 ]  . 
patients below the age of 16 / 18years pose an additional management challenge : they are both children under the responsibility of their parents and individuals with their own rights of privacy and discretion . 
depending on the national legislation , the medical staff has a critical role of giving information to and accepting decisions and consent from the right person . 1 3 742 la radiologia medica ( 2019 ) 124 : 736744 imaging thepregnant woman if pregnancy has been confirmed , justification will differ in the following three scenarios : - imaging without ionizing radiation and imaging of the extremities or the head and neck are justified as in non - pregnant women . 
as radiation exposure is absent or irrelevant , the examinations can be performed anytime . - imaging of the trunk of the body without direct radiation to the conceptus will cause some scatter radiation and a dose of well below 1mgy to the conceptus . 
examinations of this category will usually be performed when well - justified , but optimization becomes more important : the field of view should be minimized to the clinical question . 
a medical physicist should calculate the conceptus dose for intermediate or high - dose examinations of the trunk with the potential of more than 1mgy to the uterus , but not for examinations of the extremities or head . 
 termination of pregnancy should only really be considered for conceptus doses above 100mgy , depending on the phase of pregnancy [ 50 , 51 ]  . occupational exposure the first duty of a department is to inform female workers of the existence and the risks of occupational exposure , the legal duties and rights , and to train them to behave correctly to minimize exposure [ 51 ]  . 
international guidance and national laws clearly prescribe the exposure level allowed for a pregnant worker : as soon as she informs the employer of the pregnancy , the employer shall ensure employment conditions for the pregnant worker such that the equivalent dose to the unborn child is as low as reasonably achievable and unlikely to exceed 1msv during at least the remainder of the pregnancy [ 57 ]  . comparison of the radiation exposure from a diagnostic examination with other radiation exposures ( such as chest x - rays or natural background radiation ) has been proposed as a tool to communicate with parents , but this system may be misleading ; in fact , the dose of a chest x - ray is so low that comparing it with the level of dose of any other radiological procedure may be unnecessarily alarming . 
moreover , background radiation involves whole - body exposure , whereas diagnostic radiation exposures more often have regional ( more localized ) exposures . a key concept that may help in the communication of the potential risk from radiological examination is that of the lifetime baseline risk ( lbr ) and the lifetime attributable risk ( lar )  . the lbr is the general chance that everyone has of having a cancer and / or dying from cancer over the course of her / his lifetime . 
the benefit of early diagnosis and treatment must be balanced with the current understanding of latent cancer risk , compared to the age of the patient and other comorbidities . a recent who document helps support the discussion about benefits and risks of radiological examinations [ 58 ]  . taking active steps to reduce the risk of ionizing radiation during pregnancy and childhood is imperative . 
relevant changes include new definitions , a new dose limit for the eye lens , non - medical imaging exposures , procedures in asymptomatic individuals , the use and regular review of diagnostic reference levels ( including interventional procedures ) , dosimetric information in imaging systems and its transfer to the examination report , new requirements on responsibilities , the registry and analysis of accidental or unintended exposure and population dose evaluation ( based on age and gender distribution )  . 
furthermore , the directive emphasises the need for justification of medical exposure ( including asymptomatic individuals ) , introduces requirements concerning patient information and strengthens those for recording and reporting doses from radiological procedures , the use of diagnostic reference levels , the availability of dose - indicating devices and the improved role and support of the medical physics experts in imaging . keywords bss european directive euratom medical exposures radiation protection radiodiagnostic and radiotherapeutic procedures introduction the new european directive 2013 / 59 / euratom [ 1 ] , laying down basic safety standards ( bss ) for protection against the dangers arising from exposure to ionising radiation and repealing directives 89 / 618 / euratom , 90 / 641 / euratom , 96 / 29 / euratom , 97 / 43 / euratom and 2003 / 122 / euratom , is expected to have a relevant and positive impact on european radiodiagnostic and radiotherapeutic procedures . the basic safety standards take into account the new recommendations of the international commission on radiological protection ( icrp ) [ 2 , 3 ] and are revised in the light of new scientific evidence and operational experience . 
the directive was unanimously adopted by the council of the european union ( eu ) on 5 december 2013 after 4years of work by different european scientific and technical committees . 
dose limits shall not apply to medical exposures . according to the new directive , a high level of competence and a clear definition of responsibilities and tasks among all professionals involved in medical exposures are fundamental to ensure adequate protection of patients undergoing medical radiodiagnostic and radiotherapeutic procedures . 
this applies to medical doctors , dentists and other health professionals entitled to take clinical responsibility for individual medical exposures , to medical physics experts and to other professionals carrying out practical aspects of medical radiological procedures , such as radiographers and technicians in radiodiagnostic medicine , nuclear medicine and radiotherapy . furthermore , the directive requires radiation protection education , training and provision of information . the directive distinguishes between existing , planned and emergency exposure situations . 
considering this new framework , the directive covers all exposure situations and all categories of exposure , namely occupational , public and medical . the most relevant changes in the new directive in comparison with the existing ones96 / 29 / euratom [ 4 ] on the protection of workers and the general public and 97 / 43 / euratom [ 57 ] on medical exposuresare : 1 . 
new non - medical imaging exposures ( article 22 ) replacing the old medico - legal exposures other requirements of directive 2013 / 59 / euratom with high relevance to medical imaging are : 1 . 
member states shall ensure that the operational protection of exposed workers is based on optimisation of radiation protection in all working conditions , including occupational exposures as a consequence of practices involving medical exposures . regulation forradiological procedures inasymptomatic individuals a new article ( 55.2.h ) on justification has been added , concerning medical radiological procedures on asymptomatic individuals , to be performed for early disease detection . 
 such procedures should either be part of a health screening programme or require specific documented justification for that individual by the practitioner , in consultation with the referrer , following guidelines from relevant medical scientific societies and the competent authority . 
underlines the need for appropriate local reviews whenever diagnostic reference levels are consistently exceeded and requires that appropriate corrective action is taken without undue delay . article 18 of the new directive deals with education , information and training in the field of medical exposure . 
the selection of equipment required to perform radiation protection measurements ; equipment for interventional radiology installed after 6 february 2018 must have a device to inform the practitioner and those carrying out practical aspects of the medical procedures of the quantity of radiation produced during the procedure . equipment for interventional radiology and computer radiography must have a device to inform the practitioner and those carrying out practical aspects of the medical procedures of the quantity of radiation produced at the end of the procedure . 
such equipment installed after 6 february 2018 must be able to transfer the information required above to the record of the examination . all new medical radiodiagnostic equipment must provide the practitioner with the relevant parameters for assessing patient dose and where appropriate to transfer this information to the record of the examination and , if available , to the rdim systems to ensure data recording as required by the directive for the purpose of dose assessment to the population . equipment used for external beam radiotherapy , installed after 6 february 2018 , with energies greater than 1 mv , must have a device that verifies the key treatment parameters . it is the responsibility of each department of radiology , nuclear medicine and radiotherapy to ensure the correct management , verification and safe storage of exposure data for the different procedures according to international standards using the reference metrics indicated in table1 . these parameters provide information on the individual procedure , but must take into account the level of uncertainty of the data supplied or estimated . 
the uncertainty should be estimated by the appropriate medical physics expert for each modality . it should be noted that the registration of the exposure data must be provided for all procedures , such as interventional radiology procedures performed with portable or remote equipment . the exposure data , supplied by the medical radiological equipment outside radiology departments , must be managed as an integral part of the radiology information systems with 1 3 726 la radiologia medica ( 2019 ) 124 : 721727 the metrics indicated by the international standards and by the present document . for radiotherapy treatments , the dose to the tumour target and to the critical organs is an integral part of the end - oftreatment clinical report . 
exposure data deriving from the imaging used for the preparation of the treatment plan and verification must be recorded and archived according to the same procedures envisaged for diagnostic imaging . for radiometabolic therapy treatments in nuclear medicine , reference should be made to the specific document / guidelines that defines the specific fields of application for the different therapies / isotopes , provided by the manufacturer and as developed jointly by the nuclear medicine and medical physics societies . for nuclear medicine activities , that use unsealed sources , the activity administered , the radiopharmaceutical used and correction factors that allow these data to be correlated with the patients exposure must be documented . for hybrid equipment , e.g. 
pet / ct , exposure data for both diagnostic techniques must be reported . in order to guarantee the efficacy of the registration of the activity and the type of radioisotope used for nuclear medicine investigations , it is necessary , in compliance with the regulations for the good preparation of radiopharmaceuticals , that the flow of information throughout the process is also traceable and certified . the exposure data will have to be managed by carrying out appropriate tests as indicated by current standards . 
this must be implemented correctly and exhaustively by the equipment manufacturers and be updated throughout the life of the equipment used . accidental andunintended exposures : article 63 member states shall ensure that : a . 
for all medical exposures , the undertaking implements an appropriate system for the record keeping and analysis of events involving or potentially involving accidental or unintended medical exposures , commensurate with the radiological risk posed by the practice ; d . 
arrangements are made to inform the referrer and the practitioner , and the patient , or their representative , about clinically significant unintended or accidental exposures and the results of the analysis ; ( ii ) ( i ) the undertaking declares as soon as possible to the competent authority the occurrence of significant events as defined by the competent authority ; the results of the investigation and the corrective measures to avoid such events are reported to the competent authority within the time period specified by the member state ; f . 
mechanisms are in place for the timely dissemination of information , relevant to radiation protection in medical exposure , regarding lessons learned from significant events . article 63 introduces a new set of requirements for registration and analysis of accidental and unintended medical exposures . in the registration of accidental exposures , tools of incident reporting must be present to record and manage the incidents . estimates ofpopulation doses : article 64 the age and gender must now be taken into account in the distribution of individual dose estimates from medical exposures . 
the competent authorities of each member state will have responsibility for the registration and monitoring of population exposures ; for this purpose , exposure data storage systems ( rispacs ) become indispensable . 
rdim systems are useful for transferring correct and certified data to central depositories responsible for collecting and analysing the data . for such transfers , it is essential to use recognised international standards and precisely the ihe profiles for radiation exposure monitoring ( rem )  . the data to be sent to the central depositories must be certified by the practitioner responsible for the radiological system and by the medical physics expert in order to ensure the correct management of exposure data . the above - mentioned professionals must be provided with the appropriate instruments and technological resources to carry out their functions . general recommendation it is inappropriate to use the estimated effective dose parameter for an individual patient . 
this quantity refers to a standard patient ( usually reference phantom ) and should therefore not be used to derive individual radiological risk values ( icrp 103 )  . the evaluation of the radiation dose to the patient , if required or necessary , requires complex evaluation of the 1 3 la radiologia medica ( 2019 ) 124 : 721727 experimental data , and it is the exclusive competence of the medical physics expert . if the referrer wants to know the risk associated with the estimate of the radiation dose absorbed by a patient , it should be obtained from the practitioner . 
the estimate of the absorbed dose is the responsibility of the medical physics expert who will carry out a personalised dosimetric evaluation . finally , it is considered essential that authoritative sources of information are used on the complex issue of medical exposures . 
for all these activities , radiation oncologist should coordinate and collaborate with a team including different professionals : nurses , radiographers ( rtt ) , clinical engineers , information system experts , taking advantage in particular of the dosimetry expertise of the medical physicist . 
this paper describes several contemporary and emerging concerns related to radioprotection in radiation therapy including quality and safety in external beam radiotherapy and brachytherapy , foetal dose , secondary malignancies , and the safety issues related to the new techniques and treatment strategies . keywords radioprotection radiotherapy radiation exposure radiation risk management introduction in radiation therapy , unlike most other applications involving ionizing radiation , the intent is to deliver high doses of radiation to cancer , limited by the adverse effects of radiation to healthy tissues . 
radiation safety , for patients and staff , is a crucial responsibility for all members of the radiation oncology department . a high level of competence and a clear definition of responsibilities and tasks among all professionals involved in medical exposure are fundamental to ensure adequate protection of patients undergoing radiotherapy . 
this applies to medical doctors , medical physicists and other professionals carrying out practical aspects of radiotherapy procedures , such as radiographers . clinical responsibility means responsibility of a practitioner ( health professional who is entitled to take clinical responsibility for an individual medical exposure in accordance with national requirements ) for individual medical exposures , in particular : justification , optimization , clinical evaluation of the outcome , cooperation with other specialists , giving information on the risk of ionizing radiation to patients , etc . the regulatory bodies require that health professionals with responsibilities for medical exposure are specialized in the appropriate area and that they fulfil the requirements for education , training and competence in the relevant specialty . radiation oncologists ( ro ) have the full and final responsibility for treatment , follow - up and supportive care of the patient . 
they should : ensure that the exposure of normal tissue is kept as low as reasonably achievable while the required dose is delivered to the planning target volume ( the alara principle )  . establish optimized protocols and quality assurance programmes for therapeutic procedures . ensure that radiotherapeutic procedures for women who are pregnant or likely to be pregnant be avoided unless there are strong clinical indications ( otherwise life - threatening diseases ) ; assume the responsibility for the consent procedure regarding prosecution / interruption of the pregnancy , and when needed define the most efficient radiation protection strategy for the women and embryo / foetus . evaluate any radiation accident or incident from a medical point of view . for all these activities , ro should coordinate and collaborate with a team including different professionals : nurses , radiographers ( rtt ) , clinical engineers , information system experts , taking advantage in particular of the dosimetry expertise of the medical physicist . safety andquality inexternal radiotherapy andbrachytherapy the actual work up of the radiotherapy process is needed to guarantee appropriateness , quality and safety . 
over the last decade , the rapid development of new technology has significantly changed the way in which radiotherapy is planned and delivered , with the surge of a variety of new technologies and approaches . external beam radiotherapy three - dimensional computed tomography - based planning is today a standard , but more recent imaging modalities such as magnetic resonance imaging ( mri ) and positron emission tomography ( pet ) may be used . 
flattening filter - free machines , improved immobilization techniques and more sophisticated planning and data management software are the basis for the exponential increase in the use of stereotactic techniques , delivering very high fractional doses . 
 more than half of the italian linear accelerators ( linacs ) are in fact equipped [ 2 ] with cone beam or megavoltage ct on board , to verify target position before delivering the appropriate radiation dose to the target . 
the radiation exposure deriving from the ct used for the treatment plan preparation and the verification of the correct patient daily treatment position may be globally considered irrelevant for the calculation of the dose delivered to patient [ 3 ]  . 
in fact , igrt permits a large dose reduction to critical structures thanks to clinical target volume ( ctv ) planning target volume ( ptv ) margin reduction [ 4 ]  . 
therefore , even if the frequency and number of cts obtained may change according to specific disease and to different igrt strategies , such extra dose may be considered almost negligible . 
the recent clinical introduction of hybrid machines combining a linac and an on - board mri allows the ro to avoid the use of ionizing radiations to perform igrt treatments ; the same holds true for the new mri simulators . 1 3 la radiologia medica ( 2019 ) 124 : 777782 at the same time , new techniques may raise new concerns . 
for example , the use of techniques such as intensity - modulated radiotherapy ( imrt ) or volumetric modulated arc therapy ( vmat ) may expose larger volumes of normal tissue to low doses , in comparison with conventional 3dcrt . 
some authors , for example , are reluctant to use imrt for female with hodgkin lymphoma because of concerns over excessive low - dose exposure of large volumes of normal tissues such as the breast [ 6 ]  . 
however , although the risk of breast cancer is clearly dose - dependent , the relationship between lowdose radiation and carcinogenesis is more speculative [ 7 ] , as further specified in the section on radioprotection of children and during pregnancy . therefore , training of the radiation oncologists should provide sufficiently detailed knowledges and adequate skills to cope with these technical advances in external beam radiotherapy . 
however , every step of the radiotherapy work - up requires the knowledge of the natural history and biology of cancer that profoundly impact also on the dose reduction to critical organs . 
a typical example of that is the very large reduction in the treated volumes in the management of hodgkins lymphomas ( from extended fields to involved fields ) as well as for paediatric patients , avoiding unnecessary exposure of a very large amount of healthy tissues , independently from the technique used . 
for example , dose escalation aiming at better tumour control should be considered cautiously when applied to concomitant chemoradiotherapy for lung cancer [ 8 ]  . brachytherapy another effective therapeutic use of radiation is brachytherapy , in which radioactive sources are placed permanently or temporarily in or very near the target volume . 
in some cases , the ro works together with the surgeon to position brachytherapy devices , such as needles , during open surgery , but in general the surgical competence and the expertise necessary for the ro involved in this technique must be very high . 
the use of afterand / or remote - loading devices allows reducing the risks of exposure of the involved professionals ; brachytherapy is , however , more demanding from the radioprotection point of view than external beam radiotherapy . the main concept of brachytherapy is to exploit the rapid fall - off of the dose with the increasing distance from the sources . 
among the more widely used procedures , permanent implantation of encapsulated 125i and 103pd sources , colloquially called seeds , has been a common method for low dose rate ( ldr ) prostate cancer treatment since the 1990s [ 10 ]  . 
more recently , breast brachytherapy with balloon - catheters , and particularly high dose rate ( hdr ) brachytherapy , has increased significantly and , in general , adherence to recommended radiation protection standards based on the usual principles ( distance , time , shielding ) is important in the process of patient care . trained personnel must be appropriately informed and work together to ensure adequate radiation protection during the work up of this highly individualized treatment . 
for a detailed analysis of radiation protection measures in brachytherapy , the interested reader is referred to guidelines and recommendations produced by several organizations , including the american brachytherapy society ( abs ) , astro , the groupe european de curiethrapie - european society for therapeutic radiology and oncology ( gec - estro ) , the acr and the aapm [ 1113 ]  . radioprotection ofchildren andduringpregnancy ionizing radiation is a known carcinogen to which children are particularly vulnerable [ 14 ]  . 
although the mechanism of greater susceptibility is not well understood , it is likely to be linked to faster cell division in growing and developing tissues [ 15 ]  . 
 age dependence for these cancers , which are among the 1 3 780 la radiologia medica ( 2019 ) 124 : 777782 diseases for which radiation has been more often involved as a possible causative agent , is complex , and generally , the risk due to radiation is added to the overall increase in cancer risk due to aging . these considerations may have an impact on treatment planning . 
some concerns include a possibly increased risk of secondary cancers ( sc ) from imrt but accumulating evidence suggests that sc risk after imrt is likely not higher than with more traditional techniques such as 3d - crt . 
 [ 17 ] , there was no difference in the risk of leukaemia or myelodysplasia after imrt versus 3d - crt , and also the risks of other solid cancers and lymphomas did not significantly differ between imrt and 3d - crt . 
 [ 18 ] : nevertheless , this initial report remains unclear whether in addition to the other advantages of particle therapy for some tumour types it might measurably reduce clinical sc risk . 
despite the potential increased risk of sc , rt for paediatric cancer is a life - saving procedure that should not be withheld when clinically indicated . managing cancer during pregnancy is another very critical clinical situation . 
pregnant women with malignant diseases are advised to delay radiotherapy until after delivery but when this is not possible due to life - threatening tumours , it is estimated that the possible radiation effects , such as mental retardation and organ malformations probably only begin to manifest above a threshold dose of at least 500mgy [ 19 ]  . 
in effect , the dose to the embryo and foetus depend on several factors [ 21 ] : ( a ) the treatment machine used and its leakage radiation ; ( b ) the target dose : the risk of detrimental effects on the embryo / foetus is less likely when treating diseases such as lymphomas ( prescribed doses 3040gy ) compared to lung neoplasms ( prescription doses of about 60gy ) ; ( c ) the size of the radiation fields ; ( d ) the distance from the edges of the fields to the embryofoetus ; and ( e ) the use of wedges , lead blocks , compensators and other scattering objects . 
lesser leakage , lower target dose , smaller radiation fields , greater distance of the edges of the radiation fields from the embryofoetus , and avoidance of wedges and other scattering objects decrease the radiation dose to the embryofoetus . 
 radiation treatment for neoplasms of the head , neck and thoracic region with adequate uterine shielding should be only performed in centres with experience in managing patients diagnosed with cancer during pregnancy . prevention ofaccidental exposure in italy , about 230 , 000 patients per year are submitted to radiotherapy out of 366 , 000 people / year with malignant tumour ( airtum 2014 )  . 
in europe , the 2013 / 59 / euratom directive that sets the basic safety standards concerning protection against dangers arising from exposure to ionizing radiation makes a legal obligation to report and makes corrective measures for accidental and involuntary exposures [ 24 ]  . 
all reasonable measures are to be taken to minimize the probability and magnitude of accidental or unintended exposures of individuals subject to medical exposure ; 1 3 la radiologia medica ( 2019 ) 124 : 777782 2 . 
for radiotherapeutic practices , the quality assurance programme includes : a study of the risks to be prevented , an appropriate system for the record keeping and analysis of events ( follow - up ) ; 3 . 
for all medical exposures , the person responsible for the treatment machine should implement an appropriate system for the record keeping and analysis of events involving or potentially involving accidental or unintended medical exposures , commensurate with the radiological risk posed by the practice ; 4 . 
the hospital and the ro should inform the patient ( or his representative ) and the general practitioner caring for him about any clinically significant unintended or accidental exposures and about the results of analysis ; 5 . 
the undertaking ( the hospital manager ) declares as soon as possible to the competent authority the occurrence of significant events ; also , the results of the investigation and the corrective measures to avoid such events are reported to the competent authority within the time period specified by the member state ; 6 . 
mechanisms are in place for the timely dissemination of information relevant to radiation protection in medical exposure regarding lessons learned from significant events . recently , an italian intersociety consensus underlined these points and recommend to use the available incident reporting tools along with exposure data [ 26 ]  . major cases of accidental exposure in radiotherapy have only been reported a few times over many decades of modern radiotherapy practice throughout the world . 
these rare severe accidental exposures in radiotherapy were due to different causes ( accelerator software problems , incorrect decay data , miscalibration of beams , erroneous use of treatment planning system ( tps ) , computer file not updated and errors in tps data entry )  . 
in italy , the risk of potentially severe errors has been tentatively estimated at about 0.2%. reporting an incident is of little value if what happens is not investigated and analysed . 
different types of ils exist based mainly on the generic hospital incident reporting system or on radiation oncology - specific syste the first one has the advantage to address specific quality and safety concerns , but it might be not targeted for radiotherapy because of the complexity of the processes involved ; from another perspective , develop a specific rt system might be challenging . 
a possible practical option could be to integrate generic incident reporting system with specific radiation oncology report with the aim to identify the so - called sentinel event and get conclusions about patient and treatment - specific factors , to avoid further events [ 28 ]  . 
 furthermore , ilss are recommended by authoritative radiation oncology societies as the american society for therapeutic radiology and oncology ( astro )  . conclusions radiation oncology is a technologically demanding field , which is dependent on well - trained and highly skilled members of the radiation oncology teait is crucial that all members of the team maintain the proper credentials , skills and training levels , satisfying clinical competencies . for radiotherapy treatments , the delivery of radiation dose to the tumour target , minimizing the exposure to critical organs , is an integral part of the end - of - treatment clinical report . 
this software provides estimates of effective dose and doses to the other various organs . results average value of the patients position is found to be below the isocentre for 48 25mm and 29 27mm in the prone and supine position . 
it was observed that the increase in ctdi and dlp values for patients in group 1 , due to the inaccurate positioning , was estimated at about 30% and 20% for prone and supine position , respectively , while in group 2 , a decrease in ctdi and dlp values was estimated at about 16% and 18% for prone and supine position , respectively , due to an average position above isocentre . 
a dose increase ranging from 4 up to 13% was calculated with increasing the overscanned region below anal orifice . conclusion radiographers and radiologists need to be aware of dose variation and noise effects on vertical positioning and over - scanning . 
17 , 90128palermo , italy 5 department ofsurgical andmedical sciences andtranslational medicine , sapienza - university ofrome , santandrea university hospital , via di grottarossa 1035 , 00189rome , italy introduction the constantly increasing number of computed tomography ( ct ) examinations and consequent progressive increase of dose exposition of population lead clinicians and patients themselves to a growing concern about radiation dose [ 14 ]  . 
 dose exposition needs to be as low as possible without reducing the quality of ct examinations especially when x - rays are used for screening purpose [ 59 ]  . ct colonography ( ctc ) performed with multi - detector ct scanner ( mdct ) is well known as valuable alternative technique to optical colonoscopy ( oc ) in the evaluation of colon , replacing barium enema for all clinical queries [ 10 , 11 ]  . 
american college of radiologist ( acr ) and the american cancer society ( acs ) colorectal cancer advisory group suggest ctc for colorectal cancer screening vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 762767 on individual basis , also in high - risk patients if unwilling to undergo colonoscopy [ 6 ]  . msct 64 slice ( ge lightspeed vct - ser ) equipped with volara digital das and asir . european society for gastrointestinal radiology ( esgar ) and acr guidelines suggest a standardized low - dose acquisition protocol aimed to maintain dose exposure as low as reasonable achievable [ 5 , 6 ]  . 
the guidelines for screening ctc recommend a tube voltage of 120kv , tube current of 5080ma and total exposure dose for two positions ( prone and supine ) within a ct dose index volume ( ctdivol ) of 12.5mgy. 
in ctc position , operator mistake may have in higher impact compared to routine abdominal scan due to double acquisition performed with patient supine and prone . ctc acquisition protocol parameters and reconstruction techniques are routinely implemented for reducing radiation dose according to patients size ; however , some pure operator - dependent factors can significantly affect radiation exposure [ 12 ]  . different studies underlined how patient positioning with respect to gantry isocentre could increase absorbed and effective dose in thoracic and abdominal ct [ 7 ]  . 
moreover , scan length affect organ dose , if we consider that exceeding below anal orifice , in the case of ctc acquisitions , leads to irradiation of male gonads [ 1315 ]  . for all of these reasons , we focused our investigation on these two , most relevant , operator - dependent parameters ( exact patients position and precise scan length ) that can be easily corrected during examination . 
in ctc position , operators mistake may have higher impact compared to routine abdominal scan due to the double acquisition performed with patient supine and prone . materials andmethods we performed a retrospective analysis of mdct acquisition data of two separated groups of patients that underwent ctc in 2years in two different secondary referral hospitals , policlinico universitario of palermo ( group 1 ) and ospedale universitario i.c.o.t. 
of latina ( group 2 )  . both electronic medical record databases were retrospectively searched for consecutive patients who underwent ctc of the abdomen between march 2015 and may 2017 . 
patients were eligible for inclusion in the study if they underwent clinically indicated ctc and both prone and supine scans were available . our final study population consisted of 199 patients . 
both centres have similar examination protocol consisting in 2 scout scans and other 2 abdomen scans , respectively , in prone and supine position , according to esgar guidelines [ 5 ]  . care dose4d performs automatic tube current modulation in the angular and longitudinal directions and also adapts to different anatomical regions and patient sizes . 
volara digital data acquisition system ( das ) delivers high processing power for high - quality images and low - dose performance based on attenuation and mas revelation from initial scan ( ge healthcare source )  . all images were imported into commercially available software , which supports dual - energy post - processing capabilities in group 1 ( syngo dual energy , version vb 10b ; siemens healthcare ) and in osiris in group 2 . 
the last distal images to include the inferior pubic rami were considered within the protocol , while any additional images below were instead considered not necessary and potentially avoidable . ct dose reports were derived from pacs in ctdivol ( ct dose index volume ) and dlp ( doselength product )  . the ct scanner software provided ctdivol values , and according to their specifications , these values were measured according to guidelines eur16262 / 1998 [ 16 ] ( and to verify that the measured value was within 10% of the console displayed ctdivol [ 16 ] )  . in order to evaluate the estimated effective total dose and the dose to various organs , we used the ct - expo software version 2.2. 
for 1 3 764 la radiologia medica ( 2019 ) 124 : 762767 each patient , an estimate of the effective dose was achieved by using the dlp value stored inside the pacs system and the scanner obtained through the ct - expo software . 
since real patients do not match exactly the features ( in terms of weight and height ) of the standard patients , the dose values obtained through this analysis are only estimates of the real doses absorbed by patients [ 17 ]  . regarding the calculation of dose increase due to inaccurate patient positioning , we used the values of ctdi as function of the position reported in the literature [ 1821 ]  . 
in particular , we fitted those experimental values with a quadratic function ( of the type f ( x ) = a * 2 + b * x + c ) and we used the best - fit function to rescale the dose values obtained from the images . 
patient bmi data were not available as not routinely collected , and due to the retrospective nature of the study , but patient thickness of the abdomen was available from the images data [ 17 ]  . results mean estimated dose in first cohort was slightly below 5.0msv for the university hospital of palermo , while in second cohort was about 3.1msv. 
this involves a significant increase of the effective dose of about 23% as shown in fig.3. in the case of the university hospital of latina , since the patients are on average scanned above the isocentre and according to literature the positioning above the isocentre involves a dose reduction , the decrease in ctdi and dlp values , due to this positioning , was estimated at about 16% and 18% for prone and supine position , respectively [ 1820 ]  . 
if the data from both hospitals are analysed together , on average an increase in ctdi and dlp values , due to the positioning , was estimated at about 15% and 4% for prone and supine position , respectively . 
error bars correspond to 1 s.d maximum patient thicknesses were calculated from ct images in both prone and supine positions . the values related to the patients scanned in group 1 are 234 33mm and 262 38mm in the prone and supine position , respectively , whereas , these values are 270 36mm and 291 38mm in the prone and supine positions , for group 2 . 
as expected , the thickness values in prone position are smaller than those in supine position because at prone position the body weight squeezes the abdomen . furthermore , we noticed that often the acquisition of ctc exceeded the area required for the diagnostic question 1 3 la radiologia medica ( 2019 ) 124 : 762767 fig . 
error bars correspond to 1 s.d of the examination ; indeed , the acquisitions were performed by scanning also regions below the anal orifice , therefore exposing sensitive anatomical organs to ionizing radiation , especially in male patients . 
through our ct - expo software version 2.2 , we estimated effective dose to testicles and prostate in our 31 male patients , that is values ranging between 212msv ( mean 3msv ) for testicles and 428msv ( mean 9msv ) for prostate . 
in one patient , over - scanning below anal orifice was not significant ( 0mm in prone position ; 12mm in supine position ) with estimated dose of below 2msv to testicles and prostate . mean estimated dose in group 1 was slightly below 5msv , while in group 2 was about 3msv ( table1 )  . as can also be seen from fig.4 , the process of overscanning regions up to 8cm below anal orifice involves a percentage increment of effective dose of atmost 2% for female patients . discussion berrington de gonzalez [ 22 ] reported estimated mean effective dose per ctc screening study between 7 and 8msv in patients from 50 to 80years old . 
mean estimated effective dose for our patients was ~ 5msv and ~ 3msv , respectively , for two cohorts . as confirmed in recent literature , moderate - level iterative reconstruction and high - level iterative reconstruction methods have crucial role in saving dose protocols [ 18 ] , thus reducing radiation - related risks [ 2 , 22 ] , an even more essential matter concerning a screening examination as ctc . 
 however , we need to remember that even if our ct scanner is equipped with iterative reconstruction , wrong positioning leads to an unnecessary dose increase [ 19 , 20 ]  . our results show that imprecise acquisition techniques , occurring when optimized protocols for implementing the fig . 
nowadays , the acquisition technique is an important topic to take into account in the daily practice of acquisition of examinations , giving larger importance to the amount of ionizing radiation dose that is provided to the patient in routine tests [ 11 ]  . in literature , consequences of wrong positioning are well documented . 
 [ 19 ] reported significant changes in organ doses resulting from vertical positioning in paediatric chest ct ( breast dose increased up to 16% and thyroid dose up to 24% in lower positions )  . 
 [ 20 ] performed a study reviewing a group of 480 patients , showing mis - centring of 2.2cm in average below the isocentre with an increase of 23% for patient dose . 
reviewing and comparing these data to phantom testing , eurosafe imaging group observed a 22% difference in dose from 6 - cm table height difference , resulting also in contrast change and noise increase [ 21 ]  . 
two different scout views are required in ctc examination because the abdomen in obese patients may vary in supine and prone position ; in fact in prone one the abdomen may be squeezed resulting in a diverse ap and ll value from supine position . our data suggest that there is a trend for radiographers to get worst centring on prone position ( 48 25mm ) than on supine position ( 29 27mm ) , probably because a supine abdomen ct scan is far more common that prone and anyway each scanning is susceptible of errors . during our retrospective analysis , we found that scan often exceeded the area required for the ctc examination , including also regions below the anal orifice , therefore exposing sensitive anatomical organs to ionizing radiation , especially in male patients . on the basis of fig.4 which reports estimates of the effective dose increment with increasing the over - scanning below anal orifice ( calculated through the ct - expo software ) , we can conclude that an over - scanning of 2cm would cause an effective dose increment of about 6% , whereas this dose increase is of about 12% for over - scanning of 5c five centimeters is the mean value of over - scanning for the first cohort , while two centimeters is the mean value for the second cohort . furthermore , we can evaluate differences between two centres where this study was conducted . 
both table height and extension of scanning below anal orifice parameters varied significantly between the two patient groups . naturally , the best examination possible is the one that maintains an acceptable diagnostic quality with lowest dose possible , as alara principle states [ 9 ]  . 
radiologists and radiographers need to be specifically trained to perform such peculiar examination in order to reduce patient dose exposure [ 3 ]  . our study is characterized by intrinsic limitation : firstly , only two centres were involved and a retrospective analysis that can be improved with further data derived from direct modification in acquisition technique and phantom measurements . 
our patients data may be considered incomplete due to the lack of body mass index ( bmi ) , but in the patient area of interest , the abdomen , we have the data of patient thickness according to the ssde of the aapm [ 17 ]  . 
then , distance between table and centre of patient and from anal orifice to last slice was not automatized . this study was aimed to evaluate operator - related errors on positioning and centring of patients regardless of availability of iterative reconstruction methods and patient characteristics . 
it is our believe that only if you acquire and measure dosimetric data , you can understand real impact of centring errors on daily practice , especially for screening examination as ctc . dose surveillance programs as doses from computed tomography ( ct ) examinations in the uk are useful to understand and monitor ionizing radiation impact on general population . 
even if dose saving technology and continuous update of ct apparatus are essential , a significant dose reduction can be obtained from operator - dependent factors surveillance . in conclusion , radiographers and radiologists need to be aware of dose variation and noise effects on vertical positioning and over - scanning . 
more accurate training need to be achieved even so when examination protocol varies from general practice . compliance with ethical standards conflict of interest the authors declare they have no conflict of interest . ethical approval this article is a retrospective study not implying any modification in patients treatment or images protocol and is an analysis of dose data of the ct examinations performed . 
effective doses due to the radiopharmaceuticals can be estimated by multiplying the administered activities by the effective dose coefficients , while for the ct component the dose - length product can be multiplied by a conversion coefficient k . 
although the effective dose can be used to estimate and compare the risk of radiation exposure across multiple imaging techniques , clinicians should be aware that it represents a generic evaluation of the risk derived from a given procedure to a generic model of the human body . 
it cannot be applied to a single individual and should not be used for epidemiologic studies or the estimation of population risks due to the inherent uncertainties and oversimplifications involved . 
this has led to an increase in per capita annual radiation dose to the us population due to nuclear medicine procedures from 0.14msv in 1982 to 0.8 msv in 2006 [ 1 ]  . most of this rapid growth is due to the diffusion of molecular hybrid imaging procedures such as single - photon emission computed tomography / computed tomography ( spect / ct ) and positron emission tomography / computed tomography ( pet / ct ) that provide relevant functional and anatomical information [ 2 ]  . 
these hybrid systems show high sensitivity , specificity , and accuracy and also increase reader confidence and decrease inter - observer variability through more accurate localization and definition of scintigraphic findings [ 3 ]  . the spect and pet have been used for some years to obtain functional and metabolic information in a variety of pathologic conditions . 
the following introduction of ct determined a significant change , commuting spect and pet in hybrid imaging systems ( spect / ct and pet / ct ) , vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 768776 which are able to provide functional and anatomic images and to overcome the limitations of the separate modalities . 
 within a few years , hybrid imaging became one of the most powerful diagnostic tools available in a number of nuclear medicine departments across the world , and nowadays it plays a vital role in the clinicians daily workflow [ 4 ]  . hybrid imaging is used in several clinical applications , especially in oncology , since it allows a better localization of disease , its characterization before and after therapy , an accurate delineation for biopsy and therapy planning , as well as the detection of the most clinically relevant lesions [ 5 ]  . 
pet / mri hybrid scanners have been introduced into clinical practice both as separate units with a common bed and as a fully integrated unit that allows for simultaneous acquisition of pet and mri . 
although these devices are considerably more expensive than commercial pet / ct units and the clinical indications are still being developed , they have received growing attention ( fig.2 ) in regard to the reduction in radiation dose to the patient due to the elimination of the ct component [ 6 ]  . however , the hybrid systems ( figs.3 , 4 ) inevitably lead to an increase in medical radiation exposure because the radiation dose to patients is the sum of the dose due to the administered radiopharmaceutical and the dose from the ct component of the study [ 7 ]  . 
1 trend of scientific papers published in medical literature ( pubmed ) between 1989 and 2018 about spect / ct and pet / ct 1 3 770 la radiologia medica ( 2019 ) 124 : 768776 fig . 
4 example of pet / ct system radiation doses inhybrid imaging in hybrid imaging , the total radiation dose to patients is the sum of the dose due to the radiopharmaceutical used for spect / pet imaging and the dose derived from the ct component of the study . 
the combined acquisition of functional and anatomical images can substantially increase radiation exposure to patients , particularly in case of a hybrid system with diagnostic ct capabilities . in nuclear medicine , the effective dose due to the administration of radiopharmaceuticals can be calculated by multiplying the administered activity by the effective dose coefficients per unit of administered activity . 
 [ 9 ] , adapted from the icrp publication 80 [ 10 ] , and the administered activities in terms of diagnostic reference levels ( drls ) recommended by dlvo 187 / 00 [ 11 ] for spect and pet . the ct component is responsible for a considerable percentage of the patient dose of a hybrid imaging examination [ 1214 ]  . 
the dlp can also be used to obtain an approximate estimate of the effective dose to the patient , by multiplying the dlp by a conversion coefficient k specific for the anatomical region under examination ( e = k dlp )  . 
in these data , each clinical purpose was associated with a wide range of doses and there was a significant overlap in doses for different clinical purposes , thus highlighting the need for optimization in hybrid imaging . 
for most studies , the ct effective doses were less than 30% of the ones due to radiopharmaceuticals and they only exceeded the 50% mark in case of the radiopharmaceuticals administered for half - body pet / ct and meta - iodobenzylguanidine ( mibg ) spect / ct examinations . in hybrid imaging , the effective dose due to the ct component depends on the different clinical purposes such as attenuation correction ( ac ) , anatomical localization , and sometimes even diagnosis . 
 [ 17 ] , with reference to diagnostic spect / ct , reported an additional 614msv to the radiation dose of the radiopharmaceutical , depending on the field of view in z - axis . 
miller [ 20 ] showed an even lower radiation exposure for the ct component of the spect / ct examination , such as 0.47msv for an abdominal non - diagnostic localization and attenuation - correction scan . 
however , the estimated value of effective dose is subject to a high degree of uncertainty , related to the tissue - weighting coefficients that are used and their estimation of relative biologic risk [ 22 ]  . 
thus , effective dose must be interpreted as a broad , generic estimate of biologic risk , and differences of several msv do not imply a true discrepancy in biologic risk [ 23 , 24 ]  . martin [ 23 ] evaluated the inherent uncertainties in estimating effective dose to be about 40% . 
as a consequence , biologic risk should be described using broad categories : negligible , < 0.1 msv ; minimal , 0.11 msv ; very low , 110msv ; and low , 10100msv [ 23 ]  . 
he also suggested that because effective dose was defined by the icrp [ icrp 103 ] to represent an overall risk averaged over all ages and both sexes for a reference patient , neither the monte carlobased organ - dose coefficients nor the dlp - based k values [ 15 ] should be used to calculate effective dose estimates for individual patients or to predict population risks . moreover , the k coefficient is based on data averaged over many scanner makes and models and therefore cannot accurately represent a specific scanner . 
finally , since the dose coefficients are calculated using a simplified anthropomorphic patient model , all estimates of effective dose are applicable only to scans of a standard adult patient . 
considering the recent increase in the number of overweight and obese patients , the calculated values of effective dose should therefore be used with caution . in conclusion , although the effective dose can be used to estimate and compare the risk of radiation exposure across multiple imaging techniques , clinicians should be aware that table 4 radiation dose reduction methods in hybrid imaging it represents a generic evaluation of the risk derived from a given procedure to a generic model of the human body . 
it cannot be applied to a single individual and should not be used for epidemiologic studies or the estimation of population risks due to the inherent uncertainties and oversimplifications involved . dose reduction andoptimization inhybrid imaging practical ways to reduce radiation dose to patients eligible for hybrid imaging include adjustments to both the planning phase and throughout the execution of the study ( table4 )  . planning phase prior to the execution , spect / ct and pet / ct must be subject to the principle of individual justification of radiation exposure , which is stated as a sufficient net benefit when balanced against possible detriment that the examination might cause . 
both the prescriber and the nuclear medicine physician must take into account the specific objectives of the examination and the clinical data of the patient involved , in order to avoid unnecessary radiation exposure [ 11 ]  . in addition to the radiation exposure justification , the choice of the radiopharmaceutical which has shorter physical and biological half - life also permits to reduce the patient radiation dose . 
usually , 99mtc - labeled radiopharmaceuticals should be preferred due to the favorable physical properties of the radionuclide . drls are a useful tool to optimize the radiation dose to patients undergoing spect / ct or pet / ct examinations . 
the drls related to the radiopharmaceutical component of a hybrid imaging procedure are established in terms of administered activity , while the ctdivol and the dlp are used for the ct component . 
currently in italy , the drls for planar and spect radiopharmaceuticals are set at a national level in dl 187 / 00 , while for the pet radiopharmaceuticals they are defined only at a local level . 
as to the ct component , the adoption of the standard planning phase execution phase general and individual justification of the examination choice of the radiopharmaceuticals , promoting the ones with shorter physical and biological half - lives adherence to national and / or institutional dlrs patient hydration and bladder voiding detectors with higher energy resolution ( cdznte in spect / ct ) special collimators , which allow a higher sensitivity ( variable focus collimators ) iterative algorithms able to reconstruct the image with less counts without affecting spatial resolution 1 3 774 la radiologia medica ( 2019 ) 124 : 768776 diagnostic drls is not appropriate because of differences in the clinical purpose , scan range , and image quality requirements of hybrid imaging ct scan . 
two surveys have been recently conducted in switzerland and uk in order to evaluate ct doses for a wide range of pet / ct and spect / ct procedures aiming to propose national drls [ 16 , 25 ]  . hydration and bladder voiding are also important ways for dose reduction in hybrid imaging since they limit the radiation dose to the bladder , which is the critical organ subject to the higher exposure . 
these devices , mainly used in nuclear cardiology , provide a fourfold to sevenfold improvement in sensitivity thanks to higher resolution and contrast - to - noise ratio , if compared with a conventional dual - detector spect . 
a variable focus collimator was developed by siemens retaining the magnifying properties of a cone - beam collimator near the center of the field of view and eliminating the truncation artifacts at the edges of the field that are common to pinhole and focusing collimators . 
several studies conducted in different fields proved that it is possible to achieve the same image quality with a smaller number of collected counts [ 3134 ]  . similarly , also in ct imaging the introduction of iterative methods allowed significant radiation dose reduction without sacrificing image quality . 
it is now recognized iterative methods , compared with fbp method , increases detectability at a given radiation dose and allows radiation dose reduction while maintaining low - contrast detectability . in hybrid imaging , the structural information provided by ct serves different purposes such as ac , anatomical localization , and sometimes even diagnosis . 
the radiation dose due to ct can be reduced by an accurate choice of ct protocols ( i.e. , lowor high - dose ct ) and scan parameters , such as tube potential , tube load , rotation time , beam width , pitch , and reconstructed image thickness . 
as a general rule , low - dose acquisition is suggested when a recent diagnostic ct is available , in case of evaluation of treatment response , and to better study lesions showed in planar or spect images . a large number of studies covering the previously mentioned parameters have been published [ 35 ]  . 
the introduction of simulation tools allows to optimize the ct scanning parameter by adding an artificial level of noise to the ct raw data in order to simulate a lower - dose examination , without affecting the image quality . 
a useful method to reduce the dose to radiosensitive organs close to the body surface ( i.e. , mammary glands , thyroid glands , and eye lenses ) in spect / ct is the organ - based tube current modulation . 
to compensate for this reduction , the tube current in the x - ray projection from the opposite side is increased thus leaving mean image noise constant [ 27 ]  . 
in musculoskeletal imaging , uric acid material decomposition images can help identify articular deposition of uric acid crystals ( in addition to the detection of uric acid renal stones )  . 
material separation can also help detect bone marrow edema on ct in the case of trauma , algoneurodystrophy , inflammation ( osteitis ) or malignant bone marrow infiltrates , such as metastases . 
dual - source scanners are equipped with two x - ray tubes , which work at different voltages ( 70kv and 150kvp ) allowing simultaneous acquisition of images at these two different energy levels . 
the use of two x - ray tubes with different energy levels makes it possible to extract information and characterizes the chemical composition of material , based on the different degrees of x - ray beam , absorption and attenuation , according to the atomic weight electron density of the compounds [ 7 ]  . 
comparing attenuation values at high vol . : ( 0123456789 ) 1 3 1176 la radiologia medica ( 2019 ) 124 : 11751183 and low energy levels permits dect post - processing algorithms and provides the basis for material decomposition [ 8 ]  . 
the principal applications in musculoskeletal imaging include : ( 1 ) detection of urate crystals in gout or other crystal - induced arthropathies , e.g. , calcium pyrophosphate dehydrate ( cppd ) crystal arthropathy ; ( 2 ) bone marrow edema ( bme ) detection , e.g. , in the case of trauma , algoneurodystrophy or inflammation ( osteitis ) ; ( 3 ) characterization of collagenous structures , such as tendons , ligaments and intervertebral disks ; ( 4 ) minimization of metal prosthesis beam - attenuating artifacts . 
in this context , one of the most outstanding possibilities in musculoskeletal radiology is the so - called virtual non - calcium technique ( vnca technique ) , which makes it possible for edemas to be visualized , as it is necessary to detect fragility fractures [ 9 ]  . 
several technical approaches such as , sequential acquisition , rapid voltage switching , dual - source ct ( dsct ) , layer detecting , quantum countingoffer different spectral contrasts and dose efficiencies . 
three main types of algorithms are currently used to post - process dect data sets : ( 1 ) image optimization algorithms usually provide three sets of images : two sets of mono - energetic images ( typically at 80 or 100kev and 140kev ) , as well as an optimum contrast image from nonlinear blending of the low - energy images ( providing high contrast ) and high - energy images ( providing low noise ) ; ( 2 ) differentiation algorithms allow a certain material to be subtracted from the data set , or the differentiation between two materials , through color - coding , for example ; ( 3 ) quantification algorithms are based on three - material decompositions and can provide color - coded images of iodine content in post - contrast examinations [ 10 ]  . applications ofdualenergy ct tomusculoskeletal imaging dualenergy ct forevaluation oftophaceous gout gout is one of the most common forms of inflammatory arthritis , with prevalence rates above 1% across much of the world . 
dect may be a promising and noninvasive imaging method to assess and diagnose early - stage or acute gout and can help to prevent complication of the chronic late stage of the disease . 
it can directly depict monosodium urate ( msu ) deposits and may easily confirm a diagnosis of gout in patients with normal serum urate levels or exclude it in patients with hyperuricemia [ 11 ]  . 
dect is able to separate different materials , such as calcium , a high molecular weight compound , from uric acid , a low molecular weight compound , as well as cortical bone from trabecular bone . 
 the materials are then color - coded and superimposed on to standard dect images at a multimodality workstation , where multiplanar and three - dimensional volume rendering reformations may be produced and viewed 360 around any axis to best depict the msu deposits . 
this function is particularly useful in quantifying monosodium urate crystals and can be used to monitor the response to treatment and as an outcome measure in patients with gout [ 13 ]  . 
in recent years , a number of studies have shown the ability of dect to detect deposits of monosodium urate crystals with high sensitivity ( 78100% ) and specificity ( 89100% ) [ 1417 ]  . 
therefore , the use of dedicated software dect can measure tophus volumes in the extremities and may be able to demonstrate reduction in tophus volume after successful therapy [ 13 , 18 ]  . 
knee joint view displaying ( b ) urate deposits ( depicted in green ) in the popliteal groove region ( arrow head ) and synovial suprapatellar , lateral and medial recesses of both knees . 
hand / wrist view ( c ) displaying numerous urate deposits ( depicted in green ) along the metacarpophalangeal and proximal and distal interphalangeal joints and in the right wrist . 
automated quantification of urate volume is displayed at the top of each image many reports , including meta - analysis or systemic reviews , have compared us with dect for the diagnosis of confirmed or suspected gout [ 19 ]  . 
dect is a highly accurate tool for detecting the presence of monosodium urate crystals and for confirming gout or distinguishing it from other inflammatory arthritis , although the overall sensitivity of us is higher than that of dect . 
in some studies , dect has proved to be superior in synovial fluid analysis and that a number of patients could be tested false negative by synovial fluid examination alone [ 12 , 23 ]  . dualenergy ct forthedetection ofbone marrow lesions mri has been used as a method of choice for detection of bme . 
conventional ct does not permit the visualization of bone marrow , despite the implementation of numerous protocols , because it is incapable of removing the bone trabeculation and therefore provides inadequate visualization of the bone marrow cavity [ 2426 ]  . 
dect acquires two ct data sets at different energy levels simultaneously and it can characterize and differentiate various chemical elements , through the use of material decomposition protocols [ 8 , 27 ]  . 
 post - processing software is then used to remove calcium in trabecular bone by using a vnca subtraction process , thus creating vnca images [ 24 , 2830 ] , so that the bone marrow can be assessed without superposition . 
in recent years , siemens healthcare has released innovative software containing an enhanced , color - coded , dual - energy ct vnca application , so edema detected can be depicted as color imaging grayscale overlay or even 3d . 
these areas of increased bone marrow attenuation may be due to trauma ( fig.2 ) , algoneurodystrophy ( fig.3 ) , inflammation ( osteitis ) or malignant bone marrow infiltrates , such as metastases . these conditions differ only slightly in density , and there is a considerable overlap , so the differentiation cannot be realized yet , but it appears theoretically possible . 
numerous pitfalls , which can cause false - positive results , have to be recognized ; in particular , it appears difficult to evaluate areas of bone marrow immediately adjacent to cortical bone , sclerotic bone or gas due to potential artifacts [ 28 , 29 ]  . 
2 short tau inversion recovery mr images in the coronal ( a ) and sagittal ( b ) plane demonstrating diffuse bone marrow edema involving the distal right radio , within the side of fracture ( hypointense line , arrow head )  . 
coronal ( d ) multiplanar reformatted color - coded dual - energy virtual non - calcium image showed extensive bone marrow edema ( coded green ) and fracture involving the distal radio ( arrow head ) fig . 
3 short tau inversion recovery mr image in the coronal plane ( a ) demonstrating diffuse bone marrow edema involving head and neck of left femur in patient with algoneurodystrophy ( arrow head ) ; coronal multiplanar reformatted color - coded dual - energy virtual non - calcium image ( b ) reveals bone marrow signal ( depicted in green ) involving the head and neck of left femur , which corresponds to bone marrow edema on the mr image . 
the dedicated software also allows the precise calculation of the attenuation values to be referred to edema in the region of interest ( roi ) 1 3 la radiologia medica ( 2019 ) 124 : 11751183 1179 protocols to detect trauma - associated bme in the spine and extremities , malignant bone marrow infiltration [ 3134 ] and marrow edema associated with inflammatory rheumatic diseases , such as rheumatoid arthritis ( ra ) ( fig.4 ) [ 35 , 36 ] and axial spondyloarthritis [ 31 ]  . 
dect vnca reconstruction has been demonstrated as able to detect bme in traumatic fractured vertebrae and in the detection of incidental thoracolumbar vertebral compression fractures in oncological patients without previous trauma , with high diagnostic accuracy [ 4042 ]  . 
a recent study showed that the dect vnca technique is feasible in the assessment of bme in the sacroiliac joints in patients with axial spondyloarthritis ( axspa ) [ 31 ]  . 
the authors used dect with visual analysis of color - coded images and quantitative roi - based density measurements to detect sacroiliac joint bme , and mr images served as the reference standard . 
dect with the addition of color - coded vnca images to standard ct is also able to simultaneously visualize bme and minor structural lesions in the sacroiliac joints of patients with axspa [ 35 ]  . collagen analysis : ligaments , tendons andintervertebral disks dect is offering a new imaging method to visualize tendons and ligaments . 
with the use of a tissue decomposition algorithm and thanks to their relatively high density , due to the presence of hydroxylysine and hydroxyproline side chains , dect is be able to visualize soft tissue collagenous structures , such as ligaments and tendons , and recognize pathological conditions . using a dedicated software these structures can then be color - coded and fused to standard grayscale ct images . 
 recent studies [ 8 , 43 ] have demonstrated that with dect it is possible to visualize some ligament structures of the knee , such as the anterior cruciate ligament , posterior cruciate ligament , patellar ligament and fibular collateral ligament , without intraarticular contrast media injection , while others , such as the tibial collateral ligament , transversal ligament and oblique popliteal ligament , were not satisfactorily displayed for diagnostic assessment [ 43 ]  . 
althoughthe sensitivity in the detection of ligament damage is lower than for mri , dect may be considered a new imaging method in patients with mri contraindications such as in the case of pacemakers , claustrophobia and in the case of obesity . 
 dect may also play a role in the characterization of tendons , and the volume rendering technique allows anatomic localization , the relationship of the tendons to the bony structures and a better delineation of the soft tissue to be visualized . 
dect makes it possible to visualize the continuity , profile and insertion of many tendons such as the flexor pollicis longus tendon , flexor digitorum superficialis / profundus tendon , achilles tendon , extensor hallucis longus tendon and extensor digitorum longus tendon . 
4 short tau inversion recovery mr image in the sagittal plane of right foot / ankle demonstrating diffuse bone marrow edema involving the tarsal bones and the and the bases of the metatarsal bones in a patient with rheumatoid arthritis ( arrow head ) ( a ) ; sagittal multiplanar reformatted color - coded dual - energy virtual non - calcium image reveals bone marrow signal ( depicted in green ) involving the tarsal bones and the bases of metatarsal bones ( b )  . 
the dedicated software also allows the precise calculation of the attenuation values to be referred to edema in the roi 1 3 1180 la radiologia medica ( 2019 ) 124 : 11751183 dualenergy ct using iodine overlay techniques iodine mapping is an image processing technique used with dect to improve iodine contrast resolution . 
iodine mapping with dect has been clinically used for visualization of regional lung perfusion and in the diagnosis of pulmonary embolism [ 1 , 44 ] and other disorders such as metastatic renal cell carcinoma and squamous carcinoma of the head and neck [ 4547 ]  . 
recently , this innovative imaging method had been used in musculoskeletal issues and in particular to detect inflammation lesions , such as synovitis and tenosynovitis , in the peripheral joints of patients with rheumatic diseases . 
the acquired data are processed with a dedicated software , using a three - material decomposition technique that can identify iodine - containing voxels from dual - energy data and create a virtual unenhanced image . 
the authors concluded that dect iodine mapping is an image processing technique that can delineate inflammatory lesions , preserving the features of ct , such as the reliable evaluation of structural changes and may be particularly useful , not only in early diagnosis of inflammatory arthritis , but also in therapeutic assessment . 
the same authors , in another prospective study [ 49 ] , determined the feasibility of dect with an iodine overlay image ( ioi ) for evaluation of psa in the hand . 
dect is superior to mr arthrography and ct arthrography in the detection of meniscal tears , in the demonstration of suspected intraarticular hip pathology and in the assessment of glenohumeral joint cartilage [ 6 , 58 , 59 ]  . 
however , other studies and large patient trials are required to confirm the potential role of dect arthrography and its clinical efficacy . bone mineral density analysis anddetection ofmetastases promising early results have been seen for the detection of vertebral body density by dect , when compared with dual x - ray absorptiometry ( dxa ) [ 50 , 51 ]  . 
with dedicated software , using quantitative and qualitative tissue decomposition and virtual non - contrast - enhanced imaging and iodine mapping , dect may define and color - code cortical and trabecular bone and makes it possible to identify metastases as abnormal tissue composition or defects in trabecular bone [ 52 ]  . 
however , these techniques will require further investigation to validate their application in a clinical setting . metal artifact reduction techniques many studies support the use of dect in metal artifact reduction through the implementation of energy - specific post - processing , which allows a virtual monochromatic energy spectrum to be created [ 5356 ]  . 
when compared with conventional polychromatic ct imaging , dect demonstrates a lower susceptibility to beam hardening artifacts , provides superior quality and improves the visualization of the peri - prosthetic cortex , medullary bone trabeculation and adjacent soft tissue [ 53 ] , without an increase in the total radiation dose [ 8 , 57 ]  . conclusion in summary , dect represents an emerging field in clinical ct imaging . 
the ability of dect to differentiate materials of different effective atomic numbers makes several new and clinically relevant ct applications possible in the musculoskeletal field , such as detecting bme in the case of trauma , algoneurodystrophy , inflammation ( osteitis ) or malignant bone marrow infiltrates , such as metastases , and to visualize 1 3 la radiologia medica ( 2019 ) 124 : 11751183 1181 tendons and ligaments . 
recently , ultrasonography ( us ) is widely used in musculoskeletal field and us performs better or at least equally well for identification of osteophytes and morphologic degeneration of cartilage in oa patients . 
this review article explains relevant pathologic findings in oa and clinical usefulness in daily practice with us images . keywords ultrasound osteoarthritis synovitis osteophyte cartilage introduction osteoarthritis ( oa ) is the most common disorder of human joints . 
oa of the knee and hip is diagnosed in 40% of people aged > 65years in the uk , and pain is the major symptom of oa causing disability ( e.g. , difficulty in walking and climbing stairs ) [ 1 ]  . 
severity of knee oa is assessed commonly using the kellgrenlawrence system , which is a composite score combining osteophyte presence and jsn for the whole knee [ 3 ]  . 
jsn evaluation is the gold standard in daily practice and has been recommended as the best available method for assessment of jointdamage progression due to oa [ 4 ]  . 
however , conventional radiography is not sensitive to early degenerative changes of cartilage ( which are already occurring before radiographic reduction of the joint space ) and structural alterations visible on conventional radiography ( osteophytes and jsn ) appear only at the relatively late stages of oa [ 5 , 6 ]  . 
in contrast to grading using the kellgrenlawrence system , featureoriented atlas - based compartmental radiographic grading according to standards set by the osteoarthritis research society international is becoming deployed more frequently in clinical research [ 7 ]  . 
 however , radiographic jsn reflects cartilage thinning and meniscal extrusion and does not demonstrate morphologic damage to cartilage and menisci directly [ 8 , 9 ]  . currently , magnetic resonance imaging ( mri ) is being used increasingly to visualize cartilage and intra - articular vol . : ( 0123456789 ) 1 3 1102 la radiologia medica ( 2019 ) 124 : 11011111 of inflamed tissues using noninvasive imaging in daily clinical practice is important . 
although conventional radiography shows lesions affecting bone structure , detection of synovial inflammation using radiography is difficult . us and mri have been recognized as valuable imaging tools to evaluate inflammation severity in synovial tissues [ 19 , 20 ]  . 
the outcome measures in rheumatology ( omeract ) us working group defined synovial hypertrophy as abnormal hypoechoic ( relative to subdermal fat , but sometimes may be isoechoic or hyperechoic ) intra - articular tissue ( fig.1a ) that is nondisplaceable and poorly compressible and which may exhibit a doppler signal [ 21 ]  . 
for example , in ra , a high pdus is associated with pathologic findings of active synovitis , including neovascularization and intensive infiltration of inflammatory cells such as macrophage - like synoviocytes and t - helper - 17 cells [ 22 ]  . 
fluid aspiration is very important for resolving pitfalls in differential diagnoses . us assessment of cartilage has limitations since the acoustic window has only a small area of cartilage available for structures directly . 
mri is the gold standard for assessing the knee joint and is considered to be the most accurate imaging modality for assessment of knee oa [ 2 , 10 ]  . 
despite its high sensitivity , however , mri is not usually employed as an initial imaging method for knee oa and is not readily available for serial evaluation of cartilage status due to practical reasons and expense [ 11 ]  . 
ultrasound ( us ) has been less well studied , but recent high - resolution us , which can be dynamic and offer multiplane visualization , has become of great interest in oa research and daily clinical practice . 
morphologic changes in the bone surface , menisci and femoral cartilage can be depicted reliably and assessed semiquantitatively and / or quantitatively as single features upon us examination [ 1214 ]  . 
additionally , us examination is relatively inexpensive , does not involve ionizing radiation and , overall , there are no contraindications to its use [ 15 , 16 ]  . 
in this review , we describe the specific findings and utility of us for oa assessment . us findings inoa synovitis oa is regarded widely as being primarily a degenerative disorder of articular cartilage , but which is associated with synovial inflammation . 
accurate assessment of synovial inflammation is important for the early diagnosis and evaluation of disease activity not only in inflammatory arthropathies , such as rheumatoid arthritis ( ra ) , but also in oa . 
3 a normal hyaline cartilage is visualized as a homogeneously anechoic layer ( aplio i800 with 24 - mhz probe ultrahigh - frequency transducer , canon medical systems )  . 
usually , on us , an osteophyte has a posterior acoustic shadow , and the size of the osteophyte correlates with the severity of joint damage and duration of oa . 
5 an ultrasound - detected bone erosion ( asterisk ) is discontinuity of the bone surface that is visible in two perpendicular planes at metacarpal head , longitudinal dorsal scan ( a ) and transverse dorsal scan ( b ) la radiologia medica ( 2019 ) 124 : 11011111 in oa . 
however , erosive oa ( sometimes called inflammatory oa ) with more inflammatory signs , such as synovial hypertrophy , pdus and effusion , is recognized an important subtype of hand oa . 
prevalence of knee oa in older populations is increasing and leads to a lower quality of life and working disability , which has major implications for health care and the overall economy [ 29 , 30 ]  . 
highresolution us can be employed to assess the superficial structures in a knee joint , such as synovial tissue , cartilage , menisci , osteophytes and popliteal cysts ( bakers cyst )  . synovitis inknee oa synovial proliferation in knee oa is usually low grade , which can be visualized with / without synovial fluid and / or pdus , though some patients have severe synovitis as the same as in ra . 
a prospective study provided evidence that oa synovitis also generates joint - damage progression , and that usdetected effusion is a predictor of the requirement of total knee arthroplasty [ 31 ]  . 
thus , us - detected synovitis of knee oa can be a target of treatment , as well as demonstrating that us may be a useful outcome measure for studies involving inflammation treatment . cartilage inknee oa establishing clinical measurements of cartilage health and identifying changes in cartilage status are very important for evaluating the effectiveness of protocols to reduce the risk of the development and progression of knee oa [ 32 ]  . 
 although evaluation of knee cartilage by us can be limited due to the depth and lack of adequate visualization , some diseases can be detected clearly by high - resolution us . 
cartilage thickness is an important measure to detect the onset and progression of oa because structural changes in the development and progression of clinical oa are characterized by thinning and loss of cartilage . 
it has been suggested that the center of the medial femoral cartilage should be assessed to evaluate changes in cartilage morphology at the early stage of knee oa [ 36 ]  . 
therefore , accurate and effective tools to measure medial femoral cartilage thickness could be clinically useful and necessary to detect cartilage defects and monitor the treatment effects for thein diagnostic assessment of cartilage thickness , us can be an alternative measure as a clinically available and cost - effective imaging method for articular cartilage of the knee [ 37 ]  . 
we reported that us - assessed damage to the medial femoral cartilage correlated strongly with radiographic narrowing of the medial tibiofemoral joint , and that us was a sensitive imaging method for detecting cartilage damage even in the early radiographic stages of knee oa [ 26 ]  . 
although the peripheral aspects of menisci are visualized clearly , the inner regions are often poorly identified and , in particular , overall visualization of the lateral meniscus tends to be limited [ 39 ]  . 
however , some studies have reported that us assessment is a more accurate imaging method for the detection of medial meniscal extrusion ( fig.6b ) ( which is caused by knee oa and related to knee pain and radiographic medial jsn ) than radiographic assessment [ 18 ]  . 
cyst aspiration with usguided corticosteroid injection from the posterior side of the knee yielded clinical improvement and cyst - volume reduction in patients with a bakers cyst secondary to knee oa [ 45 ]  . 
thus , us examination of the popliteal fossa should be undertaken to detect a bakers cyst to prevent advancement of knee oa . differential diagnoses inknee oa other diseases that should be differentiated from knee oa are crystal - induced arthropathies such as gout and calcium pyrophosphate deposition disease ( cppd )  . 
in one study , dcs identification indicated gout with 83% sensitivity and 76% specificity and disappeared when the serum level of uric acid was < 6ml / dl [ 48 ]  . 
the dcs is differentiated from the normal cartilage interface in that of the latter as a smooth , less bright continuous interface identified only if the cartilage surface is perpendicular to the transducer sound beathe dcs of gout should be differentiated from chondrocalcinosis ( which is seen in cppd and other conditions ) , which appears as hyperechoic calcific foci within the hyaline cartilage [ 47 , 49 ]  . 
they reported that us was an accurate and reliable imaging method for detection of calcification of articular cartilage in the knee joint in patients with cppd [ 52 ]  . 
this four - point scale was : 0 = normal cartilage ( anechoic structure , normal margins of cartilage ) ; 1 = loss of anechoic structure and / or focal thinning of the cartilage layer or irregularities and / or loss of sharpness of at least one cartilage margin ; 2 = loss of anechoic structure and / or focal thinning of the cartilage layer and irregularities and / or loss of sharpness of at least one cartilage margin ; 3 = focal absence or complete loss of the cartilage layer . 
however , the prevalence of hand oa using radiography is not known because of the considerable variation between studies , which may be due to differences in types of populations , disease definitions and / or risk factors ( e.g. , genetic background or environmental exposures ) across study cohorts [ 56 ]  . 
hand oa is found frequently in the first carpometacarpal joint and 25 distal interphalangeal ( dip ) joints , but is also found in 15 proximal interphalangeal joints and 15 metacarpophalangeal joints . 
a , b longitudinal scans 1 3 1108 la radiologia medica ( 2019 ) 124 : 11011111 hyaluronic acid in patients with hand oa , and a decrease in joint thickening and pdus was found . differential diagnoses inhand oa psoriatic arthritis ( psa ) is an inflammatory arthritis that develops in 540% patients with psoriasis [ 62 ]  . 
psa affects the dip joint frequently , and arthritis in the dip joint can be confused with oa , and periosteal proliferation at the level of the dip joints goes into differential diagnosis with oa osteophytes . 
these agents are indicated for treatment of the pain associated with knee oa in patients who do not respond adequately to conservative therapies . traditionally , intra - articular injections have been carried out using anatomic landmarks to identify the correct trajectory for needle placement . 
this has been due , in large part , to the fact that the treating physician cannot visualize the area of interest directly , and variations in anatomy are common . 
furthermore , inaccurate corticosteroid injections in the knee , for example , can result in post - injection pain , crystal synovitis , hemarthrosis , joint sepsis , atrophy of articular cartilage , as well as systemic effects ( e.g. , fluid retention or exacerbation of hypertension or diabetes mellitus ) [ 66 ]  . 
therefore , identification of methods and appropriate training to aid correct placement of the needle during these procedures are warranted . fluoroscopy , ct and mri can be used to improve the accuracy of intra - articular injections . 
however , musculoskeletal us is very practical because it is rapid , safe , relatively inexpensive , emits no ionizing radiation and can be undertaken in the outpatient clinical setting [ 67 ]  . 
radiographic features of psa are characteristic and differ from those observed in ra , especially in the distribution of affected joints and in the presence of destructive changes and bone proliferation at the same time . 
the presence of radiographic bone erosions is fundamental for ra classification , according also to the more recent classification criteria ( american college of rheumatology ( acr ) / european league against rheumatism ( eular ) 2010 classification criteria ) [ 6 ] , while for psa , in the classification criteria for psoriatic arthritis ( caspar ) , radiography still remains one of the main criteria for classifying psa [ 7 ]  . assessing radiographic abnormalities is one of the most powerful means available to the clinical investigator for determining the effects of ra and has been used as a relatively objective marker in clinical trials for evaluating treatment response [ 8 ]  . 
therefore , the current gold standard for radiological evaluation of disease progression in ra is the assessment of disease progression with plain radiographs . many researches have shown that in ra , joint damage occurs within the first 2years after symptom appearance [ 1114 ]  . 
it has been demonstrated that within 4months of vol . : ( 0123456789 ) 1 3 1072 la radiologia medica ( 2019 ) 124 : 10711086 disease onset , 34.9% of patients have erosions evident on x - ray , and 54.9% were erosive at the 12th - month follow - up [ 15 ]  . with the increasing use of dmards and biological dmards ( bdmards ) , early diagnosis is now of paramount importance , and disease progression has to be assessed regularly to monitor efficacy of the treatment [ 1618 ]  . 
in these patients , effective therapy can reduce the odds of progression [ 20 , 21 ] , and both early and intensive treatment can alter the course of the disease by slowing the rate of radiographic progression [ 22 , 23 ]  . regarding psa , at the current state of the art , there is evidence supporting the concept of psa being a distinct disease from ra clinically [ 24 ] , radiologically , and pathologically [ 25 ]  . 
in psa , the presence of radiological damage has been enhanced in 47% of patients within the first 2years , and as in ra , the use of bdmards has been capable of inhibiting progression of structural damage in several randomized controlled trials [ 28 ]  . rheumatoid arthritis andpsoriatic arthritis radiographic comparison as mentioned above , despite certain similarities , the two inflammatory joint diseases show considerably different features . 
in ra , usually , the metacarpophalangeal ( mcp ) joints , the proximal interphalangeal ( pip ) joints , all wrist compartments , and the metatarsophalangeal ( mtp ) joints are the most commonly involved sites . 
in addition , joints in the midfoot and hindfoot , knees , glenohumeral joint at the shoulder , the elbow , and cervical spine can also be affected [ 29 , 30 ]  . in psa , the distribution of affected joints is more often asymmetric and oligoarticular than in ra . 
the distal interphalangeal ( dip ) joints are frequently and early involved , while in ra involvement of the dip joints , in general , is rare and more often a feature of the late disease . 
in psa , dip joints , large joints of the lower extremities , the axial spine , and sacroiliac joints are commonly affected ; the mcp and mtp joints and wrist can be involved as well . the first radiographic changes observed in ra are soft tissue swelling and juxta - articular osteopenia as bone density is reduced adjacent to the joint as a result of local synovial inflammation [ 31 ]  . 
the lack of osteoporosis , even in patients with severe destructive arthritis , is a reliable sign in the differentiation of psa from ra , although the presence of osteoporosis does not exclude psa . the erosions in ra tend to be periarticular and are often described as marginal erosions as they are close to the joint and reflect the direct mechanical action of the hypertrophied synovium and granulation tissue . 
the inflamed synovium slowly invades adjacent structures , causing damage and destruction to the cartilage and bone , leading to joint space narrowing ( jsn ) and bone erosion that can be seen on radiographs . 
the jsn in ra tends to be uniform and concentric , reflecting the generalized nature of the synovial inflammation within the joint . in psa , the early erosive changes predominate in the marginal articular areas , resembling mouse ears . 
 later , the bone appears as if it is being gnawed away , the bone surface becomes frequently irregular or jagged but still sharply delineated , whereas peripherally new bone formation may create an unclear ill - defined outline . 
the uniform reduction of joint space is the radiographic expression of cartilage loss and could be seen at any involved joint , more typically at the dip and pip joints , and more infrequently at the mcp joints . the proliferation of erosions may form irregular excrescences with a spiculated appearance . 
condensation of bone on the periosteal and endosteal surfaces accompanied by thickening of the trabeculae can cause radiodensity of an entire phalanx ( ivory phalanx ) , another manifestation of bone proliferation . 
table1 summarizes the main radiological differences between ra and psa . 1 3 la radiologia medica ( 2019 ) 124 : 10711086 table 1 radiological features that distinguish between rheumatoid arthritis and psoriatic arthritis radiographic features rheumatoid arthritis preponderance for radial sites number of erosions severity of erosions ( size ) erosion distribution dip erosions number of osteophytes severity of osteophytes ( size ) bone proliferation inflammatory changes synovitis tenosynovitis enthesitis dactylitis mutilans ( erosions on both sides of joints ) dips distal interphalangeal joints 1073 psoriatic arthritis evenly distributed radiographic scoring methods inrheumatoid arthritis as discussed above , in ra all the synovial joints can be affected but only some joints in a scoring method can be included . 
these studies indicate the importance to include feet in a scoring method assessing ra radiographic damage . the scoring systems that have been designed to evaluate radiographic changes in ra can be divided into two main groups : global and detailed . 
global scoring systems assign one score to the entire joint , taking into account all the abnormalities seen , whereas detailed systems assign scores on at least two separate variables for each joint evaluated [ 37 , 38 ]  . 
radiographic scores , such as the larsen and sharp scores [ 39 ] and their modifications [ 40 , 41 ] , are the standard methods for determining joint damage and its progression [ 42 , 43 ]  . 
table2 summarizes the main ra features included in the different radiographic scoring methods described below . sharp scoring method ( 1971 ) in 1971 , sharp and colleagues proposed a detailed scoring method for the hands and wrists that is divided into two scores , one for erosions and the other for jsn [ 44 ]  . 
the table 2 features of rheumatoid arthritis included in the sharp and in the larsen scoring systems and further modifications scoring method erosion jsn osteoporosis soft ankylosis cyst sharp ( 1971 ) larsen ( 1977 ) modified sharp ( 1985 ) kaye ( 1987 ) van der heijde / sharp ( 1989 ) + modified larsen ( 1995 ) genant ( 1998 ) ratingen score ( 1998 ) sens ( 1999 ) jsn joint space narrowing , sens simplified erosion narrowing score + = included in the scoring system ; = not included in the scoring system tissue swelling alignment / ( sub ) luxation 1 3 1074 la radiologia medica ( 2019 ) 124 : 10711086 fig . 
a joints selected in each hand for erosions : 4 pip , 5 mcp , ip , scaphoid , lunate , distal ulna , distal radius , the two components of the cmc joints of the thumb are evaluated separately ( pmc and trapeziumtrapezoid )  . 
b joints selected in each foot for erosions : the proximal and distal articular components of the mtp and ip are evaluated separately resulting in a 010 score for each joint . 
c joints selected in the hand : the cmc 3 , cmc 4 , cmc 5 are scored separately , the ip is not included , only the radio - scaphoid part of the radiocarpal joint is evaluated . 
cmc carpometacarpal , cs capitatescaphoid , ip interphalangeal joint , lun lunate , mcp metacarpophalangeal joint , mtp metatarsophalangeal joint , pip proximal interphalangeal joint , pmc proximal metacarpal , rad radius , rc radio - scaphoid , sc scaphoid , st scaphoid - trapezium , tt trapezium trapezoid , ul ulna number and selection of joints in the sharp score evolved in the years , and a modification proposed in 1985 of the sharp method [ 45 ] is now considered the standard for the method . and wrists and also for the feet . 
larsen produced a set of standard reference films to compare the grading of the joints . modified sharp method ( 1985 ) larsen scoring method ( 1977 ) the larsen method was developed by larsen etal . 
however , grade 1 can be based on soft tissue joint swelling only , which is not a real sign of structural damage and is also difficult to assess reliably . 
erosions are counted when discrete , and surface erosions are scored according to the surface area involved [ 45 ]  . 1 3 la radiologia medica ( 2019 ) 124 : 10711086 1075 fig . 
inevaluable joints were not scored and were therefore excluded from analysis . van der heijdemodified sharp scoring method ( 1989 ) the most noticeable difference in the van der heijde modification is the addition of the joints of the forefoot . 
some sites ( triquetrum for erosions and lunate triquetrum , first ip joint and radioulnar joint for jsn ) were difficult to assess in a reliable fashion , mainly due to superimposition , and often were difficult to score leading to interobserver disagreement . 
these modifications also enhance sensitivity and increase reliability . simplified erosion andnarrowing score ( sens ) ( 1999 ) the sens was developed by van der heijde [ 54 ] and is a simplified method by summing the number of eroded and narrowed joints on selected joints on hand and foot radiographs . 
it exploits the same joints of hands and feet , but as opposed to applying a semiquantitative scale of 04 for jsn and 05 for erosions , the sens simply dichotomizes ( bimodal answer modality ) whether an erosion is absent ( score 0 ) or present ( score 1 ) and whether jsn is absent ( score 0 ) or present ( score 1 )  . 
a joints selected in each hand for erosions : 4 pip , 5 mcp , the ip , the cmc of the thumb , scaphoid , distal ulna , distal radius . 
cmc carpometacarpal , csl capitatescaphoidlunate , ip interphalangeal joint , lun lunate , mcp metacarpophalangeal joint , mtp metatarsophalangeal joint , pip proximal interphalangeal joint , pmc proximal metacarpal , rad radius , rc radiocarpal , sc scaphoid st scaphoidtrapezium , ul ulna . 
the + sign represents a 0.5 increment feasibility ofthescoring methods inclinical practice an important disadvantage of the scoring methods for clinical trials is the fact that they require significant training and that scoring according to these methods is time - consuming , making these techniques unfeasible for routine clinical practice . 
the time to score seven radiographs of hands and feet was found to be 3.9min for larsen , 19min for sharp , 25min for the sharp / van der heijde method , and 9min for the ratingen method [ 58 ]  . 
the time needed is one drawback of both the sharp method and the sharp / van der heijde method ; it is related to their higher degree of detail as compared with the larsen and sens methods . radiographic scoring methods inpsoriatic arthritis the measurement of radiographic joint damage in psa is a core outcome measure in both randomized control trials for novel therapies [ 60 ] and longitudinal observational 1 3 1078 la radiologia medica ( 2019 ) 124 : 10711086 fig . 
a joints selected in each hand for erosions : 4 pip , 5 mcp , ip , scaphoid , lunate , distal ulna , distal radius , the two components of the cmc joints of the thumb are evaluated separately ( pmc and trapeziumtrapezoid )  . 
c joints selected in the hand : the cmc 3 , cmc 4 , cmc 5 are scored separately , the ip is not included , only the radioscaphoid part of the radiocarpal joint is evaluated . 
cmc carpometacarpal , cs capitatescaphoid , ip interphalangeal joint , lun lunate , mcp metacarpophalangeal joint , mtp metatarsophalangeal joint , pip proximal interphalangeal joint , pmc proximal metacarpal , rad radius , rc radio - scaphoid , sc scaphoid , st scaphoidtrapezium , tt trapezium trapezoid , ul ulna 1 3 la radiologia medica ( 2019 ) 124 : 10711086 1079 studies [ 61 ] and is included in the research agenda as a domain of interest by the outcome measures in rheumatology ( omeract ) [ 62 ]  . 
these instruments include the modified steinbrocker global scoring method , the modified sharp score ( mss ) , and the modified sharp / van der heijde score ( msvdhs ) for psa [ 36 , 63 ]  . 
as for scoring systems adopted in ra , their lowest common denominator is the large time to performoreover , their scoring requires trained observers . modified steinbrocker global scoring method this method was developed at the psa clinic at the university of toronto . 
the same joints were scored as in the original method , with the addition of the dip from 2 to 5 joints of hands [ 36 , 63 ]  . 
other radiographically detectable changes in psa , such as periostitis and tuft resorption are recorded and scored separately , but not included in the score value . sharpvan der heijdemodified scoring method forpsoriatic arthritis ( msvdhs ) the modification based on the sharpvan der heijde method for ra scores the same joints and definitions as seen in ra [ 41 ] , with the addition of the eight dip joints for erosions and the eight dip and two ip joints of the thumb for jsn . 
spars assess the same joints of the pars in a simpler manner : the grade of the combination of erosion and bony proliferation of the pars is replaced by the sum of joints with erosion and the number of joints with bony proliferation . 
there was consensus that mss and msvdhs were the optimal tool to use in randomized controlled trials ( where sensitivity to change is often the most important attribute of the outcome measure ) , but the most appropriate tool for use in longitudinal observational studies has yet to be established [ 62 ]  . 
the spars has properties which are close to the ones of the msvdhs and pars allowing a quicker calculation [ 67 ]  . 1 3 1080 table 4 features of psoriatic arthritis included in the five radiographic scoring systems for psoriatic arthritis scoring method la radiologia medica ( 2019 ) 124 : 10711086 erosion joint space narrowing bony proliferation modified steinbrocker global scoring method modified sharp score ( mss ) modified sharpvan der heijde method for psoriatic arthritis ( msvdhs ) psoriatic arthritis ratingen score ( pars ) simplified psoriatic arthritis radiographic score ( spars ) fig . 
dip distal interphalangeal joint , ip interphalangeal joint , mcp metacarpophalangeal joint , mtp metatarsophalangeal joint , pip proximal interphalangeal joint conclusion plain radiography remains the gold standard for the assessment of structural joint damage in ra and psa . 
 plain radiography can be helpful in the differentiation of ra from psa and other joint conditions , including osteoarthritis , calcium pyrophosphate deposition disease , gout , and neoplasms [ 71 ]  . 
early bone erosions are correlated with poor long - term radiographic and functional outcome , and early progression in radiographic erosions is related to future impairment in physical function [ 72 ]  . 
the presence of gross osteolysis and pencil in cup is scored separately ; if one of these abnormalities is present , the joint gets the maximum score assigned for erosion ( 5 points ) and for jsn ( 4 points )  . 
a joints selected in each hand for erosions : 4 pip , 5 mcp , ip , scaphoid , lunate , distal ulna , distal radius , the two components of the cmc joints of the thumb are evaluated separately ( pmc and trapeziumtrapezoid )  . 
b joints selected in each foot for erosions : the proximal and distal articular components of the mtp joints and ip are evaluated separately resulting in a 010 score for each joint . 
c joints selected in the hand in the msvdhs : the cmc 3 , cmc 4 , cmc 5 are scored separately , the ip is not included , only the radio - scaphoid part of the radiocarpal joint is evaluated . 
cmc carpometacarpal , cs capitatescaphoid , dip distal interphalangeal , ip interphalangeal joint , lun lunate , mcp metacarpophalangeal joint , mtp metatarsophalangeal joint , pip proximal interphalangeal joint , pmc proximal metacarpal , rad radius , rs radio - scaphoid , sc scaphoid , st scaphoidtrapezium , tt trapezium trapezoid , ul ulna of concepts concerning the severity of ra and psa and the need for tight control to prevent anatomic damage . 
it will have , also , a crucial role in many aspects of treatment in the rheumatic diseases , including identifying patients who are suitable for use of disease - modifying antirheumatic drugs ( dmards ) and biological agents ( bdmards ) , predicting patient response and relapse , and identifying true disease remission [ 17 , 19 , 71 , 73 , 74 ]  . 
a deeper insight into the mechanism of structural changes triggered by these chronic joint diseases is essential for developing therapies that can arrest , prevent , and even reverse bone and cartilage changes . even though magnetic resonance imaging ( mri ) and ultrasound ( us ) demonstrated to be more sensitive than radiographs in detecting early structural changes in joints and surrounding structures [ 75 , 76 ] , availability and costs may limit the use of these techniques in daily clinical practice . further research in the use of mri and us will lead to their proper integration with conventional radiography . 
a joints evaluated in each hand for destruction and proliferation : 4 dip , 4 pip , 5 mcp , the ip of the thumb and the wrist ( evaluated as one joint )  . 
dip distal interphalangeal joint , ip interphalangeal joint , mcp metacarpophalangeal joint , mtp metatarsophalangeal joint , pip proximal interphalangeal joint 1 3 la radiologia medica ( 2019 ) 124 : 10711086 1083 fig . 
 a joints evaluated in each hand for erosion , joint space narrowing and bone proliferation : 4 dip , 4 pip , 5 mcp , the ip of the thumb , and wrist is evaluated as one joint . 
ct is the standard reference imaging modality for visualizing bone damage , including bone erosions in ra , but lacks sensitivity for soft - tissue changes , including synovitis and tenosynovitis . 
ct has a minimal role in ra clinical trials and practice , except in selected patients where mri is contraindicated or not available or if crystal arthritis such as gout or pseudo - gout is suspected . 
mri has documented utility in diagnosis , monitoring and prognostication of patients with ra and is increasingly used for these purposes in clinical practice and particularly clinical trials . keywords magnetic resonance imaging computed tomography rheumatoid arthritis synovitis bone erosion tenosynovitis bone marrow edema diagnosis monitoring prognostication introduction early diagnosis combined with early initiation of appropriate therapy and tight control of inflammation has been recognized as essential for optimal clinical outcomes in rheumatoid arthritis ( ra ) [ 1 ]  . 
this has increased the need for powerful , sensitive techniques that at an early stage can diagnose and indicate the prognosis of patients with ra and accurately monitor the efficacy of treatment . conventional radiography is able to detect structural joint damage in patients with established disease , but is not * mikkel stergaard mo@dadlnet.dk 1 copenhagen center forarthritis research ( copecare ) , center forrheumatology andspine diseases , rigshospitalet , valdemar hansens vej 17 , 2600glostrup , denmark 2 department ofclinical medicine , university ofcopenhagen , copenhagen , denmark 3 department ofradiology , bispebjerg andfrederiksberg hospital , university ofcopenhagen , copenhagen , denmark sensitive for detecting early disease manifestations such as soft - tissue changes and bone damage at its earliest stages [ 2 ]  . computed tomography ( ct ) is a three - dimensional radiographic technique favored by the lack of projectional superimposition and by an exquisite inherent contrast between bone and soft tissue , which makes it a gold standard reference for the detection of bone damage , including bone erosion , new bone formation , calcifications and sclerosis . 
however , ct is hampered by very limited ability to visualize soft - tissue changes using conventional monochromatic ct units , but the introduction of newer ct technologies such as dual - energy or multispectral ct that allows better chemical composition quantification and tissue separation may change this in the near future . magnetic resonance imaging ( mri ) is a non - ionizing imaging technique that has excellent soft - tissue visualization and allows multiplanar tomographic imaging of the body in any plane without projectional superimposition and geometric distortions associated with projectional techniques , such as radiography . 
thus , early bone involvement and inflammatory soft - tissue changes of synovitis vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 11281141 1129 fig . 
axial pre ( a , c ) and post ( b , d ) contrast t1 - weighted images obtained before treatment ( a , b ) and at 1 - year follow - up during treatment with intraarticular glucocortocoids and methotrexate . 
 at baseline , marked post - contrast enhancement , indicating synovitis ( arrows ) , is seen , while only minimal synovitis ( arrows ) is seen at follow - up ( fig.1 ) and tenosynovitis ( fig.2 ) , which are not detectable by conventional clinical , biochemical and radiographic methods , can be directly visualized and evaluated in detail by mri [ 3 ]  . 
disadvantages of mri include relatively higher costs , longer examination times and lower availability than both radiography and ct , and except for whole - body mri ( wbmri ) , mri has restricted anatomical coverage per imaging session . 
mri , as compared to ultrasonography ( us ) , offers greater anatomical coverage than ultrasonography ( us ) does , because us cannot penetrate bone , and thus cannot visualize structures hidden in acoustic shadows . 
coronal short tau inversion recovery ( stir ) images before and 1 year after initiation of tumor necrosis factor inhibitor therapy show bone marrow edema ( osteitis , arrows ) in several carpal joints at baseline , which has disappeared at follow - up 1 3 1130 la radiologia medica ( 2019 ) 124 : 11281141 in clinical trials , the advantage of mri compared to us highly predominates , due to the possibility of standardized image acquisition , centralized reading with full blinding of both image acquisition and reading . 
in clinical practice , us is favored by providing results immediately and thus allows more rapid therapeutic decision making as well as potential for imaging - guided punctures , aspirations , biopsies and injections , but this requires a skilled ultrasonographer and in most instances high - end us equipment in the outpatient clinic [ 3 ]  . this review article will focus on the potential uses of mri and ct in the clinical management of patients with suspected or definite ra . 
after sections on technical aspects , it will describe the current knowledge on mri and ct , respectively , for early detection of ra manifestations , diagnosis of ra , monitoring of disease activity and joint damage progression and its role in determining prognosis . 
 the usefulness in both clinical trials and routine practice will be discussed . magnetic resonance imaging ( mri ) technical considerations high - resolution , t1 - weighted ( t1w ) often three - dimensional ( 3d ) imaging sequences , obtained before and after intravenous contrast injection with or without fat saturation ( fig.1 ) , and a water sensitive technique , such as t2 - weighted ( t2w ) spin echo with fs or short tau inversion recovery ( stir ) ( fig.3 ) , constitute the standard sequences obtained in ra [ 4 ]  . for evaluating structural changes , such as bone erosion , t1 - weighted images are preferred ( fig.4 ) [ 2 , 46 ]  . 
3 coronal t1w 3d gradient echo images post - contrast with axial reconstructions in the level of the wrist ( lower right ) and mcp1 ( upper right ) in a 57 - year - old male with ra for 8years show moderate flexor tendon tenosynovitis of the common flexor retinaculum of the wrist ( arrows ) that can be followed on the axial images involving the flexor pollicis longus and fifth flexor tendons . 
axial ( a ) and coronal ( b ) pre - contrast t1 - weighted images and a coronal short tau inversion recovery ( stir ) image ( c ) show a bone erosion ( long arrows ) in the metacarpal head surrounded by bone marrow edema ( osteitis , short arrows ) also be applied , e.g. , dual echo steady state ( dess ) 3d gradient echo sequences . 
new mri techniques continue to be developed , and a variety of sequences are now available for detecting free water in otherwise fatty bone marrow , indicative of inflammationso called bone marrow edema ( bme ) or osteitis . 
these include t2w spin echo with fat saturation ( t2 fs ) , proton density - weighted with fat saturation ( pdw fs ) or water - excitation ( t2we ) and short tau inversion recovery ( stir ) sequences , but also hybrid sequences , and techniques applying chemical shift - based fatwater separation , such as the dixon technique [ 8 ]  . 
the dixon technique is increasingly being employed , though t2 fs and stir sequences are still most frequent , because of their high general reliability , consistency across different manufacturers and , for stir , applicability at lower magnetic field strengths ( < 1t )  . whole - body mri is promising technique that allows imaging of the entire body in one examination , i.e. , simultaneous assessment of peripheral and axial joints and entheses in both ra [ 9 ] , spondyloarthritis and psoriatic arthritis [ 1012 ] , where it may provide an mri - based total joint inflammation count . 
improved image resolution and more validation are still needed for wbmri imaging of the small joints of the hands and feet , before the method is ready for routine use . dynamic contrast - enhanced mri ( dce - mri ) is based on the perfusion dynamics of synovial enhancement curves after contrast injection and has been found to correlate with synovial histopathological inflammation ( fig.5 ) [ 13 , 14 ]  . 
it involves no ionizing radiation or risk of malignancy , with the major contraindications being claustrophobia or the presence of any metal devices including pacemakers or clips ( though many of these are now made mri safe )  . 
the european society of urogenital radiology ( esur ) guidelines recommend no patients should be denied a well - indicated gd - enhanced mri , and those agents with highest stability ( lowest risk of nephrogenic systemic fibrosis ) ( e.g. , macrocyclic gd chelates , such as gadoterate meglumine and gadoteridol ) should be used , at the lowest diagnostic dose [ 20 ]  . mri detection ofpathology inra the clinical value of mri in ra relates primarily to its ability to evaluate the wrists , hands and feet in high detail , which is also the main focus of this section . 
mri is also useful for evaluating other anatomical sites involved by ra , such as the knees , hips , elbows and shoulders , and the axial skeleton , particularly the cervical spine . 
comparisons with miniarthroscopy and histopathological findings have documented that mri synovitis , as determined by gd - enhanced t1 - weighted mri , reflects true synovial 1 3 1132 la radiologia medica ( 2019 ) 124 : 11281141 fig . 
from left to right : coronal t1w non - fat - saturated spin echo , coronal stir , coronal 3d gradient echo fat - saturated post - contrast vibe sequence and coronal dce - mri analyzed with dynamika , image analysis group , london , uk . 
 note the severe bme on the initial stir and the moderate - to - severe synovitis ( bright enhancing tissue ) on the 3d vibe sequence as well as the high perfusion / inflammation in the dce - image ( seen as bright and white colors )  . 
this is associated with a significant reduction in the bme on the stir , a slight reduction in contrast enhancing synovitis on the vibe sequence , and a significant reduction in the perfusion in the enhancing synovium , seen as more dark red colors on the dce - mri inflammation [ 13 , 14 , 18 , 22 ]  . 
diagnostic tests are never 100% sensitive or specific , and thus , the inherent trade - off between false - negative and false - positive rates as one increases the positivity criterion for that test must be considered carefully in light of the particular role that imaging is being asked to play . 
the tendon itself may appear normal , or it may be thickened , irregular and / or have increased signal intensity within the tendon on t2w / pdw and stir images ( tendonitis ) , or with complete or incomplete tears [ 28 ]  . 1 3 la radiologia medica ( 2019 ) 124 : 11281141 1133 table 1 definitions of important ra joint pathologies [ 4 ] synovitis : an area in the synovial compartment that shows above - normal post - gadolinium enhancement ( signal intensity increase ) of a thickness greater than the width of the normal synovium mri bone erosion : a sharply marginated bone lesion , with correct juxtaarticular localization and typical signal characteristics * , which is visible in two planes with a cortical break seen in at least one plane * * * on t1 - weighted images : discontinuity of the signal void of cortical bone and loss of normal high signal intensity of bone marrow fat . 
rapid post - gadolinium enhancement suggests presence of active , hypervascularized pannus tissue in the erosion * * other focal bone lesions and variations of normal anatomy must obviously be considered , but are generally distinguishable with associated mri osteitis / bone marrow edema : a lesion * within the trabecular bone , with ill - defined margins and signal characteristics consistent with imaging and clinical findings increased water content * * * may occur alone or surrounding an erosion * * high signal intensity on t2 - weighted fat saturation or stir images and low signal intensity on t1 - weighted images mri joint space narrowing : reduced joint space width compared to normal , as assessed in a slice perpendicular to the joint surface mri tenosynovitis : peritendinous effusion * and / or tenosynovial post - contrast enhancement * * , seen on axial sequences over 3 consecutive * high signal intensity on t2 - weighted fat - saturated / stir images * * enhancement ( signal intensity increase ) is judged by comparison with t1 - weighted images obtained before and after i.v. 
gadolinium - contrast slices bone marrow edema bone marrow edema ( figs.3 , 4 ) , i.e. , the presence of mrisigns of increased water in the bone marrow compartment , is frequently detected in ra . 
whereas erosions reflect bone damage that has already occurred , bme appears to represent the link ( forerunner ) from joint inflammation to bone destruction where persistent osteitis leads to trabecular bone loss and an erosion [ 3234 ]  . enthesitis agreement for detection of bone erosions in ra wrists and mcp - joints between mri and ct , the gold standard reference for detection of bone damage , provides evidence that mri erosions represent true bone damage [ 43 , 44 ]  . erosions are also frequently seen in other inflammatory arthritidies and erosive oa and are thus not specific for ra . 
 small erosion - like lesions can also be visualized in healthy controls and oa [ 25 , 26 ] , so to avoid overestimation , using strict definitions of bone erosion and other pathologies is essential . 
as an example , bone erosions should be visible in two planes , with a cortical break visible in at least one plane to be registered ( fig.4 ) , and the normal anatomy and pitfalls should be kept in mind [ 21 , 45 , 46 ]  . 
cartilage assessment in small joints requires high image quality and resolution , but reliable semi - quantitative assessment systems have been developed [ 3739 ]  . bone erosion bone erosion ( fig.4 ) is detected with higher sensitivity with mri than radiography [ 4042 ] , and the very high level of all joints , tendons and ligaments of the cervical spine can be involved in ra , leading to spine instability or subluxations . 
the primary imaging modality is cr , but mri can provide detailed information on bone and soft - tissue abnormalities , e.g. , the pannus tissue around the odontoid process , and this can be a valuable supplement to radiographic evaluation ( fig.7 ) [ 2 , 48 , 49 ]  . 
mri erosions of the atlas and reduced subarachnoid space are associated with subsequent clinical neurological dysfunction [ 50 ] , and cord compression on mri better predicts subsequent clinical deterioration than initial clinical and radiographic features [ 51 ]  . 
a mid - sagittal 3d ct reformat of the cervical spine shows degenerative spine changes on multiple cervical levels as well as block vertebrae between c4 and c5 with approximately 5mm anterolistesis between c3 and the block vertebrae . 
bd corresponding mri of the same mid - sagittal slice , b t1 weighted image without fat saturation , c t2 - weighted image without fat saturation and d stir image showing capsular hypertrophy , small erosions in the c1 / c2 articulation and slight bone marrow edema of dens axis on the stir sequence ( arrow heads )  . 
e a lateral right - sided 3d ct sagittal reformat at the level of the articulation between massa lateraris and axis c2 showing severe erosive changes in the articulation between massa lateralis of c1 and c2 ( arrowheads ) along with degenerative changes of the upper cervical facet joints ( arrows )  . 
fh corresponding mri of the same right - sided sagittal slice , f t1 - weighted image without fat saturation , g t2 - weighted image without fat saturation and h stir showing destruction of the right - sided articulation between massa lateralis c1 and axis c2 ( arrowheads ) as well as severe bone marrow edema of the adjacent bone of the articulation on the stir sequence h ( arrows )  . 
facet joint degeneration is also noted ( open arrows ) for the use of imaging in ra clinical management state that monitoring of functional instability of the cervical spine by lateral radiograph obtained in flexion and neutral should be performed in patients with clinical suspicion of cervical involvement . 
furthermore , eular recommends that mri should be performed when the radiography is positive or specific neurological symptoms and signs are present [ 2 ]  . diagnosis longitudinal studies of undifferentiated arthritis have documented an independent predictive value of mri in the subsequent diagnosis of ra [ 52 , 53 ]  . 
in patients with undifferentiated arthritis , a prediction model , including clinical hand arthritis , morning stiffness , positive rheumatoid factor and mri bone edema score in metatarsophalangeal and wrist joints , correctly identified the development of ra or non - ra in 82% of patients [ 53 ]  . 
in another study of undifferentiated arthritis , assessment of mri tenosynovitis was helpful in predicting future ra diagnosis or disease - modifying antirheumatic drugs therapy initiation [ 54 ]  . in the acr / eular 2010 criteria for ra [ 55 ] , classification as definite ra is based on the presence of definite clinical synovitis ( swelling at clinical examination ) in 1 joint , absence of an alternative diagnosis that better explains the synovitis and achievement of a total score 6 ( of a possible 10 ) from the individual scores in four domains . 
t1 - weighted mid - sagittal mri of cervical spine ( c ) and corresponding t2 - weighted mid - sagittal image ( d ) in the same patient as ( b ) show capsular hypertrophy and erosions in the top of dens axis ( c2 , arrows ) specific for ra , and the acr / eular criteria are classification not diagnostic criteria . 
in the diagnostic process of the individual patient in routine clinical practice , it is important always to consider the clinical context ( as with any other diagnostic test ) to avoid over - diagnosis , since mri findings are not pathognomonic . 
the eular recommendations also state that when there is diagnostic doubt , cr , ultrasound or mri can be used to improve the certainty of a diagnosis of ra above clinical criteria alone . 
the recommendations also state the presence of inflammation seen with ultrasound or mri can be used to predict the progression to clinical ra from undifferentiated inflammatory arthritis [ 2 ]  . monitoring disease activity andstructural damage clinical trials andobservational cohorts to be valuable for monitoring joint inflammation and destruction , a method must be truthful , reproducible and sensitive to change . 
several randomized controlled trials have documented the superior ability of mri to discriminate the effects of different therapies in inhibiting progressive structural bone and cartilage damage [ 38 , 6163 ]  . 
the omeract erosion score is closely correlated with erosion volumes estimated by mri and ct [ 64 ]  . due to the high responsiveness and discriminatory ability , and because mri has demonstrated criterion validity for osteitis and synovitis with histology and construct validity for erosions when compared with ct , there has been a rapid increase in the use of mri in ra randomized clinical trials over the past decade . 
a report by the imaging subcommittee of the american college of rheumatology ( acr ) clinical trials task force [ 65 ] concluded that mri met the omeract validation filter for truth , discrimination and feasibility [ 66 ] and that mri best serves the purpose of achieving sensitive ascertainment of structural damage in rcts while also providing objective measures 1 3 1136 la radiologia medica ( 2019 ) 124 : 11281141 fig . 
a , b two sagittal 3d ct reformats show degenerative spine changes on multiple cervical levels as well as a block vertebra between c4 and c5 with approximately 5mm anterolistesis between c3 and the block vertebrae . 
c coronal reformat shows erosions in the left part of the dens axis ( black arrowhead ) , and there are severe erosive changes in the articulation between massa lateralis of c1 and c2 on the right side . 
e axial slice at the level of the articulation of the neck of dens axis ( * ) , in the plane corresponding to the black dotted line in c . 
white arrowheads point at severe erosive changes in the articulation between massa lateralis of c1 and c2 on the right side also seen in c of inflammatory predictors of damage [ 65 ]  . 
quantitative methods , like dynamic mri ( fig.5 ) and quantitative volume determination of synovitis , bme and erosion based on supervised machine learning techniques may also prove useful for early treatment - induced changes in joint inflammation [ 15 , 19 ]  . 
all these methods are used in clinical trials but require further validation before clinical use can be recommended . routine clinical practice the eular recommendations state that us and mri are superior to clinical examination in the detection of joint inflammation ; these techniques should be considered for more accurate assessment of inflammation [ 2 ]  . 
however , important questions remain about when such imaging is needed and when it is cost - effective to do ? there is a lack of studies to document exactly how mri should be used for this purpose , e.g. , imaging is not needed to assess disease activity if the patient has obvious clinical signs of active ra . 
for assessment of inflammation in the bone ( osteitis ) , mri is currently , however , the only available modality , and it is also the best method , except for computed tomography ( ct ) , for monitoring of progression of erosions [ 30 , 31 , 4244 , 67 , 68 ]  . 1 3 la radiologia medica ( 2019 ) 124 : 11281141 predicting disease outcome the predictive value of mri - detected pathology in wrist and / or mcp joints for subsequent radiographic progression in bilateral hands , wrists and feet is well established . 
regression analyses in 3and 5 - year follow - up in two cohorts and in clinical trials have documented that mribone edema is a strong predictor of short - term and long - term ( 3 , 5 and 11year ) radiographic progression [ 7174 ]  . thus , mri in early ra is a useful method to predict patients with potentially worse outcomes , which may assist the clinician in the choice of treatment strategy . 
in agreement with this , the eular recommendations for the use of imaging in the clinical management of ra state mri bone edema is a strong independent predictor of subsequent radiographic progression in early ra and should be considered for use as a prognostic indicator [ 2 ]  . 
this indicates that the presence of bme could be used as an inclusion criterion in clinical trials to enrich the study population for patients with high risk of structural progression . utility inclinical remission another issue of high clinical importance is whether mri is useful to predict the disease course in patients in clinical remission . 
mri synovitis and bme are found frequently in patients in clinical remission [ 75 , 76 ] , and these findings are significantly related to subsequent progressive structural damage [ 7779 ]  . 
these encouraging results are acknowledged in the eular recommendation , which states that mri and us can detect inflammation that predicts subsequent joint damage , even when clinical remission is present [ 2 ]  . mri may assist in predicting the success of tapering of biologics . 
in a cohort of routine care ra patients in sustained remission on biological disease - modifying antirheumatic drugs ( bdmards ) , the bdmard therapy was tapered according to a predefined treatment guideline . 
successful tapering was independently predicted by 1 previous bdmard , male gender , low baseline mri combined inflammation score ( synovitis , tenosynovitis and bone marrow edema ) and / or combined damage score ( erosion and joint space narrowing ) [ 80 ]  . 
the available data encourage further exploration of mri for predicting the disease course and for evaluating disease status , including defining remission . computed tomography although still limited in soft - tissue contrast , conventional ct offers fast and reliable acquisition , high resolution 1137 and multiplanar capabilities that have promoted its use in recent years . technical aspects ct image acquisition is no longer restricted to the axial plane , and its multiplanar capability is now so pronounced that many ct scans of the body are now interpreted primarily from thin - slice coronal or sagittal reconstructions in the same way as mri . 
while this is not a problem with the more distal extremities as the exposure doses are smaller and the tissue more radiation resistant , it remains an issue for spine , hip and shoulder ct . 
consequently , in most routine clinical situations , radiography provides sufficient information of a similar nature to ct for clinical decision making and x - ray is cheaper than ct and readily available ; ultrasonography is a better and cheaper way to visualize and quantify superficial softtissue pathology ; and mri offers superior soft - tissue contrast and bone marrow imaging . 
however , the technology continues to evolve and low - dose ct ( ldct ) is becoming more common with iterative reconstruction techniques which reduce the radiation exposure by about 80% [ 81 ]  . 
 exposure dose from ldct of the sacroiliac joints is similar to radiography and should probably replace radiography as a first - line test in many cases [ 82 ]  . ldct can quantify bone formation in the spine which is undetectable with radiography [ 83 ] , and cone - beam ct is a new technology that can detect bone erosion in extremities at extremely low dose [ 84 ]  . dual - energy ct ( dect ) allows the separation of calcium containing bone from the soft - tissue components , thereby allowing detection of soft - tissue changes that were previously invisible with ct . 
for example , it has recently been reported that dect can detect bone marrow edema ( bme ) with good reliability even in the small bones of the wrist in patients with ra [ 85 , 86 ]  . dect can also be used for analysis of composition of some specific tissues , most particularly urate crystals which can be detected in bone and soft tissues ( see below )  . 1 3 1138 ct inra the excellent contrast between bone and soft tissue makes ct a gold standard reference for the detection of bone damage , including erosion in ra . 
modern ct with isotropic voxel acquisition and three - dimensional visualization allows accurate detection and quantification of bone erosion with good intra - observer agreement , whereas radiography is limited by its two - dimensional projection and superimposition of structures . 
however , ct is very limited in ability to visualize soft - tissue changes and even with contrast enhancement and complex subtraction techniques ; ct is still inferior to mri and ultrasound for assessment of synovial changes such as thickening and hyperemia . 
detection of erosion on ct and mri shows very good agreement although ct is slightly more sensitive [ 43 , 67 ]  . diagnosis , monitoring andprognostication ct is not currently used in routine clinical practice for the diagnosis of ra . 
ct is used for problem solving in specific cases such as examination of the cervical spine if mri is unavailable or contraindicated . ct may potentially be useful for longitudinal assessment of damage progression [ 67 , 87 ] , and ct can detect and quantify repair of erosion [ 88 ]  . 
no ct data are available for prognostication in ra , and current use of ct in ra is very limited but since it has been shown to detect erosion reliably [ 89 ] , scoring systems are in development [ 90 ]  . conclusion mri is a sensitive imaging modality which has documented utility in diagnosis , monitoring and prognostication of patients with ra , and important new knowledge and technical improvements are continuously being acquired . 
several questions regarding the optimal use of these imaging modalities in routine practice and in clinical trials still need to be scientifically explored . compliance with ethical standards conflict of interest m has received speaker / consultant fees from abbvie , bms , boehringer - ingelheim , celgene , eli lilly , hospira , janssen , merck , novartis , novo , orion , pfizer , regeneron , roche and la radiologia medica ( 2019 ) 124 : 11281141 ucb , and research grants from abbvie , celgene , centocor , merck , and novartis . 
mb is a shareholder of image analysis group and serves as a consultant to image analysis group , eli lilly , esaote , celgene , pfizer , abbvie , carestream / canon , siemens and astrazeneca . ethical standards the manuscript does not contain clinical studies or patient data . 
this article does not contain any studies with human participants or animals performed by any of the authors . la radiologia medica ( 2019 ) 124 : 10671070 editorial musculoskeletal imaging oftheinflammatory anddegenerative joints : current status andperspectives faustosalaffi1 marinacarotti2 antoniobarile3 received : 22 january 2019 / accepted : 11 february 2019 / published online : 27 february 2019 italian society of medical radiology 2019 this special issue of la radiologia medica focuses on the broad spectrum of musculoskeletal imaging , ranging from essential concepts for inflammatory ( rheumatoid arthritis , spondyloarthritis , gout , and other crystal - induced arthropathies ) , noninflammatory ( osteoarthritis , algoneurodystrophy ) to multisystem and general pathologic conditions ( such as large - vessel vasculitis ) [ 1 ]  . 
musculoskeletal radiology continues to evolve rapidly , benefiting from advances as continually improving magnets , sequences in magnetic resonance imaging ( mri ) as well as the broader application of plain radiographs , ultrasonography ( us ) , and computed tomography ( ct ) , or dual - energy ct ( dect ) to the musculoskeletal system [ 2 ]  . 
the articles included in this focused issue have been compiled with a goal to provide a valuable resource for residents , fellows , and practicing radiologists and rheumatologists in the scope of a shared interpretation of musculoskeletal imaging examinations . 
both specialties have a duty to use imaging cost - effectively , to provide the requisite clinical information on diagnosis , prognosis , and serial assessment with the least risk and inconvenience to the patient . 
radiographic progression is one of the most important outcome measures in ra and psa clinical trials , because it reflects historical disease activity , is associated with loss of function over time , and can be reliably assessed [ 3 ]  . 
the various scoring systems for the two diseases are detailed in this issue . the potential applications of us in rheumatology have been further increased with the dawn of the third generation us machines , equipped with very high - frequency probes ( > 10mhz )  . 
these can reach a spatial resolution of less than a tenth of a millimeter and make it possible to study the finest details of the smaller joints and hand tendons which are involved early on in ra [ 58 ]  . 
in their article , ultrasound imaging in rheumatoid arthritis , emilio filippucci , md , and colleagues at the rheumatological clinic , universit politecnica delle marche , ancona ( italy ) , provide an overview of the main studies focusing on the value of us in the assessment of the patients with ra , and discussing the elementary lesions detectable by us ( synovitis , bone erosion , cartilage damage , tenosynovitis , and tendon damage ) , the scoring systems currently available , and the scanning protocols in definite clinical settings ( undifferentiated arthritis , early and long - standing ra )  . several sonographic abnormalities may be also observed in patients with osteoarthritis ( oa ) [ 9 ]  . 
federico bruno , md , and colleagues at the department of biotechnology and applied clinical sciences , university of laquila , laquila ( italy ) , in the article new advances in mri diagnosis of degenerative osteoarthropathy of the peripheral joints , provide an overview of the recent applications of advanced mri techniques for the evaluation of degenerative joint changes ( notably affecting the articular cartilage ) that could have a great potential in the early diagnosis and treatment monitoring . while conventional radiography and us remain a mainstay in rheumatology , mri and ct have become increasingly common in patient care . 
efficient methods for diagnosis , monitoring , and prognostication are essential in early ra [ 13 , 14 ]  . mikkel stergaard , md , ph.d. , and colleagues at the center for rheumatology and spine diseases , rigshospitalet , glostrup , and the department of clinical medicine , university of copenhagen , copenhagen ( denmark ) , focused on the potential uses of mri and ct in the clinical management of patients with suspected or definite ra in the article imaging in rheumatoid arthritis : the role of magnetic resonance imaging and computed tomography . 
 after sections on technical aspects , it described the current knowledge on mri and ct for early detection of ra manifestations , diagnosis of ra , monitoring of disease activity and joint damage progression , and its role in determining prognosis . sacroiliac ( si ) joints involvement is a crucial feature of inflammatory spondyloarthropathies , and mri is the gold standard imaging modality to detect si pathology [ 1518 ]  . 
maria antonietta mazzei , md , and colleagues at the department of medical , surgical and neuro sciences , diagnostic imaging , university of siena ( italy ) , in their article magnetic resonance imaging of the sacroiliac joints in spa : with or without intravenous contrast media ? , discuss the different mr imaging modalities and strategies available for the assessment of inflammatory involvement of the si joints , with a particular focus on the advantages and limits in the use of intravenous contrast media . despite the recognized fundamental role of mri in inflammatory joint pathologies , as previously discussed , mr imaging findings in clinical diagnostic criteria often lack specificity , giving potential diagnostic issues especially in the differential diagnosis with degenerative alterations [ 1820 ]  . 
to further explore this topic , in the contribution mri of the axial skeleton : differentiating non - inflammatory disease and axial spondyloarthritis a review of current concept and applications , ernesto la paglia , md , and colleagues at the department of radiology , ospedale ss . 
antonio e biagio e cesare arrigo , alessandria ( italy ) , discuss the main mri signs and the possible differential diagnoses , to improve the diagnostic performance in inflammatory spondyloarthropathies . although the role of imaging in the diagnosis , staging , and disease monitoring has been well established [ 21 ] , several anatomical variants and imaging artifacts can show unusual appearance , leading to possible diagnostic pitfalls [ 22 , 23 ]  . 
marcello zappia , md , and colleagues at the dipartimento di medicina e di scienze della salute vincenzo tiberio , universit del molise , campobasso ( italy ) , with their contribution diagnostic imaging pitfalls in rheumatology , illustrate the possible imaging pitfalls encountered in the imaging of inflammatory joint pathologies , and the available tips and tricks to recognize them . with the assistance of innovations in scanner engineering and software design , dual - energy ct ( dect ) is a recently developed advanced imaging method in the last decade . 
the principal advantages of dect over conventional ct in the musculoskeletal setting relate to the additional information provided regarding tissue composition , artifact reduction , and image optimization [ 2426 ]  . 
marina carotti , md , and colleagues at the radiology department , universit politecnica delle marche , ancona ( italy ) , focus on applications of dect to musculoskeletal imaging including gout and other crystal - induced arthropathies , virtual non - calcium images , and the study of bone marrow lesions in the application of dual - energy computed tomography in the diagnosis of musculoskeletal disordersa review of current concepts and applications . the next portion of this monograph is dedicated to multimodality musculoskeletal imaging . 
where are we now ? , marwin gutierrez , md , and colleagues at the instituto nacional de rehabilitacin , mexico city , and rheumatology section , center of excellence of rheumatology , mexico city ( mexico ) , provide an overview of the role of us in the assessment of ild in rheumatic disorders and discuss the current evidence supporting its clinical application in daily clinical practice . lvv is the most common form of primary vasculitis comprising giant cell arteritis ( gca ) , takayasu arteritis ( tak ) , and idiopathic aortitis ( ia ) [ 31 , 32 ]  . 
early diagnosis and treatment of lvv are paramount to reduce the risk of ischemic complications such as visual loss and strokes , vascular stenosis and occlusion , and aortic aneurysm formation . 
use of imaging modalities ( us , mri , ct , and [ 18f ] - fluorodeoxyglucose positron emission tomography ( pet ) ) has steadily increased to enable assessment of cranial and extracranial arteries , as well as the aorta . 
key concepts underlying lvv are covered in the article eular recommendations for the use of imaging in large vessel vasculitis in clinical practice by mohammad bardi , md , and colleagues at the department of rheumatology , university of british columbia ( canada ) , that summarizes the current evidence of imaging in patients with or suspected of having lvv and highlights the clinical implications of the eular recommendations . as guest editors , we hope that this monograph issue provides residents , fellows , and practicing radiologists and rheumatologists with a strong foundation for interpreting multimodality musculoskeletal imaging studies . 
these include quantitative analysis and several compositional techniques ( t1 and t2 relaxometry measurements and mapping , sodium imaging , delayed gadolinium - enhanced mri of cartilage dgemric , glycosaminoglycan - specific chemical exchange saturation transfer gagcest , diffusion - weighted imaging dwi and diffusion tensor imaging dti )  . 
these compositional mri techniques may have the potential to serve as quantitative , reproducible , noninvasive and objective endpoints for oa assessment , particularly in diagnosis of early and pre - radiographic stages of the disease and in monitoring disease progression and treatment effects over time . keywords mri cartilage advanced imaging t2 mapping dwi introduction degenerative osteoarthritis ( oa ) of the peripheral joints is a leading cause of musculoskeletal - related morbidity and disability in the western world [ 15 ]  . 
the pathological process of oa influences different articular structures , but the degeneration of articular cartilage represents the * antonio barile antonio.barile@cc.univaq.it 1 department ofbiotechnological andapplied clinical sciences , university oflaquila , via vetoio 1 , 67100laquila , italy 2 department ofprecision medicine , university ofcampania luigi vanvitelli , naples , italy 3 department life andhealth v . 
tiberio , university ofmolise , campobasso , italy 4 department ofradiology , scientific institute casa sollievo della sofferenza hospital , university offoggia , foggia , italy 5 department ofradiology , ospedali riuniti , universit politecnica delle marche , ancona , italy primum movens in the pathogenic phases of the disease . 
magnetic resonance imaging ( mri ) , due to its excellent soft tissue contrast resolution , is the preferred imaging tool for the evaluation of different diseases affecting the musculoskeletal system , both for diagnostic and interventional purposes [ 1126 ]  . 
in the last years , several advanced mr imaging sequences and techniques were developed to provide a global , sensitive and specific assessment of the joint degenerative processes with semiquantitative , quantitative and compositional analysis methods [ 30 , vol . : ( 0123456789 ) 1 3 1122 la radiologia medica ( 2019 ) 124 : 11211127 31 ]  . 
in this article , we review the recent advances in mri techniques and analysis to assess degenerative articular changes in the peripheral joints . applications andresults semiquantitative analysis semiquantitative mr scoring systems are based on the global morphological evaluation of pathological changes ( e.g. , alterations of articular cartilage , subchondral bone , fibrocartilages ) that affect the functional and structural joint integrity and determine the severity of the disease [ 32 ]  . 
four scoring systems were established for the knee : the whole organ magnetic resonance score ( worms ) , the knee osteoarthritis scoring system ( koss ) , the boston - leeds osteoarthritis knee scoring ( bloks ) and the moaks ( mri osteoarthritis knee score )  . 
concerning the method , in brief , the joint is divided into several articular compartments / subregions ( e.g. , medial and lateral tibia , medial and lateral femoral condyle ) , and several joint features ( e.g. , cartilage signal and morphology , synovitis , subchondral bone ) are analyzed and scored according to the severity of the involvement ( fig.1 ) [ 34 ]  . numerous studies validated the reproducibility of these scoring systems ; the moaks is currently the most used one for the knee , bringing together the advantages of all scoring systems [ 35 ]  . 
the clinical value of semiquantitative analysis was demonstrated by the presence of some specific alterations ( such as hoffa synovitis , joint effusion , medial meniscus lesions ) being associated with an increased risk of oa radiographic progression . 
other studies , using these methods for transversal and longitudinal comparisons of the disease evolution , highlighted how the presence of cartilage damage and the presence of subchondral edema correlate with an increased risk of prosthetic surgery [ 10 , 32 , 33 , 3540 ]  . quantitative evaluation quantitative assessment with mri provides a more sensitive and specific assessment of the degree of cartilage degeneration and is superior to semiquantitative techniques to evaluate structural changes [ 38 , 39 , 41 ]  . technique three - dimensional ( 3d ) , high - resolution sequences are required to image the bonecartilage interface and the cartilage surface with adequate contrast . 
this allows extracting data sets and image reconstructions to evaluate several quantitative features ( e.g. , cartilage thickness , area , volume ) as continuous variables [ 31 , 33 , 42 ]  . applications andresults studies on quantitative cartilage evaluation showed good inter - operator reproducibility at different degrees of cartilage degeneration and excellent correlation with the surgical and histological findings . 
quantitative methods have a good correlation with semiquantitative results , even if more sensitive and specific in predicting cartilage loss ( especially in small widespread defects using regional analysis ) ; for these reasons , some authors suggest a combined use of the techniques [ 31 , 3335 , 38 , 39 , 4144 ]  . 
changes in cartilage volume and thickness were used as an outcome in observational studies and trials of pharmacological treatments ( e.g. , chondroitin sulfate ) , physical therapy and rehabilitation , and surgical treatments . 
the need for dedicated software and the timeconsuming analysis is still considered a major disadvantage to the routine clinical application [ 5 , 30 , 40 , 4548 ]  . compositional analysis articular cartilage is made of chondrocytes , spersed within a matrix composed of water and a highly organized network of collagen proteoglycans ( pgs ) and glicosaminoglycans ( gags )  . 
notably , these matrix changes , however , are not apparent on standard morphological mri sequences in the early stages of oa development [ 9 , 31 , 45 , 48 , 49 ]  . 
based on the knowledge of the pathogenesis of joint degenerative processes , we know that at the histological level the biochemical changes in cartilage ultrastructure , including the reduction of proteoglycans ( pgs ) and glycosaminoglycans ( gags ) and the increase in water content , precede morphological changes . 
in particular , mr imaging techniques are based on the modification of one or more cartilaginous ultrastructural components ( e.g. , gag , pg ) [ 40 , 50 ]  . 
these techniques include the measurement of relaxation times ( t2 and t1rho mapping ) , the sodium imaging , the delayed gadolinium enhancement mri of cartilage ( dgemric ) imaging , the chemical exchange saturation transfer imaging of gag ( gagcest ) imaging and the diffusion imaging ( dwi and dti ) [ 30 , 40 , 42 , 45 , 48 , 49 , 51 ]  . techniques t2 andt1 mapping these sequences measure the t1 and t2 relaxation times ( expressed in ms ) of the molecules present at the tissue level . 
the relaxation time of t2 is measured as a function of the signal measured in multi - echo se and fse t2 - weighted images with mono or multi - exponential decay curve at the different echo times ( te ) [ 42 , 5255 ]  . t1 mapping is a compositional technique sensitive to regional changes in cartilage matrix proteoglycans fig . 
on the right , quantitative analysis through the positioning of regions of interests ( roi ) shows t2 relaxation times in the normal value range 1 3 1124 la radiologia medica ( 2019 ) 124 : 11211127 fig . 
t2 mapping color map b clearly depicts the same area in red , and quantitative analysis c confirms higher t2 relaxation time values , consistent with collagen degradation and thus chondropathy characterized by continuous resonance rf pulse . 
the main disadvantages are represented by issues related to high sar ( due to the application of long - lasting rf pulses ) and long acquisition times [ 42 , 45 ]  . a fundamental advantage of relaxation mapping sequences is that contrast medium administration is not necessary . 
in addition to dwi , diffusion tendon imaging ( dti ) can also be applied to cartilage imaging , evaluating the fractional anisotropy as a parameter of water diffusion in various spatial orientations . 
similar to other compositional sequences , diffusion and tensor parameters correlate with the collagen and proteoglycan content within the cartilage matrix and can be used as quantitative markers of cartilage degeneration . 
the loss of collagen and proteoglycan can lead to significant increases in mean diffusion ( fig.4 ) [ 40 , 42 , 43 , 49 , 51 , 55 ]  . sodium imaging ( 23na ) this compositional imaging technique is based on the detection of sodium , the positive cation linked to the negatively charged glycosaminoglycan ( gag ) of the cartilage matrix . 
axial dwi sequence b with adc map c confirms altered cartilage matrix with increased diffusion values 1 3 la radiologia medica ( 2019 ) 124 : 11211127 1125 more specifically , the concentration of sodium within the cartilage matrix is directly correlated with the concentration of gag and therefore proteoglycan . 
however , one of the limits of invivo sodium imaging of cartilage is the low intrinsic snr , caused by the low 23na mri signal compared to the one from protons [ 30 , 40 , 42 , 43 , 45 , 49 ]  . delayed gadolinium enhancement mri ofcartilage ( dgemric ) increasingly important in studies of cartilage degeneration and in testing new therapies for prevention or delaying the progression of oa , due to the recent widespread development of disease - modifying drugs and regenerative therapies ( e.g. , platelet - rich plasma , hyaluronic acid , chondrocyte implantation ) [ 5661 ]  . 
as the efficacy is closely connected with the early establishment of treatment , their use requires suitable biomarkers to provide an early diagnosis and detect signs of progression during treatment [ 62 ]  . 
advanced mri findings can represent , in this scenario , a powerful tool to understand how to best treat and manage oa and possibly will allow making a target - based therapy for every single component of the cartilage matrix [ 6366 ]  . for this imaging method , the administration of contrast medium ( gadolinium ) , injected intravenously , is necessary . 
 gadolinium is negatively charged and is rejected by positively charged gags in cartilage , while in the case of cartilage matrix degradation , the amount of contrast in cartilage tissue will be increased in an inversely related manner . 
the dgemric technique showed high sensitivity and specificity ; the routine clinical use is limited by the need for high doses of gadolinium [ 40 , 42 , 45 , 46 , 49 , 51 ]  . chemical exchange saturation transfer imaging ofgag ( gagcest ) this sequence is based on the constant transfer of labile protons between solutes ( in the case of cartilage , gags ) and water . 
the proton transfer between water molecules and the one between water and gag are different , and the difference between waterwater transfer and watergag transfer is measured as the magnetic transfer ratio . 
unfortunately , strong magnetic fields ( 7t scanners ) are required to obtain sufficient signal , thus widespread use , even in the research field , is currently limited [ 42 , 45 , 49 ]  . conclusion advanced imaging mri techniques for the study of joint degenerative pathology represent a particularly promising field of research , which has seen significant developments in recent years . 
in particular , we revise the general aspects common to most procedures and the different imagingguided interventions which can be performed around joints , soft tissues , and spine . keywords interventional radiology ultrasound musculoskeletal system tendinopathy osteoarthritis introduction imaging - guided interventional procedures have become increasingly popular in the treatment of several pathologic conditions in the musculoskeletal systebesides oncological treatments , musculoskeletal procedures can be performed to treat differentdegenerative or inflammatory conditions . according to the anatomical location to reach , interventional procedures can be guided using ultrasound , fluoroscopy , computed tomography ( ct ) , or occasionally magnetic resonance ( mr ) [ 1 ]  . 
ultrasound has certainly the great advantage of being cheap , quick , available , real time , and radiation - free and is excellent to perform interventional procedures in the soft tissues [ 24 ]  . 
however , ultrasound may be occasionally limited by deep anatomical location of the * luca maria sconfienza io@lucasconfienza.it irccs istituto ortopedico galeazzi , via riccardo galeazzi 4 , 20161milano , italy 2 s.c. 
diagnostica perimmagini ed ecografia interventistica , ospedale evangelico internazionale , 16121genova , italy 3 dipartimento di scienze biomediche perla salute , universit degli studi di milano , 20100milano , italy 4 department ofinterventional radiology , university hospital ofstrasbourg , 67000strasbourg , france 5 dipartimento di radiologia interventistica , istituto europeo di oncologia irccs , 20100milano , italy area to treat or by the interposition of bony cortex , which does not allow ultrasound beam penetration . 
fluoroscopy and ct are more useful for procedures involving the bone , with the former more useful when longitudinal precision and real - time visualization are needed and the latter preferred when axial precision is required . 
 last , mr is theoretically ideal to guide procedures in the musculoskeletal system , as it combines most of the advantages of all other modalities ; however , the limited diffusion of dedicated magnets and high costs have remarkably limited its application in clinical practice . in the setting of degenerative and inflammatory conditions , interventional procedures can be used to perform joint injections or aspirations and procedures in or around tendons and bones . 
this paper is aimed to review clinical indications and technical aspects of these kinds of procedures . general aspects some aspects should be considered when performing interventional procedures in the musculoskeletal system : disinfection : all procedures should be performed under strict sterile conditions . 
sterile material ( gloves , draping , probe covers for ultrasound procedures ) should be used to ensure the best sterility possible in the operatvol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 11121120 1113 ing environment . 
accurate skin disinfection should also be achieved using specific antiseptic solutions ( e.g. , iodineor chlorhexidine - based solutions ) ; substances to inject : according to the disease to treat , different substances can be injected . 
theycan be particulate ( e.g. , triamcinolone acetonide , with lower solubility and longer duration of action ) or non - particulate ( e.g. , dexamethasone , with shorter duration of action )  . 
steroids are not contraindicated in diabetic patients , who should expect only a transient , non - clinically significant increase in glycemia ; however , an increased risk of infections should be considered in these patients . 
 blood products ( such as platelet - rich plasma [ prp ] or autologous blood ) have the purpose of delivering in the selected site growth factors contained in platelets . 
it is commonly used for bone procedures , such as vertebroplasty and kyphoplasty . patients positioning : the patient should be positioned according to the area to treat , so standardization cannot be done . 
however , for procedures other than simple injections , we recommend having the patient laying down on the examination bed to avoid the occurrence of vasovagal reactions ; ultrasound guidance : when performing ultrasoundguided procedures , the needle can be inserted with two different approaches with respect to the ultrasound bea more commonly , the in - plane modality is used , which implies that the needle is inserted perpendicularly and on the same plane of the ultrasound beam ( i.e. , needle is inserted oblique from the short side of the probe )  . 
in some cases , however , the out - of - plane approach might be preferred , which implies that the needle is inserted almost parallel to the ultrasound beam ( i.e. , needle inserted almost vertical on the long side of the probe )  . 
this approach requires greater experience , as only the needle tip is visualized , but is helpful in those cases where joint or peri - tendinous space is limited or when lateral access is precluded . post - procedure care : in case of simple procedures performed on an outpatient basis , we generally ask the patient to remain in our department for about 30min of observation after treatment . 
other , more complex procedures require a longer hospital stay . joint procedures for all joint procedures , ultrasound should be the guidance modality of choice , as it has comparable accuracy to fluoroscopy , but it does not use ionizing radiations [ 1 ]  . 
according to each specific anatomical site , the joint should be positioned to maximize effusion visibility in the desired recess ( e.g. , humerus maximally externally rotated to squeeze anterior joint recess and inflate posterior recess )  . 
in degenerative conditions , aspiration of synovial fluid is usually performed prior to injection , with the purpose of eliminating the highest amount of synovial fluid in the joint , which may potentially interfere with the effect of the injectable . 
with the same technique , synovial biopsies can be performed to obtain tissue specimens to confirm or clarify a clinical diagnosis of inflammatory arthritis . needles should be chosen according to the specific joint . 
 for fluid aspiration , generally large - bore needles ( e.g. , 1618 gauge ) should be preferred , as they do not get obstructed by synovial tissue or intra - articular debris . 
longer spinal needles ( 9cm ) are preferred for deeper joints such as the hip , 1 3 1114 la radiologia medica ( 2019 ) 124 : 11121120 while standard length needles ( 45cm ) can be used in all other joints . 
we prefer to use 14 gauge semiautomatic tru - cut needles . for simple joint aspiration , local anesthesia is not generally required , as the stingy sensation induced by anesthesia injection is very similar to the little discomfort induced by the aspiration needle . 
of note , we do not recommend injecting anesthesia inside the joint to avoid interference with the potential bacterial load . injections intra - articular injections are generally performed to treat degenerative or inflammatory or conditions not responding to oral anti - inflammatory drugs . 
for longer term pain relief , ha and prp may be considered in patients with degenerative joint disease . soft tissue procedures drainage offluid collections superficial or deep fluid collections include abscesses , hematomas , or seromas and can be treated with imageguided percutaneous aspiration or drainage . 
abscesses may develop from surgical procedures , trauma , foreign bodies , or the spreading of contiguous or distant infections , while hematomas and seromas are usually caused by traumatic events or after surgical procedures [ 5 ]  . ultrasound is the modality of choice to guide aspiration or drainage of fluid collections . 
the direct puncture technique is generally preferred , although in some cases , the tube can be also inserted over a previously placed guide wire ( seldinger technique )  . 
 tube caliber ranges from 6 to 24 fr and is selected according to the thickness of the collected fluid ; accordingly , highly corpuscolated collections ( e.g. , hematoma ) need large caliber tubes ( 1624fr )  . prior to aspiration of hematomas , the degree of internal coagulation should be evaluated ; in particular , operators should rule out an active bleeding supplying the hematoma before puncturing it . 
in the end , if no signs of infection are noted , it is preferred not to drain the hematoma . ganglion aspiration soft tissue ganglia can occur around multiple joints throughout the musculoskeletal systethey are more frequent between 2040years of age [ 7 ] and are commonly encountered around the wrist , where they represent the most common benign soft tissue lesion [ 8 ]  . 
however , aspiration has been reported to be effective in up to 89% of cases [ 29 ] , although recurrence is frequent . due to the superficial location of ganglion cysts , the procedure is generally performed under us guidance with inplane or out - of - plane approach using a large - bore needle ( 1618 gauge )  . 
when the ganglion is completely drained , a small amount of steroid is generally injected into the cavity and a compression dressing is applied for a week to minimize the risk of recurrence . an example of ganglion aspiration is shown in fig.1. bursitis bursitis is a common reactive process characterized by thickening of the bursal walls in response to an external stimulus , which may be associated or not with increased fluid content [ 9 ]  . 
septic bursitis may also occur , being transcutaneous route the most common way of contamination [ 10 , 11 ]  . intra - bursal steroid or ha injection of aseptic bursitis is generally performed for therapeutic purposes when patients do not benefit from conservative treatment to reduce local inflammation and pain case of septic bursitis , bursal drainage is mainly indicated for diagnostic purposes and steroids should not be used , as they reduce local immunity [ 12 ]  . bursal injection is performed under us guidance , inserting a 2123 gauge needle in the bursa with an in - plane 1 3 la radiologia medica ( 2019 ) 124 : 11121120 1115 is the so - called calcific enthesopathy , and it should not be confused with calcific tendinopathy , which is a totally different entity [ 1525 ]  . histopathological evaluation reveals disordered proliferation of tenocytes , a small amount of physiologic collagen fibers , an increase of fibrous matrix , and often neovascularization [ 26 ]  . 
in the chronic phases ( at least 6months ) , inflammation may be absent , suggesting that the inflammation process is probably a trigger in the pathologic process [ 26 ]  . 
us findings include tendon thickening with fibrillary disorganization , ill - defined hypoechogenicity , and increased vascularity on power doppler examination [ 27 , 28 ]  . several therapeutic procedures can be performed under us guidance to treat tendinopathy ; they range from the widely used peri - tendinous injection of corticosteroid or low molecular weight ha , dry needling , sclerotherapy , prolotherapy , high - volume injections ( hydrodissection ) , autologous blood injection , and newest regenerative therapeutic alternatives , including prp [ 29 ] and adipose - derived mesenchymal stem cells ( ascs ) [ 30 ]  . for all procedures , the probe is placed on the affected tendon with a longitudinal approach . 
in such cases , the purpose of the infiltrative therapy will be to bring the drug ( steroid or low molecular weight ha ) exactly in the space between the sliding sheath and the tendon to achieve the maximum therapeutic effect without damaging the tendon [ 3134 ]  . 
in case of stenosing tenosynovitis ( such as de quervains disease and trigger finger ) , steroid injection can be combined with a second treatment consisting in a injection of low molecular weight ha ( about 1ml ) between the tendon and the retinaculum or tendon sheath [ 35 ]  . 
this has the twofold purpose of favoring tendon gliding and stretching the peritendinous tissues , allowing to obtain satisfactory results up to 12months [ 36 ]  . an example of tendinopathy treatment is shown in fig.3. spine procedures interventional radiology can be of great value in the treatment of several spine conditions , which may cause back pain and disability . 
us guidance should help to pierce only one side of the bursa to avoid drug leakage in the underlying structures . an example of bursal injection is shown in fig.2. tendinopathies degenerative tendinopathy is characterized by structural degenerative changes of the collagen fibers with accompanying fibrosis , associated with minimal or no inflammation signs [ 13 ]  . 
following fj injection , pain relief is generally temporary ( weeksmonths ) and usually patients come back for recurring pain such cases , if patients have reported some pain relief following fj injections , they can be treated with thermocoagulation of the fj nerves ( i.e. , rhizolysis ) which is performed under local anesthesia and imaging guidance . selective nerve root block ( snrb ) nerve root compression is another very common cause of back pain , generally associated with neuropathic manifestations with electric sensation irradiating to the lower limbs with a typical dermatomeric distribution . 
us guidance can also be used . injections can be performed in the posterior epidural space or around the nerve roots . technically , the patient lies prone or supine ( e.g. , cervical peri - radicular injection ) or prone ( dorso - lumbar epidural or peri - radicular injections ) on the bed and a 20g spinal needle is used . when injection is performed in the posterior epidural space , a co2 - test is performed to confirm the epidural location of the needle before injecting the drug . 
if co2 dissection of the posterior epidural space is not achieved ( e.g. , post - surgical fibrosis ) , few ml of contrast medium can be injected to confirm the right location of the needle . 
despite the type of agent used ( co2 or contrast medium ) , the typical epidural location is confirmed when the injected lies around the dural sac with a reversed crescent shape morphology ( i.e. , concavity of the crescent facing the dural sac )  . on the other hand , when peri - radicular injections are performed , mainly at the cervical or lumbar level , the needle tip is advanced at the posterior distal aspect of the neuroforamen and few ml of contrast medium is injected to confirm that the needle is not inside a vessel , which contraindicated the steroidal injections . 
if the needle is correctly deployed , injected contrast medium spreads around the nerve root ( rootgram ) and sometimes also in the epidural space . once checked the correct needle position , a 1 : 1 solution of non - particulated steroid ( e.g. , dexamethasone ) and local anesthetic can be injected . 
of note , the use of non - particulated steroids in this anatomical area is very important especially in the cervical spine , as inadvertent intra - arterial injection may lead to thrombosis of distal branches with fig . 
p patellar tendon , h hoffas fat pad , arrow needle tip , asterisks dry needling procedure and prp injection and increased accuracy of spine procedures and is mainly based on fluoroscopy and computed tomography ( ct )  . 
more recently , the use of us + ct / mr fusion imaging has been reported [ 37 ]  . facet joint ( fj ) injections fj osteoarthritis is a very common and frequently overlooked painful condition , which can be considered responsible for 1540% cases of low back pain ( lbp )  . 
in the end , pain is evoked by palpation of the target fj and is increased with hyperextension of the spine . in such cases , fj injections with local anesthetics or and steroids can be performed . currently , fj injections are commonly performed under fluoroscopy or ct guidance , allowing excellent visualization of typical bony landmarks to guide the procedure . 
 the patient is positioned prone on the bed , and the joint is approached directly either with posterolateral or straight posterior vertical approaches to the joint using a 20 gauge spinal needle . 
correct needle placement may be confirmed by injecting a small amount of contrast agent ( 0.2ml ) , and 1 3 la radiologia medica ( 2019 ) 124 : 11121120 1117 potentially life - threatening sequelae . 
transient anesthesia in the innervated territories of the injected root is a minor side effect of this treatment . disc procedures intervertebral discs can be affected by both degenerative and inflammatory conditions [ 39 ]  . 
regarding inflammatory conditions , the disc may be affected by infections ( i.e. , spondylodiscitis ) and discal biopsy can be performed with similar technique . the procedure can be performed either under ct or fluoroscopic guidance . 
 however , in general , most of these symptoms disappear after two weeks . an example of disc biopsy is shown in fig.4. kyphoplasty andvertebroplasty vertebral compression fractures ( vcf ) represent a cause of thoracic and lumbar spine paalthough some vcf can be due to high - energy trauma , many of them occur after lowenergy or no trauma in patients with altered bone structure , such as metastases or osteoporosis [ 4448 ]  . 
 the main issue of osteoporosis is of being a silent disease , so if patients do not undergo routine screening , a fracture may be the only sign of this condition . 
conservative treatment for osteoporotic vcf includes pain management with bracing , rest , analgesic and anti - inflammatory drugs , calcium , vitamin d , and bisphosphonates supplements [ 50 ]  . 
4 76 - year - old male patient presenting with a systemic inflammatory syndrome and lumbar pa a contrast - enhanced sagittal t1 - weighted and b stir sequences raised the suspicion of l3 - l4 spondylodiscitis ( arrows )  . 
5 90 - year - old osteoporotic female patient presenting with spontaneous sudden dorsal paa sagittal t1 - weighted and b stir sequences showed t8 and t9 vertebral compression fractures ( arrows )  . 
the patient underwent double - level vertebroplasty under conscious sedation and local anesthesia ; fluoroscopic guidance was used to c deploy a 10g bone bevelled bone trocar in each vertebral body and to d inject the polymethyl methacrylate ( pmma ) , asterisk . 
final e fluoroscopic and f cbct images confirmed the good distribution of the pmma ( from endplate to endplate in the anterior two - thirds of the vertebral body ) in each treated level approach [ 53 , 54 ]  . 
extra - osseous approaches should be avoided due to the risk of iatrogenic injuries to the thoracic / lumbar artery supplying the anterior spinal artery ( adamkiewiczs artery )  . 
kyphoplasty is a modification of vertebroplasty , characterized by the percutaneous insertion of an inflatable high - pressure compliant balloons into the fractured vertebral body with the aim of restoring the vertebral height and create a cavity inside the vertebra where the cement can be injected for vf stabilization [ 56 ]  . 
both vertebroplasty and kyphoplasty can be performed under general anesthesia , although conscious sedation is also possible [ 5759 ]  . pmma is the most frequently used cement in both procedures , although new biological materials , such as calcium phosphate and hydroxyapatite , have been introduced [ 60 ] even though there is no general agreement on their use ; for this reason , pmma is still the most commonly used agent . vertebroplasty and kyphoplasty are equivalent in terms of early and long - term pain relief ; the only advantages of kyphoplasty over vertebroplasty are ( 1 ) higher chance to restore the kyphotic deformation , thus ideally having a better long - term impact on the respiratory function ; ( 2 ) a reduced risk of extra - osseous pmma leakage . 
in most of the cases , pmma leakages remain clinically nonsignificant unless large amount of pmma leak in the antero / lateral venous plexus with subsequent massive pulmonary embolism ; or in the posterior venous plexus with subsequent compression of the spinal cord . 
however , epidural injections can be performed with chilled saline and steroids to prevent neurologic complications related to the transient exothermic pmma polymerization . 1 3 la radiologia medica ( 2019 ) 124 : 11121120 1119 an example of vertebroplasty is shown in fig.5. conclusion interventional procedures in the musculoskeletal system can be used to treat a wide variety of inflammatory and degenerative conditions . 
the ra - induced morpho - structural changes can be effectively detected and measured by us , and us findings represent an additional advantage over clinical and laboratory evaluation , showing the face of the disease ( i.e. , proliferative synovitis ) and revealing its aggressive behavior ( i.e. , presence of bone erosions not detectable by conventional radiography )  . 
the present review provides an overview of the main studies focusing on the value of us in the assessment of the patients with ra , and discussing the elementary lesions detectable by us ( synovitis , bone erosion , cartilage damage , tenosynovitis and tendon damage ) , the scoring systems currently available and the scanning protocols in definite clinical settings ( undifferentiated arthritis , early and long standing ra )  . keywords ultrasonography rheumatoid arthritis synovitis bone erosion scoring systems introduction rheumatoid arthritis ( ra ) is a chronic inflammatory autoimmune disease , mainly involving the small joints of the hands and feet . 
in fact , further evidence is still required to increase specificity , reduce false positivity and provide knowledge 1 clinica reumatologica , universit politecnica delle marche , ospedale carlo urbani , via aldo moro , 25 , 60035jesi , ancona , italy 2 dipartimento diagnostica perimmagini , ulss 6 euganea , ospedale di cittadella , casa di ricovero , 40 , 35013 , cittadella , padova , italy 3 radiology department , universit politecnica delle marche , ospedali riuniti di ancona , via conca , 71 , 60126ancona , italy vol . : ( 0123456789 ) 1 3 1088 la radiologia medica ( 2019 ) 124 : 10871100 regarding the long - term impact of ultrasonography ( us ) findings . 
thus , us is still not included in the standard procedures recommended by eular for the management of early arthritis [ 57 ]  . over the last two decades , a growing number of studies have been published aiming at investigating the role of us in the diagnosis and follow - up of patients with ra . the analysis of the obtained results indicates that a systematic use of us may provide the following additional values [ 812 ] : in ra . 
b severe joint dislocation due to volar subluxation of the proximal phalanx together with extensive synovial proliferation ( * ) and massive erosive change in the metacarpal head ( arrow )  . 
in this scenario where many publications emphasize the importance of identifying synovitis and erosions at an early stage , it is essential to know the possible pitfalls which can determine both false positives and false negatives . 
the high variability of the musculoskeletal system anatomy makes it necessary to have a correct knowledge of all anatomical complexes , in order not to confuse them with the pathology . 
moreover , the correct and standardized method of the execution and interpretation of the exams , such as ultrasound , is crucial to identifying and correctly monitoring the pathological hallmarks of the arthritis . 
this paper aims to provide an instrument to radiologists , highlighting the main imaging pitfalls in ultrasound and magnetic resonance which may be encountered in daily practice . keywords mri arthritis ultrasound enthesitis pitfalls introduction joint effusion andsynovitis arthritis and spondyloarthritis represent a heterogeneous group of inflammatory disorders characterized by the chronic inflammation of joints and enthesis . although the diagnosis is still based on clinical criteria , imaging is of fundamental help in the management of these diseases [ 1 , 2 ]  . 
salvatore hospital , university oflaquila , laquila , italy 5 department ofradiology , pineta grande hospital , castelvolturno , ce , italy synovial effusion and synovitis are the hallmarks of chronic inflammatory arthritis and both ultrasound ( us ) and magnetic resonance ( mr ) are widely used for their detection , grading and monitoring [ 3 ]  . the us synovial effusion is defined as abnormal hypoechoic or anechoic ( relatively to the subdermal fat , but sometimes it may be isoechoic or hyperechoic ) intraarticular material that is displaceable and compressible ; the us synovial hypertrophy is defined as abnormal hypoechoic ( relatively to the subdermal fat , but sometimes may be isoechoic or hyperechoic ) intraarticular tissue that is non - displaceable and poorly compressible and which may exhibit a doppler signal [ 4 ]  . 
in fact colour doppler ( cd ) and power doppler ( pd ) us are able to detect pathological synovial blood flow in the synovial tissue , which reflects the inflammatory activity in the joint [ 5 ]  . one potential us pitfall is the failure to detect intraarticular fluid collection and synovial hyperaemia in the presence of synovial hypertrophy . in the small joint the excessive pressure of the us probe might dislocate the fluid on the other side with the result of hiding it ; moreover , the transducer pressure might compress the small synovial vessels with the disappearance of vol . : ( 0123456789 ) 1 3 1168 la radiologia medica ( 2019 ) 124 : 11671174 the doppler signal . 
in addition , if the us scan is performed with the hip in internal rotation , the anterior joint capsule becomes convex anteriorly measuring more than 7mm [ 9 ]  . normal fat structures can also simulate synovial hypertrophy at us scan , such as the prefemoral fat pad . 
3 a , d normal prefemoral fat pad ( arrows ) in a lateral transverse us scan of the superolateral recess of the knee simulates synovial hyperplasia but on b axial pd fatsat sequence mr image shows normal fat low signal . 
c , d true synovial hyperplasia appears hyperechoic on c transverse us scan and shows high signal on d axial pd fatsat sequence mr ( not distinguishable from synovial fluid ) 1 3 la radiologia medica ( 2019 ) 124 : 11671174 1169 fig . 
4 short axis pdus scan of the long head biceps tendon in its bicipital groove shows : a ascending branch of the anterior humeral circumflex artery ( black arrow ) that runs laterally to the tendon ; b synovial hyperplasia ( white arrows ) with several spots of doppler ( black arrows ) and sheath fluid distension ( star )  . 
5 long axis us scan of the fourth compartment extensor tendon at the level of radiocarpal joint showing the normal extensor retinaculum of the wrist ( white arrows ) that runs perpendicular to the tendons . 
6 long axis us scan of the achilles tendon enthesis shows a several doppler spots when the ankle is positioned in plantar flexion and b no doppler signal is visible in dorsiflexion . 
even the presence of a low grade of an inflammation severity should be considered with caution when it is used for a diagnostic purpose [ 15 ]  . tenosynovitis tenosynovitis is defined on b - mode us as an abnormal anechoic and / or hypoechoic ( relatively to tendon fibres ) tendon sheath widening , related both to the presence of tenosynovial abnormal fluid and / or hypertrophy [ 16 ]  . structures that may simulate an ultrasonographic tenosynovitis are the retinacula of the extensor tendons of wrist and ankle . 
8 pdw fatsat axial mr image of the shoulder showing two tendons ( arrows ) of the long head biceps in the bicipital groove 1 3 la radiologia medica ( 2019 ) 124 : 11671174 1171 fig . 
9 longitudinal dorsal us scan of the second metacarpophalangeal joint showing : a normal pseudoerosion ( arrow ) of metacarpal bone and b true erosion of the metacarpal head with cortical discontinuities ( arrow )  . 
mc metacarpal head , p2 second proximal phalanx ankle at the level of the radiocarpal and tibiotalar joints , running over the extensor tendons more or less parallel to thein healthy subjects the extensor retinacula measure 0.61.7mm in thickness over the wrist and 0.61.4mm over the ankle [ 17 , 18 ]  . 
the normal extensor retinaculum shows a subtle fibrillar echotexture when imaged perpendicular to the sound beam and it is slightly hypoechoic compared with adjacent tendons and with the subcutaneous fat . 
10 t1w coronal mr image of the wrist showing normal cortical irregularity of the radial aspect of the capitate bone ( arrow ) at the insertion of the scaphocapitate ligament 1 3 1172 fig . 
11 stir oblique coronal mr image of the sacroiliac joint showing accessory sacroiliac joint on the right side ( white arrow ) and the iliosacral complex on the left side ( arrowheads ) with intrarticular high signal due to the presence of several vessels la radiologia medica ( 2019 ) 124 : 11671174 enthesitis the us enthesitis is defined as an hypoechoic and / or thickened insertion of the tendon close to the bone ( within 2mm from the bony cortex ) which exhibits a doppler signal if it is active and which may show erosions and enthesophytes / calcifications as a sign of structural damage [ 20 , 21 ]  . probably the most insidious pitfall during the us scan of the enthesis is the failure to detect the doppler signal due to the position of the joint . 
on the contrary of the b - mode scan , during the pd - scan of the enthesis the tendon must not have tension , in order to avoid the collapse of the vessels and the hiding of the doppler signal . 
therefore , during the pdus scan of the achilles tendon , the ankle should be positioned in plantar flexion or in neutral position ( fig.6 ) ; during the pdus scan of the quadriceps and patellar tendons , the knee should be extended [ 2224 ]  . in mr a potential mistake is related to the identification of small enthesophytes and syndesmophytes when no active inflammation is present . 
the accessory tendon frequently has an extraarticular origin from the superior capsule , rather than the intraarticular one and some authors consider this structure as an aponeurotic expansion [ 29 , 30 ]  . 
the pseudoerosion is a cortical homogeneous depression ( mean depth , 0.3mm ) in 1 3 la radiologia medica ( 2019 ) 124 : 11671174 1173 the dorsal metacarpal head , located adjacent to the hyaline articular cartilage at the site of the dorsal joint recess [ 33 ]  . 
contrast material when evaluating the response to tumor necrosis factor ( tnf ) antagonists therapy , on the extension of bone marrow oedema ( bmo ) and pathological enhancement ( osteitis / synovitis ) in the sacroiliac joints ( sijs ) on mri . materials and methods forty - three patients ( 25 females and 18 males , mean age of 54 16.60years , range 2275years ) with a clinical diagnosis of spa and active sacroiliitis at mri with i.v. 
scores difference ( improvement ) after treatment was calculated in the mri sequences both with and without contrast agent ( respectively , mean value and range 3.18 , 012 with contrast and 1.63 , 07 without contrast )  . 
inflammation at either ligament , tendon or capsular insertions , or enthesitis , is the hallmark of these diseases which consist of ankylosing spondylitis , psoriatic arthritis , arthritis / spondylitis associated with inflammatory bowel disease and reactive arthritis [ 1 ]  . 
in fact , sacroiliac joints ( sijs ) involvement is one of the key criteria for diagnosing axial spa , which usually occurs in the first stages of the vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 11421150 1143 sacroiliitis at mri with i.v. 
inclusion criteria included meeting the asas classification criteria for spa and not being treated with glucocorticoids , nonsteroidal anti - inflammatory drugs ( nsaids ) or disease modifying antirheumatic drugs for a period of at least 2months prior to the trial . 
thanks to the recent introduction of new and promising treatment options , such as tumour necrosis factor ( tnf ) antagonists therapy , which could modify the natural history of the disease , not only an early diagnosis is fundamental , but also an objective mean to evaluate response to therapy is often demanded by clinicians . 
in this respect , imaging , together with clinical and laboratory evaluation , are not only important tools to achieve an accurate diagnosis but also in the grading of disease modification , influencing both clinical management and therapy [ 2 ]  . 
conventional radiography is the most accepted imaging method for diagnosing sacroiliitis because it is relatively inexpensive , readily available , and , when it yields positive findings , very effective ; however , conventional radiography can only detect advanced structural damage , which usually requires several years to become evident and above all active inflammation cannot be assessed [ 3 ] , making this technique unsuitable to evaluate response to therapy . 
instead , magnetic resonance imaging ( mri ) can provide an early diagnosis and is able to quantify inflammatory activity showing bone marrow oedema ( bmo ) or osteitis , synovitis , which makes it ideal for monitoring disease activity too . 
thus , in 2009 , mri was included in the new assessment of spondyloarthritis international society ( asas ) criteria for the diagnosis of axial spa , in order to facilitate the identification of early features of spa before radiographic findings and in the assessment of treatment response . 
despite this , a clear definition of mri active sacroiliitis still does not exist , even if the presence of subchondral bone oedema is believed mandatory and examination without an i.v. 
 contrast material in evaluating the response to tnf antagonists therapy in the sijs , comparing the reduction grade of bmo and osteitis / synovitis , respectively , on mri without and with i.v. 
contrast agent , with bath ankylosing spondylitis disease activity index ( basdai ) before and after treatment . materials andmethods this study was approved by the institutional review board of ouruniversity hospital . 
written informed consent was obtained from all participants . patient information from december 2014 to march 2016 , 43 patients ( 25 females and 18 males , mean age of 54 16.60years , range 2275years ) with a clinical diagnosis of spa and active fig . 
2 ad unilateral sacroiliitis in a 64 - year - old woman with right upper sacrum bmo on t2 w fat - saturated images and osteitis on 3d gradient - echo t1 w images after i.v. 
of the contrast agent ( a , b ) ; after 6 months of tnf antagonists therapy the oedema was unchanged , whereas osteitis disappeared ( c , d ) subtracted from the later scans . 
the first two sequences ( t1 - weighted fse and t2 - weighted frfse ) allow the detection of structural changes like erosions and sclerosis ; t2 - weighted fat - saturated images depict inflammatory bmo as increased signal intensity and the latter sequence allows the detection of osteitis , synovitis and capsulitis . image analysis two experienced observers , a radiologist and a resident radiologist , with more than 15 and 3years experience , respectively , in musculoskeletal imaging , were blinded to the patients clinical characteristics and evaluated mri images , scoring bmo in t2 - weighted fat - saturated sequences and areas of pathological enhancement in gradient - echo t1 sequences after i.v. 
the score was semiquantitative and based on the extension and intensity of the lesions ; grade 0 = no inflammatory changes ; grade 1 = inflammation involving up to 20% of the joint area ( signal intensity changes confined to the joint space , joint capsule and erosions ) ; grade 2 - 3 = inflammation of paraarticular bone marrow involving up to 50% or over 50% of the region . 
any disagreement was discussed until a consensus was reached . 1 3 la radiologia medica ( 2019 ) 124 : 11421150 1145 statistical analysis descriptive statistics were used to describe the overall distribution of the scores ; students t test was used to compare the difference ( improvement ) between basdai scores at baseline and after treatment ; students t test and blandaltman plot were used to compare the differences between mr scores ( improvement ) at baseline and after treatment in sequences with and without contrast media . 
in contingency tables , the values are expressed as percentage of the total number of patients . results among all the 43 patients , 3 were excluded since they refused mr follow - up and 2 since therapy was prematurely interrupted for adverse dermatological reactions . 
3 ad a 41 - year - old woman with chronic bilateral sacroiliitis , represented by diffuse subchondral sclerosis involving the iliac portion of sij , developed a clinical exacerbation . 
a , b there was no bmo on t2 - weighted fat - saturated images , but a little area of synovitis in the upper left sacrum on 3d gradient - echo t1w images after i.v. 
injection of the contrast agent ; c , d after therapy an area of sclerosis was clear on both sequences 1 3 1146 table 1 summary of the scoring results of mri findings ( bmo and osteitis / synovitis ) and basdai for each patient before and after therapy la radiologia medica ( 2019 ) 124 : 11421150 patient ( n ) bone marrow oedema osteitis / synovitis basdai before after before after before after with the sacral aspect of sijs being the most commonly affected area ( more than 70% of the patients )  . 
thirtysix out of 38 patients showed an improvement on clinical assessment after therapy , assessed by the basdai : 20 out of 36 ( 55.5% ) showed reduction / disappearance of both enhancement and bmo , but the reduced grade of the first parameter was higher ; 11 out of 36 ( 30.5% ) showed reduction / disappearance of pathological enhancement , whereas bmo was unchanged ( fig.2 ) ; 2 out of 36 ( 5.5% ) showed reduction of enhancement in the absence of bmo before or after therapy ( fig.3 ) ; 3 out of 36 ( 8.3% ) and the remaining 2 out of 38 did not show modification of mri findings . 
moreover , a significant 1 3 la radiologia medica ( 2019 ) 124 : 11421150 1147 discussion inflammatory sacroiliitis is a chronic , disabling disease because of the patients pain and motions restriction due to progressive structural damages of sijs . 
in the past , several therapies have been used to treat these pathologies , for example , nonsteroidal anti - inflammatory drugs ( nsaids ) , corticosteroids and immunosuppressant drugs . 
tnf antagonists have been investigated in randomized controlled trials in patients with spa , and benefits were obtained particularly in patients with recent - onset disease ; in fact , most of response rates were higher than in controlled trials of the same drugs used to treat advanced disease , suggesting the existence of a window opportunity [ 7 ]  . 
however , the management of these therapies requires great attention in diagnosis and follow - up ; such therapy is not only expensive but may also raise the risk of developing serious infections and malignancies [ 8 , 9 ]  . 
recently , it has been shown that dose tapering of tnf antagonists is a feasible therapeutic option in patients with spa and low disease activity , without relevant changes in the clinical outcome , resulting in remarkable cost savings [ 10 ]  . 
although disease activity is usually assessed by clinical and biochemical parameters , mri may help to objectify response to therapy and therefore to optimize treatment plan for each patient [ 11 , 12 ]  . 
mri is the best imaging modality able to detect and quantify active inflammatory changes of the sijs , even if dual - energy ct ( dect ) , at present , seems to be promising in this regard , despite limited to bmo detection and quantification [ 1315 ]  . 
although several studies in the literature compared mri contrast - enhanced fat - saturated t1 weighted sequences with t2 - weighted fat - saturated sequences to diagnose active inflammatory sacroiliitis , reporting bmo sufficient for this task [ 16 , 17 ] , other studies declared that the administration of contrast medium may be beneficial by providing a different view of the lesions with another sequence , which can be helpful when mri is interpreted by inexperienced readers or when minimal changes occur [ 18 ]  . 
in particular , thanks to its higher spatial resolution , post - contrastt1 - weighted imaging is more sensitive in detecting small subchondral lesions and other imaging findings that are sometimes difficult to see without contrast material administration , such as synovitis and capsulitis [ 3 , 19 ]  . furthermore , the pathogenetic mechanism of spa has not yet been fully clarified and there are many hypotheses about the mechanism of inflammation development and damage production . 
4 box plot of scores difference ( improvement ) in the mr sequences with and without contrast agent administration difference ( p < 0.05 ) was found between the improvement calculated in the sequences with and without contrast media administration . 
 descriptive statistics show differences in score between the mri sequences with and without contrast agent , demonstrating a higher improvement in post - therapy using the former ( figs4 , 5 , 6 , table2 )  . 
5 function of empirical distribution for the improvement ; black ( mr with contrast ) , red ( mr without contrast ) 1 3 1148 la radiologia medica ( 2019 ) 124 : 11421150 and after therapy . 
moreover , in patients with chronic sacroiliitis , findings such as long - standing erosions sometimes showed nuanced bmo in absence of active inflammation signs in post - contrast sequences ; in such cases , post - contrast sequences are extremely useful to establish a proper therapeutic management . 
on the other hand , the presence of inflammation alone may not be the primary factor associated with structural damage progression , which seems to be also related to the development of fat metaplasia . 
in this regards , further studies are necessary to understand the physiopathological connections between inflammation , fat metaplasia and structural damage in spa . some limitations of our study should be noted . 
in addition , mri scores before and after treatment were correlated with basdai since ankylosing spondylitis disease activity score ( asdas ) was not available for all the patients ; in this regard asdas seems to be more closely associated with inflammatory biomarkers and may better reflect the inflammatory disease process in patients with axial spa treated with tnf inhibitors [ 3336 ]  . 
6 blandaltman plot showing the dispersion of the values from the mean suggests a difference between the mr improvement calculated on the sequences with and without contrast agent most involved area [ 20 , 21 ] , whereas others argued that the pathologic mechanism of spa is different from that of ra , since the most affected region is the enthesis [ 2226 ]  . 
 however , studies that investigated sacroiliitis through fine needle aspiration biopsy found that in the early stage of the disease , pannus formation , which is constituted by highly vascular granulation tissue formed by the inflamed synovium and / or subchondral bone marrow , in the area where bone and cartilage oppose one another , may be the pathological hallmark ; subsequently , inflammation may trigger the formation of angiogenesis with an increased microvessel density that provides a channel for inflammatory cell invasion and mediator expression , which in turn worsen the local damage to the cartilage [ 2732 ]  . this increase in angiogenesis has been shown to decrease significantly after tnf antagonist treatment , revealing the pivotal role of angiogenesis in spa . 
this statement seems to be supported by the thesis that subchondral inflammation may trigger the formation of angiogenesis with an increased microvessel density that provides a channel for inflammatory cell invasion and inflammatory mediator expression , which in turn worsen the local damage to the cartilage . 
antonio e biagio e cesare arrigo , via venezia 16 , 15121alessandria , italy 2 department ofhealth sciences ( dissal ) , university ofgenoa , c.so montegrappa 26 , genoa , italy 3 department ofmedical , surgical andneuro sciences , diagnostic imaging , azienda ospedaliera universitaria senese , university ofsiena , viale bracci 10 , 53100siena , italy introduction axial skeleton , including both the spine and the sacroiliac joints ( sijs ) , represents a diagnostic challenge both for the clinician and for the radiologist . 
in the literature , vertebral column has been the main target of study in degenerative diseases , while sijs have been the leading subject of study , especially in magnetic resonance imaging ( mri ) , in relation to spondyloarthritis ( spa )  . 
in fact , mri plays an important role in the diagnosis of axial spa by detecting sacroiliitis ( using the assessment in spondyloarthritis international society ( asas ) imaging arm ) , more sensitively and also with a better reproducibility compared with conventional radiography . 
however , more recent studies , which have included both patients with spa diagnosis and patients with non - specific low back pain ( lbp ) , showed that mri findings related to sijs , once considered specific for diagnosis of spa , are also prevalent in patients with non - specific lbp [ 16 ]  . at the same time , vertebral features specific for spa diagnosis may be underdiagnosed in the context of a large prevalence of degenerative findings ( modic , etc . ) , which indeed could mislead the assessment of spa diagnosis [ 7 ]  . 
thus , the best knowledge in the use of instrumental and diagnostic techniques , such as mri , able to identify the objective signs of inflammation , such as bone edema , that is a non - specific but very early vol . : ( 0123456789 ) 1 3 1152 la radiologia medica ( 2019 ) 124 : 11511166 finding should be desirable . 
in particular , regarding the use of mri , the main risk is overdiagnosing spa ( just as damaging as a diagnostic delay ) , scotomizing the most common degenerative diseases recognized in acute phase : pousses during discoarthrosis or fibromyalgic syndromes in which a lumbar pain with inflammatory characteristics could be present in up to 40% of cases [ 8 , 9 ]  . 
in fact , literature data report that only 36% of patients with inflammatory lumbar pain had a diagnosis of sacroiliitis based on mri findings according to asas criteria : in this sense , anamnesis and clinical history are important in differential diagnosis . 
furthermore , the diagnostic delay in diagnosing spa exposes the patient to very important consequences in terms of activity and evolution of the disease , with impaired quality of life , functional limitations and increased risk of cardiovascular events , professional economy , especially when dealing with young people with loss of workability and medical nomadism , which requires specialist advice and therapies that are inappropriate . the aim of this manuscript is to review the differences in degenerative and spa - related mri findings in spine and sijs in order to give a complete scenario in differential diagnosis of low back pain using imaging . mri andaxial skeleton : technique andassessment in order to better identify the mri findings of both degenerative and inflammatory diseases , it is important to give the definition of prescriptive and executive appropriateness and fairness in mri of axial skeleton and , moreover , the definition of spine and pelvis mri study protocols . mr examination of specific anatomical regions should be performed using dedicated spinal or body phased - array coils . in the early diagnosis of axial spa , a sijs mri is recommended . 
in clinical practice , and especially in case of doubt , spinal mri may be useful for the positive diagnosis of other diseases ( e.g. , modic type 1 , scheuermanns disease , diffuse idiopathic skeletal hyperostosis )  . 
in spa patients , the mri scan range on sagittal slices should be laterally extended to include the paravertebral structures such as facet , costotransversal and costovertebral joints that are commonly involved in spa . 
 sagittal sequences should be positioned to comprehend the entire spine including facet joints , the costovertebral and costotransverse joints ( a ) , and coronal sequences should be positioned to comprehend the entire spine too , including anterior longitudinal ligament and the posterior part of the spine to the spinous process , including the zygoapophyseal joint , the costovertebral and costotransverse joints ( b , c , d ) 1 3 la radiologia medica ( 2019 ) 124 : 11511166 1153 inflammatory findings , such as erosions , sclerosis , fat deposition and bone marrow edema ( bme ) , which is considered an objective sign of inflammation , identifying the concept of non - radiographic spondyloarthritis ( nr - spa ) in new asas criteria ( figs.3 , 4 ) [ 1 ]  . 
the use of intravenous ( i.v. ) injection of paramagnetic contrast media has proven valuable in showing the extension of inflammatory changes , and in particular in demonstrating osteitis , as well as abscesses in septic sacroiliitis or spondylodiscitis or in the assessment of neoplastic disease . 
injection of contrast media in spondyloarthropathies may be useful , although this is debatable . mri findings inthesacroiliac joints : sacroiliitis andnoninflammatory disease the special configuration and anatomy of sijs determine that they undergo morphological transformations throughout life and produce a degeneration in which extrinsic factors , such as trauma or stress ( scoliosis , dysmetria , previous surgery of the column , pregnancy , childbirth , etc . ) , or intrinsic diseases , both inflammatory or infectious ( spondyloarthropathies and , less frequently , septic arthritis ) , and , less frequently , tumor lesions , could act on their changes . normal mri anatomy the sij is one of the largest axial joints and is composed in its upper and dorsal region by a syndesmosis , which is a fibrous joint in which the bony surfaces are joined by interosseous ligaments , and a cartilaginous area , classically defined as synovium , which extends into the lower two - thirds of the ventral area , but actually has a structure of symphysis with hyaline cartilage firmly attached to the adjacent bone by fibrous tissue . 
these anatomical features could explain why the changes begin and are more relevant in the iliac aspect of the joint , while rheumatoid arthritis has a poor expression in the sijs and inflammatory changes in spondyloarthropathies show similar changes to those that occur in a symphysis . 
articular stability is provided by the intrinsic sacroiliac ligaments in the ventral region and interosseous ligaments in the dorsal area and extrinsic ligaments , which are fibrous extensions of closer muscles and contribute to capsular reinforcement [ 1013 ]  . 
axial sequences should be positioned from 1 to 2cm above the iliac crests to the femoral lesser trochanter with fov sized enough to cover the entire soft tissue in the anteroposterior direction for the study of the enthesis ( a )  . 
oblique coronal and oblique axial sequences ( b ) should be , respectively , oriented along the long axis of the sacral bone and oriented perpendicularly to the semi - coronal or along the short axis of the sacral bone , to comprehend the entire sacroiliac joint t1 - weighted sequences ( tse t1w ) and high intrinsic contrast sequences , turbo spin - echo t2 - weighted sequences ( tse t2w ) with fat suppression or short - tau inversion recovery ( stir ) sequences should be performed in order to obtain a clear detection , respectively , of both structural and 1 3 1154 la radiologia medica ( 2019 ) 124 : 11511166 fig . 
if there is only one lesion , it should be present at least on two consecutive slices ; if there is more than one lesion on a single slice , one slice may be enough ; b corresponding hypointensity for bme in coronal oblique tse t1w and structural lesions such as erosions fig . 
4 oblique axial tse t1w ( a ) and tse t2w ( b ) and spir t1w with gadolinium ( c ) and oblique coronal stir ( d ) : outcomes of inflammatory process with evolution in adipose substitution of bone marrow opposed to the sacroiliac articulations , on both sides , with fine structural irregularity of the articular line and enhancement of the anteroinferior portion degenerative changes in all joints from the age of 50 on , some type of degeneration is observed , and these changes are more profuse in women than in men of the same age and progress more rapidly in female multiparae than in nulliparous women . 
also , anatomical variants are observed and the knowledge of these variants is essential to avoid diagnostic errors ( accessory sacroiliac joint 1719% , iliosacral complex 5.89.5% , bipartite iliac bone 4.17.8% , defect semicircular articular surfaces 3.74.8% , crescent articular surface 3.5% and ossification centers of the sacral rings 1% )  . 
most of them share the fact that they are often observed in people older than 60years and some are more frequent in women ( the first of the variants in women with more than three children ) and in obese patients [ 1416 ]  . fatty bone marrow deposition , sclerosis and erosions were strongly associated with female gender , and this could be explained by a correlation between sij findings and a different load distribution in women and / or pregnancy - related physiological changes of the pelvic girdle and birth - related strain [ 17 ]  . 
in the literature , associations between bme and degenerative / load - related changes at different locations such as spine ( modic type i changes ) [ 18 , 19 ] and knee [ 20 , 21 ] are known . 
 [ 8 ] , supported by bme being more prevalent in the oldest patients in their study , assumed that bme in sijs also may be correlated with degenerative changes due to impaired chronic loading . both osteoarthritis and arthritis can present common structural changes such as subchondral sclerosis , joint clamping ( which will favor arthritis when the joint measures less than 2mm in patients under 40years of age ) and ankylosis . 
osteophytes , pneumocystis and intra - articular vacuum are typical of osteoarthritis , although arthritis without active inflammation may present intra - articular void that could be a sign of inactivity . table 1 active and structural changes of the sijs according to asas active inflammatory lesions inflammatory changes erosions are typical of chronic morphological changes of arthritis . 
on the other hand , when there are acute inflammatory signs in a spondyloarthropathy or infectious arthritis , the only test to detect inflammation with good sensitivity and specificity is mri . 
spinal spa - related mri findings are rare , and bme is the most common ( 21% ) in sijs ; furthermore , spinal inflammatory lesions suggestive of axial spa are rarely found in the absence of sacroiliitis on mri ( or radiography ) [ 3 ]  . in the literature , from 10% [ 8 ] to 36% [ 22 ] of the patients fulfill the asas criteria for spa via the imaging arm ( mri findings fulfilling the asas definition of a positive mri for sacroiliitis , with a final clinical diagnosis of axial spa ; table1 )  . 
however , whether this is an appraisal of the prevalence of patients with true spa , it remains unknown in the general population , due to the uncertainties regarding the diagnostic performance of the criteria which resemble more a classification system than proper diagnostic criteria ; therefore , their specificity might be low due to a high prevalence of a positive mri among non - specific lbp patients . 
 historically , the aim in research of mri findings in sijs focused on patients with inflammatory back pain or spa and few studies have reported on mri findings in sijs in patients with non - inflammatory back pain [ 23 ]  . 
a few casecontrol studies have reported the prevalence of bme , fatty marrow deposition , erosions and ankylosis at the sijs in patients with non - specific lbp to be 2227% [ 8 , 23 ] , 1527% [ 8 , 9 ] , 413% [ 8 , 9 ] and 0% [ 8 , 9 ] , respectively , suggesting a high specificity of ankylosis for stadium iv of spa . among patients with bme in sij , it is reported in the literature a significantly higher prevalence of erosions in patients fulfilling the asas criteria for spa ( 41% ) compared to those who did not achieve these criteria ( 20% )  . 
bone marrow edema area of high signal on t2 fs or stir images ; typically located subchondrally in the cartilaginous parts of the sijs thickening of the sijs capsule and adjacent ligaments ; it appears as hyperintense signal on t2 fs or stir images and / or hyperintense on contrast - enhanced t1 fs sequences it presents as a hyperintense signal on contrast - enhanced t1 fs images , in the synovial part of the sacroiliac joint it is seen as hyperintense signal at sacroiliac ligaments or entheses on stir or t1 fs contrast - enhanced sequences ; it could be accompanied by bme of the bony part of the entheses ( bme ) 2 . 
bone erosions subchondral area of low - intensity signal at the sijs on all sequences joint facet / subchondral bone abnormalities appearing as few , multiple , localized or contiguous low signal intensities on t1 - weighted mr images with varying signal intensity on t1 fs or t2 fs sequences 3 . 
bony bridges , ankyloses subchondral hyperintense signal on t1and t2 - weighted images , which is suppressed on fs or stir sequences bony appositions , end stage of disease with union across the joint 1 3 1156 la radiologia medica ( 2019 ) 124 : 11511166 these results support the inclusion of erosions in the definition of a positive mri finding for the classification of spa [ 6 , 2426 ]  . 
furthermore , sijs erosions were most prevalent in patients aged 1829years and bme in patients aged 3040years , whereas sijs sclerosis and fatty marrow deposition were most common in women . mri of the sijs commonly shows non - inflammatory disease in patients clinically suspected of si : 41% of patients with lbp had normal mri findings ; 36% of patients showed mri features compatible , according to asas criteria , with sacroiliitis in axial spa ( bme and erosions ) ; 21% of the patients showed bme ; and only 44% of these showed a diagnosis compatible with si according to the asas criteria ( bme and erosions )  . 
in order to detect such finding is suggested to obtain axial stir images from l5 to the lesser trochanter when performing mri of the si joint , much helpful than obtaining only semi - coronal sequences of the sijs , which may cause undetection of unexpected findings [ 22 ]  . 
5 axial tse t2w ( a ) and t1w ( b ) sequences and axial tse t2 ( c ) and corresponding sagittal stir sequences of the spine ( d ) show degenerative pathology of the articular facets . 
 articular hypertrophy with alteration of the loads on the facet with osteophytes , thinning of the joint space , bone marrow edema , soft tissue signal changes around the apophyseal joint 1 3 la radiologia medica ( 2019 ) 124 : 11511166 1157 fig . 
lumbar findings are in general more frequent than thoracic findings , and thoracic findings are reported to be more frequent than cervical findings or vice versa [ 27 ]  . 
7 sagittal stir ( a ) , coronal oblique spair t2w ( b ) , axial tse t2w ( c ) and spair t2w ( d ) demonstrate a normal aspect of sacroiliac synchondrosis in a 44 - year - old male patient with suspected sacroiliitis , compared to evidence of a left posterolateral hernia migrated posteriorly along the wall of s1 la radiologia medica ( 2019 ) 124 : 11511166 fig . 
8 transient osteoporosis of the hips in a 33 - year - old patient in the seventh month of pregnancy demonstrates a characteristic bone marrow edema of the cervico - cephalic region of both femurs , respectively , clockwise , in the axial stir sequences ( a ) , sagittal tse t1w ( b ) , coronal stir ( c ) and tse t1w ( d ) 1 3 la radiologia medica ( 2019 ) 124 : 11511166 1159 fig . 
9 coronal oblique spair t2w ( a ) and axial tse t2w ( b ) : left hemisacralized lumbosacral transition vertebra with reactive bone marrow edema and marginal sclerosis patients with degenerative changes . 
 the pathogenetic process begins with discal thinning and consequent cranial subluxation of the upper articular process onto the underlying vertebra versus inferior articular process of the overlying vertebra with alteration of the loads on the facet . 
mri findings are osteophytes , subchondral sclerosis , cysts ( fig.6 ) , thinning of the joint space , bme , fatty marrow deposition , soft tissue signal changes around the apophyseal joint . 
 inflammatory changes only 10% of patients who underwent mri for lbp show inflammatory changes in the column ( instead of 28% of patients who present inflammatory changes in sijs ) : bme / adipose replacement of somatic edges ( romanus vertebra ) , erosive changes within intervertebral space ( andersson lesions ) , chondroid metaplasia and enchondral ossification leading to syndesmophytes formation and vertebral fusion . 
 also , bme of costovertebral joints and apophyseal joint and edema in the insertion of thorny ligaments may be depicted . in particular , early spinal changes encompass erosion of vertebral corners ( romanus lesions ) causing vertebral squaring and eliciting reactive sclerosis ; these changes are caused by fig . 
10 ct axial scan in a 32 - year - old woman who had given birth to her second child 6months before affected by pain in the lower back and buttocks shows osteitis condensans ilii presenting as a bilateral sclerotic lesion , mainly in iliac bone adjacent to both sacroiliac joint with relative normal joint space ( no joint irregularity , erosions or loss of joint space is observed ) for vertebral endplate signal changes , it was noticed that the anterior location was the most common in the thoracic spine , while in the lumbar spine , widespread vertebral endplate signal changes were the most frequent . 
in the thoracic spine , the load is mainly on the anterior part of the motion segment due to the kyphosis and vice versa in the lumbar region due to the lordosis . 
 moreover , the lumbar segments have more degrees of freedom of movement , including sagittal angulation and axial rotation , making loading on the entire endplate possible in opposition to the thoracic spine , which has a limited range of motion due to the rib cage . 
the regional differences in the location of vertebral endplate signal changes found in the literature support the hypothesis that the occurrence of vertebral endplate signal changes is related to mechanical loading [ 28 ]  . 
fulfillment of the imaging arm of the asas criteria takes into account the presence of mri - documented inflammation only in the sijs , but not in the spine [ 29 ]  . 
in daily clinical practice , mri findings can be useful in confirming a clinical diagnosis of non - radiographic axial spa and detecting objective evidence of active inflammation in patients with an established diagnosis of non - radiographic axial spa . 
the literature agrees on the distribution of the dvu involvement along the spine with findings of thoracic and lumbar region of the spine being the areas most frequently affected by inflammation in axial spa [ 2934 ]  . 
according to the most recent literature , a positive mri spine ( the presence of 5 / 3 inflammatory lesion that is corner bme lesions ) was rare in patients without sacroiliitis on mri - si and x - ray - si . 
 addition of mri spine as imaging criterion to the asas axial spa criteria had a low yield of newly classified patients and is therefore not recommended [ 35 ]  . current concepts andapplication inaxial spa represents a diagnostic challenge being known that the diagnostic delay between the first symptoms and the certain diagnosis of the disease is around 8years . 
the reasons for this delay are essentially based on the difficulties of early 1 3 la radiologia medica ( 2019 ) 124 : 11511166 1161 ( defined as sacroileitis , term not properly correct because it implies a process of inflammatory nature that cannot be unveiled in traditional radiography , able to highlight structural lesions such as joint erosions and subchondral bone sclerosis )  . the new asas criteria [ 1 ] allow a more early classification and identification of the concept of nr - spa . 
it is therefore extremely important to have clinical diagnostic tools capable of addressing the diagnostic question of spa , especially of axial forms , also with a view to exploiting the therapeutic window of opportunity , which allows to obtain the best efficacy before the appearance of irreversible alterations . the recent recommendations for the use of imaging techniques in the diagnosis and management of spa in clinical practice [ 37 ] have produced ten evidence - based recommendations in monitoring of inflammation and structural damage , in outcome prediction , in response to treatment and in the detection of spinal fractures and osteoporosis . diagnostic criteria inaxial spa in general , conventional radiography of the sijs is recommended as the first imaging technique for the diagnosis of si in axial spa [ 38 ]  . 
a sagittal tse t1w image , hypointense bone marrow edema , b sagittal spair t2w image , hyperintense bone marrow edema diagnosis if the modified new york conventional criteria [ 36 ] are used , which require the obligatory presence of structural lesions of the sijs to the radiography of the pelvis fig . 
 a sagittal tse t1w image , hypointense bone marrow with sclerosis , b sagittal spair t2w image , hypointense bone marrow with sclerosis la radiologia medica ( 2019 ) 124 : 11511166 fig . 
15 a , b sagittal tse t1and t2 - weighted mr image shows typical characteristic of schmorls node located at the inferior endplate of l3 and l4 vertebrae on mr imaging . 
c sagittal stir sequence shows schmorls nodes located at the inferior endplate of l2 and superior endplate of l3 vertebrae : bone marrow edema and hemorrhagic infarction surround the herniation of the intervertebral disk nuclear material through the cartilaginous and body endplate into the body of the vertebra if the diagnosis of axial spa cannot be established by clinical characteristics and conventional radiography and there is still suspicion of axial spa , the mri of the sijs is recommended . 
injection of contrast media sequences of the spine demonstrate discitis of the l1l2 disk space with rime enhancement and osteomyelitis of the adjacent l1 and l2 vertebral bodies in patient affected by spondylodiscitis fig . 
injection of contrast media sequences of the intervertebral space demonstrate destruction of the disk space with rime enhancement and osteomyelitis of the adjacent vertebral bodies in patient affected by spondylodiscitis on mri might be observed in half the patients with nonradiographic axial spa without sijs inflammation . 
however , fulfillment of the imaging arm of the asas criteria takes into account the presence of mri - documented inflammation only in the sijs , but not in the spine . 
rheumatologists in clinical practice rightly dispute a diagnosis of axial spa even when there is a high number of spa features ( ibp , a positive family history of spa , a good response to nonsteroidal anti - inflammatory drugs , elevated c - reactive protein or erythrocyte sedimentation rate and enthesitis ) , especially when imaging is normal and patients are negative for hlab27 . 
the best knowledge in the use of instrumental methods associated with greater awareness of the clinics , supported by the irreplaceable comparison with radiologists , must be the objectives to be pursued in reducing the diagnostic delay and optimizing the care of patients with spondyloarthritis . 
18 sagittal tse t2w ( a ) and t1w ( b ) spine sequences in axial spa show a total bony bridge at the level of d10d11 ( c ) such as chondroid metaplasia phenomenon and enchondral ossification leading to syndesmophytes formation ( d ) la radiologia medica ( 2019 ) 124 : 11511166 sensitivity toward the spa , through a shared path on clinical findings and instrumental methods [ 40 ]  . response to therapy in sijs involvement in spa using the evaluation of enhancement [ 4244 ]  . the concomitant presence of pelvic enthesitis phenomena in more than one site on the mri examination of the axial skeleton during spa increases the specificity and the positive predictive value ( ppv ) in the diagnosis of spa , especially when the findings of sacroileitis are equivocal or absent . 
enthesitic sites with high ppv in the diagnosis of spa are the wings and the iliac crest and the posterior interosseous ligaments , while the great trochanter and the posterior spinal ligaments of the sacroiliac joints constitute enthesitic sites with less ppv ( fig.19 ) [ 41 ]  . other imaging techniques , in addition to conventional radiography and mri , are not usually recommended for axial spa diagnosis . 
bone scintigraphy and ultrasounds ( us ) are not recommended for the diagnosis of si in axial spa . monitoring oftheactivity intheaxial spa mri of the sijs and / or spine joints can be used to determine and monitoring disease activity in the axial spa , providing additional information to clinical and biochemical evaluations . 
in general , stir sequences are sufficient to detect inflammation and the use of contrast medium is not necessary , even though some recent articles have reported an improvement in the mri assessment of monitoring ofstructural changes intheaxial spa conventional radiography of si and / or spinal column joints can be used for the long - term monitoring of structural damage , particularly the formation of new bone , in axial spa . 
mri can provide further information . forecasting oftheoutcome / gravity intheaxial spa in patients with ankylosing spondylitis ( not with non - radiographic axial spa ) , a preliminary conventional radiograph of the lumbar and cervical spine is recommended to detect syndesmophytes , which are predictive of the development of new syndesmophytes . 
mri findings ( vertebral inflammatory lesions or adipose substitution ) could also be used to predict the development of new radiographic syndesmophytes . forecast ofthetreatment effect intheaxial spa the high inflammatory activity in mri ( bme ) , particularly in the column of patients with ankylosing spondylitis , could be predictive of a good clinical response to treatment with anti - tnf drugs in axial spa . 
thus , mri can help in the decision to initiate therapy with an anti - tnf , in addition to clinical examination and pcr . 1 3 la radiologia medica ( 2019 ) 124 : 11511166 1165 fig . 
 ( circles ) mri of the pelvis carried out with axial scan planes and stir sequences at the level of the main enthesis showingthe presence of bone edema as a sign of osteitis in patients affected by axial spondyloarthritis a possible comment on these indications , which represent an undoubted value in terms of actual practice , concerns that mri scan of the spine is not recommended for the diagnosis of axial spa . 
therefore , a global discussion on the subject through a systematic review of the literature and a meta - analysis is necessary . methods a systematic search of pubmed was performed up to april 23 , 2018 . 
studies exploring the role of pmcta in cases of sudden cardiac death and the accuracy of this method in diagnosing the cause of death compared to traditional autopsy were included . results the overall sensitivity and specificity of the seven included studies , using conventional autopsy as a reference standard , were 92% and 95% , respectively . 
however , pmcta can improve the performance of the autopsy , serving as an aid and guide in the sampling phase for histopathological investigations . keywords postmortem computed tomography angiography traditional autopsy sudden cardiac death coronary artery disease * vittorio fineschi vfinesc@tin.it 1 department ofanatomical , histological , forensic andorthopaedic sciences , sapienza university ofrome , viale regina elena 336 , 00161rome , italy irccs neuromed , via atinense 18 , pozzilli86077 , italy 3 department ofradiological sciences , oncology andpathology , sapienza university , viale regina elena 324 , 00161rome , italy introduction sudden cardiac death ( scd ) has been defined as a natural , unexpected fatal event occurring within 1h from the onset of symptoms in an apparently healthy subject or in one whose disease was not so severe as to predict an abrupt outcome . 
the incidence of scd increases with age , in line with the increase in the incidence of the coronary artery disease which , according to numerous studies , represents the pathology most frequently involved in the determinism of sudden death ( sudden coronary death ) [ 1 , 2 ]  . postmortem examination and autopsy represent reliable methods for determining the causes of scd [ 3 , 4 ]  . 
 however , due to ethical , religious , cultural , economic and organizational reasons , the number of autopsies performed vol . : ( 0123456789 ) 1 3 110 la radiologia medica ( 2019 ) 124 : 109117 is decreasing worldwide and the use of minimally invasive ancillary methods is considered preferable in some circumstances . in view of these circumstances , postmortem cardiac imaging is assuming increasing importance in the study of myocardium and coronary arteries as an aid and guide in the execution of conventional autopsy and , sometimes , as an alternative [ 5 , 6 ]  . currently , postmortem computed tomography ( pmct ) , postmortem computed tomography angiography ( pmcta ) , postmortem magnetic resonance ( pmmr ) and postmortem magnetic resonance angiography ( pmmra ) have become established methods due to numerous studies that have documented the undeniable advantages in terms of acquisition times , costs and possibility to review the data acquired at any time [ 713 ]  . a series of studies have been performed over the years to assess the efficacy of pmcta in solving cases of sudden cardiac death , even in comparison with the traditional autopsy . 
the pubmed section similar articles and the references of the selected articles were used to expand the research . inclusion andexclusion criteria studies were included if they met the following criteria : ( 1 ) population consisting of persons who died of suspected sudden cardiac death ; ( 2 ) evaluation of the accuracy of pmcta in diagnosing the cause of death and in detecting major or minor diagnostic findings ; ( 3 ) comparison of the efficacy of pmcta with the gold standard , the conventional autopsy ; ( 4 ) availability of the entire panel of true positives ( tp ) , false positives ( fp ) , false negatives ( fn ) and true negatives ( tn ) , or sufficient data to calculate themeanwhile , studies were excluded if they were : ( 1 ) case reports ; ( 2 ) not original research , such as review articles ; ( 3 ) studies conducted on fetuses . data extraction andassessment data from each included study were extracted using microsoft excel spreadsheets , including information on authors , journal , publishing years , nation , population selection criteria , sample size , gender , age , diagnostic technique under examination , reference standards , as well as results in terms of tp , fp , fn and tn . the quality of the individual studies was determined through the quadas - 2 ( quality assessment of diagnostic accuracy study ) tool , structured in four key domains : ( 1 ) patient selection ; ( 2 ) index test ; ( 3 ) reference standard ; and ( 4 ) flow and timing . 
the studies were considered at low risk of bias if the population selection criterion was clear , if the radiologist and forensic pathologist operated in double - blind , if they both had access to the same information about the case and if the interval between performing the imaging exam and the autopsy was less than 72h . 
the studies were considered at moderate risk of bias in the presence of at least one of the following conditions : ( 1 ) unclear selection , ( 2 ) knowledge by the radiologist or the forensic pathologist of the other test results , ( 3 ) latency time between the two investigations exceeding 72h . 
the studies were considered at high risk of bias in the presence of two or more of the previously indicated conditions . statistical analysis the meta - analysis was conducted using stata 13.0 ( stata corporation , college station , texas , usa ) [ 1619 ]  . 
the 1 3 la radiologia medica ( 2019 ) 124 : 109117 results in terms of tp , fp , fn and tn were used to calculate the sensitivity and specificity in each of the primary studies . 
the sensitivity and specificity values of the individual studies were graphically represented in a forest plot using the meta - disc software ( unit of clinical biostatistics , ramn y cajal hospital , madrid , spain )  . 
for the synthesis of the results on the pmcta performances , two random effect models were used : the bivariate model and the hsroc ( hierarchical summary receiver operating characteristic ) model . results included studies by applying the search strategy mentioned above , we found a total of 793 studies in pubmed . 
 the process of study inclusion is illustrated in fig.1. the sample size ranged between 5 and 38 with a total of 127 subjects ; the publication time was from 2010 to 2014 . 
the risk of bias was globally low or unclear in the four domains except for the reference standard ; the latter was at high risk in two studies because the autopsy was performed by a pathologist aware of the results of radiological investigations . 
therefore , four studies were considered at low risk of bias , one at low / undefined risk and two at moderate / undefined risk ( table2 )  . qualitative analysis ( systematic review ) bolliger etal . 
 [ 20 ] evaluated the diagnostic accuracy of a minimally invasive approach ( pmct , pmcta , ct - guided biopsy ) in 20 cases of apparently natural death of which 11 of sudden coronary death . 
 [ 23 ] radiology ( 2012 ) international journal of selective coronary pmcta 77 ( range 4292 ) 56 ( range 1580 ) whole body pmcta 16 : 4 52.3 ( range 3789 ) 21 : 2 whole body pmcta morgan etal . 
in 8 cases , there was complete agreement between the data obtained from the two tests , while in one case radiological investigations had overestimated the severity of a stenosis that was considered nonsignificant to autopsy . 
 [ 22 ] have shown a concordance between conventional autopsy and pmcta associated with ct - guided biopsy in 20 cases of sudden death after chest pain 8 of the 9 cases of sudden coronary death , the pmcta had been able to highlight the alterations responsible for the death , while in one case , early myocardial infarction of the papillary muscles had been missed . pmcta allowed an excellent visualization of the coronary arteries and a better evaluation of stenosis and occlusions . 
regarding the detection of coronary disease , pmcta and autopsy showed complete concordance in identifying at least one area of critical stenosis in each case ; however , 18 discrepancies were recorded , including 5 major and 13 minor . 
in 18 cases , 1 3 la radiologia medica ( 2019 ) 124 : 109117 both the autopsy and the pmcta showed coronary lesions that only in 11 cases were causally related to death . 
3 forest plot of specificity 1 3 114 la radiologia medica ( 2019 ) 124 : 109117 in conventional postmortem investigations , the diagnosis of coronary artery disease or ischemic heart disease is obtained after a complete autopsy followed by a gross examination of the heart and a microscopic examination with histological and immunohistochemical staining . 
nevertheless , such investigations require the experience of the pathologist and the application of a rigorous methodological approach to the complex task represented by sudden cardiac death . postmortem imaging provides a decisive contribution to forensic pathology , its importance is widely recognized , and its use is increasingly common in forensic practice [ 27 ]  . 
this consideration assumes remarkable significance in light of the importance of imaging methods in the approach to cases of sudden cardiac death [ 28 , 29 ]  . computed tomography ( ct ) is the most widely used method in modern forensic radiology . 
despite the extensive use of this type of investigation , the method presents two major limitations consisting of the low density of soft tissues and the poor visualization of vascular structures . 
in fact , in the included primary studies that required the execution of a scan without contrast medium before pmcta , pmct did not prove useful in the diagnosis of coronary heart disease , being able only to highlight findings such as cardiac tamponade or calcifications of coronary arteries . post - mortem ct angiography ( pmcta ) , on the other hand , is a minimally invasive procedure , able to show direct ( coronary stenosis or occlusions ) and indirect ( contrastographic enhancement of the myocardium as a sign of ischemic necrosis ) evidence of coronary artery disease with a much higher diagnostic accuracy than unenhanced pmct [ 30 , 31 ]  . the main advantage of pmcta is the possibility of visualizing myocardial bridging and alterations of luminal patency without altering the examined vascular structure , as it inevitably occurs during the conventional investigation . 
in fact , pmcta studies the vessels throughout their course allowing to identify even small areas of critical stenosis that could be missed by the sections carried out during the autopsy . 
in addition , pmcta has shown to be superior to autopsy in investigating the patency of coronary vessels as it is able to demonstrate through perfusion pressure the patency of calcific vessels that had been described as occluded at the autopsy . post mortem ct angiography also allows the study and three - dimensional evaluation of coronary artery bypass grafts and coronary stents , [ 32 , 33 ] facilitating the forensic pathologist in the identification of findings difficult to highlight during autopsy due to the adhesion phenomena attributable to the surgical manipulation of the pericardium and heart . 
5 bivariat model of sensitivity and specificity 1 3 la radiologia medica ( 2019 ) 124 : 109117 the main limitation of pmcta is the impossibility of providing data on the functional relevance of a stenosis , being an exclusively morphological imaging technique . 
 the included primary studies have also shown that only a relatively low percentage of ischemic myocardial necrosis was detectable by pmcta as a contrast enhancement area , documenting a sensitivity limit of this method compared to the histopathological evaluation or other imaging techniques such as pmmr . 
on the other hand , the tomographic scans are limited by the impossibility of visualizing the vessels downstream of the stenosis and by the difficulty in ascertaining the acute or chronic nature of the stenosis . 
a further doubt about the diagnostic efficacy of pmcta could derive from the possibility of a thrombus displacement following coronary artery reperfusion , with consequent failure to visualize coronary occlusion ; although the hypothesis cannot be refuted with certainty , several studies have reported as unlikely the possibility that a perfusion pressure lower than the invivo one can determine the displacement of a thrombus . the present systematic review and meta - analysis demonstrated the high accuracy of pmcta in the diagnosis of sudden coronary death . 
post mortem ct angiography represents a valid tool for the assessment of coronary arteries anatomy , for the detection of stenosis and occlusions , [ 34 ] as well as for the guide of sampling for histological examination . 
at the same time , however , this method has proved less effective in the visualization of myocardial ischemia and necrosis . there are two major limitations concerning the present study . 
first , the meta - analysis was performed on a small number of primary studies , many of which had a small population and did not provide enough information to clearly determine the quality of the study . 
therefore , even if an hsroc model was used for data analysis , the results should be interpreted carefully . for these reasons , to date , autopsy followed by histological examination remains the gold standard for the diagnosis of sudden coronary death . 
in fact , pmcta is extremely useful in cases where a minimally invasive approach is required for several reasons or in cases where a guide to myocardial and coronary sampling is required during the autopsy . in conclusion , in the field of cardiac pathology , postmortem coronary imaging is still insufficient to establish with certainty the cause of death and autopsy remains the gold standard . 
currently , in fact , no radiological technique can replace the histological examination . the value of this study is in the combination of data from several studies resulting in the evaluation of a larger cohort of subjects as well as in obtaining data more applicable to forensic practice . 
nevertheless , further studies are still required to offer better evidence on the use of pmcta as a routine investigation or as an aid to conventional autopsy . the role of pmcta in cases of sudden cardiac death is that of guidance and aid to traditional autopsy . 
in particular , this investigation can determine an increase in diagnostic performance and ensure a better evaluation of the coronary arteries as well as a more accurate coronary and myocardial sampling [ 3640 ]  . for these reasons , postmortem computed tomography angiography must be considered an ancillary method in determining the cause of sudden cardiac death , rather than an alternative to conventional investigations [ 41 , 42 ]  . authors contribution all authors contributed to manuscript drafting and critical discussion and approved the definitive version . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical standards this article does not contain any studies with human participants or animals performed by any of the authors . 
we also attempted to identify a relationship between mdct imaging and the 2010 world health organization ( who ) classification system . materials and methods we selected all patients with pathologically proven gi - nets diagnosed between january 2010 and august 2017 . 
we also analysed the relationship between mdct imaging and the 2010 who classification . results a total of 20 patients ( 13 males , 7 females , age range 3789years , mean age 69.9years ) were included in our study . 
 the majority of gi - nets ( 85% ) occurred in the small bowel and mainly in the terminal ileuforty - five percentage of our gi - nets were diagnosed after an access to emergency medical service for obstruction symptoms or gastrointestinal bleeding . 
desmoplastic reaction and lymph nodes metastases were significantly correlated with higher grade of gi - nets . conclusions the majority of gi - nets appears as intraluminal mass often associated with extra - intestinal signs . 
contrast - enhanced mdct has allowed an optimization of scan protocols , with many advantages , including ( 1 ) very rapid scan times reducing movement artefacts ; ( 2 ) contrast - enhanced images with arterial and portal phase and ( 3 ) the ability to reformat the images in thinner slices , in order to improving resolution and allowing the images to be viewed optimally in different anatomical planes . 
for these reasons , contrast - enhanced mdct is the main imaging modality for diagnosis , staging , preoperative evaluation and post - treatment follow - up of net [ 24 ]  . 
mdct images of gi - nets usually appear as a hyper - enhancing nodular mass arising from the bowel wall or as a regional uniform bowel wall thickening [ 4 , 5 ]  . 
the world health organization ( who ) classification system of 2010 attempted to grade gi - nets on the basis of their ki - 67 index and mitotic count , dividing these tumours into well - differentiated neuroendocrine tumours ( grades 1 and 2 , ki - 67 index < 2% and 320% , respectively ) , also termed carcinoid tumours , and poorly differentiated neuroendocrine carcinomas ( grade 3 , ki - 67 > 20% ) [ 610 ]  . 
we also attempted to identify a relationship between mdct imaging and the 2010 who classification system . materials andmethods patients with a query ( gastrointestinal and neuroendocrine tumours ) of the patients medical records database at the careggi hospital of florence , a radiologist resident selected all patients with pathologically proven gi - nets diagnosed between january 2010 and august 2017 . 
 all patients signed a written consent form . all ct investigations were carried out on a 64 - detector helical ct ( somatom sensation 64 , siemens healthcare , germany )  . a standard protocol was used : the patients were scanned in supine with craniocaudal breath - hold scans ; all cases underwent non - contrast and contrast - enhanced mdct scanning with a thickness of the scanning slices of 3 or 5m iodinated contrast medium ( ultravist 300 , bayer schering , berlin , germany ) was injected in the antecubital vein at a flow rate of 34ml / s using an automatic injector , immediately followed by a saline flush ( 4050ml ) with a rate of injection of 34ml / s . 
dual - phasic contrast - enhanced images were obtained during the arterial phase ( 3035s after the start of the injection ) and portal venous phase ( 7075s after initiation of the injection )  . the parameters for both non - contrast and contrastenhanced ct examination were : tube voltage , 120kv ; tube current , 200250 mas , depending on the patients size ; beam collimation , 64 0.5mm ; and rotation time , 0.4s. imaging analysis all gi - nets were retrospectively assessed for the mdct intestinal and extra - intestinal signs . 
the readers were blinded to the clinical and pathological data of all of the patients . among intestinal signs , we evaluated morphological features , classifying the lesions as the following , in accordance with the existing literature [ 4 , 5 , 11 ] : intraluminal nodular mass arising from the bowel wall and regional uniform bowel wall thickening . the extra - intestinal signs included mesenteric metastasis surrounded by a desmoplastic reaction , ascites , lymph nodes and liver metastases . 
in accordance with the existing literature [ 5 , 12 ] , we classified as mesenteric metastasis a mass - like process generally located in the mesenteric fat near to the primary tumour , which may or may not be calcified . 
these signs were classified as the classic desmoplastic reaction that could lead to kinking of the bowel , resulting in bowel obstruction , or in venous ischaemia [ 5 , 12 , 13 ]  . 1 3 96 pathological classification histopathological classification of gi - nets was made according to who 2010 guidelines [ 610 ] as : grade 1 ( g1 ) , mitotic count < 2 per 10 high - power fields ( hpf ) and / or ki - 67 2% ; grade 2 ( g2 ) , mitotic count 220 per 10 hpf and / or ki - 67 320% ; and grade 3 ( g3 ) , mitotic count > 20 per 10 hpf and / or ki - 67 > 20% . statistical methods graphpad prism v7.0 software ( graphpad software inc . , la jolla , ca , usa ) was used to perform all statistical analyses . 
 among these , eight patients did not undergo contrastenhanced mdct in the immediate preoperative period and three patients were excluded for poor mdct image quality . a total of 20 patients ( 13 males , 7 females , age range 3789years , mean age 69.9years ) were included in our study . of the 20 cases , 3 ( 15% ) were duodenal neuroendocrine tumours and 17 ( 85% ) were small - bowel neuroendocrine tumours , defined as tumours of the jejunum and of the ileum [ 14 ]  . 
three patients complained weight loss and abdominal pain as their first symptovomiting was present in two cases , and only one patient showed jaundice due to the presence of a periampullary duodenal tumour . 
finally , in two cases , gi - nets were discovered incidentally by mdct imaging studies performed during follow - up of a lung cancer and of a breast cancer , respectively , ( table1 )  . mdct andhistopathological findings mdct findings of each patient are shown in table 2 . 
axial ( a ) and coronal ( b ) contrast - enhanced mdct images in the arterial phase demonstrate a wellcircumscribed enhancing mass ( white arrows ) in the ileocecal valve 1 3 la radiologia medica ( 2019 ) 124 : 94102 fig . 
axial ( a , b ) , sagittal ( c ) and coronal ( d ) contrastenhanced mdct images in the arterial phase demonstrate multiple intraluminal masses ( white arrows ) fig . 
arterial phase ( c ) and portal venous phase ( d ) contrast - enhanced mdct show multiple hyper - vascular liver metastases that become almost isodense on the portal venous phase except for central regions of necrosis . 
5 gastrointestinal neuroendocrine carcinoma ( g3 ) in the terminal ileuaxial contrastenhanced mdct image in the portal venous phase ( a ) demonstrates a bowel wall thickening of the terminal ileum and a stellate soft - tissue nodule ( white star ) in the mesentery , typical of a desmoplastic reaction . 
there were no statistically significant differences in intestinal signs , liver metastases and ascites ( table3 )  . discussion in the last 20years , the incidence of gi - net has increased probably due to recent improvements in diagnostic techniques such as the diffusion of thin - section multi - detector ct . 
although nets can occur in any organ , approximately 6070% of these tumours arise in the gastrointestinal tract because it has the largest reservoir of neuroendocrine cells in the body [ 15 ]  . 
studies analysing data from 1973 to 1997 found that the most common site of gi - nets is the small bowel , defined as jejunum and ileum [ 16 ]  . 
our study confirmed that the majority of gi - nets ( 85% ) occurs in the small bowel and mainly in the terminal ileum . at the time of initial diagnosis , half of the patients with gi - nets have advanced disease [ 17 ] , with metastases to the regional lymph nodes , liver and , finally , bone [ 18 , 19 ]  . 
in addition , the carcinoid syndrome occurs in the presence of liver metastases because only with hepatic metastatic disease the biologically active metabolite ( 5 - hydroxy - tryptamine ) , which is otherwise inactivated in 1 3 la radiologia medica ( 2019 ) 124 : 94102 100 fig . 
6 neuroendocrine tumour ( g2 ) in the terminal ileu axial ( a ) and coronal ( c ) contrast - enhanced mdct images demonstrate a bowel wall thickening of the terminal ileum ( white arrows ) : the thickened wall is homogeneously enhanced . 
the presence of the characteristic carcinoid syndrome can facilitate nets detection , otherwise patients present non - specific symptoms , such as weight loss , bleeding or abdominal pain or are asymptomatic [ 20 , 21 ]  . 
 this result demonstrates how the radiologists should suspect gi - nets also in the emergency setting . in mdct , after administration of contrast medium , the primary tumour usually presents as a hyper - enhancing , nodular mass arising from the bowel wall or as a regional uniform bowel wall thickening [ 4 , 5 ]  . 
similar to their results , we observed intraluminal nodular mass in 75% of our patients and wall thickening in 25% . a very typical extra - intestinal finding in gi - nets is the mesenteric metastasis , which generally appears as a masslike process in the mesenteric fat near the primary tumour . 
 we observed desmoplastic reaction in 40% of cases , lymph nodes metastases in 60% , liver metastases in 45% and ascites in 35% . the who 2010 classification system is currently wellaccepted by clinicians as a simple , reproducible , and prognostic effective grading of gi - nets , since the who classification from 2017 is only for pancreatic net ( p - nets ) [ 6 ]  . 
the who classified gi - nets into well - differentiated tumours with benign or uncertain behaviour ( g1 ) ; welldifferentiated tumours with low - grade malignant behaviour ( g2 ) ; and poorly differentiated carcinomas with high - grade malignant behaviour ( g3 ) [ 610 ]  . 
in recent literature , concerning 1 3 la radiologia medica ( 2019 ) 124 : 94102 gi - nets , no correlation between imaging features and grading of pathologic classification is reported . 
our results indicated that mdct imaging features may be predictive of gi - nets classifications . in our series , all g1 tumours appeared as intraluminal mass while 50% of g3 tumours showed a bowel wall thickening as intestinal sign . 
nevertheless , no statistically significant differences were found in intestinal signs . comparing mdct and histopathological findings , we found statistically significant difference among g1 , g2 and g3 tumours in desmoplastic reaction and lymph nodes metastases . 
this is likely due to the fact that the pathological grading criteria were established based on the mitotic count and ki - 67 index , both of which reflect the proliferation and invasiveness of tumour cells [ 23 , 24 ]  . several criticisms may be raised with respect to our study . 
second , although we examined gi - nets between january 2010 and august 2017 , our sample size is rather small consisting of 20 patients because patients did not undergo contrast - enhanced mdct in the immediate preoperative period or with poor mdct image quality were excluded . 
thus , it may be possible that the distribution of grading correlation we found is different in a more general population . in conclusion , as confirmed in the literature , our study showed that the majority of gi - nets appears as intraluminal nodular mass often associated with extra - intestinal signs on mdct imaging . on the other hand , we found a statistically significant correlation between higher grade of gi - nets and extra - intestinal signs , not previously demonstrated . 
therefore , mdct imaging is a technique that may be useful in predicting the pathological classification of gi - nets . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest related to the publication of this article . 
no funding was received for this study . ethical approval all procedures performed in the studies involvement human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . 
informed consent was obtained from all individual participants included in the study . la radiologia medica ( 2019 ) 124 : 132135 radiotherapy fiducial markers implantation forprostate imageguided radiotherapy : areport onthetransperineal approach luigidecicco1 stefanobracelli1 received : 21 may 2018 / accepted : 15 october 2018 / published online : 25 october 2018 italian society of medical radiology 2018 abstract introduction in the external beam prostate cancer radiation therapy , daily gland displacement could lead to a target missing . 
the use of intra - prostatic gold fiducial markers for daily prostate position verification and correction before and during treatment delivery ( image - guided radiotherapy , igrt ) is widely used in the radiation therapy centers to accurately target the prostate . 
we report our experience in prostate fiducial markers implantation through a transperineal approach . patients and methods between september 2011 and january 2018 at our center , 101 patients underwent gold seed fiducial marker transperineal ultrasound - guided implantation for prostate igrt . 
no markers lost or migration occurred . discussion and conclusion according to our experience , prostate fiducial markers implantation through a transperineal approach is safe and should be recommended to limit the use of antibiotic therapy and patients morbidity . 
a previous turp was not related to a higher risk of loss of seeds . keywords prostate cancer radiotherapy fiducial markers image - guided radiotherapy prostate radiotherapy introduction in the external beam prostate cancer radiation therapy , daily gland displacement could lead to a target missing . 
prostate pelvic motion up to 2cm are reported , so that the use of intra - prostatic gold fiducial markers for daily prostate position verification and correction before and during treatment delivery ( image - guided radiotherapy , igrt ) is widely used in the radiation therapy centers because accurately targeting of the prostate allows margin reduction , sparing normal tissue treated and thereby reducing complications , [ 1 , 2 ] without affecting tumor control and allowing dose escalation . 
 gold fiducial markers are considered an accurate , reliable and tolerable method of daily prostate localization [ 3 ]  . the most commonly used implantation procedure involves transrectal ultrasound - guided insertion of fiducial markers , with no negligible complications such as infections , pain and rectal bleeding . we report our experience in prostate fiducial markers implantation through a transperineal approach ; it is very similar to radioactive seed implantation for low - dose - rate brachytherapy . patients andmethods * luigi de cicco luigi.decicco@yahoo.it 1 department ofradiation oncology , asst della valle olona , via a . 
da brescia , 1 , 21052bustoarsizio , va , italy between september 2011 and january 2018 at our center , after a written consent acquisition , 101 patients underwent gold seed fiducial markers transperineal ultrasound - guided implantation for prostate igrt , performed by a referring vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 132135 radiation oncologist with proper skills in prostate brachytherapy ( dcl )  . 
one marker was placed in the prostate base , one at the apex and one in the middle ; two seeds were released in a prostatic lobe and one in the other one . of each fiducial , were acquired and printed two orthogonal ultrasound images to record the correct intra - prostatic position . in one patient , one seed was detected through the prostatic capsule with a higher risk of lost , so a fourth seed was implanted into the gland . after the procedure , patients remained under observation for about 2h and were encouraged to drink water and to urinate into a container to detect a possible hematuria . 
after 2h and at least two voids , the patients were re - evaluated in relation to any dysuria , or pabefore discharge , they were instructed to contact the referring radiation oncologist if there were any complications or variation in the urinary framework or pain . the planning tc was performed at least 7days later . 
during radiation treatment , daily cone - beam ct or orthogonal kv / mv was used to verify the correct position of the target and monitor the number of the markers and distance between them . in 3 ( 7% ) patients the antiplatelet oral therapy was not interrupted ( table1 )  . all the 101 procedures of fiducial markers implantation through the perineum under transrectal ultrasound guidance were uncomplicated . 
no procedure was interrupted because of unsustainable patient pain . in one patient , that have stopped antiplatelet oral therapy 7 days before the procedure , a singleepisode of self - limiting urinary bleeding occurred just after implantation . 
all the patients , at the evaluation before discharge , reported no pain or dysuria . no rectal bleeding , no urinary obstruction or infection and no episode of hematospermia were reported in the next days . all patients started radiation therapy as planned , without any delay . during the subsequent phases of the radiation treatment , all the implanted markers were detected within the prostate gland ; no markers lost or migration grater than 2mm occurred . discussion andconclusion the insertion of intra - prostatic markers for igrt is commonly used to track the prostate gland before and during radiation treatment delivery to reduce margin around prostate , sparing surrounding organs and escalating dose to the tumor . 
the transrectal implantation way is the widely used one because this procedure is commonly carried out by urologists and this approach requires the same equipment used for the prostatic biopsies , so that , likewise to transrectal prostate biopsy , no negligible complications such as infections , pain and rectal bleeding are expected . an increasing incidence of multidrug resistant infections is reported in the transrectal prostate biopsy that is similar to the transrectal seed implantation procedure , with febrile infection rates commonly ranging from 1 to 4% despite the prophylactic antibiotic therapy [ 4 ]  . results median age of the 101 patients was 75years ( range 5384 )  . twenty - two ( 21.8% ) patients had previously been subjected to a transurethral prostate resection ( turp ) for obstructive urinary symptoms because of benign prostatic hypertrophy . forty - three ( 42.6% ) patients were in anticoagulant or antiplatelet oral therapy . 
according to their cardiovascular risk , in 13 patients of this group ( 30% ) these medications were stopped , in 27 ( 63% ) patients were replaced with low molecular weight heparin 710days before the implant , and table 1 characteristics of patients median ( age ) 75years ( range 5384 ) no of patients ( % ) previous turp in anticoagulant or antiplatelet therapy 1 . 
3 ( 7% ) turp transurethral prostate resection , ebpm low molecular weight heparin 1 3 134 la radiologia medica ( 2019 ) 124 : 132135 there are increasing concerns about the incidence of serious infection associated with this procedure , in tandem with a rise in the prevalence of rectal ciprofloxacin - resistant organisms ( cro )  . 
reported a relatively high rate of seeds loss , implanting four markers in two strands with two fiducial each one , 1cm apart ; the two strands of the two markers were located on the same plane in the middle of the prostate , at least 2cm apart from the midline . 
 all intra - prostatic fiducials were usefully used to track the prostate gland before and during treatment [ 10 ]  . the first report on the transperineal markers implantation technique was on a little group of 12 patients ; no infective episodes occurred , but a mild rectal bleeding associated with significant pain resulted from the procedure in the initial half of the series , maybe related to the lower experience of the operator in this part of the learning curve [ 12 ]  . a seed loss in igrt could compromise the correct continuation of the treatment . 
it could be related to the implantation technique or operator experience ; seed misplacing had been reported more frequently in the first group of implanted patients [ 12 ]  . 
several authors had already highlighted the importance of avoiding the urethra and releasing the seeds only when the tip of needle has sure penetrated the capsule , considering the tending of the prostate by the needle , to avoid their loss [ 2 ]  . transperineal ultrasound - guided prostate fiducial markers implantation is very similar to radioactive seed implantation for low - dose - rate brachytherapy ; seed loss through the urinary tract is a common event after prostate brachytherapy ; and it is significantly more frequent in patients with prior turp [ 13 ]  . 
no previous report regarding the implant of seeds for igrt shows the presence or absence of previous turp among the characteristics of the patients ; it could be related to rate of loss of seeds reported in these series . no gold implanted seed was lost in our experience . 
 according to our knowledge , no previous report exists on the prostate fiducial markers implantation in patients with a previous turp ; it should not be considered a contraindication to the procedure , because in our series sparing the cavity linked to the previous turp avoided the risk of seeds loss . according to the literature data , in our series the fiducial markers implantation into the prostate gland through a transperineal approach resulted in a very low rate of complications . 
avoiding to cross the rectum , the risk of rectal bleeding or infections is very limited , the use of antibiotic prophylaxis can be omitted , and the start of radiation treatment is not delayed . 1 3 la radiologia medica ( 2019 ) 124 : 132135 no antibiotic prophylaxis was adopted in our series resulting in no infective event in 101 procedures . despite the high number of patients in anticoagulant or antiplatelet therapy ( 42.6% ) , due to the high median age ( 75years ) and the consequent frequent comorbidities , a low incidence ( 1% ) of self - limiting hematuria ( a single episode ) and no case of hematospermia were recorded . our detected low risk of bleeding agrees with other bibliographic experiences previously reported ; moman etal . 
 [ 9 ] do not advise discontinuation of anticoagulant therapy to their patients because the risk of bleeding is considered inferior to the impact of possible blood clot formation , and no severe bleeding has occurred in the past years . no worsening of baseline urinary obstruction symptoms was reported by the patients . 
all the patients after the procedure referred no persistent pain our series , no systemic analgesic drug was administered before the procedure , but only local anesthesia with mepivacaine injection was enough to tolerate it ; no drug was necessary after it . according to our experience , prostate fiducial markers implantation through a transperineal approach is safe even in patients with a previous turp , in anticoagulant or antiplatelet therapy , and advanced age ; it should be also recommended to limit the use of antibiotic therapy and patients morbidity . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical standards this is a retrospective study . 
future studies are needed to confirm our findings . keywords prostate lesion magnetic resonance imaging ivim perfusion prostate cancer is the most common cancer in men ( after skin cancer )  . 
during progression switch , prostate cancer induces angiogenesis and vasculogenesis , and as a result , it shows rapid contrast enhancement and washout in dynamic contrast - enhanced mri sequences [ 5 ]  . 
it has clearly shown that diffusionweighted images with high b value ( 2000s / mm2 ) improve detection of prostate cancer , but there is an ongoing controversy in the literature related to the value of intravoxel incoherent motion ( ivim ) imaging [ 7 , 8 ]  . 
since the rate of signal attenuation resulting from perfusion is typically an order of magnitude greater than from molecular diffusion in tissues because of larger vol . : ( 0123456789 ) 1 3 88 la radiologia medica ( 2019 ) 124 : 8793 distances of proton displacement during the application of the pulses , its relative contribution to the diffusion - weighted mri signal becomes significant only at very low b values [ 9 , 10 ]  . dce - mri uses compartmental pharmacokinetic models of tracer kinetics to describe the microscopic processes leading to the distribution of molecules of contrast agent between the vascular and extravascular spaces . 
time dependence of contrast agent concentration was applied to calculate contrast agent transfer rate between blood and tissue ( ktrans ) , contrast agent back - flux rate constant ( kep ) , extravascular extracellular fractional volume ( ve ) , initial area under curve ( iauc ) and 2 values ( tofts model )  . 
due to sensitivity to contrast media , dynamic contrast - enhanced imaging table 1 acquisition parameters of the multiparametric mri protocol was not performed in 3 patients , and due to image distortion , 2 patients were excluded from the study . 
dcemri was performed with a 3d volumetric t1 - weighted gradient echo sequence using the view - sharing undersampling technique twist ( time - resolved angiography with stochastic trajectories )  . 
using an automated injector ( spectris solaris , medrad , indianola , pa ) , 0.1mmol / kg bolus of gadolinium - based contrast agent was injected intravenously , followed by 20ml saline flush , both at a rate of 2ml / s . 
mri parameters are shown in table1 . dwi data in a dicom format were transferred to a standard personal computer running an in - house - developed software package ( firevoxel , .nyu.edu / hr18 / publi c ) ( 12 )  . 
 [ 9 ] : sbs0 = ( 1 f ) exp ( bd ) + f exp [ b ( d + d ) ] where sb is the signal intensity in the pixel with diffusion gradient b , s0 is the signal intensity in the pixel without diffusion gradient , d is the true diffusion as reflected by pure molecular diffusion , f is the perfusion fraction related to microcirculation , and d * is the pseudodiffusion coefficient representing perfusion - related diffusion or incoherent microcirculation [ 12 ]  . the methods , formulas and parameters we used in our study are as follows : and prostate intraepithelial neoplasia were accepted as malignant . statistical analysis data were analyzed using the statistical package for the social sciences version 20.0 for windows ( spss inc . , chicago , il , usa )  . 
all patients underwent histopathological analysis , and cases with prostate cancer the normal peripheral zone ( pz ) was considered to show a homogeneously high - intensity signal on the t2 - weighted images . 
the voi placed ( b ) on lesion and the ktrans fusion map ( c ) and concentration curve and values ( d ) showed high ktrans , kep , ve , iauc and 2 values . 
two patients ( gleason 7 and gleason 9 ) had extraprostatic extension . discussion in our study , we found that the ivim diffusion and dynamic contrast - enhanced parameters of malignant tumors in peripheral zone and transitional zone differ . 
there was a significant difference between malignant transitional zone lesions and benignnormal transitional zone tissue for all parameters except kep , whereas there was no significant difference between malignant peripheral zone lesions and benignnormal peripheral zone tissue for ve , f and dp values . 
 the f ( perfusion fraction ) in the prostate cancer was significantly lower than that in the normal transitional and 1 3 92 la radiologia medica ( 2019 ) 124 : 8793 fig . 
the voi placed ( b ) on lesion and the ktrans fusion map ( c ) and concentration curve and values ( d ) showed low ktrans , kep , ve , iauc and 2 values . 
there were no significant differences in the normal transitional and peripheral zones , benign tissue and prostate cancer for the dp ( perfusion - related diffusion coefficient ) [ 7 ]  . 
our study showed that dp , dt and f values were significantly lower for transitional zone tumors , but dp and f values were not significantly lower for peripheral zone tumors . 
we also showed that lower dt values are important to diagnose prostate cancer in both transitional zone and peripheral zone . we also found that perfusion parameters ktrans , kep and iauc were significantly higher for peripheral zone malignant tumors , and ktrans , ve and iauc were significantly higher for transitional zone tumors . 
also found higher ktrans , kep , ve and iauc values in malignant lesions and stated that higher iauc is strongly correlated with prostate malignant lesions [ 13 ]  . this study has several limitations . 
third , we accepted premalignant lesions ( hgsil , high - grade squamous intraepithelial lesions , and asap , atypical small acinar proliferation ) as malignant that might influence our results . 
finally , we did not use endorectal coil in our study since in prostate imaging guidelines , it is only recommended but not a must . 1 3 la radiologia medica ( 2019 ) 124 : 8793 conclusion in conclusion , both restricted diffusionpseudodiffusion and increased perfusion parameters ( especially ktrans and iauc ) are important to differentiate prostate cancer from benign pathologies . 
the aim of this study was to evaluate the tissue stiffness values of pgs of hiv - infected children via swe and compare the results with the counterparts of healthy subjects . materials and methods this single - center , prospective study included the pg examinations of 23 pediatric hiv patients and 40 healthy children via grayscale ultrasound and swe . 
in addition , when the patients were separated into two groups according to the appearance of pg on grayscale ultrasound as homogeneous and heterogeneous , stiffness values were increased in the patients with homogeneous parenchymal appearance . 
swe can be used as an ultrasound - assisted noninvasive technique in this manner . keywords human immunodeficiency virus infection parotid gland shear wave elastography children introduction the association of parotid gland involvement with human immunodeficiency virus ( hiv ) infection has been reported soon after the first description of hiv infection [ 1 ]  . 
parotid gland involvement is usually presented as soft , painless , sometimes cystic , slowgrowing masses which ismostly accompanied by persistent cervical or generalized lymphadenopathy which may also be the initial manifestation of hiv infection [ 5 ]  . parotid gland ( pg ) imaging via either ultrasonography , computerized tomography ( ct ) or magnetic resonance imaging ( mri ) has long been used for the pg enlargement detection and follow - up [ 68 ]  . 
as far as we could research from english literature , no study has yet been performed regarding pg examination in hiv - infected children . in our study , we evaluated the pgs of hiv - infected children and compared the tissue stiffness values with the counterparts of healthy subjects . 
all cases in the study had sufficient image quality obtained and appropriate technique achieved , so no case was excluded from the study . grayscale ultrasound and swe measurements were completed by experienced pediatric radiologists with more than 10years of ultrasound and 5years of elastography experiences . 
the quality mode , which is identified as the propagation mode ( arrival time contour ) , is a mode in which reliable data are obtained when the lines are parallel and smooth , and the increase in distance between lines is parallel to the increase in stiffness . 
the speed of ultrasound waves was either measured in meters / second ( m / s ) or kilopascals ( kpa ) ; within seconds the result was displayed on the ultrasound screen . 
three valid measurements were recorded in kpa and m / s modes la radiologia medica ( 2019 ) 124 : 126131 values were automatically calculated by averaging 9 values for each pg . 
mannwhitney u test table 1 charactertistics of hiv - infected children parameter twenty - three children with the median age of 11 ( 117 ) years were included in the study . 
the median of cd4 counts and hiv viral load on the last patient were 267 ( 62820 ) cells / l and < 45 ( < 45896.398 ) copies / ml , respectively . 
others demonstrated lymphocytic aggregations , lymphoepithelial cysts , fatty infiltration and isolated lymphadenopathy . most of the reports point out the presence of cystic lesions in pgs of hiv - infected patients . 
increased tissue stiffness shown by swe even in the absence of those gross changes is particularly noteworthy . in our study , the patients with homogeneous pg appearance also had higher stiffness values than healthy controls . 
in the patients with heterogeneous pg appearance , swe can be used as an assistant for ultrasound . parotid gland enlargement in hiv can be due to inflammatory disorders , infections , neoplasms and benign lymphoepithelial cysts , so - called blecs . 
histologically , benign lymphoepithelial lesions with ductal metaplasia , reactive hyperplasia of an intraparotid lymph node and benign lymphoepithelial cysts are observed in pgs of patients with hiv infection [ 16 ]  . 
the second category named shear wave speed ( sws ) measurement includes transient elastography ( te ) and arfi quantification , as a point shear wave elastography ( p - swe ) , which can quantitatively calculate sws for assessing tissue stiffness . 
both p - swe and 2d - swe are based on capturing the propagation of sws induced by displacement of local tissues under the excitation of acoustic impulses from probes . 
 [ 14 ] mentioned that arfi provides a quick , easy and reliable diagnostic tool for the assessment of disease severity and progression in patients with chronic recurrent parotitis that can be seamlessly implemented into preexisting ultrasound protocols . our study is privileged since it is the first study evaluating pgs of hiv - infected children via swe . 
during routine cervical examination , pg evaluation via swe together with grayscale ultrasound can be very helpful . among the limitations of our study are the low numbers of patients , no deep lobe of pg measurements and lack of histological evaluation . 
another limitation is that elastography measurements were taken by single operator , and there was no comparison between users . conclusions pg examination of hiv - infected children via swe reveals increased tissue stiffness when compared with healthy subjects . 
swe can be used in the detection of early parenchymal 1 3 la radiologia medica ( 2019 ) 124 : 126131 changes even in the patients with homogeneous pg appearance . 
the diagnostic performance and agreement of us features between the cad system and the radiologist were compared . results sixty - two patients ( 50 f ; age 60 12years ) were enrolled : 77.4% ( 48 / 62 ) of thyroid nodules were benign , 22.6% ( 14 / 62 ) were undetermined to malignant and required follow - up or surgery . 
although it is an innovative tool with good potential , additional efforts are needed to improve its diagnostic performance . keywords computer - aided diagnosis nodule thyroid ultrasound * salvatore gitto sal.gitto@gmail.com 1 scuola di specializzazione inradiodiagnostica , universit degli studi di milano , via festa del perdono 7 , 20122milan , italy 2 scuola di specializzazione inendocrinologia e malattie del metabolismo , universit degli studi di milano , milan , italy 3 fondazione irccs istituto nazionale dei tumori , milan , italy 4 servizio di radiologia , irccs policlinico san donato , sandonatomilanese , italy 5 dipartimento di scienze biomediche perla salute , universit degli studi di milano , milan , italy 6 unit operativa di radiologia diagnostica ed interventistica , irccs istituto ortopedico galeazzi , milan , italy 7 divisione di radiologia interventistica , ieo , istituto europeo di oncologia irccs , milano , italy vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 118125 introduction thyroid nodules are detected in up to 68% of general population and malignancies are observed in 310% of cases [ 1 , 2 ]  . 
 the critical role of the radiologist is to detect and characterize thyroid nodules with ultrasound ( us ) and us - guided fine - needle aspiration biopsy ( fnab ) serving as diagnostic cornerstones [ 3 ]  . 
us and fnab ensure the identification of thyroid nodules that need to be removed , such as malignancies and indeterminate follicular lesions , also reducing the number of unnecessary surgical procedures in favor of both conservative and minimally invasive treatment of benign thyroid nodules [ 48 ]  . computer - aided diagnosis ( cad ) systems have been developed to aid the radiologist in the image interpretation , speed up the diagnostic process and reduce interobserver variability . 
it aims to help users to increase diagnostic confidence with accurate and consistent recommendations , and improve patient throughput by adopting automatic report feature [ 9 ]  . the aim of our study is to compare the diagnostic performance of this new , commercially available cad system with that of a non - computer - aided radiologist in the characterization of low - to - high suspicion thyroid nodules . materials andmethods study design andpopulation the institutional review board of our hospital approved this retrospective study and waived the need for informed consent . 
thy 1 according to the bethesda system for reporting thyroid cytopathology [ 15 ] ; and ( 2 ) oblique rather than axial thyroid nodule us image available for post - processing . us examination andfnab all us examinations and procedures were performed with the patient lying supine on the examination table and the neck in hyperextension ; right or left - deviation were used according to the nodule location . 
the right and left lobes of the thyroid gland were evaluated in axial and longitudinal planes by a radiologist with 5years of experience in diagnostic and interventional thyroid imaging using a high - end us system ( rs80a with prestige us systemsamsung medical imaging , seoul , korea ) equipped with a broadband linear - array transducer ( l312a ) that worked at a fixed frequency of 10mhz . 
cytological analysis results were available in 47days after the procedure and classified according to the bethesda system for reporting thyroid cytopathology [ 15 ]  . postprocessing andimage analysis post - processing was performed in consensus by a radiology resident and an endocrinology resident with 18 and 6months of supervised experience in thyroid imaging , respectively , both blinded to cytological results . 
for each patient , an axial b - mode image of the thyroid nodule selected for fnab was combined with s - detect for thyroid ( samsung medical imaging , seoul , korea )  . 
based on this box , it performs thyroid nodule segmentation and then allows the user to select among a series of candidates or manually modify the contours of the lesion . 
 1 3 120 la radiologia medica ( 2019 ) 124 : 118125 information regarding the presence of calcifications is available only upon user selection , and thus the operators selected it after they retrieved this information from medical imaging reports that were provided by the experienced radiologist at the time of examination and were available for review . 
k - tirads [ 17 ] , russ [ 18 ] , and american thyroid association guidelines [ 11 ] , are embedded in the us systek - tirads was chosen for rating the nodule risk of malignancy in this study . the same radiologist performing the fnab with 5years of experience in diagnostic and interventional thyroid imaging , blinded to cytological results and post - processing analysis , randomly reviewed the thyroid nodule us images previously used for post - processing . 
a recommendation on the nodule risk of malignancy was formulated and based on k - tirads . statistical analysis for statistical analysis , anonymous data were entered into a database ( microsoft excel 2010 ) and then analyzed with spss 22.0 windows version ( spss inc . , armonk , ny , table 1 lexicons for thyroid nodule us assessment provided by the cad system [ 9 ] lexicon composition echogenicity orientation margins spongiform shape calcificationsa elasticitya vascularitya a only user selection is available indication solid partially cystic cystic hyper / isoechoic hypoechoic parallel non - parallel well - defined smooth microlobulate / spiculated ill - defined appearance non - appearance ovoid to round irregular micro / macrocalcifications no calcifications stiff soft appearance non - appearance usa )  . 
interobserver agreement between the cad system and the radiologist for assessment of thyroid nodule composition , echogenicity , orientation , margins , shape , and k - tirads was measured with the kappa statistic and interpreted using landis and kochs scale [ 19 ]  . 
for both the cad system and the radiologist , the sensitivity , specificity , positive predictive value , negative predictive value and accuracy in the identification of thyroid nodule requiring follow - up or surgery were calculated . 
categorical variables were reported as absolute value and percentage ; continuous variables were reported as mean value standard deviation , as detailed in the text . results a series of 79 patients ( 20 men , 59 women ; age 61 12years ) were referred for us - guided fnab of a thyroid nodule . 
among them , 17 patients were excluded due to non - diagnostic specimen after cytological analysis ( 8 cases ) or oblique rather than axial thyroid nodule us image available for post - processing ( 9 cases )  . 
 thyroid nodule maximum diameter measured 18 7m according to the bethesda system for reporting thyroid cytopathology [ 15 ] , 77.4% ( 48 out of 62 ) of thyroid nodules were cystic or benign , 22.6% ( 14 out of 68 ) were undetermined to malignant and required follow - up or surgery ( table2 )  . post - processing was performed in all included thyroid nodules and took 4 to 5min per nodule . 
thyroid nodules were classified into the k - tirads categories 3 , 4 and 5 in 50 , 8 and 4 cases by the cad system and 35 , 18 and 9 cases by the radiologist , respectively . 
 table4 details the sensitivity , specificity , positive predictive value , negative predictive value and accuracy for both the 1 3 la radiologia medica ( 2019 ) 124 : 118125 table 2 diagnostic categories of the thyroid nodules in our population according to the bethesda system for reporting thyroid cytopathology [ 15 ] diagnostic categories table 3 us features and k - tirads assigned to each thyroid nodule by the cad system and the experienced radiologist thy 1cconsistent with cyst thy 2benign thy 3aatypia of undetermined significance or follicular lesion of undetermined signifi4 ( 6.5% ) 44 ( 70.9% ) 8 ( 12.9% ) cance thy 3ffollicular neoplasm or suspicious for a follicular neoplasm thy 4suspicious for malignancy thy 5malignant lexicon indication cad system radiologist composition echogenicity orientation margins shape k - tirads solid partially cystic cystic hyper / isoechoic hypoechoic parallel non - parallel well - defined smooth microlobulate / spiculated ill - defined ovoid to round irregular category 3 ( low suspicion ) category 4 ( intermediate suspicion ) category 5 ( high suspicion ) no . 
this thyroid nodule was undetermined ( thy 3a ) according to the bethesda system for reporting thyroid cytopathology discussion thyroid nodules are a common clinical problem with a reported prevalence up to 68% of general population . 
the vast majority consists of benign lesions not requiring intervention or follow - up , as malignancies are observed only in 310% of cases [ 1 , 2 ]  . 
specifically , the radiologist classified the orientation and margin as non - parallel and well - defined , whereas the cad system classified them as parallel and ill - defined , respectively . 
such interobserver variability reflects that described between radiologists themselves , although higher concordance was found in margin evaluation [ 22 ]  . in the literature , there are a limited number of studies using cad systems and us to characterize thyroid nodules . 
some preliminary studies based on images obtained from ceus - 3d ( contrast - enhanced three - dimensional ultrasound ) , 2d - us ( two - dimensional ultrasound ) and hrus ( high - resolution ultrasound ) reported an excellent accuracy for thyroid malignancy detection [ 2326 ]  . 
however , such studies were conducted by independent research groups on a small number of patients not enrolled in a clinical setting , using non - commercial systems impractical for real time use because of complex and long - lasting post - processing analyses [ 2326 ]  . 
patients with a decisive diagnosis of either a benign or malignant thyroid lesion obtained by fnab , core - needle biopsy , surgical specimen histopathology or very low suspicion us finding of spongiform or cystic aspect were included . 
compared the diagnostic performance of an us image - based automatic thyroid recognition system built upon a multiple - scale convolution neural network model with that of an experienced 1 3 124 la radiologia medica ( 2019 ) 124 : 118125 radiologist using the current guidelines for thyroid nodule management [ 28 ]  . 
similarly , the cad system had comparable sensitivity but lower specificity than the experienced radiologist [ 28 ]  . the difference between our results and those obtained by the aforementioned studies may be due to several reasons , firstly including the use of different reference standards . 
indeed , according to the current international guidelines , fnab should guide thyroid nodule management along with clinical data and us evaluation , and it is regarded as the best triage test for preoperative assessment of thyroid nodules [ 10 ]  . 
further , the aforementioned studies hypothesized that the cad system may help to reduce unnecessary invasive procedures requested by inexperienced users although a radiologist performed post - processing in both cases [ 27 , 28 ]  . 
this was mainly related to the selection of the segmented nodule among a series of candidates provided by the cad system or , alternatively , to the need of manually modifying the contours of the lesion . 
indeed , we required that the segmented part and the thyroid nodule matched completely in order to increase the specificity of the cad systethis differs from the study by choi etal . 
where nodule segmentation was regarded as successful if , although not perfect , the segmented part was still representative of the nodule and the maximum contour mismatch did not exceed 30% [ 27 ]  . 
further , we did not assess thyroid nodules with spongiform or cystic appearance as our study population included patients referred for fnab and such us patterns require invasive procedures only if relevant in size or for therapeutic purposes [ 10 ]  . in conclusion , at present , the cad system is less sensitive than an experienced radiologist and showed slight - to - substantial agreement with the radiologist for the characterization of thyroid nodules . 
the classic presentation is abdominal pain , diarrhoea and / or rectal bleeding , but it is not specific and highly variable and so the diagnosis usually depends on clinical suspicion and is supported by serologic and colonoscopic findings . 
while plain radiography and ultrasound can orient the diagnosis , ct allows to define the morphofunctional alterations discriminating the non - occlusive forms from the occlusive forms and in most cases to estimate the timing of ischaemic damage . 
purpose of the review is to define the role of ct in the early identification of pathological findings and in the definition of evolution of colonic ischaemic lesions , in order to plan the correct therapeutic approach , suggesting the decision of medical or surgical treatment . keywords ischaemic colitis bowel infarction abdominal pain introduction ischaemic colitis ( ic ) is the most common manifestation of vascular disorder in the gastrointestinal tract . the pathogenesis of ic depends on two different forms of vascular colonic insult : non - occlusive injury and occlusive injury [ 1 ]  . the classic presentation of ic is abdominal pain , diarrhoea and / or rectal bleeding , but it is not specific and highly variable , so the diagnosis usually depends on clinical suspicion and has to be supported by serologic , radiological and colonoscopic findings . 
the anatomic damage results in ischaemic necrosis of variable severity : the ischaemic injury may involve only the colonic mucosal and submucosal layer or result in transmural ischaemic injury [ 2 , 3 ]  . 
even if colonoscopy within 48h represents the gold standard for final * graziella di grezia graziella.digrezia@gmail.com 1 radiology department , criscuoli hospital , santangelodeilombardi , avellino , italy 2 radiology department , university ofcampania luigi vanvitelli , naples , italy 3 radiology department , casa di cura villa dei fiori , acerra , napoli , italy diagnosis and different imaging methods have been used to diagnose ic , american college of gastroenterology clinical guideline considers computed tomography ( ct ) the main technique for the non - invasive diagnosis of ic . purpose of this report is to define the role of ct in the early identification of pathological findings and in the definition of ischaemic lesions evolution in order to plan the correct therapeutic approach , suggesting the decision of medical or surgical treatment [ 4 ]  . materials andmethods we retrospectively analyzed 95 cases of surgically confirmed ischaemic colitis in patients that underwent ct scan in radiology departments of our hospitals in the period between 1 january 2007 and 31 december 2016 . 
in case of occlusive cause , arterial from venous thrombosis has been diagnosed . the following findings were assessed : the location and length of the colonic segment involved ; the appearance and degree of wall thickening ; the presence of a double - halo or target configuration ( two or three concentric rings ) ; pericolic vol . : ( 0123456789 ) 1 3 104 la radiologia medica ( 2019 ) 124 : 103108 streakiness , peritoneal fluid or blood , the presence of intramural , mesenteric or portal venous gas , and free intraperitoneal air or other relevant abdominal findings were also recorded . 
the bowel wall was considered thickened if it measured more than 3mm in diameter . results all images have been analyzed and post - processed with multiplanar and three - dimensional reconstructions . of the 95 cases of ischaemic colitis , 72 were occlusive infarction , ( 35 cases of arterial thrombosis and 37 of venous thrombosis ) and 23 were non - occlusive cases . of the 58 cases of arterial thrombosis and non - occlusive ischaemia , reperfusion damage occurred in 13 . discussion the incidence of ic reported in the literature is of 6.144 cases / 100 , 000 person years with confirmatory histopathology , but it is a underestimated value because it could be a misdiagnosed cause of acute abdomen for its wide spectrum of clinical symptoms , the non - specificity of clinical and laboratory tests and the transient course of the disease . ic is caused by an acute interruption or chronic decrease in the colonic blood supply as in general hypotension , arterial stenosis and occlusion , bowel obstruction and venous outflow obstruction . 
although ic is usually observed in the elderly , the disease is now being increasingly diagnosed in persons younger than 50years of age , with definite predisposing causes , including vasculitis , medications , cocaine use , haemodialysis and long aeroplane flight [ 57 ]  . the superior mesenteric artery ( sma ) , which perfuses the ascending and transverse colon , and the inferior mesenteric artery ( ima ) , which distributes to the descending and sigmoid colon , are the two major arteries supplying most of the colon . 
the internal iliac arteries supply the rectu the splenic flexure ( griffiths point ) and sigmoid colon ( sudecks point ) are so - called watershed areas because these regions are located between two different vascular systems and so are particularly vulnerable to ischaemic insults . 
a third potential watershed area is the right colon , where the marginal vessel is poorly developed in up to 50% of people . the pathogenesis of acute ic depends on two different forms of vascular colonic insult : occlusive injury ( fig.1 ) and non - occlusive injury ( fig.2 ) [ 8 ]  . 
these remarks are useful in considering differential diagnosis with pathology having similar imaging features , but different distribution , as crohn . the classic presentation of acute ic is abdominal pain , diarrhoea and / or rectal bleeding , but it is not specific and highly variable and so the diagnosis usually depends on clinical suspicion and is supported with serologic and colonoscopic findings [ 9 , 10 ]  . the second one consists of non - occlusive ischaemia of the bowel caused by a sudden drop in blood flow in the small vessels of the colon , generally secondary to a lowflow state . 
 thinning of the bowel wall or paper - thin wall is caused by volume loss of tissue and vessels in the bowel wall and by loss of intestinal muscular tone due to the lack of blood flow . 
the latter could be misdiagnosed because of the similarity to the physiological colonic meteorism . the unthickened or paper - thin colonic wall is found in all conditions where ischaemia is not followed by effective reperfusion ; in acute phase , this condition could be observed in all cases of nomi until reperfusion occurs and in vasculopathic patients with ima occlusion in which the riolano arcade is unable to provide for an adequate blood supply . 
 a low - flow state causes a broad reduction in blood flow , simulating a simultaneous obstruction of sma and ima ; this explains the finding of wall thickness from normal to paper - thin wall in the acute phase of non - occlusive colitis with gaping lumen . the administration of iv contrast medium allows to evaluate the bowel wall enhancement as bowel wall hypodensity on enhanced ct in case of the absence of effective reperfusion due to the bowel wall ischaemic injury . 1 3 la radiologia medica ( 2019 ) 124 : 103108 fig . 
three kinds of infarction : abdominal ct showing an arterial occlusion ( a ) , a venous thrombosis ( b ) and narrowing of the sma with poor demonstration of the branches of the arteries in a non - occlusive mesenteric ischaemia ( c , d ) in case of nomi , beyond intestinal damage , there was also a parenchymal involvement ( liver , kidney , spleen ) in ischaemic injury . 
when reperfusion occurs , findings are not discernable from the first ones . clinically , ic may be classified as two major forms : non - gangrenous ( mild ) and gangrenous ( acute fulminant )  . 
the gangrenous form is associated with transmural necrosis and requires urgent surgery . colonoscopy within 48h represents until today the gold standard for final diagnosis , but it can be non - diagnostic in emergency setting without previous preparation and it should not underestimate the risk of perforation . imaging methods too have been used to diagnose ic . 
 plain radiography may show suggestive signs of chronic disease such as thumbprinting , which appears as rounded densities along the sides of a gas - filled distended colon , dilation of the colonic loops and loss of haustration . ultrasound is able to detect abnormalities in the bowel wall and identify segmental involvement of the colon . 
it allows to evaluate bowel wall thickening , colon wall stratification , pericolic fat , free fluid and pneumatosis . today , ct scan with iv contrast media allows defining the injured colonic segment and detecting the presence of complications.ct allows determining the morphofunctional alterations discriminating the non - occlusive forms from the occlusive forms , and it allows estimating the timing of ischaemic damage . the anatomic damage results in ischaemic necrosis of variable severity ; the ischaemic injury may involve only the colonic mucosal and submucosal layer or result in transmural ischaemic injury . 
also , imaging features can vary depending on the time course and aetiology , and the radiological findings can be summarized in four main categories ( table1 )  . the pericolic fluid is a crucial finding for ic diagnosis since its evidence suggests the presence of ongoing damage , thus focusing the attention on other pathological aspects which could be misdiagnosed : in fact , in case of acute ischaemic non - occlusive ic , the dilated thinned colonic wall could be misinterpreted as a physiological colonic distension caused by the presence of intestinal gas . 
in case of reperfusion , the amount of pericolic fluid may increase or decrease , depending on the evolution of 1 3 la radiologia medica ( 2019 ) 124 : 103108 106 fig . 
ct in axial plane ( a ) and coronal reconstruction ( b ) show a non - occlusive mesenteric ischaemia with mesenteric imbibition , wall , mesenteric vessels pneumatosis and portal pneumatosis . 
ct in axial plane ( c ) and coronal reconstruction ( d ) show a non - occlusive mesenteric ischaemia with wall thickening and stratification due to reperfusion damage fig . 
in addition , perivisceral imbibition has been reported , especially in subacute phase ( a , b ) , ( f , g ) left colonic features at doublecontrast barium x - ray 1 3 la radiologia medica ( 2019 ) 124 : 103108 fig . 
 b free fluid in peritoneal cavity ( white arrows ) table 1 radiological findings of ischaemic colitis vessel findings retroperitoneal cavity findings bowel wall findings parenchymal findings vascular occlusion or stenosisfindings of defects or occlusion of the sma e ima vascular pneumatosisthe presence of gas within the mesenteric venules and portal venous system free fluid pneumoretroperitoneum wall thickness ( normal thickness < 3 mm ) wall enhancement is considered pathological in the presence of a double - halo or target configuration ( two or three concentric rings ) or when absent wall pneumatosis signs of ischaemic damage involving the liver , kidneys , spleen and pancreas the ischaemic damage and thickened walls suggesting the decision of medical or surgical treatment ( table2 ) [ 11 ]  . pathological findings and in the definition of evolution of ischaemic lesions and [ 12 ]  . however , it requires the use of ionizing radiation and an iodinate contrast agent , limiting the possibility to use this technique in a short - term follow - up . acg clinical guideline ct considers ct the main technique for the non - invasive diagnosis of acute ic and recognizes the role in follow - up of mri in the identification of the treatment depends on the severity and cause of the ischaemia , and the detection of ic signs is crucial to plan the correct therapeutic approach and reduce the reported mortality rate ( 412% )  . 
multiple embolizing agents have been described , and among them , onyx seems to be promising ; this is a liquid embolic agent , originally applied in neurointerventional radiology and recently adopted also in peripheral embolizations . 
the aim of this study is to report on a 10 - year experience of transarterial embolization of peripheral high - flow avm with onyx in terms of technical and clinical outcomes . materials and methods retrospective analysis was conducted on patients affected by high - flow avm and treated electively by transarterial embolization with onyx . 
technical and clinical success was evaluated ; follow - up was conducted 30days after the last treatment session and yearly in case of success . results sixteen patients have been included , totally 38 embolizing procedures . 
technical success was obtained in 11 patients ; at 30 - day follow - up , 15 patients showed improvements in symptoms , even those with incomplete embolization ; however , after almost 1year from treatment accomplishment , 7 patients showed relapse of symptoms and presented radiological signs of avm recurrence . 
no clinically relevant complications occurred . conclusions in this series , in accordance with previous but limited published data , onyx appeared safe and technically effective to embolize high - flow peripheral avm with transarterial approach . 
cardarelli 9 , 80131naples , italy vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 154162 introduction vascular malformations presenting with an arterial component are defined as high - flow arteriovenous malformations ( avms ) ; these vascular lesions are constituted by arteriovenous microfistulae through a vascular nidus [ 1 ]  . 
 according to the localization into or outside the central nervous system , they are divided into central and peripheral , respectively [ 2 ]  . different classification systems [ 35 ] have been proposed in the literature focusing on morphology [ 3 , 6 ] and clinical impact [ 4 ] ; however , confidence of operators in each unique angiographic avm architecture is essential in planning the proper endovascular strategy , considering also that the angioarchitecture can evolve while staging sessions of embolization . clinical presentation of high - flow avms ranges from asymptomatic to intermittent pain , tissue swelling , bleeding and even right heart failure [ 7 , 8 ]  . the surgical resection is avoided because historically it carried high rate of recurrence due to incomplete vascular eradication , so the first - choice treatment is the catheterdirected embolization [ 1 , 9 ] ; these are technically challenging procedures because of the risk of nontarget embolization and / or incomplete treatment . multiple embolizing agents can be adopted ; thanks to the technological developments promoted by industries , today interventional radiologists handle several agents , allowing a customized embolization procedure based on the vascular pattern they are facing with . 
onyx ( ev3 , irvine , california , usa ) is a nonadhesive liquid embolizing agent ; originally adopted in interventional neuroradiology , its use has been reported also in peripheral embolization for the treatment of avm , type ii aortic endoleak , portal vein embolization , aneurysms and bleedings [ 1013 ] ; it consists of a plastic polymer ( ethylene vinyl alcohol , evoh ) dissolved in a organic solvent agent ( dimethyl sulfoxide , dmso ) ; fluoroscopic visibility is obtained thanks to the tantalum powder component suspension after shaking of the evoh vial for almost 20min before usage . the aim of this study is to report on a single - center 10 - year experience transarterial high - flow avm embolization with onyx in terms of technical and clinical outcomes . materials andmethods sample this is a tertiary care - level single - center , retrospective analysis of the last 10years , from january 2007 to april 2018 , focusing on patients affected by high - flow avm and managed by transarterial embolization . 
 written informed consent was obtained the day before the procedure . based on the local picture archive and communication system ( pacs ) and electronic records , retrospective data collection included : preinterventional clinical radiological evaluations , procedural data and post - procedural clinical radiological assessment . 
in case of unavailable data on the electronic records , patients were interviewed by phone calls . preprocedural radiological evaluation included always first - level examinations ( echo color doppler ultrasound , ecd - us ) and a consequent second - level imaging ( contrastenhanced computed tomography , ct and / or magnetic resonance , mr )  . 
in all cases , diagnosis of avm was acquired before angiography . embolization technique for a comfortable intervention , procedures were conducted under local anesthesia in correspondence with the vascular access and sedatives for pain reduction ; no general anesthesia was given . standard vascular access consisted of us - guided or fluoroscopy - guided retrograde femoral puncture with 5 french sheath ( radifocus introducer ii , terumo , japan ) positioning . with a diagnostic 5 french catheter , different tip curves related to the lesion anatomy , preprocedural angiography was performed with different obliquities projections . 
 according to the best projection to visualize the avm feeder ( s ) and nidus , the 5 french catheter was then positioned as distal as possible , and then , a microcatheter was navigated into each feeding artery as close as possible to the nidus . 
to completely suck the liquids , 14 - gauge and 16 - gauge cannulas were , respectively , inserted into evoh and dmso vials ; then , the syringes were filled with air and injected into the vials to increase the pressure on the liquid content ; finally , 1 3 156 la radiologia medica ( 2019 ) 124 : 154162 fig . 
1 patient 4 , procedural angiography of a 24 - year - old woman affected by a high - flow avm of the right leg presenting with swelling and padiagnostic angiography demonstrated two arterial feeders originating from the posterior tibial artery . 
f angiographic control after onyx 34 injection showing onyx cast in the posterior portion of the avm ( white arrow , same in figure g ) and a small amount of accidental onyx reflux into the posterior tibial artery ( black arrow , same in figure g )  . 
h final angiographic control after embolization completion with onyx 34 demonstrating complete technical success with a small amount of onyx occluding the posterior tibial artery in correspondence with the middle tract ( black arrow )  . 
when evoh starts outflowing from the microcatheter , the first injection has to voluntary reflux around the microcatheter tip and should be waited for 23min ; this is intended to create a cast that avoids further evoh reflux and possible nontarget embolization . 
then , evoh injection is continued until the desired embolization is obtained ; at the end of the procedure , the dead space of 1 3 la radiologia medica ( 2019 ) 124 : 154162 fig . 
2 patient 13 , procedural angiography of a 29 - year - old woman affected by a high - flow avm of the uterus presenting with pain , bleeding and dysmenorrhea . 
ab contrast - enhanced ct ( a ) and mr ( b ) in arterial phase in axial planes showing highflow avm of the uterus fed by both uterine arteries appearing ectasic and tortuosous ( white arrows )  . 
c angiography acquired after contrast injection from pigtail catheter positioned in aorta ; uterine avm was confirmed with dominant venous drainage in the left common iliac vein ( white asterisk )  . 
at 30 - day clinical follow - up , patient showed partial improvement in symptoms ; therefore , another treatment session was planned with additional embolizing agents from both arterial ( coils and particles ) and venous districts ( sodium tetradecyl sulfate )  . 
at 1 - year clinical follow - up , the patient was clinically asymptomatic with regular period the microcatheter is cleaned with dmso , and finally , the microcatheter is retrieved in aspiration . final angiograms are acquired from the 5 french catheter . eventually , additional embolizing agents ( coils , particles , glue , sodium tetradecyl sulfate ) were needed to seal the vessels lumen or to occlude small collaterals unsuitable for onyx embolization . the arterial puncture site was closed by manual compression or mechanical hemostasis ( femoseal , terumo , japan )  . no anticoagulants were used during any of the procedures . antiinflammatories and antibiotics were prescribed for 5days after the intervention to prevent / reduce the occurence of the post - embolization syndrome . procedural outcome evaluation technical success was defined as disappearance of nidus and drainage vein ( s ) opacification at final angiograms ; in some cases , multiple treatment sessions would be performed to obtain the devascularization . clinical success was intended as improvement in symptoms without the need for additional therapies . intraand post - procedural complications were registered and evaluated according to the cirse classification system for complication [ 14 ]  . 1 3 158 la radiologia medica ( 2019 ) 124 : 154162 fig . 
3 patient 14 , procedural angiography of a 39 - year - old man affected by a wide high - flow avm of the right emi - thorax presenting with hemoptysis . 
ad diagnostic angiography showing avm refurnishment from intercostal ( a ) , bronchial ( bc ) and mammary ( d ) arteries , with multiple anastomosis ; other right thoracic intercostal arteries were involved ( not shown )  . 
 at 30 - day clinical follow - up , patient showed improvement in symptoms ; however , after two months he suffered from new hemoptysis episodes ; therefore , another two treatment sessions were performed . 
in two patients ( patients 10 and 13 ) , an additional transvenous approach was required for retrograde embolization session ; both were complex pelvic avms . microcatheters used were dmso compatible ( rebar 18 and rebar 27 , medtronic , usa ) ; however , in accordance with empirical experience as well as previous published papers [ 1517 ] , also not technically dmso - compatible microcatheters ( progreat 2.7 , terumo , japan ) were employed without problems . additional embolizing agents were required in five patients ( patients 10 , 11 , 13 , 14 and 16 )  . technical success was obtained in 68.7% ( 11 out of 16 patients ) ; in five patients ( patients 10 , 11 , 12 , 14 and 16 ) , cirse classification system for complications [ 14 ] was adopted : one episode of nontarget embolization due to evoh reflux occurred ( patient 4 ) , with consequent peroneal artery definitive embolization ; however , no long - term ischemic clinical sequelae were seen ( grade 3 )  . 
in three patients , groin swelling and hematoma , self - limiting without additional therapies , were registered ( grade 1 ) ; 4 patients who showed post - embolization syndrome up to 5days after the procedure were managed with antiinflammatory and antibiotics ( grade 1 )  . after all the procedures , a normal finding was garlic smelling present for 4872h because of dmso elimination via respiration and sweat . all patients were symptomatic before treatment ; at the follow - up visit 30days after the last embolization session , all but one patient ( patient 16 ) showed improvements in symptoms , even those with incomplete embolization ( 30day clinical success 93.7% ) ; however , after almost 1year from treatment accomplishment , 43.7% of the patients 1 3 table 1 patient data in terms of sex , age , body district involved and clinical findings patient sex age district clinics 1st and 2nd hand fingers swelling 160 m 23 tongue m 73 thigh m 68 bronchus 46 humerus m 23 humerus m 28 buttock 38 uterus pelvis eyelid pelvis 29 uterus m 39 bronchus / ribs pelvis m 29 functional impairment swelling pain bleeding functional impairment swelling swelling pain hemoptysis swelling pain swelling pain swelling pain pain bleeding functional impairment pain bleeding pain bleeding functional impairment pain bleeding functional impairment pain bleeding functional impairment hemoptysis pain bleeding functional impairment swelling pain bleeding showed relapse of symptoms and presented signs of avm recurrence at contrast - enhanced ct and / or mr . discussion onyx is a liquid embolizing agent introduced in the routine clinical practice at the beginning of 2000s by neurointerventionalists for the treatment of central avm . 
its successful usage for the treatment of peripheral avms has also been reported , by both transvascular and direct puncture techniques , in adults and children [ 1 , 5 , 9 , 10 , 1620 ] ; however , literature data about peripheral applications are still scarce and studies have been conducted in small samples . la radiologia medica ( 2019 ) 124 : 154162 it is considered mainly as an alternative to cyanoacrylate . 
 compared to glue , it presents the advantage of nonadhesive property ; its injection is therefore voluntary slow , permitting to visualize the fulfilling of the lesion without the risk of microcatheter glueing . 
recently , industry competitors have developed other new liquid nonadhesive embolizing agents in ready - to - use format and those agents have already been adopted in neurointervention [ 21 ] ; however , because of the very last development , clinical efficacy has to be still evaluated in the literature in peripheral avm treatment . specifical evoh - related complications are rare : microcatheter entrapments have been reported [ 2224 ] and these are consequences of ab estrinseco compression of onyx cast on the microcatheter in narrow and tortuous vessels after long - lasting procedures with extended segments entrapped . 
another risk to consider is skin darkening in case of subcutaneous lesions , due to tantalum deposition ; this is mainly related to direct injection technique [ 1 , 25 ]  . 
on the other hand , dmso should be handled with special care because compared to evoh it induces pain and vasospasms if injected fast , reducing evoh distalization ; patients should always be informed of the postprocedural 4872 - h garlic smelling occurring because of dmso elimination . overall , in this sample complications were scarce and of low grade according to cirse classification system , all without clinical sequelae . dmso - compatible microcatheters should be preferably adopted ; however , off - label usage of not technically compatible microcatheters has been reported without problems [ 1517 ] , as in this series . 
in some particularly challenging cases , tip - detachable microcatheters have been successfully employed , also from a transvenous approach [ 19 , 26 , 27 ]  . high - flow avm is extremely complex vascular lesions which should be treated with a customized approach based on the diagnostic angiographic findings . 
multiple embolizing agents have been adopted , and onyx is one of the most successful in the literature [ 1 , 9 , 18 , 19 ] because of its ability to form a cast penetrating the nidus , even if this is not directly reachable with microcatheter . in the peripheral body , the most frequently reported use of onyx is represented by the treatment of the type ii endoleaks of abdominal aortic aneurysms [ 11 , 12 ] ; literature data concerning peripheral avm embolization with onyx are still scarce . 1 3 la radiologia medica ( 2019 ) 124 : 154162 table 2 embolization data , technical and clinical success , follow - up patient treatment sessions additional transvenous access procedural technical success 30 - day clinical success 1 - year follow - up additional embolizing agents sts sodium tetradecyl sulfate glue coils glue coils coils particles coils particles glue coils in an initial case series published in 2004 , numan etal . 
the reason of this low success rate may be explained by the uncomplete knowledge of onyx properties at that time and since handling of this material has improved thanks to the experience acquired in neurovascular interventions . more recently in 2016 , de beule etal . 
 [ 1 ] reported their experience on peripheral avm embolizations with onyx on 28 sessions of 19 patients : compared to the data reported in this sample , they described similar post - embolization avm total occlusion rate ( 63% )  . additional agents should be considered to occlude vessels not suitable for onyx , as occurred in 31.2% of cases in this series ; comparable additional agents usage rates were reported by both de beule etal . 
 [ 19 ] reported a novel technique of retrograde transvenous onyx embolization of peripheral avm , after preliminary transarterial and transvenous flow reduction , with encouraging results . even if complete technical success is obtained , at follow - up recurrences have to be expected because of frequent recruitment of new arterial feeders . 
on the other hand , it should be underlined that even if technical success is not completely obtained at first attempt , temporary symptom relief may occur because of reduced blood flow to the nidus , as reported also by numan etal . 
 [ 18 ] ; therefore , multiple treatment sessions are usually required , and in this sample , only eight of sixteen patients have been treated once . main limitation of this study is the small number of patients although this is the reported sample with the highest number of procedures concerning peripheral avm onyx embolization ; however , it should be considered that the global incidence of the pathology is low . 
another issue is that only trained operators performed the embolizations ; indeed , considering the specific injection technique and the material cost , onyx should not be handled by short - experienced interventionalists . 1 3 162 la radiologia medica ( 2019 ) 124 : 154162 in conclusion , in this series , in accordance with previous but limited published data , onyx appeared as a safe and technically effective agent to embolize high - flow avm with transarterial approach . 
however , clinical imaging follow - up is mandatory in these patients because new feeder recruitment has to be expected ; patients should be informed of the concrete possibility of multiple treatment sessions . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . research involving human participants all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . informed consent informed consent was obtained from all individual patients included in the study . la radiologia medica ( 2019 ) 124 : 136144 radiotherapy hypofractionated radiation therapy inthemanagement oflocally advanced nsclc : anarrative review oftheliterature onbehalf oftheitalian association ofradiation oncology ( airo ) lung working group giuseppeparisi1 rosariomazzola2 patriziaciammella3 giorgiatimon3 alessandrafozza4 davidefranceschini5 federiconavarria6 alessiobruni7 marcoperna8 niccolgiajlevra2 filippoalongi2 vieriscotti8 marcotrovo1 received : 28 may 2018 / accepted : 15 october 2018 / published online : 27 october 2018 italian society of medical radiology 2018 abstract a systematic literature was performed to assess the benefit in terms of effectiveness and feasibility of hypofractionated radiotherapy ( hyport ) , with or without chemotherapy ( ct ) , in the treatment of locally advanced non - small cell lung cancer ( nsclc )  . 
we have identified all studies , published from 2007 onwards , on patients with locally advanced nsclc treated with hyport with radical intent , with a minimal dose per fraction of 2.4gy , with or without concurrent chemotherapy . 
actuarial 2 - year pfs ranged from 13 to 57.8% , and 1 , 2and 3 - year overall survival ( os ) ranged from 51.3 to 95% , from 22 to 68.7% , and from 7 to 32% , respectively . 
non - small cell lung cancer ( nsclc ) represents the majority of lung cancer diagnosis , and most of these are in locally advanced stage , half of which unresectable [ 2 , 3 ]  . 
the standard radiation scheme is 6066gy delivered in 3033 fractions [ 5 ]  . nevertheless , after standard radiation doses of crt , the risk of local recurrence remains high and 5 - year survival is poor . 
radiation therapy oncology group ( rtog ) 0617 randomized phase 3 study failed to show a beneficial effect in survival when delivering higher dose of rt at 2gy per fraction [ 8 ]  . 
a strategy to increase the biological effective dose ( bed ) of rt could be obtained using hypofractionated regimens characterized by a reduction in the overall treatment time with dose per fraction higher than 2gy [ 1113 ]  . considering the technological worldwide implementation in rt facilities and the issue of maintaining limited waiting list for the patients , hypofractionated was adopted in many centers and in several clinical settings , including lung cancer [ 14 ]  . aim of the current narrative review is to assess the benefit of hypofractionated rt , with or without concurrent chemotherapy ( ct ) , in terms of effectiveness and feasibility in the treatment of locally advanced nsclc . materials andmethods all studies included in the present review satisfied the following criteria : ( 1 ) patients with locally advanced nsclc , ( 2 ) patients treated with hypofractionated rt with radical intent , ( 3 ) with a minimal dose per fraction of 2.4gy , ( 4 ) patients treated with hypofractionated rt with or without concomitant ct , ( 5 ) studies published from 2007 onwards , ( 6 ) english manuscripts . 
disagreement was resolved by consensus ; if consensus could not be achieved , the study coordinator provided an assessment of eligibility . for data extraction , all the papers were scrutinized for the following information : study design ( retrospective , prospective ) ; number of patients ; number of patients comprise in study with concomitant ct ; oncological treatment strategy ( rt alone and / or sequential crt , versus concurrent crt ) ; total dose ; dose per fraction ; definition of acute and late toxicity profile clinical outcomes . results twenty - nine studies [ 1543 ] of hypofractionated rt in locally advanced nsclc with or without concurrent ct that met the inclusion criteria were identified , for a total of 2614 patients . 
of these , eight were phase i trials , and ten were phase ii trials . clinical outcomes innonconcurrent chemotherapy group seventeen studies [ 1531 ] , for a total of 1730 patients , without concurrent ct that met the inclusion criteria were identified . 
the most common ct agents employed were cisplatin ( 70.5% ) , predominantly in doublet with , gemcitabine or vinorelbine . 1 3 138 la radiologia medica ( 2019 ) 124 : 136144 1 3 la radiologia medica ( 2019 ) 124 : 136144 the most common rt technique was 3d - conformal radiation therapy ( 3d - crt ) ( 64.7% ) , whereas intensity - modulated rt ( imrt ) was employed in only three studies ( 23.5% ) ; in one study patients were treated with protons . 
actuarial 2 - year progression free survival ( pfs ) , which was reported in 12 studies , ranged from 13 to 57.8% , and 1 - , 2and 3 - year overall survival ( os ) ranged from 51.3 to 95% , from 22 to 68.7% , and from 7 to 32% , respectively . in only three studies ( 17.6% ) , elective lymph nodes were irradiated . 
regarding pulmonary toxicity , acute pneumonitis occured in 044% , whereas late pneumonitis occured in 047% , most commonly grade 3 . clinical outcomes inconcurrent chemotherapy group sixteen studies of hypofractionated rt [ 15 , 16 , 26 , 30 , 31 ] delivered concomitantly with ct were identified , for a total of 884 treated patients ( table2 )  . 
the most common ct agents employed were cisplatin 50% , carboplatin 25% and vinorelbine 37.5% ; other agents employed less frequently were , liposomal doxorubicin , docetaxel , gemcitabine , cetuximab and alk - inhibitors . 
acute esophagitis occurred in 041.7% , while late esophageal toxicity occurred in 08.3%. the overall incidence of acute pneumonitis ranged from 0 to 23% , whereas late pneumonitis occured in 047% , similarly to the non - concomitant ct group . discussion most of nsclc patients with locally advanced disease have poor prognosis [ 45 ]  . 
in fact , the treatment 1 3 140 la radiologia medica ( 2019 ) 124 : 136144 failure of the primary nsclc has a detrimental impact in terms of pfs , metastasis - free survival and os [ 46 ]  . to date , the standard of care is represented by rt using conventional fractionation , with concurrent or sequential platinum - based ct [ 47 ]  . 
at a median follow - up of 22.9months , 74gy given in 2gy per fractions with concurrent ct showed to be not better than 60gy for patients affected by stage iii nsclc . 
the authors concluded that high - dose conformal rt might be potentially harmful [ 8 ]  . on the other hand , it has been demonstrated that a long duration of rt in nsclc seems to be detrimental in terms of tumor control and survival , due to accelerated repopulation of tumor cells , with a loss of local control of 1.66% per day of lengthening over 6weeks [ 9 , 10 ]  . 
in the present review , we analyzed the available literature data concerning the role of hypofractionated rt in the management of locally advanced nsclc distinguishing two main groups : ( 1 ) non - concurrent ct patients and ( 2 ) concomitant ct patients . looking at the first patients category , 1 - , 2and 3 - year os ranged between 51 and 95% , 22 and 68% , and 7 and 32% , respectively ; 2 - year pfs varied from 13 to 58% . 
there was no significant difference regarding pulmonary toxicity . analyzing the clinical outcomes of hypofractionated rt administered concomitantly with ct , 3 - year os ranged from 31 to 44% , whereas 2 - year pfs varied between 19 and 58% . 
 speculatively , potential higher rates of oncological outcomes compared to conventional crt could be expected using a hypofractionation regimen in non - concurrent than in concomitant hypofractionated rt for nsclc . 
 obviously , these last assumptions need to be confirmed and a direct comparison between different fractionation schedules requires well - designed randomized studies before to draw any kind of definitive conclusion . radiobiological modelings suggest that shortened treatment schedules might increase the risk of late toxicity . 
in the available data here reported , acute esophagitis occurred in 015% in the nonconcurrent group comparing to 041.7% in the concomitant arregarding pulmonary toxicity , acute pneumonitis occurred in 044% , whereas late pneumonitis was recorded until to 47% of cases , most commonly grade 3 . 
a similar pulmonary toxicity profile was noted in the non - concomitant hypofractionated group . conclusions in summary , in the current review of the literature an extreme heterogeneity was noted in terms of the rt - treatment schedules , in terms of the adopted techniques as well as the administered drugs in combination with irradiation . 
these conditions associated with the rapid evolution of technologies and the hypothesis of association with new target agents and immunotherapy could constitute , in the future , a potential new frontier in the treatment of locally advanced nsclc . author contribution statements in our review are listed 13 authors . 
all authors approved the final manuscript . compliance with ethical standards conflict of interest all authors declare no conflict of interest . research involving human participants and / or animals this article does not contain any studies with human participants or animals performed by any of the authors . 1 3 la radiologia medica ( 2019 ) 124 : 136144 1 3 142 la radiologia medica ( 2019 ) 124 : 136144 la radiologia medica ( 2019 ) 124 : 145153 radiotherapy delta radiomics forrectal cancer response prediction withhybrid 0.35t magnetic resonanceguided radiotherapy ( mrgrt ) : ahypothesisgenerating study foraninnovative personalized medicine approach lucaboldrini1 davidecusumano1 giudittachiloiro1 calogerocas1 carlottamasciocchi1 jacopolenkowicz1 francescocellini2 nicoladinapoli2 luigiazario3 stefaniateodoli2 mariaantoniettagambacorta1 marcodespirito3 vincenzovalentini1 received : 7 june 2018 / accepted : 15 october 2018 / published online : 29 october 2018 the author ( s ) 2018 abstract the aim of this study was to evaluate the variation of radiomics features , defined as delta radiomics , in patients undergoing neoadjuvant radiochemotherapy ( rct ) for rectal cancer treated with hybrid magnetic resonance ( mr ) - guided radiotherapy ( mrgrt )  . 
an imaging acquisition protocol of 6 mr scans per patient was performed : the first mr was acquired at first simulation ( t0 ) and the remaining ones at fractions 5 , 10 , 15 , 20 and 25 . 
the variations of each feature during treatment were quantified , and the ratio between the values calculated at different dose levels and the one extracted at t0 was calculated too . 
the most predictive feature ratios in ccr prediction were the l_least and glnu ones , calculated at the second week of treatment ( 22gy ) with a p value = 0.001. 
delta radiomics approach showed promising results and the quantitative analysis of images throughout mrgrt treatment can successfully predict ccr offering an innovative personalized medicine approach to rectal cancer treatment . keywords rectal cancer radiomics delta radiomics personalized medicine innovative biotechnology mridian viewray * davide cusumano davide.cusumano@policlinicogemelli.it 1 dipartimento di diagnostica perimmagini , radioterapia oncologica ed ematologia , istituto di radiologia , fondazione policlinico a . 
gemelli irccs universit cattolica sacro cuore , rome , italy introduction significant improvements in locally advanced rectal cancer ( larc ) treatment have been met in the past two decades , and to date the typical therapeutic workflow is represented by neoadjuvant long - course radiochemotherapy ( rct ) , followed by total mesorectal excision ( tme ) [ 14 ]  . regardless of the initial disease stage , approximately 1142% of these patients achieve a pathological complete response ( pcr ) after long - course rct . 
in a very recent systematic review and pooled analysis , however , patients undergoing w&w approach showed a 3 - year overall survival of 93.5% ( against a 90.1% rate for patients with pcr ) and a non - regrowth free survival rate was of 89.2% at 3years , supporting the favourable prognostic value of ccr already supposed by the first conservative experiences of habrgama etal . 
no detection of residual lesions or the presence of a flat scar at endoscopic examination . image analysis an imaging protocol consisting in 6 mr acquisitions was applied to all patients . the first mr scan was acquired during the treatment simulation procedures ( t0gy ) , and the others were performed one every five fractions ( t11gy , t22gy , t33gy , t44gy and t55gy )  . figure1 shows the imaging protocol applied in a case where a clinical complete response was achieved . 
the red contour indicates the gtv delineation . all images were acquired on mridian using a true fast imaging ( trufi ) with steady - state precession sequence , sixteen consecutive patients ( 13 males and 3 females ) affected by larc ( stages iiaiiic ) , with a median age of 64years ( range 4986 ) were retrospectively enrolled for this study . five patients ( 31% ) showed ccr at restaging examinations ( dre and mri )  . patients characteristics are described in table1 . median time interval between end of neoadjuvant rct and restaging examinations was 62days . a total of 53 radiomics features belonging to four families ( morphological , statistical , fractal and textural based on runlength matrix ) were extracted from each raw image , without applying any image filter . a total of 318 radiomics features were therefore obtained ( 53 features extracted from the simulation image and 265 calculated as delta features )  . of these , a total of 6 simulation features and 57 delta radiomics features showed a p value < 0.05 ( wmw test ) in discriminating ccr patients from the non - responding ones . 
 table2 reports the features that are significant with the corresponding time of acquisition and p values . as reported in the tables , most of the delta radiomics features show a higher statistical significance in discriminating between ccr and not - ccr patients when compared to those extracted from the analysis of the simulation images . more specifically , three delta features ( energy , grey level non - uniformity and least axis length ) showed a statistically 1 3 148 la radiologia medica ( 2019 ) 124 : 145153 fig . 
the introduction of the described radiomics workflow can enrich the observations already available in the literature that took into account the predictive power of different tumour - related parameters during treatment : as an example , palmisano etal . 
patients undergoing ccr are indicated in light grey this significant limit has been recently overcome by the availability of mrgrt daily setup images that allow the development of imaging - based response predictors during rct treatments course with no additional burden in terms of dose to the patient , costs and procedures . indeed , the aim of this hypothesis - generating study was to explore the potentialities of the delta radiomics approach , more than to train a multiparametric prediction model , as the limited number of patients could not gather reliable conclusions . the choice of using the wilcoxonmannwhitney test to investigate the predictive value of the delta radiomics parameters was adopted considering a recent study carried out by parmar etal . , who compared 14 different feature selection methods . 
the other authors do not have conflict of interest . ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . all the definitions refer to the image biomarker standardisation initiative by zwanenburg etal . 
all patients underwent mr imaging with a 1.5 t scanner system , including diffusionweighted imaging ( dwi ) with three different b values ( b = 0 , 500 and 800s / mm2 )  . 
the entire tumor volume was manually delineated on the adc maps , using the t2 - weighted images and dwis with b = 800s / mm2 as a guide to the lesion location . 
the optimal cutoff values for adcmean and adcmedian as predictors for lymphoma were 0.83 103 mm2 / s and 0.73 103 mm2 / s , respectively . conclusions the whole - lesion adc histogram analysis of cervical lymphadenopathy may help to discriminate lymphomas from non - nasopharyngeal scc in patients with unknown clinical primary tumor . keywords diffusion - weighted imaging head and neck cancer neck nodes quantitative imaging * antonello vidiri antonello.vidiri@ifo.gov.it 1 radiology anddiagnostic imaging department , irccs regina elena national cancer institute , via elio chianesi 53 , 00144rome , italy 2 medical physics laboratory , irccs regina elena national cancer institute , via elio chianesi 53 , 00144rome , italy 3 department ofradiology , f . 
policlinico gemelli irccs , largo agostino gemelli 8 , 00168rome , italy 4 department ofotolaryngology andhead andneck surgery , irccs regina elena national cancer institute , via elio chianesi 53 , 00144rome , italy 5 department ofhematology , irccs regina elena national cancer institute , via elio chianesi 53 , 00144rome , italy 6 department ofpathology , irccs regina elena national cancer institute , via elio chianesi 53 , 00144rome , italy 7 via pieve di cadore 30 , 00135rome , italy vol . : ( 0123456789 ) 1 3 20 introduction in an era of advanced diagnostics , metastasis to cervical lymph nodes from an occult primary tumor is a rare clinical entity and ranges approximately from 1.5% to 9% of all head and neck malignancies [ 14 ]  . 
in the neck , two - thirds of cervical lymph nodes are histologically confirmed as squamous cell carcinomas ( sccs ) , followed by undifferentiated carcinoma and adenocarcinoma [ 5 ]  . 
a formal fine needle aspiration ( fnac ) is the preferred method of biopsy in a patient affected by a scc lymphadenopathy , who presents to a physician for diagnostic examination . 
in fact fnac does not allow to differentiate histological subtypes and to perform the differential diagnosis between hyperplasia and low - grade non - hodgkin lymphoma [ 7 ]  . on the basis of conventional mri alone , it is very difficult to differentiate scc from lymphoma but advances in mri acquisition techniques , as diffusion and perfusion mri , demonstrated to be useful noninvasive imaging tools for a better tumor characterization [ 8 , 9 ]  . 
several dwi studies reported that the apparent diffusion coefficient ( adc ) is helpful in the differential diagnosis of these two head and neck malignancies , because dwi typically shows different rates of microscopic water diffusion in tissues , according to different features of tumor cellularity [ 1014 ]  . 
most of these studies documented an overlap of adc values between scc and lymphoma in head and neck lesions , based on a simplistic adc analysis of mean values within manually selected regions of interest ( rois ) inside the lesion . 
however , this approach to the image analysis is not recommended by the international consensus [ 15 ] , as the roi placement is subjective , does not allow a straightforward comparison between results from different centers , besides the fact that it does not enable to address the tumor heterogeneity [ 16 , 17 ]  . the calculation of the whole - volume adc histogram , based on the distribution of the voxel - level values within the entire lesion , can improve the reproducibility of quantitative dwi , promoting a standardization of adc [ 15 , 18 ]  . 
a few studies , based on the adc histogram analysis , documented a better differentiation of head and neck lesions , as well as an improvement in grading tumors and in predicting the outcome of chemo - radiotherapy [ 8 , 1922 ]  . la radiologia medica ( 2019 ) 124 : 1926 the aim of this study was to retrospectively evaluate the potential of whole - volume adc histogram of cervical lymph node to discriminate between lymphomas and metastatic non - nasopharyngeal sccs , in patients without any evidence of a primary tumor on clinical examination . materials andmethods patient population the present retrospective study was authorized by the hospital ethics committee . 
the patients were eligible for the study if they had : ( 1 ) solitary or multiple cervical lymph node metastases , without evidence of primary tumor after initial clinical examination ; ( 2 ) a histological confirmation with fnac or biopsy ; ( 3 ) pre - treatment mri studies according to the imaging protocol described in the following section . 
 the exclusion criteria for the patients were : ( 1 ) histological confirmation of nasopharyngeal carcinoma ( npc ) or nasopharyngeal lymphoma , ( 2 ) image distortion because of strong susceptibility and / or motion artifacts on dwi , and ( 3 ) largely necrotic node . mr imaging protocol mr imaging was performed with a 1.5 t scanner system ( optima mr 450w , ge healthcare , milwaukee , wi , usa ) , using a 16 - channel receive - only rf coils : a head coil , a surface neck , and a spine coil . 
fast spin - echo t2 - weighted imaging ( fse t2wi ) on the coronal plane ( acquisition matrix 288 256 , field of view 27cm , repetition time / echo time ( tr / te ) 5901ms / 102ms , slice thickness 4mm ) was first obtained , followed by axial spin - echo t1 - weighted imaging ( se t1wi ) ( acquisition matrix 288 256 , field of view 20cm , tr / te 617ms / 8.1ms , slice thickness 3mm ) and axial fse t2wi ( acquisition matrix 288 256 , field of view 20cm , tr / te 6844ms / 105ms , slice thickness 3mm )  . 
dwis were obtained by single - shot spin - echo echo planar imaging with the following parameters : acquisition matrix , 128 128 ; field of view , 2628cm ; tr / te , 2393ms / 66.4ms ; slice thickness , 4 . 
three different b values ( b = 0 , 500 and 800s / mm2 ) were used , with all diffusionsensitizing gradients applied in three orthogonal directions to obtain trace - weighted images . 
to take the reduced signal - to - noise ratio with the largest b values into account , six signal averages were chosen for b value of 0s / mm2 , 8 for b values of 500s / mm2 and 10 for a b value of 800s / mm2 . 
the asset ( array spatial sensitivity encoding technique ) imaging option with a scan time reduction factor of 2 was used , with a resulting scan duration of 2min and 30s . 
 after gadolinium infusion ( 0.1mmol / kg ) , lava ( liver 1 3 la radiologia medica ( 2019 ) 124 : 1926 acquisition with volume acceleration ) sequences ( tr 9.8 te mfov 26 , matrix 288 288 and slice 1mm ) were performed . adc analyses the adc analyses were articulated in three main phases : 1 . 
the entire tumor volume was delineated on the adc maps , using the t2 - weighted images and dwis with b = 800s / mm2 as a guide to the lesion location . 
the adc maps and the delineated contours were loaded in the matlab workspace ( release 2013a , the math works , inc . , natick , ma , usa ) , where dedicated scripts were developed for the quantitative image analyses . 
 from the volumetric distribution of adc values , the following parameters were derived : mean ( adcmean ) , standard deviation ( adcstd ) , median ( adcmedian ) , skewness and kurtosis . the skewness is a measure of degree of asymmetry of the distribution ( skewness is negative if the distribution is skewed to the left , while positive if it is skewed to the right )  . 
medcalc software ( version 9 , mariakerke , belgium ) was used for the statistical analyses . results from february 2011 until september 2017 , a total of 47 patients were initially selected for this study . 
among them , three patients were excluded because of strong susceptibility and / or motion artifacts on dwi , two patients were excluded because of largely necrotic nodes and three patients because they had histological confirmation of nasopharyngeal scc . 
 tumor characteristics are reported in more detail in table1 . mri was able to identify a small primary tumor in the hypopharynx in three patients , while mri findings were negative for detection of primary tumor in 16 patients . 
among these 16 patients , pet - ct was able to identify a primary tumor in the palatine tonsil for three patients and in base of the tongue for one patient ; the panendoscopy and tonsillectomy were useful in three patients ( they had small foci in palatine tonsil ) , while the site of primary tumor remained unknown in 9 patients . summary statistics of adc values for the two patient groups ( lymphoma and scc ) are reported in table2 and illustrated in fig.1. 
the edges of the box are the 25th and 75th percentiles , and the horizontal lines extend from the minimum to the maximum value , outliers being plotted with cross - markers . 
areas under the roc curves ( aucs ) for the most significant adc histogram parameters are reported in table3 , with the optimal cutoff values and the corresponding sensitivity and specificity . 
the adcmean and adcmedian had the best discriminative powers for differentiating lymphoma and scc , with optimal cutoff values as predictors for lymphoma of 0.83 103 mm2 / s and 0.73 103 mm2 / s , respectively . 
to eliminate the dependence of the histogram from the between - subject differences in the volume size , voxel counts were calculated as percentages , dividing them by the total number of voxels in the whole lymph node . some illustrative cases are reported in figs.3 , 4 and 5 . discussion the differential diagnosis between scc and lymphoma represents an important issue , particularly in patients affected by a painless neck mass with unknown clinical primary tumor , who already underwent a non - diagnostic fnac [ 7 ]  . 
 in this set of patients , mri and advanced mr techniques may have a central role in the differential diagnosis of malignant lymphadenopathies , as mri techniques provide an accurate tissue characterization , both anatomical and physiological [ 17 ]  . 
however , conventional sequences are limited because of some overlapping appearance of scc metastases and lymphoma , even if lymphomas typically shows lower t2 - weighted signal intensity and more homogeneous enhancement after contrast medium infusion [ 24 ]  . 
 previous studies demonstrated the ability of dwi to differentiate scc nodes and lymphomas , the last having lower adc values , compared to sccs , with an optimal cutoff of about 0.7 103 mm2 / s [ 1014 ]  . 
this is because npc is typically characterized by high concentration of macromolecular content , high cellularity , high nuclear to cytoplasmic ratio and low content of keratin [ 26 ] , which lead to decreased diffusion coefficients compared to other anatomic sites [ 27 ]  . 
for these reasons , it has been suggested in the literature not to consider sccs as a single group but to take into account the location of the primary tumor to assess the diagnostic power of dwi and determine the optimal adc cutoff values [ 25 ]  . in the present retrospective study , we evaluated the ability of whole - volume adc histogram of cervical lymph node to discriminate lymphomas from metastatic fig . 
3 axial t2 - weighted image ( a ) in a 59 - year - old man shows a right neck mass with homogeneous and moderately high signal intensity ; the parametric maps of the apparent diffusion coefficient adc in the same anatomical location ( b ) ; adc histogram within the entire tumor volume ( c )  . 
4 axial t2 - weighted image ( a ) in a 55 - year - old man shows a right neck mass in the levels iia and iib with disomogeneous and moderate hyperintensity signal ; the parametric maps of the apparent diffusion coefficient adc in the same anatomical location ( b ) , adc histogram within the entire tumor volume ( c )  . 
5 axial t2 - weighted image ( a ) in a 67 - year - old man shows a left neck node in the level iv with homogenous and moderate high signal intensity , without extranodal extension ; the parametric maps of the apparent diffusion coefficient adc in the same anatomical location ( b ) ; adc histogram within the entire tumor volume ( c )  . 
the histogram analysis does not allow a differential diagnosis between scc and lymphoma : the adcmedian was 0.80 103 mm2 / s , suggesting a scc , while adcmean was 0.83 103 mm2 / s , which was identical to the cutoff value . 
first - order statistics , including mean , median , standard deviation , skewness and kurtosis , was calculated on the basis of the distribution of the adc values to extract some texture features of the tumor heterogeneity [ 16 ]  . 
we hypothesized that considering the entire lesion instead of a single representative roi could improve the reproducibility and promote a standardization of dwi analysis [ 15 , 18 ]  . we found that both adcmean and adcmedian of lymphomas were significantly lower than those of the sccs , consistently with previous studies [ 8 , 12 ]  . 
correspondingly , adcmean and adcmedian had good auc values with remarkably high specificity and ppv scores : the cutoff value of 0.83 103 mm2 / s for adcmean allowed us a correct diagnosis in all the scc lesions . 
lymphomas had higher skewness as well , i.e. , exhibited a shift to the left in the distribution of adc values , which is indicative of 1 3 la radiologia medica ( 2019 ) 124 : 1926 remarkably decreased water mobility , as a consequence of the high cellularity and high nucleus - to - cytoplasm ratio of lymphomas [ 12 , 20 , 28 ]  . 
these features of the adc values distribution are better exemplified in fig.2 , by a visual comparison of the averaged adc histograms among the two patients groups . nevertheless , both kurtosis and skweness did not allow a differential diagnosis between lymphomas and non - nasopharyngeal sccs : they did not significantly differ between the two patient groups , though kurtosis showed a trend toward significance . 
 [ 8 ] , who reported that the 90th percentile of adc ( adc90 ) had the best diagnostic performance , followed by adcmean and adcmedian ; however , it was not indicated whether the differences between aucs of adcmean / adcmedian and adc90 were statistically significant or not . 
the optimal adc cutoff values in this study may not be straightforwardly applicable in other centers due to differences in dwi acquisition protocols , no consensus existing about the most appropriate number and range of b values . 
moreover , the lymphoma population had a prevalence of diffuse large b cell histology and this represents a risk of bias that could have affected our results . in future investigations , we plan to study the potential of combined analyses of t2 - weighted signal intensity and contrast enhancement together with adc , as we believe that this might further improve the diagnostic ability of mri findings to clarify the nature of cervical lymph node metastases . conclusions the whole - lesion adc histogram analysis of cervical lymphadenopathy may help to discriminate lymphomas from non - nasopharyngeal scc in patients with unknown clinical primary tumor . 
the adcmean and adcmedian values had the highest diagnostic powers , showing good diagnostic accuracy with remarkably high specificity . acknowledgements we thank michele farella , elisa tommasini , pierfrancesco rinaldi for their continued technical assistance . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . 
the 145 cases of ggns were divided into pre - invasive ( pi ) group ( n = 46 ) , micro - invasive adenocarcinoma ( mia ) group ( n = 48 ) , and invasive adenocarcinoma ( iac ) group ( n = 51 )  . 
the presence of a tumourlung interface in the mia and iac groups was significantly higher than that in the pi group ( p < 0.05 ) , but no significant difference was found between the mia and iac groups . 
the presence of a pleural indentation sign in the iac group was significantly higher than that in the other two groups ( p < 0.05 ) , but no significant difference was noted between the latter two groups . 
no significant difference was found in the ggn density , vacuole sign , air bronchus sign and notch sign among the three groups . conclusions the hrct signs of ggns could be used to differentiate among pre - invasive lesions , micro - invasive lesions and invasive lung adenocarcinoma . keywords high - resolution computed tomography ground glass nodule pathologic classification lung adenocarcinoma introduction ground glass nodules ( ggns ) can be classified as pure ggns and mixed ggns , depending on whether they contain solid components [ 1 , 2 ]  . 
ordinary ct cannot display ggns or display them clearly , while * shuang - qing chen sznaonao@163.com 1 department ofoncology , the affiliated suzhou hospital ofnanjing medical university , suzhou215001 , china 2 department ofradiology , the affiliated suzhou hospital ofnanjing medical university , no . 
16 , bai - ta - xi road , suzhou215001 , jiangsuprovince , china high - resolution computed tomography ( hrct ) can improve the image quality and display ggns more clearly [ 3 , 4 ]  . in 2011 , the international association for the study of lung cancer ( iaslc ) , the american thoracic society ( ats ) and the european respiratory society ( ers ) jointly issued a new international multidisciplinary classification of lung adenocarcinoma and first proposed the term microinvasive adenocarcinoma ( mia ) [ 5 ]  . 
given the increased number of lung ggns detected and advocacy of the clinical strategy of the partial resection of pre - invasive and micro - invasive carcinoma , it is primarily important to differentiate among pre - invasive , micro - invasive and invasive adenocarcinoma lesions in the new classification [ 6 , 7 ]  . 
 the aim of the present study was to observe the relationship between hrct signs and the new pathologic classification vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 813 in ggn - like lung adenocarcinoma to explore the clinical value of hrct in the diagnosis of lung adenocarcinoma . materials andmethods patients the hrct data of 145 patients with single - onset ggns 2cm in diameter and surgically treated between january 2014 and december 2016 were retrospectively reviewed . 
 according to the 2011 edition of the international multidisciplinary classification of lung adenocarcinoma , they were classified into three groups : pre - invasive ( pi ) group ( n = 46 ) , including 21 cases of atypical adenomatous hyperplasia ( aah ) and 14 cases of adenocarcinoma insitu ( ais ) ; microinvasive adenocarcinoma ( mia ) group ( n = 48 ) ; and invasive adenocarcinoma ( iac ) group ( n = 51 )  . 
in the retrospective analysis , all patients provided informed consent requirement before enrolment , and the study was approved by the ethics committee of nanjing medical university . examination methods a philips brilliance 128 - slice spiral ct scanner was used for examination , with the patient holding their breath during the stable respirations . 
routine ct was first performed from the lung apex to the base ; after location of the nodule , high - resolution thin - section target scanning was performed under the conditions of 120kv , 299ma , 1 - mm thickness and a 1 - mm interval . 
depending on the size and density of the lesions , the window width and position were adjusted from time zero during the processing procedure to obtain the optimal images . image analysis the images were interpreted by two senior radiologists independently in a blind manner . 
density : the maximal ggn cross section was selected from the standard reconstruction images of the ggn by avoiding the blood vessel and bronchus ; three regions of interest ( rois ) were selected , the ct value was measured , and the mean value of three measurements was used for analysis . 
inside and around the ggn : tumourlung interface , pleural indentation , notch sign , air bronchus sign , vacuole sign , lobulated sign and spicule sign of the ggn . 
tumour microvascular ct imaging sign : the tumour microvascular ct imaging sign was defined when one or more vessels entered the lesion and the course of the vascular segment of the lesion was twisted and rigid . 
no significant difference was noted in the ggn density , notch sign , air bronchus sign and vacuole sign among the three groups . discussion with wider applications of hrct in recent years , the detection rate of ggn has increased obviously [ 8 , 9 ]  . 
a transverse thin - section target scanning imaging ; b coronary mpr imaging ; c pathology confirmed for iac 1 3 la radiologia medica ( 2019 ) 124 : 813 fig . 
a transverse thin - section target scanning imaging ; b sagittal mpr imaging ; c pathology confirmed for iac 1 3 12 la radiologia medica ( 2019 ) 124 : 813 first proposes the method of classification appropriate for surgical resection and biopsy , increasing the 5 - year survival rate of the complete surgical resection of pre - invasive and micro - invasive carcinoma to 100% [ 5 , 7 , 11 ]  . 
 [ 16 ] found that the size of pre - invasive ggns was significantly smaller than that of micro - invasive and invasive gnns , and the ggn size could help differentiate between pre - invasive and invasive lesions . 
the results of the present study showed that the ggn size was increased positively with the increasing degree of malignancy , and significant differences were noted in the ggn size among the three groups , findings that were consistent with those of eguchi etal . 
however , there was no significant difference in the ggn density among the three groups , suggesting that the ggn density is not an effective sign for the judgment of invasion . 
the cause may be that it is difficult to select rois when the lesion is not sufficiently large or the density is uneven . studies have shown that with the increasing degree of ggn invasiveness , the presence of lobulated , spicule and notch signs were also increased , reflecting the morphologic changes in ggn progression from a pre - invasive state to a micro - invasive / invasive state [ 17 , 18 ]  . 
it was found that , in the present study , the presence of lobulated and spiculated signs in micro - invasive and invasive adenocarcinoma was not only significantly higher than that in pre - invasive lesions but also showed a significant difference between micro - invasive and invasive lesions , indicating that these two signs have a relatively high value to assess the invasiveness and degree of invasion of gnn . 
 [ 20 ] pointed out that the presence of a rough tumourlung interface on ggns tended to indicate malignancy , likely because with the increase in the degree of invasion , the tumour cell arrangement on alveolar walls became dense and thickened , thus presenting a clearer tumourlung interface on ct imaging . 
we also found that the clarity of the tumourlung interface can be used as an indicator of pre - invasive and invasive lesions but did not seem to work effectively for the differentiation between micro - invasive and invasive adenocarcinomas . 
comparison of the tumour microvascular ct imaging signs showed that they occurred more frequently in the mia and iac groups than that in the pi group , implying that the entry of one or more vessels into the lesion and a twisted and rigid vascular running course within the lesion may suggest a greater possibility of malignancy of ggn [ 17 ]  . 
 the mechanism underlying microvascular formation remains unclear at present , possibly because of traction of the fibrous components within the lesion on the surrounding normal vessels , resulting in vascular course change . 
others have proposed that the infiltrative growth of the tumour tissue into small vessels of the lesion or interlobular infiltrative growth stimulated the growth of fibrous components , causing blood vessels to become twisted and rigid [ 21 , 22 ]  . 
in the present study , we found no significant difference between the micro - invasive and invasive groups , and further analysis is awaited concerning the details of tumour microvascular ct imaging signs . in summary , the size , lobulated sign , spicule sign , notch sign , tumourlung interface , and tumour microvascular ct imaging sign can help better understand the new classification of small ggn - like lung adenocarcinoma . 
the aim of the study is to isolate which features of magnetic resonance imaging ( mri ) - based radiomic analysis have to be considered the most significant predictors of metastasis in oncological patients with spinal bone marrow metastatic disease . materials and methods eight oncological patients ( 3 lung cancer ; 1 prostatic cancer ; 1 esophageal cancer ; 1 nasopharyngeal cancer ; 1 hepatocarcinoma ; 1 breast cancer ) with pre - radiotherapy mr imaging for a total of 58 dorsal vertebral bodies , 29 metastatic and 29 non - metastatic were included . 
a wilcoxon test was applied to the 89 features and the most statistically significant of them underwent to a stepwise feature selection , to find the best performing predictors of metastasis in a logistic regression model . 
 an internal cross - validation via bootstrap was conducted for estimating the model performance in terms of the area under the curve ( auc ) of the receiver operating characteristic . results of the 89 textural features tested , 16 were found to differ with statistical significance in the metastatic vs nonmetastatic group . 
the internal cross - validation showed an auc of 0.8141 ( 95% ci 0.68540.9427 ) in t1 images and 0.9116 ( 95% ci 0.82940.9937 ) in t2 images . conclusions the results suggest that mri - based radiomic analysis on oncological patients with bone marrow metastatic disease is able to differentiate between metastatic and non - metastatic vertebral bodies . 
the most significant predictors of metastasis were found to be based on t2 sequence and were one morphological and one textural feature . keywords vertebral metastases radiomics magnetic resonance quantitative imaging oncology radiotherapy * laura filograna laura.filograna@gmail.com 1 department ofradiation oncology gemelli - art , catholic university ofrome , school ofmedicine , foundation university hospital a . 
gemelli 8 , 00168rome , italy 4 department ofdiagnostic andinterventional radiology , molecular imaging andradiotherapy , ptv foundation , tor vergata university ofrome , viale oxford 81 , 00133rome , italy vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 5057 introduction metastatic bone disease is , unfortunately , a common event in the evolution of cancer . 
the development of metastases represents a challenge for the clinician who manage with oncological patients , because , once bone metastases occurred , cancer becomes a systemic pathology and palliative therapy is the only viable approach . the spine is the commonest site of metastases . 
in radiomics a large amount of advanced quantitative imaging features are extracted from medical images obtained with computed tomography ( ct ) , positron emission tomography ( pet ) or magnetic resonance imaging ( mri ) , with the aim to build diagnostic , prognostic and predictive models . since its origin , this image analysis method has been largely employed in the management of oncological patients . 
many other studies with radiomics - based analysis have been performed in the management of different tumors types [ 712 ]  . magnetic resonance ( mr ) imaging plays a crucial role in the characterization of spinal bone marrow disorders and in monitoring response to therapy [ 13 ]  . 
for these reasons mr is considered an imaging method of vital importance for the management of oncological patients with bone marrow metastatic disease . to our knowledge , there is only one paper using both morphological and textural features analysis on mr imaging for the study of vertebral bone marrow metastases [ 14 ]  . 
in particular , the authors analyze preand post - radiotherapy mr imaging for evaluating early response to therapy in patients with metastatic cancer , with promising results [ 14 ]  . in this paper , from pre - treatment mr images of oncological patients with diagnosed vertebral bone marrow metastases quantitative imaging features of pathological vs non - pathological vertebral bodies are extracted by t1 and t2 images and analyzed with the aim to isolate which features of mr imaging - based radiomic analysis have to be considered the most significant predictors of spinal bone marrow metastatic disease in oncological patients . materials andmethods eight patients ( 5 male , 3 female , age range 4180 aa , mean age 60years old ) with cancer ( 3 lung cancer ; 1 prostatic cancer ; 1 esophageal cancer ; 1 nasopharyngeal cancer ; 1 hepatocarcinoma ; 1 breast ) with vertebral bone marrow metastases with pre - radiotherapy mr imaging were examined . 
a vertebral body was considered metastatic on mri , if on t1 - weighted sequences the lesion appeared hypointense compared to fatty tissue , and if on t2 - weighted images it showed a variable hyperintensity [ 15 ] , with or without a hyperintense halo , due to perilesional edema [ 16 , 17 ]  . none of the vertebral bodies examined had pathological or non - pathological fracture . 
the obtained data were transferred to an automated data extraction system for morphological , statistical and textural analysis . three groups of features were used for the analysis : statistical / histogram , morphological and textural features . 
in statistical / histogram features , the three - dimensional data from contoured volume are translated to an histogram that illustrates the fractional volume in the examined structure for the range of voxel values , in this study , intensity values . 
to the histogram , statistic tests can be applied for calculating , for example , skewness , kurtosis , median , mean . 1 3 la radiologia medica ( 2019 ) 124 : 5057 fig . 
1 60 - year - old woman with breast carcinoma : the images are referred to sagittal t1 image ( a ) and the same sagittal t1 image ( b ) with the roi traced on a non - metastatic vertebral body , visualized with a particular ( non - diagnostic ) viewer built for moddicom - based radiomic analysis . 
also other more complex features such as the surface to volume ratio or descriptors of tumor compactness can be determined . finally , textural features are based on second - order statistics or co - occurrence matrix . 
by the co - occurrence matrix many advanced features can be extracted , for example , contrast , autocorrelation , correlation , cluster prominence , maximum probability , dissimilarity , sum entropy , sum variance . 
grey - level run length matrix features can describe grey - level non - uniformity , short and long run emphasis , run length non uniformity , high grey - level run emphasis , low grey - level run emphasis . with the aim to find among this large amount of features the most representative in differentiating metastatic vs nonmetastatic vertebral bodies in the study population selected , initially a total of 89 features deriving from the three groups ( statistical / histogram , morphological and textural features ) have been extracted without any kind of image filtering for 1 3 la radiologia medica ( 2019 ) 124 : 5057 fig . 
2 72 - year - old man with lung cancer : the images are referred to sagittal t1 image ( a ) and t2 fs image ( c ) and the same sagittal t1 image ( b ) and t2 image ( d ) with the roi traced on a metastatic vertebral body , visualized with a particular ( non - diagnostic ) viewer built for moddicom - based radiomic analysis . 
note presence of an ocular metastasis hypointense in t1 ( a ) and hyperintense in t2 fs image ( c ) , and the dishomogeneity of the pixel colors distribution for the presence of a focal concentration of color pixel corresponding to the metastasis t1 and t2 mri scans . 
the image processing and feature extraction was performed with the r library moddicom for radiomics analysis , a library whose aim is to support medical image analysis , feature extraction , radiobiological and clinical modeling . 
 the statistical software r version 3.4.2 was used for statistical analysis [ 18 ]  . given the set of features , their number was furtherly restricted by using a wilcoxon test , performed to investigate their statistical significance with respect to the outcome ( differentiating metastatic vs non - metastatic bodies )  . 
these two features were found the best ones for discriminating among metastatic and non - metastatic vertebral bodies in t2 weighted images , in our study population . 1 3 la radiologia medica ( 2019 ) 124 : 5057 discussion in this paper , mri - based radiomic analysis on untreated with radiotherapy oncological patients with bone marrow metastatic disease has been demonstrated as being able to differentiate between metastatic and non - metastatic vertebral bodies . 
furthermore , two features for t1 ( one morphological , and one histogram feature ) , and two for t2 images ( one morphological and one textural feature ) were identified as the best differentiating healthy and metastatic vertebral bodies in a radiomic analysis , respectively , in the two imaging data sets . the process of radiomics consists on the extraction and analysis of advanced quantitative imaging features with the aim of creating mineable databases for conducting predictive and prognostic correlations between images and patients outcome [ 17 ]  . the classical approach in imaging analysis of cancer is based on qualitative inferences ; a quantitative approach consists generally on the measurement of tumor dimensions or on a region of interest ( roi ) - based analysis . 
 unfortunately , these measurements , particularly tumor dimensions , do not reflect the tumor tissue complexity and do not generally correlate with prognosis . in opposition to the classical approach , radiomics analysis of tumor , by analyzing quantitative features related to tumors texture and morphology , offers the possibility to generating predictive models . according to kumar [ 17 ] , the radiomics process includes five steps , i.e. , ( 1 ) image acquisition and reconstruction , ( 2 ) image segmentation and rendering ( 3 ) feature extraction and feature qualification ( 4 ) databases and data sharing and ( 5 ) ad hoc informatic analyses . 
each of these steps poses some specific challenges . in the present study this process was applied on metastatic vertebral bodies , by using moddicom , an userfriendly and freely available library , able to be the platform for conducting radiomics analysis as proposed by dinapoli etal . 
the tumoral mass can be characterized by a very large amount of quantitative features that describe its complexity , belonging to the three large groups of radiomics features reported in the section materials and methods . this huge amount of features includes also some features that may not be straightly representative of the fig . 
3 in this figure , the area under the curve ( auc ) of the receiver operating characteristic ( roc ) deriving from the cross - validation via bootstrapping technique on the model constructed on t1 images ( features : center of mass shift and minimum grey level ) is illustrated fig . 
as kumar underlines [ 11 ] , often the number of radiomics features potentially included in the analysis is higher than the number of the subjects examined , particularly if it is considered that the study population included in this kind of studies , rarely is large . 
thus , the risk is to reduce the power of the study , and to augment the possibility of over fitting data . in this context , the selection of specific features best fitting the specific task in the management of the oncological patient under examination is crucial for optimizing the study . in this contribute , oncological patients with diagnosed vertebral bone marrow metastatic disease were analyzed with a radiomics approach based on mr images , according to the protocol proposed by dinapoli etal . 
in 2013 [ 14 ] tried to apply radiomics principles on preand post - radiation therapy mr examination of the spine for evaluating early therapy - induced bone marrow changes . 
in this work the authors conduct a roibased analysis on mr images related to 35 patients with spine and hip using 36 radiomics features with the aim to create a neural network - based classification system for the recognition and characterization of post - radiation bone marrow lesions detected with conventional mr imaging . 
among the 36 features , five textural ones were isolated as the optimal : the standard deviation , the difference entropy , the sum average , the entropy and the long run emphasis . in the present study 89 features for the 58 vertebral soma ( 29 metastatic and 29 non - metastatic ) examined in both t1 and t2 images were computed . 
of these features , 6 for t1 and 10 for t2 were found to differ with statistical significance in the metastatic vs non - metastatic group , and two of these , respectively , for t1 and two for t2 images have been demonstrated as predictors best performing for the characterization of vertebral bodies affected by metastases . 
moreover , one histogram feature , minimum grey level , and one textural feature , grey - level co - occurrence matrix joint variance , were found as being the best fitting features , respectively , in t1 images and t2 images . center of mass shift is the distance between the roi volume centroid and the intensity - weighted roi volume centroid , and measures the placement of high and low intensity regions within the volume . 
grey - level cooccurrence matrix joint variance is the grey - level weighted variance of joint probabilities . going beyond the specific meaning of each feature , it is important to note that the more robust results according to the p value , auc calculation to 95% ci were the two features isolated on t2 sequence ( tables1 , 2 )  . 
particularly , they were one morphological feature center of mass shift and one textural feature grey - level co - occurrence matrix joint variance . both the selected features straightly depend on the heterogeneity of signal intensity of the vertebral body examined . 
moreover , the evidence that t2 fat saturated images best describe the differences between non - metastatic and metastatic vertebral bodies can be explained by the fact that the presence of the metastatic tissue causes more important variation in the distribution of the signal intensity in t2 fat saturated images than it does occur in t1 images . 
on t2 fat saturated images , in fact , the signal hyperintensity differences are more evident with respect to the surrounding not affected bone marrow than in t1 images , as they better reflect not only the presence of the tumoral tissue , but also the perilesional edema . this study has some limitation . 
many factors , in fact , influence the signal intensity of the nonmetastatic vertebral bodies , in relation to the different percentages of adipocytes and red marrow depending on the age , eventual medicament assumption of the subjects , etc . 
a larger study population is needed to clarify the influence of these factors on the final results . in conclusion , in this study the radiomics features most representative of metastatic versus non - metastatic vertebral bodies are identified in our model . 
with the advantage to foster radiomics analysis for the study of vertebral bone marrow metastatic disease , we propose the privileged use of one morphological feature , center of mass shift , and one textural feature , grey - level co - occurrence matrix joint variance , in t2 fat saturated images . 
at 1 - month follow - up enhanced mri , complete remission was achieved in 82.6% of patients ( 38 / 46 ) , and partial remission in 17.4% ( 8 / 46 )  . 
tace combined with simultaneous rfa provides a new treatment option for solitary large hccs in which dynact has important clinical value . keywords hepatocellular carcinoma therapeutic embolization interventional radiology dynact introduction compared with other countries , large hccs ( maximal diameter > 5cm ) have a higher incidence in asia , especially in china . 
newer technologies such as the flat - panel detector digital subtraction angiography ( dsa ) system with cbct have become popular in clinical practice , and perspective , photography , dsa , and 3d reconstruction / imaging can be performed simultaneously on the same working bed using dynact [ such as the artis zee dsa system with cbct ( siemens , germany ) ] [ 9 ]  . 
 after tace under dsa , dynact can be used for scanning vol . : ( 0123456789 ) 1 3 2 la radiologia medica ( 2019 ) 124 : 17 and imaging and the puncture site and the route can then be determined . 
the use of dynact not only realizes simultaneous combination of tace and rfa , but also maximizes the synergistic effect of tace and rfa , increasing therapeutic efficacy [ 11 ]  . 
ultrasound also could be simultaneously used to guide ablation after tace [ 12 ] , and that contrast - enhanced ultrasound and fusion imaging were another advanced technique for difficult cases [ 13 ]  . 
 this study was undertaken to investigate the clinical value and safety of dynact in the treatment of solitary large hccs . materials andmethods general patient characteristics all patients were diagnosed with hcc by imaging or pathological examination and received tace with simultaneous dynact - guided rfa . 
a radio frequency of 460khz was chosen , and a rita multipolar radiofrequency ablation electrode needle ( outer tube diameter : 14g ) with nine hook - shaped bundle electrodes ( umbrella - like opening with the diameter of 5cm ) was used . treatment methods tace treatment after routine skin sterilization , local anesthesia with lidocaine was administered . 
rfa was performed immediately in the region where iodine oil deposition is poor under the guidance of dynact . dynactguided syngo iguide needle puncture dynact image acquisition lateral x - rays . 
the following parameters were used : rotation angle of c - arm : 200 ; rate of image capture : 50 frames / s ; increment in each frame : 0.5 ; x - ray dose : 0.36gy / frame ; total time : 8s ; and total number of frames : 396 . 
the inner puncture target spot was selected , and the surface puncture was marked , according to the puncture routine automatically selected by the iguide software ( fig.1b ) ; the depth of puncture was also displayed ; ( 2 ) the bulls eye position was selected , the operating lever was pushed , the c - arm was automatically fixed at the bulls eye position , and then the inner puncture site was assured to overlap the surface puncture site . 
rfa was performed using a power of 150200w and temperature of 105c for 1520mfor the large tumor size , the needle location was adjusted after ablation at a site for further ablation until the ablated area covered the edge of the tumor and its three - dimensional space . 
we studied the time to progression ( ttp ) and 1 - , 2 - , and 3 - year survival rates . response assessment technical success was defined as completion of both tace and rfa in one treatment session . 
initial tumor response was assessed by contrast - enhanced mri 1 month after treatment according to the modified response evaluation criteria in solid tumor ( m - recist ) developed by the american association for the study of liver diseases ( aasld ) [ 14 ] : complete remission ( cr ) , partial remission ( pr ) , stable disease ( sd ) , progressive disease ( pd )  . 
for a lesion close to the important organs ( peripheral lesion , subdiaphragmatic lesion , and lesion close to the intestine ) , lidocaine in normal saline was injected to separate the lesion from surrounding tissues , followed by puncture . 
after initial treatment , ten patients received a second tace or rfa during follow - up , of whom cr was achieved in seven patients ( as the remaining patients did not receive further tace or rfa due to tumor progression or personal rejection )  . 
the occurrence rate of grade 3 was 6.5% , included subcapsular hematoma of liver ( n = 2 ) , sand diaphragmatic damage with pleural effusion ( n = 1 )  . discussion in recent years , rfa has become a common nonsurgical treatment besides tace and its effectiveness has been confirmed in small hccs , comparable to that of radical treatments ( such as surgical intervention and liver transplantation )  . 
with the development of rfa instrumentation and imaging guidance equipment and the accumulation of clinical experience , rfa has been increasingly used in the treatment of large hccs [ 17 ]  . 
local tumor progression - free survival after dynact - guided tace with simultaneous rfa ( b ) 1 3 6 la radiologia medica ( 2019 ) 124 : 17 combination of tace with simultaneous rfa , providing a new , convenient , and effective treatment for large hccs . 
 [ 14 , 19 ] employed tace with simultaneous cbct - guided rfa in the treatment of large hccs in 21 patients , with a technical success rate of 100% . dynact employs ctcb using the artis zee dsa system ( simens , germany ) and can realize synchronized acquisition with flat - panel detector during c - arm rotation . 
after image reconstruction using the workstation , three - dimensional images of the target lesion and ct images of soft tissues can be obtained , which overcomes the limitations of traditional instruments . 
at 1 , 2 , and 3years after surgery , the survival rate was 89.1% ( 41 / 46 ) , 71.7% ( 33 / 46 ) , and 56.5% ( 26 / 46 ) , respectively , which is consistent with the survival rates reported by takaki etal . 
in 46 patients , no intraoperative and postoperative severe complications were observed , suggesting favorable safety of this technique . based on our findings , dynact - guided tace with simultaneous rfa appears safe and reliable for treatment of solitary large hccs . 
the advantages of this technique include simultaneous combination of tace and rfa which reduces the time interval between two treatments , thus avoiding the clearance of lipiodol and chemotherapeutics and the formation of new collateral vessels and vascular recanalization after embolization in this time interval [ 21 ]  . 
in addition , dynact - guided tace with simultaneous rfa may exert a synergistic effect , i.e. , rfa after complete lipiodol deposition may maximize the heat conduction effect of the lipiodol , and , therefore , the heat produced by the rf needle can focus on the site of lipiodol deposition , exerting maximum anti - tumor effect ; this is important for irregular large hccs because peripheral tumor is difficult to completely ablate by rfa , but rfa after complete lipiodol deposition improves transduction of heat to peripheral tissues , which reduces the risk for recurrence and metastasis [ 19 , 22 ]  . 
with rotation of the c - arm , the patients can receive rfa on the same treatment bed without movement after tace , which avoids the transfer of patients which normally occurs during traditional interventional therapy . 
finally , the instrument can provide information in a real - time manner , which is helpful for the adjustment of treatment protocol ; dynact scanning after tace can accurately display and identify lipiodol deposition in normal liver tissues , which is helpful for decision making in surgery ( i.e. , whether or not additional lipiodol injection is needed ) [ 23 ]  . of note , dynact has several limitations including : ( 1 ) the puncture should be continuously monitored by dynact , which increases the radiation exposure to clinicians ; ( 2 ) the images obtained from dynact should be subjected to mpr reconstruction in the workstation , and the reconstructed images have poor resolution and poor intensity differences between tissues as compared with those from routine ct [ 24 ]  . 
the inclusion and exclusion criteria were strict , i.e. , patients should receive complete preoperative examinations , the functional reserve of the liver should be carefully evaluated , liver - protective treatment should be administered after surgery to avoid liver failure , especially for those with poor liver reserve function , chemotherapeutics acting on cell division or proliferation were preferred , and two or more chemotherapeutics were recommended for chemotherapy [ 25 ]  . 
in addition , local analgesic treatment with lidocaine was administered during surgery because patients receiving combined treatment experience longer operative times . conclusions our results indicated that dynact - guided tace with simultaneous rfa is a safe and efficacious treatment for solitary large hccs . 
the aim of the present paper is to increase awareness , acceptance and adoption of interventional radiology procedures for the treatment of bm ; and to present the joint position of the italian college of musculoskeletal radiology and the italian college of interventional radiology . keywords bone metastasis skeletal - related events interventional radiology epidemiology andclinical manifestations ofbone metastases bone metastases ( bm ) are the most common malignant lesions of the bone , commonly involving the axial skeleton , pelvic ring and proximal extremities [ 1 ]  . 
in the usa , it is estimated that approximately 100 , 000 individuals develop bm every year [ 2 ] ; and the incidence is expected to increase in the future due to the improved survival of cancer patients . 
 the incidence of bm is particularly high in patients suffering from breast , prostate or lung cancer ; intermediate in those presenting with melanoma , renal or thyroid cancer ; and relatively low in patients presenting with gastrointestinal tumors . * roberto luigi cazzato gigicazzato@hotmail.it extended author information available on the last page of the article bm significantly impact patients clinical status due to pain , fractures , compression of nearby structures such as nerves , hypercalcemia , and often require radiation therapy ( rt ) and / or surgery , especially when spinal cord compression is noted . 
usually three types of fractures occur in cancer patients , namely pathologic fractures ( pf ) , impending fractures ( imf ) , and fractures related to bone insufficiency ( bif )  . 
lastly , bif result from bone reabsorption due to vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 3449 medical therapies ( e.g. , hormonal therapies , steroids ) and / or cytokines produced by the tumor [ 57 ]  . current treatment modalities to treat pain and prevent fractures are medical therapy ( including analgesics , bisphosphonates and denosumab ) and rt ; both allow good but far from excellent results . 
although rt is considered the best evidencebased non - interventional treatment for bm - related pain , several limitations have been observed : ( a ) ineffectiveness in case of radio - resistant tumors such as renal cancer or melanoma ; ( b ) 12 - week latency between the end of treatment and the onset of pain relief ; ( c ) overall ( complete and partial ) pain relief observed in less than 60% of patients and recurrent pain in up to 50% of those who respond by 2024weeks after the end of treatment ; ( d ) retreatment is not always possible if the maximal radiation dose has already been delivered ; ( e ) no immediate bone consolidation is provided [ 35 ]  . 
 although the recently introduced tumoricidal stereotactic body rt yields higher rates of local tumor control and pain management compared with standard external beam rt [ 9 , 10 ] , the risk of secondary fracture is significantly increased when the former technique is used [ 9 ]  . 
on the other hand , bone - targeting agents interfering with tumor - mediated osteolysis , can reduce but not completely remove the risk of sres [ 3 ]  . in this setting , interventional radiology procedures may play an important primary or complementary role to manage bm [ 1113 ] , especially in the palliative setting where pain and fracture management is necessary . the aim of the present paper is to increase awareness , acceptance and adoption of interventional radiology procedures for the treatment of bm ; and to present the joint position of the italian college of musculoskeletal radiology and the italian college of interventional radiology . interventional procedures osteoplasty indications percutaneous osteoplasty refers to polymethylmethacrylate ( pmma ) injection into weakened or fractured bone . 
on the other hand , in case of tumor compression of the spinal cord or in case of vertebral instability due to tumor involvement of the posterior elements of the vertebra , surgical decompression / stabilization should be considered as early as possible . 
this is due to the fact that pmma has limited resistance to torsion and bending ; therefore , surgical endomedullary nailing is the preferred option to achieve effective biomechanical stabilization . 
 absolute contraindications to osteoplasty are local / systemic infection , known allergy to the pmma and irreversible coagulopathy . technique the procedure can be performed under fluoroscopic and / or ct - guidance often under local anesthesia / mild sedation . 
the consolidation time of pmma is variable ( 820min ) , and its polymerization results in an exothermic reaction with temperature > 70c , which is , however , inadequate to achieve effective local tumor control . results osteoplasty has been proved to be effective for the symptomatic treatment of osteolytic tumors including multiple myeloma . 
1 a , b painful lytic metastasis of t12 in a 70 - year * old patient suffering from hepatocellular carcinoma ; the metastasis disrupted the posterior wall of the vertebral body ( white arrows )  . 
c , d percutaneous vertebroplasty was safely performed with immediate pain relief and no cement leakage ( vertebroplasty was also performed in t11 and l1 during the same interventional session due to painful pathological fractures ) fig . 
b the fracture was fixed percutaneously by means of a compressive screw ; the screw tip was fixed in distal normal bone through the injection of few ml of cement ( white arrow ) osteosynthesis indications percutaneous osteosynthesis is consistent with screw fixation to consolidate minimally / non - displaced fractures of the pelvic ring [ 21 ]  . 
the technique can also be performed in the proximal femur to consolidate imf ( i.e. , mirels score 8 ) without significant trochanteric and cortical involvement [ 22 , 23 ]  . 
accordingly , osteosynthesis should be strictly reserved for non - surgical cancer patients with limited life expectancy to provide rapid analgesia and mobilization 1 3 la radiologia medica ( 2019 ) 124 : 3449 without the need for suspension of systemic therapies , which is often necessary with surgical treatments . 
moreover , compared to surgery , risk of bleeding and infection is also significantly reduced . thermal ablation indications technique percutaneous osteosynthesis is performed under fluoroscopic and / or ct - guidance . 
due to the long procedural time ( around 2h due to challenging bone access ) , general anesthesia is usually preferred over local anesthesia / mild sedation . threaded screws are used in case of mildly displaced fractures in order to achieve maximal inter - fragment compression . 
finally , pmma - injectable screws providing distal holes are preferred in cases of advanced osteoporotic bone to enhance screw anchoring . the most challenging phase of the procedure is often the perpendicular bridging of the fracture line by means of a 1.82mmk - wire ; after which , the screw is manually advanced over the wire by means of a screwdriver until its distal tip is safely anchored within distal healthy bone , and its head abuts the cortical bone . results osteosynthesis has been shown to be effective for the symptomatic treatment of pelvic fractures or those of the proximal femur . 
in a study of 64 patients undergoing pelvic osteosynthesis alone or in combination with osteoplasty , a median pain reduction of 6 / 10 points was noted , with only two secondary fractures observed in the proximal femur [ 25 ]  . 
another similar series of 33 patients reported an effective analgesic / functional amelioration in 87.1% cases at 1 - month follow - up [ 21 ] ; nevertheless , three major and one minor complications were reported . 
moreover , although no secondary fractures were reported , unfavorable local evolution of the treated site ( i.e. , poor consolidation and / or screw loosening ) was noted in 12.5% cases on imaging follow - up ( mean 8.7month ) thus , suggesting the need for strict clinical follow - up . 
the final necrotic volume depends on the amount of energy delivered and the local tissue characteristics such as vascularization , which may be responsible for energy dissipation ( i.e. , heat / cold - sink effect ) [ 26 , 31 ]  . compared to rt , the effects of ablation are immediate and there is no limit to the number of ablative treatments that can be performed on the same tumor , which is not the case for rt as it cannot be repeated once the maximum dose of the target organ is reached . 
in the majority of cases , bone consolidation can be combined with ablation in the same session . anesthesia ( deep sedation , general anesthesia , nerve block or spinal anesthesia ) is usually required [ 32 ]  . 
antibiotic prophylaxis remains controversial although it is generally applied [ 32 ]  . techniques laser lasers deliver electromagnetic energy in the infrared wavelength through small optic fibers deployed coaxially into 18g needles . 
however , only small ablation zones can be obtained since , even when multiple fibers are activated simultaneously , ablations zones rarely exceed 23cm [ 3133 ] ; for this reason , lasers can be applied to treat only small benign bone tumors such as osteoid osteoma [ 34 ]  . radiofrequency ablation ( rfa ) rfa is probably the most commonly applied technique in bone ablation [ 31 , 33 , 35 ]  . 
for this reason , rfa cannot be used if pacemakers or other implantable electric devices are present , or in case of osteoblastic bm impeding effective conduction of the electrical current , which can flow between the electrode and the grounding pads attached on the skin of the patient ( unipolar system ) , or between two electrodes or two dipoles localized at the distal tip of the same electrode 1 3 38 la radiologia medica ( 2019 ) 124 : 3449 fig . 
f bilateral bipolar rfa was performed with thermal monitoring at the level of the posterior wall ( white arrow ) and hydro - dissection of the epidural space ( black arrow ) ; g , h in the end , vertebroplasty was performed to prevent a secondary vertebral body collapse . 
nevertheless , rfa cannot achieve ablation zones that exceed 34cm . microwave ablation ( mwa ) mwa uses electromagnetic energy , which is delivered through an antenna [ 31 , 35 , 38 ]  . 
the main drawbacks of mwa are : ( a ) oval - shaped ablations , especially with first generation antennas ; ( b ) limited experience available with bm due to the fact that the technique is relatively new [ 39 , 40 ]  . cryoablation ( ca ) ca destroys tumors using cold temperatures ( up to - 40 c ) dissipated into tissues through dedicated cryoprobes [ 3033 , 35 ]  . 
 cellular death occurs through a complex mechanism that is not completely understood , including mechanical damage to cellular membranes induced by ice crystals , osmotic changes into the tissue , endothelial injury , ischemia , and cryo - immunological effects . 
compared to the other techniques of ablation , the main advantages of ca are : ( a ) precise control of the ablation area due to adequate visualization by common imaging modalities ( especially ct or mri ) , thus allowing for precise intra - operative evaluation of tumor coverage ; ( b ) multiple probes ( up to 40 with the most recent systems , each with different sizes and shapes of ablation ) can be simultaneously activated with a final synergistic effect ; therefore , the iceball can be shaped according to tumor morphology , and large - volume bm can be treated ; ( c ) possibility of treating osteoblastic bm since the iceball can easily go through cortical and blastic bone ; ( d ) since the iceball has intrinsic anesthetic properties , the procedure is less painful compared to heat - based techniques ; accordingly , ca is preferred for the treatment of bm with soft tissue extension . 
apart from its totally non - invasive profile , mrgfus has the benefit of allowing optimal mri resolution to target bm , and allows monitoring of the temperature reached within the tumor , surrounding it as well as in interposed tissues ( except bone )  . 
on the other hand , since bone can absorb the ultrasound beam , it is possible to induce high periosteal temperatures to achieve effective pain management in case of painful bm . 
drawbacks include limited availability of the technique , long procedural time ( 23h ) , and contraindication in patients contraindicated to mri . protective measures to adopt during bone ablation thermal ablation is generally considered safe ; nevertheless , the most common complications include the unintentional ablation of nearby non - target organs , particularly nerves . 
in bone ablation , all these measures can be applied ( table1 ) alone or in combination to avoid iatrogenic injuries , in particular for nerves and sk co2 or fluids are often injected to achieve physical displacement . 
the great advantage of fluid dissection over table 1 protective measures that can be adopted during ablation advantages disadvantages fluid dissection angioplasty balloon low thermal conductivity highly soluble no renal or hepatic toxicity non - allergic low cost sterile suitable with all imaging modalities ( us , ct , and mri ) : spontaneous visualization under mri - guidance ; increased visualization under ct - guidance with 510% contrast medium dilution low cost sterile allow for temperature adjustment non gravity - dependent distribution no need for volume readjustment no cooling or warming properties distribution in anti - declivous areas unsuitable for us - guidance distribution to dependent area potential fluid overload temperature monitoring somatosensory - evoked potentials precise knowledge of the temperature in critical areas needs precise thermocouple deployment precise monitoring of electrical conduction of the tested nerve relatively expensive technically challenging unsuitable for the protection of neurological structures difficult to be performed requires dedicated trained physicians no neuromuscular blocking neither muscle relaxant agents should be administered during the procedure cannot be used continuously in order to prevent muscle fatigue no neuromuscular blocking neither muscle relaxant agents should be administered electrostimulation simple to be performed no specific training required 1 3 40 la radiologia medica ( 2019 ) 124 : 3449 co2 is that fluid temperature may allow for warming - up or cooling - down of the non - target structure during ablation . angioplasty balloon ( 510mm 2040mm ) interposition is rarely used due to the high risk of nerve damage during balloon manipulation . temperature monitoring is usually achieved directly through percutaneous deployment of thermocouples or fiber - optic sensors that can be easily combined with fluid dissection , especially when the latter alone is not deemed sufficient . 
in case of mrgfus ablation , non - invasive mrimediated temperature monitoring can be obtained . electrostimulation and somatosensory - evoked potentials allow for the functional monitoring of nerves during the ablation and are particularly useful since nerve roots are very sensitive outside the physiologic range ( 1045c ) [ 4650 ]  . 
usually , fluids ( including local anesthetic agents ) are injected within the sub - cutaneous tissue to increase its width ; additionally , sterile gloves filled with hot or cold saline can be transiently applied on the target area to mitigate skin temperature change during the ablation . results reported in patients affected by tumors that are commonly considered aggressive , such as lung cancer [ 51 ]  . 
accordingly , it is important to take into account each patients tumor biology when selecting candidates for curative ablation . a considerable number of papers provide evidence of ablation in achieving fast and effective pain relief in patients presenting with painful bm ( table3 )  . 
given the non - invasive profile of hifu , it is likely that it will obtain a prominent position in the future for the palliative management of bm . embolization indications the aim of trans - arterial embolization ( tae ) is to devascularize hyper - vascular bm and to preserve all non - target vessels . 
accordingly , tae should be as selective as possible . tae can be applied in case of hyper - vascular bm in order despite the relative few patients that can benefit from curative treatments , growing evidence is demonstrating the effectiveness of such therapeutic treatments in selected patients ( table2 )  . 
minimize the blood loss during subsequent surgery ; in this scenario , tae should be performed within 3days from surgery to reduce the risk of tumor revascularization [ 5255 ] ; table 2 results of curative ablation study histology sitea ablation modality mean tumor size ( cm ) no . 
 nsclc bone ca , rfa , 45 ( 76 ) 68% 1years 2.6% nr not reported , ca cryoablation , rfa radiofrequency ablation , mwa microwave ablation , mskmusculoskeletal , nsclcnon - small - cell lung carcinoma a study includes metastatic sites beyond bone and soft tissue ; only data related to bone and soft tissue metastases are reported [ 82 ] [ 83 ] [ 85 ] [ 51 ] mean 1 3 la radiologia medica ( 2019 ) 124 : 3449 table 3 results of palliative ablation study ablation modality no . 
permanent agents are most commonly used for preoperative tae and for palliative cases ; however , temporary agents such as gelatin sponge have also shown their efficacy before surgery [ 56 ]  . 
4 a large ( 13 cm ) painful right iliac metastasis from hepatocellular carcinoma in a 55 - year - old patient undergoing combined single - session embolization and cryoablation ; due to the metastasis , the patient was bedridden . 
 d , e cryoablation was accomplished by means of 10 cryoprobes producing a large iceball ( * ) , and with concomitant hydro - dissection to protect the femoral nerve ( white arrow )  . 
f 1 - month mri follow - up showed complete devascularization of the tumor ( * ) ; at the same time interval the patient was able to walk again 1 3 42 results arterio - venous shunts . 
lastly , particles are most commonly used for bone devascularization and the choice of particles diameter ( 401200 micron ) is dependent on vessel size and desired distal embolization . in a large series of 93 patients undergoing pre - surgical embolization of spinal tumors , the benefits of embolization have been demonstrated in terms of reduction of intra - operative blood loss , especially when hyper - vascular bm from renal cell carcinomas were treated , and extensive surgery ( corpectomy / vertebrectomy ) was performed [ 57 ]  . another series of 243 patients undergoing bm embolization reported > 50% and 97% reduction in pain and analgesic consumption , respectively . 
nevertheless , post - embolization syndrome , ischemic pain at the embolization site , paresthesia , skin breakdown , and sub - cutaneous necrosis were observed in 35% patients [ 58 ]  . preprocedure workup , clinical andtechnical requirements normal coagulation parameters , renal function and the absence of infection should be checked . 
a series of prognostic factors favouring curative treatments have been identified : oligometastatic / metachronous status ; the absence or limited cortical bone disruption ; limited bm size ( < 2cm ) ; long life expectancy ; and good patient performance status [ 59 ]  . 
patients should be offered such treatment in case of focal pain ( 4 / 10 on a 010 visual analogic scale over the 24h ) corresponding to a focal bm on cross - sectional imaging . 
 ideally , ablation should target the interface between the normal bone and the bm ; nevertheless , whenever possible , complete bm destruction should be achieved . tumor debulking can be achieved with ablation such as in the case of bm extending to surrounding soft tissues or spinal tumors growing quickly into the spinal canal . 
 nevertheless , it should be noted that local tumor control cannot be achieved if the bm has already invaded the anterior epidural space . despite the curative or palliative intent , when ablation and / or embolization are performed ( or following rt ) , bone strength is impaired ; and a substantial risk of secondary bif exists . 
accordingly , based on simple biomechanical consideration , interventional or surgical consolidation is mandatory to avoid a secondary bif , which usually occurs within the first few weeks even though cases of delayed bif have been reported [ 61 ]  . 
consolidation should ideally be performed during the same interventional session , or at the latest during the same hospital stay . followup afterinterventional treatments in the majority of cases , the main goal of percutaneous treatment is pain management [ 8 , 62 ]  . 
for this reason , many authors evaluate procedural success on the basis of clinical data by applying pain scales ( e.g. , vas scale , brief pain inventory ) or quality of life scores ( e.g. , mcgill quality of life questionnaire ) [ 39 , 40 , 6365 ]  . 
additionally , some authors also evaluate the consumption of analgesics including opioids [ 40 , 66 ] that are nevertheless limited by side effects such as constipation , sedation , and nausea . 
 clinical data are collected at least 1week after treatment ( to reduce the placebo effects or other confounding factors , such as the effects of analgesics administered during the procedure or pain related to the procedure itself ) , and thereafter , follow - up is scheduled at 1 , 3 and 6months according to the patients status and disease evolution . unless new symptoms occur , imaging follow - up is unnecessary in patients with diffuse metastatic disease who underwent palliative treatments [ 39 , 67 ]  . 
on other hand , oligometastatic patients treated with a curative intent should undergo periodic imaging follow - up to assess local tumor control ( i.e. , identification of residual viable or recurring tumor in the treated area )  . 
to avoid confounding variables , such as inflammation , the first followup study should be carried out 4 or even 812weeks after the treatment [ 40 , 59 , 70 ]  . 
finally , it is important to notice that recist criteria cannot be used in the setting of bm as recist has only been used for soft tissue metastases ; moreover , bm is often considered non - measurable [ 68 ]  . possible interactions withother treatments the application of interventional radiology for the management of bm is relatively recent . 
this may explain why no comparative studies are available between interventional treatments and rt , which still remains the widely accepted gold standard for the treatment of sres , particularly pain [ 71 ]  . 
in the few studies describing the combined use of rt and percutaneous treatments [ 63 , 66 , 72 ] , authors emphasized the achievement of better results in terms of pain relief when the combined approach was applied as compared to a single treatment modality , without significant increase in morbidity . 
the exact mechanisms through which the combined treatment relieves pain remain largely unknown , although it has been advocated that tumor microenvironment perturbation resulting from ablation may enhance the effect of rt [ 73 ]  . the development of immunotherapy has resulted in new and revolutionary advances . 
it has been postulated that this phenomenon is immune mediated and is mainly observed in patients undergoing rt 1 3 la radiologia medica ( 2019 ) 124 : 3449 fig . 
indeed , focal treatments such as rt may trigger and potentiate the effects of immunotherapeutic agents ; and it has been postulated that similar to rt , thermal ablation can also stimulate a systemic immune - mediated antitumor response [ 7578 ]  . medical therapy for bm disease relies on agents ( bisphosphonates or denosumab ) avoiding bone loss [ 3 , 7 ]  . 
to our knowledge , no side effects have been described in patients receiving such agents and interventional treatments . surgery can be the definitive solution in oligometastatic patients with slowly evolving disease and long life expectancy . 
however , there are different limitations to its use since surgical interventions carry a significant risk of infection and bleeding coupled to a long recovery time [ 67 , 71 ] ; as a result of which , a long delay to systemic therapies is expected . 
due to the complex clinical scenario regarding bm , we suggest that the most adapted therapeutic strategy should be chosen in consensus by a dedicated multidisciplinary tumor board including medical oncologists , radiation oncologists , surgeons and interventional radiologists . 
interventional treatments have several different advantages including the synergic effect with all the other non - interventional treatments , no need for significant interruption of systemic therapies , reduced morbidity and in - hospital stay , and patients fast recovery . 
due to technical and anatomic specifications , interventional treatments should be performed by trained interventional radiologists ( or by young interventional radiologists under the supervision of a senior )  . 
interventional procedures on bm are usually painful , and the interventional radiologist performing the procedure may not be able to provide alone the best intraoperative anesthesia and postoperative analgesia ; as a consequence , dedicated anesthesiology teams should be involved . 
 evaluated parameters were gender and age of the patients , size , side , homogeneity , morphology , radiodensity , mineralization , borders , relation to roots , affected tooth or teeth and location of the analyzed lesions . results of the 6340 assessed patients , 354 ( 5.6% ) harbored 362 lesions . 
radiopaque image , radicular location , round shape , homogeneous core and well - defined boundaries were the more frequent io features . conclusions our method allows to analyze io lesions with precise parameters . 
analysis of the results does not support the previously suggested theories to explain their origin , and these figures suggest that the so - called ios are developmental alterations of the bone . keywords idiopathic osteosclerosis bone lesions radiopaque osseous lesions developmental bone alterations introduction intrabony radiopaque lesions are common manifestations of neoplasia , sequelae of carious lesions , traumatic event , malignant tumors , metastasis , neoplastic and non - neoplastic entities or developmental alterations . 
many different names have been proposed for them : osteosclerosis , condensing osteitis , bone eburnation , bone scar , osteopetrotic scar , osteopetrosis , enostosis , sclerotic bone , socket sclerosis , bone whorl and dense bone island [ 124 ]  . 
other names were : focal sclerosing osteomyelitis , focal periapical osteopetrosis , focal osteosclerosis and idiopathic osteosclerosis * constantino ledesma - montes cledezma@unam.mx 1 clinical oral pathology laboratory , divisin de estudios de posgrado e investigacin , facultad de odontologa , universidad nacional autnoma de mxico , 04510mexico , d.f. , mexico 2 laboratory ofimmunology , divisin de estudios de posgrado e investigacin , facultad de odontologa , universidad nacional autnoma de mxico , 04510mexico , d.f. , mexico ( io )  . 
these synonyms were used for many years by researchers in different studies , and differences in interpretation , terminology , methodology , image features , analyzed populations and frequencies produced confusion in the literature [ 119 , 2124 ]  . in recent years , several studies on radiopaque lesions of the maxillofacial regions separated two distinct entities of different origin [ 1 , 2 , 59 , 13 , 15 , 18 , 21 , 22 ]  . 
the first is condensing osteitis , and it arises from an inflammatory process as sequelae of caries , pulpal necrosis , deep or large restorations or associated with teeth serving as abutments for fixed bridges or partial dentures . 
 a panoramic radiograph was made to all of them , and all radiographs were reviewed and discussed by two panelists ( cl - m and jchg ) , solving discrepancies by consensus and agreement . 
for stringent selection , we added parameters to those previously proposed [ 7 , 9 , 15 , 19 , 23 , 24 ] and excluded lesions presented with the following features : 1 . 
lesions associated with periodontal disease zones . in this study , we included those painless , radiopaque or mixed ( radiolucentradiopaque ) lesions , located in the tooth - bearing areas associated with vital , non - carious teeth or tooth . 
sometimes , teeth contained small restorations , and frequently , the lesions were seen separated from the root and they were not associated with inflammatory , infectious or traumatic episodes . la radiologia medica ( 2019 ) 124 : 2733 fig . 
the maxillary portion was superiorly limited by the floor of the maxillary sinus and floor of the nasal cavity ; the mandibular area was limited by the upper portion of the mandibular canal and a straight line connecting both mental foramina . 
these zones were limited by the left and right distal portions of the third molars . relation to roots and bone : for classification purposes , a modification of the geist and katz study ( 9 ) was used . 
the observed io decrease in frequency in older age groups was noticed in this study and other studies [ 1 , 2 , 4 , 5 , 8 , 9 , 11 , 24 ] , suggesting that this phenomenon may be related to the loss of bone in mature and old people . 
 as it was in other studies [ 1 , 2 , 4 , 5 , 8 , 9 , 11 , 24 ] , these findings strongly suggest that io lesions are developmental alterations or anatomic variants of the bone . in this study and previously reported studies , io lesions were more commonly found in the mandible than in the maxilla [ 113 , 1519 , 2124 ]  . 
relation to bone and roots varied widely from lesions entirely surrounded by bone , lesions in contact with the lamina dura or periodontal ligament and others entirely obscuring or covering part of the root . to date , origin of io lesions is a matter of debate . 
analyzing this theory , the resulting lesions are associated with dental trauma or more commonly to a long standing carious process and they fall in the condensing osteitis group of lesions . 
interestingly , there are reports on the microscopic features of histologically processed cases showing the presence of bone tissue and no traces of roots [ 9 , 10 ]  . 
additionally , data on the more common occurrence of io cases in the molar areas and the higher frequency of lesions without inflammatory , traumatic or other stimulatory effects do not support this proposal . 
our findings on location of the analyzed lesions suggest that ios surrounded by bone could arise from cells of osteoblastic lineage and those in contact with periodontal ligament could derive from both osteoblastic cells and noncommissioned periodontal cells . several authors suggested that ios are developmental alterations of the bone [ 1 , 4 , 5 , 8 , 9 , 11 , 12 , 24 ]  . 
the clinical care classification system [ 20 ] defines a developmental alteration as the change in or modification of age - specific normal growth standards and or developmental skills , and commonly , frequency of developmental alterations is different between the studied populations . 
in this study , we found that ios was detected in 5.6% of our patients , and in other reports [ 119 , 2124 ] io frequency varied widely in each of the studied communities . 
also , in this study , ios were common during the 2nd to 4th decades declining in older patients , their frequency also rose from 1st to 3rd decades and then decreased , and additionally , size increased in patients aged between 7 and 30years and then decreased . 
the higher io frequency in the young group compared with frequency in the old patient group , the increasing size of the radiopacities in patients up to 30years and its decrease in older patients support the possibility that io 1 3 32 la radiologia medica ( 2019 ) 124 : 2733 lesions develop and grow at early ages mainly . 
additionally , the sudden increase in io frequency from 0.8% in children to 24.3% in the 3rd decade supports this assertion , suggesting that io is more frequent during development and maturation ages . our study analyzed many io imagenologic features are poorly studied in the past . 
previous reports considered that round or oval shape is the form for io , and this study showed that a high percentage ( 20.2% ) of the studied lesions were of irregular , squared , elongated or triangular shapes . 
we found that most of them were well - defined lesions , but 21% showed ill - defined boundaries . technically , there are three entities characterized by osteosclerosis in the maxillofacial area . 
we consider that io lesions could be defined as asymptomatic , non - expansile , osteosclerotic , radiopaque sometimes mixed ( radiolucentradiopaque ) lesions , developing in the tooth - bearing area , that appear at any age , in both women and men , lacking any relationship with inflammatory , infectious or traumatic phenomena . 
io lesions should not be diagnosed when these radiopaque lesions are located in places with previous tooth extraction or surgical procedures , and they are lesions that cannot be diagnosed as any other known osteosclerotic entity . 
the second entity comprises a set of osteosclerotic lesions radiographically similar to those analyzed here , but they are observed outside the area considered in the study and they never were located in the tooth - bearing area . 
the third entity is known as condensing osteitis . in the past , it was proposed that cases resembling this lesion were related to traumatic occlusion [ 6 , 9 , 16 , 18 ]  . 
 during the study , we found few cases of radiopaque areas close to the tip of the root , associated with radicular resorption , and they were diagnosed as condensing osteitis and excluded from the study . 
these osteosclerotic lesions associated with excessive occlusal forces and root resorption do not fulfill the parameters mentioned here and should be considered examples of condensing osteitis . conclusions ios are not associated with inflammatory , traumatic or infectious stimulus . 
 the aim of this study is to investigate the causes and incidence of violence against radiographers in radiology departments of educational centers and hospitals . materials and methods in this descriptive - analytic study , violence incidence was investigated in all 121 radiographers working in radiology departments of educational centers of kermanshah in 2016 . 
data analysis was also performed using descriptive statistics and t test and chi - square tests by stata 11 software . results the results showed that 72.7% of radiographers had experienced violence in their work environment . 
the verbal violence against radiographers younger than 40 was significantly higher ( p = 0.04 ) than the age group above 40years . conclusion the incidence of verbal violence against radiographers in radiology departments is high which can be reduced by providing adequate human resource and equipment in radiology departments , re - training courses on the prevention and management of violent behavior and the suing the violent events against radiologists . keywords physical violence verbal violence radiologists of radiology department introduction any violent act whether physical or verbal against people at their workplace can be defined as violence in workplace [ 1 ]  . 
however , these acts can be also from other * sogand abbasi azizi kahkeshan1994@gmail.com 1 department ofradiology andnuclear medicine , school ofparamedicine , kermanshah university ofmedical sciences , kermanshah , iran 2 student research committee , kermanshah university ofmedical sciences , kermanshah , iran personnel in the medical staff [ 2 ]  . 
recent reports on violence in treatment departments showed that about 25% of violent incidences have occurred in this area and more than half of care centers staffs have the experience of at least one time of physical or verbal violence throughout their professional life . 
as a group of high - risk jobs , the probability of violent experience is 16 times higher for a care - health employee as compared with other services [ 5 ]  . 
occurrence of workplace violence can lead to anger , fear , frustration , humiliation , anxiety , stress , sense of disability , isolation or sometimes vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 1418 feeling of guilt or tendency to retaliate , change behavior or change of workplace [ 2 ]  . 
on a large group of italian radiologists to investigate the incidence of violent behavior , they found that despite the fact that violence against radiologists is relatively unknown , the phenomenon is significant ; and 20% of radiologists are victims of physical and verbal violence [ 2 ]  . 
a study in hong kong showed that 61% of radiographers experienced violence during past 3years , and 34% had more than 5 violent incidents , with verbal violence ( 97% ) mostly by the patients as the most common type . 
in the healthcare area , lack of policy on organizational reporting of perceived violence , lack of employees belief in the usefulness of reporting and their concern that violence and insults may be considered as evidence of their poor performance or ignorance are the most important barriers in reporting violence at work [ 11 ]  . 
considering the increasing prevalence of occupational violence in developing countries and the high rates of violence against the medical staff and its serious complications , it is necessary to carry out further studies in this field to find the ways to reduce the frequency of these incidents and their consequences . 
this information helps international organizations to propose specific legal systems for addressing and resolving violence at the workplace [ 1517 ]  . radiographers are often exposed to various forms of violence due to their direct communication with patients and their relatives and faced with many professional tensions , especially during radiography of emergency patients . 
since there has not been a study on the experiences of radiographers about occupational violence in iran , the purpose of this study is to investigate the incidence of verbal and physical violence from the patients and their relatives to the radiographers of educational centers of kermanshah university of medical science . materials andmethods the target community of this descriptive - analytical study includes all 121 radiographers of the radiology department of kermanshah university of medical sciences , kermanshah , iran , in 2017 , who had more than 1year of experience and were volunteers to participate in the study . 
the first part included the demographic data of radiographers such as age , sex , work experience , employment status and work shithe second part of the questionnaire included questions that assessed violence in terms of type , and cause of violence , as well as the response of radiographers to violence . 
the research staff referred to the radiology centers in different work shifts and explained the objectives of the study ; they distributed the anonymous questionnaire among the volunteer radiographers and then collected the completed ones . 
at the end , the collected data were analyzed by stata 11 software using descriptive statistics and inferential tests including chi - square with a significant level of p < 0.05. results in this study , all the 121 radiographers working in educational hospitals affiliated to kermanshah university of medical sciences completed the questionnaires . 
seventy - seven ( 63.6% ) of the radiographers were women and 52 of them ( 42.9% ) had below 5 - year work experience record ; and 49 radiographers ( 49.5% ) were officially registered ( table 1 )  . 
also , the findings indicated that the highest rate of violence was from accompaniments ( 73 , 64.4% ) with middle - aged age group ( 45 , 40.9% ) ( table2 )  . 
the distribution of violators in terms of age group showed that most of the violence actors were in the middle - age group . considering that each radiographers was allowed to choose several causes for physical and verbal violence , the results showed that overcrowding of patients in radiology department with the highest frequency ( 21.0% ) was the most important cause of violence against radiographers . 
the lack of available security facilities ( 12.6% ) and inadequate number of radiology personnel ( 11.7% ) are the other reasons for the emergence of violence ( table3 )  . 
similarly , considering that every radiographers was allowed to choose several options to respond to the violence , the most important table 1 frequency if violence against radiology department radiographers based on their demographic data variable group frequency number ( % ) verbal violence number ( % ) physical violence number ( % ) total number ( % ) test results experience employment status age group women > 40 510 1120 > 20 contractual contract resident official young middle - aged adults 77 ( 63.6 ) 22 ( 36.4 ) 89 ( 73.5 ) 32 ( 26.5 ) 52 ( 42.9 ) 15 ( 12.4 ) 34 ( 28.1 ) 20 ( 16.6 ) 7 ( 5.8 ) 18 ( 14.9 ) 47 ( 38.8 ) 49 ( 40.5 ) 17 ( 14 ) 48 . 
jiao and colleagues also reported that according to their study on occupational violence against nurses , 7.8% of nurses experienced physical violence and 24% had faced with verbal violence [ 17 ]  . 
it seems that educating radiographers to improve their communication skills and anger management the principles can reduce the prevalence of violence . the results of the present study showed that the verbal violence was higher toward radiographers younger than 40 . 
it seems that due to the lack of communication skills and the management of occupational violence during university studies , only the professional experience and social interactions of radiographers in the age group over 40years can lead to a significant reduction in the incidence of occupational violence against them . the results of present study showed that the most number of violence against radiographs was from the patient accompaniment . 
also , magnavita study found that patients were mostly committing violent events against radiographers ( 37.1% ) and their accompaniments and relatives ( 34.3% ) were in the next rank [ 2 ]  . 
the difference in the results seems to be due to cultural differences . the results of this study indicated that overcrowding of patients in radiology departments ( 33% ) , lack of available security facilities and shortage of personnel are the most important causes of violence against radiographers . 
showed that the long waiting for radiotherapy services , communication problems and dissatisfaction with the provided services by radiographers are the most important causes of violence [ 9 ]  . 
regarding the increasing number of patients in radiology centers , it seems that providing adequate equipment and human resources can reduce patients congestion and improve the level of patients satisfaction with the quality of radiology services . in the present study , the reaction of radiologists to the violence against them was first inviting the invader to calm down ( 21% ) ; then they asked for help from their colleagues ( 15% )  . 
since the degree of violence , the level of personnel experience , staff working hours and other influential factors can be effective in this response , the reaction of the photographers and other medical staff can be different . given the high rate of violence , more diagnostic errors are likely to occur . 
studies conducted in hong kong [ 9 ] , uk [ 19 ] , and egypt [ 20 , 21 ] show that increased violence reduces work performance and increases the number of diagnostic errors by radiologists and radiographers . 
it seems that annual education is effective in this regard and it is suggested that educational content should include evaluation and reduction of the underlying conditions of violence , violent behavior management , identification of security risks , reporting of violence and its documentation , methods of acquisition assistance insituations of violence , behavioral control , use of security equipment , etc . 
it is also necessary to address these trainings at universities and at radiologist and radiographer training units [ 1923 ]  . conclusion results of this study showed high prevalence of workplace violence against radiology department personnel in centers affiliated by kermanshah university of medical science . 
 1 3 18 la radiologia medica ( 2019 ) 124 : 1418 therefore , equipping the hospitals with adequate radiography facilities and human resources , implementing educational programs for the security staffs to enable them , holding educational courses about preventing and management of violence against radiographers as well as definite legal systems to sue the violence cases , paying serious attention to violence against the personnel and establishment of responsible centers to track the case reports of violence are necessary . compliance with ethical standards conflict of interest mohammad - rasoul tohidnia declares that he has no conflicts of interest . 
no limb amputation was witnessed . conclusions according to our experience , endovascular approach appeared as an effective treatment for popliteal artery aneurysms , as it appeared affected by a low rate of periand post - procedural complications . 
it could be proposed as treatment of choice in patients with high surgical risk . keywords popliteal artery aneurysm endovascular treatment stent graft introduction popliteal artery aneurysms ( paa ) are the most frequent peripheral aneurysms ; they could be associated with other large vessel aneurysms ; and they could be unilateral or bilateral [ 1 ]  . 
most of the time , these types of aneurysm are asymptomatic and are diagnosed by screening examinations because they present as a pulsatile mass of the popliteal fossa [ 2 ]  . 
if symptoms are present , they could vary between different degrees of ischemic symptoms of lower extremities [ 3 ] , such as claudication , distal ischemia due to embolization , or * giuseppe guzzardi guz@libero.it 1 scdu radiologia diagnostica ed interventistica , azienda ospedaliero - universitaria maggiore della carit , novara , italy 2 sc radiologia diagnostica ed interventistica , ospedale regionale u . 
di radiologia , azienda ospedaliera mater domini , catanzaro , italy 4 sc chirurgia vascolare , azienda ospedaliero - universitaria maggiore della carit , novara , italy acute limb ischemia due to aneurysm thrombosis [ 4 ] ; less frequently , they could lead to neurovascular compression [ 5 ] or they could rupture and lead to hemorrhage , usually confined in the popliteal fossa [ 6 ]  . to assess whether these aneurysms should be treated , if asymptomatic , diameter of the lesion is the most important characteristic that should be taken into consideration ; aneurysms 2cm are considered candidates for treatment [ 7 ]  . 
 symptomatic aneurysms should be treated regardless of their dimensions . until recently , surgery was considered as treatment of choice of this pathology , especially for large and symptomatic lesions , and it is performed with aneurysmectomy followed by surgical bypass . 
in the last 7years , we retrospectively analyzed 48 patients treated for paa , who were affected by severe systemic comorbidities and were therefore excluded from surgical repair . we divided patients in groups based on the length ( cut off : 70mm ) and diameter of the lesion ( cutoff : 25mm ) , on the number of runoff vessels ( 1 , 2 , or 3 ) , and on the patient symptoms ( symptomatic or asymptomatic ) to obtain homogeneous groups and to compare outcomes with anatomical and clinical feature of the aneurysm . symptomatic patients were defined as those with peripheral embolism , complete acute thrombosis , venous compression , or claudication due to the paa . 
inclusion criteria were technical feasibility ( proximal and distal landing zone of at least 2cm , no limits in length lesion ) and patency of at least one tibial vessel . exclusion criteria were age < 60 years old ; severe comorbidities ( asa > 3 ) ; contraindication to antiplatelet , anticoagulant , or thrombolytic therapy [ 10 , 11 ]  . 
patients younger than 60years old and in good clinical conditions were considered for open repair . pre - procedural planning was performed with angio - ct study in all patients ( lightspeedplus , ge , milwaukee , usa , and aquilion 64 , toshiba , tokyo , japan ) , and ct acquisition parameters were section thickness 0.625mm ; pitch 1 : 0.984 ; rotation time 0.7s ; kv 100 ; ma in between 250 and 600 depending on patient constitution . 
we injected 100ml of iodinated contrast medium ( iomeron , bracco , milano , it , 400mg / ml ) at a rate of 45ml / s , followed by a 30 - ml chaser bolus of saline solution at the same speed . post - processing mpr ( multiplanar reconstruction ) and mip ( maximum intensity projection ) were performed to assess morphological characteristics of the vessel and to choose the correct device to implant evaluating best vascular access . all procedures were performed in angiographic room , using a monoplane angiograph ( integris allura xper fd20 philips , eindhoven , the netherlands , and innova , ge , milwaukee , usa ) with local anesthesia . endovascular procedures were performed by three different operators , all having 15years of experience in angiographic procedures . la radiologia medica ( 2019 ) 124 : 7985 vascular access was performed by percutaneous anterograde common femoral artery puncture to avoid crossing the aortic carrefour ( often tortuous and calcific ) with wide and stiff devices ( from 8 to 11 french ) ; after the diagnostical angiography the aneurysm was crossed with a guidewire ( hydrophilic guidewire 0.035 , radiofocus , glidewire , terumo , tokyo , japan ) and a catheter ( 5f , cordis , miami , usa )  . 
all patients , in our case series , were treated with the same kind of endoprosthesis gore viabahn ( gore , flagstaff , az , usa ) which is characterized by a good elasticity and flexibility , allowing the positioning of these devices even in the intraarticular segment of the vessel . after graft positioning , we performed angioplasty with semi - compliant balloon at the proximal and distal end of the endoprosthesis to allow a better sealing of the aneurysmatic lesion . angiographic controls were performed after deployment and angioplasty of the stent graft both in leg extension and in knee flection to assess whether knee movements were causing graft compression , displacement , or rupture . during the procedure , we administered intravenously a weight - adjusted bolus of heparafter the procedure , all patients were treated with antiplatelet therapy with acetylsalicylic acid 100mg and clopidogrel 75mg for 1month and single antiplatelet therapy afterward . follow - up was performed with the clinical examination followed by duplex scan and / or cta at 1 , 3 , 6months after treatment and then yearly . we evaluated technical success defined as correct deployment of the graft with complete exclusion of the aneurys primary outcome of our study was the evidence of primary or secondary patency of the stent graft at follow - up ( secondary patency was defined as restored patency through the originally treated segment )  . 
we evaluated as secondary outcomes the incidence of infections of the graft and development of type i endoleak due to extension of the disease and / or migration of the stent graft that required a second intervention . evaluation of procedural costs was not performed , considering that main endpoints were clinical and the most literature regarding this subject takes into consideration mainly peripheral obstructive disease rather than peripheral aneurysmatic disease [ 12 ]  . statistical analysis data were collected and analyzed using spss 15.0 software ( spss inc , chicago , ill )  . 
patency was evaluated 1 3 la radiologia medica ( 2019 ) 124 : 7985 using kaplanmeier curves and log - rank tests with the related standard error ( se )  . table 2 procedural details results we treated 48 aneurysms , deploying a total of 79 endoprosthesis . 
mean aneurysm length was 76.3mm ( range 22200mm ) , and 24 / 48 ( 50% ) patients had an aneurysm whose length was > 70mm ; mean diameter of the lesion was 34.4mm ( range 2160mm ) , and in 27 / 48 ( 56% ) of cases the aneurysm was > 25mm of diameter . thirty - one patients ( 64.6% ) were asymptomatic , and the diagnosis was incidental during duplex scan or ct imaging performed for other reasons , while 17 ( 35.4% ) had symptoms at diagnosis ; in eight cases patients presented with mild claudication , seven had distal limb ischemia with rest pain , due to peripheral embolism from the aneurysm , and two presented symptoms due to neurovascular compression from the aneurysm . patients selected had at least one runoff vessel . 
more specifically , most of the enrolled patients had two runoff vessels ( 21 / 48 , 44% ) , seventeen ( 35% ) had three runoff vessels , while ten ( 21% ) had only one runoff vessel . all patients selected in our study were treated electively with percutaneous access and local anesthesia ( table2 )  . 
at 30 - day follow - up , we registered a total of five endograft thromboses that were treated with intra - arterial thrombolysis with complete recanalization of the prosthesis in three patients ; two patients also required angioplasty after locoregional fibrinolysis , to achieve a good stent graft patency . out of these patients , three had no other relapses and the endoprosthesis was still patent at long - term follow - up , while in two cases occurred a second graft thrombosis that required to perform a surgical bypass ( table3 )  . 
three patients presented a progression of the disease and required a reintervention to extend the coverage of the graft ; only one patient presented infection of the endoprosthesis 5months after the procedure and was treated by removing the stent and with surgical bypass . 
in our case series , we did not witness any case of type ii endoleak at shortand at long - term follow - up . in our study group , primary stent graft patency was 34 / 48 ( 70.8% ) while secondary patency was 43 / 48 ( 89.6% ) ; no limb amputation was reported . we did not find any statistically significant difference in long - term patency rate between patients with long and short lesions ( 70mm or > 70mm ) , with small or large 1 3 82 la radiologia medica ( 2019 ) 124 : 7985 fig . 
3 primary and secondary patency related to lesion length ( blue : 25mm , green : > 25mm ) [ 2 ] , it is associated with a high rate of limb amputation due to a decreased distal perfusion [ 3 ]  . studies have shown how surgical bypass patency is highly influenced by the clinical conditions of the patients . 
patients treated in elective settings showed a long - term patency of 80% while patients treated due to severe symptoms in emergency settings showed a long - term patency of only 60% [ 13 ]  . endovascular techniques for the treatment of aneurysms have developed significantly in the recent years , granting a good outcome in a high percentage of patients for both aortic and distal arteries aneurysms [ 14 , 15 ] ; therefore , endovascular treatment has been chosen in a higher percentage of patients showing suitable anatomical conditions . our exclusion criteria according to patient age were inspired by the literature [ 10 , 11 ] ; both antonello etal . 
we stretched that limit to 60years old , for selected patients , to allow surgical repair in a wider 1 3 84 la radiologia medica ( 2019 ) 124 : 7985 fig . 
4 primary and secondary patency related to number of runoff vessels ( blue : one vessel , green : two vessels , gray : three vessels ) range of patients , reserving endovascular repair to older and high - risk ones ( asa > 3 )  . main challenge of endovascular treatment of aneurysms of the popliteal artery is represented by the fact that this anatomical district is highly mobile and flexible ; therefore , devices implanted must be able to sustain a high compressive force during limb flexion , to grant a valid distal perfusion and avoid stent thrombosis [ 17 , 18 ]  . endovascular treatment of popliteal aneurysms has been performed with different techniques such as multilayer and covered stents . 
few cases of multilayer stents treatment of aneurysms have been reported in the literature [ 16 ] , which showed a variable rate of aneurysms occlusion in association with a moderate percentage of stent thrombosis ; therefore , their use has decreased over the last years . 
 [ 21 ] reported a large case series confronting open surgical repair with endovascular repair in patients with popliteal aneurysms comparing a total of 178 patients treated with surgical technique and 134 with endovascular approach ; patients of the surgical group were more frequently symptomatic , with distal limb ischemia and a decreased runoff score . 
further studies will be needed to correctly evaluate whether patients and aneurysms characteristics could predict the efficacy of the treatment , and this will require a larger number of treated cases and longer follow - up and would allow a better patient selection . compliance with ethical standards conflict of interest on behalf of all authors , the corresponding author states that there is no conflict of interest . ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . informed consent informed consent was obtained from all individual participants included in the study . la radiologia medica ( 2019 ) 124 : 5864 radiotherapy inoperable earlystage primary andearly recurrent nonsmall cell lung cancer : outcomes ofamonoinstitutional experience using amoderate hypofractionated schedule mauriziovaleriani1 mattiafalchettoosti1 lucamarinelli1 chiarareverberi1 vitalianadesanctis1 davidemollo1 lucanicosia1 received : 26 june 2018 / accepted : 20 august 2018 / published online : 3 september 2018 italian society of medical radiology 2018 abstract background patients with medically inoperable early - stage non - small cell lung cancer ( nsclc ) may beneficiate of a hypofractionated radiation therapy in order to intensificate the treatment and to reduce the number of hospital access . methods from 2007 to 2015 , 27 patients with early - stage primary or limited loco - regional recurrent ( t2a > 4cm , t2b n0 or t12 n1m0 ) nsclc were treated . 
survival outcomes were statistically favourable in patients with partial or complete response with respect to patients with stable or progressive disease , whereas stage ( n0 vs n1 ) and primary or relapse / recurrent disease not . 
standard treatment for early - stage non - small cell lung cancer ( nsclc ) is surgical resection [ 3 ] with a 5 - year overall survival ( os ) ranging between 55 and 72% in stage i disease and between 29 and 51% [ 4 ] in stage ii disease . 
t1t2a n0 patients can be treated with stereotactic radiotherapy [ 57 ] , but in t2b n0 or t12 n1 patients , stereotactic radiotherapy may be infeasible for the volume and / or for the site of the disease . 
several studies have evaluated the vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 5864 role of moderate hypofractionation [ 12 , 13 ] in order to intensify the treatment and limiting tumour repopulation [ 14 , 15 ] by reducing the total treatment time . 
based on these factors in 2007 , we started to treat early - stage primary or limited recurrent nsclc patients with a moderate hypofractionated schedule . methods andmaterials patients twenty - seven patients with early - stage primary or limited loco - regional recurrent ( t2a > 4cm , t2b n0 or t12 n1m0 ) nsclc were treated in our institution from 2007 to 2015 . 
free breathing basal ct images were matched with the maximum intensity projection ( mip ) series , with diagnostic ct and with pet - tc using automatic matching systethe gross tumour volume 1 ( gtv1 ) included the pulmonary lesion ; gtv2 included lymph nodes with a short axis diameter 1 cm and / or positive nodes at pet - tc . 
all patients were treated with 3d - crt using multiple coplanar and non - coplanar fields and received 60gy in 20 fractions of 3gy , 5 times per week delivered by a linear accelerator ( 6 - mv photon beams )  . 
time - adjusted biological effective dose ( bed ) was calculated using the following formula : bed = nd ( 1 + d / [ a / b ] ) ln2 ( t tk ) [ 16 ] , where nd is the total dose , d the dose per fraction , t the overall treatment time expressed in days , and tk the proliferation start time ( 28days )  . 
a total body contrast - enhanced ct was performed at 1month after rt completion and every 6months afterwards , a pet - tc was performed at 3months after rt and in case of suspected 1 3 60 la radiologia medica ( 2019 ) 124 : 5864 fig . 
response rates were determined using response evaluation criteria in solid tumours ( recist version 1.1 ) [ 19 ]  . statistical analysis was performed using spss , version 24.0 , software ( spss , inc , chicago , il )  . 
progression - free survival ( pfs ) was defined as the time to local / systemic progression or death and loco - regional pfs ( lr - pfs ) as the time to infield or outfield local and regional recurrence / progression . 
twelve patients achieved a complete response ( cr ) ( 44.4% ) and 8 a partial response ( pr ) ( 29.6% ) with a tumour response rate of 74% . 
also pfs and mfs were statistically better in patients with partial or complete response ( median pfs 47months , median mfs 62months ) with respect to patients with stable or progressive disease ( median pfs 3months , median mfs 10months ) with 1 3 la radiologia medica ( 2019 ) 124 : 5864 css , lr - pfs , pfs and mfs . 
thus , 13 patients ( 48.1% ) presented metastasis : 4 in controlateral lung , 2 distant nodes , 3 bone , 2 brain , 1 brain plus bone and 1 adreanal . stage ( n0 vs n1 ) and primary or relapse / recurrent diseases were not statistically significant in terms of os , patients with primary or recurrent early - stage nsclc may be managed with surgery , but for medically inoperable cases or patients who refused surgery , radiotherapy may be a valid option . stereotactic radiotherapy represents the standard treatment for low - volume n0 lesions . 
in patients not suitable to stereotactic approach for high - volume n0 or n1 disease , an intensified treatment may be evaluated in order to ameliorate results of standard dose and fractionation radiation therapy . 
in our case 60gy in 4weeks presented a bed10 [ 16 ] of 79.4gy , whereas , for example , 70gy in 35 fractions over 7weeks has a bed10 normalized with time factor of 70.9gy. therefore , frequently early - stage nsclc patients were referred for radiotherapy in presence of comorbidity , which may affect survival [ 20 , 21 ] , or for old age . 
in our cohort median age was 76years and there were some cases ( 5 / 27 patients ) that died of causes not related to the disease , but after a median of 42months . 
patients with complete or partial response presented a statistically significant better os , css , lr - pfs , pfs and mfs with respect to patients with stable or progressive disease . 
table3 summarizes the most important studies on moderate hypofractionation in nsclc . these results showed that moderate hypofractionation is well tolerated with a very low rate of high - grade toxicity . 
moreover , the results , in terms of survival , obtained in our study are promising and compared favourably with the results of the other studies despite that in some of these were enrolled patients with t1 or t2a n0 disease that currently were treated with stereotactic radiotherapy [ 57 ]  . 
a greater number of patients and a longer follow - up are necessary to confirm the results obtained with our treatment . compliance with ethical standards conflict of interest all authors declare that they have no conflict of interest . ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . informed consent informed consent was obtained from all individual participants included in the study . la radiologia medica ( 2019 ) 124 : 6578 radiotherapy late toxicity ofimageguided hypofractionated radiotherapy forprostate : nonrandomized comparison withconventional fractionation barbaraalicjajereczekfossa1 , 2 alessiasurgo2 mariannaalessandragerardi2 dariozerini2 giuliamarvaso2 deliaciardo2 stefaniavolpe1 , 2 damarispatriciarojas1 , 2 giuliariva1 , 2 ombrettaalessandro1 , 2 samanthadicuonzo2 giuseppefanetti1 , 2 paolaromanelli2 annastarzyska4 federicacattani5 raffaellacambria5 cristianafodor2 cristinagaribaldi5 chiararoman5 , 6 ottaviodecobelli1 , 7 robertoorecchia8 patrickmaisonneuve3 andreamaucieri2 received : 6 march 2018 / accepted : 28 august 2018 / published online : 15 september 2018 italian society of medical radiology 2018 abstract purpose to evaluate the incidence and predictors for late toxicity and tumor outcome after hypofractionated radiotherapy using three different image - guided radiotherapy ( igrt ) systems ( hypo - igrt ) compared with conventional fractionation without image guidance ( non - igrt )  . methods and materials we compared the late rectal and urinary toxicity and outcome in 179 prostate cancer patients treated with hypo - igrt ( 70.2gy / 26 fractions ) and 174 non - igrt patients ( 80gy / 40 fractions )  . 
5and 8 - year recurrence - free survival ( rfs ) and overall survival ( os ) were analyzed . results mean follow - up was 81months for hypo - igrt and 90months for non - igrt group . 
mainly mild late toxicity was observed : hypo - igrt group experienced 65 rectal ( 30.9% g1 / g2 ; 6.3% g3 / g4 ) and 105 urinary events ( 56% g1 / g2 ; 4% g3 / g4 )  . 
multivariate analysis showed that hypo - igrt is a predictor for late genitourinary toxicity , whereas hypo - igrt , acute urinary toxicity and androgen deprivation therapy are predictors for late rectal toxicity . 
advanced t stage and higher gleason score ( gs ) were correlated with worse rfs . conclusions a small increase in mild late toxicity , but not statistically significant increase in severe late toxicity in the hypoigrt group when compared with conventional non - igrt group was observed . 
prostate cancer radiobiological behavior ( slow proliferation rate with high sensitivity to high dose per fraction ) [ 6 , 7 ] , technological advantages in treatment planning and high precision in dose delivery and accuracy of target definition ( image - guided vol . : ( 0123456789 ) 1 3 66 la radiologia medica ( 2019 ) 124 : 6578 radiotherapy , igrt ) allowed introduction of hypofractionated radiotherapy schedules [ 1 ]  . italy ( eio no 79 )  . 
the inclusion criteria and techniques were described in our previous paper [ 11 ]  . since 2003 , we used in our department 3 - dimensional conformal ( 3d - crt ) 2 - dynamic arc radiotherapy to the dose of 80gy / 40 fractions for prostate cancer treatment [ 5 , 8 ]  . 
 in 2006 , three igrt modalities were installed : ultrasoundbased b - mode acquisition and targeting system ( bat ) , stereo x - ray imaging system ( exactrac ) and cone beam computed tomography ( ct ) ( on board imager ct )  . 
the higher incidence of the acute rectal reaction was not significant in multivariate analysis . in the present report , we analyzed the late toxicity registered in the same two - patient cohorts : this is a follow - up study of the previous publication in the urologic oncology [ 11 ]  . the aim of this report is to evaluate the incidence and predictors for late toxicity of the hypo - igrt and secondarily to analyze tumor outcome data . despite methodological limitations ( non - randomized comparison ) , such analysis might help clinicians in discussing with a patient the treatment choice and possible treatment - related toxicity . 
moreover , it creates a hypothesis for further prospective investigation . assessment ofthetoxicity andtumor outcome data after treatment , a radiation oncologist or urologist clinically evaluated patients every 6months for the first year and once a year for 10years . 
late rectal and urinary toxicities were evaluated in patients with follow - up > 3months according to the radiation therapy oncology group / european organization for research and treatment of cancer ( rtog / eortc ) criteria [ 12 ]  . biochemical failure was defined according to american society for radiation oncology ( astro ) phoenix 2005 ( nadir plus 2ng / ml at call ) [ 13 ]  . 
prostate - specific antigen ( psa ) test was performed every 3months for the first 2years after radiotherapy , every 6months for 5years and annually thereafter . clinical failure included local recurrence , regional recurrence and distant metastases : magnetic resonance imaging ( mri ) , ct , choline c - 11 positron emission tomography / ct scan ( choline pet - ct ) or prostate biopsy was used to study patients with psa increase or clinical suspect . statistical analysis in the same way as our previous study , we evaluated the occurrence of two separate radiotherapy - related toxicities ( rectal and urinary ) within the whole population ( hypoigrt and non - igrt groups ) and in hypo - igrt cohort alone . 
univariate and multivariate cox proportional hazards regression models were used to assess the impact of various patient - , tumorand treatment - related factors on late rectal and urinary toxicity . 
the actuarial cumulative incidence rates of rectal and urinary toxicities and the actuarial rfs and overall survival ( os ) rates were estimated for both groups at 5 and 8years and were calculated using kaplanmeier method . 
shortly , patients treated with hypo - igrt were significantly older , had less advanced disease and received less androgen deprivation therapy ( adt ) or previous transuretheral prostate resection ( turp )  . 
therefore , univariate and multivariate analysis ( tables3 , 4 and 5 ) was built on 349 patients including 175 patients treated with hypo - igrt . a predominance of limited g1 events was observed in both groups ( table2 )  . 
there were few g3 and g4 events in both groups ( no g5 event )  . considering high - grade rectal toxicity , all g3 events ( 11 in hypo - igrt group and 5 in non - igrt group ) were proctopathy or persistent rectal bleeding ( requiring transfusion only in two cases ) resolved with coagulation laser therapy . 
as far as high - grade urinary toxicity is concerned , in hypo - igrt group , six g3 urinary events occurred , namely severe dysuria , nicturia and frequent macroscopic hematuria requiring interruption of anticoagulants and / or cystostopic revision in three cases . 
at the last follow - up , 137 hypo - igrt patients had no evidence of disease ( ned ) and 11 were alive with disease ( awd ) and five patients died for prostate cancer ( dod )  . 
in multivariate analysis , risk of recurrence was higher in patients with t3 disease ( hr = 3.00 ; 95% ci 1.426.38 ; p = 0.004 ) and gs > 6mg / ml ( hr = 2.74 ; 95% ci 1.514.96 ; p = 0.0009 ) , while risk of death was significantly higher among patients who received neoadjuvant plus concomitant adt ( hr = 3.18 ; 95% ci 1.546.59 ; p = 0.002 ) ( table6 )  . discussion our study , including 353 patients ( divided into two cohorts of 179 and 174 patients ) , showed a small increase in the mild late urinary and rectal toxicity among prostate cancer patients treated with hypo - igrt schedule when compared to patients treated with conventionally fractionated radiotherapy without image guidance . 
 the tumor outcome univariate analysis showed a gain in 8 - year rfs in hypo - igrt group ( p = 0.0005 ) , although the os was comparable between the two groups . 
 the challenges for numerous non - igrt patients include the follow - up by the urologists ( radiotherapy toxicity might have been underestimated ) and pre - treatment staging based in many cases on the clinical evaluation only . 
the most recently treated hypo - igrt patients were followed mainly by radiation oncologists , and most probably any late event was registered . several recent clinical studies investigated hypofractionation in prostate cancer [ 1825 ] comparing toxicity and efficacy of the short schedules with the conventional fractionation radiotherapy . 
 in both cases , high precision treatment planning and dose delivery are essential for sparing adjacent normal tissue and to decrease toxicity . in this scenario , our analysis , as well as the current evidence , showed that late urinary and rectal toxicity might be increased in the moderate hypo - igrt group [ 2124 ]  . 
 [ 22 ] in a recent multicentric randomized study similarly observed an increased rectal and urinary toxicity comparing two radiotherapy fractionation schedules : 73.8gy / 41 fractions versus 70gy / 28 fractions . 
albeit the power of the randomized character of the study , they compare 3d / intensity modulated radiotherapy ( imrt ) hypofractionated and 3d / imrt conventional treatment without specification in imaging guidance . 
perhaps the selection of a unique 3d - technique and the advantage of igrt might have influenced the recurrence rate , even though the multivariate analysis connected igrt group with an increased urinary toxicity . 
 [ 20 ] randomized trial revealed no difference in late adverse reactions between hypofractionation and conventional fractionation 3d - treatment , but the authors underlined that the incidence of complications increased with time . 
in our series , the incidence of rectal and urinary events increased 2436months after treatment : the rectal toxicity remained almost constant with no great difference between the two groups , but urinary toxicity , especially in the hypo - igrt group , continues to rise . 
 indeed , the lack of significant correlation between acute and late urinary toxicities observed in our study reflects complex mechanisms involved in urinary tract functioning after radiotherapy . interestingly , use of exactrac igrt system was weakly correlated with higher rectal toxicity in the multivariate analysis . 
 [ 21 ] in a mixed risk prostate cancer population showed that late rectal and urinary toxicity was not considerably different , but they analyzed the important correlation with the urinary toxicity > g2 and compromised baseline dysfunction . 
a sub - analysis of the main severe rectal toxicity demonstrated an increase in g3 or g4 toxicity when patients underwent previous turp or used anticoagulants for cardiac dysfunctions . no difference in os in both groups was found in line with all recent evidences [ 1825 ]  . at the same time as well as imrt , stereotactic body radiotherapy ( sbrt ) for organ - confined or locally advanced prostate cancer is becoming almost standard in our clinical practice . 
the advances in technologies and the improvements in detection and characterization of prostate cancer lesions ( as the multiparametric prostate mri ) have led to increase the use of extreme hypofractionated schedules . 
furthermore , future studies on the dosimetric , clinical and biological aspects ( improvement in planning and dose distribution with use of mri - based planning , prevention of late radiotherapyinduced injury or identification of patients at risk of toxicity ) are warranted in order to improve the therapeutic index of hypofractionation . conclusions a small increase in mild late urinary and rectal toxicity in the hypo - igrt group when compared with conventional non - igrt treatment was observed with no statistically significant difference in the severe late toxicity between the two cohorts . 
no major predictor for late toxicity has been found apart from previous acute rectal toxicity and of borderline significance with the use of exactrac systethe study confirms the hypothesis that igrt allows for safe moderate hypofractionation , offering a shorter overall treatment time , an impact in term of prostate cancer rfs and providing potentially more economic health care . 
although in the last decade anthracycline - based chemotherapy single agent or in combinations has been able to improve clinical benefits , prognosis is still poor and stss represent an important unmet medical need . 
the following attempts to provide a review of literature focusing on the available data concerning novel treatments and future prospective for the management of metastatic stss . keywords soft tissue sarcomas immune - checkpoint inhibitors pembrolizumab eribulin olaratumab targeted therapy introduction soft tissue sarcomas ( stss ) represent a rare and heterogeneous group of solid tumours derived from mesenchymal progenitors and account for 1% of all adult malignancies [ 1 ]  . 
an observational study of the american college of surgeons showed that among a cohort of 4550 patients suffering from sarcomas , 46% originate from thigh , buttock and groin , 13% by * giuseppe badalamenti giuseppe.badalamenti@unipa.it 1 section ofmedical oncology , department ofsurgical , oncological , andoral sciences , university ofpalermo , palermo , italy 2 academic unit ofmedical oncology , institute forcancer treatment andresearch , policlinico hospital san martino , genoa , italy 3 department ofinternal medicine , school ofmedicine , university ofgenoa , genoa , italy upper extremities , 18% torso , 13% retroperitoneum and 9% head and neck [ 2 ]  . as classified by the world health organization ( who ) , the group of stss comprise more than 100 different histologies according to the presumptive tissue in origin [ 3 ]  . 
after the development of distant metastasis the median overall survival ( os ) is 1219months , and almost 20% of patients are still alive at 3years [ 4 ]  . 
for patients with a good performance status and sts histology that is known to have sensitivity to anthracyclines ( liposarcoma , leiomyosarcoma , synovial sarcoma , pleomorphic or undifferentiated sarcoma , malignant nerve sheath tumour , angiosarcoma ) , anthracyclinebased chemotherapy represents the standard of care for first - line setting [ 5 ]  . 
for patients ineligible to anthracycline vol . : ( 0123456789 ) 1 3 260 la radiologia medica ( 2019 ) 124 : 259265 combination due to a poor performance status or extensive comorbidity , pegylated liposomal doxorubicin , gemcitabine alone or in combination and dacarbazine are reasonable options [ 68 ]  . 
 weekly paclitaxel seems to be useful for advanced angiosarcomas [ 9 ] ; synovial sarcomas are sensitive to alkylating agents as ifosfamide [ 10 ] ; sunitinib appears to be active in patients with solitary fibrous tumour and alveolar sarcoma , clear cell sarcoma and extra - skeletal myxoid chondrosarcoma [ 1113 ] ; gemcitabine plus docetaxel regimen is active on uterine leiomyosarcomas [ 14 ]  . 
 pazopanib might be considered for stss other than liposarcomas for patients progressing on first - line chemotherapy ( including anthracyclines ) [ 16 ]  . although these treatments or chemotherapy combinations have been able to improve clinical benefits , prognosis is still poor and stss represent an important unmet medical need . continuous advances in cancer genetics and genomics have contributed to change management paradigms of stss as it occurred for other solid tumours . 
several treatments have been recently developed with the specific aim of targeting different cell pathways and immune - checkpoints that have been recognized to drive tumour progression ( table1 )  . 
 the following attempts to provide a review of literature focusing on the available data concerning novel treatments and future prospective for the management of metastatic stss . methods a literature search using pubmed was carried out with no date restriction up to november 2017 . 
a computerized search of the abstracts reported at asco and esmo library , and the clinical trial database on the website caltr ial.gov was performed to identify relevant unpublished studies and ongoing trials . 
 finally , a crosscheck references from review articles and relevant studies on the same topic were performed to confirm retrieval of all pertinent trials . eligible studies had to fulfil the following criteria : randomized prospective phase iiiii trial assessing new or recently approved treatments for patients with metastatic stss . table 1 main characteristics of clinical trials carried out on metastatic sts patients clinical trial drug histological type study design primary endpoint n . 
no language restriction was applied . olaratumab platelet - derived growth factor ( pdgf ) and pdgf receptor ( pdgfr ) are key regulators of oncogenesis process in many solid tumours , including mesenchymal stem cell differentiation , angiogenesis and tumour growth [ 17 ]  . 
olaratumab alone or in combination with doxorubicin showed antitumor activities in human sarcoma xenograft models [ 20 ]  . basing on these data and rationale , an open - label phase ib / ii trial randomized 133 patients with histologically confirmed stss diagnosis to receive olaratumab 15mg / kg on day 1 and 8 plus doxorubicin 75mg / m2 day 1 three - weekly or doxorubicin alone [ 21 ]  . 
this achievement was consistent across the subgroup analysis including pdgfr status , histological subtypes ( leiomyosarcoma versus nonleiomyosarcoma ) and number of previous treatments ( 0 versus 1 )  . 
infusion - related reaction ( 3% ) was the main cause leading to olaratumab discontinuation , whereas 5% of ejection - fraction decrease was the most common reason of discontinuation in doxorubicin group . 
the most common adverse events in the combination arm include neutropenia , nausea , fatigue , vomiting and mucositis [ 21 ]  . despite clinical trial limitation due to the small sample size and heterogeneity of stss histology , this finding is particularly notable given the little progress in improvement of median os achieved for patients suffering from metastatic stss . 
however , this small benefit was achieved at expense of higher toxicities without any improvement in median os ( hr 0.83 , p = 0.76 ) [ 22 ]  . 
os in whole population and in patients with leiomyosarcoma are the two primary objectives of the study . a phase ib trial ( nct02783599 ) is recruiting potentially 40 patients to receive olaratumab 20mg / m2 alone for day 1 and 8 of the first cycle , then olaratumab 20mg / m2 in combination with doxorubicin 75mg / m2 for the second cycle and then olaratumab 15mg / m2 on the same schedule alongside doxorubicin for the third cycle . 
the primary endpoint was to molecularly characterize the circulating tumour cell preand post - olaratumab , and to analyze pdgfr , pdgfr , and pdgf ligand changes . a phase ib / ii ( nct02659020 ) trial is enrolling patients with metastatic stss to assess safety , activity and efficacy of olaratumab in combination with gemcitabine and docetaxel . 
os is the primary endpoint . an open - label phase i trial ( nct03126591 ) is recruiting patients with metastatic stss who has progressed to standard treatment to receive olaratumab plus pembrolizumab . 
this finding may be partially explained by the occurrence of activating mutation in the gene encoding the pdgfr [ 24 ]  . most gists harbouring pdgfr are primarily resistant to standard therapy due to d842v mutation that leads tumours not to be inhibited by approved treatments [ 25 ]  . 
 given the benefits achieved by olaratumab targeting pdgfr in preclinical models [ 20 ] , a phase ii trial evaluated tumour response to olaratumab in 30 previously treated 1 3 262 la radiologia medica ( 2019 ) 124 : 259265 metastatic gists into two cohorts with or without pdgfr mutations , respectively [ 26 ]  . 
despite the limitation of this trial due to small sample size and inter - study heterogeneity to draw a definitive conclusion , these data provide the rationale for further study of pdgfr - mutant gist in larger study [ 26 ]  . eribulin eribulin , originally isolated from the marine sponge halichondria okadai , is a structurally modified analogue of halichondrin b . 
moreover , eribulin seems also to inhibit wnt / - catenin signalling , alter tumour vascularization resulting in higher perfusion and drug delivery , and reverse the epithelial to mesenchymal transition [ 28 , 29 ]  . 
given eribulins antitumor activity in preclinical xenograft models of leiomyosarcomas and fibrosarcomas , a non - randomized phase ii ( study 17 ) trial assessed the activity and safety of eribulin 1.4mg / m2 administered intravenously on days 1 and 8 every 3weeks in four independent strata of 128 patients with mesenchymal tumours ( leiomyosarcoma , liposarcoma , synovial sarcoma and other defined sts ) [ 31 ]  . 
activity was demonstrated in patients with liposarcoma ( 46.9% pfs at 12weeks , 95% ci 29.165.3% ) and leiomyosarcoma ( 31.6% pfs at 12weeks , 95% ci 17.648.7% ) [ 31 ]  . an open - label phase iii trial ( study 309 ) randomly assigned 458 patients with metastatic intermediate or high - grade liposarcoma and leiomyosarcoma previously progressed on anthracycline - based chemotherapy to receive eribulin 1.4mg / m2 day 1 and 8 or dacarbazine 8501200mg / m2 every 21days until disease progression or unacceptable toxicities [ 32 ]  . 
most common adverse events with eribulin included neutropenia ( 43 versus 24% ) , pyrexia ( 28 versus 14% ) , alopecia ( 35 versus 3% ) and peripheral sensory neuropathy [ 32 ]  . basing on these results , eribulin has been approved in the usa and europe for treatment of metastatic liposarcoma for patients who received prior anthracycline - based chemotherapy . an ongoing single - arm ( nct03331250 ) phase ii trial aims to assess the activity and effectiveness of eribulin 1.4mg / m2 on day 1 and 8 administered intravenously threeweekly in patients with unresectable or metastatic angiosarcoma and hemangioendothelioma treated with at least one prior systemic treatment . 
 primary completion date is estimated for may 31 , 2021 . immunecheckpoint inhibitors although the approval in the last decade of several drugs for the treatment of metastatic stss [ 15 , 16 ] , these therapies did not achieve a substantial cure rate , leading to the development of new agents . response to conventional radiotherapy or chemotherapy is dependent on histology because some subtypes are chemo - resistant . cancer immunotherapy is gradually taking on a key role in the management of metastatic solid tumours , preventing the development of resistant clones to traditional chemotherapy and fostering tumour recognition by the immune system [ 34 ]  . 
historically , studies using cytokines or immune adjuvants provided small benefits for patients with metastatic stss [ 35 , 36 ]  . in a randomized phase iii trial , immunotherapy with adjuvant mifamurtide , a nonspecific immune stimulator , improves os in patients with osteosarcoma [ 37 ]  . 
recently , the appeal for the improved clinical benefits in several solid tumours due to immune - checkpoint inhibitors leads to assess the safety and activity of these agents in stss . a two - cohort , single - arm , open - label phase ii trial ( sarc028 ) enrolled 84 patients with metastatic or unresectable stss ( leiomyosarcoma , poorly differentiated or dedifferentiated liposarcoma , ewings sarcoma , synovial sarcoma , undifferentiated pleomorphic osteosarcoma , dedifferentiated or mesenchymal chondrosarcoma ) previously progressed up to three lines of chemotherapy to receive pembrolizumab 200mg intravenously every 3weeks until 1 3 la radiologia medica ( 2019 ) 124 : 259265 progression or unacceptable toxicities [ 38 ]  . 
almost 18% of patients with stss had a clinically meaningful objective response , including 40% with undifferentiated pleomorphic sarcoma , 20% with liposarcoma , 10% with synovial sarcoma , and 5% with osteosarcoma . 
no patients with leiomyosarcoma had an objective response . this result is consistent with data from a phase ii trial on nivolumab in leiomyosarcomas that was stopped early because of futility , suggesting a detrimental effect of antipd1 single agent in this subset of patients [ 39 ]  . 
although the mechanism of immune resistance related to leiomyosarcoma histology is still unclear , pten loss and pi3k pathway may play a key role in this process [ 40 ]  . 
 most common adverse events were anaemia , decreased lymphocyte count and platelet count in the bone sarcoma group , whereas anaemia decreased lymphocyte count and prolonged activated partial thromboplastin time in the stss group . in an open - label multicentre phase ii trial , 85 patients with metastatic stss or osteosarcoma previously treated with standard chemotherapies were randomized to receive the anti - pd1 monoclonal antibody nivolumab with or without the anti - ctla4 ipilimumab [ 41 ]  . 
many ongoing trials are still recruiting patients with metastatic stss to assess the activity and efficacy of immune - checkpoint inhibitors in metastatic settings in combination with chemotherapy or neoadjuvant setting with radiotherapy ( nct03092323 , nct03092323 , nct03307616 )  . 
briefly , several ongoing phase i / ii clinical trials are exploring the activity of pembrolizumab in combination with doxorubicin ( nct02888665 , nct03056001 ) , axitinib ( nct02636725 ) , olaratumab ( nct03126591 )  . 
similarly , several ongoing phase i / ii trials are assessing safety and tolerability of nivolumab single agent ( nct03316274 ) or in combination with pazopanib ( nct03149120 ) , ipilimumab ( nct03219671 , nct02982486 , nct02500797 , nct02304458 , nct02428192 )  . conclusion histology - driven approach still remains the mainstay for the treatment of advanced unresectable or metastatic stss . 
although the utmost efforts in the development and quick approval of novel active and practice changing drugs [ 15 , 16 , 21 , 32 ] , prognosis is still poor [ 4 ]  . 
however , standard morphological sequences are often not sufficient to characterize the exact nature of the lesion , addressing the patient to an invasive bioptic examination for the definitive diagnosis . 
the recent technological advances with the development of functional mri modalities such as diffusion - weighted imaging , dynamic contrast - enhanced perfusion imaging , magnetic resonance spectroscopy , and diffusion tensor imaging with tractography have implemented the multiparametricity of mr to evaluate in a noninvasive manner the biochemical , structural , and metabolic features of tumor tissues . 
the purpose of this article is to review the state of the art of these advanced mri techniques , with focus on their technique and clinical application . keywords mri soft tissue tumors dwi spectroscopy dti introduction soft tissue tumors ( stt ) that include a large variety of benign and malignant lesions are of a diverse histological nature . 
incidence increases with age and is higher in males : benign lesions have an incidence of approximately 300 / 100 , 000 , while malignant ones ( that account for 1% of all malignant tumors ) about 5 / 10 , 000 per year . 
the prognosis of these diseases depends on a correct and early diagnosis , the efficacy of treatment , and accurate follow - up for monitoring of recurrence [ 1 ]  . 
beyond the clinical evaluation , imaging plays a fundamental role in the initial assessment of soft tissue tumors for a cost - effective patient management , and mr imaging is considered the modality of choice due to its intrinsic high tissue contrast and multiplanarity , as in the study of other pathologies in the musculoskeletal field [ 110 ]  . 
with the use of standard pulse sequences , it is possible to evaluate the signal intensity , growth pattern , degree of enhancement , and relationship with neighboring * antonio barile antonio.barile@cc.univaq.it 1 department ofbiotechnology andapplied clinical sciences , s . 
standard protocols should include t1and t2 - weighted sequences , with and without fat saturation , gradient echo sequences ( detection of hemorrhage ) , and post - contrast acquisitions , in at least two orthogonal planes [ 12 , 13 ]  . 
however , using standard morphological sequences alone , few soft tissue lesions have specific and pathognomonic mr characteristics ( e.g. , simple lipoma / cyst , hemangioma ) , and the differential diagnosis remains very wide for most cases ; another problem is to differentiate a benign histotype from a malignant , also often not immediate with standard imaging as some overlap exists [ 1417 ]  . 
many limitations of the standard mr sequences are also encountered in the follow - up , both in assessing the response to radiochemotherapy treatment and in identifying possible residual and recurrent tumor after surgery [ 13 ]  . 
in recent years , there has been an increased technical advancement in functional mr sequences , such as proton ( 1h ) magnetic resonance spectroscopy ( mrs ) , diffusion - weighted mri ( dwi ) , and dynamic contrast - enhanced ( dce ) mri . 
these sequences provide detailed structural and metabolic information about vol . : ( 0123456789 ) 1 3 244 la radiologia medica ( 2019 ) 124 : 243252 tumor tissue , allowing to formulate a diagnostic hypothesis as close as possible to histological diagnosis in a noninvasive way [ 13 , 2023 ]  . the purpose of this article is to review technical aspects of each of these advanced mr imaging techniques , with a particular focus on their clinical application in the characterization , differentiation of benign from malignant lesions and the assessment of response to treatment in soft tissue tumors . diffusionweighted imaging ( dwi ) dwi is a functional mri technique that provides information about tissue cellularity and cell membrane integrity , evaluating the diffusivity of water molecules ( brownian motion ) [ 2427 ]  . technique the dwi signal is given by the degree of movement of water molecules in cellular spaces ( extracellular , intracellular , transcellular , and intravascular ) , in an inversely proportional manner [ 28 ]  . 
it is important to take into account the contribution to the dwi signal of the intravascular movement of water molecules since in very vascularized lesions it can be significant [ 1 , 11 , 19 ]  . there are several dwi sequences , including spin - echo dwi , echo - planar imaging ( epi ) , and steady - state free precession sequences [ 11 ]  . 
some authors suggest that the evaluation of the minimum adc may be more accurate as it would represent the area of the greatest cellularity of the lesion [ 11 , 29 ]  . 
as mentioned earlier , it is important to take into account tissue perfusion and intravascular water component , as adc values increase in vascularized tumors , leading to overlapping in adc values between benign and malignant tumors . 
the main limits of dwi are represented by the inherent low signal - to - noise ratio and by the low spatial resolution , the artifacts from susceptibility ( artifacts in correspondence of tissues with blood products or air )  . 
for these reasons , dwi must always be interpreted in conjunction with anatomical sequences [ 15 , 19 , 29 , 30 ]  . applications the dwi signal and the adc values reflect the cellularity of the tissues , so even if there are no normal cutoff values , dwi can characterize the biological activity of the tissues . 
in the study of musculoskeletal soft tissue tumors , this means that , as a general rule , benign tumors with a low degree of biological activity will have a loss of adc signal as the b values increase , while malignant tumors ( in which the water has greater restriction in movement ) will show high intensity at high b values . 
the authors found the highest values in benign cystic tumors and the lowest values in giant cell tumors of tendon sheaths ( maybe due to their spindle - shaped stromal cells and multinucleated giant cells histological composition )  . 
 [ 30 ] , using conventional mri and dwi sequences together , obtained values of sensitivity , specificity , and accuracy of 96% , 85.7% , and 90% , respectively . 
the authors confirmed the persistence of high signal intensity with increasing b values in all malignant lesions ( muscular metastases , myxoid liposarcoma , etc . ) on the qualitative evaluation , with low adc values on quantitative analysis with increasing b values , although with differences among the various histological types . 
they also found that higher b values ( more than 800s / mm2 ) are useful to increase the contrast between benign and malignant lesions and thus reduce the number of equivocal cases . 
 [ 32 ] reported the role of dwi in the distinction between benign and malignant peripheral nerve sheath tumors ; in particular , their results showed that the minimum adc value is a better indicator of malignancy than the average adc value , due to the heterogeneity in cellularity of both benign and malignant pnsts . 
dwi sequences show values of 2.75 103 mm2 / s ( b value = 1000 ) on adc map , confirming the benign cystic nature of the lesion 1 3 246 la radiologia medica ( 2019 ) 124 : 243252 in another study [ 28 ] , mean adc of desmoid tumors was found to be significantly higher than that of malignant soft tissue tumors without overlap in the minimum adc values . dwi has proved very useful also in the assessment of treatment response to chemotherapy in soft tissue tumors . 
similarly , the evaluation of enhancement patterns can be challenging , as both granulation and scar tissues ( aspecific tissue changes after chemo / radiotherapy ) are enhancing after contrast administration , and the differentiation from the viable tumor is not always direct [ 15 ]  . 
dwi demonstrated to improve this discrimination earlier than conventional imaging , as solid tumors are characterized by high cellularity with intact cell membranes , while tissues after cytotoxic treatment show lower cellularity and membrane damage [ 15 , 22 ]  . 
dwi implements standard morphological sequences also in the evaluation of postsurgical follow - up [ 6 , 35 , 36 ] , aiding to detect residual / recurrent tumor tissue . dynamic perfusion mri dynamic perfusion mri is a functional imaging technique often used to evaluate musculoskeletal tumors , mainly to depict early intravascular and interstitial distribution of gadolinium [ 22 , 29 , 37 ]  . technique dce - mri is typically performed with multiple repeated rapid , volumetric , and gradient echo sequences acquired immediately after intravenous administration of gadoliniuat our institution , routine dynamic contrast enhancement is performed using t1 3 double fast spin - echo ( dfse ) sequences with fat saturation [ 29 ]  . 
we then perform eight 19 - s scans in fast succession , with a scan delay variable on the region to be studied but that never exceeding 20s , to ensure proper arterial - phase perfusion . 
semiquantitative methods of postprocessing involve the postprocessing of timeintensity curves using three identical rois positioned at a site of marked early tumor enhancement , in an artery , and in a healthy muscle , respectively [ 22 , 37 , 38 ]  . 
the resulting timeintensity curves provide a graphic representation of contrast perfusion , from which quantitative information ( time to enhancement , wash in , peak enhancement , and washout ) can be obtained [ 37 ]  . the curves can be classified into five types [ 22 ] : type 1 , no enhancement ( e.g. , lipoma ) ; type 2 , weak and gradual enhancement ( e.g. , benign tumors or schwannoma ) ; type 3 , rapid early enhancement followed by a plateau ( e.g. , benign vascular tumors , desmoid tumors , abscesses , and some malignant tumors , but with limited specificity ) ; type 4 , rapid early enhancement followed washout ( highly vascular tumors with small interstitial compartment such as malignant histiocytofibroma , synovial sarcoma , and leiomyosarcoma and several benign tumors such as giant cell tumor ) ; type 5 , rapid early enhancement followed by slow , gradual enhancement ( tumors with large interstitial compartments such as myxoid tumors )  . however , even if malignant lesions typically demonstrate rapid early arterial enhancement and higher slopes of enhancement compared with benign lesions , the patterns may show some degree of overlap secondary to highly vascularized benign lesions and poorly vascularized or necrotic malignant lesions [ 29 ]  . applications the main applications in the imaging of soft tissue tumors of dynamic perfusion mri are in the characterization of the lesions , identification of viable tumor areas to target biopsy , monitoring of chemotherapy treatment outcome , and differentiation of residual tumor from scarring [ 11 , 22 , 39 ]  . 
regarding soft tissue lesions , whereas perfusion mri enabled differentiation between lesions with low and high biological aggressiveness , we could never obtain typical timeintensity curves for the various histological tumor types [ 37 ]  . 
pictures a14 : lipoma of the elbow showing homogeneous hyperintense signal intensity on a t1 - weighted sequence ( a1 ) and no enhancement on qualitative evaluation after gadolinium administration ( a2 )  . 
the second subcutaneous lesion ( b14 ) shows inhomogeneous internal structure with areas of adipose signal intensity , intralesional septa , and prevalent peripheral enhancement after gadolinium injection ( b3 ) , with evidence of a type 2 signal intensity curve . 
cases c14 show a subcutaneous polilobulated lesion of the leg strictly adherent to the superficial fascia , with inhomogeneous internal structure and intense contrast enhancement after gadolinium injection ( c4 ) , with evidence of timeintensity curves types 3 and 4 ( arrow )  . 
the mri features of the lesion are consistent with a myxoid liposarcoma on dynamic contrast - enhanced mr imaging , malignant peripheral nerve sheath tumors ( mpnsts ) usually show early arterial enhancement that is much rarer in their benign counterpart ( bpnsts ) [ 23 ]  . the application of dynamic perfusion mri in guiding bioptic procedures is particularly useful in soft tissue tumors that have a significant cystic or hemorrhagic component [ 1 , 41 ] , as purely fluid areas without contrast enhancement should be avoided during biopsy , to prefer the more solid , enhanced portions . 
dynamic contrast - enhanced mri is also helpful in delineating tumor margins ; in fact , tumor enhancement is precocious and faster than that of peritumoral edema and the initial slopes of tumor and nontumor tissues differ significantly [ 11 ]  . perfusion mri is also useful for distinguishing cysts from myxoid tumors and to characterize the benign or malignant nature of myxoid lesions : cysts show no enhancement , while the myxoid component of sarcomas exhibits early and avid enhancement [ 11 ]  . 
the authors postulated that whether differentiation of low - flow from high - flow vascular malformations can be made with nearly 100% sensitivity and specificity setting a 30 - s threshold for the contrast rise time [ 43 ]  . dynamic enhancement is also showing utility in monitoring chemotherapy response , evaluating the degree of tumor necrosis and distinguishing postoperative tissue changes ( e.g. , fibrosis ) from recurrent tumor [ 1 ]  . 
direct visual ( qualitative ) inspection of mri images allows simple recognition of perfused viable tumor tissue with less intense ( slower enhancing ) areas of peritumoral edema , normal tissue , and tumor necrosis [ 11 ]  . 
 quantitatively , an at least 60% decrease in the timeintensity curve slope value indicates more than 90% of tumor necrosis and good treatment outcome [ 11 , 22 ]  . 
optimal follow - up requires three dynamic perfusion mri studies : preoperatively ( before the biopsy and before surgery ) and postoperatively ( during chemotherapy ) [ 11 ]  . 
follow - up examinations ( after 36months ) are necessary when evaluating the treatment response after radiotherapy , as radiotherapy may lead to granulation tissue with increased perfusion that cannot be distinguished by residual tumor early [ 22 ]  . magnetic resonance spectroscopy ( mrs ) mrs is a functional noninvasive imaging technique that requires no intravenous contrast administration and characterizes lesions based on their metabolic constituents [ 1 , 11 , 13 , 29 , 44 ]  . 
 higher field strengths provide a greater signal - to - noise ratio ( snr ) , with improved spectral resolution ; the main drawbacks are the increased field inhomogeneity and the consequently increased metabolite line widths [ 44 ]  . 
using the multi - voxel technique , it is possible to obtain information over a wider field of view , analyzing entire large lesions and surrounding tissue or multiple lesions with a single acquisition . 
the positioning of rois is critical to avoid areas containing bony structures , necrotic or hemorrhagic foci ( underestimation of the choline peak ) , calcifications ( increased field inhomogeneity ) , fat ( can obscure the choline peaks ) , or muscle ( overestimation of the choline peak )  . 
the enhancing portion of the tumor is targeted in roi positioning ; for tumors exhibiting weak / slow enhancement or no enhancement after 5min , the voxel is positioned at sites of delayed enhancement [ 44 ]  . applications already widely used in neuroradiology , magnetic resonance spectroscopy ( mrs ) has recently gained also a role in the musculoskeletal field . 
one of the main applications is the assessment of malignancy in musculoskeletal tumors since specific metabolites ( trimethylamine / choline - containing compounds including phosphocholine , glycerophosphocholine , and free choline ) are distinctively increased in malignant lesions , and they are considered as a marker of malignancy [ 45 ]  . 
to date , the literature results in musculoskeletal mrs investigated the qualitative analysis of the choline or trimethylamine peak as a marker for malignancy [ 21 , 44 ]  . 
in a series of russo etal . , all tumors with a mitotic index greater than 2 / 10 hpf had a positive choline peak and were malignant and correlation between 1h - mrs findings and mitotic index was high [ 16 ]  . 
3 mri axial images of a solid lesion at the level of the flexor tendons of the wrist showing a low signal intensity on t1and t2 - weighted sequences ( a , b ) and intense contrast enhancement after gadolinium injection ( c )  . 
the imaging features are consistent with a giant cell tumor of the tendon sheaths ( gctts ) positive choline peak , and also a large number of inflammatory lesions may produce a high choline peak [ 16 ]  . for example , benign peripheral nerve sheath tumors typically have detectable choline content by mrs . 
in a recent study that used mr spectroscopy to evaluate bpnsts and mpnsts , trimethylamine concentrations and the trimethylamine fraction were found to be relatively lower in the benign neoplasms , and a trimethylamine fraction threshold of 50% resulted in 100% sensitivity and 72.2% specificity [ 45 ]  . the presence of a lipid peak can be detected in the wall of abscesses , in the solid part of highly malignant lesions , and in tumors during treatment response , probably related to cell membrane turnover . 
lipid peak , however , has to be analyzed carefully , due to the possibility of lipid contamination caused by surrounding tissue [ 11 , 16 ]  . mrs evaluation of choline may be used not only for tumor characterization but also to assess treatment response , showing a choline peak reduction after chemotherapy in malignant musculoskeletal lesions [ 11 ]  . the limitation of the literature choline mrs studies up to date is that they are mostly qualitative rather than quantitative , making the distinction of metabolically active benign soft tissue tumors from malignant masses difficult . 
 so , currently , the utility of mrs is limited to its high negative predictive value [ 29 ]  . diffusion tensor imaging andtractography diffusion tensor imaging ( dti ) and tractography are feasible techniques to visualize the spatial anatomy of peripheral nerves in the presence of stt , especially in cases where the course and the involvement of major peripheral nerves are difficult to assess with standard morphological mr sequences [ 20 , 23 , 42 , 46 ]  . technique dti evaluates the three - dimensional ( 3d ) motion of protons in tissues providing quantitative data on the amount and directionality of random movement of water molecules [ 46 ]  . 
4 axial ( a ) an coronal ( b ) mr images of a rounded fusiform lesion at the level of the posterior compartment of the leg ( within the soleus muscle ) showing intense , homogeneous contrast enhancement . 
 axial dti sequence ( c ) with fiber track reconstruction ( d ) demonstrates the growth of the lesion within the nerve fibers and its relationship to thethe imaging features are consistent with the diagnosis of schwannoma limiting it in another , the so - called anisotropy . 
images are evaluated for image quality , adc of the lesion , tractography , and fractional anisotropy of nerves . applications in musculoskeletal imaging clinical practice , dti and tractography are mainly used to evaluate peripheral nerve tumors and soft tissue tumors arising around nerve structures [ 20 ]  . 
this approach is of paramount importance not only for the diagnosis when there is often great difficulty in correctly delineating the tumor from healthy nerve structures , but also for the preoperative planning , as the preservation of unaffected nerve fascicles is important to maintain neuromuscular function after surgery [ 20 ]  . 
 moreover , correlation of d sectional images with surgical findings , particularly in anatomically complex regions , can 1 3 la radiologia medica ( 2019 ) 124 : 243252 be challenging [ 46 ]  . 
in schwannomas , in which the tumor originates from the sheath of a single fascicle , leaving the main trunk of the peripheral nerve attached to the mass , dti and tractography imaging are helpful in the differential diagnosis with neurofibromas , clearly depicting the eccentric and separate tumor growth relative to the involved nerve . 
the added information obtained from these sequences , either alone or integrated , may improve multiparametric evaluations of soft tissue tumors that result in more accurate diagnosis , appropriate treatment planning , and monitoring of treatment efficacy . 
it is important to further implement these techniques to be able to use them on a routine clinical basis and to standardize furtherly acquisition protocols . compliance with ethical standards conflict of interest all authors declare that they have no conflict of interest . ethical approval this article does not contain any studies with human participants performed by any of the authors . la radiologia medica ( 2019 ) 124 : 290300 radiotherapy influence ofage andsubtype inoutcome ofoperable liposarcoma danielagreto1 calogerosaieva2 mauroloi1 francescaterziani1 lucavisani1 pietrogarlatti1 monicalorusso1 cristinamuntoni1 carlottabecherini1 julianatopulli1 domenicocampanacci3 giovannibeltrami3 guidoscoccianti3 francescomuratori3 pierluigibonomo1 isaccodesideri1 giuliofrancolini1 lorenzolivi1 received : 13 february 2018 / accepted : 2 november 2018 / published online : 12 november 2018 italian society of medical radiology 2018 abstract aim liposarcoma ( lps ) is rare tumor deriving from adipocytes . 
the aim of this study is to evaluate whether clinical characteristics , tumor and treatment - related features affect clinical outcome in patients treated with curative intent for non - metastatic liposarcoma . methods data of patients with locally advanced , non - metastatic liposarcoma treated between 1990 and 2015 were retrospectively reviewed . 
statistical analysis was performed to assess correlation between the above - cited variables and local recurrence - free survival ( dfs - lr ) , distant metastasis - free survival , overall survival ( os ) and disease - specific survival ( dss ) ; moreover , differences in clinical outcome between the two age groups were identified . results data of 186 patients were collected . 
further studies are needed to stratify patients subgroup and develop tailored treatment strategies ( i.e. , altered fractionations and different chemotherapy regimens in aggressive subtypes ) , in particular more prospective trials are needed to develop treatment guidelines in elderly sts , taking into account the frailty and the peculiarity of this subgroup . keywords liposarcoma radiotherapy soft tissue sarcoma age extended author information available on the last page of the article vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 290300 background soft tissue sarcomas ( stss ) are rare tumors accounting for about 1% of all tumors [ 1 ]  . 
liposarcoma is a heterogeneous disease characterized by different clinical behavior according to histological subtypes : ddlps showed the most aggressive clinical course in terms of disease recurrence and worse os . 
further studies are needed to stratify patients subgroup and develop tailored treatment strategies ( i.e. , altered fractionations and different chemotherapy regimens in aggressive subtypes ) , in particular more prospective trials are needed to develop treatment guidelines in elderly sts , taking into account the frailty and the peculiarity of this subgroup . 
well - differentiated liposarcomas ( wdlps ) are considered locally aggressive , with low potential for metastatic spread [ 4 ] ; however , more aggressive features of disease can be acquired in the event of local recurrence . 
 dedifferentiated liposarcoma ( ddlps ) , both de novo and secondary to progression from a well - differentiated disease , [ 4 ] , has a worse prognosis with a 41% and a 1730% rate of local recurrence and metastatic spread , respectively . 
myxoid liposarcoma ( mlps ) , the most common subtypes in pediatric population , is characterized by a particular metastatic pattern , with high predisposition to spread into fat - bearing areas , such as mediastinum , bone marrow or retroperitoneum [ 8 , 9 ]  . 
pleomorphic liposarcoma ( plps ) is the subtype with the lowest grade of differentiation characterized by poor outcomes , with an estimated risk of 3445% of local recurrence and 3257% of metastatic disease [ 10 ]  . 
approximately 40% of sts patients are diagnosed over the age of 65 [ 14 ] , and in particular , with the exception of mlps , most liposarcoma subtypes are diagnosed in adults [ 15 ]  . 
it is unclear whether differences in outcome can be explained by a more aggressive biological behavior or because in most cases elder patients do not receive full standard treatment and are not included in clinical trials [ 16 ]  . a tailored approach taking into account the specificity of disease subtype and age at presentation could be helpful in delineating therapeutic management of liposarcoma . 
the aim of this study is to evaluate whether clinical characteristics , tumorand treatment - related features affect clinical outcome in patients treated with curative intent for nonmetastatic liposarcoma . materials andmethods data of patients with locally advanced , non - metastatic liposarcoma treated between 1990 and 2015 were retrospectively reviewed . 
a baseline chest ct scan or x - ray was performed in all patients , to exclude metastatic lung disease ; contrast - enhanced magnetic resonance ( mri ) and / or ct of the primary tumor location was performed , to complete locoregional staging and define tumors relationship with surrounding anatomical structures . 
all patients were selected to undergo radical surgery ; in case of unresectable , locally advanced disease , preoperative rt for a total dose of 50gy ( 2gy per fraction for 5days a week ) was delivered , to reduce the tumor size . 
postoperative rt ( port ) was delivered in case of high - grade disease , tumor diameter > 5cm and / or deep location , for a total dose of 60gy ( 2gy per fraction for 5days a week )  . 
anthracycline - based chemotherapy was administered in case of high - risk disease ( at least two of the following prognostic factors : high grade , deep - seated tumor , large dimensions , positive surgical margins ) in association to rt . 
 treatment schedule consisted of epirubicin ( 60mg / m2 on days 12 ) and ifosfamide ( 3g / m2 on days 13 ) , repeated every 21days ( table1 )  . 
 data about age at diagnosis , sex , age , tumor location ( trunk , upper and lower limb ) and tumor depth were collected , as well as histopathological characteristics such as tumor size , 1 3 292 la radiologia medica ( 2019 ) 124 : 290300 table 1 treatment procedures chemotherapy postoperative radiotherapy preoperative radiotherapy brachytherapy epirubicin : 60mg / m2 day : 1 , 2 ifosfamide : 3g / m2 / dayday : 1 , 2 , 3 every 21days 60gy : 2gy per fraction ; 5days a week 50gy : 2gy per fraction ; 5days a week 3035gy ( brachy needles implantation ) grade ( according to fnclcc grading system [ 18 ] ) , surgical margins status . 
the pathological specimens of patients treated before 2013 were reviewed and classified according to 2013 world health organization ( who ) classification of tumors of soft tissue and bone [ 19 ]  . clinical outcome endpoints analyzed were local relapse - free survival ( lrfs ) , distant relapse - free survival ( drfs ) , overall survival ( os ) and disease - specific survival ( dss )  . 
overall survival ( os ) and dss were calculated from the date of surgery to the time of the last follow - up or death for any causes and death from cancer , respectively ; local relapse - free survival ( lrfs ) and distant relapse - free survival ( drfs ) were calculated from the surgery to the time of the local recurrence and the time of the distant metastasis development , respectively . 
wide excision , marginal surgery and intralesional excision were performed in 151 ( 81.2% ) , 25 ( 13.4% ) and 7 ( 3.8% ) patients , 1 3 la radiologia medica ( 2019 ) 124 : 290300 respectively ; three ( 1.6% ) patients , all of whom younger than 65years , underwent amputation . 
port was administered in 112 ( 60.2% ) cases : brachytherapy ( bt ) was delivered in addition to external beam ( ebrt ) in 39 patients and as an exclusive treatment in six patients . 
5 kaplanmeier curves analysis of os in all patients surgical resection remains the cornerstone of curative management of liposarcoma , as well as for other subtypes of soft tissue sarcoma . 
depending on patients and tumor characteristics , surgery is often associated with rt and / or chemotherapy . however , liposarcoma is a heterogeneous disease , with distinct subtypes and variable clinical behavior [ 15 ]  . 
in particular , the role of port in wdlps is debated , since the low risk of distant metastases could be related to a decreased clinical benefit in this subset [ 20 ]  . 
conversely , local control rates of about 97% are reported for mlps treated with surgery and rt [ 21 , 22 ] ; moreover , preoperative rt might yield excellent responses , due to the high radiosensitivity of mlps , and should always be considered when patients are not amenable to upfront surgery [ 23 , 24 ]  . due to the high heterogeneity of liposarcomas , literature data about clinical outcomes are extremely different . 
in our cohort , at a mean follow - up of 10years , about one patient out of four experienced local recurrence or metastatic relapse ; half of the patients were alive at the time of our analysis . 
authors reported rates of relapse of 1857% , 728% and 1645% in dlps , mlps and plps , respectively , and survival rates ranging between 7693% , 5464% , 4075% and 063% in wdlps , dlps , mlps and plps . 
 [ 25 ] proposed a 1 3 298 la radiologia medica ( 2019 ) 124 : 290300 liposarcoma - specific postoperative nomogram for diseasespecific survival ( dss ) , aimed to provide an individualized estimate of cancer specific mortality . 
identification of risk classes could allow to intensify treatment in certain disease subtypes with poor clinical outcomes ( i.e. , chemotherapy to reduce metastatic spread in plps ) , while overtreatment could be avoided in more favorable subgroups . 
in soft tissue sarcoma , baseline features such as age , tumor grade and surgical margins are well - known predictive factors of clinical outcome . in our series , age older than 65 was an independent prognostic factor affecting local and distant recurrence and os . 
specific genomic alterations are more likely to occur in older patients , influencing prognosis [ 30 ]  . in our report , disease characteristics and treatment modalities distribution between patients younger than 65years and the elderly were different . 
plps and ddlps that were more common in our elderly patients are well - known histopathological subtypes correlated to a worst prognosis in terms of local and distant recurrence than mlps , and the response to perioperative treatments such as radiotherapy and chemotherapy are expected inferior compared to mlps [ 15 ]  . 
preoperative radiotherapy was delivered in 12.1% of patients older than 65years compared to 30% of younger patients ( p = 0.004 ) ; multidisciplinary team in case of elderly patients usually reserve radiotherapy indication only in very high risk of disease recurrence such as positive margins , high - grade and deepsited tumors . 
 [ 32 ] analyzed 1240 localized soft tissue sarcoma patients from the french soft tissue sarcoma database , reporting that tumor grade was an independent predictor of metastasis in all the histologies ( 15.2% of patients had a diagnosis of liposarcoma ) with the exception of malignant schwannomas and rhabdomyosarcomas . 
according to a large cohort - based analysis of 1513 soft tissue sarcoma cases [ 33 ] , anthracycline - based adjuvant chemotherapy improved dmfs and os only in g3 patients . 
it could be possible that the worse outcome of patients treated with preoperative radiotherapy was due to unfavorable disease with large tumor size and deep - sited location . in our cohort , wide excision was significantly related to improved survival . 
due to the high risk of microscopic residual disease in the peritumoral tissue , the adequacy of surgical margins has been investigated by several authors as a major predictor of outcome ; its value has been confirmed in a recent study [ 37 ]  . 
liposarcoma often infiltrates surrounding soft tissue ; thus , it is possible that maximizing tumor control in the primary site might result in improved long - term disease control [ 38 ]  . finally , the safety of concomitant rt and chemotherapy administration was analyzed . 
our data are similar to those described in literature [ 39 ]  . conclusions liposarcoma is a heterogeneous disease characterized by different clinical behavior according to histological subtype : ddlps showed the most aggressive clinical course in terms of disease recurrence and worse os . 
further studies are needed to stratify patients subgroup and develop tailored treatment strategies ( i.e. , altered fractionations and different chemotherapy regimens in aggressive subtypes ) , in particular more prospective trials are needed to develop treatment guidelines in elderly sts , taking into account the frailty and the peculiarity of this subgroup . compliance with ethical standards conflict of interest none . ethical approval this article does not contain any studies with animals performed by any of the authors . informed consent informed consent was obtained from all individual participants included in the study . la radiologia medica ( 2019 ) 124 : 241242 editorial artificial intelligence : achallenge forthird millennium radiologist robertograssi1 vittoriomiele2 andreagiovagnoni3 received : 4 january 2019 / accepted : 14 january 2019 / published online : 1 february 2019 italian society of medical radiology 2019 diagnostic imaging techniques , i.e. 
they produce a large number of images , on which the radiologist writes , after a subjective and personal evaluation , essentially qualitative , the diagnosis , the so - called report . these diagnostic technologies acquire an enormous amount of numerical data in dicom format , of which only a part is visually evaluated in greyscale . 
hundreds to thousands can be derived from a statistical mathematical analysis of raw data [ 1 ]  . for some years now , throughout the world , there has been an attempt not to disperse this great mass of information in the raw data . 
the ambitious objective is to identify , thanks to complex mathematical and statistical algorithms , which information , inaccessible to simple visual analysis , can predict the evolution of the disease and the effectiveness of the therapy , but in a quantitative and objective way . artificial intelligence , machine learning , big data , radiomics are just some of the many terms that are used by researchers in the field , especially computer scientists , clinical engineers , physicists , to define their activities . 
brambilla , 3 , 50134florence , italy 3 department ofradiological sciences , azienda ospedaliero universitaria ospedali riuniti , universit politecnica delle marche , ancona , italy what is missing then and what will be the keystone to achieve these goals ? the answer may seem trivial , but it is not : the radiologists collaboration . in fact , it will never be sufficient to collect only the data , raw or in dicom format , also re - elaborated to constitute the so - called big data , but it will be necessary to correlate them with the radiological reports , so that the computer can process them and find the correlation between them and the phenotype in question . but , to do this , the computer must have a standardized report , which , in part , is also referred to as a structured report . 
it , thanks to the recognized authoritativeness , has the possibility to convey its use in a large number of structures . oncology is the first area in which to apply . 
combining the huge amount of data tagged with the radiological reports will be like inserting the plug into the socket : our radiological world will glow with a new light . 
our report will contain over time not only a subjective and qualitative interpretation of the images , our diagnosis , but also a series of numerical values , objective and quantitative features , our contribution to the prognosis , because some of them will have proved useful to predict the efficacy of the therapeutic response . 
parp inhibitors exert antitumor activity by both catalytic parp inhibition and parpdna trapping , moreover parp inhibition represents a potential synthetic lethal approach against cancers with specific dna - repair defects . 
soft tissue sarcoma ( stss ) are a heterogeneous group of mesenchymal tumors with locally destructive growth , high risk of recurrence and distant metastasis . objectives the purpuse of this review is to provide an overview of the main preclinical and clinical data on use of parpi in stss and of effect and safety of combination of parpi with irradiation . results due to numerous genomic alterations in stss , the dna damage response pathway can offer an interesting target for biologic therapy . 
although mechanisms of synergisms are not completely known , combination of radiation therapy and parp inhibitors exerts antitumor effect by accumulation of unrepaired dna damage , arrest in g2 / m , activity both on oxic and hypoxic cells , reoxygenation by effect on vessels and promotion of senescence . 
early trials have shown a good tolerance profile . conclusions the use of parp inhibitors in advanced stage stss , alone or combined in multimodal treatments , is of great interest and warrants further investigations . keywords poly ( adp - ribose ) polymerases ( parp ) soft tissue sarcoma ( stss ) dna damage response pathway radiation therapy * monica mangoni monica.mangoni@unifi.it 1 department ofbiomedical , experimental andclinical sciences mario serio , section ofradiation oncology , university offlorence , largo brambilla 3 , 50134florence , italy 2 department oforthopaedic oncology , azienda ospedaliera universitaria careggi , largo brambilla 3 , 50134florence , italy itt , istituto toscano tumori , via taddeo alderotti 26 / n , 50139florence , italy introduction poly ( adp - ribose ) polymerases ( parp ) are a large family of enzymes involved in several cellular processes , including dna single - strand break repair ( ssb ) via the baseexcision repair ( ber ) pathway [ 1 , 2 ]  . 
subsequently , parp1 leaves damaged dna , owing to the dense negative charge of padpr , and allows vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 282289 fig . 
the resulting negatively charged poly ( adp - ribose ) polymers decrease the affinity of parp1 for dna , while recruit additional dna repair proteins , leading to dna repair . 
loss of function of brca proteins ( hr deficiency ) leads to unrepaired damage ( i.e. , due to replication fork collapse ) and subsequent cell death , or to the use of alternative repair pathways , such as non - homologous end joining ( nhej )  . 
 moreover , parp - 1 , due to the inhibition that blocks autoparylation , can remain trapped onto dna damaged site , in particular during ber , and determine an obstruction to replication forks . 
in this case , if parp1 cannot cope with ssbs during this semi - conservative repair process , the accumulated unrepaired ssbs that are converted to dsbs could became highly toxic to cells . 
parp - 1 is implicated in the cellular response to dsbs by promoting hr at damaged replication forks , and repressing the nonhomologous end - joining ( nhej ) repair , 1 3 284 la radiologia medica ( 2019 ) 124 : 282289 the alternative dsb repair pathway in response of a defective hr . 
hr represents a strong determinant of response of the hr - competent cells to parp inhibition . the combination between hr deficiency and a parp inhibition can be described as a synthetic lethality , that is the cell death as result of a combination of deficiency in the expression of genes , due to mutations , epigenetic alterations , and inhibition . 
many cancers seem to be particularly sensitive to parp inhibition , such as ovarian cancers and other solid tumor with germline mutations in brca1 or brca2 , crucial proteins for hr . 
indeed , in brca1 / 2 defective cells , parpi induce an increase in ssbs , which in turn are transformed during replication in irreparable toxic dna double - strand breaks ( dsbs ) , due to the hr process defect [ 2 , 6 ]  . 
additional genetic modulators of parpi sensitivity have been identified , such as mutations in the genes encoding atm , atr or pten , and elevated parp1 expression is emerging as a measure of parpi sensitivity [ 7 , 8 ]  . parp inhibition may represent a new potential synthetic lethal approach against cancers with specific dna - repair defects . 
moreover , the parp - trapping mechanism may have a pivotal role in the synergy between a parpi and dna damaging agents [ 3 ]  . there are currently three parpi that have been fdaapproved for use : olaparib ( astra zeneca , london , uk ) , rucaparib ( clovis oncology , boulder , co , usa ) , and niraparib ( tesaro inc . , waltham , ma , usa )  . 
other parpi in active development include veliparib ( abbvie pharmaceuticals , north chicago , il , usa ) , and talazoparib ( pfizer pharmaceuticals , new york city , ny , usa ) [ 9 ]  . soft tissue sarcoma ( stss ) are a heterogeneous group of mesenchymal tumors with locally destructive growth , high risk of recurrence and distant metastasis [ 10 , 11 ]  . 
both preand post - operative radiotherapy ( rt ) are recommended for intermediate or high - grade stss and for patients with large or marginally excised , low - grade tumors . 
although exist some controlled evidences about of the benefit of chemotherapy , often are conflicting , due to the different chemosensitivity depending on the histological subtype , and insufficient to support chemotherapy as a standard administration . 
for that reason , at present , it does not exist a shared strategy and many efforts are necessary to ameliorate knowledge on subtypes genetic profile , tumorigenesis and drug activity to allow specific and individualized treatments [ 1113 ]  . 
 cytogenetic molecular analyses have allowed two different types of sarcoma to be distinguished : stss with simple genomics , associated with a simple genomic alteration , and stss with complex genomics , characterized by very complex karyotypes and a high genomic instability . 
from well - differentiated to de - differentiated histotypes , the secondary genetic mutations determine an increased genomic complexity , chromosome aberrations and loss of specific targets [ 14 , 15 ]  . 
notably , ewsfli1 gene fusion acts in a positive feedback loop to maintain the expression of parp1 , which is required for ewsfli - mediated transcription , thereby enforcing oncogene - dependent sensitivity to parp - 1 inhibition [ 16 ]  . 
thus , it is speculated that ewsfli1 transcriptional program primes ewing sarcoma cells for hypersensitivity to parpi by inducing high parp1 expression , increasing the availability of parp1 for trapping , and elevating basal dna damage [ 7 ]  . 
the ability of parpi to trap parp differs among parpi , and is not linked to their ability to inhibit parp ; veliparib is a potent inhibitor of parp enzymatic activity but a poor trapper and results less toxic than olaparib in ewing sarcoma cells [ 17 ]  . 
 the comparison of four parp inhibitors in ewing sarcoma cells showed marked differences in the single agent cytotoxicity with decreasing potency in the order of talazoparib ( ic50 10 nm ) > niraparib ( ic50 300 nm ) > olaparib ( ic50 1000 nm ) > veliparib ( ic50 10 , 000 nm ) [ 18 ]  . 
after the discovery of the ability of parpi to trap parp , that is even more toxic to cells than the accumulating dna damage , the combination of parpi and cytotoxic drugs was tested [ 20 ]  . 
the combination with a chemotherapy agent can drive accumulation of dna damage in ewing sarcoma cells , heightening the recruitment of parp1 to dna for ssb repair , and thereby driving enhanced parp1 trapping [ 7 ]  . 
the differential ability of chemotherapeutic drugs to synergize with parpi has been attributed to the type of dna lesions generated by the anticancer agents that differentially require parp1 for dna repair or affect binding of parp1 to the dna [ 18 ]  . 
consequently , the antiapoptotic protein mcl - 1 is downregulated , the proapoptotic proteins bax and bak are activated , mitochondrial outer membrane is permeabilizated and caspases and caspasedependent cell death are activated [ 18 ]  . 
mechanistically , talazoparib and temozolomide cooperated to induce apoptotic cell death , loss of mitochondrial membrane potential , caspase activation , dna fragmentation and caspase - dependent cell death [ 18 ]  . parpi have been reported to act also in concert with doxorubicin , melphalan , carboplatin , etoposide and irinotecan or its active metabolite sn38 in ewing sarcoma cells and other stss cell lines [ 18 , 2123 ]  . 
in u2os osteosarcoma cell line , knockdown of parp - 1 resulted in decreased cell proliferation , increased cell apoptosis , and g0 / g1 phase arrest and correlated with elevated chemosensitivity to cisplatin through inactivation of the erk1 / 2 signaling pathway [ 24 ]  . 
in ewing sarcoma cells , the combination of olaparib and trabectedin was found to be highly synergistic , inhibiting cell proliferation , inducing apoptosis , and the accumulation of g2 / m [ 25 , 26 ]  . 
thus , ewing sarcoma cells were the most sensitive to the combination parpi / trabectedin , preclinical data show additive or synergic activity also in large panels of stss [ 27 ] , where different degree of synergism was observed among different cells line [ 26 ]  . 
for example , in leiomyosarcoma models the combination was significantly more active than single agents , while in osteosarcoma models no further advantage was obtained if compared to trabectedin alone . 
parp1 expression or different parylation was showed to dictate the degree of the synergism [ 26 ] , i.e. , single nucleotide polymorphism in the parp1 gene ( val762ala ) is known to result in a less efficient parp1 variant [ 28 ]  . 
moreover , gene sets involved in the combination synergism suggest a specific dna - damage response / repair and cell cycle control gene signatures that might become predictive biomarkers of response [ 26 ]  . 
despite the fact that parpi as single agents exerted promising cytotoxicity against stss cells invitro [ 29 ] , they exerted limited efficacy in xenograft models [ 18 ] without improvement of survival [ 21 ]  . 
synergistic effect was observed with combining treatment of parpi with cyclophosphamide , topotecan , temozolomide , irinotecan and trabectedin [ 16 , 20 , 25 , 27 , 30 , 31 ]  . clinical data withparpi instss the first phase ii clinical trial testing single - agent parpi in ewing sarcoma patients with recurrent disease failed to show improvement in outcome [ 7 , 16 , 32 , 33 ]  . 
clinical 1 3 286 la radiologia medica ( 2019 ) 124 : 282289 data were consistent with the minimal activity observed in xenograft model , due to poor parp1 trapping invivo [ 16 ]  . 
in the recent phase i nct01286987 study , talazoparib showed single - agent antitumor activity and a tolerable safety profile in brca mutation - associated breast and ovarian cancers , and in patients with pancreatic and small cell lung cancer , however , for patients with ewings sarcoma no objective response was observed [ 34 ]  . 
a possible explanation for the discrepancy between invitro and invivo / clinical effect is that some cell lines may have been derived from patient tumor samples that were not yet chemoresistant , while all participant of clinical trials had relapse or progression after standard chemotherapy [ 32 ]  . 
actually there are two phase ii trials ( pediatric match nct03233204 and nct03155620 ) evaluating single agent olaparib in advanced tumors with defects in dna damage repair genes , including stss . 
early results showed good tolerance of the combination and in 22 patients evaluable for response 18% partial responses ( synovial sarcoma , leiomyosarcoma ) and 23% stable diseases ( liposarcoma , ewing sarcoma , solitary - fibrous - tumor , malignant peripheral nerve sheath tumor ) [ 35 ]  . parpi andirradiation ionizing radiation causes damage mainly through interaction with dna , leading to strand breaks and nucleotides damage . 
thus , there is a strong rationale in multimodal treatments combining parpi and radiation therapy ( rt ) , with described median enhancement ratio from 1 , 3 to 1 , 5 [ 4 ]  . 
the effect is greater in highly proliferating tumor cells and could be enhanced in cancers harboring defects in hr and by synthetic lethality mechanis combined treatment is of great interest in stss due to presence of genetic alteration amplifying parpi effect , moreover rt is part of standard treatment in intermediate / high - grade stss and locally advanced disease [ 12 ]  . 
the radiosensitizing effects can be observed at much lower olaparib doses than the single agent effect , suggesting a crucial role to the increased requirement of parp activity after radiation . 
in lung and pancreatic cancer cell lines combined treatment increases expression of h2ax and 53bp1 foci and upregulates expression of phosphorylated atm , atr and their respective kinases [ 39 ]  . 
either in cell culture studies or in tumor - bearing animal experiments , the primary effect of the combination treatment almost uniformly reflected growth arrest rather than cell death with little or no contribution from apoptosis , as confirmed by demonstrating that radiosensitization is not altered by caspase inhibition . 
however , the possible recovery of senescent cells and re - entry into cell 1 3 la radiologia medica ( 2019 ) 124 : 282289 cycle after prolonged arrest could lead to tumor recurrence [ 45 ]  . parpi andirradiation instss combined treatment of rt and parpi has showed radiosensitization with olaparib in ewing sarcoma , leading to synergistic increases in apoptosis and cell death in a ewsfli1 dependent manner , as shown using the comet assay . 
in addition , in a xenograft model olaparib in combination with a single dose of ionizing radiation ( 4gy ) showed delayed tumor growth ; examination of treated tumors demonstrated profound effects on dna damage , apoptosis , and proliferation [ 46 ]  . 
moderate radiosensitization in ewing sarcoma was observed also with rucaparib [ 47 ]  . our group is working on combination of radiation with olaparib , iniparib and veliparib in four human cell lines of stss ( fibrosarcoma , liposarcoma , leiomyosarcoma and rhabdomyosarcoma )  . 
in brain , veliparib ( 200mg bid ) in combination with whole brain rt ( wbrt ) in patients with metastases demonstrated an adverse event profile similar to that of wbrt alone [ 49 ]  . 
however , data are not univocal and in a phase i study on glioblastoma , association of veliparib with radiotherapy and temozolomide was found unsafe because of hematologic complications [ 50 ]  . 
in thorax , a phase i study with veliparib and rt on loco regional radiotherapy for local recurrence of triple negative breast cancer , showed comforting safety data [ 52 ]  . 
a phase i study of veliparib in combination with low - dose fractionated whole abdominal radiation therapy ( ldfwar ) in patients with peritoneal carcinomatosis demonstrated that combined treatment is a tolerable regimen with primary toxicities of myelosuppression , fatigue and gastrointestinal symptoms . 
 these studies suggest that olaparib might be a carcinogen and warrant further investigations [ 55 ]  . conclusions in conclusion , parpi are promising agents that can give advantages in stss treatment , alone or in multimodal therapies . 
notably , radiosensitizing activity of parpi is of great interest due to the large use of rt in standard protocols of stss and to the presence of genetic alteration amplifying parpi effect . 
the morphologic diagnosis of sarcoma relies on the evaluation as well as the integration of four main features : the shape of the neoplastic cells ; the pattern of growth ; the quality of the background ; the architecture of the vascular network . 
molecular genetics is increasingly used for diagnostic purposes to distinguish specific subtypes of sarcomas , to support diagnosis in non - canonical clinical presentations and also to distinguish true sarcomas from benign mimickers . 
with many exceptions , histologic typing does not provide sufficient information for predicting the clinical course of the disease and , therefore , grading systems based on histological parameters were introduced to provide a more accurate estimation of the degree of malignancy of tumors . 
 the three - tiered system devised by the french federation of cancer centers sarcoma group ( fnclcc ) systems is widely adopted ; however , several limitations exist that have led to the development of prognostic nomograms that incorporate the specific histotype as one of the relevant parameters . keywords soft tissue sarcoma immunohistochemistry molecular genetics introduction soft tissue sarcomas represent a heterogeneous group of rare malignancies with an overall incidence of around 5 / 100 , 000 / year . 
half of the cases occur in the limbs ( wherein the thigh is by far the commonest site ) , 30% occur intra - abdominally ( including the retroperitoneum ) , and 15% arise in the trunk and in the head and neck region . 
to avoid major mistakes , careful evaluation of clinical presentation and integration of immunohistochemistry and molecular genetics whenever relevant are mandatory . soft tissue sarcomas are currently classified on the basis of the 2013 who classification of soft tissue tumors [ 2 ]  . 
who classifies the different entities on the basis of vol :  . ( 1234567890 ) 1 3 la radiologia medica ( 2019 ) 124 : 266281 histomorphology and includes all available immunophenotypic and genetic data since the 2002 edition [ 3 ]  . 
the major advances introduced since 2002 can be summarized as follows : adipocytic tumors one of the major changes introduced since 2002 is represented by the use of a stricter terminological definition of well - differentiated liposarcoma , which represents the commonest of liposarcoma subtypes . 
it has been clarified that the terms atypical lipomatous tumor and well - differentiated liposarcoma are synonyms and that the latter term should only be used for lesions that occur in the retroperitoneum / mediastinum or in other anatomic sites where complete resectability is unachievable . 
the use of the term atypical lipomatous tumors for resectable lesions is justified by the fact they never recur and are most often cured by complete ( even marginal ) surgical excision . 
in 2002 , it was recognized that in dedifferentiated liposarcoma ( defined as morphological progression from well - differentiated liposarcoma to high - grade non - lipogenic sarcoma ) , also a low - grade dedifferentiation can be observed . 
to reflect the fact that both lesions actually represent the ends of a morphological spectrum of a genetically distinct histology , in 2002 myxoid and round cell liposarcoma merged in one single entity . 
in 2013 , the term round cell liposarcoma was eliminated and replaced by the label high - grade myxoid liposarcoma to underline the fact that clinical outcome depends on the amount of hypercellularity and not on the shape on neoplastic cells that can be either rounded or spindled . fibroblastic / myofibroblastic tumors a key conceptual change was represented by the inclusion of hemangiopericytoma ( hpc ) since 2002 within the chapter on solitary fibrous tumor . 
at that time , it was already clear that hpc merely represented a collection of unrelated , benign as well as malignant , sharing an hpc - like vascular network ( i.e. , the presence of dilated , thin - walled , branched blood vessels ) fig.1. 
as a logical consequence of this conceptual evolution in 2013 , the label hemangiopericytoma table 1 intermediate ( locally aggressive and / or rarely metastasizing ) and malign soft tissue tumors recognized by 2013 who classification of soft tissue tumors intermediate adipocytic tumors atypical lipomatous tumor / well - differentiated liposarcoma malignant adipocytic tumors dedifferentiated liposarcoma myxoid liposarcoma pleomorphic liposarcoma intermediate fibroblastic / myofibroblastic tumors superficial fibromatosis desmoid - type fibromatosis lipofibromatosis giant cell fibroblastoma dermatofibrosarcoma protuberans and variants solitary fibrous tumor inflammatory myofibroblastic tumor low - grade myofibroblastic sarcoma myxoinflammatory myofibroblastic tumor infantile fibrosarcoma malignant fibroblastic / myofibroblastic tumors adult fibrosarcoma myxofibrosarcoma low - grade fibromyxoid sarcoma sclerosing epithelioid fibrosarcoma intermediate so - called fibrohistiocytic tumors plexiform fibrohistiocytic tumor gant cell tumor of soft tissues malignant smooth muscle tumors leiomyosarcoma malignant skeletal muscle tumors embryonal rhabdomyosarcoma alveolar rhabdomyosarcoma pleomorphic rhabdomyosarcoma spindle cell / sclerosing rhabdomyosarcoma intermediate vascular tumors kaposiform hemangioendothelioma retiform hemangioendothelioma papillary intralymphatic angioendothelioma composite hemangioendothelioma pseudomyogenic ( epithelioid sarcoma - like ) hemangioendothelioma kaposi sarcoma malignant vascular tumors epithelioid hemangioendothelioma angiosarcoma of soft tissue malignant chondro - osseous tumors extraskeletal mesenchymal chondrosarcoma gastrointestinal stromal tumors malignant nerve sheath tumors malignant peripheral nerve sheath tumor 1 3 268 table 1 ( continued ) epithelioid malignant peripheral nerve sheath tumor malignant triton tumor malignant granular cell tumor ectomesenchymoma intermediate tumors of uncertain differentiation hemosiderotic fibrolipomatous tumor atypical fibroxanthoma angiomatoid fibrous histiocytoma ossifying fibromyxoid tumor mixed tumor myoepithelioma myoepithelial carcinoma phosphaturic mesenchymal tumor malignant tumors of uncertain differentiation synovial sarcoma epithelioid sarcoma alveolar soft parts sarcoma clear cell sarcoma of soft tissues extraskeletal myxoid chondrosarcoma ewing sarcoma desmoplastic small round cell tumor extra renal rhabdoid tumor pecoma intimal sarcoma undifferentiated / unclassified sarcoma undifferentiated spindle cell sarcoma undifferentiated pleomorphic sarcoma undifferentiated round cell sarcoma undifferentiated epithelioid sarcoma la radiologia medica ( 2019 ) 124 : 266281 organized in a perivascular pattern of growth survives within the label myopericytoma . fibrosarcoma also experienced a significant remodeling . 
whereas it is currently recognized that most superficially located fibrosarcomas actually represent examples of fibrosarcomatous dermatofibrosarcoma protuberans ( fs - dfsp ) , infantile fibrosarcoma is confirmed as a clinically , pathologically and , genetically distinct entity . 
these are low - grade fibromyxoid sarcoma , myxoinflammatory fibroblastic sarcoma , sclerosing epithelioid fibrosarcoma , and low - grade myofibroblastic sarcoma . socalled fibrohistiocytic tumors the same destiny of hpc occurred to malignant fibrous histiocytoma ( mfh ) who also totally disappeared in 2013 . 
as of today pleomorphic mfh , once the most commonly diagnosed sarcoma , is now synonymous with high - grade undifferentiated pleomorphic sarcoma and it should not exceed approximately 5% of newly diagnosed sarcomas . 
the so - called giant cell variant of mfh appears to be an heterogeneous collection of clinically as well as morphologically distinctive lesions that includes giant cell tumor of soft tissue , extraskeletal osteosarcoma , and spindle cell sarcoma ( most often leiomyosarcoma ) featuring osteoclast - like giant cells . 
as its line of differentiation remains unknown , it has also been moved to the category of mesenchymal tumors of uncertain differentiation . the existence of a broader category of undifferentiated sarcomas ( pleomorphic , epithelioid , round cell , and spindle cell ) is now fully acknowledged . 
currently , the original ( still valid ) idea generated by arthur purdy stout of the existence of lesions mainly composed of contractile cells in the last two decades , several new entities have been characterized , particularly in the intermediate malignancy category , including the kaposiform , retiform , and composite hemangioendothelioma . 
since the 2002 who classification epithelioid hemangioendothelioma ( ehe ) 1 3 la radiologia medica ( 2019 ) 124 : 266281 has been reclassified as malignant due to its considerable metastatic rate ranging between 15 and 30% . 
a novel , genetically distinct entity named pseudomyogenic hemangioendothelioma , characterized by multifocality as well as relatively indolent clinical behavior , has also been added to the group of vascular neoplasm of intermediate malignancy . tumors ofuncertain differentiation this category contains tumors without a clear line of differentiation or without a normal cellular counterpart . 
since we now know more about divergent differentiation in various sarcomas subtypes , the category of malignant mesenchymoma is also losing ground as it is currently acknowledged that heterologous differentiation may occur in context of specific entities such as malignant peripheral nerve sheath tumor ( mpnst ) and dedifferentiated liposarcoma . 
the morphologically rather elusive category of intimal sarcoma was introduced as a new entity in this group . principles ofsarcomagenesis the pathogenesis of the vast majority of soft tissue sarcomas is still unknown and most of them arise de novo without an apparent causative factor . 
in rare cases , genetic and environmental factors such as radiation , lymphedema ( secondary angiosarcoma of the breast ) , viral infections ( hhv8 infection is associated with kaposi sarcoma ) , exposure to chemicals ( vinyl chloride is linked to hepatic angiosarcoma ) , and immunodeficiency ( ebv infection in immunodeficient subjects is associated with the development of smooth muscle tumors ) have been identified as risk factors . 
it is broadly accepted that trauma does not represent a predisposing factor and that , at best , it can simply draw attention to the presence of pre - existing mass . genetic susceptibility plays a role in a minority of soft tissue sarcomas . 
the autosomal dominant lifraumeni syndrome ( wherein germline mutations of the tp53 gene occur ) has been shown to predispose to the development of malignant tumors , one - third of which are represented by bone and soft tissue sarcomas . 
recent data have shown that approximately half of patients with sarcoma have putatively pathogenic monogenic and polygenic variation in known and novel cancer genes among which are tp53 , atm , atr , brca2 , and ercc2 [ 4 ]  . in the last two decades , molecular genetics have greatly contributed to elucidate some of the molecular mechanisms associated with the development of soft tissue sarcomas [ 57 ]  . 
good examples are represented by desmoid fibromatosis ( the vast majority of which are associated with mutations of either the ctnnb1 or apc genes ) and gastrointestinal stromal tumors ( most often associated with mutation of the kit and pdgfra genes and far less often of the braf , sdh and nf1 genes )  . 
in this context , particularly relevant is the occurrence of gene copy number alterations as observed in well - differentiated / dedifferentiated liposarcoma , wherein the amplification of the mdm2 , cdk4 and hmga2 genes represents the key driver genetic event . pathologic diagnosis sarcomas are currently classified on the basis of their morphology , their immunophenotype , and their molecular status . 
for practical reasons , the classification scheme follows a histogenetic approach , even though currently it is no longer believed that a given mesenchymal neoplasm actually originates from a mature normal counterpart . 
interestingly , the list of lesions of unknown histogenesis ( i.e. , unknown line of differentiation ) has increased in size , reflecting the uncertainties surrounding the mechanisms of sarcomagenesis . microscopic observation of hematoxylinand eosinstained slides still represents the mainstay of sarcoma classification . 
any other ancillary technique ( immunohistochemistry and / or molecular pathology / genetics ) , even the most sophisticated , certainly represents an important complement to , but under no circumstances a replacement for , classic morphological observation . 
it should be also noted that macroscopic observation too plays a fundamental role , firstly in providing accurate reporting of the status of surgical margins , and secondly in guiding proper sampling and , therefore , posing the milestone for correct classification . 
the diagnosis of sarcoma relies upon the evaluation as well as the integration of four main features : despite the intrinsic challenge of sarcoma diagnosis , is possible to achieve a correct classification in most instances , provided that cases are approached following a rigorous 1 . 
the architecture of the vascular network . this approach possesses the great merit of reducing dramatically the number of diagnostic options , also allowing a rational choice of ancillary immunohistochemical and molecular tests . 
spindle cells are defined by the presence of an elongated cytoplasm , harboring oval nuclei that can be blunt - ended ( as typically seen in smooth muscle tumors ) ( fig.2a ) , tapering ( as seen in myofibroblastic tumors ) or pointed ( as seen most often in neural neoplasm )  . 
neoplastic cells exhibit extreme nuclear pleomorphismultiple prominent nucleoli are seen 1 3 la radiologia medica ( 2019 ) 124 : 266281 table 3 intermediate and malignant soft tissue neoplasm featuring spindle cell morphology table 5 malignant soft tissue neoplasm featuring round cell morphology dermatofibrosarcoma protuberans ( dfsp ) fibrosarcomatous dermatofibrosarcoma protuberans giant cell fibroblastoma angiomatoid malignant fibrous histiocytoma low - grade myofibroblastic sarcoma desmoid fibromatosis phosphaturic mesenchymal tumor gastrointestinal stromal tumor ( gist ) leiomyosarcoma solitary fibrous tumor synovial sarcoma infantile fibrosarcoma malignant peripheral nerve sheath tumor ( mpnst ) spindle cell liposarcoma spindle cell and sclerosing rhabdomyosarcoma intimal sarcoma undifferentiated spindle cell sarcoma table 4 intermediate and malignant soft tissue neoplasm featuring epithelioid cell morphology epithelioid sarcoma classic type epithelioid sarcoma proximal type malignant rhabdoid tumor malignant myoepithelioma ( myoepithelial carcinoma ) pseudomyogenic hemangioendothelioma ( can be spindled ) epithelioid hemangioendothelioma epithelioid angiosarcoma epithelioid malignant peripheral nerve sheath tumor clear cell sarcoma of soft parts clear cell sarcoma of gastrointestinal tract ( malignant gastrointestinal neuroectodermal tumor ) sclerosing epithelioid fibrosarcoma alveolar soft part sarcoma pecoma epithelioid pleomorphic liposarcoma epithelioid gist epithelioid myxofibrosarcoma epithelioid leiomyosarcoma epithelioid rhabdomyosarcoma epithelioid inflammatory myofibroblastic sarcoma undifferentiated epithelioid sarcoma 4 . 
soft tissue malignancies featuring a predominantly pleomorphic morphology are listed in table6 . ewing sarcoma cic - dux4 associated round cell sarcoma bcor - ccbn3 associated round cell sarcoma extraskeletal mesenchymal chondrosarcoma desmoplastic small round cell tumor alveolar rhabdomyosarcoma poorly differentiated round cell synovial sarcoma high - grade myxoid ( formerly round cell ) liposarcoma table 6 malignant soft tissue neoplasm featuring pleomorphic morphology pleomorphic rhabdomyosarcoma pleomorphic liposarcoma dedifferentiated liposarcoma extraskeletal osteosarcoma pleomorphic high - grade myxofibrosarcoma pleomorphic leiomyosarcoma pleomorphic malignant peripheral nerve sheath tumor undifferentiated pleomorphic sarcoma the patterns ofgrowth the pattern of growth of the neoplastic cell population is extremely important as helps to further refine the possible diagnostic options . 
the presence of osteogenic matrix represents prerequisite to the diagnosis of extraskeletal osteosarcoma . the architecture ofthevascular network blood vessels can variably organize to form a plexiform , archiform , and hpc - like architecture . 
 myxofibrosarcoma myxoinflammatory fibroblastic sarcoma low - grade fibromyxoid sarcoma myxoid liposarcoma extraskeletal myxoid chondrosarcoma ossifying fibromyxoid tumor embryonal rhabdomyosarcoma this patter has been variably labeled as chicken wire of crows feet depending on the imagination of the pathologist . 
the lesions associated with an hpc - like vascular network are listed in table8 . as already mentioned , the integration of the abovementioned morphologic features which is enable to reduce significantly the number of diagnostic options to the extent that in some instances ancillary technique may play a rather limited role . 
however , in many situations an accurate diagnosis may require a second step represented by application of a variable ( ideally relatively limited ) number of immunohistochemical stains . immunohistochemical characterization ofsoft tissue tumors immunohistochemical characterization plays a key role in the diagnostic workup of soft tissue sarcomas [ 8 , 9 ]  . 
the number of potential diagnostic markers has grown exponentially through the years ; however in consideration of the natural evolution of the field , some markers have lost their role while others have gained diagnostic relevance . 
it has to be underlined that , with some exceptions that will be discussed , the majority of classic differentiation markers tend to show good sensitivity , however , associated with rather limited specificity . 
to identify myogenic differentiation in dedifferentiated liposarcoma . myogenic differentiation markers classic myogenic markers are basically represented by smooth muscle actin , muscle specific actin , desmin , and h - caldesmon . demonstration of myogenic ( both smooth muscle and striated ) differentiation is clinically relevant in the following situations : 1 . 
to differentiate between rhabdomyosarcoma and nonrhabdomyosarcoma pediatric soft tissue tumors ( those two broad groups definitely undergo distinct systemic treatments )  . table 8 soft tissue neoplasm featuring an hpc - like vascular network benign soft tissue neoplasm featuring an hpc - like vascular network myofibroma / myofibromatosis myopericytoma deep - seated benign fibrous histiocytoma malignant soft tissue neoplasm featuring an hpc - like vascular network solitary fibrous tumor phosphaturic mesenchymal tumor synovial sarcoma extraskeletal mesenchymal chondrosarcoma malignant peripheral nerve sheath tumor infantile fibrosarcoma smooth muscle actin immunopositivity is observed in most smooth muscle tumors but also , in myofibroblastic , myoepithelial and pericytic neoplasmuscle specific actin ( hhf - 35 ) also stains smooth muscle lesions and , however , also represents a very sensitive markers of striated muscle differentiation , staining up to 90% of rhabdomyosarcomas of all subtypes ( embryonal , alveolar , and pleomorphic )  . 
h - caldesmon immunoreactivity is observed in most smooth muscle neoplasin contrast with other smooth muscle markers , it tends to be negative in myofibroblastic lesions as well as in rhabdomyoblastic neoplasm . the most specific and sensitive markers to demonstrate rhabdomyoblastic differentiation remain myogenin ( myf4 ) , a lineage restricted nuclear transcription factor involved in striated muscle differentiation . 
recently , it has been shown that myod1 ( myf3 ) , an alternative nuclear transcription factor involved with the development of striated muscle , represents the most sensitive marker for the spindle cell variant of rhabdomyosarcoma , wherein myod1 gene homozygous mutations have shown to occur . neural differentiation markers paradoxically , the best use of the prototypic schwannian differentiation markers , namely s100 protein , is achieved out of context of recognition of mpnst . 
in fact , approximately 1 3 276 la radiologia medica ( 2019 ) 124 : 266281 only 30% of malignant peripheral nerve sheath tumors ( mpnst ) exhibit s - 100 positivity , which is usually limited to less than 30% of neoplastic cells . 
it has to be stressed that s100 exhibits a distinctive multispecificity to the extent that its evaluation needs to be strictly performed in context with morphology . the best use of s100 immunostains is as follows : 1 . 
recognition of cartilaginous differentiation ( particularly useful to identify focal heterologous component in mpnst and dedifferentiated liposarcoma )  . another member of the group of the intermediate filaments , glial fibrillary acidic protein ( gfap ) is also detectable in schwann cells . 
gfap immunopositivity can also be observed in myoepithelial neoplas sox10 and h3k27me3 ( histone 3k27 trimethylation ) represent more recently introduced markers that can be used in the diagnosis of neural soft tissue neoplassox10 is involved in melanogenesis and schwannian differentiation , and has been proposed as a valid immunohistochemical marker for both melanocytic and neural neoplassox10 tends to immunostain the vast majority of benign neural mesenchymal lesion whereas when dealing with mpnst its sensitivity is much lower , with approximately 20% of cases showing positivity in a minority of neoplastic cells . 
 h3k27me expression tends to be lost in approximately half of mpnst as a consequence of homozygous inactivation of the polycomb repressive complex 2 ( prc2 )  . epithelial differentiation markers any soft tissue neoplasm featuring true epithelial differentiation or epithelioid morphology is characterized by variable expression of epithelial differentiation markers , namely cytokeratin and epithelial membrane antigen ( ema )  . 
epithelial membrane antigen also stains most epithelioid sarcomas , and 90% of synovial sarcoma ( including poorly differentiated ones ) , therefore , representing the most sensitive marker of epithelial differentiation in this context . endothelial differentiation markers demonstration of endothelial differentiation appears crucial when dealing with poorly differentiated vascular neoplasms , in particular epithelioid angiosarcoma that may often lacks overt vasoformative morphology . 
when dealing with vascular neoplasm cd34 is very sensitive , but it has to be stressed that is also expressed in half of epithelioid sarcomas in addition to an endless list of non - vascular spindle cell neoplasms . 
in the recent years , as a byproduct of new insights in the molecular pathogenesis of vascular neoplasm , a number of new diagnostic immunohistochemical markers have been made available . 
fosb expression is consistently observed in pseudomyogenic hemangioendothelioma ( associated with an fosb - serpin1 gene fusion ) as well as in subset of epithelioid hemangioma . melanocytic differentiation markers despite total lack of specificity s100 remains the most sensitive marker of melanocytic differentiation . 
hmb - 45 , melana ( mart1 ) and mitf1 all represent sensitive melanocytic differentiation ; however , their sensitivity drops significantly when dealing with sarcomatoid variants of malignant melanoma , wherein s100 is most often the only expressed diagnostic marker . 
they not only play an obvious role in the differential diagnosis of malignant melanoma , but they have also proved extremely helpful in recognizing the members of the clinically as well as morphologically heterogeneous 1 3 la radiologia medica ( 2019 ) 124 : 266281 family of pecomas , as well in the proper classification of clear cell sarcoma . other useful immunohistochemical markers there exist an increasing number of immunohistochemical markers that despite variable specificity play a major role in the differential diagnosis of soft tissue sarcomas . alk ( anaplastic lymphoma kinase ) cytoplasmic expression of the tyrosine kinase alk plays a key role in supporting the diagnosis of inflammatory myofibroblastic tumor ( imt ) , including the aggressive epithelioid subtypes . 
 unfortunately , approximately half of cases of imt lack alk expression . betacatenin nuclear expression of beta - catenin represents an extremely valuable confirmatory finding in the diagnosis of desmoid fibromatosis . 
nuclear accumulation is due to mutation of the ctnnb1 gene ( found in up to 90% of sporadic desmoid fibromatosis ) or alternatively , of the apc gene ( in the context of gardners syndrome )  . brachyury ( t ) nuclear expression of brachyury represents a key diagnostic feature in the recognition of chordoma and is very helpful in the differential diagnosis with chondrosarcoma , metastatic carcinoma and myoepithelial neoplasms . cd99 when dealing with the differential diagnosis of small round cell sarcomas , cd99 ( a cell surface glycoprotein normally expressed on thymic t cells ) represents a powerful diagnostic marker . 
importantly , immunopositivity in non - ewing tumors tends to be more diffuse , rarely matching the thick membrane staining typically observed in ewing sarcoma . cyclinb3 expression of the cell cycle regulator cyclin - b3 seems to be useful in the recognition of a small subset or poorly differentiated round cell sarcomas associated with a bbcor - cnnb3 gene fusion . sion is consistently observed in malignant rhabdoid tumors ( including atypical teratoid rhabdoid tumors of cns ) as a consequence of ini1 biallelic inactivation . 
loss of ini1 is also observed in up to 95% of both classic and proximal variants of epithelioid sarcoma wherein it tends to be associated with homozygous deletion of the ini1 locus . 
ini1 negativity has been described also in up to 70% of epithelioid mpnst , in 1035% of myoepithelial carcinoma , and in almost all renal medullary carcinoma . kit ( cd117 ) kit represents a tyrosine kinase receptor that is involved on the development of mast cells , melanocytes and interstitial cells of cajal . 
kit expression does not predict response to tyrosine kinase inhibitors and can be observed in unrelated neoplasm such as seminoma , thymic carcinoma , melanocytic neoplasm , and mast cell disorders . mdm2 ( murine double minutes ) mdm2 is the product of the mdm2 proto - oncogene , the main function of which is to promote cell proliferation via inhibition of tp53 . 
 mdm2 overexpression related to gene amplification is also observed in intimal sarcoma and in bone by low - grade osteosarcoma . muc4 ( mucin 4 ) muc4 is a high molecular weight transmembrane glycoprotein normally expressed in epithelial cells . 
the major diagnostic role of muc4 is represented by diagnostic confirmation of both low - grade fibromyxoid sarcoma and sclerosing epithelioid fibrosarcoma , wherein gene expression profiling has identified its upregulation . rb ( retinoblastoma gene product ) rb is the product of the rb tumor suppressor gene that acts as a potent negative regulator of cell cycle . 
the same phenomenon is observed also in atypical spindle cell lipomatous tumor . dog1 ( discovered ongist 1 ) dog1 represents a useful and sensitive marker for diagnosis of gist and is extremely helpful in rescuing at least half of kit - negative gist cases . ini1 ( smarcb1 ) ini1 also known as smarcb1 is a member of the baf molecular complex that contributes to the process of chromatin remodeling and plays a key role in regulating transcription of dna . 
satb2 is best used whenever h&e does not 1 3 278 la radiologia medica ( 2019 ) 124 : 266281 molecular characterization ofsoft tissue tumors tions . discriminate unequivocally between osteoid and sclerotic collagen , or when osteoid is minimally or not represented in the sample . stat6 nuclear expression of stat6 is consistently expressed in all variant of solitary fibrous tumors as a consequence of the nab2 - stat6 gene fusion . tfe3 tfe3 decorates the neoplastic cell population of alveolar soft part sarcoma ( as a consequence , the presence of the aspscr1 - tfe3 gene fusion leads to the overexpression of the tfe3 protein ) , but as other markers it is not entirely specific . 
in fact , it is expressed also in xp11 translocated renal cell carcinomas , in a small subset ( approximately 5% ) of perivascular epithelioid cell tumors ( pecoma ) , and in a small subset of epithelioid hemangioendothelioma . tle1 tle1 is a transcriptional co - repressor that inhibits wnt signaling . 
gene profiling studies have demonstrated high levels of tle1 in synovial sarcoma that can be detected immunohistochemically . wt1 nuclear expression of the c - terminus wt1 ( wilms tumor 1 ) can be used as a confirmatory tool in the diagnosis of desmoplastic small round cell tumor . the marriage of molecular genetics and soft tissue tumor pathology certainly represents one of the most fruitful events of the last decade . 
the close relationship between morphology ( including immunohistochemistry ) and molecular genetics was certified by the 2002 who classifications of bone and soft tissue tumors and further expended in the 2013 update [ 1 , 2 ]  . this integration has impacted on several aspects of pathology [ 1012 ] : 1 . 
as an example , the identification of the fosb - serpine1 gene fusion in pseudomyogenic hemangioendothelioma has greatly contributed to the credibility of this newly identified entity [ 13 ]  . 
the merging of myxoid liposarcoma and round cell liposarcoma into a single entity has been certainly supported by the detection in both lesions of the same rearrangement involving the chop gene . improvement ofdiagnostic accuracy from the diagnostic standpoint in the last two decades , it has become clear that molecular testing may add diagnostic accuracy in important subsets of challenging soft tissue tumors . 
their occurrence can be routinely assessed for diagnostic purposes via fish and / or pcr - based techniques . the diagnostic utility of molecular genetics can be best exemplified in the following situations : 1 . 
distinguishing sarcomas from benign mimickers . distinguishing specific subtypes of sarcomas the distinction of specific sarcoma subtypes is becoming increasingly important as more specific local as well as systemic treatments are being developed . 
round cell sarcomas include ewing sarcoma , desmoplastic small round cell tumor ( dsrct ) , alveolar rhabdomyosarcoma , poorly differentiated round cell synovial sarcoma ( pdss ) , cic - rearranged and bcor - rearranged undifferentiated round cell sarcomas and a minority of cases or high - grade liposarcoma featuring a round cell morphology . 
 1 3 la radiologia medica ( 2019 ) 124 : 266281 the demonstration by fish of ewsr1 , ss18 and foxo1 rearrangements in ewing sarcoma , rms and pdss , respectively , or , alternatively , of specific chimerical transcripts by pcr - based techniques or via an ngs - based approach , is of great help for achieving a correct diagnosis . 
as far as fish analysis of the ewsr1 gene is concerned a major caveat is represented by the fact that its rearrangement can occur in a variety of unrelated lesions . 
as a consequence , any result needs to be mandatorily interpreted in context with morphology and immunohistochemical findings . the identification of gene copy number variations has also proved extremely helpful . 
the perfect example is represented by dedifferentiated liposarcoma , a pleomorphic mostly ( but not exclusively ) retroperitoneal adipocytic malignancy that , in contrast with other sarcoma such as leiomyosarcoma , exhibits the tendency to recur locally with a comparatively lower rate of metastatic spread . 
in this context , the detection of mdm2 amplification by fish or quantitative rtpcr certainly represents a useful diagnostic adjunct [ 15 ]  . supporting diagnosis in noncanonical clinical presenta tions as a result of the widespread use of molecular pathology as a confirmatory diagnostic tool , the range of clinical presentations of many entities has broadened . 
molecular genetics has undoubtedly greatly contributed for instance to the identification of primary ewing sarcoma of the skin , kidney , and meninges , as well of synovial sarcomas occurring at visceral sites such as the lungs and the gastrointestinal tract . distinguishing true sarcomas from benign mimics as already mentioned , the morphological appearance of mesenchymal lesions not always reflects the clinical behavior . 
this is particularly true when dealing with lowgrade fibromyxoid sarcoma ( lgfms ) , a deceptively bland looking spindle cell mesenchymal malignancy characterized by an aggressive clinical behavior on long - term basis . 
the differential diagnosis of lgfms includes benign lesions such as perineurioma , neurofibroma , cellular myxoma , and nodular fasciitis , as well locally aggressive neoplasms such as desmoid fibromatosis . 
even if muc4 expression is currently regarded as a key diagnostic feature , the identification of fus rearrangement via interphase fish or the identification of either fus - creb3l2 or fus - creb3l2 transcripts represents an extremely useful diagnostic tool [ 16 ]  . 
 as mentioned , desmoid fibromatosis enters the differential diagnosis and it should be , therefore , noted that in addition to immunohistochemical detection of nuclear accumulation of - catenin , mutational analysis of the ctnnb1 gene may also represent a valuable diagnostic tool [ 17 ]  . identification ofmolecular predictive andprognostic markers during the last decade , several attempts have been made to determine the prognostic value of molecular genetic findings . 
both factors may unfortunately hamper a large - scale clinical application of cinsarc . an extremely important , clinically relevant exception is represented by gastrointestinal stromal tumors ( gist ) , wherein the type of mutations involving both the kit and pdgfra genes are associated with distinctive outcomes [ 19 ]  . 
 as examples , it is now well known that deletions occurring at the exon 11 of the kit gene are associated with more aggressive disease , whereas mutations of exon 18 of the pdgfra gene generally identify a more indolent clinical course . 
distinct mutation types reflect different objective response rates ( greater for kit exon 11 mutation and much lower for the so - called wild - type gist ) as well as different progression - free survival and overall survival . 
mutational analysis in gist impacts also over dose selection : in fact the progression - free survival of gist patients with kit exon 9 mutations is known 1 3 280 la radiologia medica ( 2019 ) 124 : 266281 to be significantly better in patients treated with 800mg of imatinib as compared to 400mg per day . it has also to be stressed how molecular pathology / genetics represents the most valuable tool in order to identify and validate new therapeutic targets . 
good examples are represented by alk in inflammatory myofibroblastic tumor [ 20 ] , mdm2 and cdk4 in dedifferentiated liposarcoma , the mtor pathway in malignant pecoma and lymphangioleiomyomatosis , pdgfb in dermatofibrosarcoma protuberans [ 21 ] , kdr in angiosarcoma , ntrk3 in gist [ 22 ] , and csf1 in giant cell tumor of tendon sheath [ 23 ]  . the grading ofsoft tissue sarcomas soft tissue sarcomas tend to behave aggressively and metastasize in a large percentage of cases . 
tumor size , location , depth , and histological type are all prognostic factors in terms of metastatic risk and overall survival . with many exceptions , histologic typing does not provide sufficient information for predicting the clinical course of the disease and , therefore , grading systems based on histological parameters were introduced to provide a more accurate estimation of the degree of malignancy of tumors . several different grading systems have been developed . 
 the most successful have been the three - tiered systems of the national cancer institute ( nci ) and the french federation of cancer centers sarcoma group ( fnclcc ) scheme . 
as summarized in table9 , each parameter generates a score the sum of which determines a final score that assigns the tumor to one of the three groups . prognostic nomograms that recently have incorporated the revised who classification of soft tissue tumors represent an additional valuable clinical tool [ 26 ]  . 
from one of these monograms , a freely downloadable application for portable devices termed sarculator has been also generated . in conclusion , soft tissue sarcomas represent a heterogeneous group of clinicopathologic distinct tumor entities . 
both immunohistochemistry and genetics have proved extremely valuable not only in increasing diagnostic accuracy , but also in refining classification and validating newly described tumor entity . table 9 fnlcc grading system tumor differentiation score 1 sarcoma closely resembling normal adult mesenchymal tissue sarcoma for which histologic typing is certain in myxoid variants expression may be weaker score 2 score 3 mitotic count score 1 score 2 score 3 tumor necrosis score 0 score 1 score 2 histologic grade grade 1 grade 2 grade 3 09 mitoses / 10hpf 1019 mitoses / 10hpf 20 mitoses / 10hpf no necrosis < 50% tumor necrosis > 50% tumor necrosis total score : 2 to 3 total score : 4 to 5 total score : 6 to 8 funding this study was not funded . compliance with ethical standards conflict of interest ap dei tos has received speaker honoraria from pfizer , lilly oncology ; pharmamar . 
first , because the imaging alone usually is not able to ensure a definitive diagnosis , ir has a basic role in the staging : the percutaneous biopsy is infact an irreplaceable step . 
furthermore , the proved safety and effectiveness of ir in a multiple oncologial applications prompt a wider use also in this field . keywords soft - tissue lesions sarcomas desmoid lesions interventional radiology biopsy introduction biopsy soft - tissue lesions are rarely encountered and belong to a large and heterogeneous group of different pathologies . 
the imaging features are of complex interpretation and since it is often difficult to accomplish a final diagnosis based on imaging alone [ 2 , 8 , 9 ] , biopsy becomes mandatory to obtain a histological diagnosis [ 24 , 10 ]  . in the last years , the field of pathological anatomy has tremendously developed . 
histological examinations as well as biochemical and molecular studies ( biomarkers ) play a capital role in the diagnosis and management of the great variety of lesions , in particular in the oncological field . 
once the diagnosis has been accomplished , the oncologist needs to know the biological features of the neoplastic tissue to step forward in the complex process of therapeutic decision - making . 
in fact , a successful biopsy mainly depends on the quality of its execution [ 11 ] ; also the surgical strategy depends on the technical success of a biopsy . 
 the extraction of the tissue represents a crucial process : if necrotic or healthy tissues are taken instead of vital and vol . : ( 0123456789 ) 1 3 254 la radiologia medica ( 2019 ) 124 : 253258 pathological ones , the procedure is useless or even harmful for the patient [ 12 ]  . 
nevertheless , only the radiologist has the possibility to choose among other techniques ( mri in particular ) and has familiarity with all the diagnostic imaging tools available . an interesting discussion is going on about the opportunity of performing an incisional biopsy ( ib ) and / or a core needle biopsy ( cnb ) [ 1315 ]  . 
 on other hand , cnb is a percutaneous procedure through which it is usually possible to obtain the right quantity of tissue to perform both histological examinations and molecular studies . 
cnb is performed in the radiological department under us , ct , fluoro or mr guidance thanks to specific needles that allow reaching of the lesions deeply located in the tissues with a minimum impairment of the structure . 
 complications are the same as encountered during ib , but with lower frequency ( from 0.1 to 10% , usually inferior to 5% ) [ 14 , 16 ]  . 
the same authors suggest a flowchart for the biopsy of soft tissue : ib is reserved only in limited cases ( lesions of the limbs ) in which the ( already performed ) cnb has resulted not diagnostic . 
they in fact , suggest the use of imaging - guided cnb as first choice for biopsy in the bone and soft - tissue lesions both in the spine and in the limbs . 
unlike lesions in the limbs , for the lesions located in the spine , if the first biopsy is not diagnostic , a second extraction must be performed under ct guidance , but with intraoperative frozen sections . 
in conclusion , ib should be performed only when a second extraction is required to obtain the final diagnosis , after a first , scarcely diagnostic cnb but only when we are studying soft - tissue lesions of the limbs . 
in another study [ 11 ] , the authors suggest that the diagnostic accuracy of cnb is better in case of metastatic lesions than in case of primitive ones . in conclusion , the stage of a soft - tissue lesion is defined with biopsy . 
with the exception of particular cases , the best cost - effectiveness ratio is obtained using imaging - guided cnb performed by an interventional radiologist . treatments the management of soft - tissue lesions is mainly surgical [ 24 ]  . 
nevertheless , ir can nowadays rely on a great variety of effective and safe treatments of ablation , such as radiofrequency , microwaves , cryoablation , focused ultrasounds , etc . , that are employed to treat lesions with homogeneous necrosis and extremely defined margins [ 1723 ]  . 
the safety profile of these treatments is very high because , apart from the optimal control in releasing energy , multiple systems to control the sensitive structures are available [ 28 ] : there are thermocouples to monitoring the temperature reached near sensitive structures or it is possible to move these sensitive structures , far from the region of ablation thanks to injection of air , carbon dioxide , saline or glucosate solutions . 
finally different complications , like bleeding , that can occur during the treatment could be managed immediately in the ir department [ 29 ]  . in addition , the endovascular treatments can be employed in the management of soft - tissue lesions . 
the second advantage is represented by the injection of a chemotherapeutic drug during the embolization ( chemoembolization ) that can increase the effectiveness of the drugs ( when administered systematically through a peripheral vein ) and leads to a better quality of life and longer relapse intervals [ 31 ]  . among the soft - tissue lesions , those well vascularized are particularly suitable for treatment with chemoor simple embolization : this technique consists in the puncture of a peripheral artery ( usually femoral or radial artery ) and the navigation of the arterial system until the vessels that feed the lesion : there it is possible ( thanks to specific material such as microcoils , glue or microparticles ) to occlude the vessels to obtain an ischemic necrosis of the lesion . some experiences were described for both the aims stated above : in particular a preoperative embolization 1 3 la radiologia medica ( 2019 ) 124 : 253258 of highly vascularized lesions reduces the perioperative risks and the size of the lesion , thus making the surgical intervention more complete and effective [ 32 ]  . 
the use of embolization as therapy for unresectable lesions [ 31 ] , in particular soft tissue sarcomas also was described and proved as effective : in particular , the selective transarterial chemoembolization , when performed as therapy for unresectable lesions seems to be very effective in terms of decreasing cancer pain and increasing tumor relapse intervals ; even if the authors of this study stated some limitations like a small cohort of patients and short followup , no complications were recorded and so this treatment can be considered also safe . among the ablative treatments instead [ 32 ] , one possible application of ir is represented by the treatment of desmoid tumors , where it has proven to be safe and effective [ 7 , 17 , 32 , 33 ]  . 
first , it gives the possibility to treat even large and multiple lesions in one session , creating a large area of ablation , with low risks of spreading the pathological cells . 
in case they are symptomatic and / or enlarged on serial exams , ablation can be considered as non - surgical treatment to control the disease [ 7 ]  . another field of application of ir is represented by the treatment of venous malformations or low - flow vascular malformations . 
these different types of soft - tissue lesions can be treated by ir as alternative to the injection of sclerosing agents currently widely accepted as first - line treatment [ 34 ]  . 
t1w fat sat follow - up 1 year after treatment with the disappearance of the lesions ( mrgfus ) [ 37 ] have proven to be safe and effective tools to control venous malformations or low - flow vascular malformations . 
the main advantage of ir over the injection of sclerosing agents is the possibility of visualizing the lesion during the treatment . finally , the keloid represents a further field of application of ir [ 7 ] , even if its use , however , is still under investigation and further research studies are needed . ir appears to be useful also in the treatment of soft tissue sarcoma ( sts ) [ 3 , 4 , 6 ] , a group of extremely heterogeneous neoplasms ( more than 100 sarcoma subtypes have been identified ) of mesenchymal origin that occur mainly in the soft tissue ( 80% ) and in the bone ( 20% )  . 
due to their rarity ( as said above , less than 1% of all cancers in the adult ) , specific groups of specialists have been created to develop an appropriate service for management ( sarcoma groups )  . 
radiotherapy and chemotherapy play a role also as neo - adjuvant treatments to reduce the size of the lesion and to allow a more effective and complete surgical treatment [ 3 , 6 ]  . 
to date , the role of ir is limited to the management of local recurrence or metastases : in selected cases , as for example in the oligometastatic disease , radiofrequency or cryoablation may be performed to prolong remission or reduce symptoms [ 3 , 4 , 17 , 38 ]  . finally , ir can represent a very useful and effective tool in cases of relapse of soft - tissue malignant lesions after surgery . 
in fact , should this event occur , because the malignant nature of the nodules can only be suspected on the imaging of follow - up , will be certainly not easy for the surgeon to propose to the patient an additional invasive treatment to remove lesions suspected for relapse . 
in these cases , ir may represent a valid solution : by means of ablation , in fact , it is possible to destroy the suspected nodules with a minimally invasive and repeatable procedure . 
 we employed cryoablation with excellent results in terms of complications ( none ) and control of disease in a long - term follow - up study ( 2years )  . conclusions the ir is growing up and consolidating its role in the management of the soft - tissue lesions . 
first , in fact , during the pathway towards the diagnosis , the imaging - guided biopsy is an irreplaceable step ( in particular an imaging - guided 1 3 la radiologia medica ( 2019 ) 124 : 253258 core needle biopsy , as the scientific evidence underlines )  . 
 moreover , in the therapeutical pathway , even if the surgery is considered the gold standard therapy , there are two types of procedures that are demonstrated safe and effective : the embolization , to reduce the risk of bleeding during surgery and to make the surgical procedure safer and easier and the ablation of the metastasis and of the relapses of lesions during the follow - up , to reduce the invasiveness of re - intervention procedures . compliance with ethical standards funding this study was not funded . conflict of interest all authors declare that they have no conflict of interest . ethical approval this article does not contain any studies with human participants performed by any of the authors . la radiologia medica ( 2019 ) 124 : 315322 radiotherapy radiotherapy foroligometastatic cancer : asurvey amongradiation oncologists oflombardy ( airolombardy ) , italy barbaraalicjajereczekfossa1 , 2 barbarabortolato3 mariannaalessandragerardi1 virginiamariaarienti3 stefaniaberlinghieri4 stefanobracelli5 michelabuglione6 mariangelacaputo7 gianpierocatalano8 luigifrancocazzaniga9 luigidecicco5 nadiadimuzio10 francescoromeofilippone9 andreifodor10 davidefranceschini11 paolofrata4 stefaniagottardo2 , 15 giovannibattistaivaldi12 antoniolaudati13 stefanomariamagrini6 elisamantero7 ilariameaglia12 saramorlino7 mauropalazzi3 fabiopiccoli9 paolaromanelli1 martascorsetti11 , 14 flaviaserafini13 lucianoscandolaro13 riccardovaldagni2 , 7 , 16 robertoorecchia2 , 17 paoloantognoni15 the lombardy section of the italian society of oncological radiotherapy ( associazione italiana di radioterapia oncologicalombardia , airol ) samanthadicuonzo1 received : 9 october 2017 / accepted : 5 december 2018 / published online : 15 december 2018 italian society of medical radiology 2018 abstract aims to evaluate the use of radiotherapy ( rt ) for oligometastatic cancer ( omc ) among radiation oncologists in lombardy , italy . methods and study design a survey with 12 items regarding data of 2016 was sent to all 34 lombardy rt centers . 
patient data items a total of 15.681 patients were treated in 2016 with external beam rt in 12 responding centers , and 1.087 patients were treated for omc ( 7% )  . 
brain , lymph node , lung , bone , liver and others were the most common treated sites ( 24% , 24% , 22% , 17% , 8% and 5% , respectively )  . 
the vast majority of patients ( 95% ) were treated with image - guided intensity - modulated rt or stereotactic rt . conclusions seven percent of all rt patients in lombardy are treated for omc . 
the initiative of multicenter and multidisciplinary collaboration has been undertaken in order to prepare the platform for prospective and / or observational studies in omc . keywords oligometastatic cancer lombardy radiotherapy survey stereotactic body radiotherapy introduction the concept of the oligometastatic cancer was introduced in 1995 by hellman and weichselbaum for defining a disease stage with a limited number of clinically detectable metastases [ 1 ]  . 
historically , metastasectomy * marianna alessandra gerardi marianna.gerardi@ieo.it extended author information available on the last page of the article has been performed in selected cases of low - burden metastatic cancer showing promising results in numerous series [ 46 ]  . 
new terms like metastasis - directed therapy ( mdt ) and local or lesion ablative treatment ( lat ) have been coined in order to distinguish the radical local strategies aiming to eradicate metastatic cancer cell deposits ( mdt , lat ) from palliative local therapies delivered with aim to alleviate symptoms ( palliative treatment ) [ 37 ]  . 
radiotherapy planning , delivery and verification development have allowed for the irradiation vol . : ( 0123456789 ) 1 3 316 la radiologia medica ( 2019 ) 124 : 315322 of small volumes with extreme precision so the delivery of high ablative doses with limited normal tissues exposure has become feasible . 
indeed , stereotactic radiotherapy is now frequently proposed in oligometastatic cancer patient and provides high local control and , in some cases , long - lasting progression - free and treatment - free intervals [ 79 ]  . 
moreover , the general information on their approach to oligometastatic cancer and the patient - , diseaseand treatment - related variables of the cases treated in lombardy in 2016 were also collected and analyzed . methods andstudy design a survey with 12 items regarding data of 2016 was sent to all 34 lombardy rt centers using emailing list of the lombardy section of the italian society of oncological radiotherapy ( associazione italiana di radioterapia oncologicalombardia , airo - l )  . 
all 13 centers responded to the general items , and 12 centers submitted basic patient / disease / treatment characteristics . specific items results general items five centers ( 5 / 13 , 38% ) consider oligometastatic cancer if number of metastases is less or equal to three metastases , and eight centers ( 8 / 13 , 62% ) define oligometastatic state if the number of metastases is less or equal to 5 . none of the responding centers defines as oligometastatic case a patient with more than five sites of disease . 
one of these three centers is a university - based comprehensive cancer center and two are large university - based general hospitals ( one with a dedicated cancer center )  . primary tumor included lung , prostate , breast , colorectal , kidney and other malignancies in 33% , 21% , 12% , 9% , 6% and 19% of all oligometastatic cancer cases treated with radiotherapy in lombardy in 2016 , respectively . 
although in the whole series , the lung was the most common primary tumor treated in a oligometastatic setting , followed by table 2 general items of the survey with the results from the 13 responding centers general items centers tot ( 13 ) 1 . 
in one high - volume center with particular expertise for gastrointestinal malignancy , colorectal tumor was the most common primary ( 28% of all cases )  . brain , lymph node , lung , bone , liver and others were the most common treated sites ( 24% , 24% , 22% , 17% , 8% and 5% of all sites , respectively )  . 
 liver oligometastases were treated in 3 out of 12 centers ( 25% of all facilities )  . the majority of patients ( 75% ) were treated for one metastasis , while 14% , 4% and 7% patients received radiotherapy to two , three and more than three lesions , respectively . 
in one of these high - volume centers , 33% of all patients treated for oligometastatic cancer received their therapy to more than three lesions . the vast majority of patients ( 95% ) were treated with image - guided intensity - modulated radiotherapy ( imrt ) or stereotactic radiotherapy , and 5% were treated with threedimensional conformal radiotherapy ( 3dcrt ) technique . discussion our survey showed that oligometastatic cancer is frequently treated with radiotherapy in lombardy . 
lombardy , a region of northern italy accounting for approximately 15% 1 3 la radiologia medica ( 2019 ) 124 : 315322 of the total country population ( 10 out of 61 millions of residents ) , hosts 34 radiotherapy facilities with 87 megavoltage units [ 21 ]  . 
oncology network ( rete oncologica lombarda , rol ) is well defined by the local health authorities [ 22 ] , and regular multidisciplinary meetings are held in the majority of hospitals [ 23 ]  . 
however , we think that our report is an important base to create the multi - institutional collaboration able to fully evaluate the role of ablative irradiation in this particular patient population . although radiotherapy is frequently employed in lombardy in the treatment of oligometastatic cancer , our survey shows that the definition of oligometastasis has not been established yet . 
the recent san gallen conference on the management of patients with advanced prostate cancer ( apccc 2017 ) reported that the panel of 60 experts - in - field did not reach consensus on the definition of oligometastatic disease [ 25 ] , although recent studies show the prognostic role of the number of metastasis [ 26 , 27 ]  . 
 sixty - one percent of the panelists voted for a limited number of bone and / or lymph nodes as a clinically meaningful definition of oligometastatic prostate cancer that helps in decision making ( local ablative treatment of all lesions vs systemic therapy ) , whereas the remaining panelists voted for more strict criteria for oligometastatic disease definition ; interestingly 10% of the panelists did not believe that oligometastatic prostate cancer does exist as a clinically meaningful entity at all . 
fourteen percent of panelists voted for two metastases , 66% for three metastases , and 20% of these panelists voted for five metastases as a cutoff for oligometastatic cancer [ 25 ]  . similarly , numerous fractionation schedules are employed for radiotherapy of oligometastatic cancer both in our survey and in the literature series [ 710 , 1219 , 28 , 29 ]  . 
this reflects the lack of consensus , lack of prospectively validated regimens and heterogeneity of clinical scenarios with regards to the treated volume of oligometastases ( ranging from small lymph node to large metastases ) and proximity to critical visceral organs . 
importantly , a significant percentage of oligometastatic patients receive ablative radiotherapy in previously treated areas ; this will require more cautious schedules with lower dose / fraction and lower total dose [ 30 ]  . the vast majority of lombardy centers used advanced technology ( imrt , igrt , stereotactic radiotherapy ) for treatment of oligometastatic patients . 
indeed , low - volume metastatic disease traditionally treated with conventionally fractionated palliative radiotherapy is now being managed by high - precision extreme hypofractionation with ablative / radical intent [ 31 ]  . 
 stereotactic radiotherapy is actually considered a synonym of image - guided extreme hypofractionation ( fraction doses 5gy given in 5 or 8 fractions ) [ 31 , 32 ] , even if originally this term was reserved for stereotactic target localization [ 33 ]  . 
in fact , only one center in our series routinely uses dose / fraction < 5gy to treat oligometastatic cancer , while four centers routinely use dose / fraction > 10gy . 
 only two centers routinely use regimens with more than five fractions . stereotactic radiotherapy differs from conventionally fractionated radiotherapy in terms of physical , technological and radiobiological aspects [ 3134 ]  . 
high doses of radiotherapy have different mechanisms of vascular and endothelial damage , increase in dna lethal harm and decrease in sublethal damage repair leading to major tumor cell death and high anti - tumor effect [ 31 , 35 ]  . 
high local control after stereotactic radiotherapy is constantly reported in all series , with rates exceeding 80% , quite higher than conventionally fractionated radiotherapy results , at low toxicity including mainly mild acute events and isolated g3g4 late events [ 720 , 29 , 3638 ]  . 
in recent series , stereotactic / hypofractionated radiotherapy with vmat has reached similar local control and a low toxicity rate as other techniques [ 39 , 40 ]  . numerous investigators observed favorable long - term outcome after radical local treatment of a selected group of non - small cell lung cancer patients with good performance 1 3 320 la radiologia medica ( 2019 ) 124 : 315322 status presenting with synchronous oligometastatic disease [ 41 ]  . 
such findings prompted the identification of oligometastatic stage in a new tnm staging system [ 42 ]  . apart from surprisingly high local control after stereotactic irradiation , new interaction between radiotherapy and immune system has been described [ 43 ]  . 
irradiation stimulates the release of signals and cytokines recruiting inflammatory cells into the tumor microenvironment like antigen - presenting cells that activate cytotoxic t cell function [ 43 ]  . 
in some rare cases , the anti - tumor effect of radiotherapy has been observed out of the radiation field and this phenomenon has been called abscopal effect [ 43 ]  . 
 these unknown mechanisms have opened a new field of radiotherapy investigation , including combination with new agents , in particular immunotherapy drugs [ 35 , 43 ]  . other new potential roles of stereotactic radiotherapy have been reported . 
for example , recent multicenter , randomized phase 2 study showed that local consolidative local therapy in non - small cell lung cancer patients with three or fewer metastases that did not progress after initial systemic therapy improved progression - free survival compared with maintenance therapy alone [ 44 ]  . 
for prostate cancer lymph node oligometastases , a propensity - scored matched analysis showed improvement in cancer - specific and overall survival with mdt over standard of care [ 46 ]  . 
these results suggest that aggressive local ablative therapy in oligometastatic cancer should be further explored in phase 3 trials . the level of evidence for use of stereotactic radiotherapy in oligometastatic cancer remains low , and the majority of the available studies are retrospective monoor multi - institutional series . 
numerous prospective and controlled trials have been undertaken in the recent years and will help to establish the role of stereotactic irradiation in the scenario of oligometastatic cancer and explore its full potential . 
our initiative showing that oligometastatic cancer is a significant proportion of all radiotherapy - treated patients in lombardy should help to create a platform for retrospective and prospective investigation . conclusions our survey showed that oligometastatic cancer is frequently treated with irradiation in lombardy . 
the initiative of multicenter and multidisciplinary collaboration has been undertaken in order to prepare the platform for prospective and / or observational studies in oligometastatic cancer . acknowledgements we thank the airo - lombardia , for the excellent coordination of the activities through the lombardy centers , and fondazione ieo - ccm . compliance with ethical standards conflict of interest the authors have no disclosure of potential conflicts of interest . ethical standards this article does not contain any studies with human participants or animals performed by any of the authors . informed consent this is a survey among radiation oncologists . 
therefore , a matched cohort analysis was performed in high risk soft tissue patients to analyse differences in terms of clinical outcome and toxicity between patients treated with concomitant radio - chemotherapy ( rtct ) and radiotherapy ( rt ) alone . materials and methods for each patient in rt group was selected a patient in the rtct group matching for age , t stage and grading . 
acute and late toxicity were recorded , overall survival , recurrence free survival and distant metastases free survival were analysed and compared between the two groups . results ninety patients were selected , half of patients underwent radio - chemotherapy and half received radiotherapy alone . 
 during the treatment grade 3 dermatitis was recorded in 15 ( 16.7% ) patients , 6 ( 6.7% ) patients associated chemotherapy and during follow up 12 ( 13.3% ) patients developed grade 2 late fibrosis , 3 ( 3.3% ) joint stiffness and 1 ( 1.1% ) patient experienced a bone fracture . 
at the time of analysis 15 ( 16.7% ) patients were dead , 6 ( 6.7% ) patients in the rtct group and 9 ( 10% ) patients in the rt group . 
tailored treatment has to be considered in elderly soft tissue patients to guarantee a better outcome in this high risk and fragile population . keywords soft tissue sarcoma radio - chemotherapy radiotherapy chemotherapy toxicity * daniela greto daniela.greto@unifi.it 1 department ofradiation oncology , azienda ospedaliero universitaria careggi , university offlorence , largo brambilla 3 , 50141florence , italy 2 department oforthopaedic oncology andreconstructive surgery , azienda ospedaliero universitaria careggi , university offlorence , florence , italy introduction soft tissue sarcomas ( stss ) are a rare disease accounting less than 1% of all adult neoplasms [ 1 ]  . 
however , the reported high rate of skin toxicity correlated with the association of radiotherapy and anthracycline - based chemotherapy [ 7 ] , and very few retrospective studies reporting the safety of concomitant treatment in soft tissue sarcoma result in an uncommon use of concurrent radio - chemotherapy in sts patients . 
for these reasons , the use of concurrent adjuvant radio - chemotherapy is infrequent in sts patients . the aim of our study is to compare clinical outcome and toxicity of two cohorts of patients matched for clinical and pathological characteristics treated with the association of radiotherapy and chemotherapy or radiotherapy alone for localised soft tissue sarcoma following upfront surgery . materials andmethods we retrospectively reviewed data from patients treated at our institution for soft tissue sarcoma between 1994 and 2014 . 
patients were eligible for this study in the presence of the following inclusion criteria : ( 1 ) histologically confirmed soft tissue sarcoma treated with upfront surgery ( 2 ) curative resection with negative margins ( 3 ) no evidence of distant metastases at the time of surgery ( 4 ) receiving post - operatively at least 60gy in 2gy fractions as adjuvant treatment with or without concurrent chemotherapy ( 5 ) in patients treated with chemotherapy , administration of at least three cycles of an anthracycline - based regimen , of whom at least one during the radiotherapy course . 
a matched - pair analysis was performed to compare rates of local control ( lc ) , distant metastases - free survival rates ( dmfs ) , overall survival ( os ) and treatment - related toxicities between patients receiving adjuvant radiotherapy ( rt ) and chemoradiotherapy ( crt ) following surgical excision of the primary tumour . for each patient in the crt group , a corresponding pair in rt group was identified by matching for age ( superior or inferior / equal to 65years ) , grading according to fnclcc [ 8 ] and t stage according to tnm classification system for sarcoma [ 9 ]  . 
late toxicity was recorded during followup that was performed every 3months for the first 2years , every 6months for 3years and annually thereafter . chemotherapy chemotherapy was considered associated to radiotherapy if patients received at least one cycle of chemotherapy concurrently to radiotherapy course . 
 five cycles of adjuvant chemotherapy were the therapeutic standard before 2012 , thereafter three cycles were administered when the italian sarcoma group and the spanish sarcoma group published a randomised clinical study demonstrating that three cycles of preoperative chemotherapy were not inferior to three preoperative cycles followed by two post - operative cycles of chemotherapy [ 10 ]  . granulocyte colony - stimulating factor was administered day 4 . 
statistical analysis was performed using the sem software ( silex development , mirefleurs , france )  . results patients andtreatmentrelated characteristics one hundred and fifty - eight patients met the inclusion criteria . 
 no significant differences in terms of gender , margin status and radiotherapy dose were found between the two groups . among patients who received chemotherapy , 24 ( 53.3% ) received 5 cycles , while in 21 ( 46.7% ) patients , three cycles were administered . 
none of the variables analysed was found as independent prognostic factor of local relapse at multivariate analysis . a total of 29 patients developed distant metastases , in particular 14 ( 15.6% ) patients in the rtct group and 15 ( 16.7% ) patients in the rt group . 
in the rtct group , among tumourrelated deaths , there were 3 ( 6.7% ) metastatic patients and 2 ( 4.4% ) patients with both local and distant relapse ; in the rt group 2 ( 4.4% ) patients had metastatic disease and 7 ( 15.6% ) patients suffered from both local and distant recurrence . 
 toxicity acute radiotherapy toxicity was recorded during radiotherapy course , and 15 ( 16.7% ) patients developed grade 1 3 la radiologia medica ( 2019 ) 124 : 301308 radioand chemotherapy compared to patients treated with radiotherapy alone for localised soft tissue sarcoma . 
 although our study has some limits such as the retrospective nature , the limited number of patients and the impossibility to analyse the differences between the histological subgroups due to the small population size , it provides a balanced evaluation of the impact of the chemotherapy in adjunction to radiotherapy on the outcome of localised soft tissue sarcoma patients . 
this study is one of the latest that reported the outcome of adjuvant chemoradiotherapy in stss , but before other studies investigated the role of post - operative chemotherapy in the same setting . 
a recent pooled analysis of two eortc phase iii trials [ 14 ] comparing adjuvant anthracycline - based chemotherapy to a control in localised soft tissue patients enrolled a total of 819 patients with a median follow - up of 8.2years. 
 in a large database , analysis included 3422 patients diagnosed with localised high - grade and large tumour ( > 8cm ) [ 15 ] ; the clinical choice to prescribe chemotherapy and / or radiotherapy in the perioperative setting and the clinical benefit of these treatments were investigated . 
at a median follow - up of 49months ( 2872months ) , the 5 - year overall survival was 62.1% in patients treated with radio - chemotherapy compared to 35.7% with surgery alone and 49.4% with radiotherapy or chemotherapy in the perioperative setting ( p < 0.001 ) ; mortality hr was reduced by 37% with radiotherapy and by 24% with chemotherapy . 
grade 3 dermatitis was recorded in 15 ( 16.7% ) patients ; of these , 6 ( 6.7% ) patients received chemotherapy ; local toxicity was developed at a median dose of 56.6gy ( range : 3866 )  . 
in locally advanced soft tissue sarcoma , surgery is the cornerstone of treatment and radiotherapy in preoperative or post - operative setting is complementary to conservative surgery to guarantee a local control comparable to radical surgery [ 12 ]  . the role of adjuvant chemotherapy in localised soft tissue sarcoma is still controversial [ 13 ]  . 
in most cases , randomised studies and retrospective series such as the present study that did not find a significant impact of chemotherapy in perioperative treatment of soft tissue sarcoma analysed a limited number of patients , and it could result in a not sufficient population size to be powered to observe survival difference . 
moreover , soft tissue sarcoma is a heterogeneous disease including more than 50 different histological subtypes with different prognosis and sensibility to chemotherapy ; the absence of subgroup analysis for histological subtypes could not individualise patients with unfavourable histology that could benefit from chemotherapy . 
 future studies with larger population stratified according to histology are necessary to identify high - risk patients who could benefit from chemotherapy . moreover , the caution to administered chemotherapy concurrent to radiotherapy is due to the well - known increased risk of toxicity ; historically , in breast cancer patients , the concurrent administration of anthracyclines with radiotherapy was correlated to high risk of radiation dermatitis and cardiac toxicity ; for this reason , a sequential treatment is preferred [ 16 ]  . 
if on the one hand , the early skin toxicity was well managed with supportive care , the authors correlated the higher late toxicity possibly to the 3d conformal radiotherapy technique that could not spare the dose to the organs at risk . 
the only bone fracture developed at 1year of follow - up occurred in a patient treated with radiotherapy alone ; in this case , a large volume of the femur was included in the treatment volume due to the bone involvement . 
no difference in toxicity was found between the two treatment groups . the acceptable toxicity profile of concurrent radiochemotherapy in this study could be explained first of all because in the 15.6% of patients , radiotherapy was delivered with helical tomotherapy , a high conformal technique that reduced the dose to the organs at risk [ 17 ]  . 
furthermore , with respect to other study that considers tumour size 8cm , in our study , the 22.2% of patients had a tumour smaller or equal to 5cm resulting in smaller treatment volume that correlates with a lower risk of local toxicity [ 18 ]  . 
 finally , to reduce the risk of radiation dermatitis , during the radiotherapy , contouring the treatment volume usually had a 3mm gap from patient surface to decrease the dose to the sk [ 19 ]  . if on the one hand chemotherapy did not affect clinical outcome , age was an important prognostic factor in the population studied . 
it is accepted that the incidence of cancer increases with age due to the summation of genetic and epigenetic mutations during the life that favours the carcinogenesis [ 20 ]  . 
moreover , elderly patients often had larger tumours due to late diagnosis and highergrade tumours probably associated to the higher incidence of genetic aberrations in elderly patients [ 21 ]  . 
surgery is the most important approach in stss , but in elderly population , the decreased performance status and comorbidities that could be associated with postoperative complications sometimes excluded this treatment . 
the authors concluded that elderly patients must be treated with a surgical approach such as the younger patients but that it is necessary to select elderly patients according to specific geriatric scale to guarantee the better approach to this vulnerable population . 
in particular , anthracycline - based chemotherapy increases the incidence of chemotherapy - related toxicity in 1 3 la radiologia medica ( 2019 ) 124 : 301308 elderly patients [ 23 ] , and the investigational use of novel agents with a better toxicity profile could be difficult considering the low presence of elderly patients in clinical targets . 
 the uncommon use of radiotherapy in this population is often correlated to logistical difficulties and patient refusal . in conclusion , this study did not demonstrate an increase in clinical outcome correlated with the administration of adjuvant chemotherapy in high - risk soft tissue sarcoma patients . 
older age is a poor prognostic factor in sts patients , and a tailored approach for elderly patients is necessary to guarantee an increase in clinical outcome in this unfavourable population . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical approval this article does not contain any studies with animals performed by any of the authors . informed consent informed consent was obtained from all individual participants included in the study . la radiologia medica ( 2019 ) 124 : 309314 radiotherapy intensitymodulated radiotherapy andhypofractionated volumetric modulated arc therapy forelderly patients withbreast cancer : comparison ofacute andlate toxicities albafiorentino1 fabianagregucci1 gianluisasicignano1 niccologiajlevra1 ruggeroruggieri1 sergiofersino1 stefanianaccarato1 albertomassocco2 stefaniecorradini3 filippoalongi1 , 4 rosariomazzola1 vanessafiglia1 francescoricchetti1 received : 26 january 2018 / accepted : 5 december 2018 / published online : 13 december 2018 italian society of medical radiology 2018 abstract purpose to evaluate the differences between conventional fractionated intensity - modulated radiotherapy ( imrt ) and hypofractionated ( hyport ) volumetric modulated arc therapy ( vmat ) in elderly women affected by early - stage breast cancer ( bc ) in terms of rt - related acute / late side effect . materials and methods between october 2011 and july 2015 , 80 consecutive elderly bc patients were treated with imrt for 5weeks ( 40 patients ) or hyport - vmat for 3weeks ( 40 patients )  . 
for patients receiving imrt or hyport - vmat , a total dose of 50gy ( 25 fractions ) or 40.5gy ( 15 fractions ) was prescribed to the whole ipsilateral breast , respectively . 
regarding late adverse events , only grade 1 skin toxicity was recorded . conclusion the present study showed that whole breast imrt and hyport - vmat are feasible and well tolerated in earlystage bc elderly patients and that hyport - vmat is affected by lower risk of acute and late rt - related side effects . keywords breast cancer elderly intensity - modulated radiotherapy volumetric modulated arc therapy * fabiana gregucci fabianagregucci@gmail.com 1 radiation oncology , cancer care center , ospedale sacro cuore don calabria , via don sempreboni 5 , 37034negrar , verona , italy 2 breast unit , cancer care center , ospedale sacro cuore don calabria , negrar , verona , italy 3 radiation oncology , ludwig - maximilians university ofmunich , munich , germany 4 university ofbrescia , brescia , italy introduction the standard of care for patients with early - stage breast cancer ( bc ) consists of lumpectomy followed by adjuvant whole breast radiotherapy ( rt ) , which results in a significant reduction in ipsilateral breast recurrences and bc - specific mortality [ 1 ]  . 
a recent randomized trial showed that boost delivery reduces the risk of local recurrence at 20years even in elderly patients over 60years of age : the gain is small but statistically significant and is linked to the compromise of an increased local risk of fibrosis [ 2 ]  . 
in recent decades , several randomized trials evaluated the role of hypofractionated vol . : ( 0123456789 ) 1 3 310 la radiologia medica ( 2019 ) 124 : 309314 rt ( hyport ) delivered over a period of 3weeks compared to the standard approach and showed equivalence in local control and survival without increased toxicity [ 35 ]  . 
the cancer and leukemia group b ( calgb ) 9343 trial randomized patients over 70years with er - positive bc to tamoxifen alone or tamoxifen plus adjuvant rt and showed a small but statistically significant reduction in ipsilateral breast tumor recurrence without impact on overall survival ( os ) in patients who received rt [ 8 ]  . 
moreover , elderly patients are more frequently affected by bc with favorable tumor biology and at the same time have a shorter overall life expectancy compared to younger patientstherefore , the potential benefit of adjuvant rt may be obfuscated [ 6 ]  . 
lately , technological and technical improvements in modern radiotherapy , such as intensity - modulated rt ( imrt ) or volumetric modulated arc therapy ( vmat ) , have become widespread available in clinical practice . 
the hypothetical advantages of imrt / vmat over three - dimensional conformal rt are : a better dose distribution within the target volume ( in terms of homogeneity ) and a reduction in high doses to healthy tissues and organs at risk [ 10 ]  . 
 although the new techniques significantly reduce the risk of acute toxicities and allow to deliver the boost simultaneously and integrated ( sib ) , modulated rt techniques are still not considered a standard of care as they have no impact on local control or patients quality of life [ 1113 ]  . 
based on this background , we hypothesized that elderly people , who often suffer from additional comorbidities that can influence rt - related toxicity [ 14 ] , could benefit from modulated rt techniques to reduce rt - related side effects . 
the objective of the present retrospective analysis was to investigate the differences regarding rt - related acute / late side effects between conventional fractionated imrt ( over 5weeks ) and hyport - vmat ( over 3weeks ) in elderly patients . materials andmethods between october 2011 and july 2015 , 80 consecutive elderly patients affected by bc were treated with breast irradiation after breast - conserving surgery : 40 patients received imrt over 5weeks , and 40 patients received hyport - vmat over 3weeks . 
rt began no later than 4weeks after surgery and / or by the end of adjuvant chemotherapy . radiation therapy all patients were immobilized in a supine position ( posiboard frame , civco inc . , orange city , ia , usa ) and underwent a planning computed tomography ( 3mm slide thickness ) without intravenous contrast . 
 a clinical target volume ( ctvbreast ) was obtained encompassing the glandular tissue of the breast , while the planning target volume ( ptvbreast ) was defined by adding a 5mm expansion in the transverse plane and 8mm in cranialcaudal direction to the ctvbreast . 
for patients who received imrt , a total dose of 50gy ( 25 fractions ) was prescribed to ptvbreast , while for patients receiving hyport - vmat 40.5gy ( 15 fractions ) was prescribed to ptvbreast . 
a total dose of 60gy in 25 fractions was prescribed to ptvboost for imrt , and a total dose of 48gy ( 15 fractions ) was prescribed for hyport - vmat . 
all vmat and imrt plans were created using 6 - mv photon beams commissioned for a varian trilogy linac ( varian medical system , palo alto , california , usa )  . 
for planning , approval was required : ptvbreast d95% 47.5gy for imrt and > 38.5gy for hyport - vmat , ptvboost d95% 57gy for imrt and > 45.6gy for hyport - vmat , respectively , a maximum dose ( d2% ) 107% to the ptvboost and a minimum dose to 95% of both ptvs not lower than 95% of their , respectively , prescribed doses . side effects evaluation acute and late side effects were evaluated using the radiation therapy oncology group ( rtog ) / eortc radiation morbidity scoring syste all patients were evaluated weekly during the treatment and every 34months for the first 2years of follow - up and then every 6months . 
 cosmetic results were scored by a four - grade scale ( excellent / good / fair / poor ) according to harris and colleagues 1 3 la radiologia medica ( 2019 ) 124 : 309314 ( harvard cosmetic scale ) , based on breast retraction and fibrosis , skin changes and match line effect [ 15 ]  . factors table 1 patients and tumor characteristics ( n = 80 ) statistical analysis to summarize the most relevant features of the clinical variables , descriptive statistic was performed . 
contingence tables with fishers exact test and chi - squared pearson test were utilized to evaluate on univariate analysis , association between the onset of skin toxicity and the following parameters : age ( < 75 vs 75years ) , breast volume ( < 700 vs 700cc ) and chemotherapy administration . results median follow - up time was 45 months ( range 3470 )  . 
regarding late adverse events , skin toxicity was overall mild without any grade 2 or higher toxicity , but resulted in significantly better outcome for patients treated with hyport - vmat . 
as observed in previous studies [ 17 ] , under - treatment and non - guideline - adherent treatment of elderly patients were recorded following breastconserving surgery , even if adjuvant whole breast rt is considered the standard of care . 
there are a variety of barriers why clinicians may not follow evidence - based recommendations in elderly patients , including the complaints of patients to undergo a time - consuming treatment lasting over a period of 56weeks , the associated comorbidities or the belief that bc may be more indolent in elderly patients [ 6 , 1820 ]  . 
indeed , adjuvant rt showed to decrease significantly the incidence of any recurrence without improving os in women older than 70years , as highlighted by the meta - analysis by the early breast cancer trialists collaborative group ( ebctcg ) [ 21 ]  . 
while two randomized trials demonstrated a modest reduction in local recurrence with no difference in breast cancer - specific survival in elderly women with early - stage estrogen receptor ( er ) - positive tumors receiving tamoxifen , the magnitude of benefit for er - negative tumors remains poorly understood [ 8 , 22 ]  . 
for this reason , the omission of rt as a standard - of - care option for elderly patients has to be evaluated with caution and tumor biology ; comorbidities , performance status and patient - related preferences have to be taken into account . 
over the last decades , several randomized trials showed equivalence of hyport delivered over 3weeks as compared to the standard approach in terms of local control and survival [ 35 ]  . 
nowadays , imrt / vmat is not considered the standard approach , even if they are superior to standard 3d conformal rt in terms of reduction in normal tissues irradiation and optimal target dose distribution [ 1013 , 2328 ]  . 
with this background , the hypothesis of our la radiologia medica ( 2019 ) 124 : 309314 study was that elderly patients , who often suffer from other comorbidities , could benefit from imrt / vmat in terms of reduction in acute and late toxicities . 
although there are some limitations in evaluating different fractionation schedules in terms of toxicity and the modality of the boost ( simultaneous concomitant ) , the 2 schedules represent the oldest ( 50gy in 5weeks ) and the newest ( 40gy in 3weeks ) standard fractionation for breast cancer . 
the present analysis showed the feasibility and tolerability of both techniques for bc patients aged over 70years : ( a ) all patients completed the imrt or hyport - vmat without discontinuation , and ( b ) no acute g3 or higher side effects and no late toxicity of > g1 was registered . 
however , a significant difference in terms of acute and late skin side effects was shown in favor of hyport - vmat , probably due its better dose homogeneity compared to imrt . 
 in fact , in a phase iii study published by morganti and colleagues , 332 patients were treated in three different schedules : historical control group ( standard 3d conformal rt , 50.4gy plus sequential 10gy electron boost , over 6weeks ) ; mara - 1 study ( imrt , 44gy plus concomitant 4gy boost , over 3weeks ) ; and mara - 2 study ( imrt , 50gy plus concomitant 10gy boost , over 5weeks ) [ 29 ]  . 
moreover , in a recent retrospective study , which analyzed the role of hyport in bc patients over 60years , g2 and g3 late skin toxicities were about 20 and 5% [ 30 ]  . 
in the present analysis , acute skin toxicity was very low with 25% and 0% of g2 and g3 for imrt patients and 2.5% and 0% for vmat patients , respectively . 
in fact , dose inhomogeneity was significantly associated with toxicity : in the study of morganti , simplified imrt was used for breast irradiation and 3d conformal rt for the boost , while in the study of lazzari etal . 
regarding the analysis of irradiated volume , larger ptv was associated with a greater risk of acute toxicity , as showed in several published studies [ 14 , 29 , 30 ]  . 
regarding the relationship of age and rt - related side effects , considering the low incidence of toxicity , this study confirmed that age over 75 was not considered a risk factor correlated to toxicity , as showed by several other studies [ 3133 ]  . 1 3 la radiologia medica ( 2019 ) 124 : 309314 conclusion whole breast imrt and hyport - vmat are feasible and well tolerated in elderly bc patients . 
despite its limitations ( small sample size , retrospective nature of the analysis and short follow - up ) , one hypothesis that emerges from this analysis is that in elderly bc patients , regardless of comorbidities and breast volume , hyport - vmat is associated with a lower risk of acute and late rt - related side effects . 
further studies are needed in this context . compliance with ethical standards conflict of interest the authors declare that they have no conflict of interest . ethical standards all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . informed consent informed consent was obtained from all individual participants included in the study . 
cova1 1uco di radiologia , 2uco di anatomia patologica , universit di trieste , ospedale di cattinara , strada di fiume 449 , i - 34149 , trieste , italy correspondence to : f . 
between january 2004 and june 2005 , 516 thyroid nodules in 420 patients ( 181 solitary thyroid nodules and 239 multiple nodules ) were prospectively evaluated with us , colour - doppler us and us - guided fna . 
the sensitivity , specificity , ppv , npv and overall accuracy values of grey - scale us were 46% , 73% , 34% , 82% and 67% , respectively , for solitary thyroid nodules and 35% , 72% , 14% , 90% and 68% , respectively , for multiple nodules . 
thyroid nodules cannot be accurately characterised using grey - scale us or colour - doppler us . key words thyroid nodule ultrasound colour - doppler us fine - needle aspiration biopsy riassunto obiettivo . 
nel periodo tra gennaio 2004 e giugno 2005 sono stati valutati prospetticamente con ecografia , eco - color doppler e agoaspirazione sotto guida ecografica 516 noduli tiroidei in 420 pazienti ( 181 pazienti portatori di un nodulo solitario e 239 pazienti con noduli multipli )  . 
nei pazienti con nodulo tiroideo solitario i valori di sensibilit , specificit , vpp , vpn e accuratezza diagnostica dellecografia in scala dei grigi sono risultati rispettivamente del 46% , 73% , 34% , 82% e 67% . 
thyroid cytology , as well , poses interpretation problems and requires knowledge of the functional status of the thyroid and the hormone status of the patient in order to be correctly interpreted . 
in fact , the approach to a patient with a thyroid nodule is complicated by the fact that no single diagnostic test is sufficiently specific and sensitive enough to allow correct characterisation . 
characterisation of a thyroid nodule requires a multidisciplinary approach based on clinical and laboratory assessment , us , scintigraphy and cytology [ 4 ] , all of which contribute to guiding the diagnosis and treatment decisions . 
 the purpose of this study was to evaluate , in a large , consecutive series of 516 thyroid nodules assessed by colourdoppler us and cytology , the correlation between the diagnosis of benign or malignant nodule at grey - scale and colourdoppler us and the cytological findings . 
la caratterizzazione del nodulo tiroideo richiede infatti un approccio multidisciplinare basato sulla valutazione clinica e laboratoristica , sullecografia , sulla scintigrafia e sulla citologia [ 4 ] che insieme concorrono allorientamento diagnostico , ed alla scelta terapeutica . scopo di questo lavoro stato valutare , su unampia casistica consecutiva di 516 noduli tiroidei studiati con eco - color doppler e con citologia , la correlazione tra la diagnosi ecografica ed eco - color doppler di benignit o di malignit del nodulo e i risultati della citologia . materials and methods between january 2004 and june 2005 , 516 thyroid nodules in 468 patients were prospectively studied with colourdoppler us and fine - needle aspiration ( fna ) cytology . 
the thyroid nodules were solitary in 181 patients ( 29 men , 152 women , age range 2683 years , mean age 56 ) and multiple ( n = 335 ) in 237 patients ( 30 men , 207 women , age range 2985 years , mean age 62 )  . 
 the us studies were carried out with digital us scanners ( technos mp , esaote biomedica , genoa , italy and hdi5000 , atl , washington , usa ) using broadband lineararray transducers ( 510mhz )  . 
as regards grey - scale us , the nodules were evaluated for echogenicity and echostructure , margins , the presence and characteristics of the peripheral halo , the presence of calcifications and their morphology and relationships with surrounding organs . 
per quanto riguarda lecografia in scala dei grigi sono stati considerati lecogenicit e lecostruttura del nodulo , le caratteristiche dei margini , la presenza e le caratteristiche dellalone periferico , la presenza di calcificazioni e la loro morfologia , i rapporti con gli organi contigui . 
in base alle loro caratteristiche i noduli tiroidei sono stati classificati come benigni , sospetti e maligni , secondo i criteri stabiliti in letteratura [ 2 , 59 ]  . 
all nodules show regular margins and a thin regular halo ( ac ) , have an almost entirely cystic ( a ) or predominantly solid hyperechoic echostructure ( b ) , with no calcifications ( a , b ) or with eggshell calcifications ( c )  . 
i noduli presentano margini regolari , alone periferico sottile e regolare ( a - c ) , ecostruttura prevalentemente cistica ( a ) , o prevalentemente solida con iperecogenicit ( b ) , assenza di calcificazioni ( a , b ) o calcificazioni a guscio ( c )  . 
il riscontro di un pattern di tipo i , ii , iv stato considerato segno di benignit , mentre il pattern di tipo iii stato considerato sospetto [ 1012 ]  . tutti i 516 noduli considerati sono stati sottoposti ad agoaspirazione ecoguidata con ago da 2527 g ed aspirazione forzata . 
la valutazione estemporanea stata soggettiva , senza effettuare la colorazione rapida e la visione dei vetrini al microscopio . tutti i noduli solitari della tiroide sottoposti ad agoaspirazione avevano dimensioni maggiori di 6 mi noduli cistici sono stati sottoposti ad evacuazione e ad analisi citologica del liquido aspirato . 
nei pazienti con gozzo multinodulare sono stati sottoposti ad agoaspirato il nodulo dominante e eventuali noduli con caratteristiche sospette allecografia in scala dei grigi e / o alleco color doppler . 
patterns i , ii and iv were considered to indicate benign disease , whereas pattern iii was regarded as suspicious [ 1012 ]  . all 516 nodules underwent fna biopsy with a 25to 27gauge needle and forced aspiration . 
the procedure was carried out by the radiologist in the presence of the cytologist in charge of processing the sample and evaluating its adequacy . immediate evaluation was subjective , without quick staining or viewing of the slides under the microscope . 
thyroid nodule with suspicious us features ( a ) due to the presence of irregular margins and focal absence of peripheral halo ( curved arrow ) ; cytologic findings , benign nodule . 
cytology findings were classified as inadequate , benign , indeterminate , suspicious or malignant , in accordance with published criteria [ 13 ]  . analisi statistica benigni , indeterminati , sospetti , maligni secondo i criteri riportati in letteratura [ 13 ]  . statistical analysis differences in age , gender and cytological diagnosis between the group of patients with solitary nodules and those with multinodular goitre were evaluated by using the mannwhitney test , fishers exact test and the chi - square test ( 2 )  . the reliability of grey - scale us and colour - doppler us in characterising thyroid nodules was assessed by calculating sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and diagnostic accuracy using cytology as the reference study . 
to calculate these values with regard to imaging , all nodules with benign features were considered to be negative ; all nodules with indeterminate or suspicious features were considered to be positive . with regard to cytology , cytologically benign nodules were considered negative , and indeterminate / suspicious or malignant nodules were considered positive . 
this subdivision is in fact arbitrary in that most indeterminate / suspicious lesions feature cells without atypia ; nonetheless , it reflects the need , in clinical practice , to monitor these patients both by using us to detect changes in the size or appearance of nodules and by using serial fna biopsy . 
 le differenze di et , sesso , diagnosi citologica riscontrate nel gruppo di pazienti con nodulo solitario e con gozzo multinodulare sono state valutate utilizzando il test di mannwhitney , il test esatto di fisher , e il test del chi quadrato ( 2 )  . 
lattendibilit dellecografia in scala dei grigi e delleco - color doppler nella caratterizzazione dei noduli tiroidei , utilizzando come indagine di riferimento la citologia , stata valutata calcolando i valori di sensibilit , specificit , valore predittivo positivo ( vpp ) , valore predittivo negativo ( vpn ) ed accuratezza diagnostica ( ad )  . 
per calcolare questi valori , per quanto riguarda limaging , sono stati considerati come negativi i noduli con caratteristiche benigne ; positivi tutti i noduli con caratteristiche indeterminate o sospetti per malignit . 
diagnosi citologica : nodulo maligno . risultati citologia abbiamo osservato che let media dei pazienti con noduli multipli era significativamente maggiore rispetto allet media dei pazienti con nodulo solitario ( test di mann - whitney , p < 0 , 001 )  . 
sia nei pazienti con noduli multipli che in quelli con nodulo solitario si osservata una netta prevalenza femminile ; tra i due gruppi di pazienti , peraltro , non sono state riscontrate differenze significative nella distribuzione tra maschi e femmine ( test esatto di fisher , p = 0 , 396 )  . sono risultati inadeguati 48 / 516 prelievi per valutazione citologica ( 9 , 3% ) , in particolare , 21 / 181 ( 11 , 6% ) prelievi in pazienti con nodulo solitario e 27 / 335 ( 8 , 1% ) prelievi in pazienti con noduli multipli . 
di questi noduli , 397 sono stati considerati benigni ( 85% ) , 63 sono stati considerati indeterminati ( 13% ) e 8 sospetti per malignit ( 2% )  . 
percentages are given in parentheses cytology solitary nodule multiple nodules inadequate benign indeterminate suspicious for malignancy total inadeguato benigno indeterminato sospetto per malignit totale 21 ( 11.6% ) 123 ( 67.9% ) 32 ( 17.7% ) 5 ( 2.8% ) 21 ( 11 , 6% ) 123 ( 67 , 9% ) 32 ( 17 , 7% ) 5 ( 2 , 8% ) 27 ( 8.1% ) 274 ( 81.8% ) 31 ( 9.2% ) 3 ( 0.9% ) 27 ( 8 , 1% ) 274 ( 81 , 8% ) 31 ( 9 , 2% ) 3 ( 0 , 9% ) tabella 1 agoaspirazione dei noduli tiroidei . 
i valori percentuali sono riportati tra parentesi citologia nodulo solitario noduli multipli cytology ecografia b - mode e eco - color doppler a total of 48 / 516 cytological samples ( 9.3% ) were considered inadequate ; in particular , 21 / 181 ( 11.6% ) samples from solitary nodules and 27 / 335 ( 8.1% ) samples from multiple nodules . 
differences in the distribution of cytological diagnoses among patients with solitary nodules and those with multiple nodules were statistically significant ( 2 = 13.98 , p < 0.003 ) , indicating a prevalence of benign nodules among patients with multiple nodules compared with those with solitary nodules ( table 1 )  . b - mode us and colour - doppler us of the nodules that subsequently underwent aspiration , 330 were considered benign on us ( 110 in patients with a solitary nodule and 220 in patients with multiple nodules ) , whereas 131 were considered suspicious ( 44 and 87 , respectively ) and seven malignant ( six and one , respectively )  . 
of the nodules with benign us appearance , 82% ( 90 / 110 ) of solitary nodules and 90% ( 198 / 220 ) of multiple nodules were found to be benign at cytology . 
of the nodules with suspicious us appearance , 29% ( 13 / 44 ) of solitary nodules and 13% ( 11 / 87 ) of multiple nodules were found to be indeterminate or suspicious at cytology . 
 considering nodules displaying benign features both on us and colour - doppler us , on the one hand , and those displaying suspicious features on either of the two techniques , on the other hand , 83% ( 83 / 99 ) of benign solitary allecografia sono stati considerati benigni 330 dei noduli successivamente aspirati ( 110 in pazienti con noduli solitari e 220 in pazienti con noduli multipli ) , mentre 131 sono stati considerati sospetti ( 44 e 87 rispettivamente ) e 7 maligni ( 6 e 1 rispettivamente )  . 
l82% ( 90 / 110 ) dei noduli solitari e il 90% ( 198 / 220 ) dei noduli multipli con caratteristiche di benignit allecografia in scala dei grigi sono risultati benigni alla citologia . 
il 29% ( 13 / 44 ) dei noduli solitari e il 13% ( 11 / 87 ) dei noduli multipli sospetti allecografia sono risultati indeterminati o sospetti per malignit alla citologia . 
il 71% ( 5 / 7 ) dei noduli maligni allecografia risultato indeterminato o sospetto per malignit alla citologia ( tabelle 2 e 3 )  . se si considerano da una parte i noduli che presentano caratteristiche di benignit sia allecografia che al color doppler , dallaltra tutti i noduli che presentano caratteristiche sospette in almeno una delle due tecniche , l83% ( 83 / 99 ) dei noduli solitari e l89% ( 157 / 176 ) dei noduli multipli benigni allecografia ed al color doppler risultato benigno alla citologia ( tabella 4 )  . 
tuttavia , solo il 34% ( 21 / 61 ) dei noduli solitari e il 11% ( 15 / 132 ) dei noduli multipli indeterminati o sospetti per malignit allecografia associata alla valutazione color doppler risultato indeterminato o sospetto per malignit alla citologia ( tabella 4 )  . attendibilit diagnostica dellecografia b - mode e delleco - color doppler la tabella 5 riporta i valori di sensibilit , specificit , vpp , vpn e accuratezza diagnostica dellecografia in scala dei grigi e dellecografia associata alleco - color doppler dei noduli solitari e dei noduli multipli utilizzando come indagine di riferimento la valutazione citologica . 
comparison of ultrasound ( us ) , colour - doppler us and cytology cytologya ( n = 160 ) ultrasound colour - doppler usb benign suspicious malignant pattern ii pattern iii pattern iv tabella 2 noduli tiroidei solitari : 181 pazienti , 21 dei quali ( 11 , 6% ) con prelievo citologico inadeguato . 
confronto tra laspetto ecografico dei noduli , il quadro eco - color doppler e i risultati della citologia citologiaa ( n = 160 ) ecografia eco - color dopplerb benigno sospetto maligno pattern ii pattern iii pattern iv benign indeterminate suspicious total a21 nodules with inadequate fine - needle aspiration ( c1 ) bno nodule showed a type i colour - doppler us pattern benigno indeterminato sospetto per malignit totale a21 noduli con agoaspirato inadeguato ( c1 ) b nessun nodulo presentava un pattern eco - color doppler di tipo i benign indeterminate suspicious total 22 0 a27 nodules with inadequate fine - needle aspiration ( c1 ) bno nodule showed a type i colour - doppler us pattern benigno indeterminato sospetto per malignit totale a27 noduli con agoaspirato inadeguato ( c1 ) bnessun nodulo presentava un pattern eco - color doppler di tipo i table 3 patients with multiple thyroid nodules : 239 patients , 335 nodules undergoing fine - needle aspiration biopsy , 27 ( 8.1% ) of which had inadequate cytology . 
comparison of ultrasound ( us ) , colour - doppler us , and cytology cytologya ( n = 308 ) ultrasound colour - doppler usb benign suspicious malignant pattern ii pattern iii pattern iv tabella 3 pazienti con multipli noduli tiroidei : 239 pazienti , 335 noduli aspirati , 27 dei quali ( 8 , 1% ) con prelievo citologico inadeguato . 
confronto tra laspetto ecografico dei noduli , il quadro eco - color doppler e i risultati della citologia citologiaa ( n = 308 ) ecografia eco - color dopplerb benigno sospetto maligno pattern ii pattern iii pattern iv 20 1 2 f . 
percentages are given in parentheses imaging cytology solitary nodulea ( n = 160 ) multiple nodulesb ( n = 308 ) benign indeterminate suspicious benign indeterminate suspicious benign suspicious features 83 ( 51.9% ) 40 ( 25.0% ) 15 ( 9.4% ) 17 ( 10.6% ) 1 ( 0.6% ) 4 ( 2.5% ) 157 ( 51.0% ) 117 ( 37.9% ) 19 ( 6.2% ) 12 ( 3.9% ) 0 ( 0.0% ) 3 ( 1.0% ) a21 nodules with inadequate fine - needle aspiration ( c1 ) b27 nodules with inadequate fine - needle aspiration ( c1 ) tabella 4 confronto tra le caratteristiche allimaging dei noduli tiroidei ( ecografia associata ad eco - color doppler ) e i risultati della citologia . 
i valori percentuali sono riportati tra parentesi imaging citologia nodulo solitarioa ( n = 160 ) noduli multiplib ( n = 308 ) benigno indeterminato benigno indeterminato sospetto per malignit sospetto per malignit benigno caratteri sospetti 83 ( 51 , 9% ) 40 ( 25 , 0% ) 15 ( 9 , 4% ) 17 ( 10 , 6% ) 1 ( 0 , 6% ) 4 ( 2 , 5% ) 157 ( 51 , 0% ) 117 ( 37 , 9% ) 19 ( 6 , 2% ) 12 ( 3 , 9% ) 0 ( 0 , 0% ) 3 ( 1 , 0% ) a 21 noduli con agoaspirato inadeguato ( c1 ) b 27 noduli con agoaspirato inadeguato ( c1 ) nodules and 89% ( 157 / 176 ) of benign multiple nodules on us and on colour - doppler us were found to be benign at cytology ( table 4 )  . 
nonetheless , only 34% ( 21 / 61 ) of solitary nodules and 11% ( 15 / 132 ) of multiple nodules that were indeterminate or suspicious on us and colour - doppler us were found to be indeterminate or suspicious at cytology ( table 4 )  . di una specificit compresa tra il 72% e il 73% , sensibilit e vpp sono compresi rispettivamente tra 35%46% e 14%34% . 
la valutazione della vascolarizzazione dei noduli al color doppler comporta un modesto aumento della sensibilit al prezzo di una riduzione della specificit . diagnostic reliability of b - mode us and colour - doppler us discussione table 5 shows the sensitivity , specificity , ppv , npv and diagnostic accuracy of grey - scale us and colour - doppler us for solitary and multiple nodules , using cytological assessment as the reference study . 
colour - doppler us assessment of nodule vascularity moderately increases sensitivity at the expense of specificity . the discovery of one or more thyroid nodules is a frequent occurrence in the general population . 
compresa tra 0 , 3% e 1 , 7% dei noduli nelle casistiche europee [ 3 ] , risulta pi elevata nelle casistiche americane , tra il 9% e il 13% dei noduli sottoposti a valutazione citologica [ 17 , 18 ]  . 
as regards cytology , only nodules classified as benign were considered negative ; all remaining nodules were considered positive solitary nodulea ( n = 160 ) multiple nodulesb ( n = 308 ) ultrasound ultrasound + colour - doppler us ultrasound ultrasound + colour - doppler us sensitivity specificity accuracy a21 nodules with inadequate fine - needle aspiration ( c1 ) b27 nodules with inadequate fine - needle aspiration ( c1 ) sensibilit specificit accuratezza a 21 noduli con agoaspirato inadeguato ( c1 ) b 27 noduli con agoaspirato inadeguato ( c1 ) tabella 5 valori di sensibilit , specificit , valore predittivo positivo ( vpp ) , valore predittivo negativo ( vpn ) e accuratezza diagnostica dellimaging ( ecografia ed ecografia associata ad eco - color doppler ) nei pazienti con nodulo solitario e in quelli con noduli multipli , utilizzando come indagine di riferimento la citologia . 
tutti gli altri noduli sono stati considerati positivi nodulo solitarioa ( n = 160 ) noduli multiplib ( n = 308 ) ecografia ecografia + doppler ecografia ecografia + doppler varies in different populations is estimated at 10%41% [ 14 , 15 ]  . 
the incidence of malignancy is similar among patients with solitary nodules and multiple nodules [ 2 , 17 , 18 ]  . the mean age of patients with multiple nodules in our series was significantly higher than that of patients with a solitary nodule , in keeping with the epidemiological data that show that the incidence of thyroid nodules increases with age . the high incidence of nodular thyroid disease and the low prevalence of malignancy call for a diagnostic workup capable of screening the limited number of nodules that require surgery . 
the approach is currently multidisciplinary , based on clinical and laboratory data , ultrasonography , scintigraphy and fna cytology . in patients with a solitary nodule , us permits detection of the nodule and offers guidance for the biopsy . 
its role is more complex in patients with multiple nodules , as only a lalta incidenza dei noduli tiroidei e la bassa prevalenza di malignit rendono necessario un iter diagnostico finalizzato a selezionare un numero limitato di noduli che necessitano di trattamento chirurgico . 
tale iter attualmente multidisciplinare , basato sulla clinica , i dati di laboratorio , lecografia , la scintigrafia e lagoaspirazione con esame citologico . nei pazienti con nodulo solitario lecografia consente lindividuazione del nodulo e la guida al prelievo per lesame citologico . 
il criterio di selezione comunemente utilizzato quello di sottoporre a prelievo per valutazione citologica il nodulo di maggiori dimensioni ; si tratta tuttavia di un criterio arbitrario in quanto diversi studi hanno dimostrato che non esiste correlazione tra le dimensioni del nodulo e la prevalenza di malignit [ 9 , 17 , 18 ]  . 
bench esista infatti una semeiotica ecografica ed eco - color doppler che stabilisce i criteri di benignit e di sospetto del nodulo tiroideo [ 3 , 511 ] , la sua reale attendibilit controversa , e la possibilit dellecografia di diffef . 
the current approach is to sample the largest nodule for cytology ; however , this is arbitrary in that several studies have shown that there is no correlation between nodule size and the prevalence of malignancy [ 9 , 17 , 18 ]  . 
this approach is also controversial . even though us and colour - doppler us imaging criteria exist for distinguishing benign and suspicious thyroid nodules [ 3 , 511 ] , their actual reliability has been questioned , and the ability of us to effectively differentiate benign from malignant thyroid nodules is currently being reconsidered [ 2 , 7 , 8 , 19 , 20 ]  . 
in agreement with previous reports [ 21 ] , it proved to be limited in identifying malignant nodules and was poorly predictive of disease in the case of positive findings . 
as a result , no us or colour - doppler us criteria exist that enable recognition of suspicious thyroid nodules and selection for cytology . our series revealed that grey - scale us had reasonable specificity for the characterisation of thyroid nodules and a reasonable npv both in solitary and in multiple nodules . this result does not , however , allow us to conclude that us is effective in correctly classifying a thyroid nodule as benign , because the high prevalence of benign nodules alone accounts for this , regardless of the real diagnostic effectiveness of the study . 
 in our series , the prevalence of cytologically suspicious or malignant lesions was significantly higher in patients with solitary nodules compared with those with multiple nodules . this finding conflicts with the recent literature , which reports a similar prevalence in the two groups [ 18 ]  . 
a possible explanation for this discrepancy is related to the different criteria used for selecting nodules for biopsy : in patients in our study with solitary nodules , all nodules larger than 6 mm were sampled , independent of their us and colour - doppler us features . 
owing to the poor sensitivity of us and colour - doppler us in characterising thyroid nodules , it is likely that some suspicious or malignant nodules were not sampled in patients with goitre because of their benign appearance on us and colour - doppler us . 
a similar issue has also been addressed by other authors who emphasised that , against the similar overall likelihood of patients with solitary or multiple nodules developing thyroid cancer , needle biopsy of the largest nodule will fail to identify approximately 1 / 3 malignant lesions in patients with multiple nodules [ 2 ]  . 
fna cytology is an accurate technique in characterising thyroid nodules [ 2 , 13 ] , but it suffers from a renziare efficacemente i noduli benigni della tiroide da quelli maligni attualmente ridimensionata [ 2 , 7 , 8 , 19 , 20 ]  . i risultati del nostro studio confermano la tendenza della letteratura . 
nella nostra casistica lecografia in scala dei grigi ha presentato bassa sensibilit e basso vpp nel riconoscere i noduli tiroidei maligni , ovvero , in accordo con la letteratura [ 21 ] , ha dimostrato scarsa capacit nellindividuare i noduli maligni associata ad una scarsa probabilit di malattia in caso di ecografia positiva . 
non esistono pertanto criteri ecografici ed eco - color doppler che consentono di riconoscere i noduli sospetti della tiroide e di focalizzare su di essi la valutazione citologica . la nostra casistica ha rilevato una discreta specificit dellecografia in scala dei grigi nella caratterizzazione del nodulo tiroideo ed un discreto vpn sia nei noduli solitari che nei pazienti con noduli multipli . 
questo risultato non consente tuttavia di concludere che lecografia efficace nel diagnosticare correttamente un nodulo tiroideo come benigno in quanto lalta prevalenza di noduli benigni spiega da sola questo risultato indipendentemente dalla reale efficacia diagnostica dellindagine . 
la valutazione doppler ha nella nostra esperienza comportato una ulteriore riduzione della specificit senza sostanziali variazioni del vpn . nella nostra casistica la prevalenza di lesioni sospette o maligne alla citologia stata significativamente maggiore nei pazienti con nodulo solitario rispetto ai pazienti con noduli multipli . 
una possibile spiegazione di questa discrepanza deriva dalle differenze nei criteri di selezione dei noduli biopsiati ; nei pazienti con nodulo solitario , infatti , sono stati biopsiati tutti i noduli di dimensioni > 6 mm , indipendentemente dalle caratteristiche ecografiche ed eco - color doppler ; nei pazienti con noduli multipli stato sottoposto a biopsia il nodulo pi voluminoso e eventuali noduli con caratteristiche sospette allecografia e / o alleco - color doppler . in conseguenza della scarsa sensibilit dellecografia e delleco - color doppler nella caratterizzazione dei noduli tiroidei verosimile che nei pazienti con gozzo alcuni noduli sospetti o maligni non siano stati sottoposti ad agoaspirazione , a causa delle loro caratteristiche ecografiche e color doppler di benignit . 
una simile problematica stata affrontata anche in altri lavori che sottolineano come , a fronte di una probabilit globale , analoga , per paziente con nodulo singolo e con noduli multipli , di sviluppare una neoplasia tiroidea , lagobiopsia nei pazienti con noduli multipli del solo nodulo di maggiori dimensioni comporta la mancata identificazione di circa 1 / 3 delle lesioni maligne [ 2 ]  . un limite di questo studio che come test di riferimento stata considerata la valutazione citologica . 
lagoaspirato tiroideo una tecnica accurata per la caratterizzazione dei noduli tiroidei [ 2 , 13 ] , ma gravato da una quota di prelievi inadeguati , da una piccola quota di falsi positivi e di falsi negativi , da una quota di prelievi con caratteristiche sospette che non possono essere caratterizzati come benigni o maligni e , soprattutto , da una quota rilevante di esami indeterminati [ 2 , 13 ]  . 
the reported rate of false - positive and false - negative results is less than 1% [ 2 ]  . the rate of inadequate samples depends on the manner in which cytological evaluation is carried out . 
a significant limitation to our study is that the cytologist did not perform quick staining or examine the material under the microscope immediately , as this procedure would make examination times longer , which would be incompatible with the departments workload . 
despite this limitation , the presence of the cytologist in the us room is useful and justified , as it reduces examination times , ensures correct slide preparation and allows a rough visual inspection of sample adequacy . 
in our series , the rate of inadequate samples was 9.3% , that is , lower than the rates of 15%20% usually reported in the literature [ 2 ]  . 
in our study , the sensitivity , specificity , ppv , npv and diagnostic accuracy of imaging were calculated by considering those nodules to be positive , that is , not benign . 
the decision to consider all suspicious and indeterminate nodules to be positive results in a lower sensitivity and ppv compared with the values that would be obtained if histology were taken as the reference method . 
on the other hand , only some of the thyroid nodules with indeterminate or suspicious cytology are removed by surgery , so that histological correlation is not available for those nodules . 
 the results of this study , in agreement with the most recent literature , confirm that us and colour - doppler us with the commonly accepted semiotic criteria are unable to provide the sufficiently accurate characterisation of thyroid nodules needed to select nodules for biopsy . 
whereas for single nodules the prevailing tendency is to cytologically sample all nodules larger than 6 mm , the problem is particularly serious in patients with goitre , as not all nodules can be aspirated , and the choice is necessarily based on us imaging . 
the sonologist and the clinician need to be aware of the limitations vi riportata in letteratura inferiore a 1% [ 2 ]  . la quota di prelievi inadeguati dipende dalle modalit con le quali viene eseguito lesame . 
un limite importante del nostro studio dato dal fatto che il citologo presente in sala ecografica non esegue una colorazione rapida e la visione estemporanea al microscopio del materiale ; questa procedura , infatti , comporterebbe un aumento inaccettabile dei tempi di esame , non compatibile con i carichi di lavoro del nostro servizio . 
nonostante questo limite , a nostro parere , la presenza del citologo in sala ecografica utile e giustificata in quanto riduce il tempo necessario per lesame , garantisce la corretta preparazione del vetrino e una valutazione , sia pure grossolana e ispettiva , della sua adeguatezza . 
nella nostra casistica i prelievi inadeguati sono risultati infatti del 9.3% , una quota inferiore a quella comunemente riportata in letteratura del 15%20% [ 2 ]  . la quota di noduli sospetti alla citologia che risulta istologicamente maligno varia secondo le diverse casistiche tra il 30% e il 65% [ 4 ]  . 
nel nostro lavoro , tuttavia , la sensibilit , specificit , vpp , vpn e accuratezza diagnostica dellimaging sono stati calcolati considerando questi noduli come positivi , e pertanto non benigni . 
la scelta di considerare come positivi i noduli sospetti e indeterminati comporta pertanto una riduzione dei valori di sensibilit e vpp , rispetto a quelli che si otterrebbero considerando come indagine di riferimento la valutazione istologica . 
daltra parte , solo una parte dei noduli tiroidei con citologia indeterminata o sospetta viene rimossa chirurgicamente e lesame istologico non pertanto disponibile per questo tipo di valutazione . conclusioni lapproccio alla diagnosi della patologia nodulare tiroidea necessariamente multidisciplinare . 
il ruolo nella caratterizzazione dei noduli invece stato progressivamente ridimensionato . i risultati di questo studio , in accordo con la letteratura pi recente , confermano come , utilizzando i criteri semeiologici comunemente accettati , lecografia e leco - color doppler non consentono una caratterizzazione accurata del nodulo tiroideo utile per orientare la scelta del nodulo da sottoporre a biopsia . 
mentre per quanto riguarda il nodulo singolo la tendenza prevalente quella di eseguire il prelievo citologico su tutti i noduli di dimensioni > 6 mm , il problema particolarmente importante nei pazienti con gozzo nei quali impossibile aspirare tutti i noduli e la scelta necessariamente basata sullimaging ecografico . 
in these patients , us follow - up and accurate nodule measurement is fundamental for identifying the fastest growing nodules , which require cytologic sampling regardless of size and morphological features . duazione di una quota di lesioni maligne . 
this paper describes the most appropriate mri techniques and sequences for the study of cardiovascular heart diseases on the basis of an analysis of mri studies carried out between january 2003 and june 2006 on 274 patients affected by all of the main congenital cardiovascular malformations , as well as a review of the literature . 
the advantages of mri with respect to other imaging techniques , the problems encountered and the main clinical applications and indications of mri , with special reference to the most common disease entities , are then discussed to define the role , the utility and the future perspectives of this imaging technique in the study of congenital heart diseases . key words congenital heart diseases cardiovascular magnetic resonance tetralogy of fallot riassunto negli ultimi decenni , importanti progressi nel trattamento medico e chirurgico delle cardiopatie congenite hanno reso pi elevate le aspettative di vita di questi pazienti , ma hanno anche aumentato il numero di coloro che necessitano di un monitoraggio continuo per identificare complicanze e difetti residui . 
la risonanza magnetica ( rm ) , in virt della sua non invasivit , riproducibilit ed accuratezza nel dettaglio morfologico e funzionale una metodica ideale per il follow - up di questi giovani pazienti . attraverso la valutazione della nostra esperienza di studio con rm nel periodo compreso dal gennaio 2003 al giugno 2006 , durante il quale sono stati valutati 274 pazienti affetti da tutte le principali malformazioni congenite del cuore e dei grossi vasi , e attraverso lanalisi della letteratura , sono descritte le modalit da adottare e le sequenze da impiegare per un corretto studio con rm di questa patologia . 
sono successivamente discussi i vantaggi rispetto alle altre metodiche dimaging , le problematiche da affrontare e le indicazioni ed applicazioni cliniche pi frequenti , con particolare approfondimento dei pi comuni quadri patologici studiati , definendo in conclusione lutilit e il ruolo di questa metodica nello studio delle cardiopatie congenite e le prospettive future . parole chiave cardiopatie congenite risonanza magnetica cardiovascolare tetralogia di fallot introduction introduzione congenital heart disease is undoubtedly the most common malformation , with an incidence of 0.5%1.2% of live births [ 1 ]  . 
these rates become even higher if minor cardiovascular defects are included that do not require treatment or are unle cardiopatie congenite rappresentano in assoluto la patologia malformativa pi frequente , con unincidenza variabile dallo 0 , 5% all1 , 2% dei nati vivi [ 1 ]  . 
advances in palliative and corrective surgical treatment and interventional procedures have dramatically improved patients life expectancy , leading to increasing numbers of patients who survive into adulthood [ 2 , 3 ]  . 
in addition , the continuous improvement of cardiovascular imaging techniques has contributed to the early detection of mild diseases that would otherwise go undetected and that , although not warranting immediate treatment , need to be followed up . 
at the same time , increasing numbers of patients need clinical and imaging follow - up over extended periods of time to identify possible postsurgical complications , or recurrences such as recoarctation , and to monitor a residual defect [ 6 ]  . 
whereas in the past , the role of imaging mainly involved detecting cardiac malformations and therefore providing morphological data , today , the importance of prognostic evaluation and follow - up , especially among adult patients [ 79 ] , requires larger amounts of both morphological and functional information , and the most accurate , reproducible and above all noninvasive imaging technique possible . magnetic resonance imaging ( mri ) , in particular , has made remarkable progress during the last few years regarding both acquisition speed and software design [ 10 ]  . 
mri offers important advantages over other imaging modalities in the study of congenital heart diseases : its high spatial and temporal resolution allow extremely accurate rendering of morphological and functional details , whereas its multiplanar capabilities and large field of view afford a panoramic view with unlimited access to thoracic and extrathoracic structures [ 11 ]  . 
i progressi nel trattamento chirurgico palliativo e correttivo e nelle procedure interventistiche delle cardiopatie congenite hanno determinato un netto miglioramento delle aspettative di vita dei pazienti , che nella maggior parte dei casi raggiungono let adulta [ 2 , 3 ]  . 
contemporaneamente , il continuo miglioramento delle metodiche dimaging cardiaco ha reso pi facile il riconoscimento delle forme lievi , che diversamente sarebbero passate inosservate e che , pur non necessitando un trattamento immediato , pongono il problema di una valutazione evolutiva . 
contemporaneamente aumentato il numero dei pazienti che necessita di una continua sorveglianza clinica e strumentale nel corso del tempo , sia per riconoscere precocemente eventuali complicanze post - chirurgiche sia per identificare la possibile ricorrenza di un aspetto malformativo , come nel caso della ricoartazione aortica , o infine per monitorare un difetto residuo [ 6 ]  . 
se in passato il ruolo dellimaging era prevalentemente basato sullidentificazione delle malformazioni cardiache e conseguentemente sul dato morfologico , oggi la valutazione prognostica e il follow - up sono divenuti elementi fondamentali , in particolare negli adulti [ 79 ] , richiedendo una maggiore ricchezza di informazioni , anche funzionali e una metodica dindagine il pi possibile accurata , riproducibile e soprattutto non invasiva . la rm in particolare stata protagonista negli ultimi anni di un notevole avanzamento tecnologico in termini sia di velocit delle sequenze che di software applicativi [ 10 ]  . 
la rm possiede alcuni significativi vantaggi rispetto alle altre metodiche dimaging nella valutazione delle anomalie congenite cardiovascolari : fornisce il dettaglio morfologico e funzionale con estrema accuratezza , in virt dellelevata risoluzione spaziale e temporale delle sue sequenze , mentre la multiplanarit e lampio campo di vista le consentono una visione panoramica senza limiti di accesso alle strutture toraciche ed extratoraciche [ 11 ]  . 
queste caratteristiche , e in particolare la non invasivit e lelevata riproducibilit dei parametri , la rendono una metodica ideale per lo studio delle cardiopatie congenite , considerando la progressiva evoluzione delle caratteristiche della popolazione di studio [ 2 , 12 ]  . alla luce della nostra esperienza nello studio morfologico e funzionale delle cardiopatie congenite con rm e attraverso lanalisi della letteratura , in questa review sono discusse lutilit e il ruolo della rm nella diagnosi , valutazione preoperatoria e follow - up di queste patologie malformative , del . 
cardiovascular mri is generally well tolerated by patients , despite an average scan duration of 4060 min . nonetheless , psychological reactions such as panic attacks or feelings of fear do occur , although more frequently among adults and adolescents than among children , who , on the other hand , are less cooperative ( age < 10 years )  . 
therefore , to avoid the risk of incomplete and poor - quality examinations , patients and / or their relatives need to be informed about the mri examination ( duration , intravenous line placement , need for breath - holding and immobility during image acquisition )  . 
for children aged 6 years or younger who are unable to follow breath - holding and immobility instructions , sedation or general anaesthesia are indicated . segmental analysis methodology the study of congenital heart disease calls for a systematic approach [ 7 , 13 ] , especially for the evaluation of complex congenital malformations and the definition of ambiguous situs , owing to the alteration of the relationships among the atria , ventricles and great vessels . 
an axial and , if necessary , a coronal stack of black - blood fast spin - echo ( bbfse ) images covering the entire thorax is sufficient . segmental analysis was performed in all patients , even those with a known diagnosis , because it defines the anatomical relationships between cardiac and extracardiac structures and helps identification of associated anomalies , which may have been missed on echocardiography ( limited field of view ) or cardiac catheterisation . mri examination : sequences all patients were studied with a 1.5 - tesla ( t ) mri scanner [ signa horizon , general electric medical systems ( gems ) , milwaukee , wi , usa ] equipped with a dedicated cardiac and thoracic coil ( cardiac and torsopa phased array ) , the latter used for the study of congenital aortic anomalies . 
the morphological study of congenital heart diseases was performed with a breath - hold electrocardiographic - triggered bbfse sequence [ double inversion recovery pulse , matrix 256224 , field of view ( fov ) 40 cm , echo time ( te ) 40 ms , repetition time ( tr ) 1 r - r interval ] acquired at end - inspiration and covering the entire thorax in the axial plane ( section thickness , 57 mm without gap ) and , if required , in the coronal and oblique sagittal plane ( tables 2 and 3 ) ( section thickness 45 mm , without gap )  . when cardiac anatomy was complex , the study was completed with a gradient - echo ( gre ) sequence with fast imaging employing steady - state acquisition ( fiesta ) ( gems ) , with repetition of the same axial and coronal images and , if diovascolare dellospedale s . 
tutti i pazienti sono stati precedentemente valutati mediante ecocardiografia transtoracica e / o transesofagea . tecnica di esecuzione dellindagine rm preparazione del paziente e criteri di inclusione le cause di esclusione dallindagine di rm sono state la presenza di un pace - maker ventricolare e / o un defibrillatore intra - cardiaco , clips metalliche endocraniche e forme severe di claustrofobia . 
lesame , pur avendo una durata media di 4060 minuti , normalmente ben tollerato , ma possono manifestarsi reazioni psicologiche quali attacchi di panico o sensazioni di paura , pi frequenti negli adulti e adolescenti rispetto ai bambini [ 9 ] , i quali daltra parte hanno una ridotta capacit collaborativa ( et < 10 anni )  . 
pertanto , per evitare il rischio di indagini incomplete e di ridotta qualit , occorre informare i pazienti e / o i loro familiari sulle caratteristiche dellesame ( durata , reperimento della via venosa , necessit di apnea e immobilit durante lacquisizione delle immagini )  . 
per quelli di et uguale o inferiore ai 6 anni , incapaci di seguire le istruzioni relative al respiro e allimmobilit , vi indicazione generale ad eseguire lesame in sedazione o anestesia generale . metodologia dellanalisi segmentaria un approccio sistematico nello studio delle cardiopatie congenite necessario [ 7 , 13 ] , in particolare nella valutazione delle malformazioni congenite complesse e nella definizione dei situus ambigui , data lalterazione delle connessioni tra atri , ventricoli e grandi vasi . 
una serie di immagini black - blood fast spin - echo ( bbfse ) su piano assiale e qualora necessario coronale , a copertura di tutto il torace , sono sufficienti . lanalisi segmentaria stata eseguita in tutti i pazienti , anche quando la diagnosi era nota , anche perch descrive i rapporti con le strutture extracardiache e facilita lidentificazione di eventuali anomalie associate , che possono non essere state mai rilevate dallecocardiogramma ( ridotto campo di vista ) o dal cateterismo cardiaco . esame rm : sequenze tutti i pazienti sono stati studiati con un magnete da 1 , 5 tesla ( signa horizon , general electric medical systems , gems , milwaukee , wisconsin , usa ) , con bobine dedicate cardiaca e toracica ( cardiac e torsopa phased array ) , questultima per lo studio delle anomalie congenite dellaorta . 
axial black - blood fast spinecho ( bbfse ) image shows inverted spatial relationships between the great arteries , the aorta ( ao ) is anterior and to the left of the pulmonary artery ( pa )  . 
note the ventricular trabeculations ( moderator band ) and the septal attachment of the tricuspid valve closer to the left - sided ventricle apex than the mitral valve ( arrows ) , both aspects that identify the right ventricle ( rv )  . 
note the right atrium ( ra ) and the typical finger - like appearance of the left atrial appendage ( asterisk ) identifying the left atrium ( la ) : atrioventricular discordance . 
limmagine assiale black blood fast spin - echo evidenzia uninversione dei rapporti spaziali dei grandi vasi , con laorta ( ao ) disposta a sinistra e anteriormente rispetto allarteria polmonare ( pa )  . 
si noti laccentuata trabecolatura ( banda moderatrice ) e lattacco settale della valvola tricuspide pi vicino allapice del ventricolo posto a snistra rispetto alla valvola mitrale ( frecce ) , entrambi aspetti che identificano il ventricolo destro ( rv )  . 
flow analysis with velocity - encoded cine mri ( vec - mri ) for the assessment of valvular regurgitation and stenosis and the semiquantitative study of vascular and prosthetic stenosis was performed by using a fast phasecontrast sequence ( pc apnea , gems )  . 
quantitative image analysis was performed on a commercially available workstation ( advanced windows 4.0 , gems ) using flow - analysis software ( flow , medis , leiden , the netherlands )  . 
contrast - enhanced mr angiography ( mra ) after intravenous injection ( flow velocity 12 ml / s ) of 0.1 mmol / kg gadobutrol ( gadovist , schering , berlin , germany ) or 0.2 mmol / kg gadopentate dimeglumine ( magnevist , schering ) in patients aged < 18 years was performed in 224 / 274 patients for the evaluation of pulmonary arteries [ tetralogy of fallot ( tof ) , bambini molto piccoli e i neonati sono stati studiati utilizzando la bobina per lo studio dellencefalo ( head , gems )  . lo studio morfologico delle cardiopatie congenite stato condotto con sequenze bbfse cardiosincronizzate ( doppio impulso di inversione , matrice 256224 , fov 40 cm , te 40 ms , tr = 1 intervallo r - r ) , in apnea inspiratoria , a copertura di tutto il torace su piano assiale ( spessore di strato 57 mm , intervallo 0 mm ) e , quando richiesto , coronale e sagittale ( tabella 2 e 3 ) ( spessore di strato 45 mm ed intervallo 0 mm )  . 
finally , the study and analysis of potential structural myocardial alterations ( fibrosis ) by evaluation of interstitial gadolinium accumulation ( late enhancement study ) , as in the case of ischaemic cardiopathy and other cardiomyopathies , was performed with a segmented inversion recovery fast gre sequence ( fgre , gems ) in patients with tof , congenitally corrected tga and tga with atrial switch repair ( table 2 )  . 
parameters included matrix 256160 , magini stata eseguita su una consolle di ricostruzione ( advanced windows , gems ) utilizzando un software di analisi del flusso ( flow , medis , leiden , olanda )  . 
langio - rm dopo iniezione endovenosa ( velocit di flusso 12 ml / s ; 0 , 1 mmoli / kg ) di gadobutrolo ( gadovist , schering , berlin , germania ) o , nei pazienti di et inferiore a 18 anni , di dimeglumina gadopentato ( magnevist , schering ) ( 0 , 2 mmoli / kg ) , stata effettuata in 224 / 274 pazienti per lo studio delle arterie polmonari ( tdf , ap ) , della coartazione aortica o di altre l . 
the natural history of many of these patients , such as those operated on for tof or tga , is greatly influenced by the risk of sequelae such as progressive ventricular dysfunction [ 14 , 15 ] , so that quantification of ventricular volumes and mass has become essential . 
its limitations ( operator dependence , acoustic window , use of geometric assumptions ) , however , lead to an underestimation of left ventricular volumes , with the risk of assigning patients to a different functional class in up to 44% of cases [ 16 ]  . 
 [ 17 ] in a meta - analysis of data on the quantification of cardiac volumes in a population of 164 patients derived from nine studies comparing mri and scintigraphy . mri has the advantage of calculating ventricular volumes , ejection fraction and mass without the use of geometric assumptions [ 18 ] and , with its high spatial and contrast resolution and reproducibility , it is now considered the gold standard for the evaluation of biventricular function [ 1923 ]  . 
another important component of functional analysis in the follow - up of surgically repaired congenital cardiovascular malformations is flow analysis for evaluation of pulmonary valve function and identification of residual intracardiac shunts in patients with surgical correction of tof , the measurement of pressure gradients in vascular and prosthetic conduit stenoses ( senning , mustard and fontan operations ) and the study of collateral circulation in patients with suspected recoarctation [ 24 ]  . 
since the 1990s , several authors [ 25 , 26 ] have validated this technique , demonstrating the optimal correlation with echocardiography ( colour - doppler ) in the measurement of transpulmonary , transaortic and transmitral valve flow and velocity , both in normal subjects and in patients with valvular disease , and even highlighting the superiority of mri over doppler ultrasonography when the acoustic window is limited . 
infine la ricerca e lanalisi delle possibili alterazioni strutturali ( fibrosi ) del miocardio valutando laccumulo interstiziale miocardico del gadolinio ( studio di late enhancement ) , analogamente a quanto avviene nella cardiopatia ischemica o in altre cardiomiopatie , stata effettuata con una sequenza segmented inversion recovery fast gre ( fgre , gems ) nei pazienti con tdf , tgv congenitamente corretta o tgv operata con switch atriale ( tabella 2 )  . 
i parametri includevano : matrice 256160 , fov 32 cm , tr / te 5 , 3 / 1 , 7 ms , flip angle 45 , spessore di strato 7 mm , intervallo 0 mm , nex 2 , tempo di inversione per lannullamento del segnale del miocardio normale , calcolato per ogni paziente , 230400 ms . ruolo e indicazioni cliniche della rm nella valutazione morfo - funzionale delle cardiopatie congenite il follow - up dei pazienti operati attualmente lindicazione prevalente allimpiego della rm nello studio delle cardiopatie congenite . 
per molti di questi pazienti , infatti , come quelli operati di tdf o tgv , la storia naturale grandemente influenzata dal rischio di sequele quali ad esempio una progressiva disfunzione ventricolare [ 14 , 15 ] ed elementi come il calcolo dei volumi e della massa ventricolari sono divenuti fondamentali . 
la metodica maggiormente utilizzata nella pratica clinica per il calcolo dei parametri di funzione ventricolare lecocardiografia transtoracica bidimensionale . tuttavia i suoi limiti ( operatore - dipendenza , finestra acustica , utilizzo di assunzioni geometriche ) portano ad una sottostima dei volumi ventricolari sinistri , con il rischio di attribuire i pazienti ad una classe funzionale diversa sino al 44% dei casi [ 16 ]  . 
 [ 17 ] attraverso la metanalisi dei dati relativi alla quantificazione dei volumi cardiaci di una popolazione di 164 pazienti , ricavata da 9 studi comparativi tra la rm e la scintigrafia . la rm ha il vantaggio di calcolare volumi , frazione deiezione e massa ventricolari senza assunzioni geometriche [ 18 ] e per la sua elevata risoluzione spaziale e di contrasto e riproducibilit , considerata oggi il gold - standard per la valutazione della funzione biventricolare [ 1923 ]  . unaltra importante componente dellanalisi funzionale nel follow - up delle cardiopatie congenite operate riguarda gli studi flussimetrici per la valutazione della funzione valvolare polmonare e il riconoscimento di shunts intracardiaci residui nei pazienti operati di tdf , il calcolo dei gradienti pressori nelle stenosi vascolari e dei condotti protesici ( interventi di senning , mustard e fontan ) e la valutazione dei circoli collaterali nel sospetto di ricoartazione [ 24 ]  . 
to the left : axial - orientated gradient - echo ( balanced fast field echo technique ) image ( four - chamber long - axis view ) shows severe dilation of the right cardiac sections . 
to the right : sagittal orientated gradient - echo ( balanced fast field echo technique ) image acquired on a plane behind the cardiac base identifies a remarkable dilation of the inferior vena cava ( ivc )  . 
ra right atriub velocity - encoded cine mri ( vec - mri ) images of the pulmonary trunk , systolic phase ( left ) and magnitude ( right )  . 
c flow curves of the pulmonary artery ( high ) and ascending aorta ( low ) demonstrate an evident predominance of pulmonary flow with a 3 : 1 ratio between pulmonary and systemic output , indicating a left - to - right shunt . 
to the left : maximum intensity projection reconstructed coronal image ( posterior view ) demonstrates anomalous pulmonary venous return with inferior vena cava drainage of all the right lung pulmonary venous blood through one collector only . 
a destra : limmagine sagittale obliqua gradient - echo con tecnica balanced fast field echo ( a destra ) passante per un piano subito a monte della base cardiaca , identifica unimportante dilatazione della vena cava inferiore ( ivc )  . 
c le curve di flusso dellarteria polmonare ( in alto ) e dellaorta ascendente ( in basso ) dimostrano una netta preponderanza di flusso a livello polmonare con un rapporto tra le gittate polmonare e sistemica 3 : 1 , indicativo di shunt sinistro - destro . 
a sinistra : limmagine di ricostruzione maximum intensity projection ( a sinistra ) su piano coronale ( visione posteriore ) evidenzia un ritorno venoso polmonare anomalo in vena cava inferiore di tutto il sangue venoso polmonare del polmone destro , mediante un unico collettore . 
a destra : limmagine di ricostruzione volume rendering sullo stesso piano ( visione anteriore ) definisce con maggiore chiarezza i rapporti anatomici tra la vena cava inferiore ( ivc ) e la vena polmonare destra ( rpv )  . 
the accurate description of these anatomical relationships is fundamental in surgical planning , as it allows the surgeon to reduce the procedure time [ 34 ]  . tetralogy of fallot despite the favourable outcome of surgery and improved life expectancy , the natural history of patients with tof is si autori [ 25 , 26 ] hanno validato questa tecnica dimostrandone lottima correlazione con lecocardiografia ( color doppler ) nel misurare velocit e flussi transvalvolari mitralici , aortici e polmonari sia in soggetti normali che in pazienti con patologia valvolare , evidenziandone addirittura la superiorit nei casi con limitata finestra acustica allecocardiografia , ma lunicit della rm dovuta soprattutto alla multiplanarit . 
la capacit di orientare il piano della sequenza nei tre assi dello spazio , ponendosi sempre perpendicolarmente al lume vasale , consente un campionamento pi preciso della velocit del sangue [ 27 ]  . 
 [ 28 ] hanno dimostrato una buona correlazione con i valori del cateterismo cardiaco . al pari dellecocardiografia , la completa non invasivit della rm legata al non utilizzo di mezzi di contrasto iodati e di radiazioni ionizzanti , aspetto non trascurabile nello studio dei bambini e / o dei neonati , ha portato al progressivo declino del cateterismo cardiaco diagnostico [ 29 ] e rappresenta un altro vantaggio di questa metodica , anche in considerazione della necessit di eseguire indagini seriate nel corso del follow - up . 
3a sagittal orientated gradient - echo ( balanced fast field echo technique ) image of the pulmonary infundibuludiastolic infundibular flow turbulence ( signal void ) ( arrows ) , indicating pulmonary valve insufficiency . 
b sequenza phase contrast : immagine di fase parallela al piano valvolare polmonare in fase sistolica ( a sinistra ) e diastolica ( a destra ) : il segnale bianco in sistole ( asterisco ) espressione di flusso anterogrado , il segnale nero in diastole ( asterisco ) indice di flusso retrogrado ( rigurgito valvolare )  . 
c curva di flusso valvolare polmonare : il rigurgito polmonare chiaramente visibile e quantificabile nel grafico . characterised by frequent complications and residual defects that require a careful follow - up [ 35 , 36 ]  . 
ventricular septal defects , residual or recurrent pulmonary artery stenoses , right ventricular outflow tract aneurysms , pulmonary regurgitation and consequent right ventricular dysfunction [ 27 , 37 ] represent the main issues in the natural history of patients after correction of tof . 
it has been associated with right ventricular dilation , progressive ventricular dysfunction , reduced exercise tolerance and finally increased risk for atrial and ventricular arrhythmias and sudden cardiac death [ 3941 ]  . 
laccurata descrizione di tali rapporti anatomici estremamente importante per il chirurgo in previsione di un intervento correttivo , in quanto consente di ridurre i tempi delle procedure [ 34 ]  . tetralogia di fallot nonostante la ridotta mortalit chirurgica e la migliorata aspettativa di vita , la storia naturale di questi pazienti contraddistinta da frequenti complicanze e difetti residui che richiedono un attento monitoraggio nel tempo [ 35 , 36 ]  . 
note also the remarkable dilation of the right atriuc sagittal orientated gradient - echo ( balanced fast field echo technique ) images ( short axis view ) at end diastole ( to the left ) and end systole ( to the right ) demonstrate reduced right ventricle contractility and , above all , a severe ventricular infundibulum dyskinesia ( pseudoaneurysm )  . 
d same patient : the graphic synthesises the right ventricle volumes and functional values obtained by outlining endocardial and epicardial borders in end diastole and end systole of all images acquired from the base to the cardiac apex . 
c le immagini sagittali oblique gradient - echo ( tecnica balanced fast field echo ) ( asse corto cardiaco ) in fase telediastolica ( a sinistra ) e telesistolica ( a destra ) dimostrano una ridotta capacit contrattile del ventricolo destro ed in particolare un aspetto severamente discinetico dellinfundibolo ventricolare ( pseudoaneurisma )  . 
d stesso paziente , nel grafico sono sintetizzati i valori dei volumi e dei parametri funzionali del ventricolo destro ottenuti mediante delineazione dei bordi epicardici ed endocardici nelle fasi telediastoliche e telesistoliche di tutte le immagini acquisite dalla base allapice cardiaco . 
designated cutoff values of end - diastolic and end - systolic volumes that , if exceeded , would prevent normalisation of right ventricular volumes , even after homograft placement [ 45 ]  . 
exercise mri , despite the limited availability of systems , is a useful application for the study of both ventricular function and pulmonary regurgitation and is effective in identifying a pulmonary / ventricular dysfunction not detectable at rest . 
 [ 49 ] observed an abnormal response to exercise in 15 patients with corrected tof and normal ejection fraction at rest compared with the control group , characterised by an increase in end - diastolic volume index without a significant change in ejection fraction . 
another recent mri application for the prognostic evaluation of these patients is the late enhancement study of the right ventricular myocardium , in particular the outflow tract , for the analysis of structural alterations ( fibrosis ) as an organic basis of arrhythmias and progressive right ventricular dysfunction [ 50 ]  . 
moreover , vec - mri is almost unique among noninvasive imaging techniques in its capacity to accurately measure flow velocity and pressure gradients through pulmonary stenosis , especially in pulmonary arterial ramifications [ 25 ]  . 
another application of vec - mri in this field is identification of patients with flow disparity in the pulmonary arteries , quantifying the differential contribution of the main - branch pulmonary arteries to pulmonary regurgitation [ 52 ]  . 
finally , mri identifies pulmonary - systemic palliative shunt complications ( stenosis , obstructions , leakages , dilations ) with mra and residual septal defects either directly , by visualising flow turbulence across the defect ( gre sequences ) or indirectly ( vec - mri ) , by calculating aortic and pulmonary artery flow ratio ( pulmonary stroke volume / systemic stroke volume ) [ 27 , 53 , 54 ]  . the completeness and accuracy of information provided by mri in a single examination session make it the modality of choice in the follow - up and prognostic evaluation of tof . transposition of the great arteries tga is a congenital anomaly characterised by an atrioventricular concordance and a ventriculoarterial discordance and is consequently a life - threatening condition that requires surgical correction early in life . 
 [ 45 ] hanno individuato dei valori cut - off per i volumi telediastolici e telesistolici superati i quali non si avrebbe la normalizzazione dei volumi ventricolari destri anche dopo posizionamento dellhomograft [ 45 ]  . 
la rm con test da sforzo , pur limitata dalla disponibilit delle apparecchiature , unutile applicazione dello studio sia della funzione ventricolare che del rigurgito polmonare , efficace nellindividuare una disfunzione ventricolare e / o polmonare non osservabile in condizioni di riposo . 
 [ 49 ] hanno evidenziato in 15 pazienti operati per tdf con frazione deiezione normale a riposo rispetto ai controlli , una risposta alterata allo stress fisico , caratterizzata da un aumento dei volumi telediastolici in assenza di variazioni significative della frazione deiezione . 
unulteriore recente applicazione della rm per la valutazione prognostica di questi pazienti lo studio di late enhancement del miocardio ventricolare destro , in particolare del tratto defflusso , per lanalisi delle alterazioni strutturali ( fibrosi ) come base organica delle aritmie e della progressiva disfunzione ventricolare destra [ 50 ]  . 
the diameter disparity between the two main pulmonary arteries is clearly depicted , without circumscribed stenosis of the left pulmonary artery ( lpa ) ( left pulmonary artery hypoplasia )  . 
d the graphics of the flow curves of right ( high ) and left ( low ) pulmonary arteries quantify the flow disparity between the two arteries ( left pulmonary artery hypoplasia ) and allow evaluation of their different contributions to pulmonary regurgitation . 
si apprezza meglio la disparit di calibro tra le due arterie , senza per significative stenosi circoscritte dellarteria polmonare sinistra ( lpa ) ( ipoplasia dellarteria polmonare sinistra )  . 
d i grafici delle curve di flusso dellarteria polmonare destra ( in alto ) e sinistra ( in basso ) quantificano la netta disparit di flusso tra i due rami polmonari ( ipoplasia dellarteria polmonare sinistra ) e consentono la valutazione del loro diverso contributo allinsufficienza polmonare . choice is the jatene procedure ( arterial switch ) [ 55 ]  . 
infine la rm identifica le complicanze degli shunts palliativi sistemico - polmonari ( stenosi , ostruzioni , leakage , dilatazioni ) con langio - rm e i difetti settali residui sia direttamente , visualizzando una turbolenza di flusso attraverso il l . 
a sinistra : immagine sagittale obliqua black - blood fast spin - echo : si apprezza una evidente ipoplasia dellarco distale e una riduzione di calibro moderata del tratto istmico . 
i grafici dei flussi a livello istmico ( in alto ) e diaframmatico ( in basso ) evidenziano un netto incremento dei flussi in sede aortica distale ( 36% ) indicativo di significativi circoli collaterali ( paziente in valutazione per intervento correttivo )  . monary arteries or right ventricular outflow tract [ 58 , 59 ]  . mri accurately evaluates the morphology of the great arteries after arterial switch , either with bbfse or gre sequences . 
great significance is held by mra for the study of pulmonary artery morphology and the measurement of aortic diameters . the anatomy of the coronary arteries has to be accurately considered as well , because coronary artery reimplantations may become dilated or distorted , with a risk of ostial stenosis , which was also been demonstrated in asymptomatic patients [ 60 ]  . 
le complicanze maggiori nel corso del follow - up sono : la dilatazione della radice aortica , linsufficienza valvolare aortica e le stenosi del tratto defflusso polmonare o delle arterie polmonari [ 58 , 59 ]  . 
grande importanza riveste langio - rm nello studio della morfologia delle arterie polmonari e nelle misurazioni dei diametri aortici . lanatomia delle coronarie va anchessa accuratamente considerata , in quanto i reimpianti coronarici possono dilatarsi o distorcersi con il rischio di stenosi ostiali , dimostrate anche in pazienti asintomatici [ 60 ]  . 
in questi pazienti , pur sopravvivendo sino allet adulta , il ventricolo destro rimane connesso allaorta ed sottoposto a pressioni sistemiche , favorendo la progressiva comparsa di alterazioni della funzione ventricolare sia a riposo che dopo esercizio fisico [ 15 , 62 ]  . 
essi costituiscono una popolazione la cui storia naturale caratterizzata da una percentuale di disfunzione ventricolare destra sino al 10% [ 63 , 64 ] e necessitano di continui controlli per monitorare la funzione ventricolare destra ( ventricolo destro sistemico )  . 
in aggiunta al dato funzionale la rm si avvale anche delle sequenze per lo studio del late enhancement ( tabella 3 ) che identificano eventuali alterazioni strutturali miocardiche ( fibrosi ) del ventricolo sistemico , come recentemente dimostrato da alcuni studi , che hanno inoltre evidenziato una correlazione tra la presenza e / o lestensione delle aree di iperenhancement e il grado di disfunzione ventricolare , let e alcuni parametri elettrofisiologici [ 67 , 68 ]  . 
la rm efficace sia nello studio pre - operatorio , che nel follow - up post - operatorio . lampio campo di vista e la multiplanarit consentono uno studio accurato dellanatomia spesso distorta dellaorta in sede istmica nei pazienti operati , che al contrario rendono ancora pi difficile lapproccio ecocardiografico . 
esame eseguito in anestesia generale . despite survival into adulthood , in these patients , the right ventricle remains connected to the aorta and is subjected to systemic pressures , facilitating the progressive appearance of ventricular function alterations both at rest and after exercise [ 15 , 62 ]  . 
these patients represent a population whose natural history is characterised by a rate of right ventricular dysfunction up to 10% [ 63 , 64 ] and who need periodic follow - up studies to monitor right ventricular contractile function ( systemic right ventricle )  . 
in addition to functional data , mri also makes use of late enhancement sequences ( table 3 ) that identify possible structural myocardial damage ( fibrosis ) of the systemic ventricle , as recently demonstrated by some studies , which also showed a relation between presence and / or extension of areas of hyperenhancement and the extent of ventricular dysfunction , age and some electrophysiological parameters [ 67 , 68 ]  . 
the large fov and multiplanar views permit accurate evaluation of the aortic anatomy of the isthmus , which is frequently distorted in postoperative patients and makes echocardiography even more difficult . 
even after surgical correction , these patients are exposed to the risk of developing complications such as recoarctation , patch pseudoaneurysm and hypertension secondary to residual arch hypoplasia [ 7 , 72 ]  . 
more recently , ther authors showed that mri is effective in the evaluation of the haemodynamic significance of the aortic coarctation [ 76 , 77 ] , and recommend its use above all in uncertain cases and in the presence of mild or moderate coarctation where no collaterals are seen on bbfse or mra images . 
in the latter case , mri might contribute to the identification of those patients at higher risk for medullary ischaemia if submitted to surgical replacement of coarctated aorta [ 76 ]  . 
these data demonstrate that transthoracic echocardiography has remained the dominant diagnostic imaging modality in the study of congenital heart pseudoaneurisma del patch , lipertensione da ipoplasia residua dellarco [ 7 , 72 ]  . 
laccuratezza e la riproducibilit delle misure sono importanti nel valutare gli indici di ricoartazione e nel follow - up degli aneurismi del patch prima dellintervento correttivo chirurgico o endovascolare . la grande potenzialit della rm consiste nel valutare il significato funzionale della stenosi . 
 [ 75 ] hanno dimostrato che la percentuale di incremento di flusso dallaorta discendente prossimale a quella distale correlata alla severit della stenosi e pi recentemente altri autori sottolineano laffidabilit della rm nel valutare il significato emodinamico della coartazione aortica [ 76 , 77 ] , raccomandandone limpiego soprattutto nei casi dubbi o nelle coartazioni lievi - moderate in mancanza di circoli collaterali evidenti nelle immagini bbfse o di angio - rm . 
questi dati dimostrano che lecocardiografia transtoracica ancora la metodica dindagine diagnostica dominante nello studio delle cardiopatie congenite nei bambini , in virt della sua non invasivit , semplicit e pronta disponibilit , senza considerare che la maggioranza di questi pazienti ha unottimale finestra acustica per le strutture cardiache . 
nel corso degli ultimi anni comunque emersa limportanza della rm per lo studio di questi pazienti , capace di superare i limiti dellecocardiografia nel visualizzare le strutture vascolari extracardiache e quelli del cateterismo cardiaco [ 79 , 80 ] , alternativa tradizionale allindagine ecografica , ma non esente dai rischi connessi alla sua natura invasiva . 
routine use of this technique continues to be hampered by several technical challenges ( smaller dimensions of the anatomical structures being imaged , faster heart rate ) requiring strategies to improve spatial and temporal resolution , and by the need for sedation in smaller , and thus noncooperative , children to avoid the deterioration of image quality . 
general anaesthesia with endotracheal intubation overcomes these limitations , achieving adequate sedation and airway protection and allowing acquisition of breath - hold sequences with a reduction of cardiorespiratory motion artefacts . 
several authors have advocated this approach for the study of congenital heart diseases in infants and small children , emphasising its safety and the low risk of complications [ 80 , 82 , 83 ]  . 
nevertheless , close interdisciplinary collaboration among the various specialists involved ( anaesthetist , radiologist , cardiologist ) is required to overcome the logistic and organisational difficulties associated with this approach . future perspectives although cardiac ultrasound is currently the first - line examination for the evaluation of congenital heart diseases , mri has now assumed a fundamental and well - defined role in the diagnosis and prognostic evaluation of these diseases in adults , particularly in the follow - up of postoperative patients and in surgical timing . 
in detail , mri is superior to the other imaging modalities for assessment of right ventricular function , quantification of pulmonary regurgitation in corrected tof , study of systemic ventricle and confirmation of suspected recoarctation . 
these are the main and most common indications for an mri study . it should , however , be remembered that mri is a complementary examination that does not replace echocardiography . 
in newborns and small children , echocardiography remains the primary technique , whereas mri is employed in carefully selected cases and for some specific indications [ 80 ] , even though its role is destined to gain importance in this field . 
la sedazione pu essere ottenuta con varie modalit di medicazione ed sicuramente un approccio adeguato [ 30 , 81 ] , vi sono per alcuni rischi legati alla mancata protezione delle vie aeree , mentre gli accorgimenti tecnici per evitare gli artefatti da respiro allungano notevolmente i tempi dellindagine . 
lanestesia generale con intubazione endotracheale supera questi limiti raggiungendo unadeguata sedazione , proteggendo le vie aeree e permettendo lesecuzione di sequenze in apnea respiratoria riducendo gli artefatti da movimento cardio - respiratorio . 
diversi autori hanno gi auspicato questo tipo di approccio per lo studio delle cardiopatie congenite nei neonati e nei bambini molto piccoli , sottolineandone la sicurezza e il basso rischio di complicanze [ 80 , 82 , 83 ]  . 
comunque necessaria una stretta collaborazione interdisciplinare tra i diversi specialisti coinvolti ( anestesisti , radiologi e cardiologi ) per superare le difficolt logistiche ed organizzative che questo tipo di esame pone . prospettive future nonostante lecocardiografia sia ancora oggi lindagine di prima istanza nello studio delle cardiopatie congenite , la rm ha oramai assunto un ruolo fondamentale e ben preciso nelliter diagnostico e nella valutazione prognostica di queste patologie negli adulti , in particolare nel follow - up dei pazienti operati , fornendo informazioni indispensabili per una corretta valutazione del timing chirurgico . 
nel dettaglio la rm superiore alle altre metodiche nella valutazione della funzione del ventricolo destro , nella quantificazione dellinsufficienza polmonare nei fallot operati , nello studio del ventricolo sistemico e nel confermare un sospetto di ricoartazione . 
nei neonati e nei primi anni di vita lecocardiografia resta ancora oggi la metodica primaria , mentre la rm impiegata in casi accuratamente selezionati e per alcune indicazioni specifiche [ 80 ] , anche se il suo ruolo destinato a divenire sempre pi importante in questo ambito , anche grazie alla sicurezza dellapproccio mediante anestesia generale . 
new perspectives will open , with the routine use of sequences characterised by faster acquisition times and adequate temporal and spatial resolution , such as real - time acquisitions or isotropic imaging [ 84 , 85 ]  . 
exercise mri , in particular , is able to provide additional data to the prognostic evaluation of these patients , even though the real clinical impact of these new applications needs to be evaluated in large follow - up studies . 
it is , however , essential to have an optimal knowledge of the anatomical and pathophysiological features of congenital heart diseases and their surgical treatment to be able to perform adequate mri examinations with shorter scan times and improved patient compliance . nel futuro prossimo la migliorata disponibilit di apparecchi dedicati localizzati in tutta prossimit dei reparti di afferenza e delle sale angiografiche , consentiranno un impiego progressivamente maggiore della rm . 
nuove prospettive si apriranno con limpiego routinario di sequenze con tempi di acquisizione pi rapidi e adeguata risoluzione temporale e spaziale come le acquisizioni in real time o limaging isotropico [ 84 , 85 ]  . 
in particolare la rm con test da sforzo capace di fornire dati aggiuntivi alla valutazione prognostica di questi pazienti , anche se il reale impatto sulla pratica clinica di queste nuove applicazioni dovr essere valutato mediante studi di follow - up su ampie popolazioni di pazienti . 
for each episode , we retrospectively reviewed the baseline chest radiographs obtained before the diagnosis of abpa , those obtained during the course of abpa and those obtained during follow - up . 
radiographic findings that had appeared at the time of abpa diagnosis and disappeared after treatment were considered related to abpa and thus useful for a correct diagnosis of the disease . 
radiographic findings at the time of abpa diagnosis appeared to have deteriorated in 8 / 14 cases when compared with the baseline films ; after treatment , the radiographic findings deteriorated in 6 / 14 cases and improved in 6 / 14 . 
the most significant among the radiographic signs considered ( infiltrates and mucoid impaction ) appeared at the time of abpa diagnosis in 7 / 14 and 4 / 14 cases , respectively , and in some patients , they were also present at baseline and persisted during follow - up . 
the most significant abnormalities are nonspecific and commonly seen on baseline films in cystic fibrosis without abpa and persist after treatment in most cases . key words abpa cystic fibrosis in adult patients chest x - ray riassunto obiettivo . 
sono stati individuati , tra i pazienti seguiti dal centro regionale per la fibrosi cistica , 11 soggetti ( per un totale di 14 episodi di malattia ) con diagnosi di abpa . 
per ciascun episodio stato retrospettivamente analizzato lultimo radiogramma toracico antecedente la diagnosi di abpa , quello eseguito in corso di abpa e quello successivo alla terapia , ricercando la presenza di ispessimento delle pareti bronchiali , bronchiettasie , consolidamenti , atelettasie , impatti mucoidi , linfoadenopatie , versamento pleurico e livelli idro - aerei . 
il quadro radiografico alla diagnosi di abpa era , rispetto ai precedenti , peggiorato in 8 / 14 casi ; dopo terapia i reperti toracici erano peggiorati in 6 / 14 casi e migliorati in 6 / 14 . tra i segni radiografici considerati , i reperti maggiormente significativi ( consolidamenti e impatti mucoidi ) erano comparsi , alla diagnosi di abpa , rispettivamente in 7 / 14 e 4 / 14 casi e in alcuni pazienti erano presenti gi al radiogramma di base e persistevano dopo terapia . 
i risultati evidenziano la scarsa utilit del radiogramma toracico nella diagnosi di abpa nei pazienti con fibrosi cistica : le lesioni pi significative possono essere frequentemente osservate nei pazienti fibrocistici in assenza di abpa , e spesso persistono anche dopo terapia . parole chiave abpa fibrosi cistica nelladulto rx torace v . 
the disease is caused by aspergillus fumigatus ( af ) , a ubiquitous , thermotolerant fungus with a spore size of 35 thanks to these properties , if inhaled , the fungus may cause severe damage to the respiratory systethis is particularly true in subjects affected by diseases in which increased density of secretions and / or altered mucociliary clearance facilitate the trapping of spores within the bronchial lumen , as occurs in asthma and cf . there is no evidence of airway invasion by the microorganism , but persistent colonisation of the tracheobronchial tree produces a hypersensitivity reaction with immune cell activation ( t cd4 + lymphocytes ) , humoral response ( b lymphocytes producing both local and serum immunoglobulin ige , iga and igg ) and eosinophil production [ 1 , 2 , 68 ]  . 
the intense inflammatory response damages the bronchial walls , leading to central bronchiectasis and results in the formation of mucous plugs , atelectasis and eosinophilic infiltrates . clinical presentation of abpa includes the onset or acute exacerbation of dyspnoea , fever , increase in cough and sputum and at times haemoptysis , and respiratory function is deteriorated , in particular , forced expiratory volume in 1 s ( fev1 ) ] [ 16 ]  . 
chest x - ray ( cxr ) and computed tomography ( ct ) patterns of abpa are listed in table 1 [ 16 ]  . in asthma patients , abpa is diagnosed on the basis of well - defined clinical , laboratory and radiographic criteria [ 911 ]  . 
for patients with cf , the cystic fibrosis foundation consensus conference defined the following criteria for the diagnosis of abpa in 2003 [ 1 ] : acute or subacute clinical deterioration increased total serum ige ( > 1 , 000 ng / ml ) immediate cutaneous reactivity to af antigens presence of serum precipitins to af appearance of new radiographic or computed tomography alterations ( infiltrates , mucous plugs ) nonetheless , diagnosis of abpa remains difficult in cf , as some of the criteria suggested are common manifestations of the underlying disease . 
 in fact , the clinical course of cf includes frequent episodes of nonspecific infectious exacerbation with bronchial obstruction , pulmonary infiltrates , and atelectasis , which may mimic abpa [ 12 ]  . 
an immune response to af ( involving the production of ige , iga , igg antibodies and an increase in total serum ige ) may also be noted in the absence of evident abpa [ 13 ]  . 
in addition , cxr and ct alterations seen in patients with cf ( table 2 ) are almost identical to those caused by abpa [ 1416 ]  . patients with clinical suspicion of abpa are assessed with cxr to identify possible changes in the typical radiographic pattern of cf and , above all , to look for areas of consolidation . 
 several authors have used ct , and in particular high - resolution ct ( hrct ) , for the evaluation of patients with abpa . laspergillosi broncopolmonare allergica ( abpa ) una malattia da ipersensibilit , che costituisce una grave complicanza del decorso dellasma severa e della fibrosi cistica ( fc )  . 
responsabile della patologia laspergillus fumigatus ( af ) , un fungo ubiquitario in natura che , grazie alle dimensioni delle spore ( 35 m ) e alla termotolleranza pu provocare , quando inalato , danni anche gravi a carico dellapparato respiratorio , soprattutto in soggetti affetti da patologie in cui laumentata densit delle secrezioni e / o lalterata clearance mucociliare favoriscono lintrappolamento delle spore nel lume bronchiale , come avviene nellasma e nella fc . non vi evidenza di una invasione delle vie aeree da parte del microrganismo , ma la colonizzazione persistente dellalbero respiratorio provoca una reazione da ipersensibilit con attivazione dellimmunit cellulare ( linfociti t cd4 + ) , umorale ( linfociti b produttori di ige , iga e igg sia locali che sieriche ) e degli eosinofili [ 1 , 2 , 68 ]  . 
lintensa risposta infiammatoria danneggia le pareti bronchiali , con sviluppo di bronchiettasie centrali , e determina la formazione di tappi di muco , di atelettasie e di infiltrati eosinofili . clinicamente i pazienti con abpa accusano insorgenza o aggravamento della dispnea , febbre , aumento della tosse e dellescreato , a volte emottisi ; si dimostra deterioramento della funzionalit respiratoria , specie del fev1 [ 16 ]  . 
gli aspetti rx e tc della abpa sono elencati nella tabella 1 [ 16 ]  . nei soggetti asmatici la diagnosi di abpa viene formulata in base a criteri clinici , laboratoristici e radiografici , ben codificati [ 911 ]  . 
la consensus conference della cystic fibrosis foundation ha stabilito , nel 2003 , criteri diversi per la diagnosi di abpa nei pazienti affetti da fc [ 1 ] : deterioramento clinico acuto o subacuto aumento delle ige sieriche totali ( > 1000 ng / ml ) test cutaneo immediato positivo ad antigeni af presenza di precipitine sieriche dirette contro af comparsa di nuove alterazioni rx o tc ( infiltrati , tappi di muco )  . tuttavia in questi pazienti la diagnosi di abpa rimane difficile , in quanto alcuni dei criteri proposti costituiscono comuni manifestazioni della malattia di base . nel decorso della fc sono infatti frequenti episodi di riacutizzazione su base infettiva aspecifica , con ostruzione bronchiale , comparsa di infiltrati polmonari e di atelettasie , che possono simulare il quadro della abpa [ 12 ]  . 
inoltre , possibile riscontrare una risposta immunitaria allaf ( con produzione di anticorpi ige , iga , igg e aumento del titolo sierico di ige totali ) in assenza di abpa conclamata [ 13 ]  . anche le alterazioni rilevabili al radiogramma toracico e alla tc dei pazienti con fc ( tabella 2 ) sono pressoch sovrapponibili a quelle dovute ad abpa [ 1416 ]  . il radiogramma del torace viene eseguito nei pazienti con sospetto clinico di abpa per evidenziare eventuali modificazioni dei reperti tipici della fc e , soprattutto , per ricercare lesioni consolidative . 
nella pratica clinica , per , lhrtc non fa parte dei criteri diagnostici dellabpa e viene utilizzata raramente in corso di riacutizzazione di fc per le alte dosi di radiazioni , lelevato costo e la scarsa specificit ai fini diagnostico - differenziali dei reperti che possono essere riscontrati ; pertanto il paziente viene di solito valutato solo con il radiogramma standard [ 1821 ]  . in letteratura non esistono studi che abbiano valutato sistematicamente il ruolo della diagnostica per immagini nella diagnosi di abpa nei pazienti con fc . 
scopo di questo lavoro valutare la affidabilit del radiogramma standard nella diagnosi di abpa in pazienti affetti da fc e correlare i quadri radiografici con la valutazione funzionale ( fev1 )  . materiali e metodi indeed , it is more sensitive than standard radiographs , particularly in determining the extent and severity of bronchiectasis and mucoid impaction . 
in clinical practice , however , hrct is not regarded as a diagnostic tool for abpa , and it is rarely used da luglio 1994 a maggio 2005 sono stati individuati , tra i pazienti seguiti dal centro regionale per la fibrosi cisticasettore adulti , 11 soggetti ( 9 maschi e 2 femmine con et media di 22 , 1 anni ) con diagnosi di abpa , per un totale di 14 episodi di malattia ( 3 pazienti sono andati incontro a due episodi di abpa )  . table 2 radiographic alterations ( cxr and ct ) in cystic fibrosis tabella 2 alterazioni radiografiche ( rx e ct ) nei pazienti con fc increased lung volume bronchial wall thickening bronchiectasis parenchymal consolidation mucoid impaction cystic lesions interstitial thickening diffuse nodular opacities aumento dei volumi polmonari ispessimento delle pareti bronchiali bronchiettasie addensamenti parenchimali impatti mucoidi lesioni cistiche ispessimento della trama interstiziale opacit nodulari diffuse increased lung volume bronchial wall thickening bronchiectasis ( cylindrical ) parenchymal consolidation ( peribronchial ) mucoid impaction ( isointense ) cystic lesions interstitial thickening diffuse nodular opacities aumento dei volumi polmonari ispessimento delle pareti bronchiali bronchiettasie ( cilindriche ) addensamenti parenchimali ( peribronchiali ) impatti mucoidi ( isodensi ) lesioni cistiche ispessimento della trama interstiziale opacit nodulari diffuse v . 
as a result , the patient is often examined by standard cxr only [ 1821 ]  . no previous study has systematically evaluated the role of imaging in the diagnosis of abpa in patients with cf . 
the aim of this study was to assess the reliability of standard radiography in the diagnosis of abpa in patients with cf and to correlate the radiographic findings with respiratory function ( fev1 )  . materials and methods from july 1994 to may 2005 , among the patients attending the regional cystic fibrosis centre adult section , we identified 11 subjects ( nine men and two women , mean age 22.1 years ) with a diagnosis of abpa and with a total of 14 episodes of abpa ( three patients had had two episodes )  . 
 at the time of the diagnosis , all patients presented with : bronchial obstruction , as assessed by fev1 ige > 1 , 000 ng / ml positive cutaneous reaction ( af skin - prick test ) presence of anti - af ige antibodies eosinophilia ( > 500 cells / ml3 ) peripherally and in the spupositive sputum culture for af all subjects underwent oral antifungal and cortisone treatment and radiographic follow - up . for each patient , we retrospectively assessed the last cxr obtained before the diagnosis of abpa and during a phase of apparent clinical stability ( t0 ) , the cxr obtained at the time of abpa diagnosis ( t1 ) and the follow - up cxr performed after treatment ( t2 )  . 
each cxr was assessed for the presence of bronchial wall thickening , bronchiectasis , areas of consolidation , atelectasis , mucoid impaction , lymphadenopathy , pleural effusion and air - fluid levels . 
findings were evaluated after subdividing the right and left lung fields into three zones for a total of six zones : upper , from the lung apex to the carina , middle , from the carina to the inferior pulmonary veins and lower , below the inferior pulmonary veins . 
 any cxr changes that had appeared at the time of diagnosis and disappeared after treatment were considered to be related to abpa and therefore useful for correct diagnosis of the disease . 
all patients underwent respiratory function testing ( fev1 ) at baseline , at the time of the abpa diagnosis and after treatment ; a 10% change relative to the previous result was considered significant . tutti gli 11 pazienti presentavano al momento della diagnosi : ostruzione bronchiale , valutata mediante la misurazione dei valori di fev1 ; valori di ige > 1000 ng / ml ; positivit al test cutaneo immediato ( prick test per af ) ; comparsa di ige anti - af ; eosinofilia periferica ( > 500 cell / ml3 ) e nellescreato ; positivit per af dellesame colturale dellescreato . tutti i soggetti sono stati sottoposti a terapia mediante cortisonici orali e antifungini , con successivo follow - up radiografico . di ciascun paziente stato retrospettivamente analizzato , per la presenza di ispessimento delle pareti bronchiali , bronchiettasie , consolidamenti , atelettasie , impatti mucoidi , linfoadenopatie , versamento pleurico e livelli idro - aerei , lultimo rx antecedente la diagnosi di abpa eseguito in fase di apparente stabilit clinica ( t0 ) , quello cronologicamente corrispondente al momento della diagnosi ( t1 ) e quello successivo alla terapia , in corso di follow - up ( t2 )  . 
i reperti sono stati valutati suddividendo ciascun campo polmonare in 3 zone : superiore , dallapice alla carena , media dalla carena sino a livello delle vene polmonari inferiori , e inferiore al di sotto delle vene polmonari inferiori , per un totale di 6 zone . 
lintervallo di tempo trascorso tra lesecuzione del radiogramma di base e quello alla diagnosi di abpa ( t0 - t1 ) era compreso tra un minimo di 30 giorni e un massimo di 29 , 5 mesi ( media di 11 , 2 mesi ) ; lintervallo di tempo ( t1 - t2 ) tra il radiogramma alla diagnosi di abpa e radiogramma di controllo era compreso tra un minimo di 4 , 5 mesi e un massimo di 29 mesi ( media 13 , 1 mesi )  . due radiologi hanno soggettivamente valutato per consenso se tra il primo radiogramma e quello alla diagnosi di abpa e tra quello in corso di abpa e il follow - up il quadro radiografico complessivo era stabile , migliorato o peggiorato . le modificazioni del radiogramma comparse in sincronia con la diagnosi e regredite dopo terapia sono state considerate collegate alla abpa e utili per la corretta diagnosi della malattia . 
comparivano a t1 7 nuovi consolidamenti , localizzati 1 / 7 a destra nella zona superiore e 6 / 7 a sinistra ( 2 nella zona superiore , 2 nella media e 2 in quella inferiore )  . dopo terapia i 7 consolidamenti regredivano completamente . 
at t1 , seven new areas of consolidation appeared , one located in the upper right zone and six on the left ( two in the upper zone , two in the middle zone and two in the lower zone )  . 
in one patient in apparently good clinical condition , a new area of consolidation appeared in the lower right zone after treatment . atelectasis atelectasis was present in 2 / 14 cases at t0 ( both in the upper right zone ) and remained unchanged at t1 . 
another area of atelettasie erano presenti in 2 / 14 casi nel radiogramma basale ( entrambe nella zona superiore destra ) , invariate in t1 . alla diagnosi di abpa compariva , sempre nella zona superiore destra di un altro paziente , una ulteriore area di atelettasia . 
le 3 lesioni si risolvevano dopo terapia al radiogramma di follow - up . bronchiettasie bronchiettasie erano evidenti in 9 / 14 casi prima della diagnosi di abpa e , alla diagnosi , venivano considerate invariate in 6 pazienti , aumentate in 2 casi e diminuite in 1 . 
c dopo terapia laddensamento si risolve e il quadro rx sovrapponibile a quello in t0 . atelectasis , again in the upper right zone , appeared in another patient at t1 . 
dopo terapia si evidenziava una ulteriore estensione degli impatti mucoidi in 4 casi , mentre in 3 pazienti le alterazioni regredivano parzialmente . mucoid impaction mucoid impaction was seen in 7 / 14 cases at t0 . 
at t2 , an acute exacerbation of mucoid impaction was observed in four cases , whereas it had partially disappeared in three . hilar adenopathy hilar adenopathy was present in 2 / 14 cases at t0 ( on the left in one patient and bilateral in the other ) and remained unchanged on the following radiographs . 
at t2 , the cxr picture was unchanged relative to t1 in 2 / 14 cases , had adenopatie ilari erano presenti in 2 / 14 casi ( in un paziente a sinistra e nellaltro bilateralmente ) prima della diagnosi e rimanevano invariate nei radiogrammi successivi . 
complessivamente il quadro rx alla diagnosi di abpa risultava invariato in 3 / 14 casi rispetto ai precedenti , peggiorato in 8 / 14 casi ( modicamente in 2 / 8 pazienti , discretamente in 4 / 8 , gravemente in 2 / 8 ) e migliorato in 3 / 14 casi ( modicamente in 1 / 3 e discretamente in 2 / 3 )  . 
dopo terapia i reperti toracici , rispetto alla diagnosi , erano invariati in 2 / 14 casi , modicamente peggiorati in 6 / 14 casi e migliorati in 6 / 14 casi . valutazione funzionale i valori di fev1 ( volume di flusso espiratorio al primo secondo ) riscontrati e le modificazioni a cui questi sono andati incontro al momento della diagnosi di abpa e dopo terapia sono riportati nella tabella 4 . 
c al follow - up laddensamento appare completamente deterso . 9 / 14 casi si osservava una significativa riduzione del fev1 , mentre in 5 casi i valori erano sostanzialmente immodificati . dei 9 casi andati incontro ad un significativo peggioramento alla diagnosi , 7 miglioravano nettamente dopo terapia , e uno migliorava in maniera non significativa . 
nei 5 casi invariati alla diagnosi non si osservava una variazione significativa del fev1 neppure in corso di follow - up . correlazione tra reperti radiografici e valutazione funzionale nei 9 casi che a t1 avevano subito una riduzione significativa del fev1 , il quadro rx era nettamente peggiorato in 5 casi , lievemente peggiorato in 1 caso , invariato in 2 e addirittura lievemente migliorato in un paziente . 
nei 5 casi in cui la funzionalit respiratoria era stabile alla diagnosi , si osservava un peggioramento del quadro radiografico in 2 pazienti , mentre in 2 questo era migliorato . 
dopo terapia i 7 casi in cui i valori di fev1 avevano subito un netto miglioramento e quello con variazione non significativa mostravano reperti toracici migliorati ( nettamente o lievemente ) in 6 casi , lievemente peggiorati in 1 paziente e invariati in 1 ; il paziente con funzionalit respiratoria peggiorata andava incontro ad un lieve peggioramento anche del quadro radiografico . 
nei restanti 5 casi con fev1 stabile , al follow - up le modificazioni rx erano lievemente peggiorate in 3 casi , migliorate in slightly deteriorated in 6 / 14 cases and improved in 6 / 14 casfunctional evaluation the fev1 values observed at t0 , t1 and t2 are reported in table 4 . 
of the nine cases with a significant deterioration at t1 , seven had a marked improvement at t2 and one had slight improvement ; one patient only had a further decrease in fev1 at t2 . 
among the five cases with unchanged fev1 at t1 , no significant change was observed t t2 . correlation between radiographic findings and functional evaluation among the nine cases with a significant decrease in fev1 at t1 , the corresponding radiographic picture had deteriorated markedly in five cases and moderately in one , remained unv . 
among the five cases with unchanged respiratory function at t1 , the corresponding radiographic picture had deteriorated in two and improved in two ; no change was detected in one . 
among the seven cases with markedly improved fev1 and the case with a slight improvement at t2 , the corresponding radiographic picture showed either marked or moderate improvement in six , slight deterioration in one and no change in another ; the patient with decreased fev1 at t2 also had a moderate worsening in the radiographic picture . 
at t2 , fev1 had returned to the t0 levels or increased slightly in ten cases ; the radiographic picture remained unchanged or improved moderately relative to t0 in eight cases but deteriorated in two . 
in four cases , t2 fev1 decreased relative to t0 , and this finding corresponded to an unchanged radiographic picture in three cases and deterioration in one . discussion imaging diagnosis of abpa in patients affected by cf is more complicated than in asthma patients in that radiographic abnormalities caused by the disease are also frequently observed in cf patients not affected by abpa . 
in fact , bronchial wall thickening , mucoid impaction , parenchymal thickening and bronchiectasis may be identified in cf patients both during periods of clinical stability and during phases of acute exacerbation due to bacterial infections . clinical and laboratory criteria suggested by the cystic fibrosis foundation consensus conference are also nonspecific [ 1 ] , as a possible immune response with formation of af antibodies can complicate interpretation of serological para1 e invariate in 1 . 
dopo terapia le percentuali di fev1 erano ritornate ai valori riscontrati a t0 , o addirittura lievemente pi elevati , in 10 casi ; il quadro radiografico era uguale o modicamente migliorato rispetto al radiogramma basale in 8 casi , mentre in 2 era peggiorato . 
in 4 casi i valori di fev1 al follow - up erano peggiorati rispetto a quelli iniziali ; a questi corrispondeva un radiogramma toracico sovrapponibile a t0 in 3 casi e peggiorato in 1 . discussione la diagnosi radiologica di abpa in pazienti affetti da fc risulta assai pi difficile che nei pazienti asmatici in quanto le alterazioni rx indotte dalla malattia si osservano frequentemente in pazienti fc non affetti da abpa . 
infatti , gli ispessimenti delle pareti bronchiali , gli impatti mucoidi , gli addensamenti parenchimali e le bronchiettasie si possono riscontrare nei pazienti fc sia in fase di stabilit clinica , sia in corso di peggioramento dovuto a infezioni batteriche ricorrenti . 
 anche i criteri clinico - laboratoristici proposti dalla consensus conference della cystic fibrosis foundation sono aspecifici [ 1 ] : la possibile comparsa di una risposta immunitaria con la formazione di antigeni diretti contro af pu , infatti , rendere complicata linterpretazione dei vari parametri sierologici utilizzati per la diagnosi di abpa . 
lesecuzione di un radiogramma toracico richiesta routinariamente nei pazienti con fc in cui vi sia una modificazione del quadro clinico - laboratoristico e funzionale che faccia sospettare una abpa , per ricercare dei segni che possano portare ad una conferma del sospetto diagnostico . 
the most significant radiographic findings according to the cystic fibrosis foundation consensus conference , namely , areas of consolidation and mucoid impaction , were inconstantly observed at the time of the diagnosis ( 7 / 14 and 4 / 14 cases , respectively ) and therefore have limited sensitivity . 
durante il follow - up il quadro migliorato nel 42 , 8% dei pazienti , rimasto sostanzialmente invariato nel 14 , 3% dei casi , mentre si osservato addirittura un peggioramento dei reperti radiografici nel 42 , 8% dei casi . 
i reperti maggiormente significativi , in accordo con la consensus conference della cystic fibrosis foundation , e cio i consolidamenti e gli impatti mucoidi , al momento della diagnosi , sono comparsi in maniera incostante ( rispettivamente 7 / 14 e 4 / 14 casi ) , risultando quindi poco sensibili . 
anche altre lesioni che si sono osservate con elevata frequenza in tutti i tre radiogrammi considerati sono risultate poco specifiche : lispessimento delle pareti bronchiali era presente in tutti i 14 soggetti , con peggioramento nel 50% dei casi alla diagnosi e addirittura nel 42 , 8% dei casi dopo terapia ; le bronchiettasie erano presenti nella maggior parte dei casi in tutti i tre radiogrammi eseguiti , senza subire significative variazioni nel corso del tempo . 
pleural effusion and air - fluid levels were absent in all cases , whereas adenopathy remained unchanged in the two cases in which it was seen . respiratory function tests , especially fev1 , proved to be the most reliable tool for monitoring the course of abpa . 
no similar correlation was noted between the clinical course of abpa and chest radiography . conclusions our study was undertaken to assess the reliability of chest radiographs in the diagnosis of abpa in patients affected by cf . 
the results highlight that despite its fundamental role in the diagnosis and follow - up of cf , the technique suffers considerable limitations in the diagnostic and follow - up assessment of cf patients who develop abpa . 
ct and hrct have greater sensitivity but are unable to demonstrate abnormalities that are highly specific for abpa , and they cannot be used routinely to monitor cf because of costs and radiation dose . 
il versamento pleurico e i livelli idroaerei sono risultati assenti in tutti i casi , mentre le adenopatie sono rimaste invariate nei due casi in cui erano presenti . le prove di funzionalit respiratoria , in particolare il fev1 , sono risultate essere il mezzo pi oggettivo per monitorare landamento dellabpa : alla diagnosi di abpa , corrisposto un significativo ( > 10% ) peggioramento del quadro respiratorio nel 64 , 3% dei casi , mentre si osservato un declino della funzionalit non significativo nel 35 , 7% dei casi . 
una analoga correlazione col decorso clinico non si , come gi detto , ottenuta con il radiogramma del torace . conclusioni il nostro studio stato intrapreso con lo scopo di valutare laffidabilit del radiogramma del torace nella diagnosi di abpa nei pazienti affetti da fc . 
i risultati ottenuti hanno dimostrato notevoli limiti di questa metodica , pure indispensabile per la precisazione diagnostica della fc e nel followup , nella valutazione dei pazienti con fc e insorgenza di abpa , sia alla diagnosi che nel follow - up : si infatti evidenziata una scarsa correlazione tra il quadro radiografico e landamento clinico della malattia , dalla sua acuzie alla completa risoluzione . 
bartolozzi1 1divisione di radiologia diagnostica e interventistica , dipartimento di oncologia , trapianti e nuove tecnologie in medicina 2dipartimento ad attivit integrata di endocrinologia e malattie metaboliche , 3dipartimento di chirurgia , universit di pisa , dipartimento immagini , via paradisa 2 , i - 56124 pisa , italy correspondence to : s . 
we included 60 patients with primary hyperparathyroidispreoperative first - line examinations revealed negative and doubtful ultrasound ( us ) findings in 34 and 26 cases , respectively , and negative , doubtful and positive scintigraphic findings in 19 , 20 and 21 cases , respectively . 
in 8 / 10 doubtful cases , surgery confirmed the location of the lesion in five cases , identified the ectopic location of lesions in two cases , and was negative in one case . 
le indagini di primo livello dimostravano : ecografia negativa in 34 casi e dubbia in 26 casi ; scintigrafia con mibi positiva in 21 casi , negativa in 19 casi , dubbia in 20 casi . 
in 8 casi con tc dubbia , la chirurgia ha confermato la lesione nella sede segnalata in 5 casi ed in altra sede in 2 casi , mentre in 1 caso risultata negativa . 
la tcmd rappresenta una metodica accurata nel work - up diagnostico delliperparatiroidismo primario e consente una esplorazione rapida e panoramica dellintera regione cervico - mediastinica . key words parathyroid glands primary hyperparathyroidism multidetector ct parole chiave paratiroidi iperparatiroidismo primitivo tc multidetettore introduction introduzione primary hyperparathyroidism ( phpt ) consists of an alteration in phosphate and calcium metabolism characterised by liperparatiroidismo primario ( phpt ) consiste in unalterazione del metabolismo fosfo - calcico caratterizzata da ecs . 
once considered an extremely rare disease , discovered in cases of severe bone or kidney disorders only , phpt has become the third most commonly diagnosed endocrine disorder after diabetes and thyroid dysfunction . 
the disease accounts for 32% of the causes of hypercalcaemia , and the diagnosis , which is essentially biochemical , is based on the presence of hypercalcaemia associated to high levels of pth . 
however , asymptomatic cases are also frequently encountered that have no or only mild kidney and bone disorders and mild hypercalcaemia ( < 11 mg / dl )  . the estimated prevalence of phpt in the general population is 12 cases / 1 , 000 inhabitants , with a 23 : 1 female - tomale ratio . 
it is currently estimated at approximately 30 : 100 , 000 / year , although it is thought to be rising as a result of the introduction of automated methods for serum calcium determination . 
primary hyperparathyroidism may present in a sporadic ( 90% ) or familial ( 10% ) form : multiple endocrine neoplasia ( men 12 ) , familial hypocalciuric hypercalcaemia , isolated familial hyperparathyroidism associated with jaw tumours ( hpt - jaw tumour syndrome ) or familial isolated hyperparathyroidism . diagnostic imaging of hpt is aimed to identifying the presence of one or more pathological parathyroid glands , a condition that is always associated with gland enlargement and therefore detectable with imaging techniques . 
the search should take into account possible abnormalities in the number of parathyroid glands , which are present in approximately 15% of cases ; and their possible ectopic location , above all of the inferior glands , which are ectopic in 2%8% of cases ( jugulothymic in 10%30% of cases , intrathyroidal in 3%10% and retropharyngeal , retroesophageal or mediastinal in 4%8% )  . 
 ultrasound ( us ) and 99mtc - sestamibi scintigraphy are the first - line diagnostic modalities for detection of a parathyroid lesion , as they are inexpensive , easy to perform and have good detection rates . 
the limitations of us are mainly ectopic locations especially when mediastinal the difficulty in exploring the neck in the presence of a goitre and the fact that it is operator dependent . 
scintigraphy of the parathyroid glands is affected , in terms of diagnostic accuracy , by several factors , including the presence of a goitre and the mitochondrial content of parathyroid lesions [ 2 ]  . 
in the context of hyperparathyroidism , magnetic resonance imaging ( mri ) and computed tomography ( ct ) are considered second - line modalities that are indicated in cases of persistent or recurrent disease in which us and scintigraphy are negative or doubtful . 
 as a multiplanar technique , mri has a high spatial resolution that provides excellent definition of the relationships between the lesion and surrounding structures ; in addition , it permits exploration of poorly accessible areas such as the thoracic inlet and provides good tissue characterisation . 
the reported sensitivity of mri in the detection of hyperfunctioning parathyroid glands varies widely and is heavily influenced by technological advances in this field , with variations between cessiva e parzialmente incontrollata secrezione di paratormone ( pth ) da parte di una o pi ghiandole paratiroidee iperfunzionanti determinante ipercalcemia [ 1 ]  . 
da malattia estremamente rara , diagnosticata solo in caso di grave compromissione ossea o renale , lphpt divenuto il terzo disordine endocrino pi frequentemente diagnosticato dopo il diabete e le tireopatie . 
la malattia rappresenta il 32% delle cause di ipercalcemia e la diagnosi , principalmente biochimica , si basa sulla presenza di ipercalcemia associata ad elevati livelli di pth , ma sono molto frequenti i casi asintomatici con assente o lieve compromissione renale e ossea , con ipercalcemia lieve ( < 11 mg / dl )  . la prevalenza di phtp nella popolazione generale stimata di 12 casi / 1000 abitanti , con rapporto f / m 23 : 1 ; lincidenza generalmente cresce allaumentare dellet e nella sottopopolazione delle donne in et menopausale ( al di sopra dei 50 anni ) valutata in circa 30 : 100000 / anno , ma considerata in aumento dopo lintroduzione del dosaggio della calcemia con metodiche automatiche . 
lphtp pu presentarsi in forma sporadica ( 90% ) o familiare ( 10% ) : neoplasia endocrina multipla ( men 12 ) , ipercalcemia familiare ipocalciurica , iperparatiroidismo familiare isolato associato a tumori della mandibola ( hpt - jaw tumor syndrome ) , iperparatiroidismo familiare isolato . la diagnostica per immagini delliperparatirodismo finalizzata alla individuazione di una o pi ghiandole paratiroidee patologiche , condizione che si associa sempre ad un loro aumento di volume che le rende pertanto riconoscibili alle tecniche di imaging . 
tale ricerca deve tener conto di possibili anomalie di numero delle ghiandole paratiroidee , presenti complessivamente nel 15% dei casi e di eventuali localizzazioni ectopiche , specie delle ghiandole inferiori , presenti nel 2%8% dei casi ( giugulo - timica 10%30% dei casi , intratiroidea 3%10% , retrofaringea , retroesofagea o mediastinica 4%8% dei casi complessivamente )  . 
 lecografia e la scintigrafia 99mtc - sestamibi rappresentano il primo livello diagnostico nella individuazione di una lesione paratiroidea , in quanto a basso costo , di facile esecuzione ed affidabili in termini di detection rate . 
i limiti dellecografia sono rappresentati principalmente dalle localizzazioni ectopiche , mediastinica in particolare , dalle difficolt di esplorazione del collo in presenza di gozzo e dal fatto di essere una metodica operatore dipendente . 
la scintigrafia paratiroidea risulta daltronde condizionata , in termini di accuratezza diagnostica , da alcuni fattori , essenzialmente rappresentati dal gozzo e dal contenuto mitocondriale delle lesioni paratiroidee stesse [ 2 ]  . 
nellambito dellimaging delliperparatiroidismo la rm e la tc sono considerate metodiche di secondo livello e lindicazione principale rappresentata dai casi con malattia persistente o recidiva , in cui lecografia e la scintigrafia risultano negative o dubbie . la risonanza magnetica ( rm ) in quanto tecnica multiplanare , presenta una elevata risoluzione spaziale con ottima definizione dei rapporti della lesione con le strutture circostanti , permette inoltre di esplorare aree meno agevoli quali lo stretto toracico superiore e consente una buona caratterizzazione tissutale . 
the pitfalls of mri include long examination times , the need to use different coils for the study of the neck and mediastinum , the frequent presence of movement artefacts that affect image quality and poor patient compliance . 
the use of fast sequences and intravenous contrast material ( gadolinium ) enables analysis of the enhancement pattern of a nodular lesion , with the limitation that the same analysis cannot be simultaneously performed on other lesions detected in the same cervicomediastinal field . indications for the use of ct in head and neck pathology , as in other body regions , have been greatly extended as a result of technological progress , the advent of spiral scanners and the introduction of multidetector acquisition systems . the main advantages of multislice scanners are the possibility of studying large body volumes with thin slices and improved spatial resolution during relatively short examination times and the possibility to perform multiplanar reconstructions . 
 the aim of this prospective study was to evaluate the diagnostic accuracy of mdct in the detection of a primary parathyroid lesion and define its possible role in the diagnostic workup of hyperparathyroidism . materials and methods between january 2003 and june 2006 , we studied 375 patients ( 158 men , 217 women , mean age 57 years ) with clinical , biochemical and hormonal evidence suggestive of hyperparathyroidismost patients had been referred to us by the endocrinology department for a us study of the neck to search for a parathyroid lesion . 
integration of the data provided by the two first - line imaging modalities was done by the endocrinologist , who had to decide whether to refer the patient directly for surgery or in cases in which the two techniques had provided negative , doubtful or discordant results to continue the diagnostic workup . 
when both modalities were negative or doubtful , the patients underwent a second biochemical and hormonal assessment to exclude other causes of hyperparathyroidisin the event primary hyperparathyroidism was confirmed , they proceeded to a ct study to complete the diagnostic workup . 
ct studies were also done in patients with negative us but positive scintigraphic findings , especially when these raised a suspicion of ectopic location . mdct was performed in 60 patients ( 16% ) ( 15 men , 45 women , mean age 57 years ) with clinical and laboratory confirmation of hyperparathyroidism and no history of allergic reaction to iodinated contrast material . 
forty - seven patients had phpt at first surgery , seven had phpt in a context of multiple endocrine neoplasia , five had a recurrence of phpt vi sono stati in questo campo , con variazioni comprese tra il 13% e l88% [ 3 , 4 ]  . 
tra gli svantaggi della rm vanno considerati i tempi piuttosto lunghi di esecuzione dellindagine , la necessit di utilizzare bobine diverse per lo studio del collo e del mediastino , a cui si aggiunge il frequente rilievo di artefatti da movimento che alterano la qualit dellesame e la scarsa compliance allesecuzione di tale indagine da parte di molti pazienti . 
utilizzando sequenze veloci e la somministrazione di mdc ev ( gadolinio ) possibile inoltre analizzare il comportamento contrastografico di una lesione nodulare , con il limite tuttavia di non poter effettuare contemporaneamente la stessa valutazione su altre tumefazioni eventualmente incluse nello stesso campo dindagine cervico - mediastinico . le indicazioni allo studio con tomografia computerizzata ( tc ) della patologia del capo e del collo ha subito , come del resto anche in altri distretti corporei , un importante impulso grazie allavanzamento tecnologico ed allavvento di apparecchiature di tipo spirale ed allintroduzione di sistemi di acquisizione multidetettore . 
il principale vantaggio di tali apparecchiature infatti rappresentato dalla possibilit di poter studiare ampi volumi corporei con tempi di acquisizione relativamente brevi , utilizzando uno spessore di strato sottile , a vantaggio della risoluzione spaziale e della possibilit di poter eseguire elaborazioni multiplanari . 
 scopo del nostro studio , a carattere prospettico , stato quello di valutare laccuratezza diagnostica della tc multidetettore nellidentificazione di una tumefazione primitivamente paratiroidea e di definirne il possibile ruolo nel workup diagnostico delliperparatiroidismo . materiali e metodi in un periodo compreso tra gennaio 2003 e giugno 2006 sono giunti alla nostra osservazione 375 pazienti ( 158 m , 217 f , et media di 57 anni ) nei quali i parametri clinici , bio - umorali ed ormonali deponevano per un quadro di iperparatiroidismo . 
la maggior parte dei pazienti erano stati inviati c / o la nostra struttura dalluo di endocrinologia per eseguire uno studio ecografico del collo mirato alla ricerca di una tumefazione paratiroidea . 
in tutti i casi lo studio ecografico stato eseguito in cieco , ovvero senza conoscere il risultato della scintigrafia , qualora questultima fosse stata eseguita in epoca antecedente alla nostra osservazione . lintegrazione dei dati forniti dalle due metodiche di primo livello era affidata allo specialistica endocrinologo , il quale decideva se inviare il paziente direttamente allintervento chirurgico o , nei casi in cui entrambe le tecniche risultassero negative o fornissero reperti dubbi o discordanti , di proseguire liter diagnostico - strumentale . 
il work - up diagnostico utilizzato stato il seguente : in caso di positivit dellecografia e della scintigrafia il paziente veniva inviato direttamente allintervento chirurgico ; simile atteggiamento anche in caso di scintigrafia negativa , ma di chiara positivit ecos . 
thirty patients ( 50% ) had an associated thyroid disorder : chronic thyroiditis in 12 ( 20% ) , multinodular goitre ( mng ) in 18 ( 30% ) ( volume between 26 and 85 ml , average 47 ml ) and two had already undergone total thyroidectomy for goitre . 
us findings were negative in 34 cases ( 57% ) and doubtful in 26 ( 33% ) , whereas scintigraphy was positive in 21 ( 35% ) , negative in 19 ( 15% ) and doubtful in 20 ( 33% )  . 
 us and colour - doppler us studies were carried out with new - generation scanners ( technos , esaote biomedica , genoa , italy ) equipped with 7.5to 10 - mhz electronic lineararray probes . 
additional scans were obtained with the neck in the right or left lateral position to better visualise the deep tracheooesophageal cervical region where parathyroid lesions may at times be located . 
the study was judged to be positive for a primary parathyroid lesion in the presence of a nodule displaying the following features : oval or elongated shape , extraglandular perithyroid location , low echogenicity ( expression of hypercellular tissue with few interfaces ) , deformation during lateral neck movements , colour / power doppler vascular signals that were neither peripheral ( typical of thyroid nodules ) or linear intraglandular ( typical of lymph nodes ) and possible demonstration of an arterial vascular pole [ 6 ]  . scintigraphy was performed after the intravenous infusion of 555 mbq of 99mtc - sestamibi ( 2 - methoxyisobutyl isonitrile ) , a molecule that has a high affinity for glands and cells with high metabolic turnover . 
 [ 7 ] : a positive judgement for parathyroid lesion was expressed in the presence of persistent uptake of the tracer at the cervicomediastinal level at 15 min and at 3 h after clearance from the thyroid . all ct studies were performed using multidetector equipment ( lightspeed plus , general electric medical system , milwaukee , wi , usa ) , with scans at baseline and after intravenous administration of 110130 ml nonionic iodinated contrast material ( iomeron 400 , bracco , milan , italy ) at a flow rate of 3 ml / s . 
the study protocol involved a baseline scan with 5 - mm slice thickness , 5 - mm reconstruction interval , pitch 3 ( hq mode ) , 120 kv , 200 ma and single field of view ( fov ) for exploration of the neck and mediastinum ( 2025 mm )  . 
postcontrast scans were obtained 30 s and 80 s after administration of contrast material with , in both cases , a slice thickness of 2.5 mm , a reconstruction interval 1.25 mm , pitch 3 ( hq mode ) , 120 kv , 250300 ma and the same fov as used for the baseline scans . 
the field of exploration of ct study was between the mandibular angles and the plane passing just below the tracheal bifurcation . all native axial images were transferred to a dedicated workstation ( advantage windows 4.0 , general electric medical systems ) where multiplanar reconstruction studies were performed . 
quando entrambe le metodiche risultavano negative o dubbie , il paziente subiva una seconda rivalutazione bioumorale ed ormonale , mirate ad escludere altre cause di iperparatiroidismo e , in caso di conferma del quadro , era sottoposto a completamento diagnostico con tc . 
questultima era richiesta anche in presenza di negativit ecografica , ma con reperti scintigrafici positivi , specie nei casi in cui questultimi ponevano il sospetto di localizzazione ectopica . il completamento diagnostico con tc multidetettore stato eseguito in 60 pazienti ( 16% ) ( 15 m , 45 f , et media di 57 anni ) nei quali vi era riconferma clinico - laboratoristica di iperparatiroidismo ed assenza allanamnesi di diatesi allergica al mdc iodato . 
in 47 casi si trattava di phpt al primo intervento , 7 casi di phpt in neoplasia endocrina multipla ; 5 pazienti presentavano una recidiva del quadro iperparatiroideo a distanza di un periodo di remissione dal primo intervento ; un caso infine era affetto da una persistenza della malattia dopo lintervento stesso . 
in 30 pazienti ( 50% ) era associata una tireopatia : tiroidite cronica in 12 casi ( 20% ) , gozzo multinodulare ( gmn ) in 18 casi ( 30% ) ( volume compreso tra 2685 ml , media 47 ml ) ; due pazienti erano gi stati sottoposti a tiroidectomia totale per gozzo . 
in tali pazienti il controllo ecografico risultava negativo in 34 casi ( 57% ) e dubbio in 26 ( 33% ) , mentre la scintigrafia dimostrava reperti positivi in 21 casi ( 35% ) , negativi in 19 casi ( 15% ) e dubbi in 20 casi ( 33% )  . gli studi ecografici ed eco - color doppler sono stati eseguiti con apparecchi di ultima generazione ( technos , esaote biomedica , genova , italia ) provvisti di sonde lineari elettroniche da 7 , 510 mhz . 
scansioni ecografiche aggiuntive sono state effettuate con il collo in posizione laterale destra o sinistra , al fine di visualizzare meglio la regione cervicale profonda tracheo - esofagea dove le lesioni paratiroidee possono essere a volte localizzate . 
veniva espresso un giudizio positivo per lesione primitivamente paratiroidea in presenza di un nodulo con morfologia ovoidale o allungata situato in sede peritiroidea extraghiandolare , a bassa ecogenicit ( espressione di tessuto ipercellulato e con scarse interfacce ) , eventualmente deformabile nei movimenti di lateralit del collo , provvisto di segnali vascolari intralesionali alla valutazione eco - color / power doppler , privi di distribuzione periferica ( tipico dei noduli tiroidei ) o lineare intraghiandolare ( tipico dei linfonodi ) , con la possibilit di dimostrare un polo vascolare arterioso [ 6 ]  . la scintigrafia stata eseguita mediante linfusione ev di 555 mbq di 99mtc - sestamibi ( 6 - metossi - butil - isonitrile ) , molecola che mostra spiccato tropismo per le ghiandole e le cellule ad elevato turn - over metabolico . 
1 multidetector computed tomography in primary hyperparathyroidisthe axial image ( left ) and the coronal reconstruction ( right ) performed after contrast medium administration allow identification of a small , hyperdense , nodular lesion ( arrow ) , attributable to a parathyroid lesion , located close to the right thyroid lobe and the oesophagus . 
limmagine assiale ( sinistra ) e la ricostruzione sul piano coronale ( destra ) evidenziano , dopo somministrazione di mdc , la presenza di una piccola formazione nodulare iperdensa ( freccia ) riferibile a lesione primitivamente paratiroidea , contigua alla tiroide ed alla parete postero - laterale destra dellesofago . 
le acquisizioni post - contrastografiche erano effettuate dopo 30 s e 80 s dalla somministrazione di mdc utilizzando , in entrambi i casi , uno spessore di strato di 2 , 5 mm , un intervallo di ricostruzione di 1 , 25 mm , pitch 3 ( modalit hq ) , 120 kv , 250300 ma e fov uguale alle scansioni basali . 
il campo di esplorazione dellesame tc era compreso tra gli angoli mandibolari ed il piano passante subito inferiormente la biforcazione tracheale . tutte le immagini assiali native acquisite erano inviate ad una stazione di lavoro dedicata ( advantage windows 4.0 ; ge / medical systems , milwaukee , wi ) dove venivano eseguiti studi di ricostruzione multiplanare . 
il tempo dedicato agli studi di elaborazione risultava in ogni caso inferiore a 20 minuti . la tc ha espresso un giudizio di sospetta tumefazione paratiroidea in base alla sede ( extratiroidea ) della lesione , alla sua morfologia ( ovoidale o allungata ) , alleventuale dimostrazione dei vasi afferenti alla lesione ed al grado di captazione contrastografica , confrontato questultimo con fig . 
dopo somministrazione di mdc iodato la tumefazione paratiroidea era sospettata alla tc , in entrambe le fasi di acquisizione ( arteriosa e venosa ) , in presenza di un marcato enhancement contrastografico , questultimo legato alla loro caratteristica ipervascolarizzazione , con un wash - in e wash - out pressoch sovrapponibile o solo lievemente inferiore rispetto al parenchima tiroideo . 
sulla base di tali caratteri la tc ha espresso un giudizio positivo per lesione paratiroidea in 35 ( 58% ) casi , negativo in 15 ( 25% ) casi e dubbio in 10 ( 17% )  . 
i reperti tc sono stati confrontati con i reperti dellesplorazione chirurgica . risultati lintervento chirurgico stato eseguito in 40 / 60 ( 67% ) pazienti e condotto con approccio mini - invasivo in 18 casi ( 45 ) , mentre in 22 casi ( 55% ) stata effettuata una tecnica tradizionale open . 
il peso delle ghiandole paratiroidee asportato era compreso tra 15 mg e 2400 mg , con una media di 620 mg ; il diametro massimo delle lesioni risultava compreso tra 6 mm e 38 mm ( media 17 , 5 mm )  . 
dopo lescissione della tumefazione paratiroidea stato effettuato , in tutti i casi , il dosaggio intraoperatorio del pth circolante per verificare lassenza di eventuali altre lesioni o focolai di abnorme secrezione di pth . 
in tali casi la concomitante presenza di patologia tiroidea era osservata in 5 casi ( 22% ) ( 4 gmn , 1 tiroidite cronica di hashimoto ) , mentre 1 paziente ( 4% ) era stato in precedenza sottoposto ad intervento di tiroidectomia totale ; 4 pazienti ( 17% ) erano stati sottoposti a pregressa paratiroidectomia parziale . 
in 22 pazienti si trattava di una lesione singola , mentre 1 paziente risultava affetto da 2 lesioni ; in 20 casi si trattava di lesioni originate dalle ghiandole inferiori , in 4 casi da quelle superiori . in 8 casi ( 20% ) con tc dubbia lintervento chirurgico ha identificato la lesione nella sede segnalata in 5 casi ( 62 , 5% ) ( veri positivi ) , mentre in 2 casi ( 25% ) stata identificata in altra sede ( falsi positivi )  . 
on contrast - enhanced ct , a parathyroid lesion was suspected in both arterial and venous ct acquisition phases in the presence of a marked enhancement , indicating hypervascularity , with identical or only slightly slower washin and washout compared with the thyroid parenchyma . 
on the basis of the outlined criteria , ct was positive for parathyroid lesions in 35 ( 58% ) cases , negative in 15 ( 25% ) and doubtful in 10 ( 17% )  . 
la valutazione densitometrica della lesione ( p ) contigua allarco aortico , confrontata con quella di un linfonodo ( l ) a sede cervicale , in condizioni basali ( sinistra ) e dopo 30 secondi dalla somministrazione di mdc ( destra ) , permette di dimostrare la natura paratiroidea della lesione mediastinica in base al suo diverso comportamento post - contrastografico rispetto al linfonodo cervicale . results forty out of 60 ( 67% ) patients underwent surgery , with a minimally invasive approach in 18 cases ( 45% ) and an open technique in 22 ( 55% )  . 
in 2 casi le lesioni erano localizzate nelle ghiandole inferiori , in 5 casi in quelle superiori . in 9 pazienti ( 22 , 5% ) con tc negativa il chirurgo ha identificato la lesione paratiroidea in 8 casi ( 89% ) ( falsi negativi ) , mentre in 1 caso ( 11% ) lesplorazione chirurgica risultata negativa ( vero negativo )  . 
in tali pazienti lassociazione con un quadro tireopatico era osservato in 7 casi ( 5 gmn , 2 tiroidite cronica ) ; un paziente aveva subito un pregresso intervento di tiroidectomia totale . 
in 2 casi si trattava di lesioni ad origine dalle ghiandole inferiori , in 6 casi da quelle superiori . nel complesso la tc ha riportato 28 veri positivi , 8 falsi s . 
le ricostruzioni multiplanari nel piano coronale ( sinistra ) e sagittale ( destra ) permettono di evidenziare la presenza di un nodulo paratiroideo ipervascolarizzato ( freccia ) a localizzazione retrosternale situato in contiguit con un linfonodo mediastinico ( punta di freccia )  . 
five of these cases ( 22% ) had concomitant thyroid disease [ four multinodal goitre ( mng ) , one chronic hashimoto thyroiditis ] , whereas one patient ( 4% ) had already undergone total thyroidectomy and four ( 17% ) had undergone partial parathyroidectomy . 
in 20 cases , the lesions originated from the inferior glands and in four cases from the superior glands . of eight cases ( 20% ) with doubtful ct findings , surgical exploration identified the lesion in the location suggested by ct in five cases ( 62.5% ) ( true positives ) , whereas in two cases ( 25% ) , the lesion was identified in another site ( false positives )  . 
in two cases , the lesions were located in the inferior glands and in five cases in the superior glands . of nine patients ( 22.5% ) with negative ct , surgery identified the parathyroid lesion in eight cases ( 89% ) ( false negatives ) , whereas it was negative in one case ( 11% ) ( true negative )  . 
in two cases , the lesions originated from the inferior glands and in six cases from the superior glands . overall , ct yielded 28 true positives , eight false negatives , three false positives and one true negative . 
at contrast - enhanced ct , all lesions were hyperattenuating in both acquisition phases , with similar attenuation patterns to the thyroid parenchyma in all cases of lesions smaller than 2 c larger nodules showed greater hyperdensity and greater structural heterogeneity . 
dopo somministrazione di mdc iodato si osservava in tutti i casi un aspetto iperdenso delle lesioni in entrambe le fasi di acquisizione , con un comportamento densitometrico sovrapponibile a quello del parenchima tiroideo in tutti i casi di lesioni di dimensioni inferiori ai 2 cnei casi di noduli pi grandi era possibile osservare un livello di iperdensit minore ed una maggiore disomogeneit strutturale . 
gli autori a sfavore basano la loro posizione sul fatto che un chirurgo esperto comunque in grado di localizzare la paratiroide abnorme al primo intervento nel 95% dei casi , mentre le tecniche radiologiche sono , in tal senso , meno affidabili e , solo qualora adeguatamente integrate , possono raggiungere valori di detection rate maggiori del 90% ; esse inoltre risultano gravate da una percentuale di falsi positivi valutabile in circa il 25% [ 9 , 10 ]  . 
pi concordi appaiono invece le posizioni nei casi di iperparatiroidismo recidivante o persistente nei quali il successo chirurgico nettamente inferiore , valutato in circa il 60% e dove si rende pi evidente il contributo della diagnostica per immagini [ 11 , 12 ]  . 
a tale riguardo , le cause pi frequenti di insuccesso sono rappresentate dallinesperienza degli operatori , dalla soprannumerariet ghiandolare e dalle localizzazioni ectopiche , questultime con frequenza stimata intorno al 5%20% al primo intervento e 32% nei casi di phpt recidivante [ 13 ]  . gli autori a favore della localizzazione pre - operatoria basano la loro posizione sul fatto che limaging facilita in ogni caso lintervento , ne riduce i tempi e le complicanze , permette lidentificazione delle ghiandole ectopiche , le eventuali patologie associate ( tireopatie ) e quindi aiuta il chirurgo nella pianificazione dellintervento [ 14 , 15 ]  . 
il dosaggio del pth intra - operatorio , effettuato dopo lescissione della lesione , rappresenta inoltre un valido indicatore di effettiva remissione della malattia , in quanto permette di escludere la presenza di altre ghiandole abnormi e quindi di effettuare , in s . 
opponents base their views on the fact that an experienced surgeon is able to localise abnormal parathyroid glands at initial surgery in 95% of cases , whereas imaging techniques are less reliable , reaching detection rates higher than 90% only if they are adequately integrated . in addition , imaging is burdened by a false positive rate of about 25% [ 9 , 10 ]  . 
less controversy surrounds the use of preoperative imaging in recurrent or persistent parathyroidism , in which surgical success rates are definitely lower ( approximately 60% ) and where the contribution of diagnostic imaging is more evident [ 11 , 12 ]  . 
the most frequent causes of failure are operator inexperience , presence of supernumerary glands and ectopic locations , which have an estimated incidence of 5%20% at initial surgery and of 32% in cases of recurrent phpt [ 13 ]  . 
 proponents of preoperative localisation base their view on the fact that imaging in any case facilitates the operation , reduces surgical time and complications , allows identification of ectopic glands and possible associated conditions ( thyroid disease ) and therefore helps in surgical planning [ 14 , 15 ]  . 
in addition , intraoperative pth assays , done after the lesion has been excised , provide a valuable indication of effective remission , as they allow one to exclude other abnormal glands and therefore to perform , in the absence of contralateral thyroid disease , a unilateral neck exploration only [ 16 ]  . 
on the other hand , precise preoperative localisation is a prerequisite for planning minimally invasive surgery , a technique proposed by various groups , especially in the absence of goitre [ 17 , 18 ]  . the search for a parathyroid lesion generally involves the use of noninvasive imaging techniques such as us , colourdoppler us , sestamibi scintigraphy , ct and mri . 
the diagnostic accuracy of each method mainly depends on lesion location and size , the presence of concomitant thyroid disease and operator experience [ 2 , 6 , 20 ]  . in the diagnostic workup of hyperparathyroidism , us and 99mtc - sestamibi scintigraphy are considered first - line modalities capable of differentiating cervical and extracervical lesions . 
both techniques are reliable in demonstrating a parathyroid lesion with sensitivity values , in the absence of concurrent thyroid disease of approximately 90% for each technique separately and higher when the two techniques are appropriately combined . 
the presence of associated thyroid disease and / or enlarged perithyroid cervical nodes , such as those seen in chronic thyroiditis , reduce the diagnostic capabilities of both us and scintigraphy and introduce a high rate of false positive results . 
in these cases , colour - doppler us is useful for differentiating parathyroid lesions from other nodular lesions of the neck such as thyroid nodules , abnormal lymph nodes or vessels . the proposal of some authors to use us - guided interventional techniques ( such as needle aspiration for cytology or pth assays on the aspirate ) to confirm or exclude the parathyroid origin of a cervical mass in patients with hyperparathyroidism has not been unanimously accepted . 
daltronde lesatta identificazione pre - operatoria della lesione rappresenta un requisito indispensabile per una programmazione dellintervento chirurgico con approccio mini - invasivo , tecnica questultima proposta da vari gruppi , specie in assenza di concomitante gozzo [ 17 , 18 ]  . nella ricerca di una lesione paratiroidea sono in genere utilizzate tecniche di imaging non invasive quali lecografia , leco - color doppler , la scintigrafia con sestamibi , la tc e la rm . 
laccuratezza diagnostica di ogni metodica dipende essenzialmente dalla sede e dalle dimensioni della lesione , dalla presenza o meno di concomitante patologia tiroidea e dallesperienza delloperatore [ 2 , 6 , 20 ]  . nel work - up diagnostico - strumentale delliperparatiroidismo lecografia e la scintigrafia con 99mtecnezio - sestamibi ( tc - mibi ) , sono considerate le metodiche di primo livello , capaci di selezionare le lesioni situate a livello cervicale da quelle a sede extracervicale . 
entrambe si dimostrano affidabili nel dimostrare una lesione paratiroidea , con valori di sensibilit per ciascuna di esse , in assenza di concomitante patologia tiroidea , valutata in circa il 90% , con un apporto diagnostico superiore quando le due tecniche vengono opportunamente integrate . 
la presenza di tireopatia associata e / o di linfoadenomegalie cervicali a sede peritiroidea , quali ad esempio quelle che usualmente accompagnano la tiroidite cronica , riducono la capacit diagnostica sia dellecografia che della scintigrafia ed introducono elevati falsi positivi . 
in questi casi leco - color doppler si dimostrato un utile completamento diagnostico nel differenziare le lesioni paratiroidee da altre lesioni nodulari del collo quali i noduli tiroidei , i linfonodi , i vasi . la proposta di alcuni autori di utilizzare tecniche interventistiche ecoguidate ( quali lago - aspirato per lanalisi citologica o il dosaggio del pth sullago - aspirato ) al fine di confermare o escludere la natura paratiroidea di una tumefazione cervicale in pazienti affetti da iperparatiroidismo , non da tutti condivisa , per la limpossibilit spesso da parte del citologo di distinguere le cellule paratiroidee da quelle tiroidee e per il rischio di contaminazione emorragica della loggia paratiroidea stessa ( evento questultimo possibile in considerazione del carattere ipervascolare di tali lesioni ) che pu aumentare le difficolt chirurgiche e le complicanze post - chirurgiche e , specie nei casi di approccio mini - invasivo , indurre a procrastinare lintervento stesso [ 21 , 22 ]  . dopo lesecuzione delle due indagini di primo livello si possono configurare quattro situazioni : la positivit delle due metodiche , la positivit ecografica e la negativit scintigrafica , risultati negativi per entrambe le tecniche ed infine reperti ecografici negativi , ma scintigrafici positivi . 
the same approach is adopted in the case that scintigraphy is negative but us reveals the presence of a primary parathyroid mass having typical location , morphostructural characteristics and colour - doppler pattern . in our study , multidetector ct was used as a second - line modality in cases of negative us and scintigraphy or negative us but positive scintigraphy , situations that are often accompanied by multinodular goitre or are suspicious for ectopic location . 
our series did not include patients who underwent mri as a second - line investigation , a technique we use in patients with a history of iodine allergy or those who refuse iodinated contrast material . 
of the 375 patients studied , only 16% required ct to complete the diagnostic workup , confirming that first - line modalities are able to adequately screen patients in most cases . 
in the presence of thyroid disease , ct proved to be more reliable in recognising the inferior glands ( 92% of cases ) than the superior glands ( 47% of cases )  . 
of the cases with doubtful or negative ct , 73% had lesions in the superior glands and 27% in the inferior glands . this is related to the fact that lesions arising from the inferior glands are more readily distinguished on postcontrast images , as they are hyperdense relative to extranodal tissues ( cellular fat , muscle , oesophagus or trachea )  . 
in addition , lesions originating from the superior parathyroid glands may frequently display a thin , elongated appearance , as they develop in a confined space delimited by rigid structures such as the vertebral spine or trachea posteriorly and the thyroid parenchyma anteriorly , especially if the thyroid is nodular . 
in such cases , the parathyroid lesion may easily be confused with a lobulation of the thyroid parenchyma from which it is difficult to differentiate , even on multiplanar reconstructions . 
a completely different scenario is when the parathyroid lesion needs to be differentiated from the lymph nodes , which occurs almost constantly in the presence of concomitant chronic hashimoto thyroiditis . 
in our study , this association was detected in five patients , and in all of them , ct enabled differentiation of the two lesions by revealing different enhancement patterns , a renel nostro studio abbiamo utilizzato la tc multidetettore , quale metodica di secondo livello , in caso di negativit sia dellecografia che della scintigrafia o in presenza di positivit solo scintigrafica , situazioni spesso accompagnate da un concomitante gozzo multinodulare o che facevano sospettare una localizzazione ectopica . 
in questa casistica non sono stati inclusi quei pazienti che , nellambito di un accertamento di secondo livello , sono stati studiati con rm , metodica in diversi casi da noi comunque utilizzata , come ad esempio in quei pazienti che presentavano una storia di allergia allo iodio o che rifiutavano la somministrazione di mdc iodato . 
nellambito dellampia popolazione di pazienti da noi osservata ( n = 375 ) solo nel 16% dei casi si reso necessario il completamento con tc , a conferma che nella maggior parte dei casi le metodiche di primo livello sono in grado di poter effettuare una valida selezione . 
in presenza di tireopatia la tc si dimostrata pi affidabile nel riconoscere le ghiandole della loggia inferiore ( 92% dei casi ) rispetto a quelle della loggia superiore ( 47% dei casi ) e nei casi con tc dubbia o negativa le lesioni erano localizzate alle ghiandole superiori nel 73% dei casi ed alle ghiandole inferiori nel 27% . 
questo dato in relazione al fatto che le tumefazioni ad origine dalle ghiandole inferiori possono essere pi facilmente distinte in fase post - contrastografica , in base al loro aspetto iperdenso , rispetto ai tessuti extranodulari , questi ultimi rappresentati da cellulare adiposo , muscoli , esofago o trachea . 
non infrequentemente inoltre le lesioni a partenza dalle ghiandole paratiroidee superiori possono assumere un aspetto allungato ed esile in quanto vengono a svilupparsi in uno spazio anatomico ristretto , delimitato posteriormente da strutture rigide quale la colonna vertebrale o la trachea ed il parenchima tiroideo anteriormente , specie se questultimo sede di noduli ; in tali casi la paratiroide patologica pu pertanto essere facilmente confusa con una lobatura del parenchima tiroideo dal quale risulta mal differenziale anche ricorrendo a ricostruzioni multiplanari . ben diverso il caso in cui la lesione paratiroidea deve essere differenziata dai linfonodi , situazione che si verifica pressoch costantemente in presenza di una concomitante tiroidite cronica di hashimoto . 
i linfonodi di accompagnamento della tiroidite si localizzano spesso nella stessa sede dove normalmente vengono ricercate le ghiandole paratiroidee abnormi ed il loro carattere morfo - strutturale spesso simile ; in queste situazioni sia lecografia che leco - color doppler forniscono dati incerti . 
nel nostro studio questa associazione stata rilevata in 5 pazienti ed in tutti i casi la tc ha permesso di differenziare le due lesioni sulla base di un diverso comportamento contrastografico , espressione di una diversa cellularit e vascolarizzazione . 
un valido aiuto ci stato offerto , in tal senso , soprattutto nei casi in cui le lesioni originavano dalle ghiandole inferiori , dalla individuazione angio - tc dellarteria tiroidea inferiore e dei piccoli vasi arteriosi afferenti alla lesione stessa . 
in this respect , especially in cases of lesions arising from the inferior glands , we were greatly aided by the ct - angiography identification of the inferior thyroid artery and small arteries supplying the lesion . 
even in patients with previous neck surgery ( two with total thyroidectomy and four with partial parathyroidectomy ) , ct allowed reliable identification of the lesions in 5 / 6 cases ( 83% )  . 
one limitation emerged in patients with previous total thyroidectomy , where , especially in the case of small lesions , the technique did not allow differentiation of parathyroid nodules from residual thyroid tissue on the basis of enhancement alone . 
even in this case , analysis of the vessels supplying the lesion helped our interpretation . all the ectopic lesions in our series were successfully recognised at ct , which proved to be more reliable than the older ct scanners in this respect , although very similar to them in the case of mediastinal locations . 
finally , in patients undergoing first surgery and in the four cases of recurrent phpt , ct had a sensitivity of 86% and 100% , respectively . these data are considerably higher than those reported in the literature , which range from 50% to 70% in phpt and 46% to 60% in recurrences . conclusions in conclusion , mdct proved to be an accurate second - line investigation that enabled fast exploration of the entire cervical and upper mediastinal region and was able to defining the relationships between parathyroid lesions and vascular structures and to differentiate parathyroid lesions from other cervical structures such as lymph nodes . 
 pregressa chirurgia sul collo ( 2 con tiroidectomia totale e 4 con paratiroidectomia parziale ) la tc si dimostrata affidabile ed ha permesso il riconoscimento delle lesioni in 5 / 6 casi ( 83% )  . 
nei pazienti sottoposti a pregressa tiroidectomia totale un limite della metodica si dimostrata , soprattutto nei casi di lesioni piccole , per la difficolt di poter differenziare , sulla base del solo comportamento contrastografico , i noduli paratiroidei dai residui ghiandolari tiroidei ; anche in questultimo caso tuttavia ci siamo aiutati con lanalisi dei vasi afferenti alle lesione . le lesioni ectopiche da noi osservate sono state in tutti i casi riconosciute dallesame tc , confermandosi in tal senso pi affidabile rispetto ad apparecchiature tc meno recenti , ma pressoch sovrapponibile a questultime limitatamente alle localizzazioni mediastiniche . 
martingano2 1uo di radiologia , universit magna graecia di catanzaro , campus di germaneto , viale europa , i - 88100 catanzaro , italy 2uco di radiologia , universit di trieste , ospedale di cattinara , strada di fiume 447 , i - 34149 trieste , italy correspondence to : o . 
other advantages of mri are high - contrast resolution and multiplanar view , as well as extensive coverage of the skeleton with whole - body mri ( wbmri )  . 
according to some authors much work remains to be done to improve sensitivity and specificity of mri in order to define the real clinical value of this imaging modality in the multidisciplinary management of patients with a haematological malignancy . 
this article presents recent developments and perspectives in the use of mri in oncohaematological diseases . key words haematology bone marrow magnetic resonance imaging tissue characterisation contrast enhancement whole body bone marrow diseases riassunto la risonanza magnetica ( rm ) ha aperto nuove possibilit alla radiologia diagnostica per la valutazione del midollo osseo . 
altri vantaggi della rm sono rappresentati dallelevata risoluzione di contrasto e dalla visione multiplanare , assieme ad unampia copertura dello scheletro , fino alla rm whole body ( wbmri )  . 
tuttavia la specificit delle alterazioni di segnale bassa , perci i reperti di rm devono essere integrati con la clinica e i risultati di laboratorio assieme alla valutazione ematologica ed oncologica . la rm fornisce informazioni che aiutano la diagnosi , lo staging ed il follow - up di diverse malattie del midollo osseo . 
secondo alcuni autori molto lavoro deve ancora essere fatto per migliorare la sensibilit e la specificit della rm e per definire il reale valore clinico di questa modalit di imaging nella gestione multidisciplinare del paziente con una neoplasia ematologica . questo articolo presenta i recenti sviluppi e le prospettive nelluso della rm nelle patologie oncoematologiche . parole chiave ematologia midollo osseo risonanza magnetica caratterizzazione tissutale contrast enhancement whole body malattie midollo osseo introduction introduzione in oncohaematology and haematology , diagnosis and determination of disease stage , prognostic factors and response to treatment are of remarkable value in adjusting the initial treatment to the spread and aggressiveness of the disease to avoid overor undertreatment during the course of the disease and to enable different therapies to be compared [ 13 ]  . microscopic evaluation of bone marrow specimens from blind aspirations and biopsies remains fundamental for determining diagnosis , treatment and prognosis . 
biopsy or asin ematologia ed in oncoematologia estremamente importante stabilire la diagnosi , lo stadio di malattia , i fattori prognostici e la risposta al trattamento , in modo da poter bilanciare il trattamento iniziale alla diffusione e allaggressivit della patologia , evitare sovra o sottodosaggi e comparare differenti trattamenti [ 13 ]  . 
la valutazione microscopica dei campioni di midollo osseo da aspirati eseguiti alla cieca o da biopsie mantiene il suo ruolo fondamentale nel determinare la diagnosi , la prognosi e il trattamento da intraprendere . 
furthermore , whereas many haematological disorders are known to diffusely infiltrate the axial bone marrow , the overall distribution of disease can be heterogeneous on a length scale , precluding an accurate and definitive assessment via an aspirate or a biopsy from a single site [ 46 ]  . magnetic resonance imaging ( mri ) is the only imaging technique that allows direct visualisation of bone marrow , and it is the most sensitive . 
it is a noninvasive technique that complements bone marrow aspirations and biopsies by sampling a large volume of bone marrow and providing information that aids in diagnosis , staging and follow - up of bone marrow disease [ 46 ]  . 
therefore mri findings need to be integrated with clinical and laboratory findings as well as with haematological and oncological evaluation [ 2 , 4 ]  . imaging techniques many pulse sequences are available for bone marrow mri . mr appearance of bone marrow is highly dependent on the pulse sequence used and the relative amounts of fat and water within the marrow . 
conventional spin echo ( se ) t1weighted sequence is effective for imaging bone marrow due to its capability in showing the different signal intensity of red and yellow marrow , to the relatively high sensitivity in detecting bone marrow pathology and to good image quality and relatively short imaging time [ 411 ]  . 
limitations of se t1 - weighted sequence include low specificity , as most bone marrow disorders exhibit low signal intensity , and relatively low sensitivity in detecting pathology in children due to the similarity of signal intensity characteristics of normal red marrow with the predominant low signal of bone marrow disorders with this sequence [ 1214 ]  . se t2 - weighted sequence shows good sensitivity in detecting bone marrow pathology and allows good definition of the fluid component of the lesion . 
fse t2 - weighted sequences can be combined with frequency - selective fat suppression to improve the conspicuity of marrow lesions [ 15 ]  . biopsie e gli aspirati campionano solitamente un volume non superiore a 0 , 1 cm3 rispetto ad un volume totale di midollo potenzialmente attivo superiore ai 1000 cm3 . 
inoltre , sebbene molte patologie ematologiche infiltrino diffusamente il midollo osseo assiale , la generale distribuzione della malattia pu essere eterogenea in estensione , impedendo una valutazione accurata e definitiva qualora si utilizzi un aspirato o una biopsia provenienti da una singola sede [ 46 ]  . la risonanza magnetica lunica tecnica in grado di visualizzare direttamente il midollo osseo ed la pi sensibile . una tecnica non invasiva complementare allaspirato o alla biopsia midollare che consente di studiare un ampio volume di midollo e fornisce informazioni utili per la diagnosi , lo staging ed il follow - up delle malattie del midollo osseo [ 46 ]  . 
recentemente la disponibilit di gradienti pi veloci e di lettini a rapido movimento hanno aperto la strada alla rm whole body nellindividuazione delle malattie del midollo osseo [ 710 ]  . 
dal momento che le caratteristiche di segnale delle lesioni focali e multifocali non sono specifiche per la diagnosi , la rm del midollo osseo deve essere interpretata nel contesto clinico ; pertanto i reperti di risonanza devono essere integrati con quelli clinici e laboratoristici , assieme alla valutazione ematologica ed oncologica [ 2 , 4 ]  . tecniche di imaging per limaging rm del midollo osseo sono oggi disponibili numerose sequenze . 
la sequenza spin echo ( se ) convenzionale t1 - pesata una sequenza efficace per lo studio del midollo osseo , grazie alla capacit di evidenziare la differente intensit di segnale del midollo rosso e giallo , alla capacit sufficientemente elevata di individuare la patologia del midollo osseo , alla buona qualit di immagine e al tempo di acquisizione relativamente breve [ 411 ]  . 
gli svantaggi della sequenza se t1 - pesata comprendono la bassa specificit , dal momento che la maggior parte delle malattie del midollo osseo ha bassa intensit di segnale , e la sensibilit relativamente bassa in et pediatrica , a causa della somiglianza di intensit di segnale fra il midollo rosso normale e il basso segnale delle patologie midollari in questa sequenza [ 1214 ]  . la sequenza se t2 - pesata dotata di una buona sensibilit nellindividuazione della patologia midollare e permette una buona definizione della componente fluida delle lesioni , tuttavia la qualit di immagine spesso scarsa e il tempo di acquisizione lungo . le sequenze fast se t2 pesate ( fse ) hanno ampiamente rimpiazzato le convenzionali se t2 - pesate grazie alla notevole riduzione del tempo di esame . 
tuttavia , nonostante questo vantaggio e la buona qualit di immagine , la sequenza fse t2 - pesata presenta uno svantaggio importante che ne limita limpiego , rappresentato dallelevato segnale provereduction of fat signal intensity can be obtained with many sequences , which use different mechanisms to cancel the fat signal and enhance lesion visibility . 
the most widely used fat - suppressed sequence for bone marrow imaging is the stir sequence [ 15 ]  . diffusion - weighted imaging has been recently used for vertebral bone marrow evaluation based on the assumption that interstitial water increase in bone marrow oedema due to benign compression fractures can be differentiated from restricted motion of water molecules in tumour cell infiltration [ 16 , 17 ]  . 
data published in the literature suggest that diffusion - weighted sequences are an additional tool for differentiating vertebral fractures due to osteoporotic collapse with bone marrow oedema and metastatic collapse , and this might be helpful for monitoring response to therapy in metastatic disease [ 18 ]  . selective water and fat imaging methods or imaging with and without fat suppression have been used for quantitative evaluation of signal strengths and relaxation times to obtain more specific information on the response of haematological diseases to therapy . 
in addition to imaging , 1h - mr spectroscopy of red vertebral bone marrow has been proposed for diagnosis and follow - up in patients with bone marrow malignancies such as leukaemia , lymphoma and myeloma [ 1923 ]  . after gadolinium injection , normal bone marrow shows no signal intensity changes on t1 - weighted images , and measurements show no or only little increase . 
contrast - enhanced mr imaging with opposedphase gradient - echo sequences showing haematopoietic bone marrow with low signal intensity as opposed to intermediate intensity of fat marrow has proved to be suitable for demonstrating bone marrow involvement . 
la sequenza fse t2 - pesata deve comunque essere combinata con la tecnica a soppressione selettiva del grasso , per migliorare la visibilit delle lesioni midollari [ 15 ]  . 
la riduzione di intensit del segnale del grasso pu essere ottenuta con diverse sequenze che utilizzano differenti meccanismi per cancellare il segnale del grasso e migliorare la visibilit delle lesioni . 
vi sono quattro tipi principali di sequenze a soppressione del grasso : la short ti inversion recovery ( stir ) , la saturazione selettiva , le sequenze a chemical shift e ad opposizione di fase . 
la sequenza a soppressione del grasso pi usata nellimaging del midollo osseo la sequenza stir [ 15 ]  . limaging di diffusione stato recentemente usato per la valutazione del midollo osseo vertebrale , in base al principio che laumento di liquido interstiziale nelledema del midollo osseo dovuto ad una frattura da compressione benigna differenziabile dalla ridotta mobilit delle molecole dacqua che si ha nellinfiltrazione di cellule tumorali [ 16 , 17 ]  . 
i lavori pubblicati in letteratura riportano che le sequenze di diffusione sono uno strumento addizionale per differenziare le fratture vertebrali dovute ad un crollo osteoporotico con edema midollare dal crollo metastatico e questo pu essere utile per monitorare la risposta alla terapia nella malattia metastatica [ 18 ]  . i metodi di imaging selettivo per lacqua e per il grasso , con o senza la soppressione del grasso , sono stati usati per la valutazione quantitativa dellintensit di segnale e dei tempi di rilassamento cos da ottenere informazioni pi specifiche sulla risposta alle terapie delle patologie ematologiche . 
accanto allimaging , la spettroscopia 1h - rm del midollo rosso vertebrale stata proposta per la diagnosi ed il follow - up terapeutico nei pazienti con neoplasie del midollo osseo come la leucemia , il linfoma ed il mieloma [ 1923 ]  . dopo liniezione di gadolinio il midollo osseo normale non mostra alterazioni dellintensit di segnale nelle sequenze t1 - pesate e le misurazioni dimostrano solo un lieve aumento di segnale o nessun aumento . 
limaging rm dopo contrasto con le sequenze gradient - echo ad opposizione di fase , in cui il midollo ematopoietico ha una bassa intensit di segnale , diversamente dallintensit intermedia del midollo adiposo , si dimostrato adatto ad evidenziare il coinvolgimento del midollo osseo . 
luso dellimaging dopo contrasto in risonanza magnetica , particolarmente con le sequenze dinamiche , per lo studio dei pazienti con coinvolgimento midollare nelle malattie onco - ematologiche sta aumentando . dal punto di vista del contenimento dei costi , luso del mezzo o . 
mri criteria for normal bone marrow are based not only on t1 - weighted signal intensity analysis but also on enhancement time curve ( etc ) analysis , both of which warrant further evaluation in patients with bone marrow diseases [ 2426 ]  . recently , ultrasmall , superparamagnetic iron oxide particles have been developed for evaluation of phagocytic activity of bone marrow . 
ultrasmall particles are taken up by the reticuloendothelial system of bone marrow to reduce the t1 and t2 signal of normal marrow , thus improving contrast between infiltrated and normal marrow [ 28 ]  . new developments in mri equipment allow coronal images ( se t1 - weighted and stir images ) of the entire skeleton [ whole - body mri ( wbmri ) ] to be obtained by a tablemoving technique in a matter of minutes . 
the maximal longitudinal field of view ( fov ) is 180210 c the larger fov enables head - to - toe scanning of most adult patients without repositioning of the patient [ 30 ]  . 
it is necessary to acquire t1 - weighted and stir images for different stations , from head to feet , by using the combination of moving tabletop , table extender and image - melding software [ 31 ]  . in the evaluation of bone metastatic disease , lymphoma and myeloma , sagittal images of the spine are acquired employing tse t1 , t2 and stir sequences . 
interpretation may generally take place in as little as 1015 min [ 9 ]  . mri of normal bone marrow normal bone marrow is dynamic , and this produces a wide spectrum of appearances of bone in mri . 
normal haematopoietically inactive , hypocellular , fatty or yellow marrow consists of apdi contrasto in rm deve , come minimo , avere la potenzialit di modificare la diagnosi e la prognosi . 
la decisione di impiegare il mezzo di contrasto deve essere diretta ad ottenere risposta ad uno specifico quesito diagnostico che non possa essere risolto con metodi pi semplici [ 26 , 27 ]  . lenhancement dinamico del midollo osseo normale fortemente influenzato dallet e dal contenuto di grasso . lenhancement midollare diminuisce marcatamente allaumentare dellet e con la conversione adiposa , nonostante una variabilit interindividuale . 
alcuni autori ritengono che lenhancement midollare non sia adeguatamente valutabile con le sequenze spin - echo t1 - pesate , ma possa essere analizzato utilizzando sequenze dinamiche ultraveloci , che comportano unelevata risoluzione temporale ed una forte pesatura in t1 . 
i criteri di imaging di rm per definire il midollo osseo normale non sono basati sulla sola analisi dellintensit di segnale in t1 , ma anche sui dati di analisi della curva temporale di enhancement ( etc ) , che forniscono , entrambi , informazioni aggiuntive per la valutazione del paziente con malattia del midollo osseo [ 2426 ]  . recentemente sono state sviluppate particelle superparamagnetiche di ossido di ferro di piccole dimensioni per valutare lattivit fagocitaria anche nel midollo osseo . 
le piccole particelle sono captate dal sistema reticolo - endoteliale del midollo osseo e riducono il segnale t1 e t2 del midollo normale , migliorando cos il contrasto fra midollo normale ed infiltrato [ 28 ]  . i nuovi sviluppi nellapparecchiatura di rm permettono di ottenere immagini coronali ( se t1 - pesate e stir ) dellintero scheletro ( rm whole body ) nel tempo di minuti , utilizzando una tecnica con movimento del tavolo . 
il massimo campo di vista ( fov ) longitudinale di 180210 cil fov pi grande permette una scansione dalla testa ai piedi della maggior parte dei pazienti adulti senza necessit di riposizionare il paziente [ 30 ]  . 
necessario acquisire immagini t1 - pesate e stir per le differenti stazioni , dalla testa ai piedi , usando una combinazione di lettino portapazienti movibile , estensione del lettino e software di integrazione dellimmagine [ 31 ]  . nella valutazione della malattia metastatica dellosso , del linfoma e del mieloma , vengono acquisite anche immagini sagittali della colonna vertebrale utilizzando sequenze spin - echo t1 , t2 e stir . 
le immagini vengono quindi riallineate velocemente utilizzando software dedicati per facilitare la visione panoramica immediata , migliorata dallo scorrimento individuale delle immagini in cine - loop alla workstation . proximately 15% water , 5% protein and 80% fat . 
with aging , the fat content of cellular marrow increases so that by the age 70 , cellular marrow is approximately 30% water , 60% fat and 10% protein . at birth , most of marrow contains haematopoietic cells because of the high demands for blood by the neonate . 
this is true also for the appendicular skeleton [ 1 , 2 , 4 , 1113 , 32 ]  . haematopoietic marrow hyperplasia represents a deviation from the accepted adult red - yellow pattern distribution . this variation in red marrow distribution is generally discovered as an incidental finding on routine knee mri examination . 
its prevalence varies from 0.7% to 35% of the healthy population . large residual red marrow islands represent a physiological variation of the normal adult pattern in women , obese subjects and cigarette smokers , and in marathon runners with sports anaemia [ 1 ]  . normal age - related distribution of haematopoietic and fatty marrow has to be taken into account in the interpretation of images [ 32 ]  . 
understanding the possible variability is also a prerequisite to the interpretation of mr images of bone marrow pathology . mri of abnormal bone marrow signal intensity of bone marrow reflects its major components . 
there are three mri patterns of abnormal marrow that reflect different types of histological infiltration of bone marrow : focal pattern diffuse homogeneous pattern diffuse heterogeneous ( variegated or salt - and - pepper ) pattern the patterns of bone marrow involvement are not specific [ 16 ]  . 
the focal pattern of marrow involvement is characterised by the presence of an area of normal marrow replacement , generally but not always with low signal intensity on t1 - weighted images and variable signal intensity on t2 - weighted images . 
appearance of bone marrow on mri sometimes remains normal in patients with biopsy - proven marrow disease . classification of bone marrow changes according to their pathophysiological mechanisms has gained general acceptance [ 16 ]  . 
con linvecchiamento , il contenuto adiposo del midollo cellulare aumenta cos che a 70 anni il midollo cellulare composto da 60% grasso , 30% acqua e 10% proteine . alla nascita , a causa dellelevata richiesta di sangue del neonato , la maggior parte del midollo contiene cellule ematopoietiche . 
levoluzione graduale del midollo osseo da rosso a giallo durante laccrescimento pu presentare una variabilit interindividuale , particolarmente a livello vertebrale , ma anche a livello dello scheletro appendicolare [ 1 , 2 , 4 , 1113 , 32 ]  . liperplasia del midollo ematopoietico rappresenta una deviazione dal pattern normale di distribuzione rosso - giallo delladulto . 
ampie isole residue di midollo rosso rappresentano una variazione fisiologica del pattern normale adulto nelle donne , negli obesi , nei fumatori di sigarette e nei maratoneti con anemia dello sportivo [ 1 ]  . la normale distribuzione et - correlata del midollo ematopoietico ed adiposo deve essere presa in considerazione , quando si interpretano le immagini [ 32 ] , poich la conoscenza della possibile variabilit degli aspetti in funzione dellet rappresenta un prerequisito indispensabile allinterpretazione delle immagini di rm nella patologia del midollo osseo . rm del midollo osseo patologico lintensit di segnale riflette i componenti principali del midollo osseo . 
bone marrow ischaemia is the death of bone and red cells . myeloma mm is the most common primary tumour arising in bone , accounting for 10% of haematological malignancies and 1% of all malignant diseases . 
the staging system devised by durie and salmon was based on serum haematological , urinary bence - jones protein and skeletal survey findings [ 33 ]  . more marrow lesions are detected with mri than with plan radiograph or bone scan . 
vi sono tre pattern di rm del midollo patologico , che riflettono i tipi istologici di infiltrazione del midollo osseo : pattern focale ; pattern diffuso omogeneo ; pattern diffuso eterogeneo ( aspetto variegato a sale e pepe )  . i pattern di coinvolgimento del midollo osseo non sono specifici [ 16 ]  . 
il pattern focale di coinvolgimento midollare caratterizzato dalla presenza di unarea di sostituzione del midollo normale generalmente , ma non sempre , con bassa intensit di segnale nelle sequenze t1 - pesate ed intensit di segnale variabile nelle sequenze t2 - pesate . 
laspetto del midollo osseo in rm talvolta rimane normale anche nei pazienti in cui la malattia midollare dimostrata alla biopsia . la classificazione delle variazioni del midollo osseo in base al loro meccanismo fisio - patologico generalmente aco . 
spinecho ( se ) t1 - weighted image of the hip shows the typical salt - and - pepper pattern of bone marrow ( b ) , which is better seen on the turbo short t1 inversion recovery ( t - stir ) image ( c )  . 
limmagine se t1 - pesata mostra il tipico pattern pepe e sale del midollo osseo ( b ) , che si dimostra meglio nellimmagine t - stir ( c )  . 
alla t - stir della colonna toraco - lombare si vede il pattern pepe e sale diffuso del midollo osseo e una lesione focale di l1 ( e )  . have a solitary bone plasmocytoma . 
progression to systemic disease in patients with solitary bone plasmocytoma has been attributed to growth of previously occult disease [ 5 , 35 ]  . mri should be part of the staging procedures in patients with solitary bone plasmocytoma to better assess the local extent of the tumour , define radiation portals and search for other unanticipated foci of disease . 
some authors emphasise the role of mdct in combination with mri for staging these tumours . mdct is markedly superior to conventional radiography and allows more precise assessment of bony destruction [ 36 ]  . 
other authors emphasise the role of nuclear medicine in solitary myeloma to exclude other involved sites [ 37 ]  . a well - recognised contribution of mri in the management of patients with mm is the evaluation of patients with neurological symptoms . 
in possible acute spinal cord compression , mri is the first , and often the only , examination to perform [ 3 ]  . the staging system of durie and salmon [ 33 ] developed in cettata [ 16 ]  . 
lischemia del midollo osseo la morte dellosso e delle cellule ematopoietiche . mieloma il mieloma multiplo ( mm ) il tumore primitivo dellosso pi comune , rappresentando il 10% delle neoplasie ematologiche e l1% di tutte le neoplasie . 
il sistema di stadiazione proposto da durie e salmon era basato sui reperti laboratoristici del sangue , sulle proteine di bence - jones urinarie e sulle radiografie dello scheletro [ 33 ]  . 
proposed implementation of mri in the staging system of durie and salmon [ 35 ]  . the combination of mri in the staging system of durie and salmon provided the best results with respect to survival . 
because the spine covers a large portion of the red marrow and it is the major site of involvement in patients with mm , the spine is ideally suited for staging the extent of bone marrow involvement [ 35 ]  . wbmri allows direct visualisation of actual tumour burden within the bone marrow [ 9 ] : in the stage with slight interstitial plasma cell infiltration ( < 20% in bone marrow biopsy ) , a normal - looking bone marrow signal is found in all sequences . 
 because the importance of mri for sensitive detection and prognostic significance of bone marrow infiltrates has been well demonstrated , durie and coworkers recently created the durie and salmon plus staging systems [ 39 ]  . 
concerning diagnostic imaging , mri and fluorodeoxyglucose positron emission tomography ( fdg - pet ) were included for staging patients with mm : a three - grade scale for spinal mri has been recently implemented in the clinical staging systems of durie and salmon . selection of therapy is based upon this classification [ 39 ]  . however , traditional mri protocols often do not include the skull , sternum or ribs , which are areas with large amounts of red marrow and represent frequent sites of infiltration . 
in future studies , a large series of patients and different staging systems inclusive of mri should be evaluated and compared with staging without the inclusion of mri . mri bone marrow surveys in patients with mm demonstrate the broad spectrum of involvement , treatment results , areas of potential complications and sites of focal disease for safe bone biopsy . 
recently , some authors have suggested the use of dynamic contrast - enhanced mri for diagnosing and grading bone marrow involvement in patients with lymphoproliferative disease and diffuse bone marrow infiltration as well as for evaluating treatment response [ 42 , 43 ]  . precedentemente occulta [ 5 , 35 ]  . 
la rm dovrebbe essere parte delle procedure di stadiazione nei pazienti con plasmocitoma osseo solitario per meglio determinare lestensione locale del tumore , per definire il campo dirradiazione e per ricercare altri foci di malattia non precedentemente conosciuti . 
la soppressione del grasso fornisce un elevato contrasto fra i foci tumorali ed il midollo normale . la rm pu riconoscere foci occulti dinteressamento mielomatoso e pu prevedere un decorso di malattia pi sfavorevole . 
altri autori enfatizzano il ruolo della medicina nucleare nel mieloma solitario per escludere altri siti di coinvolgimento [ 37 ]  . un contributo ben conosciuto della rm , nella gestione dei pazienti affetti da mieloma multiplo , la valutazione dei pazienti che presentano sintomi neurologici . 
daltra parte la tc permette una valutazione ottimale della distruzione ossea . nellipotesi di compressione midollare acuta la rm il primo , e spesso lunico , esame da eseguire [ 3 ]  . il sistema di stadiazione di durie e salmon [ 33 ] , sviluppato nel 1975 , ancora largamente in uso . 
dal momento che la colonna vertebrale rappresenta unampia porzione del midollo rosso e che uno dei principali siti di coinvolgimento nei pazienti con mieloma multiplo , il rachide il sito ideale per la stadiazione dellestensione del coinvolgimento midollare [ 35 ]  . la rm whole body permette la visualizzazione diretta del reale coinvolgimento tumorale allinterno del midollo osseo [ 9 ] : nello stadio di infiltrazione plasmacellulare interstiziale lieve ( < 20% alla biopsia del midollo osseo ) , in tutte le sequenze il midollo osseo presenta segnale normale . 
coronal whole - body magnetic resonance imaging ( a ) and sagittal t1 - weighted ( b ) and short t1 inversion recovery ( c ) images show normal bone marrow signals . 
istologicamente questo corrisponde ad uninfiltrazione moderata di plasmacellule ( < 20% vol alla biopsia del midollo osseo )  . lymphoma although secondary osseous involvement is relatively common in both hodgkins disease and nhl and occurs in up to 16% of cases , primary bone lymphoma is uncommon . 
lymphomatous involvement of bone marrow is seen as diffuse , primarily heterogeneous , replacement of collaboratori hanno sviluppato il sistema di stadiazione durie e salmon plus [ 39 ]  . 
per quanto riguarda limaging diagnostico , sono state incluse nella stadiazione dei pazienti con mieloma multiplo la rm e la fdg - pet : una scala a tre gradi per la rm del rachide stata aggiunta nel sistema di stadiazione clinico di salmon e durie . 
la scelta della terapia si basa su questa classificazione [ 39 ]  . tuttavia , i tradizionali protocolli di rm spesso non includono il cranio , lo sterno e le coste , che sono aree ad elevato contenuto di midollo rosso e spesso rappresentano sede di infiltrazione . 
coronal t1 - weighted ( a , b ) and short t1 inversion recovery ( stir ) ( c , d ) wholebody magnetic resonance imaging show a homogeneous decrease of signal in t1 - weighted spin - echo images and increased signal intensity on fat - suppressed images . 
la rm whole body coronale t1 - pesata ( a , b ) e stir ( c , d ) mostrano unomogenea diminuzione di segnale nelle t1 - se e un aumento di intensit nelle immagini a soppressione del grasso . 
patients with lymphoma and femoral marrow involvement seen on mri appear to have an overall lower survival rate than patients with normal femoral marrow on mri , whatever the results of blind biopsy . recently some authors have emphasised the role of wbmri as a noninvasive and nonradiation imaging method , which is used to assess stage and prognosis , select biopsy sites and monitor treatment response in patients who cannot undergo pretreatment ct - pet [ 46 , 47 ]  . leukaemias are a group of different neoplasms derived from arrested , or aberrant , development of a clone of normal haematopoietic cells . 
these immature cells proliferate progressively within bone marrow , replacing normal hematopoietic tissue and circulating within the peripheral blood being leukaemia no dimostrato che un sistema di staging esteso , che includa la rm , ha una significativa influenza sulla prognosi del paziente e sul tasso di sopravvivenza : usando il sistema di stadiazione classico di durie e salmon senza la rm , 25 di 77 pazienti sarebbero stati sottostadiati . 
sarebbe necessario valutare , in futuri studi , un grande numero di pazienti e diversi sistemi di staging che comprendano la rm e , quindi , compararli con le stadiazioni che non includono la rm . unanalisi con rm del midollo osseo nei pazienti con mieloma multiplo dimostra lampio spettro di coinvolgimento , i risultati del trattamento , le aree di potenziali complicanze ed i siti di malattia focale per una biopsia ossea sicura . 
nel iii stadio di mieloma multiplo , il coinvolgimento del midollo osseo vertebrale in rm si correla con la prognosi [ 40 , 41 ]  . vi sono prove crescenti che langiogenesi incrementa nelle neoplasie ematologiche . 
focal bone lesions are more common in acute lymphoblastic leukaemia than in acute myelogenous leukaemia [ 14 ]  . there are no definite findings to suggest that mri has a clinical role to play in patients with acute leukaemia . 
however , it is useful in patients suspected of relapse , for example , in patients believed to be in remission who re - present with bone pain or in patients at high risk of relapse in whom serial bone marrow biopsies are negative [ 1 , 2 , 5 ]  . 
accurate assessment of residti con malattia linfoproliferativa ed infiltrazione midollare diffusa ed anche per la valutazione della risposta al trattamento [ 42 , 43 ]  . linfoma sebbene il coinvolgimento osseo secondario sia relativamente comune sia nella malattia di hodgkin sia nel linfoma non hodgkin ( nhl ) , ed avvenga fino al 16% dei casi , il linfoma osseo primario non comune . 
the d - 6 lesion is slightly hyperintense on turbo short t1 inversion recovery ( t - stir ) image ( c ) , whereas the haemangioma shows low signal intensity . 
nellimmagine tse - t2 pesata gli emangiomi sono iperintensi , mentre la lesione linfomatosa appare lievemente ipointensa ( b )  . la lesione di d6 appare lievemente iperintensa nellimmagine t - stir ( c ) , mentre lemangioma ha bassa intensit di segnale . 
il tessuto solido mostra omogeneo enhancement nellimmagine spir t1 - pesata dopo iniezione intravenosa di mezzo di contrasto ( d )  . ual disease may be important for predicting outcome in patients with acute leukaemia in complete remission . 
some authors have emphasised the role of mri of femoral marrow in predicting outcome in adult patients with aml in complete remission [ 48 ]  . post - therapy changes special diagnostic problems occur in patients with cancer of the haematopoietic system during therapy . 
before evaluating posttherapy changes seen on mri of bone marrow , it is necessary to be familiar with changes that occur in each mr pattern of abnormal marrow after treatment . 
i pazienti con linfoma e coinvolgimento del midollo femorale diagnosticato in rm sembrano avere un tasso di sopravvivenza generale inferiore rispetto ai pazienti con midollo femorale normale alla rm , qualunque sia il risultato della biopsia eseguita alla cieca . recentemente alcuni autori hanno sottolineato il ruolo della rm whole body come tecnica di imaging non invasiva e priva di radiazioni per la formulazione dello stadio e della prognosi , per selezionare i siti da bioptizzare e per monitorare la risposta terapeutica nei pazienti che non possono sottoporsi ad una tc - pet prima del trattamento [ 46 , 47 ]  . leucemia le leucemie sono un gruppo di differenti neoplasie che derivano dallarresto o da un aberrante sviluppo di un clone di cellule ematopoietiche normali . 
le lesioni ossee focali sono pi comuni nella leucemia linfoblastica acuta piuttosto che nella leucemia mieloide acuta [ 14 ]  . non vi sono risultati definitivi che dimostrino che la rm abbia un ruolo nei pazienti con leucemia acuta . 
donna di 36 anni con pancitopenia ( gr 1 , 2 ; emoglobina 6 , 6 , piastrine 67000 ) da chemioterapia con temozolomide usato per trattare una neoplasia cerebrale ( oligoastrocitoma anaplastico )  . 
dopo il trattamento con g - csf ( gb 8 , 6 , emoglobina 9 , 6 g / dl , piastrine 409000 ) le immagini pesate in t1 ( b ) e stir ( c ) dimostrano una diffusa basa intensit di segnale nelle t1 e lieve aumento omogeneo dellintensit di segnale nella stir dovuto alla riconversione midollare . images predominantly inside the rt field . 
the characteristic fatty marrow appearance is not reversible for doses equal to or higher than 30 to 40 gy , because destruction of vascular sinusoids is nonreversible . changes that occur in bone marrow after chemotherapy are known from histological studies [ 4951 ]  . administration of haematopoietic growth factors may delay the fatty transformation of bone marrow or may cause reconversion of fatty to haematopoietic marrow and simulate persistent or relapsing disease [ 5254 ]  . 
colony - stimulating factor ( csf ) , in the form of granulocyte macrophage csf ( gmcsf ) and g - csf is becoming a widely used adjuvant to chemotherapy for treating many malignancies , because it dealterazioni post - terapia particolari problemi diagnostici si presentano nei pazienti con neoplasie del sistema ematopoietico durante la terapia . prima di valutare le alterazioni post - terapia che si verificano nelle immagini rm del midollo osseo , necessario avere dimestichezza con le alterazioni che avvengono nel midollo patologico dopo terapia . 
dopo uniniziale diminuzione dellintensit di segnale nelle immagini t1 - pesate ed un aumento in quelle stir , dovuto alledema ed alla necrosi midollare , si verifica la deplezione midollare . 
tuttavia , una diminuzione di enhancement dopo contrasto si osservata nel midollo osseo , non solo allinterno , ma anche fuori dal campo di radioterapia nel primo mese dopo la terapia . 
 i cambiamenti che avvengono nel midollo osseo dopo chemioterapia sono noti dagli studi isologici [ 4951 ]  . la somministrazione di fattori di crescita ematopoietici ritarda la trasformazione adiposa del midollo ematopoietico e simula una malattia persistente o recidiva [ 5254 ]  . 
i fattori di crescita , nella forma di gmcsf e di gcsf , stanno divenendo un adiuvante alla chemioterapia largamente utilizzato nel trattamento di diverse neoplasie , perch provocano una diminuzione dellincidenza di eventi neutropenici e di infezioni e riducono il numero e la durata delle ospedalizzazioni e delle terapie antibiotiche . 
si ritiene che tutti i radiologi che interpretino uno studio di rm di un paziente con neoplasia maligna debbano avere familiarit con questi aspetti del midollo osseo . nel 2002 , dalrup - link et al . 
proposero luso degli ossidi di ferro superparamagnetici per differenziare linfiltrazione tumorale dal midollo osseo ipercellulare nei pazienti con linfoma non hodgkdopo la somministrazione di particelle di ossido di ferro superparamagnetico ( spio ) si osserva unalterazione del segnale del midollo osseo vertebrale simile a quella che si verifica nel fegato e nella milza . 
immagini t1 - pesate ( a ) e stir ( b ) dopo trattamento con gsf : nel midollo osseo vi sono un basso segnale nellimmagine t1 e un incremento disomogeneo nellimmagine stir , dovuti allaumento di midollo ematopoietico . crease the incidence of neutropenic events and infections and reduces the number and length of hospitalisations and duration of antibiotic therapy . 
we believe that all radiologists interpreting mri studies of patients with primary malignant tumours should be familiar with these aspects of bone marrow . recurrent in 2002 , daldrup - link et al . 
after administration of superparamagnetic iron oxide particles ( spio ) , a signal intensity change similar to that seen in the liver and spleen is observed in vertebral bone marrow . 
it is important to note that in patients with a minor amount of diffuse tumour cell infiltration , iron oxide uptake approximated that of normal haematopoietic bone marrow [ 28 ]  . some authors recently proposed dynamic gadoterate meglumine - enhanced turbo fast low - angle shot ( flash ) mr images to evaluate the increased spinal bone marrow enhancement in patients with lymphoproliferative diseases and diffuse bone marrow infiltration and to monitor bone perfusion in the follow - up of patients receiving antiangiogenesis therapy . 
thus , dynamic contrast - enhanced mri may be helpful in diagnosing and grading bone marrow involvement in such patients and for evaluating treatment response to angiogenesis - inhibiting drugs such as thalidomide . 
initial results of a clinical histological study involving patients with mm revealed that after chemotherapy , mean microvessel density was significantly lower in responders compared with nonresponders [ 26 ]  . it is important to point out that wbmri of therapeutic response in human bone marrow was achieved without the use of contrast agents by using diffusion - weighted echo - planar mri of physiological water . 
the results encourage further investigation . conclusions although it is unlikely that mri will actually replace bone marrow biopsy for detecting bone marrow disease , mri and particularly wbmri is useful in evaluating the entire bone marrow and the number , type and location of bone marrow lesions [ 9 , 10 ]  . 
iliac crest biopsy may be negative when bone marrow infiltration is focal rather than diffuse . understanding the normal age - related changes in bone marrow signal is required for interpretation . 
although the morphological aspects are useful , pathological changes can be nonspecific , and correlation should always be made with clinical , laboratory and radiological findings . what is todays clinical role of bone marrow mri in oncohaematological disease ? infiltrazione tumorale di tipo diffuso , luptake di ossidi di ferro simile a quello del midollo osseo ematopoietico normale [ 28 ]  . alcuni autori hanno recentemente proposto le immagini dinamiche di tipo turbo flash con gadoterato meglumina come mezzo di contrasto per valutare laumentato enhancement del midollo osseo del rachide nei pazienti con malattie linfoproliferative ed infiltrazione midollare diffusa e per monitorare il follow - up dei pazienti che ricevono una terapia angiogenetica . 
perci le sequenze dinamiche dopo contrasto potrebbero essere utili nella diagnosi e nel grading del coinvolgimento midollare in questi pazienti e per valutare la risposta al trattamento con farmaci che inibiscono langiogenesi , come la talidomide . 
i risultati iniziali di uno studio clinico istologico che coinvolge i pazienti con mieloma multiplo rivelano che dopo la chemioterapia la densit media di vasi di piccolo calibro significativamente inferiore nei pazienti che rispondono alla terapia rispetto ai non responders [ 26 ]  . importante sottolineare che limaging whole body per valutare la risposta terapeutica nel midollo osseo umano era stato ottenuto senza utilizzare mezzo di contrasto , ma servendosi di imaging echo - planare di diffusione del liquido fisiologico . 
i risultati incoraggiano a continuare le ricerche . conclusioni sebbene non sia probabile che la rm possa rimpiazzare la biopsia del midollo osseo per lindividuazione della patologia midollare , la rm , e particolarmente la rm whole body , utile per valutare il midollo osseo nella sua interezza ed il numero , il tipo e la sede delle lesioni midollari [ 9 , 10 ]  . 
la biopsia della cresta iliaca pu essere negativa , quando linfiltrazione midollare non diffusa ma focale . la comprensione dei normali cambiamenti che avvengono nel segnale del midollo osseo con let necessaria per linterpretazione . 
sebbene alcuni aspetti morfologici possano essere utili , le variazioni patologiche non sono specifiche , perci sempre necessaria la correlazione fra la clinica ed i reperti radiologici e laboratoristici . quale ruolo clinico riveste oggi la rm del midollo osseo nelle malattie onco - ematlogiche ? 1 . 
la rm pu essere utilizzata per selezionare i siti di biopsia , dato che la biopsia della cresta iliaca pu risultare negativa in presenza di uninfiltrazione midollare focale e non diffusa . 
optimal marrow evaluation should include both biopsy and mri , and it is important to note that biopsy induces important changes in the mri signal and creates hot spots on bone scintigraphy . 
mri is a useful adjunct to bone marrow biopsy for patients who are candidates for bone marrow transplant and in particular for patients with lymphoma , a disease with a well - known patchy mode of infiltration of the bone marrow [ 5 ]  . mri requires someone with a great deal of experience in making choices regarding imaging conditions and evaluating findings . 
technical - methodological guidelines need to be devised , and research is under way and not only for sequences but also for a contrast medium [ 6063 ]  . wbmri has oncological applications in evaluation of skeletal metastases , myeloma and lymphoma . 
nevertheless , it is reasonable to propose that wbmri should be used as a single modality as a first step in bone marrow imaging in oncohaematological diseases [ 9 , 10 ]  . a balanced and positive relationship within the team composed of different specialists is necessary , because today in medicine , multidisciplinary patient management is mandatory . 
as in other malignancies , close liaison between clinician and radiologist is essential to determine the most appropriate use of imaging for individual patient care . te notare che la biopsia induce importanti cambiamenti del segnale di rm del midollo osseo e crea degli spot ipercaptanti alla scintigrafia ossea . 
la rm un utile complemento alla biopsia midollare per i pazienti candidati a trapianto del midollo osseo e , in particolare , per i pazienti con linfoma , una malattia di cui nota linfiltrazione midollare a chiazze [ 5 ]  . la rm richiede un operatore con molta esperienza per la scelta dei piani di studio e delle sequenze da utilizzare e per linterpretazione dei reperti . 
necessario formulare delle linee guida tecnico - metodologiche , e la ricerca ancora in corso , non solo sulle sequenze , ma anche sui mezzi di contrasto [ 6063 ]  . in ambito onco - ematologico la rm whole body trova oggi applicazione nella valutazione delle metastasi scheletriche , del mieloma , del linfoma . 
tuttavia ipotizzabile che la rm whole body debba essere usata come unica modalit di imaging di primo livello del midollo osseo nelle patologie onco - ematologiche [ 9 , 10 ]  . necessario istaurare un rapporto valido e bilanciato allinterno di un team composto da differenti specialisti , perch nella medicina odierna necessaria una gestione multidisciplinare del paziente . 
isbn 978 - 88 - 470 - 0666 - 9 . published online : 23 july 2007 this book has come a long way from the in - depth knowledge and experience acquired daily on the field by a group of radiologists who have always had a keen interest in the pathophysiology and morphodynamism of the small bowel in acute abdominal disease . through the correct interpretation of these findings , radiologists enable clinicians both to establish a diagnosis and to evaluate the time course of the event , thereby guiding them towards the most appropriate treatment strategy . the decision to use an atlas - textbook to illustrate one of the most common causes of acute abdomen , namely , small - bowel obstruction and its natural course , emerged during the itinerant courses of the urgency and emergency section of the societ italiana di radiologia medical , as this topic invariably met with great interest among course participants , who repeatedly asked for an easy and straightforward text that could serve as a quick reference . the topic lends itself particularly well to this purpose , as it presents the entire spectrum of findings that a young radiologist should be familiar with . 
a large part of the text focuses on computed tomography ( ct ) imaging , the main tool for understanding the ileum - mesentery complex . the authors fill a gap in the scientific literature on this difficult topic , which , for a correct approach to imaging , requires in - depth knowledge of the clinical pathophysiology of the disease . questo libro nasce da una profonda conoscenza ed esperienza acquisite quotidianamente sul campo da un gruppo di radiologi attenti , da sempre , agli aspetti fisiopatologici e al morfodinamismo del tenue nella patologia addominale acuta . grazie alla corretta interpretazione di questi rilievi , il radiologo offre al clinico la possibilit sia di fare diagnosi , sia di valutare la cronologia dellevento , indirizzandolo verso la decisione terapeutica idonea . la scelta di illustrare con un testo - atlante una delle cause pi frequenti di addome acuto locclusione del piccolo intestino e il suo evolversi maturata nei corsi itineranti della sezione di urgenza ed emergenza della societ italiana di radiologia medica . 
tale argomento , infatti , ha sempre suscitato interesse nei discenti , i quali hanno ripetutamente chiesto un testo facile , comprensibile e di rapida consultazione . largomento ben si presta a questo scopo , poich mostra lintero paradigma dei reperti che un giovane radiologo deve conoscere . 
in the past , when patients required a contrast - enhanced imaging study , the tendency was to select magnetic resonance ( mr ) imaging with a gadolinium ( gd ) - based contrast agent over computed tomography ( ct ) with iodinated contrast media ( cm ) due to the known nephrotoxic nature of iodinated cm , which is associated in some patients with the development of contrast - induced nephropathy ( cin )  . 
however , recently , the administration of gd - based contrast agents has been associated with a severe , potentially fatal , adverse reaction , termed nephrogenic systemic fibrosis ( nsf ) , in patients with moderate - tosevere renal insufficiency [ 1 ]  . 
in order to optimise patient outcomes , imaging of patients with ckd requires an understanding of the risk factors for both cin and nsf . key words contrast media nephrogenic systemic fibrosis renal disease contrast - induced nephropathy gadolinium riassunto sovente nei pazienti con insufficienza renale cronica si presenta la necessit di effettuare una procedura di imaging diagnostico con mezzo di contrasto ( mdc ) e il radiologo si trova a dover decidere qual la modalit di imaging pi idonea per ciascun paziente in base al grado di compromissione della funzionalit renale . 
in passato la tendenza era quella di indirizzare il paziente alla risonanza magnetica ( rm ) con chelati del gadolinio ( gd ) piuttosto che sottoporlo alla tomografia computerizzata ( tc ) con mdc iodato , data la potenziale nefrotossicit di questi agenti che in alcuni pazienti pu dar luogo a nefropatia da contrasto ( cin )  . recentemente tuttavia , la somministrazione di mezzi di contrasto per rm a base di gd in pazienti con insufficienza renale da moderata a grave stata associata alla comparsa di una malattia grave e con esito potenzialmente fatale conosciuta come fibrosi sistemica nefrogenica ( nsf ) [ 1 ]  . 
per ottimizzare la gestione del paziente con funzionalit renale compromessa da sottoporre a procedure di imaging necessaria pertanto la comprensione dei fattori di rischio per entrambe queste patologie . parole chiave mezzi di contrasto fibrosi sistemica nefrogenica insufficienza renale nefropatia da contrasto gadolinio introduction introduzione for over 10 years , when patients with renal impairment required a contrast - enhanced imaging procedure , radiologists were inclined to select magnetic resonance ( mr ) over computed tomography ( ct ) in order to preserve kidney function , as iodinated contrast media ( cm ) used in ct have been shown to increase the risk of contrast - induced nephropathy ( cin ) in that group of patients . 
cin is defined as an acute decline in renal function following administration of iodinated cm with no other known cause , is responsible for over 10% of cases of hospital - acquired renal insufficiency and is associated with significant morbidity and mortality . 
 da oltre 10 anni , quando un paziente con ridotta funzionalit renale doveva essere sottoposto ad una procedura di imaging con mezzo di contrasto ( mdc ) , il radiologo era incline a selezionare la risonanza magnetica ( rm ) invece della tomografia computerizzata ( tc ) per preservare la funzionalit renale , dal momento che noto che in quel gruppo di pazienti i mezzi di contrasto iodati usati nella tc possono aumentare il rischio di insorgenza di nefropatia da contrasto ( cin )  . 
1 a 49 - year - old man with terminal renal failure [ glomerular filtration rate ( gfr ) 10 ml / min / 1.73 m2 ] was first exposed to gadodiamide ( 0.3 mmol / kg ) during a magnetic resonance angiography study in april 2003 . 
1 paziente di sesso maschile di 49 anni con insufficienza renale terminale ( gfr 10 ml / min / 1 , 73 m2 ) stato dapprima esposto a somministrazione di gadodiamide ( 0 , 3 mmol / kg ) durante unindagine in mra nellaprile 2003 in seguito alla quale ha sviluppato unnsf progressiva con gravi variazioni cutanee e cachessia . 
il paziente deceduto 30 mesi dopo la prima somministrazione ( per gentile concessione del peter marckmann , dipartimento di of nefrologia dellospedale universitario di copenhagen , herlev , danimarca )  . recently , a severe , systemic adverse reaction nephrogenic systemic fibrosis ( nsf ) has been observed days to months following administration of some of the extracellular gadolinium ( gd ) - based mr contrast agents in patients with baseline renal insufficiency [ 1 , 2 ]  . 
thus , selection of an imaging modality in the patient with reduced renal function [ chronic kidney disease ( ckd ) stage 45 : glomerular filtration rate ( gfr ) < 30 ml / min / 1.73 m2 ] is no longer a simple decision : this same patient group has an increased risk for both cin after administration of iodinated cm and nsf after administration of gd - based mr contrast agents . 
 determination of patients at risk for cin and nsf baseline renal insufficiency is the greatest risk factor for both cin and nsf : the poorer the kidney function ( based on gfr ) , the higher the risk . 
pertanto la scelta della modalit di imaging nel paziente con funzionalit renale ridotta ( insufficienza di stadio 4 - 5 : gfr < 30 ml / min / 1 , 73 m2 ) pi complessa di un tempo , in quanto questo stesso gruppo di pazienti presenta un aumentato rischio sia per la cin dopo somministrazione di mdc iodato che per lnsf dopo somministrazione di mdc a base di gd . in questo articolo passer in rassegna le attuali conoscenze riguardo ai fattori di rischio per cin e nsf e alla loro prevenzione . individuazione dei pazienti a rischio di cin e nsf il principale fattore di rischio sia per la cin che per lnsf la presenza di insufficienza renale : maggiore il grado di compromissione della funzionalit ( in base al gfr ) e maggiore il rischio . 
tuttavia , lidentificazione e la stratificazione dei pazienti con insufficienza renale non semplice e diversi studi hanno dimostrato che lestendere a tutti i pazienti la misurazione della creatinina serica e del gfr non conveniente dal punto di vista del costo - beneficio [ 3 ]  . 
inoltre le formule usate per calcolare la funzionalit renale ( quella di cockroft - gault e quella nota come modification of diet in renal disease [ mdrd ] ) si dimostrano utili solo in pazienti con un gfr < 60 ml / min / 1 , 73 m2 e i risultati ottenuti con lapplicazione di queste due formule possono differire tra loro . il comitato per la sicurezza dimpiego dei mezzi di contrasto della societ europea di radiologia urogenitale ( esur ) raccomanda che venga effettuatata di routine la determinazione dei livelli di creatinina serica e la stima del grf in determinati gruppi di pazienti : quelli con pregressa creatininemia elevata , i diabetici trattati con metformina , i pazienti da sottoporre a somministrazione endoarteriosa del mdc e quelli con unanamnesi compatibile con livelli elevati di creatinina ( per esempio , nefropatia , storia di chirurgia renale , proteinuria , diabete mellito , ipertensione , gotta o recente assunzione di farmaci nefrotossici ) [ 4 ]  . 
una volta confermata la presenza e lentit della insufficienza renale , allora il radiologo pu essere in grado di raccomandare la modalit di imaging pi opportuna per il paziente . not cost effective [ 3 ]  . 
cockcroft - gault and modification of diet in renal disease ( mdrd ) ] are useful only in patients with a gfr < 60 ml / min / 1.73 m2 , and the results obtained with these two formulae may differ . 
once the existence and level of renal dysfunction are confirmed , radiologists can then recommend the best imaging modality for each patient . prevention of cin for many years , it has been recognised that cin develops in significantly more patients after administration of high - osmolar cm ( osmolality > 1 , 500 mosm / kg ) compared with low - osmolar cm ( osmolality < 915 mosm / kg ) [ 5 ]  . 
however , it remains controversial whether differences in the incidence of cin exist between available cm , either low - osmolar cm or isoosmolar cm ( iocm , osmolality 290 mosm / kg ) [ 6 ]  . 
a total of eight nonionic monomers ( iohexol , iomeprol , iopamidol , iopentol , ioxilan , iopromide , ioversol and iobitridol ) , one ionic dimer ( ioxaglate ) and one nonionic dimer ( iodixanol ) are currently approved for intravascular use in europe , with their level of use varying from country to country . eleven studies have compared a low - osmolar cm with an iocm . 
in seven of these head - to - head comparisons , the cm was given intra - arterially : two were favourable for the nonionic dimer , whereas the remaining five showed no statistically significant difference between the two ca systematic analysis of published papers and us food and drug administration ( fda ) reports of adverse events demonstrated that the risk of cin after intra - arterial injection was higher in patients following the nonionic monomer iohexol than following another nonionic monomer iopamidol [ 7 ]  . 
one study demonstrated a significantly lower rate of cin after administration of the nonionic monomer than the dimer , and when one pools the two almost identical studies active and impact , and looks at the patients with reduced renal function ( < 40 ml / min / 1.73 m2 ) , no patient in the monomer group developed cin ( serum creatinine increase > 44 mol / l , 4872 h ) , whereas six did so in the group who received the dimer ( p < 0.0059 ) ( [ 10 ] ; hst personal communication )  . 
thomsen : imaging patients with chronic kidney disease : cin or nsf ? prevenzione della cin per molti anni stato riconosciuto che la cin si verifica con maggiore frequenza in pazienti a cui veniva somministrato un mdc ad alta osmolarit ( osmolalit > 1 , 500 mosm / kg ) che non dopo somministrazione di un mdc a bassa osmolarit ( osmolalit < 915 mosm / kg ) [ 5 ]  . 
rimane invece controverso se esista una differenza nellincidenza di cin tra i mezzi di contrasto a bassa osmolarit e iso - osmolari ( osmolalit 290 mosm / kg ) [ 6 ]  . 
attualmente in europa sono approvati per luso intravascolare otto mezzi di contrasto monomerici nonionici ( iohexolo , iomeprolo , iopamidolo , iopentolo , ioxilan , iopromide , ioversolo , e iobitridolo ) , un dimero ionico ( ioxaglato ) , and e un dimero nonionico ( iodixanolo ) con livelli di utilizzo variabili da paese a paese . sono stati riportati 11 studi di confronto diretto tra un mdc a bassa osmolarit e uno iso - osmolare . 
in 7 di questi il mdc stato somministrato per via intrarteriosa : 2 erano favorevoli al dimero non ionico mentre i rimanenti 5 non dimostravano una differenza significativa tra i due prodotti . unanalisi sistematica della letteratura e delle segnalazioni di reazioni avverse alla food and drug administration ( fda ) ha dimostrato che il rischio di cin dopo somministrazione intrarteriosa era pi alto nei pazienti che avevano ricevuto il monomero nonionico iohexolo rispetto a un altro monomero nonionico , lo iopamidolo [ 7 ]  . per quanto riguarda la via endovenosa , nessuno studio ha riportato dei dati a favore del dimero nonionico [ 8 , 9 ] ( tabella 1 )  . 
vi invece uno studio che ha dimostrato unincidenza significativamente pi bassa di cin dopo la somministrazione del monomero nonionico rispetto al dimero , e quando si combinano i dati di due studi quasi identici , lactive e limpact , e si considerano i pazienti con funzionalit renale ridotta ( < 40 ml / min / 1 , 73 m2 ) , si riscontra che nessun paziente nel gruppo a cui era stato somministrato il monomero ha sviluppato la cin ( aumento della creatinina > 44 mol / l , dopo 4872 ore ) mentre nel gruppo che aveva ricevuto il dimero 6 pazienti la avevano sviluppata ( p < 0 , 0059 ) ( [ 10 ] , hst , comunicazione personale )  . in conclusione , controverso se esistano delle differenze di nefrotossicit tra i mezzi di contrasto a bassa osmolarit dopo somministrazione intrarteriosa , mentre sta diventando chiaro che non vi alcun vantaggio associato alluso del dimero nonionico per procedure endovenose . prevenzione dellnsf diverse osservazioni recenti suggeriscono che la somministrazione di alcuni , ma non di tutti , i mezzi di contrasto a distribuzione extracellulare a base di gd possa essere causa dello sviluppo di nsf in pazienti con insufficienza renale grave ( < 30 ml / min / 1 , 73 m2 ) o in dialisi . 
thomsen : imaging patients with chronic kidney disease : cin or nsf ? table 1 prospective , randomised trials of various low - osmolar nonionic monomeric contrast media and the isoosmolar nonionic dimer with regard to contrastinduced nephropathy in relation to intravenous injection for computed tomography . 
no other randomised , prospective trials where the contrast medium is given intravenously have been published study locm ( monomers ) iodixanol ( dimer ) definition of cin carraro et al . 
 [ 10 ] total 0 / 32 ( iopromide ) 4 / 25 ( iobiditrol ) 0 / 76 ( iomeprol ) 0 / 77 ( iopamidol ) 4 / 210 ( 2% ) 1 / 32 4 / 25 5 / 72 2 / 76 12 / 209 ( 6% ) locm , low - osmolar contrast media ; cin , contrast - induced nephropathy ; scr , serum creatinine tabella 1 studi prospettici , randomizzati , di confronto tra diversi mezzi di contrasto monomerici nonionici a bassa osmolarit e il dimero nonionico iso - osmolare in termini di incidenza di cin dopo somministrazione per procedura di tc . 
 [ 10 ] totale 0 / 32 ( iopromide ) 4 / 25 ( iobiditrol ) 0 / 76 ( iomeprol ) 0 / 77 ( iopamidol ) 4 / 210 ( 2% ) 1 / 32 4 / 25 5 / 72 2 / 76 12 / 209 ( 6% ) locm , mezzi di contrasto a bassa osmolarit ; cin , nefropatia da contrasto ; scr , creatinina sierica prevention of nsf several recent reports suggest that administration of some , but not all , of the extracellular gd - based contrast agents may be causative for development of nsf in patients with severely reduced renal function ( < 30 ml / min per 1.73 m2 ) or on dialysis . 
the onset varies in general from a few days to 3 months after exposure . the disease is characterised by scleroderma - like skin changes that mainly affect the limbs and trunk . 
strikingly , the overwhelming majority of reported cases ( ~90% ) were observed following administration of the nonionic , linear contrast agent , gadodiamide . the mechanism by which gd - based contrast agents cause nsf is not completely understood . 
these differences in stability become clinically important in patients with reduced renal function , as in va da alcuni giorni ai tre mesi dopo lesposizione al mdc . la nsf caratterizzata da cambiamenti cutanei simili alla sclerodermia che possono interessare gli arti e il tronco . 
lispessimento cutaneo pu anche progredire fino a causare la contrattura con incapacit di estensione delle articolazioni e la fibrosi pu interessare anche altri organi quali muscoli , cuore , fegato e polmoni . 
sorprendentemente , la grande maggioranza dei casi di nsf riportati ( circa il 90% ) sono stati osservati dopo la somministrazione del mdc lineare nonionico gadodiamide . il meccanismo tramite il quale i mezzi di contrasto a base di gd causano lnsf non pienamente chiarito . 
in teoria , durante questa prolungata biodisponibilit , vi una probabilit maggiore che avvenga una transmetallazione con metalli endogeni il che darebbe luogo a un deposito di gd libero a livello cutaneo , cosa che in effetti stata riscontrata nei pazienti affetti da nsf . 
theoretically , during this extended period of bioavailability , transmetallation with endogenous ions has a greater opportunity to occur , resulting in free gd deposition in the skin , which has in fact been observed to occur in nsf patients . 
furthermore , this might explain the overwhelming majority of cases with gadodiamide , as several lines of evidence suggest that gadodiamide , which utilises a linear chelate , is a relatively unstable contrast agent . gadodiamide is now contraindicated in europe for use in patients with a gfr < 30 ml / min / 1.73 m2 , those on dialysis , or those who have had or are awaiting liver transplantation [ 12 ]  . 
thomsen : imaging patients with chronic kidney disease : cin or nsf ? anche spiegare la grande preponderanza di casi osservati dopo la somministrazione di gadodiamide , che essendo costituito da un chelato lineare , un mdc relativamente instabile . attualmente in europa la gadodiamide ha la controindicazione alluso in pazienti con un gfr < 30 ml / min / 1 , 73 m2 , in quelli in dialisi e in quelli che hanno avuto o sono in attesa di trapianto di fegato [ 12 ]  . 
non sono stati riscontrati casi di nsf dopo la somministrazione esclusiva di vari mezzi di contrasto : il gadoterato di dimeglumina , il gadoteridolo , il gadobenato di dimeglumina e il gadobutrolo nonostante luso esteso di questi prodotti in centri di imaging a cui vengono inviati pazienti da dipartimenti di nefrologia . conclusions based on available evidence , baseline renal insufficiency is a significant risk factor for cin in patients undergoing contrast - enhanced ct and for nsf in patients administered a gd - based contrast agent prior to mr imaging . 
for cin , although there have been many studies performed , it is not entirely clear whether there are differences in the incidence of cin among the low - osmolar iodinated cm , whereas for nsf , contrast agent selection clearly impacts the likelihood for the development of this serious adverse event . conclusioni in base alle evidenze disponibili , la preesistenza di una insufficienza renale un fattore di rischio significativo sia per la cin in pazienti che vengono sottoposti a tc con contrasto che per lnsf in pazienti cui viene somministrato un mdc a base di gd per una procedura di rm . 
cova1 1unit clinico operativa di radiologia , 2unit clinico operativa di clinica generale e terapia chirurgica , universit degli studi di trieste , ospedale di cattinara , strada di fiume 447 , i - 34149 trieste , italy 3unit clinico operativa di anatomia patologica istopatologia e citodiagnostica , universit degli studi di trieste , ospedale maggiore , via della piet 2 , i - 34100 trieste , italy correspondence to : a . 
we retrospectively reviewed the ct scans of 26 patients who underwent surgery for histologically confirmed gist of the stomach ( 20 cases ) , the duodenum ( 1 ) , the caecum ( 1 ) , the small bowel ( 2 ) , the descending colon ( 1 ) and the rectum ( 1 )  . 
sono stati valutati retrospettivamente gli esami tc di 26 pazienti sottoposti ad intervento chirurgico con diagnosi istologica di gist localizzato allo stomaco ( 20 casi ) , al duodeno ( 1 ) , al cieco ( 1 ) , allintestino tenue ( 2 ) , al colon discendente ( 1 ) ed al retto ( 1 )  . 
although they may affect any portion of the gastrointestinal tract , they are seen with different frequencies in the different portions : 60% of gists are located in the stomach and 30% in the small bowel [ 1 ]  . they rarely arise in other locations , such as the large bowel or duodenudiscovery of a gist at the oropharyngeal level is extremely rare [ 2 ]  . 
an uncommon association between crohns disease of the terminal ileum and a high risk of gist development in this anatomical district region has also been reported [ 5 ]  . immunohistochemically , gists are defined as tumours that express a particular cell - surface tyrosine kinase , the cd117 antigen , whose overexpression is directly related to tumour aggressiveness . 
symptoms are nonspecific and essentially depend on tumour size [ 7 ] : small gists are normally asymptomatic and discovered incidentally during diagnostic procedures or surgery performed for other purposes ; these amount to approximately one third of all gist diagnoses . 
the most common clinical presentation of gist is gastrointestinal bleeding related to mucosal ulceration [ 7 ] , which may be chronic with associated anaemia or acute requiring emergency treatment ( approximately 40% of cases of bleeding ) [ 8 ]  . 
occlusion is seen in small - bowel tumours only , and perforation is always rare . the diagnosis is often incidental and , to a small extent only , based on endoscopic procedures , given the predominantly extramucosal growth pattern of gists . 
the aim of this study was to identify the ct patterns of gist and assess the techniques efficacy in identifying those lesions at a higher risk for malignancy , with a view to highlighting those patterns that may prove useful in treatment planning . materials and methods we retrospectively reviewed the ct studies performed between the year 2000 and 2005 in 26 patients ( 12 men , 14 women , mean age 67.6 years ) who underwent surgery for histologically proven gist located in the stomach ( 20 cases ) , the duodenum ( 1 ) , the small bowel ( 2 ) , the caecum ( 1 ) , the descending colon ( 1 ) and the rectum ( 1 )  . 
la loro massima incidenza si osserva fra la quinta e la sesta decade ; pur interessando tutto il tratto gastrointestinale si presentano con differente frequenza nei diversi tratti : nel 60% dei casi sono localizzati a livello dello stomaco , nel 30% al piccolo intestino [ 1 ] , raramente in altri distretti quali colon o duodeno . 
 stata descritta una rara associazione fra il morbo di crohn a carico del tratto terminale dellileo ed un elevato rischio di insorgenza di gist in tale distretto anatomico [ 5 ]  . dal punto di vista immuno - istochimico vengono classificati come gist le forme che esprimono sulla superficie cellulare una particolare tirosin - chinasi : lantigene cd117 , la cui sovra - espressione direttamente correlabile con la malignit della patologia . 
lespressione di un altro marcatore immunologico , il cd34 [ 6 ] appare essere correlato con una maggior propensione della neoplasia alla diffusione secondaria , in quanto codificante per una particolare molecola di adesione . 
la sintomatologia aspecifica e dipende essenzialmente dalla dimensione della neoplasia [ 7 ] : i gist di piccole dimensioni sono solitamente asintomatici e sono diagnosticati generalmente in maniera occasionale durante indagini diagnostiche od interventi chirurgici non correlati con tale patologia ; tali reperti ammontano a circa un terzo del totale . 
la manifestazione clinica pi comune del gist il sanguinamento causato dallerosione della mucosa [ 7 ] , che pu essere cronico con associata anemia oppure acuto e richiedere un trattamento demergenza ( in circa il 40% dei casi di sanguinamento ) [ 8 ]  . 
solamente nel caso di lesioni localizzate al piccolo intestino locclusione frequente ; in tutti i casi rara la perforazione . la diagnosi , spesso occasionale , si basa solo limitatamente su procedure endoscopiche , dato lo sviluppo prevalentemente extramucoso dei gist ; a queste si aggiungono le indagini contrastografiche tradizionali , lecografia e la tomografia computerizzata ( tc ) [ 9 ]  . 
lo scopo di questo lavoro quello di riportare gli aspetti in tc dei gist e di valutare lefficacia dellindagine nellidentificazione delle lesioni a pi elevato rischio di malignit con lobiettivo di evidenziare alcuni aspetti che possano essere utili nella pianificazione della terapia . materiali e metodi sono stati valutati retrospettivamente gli esami tc eseguiti dal 2000 al 2005 in 26 pazienti ( 12 maschi , 14 femmine , et media 67 , 6 anni ) sottoposti ad intervento chirurgico con diaa . 
images were acquired with a 5 - mm slice thickness , 5 - mm reconstruction interval and 7 - mm table feed ( pitch 1.4 ) after oral administration of gastrografin ( nine patients ) or water ( 17 patients ) , in the case of suspected gastric location , with scans obtained at baseline and 70 s and 180 s ( portal and late phase ) after the intravenous injection of 120140 cc of iodinated contrast material ( iomeprolo 300 mgi / ml , iomeron 300 , bracco , milan , italy )  . 
ct images were processed on a dedicated workstation ( easyvision 4.3 , philips medical systems , the netherlands ) where multiplanar ( mpr ) and three - dimensional 3d reconstructions were generated by using the virtual colonoscopy software package . radiographic contrast studies were obtained by using a remote - controlled x - ray system ( omnidiagnost eleva di 1.0 , philips medical systems , the netherlands )  . 
endoscopic us was performed with a hitachi eub 525 fm scanner ( hitachi medica systems europe , wiesbaden , germany ) equipped with a 7.5 - mhz radial probe for endoscopic us . 
administration of contrast medium presence of calcification presence of ulcerations signs of invasion of neighbouring structures ( lack of clear cleavage plane between the mass and the surrounding structures ) distant metastasis lesions were classified in accordance with the literature as benign , borderline or malignant on the basis of their size and mitotic activity : lesions smaller than 5 cm with a mitotic index less than 5 / 50 high - power fields are considered benign . 
 pathological diagnosis of gist was obtained by means of immunohistological staining of tissue sections and detection of antibodies against the cd117 antigen . results ct characteristics of the lesions and their histological correlations are summarised in table 1 . 
overall , histological examination revealed ten benign lesions ( < 5 cm ) , six borderline lesions ( based on size > 5 cm ) and ten malignant lesions that were infiltrating and very large ( > 9 cm )  . 
only one case showed microcalcification gnosi istologica di gist localizzato allo stomaco ( 20 casi ) , al duodeno ( 1 ) , allintestino tenue ( 2 ) , al cieco ( 1 ) , al colon discendente ( 1 ) ed al retto ( 1 )  . 
gli esami tc sono stati eseguiti utilizzando unapparecchiatura tc spirale a singolo strato tomoscan ave 1 ( philips medical systems , olanda )  . le immagini , sono state acquisite con spessore di scansione di a 5 mm , intervallo di ricostruzione di 5 mm ed avanzamento del tavolo di 7 mm ( pitch 1 , 4 ) , dopo somministrazione di gastrografin ( 9 pazienti ) o acqua per os ( 17 pazienti ) , in caso di sospetto clinico di localizzazione gastrica del tumore , con scansioni dirette e durante liniezione endovena di 120140 cc di mdc organoiodato ( iomeprolo 300 mgi / ml , iomeron 300 , bracco , milano , italia ) a 70 s e 180 s ( fase portale e tardiva ) dallinizio della iniezione del mdc . 
le immagini tc sono state rielaborate mediante una workstation dedicata ( easyvision 4.3 , philips medical systems , olanda ) con la quale si sono effettuate ricostruzioni multiplanari ( mpr ) e ricostruzioni 3d mediante il pacchetto software di colonscopia virtuale . le indagini contrastografiche tradizionali sono state eseguite con un apparecchio telecomandato omnidiagnost eleva di 1.0 ( philips medical systems , olanda )  . 
le indagini ecoendoscopiche sono state effettuate mediante un unit ecografica hitachi eub 525 fm ( hitachi medica systems europe , wiesbaden , germania ) equipaggiata con sonda per ecoendoscopia radiale da 7 , 5 mhz . 
per evidenziare gli aspetti tc delle lesioni utili nella pianificazione terapeutica sono stati considerati i seguenti parametri : sede ; dimensioni ; caratteristiche densitometriche prima e dopo la somministrazione di mdc ev ; presenza di calcificazioni ; presenza di ulcerazioni ; segni di infiltrazione delle strutture anatomiche circostanti ( mancanza di un piano di clivaggio evidente in tc fra la massa e le strutture vicine ) ; metastasi a distanza . in accordo con la letteratura le lesioni sono state classificate come benigne , borderline o maligne sulla base delle loro dimensioni e dellindice mitotico : lesioni al di sotto dei 5 cm con un indice mitotico minore di 5 / 50 campi ad alto ingrandimento sono da considerarsi benigne , sono invece da ritenere borderline lesioni con lo stesso indice mitotico ma con dimensioni maggiori di 5 cm , le lesioni con indice mitotico maggiore di 5 / 50 campi ad alto ingrandimento sono da ritenersi maligne qualunque sia la loro dimensione [ 9 ]  . la diagnosi anatomopatologica di gist stata ottenuta mediante tipizzazione immunistologica di preparati della lesione con la dimostrazione di positivit per gli anticorpi rivolti contro lantigene cd117 . risultati le caratteristiche delle lesioni osservate in tc ed i loro correlati istologici sono state riassunte nella tabella 1 . 
4a - d scansioni tc assiali dirette ( a ) e durante iniezione di mdc , fase precoce ( b ) e tardiva ( c ) : processo espansivo solido omogeneo con scarso e tardivo enhancement ( frecce )  . 
d reperto istologico : cellule fusate stipate disposte in fasci variamente orientati . lesioni benigne ( < 5 cm ) , 6 borderline ( in relazione alle dimensioni > 5 cm ) e 10 maligne a carattere infiltrante e comunque , di notevoli dimensioni ( > 9 cm )  . 
laspetto tc pi frequente dei gist dato dalla presenza di una lesione esofitica , spesso di cospicue dimensioni ( oltre i 10 cm ) , a contorni lobulati [ 10 ]  . 
anche nella nostra esperienza ci siamo trovati di fronte a lesioni di dimensioni notevoli ( in 16 casi superiori a 5 cm ed in 10 di questi addirittura superiori ai 9 cm ) ; questo perch queste neoplasie raramente sono stenosanti e tendono ad avere una crescita eccentrica , senza quindi manifestare una sintomatologia di tipo ostruttivo se non tardivamente . per tale motivo a volte vengono diagnosticate in maniera occasionale [ 11 ]  . per quanto riguarda la densit , essa risulta essere spesso disomogenea in relazione alla presenza di aree emorragiche e / o necrotiche , specie nelle lesioni di maggiori dimensioni ( segno di torricelli - bernoulli ) , nonch di spazi cistici [ 12 ]  . nella nostra esperienza in 16 casi su 26 le lesioni presentavano densit disomogenea alle scansioni dirette ed in maniera pi evidente dopo mdc , in relazione alla presenza di componenti necrotiche lenhancement delle aree vascolarizzate risultava globalmente scarso , quantomeno in fase tardiva . 
si trattava peraltro prevalentemente di tumori di dimensioni medio - grandi , mentre nei pochi casi di lesioni piccole ed in una minima percentuale di quelle grandi si evidenziata in tc una struttura omogenea associata ad un enhancement pi intenso . 
in linea di massima si trattava di lesioni successivamente classificate come benigne o borderline allesame istologico , prive di significative aree necroticoemorragiche . la presenza di calcificazioni un reperto incostante ed aspecifico , apprezabile pi di frequente a livello delle metastasi epatiche [ 13 , 14 ]  . 
non sempre la tc in grado di evidenziarle , specie se si utilizza unapparecchiatura monostrato , come nel nostro caso , in cui alcune minute calcificazioni evidenziabili al tavolo operatorio non sono state evidenziate in tc . lulcerazione della lesione rappresenta un indice di malignit ; non sempre per possibile evidenziare tale reperto in tc , specie se molto superficiale , come nella nostra cafig . 
6a - c enhanced computed tomography ( ct ) : expansile mass originating from the right colon and infiltrating the kidney and fat tissue ( a , b )  . 
6a - c scansioni tc assiali dopo somministrazione di mdc : processo espansivo solido a partenza dal colon destro , infiltrazione del rene e del tessuto adiposo ( a , b )  . 
this is because these tumours rarely cause luminal narrowing and tend to have an eccentric growth pattern , so that symptoms secondary to occlusion only arise in the advanced stages of disease . 
 the ct density of gists is often inhomogeneous owing to the presence of haemorrhagic and / or necrotic areas , especially in larger lesions ( torricelli - bernoulli sign ) , and of cystic spaces [ 12 ]  . 
in our study , 16 out of 26 lesions exhibited inhomogeneous density on the unenhanced baseline scans and more marked inhomogeneity on the contrast - enhanced scans as a result of the presence of necrotic components that made enhancement of the vascular areas generally poor , at least in the late phase . 
these were predominantly medium to large size tumours , whereas the few cases of small lesions and a very small proportion of large lesions exhibited homogenous density associated with brighter enhancement . 
most of these lesions were histologically classified as benign or borderline and without significant areas of necrosis or haemorrhage . the presence of calcification is an inconstant and nonspesistica in cui tale reperto stato evidenziato in soli 2 casi su 6 [ 15 , 16 ]  . linfiltrazione della parete del viscere pu dar luogo raramente a focali dilatazioni del lume , causate dalla veloce crescita della neoplasia o anche dallinfiltrazione del plesso mesenterico con conseguente ectasia da atonia causata da denervazione [ 12 ]  . 
a volte per tali segni sono riconoscibili solamente allintervento chirurgico ( nella nostra casistica in 4 casi su 10 di forme infiltranti la tc non ha rilevato segni di infiltrazione locoregionale )  . 
 verosimile che limpiego di apparecchiature multistrato consenta di evidenziare con maggior frequenza e precisione questo segno , molto importante ai fini prognostici e terapeutici . in uno solo dei 26 casi valutati sono state riscontrate metastasi epatiche , ipodense alla scansione diretta e con discreto enhancement dopo liniezione di mdc . 
ct is not always able to demonstrate calcifications , above all if a single - slice scanner is used , as was the case in our study in which some tiny calcifications seen at surgery were missed by ct . 
however , not always is it possible to demonstrate this finding on ct , especially if the ulceration is very superficial , as occurred in our series where ct identified this finding in only two cases out of six [ 15 , 16 ]  . 
 invasion of the bowel wall may occasionally give rise to focal luminal dilatation related to rapid tumour growth or infiltration of the mesenteric plexus , with ectasia caused by loss of tone due to denervation [ 12 ]  . 
in some cases , however , these signs are only recognised at surgery ( in our series , ct failed to detect signs of locoregional invasion in four out of ten cases )  . 
it is likely that the use of multislice ct scanners will allow more frequent and better depiction of this sign , which is very important for prognostic and therapeutic purposes . in only one case out of 26 was liver metastases detected . these were hypodense on the baseline scan and showed moderate enhancement after the injection of contrast material . 
liver metastases are usually small and characterised by low density on baseline scans and intense vascularity and homogeneous enhancement in the portal phase showing complete washout in the late phase , with resultant hypodensity relative to the hepatic parenchyma . 
the presence of ascites is also quite rare and more often related to the outcome of chemotherapy , in particular to the liver failure caused by the treatment [ 17 , 18 ]  . the definition of benign or malignant gist is complex in that these tumours present a wide variability of malignant potential , which is established on the basis of the pathological and immunohistochemical findings . 
tumours larger than 5 cm are generally considered to have the highest malignant potential . other ct findings that point to a diagnosis of malignancy are inhomogeneous enhancement with central low density due to tissue necrosis ( in contrast to benign lesions that tend to enhance homogeneously ) , the presence of ulcerations or signs of locoregional infiltration and / or distant metastasis . 
it should , however , be noted that gists with a mitotic index greater than five mitoses per 50 high - power fields should be classified as malignant regardless of their size at imaging [ 19 ]  . surgery is the treatment of choice , and radical resection of the gist is considered to be the most important prognostic factor for cure in nonmetastatic disease . 
lymphadenectodentensit delle stesse alla scansione diretta , da intensa vascolarizzazione ed omogeneo enhancement in fase portale con completo wash - out in fase tardiva risultando cos nuovamente ipodense rispetto al parenchima . 
esse divengono invece ipovascolarizzate in tutte le fasi dopo trattamento chemioterapico . le metastasi a livello mesenterico sono comuni in corso di recidiva della patologia od in seguito a disseminazione della massa neoplastica escissa ; esse sono di difficile riconoscimento in quanto spesso di piccole dimensioni ed a volte si collocano a distanza dalla lesione primitiva . 
la presenza di metastasi linfonodali non caratteristica comune della lesione , pertanto la dimostrazione di linfoadenopatie satelliti deve far prendere in considerazione la presenza di una lesione di differente natura . 
la presenza di ascite anchessa piuttosto rara ed pi spesso riconducibile agli esiti dei trattamenti chemioterapici in relazione alla insufficienza epatica indotta dal trattamento [ 17 , 18 ]  . la definizione di benignit o malignit dei gist complessa in quanto questi tumori presentano una grande variabilit del grado di malignit , che viene stabilito sulla base dellaspetto anatomopatologico ed immunoistochimico . 
generalmente i tumori di dimensioni superiori ai 5 cm sono quelli a pi alto potenziale di malignit . le altre caratteristiche tc che orientano verso una diagnosi di malignit sono rappresentate dallenhancement disomogeneo con ipodensit centrale data dalla necrosi tissutale ove invece le forme benigne prediligono enhancement omogeneo e dalla presenza di ulcerazioni o di segni di infiltrazione locoregionale e\o metastasi a distanza . 
va in ogni caso osservato come i gist che presentino dal punto di vista istologico un indice mitotico maggiore di 5 mitosi su 50 campi ad alto ingrandimento ( hpf ) siano da classificare come maligni indipendentemente dalle dimensioni della neoplasia allimaging [ 19 ]  . la terapia chirurgica del gist rappresenta il trattamento elettivo e la resezione totale del tumore considerato il fattore prognostico maggiore nella guarigione dei casi non metastatici . 
lobiettivo della terapia chirurgica lasportazione della massa in toto compresa la sua capsula , e con margine di sezione istologicamente libero da neoplasia , mentre la linfoadenectomia non indicata cos come la peritonectomia , data la bassa metastatizzazione a questo livello ; tuttavia la resezione chirurgica viene spesso condizionata dalla localizzazione e dalla grandezza della massa stessa [ 20 , 21 ]  . la tecnica laparoscopica nel trattamento chirurgico dei gist gi stata utilizzata , tuttavia viene riportata in pochi studi , mentre si dimostrata vantaggiosa nel trattamento delle lesioni sanguinanti dello stomaco , consentendo , infatti , una minima manipolazione della massa . mentre per le lesioni di piccole dimensioni o con scarsa aggressivit locale il trattamento di elezione stato ed solo chirurgico , invece fino a poco tempo fa il trattamento adiuvante nel caso di tumori di maggiori dimensioni e di metastasi a distanza era effettuato mediante la chemioterapia e la radioterapia . 
 whereas for small lesions or lesions with limited local aggressiveness the treatment of choice has always been and still is surgery , until recently , patients with larger lesions and those with distant metastases received adjuvant chemotherapy or radiotherapy . 
nowadays , instead , the most innovative approach relies on the use of imatinib , a molecule that acts on a tyrosine - kinase transmembrane receptor to promote apoptosis and inhibit cell proliferation in gist . 
the results achieved to date in the treatment of gist with imatinib have been encouraging in terms of both tumour metastases and shrinkage of the tumour mass [ 22 ]  . no specific indications exist for the use of radiotherapy as either primary or adjuvant treatment . 
in principle , radiotherapy can be used to sterilise possible microscopic disease foci in all those cases at a significant risk for postoperative recurrence and in cases of macroscopic residual tumour or local relapse . 
however , larger studies are needed to assess the real impact of radiotherapy on local disease control and patient survival [ 23 ]  . as for the use of chemotherapy , the oncological literature from the preimatinib days reports cumulative data regarding leiomyosarcomas as a single histological entity without distinguishing by site of occurrence . 
the data on chemotherapy in gist are therefore extrapolated from papers reporting the results of chemotherapy in soft - tissue sarcomas of the adult . the literature shows that antineoplastic chemotherapy does not play a prominent role in the treatment of gastrointestinal stromal tumours . 
clinical response was 10% and 5% in first - line and second - line treatment , respectively , that is , considerably lower than commonly observed in other softtissue sarcomas where response is 40% and approximately 30% in leiomyosarcomas . 
today , following the demonstration of the efficacy of imatinib , chemotherapy plays no more than a marginal role in the treatment of gists . in view of these issues related to treatment options , the role of ct needs to be emphasised . 
our review of 26 cases of gist examined with ct has demonstrated that the large size of these tumours and signs of locoregional infiltration are suggestive of lesions with a high malignancy potential , whereas the presence of distant metastasis is a clear indicator of malignancy , as in other neoplastic lesions . 
i risultati fino ad ora ottenuti nel trattamento di questo tipo di patologia mediante limatinib sono stati promettenti sia nei confronti dei tumori metastatici sia nella riduzione dimensionale delle masse [ 22 ]  . non esistono a tuttoggi indicazioni specifiche circa lutilizzo della radioterapia n come trattamento primario n come terapia adiuvante . 
in linea di principio la radioterapia pu essere utilizzata in tutti quei casi che presentano un rischio significativo di recidiva post - chirurgica allo scopo di sterilizzare eventuali foci microscopici di malattia , e nei casi di residuo macroscopico o di recidiva locale . 
tuttavia , sono necessari studi pi appropriati al fine di valutare il reale impatto della radioterapia sul controllo locale della malattia e sulla sopravvivenza [ 23 ]  . per quanto riguarda limpiego della chemioterapia , la letteratura oncologica nellera pre - imatinib riporta dati cumulativi riguardanti i leiomiosarcomi come entit istologica senza distinguerli per sede dinsorgenza , n esistono pubblicazioni specifiche sui gist , se si esclude un recente studio retrospettivo dellitalian sarcoma group [ 24 ]  . 
i dati sul trattamento chemioterapico dei gist sono pertanto estrapolati da pubblicazioni riportanti i risultati relativi al trattamento chemioterapico dei sarcomi delle parti molli delladulto . dalla letteratura emerge come la chemioterapia antiblastica non abbia avuto un ruolo determinante nel trattamento dei tumori stromali gastrointestinali . 
le risposte cliniche sono state del 10% e del 5% rispettivamente in prima linea e seconda linea di trattamento , percentuali diverse da quelle comunemente osservate negli altri sarcomi dei tessuti molli in cui il tasso di risposte del 40% e nel sottogruppo dei leiomiosarcomi , del 30% circa . 
oggi , in seguito alla dimostrata efficacia dellimatinib nella terapia dei gist , la chemioterapia riveste un ruolo del tutto marginale nel trattamento di questi tumori . sulla base di queste problematiche legate alle possibilit terapeutiche va sottolineato il ruolo della tc . 
la revisione dei 26 casi di gist valutati in tc ha evidenziato come le notevoli dimensioni ed i segni di infiltrazione dei tessuti locoregionali siano indicativi di lesioni con elevato potenziale di malignit , mentre la presenza di metastasi a distanza rappresenta un chiaro indicatore di malignit , come per altro in quasi tutte le forme neoplastiche . 
nei casi in cui vengano evidenziati tumori di grandi dimensioni e / o segni di infiltrazione possibile impostare una opportuna terapia adiuvante prima dellintervento , consentendo cos una maggior radicalit chirurgica al fine di migliorare la prognosi del paziente . 
levidenza alla tc di lesioni secondarie gi al momento della diagnosi consente invece di indirizzare il paziente verso la terapia medica basata sullutilizzo dellimatinib . le altre caratteristiche morfologiche e di enhancement a . 
it is therefore indispensable in clinical staging and treatment planning , as well as being useful in the follow - up of patients treated with either surgery or pharmacological therapy ( imatinib )  . conclusioni in conclusione , la tc in virt della sua elevata panoramicit e risoluzione di contrasto fornisce informazioni determinanti nel riconoscimento e nella caratterizzazione dei reperti radiologici caratteristici dei gist . 
paganelli3 1facolt di medicina e chirurgia , universit degli studi di milano , via festa del perdono 7 , i - 20122 milano , italy 2divisione di radiologia , 3divisione di medicina nucleare , 4divisione di oncologia medica , 5divisione di epidemiologia e biostatistica , istituto europeo di oncologia irccs , via ripamonti , 435 , i - 20141 , milano , italy correspondence to : m . 
tcmd e pet / tc hanno mostrato alta sensibilit e specificit nella diagnosi di recidiva locale di tumore del retto . entrambe le metodiche sono state ugualmente accurate nella diagnosi di metastasi epatiche . 
la tcmd ha mostrato maggiore sensibilit e valore predittivo positivo nella diagnosi di metastasi polmonari . key words pet / ct computed tomography rectal cancer recurrence fluorodeoxyglucose parole chiave pet / tc tomografia computerizzata carcinoma del retto recidiva fluorodesossiglucosio m . 
the percentage of patients who develop local or distant recurrence following curative surgical resection is relatively high , probably due to the presence of neoplastic foci that remain undetected at the time of surgery [ 1 ]  . 
more than one third of patients with colon or rectal cancer develop hepatic metastases [ 2 ] and / or pulmonary metastases [ 3 ] , and this typically occurs within 2 years after surgery [ 4 ]  . 
the early diagnosis of local or distant recurrence is important in that further surgery , radiotherapy and chemotherapy , separately or combined in an integrated multidisciplinary approach , may improve survival and quality of life . 
unfortunately , no standardized follow - up strategy has been yet developed for patients with rectal cancer treated with curative resection [ 5 ]  . to our knowledge no previous study has compared the role of multidetector computed tomography ( mdct ) and that of f18 - fluorodeoxyglucose positron emission tomography - computed tomography ( fdg - pet / ct ) in the follow - up of these patients . 
the aim of this retrospective study was to compare the diagnostic value of mdct and fdg - pet / ct in the diagnosis of local recurrence and distant metastases after radical surgery for rectal cancer . materials and methods patients our institutional guidelines for the follow - up of patients after rectal cancer resection recommend clinical assessment every 46 months [ including serum carcinoembryonic antigen ( cea ) testing ] , abdominal and pelvic mdct every 6 months , chest radiography every year and colonoscopy within 1 year of surgery and every 23 years thereafter . 
this retrospective study included all patients treated with radical surgery for rectal cancer between 2000 and 2004 and who underwent fdg - pet / ct for a suspicion of local or distant recurrence aroused by clinical follow - up or mdct findings in accordance with our guidelines . 
fdg - pet / ct and mdct were performed within 30 days ( mean 22 days ) of one another . the patients had undergone radiotherapy or chemotherapy depending on initial disease stage in accordance with the following indications : patients without sigmoid involvement , with an initial stage greater than t2 , or with n + and any t stage underwent radiotherapy before or after surgery , in combination with various chemotherapy schedules . 
la percentuale di pazienti che sviluppano recidiva locale o a distanza dopo la resezione chirurgica considerata curativa relativamente elevata , probabilmente a causa della presenza di focolai neoplastici non evidenziati al momento dellintervento chirurgico [ 1 ]  . 
pi di un terzo dei pazienti con tumore del colon o del retto sviluppa metastasi epatiche [ 2 ] , e / o metastasi polmonari [ 3 ] , e questo si verifica di solito entro 2 anni dallintervento chirurgico [ 4 ]  . 
la diagnosi precoce di recidiva locale o a distanza importante perch un ulteriore approccio chirurgico , radioterapico e chemioterapico , ciascuno singolarmente o associati in un approccio multidisciplinare integrato , possono migliorare la sopravvivenza e la qualit di vita . 
purtroppo , non esiste ancora una strategia di follow - up standardizzata per i pazienti con tumore del retto sottoposti a intervento chirurgico curativo [ 5 ]  . per quanto a nostra conoscenza dalla letteratura , nessuno studio ha finora confrontato il ruolo della tc multistrato ( tcmd ) e della pet / tc come indagini di follow - up in questi pazienti . 
lo scopo di questo studio retrospettivo stato quindi quello di confrontare la validit diagnostica della tcmd e della pet / tc nella diagnosi di recidiva locale e di metastasi a distanza in pazienti sottoposti a chirurgia radicale di tumore del retto . materiali e metodi pazienti le linee - guida attualmente in uso presso il nostro istituto per il follow - up di pazienti con tumore del retto operato , prevedono una valutazione clinica ogni 46 mesi ( che include dosaggio del cea ematico ) , una tcmd addominopelvica ogni 6 mesi , una radiografia del torace annuale , una colonscopia entro un anno dalla chirurgia e poi ogni 23 anni . 
sono stati inclusi in questo studio retrospettivo tutti i pazienti sottoposti a intervento di chirurgia radicale per tumore del retto tra il 2000 e il 2004 , che sono stati sottoposti ad una pet / tc per il dubbio di recidiva locale o a distanza sollevato da dati clinici o dalla tcmd previsti nel follow - up , secondo quanto indicato dalle nostre linee guida . 
i pazienti inclusi nello studio erano stati sottoposti a radioterapia o chemioterapia in base al loro stadio iniziale secondo le seguenti indicazioni : in assenza di coinvolgimento del sigma , con uno stadio iniziale superiore a t2 o qualunque t con n + , i pazienti sono stam . 
the scans were obtained during a single breath - hold with the following parameters : contiguous acquisition of 2.5 - mm - thick slices , field of view 320280 mm , 120 kv , 240300 ma , 0.8 - s rotation , matrix 512512 and standard reconstruction algorithm . fdg - pet / ct before administration of fdg , all patients fasted for 6 h and underwent blood glucose tests . 
images were acquired with an integrated pet / ct device ( discovery ls , ge medical systems ) composed of an advance nxi pet scanner and an 8 - slice light speed plus ct scanner . 
the axes of the two systems were mechanically aligned to enable transfer of the patient , lying supine , from the ct gantry to the pet scanner with a 60 - cm table shithe pet / ct data were coregistered on a computer . 
ct acquisition parameters were 120 kv , 80 ma , tube rotation time 0.8 s , pitch 1 , 5 and slice thickness 5 mm and were matched with pet image thickness . 
pet data were interactively reconstructed with segmented attenuation correction with the use of ct images [ 6 ]  . the segmented images were displayed as means by the xeleris software ( ge medical systems )  . 
in patients with excessive residual urine , a pelvic acquisition was obtained after positioning a 3 - lumen foley catheter . image interpretation the original reports were considered as the result of the examination . 
any new tissue arising at , or close to , the site of the ti sottoposti a radioterapia prima o dopo la chirurgia , in associazione a chemioterapia secondo vari schemi . 
tutti i pazienti inclusi in questo studio avevano una tc torace - addome - pelvi eseguita per stadiazione prima della chirurgia . nessun paziente affetto da diabete stato incluso in questo studio retrospettivo . diagnostica per immagini tcmd gli esami tc sono stati eseguiti con tc 16 detettori ( lightspeed scanner , general electric medical systems , milwaukee , wi , usa ) , dalle basi polmonari alla sinfisi pubica . 
le scansioni sono state acquisite 70 secondi ( addome superiore ) e 150 secondi ( pelvi ) dopo linizio delliniezione ev di 2 , 0 ml / kg di mdc iodato 350 mg / ml , somministrato con un flusso di 2 ml / s . 
prima dellesame i pazienti hanno bevuto 23 bicchieri dacqua e svuotato la vescica per evitare artefatti causati dalle urine . le immagini sono state acquisite con uno scanner pet / tc integrato ( discovery ls , ge medical systems , milwaukee , wi , usa ) che consiste in un advance nxi pet scanner e un 8 - slice light speed plus ct scanner . 
gli assi dei due sistemi sono stati allineati meccanicamente per muovere il paziente , in posizione supina , dal gantry della tc alla pet , con uno shift del tavolo di 60 clesame pet / tc stato co - registrato su supporto hardware . 
i parametri di acquisizione dellesame tc sono : 120 kv , 80 ma , tempo di rotazione del tubo 0 , 8 s , pitch 1 , 5 , e spessore di strato 5 mm , e sono stati accoppiati allo spessore delle immagini pet . 
subito dopo lacquisizione delle immagini tc , sono state acquisite le immagini pet per coprire lo stesso campo di vista assiale . lacquisizione , in direzione caudo - craniale , stata fatta in 2d ( 4 minuti / posizione del lettino ) , circa 60 minuti dopo liniezione ev di 5 mbq / kg di fdg . 
no quantitative study of standardized uptake value ( suv ) was performed on fdg - pet / ct data because no cut - off value has been established that is definitively indicative of the presence of neoplastic tissue in patients with rectal cancer . 
therefore , a qualitative image analysis was used , which is equivalent to quantitative analysis [ 7 ]  . mdct and fdg - pet / ct results were then independently compared with histology of biopsy or surgical specimens or with the outcome of at least 2 years follow - up . 
sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) for the diagnosis of local or distant recurrence were determined for mdct and fdg - pet / ct . confidence intervals for sensitivity for local recurrence were calculated for each method and then compared . results this study considered 67 patients treated with surgery for rectal carcinoma : 25 had undergone radiotherapy ( 20 before and 5 after surgery ) , 23 had undergone chemotherapy ( 5 before and 18 after surgery ) and 19 had undergone both radiotherapy and chemotherapy after surgery . 
of these , 15 ( 22% ) had local recurrence , 17 ( 25% ) had hepatic metastases ( 7 of whom had local recurrence as well ) and 8 ( 12% ) had pulmonary metastases ( 2 of whom had local recurrence as well )  . local recurrence local recurrence occurred in 15 / 67 patients ( 22% )  . 
initial disease stage of patients with local recurrence was one t2n1 , three t3n0 , five t3n1 , one t3n2 , one t4n0 and one unknown stage because the patient came to our observation after undergoing surgery at another hospital . 
on the basis of initial disease stage , 6 / 15 patients underwent adjuvant chemotherapy , 5 / 15 underwent chemotherapy before and after surgery and 4 / 15 underwent radiotherapy and chemotherapy . the presence of recurrence was suspected on the basis of the clinical data in three patients , increased cea levels in two patients and radiological or endoscopic follow - up in ten patients . 
the first technique to produce a diagnosis of local recurrence was mdct in 6 / 15 patients , fdg - pet / ct in 4 / 15 and colonoscopy in 5 / 15 . both mdct and fdg - pet / ct yielded true negative diagnoses of local recurrence in 51 patients . 
mdct yielded one false positive restata eseguita una acquisizione della regione pelvica dopo posizionamento di catetere di foley a triplo lume . interpretazione delle immagini il referto originale stato considerato come risultato dellesame . 
qualunque nuovo tessuto in corrispondenza della anastomosi chirurgica , o in area ad essa adiacente in ambito pelvico , qualora presentasse caratteristiche di asimmetria , irregolarit , disomogeneo enhancement contrastografico o modificazione rispetto a esami precedenti stato considerato come recidiva locale . 
sui dati della fdg - pet / ct non stato effettuato nessuno studio quantitativo del valore standard di uptake ( standardized uptake value : suv ) perch in letteratura non vi dimostrazione di un valore di cut - off che sia con certezza indicativo della presenza di tessuto neoplastico in pazienti con tumore del retto . pertanto stata usata unanalisi qualitativa delle immagini , che sarebbe paragonabile ad una analisi quantitativa [ 7 ]  . i risultati di tcmd e pet / tc sono stati confrontati in modo indipendente con listologia ( ottenuta con biopsia o con chirurgia ) , o con i risultati di un follow - up minimo di due anni . 
sensibilit , specificit , valore predittivo positivo ( vpp ) e valore predittivo negativo ( vpn ) nella diagnosi di recidiva locale o a distanza sono stati calcolati per tc e pet / tc . 
gli intervalli di confidenza nella sensibilit diagnostica di recidiva locale sono stati calcolati per entrambe le metodiche e confrontati . risultati in questo studio sono stati inclusi 67 pazienti operati per carcinoma del retto : 25 sono stati sottoposti a radioterapia ( 20 prima e 5 dopo la chirurgia ) , 23 a chemioterapia ( 5 prima e 18 dopo la chirurgia ) , e 19 sia a radioterapia che a chemioterapia dopo la chirurgia . 
di questi : 15 ( 22% ) hanno sviluppato recidiva locale , 17 ( 25% ) hanno sviluppato metastasi epatiche ( 7 dei quali avevano anche recidiva locale ) , 8 ( 12% ) hanno sviluppato metastasi polmonari ( 2 dei quali avevano anche recidiva locale )  . recidiva locale la recidiva locale si verificata in 15 / 67 pazienti ( 22% )  . 
la stadiazione iniziale dei pazienti con recidiva locale era : un t2n1 , tre t3n0 , cinque t3n1 , un t3n2 , un t4n0 , uno non noto perch il paziente giunto alla nostra osservazione dopo lintervento eseguito in un altro ospedale . 
sensitivity , specificity , ppv and npv in the diagnosis of local recurrence were 100% , 98% , 93% and 100% for mdct , and 93% , 98% , 93% and 98% for fdgpet / ct , respectively . 
the difference in sensitivity between the two techniques was not statistically significant , as demonstrated by the overlapping confidence intervals ( 0.781.0 for mdct and 0.681.0 for fdg - pet / ct )  . hepatic metastases during the follow - up period , 17 / 67 patients ( 25% ) developed hepatic metastases . 
initial disease stage in patients with chemioterapia adiuvante , 5 / 15 a chemioterapia prima e dopo chirurgia , 4 / 15 a radio e chemioterapia . la presenza di recidiva stata suggerita in 3 pazienti da dati clinici , in 2 pazienti da incremento del marcatore tumorale ( cea ) , in 10 pazienti da esami di follow - up radiologico o endoscopico . 
la mediana del tempo intercorso tra chirurgia e recidiva locale stata di 22 mesi ( range 1248 mesi )  . la prima modalit che ha posto diagnosi di recidiva locale stata la tcmd in 6 / 15 pazienti , la pet / tc in 4 / 15 e la colonscopia in 5 / 15 . sia tcmd che pet / tc hanno fornito risultati veramente negativi per recidiva locale in 51 pazienti . 
the wall thickening limited to the left portion of the rectal wall , close to the suture line , was reported to be a possible local recurrence ( a )  . 
sensibilit , specificit , vpp e vpn nella diagnosi di recidiva locale sono risultati rispettivamente 100% , 98% , 93% e 100% per la tcmd , e 93% , 98% , 93% e 98% per la pet / tc . 
la differenza di sensibilit tra le due metodiche non stata statisticamente significativa , come mostrato dalla sovrapposizione degli intervalli di confidenza ( 0 , 781 , 0 per la tcmd , e 0 , 681 , 0 per la pet / tc )  . metastasi epatiche durante il periodo di follow - up 17 / 67 pazienti ( 25% ) hanno sviluppato metastasi epatiche . 
la stadiazione iniziale nei pazienti con metastasi epatiche era : t2n0 in 4 pazienti , t2n1 in 3 pazienti , t3n0 in 4 pazienti , t3n1 in 5 pazienti , t3n2 in 1 paziente . 
sensibilit , specificit , vpp e vpn nella diagnosi di metastasi epatiche stata del 100% per entrambe le metodiche . metastasi polmonari metastasi polmonari sono state riscontrate in 8 / 67 pazienti ( 12% )  . 
la tcmd ha posto la prima diagnosi di metastasi polmonari in 6 / 8 casi in cui le lesioni sono state messe in evidenza esaminando con finestra da polmone le scansioni effettuate alle basi polmonari ; la pet / tc in 2 / 8 casi . 
circa il 25% delle recidive da carcinoma del colon - retto sono recidive locoregionali e il 15%20% sono metastasi a distanza potenzialmente resecabili con intento curativo [ 9 ] : una diagnosi precoce che consenta una chirurgia con intento curativo dovrebbe essere lobiettivo del follow - up [ 10 ]  . 
infatti , la riduzione di mortalit legata alla diagnosi precoce di recidiva extra - intestinale dimostrata dai programmi di follow - up intensivi stata equivalente a quella ottenuta dalla terapia adiuvante nei pafig . 
2a , b multidetector computed tomography ( mdct ) performed 18 months after rectal cancer resection and radiotherapy shows irregular and inhomogeneous thickening of the anastomotic wall with dense tissue in the surrounding fat and irregular shape of the suture ( a )  . 
2a , b tcmd , eseguita 18 mesi dopo resezione di carcinoma rettale e radioterapia , che mostra ispessimento irregolare e disomogeneo del tratto anastomotico , con tessuto denso nel grasso perirettale e irregolarit della sutura ( a )  . 
la diagnosi di recidiva locale stata poi confermata anche dalla pet / tc ( b )  . hepatic metastases was t2n0 in four , t2n1 in three , t3n0 in four , t3n1 in five and t3n2 in one . 
the first diagnosis of pulmonary metastasis was provided by mdct in 6 / 8 cases , in which the lesions were visualized by examining the lung base scans with a lung window setting ; fdg - pet / ct provided the first diagnosis in 2 / 8 cases . 
this is comparable to the 30%40% recurrence rate in the first 3 years after surgery reported in the literature [ 8 ]  . approximately 25% of colorectal carcinoma recurrences are locoregional , and 15%20% are potentially resectable distant metastases [ 9 ]  . 
the reduction in mortality resulting from the early diagnosis of extraintestinal recurrence demonstrated by intensive followup programmes was equivalent to that obtained by adjuvant treatment in patients with advanced cancers [ 11 ]  . 
i programmi di follow - up per i pazienti operati per tumore colo - rettale , che includono visita clinica , valutazione del cea ematico , radiografia del polmone , imaging del fegato e colonscopia , migliorano la sopravvivenza , ma non chiaro quale sia la frequenza ottimale degli esami e delle visite [ 12 ]  . 
una metanalisi degli studi prospettici che hanno dimostrato una riduzione del 9%13% della mortalit a 5 anni nei pazienti inclusi in programmi di follow - up intensivo , afferma che i programmi di follow - up intensivo sono economicamente giustificati e andrebbero inclusi nella pratica clinica [ 11 ]  . 
a meta - analysis of prospective studies demonstrating a 9%13% decrease in 5 - year mortality rates in patients included in intensive follow - up programmes [ 11 ] stated that intensive follow - up programmes are economically justified and should become normal clinical practice [ 11 ]  . nevertheless , due to the lack of certainties in this respect , specialised cancer centres adopt different strategies for the management of these patients . 
in the event of increased cea levels , associated with negative results on other follow - up tests , pet / ct should be recommended [ 10 ] given that it plays an important complementary role in the diagnosis of local and distant recurrence [ 4 , 13 , 14 ] and may play a role in subsequent patient management [ 15 , 16 ]  . 
previous studies have shown that the sensitivity and specificity of pet / ct for differentiating between benign and malignant presacral abnormalities reach 100% and 96% , respectively [ 17 ]  . 
in the same study , one third of extrahepatic metastases were missed at ct ( 64% sensitivity ) , whereas pet / ct failed to detect extrahepatic lesions in 11% of cases only ( 89% sensitivity ) [ 18 ]  . 
in a prospective study performed on 102 patients with colorectal carcinoma , pet altered the treatment strategy in 56% of patients as a direct result of unexpected findings [ 15 ]  . 
our retrospective study did not evaluate the mri examinations because mri is not yet part of the standard followup programme . in our study population , fdg - pet / ct and mdct had similar sensitivity , specificity , ppv and npv values for the diagnosis of local recurrence . 
this may be due to the fact that the interpretation of ct images normally relies on dimensional criteria , with large masses usually being interpreted as malignant , even though some are fibrotic or inflammatory tissue [ 17 ]  . 
pet / ct false positive results have been reported to correlate with local inflammatory processes or with the effects of radiotherapy , especially when performed less than 6 months earlier [ 20 ]  . this could explain our only false positive result at fdgpet / ct , which concerned a patient who had never undergone adjuvant therapy after surgery and who had a postoperative reparative process of the bowel wall . in this retrospective study , mdct had greater specificity for the diagnosis of local recurrence than that reported by other studies [ 18 , 19 ]  . 
whereas at the time of reporting the nuclear medicine physicians had just started interpreting pet / ct images , the radiologists had several years experience reading mdct images . cologici specializzati seguono diverse strategie di gestione di questa tipologia di pazienti . 
in caso di aumento del cea , con altri esami di follow - up negativi , la pet / tc dovrebbe essere consigliata [ 10 ] perch svolge un importante ruolo complementare nella diagnosi di recidiva locale e a distanza [ 4 , 13 , 14 ] e pu avere un ruolo nella successiva gestione del paziente [ 15 , 16 ]  . 
studi precedenti hanno mostrato che la sensibilit e specificit della pet / tc nel differenziare le anomalie presacrali benigne da quelle maligne raggiungono rispettivamente valori del 100% e 96% [ 17 ]  . 
nello stesso studio , un terzo delle metastasi extra - epatiche non sono state diagnosticate dalla tc ( sensibilit del 64% ) , mentre la pet / tc ha fallito nella diagnosi di lesioni extraepatiche solo nell11% dei casi ( sensibilit dell89% ) [ 18 ]  . 
 [ 19 ] hanno suggerito che la fdg - pet superiore alla tc e alla rm nel prevedere la risposta a trattamenti multimodali pre - chirurgici in tumori rettali localmente avanzati . 
 nella popolazione di pazienti inclusa in questo studio , la pet / tc e la tcmd hanno mostrato valori paragonabili di sensibilit , specificit , vpp e vpn nella diagnosi di recidiva locale . 
questo pu essere dovuto al fatto che linterpretazione delle immagini tc solitamente legata a criteri dimensionali , cosicch grandi masse sono di solito interpretate come maligne , anche se alcune di esse rappresentano solo tessuto fibrotico o flogistico [ 17 ]  . 
i falsi positivi della tc / pet sono stati descritti come correlati a processi flogistici locali o agli effetti della radioterapia , in particolare se tali trattamenti erano stati effettuati meno di 6 mesi prima [ 20 ]  . 
questo potrebbe spiegare il solo caso falsamente positivo della pet / tc nella diagnosi di recidiva locale , che ha riguardato un paziente che non aveva ricevuto nessuna terapia adiuvante dopo la chirurgia , con esiti post - chirurgici sulla parete intestinale . 
 in questo studio retrospettivo , la tcmd ha mostrato una specificit nella diagnosi di recidiva locale superiore rispetto a quanto riportato da altri studi [ 18 , 19 ]  . 
mentre infatti i medici nucleari , al tempo della refertazione degli esami considerati , avevano appena iniziato lesperienza della interpretazione delle immagini pet / tc , i radiologi avevano diversi anni di esperienza nellinterpretazione delle immagini tcmd . i risultati falsamente negativi della pet / tc sono di solito legati alle dimensioni della lesione ( meno di 5 mm ) e / o alla chemioterapia in corso al momento dellesame . 
in our study , the only fdg - pet / ct false negative result concerned a 5 - mm lesion , which appeared 7 months after the end of the chemotherapy course . 
in this study , mdct and fdg - pet / ct showed the same level of accuracy in diagnosing hepatic metastases , yielding no false positive or false negative results , whereas previous studies have reported different specificity values for ct and pet / ct ( 70% and 90% , respectively ) [ 18 ]  . 
this difference could depend on the better quality of ct images obtained with the multidetector technique , which allows for optimal contrast enhancement and spatial resolution . eight patients ( 12% ) in our study developed pulmonary metastases , and only two of them had local recurrence . 
 [ 21 ] demonstrated that pulmonary metastases also occur in patients with low - stage initial disease ; however , the patients with pulmonary metastases in our study had advanced - stage initial disease ( t2n1 and t4n0 )  . 
in our study , fdg - pet / ct failed to identify two cases of pulmonary metastases ( false negative results ) , one of which was very small ( 5 mm )  . 
in our experience , mdct proved to have a high level of accuracy in the diagnosis of pulmonary metastases , above all due to a multidisciplinary radiological - oncological approach . when reporting ct examinations , particular attention is paid to the patients clinical data , and the scans are always compared to previous ones ( potter kc et al . , european congress of radiology 2005 , vienna )  . 
therefore , positive findings on lung ct , in particular when the images are compared with previous negative scans , may be considered extremely accurate . our study has some limitations . 
no statistical analysis was performed to determine sample suitability , and our series is probably too small to indicate whether fdg - pet / ct or mdct is more accurate in the diagnosis of local or distant recurrence of operated rectal cancer . 
another limit lies in patient selection : as this was a retrospective study , patients did not undergo mdct and fdg - pet / ct at the same time , as would have been the case if these examinations had been part of a standard protocol . 
this could mean that one technique , in this case mdct , may have failed to detect something that was later diagnosed by the other technique , in this case fdg - pet / ct . 
this limitation would have been avoided with a prospective design , but this would have involved greater exposure and costs in the absence of proof of the usefulness of pet / ct . 
for this reason , we decided to compare the diagnostic value of the two techniques retrospectively , regardless of the risk of encountering these limitations . di 5 mm , sviluppatasi 7 mesi dopo il completamento della chemioterapia . 
in questo studio tc e pet / tc hanno mostrato la stessa accuratezza nella diagnosi di metastasi epatiche , in assenza di falsi risultati , mentre studi precedenti avevano mostrato specificit differenti per tc e pet / tc ( 70% e 90% rispettivamente ) [ 18 ]  . 
in questo studio la pet / tc non ha identificato 2 casi di metastasi polmonari ( casi falsi negativi ) , uno dei quali era una lesione molto piccola ( 5 mm )  . 
infatti , durante la refertazione degli esami tc , una elevata attenzione viene prestata alla clinica e gli esami sono sempre confrontati con i precedenti ( potter kc et al . , european congress of radiology 2005 , vienna )  . 
questo risulta particolarmente utile per lesioni polmonari piccole , come quelle che possono essere perse dallesame pet / tc . quindi , i risultati positivi della tc del polmone , in particolare quando i reperti sono confrontati con esami precedentemente negativi , possono essere considerati molto accurati . questo studio ha alcuni limiti . 
non stata eseguita una analisi statistica per determinare lappropriatezza del campione e la nostra casistica probabilmente insufficiente per indicare quale esame diagnostico , tra la pet / tc e la tcmd , sia pi accurato nella diagnosi di recidiva locale o a distanza di tumori del retto operati . 
un altro limite risiede nella selezione dei pazienti : essendo questo uno studio retrospettivo , i pazienti non hanno eseguito tcmd e pet / tc allo stesso tempo , come sarebbe opportuno se tali esami facessero parte di un protocollo standardizzato . 
questo rende possibile che ad una modalit , in questo caso alla tcmd , sia sfuggito qualcosa che stato poi diagnosticato dallaltra modalit , in questo caso la pet / tc . 
questo limite sarebbe stato evitato con una selezione dei pazienti di tipo prospettico , ma ci avrebbe comportato una esposizione ed un costo aggiuntivi , in assenza di una dimostrazione certa dellutilit dellesame pet / tc . 
per tale motivo , abbiamo deciso di confrontare il valore diagnostico delle due modalit in modo retrospettivo , nonostante il rischio di incorrere in tali limiti . conclusions in conclusion , the diagnostic value of fdg - pet / ct and mdct in the follow - up of patients operated on for rectal cancer is comparable , and the advantages of fdg - pet / ct in conclusione , il valore diagnostico attuale di pet / tc e tcmd nel follow - up di pazienti operati di tumore del retto paragonabile , i vantaggi della pet / tc sono la possibilit di valutare con un singolo esame tutto il corpo , la pi bassa conclusioni m . 
in most specialised centres , the follow - up of patients after surgery for rectal carcinoma includes mdct scans every 6 months : we believe the scan should be extended to the chest given the relatively high incidence of pulmonary metastases . 
at present , fdg - pet / ct should only be used to solve any diagnostic doubt resulting from mdct , or in the event of discrepancy between clinical and imaging findings . esposizione alle radiazioni e la possibilit , in futuro , di eseguire un unico esame pet ed una tc con mdc , somministrato per via endovenosa [ 2325 ]  . 
il follow - up dei pazienti operati per carcinoma rettale prevede , nella maggioranza dei centri specialistici , lesecuzione di tcmd ogni 6 mesi : riteniamo che lesame dovrebbe essere esteso al torace , per la relativamente alta incidenza di metastasi polmonari . 
krestin2 1dipartimento di radiologia e dipartimento cuore , imaging cardiovascolare non invasivo , azienda ospedaliera di parma , italy 2dipartimento di radiologia e cardiologia , erasmus medical center , rotterdam , holland 3dibimel , sezione di scienze radiologiche , universit di palermo , italy 4dipartimento di radiologia , universit degli studi di verona , italy 5dipartimento di radiologia , universit degli studi di trieste , italy correspondence to : f . 
cademartiri , viale rustici , 2 , i - 43100 , parma , italy , tel . : + 39 - 052 - 1961833 , e - mail : filippocademartiri@hotmail.com received : 20 august 2006 / accepted : 2 october 2006 / published online : 23 july 2007 abstract the goal of this article is to illustrate the main invasive and noninvasive diagnostic modalities to image the vulnerable coronary plaque , which is responsible for acute coronary syndrome . 
the main epidemiologic and histological issues are briefly discussed in order to provide an adequate background . comprehensive coronary atherosclerosis imaging should involve visualization of the entire coronary artery tree and plaque characterization , including three - dimensional morphology , relationship with the lumen , composition , vascular remodelling and presence of inflammation . 
in particular , we describe multislice computed tomography , which at present seems to be the most promising noninvasive tool for an exhaustive image - based quantification of coronary atherosclerosis . key words coronary artery disease imaging vulnerable plaque multislice computed tomography ct riassunto con questo articolo si vogliono illustrare le principali metodiche di imaging invasivo e non invasivo , che si prefiggono di identificare la placca vulnerabile coronarica , responsabile delle sindromi coronariche acute . 
un imaging onnicomprensivo della malattia aterosclerotica coronarica dovrebbe essere in grado di visualizzare lintero albero coronarico e caratterizzare la placca nei suoi vari aspetti quali la morfologia tridimensionale , il rapporto con il lume , la composizione tessutale , il rimodellamento vascolare e la presenza di infiammazione . 
nessuna tecnica riesce singolarmente a fornire un quadro talmente completo e nessuna modalit disponibile identifica in modo inequivocabile la placca vulnerabile . particolare attenzione stata rivolta alla tomografia computerizzata multistrato , che sembra , al momento , la tecnica pi promettente nel panorama delle metodiche non invasive per la quantificazione complessiva per immagini della malattia aterosclerotica coronarica . parole chiave coronaropatia aterosclerotica imaging placca vulnerabile tomografia computerizzata multistrato ct introduction introduzione atherosclerosis is a degenerative inflammatory process that affects artery walls . 
this classification reflects the natural history of the disease , which is asymptomatic in the initial stages laterosclerosi un processo degenerativo - infiammatorio che interessa la parete dei vasi arteriosi . 
in the past , vascular stenosis was considered the hallmark of the disease . currently , emphasis is placed on the atherosclerotic plaque , which represents the pathological substrate for vascular stenosis [ 3 , 4 ]  . the introduction over the past 10 years of sophisticated ultrasound ( us ) probes mounted on angiographic catheters has enabled in vivo visualisation of atherosclerotic plaque within the coronaries . 
the aim of this review is to illustrate the results provided by invasive and noninvasive imaging modalities and discuss the advantages and limitations , with special reference to multislice computer tomography ( msct )  . tica , mentre negli stadi pi avanzati progredisce in maniera variabile . 
viene dato attualmente risalto alla placca aterosclerotica , che rappresenta il substrato anatomo - patologico della stenosi vascolare [ 3 , 4 ]  . nellultimo decennio lintroduzione di sofisticate sonde ecografiche applicate a cateteri angiografici ha permesso la visualizzazione in vivo della placca aterosclerotica nelle coronarie . 
lo scopo del lavoro mostrare i risultati ottenuti dalle tecniche di imaging invasivo e non invasivo , illustrandone i vantaggi ed i limiti , con particolare attenzione per la tomografia computerizzata multistrato ( tcms )  . epidemiologia le malattie cardiovascolari sono la prima causa di morte nei paesi industrializzati e prevedibilmente lo diventeranno anche nei paesi in via di sviluppo . 
nei prossimi decenni la mortalit per cardiopatia ischemica destinata ad aumentare dell80% nelle donne e del 100% negli uomini , con un aumento pi marcato nei paesi in via di sviluppo rispetto ai paesi industrializzati [ 5 ]  . 
secondo le pi recenti statistiche dellamerican heart association , laterosclerosi delle coronarie ha una prevalenza del 6 , 9% nella popolazione e un tasso di mortalit per infarto di 177 , 8 per 100000 abitanti [ 6 ]  . 
i dati europei pi recenti riportano un tasso di mortalit di 125 per gli uomini e di 63 per le donne , seppure con unampia variabilit regionale per la presenza di gradienti a direzione nord - sud ed est - ovest . 
litalia ha un tasso di mortalit di 105 per gli uomini e di 51 per le donne [ 710 ]  . i sistemi sanitari si trovano a fronteggiare i costi dovuti allaumento della prevalenza della malattia e delle correlate procedure invasive . 
vi sono , pertanto , delle forti aspettative nei riguardi delle tecniche panoramiche non invasive , che potrebbero sostituire la diagnostica invasiva e fornire una diagnosi precoce di aterosclerosi coronarica , con impatto sul trattamento , la prognosi e il costo complessivo . biologia e fisiopatologia la prima manifestazione di aterosclerosi consiste in un accumulo di materiale lipidico visibile allesame macroscopico che prende il nome di stria lipidica ( fatty streak lesion )  . 
da questo nucleo iniziale , con gli anni , prende forma la placca aterosclerotica matura , la quale consiste in un core lipidico 03 cademartiri 23 - 07 - 2007 11 : 40 pagina 639 epidemiology cardiovascular disease is the leading cause of death in industrialised countries and is emerging as a major cause of death in developing countries . 
over the next few decades , deaths due to ischaemic heart disease are expected to increase by 80% among women and 100% among men , with a more marked rise in developing countries compared with developed countries [ 5 ]  . 
according to the most recent american heart association statistics , coronary atherosclerosis has a prevalence of 6.9% in the general population and a mortality rate due to infarction equal to 177.8 per 100 , 000 inhabitants [ 6 ]  . 
recent data for europe report a mortality rate of 125 among men and 63 among women , but with wide regional variations due to north - south and east - west gradients . 
high expectations are therefore placed on noninvasive panoramic imaging modalities that could replace invasive diagnostic techniques and provide an early diagnosis of coronary atherosclerosis , with impact on treatment , outcomes and overall costs . biology and pathophysiology the first manifestation of atherosclerosis is the accumulation of fatty material , which is grossly visible and is called fatty - streak lesion . 
this means that coronary stenosis is the expression of advanced disease and that the disease needs to be characterised at an earlier stage [ 14 ]  . acute coronary events manifest when atherosclerotic plaque ruptures and blood comes into contact with the plaques lipid content , which is a potent procoagulant . plaque responsible for coronary thrombosis is defined as a culprit plaque at conventional coronary angiography ( cca )  . 
the new frontier is instead to characterise stable plaque that is likely to become complicated , known as vulnerable plaque , before the acute event occurs [ 15 ]  . culprit plaque is characterised on the basis of autopsy studies [ 1618 ] , and the study of vulnerable plaque relied on the experience gained from these studies . 
da ci si evince che la stenosi coronarica espressione di un processo patologico in fase avanzata e sorge quindi la necessit di caratterizzare la malattia in una fase pi precoce [ 14 ]  . levento coronarico acuto si manifesta quando una placca aterosclerotica si fissura e il sangue viene a contatto con il contenuto lipidico , il quale ha un elevatissimo potere procoagulante . 
la nuova ambiziosa frontiera quella di caratterizzare , invece , una placca stabile suscettibile di complicazione , definita placca vulnerabile , prima dellevento acuto [ 15 ]  . la placca culprit stata caratterizzata a partire da studi autoptici [ 1618 ]  . 
 [ 19 ] in seguito hanno identificato la placca vulnerabile come incline alla rottura , caratterizzata da un core lipidico ricoperto da un sottile cappuccio fibroso , e da cellule infiammatorie . 
 [ 20 , 21 ] una capsula fibrosa assottigliata , con spessore inferiore ai 65 m , fa perdere alla placca le caratteristiche di stabilit e la rende incapace di sopportare lo stress circonferenziale con successiva rottura . 
i criteri maggiori sono : linfiammazione ( presenza di monociti , macrofagi , linfoun ampio core lipidico con un sottile cappuccio fibroso lesposizione dellendotelio con liniziale aggregazione citi t ) delle piastrine lulcerazione della placca e una stenosi > 90% . 
 nonostante la placca vulnerabile sia lelemento primariamente responsabile dellulcerazione , anche le caratteristiche del sangue , se inclini alla trombosi ( sangue vulnerabile ) , e le caratteristiche del miocardio , se inclini allaritmia ( miocardio vulnerabile ) , dovrebbero essere tenute in considerazione nella valutazione del rischio coronarico , portando allidentificazione del paziente vulnerabile ad alto rischio di evento cardiaco acuto [ 28 ]  . 03 cademartiri 23 - 07 - 2007 11 : 40 pagina 640 f . 
 [ 20 , 21 ] , plaque with a thin fibrous cap , less than 65 - m thick , loses its stability and becomes unable to withstand circumferential stress , with subsequent rupture . 
la visione planare dellalbero coronarico ( luminografia ) consente lesecuzione di una stima del lume vascolare ( quantitative coronary angiography , qca ) , ma non fornisce indicazioni sullaspetto delle pareti vascolari e leventuale presenza di placche [ 31 ]  . 
secondo una metanalisi il 49% delle lesioni colpevoli , ovvero responsabili di infarto miocardio acuto ( ima ) causato da stenosi non significative inferiori al 50% [ 32 ]  . 
molte di queste metodiche sono invasive ovvero basate sullintroduzione di un catetere , come lecografia intravascolare ( ivus ) , che considerata il gold standard per limaging di placca . ecografia intravascolare livus una tecnica invasiva in grado di fornire immagini assiali del lume e della parete dei vasi , superando la visione 03 cademartiri 23 - 07 - 2007 11 : 40 pagina 641 f . 
2 erosion of a pathological intimal thickening and rupture of a thin fibrous cap atheroma may lead to thrombosis and result in an acute coronary syndrome , or it may heal . 
2 lerosione di un patologico ispessimento intimale e lulcerazione di un ateroma con sottile cappuccio fibroso possono provocare una trombosi ed originare una sindrome coronarica acuta oppure andare incontro a guarigione . 
planar visualisation of the coronary tree ( luminography ) allows estimation of the vascular lumen [ quantitative coronary angiography ( qca ) ] but does not provide information on the appearance of the vessel walls and the possible presence of plaque [ 31 ]  . 
according to a meta - analysis , 49% of culprit lesions responsible , that is , for acute myocardial infarction ( ami ) are related to nonsignificant stenosis less than 50% [ 32 ]  . 
ivus uses a catheter fitted with a miniature rotating transducer in mechanical systems or made up of an annular array of crystals ( up to 64 elements ) in electronic systems . 
frequencies employed are in the range of 20 and 40 mhz , with an axial resolution between 100 and 200 lateral resolution ranges from 200 to 250 m , depending on image depth . 
once inside the vessel , the catheter is withdrawn at a constant speed by means of a motorised mechanism while the study is recorded on a videotape . on ivus , the arterial wall usually displays a trilaminar appearance corresponding to the wall components : the intima , media and adventitia . 
ivus enables an accurate quantitative assessment of the vessel contours , luminal area , atheroma size ( which is relatively hyperechoic to the lumen and media ) , calcification , stent diameter and areas of remodelling [ 37 ]  . ivus may demonstrate the presence of an appreciable atherosclerotic burden in angiographically intact vessels , so this imaging modality may be considered useful for predictplanare dellangiografia . 
lapparecchiatura ivus si avvale di un catetere provvisto di un trasduttore miniaturizzato , rotante nei sistemi meccanici oppure costituito da una matrice anulare di cristalli ( fino a 64 elementi ) nei sistemi elettronici . 
le frequenze impiegate sono comprese tra 20 e 40 mhz , con una risoluzione assiale tra 100 e 200 la risoluzione laterale varia invece tra 200 e 250 m , a seconda della profondit di immagine . 
una volta posizionato allinterno del vaso il catetere viene ritirato con un meccanismo motorizzato a velocit costante e lo studio viene registrato su nastro . la parete arteriosa allindagine ivus mostra solitamente un aspetto trilaminare corrispondente ai componenti della parete : lintima , la media e lavventizia . 
livus consente unaccurata valutazione quantitativa dei contorni dei vasi , dellarea del lume , delle dimensioni dellateroma ( relativamente iperecogeno rispetto al lume e alla media ) , delle calcificazioni , del diametro degli stent e delle aree di rimodellamento [ 37 ]  . livus pu dimostrare la presenza di un apprezzabile carico aterosclerotico in vasi angiograficamente indenni e pertanto questa modalit di imaging potrebbe essere considerata utile nel predire il rischio di un evento acuto [ 38 ]  . sebbene livus consenta una dettagliata visione della parete dei vasi , linterpretazione della composizione di placca verte esclusivamente sulla semplice valutazione qualitativa degli echi . 
in particolare la visualizzazione delle placche inficiata da quelli da riverberazione ( ring - down ad anello , blood speckle ) , a meno che non si provveda a diminuire i guadagni , pur con il rischio di provocare unattenuazione del segnale nei tessuti bersaglio [ 37 ]  . numerosi studi in vivo mostrano risultati promettenti circa le effettive capacit di visualizzazione della placca vulnerabile e la sua correlazione con le sindromi coronariche acute [ 4246 ]  . 
in particolare , uno studio esteso alle tre coronarie ha evidenziato come nei pazienti con sindrome co03 cademartiri 23 - 07 - 2007 11 : 40 pagina 643 ing the risk of an acute event [ 38 ]  . 
hypoechoic plaque is defined as soft plaque , not in relation to its tissue composition but to the characteristics of it acoustic signal relative to the adventitia [ 39 , 40 ]  . 
calcific plaque is , instead , characterised by hypereflective echoes with the posterior shadow cone [ 41 ]  . in fact , mixed - composition plaque with a corresponding mixed sonographic appearance is very common . 
in particular , plaque visualisation is limited by reverberation artefacts ( ring - down , blood speckle ) unless gain is decreased , even though there is a risk of attenuating the signal in the target tissues [ 37 ]  . several in vivo studies have shown promising results as to the techniques effective ability to visualise vulnerable plaque and its correlation with acute coronary syndromes [ 4246 ]  . 
in particular , one study extended to the three coronary arteries showed that ivus can identify multiple ulcerated plaque disseminated along the coronary tree independently of the culprit lesion in patients with acute coronary syndrome [ 47 ]  . 
however , differentiation of the lipid core ( area of reduced echogenicity ) from the fibrous tissue is still difficult , as the thickness of the fibrous cap is below the techniques spatial resolution . 
a new ivus - related technology , called virtual histology , has recently been introduced that generates colour maps of plaque composition on the basis of the amplitude and frequency of the echoes [ 4850 ]  . 
though considered the reference standard for plaque evaluation , ivus cannot be routinely used because of its invasiveness , which implies a 1%3% complication rate [ 51 , 52 ] , the cost of materials and the time required for a single procedure . 
in particular , the excessive length of the examination does not allow for a panoramic assessment of all of the coronary arteries , and therefore , the study of plaque is somewhat segmental . 
currently , ivus is considered supplemental though with independent value to angiography , which is particularly useful in ostial or bifurcation stenosis , in the assessment of diffusely atherosclerotic vessels and in the visualisation of eccentric plaques [ 37 ]  . 
ivus has received the approval of the us food and drug administration ( fda )  . elastography elastography is an ivus - related technique that relies on the different mechanical properties of tissues . 
in particular , by applying a known pressure and recording the strain produced , one can define the heterogeneity of the tissue of an atherosclerotic plaque with high reliability [ 53 ]  . 
tuttavia , ancora ardua una differenziazione del core lipidico ( area a ridotta ecogenicit ) dal tessuto fibroso , dal momento che lo spessore del cappuccio fibroso al di sotto della risoluzione spaziale della metodica . 
recentemente stata introdotta una nuova tecnologia ivus - relata , definita istologia virtuale , che , sulla base dellampiezza e della frequenza degli echi , riesce ad elaborare mappe a colori della composizione di placca [ 4850 ]  . 
livus pur essendo considerata lo standard per la valutazione della placca non pu essere utilizzata routinariamente a causa della sua invasivit , che implica un tasso di complicanze dell1%3% [ 51 , 52 ] , dei costi dei materiali e del tempo necessario per una singola procedura . 
allo stato attuale , pur con un valore indipendente , livus considerata supplementare allangiografia , di particolare utilit nello studio delle stenosi ostiali o di biforcazione , nella valutazione dei vasi diffusamente interessati da aterosclerosi , nella visualizzazione delle placche eccentriche [ 37 ]  . livus ha ricevuto lapprovazione da parte della food and drug admistration ( fda )  . elastografia lelastografia una tecnica ivus - relata che sfrutta le diverse propriet meccaniche dei tessuti . 
studi su pazienti con angina stabile , angina instabile e ima hanno evidenziato caratteristiche di deformabilit delle placche significativamente diverse [ 54 ]  . termografia a differenza delle altre metodiche invasive che forniscono una visualizzazione diretta , la termografia ha come obiettivo quello di caratterizzare la placca dal punto di vista metabolico - funzionale . 
in particolare , condizioni di flogosi localizzata , e quindi di incremento della temperatura , si associano ad instabilit della placca : possibile misurare queste variazioni di temperatura con dei sensori applicati a cateteri ( sensibilit termica 0 , 05c , risoluzione spaziale 500 m ) [ 55 , 56 ]  . 
il riscontro di valori di temperatura significativamente diversi in pazienti con infarto miocardico rispetto a pazienti con angina stabile la dimostrazione di placche in differente stato metabolico [ 57 ]  . 
linformazione aggiuntiva sullo stato flogistico della placca risulta di grande importanza se associata ad una metodica visual - based . 03 cademartiri 23 - 07 - 2007 11 : 40 pagina 644 f . 
in particular , local inflammatory states , and consequent rise in temperature , are associated with plaque instability : these temperature variations can be measured by sensors applied to catheters ( thermal sensitivity 0.05c , spatial resolution 500 m ) [ 55 , 56 ]  . 
the finding of significantly different temperature values between patients with myocardial infarction and patients with stable angina is proof that plaque is in a different metabolic state [ 57 ]  . 
additional information on the inflammatory status of plaque is of great value when associated with a visual - based method . angioscopy vascular angioscopy is the only technique allowing direct visualisation of the endothelium and superficial components of atherosclerotic plaque [ 58 ]  . 
in addition , a temporary occlusion of the vessel needs to be induced in order to view the lesion , as the light signal is heavily affected by the presence of blood . 
angioscopy has been approved for clinical use in japan only . optical coherence tomography optical coherence tomography reconstructs images on the basis of the codification of an image reflected in a similar manner to us , with the difference that infrared rays are used instead of us . 
currently , it is the only technique capable of accurately visualising fibrous caps at risk of rupture ( 65 m ) and identifying a complicated lesion [ 62 ]  . 
its major limitation is the complexity of the procedure : a transient ischaemia of approximately 20 s has to be induced , as electromagnetic waves are heavily attenuated by blood . 
another limitation is its poor penetration depth ( 12 mm ) , which does not allow assessment of the entire thickness of the vessel [ 63 ]  . spectroscopy raman spectroscopy is based on the phenomenon described by the indian physicist raman , whereby the inelastic collilangioscopia vascolare lunica tecnica che permette una visione diretta dellendotelio e delle componenti superficiali della placca aterosclerotica [ 58 ]  . 
lutilizzo in ambito clinico approvato solamente in giappone . optical coherence tomography la tomografia a coerenza ottica ricostruisce le immagini a partire dalla codifica di un segnale riflesso in modo analogo allecografia , con la differenza che al posto degli ultrasuoni si utilizzano i raggi infrarossi . 
unaltra limitazione costituita dalla scarsa penetrazione in profondit ( 12 mm ) che non consente una valutazione a tutto spessore del vaso [ 63 ]  . spettroscopia la spettroscopia raman si fonda sul fenomeno , evidenziato dallomonimo fisico indiano , per il quale dalla collisione anelastica tra un fotone ( con compresa tra 750 e 850 nm ) e una molecola scaturiscono emissioni di luce a differente frequenza [ 64 ]  . 
la spettroscopia raman ha indiscutibili potenzialit per lo studio dellaterosclerosi in termini di composizione chimica , ma non fornisce alcuna informazione sulla morfologia delle arterie . la spettroscopia che utilizza i raggi del vicino infrarosso , con compresa fra 750 e 2500 nm , garantisce una profondit di penetrazione fino a 2 mmoreno et al . 
 [ 68 ] hanno dimostrato in vitro la presenza di uno spettro caratteristico per le placche vulnerabili , riportando sensibilit e specificit molto elevate per i componenti lipidici della placca . 03 cademartiri 23 - 07 - 2007 11 : 40 pagina 645 sion of a photon ( with between 750 and 850 nm ) and a molecule generates light emissions with different frequencies [ 64 ]  . 
a catheter ( ~1 mm ) emits a laser beam focused on the tissue under examination , and the reflected beam contains the spectrum of the chemical structures present in the tissue . 
raman spectroscopy has indisputable potential for the study of atherosclerosis in terms of chemical composition but provides no information on arterial morphology . near - infrared spectroscopy , with between 750 and 2500 nm , has a penetration depth up to 2 m moreno et al . demonstrated the presence of a characteristic spectrum for vulnerable plaque in vitro , reporting very high sensitivity and specificity for the lipid plaque components [ 68 ]  . 
the ex vivo analysis conducted on carotid plaque characterised as stable and vulnerable demonstrated significantly different absorption spectra [ 69 ]  . noninvasive modalities magnetic resonance imaging magnetic resonance imaging ( mri ) was the first noninvasive modality whose potential for tissue characterisation was applied to the study of atherosclerotic plaque [ 70 ]  . 
the use of dedicated coils has made it possible to achieve excellent spatial resolution ( 400400 m ) and to assess the fibrous cap in carotid atheromas [ 71 ]  . 
the sequences employed for luminography are threedimensional ( 3d ) turbo field - echo sequences with prospective echocardiogram ( ecg ) triggering and without contrast administration ; the spatial resolution of these sequences is 1.91.21.2 mstudy of the vessel wall involves the use of double inversion recovery ( ir ) black - blood fast spin echo ( fse ) sequences characterised by short radiofrequency pulses and preliminary inversion pulse , which ensure optimal suppression of the endovascular signal and minimise artefacts due to vessel motion [ 72 ]  . 
contrast - enhanced sequences with gadolinium - diethylenetriaminepentaacetic acid ( gd - dtpa ) or ultrasmall superparamagnetic iron oxide ( uspio ) ( selectively taken up by macrophages in inflamed unstable plaque ) have also been tested and shown to improve the sensitivity of the method [ 73 ]  . 
il calcio si presenta marcatamente ipointenso in t1 , dp e t2 . lutilizzo di bobine dedicate ha permesso di raggiungere unottima risoluzione spaziale ( 400400 m ) e di valutare il cappuccio fibroso a livello delle carotidi [ 71 ]  . 
a livello delle coronarie i risultati non sembrano , per ora , altrettanto convincenti perch le coronarie sono vasi di ridotte dimensioni , a decorso tortuoso ed in continuo movimento . 
attualmente per la luminografia sono utilizzate sequenze 3d turbo field - echo con trigger ecg prospettico senza mezzo di contrasto che presentano una risoluzione spaziale di 1 , 9 1 , 2 1 , 2 mlo studio di parete prevede lutilizzo di sequenze a sangue nero ( dual - ir black - blood fse ) , caratterizzate da brevi impulsi di radiofrequenza e preliminare impulso di inversione , che garantiscono unottima soppressione del segnale endovascolare e minimizzano gli artefatti dovuti al movimento dei vasi [ 72 ]  . 
sono state sperimentate anche sequenze con mezzo di contrasto gadolinio - dtpa o uspio ( selettivamente fagocitato dai macrofagi nelle placche infiammate instabili ) , che hanno migliorato la sensibilit della metodica [ 73 ]  . 
unaltra opportunit offerta dalla rm consiste nella soppressione del segnale proveniente dal tessuto adiposo epicardico che permette una migliore visualizzazione della parete vascolare [ 74 ]  . in letteratura sono presenti , ad oggi , poche esperienze che documentano la caratterizzazione della placca aterosclerotica coronarica umana in vivo [ 75 , 76 ]  . 
in particolare lintroduzione di bobine intravascolari ha permesso il miglioramento della risoluzione spaziale fino a 150 m in un campo magnetico di 4 , 7 t , ampliando le possibilit di caratterizzazione tissutale [ 78 , 79 ]  . 
si prospettano tuttavia tempi piuttosto lunghi per una possibile applicazione clinica . medicina nucleare il punto di forza della medicina nucleare consiste nella capacit di evidenziare lattivit metabolica della placca e di fornire informazioni funzionali complementari a quelle delle metodiche radiologiche . 
in particular , the introduction of intravascular coils has improved spatial resolution up to 150 m in a 4.7 - t magnetic field , extending the potential for tissue characterisation [ 78 , 79 ]  . 
it will , however , take years before possible clinical applications begin . nuclear medicine the strength of nuclear medicine is its ability to visualise the metabolic activity of a plaque and provide complementary functional information to that offered by radiological modalities . 
the leading modality in nuclear medicine is currently positron emission tomography ( pet ) , which acquires images with a spatial resolution up to 45 mm , thereby improving on the performance of single photon emission computed tomography ( spect ) , which has a resolution of 1015 mm [ 81 ]  . 
at the level of the carotids , atherosclerotic plaque has been shown to accumulate the radionuclide 18f - fluorodeoxyglucose , which competes with glucose for uptake by metabolically active cells [ 82 ]  . 
major correlations have also been demonstrated with atherosclerotic plaque vulnerability [ 86 , 87 ]  . multislice computed tomography the first applications of ct to the study of the heart date back to the early 1990s . 
these studies used fourth - generation ct , electron beam computed tomography ( ebct ) , at the time the only technology with a temporal resolution capable of acquiring images synchronised with the cardiac cycle . these experiences were aimed at visualising coronary calcium and allowed a direct assessment of the coronary tree [ 88 ]  . the method had high sensitivity [ 89 ] and allowed definition of a calcium - score parameter that could quantify disease severity [ 90 ]  . 
assessment of the patients biochemical parameters and history ( framingham global risk score ) underestimates risk and results in a wide lit strettamente correlata al grado di attivit metabolica della placca [ 80 ]  . 
la metodica di punta della medicina nucleare attualmente la positron emission tomography ( pet ) che ha la capacit di acquisire immagini con risoluzione spaziale fino a 45 mm , migliorando la performance della single photon emission tomography ( spect ) , che ha una risoluzione di 1015 mm [ 81 ]  . 
nelle carotidi , stata documentata la localizzazione del radionuclide 18f - fluorodesossiglucosio , che compete con il glucosio nei processi di uptake da parte di cellule metabolicamente attive , a livello delle placche aterosclerotiche [ 82 ]  . 
per migliorare la performance della tecnica sono stati , di recente , sviluppati scintillatori intravascolari al fine di garantire una pi elevata sensibilit [ 83 ]  . le applicazioni della medicina nucleare si estendono allimaging molecolare e in particolare allo studio della neoangiogenesi . 
sono state dimostrate importanti correlazioni con la vulnerabilit della placca aterosclerotica [ 86 , 87 ]  . tomografia computerizzata multistrato le prime applicazioni della tomografia computerizzata ( tc ) allo studio del cuore risalgono allinizio degli anni 90 . 
questi studi utilizzavano apparecchi tc di iv generazione , il tomografo a fascio di elettroni ( electron beam computed tomography , ebct ) , allora lunica tecnologia con risoluzione temporale in grado di acquisire immagini sincronizzate con il ciclo cardiaco . 
la metodica era caratterizzata da unelevata sensibilit [ 89 ] tanto da consentire la formulazione di un parametro di calciumscore , in grado di quantificare lo stato di malattia [ 90 ]  . attualmente il calcium score sembra avere valore nellinquadramento del rischio cardiovascolare [ 91 ]  . 
il ruolo del calcium score resta ancora discusso e , comunque , la posizione ufficiale dellultimo consensus document non favorevole allutilizzo dellebct a scopo di screening [ 9295 ]  . lebct non tuttavia in grado di visualizzare le altre componenti della placca aterosclerotica . 
parallelamente , la tcms si rivelata affidabile nel rilevare la presenza del calcio e offre inoltre la possibilit di visualizzare lesioni non calcifiche 03 cademartiri 23 - 07 - 2007 11 : 40 pagina 647 proportion of patients with intermediate risk . 
the role of the calcium score is still debated , and the official position of the latest consensus document does not favour the use of ebct as a screening tool [ 9295 ]  . ebct is not , however , able to visualise the other components of atherosclerotic plaque . 
recent technological developments ( 64 - slice ct ) have provided msct with a spatial resolution of 0.30.30.4 mm3 ( isotropic voxel ) associated with a temporal resolution of 165 ms ( with 180 acquisition protocol )  . 
the contrast material has to provide adequate intravascular opacification and must therefore have a high iodine concentration ( 350400 mgi / ml ) and reach the coronary district with a high flow rate ( 45 ml / s ) [ 101 ]  . 
for the best vascular attenuation to be obtained , the beginning of the scan should be synchronised with the arrival of the contrast material by using the bolus tracking technique [ 102 ]  . 
the reconstruction algorithm exploits the possibility of retrospectively synchronising the data with a specific phase of the cardiac cycle expressed as a percentage of the r - r interval or as a distance in milliseconds from the subsequent r wave . 
this type of study cannot be viewed and evaluated on film , as the complexity and tortuousness of the structures being studied is such that the process would require printing out an excessive number of images . the diagnostic performance of msct is considered excellent in the evaluation of coronary stenosis . 
sixteen - slice scanners have a sensitivity between 82% and 95% , a specificity between 93% and 98% and a negative predictive value ( npv ) of 97%99% [ 103105 ]  . 
i recenti avanzamenti tecnologici ( tc a 64 strati ) hanno conferito alla tcms una risoluzione spaziale di 0 , 30 , 30 , 4 mm3 ( voxel isotropico ) associata ad una risoluzione temporale di 165 ms ( con protocollo di acquisizione a 180 )  . il protocollo usato attualmente per la valutazione delle stenosi coronariche prevede lutilizzo dei seguenti parametri : scansione con 120 kv e 800900 mas , ricostruzione dellimmagine con spessore di strato di 0 , 60 , 75 mm e incremento di ricostruzione di 0 , 30 , 4 mm [ 97100 ]  . 
pertanto la concentrazione iodica deve essere elevata ( 350400 mgi / ml ) e arrivare nel distretto coronarico con alta velocit di flusso ( 45 ml / s ) [ 101 ]  . 
lalgoritmo di ricostruzione sfrutta la possibilit di sincronizzare retrospettivamente i dati con una particolare fase del ciclo cardiaco espressa come percentuale dellintervallo r - r oppure come distanza in ms dalla successiva onda r . 
non pensabile osservare e valutare questo tipo di esame su pellicola perch le strutture in studio hanno unanatomia talmente complessa e tortuosa che richiederebbe la stampa di un numero troppo elevato di immagini . la performance diagnostica della tcms considerata eccellente nella valutazione delle stenosi coronariche . 
gli apparecchi a 16 strati mostrano una sensibilit fra l82% e il 95% , una specificit fra il 93% e il 98% e un valore predittivo negativo fra il 97% e il 99% [ 103105 ]  . 
 [ 111 ] hanno mostrato che le caratteristiche ivus delle lesioni correlano con lattenuazione tcms nel 78% delle sezioni contenenti placche ipoecogene ( placche soft ) , nel 78% delle sezioni contenenti placche iperecogene ( placche fibrose ) , nel 95% delle sezioni contenenti placche calcifi03 cademartiri 23 - 07 - 2007 11 : 40 pagina 648 f . 
le corrispondenti immagini assiali mostrano lesioni di diversa densit ( c , d )  . tures correlated with msct attenuation in 78% of sections containing hypoechoic plaque ( soft plaque ) , in 78% of sections containing hyperechoic plaque ( fibrous plaque ) and in 95% of sections containing calcified plaque . 
in 484 of the 525 sections examined ( 92% ) , atherosclerotic lesions were correctly excluded , whereas the remaining 8% were incorrectly classified as containing prevalently noncalcified plaque ( 36 / 41 sections ) , owing to adjacent calcification , stents or image quality . 
reports a sensitivity of 82% ( 41 / 50 segments with plaque ) and a specificity of 88% ( 29 / 33 intact segments ) relative to ivus , although sensitivity is only 53% for noncalcified plaque . 
visualisation of noncalcified plaque is limited by the size of the plaque and vessel , accuracy improving when the analysis is restricted to the proximal segments ( tables 2 and 3 ) [ 112 ]  . 
in 484 delle 525 sezioni esaminate ( 92% ) , le lesioni aterosclerotiche sono state correttamente escluse , mentre il restante 8% , stato erroneamente classificato come contenente placche prevalentemente non calcifiche ( 36 / 41 sezioni ) a causa delle calcificazioni attigue , degli stents e della qualit di immagine . 
 [ 112 ] riporta rispetto allivus una sensibilit dell82% ( 41 / 50 segmenti con placche ) e una specificit dell88% ( 29 / 33 segmenti indenni ) , sebbene per le placche non calcifiche la sensibilit sia solamente del 53% . la visualizzazione delle placche non calcifiche limitata dalle dimensioni della placca e del vaso , con una migliore accuratezza quando si restringe lanalisi ai segmenti prossimali ( tabelle 2 e 3 ) [ 112 ]  . 
al riguardo i dati presenti in letteratura concordano : le placche dei pazienti con evento acuto hanno densit media minore di quella dei pazienti con angina stabile [ 114116 ]  . 03 cademartiri 23 - 07 - 2007 11 : 40 pagina 649 f . 
le immagini assiali mostrano il lume normale a monte della lesione ( d ) , la placca ( e , f ) a livello della biforcazione e dellada prossimale . table 2 in vivo assessment of coronary atherosclerotic plaque : multislice computed tomography ( msct ) vs . 
intravascular ultrasound ( ivus ) study msct scanner sensitivity ( % ) specificity ( % ) noncalcified soft fibrous calcified total schroeder [ 110 ] leber [ 111 ] achenbach [ 112 ] schoenhagen [ 129 ] moselewski [ 128 ] schroeder [ 110 ] leber [ 111 ] achenbach [ 112 ] schoenhagen [ 129 ] moselewski [ 128 ] a30 / 40 plaques visualised on msct ; bextrapolated data ( 299 / 350 plaques correctly classified ) ; c37 / 46 segments examined tabella 2 valutazione delle placche aterosclerotiche coronariche in vivo : confronto tcms vs ivus studio tcms scanner sensibilit ( % ) specificit ( % ) non calcifiche soffici fibrose calcifiche totale a34 / 40 placche visualizzate dalla tcms ; bdati estrapolati ( 299 / 350 placche correttamente classificate ) ; c37 / 46 segmenti esaminati 03 cademartiri 23 - 07 - 2007 11 : 40 pagina 650 f . 
however , the hu score suggested for noncalcified plaque does not take into account the overlap in density values between fibrous and lipid plaque , as shown by ex vivo and in vivo studies , or the misdiagnosis of thrombi , which have a similar attenuation value [ 120 ] ( tables 4 and 5 )  . 
a phantom study has demonstrated that when used at different concentrations ( 1 : 30 , 1 : 40 , 1 : 50 ) , the iodinated contrast material required to produce adequate density gradients between plaque and blood leads to changes in plaque density [ 121 ]  . 
density ranges defined for soft , fibrous and calcified plaque are therefore unlikely to have an absolute value . nel tentativo di perfezionare la tecnica , sono stati pubblicati vari studi ex vivo con il chiaro vantaggio di un modello non in movimento e del confronto istologico [ 117 ]  . 
 [ 118 ] riportano una significativa differenza in densit ( p < 0 , 01 ) tra placche lipidiche ( 479 hu ) e placche fibro - calcifiche ( 10428 hu )  . 
tuttavia lo score in uh suggerito per la placca non calcifica non tiene conto della sovrapposizione dei valori di densit per le placche fibrose e lipidiche , evidenziato dagli studi ex vivo ed in vivo , e della diagnosi non corretta dei trombi che hanno un analogo valore di attenuazione ( tabella 4 e 5 ) [ 120 ]  . 
da uno studio su fantoccio emerge che il mdc iodato , necessario per creare unadeguata differenza di densit tra placche e sangue , determina , utilizzando diverse concentrazioni ( 1 : 30 , 1 : 40 , 1 : 50 ) , una variazione della densit di placca [ 121 ]  . 
non quindi verosimile che gli intervalli di densit definiti per le placche soffici , fibrose e calcifiche abbiano un valore assoluto . discussion discussione diagnosis of a disease entity in the preclinical phase has become possible in several fields of medicine as a result of imla diagnosi di unentit nosologica in fase pre - clinica oggi possibile in svariati campi della medicina , grazie alla miglio03 cademartiri 23 - 07 - 2007 11 : 40 pagina 651 f . 
atherosclerotic disease is characterised by a different and , in many respects , unclear or unexplored natural course : traditionally regarded as a degenerative disease , it is now believed to be closely related to a generalised inflammatory process [ 123 , 124 ]  . the study of vulnerable plaque is still in its very early stages , and no large - scale prospective studies have been carried out . 
one can nonetheless legitimately consider the current role and future prospects of the techniques that are being applied to the characterisation of coronary atherosclerotic plaque and highlight their potential and limitations . ivus is a well - known , well - tested modality for which a consensus document has been produced [ 37 ]  . 
in addition , development and application will probably be limited to a handful of referral centres due to the high professionalism required for operators and the very high cost of materials . the invasiveness and risks associated with these procedures could also hinder large prospective studies from being conducted . application of noninvasive modalities to the study of the coronaries is more recent [ 75 , 126 ]  . 
there is no agreement on the true potential of msct as to size estimation , with some reports stating that volume is underestimated and others that it is overestimated [ 112 , 128 ]  . 
according to recent publications , msct allows the study of positive vascular remodelling , an event that seems to delay the development of stenosis , on one hand , and to be associated with acute coronary syndromes , on the other [ 129131 ]  . 
the search for a protocol optimised for plaque evaluation and the development of software enabling an accurate and reproducible characterisation could represent valid solutions to these problems . no single imaging modality is currently able to detect and characterise vulnerable coronary plaque unequivocally ( table 6 )  . 
technological issues aside , the success of such a complex goal falls into a broader context of clinical imaging , which requires a careful selection of high - risk patients to be studied with such a specific type of examination . 
la malattia aterosclerotica caratterizzata da una storia naturale diversa e per molti aspetti ancora poco chiara o inesplorata : da sempre considerata una malattia degenerativa , oggi ritenuta fortemente correlata con un processo infiammatorio generalizzato [ 123 , 124 ]  . lo studio della placca vulnerabile in una fase preliminare , mancando studi prospettici su un vasto campione . 
si prefigurano possibilit di sviluppo e applicazione in pochi centri specialistici per la grande professionalit richiesta agli operatori e per lelevatissimo costo dei materiali . linvasivit e i rischi connessi a queste procedure potrebbero inoltre ostacolare lesecuzione di ampi studi prospettici . lapplicazione delle metodiche non invasive allo studio delle coronarie pi recente [ 75 , 126 ]  . 
non vi ancora accordo circa le reali potenzialit della tcms nello stimare le dimensioni : per alcuni ricercatori il volume viene sottostimato , mentre per altri viene sovrastimato [ 112 , 128 ]  . 
la tcms secondo alcune recenti pubblicazioni permette lo studio del rimodellamento vascolare positivo , fenomeno che da un lato sembrerebbe ritardare lo sviluppo delle stenosi e dallaltro essere associato a sindromi coronariche acute [ 129131 ]  . 
la ricerca di un protocollo desame ottimizzato per la valutazione della placca e lo sviluppo di un software , che consenta alloperatore unaffidabile e riproducibile caratterizzazione , potrebbero essere valide soluzioni a queste problematiche . attualmente nessuna metodica di imaging capace di identificare e caratterizzare in modo inequivocabile la placca vulnerabile coronarica ( tabella 6 )  . 
al di l dellaspetto tecnologico , la riuscita di un progetto cos complesso si inserisce in un pi vasto contesto di imaging clinico nel quale appare fondamentale la selezione del paziente ad alto rischio da sottoporre ad un tipo di esame talmente specifico . solo questo tipo di confronto potr validare lefficacia di 03 cademartiri 23 - 07 - 2007 11 : 40 pagina 653 f . 
characterisation of an inflammatory state with assessment of inflammatory markers ( c - reactive protein , fibrinogen , interleukin 6 , tumour necrosis factor - ) could help to identify the vulnerable patient . 
imaging techniques , primarily noninvasive and panoramic techniques such as msct , would come into play at a later stage to evaluna tecnica e la sua superiorit sulle concorrenti . in un ipotetico algoritmo diagnostico , la selezione del paziente ad alto rischio ( sintomatico o asintomatico ) costituirebbe , infatti , il punto di partenza . 
la caratterizzazione dello stato infiammatorio con valutazione dei marcatori della flogosi ( proteina c reattiva , fibrinogeno , interleuchina 6 , tnf - ) potrebbe favorire lidentificazione del paziente vulnerabile . 
le tecniche dimaging , primariamente quelle non invasive e panoramiche come la tcms , entrerebbero in scena in un secondo tempo per valutare lo stato di malattia co03 cademartiri 23 - 07 - 2007 11 : 40 pagina 654 f . 
a rteriosa d ieta e stile di v ita terapia tc m ultistrato ( ? ? ) stenosi p lacca culprit m etodiche invasive placca vulnerabile t rattam ento t rattam ento ( ? ) t erapia m edica ottim izzata fig . 
5 algoritmo diagnostico del paziente vulnerabile e possibile ruolo delle metodiche di imaging non invasive tcms . uate the severity of coronary artery disease by quantifying plaque burden without concentrating on the single vulnerable plaque . 
there is a need to reconsider the concept of vulnerable plaque : rather than simply an entity responsible for the acute event , it is the expression of a disease state that involves the entire coronary tree in the clinical setting of a vulnerable patient . 
image - based msct quantification of coronary atherosclerotic disease seems to be an achievable goal , which will require the close collaboration of radiologists and cardiologists in order to optimise the technique and identify its limitations and advantages . conclusioni la ricerca della placca vulnerabile una grande sfida , che presenta notevoli problematiche , in termini di scelta della tecnica ( risoluzione insoddisfacente ) , selezione del paziente ed eventuale trattamento . 
macchi , via borri 57 , i - 21100 varese , italy 2responsabile medico football club internazionale , milano , italy 3clinica ortopedica e traumatologica delluniversit , irccs policlinico s . 
 + 39 - 0332 - 278967 , fax + 39 - 0332 - 278510 , e - mail : eugegeno@tin.it received : 11 november 2006 / accepted : 26 december 2006 / published ondine : 23 july 2007 abstract purpose . 
in all athletes ( nine first - grade lesions , 11 second - grade lesions ) , by using contrast agent intravenous injection done after 20 days , the appearance of contrast spots affecting part or all the lesioned area were observed . 
us with a second - generation contrast agent , thanks to the neoangiogenesis identification , allows recognition , individuation and monitoring the repair processes in the muscle lesion and allows estimation of when athletes can return to competitive activity . 
in tutti gli atleti ( 9 con lesioni primo grado ; 11 di secondo grado ) , dopo iniezione endovenosa di mdc si osservata a 20 giorni la comparsa di spots di contrasto che interessavano tutta o parte dellarea lesionata ; in 8 lesioni di secondo grado permaneva una zona centrale emorragica . 
lecografia con mdc ecografico di ii generazione , attraverso lidentificazione dei vasi neoformati consente di riconoscere , individuare e monitorare i processi riparativi in sede di lesione muscolare e quindi di stabilire i tempi di ripresa dellattivit agonistica dellatleta con ovvia riduzione delle recidive e delle complicanze . parole chiave ecografia mezzo di contrasto di ii generazione atleti professionisti lesioni muscolari introduction introduzione e.a. 
often , us is utilised only to determine the onset of complications , whereas the evaluation of functional recovery has been mainly limited to clinical examinations [ 2 ]  . 
in professional athletes , the appropriate time to return to competitive activity is scheduled to reduce the risk of lesion complications and relapse . the aim of this study was to evaluate the role of second - generation us contrast media ( cm ) in the follow - up of strain injuries in professional athletes , to check muscle repairing processes and to determine when it is appropriate for athletes to return to competitive performance . materials and methods twenty high - performance athletes affected by indirect strains of the medial gastrocnemius ( 3 cases ) , lateral gastrocnemius ( 2 cases ) , soleus ( 2 cases ) , femoral biceps ( 2 cases ) , semimembranous ( 2 cases ) , semitendinous ( 1 case ) , longus adductor ( 2 cases ) and rectus femoral ( 2 cases ) were examined . 
patients were 18 men and two women aged between 18 and 34 ( mean age : 26 years ) ; 11 were football players , seven were practicing athletes and two were volleyball players . 
each patient was studied during the 48 h following the strain with a basal us examination ( esaote technos mpx , esaote biomedica , genoa , italy ) with a high - frequency linear probe ( 413 mhz )  . our study was based on the analysis of nine first - degree and 11 second - degree strains [ 1 ]  . 
follow - up was carried out in three different temporal phases : phase 1 ( 20 days after trauma ) , phase 2 ( 40 days after trauma ) and phase 3 ( 60 days after trauma )  . 
the process consisted of an iv bolus injection of 5 ml of cm ( 8l / ml of sulfur hexafluoride microbubbles ) obtained through the reconstitution of 25 mg of freeze - dried powder with 5 ml of sodium chloride 0.9% weight / volume , followed by a bolus injection of 5 ml of normal saline . 
in all cases , cm was injected in bolus and only images obtained within 2040 s from the start injection of ly images obtained within 2040 s from the start injection of cm were considered in order to identify the arterial phase . we finally evaluated the distribution , site and intensity of the contrast spots . all examinations were evaluated by two operators ( eag , lc ) separately at the same time . 
at the time of each evaluation , a subjective assessment of pain was performed using the visual analogue scale ( vas ) evaluation lecografia nella diagnostica delle lesioni muscolari traumatiche ormai universalmente riconosciuta come metodica di prima istanza [ 18 ] , mentre considerato secondario il suo ruolo nella valutazione dei processi riparativi [ 2 ] ; dalla maggioranza degli autori lecografia stata utilizzata quasi esclusivamente per individuare linsorgenza di complicanze , mentre la valutazione del recupero funzionale stata principalmente delegata al giudizio clinico [ 2 ]  . 
scopo del lavoro quello di studiare le potenzialit del mezzo di contrasto ( mdc ) ultrasonografico di seconda generazione nel follow - up delle lesioni distrattive in atleti professionisti , al fine di monitorare levoluzione dei processi riparativi del muscolo e quindi di individuare i tempi pi opportuni per la ripresa dellattivit agonistica . materiali e metodi sono stati esaminati 20 atleti ad elevata performance con lesioni muscolari distrattive indirette allarto inferiore dei seguenti muscoli : gastrocnemio mediale ( 3 casi ) , gastrocnemio laterale ( 2 casi ) , soleo ( 2 casi ) , bicipite femorale ( 6 casi ) , semimembranoso ( 2 casi ) , semitendinoso ( 1 caso ) , adduttore lungo ( 2 casi ) , retto - femorale ( 2 casi )  . 
rientravano nello studio 18 maschi e 2 femmine di et compresa tra 18 e 34 anni ( et mediana 26 anni ) ; dei quali 11 giocatori di calcio , 7 praticanti atletica leggera , 2 pallavolisti . 
alla diagnosi tutti i pazienti sono stati studiati con ecografia basale entro 48 ore dallevento traumatico mediante apparecchiatura esaote technos mpx ( esaote biomedica , genova , italia ) con sonda lineare ad alta frequenza ( 413 mhz )  . 
 nella serie del nostro studio il grading delle lesioni stato formulato in tutti i casi con il solo esame ecografico basale ed abbiamo considerato 9 lesioni distrattive di primo grado e 11 di secondo grado [ 1 ]  . 
il follow - up stato condotto in 3 diverse fasi temporali rispetto al trauma : fase 1 a 20 giorni , fase 2 a 40 giorni , fase 3 a 60 giorni . 
il controllo comprendeva lo studio in condizioni basali con sonda lineare ad alta frequenza ( 413 mhz ) e dopo iniezione endovenosa ( ev ) di mdc ultrasonografico di seconda generazione ( sonovue , bracco , milano , italia ) con sonda lineare a basso indice meccanico ( 38 mhz ) , previo consenso informato . 
la procedura prevedeva liniezione ev a bolo di 5 ml di dispersione iniettabile di mdc ( 8 l / ml di microbolle di esafluoruro di zolfo ) ottenuta dalla ricostituzione di 25 mg di polvere liofilizzata con 5 ml di cloruro di sodio 0 , 9% peso / volume , seguita dalliniezione a bolo di almeno 5 ml di soluzione fisiologica . 
also in these patients , the vas evaluation ( vas = 0 ) showed the absence of pain , but these athletes were not allowed to resume their competitive activity . discussion muscle - strain lesions are becoming more frequent in sports [ 4 , 9 ] ; in particular , they are highly frequent in professional athletes due to the major work muscles are required to bear [ 5 , 10 ]  . 
us is the main method used to evaluate muscle strains : it is widely employed for diagnosis [ 2 , 4 , 7 , 8 ] but less so during follow - up [ 11 ]  . 
consequently , they also allow neoformed vessels to be located , which indeed are not visible by power - doppler , which can only locate large - diameter vessels [ 12 ]  . 
to better understand the importance of evaluating the microcirculation in the follow - up of muscle lesions , it is necessary to consider the pathological processes taking place after a traumatic event . 
anche in questi casi con il sistema di valutazione ( vas = 0 ) vi era assenza di dolore , ma si ritenuto di non inviare gli atleti alla ripresa dellattivit agonistica . discussione i traumi muscolari sono evenienze sempre pi frequenti nelle attivit sportive [ 4 , 9 ] ; in particolare negli atleti professionisti le lesioni distrattive mostrano elevata incidenza in relazione alla maggiore richiesta funzionale [ 5 , 10 ]  . 
la diagnosi di lesione muscolare prevalentemente clinico - anamnestica ; il ruolo della diagnostica per immagini quello di confermare la sede della lesione e di stabilirne il grading [ 2 ]  . 
lecografia la metodica di prima istanza nella patologia traumatica muscolare , utilizzata ormai di routine in fase diagnostica [ 2 , 4 , 7 , 8 ] e meno frequentemente durante il follow - up [ 11 ]  . 
b same patient : image obtained using a low - mechanical - index probe with contrast - tuned imaging contrast - specific software ( esaote biomedica , genoa , italy )  . 
c dopo iniezione di mdc , omogenea distribuzione degli spots di contrasto nei capillari neoformati ( frecce )  . microbolle contenenti gas che , introdotte per via endovenosa , esaltano gli echi del torrente circolatorio e consentono quindi di individuare le strutture vascolari compresi i vasi di piccolo calibro , fino al letto capillare e perci anche i vasi neoformati non visibili con metodiche power doppler , anche se particolarmente sensibili e di ultima generazione , in grado di evidenziare unicamente vasi di calibro superiore [ 12 ]  . 
per comprendere quanto sia importante riconoscere il microcircolo nel follow - up delle lesioni muscolari necessario fare un cenno ai processi anatomo - patologici che si succedono dopo levento traumatico . 
immediatamente dopo il danno muscolare viene indotto un processo infiammatorio con vasodilatazione ed edema , da alterazione della permeabilit vascolare , associato a sanguinamento pi o meno abbondante a seconda della sede e del grado della lesione . successivamente si assiste ad organizzazione dellematoma con formazione del coagulo che viene progressivamente colonizzato da parte del tessuto di granulazione ( vasi neoformati e cellule ) , e alla riduzione volumetrica della lesione . 
this process is followed by haematoma organisation and subsequent clot , which is progressively invaded by granulation tissue ( neoformed vessels and cells ) , followed by the release of substances from blood cells and the removal of damaged muscle cells . 
2a - i phase 1 : a second - degree femoral biceps lesion : in b - mode , the lesion appears as an inhomogeneous solid area ( empty white arrows )  . 
b same patient : image obtained using a low - mechanical - index probe with contrasttuned imaging ( cnti ) contrast - specific software ( esaote biomedica , genoa , italy )  . 
c after contrast - medium ( cm ) injection , an intense but inhomogeneous enhancement with peripheral spots ( arrows ) can be observed , which defines the residual organised haematoma ( asterisk )  . 
phase 2 : d second - degree femoral biceps lesion : in b - mode , the lesion appears as a solid , inhomogeneous , small - sized area ( empty white arrows )  . 
f after cm injection , an inhomogeneous enhancement with peripheral spots of minor intensity ( arrows ) and without haemorrhagic residues can be observed . phase 3 : g femoral biceps second - degree lesion : in b - mode , the solid , inhomogeneous area is even more reduced and almost not detectable . 
2a - i fase 1 : a lesione di ii grado del bicipite femorale , esame b - mode , area ipoecogena disomogenea in sede di lesione ( frecce bianche vuote )  . 
fase 2 : d lesione di ii grado bicipite femorale , esame b - mode , area solida disomogenea di dimensioni ridotte in sede di lesione ( frecce bianche vuote ) , e stesso paziente , immagine ottenuta con sonda a basso indice meccanico con modulo contrasto ( cnti ) attivato ( esaote biomedica , genova , italia )  . 
c after contrast - medium ( cm ) injection , the fluid collection is inhomogeneously surrounded by contrast spots , which indicate neoformed capillaries ( arrows ) ; in fact an oval area is present , which is not refillable , and is indicative of an organised haematoma ( asterisks )  . 
e same patient : image obtained using a low - mechanical - index probe with contrast - tuned imaging ( cnti ) contrast - specific software ( esaote biomedica , genoa , italy )  . 
f after cm injection , the fluid collection is inhomogeneously surrounded by contrast spots , which indicate neoformed capillaries ( arrows ) ; a residual oval collection is present , which is not refillable and is indicative of an organised haematoma smaller in size ( asterisks )  . 
phase 3 : g medial gastrocnemius second - degree lesion : in b - mode , the fluid collection is almost reabsorbed ; an echoic solid area at the lesion point is still present ( asterisks )  . 
i after cm injection , a rich and inhomogeneous contrast spot distribution can be observed , indicating the presence of neoformed capillaries and that the scar is still not stable . 
3a - i fase 1 : a lesione di ii grado del gastrocnemio mediale , esame b - mode , residua ampia raccolta fluida ( asterischi )  . , b stesso paziente , immagine ottenuta con sonda a basso indice meccanico con modulo contrasto ( cnti ) attivato ( esaote biomedica , genova , italia )  . 
c dopo iniezione di mdc , la raccolta fluida disomogeneamente circondata da spots di contrasto che indicano la presenza di capillari neoformati ( frecce ) , infatti presente area ovalare che non si riempie riferibile a ematoma organizzato ( asterischi )  . 
f dopo iniezione di mdc , la raccolta fluida disomogeneamente circondata da spots di contrasto che indicano la presenza di capillari neoformati ( frecce ) , residua raccolta ovalare che non si riempie riferibile a ematoma organizzato di dimensioni nettamente ridotte ( asterischi )  . 
fase 3 : g lesione di ii grado del gemello mediale , esame b - mode , riassorbita la raccolta fluida , permane area iso / ipoecogena solida in sede di lesione ( asterischi )  . 
i dopo iniezione di mdc , omogenea e ricca distribuzione degli spots di contrasto ( frecce ) che indicano la persistenza di capillari neoformati , segno di mancata stabilizzazione della cicatrice . fico di ii generazione nel follow - up delle lesioni muscolari ha consentito di correlare i reperti ecografici ai quadri anatomo - patologici , attraverso lidentificazione della neo - angiogenesi tipica del tessuto di granulazione ed ha documentato levoluzione dei processi riparativi , attraverso le modie.a. 
in our study , cm allowed us to correlate us findings with pathological patterns and to document the evolution of the repair processes by demonstrating changes in the haemorrhagic area and its progressive replacement by solid granulation tissue , rich in neoformed microvessels . at the first evaluation after 20 days ( phase 1 ) , unenhanced us showed the presence of solid echoic tissue in all patients . the tissue was inhomogeneous in the area of the lesion , with different degrees of reabsorption of the fluid collection described earlier . 
data from the literature suggest that when solid reparative tissue appears in the area of the lesion and replaces the haemorrhagic content , athletes may resume moderate sports activity [ 14 ]  . 
it is actually not possible to evaluate this phase using us only , because it is not easy to detect the muscle tissues initial organisation around the lesion , and us alone does not have the ability to distinguish a clot from granulation tissue [ 14 ]  . 
therefore , it has been reported that the appropriate time for athletes to resume their sports activity should be determined on the basis of the results of the clinical examination or when symptoms disappear . 
in general , a period of 46 weeks should elapse between the trauma and resumption of activity [ 3 ]  . in our study , after 20 days ( phase 1 ) , the use of cm enabled us to differentiate between the peripheral neoangiogenesis , which is the reparative granulation tissue , and the central solid tissue , which is the organised haematoma without neoangiogenesis . 
in some cases , a thin intermediate band with rare contrast spots was detected between vascularised and nonvascularised tissue ; we considered this band as an initial invasion of reparative tissue from the periphery to the organised haematoma . 
at the first examination after 20 days , cm did not change the therapeutic management , even if it allowed detection of angiogenesis , in the granulation tissue , which is indicative of the repair process . 
in fact , different quantities of cm spots in different areas of the lesion were still present , even though most cases showed a reduction of us signal intensity , and the presence of this pattern was considered a contraindication for the athletes to resume competitive activity . 
at this time , the subjective evaluation was not completely negative , and all \athletes referred to moderate discomfort at the lesion site . muscle - lesion recovery coincides with reabsorption of the neoformed capillary vessels . 
indeed , in 17 / 20 patients examined , we observed the progressive reduction of neoformed vessels until total disappearance of vascular spots 60 days after the trauma ( phase 3 ) ; moreover the 3 / 20 patients with residual vascular spots presented a negative clinical outcome ( vas = 0 )  . 
secondo la letteratura la ripresa di una limitata attivit sportiva non sarebbe controindicata nei pazienti in cui larea di lesione sostituita da tessuto riparativo , quindi nei casi in cui non sia ecograficamente pi rilevabile la componente emorragica in sede di lesione [ 14 ]  . 
in realt determinare questa fase con lecografia tradizionale quasi impossibile , risulta particolarmente complicato evidenziare liniziale organizzazione di tessuto muscolare sano ai bordi della lesione , mentre non esiste semeiotica ecografica che consenta di distinguere un coagulo dal tessuto di granulazione [ 14 ]  . 
pertanto , sulla base di tali considerazioni , attualmente gli autori delegano la scelta della ripresa dellattivit sportiva allesame clinico , considerando come fattore positivo la scomparsa della sintomatologia ; pi in generale viene individuato un periodo non minore di 46 settimane dallevento traumatico [ 3 ]  . seguendo il nostro protocollo , nel follow - up a 20 giorni ( fase 1 ) , liniezione ev di mdc ultrasonografico ha permesso di distinguere una componente , solitamente periferica , dotata di importante microvascolarizzazione ( angiogenesi ) corrispondente al tessuto di granulazione di significato riparativo , dalla componente solida , centrale , riferibile a residuo ematoma organizzato ( assenza di angiogenesi )  . 
in alcuni casi si evidenziato inoltre , tra tessuto vascolarizzato e tessuto non vascolarizzato , una sottile fascia intermedia in cui il segnale del mezzo di contrasto appariva sporadico e che abbiamo interpretato quale segno di iniziale colonizzazione del tessuto riparativo dalla periferia verso lematoma organizzato . 
al primo controllo ecografico a 20 giorni , anche lutilizzo di mdc non modifica in modo significativo il comportamento terapeutico pur consentendo di individuare i fenomeni di angiogenesi che caratterizzano il tessuto di granulazione , sempre presente nei processi riparativi . 
il significato prognostico del mdc si appalesa nei controlli a 40 giorni ( fase 2 ) : infatti in tutti i pazienti residuavano spots diffusi o periferici di mdc , seppur nella maggior parte con riduzione dellintensit del segnale ultrasonografico ; tale comportamento contrastografico ha controindicato il ritorno allattivit agonistica . la guarigione di una lesione muscolare coincide con il riassorbimento dei capillari neoformati ; nella nostra serie di pazienti abbiamo assistito alla progressiva riduzione dei vasi neoformati nel tempo fino alla pressoch completa scomparsa degli spots vascolari in 17 / 20 pazienti a 60 giorni dalla lesione ( fase 3 ) , peraltro nei 3 pazienti nei quali erano presenti residui spots vascolari la clinica era negativa . 
alla luce di quanto osservato possiamo considerare il mdc ecografico un valido indicatore della attivit tissutale con ovvia ricaduta clinica ; infatti in presenza di residua vascolarizzazione dellarea di lesione da parte di capillari neoformati , sconsigliabile la ripresa dellattivit agonistica , poich il processo di guarigione non si pu ritenere completamente concluso . nel follow - up con ecografia senza mdc delle lesioni muscolari il riempimento da parte di tessuto solido , disomogee.a. 
in highdegree lesions or in those not correctly treated , however , the process can turn into scar tissue , whose entity is proportional to the initial seriousness of the lesion [ 1 , 5 ] , to the length of recovery and to the precociousness of the resumption of sports activity . 
in these situations , the us pattern indicates complete repair of muscle injury , and it could indicate that athletes can resume sports activity . nevertheless , in contrast - enhanced examinations carried out in patients who were diagnosed with complete lesion repair by unenhanced us examinations , we still observed the presence of many small neoformed vessels , which clearly indicated the presence of active reparative tissue . 
il processo riparativo ecogeno nelle lesioni meno estese pu completarsi con la restitutio ad integrum del muscolo lesionato o pu , soprattutto nelle lesioni di grado elevato o non adeguatamente trattate , trasformarsi in tessuto fibrocicatriziale la cui entit proporzionale alla gravit della lesione iniziale [ 1 , 5 ] , alla lunghezza del processo di guarigione , e alla precocit del ritorno allattivit sportiva . 
 in entrambe le situazioni tale pattern ecografico espressione della avvenuta riparazione del danno muscolare e indicherebbe la possibilit di ritorno alla attivit sportiva . tuttavia anche nei casi di avvenuta riparazione ecografica della lesione si osservato durante lindagine con mdc tessuto ricco di piccoli vasi neoformati che indicavano inequivocabilmente la presenza di tessuto riparativo in fase di attivit ; noi riteniamo quindi che la presenza di angiogenesi , seppur residua , sia espressione di una lesione riparata , ma non stabile . 
facilmente intuibile perci come , soprattutto negli atleti i cui muscoli sono sottoposti ad un significativo sovraccarico funzionale , tale rilievo rivesta notevole importanza prognostica , in quanto un muscolo ancora sede di fenomeni riparativi , se sottoposto a carichi massimali , presenta un elevato rischio di recidiva di lesione . 
we believe that this method , especially when used for professional athletes , is the only one that can provide the physician with the correct information regarding lesion staging in order to determine with greater accuracy the appropriate time for functional recovery of sports activity to reduce relapses and complications . in conclusione , possiamo affermare che il mdc ecografico di ii generazione consente di riconoscere i processi di angiogenesi tipici di tutti i processi riparativi , tale caratteristica pu e deve essere sfruttata per ottenere informazioni fino ad oggi non cos dettagliate sulla evoluzione dei processi riparativi ed il grado di guarigione del muscolo lesionato . 
forty - one patients ( mean age 30 years ) affected by chondral defects of the knee ( 27 patients ) and ankle joint ( 14 patients ) who underwent arthroscopic autologous osteochondral grafting were studied 6 months and 1 year postoperatively with mri . 
cartilage repair after chondrocyte implantation was studied by assessing the degree of defect filling , graft integration , graft signal intensity , integrity of the subchondral lamina and trabecular oedema underneath the gramr findings were correlated with clinical data . 
postoperative mri evaluation at 6 months demonstrated complete filling of the osteochondral defect in 12 / 41 cases , complete integration in 18 / 41 , mild hyperintensity in 28 / 41 , intact subchondral lamina in 38 / 41 and trabecular oedema in 11 / 41 . postoperative mri evaluation at 1 year demonstrated complete filling of the osteochondral defect in 9 / 41 patients , complete integration in 22 / 41 , mild hyperintensity in 23 / 41 , intact subchondral lamina in 36 / 41 and trabecular oedema in 8 / 41 . 
sono state esaminate con rm a sei mesi ed un anno dallintervento , 41 lesioni cartilaginee del ginocchio ( 27 ) e tibio - tarsiche ( 14 ) , trattate per via artroscopica con impianto di condrociti autologhi su supporto tridimensionale di acido ialuronico . 
levoluzione dellimpianto stata studiata analizzando il grado di colmatura del difetto osteocondrale , il grado di integrazione , lintensit di segnale della cartilagine trapiantata , lintegrit della lamina corticale subcondrale e ledema trabecolare . 
a sei mesi : completo grado di colmatura in 12 / 41 pazienti , integrazione completa dellimpianto in 18 / 41 , modesta iperintensit del segnale in 28 / 41 , lamina subcondrale intatta in 38 / 41 , edema trabecolare in 11 / 41 . 
ad un anno : completo grado di colmatura in 9 / 41 pazienti , integrazione completa dellimpianto in 22 / 41 , modesta iperintensit del segnale in 23 / 41 , lamina subcondrale intatta in 36 / 41 , edema trabecolare in 8 / 41 . 
la valutazione rm presenta elementi prognostici in termini di buon attecchimento dellimpianto . parole chiave risonanza magnetica trapianto di condrociti lesioni cartilaginee introduction introduzione articular cartilage injuries are one of the most common injuries seen in orthopaedic practice [ 1 ]  . 
treatment of such injuries is difficult , as articular cartilage has a limited capacity le lesioni della cartilagine articolare sono le lesioni di pi frequente riscontro in ortopedia [ 1 ]  . 
the most frequently used surgical procedures aim to promote the growth of repair tissue at the level of the chondral defect by means of chondroplasty or chondroabrasion , associated , if necessary , with subchondral perforation or microfracture [ 3 ]  . 
fibrocartilaginous repair tissue has different biochemical and bioelastic properties from that of hyaline cartilage , and the result achieved with these techniques is merely to delay the onset of degenerative osteoarthritis [ 4 ]  . autologous chondrocyte implantation ( aci ) is the first technique that aims to restore the articular surface with hyaline - like cartilage . 
the original technique involved applying to the injury cultures of autologous chondrocytes arthroscopically harvested from non - weight - bearing articular areas ; adhesion to the defect is ensured by a periosteal flap also harvested from the patient [ 5 ]  . 
among the various biomaterials , two are the most commonly used : hyalograft c , which employs hyaluronic - acid scaffolds , and maci ( matrix - induced aci ) , which employs type 1 and type 3 collagen scaffolds [ 8 ]  . 
follow - up after aci can be performed by using several diagnostic procedures , such as clinical assessment , direct visualisation of the graft with arthroscopy with the possibility of obtaining biopsies or indirect visualisation with magnetic resonance imaging ( mri ) [ 9 ]  . 
all subjects were treated with aci with a 3d hyaluronic - acid scaffold ( hyalograft c , fidia advanced biopolymers srl , abano terme , italy ) and underwent clinical assessment by an orthopaedic surgeon and mri evaluation by a musculoskeletal radiologist . the mri studies were performed at 6 and 12 months postoperatively using a 1.5 - tesla ( t ) unit ( symphony , siemens , erlangen , germany ) and the following sequences : sagittal t1 spin echo ( tr : 463 ms ; te : 17 ms ; flip angle : 150 ; fov : 180 ms ; matrix : 240320 ; slice thickness : 4 mm ; number of acquisitions : 2 ) ; sagittal dual fast spin echo ( fse ) ( tr : 600 ms ; te : 18 ms ; flip angle : 20 ; fov : 150 ms ; matrix : 192256 ; slice thickness : 4 mm ; number of acquisitions : 2 ) and coronal and sagittal flash t1 fat - sat ( tr : 500 ms ; te : 8 ms ; flip angle : 65 ; fov : 200 ms ; matrix : 256256 ; slice thickness : 4 mm ; number of acquisitions : 2 )  . each mri study was assessed for : the degree of filling of the osteochondral defect ; the degree of graft integration ; signal intensity ; the integrity of the subchondral lamina ; and trabecular oedema . 
i trattamenti chirurgici pi utilizzati mirano alla crescita di tessuto riparativo a livello del difetto condrale mediante tecniche di condroplastica / condroabrasione , associate o meno a perforazioni subcondrali o microfratture [ 3 ]  . 
il tessuto fibro - cartilagineo di riparazione presenta caratteristiche biochimiche e bioelastiche differenti dalla cartilagine ialina ed il risultato che si riesce ad ottenere con queste metodiche soltanto un ritardo nel progressivo sviluppo del processo artrosico [ 4 ]  . limpianto di condrociti autologhi ( aci ) rappresenta la prima tecnica che si propone di ripristinare la superficie articolare con tessuto cartilagineo di tipo ialino . 
la tecnica originale prevede di applicare sulla lesione colture di condrociti autologhi prelevati per via artroscopica da zone articolari non soggette a carico ; ladesione alla zona innestata viene assicurata da un lembo periostale prelevato dal paziente [ 5 ]  . 
tra i vari biomateriali due sono quelli pi utilizzati : hyalograft c e maci ( matrix - induced autologous chondrocytes implantation ) , nel primo caso i supporti sono costituiti da acido ialuronico ( hyalograft c ) , nel secondo da collagene di tipo i e iii ( maci ) [ 8 ]  . 
il follow - up dei trapianti di condrociti autologhi pu essere effettuato mediante diversi procedimenti diagnostici , quali la valutazione clinica , la visualizzazione diretta dellarea sottoposta a trapianto mediante artroscopia , con possibilit di effettuare biopsie , o la visualizzazione indiretta mediante risonanza magnetica ( rm ) [ 9 ]  . scopo del lavoro quello di valutare , mediante rm , levoluzione dellimpianto di condrociti autologhi su supporto tridimensionale di acido ialuronico e correlare tale evoluzione con landamento clinico . materiali e metodi dal 2002 al 2005 sono stati esaminati , consecutivamente , 41 pazienti ( 30 maschi e 11 femmine ) di et compresa fra i 17 anni ed i 50 anni ( et media 35 , 2 anni , range 1750 ) ; di questi , 27 erano affetti da una lesione a carico della cartilagine dellarticolazione del ginocchio , tutte interessanti il compartimento femoro - tibiale , e 14 da una lesione a carico della cartilagine dellarticolazione tibio - tarsica . 
graft integration was considered complete when the graft showed continuity with the adjacent native cartilage and incomplete when a linear fissure - like lesion or a larger defect could be seen . 
signal intensity of the graft was considered isointense if the graft had the same signal intensity as the adjacent cartilage and hyperintense or hypointense if its signal intensity was higher or lower . 
 each variable was given a score : degree of filling ( score from 0 to 1 : 0 = complete , 1 = incomplete ) , graft integration ( score from 0 to 1 : 0 = complete , 1 = incomplete ) , signal intensity ( score from 0 to 3 : 0 = isointense , 1 = moderately hyperintense , 2 = markedly hyperintense , 3 = hypointense ) , subchondral lamina ( score from 0 to 1 : 0 = intact , 1 = not intact ) and oedema ( score from 0 to 1 : 0 = absent , 1 = present )  . 
the best score , indicative of optimal graft quality according to the literature [ 9 ] is 0 , whereas the worst score is 7 ( 0 = excellent integration , 12 = good integration , 35 = fair integration , 67 = poor integration )  . 
of the 14 patients with injuries of the ankle - joint cartilage , 5 / 14 ( 35.7% ) had excellent results , 7 / 14 ( 50% ) had good results and only 2 / 14 ( 14.3% ) had fair results . 
il grado di colmatura stato valuto in base allo spessore del tessuto trapiantato : stato considerato completo quando la cartilagine trapiantata aveva lo stesso spessore della cartilagine nativa , incompleto quando il tessuto trapiantato era pi sottile rispetto a quello della cartilagine nativa , mentre stato definito ipertrofico quando lo spessore dellimpianto era superiore rispetto a quello della cartilagine nativa . 
lintegrazione stata considerata completa quando limpianto non presentava soluzioni di continuo con la cartilagine adiacente , incompleta quando era visibile o una lesione lineare simile a una fessura oppure un difetto pi ampio . 
per quanto riguarda lintensit del segnale , limpianto stato considerato isointenso se appariva della stessa intensit della cartilagine adiacente , iperintenso o ipointenso se presentava , rispettivamente , unintensit maggiore o minore rispetto alla cartilagine nativa . 
infine stata valutata la presenza o lassenza di edema a livello dellosso subcondrale adiacente alla cartilagine trapiantata . ad ogni parametro esaminato stato assegnato un punteggio predefinito : grado di colmatura ( score da 0 a 1 : 0 = colmatura completa , 1 = colmatura incompleta ) , grado di integrazione ( score da 0 a 1 : 0 = integrazione completa , 1 = integrazione incompleta ) , intensit ( score da 0 a 3 : 0 = isointensit , 1 = moderata iperintensit , 2 = marcata iperintensit , 3 = ipointensit ) , integrit della lamina ( score da 0 a 1 : 0 = lamina intatta , 1 = lamina non intatta ) , edema ( score da 0 a 1 : 0 = assenza di edema , 1 = presenza di edema )  . 
grado di colmatura completo ( a ) : lo spessore dellinnesto uguale allo spessore della cartilagine nativa ; grado di colmatura incompleto ( b ) : lo spessore dellinnesto inferiore allo spessore della cartilagine adiacente . table 1 osteochondral defect filling and graft integration at 6 and 12 months after autologous chondrocyte implantation defect filling complete incomplete hypertrophic graft integration complete incomplete 6 months 12 months 6 mesi 12 mesi tabella 1 grado di colmatura e grado di integrazione a 6 mesi ed a 12 mesi dopo trapianto di condrociti autologhi grado di colmatura completo incompleto ipertrofico grado di integrazione completo incompleto one - year follow - up defect filling was complete in 9 / 41 patients ( 21.9% ) , incomplete in 30 / 41 ( 73.2% ) and hypertrophic in 2 / 41 ( 4.9% ) ( table 1 )  . 
la cartilagine trapiantata risultata essere isointensa in 3 / 41 pazienti ( 7 , 3% ) , moderatamente iperintensa in 28 / 41 pazienti ( 68 , 3% ) , marcatamente iperintensa in 7 / 41 pazienti ( 17 , 1% ) , ipointensa in 3 / 41 pazienti ( 7 , 3% )  . 
una sua lesione espressione di un esito sfavorevole del trapianto . table 2 subchondral lamina at 6 and 12 months after autologous chondrocyte implantation subchondral lamina intact not intact 6 months 12 months 6 mesi 12 mesi tabella 2 caratteristiche della lamina subcondrale a 6 mesi ed a 12 mesi dopo trapianto di condrociti autologhi lamina subcondrale intatta non intatta follow - up a 1 anno tients ( 63% ) with injuries of the knee cartilage had a total score between 95 and 100 on the tegner and lysholm activity scale , 8 / 27 ( 29.6% ) had scores between 84 and 94 and 2 / 27 ( 7.4% ) had scores below 84 . 
of the 14 patients with injuries to ankle - joint cartilage , 10 / 14 ( 71.4% ) had excellent results , 3 / 14 ( 21.4% ) had good results and only 1 / 14 ( 7.2% ) had fair results . 
 the number of patients with poor clinical functional outcome at 1 year was 14 / 41 ( 34.1% ) , eight of whom belonged to the group with knee - cartilage defects and six to the group with ankle cartilage defects . 
graft integration was incomplete in 10 / 14 ( 71.4% ) and complete in tilagine dellarticolazione di ginocchio 8 / 27 pazienti ( 29 , 6% ) hanno ottenuto un punteggio totale tra 95100 ( funzionalit normale ) del protocollo di tegner lysholm , 15 / 27 pazienti ( 55 , 6% ) un punteggio tra 84 e 94 ( sintomatologia solo nelle attivit pesanti ) ed infine 4 / 27 ( 14 , 8% ) un punteggio sotto gli 84 punti ( sintomi nella quotidianit )  . 
dei 14 pazienti con lesioni della cartilagine dellarticolazione tibiotarsica , 5 / 14 ( 35 , 7% ) hanno ottenuto una valutazione eccellente , 7 / 14 ( 50% ) una valutazione buona e solo 2 / 14 ( 14 , 3% ) una valutazione sufficiente . il grado di colmatura risultato completo in 9 / 41 pazienti ( 21 , 9% ) , incompleto in 30 / 41 pazienti ( 73 , 2% ) , ipertrofico in 2 / 41 pazienti ( 4 , 9% ) ( tabella 1 )  . 
il grado di integrazione risultato completo in 22 / 41 pazienti ( 53 , 7% ) , incompleto in 19 / 41 pazienti ( 46 , 3% ) ( tabella 1 )  . 
la lamina subcondrale risultata intatta in 36 / 41 pazienti ( 87 , 8% ) , non intatta in 5 / 41 pazienti ( 12 , 2% ) ( tabella 2 )  . 
la cartilagine trapiantata risultata essere isointensa in 8 / 41 pazienti ( 19 , 5% ) , moderatamente iperintensa in 23 / 41 pazienti ( 56 , 1% ) , marcatamente iperintensa in 7 / 41 pazienti ( 17 , 1% ) , ipointensa in 3 / 41 pazienti ( 7 , 3% )  . 
dei 14 pazienti con lesioni della cartilagine dellarticolazione tibio - tarsica , 10 / 14 ( 71 , 4% ) hanno ottenuto una valutazione eccellente , 3 / 14 ( 21 , 4% ) una valutazione buona e solo 1 / 14 ( 7 , 2% ) una valutazione sufficiente . i casi ad andamento clinico - funzionale peggiore a distanza di 1 anno sono risultati 14 / 41 ( 34 , 1% ) , di cui 8 casi appartenevano al gruppo con lesioni a carico dellarticolazione del ginocchio e 6 al gruppo con lesioni a carico dellarticolazione tibio - tarsica ; in questi il grado di colmatura risultato incompleto in 12 / 14 casi ( 86% ) , completo in 2 / 14 ( 14% ) ; lintegrazione risultata incompleta in 10 / 14 ( 71 , 4% ) , completa in 4 / 14 ( 28 , 6% ) ; limpianto risultato iperintenso in 10 / 14 ( 71 , 4% ) , isointenso in 1 / 14 ( 7 , 2% ) , ipointenso in 3 / 14 ( 21 , 4% ) ; la lamina subcondrale risultata non intatta in 9 / 14 casi ( 64 , 3% ) , intatta in 5 / 14 ( 35 , 7% ) ; ledema subcondrale risultato assente in 10 / 14 ( 71 , 4% ) , presente in 4 / 14 ( 28 , 6% )  . la rm utilizzata di routine nello studio delle lesioni della cartilagine articolare , sia in fase di diagnosi , sia per il successivo monitoraggio delle lesioni dopo il trattamento chirurgico ; in particolare la rm indicata nel valutare se la cartilagine trapiantata presenti caratteristiche del tutto sovrapponibili alla normale cartilagine articolare di tipo ialino [ 1215 ]  . nello studio della cartilagine articolare si utilizzano sequenze specifiche , che determinano un adeguato contrasto tra la cartilagine e le strutture adiacenti . 
le sequenze pi frequentemente utilizzate nel follow - up dei trapianti di condrociti autologhi sono rappresentate da sequenze fast spin echo t2 - dipendenti ( fse ) e sequenze gradient echo ( gre ) con soppressione del grasso e ricostruzioni in tre dimensioni [ 1620 ]  . 
le sequenze t2 - dipendenti , invece , determinano un basso segnale della cartilagine articolare contrapposto ad un alto segnale del liquido sinoviale , e permettono unaccurata valutazione della struttura della cartilagine articolare , sia essa normale o danneggiata [ 16 , 19 , 21 , 22 ]  . 
inoltre le sequenze fse t2 - dipendenti non sono alterate da eventuali artefatti magnetici , che si possono creare in pazienti sottoposti precedentemente ad interventi chirurgici a livello dellarticolazione in esame . con il miglioramento delle tecniche di imaging si sono potuti definire i parametri da analizzare per ogni esame espletato , in modo tale che la valutazione del tessuto di riparazione sia la pi oggettiva possibile . 
6 clinical outcome at 6 and 12 months after autologous chondrocyte implantation in the group with lesions of the ankle joint evaluated by the american orthopaedic food and ankle society scale ( aofas )  . 
6 andamento clinico a 6 mesi e a 12 mesi dei trapianti di condrociti autologhi nel sottogruppo con lesioni a carico dellarticolazione tibio - tarsica valutato secondo il protocollo aofas . articular cartilage is evaluated by using specific sequences that produce adequate contrast between the cartilage and the surrounding structures . 
the most frequently used in the follow - up of aci are t2 - weighted fse and gradient echo ( gre ) sequences with fat suppression and 3d reconstructions [ 1620 ]  . 
gre sequences are characterised by a relatively high signal intensity of cartilage in contrast with a low signal intensity of the surrounding tissues ; 3d gre sequences enable precise determination of cartilage thickness and surface . t2 - weighted sequences , on the other hand , generate a low signal intensity of articular cartilage in contrast to a high signal intensity of synovial fluid and allow accurate evaluation of the structure of both healthy of damaged articular cartilage [ 16 , 19 , 21 , 22 ]  . 
 improvements in imaging techniques have made it possible to establish which parameters should be assessed in each examination so as to make evaluation of repair tissue as obpianto ed infine lo stato della lamina e dellosso subcondrale [ 23 ]  . 
il trapianto si considera infatti riuscito qualora il tessuto appaia dello stesso spessore della cartilagine adiacente e la superficie sia liscia [ 16 ] , i margini non mostrino soluzioni di continuo con la cartilagine nativa , losso subcondrale a livello dellarea sottoposta a trapianto sia anchesso liscio , il midollo osseo abbia la stessa intensit di segnale del normale midollo osseo e il tessuto trapiantato risulti isointenso rispetto alla cartilagine non sottoposta a trapianto [ 16 , 17 ]  . lassegnazione di un punteggio prestabilito ad ogni parametro analizzato rappresenta un ulteriore criterio di oggettivit nella valutazione della riuscita del trapianto di condrociti autologhi . 
scale di valutazione simili sono gi proposte in alcuni lavori presenti in letteratura , come il lavoro di handerson del 2003 o nel lavoro , ancora pi recente di tratting del 2005 [ 9 , 24 ]  . nel nostro studio si sono voluti correlare i parametri rm accettati in letteratura [ 22 , 24 ] come indicativi di attecchif . 
mr images allow an accurate assessment of graft thickness , graft integration with the native cartilage , graft signal intensity and the state of the subchondral lamina and bone [ 23 ]  . 
graft implantation is considered successful when the repair tissue has the same thickness as the adjacent cartilage and has a smooth surface [ 16 ] , the edges of the graft are in continuity with the native cartilage , the subchondral bone beneath the graft is also smooth , bone marrow has the same signal intensity as normal marrow and the graft appears isointense relative to the native cartilage [ 16 , 17 ]  . use of a preestablished scoring system in the analysis of each parameter makes assessing the success of an aci procedure more objective . 
in 2005 [ 9 , 24 ]  . in our study , we attempted to correlate the established mri parameters [ 22 , 24 ] for assessment of successful graft incorporation with the clinical data to evaluate whether there is a true clinical - mri correlation and to identify the most significant parameters for the follow - up . 
defect filling and graft integration are the two parameters that reflect repair tissue growth at the defect site and its good incorporation ; if incomplete , they indicate poor graft outcome , which translates into worse clinical outcomes . 
this is confirmed by three patients in our study ( table 1 ) who had a good clinical outcome at 6 months but a worsening of symptoms at 12 months , at which time mri showed incomplete defect filling or , in one case , graft hypertrophy . 
a similar situation occurred in trattnig et al.s study [ 24 ] : in two out of 20 cases , the degree of osteochrondral defect filling worsened , and this was considered a poor prognostic sign . 
a change was seen in this parameter at 12 months with respect to the previous mri study ( table 2 ) : the subchondral lamina was not intact in five cases as against the three cases seen at 6 months . 
however , moderate signal hyperintensity in the early postoperative period does not preclude the possibility that the signal will return to normal , as was seen in five cases in our series . subchondral bone oedema was absent in 71.4% of cases with poor clinical - functional outcome . 
as has been previmento ben riuscito con i dati clinici , per valutare se esista realmente una correlazione clinico - rm e per identificare i parametri pi significativi nel follow - up . 
il grado di colmatura e il grado di integrazione sono due parametri che riflettono la crescita del tessuto di riparazione nel sito di impianto ed il suo buon attecchimento ; quando risultano essere incompleti , sono indicativi di un esito sfavorevole del trapianto che si traduce in un peggiore andamento clinico . 
a conferma di ci 3 casi del nostro studio ( tabella 1 ) , caratterizzati da buon andamento clinico ai controlli a 6 mesi , hanno invece presentato ai controlli a 12 mesi un peggioramento della sintomatologia e allesame rm si riscontrato un grado di colmatura incompleto o in un caso ipertrofia della cartilagine trapiantata . 
 [ 24 ] presente una situazione analoga : in 2 casi su 20 esaminati si riscontrato un peggioramento nel grado di colmatura del difetto osteocondrale ; tale peggioramento stato considerato un segno prognostico negativo per la riuscita del trapianto . 
nello stesso studio , in 3 pazienti si assistito ad un miglioramento del grado di integrazione da incompleto a completo con conseguente buona riuscita del trapianto effettuato . nel 64 , 3% dei casi ad andamento clinico - funzionale peggiore la lamina subcondrale risultata non intatta . 
la lamina subcondrale di norma non viene intaccata durante gli interventi di impianti di condrociti autologhi e quindi dovrebbe apparire intatta allo studio rm , come risultato nella maggior parte dei casi nel nostro studio . 
si assistito , inoltre , ad una modificazione di tale parametro nei controlli a 12 mesi rispetto ai controlli precedenti ( tabella 2 ) : in 5 casi , infatti , la lamina subcondrale risultata non intatta contro i 3 casi rilevati ai controlli a 6 mesi . 
quindi anche tale parametro pu eventualmente essere espressione di un esito sfavorevole del trapianto . nel 71 , 4% dei casi ad andamento clinico - funzionale peggiore la cartilagine trapiantata risultata essere iperintensa rispetto alla cartilagine nativa . 
a nostro avviso lintensit del segnale manifestazione del grado di maturit del tessuto trapiantato , in particolare se messo in relazione con la cartilagine adiacente , rispetto alla quale dovrebbe risultare isointensa . 
tuttavia una moderata iperintensit di segnale nelle fasi iniziali dopo lintervento non preclude una normalizzazione del segnale , come si potuto constatare in 5 casi della nostra casistica . ledema a livello dellosso subcondrale , infine , risultato assente nel 71 , 4% dei casi di questo studio ad andamento clinico - funzionale peggiore . 
come gi descritto in letteratura [ 12 , 23 ] , la presenza di edema anchessa la manifestazione del normale processo di maturazione della cartilagine sottoposta a trapianto , sinonimo del rimodellamento e f . 
this would appear to be confirmed by the five patients ( table 3 ) who had no oedema and a positive clinical evaluation at 6 months but had subchondral oedema and a clinical evaluation that was either negative ( three cases ) or positive ( two cases ) for vigourous activities only at 12 months . 
il rilievo sembrerebbe dimostrato dai cinque casi ( tabella 3 ) che a 6 mesi erano caratterizzati da assenza di edema e concomitante clinica positiva , mentre ai controlli a 12 mesi si presentavano con edema a livello dellosso subcondrale e clinica negativa in 3 casi o positiva solo per le attivit pi pesanti nei rimanenti 2 casi . 
lassenza di edema quindi indice di una carenza nel processo di maturazione , che determina un peggior andamento clinico . conclusions mri proved to be fundamental in the follow - up of aci procedures , as it enables accurate and noninvasive evaluation of the state of the cartilage grathe parameters degree of defect filling , degree of graft integration and graft signal intensity allow the repair tissue maturation process to be monitored until it displays the typical characteristics of hyalinelike articular cartilage . 
detection of the findings absence of oedema and nonintact subchondral lamina during the early follow - up period has a negative prognostic value . conclusioni la rm si dimostrata fondamentale nel follow - up dei trapianti di condrociti autologhi , in quanto consente unaccurata e non invasiva valutazione dello stato della cartilagine trapiantata . 
i parametri grado di colmatura e di integrazione ed intensit della cartilagine trapiantata permettono di monitorare il processo di maturazione del tessuto trapiantato , fino allacquisizione da parte di questultimo delle caratteristiche definitive tipiche della cartilagine articolare di tipo ialino . 
sorrentino , via san lorenzo 291 / a , i - 90146 palermo , italy , tel . : + 39 - 91 - 6552335 , fax : + 39 - 91 - 6552337 , e - mail : drdiving@yahoo.it received : 19 september 2006 / accepted : 24 november 2006 / published online : 23 july 2007 abstract purpose . 
hrus is a fairly reliable technique in the detection , characterisation and differentiation of the different forms of meniscal cyst . key words meniscal cyst high - resolution ultrasonography knee meniscus riassunto obiettivo . 
lhrus una metodica abbastanza affidabile nellidentificazione , nella valutazione delle caratteristiche semeiologiche e nella distinzione delle differenti forme delle cisti meniscali . parole chiave cisti meniscale ecografia ad elevata risoluzione ginocchio menisco introduction introduzione meniscal cysts are relatively uncommon lesions that are often clinically unrecognised . 
they have a reported incidence ranging from 1%2% to 7%8% in the different arthroscopic and le cisti meniscali sono una patologia abbastanza infrequente , spesso clinicamente misconosciuta , con unincidenza variabile tra l1%2% ed il 7%8% secondo diverse casistiche f . 
in most cases , they are accompanied by mucoid degeneration and horizontal cleavage tear , with infiltration of synovial fluid into the capsular - meniscal complex [ 1 ]  . 
meniscal cysts may manifest either as palpable masses of the knee joint or else be diagnosed at imaging , especially with magnetic resonance imaging ( mri ) performed to assess injuries to meniscal and ligamentous structures . 
within the field of knee - joint imaging , high - resolution ultrasound ( hrus ) is considered the modality of choice for the evaluation of extraarticular disorders on account of its high diagnostic accuracy [ 24 ]  . 
in the study of intra - articular lesions , including meniscal and cruciate ligament alterations , however , the results of us are conflicting because of methodological and anatomotopographic difficulties that make its clinical use controversial and often limited [ 3 , 5 , 6 ]  . 
the aim of this study was to assess the ability of hrus to detect parameniscal cysts and compare the mri and hrus appearance of the various types of meniscal cyst . materials and methods over a 2 - year period , 1 , 857 patients ( 989 men ; mean age 43 years ) underwent mri for either traumatic or degenerative knee disorders . 
mri was performed using a 0.5 - tesla ( t ) superconductive magnet ( vectra , ge medical systems , milwaukee , wi , usa ) with a limb - dedicated transmit / receive coil . 
spin - echo ( se ) t1 ( tr 500 ms , te 20 ms ) and gradientecho ( ge ) t2 * ( tr 320 ms , te 20 ms , flip angle 30 ) sequences were obtained in the sagittal , coronal and axial planes with 3 - mm slice thickness and 1 - mm interval . 
the acquisition matrix was 192256 , reconstruction 256256 and field of view ( fov ) ranging from 15 cm to 20 cmri were jointly evaluated by two radiologists ( fs , an )  . 
all hrus exams were performed by a single radiologist ( ai ) blinded to the mri results , with au5 ( esaote biomedica , genoa , italy ) and hdi 5000 ( atl , bothell , wa , usa ) devices , using high - resolution linear multifrequency transducers ( 712 mhz )  . 
the exam was performed with the patient in the supine position with 3040 knee flexion for identification of the anterior horn of the meniscus and in the prone position with the knee extended knee to examine the posterior horn . 
the scans obtained corresponded to precise anatomical landmarks , taking into account the spatial orientation of the structures to be examined . in order to avoid obliquity artefacts , scans were performed parallel to the femur long axis and absolutely perpendicular to the base of the meniscus . 
nella maggior parte dei casi , si accompagnano a degenerazione mucoide e fissurazione orizzontale del menisco , con infiltrazione di liquido sinoviale sino alla connessione menisco - capsulare [ 1 ]  . 
le cisti meniscali possono manifestarsi o come masse palpabili in relazione con larticolazione del ginocchio o possono venire diagnosticate allimaging , prevalentemente con risonanza magnetica ( rm ) , eseguita nello studio della patologia traumatica delle strutture meniscali e legamentose . 
nellambito della diagnostica per immagini dellarticolazione del ginocchio , lecografia ad elevata risoluzione ( hrus ) viene odiernamente considerata metodica di prima istanza nella valutazione delle affezioni extrarticolari , grazie allelevata accuratezza diagnostica dimostrata [ 24 ]  . 
nello studio della patologia intrarticolare , che include le alterazioni dei menischi e dei legamenti crociati , lecografia , in relazione alle relative difficolt di approccio metodologico e anatomo - topografico , che ne rendono controversa e spesso limitata la sua utilizzazione clinica , mostra spesso risultati molto contrastanti [ 3 , 5 , 6 ]  . 
la rm , di contro , presenta elevata accuratezza diagnostica , con valori del 90%95% nella diagnosi di lesione meniscale e del 95%100% in quella dei legamenti crociati [ 7 , 8 ]  . 
inoltre stata effettuata una comparazione tra laspetto rm di varie tipologie di cisti meniscali con quello hrus . materiali e metodi in un periodo di 2 anni , sono stati sottoposti ad rm del ginocchio per patologia traumatica o degenerativa 1857 pazienti ( 989 uomini ; et media 43 anni )  . 
le indagini rm sono state eseguite utilizzando ununit a magnete superconduttivo da 0 , 5 t ( vectra , ge medical systems , milwaukee , usa ) impiegando una bobina trasmittente / ricevente dedicata per estremit . 
sono state utilizzate sequenze spin - echo ( se ) t1 ( tr = 500 ms ; te = 20 ms ) e gradient - echo ( ge ) t2 * ( tr = 320 ms ; te = 20 ms ; angolo di ribaltamento = 30 ) secondo i piani di scansione sagittale , coronale ed assiale , con spessore di strato di 3 mm ed intervallo di 1 msono state utilizzate una matrice di acquisizione di 192256 e una di ricostruzione di 256256 , con un campo di vista ( fov ) variabile da 15 a 20 cle immagini rm sono state valutate in consenso da due autori ( fs , an )  . 
lo studio hrus stato eseguito in cieco dal medesimo radiologo ( a.i. ) , non a conoscenza della presenza di una cisti parameniscale , con apparecchiature au5 ( esaote biomedica , genova , italia ) e hdi 5000 ( atl , bothell , wa , usa ) utilizzando sonde lineari multifrequenza ( 712 mhz ) ad elevata f . 
in the control group , an equivalent number of patients was recruited , and four patients with cyst - like formations diagnosed as synovial ganglia at mri were purposely included . under normal conditions , hrus allows identification of the meniscal fibrocartilage , which appears as a homogeneously hyperechoic triangle , with the base towards the surface and the apex along the intra - articular direction . 
in two cases , the cyst was wrongly characterised as a synovial ganglion because of its large size ( mean diameter 4.3 cm ) , which did not allow us to appreciate the continuity between the meniscal alteration and the cyst - like formation . 
in none of the cases examined did mri or hrus depict erosions of the surrounding bone structures . surgery and histology confirmed the diagnosis established with mri , which considered to be the gold standard . discussion meniscal cysts are a generally uncommon finding first described by nicase in 1883 and later by ebner in 1904 [ 10 ]  . meniscal cysts are prevalently detected in 20to 40 - year - old men and more frequently in the lateral than medial meniscus , risoluzione . 
lesame stato condotto con paziente in posizione supina , con ginocchio in flessione di circa 3040 , per lidentificazione del corno meniscale anteriore ed in posizione prona con ginocchio esteso , per la valutazione del corno posteriore . 
al fine di evitare artefatti da obliquit sono state effettuate scansioni parallele allasse maggiore del femore e perfettamente perpendicolari alla base del menisco visualizzato . lapplicazione di un leggero stress in valgismo ha facilitato la visualizzazione del corno anteriore della fibrocartilagine meniscale mediale , mentre un leggero stress in varismo ha consentito la visualizzazione del corno anteriore controlaterale . 
i criteri utilizzati per la diagnosi di cisti meniscale sono stati : ( 1 ) identificazione di formazione con caratteristiche semeiologiche ecografiche fluide o miste tipo complex - mass ; ( 2 ) dimostrazione dellesistenza di un rapporto di continuit tra la formazione cistica , larticolazione e la relativa fibrocartilagine meniscale ; ( 3 ) alterazione meniscale lineare in continuit con la formazione cistica . 
tutti i pazienti sono stati sottoposti ad intervento chirurgico e le formazioni resecate sono state sottoposte ad esame istologico . risultati in 52 pazienti lindagine rm ha consentito lidentificazione di una cisti parameniscale . 
in due casi perch erroneamente caratterizzate come ganglio sinoviale a causa delle ampie dimensioni ( diametro medio 4 , 3 cm ) della formazione che non consentivano di apprezzare la continuit tra lalterazione meniscale e la formazione con aspetto cistico , e nellultimo caso , la cisti parameniscale non stata correttamente identificata perch di piccole dimensioni e localizzata in sede profonda posteromediale . 
nel gruppo controllo lhrus ha escluso in tutti i casi la presenza di una cisti meniscale e ha consentito la corretta identificazione e caratterizzazione dei 4 casi con ganglio cistico , le cui dimensioni erano comprese tra 2 cm e 3 clhrus ha manifestato nella detezione delle cisti parameniscali una sensibilit del 94 , 23% , una specificit del f . 
this triangle is separate from the femoral and tibial cortical border represented by a hyperechoic layer and from the hyaline cartilage , which appears as a thin , homogeneous hypoechoic band . 
questo triangolo separato dal profilo corticale osseo femorale e tibiale rappresentato da una stria regolare iper - riflettente , dalla cartilagine ialina di rivestimento articolare , che appare come una sottile banda ipoecogena . 
nella sede anatomica meniscale , si evidenzia una formazione simil - cistica che presenta morfologia a cuori di carta da gioco , a margini netti con tramite fistoloso intra - meniscale . 
ampia formazione simil - cistica ( diametro 4 , 5 cm ) con componente corpuscolata , a margini netti , con tramite fistoloso intra - meniscale sul versante interno distale . 
lintervento chirurgico e listologia hanno confermato la diagnosi rm considerata nel nostro studio come gold standard . discussione le cisti meniscali , in genere , non sono di frequente riscontro e furono descritte per la prima volta da nicase nel 1883 e successivamente da ebner nel 1904 [ 10 ]  . 
le cisti meniscali si evidenziano nella maggior parte dei casi in uomini tra i 20 e i 40 anni , con prevalente coinvolgimento del menisco laterale rispetto al mediale , con rapporto variabile tra 3 : 1 e 7 : 1 [ 1 ] ; esistono , tuttavia , alcuni studi che riportano differenti rapporti di incidenza , con uguale incidenza in entrambi i menischi o con prevalente coinvolgimento del menisco mediale [ 11 ]  . 
la tesi pi accreditata quella microtraumatica iterativa che in associazione alla critica vascolarizzazione del menisco inducono processi degenerativi rappresentati da fenomeni di vacuolizzazione , colliquazione e trasformazione mucoide che partendo dal centro della fibrocartilagine meniscale si portano verso la superficie in direzione capsulare . 
la cisti parameniscale la forma pi frequentemente riscontrata [ 1 , 10 ] ed rappresentata da una raccolta fluido - sinoviale che , tramite la lesione meniscale , si fa strada attraverso i tessuti circostanti [ 1 ]  . 
la sua estrinsecazione pu avvenire : anteriormente , a livello del cuscinetto adiposo sottopatellare , lateralmente , in prossimit del legamento collaterale , posteriormente , tra questo ed il tendine popliteo e medialmente , nel cavo popliteo od in prossimit della zampa doca . 
la diagnosi ecografica di cisti parameniscale si basa sulla identificazione della raccolta fluida contraente stretti rapporti di continuit con la cavit articolare ed , in particolare , con la corrispondente fibrocartilagine meniscale da cui si origina . 
nel presente studio lhrus ha consentito nel 94 , 23% dei casi la corretta identificazione delle cisti parameniscali e le limitazioni sono derivate dalle ampie dimensioni e dalla localizzazione posteriore - profonda . 
come evidenziato nella nostra casistica lecostruttura delle cisti molto variabile e dipende dalla viscosit del fluido sinoviale e dal tempo di comparsa ; leventuale presenza di detriti o di formazioni ecogene nel suo contesto sono in genere messe in relazioni a microframmenti derivanti dal menisco degenerato . 
this leakage may be directed either anteriorly towards the subpatellar fat pad , laterally near the collateral ligament , posteriorly between the collateral ligament and the popliteal tendon or medially within the popliteal fossa or near the anserine bursa . 
meniscal cyst size varies considerably and may also be very large , as seen in our series , with the formation of a pseudocapsule and in some cases internal septation . 
us diagnosis of a parameniscal cyst is based on identification of a fluid collection in close continuity with the articular cavity and , in particular , with the corresponding meniscal fibrocartilage from f . 
as seen in our series , cyst echostructure varies widely and depends on synovial fluid viscosity and the time of appearance ; the possible presence of debris or echogenic masses within the cyst are generally related to microfragments from the degenerated meniscus . 
on the basis of their experience with 13 patients , seymour and lloyd reported that the echostructural variability does not allow adequate diagnostic accuracy and creates problems in the differential diagnosis with other lesions such as cystic ganglia or soft tissue tumours [ 14 ]  . 
in contrast , in our experience , echostructure did not have a negative effect on diagnostic accuracy , and differentiation between parameniscal cysts and cystic ganglia was influenced by the size of the cysts which , when larger than 4 cm , prevented identification of the connection with the meniscus . 
it has been reported that very large cysts are associated with erosion of the cortical bone profile [ 10 , 15 , 16 ] , although this was never detected in our series . 
longitudinal scans were best for visualising the entire meniscal cyst and detecting the connection with the adjacent meniscal fibrocartilage , whereas axial scans proved useful for assessing extension of the lesion . conclusions hrus proved to be a reliable technique in the presumptive diagnosis of meniscal cysts , as it allowed both identification and characterisation of their imaging features . 
hrus does not usually play a role in the diagnostic workup of meniscal lesions , which is carried out by ct and especially mri , two techniques that allow correct identification of the lesion and precise definition of its size . it should nonetheless be emphasised that during an examination to assess superficial tendon and / or ligament structures , hrus can potentially allow the incidental discovery of a meniscal cyst , leading to a presumptive diagnosis useful in the clinical assessment of patients . dellaspetto ecostrutturale non consenta una adeguata accuratezza diagnostica , con problemi di diagnosi differenziale con altre entit patologiche come i gangli cistici o i tumori dei tessuti molli [ 14 ]  . 
nella nostra esperienza , lecostruttura non ha influenzato negativamente laffidabilit diagnostica della metodica e la diagnosi differenziale tra cisti parameniscali e gangli cistici stata inficiata dalle dimensioni , superiori ai 4 cm , che non consentono lidentificazione del tramite con il menisco . 
in presenza di cisti di cospicue dimensioni , stata segnalata la presenza di erosione del profilo corticale osseo [ 10 , 15 , 16 ] , che , tuttavia , nella nostra casistica , non mai stata rilevata . 
le scansioni longitudinali si sono dimostrate le pi idonee ad evidenziare le cisti meniscali in tutto il loro decorso , con dimostrazione del tramite con ladiacente fibrocartilagine meniscale , mentre le scansioni assiali sono servite a verificare lesatta estensione della lesione . conclusioni lhrus si dimostrata una metodica affidabile nella diagnosi presuntiva delle cisti meniscali consentendone sia lidentificazione che la valutazione delle caratteristiche semeiologiche . 
nella nostra esperienza , anche se limitata , la hrus ha mostrato una elevata accuratezza diagnostica possedendo una sensibilit del 94 , 23% , una specificit del 100% , un valore predittivo positivo del 100% e un valore predittivo negativo del 94 , 54% . 
nel corretto iter diagnostico della patologia meniscale del ginocchio la hrus abitualmente non svolge un ruolo , che deve essere assolto dalla tc ed in particolare dalla rm , metodiche che consentono la corretta identificazione della patologia ed un preciso bilancio di estensione . 
all rotational studies were conducted by selective cannulation of the vessel supplying the lesion ( internal carotid artery or vertebral artery ) with a single injection of 20 cc of contrast agent after diagnostic angiography in anterior - posterior ( ap ) and laterolateral ( ll ) views . 
three - dimensional ra enabled accurate definition of site , orientation , morphology and size of the sac and its relationship with the parent arteries and helped us choose the most appropriate angulation of the c - arm for guiding and controlling the embolisation procedure . 
based on our experience before ra equipment became available and in agreement with the literature , we believe that 3dra improves the identification of all lesions and helps refine the choice of the most suitable embolisation material and technique . 
lo studio rotazionale stato sempre eseguito mediante cateterizzazione selettiva del vaso afferente ( carotide interna o arteria vertebrale ) con singola iniezione di 20 cc di mdc dopo angiografia diagnostica nelle due proiezioni ortogonali . 
mediante la 3dra in ogni caso stato possibile definire in maniera accurata la sede , lorientamento , la morfologia e le dimensioni della sacca , il rapporto con i vasi parenti e la scelta dellidonea angolazione dellarco a c ai fini della guida e del controllo dellembolizzazione . 
in base alla nostra esperienza precedente alla disponibilit di un angiografo rotazionale , e in accordo con i dati della letteratura , riteniamo che la 3dra consenta di individuare con maggiore accuratezza tutte le lesioni e di scegliere con maggiore precisione il materiale pi idoneo per la procedura , la tecnica di embolizzazione , nonch di ridurre sensibilmente il numero di proiezioni e quindi la dose di esposizione e di mdc al paziente , con una verosimile riduzione dei tempi di trattamento , dei rischi e delle complicanze . key words cerebral angiography intracranial aneurysm embolization , therapeutic imaging , three - dimensional parole chiave angiografia cerebrale aneurismi intracranici embolizzazione terapeutica imaging tridimensionale a . 
subarachnoid haemorrhage ( sah ) due to aneurysm rupture has an incidence of 1025 / 100 , 000 / year and is a devastating event associated with high morbidity and mortality from rebleeding and vasospasm [ 1 ]  . management of intracranial aneurysms has been facilitated by constant improvements in diagnostic and therapeutic procedures , which have recently led to an increased use of noninvasive imaging [ computed tomography ( ct ) angiography and magnetic resonance ( mr ) angiography ] and percutaneous endovascular treatment rather than conventional surgery [ 2 , 3 ]  . 
the technique and outcome of cerebral embolisation procedures are closely dependent on an accurate preliminary assessment of the morphology and size of the aneurysmal sac and of its relationship with parent vessels . the aim of this study was to assess the systematic use of 3d rotational angiography ( 3dra ) in the diagnosis and preoperative evaluation of cerebral aneurysms with a view to planning endovascular treatment . la prevalenza degli aneurismi cerebrali nella popolazione generale stimata tra 0 , 2% e 6 , 8% . 
lemorragia subaracnoidea ( esa ) da rottura di aneurisma , con unincidenza di 1025 / 100000 / anno , rappresenta un evento devastante con elevata morbilit e mortalit legate soprattutto al risanguinamento e al vasospasmo [ 1 ]  . la gestione dellaneurisma intracranico oggi favorita da un evidente e progressivo miglioramento delle procedure diagnostiche e terapeutiche che nellultimo decennio hanno permesso un crescente orientamento verso limaging non invasivo ( angio - tc e angio - rm ) e il trattamento endovascolare percutaneo rispetto alla chirurgia tradizionale [ 2 , 3 ]  . 
la tecnica di embolizzazione cerebrale e il suo esito sono strettamente dipendenti da una preliminare accurata valutazione morfo - dimensionale della sacca aneurismatica e dei suoi rapporti con i vasi parenti . lobiettivo del presente lavoro quello di valutare lutilit dellimpiego sistematico dellangiografia rotazionale 3d ( 3dra ) nella diagnostica preliminare al trattamento endovascolare degli aneurismi cerebrali , ai fini della pianificazione della procedura . materials and methods thirty - five consecutive aneurysms in 32 patients [ 20 females and 12 males ; age range 3 months ( one case ) to 85 years ; mean age 49.3 years ] were studied and treated using 3dra ( philips allura xper fd 20 single - plane ra system with a flat - panel detector ) between march 2005 and march 2006 . 
fifteen of the aneurysms were treated after sah , whereas 20 had been discovered incidentally at mr performed for different diagnostic indications , or identified as the cause of cranial nerve deficits or embolic ischaemic events . 
the aneurysms were located at the anterior communicating artery or between segments a1 and a2 of the anterior cerebral artery ( seven cases ) , at the origin of the posterior communicating artery ( six cases ) , at the origin of the anterior choroidal artery ( two cases ) , in the ophthalmic segment of the internal carotid artery ( six cases ) , at the internal carotid bifurcation ( three cases ) , at the basilar artery apex ( five cases ) , in the cavernous segment of the internal carotid ( two cases ) and at the bifurcation of the middle cerebral artery ( four cases )  . 
 rotational angiography was performed in all cases via transfemoral access and selective catheterisation of the feeding vessel ( internal carotid or vertebral artery ) with a single injection of 1520 cc nonionic iodinated contrast material at a flow rate of 34 ml / s and 240 rotation of the c - arm with the propeller technique . 
in all cases , the rotational phase was preceded by a preliminary diagnostic 2d acquisition in two orthogonal views [ anterior - posterior ( ap ) and materiali e metodi sono stati studiati e trattati consecutivamente 35 aneurismi con luso di 3dra ( angiografo rotazionale monoplano philips allura xper fd 20 con flat - detector ) in 32 pazienti tra marzo 2005 e marzo 2006 . 
di questi , 15 sono stati trattati dopo esa , mentre 20 aneurismi costituivano reperto occasionale in pazienti sottoposti a rm cerebrale per diversi quesiti diagnostici o rappresentavano la causa di deficit di nervi cranici o eventi ischemici su base embolica . 
la sede degli aneurismi era rispettivamente a livello dellarteria comunicante anteriore o tra i segmenti a1 e a2 dellarteria cerebrale anteriore ( 7 casi ) , allorigine dellarteria comunicante posteriore ( 6 casi ) , allorigine dellarteria corioidea anteriore ( 2 casi ) , nel tratto oftalmico della carotide interna ( 6 casi ) , alla biforcazione di carotide interna ( 3 casi ) , sullapice di basilare ( 5 casi ) , nel tratto cavernoso della carotide interna ( 2 casi ) e alla biforcazione di arteria cerebrale media ( 4 casi )  . lo studio angiografico rotazionale stato sempre eseguito tramite accesso transfemorale e mediante cateterizzazione selettiva del vaso afferente alla lesione ( carotide interna o arteria vertebrale ) con singola iniezione di 1520 cc di mdc iodato non ionico , flusso a 34 ml / s e rotazione dellarco di 240 in modalit propeller . 
all diagnostic examinations were performed with the patient under general anaesthesia , as they formed part of the following interventional procedure and as this ensured total immobility , which is crucial for high diagnostic quality , accuracy and reliability of the examination . 
la fase rotazionale in tutti i casi stata preceduta da una preliminare fase diagnostica 2d nelle due proiezioni ortogonali ( ap e ll ) utilizzata anche per la valutazione panoramica del circolo endocranico . 
in tutti i casi lesame stato eseguito in anestesia generale in quanto parte integrante della successiva fase terapeutica e per le condizioni di assoluta immobilit del paziente , imprescindibili ai fini di una elevata qualit diagnostica e quindi accuratezza e affidabilit dellesame . 
immediatamente al termine dellacquisizione rotazionale , dopo ricezione automatica della serie angiografica non sottratta attraverso una connessione di rete , il software dellapparecchiatura ( workstation integris 3d - ra ) in pochi secondi ha prodotto automaticamente il modello volumetrico in 3d con matrice di 5123 sul quale sono state poi scelte ricostruzioni in volume rendering ( vr ) , gradient rendering ( gr ) , shaded surface ( ss ) o maximum intensity projection ( mip ) a seconda delle informazioni che necessitava il singolo caso . 
1a - d a 3 - month - old child with a giant aneuryson 2d angiograms , it is difficult to determine the precise site of origin of the lesion ( a )  . 
three - dimensional gradient rendering ( b ) and volume rendering ( c ) reconstructions allow accurate localisation of the origin between the right a1 segment and the anterior communicating artery , which appears to be a plexiform variant ( arrow )  . the relationship of the lesion with the adjacent vessels is also clear . 
le ricostruzioni 3d in gr ( b ) e vr ( c ) permettono di localizzare con accuratezza lorigine della lesione situata tra a1 di destra e larteria comunicante anteriore che appare di tipo plessiforme ( freccia )  . 
2a - d two - dimensional angiography ( a ) in a patient with an aneurysm of the anterior communicating artery ( black arrow ) and ophthalmic internal carotid artery ( ica ) ( white arrow ) treated after subarachnoid haemorrhage probably due to the first aneurysthe volume rendering 3d images ( b , c ) clearly show the morphology and orientation of the lesions . 
2a - d angiografia 2d ( a ) in paziente con aneurisma di arteria comunicante anteriore ( freccia nera ) e di ci oftalmica ( freccia bianca ) dopo esa verosimilmente dal primo . 
questultima valutazione stata resa possibile grazie allautomatica rappresentazione dei gradi di angolazione in senso cranio - caudale e laterale del tubo a c riportati nello spazio in alto a sinistra sullimmagine 3d ( visibile nelle figure ) dopo scelta da parte delloperatore delladeguata proiezione di lavoro sul modello stesso . 
a aneurisma di ci oftalmica ; b , c aneurisma allorigine della comunicante posteriore sui quali sono misurati i diametri della sacca e del colletto . la selettiva opacizzazione del vaso arterioso afferente alla lesione senza sovrapposizione di vasi venosi e il celere e non sempre necessario lavoro di post - processing manuale per la sottrazione delle strutture ossee del cranio hanno permesso in ogni caso di ottenere un rapido e accurato imaging volumetrico in tempi stimati mediamente intorno ai 5 minuti . 
sulla console sono state quindi eseguite misurazioni e valutazioni necessarie ai fini della procedura endovascolare in tempi variabili a seconda della complessit dei singoli casi , ma non superiori ai 57 minuti . tra le possibili modalit di ricostruzione 3d sono state utili la vr e la gr per rispondere a tutti i quesiti richiesti dalla successiva fase di embolizzazione . 
non si sono mai verificate complicanze maggiori , mentre per quanto riguarda le complicanze minori ( 1 caso di embolia temporale periferica risolto con fibrinolisi loco - regionale , 2 casi di trombosi precoce della lesione con evidenza di parziale ricanalizzazione nei successivi controlli di follow - up e 1 caso in cui si verificato un lieve bulging di spirali attraverso il colletto aneurismatico ) , tali eventi non erano conseguenti a mancata accuratezza della diagnostica preliminare . among the possible 3d reconstructions , vr and gr were most helpful in providing all the information required for the subsequent embolisation procedure . 
by further modifying the model projection ( c ) , one can clearly distinguish the course and origin of the posterior communicating artery ( arrowhead ) in relation to the aneurysm ( arrow )  . 
vr reconstruction ( d ) shows a wide - necked aneurysm ( arrow ) of the posterior communicating artery ( arrowhead ) ; another projection ( e ) enables identification of a pouch ( arrow ) on the sac and the origin of the communicating artery ( arrowhead ) from the sac itself . 
4a - h alcuni casi in cui le immagini 3d permettono di definire con accuratezza morfologie complesse di sacche aneurismatiche e di stabilire i rapporti della lesione con i vasi parenti . 
modificando ulteriormente la proiezione del modello ( c ) possibile distinguere chiaramente il decorso e lorigine della comunicante posteriore ( punta di freccia ) in rapporto allaneurisma ( freccia )  . 
in d la ricostruzione in vr mostra un aneurisma ( freccia ) di comunicante posteriore ( punta di freccia ) con ampio colletto ; con unaltra proiezione del modello ( e ) possibile individuare una tasca accessoria ( freccia ) sulla sacca e lorigine dellarteria comunicante ( punta di freccia ) dalla sacca stessa . 
i gradi di angolazione del tubo a c forniti nellimmagine in alto a sinistra , dopo aver scelto la proiezione di lavoro per lembolizzazione sul modello volumetrico , permettono di orientare il tubo nella medesima proiezione per guidare la procedura su entrambe le lesioni ( freccia ) ( b ) e controllare il risultato finale che appare ottimale ( freccia ) ( c )  . discussione langiografia a sottrazione digitale rappresenta ancora oggi lesame gold standard per lidentificazione e laccurata valutazione degli aneurismi intracranici . 
limaging non invasivo , e in special modo langio - tc , con il miglioramento delle apparecchiature e dei software di ricostruzione bie tridimensionale , trova sempre maggior applicazione nelliter diagnostico di questa patologia anche ai fini della valutazione per la scelta terapeutica di tipo chirurgico o endovascolare [ 4 ]  . tuttavia , grazie allimpiego selettivo del mezzo di contrasto , alla panoramicit e alla risoluzione spaziale dellesame angiografico , il corretto e affidabile studio dellaneurisma ancora appannaggio di questa metodica , soprattutto nella valutazione preliminare alla procedura di embolizzazione endovascolare e ai fini della sua pianificazione [ 5 ]  . i limiti della metodica angiografica 2d tradizionale nella valutazione pre - interventistica sono rappresentati in molti casi dalla necessit di numerose proiezioni con relative multiple iniezioni di mdc trans - catetere ed esposizioni a serie radiografiche per una corretta definizione morfologica della lesione vascolare , del colletto aneurismatico e dei suoi rapporti con i vasi endocranici . 
non raramente le multiple proiezioni non permettono comunque una esaustiva descrizione di tali dati per sovrapposizioni strutturali non dissociabili a causa dellanatomia vascolare o dei limiti di escursione del tubo a c . 
langiografia tradizionale inoltre non consente misurazioni quantitative dimensionali della sacca , del colletto e dei vasi parenti , estremamente utili soprattutto per la scelta del tipo e delle dimensioni delle spirali metalliche da utilizzare . discussion digital subtraction angiography remains the gold standard examination for the detection and evaluation of intracranial aneurysms . 
noninvasive imaging , in particular ct angiography , with the improvements in hardware and software for 2d and 3d image reconstruction , is progressively gaining ground in the diagnostic workup of cerebral aneurysms and a view to guiding decisions concerning surgical or endovascular management [ 4 ]  . 
however , thanks to the selective use of contrast material , the panoramic view and the spatial resolution afforded by angiography , angiography is still relied upon to provide an accurate and reliable study of the aneurysm , above all for preoperative evaluation before endovascular embolisation and for procedure planning [ 5 ]  . the limitations of conventional 2d angiography in procedure planning are the frequent need to obtain multiple projections with repeated injections of contrast material and increased radiation dose to the patient in order to obtain correct morphological depiction of the aneurysm , aneurysa . 
sulla proiezione laterale ( b ) si osserva unicamente una irregolarit marginale di dubbia interpretazione che si proietta sul profilo superiore del segmento cavernoso della carotide interna ( nel cerchio )  . 
la ricostruzione 3d in vr ( c ) grazie alla sua multiproiettivit identifica un piccolo aneurisma del medesimo segmento della carotide interna e ne mostra chiaramente la morfologia senza necessit di ricorrere a ulteriori iniezioni di mdc e proiezioni angiografiche . mal neck and relationships with intracranial vessels . 
in many cases , these multiple acquisitions fail to provide a comprehensive depiction due to the superimposition of structures that cannot be discerned because of vascular anatomy or limited excursion of the c - armoreover , conventional angiography does not allow quantitative determinations of the size of the sac , neck or parent vessels , which are especially helpful in selecting the type and size of coils to be used for embolisation . in our experience , albeit in a small number of patients , these limitations were always fully overcome by the use of rotational sequences with 3d reconstructions . 
this result was related to the high spatial resolution and selective use of contrast material of angiography compared to the noninvasive modalities , and to the multiple views afforded by the 3d model , which , unlike conventional angiography , allowed complete discrimination of superimposed vascular structures and thus the confident identification and interpretation of aneurysms . similar findings were also reported by other authors [ 610 ] , who confirmed the diagnostics achieved by ra thanks to the major technological developments introduced since its first applications in the 1970s [ 11 , 12 ]  . 
in agreement with these authors , we found that crucial factors in planning the endovascular procedure and ensuring an optimal outcome were the ability to measure the aneurysm quantitatively for selection of the first coil and to determine carm angulation for the appropriate working projection . 
questo dato stato correlato allelevata risoluzione spaziale e allimpiego selettivo del mezzo di contrasto dellesame angiografico rispetto alle metodiche non invasive , e sostanzialmente alla multiproiettivit del modello volumetrico che , rispetto allangiografia tradizionale stessa , ha permesso la completa dissociazione delle strutture vascolari sovrapposte e quindi la sicura interpretazione e lidentificazione degli aneurismi . 
 tali constatazioni sono riportate anche nelle casistiche di altri autori [ 610 ] , i quali confermano la validit diagnostica dellangiografia rotazionale dopo limportante sviluppo consentito dallevoluzione tecnologica dai tempi delle sue prime applicazioni negli anni 70 [ 11 , 12 ]  . 
ai fini della pianificazione del trattamento endovascolare e dellottimale risultato terapeutico , come per gli autori suddetti , risultata fondamentale la possibilit della misurazione quantitativa dellaneurisma per la scelta della prima spirale da rilasciare nella sacca e quella di stabilire langolazione del tubo a c per lidonea proiezione di lavoro . 
7a , b angiographic volume rendering 3d standard reconstruction ( a ) showing an aneurysm of the anterior communicating artery with its neck larger than the sac and the a2 segments of the anterior cerebral arteries originating from the neck itself . 
7a , b ricostruzione angiografica 3d standard in vr ( a ) che mostra un aneurisma di comunicante anteriore con colletto pi ampio della sacca e dal quale emergono i tratti a2 delle cerebrali anteriori . 
 with regard to analysis of results obtained with 3dra , and in partial agreement with recently published analyses [ 1315 ] , there was no case of significant overor undersizing of the aneurysm due to intrinsic limitations of the 3d reconstruction software . 
in our series , this finding was always confirmed when the first coil was positioned , as in all cases its size corresponded with the measurements made on the 3d model by the postprocessing software . 
in some cases ( 5 / 35 ) , the 3d reconstruction software produced artefactual images of the course or emergence of vessels relative to the aneurysm sac , consisting mostly of marginal fusion between single adjacent structures . 
these artefacts have been described for the various digital acquisition and 3d reconstruction modalities and are chiefly related to the spatial resolution of the single machines and degree of geometric distortion intrinsic to the acquisition modality [ 16 , 17 ]  . 
questo dato trova peraltro parziale conferma nei lavori suddetti e la sua verifica , nei nostri casi , stata ottenuta sempre dopo il posizionamento della prima spirale , le cui dimensioni hanno corrisposto in ogni singolo caso alle misure elaborate tramite il software di post - processing sul modello 3d dellaneurisma . 
in alcuni casi ( 5 / 35 ) la ricostruzione 3d ha prodotto immagini artefattuali di decorso o emergenza di vasi in rapporto con la sacca aneurismatica rappresentate prevalentemente da fusione marginale tra singole strutture adiacenti . 
tali artefatti sono descritti nelle varie modalit di acquisizione digitale e ricostruzione 3d e legati principalmente alla risoluzione spaziale delle singole apparecchiature e al grado di distorsione geometrica intrinseca alla modalit di acquisizione [ 16 , 17 ]  . 
in such conditions , an additional 2d angiographic acquisition after selective contrast injection and in the most appropriate projection , as determined on the 3d model , may be useful to provide superimposable images , allowing accurate comparison and diagnostic supplementation . finally , in agreement with other authors [ 18 ] , we believe that the acquisition technique that uses propeller rotation is more useful than the standard technique because it allows less contrast material to be administered for each rotation and a broader rotational arc of the c - arm , with high image quality and shorter acquisition times . va , dopo iniezione selettiva di mdc , secondo la proiezione pi utile determinata sul modello 3d in modo da ottenere immagini di integrazione diagnostica sovrapponibili per un idoneo confronto . in accordo con altri autori [ 18 ] , infine , riteniamo che la tecnica di acquisizione rotazionale in modalit propeller sia vantaggiosa rispetto a quella tradizionale , in quanto permette una ridotta quantit di mdc somministrato per singola rotazione e un pi ampio range di escursione rotazionale del tubo a c con elevata qualit delle immagini in tempi di acquisizione pi rapidi . conclusions in comparison with conventional 2d angiography , 3dra allowed a more confident choice of embolisation technique and materials ( type and angulation of the microcatheter , type and diameter of metal coils ) and a drastic reduction in the number of projections required for evaluating the lesion , leading to lower radiation dose and less contrast material to the patient . the 3d reconstructions enabled the correct identification of some small aneurysms that had doubtful findings on conventional imaging modalities . 
in addition , it allowed accurate evaluation of the lesions , thereby helping us in our choice of embolisation materials and technique and improving our results with regard to aneurysmal sac exclusion and coil deployment , with only one case of slight bulging of the coil through the aneurysm neck . 
finally , 3dra allowed for shorter procedure times and fewer risks and complications related to long procedure times and inaccurate choice of materials . we therefore believe this technique should become an integral part of angiographic diagnostics for planning endovascular treatment of cerebral aneurysms . conclusioni la 3dra , rispetto alla diagnostica 2d tradizionale , ha permesso di scegliere con maggiore precisione il materiale pi idoneo per la procedura ( tipo e curvatura del microcatetere , diametro e tipologia delle spirali metalliche ) , la tecnica di embolizzazione , e di ridurre drammaticamente il numero di proiezioni necessarie alla valutazione della lesione vascolare consentendo pertanto una equivalente riduzione della dose di esposizione e mdc al paziente . grazie alle ricostruzioni 3d sono stati correttamente individuati alcuni piccoli aneurismi , la cui presenza era dubbia con le metodiche di imaging tradizionali . 
inoltre , la possibilit di valutazioni accurate per la scelta del materiale e della tecnica ha consentito di ottenere risultati migliori in termini di esclusione della sacca aneurismatica e di disposizione delle spirali allinterno della sacca stessa , con un solo caso di lieve bulging di spirale attraverso il colletto dellaneurisma . 
la 3dra ha permesso infine la riduzione dei tempi di trattamento , dei rischi e delle complicanze per il paziente , legati sia ai tempi dintervento che alluso e alla scelta dei materiali . 
faccioli , tel : + 39 - 045 - 8124301 , fax : + 39 - 045 - 8277808 , e - mail : nfaccioli@sirm.org received : 01 september 2006 / accepted : 12 february 2007 / published online : 21 september 2007 abstract purpose . 
the aim of this study was to perform a cost analysis of contrast - enhanced ultrasonography ( ceus ) in the study of benign focal liver lesions ( bfll ) with indeterminate appearance on ultrasonography ( us )  . 
this cost analysis shows that ceus is the least expensive second - line modality after baseline us for the diagnosis of bfll . key words cost analysis contrast - enhanced ultrasonography liver benign focal lesions computed tomography riassunto obiettivo . 
i pazienti sono stati 398 , aventi 213 angiomi , 41 iperplasie focali nodulari ( fnh ) , e 154 pseudolesioni ( aree di risparmio di steatosi , aree di steatosi focale )  . 
per la diagnosi di angiomi nel 2002 abbiamo risparmiato 1406 , 97 , nel 2003 5315 , 22 , nel 2004 10317 , 78 , nel 2005 9536 , 13  . 
questo studio sullanalisi dei costi ha dimostrato che la ceus la pi conveniente tecnica di secondo livello dopo lecografia basale per la diagnosi di lesioni focali epatiche benigne . parole chiave analisi dei costi ecografia con mezzo di contrasto fegato lesioni focali benigne tomografia computerizzata n . 
when a focal liver lesion is incidentally discovered on baseline ultrasound ( us ) , the standard second - line examination , where required , is contrast - enhanced ct [ 35 ]  . the use of ceus has been suggested as a valuable diagnostic alternative to ct [ 15 ]  . 
 our analysis considers the costs of radiology services from the point of view of the producer ( the hospital ) whose aim is to provide the patient with the best and most cost - effective clinical approach . 
our study presents a cost - identification analysis as performed in previous studies [ 6 , 7 ] , namely , a partial assessment aimed at determining the impact of ceus and ct on costs . 
the aim of his study is therefore to analyse the impact on costs to the radiology department of the use of ceus rather than ct for the study of benign focal liver lesions ( bfll ) with indeterminate appearance on baseline us over a period of 4 years . materials and methods out of 2 , 140 ceus examinations performed between 2002 and 2005 , we retrospectively reviewed 1 , 187 studies of the liver . 
a decision - making model was constructed for patients with suspicious lesions on baseline us , which turned out to be bfll ( angiomas ; focal nodular hyperplasias ; and pseudolesions , areas of fatty sparing or focal fatty areas ) in 398 patients . 
the criteria by which a focal lesion was classified as indeterminate were the absence of the typical signs of angioma ( a homogeneously hyperechoic lesion with sharp margins ) or the absence of the typical signs of focal fatty area or focal fatty sparing ( a hyperechoic or hypoechoic lesion with sharp or blurred margins located in a typical perihilar or perivascular site )  . 
we analysed the cost effectiveness of ceus based on the need to follow up the studies with a ct scan ; that is , we considered the number of cases in which the information provided by ceus proved conclusive for the diagnosis . 
i progressi tecnologici hanno fatto s che tale indagine possa garantire una specificit elevata , anche se la letteratura riporta un range assai ampio ( tra 85% e 95% ) utilizzando di volta in volta la tomografia computerizzata ( tc ) , la risonanza magnetica ( rm ) o , meglio , listologia come gold standard [ 2 ]  . 
molti lavori hanno infatti preso in considerazione lapporto della ceus nei processi diagnostici e hanno sottolineato i suoi benefici per il trascurabile costo biologico intrinseco della metodica [ 2 , 3 ]  . 
attualmente , dopo il riscontro incidentale di una lesione focale epatica allecografia basale lesame standard di secondo livello , dove richiesto , consiste in una tc con mezzo di contrasto [ 35 ]  . 
in letteratura lutilizzo della metodica ceus segnalata come valida alternativa diagnostica alla tc [ 15 ] ; in caso di persistenza del sospetto si ricorre quindi alla tc , ma solo come eventuale terzo stadio delliter diagnostico . 
 la nostra analisi considera i costi di totale pertinenza dei servizi di radiologia dal punto di vista del produttore ( lazienda ospedaliera ) , il quale ha lobiettivo di fornire al paziente lapproccio clinico migliore e con il miglior rapporto costo - efficacia . 
lanalisi dei costi effettuata del tipo cost - identification cos come in altri lavori [ 6 , 7 ] , ovvero una valutazione parziale con lobiettivo di determinare , analizzando le due indagini considerate ( ceus e tc ) gli effetti sui costi . 
scopo di questo lavoro quindi lanalisi dellimpatto sui costi di pertinenza dei servizi di radiologia dellutilizzo della ceus in luogo della tc nello studio delle lesioni focali epatiche benigne ( lfeb ) ad aspetto non esaustivo dopo ecografia basale ( us ) , in un arco temporale di quattro anni . materiali e metodi su un totale di 2140 esami ceus eseguiti tra il 2002 ed il 2005 sono stati retrospettivamente considerati 1187 esami ceus del fegato , sui quali stato costruito un modello decisionale per pazienti con lesioni sospette allecografia basale rivelatesi poi lfeb ( angiomi , iperplasie focali nodulari , pseudolesioni : aree di risparmio di steatosi o aree di steatosi focale ) , per un totale di 398 pazienti . 
i criteri che hanno fatto classificare una lesione focale come indeterminata sono stati lassenza di aspetto tipico di angioma ( lesione a margini netti , omogeneamente iperecogena ) , o lassenza di aspetto tipico di aree di steatosi focale o di risparmio di steatosi ( lesione a margini netti o sfumati ipero ipoecogene nelle sedi tipiche perilari / perivascolari )  . 
all lesions were followed up for 22 months on average ( range : 836 months ) , and in no case was the initial ceus diagnosis changed during follow - up . as for evaluation of the economic impact from the hospitals point of view , the resources absorbed by the examinations were calculated by defining the unit cost in euros of each type of resource used . 
the resources assessed were equipment costs ( purchase and service contract costs ) , cost of supplies ( contrast agents , saline solution , medical supplies , films ) and staff costs ( radiologist , technician / nurse , administrative staff )  . ceus technique all examinations were carried out by radiologists with more than 5 years experience with a us device equipped with contrast - specific software that uses a harmonic frequency with low acoustic pressure ( 24 mhz coherent contrast imaging or cadence contrast pulse sequencing ; mechanical index 0.2 ; 1213 frames / s )  . 
insonation of the focal lesion was continuous , with dynamic observation of the transition between the unenhanced and the contrast - enhanced phase . the lesion enhancement pattern was compared with that of normal parenchyma and classified according to the literature [ 1 , 5 ]  . ct technique liver ct was performed with a single - detector device using a four - phase technique . 
spiral acquisition was performed with a fixed delay from the start of the injection , after 30 s ( arterial phase ) , after 75 s ( portal phase ) and after 4 min ( equilibrium phase )  . 
scan parameters for all phases were the following : collimation 5 mm , table feed 7.5 mm per rotation , reconstruction interval 5 mall ct studies were performed within 2 weeks from us . economic analysis the differential costs ( defined as the sum of equipment costs , cost of supplies and staff costs ) of ceus and ct were analysed according to the methods in use at our institute . three components of the differential costs were considered : ( 1 ) equipment costs ( depreciation and service contracts ) ; ( 2 ) cost of supplies and related services ; ( 3 ) staff costs . 
the cost of supplies and equipment are inclusive of 20% value added tax . the common costs of productive factors within the radiology department , that is , factors supporting all diagnostic seguire le indagini con un esame tc , ovvero considerando in quanti casi le informazioni ricavate dal mezzo di contrasto ecografico sono risultate esaustive in termini di iter diagnostico . 
nei pazienti con lfeb si sono riscontrati : 213 angiomi , 41 iperplasie focali nodulari ( fnh ) e 154 pseudolesioni ( aree di risparmio di steatosi , aree di steatosi focale ) ; i pazienti con lesioni benigne multiple sono stati considerati unitariamente , in quanto il protocollo dindagine non ha subito variazioni . tutti i pazienti hanno effettuato un esame ceus ( 36 nel 2002 , 113 nel 2003 , 125 nel 2004 , 124 nel 2005 ) e per 98 di questi pazienti si resa necessaria anche lesecuzione di un esame tc ( 9 nel 2002 , 41 nel 2003 , 21 nel 2004 , 27 nel 2005 )  . 
tutte le lesioni sono state seguite in follow - up per un periodo medio di 22 mesi ( range 836 mesi ) , e in nessun caso la diagnosi ceus iniziale mutata nel corso del follow - up . per la valutazione dellimpatto economico dal punto di vista dellazienda ospedaliera sono state calcolate le risorse consumate per svolgere gli esami , definendo per ogni tipologia di risorsa utilizzata il costo unitario in euro ( ) ; le risorse valutate sono state : costo delle apparecchiature ( prezzo di acquisto e manutenzione ) , costo dei materiali ( mezzi di contrasto , soluzione fisiologica , materiale sanitario , pellicole ) , costo del personale ( tempo medico , tecnicoinfermieristico , amministrativo )  . tecnica desame ceus tutte le indagini sono state eseguite da medici radiologi con pi di 5 anni di esperienza su un apparecchio ecografico , dotato di software contrasto specifico che utilizza una frequenza armonica a bassa pressione acustica ( 24 mhz coherent contrast imaging o cadence contrast pulse sequencing ; indice meccanico 0 , 2 ; 1213 frames / s )  . 
un bolo di 2 , 4 ml ( mezza dose ) di mezzo di contrasto di seconda generazione , sonovue ( bracco , milano , italia ) , stato iniettato endovena seguito da un bolo di soluzione salina di 5 ml . linsonazione della lesione focale stata continua con osservazione dinamica del passaggio tra la fase non contrastografica e quella contrastografica . 
il pattern di impregnazione della lesione stato confrontato con quello del normale parenchima e classificato secondo letteratura [ 1 , 5 ]  . tecnica desame tc lo studio tc del fegato stato espletato mediante apparecchio a singolo detettore e tecnica quadrifasica . 
lacquisizione spirale avvenuta con ritardo fisso dallinizio delliniezione a 30 s ( fase arteriosa ) , a 75 s ( fase portale ) e a 4 min ( fase di equilibrio )  . 
 when evaluating differential costs , we confined ourselves to the technical act , disregarding any other costs related to patient management , as done in previous studies [ 6 ]  . 
inclusion of the latter would have required an explicit description of the relations between the radiology department and other departments of our university hospital and , with regards to inpatients , the role played by imaging studies on the therapeutic process . 
therefore , our analysis was limited to the costs incurred by the radiology department , enabling us to make comparisons over time ( with reference to budget targets ) , between volumes ( increasing or decreasing workloads ) and across space ( with reference to other hospitals ) [ 6 ]  . 
 the cost of fixed - asset utilisation ( technology ) was related to the time of use , and the cost of radiological , technical and nursing staff was related to their specific activity measured on the basis of activity time . 
 economic savings were calculated by multiplying the number of lesions not requiring ct scans by the cost of a single ct examination : from this , we subtracted the differential cost of ceus compared with baseline us , i.e. 
the calculation can be represented as follows , where ct , ceus and us indicate their respective costs ( table 1 ) : ctn [ ( ceusus ) n ]  . results costs of ceus equipment costs ( depreciation and service contract ) we considered the cost of our us scanner . 
sono state prese in considerazione 3 componenti del costo differenziale , e precisamente : ( 1 ) costi delle apparecchiature ( ammortamenti e manutenzione ) ; ( 2 ) costi dei materiali e servizi connessi ; ( 3 ) costo del personale . 
i costi di materiali e apparecchiature sono comprensivi di iva al 20% . non sono stati calcolati i costi comuni dei fattori produttivi interni allistituto di radiologia , cio quei fattori che garantiscono unattivit di supporto a tutti gli atti diagnostici svolti nel reparto , poich si tratta di costi che non si modificano al variare del numero totale di esami svolti nel periodo annuo e che , essendo specifici per ogni struttura , rappresentano una variabile indipendente . il costo delle apparecchiature stato calcolato in base al tempo medio di utilizzo per esame delle stesse , valutando il costo dacquisto ed il calcolo dellammortamento . 
il calcolo dellammortamento stato effettuato su base temporale , a valore annuo costante . la durata della vita lavorativa delle apparecchiature radiologiche stata considerata di un tempo tecnologico massimo di otto anni . 
il tempo medico richiesto per lo svolgimento degli esami , considerato conforme al carico di lavoro standard del nostro istituto di radiologia , stato ricavato dal documento sirm per la determinazione dei volumi di attivit e produttivit dei medici radiologi [ 8 ] : tale valore risulta pari a 23 minuti per lo svolgimento di un esame us , 36 minuti per lo svolgimento di un esame ceus e 45 , 6 minuti per un esame nella valutazione del costo differenziale ci siamo limitati a considerare latto tecnico , ignorando tutti gli altri costi legati alla gestione del paziente , cos come in altri lavori [ 6 ] ; ci infatti avrebbe comportato lesplicitazione di tutti i rapporti tra la radiologia e gli altri reparti dellazienda ospedaliero - universitaria e , per i pazienti ricoverati , anche del ruolo che le indagini radiologiche hanno sul processo di cura . 
nella nostra analisi abbiamo quindi introdotto esclusivamente i costi dellistituto di radiologia : cos facendo possibile un confronto nel tempo ( rispetto a obiettivi di budget ) , nel volume ( secondo laumento o la diminuzione dei carichi di lavoro ) e nello spazio ( rispetto ad altre aziende ospedaliere ) [ 6 ]  . 
 il costo di utilizzo dei capitali fissi ( cio la tecnologia impiegata ) legato al tempo di impiego , cos come il costo del personale medico , tecnico e infermieristico legato alle specifiche attivit che li vedono coinvolti mediante misure del tempo di attivit . 
il costo del tempo medico , secondo i dati forniti dalla gestione risorse della nostra direzione sanitaria , risulta pari a 1 , 02 al minuto , mentre per quanto riguarda le figure professionali del tecnico di radiologia e dellinfermiere , di 0 , 37 al minuto . 
 il risparmio economico stato calcolato moltiplicando il numero delle lesioni che non hanno richiesto un esame tc per il costo di un singolo esame tc : da questo prodotto abbiamo sottratto il costo differenziale di un esame ecografico n . 
prendendo in considerazione la singola giornata lavorativa dellecografo pari a 12 ore e valutando in 36 minuti il tempo in cui lapparecchiatura impegnata per un esame delladdome , emerge un ecosto apparecchiature per esame di 8 , 43  . costi dei materiali e servizi connessi lesecuzione di un esame ceus prevede in media lutilizzo di : 2 , 4 ml ( mezza dose ) di mezzo di contrasto ecografico sonovue , per un costo di 30 , 68 a paziente , dato il costo confezione di 61 , 36 ; in media 4 pellicole di 2025 cm dal costo di 2 , 98 luna , per un totale di 11 , 92 ; una ago - cannula da 18 g ( 0 , 44 ) ; 13 ml in media di gel per ultrasuoni ( 0 , 41 per flacone da 260 ml , in media bastevole per 20 esami , quindi con un costo per esame di 0 , 02 ) e 5 ml di soluzione fisiologica ( meno di 0 , 01 , in quanto la confezione da 250 ml ha un costo di 0 , 21 e quella da 500 ml un costo di 0 , 31 ) , entrambi dal costo trascurabile . globalmente quindi risulta un costo variabile per esame di 43 , 04  . costo del personale limpegno temporale del medico dovuto al tempo preliminare allesame legato al raccordo anamnestico e al consenso informato , allesecuzione dellesame , allanalisi delle immagini e alla refertazione . 
we did not consider the actual time worked per year including days off work , as we were only analysing mean staff costs for each examination . for the baseline us scan , we calculated the same equipment costs , lower supplies costs two films instead of four and a radiological and nursing staff time of 23 min [ 8 ]  . 
non stato necessario considerare il tempo lavorativo effettivo annuo , comprese le assenze per congedo ordinario , poich abbiamo considerato solo il costo del tempo medio per esaper lesame ecografico basale stato calcolato il medesimo costo apparecchiature , un costo materiali inferiore , comprendente solo due pellicole , e un tempo medico e infermieristico di 23 min [ 8 ]  . 
da quanto esposto risulta che , sommando tutti i parametri considerati , il costo di un esame ceus del parenchima epatico presso il nostro istituto di 101 , 51 , con un costo differenziale di 55 , 15 rispetto allecografia basale ( tabella 1 )  . costi della tomografia computerizzata costi delle apparecchiature ( ammortamenti e manutenzioni ) il costo di acquisto dellapparecchiatura tc da ammortizzare risultato di 1032913  . 
essa viene utilizzata dallistituto di radiologia con contratto di manutenzione dal costo pari a 15000 , 00 annui , che , sommati alla quota di ammortamento di costo annua e considerando un anno di garanzia risulta di 24375 , 00  . 
prendendo in considerazione la singola giornata lavorativa della tc , pari a 12 ore , e quantificando in 45 , 6 minuti il tempo in cui lapparecchiatura impegnata per un esame delladdome , emerge un costo apparecchiature per esame di 68 , 27  . costi dei materiali e servizi connessi si impiegano in media 110 ml di mezzo di contrasto ; impiegando in ugual misura 4 tipi di mezzo di contrasto uroangiografici non ionici monomeri , il cui costo medio per flacone di 200 ml di 56 , 06 , il costo medio per esame quindi di 30 , 80 ; nella documentazione dellesame tc previsto luso di quattro pellicole 3543 cm dal costo unitario di 7 , 93 , per cui il costo medio delle pellicole per esame quantizzabile in 31 , 72 ; una ago - cannula da 18 g ( 0 , 44 )  . 
inoltre in circa lo 0 , 5% dei pazienti sottoposti a esami tc , essendoci unanamnesi allergica positiva , si esegue anche una preparazione farmacologica ( a base di cortisonici e antiistaminici ) per un costo di 4 , in media 0 , 04 in pi per ogni esame . 
in addition , approximately 0.5% of patients have a history of allergy and require pharmacological preparation ( cortisone and antihistamines ) costing 4 euros , leading to an additional 0.04 euros on average for each examination . 
overall , the variable costs per examination were euros 62.96. staff costs radiologist time covers different activities : review of the patients medical history , obtaining informed consent , planning the examination after patient preparation , acquiring the images and viewing them on the monitor and reporting . 
this description shows that the differential cost of a ct scan at our institute is 211.48 euros ( table 1 )  . technicians assistance radiology the external costs all patients imaged by contrast - enhanced studies with organic iodinated contrast agents , as in ct , undergo a blood test to determine serum creatinine , nitrogen and bilirubin , for an overall cost of approximately 5 euros per examination . this cost does not fall under the examination costs , but ct inevitably has a greater impact than ceus on costs external to the radiology department [ 7 ]  . 
per quanto riguarda il costo unitario legato alla professione infermieristica necessaria allo svolgimento dello stesso di 0 , 37 al min , e quindi di 16 , 87 il costo per esame . 
a differenza dellesame ceus necessaria anche la collaborazione del tecnico sanitario di radiologia medica ( tsrm ) ( 0 , 37 al min ) che comporta un costo aggiuntivo di 16 , 87  . 
da quanto esposto risulta come , sommando tutti i parametri considerati , il costo differenziale di una indagine tc risulti presso il nostro istituto di 211 , 48 ( tabella 1 )  . costi esterni in tutti i pazienti sottoposti a indagini contrastografiche che prevedono limpiego di un mezzo di contrasto organo iodato , quale lesame tc , viene effettuato un prelievo ematico per valutare i livelli sierici di creatinina , azotemia e bilirubinemia per un costo complessivo di circa 5 a esame . 
questo costo non rientra tra i costi dellesame , per inevitabilmente la tc viene a pesare maggiormente sui costi esterni alla struttura radiologica rispetto a un esame ceus [ 7 ]  . risparmio per esami ceus e tc per la diagnosi di angiomi nel 2002 abbiamo compiuto 4 tc / 13 ceus , il risparmio risultato stato di 1406 , 97  . 
in totale nei 4 anni il risparmio per le diagnosi di angioma stato di 26576 , 10  . per diagnosticare iperplasie focali nodulari nellanno 2002 abbiamo effettuato 1 tc / 2 ceus , il risparmio non stato significativo . 
in totale nei 4 anni passati il risparmio per le diagnosi di iperplasia focale nodulare stato di 3126 , 60  . per diagnosticare pseudolesioni nel 2002 abbiamo effettuato 4 tc / 22 ceus , risparmiando 2813 , 94  . 
nel 2003 abbiamo effettuato 17 tc / 50 ceus , risparmiando 5158 , 89  . nel 2004 abbiamo effettuato 5 tc / 38 ceus , risparmiando 5158 , 89  . 
 the introduction of ceus for the diagnosis of bfll with indeterminate appearance on baseline us enabled savings of 4 , 377.24 euros in 2002 , 11 , 255.76 euros in 2003 , 16 , 258.32 euros in 2004 and 15 , 164.01 euros in 2005 ; thus , in the 20022005 period , we saved 47 , 055.33 euros ( table 3 )  . discussion the effectiveness and efficiency of production processes are common concerns in italian health care agencies , particularly now that expenditure responsibilities are being transferred to the regions [ 9 ]  . 
our cost - analysis of ceus , unique in its kind , makes use of techniques commonly used in industry that ensure a methodological rigour that reduces the approximations of conventional cost - calculation methods [ 1016 ]  . the data that emerged from our analysis cannot be easily transferred to other radiology departments , as they reflect the unique situation of our facility at a specific point in time , and the costs incurred by other facilities may be very different as a result of different equipment costs and different organisational choices that may affect the cost of supplies or staff costs . 
the value of this analysis is that it proposes a model that can be applied to other settings and can serve as a stimulus for similar comparisons of the costs of two techniques . 
as previously suggested [ 7 ] , it is clear that cost analysis is merely the first step in a cost - effectiveness analysis that encompasses diagnostic benefits , including effects on treatment and biological costs . 
 the high diagnostic accuracy of ceus in the evaluation of bfll is well established , thanks to the diagnostic power of the dynamic and sinusoidal phases [ 15 ]  . 
the performance of ceus and ct in the characterization of bfll is similar despite the differences between the techniques . ceus nonetheless has a lower cost , and our department achieved savings of 47 , 055.33 euros over 4 years . 
the current diagnostic workup for incidentally detected liver lesions with a suspected benign nature includes baseline us , if necessary followed by ct . ceus can be inserted after baseline us to avoid the subsequent ct examination . 
our study considered cases in which ceus , performed as a complement to baseline us , enabled a definitive diagnosis , thus avoiding the use of ct and the differential cost related to its use . 
this distinction does not apply to malignant lesions , which , once detected , are compulsorily imaged by ct for oncological staging ; this explains why malignant lesions were not considered in our study . furthermore , with regard to biological costs , ceus undiscussione lefficacia e lefficienza dei processi produttivi sono argomenti avvertiti quotidianamente nelle aziende sanitarie , soprattutto in questa fase di regionalizzazione delle responsabilit di spesa [ 9 ]  . 
lanalisi dei costi ceus da noi effettuata in questo campo applicativo fa riferimento a tecniche ampiamente utilizzate in campo industriale che garantiscono un rigore che riduce le approssimazioni dei metodi tradizionali di calcolo dei costi [ 1016 ] , e non risulta comparabile a nessun lavoro in letteratura . 
i dati che emergono dalla nostra analisi non sono facilmente esportabili ad altre unit operative : essi rappresentano una fotografia della situazione nella nostra struttura in un certo momento storico , ma evidente che in altre strutture i costi possono essere estremamente diversi , sia per il costo differente delle apparecchiature utilizzate , sia per differenti scelte organizzative che possono incidere sui costi dei materiali o sui costi del personale . 
come gi suggerito [ 7 ] , chiaro che unanalisi dei costi rappresenta solo un primo tassello per giungere a unanalisi costo - efficacia , che consideri quindi da un lato i benefici diagnostici , includendo le ricadute terapeutiche , e dallaltro i costi biologici . 
 lelevata accuratezza diagnostica della ceus nella valutazione delle lfeb ormai un dato acquisito , in virt del potere diagnostico della fase dinamica e della fase sinusoidale [ 15 ]  . 
ceus e tc hanno performance simili nella caratterizzazione delle lfeb , sebbene le due metodiche abbiano differenti peculiarit : la ceus comunque associata a un costo inferiore , visto il risparmio di 47055 , 33 ottenuto nella nostra struttura nellarco di quattro anni . 
il nostro studio considera i casi in cui la ceus , espletata a completamento dellesame ecografico basale , ha condotto alla diagnosi definitiva risparmiando cos il ricorso a un esame tc e quindi il costo differenziale relativo . 
questa distinzione viene meno per le lesioni maligne per le quali , in seguito alla loro identificazione , si ricorre obbligatoriamente alla tc per esigenze di stadiazione oncologica ; per questo motivo esse non sono state considerate nel nostro studio . 
 non vi dubbio inoltre che , per quanto riguarda i costi biologici , la ceus offra chiari vantaggi rispetto alla tc per lassenza di radiazioni ionizzanti , nonch per lutilizzo di un mezzo di contrasto a microbolle con un profilo di tollerabilit migliore rispetto a un prodotto organo - iodato . 
these factors prompted us to replace ct with ceus as a second - line diagnostic procedure in the study of suspected benign lesions on us , despite the fact that ceus does not offer the same panoramic view as ct and is burdened by a small proportion of technical failures related to meteorism or obesity ( approximately 4% )  . 
 the use of ceus for the diagnosis of bfll over 4 years increased by 217% per year , and in 2005 , ceus was carried out in all patients with suspected benign lesions on us , reserving ct for only 17% ( 7 / 41 ) of thethe ceus examination , performed as a complement to baseline us during the same session , simplifies the diagnostic process , especially in terms of management . 
however , the radiologist needs to be perfectly aware of focal liver lesion semiotics during dynamic imaging , and a remote risk of anaphylactic reactions to us contrast material has been reported . the limitations of this study are related to the inclusion criteria : we arbitrarily decided to exclude malignant or suspicious lesions , which always require a second examination for confirmation or staging . 
the reason for this choice was that if malignant lesions had been included , the ceus study would have necessarily represented an additional cost in the diagnostic workup , and no other study would have been avoided . sostituzione della tc con la ceus come diagnostica di secondo livello nello studio delle lesioni ecograficamente sospette , anche se questultima metodica non garantisce la stessa panoramicit di esame ed gravata da una piccola percentuale di incompetenza tecnica , da meteorismo , o da obesit ( 4% circa )  . 
 lincremento di utilizzo diagnostico della ceus nella diagnosi di lfeb nel nostro istituto nellarco di 4 anni stato infatti del 217% annuo ; inoltre , nel 2005 la ceus stata effettuato in tutti i pazienti con lesioni sospette benigne allus , riservando la tc solo al 17% ( 7 / 41 ) di essi . 
midiri sezione di scienze radiologiche del dipartimento di biotecnologie mediche e medicina legale , via del vespro 127 , i - 90100 palermo , italy correspondence to : lo re , tel . : + 39 - 091 - 6518889 ; + 39 - 328 - 4553761 , fax : + 39 - 091 - 6552305 , e - mail : giuseppe.lore12@tin.it received : 28 august 2006 / accepted : 4 december 2006 / published online : 21 september 2007 abstract objective . 
sono stati esaminati 28 pazienti con diagnosi ileocoloscopica ed istologica di morbo di crohn ( mc ) , 6 pazienti con ileoconoscopia negativa per mc sono stati utilizzati come gruppo di controllo . 
le - tcmd con somministrazione orale di soluzione isotonica consente di valutare con elevata accuratezza le alterazioni dellintestino tenue in pazienti affetti da mc , dimostrandosi valida alternativa agli studi radiologici convenzionali , specie nei pazienti che rifiutano lintubazione naso - digiunale . key words gastrointestinal diseases small bowel multidetector ct parole chiave patologie gastrointestinali intestino tenue tc multidetettore introduction introduzione crohns disease ( cd ) is a chronic inflammatory disease that may affect any portion of the gastrointestinal tract and that requires the use of specific techniques for diagnosis , complication detection and follow - up . 
among the numerous techniques that have been used in the diagnosis and follow - up of patients with cd , the most important are conventional enteroclysis , ultrasound ( us ) , computed tomography ( ct ) and il morbo di crohn ( mc ) una malattia infiammatoria cronica che pu interessare il tratto gastroenterico in tutta la sua estensione , e che necessita di metodiche dedicate per la diagnosi di malattia per la ricerca di complicanze , per il follow - up . 
its main limitations include the high radiation dose [ 2 ] , in part related to the need to use fluoroscopy guidance for placement of the nasojejunal tube [ 3 ] , the invasiveness of the intubation procedure and an inability to provide direct information on possible extraparietal involvement and / or possible complications . 
us is a valuable technique for the diagnosis and follow - up of cd [ 4 , 5 ] , partly due to the possibility of using sonographic contrast media [ 6 ] , as it allows direct evaluation of the diseased bowel loops and the degree of inflammatory activity and detection of complications . 
however , in addition to the limitations inherent to us , that is , the presence of bowel gas and operator dependency , further problems include the length of the examination , quantified by parente et al . 
as 2060 min per exam , and the risk of underestimating low - grade stenoses [ 7 ]  . ct , with or without bowel distension ( ct enteroclysis ) , has been proposed as a modality allowing accurate assessment of bowel involvement and , more recently , its performance has been considerably improved by the use of the newer multidetector ct scanners [ 815 ]  . 
reported that mri could be considered an alternative to ct , as its dynamic capabilities provide additional information in the absence of ionising radiation or iodinated contrast media [ 8 ]  . data from the literature demonstrate that mri does not suffer true limitations in the evaluation of patients with cd ; however , mri is less readily available and more expensive . 
 the aim of our paper is to propose an mdct - enteroclysis technique that does not require nasojejunal intubation and to evaluate the techniques diagnostic capabilities in patients affected by cd . materials and methods between june and december 2005 , we studied 28 patients ( 15 women , 13 men ; mean age 38 years ) by mdct enteroclysis at our department . 
the control group consisted of 15 patients with ileocolonoscopy negative for cd who underwent mdct enteroclysis for other reasons ( eight for intestinal bleeding , five for malabsorption syndrome and two for suspected bowel neoplasm )  . 
all patients were instructed to follow a low - residue diet during the 3 days prior to the examination , and on the third day , they received 4 , 000 ml of water and selg - esse 1000 ( promefarm , milan , italy )  . 
this was necessary to eliminate residual faeces , avoid reflux of faeces into the small fia , la tomografia computerizzata ( tc ) e la risonanza magnetica ( rm )  . lenteroclisi rx costituisce lesame radiologico principale nella diagnosi del mc presentando alta sensibilit e specificit [ 1 ]  . 
le principali limitazioni di questa tecnica sono riconoscibili nellalta dose di esposizione per il paziente [ 2 ] , anche a causa della necessit dellutilizzo della guida fluoroscopica per il posizionamento del sondino naso - digiunale [ 3 ] , nellinvasivit conseguente allintubazione naso - digiunale , e nellincapacit di fornire informazioni dirette sulleventuale interessamento extraparietale e / o eventuali complicanze . 
lecografia rappresenta una valida metodica per la diagnosi e il follow - up del mc [ 4 , 5 ] , anche in relazione al possibile utilizzo dei mezzi di contrasto ( mdd ) ecografici [ 6 ] , consentendo la valutazione diretta delle anse intestinali coinvolte dal processo flogistico , oltre che del suo grado di attivit e delleventuale presenza di complicanze di malattia . 
tuttavia , oltre ai limiti propri della metodica ecografica la presenza di gas nelle anse e la dipendenza dalloperatore altre limitazioni allesecuzione dellesame sono da riconoscere nei lunghi tempi di esecuzione , quantificati da parente in un range di 2060 minuti per esame , e nella possibilit di sottostimare stenosi di basso grado [ 7 ]  . 
la tomografia computerizzata ( tc ) , senza o con distensione della anse intestinali ( enteroclisi tc ) , si proposta come metodica in grado di valutare accuratamente linteressamento intestinale in questi pazienti , e , pi recentemente , numerosi progressi sono stati raggiunti dallutilizzazione dei pi evoluti tomografi multidetettore [ 815 ]  . 
altra tecnica ampiamente utilizzata nei pazienti affetti da mc la risonanza magnetica ( rm ) , dotata di elevata risoluzione di contrasto per i tessuti molli e della possibilit di valutazioni multiplanari [ 1618 ]  . 
hanno evidenziato come la rm possa essere considerata una tecnica alternativa allesame tc , capace di fornire ulteriori informazioni anche grazie alla possibilit di eseguire studi dinamici , in assenza di esposizione a radiazioni ionizzanti e a mdc iodati [ 8 ]  . 
i dati della letteratura dimostrano come lo studio rm non mostri reali limiti nella valutazione dei pazienti affetti da mc rispetto allo studio tc , tuttavia bisogna ricordare la minore disponibilit di tomografi a rm e il maggiore costo dellesame . 
 scopo del nostro lavoro quello di proporre una metodologia di esecuzione dellenteroclisi tc multidetettore ( e - tcmd ) senza intubazione naso - digiunale , oltre che quello di valutarne la capacit diagnostica nei pazienti affetti da morbo di crohn ( mc )  . materiali e metodi dal giugno al dicembre 2005 , 28 pazienti ( 15 femmine , 13 maschi ; et media 38 anni ) sono stati sottoposti a e - tcmd presso il nostro dipartimento . 
mdct was performed after the oral administration of approximately 2 , 000 ml of water and macrogol 4 , 000 ( polyethylene glycol , promefarm , milan , italy ) 6090 min before the examination . 
to reduce bowel peristalsis , patients were administered 20 mg of hyoscine - n - butyl bromide ( buscopan , boehringer ingelheim , italy ) iv 10 min before the ct scan . all patients were studied with a 40 - slice mdct scanner ( brilliance 40 , philips medical system , cleveland , oh , usa )  . 
the examinations were performed before and 70 s after the iv administration of 1.5 ml / kg of iodinated nonionic contrast material ( iomeprol , iomeron 400 , bracco , italy ) at a flow rate of 2.53.5 ml / s , using the following scan parameters : collimation , 400.625 mm ; gantry rotation time , 420 ms ; slice thickness , 1.5 mm ; slice increment , 0.7 mm ; 140 kv ; 250 mas . 
data on the acquired volume were transferred to a dedicated workstation where , besides the axial images , multiplanar ( mpr ) , maximum intensity projection ( mip ) and volume rendering ( vr ) reconstructions were visualised . in each patient , we assessed bowel wall thickness ( reference value < 4 mm ) [ 15 ] , layering , contrast enhancement , ulceration , bowel diameter ( reference value < 3 cm ) [ 15 ] , enhancement of the surrounding fat , enlargement of the afferent vasa recta , nodal involvement ( reference value : maximum transverse diameter > 15 mm ) and presence of complications ( e.g. intraand extraperitoneal fistulae or abscesses )  . 
in 19 patients ( 67.9% ) , the mucosa of the thickened loops enhanced intensely after contrast administration , with engorgement of the mesenteric vasa recta ( comb sign )  . 
none of the patients in the conzione ileale e rifiuto allintubazione naso - digiunale ; criteri di esclusione sono stati : insufficienza renale ( con valore di creatininemia maggiore a 2 mg / dl ) , stato di gravidanza o allergia nota al mdc . 
come gruppo di controllo sono stati valutati 15 pazienti con reperto negativo allileocolonscopia , che hanno eseguito le - tcmd per altra causa ( 8 pazienti con sanguinamento intestinale , 5 pazienti con sindrome da malassorbimento e 2 pazienti con sospetta neoplasia intestinale )  . 
in tutti i casi stata consigliata una dieta priva di scorie nei tre giorni precedenti , e al terzo giorno sono stati somministrati 4000 ml circa di una soluzione di acqua e selg - esse 1000 ( promefarm , milano , italia )  . 
questa preparazione si resa necessaria per eliminare i residui fecali , evitare leventuale reflusso di feci nellintestino tenue , e permettere un pi rapido transito del mezzo di contrasto ( mdc ) negativo endoluminale . 
le - tcmd stato eseguito previa somministrazione orale di circa 2000 ml di una soluzione di acqua e macrogol 4000 ( polietilenglicole , promefarm , milano , italia ) , nei 6090 minuti precedenti . 
per ridurre lattivit peristaltica intestinale , 10 minuti prima dellesecuzione della scansione tc sono stati somministrati per via endovenosa 20 mg di n - butilbromuro di joscina ( buscopan , boehringer ingelheim , italia )  . tutti i pazienti sono stati studiati con un tomografo multidetettore a 40 canali ( brilliance 40 , philips medical system , cleveland , ohio , usa )  . 
in tutti i pazienti lesame stato eseguito prima e dopo 70 secondi dalla somministrazione endovenosa ( e.v. ) di 1 , 5 ml / kg di mdc iodato non ionico ( iomeprolo , iomeron 400 , bracco , italia ) con flusso di 2 , 53 , 5 i seguenti parametri : collimazione ml / s , utilizzando 400 , 625 mm , velocit di rotazione del tubo radiogeno 420 millisecondi , spessore di stato 1 , 5 mm , incremento di strato 0 , 7 mm , 140 kv , 250 mas . 
i dati relativi al volume acquisito sono stati trasferiti su una stazione di lavoro dedicata , e , oltre alle immagini assiali , sono state visualizzate ricostruzioni multiplanari ( mpr ) , mip e volume rendering ( vr )  . 
in ogni paziente sono stati valutati : spessore ( valore di riferimento < 4 mm ) [ 15 ] , stratificazione , enhancement post - contrastografico e presenza di eventuali lesioni ulcerative della parete intestinale , calibro delle anse ( valore di riferimento < 3 cm ) [ 15 ] , imbibizione del tessuto adiposo circostante , aumento di calibro dei vasa recta afferenti , interessamento linfonodale ( valore di riferimento : diametro trasverso massimo > 15 mm ) e presenza di complicanze ( es . : tramiti fistolosi o ascessi intra ed extraperitoneali )  . 
 risultati in nessun caso si sono avuti episodi di nausea e / o vomito e tutti i pazienti hanno giudicato accettabile sia la preparazione catartica che la somministrazione orale di polietilenglicole . 
sensitivity and specificity of mdct enteroclysis were 92.8% and 100% , respectively , with a positive predictive value ( ppv ) of 100% and a negative predictive value ( npv ) of 88.2%. 
the main symptoms are diarrhoea , abdominal pain and subocclusion . the development of fistulae ( enteroenteric , enteroabdominal or enterocutaneous ) or severe complications such as bowel occlusion , perforation or intraand extraintestinal abscesses occurs only late in the disease . 
in 19 pazienti ( 67 , 9% ) la mucosa delle anse patologicamente ispessite appariva dotata di intenso enhancement dopo mdc e.v. , con aumento di calibro dei vasa recta mesenteriali ( segno del pettine )  . 
conventional enteroclysis , with its high sensitivity and specificity , has been the most widely used technique for identifying the sites of disease ( often multifocal ) and assessing possible complications . 
among the major limitations of conventional enteroclysis is an inability to provide direct information on the degree of extramural involvement and the presence of complications . to overcome these limitations , investigators proposed the use of ct enteroclysis . 
the first ct studies of the small bowel , in the 1980s and 1990s , demonstrated that ct had a good diagnostic accuracy for identifying the typical signs of disease on the bowel wall and possible complications [ 812 , 18 , 19 ]  . 
to distend the bowel loops , several types of intraluminal contrast media have been used : positive , such as iodinated contrast agents [ 9 ] , negative , such as 0.5% methylcellulose [ 24 ] or , less commonly , water or lipid contrast media [ 25 ]  . controllo erano presenti segni da riferire a malattia infiammatoria dellintestino tenue . 
dai dati del nostro studio la sensibilit e la specificit delle - tcmd sono state rispettivamente del 92 , 8% e 100% ( vpp = 100% ; vpn = 88 , 2% )  . 
solo tardivamente si manifestano tramiti fistolosi ( entero - enterici , entero - addominali o entero - cutanei ) o complicanze gravi come occlusione intestinale , perforazione o formazione di ascessi intraintestinali ed extraintestinali . 
1a contrast - enhanced axial multidetector computed tomography ( mdct ) enterography image shows a segmental luminal narrowing with mural thickening and layering ( * ) of the distal ileum and engorged mesenteric vessels ( arrow )  . 
1a limmagine sul piano assiale dimostra riduzione di calibro e ispessimento parietale di unansa dellileo distale con stratificazione parietale ( * ) e aumento di calibro dei vasi mesenterici ( freccia )  . 
3a - c axial images ( a , b ) show mesenteric lymphadenopathy ( arrowhead ) , engorged mesenteric vessels ( arrow ) and mural thickening ( * ) of the distal ileuc same patient . 
3a - c le immagini assiali ( a , b ) dimostrano linfoadenomegalie mesenteriche ( testa di freccia ) , aumento di calibro dei vasi mesenterici ( freccia ) e ispessimento parietale ( * ) dellileo distale . 
lileocolonscopia rappresenta il gold standard per la valutazione dellileo nel sospetto di malattia infiammatoria , ma questa tecnica invasiva , non in grado di esplorare la totalit dellintestino tenue e non fornisce informazioni riguardanti leventuale coinvolgimento extraparietale del mc . 
lenteroclisi convenzionale ha rappresentato , per la sua elevata sensibilit e specificit , la tecnica maggiormente utilizzata sia per il riconoscimento delle localizzazioni della malattia , spesso multifocale , sia per la valutazione delle complicanze . 
i primi studi tc dellintestino tenue , eseguiti negli anni 80 e 90 , hanno dimostrato come lesame di tomografia computerizzata sia in grado di identificare con buona accuratezza diagnostica le manifestazioni proprie della malattia a carico della parete intestinale e le eventuali complicanze [ 812 , 18 , 19 ]  . 
si quindi pensato di unire la tecnica dellenteroclisi rx [ 20 ] , con intubazione naso - digiunale e somministrazione di metilcellulosa , a quella della tc [ 2124 ]  . 
per la distensione delle anse intestinali sono stati utilizzati diversi tipi di mdc endoluminali , positivi , come i mdc iodati [ 9 ] , o negativi come la metilcellulosa allo 0 , 5% [ 24 ] o , meno frequentemente , lacqua o mdc lipidici [ 25 ]  . 
 una nuova spinta allo studio tc delle patologie dellintestino stata fornita dalla commercializzazione delle nuove apparecchiature tc multidetettore , caratterizzate da una pi elevata risoluzione temporale , che rende possibile lacquisizione del volume addominale in una singola apnea , e da pi elevata risoluzione spaziale lungo lasse z . 
4 immagine assiale che dimostra una irregolarit con ulcerazioni ( testa di freccia ) del profilo di parete , che appare inoltre ispessita ( * ) , dellileo terminale . the advent of the new multidetector ct scanners added momentum to ct imaging of bowel disease , as these have a higher temporal resolution , allowing the acquisition of the entire abdominal volume in a single breath - hold and a highg . 
6 contrast - enhanced axial multidetector computed tomography enterography shows marked abnormal wall thickening and mucosal enhancement in preterminal ( arrow ) and terminal ileum ( arrowhead ) consistent with crohns disease . 
inoltre possibile dimostrare la presenza di una fistola entero - enterica tra il colon e lileo ( * )  . mpr e vr dotate di maggiore dettaglio anatomico , fornendo una iconografia che si avvicina maggiormente a quella della radiologia convenzionale , pi familiare agli specialistici non radiologi [ 26 ]  . 
per ottenere una buona valutazione dellintestino dunque necessario distendere le anse intestinali con mdc endoluminali , somministrabili sia tramite intubazione naso - digiunale che per via orale [ 27 ]  . lintubazione naso - digiunale per scarsamente tollerata dai pazienti , impedendo in alcuni casi lesecuzione stessa dellesame . 
dimostrano la stratificazione parietale e lenhancement post - contrastografico dellileo distale sede di flogosi attiva ( freccia ) e la presenza di una fistola fra lileo ( testa di freccia bianca ) e il sigma ( testa di freccia gialla )  . 
this results in an isotropic voxel that allows the generation of mpr and vr with finer anatomical detail and the production of images that are closer to those of conventional radiology and therefore more familiar to the referring specialists [ 26 ]  . 
to achieve a good evaluation of the bowel , the bowel needs to be distended with intraluminal contrast media , which may be administered either by nasojejunal intubation or orally [ 27 ]  . nasojejunal intubation is poorly tolerated by patients , precluding execution of the examination in some cases . 
our experience demonstrates that the oral administration of a negative intraluminal contrast agent produces adequate bowel distension , especially of the ileum , and is well tolerated by the patient . 
in the first case , some intestinal segments would be inadequately distended and thus poorly assessable , whereas in the second case , excessive gastric distension could cause vomiting as well as prevent adequate bowel distension . 
to ensure that the proximal small bowel is well distended , it may be useful to administer the last 250500 ml of water and polyethylene glycol solution 1520 min before the examination or before dellileo , con buona tollerabilit da parte del paziente . 
i pazienti sono stati istruiti a bere la soluzione lentamente e in modo continuo , evitando cos sia una discontinuit della colonna idrica dellintestino tenue che la possibile sovradistensione gastrica . 
nel primo caso alcuni tratti di intestino si presenterebbero non adeguatamente distesi , e quindi difficilmente valutabili , nel secondo caso leccessiva distensione gastrica potrebbe stimolare il vomito da parte del paziente oltre che impedire , ancora una volta , una adeguata distensione delle anse intestinali . 
la somministrazione degli ultimi 250500 ml della soluzione di acqua e polietilenglicole 1520 minuti prima dellesecuzione dellesame , e comunque prima della somministrazione del n - butilbromuro di joscina , permetterebbe una adeguata distensione anche delle anse prossimali dellintestino tenue . 
infatti bench la fase arteriosa avrebbe potuto permettere una migliore valutazione dellenhancement mucoso della parete intestinale sede di flogosi , precedenti studi in letteratura hanno sottolineato come lesecuzione di ununica fase post - contrastografica compresa fra 40 e 70 secondi dallinfusione del mdc consenta una adeguata valutazione dellenhancement postg . 
in fact , although the arterial phase might have allowed better evaluation of mucosal enhancement , previous studies have reported that the acquisition of a single postcontrast phase 4070 s after contrast administration enables adequate evaluation of mural enhancement . 
for the assessment of nodal enlargement , we decided to use an arbitrary size threshold , recording only lymph nodes with a transverse diameter greater than 15 mm , given that the visualisation of mesenteric nodes is not necessarily linked to disease . 
also , node size may vary widely , and no reference size criteria have been established [ 31 ]  . use of mpr , mip and vr images proved helpful both in identifying the degree and extent of cd and in detecting complications . 
in particular , mpr and mip images reconstructed in the coronal and coronal oblique plane , with different thicknesses depending on the region and the findings to be analysed , were useful for identifying perivisceral lymphadenopathy and evaluating the extent of disease and complications . 
in evaluating complications , the possibility of having mpr reconstructions in operator - defined planes allows better identification of possible enteroenteric or enterocutaneous fistulae , as well as improved assessment of the extension of fistulae and stenotic segments . 
per quanto concerne il riscontro di linfoadenomegalie abbiamo deciso di utilizzare una soglia dimensionale arbitraria evidenziando solo i linfonodi con diametro trasverso massimo maggiore a 15 minfatti , la presenza di linfonodi mesenterici non sempre si associa alla presenza di malattia e la dimensione dei linfonodi pu essere estremamente variabile , non potendo essere utilizzato alcun criterio dimensionale [ 31 ]  . il ricorso alle immagini mpr , mip e vr stato utile sia nella identificazione del grado e della estensione della malattia infiammatoria intestinale , sia nella identificazione delle complicanze . 
in particolare modo , le immagini mpr e mip ricostruite secondo i piani coronale e coronale obliquo , con spessori diversi in base alla regione e ai reperti analizzati , sono state utili nella identificazione delle linfoadenomegalie periviscerali , nella valutazione dellestensione della malattia e nella identificazione delle complicanze . 
in questo ultimo caso , la possibilit di ottenere ricostruzioni mpr secondo piani definiti dalloperatore , ha consentito una migliore identificazione di eventuali tramiti fistolosi entero - enterici ed enterocutanei , oltre che una migliore valutazione dellestensione sia degli stessi tramiti fistolosi sia di eventuali segmenti stenotici del piccolo intestino . 
per quanto riguarda i due pazienti negativi per mc allindagine e - tcmd , riteniamo che lo stadio di malattia molto precoce , caratterizzato allesame endoscopico dalla presenza di scarse ulcerazioni aftoidi dellultima ansa , non abbia determinato delle alterazioni dellanatomia della parete viscerale tali da renderli evidenziabili allesame e - tcmd . conclusioni conclusions mdct enteroclysis with oral administration of isotonic solution is an easy - to - perform technique that has good diagnostic accuracy for evaluating small - bowel alterations , extraparietal complications and mesenteric involvement in patents with cd . 
 le - tcmd con somministrazione orale di soluzione isotonica si dimostrata una tecnica di facile attuazione che consente di valutare con elevata accuratezza diagnostica le alterazioni del piccolo intestino nei pazienti affetti da mc , nonch la presenza di complicanze extraparietali e il grado di interessamento mesenteriale , dimostrandosi valida alternativa agli studi radiologici convenzionali , specie nei pazienti che rifiutano lintubazione naso - digiunale . 
planning , which was performed with the neuronavigator , was based on analysis of the location of the course of the main white matter tracts adjacent to the lesion ( pyramidal tract , optic radiation and arcuate fasciculus )  . 
overall , 40 / 75 tracts studied had no anatomical relation with the tumour , were not displaced by the tumour or could not be visualised in their entire course . 
analysis of the remaining 35 white matter tracts led to an a priori change in the surgical approach for corticotomy in four patients ( 16% ) , with no disagreement between the two neurosurgeons and an impact on the extent of resection during surgery in 17 ( 68% ) , thus an overall impact on the surgical procedure in 80% of cases . 
mediante un software dedicato , sono state ottenute le relative mappe colorimetriche e ricostruite in tre dimensioni le traiettorie dei fasci di sostanza bianca adiacenti alla neoformazione che sono state elaborate per la pianificazione preoperatoria . 
la pianificazione , effettuata mediante il neuronavigatore , si basata sullanalisi della localizzazione e del decorso dei principali fasci di sostanza bianca adiacenti la lesione ( fascio cortico - spinale , radiazione ottica e fascicolo arcuato )  . 
globalmente , dei 75 fasci considerati , 40 non avevano rapporto anatomico con il tumore , non venivano dislocati dallo stesso o non risultavano visualizzabili in tutto il loro decorso . 
despite the high incidence of cases in which the lesion is responsible for changes that hinder the reconstruction of white matter tracts , the technique can change the surgical approach for corticotomy , defines the extent of resection and leads to some change in the procedure in 80% of cases . 
the improvement of pre - existing symptoms and the absence of new symptoms in the postoperative phase , in our opinion , confirms the value of the technique . key words intra - axial brain tumors mri diffusion tensor imaging tractography preoperative planning intraoperative assessment chirurgico di corticotomia in 4 pazienti ( 16% ) senza casi di discrepanza nel giudizio tra i due neurochirurghi e un impatto sulla definizione dei limiti di resezione durante lintervento in 17 ( 68% ) , risultando complessivamente di impatto sulla procedura chirurgica nell80% dei casi . 
nonostante lelevata incidenza di casi in cui la patologia determina modificazioni che inficiano la possibilit di ricostruire i fasci di sostanza bianca , la tecnica modifica lapproccio chirurgico di corticotomia , consente di definire i limiti di resezione e determina globalmente un cambiamento dellintervento nell80% dei casi . 
il miglioramento della sintomatologia pre - esistente e lassenza di nuovi sintomi in fase post - operatoria conferma , a nostro avviso , la sua validit . parole chiave tumori cerebrali intra - assiali rm tensore di diffusione trattografia pianificazione pre - operatoria valutazione intra - operatoria introduction introduzione magnetic resonance imaging ( mri ) has , for years , been used in assessment , localisation and patterns of intracranial tumours . 
the possibility of combining the two techniques has led to implementing the results in preoperative planning for patients with intracranial tumours , thus enabling greater extension of the resection margins [ 4 ]  . it has also been recently demonstrated that tractography data can be integrated in a standard system of neuronavigation , thus offering surgeons intraoperative visualisation of the main white matter tracts [ 5 ]  . 
knowing the position these tracts during surgery can help prevent significant functional damage linked to overextensive resection [ 6 , 7 ] and help modify the approach to the tumour . the aims of our study were to : assess the possibility of identifying precise white matter tracts situated in proximity to an intracranial tumour define the anatomical and topographical relations between the same white matter tracts and the tumour verify the possibility of integrating tractographic images in the context of a stack of three - dimensional ( 3d ) la risonanza magnetica ( rm ) da anni routinariamente utilizzata per la valutazione , la localizzazione e la caratterizzazione dei tumori cerebrali . 
analogamente , si di recente proposto limpiego del tensore di immagine di diffusione ( dti ) come tecnica per identificare in vivo i tratti di sostanza bianca cerebrale [ 13 ]  . 
la possibilit di combinare le due tecniche ha permesso di implementare i risultati nella pianificazione pre - operatoria di pazienti con tumori cerebrali , consentendo una maggiore estensione dei margini di resezione [ 4 ]  . 
 stato inoltre dimostrato in tempi recenti che i dati della trattografia possono essere integrati in un sistema standard di neuro - navigazione , permettendo una visualizzazione intra - operatoria dei principali fasci di sostanza bianca [ 5 ]  . conoscere la posizione dei principali tratti di sostanza bianca durante lintervento chirurgico pu aiutare a prevenire danni funzionali rilevanti legati a resezioni troppo estese [ 6 , 7 ] e modificare lapproccio alla neoplasia . obiettivi del nostro lavoro sono stati : valutare la possibilit di identificare definiti fasci di sostanza bianca , localizzati in prossimit di un tumore cea . 
tutti i pazienti affetti da tumore cerebrale nel periodo suddetto sono stati infatti studiati anche mediante valutazione della trattografia con rm ; questa stata successivamente valutata in sede pre - operatoria per la pianificazione neurochirurgica , e intra - operatoria , nellambito della neuronavigazione , per la definizione dei margini di resezione . 
undici di questi pazienti erano portatori di glioblastoma multiforme ( gbm ) , 6 metastasi singole , 3 gliomi di iii grado , 4 gliomi di ii grado , un linfoma . 
tutti i pazienti sono stati valutati clinicamente sia in fase preche post - operatoria , a 1 mese dallintervento ( tabella 1 ) per definire la presenza di sintomi riferibili a un coinvolgimento dei fasci mielinici oggetto dello studio e verificare quindi , dal punto di vista clinico , lintegrit degli stessi in seguito allintervento . la valutazione clinica stata eseguita sempre dagli stessi neurochirurghi che hanno classificato i sintomi prima e dopo lintervento come peggiorati ( comparsa di nuovi deficit o aggravamento dei precedenti ) , stazionari o migliorati . protocollo di studio rm pre - operatorio il protocollo di studio pre - operatorio prevedeva lacquisizione , presso la rm del reparto di neuroradiologia del nostro istituto , di sequenze finalizzate a caratterizzare la lesione e a mettere in evidenza i rapporti con le strutture circostanti . 
sono state acquisite sequenze t2 , flair , t1 volumetriche isotropiche ( mprage ) prima e dopo somministrazione endovenosa di mezzo di contrasto ( mdc ) paramagnetico , sequenze di diffusione , perfusione e tensore a 6 direzioni . 
lo studio del tensore di diffusione stato effettuato mediante 6 direzioni non collineari ( valore di b = 0 e 1000 s / mm2 ) con sequenze echomaterials and methods patients the population studied was prospectively selected from all patients scheduled to undergo brain neurosurgery in the mrequipped operating theatre ( brain suite , brain lab ) of our institution between april and october 2006 . 
all patients suffering from brain tumours in this period were also studied with mr tractography , which was then assessed preoperatively for surgical planning and intraoperatively for neuronavigation to define the extent of resection . 
all patients were clinically assessed both in the preand postoperative phase 1 month after surgery ( table 1 ) to establish the presence of symptoms related to involvement of white matter tracts studied and therefore to clinically verify their integrity following surgery . clinical assessment was performed by the same neurosurgeons , who compared symptoms prior to and after surgery and classified them as worsened ( onset of new deficits or worsening of previous symptoms ) , unchanged or improved . preoperative mr protocol the preoperative study protocol involved the acquisition , at the mr suite of the neuroradiology division of our institute , of sequences designed to characterise the lesion and show its relations with the surrounding structures . 
the following sequences were acquired : t2 - weighted sequences , fluid attenuated inversion recovery ( flair ) , isotropic volumetric t1 - weighted magnetisation - prepared rapid acquisition gradient echo ( mprage ) sequences before and after intravenous administration of paramagnetic contrast material , and diffusion , perfusion and tensor sequences in six directions . 
the filter tracking technique involved the 3d reconstruction of the white matter tracts using a fractional anisotropy ( fa ) threshold of 0.2 and an angle between consecutive vector lines greater than 35 . 
the positioning of the roi for fibre tracking varied according to the trajectory of the fibres to be reconstructed ( posterior limb of the internal capsule for the pyramidal tract , lateral geniculate nucleus for the optic radiation and centrum semiovale for the arcuate fasciculus ) [ 1113 ]  . 
once the white matter tracts to be studied had been obtained , the volume of the neoplasm was reconstructed three dimensionally to assess relations with the contiguous structures and possible displacement with adjacent white matter tracts . 
mean data processing time for each tract was 23 m data processing was performed double blind by the same two experienced neuroradiologists ( with the possibility of agreement with a third neuroradiologist in the event of disagreement )  . 
the neuroradiologists also assessed whether the white matter tracts were in relation to the neoplasm ( distance from the margins of altered signal ; in the case of stage ii gliomas , less than 1 cm ) , whether and in which direction they were displaced and whether the bundle could be clearly visualised in its course in proximity to the tumour . tractography results were saved in a file containing the x / y / z coordinates for each fibre . 
after rigid registration of the b = 0 images with the anatomical volumetric package , and after having verified that there were no discrepancies between data ( differences greater than 3 mm ) in the region of the tumour , white matter tracts could be displayed in standard anatomical images . 
overall time for this data processing , which was generally performed the day prior to surgery , had a mean duration of 30 min . preoperative mr study in brain suite during the preoperative mr study in the brain suite , we acquired volumetric isometric t1 - weighted images with the same characteristics as those previously obtained without paramagnetic contrast material administration . 
these sequences were the basis for subsequent fusion with the preoperative images and had the aim of recognising the markers . total acquisition time of preoperative images for surgical planning was therefore considerably limited and on average was less than 10 min , centring included . to simplify mr data registration in the navigation system , we used a multichannel coil containing stereotactic markers that are read by the neuronavigator camera and then registered by the neuronavigator itself . 
la tecnica del fiber tracking prevedeva la ricostruzione tridimensionale delle fibre di sostanza bianca utilizzando una soglia di fa di 0 , 2 , e un angolo di elaborazione superiore a 35 . 
il posizionamento delle roi per il fiber tracking variava a seconda delle traiettorie delle fibre da ricostruire ( braccio posteriore della capsula interna per il fascio piramidale , ganglio genicolato per la radiazione ottica e centro semiovale per il fascicolo arcuato ) [ 1113 ]  . 
una volta ottenuto il fascio di sostanza bianca da valutare , stato ricostruito tridimensionalmente il volume della neoformazione per verificarne i rapporti di contiguit e leventuale dislocazione con le fibre di sostanza bianca adiacenti . 
gli stessi hanno inoltre valutato se il fascio mielinico avesse un rapporto di contiguit con la neoplasia ( distanza dai margini di potenziamento o di alterato segnale , in caso di glioma di ii grado , inferiore a 1 centimetro ) , se e in quale direzione fosse dislocato , e infine se il fascio fosse o meno chiaramente visualizzabile nel suo decorso in prossimit del tumore . 
dopo una registrazione rigida delle immagini b = 0 con il pacchetto volumetrico anatomico e dopo aver verificato che non esistessero discrepanze tra i dati ( differenze superiori a 3 mm ) nella zona del tumore , le ricostruzioni delle fibre possono essere visualizzate nel contesto delle immagini anatomiche . 
il tempo globale di questa elaborazione , generalmente eseguita il giorno antecedente allintervento , ha una durata media di circa 30 minuti . studio rm pre - operatorio in brain suite nel corso dello studio rm pre - operatorio in brain suite sono state acquisite immagini t1 volumetriche isotropiche con le medesime caratteristiche di quelle ottenute in precedenza senza somministrazione di mdc paramagnetico . 
as the tumours were prevalently high grade and neurosurgery aimed at debulking rather than total resection , this only occurred in two cases . assessment of planning on neurosurgical outcome the following methodology was adopted to assess the practical impact that the dti information had on surgical planning and procedure performance . 
in the prospective phase , two neurosurgeons ( lf , gda ) were asked to independently assess , and in the event of discordance assess in agreement , the zione delle immagini pre - operatorie per la preparazione del planning in sala operatoria pertanto notevolmente limitato e non supera mediamente , compresi i centraggi , i 10 minuti . per rendere pi facile la registrazione dei dati rm nel sistema di navigazione , viene utilizzata una bobina multi - canale allinterno della quale sono presenti dei marker stereotassici che vengono letti dalla telecamera del neuronavigatore e quindi registrati dal navigatore stesso . 
results obtained in the two phases of the study were tabled as positive or negative for the two parameters ( corticotomy and resection margins ) ( table 2 )  . 
trattandosi prevalentemente di tumori di alto grado in cui la resezione totale non lobiettivo finale della neurochirurgia ( prevalendo il concetto di citoriduzione ) , questa eventualit si realizzata solo in 2 casi . valutazione del planning sullimpatto neurochirurgico per valutare limpatto pratico che linformazione dellimaging del tensore ha avuto sulla programmazione e a . 
identification and relations of the tracts studied with respect to the tumour patient optic radiation pyramidal tract arcuate fasciculus dem slightly displaced inferiorly superiorly displaced incompletely displayed not in contact but inferiorly displaced medially and superiorly displaced medially and inferiorly displaced , in contact no relation proximal portion as a deep landmark medially displaced , not in contact no relation no relation superiorly and medially displaced , in contact superiorly and anteriorly displaced , in contact no relation superiorly displaced , in contact no relation inferiorly displace , not in contact laterally displaced superiorly displaced medially displaced , in contact medially and superiorly displaced , in contact superiorly displaced medially displaced , in contact medially and superiorly displaced , in contact medially displaced distal portion poorly displayed anteriorly and medially displaced , in contact no relation not in contact , cranial portion poorly displayed no relation no relation no relation no relation medially displaced no relation no relation medially displaced , in contact no relation displaced not displayed in temporal portion no relation not displayed in temporal portion not displayed normal and in contact anteriorly no relation not displayed in the frontal portion no relation poorly displayed in the frontal portion poorly displayed in the temporal portion in contact , not displaced no relation medially displaced , in contact displaced after the temporal portion , in contact with the frontal portion not displayed in the temporal portion medially displaced , in contact posteriorly displaced , not in contact no relation not in contact , poorly displayed cranially no relation no relation no relation displayed only in the frontal portion no relation not displayed anteriorly and medially displaced superiorly displaced and poorly slightly displaced medially , in contact displayed no relation not displayed displayed only in the frontal portion slightly displaced anteriorly slightly displaced anteriorly slightly displaced anteriorly no relation not displayed results integration of tractography data in the volumetric data set for the purposes of neuronavigation was technically possible in all cases . 
in the areas of interest ( where the tumour was in relation to white matter tracts ) there was a correspondence between the b = 0 images and the 3d images , with a deviation that did not exceed 3 mthis deviation tended to increase in the more cranial and caudal portions of the brain , which nonetheless were not assessed , as all tumours were central . table 2 summarises relations between white matter tracts studied and the tumour . 
in una prima fase , prospettica , stato chiesto a 2 neurochirurghi ( lf , gda ) di valutare , in cieco e con consenso in caso di discrepanza , il tipo di approccio chirurgico ( sede della corticotomia ) stabilito sulla base delle immagini rm convenzionali prima e dopo contrasto e sulle stesse immagini integrate con limaging trattografico ( con valutazione del fascio cortico - spinale , della radiazione ottica e del fascicolo arcuato )  . 
in una seconda fase ( retrospettiva ) stato chiesto al neurochirurgo operatore di descrivere se la valutazione delle immagini trattografiche riportate sulla neuronavigazione avesse contribuito a definire i limiti di resezione della lesione . 
i risultati ottenuti nelle due fasi dello studio sono stati tabulati in termini positivo / negativo per i due parametri ( corticotomia e limiti di asportazione ) ( tabella 2 )  . 
nelle zone di interesse ( aree di rapporto anatomico tra il fascio mielinico e il tumore ) esisteva una corrispondenza tra le immagini b = 0 e le 3d con una deviazione che non ha mai superato i 3 m questa deviazione tendeva ad aumentare nelle porzioni pi craniali e caudali del cervello , non oggetto tuttavia della valutazione trattandosi in tutti casi di tumori centrali . 
venti dei 75 fasci considerati ( fascio piramidale , arcuato e radiazione ottica per 25 pazienti ) non avevano rapporti con la neoplasia ; 17 ( 23% del totale e 30% dei fasci aventi rapporto anatomico con il tumore ) non sono stati visualizzati ( anche parzialmente ) in relazione allanisotropia indotta dal tumore lungo il loro decorso . 
al controllo a 1 mese un paziente , precedentemente asintomatico , riferiva un disturbo del linguaggio a tipo disfasia sensoriale , riferibile verosimilmente a un danno del fascicolo arcuato ( da notare come , in questo paziente , non fosse stato possibile documentare in tutto il suo decorso questo fascio mielinico per le modificazioni indotte dalledema peri - lesionale ) ; nei restanti 24 la sintomatologia era invariata , con tendenza al miglioramento in 14 dei 17 pazienti sintomatici per coinvolgimento dei fasci mielinici segnalati ( tabella 1 )  . 
una asportazione estesa del tumore pu ridurre il rischio di eventuale recidiva ( specie nei gliomi di basso grado di malignit ) e consente una maggiore efficacia nelle successive terapie radianti o chemioterapiche . 
at 1 - month follow - up , one patient previously asymptomatic reported a speech disorder ( transcortical sensory dysphasia ) , most likely due to damage to the arcuate fasciculus ( it is worth noting that depiction of the entire course of this white matter tract was not possible in this patient due to changes induced by perilesional oedema )  . in the remaining 24 patients , symptoms remained unchanged , with a tendency to improvement in 14 / 17 patients reporting symptoms related to involvement of white matter tracts studied ( table 1 )  . discussion neurosurgery for a brain tumour is a trade - off between maximum surgical resection on the one hand and maximum sparing of functions on the other . 
these techniques have proved useful in establishing relations between the tumour and the surrounding functional cortical areas ( fmri ) and the state of some white matter tracts ( dti )  . 
knowledge of the structural integrity and location of certain white matter tracts with respect to an intracranial lesion is crucial for neurosurgical planning , both for defining the surgical access point and identifying the extent of tumour resection . dti is a significant advancement in the field of diagnostic imaging . 
it is , in fact , the only method capable of displaying cerebral white matter tracts in vivo , and it has been shown that this knowledge can assist the neurosurgeon in preoperative planning [ 1 ]  . 
although this technique has been available for several years , few studies have systematically analysed the impact dti data has on surgical planning and , more importantly , on surgical success [ 5 , 14 ]  . 
the main aim of the present study was , therefore , to assess how often dti data changes the approach to corticotomy and the extent of tumour resection . our study confirms that dti can be used to identify tumour location and its relations with important white matter linguaggio , migliora decisamente la qualit di vita di questi pazienti . 
questi strumenti si sono dimostrati utili per determinare la relazione tra il tumore e le aree funzionali corticali limitrofe ( fmri ) e lo stato di alcune fibre di sostanza bianca ( dti )  . 
la conoscenza dellintegrit strutturale e la localizzazione di alcuni fasci mielinici rispetto a una patologia cerebrale cruciale ai fini del planning neurochirurgico sia per definire laccesso chirurgico alla lesione che per identificare i limiti di resezione dello stesso . 
infatti il solo metodo che pu visualizzare tratti di sostanza bianca nel cervello in vivo , ed stato dimostrato che questa conoscenza pu aiutare il neurochirurgo nel planning pre - operatorio [ 1 ]  . nonostante questa tecnica abbia ormai qualche anno , pochi studi hanno analizzato in modo sistematico quale impatto abbia effettivamente linformazione fornita dal dti sul planning e , in particolare , sul successo chirurgico [ 5 , 14 ]  . nello specifico , luso pi importante del dti quello di confermare pre - operatoriamente lintegrit e la localizzazione dei tratti dislocati . 
lo scopo principale del presente studio stato appunto quello di valutare in che percentuale le informazioni prodotte dalla trattografia modifichino le modalit di un intervento neurochirugico sia nellapproccio alla corticotomia che nella definizione dei limiti di resezione . 
 il nostro studio ha confermato che il dti pu essere utilizzato per rilevare la localizzazione del tumore in rapporto a fibre importanti di sostanza bianca quali il fascio cortico - spinale , la radiazione ottica e il fascicolo arcuato in pazienti con tumori cerebrali . 
in accordo con i dati riportati da questo autore , anche nel nostro studio gli errori di registrazione non superavano i 3 mm , limite che considerato nel range dellintegrazione tra fmri e dati della neuronavigazione [ 7 , 16 ]  . 
questo margine pu tuttavia variare in aree del cervello non centrali in relazione alla distorsione tipica delle immagini echoplanari in prossimit del basicranio e della volta . una seconda considerazione deve essere fatta sulla possibilit che il tumore determini modificazioni dellanisotropia adiacente in grado di inficiare la possibilit di visualizzazione di un fascio mielinico . 
non oggetto del presente studio la ben nota difficolt di definire il significato anatomico e prognostico di tale interruzione visiva delle fibre ( se da infiltrazione , edema o distruzione delle stesse ) e la nostra attenzione si indirizzata solo alla identificazione dei fasci a . 
nell immagine a sono stati rappresentati in 3d il tumore ( giallo ) , il fascio piramidale ( rosso ) , la radiazione ottica ( viola ) , il fascicolo arcuato ( verde )  . 
images a , b show in 3d the tumour ( yellow ) , the pyramidal tract ( red ) , the optic radiation ( green ) and the t1 - weighted magnetic resonance images following contrast enhancement in the coronal and sagittal planes . 
nelle immagini a , b sono stati rappresentati in 3d il tumore ( giallo ) , il fascio piramidale ( rosso ) , la radiazione ottica ( verde ) e le immagini rm pesate in t1 dopo mdc sui piani coronali e sagittali . 
in agreement with the data reported by these authors , registration errors in our study did not exceed 3 mm , a limit considered to be in the range of integration between fmri and neuronavigation data [ 7 , 16 ]  . to define a safety margin , nimsky proposed a distance of 5 mm as reliable for the pyramidal tract . 
this margin may nonetheless vary in noncentral areas of the brain due to the typical distortion of echoplanar images in proximity to the skull base and vault . secondly , the possibility that the tumour causes adjacent anisotropy changes capable of impeding visualisation of white matter tracts needs to be considered . 
our study did not consider the well - known difficulty in defining the anatomical and prognostic significance of this visual interruption of fibres ( caused by infiltration , oedema or fibre destruction ) , as it focused only on identifying specific tracts . 
more comforting results were obtained for analysis of the pyramidal tract ( not displayed and in only its distal portion in only 16% of cases ) and the optic radiation ( not displayed in only 5% of cases )  . 
as expected , due to limited perifocal oedema , there were no documented cases of anisotropy impeding visualisation of white matter tracts in low - grade tumours . despite these intrinsic limitations , dti associated with 3d tractography for the study of white matter tracts in the brain proved highly useful for preoperative planning . 
in a recent study , they noted that the pyramidal tract shift that occurs after resection of a large part of the tumour can be as high as 8 mm [ 5 ]  . 
this observation was confirmed in our study in the two cases in which tractography was repeated during surgery . this underlines that in some cases , when tumour resection is extensive , dti images need to be repeated . 
il fascicolo arcuato risultato quello maggiormente coinvolto e considerando i casi in cui il tumore avesse un rapporto di contiguit con questo fascio la sua identificazione stata possibile solo nel 12% dei casi . 
risultati decisamente pi confortanti sono stati ottenuti dallanalisi del fascio piramidale ( non visualizzabile e solo nella sua porzione pi distale solo nel 16% dei casi ) e della radiazione ottica ( non visualizzata solo nel 5% dei casi )  . 
come atteso , in relazione allo scarso edema perifocale , non sono mai state documentate alterazioni dellanisotropia in grado di inficiare la visualizzazione dei fasci mielinici nelle forme a basso grado di malignit . nonostante questi limiti intrinseci , la tecnica del tensore di diffusione , associata alla trattografia tridimensionale per lo studio dei fasci di sostanza bianca cerebrale , risultata di grande utilit nella pianificazione pre - operatoria . 
dai risultati dal nostro studio , emerso che nel 16% dei casi analizzati , la tecnica ha modificato a priori lapproccio chirurgico di corticotomia , e nel 68% dei casi la conoscenza della posizione dei fasci mielinici ha posto indicazione sui limiti di resezione , con una modificazione globale della procedura neurochirurgica dell80% . 
di recente lo stesso autore ha sottolineato come lo spostamento del fascio piramidale che si realizza dopo lasportazione di buona parte del tumore possa raggiungere gli 8 mm [ 5 ]  . 
tale incidenza potrebbe inoltre essere pi elevata nei tumori con basso grado di malignit , in cui la resezione completa pi rilevante e che rappresentano una esigua percentuale della nostra casistica cos come di quella di nimsky [ 5 ]  . lattendibilit delle immagini trattografiche nella definizione di un fascio mielinico non una problematica di semplice risoluzione . 
 [ 20 ] , there is insufficient agreement between tractography and intraoperative subcortical electrostimulation , whereas another study [ 21 ] suggests there is good agreement between the two techniques . 
although their case series focused on low - grade glial tumours , gambini et al [ 22 ] showed a correspondence between the sites of intraoperative subcortical neurostimulation and tractography in 84% of motor tracts and 79% of language circuits . 
this means that assessment is possible only when resection is near or in the context of the white matter tract , which according to some authors may lead to postoperative transitory ( 37% ) or permanent ( 7% ) neurological deficit . 
in addition , another study [ 23 ] points out that in 50% of cases , nonsubcortical tracts cannot be identified , thus rendering comparison between intraoperative tractography and electrophysiological mapping even more complex . 
comparison is further complicated by the above - mentioned possible brain shift as a consequence of tumour resection . in our study , whereas the possibility of an intraoperative neurophysiological acquisition was not available , we assessed the effective preservation of white matter tracts on the basis of the clinical assessment performed 1 month after surgery . 
indeed , in this patient , the arcuate fasciculus was not displayed in its entirety due to changes induced by perilesional oedema . all remaining patients displayed no new neurological symptoms , and in more than 80% there was an improvement of the preexisting symptoms of neurological deficit . 
 [ 22 ] , seppure su una casistica di tumori gliali di basso grado , hanno dimostrato una corrispondenza tra i siti di neurostimolazione sottocorticale intraoperatoria e la trattografia dell84% per il fascio motorio e del 79% per i circuiti del liguaggio . 
questo significa che la valutazione possibile solo quando la resezione in prossimit o addirittura nel contesto del fascio mielinico potendo , secondo alcuni autori , determinare linsorgenza di deficit neurologici post - operatori transitori ( 37% ) o permanenti ( 7% )  . 
inoltre , secondo altri autori [ 23 ] non possibile identificare alcuna via sottocorticale nel 50% dei casi , rendendo il paragone tra la trattografia intraoperatoria e il mappaggio elettrofisiologico ancora pi complesso . 
a complicare ulteriormente tale confronto bisogna infine considerare la citata possibile dislocazione delle strutture cerebrali in conseguenza della resezione chirurgica . nel nostro studio , non disponendo della possibilit di una acquisizione neurofisiologica intraoperatoria , abbiamo valutato leffettiva preservazione dei fasci mielinici sulla base della valutazione clinica a distanza di tempo . 
questi dati sono in accordo con quanto riportato recentemente da nimsky [ 24 ] in una casistica di 137 gliomi cerebrali in cui lautore , utilizzando la trattografia intraoperatoria con una metodologia sovrapponibile alla nostra ha documentato la comparsa di nuova sintomatologia neurologica dopo lintervento chirurgico in 4 pazienti ( 2 , 9% della casistica )  . sebbene su una casistica limitata e su prevalente patologia ad alto grado di malignit , evidente , dai nostri risultati , che questo tipo di metodica un elemento di rilievo per una corretta pianificazione pre - operatoria e deve essere considerato un avanzamento importante della moderna diagnostica neuroradiologica per la pianificazione chirurgica di pazienti affetti da neoplasia cerebrale . conclusioni conclusions mr tractography offers the neurosurgeon an anatomical panoramic view and the opportunity for improved planning of surgical resection of intracranial lesions . 
despite the high incidence of cases in which the lesion produces changes that impede reconstruction of white matter tracts ( 30% of cases , with the arcuate fasciculus especially affected ) , the technique la tecnica della trattografia con rm offre al neurochirurgo una nuova panoramica anatomica che permette una migliore pianificazione della procedura chirurgica di resezione di patologia cerebrale . 
nonostante lelevata incidenza di casi in cui la patologia determina modificazioni che inficiano la possibilit di ricostruire i fasci di sostanza bianca ( 30% dei casi e specie nel fascicolo arcuato ) , la tecnica modifica lapa . 
algarotti 8 , i - 00137 roma , italy , tel . : + 39 - 349 - 1946942 , e - mail : stepieri@excite.it received : 25 september 2006 / accepted : 25 january 2007 / published online : 21 september 2007 abstract purpose . 
from 19992005 we treated 164 patients with massive pulmonary emboliswe used the same angiographic catheter for mechanical fragmentation and for administration of the fibrinolytic agent ( 2472 h )  . 
after fragmentation with the angiographic catheter , we observed four types of haemodynamic behaviour : in 61 patients ( 41.4% ) , mean pulmonary artery pressure fell rapidly below 30 mmhg ; in 38 patients ( 23.1% ) , two passes were required to achieve the same result ; in 32 patients ( 19.5% ) three passes were required . 
mechanical fragmentation with the angiographic catheter and administration of fibrinolytic agents effectively brought about a rapid improvement in patients clinical status by moving the embolus towards the periphery . key words pulmonary embolism mechanical fragmentation pharmacological thrombolysis riassunto obiettivo . 
la procedura interventistica stata effettuata direttamente con lo stesso catetere angiografico , sia per la terapia meccanica , che per linfusione del farmaco fibrinolitico nei giorni successivi ( 2472 ore )  . 
la frammentazione meccanica stata effettuata con un solo passaggio del catetere angiografico in 61 ( 41 , 4% ) pazienti e la pressione polmonare media scesa rapidamente al di sotto del valore di 30 mmhg . 
in altri 38 ( 23 , 1% ) casi si reso necessario ricorrere a due passaggi per raggiungere lo stesso risultato ; in 32 pazienti ( 19 , 5% ) si dovuto ricorrere a pi di 3 passaggi . 
la frammentazione meccanica con il semplice catetere angiografico si dimostrata valida e sicura per imprimere un brusco miglioramento delle pressioni polmonari e del generale stato clinico del paziente , mobilizzando lembolo polmonare dalla sua iniziale sede centrale . 
agresti : hybrid treatment with angiographic catheter in massive pulmonary embolism : mechanical fragmentation and fibrinolysis introduction introduzione pulmonary thromboembolism is a relatively common clinical condition [ 1 ]  . 
administration of fibrinolytic agents alone , either systemically or locally via a catheter , may fail to resolve the clinical picture [ 3 ] ; in fact , the severe haemodynamic condition needs to be corrected rapidly by lowering the mean pulmonary artery pressure and improving heart function [ 4 ]  . in 1995 , a technique for pulmonary thrombus fragmentation with the use of a modified pigtail catheter was introduced [ 5 ]  . 
in addition , the few published studies , which are based on small patient populations , show that there is not a great deal of experience with the use of such devices in this highly select field [ 7 , 8 ]  . 
special expertise is also required , and not all interventional radiologists may possess such expertise [ 9 ]  . our approach in cases of massive pulmonary embolism has been to systematically use a simple hybrid treatment involving manual fragmentation of the thrombus / embolus with a conventional angiographic catheter in association with fibrinolytic therapy administered via the same catheter over the following 3 days . 
the aim of the paper is to report the clinical and therapeutic considerations developed with our comparatively large patient population . materials and methods between 1990 and 2005 , 410 patients ( mean age 68 years , range 3578 ) were referred to our division for treatment of severe pulmonary thromboembolis of these , 164 were haemodynamically unstable due to the presence of a massive embolism , and this group was systematically treated by mechanical fragmentation of the thrombus and administration of fibrinolytic agents . the diagnosis of pulmonary embolism was made with computed tomography ( ct ) angiography . 
inclusion criteria for the hybrid combined mechanical and fibrinolytic treatment were severe hypotension ( ap < 90 mmhg ) and shock , angiographic confirmation of massive pulmonary embolism with involvement of the central pulmonary arteries and / or a pulmonary artery with flow diversion towards the contralateral side , and mean pulmonary artery pressure greater than 35 mmhg . hybrid treatment consists of a diagnostic phase and an interventional phase . 
nella variante massiva , lembolia polmonare presenta un grave quadro clinico , che pu mettere a repentaglio la vita del paziente e necessita di una rapida soluzione terapeutica [ 2 ]  . 
la sola somministrazione di farmaci fibrinolitici , per via sistemica o diretta localmente con il catetere , pu non risolvere il quadro clinico [ 3 ] ; c la infatti la necessit di migliorare il grave quadro emodinamico , con labbassamento della pressione polmonare media e il miglioramento del lavoro cardiaco [ 4 ] , in tempi estremamente rapidi . nel 1995 , stata introdotta la tecnica di frammentazione del trombo polmonare con limpiego di un catetere pig - tail modificato [ 5 ]  . 
successivamente , sono stati presentati anche altri presidi meccanici con lo scopo di diminuire i tempi della terapia fibrinolitica e di migliorarne lefficacia [ 6 ] , non solo sul versante arterioso . 
i vari presidi commerciali per , soprattutto per i loro costi , non sempre sono disponibili in una sezione di radiologia interventistica ; inoltre , dai pochi articoli presenti in letteratura , basati su una casistica molto limitata , si evince che c una limitata esperienza di un loro impiego in questo campo molto selezionato [ 7 , 8 ]  . 
richiesta inoltre una particolare perizia , che pu non essere patrimonio di tutti i radiologi interventisti [ 9 ]  . nei gravi quadri clinici abbiamo impiegato sistematicamente un semplice trattamento ibrido : una frammentazione manuale del trombo / embolo , effettuata sempre con un semplice catetere angiografico , associata a una terapia fibrinolitica , praticata dallo stesso catetere per i successivi 3 giorni . 
obiettivo del lavoro stato quello di riportare , sulla base di una ampia casistica , le considerazioni cliniche e terapeutiche maturate durante questa esperienza . materiali e metodi tra il 1990 e il 2005 , sono giunti al nostro servizio 410 pazienti per il trattamento di una tromboembolia polmonare grave : let media stata di 68 anni ( range 3578 ) ; tra di loro , 164 erano in condizioni dinstabilit emodinamica per la presenza di una embolia massiva , per cui stato applicato sistematicamente il trattamento meccanico di frammentazione del trombo seguito da infusione di farmaci fibrinolitici . la diagnosi di embolia polmonare era stata effettuata con angio - tc . 
i fattori di rischio maggiormente evidenziati erano la presenza di un intervento chirurgico nellanamnesi recente ( 48 pazienti ; 29 , 2% ) , una prolungata immobilizzazione ( 33 pazienti ; 20% ) , una sindrome dipercoagulabilit ( deficit di proteina c , antitrombina iii ; 51 pazienti ; 31% ) , combinazione di pi fattori ( 32 pazienti ; 19 , 5% )  . 
agresti : hybrid treatment with angiographic catheter in massive pulmonary embolism : mechanical fragmentation and fibrinolysis tially performed with brachial access using the venous cannula previously inserted at the time of patient admission ; when that was not possible , a femoral access was used . 
after administering local anaesthesia and substituting the needle cannula with a 7 - fr introducer sheath ( 4560 cm , arrow , reading , usa ) , we inserted the hydrophilic guidewire ( jtip , 180 cm , 3 cm floppy tip ; terumo , gadagroup , rome , italy ) , and then over this the angiographic catheter ( 145 pigtail catheter , 120 - cm long , 6 - fr diameter ; cordis , cordis europe , roden , the netherlands )  . 
the catheter enabled us to both measure the mean pulmonary artery pressure and administer the contrast material ( 40 ml at 20 ml / s in the left and right pulmonary arteries and 30 ml at 15 ml / s in the lobar branches ; optiray 300 , tyco healthcare spa , milan , italy ) to assess both the angiographic picture and guide the interventional procedure . the interventional phase of the hybrid treatment involved administration of 300 , 000 iu of urokinase ( urochinasi , ph&t spa , milan , italy ) directly via the pigtail catheter and mechanical fragmentation of the thrombus / embolus . 
the tip of the stiff portion of the guidewire was brought to about 1 cm from the beginning of the distal portion of the pigtail catheter to provide the necessary stiffness to the entire device . 
under direct fluoroscopic control , the angiographic catheter was rotated clockwise inside the pulmonary artery ( 510 rotations ) while at the same time being advanced or withdrawn over the guidewire in a craniocaudal direction . 
when required , mechanical fragmentation was repeated , with further cycles of ten rotations at a time , progressively moving peripherally ( lobar arteries ) or to the contralateral lung if pulmonary artery pressure failed to drop to below 30 mmhg . immediate outcome of the fragmentation procedure was assessed on the basis of the constant measurement of mean pulmonary artery pressure , the findings of pulmonary arteriography and the general haemodynamic status ( restored normotension )  . 
outcome was considered good when mean pulmonary artery pressure fell to 25 mmhg with the first pass of the catheter with clearance of the embolus from the central pulmonary arteries and the re - establishment of systemic arterial pressure above 90 mmhg . 
outcome was moderate if pulmonary artery pressure fell to 30 mmhg and poor when it only fell to 35 mmhg . in the following days , regardless of the pressure level reached after fragmentation with the pigtail catheter , fibrinolytic therapy was instituted in all patients . 
after having replaced the introducer sheath with a shorter one ( 8 fr , 11 cm ) , 50 , 000 iu / h urokinase was administered directly via the angiographic catheter , the tip of which was placed in the branches still affected by the fragments of the pulmonary embolis treatment was continued for 4872 h , with assessment of coagulation parameters every 34 h [ fibrinogen and international normalised ratio ( inr ) ] and angiographic and haemodynamic follow - up ever 24 h . 
in anestesia locale , dopo aver sostituito lago cannula con un introduttore con valvola 7 fr ( lungo 4560 cm , arrow , reading , usa ) , stato prima fatto scorrere il filo guida idrofilico ( punta a j , lungo 180 cm , punta floppy di 3 cm terumo , gadagroup , roma , italia ) e poi , su questo , il catetere angiografico ( pig - tail angolato a 145 , lungo 120 cm , calibro 6 fr cordis , cordis europe , roden , olanda )  . 
con questo stato possibile sia misurare la pressione polmonare media , sia iniettare mezzo di contrasto ( 40 ml a 20 ml / s nelle arterie polmonari destra e sinistra , 30 ml a 15 ml / s nei rami lobari optiray 300 , tycohealthcare spa , milano , italia ) sia per valutare il quadro angiografico , sia per indirizzare la procedura interventistica . la fase interventistica del trattamento ibrido , dopo infusione in bolo di 300000 ui di urochinasi ( urochinasi , ph&t spa , milano , italia ) direttamente dal catetere angiografico , prevedeva la frammentazione meccanica del trombo / embolo . nel catetere angiografico stata inserita la guida idrofilica per la parte caudale , di maggior supporto ; la punta della porzione rigida del filo guida stata portata fino a 1 cm circa dallinizio della porzione distale del catetere pig - tail , per fornire la necessaria consistenza a tutto il presidio . 
sotto diretto controllo fluoroscopico , il catetere angiografico stato girato in senso orario nellarteria polmonare ( 510 rotazioni ) , associandovi contemporanei movimenti di traslazione in senso craniale e caudale . 
ove necessario , la frammentazione meccanica veniva ripetuta , con altri cicli di 10 rotazioni per volta , progredendo sempre pi perifericamente ( arterie lobari ) , o aggredendo il polmone controlaterale se la pressione polmonare non scendeva sotto i 30 mmhg . il controllo degli esiti immediati della procedura di frammentazione veniva effettuato con la costante misurazione della pressione polmonare media , con le risultanze dellarteriografia polmonare e con il quadro emodinamico generale ( ristabilimento della normotensione )  . 
veniva considerato un buon risultato terapeutico il raggiungimento di un valore di pressione polmonare di 25 mmhg gi al primo passaggio , con contemporanea disostruzione delle arterie polmonari pi centrali e il ristabilimento di una pressione arteriosa sistemica oltre i 90 mmhg ; discreta era considerata la discesa della pressione nellarteria polmonare a 30 mmhg ; scarso era considerato il raggiungimento di un livello di 35 mmhg . in tutti i pazienti , indipendentemente dal valore pressorio raggiunto dopo la frammentazione meccanica con il catetere angiografico , dopo aver sostituito lintroduttore con valvola con uno pi corto ( 8 fr , 11 cm ) , la terapia veniva continuata , nei giorni successivi , con la somministrazione di farmaci fibrinolitici ( urochinasi 50000 ui / h ) direttamente dal catetere angiografico , la cui punta era posizionata nei rami ancora maggiormente impegnati dai frammenti dellembolia polmonare . 
agresti : hybrid treatment with angiographic catheter in massive pulmonary embolism : mechanical fragmentation and fibrinolysis results patients were treated within 812 h of onset of symptoms . there were 122 cases ( 74.3% ) of brachial access , 38 ( 23% ) of femoral access and only four cases of jugular access . mean pulmonary artery pressure prior to the diagnostic phase was 46 mmhg ( 5239 mmhg )  . 
injection of fibrinolytic agent alone , before contrast injection , did not substantially decrease the pulmonary artery pressure values ( from 46 to 45.1 mmhg ) , and the difference was not considered to be statistically significant . 
pulmonary artery involvement was right - sided in 62 patients , left - sided in 68 and bilateral in 34 . mechanical fragmentation was performed with a single cycle ( ten rotations of the catheter ) in 68 patients ( 41.4% ) , with mean pulmonary artery pressure dropping rapidly below 30 mmhg , haemodynamic parameters improving substantially and subjective symptoms improving ( less feeling of anxiety and oppression )  . 
labial cyanosis had disappeared , patients were able to lie supine without dyspnoea and no longer needed supplementary oxygen . two fragmentation cycles ( 20 rotations of the catheter ) were required in 38 patients ( 23.1% ) before mean pulmonary artery pressure fell below 30 mmhg . 
however , patients continued to be dependent on oxygen , with the appearance of dyspnoea at the slightest physical exertion . in 26 patients ( 15.8% ) , despite more than six cycles being performed , mean pulmonary artery pressure at no time fell below 35 mmhg . 
in all of these patients , the pulmonary embolism had simultaneously involved both pulmonary arteries la valutazione dei parametri della coagulazione ogni 34 ore ( fibrinogeno e inr ) e il controllo angiografico ed emodinamico ogni 24 ore . 
non sono stati considerati in questo studio i pazienti trattati anche con un trombolizzatore meccanico . risultati i pazienti , giunti nellarco di 812 ore dalla comparsa della sintomatologia , sono stati trattati con accesso brachiale in 122 casi ( 74 , 3% ) , femorale in 38 ( 23% ) e giugulare solo in 4 casi . 
la pressione polmonare media , misurata prima di iniziare la fase diagnostica , era di 46 mmhg ( 5239 mmhg )  . la sola iniezione del farmaco fibrinolitico , prima diniettare il mezzo di contrasto , non ha mai sostanzialmente modificato i valori delle pressioni polmonari : si passati da 46 a 45 , 1 mmhg : tale differenza di pressione non stata giudicata statisticamente significativa . 
il coinvolgimento delle arterie polmonari era preferenzialmente destro in 62 pazienti , sinistro in 68 , bilaterale in 34 . la frammentazione meccanica stata effettuata con un solo passaggio ( 10 rotazioni del catetere ) in 68 pazienti ( 41 , 4% ) ; in questi la pressione polmonare media scesa rapidamente al di sotto del valore di 30 mmhg , con un miglioramento sostanziale dei parametri emodinamici e un miglioramento della sintomatologia soggettiva ( miglioramento del senso dangoscia e di oppressione )  . 
 in altri 38 ( 23 , 1% ) si reso necessario ricorrere a due passaggi di frammentazione ( 20 rotazioni del catetere ) prima di verificare labbassamento della pressione polmonare media sotto i 30 mmhg . 
after mechanical fragmentation with angiographic the catheter , a sudden drop in mean pulmonary artery pressure is recorded , despite peripheral dispersion of the embolus fragments ( b )  . 
la preventiva arteriografia polmonare mostra unembolia polmonare massiva con interessamento di entrambe i polmoni ( a ) : il quadro , subostruttivo a destra e ostruttivo a sinistra , si associa a una pressione polmonare media di 46 mmhg . 
dopo la frammentazione meccanica con il catetere angiografico si registra un brusco abbassamento della pressione polmonare media , nonostante la dispersione periferica di frammenti di emboli ( b )  . 
mechanical fragmentation with the same angiographic catheter leads to a coarse rupture of the embolus with dislocation in peripheral segmental branches but with substantial immediate haemodynamic improvement ( mean pulmonary artery pressure 42 mmhg ) ( b )  . fragmentation is repeated in the upper lobe ( c )  . 
the result after 72 h of therapy is the near complete reperfusion of the right lung , where some peripheral perfusion defects remain in the lower lobe ( d )  . 
in six other patients , administration was suspended due to a further drop in serum fibrinogen . discussion acute massive pulmonary embolism is a severe and potentially fatal clinical condition [ 10 ]  . 
agresti : hybrid treatment with angiographic catheter in massive pulmonary embolism : mechanical fragmentation and fibrinolysis cardiac conditions and the progressive onset of right heart failure [ 11 ]  . 
criteria for distinguishing massive pulmonary embolism are the presence of systemic arterial hypotension ( < 90 mmhg ) and cardiogenic shock ( hypoperfusion , hypoxia , oliguria , altered level of consciousness ) [ 3 ]  . 
survival is therefore dependent on rapid recanalisation of the pulmonary artery and quick reduction of right heart workload . whereas the use of surgery is precluded in acute forms [ 3 , 12 , 13 ] because of the severity of the patients conditions , the invasiveness of the procedure and the rapidity required thrombolytic treatment alone is a valid supplement to heparin therapy for treating pulmonary embolisit has shown fig . 
al termine della terapia fibrinolitica di 72 ore , permangono difetti di perfusione in entrambi i polmoni ( c ) , con una pressione polmonare media rimasta a valori di 30 mmhg . terzo giorno di trattamento , per insufficienza cardiaca . 
in 16 pazienti stato impiantato un filtro cavale per levidenza di una trombosi residua a livello degli arti inferiori . non abbiamo registrato episodi di emorragia nella sede di puntura , mentre in 32 casi ( 19 , 5% ) labbassamento del livello di fibrinogeno al di sotto del livello di sicurezza ( 125 mg / dl ) ha imposto il dimezzamento della dose somministrata ogni ora , quale misura precauzionale ; in altri 6 pazienti la somministrazione stata sospesa per lulteriore abbassamento della fibrinogenemia . discussione lembolia polmonare acuta massiva , o maggiore , un grave quadro clinico , che pu mettere a repentaglio la vita del paziente [ 10 ]  . 
i pazienti sono a serio rischio di decesso nellarco di poche ore , in relazione alle preesistenti condizioni cardiache e per il progressivo instaurarsi di una insufficienza del cuore destro [ 11 ]  . 
i criteri per differenziare lembolia polmonare in forma massiva sono la presenza dipotensione arteriosa sistemica ( < 90 mmhg ) e di shock cardiogeno ( ipoperfusione , ipossia , oliguria , livello di coscienza ) [ 3 ]  . 
mechanical fragmentation with repeated passes only succeeds in breaking the embolus into very large pieces , which occupy the peripheral vessels ( b ) and almost totally exclude them from the circulation . 
larteriografia polmonare mostra la presenza di un grosso embolo nellarteria polmonare sinistra ( a ) ; la frammentazione meccanica con passaggi ripetuti in grado di rompere lembolo in parti molto grandi , che impegnano i vasi periferici ( b ) , escludendoli quasi totalmente dalla circolazione . 
la terapia fibrinolitica in grado di apportare limitati benefici ai frammenti centrali , mentre risolutiva solo per quelli pi periferici ( c )  . quindi , dipende dalla rapidit di ricanalizzazione dellarteria polmonare e dal brusco abbassamento del carico di lavoro a cui sottoposto il cuore destro . se il trattamento chirurgico improponibile nelle forme acute [ 3 , 12 , 13 ] vista la gravit del quadro clinico , la traumaticit dellatto e la rapidit richiesta la sola terapia trombolitica una valida aggiunta alla terapia eparinica per trattare lembolia polmonare . 
si dimostrata valida nel ridurre la mortalit e le recidive [ 14 ] , ma richiede tempo , dosi farmacologiche elevate e assistenza intensiva . viceversa , la frammentazione meccanica ha il vantaggio di conseguire una rapida ricanalizzazione dei vasi polmonari ; il razionale della procedura si basa sul fatto che il volume dellembolo relativamente largo se confrontato con le sole arterie polmonari principali ; viceversa molto pi piccolo se confrontato con il letto polmonare periferico . 
inoltre , la dispersione dellembolo nei rami di suddivisione periferica riduce drasticamente la pressione polmonare , aumenta il flusso disponibile per lo scambio gassoso in rami funzionalmente esclusi in precedenza , diminuisce le resistenze periferiche e il lavoro del cuore destro , migliorando lemodinamica generale . 
non ultimo , la frammentazione dellembolo incrementa la superficie di azione per il farmaco fibrinolitico , accelerando la lisi [ 4 ]  . la procedura di frammentazione stata introdotta nel to be effective in reducing mortality and recurrences [ 14 ] , although it does require time , high doses of fibrinolytic agents and intensive care . in contrast , mechanical fragmentation has the advantage of achieving rapid recanalisation of the pulmonary vessels . the rationale behind the procedure is based on the fact that the embolus is relatively large when compared with the main pulmonary arteries alone , although much smaller when coms . 
in addition , dispersion of the embolus towards the peripheral branches drastically reduces pulmonary artery pressure , increases the flow available for gas exchange in the branches functionally excluded previously , and lowers peripheral resistance and workload of the right heart , thus improving general haemodynamics . 
moreover , fragmentation of the embolus increases the surface area upon which the fibrinolytic agents can act , thus accelerating lysis [ 4 ]  . mechanical fragmentation with a special catheter was introduced in 1995 [ 5 ]  . 
the rotational movement of the tip of the modified pigtail catheter acts directly on the embolus within the pulmonary arteries , causing fragmentation and peripheral dislocation of the smaller fragments . 
in contrast , we employed a conventional pigtail catheter , using the stiff portion of the guidewire to provide greater support to the peripheral or coiled portion of the catheter . 
in this way , we were able to increase the correspondence between the rotational motion of the catheter tip and the motion imparted externally by the hands of the operator . 
in addition , both during and at the end of the rotations , we were able to verify the drop in mean pulmonary artery pressure values , improvement of haemodynamic state and peripheral dislocation of the smaller emboli in real time . 
other studies have reported similar behaviour using conventional angiographic catheters [ 4 , 1519 ] or devices for mechanical thrombolysis [ 6 , 20 , 21 ]  . the different behaviour of the emboli that we encountered with pigtail catheter fragmentation has also been reported in other studies : composition and morphology of emboli varies according to patient age [ 22 ]  . 
the stabilisation period of a thrombus varies from 2 to 33 days ( mean 10 ) , with the process being slower in more compromised patients [ 26 , 27 ]  . 
the different response of the emboli to mechanical fragmentation prompts a number of considerations that may have repercussions for the later management of the patient . on the basis of our experience and several studies published in the literature [ 6 , 16 , 17 , 28 ] , pulmonary emboli and their behaviour following mechanical fragmentation can be divided into four classes : type i : recently formed ( < 5 days ) and unorganised thrombi which have recently embolised . 
further haemodynamic improvement , with the re - establishment of near normal pulmonary artery pressure values , obtained with the administration of fibrinolytic agents over the following days , was further indirect confirmation of the recent formation of the clots and therefore of a greater fragility of the embolic material . 
it is likely that 1995 , con lausilio di un catetere speciale [ 5 ] ; il movimento rotatorio della porzione finale del catetere angiografico modificato agisce direttamente sullembolo , allinterno delle arterie polmonari , causando una frammentazione e una dispersione periferica dei frammenti pi piccoli . 
viceversa , noi abbiamo impiegato un comune catetere angiografico , fornendo un maggior supporto alla porzione pi periferica del catetere , quella arrotolata , con la porzione pi rigida del filo guida ; in questo modo stato possibile anche aumentare la corrispondenza tra movimento rotatorio della punta del catetere e quello impresso esternamente dalle mani delloperatore . dopo alcune torsioni della coda del catetere possibile seguire , sotto diretto controllo fluoroscopico , il movimento rotatorio della punta , in grado di frammentare lembolo . 
inoltre , sia durante che al termine delle rotazioni , stato possibile verificare , in tempo reale , il decremento dei valori di pressione polmonare media , il miglioramento dello stato emodinamico del paziente e la dispersione periferica degli emboli pi piccoli . 
anche altri autori hanno riscontrato lo stesso comportamento utilizzando comuni cateteri angiografici [ 4 , 1519 ] o i presidi per la trombolisi meccanica [ 6 , 20 , 21 ]  . il differente comportamento degli emboli , che abbiamo riscontrato con la semplice frammentazione del catetere angiografico , gi stato segnalato da altri autori : la composizione e la morfologia degli emboli varia con let del paziente [ 22 ]  . inoltre , i trombi tendono a retrarsi poco dopo che si sono formati ; i filamenti di fibrina vengono accollati , il siero viene espulso dal coagulo [ 23 ]  . 
con la retrazione o la compressione meccanica , il coagulo perde oltre il 90% delliniziale contenuto di plasminogeno e diventa sempre pi resistente al trattamento trombolitico [ 24 , 25 ]  . 
sulla base della nostra esperienza , confortata anche dalle esperienze di altri autori [ 6 , 16 , 17 , 28 ] possibile suddividere gli emboli polmonari e il loro comportamento dopo frammentazione meccanica in 4 classi : tipo i : pazienti con trombi di recente formazione ( < 5 giorni ) , non organizzati , che hanno embolizzato di recente . 
abbiamo registrato questa evenienza in 68 pazienti ( 41% ) ; sono quelli che hanno risposto meglio alla frammentazione meccanica con il solo catetere angiografico ; la brusca discesa dei valori pressori durante o al termine della frammentazione ne stata la diretta conseguenza . 
lulteriore miglioramento emodinamico , con ritorno ai valori quasi normali di pressione polmonare , ottenuto dopo linfusione di farmaco fibrinolitico nei giorni successivi , ha fornito lulteriore conferma indiretta della loro recente formazione e quindi di una maggiore friabilit del materiale embolico . 
probabilmente , tali emboli sarebbero andati incontro a risoluzione spontanea nei mesi successivi , ma abbiamo preferito comunque impiegare il farmaco fibrinolitico per aiutare il paziente a risolvere lepisodio acuto e diminuire il rischio dinsorgenza di una ipertensione polmonare . tipo ii : pazienti con trombi relativamente recenti ( < 10 giorni ) , o inizialmente organizzati , che hanno embolizzato s . 
agresti : hybrid treatment with angiographic catheter in massive pulmonary embolism : mechanical fragmentation and fibrinolysis these emboli would have spontaneously resolved over the following months , but we preferred to use the fibrinolytic agent to help the patient overcome the acute episode and reduce the risk of developing pulmonary hypertension . type ii : relatively recent ( < 10 days ) or initially organised thrombi that have recently embolised . 
once the proximal portion of the embolus has been damaged , the fragments that break away are coarser than the previous ones and , importantly , occupy the segmental branches and alter pulmonary perfusion . 
in these cases , continuing therapy in the following days with the administration of fibrinolytic agents is fundamental for attacking the softer portions of the embolus impacted in the segmental branches . 
in our experience , this type of embolism was seen in 38 patients ( 23% )  . type iii : more organised and less recent ( < 15 days ) thrombi , where mechanical fragmentation with the angiographic catheter required a greater number of passes before achieving a significant decrease in mean pulmonary artery pressure . 
the course in these patients , where the severe clinical picture is not completely resolved , will most likely be characterised by the onset of progressive pulmonary hypertension [ 29 ]  . type iv : older ( > 15 days ) and significantly organised thrombi . 
once they have broken away from the deep venous circulation , they present a very hard proximal portion of the embolus ( the part directed towards the guidewire ) , which cannot easily be fragmented with the simple movement of the angiographic catheter or be pierced with the guidewire . 
only 26 patients ( 15.8% ) had this type of embolis these patients required greater clinical care ( the placement of 17 inferior vena cava filters ) and received the least benefits from fibrinolytic treatment ( persistence of high levels of pulmonary artery pressure at the conclusion of the 3 - day therapy )  . continuous infusion of the fibrinolytic agent after mechanical fragmentation is performed because it is able to further improve haemodynamic condition by exploiting the greater surface area exposed to the fibrinolytic agent obtained by fragmentation of the embolus and the pressure difference . 
in questi casi , la prosecuzione della terapia , nei giorni seguenti , con la somministrazione del farmaco fibrinolitico di fondamentale importanza per aggredire le porzioni pi soffici dellembolo , quelle incuneate nei rami segmentali . 
nella nostra esperienza a questo gruppo di pazienti sono riconducibili 38 casi ( 23% )  . tipo iii : pazienti con una storia di trombi maggiormente organizzati , di data pi antica ( < 15 giorni ) , dove la frammentazione meccanica con catetere angiografico richiede un numero superiore di passaggi , prima di ottenere un significativo abbassamento della pressione polmonare media . 
nei giorni successivi , lazione fibrinolitica in grado di apportare solo lievi miglioramenti angiografici ( si sono mantenuti grossolani difetti di perfusione ) , emodinamici ( la pressione polmonare media non scesa sotto i 28 mmhg ) e clinici ( i pazienti seppure senza vistosa dispnea , a riposo continuavano ad avere una dipendenza dallossigeno e compariva dispnea al minimo impegno fisico )  . 
in questi pazienti , con risoluzione incompleta del grave quadro clinico , molto probabilmente levoluzione sar caratterizzata dallinstaurarsi di una progressiva ipertensione polmonare [ 29 ]  . tipo iv : pazienti con trombi di vecchia data ( > 15 giorni ) , notevolmente organizzati , che una volta staccatisi dal circolo venoso profondo presentano la porzione prossimale dellembolo , quella rivolta verso il filo guida , molto dura , consistente , difficili da risolvere con il semplice movimento di frammentazione operato dal catetere angiografico o da attraversare con lo stesso filo guida . 
sono i pazienti che hanno richiesto un maggiore impegno assistenziale ( impianto di 16 filtri cavali ) e che hanno risentito meno dellazione del farmaco fibrinolitico ( persistenza di valori elevati di pressione polmonare al termine dei tre giorni di cura )  . laggiunta del farmaco fibrinolitico in infusione continua , dopo la frammentazione meccanica , trova il suo razionale nel presupposto che possibile migliorare ulteriormente la condizione emodinamica del paziente , sfruttando la maggiore superficie dazione per il farmaco ottenuta dopo la frammentazione dellembolo e la differenza di pressione . 
infatti , in presenza di embolia polmonare massiva , in prossimit dellembolo ostruente si forma un flusso vorticoso ; ogni liquido infuso prossimalmente allembolo occludente ha un contatto temporaneo con i suoi confini e viene deviato e diluito nellarteria polmonare controlaterale non occlusa . 
agresti : hybrid treatment with angiographic catheter in massive pulmonary embolism : mechanical fragmentation and fibrinolysis fluid infused proximally to the obstructive embolus only temporarily comes into contact with its borders and is deviated and diluted in the nonobstructed contralateral artery . 
this enables the agent to be brought into contact with the fragments of the embolus , the surface area of which is now greater , particularly after removal of the external and less fragile portion [ 6 ]  . 
it should be borne in mind that at first , after initial fragmentation with the catheter , the haemodynamic situation can worsen following dislocation of the thrombus / embolus fragments towards the peripheral portions of the pulmonary branches , which may lead to a rise in mean pulmonary artery pressure . 
 our retrospective study identified four classes of haemodynamic behaviour after the hybrid treatment of massive pulmonary embolisit would be interesting to conduct a prospective study aimed at comparing the various therapeutic combinations according to the haemodynamic behaviour encountered in the patient after initial fragmentation with the pigtail catheter ( mechanical fragmentation and systemic heparin therapy alone , fragmentation and locoregional fibrinolytic therapy , fragmentation , emboli aspiration and locoregional fibrinolytic therapy , and fragmentation with aspirating devices and locoregional fibrinolytic therapy ) to confirm the orientations of patient management in order to more correctly define the type of hybrid therapy to systematically apply . monitoring pressure levels and angiographic findings in the acute phase only enabled us to confirm the validity of hybrid treatment in massive pulmonary embolishowever , it did not provide useful indications on the patients progression , onset or otherwise , of pulmonary hypertension or the validity of the approach adopted with respect to systemic pharmacological therapy . 
this also suggests the need for a prospective study to compare results of hybrid therapy with those obtained with systemic or locoregional fibrinolytic therapy . conclusion in conclusion , in patients with massive pulmonary embolism , mechanical fragmentation with a conventional pigtail angiographic catheter and the addition of intrapulmonary fibrinolysis proved capable of rapidly improving severe cardiopulmonary insufficiency . 
the treatment , therefore , is a viable and minimally invasive option capable of producing surprising results : it enables the patient to return to normal or near - normal mean pulmonary artery pressure values extremely rapidly . currently , no single solution is available for the manage [ 24 , 30 ]  . 
questo consente di portare il farmaco verso i frammenti di embolo , la cui superficie esposta allazione del fibrinolitico ora maggiore , soprattutto dopo aver rimosso la porzione pi esterna , meno friabile [ 6 ]  . 
occorre segnalare che , in un primo momento , dopo liniziale frammentazione con il catetere , la situazione emodinamica pu peggiorare in seguito alla dislocazione dei frammenti del trombo / embolo verso le porzioni pi periferiche dei rami polmonari ; tale evento pu comportare un innalzamento della pressione polmonare media . 
sarebbe interessante uno studio prospettico , che mettesse a confronto le varie combinazioni terapeutiche a seconda del comportamento emodinamico riscontrato nel paziente dopo liniziale frammentazione con il catetere angiografico ( solo frammentazione meccanica e terapia eparinica sistemica ; frammentazione e terapia fibrinolitica locoregionale ; frammentazione , aspirazione emboli e terapia fibrinolitica locoregionale ; frammentazione con presidi ad aspirazione e terapia fibrinolitica locoregionale ) per ottenere le conferme sugli indirizzi della gestione successiva del paziente , in modo da definire pi correttamente il tipo di terapia ibrida da applicare poi sistematicamente . il controllo dei valori pressori e delle risultanze dei quadri angiografici solo in fase acuta ci ha consentito di confermare la validit del trattamento ibrido nella embolia polmonare massiva , ma non ci fornisce utili indicazioni sullevoluzione successiva del paziente , sullinstaurarsi o meno dellipertensione polmonare , sulla validit dellopzione terapeutica effettuata rispetto al trattamento farmacologico sistemico . 
anche in questo caso si rende necessario uno studio prospettico per confrontare i risultati di questa impostazione terapeutica ibrida con quelli ottenuti con la sola fibrinolisi , sistemica o locoregionale . conclusione in conclusione , nei pazienti con embolia polmonare massiva la frammentazione meccanica con limpiego di un comune catetere angiografico pigtail , e laggiunta della successiva fibrinolisi intrapolmonare , si dimostrata in grado di migliorare rapidamente il grave quadro dinsufficienza cardiopolmonare che affliggeva il paziente . 
rappresenta quindi una opzione terapeutica reale , minimamente invasiva , con risultati sorprendenti : garantisce al paziente il ritorno a valori di pressione polmonare media normali o compatibili con la vita , in tempi estremamente rapidi . attualmente non disponibile una soluzione unica per la gestione di tutte le sfaccettature con cui si possono presentare i pazienti con embolia polmonare massiva . 
agresti : hybrid treatment with angiographic catheter in massive pulmonary embolism : mechanical fragmentation and fibrinolysis ment of all presentations of massive pulmonary embolism . the most recent guidelines suggest the use of mechanical fragmentation only in the most severe forms [ 31 , 32 ]  . 
our study seems to support this approach : when the embolus fails to immediately respond to mechanical fragmentation with a conventional angiographic catheter , other therapeutic options should be undertaken , including the use of mechanical thrombectomy devices . 
this suggests a greater commitment on the part of the vascular and interventional radiologist , who needs to be well versed in the use of such devices , as well as an economic obligation on the part of the hospital , which needs to consider these devices as lifesaving in such exceptional circumstances . linee guida suggeriscono limpiego della frammentazione meccanica solo nelle forme di embolia pi gravi [ 31 , 32 ] ; la nostra esperienza di trattamento ibrido , meccanico e farmacologico , dellembolia polmonare massiva , sembra avvalorare tale impostazione : quando lembolo non risponde subito alla frammentazione meccanica con il semplice catetere angiografico , dovrebbero essere per impiegate anche altre opzioni terapeutiche , tra cui i presidi meccanici . 
patients had been referred for suspected encephalitis ( n = 2 ) , dural sinus thrombosis ( n = 1 ) and multiple sclerosis ( n = 3 )  . mri examinations were repeated after 4 , 8 and 12 weeks and in two cases also after 6 and 9 months . 
to our knowledge and according to previous reports , the fact that these lesions are detected in a relatively large number of conditions with heterogeneous etiopathogenetic factors leads to the hypothesis that a common underlying pathophysiological mechanism that , considering signal characteristic , reversibility and white matter location , could be represented by vasogenic oedema . key words magnetic resonance imaging corpus callosum splenium focal lesions riassunto obiettivi . 
in 6 pazienti sottoposti a indagini tc e rm sono state riscontrate incidentalmente lesioni focali non emorragiche , isolate , nel contesto dello splenio del corpo calloso . i pazienti giungevano alla nostra osservazione con sospetto clinico di patologia infettiva ( 2 ) , trombotica ( 1 ) , demielinizzante ( 3 ) dellencefalo . 
secondo la nostra esperienza e quella di altri autori , essendo il riscontro di tali lesioni comune a un ampio spettro di condizioni patologiche con fattori eziopatogenetici eterogenei , ipotizzabile che attraverso un comune , complesso meccanismo fisiopatologico tali fattori possano creare squilibri responsabili della comparsa di edema vasogenico che si esprime in unalterazione del segnale rm nello splenio del corpo calloso . parole chiave risonanza magnetica corpo calloso splenio lesioni focali m . 
morphological and signal features of these lesions are usually unambiguous : they appear hyperintense in long tr sequences [ t2 - weighted and fluid attenuated inversion recovery ( flair ) ] and either slightly hypointense or insignificant in t1 . 
lesions are usually divided into two types according to extent and shape : oval , circumscribed , with well - defined borders and located in the middle portion of the splenium ; or wider , with less regular borders and involving the entire spleniu because such findings have been detected in a large number of clinical conditions , there is still controversy regarding their pathogenesis . 
the authors postulated that the lesion aetiopathogenesis and evolution could be related to medications used during treatment , as they were transient in nature and disappeared upon suspension of phenytoin and vigabatrin [ 3 ]  . 
in subsequent studies , several authors reported the detection on mri of transient focal cerebral abnormalities with similar features in patients with no evidence of seizures who were undergoing examination due to suspected viral encephalitis [ 4 ] , rotavirus encephalopathy [ 5 ] , acute cerebellitis [ 6 ] , low - grade glioma [ 7 ] , haemolyticuraemic syndrome [ 8 ] , salmonella - enteritidis - related encephalitis [ 9 ] and high - altitude cerebral oedema [ 10 ]  . although the majority of lesions were described in patients treated for seizures , the same findings were described in a wide spectrum of clinical conditions , suggesting that it is inappropriate to view them as a clearly defined disease pattern . 
rather than ascribing the findings to a distinctive pathology , it is more reasonable to believe that they represent a common specific pathophysiological condition developing in the splenium of the cc during different clinical situations . the aim of this study was to analyse the possible pathogenetic conditions responsible for the appearance of such lesions , attempting to provide an explanation and clinical correlation based upon our experience and on the published data . materials and methods six patients with transient focal lesions in the splenium of the cc were studied in our university department of radiological sciences . 
le caratteristiche morfologiche e di segnale di tali lesioni risultano generalmente univoche : appaiono iperintense nelle sequenze a tr lungo ( t2 pesate e flair ) mentre in quelle pesate in t1 possono manifestare una lieve ipointensit o risultare non apprezzabili ; non stato descritto un definito enhancement dopo iniezione di mezzo di contrasto ( mdc ) paramagnetico . 
per quanto concerne la morfologia e lestensione di tali alterazioni di segnale ne vengono descritte sostanzialmente due tipi : ovoidale , circoscritta , ben definita e mediana , oppure meno definita pi ampia e diffusa , estesa a interessarne lintera superficie . 
 [ 2 ] tali reperti venivano incidentalmente identificati in pazienti epilettici e gli autori formulavano lipotesi di una eziopatogenesi e unevoluzione correlate al relativo trattamento terapeutico , data la caratteristica transitoriet di queste alterazioni e la loro scomparsa alla sospensione di farmaci quali la fenitoina , la vigabatrina etc . 
in seguito , diversi lavori hanno riportato il riscontro di analoghe lesioni con le medesime caratteristiche in corso di condizioni differenti dallepilessia quali encefaliti virali [ 4 ] , encefalopatie da rotavirus [ 5 ] , cerebelliti acute [ 6 ] , glomi di basso grado [ 7 ] , sindrome emolitico - uremica [ 8 ] , encefaliti associate alla salmonella enteritidis [ 9 ] ed edema cerebrale da altitudine [ 10 ]  . in realt , anche se i riscontri pi frequenti sono stati descritti in pazienti trattati per crisi epilettiche di varia tipologia , il fatto che un range cos ampio di condizioni patologiche di base sottenda lo stesso reperto di imaging suggerisce limpossibilit di etichettare questo tipo di lesione quale pattern di una singola condizione clinica . 
pi ragionevole ritenere tali aspetti non tanto lespressione di una patologia piuttosto che di unaltra , ma di determinate condizioni fisiopatologiche che si sviluppano nel contesto dello splenio quale comune denominatore di unampia e variabile gamma di situazioni che ne condizionano la genesi . lo scopo di questo studio pertanto arrivare a formulare delle ipotesi circa le possibili condizioni patogenetiche responsabili della comparsa di tali alterazioni , oltre che identificarne il significato e le eventuali correlazioni cliniche in base alla nostra personale esperienza e ai dati riportati finora in letteratura . materiali e metodi presso listituto di scienze radiologiche delluniversit degli studi di sassari sono state riscontrate in 6 pazienti lesioni transitorie localizzate a livello dello splenio del corpo calloso . 
after the first mri scan , all patients underwent an electroencephalogram ( eeg ) , a complete blood count , and cerebral spinal fluid ( csf ) analysis to investigate the possible presence of pathogenic agents . 
 a 16 - year - old boy with a clinical history of hypopituitarism was referred to our department by the infectious disease ward , with a clinical suspicion of viral encephalitis . 
as this finding was nonspecific and therefore did not allow a definite diagnosis , the patient underwent further clinical investigations . a 22 - year - old woman affected by depressive crises was referred to us because of intense headaches . 
the patient was admitted to the neurology department for further diagnostic tests . a 16 - year - old boy with a clinical history of eczematoid psoriasis and intense frontotemporal headaches developed a faciobrachiocrural hemiparesis . 
in tutti i pazienti , gi sottoposti ad una tc cranio , stata effettuata una rm dellencefalo ( shimadzu 1t philips intera achieva nova s 1 , 5t ) con studio sui piani assiale , coronale e sagittale in tecnica se , tse e ir e sequenze pesate in dp , t2 , flair , diffusione e in t1 prima e dopo iniezione di mdc paramagnetico . in tutti i casi le indagini sono state ripetute dopo 4 , 8 e 12 settimane . 
successivamente al primo esame rm , in tutti i pazienti sono stati inoltre effettuati un esame emocromocitometrico , il prelievo del liquor per la ricerca di eventuali microrganismi patogeni ed un elettroencefalogramma . caso 1 un ragazzo di 16 anni , con storia di ipostatuarismo , giunge alla nostra osservazione , inviato dal reparto di malattie infettive , con sospetto clinico di encefalite virale . 
laspecificit del reperto non consente al momento valide ipotesi diagnostiche , per cui il paziente , sempre in regime di ricovero , viene sottoposto a ulteriori accertamenti clinico - laboratoristici . caso 2 una ragazza di 22 anni , con notizie anamnestiche di crisi depressive , giunge alla nostra osservazione inviata dalla clinica neurologica per cefalea gravativa . 
lesame rm dellencefalo non documenta reperti di significato patologico in ambito parenchimale , mentre evidenzia una dubbia alterazione del normale segnale di flusso nella porzione laterale dei seni traversi , bilateralmente , che rende necessaria una ulteriore valutazione mediante angiografia digitale . 
a circa 3 mesi di distanza , la ricomparsa della sintomatologia rende necessario un secondo esame rm che documenta , nellambito dello splenio del corpo calloso , unarea di alterato segnale priva di enhancement dopo mdc . 
la paziente viene quindi nuovamente ricoverata presso listituto di clinica neurologica per ulteriori accertamenti diagnostici . caso 3 un ragazzo di 16 anni , con precedenti di psoriasi eczematoide e di cefalea gravativa subcontinua a sede fronto - temporale , giunge presso la clinica neurologica per linsorgenza di emiparesi facio - brachio - crurale . 
la tc dellencefalo dimostra la presenza di empiemi multipli sia subdurali che extradurali , associati a marcati fenomeni flogistici delle cavit sinusali paranasali a livello frontale , etmoidale e mascellare . 
la successiva indagine rm dellencefalo mette in evidenza unarea di alterato segnale nel contesto dello splenio del corpo calloso , priva di enhancement , di dubbio significato demielinizzante o neoplastico astrocitario in fase iniziale . 
si decide pertanto di sottoporre la paziente a ulteriori accertamenti diagnostici clinico - laboratoristici . caso 4 caso 5 area of altered signal with no contrast enhancement in the splenium of the cc , which was suspected to be a demyelination - related lesion or an early stage astrocytoma . 
 a 34 - year - old man presenting with a 4 - day history of high fever and chills and vomiting underwent an emergency ct scan , which yielded negative results . 
the mri exam failed to show any sign of infectious disease , the only significant finding being a small area of altered signal in the splenium of the cc similar to the one described in the other cases . 
 un uomo di 34 anni con febbre elevata da 4 giorni associata a brividi e , successivamente , a vomito , viene ricoverato presso la divisione di medicina generale dellospedale civile di olbia , dove una tc dellencefalo fornisce esito negativo . 
presenza di empiemi in regione frontale dopo iniezione di mdc paramagnetico ( c )  . fatta eccezione per unarea di alterato segnale localizzata nello splenio del corpo calloso , priva di impregnazione dopo mdc . 
il paziente prosegue quindi il ricovero per essere sottoposto a tutti gli ulteriori accertamenti diagnostici del caso . caso 6 una ragazza di 17 anni , con storia di appendicite operata , lamenta da un mese deficit visivi , parestesie agli arti superiori , localizzate prevalentemente a destra e associate a cefalea . 
la paziente stata dunque ricoverata per ulteriori accertamenti . in tutti i pazienti le lesioni individuate a livello dello splenio del corpo calloso si sono rivelate focali , prive di enhancement dopo mdc e reversibili . 
i dati clinici sono stati analizzati retrospettivamente con particolare attenzione ai risultati laboratoristici e alla terapia medica effettuata da ogni singolo paziente al fine di cercare di determinare leziologia delle lesioni . 
in 4 pazienti le lesioni sono scomparse congiuntamente alla remissione completa della patologia di base , mentre in due pazienti le lesioni sono perdurate fino a 6 e 9 mesi di distanza dal primo rilievo . 
in 4 pazienti , i valori medi del coefficiente apparente di diffusione ( adc ) misurati a livello della lesione sono risultati di a 17 - year - old girl with a clinical history of surgery for appendicitis presented with a 1 - month history of visual deficiency , upper limb paraesthesia particularly on the right side associated with headache . 
she was then hospitalised for further clinical investigations . risultati in all patients , the lesions detected in the splenium of the cc were transient , focal and with no contrast enhancement . clinical data were analysed retrospectively with special atm . 
3a - e axial fluid attenuated inversion recovery ( a ) , t2 ( b ) and diffusion - weighted ( c ) images show a well - defined focal high signal with low signal in the adc map ( e )  . 
3a - e nel contesto dello splenio del corpo calloso le immagini flair ( a ) , pesate in t2 ( b ) e diffusione ( c ) mostrano una lesione iperintensa , focale e ben definita , con basso segnale nella mappa adc ( e ) e senza un definito enhancement dopo iniezione di mdc paramagnetico ( d )  . tention to the laboratory findings and type of medical treatment received with the aim of identifying the aetiology of such lesions . 
in four patients , mean apparent diffusion coefficient ( adc ) values detected within the splenial lesion were 0.3810 - 3mm2 / s , lower if compared with adc values of frontal grey matter ( 0.9810 - 3mm2 / s ) , frontal white matter adc ( 0.9010 - 3mm2 / s ) and csf ( 3.4410 - 3mm2 / s )  . 
during follow - up , complete resolution of the splenial lesion corresponded to an increase in adc values to those of the frontal white matter ( 0.8610 - 3mm2 / s ) ( table 1 )  . the patient was admitted to the infectious disease ward and case 1 0 , 3810 - 3mm2 / s , inferiori se confrontati ai valori adc della sostanza grigia ( 0 , 9810 - 3mm2 / s ) , della sostanza bianca ( 0 , 9010 - 3mm2 / s ) frontali e del liquor ( 3 , 4410 - 3mm2 / s )  . 
nei controlli effettuati a distanza si evidenziava un recupero dei valori adc comparabili con quelli della sostanza bianca frontale ( 0 , 8610 - 3mm2 / s ) ( tabella 1 )  . caso 1 il paziente , ricoverato presso il reparto di malattie infettive , viene sottoposto a terapia con lovirax per circa una settimana . 
the first mri scan after 4 weeks demonstrated full normalisation of the splenium of the cc , a finding that was also confirmed by subsequent examinations after 8 and 12 weeks . 
a distanza di 4 settimane viene nuovamente eseguita una rm dellencefalo nella quale non appare pi evidente larea descritta a livello dello splenio del corpo calloso , quadro poi confermato anche nei successivi controlli a 8 e 12 settimane . caso 2 caso 3 il giorno successivo allindagine rm il paziente manifesta due episodi convulsivi generalizzati ; viene quindi trasferito nel reparto di rianimazione dove la terapia di base viene integrata con antibiotici aggiuntivi e barbiturici e al paziente praticata assistenza sotto il profilo respiratorio con controllo delle crisi immediato e remissione dello stato febbrile . il paziente quindi sottoposto nuovamente a una tc dellencefalo che si dimostra immodificata rispetto alla precedente . nei giorni seguenti si assiste a un progressivo recupero della forza a carico dellarto inferiore sinistro e alla ripresa della deambulazione . 
a 4 settimane di distanza la persistenza di alterazioni di lato a un esame eeg suggerisce lesecuzione di una indagine rm dellencefalo in cui , accanto a un residuo ascessuale in sede frontale destra , apprezzabile m . 
riesaminato a distanza di qualche mese , si evidenzia la scomparsa completa delle lesioni ascessuali nonch dellarea di iperintensit nel corpo calloso . nel corso di un breve ricovero presso listituto di clinica neurologica la paziente va incontro rapidamente a progressivo miglioramento della sintomatologia , per cui viene dimessa dopo qualche giorno . 
a distanza di 4 , 8 e 12 settimane la ripetizione dellindagine rm mostra un quadro sostanzialmente negativo , fatta eccezione per larea di alterato segnale evidenziata alla prima osservazione , che permane invariata . 
un ulteriore controllo a 3 mesi dallultima osservazione ( 6 mesi dallesordio ) evidenzia la scomparsa della lesione callosa . caso 4 caso 5 durante il ricovero si rileva una leucocitosi neutrofila allesame emocromocitometrico ; lesame del liquor non evidenzia parametri al di fuori della norma . 
gli esami rm dellencefalo ripetuti a 4 , 8 e 12 settimane , in linea con il dato clinico , danno esito negativo , evidenziando peraltro anche la scomparsa dellarea focale di iperintensit localizzata nello splenio del corpo calloso . to regain the ability to walk . 
four weeks later , the persistence of side alterations observed during an eeg prompted the performance of a second mri scan , which showed a residual abscess in the right frontal region associated with a reduction in size and conspicuity of the focal area in the splenium of the cc . 
 ( c ) immagine in t1 dopo iniezione di mdc . caso 6 la paziente mostra un rapido miglioramento clinico e il successivo controllo rm risulta pressoch sovrapponibile al precedente , con lesione dello splenio rimasta invariata . 
a tale proposito risulterebbe opportuna qualche considerazione . secondo quanto riportato da alcuni autori , la completa remissione clinica delle patologie di base conduce a una totale scomparsa delle lesioni [ 4 , 16 ] ; altri correlano tale evenienza a una modificazione del trattamento farmacologico , lesion , which appeared to be unchanged compared to the initial examination . 
after a further 3 months ( 6 months after symptom onset ) , another mri was performed , which demonstrated the complete disappearance of the splenial lesion . case 5 case 6 during hospitalisation , the patients blood tests revealed the presence of a neutrophil leucocytosis , but csf analysis was negative for pathogens . 
mri scans performed 4 , 8 and 12 weeks later were consistent with the clinical findings and presented no abnormalities , confirming normalisation of the focal area located in the splenium of the cc . the patient had a rapid clinical improvement , and the following mri scan was almost superimposable to the previous m . 
6a - e la lesione descritta nel contesto dello splenio del corpo calloso non appare pi apprezzabile nelle immagini pesate in flair ( a ) , t2 ( b ) , diffusione ( c ) e mappa adc ( e ) ; ( d ) t1 dopo iniezione di mdc paramagnetico . one with persistence of the splenial lesion . 
several pathological conditions such as tumours , ischaemia , leukodystrophy , multiple sclerosis , hiv - related encephalopathy and marchiafava - bigspecialmente nei pazienti epilettici , sia che questa si traduca in una interruzione definitiva del farmaco , sia che il farmaco stesso venga sospeso temporaneamente [ 13 , 17 ]  . 
nella nostra esperienza , i pazienti studiati non avevano precedenti di trattamento con farmaci antiepilettici , e , in due casi , le lesioni non sono scomparse congiuntamente alla remissione della sintomatologia . 
rimane dunque aperta la discussione su quanto tale reversibilit possa essere correlata a queste condizioni . altra importante caratteristica rappresentata dallassenza di definite manifestazioni neurologiche correlabili alle lesioni , aspetto questo tuttavia non ancora del tutto chiarito . 
questa ipotesi viene supportata dal fatto che sia nei nostri pazienti , sia in altri studi [ 17 , 18 ] , le lesioni focali perduravano anche a remissione clinica completa , tale che i pazienti sottoposti a controlli nel tempo si dimostravano assolutamente asintomatici . 
however , mri findings associated with these conditions appear to be different from those described in our patients or reported in literature , which present some major common features that make their detection peculiar , the most important of which is their particular transient nature . according to some authors , complete clinical regression of the underlying condition leads to the disappearance of these lesions [ 4 , 16 ] ; others correlate this event with changes in pharmacological treatment ( both temporary and permanent interruption of drug administration ) , especially in epileptic patients [ 13 , 17 ]  . 
in our experience , none of the patients had ever received antiepileptic drugs and , in two cases , the lesions did not disappear when the symptoms regressed , making the correlation between reversibility and symptom relief controversial . another important feature is represented by the lack of specific neurological symptoms related to the lesions , even if this aspect is still not completely clear . 
this hypothesis is supported by the fact that both in our patients and in the cases reported in the literature [ 17 , 18 ] , the focal lesion persisted even after full clinical remission when , during follow - up examinations , patients appeared to be asymptomatic . 
 [ 16 ] postulated that whereas in these patients multiple foci of parenchymal signal alteration on t2 and dw could have indeed been responsible for clinical manifestations , it is possible that such alterations had completely regressed before an mri scan could be performed , making their detection elusive . 
however , it is debatable whether the patient would benefit more from the maintenance of a treatment protocol targeting the underlying pathology or from therapy conversion aimed solely at improving mri findings , which are not connected with any symptowe believe that the disappearance of the lesions is not directly connected to any clinical change , especially considering the remission of symptoms . 
 in addition to reversibility and accompanying lack of symptoms , these lesions share further features : their focal shape and lack of contrast enhancement after injection of paramagnetic contrast mediuthere is stronger agreement on the latter characteristics in the literature . 
to date , there are no reports describing nonenhancing transient and focal lesions in the splenium of the cc unassociated with other abnormalities either in the white or in the grey matter . 
the aetiopathogenesis of these peculiar lesions is the [ 16 ] tuttavia ipotizzano che nei pazienti possano essere presenti foci multipli di alterazione del segnale nel parenchima cerebrale , in t2 e in diffusione , responsabili delle manifestazioni cliniche , ma che questi possano essere andati comunque incontro a remissione completa nel periodo antecedente lesecuzione dellindagine rm non risultando pertanto documentabili . 
tuttavia rimane da stabilire se il beneficio maggiore per il paziente sia quello di mantenere una terapia farmacologica rivolta a curare una patologia di base , interromperla o , ancora , variarla al fine di migliorare un quadro rm che non correlabile ad alcun aspetto sintomatologico . a nostro avviso , infatti , la scomparsa delle lesioni non corrisponde ad alcuna modificazione nella clinica dei pazienti , peraltro gi asintomatici , mentre una variazione nel trattamento farmacologico potrebbe avere ripercussioni sullandamento della patologia di base per la quale i pazienti con tali lesioni sono giunti allo studio rm . oltre alla reversibilit e alla presunta asintomaticit , queste lesioni sono accomunate dallaspetto focale e dallassenza di enhancement dopo somministrazione di mdc . 
su queste due caratteristiche gli autori appaiono pi concordi . non sono infatti riportati lavori in letteratura con descrizione di lesioni focali dello splenio del corpo calloso con carattere di reversibilit , associate ad altre aree di alterato segnale sia nella sostanza bianca che nella sostanza grigia e con assenza di enhancement dopo somministrazione di mdc paramagnetico . 
nella nostra esperienza , in nessuno dei pazienti studiati stato riscontrato un definito enhancement , in linea dunque con i dati riportati attualmente in letteratura . pi vivace , invece , sembra essere la querelle sulle ipotesi eziopatogenetiche e fisiopatologiche di queste particolari alterazioni . 
utile ricordare in primo luogo le varie patologie con le quali tali aspetti possono entrare in diagnosi differenziale : sm , neoplasie , infarto , leucodistrofia ed encefalopatia hiv - correlata possono presentare reperti simili allindagine rm , ma non mostrano quella reversibilit e quellassenza di enhancement caratteristiche invece dei quadri da noi descritti . 
it is useful to recall the diverse conditions that these lesions could mimic : multiple sclerosis , tumours , infarction , leukodystrophy and hiv - related encephalopathy could all yield similar findings on an mri scan but would not show reversibility or lack of contrast enhancement . 
acute disseminated encephalomyelitis is more likely to be multicentric and bilateral , and some foci may show a diffuse or weak uptake of contrast depending on the grade of activity . 
early stage vascular ischaemic lesions may appear similar , but they always leave their mark on the affected brain tissue . as stated , several hypotheses have been put forth to explain the genesis of such abnormalities . 
in addition , dilantin may interfere with the intestinal absorption of dietary folate , causing a deficiency that has been linked to encephalopathy , cerebellar atrophy , myelopathy and peripheral neuropathy [ 21 ]  . 
 [ 4 ] supposed that intramyelinic oedema or macromolecular or phlogistic cell infiltration may cause similar alterations in a particular region of the brain , namely , in the cc . 
in this phenomenon , an increase in central nervous system ( cns ) inflammatory cytokines , such as interleukin - 6 , may play an important role with regard to the pathogenesis leading to mitochondrial dysfunction with lactic acidosis in the cc [ 25 ]  . 
nevertheless , all these theories require further clarification that , according to the authors , could be provided by future studies utilising mr spectroscopy techniques . a different hypothesis has been put forward by mirsattari et al . 
according to their view , such an abrupt withdrawal of pharmaceutical treatment could generate a hydric imbalance linked to alteration of the arginine - vasopressin ( avp ) syste it has been demonstrated that the avp system mediates regional cerebral flow [ 27 ] , affects brain hydric content and possibly plays a role in generating oedema [ 28 , 29 ]  . 
carbamazepine may potentiate avp antidiuretic effects [ 31 , fondamentale nella genesi di tali lesioni , pur riconoscendo che il meccanismo attraverso il quale queste vengono a formarsi ancora poco chiaro . 
inoltre il farmaco potrebbe interferire con lassorbimento intestinale di folati , il cui deficit stato collegato a encefalopatia , atrofia cerebellare , mielopatia e neuropatia periferica [ 21 ]  . 
secondo studi sperimentali , nei trattamenti a lungo termine anche la vigabatrina sarebbe responsabile di un edema intramielinico della sostanza bianca , reversibile in poche settimane dopo la sospensione del farmaco senza effetti residui [ 2224 ]  . 
 [ 4 ] suppongono che un edema intramielinico o un infiltrato flogistico di cellule infiammatorie e macromolecole possano produrre questo tipo di lesione in unarea preferenziale dellencefalo , identificata per lappunto nello splenio del corpo calloso , ipotizzando una specifica affinit dei recettori degli assoni dello splenio per antigeni virali . 
in tale meccanismo sembra possa giocare un ruolo laumento delle citochine infiammatorie del snc ( il - 6 ) che producono una disfunzione mitocondriale con conseguente acidosi lattica a livello dello splenio [ 25 ]  . 
ci verrebbe indotto attraverso un meccanismo di adattamento organico a seguito di una prolungata terapia con farmaci antiepilettici , la cui brusca interruzione provocherebbe degli squilibri nel bilancio idrico dovuti ad alterazioni nel sistema vasopressinaangiotensina ( avp )  . 
 stato dimostrato che il sistema avp regola il flusso cerebrale regionale [ 27 ] , influenza il contenuto di acqua nellencefalo e pu contribuire alla formazione delledema [ 28 , 29 ]  . 
 [ 26 ] , potrebbe indurre un aumento dei livelli serici dellavp con una conseguente alterazione dellequilibrio idrico encefalico che provocherebbe un edema citotossico in una regione vulnerabile come lo splenio del corpo calloso vascolarizzato solamente da rami terminali delle arterie cerebrali posteriori . 
this alteration would in turn induce cytotoxic oedema in a vulnerable region like the splenium of the cc , which is supported only by the terminal branches of posterior cerebral arteries . 
according to the authors , the reversible t2 signal hyperintensity of the white matter without simultaneous grey matter involvement strongly suggests that vasogenic oedema may precipitate such lesions [ 33 ]  . 
in fact , white matter , which has a lower fibre density , offers weaker resistance to oedema compared with grey matter , which is conversely more sensitive to the imbalance between energy demand and relative response and is more likely to be affected by cytotoxic oedema shown on t1 - weighted sequences [ 34 ]  . 
further evidence of this theory is provided by the fact that cytotoxic oedema does not respond to steroid therapy as does vasogenic oedema [ 35 ] , which leaves the brain tissue unaffected after resolution . 
an explanation for this pathogenic mechanism may be a blood - brain barrier alteration due to a cerebral capillary hydrostatic pressure increase , which is commonly observed in conditions such as hypertensive encephalopathy , preeclampsia , seizures , acute intermittent porphyria , toxic effects of cyclosporine , and migraine [ 3638 ]  . 
several factors may interfere with the complex balance of the bloodbrain barrier , such as neurotransmitters and neuromodulators , cyclic nucleotides , nitric oxide , histamine , and cytokines and other factors released by the interaction of white cells and endothelium [ 3941 ]  . 
some of these might be altered as a result of a hypoxemic condition [ 42 ] , and others , such as atrial natriuretic peptide , may be affected by acute mountain sickness [ 43 , 44 ]  . 
 [ 10 ] most closely resemble our own view , namely that transient focal lesions in the splenium of the cc described in our five patients may be related to vasogenic oedema which , albeit through complex and diverse mechanisms , represents the final common denominator among several pathological conditions . 
to validate this theory , in four patients , we detected low mean adc values within the splenial lesion , lower if compared with adc values of frontal grey matter , frontal white matter and csf . mean adc increased to normal values on subsequent examinations ( table 1 )  . conclusions transient signal alterations in the splenium of the cc detectsolo uno su cinque stato trattato con gardenale . 
 [ 10 ] descrivono la presenza di lesioni focali transitorie nello splenio del corpo calloso con le medesime caratteristiche rm da noi descritte in pazienti con edema cerebrale da altitudine . 
secondo gli autori la reversibile iperintensit di segnale in t2 della sostanza bianca senza contemporaneo coinvolgimento della sostanza grigia , suggerisce fortemente che ledema vasogenico sia il fattore alla base di tali lesioni [ 33 ]  . 
la sostanza bianca infatti , avendo una minor densit di fibre , offre una minore resistenza alledema di quanta ne esprima la sostanza grigia , che invece si dimostra pi sensibile allo squilibrio tra richiesta di energia cellulare e relativo apporto , andando quindi incontro pi facilmente alledema citotossico maggiormente apprezzabile su sequenze t1 pesate [ 34 ]  . 
unulteriore evidenza di quanto descritto fornita dal fatto che ledema citotossico non risponde alla terapia steroidea , a differenza di quello vasogenico [ 35 ] che tende inoltre a lasciare integro , senza reliquati , il parenchima encefalico dopo il suo riassorbimento . 
la spiegazione di tale meccanismo patogenetico potrebbe essere ricercata in unalterazione della barriera ematoencefalica per aumento della pressione idrostatica capillare a livello cerebrale , come osservabile in condizioni quali lencefalopatia ipertensiva , la tossiemia in gravidanza , le crisi epilettiche , la porfiria acuta intermittente o gli stati tossici da ciclosporine [ 3638 ]  . 
il complesso equilibrio della bee pu essere influenzato da diversi fattori quali neurotrasmettitori e neuromodulatori , ossido nitrico , istamina , citochine e altri , rilasciati dallinterazione tra globuli bianchi ed endotelio [ 3941 ]  . 
alcuni tra questi risultano alterati nelle condizioni di ipossia [ 42 ] mentre altri , quali lormone natriuretico atriale , possono risultarlo in corso di malessere acuto da altitudine [ 43 , 44 ]  . tra tutte le ipotesi eziopatogenetiche formulate , quelle proposte da hackett et al . 
 [ 10 ] sembrano coincidere maggiormente con la nostra linea di pensiero , secondo la quale le lesioni focali transitorie dello splenio descritte nei 6 pazienti possono essere ricondotte a una condizione di edema vasogenico che , attraverso i pi svariati e complessi meccanismi , potrebbe rappresentare lespressione ultima comune di condizioni patologiche differenti . 
a supporto di tale ipotesi vi il rilievo , in 4 pazienti , a livello della lesione , di bassi valori di adc , inferiori sia a quelli della sostanza grigia che della sostanza bianca frontali e del liquor , e la loro successiva normalizzazione nei controlli a distanza ( tabella 1 )  . conclusioni le alterazioni transitorie del segnale nello splenio del corpo calloso in corso di esami rm dellencefalo sono divenute , nellultimo decennio , un reperto sempre pi frequentemente descritto da numerosi autori . 
ci che nella letteratura stessa appare ancora estremamente variabile la formulazione univoca di unipotesi di natura , date le molteplici condizioni patologiche nel corso delle quali tali alterazioni vengono riscontrate . 
however , none of the patients in our study had undergone antiepileptic treatment , and similar lesions have been detected in patients affected by a wide spectrum of substantially different diseases . 
further research is therefore needed to identify common pathophysiological mechanisms . it seems reasonable to believe that through multiple and complex mechanisms , a number of pathological conditions can create a particular organic situation responsible for the imaging findings , which amount to the final common expression of different pathogenetic factors . 
as described , seizures , antiepileptic drugs , cerebral infections or high - altitude sickness all interfere with the function of the avp system , leading to vasogenic oedema characterised by an mri signal alteration in the splenium of the cc . 
this view explains the lesions transience , as the interruption of the noxa patogena ( seizures , antiepileptic drugs , infection ) leads to the progressive reduction and ultimate disappearance of the abnormal finding . 
in our opinion , this aetiopathogenetic explanation also better accounts for other features , such as the lack of symptoms , the focal shape of the lesions and the absence of contrast enhancement . the area in which such lesions have been typically found may be linked to the anisotropic diffusion in the cc , which , as reported by neeraj et al . 
 [ 45 ] , could be ascribed to a combination of several factors , such as tighter packing of axons , less - permeable myelin sheaths , fewer obliquely oriented axons , altered radius of individual axons and the presence ( or absence ) of collagenous perivascular alae . 
 in conclusion , we believe that the transient focal lesions in the splenium of the cc are an expression of a common aetiopathogenetic denominator , namely , vasogenic oedema induced by multiple pathological conditions through a mechanism involving and affecting the blood - brain barrier . va maggior convergenza dopinione quella che fa riferimento allazione di alcuni farmaci antiepilettici . 
piuttosto arduo ritenere di per s alla base di una stessa lesione differenti fattori eziologici . sembrerebbe quindi opportuno rivolgere particolare attenzione alla ricerca di meccanismi fisiopatogenetici comuni . pi ragionevole ritenere , infatti , che una serie pi o meno ampia di condizioni patologiche contribuisca , attraverso i pi svariati e complessi meccanismi , a creare una particolare situazione organica responsabile della comparsa delle alterazioni descritte quale espressione ultima e comune di differenti fattori eziopatogenetici . 
come descritto in precedenza , le crisi epilettiche , i farmaci antiepilettici , le infezioni cerebrali o laltitudine vanno tutti a interagire con il sistema avp , inducendo un edema vasogenico che si esprime in unalterazione del segnale rm nello splenio del corpo calloso . 
ci spiega la reversibilit nel tempo delle lesioni , visto che al cessare della noxa patogena ( crisi , farmaco , infezione ) la lesione tende progressivamente a ridursi divenendo sempre meno apprezzabile per poi infine scomparire . 
 [ 45 ] , dalla combinazione di diversi fattori quali giunzioni assonali pi strette , guaine mieliniche meno permeabili o pi grosse , minore quantit di assoni orientati obliquamente o presenza nel corpo calloso di strutture diverse dalle guaine mieliniche . 
dagostino5 1department of oncology , surgical unit , universit di palermo , policlinico universitario , via del vespro 129 , i - 90127 palermo , italy 2department of radiology domenico noto , hospital of sciacca , via pompei , i - 92019 sciacca , italy 3department of pathology , 4department of surgery , 5department of radiological sciences , universit di palermo , policlinico universitario , via del vespro 129 , i - 90127 palermo , italy correspondence to : f . 
ph monitoring showed sensitivity of 71% , specificity of 31% , positive predictive value ( ppv ) of 53% and negative predictive value ( npv ) of 50% ; when middle - proximal refluxes only were considered , sensitivity decreased to 45% and specificity increased to 55% . 
furthermore , the association between reflux and oesophagitis demonstrated by the chi - square ( 2 ) test proved to be statistically significant both for wst and ph monitoring , whereas the association between reflux and barretts oesophagus was not significant for either wst or for ph monitoring . 
ph - metria ha presentato sensibilit del 71% , specificit del 31% , valore predittivo positivo ( ppv ) del 53% e valore predittivo negativo ( npv ) del 50% ; la sua specificit aumenta ( 55% ) considerando solamente i reflussi medioprossimali , ma diminuisce la sensibilit ( 45% )  . 
il wst un test semplice e poco costoso , facilmente realizzabile , che pu essere utile nella diagnosi di gerd ; la sua positivit pu essere un indicatore per selezionare i pazienti da avviare allendoscopia con biopsia . parole chiave water siphon test reflusso gastroesofageo ph - metria studio baritato dellesofago e . 
twentyfour - hour ph monitoring is generally considered the gold standard for detection of acid gastroesophageal reflux [ 36 ]  . nevertheless , its use is limited , as it is invasive , expensive , time consuming , not always easily tolerated by the patients and not always readily available [ 7 ]  . 
oesophageal endoscopy is usually performed when severe symptoms are present or when a complication is suspected and systematic biopsies must be taken to confirm oesophagitis , or to detect barretts oesophagus [ 9 ]  . 
radiological reflux evaluation , which was proposed in the early 1950s , is also used to diagnose the morphological aspects of the disease , such as stenosis and the presence of hiatal hernia ( hh ) [ 10 , 11 ]  . however , barium studies have generally been considered insensitive for reflux detection as an event which correlates to the disease , and associated provocative manoeuvres , such as the water siphon test ( wst ) , have been thought to overestimate reflux when compared with ph monitoring [ 1214 ]  . 
several other reports indicate that radiological evaluation can be used as a screening test when gastroesophageal reflux is suspected and its sensitivity and specificity are increased by the use of wst [ 1518 ]  . 
in addition , comparison was made between results of wst and ph monitoring with regards to the possibility of identifying subjects with gerd complicated by oesophagitis , barretts oesophagus or intestinal metaplasia . materials and methods patients we reviewed a series of 160 patients , 77 men and 83 women , with ages ranging from 1974 ( mean 44 ) years referred to the oesophageal surgery unit between january 2002 and december 2005 for evaluation of suspected gerd . 
solitamente , nel sospetto di gerd , vengono utilizzati test diagnostici quali lendoscopia e la ph - metria [ 2 ] , questultima considerata il gold standard per lindividuazione di reflusso gastroesofageo acido [ 36 ]  . 
tuttavia limpiego della ph - metria limitato a causa della sua invasivit , del suo costo , della sua durata , della scarsa tolleranza e della ridotta disponibilit sul territorio [ 7 ] ; inoltre la ph - metria pu misurare lesposizione acida della mucosa esofagea ma non pu rilevare gli episodi di reflusso con ph > 4 ( cio reflusso non acido ) e , per questo , non si pu considerare un test perfetto [ 8 ]  . 
lendoscopia esofagea viene riservata a pazienti con sintomi severi o con sospette complicanze della malattia da reflusso quali lesofagite e / o lesofago di barrett , per la cui diagnosi necessario effettuare biopsie multiple della mucosa [ 9 ]  . 
la valutazione radiologica , che fu proposta sin dallinizio degli anni cinquanta , solitamente usata per diagnosticare le alterazioni morfologiche della malattia , quali stenosi ed esofagiti , e la presenza di ernia iatale [ 10 , 11 ]  . 
lo studio con bario stato generalmente considerato scarsamente sensibile per la rilevazione di reflusso come evento correlato alla malattia ; inoltre si considera che le manovre di provocazione associate , come il water siphon test ( wst ) , sovrastimino il reflusso rispetto alla ph - metria [ 1214 ]  . 
tuttavia , parecchi altri lavori indicano che lo studio radiologico pu essere usato come test di screening nel caso di sospetto di reflusso gastroesofageo e che il wst incrementi i valori di sensibilit e di specificit dellesame [ 1518 ]  . 
 materiali e metodi pazienti abbiamo effettuato una valutazione retrospettiva della nostra serie di 160 pazienti 77 uomini e 83 donne con et compresa tra 19 e 74 anni ( et media : 44 anni ) , avviati allunit di chirurgia esofagea fra gennaio 2002 e dicembre 2005 per sospetta gerd . 
after an overnight fast , patients were given two mouthfuls of barium sulphate suspension ( 60% weight / volume ) in the upright left oblique posterior and in the prone right oblique anterior projections with a bolster placed under the abdomen . 
the patients were then asked to lie on their left side for 30 s , and wst was performed with the patients lying supine while continuously drinking 80 ml of water through a straw and rolling from the supine to the right lateral position . 
grading was as follows : distal ( refluxed column of barium in the lower 5 cm of the oesophagus ) , middle ( reflux up to the tracheal carrefour ) and proximal ( reflux up to pharyngoesophageal junction )  . motility study all motility studies were performed by one of the authors . oesophageal manometry was performed transnasally using an appropriate probe ( marquat c47 ) with an open - ended tip and four radial paths with a bearing point every 5 cperfusion equipment included a nitrogen infusion pump ( model 745 - 0100 , international biomedical inc ) , and data were recorded by an autocalibrating polygraph ( narco bio system mms 200 ) connected to a pc by software for automatic data analysis . 
manometric findings of this condition were two pressure peaks , intrathoracic pressure inversion point , short oesophagus , variable plateau pressure , dystonic high - pressure zone ( hypoor hypertonic ) and double respiratory reversal . twenty - four - hour ph monitoring all ph studies were performed by one of the authors . 
patients underwent 24 - h ambulatory ph monitoring after an overnight fast ; any medication such as prokinetics or proton pump inhibitors ( ppi ) were suspended for at least 7 days before the test . 
after standard calibrations at 37c in ph 7 and ph 1 buffer solutions , the ph catheter was inserted nasally in order to place the proximal electrode 5 cm above the manometrically determined proximal border of the lower oesophageal sphincter and the distal electrode in the proximal stomach . 
during the 24 - h period of ph tro 2 settimane e sono state interpretate indipendentemente e senza alcuna conoscenza dei risultati degli altri test . cineradiografia digitale e water siphon test tutti gli studi dinamici con bario ( cineradiografia ) sono stati eseguiti da due autori impiegando un telecomandato digitale con arco a c ( polifunzionale eurocolumbus tr3d , milano , italia ) con intensificatore di brillanza da 16 pollici e con sequenze di acquisizione di 6 immagini al secondo con matrice 5121024 , secondo una tecnica standardizzata . 
dopo 12 ore di digiuno sono stati somministrati ai pazienti due sorsi di sospensione di solfato di bario 60% peso - volume rispettivamente in posizione ortostatica e proiezione obliqua anteriore destra e in posizione prona e in proiezione obliqua posteriore sinistra dopo posizionamento di un piccolo cuscino radiotrasparente sotto laddome . 
dopo circa 30 secondi di decubito sul fianco sinistro veniva eseguito il wst : il paziente , in posizione supina , assumeva in modo continuato da una cannuccia circa 80 ml di acqua , passando poi dalla posizione supina al decubito laterale destro . 
i reperti manometrici di questa condizione erano il doppio picco pressorio , il punto di inversione della pressione toracica , lesofago corto , il variabile plateau pressorio , la zona di alta pressione distonica ( ipotonica o ipertonica ) , la doppia inversione respiratoria . ph - metria 24 ore tutti i test ph - metrici sono stati eseguiti da uno degli autori . i pazienti sono stati sottoposti a ph - metria 24 ore durante un ricovero in day hospital e dopo un digiuno di almeno 12 ore ; tutti i trattamenti farmacologici , con procinetici e / o inibitori di pompa protonica , sono stati sospesi per almeno 7 giorni prima del test . 
 endoscopy all endoscopic procedures were performed by one of the authors using an olympus videoendoscope ( gif q160 , tokyo , japan ) following the same criteria for all examinations . 
the squamous - columnar junction was identified and the normal z - line or the presence of columnar - lined oesophagus , suspected barretts oesophagus , short ( tongued or < 3 cm ) or long ( 3 cm ) segment , were defined . 
in all patients , four - quadrant biopsies from the distal oesophageal mucosa , at least 1 cm proximal to the gastroesophageal junction , were taken when a normal z - line was identified , and multiple biopsies of the long or short columnar - lined mucosa were performed to detect intestinal metaplasia ( im ) to determine barretts oesophagus . 
four - quadrant biopsies were also taken from the columnar mucosa at least 1 cm distal to the gastroesophageal junction , but the results are beyond the scope of this study . 
all biopsies were immediately fixed in 10% buffered formalin , numbered and sent to the same pathologist , who was unaware of the endoscopic diagnosis . histological analysis all histological analyses were performed by one of the authors . 
all the biopsy samples were fixed in 10% buffered formalin and embedded in paraffin and 5 - m sections were obtained and stained with haematoxylin - eosin and alcian blue periodic acid - schiff to identify goblet cells . 
histological criteria for evidence of reflux oesophagitis included one of the following findings : increase in the basal cell layer thickness > 20% , papillae extending through more than 66% of the epithelial thickness , presence of balloon cells , neutrophils ( > 15 high power field ) or eosinophils , dilatation and congestion of capillary vessels and intraepithelial haemorrhage . 
im was defined as the presence of intestinal - type goblet cells . statistical analysis data were analysed using the sas / stat 8.0 package ( sas institute inc . , cary , nc , usa )  . 
sensitivity , specificity and positive ( ppv ) and negative ( npv ) predictive values of the wst in revealing the possible presence of gastroesophageal reflux were calculated using ph monitoring as the gold stanin antimonio distanziati 15 cm luno dallaltro e un elettrodo di riferimento interno ( zinetics 24 , medtronic , italia )  . 
dopo calibrazione in soluzioni tampone a ph 7 e a ph 1 , il catetere stato inserito per via nasale in modo da posizionare lelettrodo prossimale 5 cm sopra il bordo superiore dello sfintere esofageo inferiore , identificato manometricamente , e lelettrodo distale nello stomaco prossimale . 
durante il periodo di 24 ore di monitoraggio del ph , il paziente veniva mandato a casa e invitato a prendere nota di eventuali sintomi , degli orari dei pasti , di riposo e di risveglio , e comunque incoraggiato a svolgere le proprie attivit giornaliere . 
i dati registrati , scaricati in un pc e analizzati dai programmi specifici multigram gi edition 94 o polygram net , forniti da medtronic italia , sono stati inseriti in una tabella di punteggio ( demeester score ) e un punteggio superiore a 14 , 8 fu considerato anormale [ 3 ]  . 
 endoscopia tutte le procedure endoscopiche sono state eseguite da uno degli autori utilizzando un videoendoscopio olympus ( gif q160 , tokyo , giappone ) e seguendo lo stesso criterio per tutti gli esami . 
per la classificazione delle esofagiti stata utilizzata la classificazione endoscopica di los angeles [ 19 ]  . stata identificata la linea z normale o la presenza di mucosa colonnare estendentesi al di sopra della giunzione gastroesofagea , sospetta per esofago di barrett corto ( a fiamma o < 3 cm ) o lungo ( 3 cm )  . 
in presenza di una linea z normale sono state eseguite 4 biopsie nella mucosa dellesofago distale ( una per quadrante almeno a distanza di un centimetro , prossimali alla giunzione gastroesofagea ) ; in presenza di mucosa colonnare estendentesi prossimalmente alla giunzione gastroesofagea , sono state eseguite biopsie multiple per confermare istologicamente lesofago di barrett . 
tutte le biopsie sono state fissate in formalina tamponata al 10% , numerate e inviate allo stesso patologo , ignaro della diagnosi endoscopica . analisi istologica tutte le analisi istologiche sono state eseguite da uno degli autori . 
tutti i campioni bioptici sono stati fissati in formalina tamponata al 10% , inclusi in paraffina , e le sezioni di 5 micron ottenute sono state colorate con ematossilina - eosina e alcian blue periodic acid - shiff per identificare le cellule goblet . 
i criteri istologici di evidenza di esofagite da reflusso includevano uno dei seguenti parametri : incremento > 20% dello spessore dello strato basale , altezza delle papille > 66% dello spessore epiteliale , presenza di cellule balloon , neutrofili ( > 15 high power field ) o eosinofili , dilatazione e congestione dei capillari , emorragie intraepiteliali . 
sensitivity , specificity and npv and ppv of wst and ph monitoring in revealing the possible presence of oesophagitis ( gold standard : endoscopy ) , barretts oesophagus ( gold standard : endoscopy + biopsy ) and im ( gold standard : histological analysis ) were then calculated . 
ph monitoring wst was positive in 112 patients ( 70% ) , but only 59 / 112 ( 52.67% ) also had positive ph monitoring ; furthermore 24 / 48 patients with a negative wst had a positive ph monitoring ( table 1 )  . 
of the 112 patients with a positive wst , 72 had middle ( 44 / 72 ) and proximal ( 28 / 72 ) reflux , but only 37 of these had positive ph monitoring . 
ph monitoring in the diagnosis of oesophagitis sixty - one out of 160 patients had endoscopic findings of complicated gerd such as oesophagitis also associated to table 1 water siphon test ( wst ) vs . 
ph - metria 24 ore numero di test ph - metria positivi negativi wsta positivi wsta negativi wstb positivi a , considerati globalmente ; wstb , solo reflussi medi e prossimali endoscopico , stata definita dalla presenza di cellule intestinali tipo goblet . 
 analisi statistica i dati sono stati analizzati impiegando il pacchetto software sas / stat 8.0 ( sas institute inc.100 sas campus drive cary , nc27513 - 2414 , usa )  . 
sono stati calcolati i valori di sensibilit , specificit , valore predittivo positivo ( vpp ) e negativo ( vpn ) del wst , rispetto alla ph - metria ( gold standard ) , ai fini della individuazione del reflusso gastroesofageo . 
inoltre sono stati calcolati i valori di sensibilit , specificit valore predittivo , positivo e negativo di wst e ph - metria nella individuazione di possibile presenza di esofagite ( gold standard : endoscopia ) , di esofago di barrett ( gold standard : endoscopia con biopsia ) e di metaplasia intestinale ( gold standard : esame istologico )  . 
stato infine eseguito il test del chi - quadro per valutare lattendibilit statistica dellassociazione tra presenza di reflusso dimostrato al wst e alla ph - metria con presenza di esofagite , esofago di barrett e metaplasia intestinale ; stato considerato statisticamente significativo un valore di p0 , 05 . risultati in tutti i pazienti sottoposti a cineradiografia digitale era presente unernia iatale . 
ph - metria centododici pazienti presentavano un wst positivo ( 70% ) , ma solamente 59 di questi ( 52 , 67% ) avevano anche una phmetria positiva ; inoltre 24 dei 48 pazienti con wst negativo avevano una ph - metria positiva ( tabella 1 )  . 
settantadue dei 112 pazienti con wst positivo avevano un reflusso medio ( 44 pz ) o prossimale ( 28 pz ) , ma solamente 37 presentavano una ph - metria positiva . 
considerando solamente i pazienti con reflusso medio e prossimale al wst , la sensibilit diminuisce ( 45% ) ma la specificit aumenta ( 55% )  . sessantuno dei 160 pazienti avevano segni endoscopici di malattia da reflusso complicata da esofagite anche associata a esofago di barrett : 48 / 61 di questi pazienti avevano un wst positivo mentre solamente 42 avevano una ph - metria positiva come mostrato in tabella 2 . 
nel confronto tra la capacit di wst e ph - metria di individuare i pazienti con esofagite , avendo come gold standard lendoscopia , si rileva che il wst ( considerati tutti i pazienti positivi ) mostra una sensibilit maggiore ( 79% ) ma valori di specificit ( 35% ) , vpp ( 43% ) e vpn ( 73% ) inferiori rispetto alla ph - metria ; qualora poi si a , considered overall ; wstb , considering only middle and proximal refluxes wst vs . 
vpp , valore predittivo positivo ; vpn , valore predittivo negativo . sensitivity % specificity % ppv % npv % wst overall ph monitoring wst middle / proximal accuracy of wst and ph - monitoring in patients with barretts esophagitis accuratezza del wst e della ph - metria in pazienti con esofago di barret sensitivity % specificity % ppv % npv % wst overall ph monitoring wst middle / proximal fig . 
vpp , valore predittivo positivo ; vpn , valore predittivo negativo . barretts oesophagus : 48 out of 61 patients with complicated gerd had a positive wst , whereas 42 had positive ph monitoring ( table 2 )  . 
if the presence of oesophagitis at endoscopy was considered as evidence of gerd , wst , considered overall , showed a higher sensitivity ( 79% ) but lower specificity ( 35% ) , ppv ( 43% ) and npv ( 73% ) than did ph monitoring . 
in this group of 24 patients with considerino solamente i reflussi medio - prossimali la sensibilit diminuisce ( 61% ) , ma la specificit ( 65% ) il vpp ( 51% ) e il vpn ( 73% ) aumentano , come mostrato in figura 1 . 
in questo gruppo di 24 pazienti , con presenza di mucosa colonnare allendoscopia , esofago di barrett corto o lungo , e presenza di metaplasia intestinale allesame istologico , il wst risultato positivo in 21 pazienti , mentre la ph - metria risultava positiva solamente in 13 pazienti come mostrato in tabella 2 . 
la valutazione statistica del wst fa rilevare valori di sensibilit ( 88% ) , vpp ( 19% ) e vpn ( 94% ) superiori alla ph - metria ma valoe . 
vpp , valore predittivo positivo ; vpn , valore predittivo negativo . endoscopic findings of either long or short columnar - lined mucosa and im findings at histological analysis , wst was positive for reflux in 21 , whereas ph monitoring was positive for reflux in 13 only , as shown in table 2 . 
overall , statistical analysis of wst showed higher sensitivity ( 88% ) , ppv ( 19% ) and npv ( 94% ) and lower specificity ( 33% ) than did ph monitoring . 
considering only middle and proximal reflux at wst , sensitivity decreased ( 54% ) , but specificity increased and was higher ( 57% ) than ph monitoring ( 49% ) , as shown in figure 2 . 
overall in this group of patients , wst showed a higher sensitivity ( 89% ) , ppv ( 36% ) and npv ( 90% ) and lower specificity ( 37% ) than did ph monitoring , but considering only middle and proximal reflux , statistical analysis of wst showed all values higher than ph monitoring , as shown in figure 3 . 
assumendo come popolazione di controllo i pazienti risultati negativi allesame endoscopico e applicando il test del chi - quadro , il wst ( reflusso medio - possimale ) rileva reflussi statisticamente correlati ( p = 0 , 002 ) alla presenza di metaplasia intestinale mentre lo stesso non avviene per la ph - metria ( p = 0 , 3014 ) discussione si ritiene che la diagnosi di gerd possa essere fatta sulla e . 
recently , however , different tests ( 24 - h ph monitoring , endoscopy , radiological study , etc . ) have commonly been used for this frequent disorder , each providing information regarding a different aspect of the disease . 
it is our opinion that in patients with symptoms of gerd , the presence of hh should always be investigated . cineradiography is a noninvasive , useful and reliable test to define the anatomical disorder of the gastroesophageal junction , which , in our own study , was confirmed in 95% by one test only ( motility study or endoscopy ) and in 83% by two tests ( motility study and endoscopy )  . 
 the results of this study demonstrate that wst can be useful for the detection of gastroesophageal reflux , even though it must be borne in mind that it only accounts for a mechanical event and does not provide any information about the chemical changes in the oesophagus . 
taking a positive ph study as evidence of reflux , the sensitivity of wst in predicting gerd is high when considered from an overall point of view , but specificity increases only when middle and proximal refluxes are considered , even though sensitivity decreases . 
nevertheless , the finding of 25% of complicated gerd at endoscopy in patients with a positive wst and negative ph monitoring for reflux clearly shows that ph monitoring is not a perfect standard . 
these data show that rabase della storia clinica del paziente ed generalmente accettato che possa essere indicativa per gerd la risposta positiva alla terapia con farmaci inibitori di pompa protonica . nella diagnosi di gerd , sono stati impiegati test differenti ( ph - metria 24 ore , endoscopia , studio radiologico , ecc . ) ognuno dei quali d informazioni diverse rispetto a differenti aspetti della malattia . 
nostra opinione che , nei pazienti con sintomi di gerd , debba essere sempre ricercata la presenza di ernia iatale , e che lesame radiologico sia un test utile , non invasivo , ma attendibile nel definire lalterazione anatomica della giunzione gastroesofagea ; infatti , nel nostro studio , la diagnosi radiologica di ernia stata confermata nel 95% dei casi da un solo test ( manometria o endoscopia ) e nell83% dei casi da due test ( manometria ed endoscopia )  . i risultati di questo studio dimostrano come il wst possa essere utile nella diagnosi di reflusso gastroesofageo , anche se va tenuto presente che il test , quando positivo , rappresenta solamente levento meccanico del reflusso non fornendo , peraltro , alcuna informazione sulle variazioni chimiche allinterno del lume esofageo . 
assumendo come gold standard per reflusso la positivit alla ph - metria , il wst , globalmente considerato , presenta una elevata sensibilit , ma la specificit aumenta soltanto quando sono considerati come positivi i reflussi medio - prossimali anche se , in questo caso , la sensibilit diminuisce . 
tuttavia , il riscontro di 25% di gerd complicata allendoscopia in pazienti con wst positivo e ph - metria negativa per reflusso indica chiaramente che la ph - metria non un test perfetto . 
one study reported 92% sensitivity 0% specificity [ 20 ] , whereas another reported quite different values , with sensitivity at 70% and specificity at 74% [ 21 ]  . 
this fact would also seem to be confirmed by another recently published study [ 22 ] , which , however , took into consideration only 28 patients with refluxes either at or above the thoracic inlet that were either spontaneous or induced by provocative manoeuvres in the recumbent position . 
in this group of patients , the radiological study showed specificity of 100% and sensitivity of 64% . the difference found in our study , with 45% sensitivity and 55% specificity , is due both to the larger number of patients involved and to the fact that we considered positive those with middle - proximal refluxes . 
 our experience , however , is similar to that reported in the recent literature with regard to the high reliability of a radiologically demonstrated proximal reflux in the diagnosis of gerd and in laryngopharyngeal reflux [ 23 ]  . 
if middle and proximal refluxes are considered , wst accuracy is similar to ph monitoring in predicting oesophagitis , with a significant association between reflux and oesophagitis both for wst and for ph monitoring . 
moreover , in our study , wst seemed more reliable than ph monitoring in predicting barretts oesophagus , even though we did not demonstrate a significant association between reflux demonstrated radiologically or with ph monitoring and this disease . 
fewer than 50% of patients had complicated gerd , showing that objective criteria are necessary to suspect and detect the complication . from this point of view , a highly positive wst might suggest endoscopy with biopsies to detect a complication of gerd , as suggested by the presence of 15% of barretts oesophagus and 13% of im in an endoscopically normal oesophageal mucosa . 
with regard to these conditions , wsts higher accuracy might be explained by the fact that im might be correlated to the presence of bile in the oesophageal refluxate , which might disguise the true acid exposure , resulting in false negative results at ph monitoring [ 8 ]  . 
uno studio ha riportato valori di sensibilit del 92% e di specificit pari a zero [ 20 ] , mentre un altro ha riportato valori totalmente differenti ( sensibilit del 70% , e specificit del 74% ) [ 21 ]  . 
questo dato sembra confermato anche da un recente lavoro [ 22 ] che ha preso in considerazione un campione , seppur piccolo , di 28 pazienti con reflusso fino allimbocco toracico , insorto spontaneamente o dopo test di provocazione . 
la differenza riportata nel nostro studio ( sensibilit del 45% e specificit del 55% ) da attribuire al campione pi ampio di pazienti e al fatto di avere considerato come positivi i pazienti con reflusso medio - prossimale . 
 la nostra esperienza simile a quanto riportato recentemente in letteratura riguardo alla elevata attendibilit di un reflusso prossimale radiologicamente dimostrato nella diagnosi di gerd in pazienti con reflusso laringo - faringeo [ 23 ]  . se si considerano come positivi solamente i reflussi medioprossimali , laccuratezza del wst simile a quella della phmetria nel predire lesofagite , con significativa associazione statistica tra reflusso ed esofagite sia per wst che per phmetria . 
inoltre nel nostro studio il wst sembra pi attendibile della ph - metria nel predire lesofago di barrett , anche se non possibile dimostrare una associazione statisticamente significativa tra reflusso dimostrato radiologicamente o con ph - metria e tale patologia . 
nella nostra casistica , tuttavia , meno del 50% dei pazienti ha avuto una gerd complicata , il che indica che sono necessari criteri oggettivi che facciano sospettare e rilevare la complicanza . 
 da questo punto di vista un wst altamente positivo potrebbe suggerire lindicazione a unendoscopia con biopsie per rilevare una gerd complicata , come dimostrato , nella nostra esperienza , dalla presenza nel 15% dei casi di esofago di barrett e nel 13% di metaplasia intestinale in esofago normale allendoscopia . 
in questi casi la maggiore accuratezza del wst potrebbe essere spiegata dal fatto che la metaplasia intestinale potrebbe essere correlata alla presenza di bile nel materiale refluito in esofago , con effetto tampone dellacido e conseguente risultato falsamente negativo alla ph - metria [ 8 ] ; di contro il wst , mostrando il reflusso del contenuto gastrico in esofago , rappresenta radiologicamente levento meccanico indipendentemente dalla sua componente chimica . conclusions in our opinion , wst is no substitute for 24 - h ph monitoring which , although more expensive , time - consuming and invapossiamo affermare che il wst non sostituisce la ph - metria 24 ore la quale , anche se pi costosa , pi lunga e pi invasiva , deve essere usata per valutazioni quali - quantitative sul conclusioni e . 
on the other hand , wst associated with the barium test could be used as a first choice in patients with suspected gerd , as it is an inexpensive , rapid and noninvasive procedure that is simple to perform , readily available , well tolerated by patients and correlates fairly well with symptoms of gerd . 
in our experience , wst , which shows gerd as a mechanical event , is more reliable than ph monitoring in predicting complicated gerd , especially when barium refluxes extending as far as the tracheal carrefour or the pharyngoesophageal junction are taken into account . 
in patients with symptoms of gerd , wst positive for reflux , especially if a middle or proximal one , might be an indication for endoscopy with multiple biopsies . reflusso ed essenziale in pazienti con presentazione atipica di gerd , in pazienti con insufficiente risposta alla terapia con inibitori di pompa protonica e in pazienti candidati allintervento chirurgico . 
per contro , lesame radiografico con wst pu essere utilizzato in pazienti con sospetta gerd come indagine di prima scelta in quanto procedura economica , veloce e non invasiva , semplice da effettuare , facilmente disponibile , ben tollerata dai pazienti e abbastanza ben correlata con i sintomi di gerd . 
nella nostra esperienza , il wst , che dimostra levento meccanico della malattia da reflusso , pi attendibile della ph - metria nel predire le complicanze della malattia , particolarmente quando il bario refluito si estende fino al carrefour tracheale o alla giunzione faringoesofagea . 
in pazienti con i sintomi di gerd , un wst positivo per reflusso , in particolare se medio - prossimale , potrebbe essere unindicazione per una endoscopia con biopsie multiple . 
fugazzola1 1cattedra di radiologia , 2chirurgia vascolare , 3servizio di anestesia , universit degli studi dellinsubria , azienda ospedalierouniversitaria ospedale di circolo e fondazione macchi , viale borri 57 , i - 21100 varese , italy correspondence to : d . 
between march 1999 and march 2004 , 15 isolated iaas in 13 patients ( mean age : 71.8 years ) were selected for endovascular repair by means of a covered stent or stent - graft : 12 were in the common iliac artery ( 2 with the proximal end 12 mm from the aortic bifurcation and 2 involving the distal hypogastric artery ) , and three were in the external iliac artery . 
follow - up ( mean duration : 25 months , range : 660 months ) in the remaining 11 patients , affected by 13 aneurysms , showed aneurysm exclusion in nine cases and progressive shrinkage of the aneurysmal sac in four cases , whereas in the other five , the size of the aneurysm remained unchanged . 
in a patient with bilateral iaa , bilateral proximal endoleaks were observed after 2 years , and the patient was treated with a bifurcated aortic stent - grain another patient with a large aneurysm , a left aortofemoral bypass became necessary after 2 months because of stent - graft dislodgement . 
however , a longer follow - up and larger patient series are needed to verify the long - term efficacy of this form of treatment . key words iliac aneurysm endovascular therapy arteries stents interventional procedures riassunto obiettivo . 
tra marzo 1999 e marzo 2004 sono stati selezionati per lesclusione endovascolare mediante stent ricoperto o endoprotesi 15 aneurismi isolati dellaai in 13 pazienti ( et media 71 , 8 anni ) : 12 dellarteria iliaca comune ( di cui 2 con estremo prossimale distante 12 mm dalla biforcazione aortica e 2 coinvolgenti distalmente larteria ipogastrica ) e 3 dellarteria iliaca esterna . 
il follow - up ( durata media 25 mesi , range 660 mesi ) , disponibile nei restanti 11 pazienti , portatori di 13 aneurismi , ha dimostrato lesclusione dellaneurisma in 9 casi , con la progressiva riduzione volumetrica della sacca in 4 , mentre negli altri 5 la sacca rimasta immodificata . 
in un paziente portatore di aneurismi isolati dellaai bilaterale si osservato un endoleak prossimale di entrambi gli aneurismi a 2 anni , trattato pertanto con endoprotesi aortica biforcata ; in una paziente con un voluminoso aneurisma si resa necessaria la conversione in by - pass aorto - femorale sinistro a due mesi di distanza per dislocazione di pi endoprotesi embricate ; in un paziente , infine , a due anni si rilevato un endoleak rifornito dallarteria ipogastrica , che non stato trattato . 
tuttavia necessario un follow - up pi protratto e una casistica pi numerosa per verificare lefficacia a lungo termine di questo tipo di trattamento . parole chiave aneurisma iliaco terapia endovascolare arterie stents procedure interventistiche introduction introduzione d . 
specific symptoms resulting from mural thrombosis with distal microembolism , rupture or local compression of venous and nervous structures and the urinary tract are relatively infrequent [ 46 ]  . although data on the natural history of isolated iaas are limited owing to the rare occurrence of these aneurysms , expansile growth and subsequent rupture has been documented by various authors [ 2 , 3 , 5 ]  . 
the treatment of choice for isolated aneurysms of the common or external iliac artery with diameter greater than 3 cm is surgical bypass grafting , even if the patient is asymptomatic [ 2 , 5 ]  . 
as occurs with abdominal aortic aneurysms , surgical repair of isolated iaas is considered major surgery , associated with mortality rates of 7%11% for elective procedures and 30%50% in emergency settings [ 5 ]  . 
 the purpose of this paper is to describe our experience in a series of 15 aneurysms involving the common and / or external iliac arteries in 13 patients treated with stent - grafts and to compare our results with those reported in the literature . materials and methods between march 1999 and march 2004 , 160 abdominal aortic and iliac aneurysms were treated by endovascular repair at our institution . 
in particular , 127 aneurysms were either aortic or aortoiliac ; 4 involved the hypogastric artery , 14 were anastomotic pseudoaneurysms ( 9 distal , 5 proximal ) and 15 were isolated iaas . 
the series considered in this study comprised 13 patients ( 10 men and 3 women ) aged between 55 and 91 years ( mean age 71.8 years ) with either unilateral or bilateral iaas selected for endovascular repair using stentgrafts ( table 1 )  . 
twelve were in the common iliac artery ( 2 in the proximal end 12 mm from the aortic bifurcation and 2 involving the distal hypogastric artery ) and 3 in the external iliac artery . 
hypogastric artery aneurysms ( n = 4 ) treated by coil embolisation upstream or downstream from the aneurysm without stent deployment were not considered in this study . two patients were treated as an emergency : one due to haemorrhagic shock following aneurysm rupture , and the othgli aneurismi isolati dellasse arterioso iliaco ( aai ) sono rari , rappresentando circa il 2%7% di tutti gli aneurismi addominali [ 13 ]  . 
la loro eziologia principalmente ateromasica ; le altre cause ( infettive ; in corso di gravidanza ; malattie del collagene come la sindrome di marfan ) sono assai meno frequenti . 
la maggior parte degli aneurismi isolati dellaai asintomatica e viene pertanto diagnosticata in maniera occasionale mediante ecografia , tomografia computerizzata ( tc ) o angiografia ; i sintomi specifici , conseguenti allo sviluppo di una trombosi murale con microembolia distale , alla rottura e alla compressione locale su strutture venose , nervose e vie urinarie , sono piuttosto infrequenti [ 46 ]  . bench i dati sulla storia naturale degli aneurismi isolati dellaai siano limitati a causa della loro relativa rarit , la crescita espansiva e la conseguente rottura stata documentata in diversi studi [ 2 , 3 , 5 ]  . 
il trattamento di scelta nella dilatazione aneurismatica isolata dellarteria iliaca comune o esterna lintervento chirurgico mediante il confezionamento di un by - pass quando il diametro dellaneurisma supera i 3 cm , anche se il paziente asintomatico [ 2 , 5 ] ; per contro , nella patologia dilatativa dellarteria ipogastrica lintervento chirurgico tecnicamente pi impegnativo , soprattutto in considerazione della posizione profonda nella pelvi di tale vaso . analogamente a quanto avviene per gli aneurismi dellaorta addominale , gli interventi per aneurismi isolati dellaai sono considerati interventi di chirurgia maggiore , associati a un tasso di mortalit del 7%11% in elezione e del 30%50% in condizioni di urgenza [ 5 ]  . 
 scopo del presente lavoro descrivere la nostra esperienza in una serie di 15 aneurismi a carico dellarteria iliaca comune e / o esterna in 13 pazienti trattati con endoprotesi e confrontare i risultati ottenuti con quelli riportati in letteratura . 
 materiali e metodi nel periodo compreso tra marzo 1999 e marzo 2004 presso il nostro istituto sono stati sottoposti a trattamento endovascolare 160 aneurismi dellaorta addominale e degli assi iliaci ; in particolare , 127 erano aortici o aorto - iliaci ; 4 erano di pertinenza dellarteria ipogastrica , 14 erano pseudoaneurismi anastomotici ( 9 distali , 5 prossimali ) e 15 aneurismi isolati dellaai . 
la casistica oggetto di questo lavoro si riferisce a 13 pazienti ( 10 maschi e 3 femmine ) , di et compresa tra 55 e 91 anni ( et media di 71 , 8 anni ) , con aneurismi dellaai monoo bilaterali selezionati per lesclusione endovascolare mediante endoprotesi ( tabella 1 )  . 
complessivamente , si trattava di 15 aneurismi : 12 dellarteria iliaca comune ( di cui 2 con estremo prossimale distante circa 12 mm dalla biforcazione aortica e 2 coinvolgenti distalmente larteria ipogastrica ) e 3 dellarteria iliaca esterna , con diametro medio di 4 cm ( range 2 , 58 ) e lunghezza media di 3 , 2 cm ( range 27 )  . 
all patients had associated cardiorespiratory disease with high surgical and anaesthetic risk . preprocedural planning was performed with spiral ct angiography ( except in 1 case ) to evaluate the morphology , size , and anatomical relationships of the aneurysm and the existence of a portion of healthy artery upstream and downstream from the aneurysm ( proximal and distal necks )  . 
this information is indispensable for deciding whether the procedure is feasible and for determining the diameter and length of the stent - grato better determine the length of the stentgraft , also in consideration of the tortuosity of the iliac axes , during the same session , we performed a preliminary angiographic scan using a marked catheter ( 5f optimed pigtail catheter , ettingen , germany )  . twelve patients were treated in the angiography suite under local anaesthesia ( 2% lidocaine ) with and anaesthetist in attendance and monitoring of vital parameters ( heart rate , arterial pressure , oxygen saturation )  . 
subsequently , we used conical endoprostheses , as in the prosthetic branch and extensions of aortobiiliac stent - grafts ( excluder ) , or metal alloy stentgrafts ( wallgraft ) , which have different calibres at their ends and are therefore best suited to address the 2to 3 - mm discrepancy between the diameter of the proximal and distal fixation points along the iliac axis . 
one case required deployment of two stent - grafts owing to unforeseeable shortening of the wallgraft ; another case required three excluder extensions to exclude a large aneurysm . after deployment , we dilated the wallgraft stent - graft and the passager covered stent with an angioplasty balloon ( powerflex cordis , miami , fl , usa ) and the excluder stent - grafts with a latex balloon ( equalizer boston scientific ) to ensure sealing of the proximal and distal fixation sites and complete exclusion of the aneurysm , which was checked by postprocedural angiography . 
 due pazienti sono stati trattati in urgenza : un paziente per shock emorragico conseguente alla rottura dellaneurisma , unaltra paziente perch aveva sviluppato una fistola artero - venosa tra i vasi iliaci con insufficienza cardiaca da alta portata ; nei rimanenti casi asintomatici la diagnosi stata occasionale nel corso di esami ecografici , tc o angiografici espletati per altri motivi . 
tutti i pazienti erano affetti da patologie associate cardiorespiratorie a elevato rischio chirurgico e anestesiologico . il planning pre - procedura stato effettuato mediante angio - tc spirale ( tranne in 1 caso ) per valutare la morfologia , le dimensioni , i rapporti anatomici e lesistenza di una porzione di arteria sana a monte e a valle dellaneurisma ( colletti prossimale e distale ) , informazioni indispensabili per decidere la fattibilit della procedura e , in secondo luogo , il diametro e la lunghezza della protesi . 
per una pi precisa valutazione della lunghezza della endoprotesi , anche in relazione alla tortuosit degli assi iliaci , stato espletato uno studio angiografico preliminare , nella stessa seduta della procedura interventistica , con catetere centimetrato ( pigtail 5 f optimed , ettingen , germania )  . dodici pazienti sono stati trattati in sala angiografica in anestesia locale ( lidocaina al 2% ) , in assistenza anestesiologica con monitoraggio dei parametri vitali ( frequenza cardiaca , pressione arteriosa , saturazione di ossigeno ) mediante approccio percutaneo transfemorale omolaterale alla lesione con introduttori da 10 - 12 f . 
solo la prima procedura stata effettuata con uno stent ricoperto ( passager ) ; successivamente sono state utilizzate endoprotesi coniche tipo braccio protesico ed estensioni di endoprotesi aorto - bisiliaca ( excluder ) o endoprotesi in lega metallica ( wallgraft ) che abbracciano calibri diversi alle estremit e che quindi si adattano meglio alla discrepanza di 23 mm tra il diametro dellancoraggio prossimale e quello dellancoraggio distale lungo lasse iliaco . 
in the remaining 11 patients with a total of 13 aneurysms the follow - up imaging studies ( mean follow - up : 25 months , range : 660 ) demonstrated stent - graft patency and exclusion of the aneurysm in 8 / 11 patients , for a total of 9 / 13 aneurysms ( cases 18 in table 1 )  . 
nel periodo postoperatorio stata somministrata eparina in infusione endovenosa per 48 ore e quindi eparina a basso peso molecolare per almeno un mese ; successivamente si fatto ricorso alla terapia antiaggregante domiciliare quoad vitam ( 120 mg / die di ticlopidina o di acido acetilsalicilico )  . il follow - up stato espletato mediante esame eco - colordoppler prima della dimissione e con controlli angio - tc seriati a 3612 mesi dal trattamento e , successivamente , una volta allanno , per escludere riperfusioni e per valutare le dimensioni della sacca aneurismatica . 
 risultati in tutti i casi trattati si ottenuta la completa e immediata esclusione della sacca aneurismatica , documentata al controllo angiografico effettuato al termine della procedura ( successo tecnico primario del 100% )  . 
c postprocedural angiogram : complete exclusion of the aneurysmal sac after placement of a stent - graft in the common iliac artery and deployment of platinum coil in the hypogastric artery . 
d ct angiography at 2 years ( volume rendering reconstruction ) : complete exclusion of the aneurysmal sac , the conical shape of the stentgraft and the platinum coil in the hypogastric artery are visible . 
e ct angiography at 2 years ( curved multiplanar reconstruction ) : the aneurysmal sac has almost disappeared and the stent appears patent , without intimal hyperplasia . fig , 1a angio - tc : aneurisma isolato dellarteria iliaca comune di destra di 3 , 4 cm di diametro , con coinvolgimento dellarteria ipogastrica . 
b angiografia pre - procedura : aneurisma isolato dellarteria iliaca comune di destra che coinvolge lemergenza dellarteria ipogastrica e larteria iliaca esterna c angiografia post - procedura : posizionata endoprotesi in arteria iliaca comune e spirale in platino in arteria ipogastrica con completa esclusione della sacca aneurismatica . 
d angio - tc a 2 anni ( ricostruzione vr ) : completa esclusione della sacca aneurismatica , riconoscibile la configurazione conica dellendoprotesi e la presenza di spirale in arteria ipogastrica . 
2a computed tomography ( ct ) angiography : isolated aneurysm of the right common iliac artery with a wide eccentric rim of thrombosis measuring 5.2 cm in its greater axis . 
2a angio - tc : aneurisma isolato dellarteria iliaca comune di destra con vasta banda di trombosi eccentrica a maggior asse di 5 , 2 cb angiografia pre - procedura : dilatazione aneurismatica con colletto prossimale di circa 2 c larteria ipogastrica destra appare steno - occlusa allorigine ( freccia )  . 
d angio - tc a 2 anni ( immagine assiale ) : la sacca aneurismatica appare completamente esclusa e persiste immodificata per dimensioni ( 5 , 2 cm )  . ni embricate di endoprotesi di nitinol della lunghezza di 710 cm , si sono scardinate non avendo un valido sostegno nel contesto della sacca aneurismatica , voluminosa , particolarmente estesa in lunghezza . 
non disponibile un follow - up pi prolungato , essendo il paziente deceduto a 3 anni dal trattamento . in sintesi , 2 / 13 pazienti sono stati esclusi dal follow - up ; nei restanti 11 si riportato un successo clinico in 8 / 11 ( con efficace esclusione di 9 aneurismi )  . discussione gli aneurismi isolati dellaai sono rari [ 7 , 8 ] , e la loro storia naturale caratterizzata da un alto rischio di rottura [ 5 , 9 ]  . 
la crescita dellaneurisma si verifica nel 36% dei pazienti con una velocit di 4 mm allanno ; tuttavia , la dimensione critica di rottura non stata ancora definita [ 10 ]  . 
in 36% of patients , the aneurysm grows at a rate of 4 mm / year , but a critical size for rupture has yet to be been defined [ 10 ]  . 
however , because the signs of impending rupture are difficult to detect and the risk of death from a ruptured aneurysm is particularly high , some authors prefer to operate even aneurysms smaller than 3 cm [ 5 , 9 ]  . 
surgical repair after aneurysm resection and bypass graft deployment is nonetheless a complex procedure , with mortality rates ranging from 7% to 13% [ 5 , 11 ] and a significant rate of complications such as peripheral ischaemia , haemorrhage , bypass infections and ureteral lesions [ 5 ]  . 
endovascular deployment of stent - grafts is a sufficiently safe , alternative approach to surgery that has been shown to be feasible in animals and humans [ 12 , 13 ]  . from the literature , it is clear that experiences of endovascular treatment with covered stents of iaas are limited to case reports and case series with short - term and mid - term follow - up [ 1428 ]  . 
the published results indicate clinical success in 78.9% [ 28 ] to 97% [ 18 , 19 , 25 ] , with 100% success reported for some small series with short - term follow - up [ 15 , 16 ]  . 
in our series , one of the largest published nationally and with the longest mean follow - up period ( 25 months ) [ 22 , 25 , 26 ] , clinical success was achieved in 72% of patients ( 69% of iaas ; in particular , it should be noted that six of the nine successfully treated aneurysms were 3 cm )  . 
this relatively low success rate is justified by several factors : in one patient with a bilateral aneurysm of the common iliac artery with a fairly short proximal neck ( 12 mm ) , the stent - grafts excluded the aneurysms only temporarily ; subsequently , progressive dilatation of the aortic bifurcation caused migration of both stent - grafts , along with appearance of a bilateral type 1 endoleak , requiring deployment of a bifurcated stent - graft after 30 months . 
today , we would consider this case by the same standards as an aortobiiliac aneurysm , that is , deserving treatment with a bifurcated stent - grain one patient with a large , particularly long , aneurysm that communicated with the iliac vein , treatment might have been successful had we used an adequately long stent - graft ( not commercially available at the time ) ; the procedure was , however , carried out as an emergency and allowed elective bypass surgery to be performed after 2 months . 
 no complications , such as stent - graft thrombosis [ 20 , 24 ] , significant intimal hyperplasia [ 19 ] or distal embolisation [ 15 , 21 , 23 , 27 ] were observed . 
in contrast to other series [ 15 , 17 , 23 , 27 ] , there were no cases of intestinal ischaemia or gluteal claudication , probably because the hypogastric trattamento chirurgico elettivo indicato per la maggior parte dei pazienti ed raccomandato per aneurismi asintomatici di diametro > 3 cm , tenuto conto che quelli pi piccoli hanno di solito una buona prognosi [ 2 ] ; tuttavia , data la difficolt di trovare dei segni di imminente rottura e lelevato rischio di morte nel caso questa evenienza si verificasse , alcuni autori preferiscono intervenire anche per dimensioni inferiori [ 5 , 9 ]  . 
la ricostruzione chirurgica dopo resezione dellaneurisma e interposizione di un by - pass per una procedura complessa con tassi di mortalit del 7%13% [ 5 , 11 ] , accompagnati da significative complicanze operatorie quali ischemia periferica , emorragia , infezione del by - pass e lesioni delluretere [ 5 ]  . 
 dalla revisione della letteratura si evince che esperienze relative al trattamento endovascolare con stent ricoperti degli aneurismi dellaai sono limitate a case report e a casistiche con un follow - up a breve e medio termine [ 1428 ] ; i risultati pubblicati riportano un successo clinico variabile dal 78 , 9% [ 28 ] al 97% [ 18 , 19 , 25 ] , con risultati del 100% riferiti da alcuni autori per serie numericamente contenute e con follow - up a breve termine [ 15 , 16 ]  . 
nella casistica personale , che una delle pi numerose tra quelle pubblicate a livello nazionale e con follow - up medio pi lungo ( 25 mesi ) [ 22 , 25 , 26 ] , il successo clinico stato ottenuto nel 72% dei pazienti ( 69% degli aneurismi dellaai : in particolare va segnalato che dei 9 aneurismi trattati con successo , 6 presentavano un diametro 3 cm )  . 
in un paziente , portatore di aneurisma bilaterale dellarteria iliaca comune con colletto prossimale piuttosto corto ( 12 mm ) , le protesi hanno escluso solo temporaneamente gli aneurismi ; peraltro la malattia per la progressiva dilatazione della biforcazione aortica ha provocato la migrazione di entrambe le endoprotesi con comparsa di endoleak bilaterale di tipo i , ci che ha reso necessario il posizionamento a 30 mesi di distanza di una endoprotesi biforcata : tale paziente stato da noi trattato nel 1999 , allinizio della nostra esperienza ; oggi considereremmo questo caso sin dal primo approccio alla stessa stregua di un aneurisma aorto - bis - iliaco , meritevole pertanto di trattamento mediante unendoprotesi biforcata . 
in una paziente , portatrice di aneurisma voluminoso , particolarmente esteso in lunghezza e comunicante con la vena iliaca , il trattamento avrebbe potuto avere successo se si fosse impiegata ununica endoprotesi di lunghezza adeguata ( allora peraltro non disponibile in commercio ) ; la procedura comunque , espletata in urgenza , ha consentito di effettuare , a distanza di 2 mesi , lintervento chirurgico di by - pass in elezione . 
 non si sono verificate complicanze quali trombosi dellendoprotesi [ 20 , 24 ] , iperplasia intimale di significativa entit [ 19 ] , embolizzazione distale [ 15 , 21 , 23 , 27 ] ; non si d . 
abbiamo riscontrato uno pseudoaneurisma della arteria femorale comune , trattato con successo mediante iniezione percutanea di trombina . conclusions conclusioni endovascular repair of iaas has the advantage of being a minimally invasive procedure that can be proposed not only for patients with contraindications for conventional surgery but also for those who are eligible for bypass surgery , as the incidence of complications is lower . 
in small aneurysms , instead , it can be proposed as a definitive procedure , even though longer follow - up periods are needed to establish its efficacy in the long term . il trattamento endovascolare degli aneurismi dellaai possiede i vantaggi delle procedure mini - invasive ed proponibile non solo nei pazienti in cui la chirurgia tradizionale controindicata , ma anche in quelli che possono affrontare lintervento di by - pass per la minore incidenza di complicanze ; inoltre , non preclude lintervento chirurgico , che pu venire effettuato in un secondo tempo . 
fugazzola1 1vascular and interventional radiology , department of radiology , 2department of urology , 3anesthesiology , university of insubria , viale borri 57 , i - 21100 varese , italy correspondence to : g . 
over the last year , we replaced 27 double - j ureteral stents in 20 patients ( 10 men and 10 women ; mean age 67.7 years , range 4383 ) ; 15 / 20 patients had a native kidney , 3 / 20 had a transplanted kidney and 2 / 20 had a ureteroileal conduit . 
all stents were grasped with a gooseneck snare under fluoroscopic control , and the distal end was withdrawn just outside the urethra ; then a wire was advanced through the stent lumen and positioned in the renal pelvis . 
nel corso dellultimo anno abbiamo sostituito 27 stent ureterali in 20 pazienti ( 10 maschi e 10 femmine ; et media 67 , 7 anni , range 4383 ) ; 15 / 20 in reni nativi ; 3 / 20 in pazienti portatori di trapianto renale e 2 / 20 in pazienti con uretero - ileo - cutaneostomia . 
several approaches have been described for retrieval and replacegli stent ureterali sono un dispositivo ampiamente utilizzato nel trattamento delluropatia ostruttiva ; possono essere posizionati per via discendente transnefrostomica o ascendente mediante cistoscopia . 
il management degli stent richiede la sostituzione circa ogni 6 mesi al fine di prevenirne lostruzione e linfezione , che sono evenienze tra loro correlate e relativamente frequenti [ 1 ]  . 
 the purpose of this study was to evaluate the feasibility and results of an interventional radiology technique for transurethral retrieval and replacement of ureteral stents under fluoroscopic control with the aim of proposing the method as an alternative to cystoscopic replacement . te diverse esperienze riguardati la rimozione e la sostituzione di stent ureterali con guida fluoroscopica mediante approccio percutaneo transrenale [ 24 ] o con guida cistoscopica per via transuretrale [ 5 ] ; sono stati riportati solo pochi casi di rimozione e sostituzione per via retrograda senza guida cistoscopica [ 3 , 4 , 68 ]  . 
la sostituzione transrenale una procedura con un rischio di sanguinamento non trascurabile legato al ripetuto passaggio di dispositivi di elevato calibro attraverso il parenchima renale [ 24 ] ; la sostituzione ascendente cistoscopica prevede lutilizzo di stent di calibro non superiore a 6 f , per motivi tecnici legati al diametro del canale operativo del cistoscopio ; tali stent vanno quindi incontro a occlusione pi frequentemente rispetto a quelli posizionati per via discendente [ 5 ]  . 
 scopo del nostro lavoro valutare fattibilit e risultati di una tecnica di radiologia interventistica di rimozione e sostituzione transuretrale degli stent ureterali con guida fluoroscopica , al fine di proporre la metodica come alternativa alla sostituzione cistoscopica . materials and methods materiali e metodi transplants , three kidney over the past year , we replaced 27 double - j ureteral stents in 20 patients ( 10 men and 10 women ; mean age 67.7 years , range 4383 ) ; 13 / 20 patients underwent unilateral stent replacement and 7 / 20 underwent bilateral replacement . 
stents had been implanted antegrade ( 9 cases ) or retrograde ( 18 cases ) to treat malignant ureteral obstructions ( 14 cases : five gynaecological tumours , three bladder carcinomas , three prostate carcinomas and three colorectal carcinomas ) or fibrotic obstructions ( six cases : ureteroileal conduits and one postoperative fibrosis )  . 
all procedures were carried out with the patients under sedation and analgesia ( with propofol , fentanyl and midazolam in appropriate doses for the patients weight , the length of the procedure and the patients pain threshold )  . 
after accurate disinfection , the bladder was catheterised with a vascular introducer sheath ( calibre : 7 f ; length : 15 cm for women and 23 cm for men ) ( cordis , miami , fl , usa )  . 
the bladder was distended with a 3 : 1 solution of iodinated contrast material and saline to reduce pain and facilitate intravesical manoeuvres ; the dilution was chosen to obtain sufficient bladder opacification to provide a landmark for the fluoroscopic manoeuvres without affecting visualisation of the devices . 
the distal end of the ureteral stent was grasped with a 6 - f gooseneck snare catheter with an 18 - mm loop ( hooker , meditalia , biomedica , modena , italy ) under fluoroscopic guidance nellultimo anno abbiamo sostituito 27 stent ureterali con configurazione a doppio j in 20 pazienti ( 10 maschi e 10 femmine ; et media 67 , 7 anni , range 4383 ) ; in 13 / 20 pazienti stata effettuata una sostituzione monolaterale e in 7 / 20 pazienti bilaterale ; in 15 / 20 pazienti la sostituzione stata effettuata in uretere nativo , 3 / 20 pazienti erano portatori di trapianto renale e 2 / 20 di uretero - ileo - cutaneostomia . 
gli stent erano stati posizionati per via anterograda ( in 9 casi ) o retrograda ( in 18 casi ) per trattare ostruzioni ureterali maligne ( 14 casi : 5 tumori ginecologici , 3 carcinomi vescicali , 3 carcinomi prostatici e 3 carcinomi del colon - retto ) o fibrose ( 6 casi : 3 trapianti renali , 2 uretero - ileo - cutaneostomie e 1 post - chirurgica )  . 
tutte le procedure sono state effettuate con sedazione e analgesia ( ottenuta mediante propofol , fentanyl e midazolam in dosi adeguate al peso del paziente , alla durata della procedura e alla tollerabilit del dolore da parte del paziente )  . 
dopo asepsi accurata stato eseguito il cateterismo vescicale utilizzando un introduttore vascolare ( calibro : 7 f ; lunghezza : 15 cm per le femmine e 23 cm per i maschi ) ( cordis , miami , fl , usa )  . 
after stent withdrawal , the hydrophilic guidewire was exchanged with a superstiff device ( amplatz , boston scientific , ratingen , germany ) by using a straight 5 - f catheter ( glidecath , terumo , tokyo , japan )  . 
in one patient with a ureteroileal conduit , the stent could not be withdrawn , as incrustations and adhesions due to recurrent infecstato ottenuto un successo tecnico in 26 / 27 casi . 
in un paziente con uretero - ileo - cutaneostomia non stata possibile la retrazione dello stent , il quale risultava adeso alle pareti ureterali a causa di incrostazioni e aderenze causate da ing . 
durante il follow - up ( 116 mesi , medio 6 , 7 ) , abbiamo osservato 4 ostruzioni di stent trattate con una ulteriore sostituzione retrograda con guida fluoroscopica . discussion ureteral stents are used to maintain ureteral patency in various benign or malignant conditions . 
cystoscopic retrograde replacement has been widely described [ 5 ] ; for cases of failure of this approach , various authors have reported antegrade transrenal replacement [ 24 ]  . a limited number of studies have reported on retrograde replacement under fluoroscopic control , with success rates of 97%100% . 
fluoroscopically guided retrograde removal and replacement with snare catheters may be used when the cystoscopic approach is unfeasible or fails ( patients with frozen pelvis or bladder - neck sclerosis in whom cystoscope manipulation may be difficult ; malignant obstructions involving the ureteral papilla ; patients with ankylosis who are unable to assume the lithotomy position for cystoscope insertion ; patients with bleeding - prone bladder neoplasms because the introduction of cystoscopy instruments that are larger in calibre than those used in fluoroscopic removal carries a higher risk of bleeding ; patients with a urostomy )  . 
in such cases , the advantage of fluoroscopically guided replacement lies in the smaller calibre of the devices ( 68 f ) , which are easier to manipulate inside the bladder and consequently involve a lower risk of bleeding in neoplastic disease . 
in addition , angiographically guided recanalisation of the stent being removed allows maintenance of ureteral - tract patency , which is monitored by fluoroscopy , and the ability to check that the distal extremity of the new stent has reached the renal pelvis . 
because the male urethra is longer and grasping and withdrawing of the stent may be more difficult , special care must be taken when performing these manoeuvres in men [ 6 , 8 ]  . 
in our experience , we used commercial gooseneck snare catheters ; some authors have described the use of homemade snares consisting of a catheter and a hydrophilic guidewire , which , albeit more difficult to manipulate , reduces the cost of the procedure [ 6 ]  . 
forceps - type devices have seldom been used and appear to be poorly suited discussione gli stent ureterali sono utilizzati per mantenere la perviet della via urinaria in diverse patologie benigne e maligne . 
la sostituzione retrograda con guida cistoscopica stata ampiamente descritta [ 5 ] ; in caso di fallimento di tale approccio stata riportata da diversi autori la sostituzione anterograda per via transrenale [ 24 ]  . sono state descritte poche esperienze riguardanti la sostituzione retrograda con guida fluoroscopica con tassi di successo compresi tra 97%100% [ 3 , 4 , 68 ]  . 
la sostituzione retrograda fluoroscopica , mediante catetere a laccio , pu essere utilizzata in caso di non fattibilit o di fallimento dellapproccio cistoscopico ( pazienti con pelvi congelata o con collo vescicale sclerotico nei quali risulta difficoltoso manovrare il cistoscopio ; ostruzioni maligne coinvolgenti la papilla ureterale ; pazienti con anchilosi che non possono essere messi in posizione ginecologica per lintroduzione del cistoscopio ; pazienti con neoplasie facilmente sanguinanti che coinvolgono la vescica , in quanto lintroduzione dei dispositivi cistoscopici , di calibro maggiore rispetto a quelli per la rimozione fluoroscopica , ha un maggior rischio di sanguinamento ; pazienti con urostomie )  . 
il vantaggio della sostituzione con guida fluoroscopica , in questi casi , legata al minor calibro dei dispositivi introdotti ( 68 f ) che risultano pi manovrabili allinterno della vescica e comportano quindi minori rischi di sanguinamento in caso di patologia neoplastica . 
inoltre la ricanalizzazione dello stent da rimuovere mediante guida angiografica consente di mantenere la canalizzazione delluretere , che viene controllata mediante fluoroscopia , e di verificare che lestremo distale del nuovo stent abbia raggiunto la pelvi renale ; tale manovra di fondamentale importanza soprattutto in pazienti con stenosi severe da patologia maligna [ 6 ]  . 
nei pazienti di sesso maschile , a causa della maggiore difficolt nellafferrare e retrarre lo stent in relazione alla lunghezza delluretra , deve essere osservata una particolare attenzione durante lespletamento di queste manovre [ 6 , 8 ]  . 
nella nostra esperienza abbiamo utilizzato cateteri a laccio tipo goose - neck commerciali ; alcuni autori hanno riportato lutilizzo di lacci home made , costituiti da un catetere e una guida idrofilica , che risultano meno maneggevoli ma consentono una riduzione dei costi della procedura [ 6 ]  . 
stent replacement in patients with a kidney transplant does not normally pose special problems , although more accurate asepsis needs to be maintained because of the risk of upper urinary tract contamination in subjects receiving immunosuppressant therapy . 
in this case , we hypothesised that encrustations related to intestinal secretions or recurrent upper urinary tract infections caused the stent to adhere to the ureteral walls , preventing its withdrawal . 
retrograde replacement may also prove unfeasible in the case of proximal stent migration into the renal pelvis with distal end at ureteral level . a further advantage is that pyelographic intraprocedural monitoring with intracavitary injection of contrast material can be performed in addition to fluoroscopic control to ensure more precise and safer positioning of the cranial extremity of the stent and easier negotiation of possible ureteral kinks or bends . raramente utilizzati e paiono poco adatti a questo tipo di procedura in relazione al piccolo calibro dei bracci [ 2 ]  . il rischio di sanguinamento dellapproccio retrogrado minore rispetto alla sostituzione anterograda a causa dellelevato calibro dei dispositivi che in questultimo caso devono attraversare il parenchima renale ; tale approccio deve quindi essere riservato , secondo la nostra opinione , alla eventualit di fallimento della sostituzione retrograda . 
la sostituzione in pazienti con trapianto renale generalmente non pone particolari problemi , ma necessario osservare unasepsi pi accurata in relazione al rischio di contaminazione delle alte vie urinarie in soggetti in terapia immunosoppressiva . 
 lunico caso di insuccesso della rimozione , nella nostra esperienza , si verificato in presenza di derivazione urinaria ( uretero - ileo - cutaneostomia ) ; in questo caso stato ipotizzato che le incrostazioni causate dalle secrezioni intestinali o dalle ricorrenti infezioni delle alte vie urinarie che si erano verificate in questo paziente avessero causato ladesione dello stent alle pareti ureterali impedendone la retrazione . 
la sostituzione retrograda pu risultare non fattibile anche in caso di dislocazione prossimale dello stent in pelvi renale con estremo distale a livello delluretere . inoltre la possibilit di effettuare , oltre al controllo fluoroscopico , anche un controllo intra - procedurale pielografico mediante iniezione intracavitaria di mezzo di contrasto , permette un posizionamento pi preciso e sicuro dellestremo craniale dello stent , superando pi facilmente anche eventuali inginocchiamenti o kinking delluretere . conclusion conclusione in conclusion , transurethral fluoroscopically guided replacement of dysfunctioning ureteral stents may be considered a safe and effective alternative to cystoscopic replacement , as it is just as acceptable to the patient and allows insertion of larger stents with better long - term patency rates . 
in cases where the cystoscopic approach is unfeasible , transurethral fluoroscopically guided replacement should be preferred to transrenal replacement because it is less invasive , avoiding passage through the renal parenchyma and eliminating the risk of bleeding . in conclusione , la procedura pu essere considerata unalternativa sicura ed efficace alla sostituzione per via cistoscopica degli stent ureterali non funzionanti , in quanto almeno altrettanto compliante per il paziente e permette il posizionamento di stent di calibro maggiore , con una migliore perviet a lungo termine . 
in caso di non fattibilit dellapproccio cistoscopico , la sostituzione retrograda preferibile alla sostituzione transrenale per la minore invasivit , poich evita lattraversamento del parenchima renale eliminando il rischio di sanguinamento a esso correlato . 
pupillo , via della mainetta , 88d , i - 67010 coppito , laquila , fax : + 39 - 086 - 4273184 , e - mail : pupillina@libero.it received : 05 september 2006 / accepted : 12 january 2007 / published online : 21 september 2007 abstract purpose . 
magnetic resonance imaging ( mri ) was performed using a 1.5 - t magnet with a phased - array coil and acquisition of t2 - weighted single - shot fast spin echo ( ssfse ) half fourier sequences before intravenous administration of gadolinium , and t1 - weighted fast spoiled gradient ( fspgr ) fat - saturated sequences before and after contrast administration . 
before the examination , patents received oral polyethylene glycol ( peg ) ( 1 , 000 ml for adults ; 10 ml / kg of body weight for children )  . 
abbiamo studiato un campione di 50 pazienti con diagnosi istologica di morbo di crohn ( 37 in fase attiva di malattia ; 13 in remissione clinica ) ; abbiamo utilizzato un magnete da 1 , 5 t , bobina phased array e sequenze t2 dipendenti ssfse half fourier in fase precontrastografica e t1 dipendenti fspgr fat - sat in fase pree post - contrastografica . 
prima di eseguire lesame stato somministrato per os glicole polietilenico ( peg ) ( 1000 ml per adulti ; 10 ml / kg del peso corporeo per bambini )  . abbiamo tracciato roi ( region of interest ) per valutare lintensit del segnale nella parete intestinale normale e patologica e la percentuale dincremento nel tempo . 
in caso di remissione della malattia dopo terapia , per contro , si ha una significativa riduzione dellincremento di segnale , di poco aumentato rispetto a quello delle anse normali . 
lo studio dinamico con risonanza magnetica ( rm ) ci consente di quantificare lattivit infiammatoria locale della parete intestinale . parole chiave morbo di crohn risonanza magnetica cdai introduction introduzione crohns disease is an idiopathic inflammatory disease that may affect any portion of the gastrointestinal tract from the mouth to the anus . 
assessment of disease activity , instead , is based on laboratory parameters and disease activity indexes , among which the gold standard is the crohns disease activity index ( cdai ) , which assigns a score to each sign or symptom ( table 1 ) to enable an overall clinical assessment [ 14 ]  . 
 the diagnosis of crohns disease essentially relies on ileocolonoscopy and radiographic barium studies of the no tenue coinvolto nell80% dei pazienti , con una netta predisposizione della malattia per lileo terminale . 
la patologia interessa anche il colon nel 50% dei pazienti , mentre nel 15%20% dei casi si verifica interessamento isolato del colon e nel 2%3% si osserva una patologia perianale isolata . 
 i pazienti affetti dal morbo di crohn non rappresentano una popolazione omogenea , per cui , per poter meglio orientare le scelte terapeutiche , si reso necessario un sistema di classificazione della patologia basato sulla severit e sullestensione della stessa . 
lattivit di malattia viene invece valutata utilizzando , oltre ai parametri laboratoristici , indici di attivit di malattia tra i quali il gold standard considerato il crohns disease activity index ( cdai ) , che attristool count summed daily for 7 days sum of 7 days of daily ratings as : 0 = none ; 1 = mild ; 2 = moderate ; 3 = severe sum of 7 days of daily ratings as 0 = generally well ; 1 = slightly below par ; 2 = poor ; 3 = very poor ; 4 = terrible any of the following present during the previous 7 days : a . 
fever > 100f 0 = no ; 1 = yes 0 = none ; 2 = questionable ; 5 = definite 47 - hematocrit ( males ) 42 - hematocrit ( females ) 100 [ 1 - ( body weight / standard weight ) ] table 1 crohns disease activity index variable scale liquid or very soft stools abdominal pain general well - being features of extraintestinal disease opiates for diarrhoea abdominal mass hematocrit % body weight below standard tabella 1 indice di attivit del morbo di crohn variabili scala feci liquide o molto morbide dolore addominale benessere generale manifestazione extraintestinali somma di una valutazione quotidiana per 7 giorni somma di una valutazione quotidiana per 7 giorni : 0 = buono ; 1 = lieve ; 2 = moderato ; 3 = severo somma di una valutazione quotidiana per 7 giorni : 0 = buono ; 1 = lievemente sotto la media ; 2 = basso ; 3 = molti basso ; 4 = scarso alcuni dei seguenti aspetti nei precedenti 7 giorni : a . 
febbre > 100f 0 = no ; 1 = si 0 = assente ; 2 = dubbia ; 5 = sicura 47 - ematocrito ( maschi ) 42 - ematocrito ( femmine ) oppiacei per la diarrea masse addominali ematocrito % di peso corporeo sotto la media 100 [ 1 - ( peso corporeo / peso standard ) ] weight 20 each punteggio 20 ognuna v.a. 
sonography is widely used in patients with acute manifestations of disease ; it is able to demonstrate indirect signs of inflammation and , with use of colour power - doppler imaging , it can identify increased wall vascularity . 
spiral computed tomography ( ct ) and ct enteroclysis are valuable tools for detecting stenosis and complications of crohns disease . the diagnostic accuracy of this examination is in part constrained by the difficulty in obtaining adequate lumen distension , a situation that does not allow for correct bowel wall measurement , and by the subjective nature of the assessment of wall enhancement . 
in addition , the use of ionising radiation is not to be underestimated . magnetic resonance imaging ( mri ) is a powerful tool in the diagnosis and follow - up of intestinal diseases [ 822 ]  . 
the technique does not make use of ionising radiation , an important feature if we consider that crohns disease predominantly affects young subjects [ 15 , 2435 ]  . the aim of our study was to assess the possibility of quantifying local inflammatory activity in patients with crohns disease by means of a dynamic study of contrast uptake by the intestinal wall after oral administration of an isosmolar solution to distend the small bowel [ 20 , 36 , 37 ]  . materials and methods fifty patients ( 30 females and 20 males ; age range 871 years , average age 26.3 years ) with crohns disease diagnosed by endoscopic examination and biopsy was studied by mri before and after administration of contrast material . thirty - seven patients were in the active phase of disease ( 25 in the early phase ; 12 in the reactivation phase ) ; 13 were in the remission phase after treatment . 
only five patients had undergone surgery prior to the mri examination ( almost 1 year earlier ) : three had ileocolic resection with terminoterminal anastomosis , and two had ileal resection with sparing of the ileocaecal valve and laterolateral ileoileal anastomosis . 
about 25 min before the examination , patients were administered an oral contrast agent , polyethylene glycol ( peg ) ( 1 , 000 ml for adults ; 10 ml / kg body weight for children ) to distend the small bowel [ 21 , 30 , 38 ]  . 
the mri examination [ 39 , 40 ] was performed with : t2 - weighted single shot fast spin echo ( ssfse ) half fourier sequences [ te : 96.1 ; tr : ( cid : 2 ) ; field of view ( fov ) : 40 40 cm ; matrix : 256 160 ; slice thickness : 5 mm ; acbuisce un punteggio a ogni segno o sintomo ( tabella 1 ) , permettendo cos una valutazione clinica globale [ 14 ]  . 
lecografia viene ampiamente utilizzata soprattutto in pazienti con manifestazioni acute ; in grado di dimostrare segni indiretti di flogosi e , con luso del modulo color power doppler capace di rilevare lincremento della vascolarizzazione parietale . 
la tomografia computerizzata ( tc ) e il clisma - tc mostrano con accuratezza le stenosi e le complicanze del morbo di crohn . laccuratezza diagnostica di tale indagine comunque in parte limitata dalla difficolt a ottenere unadeguata distensione del lume ( tc spirale ) , situazione che non permette la corretta misurazione della parete viscerale , e dalla soggettivit nel valutare il grado di accumulo di contrasto parietale ; inoltre il paziente sottoposto a radiazioni ionizzanti . la risonanza magnetica ( rm ) rappresenta un valido strumento per superare questi limiti nella diagnosi e nel followup delle patologie intestinali [ 822 ]  . 
tale tecnica non espone il paziente a radiazioni ionizzanti , fattore da non sottovalutare dato che la popolazione colpita dalla patologia prevalentemente costituita da persone giovani [ 15 , 2435 ]  . scopo del nostro lavoro stato valutare se possibile determinare il grado di attivit infiammatoria locale in pazienti affetti da morbo di crohn studiando dinamicamente laccumulo di contrasto della parete intestinale dopo somministrazione per via orale di una soluzione isosmotica in grado di produrre distensione intestinale [ 20 , 36 , 37 ]  . materiali e metodi un campione di 50 pazienti ( 30 di sesso femminile e 20 di sesso maschile ; di et compresa tra 8 e 71 anni ; et media 26 , 3 anni ) , con diagnosi di morbo di crohn dimostrata mediante esame endoscopico associato a biopsia , stato sottoposto a esame di rm senza e con mezzo di contrasto paramagnetico . in 37 dei pazienti esaminati la malattia di crohn si presentava in forma attiva ( 25 pazienti erano nella forma primitiva di malattia , 12 pazienti erano in fase di riacutizzazione ) ; 13 pazienti si presentavano in fase di remissione di malattia dopo terapia . 
solo 5 dei pazienti esaminati erano stati precedentemente ( almeno 1 anno prima ) trattati chirurgicamente , 3 mediante resezione ileo - colica con anastomosi termino - terminale e 2 mediante resezione ileale con conservazione della valvola ileo - cecale e anastomosi ileo - ileale latero - laterale . 
peg has the same signal intensity as water , making the distended intestinal lumen appear hyperintense on t2weighted sequences and hypointense on t1 - weighted sequences . image evaluation images were assessed for bowel wall thickness at the terminal ileum , significant and nonsignificant luminal stenosis , and intra - abdominal abscess or fluid collections . 
moreover , after the mri examination , regions of interest ( rois ) of 57 mm2 were placed over the diseased loop and healthy bowel wall to study signal intensity and percentage increase over time and thus quantitatively assess bowel wall vascularity and disease activity [ 41 ]  . 
 spearmans test was used to correlate each patients cdai score with the time required to reach a plateau phase and the percentage of maximum enhancement of the diseased bowel [ 14 , 42 , 43 ]  . 
the software used for statistical analysis was stata ( version 8.2 ; stata corp ; college station ; tx , usa )  . results in 44 ( 88% ) out of 50 patients , we observed significant thickening of the bowel wall at the level of the terminal ileum . more specifically , in 41 out of 44 patients who had not undergone surgery and had a histological diagnosis of crohns disease , the mri study showed significant wall thickening at 1 , 5 t ( slow rate 120 mt / m / s ) , bobina phased array . 
per distendere le anse intestinali abbiamo somministrato per os a ogni paziente , circa 25 minuti prima dellinizio dellesecuzione dellesame , glicole polietilenico ( peg ) ( 1000 ml nei pazienti in et adulta ; 10 ml / kg nei pazienti in et pediatrica ) [ 21 , 30 , 38 ]  . 
inoltre , prima di iniziare lesame abbiamo iniettato per via intramuscolare 1 fiala ( 10 mg / ml ) di butilscopolamina ( buscopan ) per ridurre la peristalsi intestinale [ 31 , 39 ]  . 
lo studio rm [ 39 , 40 ] stato condotto con : sequenze t2 dipendenti di tipo ssfse ( single shot fast spin echo ) half fourier ( te : 96 , 1 ; tr : ( cid : 2 ) ; fov : 40 40 cm ; matrice : 256 160 ; spessore : 5 mm ; tempo di acquisizione : 18 s in apnea ) in fase precontrastografica su piani assiali e coronali o obliqui per consentire lindividuazione dellultima ansa ileale ; una volta individuato il piano ottimale , abbiamo ripetuto la sequenza con matrice pi alta ( 512 256 ) ; sullo stesso piano abbiamo utilizzato sequenze t1 dipendenti di tipo fspgr ( fast spoiled gradient echo ) con saturazione del grasso ( te : 1 , 3 ; tr : 200 ; fov : 40 40 cm ; matrice : 256 160 ; spessore : 5 mm ; tempo di acquisizione : 16 sec in apnea ) in fase precontrastografica e ripetute in serie 6 volte ( le prime 3 luna di seguito allaltra , le altre a distanza di 40 secondi dalla fine della precedente ) dopo somministrazione endovenosa di 0 , 1 mmol / kg di gadolinio . tutti i pazienti , anche quelli in et pediatrica , hanno ben tollerato lesame di rm collaborando adeguatamente con lattivit respiratoria . 
in tutti si avuta unadeguata visualizzazione dellintestino grazie allutilizzo del peg , che ha consentito di ottenere buoni livelli di distensione intestinale ; stato possibile cos ottenere informazioni su tutto lintestino e nel dettaglio sullultima ansa ileale . 
il peg ha presentato un comportamento sovrapponibile a quello dellacqua per quel che riguarda lintensit di segnale , facendo apparire il lume intestinale , disteso da tale soluzione isosmotica , iperintenso nelle sequenze t2 dipendenti e ipointenso nelle sequenze t1 dipendenti . sulle immagini acquisite abbiamo valutato lo spessore della parete intestinale a livello dellultima ansa ileale , la presenza di stenosi significativa o non significativa del lume , la presenza di raccolte ascessuali o di versamento libero endoaddominale , e abbiamo definito come patologiche le anse con uno spessore parietale 4 mm e come significative solo le stenosi associate a dilatazione a monte . 
dopo lesecuzione dellesame rm sono state posizionate delle roi ( region of interest ) delle dimensioni comprese tra 5 e 7 mm2 di area a livello dellansa patologica e a livello della parete intestinale sana al fine di valutare lintensit del segnale e la percentuale dincremento dello stesso nel tempo , ottenendo cos una valutazione quantitativa statistical evaluation analisi delle immagini v.a. 
in 13 out of 44 patients ( 29.5% ) , mri showed bowel wall thickening in other portions of the gasdellincremento di vascolarizzazione della parete intestinale e quindi dellattivit di malattia [ 41 ]  . 
 in 8 pazienti la rm ci ha permesso di esaminare alterazioni a carico del tessuto adiposo mesenteriale , che nelle sequenze t2 dipendenti si presentava con disomogenea iperintensit del segnale . 
 in 41 pazienti ( 82% ) , dopo somministrazione endovenosa di mezzo di contrasto ( 0 , 1 mmol / kg di gadolinio ) si avuta una elevata contrastografia a livello del segmento parietale intestinale ispessito . 
in tutti i 41 pazienti si avuto un incremento contrastografico a tutto spessore pi evidente negli strati pi interni ( mucosa ) , ma solo in 36 pazienti ( 72% ) a tale uptake corrispondeva un cdai > 150 . nel nostro studio abbiamo ottenuto una correlazione positiva , seppur non statisticamente significativa , tra il tempo necessario per raggiungere il plateau e i valori di cdai ottenuti ( r = 0 , 012 , p = 0 , 01 )  . 
successivamente abbiamo statisticamente correlato i valori percentuali di massima impregnazione di mezzo di contrasto a livello della parete intestinale patologica calcolando il coefficiente di correlazione di spearman e ottenendo un valore positivo e statisticamente significativo ( r = 0 , 73 )  . 
in these 41 patients , we observed full - thickness enhancement , more evident at the mucosa level , but in only 36 patients ( 72% ) did this uptake correspond to cdai > 150 . 
therefore , the two methods may be used interchangeably . discussion in patients with active crohns disease , the earliest and most common alteration in the intestinal mucosa is neutrophil - mev.a. 
contrast - enhanced axial t1 - weighted fast spoiled gradient fat - saturated image ( a ) shows marked contrast uptake by the diseased bowel wall , especially at the mucosal level . 
two regions of interest were placed on the image : one over the diseased intestinal wall ( red ) , and one over the healthy intestinal wall ( green )  . 
di gadolinio in cui si evidenzia marcato potenziamento parietale del tratto di intestino ispessito prevalente a livello dello strato mucoso ; in tale immagine sono poste delle roi : una sul segmento patologico ( rossa ) e una su un segmento sano ( verde )  . 
contrast - enhanced axial t1 - weighted fast spoiled gradient fat - saturated image ( a ) shows marked contrast uptake by the diseased bowel wall , especially at the mucosal level . 
regions of interest were placed on a vessel ( light blue ) , on a healthy bowel loop ( green ) and on a diseased loop ( red )  . 
di gadolinio che documenta prevalente impregnazione di mezzo di contrasto degli strati mucosi ; sono posizionate roi a livello di un vaso ( celeste ) , di unansa sana ( verde ) e di unansa patologica ( rossa )  . 
il grafico intensit / tempo ( b ) mostra il diverso uptake contrastografico nel tempo tra strutture vascolari ( linea tratteggiata celeste ) , segmento intestinale sano ( linea tratteggiata verde ) e segmento intestinale patologico ( linea tratteggiata rossa )  . discussione diated injury , whereas the earliest gross alterations due to inflammation are slight hyperperfusion and mucosal oedema . as the disease progresses , the bowel wall becomes thickened , fibrous and stenotic . 
the search for parameters capable la pi precoce e frequente alterazione della mucosa osservabile nellintestino di un paziente con morbo di crohn in fase attiva il danno criptico mediato da neutrofili . 
il razionale nasce da unintrinseca capacit di questa tecnica nellidentificazione dellacqua libera , e quindi dellinfiammazione , e nel fatto che liperemia della parete intestinale si traduce in un aumento dellaccumulo di contrasto a livello del segmento intestinale interessato dalla patologia . 
precedenti lavori hanno dimostrato la possibilit di tali rilievi , ma risultavano limitati da uninadeguata distensione del viscere che limitava le potenzialit diagnostiche di tale indagine ; lutilizzo dei mezzi di contrasto orali ha superato questa limitazione [ 24 , 30 , 31 ]  . 
paragonabile allacqua come intensit di segnale in rm , va considerato come mezzo di contrasto bifasico [ 30 , 31 ] , comportamento utile sia in fase pre - contrastografica , dove nelle sequenze t2 dipendenti fa apparire il lume viscerale che lo contiene disteso e iperintenso , e quindi ben differenziabile dalla parete intestinale che appare ipointensa , sia in fase post - contrastografica nelle sequenze t1 dipendenti con saturazione del grasso , dove il lume viscerale disteso dal peg appare ipointenso e differenziato in maniera ottimale dalla mucosa e dalla sottomucosa , le quali mostrano fisiologicamente una sottile rima di potenziamento contrastografico o , in caso di attivit infiammatoria locale , marcato accumulo di contrasto in relazione alla dilatazione dellinterstizio . 
 nello studio del morbo di crohn informazione clinicamente e prognosticamente di primaria importanza lidentificazione del grado di attivit infiammatoria locale ; questo fattore consentir al clinico di stabilire una terapia specifica e differenziata per ogni singolo paziente [ 14 , 38 , 39 , 42 , 44 ]  . 
per valutare il grado di attivit infiammatoria di parete oltre ai parametri laboratoristici che si controllano regolarmente per monitorare la malattia quali la ves , la conta dei globuli bianchi , hb , albumina , 2 globuline , ferro nel siero , pcr , 1 glicoproteina e 1 antitripsina possono essere utilizzati indici numerici di attivit di malattia , la cui valutazione per potrebbe risultare complessa e richiedere tempo [ 45 ]  . 
quello pi attendibile , da noi utilizzato nel nostro studio per correlarvi i dati quantitativi ottenuti con lo studio mediante rm , il cdai [ 1 , 2 , 15 , 46 ] , che si basa sulla valutazione di 8 variabili ( tabella 1 )  . 
il range degli score da 0 a 600 , con un cut off tra patologia e normalit a 150 , e tra malattia attiva e malattia di grado severo a 450 . 
the rationale lies in the techniques ability to identify free water and consequently inflammation and in the fact that bowel wall hyperperfusion corresponds to increased enhancement of the diseased bowel segment . 
previous studies have demonstrated this ability of mri but were limited by inadequate bowel distension , a limitation now overcome by the use of oral contrast agents [ 24 , 30 , 31 ]  . the oral contrast agent used in our study was peg , an agent consisting of a water solution of polyethylene glycol ( 70 g dissolved in 1 l of water ) , which once ingested prevents the water molecules bonded to it from being absorbed by the intestinal mucosa . 
moreover , because it is not fermentable and cannot be attacked by the bacterial flora , it flows rapidly through the jejunal - ileal loops to the caecumri signal intensity characteristics of peg are similar to those of water , so it can be considered a biphasic oral contrast agent [ 30 , 31 ]  . 
this behaviour proves useful both before contrast injection , when the intestinal lumen appears hyperintense in t2weighted sequences and easily differentiated from the hypointense intestinal wall , and after contrast injection , when the intestinal lumen appears hypointense in t1 - weighted sequences with fat suppression and well differentiated from the mucosal and submucosal layers , which physiologically display a thin rim of enhancement and , pathologically , high contrast enhancement related to interstitial dilatation . 
 in the study of crohns disease , it is clinically and prognostically fundamental to identify the degree of local inflammatory activity , as this information will help the clinician decide on specific treatment for each patient [ 14 , 38 , 39 , 42 , 44 ]  . the aim of our study was to investigate whether it was possible to quantify the degree of disease activity . 
to assess the degree of inflammatory activity , numerical indexes of disease activity can be used [ 45 ] in addition to the laboratory parameters commonly used during follow - up , such as erythrocyte sedimentation rate , white blood cell count , haemoglobin , albumin , 2 globulin , serum iron , c - reactive protein , 1 glycoprotein and 1 antitrypssuch indexes are particv.a. 
the most reliable index and the one used in our study to correlate the quantitative data provided by mri was the cdai [ 1 , 2 , 15 , 46 ]  . 
the limitations of this index are interobserver variability , inclusion of subjective variables such as general well - being and intensity of abdominal pain and the fact that it is based on a diary completed by the patients for 7 days before assessment , a requirement that precludes its use in everyday practice . 
 mri is able to not only easily identify the presence of bowel wall inflammation through assessment of contrast enhancement [ 14 , 29 , 39 , 47 ] but also to quantify this by means of roi evaluation of percentage changes in signal intensity . 
mri has a potentially high diagnostic value in assessment of local inflammatory activity in patients with crohns disease [ 14 , 20 , 36 , 37 , 42 , 47 ]  . 
besides enabling identification of the diseased small bowel segment by accurately visualising luminal stenosis , wall thickening and dilatation of the intestinal loops , mri allowed us to quantify the degree of inflammatory activity in all patients , providing crucial information for treatment planning [ 7 ]  . 
moreover , most of the results obtained with mri had a good correlation with clinical data and cdai scores . conclusions mri is a potentially valuable procedure in assessment of the small bowel in patients with suspected crohns disease or monitoring patients after treatment . 
it is easily reproducible and well tolerated , even by children [ 34 , 38 ] , provides both panoramic and detailed views and allows identification of changes in signal intensity or bowel wall thickness . 
therefore , it can be considered a very useful diagnostic tool that provides data on site , extent , degree of inflammatory activity and complications in patients with histologically proven crohns disease , without the use of ionising radiation [ 11 , 23 , 35 , 49 , 50 ]  . 
its limitations lie in poor depiction of wall alterations such as ulcers or fissures , which are better demonstrated by conventional small - bowel enteroclysis [ 25 , 30 , 48 , 50 ]  . 
finally , dynamic mri enables quantitative analysis la variabilit interosservatore , variabili soggettive del paziente , quali il benessere generale e lintensit del dolore addominale , e anche il fatto che il calcolo si effettua dopo una valutazione per 7 giorni del paziente , il che ne impedisce lutilizzo nella pratica quotidiana . 
inoltre , come gi detto , non accurato in pazienti con fistole e stenosi , e non utilizzabile in pazienti che hanno subito una estesa resezione o stomia ileo - colica . 
nonostante questi limiti , allo stato attuale il cdai da considerarsi ancora il gold standard per la valutazione dellattivit di malattia [ 46 ]  . la rm ci consente di identificare agevolmente la presenza di infiammazione a carico della parete intestinale mediante la valutazione dellaccumulo di mezzo di contrasto [ 14 , 29 , 39 , 47 ] , oltre ad una possibile valutazione quantitativa della stessa mediante la valutazione su volumi campione ( roi ) della variazione percentuale del segnale . 
sono per presenti limiti nella visualizzazione di minime alterazioni superficiali di parete , vista la modesta risoluzione spaziale della tecnica [ 30 , 48 ]  . questa indagine si propone come esame potenzialmente a elevato contributo diagnostico nella valutazione dellattivit infiammatoria locale in pazienti con morbo di crohn [ 14 , 20 , 36 , 37 , 42 , 47 ]  . 
in definitiva , la rm ci ha consentito oltre allidentificazione corretta dei diversi segmenti patologici del piccolo intestino , con una ottima rappresentazione sia del lume stenotico , sia dellispessimento parietale , sia della dilatazione delle anse a monte una valutazione quantitativa del grado di attivit infiammatoria in tutti i casi , utile per la successiva pianificazione terapeutica [ 7 ]  . 
inoltre , in una buona percentuale i risultati ottenuti con lo studio mediante rm si sono dimostrati ben correlabili col dato clinico e numerico ottenuto mediante il calcolo del cdai . 
 conclusioni la rm unindagine di grande interesse nello studio dellintestino nei pazienti con sospetto morbo di crohn , o nel caso di follow - up post - terapia , perch facilmente riproducibile , ben tollerata anche dai pazienti pediatrici [ 34 , 38 ] , panoramica e di dettaglio ; inoltre , consente di identificare alterazioni di segnale o di spessore a carico della parete intestinale . 
pertanto pu senza dubbio rappresentare uno strumento diagnostico estremamente utile nel fornire , senza lutilizzo di radiazioni ionizzanti , informazioni circa la localizzazione , lestensione , il grado di attivit infiammatoria intestinale ed eventuali complicanze in pazienti con diagnosi istologica di morbo di crohn [ 11 , 23 , 35 , 49 , 50 ] , pur nella consapevolezza dei limiti che la metodica presenta nellevidenziare eventuali alterazioni di parete quali ulcerazioni e fissurazioni , meglio identificabili col clisma del tenue [ 25 , 30 , 48 , 50 ]  . 
the aim of this study was to assess the reliability of the sonographic breast imaging reporting and data system ( birads ) classification in differentiating benign from malignant breast masses . 
all lesions were classified according to the sonographic bi - rads lexicon . sensitivity , specificity , accuracy , positive predictive value ( ppv ) and negative predictive value ( npv ) for the sonographic birads lexicon and ppv and npv for each lesion category and each sonographic descriptor were calculated . 
il nostro studio conferma che la classificazione ecografica bi - rads costituisce un accurato sistema per la descrizione e il management delle lesioni mammarie . parole chiave mammella nodulo ecografia diagnosi introduction introduzione in the past 15 years , ultrasound ( us ) has increasingly proved to be indispensable in the study of breast masses , above all as a result of continuing technological advances . 
image quality has been remarkably improved by the introduction of highfrequency probes , harmonic imaging , colourand powerdoppler applications and the use of sonographic contrast menegli ultimi 15 anni lecografia si dimostrata metodica sempre pi indispensabile nello studio delle lesioni mammarie grazie soprattutto agli incessanti avanzamenti tecnologici . 
although breast us was initially used chiefly to distinguish between solid and cystic lesions , its use for characterising solid masses and identifying suspicious findings for biopsy and benign lesions for follow - up has gained increasing interest [ 713 ]  . 
the lexicon introduced by the bi - rads system includes sonographic descriptors referring to lesion shape , orientation , margins , boundary , echo pattern , posterior acoustic features and surrounding tissue . 
on the basis of these descriptors , each lesion is assigned a level of suspicion , which is associated with a recommendation for management ( table 1 ) [ 19 ]  . the purpose of this study was to assess the reliability of the bi - rads system in differentiating between benign and malignant solid breast masses . 
in detail , the aims of the study were to : evaluate the diagnostic accuracy of bi - rads in differentiating benign from malignant breast masses determine sensitivity , specificity , prevalence , and negadellimpiego di mezzi di contrasto ecografici [ 16 ]  . 
bench lecografia mammaria sia stata adoperata inizialmente con lo scopo di distinguere le lesioni solide da quelle liquide , si sviluppato un interesse sempre crescente nellutilizzare gli ultrasuoni per la caratterizzazione delle lesioni solide , per identificare le immagini sospette da sottoporre a biopsia da quelle benigne da inviare al follow - up [ 713 ]  . 
la terminologia introdotta dal sistema bi - rads include descrittori ecografici in riferimento alla forma , allorientamento , ai margini , allinterfaccia , allecogenicit , alle caratteristiche acustiche posteriori e alle alterazioni del parenchima circostante . 
sulla base di questi descrittori ogni lesione viene assegnata ad una classe di sospetto che prevede il successivo comportamento diagnostico - terapeutico ( tabella 1 ) [ 19 ]  . lo scopo di questo studio stato quello di determinare lattendibilit del sistema bi - rads nel differenziare le lesioni solide della mammella in benigne e maligne . 
in particolare le finalit di questo studio sono state : valutare laccuratezza diagnostica del sistema bi - rads nel differenziare le lesioni mammarie benigne da quelle maligne ; definire sensibilit , specificit , prevalenza , valori predittivi negativi e positivi per ogni classe di sospetto ; valutare i valori predittivi positivi e negativi dei vari dematerials and methods studiare il valore diagnostico della diversa associazione us examinations were prospectively performed on all consecutive breast masses undergoing biopsy at our department between november 2004 and march 2006 . 
in some cases fnac was performed at the request of the patient or the referring physician . us imaging was done with a siemens antares device with a linear - array high - frequency transducer ( 1013 mhz )  . the examination involved multiple scans of both breasts and the axillary region and focused on the study of the morphological features of the lesion to be aspirated . 
 all lesions underwent us - guided fnac ( 2125 gauge )  . cytology reports were considered reliable only when they documented a specific diagnosis of malignancy or benignity . cytology was not considered reliable if there was discordance between the sonographic findings and the cytological sample , if the sample was inadequate or if it was found to contain only normal parenchymal cells . 
a specific negative cytological result was defined as no evidence of atypical cells and findings compatible with a diagnosis of complicated cyst , fibroadenoma , galactocele , liponecrosis , granuloma and apocrine metaplasia . 
cases with specific negative cytology were scheduled for short - interval follow - up every 6 months for at least 2 years . all us examinations and fnac were done by two radiologists expert in breast imaging ( mc and pb ) who described and classified the lesions according to the bi - rads system at the time of the examination , without knowing the histopathological results . 
lesions displaying all of the signs suggestive of a benign lesion were assigned to bi - rads category 3 ( oval or round shape , parallel orientation , circumscribed margins , abrupt interface , enhancement or absence of posterior acoustic features , absence of surrounding tissue alterations )  . 
it was decided to include only this type of lesion into category 3 , as inclusion of masses with a single suspicious sign in a previous study [ 20 ] had reduced the npv and increased the ppv by more than 2% . 
in alcuni casi lago - aspirato stato eseguito per volont dei medici curanti o delle pazienti . lo studio ecografico stato eseguito usando un apparecchio siemens antares con trasduttore lineare ad alta frequenza ( 1013 mhz )  . 
lesame ecografico ha incluso lo studio di entrambe le mammelle e della regione ascellare secondo scansioni multiple e si concentrato sullo studio delle caratteristiche morfologiche della lesione da ago - aspirare . tutte le lesioni sono state sottoposte ad ago - aspirato ecoguidato eseguito con ago sottile ( 2125 g )  . 
lesame citologico non stato giudicato attendibile se vi era discordanza tra i risultati dellimmagine ecografica e del prelievo citologico , se il prelievo era insufficiente o se in esso erano state individuate solo cellule parenchimali normali . 
stato considerato un risultato negativo specifico un esame citologico privo di cellule atipiche e compatibile con una diagnosi di cisti complicata , fibroadenoma , galattocele , liponecrosi , granuloma e metaplasia apocrina . 
nei casi di diagnosi citologica negativa specifica la lesione stata inviata ad uno stretto follow - up : ogni 6 mesi per almeno due anni . tutti gli esami ecografici e i successivi ago - aspirati sono stati eseguiti da due radiologi dedicati alla diagnostica senologica ( gli autori mc e pb ) che le hanno descritte e classificate secondo il sistema bi - rads al momento dellesame senza conoscere i risultati istopatologici . 
lesions were assigned to category 4 if they did not meet the requirements for benignity and did not show the combination of at least three suspicious signs , therefore having indeterminate appearance . 
a definitive diagnosis of atypical hyperplasia was classed as a malignant lesion , whereas a definitive diagnosis of typical hyperplasia was classed as a benign lesion . statistical analysis specificity , sensitivity , accuracy , ppv and npv of the birads classification system were calculated by grouping category 3 lesions ( probably benign ) among benign lesions , and category 4 ( probably malignant ) and category 5 ( definitely malignant ) among malignant lesions . 
the ppv and npv of each descriptor was obtained as follows : ppv : number of malignant lesions per sonographic descriptor npv : number of benign lesions per sonographic descripthe 2 and fischer exact tests were used to determine significant differences between the proportions of malignant and benign lesions in the following groups : upper outer quadrant of left breast , including the border areas ( intersection of outer quadrants and upper quadrants ) ( present / absent ) shape ( irregular , oval , round ) diameters less or greater than 1.4 cm orientation ( nonparallel , parallel ) presence or absence of circumscribed margins type of boundary ( abrupt and echogenic halo ) hypoechoic pattern ( presence / absence ) posterior acoustic features ( enhancement , shadowing , none ) alterations of the surrounding tissue ( presence / absence ) category ( 3 , 4 , 5 ) ratio between horizontal and vertical diameter greater or less than 1 cm . to assess the predictive factors for malignant lesions we constructed a logistic regression model . 
the variables included were : shape ( oval ) noncircumscribed margins echogenic halo hypoechoic pattern posterior acoustic features ( enhancement ) surrounding tissue alterations category ( category 3 ) orientation and age ( age group < 53 years )  . regression analysis was conducted using the stepwise procedure with backward elimination ; the hosmer and lemeshow test was used to evaluate the goodness of the model . 
stato deciso di comprendere nelle classe iii solo questo tipo di lesioni dato che in un precedente studio [ 20 ] la presenza di formazioni con un unico segno sospetto in classe iii ne aveva abbassato il valore predittivo negativo e innalzato il valore predittivo positivo oltre il 2% . 
alla iv classe sono state assegnate tutte le lesioni che non soddisfacevano i criteri per le lesioni benigne e che non presentavano la combinazione di almeno tre segni suggestivi di malignit , e che perci apparivano indeterminate . 
una diagnosi definitiva di iperplasia atipica stata considerata come lesione maligna ; per contro una diagnosi definitiva di iperplasia tipica stata considerata come lesione benigna . analisi statistica stata calcolata la specificit , la sensibilit , laccuratezza , il valore predittivo positivo e negativo ( vpp e vpn ) del sistema di classificazione bi - rads , includendo le lesioni di iii classe ( lesioni giudicate probabilmente benigne ) nel gruppo delle lesioni benigne , quelle di iv ( lesioni giudicate probabilmente maligne ) e di v classe ( lesioni giudicate francamente maligne ) nel gruppo delle lesioni maligne . 
patients with bi - rads category 3 and 4 lesions , for which fnac documented a specific benign lesion , were advised to repeat the sonographic examination every 6 months for at least 2 years . mean patient age was 53.2 ( range : 1990 ) years . 
mean lesion dimensions were : horizontal diameter 13.9 mm ( range 390 mm ) and vertical diameter 10.3 mm ( range 380 mm )  . on the basis of the bi - rads classification system , our cases were classed as follows : 63 in category 3 , 94 in category 4 and 135 in category 5 . 
no cases were assigned to categories 0 , 1 , 2 or 6 , in keeping with the inclusion criteria . cytology and histology revealed 168 malignant lesions and 124 benign lesions . 
of the irregular - shaped lesions , 28 were assigned to category 4 ( percentage of category 4 lesions with irregular shape : 29.7% ) and 130 to category 5 ( percentage of category 5 lesions with irregular shape : 96.2% ) ( table 3 )  . costruito un modello di regressione logistica . 
le variabili inserite nel modello sono state : forma ( con riferimento alla caratteristica ovalare ) margini non circoscritti alone iperecogeno ipoecogenecit caratteristiche posteriori ( con riferimento alla caratteristica enhancement ) alterazioni del parenchima circostante classe ( con riferimento la classe iii ) orientamento ed et ( con riferimento al gruppo det < 53 anni )  . lanalisi di regressione stata condotta utilizzando la procedura stepwise con eliminazione a ritroso ( backward elimination ) ; la bont del modello stata valutata utilizzando il test di hosmer e lemeshow . 
le dimensioni medie delle lesioni sono state : diametro orizzontale medio 13 , 9 mm ( range 390 mm ) , diametro verticale medio 10 , 3 mm ( range 380 mm )  . basandoci sulle definizioni proposte dal sistema bi - rads , i nostri casi sono stati classificati nel modo che segue : 63 in classe iii , 94 in classe iv , e 135 in classe v . 
il sistema ecografico bi - rads ha mostrato il 77 , 0% di accuratezza , il 98 , 2% di sensibilit , il 48 , 4% di specificit , con un vpp e un vpn rispettivamente del 72 , 0% e del 95 , 2% ( tabella 2 )  . centocinquantotto casi presentavano morfologia irregolare , 100 ovalare e 34 rotondeggiante . 
delle 158 lesioni con morfologia irregolare , 134 erano maligne e 24 benigne per un vpp e un vpn rispettivamente dell84 , 8% e del 15 , 2% . delle lesioni con morfologia irregolare 28 sono state assegnate alla classe iv ( percentuale di lesioni della iv classe che presentavano morfologia irregolare : 29 , 7% ) e 130 alla classe v ( percentuale di lesioni della classe v con morfologia irregolare : 96 , 2% ) ( tabella 3 )  . i margini erano circoscritti in 80 casi , indistinti in 106 , angolari in 63 , microlobulati in 7 , spiculati in 36 . 
lesame istologico ha confermato una lesione benigna ( fibroadenoma )  . angular in 63 , microlobulated in seven and spiculated in 36 . only eight of 80 lesions ( 10.0% ) with circumscribed margins were found to be malignant , with npv of 90.0%. 
only seven cases had microlobulated margins , and all of table 3 shape descriptor in relation to the breast imaging reporting and data system classification tabella 3 distribuzione della morfologia in relazione alla classificazione bi - rads total totale category diagnosis round oval irregular totals b . 
2 small , round , hypoechoic mass with indistinct margins , abrupt interface , and absence of posterior acoustic features ( breast imaging reporting and data system category 4 )  . 
si trattava di un carcinoma duttale invasivo . ipoecogene , 134 appartenevano alla v classe ( oltre il 99% di tutte le lesioni di classe v ) , 71 alla classe iv ( il 75 , 5% di tutte le lesioni di classe iv ) e 57 alla classe iii ( il 90% di tutte le lesioni di classe iii )  . 
tale parametro si dimostrato essere inversamente proporzionale alla classe di sospetto ( da 1 , 82 a 1 , 12 )  . dei 292 casi 200 presentavano uninterfaccia netta e 92 un alone iperecogeno . 
di 292 casi , 167 ( il 42 , 8% ) hanno mostrato assenza di segni acustici posteriori e la gran parte di queste lesioni stata assegnata alla iv classe . 
su 22 lesioni che presentavano rinforzo acustico posteriore , 16 si sono rivelate benigne ( vpn del 72 , 7% ) e 6 maligne ( 3 poste in iv classe , 1 in iii e 2 in v )  . 
of the 262 hypoechoic lesions , 134 were category 5 ( more than 99% of all category 5 lesions ) , 71 were category 4 ( 75.5% of all category 4 lesion ) and 57 were category 3 ( 90% of all category 3 lesions )  . 
solo 18 lesioni ( 17 maligne e 1 benigna ) hanno mostrato distorsione del parenchima circostante ( tabella 9 )  . la maggior parte dei casi assegnati alla iv classe presentavano ecostruttura ipoecogena e margini indistinti . 
come si osserva nella tabella 10 , lunica variabile che non ha riportato differenze significative tra i due gruppi di lesioni lassociazione della lesione maligna con il quadrante supero - esterno della mammella sinistra , che presenta un livello di p = 0 , 669 . relativamente ai risultati dellanalisi multivariata , la regressione logistica ha permesso di valutare la stima del rischio di avere una lesione maligna , sotto forma di odds ratio ( or ) ed i relativi intervalli di confidenza al 95% ( ic 95% )  . utilizzando questa metodica , per valori di or superiori ad 1 si considera che il fattore di esposizione sia un fattore di rischio , mentre per valori inferiori a 1 si considera che il fattore di esposizione sia un fattore protettivo . 
fra le covariate inserite nel modello quelle che sono risultate avere un rischio significativamente maggiore di avere lesioni maligne ( or significativamente maggiore di 1 ) sono : la morfologia rotondeggiante rispetto alla ovalare , al limite della significativit con p = 0 , 52 la morfologia irregolare rispetto alla ovalare con p = 0 , 008 lassenza di caratteristiche acustiche posteriori rispetto allenhancement con p = 0 , 025 infine le classi 4 e 5 rispetto alla 3 , con rispettivamente p = 0 , 001 e p < 0 , 001 . il test di hosmer e lemeshow , con un p = 0 , 37 , indica che il modello utilizzato nella regressione buono per predire la fig . 
relativamente alla forma , il rischio di avere una lesione maligna particolarmente elevato nelle donne che presentano una forma irregolare ( rischio pi di 3 , 5 volte maggiore rispetto a coloro che hanno una lesione con forma ovalare ) , mentre coloro che presentano una lesione rotondeggiante hanno un rischio che tale lesione sia maligna poco pi di 2 , 5 volte rispetto a coloro che hanno una lesione con forma ovalare . 
of the 22 lesions with posterior enhancement , 16 proved to be benign ( npv 72.7% ) and six malignant ( three in category 4 , one in category 3 and two in category 5 )  . 
only 18 lesions ( 17 malignant and one benign ) showed parenchymal distortion ( table 9 )  . the majority of cases assigned to category 4 had hypoechoic echo pattern and indistinct margins . 
as can be seen in table 10 , the only variable that did not differ significantly between the two groups was location of the malignant lesion in the upper outer quadrant of the left breast , for which the p value was 0.669. with regard to multivariate analysis , logistic regression was used to estimate the risk of having a malignant lesion , in the form of odds ratio ( or ) and corresponding 95% confifig . 
si trattato di un caso falso positivo . table 9 surrounding tissue alterations in relation to the breast imaging reporting and data system classification tabella 9 alterazioni del parenchima circostante secondo la classificazione bi - rads category diagnosis no alterationssurrounding tissue alterations totals b , benign ; m , malignant classe diagnosi nessuna alterazione alterazioni del parenchima circostante totale b , benigno ; m , maligno total totale m . 
of the covariates included in the model , those associated with a significantly higher risk of having a malignant lesion ( or significantly > 1 ) are : round rather than oval shape , almost significant with p = 0.52 irregular rather than oval shape ( p = 0.008 ) absence of posterior acoustic features rather than enhancement ( p = 0.025 ) categories 4 and 5 rather than category 3 , with p = 0.001 and p < 0.001 , respectively the hosmer and lemeshow test , with a p = 0.37 , indicates that the model used in regression was good for predicting the outcome variable of having a malignant lesion . multivariate analysis clearly showed that the risk of having a malignant lesion is particularly high in women with senza caratteristiche acustiche posteriori rispetto al quelle che presentano enhancement ( tabelle 10 , 11 )  . discussione per la frequente sovrapposizione di segni benigni e maligni , i noduli mammari studiati ecograficamente vengono spesso sottoposti a biopsia per determinarne la natura [ 912 ]  . 
il gran numero di biopsie eseguite su lesioni benigne o per desiderio delle pazienti o per lincertezza del medico o per lindicazione di alcuni protocolli , rappresenta un ulteriore problema [ 21 ]  . 
infatti , un eccessivo numero di biopsie non necessarie ha un effetto svantaggioso determinando laumento dei costi dei programmi di screening e di tutela della salute pubblica [ 10 , 21 , 22 ]  . lecografia mammaria non sfruttata al massimo delle sue potenzialit se il suo utilizzo viene limitato alla sola capacit di differenziare le lesioni liquide da quelle solide o alm . 
the p values for the variable category show higher statistical significance . with regard to shape , the risk of having a malignant lesion is particularly high in women with irregularly shaped lesions ( 3.5 times higher risk compared with those with an oval lesion ) , whereas women with a round lesion are a little more than 2.5 times more likely to have a malignant lesion than those with an oval lesion . 
al fine di diminuire il numero di biopsie non necessarie , lecografia dovrebbe tendere ad una sempre pi precisa caratterizzazione delle lesioni [ 6 , 7 , 12 ]  . 
tale sistema aiuta il radiologo nella descrizione delle caratteristiche ecografiche e nellassegnazione ad una classe definitiva di appartenenza alla quale associato il trattamento clinico pi appropriato per la paziente [ 19 ]  . 
se questi criteri fossero strettamente applicati a partire dalliniziale referto radiologico , il numero delle biopsie per le lesioni benigne potrebbe essere ridotto . questo studio conferma lalta sensibilit ( e cio lidentificazione di lesioni maligne in pazienti con tumore al seno , 95 , 3% ) e lalto valore predittivo negativo ( e cio lidentificazione dei veri negativi in donne senza malattia , 92 , 3% ) del sistema ecografico bi - rads . 
performance of too many unnecessary biopsies has detrimental effects that increase the cost of screening and public health protection programmes [ 10 , 21 , 22 ]  . breast us is not used to its full potential if it is limited to the differentiation of cystic and solid lesions or the study of particularly dense breasts . 
 [ 7 ] nella loro ampia casistica hanno riportato un 98 , 4% di sensibilit , un 67 , 8% di specificit , un 38% di vpp , un 99 , 5% di vpn e un 72 , 9% di accuratezza . 
nello studio di stavros , il vpp era significativamente pi basso rispetto al nostro ( 38% vs 72% ) , probabilmente in relazione alla diversa prevalenza di tumori mammari ( 17% vs 57% ) e ai diversi criteri di selezione delle pazienti . 
nel nostro studio laccuratezza diagnostica dellecografia ( 77 , 0% ) nella differenziazione delle lesioni mammarie benigne e maligne attraverso luso del sistema classificativo bi - rads stata simile ai risultati ottenuti nello studio di stravos . nel nostro studio il vpn per la iii classe stato pari al 95 , 3% . 
in our study , the diagnostic accuracy of us ( 77.0% ) in differentiating benign from malignant breast lesions through the use of the bi - rads classification system was similar to that obtained by stavros et al . 
because only lesions studied by cytology and / or histology were included in this study , only a small number of women with probably benign lesions and with a known diagnosis were included , and in these cases , the decision to perform cytology was made by the patient or the referring physician . the risk of malignancy for category 3 lesions should be lower than 2% . 
if the apparently benign mass increases in size during follow - up , lesion classification is changed to category 4 and a biopsy becomes appropriate [ 19 , 22 ]  . 
solo le lesioni che sono state sottoposte allesame citologico e / o istologico sono state inserite in questo studio , perci solo un piccolo numero di donne con delle lesioni probabilmente benigne e con diagnosi nota sono state incluse . 
in questi casi lesame citologico stato eseguito per scelta della paziente o del medico curante . il rischio di malignit per le lesioni assegnate alla classe iii dovrebbe essere inferiore al 2% . 
se la massa apparentemente benigna aumenta di dimensioni durante il follow - up , si modifica trasformandosi in una lesione di classe iv , perci lesame bioptico a questo punto appare appropriato [ 19 , 22 ]  . 
nessuna delle lesioni della nostra casistica attribuite alla iii classe ha mostrato modificazioni morfologiche o dimensionali durante il follow - up ( anche se non abbiamo il follow - up completo di tutte le pazienti )  . nel nostro studio , i vpp per le classi iv e v sono stati rispettivamente del 48 , 9% e 88 , 1% . 
in questa categoria lorientamento e le caratteristiche morfologiche costituiscono i segni pi validi per differenziare le lesioni benigne da quelle maligne . solo un piccolo numero di casi posti in iv classe ha mostrato alone iperecogeno o segni acustici posteriori e in nessun caso si sono documentate alterazioni del parenchima circostante . comunque questi criteri non sono stati sufficienti per discriminare le lesioni maligne da quelle benigne ed stato sempre necessario ricorrere alla biopsia . 
delle lesioni assegnate alla v classe ( 135 ) , 119 si sono rivelate maligne e 16 benigne . queste lesioni hanno presentato almeno tre segni sospetti per la malignit : morfologia irregolare , margini non circoscritti , orientamento anti - parallelo , alone iperecogeno e presenza di sbarramento acustico posteriore o di alterazioni del tessuto circostante . 
these lesions displayed at least three suspicious signs : irregular shape , noncircumscribed margins , nonparallel orientation , echogenic halo and presence of posterior shadowing or alterations of the surrounding tissue . lesion morphology is one of the most reliable criteria for differentiating benign and malignant breast masses . 
round shape proved to be a nonspecific sign . margin evaluation revealed that only 36 lesions had spiculated margins , and these were classified as category 4 or 5 . the relatively small number of masses with spiculated margins is probably due to the small size of the lesions studied ( mean diameter among category 5 lesions was less than 13 mm ) , the introduction of new margin descriptors ( angular and microlobulated ) and the fact that an echogenic halo often reflects the presence of spiculations that are too small to be detected on us . 
hypoechoic pattern had a lower ppv compared with irregular shape or noncircumscribed margins , but this variable increases in reliability if it is associated with other suspicious signs . in agreement with other authors [ 5 , 2427 ] , lesion shape , margins and echo pattern were the most significant factors in the differential diagnosis between benign and malignant lesions . 
the variable orientation was closely related to lesion shape ; generally , all oval lesions tended to have parallel orientation . irregular morphology was often found to be associated with nonparallel orientation . 
in our study , parallel orire risultata associata con il pi alto vpp ( 84 , 8% ) , mentre la morfologia ovalare con il pi alto vpn ( 80 , 0% )  . 
il relativo basso numero di masse a margini spiculati probabilmente dovuto alle piccole dimensioni delle lesioni studiate ( il diametro medio delle lesioni poste in classe v stato inferiore a 13 mm ) , allintroduzione di nuovi descrittori dei margini ( angolari e microlobulati ) e al fatto che lalone iperecogeno in molti casi espressione della presenza di spicule troppo piccole per essere visualizzate ecograficamente . 
leco - pattern ipoecogeno ha mostrato un vpp pi basso rispetto alla morfologia irregolare o ai margini non circoscritti , ma lattendibilit di questo parametro aumenta se si associa ad altri segni di sospetto . in accordo con altri autori [ 5 , 2427 ] la morfologia , i margini e leco - pattern rappresentano i fattori pi significativi per la diagnosi differenziale benigno / maligno in ecografia . 
il vpp del segno antiparallelo stato del 71 , 5% ( 85 , 9% per le lesioni poste in classe v e 66 , 6% per quelle poste in classe iv )  . 
 [ 25 ] nel valutare gli aspetti caratteristici dei noduli maligni hanno incluso come parametro un rapporto tra diametro trasversale e antero - posteriore pari o inferiore a 1 , 4 . 
i nostri risultati confermano questo valore come soglia significativa per assegnare la lesione alla classe iii ( 1 , 82 ) o alle classi iv e v ( 1 , 46 e 1 , 12 )  . 
linterfaccia netta di contro ha mostrato un vpn pari al 73 , 5% . la presenza di sbarramento acustico posteriore il risultato dellattenuazione degli ultrasuoni da parte della reazione desmoplastica tipica del cancro mammario [ 6 ]  . 
despite the fact that posterior attenuation is more commonly associated with malignant breast lesions , this sign may also be seen in benign lesions [ 6 , 28 ]  . 
in agreement with other authors [ 7 , 24 ] , both types of transmission were considered nonspecific signs . most of our cases showed no alteration of the surrounding tissue , probably as a result of the small size of lesions . 
only 17 tumours had distortion of the surrounding tissue or skin thickening . conclusions our study confirmed that the bi - rads classification system proposed by the acr is an accurate system for the description and management of breast masses . 
in the analysis of our results , we noted that some sonographic signs , such as hypoechoic pattern and indistinct margins , are often less predictive of malignancy if compared with irregular shape ; spiculated , angular or microlobulated margins ; echogenic halo ; and posterior acoustic shadowing . 
on the other hand , some sonographic signs normally associated with benign lesions , such as round shape , abrupt interface and the absence of posterior acoustic signs , are often seen in malignant lesions . oval shape , parallel orientation and circumscribed margins are the signs that best characterise benign lesions . however , regardless of ppv or npv of each sign , the heterogeneous nature of tumours calls for a global assessment based on the combination of multiple signs . 
this is the only method that achieves a more reliable discrimination between benign lesions or lesions with a low likelihood of malignancy ( displaying a combination of signs predictive of benignity ) and malignant lesions or lesions with some likelihood of malignancy ( displaying a combination of signs predictive of malignancy )  . if judgement is based on a meticulous description of lesions and the correct combination of signs , minimally or highly suspicious lesions requiring biopsy may be distinlesioni hanno rivelato la presenza di sbarramento acustico posteriore : il vpp per questo segno stato del 73 , 8% ( 82 , 35% per la classe v e 66 , 66% per la classe iv )  . 
malgrado lattenuazione posteriore sia un segno ecografico pi comunemente associato a lesioni mammarie maligne , questo stesso reperto ecografico pu essere rilevato anche in lesioni benigne [ 6 , 28 ]  . 
bench la presenza di sbarramento sia preoccupante come segno ecografico per la possibile malignit della lesione , noi riteniamo che n una normale trasmissione degli ultrasuoni n una trasmissione con enhancement possa essere usata come elemento di rassicurazione circa la benignit della lesione . 
in accordo con altri autori [ 7 , 24 ] , entrambe le modalit di trasmissione vengono considerate segni aspecifici . la maggior parte dei nostri casi non ha mostrato alterazioni del parenchima circostante probabilmente in relazione alle piccole dimensioni delle lesioni . 
solo 17 tumori hanno presentato distorsione del parenchima circostante o ispessimento cutaneo . conclusioni il nostro studio ha confermato che il sistema di descrittori bi - rads proposto dallacr costituisce un accurato sistema per la descrizione e il management delle lesioni mammarie . nellanalisi dei nostri risultati abbiamo notato che alcuni segni ecografici come lipoecogenicit e i margini indistinti sono spesso meno predittivi di malignit se confrontati con la forma irregolare , i margini spiculati , angolari o microlobulati , lalone iperecogeno e lo sbarramento acustico posteriore . 
daltra parte alcuni segni ecografici abbinati generalmente alle lesioni benigne hanno spesso dimostrato di essere associati anche a lesioni maligne come , nello specifico , la morfologia rotondeggiante , linterfaccia netta e lassenza di segni acustici posteriori . 
la morfologia ovalare , lorientamento parallelo , i margini circoscritti sono i segni che caratterizzano meglio le lesioni benigne . a prescindere dal valore predittivo positivo o negativo di ogni segno , a causa delletereogeneit delle forme tumorali , necessaria una valutazione complessiva della lesione basata sullassociazione di molteplici segni . 
questo lunico modo per distinguere con maggior attendibilit le lesioni benigne o quelle con bassa probabilit di essere maligne ( per le quali esiste unassociazione di segni predittivi di benignit ) da quelle maligne o con pi o meno alta probabilit di malignit ( per le quali esiste unassociazione di pi segni predittivi di malignit )  . basandoci su una scrupolosa descrizione delle lesioni e su una corretta associazione di diversi segni , le lesioni minimamente o altamente sospette che richiedono lesecuzione di una biopsia possono essere distinte dalle lesioni probabilm . 
it is , in fact , very unlikely that an apparently benign lesion will be found to be malignant , and the reason for this is the high npv of us . 
we believe that only through experience and a careful periodic review of cases is it possible to achieve an accurate interpretation of sonographic descriptors . mente benigne che possono essere sottoposte ad un stretto follow - up . 
da una casistica di 468 pazienti con diagnosi istologica di neoplasia mammaria maligna sottoposte a rmm , abbiamo estrapolato 27 casi ( 5 , 7% ) di neoplasie rare , confermate alla diagnosi istologica definitiva : 4 ( 0 , 9% ) carcinomi papillari intracistici , 4 ( 0 , 9% ) carcinomi papillari intraduttali , 5 ( 1 , 0 % ) carcinomi papillari invasivi , 2 ( 0 , 4% ) carcinomi midollari , 7 ( 1 , 5% ) carcinomi mucinosi , 3 ( 0 , 6% ) carcinomi tubulari , 1 ( 0 , 2% ) carcinoma tubulo - lobulare e 1 ( 0 , 2% ) tumore desmoide . 
di frequente le neoplasie maligne rare della mammella , verosimilmente a causa dellelevato grado di differenziazione e delle peculiarit istologiche , presentano alla rmm caratteristiche morfologiche e di enhancement poco sospette e vengono classificate come lesioni dubbie / indeterminate . key words breast magnetic resonance mammography breast rare neoplasms parole chiave mammella risonanza magnetica mammaria tumori mammari rari introduction introduzione breast cancer is a major public health issue worldwide . 
according to estimates , in 2002 , there were more than one million new breast cancer diagnoses , more than 400 , 000 deaths il tumore della mammella attualmente uno dei principali problemi di salute pubblica su scala mondiale . 
breast cancer is an extremely heterogeneous disease , with a wide variety of clinical presentations and stages at presentation , biological behaviour , histological features , imaging findings , responses to treatment and prognostic features . 
the two most common histological types of breast cancer are ductal and lobular carcinomas , constituting approximately 75% and 15% of all breast neoplasms , respectively [ 2 ]  . 
this may explain why almost all of the literature about breast neoplasms is focused on the epidemiological , pathological , imaging and clinical features of these histological types , whereas much less is known about the rarer types of breast cancer , which together account for about 10% of all cases [ 3 ]  . over the last few years , magnetic resonance mammography ( mrm ) has gained increasing importance in the field of breast imaging . 
scientific evidence from many large - scale trials has confirmed the high efficacy of mrm for the diagnosis of primary and recurrent breast cancer , with reported sensitivity approaching 100% [ 46 ]  . 
the most important indications for mrm are cancer detection in young women with a genetic - hereditary risk of breast carcinoma [ 7 , 8 ] , in patients with carcinoma of unknown primary ( cup ) syndrome [ 9 , 10 ] and in women with breast implants [ 11 ] ; characterisation of equivocal mammographic and / or sonographic findings [ 12 ] ; local staging before surgical intervention [ 1315 ] or as preand post - neoadjuvant chemotherapy examination [ 16 , 17 ] and follow - up after breast - conservative surgery / radiotherapy [ 1820 ]  . 
to make the most of this diagnostic tool , now that its use is becoming increasingly widespread , it is imperative to be familiar with the mrm appearance of breast cancer of cases showing both usual and unusual semiological features . whereas much is known about the mrm appearance of the most frequently encountered histological breast cancer subtypes , ductal and lobular invasive carcinomas [ 21 ] , much less is known about mrm appearance of rare histological types [ 2226 ]  . 
on the other hand , rare tumours constitute a remarkable proportion of breast neoplasms and can be found rather frequently in some groups of women who might take particular advantage of mrm , namely , carriers of brca 1 germline mutations ( medullary carcinoma ) [ 27 ] , patients with nipple discharge ( papillary carcinoma ) [ 28 , 29 ] and women with gardners disease ( desmoid tumour ) [ 30 ]  . the purpose of this study is to describe mrm features of rare malignant neoplasms of the breast , utilising data from the literature and from our experience . materials and methods patients out of 468 patients with a breast malignancy who between september 2003 and june 2006 underwent preoperative ne di nuovi casi , oltre 400000 sono state le morti causate dalla neoplasia mammaria e pi di 4 milioni di donne ne sono attualmente affette [ 1 ]  . 
la patologia neoplastica mammaria estremamente eterogenea , abbracciando una vastissima gamma di modalit e stadi di presentazione , comportamento biologico , caratteristiche istologiche , aspetto radiologico , risposta ai vari tipi di trattamento e caratteristiche prognostiche . 
ci spiega perch la quasi totalit della produzione medica scientifica in ambito senologico sia incentrata sulla valutazione degli aspetti epidemiologici , patologici , radiologici e clinici dei suddetti istotipi e molto minore sia stata lattenzione per istotipi pi rari che , nel complesso , costituiscono il 10% delle neoplasie mammarie [ 3 ]  . negli ultimi anni , la risonanza magnetica mammaria ( rmm ) ha guadagnato un ruolo sempre pi importante nella diagnostica senologica . 
numerosi studi su larga scala hanno infatti messo in evidenza lelevata efficacia della rmm nellidentificazione di carcinomi mammari , sia primitivi che esito di recidive , con valori di sensibilit che possono raggiungere il 100% [ 46 ]  . 
in particolare le sue applicazioni principali sono lidentificazione di neoplasie mammarie in giovani donne ad alto rischio genetico [ 7 , 8 ] , in pazienti con cup syndrome [ 9 , 10 ] e con protesi mammarie [ 11 ] , la caratterizzazione di reperti mammografici ed ecografici di incerto significato diagnostico [ 12 ] , il bilancio di estensione di neoplasie gi istologicamente diagnosticate [ 1315 ] o nella valutazione pree post - chemioterapia neoadiuvante in caso di tumori localmente avanzati [ 16 , 17 ] e il follow - up post chirurgia conservativa / radioterapia [ 1820 ]  . 
per poterne sfruttare a pieno le potenzialit , oggi che questa metodica si sta diffondendo su larga scala , fondamentale che i radiologi entrino sempre pi in confidenza con la semeiotica della rmm , imparando a riconoscere sia gli aspetti tipici che atipici orientativi verso neoplasie mammarie . sebbene al momento attuale siano gi noti molti aspetti relativi alla semeiotica rmm delle neoplasie mammarie pi frequenti carcinoma duttale e lobulare infiltrante [ 21 ] sono molto meno conosciute le caratteristiche delle neoplasie maligne rare della mammella [ 2226 ]  . 
all 5 invasive papillary carcinomas were histological grade 2 ( according to elston - ellis ) , both medullary carcinomas were grade 3 , 3 / 7 mucinous carcinomas were grade 1 and 4 / 7 were grade 2 , all three tubular carcinomas were grade 1 and the tubulo - lobular carcinoma was grade 2 . with respect to hormone receptor status , 20 ( 74% ) patients had estrogen - receptor - positive / progesterone - receptor - positive ( er + / pr + ) tumours , 3 ( 11% ) had er - / prtumours , 1 ( 4% ) had an er + / prtumour and in 3 ( 11% ) cases hormone receptor profile was assessed . 
patients age ranged from 33 and 77 ( mean age 60 ) years . biopsies were performed under sonographic guidance in 25 cases , by using a 14 - gauge automated or semiautomated device ; and under stereotactic guidance in two cases , by an 11 - gauge directional vacuum - assisted biopsy probe . 
histological diagnoses were confirmed by pathological analysis of the surgical specimens ( 24 lumpectomies and 3 mastectomies )  . mrm technique in 20 cases , mrm was performed with a 1.5 - t system ( magnetom , avanto , siemens medical system , erlangen , germany ) with a dedicated bilateral breast surface coil and the patient in prone position . 
t1 - weighted images were acquired using a 3d fast low - angle shot ( flash ) pulse sequence with the following imaging parameters : tr / te 15 / 4.7 ms ; flip angle 25 ; matrix 197448 ; field of view 350175 mm ; section thickness 1.8 mm ; scanning time 88s . 
in the remaining seven cases , mrm scans were obtained using a 1 - t system ( magnetom impact , siemens , erlangen , germany ) with a dedicated bilateral breast surface coil and the patient in prone position . 
t1 - weighted 3d flash images were obtained with imaging parameters of tr / te 14 / 7 ms ; flip angle 25 ; matrix 192256 ; field of view 320160 mm ; section thickness 2.5 mm ; acquistition time 84s . gadopentetate dimeglumine ( magnevist , schering berlin , germany ) was administered iv as a bolus injection at a dose of 0.1 mmol / kg body weight at a flow rate of 2 ml / s followed by a 20 - ml saline flush . 
the images obtained underwent postprocessing : subtraction of the precontrast images from the postcontrast ones , and multiplanar reconstrucmateriali e metodi pazienti da 468 pazienti con neoplasia mammaria che , tra settembre 2003 e giugno 2006 , sono state sottoposte a rmm preoperatoria per bilancio di estensione , sono stati estrapolati 27 ( 5 , 7% ) casi di neoplasia mammaria maligna di istotipo raro ( secondo la classificazione who )  . 
in particolare : 13 ( 2 , 8% ) carcinomi papillari di cui 4 ( 0 , 9% ) carcinomi papillari intracistici , 4 ( 0 , 9% ) carcinomi papillari intraduttali , 5 ( 1 , 0 % ) carcinomi papillari invasivi ; 12 ( 2 , 5% ) varianti di carcinoma duttale infiltrante ( cdi ) di cui 2 ( 0 , 4% ) carcinomi midollari , 7 ( 1 , 5% ) carcinomi mucinosi e 3 ( 0 , 6% ) carcinomi tubulari ; 1 ( 0 , 2% ) carcinoma tubulo - lobulare e 1 ( 0 , 2% ) tumore desmoide ( fibromatosi extra - addominale )  . 
i 5 carcinomi papillari invasivi presentavano tutti grado istologico 2 secondo elston ellis , i 2 carcinomi midollari grado 3 , 3 / 7 carcinomi mucinosi grado 1 e 4 / 7 grado 2 , tutti i carcinomi tubulari grado 1 ed il carcinoma tubulo - lobulare grado 2 . lo stato recettoriale in 20 ( 74% ) pazienti era positivo sia per estrogeni che per progesterone , in 3 ( 11% ) era negativo , in 1 ( 4% ) caso positivo per estrogeni e negativo per progesterone e in 3 ( 11% ) casi non stato valutato . 
lesame istologico dei linfonodi ascellari ha evidenziato metastasi linfonodali in 2 casi ( 7% ) , entrambi carcinomi midollari . let delle pazienti risultava compresa tra 33 e 77 anni , con media di 60 anni . 
 la diagnosi di neoplasia mammaria stata posta mediante biopsia percutanea con prelievo microistologico sotto guida ecografica in 25 casi ( ago da 14 g , dispositivi di prelievo automatici o semi - automatici ) e sotto guida stereotassica in 2 casi ( mammotome 11 g ) e confermata sul campione operatorio ( 24 quadrantectomie e 3 mastectomie )  . esame rmm in 20 pazienti lesame stato effettuato con un magnete da 1 , 5 t ( magnetom , avanto , siemens medical system , erlagen , germania )  . 
stata eseguita una sequenza flash 3d utilizzando i seguenti parametri : tr / te = 15 / 4 , 7 ms ; flip angle 25 ; matrice 197448 pixel ; fov 350175 ; spessore 1 , 8 mm ; tempo di acquisizione 88 secondi . 
nelle rimanenti 7 pazienti la rmm stata eseguita con un sistema a 1 t ( magnetom impact , siemens , erlangen , germania ) , anche in questo caso in posizione prona e con bobina di superficie bilaterale dedicata . 
sono state eseguite sequenze flash 3d con i seguenti parametri : tr / te = 14 / 7 ms ; flip angle 25 ; matrice 192256 pixel ; fov 320160 ; spessore di 2 , 5 mm ; tempo di acquisizione 84s . in tutti i casi le immagini sono state acquisite sul piano coronale in una acquisizione precedente e in cinque successive alliniezione endovenosa di 0 , 1 mmol di gd - dtpa / kg di peso corporeo alla velocit di 2 ml al secondo , seguita a . 
in particular , morphological criteria included shape ( round , oval , dendritic , irregular ) ; borders ( well - defined , ill - defined ) ; pattern of contrast enhancement [ homogeneous , inhomogeneous and rim - like ( stronger enhancement of the peripheral portion of the lesion and delayed , slight enhancement of tumour core )  . 
kinetic criteria were enhancement rate that is the relative signal intensity increase that occurs in the first postcontrast minute ( less than 50% , between 50% and 100% , more than 100% ) and morphology of time - intensity curves ( continuous increase , type i ; plateau , type ii ; washout , type iii )  . 
the readers indicated a level of diagnostic suspicion and classified each lesion in one of five classes according to the breast imaging reporting and data system ( bi - rads ) [ 32 ]  . results one intracystic papillary carcinoma and one invasive papillary carcinoma were not detected at mrm . 
the former had been identified at mammography as a cluster of pleomorphic microcalcifications ( 10 mm ) , whereas the latter was a tiny ( 5 mm ) , hypoechoic , nonvascular mass unidentifiable at mammography . 
pathological examination of surgical specimens revealed the presence of neoplasms in both cases . the morphological and kinetic features of papillary neoplasms proved to be quite variable ( table 1 )  . 
enhancement rates were variable ( less than 50% , 33% ; between 50% and 100% , 33% ; and more than 100% , 33% ) , and time - intensity curves were type i in 1 case ( 33% ) and type ii in 2 cases ( 66% )  . intraductal papillary carcinomas presented as spiculated or irregular masses in 3 cases ( 75% ) and as an oval - shaped mass in 1 case ( 25% ) , with well - defined margins in 75% of cases and homogeneous enhancement pattern in 50% and inhomogeneous in the remaining cases . 
in all cases , the enhancement rate was less than 100% ( in 3 cases less than 50% ) , with type i ( 50% ) and ii kinetic curves ( 50% )  . the 4 ( 80% ) invasive papillary carcinomas detectable at mrm , demonstrated a wide range of shapes ( oval 50% , spiculated 25% and irregular 25% ) , with mainly ill - defined margins ( 75% ) and inhomogeneous ( 75% ) or rim - like ( 25% ) enhancement patterns . 
posizionando una regione dinteresse ( roi ) sulle focalit evidenti sono state costruite curve delle variazioni di intensit di segnale / tempo . valutazione delle immagini le immagini sono state valutate in consenso da due radiologi esperti in diagnostica senologica . 
in particolare i parametri morfologici descritti includevano : forma della lesione ( rotondeggiante ; ovalare ; spiculata ; irregolare ) , margini ( ben definiti ; mal definiti ) e pattern di enhancement ( omogeneo ; disomogeneo ; rim - like enhancement accumulo di mezzo di contrasto ( mdc ) prevalentemente nella porzione periferica della lesione , con aspetto ad anello , ed eventuale riempimento tardivo della porzione pi centrale ) ; mentre quelli cinetici comprendevano lenhancement rate , cio lincremento relativo dellintensit di segnale nel primo minuto dopo la somministrazione di mdc ( inferiore al 50% , compreso tra 50% e 100% , superiore al 100% ) e laspetto della curva cinetica intensit - tempo ( incremento progressivo e graduale , tipo i ; rapido wash in e successivo plateau , tipo ii ; rapido wash in e wash out , tipo iii )  . 
i lettori hanno inoltre classificato le lesioni in base al grado di sospetto radiologico , secondo una scala da 1 a 5 in relazione al breast imaging reporting and data system ( bi - rads - mri ) [ 32 ]  . risultati uno dei carcinomi papillari intracistici e uno dei carcinomi papillari invasivi non sono risultati identificabili alla rmm . nel primo caso la lesione si presentava mammograficamente come un focolaio di microcalcificazioni a morfologia plemorfa con estensione di 10 mm , mentre nel secondo caso si trattava di una minuta ( diametro di 5 mm ) formazione solida ipoecogena , non vascolarizzata , non evidente alla mammografia . 
lenhancement rate risultato variabile ( inferiore al 50% , 33% ; compreso tra 50% e 100% , 33% ; superiore a 100% , 33% ) e la curva cinetica risultata di tipo i in 1 ( 33% ) caso e di tipo ii in 2 ( 66% ) casi . i carcinomi papillari intraduttali si sono presentati con forma spiculata o irregolare in 3 ( 75% ) casi e ovalare in 1 ( 25% ) caso , margini ben definiti nel 75% dei casi e un pata . 
in the upper quadrants of the left breast , an oval , enhancing mass with well - defined borders ( enhancement rate 108% , time - signal intensitive curve type ii ) is shown . 
al passaggio tra i quadranti superiori della mammella sinistra si apprezza unarea focale ovalare a margini ben definiti di intenso enhancement ( enhancement rate : 108% , curva intensit / tempo di tipo ii )  . 
as a result of these features , this lesion was classified as bi - rads 4 . the desmoid tumour appeared on mrm as an irregular focal area with well - defined borders , mildly and inhomogeneously hyperintense after contrast administration ( enhancement rate less than 50% and type i kinetic curve ) , with nonsuspicious appearance ( bi - rads 2 ) ( table 3 )  . tern denhancement omogeneo nella met dei casi e disomogeneo nella rimanente met . 
lenhancement rate risultato in tutti i casi inferiore al 100% ( in 3 casi inferiore al 50% ) , con curve di tipo i ( 50% ) e ii ( 50% )  . i 4 ( 80% ) carcinomi papillari invasivi identificabili alla rmm hanno dimostrato morfologia variabile ( ovalare , 50% ; spiculata , 25% ; irregolare , 25% ) e , nella gran parte dei casi ( 75% ) , margini mal definiti e pattern denhancement disomogeneo ( 75% ) o tipo rim ( 25% )  . 
in 3 / 4 neoplasie papillari invasive lenhancement rate era superiore al 100% con curva cinetica di iii tipo , mentre nel quarto caso ( 25% ) lincremento iniziale di enhancement era compreso tra 50% e 100% e la curva era di tipo ii . le neoplasie papillari sono state classificate secondo il bi - rads come segue : 1 / 4 ( 25% ) carcinoma papillare intracistico e 1 / 5 ( 20% ) carcinoma papillare invasivo come birads 1 ; 1 / 4 ( 25% ) carcinoma papillare intraduttale come bi - rads 2 ; 2 / 4 ( 50% ) carcinomi papillari intracistici e 3 / 4 ( 75% ) intraduttali come bi - rads 3 ; 1 / 4 ( 25% ) carcinoma papillare intracistico e 2 / 5 ( 40% ) invasivi come bi - rads 4 e , infine , 2 / 5 ( 40% ) carcinomi invasivi come bi - rads 5 . le caratteristiche morfologiche e cinetiche delle varianti di carcinoma duttale infiltrante sono risultate piuttosto eterogenee , con differenze consistenti tra i diversi sottotipi ( tabella 2 )  . 
nessuna delle neoplasie di questo gruppo ha presentato una curva dinamica di tipo iii . i lettori che hanno esaminato i casi hanno espresso un grado di sospetto bi - rads 3 per 3 ( 43% ) dei carcinomi mucinosi e per tutti ( 100% ) i carcinomi tubulari , bi - rads 4 per 4 ( 57% ) neoplasie mucinose e bi - rads 5 per entrambe a . 
in the outer quadrants of the left breast ( arrow ) an irregular , inhomogeneously enhancing mass with ill - defined margins ( enhancement rate 112% , time - signal intensity curve type ii ) can be appreciated . 
al passaggio tra i quadranti esterni della mammella sinistra ( freccia ) evidente unarea focale a morfologia irregolare e margini mal definiti di disomogeneo e intenso enhancement ( enhancement rate : 112% , curva intensit / tempo di tipo ii )  . 
in the upper - outer quadrant of the right breast ( arrow ) , an irregular , mildly enhancing mass ( enhancement rate 70% , time - signal intensity curve type i ) is shown . 
a focal round area with well - defined margins and hypointense central core and peripheral , mild enhancement ( rim - like enhancement ) ( enhancement rate 45% , time - signal intensity curve type i ) is shown in the upper - outer quadrant of the left breast ( arrow )  . 
al quadrante superoesterno della mammella sinistra ( freccia ) , si evidenzia unarea focale a morfologia rotondeggiante e margini ben definiti , con porzione centrale ipointensa e orletto periferico lievemente iperintenso ( rim - like enhancement ) ( enhancement rate : 45% , curva intensit / tempo tipo i )  . 
in the upper quadrants of the left breast , an irregular , ill - defined , inhomogeneously hyperintense mass ( enhancement rate 74% , dynamic curve type ii ) is demonstrated . 
5 carcinoma tubulo - lobulare di grado 2 . nei quadranti superiori della mammella sinistra apprezzabile unarea irregolare a margini poco definiti , disomogeneamente iperintensa ( enhancement rate : 74% , curva cinetica tipo ii )  . 
lesions are identified because they enhance after iv injection of contrast agent , and mrm identifies the angiogenic focus rather than the tumour focus itself [ 21 , 33 , 34 ]  . 
these pathophysiological features seem to account for kinetic signs considered highly suggestive for invasive carcinoma , such as strong enhancement after contrast material injection and decrease of signal intensity in the early postcontrast period ( washout phenomenon )  . 
for example , 48% of lesions identified at mrm had an oval or round shape and 60% smooth margins , both of which are features typically associated with solid benign lesions , such as fibroadenomas [ 43 ] or papillomas , or small foci of fibrocystic change . 
moreover , only 28% and 12% of lesions , repregnazione , con protratta persistenza delliperintensit di segnale ( enhancement rate compreso tra 50% e 100% , curva cinetica di tipo ii ) ( tabella 3 )  . 
in considerazione di tali aspetti i due lettori hanno espresso un grado di sospetto diagnostico bi - rads 4 . laspetto del tumore desmoide risultato quello di unarea focale a morfologia irregolare e a margini netti , lievemente e disomogeneamente iperintensa dopo la somministrazione del mdc ( enhancement rate < 50% e curva cinetica di tipo i ) , presentando un aspetto poco sospetto ( bi - rads 2 ) ( tabella 3 )  . la valutazione globale degli aspetti morfologici e cinetici delle 27 neoplasie di istotipo raro riassunta in tabella 4 . nel complesso , il grado di sospetto espresso dai due lettori per le neoplasie esaminate risultato il seguente : 7 , 5% birads 1 , 7 , 5% bi - rads 2 , 40% bi - rads 3 , 30% bi - rads 4 e 15% bi - rads 5 ( tabella 5 )  . discussione le basi fisiopatologiche della rmm sono lincremento della vascolarizzazione ( densit vascolare ) nella sede della neoplasia , che conduce a un accumulo del mdc , e una aumentata permeabilit capillare , che determina una rapida fuoriuscita di mdc nellinterstizio tumorale . 
per tali motivi la lesione mammaria viene identificata in seguito al netto incremento di intensit rispetto ai tessuti circostanti dopo somministrazione di mdc , con dimostrazione , da parte della rmm , del focolaio angiogenetico piuttosto che della focalit tumorale di per s [ 21 , 33 , 34 ]  . 
questi aspetti fisiopatologici sono in parte in grado di spiegare le caratteristiche cinetiche considerate suggestive di neoplasia mammaria , in particolare lelevato incremento di intensit di segnale dopo mdc e la rapida dismissione dello stesso ( fenomeno descritto dalla curva cinetica tipo wash out )  . 
as a consequence , less than 50% of tumours were properly classified as bi - rads 4 and 5 , whereas 7.5% were interpreted as benign ( bi - rads 2 ) and 40% as probably benign ( bi - rads 3 )  . 
 il valore diagnostico di tali aspetti stato valutato da numerosi autori [ 31 , 3538 ] che , pur sottolineandone lelevato valore predittivo positivo , hanno riscontrato la possibilit che alcune neoplasie mammarie , in particolare le neoplasie intraduttali , i carcinomi lobulari e alcuni istotipi rari , in relazione alle loro specifiche caratteristiche istologiche e pattern di crescita , possano presentarsi in rmm con aspetti suggestivi per formazioni benigne piuttosto che maligne [ 36 , 39 ] o , in alcuni casi , non essere addirittura identificabili [ 4042 ] , originando dei falsi negativi . i risultati della nostra esperienza si pongono in linea con tali osservazioni , dimostrando che le neoplasie mammarie di istotipo raro tendono frequentemente a presentare caratteristiche suggestive di benignit ( 47 , 5% ) o addirittura a non essere riconoscibili alla rmm ( 7 , 5% )  . 
infatti ben il 48% delle neoplasie identificabili alla rmm presentava forma ovalare o rotondeggiante e il 60% margini netti , entrambi aspetti morfologici tipicamente associati a formazioni solide benigne come fibroadenomi [ 43 ] o papillomi , o a piccoli foci di mastopatia fibroso - cistica . 
inoltre solo il 28% e il 12% delle lesioni esaminate , alla valutazione dinamica , hanno dimostrato , rispettivamente , un enhancement rate superiore al 100% e una curva cinetica di tipo iii , altamente suggestivi per neoplasie mammarie maligne . 
come conseguenza di tali modalit di presentazione , meno della met delle lesioni stata classificata correttamente come bi - rads 4 e 5 , mentre il 7 , 5% e il 40% delle neoplasie sono state interpretate rispettivamente come benigne ( bi - rads 2 ) e probabilmente benigne ( bi - rads 3 )  . in particolare , si sono presentati con aspetti poco suggestivi per malignit i tumori papillari intracistici e intraduttali verosimilmente in relazione alla scarsa vascolarizzazione e alle caratteristiche di non invasivit , i carcinomi mucinosi , tubulari e il tumore desmoide . 
a spiegazione dellaspetto presentato dai carcinomi mucinosi , pu essere invocata , come gi commentato in letteratura [ 22 ] , la presenza di abbondante quantit di mucina che rende difficoltosa e lenta la diffusione del mdc allinterno della massa neoplastica . 
labbondante componente fibrosa [ 44 ] potrebbe invece essere responsabile degli aspetti rmm poco sospetti presentati dai carcinomi tubulari [ 45 ] e dei tumori desmoidi [ 46 ]  . 
alcune delle varianti rare di cdi , in particolare i carcinomi mucinosi e i tubulari , e i carcinomi papillari sono estremamente ben differenziati e sono associati a minimo potenziale metastatico e a buona prognosi [ 2 , 48 ]  . 
finally , a relevant role might be played , as hypothesised by mussurakis et al . [ 47 ] , by the high degree of differentiation of these tumours . some special types of idc , such as mucinous and tubular carcinomas and papillary carcinomas , are highly differentiated and are associated with low metastatic potential and a good prognosis [ 2 , 48 ]  . 
most of the tumours we evaluated were grade 1 or 2 , with a positive hormone receptor profile . the only exception was the appearance of medullary carcinomas , which exhibited mrm characteristics highly suggestive of malignancy ( both were classified as bi - rads 5 ) and had , in agreement with the literature [ 2 ] , more aggressive biological features histological grade 3 and negative hormone receptor status than other rare tumours . bi - rads 1 bi - rads 2 bi - rads 3 bi - rads 4 bi - rads 5 bi - rads 1 bi - rads 2 bi - rads 3 bi - rads 4 bi - rads 5 conclusioni 2 ( 7.5% ) 2 ( 7.5% ) 11 ( 40% ) 8 ( 30% ) 4 ( 15% ) 2 ( 7 , 5% ) 2 ( 7 , 5% ) 11 ( 40% ) 8 ( 30% ) 4 ( 15% ) tabella 5 tabella riassuntiva relativa alle classi breast imaging reporting and data system ( bi - rads ) totale ( percentuale ) conclusions analysis of mrm appearance of rare tumours did not reveal any distinctive feature . 
these findings strengthen the concept that mrm must be interpreted in conjunction with the other breast imaging diagnostic examinations and that it cannot replace breast biopsy . dallanalisi delle caratteristiche rmm di tumori di istotipo raro non sono emersi aspetti peculiari , e il dato pi rilevante risultato essere la tendenza di questi a presentarsi come lesioni benigne o poco sospette , o a non essere identificabili allesame rmm . 
bazzocchi1 1istituto di radiologia , azienda ospedaliero - universitaria di udine , via colugna 50 , i - 33100 udine , italy 2diagnostica senologica , aou careggi , viale pieraccini 17 , i - 50139 firenze , italy correspondence to : v . 
all patients underwent mammography , high - frequency broadband us and percutaneous breast biopsy with a 14 - gauge needle and a mean number of five samples ( range 47 passes )  . 
twenty - seven out of 65 dcis at core biopsy were found to have an invasive or microinvasive component at surgical excision , leading to rate of histological underestimation of core biopsy of 41.5%. 
although some histological features ( such as stromal fibrosis , periductal inflammatory infiltrate , high nuclear grade ) can suggest the presence of an invasive component , the sonographic appearance of dcis cannot be used to predict the cases that are underestimated on us - guided core biopsy . nevertheless , a sonographically detectable solid component , either inside dilatated ducts or associated with microcalcifications , and a size greater than 20 mm are frequently associated with the presence of an invasive component . key words ductal carcinoma in situ breast neoplasms diagnosis us breast biopsy riassunto obiettivi . 
tutte le pazienti hanno eseguito lesame mammografico e successivamente ecografico con sonda ad alta frequenza a larga banda , e la biopsia percutanea con aghi da 14 g con un numero medio di 5 frustoli ( range 47 )  . 
ventisette / 65 lesioni con diagnosi bioptica percutanea di cdis sono risultate associate a una componente infiltrante o microinfiltrante allesame istologico definitivo , ottenendo pertanto una sottostima istologica della core - biopsy pari al 41 , 5% . 
lelevata incidenza di sottostime istologiche alla core - biopsy impone un atteggiamento oltremodo prudente di fronte a una diagnosi di cdis effettuata con la core - biopsy stessa . sebbene alcune caratteristiche istologiche presenti nei frustoli bioptici prelevati ( quali la fibrosi stromale , linfiltrato infiammatorio periduttale , lalto grado nucleare ) possano suggerire la presenza di una componente invasiva , le caratteristiche ecografiche dei cdis non possono essere utilizzate per predire i casi che vengono sottostimati alla biopsia percutanea eco - guidata . 
esso viene considerato un potenziale precursore del carcinoma invasivo , a differenza del carcinoma lobulare in situ ( clis ) considerato piuttosto un indicatore di alto rischio per lo sviluppo di tumore alla mammella omolaterale o controlaterale [ 1 , 2 ]  . 
il carcinoma duttale in situ ( cdis ) istologicamente rappresentato da un gruppo eterogeneo di lesioni che presentano differenti caratteristiche istologiche , pattern di crescita , presentazione clinica e comportamento biologico [ 1 ]  . 
lintroduzione dello screening mammografico ha determinato il sempre pi frequente riconoscimento di lesioni subcliniche : il cdis rappresenta il 15%20% di tutti i tumori mammari , e il 25%56% di tutti i tumori clinicamente occulti [ 3 , 4 ]  . 
 la mammografia rappresenta lindagine pi sensibile e pi accurata per la diagnosi dei cdis , con una riduzione della sua accuratezza diagnostica solo nei casi di cdis non associati a microcalcificazioni [ 5 , 6 ]  . 
alla mammografia , dal 62% al 98% dei cdis vengono diagnosticati grazie alla presenza delle microcalcificazioni , mentre solo il 2%23% si manifesta come masse focali o asimmetrie di densit , o come distorsioni architetturali [ 58 ]  . 
in particolare , lecografia potrebbe svolgere potenzialmente alcuni ruoli nella valutazione dei cdis , includendo anche i casi che non si manifestano sotto forma di microcalcificazioni [ 11 ] : lecografia pu essere utilizzata per visualizzare ampi ( > 10 mm ) focolai di microcalcificazioni con forte sospetto di malignit e pu essere utilizzata come guida nelle procedure di biopsia percutanea ; lecografia pu giocare un ruolo importante nella valutazione dei cdis non calcifici , non solo nella identificazione di queste lesioni ma anche nella valutazione della loro estensione ; lecografia pu essere usata per rilevare cdis mammograficamente occulti nelle pazienti con elevata densit mammaria [ 11 , 1517 ]  . tra le varie tecniche di prelievo percutaneo per la caratterizzazione delle lesioni mammarie , ha avuto particolare introduction introduzione ductal carcinoma in situ ( dcis ) is defined as a proliferation of malignant epithelial cells within the ducts and lobules of the breast parenchyma yet without microscopic evidence of invasion through the basement membrane into the periductal stromal tissue . 
it is considered a potential precursor of invasive carcinoma , unlike lobular carcinoma in situ ( lcis ) , which is instead regarded as a high - risk lesion for the development of cancer in the same or contralateral breast [ 1 , 2 ]  . histologically , dcis is not a single entity but a heterogeneous group of lesions that differ in their histopathologic features , growth pattern , clinical presentation and biological behaviour [ 1 ]  . before the widespread diffusion of mammographic screening , dcis was rarely detected , and accounted for only 0.8%5% of all breast cancers [ 3 ]  . 
the introduction of mammographic screening has significantly increased the frequency with which clinically occult breast neoplasms are diagnosed , and dcis now accounts for 15%20% of all detected breast cancers and for 25%56% of all clinically occult cancers [ 3 , 4 ]  . 
 mammography is the primary and most sensitive tool for the detection of dcis , demonstrating a poor performance only in the absence of microcalcifications [ 5 , 6 ]  . 
although mammography identifies most cases of dcis , 6%23% of dcis lesions are not mammographically detectable [ 59 ]  . some recent studies have examined the role of us in the evaluation of mammographically detected microcalcifications [ 1014 ]  . 
in particular , us could have several potential roles in the evaluation of dcis , including those without calcifications [ 11 ] : it can be a useful diagnostic tool in the case of large ( > 10 mm ) clusters of microcalcifications that are highly suspicious for malignancy and can be used as a guide for interventional procedures . it may play an important role in the evaluation of noncalcified dcis , not only in detecting the lesion but also in evaluating its extent . it can be used to reveal mammographically occult dcis in patients with dense breasts [ 11 , 1517 ]  . percutaneous breast biopsy with automatic devices is widely considered one of the most effective techniques for breast lesion characterisation . 
core needle biopsy ( cnb ) is a simple , fast ( with real - time visualisation of needle position during the procedure ) and comfortable technique ( the patient is supine and breast compression is not required ) , and it is v . 
however , in some situations , cnb may underestimate the lesion , as in cases of atypical ductal hyperplasia ( adh ) on cnb that turn out to be dcis on surgical biopsy or dcis on cnb that are found to be invasive ductal carcinomas at final histology on the surgical specimen [ 12 , 13 , 1820 ]  . the purposes of our study were to retrospectively review our series of lesions with a cnb diagnosis of dcis and compare the results with those obtained after surgery and to describe the sonographic appearances of both pure dcis and dcis with an invasive component with a view to identifying possible sonographic features that might be useful in the case of cnb underestimation . materials and methods between 2000 and 2005 , 2 , 423 patients underwent mammography , us and breast cnb under us guidance at our institution . 
all 65 patients with dcis diagnosed at us - guided cnb presented spontaneously to our institution . among these , 44 were asymptomatic and were undergoing routine mammography and sonography , whereas 21 had a palpable nodule ( three with bloody nipple discharge and one with pagets disease )  . 
us examinations were performed with a broadband 5to 12 - mhz linear - array transducer ( hdi 5000 philips - advanced technology laboratories , bothell , wa , usa ) and a broadband 10to 13 - mhz linear - array transducer ( technos esaote )  . 
 sonograms were reviewed for : masses : us features ( hypoechoic , inhomogeneous ) , shape ( round , oval ) , size and margins ( regular or irregular or microlobulated ) architectural distortion ductal changes ( dilatation , extension , duct filled with hyperechoic spots , etc . ) microcalcifications on the basis of mammographic and sonographic features , lesions were classified according to the breast imaging reporting and data system ( bi - rads ) of the american college of radiology [ 21 ] ; in cases of disagreement between the two modalities , we considered the higher bi - rads category . all dcis were sonographically detectable , so the biopsy procedures were performed under us guidance . 
cnb was performed by using an automated gun system ( bard , magnum biopsy instrument , covington , ga , usa ) with 17 - mm sample window and 23 - mm throw or by using a semiautomatic biopsy gun ( precisa , hospital service ) with a 14 - gauge needle . 
la core - biopsy una metodica semplice , rapida ( con la visualizzazione della posizione dellago in tempo reale ) , confortevole per la paziente ( che viene posizionata supina senza necessit di comprimere la mammella ) , meno invasiva e meno costosa della biopsia chirurgica . 
esistono tuttavia delle situazioni nelle quali la diagnosi ottenuta con la biopsia percutanea rappresenta una sottostima istologica , in quanto lesioni diagnosticate come iperplasia duttale atipica ( ida ) possono poi risultare dei cdis o , ancora , lesioni diagnosticate come cdis possono parimenti risultare dei carcinomi duttali infiltranti alla verifica istologica definitiva dopo escissione chirurgica [ 12 , 13 , 1820 ]  . scopo del nostro lavoro stato rivalutare retrospettivamente la nostra casistica riguardante le lesioni mammarie con diagnosi istologica percutanea di cdis , mettendo a confronto i risultati della biopsia percutanea con quelli ottenuti dalla escissione chirurgica , e descrivere le caratteristiche ecografiche del cdis puro e del cdis con componente invasiva , valutando se alcuni aspetti ecografici possono aiutare nella identificazione dei casi sottostimati alla biopsia percutanea . materiali e metodi dal 2000 al 2005 , 2423 pazienti sono state sottoposte a mammografia , ecografia mammaria ( us ) e biopsia mammaria ecoguidata . 
nel contesto di tali diagnosi bioptiche , 65 casi ( 6 , 7% ) erano cdis e 903 ( 93 , 3% ) carcinomi invasivi . le lesioni diagnosticate come cdis alla biopsia percutanea eco - guidata sono state riscontrate in donne che si sono presentate spontaneamente presso il nostro istituto . 
in particolare , 44 pazienti asintomatiche eseguivano i periodici controlli mammografici ed ecografici , mentre 21 pazienti presentavano una massa clinicamente palpabile ( in tre di esse si associava anche una secrezione ematica dal capezzolo , in una paziente una malattia di paget del capezzolo )  . 
abbiamo valutato retrospettivamente soltanto le 65 lesioni diagnosticate come carcinoma duttale in situ , al fine di analizzare le caratteristiche ecografiche e valutare lapporto della core - biopsy con guida ecografica . 
lecografia mammaria stata eseguita mediante sonda lineare broadband da 512 mhz ( hdi 5000 philips - advanced technology laboratories , bothell , wa , usa ) e sonda lineare broadband da 1013 mhz ( technos esaote )  . le ecografie mammarie sono state riviste per la valutazione di : masse : sono state riportate lecogenicit ( ipoecogene , disomogenee ) , la morfologia ( rotonda , ovale ) le dimensioni e le caratteristiche dei margini ( regolari o irregolari o microlobulati ) ; distorsioni architetturali ; v . 
all biopsies were performed with the patient in a supine or supine - oblique position . the mean number of core samples obtained for each lesion was five ( range 47 passes )  . 
le core - biopsies sono state eseguite con dispositivi di prelievo automatico ( bard , magnum biopsy instrument , covington , ga , usa ) o semi - automatico ( precisa , hospital service ) , entrambi con finestra di prelievo di 17 mm e corsa del mandrino di 23 mm , con aghi trancianti da 14 gauge . 
la tecnica utilizzata per eseguire le biopsie ecoguidate con sistema automatico gi stata descritta in precedenza [ 18 , 22 , 23 ]  . tutte le biopsie sono state eseguite con paziente supina o in posizione supina - obliqua , con approccio laterale per le lesioni localizzate ai quadranti esterni , e con approccio mediale per le lesioni localizzate ai quadranti interni . il numero medio dei frustoli bioptici ottenuti per ogni lesione stato di 5 ( intervallo da 4 a 7 passaggi )  . 
tutte le biopsie sono state precedute da uniniezione locale di 5 cc di anestetico ( 2% di mepivacaina , astra zeneca ) attraverso ago da 26 g ( terumo europe nv , leuven , belgio )  . il consenso informato e lanamnesi sulla coagulazione sono stati ottenuti prima della biopsia mammaria , e le eventuali terapie anticoagulanti sono state sospese 3648 ore prima del prelievo . 
i risultati istopatologici della nodulectomia o della mastectomia sono stati confrontable 1 correlation between percutaneous core - biopsy and histologic diagnosis at surgical excision final diagnosis : pure dcis final diagnosis : dcis with invasive component final diagnosis : benign 35 ( 53.9% ) 3 ( 4.6% ) 27 ( 41.5% ) 7 microinvasive 20 invasive tabella 1 confronto tra diagnosi bioptica core - biopsy e diagnosi istologica definitiva ( pezzo operatorio ) diagnosi bioptica core - biopsy diagnosi definitiva ca invasivi diagnosi definitiva benigna cdis 65 35 ( 53 , 9% ) 3 ( 4 , 6% ) 27 ( 41 , 5% ) 7 mic 20 infiltranti percutaneous corebiopsy dcis 65 dcis , ductal carcinoma in situ diagnosi bioptica core - biopsy cdis , carcinoma duttale in situ v . 
1a , b pure ductal carcinoma in situ ( dcis ) ( percutaneous biopsy / surgical excision concordance ) , occult at mammography , in a 48 - year - old asymptomatic patient . 
ventinove su 38 cdis puri ( 76 , 3% ) erano visibili alla mammografia , e il loro aspetto predominante era rappresentato da microcalcificazioni isolate in 22 casi ( 75 , 9% ) , opacit associate o non a microcalcificazioni in 7 casi ( 24 , 1% )  . 
la maggior parte delle lesioni ( 23 / 38 , 60 , 5% ) sono state classificate come sospette allecografia ( 15 / 38 , 39.5% con bi - rads 4 ) o altamente suggestive di malignit ( 8 / 38 , 21% con bi - rads 5 )  . 
2a , b pure ductal carcinoma in situ ( dcis ) ( percutaneous biopsy / surgical excision concordance ) in a 56 - year - old patient , with a palpable mass in the left breast . 
a lecografia dimostra la presenza di una formazione ovalare ipoecogena , a margini in gran parte regolari , al cui interno si apprezzano alcuni spot iperecogeni , corrispondenti alle microcalcificazioni . 
most of these lesions ( 23 / 38 , 60.5% ) were classified as suspicious ( 15 / 38 , 39.5% , birads 4 ) or highly suspicious ( 8 / 38 , 21% , birads 5 ) for malignancy . 
the us features of dcis with invasive component were : solid round masses , hypoechoic , microlobulated , not calcified , highly vascular in 17 cases ( 63% ) solid oval masses , hypoechoic , microlobulated , with internal hyperechoic spots corresponding to microcalcifications in six cases ( 22% ) fig . 
3a , b pure ductal carcinoma in situ ( dcis ) ( percutaneous biopsy / surgical excision concordance ) , occult at mammography , in a 46 - year - old patient with bloody nipple discharge . 
3a , b cdis puro ( concordanza core - biopsy / chirurgia ) occulto alla mammografia , in donna di 46 anni con secrezione spontanea siero - ematica . a lecografia dimostra la presenza di strutture duttali dilatate , con pareti lievemente ispessite e contenenti materiale ecogeno di tipo necrotico . 
tutte le lesioni sono state classificate come sospette per malignit ( bi - rads 4 in 20 / 27 casi , 74% ) o altamente suggestive per malignit ( bi - rads 5 in 7 / 27 casi , 26% ) ( tabella 5 )  . 
 considerando come casi di sottostima istologica solo quelli con infiltrazione ( escludendo cio i cdis microinvasivi , dal momento che le lesioni microinvasive , confrontate con i cdis puri , non differiscono per tasso di recidiva locoregionale , per tasso di sopravvivenza libera da malattia e sopravvivenza totale ) la percentuale di sottostima si ridurrebbe dal 41 , 5% al 31% ( 20 su 65 lesioni )  . la mammografia rappresenta lindagine pi importante e pi sensibile per lidentificazione dei cdis associati a mifig . 
lecografia ( us ) ha avuto finora un ruolo marginale nella diagnosi dei cdis a causa della scarsa visibilit ecografica delle microcalcificazioni , specialmente nel caso di microcalcificazioni isolate o di microcalcificazioni che si sviluppano nel contesto di un parenchima fibro - ghiandolare diffusamente iperecogeno . negli ultimi anni , lintroduzione delle nuove sonde broadband , lineari ad alta frequenza , con maggior risoluzione spaziale e maggior risoluzione di contrasto ( la cos detta ecografia ad alta risoluzione ) ha permesso di incrementare la sensibilit e specificit dellus , e la confidenza diagnostica delloperatore [ 2426 ]  . 
daltra parte ben noto in letteratura che dal 6% al 23% dei cdis risultano mammograficamente occulti [ 4 , 5 , 7 , 9 , 27 ] , percentuale corrispondente al 21 , 5% ( 14 / 65 ) nella nostra casistica . 
essa risulta inoltre meglio tollerata dalle pazienti , rispetto alla guida stereotassica , poich la posizione supina risulta pi confortevole , senza necessit di comprimere la mammella , e la visualizzazione in tempo reale dellago bioptico permette di prelevare in ogni sede della mammella , incluse le regioni periferiche lungo il prolungamento ascellare o la parete toracica in caso di esiti di mastectomia . 
i suoi risultati sono estremamente validi , e se applicata con una metodologia adeguata ( utilizzando aghi da 14 g , con lunga escursione del mandrino , con un minimo di 5 prelievi ) in grado di fornire risultati paragonabili a quelli della biopsia chirurgica , considerata finora il gold standard di riferimento . nella letteratura , considerando le procedure eseguite sia con guida ecografica che mammografica , la concordanza con la biopsia chirurgica compresa tra l80% e il 96% . nella nostra serie consecutiva di 2423 core - biopsy ecoguidate con ago da 14 g , in 65 casi stato diagnosticato un cdis , mentre allesame istologico post - chirurgico stata riscontrata la presenza di una componente infiltrante in 27 casi ( 41 , 5% )  . 
 nonostante lelevata concordanza tra i risultati istologici ottenuti con la scnb ( stereotactic core needle biopsy ) e la successiva escissione chirurgica , molti autori hanno riportato percentuali variabili ( dal 15% al 36% ) di cdis senza fig . 
allecografia si evidenzia piccolo nodulo ( 5 mm ) , tenuemente ipoecogeno , microlobulato , con buona trasmissione del segnale acustico posteriore ; si riconosce tenue alone iperecogeno perilesionale come da reazione desmoplastica . 
chirurgia : cdis con focolai di infiltrazione stromale . ductal changes ( focal dilatation with intraductal hypoechoic necrotic - like material , associated with microcalcifications ) in four cases ( 15% ) at us , most lesions ranged in size from 10 to 20 mm ( 13 / 27 , 48% ) , 11 were > 20 mm ( 41% ) and three < 10 mm ( 11% ) ( table 4 )  . 
all lesions were classified as suspicious ( birads 4 in 20 / 27 cases , 74% ) or as highly suspicious ( bi - rads 5 in 7 / 27 cases , 26% ) for malignancy ( table 5 )  . considering only dcis with invasive component demonstrated at surgical excision ( thus excluding microinvasive dcis , as they have a similar locoregional recurrence rate , disease - free survival and overall survival rate to pure dcis ) , the underestimation rate is reduced to 31% ( 20 / 65 lesions )  . discussion mammography is the most important and sensitive technique for the detection of dcis associated with microcalcifications . 
moreover , it shows the number and extent of microcalcification clusters and can be used as a guide for biopsy . until now , us had only a marginal role in the diagnosis of v . 
over the last few years , the introduction of new broadband highfrequency linear - array transducers with higher spatial and contrast resolution ( so called highresolution sonography ) has improved the techniques sensitivity and specificity and the operators diagnostic confidence [ 2426 ]  . 
in our series , 21.5% ( 14 / 65 ) of dcis was mammographically occult , in line with the percentage of 623% reported in the literature [ 4 , 5 , 7 , 9 , 27 ]  . 
us , performed by expert physicians with high - resolution linear - array broadband transducers with 10to 13 - mhz frequency , may be considered complementary to mammography for the detection of dcis , especially for those not associated with microcalcifications , and for the evaluation of the ductal extent of dcis in dense breasts [ 11 ]  . 
us - guided cnb offers many advantages over surgical biopsy , as it is fast , less costly and less invasive ( no surgical scars responsible for mammographic and sonographic distortion )  . 
moreover , it is more comfortable than stereotactic biopsy ( no breast compression is required ) and , thanks to the real - time visualisation of the needle probe , allows tissue retrieval from all breast regions , including peripheral regions such as the axillary tail and chest wall in the case of a previous mastectomy . 
if correctly performed ( using 14 - gauge needles , long throw and at least five tissue samples ) , its results are as satisfying as those of excisional biopsy , which is still considered the gold standard procedure . in literature , the concordance between cnb ( performed both under sonographic and mammographic guidance ) and surgical excision ranges from 80% to 96% . 
our study revealed an underestimation rate of 41.5% , with surgical excision demonstrating microinfiltration ( invasive foci 1 mm ) or infiltration in 27 lesions ( seven dcis were microinvasive and 20 dcis showed invasive components at definitive histology )  . most investigations of the accuracy of cnb in the diagnosis of dcis focus on stereotactically rather than sonographically guided procedures . 
despite the high concordance between stereotactic core needle biopsy ( scnb ) and surgical excision , many authors have reported variable ranges ( 15%36% ) of pure dcis diagnosed on scnb later upgraded to invasive carcinomas after lumpectomy [ 2834 ]  . in a paper comparing the outcome of us - guided cnb and vacuum - assisted biopsies ( vab ) for the diagnosis of dcis , philpotts et al . 
 [ 19 ] found no significant differences in the outcomes of us - guided core biopsies performed with the 14gauge automated gun compared with those performed with the 11 - gauge vacuum - assisted device . 
in particular , no differences were reported regarding the number of false - negative cancers , underestimation , complications or recommendations aspetti di infiltrazione stromale alla scnb , risultati poi delle lesioni infiltranti alla successiva nodulectomia [ 2834 ]  . 
 [ 19 ] effettuano un confronto anche con i risultati della vacuumassisted biopsy ( vab ) , senza riportare significative differenze di accuratezza tra le diagnosi ottenute con il sistema automatico ( ago 14 g ) e quelle ottenute con il sistema vab ( ago 11 g )  . 
in particolare , gli autori riportano risultati della cb comparabili con quelli della vab per quanto concerne il numero di carcinomi falsi - negativi e di sottostime istologiche , a parit di complicanze . 
essi ritengono inoltre che non vi siano differenze sostanziali nel rinvio a re - biopsia ( o biopsia chirurgica ) fra cb e vab , in relazione alle problematiche concernenti la discordanza tra quadro radiologico e istologico , in caso di diagnosi di lesione proliferativa ad alto rischio ( radial - scar , iperplasia duttale atipica , clis , papilloma ) o in caso di biopsia inadeguata ( evento questultimo abbastanza raro per entrambe le metodiche )  . inoltre , il numero di frustoli considerato sufficiente per la diagnosi di un nodulo ecografico solitamente inferiore con lutilizzo della biopsia ecoguidata ( 4 - 5 frustoli ) , dal momento che il prelievo avviene in tempo reale con la visualizzazione dellago , rispetto alla biopsia sotto guida stereotassica che necessita di pi di 10 frustoli ( soprattutto in presenza di microcalcificazioni ) [ 19 ]  . 
il sistema di biopsia vab a guida ecografica , caratterizzato dallinserimento manuale dellago allinterno della lesione senza il sistema di avanzamento automatico , potrebbe invece essere utilizzato nei casi di noduli ecografici profondi o nelle lesioni di difficile visualizzazione , al fine di ridurre il rischio di perforazione della gabbia toracica [ 19 ]  . per quanto concerne la biopsia sotto guida stereotassica ( scnb ) , lee et al . 
 [ 35 ] , nella loro casistica di 59 cdis diagnosticati alla scnb , hanno presentato un tasso di sottostima pari al 18% ( 6 / 34 ) tra i cdis biopsiati con sistema vab con ago da 11 g e pari al 44% ( 11 / 25 ) tra i cdis biopsiati con sistema di prelievo automatico con ago da 14 g . 
 [ 36 ] , nella loro casistica di 40 lesioni consecutive diagnosticate come cdis utilizzando la guida stereotassica , hanno ottenuto valori di sottostima sostanzialmente pi bassi con il vacuum - assisted device da 11 g ( 15% , 3 / 20 ) rispetto al sistema automatico da 14 g ( 35% , 7 / 20 )  . 
la maggiore accuratezza nella diagnosi istologica percutanea dimostrata dai sistemi vab con ago da 11 g si pu spiegare con il ricorso ad aghi di maggior calibro , che consentono il prelievo di una maggiore quantit di tessuto , e con il maggior numero di prelievi bioptici ottenuti . 
in letteratura , molti autori per ritengono che i sistemi di vacuum - assisted biopsy ( vab ) possano ridurre , ma non eliminare completamente , il numero delle sottostime istologiche percutanee nelle diagnosi di cdis [ 2834 ]  . i motivi delle sottostime istologiche percutanee riscontrate anche con i sistemi vab da 11 g sarebbero legati al fatto che le calcificazioni , ossia il target di prelievo delle procedure stereotassiche , non corrispondono effettivamente alle aree di tumore invasivo che , cos , potrebbero non essere adeguatamente incluse allinterno dei frustoli calcifici prelevati . 
in fact , a second biopsy , or better surgical biopsy , is warranted in all cases of radiologicalpathological discordance , core biopsy diagnosis of high - risk proliferative lesions ( radial scar , atypical ductal hyperplasia , lcis , papillary lesion ) or inadequate sampling ( occurring quite infrequently with both modalities )  . the minimum number of specimens needed for a histological diagnosis is usually lower for us - guided biopsy ( 45 specimens ) than for stereotactic biopsy ( more than 10 specimens ) , particularly in the presence of microcalcifications [ 19 ]  . 
us - guided vab , with the transducer manually introduced into the lesion without shooting through automated systems , might be used in cases of deep nodules or low visibility in order to reduce the risk of chest wall perforation [ 19 ]  . with regard to scnb , a study by lee et al . 
 [ 35 ] on 59 dcis diagnosed by scnb reported an 18% ( 6 / 34 ) underestimation rate for vab with 11 - gauge needles and a 44% ( 11 / 25 ) underestimation rate for an automated system with 14 - gauge needles [ 35 ]  . 
similar results were reported by won et al . , with underestimation rates for stereotactic vab of 15% and of 35% with 11 - gauge and 14 - gauge needles , respectively [ 36 ]  . 
in the literature , vab is regarded as a useful diagnostic tool , able to reduce but not completely avoid underestimation in the diagnosis of dcis [ 2834 ]  . 
 however , the reasons for the underestimations produced even by the 11 - gauge vab systems are related to microcalcifications , which the biopsy aims to target , not being contained within the invasive tumoral component . 
the most frequent appearances were : mass without microcalcifications mass with microcalcifications isolated microcalcifications it is also important to consider : ductal changes dimensions mass without microcalcifications this was the most frequent appearance of dcis and was mainly a solid nodule with microlobulated boundaries and guardanti laccuratezza della diagnosi istologica percutanea su focolai di microcalcificazioni , quando vengano utilizzati dispositivi di prelievo automatico , in relazione alla esiguit del materiale bioptico fornito dagli aghi 14 g ( materiale che talvolta risulta anche frammentato ) e pertanto non sempre rappresentativo dellintera lesione che si desidera sottoporre a biopsia . 
gli aspetti principali da considerare sono rappresentati dal riscontro di : massa senza microcalcificazioni massa con microcalcificazioni microcalcificazioni isolate . va inoltre considerato leventuale pattern di presentazioaspetti duttali dimensioni . massa senza microcalcificazioni si tratta dellaspetto ecografico di cdis riscontrato nella nostra casistica pi frequentemente , caratterizzato da una massa solida , microlobulata , non calcifica ( riconosciuta nel 47 , 4% dei cdis puri e nel 63% dei cdis invasivi )  . 
tale aspetto quello comunemente riscontrato nei cdis non associati a microcalcificazioni , che istologicamente corrispondono al pattern cribriforme , micropapillare , papillare o solido , e che si accrescono senza formare n necrosi n calcificazioni , senza reazione desmoplastica ( corrispondente a fenomeni di fibrosi , sclerosi o fenomeni cicatriziali ) ; questultima caratterizza invece lassorbimento acustico posteriore tipico dei carcinomi invasivi aggressivi . 
molti autori hanno sottolineato la possibilit di dimostrare le microcalcificazioni grazie allecografia ad alta risoluzione , dove appaiono come piccoli spot iperecogeni , in genere non associati allassorbimento acustico posteriore in relazione alle loro ridotte dimensioni [ 13 , 24 , 26 ]  . 
alcuni autori hanno sottolineato inoltre che le calcificazioni associate a lesioni maligne sono pi facilmente visibili allus poich si sviluppano allinterno di un nodulo solido , mentre la maggior parte delle calcificazioni benigne si forma nel parenchima mammario fibro - ghianv . 
dcis not associated with microcalcifications correspond histologically to cribriform , micropapillary , papillary or solid patterns that grow without forming necrosis , calcifications or desmoplastic reaction ( fibrosis , sclerosis , scarring ) ; a desmoplastic reaction may explain the presence of posterior acoustic shadowing in aggressive invasive cancers . 
 mass with microcalcifications microcalcifications , set within solid masses , represent the second most frequent us pattern ( found in 23.7% of pure dcis and in 22% of dcis with invasive component )  . 
many authors have recently emphasised the possibility of identifying microcalcifications through high - resolution sonography , where they appear as small , hyperechoic spots without posterior acoustic shadowing due to their small size [ 13 , 24 , 26 ]  . 
calcifications associated with malignant lesions are easily detected on us , as they develop within a solid nodule , whereas most benign calcifications develop in hyperechoic fibroglandular breast parenchyma and are more difficult to identify [ 11 ]  . isolated microcalcifications isolated microcalcifications within normal breast parenchyma were found in only four cases ( 10.5% ) of pure dcis but in no case of invasive dcis . 
usually , it is very difficult to depict small clusters of microcalcifications at us , especially if they are < 5 mm in size , as these small hyperechoic spots are not associated with posterior acoustic shadowing and can be confused with hyperechoic us tissue interfaces or with speckle artefacts [ 24 , 26 ]  . 
the microcalcifications more easily depicted by us are large ( > 10 mm ) clusters of packed , highly suspicious ( bi - rads 5 ) calcifications with segmental distribution [ 13 ]  . 
 [ 38 ] , who defined the extension of tumoral cells towards the nipple as ductal diffusion and the extension of tumoral cells in the opposite direction as a branching pattern . 
they interpreted this branching pattern as the expression of the diffusion of a proliferative process through the ductal system and correlated it to a high probability of malignancy [ 38 ]  . 
in fact , intraductal vascularisation dolare iperecogeno e pertanto pi difficile da identificare [ 11 ]  . microcalcificazioni isolate la presenza di microcalcificazioni isolate nel contesto del parenchima mammario normale stata evidenziata solo in 4 casi ( 10 , 5% ) di cdis puri , ma in nessun caso di cdis invasivo . 
di solito molto difficile individuare piccoli focolai di microcalcificazioni allus , soprattutto quelli con dimensioni < 5 mm , dal momento che questi piccoli spot iperecogeni non associati ad assorbimento acustico posteriore possono essere confusi con le interfacce acustiche iperecogene del tessuto o con i cosiddetti speckle artifacts [ 24 , 26 ]  . 
le microcalcificazioni che hanno maggior probabilit di essere viste allus sono rappresentate da focolai di microcalcificazioni con dimensioni > 10 mm , con numerosi spot calcifici allinterno del focolaio , caratterizzati da una distribuzione segmentale e da un elevato sospetto di malignit ( bi - rads 5 ) [ 13 ]  . 
 [ 38 ] hanno descritto gi questi tipici aspetti duttali , definendo come diffusione duttale lestensione delle cellule tumorali verso la regione del capezzolo , e come pattern ramificato lestensione delle cellule tumorali in direzione opposta rispetto al capezzolo . 
gli autori hanno interpretato questo pattern ramificato come lespressione della diffusione del processo proliferativo lungo il sistema duttale , correlandolo ad una maggior probabilit di malignit [ 38 ]  . va sottolineato inoltre lapporto del color - doppler nel definire la natura del contenuto dei dotti dilatati . 
nevertheless , this underestimation rate appears to be irrelevant in relation to the total number of breast neoplasms diagnosed on cnb ( 27 / 968 , 2.8% ) and seems not to affect patient outcome , although this statement should be further confirmed . 
we found no sonographic pattern specific for invasive component , although features such as a solid component , either inside dilatated ducts or together with microcalcifications , and a size > 20 mm are more frequently associated with infiltration . criterio dimensionale per quanto riguarda invece il criterio dimensionale , la maggior parte delle lesioni riscontrate presentava dimensioni comprese tra 10 e 20 mm ( 58% dei cdis puri , 48% dei cdis sottostimati ) ; tuttavia va sottolineato che il 40% dei cdis puri presentava dimensioni inferiori ai 10 mm , mentre il 41% dei cdis invasivi presentava dimensioni pi grossolane , superiori ai 20 mpertanto possiamo affermare che il riscontro ecografico di lesioni con dimensioni > 20 mm pu far sospettare pi frequentemente la presenza di una componente invasiva . conclusioni la diagnosi di cdis alla cb sotto guida ecografia gravata da un numero elevato di sottostime istologiche ( 41 , 5% )  . 
daltro canto , questa percentuale di sottostima sembra essere irrilevante sul totale dei cancri diagnosticati alla cb ( 27 / 968 , 2 , 8% ) e inoltre il tasso di sottostima istologica sembra non influenzare loutcome delle pazienti , nonostante tale affermazione necessiti di ulteriori studi per essere confermata . 
non esistono pattern ecografici specifici per la diagnosi della componente infiltrante , anche se la presenza della componente solida visibile ecograficamente , sia allinterno di immagini duttali che nei casi associati a calcificazioni , e le dimensioni superiori a 20 mm delle lesioni , si associno pi frequentemente a infiltrazione . 
carriero scdu radiologia , sezione di risonanza magnetica , ao maggiore della carit , universit del piemonte orientale , c.so mazzini 18 , i - 28100 novara , italy correspondence to : a . 
contrast - enhanced mra , even with a low - field ( 0.5 - t ) unit , is a feasible , sensitive and accurate technique for the study of the renal arteries of the transplanted kidney . key word renal transplant renal artery stenosis angio - mri riassunto obiettivo . 
langio - rm con contrasto iniettato con tecnica a bolo di contrasto , per lo studio delle arterie renali dei reni trapiantati , una metodica fattibile dal punto di vista pratico , sensibile e con unottima accuratezza diagnostica , anche con apparecchiatura rm a basso campo ( 0 , 5 t )  . parole chiave trapianto renale stenosi dellarteria renale angio - rm introduction introduzione in 2005 alone 213 kidney transplants from cadaver donors were performed in the italian region of piedmont , 80 of solo nel 2005 in piemonte sono stati effettuati 213 trapianti di reni da donatore cadavere , e nella nostra struttura ospe1026 09 stecco 19 - 10 - 2007 13 : 41 pagina 1027 a . 
in either case , well - defined images are required to evaluate the presence , site , degree and extent of stenosis , information that facilitates the choice between endovascular or surgical treatment and allows more invasive radiological techniques to be avoided . colour - doppler ultrasound ( us ) is the easiest and fastest imaging tool for diagnosing possible vascular complications of the kidney graft [ 9 , 10 ]  . 
the current gold standard is digital subtraction angiography ( dsa ) [ 11 , 12 ] , which has proved to provide the best characterization of arterial stenosis and atheromas , especially since the introduction of the three - dimensional rotational technique . 
other reasons include potential nephrotoxicity of the contrast material , need for percutaneous access , the use of ionising radiation and possible complications that may occur during the procedure that carry a low but definite risk of mortality ( 0.02% ) [ 13 , 14 ]  . some authors [ 1517 ] have evaluated the role of magnetic resonance angiography ( mra ) in the detection and characterisation of arterial stenosis , describing it as a noninvasive method that relies on intravenous injection of gadolinium and does not require ionising radiation or percutaneous access . 
the aim of our study was to assess interobserver variability , sensitivity , specificity and diagnostic accuracy of contrast - enhanced low - field mri in the evaluation of arterial stenosis of the transplanted kidney by analysing both source images and digitally reconstructed images . materials and methods patients we studied 49 patients ( 31 men , 18 women ; age 2473 years ; all cadaver - donor kidney recipients ) who underwent a total of 64 mra examinations for clinical suspicion of renal artery stenosis based on : the presence of non - rejection - related renal failure , alterations identified on colour - doppler us and hypertension of unknown origsome of these patients were undergoing a series of follow - up studies owing to difficult clinical situations . 
in entrambi i casi si necessita di immagini ben definite per valutare la presenza , il sito , il grado e lestensione della stenosi e questo rende pi facile anche la scelta del trattamento endovascolare o chirurgico , e ancora permette di evitare indagini radiologiche pi invasive . leco - color doppler lo strumento pi facile e veloce da utilizzare per diagnosticare eventuali complicazioni vascolari nei reni trapiantati [ 9 , 10 ] , ma allo stesso tempo una metodica operatore dipendente , caratterizzata da una variabilit durante lesecuzione e quindi dallimpossibilit , in alcuni casi , di evidenziare totalmente o parzialmente larteria renale . 
attualmente il gold standard radiologico langiografia a sottrazione digitale ( dsa ) [ 11 , 12 ] , che sin dallintroduzione dellangiografia volumetrica rotazionale , si dimostrata capace di caratterizzare al meglio le stenosi arteriose e le placche ateromatose . 
questa tecnica angiografica necessita per di iniezione intra - arteriosa di mezzo di contrasto iodato , e per questo e non solo considerata invasiva ; inoltre bene ricordare la potenziale nefrotossicit del mdc , la necessit di un accesso percutaneo , lutilizzo di raggi x , e le possibili complicanze durante la procedura che presentano un seppur basso ( 0 , 02% ) rischio di mortalit [ 13 , 14 ]  . alcuni autori [ 1517 ] hanno valutato il ruolo dellangiografia con risonanza magnetica ( arm ) nella visualizzazione e caratterizzazione delle stenosi arteriose considerandola una metodica non invasiva per lutilizzo intravenoso di gadolinio , lassenza di raggi x e di accesso percutaneo ; tutti hanno per utilizzato un tomografo rm a medio - alto campo magnetico ( 1 - 1 , 5 tesla ) , senza menzionare le possibilit di utilizzo di un tomografo rm a basso campo ( 0 , 5 t )  . 
per questo il nostro studio mira a valutare la variabilit tra gli esaminatori , la sensibilit , la specificit e laccuratezza diagnostica della tomografia rm a basso campo con mdc , nella valutazione delle stenosi arteriose del rene trapiantato , con losservazione sia delle immagini originali , sia delle ricostruzioni digitali . materiali e metodi pazienti sono stati esaminati 49 pazienti ( 31 maschi e 18 femmine , di et compresa tra i 24 e i 73 anni , tutti sottoposti a trapianto renale da donazione da cadavere ) , sottoposti ad un totale di 64 arm per sospetto clinico di stenosi dellarteria renale , dovuto a : presenza di insufficienza renale non da rigetto ; alterazioni evidenziate mediante eco - color doppler ; ipertensione di origine sconosciuta . 
a bolus of 1 ml of gadolinium - benzyloxypropionictetraacetate ( gdbopta ) ( multihance , bracco , italy ) was infused via an automatic injector ( spectris , medrad , pittsburgh , pa , usa ) at a rate of 2.0 ml / s , followed by 20 ml of saline solution . 
during the bolus test , a single sagittal section was acquired with the multiphase technique using a fast - gradient t1 sequence with high temporal resolution ( tr / te min / 2.6 ; flip angle 60 ; ta 30s ; slice 10 - mm ; fov 40 cm ; matrix 224128 ; nex 1 ) , positioned along the course of the aorta , after a preliminary localization sequence in the coronal plane . 
then we proceeded with a scan using fast gradient echo sequences ( fgre - 3d ; tr / te / inversion time min / 1.9 / 41 ; flip angle 50 ; ta 27s ; thickness 5 mm ; fov 42 cm ; matrix 320160 , nex 1 ) with fat suppression . 
this pulse sequence is characterised by optimal signal - to - noise ratio ( snr ) and rotation time ( tr ) , with centric k - space encoding that improves contrast between vascular structures and especially snr . 
fat suppression ( inversion time = 41 ms ) does not require additional digital subtraction of contrast images from the mask image but nevertheless provides suppression and deletion of nonvascular structures . post - processing with 3d algorithms was performed on a workstation ( advantage windows 4.0 , ge , milwaukee , il , usa ) by a third operator to create maximum intensity projection ( mip ) and multiplanar reconstructions ( mpr )  . 
mip images were obtained with 20 rotations at 18 on the longitudinal axis to cover 360 of the volume acquired ; mpr images were generated to visualise simultaneously three windows displaying sagittal , coronal and axial views . 
 to evaluate possible stenoses of the iliac arteries , patients also underwent dsa with a digital subtraction philips integris v5000 unit ( philips , eindhoven , the netherlands ) , during which multiple boluses ( 20 ml ) of noniodinated contrast medium ( iomeron 300 , bracco ) were injected into the abdominal aorta at a rate of 10 ml / s . 
patients with suspected stenosis of the anastomosis or external iliac artery were administered a bolus of 810 ml of contrast material in the iliac artery at a rate of 3 ml / s . 
images were acquired in the anteroposterior and oblique positions ( 30 )  . image analysis two radiologists ( po and as ) evaluated the three mra images sets ( mip , mpr and raw data ) , which had been processed by a fourth - year radiology resident blinded to patients clinical conditions and dsa results . 
images were evaluated using a preprinted form on which readers had to indicate whether stenosis was present and the arterial branch involved , divided into three segments ( preanastomotic , anastomotic and postanastomotic )  . 
where a stenosis was identi1028 ( 22 , 4% ) sono stati inoltre sottoposti a dsa , anche pi volte per il lungo follow - up o per delle ristenosi dopo angioplastica : tutti i pazienti hanno firmato il consenso informato prima dellesame . tecnica dellimmagine le scansioni rm sono state acquisite grazie ad una bobina phased - array ( body - flex ii ) e ad uno scanner a 0 , 5 t ( signa contour , ge , milwaukee , il , usa ) , con gradienti aggiornati . 
stato eseguito un test ( bolus track ) di infusione per il calcolo del tempo di circolo , con 1 ml di gadolinium - benzyloxypropionictetraacetato ( gd - bopta ) ( multihance , bracco , italia ) , con velocit di infusione di 2 , 0 ml / s , seguito dallinfusione di 20 ml di soluzione salina ; tutto tramite iniettore automatico ( spectris , medrad , pittsburg , pa , usa )  . durante liniezione del bolus test stata acquisita con tecnica multifasica una singola sezione nel piano sagittale con sequenza fast - gradient t1 ad elevata risoluzione temporale ( tr / te min / 2 , 6 ; flip angle 60 ; ta 30 s ; slice 10 mm ; fov 40 cm ; matrice 224128 ; nex 1 ) , posizionata lungo il decorso dellaorta , dopo una preliminare sequenza di localizzazione sul piano coronale . 
dopo aver calcolato il tempo di circolo e pianificato le sequenze angio - rm con mdc , sono state iniettati 30 ml di gd - bopta , seguiti da 20 ml di soluzione salina alla velocit di 2 , 0 ml / s . 
questa sequenza ad impulsi caratterizzata da valori di snr e tr ottimizzati , con una fase di codifica centrica del - spazio , al fine di ottimizzare il contrasto tra le strutture vascolari e soprattutto il rapporto segnale - rumore . 
la tecnica di soppressione del grasso ( tempo di inversione = 41 ms ) non richiede altre sottrazioni digitali delle immagini con contrasto dalla maschera , ottenendo comunque la soppressione e la cancellazione delle strutture non vascolari . la rielaborazione con algoritmi 3d stata effettuata da un terzo operatore con una workstation ( advantage windows 4.0 , ge , milwaukee , il , usa ) , al fine di creare delle ricostruzioni mip e multiplanari ( mpr )  . 
le immagini mip sono state ottenute con venti rotazioni di 18 attorno allasse longitudinale in modo da coprire i 360 del volume acquisito ; le ricostruzioni mpr sono state effettuate in modo da ottenere nella stessa schermata contemporaneamente tre finestre con visione sagittale , coronale ed assiale . al fine di confermare eventuali stenosi delle arterie iliache , i pazienti sono stati inoltre sottoposti a dsa con angiografo digitale tipo integris v5000 ( philips , eindhoven , olanda ) , durante la quale stato iniettato un bolo ( 20 ml ) di mdc non iodato ( iomeron 300 bracco ) attraverso un catatere posizionato in aorta addominale sottorenale e con una velocit di infusione di 10 ml / s . 
ai pazienti con sospetta stenosi a livello dellanastomosi o della arteria iliaca esterna , durante la asd sono stati somministrati 810 ml di mdc in bolo nellarteria iliaca esterna ad una velocit di 3 ml / s . 
dsa results were regarded as the gold standard against which mra results were compared . statistical analysis statistical analyses were performed with excel ( microsoft , redmond , wa , usa )  . 
le valutazioni delle immagini sono state eseguite utilizzando un modulo cartaceo a risposte obbligate , ovvero determinando la presenza o meno della stenosi e considerando il ramo arterioso diviso in tre sezioni : pre - anastomotica , anastomotica e post - anastomotica . 
quando stata identificata una stenosi stata eseguita una misurazione della stessa ( a ) ed stato calcolato il rapporto tra il restringimento e il diametro dellarteria ( b ) nel tratto a valle pi vicino non coinvolto dalla stenosi : 100x ( 1a / b ) [ 18 , 19 ]  . 
i pazienti sono stati classificati come portatori di stenosi lieve ( 0%39% ) , moderata ( 40%69% ) o severa ( > 70% ) [ 20 ] , ma solo il riscontro di una stenosi grave stato considerato emodinamicamente significativo e quindi in tal caso lesame stato considerato positivo . 
le immagini dsa sono state valutate invece da un esperto radiologo interventista ( r.f. ) sempre mantenendo la soglia del 70% , per considerare lesame angiografico positivo . abbiamo considerato i risultati dellangiografia come gold standard e confrontato gli stessi con i risultati della angio - rm . analisi statistiche of the 49 patients enrolled , 36 underwent a single mra examination , 11 underwent two examinations and two underwent three , for a total of 64 mra examinations . 
la dsa conferma la presenza di entrambe le stenosi , quella craniale > 70% , laltra emodinamicamente non significativa . 1029 09 stecco 19 - 10 - 2007 13 : 41 pagina 1030 a . 
lanalisi finale si basata su 70 scansioni per 210 tratti vascolari e per un totale di 630 immagini ( mip , mpr , dati sorgente )  . undici pazienti ( 4 dei quali hanno ripetuto due volte lesame ) , sono stati sottoposti a 15 angiografie digitali , al fine di confermare e possibilmente trattare la stenosi . 
un esame arm stato scartato per insufficiente acquisizione dei dati che non hanno permesso la ricostruzione completa del peduncolo vascolare ; mentre un altro paziente , sottoposto a due angiografie , stato scartato dallo studio per motivi statistici in quanto la stenosi era dovuta a fistola post - bioptica . per due pazienti abbiamo considerato quattro esami ( per la presenza di due diverse stenosi e per la presenza di due arterie renali ) ; quindi un totale di 14 angiogrammi digitali renali hanno mostrato 42 sezioni vascolari ( sempre divise in pre - anastomotiche , anastomotiche , post - anastomotiche )  . 
la sensibilit , la specificit e laccuratezza diagnostica delle immagini mip , mpr e partizionali , nella determinazione della presenza e dellentit della stenosi , sono riportare nella tabella 3 : laccordo tra i due radiologi risultato elevato ( = 0 , 7976 )  . 
the latter patient was subsequently followed up by colour - doppler us and computed tomography ( ct ) , which showed that the anastomosis had not suffered damage from exposure to the magnetic field . 
the final analysis was based on 70 scans for 210 vascular segments for a total of 630 images ( mip , mpr , raw data )  . eleven patients ( four with two mra scans ) underwent 15 dsa examinations to confirm and possibly treat the stenosis . one angiogram was discarded due to inadequate data acquisition preventing complete reconstruction of the vascular pedicle ; another patient , who underwent two angiographic studies , was excluded for statistical reasons , as the stenosis was due to a postbiopsy fistula . 
sensitivity , specificity and diagnostic accuracy of the mip , mpr and raw data image sets for evaluation of the presence and severity of stenosis are shown in table 3 . 
the role of mra in the evaluation of vascular complications of the kidney graft is pazienti con complicanze post - trapianto rena , fondamentale in un momento diagnostico dove lo strumento di diagnosi deve arrecare minor danno possibile ad un parenchima renale gi sofferente ; pertanto langiografia dovrebbe essere limitata ai casi con elevato sospetto clinico e radiologico . tra le varie complicanze , vascolari e non , precoci e tardive , che possano interessare un rene trapiantato , la stenosi dellarteria renale la pi frequente [ 25 ]  . 
il ruolo dellarm nella valutazione delle complicanze vascolari del rene trapiantato noto , come anche lutilit dellabbinare una valutazione arm con tecnica a contrasto di fase , a quella a bolo di contrasto [ 22 ]  . 
tutti gli studi precedenti hanno preso in esame casistiche studiate con magneti da 1 t o 1 , 5 t ; non invece chiaro allo stato se sia utile e che impatto abbiano le immagini ottenute con magneti a basso campo ( 0 , 5 t )  . 
all previous studies have been conducted on patients imaged with 1 - t or 1.5 - t equipment , so the usefulness and impact of images obtained with low - field ( 0.5 - t ) magnets has not been clarified . 
clearly with this type of magnet , the possibility of conducting studies with phase - contrast sequences is limited by the length of the scan , which increases the time during which the patient must lie still , with motion artefacts consequently reducing overall examination quality . 
phase - contrast mra and bolus - contrast mra can both be performed on low - field mri scanners with fast - gradient sequences . bolus - contrast mra , even with a 0.5 - t unit , may be diagnostic in the evaluation of renal arteries . 
sia larm con tecnica pc che larm con tecnica a bolo di contrasto sono entrambe eseguibili con un tomografo rm a basso campo con sequenze di tipo fast - gradient . larm a bolo di contrasto pu essere diagnostica nella valutazione delle arterie renali , anche usando un magnete a 0 , 5 t . 
solo il 2 , 7% degli esami arm non sono risultati diagnostici nel nostro studio ; al contrario nel 97 , 3% dei casi siamo riusciti ad ottenere una valutazione diagnostica sullo stato delle arterie del rene trapiantato . 
in our experience , although bolus - contrast mra was not particularly specific in detecting the stenosis , it proved to have higher specificity in quantifying the lesion . as reported in the literature , raw data image sets play a fundamental role given that they are both sensitive and specific and are routinely evaluated to complete the analysis of mip images in cases of arterial dissection , nonocclusive thrombus , in the presence of metal clips and venous contamination [ 2326 ] biamo rilevato delle differenze tra le ricostruzioni mip , mpr e le immagini partizionali ( sorgente , grezze ) , in termini di identificazione della stenosi ; le immagini partizionali si sono rivelate pi sensibili ( 75% vs 62 , 5% ) , egualmente specifiche e con una maggior accuratezza diagnostica ( 75% vs 71 , 43% ) nello stabilire il grado di severit della stenosi . 
secondo la nostra esperienza , larm a bolo di contrasto non si dimostrato un esame molto specifico nellidentificare le stenosi , al contrario invece presentando una maggiore specificit nella sua quantificazione . come gi riscontrato in letteratura le immagini partizionali hanno un ruolo fondamentale , essendo sia sensibili che specifiche , e vengono routinariamente analizzate perch completano lanalisi delle mip nei casi di dissezione arteriosa , di trombo non occlusivo , in presenza di clips metalliche e in overlap venoso [ 2326 ] in conclusione , i nostri risultati dimostrano che larm 1033 09 stecco 19 - 10 - 2007 13 : 41 pagina 1034 a . 
historically , cacs was introduced with electron beam computed tomography ( ebct ) , but in the last 30 years , many changes have occurred in ct , where the development of multidetector spiral technology has made reliable the noninvasive study of the heart and coronary arteries . 
correlation studies with intravascular ultrasound ( ivus ) and histology have demonstrated the capability of multidetector ct ( mdct ) to provide information useful for characterising atherosclerotic plaque in a noninvasive manner . 
this has shifted the interest from heavily calcified deposits to plaque with a low - density core and small , superficial calcified nodules , features more frequently present in atherosclerotic plaque prone to rupture and responsible for acute coronary events ( culprit lesions )  . 
the purpose of this review article is to summarise the recent evolution and revolution in the field of ct , strengthen the importance of a coronary ct study not limited to cacs evaluation and cad grading but also used to obtain information about plaque composition , and to improve stratification of the patient at risk for acute coronary events . key words coronary artery calcium score coronary calcium screening multidetector computed tomography intravascular ultrasound riassunto la deposizione di calcio lungo le pareti delle arterie coronarie un indicatore biologico della malattia aterosclerotica e la sua quantit potrebbe riflettere la severit della malattia coronarica ( cad )  . 
in molti lavori della letteratura stato riconosciuto al coronary artery calcium score ( cacs ) un ruolo di strumento di screening della malattia coronarica ; storicamente esso stato introdotto con la electron beam - ct ( ebct ) , ma negli ultimi anni la tumultuosa evoluzione / rivoluzione tecnologica in ambito della tomografia computerizzata ( tc ) , con lo sviluppo di scanner spirali multidetettore , ha reso possibile lo studio non invasivo del cuore e delle coronarie anche con questa metodica . 
studi di correlazione con lecografia endovascolare ( ivus ) e listologia hanno recentemente documentato la possibilit di ottenere mediante tcmd ed in maniera non invasiva informazioni utili ai fini della caratterizzazione della placca aterosclerotica , spostando lattenzione dai marcati depositi di calcio alle placche coronariche con core a bassa densit e piccoli noduli calcifici superficiali , reperti pi frequentemente presenti nelle lesioni ateromasiche a rischio di rottura e responsabili , pertanto , di eventi coronarici acuti ( culprit lesion )  . 
lo scopo di questa review di riassumere levoluzione verificatasi recentemente nel campo della tomografia computerizzata , sottolineando limportanza di uno studio angiografico non invasivo delle coronarie con tcmd non limitato solamente alla semplice valutazione del cacs ed al grading delle lesioni , ma utile anche al fine di ottenere informazioni relative alla composizione della placca e migliorare , quindi , la stratificazione del paziente a rischio di evento coronarico acuto . parole chiave coronary artery calcium score screening del calcio coronarico tomografia computerizzata multidetettore ecografia intravascolare r . 
bonomo : coronary artery calcium score : has anything changed ? introduction introduzione in the last 30 years , diagnostic imaging , and in particular computed tomography ( ct ) , has been characterised by dramatic progress , regarded both as evolution and revolution . from its introduction in 1971 until 1989 , the hardware component of ct scanners evolved from the first generation of mechanical scanners , developed by g.n. 
furthermore , in the last 10 years , we have witnessed a rapid and tumultuous evolution of spiral ct scanners with the introduction and development of the multidetector spiral technology , from the first dual - slice ct scanner ( 1992 ) , through the four ( 1998 ) , eight - , 12 - , 16 ( 2003 ) , 40and currently 322 ( flying focal spot technology ) or 64 - detector - row ct scanner ( 64 - dct ) ( 20042005 )  . at the same time , this evolution has been accompanied by a revolution in the field of ct imaging , both in acquisition or reconstruction time and in image quality . 
cormack , alla tc spirale a singolo strato ( tcss ) , che ha rappresentato la vera pietra miliare sia per la tc in senso lato che per la tecnica di angio - tc . 
inoltre , nellultimo decennio siamo stati tutti testimoni della rapida e forse sin troppo tumultuosa evoluzione degli scanner tc spirale con lintroduzione e sviluppo della tecnologia multi - detettore , dai primi scanner a due strati ( 1992 ) , poi quelli a quattro ( 1998 ) , otto , dodici , sedici ( 2003 ) , quaranta , e recentemente ( 20042005 ) a 322 ( flying focal spot technology ) o 64 file di detettori ( 64tcmd )  . 
contemporaneamente , tale evoluzione stata accompagnata da una reale rivoluzione nel campo dellimaging tc , sia per quanto riguarda il tempo di acquisizione o ricostruzione delle immagini che per la qualit diagnostica di queste ultime . 
for these reasons , mandatory requisites for dedicated diagnostic hardware are high temporal and spatial resolution , an acquisition time well - matched with the apnoea compliance of the patient and dedicated and accurate ecg synchronisation . historically , cardiac ct imaging started with electron beam ct ( ebct ) [ 1117 ] , characterised by the highest temporal resolution ( 100 ms ) but with a limited spread , mostly in european countries , fundamentally due to the high cost and to a restricted application in examination of different anatomic regions ( thorax ; abdomen )  . 
one of the main results achieved by ebct in cardiac imaging has been noninvasive assessment of calcium deposits along the coronary artery walls , making noninvasive assessment of coronary atherosclerotic plaque burden reliable through measurement of a calcium score index , the agatston score ( as ) [ 18 ]  . 
given that calcium deposition is a surrogate biomarker of atherosclerotic disease , and given the strict correlation between the amount of calcium in the artery wall , disease severity , the likelihood of high - grade stenoses and the high negative predictive value ( npv ) of the score , supporters of cacs as a screening tool for coronary artery disease ( cad ) consider that cacs could be useful for evaluating subjects with more than one conventional risk factor for atherosclerotic disease [ 19 ] , for patients with atypical chest pain , for clinical decision making jointly to the conventional risk factors and treatment monitoring . 
furthermore , it has been demonstrated that a high cacs could increase the risk in patients classified as at intermediate - risk from the framingham risk score [ 20 ]  . 
some authors [ 21 ] suggest using the cacs to substitute the age factor among the conventional risk factors ; in fact , although the cacs increases with the age , age alone is not a real risk factor for coronary calcium but is a cumulative measure of exposure to conventional risk factors . prospective studies conducted on large cohorts of subjects [ 22 , 23 ] could probably explain in the coming years the real meaning and the possible utility of cacs . 
the significance of the cacs has been summarised by the expert consensus document on ebct for the diagnosis and prognosis of cad [ 36 , 37 ] , as briefly reported in table 1 . 
per tali motivi , requisiti imprescindibili di un hardware diagnostico dedicato allo studio delle coronarie sono essenzialmente una elevata risoluzione sia spaziale che temporale , una durata della scansione compatibile con la compliance respiratoria del paziente ed un dedicato e quanto pi preciso software di cardiosincronizzazione . storicamente , la cardio - tc nasce con la electron beamct ( ebct ) [ 1117 ] , caratterizzata dalla maggior risoluzione temporale ( 100 ms ) , ma con limitata diffusione territoriale , soprattutto in ambito europeo , essenzialmente per i suoi elevati costi e per la ristretta applicabilit nello studio di differenti regioni anatomiche ( torace , addome )  . 
uno dei principali risultati ottenuti con la ebct nellimaging cardiaco stata la visualizzazione dei depositi di calcio lungo le pareti delle arterie coronarie , rendendo cos possibile la valutazione non invasiva del carico aterosclerotico complessivo mediante la determinazione di uno score quantitativo del calcio presente , lo score di agatston ( as ) [ 18 ]  . 
il principale scopo del calcium score coronarico ( cacs ) la visualizzazione e quantificazione del calcio coronarico ai fini dellidentificazione dei soggetti asintomatici ad alto rischio di evento coronarico acuto . 
essendo il calcio un marcatore biologico della malattia aterosclerotica e data la stretta correlazione tra il quantitativo di calcio presente nella parete arteriosa , la severit della malattia e la probabilit di stenosi severe ed , infine , dato lelevato valore predittitivo negativo ( vpn ) dello score , i sostenitori del cacs come strumento di screening della malattia coronarica ( cad ) considerano che il cacs possa essere quindi utile nella valutazione dei soggetti con pi di un fattore di rischio ( fr ) convenzionale per malattia aterosclerotica [ 19 ] , nei pazienti con dolore toracico atipico o nellassumere particolari decisioni cliniche unitamente alla valutazione dei fr convenzionali o nel monitoraggio del trattamento ( statine )  . 
inoltre , stato dimostrato che un elevato valore di cacs possa effettivamente aumentare il rischio in quei pazienti classificati a rischio intermedio secondo lo score di framingham [ 20 ]  . 
qualcuno ipotizza [ 21 ] di impiegare il cacs come alternativa al fattore et tra i fr convenzionali della malattia aterosclerotica ; infatti , sebbene il cacs aumenti con let , la sola et non costituisce un reale fattore di rischio per laccumulo di calcio coronarico , rappresentando una misura cumulativa dellesposizione ai fr convenzionali . studi prospettici attualmente condotti su ampie coorti di soggetti [ 22 , 23 ] potranno probabilmente spiegare nei prossimi anni il reale significato e leventuale utilit clinica dello screening del calcio coronarico . 
in effect , these nonsignificant lesions are often the real culprit plaque and are responsible for the acute onset of coronary events because of the greater weakness of their components and structure . 
these details , together with the small but always present risks [ 43 , 44 ] and the high percentage ( up to 30% ) of ca examinations performed with simple diagnostic purposes and not followed by interventional procedures ( ptca / stenting ) , should prompt the search alternative , noninvasive diagnostic tools for assessing coronary arteries in well - selected patients . as the reliability of mdct in detecting and grading coronary artery stenoses has been established [ 110 ] , the challenge now is to define the ability of this diagnostic tool to distinguish coronary artery plaque prone to rupture or with findings suspected for instability and potentially responsible of acute coronary events [ 4446 ]  . 
studies using intravascular ultrasound ( ivus ) have illustrated the association between the pattern of distribution of coronary artery calcifications ( step and shot technique ) o quella spirale ( volumetric ) ; la seconda caratterizzata da una maggiore riproducibilit , essenzialmente dovuta alla minore incidenza di effetti di volume parziale , ma limitata dalla maggiore dose di esposizione del paziente [ 34 , 35 ]  . 
inoltre , in entrambe le modalit di esecuzione , la tcmd caratterizzata da maggiore riproducibilit sia inter - scan che inter - osservatore del ms versus vs e as [ 28 , 34 ] con differenze significative anche in pazienti con frequenza cardiaca 70 battiti per minuto ( bpm ) [ 29 ]  . 
modified from [ 42 ] major criteria inflammation thin fibrous cap large lipid corea superficial plaque erosion superficial platelet aggregation severe stenosis ( > 90% ) a minor criteria small superficial calcified nodulea haemorrhagic foci inside plaque endothelial dysfunction positive remodellinga shimmering yellow aassessable with multidetector computed tomography shimmer tabella 2 placca vulnerabile : criteri maggiori e minori . 
modificata da [ 42 ] criteri maggiori infiammazione sottile cappuccio fibroso criteri minori piccolo noduli calcifici superficialia foci emorragici nel contesto della placca disfunzione endoteliale rimodellamento positivoa ampio core lipidicoa erosione superficiale della placca aggregazione superficiale piastrinica riflesso giallastro stenosi severa ( > 90% ) a a valutabile con la tcmd and different clinical settings , such as acute myocardial infarction ( ami ) , unstable angina pectoris ( uap ) and stable angina pectoris ( sap ) [ 47 ]  . 
this mechanism is often responsible for the lack of symptoms , because the diameter enlargement initially prevents any significant lumen reduction and consequently preserves the distal blood flow [ 49 ] but with greater weakness of these plaques and a risk of rupture . 
otherwise , in sap the remodelling process is more frequently negative because of a fibrosing process , and it is often accompanied by significant lumen reduction , presence of symptoms and plaque stability . nowadays , state - of - the - art mdct scanners are able to acquire data with isotropic submillimetre spatial resolution [ 50 ]  . 
correlation studies with ivus and histology [ 5153 ] have demonstrated the capacity of mdct to noninvasively provide information useful for characterising atherosclerotic plaque , shifting the interest from heavy coronary calcium deposits for identification of patients at risk , to plaque with low - density core and small superficial calcified nodules , features more frequently present in atherosclerotic plaque prone to rupture and therefore responsible for acute coronary events . affette da stenosi non significative , in particolare con stenosi 70% . 
in effetti , tali lesioni non emodinamicamente significative sono nella maggioranza dei casi le reali culprit lesion ( lesioni colpevoli ) , responsabili dellinsorgenza acuta dellevento coronarico a causa della maggiore debolezza / friabilit delle loro componenti e struttura . 
le caratteristiche delle placche a rischio di rottura sono state precedentemente ed estesamente descritte [ 42 ] con la distinzione di criteri maggiori e minori ( tabella 2 )  . 
questi particolari in associazione ai rischi , rari ma pur sempre presenti [ 43 , 44 ] e alla significativa percentuale ( sino al 30% ) di esami coronarografici eseguiti per semplice finalit diagnostica e non seguiti pertanto da procedure interventistiche ( ptca / stenting ) dovrebbero obbligare , in pazienti adeguatamente selezionati , a cercare alternative diagnostiche non invasive per la valutazione del circolo coronarico . essendo stata gi definita la possibilit di visualizzare e definire lentit delle stenosi coronariche mediante tcmd [ 110 ] , la sfida pi ambiziosa si sposta attualmente alla definizione della capacit di tale metodica diagnostica di distinguere tra le placche coronariche quelle a rischio di rottura / complicanza o con reperti sospetti per instabilit e , quindi , potenzialmente responsabili di eventi coronarici acuti [ 4446 ]  . 
in studi condotti con ecografia endovascolare ( ivus ) , stata dimostrata lassociazione tra pattern di distribuzione delle calcificazioni coronariche e differenti quadri clinici , quali linfarto miocardico acuto ( ami ) , langina pectoris instabile ( uap ) e quella stabile ( sap ) [ 47 ]  . 
inoltre , il processo aterosclerotico negli eventi coronarici acuti frequentemente accompagnato da fenomeni di rimodellamento positivo del vaso [ 48 ] ; tale meccanismo spesso responsabile della mancanza di sintomatologia in quanto laumento del diametro vasale complessivo limita inizialmente leffetto della placca ateroslcerotica sullemodinamica locale , preservando cos distalmente il flusso ematico [ 49 ] , ma la debolezza delle componenti della placca stessa sono alla base del rischio di rottura e complicanza . 
4 intravascular ultrasound studies [ 47 ] have described the association between the distribution pattern of coronary artery calcifications and different clinical settings , such as acute myocardial infarction ( ami ) , unstable angina pectoris ( uap ) and stable angina pectoris ( sap )  . 
4 stato riportata [ 47 ] , da studi condotti mediante ecografia intravascolare ( ivus ) , lassociazione tra diversi pattern di distribuzione del calcio coronarico e differenti quadri clinici quali linfarto miocardico acuto ( ami ) , langina pectoris instabile ( uap ) e langina pectoris stabile ( sap )  . 
the multidetector computed tomography ( mdct ) curved multiplanar reconstruction ( mpr ) ( b ) of the cx shows lesion length , whereas axial reconstructions ( 13 ) of the vessel demonstrate the eccentric noncalcified plaque and severe lumen reduction ( arrowheads )  . 
la ricostruzione mpr curvata ( b ) della cx ben documenta la lunghezza della lesione , mentre le immagini ricostruite perpendicolarmente allasse del vaso ( 13 ) documentano la placca eccentrica non calcifica e la marcata riduzione del lume residuo ( teste di freccia )  . 
6a - e curved multiplanar reconstruction ( mpr ) image ( a ) of a concentric , noncalcified plaque in the proximal segment of the anterior descending artery ( ada ) ( arrowheads ) distally treated 1 year earlier with percutaneous transluminal coronary angioplasty ( ptca ) and stenting . 
the mpr image axial to the vessel ( b ) shows lumen reduction and the presence of a concentric , noncalcified tissue ( arrowheads ) around the residual vessel lumen . 
mpr reconstruction ( c ) of the circumflex coronary artery ( cx ) with a segmental mixed atherosclerotic lesion , as demonstrated by the axial image ( d ) with eccentric calcified nodule and small amount of noncalcified tissue ( arrowhead ) between calcium and residual lumen . 
limmagine ricostruita perpendicolarmente allasse del vaso ( b ) ben documenta la riduzione del lume e la presenza di tessuto concentrico ipodenso non calcifico ( teste di freccia ) attorno al lume residuo opacizzato . 
ricostruzione mpr - curvata della ada ( e ) con tre lesioni eccentriche non significative composte da noduli calcifici superficiali ( frecce )  . table 3 computed tomography ( ct ) density value of different components of atherosclerotic plaque feature ct density value ( hu ) recent thrombus lipid plaque fibrous plaque calcium plaque ~100 > 130 tabella 3 placca calcifica > 130 spostato linteresse , ai fini dellidentificazione del paziente a rischio , dai marcati depositi di calcio lungo le pareti delle coronarie alle placche con core ipodenso ( core lipidico ) e piccoli noduli calcifici superficiali , reperti maggiormente presenti nelle placche a rischio di rottura / complicanza e , quindi , responsabili dellinsorgenza di eventi coronarici acuti . 
 conclusione in conclusione , diversamente da quanto si pensasse in precedenza quando per la visualizzazione delle coronarie era a disposizione solamente la ebct ed essa era utilizzata essenzialmente per la valutazione del quantitativo di calcio , r . 
bonomo : coronary artery calcium score : has anything changed ? conclusion in conclusion , unlike in the past when the only available means to visualise coronary arteries was ebct which was used to assess and screen for coronary calcium , the use of mdct should probably induce us to change some of our opinions on the basis of new features . 
in fact , mdct with retrospective ecg gating has made the noninvasive study of coronary arteries reliable , with a real role in clinical practice mostly thanks to its high npv for the detection of significant cad . 
in the near future , it could probably play an important clinical role for a more accurate stratification of atherosclerotic risk , no longer simply based on visualisation and quantification of calcium deposits but prevalently based on direct , noninvasive visualisation of coronary plaque and characterisation of their components . limpiego dei nuovi scanner tcmd dovrebbe probabilmente indurci a modificare alcuni concetti sulla base dei nuovi reperti che siamo attualmente in grado di ricercare nel corso dello studio angio - tc del circolo coronarico . 
sardanelli universit degli studi di milano , dipartimento di scienze medico - chirurgiche , servizio di radiologia , irccs policlinico san donato , via morandi 30 , i - 20097 san donato milanese , milano , italy correspondence to : a . 
at the department of radiology of the policlinico san donato ( university of milan ) , breast mr is routinely performed at 1.5 t as follows : 36 - slice axial 2d shorttime inversion - recovery ( stir ) sequence ; 128 - partition 3d gradient - echo coronal sequence ( 1 - mm3 isotropic voxel ) before and after rapid automatic intravenous injection of 0.1 mmol / kg of gd - dota ( one precontrast and four postcontrast phases )  . postprocessing includes temporal subtraction ( postcontrast minus precontrast ) , maximum intensity projections ( mips ) , percent enhancement - to - time curves for small regions of interest , and axial and / or sagittal multiplanar reconstructions . 
we select only four mips of an early subtracted dynamic phase : one axial similar to craniocaudal x - ray mammographic views , one coronal , and two lateral similar to lateral 90 x - ray mammographic views . 
for each lesion described in the report , we select five items , including three images , one graph , and one table : stir image , precontrast and subtracted postcontrast images ( morphology ) , percent enhancement - to - time curves and a table of raw data generating the curves ( dynamics )  . 
the selected items range usually from four ( no detected lesion ) to 14 ( one lesion , studied also with 1h - mrs ) , to 34 ( five lesions , one of them studied also with 1h - mrs )  . 
presso il nostro dipartimento di radiologia le indagini di rm mammaria sono routinariamente effettuate a 1 , 5 t secondo il seguente protocollo : sequenza assiale 2d short - time inversion - recovery ( stir ) , 36 strati ; sequenza coronale 3d gradient - echo ( 128 partizioni ; voxel isotropico , 1 mm3 ) prima e dopo somministrazione automatica rapida di 0 , 1 mmol / kg di gd - dota ( una fase precontrasto e quattro postcontrasto )  . 
 il postprocessing include : sottrazione temporale ( postcontrasto meno precontrasto ) , ricostruzioni con algoritmo di massima intensit ( mip ) , curve di enhancement percentuale in funzione del tempo per piccole regioni di interesse e ricostruzioni multiplanari assiali e / o sagittali . 
tale protocollo genera oltre 1200 immagini per indagine . tra queste operiamo una ridotta selezione che include quattro mip di una fase dinamica precoce con sottrazione : una assiale simile alle proiezioni mammografiche cranio - caudali , due laterali simili alle proiezioni mammografiche laterali 90 e una coronale . per ciascuna lesione descritta nel referto selezioniamo cinque oggetti che includono tre immagini , un grafico e una tabella : immagine stir , immagini precontrasto e postcontrasto sottratte ( valutazione morfologica ) , curve di enhancement percentuale in funzione del tempo e tabella dei dati grezzi che generano le curve ( valutazione dinamica )  . 
se stata effettuata spettroscopia protonica , si aggiungono altri 5 oggetti : due spettri elaborati ( valutazione metabolica ) e le tre immagini di localizzazione del volume di interesse spettroscopico . 
il numero degli oggetti selezionati oscilla usualmente da quattro ( nessuna lesione evidenziata ) a 14 ( una lesione , studiata anche con spettroscopia protonica ) , a 34 ( cinque lesioni , una delle quali studiata anche con spettroscopia protonica )  . 
la rm mammaria pu essere sintetizzata presentando solo una minima frazione di tutte le immagini generate . parole chiave mammella risonanza magnetica ( rm ) spettroscopia a risonanza magnetica introduction introduzione magnetic resonance ( mr ) imaging of the breast has gained an important role in breast cancer diagnosis thanks to a very high sensitivity for at least five important indications : presurgical local staging and treatment planning , suspected recurrence in the operated breast , evaluation of the effect of neoadjuvant therapy , search for occult primary breast cancer with known metastases , and surveillance of women at high genetic - familial risk of breast cancer [ 1 ]  . 
however , some authors propose proton mr spectroscopy ( 1h - mrs ) to further increase the ability to characterise the lesion , with a gain in specificity [ 36 ]  . in breast mr , we daily face a general problem for crosssectional imaging . 
the development of hardware , software , and more and more sophisticated postprocessing techniques enable us to get native , postprocessed , and reconstructed images , including three - dimensional ( 3d ) representations . these are of great interest not only for radiologists but also for surgeons , radiation therapists , and oncologists , as it has been shown for fields other than mr or breast diseases [ 79 ]  . 
in our opinion , the key point is the ability of radiologists to manage a great deal of information ( i.e. , a huge number of images ) produced during a state - of - the - art examination , in order to provide other physicians selected information as an efficient clinical synthesis . 
 we present here a proposal to summarise a complete breast mr examination presenting contrast - enhanced breast 3d maximum intensity projections ( mips ) and a reduced number of images containing morphologic , dynamic , and metabolic information of lesions described in the report . la rm mammaria ha ormai acquisito un ruolo rilevante nella diagnosi dei tumori mammari in virt della sua elevata sensibilit . 
almeno cinque importanti indicazioni sono definite : stadiazione prechirurgica e pianificazione del trattamento ; sospetta recidiva nella mammella operata ; valutazione delleffetto della terapia neoadiuvante ; ricerca di tumore mammario primitivo occulto in paziente con note metastasi ; sorveglianza delle donne ad alto rischio genetico - familiare di tumore mammario [ 1 ]  . 
sebbene lintegrazione di parametri morfologici e dinamici consenta di ottenere livelli relativamente elevati di specificit [ 2 ] , alcuni autori propongono lutilizzo della spettroscopia protonica per elevare le capacit di caratterizzazione lesionale con un guadagno in specificit [ 36 ]  . in rm mammaria ci troviamo ad affrontare quotidianamente un problema generale per le attuali tecniche tomografiche . 
lo sviluppo di hardware , software e tecniche di postprocessing sempre pi sofisticate ci consente di ottenere immagini native , elaborate e ricostruite comprese le rappresentazioni tridimensionali ( 3d ) di grande interesse non solo per i radiologi ma anche per i chirurghi , i radioterapisti e gli oncologi , come stato dimostrato anche in ambiti differenti da quello rm e delle applicazioni senologiche [ 79 ]  . 
1h - mrs is performed with one ( rarely two or three ) single - voxel point - resolved spin - echo sequence with spectral lipid and water suppression ( tr / te 1 , 500 / 136 ms ; bandwidth 1 khz ; 512 excitations ; acquisition time 13 min )  . 
1h - mrs postprocessing is performed using relatively standardised protocols , including fourier transform , filters ( exponential , gaussian , hennings , rectangular ) , phase and baseline correction , and curve fitting aimed at calculating the area under the investigated compound peaks . 
identification of the different compound peaks is obtained considering the position of their central frequency on the parts per million ( ppm ) axis . thus , at the end of the examination , we have generated 36 predynamic images , 640 native and 512 subtracted images of the dynamic study , and a variable number of multiplanar reconstructions , for a total of more than 1 , 200 images . 
spettroscopia protonica : una ( raramente due o tre ) acquisizioni con sequenza a voxel singolo point resolved spin - echo con soppressione spettrale dei lipidi e dellacqua ( tr / te , 1500 / 136 ms ; banda passante , 1 khz ; 512 eccitazioni ; tempo di acquisizione , circa 13 minuti )  . 
il postprocessing del segnale spettroscopico avviene mediante luso di protocolli relativamente standardizzati che includono la trasformata di fourier , lutilizzo di filtri ( esponenziale , gaussiano , di henning , rettangolare ) ; la correzione della fase e della linea di base ; la costruzione di una curva finalizzata al calcolo dellarea sottesa dal picco dei composti indagati . 
il riconoscimento dei picchi avviene in funzione della frequenza centrale sullasse delle parti per milione ( ppm )  . alla fine dellindagine , abbiamo cos generato 36 immagini predinamiche , 640 immagini native e 512 sottratte dello studio dinamico e un numero variabile di ricostruzioni multiplanari per un totale di oltre 1200 immagini . 
breast magnetic resonance imaging is presented using only maximum intensity projections of the first subtracted images ( a axial ; b coronal ; c right lateral ; d left lateral )  . 
conclusioni del referto : nessun focolaio sospetto di contrast enhancement in entrambe le mammelle . we select a small series of images that can be printed or stored on a digital support , e.g. 
3d mips of the optimal ( considering the best visualisation of the possible lesions ) dynamic subtracted sequence ( 4in - 1 format , if they are printed ) : a . 
selected images for each detected lesion specifically described in the report ( from 20 - in - 1 to 30 - in - 1 film format , if they are printed ) : a . 
immagini selezionate per ciascuna lesione individuata e specificatamente descritta nel referto ( formato da 20 - in - 1 a 30 - in - 1 , se sono stampate ) : a . 
2a - d a 65 - year - old asymptomatic woman with positive x - ray mammography and ultrasound in the lower inner quadrant of the right breast ( not shown )  . 
breast magnetic resonance imaging maximum intensity projections are presented ( a axial ; b coronal ; c right lateral ; d left lateral ) , with multiple foci of contrast enhancement in the lower inner quadrant of the right breast and a small focus of contrast enhancement in the lower inner quadrant of the left breast . 
le mip della rm dinamica ( prima fase contrastografica sottratta ; a , assiale ; b , coronale ; c , laterale destra ; d , laterale sinistra ) evidenziano multipli focolai di contrast enhancement al quadrante infero - interno della mammella destra e un piccolo focolaio di contrast enhancement al quadrante infero - interno di sinistra . 
postprocessed proton spectrum without ( f1 ) and with ( f2 ) calculation of the integral under the choline - containing compounds peak , obtained for the volume of interest including the largest malignant focus located on sagittal ( f3 ) , coronal ( f4 ) and axial ( f5 ) reference images . 
comment : by integrating morphologic and dynamic criteria , the five foci in the right breast were classified as malignant lesions , whereas the focus in the left breast was classified as a probably benign lesion . 
sar tuttavia opportuno che il clinico sia guidato alla valutazione di un limitato numero di immagini selezionate dal radiologo . results risultati the selected items usually range from four ( no detected lesion ) , to 14 ( one lesion , studied with 1h - mrs also ) , to 34 ( five lesions , one studied with 1h - mrs also )  . 
the percentage of items presented with the report compared with the total number of generated images is equal to 0.33% ( 4 / 1 , 200 ) , 1.17% ( 14 / 1 , 200 ) , and 2.83% ( 34 / 1 , 200 ) , respectively . 
thus , breast mr imaging and 1h - mrs can be effectively summarised by presenting a minimal fraction of all generated images . il numero degli oggetti selezionati oscilla usualmente da quattro ( nessuna lesione evidenziata ) a 14 ( una lesione , studiata anche con spettroscopia ) , a 34 ( cinque lesioni , una delle quali studiata anche con spettroscopia )  . 
la percentuale di oggetti presentati rispetto al totale delle immagini pari allo 0 , 33% ( 4 / 1200 ) , 1 , 17% ( 14 / 1200 ) e 2 , 83% ( 34 / 1200 ) , rispettivamente . la rm mammaria ( imaging dinamico e spettroscopia ) pu quindi essere efficacemente sintetizzata presentando solo una minima frazione di tutte le immagini generate . discussion using cross - sectional high - resolution imaging techniques , an heavy bulk of images is increasingly obtained . 
the challenge is to convey the diagnostic message to other physicians selecting a small number of images . subtracted contrast - enhanced 3d mips of the breasts can easily and quickly give a general evaluation of breast vasculature and show possible parenchymal foci of contrast enhancement . 
in our opinion , this approach manages to link the new cross - sectional imaging modality , mr , to a known background ( xray mammography is the standard imaging modality of reference for all physicians dealing with breast diseases ) , adding the coronal view to complete the 3d information . moreover , using an isotropic ( 1 - mm3 voxel ) imaging protocol , the same high spatial resolution is obtained in lateral and axial mips . 
finally , we should take into account that an asymmetrical increased breast mip map was demonstrated to be frequently associated with ipsilateral invasive cancers [ 11 , 12 ]  . when one ore more enhancing foci is specifically evaluated and reported , we present a small selected series of images demonstrating its morphology , dynamics , and metabolismorphology ( shape , borders , signal intensity , and contrast material internal uptake distribution ) is displayed 1066 discussione lutilizzo di tecniche tomografiche ad alta risoluzione genera sempre pi frequentemente un enorme numero di immagini . 
la sfida che occorre raccogliere riuscire a trasmettere il messaggio diagnostico operando la selezione di un numero di immagini molto pi ridotto . le proiezioni mammarie mip 3d delle scansioni contrastografiche sottratte forniscono una rappresentazione rapida e semplificata che rende possibile una valutazione generale della vascolarizzazione mammaria e il riconoscimento di eventuali focolai parenchimali di contrast enhancement . 
a nostro avviso , questo approccio riesce a collegare la nuova modalit senologica tomografica , la rm , a un riferimento ben noto ( la mammografia rimane la tecnica standard per tutti gli specialisti dellambito senologico ) , aggiungendo altres la proiezione coronale a completamento dellinformazione 3d . peraltro , utilizzando un protocollo di studio dinamico isotropico con voxel di 1 - mm3 , si ottiene la medesima elevata risoluzione spaziale anche nelle mip laterali e assiale . 
le immagini mip possono essere concepite come mappe che si aggiungono alla mammografia , soprattutto in caso di discordanza dimensionale del parametro t ( es . : in presenza di un tumore invasivo associato a estensiva componente intraduttale ) o come strumento esclusivo , quando la lesione mammograficamente occulta e visibile solo alla rm . 
metabolism is displayed through the 1h - mrs spectrum associated with three - plane orthogonal images localising the voi . due to the main commitment to cancer detection in breast imaging , it is not surprising that when no suspicious or clinically relevant abnormality is found , only four images can summarise 1 , 192 precontrast and dynamic images : four localizer , 36 predynamic , 640 native , and 512 subtracted of the dynamic study ( note that if multiplanar reconstructions are obtained , a total of 1 , 200 images is reached and probably outnumbered )  . 
 at our hospital , surgeons and oncologists have showed a progressive interest in breast mr after looking at this small series of images we select and present to thepreviously , they were discouraged with the large number of films they received for a breast mr examination . 
none of the colleagues , with the exception of radiologists dedicated to breast mr , can extract clinically useful information from this bulk of data in a small time interval , even if a highquality mr study is done ( the higher examination quality , probably the larger number of images )  . 
printed on films , stored on cd - rom , or also sent through pacs , only a reduced number of selected images can be useful to surgeons , oncologists , radiotherapists , or other physician dealing with breast diseases . the radiologists ability to recognise and characterise breast lesions with mr on the basis of morphology , dynamics , and metabolism must be made available to other physicians while offering a high level of synthesis . 
daltra parte cinque - dieci immagini sono ampiamente sufficienti per mostrare le caratteristiche utili alla caratterizzazione di ciascuna eventuale lesione . chirurghi e oncologi del nostro ospedale hanno mostrato un progressivo interesse verso la rm mammaria osservando soltanto la ridotta serie di immagini che non presentiamo loro . in precedenza , essi si trovavano in difficolt di fronte al grande numero di immagini ( e di pellicole ) che ricevevano per una indagine rm mammaria . 
linvio al clinico di tutta la serie completa delle immagini troppo e troppo poco al tempo stesso : nessuno dei colleghi , con la sola eccezione dei radiologi con esperienza in rm mammaria , sarebbe in grado di estrarre informazione clinicamente utile in tempi ragionevoli da questo ammasso di dati , anche se lindagine rm fosse di elevata qualit ( anzi , pi elevata la qualit dellindagine , tendenzialmente maggiore sar il numero delle immagini )  . 
stampate su pellicola , archiviate su cd - rom o anche inviate mediante pacs , solo un ridotto numero dimmagini sono utili a chirurghi , oncologi , radioterapisti o agli altri colleghi dellambito senologico . labilit del radiologo nel riconoscere e caratterizzare le lesioni mammarie con rm in base a criteri morfologici , dinamici e metabolici deve essere messa a disposizione dei colleghi offrendo un elevato livello di sintesi . 
between march 2001 and june 2003 , cmi was performed on 40 patients ( 27 men and 13 women , age 2358 years , median 41 years ) affected by irreparable medial meniscal lesions . 
all patients underwent mri follow - up at 6 months and 1 year and 16 patients 2 years after the operation ; 12 patients underwent second - look arthroscopy with implant biopsy . all mri examinations were performed with a 1.5 - t unit using ge t2 * , spin - echo ( se ) t1 , and fatsat fast spin - echo ( fse ) dp and t2 - weighted sequences , with different orientations . 
mri follow - up at 6 months showed cmi shape and size to be normal ( type 3 ) in 35 / 40 patients and type 2 in 5 / 40 patients . cmi signal intensity was type 1 in 32 / 40 patients and type 2 in 8 / 40 . 
at 24 months , cmi size was type 3 in 9 / 16 patients , type 2 in 6 / 16 , and type 1 in one patient in whom the implant could not be identified , as it had been totally resorbed . 
quaranta pazienti , 27 maschi e 13 femmine ( di et compresa tra 23 e 58 anni , et mediana : 41 anni ) , sottoposti a impianto di menisco protesico mediale per via artroscopica , hanno eseguito follow - up con rm a 6 mesi e a 1 anno e solo 16 pazienti a 2 anni dallintervento ; in 12 casi stato eseguito second look artroscopico con prelievo bioptico dellimpianto . 
in 2 / 40 pazienti vi era versamento reattivo sinoviale . nel controllo a 12 mesi la morfologia e le dimensioni dellimpianto erano normali ( tipo 3 ) in 33 / 40 pazienti , mentre erano di tipo 2 in 7 / 40 . 
nel controllo a 24 mesi in 9 / 16 pazienti le dimensioni del cmi erano di tipo 3 , in 6 / 16 di tipo 2 ; in 1 pazienti limpianto non risultava identificabile , totalmente patients . 
la rm consente di monitorare nel tempo le variazioni morfologiche e strutturali del cmi , riteniamo che il follow - up possa essere prolungato altre i 2 anni , fino alla stabilizzazione del cmi e alla completa risoluzione delle aree di edema della spongiosa ossea subcondrale . 
nellunico caso di evoluzione sfavorevole del cmi non si sono individuati segni diretti o indiretti correlabili . parole chiave ginocchio impianto di menisco collagenico risonanza magnetica artro - rm menisco operato introduction introduzione meniscal fibrocartilage is a cushioning tissue essential for joint stability in that it controls the transmission of forces and distributes loads across the joint [ 1 , 2 ]  . 
the best treatment for meniscal injury is to preserve the meniscus by suturing the lesion . however , in many cases , meniscal injuries are irreparable and require partial or total meniscectomy [ 4 ]  . 
a tissue engineering technique , however , has provided a prosthetic material with ideal properties : a biocompatible absorbable collagen meniscus derived from bovine achilles tendons , which promotes new meniscal tissue formation [ 813 ]  . the benefits of magnetic resonance imaging ( mri ) in the diagnosis and follow - up of meniscal injuries have already been described in the literature [ 1418 ]  . 
the purpose of our study was to identify mri patterns that could be used to define the evolution of collagen meniscal implants ( cmi ) and correlate them with the clinical and histological data ( from patients undergoing second - look arthroscopy )  . la fibrocartilagine meniscale un interpositore indispensabile alla stabilit articolare , controlla la trasmissione delle forze e distribuisce il carico dellarticolazione [ 1 , 2 ] , ma noto da molto tempo che la perdita di materiale meniscale porta a progressiva degenerazione condrale ed osteocondrale con grave alterazione della funzionalit articolare [ 1 , 35 ]  . 
tuttavia in molti casi le lesioni meniscali non sono riparabili pertanto si esegue una meniscectomia parziale o totale [ 4 ] : per questo motivo la sostituzione del tessuto meniscale asportato con un menisco protesico potrebbe essere una soluzione per prevenire i fenomeni degenerativi [ 6 , 7 ]  . 
con una tecnica di ingegneria tissutale stato ottenuto un materiale protesico con caratteristiche ottimali : un menisco collagenico , derivato da tendine dachille bovino , biocompatibile e riassorbibile che induce la formazione di nuovo tessuto meniscale [ 813 ]  . sono state gi descritte in letteratura i vantaggi della risonanza magnetica ( rm ) nella diagnosi e nel follow - up delle lesioni meniscali [ 1418 ]  . 
scopo del nostro lavoro individuare patterns rm idonei a definire levoluzione dellimpianto collagenico ( cmi ) , comparati con i dati clinici ed istologici ( ottenuti nei pazienti sottoposti a second - look artroscopico )  . materials and methods materiali e metodi mri follow - up was performed on 40 patients ( 27 men and 13 women ; age range 2358 years ; median age 41 years ) treated with bovine collagen meniscus implants positioned via arthroscopy . 
in all cases , a medial prosthetic meniscus was implanted , in the right knee in 24 pail follow - up con risonanza magnetica ( rm ) stato eseguito in 40 pazienti di et compresa tra 23 e 58 anni ( et mediana 41 anni ) , 27 maschi e 13 femmine , sottoposti a impianto di menisco collagenico bovino ( cmi ) per via artroscopica . 
twenty - one patients underwent combined surgical procedures [ 16 anterior cruciate ligament repairs ; two microfractures to treat chondral lesions of the patella and internal femoral condyle , respectively ; two autologous chondrocyte implantations in the medial femoral condyle ( mfc ) ; one valgus tibial osteotomy ]  . all patients underwent clinical and mri follow - up at 6 months and 1 year ; only 16 patients were followed up for 2 years . 
patient selection for second - look arthroscopy was based on clinical findings ( persisting pain ; joint trauma with possible damage to the implant ) and the need for an arthroscopic procedure for associated disorders ( harvesting and implantation of autologous chondrocytes ; removal of tibial plateau from valgus osteotomy )  . 
on the basis of these characteristics , we identified three patterns that were classified from 1 to 3 , with higher scores reflecting patterns more closely resembling those of the normal meniscus ( table 1 )  . 
in 21 casi stata eseguita chirurgia associata ( 16 ricostruzioni del legamento crociato anteriore ; 2 microfratture per trattamento di patologia condrale rispettivamente rotulea e del condilo femorale interno ; 2 impianti di condrociti autologhi a livello del condilo femorale mediale ; 1 osteotomia tibiale devarizzante )  . tutti i pazienti sono stati sottoposti a follow - up clinico e radiologico , mediante esame rm a 6 mesi e a 1 anno , solo 16 pazienti sono rientrati nel follow - up a 2 anni dallintervento . 
la selezione dei pazienti da sottoporre al second look stata effettuata in base a dati clinici ( persistenza della sintomatologia dolorosa ; trauma dellarticolazione con possibile lesione dellimpianto ) e in base alla necessit di sottoporre i pazienti ad un intervento artroscopico per patologia associata a quella del menisco ( prelievo e impianto di condrociti autologhi ; rimozione di placche tibiali da osteotomia devarizzante )  . 
in base a queste caratteristiche abbiamo individuato tre tipi di patterns che abbiamo classificato da 1 a 3 , con valore tanto pi alto quanto pi queste si avvicinano alle caratteristiche del menisco normale ( tabella 1 )  . 
per quanto riguarda i criteri indiretti abbiamo preso in considerazione lo stato del rivestimento condrale nella sede dellimpianto ( condilo femorale e piatto tibiale rispettivamente interno ed esterno ) stimando il grado di erosione cartilaginea considerando una percentuale superiore o inferiore al 50% e leventuale presenza di segni di sofferenza a carico della spongiosa ossea subcondrale corrispondente allimpianto . 
tutte le valutazioni sono state elaborate , indipendentemente , da due operatori ( eg , mga )  . analisi statistica nel confronto di proporzioni , visto il numero ridotto di casi , per la valutazione della morfologia del cmi si utilizzato il test esatto di fisher . 
nel confronto di proporzioni di tabelle superiori a 22 si fatto ricorso al test 2 per la valutazione dellintensit di segnale del cmi in tutti i controlli . risultati follow - up a 6 mesi nel controllo rm a 6 mesi stato evidenziato il cmi in tutti i casi esaminati . 
sono stati identificati 3 casi ( 7 , 5% ) di patologia condrale a livello del condilo femorale mediale , in 1 caso associata a lesione condrale a carico del piatto tibiale corrispondente . 
in 1 / 40 si osservato esteso edema della spongiosa del condilo femorale esterno , non in relazione allimpianto di pertinenza del menisco interno ; lalterazione di segnale stata considerata come algodistrofia , totalmente regredita gi al controllo a 12 mesi . 
collagen meniscus implant ( cmi ) size is identical to that of the normal meniscus : type 3 ( arrow ) ; the sagittal images obtained with fat - suppressed t2 / dp show an inhomogeneous and marked increase in signal intensity : type 1 ( arrow )  . 
b same patient : follow - up at 12 months . on the t2 / dp - weighted sagittal images with fat suppression , scaffold size is type 3 ( arrow ) , but signal intensity is reduced compared with the previous examination ; signal characteristics are type 2 ( arrow )  . 
scaffold size is still type 3 ( arrow ) , but signal intensity is reduced with respect to the examination at 12 months : signal characteristics are type 3 ( arrow )  . 
il cmi presenta dimensioni sovrapponibili a quelle del menisco normale : tipo 3 ( freccia ) , nelle immagini sagittali ottenute in t2 / dp con soppressione del grasso si osserva disomogeneo e marcato aumento dellintensit di segnale : tipo 1 ( freccia )  . 
nelle immagini sagittali ottenute in t2 / dp con soppressione del grasso il complesso mostra dimensioni di tipo 3 ( freccia ) , ma si osserva riduzione dellintensit di segnale rispetto al controllo precedente : le caratteristiche di segnale sono di tipo 2 ( freccia )  . 
il complesso mantiene dimensioni di tipo 3 ( freccia ) , ma si osserva riduzione dellintensit di segnale rispetto al controllo a 12 mesi : le caratteristiche di segnale sono di tipo 3 ( freccia )  . 
nelle immagini sagittali se t1 ottenute dopo iniezione intra - articolare di mdc il cmi mostra dimensioni di tipo 3 ( freccia ) , limpianto integrato , non vi infiltrazione di mdc . showed subchondral bone marrow oedema , which had completely regressed in 5 / 8 patients . 
in the t2 / dp sagittal images with fat suppression , collagen meniscus implant ( cmi ) size is type 3 , and the signal intensity has inhomogeneously increased , reaching type 1 ( white arrow )  . 
nelle immagini sagittali ottenute in t2 / dp con soppressione del grasso le dimensioni del cmi sono di tipo 3 e lintensit di segnale disomogeneamente aumentata , di tipo 1 ( freccia bianca )  . 
il complesso mostra riduzione delle dimensioni del cmi e diviene di tipo 2 ( freccia bianca ) , ma si osserva riduzione delle dimensioni del cmi che diviene di tipo 2 ( freccia bianca )  . 
3a , b on the t2 / dp - weighted and fat - suppressed coronal images , the interface between the prosthetic meniscus and the native meniscus is indicated by a sharp hyperintense line both at 6 ( white arrows in a ) and 12 ( white arrows in b ) months . 
a 12 mesi in nessun caso abbiamo riscontrato reazione sinoviale ( tabella 3 )  . follow - up a 24 mesi nel controllo a 24 mesi la valutazione relativa alla morfologia e allintensit di segnale dellimpianto stata effettuata in base alla valutazione combinata dellesame rm convezionale e dellartro - rm . 
in 11 / 15 pazienti il complesso presentava caratteristiche di segnale di tipo 2 , mentre in 4 / 15 lintensit del complesso era sovrapponibile a quella del menisco normale , di tipo 3 ( fig . 6a ) , confermando la progressiva riduzione dellintensit di segnale osservata al controllo precedente . 
attraverso liniezione di mdc intra - articolare abbiamo potuto stimare il grado di erosione in 1 / 5 > 50% , sottostimato allindagine basale ( fig . 7a , b ) e in 4 / 5 < 50% . 
in 11 / 15 patients , the complex showed type 2 signal characteristics , whereas in 4 / 15 , signal intensity was identical to that 1042 table 3 indirect signs : results at 6 , 12 , and 24 months follow - up chondral pathology subchondral bone marrow oedema synovial reaction e . 
in no case was synovial reaction observed ( table 3 )  . in the 12 patients who underwent second - look arthroscopy and biopsy , a progressive reduction in implant size ( from 25% to 90% ) was identified in the posterior horn region . 
in nessun caso si osservata reazione sinoviale ( tabella 3 )  . nei 12 pazienti sottoposti a second look artroscopico con prelievo bioptico si rilevata una progressiva riduzione delle dimensioni ( da un minimo del 25% ad un massimo del 90% ) dellimpianto a livello del corno posteriore . 
solo in 1 paziente limpianto risultava totalmente riassorbito ( a conferma del rilievo rm ) , con frammenti liberi della protesi . per quanto riguarda lanalisi statistica dei nostri risultati , nella valutazione della morfologia del cmi il test esatto di fisher non ha evidenziato differenze statisticamente significative tra 6 e 12 mesi , mentre la differenza stata significativa tra 6 e 24 mesi ( p = 0 , 033 )  . 
nellanalisi della differenza della sofferenza sottocondrale i dati a 6 mesi confrontati con quelli a 12 mesi , valutati con il test esatto di fisher , mostrano un trend di significativit ( p = 0 , 09 )  . discussione le lesioni meniscali sono di frequente riscontro , rappresentano circa i 2 / 3 di tutte le lesioni del ginocchio , spesso associate a patologie capsulo - legamentose e cartilaginee [ 3 , 18 ]  . in passato la meniscectomia mirava a risolvere la sintomatologia dolorosa , ma nel tempo larticolazione era sottoposta ad anomali carichi sulle superfici articolari e conseguente degenerazione cartilaginea che culminava osteoartrosi [ 4 ]  . 
nel 1992 stato proposto per la sperimentazione sulluomo il cmi che ha dimostrato , dai primi studi a medio termine , risultati incoraggianti [ 3 , 611 , 20 , 21 ]  . 
t2 / dp - weighted sagittal scan with fat suppression : both size and signal intensity are unchanged ( white arrow ) , and a sharp interface can be seen between the prosthetic and native meniscus ( small arrows )  . 
scansione sagittale t2 / dp con soppressione del grasso : le dimensioni del cmi sono di tipo 2 e lintensit di segnale di tipo 2 ( freccia bianca )  . 
scansione sagittale t2 / dp con soppressione del grasso : sia le dimensioni che lintensit di segnale sono invariati ( freccia bianca ) , si evidenzia netta interfaccia tra il menisco protesico e il menisco nativo ( frecce piccole )  . 
in the past , meniscectomy was used to treat pain , but with time , the discussion 1044 cmi ha lo scopo di ripristinare la perdita di sostanza meniscale e favorire i processi di rigenerazione meniscale in caso di meniscectomia parziale [ 711 , 13 , 20 , 21 ]  . 
a t2 / dp - weighted sagittal image with fat suppression : collagen meniscus implant ( cmi ) signal intensity is identical to that of the normal meniscus ( type 3 ) ( white arrow ) , and an interface is visible between the implant and the native meniscus ( small white arrow )  . 
a scansione sagittale t2 / dp con soppressione del grasso : lintensit di segnale del cmi sovrapponibile a quella del menisco normale , di tipo 3 ( freccia bianca ) e si osserva interfaccia tra limpianto e il menisco nativo ( freccia bianca piccola )  . 
a spin - echo t1 - weighted sagittal scan obtained after intra - articular injection of contrast agent : chondral erosion of the internal femoral condyle involving more than 50% of the cartilage thickness ( empty black arrow )  . 
scansione sagittale se t1 , ottenuta dopo iniezione intra - articolare di mdc : erosione cartilaginea del condilo femorale interno che interessa pi del 50% dellintero spessore della cartilagine ( freccia nera vuota )  . 
the purpose of cmi is to compensate for the loss of meniscal tissue and promote meniscal regeneration after partial meniscectomy [ 711 , 13 , 20 , 21 ]  . 
the data provided by morphological and ultrastructural studies have shown that cells are able to migrate within the prosthetic implant , grow , and produce new extracellular matrix [ 8 , 9 , 11 , 20 , 21 ]  . 
a vast body of literature exists on the role of mri with and without contrast material in patients after meniscectomy [ 16 , 17 , 18 , 2226 ] , whereas no studies have investigated the role of mri in patients who have undergone implantation of bovine collagen menisci . in our study , the evaluation of the direct criteria revealed that morphology and size were substantially preserved and similar to those of the normal meniscus in most patients ( 87.5% at 6 months ; 82.5% at 12 months ; 56.25% at 24 months )  . 
in particular , implant height reduction is to be attributed to compressive forces acting on the knee during walking [ 3 ] and does not seem to jeopardise integration of the cmi and native meniscus . the few cases of synovial reaction ( only two cases at 6 months ) and its complete resolution during follow - up reflect the arthroscopic finding that no inflammatory processes affected the implant site , confirming the safety and biocompatibility of the prosthetic material . in our experience , data implant / meniscus complex integration are more controversial : contrary to our expectations , data from the first examination at 6 months may be considered either insufficient or nonsignificant . 
identification of an interface between the residual meniscus and the implant is the only sign that can help distinguish the two components and might be considered an evolutionary step in the integration between the cmi and the native meniscus . unfortunately , however , our data show that the lack of an interface has no significance with regard to an unfavourable implant outcome , as confirmed by the case of complete resorption of the implant at 2 years despite the interface having being identified at 12 months . 
 mri follow - up showed a progressive reduction in implant signal intensity : at 6 months , it was strongly hyperintense in 80% , with a drastic reduction at 12 months ( 35% ) , but no implant had a signal intensity of normal meniscus at either 6 or 12 months . 
the ultrastructural explanation for this lies in the progressive maturation of the newly formed tissue that experiences a reduction in the number of vessels and increased arrangement of collagen cells and fibrils into perpendicular planes [ 3 , 13 ]  . 
in our view , the interpretation of signal patterns of implant / meniscus complexes is still controversial , as only 26.6% of patients showed a signal intensity similar to that of the native meniscus at 24 months . 
ampia la letteratura sul ruolo della rm con e senza mezzo di contrasto nel paziente meniscectomizzato [ 16 , 17 , 18 , 2226 ] , mentre non sono presenti contributi sul ruolo della rm nei soggetti sottoposti ad impianto di menisco collogenico bovino . nella nostra serie la valutazione dei criteri diretti ha permesso di rilevare la sostanziale conservazione di morfologia e dimensioni simili a quelle del menisco normale nella maggior parte dei pazienti ( 87 , 5% dei pazienti a 6 mesi ; 82 , 5% dei pazienti a 12 mesi ; 56 , 25% dei pazienti a 24 mesi )  . 
la riduzione progressiva delle dimensioni documentata con rm ( 37 , 5% dei pazienti a 24 mesi ) confermata dai dati forniti nel corso del second look artroscopico effettuato sugli stessi pazienti . 
in particolare la riduzione in altezza dellimpianto da attribuire alle forze meccaniche in compressione sviluppate sul ginocchio durante la marcia [ 3 ] , e non sembra compromettere lintegrazione cmi - menisco nativo . lo scarso riscontro di reazione sinoviale ( solo 2 casi a 6 mesi ) e la completa risoluzione nel follow - up corrisponde al dato artroscopico di assenza di fenomeni infiammatori a livello dellimpianto , confermando la sicurezza e biocompatibilit del materiale protesico utilizzato . nella nostra esperienza riteniamo pi controverse le informazioni sullintegrazione del complesso impianto / menisco : queste possono essere ritenute scarse o non significative , al contrario di quanto si prevedeva nel primo controllo a 6 mesi . 
lidentificazione di una interfaccia tra il menisco residuo e limpianto lunico segno che pu aiutare a distinguere le due componenti e potrebbe essere considerato evolutivo dei processi di integrazione tra il cmi ed il menisco nativo , ma , sfortunatamente , alla luce dei nostri dati , lassenza di interfaccia non significativa per una evoluzione sfavorevole del complesso . 
questa affermazione sembra avvalorata dal fatto che in un caso , seppur in presenza di interfaccia dimostrata al controllo a 12 mesi , si potuto evidenziare il completo riassorbimento del complesso nel controllo a 2 anni dallintervento ; il dato stato confermato dallartroscopia , infatti in sede di impianto non si riscontrato tessuto simil - meniscale . il follow - up con rm ha rilevato progressiva riduzione dellintensit di segnale dellimpianto : a 6 mesi nell80% dei casi limpianto risultava marcatamente iperintenso con drastica riduzione a 12 mesi ( 35% ) , ma sia a 6 che a 12 mesi nessun impianto mostrava intensit di segnale sovrapponibile a quella del menisco normale . 
il dato si spiega dal punto di vista ultrastrutturale con la progressiva maturazione del tessuto neoformato che va incontro alla riduzione del numero dei vasi e a maggiore organizzazione in piani ortogonali delle cellule e delle fibrille collageniche [ 3 , 13 ]  . 
alla luce della nostra esperienza rimane controversa linterpretazione dellevoluzione del segnale del complesso impianto / menisco , in quanto solo il 26 , 6% dei pazienti a 24 mesi mostra intensit di segnale sovrapponibile a quella del menisco nativo , tale valutazione ci induce a sospettare che a 2 anni , in una buona percentuale , il complesso non abbia completato il processo di maturazione o rigenerazione tissutale e quindi non possiamo ritenere concluso il follow - up . us to suspect that in most cases , the complex has not yet completed the process of tissue maturation or regeneration after 2 years and that consequently , we cannot consider the follow - up to be concluded . 
 the purpose of the bovine collagen meniscus implant is to restore the physiological thickness of the fibrocartilage cushion in order to re - establish correct distribution of articular loads and prevent possible cartilage damage from the wear caused by altered biomechanical loads . 
the aim of mri is therefore not only to assess the meniscus / implant complex but to identify possible early signs of chondral and subchondral disorders that may herald possible failure . chondral lesions have been shown to remain substantially unchanged at imaging 5 years after implantation [ 27 ]  . 
in our series , the chondral alterations observed at 6 months remained unchanged at 12 months , in agreement with the literature [ 27 ] , but two new cases of chondral lesions were identified at follow - up after 24 months . 
in our series , follow - up at 24 months included the use of intra - articular contrast material , which significantly increases the rate of detection of small chondral lesions [ 26 ]  . 
it may therefore be advisable to use intra - articular contrast material as early as the first follow - up examination at 6 months , as it is very likely that the chondral lesions that appeared after 2 years were in fact present earlier but could not be identified with conventional mri . 
 we can reasonably state that the first follow - up at 6 months does not enable us to establish any certain correlation between associated chondral lesions and subchondral bone marrow oedema . 
in most cases , the signal alteration of the spongy bone decreases or disappears at later follow - up examinations , and no direct correlation can be identified between subchondral bone marrow oedema , chondral lesions and possible implant failure . 
in fact , in the only case with implant resorption , which occurred between 12 and 24 months , the previous examinations showed neither subchondral bone marrow oedema nor chondral lesions . 
 finally , we can conclude that mri is a valuable modality for the follow - up of patients who have undergone collagen meniscus implants and that , in light of our results , intra - articular contrast material should preferably be injected at the first follow - up examination to identify any chondral lesions as early as possible . 
we believe that only the follow - up can allow one to express certain judgements on implant integration , a process that can be considered concluded only when complete stabilisation of the complex has been demonstrated . 
this corresponds to the finding of markedly low signal intensity typical of the meniscus and complete resolution of bone marrow oedema , a process that could take more than 2 years in some patients . 
la rm non ha quindi il solo compito di valutare il complesso menisco / impianto , ma ha lo scopo di individuare la presenza di segni precoci di sofferenza condrale e sotto - condrale che possano prevedere leventuale fallimento terapeutico . 
stata descritta sostanziale stabilit nel tempo delle lesioni condrali nei controlli strumentali a 5 anni dallimpianto [ 27 ] , nella nostra serie le alterazioni condrali osservate al controllo a 6 mesi si sono mantenute immodificate a 12 mesi , in accordo con quanto riportato in letteratura [ 27 ] , ma si sono osservati 2 nuovi casi di lesioni condrali al controllo a 24 mesi . 
questa discrepanza tra gli studi potrebbe essere spiegata con la differente tecnica utilizzata , infatti nella nostra casistica i controlli a 24 mesi prevedevano lutilizzo di mdc intra - articolare che incrementa sensibilmente il riconoscimento delle piccole lesioni condrali [ 26 ]  . 
tali considerazioni sono sufficienti a concludere che forse preferibile associare il mdc intra - articolare gi al primo controllo a 6 mesi , poich , molto verosimilmente , le nuove lesioni condrali comparse a 2 anni si sarebbero potute riconoscere pi precocemente , infatti tali alterazioni non erano identificabili allesame basale ed in un paziente con lartro - rm stato possibile modificare il grading della lesione condrale . con buona approssimazione si pu affermare che al primo controllo a 6 mesi non possibile stabilire una sicura correlazione tra le lesioni condrali associate e la sofferenza della spongiosa sotto - condrale . 
lalterazione di segnale della spongiosa decresce o scompare nella maggior parte dei casi con lavanzare del follow - up ed inoltre non possibile stabilire una diretta correlazione tra la sofferenza della spongiosa , le lesioni condrali e leventuale fallimento dellimpianto ; infatti nellunico caso di riassorbimento dellimpianto , avvenuto tra i 12 e 24 mesi , nei controlli precedenti non vi erano aree di sofferenza della spongiosa sottocondrale e lesioni condrali , quindi tali alterazioni possono essere considerate segni indiretti correlati alla sintomatologia dolorosa e alla stabilit dellarticolazione , ma non segni predisponesti al fallimento dellimpianto . in conclusione si pu affermare la validit del follow - up strumentale con rm nei pazienti sottoposti ad impianto con menisco collagenico e che alla luce di quanto emerso sia preferibile iniettare il mdc intra - articolare gi al primo controllo , al fine di individuare pi precocemente eventuali sofferenze condrali . 
noi riteniamo che solo il follow - up pu esprimere giudizi sicuri sullintegrazione dellimpianto e questo pu ritenersi concluso solo quando si sia dimostrata la completa stabilizzazione del complesso che corrisponde al raggiungimento delliposegnale caratteristico del menisco e la completa risoluzione delledema della spongiosa , quindi in taluni pazienti potrebbe essere giustificato il prolungarsi dei controlli oltre i 2 anni . 
albrizio , the queen elizabeth hospital , kings lynn , norfolk , pe30 4et , uk , tel . : + 44 - 0155 - 3766649 , fax : + 44 - 0135 - 5584473 , e - mail : contact@drmauroalbrizio.it received : 23 november 2006 / accepted : 26 january 2007 / published online : 19 october 2007 abstract ppurpose . 
lintroduzione del wells score non ha prodotto una variazione statisticamente significativa del tasso di ctpa positive . parole chiave ctpa wells embolia polmonare metodi predittivi introduction introduzione there is ample evidence in the literature to support computed tomography pulmonary angiography ( ctpa ) as the first - line imaging investigation for suspected pulmonary embolism ( pe ) [ 16 ]  . 
with the widespread availability of multislice ct scanners , ctpa has effectively rendered conventional pulmonary angiography obsolete , despite limited sensitivity in the visualisation of subsegmental clots [ 7 ]  . 
ctpa offers the significant advantage of identifying a wide range of chest diseases , thereby facilitating differential diagnosis [ 8 , 9 ]  . the advent of multislice and multiplane technology has la validit della ctpa quale strumento di scelta per la diagnosi di embolia polmonare ( pe ) riportata in diversi studi scientifici [ 16 ]  . 
nel regno unito , la disponibilit di ctpa su tutto il territorio nazionale ha reso obsoleto luso dellangiografia polmonare nonostante lefficacia limitata della ctpa nel rilevare la presenza di emboli di piccola taglia [ 7 ]  . 
studies have even shown that patients with clinically suspected acute pe , no symptoms or signs of deep venous thrombosis ( dvt ) and negative ctpa generally have a good clinical outcome at 3 months and that it is probably safe to withdraw anticoagulation treatment in such cases [ 1 , 13 ]  . according to a meta - analysis by moores et al . 
for this reason , ctpa is often applied as part of a screening algorithm that includes predictive rules [ 14 , 15 ] and other diagnostic tests such as d - dimers , lower - extremity ultrasonography and lung scintigraphy [ 1619 ]  . 
it is an important step in contemporary diagnostic strategies aiming to limit the number of unnecessary imaging investigations . the two most widely used prediction models for pe described in the literature are the canadian prediction rule ( the wells score ) and the geneva prediction rule . 
we present an audit that aims to evaluate the effect of the protocol on the rate of positive ctpas . materials and methods at our hospital all ctpas are requested on a standard x - ray form , usually filled in by an accident and emergency ( a&e ) or ward doctor and signed by a consultant . 
the local protocol uses a slice thickness of 3.2 mm , a pitch of 1.25 and a reconstruction interval of 1.6 mm ; 100 ml of 300 mg i / ml iohexol are administered at a rate of 3 ml / s through an 18 - gauge antecubital venous cannula . 
ctpa is performed in arrested inspiration starting at the lung bases and covering the entire thorax . a list of all patients who underwent ctpa from august 2004 to january 2005 and from march 2005 to august 2005 was obtained from the computerised radiology information syste using a sequential method , we selected 100 cases from the preimplementation period ( that is , prior to february 2005 ) and 100 from the postimplementation period ( that is , after february 2005 )  . 
mean age was 65 years ( 63 in the preimplementation period and 67 in the postimplementation period ) ranging from 18 to 91 years . lavvento delle ctpa multislice e multiplane ha poi ulteriormente affinato laccuratezza dellimmagine permettendo una diagnosi pi precisa [ 10 ]  . 
al momento la letteratura conferma che la ctpa offre una sensibilit complessiva del 77% ( 95% per embolie segmentali e 89% per embolie pi voluminose ) e una specificit dell89% [ 12 ]  . 
sulla base di questi risultati alcuni studiosi hanno addirittura affermato che i pazienti con sospetta embolia polmonare , ma senza sintomi di trombosi delle vene profonde degli arti inferiori ( dvt ) e con una ctpa negativa hanno generalmente un risultato clinico soddisfacente a 3 mesi e che quindi plausibile , in tali casi , la sospensione del trattamento anticoagulante [ 1 , 13 ]  . dunque esistono numerose evidenze scientifiche che confermano laccuratezza della ctpa nella diagnosi di embolia polmonare . 
secondo la meta - analisi di moores [ 1 ] , ogni volta che viene sospettata unembolia polmonare , il paziente ha una probabilit di conferma radiologica che varia tra il 15 , 4% e il 37 , 4% . 
per questo motivo la ctpa spesso parte di algoritmi diagnostici che includono metodi predittivi [ 14 , 15 ] e altri strumenti come i d - dimeri , lecografia delle vene degli arti inferiori e la scintigrafia polmonare [ 1619 ]  . 
questa suddivisione pu risultare estremamente utile se ci si pone lobiettivo di limitare il numero di pazienti esposti inutilmente a radiazioni . i due modelli predittivi pi usati per lembolia polmonare sono il canadian prediction rule ( o wells score ) e il geneva prediction rule . 
lo scopo di questo studio dunque quello di valutare gli effetti di tale protocollo sul tasso di ctpa positive per embolia polmonare . materiali e metodi nella nostra struttura ospedaliera , le ctpa sono richieste a mezzo di un modulo compilato da personale medico di qualunque livello , tuttavia sempre necessaria la firma di un primario . 
la scansione viene effettuata a 3 , 2 mil mezzo di contrasto ( iohexol ) somministrato ad una velocit di 3 ml / secondo utilizzando un ago cannula di 18 g generalmente in una vena antecubitale . 
mizzi : rate of computed tomography pulmonary angiographies ( ctpa ) positive for pulmonary embolism and predictive scores results table 1 shows the demographic distribution of patients before and after protocol implementation . 
tables 2 and 3 summarise the sources of referral for ctpa : the majority of referrals to the radiology department ( 58% of the total 200 patients ) came from an acute medical ward both in the pre - implementation and post - implementation periods , but it is not possible to define whether these patients had arrived on ward following a first assessment at the a&e or by their general practitioner . 
positive findings other than pe were seen in up to 62% of ctpas . discussion since february 2004 , patients presenting to our institution with a high clinical probability for pe ( as demonstrated by the wells score ) have been treated with low molecular weight heparin and only subsequently assessed by ctpa . 
in patients with an intermediate or low clinical probability of pe , ddimers are assayed : if d - dimers are elevated , the patient proceeds to ctpa , whereas if they are negative , pulmonary embolism is reliably excluded and ctpa is not performed . 
however , there was no significant difference in the rate of positive ctpas before and after protocol implementactpa poi effettuata in ispirazione partendo dalla base del torace . utilizzando il database del dipartimento di radiologia abbiamo ottenuto la lista dei pazienti sottoposti a ctpa tra agosto 2004 e gennaio 2005 ( periodo precedente allintroduzione del nuovo protocollo ) e tra marzo 2005 e agosto 2005 ( periodo successivo allintroduzione del protocollo )  . usando un metodo sequenziale , abbiamo selezionato 100 casi nel periodo precedente lintroduzione del protocollo e 100 casi nel periodo successivo , per un totale di 200 pazienti . 
let di tali pazienti variava da 18 a 91 anni con una mediana di 65 ( 63 nel periodo precedente allintroduzione del protocollo e 67 nel periodo successivo )  . risultati la tabella 1 rappresenta la distribuzione demografica dei pazienti prima e dopo lintroduzione del protocollo . 
la provenienza della richiesta di consulenza radiologica rappresentata nelle tabelle 2 e 3 : la maggior parte delle richieste di ctpa ( 58% del totale ) proveniva da un reparto di medicina sia prima che dopo lintroduzione del protocollo . 
lortopedia risultava essere la specialit chirurgia con pi alta percentuale di richieste di consulenza radiologica , ma anche quella con pi alto tasso di ctpa negative . sul totale di 200 pazienti con sospetta embolia polmonare , la conferma radiologica stata ottenuta in 50 casi ( 25% del totale ; 23% nel periodo antecedente lintroduzione del wells score e 27% nel periodo successivo )  . 
nel 62% dei table 1 demographic distribution of patients before and after protocol implementation pre - implenentation period patients with confirmed pte post - implementation period patients with confirmed pte n . 
patients males / females age ( mean ) number of d - dimer requests 4555 tabella 1 distribuzione demografica dei pazienti prima e dopo lintroduzione del protocollo periodo antecedente pazienti con conferma periodo successivo alllintroduzione diagnostica di pe del protocollo allintroduzione del protocollo pazienti con conferma diagnostica di pe 1211 1211 2664 2664 1215 1215 n . 
mizzi : rate of computed tomography pulmonary angiographies ( ctpa ) positive for pulmonary embolism and predictive scores table 2 source of referral for ctpa source of referral pre - implementation post - implementation casi la ctpa ha consentito di pervenire ad una diagnosi diversa da quella di embolia polmonare . discussione a partire da febbraio 2004 , nella nostra clinica i pazienti con alta probabilit di essere affetti da embolia polmonare sono trattati con eparina a basso peso molecolare e solo successivamente allinizio della terapia sono sottoposti a ctpa . 
per i pazienti con probabilit medio - bassa il valore dei d - dimeri a guidare il management successivo : se i ddimeri sono elevati , il paziente viene sottoposto a ctpa , se i d - dimeri sono negativi la diagnosi di pe si pu considerare esclusa e la ctpa non viene considerata come strumento diagnostico . nel nostro studio la percentuale di pazienti con confermata embolia polmonare variata dal 23% al 27% con lintroduzione del wells score . 
se consideriamo il range della meta - analisi di moores [ 1 ] possiamo confermare che i risultati ottenuti sia nel periodo antecedente lintroduzione del wells score sia in quello successivo non si discostino affatto dallintervallo di valori rilevato . ciononostante , a livello puramente statistico , lintroduzione del wells score non ha portato ad un cambiamento significativo nel numero di pazienti con diagnosi di pe confermata . 
tale risultato pu avere una duplice spiegazione : ( i ) il protocollo ( e quindi il wells score ) non offre nessun vantaggio predittivo - diagnostico rispetto al semplice esame tabella 2 provenienza della richiesta di ctpa provenienza della richiesta di ctpa ( reparti ) periodo antecedente periodo successivo allintroduzione del protocollo ( % ) allintroduzione del protocollo ( % ) medical ward geriatrics general surgery orthopaedics thoracic surgery medical out - patient 3 unit itu and ccu oncology gynaecology medicina ( reparto ) pronto soccorso geriatria chirurgia generale 2 ortopedia chirurgia toracica 2 medicina ( clinica ) terapia intensiva oncologia ginecologia table 3 post - operative patients general surgery thoracic gynaecology orthaopaedic total tabella 3 pazienti post - operatori source chirurgia generale chirurgia toracica ginecologia ortopedia totale source pre - implementation ( % ) pre - implementation ( % ) post - implementation ( % ) post - implementation ( % ) total ctpas positive for pe total ctpas positive for pe periodo antecedente alllintroduzione del protocollo ( % ) periodo antecedente alllintroduzione del protocollo ( % ) periodo successivo allintroduzione del protocollo ( % ) periodo successivo allintroduzione del protocollo ( % ) numero totale di ctpa positive per pe numero totale di ctpa positive per pe m . 
this suggests one of two possibilities : ( 1 ) the protocol ( the wells score ) offers no significant predictive - diagnostic advantage over plain clinical judgement , or ( 2 ) the protocol exists in theory but is not being adhered to in clinical practice . it is clear that the number of patients recruited in this study is not sufficiently large to refute the validity of the wells score , which has been widely validated by the literature and whose predictive value can be therefore assumed . the most likely reason for the results of this audit remains the second possibility : the protocol may not have been adequately applied in clinical practice . 
if this is true , we believe there might be a potential for increasing further the rate of positive ctpas and thus reducing the number of patients undergoing unnecessary scans . an example of how this could be achieved is the introduction of a specific mandatory pro - forma to be submitted whenever a request for ctpa is made . 
the pro - forma would include details of clinical probability ( wells score ) , d - dimer levels ( when appropriate ) and make reference to the hospital protocol ; it would also represent a legal document attesting to the need to expose the patient to ionising radiation as well as being an excellent tool for further research . clinico , ( ii ) il protocollo esiste in teoria , ma non stato applicato sufficientemente nella pratica clinica . 
va sottolineato che il numero di pazienti utilizzato per questo studio non abbastanza grande da consentire di confutare la validit del wells score confermata invece da una larga porzione di letteratura scientifica . 
se tale interpretazione corretta si pu considerare potenzialmente possibile un ulteriore incremento del numero di ctpa positive con lobiettivo di ridurre il numero di pazienti esposti inutilmente a radiazioni . un esempio di come tale risultato possa essere ottenuto lintroduzione di un pro - forma obbligatorio da compilare ogni qualvolta una richiesta di ctpa viene avanzata . 
tale pro - forma conterrebbe riferimenti al wells score , ai d - dimeri ( se necessari ) e al protocollo ospedaliero in s e sarebbe anche un documento legale comprovante la necessit di esporre il paziente a radiazioni nonch un ottimo strumento per lo svolgimento di ulteriori indagini scientifiche . conclusions the results of this audit show that the rate of positive ctpas at our hospital lies well within the accepted range . 
simplification of the scoring criteria and the introduction pro formas are recommended . conclusioni i risultati di questo studio dimostrano che il tasso di ctpa positive rientra nel range indicato in letteratura come accettabile . 
zobel1 1centro interdisciplinare per la ricerca bio - medica , department of radiology , 2department of oncology , universit campus bio - medico di roma , via longoni 47 , i - 00155 roma , italy correspondence to : c.c. 
analysis of the cases observed for the first time during the 19962000 period showed osteolytic lesions in 53.6% ( 15 / 28 ) , osteosclerotic lesions in 32.1% ( 9 / 28 ) and mixed lesions in 14.3% ( 4 / 28 )  . 
tra il 1996 e il 2005 , 468 pazienti con carcinoma della mammella sono stati sottoposti presso il nostro dipartimento ad una tc di stadiazione o per follow - up . 
in pazienti con metastasi ossee , la tc ha rilevato 18 pazienti con lesioni osteolitiche ( 30% ) , 32 con lesioni osteoaddensanti ( 53 , 3% ) e 10 ( 16 , 7% ) con lesioni miste . 
lanalisi dei casi osservati per la prima volta nel periodo compreso tra il 1996 e 2000 , ha mostrato lesioni osteolitiche nel 53 , 6% dei casi ( 15 / 28 ) , lesioni osteoaddensanti nel 32 , 1% dei casi ( 9 / 28 ) e miste nel 14 , 3% dei casi ( 4 / 28 ) mentre i risultati erano rispettivamente del 9 , 4% ( 3 / 32 ) , 71 , 9% ( 23 / 32 ) e 18 , 7% ( 6 / 32 ) per gli stessi gruppi nel periodo compreso tra il 2001 e 2005 . 
lanalisi istologica di tutti i casi evidenziava la presenza di carcinoma duttale infiltrante nel 81 , 9% dei casi , di carcinoma lobulare infiltrante nel 11 , 2% dei casi , di carcinoma duttale lobulare misto nel 3 , 7% dei casi e nel 3% dei casi di carcinoma midollare . 
imaging of bone metastases in breast cancer is an essential step to stage the disease , plan a therapeutic intervention and monitor response to treatment , whether chemotherapy , radiotherapy or endocrine therapy . 
numerous imaging modalities are now used , such as conventional x - rays , bone scintigraphy , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , positron emission tomography ( pet ) and single photon emission ct ( spect )  . 
however , a standard diagnostic and follow - up protocol has not been established in clinical practice , and meta - analyses have not been possible because of the heterogeneity of imaging data . moreover , the response evaluation criteria to solid tumours ( recist ) system always considered bone disease as an unmeasurable entity until hamaoka et al . 
revised those criteria by including ct and mri findings , highlighting the contribution of these techniques above all in the assessment of bone response to therapy [ 4 ]  . compared with conventional x - rays where at least 30% variation of mineral content is necessary to reach the detection threshold [ 5 ] , ct shows areas of decreased or absent density ( osteolytic , due to the destruction of bone matrix ) or hyperdense areas as in sclerotic bone lesions ( osteosclerotic ) , confirming the positive finding of bone scan hypermetabolic areas , with a relatively low cost in the context of whole - body follow - up study and a higher sensitivity ranging from 71% to 100% [ 6 ]  . 
ct provides the most accurate morphological images of the bone , allowing visualisation of compact and trabecular bone and tumour margins and dimensions [ 4 ] ; on follow - up studies , it depicts the presence of sclerosis in the context of lytic lesions , thus allowing a better evaluation of response to treatment [ 4 ]  . 
 even though single - slice ct has had limited value in the evaluation of multiple bone sites , the advent of multislice and spiral ct has allowed whole - body scans in a very short time . 
lavvento dei bifosfonati di terza generazione pu aver cambiato laspetto tc delle metastasi ossee da carcinoma della mammella . parole chiave tomografia computerizzata carcinoma della mammella metastasi ossee acido zoledronico il carcinoma della mammella caratterizzato da una alta prevalenza di metastasi ossee : dal 30% al 85% dei pazienti con metastasi da cancro della mammella presenta un coinvolgimento secondario osseo durante il corso della patologia [ 1 , 2 ] e losso rappresenta il primo sito di metastasi dal 26% al 50% dei casi [ 2 , 3 ]  . 
limaging delle metastasi ossee nel carcinoma della mammella rappresenta una tappa essenziale nella stadiazione della patologia , nella pianificazione di un adeguato intervento terapeutico e nel controllo della risposta al trattamento , sia esso chemioterapico , radioterapico o endocrino . 
numerose tecniche di imaging sono oggi utilizzate e comprendono la radiologia tradizionale , la scintigrafia ossea , la tomografia computerizzata ( tc ) , la risonanza magnetica ( rm ) , la tomografia ad emissione di positroni ( pet ) e la tomografia computerizzata a singola emissione di positroni ( spect )  . 
tuttavia , non stato ancora stabilito un protocollo standard per la diagnosi ed il follow - up nella pratica clinica e studi di metanalisi non sono stati possibili soprattutto per la grande eterogeneit dei dati radiologici . 
 [ 4 ] hanno modificato questi criteri includendo le informazioni derivanti dalle immagini tc e rm , evidenziando il miglioramento che queste tecniche apportano , specialmente nella valutazione della risposta dellosso alla terapia . rispetto alla radiologia tradizionale , dove per evidenziare una lesione necessaria una perdita di almeno il 30% del contenuto minerale dellosso [ 5 ] , la tc mostra aree di densit assente o diminuita ( osteolitiche ) , dovuta alla distruzione della matrice ossea , o aree iperdense come da lesioni ossee sclerotiche ( osteoaddensanti ) , che confermano la presenza di aree di ipercaptazione alla scintigrafia , con un costo relativamente basso nel contesto di un follow - up total body e con una sensibilit che varia dal 71% al 100% [ 6 ]  . 
la tc fornisce le immagini pi accurate della morfologia ossea , permettendo la visualizzazione dellosso compatto e trabecolare , del margine e delle dimensioni del tumore [ 4 ]  . 
lytic lesions , reducing the bone resistance to load or tangential forces , are strongly linked to skeletal - related events , including pathological fractures , radiation therapy , bone surgery or spinal cord compression [ 9 ]  . 
 the advent of new ct equipment and the routine use of ct scans in the follow - up of breast cancer patients may have modified the real incidence of bone metastases and their radiological appearance in comparison with the past . 
here we present a retrospective analysis of the radiological appearance of bone metastases in patients with advanced breast cancer referred to our department through 19962005 in which we observed a predominant osteosclerotic pattern on ct scans . 
images were obtained with a single - row ct ( high speed general electric , 19962003 ) and with 16 - row spiral ct scanner ( somatom sensation 16 , siemens , 20032005 )  . 
ct studies were performed before and during bolus administration of nonionic iodinated contrast agent at a concentration of 350 mgi / ml ( iobitridol xenetix , guerbet , france )  . 
the malignant nature of the lesions was determined by considering progression , the presence of nel contesto di lesioni litiche , consentendo cos una migliore valutazione della risposta al trattamento [ 4 ]  . sebbene la tc a singolo strato ha avuto un valore limitato nella valutazione di siti multipli di metastasi , lavvento della tc multistrato e spirale permette di effettuare oggi uno studio di tutto lo scheletro osseo in poco tempo . 
la tc totalbody fa parte oggi degli esami di routine nel protocollo di follow - up dei pazienti oncologici e la possibilit di valutare lintero volume corporeo , usando la finestra per losso , ha dato alla tc un ruolo molto importante nel diagnosticare lesioni ossee e nel valutare la risposta alla terapia [ 7 , 8 ]  . 
le lesioni litiche , riducendo la resistenza dellosso alle forze tangenziali e di carico , sono fortemente correlate ad eventi scheletrici definiti sre ( skeletal related events ) , e che includono linsorgenza di fratture patologiche , terapia radiante dellosso , e terapia chirurgica ortopedica [ 9 ]  . 
le lesioni miste rappresentano daltra parte uno stadio intermedio tra le lesioni litiche e le blastiche . lavvento di nuove tecniche tc e luso di routine della tecnica tc nei protocolli di follow - up del carcinoma della mammella potrebbero aver modificato la reale incidenza delle metastasi ossee e il loro quadro radiologico rispetto al passato . 
in questo lavoro presentiamo una analisi retrospettiva del quadro radiologico delle metastasi ossee nelle pazienti affette da carcinoma della mammella in stadio avanzato osservati nel nostro dipartimento nel periodo compreso tra il 1996 e 2005 , nei quali abbiamo osservato un aspetto di tipo prevalentemente osteoaddensante allesame tc . 
tra il 1996 e il 2005 , 468 donne con una diagnosi di carcinoma della mammella sono state sottoposte ad un esame tc nel nostro dipartimento , sia per effettuare una prima stadiazione , sia per follow - up . 
dal 2000 sono giunte allosservazione nel nostro dipartimento donne affette da carcinoma della mammella che provenivano dal programma di screening effettuato seguendo le linee guida dellautorit regionale di sanit del lazio . 
le immagini sono state ottenute con tc a singolo strato ( high speed general electric 19962003 ) e con tc spirale a 16 strati ( somatom sensation 16 , siemens , 20032005 )  . 
le immagini sono state acquisite prima e durante somministrazione di mezzo di contrasto , alla concentrazione di 350 mgi / ml ( iobitridol , xenetix , guerbet , francia )  . 
negli studi di follow - up , le pazienti venivano sottoposte di routine ad un esame tc ogni sei mesi nel primo anno e ogni dodici mesi nei successive cinque anni . lesame tc ha evidenziato metastasi sistemiche in 142 / 468 pazienti , 60 delle quali con coinvolgimento osseo , confermato dalla scintigrafia ossea . 
due esperti radiologi hanno osservato indipendentemente gli esami tc delle pazienti con carcinoma della mammella , riportando su un file di microsoft excel laspetto delle metastasi ossee ( litiche , miste o addensanti ) e la loro localizzazione . 
also , grading of breast cancer at histology failed to correlate with radiological appearance of bone metastases at initial diagnosis . since 2001 , 20 out of 47 patients received zoledronic acid i.v. 
 ( zometa , novartis pharma stein ag , switzerland ) ( 4 mg every 28 days )  . higher prevalence of osteosclerotic lesions we performed a temporal distribution curve of lytic , mixed and sclerotic metastases and observed an increased prevalence of sclerotic changes since 2000 . 
women undergoing mastectomy , with a histological grade defined according to the nottingham method and a stage according to the tnm classification , were 68.3% ( 41 / 60 )  . 
the prevalence of osteosclerotic lesions at higher ptn did not change significantly , whereas osteolytic lesions became significantly more abundant . evaluation and interpretation of mixed lesions ( meaning a mixture of lytic and blastic lesions in the same patient or a mixture of lytic and sclerotic component in the same lesion ) was difficult due to the limits of a retrospective observation . association between bone metastasis and zoledronic acid treatment zoledronic acid was introduced in the chemotherapy protocols in 2001 . 
in pazienti con metastasi ossee , la tc ha rilevato 18 pazienti con lesioni osteolitiche ( 30% ) , 32 con lesioni osteoaddensanti ( 53 , 3% ) e 10 con lesioni miste ( 16 , 7% )  . 
lesame istologico di tutti i casi ha evidenziato l81 , 9% di carcinoma duttale infiltrante ; l11 , 2% di carcinoma lobulare infiltrante : il 3 , 7% di carcinoma misto duttale - lobulare ed il 3% di carcinoma midollare . 
non stata trovata correlazione tra il grading istologico del carcinoma della mammella alla prima diagnosi e laspetto radiologico delle metastasi alla prima diagnosi . a partire dal 2001 , 20 dei 47 pazienti sono stati trattati con somministrazione endovenosa di acido zoledronico ( zometa , novartis pharma stein ag , svizzera ) 4 mg ogni 28 giorni . incremento della prevalenza delle lesioni osteoaddensanti abbiamo analizzato le curve di distribuzione nel tempo delle lesioni metastatiche litiche , miste e osteoaddensanti ed abbiamo osservato un aumento della prevalenza delle modificazioni sclerotiche a partire dal 2001 . 
paragonando la distribuzione delle metastasi ossee tra i due gruppi , abbiamo trovato un valore chi quadro molto alto con una differenza statisticamente significativa ( p < 0 , 001 ) nella direzione di una pi alta prevalenza di lesioni ostreoaddensanti . correlazione tra metastasi ossee e stadio del carcinoma della mammella alla prima diagnosi non abbiamo trovato una correlazione statisticamente significativa tra laspetto radiologico delle metastasi ossee ed il tipo istologico del tumore primitivo . 
le donne sottoposte ad un intervento chirurgico di mastectomia , con un grado istologico definito secondo il metodo di nottingham e la stadiazione definita secondo la classificazione tnm , erano il 1053 c.c. 
we found no correlation with sclerosis of bone metastases when standard chemotherapy agents , antiestrogens and chemotherapy plus monoclonal antibodies ( trastuzumab , herceptin ; roche , basel , switzerland ) were considered . 68 , 3% ( 41 / 60 )  . 
le pazienti con una stadiazione pt1n0 e pt1n1 - 3 alla prima diagnosi presentavano rispettivamente il 75% e il 50% di lesioni osteoaddensanti , mentre non erano evidenti lesioni litiche . 
test 2 mostra una differenza statisticamente significativa ( p < 0 , 05 ) acido zoledronico no acido zoledronico totale discussion bone metastases are osteostructural dysmorphisms due to secondary localisation of tumour cells following systemic dissemination through the blood and / or lymphatic vessels . in the case of breast cancer , they have been described to be mainly osteolytic ( up to 50% ) , as areas of bone resorption , and , more rarely , as osteosclerotic , as areas of bone deposition , or mixed [ 1 , 12 ]  . 
this phenomenon has been explained by the activation of osteoclastic activity in the bone tissue stimulated by a paracrine effect of substances produced by the tumour cells , such as the parathyroid - hormone - related protein ( pthrp ) [ 13 ] , interleukin - 8 ( il - 8 ) [ 14 ] , il - 11 and vascular endothelial growth factor ( vegf ) , as well as bonederived mediators such as tumour growth factor ( tgf ) - beta [ 15 ] and insulin - like growth factor - 1 ( igf - 1 ) [ 16 ]  . 
lanalisi degli effetti sullaspetto delle metastasi ossee dovuti alla somministrazione di acido zoledronico stata effettuata dividendo le pazienti del gruppo 20012005 in due gruppi : quelle trattate con acido zoledronico ( n = 20 ) e quelle non trattate ( n = 27 )  . 
un aumento statisticamente significativo delle lesioni osteoaddensanti stato osservato nel gruppo trattato con acido zoledronico ( p < 0 , 05 ) ( tabella 1 )  . durante la ristadiazione effettuata mediante tc condotta sulle stesse pazienti , una variazione sclerotica progressiva stata inoltre osservata durante i trattamenti chemioterapici che comprendevano la somministrazione ev di acido zoledronico . 
non abbiamo trovato una correlazione statisticamente significativa tra sclerosi delle metastasi ossee ed altri trattamenti farmacologici : agenti chemioterapici standard , antiestrogeni e chemioterapia standard con associazione di anticorpi monoclonali ( trastuzumab , herceptin , roche , basilea , svizzera )  . discussione le metastasi ossee si possono definire come dismorfismi osteostrutturali determinati dalla localizzazione secondaria delle cellule tumorali in seguito a disseminazione sistemica per via ematica o linfatica : nel caso del carcinoma della mammella sono state descritte come lesioni prevalentemente litiche ( in pi del 50% dei casi ) e , soltanto raramente , come lesioni osteoaddensanti in quanto aree di deposizione ossea o come lesioni miste [ 1 , 12 ]  . 
questo fenomeno stato spiegato dallattivazione osteoclastica del tessuto osseo , stimolata dalleffetto indotto da sostanze paracrine prodotte da cellule tumorali come le proteine producenti ormone analogo del pth ( pthrp ) [ 13 ] , interleuchina 8 ( il - 8 ) [ 14 ] , interleuchina 11 e fattore di crescita vascolare endoteliale ( vegf ) e mediatori derivati dallosso come il tgfbeta [ 15 ] e il fattore di crescita analogo dellinsulina di tipo i ( igf - 1 ) [ 16 ]  . 
lattivazione degli osteoclasti come fattore principale determinante losteolisi , stato confermato dallevidenza che gli osteoclasti sono stati trovati in prossimit dei depositi di tumore osseo alla microscopia elettronica e dal fatto che le lesioni ossee di tipo osteolitico presentano delle lacune di riassorbimento osteoclastico ben riconoscibili . 
inoltre , i farmaci in grado di inibire lattivit osteoclastica ( ad esempio bifosfonati , plicamicina , e nitrato di gallio ) sono attivi contro tutti i tipi di metastasi ossea [ 12 ] e riducono inoltre la ipercalce1055 c.c. 
una donna di 51 anni con carcinoma della mammella ( carcinoma duttale infiltrante , pt2n1 ) sottoposta a mastectomia e chemioterapia adiuvante , ricevette diagnosi di metastasi osteolitiche nel 1999 . durante trattamento con acido zoledronico , le lesioni litiche vertebrali mostrano progressiva sclerosi , fino a coinvolgere completamente la lesione . 
a e d immagini tc ottenute nel marzo 2004 ; b ed e stesso livello delle immagini a e d rispettivamente , ottenute sei mesi dopo ; c ed f stesso livello delle immagini a e d rispettivamente , ottenute dodici mesi dopo . well - recognisable osteoclast resorption lacunae . 
on the other hand , it has been shown that breast cancer cells may directly promote bone resorption in vitro , producing both mineral release and bone matrix degradation , when added to devitalised bone [ 17 ] , leading to the hypothesis of a possible convergence of the two mechanisms in the origin of osteolysis . the most common sites of bone involvement include axial bones such as vertebrae and pelvis , possibly because of the high vascularity and abundant presence of red bone marrow , even though ribs , skull and femur may also be involved [ 7 , 8 , 18 ]  . ct is very useful in detecting bone metastases and assessing their response to treatment [ 7 , 8 ]  . 
daltra parte , tuttavia , stato dimostrato che le cellule neoplastiche del carcinoma della mammella possono promuovere direttamente il riassorbimento osseo in vitro , determinando il rilascio di prodotti di degradazione della matrice ossea , quando aggiunti ad osso devitalizzato [ 17 ] , permettendo cos di ipotizzare una possibile convergenza dei due meccanismi allorigine dellosteolisi . le sedi pi comuni di coinvolgimento dellosso comprendono lo scheletro assiale , tra cui le vertebre e la pelvi , verosimilmente a causa dellabbondante vascolarizzazione e della presenza di midollo ematopoietico , anche se possibile il coinvolgimento di coste , cranio e femore [ 7 , 8 , 18 ]  . la tc molto utile nellindividuare metastasi ossee e nel valutare la risposta al trattamento [ 7 , 8 ]  . 
tuttavia il progredire della lisi ossea o la comparsa di nuove aree di osteolisi , che possono presentarsi o allinterno di aree di sclerosi o in regioni miste o come au1056 c.c. 
a 68 - year - old woman received a diagnosis of breast cancer ( pt1n0 ) in 1993 and follow - up studies were negative until osteolytic lesions were diagnosed in 2004 ( a , c )  . 
the significance of these distribution changes is not clear , but some hypotheses can be suggested . in the last year bone metastases have been diagnosed earlier than in the past , also thanks to the advent of breast cancer screening programmes that have led to earlier therapeutic approaches . 
even though a correlation study , in respect to the stage of disease at the time of diagnosis , was not possible due to the small size of groups , the trend observed is worth of further analysis on larger populations . response to chemotherapy treatments has been correlated with sclerosis of bone metastases , even though a clear association between cytotoxic drugs and osteosclerotic lesions has never been demonstrated . 
adding bisphosphonates to chemotherapy protocols has become a standard strategy in mento delle dimensioni originarie di una lesione blastica , potrebbero rappresentare un indice di progressione di malattia . nel nostro studio retrospettivo abbiamo rilevato una alta prevalenza di lesioni ossee metastatiche osteoaddensanti in pazienti affette da carcinoma della mammella nel periodo compreso tra il 20012005 rispetto a quelle del periodo compreso tra il 19962000 ( 71 , 9% versus 32 , 1% )  . 
il significato del cambiamento di questa distribuzione non ancora chiaro ma possono comunque essere avanzate alcune ipotesi . nellultimo periodo le metastasi ossee sono state diagnosticate pi precocemente grazie ai programmi di screening , consentendo trattamenti pi precoci . 
sebbene non sia possibile uno studio di correlazione rispetto alla precocit della diagnosi , a causa del campione troppo piccolo , landamento osservato appare meritevole di ulteriori analisi su un campione di maggiori dimensioni . la risposta al trattamento chemioterapico stata correlata con la sclerosi delle metastasi ossee anche se una chiara associazione tra i farmaci citotossici e le lesioni di tipo osteoaddensante non stata trovata . 
bisphosphonates are accumulated in the site of metabolically active bone and play a complex role on bone metastasis , modifying the osteoblastosteoclast equilibrium [ 20 ]  . different functions have been ascribed to biphosphonates and especially to zoledronic acid , such as a direct antitumoural effect and antiosteoclastic and antiangiogenetic activity . 
the first is explained by the proapoptotic effect played against the tumour cells , inhibiting their growth and possibly their invasion through the endothelium [ 2124 ]  . the antiosteoclastic activity is realised through inhibition of formation , migration and activity of osteoclasts [ 25 , 26 ] and by inducing osteoclastic apoptosis [ 27 ]  . 
administration of 4 mg of zoledronic acid [ 28 ]  . a macroscopic effect of zoledronic acid through change ct appearance of bone metastases could be related to all three of these effects on bone metabolism , with sclerosis of osteolytic lesions explained by a reduction of osteoclastic activity and a decrease in microvascular perfusion of bone lesions . the correlation between zoledronic acid and osteosclerotic lesions due to metastatic breast cancer brings new insights into the mechanisms of response to therapy of bone metastases . 
a prospective analysis conducted on follow - up studies , comparing ct appearance of bone metastases before and after zoledronic acid treatment , will clarify some of the questions raised by these preliminary data . minato una riduzione definitiva dellincidenza di eventi scheletrici ( sre ) [ 19 ]  . 
lacido zoledronico un bifosfonato di terza generazione , con una attivit clinica stimata 100 volte maggiore dei bifosfonati di prima generazione ( ad esempio etidronato , clodronato ) , inibendo il riassorbimento osseo anche a concentrazioni micromolari . 
i bifosfonati si accumulano nelle sedi ossee metabolicamente attive e giocano un ruolo complesso sulle metastasi ossee , modificando lequilibrio tra osteoblasti e osteoclasti [ 20 ]  . funzioni diverse sono state attribuite ai bifosfonati ed in modo particolare allacido zoledronico , tra questi un effetto antitumorale diretto , una attivit anti - osteoclastica ed una attivit anti - angiogenica . 
leffetto anti - angiogenico supportato dallevidenza di una riduzione significativa dei livelli sierici di vegf dopo la somministrazione ev di 4 mg di acido zoledronico [ 28 ]  . un effetto macroscopico dellacido zoledronico evidenziabile attraverso la modificazione dellaspetto tc delle metastasi ossee potrebbe essere correlato a tutti e tre questi effetti sul metabolismo osseo con conseguente sclerosi delle lesioni osteolitiche dovute ad una riduzione dellattivit osteoclastica , ed ad una riduzione della perfusione vascolare nelle lesioni ossee . la correlazione tra acido zoledronico e le lesioni osteoaddensanti da metastasi della mammella apre nuove prospettive nellanalisi del meccanismo di risposta alla terapia delle metastasi ossee . 
radiologia , roma , italy 2istituto di medicina del lavoro delluniversit cattolica del sacro cuore , largo gemelli 8 , i - 00168 roma , italy 3medico del lavoro , roma , italy 4u.o. 
regardless of the outcome of legal proceedings , physicians who are sued usually perceive the claim as an assault on their integrity and may suffer psychological or physical effects known as malpractice stress syndrome . 
radiological errors were purportedly related to occupational discomfort , and the latter variable was significantly associated with work dissatisfaction and a low level of psychological and physical well - being . 
i radiologi hanno una buona conoscenza del fenomeno della cosiddetta malpractice medica e della cause che contribuiscono ad enfatizzarlo , cos come delle condizioni lavorative che causano stress da lavoro , insoddisfazione e aumento degli errori ; mostrano per una insufficiente padronanza delle tecniche di clinical risk management e della dottrina del consenso informato . 
analysis of reports presented by radiologists to the professions insurance agency between 1993 and 2005 to safeguard themselves from legal proceedings of civil liability shows that about one half of insured radiologists have received or will shortly receive a claim for compensation or will be called to face criminal proceedings related to their professional activity performed in the last decade . 
in one third of these cases , claims refer to events involving the death of the patient , which produces requests for very high compensation payments and possible severe criminal repercussions [ 1 ]  . apart from the legal consequences , however , litigation can become a stress factor for the physician known as malpractice stress . 
we therefore felt the need to perform a study in italy regarding the consequences that compensation claims for civil liability and criminal proceedings have on the well - being and professional conduct of radiologists . le denunce contro i medici per presunta malpractice sono un fenomeno di rilievo anche in italia . 
lanalisi delle segnalazioni presentate dai radiologi allistituto assicuratore tra il 1993 ed il 2005 per tutelarsi da azioni di responsabilit civile indica che circa la met dei radiologi assicurati ha ricevuto o sta per ricevere una richiesta di risarcimento o una citazione penale relativa allattivit professionale svolta nellultimo decennio . 
in un terzo dei casi le denunce si riferiscono ad eventi nei quali si verificato il decesso del paziente ; ci determina richieste di risarcimento di importo molto elevato e possibili pesanti ripercussioni penali [ 1 ]  . al di l delle conseguenze legali , lazione di responsabilit pu rappresentare per il sanitario un fattore di stress , il cosiddetto stress da malpractice . 
che la denuncia per responsabilit civile rappresenti per il medico un vero e proprio fattore di stress stato intuito sin dalla fine degli anni 80 , dai ricercatori americani [ 2 ] ; studi recenti dimostrano come il timore di denunce sia in grado di influenzare i comportamenti dei medici e le loro scelte professionali [ 35 ]  . 
ci parso necessario , pertanto , avviare anche nel nostro paese unindagine sulle conseguenze che le richieste di risarcimento per responsabilit civile e le denunce penali esplicano sul benessere e sui comportamenti professionali dei medici radiologi . materials and methods radiologists attending two radiology and oncology radiotherapy congresses were invited to anonymously respond to a questionnaire that explored their knowledge and opinions regarding malpractice , their personal experience of litigation for alleged malpractice and how they coped with the event . the questionnaire comprised two parts : one devised ad hoc for the study and one derived from previously published experiences . 
the first part of the questionnaire asked respondents to provide some personal data ( gender , age range , years of professional experience , professional role or qualification , type of public or private professional activity ) and included a series of questions relating to malpractice and the causes of clinical error . 
opinions regarding malpractice were investigated through a series of 24 statements : physicians were invited to express their level of agreement to each statement . replies were graded from ( 1 ) strongly disagree to ( 5 ) strongly agree , according to a 5 - point likert scale . 
in this case , the scale ranged from ( 1 ) totally irrelevant to ( 5 ) highly relevant . the second part of the questionnaire contained the italian version of several standardised questionnaires modified to render them appropriate to the sample being studied . 
with these tools we aimed to assess ( 1 ) consequences of litigation , ( 2 ) level of job satisfaction and ( 3 ) level of individual 1070 materiali e metodi i radiologi partecipanti a due congressi di radiologia e di radioterapia oncologica sono stati invitati a compilare un questionario anonimo , che indagava le conoscenze ed opinioni riguardanti il fenomeno della malpractice , la personale esperienza di denunce per presunta malpractice e il vissuto relativo a tale evento . 
la prima parte del questionario richiedeva lindicazione di alcuni dati anagrafici ( sesso , classe di et , anni di esperienza professionale , ruolo o qualifica professionale , tipo di attivit professionale pubblica o privata ) e conteneva una serie di domande relative alla malpractice ed alle cause di errore medico . 
le opinioni relative al fenomeno della malpractice sono state indagate proponendo ai medici radiologi una serie di 24 affermazioni ed invitandoli ad esprimere il grado di consenso con ciascuna affermazione . 
la risposta era graduata da ( 1 ) non sono affatto daccordo a ( 5 ) sono del tutto daccordo , secondo una scala analogico verbale - numerica di likert . 
this questionnaire was developed to describe the consequences of an assault , a threat or another form of harassment and was used in the italian version to assess the consequences of violent actions on health care workers [ 7 , 8 ]  . 
in this case , only the section regarding harassment was considered , which involves ten possible responses ( fear , distress , anger , anxiety , humiliation , guilt , disappointment , helplessness , physical harm or no harm )  . 
the overall score , which is obtained by adding up the scores to all of questions and therefore ranges from 7 to 70 points , expresses the degree of job satisfaction . 
psychological and physical well - being at the time of the study was assessed using the goldberg general health questionnaire [ 11 ] in the 12 - item version ( ghq - 12 ) , which is recommended as a valid screening test for psychological distress [ 12 ]  . 
each of the ghq - 12 questions offers the choice of four responses , which can either be interpreted in a bimodal fashion or with an increasing score in the manner of a likert scale . 
adoption of the former or the latter method influences only the frequency of subjects classified as possible cases but not the validity of the results at the group level [ 12 ]  . bearing this in mind , we chose to use a 4 - point likert scale , because this method produces results better approaching normal distribution . 
the final score ranges from 12 to 48 points ; low values correspond to complete well - being , whereas higher values are indicative of a state of psychological tension . we distributed 350 questionnaires at the two congresses : 321 were returned , of which seven were discarded as being incomplete . 
a total of 314 physicians therefore responded to the questionnaires , equivalent to a response rate of 89.7%. data were analysed with the spss software package ( statistical package for social sciences )  . 
analysis of the frequency of the individual variables was conducted using descriptive statistics , whereas comparison between groups was performed using the mann - whitney u test for nonparametric data . 
this technique derives principal components , giving rise to correlation matrices that indicate strength of association among the variables . la seconda parte del questionario conteneva la versione italiana di alcuni questionari standardizzati , con opportune modificazioni che li rendevano adeguati alla casistica in oggetto . 
mediante tali strumenti ci si proposti di valutare ( a ) le conseguenze della denuncia ; ( b ) il grado di soddisfazione ricavata dallattivit professionale ; ( c ) il livello individuale di benessere psico - fisico al momento dellindagine . 
tale questionario stato messo a punto allo scopo di descrivere le conseguenze di una aggressione , di una minaccia o di unaltra forma di molestia ed stato usato , nella versione italiana , per valutare le conseguenze di azioni violente sul personale sanitario [ 7 , 8 ]  . in questo caso stata impiegata ovviamente solo la sezione relativa alle conseguenze delle azioni moleste , che consta di dieci possibili risposte ( paura , angoscia , rabbia , ansia , umiliazione , senso di colpa , delusione , senso di essere indifeso , danni fisici , o nessun danno )  . 
il livello di soddisfazione ricavata dal lavoro stato indagato mediante il questionario messo a punto da warr [ 9 ] , nella versione specificamente impiegata per indagini su popolazioni mediche [ 10 ]  . 
lo stato di benessere psicofisico al momento dellindagine stato valutato tramite il general health questionnaire di goldberg [ 11 ] , nella versione in 12 domande che stata raccomandata come valido test di screening per il distress psicologico [ 12 ]  . 
ciascuna delle domande del ghq - 12 prevede quattro risposte , che possono essere interpretate con un punteggio dicotomo , o con un punteggio crescente come una comune scala likert . ladozione di uno o dellaltro metodo influenza solo la frequenza di soggetti classificabili come possibili casi , ma non la validit dei risultati a livello di gruppo [ 12 ] ; in considerazione di ci , abbiamo impiegato in questo lavoro la scala likert in 4 punti , perch in tal modo i risultati approssimano meglio la distribuzione normale . 
il punteggio finale varia da 12 a 48 punti ; valori bassi corrispondono al pieno benessere , mentre valori elevati esprimono uno stato di tensione psicologica . nelle diverse sedi congressuali sono stati distribuiti 350 questionari ; i medici ne hanno restituiti 321 e di questi 7 sono stati scartati perch incompleti . 
complessivamente , quindi , hanno risposto 314 medici , pari all89 , 7% degli intervistati . i dati sono stati analizzati mediante il software statistico spss ( statistical package for social science )  . 
per lanalisi delle frequenze delle singole variabili sono state utilizzate le statistiche descrittive ; i confronti tra gruppi sono stati effettuati con il test u di mann whitney per dati non parametrici . stata impiegata lanalisi della regressione logistica ( forward stepwise conditional logistic regression ) per la selezione dei fattori predittivi nel caso di variabili dipendenti binarie , come ad esempio lavvenuta denuncia per malpractice . 
a large majority were men ( 251 men , 79.9% ; 63 women ) , and nearly all were practising in the public sector ( 289 physicians , > 92% )  . 
physicians practising within the national health system fell into three categories : 20% were department heads ( 59 ) , 19% were division heads ( 56 ) and the remaining 61% were staff physicians ( 174 )  . 
demographics of the sample are summarised in table 1 . one third of physicians ( 105 , 33.4% ) responded that they had been sued for civil or criminal liability with a request for compensation at some time . 
there were no significant differences between radiotherapists and radiologists with regard to demographic variables ( gender , age ) , or work - related variables ( experience , role , public or private sector )  . 
the mean number of consequences of litigation indicated by radiologists was 2.8 , whereas only 11.3% of respondents reported not having suffered any harm . with regard to the consequences malpractice litigation had on their professional practice , 39.0% of respondents admitted having altered their professional conduct . 
indeed , the mean number of consequences and the frequency of each consequence of malpractice litigation was the same in the two groups when compared using the mann - whitney u test . opinions on malpractice respondents were asked to express their opinions on the subject of malpractice , and their responses demonstrated that they were aware of the risk of civil and criminal proceedings . in fact , 246 ( 78.3% ) stated they strongly agreed with the statement : the radiologist is exposed to civil and criminal 1072 riabile dipendente . 
la seconda variabile selezionata tramite il coefficiente di correlazione parziale pi elevato , e candidata per linserimento ; se ne valuta per anche luscita dal modello , con un valore di significativit predefinito ( p = 0 , 10 )  . 
mediante tale statistica si procede allestrazione di componenti principali , dando luogo a matrici di correlazione che indicano la forza di associazione delle variabili tra di loro . risultati hanno partecipato 314 medici , di cui 108 specialisti in radioterapia e 206 specialisti in radiologia in attivit lavorativa . 
i soggetti esaminati risultavano prevalentemente di sesso maschile ( 251 maschi , pari al 79 , 9% , 63 femmine ) , e quasi tutti operanti nel settore pubblico ( 289 medici , oltre il 92% )  . i medici dipendenti del ssn erano per il 20% direttori di unit operativa ( 59 unit ) , per oltre il 19% responsabili di struttura semplice ( 56 unit ) , per il rimanente dirigenti medici ( 174 unit , 61% )  . 
la classe di et pi frequente risultata quella compresa tra 46 e 50 anni ( 27 , 7% ) , la categoria di esperienza lavorativa pi frequente quella superiore a 26 anni ( 22 , 6% )  . 
 un terzo dei medici ( 105 soggetti , pari al 33 , 4% ) ha risposto nel questionario di avere subito in passato una denuncia civile o penale con richiesta di risarcimento . 
il gruppo dei radioterapisti non presentava differenze significative con quello dei radiologi per nessuna delle variabili anagrafiche ( genere , et ) n per quelle legate al lavoro ( esperienza , ruolo , attivit nel settore pubblico o privato )  . 
anche la prevalenza delle denunce risultata uguale nei due gruppi , se confrontata con il test u di mann whitney ( p > 0 , 19 ) ( tabella 1 )  . lanalisi di regressione logistica ha dimostrato che il solo fattore anagrafico significativamente associato con loccorrenza di denunce let ( p < 0 , 0001 ) , mentre gli altri fattori ( genere , anni di esperienza , tipo di struttura pubblica o privata , posizione gerarchica ) non sono correlati ( tabella 2 )  . conseguenze della denuncia i radiologi e radioterapisti che avevano subito una denuncia per presunta malpractice sono stati invitati a riferire le sensazioni provate in relazione allaccaduto . 
apprendere di essere stati denunciati ha indotto in molti radiologi : sensazione di essere indifeso ( 39 , 0% ) , delusione ( 32 , 4% ) , angoscia ( 32 , 4% ) , umiliazione ( 19 , 0% )  . 
significance calculated with the two - tailed mann - whitney u test for nonparametric data entire group number ( % ) radiologists number ( % ) radiotherapists number ( % ) significance p value 251 ( 79.9 ) 167 ( 81.1 ) role department head division head staff physician other tabella 1 caratteristiche anagrafiche del campione esaminato e confronto tra i due sottogruppi ( radiologi e radioterapisti )  . 
al contrario , solo l11 , 3% dichiara di non avere riportato alcun danno . per quanto riguarda le conseguenze che la denuncia ha avuto sulla condotta professionale , il 39 , 0% degli intervistati ha ammesso di avere cambiato la propria condotta . 
the first was statistically associated with seven of the eight causes ( excessive workloads , insufficient time , obsolete equipment , lack of opportunity to consult with colleagues , inadequate continuing education , poor work organisation , tense and uncooperative work climate ) , whereas the second component was correlated only with imprecise requests from referring physicians . the two components have a clear logical construct in that the first expresses distress related to the workplace and the second expresses distress related to the external environment . by adding the responses to the seven questions making up the first component , an indicator of workplace distress can la maggior parte dei professionisti intervistati ritiene che il rischio di denuncia non riguardi solo i meno preparati ( 74 , 2% di pareri contrari ad una affermazione in tal senso , contro il 15 , 6% di favorevoli ) , e che le denunce siano imprevedibili e indipendenti dalla perizia personale ( 73 , 2% di approvazione a questa proposizione ) e possano colpire chiunque ( il 92 , 7% si pronuncia in tal senso )  . 
la prima di tali domande registra una differenza molto significativa ( p < 0 , 005 ) tra coloro che hanno subito una denuncia e gli altri , con i primi pi decisi a negare che il rischio di denuncia riguardi solo i meno preparati . in larga prevalenza , gli intervistati ritengono che gli anziani siano pi esperti dei giovani ( 65 , 4% favorevoli , 14 , 6% incerti , 20% contrari a questa affermazione )  . 
sono incerti sullaffermazione che i giovani possano essere pi aggiornati ( 8 , 9% del tutto daccordo , 43 , 9% parzialmente daccordo , 23 , 6% incerto , 23 , 5% contrario ) , ma tendono a pensare che i giovani facciano pi errori degli anziani ( 49 , 7% daccordo , 28% incerto , 22 , 3% contrario )  . nel complesso , la larga maggioranza del campione ( 73% ) si dichiara preoccupata di poter essere denunciata ; tale preoccupazione significativamente maggiore nel gruppo di chi ha gi subito una denuncia ( p < 0 , 018 )  . 
i radiologi / radioterapisti ritengono che il rischio di denunce spinga a modificare in senso difensivo la pratica medica ( 86 , 6% daccordo o del tutto daccordo )  . le cause delle denunce il fenomeno della malpractice medica sarebbe sostenuto da una serie di fattori ( tabella 3 ) , tra i quali i radiologi annoverano , in ordine di frequenza , lorientamento dei massmedia in senso non favorevole ai medici ( 65 , 3% del tutto daccordo , 23 , 2% daccordo , solo 5 , 7% contrario ) , la possibilit di ottenere cospicui risarcimenti ( 49% del tutto daccordo , 35% daccordo , 6% contrario ) e le dichiarazioni di altri medici ( 81% daccordo , 9% contrario )  . 
tra i fattori favorenti il rischio di denunce vengono indicati anche i cattivi rapporti con i pazienti ( concorda oltre il 71% ) e le pretese eccessive dei pazienti ( concorda il 60% )  . 
meno della met ( 40 , 5% ) attribuisce laumento delle denunce genericamente allo sfascio della sanit , mentre una percentuale analoga di specialisti ( 37 , 9% ) contrario a tale affermazione , ed il rimanente 21 , 7% incerto . alcune di queste risposte risentono significativamente delle esperienze personali . 
i medici che hanno subito una denuncia indicano con frequenza significativamente maggiore rispetto agli altri le responsabilit di altri medici ( p < 0 , 0001 ) , il ruolo dei mass media ( p < 0 , 002 ) , la ricerca di risarcimenti ( p < 0 , 019 )  . lerrore diagnostico tra i fattori che concorrono a determinare lerrore in radiologia e radioterapia ( tabella 4 ) vengono annoverati , in ordine di consenso , i ritmi di lavoro eccessivi ( vengono definiti molto importanti dal 67% degli intervistati e concorda sullimportanza un ulteriore 30% ) , i tempi insufficienti per la refertazione per i radiologi , o per i radioterapisti lelabo1075 a . 
it should be borne in mind that in identifying the causes of error , there were no significant differences between those who had been involved in civil or criminal proceedings and those who had not . possible solutions despite the fact that according to respondents the risk of malpractice litigation would lead radiologists to adopt defensive practices , only 34.7% believe that suggesting further examinations is an appropriate method for avoiding litigation , whereas 51.6% were not in favour of this type of behaviour . most respondents felt that constant adherence to guidelines , even when not convinced of their usefulness , would help to avoid malpractice litigation ( 51% ) , even though 18% were uncertain and 31% did not agree . 
in contrast to these last findings , with regard to the need or otherwise of informing the patient of the results of the radiological examination , 34% of radiologists believe it is better not to inform the patient in the event of an unfavourable diagnosis , because the patient may not be prepared to face such a diagnosis . 
a further 15% were uncertain how to respond , so the percentage of those who would always inform the patient even in the event of an unfavourable diagnosis fell to about 50% . opinions regarding the usefulness of clinical risk management techniques were divided . 
in this context , there was also a certain difference between those who have and those who have not been sued for alleged malpractice , with the former expressing greater faith in clinical risk management . 
tale disagio risulta inversamente correlato con la soddisfazione dal lavoro e con il livello di benessere psicofisico : al crescere del disagio per lambiente di lavoro si associano bassi livelli di soddisfazione ( correlazione inversa , coefficiente di segno negativo ) ed elevati livelli di malessere psicologico , misurati dal ghq - 12 ( correlazione diretta , coefficiente di segno positivo ) ( tabella 6 )  . 
importante sottolineare che nellidentificazione delle cause di errore non emergono differenze significative tra coloro che avevano subito una denuncia civile o penale rispetto a quelle degli altri colleghi non denunciati . le possibili soluzioni nonostante il fatto che secondo gli intervistati il rischio di denunce avrebbe spinto molti radiologi a modificare in senso difensivo la propria pratica , solo il 34 , 7% ritiene che per evitare denunce sia bene suggerire lesecuzione di ulteriori accertamenti , mentre il 51 , 6% contrario a tale atteggiamento . seguire sempre le linee guida , anche se non si convinti della loro reale utilit , consentirebbe di evitare il contenzioso secondo la maggior parte degli intervistati ( 51% ) , anche se , a questo riguardo , si rileva il 18% di incerti ed il 31% di contrari . 
questa domanda registra una differenza significativa tra coloro che hanno subito una denuncia e gli altri ( p < 0 , 038 ) , con una maggiore propensione a seguire acriticamente le linee guida da parte dei primi . con riferimento al consenso informato , l84 , 7% ritiene necessario raccoglierlo nei casi in cui la tecnica radiologica potrebbe comportare problemi per il paziente ( si noti che circa il 13% dei radiologi decisamente contrario a chiedere il consenso ) , mentre il 97 , 5% ritiene necessario informare in modo completo i pazienti . 
in us hospitals , the radiology department is among those that receive the highest number of claims for compensation due to diagnostic error [ 15 ] , and payments for incorrect radiological diagnosis are amongst the highest [ 16 ]  . 
the most recent data regarding the italian situation show that the risk of litigation for civil liability has become as significant for italian radiologists as it is in the united states , albeit with a lower incidence [ 1 ]  . considerations regarding the relevance of the problem led us to investigate the phenomenon among italian radiologists to determine their opinions on the topic and to assess the effects that civil and criminal proceedings have on physician health . 
as far as we know , this is the first attempt to shed light on the topic . this study has the limitation of not having been performed on a randomised population . 
nonetheless , the high representation of one category ( radiotherapists ) with respect to the national total , and the fact that there were no significant differences between responses provided by radiotherapists and those provided by radiologists , suggests that the study faithfully reflects the real situation . our study takes into consideration two different albeit closely related features : diagnostic errors and malpractice litigation , with the aim of assessing the consequences of these features on the health and professional behaviour of the study population . 
due to reasons linked to how errors are detected , data regarding medical error is highly uncertain italy there have been a number of estimates based on patient reporting , which place radiologists in a comparatively better situation than other specialists ( table 7 )  . 
the technical committee on clinical risk of the ministry of health , however , has dedicated special attention to the problem of error in radiology [ 18 ] , which is a clear sign of the significance of the problem . in our study , physicians who responded to the questionnaire clearly demonstrated their awareness of strategies for reducing radiological errors , particularly those which reason [ 19 ] classifies as latent errors errors that are inherent to the workplace . 
adequate time and workload and the availability of human resources and appropriate equipment are undoubtedly conditions that can enhance a policy of limiting the risk of clinical error in radiology as in other medical fields . our study appears to demonstrate a widespread and alarming situation of discomfort caused by the excessive workloads and insufficient time for reporting results or formulating treatment plans , poor work organisation and a noncooperative and tense working climate . 
added to this are staff shortages , which in many cases render consulting with colleagues for a second opinion practically impossible ; obsolete radiological equipment ; and the inability to be sufficiently up to date at the professional level . 
even though these factors were examined with reference to an abstract situano di comunicare al paziente lesito della indagine radiologica , il 34% dei radiologi ritiene che sia meglio non informare il paziente in caso di diagnosi infausta , perch questi potrebbe non essere pronto a riceverla , ed un ulteriore 15% palesa incertezze a questo quesito , per cui la percentuale di quanti informerebbero in ogni caso il paziente dei risultati dellindagine radiologica infausta scende al 50% circa . i pareri sullutilit delle tecniche di clinical risk management sono divisi . 
centodieci radiologi ( il 35% ) manifestano incertezza e i rimanenti sono equamente divisi tra quanti ritengono utili le tecniche di gestione del rischio clinico ( 33 , 1% ) e quanti invece temono che la segnalazione sistematica spontanea di errori e quasi errori ( near miss events ) possa aumentare il rischio di denunce contro i medici ( 31 , 9% )  . 
a questo proposito si manifesta anche una certa divaricazione tra chi ha subito una denuncia e chi no , con una dichiarata maggiore fiducia dei primi nella gestione del rischio clinico ; la differenza , tuttavia , non raggiunge significativit statistica ( p > 0 , 07 )  . oltre la met degli intervistati ritiene necessario un intervento del legislatore per limitare le denunce contro i medici ( concorda il 64 , 6% ) ; tale ipotesi vede maggiormente favorevoli coloro che hanno gi subito una denuncia ( p < 0 , 024 )  . discussione la diagnostica per immagini in usa la specialit a pi alto rischio di denunce per malpractice , dopo le discipline chirurgiche [ 14 ]  . 
sempre in usa in ospedale , il dipartimento di radiologia tra quelli che ricevono il maggior numero di denunce per errore diagnostico [ 15 ] e gli indennizzi per errata diagnosi radiologica sono tra i pi elevati in assoluto [ 16 ]  . 
i pi recenti dati riferiti al nostro paese consentono di affermare che il rischio di denuncia per responsabilit civile ha assunto per i radiologi italiani rilievo paragonabile a quello che si osserva negli stati uniti , anche se con incidenza inferiore [ 1 ]  . le considerazioni sulla rilevanza del problema ci hanno spinto ad indagare tra i medici radiologi italiani , al fine di conoscere le loro opinioni sullargomento , e di valutare gli effetti che le denunce civili e penali hanno sulla salute del medico . 
a nostra conoscenza , questo il primo tentativo di fare luce sullargomento . questo studio ha la limitazione di non essere stato svolto su una popolazione randomizzata ; tuttavia , lelevata rappresentazione di una categoria ( quella dei radioterapisti ) rispetto al totale nazionale , e il fatto che non emergano significative differenze nelle risposte fornite dai radioterapisti rispetto a quelle dei radiologi , inducono a ritenere che lo studio fornisca una buona rappresentazione della realt . la nostra ricerca ha preso in considerazione due aspetti diversi , anche se tra loro strettamente correlati : quella dellerrore diagnostico , e quello delle denunce per malpractice , al fine di valutare le conseguenze sullo stato di salute e sui comportamenti professionali . 
this is confirmed by the fact that the discomfort measured in this way is significantly and inversely correlated with job satisfaction ( measured by the warr questionnaire ) and directly correlated with the ghq - 12 score , which measures the level of psychological and physical well - being at the time of the study . 
it is recognised that caution is required when dealing with cross - sectional studies , which by definition are unable to provide information regarding causation . however , it appears likely that the experience of difficult evitato in assoluto , ma la sua frequenza pu e deve essere controllata e ridotta [ 17 ]  . 
i dati relativi agli errori medici sono , per considerazioni legate alle modalit di rilevazione degli errori stessi , estremamente aleatori ; nel nostro paese esistono alcune stime , basate sulle segnalazioni dei pazienti , che pongono la radiologia in una situazione comparativamente migliore di altre specialit ( tabella 7 )  . 
la commissione tecnica sul rischio clinico del ministero della salute ha tuttavia dedicato una specifica attenzione al problema dellerrore in radiologia [ 18 ] a testimonianza del rilievo del problema.nella nostra ricerca , i medici intervistati hanno dimostrato di sapere con chiarezza quali possano essere le strategie per la riduzione degli errori radiologici , soprattutto di quelli che reason [ 19 ] classifica come errori latenti , 1078 a . 
while taking great care in interpreting the data , the association of litigation with radiologist age alone and not with other associated variables , such as years of professional experience or hierarchical role in the healthcare system , suggests that ageing and the degenerative conditions associated with it may affect the radiologists ability to interpret radiological findinsiti nellorganizzazione del lavoro : la congruit dei tempi e dei ritmi di lavoro e la disponibilit di risorse umane e strumentali adeguate , sono sicuramente le condizioni che possono favorire una politica di controllo del rischio di errori clinici in radiologia come nelle altre discipline mediche . dal nostro lavoro pare emergere una condivisa e allarmante situazione di disagio per i ritmi di lavoro i tempi insufficienti per la refertazione o per lelaborazione dei piani di cura , la cattiva organizzazione del lavoro , il clima lavorativo non collaborativo o carico di tensioni . 
a ci si aggiunge la carenza di organico , che determina in molti casi limpossibilit di consultare i colleghi per ottenere una seconda opinione , lobsolescenza delle macchine radiologiche , limpossibilit di un sufficiente aggiornamento profes1079 a . 
this is , of course , merely a hypothesis at this stage and would need to be verified on the basis of larger and possibly longitudinal studies . clinical error is often at the origin of malpractice claims , even though there is no biunique correspondence between the two phenomena , such that there may be both serious errors that do not give rise to litigation and litigation not substantiated by errors . it is often claimed that the term malpractice is often inappropriately used , as most legal proceedings against physicians are groundless and are resolved with acquittal . 
in any event , the blame associated with negligence , or alleged negligence , can significantly affect the physician involved . physicians sued for alleged malpractice perceive the proceedings as a threat to their integrity and can react in a dysfunctional fashion [ 20 ]  . 
these ailments can include a sense of isolation , low self - esteem , tension , depression , insomnia and other emotional problems , including anxiety attacks , irritability , frustration , asthenia , a reduction in sexual desire , loss of appetite and apathy . our study confirms that radiologists involved in proceedings for civil or criminal liability present a series of psychological and physical symptoms related to the traumatic event , symptoms that are very similar to those experienced after physical assault and that in some cases can give rise to posttraumatic stress disorder [ f43.1 according to the diagnostic 1080 sionale . 
anche se tali fattori sono indagati con riferimento ad una situazione astratta , intuibile che ciascuno dei radiologi nel rispondere alle domande abbia fatto riferimento soprattutto alla sua propria esperienza professionale . 
a conferma di ci , si osserva che il disagio cos misurato si correla significativamente , e in modo inverso , con la soddisfazione dal lavoro ( misurata dal questionario di warr ) e direttamente con il punteggio del ghq - 12 che misura il livello di malessere psicofisico al momento dellindagine . 
pur con la cautela che si deve osservare in tutte le indagini trasversali , che per definizione non consentono di inferire sulla causalit , suggestiva lipotesi che lesperienza di condizioni lavorative difficili e conflittuali ed il disagio lavorativo che ne consegue , oltre a determinare un aumento degli errori , siano allorigine di una minore soddisfazione dei radiologi e di un eccessivo stress professionale che si traduce in una alterazione dei livelli di benessere psicofisico . tra le possibili cause di errore clinico occorre ricordarne una che , seppure non direttamente indagata nel nostro questionario , ha dimostrato una significativa associazione con loccorrenza di denunce : let anagrafica . 
pur con ogni cautela interpretativa , lassociazione delle denunce solo con let del radiologo e non con grandezze ad essa correlate , come gli anni di esperienza professionale o il ruolo gerarchico nellorganizzazione sanitaria , inducono a formulare lipotesi che linvecchiamento e le condizioni morbose degenerative ad esso associate possano determinare alterazioni a . 
only a minority of respondents indicated the absence of symptoms , and it is difficult to say whether they were subjects particularly resistant to this type of stress or whether they were subjects in whom the process of denial was particularly strong . regardless , our study shows that being sued for alleged malpractice leads to a number of significant changes in ones opinion of the problem and of the measures enacted to protect oneself . 
many physicians who had been sued for alleged malpractice reported a change in their work behaviour , with the adoption of defensive practices that may even lead to refusal of seriously ill patients or patients perceived as dangerous , or to less empathy and at times aversion towards such patients . 
some radiologists in the united states have abandoned certain procetable 6 correlation between work distress ( principal component 1 of causes of error ) , job satisfaction ( warr scale ) and psychological and physical well - being [ general health questionnaire ( ghq ) - 12 ]  . 
correlations with a positive sign ( + ) indicate that one variable increases as another increases , whereas a negative ( ) sign indicates an inverse correlation where high values of one variable are associated with low values of the other disagio ghq - 12 warr pearson correlatino sig . 
in grassetto le correlazioni significative . le correlazioni di segno positivo indicano che una variabile cresce al crescere dellaltra , quelle di segno negativo indicano una correlazione inversa , valori alti di una variabile si associano con valori bassi dellaltra ghq - 12 pearson + 0 , 161 ( * ) disagio coefficiente di correlazione sig . 
si tratta , al momento , solo di unipotesi che dovr essere verificata sulla base di studi di maggiori dimensioni , possibilmente a carattere longitudinale . lerrore clinico spesso allorigine delle denunce per malpractice , anche se tra i due fenomeni non vi corrispondenza biunivoca , potendo esistere sia errori gravi che non danno luogo a denunce , che denunce non sostanziate da errori . da pi parti si sostiene che il termine malpractice usato spesso in modo improprio , giacch la gran parte delle azioni di contenzioso contro i medici sono infondate , e i processi si risolvono con assoluzioni . 
generally speaking , however , in numerous cases , the attention required to follow the proceedings , the lengthy times required to prepare the defence and the constant rumination over the event place the physician in social isolation , which can even disrupt family relations . the cross - sectional nature of our study , which was based on the reporting of consequences to events at times quite remote , did not enable assessment of the effects malpractice stress can have on current health . 
it is nonetheless clear that this specific stress factor is added to the numerous causes of professional strain , many of which are specific to the medical profession , such as coping with the suffering and death of patients and the difficulty in providing a service of constant quality in a system of reduced resources [ 22 ]  . 1082 pria integrit e possono reagire in modo disfunzionale [ 20 ]  . indipendentemente dallesito del confronto giudiziario , il medico pu sperimentare una serie di segni e sintomi , che vanno sotto il nome di sindrome dello stress da malpractice o malpractice stress sindrome ( mss )  . 
tali disturbi comprendono : senso di isolamento , riduzione dellautostima , tensione , depressione , insonnia ed altri disturbi emozionali , a volte attacchi di angoscia , irritabilit , frustrazione , talora astenia , riduzione della libido , inappetenza ed apatia . nella nostra indagine si conferma che i radiologi sottoposti a denunce di responsabilit civile o penale hanno presentano una serie di sintomi psicologici e fisici in relazione con tale evento traumatico , del tutto analoghi e sovrapponibili a quelli che si verificano dopo unaggressione fisica e che possono dare luogo , in alcuni casi , ad un disturbo posttraumatico da stress ( condizione morbosa classificata f43.1 nel dsm - iv - tr ) [ 21 ]  . 
solo una minoranza non riferisce sintomi ma , allo stato , non si pu dire se si tratti di soggetti particolarmente resistenti a tale tipo di stress o di casi in cui il sistema di negazione particolarmente strutturato . comunque si osserva nella nostra ricerca che lessere stati denunciati induce alcuni significativi cambiamenti delle proprie opinioni sul problema della malpractice e sulle misure messe in atto per difendersi da essa . 
molti tra i medici denunciati riferiscono un cambiamento delle proprie abitudini di lavoro , con ladozione di pratiche difensive che talora giungono al rifiuto di pazienti particolarmente gravi o percepiti come pericolosi o una ridotta empatia e talora avversione verso taluni pazienti . 
negli stati uniti stato osservato che alcuni tendono ad abbandonare determinate procedure , sulle quali hanno minore controllo o che ritengono possano esporli maggiormente a contenzioso : il caso , ad esempio , della mammografia [ 3 , 5 ]  . 
fortunatamente , nella maggior parte dei casi la controversia che ha dato luogo alla denuncia pu essere risolta in sede extragiudiziale , o non passa il vaglio delle indagini preliminari . 
ma in generale , in non pochi casi lattenzione posta al processo , i lunghi tempi necessari per organizzare la difesa e la ruminazione sullaccaduto pongono il medico in una situazione di isolamento sociale che pu talora giungere a turbare anche le relazioni familiari . il carattere trasversale del nostro studio , basato sul recupero anamnestico delle conseguenze di eventi talora remoti , non consente di valutare il rilievo che lo stress da malpractice esplica sullo stato di salute attuale . 
tuttavia evidente che questo specifico fattore di stress si aggiunge alle molteplici cause di tensione o strain professionale , molte delle quali specifiche della professione medica , come il confronto con le sofferenze e la morte dei pazienti e le difficolt nel fornire un servizio di qualit costante in un contesto di riduzione delle risorse [ 22 ]  . poich la possibilit di confrontarsi efficacemente con tale tipo di stress discende direttamente dal grado di consapea . 
however , it does appear that radiologists hold little faith in the techniques of clinical risk management and are not fully aware of the need for informed consent and the relative legal implications . 
such training would need to begin at university and be followed up in specialty courses , where ideally each physician would be made aware of the problems related to clinical error and the procedures aimed at minimising and managing it . 
similarly desirable would be a commitment to continuing education , possibly to be organised and carried out with the assistance of scientific and professional associations , to enable radiologists and radiotherapists to compare their experiences and , through such comparison , improve their own abilities for limiting error and its consequences . note : data regarding the opinions of radiotherapists were partially communicated at the xvi national congress a.i.r.o. , associazione italiana radioterapia oncologica . 
malpractice in radiation oncology : the physicians experience volezza del problema e dal possesso delle corrette tecniche di controllo , occorre domandarsi se i radiologi italiani siano culturalmente attrezzati per controllare le problematiche dellerrore diagnostico e lo stress da malpractice . in base ai dati forniti da questa indagine possibile dire che i radiologi italiani hanno una sufficiente conoscenza del tema delle controversie legali e dei fattori che possono determinare un aumento della frequenza degli errori medici . non sembra per che essi credano alla efficacia delle tecniche del clinical risk management , n che siano pienamente consapevoli della necessit del consenso informato e della relativa giurisprudenza . 
tale formazione dovrebbe essere iniziata nelluniversit e segnatamente nei corsi di specializzazione , nei quali sarebbe auspicabile che ciascun medico fosse messo a conoscenza delle problematiche legate allerrore clinico e delle procedure tendenti a minimizzarlo e gestirlo . sarebbe poi opportuno un impegno di aggiornamento permanente , da operarsi eventualmente con il concorso delle societ scientifiche e degli ordini professionali , per consentire agli specialisti radiologi e radioterapisti di confrontare le proprie esperienze e migliorare in questo confronto la propria capacit di controllo sullerrore e sulle sue conseguenze . i dati relativi alle opinioni dei radioterapisti sono stati parzialmente comunicati al xvi congresso nazionale a.i.r.o. , associazione italiana radioterapia oncologica , lecce , 21 - 24 ottobre 2006 . 
particularly interesting in view of the expected future developments is the section on contrast - enhanced sonography for the study of tumour neoangiogenesis . the second chapter is devoted to the examination technique and basic sonographic semiotics . 
of great value to radiologists in training , the chapter is intended as a necessary step to allow readers to fully understand the clinical issues discussed in chapter three . the third chapter is the heart of the book . 
as a result , this chapter truly meets the needs of ultrasound radiologists who are faced with a whole range of oncological issues in their daily clinical practice . the fourth chapter deals with ultrasound - guided interventional procedures in oncology . 
one section is given over to the description , complete with technical details , of diagnostic procedures such as fine - needle aspiration or biopsy of superficial and deep lesions , and the positioning of drains . 
the final section focuses on the use of ultrasound to assess and monitor lesions after treatment . in conclusion , this book is a complete , exhaustive and easy - toread textbook on ultrasound in oncology . lopera in volume singolo si propone come un aggiornato , completo ed esauriente testo - atlante di ecografia focalizzato sulle applicazioni cliniche della metodica in campo oncologico . la capacit di sintesi degli autori permette di ritrovare nelle 395 pagine di questo libro la trattazione di tutte quelle problematiche che , nella pratica clinica , il medico ecografista si trova ad affrontare in campo oncologico sia con intenti diagnostici che terapeutici . il libro articolato in quattro capitoli . il primo capitolo presenta le possibilit della moderna ecografia in oncologia . 
particolarmente interessante per gli attesi sviluppi futuri il paragrafo dedicato in questo capitolo allo studio della neoangiogenesi tumorale con impiego di mezzi di contrasto in ecografia . il secondo capitolo dedicato alla metodologia dindagine e alla semeiotica ecografica elementare . 
questo capitolo rappresenta un passaggio obbligato , voluto dagli autori , per poter comprendere appieno le problematiche cliniche successivamente trattate nel capitolo tre . il terzo capitolo dunque il cuore del libro . 
il capitolo quindi realmente capace di soddisfare le richieste del medico ecografista per fronteggiare le svariate problematiche cliniche oncologiche della pratica clinica quotidiana . il quarto capitolo tratta le procedure interventistiche ecoguidate in campo oncologico . 
viene dedicato spazio , con particolari di tecnica , alle procedure diagnostiche quali lagoaspirazione o la biopsia di lesioni superficiali e profonde nonch al posizionamento di drenaggi . vengono quindi descritte le possibilit di guida ecografica di procedure di trattamento delle lesioni epatiche quali la alcolizzazione e la termoablazione . 
mollet1 1dipartimento di radiologia e cardiologia , erasmus medical center , rotterdam , holland 2dipartimento di radiologia e di cardiologia , azienda ospedaliero - universitaria di parma , viale rustici , 2 , i - 43100 parma , italy 3dipartimento di radiologia , universit degli studi di verona , italy 4unit di riabilitazione cardiovascolare , fondazione don gnocchi onlus , parma , italy 5dibimel , sezione di scienze radiologiche , universit di palermo , palmermo , italy 6dipartimento di cardiologia , ospedale versilia , lido di camaiore viareggio , viareggio , italy correspondence to : f . 
we studied 72 patients ( 38 men , 34 women , mean age 53.98.0 years ) with atypical or typical chest pain and stratified in the lowto intermediate risk category . msct - ca ( sensation 64 cardiac , siemens , germany ) was performed after iv administration of 100 ml of iodinated contrast material ( iomeprol 400 mgi / ml , bracco , italy )  . 
msct - ca scored 15 false positives on a per - segment base , which affected only marginally the per - patient performance ( only one false positive )  . 
we concluded that 64 - slice ct - ca is a diagnostic modality with high sensitivity and negative predictive value in patients at low to intermediate risk . key words multislice computed tomography ct coronary angiography conventional coronary angiography coronary artery disease 64 - slice ct low cardiovascular risk riassunto obiettivo . 
sono stati studiati 72 pazienti ( 38 maschi , 34 donne , et media 53 , 98 , 0 anni ) che presentavano dolore toracico atipico o angina pectoris stabile e che venivano stratificati nella categoria del rischio basso - intermedio . 
sensibilit , specificit , valore predittivo positivo e negativo dellac - tc nella determinazione delle stenosi significative utilizzando unanalisi per segmenti sono risultate , rispettivamente , del 100% , 98 , 6% , 71 , 2% e 100% . 
this is related to the fact that validation studies have been performed on candidates for conventional coronary angiography ( cag ) and therefore on patients with typical symptoms , a high risk level and a positive exercise tests . 
the recent guidelines on stable angina of the european society of cardiology have added ct to the diagnostic algorithm and , with a low level of evidence ( iib ) , suggest that the technique be used in patients with low to intermediate risk and with doubtful stress test results or inability to undergo stress testing [ 18 ]  . very few studies have investigated the diagnostic performance of msct - ca in patients at low to intermediate risk [ 19 ]  . 
the aim of this study was to evaluate the diagnostic accuracy of 64 - slice msct - ca for detecting significant stenoses in a population of patients at low to intermediate risk . materials and methods patient population between march 2005 and march 2006 , we recruited 72 patients ( 38 men , 34 women , mean age 53.98.0 years ) with a low or intermediate cardiovascular risk atypical chest pain ( 26 / 72 ; 36.1% ) or exertional angina ( 46 / 72 ; 63.9% ) , and positive , doubtful or inconclusive stress electrocardiogram ( ecg ) who were scheduled for cag to determine the presence and extent of coronary disease . 
only patients with sinus rhythm , those who had never undergone percutaneous angioplasty or surgical by - pass grafting and those who were able to hold their breath for at least 12 s were included in the study . 
our institutions ethics committee approved the study protocol , and all patients provided their informed consent . patients with a heart rate ( hr ) above 65 bpm were administered , if no contraindications were present , a single oral dose patient preparation langiografia coronarica ( ac ) con tomografia computerizzata ( tc ) spirale multistrato ( ac - tc ) una nuova modalit diagnostica per la rilevazione delle stenosi coronariche significative ( riduzione del calibro del lume > 50% ) [ 17 ]  . lelevata sensibilit e valore predittivo negativo costituiscono uno dei dati pi coerenti nella recente letteratura di questa metodica [ 317 ]  . 
le recenti linee guida della societ europea di cardiologia sullangina stabile introducono la tc nellalgoritmo diagnostico e con un livello basso di evidenza ( iib ) suggeriscono che la tc possa essere utilizzata nei pazienti a rischio basso - intermedio nei quali i test provocativi siano dubbi o non eseguibili [ 18 ]  . 
scopo dello studio di valutare laccuratezza diagnostica della ac - tc a 64 strati nella a rilevazione di stenosi significative in una popolazione di pazienti a rischio basso - intermedio . materiali e metodi popolazione studiata dal marzo 2005 al marzo 2006 , sono stati arruolati 72 pazienti ( 38 maschi , 34 femmine , et media 53 , 98 , 0 anni ) con rischio cardiovascolare basso o intermedio , con dolore toracico atipico ( 26 / 72 ; 36 , 1% ) o angina da sforzo ( 46 / 72 ; 63 , 9% ) , e ecg da stress positivo / dubbio / inconclusivo , programmati per lesecuzione di unangiografia coronarica convenzionale allo scopo di determinare la presenza e lestensione della malattia coronarica . 
solo i pazienti con ritmo sinusale , mai sottoposti ad angioplastica percutanea o ad intervento chirurgico per il posizionamento di by - pass e capaci di trattenere il respiro per almeno 12 secondi , sono stati inclusi nello studio . 
i pazienti nei quali esistevano delle controindicazione assolute alla somministrazione endovenosa di mezzo di contrasto iodato ( ad esempio , allergia nota , insufficienza renale o disordini tiroidei ) sono stati esclusi dallo studio . 
a few minutes before the scan , patients received sublingual isosorbide dinitrate ( carvasin 5 mg , wyeth lederle )  . scan protocol and image reconstruction the ct study was performed with a 64 - slice scanner ( sensation 64 , siemens , forchheim , germany )  . 
images were reconstructed with the following parameters : effective slice thickness 3 mm , reconstruction increment 1.5 mm , field of view ( fov ) 150180 mm , dedicated convolution filter for the calcium score . 
 dedicated software ( windose , medical physics institute , erlangen , germany ) was used to calculate the radiation dose delivered during the angiographic scan ( mean value 15.2 msv for women and 21.4 msv for men )  . 
patients were administered 100 ml of iodinated contrast material ( iomeprol , iomeron 400 , bracco , milan ) at a rate of 5 ml / s via an automatic injector ( stellant , medrad , pittsburgh , pa , usa ) connected to a 20 - gauge needle cannula previously positioned in an antecubital vein [ 5 ]  . to optimise opacification of the coronary arteries , the bolus - tracking technique ( care bolus , siemens , forchheim , germany ) was used to synchronise the arrival of the contrast agent in the coronaries with the beginning of the scan [ 5 , 20 ]  . 
the temporal windows used were midand end diastolic ( from 300 to 450 ms before the following r wave and / or from 60% to 70% of the r - r interval )  . 
ai pazienti con una frequenza cardiaca superiore a 75 bpm stata somministrata per via orale una dose addizionale di 1 mg di lorazepam ( temesta , wyeth - ayerst )  . 
pochi minuti prima della scansione stato somministrato isosorbide dinitrato sublinguale ( carvasin 5 mg , wyeth lederle )  . protocollo di scansione e ricostruzione delle immagini per lo studio mediante tc stato utilizzato uno scanner a 64 strati ( sensation 64 , siemens , forchheim , germania )  . 
le immagini sono state ricostruite con i seguenti parametri : spessore di strato effettivo 3 mm , incremento di ricostruzione 1 , 5 mm , campo di vista ( fov ) 150180 mm , filtro di convoluzione dedicato per il calcium score . 
non stata utilizzata la modulazione prospettica dellamperaggio al fine di non compromettere leventuale necessit di eseguire ricostruzioni con finestra temporale posizionata in fase tele - sistolica . utilizzando un software dedicato ( windose , istituto di fisica medica , erlangen , germania ) stata calcolata la dose di radiazioni ionizzanti alla quale i pazienti sono stati esposti durante la scansione angiografica ( valore medio 15 , 2 msv per le donne e 21 , 4 msv per gli uomini )  . 
la tecnologia con doppia macchia focale oscillante sullasse z ( flying focal spot ) permette di sovra - campionare due volte per ogni posizione angolare del tubo radiogeno le 32 file di detettori dello spessore minimo di 0 , 6 mquesta geometria di scansione consente di ottenere unimmagine con voxel isotropico di 0 , 4 mm3 . 
the reconstruction algorithm applied acquires the data during a single heartbeat obtained during half a rotation of the x - ray tube . quantitative coronary angiography ( cag ) cag was performed within 2 weeks of the msct - ca study . 
a single observer , unaware of the msct - ca results , identified the coronary segments by using a 17 - segment classification modified from that provided by the american heart association ( aha ) [ 21 ]  . 
all segments , regardless of diameter , were included in the comparison with msct - ca . segments were classed as nonsignificantly stenotic ( normal or with wall irregularities or with < 50% lumen reduction ) or significantly stenotic ( 50% lumen reduction )  . 
cases of disagreement between the two observers were resolved by consensus . image quality was assessed on each segment and classified as good ( no motion artefacts or severe calcification ) , adequate ( presence of artefacts degrading the image but not preventing evaluation with an acceptable level of diagnostic confidence ) or poor ( artefacts allowing image evaluation with low diagnostic confidence )  . statistical analysis the diagnostic performance of msct - ca for detecting significant atherosclerotic coronary lesions , using cag as the reference standard , is reported below in terms of sensitivity , specificity and positive and negative predictive value , with 95% confidence intervals ( cis ) calculated using binomial expansion . 
i dati della scansione angiografica sono stati ottenuti durante una singola acquisizione della durata di 12 , 20 , 9 s . sono state effettuate delle ricostruzioni retrospettive basate sul segnale ecg per ottenere una qualit dellimmagine priva di artefatti da movimento . 
le finestre temporali utilizzate sono state la fase mesoe tele - diastolica ( da 300 a 450 ms prima della successiva onda r e / o dal 60% al 70% dellintervallo r - r )  . 
quando ritenuto necessario ( ad esempio , in caso di persistente movimento cardiaco residuo che riduce la qualit diagnostica dellimmagine ) sono state analizzate altre ricostruzioni in differenti finestre temporali di ricostruzioni del ciclo cardiaco , generalmente dal 25% al 35% dellintervallo r - r ( oppure + 225 ms , + 275 ms , e + 325 ms dopo la precedente onda r )  . 
lalgoritmo di ricostruzione utilizzato preleva i dati derivanti da un singolo battito cardiaco ottenuto durante met rotazione del tubo radiogeno . angiografia coronarica quantitativa ( cag ) la cag stata eseguita entro 2 settimane dallac - tc . 
un singolo osservatore , non a conoscenza dei risultati dellactc , ha identificato i segmenti coronarici utilizzando una classificazione in 17 segmenti modificata da quella fornita dallamerican heart association [ 21 ]  . 
i segmenti sono stati classificati come non significativamente stenotici ( normali o con irregolarit della parete o con riduzione del diametro luminale < 50% ) o significativamente stenotici ( riduzione del lume coronarico 50% )  . le stenosi coronariche sono state quantificate in due proiezioni ortogonali utilizzando un algoritmo di misurazione validato per langiografia coronarica ( caas , pie medical , maastricht , olanda )  . valutazione dellimmagine di ac - tc tutte le scansioni sono state analizzate indipendentemente da due osservatori , che non erano a conoscenza dei risultati dellangiografia coronarica convenzionale . 
most patients with at least one stenosis > 50% ( 13 / 71 , 18% ) had single - vessel disease . table 2 shows the additional data provided by msct . coronary calcium scores according to agatston were very low ( 47.790.4 ) , reflecting the low disease prevalence in the study population and the low coronary disease burden . 
oral - blockers ( used in over 90% patients ) before the examination were effective in reducing mean hr by approximately 10 bpm ( from approximately 70 bpm to 60 bpm during the angiographic scan )  . interobserver variability , expressed as a k - value , for detecting significant lesions was 0.98 ( corresponding to excellent on a semiquantitative scale )  . 
image quality on a per - segment basis was classified as good in 98.5% of cases , moderate in 1.2% and poor in 0.4%. a total of 1 , 098 segments were included in the comparison with cag . 
la maggior parte dei pazienti con almeno una stenosi > 50% ( 13 / 71 ; 18% ) erano mono - vasali . in tabella 2 vengono riportate multiple informazioni ulteriori ricavate dalla tc . 
il calcium score coronarico secondo agatston risultato molto basso ( 47 , 790 , 4 ) , rispecchiando la bassa prevalenza di malattia nella popolazione ed il basso carico di malattia coronarica . 
leffetto della terapia beta - bloccante orale ( utilizzata in oltre il 90% dei pazienti ) prima dellesame si dimostrato efficace nel ridurre in media di circa 10 bpm la frequenza cardiaca media ( da circa 70 bpm a circa 60 bpm durante la scansione angiografica )  . le variabilit inter - osservatore , espressa come valori di k , per lindividuazione delle lesioni significative risultata 0 , 98 ( corrispondente a ottima su una scala semi - quantitativa )  . 
la qualit delle immagini su base segmentale stata classificata come buona nel 98 , 5% , moderata nel 1 , 2% e bassa nel 0 , 4% . sono stati inclusi per il confronto con langiografia coronarica convenzionale 1098 segmenti . 
il numero di falsi positivi ha un impatto particolarmente evidente nella valutazione per segmento ( n = 15 ) e per vaso ( n da 2 a 5 eccetto che per il tronco comune ) , mentre nella valutazione per paziente ( n = 1 ) non si evidenzia particolarmente . se la ac - tc fosse stata utilizzata come gate - keeper per langiografia coronarica , sarebbe stata indotta una cag in un paziente che non avrebbe poi dimostrato stenosi significative , mentre tutti i pazienti con stenosi sarebbero correttamente stati inviati alla cag ( tabella 4 )  . 
nessun paziente con malattia coronarica significativa sarebbe erroneamente stato classificato come senza stenosi coronariche significative . discussione lac - tc con apparecchiature di ultima generazione ( 64 strati ) permette una valutazione dellintero albero coronarico ( vasi principali e loro rami collaterali e marginali ) [ 317 ]  . 
gli studi di validazione condotti fino a questo momento si sono focalizzati su popolazioni di pazienti ad elevata probabilit pre - test di malattia coronarica ( range 50%80% circa )  . 
no patient with significant coronary disease would have been misclassified as not having significant coronary stenoses . discussion msct - ca with the latest - generation scanners ( 64 slice ) allows evaluation of the entire coronary tree ( main vessels and their collateral and marginal branches ) [ 317 ]  . 
infatti , sia nelle recenti linee guida della societ europea di cardiologia [ 18 ] , sia in un recente documento di consenso sullappropriatezza delle indicazioni della cardio - tc e cardio - rm [ 22 ] , viene suggerito che un utilizzo congruo della metodica sarebbe nei casi in cui il paziente sia a rischio basso - intermedio ed i test provocativi siano dubbi , non fattibili o inconclusivi . questa popolazione non stata ancora studiata approfonditamente nella letteratura nota se non in una esperienza di nikolaou et al . 
the sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) are expressed as percentages , followed by 95% confidence intervals ( cis ) calculated with binomal expansion in parentheses . tp , true positive ; tn , true negative ; fp , false positive ; fn , false negative ; rca , right coronary artery ; lmt , left main trunk ; lad , left anterior descending artery ; lcx , left circumflex artery tabella 3 valori di accuratezza diagnostica della tc a 64 strati 1045 sensibilit specificit il criterio di valutazione la stenosi coronarica significativa che corrisponde ad una riduzione del lume 50% . 
i valori di sensibilit , specificit , valore predittivo positivo ( vpp ) e valore predittivo negativo ( vpn ) , sono espressi in percentuale seguiti da parentesi con gli intervalli di confidenza del 95% calcolati con espansione binomiale . vp , veri positivi ; vn , veri negativi ; fp , falsi positivi ; fn , falsi negativi ; acd , arteria coronaria destra ; tcs , tronco comune sinistro ; ada , arteria coronaria discendente anteriore ; acx , arteria coronaria circonflessa f . 
il diagramma mostra come i pazienti si sono distribuiti sulla base delle informazioni diagnostiche della coronarografia tc ( ac - tc )  . i pazienti con ac - tc negativa sono poi risultati tutti negativi alla coronarografia convenzionale . 
both the recent guidelines of the european society of cardiology [ 18 ] and a recent consensus statement on the appropriateness criteria for cardiac ct and cardiac magnetic resonance imaging ( mri ) [ 22 ] suggest that appropriate use of msct - ca would be in cases of patients at low to intermediate risk and doubtful , unfeasible or inconclusive stress tests . 
rimane comunque preservato anche in questo lavoro lelevato valore predittivo negativo [ 19 ]  . nel nostro studio esistono diversi elementi di innovazione . il primo riguarda la tecnologia a 64 strati non precedentemente decritta in questo campo di applicazione clinica . 
however , this study focusing on a low - risk population had the problem that , although a reasonable number of patients were enrolled ( n = 72 ) , the 6.9% disease prevalence ( number of positive cases = 5 ) did not make it possible to transfer the results into clinical practice ( very wide cis ) [ 19 ]  . 
lalbero coronarico risultato esplorabile in toto in tutti i pazienti ed i falsi positivi sono stati tutti generati in pazienti con maggiore carico di malattia dove altre lesioni significative ( > 50% ) erano gi presenti . 
sections obtained orthogonally to the vessel axis in the planes shown in c ( 1 , 2 and 3 ) show the portion above the stenosis , the stenosis , and the portion below the stenosis . 
le sezioni ortogonali allasse del vaso effettuate sui piani indicati in c ( 1 , 2 e 3 ) indicano in tratto a monte , la stenosi , ed il tratto a valle della lesione . 
the entire coronary tree was assessable in all patients , and false positive results all occurred in patients with a greater disease burden , where other significant lesions ( > 50% ) were already present . false positive results had a particularly evident impact in the evaluation per segment and per vessel but not in the evaluation per patient . 
this is explained by the fact that most false positive results occurred in patients with a greater disease burden who already had other significant stenoses located in other coronary segments ( that is , in the seven patients with multivessel disease , equal to 10% of the total population )  . calcifications did not produce excessive artefacts in the population studied , whose calcium score was generally low ( lower mean age and more female patients compared with other studies )  . 
this , together with the relatively low prevalence of disease , is likely to account for the optimal results achieved in the per - segment , per - vessel and per - patient evaluation . 
in fact , in our population , only five significant lesions were present in the distal segments ( according to the coronary segmental classification aha : 4 - 8 - 9 - 10 - 14 - 15 )  . 
this explanation is also suple calcificazioni non sono state motivo di eccessivi artefatti nella popolazione studiata che era caratterizzata da un basso calcium score globale ( et media pi bassa i altri studi e popolazione femminile molto pi rappresentata )  . 
la malattia nei vasi distali molto pi probabile nei pazienti ad alto rischio ed in particolare nei diabetici ( che sono stati esclusi dalla nostra popolazione )  . infatti , nella nostra popolazione solo 5 lesioni significative erano presenti nei segmenti distali ( secondo la classificazione dei segmenti coronarici aha : 4 - 8 - 9 - 10 - 14 - 15 )  . 
questa spiegazione sostenuta anche dalla elevatissima concordanza tra gli osservatori , sostenuta dalla bassa prevalenza di malattia ( vedi anche valore medio di calcium score )  . come valore aggiunto della ac - tc abbiamo voluto includere la valutazione dei parametri volumetrici del ventricolo sinistro estrapolati contestualmente allesame angiografico . 
however , this reduces the systems flexibility in the image reconstruction phase , as it is no longer possible to use end - systolic reconstructions , which in many cases are diagnostically decisive ( in our population , the operators found it necessary to also use the systolic phase in 54% of cases , which in 7% of cases was recognised as the principal reconstruction phase )  . the use of - blockers may be considered a limitation , even though it is still not clear how many patients have to be excluded from msct - ca in clinical practice because of contraindications to the administration of - blockers combined with impossibility of administering other negative chronotropic agents . 
patients with an initial heart rate above 70 bpm received a dose of - blockers before the scan , which allowed mean hr to be lowered to 60.6 bp future improvements in temporal resolution will progressively reduce the need for b - blockers . 
reports have already been published on the use of dual - source ct systems , characterised by a temporal resolution of 83 ms [ 3436 ]  . ottenere una ragionevole prevalenza di malattia ed una realistica rappresentazione della pratica clinica abbiamo dovuto includere pazienti a rischio basso - intermedio con ecg stress dubbio - inconclusivo , ma anche quelli con test provocativo positivo . 
in aggiunta , questa costituisce la popolazione nella quale , essendo la probabilit di malattia bassa , i test provocativi hanno una performance ridotta [ 30 , 31 ]  . 
inoltre , la presenza di sintomi tipici o atipici non un fattore condizionante la mortalit dei pazienti sottoposti a stress ecg [ 32 ]  . la dose di radiazioni stimata durante lac - tc ( 15 , 221 , 4 msv ) superiore a quella di una cag . 
lesposizione a radiazioni ionizzanti pu essere ridotta utilizzando la modulazione prospettica della corrente del tubo radiogeno [ 33 ]  . tuttavia , in questo modo , si riduce la flessibilit del sistema in fase di ricostruzione delle immagini . 
non pi possibile infatti utilizzare a fase tele - sistolica di ricostruzione che in molti casi pu risultare dirimente dal punto di vista diagnostico ( nella nostra popolazione stato necessario secondo gli operatori utilizzare anche la fase sistolica nel 54% dei casi e nel 7% dei casi la fase sistolica era riconosciuta come la fase principale di ricostruzione )  . lutilizzo di - bloccanti pu essere considerato una limitazione anche se ad oggi non stato chiarito quanti siano nella pratica clinica i pazienti che devono essere esclusi dalla scansione mediante ac - tc sulla base del criterio della contro - indicazione alla somministrazione dei - bloccanti concomitante alla impossibilit di somministrare altri farmaci cronotropi negativi . 
i pazienti con una frequenza cardiaca iniziale al di sopra di 60 bpm hanno ricevuto una dose di - bloccanti prima della scansione che ha permesso di ridurre la frequenza cardiaca media a 60 , 6 bpi miglioramenti futuri in termini di risoluzione temporale renderanno progressivamente meno necessario lutilizzo di betabloccanti . 
in questo campo siamo gi a conoscenza di esperienze mediante apparecchiature a doppia sorgente caratterizzate da risoluzione temporale in hardware di 83 ms [ 3436 ]  . conclusioni conclusions noninvasive 64 - slice msct - ca is a technique with a high diagnostic accuracy in the detection of significant coronary stenoses in patients with atypical pain or stable angina and low to intermediate risk . 
given the low disease prevalence in this population , msct - ca may become an ideal screening tool to exclude the presence of critical coronary disease and avoid the use of cag in a subset of patients . lac - tc non invasiva a 64 strati una tecnica ad elevata accuratezza diagnostica nellidentificazione delle stenosi coronariche significative in pazienti con dolore atipico o angina stabile con rischio basso - intermedio . 
am j cardiol 98 : 145148 16 garcia mj , lessick j , hoffmann mh ( 2006 ) accuracy of 16 - row multidetector computed tomography for the assessment of coronary artery stenosis . 
ropers d , rixe j , anders k et al ( 2006 ) usefulness of multidetector row spiral computed tomography with 64x 0.6mm collimation and 330 - ms rotation for the noninvasive detection of significant coronary artery stenoses . 
fox k , garcia ma , ardissino d et al ( 2006 ) guidelines on the management of stable angina pectoris : executive summary : the task force on the management of stable angina pectoris of the european society of cardiology . eur heart j 27 : 13411381 19 . 
nikolaou k , rist c , wintersperger bj et al ( 2006 ) clinical value of mdct in the diagnosis of coronary artery disease in patients with a low pretest likelihood of significant disease . 
cademartiri f , nieman k , van der lugt a et al ( 2004 ) intravenous contrast material administration at 16 - detector row helical ct coronary angiography : test bolus versus bolus - tracking technique . 
austen wg , edwards je , frye rl et al ( 1975 ) a reporting system on patients evaluated for coronary artery disease . report of the ad hoc committee for grading of coronary artery disease , council on cardiovascular surgery , american heart association . 
hendel rc , patel mr , kramer cm et al ( 2006 ) accf / acr / scct / scmr / asnc / nasci / scai / sir 2006 appropriateness criteria for cardiac computed tomography and cardiac magnetic resonance imaging : a report of the american college of cardiology foundation quality strategic directions committee appropriateness criteria working group , american college of radiology , society of cardiovascular computed tomography , society for cardiovascular magnetic resonance , american society of nuclear cardiology , north american society for cardiac imaging , society for cardiovascular angiography and interventions , and society of interventional radiology . 
juergens ku , grude m , fallenberg em et al ( 2002 ) using ecg - gated multidetector ct to evaluate global left ventricular myocardial function in patients with coronary artery disease . ajr am j roentgenol 179 : 15451550 24 . 
juergens ku , grude m , maintz d et al ( 2004 ) multi - detector row ct of left ventricular function with dedicated analysis software versus mr imaging : initial experience . 
schuijf jd , bax jj , jukema jw et al ( 2004 ) noninvasive angiography and assessment of left ventricular function using multislice computed tomography in patients with type 2 diabetes . diabetes care 27 : 29052910 26 . 
heuschmid m , rothfuss jk , schroeder s et al ( 2005 ) assessment of left ventricular myocardial function using 16 - slice multidetector - row computed tomography : comparison with magnetic resonance imaging and echocardiography . 
salm lp , schuijf jd , de roos a et al ( 2006 ) global and regional left ventricular function assessment with 16detector row ct : comparison with echocardiography and cardiovascular magnetic resonance . 
torricelli1 1cattedra e servizio di radiologia i , dipartimento integrato dei servizi diagnostici e per immagini , 2cattedra e servizio di cardiologia , dipartimento integrato emergenza urgenza , universit degli studi di modena e reggio emilia , azienda ospedaliero - universitaria policlinico di modena , via del pozzo 71 , i - 41100 modena , italy correspondence to : g . 
the gadolinium - enhanced images were evaluated using a segmental model with two different methods : a visual score on a 5 - point scale ( 0 no hyperenhancement , 4 hyperenhancement > 76% of myocardial wall ) and a quantitative analysis based on the manual tracing of infarct contours with automatic threshold analysis . 
le immagini post - gadolinio sono state valutate utilizzando un modello segmentale con 2 metodi : uno score visuale con scala a 5 punti ( 0 = assenza di enhancement , 4 = enhancement > 76% della parete ) ed unanalisi quantitativa disegnando manualmente larea dellinfarto con correzione automatica dei contorni . 
dei 1280 segmenti , 322 ( 25 , 1% ) presentavano contrattilit alterata con enhancement in 327 ( 25 , 5% ) valutati visivamente e in 414 ( 32 , 3% ) quantitativamente . 
tra i segmenti con normale o lieve ipocinesia l89 , 2% aveva uno score di enhancement 1 , mentre l80 , 2% dei segmenti discinetici o acinetici aveva uno score 3 . 
lanalisi visuale del delayed enhancement un approccio pi rapido e sufficiente per definire lestensione transmurale dellinfarto ; inoltre ha unalta correlazione con la disfunzione contrattile . parole chiave imaging risonanza magnetica infarto miocardio mezzi di contrasto disfunzione ventricolare g . 
in acute infarction , contrast washin and wash - out kinetics as well as altered integrity of myocyte and sarcolemmal membranes may play an important role [ 4 ]  . 
in the setting of chronic infarction , the areas of scarring and fibrosis alter the kinetic behaviour and distribution of gadolinium as the extracellular space increases [ 5 ]  . 
estimating viability and scar tissue is important for guiding treatment in patients with ischaemic heart disease , and the correlation between myocardial injury and hyperenhancement on cemri is very close [ 6 ]  . 
furthermore , ce - mri is also useful in assessing the transmural extent of infarction , information often needed by the cardiologist to decide whether or not the patient will benefit from revascularisation . 
as the first method may be less accurate and the latter may be time consuming , the aim of our study was to demonstrate the feasibility of visual analysis compared with quantitative evaluation . 
exclusion criteria were recent ( < 1 month ) myocardial infarction , arrhythmias and general contraindications to mri ( e.g. , pacemakers , intracranial clips , claustrophobia )  . all patients gave written informed consent to participate in the study , and the local ethics committee approved the study protocol . 
 study protocol the study protocol included a cine - mri study at rest in three planes ( two chambers , four chambers and multiple short axes ) to analyse regional and global left ventricular function , followed by ce - mri to evaluate the presence of infarction . all images were acquired using a five - element synergy coil during breath - holds and were gated to the electrocardiogra noto che la risonanza magnetica con contrasto ( cemri ) permette una precisa delineazione dellestensione e della transmuralit dellinfarto miocardico con una elevata risoluzione spaziale . 
il meccanismo dellenhancement miocardico nella sede dellinfarto duplice , sia legato alla rottura delle membrane dei miociti che permette la diffusione del contrasto paramagnetico a base di gadolinio nello spazio intracellulare , sia per espansione dello spazio interstiziale per deposizione di collagene [ 13 ]  . 
nellinfarto acuto le cinetiche di wash - in e wash - out del mezzo di contrasto come anche lalterata integrit delle membrane dei miociti e del sarcolemma , giocano un ruolo importante [ 4 ]  . in sede di infarto cronico le aree di cicatrice e fibrosi portano ad una modificazione del comportamento di cinesi e di distribuzione del gadolinio allaumentare dello spazio intracellulare [ 5 ]  . 
stimare la presenza di tessuto vitale e necrotico importante e necessario per guidare il follow - up dei pazienti con cardiopatia ischemica ; la correlazione tra danno miocardico e iper - enhancement tissutale alta [ 6 ]  . 
inoltre , la cemri utile nel valutare la transmuralit dellinfarto , che rappresenta una informazione necessaria al cardiologo per impostare la terapia e selezionare i pazienti che trarrebbero beneficio da eventuale rivascolarizzazione . lanalisi delle immagini ottenute con sequenze di cemri pu essere effettuata visivamente o quantitativamente [ 79 ] e , dato che il primo metodo potrebbe risultare meno accurato ed il secondo pi laborioso , lo scopo del nostro studio di dimostrare la fattibilit della valutazione visuale rispetto a quella quantitativa . 
oltre a ci , abbiamo correlato il grado di iper - enhancement con le alterazioni contrattili a riposo . materiali e metodi popolazione abbiamo valutato 80 pazienti ( et media di 63 , 18 anni , 76 , 4% maschi ) , con nota cardiopatia ischemica cronica e precedente infarto ( almeno 1 mese prima dello studio )  . 
i criteri di esclusione erano : infarto miocardico recente ( < 1 mese ) , aritmie e controindicazioni generali allesecuzione di risonanza magnetica ( come presenza di pace - maker , clips intracraniche , claustrofobia )  . il consenso informato stato ottenuto da tutti i pazienti arruolati nello studio . 
i pazienti sono stati sottoposti a risonanza magnetica cardiaca 1 , 5 tesla ( philips intera , best , olanda )  . protocollo di studio il protocollo di studio include lo studio funzionale di risog . 
depending on the size of the heart , 1012 cine short - axis views were obtained from apex to base with a bffe sequence during breath - holds of approximately 15 s . 
delayed - enhancement images were acquired approximately 15 min after administration of intravenous contrast material [ 0.1 mmol / kg , gadoliniumtetraazacyclododecane tetraacetic acid ( dota ) , dotarem ] with a t1 - weighted turbo fast low - angle shot sensitivity - encoded sequence with a segmented 180 inversion recovery preparatory pulse . 
the inversion time ( ti ) that permits complete suppression of normal myocardium signal was individually adapted and typically ranged from 260 ms to 320 ms . the optimum ti to null healthy myocardium and highlight the evidence of infarcted tissue , where gadolinium accumulates , was found by using a series of real - time images obtained in the same plane with variable inversion time ( look - locker sequences )  . 
each segment was assigned a wall - motion score : 0 = normokinesia ; 1 = mild hypokinesia ; 2 = severe hypokinesia ; 3 = akinesia ; 4 = dyskinesia . global function was evaluated by outlining endocardial and epicardial borders manually on the short - axis images with cardiac software ( cardiac analysis , viewforum 4.1 , philips , nl )  . 
left ventricle ( lv ) end - systolic and end - diastolic volumes and ejection fraction were calculated . to visually quantify infarct size on the delayed - enhancement mr images , each segment was assigned a visual score on a 5 - point scale ( 0 = no hyperenhancement , 1 = hyperenhancement 1%25% of lv wall thickness , 2 = hyperenhancement 26%50% of lv wall thickness , 3 = hyperenhancement 51%75% of lv wall thickness , 4 = hyperenhancement > 76% of lv wall thickness )  . quantitative analysis involved manually tracing the lv endocardial and epicardial borders and placing two regions of interest ( rois ) : one at the centre of the infarction area , and the other at the centre of normokinetic healthy myocardiuscar tissue is then automatically delineated based on a threshold signal intensity ( si ) derived from the mean intensity of infarcted and normal myocardiu the threshold was considered to be ( si of infarction + si of normal myocardium ) / 2 . 
therefore , myocardium exhibiting a higher signanza magnetica a riposo su tre i piani cardiaci ( due camere , quattro camere e multipli assi corti ) per analizzare la funzione ventricolare regionale e globale ( cine - mri ) , seguito da lo studio con contrasto ( cemri ) per valutare la presenza di infarto . 
tutte le immagini sono state acquisite con il mantenimento dellapnea e sincronizzazione cardiaca utilizzando una bobina synergy a 5 canali . la cinesi ventricolare sinistra stata valutata utilizzando sequenze bilanciate fast field echo ( b - ffe ) sui tre piani : due camere , quattro camere e asse corto del cuore . 
le immagini tardive di delayed enhancement sono state acquisite circa 15 minuti dopo somministrazione di contrasto endovenoso ( 0 , 1 mmol / kg , gadolinium - dota , dotarem ) utilizzando una sequenza t1 pesata turbo gradient echo con un impulso di inversione preparatorio di 180 . 
lottimale ti per annullare il segnale del miocardio normale ed evidenziare il segnale del tessuto infartuato , dove il gadolinio si accumula , stato selezionato utilizzando una serie di immagini ottenute in tempo reale su uno stesso piano con tempi di inversione variabili ( sequenza look - locker )  . 
i parametri applicati per la sequenza ir - ge sono stati i seguenti : tr / te 4 , 4 / 1 , 32 ms , fa 15 , ti 260320 ms , sense 2 , fattore tfe 14 , spir soppressione del grasso , matrice 256 , spessore di fetta 8 analisi dei dati la funzione regionale e globale del ventricolo sinistro stata valutata sulle immagini di cine - mri secondo il modello segmentale a 16 segmenti derivato da quello a 17 segmenti di cerqueira [ 10 ] , ottenendo 1280 segmenti ( 16 segmenti per 80 pazienti )  . 
ad ogni segmento stato attribuito uno score di cinesi definito come wall motion score ( wms ) variabile da 0 = normocinesi , 1 = ipocinesia lieve , 2 = severa ipocinesia , 3 = acinesia , 4 = discinesia . la funzione globale stata ottenuta delineando i bordi endocardici ed epicardici sulle immagini in asse corto utilizzando un software dedicato cardiaco ( cardiac analysis , viewforum 4.1 , philips , olanda )  . 
per quantificare visivamente lentit dellinfarto sulle immagini di cemri stato assegnato ad ogni segmento un punteggio visuale in base a una scala da 5 punti variabile da : 0 = asg . 
in both the qualitative and quantitative approaches , areas of persistent microvascular obstruction were considered part of the infarction and were included manually in the enhanced region in the quantitative approach . delayed - enhancement images were scored visually by two radiologists with different experience ( one board - certified radiologist and one resident radiologist )  . 
cardiac medical therapy was continued during the study and consisted of angiotensin - converting enzyme ( ace ) inhibitors in 62 patients and beta - blockers in 68 . cardiac mri analysis mean lv ejection fraction was 49.314.9%. 
among segments with normal or mild hypokinesia , 89.2% had a delayed enhancement score 1 , whereas 80.2% of akinetic or dyskinetic segments had a score 3 , confirming the relationship between transmurality of infarction and contractile impairment ( table 1 )  . there was almost perfect agreement between the visual senza di iper - enhancement , 1 = iper - enhancement 1%25% dello spessore ventricolare sinistro , 2 = iper - enhancement 26%50% dello spessore ventricolare sinistro , 3 = iperenhancement 51%75% dello spessore ventricolare sinistro , 4 = iper - enhancement > 76% dello spessore ventricolare sinistro . lanalisi quantitativa stata basata sulla delineazione manuale dei contorni endocardici ed epicardici del ventricolo sinistro e successivamente sono state posizionate due regioni di interesse ( roi ) : una nel centro del miocardio infartuato e una nel centro di miocardio sano normocinetico . 
da ci , segue la delineazione automatica del tessuto infartuato sulla base di un valore di intensit di segnale ( si ) soglia derivante dalla media delle intensit del tessuto infartuato e normale . 
ci consegue che il miocardio che presenta un valore di intensit seriore al valore soglia considerato infartuato . il software permette di ottenere anche grafici ad occhio di bue , di facile lettura nel valutare la transmuralit dellinfarto . 
le aree di ostruzione microvascolare persistente sono state considerate in entrambi gli approcci come parte della zona infartuata ed incluse manualmente nella zona di enhancement durante la valutazione quantitativa . le immagini di delayed enhancement sono state esaminate due radiologi con differente esperienza ( un medico radiologo esperto e un medico radiologo in formazione ) ; per calcolare la variabilit inter - osservatore dellapproccio visuale sono stati considerati 20 pazienti . 
la variabilit intraosservatore stata calcolata sui medesimi 20 pazienti che sono stati rivalutati dal medico radiologo esperto dopo un mese . valutazione statistica la valutazione statistica stata effettuata ( software stata ) considerando la valutazione quantitativa come gold standard : le concordanze tra lanalisi visuale e quantitativa dellinfarto e tra lo score di cinesi segmentale e lanalisi visuale dellinfarto sono state calcolate usando il valore  . 
un valore di p < 0 , 05 stato considerato statisticamente significativo . risultati popolazione dei pazienti la popolazione in studio era composta da 80 pazienti consecutivi , la maggior parte dei quali maschi ( 76 , 4% ) , con unet media di 63 , 18 anni . 
there was a moderate but statistically significant correlation between the visual scar score and the segmental wall - motion score , the value being 0.58 , with se 0.025 and p < 0.01. among normal or mild hypokinetic segments ( score : 01 ) , 89.2% had a visual score 1 , and among dyskinetic or akinetic segments ( score : 34 ) , 80.2% had a visual score 3 ( tables 3 and 4 )  . 
interand intraobserver agreement of the visual score were both excellent : 89% and 94% , respectively . discussion cardiac ce - mri is now considered the gold standard in zima convertitore dellangiotensina in 62 pazienti e betabloccanti in 68 pazienti ; essa non stata interrotta durante lo studio . analisi di risonanza magnetica cardiaca la frazione di eiezione media era di 49 , 314 , 9% . 
tra i segmenti con cinesi normale o lieve ipocinesi , l89 , 2% presentava uno score 1 , mentre 80 , 2% dei segmenti acinetici o discinetici presentava uno score3 , confermando la relazione tra la transmuralit dellinfarto e la disfunzione contrattile ( tabella 1 )  . si riscontrata una pressoch perfetta concordanza tra il metodo visuale e quantitativo con un valore di 0 , 92 con errore standard ( se ) di 0 , 023 e p < 0 , 01 ( tabella 2 ) ; lanalisi visuale ha sottostimato 104 ( 8 , 1% ) e sovrastimato 19 ( 1 , 5% ) segmenti . 
inoltre stata trovata una correlazione moderata ma statisticamente significativa , tra lo score visuale e lo score di motilit segmentale con valori di 0 , 58 con se di 0 , 025 e p < 0 , 01 . 
tra i segmenti con cinesi normale o lievemente depressa ( score : 01 ) , l89 , 2% ha score visuale 1 e tra i segmenti discinetici o acinetici ( score : 34 ) , l80 , 2% ha score visuale 3 ( tabelle 3 , 4 )  . 
le concordanze interad intra - osservatore dello score visuale erano entrambe eccellenti : 89% e 94% rispettivamente . discussione la risonanza magnetica cardiaca con contrasto attualmente considerata il gold standard per la definizione della presenza di infarto miocardico e ci dovuto alla sua alta capacit di visualizzare il tessuto necrotico dopo somministrazione di gadolinio . 
numerosi studi hanno confermato alta concordanza tra la distribuzione di gadolinio e lestensione dellinfarto valutato mediante analisi istologica , suggerendo che larea di miocardio che presenta enhancement costituita da tessuto cicatriziale [ 4 ]  . lestensione dellinfarto , come evidenziato da cerm , cosiderata uno dei fattori prognostici principali da valutare nei pazienti con cardiopatia ischemica , in quanto si correla direttamente al rimodellamento ventricolare sinistro . 
per definire precisamente quanto del tessuto cardiaco danneggiato , i metodi quantitativi proposti in letteratura sono molteplici e variano a seconda del software di post - processing impiegato . lestensione del tessuto cicatriziale e la sua distribuzione transmurale sono fattori prognostici importanti , dato che un g . 
many different studies have confirmed the close correlation between gadolinium distribution and extent of infarction as assessed by histological examination , suggesting that what hyperenhances in delayed - enhancement imaging is scar tissue [ 4 ]  . 
infarction extent , as depicted by ce - mri , is now considered one of the main prognostic factors in patients with ischaemic heart disease , as it correlates directly with left ventricular remodelling . 
many quantitative methods , depending on the scanners and postprocessing software used , have been proposed in the literature to define precisely how much heart tissue is infarcted . the extent of scar tissue and its transmural distribution are important prognostic factors , given that a large infarct but with circumferential distribution can cause less contractile dysfunction than one that is small but transmural . 
moreover , it is well known that in dysfunctional segments , the likelihood of improvement in regional contractility after revascularisation decreases progressively as the transmural infarto esteso ma con una distribuzione circonferenziale pu causare una disfunzione contrattile minore di un infarto di minore estensione longitudinale , ma transmurale . 
inoltre riconosciuto che nei segmenti con alterazione della cinesi , la probabilit di miglioramento contrattile post - rivascolarizzazione diminuisce progressivamente con laumentare dellestensione transmurale della zona di iper - enhancement [ 11 ]  . i nostri risultati mostrano che la valutazione visuale del tessuto cicatriziale correla molto bene con quella quantitativa , considerando sia i segmenti interessati sia la loro corrispondente transmuralit . 
oltre a ci , lanalisi visuale ha alta concordanza sia interche intra - osservatore , quindi pu essere considerata un approccio altamente riproducibile per definire in modo semplice e rapido lestensione e la transmuralit dellinfarto . tuttavia , bisogna riconoscere che il metodo quantitativo permette di ottenere lesatta quantificazione dellinfarto esprimendo tale dato come percentuale o come grammi di tessuto danneggiato rispetto al miocardio sano , in modo tag . 
the visual score may therefore be considered a timesaving and reproducible approach to easily define infarct extent and transmurality . however , it must be noted that the quantitative method provides exact quantification of infarction in terms of percentage or grams of damaged tissue with respect to normal myocardium , allowing one to infer the amount of healthy myocardium , which is another important parameter in defining prognosis . 
our software permits us to define a threshold intensity of scar tissue based on the different intensities of normal and hyperenhanced myocardium , so that damaged tissue can be automatically defined and quantified . 
it does not , however , have the recently reported postprocessing correction algorithms , such as the feature analysis and comle da poter dedurre lentit di tessuto normale , che risulta essere un altro importante parametro per definire la prognosi di tali pazienti . 
il software impiegato nel presente studio consente di fissare un valore soglia basandosi sulla differenza di intensit di segnale tra tessuto infartuato e miocardio remoto ; tale approccio consente di definire e quantificare automaticamente il tessuto danneggiato . 
nonostante ci , questo software non possiede gli algoritmi di correzione recentemente riportati in letteratura , come lalgoritmo di feature analysis and combined thresholding ( fact ) [ 8 , 9 ] , che , proposto su animali e in una piccola coorte di pazienti , permette di escludere gli errori di volume parziale , di segmentazione imperfetta e gli errori di calcolo automatico dovuti per esempio alla presenza di aree di ostruzione microvascolare nel contesto dellarea infartuata . tutto ci sottolinea la necessit di nuovi algoritmi che siano facilmente utilizzabili e che quantifichino accuratamente la presenza di infarto nelle immagini ottenute per lo studio del delayed enhancement , dato che questa tecnica la metodica non invasiva con pi alta risoluzione total totale g . 
this highlights a need for new , easy - touse algorithms that can accurately quantify the extent of infarction on delayed - enhancement imaging , a noninvasive technique providing the highest spatial resolution currently available for the assessment of viability . the software algorithm employed in our study allowed us to identify , in a few cases , subendocardial infarction that had been visually underestimated . 
moreover , it better defined the limits between score 3 and 4 transmural extent that , although not affecting prognosis if taken alone , can in fact change the final evaluation and patient management when integrated with the other clinical and functional parameters . 
 not surprisingly , there was good agreement between the visual scar score and wall - motion score , as segments with little or no infarction had no contractile impairment , whereas segments with infarct transmurality > 50% had variable contractile dysfunction . 
the less - than - excellent concordance ( index 0.57 ) may be due to a direct or reverse tethering effect of the adjacent healthy myocardium . we can conclude that visual analysis of delayed enhancement is a timesaving approach that is adequate for assessing infarct extent and transmurality and that has a close correlation with wall - motion abnormalities . 
quantification of scar tissue may also be important in patients with intermediate transmurality , who should be referred for coronary angioplasty to improve the viable but stunned or hibernated myocardiua work in progress is to determine the amount of scar tissue before and after coronary revascularisation to assess possible shrinkage of infarction with respect to healthy myocardium . our study demonstrated that the qualitative approach is sufficient in routine clinical practice . 
however , another application could be to use both methods to evaluate infarct size before and after drug therapy in order to further validate the visual analysis approach in an experimental setting . 
further studies are needed above all to compare the amount of scar tissue before and after revascularisation to evaluate whether there is effective improvement due to infarct shrinkage or whether the exact quantity of damaged tissue is predictive of future response to revascularisation . spaziale attualmente disponibile per valutare la vitalit tissutale . lalgoritmo utilizzato dal nostro software ha permesso di identificare in alcuni casi la presenza di infarto sub - endocardico , che visivamente era stato sottostimato ; inoltre ha definito meglio i limiti tra estensione transmurale con score 3 e 4 che da sola non influenza la prognosi del paziente ma che integrata con gli altri parametri clinici e funzionali , pu modificare la valutazione finale ed il trattamento del paziente . naturalmente , abbiamo riscontrato una buona concordanza tra lo score visuale e lo score di cinesi , dato che segmenti senza infarto o con una piccola quantit di danno miocardico non presentavano disfunzione contrattile , mentre segmenti con una transmuralit infartuale maggiore del 50% mostravano gradi variabili di alterazione della cinesi . la concordanza non eccellente ( indice di 0 , 57 ) potrebbe essere dovuta alleffetto di trascinamento diretto e / o inverso del miocardio sano adiacente . possiamo concludere che lanalisi visuale del delayed enhancement un approccio meno laborioso e sufficiente per valutare la transmuralit e lestensione spaziale dellinfarto ; inoltre ha una notevole correlazione con le alterazioni contrattili . 
la quantificazione del tessuto cicatriziale potrebbe essere importante in pazienti con una transmuralit intermedia che dovrebbero essere indirizzati verso una procedura invasiva mediante coronarografia tradizionale per migliorare il miocardio vitale , ma stordito od ibernato . 
una studio in corso di calcolare la quantit di tessuto danneggiato prima e dopo rivascolarizzazione coronarica in modo tale da valutare una eventuale diminuzione dellinfarto rispetto al corrispondente tessuto miocardico sano . il significato di questo studio che lapproccio qualitativo risulta essere sufficiente nella routine clinica , ma certo in campo di ricerca unaltra applicazione potrebbe essere di valutare le dimensioni infartuati prima e dopo terapia farmacologia con entrambi i metodi , per validare anche in campo sperimentale lanalisi visuale . 
midiri2 , 5 1dipartimento di radiologia e dipartimento cuore , imaging cardiovascolare non invasivo , azienda ospedaliera di parma , via gramsci 14 , i - 43100 parma , italy 2dipartimento di radiologia e cardiologia , erasmus medical center , rotterdam , holland 3istituto di radiologia , universit degli studi di verona , verona , italy 4istituto di radiologia , ospedale universitario cattinara , trieste , italy 5dibimel , sezione di scienze radiologiche , universit di palermo , palermo , italy correspondence to : f . 
six hundred and seventy patients undergoing msct - ca with 16 - slice and 64 - slice ct scanners for suspected atherosclerotic disease of the coronary arteries were retrospectively reviewed . 
a significant number of noncardiac findings might have been missed in msct - ca scans ; the appropriate approach should be as a team trained in cardiology and radiology . key words multislice ct coronary angiography collateral findings incidental findings riassunto obiettivo . 
sono stati riscontrati 1234 rilievi accessori , divisi in 332 ( 26 , 9% ) non significativi , 821 ( 66 , 53% ) significativi e 81 ( 6 , 56% ) in obbligo di ulteriori accertamenti . 
lapproccio appropriato dovrebbe prevedere la lettura degli esami da parte di un team esperto in cardiologia e radiologia . parole chiave angiografia coronarica mediante tc multistrato reperti collaterali reperti incidentali f . 
noninvasive vascular imaging represents an important clinical application , as multislice ct coronary angiography ( msct - ca ) has proven to be a reliable alternative to conventional digital subtraction angiography [ 25 ]  . 
these technical advances permit a reliable study of coronary arteries to quantify calcium deposits among coronary plaques [ 6 ] and to detect significant stenosis [ 7 , 8 ] , anatomical variants of the anatomy of the coronaries [ 9 ] and functional evaluation of the heart [ 10 ]  . 
consequently , msct - ca has shown to be the technique of choice in patients with lowto intermediate risk of acute chest pain [ 11 ]  . msct - ca appears to be an excellent tool for improving diagnostic accuracy in patients with chest pain and moderate pretest probability of coronary artery disease ( cad ) and negative or equivocal findings on exercise stress testing . 
on the other hand , the wide availability of these scanners has led to a constant increase of these studies , with the emerging problem of extracardiac collateral findings revealed during msct - ca . 
sometimes these findings are of primary importance for the patients health and reaching a correct diagnosis . the aim of this study was to further investigate this aspect of msct - ca through the analysis of collateral findings . materials and methods patient population six hundred and seventy consecutive patients ( 380 men and 290 women , mean age 59.710.2 years ) undergoing msctca due to suspected cad between january 2004 and january 2005 were retrospectively studied in a single - centre study . 
four hundred and forty - six examinations were performed using a 16 - slice ct scanner ( sensation 16 , siemens , forchheim , germany ) and 224 with a 64 - slice scanner ( sensation 64 , siemens , forchheim , germany )  . inclusion and exclusion criteria inclusion criteria for the msct - ca examination were regular heart rate < 65 beats per minute ( bpm ) and ability of the patient to hold the breath for 20 s ( 16 - slice scanner only )  . exclusion criteria were refusal to provide informed consent , prior adverse reactions to iodinated contrast material , kidney failure ( serum creatinine > 120 mmol / l ) , possible pregnancy , impaired respiratory function , unstable clinical condition or marked heart failure , prior coronary angioplasty or coronary surgery and nondiagnostic examination quality . 
the local ethics committee approved the study , and all patients provided informed consent . la tecnologia multistrato tc ha permesso lapplicazione della tomografia computerizzata per lo studio del cuore e delle arterie coronarie [ 1 ]  . 
lo sviluppo di apparecchiature a 16 e 64 strati permette attualmente lo studio del cuore in tempo ridotto e con un aumento sostanziale in termini di risoluzione temporale e spaziale . 
queste evoluzioni tecnologiche permettono uno studio affidabile dellalbero coronarico per la quantificazione dei depositi di calcio a livello delle placche coronariche [ 6 ] , per la ricerca di stenosi significative [ 7 , 8 ] , varianti anatomiche [ 9 ] e addirittura per lo studio funzionale del cuore [ 10 ]  . 
conseguentemente , la ac - tcms ha dimostrato di essere la tecnica di prima scelta nei pazienti con dolore toracio acuto e profilo a basso - intermedio rischio [ 11 ]  . 
la ac - tcms sembra essere uneccellente metodica per migliorare laccuratezza diagnostica nei pazienti con dolore toracico , moderata probabilit pretest di coronaropatia e rilievi negativi o dubbi nei vari stress test . 
daltra parte la larga disponibilit odierna di queste apparecchiature ha portato ad un aumento costante degli esami effettuati , facendo emergere il problema di reperti collaterali dimostrati durante questi esami . 
alle volte tali rilievi possono essere di importanza primaria per la salute del paziente e permettono un corretto inquadramento diagnostico . quindi lo scopo del nostro studio consiste nellapprofondire questo aspetto della tc delle coronarie attraverso lanalisi dei reperti collaterali osservati . materiali e metodi popolazione di studio sono stati valutati retrospettivamente mediante uno studio monocentrico 670 pazienti consecutivi ( 380 maschi e 290 femmine , et media 59 , 710 , 2 anni ) sottoposti a ac - tcms per sospetta patologia delle coronarie nel periodo compreso tra gennaio 2004 e gennaio 2005 . 
quattrocentoquarantasei di tali indagini sono state eseguite mediante apparecchiatura tc a 16 strati ( sensation 16 , siemens , forchheim , germania ) , e 224 con tc a 64 strati ( sensation 64 , siemens , forchheim , germania )  . criteri di inclusione ed esclusione i criteri di inclusione per gli esami ac - tcms erano costituiti da un ritmo cardiaco < 65 battiti / minuto ( bpm ) e la capacit di trattenere lapnea inspiratoria per 20 s ( solamente per apparecchiatura 16 - strati )  . 
i criteri di esclusione erano rappresentati da : rifiuto al consenso informato per lo studio , precedenti reazioni avverse al mezzo di contrasto iodato , insufficienza cardiaca , ( creatininemia > 120 mmol / l ) , gravidanza , insufficiente funzionalit respiratoria , condizioni clif . 
patients were scanned in the supine position during breath hold from the level of the pulmonary arteries through the very cranial part of the abdomen in order to visualise the base of the heart . 
in patients with a known coronary artery bypass graft , the scan was extended cranially from the level of the aortic arch through the base of the heart . scan protocol we intravenously administered 80100 ml of nonionic iodinated contrast material ( iomeprol 400 mg / ml , iomeron , bracco , milan , italy ) at a flow rate of 45 ml / s , followed by 40 ml of saline solution at the same rate . 
the contrast material was administered with a dual - syringe automatic injector ( stellant , medrad , pittsburgh , pa , usa ) connected to an 18 - gauge cannula placed in an antecubital vescan synchronisation with contrast material passage was performed with the bolus tracking technique ( care bolus , siemens , forchheim , germany ) [ 2 ]  . 
scan and reconstruction parameters for the 16and 64 - slice scanners are summarised in table 1 . data processing a retrospective electrocardiogram ( ecg ) - gated technique was used for image reconstruction . 
data sets were reconstructed during the mid - to - end diastolic phase , with reconstruction windows set at 300 ms to 450 ms before the next r wave or 60%70% of the rr interval . 
if mildly irregular heart rates were encountered , such as bundle branch blocks or premature beats , the temporal variability in the reconstruction phase was compensated for manually with ecg editing . 
inaccurate detection of the r waves was corrected , and temporal windows applied to premature beats were deleted , filling the next diastolic interval with one or more temporal windows to avoid data gaps . the reconstructed slice thickness was 0.75 mm , with a reconstruction increment of 0.4 mall ct data sets were filtered with a medium - soft convolution kernel . in addition , images were reconstructed with 1 - mm thickness and reconstruction increment of 0.6 mm , with an axial field of view ( fov ) collimated to the patients chest using standard mediastinal window ( 350 w / 50 c ) and lung window ( 1 , 400 w / 600 c ) levels to evaluate soft tissue and lung parenchyma . image evaluation all scans were reviewed by two radiologists ; data sets obtained with a large fov were reviewed in mediastinal and lung windows . 
il comitato etico locale ha approvato lo studio e tutti i pazienti esaminati hanno fornito il consenso informato . preparazione del paziente per i pazienti studiati con apparecchio a 16 e 64 strati in caso di frequenze cardiache uguali o superiori a 65 bpm , sono stati somministrati 100 mg di metoprololo unora prima dellesecuzione dellesame . 
i pazienti sono stati studiati in posizione supina in apnea inspiratoria dal livello delle arterie polmonari fino alla porzione prossimale delladdome in modo tale da comprendere anche la base del cuore . 
in pazienti con bypass il limite della scansione stato innalzato cranialmente alla porzione iniziale dellarco aortico fino alla base del cuore . protocollo di scansione abbiamo somministrato per via endovenosa 80100 ml di mezzo di contrasto non ionico iodato ( iomeprol 400 mgl / ml , iomeron , bracco , milano , italia ) con un flusso di 45 ml / s , seguito da 40 ml di soluzione salina allo stesso flusso . 
il mezzo di contrasto stato introdotto tramite un iniettore automatico a doppia pompa ( stellant , medrad , pittsburgh , pa , usa ) connesso ad una vena antecubitale connessa con un agocannula da 18 g . 
la sincronizzazione della scansione con il passaggio del mezzo di contrasto stata eseguita mediante la tecnica del bolus tracking ( care bolus , siemens , forchheim , germania ) [ 2 ]  . 
i parametri di acquisizione e di ricostruzione per gli apparecchi a 16 e 64 - strati sono riassunti in tabella 1 . elaborazione dei dati per la ricostruzione delle immagini stata utilizzata una tecnica retrospettica ecg - gated . 
i pacchetti di dati sono stati ricostruiti durante la fase meso / tele - diastolica , con finestre di ricostruzione da 300 ms fino a 450 ms dalla successiva onda r o 60% fino a 70% dellintervallo r - r . 
in caso di qualit delle immagini non sufficiente sono state eseguite ricostruzioni aggiuntive durante la fase tele - sistolica ( 25% fino a 35% dellintervallo r - r )  . 
la ricerca non accurata delle onde r stata corretta e le finestre temporali applicate ai battiti ectopici sono state eliminate , compensando il successivo intervallo diastolico con uno o pi finestre . 
the clinical importance of noncardiac findings was divided into three categories : minor ( no clinical meaning ; not necessary to report ) , mild ( uncertain clinical meaning ; preferably mentioned in the report ) , major ( compulsory to be further investigated ; mandatory to report )  . 
among significant - disease patients , medical records were reviewed to check the clinical followup , subsequent examinations or surgical procedures of noncardiac abnormalities in the 6 months after msct - ca . 
the clinical statistics results in aggiunta , le immagini sono state ricostruite con uno spessore di fetta di 1 mm e un incremento di ricostruzione di 0 , 6 mm con un campo di vista assiale collimato al torace del paziente utilizzando le finestre mediastiniche ( 350 w / 50 c ) e polmonari ( 1400 w / 600 c ) per valutare i tessuti molli ed il parenchima . valutazione delle immagini tutte le scansioni sono state analizzate da 2 radiologi . 
limportanza clinica dei reperti collaterali non cardiaci stata distinta in 3 categorie : minori ( senza significato clinico ; senza necessit di essere segnalato ) , intermedio ( significato clinico incerto ; preferibilmente menzionato nel referto ) , maggiore ( necessari ulteriori accertamenti , indispensabile la segnalazione sul referto )  . 
i pazienti con malattie importanti sono stati tenuti in follow - up alla ricerca di ulteriori accertamenti , o procedure chirurgiche nei 6 mesi successivi alla angiografia coronarica mediante tcms . tabella 1 parametri di scansione nella angiografia coronarica tc : 16 strati e 64 strati f . 
among a total of 1 , 234 findings , 417 ( 33.79% ) were detected in the lungs , 665 ( 53.88% ) and in the mediastinum , 74.13% of which were mediastinal lymph nodes ( table 3 )  . findings were then subdivided into three main categories : minor , mild and major . 
among the 1 , 234 additional findings recorded , 81 patients ( 12.08% ) revealed major findings , 439 ( 65.5% ) had mild findings and 12 ( 1.8% ) had only minor findings . 
in the other case , msct - ca permitted incidental diagnosis of multiple lytic lesions located at the body and peduncles of two thoracic vertebrae in a 66 - year - old man with suspected atherosclerotic disease who underwent a 16slice msct - ca . 
le frequenza dei sintomi , fattori di rischio , e qualsiasi reperto collaterale osservato sono state espresse in valori assoluti e percentuali . risultati tra i 670 esami effettuati , l85% ha dimostrato malattie delle arterie coronarie . 
given that the msct scans were done to detect atherosclerotic disease as part of various research protocols , it is remarkable that accidental pathological changes in organs and structures , which were not the target organs of the examination , were found almost in every patient . 
tra 1234 reperti addizionali riscontrati , 81 pazienti ( 12 , 08% ) hanno dimostrato reperti maggiori , 439 ( 65 , 5% ) avevano reperti intermedi e 12 ( 1 , 8% ) avevano solo reperti minori . 
the three categories identified were : minor ( no clinical meaning ; not necessary to report ) , mild ( uncertain clinical meaning ; preferably mentioned in the report ) , major ( compulsory to be further investigated ; mandatory to report ) svc , superior vena cava ; r , right ; l , left dental extracoronary findings in msct - ca , the detection of malignant disease in one case and the possibility of justifying dyspnoea and properly treat the patient in the other case , emphasise the importance of accurate and complete reading of the investigation [ 12 ]  . there is very little in the literature about the prevalence of noncardiac abnormalities in cardiac ct studies [ 1315 ]  . 
the higher prevalence of lesion in our study is mainly due to a very systematic and accurate analysis of all kind of possible abnormalities within the scan range ; for this reason , only 138 ( 20.6% ) patients were completely without any additional finding . 
se consideriamo solamente i reperti maggiori riportiamo una prevalenza del 45 , 6% . gli esami tc del cuore vengono eseguiti con una frequenza in costante aumento nei vari paesi come metodi di f . 
le tre categorie identificate sono : minori ( senza significato clinico , senza necessit di essere segnalato ) , intermedio ( significato clinico incerto ; preferibilmente menzionato nel referto ) , maggiore ( necessari ulteriori accertamenti , indispensabile la segnalazione sul referto ) vcs , vena cava superiore ; d , destra ; s , sinistra malignant disease were revealed . 
the authors reviewed the scans done with a fov of 350 mm , never imaging the entire chest , and thus the most lateral parts of the lungs could not have been investigated in some patients . 
dato che gli esami tc sono stati eseguiti per ricercare malattie aterosclerotiche come parte di vari protocolli di ricerca , un fatto considerevole che sono stai riscontrati reperti accessori in quasi tutti i pazienti . 
tale percentuale cos elevata di rilievi addizionali , il riconoscimento di patologia maligna in un caso e la possibilit di giustificare la dispnea e quindi trattare appropriatamente il paziente nellaltro caso , enfatizza limportanza di una lettura accurata e completa dellindagine [ 12 ]  . in letteratura corrente vi sono pochissimi lavori circa la prevalenza di anomalie non cardiache in studi cardiaci f . 
of 1 , 812 consecutive patients undergoing ebct cardiac studies , the authors reviewed only the mediastinal windows but also encountered cardiac findings such as mitral valve or pericardiac pathologies . 
moreover , abnormalities found in that study were a large number of minor , relatively insignificant , abnormalities , such as scars , granulomas , atelectasis , degenerative arthritis and rib fractures . 
la prevalenza maggiore di lesioni nel nostro studio soprattutto dovuta ad unanalisi molto accurata e sistematica di tutte le possibili anomalie allinterno del range di scansione ; per tale ragione solo 138 ( 20 , 6% ) pazienti si sono dimostrati completamente senza malattia . 
nel suo studio , onuma ha descritto che il 22 , 7% dei pazienti aveva un reperto non significativo che necessitava un lavoro addizionale e sono stati riscontrati solo 4 casi ( 0 , 8% ) di patologia neoplastica maligna . 
gli autori hanno revisionato le scansioni effettuate con un campo di vista di 350 mm , mai comprendente lintero torace , e quindi le porzioni pi laterali dei polmoni in alcuni pazienti non sono state studiate , asserendo che non necessario ricoprire lintero torace in quanto rappresenta un lavoro in pi . 
noi abbiamo analizzato lintera sezione toracica acquisita e ricostruita in modo specifico processato a partire dai dati grezzi , fatto che rappresenta effettivamente un lavoro aggiuntivo per loperatore , ma pu rivelare una grande quantit di reperti aggiuntivi medi e maggiori che altrimenti non sarebbero scoperti . un altro lavoro significativo stato pubblicato da hunold et al . 
in 1812 pazienti consecutivi sottoposti a ebct studi del cuore , gli autori hanno revisionato solamente le finestre mediastiniche , ma hanno annoverato nella casistica anche reperti cardiaci come la presenza di valvole mitraliche o patologie pericardiche . 
inoltre le anomalie incluse nel loro studio comprendevano un elevato numero di reperti minori relativamente privi di significato pratico come cicatrici , granulomi , atelettasie , artriti degenerative o esiti di fratture costali . 
nel nostro studio abbiamo descritto anche lesioni minori che rappresentano 332 su 2198 , 15 , 1% di tutte le lesioni descritte . come gi notato da onuma , hunold nel suo lavoro non ha revisionato le finestre polmonari e perci il numero effettivo di noduli polmonari presenti nei pazienti esaminati sconosciuto . 
2a , b vertebral metastases : incidental finding of multiple lytic lesions located at the body and peduncles of two thoracic vertebrae ( arrows in a and b ) in a 66 - year - old man with suspected atherosclerotic disease who underwent a 16 - slice computed tomography coronary angiography . 
diagnosi accidentale di multiple lesioni litiche localizzate al corpo e peduncoli di due vertebre toraciche ( frecce in a e b ) in un maschio di 66 anni con sospetta coronaropatia sottoposto a ac - tcms . 
in our study , we discovered seven patients with pulmonary nodules > 1 cm , two with lung cancer and two with lung metastasis , but these pathologies were already known and in follow - up . although in clinical practice , it has been proposed that msct - ca may be interpreted by cardiologists who evaluate only the heart and the coronaries [ 18 ]  . 
this practice should be discouraged on the basis of our large series of cases , as the prevalence of extracardiac findings is extremely high so that they will need further investigation . in some cases , the operator may keep the fov as small as possible to include the heart only . 
for this reason , if the operator looks at the coronaries only , some part of the radiation dose given to the patient is wasted , eliminating the opportunity , as our results confirm , to find what might be important additional findings . how ethical it is to ignore or overlook these anatomical regions is a controversy based on a turf battle . 
in addition , the patients interest should always be preserved , and any finding regarding his / her health should be reported . con noduli polmonari superiori ad 1 cm di cui 2 con neoplasia al polmone e 2 pazienti con metastasi polmonari ( questa patologie erano gi note e in follow - up )  . sebbene nella pratica clinica sia stato proposto che la tc cuore potrebbe essere interpretata solamente da cardiologi per la valutazione dellalbero coronario [ 18 ] , questo approccio dovrebbe essere scoraggiato sulla base della nostra casistica dal momento che la prevalenza di reperti extra cardiaci risulta estremamente elevata e parecchie volte necessita di ulteriori indagini . in alcuni casi loperatore potrebbe tenere il campo di vista il pi stretto possibile in modo da includere solamente il cuore . 
tale approccio corretto nellintento della visualizzazione del solo albero coronarico , comunque le informazioni disponibili per lo studio delle aree periferiche al cuore esistono e sono contenute nei dati grezzi . 
per tale ragione se loperatore si concentra solo sulle coronarie , una parte della dose somministrata al paziente viene sprecata , annullando lopportunit , come i nostri risultati confermano , di scoprire eventuali reperti aggiuntivi . 
riteniamo che allapice dellinteresse vi sia la salute del paziente e qualsiasi reperto che riguardi la sua salute sia da riportare nel referto . conclusions conclusioni in conclusion , a significant number of noncardiac findings can be detected in msct - ca scans . 
we studied 120 patients with suspected small - bowel disease by 16 - slice mdct after oral administration of a polyethylene glycol solution ( n = 56 ) or methylcellulose via a nasojejunal tube ( n = 64 )  . 
fifteen patients were found to be affected by small - bowel neoplasm ( six had non - hodgkins lymphoma , three had carcinoid tumour , two had peutz - jeghers syndrome , two had adenocarcinoma , two had melanoma metastases , one had lipoma )  . in the remaining patients , 58 cases of crohns disease and seven miscellaneous diseases were detected . 
mdct performed after bowel - loop distension with low - density contrast material and iv administration of iodinated contrast agent is a reliable method for diagnosing and staging small - bowel neoplasms . key words neoplasms small bowel enteroclysis multidetector - ct riassunto obiettivo . 
sono stati studiati 120 pazienti con sospetta patologia tenuale mediante tc multidetettore ( 16 strati ) previa somministrazione di polietilenglicole per via orale ( n = 56 ) o di metilcellulosa attraverso sondino naso - digiunale ( n = 64 )  . 
lesame tc stato eseguito prima e durante infusione di 130 ml di mdc iodato con flusso di 3 ml / s ed inizio delle scansioni dopo 40 secondi dallinizio della somministrazione . 
quindici pazienti sono risultati affetti da neoplasia dellintestino tenue ( 6 pazienti con linfoma non hodgking , 3 con carcinoide , 2 con sindrome di peutz jeghers , 2 con adenocarcinoma , 1 con metastasi da melanoma e 1 con lipoma )  . in 58 pazienti la diagnosi era di morbo di crohn , in 7 pazienti erano presenti patologie varie . 
la tc multidetettore effettuata previa distensione delle anse con mdc ipodenso ed infusione ev di mdc iodato una metodica utile nella diagnosi e stadiazione delle neoplasie tenuali . parole chiave neoplasie intestino tenue enteroclisi tc multidetettore introduction introduzione primary tumours of the small bowel account for only 5% of all gastrointestinal tumours , and this is quite surprising considering the length , extensive surface and structural diversity of the small intestine . 
 le neoplasie primitive dellintestino tenue rappresentano solo il 5% di tutti i tumori gastrointestinali e questa unevenienza sorprendente se consideriamo la notevole lunghezza del tenue , la sua vasta superficie e la diversit degli elementi strutturali . 
an interesting , though not very recent , study analysed data of all patients with a diagnosis of malignancy of the small bowel over 21 years [ 1 ] , evaluating time from symptom onset to the first medical contact and the time from medical contact to diagnosis . 
the authors demonstrated that the average delay in diagnosis attributable to the patient was less than 2 months , the delay attributable to the physician not ordering the appropriate diagnostic test was 8.2 months , and that attributable to the radiologist failing to make the diagnosis was 12 months . 
as a consequence , the greatest delay in diagnosis occurred after the patient had sought medical help and was the result of incorrect radiological interpretations or false - negative results . three quarters of the patients observed in that study had advanced disease at the time of diagnosis . small - bowel barium studies ( barium follow - through ) and small - bowel enema ( or enteroclysis ) are standard examinations when either neoplastic or inflammatory disease of the small bowel is suspected . 
these examinations visualise the gastrointestinal tract from the inside and therefore allow accurate evaluation of the normal features and possible alterations of the mucosa and lumen , but they provide only indirect information on the intestinal wall and adjacent extraintestinal structures [ 2 ]  . 
tomographic techniques , such as ultrasound and computed tomography ( ct ) , directly depict both the wall ( allowing evaluation of thickness and structural characteristics ) and adjacent structures ( mesentery , adjacent fibrofatty tissue , lymph nodes , peritoneal spaces )  . 
additionally , as this modality is noninvasive and involves no exposure to ionising radiation , it is ideal in patients requiring prolonged follow - up ( chronic inflammatory diseases ) or in children , or to confirm or rule out clinical suspicion , reserving the other diagnostic methods for strongly suspicious cases or cases with positive ultrasound [ 3 ]  . 
introduction of the multidetector technique is also modifying the diagnostic workup of patients with suspected small - bowel disease [ 48 ] , but only few reports have described the use of this method in patients with small - bowel neoplasms [ 912 ]  . the purpose of our study was to evaluate the diagnostic potential of multidetector ct ( mdct ) in the study of smallbowel tumours after distension of the small bowel with a low - density contrast agent . materials and methods study population we examined 120 consecutive patients with a definite , strongly suspected or suspected diagnosis of small - bowel 1014 locclusione intestinale il sintomo di esordio nel 36% dei pazienti [ 1 ]  . nonostante si sia verificato negli ultimi 40 anni un notevole miglioramento sia della chirurgia che dellimaging diagnostico , la sopravvivenza dei pazienti con malignit primitiva dellintestino tenue non ha mostrato un parallelo miglioramento , principalmente in rapporto allo stadio avanzato del tumore al momento dellintervento chirurgico . 
anche se non recente , un interessante studio ha analizzato i dati di tutti i pazienti con diagnosi di malignit dellintestino tenue in un periodo di 21 anni [ 1 ] , valutando lintervallo di tempo che intercorreva tra linizio dei sintomi ed il primo contatto medico e tra il contatto medico e la diagnosi , e ha dimostrato che il ritardo diagnostico medio attribuibile al paziente era inferiore a 2 mesi ; quello attribuibile al medico per mancata richiesta di un test diagnostico appropriato era di 8 , 2 mesi ; quello attribuibile al radiologo per mancata diagnosi era 12 mesi . 
tra i pazienti osservati in questo studio , tre quarti di essi aveva una malattia avanzata al momento della diagnosi . il tenue baritato per os ( o transito intestinale ) e il clisma del tenue ( o enteroclisi ) sono gli esami che solitamente vengono effettuati nel sospetto di patologia dellintestino tenue , sia neoplastica che infiammatoria . 
essi visualizzano il tratto gastroenterico dallinterno e quindi possono valutare con accuratezza i caratteri normali e le eventuali alterazioni della mucosa e del lume del viscere , ma forniscono informazioni solo indirette sulla parete intestinale e sulle strutture extraintestinali adiacenti [ 2 ]  . 
le tecniche tomografiche , quali ecografia e tomografia computerizzata ( tc ) , consentono invece la visualizzazione diretta sia della parete ( valutandone spessore e caratteri strutturali ) sia delle strutture extraviscerali adiacenti ( mesentere , cellulare fibroadiposo periviscerale , linfonodi , spazi peritoneali )  . 
lesame ecografico ha anche la prerogativa di visualizzare tono e attivit contrattile dei segmenti tenuali , esaminati in condizioni assolutamente fisiologiche , ed essendo tecnica non invasiva e priva di esposizione a radiazioni ionizzanti , si propone come mezzo ideale qualora sia necessario un follow - up prolungato nel tempo ( pazienti con patologia infiammatoria cronica ) oppure in et pediatrica , oppure per avvalorare o al contrario escludere un sospetto clinico riservando altre metodiche diagnostiche solo ai casi risultati effettivamente sospetti o positivi allecografia [ 3 ]  . 
definite diagnoses were based on histological examination of the biopsy specimen ; strongly suspected diagnoses were based on a recent contrast - enhanced study of the small bowel ( small - bowel enema or barium study ) that was positive for small - bowel disease ; suspected diagnoses were based on the presence of clinical signs and symptoms ( diarrhoea , abdominal pain , bleeding ) indicative of small - bowel disease . 
after methylcellulose infusion , the ct scan was performed before and during iv administration of iodinated contrast material , with image acquisition from the diaphragm to the perineum during a single inspiration . 
the contrast - enhanced ct study was acquired 40 s after iv administration of 130150 ml of contrast material ( ultravist 370 , schering ag , berlin , germany ) injected at a rate of 3 ml / s . 
il consenso informato stato ottenuto da tutti i pazienti . tecnica prima dellesame tutti i pazienti venivano sottoposti a preparazione intestinale secondo il seguente schema : due giorni prima dellesame , dieta povera di frutta e verdura ; il giorno prima , 150 mg di una miscela di sennosidi a e b con una tazza di t zuccherato alle ore 8 : 00 ; alle 13 : 00 , dieta semiliquida ; alle 17 : 00 , 15 mg di solfato di magnesio in 3 / 3 4 / / di un bicchiere di acqua tiepida seguita dallassunzione di 3 litri di acqua nelle 45 ore successive ; alle 21 : 00 , una tazza di brodo caldo ; dalle 21 : 00 in poi , digiuno . cinquantasei pazienti ottenevano una distensione dellintestino mediante somministrazione orale di 1 , 52 , 0 l di una soluzione iso - osmolare di polietilen - glicole somministrata in dosi uguali di 100 ml a partire da 45 minuti prima dellesame tc ( enterografia - tc con peg o peg - tc )  . 
sessantaquattro pazienti sono stati sottoposti al posizionamento sotto guida fluoroscopica di un sondino naso - digiunale 1216 f cui seguita liniezione manuale di 1 , 52 , 5 l di metilcellulosa prima dellesame tc ( enteroclisi tc o e - tc )  . al fine di evitare spasmi , ottenere una omogenea distensione dellintestino tenue , ridurre il disagio e la tensione addominale , veniva somministrato endovena ai pazienti un farmaco anticolinergico ( n - butilscopolamina ) ; somministravamo 10 mg di farmaco quando il paziente cominciava ad avvertire dolore addominale ( generalmente dopo la somministrazione di 11 , 5 l di metilcellulosa o di peg ) , 10 mg subito prima dellacquisizione delle immagini tc . tutti i pazienti venivano studiati con tc multidetettore a 16 strati ( lightspeed pro16 , ge medical system , milwakee , usa ) , con i seguenti parametri di scansione : collimazione 1 , 25 mm , velocit di scorrimento del tavolo 13 , 75 mm / rot , 120 kw , mas 500 , pitch 1 , 375 , tempo di rotazione 0 , 6 s . 
al termine dellinfusione di metilcellulosa , lesame tc veniva eseguito prima e durante la somministrazione di mdc iodato ev , con acquisizione delle immagini dal diaframma al perineo , durante una singola inspirazione . 
lacquisizione delle immagini avveniva in decubito supino tranne che nei pazienti sottoposti ad enteroclisi tc nei quali si verificava vomito al termine o durante linfusione di metilcellulosa ; in tali pazienti lassunzione del decubito prono 1015 l.m. 
a welldistended loop was defined as pathological if thinner than 3 m pathological loops were assessed for wall - thickening characteristics ( symmetrical / asymmetrical , circumferential / eccentric ) , degree and pattern of parietal enhancement after contrast administration ( homogeneously hyperdense or hypodense , stratified , inhomogeneous , target sign with alternating layers of hyperdensity and hypodensity )  . 
we also considered the presence of associated signs : perienteric stranding ( loss of the normal interface between the loop wall and the mesentery ) ; comb sign ( mesenteric hypervascularity with vascular dilatation , tortuosity , hypertrophy and increased spacing , dilatation of the vasa recta ) ; fibrofatty proliferation ( increased attenuation of mesenteric fat ) ; complications such as abscesses ( round or oval masses with fluid density , delimited by a hyperdense wall and possibly containing air ) , fistulae ( communication between two contiguous organs or with the skin ) or phlegmons ( poorly defined mesenteric densities ) ; enlarged lymph nodes ; reduced lumen size with possible upstream dilatation ; extravisceral masses ; and others . the 2 statistical test was used to confirm the statistical significance of the difference between bowel - loop distension in patients studied with peg - ct and those studied with ct enteroclysis . 
a significance level of 95% was considered adequate [ 13 ]  . results examinations were successfully completed in all patients . vomiting occurred in 7 / 64 ( 11% ) patients studied with ct enteroclysis . with reference to the degree of loop distension in patients studied with peg - ct , complete distension of the proximal jejunum was found in 35 / 56 ( 62% ) , of the distal jejunum in 42 / 56 ( 76% ) , of the proximal ileum in 47 / 56 ( 84% ) , of the distal ileum in 47 / 56 ( 84% ) and of the terminal ileum in 43 / 56 ( 78% ) patients . 
in patients studied with ct enteroclysis , complete distension of the proximal jejunum was found in 50 / 64 ( 79% ) cases , of the distal jejunum in 53 / 64 1016 serviva ad ostacolare il reflusso di metilcellulosa nello stomaco e a ridurre il rischio di inalazione del vomito , favorendo pertanto leffettuazione dellesame stesso . 
lo studio tc con il contrasto veniva acquisito 40 secondi dopo la somministrazione ev di 130150 ml di mezzo di contrasto ( ultravist 370 , schering ag , berlino , germania ) , iniettato alla velocit di 3m / s ; se ritenuto opportuno , lesame veniva completato con studio del parenchima epatico in fase portale e tardiva . con una computer workstation ( advantage windows , ge medical system ) le immagini assiali venivano rielaborate con ricostruzioni in 2d , coronali ed eventualmente sagittali . analisi delle immagini tc due radiologi esperti in radiologia gastro - intestinale , non coinvolti nelleffettuazione dellesame , hanno rivisto le immagini in consenso senza essere a conoscenza dei dati clinici e chirurgici , e hanno valutato grado di distensione delle anse tenuali , numero e sede delle anse patologiche . il grado di distensione delle anse stato classificato con una scala soggettiva a 4 punti ( 0 : distensione assente ; 1 : incompleta ; 2 : parziale ; 3 : completa )  . 
unansa ben distesa stata definita patologica se di spessore superiore a 3 mdi essa abbiamo valutato le caratteristiche dellispessimento parietale ( simmetrico / asimmetrico , circonferenziale / eccentrico ) , il grado e le caratteristiche dellenhancement parietale dopo mdc iodato : omogeneamente iperdenso o ipodenso , stratificato , disomogeneo , segno del bersaglio ( o target sign : strati alternati di ipered ipodensit )  . 
abbiamo inoltre considerato la presenza di segni associati : stranding perienterico ( perdita della normale interfaccia tra la parete dellansa e il mesentere ) ; segno del pettine ( o comb sign , cio ipervascolarizzazione del mesentere coinvolto da flogosi che si manifesta come dilatazione delle arterie mesenteriche , tortuosit , ipertrofia e maggiore ampiezza , dilatazione dei vasi retti ) ; proliferazione fibroadiposa ( o fibrofatty proliferation : aumento dei valori di attenuazione del grasso mesenterico ) ; complicanze , come ascessi ( masse a densit fluida rotondeggianti o ovoidali , delimitate da parete iperdensa ; eventuale presenza di aria allinterno della massa ) , fistole ( comunicazioni tra due organi attigui o comunicazioni con la cute ) e flemmoni ( densit mesenteriali scarsamente definite ) ; linfoadenomegalie ; riduzione di calibro del lume con eventuale dilatazione a monte ; masse extraviscerali ; altro . per la verifica della significativit statistica della differenza tra la distensione delle anse intestinali nei pazienti studiati con peg - tc e in quelli studiati con enteroclisi - tc , si adottato il test statistico del 2 . 
si considerato adeguato un livello di significativit pari al 95% [ 13 ]  . ( 83% ) , of the proximal ileum in 58 / 64 ( 90% ) , of the distal ileum in 59 / 64 ( 92% ) and of the terminal ileum in 58 / 64 ( 90% ) patients . 
an additional dose of 250300 ml of methylcellulose or peg was administered at the end of the unenhanced scans in 5 / 64 ( 8% ) patients and in 8 / 56 ( 14% ) patients , respectively . distension of the proximal jejunum and terminal ileum was found to differ significantly , as confirmed by the 2 test ( with 4.10 degrees of freedom ) in patients studied with ct enteroclysis and those studied with peg - ct . 
in other words , distension of the proximal jejunum and the terminal ileum was significantly better in patients studied with ct enteroclysis compared with those studied with peg - ct . 
the first iv dose of 10 mg was usually administered after 11.5 l of methylcellulose or peg . fifteen out of 120 patients ( 12.5% ) had small - bowel neoplasms ( table 1 )  . 
vomito si verificato in 7 / 64 ( 11% ) pazienti studiati con enteroclisi tc . in riferimento al grado di distensione delle anse , nei pazienti studiati con peg - tc si riscontrata una distensione completa del digiuno prossimale in 35 / 56 ( 62% ) pazienti , del digiuno distale in 42 / 56 ( 76% ) pazienti , dellileo prossimale in 47 / 56 ( 84% ) pazienti , dellileo distale in 47 / 56 ( 84% ) pazienti e dellultima ansa in 43 / 56 ( 78% ) pazienti . nei pazienti studiati con enteroclisi tc si riscontrata una distensione completa del digiuno prossimale in 50 / 64 ( 79% ) pazienti , del digiuno distale in 53 / 64 ( 83% ) pazienti , dellileo prossimale in 58 / 64 ( 90% ) pazienti , dellileo distale in 59 / 64 ( 92% ) pazienti e dellultima ansa in 58 / 64 ( 90% ) pazienti . 
2 computed tomography enteroclysis shows minimal hypodense wall thickening of a few jejunal loops ( inside of octagon ) ; several lymphadenopathies ( inside the triangle ) are also evident . 
2 lenteroclisi tc documenta minimo ispessimento parietale ipodenso di alcune anse digiunali ( allinterno dellottagono ) ; limitrofe ad esse sono evidenti diverse linfoadenomegalie ( allinterno del triangolo )  . 
in altre parole la distensione del digiuno prossimale e dellultima ansa ileale sono risultate migliori in modo statisticamente significativo nei pazienti studiati con enteroclisi tc rispetto a quelli studiati con peg - tc . 
la prima dose di 10 mg ev stata solitamente somministrata dopo la somministrazione di 11 , 5 l di metilcellulosa o di peg . quindici / 120 ( 12 , 5% ) pazienti sono risultati affetti da neoplasia dellintestino tenue ( tabella 1 ) ; in particolare abbiamo riscontrato un tumore primitivo benigno in 4 / 15 ( 27% ) pazienti , un tumore primitivo maligno in 10 / 15 ( 67% ) , una localizzazione metastatica da melanoma in 1 / 15 ( 6% )  . 
4 tc con peg : formazione polipoide dellultima ansa ileale ( freccia grande ) in paziente affetta da sindrome di peutz - jeghers ; altri polipi pi piccoli ( frecce piccole ) sono evidenti vicino ad essa . discussione discussion small - bowel neoplasms are uncommon tumours that are often small and difficult to identify and therefore constitute a major diagnostic challenge for radiologists . 
il clisma del tenue ( effettuato previa somministrazione di bario e metilcellulosa tramite sondino naso - digiunale ) e il tenue baritato per os ( solo bario somministrato per via orale ) sono gli esami che vengono solitamente effettuati nel sospetto di patologia neoplastica dellintestino tenue . 
these findings are similar to those of a previous study that attributed a sensitivity of 44% to barium follow - through studies in detecting actual neoplasms in the small bowel [ 15 ]  . 
the results of these studies indicate that small - bowel enema is far more sensitive than barium follow - through in identifying small - bowel tumours , because it can diagnose tumours at an early stage and consequently influence their prognosis . over the last few years , however , the introduction of ct associated with iodinated contrast material and small - bowel distension has modified the diagnostic approach to patients with suspected small - bowel disease [ 9 , 1620 ]  . 
reperto operatorio : metastasi da melanoma . ricano ha comparato il valore del clisma del tenue con quello del tenue baritato per os nella dimostrazione di tumori maligni primitivi del tenue [ 14 ]  . 
i risultati ottenuti da questi studi indicano che il clisma del tenue molto pi sensibile del tenue maritato per os nellindividuare i tumori dellintestino tenue , in quanto pu diagnosticare tumori in stadio precoce e di conseguenza influenzare la prognosi . negli ultimi anni , tuttavia , lintroduzione della tc associata alla somministrazione di mezzo di contrasto iodato e alla distensione delle anse tenuali , ha modificato lapproccio diagnostico al paziente con sospetta patologia tenuale [ 9 , 1620 ]  . 
segni tc caratteristici di una neoformazione maligna sono lispessimento della parete eccentrico o asimmetrico , un contorno ( sia interno che esterno ) lobulato e / o la presenza di una massa della densit dei tessuti molli superiore ai due cm , estesa dal lume alla superficie sierosa [ 18 , l.m. 
la recente introduzione della tc multidetettore ha migliorato la sensibilit della metodica nel rilievo di piccole lesioni neoplastiche [ 5 , 9 , 11 , 12 ] , nonch la qualit delle ricostruzioni 2d e 3d , permettendo di ottenere immagini gradite al clinico e al chirurgo perch utili alla pianificazione chirurgica [ 5 , 19 ]  . un problema dello studio tc dellintestino tenue la mancanza di una standardizzazione della tecnica in letteratura ove non esiste un accordo su quale sia il tipo di contrasto da impiegare per la distensione delle anse , ipodenso [ 2527 ] o iperdenso [ 28 ] , e sulla modalit di somministrazione , per via orale a dose unica , multipla o frazionata [ 23 , 29 ] oppure tramite sondino naso - digiunale con pompa di infusione [ 20 , 30 , 31 ] o senza pompa di infusione [ 19 , 27 ]  . 
i mdc iperdensi sono pi noti e sperimentati , consentono di delimitare le anse intestinali e stabilire se un processo espansivo intrao extraluminale , ma non consentono , per lelevata densit , una accurata valutazione delle caratteristiche di parete ed interferiscono con eventuali ricostruzioni 3d simil - angiografiche . 
i mezzi di contrasto ipodensi sono preferibili a quelli iperdensi perch permettono una migliore rappresentazione dellenhancement della parete compresa tra lipodensit del fluido endoluminale e lipodensit del tessuto adiposo extraluminale [ 26 ] e non interferiscono con le ricostruzioni similangiografiche . 
come mezzi di contrasto ipodensi sono stati proposti per via orale acqua , aria , emulsioni di olio , volumen o peg , oppure metilcellulosa o acqua tramite sondino naso - digiunale [ 12 , 19 , 20 , 2527 , 3032 ]  . le emulsioni di olio forniscono valori di densit pi negativi rispetto allacqua , ma a causa del sapore non sono apprezzate dai pazienti e lelevato costo limita il loro routinario impiego clinico . 
lacqua ha il vantaggio della economicit , della buona accettabilit da parte del paziente , di consentire laccurata valutazione della parete intestinale e del suo enhancement , di non interferire con eventuali ricostruzioni 3d simil - angiografiche , ma viene rapidamente assorbita e tende a stimolare la peristalsi , non garantendo una adeguata distensione del digiuno distale e dellileo . 
laria , somministrata o mediante assunzione orale di sostanze effervescenti a rilascio ritardato o , per lo studio delle ultime anse ileali , per via rettale , ha ottenuto risultati soddisfacenti sulla dimostrazione di patologie intraluminali ed intramurali , anche se leccessiva differenza tra la sua densit ( 800 hu ) e quella della parete intestinale dopo mdc ( da + 100 a - 135 hu ) determina spesso comparsa di artefatti che ne hanno limitato limpiego . 
7a , b polyethylene glycol computed tomography shows ( a ) an intraluminal mass ( black arrow ) with fat density associated with loop invagination ( white arrow )  . 
no agreement exists in the literature on the type of contrast material to be used for bowel distension low density [ 2527 ] or highdensity [ 28 ] or regarding administration protocol oral in a single dose or in multiple or split doses [ 23 , 29 ] , or via a nasojejunal tube with [ 20 , 30 , 31 ] or without [ 19 , 27 ] an infusion pump . 
they allow intestinal loops to be delimited and help verify whether an expansive process is intraor extraluminal , but they do not allow , given their high density , accurate evaluation of wall characteristics , and they interfere with possible angiographylike 3d reconstructions . 
low - density agents are to be preferred because they allow better depiction of wall enhancement between the hypodensity of the intraluminal fluid and the hypodensity of the extraluminal fat tissue [ 26 ] and do not interfere with angiography - like reconstructions . 
proposed low - density contrast agents are water , air , oil emulsions , volumen or peg for oral use , and methylcellulose or water administered through a nasojejunal tube [ 12 , 19 , 20 , 2537 , 3032 ]  . 
oil emulsions provide lower density values than do water , but because of their taste , patients do not appreciate them , and their high cost limits their routine clinical use . 
water is cheap , well - tolerated by patients , allows accurate evaluation of the intestinal wall and its enhancement , and does not interfere with any 3d angiography - like reconstructions ; however , it is absorbed rapidly and tends to stimulate peristalsis , failing to ensure adequate distension of the distal jejunum and the ileuair , administered either orally through delayed - release effervescent substances or rectally , for the study of the terminal ileum , has produced satisfactory results in the demonstration of intraluminal and intramural diseases . however , the excessive difference between its density ( 800 hu ) and that of the enhanced intestinal wall ( + 100135 hu ) often gives rise to artefacts , which have limited its use . 
 a common drawback of oral contrast agents is that they fail to ensure a suitable and uniform distension of all smallbowel loops , giving rise to significant problems in differenti1022 fig . 
8 enteroclisi tc ( scansione ottenuta in posizione prona ) : ultima ansa ileale ed ileo distale a pareti ispessite ; in particolare evidente laspetto target sign dellultima ansa ileale ( freccia grande ) associato a comb sign ( frecce piccole )  . li tra ispessimenti parietali reali o insufficiente distensione / spasmo viscerale . 
il problema viene superato , a prezzo di maggiori invasivit , tempo e costo , dallenteroclisi - tc , metodica sviluppata nei primi anni 90 , in cui , prima dellesecuzione dellesame tc senza e con mdc ev , quantit variabili ( 20002750 cc ) di mdc ipodenso ( metilcellulosa o acqua ) o iperdenso ( diatrizoato di sodio al 4%15% , solfato di bario all1% ) vengono infuse manualmente o con pompa peristaltica attraverso sondino naso - digiunale [ 12 , 19 , 20 , 27 , 30 , 31 ]  . nel nostro lavoro abbiamo usato entrambe le tecniche ed abbiamo preferito usare due mezzi di contrasto ipodensi in modo da ottenere un ottimale studio della parete compresa tra lipodensit del lume opacizzato dal contrasto e lipoating between real wall thickenings and inadequate distension or spas the problem can be overcome although with greater invasiveness , time and costs by using ct enteroclysis , a method developed in the early 1990s in which variable amounts ( 2 , 0002 , 750 cc ) of low - density ( methylcellulose or water ) or high - density ( 4%15% sodium diatrizoate , 1% barium sulphate ) contrast material are infused by hand or with a peristaltic pump through a nasojejunal tube before the ct scan with and without iv contrast material [ 12 , 19 , 20 , 27 , 30 , 31 ]  . in our study , we employed both techniques , and we preferred to use two low - density contrast agents in order to obtain an optimal study of the wall between the hypodensity of the enhanced lumen and the hypodensity of the extravisceral fat . 
we chose methylcellulose for ct enteroclysis and peg for ct enterography , the former already widely employed both in small - bowel enema and in ct [ 19 , 20 , 30 , 31 ] , the latter mainly used in magnetic resonance imaging [ 33 , 34 ] and more rarely in ct [ 26 ]  . 
in contrast to other authors [ 21 , 31 ] , we preferred to administer the hypotonic agent in two doses of 10 mg , the first when the patient felt abdominal pain ( generally after receiving 11.5 l of methylcellulose or peg ) , the second immediately before the ct scan . 
 if we compared the degree of loop distension obtained using ct enteroclysis and peg enterography in our study , we found that the degree of distension was better with enteroclysis compared with the simple oral administration of contrast material , particularly with regard to the jejunu this finding might , theoretically , lower the sensitivity of ct in detecting jejunal neoplasms if the loop is not well distended . 
in our study , we identified ten jejunal neoplasms ( table 1 ) , five studied with ct enteroclysis and five with peg - ct , and all were well depicted . 
regardless of the technique used , in all neoplastic patients , we obtained excellent characterisation of wall masses with exophytic and intraluminal growth patterns . as for the iodinated contrast material , there is no agreement in the literature over the timing of administration : some prefer a 25 - s arterial phase [ 31 ] , others a late arterial phase [ 20 , 28 ] , yet others a single phase of 60 / 70 s [ 35 ]  . 
in our study , the ct investigation was performed 40 s after the start of infusion to obtain a study of the loops in the arterial phase , possibly followed by a study of the liver parenchyma in the portal and late phases . 
iodinated contrast material administration helped us to better define the wall thickness and enhancement pattern , to optimise lesion characterisation , to differentiate between lymphadenopathy and wall masses and to stage the neoplasms . 
abbiamo scelto la metilcellulosa per lenteroclisi tc e il peg per lenterografia tc , la prima gi ampiamente impiegata sia nel clisma del tenue che nellesame tc [ 19 , 20 , 30 , 31 ] , il secondo usato principalmente in rm [ 33 , 34 ] , pi raramente in tc [ 26 ]  . poich non abbiamo una pompa per linfusione , abbiamo somministrato manualmente la metilcellulosa , cercando di ottenere uniniezione costante e continua . 
la tc veniva eseguita al termine dellinfusione ; se le scansioni di base senza contrasto dimostravano una non ottimale distensione delle anse , somministravamo unulteriore dose di metilcellulosa . inoltre , abbiamo usato un ipotonizzante , somministrato ev in modo da avere una maggiore rapidit dazione , con la finalit di ottenere unomogenea distensione delle anse e ridurre il grado di disagio del paziente . 
a differenza di altri autori [ 21 , 31 ] , abbiamo preferito somministrare lipotonizzante in due dosi di 10 mg , la prima quando il paziente lamentava dolore addominale ( generalmente dopo la somministrazione di 11 , 5 l di metilcellulosa o di peg ) , la seconda subito prima dellesame tc . 
seguendo questi accorgimenti , in uno studio precedente abbiamo dimostrato di potere ottenere anche senza pompa di infusione una buona distensione delle anse tenuali [ 19 ]  . se nel nostro lavoro confrontiamo il grado di distensione delle anse ottenuto con le due metodiche , enteroclisi tc ed enterografia con peg , riscontriamo che il grado di distensione migliore con tecnica enteroclisi rispetto alla semplice somministrazione del mezzo di contrasto per via orale , in particolare nel digiuno . 
nel nostro studio abbiamo dimostrato 10 localizzazioni neoplastiche digiunali ( tabella 1 ) , 5 studiate con enteroclisi tc , 5 studiate con peg - tc , e in tali pazienti abbiamo ottenuto una buona rappresentazione della patologia neoplastica . 
indipendentemente dalla tecnica impiegata , in tutti i pazienti neoplastici abbiamo ottenuto uneccellente caratterizzazione delle masse parietali sia a sviluppo esofitico che endoluminali . per quanto riguarda il mezzo di contrasto iodato , non esiste un accordo in letteratura sul tempo di somministrazione : alcuni preferiscono una fase arteriosa a 25 secondi [ 31 ] , altri una fase arteriosa tardiva [ 20 , 28 ] , altri una singola fase a 6070 secondi [ 35 ]  . 
nel nostro studio lesame tc stato effettuato dopo 40 secondi dallinizio dellinfusione iodata , in modo da ottenere uno studio delle anse in fase arteriosa con eventuale completamento di studio del parenchima epatico in fase portale e tardiva . 
la somministrazione del contrasto iodato ci ha permesso di definire meglio lo spessore della parete e le caratteristiche del contrast enhancement parietale , di ottimizzare la caratterizzazione delle lesioni , di differenziare tra linfoadenopatie e masse parietali , e di stadiare la patologia neoplastica . 
rispetto ad esse la tc ha il vantaggio di definire lestensione reale delle lesioni parietali , leventuale estensione transmurale , il grado di coinvolgimento del mesentere e le localizzazioni a distanza . 
in our study , for example , we identified extramural extension in five patients and nodal enlargement in the six cases of lymphoma and the case of adenocarcinoma ; hepatic metastases were present in one case of carcinoid tumour associated with calcified lymph nodes and hypervascular lymphadenopathy . 
however , ct was unable to depict the fine details of the mucosal surface and intramural structures that only barium studies can demonstrate [ 8 , 19 , 30 ]  . 
this factor represents a significant limitation , particularly in diseases where the mucosal layer integrity needs to be demonstrated . in conclusion , multidetector ct , performed after distension of the small bowel with low - density contrast material and after infusion of iodinated contrast material , is a useful method in the diagnosis and staging of small - bowel neoplasms . 
it requires a scrupulous technique and , in selected cases , should be integrated with conventional radiological methods . stro studio , ad esempio , abbiamo riscontrato unestensione extramurale in 5 pazienti , linfoadenomegalie nei 6 casi di linfoma e nel caso di adenocarcinoma ; metastasi epatiche erano presenti in un caso di carcinoide ove si associavano anche linfonodi calcifici e linfoadenomegalie ipervascolarizzate . 
tuttavia , la tc non pu fornire quei fini dettagli della superficie mucosa e delle strutture intramurali che solo gli esami con contrasto baritato possono dimostrare [ 8 , 19 , 30 ]  . 
tale dato costituisce una limitazione importante della metodica , in particolare nelle patologie in cui sia necessario dimostrare leventuale integrit dello strato mucoso . in conclusione possiamo affermare che la tc multidetettore , effettuata previa distensione delle anse tenuali con mezzo di contrasto ipodenso e dopo infusione di mezzo di contrasto iodato , una metodica utile nella diagnosi e stadiazione della patologia neoplastica tenuale . 
sixty patients were evaluated with 64row cta ( vct , general electric healthcare , milwaukee , wi , usa ) with a collimation of 0.625 mm after the injection of iodinated nonionic contrast material ( 4 ml / s )  . 
the common hepatic artery was normal in 60% of patients . the inferior pancreaticoduodenal arteries were either absent or not assessable in 8.3% of cases and there was a double trunk in 5% , a common trunk in 83.3% and a single vessel in 3.3%. 
the techniques high sensitivity allowed us to observe that the prevalence of vascular abnormalities is higher than that reported in the literature . key words mdct angiography hepatic arteries anomalies abdominal vessels riassunto obiettivo . 
sono stati valutati 60 pazienti sottoposti a studio con angio - tc a 64 strati , utilizzando una collimazione submillimetrica ( 0 , 625 ) e previa somministrazione di mezzo di contrasto iodato non ionico ad alto flusso ( 4 ml / s )  . 
le arterie pancreatico - duodenali inferiori risultavano assenti o non valutabili nel 8 , 3% , era presente un doppio tronco nel 5% , un tronco comune nel 83 , 3% , ed un unico vaso nel 3 , 3% . 
questa elevata sensibilit ha permesso di rilevare come il numero di anomalie vascolari risulti pi elevato di quello riportato in letteratura . parole chiave angiografia mdct anomalie delle arterie epatiche vasi addominali introduction introduzione technological advances in multidetector computed tomography ( mdct ) and hardware and software systems have made gli sviluppi tecnologici della tomografia computerizzata multidetettore , dei sistemi hardware e dei software applicar . 
this makes the technique indispensable for surgeons , for example , in planning liver transplantation surgery or , more commonly , in fashioning intestinal anastomoses , the success of which is dependent on adequate vascularity . 
in addition , several regions , such as the splenic fissure of the transverse colon , show major variations in both the distribution and calibre of vessels , which often affect the outcome of surgery . our study aimed to assess the accuracy of 64 - row cta in studying vascular anatomy and evaluating the prevalence of anatomical variations in the origin and collateral branches of the coeliac trunk and superior and inferior mesenteric arteries . 
our findings were compared with those of previous studies based on other methods ( conventional angiography and anatomical dissection )  . materials and methods patient population between march and june 2006 , 350 patients were studied by multidetector spiral ct at our department . 
from among these 150 patients , a radiologist ( cndc ) not involved in subsequent image analysis prospectively and consecutively selected 60 patients meeting the following inclusion criteria : performance of a ct scan in the arterial and venous phase for a variety of clinical indications , from inflammatory ( crohns disease , prostatitis ) to oncological diseases ( breast cancer , lung cancer , prostate cancer , colon cancer )  . 
exclusion criteria were the presence of any condition likely to affect normal vascular anatomy , such as gastric resection surgery ; extended jejunoileal resections ; colonic resections ; anterior rectal resection ; partial pancreaticoduodenectomy ; bariatric surgery ; liver , pancreas , bowel or multiorgan transplants ; and major hepatic resections . 
patients were also excluded if they had conditions that could modify normal arterial diameter and blood flow , as this would affect the study of collateral circulation between the coeliac trunk and the superior mesenteric artery and between the superior and inferior mesenteric artery . 
in detail , patients were excluded if they had stenotic or aneurysmal disease of the arteries to be studied , severe aortic atherosclerosis , arteritis with possible involvement of the vessels being studied ( kawasakis disease , polyarteritis nodosa , takayasus arteritis , churg - strauss syndrome ) present for at least 5 years , severe untreated abdominal aorta aneurysms or aortic dissection . 
patients who had undergone splenectomy , minor hepatic resections , partial jejunoileal resections or had ascending or thoracic aorta disorders were included because the vascular changes associated with these conditions were irrelevant for the purposes of the study . tivi , hanno reso langiografia con tc spirale multidetettore il test diagnostico di scelta per lo studio non invasivo dei vasi splancnici [ 1 ]  . 
le tc di ultima generazione a 64 strati permettono lesecuzione di esami ad altissima risoluzione spaziale ( fino a 0 , 4 mm ) con risoluzione temporale di pochi secondi . 
si pensi ai vantaggi che ci determina in fase di programmazione di interventi di trapianto del fegato o , pi frequentemente , nellesecuzione di anastomosi intestinali , per la cui corretta tenuta fondamentale una adeguata vascolarizzazione . 
inoltre , alcuni distretti , come la flessura splenica del colon trasverso , presentano unimportante variabilit sia nella distribuzione che nel calibro vasale , che spesso inficiano la buona riuscita dellintervento . il nostro studio si prefisso lo scopo di valutare quanto langio - tc a 64 strati sia adeguata nello studio dellanatomia vascolare e nella valutazione dellincidenza delle varianti anatomiche del tripode celiaco , delle arterie mesenteriche superiori ed inferiori , sia per quanto riguarda la loro origine dallaorta sia per quanto riguarda i loro rami collaterali ; tali risultati sono stati confrontati con quelli presenti in letteratura ottenuti con altre metodiche ( angiografia e dissezione anatomica )  . materiali e metodi popolazione di pazienti nel periodo compreso tra marzo e giugno del 2006 , presso il nostro istituto sono stati sottoposti a tc spirale multidetettore 350 pazienti , dei quali 150 per tc total body o addomepelvi . 
tra questi ultimi 150 casi , un radiologo ( c.n.d.c ) non coinvolto nella successiva fase di lettura ha selezionato prospetticamente e consecutivamente 60 pazienti che rientravano nei seguenti criteri di inclusione : esecuzione di uno studio in fase arteriosa e venosa per indicazioni cliniche di varia natura da quelle di origine infiammatoria ( morbo di crohn , prostatiti ) ad indicazioni di tipo oncologico ( tumore della mammella , tumore del polmone , tumore della prostata , tumore del colon )  . 
i criteri di esclusione riguardavano tutte quelle condizioni che potevano modificare la normale anatomia vascolare come : interventi chirurgici di resezione gastrica ; resezioni digiuno - ileali estese ; resezioni coliche ; resezione anteriore del retto ; duodeno - cefalopancreasectomia ; chirurgia bariatrica ; trapianti di fegato , pancreas , intestino o trapianti multiviscerali ; resezioni epatiche maggiori . 
sono stati esclusi anche i pazienti affetti da condizioni patologiche che potevano modificare il normale calibro e flusso arterioso alterando lo studio dei circoli collaterali tra tripode celiaco ed arteria mesenterica superiore e tra arteria mesenterica superiore ed inferiore . 
thus , a patient weighing 80 kg will require the injection of 40 gi ( approximately 115 ml of contrast medium followed by 40 ml of saline solution ) ( xenetix 350 , guerbet , france )  . 
the injection rate , identical for contrast bolus and saline solution , should be high , as arterial enhancement is directly dependent on flow rate ; in our study , a flow rate of 4.0 ml / s was used routinely . 
we used multiplanar reconstructions ( mprs ) in the three spatial planes and three - dimensional reconstructions using maximum intensity projection ( mip ) and volume rendering ( vr )  . 
images were reconstructed and analysed interactively by two radiologists working in consensus , who wrote up the reports on a specific computerised forfor the study of variation of the hepatic arteries , we adopted the michels classification ( table 1 )  . 
sono stati inclusi nello studio i pazienti sottoposti a splenectomia , resezioni epatiche minori , resezioni digiunoileali segmentarie e patologie dellaorta ascendente o toracica dato che le modificazioni vascolari associate a tali quadri patologici risultano ininfluenti ai fini dello studio . tecnica di studio le immagini tc sono state ottenute mediante unapparecchiatura tc multidetettore a 64 strati ( vct , general electric healthcare , milwaukee , usa ) con matrice simmetrica composta da 64 file di detettori , ciascuno dello spessore minimo di 0 , 625 mm utilizando una collimazione di 0 , 625 . 
la somministrazione di mdc risulta un elemento fondamentale per la buona riuscita dellesame : per liniezione del mezzo di contrasto stato utilizzato un iniettore a doppia testa ( ezem , empower cta , ny , usa ) che consente di iniettare un bolo compatto di mezzo di contrasto iodato seguito da una spinta di un bolo di soluzione fisiologica ( nacl 0 , 9% )  . 
la quantit di iodio necessaria per una buona opacizzazione dei vasi mesenterici , espressa in grammi di iodio , si pu ottenere dividendo per due il peso del paziente ( gi = bw / 2 dove g = grammi ; i = iodio ; bw = body weight = peso del paziente )  . 
pertanto per un paziente di 80 kg si devono iniettare circa 40 g di iodio ( circa 115 ml di mdc seguito da 40 ml di soluzione fisiologica ) ( xenetix 350 ; guerbet , francia )  . 
la velocit di iniezione , identica per il bolo di mezzo di contrasto e per quello di soluzione fisiologica , deve essere elevata in quanto lenhancement vascolare arterioso direttamente dipendente dalla velocit del flusso ; nella nostra esperienza abbiamo utilizzato routinariamente una velocit di flusso di 4 , 0 ml / s . 
il tempo di ritardo dallinizio della scansione stato stabilito mediante una tecnica di identificazione automatica del bolo ( bolus tracking ) posizionando la regione di interesse per il calcolo della densit a livello dellaorta al passaggio toraco - addominale ; linizio della scansione avviene ad una densit di 150 uh . lanalisi delle immagini stato eseguito su console dedicata ge equipaggiata con software di ricostruzione advantage windows 4.2 utilizzando ricostruzioni multiplanari sui tre piani dello spazio e ricostruzioni tridimensionali utilizzando algoritmi di ricostruzione di massima intensit ( mip ) e di resa di volume ( volume rendering )  . 
le analisi statistiche sono state effettuate mediante il software statistical package for the social sciences version 14.0 ( spss , chicago , illinois , usa )  . risultati dei pazienti studiati 29 / 60 ( 48 , 3% ) erano di sesso femminir . 
left hepatic artery from left gastric artery and right hepatic artery from superior mesenteric artery common hepatic artery from superior mesenteric artery common hepatic artery from left gastric artery viii anatomia normale epatica sinistra da gastrica sinistra epatica destra da arteria mesenterica superiore epatica sinistra da gastrica sinistra + epatica destra da arteria mesenterica superiore epatica sinistra acc . 
in 5% of cases , there was no common hepatic artery , and the left hepatic artery originated from the left gastric artery and the right hepatic artery from the superior mesenteric artery ( michels iv )  . 
a arteria gastrica sinistra ; b tronco spleno - epato - mesenterico ; c tronco spleno - epatico ; d arteria splenica ; e arteria epatica comune ; f arteria epatica propria ; g arteria epatica sinistra ; h arteria epatica destra ; i arteria epatica destra accessoria che origina dallarteria pancreatico - duodenale inferiore , primo ramo dellarteria mesenterica superiore . j arteria gastroduodenale ; k arteria mesenterica superiore . le e 31 / 60 ( 51 , 7% ) di sesso maschile . 
let era compresa tra i 26 e gli 80 anni ( et media 60 , 314 , 6 )  . superior mesenteric artery tronco celiaco the superior mesenteric artery arose from the aorta at l1 in il tronco celiaco nel 96 , 7% dei casi originava dallaorta r . 
nel 56 , 7% dei pazienti il tripode presentava una normale triforcazione ; nel 10% era presente unarteria epatica sinistra che originava dallarteria gastrica di sinistra ; nel 16 , 7% vi era solo larteria epatica sinistra mentre larteria epatica destra nasceva dalla mesenterica superiore ; nel 5% dei casi vi era lassenza dellepatica comune con lepatica sinistra che originava dalla gastrica sinistra e lepatica destra dallams ; nell1 , 7% stata riscontrata unarteria gastrica sinistra originare direttamente dallaorta ; nell1 , 7% si osservava la gastrica sinistra originare dallaorta e la epatica destra dalla ams ; nell1 , 7% vi era lassenza dellarteria epatica comune con lepatica sinistra che nasceva dalla colica media e lepatica destra dallarteria mesenterica superiore ; nell1 , 7% si osservava una gastrica sinistra originare dallarteria splenica ; sempre in un 1 , 7% vi era una origine separata dei vasi del tripode celiaco ; nell1 , 7% dei casi era presente unepatica sinistra accessoria che originava dalla gastrica sinistra ; infine sempre in un 1 , 7% si registrava lassenza dellarteria epatica comune con lepatica sinistra che originava dalla gastrica sinistra e lepatica destra dallarteria mesenterica superiore . arterie epatiche per quanto riguarda la variabilit delle arterie epatiche abbiamo seguito la classificazione di michels ( tabella 1 )  . larteria epatica comune risultata normale nel 60% dei casi ( michels i )  . 
le arterie pancreatico - duodenali inferiori risultavano assenti o non valutabili nel 8 , 3% , era presente un doppio tronco nel 5% , un tronco comune nel 83 , 3% , ed un unico vaso nel 3 , 3% . 
le arterie sigmoidee variavano in numero da un minimo di 1 ad un massimo di 3 con una media di 1 , 90 , 39 , nel 13 , 3% dei casi era presente una sola arteria sigmoidea , nell83 , 3% erano due , e nel 3 , 3% risultavano essere tre . 
larcata di riolano risultata valutabile nel 31 , 7% dei casi , e di questi era sviluppata nel 68 , 4% . discussione lo studio effettuato risulta uno dei primi in letteratura condotti in fase preliminare con angio - tc a 64 strati per lo studio delle variabilit anatomica del tripode celiaco , dellarteria mesenterica superiore ed inferiore , e dei loro vasi collaterali . 
il confronto tra i nostri dati e quelli presenti in letteratura presenta alcune convergenze ed alcune divergenze che necessitano di unanalisi dettagliata ( tabella 2 )  . la frequenza del tripode che origina in maniera classica dallaorta risultata leggermente pi alta rispetto alla letteratura [ 2 ] , tale risultato dovuto probabilmente al numero di pazienti non elevato ; mentre la frequenza delle anomalie riscontrate ( tronco celiaco mesenterico ed assenza del tripode ) fedele alla letteratura . 
4a - g three - dimensional volume - rendered computed tomography angiography image showing the absence of a common hepatic artery , with the right hepatic artery arising from the superior mesenteric artery and the left hepatic artery from the inferior pancreaticoduodenal artery . 
these arteries originated from the superior mesenteric arteries in 86.7% of cases , from the middle colic artery in 8.3% , from the right colic artery in 3.3% and from the arc of buhler in 1.7%. 
there were no further superior mesenteric artery collaterals . inferior mesenteric artery the inferior mesenteric artery originated from the aorta at the level of the lower third of l3 in 100% of cases . 
the sigmoid arteries ranged in number from one to three ( mean 1.90.39 ) ; there was a single sigmoid artery in 13.3% of cases , two in 83.3% , and three in 3.3%. 
the riolan arcade was assessable in 31.7% of cases and developed in 68.4%. discussion this study is one of the first preliminary studies conducted with 64 - row cta to investigate anatomical variations of the coeliac trunk and superior and inferior mesenteric arteries and their collateral vessels . 
 the incidence of coeliac trunks with a normal origin from the aorta was found to be slightly higher than reported in the literature [ 2 ] , probably as a result of the small size of our study sample . 
our findings with regard to the origin of the superior and inferior mesenteric arteries confirmed the very low incidence of abnormalities . the literature reports a higher frequency of coeliac trunks with normal anatomy and collaterals than observed in our study [ 3 ]  . 
this result may be explained by the small number of patients , as previously noted , but also by the greater sensitivity of 64 - row ct in detecting minor abnormalities , such as accessory hepatic arteries , that are more difficult to visualise on conventional angiography . 
the reported incidence of left gastric arteries originating from the aorta is 10% [ 4 ] , whereas the incidence in our study was substantially lower . the splenic artery with its collaterals was the most constant vessel to arise from the coeliac trunk . 
compared to the anatomy of the hepatic arteries reported by other angiographic or anatomical studies [ 511 ] based on the michels classification , the common hepatic artery originated normally from the coeliac trunk , with an incidence similar to that reported in the literature . 
abnormalities of the origin of the left hepatic artery ( michels ii ) had a higher prevalence than previously reported ; the prevalence of right hepatic arteries arising from the superior mesenteric artery was also higher than reported in the literature . 
the absence of the common hepatic artery , with the left hepatic artery originating from the left gastric artery and the right hepatic artery from the superior mesenteric artery ( michels iv ) , was also more common in our study than reported in the literature . 
conventional angiography is unable to depict small arteries such as accessory or aberrant hepatic arteries ( michels ii , iii and iv ) , which means that the method may underestimate the true prevalence of these vessels [ 5 ]  . 
the reason for this is that in conventional angiography , correct vessel cannulation is operator dependent , and it is technically difficult to opacify colportata in letteratura pi alta rispetto a quella del nostro studio [ 3 ] , tale risultato pu essere la conseguenza di un bias dovuto al basso numero di pazienti studiati come gi riportato ; tuttavia pu essere anche ricondotto alla maggiore sensibilit della tc a 64 strati nel rilevare le piccole anomalie , come le arterie epatiche accessorie che pi difficilmente si rilevano negli studi agiografici classici . 
dalla comparazione dellanatomia delle arterie epatiche rilevate con altri studi angiografici o anatomici [ 511 ] basati sulla classificazione di michels abbiamo rilevato che larteria epatica comune nel nostro studio origina regolarmente dal tripode celiaco , con una frequenza che concorda con la letteratura . 
le anomalie di origine dellarteria epatica sinistra ( michels ii ) , presenta una prevalenza maggiore rispetto alla media della letteratura ; anche per lepatica destra che nasce dallarteria mesenterica superiore stata riscontrata una maggiore prevalenza rispetto alla media della letteratura . 
anche lassenza dellepatica comune con lepatica sinistra che originava dalla gastrica sinistra e lepatica destra dallarteria mesenterica superiore ( michels iv ) , presentava una prevalenza maggiore nei nostri risultati rispetto alla letteratura . questa maggiore frequenza delle anomalie nel nostro studio riteniamo possa essere dovuta allalta sensibilit della metodica che permette la visualizzazione anche di vasi di piccolo calibro . 
infatti langiografia tradizionale mostra una certa difficolt nel riconoscimento di arterie di piccolo calibro come le arterie epatiche accessorie o aberranti ( michels ii , iii e iv ) e quindi tale metodica pu sottostimare la reale prevalenza di questi vasi [ 5 ]  . 
questo da ricondursi al fatto che nellangiografia tradizionale il corretto incannulamento dei vasi risulta operatore dipendente , con una certa difficolt tecnica nellopacizzazione dei rami collaterali a bassa portata ematica . 
viceversa la nostra serie si dimostrata pi efficace nel riconoscimento di tali tronchi grazie alla risoluzione spaziale submillimetrica della tc a 64 strati ed alla tecnica del bolus tracking , che rende possibile un perfetto timing nellopacizzazione dei vasi . 
non sono state rilevate anomalie dei tipi vi , vii , ix e x mentre in letteratura riportata una prevalenza media rispettivamente di 3 , 1%2 , 2% , 0 , 4%0 , 4% , 2 , 7%1 , 2% e 0 , 07%0 , 2% . 
come il nostro studio , anche altri lavori mostrano una elevata prevalenza di casi non classificati ; riteniamo che questo sia sempre da ricondurre allalta sensibilit della metodica da noi utilizzata . 
inoltre da sottolineare come in tutti gli studi analizzati nessuno riporti la presenza dellanomalia tipo x di michels ( epatica comune da gastrica sinistra ) , questo ci porta a concludere che a fronte dei notevoli sviluppi r . 
in contrast , recognition of these vessels was more effective in our series as a result of the submillimetre spatial resolution of 64 - row ct and the bolus tracking technique , which allows perfect timing for vessel opacification . 
this leads us to think that , given the developments in vascular imaging that enable dellimaging vascolare che permette oramai la visualizzazione pressoch di tutti i vasi addominali , sarebbe forse il momento di aggiornare la classificazione di michels inserendo anche quei casi non riportati da lui , ma che presentano una discreta frequenza , come per esempio lepatica comune che nasce direttamente dallaorta o lepatica sinistra dalla colica media . 
tali anomalie non classificate risultano , daltra parte di fondamentale importanza per una corretta programmazione chirurgica . per quanto riguarda larteria mesenterica inferiore , non stata riscontrata nessuna arteria di testut , riteniamo a causa del ridotto numero di pazienti visto che la sua presenza in letteratura riportata solo aneddotticamente senza una precisa percentuale [ 12 ]  . le arterie pancreatico - duodenali inferiori sono risultati i tronchi pi difficili da valutare . 
these unclassified anomalies are fundamental for correct surgical planning . as regards the inferior mesenteric artery , we found no case of testut artery , possibly because of the small study sample , given that its presence has only been reported anecdotally without precise percentages [ 12 ]  . the inferior pancreaticoduodenal arteries proved to be the most difficult to evaluate . 
our findings were in agreement with previous series as regards the manner of origin of these arteries ( single trunk , double trunk [ 13 , 14 ] , single anterior branch [ 15 ] , not assessable or absent )  . 
our data on the origin of the inferior pancreaticoduodenal arteries indicate a higher frequency of abnormalities compared with previous studies , in which the frequency of their origin from the superior mesenteric arteries ranges from 1.8% to 50% depending on the method used , with higher rates with imaging than with autopsy studies [ 16 ]  . 
the frequency of the presence of the arc of buhler , which courses between the superior mesenteric artery and the coeliac trunk and between the superior mesenteric artery and the splenic artery , was similar to that reported in the literature [ 17 ]  . the number of jejunal and ileal arteries was lower than that reported in the literature [ 18 , 19 ]  . 
on ct , it is impossible to distinguish clearly the upper and lower group , as one cannot infer when the superior mesenteric artery enters the root of the mesentery . this subdivision was made arbitrarily by assuming that the presence of a greater distance than observed between the first arteries marked the transition from the upper to the lower group . 
the origin of the right colic artery from the superior mesenteric artery was substantially more frequent compared with that reported in the literature ( 10.7% of cases ) [ 20 ]  . 
the middle colic artery was visualised with a high frequency in our study compared with other studies , where it was reported to be extremely variable ( from 36% to 98% ) [ 21 , 22 ] , with evidence of the middle colic artery arising from the right colic artery in 53% of cases [ 23 ]  . even this highly discordant finding may be due to the small number of patients studied . we found no collaterals to the superior mesenteric artery . among the collaterals to the inferior mesenteric artery , the left colic artery was always present . 
i dati riscontrati riferiti alle anomalie di origine delle arterie pancreatico - duodenali inferiori mostrano una frequenza superiore rispetto alla letteratura in cui la nascita dallarteria mesenterica superiore varia dall1 , 8% al 50% a seconda delle metodiche usate , con valori pi elevati per quelle radiologiche rispetto alle dissezioni [ 16 ]  . 
lorigine delle arterie pancreatico - duodenali inferiori dalla colica media e dalla colica destra in letteratura viene considerata aneddottica mentre nel nostro caso si riscontrata con una frequenza relativamente alta ( 3 , 3%8 , 3% )  . 
la frequenza della presenza dellarco di buhler , il cui decorso stato identificato tra larteria mesenterica superiore ed il tripode celiaco e tra lams e larteria splenica , risultata paragonabile a quella presente in letteratura [ 17 ]  . il numero delle arterie digiuno - ileali risultato inferiore a quello riportato in letteratura [ 18 , 19 ] : riteniamo che ci sia dovuto al fatto che i piccoli rami terminali che irrorano la parte finale dellileo possano essere misconosciuti a causa della sovrapposizione della arcate ileali . 
allesame tc non possibile dividere in maniera esatta il gruppo superiore dallinferiore visto che non si pu dedurre quando larteria mesenterica superiore entra nella radice del mesentere . tale suddivisione stata effettuata in maniera arbitraria assumendo che la presenza di una distanza maggiore di quella osservata tra le prime arterie segnasse il passaggio dal gruppo superiore a quello inferiore . 
langio - tc ha dimostrato di poter rilevare anche i piccoli rami delle arterie digiunoileali arrivando a visualizzare anche la seconda serie di arcate intestinali . larteria ileocolica e la colica destra si sono mostrate estremamente costanti ( 100% dei casi )  . 
lorigine della colica destra dalla mesenterica superiore risulta con una frequenza sensibilmente superiore rispetto alla letteratura ove si riscontra solo nel 10 , 7% dei casi [ 20 ]  . 
larteria colica media stata rilevata con unalta frequenza nel nostro studio rispetto alla letteratura ove la sua presenza risulta estremamente variabile a seconda degli studi eseguiti ( tra il 36% al 98% ) [ 21 , 22 ] con evidenza nel 53% dei casi della nascita dellarteria colica media dalla colica destra [ 23 ]  . 
il numero delle arterie sigmoidee ( da minimo di 1 ad un massimo di 3 nella nostra esperienza ) , come gi riportato in letteratura , correlato alla lunghezza e mobilit del sigma . il dato sulla presenza e sviluppo dellarcata di riolano risulta compatibile con i dati in letteratura , ove riportata unampia variabilit di questo collaterale ( dal 9 , 6% al 99 , 97% ) [ 24 , 25 ] .una precisa conoscenza della sua prevalenza fondamentale , soprattutto in ambito chirurgico . come gi riportato durante la discussione , bisogna sottolineare come i dati rilevati vadano interpretati tenendo anche presente i limiti del nostro studio : in primo luogo il relar . 
 as stated , our data should be interpreted within the limitations of the study : first , the relatively small number of patients studied , which might affect the statistical frequency of given abnormalities ; and second , the lack of a direct correlation with a diagnostic reference standard such as conventional angiography . conclusions sixty - four - row cta is superior to conventional angiography in terms of diagnostic accuracy , safety , patient compliance and examination time . 
our study demonstrates that the new 64 - row ct scanners have reached a high definition in the study of mesenteric vessels , in part thanks to bolus tracking techniques , allowing the visualisation of small vessels and accessory arteries that are difficult to identify with other methods . our findings indicate a larger number of vascular abnormalities than reported in the literature with other modalities . if confirmed , this result would call for a review of the old classifications , such as the michels classification for the hepatic arteries , which although still in use , underestimate the real prevalence of aberrant vessels . 
similarly , we found abnormalities not contained in the michels classification , such as the right hepatic artery originating from the right or middle colic artery . besides replacing conventional angiography in vascular imaging , 64 - row cta also represents an innovation in surgical planning . 
the possibility of performing three - dimensional reconstructions that provide the surgeon with a detailed map of the abdominal vasculature reduces the risk of iatrogenic injuries or ischaemia caused by the ligation of aberrant vascular trunks . our study demonstrated that 64 - row cta can assess the calibre of anastomotic circulations , such as the riolan arcade and the arc of buhler , and therefore be used to evaluate the possibility of using these vessels for intestinal anastomoses . 
 in conclusion , 64 - row cta not only offers substantial advantages in the field of vascular imaging but also promises important developments in anatomical knowledge and surgical planning , becoming the first - line noninvasive modality for the study of abdominal vessels . tivamente basso numero di pazienti studiati , che potrebbe influenzare la frequenza statistica di determinate anomalie riportate ed in secondo luogo , la mancanza di confronto diretto con tecniche diagnostiche standard di riferimento come langiografia tradizionale . conclusioni langio - tc a 64 strati risulta essere una metodica che per adeguatezza diagnostica , sicurezza , compliance per il paziente e velocit di esecuzione risulta superiore allangiografia convenzionale . 
il nostro lavoro dimostra lalta definizione raggiunta dalle nuove tc a 64 strati nello studio dei vasi mesenterici , grazie anche alle metodiche di bolus tracking , permettendo di visualizzare piccoli vasi ed arterie accessorie , difficilmente identificabili con altre metodiche . nel nostro studio stato rilevato un numero di anomalie vascolari pi alto di quello riportato in letteratura con altre metodiche ; tale risultato se confermato renderebbe addirittura necessario aggiornare le vecchie classificazioni , come quella di michels sulle arterie epatiche , utilizzate fino ad ora , in cui presente una sottostima della reale frequenza dei vasi aberranti . 
del maschio1 1department of radiology , 2department of surgery , 3department of gastroenterology , 4department of pathology , vita - salute university , san raffaele hospital , via olgettina 60 , i - 20132 , milan , italy correspondence to : s . 
twenty - nine endocrine pancreatic lesions with definitive morphological and immunohistochemical characterisation after surgical treatment ( n = 24 ) or fine - needleaspiration cytology under endoscopic ultrasonography guidance ( n = 5 ) were retrospectively evaluated . 
endocrine tumour and normal pancreas attenuation and the mean absolute tumour - to - gland attenuation difference were measured in each phase , and data were analysed with students t test . 
in our experience , the pancreatic phase can replace the arterial phase in the evaluation of pancreatic endocrine tumours . key words pancreas spiral computed tomography endocrine system neoplasms riassunto obiettivo . 
sono state valutate retrospettivamente 29 lesioni endocrine pancreatiche , con diagnosi definitiva alla valutazione morfologica e immuno - istochimica , dopo trattamento chirurgico ( n = 24 ) o prelievo citologico sotto guida ecoendoscopica ( n = 5 )  . 
le immagini ottenute in ciascuna fase sono state interpretate da due radiologi , esperti in patologia pancreatica , alloscuro delle diagnosi definitive , valutando le densit di ghiandola pancreatica e tumore e la differenza di attenuazione tumore - ghiandola in valore assoluto , in ogni fase ; i dati ottenuti sono stati analizzati con il test t di student . 
per entrambi i radiologi il valore assoluto della differenza di attenuazione tumore - ghiandola in fase pancreatica ( 4053 hu e 3456 hu ) risultata significativamente maggiore ( p < 0 , 05 ) di quella ottenuta in fase arteriosa ( 3138 hu e 2643 hu )  . 
sct has variable sensitivity ( 47%72% ) [ 13 ] in the detection of endocrine pancreatic tumours , and this sensitivity significantly decreases in the evaluation of small ( < 2 cm ) lesions ( 12% ) [ 2 ]  . 
most of them [ 46 ] have used 4or 8 - row ct scanners and analysed a limited number of lesions and mostly a specific subtype of endocrine tumours ( insulinomas ) , or they have evaluated endocrine lesions together with pancreatic or periampullary tumours [ 7 , 8 ]  . 
 the dual - phase technique , with a pancreatic and portal phase , has been reported [ 1013 ] to be the best choice for detection and staging of pancreatic ductal adenocarcinoma with both sct and mdct . 
in the assessment of pancreatic endocrine tumours , especially insulinomas , the dual - phase ct technique has mostly been reported [ 1 , 4 , 5 , 1416 ]  . 
with mdct , the reported biphasic technique consists of either an early or late arterial phase [ 4 , 14 ] followed by a portal phase [ 4 ]  . 
it has been reported that the pancreatic phase may replace the arterial phase in insulinoma protocols , but further studies have been advocated to asses this issue [ 4 ]  . 
a recent paper has reported on the use of a dual - phase technique with an arterial phase ( at 25 s ) and an early portal phase ( at 5560 s ) , stating that pancreatic endocrine tumours , especially small ones , are better detected during the arterial phase of enhancement [ 9 ] , but the number of patients included in the study was not indicated . the aim of our study was to assess whether the pancreatic phase may replace the arterial phase in the evaluation of endocrine pancreatic tumours , as suggested in the literature [ 4 ] and already accepted for pancreatic adenocarcinomas [ 1013 ] using a 16 - row ct scanner with triple - phase technique . materials and methods patients we retrospectively analysed the ct examinations of 23 patients with a definitive diagnosis of pancreatic endocrine tumour . 
the patients underwent surgical resection ( n = 20 ) or fine - needle - aspiration cytology ( fnac ) with endoscopic ultrasonography ( eus ) guidance ( n = 3 ) , which led to a definitive diagnosis of pancreatic endocrine tumour . 
 a total of 29 lesions were identified ; six patients had multifocal disease ( two lesions per patient ) and two were affect1000 il valore della tomografia spirale computerizzata ( sct ) nella valutazione dei tumori endocrini pancreatici gi stato riportato in vari studi [ 18 ] ; la sensibilit di questa metodica nella ricerca delle lesioni endocrine pancreatiche variabile ( range 47%72% ) [ 13 ] , con una significativa riduzione ( 12% ) nella valutazione di lesioni di piccolo diametro ( < 2 cm ) [ 2 ]  . 
la maggior parte di questi studi [ 46 ] utilizza una tecnologia a 4 o 8 detettori e si occupano di un numero limitato di lesioni , soprattutto di insulinomi , ossia solo di una specifica categoria di tumori endocrini , o analizzano le lesioni endocrine con altri tumori pancreatici o periampollari [ 7 , 8 ]  . 
la pubblicazione pi recente , che tratta specificamente i tumori endocrini pancreatici , un education exhibit in cui viene utilizzato uno scanner a 64 detettori [ 9 ]  . la tecnica bifasica , con acquisizione delle immagini in fase pancreatica e portale , considerata [ 1013 ] la scelta migliore per lidentificazione e la stadiazione delladenocarcinoma duttale , sia in sct che in mdtc . 
considerando la mdct la tecnica bifasica descritta consiste in una fase arteriosa , alternativamente precoce o tardiva [ 4 , 14 ] , seguita da una fase portale [ 4 ]  . 
la tecnica trifasica di acquisizione ( con fase arteriosa , pancreatica e portale ) stata utilizzata solo in pochi pazienti [ 4 , 6 ] ; in letteratura stato riportato come la fase pancreatica possa sostituire la fase arteriosa nei protocolli di studio degli insulinomi , ma questa ipotesi necessita di essere confermata in ulteriori studi [ 4 ]  . 
un recente lavoro afferma che , utilizzando la tecnica bifasica con fase arteriosa ( a 1520 secondi ) e portale precoce ( 5560 secondi ) , le lesioni endocrine pancreatiche siano meglio identificabili in fase arteriosa , specie quelle di piccole dimensioni , ma non viene specificato il numero di pazienti reclutati nello studio [ 9 ]  . scopo di questo studio determinare se la fase pancreatica possa sostituire la fase arteriosa nella valutazione delle lesioni endocrine pancreatiche , come suggerito in letteratura [ 4 ] e gi accettato per ladenocarcinoma pancreatico [ 1013 ] , utilizzando uno scanner a 16 detettori e la tecnica di studio trifasica . materiali e metodi pazienti sono state retrospettivamente analizzate indagini tc di 23 pazienti con diagnosi definitiva di tumore endocrino pancreatico ; tutti gli esami tc sono stati effettuati dal dicembre 2004 allagosto 2006 presso la nostra unit operativa di radiologia . 
ten of 23 patients ( 44% ) had functioning pancreatic endocrine tumours with the typical symptoms of a hormone - secreting lesion : hypoglycaemia ( n = 7 ) , skin rash ( n = 1 ) , cushings syndrome ( n = 1 ) and diarrhoea and dehydration ( n = 1 )  . 
six had nonspecific symptoms ( 26% ) : mesogastric pain ( n = 4 ) and left upper abdominal pain ( n = 2 )  . the remaining seven patients ( 30% ) were asymptomatic , and the pancreatic mass was detected during sonographic evaluation performed for annual follow - up of gallbladder ( n = 3 ) and kidney stones ( n = 4 )  . 
 histological evaluation our series was composed of 24 histological specimens obtained at surgery and five cytological specimens obtained by eus - guided fnac ; the 24 histological specimens were analysed according to the world health organization ( who ) criteria [ 17 ]  . 
the results of morphological and immunohistochemical evaluation and correlation with symptoms are shown in table 1 . mdct technique all examinations were conducted with a 16 - row ct scanner ( aquilion 16 , toshiba , tokyo , japan )  . 
for the unenhanced acquisition , a scan from the top of the liver to the aortic bifurcation was performed with the following parameters : kv 120 ; ma 250 ; gantry rotation time 0.5 s ; hp 15.0 ; beam pitch 0.9 ; couch speed 30 mm / rotation ; acquisition thickness 2 mm ; reconstruction index 2 m the triple - phase technique consists of an acquisition during the arterial , pancreatic and portal phases . 
for contrast administration , a 20 - gauge plastic intravenous catheter was placed in an antecubital vein , and the line was connected to a power injector ( stellant d , medrad inc . , indianola , ia < usa )  . 
a total of 2 ml / kg of body weight of low osmolarity iopamidol ( ultravist 370 , schering ag , berlin , germany ) , ordinarily used for thorax and abdominal ct at our institution , was administered at a rate of 3.64.0 ml / s , followed by 30 ml of normal saline solution at the same rate ( stellant d , medrad inc . , indianola , usa )  . the parameters of the triple - phase acquisition were : kv 120 ; ma 250 ; gantry rotation time 0.5 s ; hp 15.0 ; beam pitch 0.9 ; couch speed 30 mm / rotation ; acquisition thickness 1 mm ; reconstruction index 0.8 mscan extension was the same as for unenhanced scans . 
if a study of the thorax and abdomen was required , the portal scan started from the apex of the lungs and extended to the pelvis . to define the correct timing of the arterial phase , a bolusgendo ad una diagnosi definitiva di tumore endocrino del pancreas . sono state quindi identificate 29 lesioni ; in 6 pazienti stata osservata una malattia multifocale ( 2 lesioni per paziente ) e 2 di essi erano affetti da neoplasie endocrine multiple tipo i ( men - 1 )  . 
dieci / 23 ( 44% ) pazienti erano affetti da tumori endocrini pancreatici funzionanti e presentavano sintomi tipici correlati allipersecrezione ormonale : ipoglicemia ( n = 7 ) ; rush cutaneo ( n = 1 ) ; sindrome di cushing ( n = 1 ) ; diarrea e disidratazione ( n = 1 )  . 
tredici / 23 ( 56% ) pazienti erano affetti da tumori endocrini pancreatici non funzionanti ; 6 pazienti ( 26% ) presentavano sintomi aspecifici : dolore in mesogastrio ( n = 4 ) , dolore in epigastrio ( n = 2 )  . 
gli altri 7 pazienti ( 30% ) erano asintomatici e la masse pancreatiche erano state rilevate durante una valutazione ecografica effettuata per un controllo annuale di colelitiasi ( n = 3 ) e per valutazione di calcoli renali ( n = 4 )  . 
il consenso informato stato ottenuto in tutti i 23 pazienti prima di sottoporsi a indagine tc multidetettore con tecnica trifasica , secondo un protocollo approvato dal comitato etico del nostro istituto . valutazione istologica i nostri reperti erano composti da 24 campioni istologici , ottenuti da trattamento chirurgico e da 5 campioni citologici ottenuti mediante agoaspirato sotto guida ecoendoscopica ; i reperti istologici sono stati analizzati secondo i criteri who del 2000 [ 17 ]  . 
i risultati della valutazione morfologica ed immuno - istochimica e la correlazione con i sintomi sono presentati nella tabella 1 . tecnica di acquisizione mdct tutte le 29 lesioni sono state studiate con mdct a 16 detettori ( aquilion 16 , toshiba , tokyo , giappone )  . 
nei 30 minuti prima dellesame sono stati somministrati per via orale 7501000 ml di acqua ; stata quindi effettuata una scansione senza mezzo di contrasto , seguita da una acquisizione trifasica . 
nellacquisizione senza mezzo di contrasto si effettuata una scansione dallapice del fegato sino alla biforcazione aortica con i seguenti parametri : 120 kv , 250 ma , gantry rotation time : 0 , 5 s , spessore di acquisizione 2 mm16 file di detettori , helical pitch ( hp ) : 15 , 0 , beam pitch : 0 , 9 , velocit del tavolo 30 mm / rotazione , spessore di ricostruzione : 2 mm , con intervallo di ricostruzione di 2 mm . la tecnica trifasica consiste in acquisizioni in fase arteriosa , fase pancreatica e portale . 
per linfusione ev di mdc stato utilizzato un accesso venoso periferico del calibro di 20 gauge , posizionato a livello della superficie volare dellavambraccio o del gomito ; stato somministrato un totale di 2 ml / kg di peso corporeo di iopamidolo ( ultravist 370 , schering ag , berlin , germania ) , routinariamente utilizzato per indagini tc torace e addome nel nostro istituto , seguito da 30 ml di soluzione fisiologica ; entrambe le infusioni sono state eseguite con un flusso di 3 , 64 , 0 ml / s grazie a un iniettore automatico a doppia siringa ( stellant d , medrad inc . , indianola , usa )  . i parametri dellacquisizione trifasica erano : 120 kv , 250 ma , rotation time : 0 , 5 s , spessore di acquisizione : 1 mm16 1001 s . 
nei casi in cui era richiesto uno studio di torace e addome , la scansione portale iniziava dellapice del polmone e si estendeva sino alla pelvi . per stabilire il timing corretto della fase arteriosa , stata utilizzata la tecnica del bolus tracking . 
la regione di interesse ( roi ) stata posizionata a livello dellaorta addominale , allaltezza di piani passanti per lorigine del tronco celiaco ; stato definito il valore di densit da raggiungere per la partenza automatica dellacquisizione in fase arteriosa aggiungendo 70 hu alla densit basale in aorta . 
le fasi pancreatiche e portali sono state iniziate , rispettivamente , 45 e 70 secondi dopo liniezione del mezzo di contrasto , circa 25 e 50 secondi dopo la fase arteriosa , secondo quanto riportato in letteratura [ 13 ]  . interpretazione delle immagini le immagini ottenute durante ciascuna fase sono state internonfunctioning tumours well - differentiated endocrine tumour with benign behaviour well - differentiated endocrine carcinoma well - differentiated endocrine tumour well - differentiated endocrine tumour with benign behaviour well - differentiated endocrine tumour well - differentiated endocrine tumour well - differentiated endocrine tumour with benign behaviour well - differentiated endocrine tumour with benign behaviour well - differentiated endocrine tumour with uncertain behaviour well - differentiated endocrine tumour with uncertain behaviour well - differentiated endocrine carcinoma well - differentiated endocrine carcinoma poorly differentiated endocrine carcinomapan - in2 poorly differentiated endocrine carcinomapan - in2 poorly differentiated endocrine carcinoma poorly differentiated endocrine carcinoma well - differentiated endocrine carcinoma well - differentiated endocrine tumour with uncertain behaviour pan - in2 , pancreatic intraephitelial heoplasia type 2 tracking technique was used : a region of interest ( roi ) was positioned on the abdominal aorta , near the origin of the coeliac trunk , and the scan was triggered at the threshold aortic attenuation ( 70 hu above baseline )  . 
the pancreatic and portal phases started 45 and 70 s , respectively , after the start of the injection of contrast medium , about 25 and 50 s , respectively , after the arterial phase , as suggested in the literature [ 13 ]  . image interpretation images obtained during each phase were interpreted separately by two senior radiologists experienced in pancreatic pathology and who were blinded to the histopathological results . 
in addition , the arterial and pancreatic phase images were assessed for the presence of arterial variation and hypervascular liver metastases . postprocessing was performed using 2d ( multiplanar reconstructions ) and 3d ( maximum intensity projection ) reconstructions to demonstrate the pancreatic pertinence of possible exophytic lesions and evaluate the presence or ab1002 s . 
surgical treatment was considered the gold standard in the evaluation of vascular involvement and arterial variations . the endocrine pancreatic tumours included in our study were divided into three groups on the basis of their size : group a ( diameter 2 cm ) , group b ( diameter from 2 to 4 cm ) , group c ( diameter 4 cm )  . 
attenuation of the normal pancreatic parenchyma and the tumour , expressed in hounsfield units , in all phases ( unenhanced , arterial , pancreatic and portal ) was measured using a circular roi . 
essi hanno analizzato le immagini ottenute in tutte le fasi su work station dedicata ( vitrea2 , vital images inc . , minnetonka , minnesota , usa ) , rilevando la presenza di lesioni pancreatiche focali e definendo le loro dimensioni e il tipo di pattern contrastografico tumorale ( ipervascolare , ipovascolare o eterogeneo )  . 
inoltre hanno valutato la presenza di anomalie vascolari arteriose e di metastasi epatiche nella fasi arteriosa e pancreatica . il post - processing stato effettuato attraverso luso di ricostruzioni 2d ( ricostruzioni multiplanari : mpr ) e 3d ( maximum intensity projection : mip ) , per dimostrare la pertinenza pancreatica di eventuali lesioni esofitiche e per valutare la presenza eventuale di coinvolgimento vascolare , secondo i criteri espressi in letteratura [ 18 , 19 ] e di eventuali anomalie vascolari arteriose . 
comparison of attenuation differences between the tumour and normal pancreas in different phases of contrast enhancement and of the mean absolute tumour - to - gland attenuation difference in the arterial , pancreatic and portal phases were performed with the paired students t test . 
finally , a statistical analysis of the mean absolute tumour - to - gland attenuation difference in the arterial and pancreatic phases was performed in hypervascular lesions only and in group a lesions ( diameter 2 cm ) only . results all mdct examinations were of diagnostic quality , and all pancreatic lesions were detected on both the arterial and pancreatic phases . 
stata misurata lattenuazione del tumore e del pancreas normale , espressa in hu , in tutte le fasi effettuate ( basale , arteriosa , pancreatica e portale ) , utilizzando una roi circolare . 
le regioni di interesse sono state accuratamente posizionate allinterno dei margini dellintera lesione ; nel caso in cui la lesione fosse disomogenea , le regioni di interesse sono state collocate in modo da includere lintera lesione , senza escludere le componenti tumorali dotate di diversi valori di attenuazione tc . 
quindi stata misurata la differenza media di attenuazione tumore - ghiandola , espressa in valore assoluto , nelle fasi arteriosa , pancreatica e portale . stato analizzato il contributo delle fasi arteriosa e pancreatica alla confidenza diagnostica di ciascun radiologo nel riconoscere le lesioni pancreatiche ipero ipovascolari , le lesioni eterogenee , le anomalie vascolari arteriose e le metastasi ipervascolari epatiche ; stato utilizzato uno schema valutativo basato su una scala di 4 punti che riflettono lapporto soggettivo di ogni fase alla confidenza diagnostica ( 3 = contributo elevato , 2 = contributo medio , 1 = contributo basso , 0 = nessun contributo )  . analisi statistica i dati , distribuiti secondo una normale continua , sono stati presentati come media deviazione standard ( sd )  . 
i confronti tra le differenze di attenuazione tra il tumore e il pancreas normale nelle diverse fasi e tra le differenze medie assolute di attenuazione tumore - ghiandola nelle fasi arteriose , pancreatiche e portali sono stati effettuati con il test t di student . 
i dati registrati da ciascun radiologo sono stati analizzati separatamente e quindi i dati raccolti da entrambi i radiologi sono stati infine confrontati per valutare la concordanza diagnostica , in particolare per valutare leventuale presenza di una differenza significativa tra i 2 osservatori . 
infine lanalisi statistica stata effettuata considerando il valore assoluto della differenza di attenuazione media tra tumore e ghiandola pancreatica , in fase arteriosa e pancreatica , nelle sole lesioni ipervascolari e nelle sole lesioni di gruppo a ( diametro 2 cm )  . risultati tutte le indagini tc erano di qualit diagnostica e tutte le lesioni pancreatiche sono state rilevate in entrambe le fasi , arteriosa e pancreatica . 
il diametro medio delle lesioni pancreatiche era 36 , 8 mm ( range : 898 mm ) ; esse erano localizzate a livello del processo uncinato ( n = 5 ) , a livello della testa ( n = 8 ) , a livello del corpo ( n = 8 ) e a livello della coda ( n = 8 ) pancreatici . 
1a - d transverse computed tomography images in a 63 - year - old woman with hypervascular , insulin - secreting , well - differentiated endocrine tumour with benign behaviour in pancreatic tail ( arrow )  . 
a image of the tumour obtained in the unenhanced phase , with tumour - to - gland attenuation difference of 5 hu for the first observer and 1 hu for the second . 
b image of tumour obtained in the arterial phase , with tumour - to - gland attenuation difference of 52 hu for the first observer and 46 hu for the second . 
c image of tumour obtained in the pancreatic phase , with tumour - to - gland attenuation difference of 89 hu for the first observer and 85 hu for the second . 
1a - d immagini tc assiali in una donna di 63 anni affetta da un tumore endocrino ipervascolare ben differenziato secernente insulina con comportamento benigno nella coda pancreatica ( freccia )  . 
a immagine del tumore ottenuta nellacquisizione senza mezzo di contrasto , con una differenza di attenuazione tumore vs ghiandola di 5 hu per il primo osservatore e 1 hu per il secondo . 
b immagine del tumore ottenuta nella fase arteriosa , con una differenza di attenuazione tumore vs ghiandola di 52 hu per il primo osservatore e 46 hu per il secondo . 
c immagine del tumore ottenuta nella fase pancreatica , con una differenza di attenuazione tumore vs ghiandola di 89 hu per il primo osservatore e 85 hu per il secondo . 
d immagine del tumore ottenuta nella fase portale , con una differenza di attenuazione tumore vs ghiandola di 43 hu per il primo osservatore e 34 hu per il secondo . diameter was 21 ( range 762 ) m no hypovascular liver metastases were identified . 
a image of the tumour obtained in the unenhanced phase , with tumour - to - gland attenuation difference of 0 hu for the first observer and 12 hu for the second . 
b image of tumour obtained in the arterial phase , with tumour - to - gland attenuation difference of 41 hu for the first observer and 53 hu for the second . 
c image of the tumour obtained in the pancreatic phase , with tumour - to - gland attenuation difference of 46 hu for the first observer and 50 hu for the second . 
d image of the tumour obtained in the pancreatic phase , with tumour - to - gland attenuation difference of 8 hu for the first observer and 10 hu for the second . 
2a - e immagini assiali e ricostruzione coronale in una donna di 32 anni affetta da un tumore endocrino ipovascolare ben differenziato secernente insulina con comportamento incerto a livello del passaggio corpo - coda pancreatico ( freccia )  . 
a immagine del tumore ottenuta nellacquisizione senza mezzo di contrasto , con una differenza di attenuazione tumore vs ghiandola di 0 hu per il primo osservatore e 12 hu per il secondo . 
b immagine del tumore ottenuta nella fase arteriosa , con una differenza di attenuazione tumore vs ghiandola di 41 hu per il primo osservatore e 53 hu per il secondo . 
c immagine del tumore ottenuta nella fase pancreatica , con una differenza di attenuazione tumore vs ghiandola di 46 hu per il primo osservatore e 50 hu per il secondo . 
d immagine del tumore ottenuta nella fase portale , con una differenza di attenuazione tumore vs ghiandola di 8 hu per il primo osservatore e 10 hu per il secondo . 
e la ricostruzione coronale mostra lorigine di questo tumore dal passaggio corpo - coda pancreatico . mente un coinvolgimento vascolare , confermato dallesplorazione chirurgica , in 5 pazienti : infiltrazione dellarteria e della vena splenica ( n = 1 ) , della vena splenica ( n = 1 ) , della vena mesenterica superiore ( n = 1 ) , della vena porta ( n = 1 ) ; ( 20% ) hypervascular , none hypovascular , four ( 80% ) heterogeneous . data obtained on the attenuation of normal pancreas and endocrine tumour by both radiologists are shown in table 2 . for the first observer , mean absolute tumour - to - gland attenuation was greatest on images obtained in the pancreatic ( 4053 hu ; p < 0.001 ) and arterial ( 3138 hu ; p = 0.02 ) phase when each of these values was compared with that in the portal phase ( 26 hu29 )  . 
3a - d transverse computed tomography images in a 40 - year - old woman with heterogeneous vasoactive - intestinal - peptide - secreting , well - differentiated endocrine tumour with uncertain behaviour in the pancreatic uncinate process ( arrow )  . 
a image of the tumour obtained in the unenhanced phase , with tumourto - gland attenuation difference of 3 hu for the first observer and 29 hu for the second . 
b image of the tumour obtained in the arterial phase , with tumour - togland attenuation difference of 67 hu for the first observer and 89 hu for the second . 
c image of the tumour obtained in the pancreatic phase , with tumourto - gland attenuation difference of 100 hu for the first observer and 115 hu for the second . 
3a - d immagini assiali in una donna di 40 anni affetta da un tumore endocrino disomogeneo ben differenziato secernente vip con comportamento incerto nel processo uncinato ( freccia )  . 
a immagine del tumore ottenuta nellacquizione senza mezzo di contrasto , con una differenza di attenuazione tumore vs ghiandola di 3 hu per il primo osservatore e 29 hu per il secondo . 
b immagine del tumore ottenuta nella fase arteriosa , con una differenza di attenuazione tumore vs ghiandola di 67 hu per il primo osservatore e 89 hu per il secondo . 
c immagine del tumore ottenuta nella fase pancreatica , con una differenza di attenuazione tumore vs ghiandola di 100 hu per il primo osservatore e 115 hu per il secondo . 
d immagine del tumore ottenuta nella fase portale , con una differenza di attenuazione tumore vs ghiandola di 74 hu per il primo osservatore e 74 hu per il secondo . gland attenuation was greatest on images obtained in the pancreatic phase ( 3456 hu ; p < 0.001 ) when compared with that in the portal phase ( 2640 hu )  . 
il numero medio di metastasi ipervascolari era 11 ( range 221 ) , localizzate sia nel lobo destro sia nel lobo sinistro ; il loro diametro medio era 21 mm ( range 762 mm )  . 
le anomalie vascolari arteriose e le metastasi epatiche ipervascolari sono stati rilevati da entrambi i radiologi , sia in fase arteriosa che in fase pancreatica , senza differenze . sono state rilevate anomalie vascolari arteriose in 6 pazienti ( 26% ) : in 1 caso larteria epatica originava dallarte1007 s . 
4a - d transverse computed tomography images in a 51 - year - old man with heterogeneous , glucagons - secreting , well - differentiated endocrine carcinoma in the pancreatic body ( arrow ) with hypervascular liver metastases . 
a image of the tumour obtained in the unenhanced phase , with tumour - to - gland attenuation difference of 16 hu for the first observer and 18 hu for the second . b image of the tumour obtained in the arterial phase , with tumour - to - gland attenuation difference of 42 hu for the first observer and 35 hu for the second . 
4a - d immagini assiali in un uomo di 51 anni affetto da un carcinoma endocrino disomogeneo ben differenziato secernente glucagone nel corpo del pancreas ( freccia ) con metastasi epatiche ipervascolari . 
a immagine del tumore ottenuta nellacquisizione senza mezzo di contrasto , con una differenza di attenuazione tumore vs ghiandola di 16 hu per il primo osservatore e 18 hu per il secondo . 
b immagine del tumore ottenuta nella fase arteriosa , con una differenza di attenuazione tumore vs ghiandola di 42 hu per il primo osservatore e 35 hu per il secondo . 
c immagine del tumore ottenuta nella fase pancreatica , con una differenza di attenuazione tumore vs ghiandola di 77 hu per il primo osservatore e 85 hu per il secondo . 
questi reperti sono stati confermati allesplorazione chirurgica . tredici lesioni erano incluse nel gruppo a : 11 ( 85% ) erano ipervascolari , 2 ( 15% ) ipovascolari , nessuna era disomogenea . 
cinque lesioni erano incluse nel gruppo c : 1 ( 20% ) era ipervascolare , nessuna era ipovascolare , 4 ( 80% ) erano disomogenee . i dati ottenuti da entrambi i radiologi , analizzando latte1008 s . 
analizzando i dati del primo osservatore , il valore assoluto medio della differenza di attenuazione tra la lesione e il pancreas sano era maggiore nelle immagini ottenute nella fase pancreatica ( 4053 hu ; p < 0 , 001 ) e nella fase arteriosa ( 3138 hu ; p < 0 , 02 ) , quando ciascuno di questi valori era confrontato con quello della fase portale ( 26 hu29 )  . 
era presente una differenza statisticamente significativa ( p = 0 , 02 ) tra i valori assoluti medi di differenza di attenuazione tra la lesione e il pancreas sano nella fase arteriosa e pancreatica . 
per il secondo osservatore , il valore assoluto medio della differenza di attenuazione tra la lesione e il pancreas era maggiore nelle immagini ottenute nella fase pancreatica ( 3456 hu ; p < 0 , 001 ) , quando confrontato con quello in fase portale ( 2640 hu )  . 
non cera una differenza statisticamente significativa ( p = 0 , 086 ) tra il valore assoluto medio della differenza di attenuazione tra la lesione e il pancreas sano in fase arteriosa ( 2643 hu ) e quello in fase portale . 
anche in questo caso , stata trovata una differenza statisticamente significativa ( p = 0 , 001 ) tra i valori assoluti medi di differenza di attenuazione tra la lesione e il pancreas sano in fase arteriosa e pancreatica . 
non cera una differenza statisticamente significativa nei dati espressi da ciascun radiologo ( media p = 0 , 4 , range : 0 , 1670 , 991 )  . per il primo radiologo il valore assoluto medio della differenza di attenuazione tra la lesione e il pancreas sano era maggiore in fase pancreatica rispetto al valore in fase arteriosa , sia nelle lesioni ipervascolari ( 5833 hu vs 4822 hu , p = 0 , 21 ) , sia nelle lesioni di gruppo a ( 5923 hu vs 4524 hu , p = 0 , 11 )  . 
anche per il secondo radiologo il valore assoluto medio della differenza di attenuazione tra la lesione e il pancreas sano era maggiore in fase pancreatica rispetto al valore in fase arteriosa , sia nelle lesioni ipervascolari ( 5940 hu vs 4338 hu , p = 0 , 01 ) , sia nelle lesioni di gruppo a ( 6141 hu vs 4030 hu , p < 0 , 01 )  . i risultati espressi riguardo al contributo soggettivo delle fasi arteriosa e pancreatica alla confidenza diagnostica sono riportati nella tabella 3 . 
il contributo medio alla confidenza diagnostica delle fasi arteriosa e pancreatica stato rispettivamente di 2 , 11 , 2 e 3 , 00 . discussione per valutare se la fase pancreatica possa sostituire la fase arteriosa nella valutazione di tumori endocrini pancreatici , come suggerito in letteratura [ 4 ] , sono state analizzate da 2 radiologi , separatamente , le differenti attenuazioni della ghiandola pancreatica sana e del tumore , specialmente il valore assoluto della differenza media di attenuazione tra tumore e pancreas sano , in ciascuna delle fasi della tecnica trifasica di acquisizione con mdct . 
5a , b two - dimensional reconstructions in a 57 - year - old woman with hypovascular adrenocorticotropin - hormone - secreting , functioning , well - differentiated endocrine carcinoma in the pancreatic head ( ring )  . 
similar determinations have already been reported for pancreatic adenocarcinoma for both spiral and multidetector ct [ 1215 ] , indicating that routine acquisition of images in the arterial phase is not necessary for the detection of the pancreatic adenocarcinoma [ 13 ]  . 
the latest paper reported that pancreatic endocrine tumours , especially small ones , were better detected during the arterial phase of enhancement [ 9 ] , but the study did not consider the pancreatic phase . no statistical determinations have been done to asses whether the pancreatic phase is better than the arterial phase in the evaluation of endocrine pancreatic tumours . 
the novelty of our study was to compare and analyse statistically the data obtained in the arterial and pancreatic phases , using a triple - phase technique , in 23 patients affected by 29 confirmed endocrine pancreatic lesions . 
from a previous study [ 13 ] , we know that aortic enhancement is similar during the arterial and pancreatic phases but decreases in the portal phase ; this means that the evaluation of major peripancreatic arteries can be performed in the pancreatic phase as well as in the arterial phase . 
the portal phase is necessary to evaluate liver the parenchyma and major peripancreatic veins . tumour detection depends on attenuation characteristics of the normal pancreatic gland and the tumour , as already reported for pancreatic adenocarcinoma [ 13 ]  . 
pancreatic endocrine tumours can have different types of enhancement ( hypervascular , hypovascular or heterogeneous lesion ) ; in our experience we found all these enhancement patterns , with a majority of hypervascular lesions ( 55% )  . 
in our study , mean attenuation of the normal pancreatic gland and tumour was greater in the pancreatic phase than in the arterial and portal phases for both observers ; even the mean absolute tumour - to - gland attenuation difference was greater in the pancreatic phase for both radiologists , without any statistical difference between theeven in the case of a hypervascular pattern or small lesions ( < 2 cm ) , the mean absolute tumourto - gland attenuation difference was greater in the pancreatic phase for both radiologists , suggesting that these two types of lesions can be better detected in the pancreatic phase . 
the subjective contribution of the pancreatic phase to diagnostic confidence was as high as that of the arterial phase in the detection of hypervascular lesions , hypervascular liver metastases and arterial variations , whereas the pancreatic phase provided a greater contribution to the diagnostic confidence of hypovascular and heterogeneous lesions . 
as already demonstrated for pancreatic adenocarcinoma [ 13 ] , the arterial phase may be considered unnecessary and unhelpful . nellarticolo pi recente [ 9 ] si sostiene che i tumori endocrini pancreatici , specie se di piccole dimensioni , sono pi evidenti durante la fase arteriosa , ma la fase pancreatica non viene considerata . comunque , non stata mai effettuata alcuna determinazione statistica per stabilire se la fase pancreatica sia migliore rispetto a quella arteriosa nella valutazione dei tumori endocrini pancreatici . 
la novit di questo studio confrontare i dati ottenuti in fase arteriosa e in quella pancreatica , utilizzando una tecnica trifasica , in 23 pazienti affetti da 29 lesioni endocrine pancreatiche confermate , effettuando unanalisi statistica . 
da un precedente studio [ 13 ] sappiamo che lenhancement contrastografico aortico simile durante la fase arteriosa e la fase pancreatica , ma diminuisce durante la fase portale ; questo significa che la valutazione delle maggiori arterie peripancreatiche pu essere effettuata nella fase pancreatica cos come in quella arteriosa . la fase portale invece necessaria per valutare il parenchima epatico e le maggiori vene peripancreatiche . la detezione del tumore dipende dalle caratteristiche di attenuazione della ghiandola pancreatica sana e del tumore , cos come gi evidenziato per ladenocarcinoma pancreatico [ 13 ]  . 
il tumore endocrino pancreatico pu presentare differenti quadri di pattern contrastografico ( ipervascolare , ipovascolare , disomogeneo ) ; nella nostra esperienza , abbiamo osservato tutte queste caratteristiche di enhancement , con una prevalenza del pattern ipervascolare ( 55% )  . 
nei dati ottenuti , lattenuazione media della ghiandola pancreatica normale e del tumore maggiore nella fase pancreatica rispetto a quelle arteriosa e portale , per entrambi gli osservatori ; inoltre il valore assoluto della differenza di attenuazione media tumore - ghiandola maggiore nella fase pancreatica , per entrambi i radiologi , senza differenze statisticamente significative tra di loro . 
anche nei casi di lesioni ipervascolari o lesioni di piccole dimensioni ( < 2 cm ) , la differenza di attenuazione media tumore - ghiandola , in valore assoluto , maggiore in fase pancreatica , per entrambi i radiologi ; i nostri risultati perci suggeriscono che questi 2 tipi di lesione possono essere rilevati meglio in fase pancreatica . 
 + 39 - 011 - 6336311 , fax : + 39 - 011 - 6960310 , e - mail : andrea_veltri@infinito.it received : 18 july 2006 / accepted : 20 august 2006 / published online : 20 april 2007 abstract purpose . 
colour - doppler us enabled early detection and treatment of all vascular complications ( 9 / 40 , 22.5% of patients ; 13 complications in nine patients , eight arterial and five portal complications ; 1.4 for each patient with complications )  . 
us also detected biliary complications ( 11 patients , 27.5% : three cases of segmental ducts excluded from the anastomosis , four cases of stenosis of the biliodigestive anastomosis , one lithiasis , three stenoses associated with lithiasis ) , which were successfully managed in 75% of the cases treated with interventional radiology procedures ( percutaneous bilioplasty and / or lithotripsy )  . 
the radiologists role is fundamental for early sonographic diagnosis of post - olt complications in children . vascular complications are often associated in a single patient , and early treatment may improve the prognosis . 
le complicanze vascolari ( 9 / 40 pazienti , 22 , 5% ; 13 complicanze in 9 pazienti , 8 arteriose e 5 portali , 1 , 4 per paziente complicato ) sono state rilevate precocemente con leco - colordoppler , ricorrendo a tc o angiografia solo in rarissimi casi e consentendone il trattamento tempestivo . 
anche le complicanze biliari ( 11 pazienti , 27 , 5% : 3 dotti esclusi primariamente dallanastomosi , 4 stenosi dellanastomosi biliodigestiva , 1 litiasi e 3 stenosi con litiasi ) sono state evidenziate allecografia e trattate con successo nel 75% dei casi sottoposti a procedure di radiologia interventistica ( bilioplastica percutanea e / o trattamento della litiasi )  . 
la radiologia interventistica consente di affrontare in modo sicuro ed efficace molte complicanze biliari . key words paediatric liver transplantation split doppler ultrasound complications parole chiave trapianto di fegato pediatrico split eco - colordoppler complicazioni p . 
the main indications for surgery are cholestatic diseases ( particularly biliary atresia , the most common cause of olt ) , followed by hepatic cirrhosis and metabolic disease [ 1 ]  . 
in most cases , olt was performed for biliary atresia ( 23 / 40 , 57.5% ) , in four for tumours ( 10% , three hepatoblastomas and one hemangioendothelioma ) , in four ( 10% ) for congenital hepatic fibrosis , and in four ( 10% ) for metabolic diseases ( one case of type 1 oxalosis , one of methyl malonic acidaemia , one of fulminant wilsons disease and one of type 1a glycogenosis )  . 
in the remaining five patients , olt was performed because of cystic fibrosis , portocaval malformation , type 2 progressive familial intrahepatic cholestasis , posthepatitis cirrhosis ( due to hepatitis b and delta viruses ) and secondary biliary cirrhosis . 
four of these 40 patients ( 10% ) required a subsequent retransplantation : in three cases ( 7.5% ) due to vascular complications ( two hepatic artery thromboses on day 12 and day 69 , respectively ; one hepatic artery thrombosis associated with portal thrombosis on day 16 ) , and one due to biliary complications arising almost 11 months after the first operation ( table 1 )  . in 21 / 44 cases ( 47.7% ) , the entire liver was transplanted ; this was associated with a simultaneous kidney transplantation in four patients . 
le principali indicazioni allintervento sono le patologie colestatiche ( in particolare latresia delle vie biliari , causa pi frequente di olt ) , seguite dalla cirrosi epatica e dalle malattie metaboliche [ 1 ]  . 
la trombosi venosa portale non costituisce una controindicazione assoluta allolt , ma rende lintervento chirurgico molto pi complesso . le indagini radiologiche hanno due principali campi di applicazione : da un lato il bilancio pre - operatorio , per una corretta selezione dei pazienti , dallaltro il monitoraggio intrae soprattutto post - operatorio [ 25 ]  . 
tante sono le sfide per il radiologo nella sorveglianza dellolt in et pediatrica , anche se gi coinvolto in un programma di olt delladulto ; particolari problematiche , infatti , comportano la necessit di una sua maggiore disponibilit : il coinvolgimento gi in sala operatoria , la necessit di conoscenze chirurgiche appropriate , la minore collaborazione da parte del paziente e la maggiore assiduit dei controlli nel postolt precoce , la maggiore attenzione agli aspetti radioprotezionistici , fino alla massima abilit nelle procedure diagnostiche e terapeutiche mini - invasive . lo scopo di questo lavoro consiste nel riesaminare la casistica del centro trapianti epatici di torino , che conduce un programma di olt pediatrico in associazione a quello assai pi consolidato delladulto , per analizzare , nella nostra personale esperienza , limpegno e limportanza del medico radiologo nella gestione del paziente nel post - olt . materiali e metodi nel periodo compreso tra ottobre 1999 e settembre 2005 sono stati eseguiti presso il centro trapianti epatici del nostro ospedale 44 trapianti epatici pediatrici in 40 bambini , 20 maschi e 20 femmine , di et compresa tra 4 mesi e 14 anni ( media 4 , 6 anni )  . 
lolt stato eseguito nella maggior parte dei casi per atresia delle vie biliari ( 23 su 40 , 57 , 5% ) , in 4 per patologia tumorale ( 10% , 3 epatoblastomi e 1 emangioendotelioma ) , in 4 ( 10% ) per fibrosi epatica congenita , mentre 4 pazienti ( 10% ) erano affetti da malattia metabolica ( un caso di ossalosi di i tipo , uno di acidemia metilmalonica , uno di malattia di wilson fulminante e un caso di glicogenosi di tipo ia ) ; nei restanti 5 pazienti lolt stato eseguito rispettivamente per un caso di fibrosi cistica , una malformazione porto - cavale , un caso di colestasi intraepatica familiare progressiva di tipo 2 , una cirrosi post - epatitica ( da virus dellepatite b e delta ) e , infine , una cirrosi biliare secondaria . 
in 4 di questi 40 pazienti ( 10% ) stato successivamente necessario il ritrapianto , in tre casi ( 7 , 5% ) per complicanze vascolari ( due trombosi dellarteria epatica , in dodicesima e sessantanovesima giornata , e un caso di trombosi dellarteria epatica associata a trombosi portale , in sedicesima giornata ) e nellultimo , dopo quasi 11 mesi dal p . 
within these timeframes , all radiologically detectable complications were evaluated , primo intervento , per complicanze biliari ( tabella 1 )  . in 21 / 44 casi ( 47 , 7% ) stato trapiantato il fegato intero , associato a trapianto contemporaneo di rene in 4 pazienti ; in 22 casi ( 50% ) stato trapiantato il lobo epatico sinistro ( in prevalenza ii e iii segmento , solo raramente associati al lobo quadrato e / o caudato per esigenze anatomiche o chirurgiche ) e in un solo caso ( 2 , 3% ) il lobo destro . 
nel 95 , 5% dei casi lolt stato eseguito da donatore cadavere , mentre in 2 pazienti ( 4 , 5% ) il fegato split proveniva da donatore vivente . lo studio stato condotto retrospettivamente , analizzando dettagliatamente tutti gli esami radiologici diagnostici e interventistici eseguiti presso il nostro istituto nel periodo p . 
in all three cases , the vascular anastomoses were surgically reviewed before completing the operation . us scans performed on the first post - operative days almost constantly revealed right pleural effusions and ascites , as well as mainly perihepatic fluid collections . 
finally , the us reports described acceleration of arterial flow to the hilum in one patient and flow acceleration at the portal anastomosis in seven patients , which resolved spontaneously during the follow - up period . three patients with hepatic artery thrombosis ( including the case associated with portal thrombosis ) required retransplantation , whereas surgical review of the anastomosis proved sufficient in the three remaining cases , as in one of the cases of portal thrombosis . biliary complications included three cases of bile leaks caused by the exclusion of ducts from the anastomosis , which required repeat surgery , and five cases of more - orless pronounced dilatation of intrahepatic bile ducts , which was treated early by biliary drainage in one patient only . during the first 3 months after discharge , 237 colourdoppler us examinations ( 5.9 exams per patient on average ) , one ct study , one arteriography , one cavography , 19 hepatic biopsies , three abdominal two paracenteses , drainages ( all of which were us - guided ) , 15 transhepatic percutaneous cholangiographies , five bilioplasties of the biliary - digestive anastomosis ( bda ) , and two lithotripsies were performed . 
it confirmed the other two haemodynamically significant portal stenoses and the persistence of the segmental portal thrombosis ( detected during the hospital stay and unchanged throughout the follow - up ) , and demonstrated normalisation in the eight cases of arterial or portal flow acceleration only in the anastomosis . 
usguided peritoneal punctures included two cases of us - guided paracenteses due to abundant ascites , one case of drainage tube placement in a patient with an interhepatic diaphragmatic collection , and two cases of diagnostic needle puncture performed to investigate two loculated abdominal collections ( one subphrenic and the other in morrisons pouch ) in which fluid culture excluded a bacterial superinfection . 
in three patients , us confirmed the stability of a risultati complessivamente , durante la fase di ricovero , sono stati eseguiti 622 studi us , 18 dei quali intraoperatori , con una media di 1 esame ogni 1 , 3 giorni ; inoltre , sono state effettuate 4 tc con mdc e.v. , 1 studio rm , 1 arteriografia , 4 biopsie epatiche ecoguidate , 4 drenaggi di raccolte addominali e 2 toracentesi , sempre sotto guida us , e 7 colangiografie percutanee transepatiche . lecografia intraoperatoria ha rilevato 1 caso di ipoperfusione arteriosa , una vera e propria trombosi dellarteria epatica e 1 caso di inversione di flusso nel ramo portale ( per arterializzazione vicaria , secondaria a ipoperfusione da difetto tecnico dellanastomosi venosa ) ; in tutti i 3 casi stata eseguita la revisione chirurgica delle anastomosi vascolari prima di terminare lintervento . nei controlli ecografici durante le prime giornate postoperatorie versamento pleurico destro e ascite , cos come raccolte fluide prevalentemente periepatiche , sono risultate pressoch costanti . 
infine , nei referti us sono state genericamente descritte in 1 paziente accelerazione del flusso arterioso allilo e in 7 accelerazione del flusso in corrispondenza dellanastomosi portale , poi risoltesi spontaneamente nel follow - up . 
in 3 pazienti con trombosi dellarteria epatica ( compresa quella associata a trombosi portale ) , stato necessario il ritrapianto , mentre negli altri 3 casi stata sufficiente la revisione chirurgica dellanastomosi , cos come in 1 caso di trombosi portale . per quanto concerne le complicanze biliari , in 3 casi si sono verificati spandimenti biliari secondari alla presenza di dotti esclusi dallanastomosi , che hanno richiesto un nuovo intervento chirurgico , mentre in 5 casi stata dimostrata una dilatazione pi o meno accentuata delle vie biliari intraepatiche , che in un solo paziente stata trattata precocemente con il posizionamento di un drenaggio biliare . considerando la fase successiva alle dimissioni , ma limitatamente ai primi 3 mesi , sono stati eseguiti un totale di 237 eco - color - doppler ( con una media di 5 , 9 esami per paziente ) , 1 tc , 1 arteriografia , 1 cavografia , 19 biopsie epatiche , 2 paracentesi e 3 drenaggi di raccolte addominali ( tutti ecoguidati ) , 15 colangiografie percutanee transepatiche , 5 bilioplastiche dellanastomosi bilio - digestiva ( abd ) e 2 litotripsie . 
in tale periodo lesame us ha documentato la stabilizzazione di 2 pregresse stenosi arteriose e di 2 stenosi anastomotiche portali asintomatiche , ha confermato le altre 2 stenosi portali , emodinamicamente significative , e la persistenza della trombosi portale segmentaria ( rilevata durante il ricovero e rimasta invariata per tutta la durata del follow - up ) , e ha rilevato la normalizzazione del reperto negli 8 casi di sola accelerazione del flusso arterioso o portale in sede di anastomosi . 
in two other cases , the us finding associated with laboratory abnormalities prompted the use of percutaneous cholangiography , which identified bda stenosis , in one case associated with microlithiasis and with further stenoses of the intrahepatic bile ducts . 
in one patient , however , the anastomotic stenosis recurred associated with a stenosis at the intersection of the iiand iiidanza dellascite , in 1 paziente stato posizionato per alcuni giorni un catetere di drenaggio nella compagine di una raccolta interepato - diaframmatica e in 2 si proceduto allagocentesi diagnostica di altrettante raccolte addominali saccate ( una subfrenica e una nel recesso di morison ) , laddove lesame colturale ha escluso in ambedue i casi la sovrainfezione batterica . 
in 3 pazienti lecografia ha confermato la stabilit di un precedente riscontro di dilatazione delle vie biliari , ritenuto privo di significato clinico , mentre in altri 2 il reperto us , in associazione con lalterazione dep . 
further complications occurred in this stage of the follow - up : one lymphoproliferative disease ( which completely remitted by discontinuing immunosuppressive treatment for 6 months ) , one case of hepatic granulomas and reactive hilar lymph nodes ( both subjected to percutaneous us - guided needle biopsy ) due to infection by bartonella henselae ( cat - scratch disease transmitted by the donor ) and one gaseous embolisation of the portal trunk secondary to enteritis . in the third stage ( more than 3 months from discharge ) , us and transhepatic percutaneous cholangiography identified four new cases of bda stenosis , two associated with biliary lithiasis . 
in another patient , about 3 years after olt , the repeated us detection of biliary ectasia led us to perform mr cholangiography , which documented multiple filling defects at the level of the confluence and common bile duct . 
in addition to confirming this finding , percutaneous cholangiography also enabled bda bilioplasty and lithotripsy to be performed , with good long - term results . one of the two patients with haemodynamically significant portal stenoses underwent surgical review of the anastomosis . 
however , subsequent follow - up examinations revealed the absence of post - anastomotic flow and the onset of chronic liver disease ( with right - lobe hypotrophy and leftlobe hypertrophy ) as well as almost exclusively arterial perfusion . 
five cases necessitated surgery , four for refashioning the bda and one for retransplantation . at the end of the observation period , the patient survival rate was 100% and the organ survival rate was 91% . the considerable advances in surgery and radiology since 1963 ( the year of the first paediatric liver transplantation ) have significantly increased post - olt survival [ 610 ]  . among recent studies , evrard et al.s [ 11 ] retrospective rediscussion gli esami di laboratorio , ha indotto allesecuzione della colangiografia percutanea ; questa ha dimostrato la stenosi dellabd , in un caso associata a microlitiasi e a ulteriori stenosi dei dotti biliari intraepatici . 
ulteriori complicanze occorse in questa seconda fase di follow - up sono state una malattia linfoproliferativa ( poi completamente regredita sospendendo la terapia immunosoppressiva per 6 mesi ) , un caso con granulomi epatici e linfonodi ilari reattivi ( entrambi sottoposti ad agobiopsia percutanea ecoguidata ) da infezione da bartonella henselae ( malattia da graffio di gatto , trasmessa dal donatore ) e unembolizzazione gassosa del tronco portale secondaria a unenterite . nella terza fase ( oltre i tre mesi dalla dimissione ) , infine , us e colangiografia percutanea transepatica hanno dimostrato 4 nuovi casi di stenosi dellabd , 2 dei quali associati a litiasi biliare . 
in un ulteriore paziente , a distanza di circa 3 anni dallolt , il ripetuto rilievo us di ectasia delle vie biliari ha portato allesecuzione di una colangio - rm , che ha dimostrato multipli difetti di riempimento a livello della confluenza e del dotto epatico comune . 
la colangiografia percutanea , oltre a confermare il reperto , ha consentito lesecuzione di una bilioplastica dellabd e di una litotripsia , con buon risultato a distanza . quanto alle 2 stenosi portali emodinamicamente significative , in 1 paziente stata eseguita la revisione chirurgica dellanastomosi ; i controlli successivi , tuttavia , hanno evidenziato lassenza di flusso post - anastomotico e il progressivo sviluppo di unepatopatia cronica ( con ipotrofia del lobo destro e ipertrofia del sinistro ) , in presenza di una perfusione pressoch esclusivamente arteriosa . 
nellaltra paziente con precedente evidenza di stenosi portale si assistito , in questo terzo periodo , alla progressione della stessa in occlusione , con successivo tentativo di disostruzione / angioplastica percutanea , scarsamente efficace . nel complesso , durante lintero follow - up , le complicanze vascolari maggiori sono state in totale 13 , concentrate in 9 pazienti ( 22 , 5% ; 1 , 4 complicanze per ogni paziente complicato ) : 6 trombosi dellarteria epatica ( 2 associate a trombosi anche portale , una precoce e una tardiva , dopo la revisione dellarteria ) , 2 stenosi arteriose , 3 trombosi portali isolate ( 1 parziale e 2 complete ) ( tabella 2 )  . 
in 11 pazienti ( 27 , 5% ) si sono verificate complicanze biliari ; in 3 di questi sono stati precocemente diagnosticati dotti esclusi ; gli altri sono stati 4 stenosi della abd , 1 caso di litiasi e sludge biliare , e infine 3 pazienti con concomitanza di stenosi dellabd e sludge e litiasi biliare . 
 [ 12 ] reported survival rates of 88% , 82% and 82% at 1 , 3 and 5 years , respectively , in 249 paediatric olts carried out between 1986 and 1998 . 
our study , although our series was small due to our relatively short experience with paediatric olt ( dating from 1999 only ) , showed survival rates of 100% at 1 , 3 and 5 years . 
the results of our analysis allow us to state that diagnostic and interventional radiology also contributed to this success rate . in contrast , the rate of retransplantations at our centre ( 10% ) is similar to that reported by other recent studies . 
for example , the hamburg centre reported a retransplantation rate of 11.8% among the 170 paediatric olts performed between 2001 and 2004 , with a survival rate of 97% up to january 2005 [ 13 ]  . the incidence of the main post - olt biliary complications in our series was also in keeping with those reported by the main international liver transplantation centres . 
this may be related to sample size ( reference studies were considerably larger than ours ) but also to the learning curve of the staff involved in the paediatric olt programme , which only began in 1999 at our centre as opposed to the early 1980s at the main international centres . 
nonetheless , the successful management of complications in our series could also in part be ascribed to the radiologist , who played a central role in the early diagnosis and treatment of some of the complications . discussione grazie ai notevoli progressi medico - chirurgici , ma anche di quelli delle discipline radiologiche , dal 1963 ( anno del primo trapianto epatico pediatrico ) a oggi si assistito a un significativo incremento della sopravvivenza post - olt [ 610 ] ; infatti , tra le pi recenti casistiche , evrard et al . [ 11 ] riportano , dopo analisi retrospettiva di 500 olt pediatrici effettuati tra il 1984 ed il 2000 , una percentuale di sopravvivenza a 1 , 5 e 10 anni rispettivamente dell85% , 81% e 79% , mentre migliazza et al . 
 [ 12 ] , in 249 olt pediatrici eseguiti tra il 1986 ed il 1998 , hanno evidenziato tassi di sopravvivenza a 1 , 3 e 5 anni rispettivamente dell88% , 82% e 82% . 
nella nostra casistica , per quanto composta da un numero di pazienti ancora limitato vista la recente esperienza nel settore pediatrico ( datata solo dal 1999 ) , le percentuali di sopravvivenza a 1 , 3 e 5 anni sono state finora tutte del 100% ; alla luce dei risultati dalla nostra analisi , ragionevole pensare che a questo risultato abbia contribuito anche la radiologia diagnostica e interventistica . sovrapponibile ad altri studi di recente pubblicazione , invece , la percentuale di re - olt effettuati nel centro , attestata a oggi al 10% . 
per citare un esempio , il centro di amburgo riporta , nellambito di una casistica di 170 olt pediatrici eseguiti tra il 2001 e il 2004 , un tasso di ritrapianti dell11 , 8% , con una sopravvivenza del 97% al gennaio 2005 [ 13 ]  . anche per quanto riguarda lincidenza delle principali complicanze biliari nel post - olt , i risultati del nostro studio sono paragonabili a quelli riportati in letteratura dai principali centri internazionali di trapianto epatico , mentre lincidenza delle complicanze vascolari appare maggiore , risultando attualmente del 22 , 5% ( tabella 3 )  . 
la motivazione di questo secondo dato potrebbe essere legata alle dimensioni del campione ( le casistiche di confronto sono notevolmente pi ampie della nostra ) , ma anche alla curva di apprendimento dello staff medico - chirurgico coinvolto nel programma di olt pediatrico , iniziato come detto nel 1999 , rispetto agli inizi negli anni ottanta dei principali centri internazionali . ciononostante , la buona gestione delle complicanze nella nostra serie potrebbe essere in parte attribuibile anche al radiologo , visto il ruolo da lui assunto nella diagnosi precoce e nel trattamento di alcune delle complicanze stesse . limportanza delleco - color - doppler nella diagnosi precoce delle complicanze vascolari post - olt , infatti , sancita ormai da numerose esperienze cliniche [ 4 , 2023 ]  . 
questa metodica , non invasiva ed eseguibile anche al letto del malato , indicata nellimmediato periodo post - operatorio dei pazienti pediatrici con frequenza minima giornaliera , ma spesso anche pi assidua , specie nei casi che necessitano di uno stretto monitoraggio per la complessit dellintervento chirurgico ( anastomosi multiple o a rischio , difficolt di perfusione intraoperatoria , ecc . ) o per condizioni cliniche pi precarie . 
this noninvasive study , which can be performed at the patients bedside , if need be , should be performed with at least daily frequency in the immediate postoperative follow - up of paediatric patients . 
this frequency should be increased in cases requiring close monitoring because of the complexity of the operation ( multiple or risk anastomoses , intraoperative perfusion difficulties , etc . ) or because of a less stable clinical condition . 
in particular , the earliest possible diagnosis of hepatic artery thrombosis , one of the most feared complications due to the high mortality rates and the frequent need for immediate retransplantation , is a determinant for prognosis [ 2428 ]  . 
close radiological monitoring in the first few days post - olt is thus essential for detecting thrombosis at an early stage , when surgical review of the hepatic artery can resolve the thrombosis , thereby reducing parenchymal necrosis and especially ischaemic biliary damage , which may in turn eventually lead to severe biliary complications . 
in our experience , colour - doppler us provided early diagnosis of arterial thrombosis in six patients who were still asymptomatic : immediate surgical review of the anastomosis proved adequate in three of them , thereby improving their prognosis . 
in children , ct angiography ( which has supplanted angiography in the diagnosis of vascular complications , unless this is used to plan endovascular treatments ) is usually reserved for the study of cases with uncertain us findings , as it involves a considerable radiation dose and increased risk of kidney failure due to the use of intravenous iodinated contrast material [ 29 ]  . 
the other radiological modalities can be used after colour - doppler us , as needed and with lower frequency , in ascending order of invasiveness : contrastenhanced us , ct , mri and angiography , the latter being limited to cases requiring percutaneous treatment [ 4 ]  . on the basis of our personal experience , we stress the importance of a correct and integrated interpretation of us findings to avoid dangerous false positive results . 
this finding is mostly related to a difference in calibre between the donors and the receivers vessels , to a transient oedematous reaction ( as confirmed by the gradual spontaneous resolution on subsequent us scans ) or , finally , to kinking of the vascular anastomosis . 
lo stretto monitoraggio radiologico nelle prime giornate postolt dunque fondamentale per riconoscere la trombosi in fase iniziale , quando la revisione chirurgica dellarteria epatica pu essere risolutiva , riducendo la necrosi del parenchima epatico e soprattutto il danno ischemico alle vie biliari , foriero di successive complicanze biliari gravi . 
nella nostra esperienza , lesame eco - color - doppler ha diagnosticato precocemente la trombosi arteriosa in 6 pazienti ancora asintomatici , in 3 dei quali risultata sufficiente limmediata revisione dellanastomosi chirurgica , con evidente miglioramento della prognosi . 
nel paziente pediatrico langiotc ( che ha ormai soppiantato langiografia nella diagnosi delle complicanze vascolari , se non in previsione di trattamenti endovascolari ) viene di norma riservata allo studio dei casi di incerta interpretazione ecografica , comportando una notevole irradiazione e un aumentato rischio di insufficienza renale per la somministrazione endovenosa di mdc iodato [ 29 ]  . 
seguono , quando necessario e con frequenza di impiego nettamente inferiore , le altre metodiche radiologiche in ordine crescente di invasivit : lecografia con mdc , la tc , la rm e langiografia , questultima limitatamente ai casi in cui si prospetti un trattamento percutaneo [ 4 ]  . anche in base allesperienza personale , ci preme per sottolineare limportanza di una corretta e integrata interpretazione dei reperti us , onde evitare pericolosi falsi positivi . 
infatti , nellimmediato post - operatorio , relativamente frequente ( 20% nella nostra casistica ) il riscontro di unaccelerazione di flusso in sede anastomotica , soprattutto portale ; tale reperto per lo pi legato a una differenza di calibro tra vaso del donatore e quello del ricevente o a una temporanea reazione edematosa tissutale ( come confermato dalla progressiva risoluzione spontanea nei successivi controlli us ) , o infine a unangolatura dellanastomosi vascolare ( kinking )  . 
in questi casi , si devono effettuare controlli pi ravvicinati per monitorare le ripercussioni emodinamiche sulla circolazione intraepatica ed evidenziare eventuali segni precoci di sofferenza del parenchima epatico , o prevenire la progressione della stenosi fino alla tromp . 
in these cases , close monitoring is needed to check the haemodynamic effects on the intrahepatic circulation and detect any early signs of parenchymal distress , as well as to prevent the stenosis developing into a thrombosis ( as unfortunately occurred in two of our patients as a result of the surgeons reluctance to treat the complication in the absence of clinicallaboratory signs )  . as regards biliary complications , leaks from open bile ducts call for immediate reoperation . 
in centres providing a highly qualified interventional radiology service , interventional radiology plays a central role as the initial approach in bda or biliary stenoses , lithiasis and postbiopsy haematobilia . 
it also represents the only alternative to surgery , given that fashioning of the bda on the roux loop ( always adopted in paediatric olt ) makes endoscopic retrograde cholangiopancreatography technically impossible [ 3033 ]  . 
la radiologia interventistica , nei centri in cui sia disponibile un servizio di comprovata esperienza , assume invece un ruolo fondamentale come primo approccio nelle stenosi dellabd o dei dotti biliari , nella litiasi e nella emobilia post - bioptica , costituendo lunica alternativa alla chirurgia dal momento che il confezionamento dellabd su ansa alla roux ( costantemente adottata nellolt pediatrico ) rende tecnicamente impossibile lesecuzione di una colangiopancreatografia retrograda endoscopica [ 3033 ]  . 
in letteratura , la loro incidenza molto variabile ; ad esempio , kling et al . [ 34 ] riportano una percentuale pari al 33% , ben superiore al 7% segnalato da kim et al . 
nella loro esperienza [ 13 ]  . diversamente dalle stenosi biliari intraepatiche diffuse , che sono generalmente dovute a un danno ischemico , immunologico o infettivo , le stenosi dellabd sono generalmente il risultato della formazione di tessuto fibroso a livello anastomotico [ 35 ]  . 
in paediatrics , transhepatic percutaneous cholangiography and biliary drainage demand a higher degree of expertise and skill ( above all to perform the puncture , which always requires close us monitoring ; fig . 6 ) because of the small calibre of the intrahepatic biliary branch . 
a more recent study by the same author [ 37 ] on the percutaneous dilatation of paediatric post - olt stenoses reported a 25% rate of stenotic recurrences ( after one or more bilioplasty procedures ) requiring surgical review or retransplantation , and 15% of cases necessitating a stent to ensure anastomosis patency . in our study , interventional radiology procedures were successful in 75% of treated cases , with long - term patency of the bda and intraand extrahepatic bile ducts and absence of sludge or biliary lithiasis . 
in one patient , the ischaemic damage to the entire biliary system was such that repeated restenoses followed numerous bilioplasty attempts si biliare , le procedure interventistiche danno i migliori risultati a distanza . 
 [ 36 ] hanno segnalato la mancata opacizzazione dellalbero biliare nel 4 , 2% dei casi . un pi recente studio del medesimo autore [ 37 ] sulla dilatazione percutanea delle stenosi nel post - olt pediatrico riporta un 25% di recidive stenotiche ( dopo una o pi bilioplastiche ) tali da richiedere la revisione chirurgica o il ritrapianto , e un 15% di casi in cui stato necessario il posizionamento di uno stent per mantenere la perviet dellanastomosi . 
 anche nel nostro studio le manovre di radiologia interventistica sono state risolutive nel 75% dei casi trattati , con perviet a lungo termine dellabd e delle vie biliari intraed extra - epatiche , e assenza di sludge o litiasi biliare . 
in una paziente il danno ischemico allintero sistema biliare stato tale che numerosi tentativi di bilioplastica , eseguiti con successo nellimmediato , sono stati seguiti costantemente da restenosi ; si quindi tentata una revisione chirurgica , ma alla p . 
in the last case , however , cholangiography was successfully performed , but despite various attempts , the stenosis could not be negotiated , so the bda was refashioned . the greatest risk in repeating these procedures , which is compounded by the performance of other examinations using ionising radiation before or after olt , is the induction of subsequent neoplasms ( particularly haematological ) as an expression of stochastic damage . 
in order to reduce the risk as far as possible and limit the number of parenchymal punctures , us guidance is commonly used at our centre in the early steps of biliary drainage ( from transhepatic percutaneous cholangiography to catheter placement ) , followed by minimal short periods of pulsed radioscopy . fine stato necessario il ritrapianto . 
nellultimo caso , invece , stata eseguita con successo la colangiografia ma , nonostante diversi tentativi , non si riusciti a valicare la stenosi , per cui si ricorsi al riconfezionamento dellabd . il rischio maggiore insito nella ripetizione di queste procedure , incrementato dalleventuale esecuzione nel pree / o post - olt di ulteriori indagini radiologiche impieganti radiazioni ionizzanti , linduzione di successive neoplasie ( soprattutto ematologiche ) come espressione di danno stocastico . 
al fine di ridurre il pi possibile questo rischio , entrato ormai nella pratica comune del nostro istituto limpiego della guida ecografica nelle prime fasi del drenaggio biliare ( dalla colangiografia percutanea transepatica fino al posizionamento del catetere ) , anche per limitare il numero di punture parenchimali ; successivamente si utilizzano al minimo brevi periodi di radioscopia pulsata . conclusions conclusioni if the success of olt depends in part on the early diagnosis and appropriate treatment of complications , in our experience albeit with a small series the radiologist proved irreplaceable in all stages . 
by integrating the information obtained with the various diagnostic methods ( including liver biopsy carried out by the radiologist with us guidance ) , the radiologist contributes greatly to every diagnostic conclusion and participates actively in therapeutic procedures . 
in particular , interventional radiology techniques allow safe and minimally invasive postoperative complication management , often avoiding further reoperations and contributing to improved prognosis of these young patients . se il successo dellolt epatico dipende in parte dalla diagnosi precoce e dal trattamento appropriato delle complicanze , nella nostra pur limitata esperienza il medico radiologo si dimostrato da subito insostituibile . 
infatti , integrando le informazioni ottenute dalle varie metodiche ( compresa la biopsia epatica , eseguita sempre dal radiologo con guida ecografica ) , egli fornisce allo staff del trapianto un apporto fondamentale per ogni conclusione diagnostica e partecipa attivamente alla fase terapeutica . 
giovanni battista di torino , c.so bramante 88 / 90 , i - 10126 torino , italy , tel . : + 39 - 011 - 6333574 , e - mail : gciccarelli@molinette.piemonte.it received : 21 may 2006 / accepted : 18 october 2006 / published online : 20 april 2007 abstract purpose . 
definitive histological assessment of margin status , including status after reexcision , was infiltrated margins in 23 patients ( 23% ) and clear margins in 79 patients ( 77% )  . definitive histological assessment in 12 / 19 patients ( 63.15% ) with intraoperative reexcision , confirmed margin infiltration of the first specimen . 
in tutti i casi sono state eseguite le radiografie dei pezzi anatomici nelle due proiezioni ortogonali con ingrandimento diretto per valutare : presenza / assenza della lesione ; posizione della lesione nel pezzo anatomico ; direzione verso cui effettuare un eventuale ampliamento nei casi in cui la lesione era in prossimit di un margine . 
il confronto tra le diagnosi radiologiche e istopatologiche prima degli ampliamenti , quando effettuati , ha dato valori di sensibilit e di specificit rispettivamente del 66% e 86% e valore predittivo positivo e negativo , rispettivamente , del 74% e 81% . 
la valutazione istologica definitiva dei margini , considerando anche gli ampliamenti quando effettuati , risultata : margini infiltrati in 23 pazienti ( 23% ) e indenni in 79 casi ( 77% )  . in 12 / 19 casi ( 63 , 15% ) con ampliamento estemporaneo lesame istologico definitivo conferm che i margini del primo pezzo anatomico erano infiltrati : in queste pazienti lampliamento estemporaneo stato decisivo . 
la radiografia del pezzo operatorio risultata attendibile nel rilevare i margini non infiltrati ( valore predittivo positivo 74% ) e ci ha consentito di ridurre la percentuale di secondi interventi dal 31% al 20% . 
these lesions require excisional surgery after preoperative wire localisation to ensure the removal of a portion of parenchyma with the lesion exactly at the centre of the specimen [ 1 , 2 ]  . 
in these cases , for which breast - conserving surgery is used in part to guarantee an optimal cosmetic outcome , it is fundamental to assess the resection margins to prevent the risk of reintervention and / or local recurrence [ 3 , 4 ]  . 
correct surgical excision is dependent on the use of intraoperative specimen radiography in the two orthogonal projections to ensure an adequate amount of tissue has been removed [ 5 , 6 ]  . the aim of this study was to evaluate the diagnostic reliability of specimen radiography for the assessment of resection margins in breast - conserving surgery for impalpable lesions to avoid the need for reintervention to achieve clear margins . materials and methods this was a retrospective review of 123 consecutive patients ( age range 4187 years ) who between january and december 2004 underwent breast - conserving surgery for impalpable breast lesions detected on clinical and / or screening mammography . 
in all cases , the preoperative radiological and cytohistological diagnosis indicated suspicious lesions or definitely positive lesions ( r4 / 5 , c4 / 5 , b4 / 5 )  . 
all lesions were preoperatively marked with vegetable charcoal to allow the surgeon to correctly locate and excise the lesion . before concluding the operation , the surgeon sent the excised specimen to the radiology department for radiography : suture threads were applied on each surgical specimen in the operating room to identify and orient the resection margins ( one thread for the areolar margin ; two threads for the medial or lateral margin ; three threads for the cranial or caudal margin )  . all radiographs were obtained in the two orthogonal planes . 
specimen radiography was performed on an analogical mammography unit senographe dmr v2 ( ge ) using the direct magnification technique ( 1 : 5 or 1 : 8 ) , appropriate compression , kilovolt ( kv ) between 25 and 28 and milliampere second ( mas ) between 10 and 20 depending on specimen size . 
the images were printed using kodak min r film and a dedicated automatic developer kodak mini loader 2000 p . specimen radiographs were compared with the baseline mammograms to assess adequacy of the excision . 
the parameters considered in the radiological reports were : presence / absence of the lesion position of the lesion within the specimen ( relative to the direction in which to extend the excision in the case of resection margins ) close margins la crescente diffusione della mammografia clinica e dei programmi di screening ha portato allincremento dei reperti di lesioni mammarie sospette non palpabili da sottoporre ad intervento chirurgico previa localizzazione preoperatoria per consentire lexeresi chirurgica di una porzione di parenchima mammario con la lesione esattamente centrata nella compagine [ 1 , 2 ]  . 
in questi casi , in cui lapproccio chirurgico di tipo conservativo anche per ottenere risultati estetici ottimali , importante la valutazione dei margini di resezione per evitare il rischio di reinterventi di radicalizzazione e / o di recidive locali [ 3 , 4 ]  . 
una corretta escissione chirurgica non pu prescindere dallesecuzione , durante lintervento , della radiografia del pezzo operatorio effettuato nelle due proiezioni ortogonali per avere una ragionevole certezza di adeguata escissione [ 5 , 6 ]  . scopo del presente lavoro quello di valutare lattendibilit diagnostica della radiografia del pezzo operatorio nellanalisi dei margini di resezione degli interventi conservativi di resezione mammaria in lesioni non palpabili per evitare , nei casi di margini infiltrati , di dover programmare un secondo intervento chirurgico per radicalizzare la lesione . materiali e metodi si tratta di unanalisi retrospettiva effettuata in 123 pazienti consecutive , di et compresa tra 41 e 87 anni che , da gennaio a dicembre 2004 , sono state sottoposte a interventi conservativi per lesioni mammarie non palpabili diagnosticate con esami di senologia clinica e / o di screening mammografico . 
in tutti i casi la diagnosi radiologica e cito - istologica preoperatoria stata di lesioni sospette o francamente positive per lesioni neoplastiche ( r4 / 5 , c4 / 5 , b4 / 5 ) : in tutti i casi , in previsione dellintervento chirurgico , stata posizionata una traccia di carbone vegetale per consentire al chirurgo una corretta localizzazione della lesione e conseguente adeguata asportazione . il chirurgo , prima di considerare concluso lintervento , ha inviato in radiologia il pezzo anatomico asportato per lesecuzione della radiografia : su ogni frammento chirurgico sono stati applicati , in sala operatoria , alcuni fili di sutura per identificare i margini di resezione e consentirne un corretto orientamento spaziale ( 1 filo margine areolare ; 2 fili margine mediale o laterale ; 3 fili margine craniale o caudale )  . le radiografie sono sempre state effettuate secondo i due piani ortogonali : dapprima il pezzo operatorio stato posizionato sul piatto del mammografo orientato con i fili di repere come per effettuare una proiezione cranio - caudale . successivamente il pezzo stato ruotato lateralmente di 90 per effettuare una proiezione latero - laterale . 
le radiografie del pezzo operatorio sono state effettuate utilizzando un mammografo analogico senographe dmr v2 ( ge ) , con la tecnica dellingrandimento diretto ( 1 : 5 o 1 : 8 ) , con appropriata compressione , kv compresi tra 25 e 28 e mas tra 10 e 20 in rapporto alle dimensioni del pezzo operatorio : le img . 
after radiography , the surgical specimen was sent to the pathologist for histological assessment . evaluation of the diagnostic reliability of specimen radiography was performed by comparing the radiographic and histopathological diagnoses : at histology , the resection margins were considered to be infiltrated when the tumour was located on the sectioned surface or when the distance between the lesion and the resection margin was 2 m results lesion histology table 1 shows the definitive histological diagnoses in the 123 patients considered : as one patient had both a benign and a malignant lesion , the series comprised a total of 124 lesions . 
the diagnostic reliability of specimen radiography for the evaluation of resection margins was retrospectively assessed for 102 malignant lesions . lesion size magini sono state stampate utilizzando pellicole kodak min r e sviluppatrice automatica dedicata kodak mini loader 2000 p . 
i paramenti valutati e riportati nel referto radiologico sono stati : presenza / assenza della lesione ; posizione della lesione allinterno del pezzo anatomico ( in rapporto ai margini di resezione ) ; direzione verso cui effettuare un allargamento nei casi in cui la lesione si presentava in prossimit di un margine . nei casi in cui la lesione risultava presente ed in sede centrale si data indicazione al chirurgo di concludere lintervento ; nei casi in cui invece la lesione risultava in prossimit di un margine stato comunicata la direzione verso cui effettuare un allargamento dellescissione prima di considerare concluso lintervento . 
al termine dellesame radiologico il pezzo anatomico stato inviato al patologo per lesame istologico . la valutazione relativa allattendibilit diagnostica dei reperti radiologici stata fatta confrontando le diagnosi radiologiche con quelle istopatologiche : istologicamente i margini di resezione sono stati considerati infiltrati nei casi di lesione neoplastica in corrispondenza di una delle superfici di taglio ovvero nei casi in cui la distanza tra la lesione e il margine di resezione era 2 mm . infiltrating lesions had a diameter ranging from 6 to 20 mm ; extension of calcifications in the ductal carcinomas in situ varied between 3 and 80 mm . risultati istologia delle lesioni intraoperative radiography of resection margins and indication for immediate reexcision in 34 patients , specimen radiography raised a suspicion of infiltrated resection margins : the excision was extended intranella tabella 1 sono riportate le diagnosi istologiche definitive delle 123 pazienti oggetto di questo studio : poich in una paziente vi era concomitanza di una lesione benigna e di una maligna , la casistica composta , complessivamente , da 124 lesioni . 
lattendibilit diagnostica della radiografia del table 1 final pathological diagnosis of the 124 lesions tabella 1 istologia delle 124 lesioni benign lesions : 22 sclerosing adenosis : 8 fibroadenomas : 7 papillomas : 3 fibroadenosis : 2 hyperplasia : 2 lesioni benigne : 22 adenosi sclerosante : 8 fibroadenomi : 7 papillomi : 3 fibroadenosi : 2 iperplasia : 2 malignant lesions : 102 infiltrating carcinomas : 61 multifocal infiltrating carcinomas : 6 ductal carcinomas in situ : 35 lesioni maligne : 102 carcinomi infiltranti : 61 carcinomi infiltranti multifocali : 6 carcinomi duttali in situ : 35 g . 
in 68 cases , specimen radiography did not raise a suspicion of infiltrated margins : the excision was extended intraoperatively as a precautionary measure in eight cases , whereas it was not extended in the remaining 60 cases ( table 2 )  . 
there were 13 false negatives [ six ductal carcinoma in situ ( dcis ) , three infiltrating multifocal carcinomas and four infiltrating carcinomas ] and nine false positives , leading to sensitivity and specificity values of 66% and 86% , respectively , and positive and negative predictive values of 74% and 81% , respectively . histological assessment of resection margins overall , histological assessment of resection margins for the entire series , including the cases of immediate reexcision , yielded a diagnosis of positive margins in 23 patients ( 23% ) and negative margins in the remaining 79 cases ( 77% )  . 
in 21 / 34 cases ( 61.67% ) in which intraoperative specimen radiography was suspicious for margin involvement , definitive histology demonstrated negative resection margins : 19 / 21 underwent intraoperative reexcision , as suggested by the radiologists , whereas the remaining 2 / 21 cases did not . 
in 12 / 19 cases ( 63.15% ) with intraoperative reexcision , histological assessment confirmed that the margins of the first surgical specimen were positive ; therefore , in these patients , intraoperative reexcision enabled complete removal of the lesion during the same surgical procedure . 
the reasons for not following the radiologists advice in these 8 / 13 cases were deep infiltration towards the pectoralis fascia in three cases and procedures scheduled as excisional biopsies in extensive ( 48 cm ) intraductal carcinomas with a suspicion of multifocality , later confirmed by definitive histology . 
in 13 / 68 cases ( 19.11% ) with an intraoperative radiographic diagnosis of clear margins , histology revealed that the margins were in fact infiltrated : in four of these cases the surgeon decided to extend the excision intraoperatively as a precautionary measure ; this resulted in complete removal of the lesion during the same operation in three cases , whereas a second surgical procedure was scheduled in one case . 
vi sono stati 13 falsi negativi ( 6 cdis , 3 infiltranti multifocali e 4 infiltranti ) e 9 falsi positivi con valori di sensibilit e specificit rispettivamente del 66% e 86% e valore predittivo positivo e negativo del 74% e 81% . valutazione istologica sui margini di resezione sul totale della casistica la valutazione istologica dei margini di resezione , considerando anche gli ampliamenti , quando effettuati , stata di margini infiltrati in 23 pazienti ( 23% ) mentre nei restanti 79 casi ( 77% ) i margini sono risultati indenni allesame istologico definitivo . 
in 21 / 34 casi ( 61 , 67% ) in cui la diagnosi radiologica intraoperatoria pose il sospetto di margini infiltrati , lesame istologico finale dimostr margini di resezione indenni : 19 / 21 hanno avuto un ampliamento estemporaneo cos come consigliato dai radiologi mentre i restanti 2 / 21 casi , non ampliati , avevano margini negativi . 
in 12 / 19 casi ( 63 , 15% ) con ampliamento estemporaneo , lesame istologico conferm che i margini del primo pezzo anatomico erano positivi : in queste pazienti , pertanto lampliamento estemporaneo ha consentito di radicalizzare le lesioni nella stessa seduta operatoria . 
in questi 8 / 13 casi lindicazione radiologica non fu seguita in quanto : in 3 casi vi era infiltrazione in profondit , verso la fascia del muscolo pettorale mentre nei restanti 5 soggetti lintervento era stato programmato come biopsia escissionale in lesioni g . 
in contrast , of the 70 patients who did not have intraoperative reexcision , 17 ( 24% ) required further treatments to achieve complete lesion ablation : 14 patients underwent repeat surgical procedures and three , with deep lesions infiltrating the pectoralis fascia , were treated with chemoand / or radiotherapy . estese ( da 4 a 8 cm ) rappresentate da carcinomi intraduttali sospettate essere lesioni multifocali cos come confermato dallesame istologico definitivo . 
in 13 / 68 casi ( 19 , 11% ) con una valutazione radiologica intraoperatoria di margini indenni lesame istologico ha rilevato , invece , margini di resezione infiltrati : in 4 di questi casi stato effettuato un prudenziale ampliamento estemporaneo deciso dal chirurgo che in 3 casi ha consentito di radicalizzare la lesione nella stessa seduta mentre in 1 caso stato necessario programmare un secondo intervento . 
in 55 / 68 casi ( 80 , 88% ) con valutazione radiologica intraoperatoria di margini indenni , lesame istologico definitivo ha confermato i reperti radiologici ( fig . 4 ) : in 4 di questi casi , con margini negativi , il chirurgo ha comunque effettuato un ampliamento dellescissione anche se non consigliata dai reperti radiologici . discussion the rates of reintervention to achieve complete excision of a lesion after a histological diagnosis of positive resection margins are reported to be 30%85% among women who have undergone breast - conserving surgery [ 7 , 8 ]  . 
to reduce these rates , much emphasis has been placed on the intraoperative assessment of resection margins [ 9 ] , even though the risk of local recurrence is not solely correlated with margin status . 
in altre 3 pazienti ( 3% ) con margini di resezione infiltrati in profondit , verso la fascia del muscolo pettorale , il controllo dei margini di resezione infiltrati stato effettuato con radio e / o chemioterapia . 
fragment of mammary parenchyma ( left q - 1 ) containing the lesion identified at baseline : spiculations are seen close to the section surface in the lower dorsal position . 
radiografia pezzo operatorio ( a proiezione cranio caudale ; b proiezione laterale ) : frammento di parenchima mammario ( q1 sinista ) nella cui compagine si rileva la lesione riconosciuta nellesame di base con alcune spicule marginali in prossimit della superficie di taglio in sede dorsale inferiore . 
 [ 18 ] , in a study comprising only dcis , state that intraoperative radiography substantially reduces reintervention rates ( 22% ) and report achieving complete excision in 84% of patents who underwent intraoperative reexcision . 
nonetheless , the authors also state that radiological assessment alone is not sufficient , above all in ductal carcinomas in situ : in these cases , radiography may produce a substantial rate of false negative results , especially when calcifications are lacking due to the absence of necrosis . 
in addition , the authors conclude that only the long - term follow - up of these patients will be able to establish whether intraoperative assessment of margin status and immediate reexcision are truly useful in reducing local recurrence rates . in our study , the sensitivity , specificity and diagnostic accuracy of intraoperative specimen radiography before immediate reexcision , when done , were 66% , 86% and 78% , respectively , with a positive predictive value of 74% and a negative predictive value of 81% . 
 intraoperative reexcision modified margin status in in 17 ( 24% ) programmare ulteriori trattamenti per radicalizzare le lesioni : 14 pazienti sono state rioperate e 3 , con lesioni profonde infiltranti la fascia del muscolo pettorale , sono state trattate con chemioe / o radioterapia . discussione in letteratura sono riportate percentuali di reinterventi comprese tra il 30% e l85% in pazienti precedentemente sottoposte a chirurgia conservativa della mammella per radicalizzare lesioni quando lesame istologico dimostra margini di resezione infiltrati [ 7 , 8 ]  . 
per ridurre questa percentuale si data molta importanza alla valutazione intraoperatoria dei margini di resezione [ 9 ] anche se il rischio di recidiva locale non da correlare unicamente allo stato dei margini del pezzo operatorio . 
in particolare stato dimostrato che , specie nei carcinomi duttali in situ , la mammografia pu sottostimarne lestensione [ 1012 ] in rapporto alla multifocalit di questo istotipo , specie nelle pazienti di giovane et [ 13 ]  . 
in letteratura alcuni autori hanno riportato la scarsa utilit della radiografia del pezzo operatorio negli interventi di lesioni mammarie non palpabili : in alcuni lavori [ 14 , 15 ] sono state effettuate le radiografie in ununica proiezione e , in unaltro [ 16 ] , senza la tecnica dellingrandimento diretto . altri autori [ 17 ] sostengono invece di aver ridotto la percentuale di reinterventi , dal 12% al 5% , nelle pazienti sottoposte a chirurgia conservativa grazie alla valutazione rag . 
fragment of mammary parenchyma ( right q12 ) with microcalcifications close to outer marghistological examination : multiple foci of well - differentiated cribriform ductal carcinoma in situ ( dcis ) with foci in the axially portion . 
radiografia pezzo operatorio ( a proiezione cranio caudale ; b proiezione laterale ) : frammento di parenchima mammario ( q1 - 2 destra ) con alcune microcalcificazioni in prossimit del margine esterno . 
on the other hand , the radiological indication for immediate reexcision resulted in overtreatment in seven patients , with a probable impact on the final cosmetic outcome due to the removal of excessive breast tissue . the 13 false negative cases at intraoperative radiography were six intraductal carcinomas without microcalcification on the resection margins and seven infiltrating ductal carcinomas , three of which were multifocal . 
overall , our rate of reintervention due to infiltrated resection margins after breastconserving surgery ( 20% ) , which included both intraductal carcinomas and infiltrating ductal carcinomas , is in line with previous reports . 
the use of digital mammography , so far diologica intraoperatoria dei margini di resezione effettuata nelle due proiezioni ortogonali e con la tecnica dellingrandimento diretto ( 1 , 9 )  . 
 [ 18 ] , in una casistica rappresentata solo da carcinomi duttali in situ , affermano che la radiografia intraoperatoria riduce sensibilmente la percentuale di reinterventi ( 22% ) e riportano una percentuale dell84% di pazienti che , sottoposte ad un ampliamento dellescissione nella stessa seduta operatoria , hanno avuto un intervento radicale . 
nonostante questi dati , gli autori affermano peraltro che la valutazione radiologica non sufficiente , specie per quanto riguarda i carcinomi duttali in situ : lesame radiografico in questi casi pu essere gravato di una sensibile percentuale di falsi negativi specie nei casi in cui , per assenza di necrosi , mancano le microcalcificazioni . 
3a , b allargamento richiesto per sospetto radiologico di margini di resezione infiltrati . radiografia pezzo operatorio ( a proiezione cranio caudale ; b proiezione laterale ) : frammento di parenchima mammario ( q1 - 2 destra ) nella cui compagine si rilevano tutte le microcalcificazioni riscontrate nellesame di base : alcune microcalcificazioni si proiettano in prossimit del margine posteriore . 
esame istologico : carcinoma duttale infiltrante con margini di resezione indenni . della valutazione intraoperatoria dei margini di resezione e della rescissione estemporanea nel ridurre la percentuale di recidive locali . nel nostro studio i valori di sensibilit , specificit e accuratezza diagnostica dellesame radiologico del pezzo operatorio prima degli ampliamenti , quando effettuati , sono risultati essere , rispettivamente , del 66% , 86% e 78% con un valore predittivo positivo del 74% e valore predittivo negativo dell81% . 
la valutazione radiologia intraoperatoria stata utile per i chirurghi che hanno effettuato ampliamenti di escissione nella maggior parte dei casi in cui stata posta lindicazione radiologica . limited to a small number of centres due to its high costs , is likely to improve the results of the assessment of both intraductal and infiltrating ductal lesions , as previously reported [ 19 , 20 ] , on account of its high - contrast resolution , which is markedly higher than that of analogical mammography . conclusions it is generally accepted that during surgery for impalpable breast lesions , specimen radiography should be performed g . 
per contro , sulla base delle indicazioni radiologiche , in 7 pazienti vi stato un sovratrattamento che , verosimilmente , ha inciso sugli esiti estetici finali a causa di una eccessiva exeresi di parenchima mammario . i 13 casi che sono risultati falsi negativi alla radiografia intraoperatoria erano rappresentati da 6 carcinomi intraduttali senza microcalcificazioni sui margini di resezione e da 7 carcinomi duttali infiltranti di cui 3 multifocali . 
complessivamente nella nostra casistica , composta sia da carcinomi intraduttati sia da carcinomi duttali infiltranti , la percentuale di reinterventi per margini di resezione infiltrati dopo chirurgia conservativa ( 20% ) rientra in quanto riportato dalla letteratura . 
radiografia pezzo operatorio : frammento di parenchima mammario ( q1 sinistra ) che presenta , in posizione centrale , tutto il gruppo di microcalcificazioni rilevato nellesame di base con margini di resezione giudicati radiograficamente indenni . 
this ensures reasonable certainty of the adequacy of the excision , with a consequent reduction in reinterventions to achieve complete excision and in the risk of local recurrences . our results , which allowed us to reduce our rate of reintervention from 31% to 20% , in agreement with previous reports , are , however , susceptible to improvement to reduce the rate of false negatives due to the seven infiltrating ductal carcinomas . 
mazzarella , istituto di radiologia universitaria , azienda ospedaliero - universitaria santa maria della misericordia di udine , universit degli studi di udine , i - 33100 udine , tel . : + 39 - 0432 - 559266 , fax : + 39 - 0432 - 559867 , e - mail : radiologia.segr@med.uniud.it received : 28 july 2006 / accepted : 22 november 2006 / published online : 20 april 2007 abstract the introduction of systems for automated reading in mammography has been proposed to improve the sensitivity [ computer - aided detection ( cade ) systems ] and , more recently , the specificity [ computer - aided diagnosis ( cadi ) systems ] of the test . 
therefore the role of these systems in clinical practice is still debated , and their real contribution to the overall management of the diagnostic process is not yet clear . key words mammography breast cancer screening cade cadi artificial intelligence computer vision riassunto lintroduzione di sistemi di lettura automatizzata in mammografia stata da tempo proposta per aumentare la sensibilit del test ( sistemi cade , computer - aided detection ) e , pi recentemente , la sua specificit ( sistemi cadi , computer - aided diagnosis )  . 
tali sistemi sono ancora oggetto di discussione . molti studi hanno dimostrato che ne traggono giovamento i lettori in formazione e che , dal confronto con il computer , i radiologi sono indotti a migliorare la propria performance . 
il ruolo di tali sistemi quindi ancora dibattuto e il loro reale contributo al management complessivo del processo diagnostico non ancora chiaro . parole chiave mammografia tumore della mammella screening cade cadi intelligenza artificiale visione computerizzata introduction introduzione breast cancer is the second most common cause of cancer death in women [ 1 ]  . 
in fact , some of these unappreciated or unrecognised very subtle signs may have been seen by the readers but dismissed as being insignificant , but others may be strongly suspicious [ 24 ]  . 
thus , the basis of the problem is not having detected a potential abnormality or having failed in interpreting it ( that is , having misunderstood its nature )  . 
 therefore , a lesion can be missed owing , on the one hand , to mistakes in the visual search ( responsible for 30% of failures ) , and on the other hand , to mistakes in interpretation ( 70% of failures ) [ 6 ]  . 
the former may still be defined as perceptual errors ( the radioloists gaze lands on the location of the lesion for a time insufficient for its recognition ) and the latter as decision - making errors ( the lesion is detected , but a covert negative decision is made ) [ 7 ]  . different methods to reduce the likelihood of errors have been analysed : in the first place , double reading , which provides either double perception or double interpretation of lesions . 
it has been demonstrated that a single radiologist is more accurate when reading mammograms methodically than quickly and that two observers achieve an improvement in detection rate of 5%15% [ 810 ]  . 
furthermore , different readers miss different cancers , as is evidenced by the success of double reading , in which two readers independently read the films [ 11 ]  . 
the most accurate method of interpretation is double reading with arbitration , in which a third reader reviews cases about which the two readers disagree [ 11 , 12 ]  . 
this is a complex and time - consuming method , so that double reading , if performed without an efficient system , may be not only cost ineffective but even expensive and counterproductive . the development of computerised systems as second reader is an alternative . 
researchers have been developing algorithms to detect mammographic abnormalities for more than 30 years with the aim of either automating mammographic interpretation or , more realistically , providing a tool that will enhance human film - reading accuracy . 
a benchmark against which the efficacy of such tools can be measured is whether or not the accuracy of an individual reader could be improved to the extent that double reading was no longer necessary , as this would alleviate any shortage of film readers [ 5 ]  . 
the computer can detect cancers missed by the human reader , but not at the cost of producing too many false positives and thus an unacceptable amount of recalls . strutturale della mammella , sottigliezza dei reperti , affaticamento del radiologo [ 4 ]  . molti studi riportano che fino al 70% dei cancri persi allo screening sono visibili retrospettivamente sulla mammografia precedente [ 3 ]  . 
la mancata individuazione da parte dei radiologi condizionata dalla loro capacit di percezione , a sua volta influenzata dalla propria consapevolezza della prevalenza del cancro e dalla propria soglia decisionale [ 5 ]  . 
infatti , alcuni segni non valorizzati e / o riconosciuti , molto sottili , possono essere stati notati dai lettori e considerati non significativi , ma altri possono essere chiaramente sospetti [ 24 ]  . 
quindi il problema si basa sul non avere individuato una potenziale anormalit , oppure sulla mancanza di una sua corretta interpretazione ( ovvero non avere inquadrato la sua natura )  . da un lato , pertanto , una lesione pu non essere identificata per errori di strategia di ricerca ( responsabili del 30% delle mancate identificazioni ) , dallaltro per errori di interpretazione ( 70% degli errori ) [ 6 ]  . 
i primi possono essere ancora definiti come errori di percezione ( lo sguardo si posa sulla lesione , ma per un tempo insufficiente a riconoscerla ) ed i secondi come errori decisionali ( la lesione individuata , ma viene interpretata erroneamente come reperto negativo ) [ 7 ]  . sono stati analizzati diversi metodi per ridurre la probabilit di errore , in primo luogo la doppia lettura , che consente di acquisire o una doppia percezione della lesione o una sua doppia interpretazione . 
stato dimostrato che un singolo lettore che legge le mammografie metodicamente pi accurato di uno che le legge rapidamente ; tuttavia due lettori ottengono comunque un incremento del tasso di individuazione del 5%15% [ 810 ]  . 
il pi accurato metodo di interpretazione la doppia lettura con arbitraggio , in cui un terzo lettore rivede i casi su cui c disaccordo tra i primi due [ 11 , 12 ]  . 
tale metodologia comunque complicata e allunga i tempi , cosicch , se attuata senza un sistema efficiente , pu essere non solo non economica , ma addirittura dispendiosa e controproducente . unalternativa lo sviluppo di sistemi computerizzati che fungano da secondo lettore . 
i ricercatori da pi di 30 anni stanno elaborando algoritmi che individuino anormalit mammografiche , allo scopo di automatizzarne linterpretazione o , pi realisticamente , fornire uno strumento che potenzi laccuratezza umana di lettura dimmagine . 
un punto di riferimento rispetto al quale si pu misurare lefficacia di questi strumenti la loro capacit di migliorare laccuratezza di un singolo lettore a tal punto che non sia pi necessaria la doppia lettura , il che porrebbe rimedio alla scarsit di lettori , eliminando il secondo lettore [ 5 ]  . 
il computer potrebbe identificare cancri sfuggiti al lettore umano , ma non al prezzo di introdurre troppi falsi positivi e quindi un numero inaccettabile di richiami . le tecniche di computer - aided detection ( cade ) sono state applicate a una variet di immagini mediche , ma sono usate pi ampiamente in mammografia , dove i segni di cancro della mammella in stadio iniziale sono spesso molto fini . 
consigliabile che il radiologo legga la mammografia prima di vedere i risultati del cad e , solo successivamente , guardi i suggerimenti del computer e rivaluti le aree segnalate , in modo da rifiutarle o considerarle per richiamare la paziente . 
lo scopo del cad quello di attirare lattenzione del lettore su regioni potenzialmente anormali e di aumentare il grado di sospetto per segni di anormalit che sono stati visti , ma scartati come probabilmente normali . 
i sistemi computerizzati di analisi mammografica possono essere classificati in due categorie : sistemi cade ( computer - aided detection ) : identificano in modo computerizzato possibili anomalie mammografiche ; sistemi cadi ( computer - aided diagnosis ) : aiutano il radiologo a definire il grado di sospetto di una lesione identificata . in entrambi i casi il presupposto rappresentato dallanalisi computerizzata delle mammografie . analisi computerizzata delle mammografie le immagini mammografiche digitali possono essere analizzate dai sistemi computerizzati attraverso due processi , spesso interconnessi tra loro : visione computerizzata ; intelligenza artificiale . la visione computerizzata comincia con uno stadio di processazione [ 16 ] : le caratteristiche importanti dellimmagine vengono esaltate , quelle secondarie vengono de - enfatizzate . segue una fase detta di segmentazione , che separa le immagini in regioni con caratteristiche simili . 
degli algoritmi computerizzati , attraverso gli indici numerici ottenuti , fanno una stima della probabilit di malignit di una lesione . le tecniche di intelligenza artificiale sono utilizzate per convertire le misure derivanti dalla visione computerizzata in risultati diagnostici , grazie a reti neurali artificiali applicate agli algoritmi per la identificazione o caratterizzazione di lesioni sulla mammografia . 
le reti neurali artificiali sono programmi computerizzati che identificano relazioni tra i parametri di input e gli output desiderati ( come i reperti mammografici e la loro probabilit di malignit )  . 
1 esempio di segnalazioni di un sistema di computer - aided detection ( cade ) : le microcalcificazioni sono indicate da rettangoli , le masse da cerchi . computer - aided detection ( cade ) techniques have been applied to a variety of medical images but are used most widely in mammography , where signs of early breast cancer are often very subtle . 
the radiologist is expected to read mammograms before viewing the cad results and only later to look at the cad prompts , reviewing prompted areas to either dismiss them or to take them into consideration for recalling patients . 
the purpose of cad is to attract the readers attention to potentially abnormal regions and to increase the degree of suspicion associated with signs of abnormality that had previously been seen but dismissed as probably normal by the reader . 
computerised systems for mammographic analysis may be classified in two categories : cade systems , which perform a computerised detection of possible mammographic abnormalities computer - aided diagnosis ( cadi ) systems , which help radiologists define the grade of suspiciousness of a detected lesion . 
in both cases , the basis lies in the computer analysis of mammograms . computer analysis of mammograms mammographic images in digital format can be analysed by computer systems using two kinds of techniques , often enmeshed with each other : m . 
computer algorithms estimate the likelihood of malignancy of a lesion , thanks to the numerical indices generated . artificial intelligence techniques are used to merge computer vision measures into diagnostic output , thanks to artificial neural networks applied in algorithms for the detection or characterisation of lesions in mammography . 
different forms of artificial intelligence can also be combined to classify abnormalities more accurately than any individual approach [ 18 ]  . in summary , the information contained within images undergoes ( 1 ) elaboration , ( 2 ) extraction of parameters about image features and ( 3 ) classification of these parameters in terms of probability of lesion . 
the chosen threshold value will affect the systems sensitivity and specificity : the lower it is , the higher its sensitivity and the lower its specificity ( more false positives )  . so , classification of lesions results from a compromise between threshold value and an acceptable false positive rate [ 19 ]  . cade systems ( computer - aided detection ) cade systems localise on image areas of potential abnormalities , leaving the final decision to the radiologist . 
nel leggere le immagini , la rete neurale artificiale attribuisce ad ogni elemento un valore numerico , che pi alto pi sospetto ; viene fissata una soglia per tale valore , oltre la quale la regione corrispondente classificata come sospetta . 
la soglia prescelta condizioner sensibilit e specificit del sistema : pi essa bassa , maggiore sar la sensibilit e minore la specificit ( aumento dei falsi positivi ) , e viceversa . 
la classificazione delle lesioni deriva quindi da un compromesso tra soglia e numero di falsi positivi ritenuto accettabile [ 19 ]  . sistemi cade ( computer - aided detection ) i sistemi cade localizzano sulle immagini aree di potenziale anormalit , lasciando la decisione finale al radiologo . 
sono stati messi a punto molteplici algoritmi con questo scopo , volti ad individuare masse o calcificazioni . identificazione di masse la presenza di una massa il pi importante segno mammografico di tumore . 
regions labelled as suspicious by detection algorithms are not necessarily malignant . a number of algorithms are designed to detect particular kinds of masses , such as spiculated masses , because of their high likelihood of malignancy . 
the edge orientation can be computed in many different ways : using standard deviation of a local edge orientation histogram and spatial filters [ 22 ] ; statistical analysis of a map of pixel orientations computed at three spatial scales [ 23 ] ; extracting four features for each pixel at a multiresolution representation of mammograms using the discrete wavelet transform [ 24 ]  . other algorithms have been developed for the detection of not only spiculated masses . 
suspicious regions can be segmented by adaptive grey - level thresholding and then classified by a fuzzy binary decision tree , using features based on shape , size and contrast [ 21 ]  . 
mammograms can be divided into three categories ranging from fatty to dense tissue by histogram analysis techniques ; multiple threshold values are used to detect potential masses [ 25 ]  . 
pixel - based methods have the disadvantage of being computationally intensive . in region - based detection methods , regions of interest ( rois ) are extracted by segmentation or filtering technique . then , features describing important information ( such as shape and texture ) are extracted for each region , which is classified as suspicious or not . 
several methods are based on matched filtering : the image is filtered with a filter used as a model for a mass , and the output should correspond to the central dense nucleus in the hypothetical mass ; thus rois are defined . 
such methods can use an iris filter [ 27 ] , an adaptive densityweighted contrast - enhancement filter to enhance objects and suppress background structures , coupled with a simple edge detector [ 28 ] , or a single difference of gaussian ( dog ) filter to detect masses of approximately 1 cm in diameter , then classified by features based on size , contrast , circularity and laws texture features [ 29 ]  . 
lorientamento del margine pu essere calcolato in molti modi diversi : usando la deviazione standard di un istogramma di orientamento locale del margine e filtri spaziali [ 22 ] ; con lanalisi statistica di una mappa dellorientamento dei pixel calcolata su tre scale spaziali [ 23 ] ; estraendo quattro caratteristiche per pixel , su una rappresentazione mammografica a risoluzione multipla usando la trasformata wavelet discreta [ 24 ]  . sono stati sviluppati altri algoritmi per lidentificazione non solo di masse spiculate . 
le regioni sospette possono essere segmentate per mezzo di valori soglia di grigio di tipo adattativo e quindi classificate con un albero decisionale binario di tipo fuzzy , usando caratteristiche basate su forma , dimensioni e contrasto [ 21 ]  . 
si possono dividere le mammografie in tre categorie , da tessuto adiposo a tessuto denso , con tecniche di analisi di istogramma ; si usano multipli valori soglia per individuare potenziali masse [ 25 ]  . 
i metodi basati su pixel hanno lo svantaggio di richiedere unelevata complessit di calcolo . nei metodi basati su regione , le regioni di interesse ( roi , region of interest ) vengono estratte con tecniche di segmentazione o di filtro . 
molti metodi si basano sul filtraggio combaciato : allimmagine si applica un filtro che funge da modello di una massa e il risultato dovrebbe corrispondere al nucleo centrale denso dellipotetica massa ; in tal modo si definiscono delle regioni di interesse . 
questi metodi possono usare : un filtro a iride [ 27 ] ; un filtro adattativo di esaltazione del contrasto , per esaltare gli oggetti e sopprimere le strutture dello sfondo , associato ad un rivelatore di margini [ 28 ] ; un filtro dog ( difference of gaussian ) singolo , per individuare masse di circa 1 cm , poi classificate in base a dimensioni , contrasto , circolarit e caratteristiche strutturali laws [ 29 ]  . 
i metodi basati su regione sono vantaggiosi , dato che tengono conto delle dimensioni e della morfologia del reperto e richiedono processi di calcolo meno complessi dei processi basati su pixel . 
sia i metodi basati su pixel , sia quelli basati su regione hanno il limite di non compiere unanalisi su una gamma continua di scale , mentre le lesioni tumorali sono fenomeni biologici stocastici , che nelle immagini corrispondono a varie strutture e dimensioni distribuite su gamme di scale spaziali [ 20 ]  . i metodi sopra descritti compiono unanalisi su di una singola immagine mammografica . 
both pixel - based and region - based methods have the limitation that the analysis is not made over a continuous range of scales , whereas cancerous lesions are stochastic biological phenomena , corresponding , on images , to various structures and sizes over ranges of spatial scales [ 20 ]  . previously described methods perform analysis on a single mammograsome researchers have developed methods that use multiple images , analysing the symmetry between corresponding mammograms of each breast [ 34 ] or comparing current and prior images [ 35 ]  . 
nevertheless , such methods require difficult preprocessing steps . classification of suspicious regions the classification of suspicious regions as a mass or normal tissue is the second stage of mass - detection algorithms ; its purpose is to reduce the number of false positives . 
for this task , a neural network [ 36 ] or a template - matching technique [ 37 ] can be used . detection of microcalcifications microcalcifications are small calcium deposits due to benign or malignant processes . 
their main characteristics are size , shape or morphology , number and distribution . their size varies from 0.1 mm to 1 mm ( average 0.3 mm ) ; very small ones may be missed , especially if dense breast parenchyma overlaps . 
a number of effective methods have been designed for their detection based on localisation of high spatial frequencies ( wavelet transform ) falling within the range corresponding to microcalcifications [ 20 ]  . 
the effectiveness of the wavelet transform in the detection of microcalcifications , viewed as point discontinuities ( bright dots in the grey background of fat on usual images ) , is not found in the detection of masses , viewed as edges [ 3841 ]  . other multiscale and nonwavelet - based methods have been developed [ 42 , 43 ] based on the fact that calcifications have much higher intensity values than the surrounding tissue , but they are more likely to fail in detecting microcalcifications in dense background tissue . 
as for mass - detection methods , also for microcalcifications , a number of techniques to reduce false positives have been developed by classifying rois as containing microcalcifications or normal tissue [ 20 , 44 ]  . 
an enhancement stage ( global or local ) often messo a punto metodi che utilizzano immagini multiple , analizzando la simmetria fra le corrispondenti mammografie dei due lati [ 34 ] o paragonando le immagini attuali con le precedenti [ 35 ]  . 
tali metodi si basano per su complicati passaggi di pre - processing . classificazione delle regioni sospette la classificazione delle regioni sospette come massa o tessuto normale rappresenta il secondo passaggio degli algoritmi di identificazione ; il suo scopo ridurre il numero di falsi positivi . 
a tal fine si pu usare una rete neurale [ 36 ] o una tecnica di corrispondenza a una sagoma [ 37 ]  . identificazione di microcalcificazioni le microcalcificazioni sono piccoli depositi di calcio dovute a processi benigni o maligni . 
le dimensioni variano da 0 , 1 mm a 1 mm ( in media 0 , 3 mm ) ; quelle pi piccole possono sfuggire , specialmente se si sovrappone parenchima mammario denso . 
sono stati elaborati molti metodi efficaci per la loro identificazione , basati sulla localizzazione di frequenze spaziali elevate ( trasformata wavelet ) che rientrano nel range di rappresentazione delle microcalcificazioni [ 20 ]  . 
lefficacia della trasformata wavelet nel riconoscimento delle microcalcificazioni , viste come discontinuit puntuali ( punti bianchi sullo sfondo grigio del grasso nella rappresentazione usuale ) , non trova invece riscontro nella ricerca delle masse , che si manifestano come discontinuit a fronte [ 3841 ]  . sono stati proposti metodi di altro tipo , multi - scala e non basati sulla trasformata wavelet [ 42 , 43 ] , che sfruttano il fatto che le calcificazioni hanno valori di intensit molto pi alti del tessuto circostante , ma che hanno maggiore probabilit di fallire in caso di microcalcificazioni su uno sfondo di tessuto denso . 
come per i metodi di ricerca delle masse , anche per le microcalcificazioni si sono elaborate delle tecniche per ridurre i falsi positivi , attraverso la classificazione delle roi come contenenti microcalcificazioni o tessuto normale [ 20 , 44 ]  . 
il limite di questi metodi che essi esaltano non solo le calcificazioni , ma anche la struttura di sfondo e il rumore [ 20 ]  . sistemi cadi ( computer - aided diagnosis ) una volta identificato un reperto come potenzialmente anormale , bisogna caratterizzarlo , per poter decidere la gestione della paziente . 
gli scopi dei sistemi cadi sono : evitare che il radiologo classifichi come benigna una lesione maligna ; ridurre il numero di biopsie consigliate per lesioni in realt benigne ( ma senza ridurre la sensibilit )  . 
in such methods , the limitation is that they enhance not only calcifications but also background structure and noise [ 20 ]  . cadi systems ( computer - aided diagnosis ) once a finding has been identified as potentially abnormal , it must be characterised to allow patient management decision making . 
the aims of cadi systems are to prevent a malignant lesion from being characterised as benign by the radiologist and to reduce the number of biopsy recommendations for benign lesions ( but with no reduction of sensitivity )  . 
to characterise an roi as benign or malignant , a number of roi features ( extracted by computer vision techniques ) are used by artificial intelligence techniques ( such as artificial neural networks )  . the output of cadi systems can take different forms , such as the estimated likelihood of malignancy or a visual aid for the radiologist in decision making ( by displaying a variety of known lesions similar to the one at hand ) [ 4 ]  . diagnosis of masses masses are characterised by their shape ( round , oval , lobular , irregular ) and margins or boundary ( circumscribed , microlobulated , obscured , indistinct , spiculated ) [ 46 ]  . 
usually , the diagnostic process consists of three stages : ( 1 ) segmentation of mass boundary in roi , ( 2 ) feature extraction and ( 3 ) classification [ 20 ]  . segmentation mass segmentation may be manual , semiautomated or fully automated . 
the area overlap measure is used to report the performance of segmentation algorithms : it is the ratio of the area segmented automatically to the area segmented manually by an experienced radiologist . 
however , as there is interobserver variability in the manual segmentation by radiologists , it is more appropriate to evaluate whether the variance between the boundaries marked by a radiologist and a computer falls within the variance between radiologists [ 20 , 47 , 48 ]  . 
other segmentation algorithms can use fuzzy region - growing methods , shape and texture features based on grey - level cooccurrence matrices and region - growing techniques based on radial gradient or on simple probabilistic models [ 20 ]  . feature extraction several features extracted for diagnosis concern morphology and texture . 
textural features have been designed to capture important differences between malignant and benign masses that may not be evident to the huroi benigna o maligna , varie caratteristiche della roi ( estratte con tecniche di visione computerizzata ) vengono utilizzate da sistemi di intelligenza artificiale ( come reti neurali artificiali )  . loutput dei sistemi cadi pu essere di vario tipo , come una stima della probabilit che una lesione sia maligna oppure un aiuto visivo per il radiologo nel prendere una decisione ( mostrando una variet di lesioni note , simili a quella sotto esame ) [ 4 ]  . diagnosi di masse le masse si caratterizzano per la loro forma ( rotondeggiante , ovalare , lobulata , irregolare ) e per i margini ( circoscritti , microlobulati , mascherati , indistinti , spiculati ) [ 46 ]  . 
la misura dellarea di sovrapposizione si usa per esprimere la performance degli algoritmi di segmentazione : essa il rapporto tra larea segmentata automaticamente e quella segmentata manualmente da un radiologo esperto . 
tuttavia , essendoci variabilit interosservatore nella segmentazione manuale dei radiologi , pi appropriato valutare se la varianza tra i confini demarcati da un radiologo e quelli definiti da un computer rientra nella varianza tra radiologi [ 20 , 47 , 48 ]  . 
altri algoritmi di segmentazione possono usare : metodi fuzzy region growing ; caratteristiche morfologiche e strutturali basate su matrici di concomitanza di livelli di grigio ; tecniche region growing basate su gradiente radiale o su modelli probabilistici semplici [ 20 ]  . estrazione di caratteristiche molte delle caratteristiche estratte per la diagnosi riguardano morfologia e struttura . 
molti metodi calcolano queste caratteristiche a partire da matrici di concomitanza di livelli di grigio ( analisi di densit ) [ 20 ]  . classificazione le tecniche pi usate per classificare le masse come benigne o maligne impiegano reti neurali artificiali o lanalisi discriminante lineare . 
a number of methods compute these features from grey - level cooccurrence matrices ( density analysis ) [ 20 ]  . diagnosi di microcalcificazioni classification the most used techniques for classifying masses as benign or malignant employ artificial neural networks or linear discriminant analysis . 
other methods have also been considered for classifying masses into other categories : distinction between round , nodular or stellate masses ; classification of masses as fibroadenoma , cyst or cancer ; characterisation of the roughness of tumour boundaries [ 20 , 49 ]  . le microcalcificazioni in mammografia possono apparire come individuali o raggruppate in cluster ( tre o pi microcalcificazioni in unarea di 1 cm2 )  . 
the following steps are ( 1 ) segmentation of individual microcalcifications , ( 2 ) feature extraction and ( 3 ) classification [ 46 , 20 ]  . segmentation of individual microcalcifications segmentation is based on the analysis of the properties of individual calcifications ; it is difficult to perform due to the small size of microcalcifications , and probably it is of low interest , as the properties of the whole cluster are more useful for diagnosis than those of individual microcalcifications [ 20 ]  . feature extraction the features to be considered regard morphology and texture . 
morphological features depend on the effectiveness of the segmentation algorithsome common cluster features are number of microcalcifications , mean diameter of microcalcification area , standard deviation ( sd ) of their contrast and number of microcalcifications per unit area [ 20 ]  . classification a variety of methods for automatically classifying microcalcifications clusters as benign or malignant have been developed . 
thus , researchers have developed algorithms that do not use shape features but , for example , image structure features or cluster features [ 53 ]  . la segmentazione si basa sullanalisi delle propriet delle singole calcificazioni , difficile da eseguirsi per le loro piccole dimensioni e , verosimilmente , di scarso interesse , data la maggior importanza , ai fini diagnostici , rivestita dallintero cluster , piuttosto che dal singolo elemento [ 20 ]  . estrazione di caratteristiche le caratteristiche da considerare sono morfologiche e strutturali . 
alcune propriet frequentemente considerate per i cluster sono : numero delle microcalcificazioni , diametro medio dellarea delle microcalcificazioni , deviazione standard del loro contrasto , numero di microcalcificazioni per unit di superficie [ 20 ]  . classificazione esistono vari metodi per classificare automaticamente le microcalcificazioni come benigne o maligne . 
nel valutare la performance di un algoritmo cad , importante tener conto della sua sensibilit e specificit , oltre che del tipo di casi che costituiscono il database sotto esame . 
when evaluating the performance of a cad algorithm , it is important to consider its sensitivity and specificity , as well as the nature of the cases in the database under study . 
the performance of cad systems can be evaluated in two ways : by measuring the algorithy performance or by monitoring the performance of readers using the system [ 15 ]  . evaluation of algorithm performance to evaluate cad algorithm performance , algorithms are applied to a set of images , and the response to known truth is measured . 
in practice , a combination of random sampling and selection provides the best compromise between efficiency and accuracy . truth about lesion type is determined by means of pathology reports ; truth about lesion location on mammograms is usually obtained by asking expert mammographic film readers to annotate the images . 
correspondence between the truth and the algorithm response can be evaluated by using free - response receiver operating characteristic ( froc ) curves ( plots of sensitivity versus the number of false positives per image )  . 
this provides a good method of comparing algorithms [ 15 ]  . evaluation of the performance of readers using cad in addition to measuring algorithm performance , it is important to measure the performance of readers using cad . 
in retrospective studies , the inclusion of prior cases ( the previous screening films of subsequently detected cancers ) and sibilit pu non riflettere veramente una buona performance , se nella valutazione sono stati inclusi solo casi con anormalit ovvie . 
la performance dei sistemi cad pu essere valutata in due modi : misurando la performance degli algoritmi o quella dei lettori che li usano [ 15 ]  . valutazione della performance di un algoritmo per valutare la performance di un algoritmo cad , esso viene applicato a un set di immagini e si misura la corrispondenza alla verit nota . 
nella pratica , il miglior compromesso tra efficienza e accuratezza si ottiene combinando campionamento randomizzato e selezione . la verit sul tipo di lesione si determina in base ai referti anatomopatologici , mentre quella sulla sede mammografica delle lesioni si ottiene chiedendo a lettori esperti di mammografie di annotare le immagini . 
la corrispondenza tra verit e risposta dellalgoritmo pu essere valutata con le curve froc ( free - response receiver operating characteristic curves : diagrammi che rappresentano la sensibilit in funzione del numero di falsi positivi per caso )  . 
questo un buon metodo per paragonare gli algoritmi [ 15 ]  . valutazione della performance dei lettori con cad oltre a misurare la performance degli algoritmi , importante misurare quella dei lettori che utilizzano il cad . 
comunque , negli studi retrospettivi molto importante linclusione dei precedenti ( le mammografie di screening antecedenti a quelle della diagnosi di tumore ) e la conoscenza dello stato di tutti i casi ( referti anatomopatologici per le lesioni ; almeno una mammografia di screening negativa per i casi normali )  . 
al contrario , gli studi prospettici simulano ci che accade nella vita reale e nellattivit clinica ; tuttavia , la selezione randomizzata pi laboriosa per i lettori , dato che essi devono vedere un alto numero di casi normali per raggiungere un numero di tumori statisticamente significativo . 
on the contrary , prospective studies simulate what happens in real life and in clinical work ; nevertheless , random selection is more labour intensive for the readers , as they must read a large number of normal cases to reach a statistically significant amount of cancers . 
in both retrospective and prospective studies , if cases used for evaluation without cad and with cad are not the same , the two sets should be equivalent . readers in screening mammography are known to have variability in their performance [ 54 ]  . 
in studies conducted consecutively , comparing unprompted performance over a period with prompted performance over the following period , it should be noted that the readers will be more experienced in the second period . 
in studies in which readers differ in the unprompted and prompted conditions , a bias could also be due to the readers experience , performance and degree of training before starting the trial ( to achieve stable performance ) [ 15 ]  . trials on cade systems several trials have been performed to evaluate the effectiveness of cade systems , with the aim of introducing them into the clinical practice of screening mammography . 
on a set of 286 and 377 missed cancers , respectively , at a retrospective blinded analysis , a sensitivity of 79% [ 3 ] and 75.4% [ 55 ] was calculated for the radiologists without cad , with a false negative rate of 21% [ 3 ]  . 
the conclusions of both studies were that the cad system marked most of cancers missed at screening , so its use theoretically would reduce the false negative rate , particularly for lesions presenting as microcalcifications . 
furthermore , a cad system would significantly improve the radiologists sensitivity and , therefore , be useful in screening mammography , by reducing the number of missed cancers and helping in earlier cancer detection . 
who , in a retrospective study published in 2004 [ 56 ] , found that using two different cad systems tested on 120 mammograms , lutazione senza e con cad non sono gli stessi , i due set dovrebbero essere equivalenti . si sa che la performance dei lettori nello screening mammografico variabile [ 54 ]  . 
negli studi effettuati sequenzialmente , confrontando la performance senza cad nellarco di un periodo con quello con cad nel periodo successivo , si dovrebbe tener conto che i lettori , nel secondo periodo , avranno acquisito maggior esperienza . 
negli studi in cui i lettori con e senza cad sono diversi , un bias potrebbe anche essere dovuto alla diversit tra di loro per esperienza , performance e grado di addestramento che deve essere effettuato prima di iniziare lo studio ( per raggiungere una performance stabile ) [ 15 ]  . studi sui sistemi cade molteplici studi sono stati condotti per valutare lefficacia dei sistemi cade , al fine della loro introduzione nella pratica clinica di screening mammografico . 
la sensibilit stimata per un radiologo con il cad stata quindi del 91 , 4% ( pi alta del 21 , 2% rispetto alla sensibilit senza cad ) [ 55 ]  . 
le conclusioni di entrambi gli studi sono state che il sistema cad ha segnalato la maggior parte dei cancri persi allo screening , quindi il suo uso teoricamente ridurrebbe il tasso di falsi negativi , in particolare per le lesioni che si presentano come microcalcificazioni . 
inoltre , un sistema cad migliorerebbe in modo significativo la sensibilit del radiologo e , quindi , sarebbe utile nella mammografia di screening , riducendo il numero di cancri persi e favorendo la pi precoce scoperta dei tumori . 
 [ 57 ] in 2005 retrospectively investigated the impact of cad on tumours that , undergoing double reading , had been missed by one of the two readers : in a series of 33 such lesions and 75 negative cases , sensitivity was 51.5% for cad alone , 62.5% for radiologists alone and 86.2% for radiologists with cad . 
in particular , for those lesions they had originally missed , radiologists showed a sensitivity of 58.3% without cad and of 100% with cad ( meaning that about half of missed cancers would be detected )  . 
these conclusions are the best obtained theoretically , assuming that the readers would correctly respond to all prompts of the cad systein routine use of cad , as analysed below , this would not happen , and readers are more likely to dismiss the majority of cad prompts instead of correctly responding to the prospective trials are the most informative studies because they are applied to clinical practice . 
the increase in sensitivity related to the use of cad is more realistic in these studies , and the effect of cad systems on recall rates , essential for screening programmes , can be evaluated . 
a subsequent prospective clinical part of the burhenne study [ 3 ] showed no increase in the recall rates using the cad system . this is explained by the fact that cad brings possible lesions to the attention of the radiologist who reevaluates them , dismissing false positive marks . 
over a 12month period , each of 12 , 860 screening mammograms was interpreted by an experienced breast radiologist , first without cad and immediately after using cad , and the effects of cad were evaluated . 
the authors concluded that the use of cad in screening mammography increases the detection of early stage cancers without undue effects on the recall rate or positive predictive value for biopsy . 
 in 2004 , the results of a prospective trial were published showing the effects on recall rate and breast cancer detection rate after the introduction of a cad system into a clinical radiology practice , including all screening mammograms performed during 2000 , 2001 and 2002 [ 59 ]  . 
in this study , not all cases were analysed without and with cad ; the results lettura , erano stati persi da uno dei due lettori : su di un set costituito da 33 di queste lesioni e 75 casi negativi , la sensibilit stata per il cad da solo del 51 , 5% , per i radiologi da soli del 62 , 5% e per i radiologi con cad dell86 , 2% . 
in particolare , per le lesioni che in origine essi avevano perso , i radiologi hanno avuto una sensibilit senza cad del 58 , 3% , con cad del 100% ( si recupererebbero quindi circa la met dei cancri persi )  . 
nellimpiego routinario del cad , come si dir pi avanti , ci non accadrebbe ed pi probabile che i lettori rifiutino la maggior parte dei suggerimenti del cad , anzich rispondere correttamente ad essi . gli studi prospettici sono i pi informativi , perch applicati alla pratica clinica . 
in questi studi , laumento in sensibilit correlato alluso del cad pi realistico e si pu valutare leffetto del sistema cad sul tasso di richiami , essenziale nei programmi di screening . 
questo studio ha dunque mostrato che il cad pu ridurre il tasso di falsi negativi senza aumentare quello di richianel 2001 stato pubblicato il primo studio prospettico che valutava gli effetti del cad sulla lettura delle mammografie di screening in un centro senologico di comunit [ 58 ]  . nellarco di 12 mesi , 12860 mammografie di screening sono state lette ognuna da un radiologo esperto di mammella , prima senza cad e , subito dopo , con cad , e se ne sono valutati gli effetti . 
si osservato : un aumento accettabile del tasso di richiami ( dal 6 , 5% al 7 , 7% , incremento dovuto per met a microcalcificazioni ) ; nessuna modificazione del valore predittivo positivo della biopsia ( 38% ) ; un incremento del 19 , 5% del numero di cancri trovati ; un incremento ( dal 73% al 78% ) della proporzione di tumori scoperti in stadio precoce ( 0 e i )  . 
il fatto che il valore predittivo positivo della biopsia non sia stato influenzato stato spiegato col motivo che le biopsie venivano consigliate , correttamente , sulla base di reperti clinici e dellimaging al momento del richiamo e non considerando se il cad aveva segnalato o meno la lesione . gli autori hanno concluso che lutilizzo del cad nello screening mammografico migliora lindividuazione dei tumori in stadio precoce , senza effetti negativi sul tasso di richiami n sul valore predittivo positivo della biopsia . nel 2004 sono stati pubblicati i risultati di uno studio prospettico che mostrava gli effetti sul tasso di richiami e sul tasso di scoperta di tumori al seno dopo lintroduzione di un sistema cad nella pratica clinica radiologica , includendo tutte le mammografie di screening effettuate nel corso del 2000 , 2001 e 2002 [ 59 ]  . 
in questa analisi non si leggevano tutti i casi senza e con cad ; si sono confrontati i risultati dei casi letti da 24 radiologi senza cad con quelli di un diverso set di mammografie lette con il cad . 
gli esiti senza e con cad erano simili , sia per il tasso di richiami ( 11 , 39% contro 11 , 40% ) sia per il tasso di scoperta dei tumori ( 3 , 49% contro 3 , 55% per 1000 esami di screening )  . 
these observations clearly contrasted with those of freers and ulisseyss trial [ 58 ] in which mammograms were read without cad and immediately reviewed with cad : the authors suggest that such a protocol may have introduced a lower level of vigilance during the initial interpretation . 
 [ 59 ] analysis may have some bias : besides the presence or absence of cad , other differences may have existed between the two groups , such as differences in the number of cases read by certain readers or higher levels of vigilance in the cad group induced by the awareness of participating in a trial . another prospective trial evaluated the effect of cad on screening mammogram interpretation to determine whether the outcome was different from that on clinical mammography [ 61 ]  . 
the third prospective trial [ 60 ] by the same authors was designed to evaluate the impact of cad both on a single reading before and after prompting ( as in freers and ulisseys trial [ 58 ] ) and on two distinct sets of cases ( as in gur et al.s trial [ 59 ] )  . 
there was an increase in recall rate in the cad group compared with the non - cad group but without a statistically significant increase in detection rate , suggesting a negative impact on specificity . 
 [ 59 ] : the outcomes of 19 , 402 screening mammograms interpreted with cad over a 2 - year period were compared with those of 7 , 872 films interpreted without cad freer [ 58 ] , in cui le mammografie venivano lette senza cad e immediatamente rilette con cad : gli autori suggeriscono che un simile protocollo pu avere indotto un minor grado di vigilanza nellinterpretazione iniziale . 
 [ 59 ] pu avere qualche bias : oltre alla presenza o assenza del cad , tra i due gruppi possono esserci state altre differenze , come diversit nel numero di casi visti da alcuni lettori , o livelli di vigilanza maggiori nel gruppo con cad , indotti dalla consapevolezza di stare partecipando a un trial . un altro studio prospettico ha valutato leffetto del cad sullinterpretazione delle mammografie di screening , per capire se esso fosse diverso da quello sulle mammografie cliniche [ 61 ]  . 
il tasso di richiami dopo cad stato del 9 , 9% , leggermente superiore a quello di un simile periodo precedente alluso del cad ( 8 , 4% )  . 
il terzo studio , prospettico , degli stessi autori [ 60 ] stato disegnato per valutare limpatto del cad sia sulla singola lettura prima e dopo segnalazione ( come nel lavoro di freer e ulissey [ 58 ] ) , sia su due distinti set di casi ( come nello studio di gur [ 59 ] )  . 
si osservato un aumento nel tasso di richiami per il gruppo con cad rispetto al gruppo senza cad , senza tuttavia un significativo miglioramento del tasso di identificazione , indicando un effetto negativo sulla specificit . 
in definitiva , nessuno di questi tre studi ha dimostrato un effetto statisticamente significaivo del cad . recentemente un trial ha valutato in modo prospettico leffetto del cad su di un programma di screening mammografico regionale , confrontando i risultati annuali in periodi pre - cad e cad [ 65 ] e paragonando le conclusioni a quelle dei principali studi prospettici condotti in precedenza . 
il disegno dello studio era simile a quello di gur [ 59 ] : esito di 19402 mammografie lette con cad nellarco di 2 anni confrontato con quello di 7872 mammografie lette senza cad nellanno precedente . 
il tasso di riconoscimento nello screening con cad aumentato del 16 , 1% , in accordo con freer e ulissey ( aumento del 19 , 5% ) [ 58 ] , ma in contrasto con gur ( aumento del 1 , 7% ) [ 59 ]  . 
si osservato un aumento , seppur minore , nel tasso di richiami ( 8 , 1% ) , tasso di biopsie ( 6 , 7% ) e valore predittivo positivo delle biopsie ( 8 , 8% )  . gli autori hanno sottolineato alcuni bias del loro studio . data la rarit di tumori nella popolazione sotto screening , per osservare variazioni statisticamente significative servono m . 
the authors concluded that the increase in the proportion of early stage invasive cancers and younger patient age at diagnosis with cad are clinically and statistically significant , indicating the benefit of cad . if the performance of cad systems in the different type of lesions is analysed in detail , it appears to be clear that cad is more sensitive for lesions presenting as microcalcifications than for lesions presenting as masses . 
table 2 shows the results of the different studies comparing the two type of lesions , showing that cad sensitivity for microcalcifications ranges between 86% and 100% , whereas for masses it ranges between 67% and 87% [ 3 , 5861 , 63 , 64 , 6668 ]  . the only surprising finding was reported by birdwell et al . [ 61 ] : in their study , both cancers detected only by cad and missed by screening mammographic reading were masses ; these two lesions were clinically palpable and therefore would not have been missed at clinical evaluation . recently ( 2005 ) , an analysis evaluated the performance of a cad system for the detection of breast cancer based on mammographic appearance and histopathology [ 66 ]  . 
the prognosis was better for masses than for calcifications ( in small invasive tumours ) [ 70 ] , for ductal carcinoma in situ than invasive lesions and for smaller than larger tumours [ 71 ]  . 
so , even though mammographic appearance and histopathology did not appear to affect the overall performance of the cad system ( which showed a high sensitivity in any case ) , results were better for microcalcifications , and the detection rate was higher for dcis ( ductal carcinoma in situ ) and invasive lobular carcinomas . 
this indicated that cad may have an important role in reducing the occurrence of missed cancers , even for lesions that are difficult to detect , and in influencing prognosis . 
trial [ 65 ] in which the detection rate of small invasive cancers ( 1.0 cm or less ) increased ( 164% ) , especially for those with an associated dcis component . 
le differenze osservate nei due gruppi potrebbero essere dovute alla diversa distribuzione tra screening di prevalenza e screening di incidenza ( i tassi di diagnosi sono maggiori nel primo )  . 
comunque gli autori hanno concluso che con il cad vi un aumento clinicamente e statisticamente significativo nella percentuale di cancri diagnosticati in stadio pi precoce e in pazienti pi giovani , affermando il beneficio apportato dal cad . analizzando in dettaglio la performance del cad su diversi tipi di lesioni , sembra chiaro che il sistema pi sensibile per le microcalcificazioni che per le masse . 
la tabella 2 mostra i risultati dei vari studi relativamente ai due tipi di lesioni , mostrando che la sensibilit del cad per le microcalcificazioni va dall86% al 100% , mentre per le masse varia tra il 67% e l87% [ 3 , 5861 , 63 , 64 , 6668 ]  . 
 [ 61 ] : nel loro studio entrambi i cancri visti dal solo cad e persi allo screening erano masse ; comunque queste due lesioni erano clinicamente palpabili e quindi non sarebbero state perse allesame clinico . recentemente ( 2005 ) stata condotta unanalisi della performance del cad nellindividuare il tumore mammario in relazione al suo aspetto mammografico e alla sua istologia [ 66 ]  . 
la prognosi migliore per le masse rispetto alle microcalcificazioni ( nel caso di piccoli tumori invasivi ) [ 70 ] , per il carcinoma duttale in situ rispetto alle lesioni invasive , per i tumori piccoli rispetto ai grandi [ 71 ]  . per quanto riguarda listologia , la sensibilit del cad stata del 95% per il carcinoma duttale in situ , del 95% per il carcinoma lobulare invasivo , dell85% per il carcinoma duttale invasivo e del 90% per il carcinoma mammario invasivo ( ossia con caratteristiche duttali e lobulari )  . 
quindi , pur sembrando che laspetto mammografico e listologia non abbiano influenzato la performance complessiva del cad ( che ha mostrato in ogni caso unalta sensibilit ) , i risultati sono stati migliori per le microcalcificazioni , e il tasso di individuazione stato pi alto per i carcinomi duttali in situ e lobulari invasivi . 
ci indica che il cad pu avere un importante ruolo nel ridurre la frequenza dei cancri persi , anche per lesioni difficili da vedere , e nellinfluenzare la prognosi delle pazienti . 
questi risultati sono stati confermati anche dallo studio di cupples [ 65 ] , in cui il tasso di diagnosi di piccoli tumori invasivi ( 1 , 0 cm o meno ) aumentato ( 164% ) , specialmente per i tumori con associata una componente di carcinoma duttale in situ . 
il tasso di diagnosi di tumori in stadio precoce aumentato per lesioni corrispondenti a piccoli cluster di microcalcificazioni o a piccole masse , o entrambi , avvalorando lipotesi che gli algoritmi cad sono utili per le specifiche morfologie per cui sono stati progettati . stato valutato limpatto sul cad della densit mammaria ( un importante fattore che limita la sensibilit della mammografia ) [ 67 , 68 ]  . 
anyway overall cad detection of breast cancer proved not to be affected by breast density , suggesting that cad may be particularly advantageous in patients with dense breasts . assuming at this point that cad increases the sensitivity of readers in cancer detection , the correct timing in using cad during reading remains to be defined . 
initially , cad was supposed to be used before the radiologists reading in a so - called prereading session , with the aim of dismissing all negative , unprompted cases and to select only cases with prompts to be verified by the radiologist . 
tuttavia , nel complesso , il cad non si dimostrato influenzato dalla densit mammaria nella scoperta di tumori , sembrando quindi particolarmente vantaggioso per le pazienti con seni densi . assumendo , a questo punto , che il cad aumenti la sensibilit dei lettori nellindividuare i tumori , restano da definire i tempi corretti del suo utilizzo durante la lettura . 
inizialmente il cad era inteso come strumento da utilizzare prima della lettura del radiologo , in una sessione cosiddetta pre - lettura , allo scopo di scartare tutti i casi negativi non marcati , e di selezionare solo i casi con segnalazioni , che il radiologo doveva poi verificare . 
infatti , il cad ha perso dal 14% al 16% dei tumori trovati dai radiologi [ 3 , 61 ] , rimarcando il fatto che esso non pu sostituire il radiologo , ma rappresenta un metodo aggiuntivo . 
quindi , come precedentemente sottolineato , il cad dovrebbe essere considerato solo come unalternativa al secondo lettore ; i risultati del cad dovrebbero essere valutati dal radiologo dopo una prima lettura cieca , con lo scopo di riconsiderare aree potenzialmente sospette segnalate dal cad . comunque , questo protocollo presenta di per s delle lim . 
in fact , cad missed 14%16% of cancers detected by the radiologists [ 3 , 61 ] , emphasising that a cad system cannot replace the radiologist but is an adjunct method . 
therefore , as previously highlighted , cad should be considered an alternative to the second reader ; cad results should be viewed by the radiologist after a prior blind reading in order to reconsider areas potentially suspicious prompted by cad . 
in their 2004 study [ 56 ] in which a performance comparison was made not only between two different cad systems but also between cad and a simulation of independent double reading obtained by pairing up the single - radiologist conventional readings . 
this was explained by considering that , whereas double reading may correct certain systematic reading limitations of the single radiologists , in cad reading the readers diagnostic criteria are unchanged . 
 furthermore , the reaction of the radiologist to the several prompts it not obvious : too many prompts may distract the reader instead of attracting his attention , resulting in a nonoptimal use of cad sensitivity . 
this is an automation bias of omission ( failures to respond to system irregularities or events because automated devices fail to detect or indicate them ) that results from cognitive vigilance decrements [ 72 , 73 ]  . 
their data indicated that readers learnt to recognise patterns in shape , frequency and location that characterised false positive prompts and used this to assess the mammogram a priori to decide how much effort to invest in analysing it . 
according to the authors , this is an intuitive reaction , essential for establishing trust in system performance , but it requires a cognitive cost . in a study performed by zheng et al . , the false positive rate seemed to influence the readers ability to correctly classify prompts , but not significantly . 
una di queste stata evidenziata da ciatto et al . nel loro studio del 2004 [ 56 ] , nel quale , oltre a confrontare la performance di due diversi sistemi cad , la si anche paragonata con una simulazione di doppia lettura indipendente ottenuta combinando in coppie le letture convenzionali singole : la doppia lettura risultata pi sensibile , a parit di specificit , della lettura singola con cad . 
ci stato spiegato col fatto che , mentre la doppia lettura permette di correggere eventuali limiti sistematici di interpretazione dei singoli radiologi , nella lettura con cad i criteri diagnostici del lettore rimangono gli stessi . inoltre , la reazione del radiologo a numerose segnalazioni non scontata ; troppi suggerimenti possono distrarre il lettore , anzich attirarne lattenzione , comportando un uso non ottimale della sensibilit del cad . 
taylor e ulissey [ 60 ] hanno notato che unalta percentuale di tumori correttamente segnalati non sono stati richiamati : la spiegazione data che il lettore si sia fidato della sua prima impressione di benignit . la presenza di troppi segnali falsi positivi pu avere effetti negativi sulla performance dei radiologi . 
molte segnalazioni possono essere percepite come distraenti e , a volte , la loro assenza pu essere considerata pi informativa della loro presenza : questo un automation bias di omissione ( mancata risposta a irregolarit di sistema o a eventi , dovuta al fatto che strumenti automatici falliscono nel vederli o indicarli ) che deriva da cali di vigilanza cognitiva [ 72 , 73 ]  . 
secondo gli autori , questa una reazione intuitiva , base essenziale per riporre fiducia nella performance del sistema , ma che richiede un costo cognitivo . in uno studio condotto da zheng et al . , il tasso di falsi positivi parso influenzare labilit dei lettori nel classificare correttamente le segnalazioni , ma non in modo significativo . in questo trial , la performance dei radiologi nellindividuare fini anormalit mammografiche , in condizioni sperimentali , stata testata usando quattro sistemi cad a differenti livelli di tasso di veri positivi e falsi positivi ( quattro combinazioni di tassi alti e bassi ) [ 75 ]  . 
i risultati hanno mostrato come la performance dellosservatore fosse influenzata da quella intrinseca del sistema cad : sistemi ad alta performance possono migliorarla , sistemi a bassa performance peggiorarla . comunque , allaumentare del numero di segnalazioni si osservato un certo incremento del tasso di scarto di veri positim . 
the results showed that observer performance was affected by the inherent performance of the cad system : a high - performance system can improve it , a low - performance system can degrade it . 
once the abnormality was detected , the ability of the observer to classify it as benign or malignant was not significantly affected by the cuing mode . it is important to consider that the false positive rate of a cad system influences the users confidence in it . 
according to manufacturers experience , a false positive rate of nearly three per case is hardly accepted by the users [ 76 ] , and the highest rate tolerable is of about two false marks per case . 
as it has been observed that radiologists use prompts to economise on the effort required to deal with false positive prompts , the training session must provide balance between making an a priori assessment of prompt significance and carefully examining each prompted region . 
radiologists need training to make the best use of cade , that is , using prompts only as an aid for detection and not as evidence for interpretation [ 74 ]  . trials on cadi systems mammography has a poor positive predictive value , so that 65%85% of breast biopsies are performed on benign lesions [ 77 ]  . 
the goal is to reduce the number of false positives , maintaining a high rate of cancer detection . for microcalcifications , algorithms for their detection are better than algorithms for their diagnosis . 
a study published in 2002 compared the performance of a cadi system for diagnosis of previously detected lesions based on radiologistextracted findings on masses and calcifications [ 81 ]  . 
una volta individuata unanormalit , labilit dellosservatore nel classificarla come benigna o maligna non era significativamente influenzata dalla modalit di suggerimento . importante considerare che il tasso di falsi positivi del sistema cad influenza la fiducia degli utenti in esso . 
secondo lesperienza dei costruttori , un tasso di falsi positivi di quasi tre per caso difficilmente accettato dallutente [ 76 ] , e il pi alto tasso tollerabile di circa due per caso . 
queste considerazioni suggeriscono che i sistemi cad implicano il rischio di effetti dannosi sulla performance dei lettori , che si pu ridurre addestrando i radiologi alluso dello strumento . una prima esposizione ai suggerimenti del cad senza alcuna spiegazione pu condurre alla perdita di fiducia nel mezzo e a mancanza di attenzione verso segnalazioni corrette . 
poich si visto che i radiologi usano le segnalazioni per economizzare sullo sforzo di dover gestire i falsi positivi , laddestramento deve permettere di raggiungere un equilibrio tra definizione a priori del significato di una segnalazione e attento esame di ogni area marcata . 
i radiologi necessitano di un training per fare il migliore uso del cade , ossia usarne i suggerimenti solo come aiuto per lindividuazione , e non come evidenza per linterpretazione [ 74 ]  . studi sui sistemi cadi la mammografia ha uno scarso valore predittivo positivo , cosicch il 65%85% delle biopsie mammarie sono fatte su lesioni benigne [ 77 ]  . 
questo accresce il peso emozionale , fisico ed economico che grava su pazienti e cliniche [ 78 , 79 ] e riduce lefficacia dello screening mammografico [ 80 ]  . 
lobiettivo ridurre il numero di falsi positivi , mantenendo un alto tasso di individuazione dei tumori . per le microcalcificazioni , gli algoritmi per lindividuazione sono migliori di quelli per la diagnosi . 
il passaggio pi difficile la segmentazione delle calcificazioni : sarebbero desiderabili caratteristiche tali da impedire errori di segmentazione , cos come caratteristiche di cluster che colgano la disposizione spaziale delle calcificazioni . 
uno studio pubblicato nel 2002 ha comparato la performance di un sistema cadi nel classificare lesioni , precedentemente individuate , in base alle caratteristiche mammografiche di masse e calcificazioni estratte da un radiologo [ 81 ]  . 
la presenza di calcificazioni sovrapposte a una massa pu influenzare la classificazione di quella massa , per cui masse e calcificazioni dovrebbero essere considerate separatamente quando si valuta un sistema cadi . 
these new cases ( not used for classifier training and development and thus unknown to it ) are referred to as validation cases , and the evaluation of new data is referred to as classifier validation [ 82 ]  . 
these results were considered a preclinical success of the classifier as a potential aid in reducing the number of biopsies performed on benign masses and are consistent with those reported by other authors [ 83 ]  . 
a further analysis of the same data [ 85 ] evaluated the effects of cadi on interobserver variability in interpretation of mammograms by comparing decisions without and with cadi on clustered microcalcifications : by the use of cadi , variability was reduced by 46% , eliminating the disagreement between radiologists in two thirds of cases . also , in the classification of masses , cadi systems showed good results [ 86 ]  . 
on this topic , in 2002 , the first study was published in which the cases for interpretation were unknown both to the radiologist and to the computer : cases were distinct ( independent ) from those used for the training of the classification algorithm [ 87 ]  . 
in addition , it was observed that cadi can potentially reduce the difference in accuracy between community radiologists and mammographers ( improvements were larger for the former )  . in spite of these promising results , the commercialisation of such systems has been delayed , probably due to the difficulty in obtaining fda approval . 
at present , it may be hard to gain clear evidence from large clinical trials that cadi can improve radiologists specificity in identifying benign lesions , without any decrease in sensitivity , for malignant cases . 
this is partly due to the fact that cadi outputs are commonly given by a number , such as the percentage for likelihood of malignancy ( whereas cade marks the location of a suspicious lesion )  . 
oggi nessun sistema ancora stato promosso alla commercializzazione dalla fda ( food and drug administration ) americana [ 20 ]  . solo recentemente si cominciato a testare sistemi cadi , estesamente allenati , su dati nuovi non usati precedentemente . 
questi nuovi casi ( non usati nel training e sviluppo del classificatore e ad esso sconosciuti ) vengono chiamati casi di validazione , e la valutazione di nuovi dati si chiama validazione del classificatore [ 82 ]  . 
 [ 84 ] riportano che , per la classificazione dei cluster di microcalcificazioni , laccuratezza ( espressa come area sotto la curva roc ) migliorata con luso del cadi dallo 0 , 61 allo 0 , 75 . 
unulteriore analisi degli stessi dati [ 85 ] ha valutato gli effetti del cadi sulla variabilit interosservatore nellinterpretazione di mammografie , confrontando le decisioni senza e con cadi riguardo a cluster di microcalcificazioni : con luso del cadi , la variabilit si ridotta del 46% , eliminando il disaccordo tra radiologi in due terzi dei casi . anche nella classificazione delle masse i sistemi cadi hanno mostrato buoni risultati [ 86 ]  . 
a tale riguardo , nel 2002 stato pubblicato il primo studio in cui i casi da interpretare erano sconosciuti sia al radiologo che al computer : i casi erano distinti ( indipendenti ) da quelli usati per allenare lalgoritmo di classificazione [ 87 ]  . 
questo studio ha evidenziato un miglioramento dei risultati dei radiologi con luso del cadi : larea sotto la curva roc aumentata dallo 0 , 93% allo 0 , 96% , la sensibilit dal 94% al 98% . 
non si riscontrata differenza statisticamente significativa nella specificit : il computer non ha mostrato effetti sul numero di casi benigni mandati alla biopsia . in aggiunta , si osservato che il cadi potenzialmente riduce la differenza di accuratezza tra radiologi di comunit e mammografisti ( i primi hanno ottenuto maggiori benefici )  . nonostante questi risultati promettenti , la commercializzazione di tali sistemi ritardata , probabilmente per la difficolt a ottenere lapprovazione dellfda . 
pu essere al momento difficile trarre chiara evidenza , da ampi studi clinici , che il cadi permette di migliorare la specificit dei radiologi nellidentificare lesioni benigne senza ridurre la sensibilit per quelle maligne . 
ci in parte dovuto al fatto che i risultati del cadi sono comunemente espressi da un valore numerico , come la percentuale di probabilit di malignit ( mentre il cade evidenzia la sede di una lesione sospetta )  . 
i radiologi non hanno familiarit con questi valori , dato che finora le loro diagnosi si sono basate su allenamento ed esperienza clinica di percezione visiva di tanti casi , e sulla comprensione di m . 
comunque , possibile e auspicabile che , in futuro , i radiologi prendano familiarit con questo tipo di risultati [ 83 ]  . commercial cad systems at present , four cad systems have obtained fda approval and are commercially available . 
no cadi system has been approved by the fda . r2 technology sistemi cad in commercio attualmente , quattro sistemi cade hanno ottenuto lapprovazione dalla fda e sono disponibili in commercio . 
nessun sistema cadi stato approvato dalla fda . r2 technology r2 technologys image checker was the first commercial cade system approved by the fda , in 1998 [ 88 ]  . 
pilot studies demonstrated 87% and 90.7% sensitivity for masses and microcalcification clusters , respectively . cad and digital mammography cad analysis has most frequently been applied by digitising conventional film - screen mammograms . 
this requires an intermediate step for scanning the film into a laser scanner limage checker della r2 technology stato il primo sistema cade approvato dalla fda , nel 1998 , e commercializzato [ 88 ]  . 
 stato riportato che limage checker m1000 system , version 1.0 , segnala correttamente il 98 , 0% delle microcalcificazioni , il 74 , 7% delle masse e l83 , 4% di tutte le lesioni . intelligent system software la fda nel 2002 ha approvato il sistema cade della issi , il mammoreader , per lindividuazione di cluster di microcalcificazioni , masse , lesioni spiculate , distorsioni architetturali e densit asimmetriche . 
la sensibilit complessiva della prima versione del sistema approvata dalla fda era dell89 , 3% ( del 91 , 0% per i tumori con laspetto di microcalcificazioni , dell87 , 4% nei casi di masse maligne )  . cadx medical systems il secondlook della cadx medical systems ha ricevuto lapprovazione dalla fda nel 2002 [ 90 ]  . 
la prima versione approvata aveva una sensibilit dell85% per tumori trovati allo screening ( combinazione di masse e microcalcificazioni )  . eastman kodak company il kodak mammography cad engine stato approvato dalla fda nel 2004 [ 91 ]  . 
gli studi iniziali hanno mostrato una sensibilit dell87% e del 90 , 7% rispettivamente per masse e microcalcificazioni . cad e mammografia digitale lanalisi cad stata pi frequentemente applicata a mammografie analogiche digitalizzate . 
some authors noted that adaptive modelling with cad combined with digital mammography might yield greater diagnostic performance compared with nonadaptive cad methods for detection of masses [ 31 , 92 , 93 ]  . 
alcuni autori hanno notato che modelli cad adattativi combinati con la mammografia digitale potrebbero condurre a una migliore performance diagnostica , rispetto a metodi cad non adattativi , per lindividuazione di masse [ 31 , 92 , 93 ]  . 
il sistema image checker ha ricevuto dalla fda lapprovazione per combinare la sua tecnologia cad direttamente con il sistema di mammografia digitale fabbricato dalla general electric [ 92 ]  . unsolved problems screening mammography was the first radiologic branch for which the fda , in 1998 , approved a clinical cad system . since then , various studies have been performed to evaluate the use of cad in clinical practice with not completely concording results . 
currently , cade in mammography is accepted as a second reader : the radiologist first searches the mammograms for abnormalities , then the computer marks additional suspicious regions and the final decision is made by the radiologist . 
the ultimate results of studies of cad algorithms rest on problemi aperti la mammografia di screening stata la prima branca radiologica per la quale , nel 1998 , la fda ha approvato un sistema cad clinico . 
correntemente , il cade in mammografia accettato come secondo lettore : prima il radiologo cerca anormalit sulla mammografia , poi il computer indica ulteriori aree sospette e il radiologo prende la decisione finale . 
roc studies are well understood in certain contexts , but they are not immediately applicable to cad research . several other methods are available , but the choice is not easy . 
da incoraggiare unulteriore ricerca sui metodi di valutazione e sul loro uso . possible coming improvements cade and cadi system promise to be helpful in screening mammography , but their actual benefit has still to be assessed . 
probably , in the near future , algorithms for computer detection and classification of suspicious regions on mammograms will be further improved , with a progressive increase in the sensitivity and specificity of these systems . 
another promising technical issue is the development of interfaces for the application of cad technology directly to digital mammography systems , making easier the use of such devices and improving their performance . 
thus , effectiveness of screening mammography in the early diagnosis of breast cancer would improve , with further reduction of mortality . possibili sviluppi futuri i sistemi cade e cadi sono promettenti ausili alla mammografia di screening , ma il loro reale beneficio deve ancora essere definito . 
probabilmente , nel prossimo futuro , gli algoritmi per lindividuazione e la classificazione di regioni sospette sulle mammografie verranno ulteriormente migliorati , con progressivo incremento della loro sensibilit e specificit . 
un altro campo di possibile progresso tecnico lo sviluppo di interfacce per lapplicazione della tecnologia cad direttamente ai mammografi digitali , che ne rendano pi semplice lutilizzo e ne migliorino la performance . 
infine , via via che questi sistemi saranno progressivamente applicati allo screening routinario , i radiologi acquisiranno maggiore confidenza con essi , traendone il massimo beneficio in termini di efficienza . 
blanks rg , wallis mg , moss sm ( 1998 ) a comparison of cancer detection rates achieved by breast cancer screening programmes by number of readers , for one and two view mammography : results from the uk national health service breast screening programme . 
dinnes j , moss s , melia j et al ( 2001 ) effectiveness and cost - effectiveness of double reading of mammograms in breast cancer screening : findings of a systematic review . 
cova1 1struttura complessa di radiologia , 2struttura semplice di chirurgia vascolare , azienda ospedaliero - universitaria ospedali riuniti di trieste , strada di fiume 447 , i - 34149 trieste , italy correspondence to : f . 
pozzi mucelli , tel : + 39 - 040 - 3994372 , fax : + 39 - 040 - 3994500 , e - mail : fabio.pozzimucelli@alice.it received : 10 august 2006 / accepted : 22 november 2006 / published online : 20 april 2007 abstract purpose . 
the proximal endoleaks were treated by cuff deployment , whereas the distal endoleak was treated with a bifurcated graof the two patients with type ii endoleak , one was treated by translumbar puncture and coil embolisation , and the other was treated by superselective embolisation of the lumbar feeding vessel with nonresorbable particles . 
of the three patients with type iii endoleak , two were treated by deploying an aortouniiliac endograft inside the bifurcated graft and the other by implanting a cuff to restore continuity between the graft body and the contralateral limb . 
significant endoleaks should be treated promptly . endovascular treatment can be done with different techniques , but success in not constant due to adverse anatomical conditions and technical difficulties . key words endovascular treatment abdominal aorta aneurysm endoleak riassunto obiettivo . 
dei primi 5 , 4 erano prossimali e 1 distale e sono stati trattati con il posizionamento di cuffie prossimali ; lel distale stato trattato mediante posizionamento di protesi biforcata . 
laltro caso stato trattato mediante embolizzazione dopo cateterismo superselettivo dellafferenza lombare con agente embolizzante . dei tre casi con el di tipo 3 , 2 sono stati trattati mediante posizionamento di protesi aorto - monoiliaca e in un caso stata interposta una cuffia per ripristinare la continuit tra corpo principale e gamba contro - laterale . 
il trattamento endovascolare possibile con diverse modalit , tuttavia il successo tecnico non costante a causa di situazioni anatomiche sfavorevoli e di possibili difficolt tecniche . parole chiave trattamento endovascolare aneurisma aorta addominale endoleak f . 
this complication is usually readily recognised on follow - up imaging studies , especially with computed tomography ( ct ) but also with ultrasound ( us ) and magnetic resonance imaging ( mri ) , and is known as endoleak , a term denoting persistent blood flow within the aneurysm sac outside the endograft . this may occur as a result of different mechanisms : ( 1 ) inadequate seal between the endograft and the aortic wall at the proximal or distal attachment sites ( proximal or distal type i endoleak ) , ( 2 ) retrograde reperfusion of the aneurysm sac by collateral branches ( lumbar arteries or inferior mesenteric artery ) ( type ii endoleak ) , and ( 3 ) incorrect junction of the graft components ( main body and contralateral limb or main body and proximal or distal cuff ) ( type iii endoleak )  . 
other less common types of endoleak include type iv , which occurs due to excessive graft porosity , and type v , also named endotension , in which imaging studies identify sac expansion without a detectable leak [ 1 ]  . the aim of this paper is to describe our experience with endoleaks and illustrate the strategies employed to resolve them and their effectiveness . materials and methods from january 2000 to june 2006 , 134 patients ( 118 men , 16 women ; mean age 75.1 , range 5891 years ) underwent endovascular repair of an abdominal aorta aneurysm by deployment of a bifurcated aortic endograft ( 123 / 134 ) or an aortouniiliac endograft with occlusion of the contralateral iliac artery and a femorofemoral bypass ( 11 / 134 )  . 
all interventional procedures were carried out in an operating room with portable c - arm equipment ( eurocolumbus , milan , italy ) by an interventional radiologist , a vascular surgeon and an anaesthetist after surgical exposure of both femoral arteries . the patients received spinal or general anaesthesia . 
all patients underwent ct follow - up examinations at 1 , 6 , 12 and 24 months and yearly thereafter . il trattamento endovascolare degli aneurismi dellaorta addominale mediante il posizionamento di endoprotesi aortiche diventata un opzione terapeutica sempre pi utilizzata nella pratica clinica per la scarsa invasivit della procedura che la rende eseguibile anche in pazienti con controindicazioni allintervento tradizionale ( et avanzata , patologie concomitanti , addome ostile per pregressi interventi )  . 
tuttavia uno dei talloni dachille di questo intervento dato dalla sua incapacit di escludere completamente la sacca aneurismatica nella totalit dei casi con conseguente possibile aumento della pressione allinterno della sacca e sua possibile rottura . tale complicanza viene riconosciuta agevolmente nella maggior parte dei casi al follow - up mediante diagnostica per immagini , soprattutto con tomografia computerizzata ( tc ) ma anche mediante ecografia e risonanza magnetica e va sotto nome di endoleak ( el ) , termine che non appare traducibile in italiano con ununica parola , ma che sta a significare la persistenza di flusso ematico nella sacca aneurismatica al di fuori dellendoprotesi . 
esistono inoltre ulteriori due tipi di el meno frequenti che sono lel di tipo 4 da eccessiva porosit del tessuto protesico e lel di tipo 5 detto anche endotension : questo un tipo di el particolare in quanto limaging non evidenzia la riperfusione attiva della sacca aneurismatica , ma solo laumento di dimensioni della sacca [ 1 ]  . scopo di questo articolo di descrivere la nostra esperienza con tale tipo di complicanza e di illustrare le soluzioni adottate e la loro efficacia . materiali e metodi dal gennaio 2000 a giugno 2006 , 134 pazienti ( 118 m , 16 f ; et media : 75 , 1 ; range 5891 ) sono stati sottoposti a trattamento endovascolare per lesclusione di un aneurisma dellaorta addominale mediante posizionamento di endoprotesi aortica biforcata ( 123 / 134 ) o aorto - monoiliaca con occlusione dellarteria iliaca contro - laterale mediante occlusore e confenzionamento di by pass femoro - femorale ( 11 / 134 ) utilizzando diversi devices tra quelli disponibili sul mercato ( 91 casi : talent , medtronic , sunnyvale , ca , usa ; 26 casi : excluder , wl gore & associates , flagstaff , az , usa ; 13 casi : aneurx , medtronic , sunnyvale , ca , usa ; 1 caso : life path , edwards lifesciences , irvine , ca , usa ; 1 caso : aorfix , lombard medical , oxon , uk ) ( tabella 1 )  . 
both patients were successfully treated with conventional surgery a few days later . ct follow - up at 1 , 6 , 12 , 24 and 36 months in the 132 cases successfully treated by endograft deployment demonstrated the development of type i , ii and iii endoleaks in 22 patients ( 16% )  . 
twelve cases were primary endoleaks ( that is , present at the first follow - up study ) , whereas ten cases were secondary ( apparent at later follow - up studies )  . 
twelve of these endoleaks were not treated due to small size : four regressed spontaneously , two remained small and are stable and three remained small but are being monitored ; three patients were lost to follow - up ( two patients died from unrelated causes , and one is no longer traceable )  . 
of the ten patients undergoing endovascular treatment , five had type i endoleaks ( four proximal and one distal , the latter in the patient implanted with a straight graft at another centre ) , two had type ii and three had type iii endoleaks ( table 1 )  . with regard to type i endoleaks , exclusion was initially attempted in one case by prolonged dilatation with a valvuloplasty balloon catheter ( up to 30 minutes ) , leading to parproximal cuff proximal cuff proximal cuff bifurcated endograft proximal cuff translumbar embolisation superselective embolisation aortouniiliac graft aortouniiliac graft intermediate cuff outcome successful unsuccessful unsuccessful successful successful unsuccessful successful unsuccessful successful successful cuffia prossimale cuffia prossimale cuffia prossimale endoprotesi biforcata cuffia prossimale embolizzazione translombare embolizzazione superselettiva protesi aortomonoiliaca protesi aortomonoiliaca cuffia intermedia successo insuccesso insuccesso successo successo insuccesso successo insuccesso successo successo procedure sono state effettuate in sala operatoria utilizzando un apparecchiatura portatile con arco a c ( eurocolumbus , milano , italia ) in anestesia spinale o generale dopo esposizione chirurgica di entrambe le arterie femorali comuni da un equipe costituita oltre che dal radiologo interventista dal chirurgo vascolare e dallanestesista . 
tutti i pazienti sono stati sottoposti a ripetuti esami tc di controllo a 1 mese dallintervento , quindi a 6 , 12 , 24 mesi e dopo ogni anno . risultati il posizionamento dellendoprotesi stato possibile in 132 / 134 casi . 
in entrambi i casi a distanza di alcuni giorni stato effettuato lintervento convenzionale con successo . il controllo tc effettuato entro 1 mese dal trattamento endovascolare e quindi a 6 , 12 , 24 , 36 mesi nei 132 casi portati a termine con successo ha evidenziato la comparsa di el di tipo 1 , 2 e 3 in 22 pazienti ( 16% )  . 
in 12 casi si trattava di el primari ( presenti cio al primo controllo ) mentre in 10 casi erano el secondari ( insorti cio nei successivi controlli ) dodici di tali el non sono stati trattati perch di piccole dimensioni : 4 di questi sono spontaneamente regrediti , 2 sono stabili e sempre di dimensioni contenute e 3 sempre di picf . 
a proximal cuff was subsequently implanted , but this technique also failed to achieve complete exclusion of the endoleak , so the patient underwent surgery to remove the graft and fashion an aortobiiliac bypass . 
three other cases of proximal type i endoleak were directly treated by proximal cuff deployment : in one case , a single cuff was sufficient to exclude the endoleak , whereas in another case , the first cuff failed to achieve effective sealing at the proximal end and a further cuff was added in another session , which succeeded in completely excluding the endoleak . 
despite correct positioning , this cuff was unable to completely exclude the endoleak due to aortic tortuosity , so this patient also had a surgical conversion at a later date . the single case of distal type i endoleak arose , as stated , in a patient treated at another centre by deployment of a cole dimensioni sono sotto osservazione ( 3 sono andati persi al follow - up : 2 pazienti deceduti per cause non correlate alla patologia aneurismatica ; 1 paziente non pi reperibile )  . in 11 casi di el di entit pi significativa si proceduto ad un trattamento endovascolare in 10 casi ( combinato in 1 caso ) e chirurgico in 1 caso . 
di questi 10 casi di el trattati per via endovascolare 5 erano el di tipo 1 : 4 a sede prossimale ed 1 a sede distale ( il caso trattato altrove con protesi retta ) , 2 di tipo 2 e 3 di tipo 3 ( tabella 1 )  . per quanto riguarda gli el di tipo 1 in un caso stato tentato preliminarmente di ottenere la sua esclusione mediante f . 
successivamente si proceduto al posizionamento di una cuffia prossimale , ma anche questo trattamento non stato in grado di determinare la risoluzione completa dellel per cui il paziente stato sottoposto ad intervento chirurgico con rimozione della protesi e confezionamento di by pass aortobisiliaco . 
altri 3 casi di el di tipo 1 prossimale sono stati trattati direttamente con posizionamento di una cuffia prossimale : in un caso stato sufficiente il posizionamento di ununica cuffia per ottenere la risoluzione dellel , in un secondo caso la prima cuffia non stata sufficiente a determinare la corretta sigillatura prossimale della protesi per cui in una seconda seduta stata aggiunta una seconda cuffia con risoluzione completa dellel . 
nel terzo caso , in paziente monorene , a distanza di 6 mesi dal posizionamento della protesi vi fu la comparsa di un el di tipo 1 per parziale scivolamento caudale della protesi . 
a , b computed tomography ( ct ) : a large endoleak ( * ) is visible laterally to the distal portion of the straight endograft . b the maximum intensity projection ( mip ) image depicts well the graft angulation . 
in the first case , follow - up ct demonstrated sac reperfusion both anteriorly and posteriorly to the graangiography demonstrated that the anterior reperfusion was supplied by the inferior mesenteric artery . 
because this endoleak had significantly increased after 1 year , we attempted embolisation via direct translumbar puncture and occlusion by means of gianturco coils and an embolising agent ( spongostan )  . 
despite the partial failure , the patient was not converted to surgery because of poor general condition ; however , follow - up ct at approximately 1 year demonstrated no further expansion of the aneurysm sac . 
the third case of type iii endoleak occurred in a patient who had been implanted with a new concept endograft ( lifepath , edwards ) compared with those normally used . 
in this case , use of a double femoral - brachial approach and a snare device enabled us to restore continuity between the main body and via translombare e occlusione dello stesso mediante posizionamento di spirali di gianturco e agente embolizzante ( spongostan )  . 
nonostante tale trattamento a distanza di 24 ore si ebbe la rottura della sacca aneurismatica e il paziente venne sottoposto a intervento chirurgico in urgenza con rimozione dellendoprotesi e confezionamento di by pass aorto bisiliaco . 
in entrambi i casi si proceduto al posizionamento di una protesi aortomonoiliaca ( talent ) allinterno della protesi biforcata , quindi a posizionamento di occlusore a livello dellarteria iliaca comune controlaterale e confezionamento di bypass femoro - femorale . 
il paziente , nonostante il parziale insuccesso , non stato sottoposto ad ulteriore conversione chirurgica a causa delle condizioni generali scadenti , tuttavia ad un controllo tc a distanza di un anno circa , non si osservato pi alcun incremento della sacca aneurismatica . 
nel terzo caso di el di tipo 3 questo si verificato in un paziente in cui era stata utilizzata una protesi di concezione diversa ( lifepath edwards ) rispetto a quelle comunemente utilizzate . 
a a small endoleak is located anteriorly to the gra b angiography shows that the endoleak is due to anastomosis between the right gluteal superior artery and the lumbar artery . 
d tc di controllo dopo 12 mesi dallintervento : scomparsa dellendoleak . mento dei diametri della sacca aneurismatica , latteggiamento corretto di vigile attesa in quanto ai controlli successivi si pu ottenere una sua spontanea regressione [ 4 ]  . 
come si pu vedere dallesperienza qui riportata , il trattamento risolutivo pu essere diverso da caso a caso e va scelto in base a diversi criteri , quali le condizioni generali del paziente , la conformazione della protesi e le curvature dellaorta . 
a computed tomography ( ct ) , maximum intensity projection ( mip ) image : evidence of an anterior endoleak ( arrow ) between the proximal cuff and the main graft body . 
c angiografia intra - operatoria : il controllo dopo rilascio dellendoprotesi monoiliaca dimostra la persistenza di una dilatazione dellaorta nel tratto prossimale dovuto allincompleta sigillatura dellendoleak . tipo 1 lopzione teoricamente pi efficace laggiunta di una cuffia prossimale o distale per ottenere una adeguata sigillatura tra protesi e parete aortica . 
questo tipo di trattamento nella nostra esperienza tuttavia non sempre si rivelato efficace e ben in 3 / 4 casi in cui stato utilizzato la prima cuffia non stata sufficiente ad ottenere il successo tecnico e si dovuto in una seconda seduta aggiungere unulteriore cuffia per ottenere lesclusione dellel . 
nel caso di oversizing della cuffia si pu avere infatti che il tessuto protesico non sia correttamente disteso , ma presenti delle piccole ripiegature che consentirebbero il passaggio del sangue attraverso esse e quindi continuerebbero il rifornimento dellel . 
in medium endoleaks with enlarging aneurysm sac , treatment should be considered [ 5 ]  . as can be seen from our experience , treatment will differ from case to case and should be chosen on the basis of sevf . 
c tc di controllo dopo posizionamento di estensione protesica tra corpo principale e gamba controlaterale migrata caudalmente : risoluzione del quadro . eral criteria , including the patients general condition , graft configuration and aorta tortuosity . 
the most effective treatment for type i endoleak is ideally the insertion of an additional proximal or distal cuff to achieve an adequate seal between the graft and the aorta wall . 
in our experience , however , this form of treatment did not always prove effective , and as many as 3 / 4 cases required reintervention to implant an additional cuff and achieve endoleak exclusion . 
this option , however , is not always technically feasible due to the complexity of the catheterisation procedure , and it is not always effective in the long term [ 6 ]  . in our experience , the procedure was attempted twice , in one case with success whereas in the other case it was abandoned due to technical difficulty in achieving superselective catheterisation of the intermesenteric arcade . 
another possible technique involves a direct translumbar approach to the aneurysm sac , usually under ct guidance , and filling of the sac with coils or glue or thrombin [ 7 ]  . 
other authors [ 9 ] have reported very promising results with preventive embolisation of the aneurysm sac with fibrin glue provided that care is taken to prevent distal migration of the embolising agent . 
in our study , we also considered disconnection between the proximal cuff and the main graft body as type iii endoleaks ; however , such a situation has not been codified and may well be regarded as a type i endoleak . 
there are two main treatment options for type iii endoleaks : if the main graft body has become disconnected from a graft limb , attempts should be made to restore continuity of the graft components by adding an adequately sized cuff ; if the disconnection is between the proximal cuff and the main graft body the , options are either to fill the vascular component of the sac with metal coils or to implant an aortouniiliac graft inside the previous endograft , exclude the contralateral branch with an occluder and subsequently fashion a femorofemoral bypass [ 10 ]  . in our series , endoleaks occurred more frequently with talent endografts than with other devices ( aneurx , excluder )  . 
however , we believe this to be due to the fact that talent endografts tend to be used in more complex cases ( aneurysms with short or angulated necks , large size and wall calcifications ) rather than to a limitation of the talent endograft itself . 
our experience also shows that endoleaks occurred when we implanted different endografts from those commonly used , and this could be the consequence of inadequate experience with such devices . four out of 134 patients ( 2.9% ) treated with endograft implantation underwent surgical conversion . 
to these we should add the two patients in whom endovascular treatment failed and the one who underwent immediate surgical conversion owing to coverage of both renal arteries by a proximal cuff that had migrated cranially . 
this figure is , however , low if compared with other authors , who report a surgical convervece si soprasseduto a causa delle difficolt tecniche a ottenere il cateterismo superselettivo dellarcata di riolano . 
in alternativa a questa tecnica si pu utilizzare lapproccio diretto alla sacca aneurismatica per via translombare , in genere mediante guida tc e riempimento della sacca con spirali o colla o trombina [ 7 ]  . 
in alternativa alla puntura diretta per via translombare recentemente stato proposto lapproccio per via transcavale con risultati incoraggianti [ 8 ]  . altri autori [ 9 ] hanno proposto lembolizzazione preventiva della sacca aneurismatica con colla di fibrina ponendo attenzione a evitare la migrazione distale dellagente embolizzante con risultati estremamente promettenti . gli el di tipo 3 si hanno quando vi una disgiunzione tra corpo principale protesico ed estensione protesica o quando c un difetto protesico [ 1 ] ; nella nostra esperienza abbiamo considerato el di tipo 3 anche levenienza di disgiunzione tra cuffia prossimale e corpo principale protesico non essendo in realt codificata questa situazione ma potendosi in realt inquadrare anche come el di tipo 1 . 
nellel di tipo 3 le opzioni sono fondamentalmente 2 : nel caso di disgiunzione tra corpo principale ed estensione protesica bisogna cercare di ricostituire la continuit tra le componenti protesiche mediante laggiunta di una cuffia di calibro adeguato mentre nel caso di el tra cuffia prossimale e corpo principale le opzioni sono o il riempimento con spirali metalliche della componente vascolarizzata della sacca o il posizionamento di unendoprotesi aortomonoiliaca allinterno della precedente con esclusione della branca controlaterale mediante dispositivo occlusore e successivo bypass femorofemorale [ 10 ]  . in questa casistica gli el si sono manifestati pi frequentemente con la protesi talent rispetto ad altri tipi di protesi utilizzate ( aneurx , excluder ) , tuttavia riteniamo che questo non sia conseguente a un limite della protesi talent quanto al fatto che tale tipo di protesi viene utilizzata in casi pi complessi rispetto alle altre protesi : colletti corti , angolati , di calibro elevato e con calcificazioni parietali . 
dalla nostra esperienza emerge inoltre che abbiamo avuto el quando sono state utilizzate protesi diverse da quelle abituali e questo pu essere conseguenza di una inadeguata esperienza degli operatori con tali materiali . complessivamente nella nostra esperienza 4 pazienti su 134 trattati mediante posizionamento di endoprotesi sono andati incontro a conversione chirurgica pari pertanto al 2 , 9% . 
a questi 4 casi vanno aggiunti i 2 casi nei quali il trattamento endovascolare non era tecnicamente riuscito e un caso che andato incontro a conversione chirurgica immediata per la copertura di entrambe le arterie renali da parte di una cuffia prossimale migrata cranialmente per cui abbiamo avuto complessivamente 7 conversioni chirurgiche ( una acuta e 6 tardive ) su 134 casi ( 4 , 4% )  . 
 conclusions in conclusion , our experience shows that endovascular treatment of abdominal aorta aneurysms is feasible in a large proportion of patients , but success is not guaranteed in all cases ; in addition , the treatment of endoleaks , the most common complication of endovascular repair , although possible with a variety of endovascular techniques , does not always allow definitive resolution of the problevery careful selection of candidates and the use of endografts with proven longterm effectiveness on large patient series are required . portato dalleurostar data registry centre , che riporta un incidenza globale di reinterventi pari all8 , 7% [ 12 ]  . conclusioni in conclusione , riteniamo che dalla nostra esperienza emerga che il trattamento endovascolare di esclusione degli aneurismi dellaorta addominale sia una tecnica eseguibile in una ampia casistica di pazienti portatori di aneurismi tuttavia il successo non garantito nella totalit dei casi ed anche il trattamento della complicanza pi frequente , vale a dire lel , sebbene trattabile per via endovascolare e con diverse modalit , non sempre garantisce la risoluzione definitiva del problema . 
forty - two mri exams were performed with intravascular contrast media in 21 rats : tumours were induced by subcutaneous injection of colon carcinoma cells in 7 rats and mammary adenocarcinoma cells in 14 rats . 
perfusion and permeability parameters of the implanted tumours were evaluated by using two contrast media ( b22956 / 1 and gd - dtpa37albumin ) to establish response to treatment with two different antiangiogenic drugs ( tamoxifen and su6668 )  . 
in the mammary carcinoma experiment , vascular permeability measured by means of b22956 / 1 in the animals treated with the antiangiogenic drug ( 0.00433170.0040418 ml / min - 1 / ml - 1 ) was significantly less than in untreated animals ( 0.00904600.0043680 ml / min - 1 / ml - 1 ) , whereas no significant difference was observed with gd - dtpaalbumin ( 13.1413.94 ml / min - 1 / ml - 1 in treated animals and 18.0711.92 ml / min - 1 / ml - 1 in untreated animals )  . 
sono stati eseguiti 42 esami rm in 21 ratti di laboratorio a cui in 7 erano state preventivamente impiantate cellule di carcinoma del colon e in 14 cellule di adenocarcinoma mammario . 
sono stati valutati i parametri perfusionali e di permeabilit sul tumore impiantato mediante due mezzi di contrasto ( b22956 / 1 e albuminagd - dtpa37 ) al fine di stabilire la risposta al trattamento con due diversi farmaci antiangiogenici ( tamoxifen e su6668 )  . 
per lesperimento con cellule di adenocarcinoma mammario la permeabilit vascolare media misurata mediante il b22956 / 1 negli animali trattati con farmaco antiangiogenico ( 0 , 00433170 , 0040418 ml min - 1 ml - 1 ) significativamente minore che nei controlli ( 0 , 00904600 , 0043680 ml min - 1 ml - 1 ) , mentre nessuna differenza statisticamente significativa apprezzabile con gddtpaalbumina ( 13 , 1413 , 94 ml min - 1 ml - 1 nei trattati e 18 , 0711 , 92 ml min - 1 ml - 1 nei controlli )  . 
one of the major technical problems in the study of angiogenesis is the lack of methods ensuring significant , quantitative and repeatable measurements of the angiogenic process given the large number of techniques providing numerous but not necessarily comparable data . 
radiological research has also investigated the properties of the microcirculation , the anatomical - function unit of the vascular system , to increase knowledge of tumour characteristics and vascularity . animal models of human disease are fundamental for understanding the pathogenic mechanisms of diseases , especially during the different phases of pharmacological experiments . magnetic resonance imaging ( mri ) , with its multiple parameters and high spatial resolution that can reach 50100 m with specific scanners , has an recognised role in characterising animal models in vivo in preclinical studies [ 18 ] , thanks to its ability to provide metabolic , physiological and functional information . 
examples include evaluation of the functional response of the brain to stimuli or drugs [ 1 ] , assessment of organ viability after transplantation [ 2 ] , evaluation of cerebral blood volume and flow [ 3 ] , and characterisation of tumour neoangiogenesis present in tumours lager than 1 mm3 [ 46 , 9 , 10 ]  . 
imaging techniques of angiogenesis have potentially extended the clinical applications of mri , and their development has been triggered by advances in anticancer treatments , many of which aim to inhibit tumour microcirculation . 
intravascular contrast media can be used to measure both endothelial permeability and intravascular blood volume , both of which are related to angiogenesis : the first can be quantified as of the earliest stages of tumour development and the second only once the microcirculation has formed . 
 the aim of our work was to evaluate the applications of mri and in particular of dynamic contrast - enhanced mri ( dce - mri ) to the study of tumour microcirculation by using animal models assessed before and after treatment with antiangiogenic drugs . materials and methods we assessed the results of the use of dce - mri in two experiments conducted to test the effectiveness of two antiangiogenic drugs : tamoxifen , an oestrogen receptor ligand commonly used in hormone - dependent breast cancer treatment [ 11 ] , and su6668 , an inhibitor for vascular endothelial growth factor ( vegf ) receptors [ 12 ]  . 
in the first experiment , mammary adenocarcinoma cells ( american type culture collection ) were implanted subcutaneously in the flank of 14 la patologia neoplastica un importante campo di ricerca nellambito delle alterazioni microvascolari , al fine di studiare le correlazioni tra neoangiogenesi tumorale e grado di aggressivit dei tumori . 
uno dei pi rilevanti problemi tecnici nello studio dellangiogenesi la mancanza di metodiche che garantiscano misure significative , quantitative e ripetibili del processo angiogenico , a causa del gran numero di tecniche che forniscono una moltitudine di dati , non necessariamente comparabili . 
anche in campo radiologico si studiano le propriet del microcircolo , unit anatomo - funzionale del sistema vascolare , per migliorare la conoscenza delle caratteristiche e del grado di vascolarizzazione dei tumori . 
la risonanza magnetica ( rm ) , grazie alla molteplicit dei parametri e allalta risoluzione spaziale che pu raggiungere 50100 micron mediante scanner specifici , ha un ben riconosciuto potere nella caratterizzazione dei modelli animali in vivo negli studi pre - clinici [ 18 ] , in quanto consente di offrire informazioni metaboliche , fisiologiche e funzionali . 
esempi sono la valutazione della risposta funzionale del cervello , indotta da stimoli o farmaci [ 1 ] , la valutazione della vitalit degli organi dopo trapianto [ 2 ] , la valutazione del volume e del flusso ematico cerebrale [ 3 ] , e la caratterizzazione della neoangiogenesi tumorale , presente per volumi tumorali maggiori di 1 mm3 [ 46 , 9 , 10 ]  . 
le tecniche di imaging dellangiogenesi hanno potenzialmente esteso le applicazioni cliniche della rm ed il loro sviluppo stato spinto dai progressi nelle terapie antitumorali , molte delle quali hanno come bersaglio linibizione del microcircolo . 
mediante i mdc intravascolari si possono misurare sia la permeabilit dellendotelio che il volume ematico intravasale ; questi parametri sono entrambi correlati con langiogenesi : il primo quantificabile gi allinizio della crescita tumorale , il secondo solo quando il microcircolo formato . lo scopo del nostro lavoro di valutare le applicazioni della rm ed in particolare della risonanza magnetica dinamica con mezzo di contrasto ( dynamic contrast enhancement magnetic resonance imaging : dce - mri ) nello studio del microcircolo tumorale attraverso limpiego di modelli animali , valutati pree post - trattamento con farmaci antiangiogenici . materiali e metodi sono stati valutati i risultati della applicazione della tecnica dce - mri in due esperimenti compiuti per testare la validit di due farmaci antiangiogenici : il tamoxifen , un ligando del n . 
the animals were divided into two groups ( treated and controls ) : treated rats ( n = 7 ) received 6 mg / kg of tamoxifen daily , whereas control rats ( n = 7 ) received the propylene glycol vehicle . ten days after implantation , all animals were imaged by mri with b22956 / 1 and 24 h later with gd - dtpa37albumin . in the second experiment , human colon carcinoma cells , ht - 29 , were implanted subcutaneously in the flanks of ten nu / nu rats ( harlan , san pietro , italy ) weighing approximately 25 g . 
the first group ( n = 5 ) received a single dose of su6668 ( sugen inc . , san francisco , ca , usa ) , whereas the second group ( n = 5 ) received the vehicle only . 
 b22956 / 1 ( bracco , milan , italy ) , an experimental proteinbinding gadolinium chelate with a half - life of approximately 2030 min , was injected into the rats tail vein at a dose of 50 mol / kg . 
gd - dtpa37albumin , a macromolecular , t1relaxing , blood - pool contrast agent with a half - life of 3 h , was injected at a dose of 30 mol / kg in both experiments . images were acquired with a biospec scanner ( bruker , karlsruhe , germany ) equipped with a 4.7 - t , 33 - cm bore , horizontal magnet ( oxford ltd . , oxford , uk )  . 
the rats were anaesthetised with halothane and placed prone in a birdcage coil with a 7 - cm diameter ( the diameter was 3.5 cm for the animal experiments )  . 
coronal and transverse t2 - weighted fast spin echo ( fse ) sequences rapid acquisition with relaxation enhancement ( rare ) , te = 70 ms ] were used to locate the tumour and extratumoural tissue . 
 [ 13 ] in which the time dependence of the signal is converted into time dependence of the concentration of contrast medium and analysed by using pharmacokinetic models : the analysis is repeated pixel by pixel to obtain parametric maps [ 6 , 1315 ]  . 
in summary , measurement of the intensity signal in the tissue being examined ( tumoral and healthy ) can provide parameters for permeability ( kps : expresses the ability of macromolecules to extravasate into the interstitial space ) and perfusion ( fpv : expresses the fraction of plasma in millilitres per cubic centimetre of tissue , a measure of the vascular density )  . 
after the mri study , the animals were killed and the tumours excised ; 7 - m sections in planes corresponding to the mri study were then obtained that were stained with the vessel marker cd31 to obtain a mean vessel count . 
 recettore degli estrogeni , comunemente usato nella terapia ormono - dipendente del carcinoma mammario [ 11 ] , ed il su6668 , un inibitore recettoriale del fattore di crescita vasculo - endoteliale ( vascular endothelial growth factor : vegf ) [ 12 ]  . 
nel primo esperimento , cellule di adenocarcinoma mammario ( american type culture collection ) sono state impiantate sottocute nel fianco di 14 ratti ( charles river laboratories , italia ; peso tra i 76 g ed i 150 g )  . 
gli animali sono stati suddivisi in due gruppi ( trattati e controllo ) : ai ratti trattati ( n = 7 ) stata giornalmente somministrata una dose di tamoxifen di 6 mg / kg , mentre ai ratti del gruppo di controllo ( n = 7 ) stato somministrato solo il veicolo propilene - glicole . 
tutti gli animali sono stati esaminati mediante rm 10 giorni dopo limpianto usando come mezzo di contrasto ( mdc ) il b22956 / 1 e 24 ore dopo il gd - dtpa37albumina . nel secondo esperimento , cellule di colon carcinoma ht29 umano sono state impiantate sottocute nel fianco di 10 topi nu / nu ( harlan , san pietro , italia ) , di circa 25 g . 
i primi ( n = 5 ) hanno ricevuto una singola dose di su6668 ( sugen inc , san francisco , ca , usa ) ; i secondi hanno ricevuto solo il veicolo ( n = 5 )  . tutti gli animali sono stati monitorati tramite rm prima e 24 h dopo trattamento , mediante gd - dtpa37albumina . il b22956 / 1 ( bracco , milano , italia ) un chelato del gadolinio ancora in fase sperimentale , ligando per le proteine , a emivita plasmatica di circa 2030 minuti , ed stato somministrato nella vena della coda dei ratti in dose di 50 mol / kg . 
il gd - dtpa37albumina un mdc macromolecolare t1 rilassante intravascolare ( blood pool ) con emivita di circa 3 ore , iniettato in dose di 30 mol / kg in entrambi gli esperimenti . le immagini sono state acquisite mediante uno scanner biospec ( bruker , karlsruhe , germania ) equipaggiato con un magnete orizzontale operante a 4 , 7 t con unapertura da 33 cm ( oxford ltd , oxford , gran bretagna )  . 
gli animali sono stati anestetizzati mediante alotano e posti in posizione prona in una bobina di tipo bird - cage di 7 cm di diametro ( 3 , 5 cm di diametro per gli esperimenti fatti sul topo )  . 
sono state acquisite sequenze coronali e trasversali fast spin echo t2 - weighted ( rare , teeff = 70 ms ) per la localizzazione del tumore e dei tessuti extratumorali . 
successivamente stata acquisita una serie dinamica di immagini 3d spoiled - gradient echo ( spgr ) con i seguenti parametri : tr / te = 50 / 3 , 5 ms , flip angle = 90 , matrice 25612816 , fov 842 , 4 cm3 ( corrispondente a 0 , 310 , 31 mm2 e 1 , 5 mm di spessore di fetta ) , nex = 1 ; tempo di acquisizione per immagine 3d = 104 s ; dopo somministrazione di mdc sono state acquisite scansioni dinamiche di 22 immagini con 30 s di intervallo per un totale di circa 50 minuti a esame . 
 [ 13 ] , in cui la dipendenza temporale del segnale viene trasformata nella dipendenza temporale della concentrazione del mdc e analizzata mediante modelli farmacocinetici : lanalisi viene ripetuta pixel per pixel , ricavandone delle mappe parametriche [ 6 , 1315 ]  . 
in particular , this was evident at early time points after the injection , whereas the difference tended to decrease at later time points because of the persistence in the inner area of extravasated contrast material that is not drained . 
analysis of the r1tumour / r1blood ratio ( ratio between the variation in longitudinal relaxation rate in the tumour and blood ) in a region of interest ( roi ) selected on the rim of the tumour in the group treated with tamoxifen and in the control group enabled us to visually monitor the effect of therapy [ 11 ]  . 
without going into detail on the experimental theory , we were able to demonstrate that for a blood - pool contrast medium , r1tumour / r1blood is expressed by a linear relationship as a function of time ; the intercept of this line represents plasma volume ( fpv ) , and the slope represents permeability ( kps ) [ 15 ]  . 
dopo lesame rm gli animali sono stati sacrificati , il tumore resecato , e ne sono state ottenute sezioni da 7 m secondo piani corrispondenti alle immagini rm , marcate poi con cd31 , marcatore vasale , per ottenere la conta media dei vasi . 
lanalisi vasale delle sezioni stata ottenuta tramite software image proplus 4.0. risultati nel primo esperimento , in cui abbiamo studiato leffetto del tamoxifen sul modello di tumore mammario , abbiamo ottenuto una mappa dellenhancement della regione tumorale in una scala di falsi colori , sovrapposta allimmagine anatomica in scala di grigio . 
in particolare ci evidente a tempi brevi dopo liniezione , mentre la differenziazione tende a diminuire tardivamente a causa della permanenza nella zona interna del mezzo extravasato che non viene drenato . 
2a - f enhancement on an experimental model of colon carcinoma implanted in rats with the administration of gd - dtpa37albumin : early ( 3.3 min ) ( a ) and late ( 46.4 min ) ( b ) enhancement before treatment and posttreatment ( 24 h ) , with the same timing , after su6668 injection ( d , e )  . 
2a - f intensit di segnale ottenuto in un modello sperimentale di colon carcinoma umano impiantato su topi nudi mediante gd - dtpa37albumina : prima della somministrazione di una dose di su6668 in condizioni di enhancement precoce ( a ) e tardivo ( b ) rispettivamente 3 , 3 min e 46 , 4 min dopo liniezione di mdc , e post - trattamento 24 ore dopo la somministrazione del farmaco ( d , e ) con la stessa tempistica . 
we therefore observed that , in this specific histological tumour type , mammary adenocarcinoma , only the dce - mri technique that employs b22956 / 1 was able to quantify changes in vascular permeability , whereas the study performed with gd - dtpaalbumin was unable to reveal any effect . 
even in this tumour model , the increase in signal intensity occurred predominantly in the tumour periphery ; comparison of imaging and histological data ( not shown ) clearly shows that the enhancing outer portion corresponds to an area of highly vascular viable tissue , whereas the inner portion corresponds to predominantly necrotic tissue . 
comparison of the images of the same tumour obtained before and 24 h after administration of a single dose of su6668 revealed a substantial decrease in enhancement both in the viable area and , to a lesser extent , in the inner portion . 
senza entrare nel dettaglio della teoria sperimentale stato possibile dimostrare che , per un mdc blood pool il r1tumore / r1sangue espresso da una relazione lineare in funzione del tempo ; lintercetta di questa linea rappresenta il volume plasmatico ( fpv ) , e la pendenza rappresenta la permeabilit ( kps ) [ 15 ]  . 
quantitativamente infatti la permeabilit vascolare media misurata mediante il b22956 / 1 negli animali trattati ( 0 , 00433170 , 0040418 ml min - 1 ml - 1 ) significativamente minore che nei controlli ( 0 , 00904600 , 0043680 ml min - 1 ml - 1 ) , mentre nessuna differenza statisticamente significativa apprezzabile con gddtpaalbumina ( 13 , 1413 , 94 ml min - 1 ml - 1 nei trattati e 18 , 0711 , 92 ml min - 1 ml - 1 nei controlli )  . 
abbiamo quindi osservato che , in questo particolare istotipo tumorale , ladenocarcinoma mammario , solo la tecnica di dce - mri mediante b22956 / 1 in grado di quantificare le modificazioni di pern . 
3 r1tumore in una roi periferica di colon carcinoma , come quello mostrato in figura 2 in condizioni pre - trattamento ( ) e 24 ore dopo la somministrazione di su6668 ( ( cid : 2 ) )  . 
si noti leffetto del farmaco sulla pendenza della curva ( permeabilit vascolare ) e sullintercetta ( volume ematico )  . lanalisi quantitativa compiuta sullintero gruppo sperimentale ha mostrato una riduzione rispettivamente del 51% e del 59% su questi due parametri . single dose of su6668 , which allows visual monitoring of the effect of treatment . 
the histological sections shown in figure 2c , f belong to a treated animal and a control , respectively , and show that the number of vessels in the treated animal is far smaller than in the control . 
in particular , tumour microcirculation is characterised by some structural vessel abnormalities , such as sudden changes in calibre , endothelial fenestrations and interruptions in the basal membrane , which are reflected in functional alterations such as increased transcapillary permeability , irregular flow and arteriovenous shunting [ 5 , 7 ]  . 
tumour size has long been used as a parameabilit vascolare , mentre lesame eseguito con gddtpaalbumina non in grado di evidenziare alcun effetto . nel secondo modello sperimentale , in cui sono state utilizzate cellule di colon carcinoma umano , lenhancement ottenuto presentato in figura 2 . 
anche in questo modello tumorale laumento dellintensit di segnale avviene principalmente nella zona periferica del tumore ; attraverso il confronto tra le immagini e i dati istologici ( non mostrati ) , appare chiaro che la parte esterna che subisce enhancement corrisponde ad una zona di tessuto vitale altamente vascolarizzato , mentre la parte pi interna corrisponde a tessuto principalmente necrotico . 
dopo somministrazione della molecola su6668 , confrontando le immagini dello stesso tumore prima e 24 h dopo la somministrazione di una singola dose di farmaco , abbiamo riscontrato una sostanziale diminuzione dellenhancement sia nella zona vitale che , in misura minore , nella zona interna . i grafici mostrati in figura 3 riportano landamento di r1tumore ( che equivalente a r1tumore / r1sangue essendo r1sangue approssimativamente costante per gd - dtpaalbumina ) in una roi selezionata sul bordo del tumore rispettivamente prima e 24 ore dopo la somministrazione di una singola dose di su6668 , permettendo di monitorare visivamente leffetto della terapia . 
come si vede dalla figura , la somministrazione di su6668 ha un effetto sia sulla permeabilit ( pendenza ) che sul volume ematico ( intercetta ) : i valori medi di perfusione e permeabilit per tutti gli animali studiati nel modello sperimentale di colon carcinoma , misurati nella parte periferica del tumore , dopo somministrazione di farmaco antiangiogenico sono diminuiti del 51% ( da 5 , 21 , 1 a 2 , 50 , 8 ml / 100 ml di tessuto ) e del 59% ( da 0 , 001655 , 1 a 0 , 00674 , 8 ml min - 1 ml - 1 di tessuto ) rispettivamente . 
in particolare , il microcircolo tumorale caratterizzato da alcune anomalie strutturali dei vasi , quali variazioni improvvise di calibro , fenestrature dellendotelio ed interruzioni della membrana basale , che si riflettono in alterazioni funzionali come aumento della permeabilit transcapillare , irregolarit di flusso e formazione di shunt artero - venosi [ 5 , 7 ]  . 
le dimensioni tumorali sono state per lungo tempo utilizzate come parametro di valutazione della risposta farmacologica : esistono tuttavia dei limiti a questo criterio nella valutazione della risposta a farmaci antiangiogenici , i quali inibiscono la vascolarizzazione prima che ne conseguano effetti sulle dimensioni tumorali [ 7 , 8 ]  . 
4 rappresentazione anatomica del microcircolo . arteriola / arteriole shunt av / arterovenous shunt venula / venule capillari / capillaries metarteriola / metarteriole venula / venule blood flow meter for evaluating response to treatment : this criterion is , however , limited for the evaluation of response to antiangiogenic drugs , which inhibit vascularity before affecting tumour size [ 7 , 8 ]  . 
numerous studies point to perfusion as the most important quantifiable parameter correlated to tumour growth , as intratumoural microvessel density , which is directly proportional to perfusion , is related to indices of tumour aggressiveness , such as high presenting stage and / or shorter patient survival [ 13 , 14 ]  . dce - mri is based on postprocessing methods that employ specific algorithms for deriving physiodynamic parameters that are useful in assessing tumour microvascularity in vivo and monitoring antiangiogenic therapy . 
mris of an organ or mass are acquired before and at various time intervals after the administration of contrast material : this makes it possible to describe the signal - intensity changes due to perfusion by means of a time - intensity curve . 
dce - mri techniques have been used in experimental studies to monitor the microvascular characteristics of tumours in vivo and to analyse the therapeutic effect of drugs [ 5 , 7 , 8 , 15 ]  . 
preclinical studies have shown that dce - mri , though not yet commonly used as a diagnostic technique , is the most accurate method for identifying microcirculation characteristics , in particular when associated with intravascular contrast material [ 510 , 1418 ]  . 
because less than 5% of blood - pool agents leaks out of tumour vessels in a single pass , these agents can be used to measure both endothelial permeability and intravascular considerazioni anatomo - fisiologiche . 
in base alle esperienze di molti autori il parametro quantificabile pi importante correlato allo sviluppo tumorale la perfusione ; infatti la densit microvasale intratumorale , direttamente proporzionale alla perfusione , correlata ad indici di aggressivit come lelevata stadiazione alla presentazione e / o la diminuita sopravvivenza del paziente [ 13 , 14 ]  . 
la dce - mri una tecnica basata su metodi di post - processing che si avvalgono di specifici algoritmi per ricavare parametri fisiodinamici , utili nella valutazione in vivo della microvascolarizzazione tumorale ed in particolare nel monitorare la terapia antiangiogenica . 
le immagini rm di un organo o di una massa sono acquisite prima e a vari intervalli di tempo dopo la somministrazione di mdc : in questo modo possibile descrivere il mutamento nellintensit di segnale dovuto alla perfusione attraverso una curva tempo - intensit . 
le tecniche di dcemri sono state utilizzate , in studi sperimentali , per monitorare in vivo le caratteristiche microvascolari dei tumori , e nella caratterizzazione delleffetto terapeutico di farmaci [ 5 , 7 , 8 , 15 ]  . 
studi pre - clinici hanno dimostrato che la dce - mri , anche se non ancora di comune attivit diagnostica , la metodica pi accurata nella identificazione delle caratteristiche del microcircolo , in particolare mediante mdc intravascolare [ 510 , 1418 ]  . 
i mdc intravascolari in un singolo passaggio fuoriescono dai vasi tumorali in misura < 5% , perci con essi si possono misurare sia la permeabilit dellendotelio che il volume ematico intravasale , sfruttando la maggiore permanenza allinterno dei vasi e la lenta diffusione nellinterstizio rispetto ai mdc convenzionali . 
the parameters that can be obtained with this technique , permeability and intravascular blood volume , are both correlated with angiogenesis : the first can be quantified as of the earliest stages of tumour development , the second only when the microcirculation has formed . 
in all our experiments , we were able to distinguish at least two areas : a peripheral area that is viable and highly vascular , and an internal area that is less vascular and predominantly necrotic . 
the parametric distributions in fact have bimodal characteristics , reflecting segmentation of the tumour into a core and a peripheral area , for which a high capillary density correlates with histological malignancy grade . 
other authors have calculated the two neoangiogenesis parameters ( fpv and kps ) on human tumour models implanted in rats , with the aim of obtaining quantitative anatomical maps and correlating them with immunocytochemical and ultrastructural data [ 6 , 14 , 15 ]  . 
a further remark regards the importance of the distribution analysis of the parameters kps and fpv for evaluating the presence of necrosis is necessary : conventional imaging modalities are limited in quantifying necrosis in the tumour mass , whereas the quantitative approach used in recent studies can be exploited to establish the effects of physical - chemical treatments on the tumour [ 6 , 15 , 17 ]  . 
microcirculation heterogeneity was demonstrated by means of tumour segmentation in our experiments as well , which identified distinct areas of different angiogenesis on the basis of dynamic parameters correlated to the microvascular structure and function . 
it has been demonstrated that with macromolecular contrast agents this ratio follows a linear trend over time ; the intercept and the slope are proportional to the blood volume and vascular permeability , respectively , of the region being studied [ 11 , 19 , 20 ]  . in our experience dce - mri also proved very effective for monitoring the therapeutic effect after antiangiogenic treatment . 
in particular , in the experimental animal model of colon carcinoma cells , results could be seen as early as 24 h after administration , when no change in tumour volume had yet occurred . 
it has been observed that newly formed tumour microcirculation is impermeable to macromolecules , and reduction in permeability is an excellent surrogate marker of the biological action of drugs [ 21 ]  . 
finally , in the experiment with tamoxifen , we verified that the contrast agent b22956 / 1 , thanks to its binding with plasma proteins and therefore intermediate behaviour between a low - molecularweight agent and a pure macromolecular one , seems to have the right size and extravasation capability to be suitable for testing the effects of tamoxifen on the microcirculation . tenibili con questa tecnica , permeabilit e volume ematico intravasale , sono entrambi correlati con langiogenesi : il primo quantificabile gi allinizio della crescita tumorale , il secondo solo quando il microcircolo formato . il nostro lavoro dimostra come le tecniche dce - mri permettano di individuare allinterno della massa tumorale aree a diversa perfusione : in tutti gli esperimenti abbiamo potuto distinguere almeno due aree , una periferica , vitale e altamente vascolarizzata , ed una interna meno vascolarizzata e principalmente necrotica . 
le distribuzioni parametriche infatti presentano caratteristiche bimodali , riflettendo la segmentazione del tumore in una regione nucleare ed in una regione periferica , per la quale unalta densit capillare correla al grado istologico di malignit . 
altri autori in letteratura hanno calcolato i due parametri espressione di neoangiogenesi ( fpv e kps ) su modelli tumorali umani impiantati nel ratto , al fine di ottenere mappe quantitativo - anatomiche e correlarle con le informazioni immunocitochimiche ed ultrastrutturali [ 6 , 14 , 15 ]  . 
una ulteriore osservazione va compiuta sullimportanza della analisi distributiva dei parametri kps e fpv per valutare la presenza di necrosi : le metodiche radiologiche convenzionali riescono a fatica ad quantificare la necrosi nella massa neoplastica , mentre lapproccio quantitativo , utilizzato nei lavori recenti , pu essere sfruttato per stabilire gli effetti dei trattamenti fisico - chimici sulla massa tumorale [ 6 , 15 , 17 ]  . 
leterogeneit del microcircolo viene infatti dimostrata attraverso la segmentazione tumorale anche nei nostri esperimenti , i quali individuano aree distinte a diversa angiogenesi sulla base dei parametri dinamici correlati alla struttura ed alla funzione microcircolatoria . appare inoltre interessante confrontare landamento con il tempo del rapporto r1tumore / r1sangue , in particolare nella zona vitale e periferica . 
stato dimostrato che questo rapporto , con i mdc macromolecolari , ha un andamento lineare con il tempo ; lintercetta e la pendenza sono proporzionali rispettivamente al volume ematico ed alla permeabilit vascolare della regione esaminata [ 11 , 19 , 20 ]  . la dce - mri , nella nostra esperienza , si dimostrata molto efficace anche per monitorare leffetto terapeutico post - trattamento antiangiogenico . 
in particolare , nel modello sperimentale animale di cellule di colon carcinoma , possibile ottenere un riscontro gi dopo 24 ore , quando non si ha ancora nessuna modificazione del volume tumorale . 
stato infatti osservato che il microcircolo tumorale neoformato iperpermeabile alle macromolecole , e la riduzione della permeabilit un eccellente marker surrogato di attivit biologica dei farmaci [ 21 ]  . 
in ultimo , nellesperimento con il tamoxifen , abbiamo verificato come il mdc b22956 / 1 , grazie al legame con le proteine plasmatiche , e quindi con un comportamento intermedio tra un mezzo di contrasto a basso peso molecolare ed un macromolecolare puro , sembra avere la dimensione giusta e la capacit di extravasare , adatta a testare gli effetti di questo farmaco antiangiogenico sul microcircolo . 
 cisivo nel monitoraggio della terapia antitumorale [ 2225 ]  . conclusions dce - mri dedicated to the study of the microcirculation enables a noninvasive evaluation of tumour size , characteristics and vascularity . 
the technique may help identify the passage of a tumour from a quiescent to an aggressive phenotype early on and therefore enable initiation of adequate oncological treatment and quantification of changes in the microcirculation during treatment . 
mri and the development of quantitative mri methods provide a new concept of imaging , one in which emphasis is placed not only on morphology but also on biological properties of tissues to allow assessment of response to anticancer treatments . 
questo pu servire ad individuare precocemente il passaggio di un tumore da un fenotipo quiescente ad uno aggressivo , e quindi ad impostare un trattamento oncologico adeguato , quantificando le variazioni del microcircolo durante il trattamento . 
zuiani1 1institute of radiology , udine university , via colugna 50 , i - 33100 udine , italy 2institute of medical statistics , department of medical and morphological research , udine university , p.le kolbe 3 , i - 33100 udine , italy 3liver transplantation unit . 
this study was designed to establish whether the measurement of apparent diffusion coefficients ( adcs ) is clinically accurate in diagnosing liver fibrosis in a selected series of cirrhotic patients . 
twenty - eight cirrhotic patients ( mean age 58.1 years ) with histologically proven liver fibrosis and 29 healthy controls ( mean age 43.8 yeas ) underwent liver diffusion - weighted magnetic resonance ( mr ) using a 1.5 - tesla ( t ) magnet equipped with a phased - array coil . 
diffusion studies with parallel imaging [ generalized autocalibrating partially parallel acquisitions ( grappa ) ] were performed within a single breath - hold using a single - shot spin - echo echo - planar sequence ( te 74 ms ) using four b values : b = 0 , 150 , 250 and 400 s / mm2 . 
diffusion - weighted mri is an accurate tool in evaluating advanced liver fibrosis if an optimised single - shot spinecho echo - planar sequence with maximum intermediate b value is used . 
ventotto pazienti cirrotici ( et media 58 , 1 anni ) con fibrosi epatica comprovata allistologia e 29 controlli sani ( et media 43 , 8 anni ) sono stati sottoposti a rm pesata in diffusione utilizzando un magnete da 1 , 5 t equipaggiato con una bobina di superficie phased - array . 
lo studio in diffusione con parallel imaging ( grappa ) stato effettuato in singola apnea mediante una sequenza single - shot spin echo echo - planare ( te 74 ms ) , applicando quattro b - values : b = 0 , 150 , 250 and 400 s / mm2 . 
ladc medio risultato significativamente inferiore nei fegati cirrotici rispetto a quelli dei controlli ( 1 , 110 , 16 vs 1 , 540 , 1210 - 3 mm2 / s ) ( p < 0 , 0001 )  . 
lanalisi receiving operating characteristic ( roc ) ha mostrato unarea sotto la curva ( auc ) di 0 , 96 ( ci 95% : [ 0 , 87 ; 0 , 94 ] ) , dimostrando pi elevate sensibilit e specificit ( rispettivamente , 92 , 9% and 100% ) in corrispondenza di un cut - off adc di 1 , 3110 - 3 mm2 / s . 
limaging rm pesato in diffusione ( diffusionweighted mri ) uno strumento accurato nella valutazione della fibrosi epatica avanzata utilizzando una sequenza single - shot spin echo echo - planar ottimizzata con un massimo b - value di valore intermedio . 
a major focus of current clinical hepatology is the search for a technique providing noninvasive diagnosis and quantification of liver fibrosis [ 13 ]  . fibrosis complicates chronic liver diseases and progresses through different histological stages to final cirrhosis . 
noninvasive alternatives to biopsy such as transient elastography [ 15 ] , or biochemical scores such as the aspartate transaminase to platelets ratio index ( apri ) or fibrotest [ 16 , 17 ] , have been proposed , but their clinical role has yet to be established . 
several reports show that mean adcs measured by dw - mri are significantly lower in cirrhotic livers compared with noncirrhotic livers , as expected if the fibrotic distortion of lobular architecture restricts water molecule motion [ 612 , 1820 ]  . 
nevertheless , most of these studies were not targeted to selected series of patients , and the technical parameters employed in the different experiences varied widely in imaging hardware and sequence parameters , leading to discrepancies in mean adc values . 
the purpose of this study was to investigate the overall accuracy of a dw technique optimised to provide more reproducible results in diagnosing liver fibrosis in cirrhotic patients . materials and methods patients from march 2005 to april 2006 , 57 subjects were recruited for this cross - sectional study . 
twenty - eight patients were affected by liver cirrhosis ( 21 male and 7 female , age range 4174 years , mean 58.1 ) , with child - pugh class a in 24 cases , class b in 3 cases and class c in 1 case . 
al contrario , solo pochi lavori sono disponibili dedicati alle epatopatie diffuse in casistiche selezionate [ 1012 ]  . in ogni caso , uno dei maggiori argomenti di studio della epatologia corrente la ricerca di un mezzo di diagnosi e di quantificazione non invasiva della fibrosi epatica [ 13 ]  . 
la fibrosi complica le epatopatie croniche e progredisce , attraverso differenti stadi istologici , verso una cirrosi finale . lentit della fibrosi determinata , attualmente , attraverso la biopsia percutanea , associata con complicanze maggiori nello 0 , 3% dei pazienti e con una mortalit dello 0 , 018% [ 14 ]  . 
sono state proposte alternative non invasive alla biopsia , come la transient elastography [ 15 ] o scores biochimici quali lapri index o il fibrotest [ 16 , 17 ] , ma il loro ruolo clinico deve essere ancora stabilito . 
nondimeno , la maggior parte di questi studi non mirata su casistiche selezionate ed i parametri tecnici impiegati nelle varie esperienze sono tanto differenti , in termini di hardware e di parametri di sequenza , da determinare discrepanze nei valori degli adc medi . per quanto a nostra conoscenza , nessun autore , in precedenza , ha investigato laccuratezza della misurazione delladc nella diagnosi di fibrosi epatica avanzata . 
twentynine subjects were healthy blood - donor volunteers ( 21 men and 8 women , age range 2052 years , mean 43.8 ) , without previous history of significant systemic or liver diseases and / or known liver focal lesions . 
body mass index ( bmi ) was in the range of 1924 kg / m2 ( mean 20.5 ) , and laboratory indexes [ alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , bilirubin , glutamyl transferase ( gt ) , alkaline phosphatase ( alp ) , cholesterol level and hcv and hbv markers ] were within normal limits at the time of the last blood donation . 
subjects not fulfilling the above criteria and with concomitant alcohol and / or drug use were excluded from the study . magnetic resonance imaging mr examinations were performed using a 1.5 - tesla ( t ) system ( magnetom avanto ; siemens , erlangen , germany ) equipped with high - performance gradients ( maximum amplitude 45 mt / m )  . 
a six - element phased - array surface coil was used in conjunction with the built - in spine - array coil to improve signal reception ( total imaging matrix , siemens )  . a preliminary routine , unenhanced mr protocol was performed to select the anatomical region to examine and assess the presence of focal and / or diffuse liver signal alterations . the protocol included a true fast imaging with steady - state precession ( true - fisp ) sequence intermediate - weighted ( tr 3.69 ms , te 1.85 ms , flip angle 43 , matrix 220256 , fov 350350 mm , slices 20 , thickness 6.5 mm , intersection gap 30% , 1 average , acquisition time 11 s ) in the coronal plane followed by a multi - breath - hold , t1 - weighted , spoiled gradient echo ( spgre ) sequence , acquired in phase ( te 5.04 ms ) and out of phase ( te 2.38 s ) ( tr 100 ms , flip angle 70 , matrix 180256 , fov 350256 mm , slices 27 , thickness 6 mm , intersection gap 20% , acquisition time 40 s ) and a multibreath - hold t2 - weighted turbo spin - echo short - time inversion recovery ( tse - stir ) sequence ( tr 3770 ms , te 134 ms , ti 140 ms , matrix 154256 , fov 360270 mm , slices 27 , thickness 6 mm , intersection gap 20% , acquisition time 68 s ) , both in the transverse plane . 
a dynamic enhanced study using 0.1 ml / kg of gadobenate dimeglumine ( multihance , bracco , italy ) was subsequently performed in cirrhotic patients to exclude hypervascular , malignant focal lesions . 
era disponibile una precedente dimostrazione istologica di fibrosi epatica , documentante uno score ishak 6 , con massimo intervallo fra biopsia epatica ed mri di 3 anni ( media 1 , 3 anni )  . 
ventinove soggetti erano volontari sani , donatori di sangue ( 21 maschi ed 8 femmine , range di et 2052 anni , media 43 , 8 ) , senza storia di precedenti , significative malattie sistemiche ed epatiche e / o di lesioni epatiche focali note . 
il body mass index era compreso nellintervallo di 1924 kg / m2 ( media 20 , 5 ) , mentre gli indici di laboratorio ( alt , ast , bilirubina , gt , fosfatasi alcalina , colesterolemia , markers hcv ed hbv ) risultavano nel range di normalit al tempo dellultima donazione di sangue . 
i soggetti non rispondenti alle caratteristiche citate e con concomitante assunzione di alcool e / o farmaci sono stati esclusi dallo studio . imaging rm gli esami rm sono stati effettuati utilizzando un sistema a 1 , 5 t ( magnetom avanto , siemens , erlagen , germania ) , equipaggiato con gradienti altamente performanti ( massima amplitude di 45 mt / m )  . 
stata utilizzata una bobina phasedarray di superficie a 6 elementi , lavorante in congiunzione con la bobina spine implementata nel lettino porta - paziente , al fine di incrementare la ricezione del segnale ( total imaging matrix , siemens )  . stato effettuato uno studio preliminare di routine , senza mdc , allo scopo di selezionare la regione anatomica da esaminare e di evidenziare la presenza di alterazioni del segnale focali e / o diffuse a livello epatico . 
adc derives from linear regression analysis of the signal intensity measured at each gradient application following the equation : adc = ln s / s0 / ( b0 - b ) where b is the gradient factor , s is the signal intensity after application of the diffusion gradient , and s0 is the signal intensity at b = 0 s / mm2 . 
in this equation , is the gyromagnetic ratio , g and are the strength and the duration of the diffusion gradient pulse , respectively , and is the separation between the diffusion gradient pulses . 
further technical parameters were : tr ( cid : 2 ) , te 74 ms , epi factor 102 , matrix 192154 , fov 350230 mm , slices 15 , thickness 10 mm , interslice gap 38% , average 1 , bandwidth 1 , 532 hz / pixel , acquisition time 16 s . 
the sequence was obtained within a single breath in the transverse plane . phantom study a preliminary phantom study was performed to validate our system by applying the dw sequence with b = 0 , 150 , 250 , 400 , 600 and 800 s / mm2 to two tubes filled with 500 ml of water and acetone at ambient room temperature ( 21c )  . 
adcs of water and acetone were measured on corresponding adc maps . image analysis to eliminate the influence of liver diseases other than cirrhosis on adc measurement , two radiologists ( mb , cz ) blinded to the diagnosis and who were not readers of the dw images retrospectively reviewed the images . 
subjects with findings of previously unknown suspicious focal liver lesions were excluded from measurement , as were controls with diffuse / focal signal decrease due to steatosis and / or iron deposition on t1 - weighted out - of - phase and t2 - weighted images , respectively . 
steatosis and / or iron deposition was tolerated in cirrhotic patients as coexpression of disease [ 22 ]  . after application of the dw sequence , we obtained a set of images corresponding to each b value applied and an adc map , automatically calculated by a commercially available software ( leonardo syngo , siemens , germany )  . quantitative analysis of the adcs of liver parenchyma was performed by placing a circular region of interest ( roi ) of standard dimensions ( 1 cm2 ) on the adc map in right liver segments to avoid artefacts from abdominal wall and vascular motion . 
the roi was placed far from visible vascular and plicazione di due gradienti di diffusione ( motion - probing ) , prima e dopo limpulso a 180 e dalla acquisizione dei dati con un read - out epi [ 21 ]  . 
i gradienti motion probing erano unidirezionali , applicati lungo la direzione della codifica di strato ( asse z ) , in virt della dimostrata isotropicit del fegato [ 6 ]  . 
il valore delladc deriva da una analisi di regressione lineare dellintensit di segnale misurata ad ogni applicazione del gradiente , secondo la seguente equazione : adc = ln s / s0 / ( b0 - b ) , ove b il fattore di gradiente , s lintensit di segnale dopo lapplicazione del gradiente di diffusione , e s0 lintensit di segnale per b = 0 s / mm2 . 
in questa equazione il rapporto giromagnetico ; g e rappresentano , rispettivamente , la forza e la durata dellimpulso del gradiente di diffusione , e lintervallo fra gli impulsi dei gradienti di diffusione . 
gli ulteriori parametri tecnici erano : tr ( cid : 2 ) ; te 74 ms ; epi factor 102 ; matrice 192154 ; fov 350230 mm ; sezioni 15 ; spessore 10 mm ; gap 38% ; medie 1 ; bandwidth 1532 hz / pixel ; tempo di acquisizione 16 s . stato utilizzato un riempimento del k - spazio sequenziale in modalit partial - fourier 7 / 8 . 
la saturazione selettiva del segnale del grasso stata impiegata sistematicamente per sopprimere gli artefatti da chemical - shift . la sequenza stata acquisita in una sola apnea lungo il piano assiale . studio su fantoccio per validare il nostro sistema stato effettuato uno studio preliminare su fantoccio , applicando , a temperatura ambiente ( 21c ) , una sequenza dw con b = 0 , 150 , 250 , 400 , 600 ed 800 s / mm2 a due contenitori riempiti con 500 ml di acqua ed acetone e sottoponendo i dati ad una analisi di regressione lineare del log naturale dellintensit di segnale versus i b - values applicati . 
era stato stabilito di escludere dalla misurazione i soggetti con rilievo di lesioni focali non note sospette per malignit , cos come i controlli con decremento diffuso / focale del segnale riferibili a steatosi e / o a depositi emosiderinici , rispettivamente nelle immagini t1 - pesate in opposizione di - fase e t2pesate . 
1a , b to measure apparent diffusion coefficients ( adcs ) , three circular regions of interest ( rois ) were placed over areas of homogeneous parenchyma of right liver segments on b = 0 s / mm2 images ( a ) and then transported on the corresponding slices of the adc map ( b ) with a copy - paste operation . 
1a , b al fine di misurare i coefficienti di diffusione apparente ( adcs ) sono state posizionate , sulle immagini ottenute con b = 0 s / mm2 , tre regioni di interesse circolari ( rois ) in zone omogenee di parenchima epatico dei segmenti di destra ( a )  . 
it was decided to measure adcs only on good - quality images and in homogeneous areas of parenchyma not affected by major artefacts due to chemical shift , magnetic susceptibility , abdominal wall or vascular motion . 
 statistical analysis the shapiro - wilk test was used to check for data normality . adcs of cirrhotic patients and controls were compared using a t test or mann - whitney u test , depending on the normality check . 
positive predictive value ( ppv ) and negative predictive value ( npv ) were calculated assuming the prevalence of cirrhosis in the general population of our country ( 1% ) [ 23 ]  . 
statistical analysis was performed using medcalc statistical software , version 8.1.0 ( mariakerke , belgium )  . results phantom study the natural log of the signal intensities of water and acetone showed linear regression vs . 
lanalisi quantitativa degli adcs del parenchima epatico stata effettuata posizionando una regione di interesse ( roi ) circolare , di dimensioni standard ( 1 cm2 ) sulla mappa adc , nei segmenti epatici di destra , per evitare artefatti da motilit della parete addominale e vascolari . 
stato stabilito di misurare gli adcs solo nelle immagini di buona qualit ed in regioni omogenee di parenchima , prive di rilevanti interferenze di artefatti dovuti a chemical - shift , suscettibilit magnetica , motilit addominale o vascolare . analisi statistica la normalit dei dati stata verificata con il test di shapirowilk . 
il valore predittivo positivo ( ppv ) ed il valore predittivo negativo ( npv ) sono stati calcolati assumendo la prevalenza della cirrosi epatica nella popolazione generale del nostro paese ( 1% ) [ 23 ]  . 
2a , b graphs for apparent diffusion coefficients ( adcs ) of water ( a ) and acetone ( b ) show linear regression of the natural log of signal intensity vs . 
2a , b il grafico dei coefficienti di diffusione apparente ( adcs ) di acqua ( a ) ed acetone ( b ) mostra una regressione lineare del log naturale dellintensit di segnale versus i bvalues applicati . 
lanalisi statistica si avvalsa del software medcalc statistical , versione 8.1.0 ( mariakerke , belgio )  . patients and controls no focal liver lesions suspected for malignancy were detected in the 57 subjects . 
3a - d utilizzando una sequenza single shot spin echo echo - planare sono state ottenute immagini progressivamente pesate in diffusione di buona qualit , come evidente comparando la stessa sezione ottenuta con b = 0 s / mm2 ( a ) , b = 150 s / mm2 ( b ) , b = 250 s / mm2 ( c ) e b = 400 s / mm2 ( d )  . 
image quality was good , and in no case the presence of mild artefacts due to magnetic susceptibility or abdominal - wall motion precluded adc measurement , because they occurred in the same location , far from the right liver lobe . 
4 scatterplot of apparent diffusion coefficients ( adcs ) of cirrhotic patients ( child - pugh class a = 24 , b = 3 , c = 1 ; ishak score 6 = 28 ) and healthy volunteers ( n = 29 )  . 
as the reliability of routine laboratory tests , serum markers , quantitative liver function tests and methods such transient elastography in the diagnosis and quantification of liver fibrosis has yet to be established , percutaneous biopsy remains the gold standard [ 13 , 25 ]  . 
la qualit delle immagini dw risultata buona , ed in nessun caso la presenza di modici artefatti dovuti alla suscettibilit magnetica od alla motilit della parete addominale ha precluso la misurazione degli adcs , poich essi sono occorsi nelle medesime sedi , lontane dal lobo epatico destro . 
gli adcs sono risultati significativamente inferiori nei pazienti cirrotici rispetto ai controlli : 1 , 110 , 16 versus 1 , 540 , 12 10 - 3 mm2 / s , rispettivamente ( test t , p < 0 , 0001 ) ( tabelle 1 , 2 )  . 
laccuratezza complessiva stata stimata pari a 96 , 4% ( 55 / 57 ci 95% : [ 91 , 2% ; 99 , 9% ] )  . discussione la fibrosi rappresenta un marker istologico dellepatite cronica ( determinante il cosiddetto staging della malattia ) , che trova nella cirrosi il suo stadio finale , virtualmente irreversibile , in tutti gli score istologici utilizzati nella pratica clinica [ 24 ]  . 
poich laffidabilit nella diagnosi e nella quantificazione della fibrosi epatica dei test di laboratorio routinari , dei markers sierici , dei test quantitativi della funzionalit epatica e di metodi quali la transient elastography rimangono da stabilirsi , la biopsia percutanea rimane il gold standard [ 13 , 25 ]  . 
fatta eccezione per uno studio [ 27 ] nel quale stato utilizzato limaging rm turboflash ( siemens , erlagen , germania ) , tutti gli studi precedenti hanno impiegato una sequenza echo - planare [ 612 , 1820 ] , la maggior parte ricorrendo alla variante single - shot spin echo . 
tuttavia , gli artefatti dovuti al chemical - shift , alla suscettibilit magnetica ed alla motilit fisiologica compromettono ancora la qualit dellimmagine [ 26 ] e rischiano di alterare il posizionamento delle roi . 
for such reasons , dw - mri has been proposed as a noninvasive potential tool for the evaluation of liver fibrosis . the application of this technique to the upper abdomen has become possible with the advent of echo - planar imaging because of faster acquisition times , which reduce the effect of gross physiological motion [ 26 ]  . 
except for one study [ 27 ] in which turbo flash ( siemens , erlangen , germany ) was used , all previous studies were designed with an echo - planar mr sequence [ 612 , 1820 ] , mostly with the single - shot spin - echo variant . 
moreover , reducing acquisition time by means of parallel imaging ensured coverage of the entire liver , with no interference of artefacts on overall image quality and roi positioning . minato gli artefatti da chemical - shila riduzione del tempo di acquisizione grazie allimaging parallelo , inoltre , ha assicurato lacquisizione dellintero volume del fegato senza linterferenza di artefatti sulla qualit complessiva delle immagini e sul posizionamento delle roi . 
 [ 28 ] hanno dimostrato che il parallel imaging non compromette laccuratezza della misurazione delladc e che il suo uso corrisponde , paradossalmente , ad una migliore qualit dellimmagine , nonostante il teorico decremento del rapporto segnale / rumore . 
 [ 29 ] , i quali hanno utilizzato una sequenza finger - pulse - triggered echoplanare , negli studi precedenti le immagini dw sono state acquisite in una singola apnea per ogni b - value adottato [ 612 , 1820 ]  . 
in questa esperienza abbiamo ottenuto tutti i set di immagini corrispondenti ai b - values applicati ( 0 , 150 , 250 e 400 s / mm2 ) in una singola apnea inspiratoria , minimizzando gli effetti di section misregistration . 
 [ 28 ] demonstrated that parallel imaging does not compromise the accuracy of adc measurement and could correspond to a paradoxically improved image quality despite the theoretical decrease in the signal - to - noise ratio ( snr )  . 
 [ 29 ] who used a finger - pulsetriggered echo - planar sequence , all previous studies acquired dw images within a breath - hold for each adopted b value [ 612 , 1820 ]  . 
we obtained all sets of images corresponding to the b values applied ( 0 , 150 , 250 and 400 s / mm2 ) within a single breath - hold , minimising section misregistration . 
although the ideal te of a dw sequence should approximate the t2 relaxation time of the liver parenchyma ( 466 ms ) [ 30 ] to maximise the snr , tes in previous experiences were in the range of 54123 ms [ 612 , 1820 ]  . 
nonostante il te ideale di una sequenza dw debba approssimare il tempo di rilassamento t2 del parenchima epatico ( 466 ms ) [ 30 ] al fine di massimizzare il snr , i tes nelle precedenti esperienze erano compresi in un range di 54123 ms [ 612 , 1820 ]  . 
nel presente studio stato utilizzato un te di 74 ms come compromesso fra lottenimento di immagini di buona qualit acquisite in tempi rapidi ed una misurazione pi affidabile delladc utilizzando b - values pi alti . lo studio in diffusione del fegato poggia sullipotesi che la distorsione dellarchitettura lobulare , dovuta allaccumulo di fibre di collagene strettamente legate e povere di protoni , restringa il moto browniano dellacqua allinterno di un parenchima fibrotico . 
solo due studi si sono incaricati di comparare gli adcs dei pazienti cirrotici con quelli dei controlli , ma utilizzando una analisi retrospettiva [ 12 ] od impiegando una bobina body , responsabile di immagini caratterizzate da un basso snr [ 11 ]  . 
 dw - mri of the liver relies on the hypothesis that architectural lobular distortion due to the tightly bound and proton - poor collagen fibres restricts water brownian motion within liver fibrotic parenchyma . 
the most influencing factor in explaining these discrepancies is the set of adopted b values , with maximum b value varying from 55 to 1 , 200 s / mm2 [ 6 , 7 ]  . 
as a general rule , lower b values correspond to higher mean adcs , assuming an overestimation due to signal contamination from other intravoxel incoherent motions ( ivim ) , che ladc medio risulta significativamente inferiore nel primo gruppo . 
 gli adcs medi variavano ampiamente da 0 , 600 , 19 a 2 , 711 , 6210 - 3 mm2 / s nei pazienti cirrotici e da 0 , 690 , 31 a 3 , 551 , 7510 - 3 mm2 / s nei soggetti sani [ 6 , 20 ] , a causa delle differenze nellhardware rm e nel profilo delle sequenze . il fattore pi influente nello spiegare simili discrepanze il set di b - values adottati , con un massimo b compreso fra 55 e 1200 s / mm2 [ 6 , 7 ]  . 
come regola generale , b - values pi bassi corrispondono ad adcs medi pi elevati , verosimilmente sovrastimati a causa della contaminazione del segnale da parte di altri moti incoerenti intravoxel ( ivim ) , e particolarmente dalla microperfusione sanguigna . 
in the present study , a maximum b = 400 s / mm2 was adopted to prevent signal degradation related to higher b values . the aim of this study was to assess whether adc measurement using an optimised dw - mri technique is an accurate tool for diagnosing advanced liver fibrosis . 
to our knowledge , this is the first study to evaluate the accuracy of dwmri in diagnosing advanced liver fibrosis in a selected series of patients using an optimised protocol . 
come atteso , gli adcs ottenuti erano significativamente inferiori nei fegati cirrotici rispetto a quelli dei controlli : 1 , 110 , 16 versus 1 , 540 , 1210 - 3 mm2 / s , e sovrapponibili a quelli precedentemente riportati usando set di b - values simili [ 8 , 18 ]  . 
per quanto a nostra conoscenza , questo il primo studio ad indagare laccuratezza della rm in diffusione nel diagnosticare la fibrosi epatica avanzata su una casistica selezionata ed utilizzando un protocollo ottimizzato . 
la sensibilit e la specificit sono risultate del 92 , 9% e del 100% , rispettivamente , in corrispondenza di una soglia adc di discriminazione fra fegati severamente fibrotici e fegati sani di 1 , 3110 - 3 mm2 / s . 
in corrispondenza di una soglia di 1 , 3110 - 3 mm2 / s la sensibilit e la specificit sono risultate , rispettivamente , del 92 , 9% e del 100% . spondence with an adc cutoff of 1.3110 - 3 mm2 / s to discriminate between severely fibrotic and healthy livers . 
our results suggest that dw - mri is a promising technique for evaluating fibrosis , but before dw - mri can be considered as a possible substitute for liver biopsy , it is mandatory to determine whether there is a correspondence between adc and histological grading and , if so , whether it has clinical relevance in quantifying liver fibrosis before cirrhosis development . 
nevertheless , latter authors used a very low diffusion weighting , with a maximum b value of 250 s / mm2 . a potential limitation of this study is the relatively low diffusion weighting due to the intermediate maximum b factor ( 400 s / mm2 )  . 
 [ 32 ] suggested using higher b values to approximate the adc to the true diffusion coefficient , because at lower b values , the influence of perfusion significantly contributes to the adcs of abdominal organs . 
the present study does not demonstrate that the influence of the true diffusion coefficient at lower b values is sufficient for determining adc differences between normal and fibrotic livers , because the diagnostic accuracy of dw - mri using higher b values remains undetermined . 
 dw sia una tecnica promettente nel valutare questa condizione , ma per candidarla alla sostituzione della biopsia epatica mandatario determinare se esista una corrispondenza tra adc e staging istologico e se abbia rilevanza clinica nel quantificare la fibrosi epatica prima dello sviluppo di una cirrosi . 
questi ultimi autori , tuttavia , hanno utilizzato una pesatura in diffusione molto bassa , con un massimo b - value di 250 s / mm2 . una limitazione potenziale di questo studio la limitata pesatura in diffusione dovuta al massimo fattore b adottato ( 400 s / mm2 ) , di valore intermedio . 
 [ 32 ] hanno suggerito di utilizzare b - values pi elevati per approssimare ladc al coefficiente di diffusione vero , perch con b - values inferiori linfluenza della perfusione contribuisce significativamente nella determinazione degli adcs degli organi addominali . 
nondimeno , comparando fegati normali e cirrotici questi autori non hanno trovato significative differenze n nei rispettivi adcs ( 0 , 870 , 26 versus 0 , 840 , 2210 - 3 mm2 / s ) , n nel coefficiente di diffusione vero , n nella frazione di perfusione calcolata con una appropriata analisi matematica . 
although the effects of ageing on the human liver has not been clearly determined , in no case was progressive liver fibrosis identified as a marker of liver ageing [ 33 , 34 ] , suggesting that the degree of physiological liver fibrosis ( and , consequently , of the measured adcs ) does not vary significantly with age . 
sebbene gli effetti dellinvecchiamento sul fegato umano non siano stati completamente chiariti , in nessun caso la progressione della fibrosi epatica stata identificata come marker dellinvecchiamento [ 33 , 34 ] , suggerendo che il grado di fibrosi fisiologica ( e , di conseguenza , degli adcs misurati ) non vari significativamente con lavanzare dellet . conclusions conclusioni in conclusion , adc measurement is reproducible and rapid and unaffected by interferences from artefacts related to echo - planar imaging . 
nevertheless , because of the relatively high contribution of perfusion to determining the adc with the set of b values employed in the present study , the influence of the different intravoxel incoherent motions on determining the reported high diagnostic performance needs to be investigated . 
ciononostante , a causa del contributo relativamente elevato della perfusione nel determinare ladc con il set di b - values impiegato nel presente studio , deve essere ulteriormente chiarito il peso dei differenti ivims nella determinazione della riportata performance diagnostica . 
venezia1 1department of radiology , 2departement of oncology , siena university , policlinico santa maria , viale bracci 2 , i - 53100 siena , italy 3department of radiology , university la sapienza , viale regina elena 324 , i - 00161 rome , italy correspondence to : f.s. 
ferrari , tel : + 39 - 057 - 7233119 , fax : + 39 - 05 - 7744496 , e - mail : ferrari@unisi.it received : 17 july 2006 / accepted : 18 october 2006 / published online : 20 april 2007 abstract purpose . 
la tomografia computerizzata ha evidenziato necrosi completa nel 78% dei noduli trattati con la e nel 94% di quelli trattati con rfa ; lintervallo libero da malattia stato 16 , 508 , 1 mesi . 
i pazienti trattati con rfa hanno mostrato sopravvivenza pi lunga rispetto a quelli trattati con la , sebbene questa differenza non sia statisticamente significativa ( p = 0 , 3299 )  . 
la e rfa si sono dimostrati egualmente efficaci . tuttavia , rfa consente di ottenere migliori risultati nei pazienti con noduli pi piccoli e nei pazienti child - pugh a . parole chiave epatocarcinoma termoablazione laser ablazione con radiofrequenza ( rfa ) introduction introduzione hepatocellular carcinoma ( hcc ) is the fifth most common malignancy worldwide , with a 5.4% cancer mortality rate [ 1 , 2 ]  . 
cirrhosis is a precancerous condition with an annual rate of mail carcinoma epatocellulare ( hcc ) la quinta neoplasia maligna pi comune nel mondo , con un tasso di mortalit per cancro del 5 , 4% [ 1 , 2 ]  . 
in italia ha unincidenza di 7 , 5 / 100000 fra i maschi e di 2 , 4 / 100000 per le femmine . lepatite b e lepatite c sono le cause principali che determinano lo sviluppo di un hcc . 
the exact indications for each type of thermal ablation procedure still need to be clearly defined , so that the choice of ablation procedure is currently based on several factors . 
features of the technique , such as the size and shape of the ablation zone it generates , features of the hcc lesion such as size , location and number of nodules , and the availability of image - guided systems are all important factors in decision making [ 6 , 7 ]  . 
however , according to the current guidelines for the management of hcc , radiofrequency ablation ( rfa ) should be considered the standard ablation procedure for early unresectable hcc [ 8 ]  . 
 the aim of this study was to compare laser ablation ( la ) and rfa in treating small hcc and to evaluate the clinical outcomes in terms of long - term survival . 
 materials and methods patients from january 2003 to december 2005 , 81 cirrhotic patients ( 56 men and 25 women ) with a diagnosis of hcc and aged between 51 and 82 years ( average : 69.3 ) were included in this prospective trial . 
the hcc nodules were diagnosed by histological analysis of samples obtained with ultrasound ( us ) - guided biopsy using an 18 - gauge needle ( biomol bm 1820 , hs hospital service spa , italy )  . 
prior to any treatment , patients were informed of the benefits and risks of the treatments being proposed , and all patients signed a written consent for as prescribed by current laws , privacy and confidential treatment were assured for all patients . all patients were selected during follow - up of cirrhosis performed by upper abdominal us that had revealed the hcc nodules . 
histopathologic exams performed on all patients revealed a viral hepatitis origin in 79 patients ( 51 hcv , 17 hbv , 11 hbv + hcv ) and an alcoholic origin in two patients . before treatment , the patients were subdivided into groups using the child - pugh classification system ( 59 in child - pugh class a ; 22 in child - pugh class b )  . 
there were a total of 95 hcc nodules ( mean 1.17 per patient ) , with two nodules in 14 patients and mean diameter of 27.9 mm ( range 1040 mm )  . alpha - fetoprotein ( fp ) measurements before treatment were 474.6 ng / l on average ( range 3.57 , 720 ng / l ) ( table 1 )  . 
 forty - one patients were treated with la and 40 with rfa for a total of 128 sessions ( 66 la and 62 rfa ) ; 44 of 81 ( 54% ) underwent only a single session , whereas the other 37 ( 46% ) underwent two or three sessions , for a mean number of sessions per patient of 1.58 and a mean number of sessions per lesion of 1.34 ( table 2 )  . 
treatment groups were homogeneous with regard to hcc nodule dimensions , clinical / laboratory conditions ( with homogenous distribution of child - pugh class a and b ) , and mean age and male - to - female ratio ( table 1 )  . 
i soggetti con maggiore rischio sono quelli con infezione combinata da hbv e da hcv ; altri fattori di rischio includono let maggiore di 50 anni ed il sesso maschile [ 5 ]  . lablazione termica percutanea un trattamento accettato per i pazienti con piccolo hcc non chirurgicamente asportabile . 
le caratteristiche della tecnica ablativa ( tipo e dimensione della zona di necrosi che essa produce ) , le caratteristiche dei noduli di hcc ( dimensioni , sede e numero , disponibilit dei sistemi immagine - guidati ) , rivestono unimportanza considerevole in questa scelta [ 6 , 7 ]  . 
secondo le linee - guida correnti per il trattamento dellhcc , la termoablazione con radiofrequenza ( rfa ) dovrebbe essere considerata come il trattamento standard per lhcc non resecabile [ 8 ]  . lo scopo di questo lavoro confrontare la termo - ablazione laser ( la ) e la rfa nel trattamento del piccolo hcc e valutarne il risultato clinico in termini di sopravvivenza . materiali e metodi pazienti da gennaio 2003 a dicembre 2005 , 81 pazienti cirrotici ( 56 uomini e 25 donne ) portatori di hcc , di et compresa fra 51 e 82 anni ( media : 69 , 3 ) , sono stati inseriti nel gruppo di studio . 
i noduli di hcc erano stati diagnosticati istologicamente su campioni ottenuti mediante agobiopsia ecoguidata utilizzando un ago da 18 gauge ( biomol bm 1820 , hs hospital service spa , italia )  . 
in accordo con le leggi vigenti , la segretezza ed il trattamento dei dati personali sono stati assicurati a tutti i pazienti . tutti i pazienti del nostro studio sono stati selezionati nellambito del follow - up per cirrosi epatica ottenuto con ecografia delladdome superiore che ha evidenziato i noduli di hcc . 
lesame istopatologico eseguito per tutti i pazienti ha rivelato una natura post - epatitica della cirrosi in 79 pazienti ( 51 hcv , 17 hbv , 11 hbv + hcv ) e una natura alcolica in 2 pazienti . 
prima delle procedure di ablazione percutanea i pazienti sono stati divisi in due sottogruppi sulla base dello stadio della cirrosi epatica secondo child - pugh ( 59 pazienti in stadio child - pugh a e 22 in child - pugh b )  . 
il numero totale di noduli di hcc era 95 ( 1 , 17 in media per paziente ) , con 2 noduli in 14 pazienti ed un diametro medio delle lesioni di 27 , 9 millimetri ( range 1040 mm )  . 
other inclusion criteria were a performance status between 0 and 2 , cardiac and pulmonary function between 0 and 2 [ world health organization ( who ) ] and age ranging from 19 to 82 years . 
i sottogruppi di trattamento erano omogenei sia come di dimensioni dei noduli di hcc che come condizioni clinico / laboratoristiche ( con distribuzione delle classi a e b secondo child - pugh pressoch omogenea ) cos come in termini di et media e di rapporto maschi / femmine ( tabella 1 )  . 
patients with evidence of vascular tumour extension ( portal vein , one of two main portal branches or hepatic vein thrombosis ) , invasion of the main bile duct or extrahepatic metastases on computed tomography ( ct ) during the staging phase were excluded . 
multislice ct evaluation ( light speed 16 ct , ge medical systems , milwaukee , wi , usa ) was performed with three - phase technique ( 30 , 65 and 180 s ) following i.v. 
 pretreatment evaluation each patient underwent blood chemistry testing ( total and fractionated bilirubin , quicks time inr , albumin , platelets , ammonia , fp , ferritin and hepatic markers , if not already available ) , cardiac function and performance status testing . overall assessment of coagulation status was performed within 7 days of the percutaneous procedures , and all patients were asked about possible drug allergies and use of platelet aggregation inhibitors . 
the contrast agent , at a dose of 4 g , was administered as a bolus injection ( 23 s ) at a concentration of 400 mg / ml , and images were acquired at various time intervals : 1015 s , 2030 s and 1 , 2 , 3 , 4 , 5 and 6 min after the injection . 
with these time intervals , arterial , portal , and criteri di inclusione e di esclusione sono stati selezionati i pazienti con un nodulo unico di hcc delle dimensioni massime di 40 millimetri o con multipli noduli ( al massimo 3 ) ciascuno del diametro non superiore a 30 millimetri , non precedentemente trattati . 
gli altri criteri di inclusione sono stati un performance status tra 0 e 2 , una funzionalit cardiaca e polmonare fra 0 e 2 ( who ) e let ( 1982 anni )  . 
il rilievo del tempo di protrombina < 40% ( international normalized ratio , inr > 1 , 99 ) e / o numero di piastrine < 40000 / ml hanno determinato il rinvio delle procedure percutanee fino al ripristino dei valori adeguati in 22 pazienti . 
sono stati esclusi i pazienti che , sulla base di un esame di tomografia computerizzata ( tc ) eseguito durante la fase di stadiazione della malattia , hanno mostrato estensione vascolare del tumore ( vena porta , uno dei due rami portali principali o trombosi delle vene sovraepatiche ) , interessamento della via biliare principale o metastasi extraepatiche . la valutazione tc multislice ( light speed 16 ct , ge mdical systems , milwakee , usa ) stata ottenuta con tecnica trifasica ( 30 , 65 e 180 s ) dopo somministrazione endovenosa di bolo di 150 ml di mezzo di contrasto iodato ( ultravist 370 , schering , berlino , germania )  . 
i parametri principali dellesame erano : tempo di rotazione 0 , 7 secondi , pitch di 0 , 938 : 1 , collimazione 2 , 5 millimetri e velocit di scorrimento del tavolo 18 , 75 millimetri per rotazione dellelica . 
 before treatment , all patients also underwent a multislice ct scan ( light speed 16 ct , ge medical systems ) with the three - phase technique ( 30 , 65 and 180 s ) following an intravenous injection of a 150 - ml bolus of iodinated contrast medium ( ultravist 370 , schering )  . 
catheter inserted in an antecubital vethe time interval between pretreatment ct and the ablative procedure was 12 weeks . methods both la and rfa were performed under general anaesthesia and us guidance . 
depending on the hcc nodule size , up to four fibreoptics were positioned inside the lesion and then 5 w was applied for an exposure time of 6 min to reach 1 , 800 joules per fibre . 
at the end of the session , the fibres were withdrawn with the laser still on in order to induce hyperthermia along the needle retraction path and avoid seeding of neoplastic cells . 
 rfa was performed using a cool - tip radiofrequency system generator ( valleylab , tyco healthcare , bolder , co , usa , distributed by hs hospital service spa , aprilia , lt , italy )  . 
single electrodes with lengths of 15 , 20 and 25 cm and an active segment of 2 or 3 cm were used for hcc nodules 3 cm , whereas multiple cluster ( three - prong ) electrodes with lengths of 15 or 20 cm and an active segment of only 2.5 cm were used for hcc nodules > 3 cthe electrodes were cooled by means of an internal circuit fed by a peristaltic pump . 
 evaluation of treatment efficacy twenty - four to 48 h after treatment , all patients underwent liver us followed by triphasic ct evaluation , as described above , within 15 days . 
based on ct findings , tumour ablation ( treatment efficacy ) was defined as complete when no contrast uptake was seen in the ablation zone during the early arterial phase , or incomplete if the ablation zone appeared smaller than the initial hcc nodule and contrast uptake was seen within its margins . 
la valutazione globale dello stato coagulativo stata eseguita entro sette giorni delle procedure percutanee e tutti i pazienti sono stati interrogati riguardo ad eventuali allergie farmacologiche e ad eventuali assunzioni di farmaci antiaggreganti . 
il mezzo di contrasto , nella dose di 4 g , stato somministrato a bolo ( in 23 s ) in concentrazione di 400 mg / ml e le immagini sono state acquisite a vari intervalli di tempo , a partire da 1015 s , a 2030 s e a 1 , 2 , 3 , 4 , 5 e 6 minuti dopo liniezione . 
utilizzando questi intervalli sono state ottenute le fasi arteriosa , portale e parenchimale tardiva . nellambito della valutazione pre - trattamento percutaneo , tutti i pazienti hanno eseguito un esame tc multislice ( light speed 16 ct , ge medical systems , milwakee , usa ) con tecnica trifasica ( 30 , 65 e 180 s ) dopo somministrazione endovenosa di bolo di 150 ml di mezzo di contrasto iodato ( ultravist 370 , schering , berlino , germania )  . 
i parametri principali dellesame erano : tempo di rotazione 0 , 7 secondi , pitch 0 , 938 : 1 , collimazione 2 , 5 millimetri e velocit di scorrimento del tavolo 18 , 75 millimetri per rotazione dellelica . stata ottenuta soltanto una fase arteriosa ; il mezzo di contrasto stato iniettato a 4 ml / s tramite un accesso venoso da 18 gauge in una vena antecubitale del braccio . 
lintervallo di tempo fra la tc ed il trattamento ablativo variato fra 1 e 2 settimane . metodi sia la la che la rfa sono state effettuate in anestesia generale e sotto guida ecografica . la la stata effettuata usando una fonte di luce al neodymium yttrium - aluminium - garnet , nd - yag ( deka - mela , elen , firenze , italia ) , con una lunghezza donda continua di 1 , 064 tale sorgente stata convogliata con un beamsplitter ottico ( smart 1064 hcc , deka - mela , elen , firenze , italia ) munito di 4 fibre ottiche di quarzo di 300 m , flessibili , a punta piatta , sterili e prive del rivestimento esterno nel loro tratto terminale . 
a seconda della dimensione del nodulo di hcc sono state posizionate fino a 4 fibre nella lesione impiegando 5 w per 6 minuti in modo da erogare 1800 joules per fibra . 
terminato il trattamento , le fibre sono state retratte , con il laser acceso , in modo da indurre ipertermia durante il tragitto di ritorno dellago per evitare un possibile seeding di cellule neoplastiche . la rfa stata realizzata impiegando un generatore cool tip radiofrequency system ( valleylab , boulder , tyco healthcare , colorado , usa )  . 
sono stati utilizzati elettrodi a radiofrequenza closet - tip , tutti rigorosamente monouso , da 17 gauge , raffreddati internamente ( hs hospital service , aprilia , latina , italia ) , di tipo singolo con lunghezze di 15 , f.s. 
comparison of techniques and long - term results tumour ablation , patients underwent additional treatment sessions ( 33 in total : 21 for the la group and 12 for the ra group ) , whereas in the case of complete tumour ablation , patients were scheduled for follow - up . 
at 1 month and every 3 months thereafter , patients underwent alkaline phosphatase , total and fractionated bilirubin , transaminases , gamma glutamyl transpeptidase ( gt ) , carcinoembryonic antigen ( cea ) , albumin , quicks time and inr , complete blood count ( cbc ) and platelets and fp testing . the child - pugh class was reviewed , and upper abdominal ultrasound was performed to assess portal flow . 
 at 3 months and every 6 months thereafter , patients underwent triphasic ct for evaluation of the ablation zone and detection of local tumour progression and / or new hcc lesions . 
in evaluating disease recurrence , we distinguished between local tumour progression ( the presence of neoplastic tissue within the same liver segment as that of a treated hcc nodule ) and new hcc lesion ( the development of nodules in a different liver segment )  . 
in segment iv , neoplastic tissue arising farther than 5 cm from the site of the initial hcc was regarded as a new hcc lesion rather than as local tumour progression . 
 complications 20 , 25 cm e segmento attivo di 2 o 3 cm ( per noduli fino a 3 cm ) ; per i noduli pi grandi di 3 cm stato impiegato un tipo cluster ( elettrodo a tre punte ) con lunghezze di 15 o 20 cm e segmento attivo di 2 , 5 cgli elettrodi sono stati raffreddati per mezzo di un circuito interno alimentato da una pompa peristaltica . 
durante lablazione termica la pompa peristaltica , instillando acqua a 0c , ha permesso di mantenere la punta dellago ad una temperatura di 2025c . ogni seduta di rfa ha richiesto 1012 minuti al massimo della potenza , mantenendo limpedenza del circuito entro i 10 . 
alla fine del trattamento gli elettrodi sono stati estratti innalzando la temperatura per sterilizzare il percorso ed evitare il seeding cellulare . valutazione dellefficacia del trattamento a distanza di 2448 ore dalla conclusione della sessione di trattamento tutti i pazienti sono stati sottoposti ad una ecografia del fegato ed entro 15 giorni ad una tc con tecnica trifasica , come descritto prima . 
sulla base dei risultati tc , lablazione del tumore ( efficacia del trattamento ) stata definita come completa quando nella fase precoce arteriosa non stato osservata alcuna impregnazione di mezzo di contrasto nella zona di ablazione , o incompleta se la zona di ablazione risultata pi piccola rispetto al nodulo iniziale di hcc e ha presentato impregnazione allinterno entro i margini della zona di ablazione . 
nel caso di ablazione incompleta , i pazienti sono stati sottoposti ad ulteriori sessioni ( 33 in tutto , 21 per il gruppo la e 12 per il gruppo rfa ) , mentre nel caso di ablazione completa , i pazienti sono stati inclusi nel protocollo di follow - up . complications were classified according to the society of interventional radiology ( sir ) grading system [ 9 ]  . 
 follow - up statistical analysis all patients were followed up , and all data ( personal , clinical , laboratory and biopsy ) were collected and entered into a database . 
al primo mese e successivamente ogni tre mesi , sono stati dosati la fosfatasi alcalina , la bilirubina totale e frazionata , le transaminasi , le gamma glutamil - traspeptidasi ( gt ) , lantigene carcinoembrionale ( cea ) , lalbumina , il tempo di quick e linr , lemocromo con le piastrine e lfp . 
la classe child - pugh stata rivalutata ed stata effettuata unecografia delladdome superiore per controllare il flusso portaal terzo mese , ed in seguito ogni 6 mesi , stata eseguita una tc con tecnica trifasica per studiare la zona di ablazione , linsorgenza di recidiva locale e / o di nuove lesioni . 
nella valutazione complessiva della ripresa di malattia stata distinta la recidiva locale ( la presenza di tessuto neoplastico nel segmento epatico del nodulo di hcc sottoposto a trattamento ) , dalla nuova lesione di hcc ( linsorgenza di noduli in segmenti epatici diversi da quello del trattamento )  . 
nel iv segmento epatico , la rilevazione di tessuto neoplastico ad una distanza superiore a 5 centimetri dal nodulo iniziale di hcc stata considerata come un nuovo nodulo di hcc piuttosto che una recidiva locale . 
comparison of techniques and long - term results table 3 complete tumour ablation achieved during the first session and incidence of disease recurrence ( local tumour progression and new tumour foci ) showing disease - free interval sample disease recurrence complete tumour ablation no . 
in addition to the first session , scheduled for all of the 95 nodules , 33 additional sessions were performed : 13 of these ( 39.4% ) were required to achieve complete tumour ablation , 15 ( 45.4% ) to treat local tumour progression and 5 ( 15.2% ) to treat new hcc lesions . 
of these , 60% appeared 1224 months after treatment and the remaining 40% within the first 12 months ; no local tumour progression was observed later than 24 months after treatment . 
tutte le altre complicanze sono state considerate come minori . analisi statistica tutti i pazienti sono stati seguiti nel tempo e tutti i dati ( personali , clinici , di laboratorio e bioptici ) sono stati raccolti ed stato creato un database per questo studio . 
le curve di sopravvivenza sono state ottenute mediante il metodo kaplan - meier e le differenze tra di esse sono state analizzate attraverso il log - rank test . lanalisi dei fattori prognostici stata effettuata con il metodo di regressione di cox . 
comparison of techniques and long - term results sessions 1 session additional sessions complete tumour ablation local tumour progression new tumour foci total table 4 additional sessions total la , laser ablation ; rfa , radiofrequency ablation tabella 4 trattamenti aggiuntivi totale la , laser ablation ; rfa , radiofrequency ablation totale trattamenti prima sessione trattamenti aggiuntivi necrosi tumorale recidive locali nuovi noduli totale fig . 
1 incidenza complessiva di ripresa di malattia suddivisa per tipo di trattamento . tected during follow - up were both treated with the same technique as used in the original treatment . 
 during the weeks following the first session , fp levels returned to normal in all patients in whom treatment had been successful ; in cases of local tumour progression or new hcc foci , a new elevation of fp levels occurred . 
 cumulative survival rates were calculated for child - pugh a and b groups and for the la and rfa treatment groups with the kaplan - meier method ( table 5 )  . 
 univariate analysis of survival rates revealed statistically significant differences between child - pugh a and b groups ( p < 0.0001 ) , hcc nodules 2525 mm ( p = 0.0001 ) and patients with one hcc nodule vs . 
ottantadue noduli ( 86% ) hanno richiesto una singola seduta , mentre per gli altri 13 ( 14% ) stato effettuato pi di un trattamento ( tabella 3 )  . 
oltre alla prima sessione , prevista per tutti i 95 noduli , sono state effettuate 33 sessioni supplementari : 13 di queste ( 39 , 4% ) sono state necessarie per realizzare lablazione completa del tumore , 15 ( 45 , 4% ) per trattare la progressione locale del tumore e 5 ( 15 , 2% ) per trattare i nuovi noduli di hcc . 
ventuno sessioni supplementari sono state necessarie per il gruppo della la e 12 per il gruppo della rfa ( tabella 4 )  . la necrosi completa stata raggiunta nel 78% dei noduli trattati con la e nel 94% di quelli trattati con rfa . 
lintervallo libero da malattia risultato 16 , 508 , 1 mesi ( 15 , 457 , 99 per i pazienti trattati con la e 17 , 788 , 52 mesi per quelli sottoposti a rfa ) ( tabella 3 )  . sono stati osservati quindici casi di recidiva locale ( 15 , 8% di tutti i noduli trattati )  . 
il 60% di questi si sono manifestati fra 12 e 24 mesi dopo il trattamento , il restante 40% entro i primi 12 mesi ; nessuna recidiva locale si osservata oltre i 24 mesi . 
il 20% di questi pazienti ha presentato una nuova lesione tra 12 e 24 mesi e l80% tra 24 e 36 mesi ( intervallo libero da malattia 25 , 44 , 33 mesi )  . 
sia le recidive locali che i nuovi noduli individuati durante il follow - up sono stati trattati con la stessa metodica del primo trattamento . nel corso delle settimane successive alla fine del primo trattamento , i livelli sierici di fp sono rientrati nella norma in tutti i pazienti trattati con successo ; nei casi di ripresa di malattia o di comparsa di nuovi noduli di hcc si verificato un nuovo incremento dei valori di fp . 
comparison of techniques and long - term results table 6 log - rank test of patients age , gender , child - pugh class , maximum nodule diameter , number of hepatocellular carcinoma ( hcc ) nodules and type of treatment ( child - pugh class stratifications and max ) variable log - rank test child - pugh : a ( n = 59 ) vs . 
il modello cox indica che la classe child - pugh il fattore di sopravvivenza prognostico pi importante . discussione in uno studio prospettico , la rfa e liniezione percutanea di etanolo ( pei ) sono stati messi a confronto nel trattamento di 86 pazienti con hcc , unico o multifocale , delle dimensioni 30 mm ; lablazione completa stata ottenuta in 47 noduli su 52 totali trattati con rfa ( 90% ) ed in 48 su 60 noduli sottoposti a pei ( 80% ) , con una media rispettivamente di 1 , 2 e 4 , 8 sessioni [ 10 ]  . 
 [ 11 ] hanno effettuato uno studio randomizzato per confrontare lefficacia della rfa e della pei nel trattamento dei noduli di hcc 50 millimetri , in pazienti con al massimo 3 noduli e quadro di cirrosi compensata . 
dopo 2 anni di follow - up i pazienti tratti con rfa hanno mostrato una pi bassa percentuale di recidive locali ed un periodo libero da malattia pi lungo rispetto ai pazienti trattati con pei [ 11 ]  . 
 [ 12 ] hanno valutato lefficacia e la sicurezza della rfa in 88 pazienti per un totale di 101 noduli di hcc con diametro 35 mgli autori hanno osservato un tasso cumulativo di sopravvivenza ad 1 , 3 e 5 anni rispettivamente dell89% , del 62% e del 33% con un periodo libero da malattia medio di 19 mesi . 
inoltre , negli studi concernenti la rfa , i pazienti con noduli in prossimit dellilo epatico o della colecisti sono stati generalmente esclusi , sebbene secondo pacella [ 15 ] in questi casi sarebbe possibile la termoablazione mediante la con fibre sottili . per quanto riguarda il numero di sessioni di trattamento , i nostri risultati appaiono simili a quelli ottenuti da livraghi fig . 
3 cumulative survival curves ( kaplan - meier with rectilinear interpolation ) in relation to diameter of hepatocellular carcinoma ( hcc ) nodule : 25 mm ( n = 28 ) vs . 
 discussion in one prospective study , rfa and percutaneous ethanol injection ( pei ) were compared in 86 patients with single or multiple hcc 30 mcomplete tumour ablation was achieved in 47 out of 52 tumours treated with rfa ( 90% ) and in 48 out f.s. 
 [ 10 ] ( rfa con elettrodi raffreddati internamente ) , che hanno segnalato un valore di circa 1 , 2 sessioni per nodulo , con possibilit di trattare i piccoli noduli di hcc in una singola seduta nella maggior parte dei casi . nella nostra esperienza , la capacit della rfa di indurre ablazione tumorale completa nella prima sessione stata del 94% . 
confrontando questo valore con quello ottenuto mediante la la ( 78% ) , occorre notare che vi era una piccola differenza nel diametro medio dei noduli di hcc ( 28 , 9 millimetri nel gruppo la contro 26 , 7 millimetri nel gruppo rfa )  . 
la capacit di ablazione risulta effettivamente collegata alle dimensioni del tumore e alla semplicit del trattamento ( tempo pi breve e uso di singoli aghi anzich di aghi a grappolo )  . 
utilizzando la rfa con elettrodi raffreddati internamente nel trattamento di 51 noduli di hcc di dimensioni medio - grandi ( 3050 millimetri ) , livraghi et al . [ 16 ] hanno riportato le seguenti percentuali di ablazione : in 36 noduli ( 71% ) stata ottenuta completa ablazione , in 12 noduli ( 24% ) il 90%99% di ablazione e in 3 noduli ( 6% ) la percentuale di ablazione stata soltanto del 50%89% . 
tuttavia , noi consideriamo una percentuale pi bassa o pi alta del 90% di basso valore indicativo per la prognosi del paziente , che in ogni caso deve essere sottoposto ad ulteriori trattamenti . non abbiamo osservato complicanze durante le sessioni di trattamento . 
le complicanze minori ( dolore , febbre , versamento pleurico , ematomi sottocapsulari ) sono state da noi rilevate soltanto raramente ed in ogni caso sono state di importanza secondaria , in accordo con i dati pubblicati ( < 5% ) [ 12 , 17 ]  . il nostro parere , in accordo con le linee guida di barcellona [ 18 ] , di considerare lintero periodo libero da malattia , che nella nostra esperienza risultato essere di 17 , 78 mesi per i pazienti trattati con rfa , in linea con i risultati pubblicati . 
nel nostro studio abbiamo considerato come recidiva locale il riscontro di tessuto neoplastico nello stesso segmento del nodulo trattato , perch non proponibile un cut - off di distanza in mm , ad eccezione del iv segmento . 
un recente lavoro di harrison et al . [ 19 ] sullosservazione di ripresa di malattia mette in evidenza come la recidiva sia un evento frequente in pazienti con hcc non resecabile trattati con rfa . 
le percentuali di sopravvivenza osservate nei pazienti trattati con rfa sono sovrapponibili a quelle di altri studi analoghi [ 11 , 12 ]  . la stratificazione in sottogruppi child - pugh ha mostrato come la malattia epatica di base sia determinante ai fini della sopravvivenza . 
after a 2 - year follow - up , patients treated with rfa had a lower rate of local recurrence and a longer disease - free interval compared with those treated with pei [ 11 ]  . 
evaluated the efficacy and safety of rfa in 88 patients with 101 hcc nodules 35 mthe authors reported a cumulative survival rate of 89% , 62% and 33% at 1 , 3 and 5 years , respectively , and an average disease - free interval of 19 months . 
moreover , studies of rfa have generally excluded patients with lesions near the hepatic hilum or the gallbladder , whereas according to pacella et al . , these patients can be treated by la with thin fibres [ 15 ]  . as regards the number of treatment sessions , our results are similar to those of livraghi et al . 
 [ 10 ] ( who used rfa with internally cooled electrodes ) , who reported approximately 1.2 sessions per lesion , with most small hcc nodules being treated in a single session . 
6 cumulative survival curves ( kaplan - meier with rectilinear interpolation ) in relation to treatment , stratified by child - pugh class : a : radiofrequency ablation ( ra ) ( n = 28 ) vs . 
in our experience , minor complications ( such as pain , fever , pleural effusion , subcapsular haematoma ) were rare and of little significance , in agreement with previous studies ( < 5% ) [ 12 , 17 ]  . 
 in accordance with the barcelona guidelines [ 18 ] , we consider the entire disease - free interval , which was 17.78 months for patients treated with rfa in our study , in line with current data . 
local tumour progression was considered as any neoplastic tissue seen in the same segment as that of a previously treated hcc nodule , as no cutoff distance can be proposed ( with the exception of segment iv )  . 
however , the biological features of this tumour ( that is , an ab initio organ pathology ) make it clear that disease recurrence is not directly related to the procedure itself . 
una zona di ablazione pi grande o uguale in dimensioni al nodulo trattato stata ottenuta in 89 noduli su un totale di 92 ( 97% ) con una media di 1 , 1 sedute di trattamento per nodulo . 
durante il followup , 5 degli 89 noduli , in cui era stata ottenuta ablazione completa ( 6% ) , hanno mostrato una recidiva locale ( periodo libero da malattia medio di 14 , 8 mesi ) ed in 36 pazienti su 74 ( 49% ) sono stati osservati nuovi noduli di hcc ( periodo libero da malattia medio di 17 , 7 mesi )  . 
il tasso cumulativo di sopravvivenza a 1 , 3 e 5 anni stato rispettivamente del 99% , 68% e del 15% , senza significative differenza tra il gruppo child - pugh a ed il gruppo child - pugh b . 
sono state osservate soltanto 3 complicanze minori e nessuna complicanza maggiore . confrontata con la pei , la la presenta un rischio di impianto di cellule neoplastiche ( seeding ) pi basso e spesso riesce ad indurre la necrosi completa in una sola seduta di trattamento [ 20 ]  . 
 [ 21 ] hanno confrontato i risultati a distanza della la , della pei e della embolizzazione arteriosa transcatetere ( tace ) nel trattamento del piccolo hcc in 131 pazienti cirrotici . 
i pazienti sottoposti a la hanno mostrato una sopravvivenza ( statisticamente significativa ) pi lunga di quella dei pazienti trattati con tace e pei , cos che la la si dimostrata come il trattamento pi efficace per la sua ridotta invasivit , il numero limitato di sedute , la necrosi completa in quasi tutti i casi e la migliore sopravvivenza [ 21 ]  . 
an ablation zone larger than or the same size as the lesion was achieved in 89 of 92 nodules ( 97% ) , with an average of 1.1 sessions for each hcc . 
the cumulative survival rates were 99% , 68% and 15% at 1 , 3 and 5 years , respectively , without significant differences between childpugh class a and b patients . 
patients undergoing la had a statistically significant longer survival than those treated with tace and pei , so la proved to be the most effective treatment owing to its minimal invasiveness , reduced number of sessions , complete necrosis in almost all cases and better survival rates [ 21 ]  . 
in termini di tasso di sopravvivenza , la la si dimostrata come tecnica migliore rispetto sia alla pei che alla tace , con risultati sovrapponibili a quelli del trattamento combinato [ 22 ]  . il nostro gruppo di pazienti trattato con la stato sottoposto ad una media di 1 , 46 sessioni / nodulo di hcc , un valore maggiore rispetto a quello del gruppo rfa . 
se consideriamo la ripartizione delle sessioni aggiuntive di trattamento ( tabella 4 ) , possiamo notare che si sono rese necessarie 10 sedute per ottenere la completa ablazione nel gruppo la , mentre 3 sessioni sono state necessarie nel gruppo rfa . 
abbiamo osservato che nei noduli di hcc delle dimensioni di 34 cm stato pi difficile ottenere una completa ablazione in una sola seduta di trattamento utilizzando la la rispetto alla rfa , che ha il vantaggio di disporre degli aghi a grappolo . 
 [ 23 ] hanno rilevato che nei noduli di hcc trattati con la , il follow - up con tc ha mostrato ablazione completa di tutti i noduli < 40 mm , ma non in tutti i noduli > 50 m ferrari et al . 
 [ 22 ] hanno riportato che nel trattamento del grande hcc con la la vi sono differenze significative sia riguardo al tasso di sopravvivenza che nellablazione tumorale completa a favore dei pazienti con noduli di hcc < 50 mm rispetto a quelli con noduli 50 m nella nostra esperienza , il tasso di progressione tumorale locale e dei nuovi noduli di hcc si rivelato rispettivamente pi alto e pi basso in confronto ai dati presenti in letteratura [ 15 ]  . 
comparison of techniques and long - term results table 7 cox model for prognostic survival factors variable regression coefficient exp ( coefficient ) global 2 tabella 7 analisi multivariata secondo il modello di regressione di cox dei fattori prognostici sulla sopravvivenza regressione coefficiente exp ( coefficiente ) 2 globale p value exp , exponential ; hcc hepatocellular carcinoma child - pugh class largest diameter no . 
 our group of patients treated with la underwent an average of 1.46 sessions per hcc nodule , more than with rfa . if we consider the distribution of additional sessions ( table 4 ) , we can see that the la group required ten procedures to achieve complete tumour ablation , whereas the rfa group required three sessions . 
this difference is due to the lower rate of complete tumour ablation ( 78% ) obtained with la . we observed that in hcc nodules measuring 34 cm , it is more difficult to achieve complete ablation in a single la session compared with rfa , which has the advantage of using cluster needles . 
reported that in hcc nodules treated with la , ct follow - up demonstrated complete ablation of all lesions < 40 mm but not in all nodules > 50 mm [ 23 ]  . 
reported that in the treatment of large hcc with la , there are significant differences in terms of both survival and complete tumour ablation , with better results being achieved in patients with hcc nodules < 50 mm compared with those > 50 mm [ 22 ]  . 
 in our study , we had a higher rate of local tumour progression rate and a lower rate of new hcc nodules compared with previous reports [ 15 ]  . 
the survival rate of the la group was 88% , 56% and 23% at 12 , 36 and 60 months : compared with pacellas findings , our patients had a lower survival rate at 12 and 24 months but a higher survival rate at 60 months [ 15 ]  . 
il tasso di sopravvivenza del gruppo di pazienti trattati con la stato dell88% , 56% e 23% rispettivamente a 12 , 36 e 60 mesi ; il confronto con lo studio di pacella [ 15 ] mostra che a 12 e 24 mesi i nostri pazienti hanno un tasso di sopravvivenza pi basso , mentre risultato pi alto a 60 mesi . 
questa differenza potrebbe essere dovuta al fatto che i noduli di hcc del gruppo di studio di pacella erano in media pi piccoli dei nostri ( 24 mm vs 29 mm ) e che leffetto terapeutico si dimostrato nel breve periodo ; probabile che i pazienti vissuti pi a lungo fossero portatori di neoplasie meno aggressive . abbiamo confrontato i risultati a lungo termine della la e della rfa nel trattamento del piccolo hcc . 
i due gruppi erano omogenei senza nessuna sostanziale differenza di et , sproporzione fra maschi e femmine , classe child - pugh , numero dei noduli di hcc e valori sierici di fp . 
il diametro medio dei noduli di hcc trattati con rfa era di 26 , 7 mm , mentre quello dei noduli trattati con la era di 28 , 9 mil tasso di ablazione tumorale completa stato del 78% per la e del 94% per rfa . 
tuttavia necessaria una certa esperienza nellapproccio percutaneo ecoguidato . in accordo con i dati presenti in letteratura , il maggior numero di recidive stato osservato nei primi 2 anni successivi alla termo - ablazione , ma non si sono osservate differenze significative tra i due gruppi di trattamento . 
the mean diameter of the hcc nodules treated with rfa was 26.7 mm , whereas that of the nodules treated with la was 28.9 plications , as both methods are safe . 
these data , however , clearly refer to a small study group , and a larger number of patients and a finer stratification might have revealed significant differences ( though probably only as regards age and fp values )  . 
the latter p value is so high that it deserves consideration : in patients with more severe cirrhosis , the survival rate was conditioned by the underlying disease independent of treatment . 
it is likely that , used in smaller nodules , rfa is able to generate an optimal safety margin ( demonstrated by the higher rate of complete ablation ) while causing minimal damage to the liver parenchyma , already compromised by cirrhosis . 
 [ 22 ] sostengono che il paziente con un nodulo singolo di hcc avr un tasso di sopravvivenza pi alto rispetto a quello con hcc multifocale indipendentemente dalle dimensioni del nodulo . 
probabile che nel paziente con hcc multifocale il comportamento biologico sia differente rispetto al singolo nodulo di hcc , mostrando un andamento peggiore sia in termini di sopravvivenza che di ripresa di malattia . daltra parte , let , il sesso , i valori di fp pre - trattamento non sono rilevanti in termini di sopravvivenza ( tabella 6 )  . 
chiaro che questi dati si riferiscono ad un piccolo gruppo di studio ; presumibilmente con un numero pi alto di pazienti e con una pi fine stratificazione sarebbe stato possibile osservare alcune differenze significative ( anche se probabilmente solo per let ed i valori di fp )  . 
nel confronto dei tassi globali di sopravvivenza fra i pazienti trattati con le 2 tecniche non sono state osservate differenze statisticamente significative ( p = 0 , 3299 ) anche se la figura 5 e la tabella 5 mostrano che la rfa presenta tassi di sopravvivenza pi alti rispetto alla la . 
lo studio stratificato allappartenenza ad una classe child - pugh ha dimostrato che ci sono differenze statisticamente significative fra la sopravvivenza dei pazienti child - pugh a trattati con rfa e quelli trattati con la ( p = 0 , 0174 )  . 
questo ultimo dato ha un valore cos elevato che deve far riflettere : nei pazienti con una condizione cirrotica pi grave , il tasso di sopravvivenza stato determinato sostanzialmente dalla patologia di base qualunque sia stato il trattamento effettuato . 
la stratificazione dei pazienti in sottogruppi in relazione alla dimensione del nodulo di hcc ( noduli di hcc 25 mm vs noduli > 25 mm ) ha mostrato analoghi risultati : nel gruppo di pazienti con noduli di hcc pi piccoli , quelli trattati con rfa hanno un tasso di sopravvivenza pi alto rispetto a quelli trattati con la ( p = 0 , 0818 ) , mentre questa differenza non si osserva nel gruppo di pazienti con noduli di hcc pi grandi ( p = 0 , 7605 )  . 
 probabile che nel trattamento dei noduli di hcc pi piccoli la rfa riesca ad indurre un margine di sicurezza ottimale ( lo dimostra la pi alta percentuale di ablazione tumorale completa ) creando allo stesso tempo il minor danno possibile al parenchima epatico , compromesso dalla malattia cirrotica . conclusioni nella nostra esperienza , la la e la rfa si sono rivelate trattamenti ugualmente efficaci . 
fifty - two patients underwent us examinations for bloody nipple discharge , palpable retroareolar masses , retroareolar opacities or ductal pattern on mammography . us enabled visualisation of mammary - duct ectasia ( simple or pseudocystic , retroareolar and / or peripheral ) and focal masses ( endoluminal or periductal , with ill - defined or regular margins )  . all patients with nipple discharge underwent cytological evaluation . 
in 38 / 52 cases , us diagnosed mammary - duct ectasia and in 30 / 52 cases , the presence of focal masses ( mean size 7 mm )  . 
lus ha permesso lindividuazione dei dotti ectasici ( ectasie duttali semplici o pseudocistiche , retroareolari e / o periferiche ) e di eventuali noduli ( intraluminali e periduttali , con margini regolari o sfumati )  . 
pu considerarsi indagine complementare alla galattografia o sua valida alternativa nei casi in cui questa non risulti effettuabile . key words breast galactography ultrasonography papilloma benign ductal disease parole chiave mammella galattografia ecografia papilloma patologia duttale benigna introduction introduzione breast ultrasonography ( us ) is a widely used , noninvasive , easily reproducible and inexpensive imaging modality . 
rappresenta un valido supporto diagnostico alla mammografia , di cui incrementa i valori di sensibilit e specificit ; inoltre lecografia mammaria ormai considerata come indagine di primo livello nello studio di donne al di sotto dei 35 anni di l . 
multiple papillomas of terminal ducts in young patients are less common though important forms , as they may degenerate in 25% of cases and are considered precursors to ductal carcinoma in situ ( dcis ) [ 8 ]  . 
duct size and the intraductal productive process are fundamental for visualising these diseases : papillomas often cannot be characterised by using mammography alone , and a suspicion of papilloma calls for further investigations , such as galactography and / or breast us , also in view of the patients age [ 9 , 10 ]  . the aim of this study was to evaluate , on the basis of preliminary data derived from 13 months of work , the diagnostic contribution of breast us in the study of patients with suspected ductal disease by measuring the techniques sensitivity , specificity and positive and negative predictive values . materials and methods all breast us examinations performed at the policlinico umberto i of la sapienza university in rome between january 2005 and february 2006 were reviewed , and 52 women ( age range 2874 years ; mean 45 years ) with clinical or imaging findings suspicious for ductal disease were selected . inclusion criteria were as follows ( table 1 ) : 1 . 
presence of nodular opacity in the retroareolar region and / or of prominent retroareolar ducts on mammography [ 11 ] forty - five patients had undergone preliminary mammography , with the following results : 12 showed only signs of prominent retroareolar ducts , which were bilateral in seven and unilateral in five ; 13 patients had nodular opacities and 20 had negative mammograms . 
il ruolo di tale metodica si evoluto negli anni : dapprima utilizzata per la sola diagnosi di natura , solida o liquida , di una lesione ora ha assunto unimportanza fondamentale nella pratica diagnostica come guida , ad esempio , di procedure interventistiche come lago - aspirato , la biopsia con ago sottile e la localizzazione pre - bioptica di una lesione diagnosticata allesame mammografico [ 5 , 6 ]  . una grande variet di lesioni mammarie viene classificata con il termine di patologia duttale della mammella indicando con tale termine la patologia che interessa i dotti di maggior calibro . 
la maggior parte delle patologie duttali sono di tipo benigno e comprendono : lectasia duttale , il papilloma intraduttale singolo , i papillomi multipli , la papillomatosi del capezzolo e la papillomatosi giovanile [ 7 ]  . 
si manifesta nella maggior parte dei casi con secrezione sierosa o siero - ematica dal capezzolo , non una lesione precancerosa , ma tende a recidivare [ 9 ]  . 
i papillomi multipli , a carico dei dotti terminali e nei pazienti di giovane et , sono meno frequenti ma sono forme importanti perch possono degenerare nel 25% dei casi e sono considerati i precursori del carcinoma duttale in situ ( cdis ) [ 8 ]  . 
per ottenere la visualizzazione di tali patologie sono importanti le dimensioni del dotto e del processo produttivo intraduttale : spesso il papilloma non pu essere caratterizzato mediante il solo esame mammografico ed in caso di sospetto si rende necessaria la prosecuzione delle indagini con la galattografia e / o con lesecuzione dellecografia mammaria , anche in considerazione dellet della paziente [ 9 , 10 ]  . scopo del nostro lavoro valutare , in base ai dati preliminari ottenuti in 13 mesi di ricerca , il contributo diagnostico dellecografia mammaria nello studio di pazienti con sospetta patologia duttale della mammella calcolando i valori della sensibilit , specificit , il valore predittivo positivo ed il valore predittivo negativo . materiali e metodi sono state rivalutati tutti gli esami ecografici della mammella effettuati presso il policlinico umberto i di roma , universit la sapienza , dal gennaio 2005 al febbraio 2006 e sono state selezionate 52 pazienti di sesso femminile , di et compresa tra 28 e 74 anni ( et media di 45 anni ) che presentavano anamnesi sospetta per patologia duttale mammaria . 
all patients had undergone nipple discharge cytology , which was negative for the presence of cancer cells ( c2 ) in ten cases , and showed abnormalities suspicious for the presence of cellular atypia ( c3 ) or malignancy ( c4 ) in seven . 
 in group b ( patients without discharge ) , 23 / 35 patients presented with a palpable mass in the retroareolar region , associated with the typical signs of inflammation in two cases ; in 12 cases , prominent retroareolar ducts were present , associated with a retroareolar nodular opacity in one . breast us had been performed by radiologists with experience in breast us , with aloka prosound ssd - 5500 and siemens antares us scanners equipped with high - frequency linear - array transducers ( 7.510 , 1013 mhz ) for superficial tissues . 
radial and antiradial us scans were performed , with attention being paid to the entire ductal tree and following the lactiferous ducts from the retroareolar region towards the most peripheral branches , when visible . 
us allowed for differentiation between a normally appearing ductal tree in the retroareolar and peripheral regions and mammary - duct ectasia ( ducts larger than 2 mm in diameter )  . 
presenza di opacit nodulare nella regione retroareolare e / o di dotti retroareolari prominenti allesame mammografico [ 11 ]  . quarantacinque pazienti avevano effettuato preliminarmente lesame mammografico : in 12 la mammografia aveva riconosciuto unicamente aspetti riferibili ad una condizione di prominenza dei dotti retroareolari che in 7 casi era bilaterale e monolaterale in 5 ; in 13 erano presenti opacit nodulari retroareolari ed in 20 casi la mammografia risultava negativa . 
le 52 pazienti selezionate per il nostro studio sono state suddivise in due gruppi : il primo comprendeva 17 donne con secrezione ematica o sieroematica dal capezzolo ( gruppo a ) mentre laltro includeva 35 pazienti che non presentavano secrezione dal capezzolo ( gruppo b )  . nel gruppo a ( pazienti con secrezione dal capezzolo ) in 7 / 17 pazienti era stata effettuata la galattografia . 
le restanti 10 pazienti non avevano eseguito tale esame per i seguenti motivi : rifiuto in 5 pazienti , impossibilit nellincannulare il dotto in 4 e rottura del dotto incannulato in 1 paziente . tutte le pazienti avevano effettuato esame citologico della secrezione che mostrava in 10 casi reperto negativo per cellule tumorali ( c2 ) e in 7 alterazioni dubbie per la presenza di atipie cellulari ( c3 ) o sospette per malignit ( c4 )  . nel gruppo b ( pazienti senza secrezione ) in 23 / 35 pazienti era apprezzabile una tumefazione palpabile in sede retroareolare che in 2 casi si associava a segni caratteristici per la presenza di processo flogistico ; in 12 casi era presente prominenza dei dotti in sede retroareolare che in 1 caso si associava alla presenza di opacit nodulare in sede retroareolare . gli esami ecografici erano stati effettuati da operatori con specifica esperienza in diagnostica ecografica mammaria con ecografi aloka prosound ssd - 5500 e siemens antal . 
of patients us features definitive diagnostic categories negative mammary - duct ectasia benign nodules equivocal nodules negativi ectasie noduli benigni noduli dubbi tabella 2 classificazione ultrasonografica nelle categorie diagnostiche definitive n pazienti rilievi ecografici categorie diagnostiche definitive pattern ( either simple or pseudocystic ) or duct dilation associated with a solid nodular finding . 
on the basis of the relation between the nodule and the duct , nodules were distinguished into intraductal or periductal , and each nodule was assessed for margins ( welldefined and regular , or irregular and ill - defined ) , structure ( homogeneous or inhomogeneous ) , echogenicity ( hyperechoic , isoechoic or hypoechoic ) , posterior acoustic features ( posterior shadowing or enhancement ) and vascularity . 
after the scan , each patient was assigned a definitive diagnostic category in accordance , with the us classifications shown in table 2 [ 12 ]  . patients with normal ductal - tree findings on us ( negative finding : u1 ) or with benign lesions ( u2 ) , such as isolated duct ectasia or well - defined and regular nodules , were monitored by short - interval clinical and us follow - up ( at 3 , 6 and 12 months )  . 
patients with either equivocal findings ( u3 ) or findings suspicious for malignancy ( u4 ) underwent surgical biopsy : these included patients with ill - defined or irregular nodules and those with us evidence of a nodular mass , associated with abnormalities at discharge cytology that were either equivocal ( c3 ) or suspicious for malignancy ( c4 )  . 
the us diagnosis was later correlated with the histological findings in patients who had undergone surgical biopsy and with findings of the clinical and us follow - up in patients with conditions not considered suspicious for malignancy . 
the diagnostic accuracy , sensitivity , specificity , positive ( ppv ) and negative ( npv ) predictive values were calculated to establish the role of breast us in the diagnosis of ductal disease . results in group a ( 17 women with single - duct nipple discharge ) , galactography performed on seven patients showed a dilation res equipaggiati con trasduttori lineari ad alta frequenza ( 7 , 510 , 1013 mhz ) per i tessuti superficiali . 
sono state effettuate scansioni radiali ed antiradiali al capezzolo , prestando attenzione a tutto lalbero duttale seguendo i dotti galattofori a partire dalla regione retroareolare fino alle diramazioni pi periferiche , quando visibili . 
lesame ecografico ha permesso la distinzione tra la presenza di un regolare aspetto del disegno duttale in sede retroareolare e periferica e la presenza di ectasia duttale ( dotto di calibro superiore a 2 mm )  . 
in base al rapporto tra nodulo e dotto si sono distinti noduli intraduttali e periduttali e per ognuno sono stati definiti i margini ( netti e regolari o irregolari e sfumati ) , lecostruttura ( omogenea o disomogenea ) , lecogenicit ( iperecogena , isoecogena o ipoecogena ) , il comportamento posteriore del segnale acustico ( attenuazione acustica o rinforzo posteriore ) e la vascolarizzazione . 
al termine della scansione ecografica per ogni paziente stata scelta una categoria diagnostica conclusiva secondo le indicazioni della charta senologica , riportata in tabella 2 [ 12 ]  . le pazienti che allesame ecografico presentavano un regolare aspetto del disegno duttale ( reperto negativo : u1 ) o mostravano lesioni con caratteristiche di benignit ( u2 ) ad esempio una condizione di ectasia duttale isolata o unimmagine nodulare con margini netti e regolari sono state seguite con follow - up ravvicinati nel tempo ( 3 , 6 , 12 mesi ) , di tipo clinico ed ecografico . 
sono invece state sottoposte a biopsia chirurgica sia le donne in cui lesame ecografico era caratterizzato dalla presenza di reperti dubbi ( u3 ) o sospetti per malignit ( u4 ) : immagini nodulari con margini sfumati o l . 
breast us in this group of patients demonstrated a regular ductal tree pattern in three cases , mammary - duct ectasia of a portion of the ductal tree in nine cases and ductal dilation with nodules in five cases . 
at the time of writing , these patients had undergone clinical and sonographic follow - up at 3 - , 6and 12 - month intervals ( mean interval 6 months ) , with the following results : whereas the us pattern remained stable , the clinical findings had changed over time ( disappearance of nipple discharge in eight patients , reduced amount of discharge in two , and no significant change in symptoms in two )  . 
these patients underwent clinical and sonographic follow - up at 3 , 6 and 12 months , showing no substantial changes over time . in the remaining 25 patients , breast us identified 25 nodules , ten of which were isolated and 15 associated with mammary - duct ectasia : nineteen had well - defined regular margins ( 76% ) , and six had ill - defined or irregular margins irregolari , sia quelle in cui il reperto di una formazione nodulare messa in evidenza allesame ecografico si associava al risultato dellesame citologico della secrezione dal capezzolo caratterizzato da alterazioni dubbie ( c3 ) o sospette per malignit ( c4 )  . 
la diagnosi ottenuta con lesame ecografico stata successivamente confrontata con i risultati istologici , nelle pazienti sottoposte a biopsia chirurgica , e con i reperti riscontrati ai successivi follow - up clinico ed ecografico nelle pazienti con patologia considerata dagli operatori ecografici non sospetta per malignit . 
sono state calcolate laccuratezza diagnostica , la sensibilit , la specificit , il valore predittivo positivo ( vpp ) e negativo ( vpn ) dellecografia per definirne il ruolo nella diagnosi della patologia duttale mammaria . risultati nel primo gruppo di pazienti ( 17 donne con secrezione monorifiziale dal capezzolo ) , la galattografia eseguita in 7 pazienti dimostrava in 6 la dilatazione di una porzione dellalbero duttale a cui si associava in 2 pazienti un difetto di riempimento di un lume duttale , mentre in un caso non erano evidenti anomalie del dotto . 
lesame ecografico di questo gruppo di pazienti metteva in evidenza in 3 casi un regolare aspetto del disegno duttale , in 9 una condizione di ectasia duttale di una porzione dellalbero duttale ed in 5 pazienti la presenza di dilatazione duttale nel cui contesto si rilevavano noduli . 
1a , b a 48 - year - old woman with serosanguineous left - nipple discharge and cytological examination of the discharge negative for malignant cells ( c2 ) , without palpable lesions , underwent mammography and breast ultrasonography ( us )  . 
a mammogram shows a left fatty breast with a typical ductal pattern with prominent dilated ducts in the retroareolar region [ breast imaging reporting and data systems ( birads ) 2 ]  . 
1a , b una donna di 48 anni , con secrezione sieroematica dal capezzolo sinistro e citologia della secrezione negativa per cellule maligne ( c2 ) , senza lesioni palpabili , ha eseguito mammografia e successivamente valutazione ecografica . 
2a , b a 41 - year - old woman with serosanguineous left - nipple discharge and equivocal discharge cytology for the presence of some cellular atypia ( c3 ) , without palpable masses , underwent mammography and ultrasound ( us )  . 
a mammography shows heterogeneously dense breast tissue [ breast imaging reporting and data systems ( birads ) 3 ] , without mammographic evidence of malignancy ( birads final diagnostic category 2 )  . 
b us shows ductal ectasia in the left retroareolar region , with an associated image of a solid hypoechoic nodule , with ill - defined margins ( 9 mm ) inside a dilated mammary duct ( u3 )  . 
2a , b una donna di 41 anni con secrezione sieroemorragica dal capezzolo di sinistra e con citologia della secrezione dubbia per presenza di atipie cellulari ( c3 ) , senza lesioni palpabili ha eseguito la mammografia e successivamente la valutazione ecografica . 
a la mammografia evidenzia mammelle eterogeneamente dense ( bi - rads 3 ) , senza evidenza di presenza di malignit ( categoria diagnostica definitiva bi - rads 2 )  . 
b allecografia a sinistra , in sede retroareolare , si osserva la presenza di ectasia duttale con unimmagine nodulare solida ipoecogena ( 9 mm ) a margini sfumati nel contesto di un dotto mammario dilatato ( u3 )  . 
of the 19 well - defined nodules , 17 underwent sonographic and clinical follow - up at 3 , 6 , and 12 months , with no changes being recorded at follow - up us . 
questi casi sono stati seguiti esclusivamente mediante follow - up clinico ed ecografico a 3 , 6 , 12 mesi di distanza senza evidenza di sostanziali modifiche del quadro nel tempo . 
nelle restanti 25 pazienti lecografia ha identificato 25 noduli , di cui 10 isolati e 15 associati ad una condizione di ectasia duttale : 19 a margini netti e regolari ( 76% ) e 6 a margini sfumati o irregolari ( 24% )  . 
tutti i 19 casi con caratteristiche di benignit non hanno dimostrato modifiche dal punto di vista ecografico ai successivi follow - up a distanza di 3 , 6 , 12 menella tabella 3 viene riportato lo schema riassuntivo comprendente i reperti ecografici e liter clinico - strumentale a cui si sono sottoposte le 52 pazienti in esame . 
complessivamente lesame ecografico ha evidenziato in 14 casi la presenza di un aspetto regolare dei dotti ( in 10 casi con presenza di noduli e in 4 casi con assenza di alterazioni ecostrutturali associate ) e in 38 casi una condizione di ectasia duttale ( in 20 casi con presenza di noduli e in 18 casi con assenza di alterazioni ecostrutturali associate )  . 
3a , b a 32 - year - old woman complaining of a tender , palpable mass in the retroareolar region of the right breast , with typical signs of inflammation . 
us demonstrates in the right retroareolar region a periductal mass with irregular shape ( grossly oval ) , ill - defined margins and heterogeneous echostructure , in which some debris is visualised ( u3 )  . 
gli us dimostrano nella regione retroareolare destra una massa periduttale ( 12 mm ) con forma irregolare ( grossolanamente ovale ) , margini mal definiti ed ecostruttura disomogenea , nel cui contesto era riconoscibile materiale corpuscolato ( u3 )  . 
il quadro ecografico unitamente alla clinica erano indicativi per formazione ascessuale ; la paziente ha effettuato follow - up dopo adeguata terapia antinfiammatoria ed antibiotica . mensione media risultava di circa 7 mm ( 418 mm )  . 
complessivamente nella nostra casistica , sono state sottoposte a biopsia chirurgico 10 / 52 pazienti che allesame ecografico presentavano formazioni nodulari con caratteristiche morfologiche di sospetta malignit ( u4 )  . 
i risultati di tale procedura erano i seguenti : 5 papillomi singoli , 1 papilloma singolo con focolaio di cdis , 2 casi di papillomi multipli del capezzolo e 1 papillomatosi ; in una sola paziente lesame ha messo in evidenza la presenza di un fibroadenoma . 
le pazienti che allesame ecografico presentavano un regolare aspetto del disegno duttale ( reperto negativo : u1 ) o mostravano lesioni con caratteristiche di benignit ( u2 ) ad esempio una condizione di ectasia duttale isolata o unimmagine nodulare con margini netti e regolari sono state seguite con follow - up ravvicinati nel tempo ( 3 , 6 , 12 mesi ) , di tipo clinico ed ecografico . nella analisi statistica sono state considerate vere positive ( vp ) tutte le pazienti in cui lesame ecografico ha correttamente individuato la patologia mettendo tali reperti a confronto sia con i risultati dellesame istologico , a cui venivano sottoposte 10 pazienti , sia con i reperti del follow - up ecografico e clinico , nelle donne in cui la patologia individuata non presentava caratteristiche ecografiche sospette per malignit ( ad esempio lectasia duttale o la presenza di una formazione nodulare con margini netti e regolari )  . 
sono state definite invece vere negative ( vn ) le donne in cui lecografia non aveva messo in evidenza alcuna patologica ( assenza di anomalie duttali ) comparando tali reperti con i riscontri ecografici e clinici ottenuti nei successivi controlli a ogy ( fnac ) ; 5 / 6 cases necessitated further investigation by surgical biopsy due to cytology results that were equivocal ( cellular atypia ) or frankly suspicious for malignancy . 
none of the 19 cases of benign disease showed any change at 3 - , 6 - , and 12 - month follow - up us examinations . table 3 summarises the sonographic findings and clinicalimaging examinations performed on the 52 patients . 
overall , sonography demonstrated the presence of a normal ductaltree pattern in 14 cases ( in ten cases associated with nodules and in four cases without any associated structural changes ) and a condition of mammary - duct ectasia in 38 cases ( in 20 cases associated with nodules and in 18 cases without any associated structural changes )  . 
3a , b a 39 - year - old woman with sanguineous right - nipple discharge and equivocal discharge cytological for the presence of some cellular atypia ( c3 ) , without palpable masses , underwent mammography and ultrasonography ( us )  . 
a at mammography , the breast tissue is extremely dense [ breast imaging reporting and data systems ( birads ) 4 ] , and there is a need for additional imaging assessment ( birads final category 0 )  . 
b at us , a ductal ectasia is visible in the right retroareolar region , with some associated images of hypoechoic solid nodules with ill - defined margins ( 45 mm ) inside a dilated mammary duct ( u3 )  . 
4a , b una donna di 39 anni con secrezione emorragica dal capezzolo di destra e con citologia della secrezione dubbia per presenza di atipie cellulari ( c3 ) , senza lesioni palpabili ha effettuato mammografia ed ecografia . 
a la mammografia mostra mammelle a struttura estremamente densa ( bi - rads 4 ) , che necessitano di completamento diagnostico con altri esami strumentali ( categoria finale bi - rads 0 )  . 
b allecografia a destra , in sede retroareolare , si osserva la presenza di ectasia duttale con alcune immagini nodulari solide ipoecogene rispetto al parenchima mammario ( 45 mm ) a margini sfumati nel contesto di un dotto dilatato ( u3 )  . 
la successiva biopsia chirurgica ha confermato la presenza di papillomi multipli . neously hypoechoic in 15 cases , isoechoic in eight , inhomogeneously hypoechoic in five and homogeneously hyperechoic in two . 
overall , ten patients who had suspicious ( u4 ) nodular masses at sonography underwent surgical biopsy , with the following results : five solitary papillomas , one solitary papilloma with a dcis focus , two cases of multiple nipple papillomas and one papillomatosis ; fibroadenoma was diagnosed in one patient only . 
nel nostro studio sono stati riscontrati 46 vp e 3 vn ; in 1 solo caso , classificato come falso negativo ( fn ) , il reperto ecografico non mostrava immagini riferibili a patologia duttale sebbene il risultato dellesame citologico della secrezione dal capezzolo effettuato ad un controllo successivo a 12 mesi per la persistenza della sintomatologia mostrasse unalterazione dubbia per la presenza di atipie cellulari . 
in 2 casi ( falsi positivi , fp ) lecografia metteva in evidenza una formazione nodulare a margini sfumati che ai successivi approfondimenti , con prelievo con ago sottile in un caso e biopsia chirurgica nellaltro , risultavano essere due lesioni di natura benigna . 
lesame ecografico ha dimostrato una sensibilit del 97% , una specificit del 60% , un vpp del 95.8% , un vpn del 75% ed unaccuratezza diagnostica del 94% . discussione recentemente alcuni studi hanno sottolineato limportanza e lutilit dellimpiego dellecografia nello studio della patologia mammaria non solo come strumento in grado di definire la natura solida o liquida di una lesione ed eventuall . 
instead , we considered as true negatives ( tn ) all patients with a correct sonographic diagnosis of absence of pathological findings ( no ductal abnormalities ) confirmed by sonographic and clinical follow - up at 3 , 6 and 12 months . 
in one case only , classified as a false negative ( fn ) , sonography failed to demonstrate evidence of ductal disease , although nipple discharge cytology performed at 12 months due to persisting symptoms showed changes suggestive of cellular atypia . 
in two false positive ( fp ) cases , sonography demonstrated ill - defined nodular masses that proved to be benign at fnac in one case and at surgical biopsy in the other . 
sonography had 97% sensitivity , 60% specificity , 95.8% ppv , 75% npv and 94% diagnostic accuracy . discussion several studies recently emphasised the importance and usefulness of breast us in the study of breast diseases , not only mente di guidare la mano delloperatore nella esecuzione di manovre mini - invasive ( agoaspirati e agobiopsie ) , ma anche quale prima indagine di diagnostica strumentale nello studio del seno denso giovanile [ 3 , 4 , 8 , 13 ]  . in particolare alcuni autori hanno dimostrato la capacit dellecografia di poter effettuare la diagnosi della patologia duttale benigna e la diagnosi differenziale con la patologia maligna della mammella [ 1417 ]  . 
tra le condizioni patologiche benigne pi comuni sono comprese il papilloma mammario sia singolo ( spesso localizzato in sede retroareolare in contiguit con i dotti galattofori principali ) che multiplo ( a localizzazione pi periferica ad origine dai dotti lobari terminali ) e la papillomatosi giovanile , definita come singola entit patologica dal 1980 che si presenta nelle giovani donne in unet compresa tra i 23 ed i 32 anni [ 1822 ]  . 
 [ 7 ] riportano che il papilloma solitario si associ ad un incremento del rischio del 1 , 5%2% di sviluppare un carcinoma e ne consigliano lescissione chirurgica in tutti i casi diagnosticati , mentre cardenosa e eklund [ 23 ] dimostrano nel suo articolo che il papilloma solitario originantesi in sede centrale non debba l . 
in particular , some authors have demonstrated the ability of breast us to provide a diagnosis of benign ductal disease and differentiate it from breast malignancy [ 1417 ]  . 
among the most common benign diseases are solitary papilloma ( often retroareolar , close to the main lactiferous ducts ) , multiple papilloma ( more peripheral and originating from the terminal lobar ducts ) , and juvenile papillomatosis , described as a discrete entity in 1980 , which appears in young women 2332 years old [ 1822 ]  . 
 [ 7 ] reports that solitary papilloma is associated with a 1.5%2% increased risk of developing carcinoma , and surgical excision is suggested in all cases , whereas cardenosa and eklund [ 23 ] states that solitary papillomas originating from a central site should not be considered precancerous . the most common clinical manifestation of benign ductal diseases is the presence of serous or serosanguineous nipple discharge ( 64%88% of patients with papilloma )  . unilateral or bilateral nipple discharge may be associated with papilloma , an inflammatory process , or a breast carcinoma [ 20 , 24 ]  . 
one of the most important problems in diagnostic imaging is the poor sensitivity of mammography in the diagnosis of benign ductal disease ( mammography is negative in approximately 60% of cases )  . 
mammography may visualise well - circumscribed round masses prevalently near dilated ducts in the retroareolar region or else different forms of calcification ( round , eggshell , but also microcalcification clusters , which are difficult to differentiate from breast cancer ) ; however , correct mammographic visualisation is dependent on lesion size , which must be at least 1 cm in diameter [ 7 ]  . galactography is a valuable adjunct for the diagnosis of benign ductal disease , as it detects dilated and obstructed lactiferous ducts and / or of filling defects [ 2527 ]  . 
some galactographic findings are , however , nonspecific and do not permit differentiation between benign ductal disease and breast carcinoma , especially when areas of distortion or ill - defined ductal margins are depicted [ 7 , 28 ]  . 
other problems linked to the use of galactography include the management of young patients ( age < 35 years ) with nipple discharge , the inability or impossibility to cannulate a lactiferous duct or the patients refusal to undergo this test [ 28 ]  . in our experience , in six patients who were studied by breast us and galactography , there was concordance between the two techniques in the diagnosis of ductal disease , although us provided more detailed information on duct walls and the presence of nodular masses associated with duct dilation . 
furthermore , in the ten patients in whom galactography could not be performed , us provided a diagnosis of ductal disease that had been suspected on the basis of the clinical examination alone . in our study , in all cases confirmed by biopsy , us enabled the detection of ill - defined nodules with equivocal features , probably malignant or suspicious for malignancy , essere considerato come una lesione precancerosa . la manifestazione clinica pi frequente della patologia duttale benigna la presenza di secrezione sierosa o sieroematica dal capezzolo ( 64%88% delle pazienti con papilloma )  . 
la secrezione dal capezzolo , monoo bilaterale , pu per associarsi sia alla presenza di un papilloma sia ad un processo flogistico , sia ad un carcinoma mammario [ 20 , 24 ]  . una delle problematiche pi importanti nella diagnostica per immagini data dalla scarsa sensibilit dellesame mammografico alla diagnosi della patologia duttale benigna ( la mammografia risulta negativa in circa il 60% delle lesioni )  . 
alla mammografia si possono vedere delle masse circoscritte rotondeggianti prevalentemente in sede retroareolare in prossimit di dotti dilatati o la presenza di calcificazioni di varia morfologia ( rotondeggianti , ad uovo , ma anche come un cluster di microcalcificazioni , in questultimo caso di difficile diagnosi differenziale con il carcinoma mammario ) , ma per ottenerne la visualizzazione le lesioni devono raggiungere almeno un centimetro di diametro [ 7 ]  . lutilizzo della galattografia rappresenta un supporto nella diagnosi di tali lesioni tramite la visualizzazione di dotti galattofori dilatati ed ostruiti e / o di difetti di riempimento [ 2527 ]  . 
alcuni reperti galattografici sono per aspecifici , non permettendo di effettuare una diagnosi differenziale tra patologia duttale benigna e carcinoma mammario soprattutto nel caso in cui vengano individuate aree di distorsione o irregolarit dei margini di un dotto [ 7 , 28 ]  . 
altre problematiche allutilizzo della galattografia sono ad esempio il management di pazienti giovani ( et < 35 anni ) con secrezione dal capezzolo , lincapacit o limpossibilit nellincannulare un dotto galattoforo e il rifiuto della paziente a sottoporsi a tale esame [ 28 ]  . nella nostra esperienza in 6 casi sottoposti sia ad ecografia che a galattografia le due metodiche sono risultate concordi per la diagnosi di patologia duttale , ma lecografia ha in ogni caso fornito informazioni maggiori per quanto riguarda le pareti duttali e leventuale presenza di immagini nodulari associate al quadro di dilatazione dei dotti . 
inoltre nei 10 casi in cui non stato possibile effettuare la galattografia , lesame ecografico ha permesso di porre diagnosi di patologia duttale sospettata soltanto da rilievi clinici . nel nostro studio in tutti i casi che sono stati successivamente verificati con lesame bioptico lecografia ha permesso di riconoscere un nodulo a margini sfumati con caratteristiche dubbie , probabilmente maligne o sospette per malignit delle dimensioni medie di circa 7 min questi casi lecografia mediante lindividuazione della lesione ha permesso lesecuzione preventiva di un ago - aspirato e / o il reperage della lesione per la procedura bioptica . 
nei casi in cui le indagini si sono fermate allesame ecografico , lecografia stata importante per differenziare lectasia duttale associata alla presenza di noduli con caratteristiche di benignit dallectasia duttale semplice o pseudocistica , aspetti confermati poi nel successivo follow - up . 
in ogni caso pertanto lecografia stata in grado di evidenziare una patologia duttale che era stata sospettata clinicamente e / o mammograficamente ed ha permesso di differenziare la presenza di alterazioni che necessitano del solo follow - up da quelle che necessitano di exeresi chirurgica . 
in those cases studied by breast us only , the technique enabled the differentiation between mammary - duct ectasia associated with the presence of benign nodules and simple or pseudocystic - duct ectasia , findings that were later confirmed at follow - up . 
therefore , us proved constantly able to visualise ductal disease that had been suspected on clinical or mammographic examination and allowed us to distinguish between abnormalities requiring follow - up alone and those necessitating surgical excision . the main limitation of us was its inability to recognise ductal lesions as such where the duct was not ectatic and , more importantly , to relate nodules to the duct when these were in the most peripheral portion of the ductal tree . 
the limitations of our study lie both in the small number of patients ( n = 52 ) studied over a 13 - month period and in the limited number of cases ( ten patients ) in which the us findings could be correlated with the definitive histological diagnosis ; this was due to the impossibility of taking a biopsy in cases of clearly benign disease . 
all patients whose sonographic findings were considered not to be suspicious for malignancy and who therefore did not undergo biopsy were monitored by short - interval clinical and sonographic followup ( 3 , 6 and 12 months ) in order to minimise the risk of incorrect diagnoses provided by the first us examination . 
as regards the small number of patients enrolled , it should be noted that our work was based on preliminary data and that the final results will be presented and possibly published at a later date , as they are still being verified . however , despite the above - mentioned limitations , our study achieved an encouraging and statistically significant ppv ( 95.8% ) and npv ( 75% )  . 
the ppv indicated the percentage of cases in which us correctly identified the patients with ductal disease among those with positive sonographic findings and npv the number of patients not affected by ductal disease among those with negative us scans . 
in our study , breast us applied to the study of mammary - duct disease had 97% sensitivity and 60% specificity and a good level of diagnostic accuracy ( 94% )  . despite the limitations of this technique ( which is operator dependent and not panoramic ) , breast us performed on carefully selected women with suspected ductal disease proved to be a valuable and accurate diagnostic tool able to identify nodular lesions and / or mammary - duct ectasia , to differentiate intraductal and extraductal nodules , and to enable a diagnosis of solid lesions with benign features , requiring follow - up alone , and of nodules with equivocal or suspicious features , necessitating surgical biopsy . conclusions our preliminary data enabled us to consider high - frequency breast us as a particularly useful tool for the diagnosis of ductal disease , as it allows visualisation of dilated lactiferous ducts and of any nodules inside theif we consider that it is la metodica ecografica stato la capacit di riconoscere immagini nodulari di pertinenza duttale quando il dotto non risultava ectasico e soprattutto di riconoscere come di riferimento duttale formazioni nodulari situate nella porzione pi periferica dellalbero duttale . i limiti del nostro studio sono stati sia lutilizzo di un numero non elevato di pazienti ( n = 52 ) in un lasso di tempo di circa 13 mesi , sia lesiguit di casi ( 10 pazienti ) in cui si avuta una verifica tra i reperti ecografici ed i risultati istologici definitivi ; questultimo evento dovuto allimpossibilit di esecuzione di prelievo bioptico in condizioni patologiche di natura chiaramente benigna . 
a tal proposito tutte le pazienti i cui reperti ecografici venivano ritenuti dagli operatori non sospetti per patologia maligna , non sottoposte quindi a verifica bioptica , sono state comunque seguite con follow - up clinico ed ecografico ravvicinati nel tempo ( 3 , 6 , 12 mesi ) per minimizzare leventualit di essere incorsi in errore diagnostico con il primo esame ecografico . 
in merito allesiguit del numero delle pazienti prese in esame nel nostro lavoro , dobbiamo sottolineare che si tratta di uno studio che si basa su dati preliminari , i cui risultati definitivi verranno esposti ed eventualmente pubblicati in tempi successivi in quanto ad oggi ancora sottoposti a verifica . seppur , quindi , il nostro studio sia parzialmente limitato da le condizioni precedentemente elencate , abbiamo ottenuto comunque valori incoraggianti e statisticamente significativi , di vpp ( 95 , 8% ) e di vpn ( 75% ) che indicano nel primo caso in quale percentuale di casi la metodica ecografia ha correttamente individuato le pazienti realmente affette da patologia duttale tra le positive allecografia e nel secondo le pazienti realmente non affette tra quelle negative con la metodica stessa . 
lesame ecografico applicato alla studio della patologia duttale della mammella ha dimostrato nel nostro lavoro , una sensibilit e specificit rispettivamente del 97% e del 60% ed un buon valore dellaccuratezza diagnostica : 94% . pur con i limiti connessi alla metodica ecografica stessa ( operatore - dipendente e non panoramica ) lecografia eseguita in gruppi di donne strettamente selezionate per il sospetto di una patologia duttale si dimostrata un valido ed accurato strumento diagnostico : essa infatti in grado di individuare le lesioni nodulari e / o le ectasie duttali , di differenziare i noduli intraduttali da quelli extraduttali ed infine in grado di postulare un giudizio diagnostico sulle lesioni solide che presentano caratteristiche di benignit , per le quali corretto il follow - up e noduli che presentano caratteristiche diagnostiche dubbie o sospette per i quali necessario il ricorso alla biopsia chirurgica . conclusioni in base ai dati da noi preliminarmente ottenuti possibile quindi definire lecografia mammaria ad elevata frequenza uno strumento di elevata utilit nella diagnosi della patologia duttale in quanto permette la visualizzazione dei dotti galattofori dilatati e di immagini nodulariformi eventualmente presenti nel loro contesto . 
in these cases , breast us may prove very useful , not only as an adjunct to galactography , but also as a tool for classifying nodular lesions and detecting cases of mammary - duct ectasia when galactography cannot be performed . le , lecografia pu essere definita un esame strumentale complementare alla galattografia o una sua valida alternativa nei casi in cui questultima non risulti effettuabile . 
marini1 1dipartimento di scienze radiologiche , 2dipartimento di ostetricia e ginecologia , universit degli studi di roma la sapienza , via regina elena 324 , i - 00161 roma , italy correspondence to : a . 
halffourier single - shot turbo spin - echo ( haste ) , true fast imaging with steady precession ( fisp ) , t1 - weighted fast low angle shot ( flash ) two - dimensional ( 2d ) and diffusion - weighted ( dw ) sequences with apparent diffusion coefficient ( adc ) were obtained . 
abbiamo trovato una correlazione tra rapporto matrice germinale / diametro biparietale ( dbp ) ed et gestazionale e tra area germinativa e corticale quando espresse in rapporto al diametro intraoculare esterno ( dioe )  . 
la rm pu valutare in modo affidabile la maturazione encefalica nel periodo fetale . parole chiave risonanza magnetica sviluppo encefalico parametri biometrici girazione - solcazione mielinizzazione introduction introduzione central nervous system ( cns ) abnormalities are among the most common congenital malformations , but their early detection during pregnancy remains particularly challenging despite the considerable advances made in this field . 
alle anomalie di sviluppo del sistema nervoso centrale rappresentano le malformazioni congenite pi comuni la cui diagnosi precoce , nel corso della gravidanza , rimane particolarmente complessa nonostante i notevoli progressi coml . 
 the use of magnetic resonance imaging ( mri ) in the study of brain development disorders has shown that the technique offers new diagnostic possibilities in understanding the connections between aetiological factors and disorders of foetal brain maturation [ 2 ]  . 
furthermore , the development of fast sequences , in particular half - fourier singleshot turbo spin - echo ( haste ) sequences and steady - state free precession ( true fisp ) , with very short scan times , has revolutionised mri . 
a wide range of fast sequences is now available , including echoplanar sequences , t1and t2weighted fast gradient - echo ( fgre ) sequences and the more recent diffusion - weighted ( dw ) sequences [ 3 ]  . few studies have used mri to analyse foetal brain maturation patterns , but it has been reported that cortical gyration and sulcation reflect cerebral maturation [ 4 , 5 ] and that both are closely correlated with gestational age ( ga ) [ 6 , 7 ]  . 
in addition , mri can also provide a reliable evaluation of the orderly and predictable changes in cerebral parenchymal layering and myelination [ 8 ] that reflect the maturation process . 
 the aim of this prospective study was to establish the normal pattern of development and maturation of the foetal brain according to ga and offer an overview of the numerous possibilities of mri . materials and methods patient population and acquisition protocol the study protocol was approved by our institutions ethics committee . 
at the time of the examination , ga , as determined by a us scan within the 12th gestational week ( gw ) ranged from week 19 to week 37 ; more specifically , the distribution of foetuses according to ga was 35 second - trimester foetuses and 21 third - trimester foetuses ( table 1 )  . 
written informed consent was obtained from all patients . our study included patients referred to us for maternal complications during the pregnancy or for suspected foetal abnormalities detected on a previous us scan that had , however , demonstrated normal cns development . 
maternal complications ( 25 cases ) included uterine leiomyomas , suspected deep vein thrombosis , hydronephrosis , epilepsy , and placental abnormalities , whereas foetal abnormalities ( 31 cases ) included hydronephrosis , large bladder , abnormal amount of amniotic fluid and cardiac and pulmonary malformations . normal brain development was confirmed by foetal mri , postnatal neurological examination or postnatal autopsy ( after the legal limit of pregnancy or spontaneous abortion )  . the mri study was performed with a 1.5 - tesla ( t ) unit ( siemens vision , avanto ) , with the patient lying supine , or piuti in questo campo . 
sebbene lecografia sia la modalit dimaging prenatale utilizzata di routine , essa presenta dei limiti nellaccertamento particolareggiato dello sviluppo cerebrale che , come noto , correlato alla funzionalit cerebrale e alla prognosi neurologica [ 1 ] , come nel caso di anomalie della migrazione neuronale e malattie della sostanza bianca . lutilizzo della risonanza magnetica ( rm ) per lo studio dei disordini dello sviluppo encefalico ha dimostrato che questa metodica pu offrire nuove possibilit diagnostiche nella comprensione dei collegamenti esistenti tra fattori eziologici e disturbi della maturazione cerebrale fetale [ 2 ]  . inoltre lo sviluppo di sequenze rapide , in particolare delle sequenze half fourier single - shot fast spin - echo ( haste ) e steady state free procession ( truefisp ) , con tempi di scansione molto brevi , ha rivoluzionato radicalmente limaging in rm . 
ad oggi sono disponibili una vasta gamma di sequenze veloci , incluse le sequenze eco planari , fast gradienteco t1 e t2 pesate e le pi recenti sequenze pesate in diffusione [ 3 ]  . pochi autori hanno analizzato il pattern di maturazione cerebrale del feto mediante rm ; stato riportato che lo sviluppo dei giri corticali cerebrali e la formazione dei solchi riflettono la maturazione cerebrale [ 4 , 5 ] e che entrambi sono intimamente correlati allet gestazionale [ 6 , 7 ]  . 
inoltre con la rm possibile valutare in modo affidabile anche i cambiamenti nella stratificazione del parenchima cerebrale e nella mielinizzazione che procedono attraverso fasi ordinate e prevedibili [ 8 ] , corrispondenti al processo di maturazione . obiettivo di questo studio stato stabilire , in modo prospettivo , il normale pattern di sviluppo e di maturazione del cervello fetale in relazione allet gestazionale offrendo una panoramica sulle vaste possibilit di utilizzo della rm . materiali e metodi popolazione di pazienti e protocollo di acquisizione il protocollo di studio stato approvato dal comitato etico del nostro policlinico . 
al momento dellesame let gestazionale ( ega ) , determinata da uno studio ultrasonografico eseguito entro la 12a settimana di gestazione ( sg ) , variava tra la 19a e la 37a settimana ; in particolare la distribuzione dei feti in relazione allega era la seguente : 35 feti al secondo trimestre e 21 al terzo trimestre ( tabella 1 )  . 
da tutte le pazienti fu ottenuto il consenso informato in forma scritta . il nostro studio includeva pazienti rivoltesi alla nostra attenzione in seguito a complicazioni di origine materna insorte durante la gravidanza o a sospette anomalie fetali , individuate mediante precedente esame ecografico il quale dimostrava tuttavia un normale sviluppo del sistema nervoso centrale ; per quanto riguardo le anomalie materne ( 25 casi ) esse includevano leiomiomi uterini , sospetta trombosi venol . 
of foetuses gestational week 2nd trimester 3rd trimester 2 trimestre 3 trimestre tabella 1 distribuzione della popolazione in relazione allet gestazionale settimana di gestazione n casi in the left lateral decubitus if she was unable to tolerate the supine position ( for example , caval compression )  . 
la normalit dello sviluppo cerebrale fu confermata dalla rm fetale , da un esame neurologico negativo eseguito dopo la nascita o dallautopsia post - natale ( dopo il termine legale della gravidanza o aborto spontaneo )  . lo studio rm stato eseguito con un magnete da 1 , 5 t ( siemens vision , avanto ) , con la paziente in posizione supina o in decubito laterale sinistro nei casi in cui la madre non tollerava la posizione supina ( es : compressione cavale ) ; alle donne era permesso di rilassarsi allinterno del magnete per qualche istante prima dellesame per ridurre il movimento spontaneo fetale . 
stato utilizzato il seguente protocollo : sequenze t2 pesate half - fourier single shot fast se ( haste , te = 90 , flip angle = 150 , slice thickness = 4 mm , gap = 0 , 25 , fov = 360 mm , tempo di acquisizione = 27 s ) per lo studio delle strutture superficiali , quali giri e solchi ; sequenze true fisp breath hold ( tr / te = 4 , 8 / 2 , 3 , flip angle = 70 , slice thickness = 6 mm , gap = 0 , 30 , fov = 350 mm , tempo di acquisizione = 14 s ) con segnale intermedio t1 - t2 e poco sensibili agli artefatti di movimento ; sequenze fast gradient echo t1 pesate con e senza saturazione del segnale del grasso ( flash 2d : tr / te = 362 / 4 , 8 , slice thickness = 4 mm , gap = 0 , 20 , fov = 350 mm , tempo di acquisizione = 19 s ) , utilizzate per completare lo studio cerebrale ; sequenze flair usate per fornire informazioni addizionali sullencefalo fetale ( per esempio il diametro dei ventricoli laterali o della cisterna magna ) che creano un maggiore contrasto con le strutture adiacenti agli spazi liquorali ; le immagini furono acquisite secondo piani assiali , coronali e sagittali ortogonali allencefalo fetale ; sequenze single - shot eco - planari pesate in diffusione su un piano assiale parallelo alla linea bicommessurale ( tr / te : 3600 / 92 , slice thickness = 3 mm , gap = 0 , 30 , fov = 280 mm , matrice 128128 , tempo di acquisizione = 45 s ; con valori di b compresi tra 400 e 700 ms / mm2 ) con gradienti applicati secondo i tre assi ortogonali dello spazio ( x , y , z ) e successiva ricostruzione delle mappe di adc . 
we used a lower b value ( 700 ms / mm2 ) than normally used in adults because of the fast water diffusion rates in the foetal brain . overall examination time was 1520 min , in part owing to foetal movements that forced us to repeat the acquisition in some cases . 
 image analysis mris of all foetuses were analysed in consensus by two expert radiologists who evaluated the biometric parameters and the thickness of the developmental areas of the brain cortex ( germinal matrix , intermediate layer and cortical mantle )  . 
the study of cerebral biometry included measurement of the various structures : biparietal diameter ( bpd ) at the level of the temporal ventricular horns , frontooccipital diameter ( fod ) on the midsagittal scan , transverse cerebellar diameter ( tced ) on the axial images at the level of the posterior fossa , internal and external intraocular diameters ( iiod , eiod ) and lateral ventricular diameter ( lvd ) on the axial or coronal scans , and the cm on the sagittal scans . 
moreover , we calculated the ventricle to brain ratio ( lvd / bpd ) , measured the developmental areas of the cerebral cortex ( thickness of the germinal matrix and cortical mantle at the level of the lateral ventricles on the axial or coronal images ) , and assessed parenchymal layering in the periventricular area , gyrus and sulcus formation , and myelination . 
for the adc maps , regions of interests ( roi ) were manually placed on the frontal and occipital white matter and on the grey matter of the basal ganglia . 
lo studio della biometria cerebrale includeva la misurazione di diverse strutture : diametro biparietale ( dbp ) sulle scansioni coronali a livello dei corni temporali dei ventricoli , il diametro fronto - occipitale ( dfo ) sulla scansione sagittale mediana , il diametro cerebellare trasverso ( dtce ) sulle scansioni assiali a livello della fossa posteriore , i diametri oculari interni and esterni ( dioi , dioe ) e i diametri dei ventricoli laterali ( dvi ) sulle scansioni assiali o coronali e la cisterna magna ( cm ) su quelle sagittali . 
stato inoltre calcolato il rapporto ventricolo - cerebrale ( dvi / dbp ) , effettuata la misurazione delle aree di sviluppo della corteccia cerebrale ( spessore della matrice germinale e del mantello corticale sulle scansioni assiali o coronali a livello ventricoli laterali ) , valutate la stratificazione del parenchima in corrispondenza dellarea periventricolare , la girazione - solcazione e la mielinizzazione . 
per quanto riguarda le mappe di adc sono state posizionate manualmente delle regioni di interesse ( roi ) in corrispondenza della sostanza bianca frontale e occipitale , e della sostanza grigia dei gangli della base . 
tutte le misurazioni sono state messe in relazione allet gestazionale ; le misurazione delle aree di sviluppo sono state espresse sia come valore assoluto sia come rapporto rispetto ai parametri biometrici cerebrali . analisi statistica lanalisi statistica stata effettuata con la determinazione del coefficiente di correlazione di spearman . 
per valori al di sopra di questi non vi una correlazione significativa tra le variabili esaminate . risultati i dati relativi alle correlazioni tra i diversi parametri biometrici e lepoca gestazionale mostrata in tabella 2 : tutti i parametri biometrici misurati sulle immagini rm , eccetto per il diametro dei ventricoli laterali ( coefficiente di correlazione : 0 , 069 , p = 0 , 712 ) e della cisterna magna ( coefficiente di correlazione : 0 , 340 , p = 0 , 088 ) , correlavano con let gestazionale con un indice di correlazione r compreso tra 0 , 7 e 0 , 6 ( p < 0 , 05 ) per i differenti indici biometrici ; in particolare la correlazione con let gestazionale si dimostrata pi forte per il dioe e il dbp che mostravano anche la crescita pi costante durante la gestazione . il periodo in cui stato possibile riconoscere la stratificazione parenchimale riassunto nella tabella 3 : 1719 settimane , lencefalo presentava una superficie liscia ed era possibile differenziare due strati . 
dopo la 22a settimana , in tutti i feti erano riconoscibili tre strati che comprendevano la corteccia immatura , lo strato intermedio e la matrice germinale : nelle immagini t1 pesate la zona intermedia appariva ipointensa mentre la zona germinale e la corteccia mostravano alta intensit di segnale ; mentre nelle immagini pesate in t2 questi strati presentavano un comportamento opposto e nelle immagini pesate in diffusione la zona germinale e l . 
non era presente una correlazione tra the gestational periods during which parenchymal layering became detectable are summarised in table 3 : at weeks 1719 , the brain had a smooth surface , and two layers could be differentiated . 
inoltre , stata trovata una correlazione negativa tra rapporto matrice germinale / dbp ed et gestazionale , come pure tra dvi / dioe ed et gestazionale ( coefficiente di correlazione : 0 , 559 , p = 0 , 001 )  . nei nostri casi la mielinizzazione fu osservata a partire dalla 23a settimana di gestazione quando in corrispondenza del tegmento stato possibile identificare unarea di elevato segnale sulle immagini t1 - pesate ; tutti i feti dopo la 30a sg mostravano la presenza di mielina a livello del margine posteriore della capsula interna . 
early mri studies were limited by foetal motion artefacts due to the long scanning times , but the introduction of fast imaging sequences has enabled the accurate study of foetal brain maturation , with excellent tissue contrast resolution and without the need for foetal sedation . 
mri now offers several advantages with respect to us , such as multiplanar imaging independent of foetal head position and better contrast between the brain and the cerebrospinal fluid . as a consequence , mri plays several roles , including confirming equivocal us findings , detecting other abnormalities that may affect foetal outcome or delivery management and lutilizzo della rm come strumento complementare allecografia prenatale stato riportato per la prima volta in letteratura nel 1983 [ 16 ]  . 
inizialmente gli studi con risonanza sono stati limitati dagli artefatti da movimento dovuti al lungo tempo di scansione , ma lintroduzione di sequenze veloci ha permesso di studiare la maturazione fetale cerebrale in modo accurato con un eccellente risoluzione di contrasto tra i tessuti senza la necessit di sedazione fetale . 
lo sviluppo e la maturazione avvengono secondo una stadiazione prevedibile che sulle immagini rm caratterizzata dallo spessore della matrice germinale e corticale , dalla comparsa dello strato intermedio nella corteccia , dalla girazione , dalla solcazione e dalla mielinizzazione [ 9 ]  . molti degli studi sulla risonanza in epoca prenatale sono stati eseguiti per descrivere la maturazione cerebrale nei feti normali focalizzandosi sulla descrizione della comparsa della solcazione , girazione e mielinizzazione [ 7 , 10 , 11 ]  . 
nel nostro studio , in accordo con studi precedenti , abbiamo trovato che la stratificazione del parenchima cerebrale si correla con let gestazionale come pure lo sviluppo di solchi e scissure . 
prima della 17a sg , la risoluzione spaziale non ci ha permesso di identificare le cellule migranti , mentre tra la 17a e la 19a sg stato possibile differenziare lo strato intermedio dalla matrice germinale in meno del 25% dei feti . 
 [ 13 ] , che descrissero tre strati tra la 20a e la 26a sg . abbiamo voluto fornire inoltre punti di riferimento per i parametri cerebrali misurabili cos da poter essere utilizzati per stabilire la normalit della maturazione cerebrale ; la rm si dimostrata una metodica accurata nella valutazione di indici cerebrali , talora difficili da misurare mediante fig . 
la mappa di adc mostra le rois tracciate su unimmagine assiale a livello dei gangli basali , della sostanza bianca frontale e occipitale . supporting the planning of foetal surgery [ 17 ]  . 
 the aim of our study was to verify whether it was possible to establish parameters of normalcy during cerebral development and maturation to be used in the diagnosis and management of cases of suspected foetal brain abnormality . 
limmagine t2 haste sagittale mostra lo sviluppo della girazione e della solcazione . lesame ultrasonografico , come il dioi , il dioe , e il dvi , ed ha inoltre permesso la valutazione dello spessore della zona corticale e germinale . 
va rilevato che la valutazione dei diametri cranici risultata pi semplice e ben definita rispetto alla valutazione del parenchima cerebrale , resa pi difficile dagli effetti di volume parziale che possono essere prodotti dallutilizzo di strati di 4 o 6 mm di spessore ; ci rende , peraltro , pi complessa lidentificazione di sottili anomalie soprattutto a livello del mantello corticale . 
comunque , i nostri risultati dimostrano che lo spessore della matrice corticale correlato sia con let gestazionale sia con i parametri biometrici cerebrali , con una correlazione che maggiore nei feti al ii trimestre di gravidanza , indicando un progressivo aumento del mantello corticale mano a mano che aumenta il volume encefalico . 
al contrario , lo spessore della matrice germinale non correlava n con let gestazionale n con alcun parametro biometrico . al fine di comparare i diversi parametri ottenuti abbiamo normalizzato le aree misurate in rapporto a diversi parametri biometrici , identificando come pi costanti il dbp ed il dioe . 
le aree di sviluppo cerebrale germinativa e corticale non risultano correlate tra di loro quando considerate in termini di spessore assoluto , ma lo diventano positivamente quando vengono valutate in rapporto a parametri di crescita costanti come i suddetti dbp e dioe . tale dato indicherebbe che , in relazione alle variazioni di volume del cervello , le due aree mantengono un rapporto di crescita costante . 
tuttavia larea germinativa rispetto al volume del cervello ( espressa quindi come rapporto area germinativa / dbp ) tendeva a ridursi progressivamente con il progredire dellepoca gestazionale , in particolare questo rapporto decresceva a partire dalla 15a sg . dopo una fase di crescita esponenziale dellarea germinativa infatti si ha una fase di crescita costante , come dimostrato dallinversione del rapporto con larea corticale / dioe ( o dbp ) , e quindi una riduzione della crescita in termini di volume del cervello in relazione allepoca gestazionale . 
limmagine t2 haste sagittale mostra lo sviluppo della girazione e della solcazione . cortical matrix , the appearance of the cortical intermediate zone , gyration , sulcation and myelination [ 9 ]  . 
before week 17 ga , spatial resolution was insufficient for detecting migrating cells , whereas between weeks 17 and 19 the intermediate zone of the germinal matrix was distinguishable in fewer than 25% of foetuses . 
 [ 13 ] , who described the appearance of the three layers between weeks 20 and 26 ga . we also sought to provide reference values for measurable cerebral parameters that could be used to establish normal brain maturation . 
mri enabled accurate evaluation of cerebral indices that may be difficult to measure with us , such as iiod , eiod and lvd , and allowed assessment of thickness of the cortical and germinal zones . 
it should , however , be noted that it was easier to evaluate cranial diameters than cerebral parenchyma owing to the partial volume effects generated by the use of section thicknesses of 4 mm or 6 min addition , such thickenesses also make it more difficult to identify subtle abnormalities , especially at the level of the cortical mantle . 
the germinal and cortical areas are not correlated with each another when considered in terms of absolute thickness , whereas they are positively correlated when they are evaluated in relation to constant growth parameters , such as the bpd and eiod . 
however , when considered in relation to brain volume , the germinal area ( expressed as germinal area / bpd ratio ) tended to decrease progressively with increasing ga , in particular , from week 15 ga onwards . 
 in fact , after an initial exponential growth , the germinal area goes through a phase of constant growth , as demonstrated by the inversion of the ratio with the cortical area / eiod ( or bpd ) , followed by a phase of reduced growth in terms of brain volume relative to ga . 
a similar progressive reduction was found for the lateral ventricular diameter expressed in relation to eiod and ga : in other words , with increasing ga the ventricles have a lower growth rate with respect to the remaining cerebral parenchyma . 
the volume of the lateral ventricles increases gradually before reaching a peak at 23 weeks and gradually decreasing with ga , starting from week 15 ga with the exponential increase in the thickness of the cerebral mantle . 
 imaging aspects given the small size of the foetal brain , an accurate study requires the smallest possible fov to obtain good images of the region of interest ( roi ) , with foetal anatomy filling the fov , especially for foetuses smaller than 24 weeks ga ; in addition , the maternal body and possible overlap artefacts should also be considered . 
 to visualise and measure the landmarks of the normal foetal brain , we used t2 - weighted haste sequences , which are known to be excellent at all gas [ 25 , 26 ]  . 
in our study , the true fisp sequences proved to be essential for studying cerebral maturation in the third trimester , in particular for visualising cerebral parenchyma and myelination , thanks to fast scan times , high snr , and high t1 / t2 contrast . 
 for the study of myelination , t1 - weighted fgre sequences ( flash 2d ) with and without fat suppression can provide important additional information , as they yield highquality images above all when acquired during maternal la matrice germinale si riduce rispetto al volume cerebrale totale , come riportato da studi precedenti [ 14 , 15 ]  . 
in maniera simile abbiamo riscontrato una diminuzione progressiva del diametro intraventricolare espresso in relazione al dioe e lepoca gestazionale , ovvero con il progredire della gestazione i ventricoli crescono meno rispetto al restante parenchima cerebrale . 
il volume dei ventricoli laterali aumenta gradualmente e raggiunge il picco a 23a sg ; al contrario , il rapporto tra diametro dei ventricoli laterali e spessore corticale , a partire dalla 15a sg decresce gradualmente con lavanzare della gestazione dato laumento in modo esponenziale dello spessore del mantello cerebrale . aspetti radiologici uno studio accurato dellencefalo fetale , date le sue piccole dimensioni , necessita del pi piccolo fov possibile , in modo da ottenere delle buone immagini della regione dinteresse , con lanatomia fetale che riempie il fov , soprattutto per i feti pi piccoli della 24a sg ; inoltre va considerato anche il corpo materno che pu creare artefatti da sovrapposizione . per visualizzare e misurare i punti di riferimento dellencefalo fetale normale , abbiamo usato sequenze t2 haste pesate , eccellenti in tutte le et gestazionali [ 25 , 26 ]  . 
nel nostro studio le sequenze truefisp sono state essenziali per lo studio della maturazione cerebrale nel terzo trimestre , in modo particolare nella visualizzazione del parenchima cerebrale e nella mielinizzazione grazie alla rapidit della scansione , allalto rapporto segnale / rumore , allelevato contrasto t1 / t2 . 
queste sequenze suppliscono con un buon contrasto tissutale il fov relativamente pi grande che pu limitare la visualizzazione dei dettagli pi piccoli [ 27 , 28 ]  . per lo studio della mielinizzazione le sequenze fast gradient - echo t1 - pesate ( flash 2d ) con e senza saturazione del grasso possono aggiungere importanti informazioni fornendo immagini di elevata qualit soprattutto quando acquisite in breath - hold materno . 
le modificazioni biochimiche che avvengono durante la gravidanza si riflettono nelle variazioni di intensit di segnale : laumento in contenuto di colesterolo e glicolipidi che accompagna la maturazione della mielina e laumento in densit cellulare porta ad uniperintensit di segnale nelle immagini t1 pesate . 
the biochemical changes occurring during pregnancy are reflected in variations in signal intensity : the increase in cholesterol and glycolipid content that accompanies myelin maturation and increased cell density results in high signal intensity on t1 - weighted images . 
this problem is often avoided by using flash 2d sequences with fat suppression ; in fact , because the foetal brain has a high content of relatively free water , fat suppression heightens the contrast between free water and the tightly bound water of myelin , the cortex and the basal ganglia [ 29 , 30 ]  . 
recently , dw sequences proved useful for the study not only of some brain diseases but also of brain development , allowing investigators to establish adc values of the normal foetal brain that add information on normal brain development [ 1820 , 24 ]  . 
in addition , snr and spatial resolution are not as good as those obtained in postnatal imaging . the immature brain is characterised by a high water content and relatively fast molecular diffusion . 
because the nonmyelinated foetal brain has lower diffusion anisotropy than the myelinated adult brain , during the second trimester , it is essential to generate isotropic diffusion images rather than to apply the diffusion gradient in a single direction . on dw images , the foetal brain is characterised by a low signal intensity in the subcortical white matter and a higher signal intensity in the cortex and deep grey matter . 
the mean adc value for white matter is higher than that of grey matter of the basal ganglia , as demonstrated by our study , because the basal ganglia are composed of densely packed cells compared with the nonmyelinated white matter , which contains larger amounts of interstitial water . 
as the brain matures , the adc values of the white matter decrease gradually as a result of myelin maturation ( hydrophobic ) and increase in relative anisotropy , as intracellular water tends to move along the length of the axon . 
this cerebral maturation is associated with an increase in the anisotropy index of white matter , which is appreciable during the so - called premyelinating state [ 21 , 23 ]  . 
anisotropy increases steadily between premyelination and postmyelination phases , making it possible to observe white matter maturation a few weeks before any change in signal intensity becomes evident in the t1 - t2 sequences . 
one of the main limitations of this study was the lack of data on the relative anisotropy of white matter during this period . conclusions foetal mri may provide a useful diagnostic tool for assessment of cns diseases in utero , not only because of its greater diagnostic capabilities in detecting structural malformations of the cerebral parenchyma or ventricles , but also for its ability to assess the steps in the development and matlelevata densit cellulare e quella microvascolare sono fattori importanti per limaging pesato in diffusione ( dwi ) utile nel nostro studio per visualizzare la maturazione della sostanza grigia e bianca . 
recentemente le sequenze dw si sono dimostrate importanti non solo in alcune malattie cerebrali ma anche per lo sviluppo dellencefalo , consentendo di stabilire anche i normali valori di adc del cervello fetale che aggiungono informazioni sul normale sviluppo cerebrale [ 1820 , 24 ]  . 
questo tipo di studio pi difficile di quello post - natale a causa della degradazione di qualit dellimmagine per artefatti da movimento materno - fetali e gli effetti di pulsatilit dellaorta . 
inoltre il rapporto segnale - rumore e la risoluzione spaziale sono peggiori di quelli ottenuti con limaging post - natale . lencefalo immaturo caratterizzato da un alto contenuto di acqua e da una diffusione molecolare relativamente rapida . 
dato che lencefalo fetale non mielinizzato ha una minore anisotropia di quello mielinizzato delladulto , durante il 2 trimestre essenziale generare immagini in diffusione isotropiche piuttosto che applicare il gradiente di diffusione in una sola direzione . nelle immagini dw lencefalo fetale caratterizzato da una bassa intensit di segnale a livello della sostanza bianca sottocorticale e da una maggiore intensit di segnale allinterno della corteccia e della sostanza grigia profonda ; mentre il valore medio di adc per la sostanza bianca pi alto rispetto a quello della sostanza grigia dei gangli basali , come dimostrato nel nostro studio , poich questi ultimi sono costituiti da cellule densamente impacchettate rispetto alla sostanza bianca non mielinizzata , che contiene una maggiore quantit di acqua nellinterstizio . 
con la maturazione encefalica si assiste ad un graduale declino nei valori di adc della sostanza bianca , che pu essere spiegato con la maturazione della mielina che idrofobica , e con un aumento dellanisotropia relativa poich la direzione del movimento dellacqua intracellulare preferenzialmente lungo la lunghezza dellassone . 
questa anisotropia cresce costantemente dalla fase premielinizzante a quella mielinizzante , cosicch stato possibile osservare la maturazione della sostanza bianca qualche settimana prima che i cambiamenti di segnale risultassero evidenti anche nelle sequenze t1 - t2 . uno dei limiti principali di questo studio stata proprio la mancanza di dati riguardo lanisotropia relativa della sostanza bianca in questo periodo . conclusioni la rm fetale pu rappresentare un importante supporto diagnostico clinico nella valutazione delle patologie del snc in utero , non solo per la maggiore capacit diagnostica nelle malformazioni strutturali del parenchima o dei ventricoli cerebrali , ma anche per la possibilit di valutare le tappe di sviluppo e maturazione del cervello fetale . 
further studies on larger population samples are needed to validate our findings and identify more reliable reference curves . developments in mri technology , including dw imaging , will further expand the role of foetal mri , making it an indispensable tool in prenatal medicine . verse epoche di gravidanza ha portato alla identificazione di alcuni parametri che appaiono idonei come indici di adeguato sviluppo e maturazione cerebrale . 
fifty - seven patients showing an occlusive or aneurysmal disease of the abdominal aorta and iliac arteries underwent intraarterial digital subtraction angiography ( dsa ) equipped with the ra function and a 3d workstation . 
we found that 3dra is able to generate images of the examined vessels with a very effective 3d appearance ; furthermore , it is able to create images of the lumen and wall of the vessel through two different modalities : endoviews and cross sections . 
different aspects of the arteries can be demonstrated in the angioscopic elaborations : the normal and stenotic lumen , artery bifurcations , the collateral vessel origins and the severity and extensions of atheromatous calcifications and their relationships to the vessel wall . 
we present a summary of the different radiological findings that can be demonstrated with this new imaging technique . key words rotational angiography three - dimensional angiography virtual reality iliac arteries riassunto obiettivo . 
cinquantasette pazienti portatori di patologia steno - ostruttiva o aneurismatica del distretto arterioso aortoiliaco sono stati sottoposti ad angiografia digitale con apparecchiatura in grado di effettuare acquisizioni rotazionali e con consolle di elaborazione tridimensionale di tali acquisizioni . 
langiografia rotazionale tridimensionale capace di generare immagini dei vasi indagati con un reale effetto tridimensionale ; inoltre in grado di creare immagini del lume e della parete vasale con due diverse modalit : angioscopia virtuale e sezioni ortogonali . 
in questo lavoro viene presentata una rassegna di diversi reperti radiologici che vengono forniti da questa nuova modalit di imaging . parole chiave angiografia rotazionale angiografia tridimensionale realt virtuale arterie iliache f . 
msct and mra are able to display arteries in different ways and , in addition to the axial plane , they are able to visualise arteries with multiplanar reformations ( mpr ) , maximum intensity projections ( mip ) , shaded surface displays ( ssd ) and volume rendering ( vr ) mode . 
furthermore , one of the most effective ways to visualise arteries is by means of virtual angioscopy , a technique that is quite easily achievable with modern workstations connected to the msct or mra unit . 
 however , in recent years , digital subtraction angiography ( dsa ) has also increased its ability to depict arteries , an improvement made possible by the introduction in clinical practice of rotational angiography ( ra ) and of its natural development three - dimensional rotational angiography ( 3dra )  . 
this new method is based on the three - dimensional ( 3d ) representation of vessel structures obtained from a single intraarterial injection of contrast mediuthe acquisition is performed using a motorised angiography c arm around the structure under examination . 
this technique was originally created to investigate intracranial vessels [ 69 ] , but increasing numbers of studies have also reported its use at the extracranial level [ 1017 ]  . 
the 3d images obtained in this way can be displayed with typical 3d viewing modes , such as vr , volume colour rendering ( vcr ) , shaded surface display ( ssd ) , gradient rendering ( gr ) , maximum intensity projection ( mip ) and ray sualso , 3dra is able to generate 3d views of the lumen wall of the investigated vessels from the inside with an effective virtual angioscopic appearance [ 18 ]  . another advantage is the possibility of matching the 3d ssd reconstruction of the calcified structures present at the same level using the data obtained from the rotational acquisition performed before contrast medium injection . 
 the purpose of this paper is to present some of the more negli ultimi anni si assistito ad una significativa evoluzione nella valutazione delle arteriopatie periferiche grazie ai sensibili miglioramenti di alcune modalit diagnostiche quali leco color doppler , la tomografia computerizzata multistrato ( tcms ) e langiografia mediante risonanza magnetica ( angio - rm ) che hanno il significativo vantaggio di fornire informazioni simili allangiografia convenzionale ma con inferiori rischi per il paziente . 
la tcms e langio - rm sono in grado di dimostrare le arterie con differenti modalit e oltre al piano assiale esse forniscono rielaborazioni bidimensionali multiplanari , o tridimensionali quali le elaborazioni mip ( maximum intensity projection ) , ssd ( shaded surface display ) o vr ( volume rendering )  . 
inoltre una delle modalit di visualizzazione delle arterie pi efficace la possibilit di generare immagini di angioscopia virtuale e questo ottenibile piuttosto facilmente con le moderne workstation collegate alle tcms ma anche alle apparecchiature di risonanza magnetica . 
con questa tecnica possibile creare immagini del lume interno dei vasi e navigare lungo il vaso utilizzando il set di dati ottenuto dalle acquisizioni della tcms e della risonanza magnetica [ 15 ]  . 
 [ 3 ] langioscopia virtuale fornisce una dettagliata rappresentazione del lume e consente di comprendere leffetto della malattia su di esso . tuttavia in questi ultimi anni anche langiografia digitale stata in grado di aumentare la sua capacit di rappresentazione delle arterie e questo grazie allintroduzione nella pratica clinica dellangiografia rotazionale e della sua naturale evoluzione , langiografia rotazionale tridimensionale ( ar3d )  . 
questo nuovo metodo si basa su una rappresentazione tridimensionale ( 3d ) della struttura vasale ottenuta da una singola iniezione intra - arteriosa di mezzo di contrasto . lacquisizione viene assunta utilizzando un arco a c motorizzato che ruota intorno alla struttura da esaminare . 
le immagini 3d cos ottenute possono essere visualizzate con le tipiche modalit di rappresentazione tridimensionale : volume rendering , shaded surface display ( ssd ) , gradient rendering ( gr ) , maximum intensity projection , sum , ecc . 
un altro vantaggio la possibilit di sovrapporre allelaborazione tridimensionale dei vasi ( in ssd ) lelaborazione 3d delle strutture calcifiche presenti allo stesso livello utilizzando i dati dellacquisizione rotazionale eseguita prima delliniezione del mezzo di contrasto . 
the procedures were performed in all cases on an in - patient basis after obtaining the appropriate informed consent . the examinations were performed with an angiographic and interventional system ( philips integris allura , philips medical systems , eindhoven , the netherlands ) , equipped with the ra option and connected to a workstation capable of displaying 3d reconstructions ( dell computer , round rock , tx , usa )  . 
in all cases , the examinations were performed using nonionic contrast material at a concentration of 350 mgi / ml ( iomeron , bracco , milan , italy )  . 
the standard procedure involved the placement of a straight or pigtail high - flow catheter with an arterial retrograde common femoral or brachial approach at the level of the abdominal suprarenal aorta in the case of investigation of an abdominal aortic aneurysm , and in the subrenal aorta in the case of evaluation of lesion to the iliac artery . 
the amount and flow - rate of contrast material for ra were 6472 ml at 89 ml / s based on a subjective evaluation of vessel size at preliminary angiography . 
in the case of investigations of vessels under the diaphragm , the rotational angioacquisition was done using the roll scan programme with the c arm placed lateral to the patient . 
the fundamental requirement for a correct clinical result is to place the vessel to be investigated at the isocenter of the systetwo ras are performed : a mask run to evaluate calcification and a contrast run made with the injection parameters mentioned above . 
 in un unica immagine , il lume vasale , la placca calcifica , ma possiamo anche visualizzare stent o altri dispositivi . scopo di questo lavoro di illustrare gli aspetti endovasali normali e patologici pi interessanti che si possono osservare con questa nuova modalit nella valutazione del distretto aorto - iliaco . materiali e metodi nel periodo compreso tra gennaio 2003 e dicembre 2005 , 57 pazienti ( 40 m , 17 f ; et : 4987 et media 70 , 8 ) sono stati sottoposti ad angiografia digitale del distretto arterioso aorto - iliaco per una lesione stenotica o aneurismatica precedentemente riconosciuta alla valutazione clinica o alleco color doppler , tc e / o rm . 
complessivamente sono stati eseguiti 46 esami del distretto iliaco e 11 esami dellaorta addominale mediante angiografia rotazionale del tratto sede di patologia e successive elaborazioni 3d . le procedure sono state portate a termine in tutti i casi in regime di ricovero ospedaliero , dopo aver ottenuto il consenso informato . 
stato utilizzato un sistema per angiografia e radiologia interventistica philips integris allura ( philips medical systems , best , olanda ) , equipaggiato con lopzione per lesecuzione delle acquisizioni rotazionali , connesso a una workstation in grado di fornire elaborazioni 3d ( dell computer corp , round rock , usa )  . 
la procedura standard si basa sullutilizzo di un approccio arterioso femorale retrogrado o un approccio brachiale e il posizionamento di un catetere ad elevato flusso diritto o pig - tail con estremit distale a livello di aorta soprarenale in caso di valutazione di un aneurisma aortico sottorenale e con estremit distale infrarenale in caso di studio di una lesione stenosante del distretto iliaco . 
il quantitativo totale di mdc e il flusso per lacquisizione rotazionale variano rispettivamente tra 64 e 72 ml a 89 ml / s in base ad una valutazione soggettiva delle dimensioni dei vasi alla preliminare angiografia . 
quindi si eseguono due acquisizioni rotazionali : una con funzione di maschera che risulta utile per la valutazione delle calcificazioni e unacquisizione durante liniezione del mdc con i parametri di iniezione sopraricordati . i due set di immagini bidimensionali sono quindi trasferiti alla workstation 3d . 
 the calcification detection data set is based on a rotational scan during which low kilovolt ( kv ) numbers are used to decrease the x - ray penetration rate and increase calcification visualisation . 
if any additional geometrical assessment is desired , a subreconstruction is made and the measurement tool is used to perform the measurements of , for example , plaque thickness or remaining lumen diameter . 
angioscopic evaluations can be obtained in two ways : ( 1 ) by using the automatic vessel analysis tool or ( 2 ) by using the cut - plane technique . 
 automatic vessel analysis tool this is a postprocessing function available on the workstation , which enables the operator to obtain different types of data of the examined vessel : evaluation of the diameter and area of the vessel and the degree of stenosis with graphic representation virtual stenting : 3d visualisation of a stent / stent - graft in the site of the stenosis and of how it fits to the vessel [ 15 ] endoview : 3d visualisation of the inside lumen of the vessel . 
tali correzioni sono necessarie a causa della morfologia curva dellintensificatore di immagini e dellinfluenza del campo magnetico ambientale . la correzione delle geometrie viene utilizzata per migliorare tutte le imperfezioni presenti nella acquisizione rotazionale causate da una traiettoria circolare non ideale . 
il risultato finale dellelaborazione da considerarsi privo di distorsioni . le elaborazioni 3d possono essere rappresentate con quattro differenti modalit di visualizzazione : mip , gr , ssd e vr e queste ultime due vengono utilizzate come standard . oltre alla rappresentazione 3d delle arterie , questa workstation in grado di eseguire accurate misure dei diametri vasali e ottenere rappresentazioni endoluminali virtuali dei vasi in esame . 
questultima fornisce una visualizzazione della parete interna vasale che viene utilizzata per la valutazione virtuale del lume vasale . vi sono inoltre degli ulteriori programmi che consentono la sovrapposizione tridimensionale delle due acquisizioni ( senza e con mdc ) e che utilizzano un programma che compensa gli artefatti da movimento . 
ovviamente essa in grado di dimostrare altrettanto efficacemente dispositivi endovascolari sufficientemente radiopachi . il riconoscimento delle calcificazioni si basa sullutilizzo in corso di acquisizione rotazionale di bassi valori di voltaggio con lo scopo di ridurre la penetrazione dei raggi x e di incrementare pertanto la visualizzazione delle calcificazioni . durante lacquisizione rotazionale iniziale , le calcificazioni non sono riconoscibili ad una valutazione effettuata a occhio nudo . 
b ar3d : shaded surface display ( ssd ) dellasse iliaco : lanalisi vasale automatica definisce il tratto da analizzare ( linea blu ) e il segmento giallo definisce il punto di maggior stenosi . 
with this renti tipi di informazioni sul vaso esaminato : valutazione del diametro e dellarea del vaso , il grado di stenosi con la corrispettiva rappresentazione grafica ; stenting virtuale : la visualizzazione 3d dello stent / endopprotesi nel sito della stenosi ed il suo adattamento al vaso [ 15 ] ; endoview : la visualizzazione 3d del lume del vaso . 
una volta che abbiamo deciso dove cominciare e finire la nostra indagine nel vaso patologico , con un semplice click del mouse posizioniamo i due anelli che delimitano linizio e la fine della valutazione e lo f . 
a ar3d con contemporanea visualizzazione delle strutture ossee in un caso di aneurisma bilaterale delle arterie iliache b un piano di taglio pu essere visualizzato e modificato nel suo orientamento : tutte le parti dal lato del piano rosso possono essere rimosse . 
b il colore dellarteria non si modifica a livello della stenosi . modality , by simply moving the mouse , a line is deployed and part of the artery can be easily removed . results analysis of 3d virtual angioscopy from 3dra enables different aspects of the investigated vessels to be visualised . the internal normal lumen wall of the artery is shown in virtual angioscopy with a pink colour in contrast to the red colour of the external lumen wall . 
con questa modalit , semplicemente muovendo il mouse si traccia una linea e parte dellarteria pu essere facilmente rimossa . langioscopia virtuale tridimensionale con ar3d offre la possibilit di visualizzare differenti aspetti dei vasi studiati . lume normale e lume stenotico . 
in questo tipo di valutazione la modalit sezione ortogonale risultata particolarmente efficace . stent e dispositivi vari abbiamo studiato pazienti nei quali erano presenti stent in arteria iliaca o endoprotesi a livello addominale . 
usando la modalit visualizzazione placche calcifiche questi devices sono stati correttamente visualizzati ed in particolare le calcified plaques in the iliac arteries were observed in 32 / 46 of the examined iliac cases . 
d la sezione ortogonale dimostra placche calcifiche ( strutture bianche ) che circondano il lume ( linea rossa ) e sezione ortogonale : placche calcifiche esterne al lume vasale . quantify lesions in aortoiliac arteries with high sensitivity and specificity and are less invasive with fewer risks [ 13 ]  . nevertheless , once the diagnosis has been made , a high percentage of patients may be treated endovascularly , and this must be done in an angiography suite . 
recent papers have demonstrated the possible contribution of 3dra in the evaldiscussione langiografia ha ridotto il proprio ruolo nella diagnosi delle malattie delle arterie periferiche come a livello delle arterie carotidi , dellaorta addominale , delle iliache e delle arterie infrainguinali poich la moderna diagnostica per imf . 
8a , b aortic stent - graa three - dimensional rotational angiography ( 3dra ) in a case of bifurcated aortic stent - graft ( excluder - gore )  . 
a 3dra with calciview : the small white points ( arrows ) at the origin and in the distal portion of the right common iliac artery represent the radiopaque markers of a nitinol stent ( luminexx - bard )  . 
a ar3d con visualizzazione delle placche calcifiche : i piccoli punti bianchi ( frecce ) allorigine ed al tratto distale dellarteria iliaca comune destra rappresentano i markers radiopachi dello stent in nitinol ( luminexx - bard )  . 
c catetere centimetrato in aorta addominale ectasica . magini utilizza modalit come eco color doppler , tc multistrato e angio - rm che sono capaci di rilevare e quantificare lesioni delle arterie aorto - iliache con unalta sensibilit e specificit e invasivit inferiore con minori rischi per i pazienti [ 13 ]  . 
ci nonostante , una volta fatta la diagnosi , in unalta percentuale dei casi , i pazienti possono essere trattati per via endovascolare e questo viene fatto in sala angiografica . 
a the preliminary angiogram shows a suboptimal opacification of the iliac arteries and a high degree of noise due to an unusual amount of bowel gas ( patient with previous total laryngectomy )  . 
d in the lower part of the figure , the catheter is visible with some artefacts mainly due to catheter movement during the rotational acquisition ( yellow arrow )  . 
a il preliminare angiogramma dimostra unopacizzazione sub - ottimale dellarteria iliaca ed un certo grado di rumore a causa di una inusuale quantit di aria nellintestino ( paziente operato di laringectomia totale ) b anche la rielaborazione 3d e langioscopia virtuale dimostrano un superiore livello di rumore sulla parete vasale . 
distalmente il catetere non pu essere separato dalle placche calciche ( freccia blu )  . uation of intracranial and extracranial arteries [ 618 ] , together with the usefulness of the technique in planning some types of endovascular treatment such as stent deployment [ 1315 ]  . 
in this study , we have analysed the contribution of virtual angioscopic reconstructions starting from 3dra and have shown that the quality of these images is satisfactory in a high percentage of ed extracraniche [ 617 ] e il contributo di questa tecnica nella programmazione di alcuni tipi di trattamenti endovascolari cosi come nel posizionamento degli stent [ 1315 ]  . langioscopia virtuale una nuova modalit di imaging dei vasi arteriosi e al momento poche pubblicazioni hanno focalizzato lattenzione su di essa e molti lavori fanno riferimento ad angioscopie virtuali eseguite con tc multistrato o con angio - rm . 
 zionale e riteniamo di aver dimostrato che la qualit delle immagini soddisfacente in unalta percentuale dei casi e che questa tecnica sia in grado di fornire ulteriori informazioni . conclusions we believe that the role of angioscopic imaging of arteries still needs to be defined , but in selected cases , the use of these images increases the confidence with which endovascular interventions are carried out . 
some limitations must , however , be taken in account when virtual angioscopy is done with 3dra : slightly longer examination time due to the operative protocol of rotational angiography inadequate results in cases of significantly aneurysmal arteries where the contrast medium opacification tends to be insufficient and the flow too slow to correctly opacify all the arteries of interest . 
 other limitations of virtual angioscopy images performed using all the techniques are the inability to correctly depict stenoses due to soft plaques because currently , only hyperdense plaque components are reconstructed in 3d elaborations . 
 conclusioni in conclusione noi riteniamo che , anche se il ruolo dellimaging angioscopico delle arterie deve essere ancora definito , in casi selezionati questo tipo di immagini aumenta la confidenza con cui gli interventi endovascolari sono eseguiti . 
crusco , via antica vena 18 , i - 06100 perugia , italy , tel . : + 39 - 075 - 393044 , fax : + 39 - 075 - 5783507 , e - mail : fcrusco@sirm.org received : 20 may 2006 / accepted : 12 september 2006 / published online : 20 april 2007 abstract purpose . 
conventional digital subtraction angiography ( dsa ) still represents the criterion standard for the diagnosis of vertebral artery dissection ( vad ) , but the main drawbacks of this technique include invasiveness , patient discomfort and risk of complications . we evaluated the potential of multidetector computed tomography angiography ( cta ) as a noninvasive tool providing highresolution images of the arterial lumen and wall by comparing the diagnostic accuracy of cta and colour - doppler ultrasonography ( cdus ) in detecting acute vad . 
we retrospectively reviewed 15 cases of vad in 15 patients ( five men and ten women , age range 2858 years ) who came to our attention between august 2001 and september 2005 . 
the diagnosis was made on the basis of appropriate clinical presentation , absence of atherosclerotic disease in the cerebrovascular circulation and evidence of distinctive ct features , which were subsequently confirmed by conventional angiography used as reference standard . 
the cdus examinations revealed ten out of 15 vad , with a sensitivity of 66% , a specificity of 60% , a positive predictive value of 55.5% and a negative predictive value of 70.5%. 
in five cases , cdus revealed nonspecific wall and flow alterations ; in eight patients , high resistance obstructive flow ; and in two patients , intimal flap with demonstration of the true and false lumen . 
the differences in cta and cdus sensitivity ( 100% vs 66% ) , specificity ( 95% vs 60% ) , and overall diagnostic accuracy ( 97% vs 62.8% ) , assessed by cross tabulations and compared by using the mcnemars riassunto obiettivo . 
langiografia con sottrazione dimmagine ( dsa ) rappresenta la metodica di riferimento nella diagnosi di dissezione acuta dellarteria vertebrale ( vad ) , tuttavia i principali limiti di questa tecnica comprendono linvasivit , il disagio per il paziente e il rischio di complicanze . 
abbiamo revisionato retrospettivamente 15 vad in 15 pazienti ( 5 uomini e 10 donne , et compresa tra 2858 anni ) , giunti alla nostra osservazione nel periodo agosto 2001settembre 2005 . 
la diagnosi stata posta in base ad una sintomatologia clinica appropriata , in base allesclusione di una malattia aterosclerotica del circolo cerebrovascolare e in base alla presenza di alcuni segni tomodensitometrici specifici , successivamente confermati con dsa , considerata come metodica gold standard . 
lecd risultata diagnostica in 10 / 15 vad angiograficamente dimostrate con risultante sensibilit del 66% , specificit del 60% , valore predittivo positivo del 55 , 5% e valore predittivo negativo del 70 , 5% . 
in 5 casi , con esame ecd , si sono evidenziate alterazioni parietali e flussimetriche non specifiche , in 8 pazienti flusso ostruttivo ad alta resistenza e in 2 pazienti stato possibile visualizzare un flap intimale con rilievo dei lumi vero e falso . 
la cta si dimostrata diagnostica in tutti i 15 casi di vad , con risultante sensibilit , specificit , valore predittivo positivo e valore predittivo negativo rispettivamente del 100% , 95% , 93 , 7% f . 
used as a complement to unenhanced brain ct , it has the advantage of being readily available and easy to perform . key words multidetector ct doppler ultrasonography vertebral artery dissection e 100% . 
la cta si dimostrata tecnica sensibile per la diagnosi di vad ; effettuata a completamento di uno studio tc dellencefalo senza mdc , ha il vantaggio di essere rapidamente disponibile e semplice da eseguire . parole chiave angio - tc eco color doppler vertebral artery dissection introduction introduzione acute vertebral artery dissection ( vad ) is one of the recognised causes of posterior circulation stroke and subarachnoid haemorrhage , with a reported annual incidence of 11.5 cases per 100 , 000 and no significant gender predilection . prompt detection of the condition , especially in young and middle - aged subjects , who account for 20% of cases , is mandatory [ 13 ]  . 
among neuroimaging tests , conventional digital subtraction angiography ( dsa ) has long been the criterion standard for diagnosis and followup of vad [ 4 ] , but this technique is not without risk . 
however , dsa is still irreplaceable in evaluating complications and providing a panoramic view of the vascular tree for planning surgical or endovascular treatment [ 5 ]  . colour - doppler ultrasonography ( cdus ) is the first - line screening tool for assessing the epiaortic vessels in patients with suspected dissection , but its major drawback is the lack of high specificity . 
head and neck imaging has recently seen major changes with the advent of the latest generation multidetector computed tomography ( mdct ) scanners with shorter examinations times , thinner sections and higher resolution . 
 materials and methods we retrospectively reviewed 15 cases of vad in 15 patients ( five men and ten women , age range 2858 years ) who came to our attention between august 2001 and september 2005 . the diagnosis was based on appropriate clinical presentation , absence of atherosclerotic disease in the cerebrovascular circulation and the presence of distinctive ct features subsequently confirmed by conventional angiography used as the reference standard . 
all patients with clinical suspicion of la dissezione acuta dellarteria vertebrale ( vad ) rappresenta una delle cause riconosciute di ictus nel territorio del circolo posteriore e di emorragia subaracnoidea con un incidenza annuale che si attesta intorno a 11 , 5 casi per 100000 abitanti / anno senza significative differenze di sesso . 
tra le indagini di neuroimaging lesame angiografico ( dsa ) considerata la metodica di riferimento per la diagnosi e il follow - up della vad [ 4 ] , ma tale tecnica non scevra da rischi . 
i limiti maggiori sono rappresentati dallinvasivit e soprattutto dallincapacit di analizzare direttamente la parete vasale , inoltre nel caso di lievi alterazioni parietali di media entit non completamente stenosanti lo studio pu essere non diagnostico . 
tuttavia la dsa rimane ancora insostituibile per lo studio delle complicanze e per una visione panoramica dellalbero vascolare , fornendo gli elementi necessari per pianificare gli interventi di tipo chirurgico e / o endovascolare [ 5 ]  . leco color doppler ( ecd ) rappresenta attualmente lindagine di screening di prima istanza dei vasi epiaortici nei pazienti con sospetto clinico di dissezione , tuttavia lo svantaggio maggiore la mancanza di elevata specificit . 
lapproccio allimaging del capo e del collo completamente cambiato negli ultimi anni grazie allavvento dei tomografi multistrato di ultima generazione , capaci di eseguire studi con tempi di scansione minori , con collimazioni pi sottili , con risoluzione pi elevata . 
scopo del presente studio quello di confrontare laccuratezza diagnostica dellecd e della cta nella diagnosi di dissezione acute dellarteria vertebrale . materiali e metodi abbiamo revisionato retrospettivamente 15 vad in 15 pazienti ( 5 uomini e 10 donne , et compresa tra 2858 anni ) , giunti alla nostra osservazione nel periodo agosto 2001 - setf . 
the main clinical presentations leading to imaging were posterior cervical pain and occipital headache in ten patients , visual impairment and facial nerve deficits in two patients , dysphagia with unilateral cerebellar ataxia in one patient and sensory and motor deficits in two patients . 
a region of interest ( roi ) was positioned in the carotid artery , and a threshold of 80 hu was selected , with a fixed delay of 6 s after beginning of the acquisition . 
in selected cases , we performed late - phase acquisitions after 34 m volume data were acquired in 3040 s by using a nominal section thickness of 1.25 mm , a table feed / rotation of 7.5 mm , pitch 1.5 and a gantry rotation period of 0.8 s , 120 kv and 230 ma . 
the axial - source ct images were postprocessed into thin - slab maximum intensity projection ( mip ) and three - dimensional ( 3d ) volume rendered ( vr ) reconstructions by using a dedicated workstation ( advantage windows 4.0 , ge medical systems )  . 
all cta images were evaluated by two expert neuroradiologists ( rs , fp ) who were aware of the clinical data but blinded to the us diagnosis . cases of discordant interpretation were solved by consensus . the time required for image interpretation was around 15 mcerebral angiography was done by two expert interventional neuroradiologists ( fp , am ) using dsa ( integris bn 3000 , philips medical systems , best , the netherlands )  . 
ct and dsa diagnostic criteria for vad were increased vessel calibre with aneurysmal pattern ( focal or fusiform dilatation with or without proximal or distal stenosis ) , evidence of a stenoocclusive pattern with tapering stenosis and direct visualisation of false lumen or segmental or long stenosis . 
la diagnosi stata posta in base ad una sintomatologia clinica appropriata , in base allesclusione di una malattia aterosclerotica del circolo cerebrovascolare e in base alla presenza di alcuni segni tomodensitometrici specifici , successivamente confermati con dsa , considerata come metodica gold standard . 
il quadro clinico desordio era caratterizzato da dolore cervicale posteriore e / o cefalea occipitale in 10 pazienti , deficit visivo e del faciale in 2 pazienti , disfagia ed atassia cerebellare unilaterale in un paziente , deficit sensitivi e motori in 2 pazienti . 
nessun paziente , al momento della diagnosi , presentava fattori di rischio per patologia vascolare n connettivopatie o presenza di potenziali sorgenti emboligene . tutti gli esami ecd ( acuson , aspen , usa ) sono stati eseguiti da un operatore esperto ( g.c. ) con sonda lineare ad alta frequenza 7 , 512 mhz , scansioni longitudinali e assiali sulle arterie vertebrali , valutazione della direzione di flusso e dello spettro doppler . 
la cta , effettuata con apparecchiatura 4 - canali ( lightspeed , qxi , ge medical systems , milwaukee , wi , usa ) , a complemento di uno studio diretto senza mdc dellencefalo , stata eseguita mediante iniezione ev di mdc iodato non - ionico a bolo singolo con volume di circa 140 ml ( visipaque320 , amersharm health , gran bretagna ) con flusso di 4 , 5 ml / s . 
per ottimizzare il contrast enhancement , lo scan delay stato selezionato in base a tecnica semiautomatica di bolus tracking ( smart prep ) dopo posizionamento di una roi in carotide , scelta di una soglia di 80 uh predefinita con ritardo fisso di 6 secondi dallinizio della scansione . 
in alcuni casi sono state inoltre eseguite delle scansioni in fase tardiva dopo circa 34 minuti . i dati volumetrici sono stati acquisiti durante unapnea di 3040 secondi , utilizzando spessore effettivo di strato di 1 , 25 mm , velocit letto portapazienti / rotazione 7 , 5 mm , pitch 1 , 5 , periodo di rotazione tubo radiogeno - detettori 0 , 8 s , kv 120 e ma 230 . 
nel post - processing abbiamo ricostruito , a partire dalle immagini assiali , immagini 3d - mip thin slab e 3d - vr utilizzando una workstation dedicata ( advantage windows 4.0 , ge medical systems )  . 
criteri diagnostici cta e dsa di dissezione sono stati : visibilit di un calibro vasale aumentato con pattern aneurismatico ( dilatazione settoriale o fusiforme con o senza stenosi prossimale o distale ) , visibilit di un pattern steno - occlusivo con assottigliamento progressivo del lume vasale , visualizzazione diretta del doppio lume , stenosi segmentale o lunga . 
concordance between readers 1 and 2 for the detection of vad by cta was calculated with the cohen k value according to the formula k = io - ie / 1 - ie , where io is the observed concordance and ie the expected concordance . analisi statistica trattamento con terapia anticoagulante e / o antiaggregante e / o procedura interventistica , stato effettuato con ecd e cta a 6 e 9 mesi . results among the 15 vad , the most common findings demonstrated by angiography were a stenoocclusive pattern in 11 patients ( three tapering stenoses with distal occlusion , four dissecting membrane with true and false lumen and visible intimal flap and four irregular segmental or long stenoses with string - sign appearance )  . 
four patients had aneurysmal an angiographic pattern with focal dilatation of 22.8 cten of the 15 vad ( 66% ) were located at segment v4 , three ( 20% ) at segment v3 and two ( 13% ) at segment v2 . 
of the four patients with aneurysmal angiographic pattern , three were treated with balloon or coil embolisation and the remaining one was managed conservatively . cdus revealed ten out of 15 vad , with a sensitivity of 66% , specificity of 60% , positive predictive value of 55.5% and negative predictive value of 70.5%. 
among the true positive cases , in eight , the diagnosis was based on indirect signs , such as high resistance obstructive flow with severely diminished systolic and absent or low diastolic flow ( figs . 1e , f , 2e )  . 
in the other two patients , the diagnosis was based on the evidence of the dissecting membrane with visualisation of the true and false lumen . cta enabled the correct identification of all 15 vad . the reported sensitivity , specificity , positive predictive value and negative predictive value were 100% , 95% , 93.7% and 100% , respectively . 
differences in cta versus cdus sensitivity ( 100% vs 66% ) , specificity ( 95% vs 60% ) and diagnostic accuracy ( 97% vs 62.8% ) were significant ( p value < 0.05 ; mcnemar test )  . 
la concordanza interosservatore per la detezione di vad con cta stata calcolata utilizzando il k statistico di cohen , in accordo alla formula : k = io - ie / 1 - ie , dove io la concordanza osservata e ie la concordanza attesa . risultati i reperti semeiologici riscontrati angiograficamente tra le 15 dissezioni rilevate sono stati : un pattern di tipo steno - occlusivo in 11 pazienti ( tre stenosi con assottigliamento progressivo del vaso fino allocclusione distale , segno della fiamma , quattro dissezioni vere e proprie con visualizzazione del doppio lume e del relativo flap intimale , quattro stenosi segmentarie irregolari o lunghe con aspetto string sign ) ed pattern era tipo aneurismatico in 4 pazienti , con dilatazione focale del lume vasale tra 2 e 2 , 8 cdieci dell1e 15 dissezioni ( 66% ) erano localizzate a livello del segmento v4 , 3 ( 20% ) a livello del segmento v3 e 2 ( 13% ) in corrispondenza del segmento v2 . 
tutti i pazienti con dimostrata dissezione steno - occlusiva hanno ricevuto un trattamento terapeutico anticoagulante o antiaggregante ; in tutti i casi si ottenuta una guarigione clinica pressoch completa con ricanalizzazione del vaso nei controlli a 69 mesi . 
dei 4 pazienti che presentavano pattern aneurismatico , tre sono stati sottoposti a trattamento percutaneo di angioplastica o embolizzazione con spirali , un paziente stato trattato conservativamente . lecd risultata diagnostica in 10 / 15 vad con una riportata sensibilit del 66% , specificit del 60% , valore predittivo positivo del 55 , 5% , e valore predittivo negativo del 70 , 5% . 
la diagnosi stata posta nei rimanenti 2 casi attraverso la visualizzazione diretta del doppio lume ; i dati statistici sono riportati in tabella 1 . la cta ha identificato correttamente tutte 15 le vad , con valori di sensibilit , specificit , valore predittivo positivo e valore predittivo negativo rispettivamente del 100% , 95% , 93 , 7% , 100% . 
1a - f dissezione spontanea della va in donna di 45 anni , che presentava cefalea occipitale , ad esordio acuto , con cervicalgia seguito da deficit sensitivo dellarto superiore . 
alla valutazione doppler si rileva flusso stenoostruttivo ad alta resistenza con riduzione del picco sistolico e con assente componente diastolica ( e , f )  . tissue disorders associated with an underlying structural defect of the arterial wall , such as marfan syndrome , ehlers danlos syndrome , fibromuscular dysplasia , pseudoxanthoma elasticum and osteogenesis imperfecta type ii , whereas enviparietali che mimavano una stenosi filiforme . 
tra cta e ecd sono risultate statisticamente significative ( p < 0 , 05 ) le differenze tra i valori sensibilit ( 100% vs 66% ) , specificit ( 95% vs 66% ) e accuratezza diagnostica ( 97% vs 62 , 8% )  . 
anomalie di lunghezza e decorso dei vasi quali kinkinge coiling sembrano incrementare il rischio di una dissezione [ 6 , 7 ]  . in ogni modo le cause di dissezione arteriosa sono attribuibili alla lacerazione dellintima oppure alla rottura dei vasa vasorum , con formazione di un falso lume o di un ematoma intramurale a seconda che ci sia o meno una comunicazione diretta con il lume vasale . 
se il versamento emorragico rimane confinato tra lamina elastica interna e tonaca media si ha stenosi oppure ostruzione del vaso , mentre se il danno si estende a coinvolgere lavventizia si pu avere un rigonfiamento sacculare o fusiforme negli strati pi esterni della parete ( aneurisma dissecante )  . 
la localizzazione pi frequente dellaneurisma dissecante nella regione del segmento v4 , in virt a tale livello di una lamina elastica interna spessa , dellassenza della lamina elastica esterna , di unavventizia sottile e di poche fibre elastiche nella tunica media [ 8 ]  . 
tuttavia la localizzazione pi frequente della dissezione , qualsiasi sia il pattern di presentazione , a livello del tratto extracranico , ed in particolare a livello del segmento v3 , perch mobile e maggiormente esposto a traumi anche lievi [ 14 ]  . 
axial and volume rendered ( vr ) computed tomography angiography ( cta ) ( a , b ) of the left v4 segment shows increased external diameter with dissection ( red arrow )  . 
le immagini assiali e le ricostruzioni vr ( a , b ) del segmento v4 di sinistra mostrano occlusione completa con aumento complessivo del calibro vasale su base dissecativa ; le corrispondenti immagini ecografiche dimostrano un flusso distale retrogrado dal vaso controlaterale ( c )  . ronmental factors include hypertension , tobacco use , oral contraceptives , abuse of sympathicomimetic drugs , events associated with hyperextension or rotation of the neck ( yoga , coughing , vomiting , sneezing ) , chiropractic manipulations and recent history of respiratory tract infection and traumatic events . 
arterial kinking or coiling seems to increase the risk of spontaneous dissection [ 6 , 7 ]  . pathological causes of the dissection are related to tearing of the intima or rupture of vasa vasorum , with formation of false lumen or an intramural haematoma , depending on the presence or absence of direct communication with the vessel lumen . 
in our patients , 13 / 15 dissections ( 86.6% ) occurred at segments v3 and v4 , in agreement with previous reports [ 9 ]  . typical clinical manifestations of vad include posterior cervical pain spreading to the shoulder and headache in the occipital area ; ischaemic symptoms are common and may be related to involvement of lateral medulla ( wallenbergs syndrome ) , thalamus and cerebral or cerebellar hemispheres . despite its relatively poor specificity ( 60% in our series ) , tendency to overestimate stenoses and inability to assess part of the cervical and intracranial va , cdus is the fastest and simplest method for the initial evaluation of patients with suspected vad and for the follow - up . 
typical us findings , such as irregular stenosis , dissecting membrane , localised increase in diameter of the artery , pseudoaneurysm , intramural haematoma and tapering stenosis with distal occlusion are detectable in more than 90% of cases [ 10 , 11 ]  . 
sonographic diagnosis of dissection of the third and fourth segments is based on indirect signs such as absence of flow , high resistance obstructive flow with severely diminished systolic and absent or low diastolic flow ( staccato flow ) , and diminished peak systolic velocity with at least 50% reduction compared with the contralateral side [ 11 ]  . cta is an important alternative in diagnostic screening , as it is able to provide high - resolution and high - contrast images of the arterial lumen and wall , allowing recognition of both vascular alterations and possible thromboembolic complications [ 12 ]  . 
the most reliable diagnostic ct criteria , consistent with the dsa il tipico quadro clinico di vad caratterizzato da dolore cervicale posteriore con irradiazione alla spalla e / o cefalea occipitale ; i sintomi ischemici sono comuni e interessano in maniera particolare la porzione laterale del midollo spinale ( manifestandosi in tal caso come sindrome di wallenberg ) , il talamo e gli emisferi cerebellari e cerebrali . 
nonostante la relativa bassa specificit ( 60% nella nostra casistica ) , la tendenza a sovrastimare il grado di stenosi e limpossibilit di esplorare parte del tratto cervicale e intracranico della va , lecd la tecnica pi rapida e semplice per liniziale inquadramento diagnostico dei pazienti con sospetta vad , e per il follow - up degli stessi . 
le tipiche alterazioni semeiologiche ecografiche ( lesioni di tipo steno - ostruttivo , alterazioni di parete con possibilit di visualizzare il flap intimale , incremento dimensionale segmentario del vaso occluso , lo pseudoaneurisma , lematoma intramurale e la stenosi filiforme con occlusione distale del vaso ) , sono riscontrabili in pi del 90% dei casi [ 10 , 11 ]  . 
la diagnosi ecografica di dissezione con localizzazione a livello dei segmenti v3 e v4 , pu essere basata su segni indiretti come lassenza di flusso , un picco sistolico seguito da flusso diastolico ridotto e / o assente , indicativo di alte resistenze distali ( staccato flow ) , un picco di velocit sistolica ridotto di almeno il 50% rispetto ai valori di picco del vaso controlaterale [ 11 ]  . la cta rappresenta unimportante alternativa , nella fase di screening diagnostico , in quanto fornisce immagini ad alta risoluzione ed alto contrasto del lume e della parete arteriosa , consentendo di riconoscere sia le alterazioni vascolari che le possibili complicanze trombo - emboliche [ 12 ]  . 
i criteri pi affidabili per la diagnosi , congrui a quelli della dsa , riflettono lanalisi del lume arterioso residuo , la sede della dissezione e la misurazione del calibro vasale complessivo ; nella nostra serie tutte e 15 le dissezioni presentavano aumento dimensionale segmentario del vaso occluso . 
in our series cta yielded a sensitivity of 100% , a specificity of 95% , a positive predictive value of 93.7% , a negative predictive value of 100% and a diagnostic accuracy of 97% . a limitation of our study was the lack of correlation with mri . 
the value of mri lies in its particularly high resolution in the posterior fossa and the capability for direct visualisation of intramural haematomas and other luminal abnormalities , such as aneurysmal dilatation , intimal flap or occlusion . the typical mri appearance of an intramural haematoma consists of an increased external diameter of the artery , an eccentric region of high signal intensity and narrowing of the arterial lumen . 
the region of high signal intensity is due to the methemoglobin blood products within the haematoma . for this reason , mri is superior to cta in depicting a subacute or chronic intramural haematoma [ 13 ]  . the differential diagnosis of vad , especially with stenoocclusive pattern with tapering stenosis , is sometimes difficult . 
these cases require accurate evaluation of patients history , neurologic examination , laboratory tests ( blood screening and coagulation test ) and imaging examinations [ echocardiography ( ecg ) , cdus of the epiaortic vessels ] to exclude potential cardioembolic sources , atheromas , vasculitis , hypoplasia or dysplasia and vasospasm . conclusions in conclusion , the results of our experience confirm previous findings [ 12 ]  . 
with this technique , visualisation of the intramural haematoma combined with staccato high - resistance obstructive flow with severely diminished systolic and absent or low diastolic flow , strongly indicate the presence of spontaneous vad . 
however , because of the frequent nonspecificity of cdus findings , patients with a clinical suspicion of vad and inconclusive or normal duplex scan findings should be further investigated by cta . 
 stra casistica sono stati riportati per la cta valori di sensibilit del 100% , specificit del 95% , un valore predittivo positivo del 93 , 7% , un valore predittivo negativo del 100% e unaccuratezza diagnostica del 97% . un limite del nostro studio stato la mancata correlazione tra cta e rm . 
la rm presenta infatti una migliore risoluzione a livello della fossa cranica posteriore , ed ha il vantaggio di visualizzare direttamente lematoma intramurale , la dilatazione aneurismatica , il flap intimale e locclusione . lematoma intramurale evidenziabile in rm sotto forma di un aumento del diametro vasale esterno , come ispessimento parietale eccentrico iperintenso che circonda a semiluna o a bersaglio il lume vascolare ristretto . 
per tale motivo , la rm distingue meglio , rispetto alla cta , tra ematoma intramurale subacuto e / o cronico [ 13 ]  . i pattern steno - occlusivi di dissezione , con stenosi filiforme associata , sono alcune volte di dubbia interpretazione , necessario pertanto unaccurata valutazione della storia dei pazienti , un attento esame neurologico , il ricorso ad esami laboratoristici ( esame emocromocitometrico , ves , test di emocoagulazione ) e strumentali ( ecg , ecocardiografia , ecd dei vasi epiaortici ) per escludere potenziali sorgenti emboligene cardiache , alterazioni ateromasiche o quadri vasculitici , ipoplasie , displasie e vasospami . conclusioni i risultati della nostra esperienza confermano quanto riportato in letteratura negli ultimi anni [ 12 ]  . 
in particolare , lecd risulta la tecnica pi rapida e semplice per liniziale inquadramento diagnostico , per una rapida visione dei vasi extracranici del collo , e per il follow - up . 
con questa tecnica la visualizzazione diretta dellematoma intramurale associata ad un flusso staccato , steno - ostruttivo , ad alta resistenza con picco di velocit sistolica ridotto , seguito da un flusso diastolico ridotto o assente , fortemente indicativa di una dissezione arteriosa spontanea . 
tuttavia a causa dellaspecificit dei reperti ecografici , pazienti con sospetto clinico di dissezione , in presenza di rilievi ecografici negativi o dubbi , dovrebbero essere sottoposti ad esame cta . 
i - 00168 rome , italy 2deptartment of radiology , neuroradiology section , ucsf university of california san francisco , san francisco , ca , usa correspondence to : a . 
by providing quantitative measurements of cerebral blood flow ( cbf ) and cerebral blood volume ( cbv ) , dynamic perfusion computed tomography ( p - ct ) allows visualisation of cerebral autoregulation mechanisms and represents a fast , available and reliable imaging option for assessing cerebral perfusion . 
thanks to its feasibility in emergency settings , p - ct is considered most useful , in combination with ct angiography , in acute ischaemic patients , as it is able to provide a fast and noninvasive assessment of cerebral perfusion impairment . 
in addition , p - ct can play a diagnostic role in other types of cerebrovascular disease to assess functional reserve , and in intracranial neoplasms , where it has a role in diagnosis , grading , biopsy guidance , and follow - up during treatment . 
this article illustrates the principles , technique and clinical applications of p - ct cerebral perfusion studies . key words brain ischemia brain neoplasms brain injury hemodynamics perfusion ct riassunto lo studio dellemodinamica cerebrale , ottenuto con le metodiche di imaging , con le sue varie ed attuali applicazioni , genera interesse crescente . 
la tc perfusione dinamica ( p - tc ) permette una valutazione quantitativa del flusso cerebrale ematico ( cbf ) e del volume cerebrale ematico ( cbv ) , offrendo cos una visualizzazione diretta dei meccanismi di autoregolazione cerebrale , e si pone come una valida alternativa ad altre modalit di misurazione della perfusione cerebrale , rispetto alle quali ha il maggior vantaggio di essere una tecnica prontamente disponibile ed accessibile , in condizioni di emergenza , nella maggior parte dei centri medici . 
per tale ragione la p - tc utile soprattutto nellischemia cerebrale acuta , condizione in cui , associata allangio - tc , offre in maniera rapida e non - invasiva , la valutazione eziologica dellipoperfusione , nonch delle sue ripercussioni emodinamiche e fisiopatologiche sul parenchima cerebrale . 
inoltre la p - tc trova utile impiego in pazienti con altre patologie cerebro - vascolari e per la diagnosi , il grading , la guida alle procedure bioptiche , ed il controllo durante la terapia , dei tumori intra - cranici . 
questo articolo si propone di riassumere i principi , la tecnica e le principali applicazioni cliniche degli studi di perfusione cerebrale basati sulla metodica tc . parole chiave ischemia cerebrale neoplasie cerebrali trauma cranico perfusione tc introduction introduzione rapid technological advances in functional imaging techniques have extended the scope of radiology , and in particular of neuroradiology , beyond the boundaries of morphological imaging . 
perfusion studies can now be carried out with magnetic resonance imaging ( mri ) and computed tomography ( ct ) as well as with the gold standard modalities used until now , such as positron emission tomography ( pet ) , single - photon emission tomography ( spect ) il rapido progresso tecnologico applicato alle tecniche di imaging funzionale ha spinto la radiologia in generale , e la neuroradiologia in particolare , oltre i confini della morfologia ; in questo contesto genera crescente interesse la valutazione a scopo diagnostico dellemodinamica cerebrale ( perfusione cerebrale )  . 
gli studi di perfusione ottenuti con rm e tc si sono affiancati alle metodiche fino ad ora impiegate e considerate gold standard , quali la tomografia ad emissione di positroni ( pet ) , la tomografia ad emissione di singolo fotone ( spect ) e la xenon - tc , con 1225 a . 
this is because the introduction of thrombolytic therapy for the treatment of stroke has highlighted the need for a fast and readily available technique capable of identifying the perfusion defect and evaluating its extent and severity [ 2 , 3 ]  . 
 in patients with acute stroke undergoing emergency evaluation , dynamic perfusion ct ( p - ct ) has been proposed as a particularly useful technique after unenhanced brain ct ( which rules out intracranial haemorrhage ) on account of its ease of use , reproducibility of quantitative measurements , ready availability , limited cost and tolerability [ 3 , 4 ]  . 
moreover , the use of p - ct may be extended to the evaluation of the cerebrovascular reserve in patients with haemodynamically significant stenoses of the intraor extracranial arteries even with acetazolamide challenge in potential candidates for temporary or permanent occlusion of the internal carotid artery ( balloon occlusion test ) [ 5 ] , or in patients with subarachnoid haemorrhage at risk of vasospasm [ 6 ]  . 
in addition to acute and chronic cerebrovascular disease , a further application of p - ct is the measurement of cerebral blood volume ( cbv ) and microvascular permeability ( ps ) in brain tumours [ 7 ]  . general technique p - ct derives information on brain haemodynamics by analysing the first passage through the cerebral vessels of an intravenous contrast bolus . 
because there is a direct linear relation between the concentration of contrast material and density , passage of contrast bolus results in an increase in density of the areas being examined that is proportional to the amount of contrast material present in the blood vessels . the blood - brain barrier ( bbb ) prevents the contrast material from spreading into the interstitium , so that under normal conditions the increase in density is only transient , occurring during the first intravascular passage of the bolus . 
p - ct is based on the physical - mathematical tracer kinetic model [ 8 ] , which assumes that the contrast bolus is instantaneous , is introduced into a single vessel , passes through a capillary network , remains totally intravascular and flows out through a single venous conduit . p - ct can be performed on any spiral ct scanner capable of cine mode scans , with the aid of an automatic injector . the anatomical coverage of p - ct along the craniocaudal axis is limited , as the scan is obtained without table motion at 510 mm for single - detector devices , at 20 mm for multidetector devices ( 232 ) and at 40 mm for the recent 64 - detector - row scanners . 
for this reason , the anatomical level to be studied is selected , generally on the scout view or initial morphological scan , and the perfusion study performed at that location using a cine scan without table motion . 
one possible study protocol , applicable to both singleand multislice scanners ( 232 slices ) involves 1 rotation / s for 50 s , 1226 relativi vantaggi e limiti [ 1 ]  . 
infatti lintroduzione della terapia trombolitica nel trattamento dellictus ischemico acuto ha reso evidente il bisogno di una tecnica rapida e prontamente disponibile per lidentificazione del deficit perfusionale e la valutazione della sua estensione e della sua entit [ 2 , 3 ]  . nei pazienti con ictus , in condizioni di emergenza , dopo la tc cerebrale di base senza mdc ( che esclude lemorragia intra - cranica ) la tc perfusione dinamica ( p - tc ) , per semplicit e rapidit di esecuzione , riproducibilit di misurazioni quantitative , disponibilit sul territorio , costi contenuti e tollerabilit da parte dei pazienti , si propone come metodica di particolare interesse ed utilit [ 3 , 4 ]  . 
la p - tc pu essere effettuata velocemente con qualsiasi apparecchiatura spirale , e le mappe di perfusione possono essere ottenute in breve tempo mediante una workstation dotata di apposito software . 
limpiego della p - tc pu inoltre essere esteso alla valutazione della riserva cerebro - vascolare in pazienti con stenosi arteriose emodinamicamente significative intrao extra - craniche anche con stress - test allacetazolamide in potenziali candidati ad occlusione temporanea o definitiva dellarteria carotide interna ( balloon occlusion test ) [ 5 ] , o in pazienti con emorragia sub - aracnoidea , a rischio di vasospasmo [ 6 ]  . 
poich esiste una relazione proporzionale lineare diretta tra concentrazione del mdc e densit , il passaggio del bolo di mdc induce un aumento della densit nelle aree esaminate , proporzionale alla quantit di mdc presente nei vasi . 
la presenza della barriera emato - encefalica ( bee ) impedisce al mdc di diffondersi dai vasi allinterstizio , cosicch , in condizioni normali , laumento di densit solo transitorio , durante il suo primo passaggio intra - vascolare . 
la p - tc basata sul modello fisico - matematico del tracciante cinetico ( kinetic tracer model ) [ 8 ] , il quale assume che il bolo di mdc sia pressoch istantaneo , sia immesso in un vaso unico , passi attraverso una rete capillare , rimanga totalmente intra - vascolare , e defluisca da un singolo collettore venoso . la p - tc pu essere effettuata su qualsiasi apparecchiatura tc con tecnologia spirale che sia in grado di effettuare scansioni in modalit cine , con lausilio di un iniettore automatico . 
la copertura anatomica della p - tc lungo lasse cranio - caudale limitata , poich la scansione si effettua a tavolo fermo , a 510 mm per le apparecchiature spirali a strato singolo , a 20 mm per le apparecchiature tc multia . 
the short scan delay is aimed at the acquisition of a first baseline scan before arrival of bolus and is applied independent of the patients cardiocirculatory condition or type of ct device used . 
for a four - detector - row scanner but also possible with other multislice devices allows the effective radiation dose to be contained below that calculated for a conventional brain ct study [ 10 , 11 ]  . 
lower milliampere settings result in a reduced signal - to - noise ratio ( snr ) and poorer densitometric resolution , with consequently less precision of the parametric measurements ( expressed by increased standard deviation values ) ; nevertheless , it represents a reasonable compromise between radiation - dose containment and diagnostic quality of the examination . 
use of the automatic injector and , if a dual syringe injector is available , of a saline flush after the contrast bolus allows administration of a fast and compact bolus [ 11 ]  . 
the images , generally acquired with 5 - mm thickness to avoid beam - hardening artefacts , are reconstructed with a thicknesses of 10 mm to increase the snr , and at 0.5 - s intervals to increase temporal resolution . data acquired during the cine scan are then analysed on a workstation running dedicated postprocessing software that generates and analyses the time - density curves . 
a region of interest ( roi ) is placed on an artery in the images to view the time - density curve , which reflects the compactness of the bolus . 
it is important to note that , although ideally one might expect the arterial curve to fall back to baseline density levels after the first pass of the bolus , in practice , even when the bolus is sufficiently compact and the bbb intact , the curve tends to remain on a plateau varying height above baseline ( usually 10%30% of the peak )  . 
the maximum slope model approximates the cerebral blood flow ( cbf ) value from the slope of the time - density curve , calcustrato , dotate di multiple file di detettori ( da 2 a 32 ) , a 40 mm per le recenti tc multidetettore a 64 strati . 
per tale ragione si seleziona il livello anatomico da studiare , generalmente sulla scout view o su una scansione morfologica iniziale , ed al livello selezionato si esegue lo studio di perfusione , che consiste in una scansione cine , a tavolo fermo . 
un possibile modello di protocollo di studio , applicabile con apparecchiature tc a strato singolo o multiplo ( 232 strati ) , prevede 1 rotazione / s per 50 s , con 57 secondi di ritardo rispetto alliniezione attraverso una cannula 1820 g , in una vena antecubitale , di 4050 ml di mdc iodato non ionico ( 300370 mg% ) a 45 ml / s [ 9 ]  . 
il breve ritardo tra liniezione del mezzo di contrasto e linizio della scansione finalizzato ad ottenere lacquisizione di una linea di base , prima dellarrivo del bolo , ed applicabile indipendentemente dalla condizione cardio - circolatoria del paziente e dal tipo di apparecchiatura tc utilizzata . 
 [ 10 , 11 ] per una apparecchiatura tc multistrato con quattro file di detettori , ma applicabile anche ad altri tipi di tc multistrato , permette di contenere la dose effettiva di radiazioni al di sotto di quella calcolata per un esame tc del cranio convenzionale . 
la limitazione dellamperaggio causa una diminuzione del rapporto segnale / rumore ed una ridotta capacit di risoluzione densitometrica , cui consegue una ridotta precisione delle misurazioni parametriche ( espressa dagli aumentati valori di deviazione standard ) , ma rappresenta un ragionevole compromesso tra contenimento della dose di radiazioni e qualit diagnostica dellesame . 
luso delliniettore automatico e , se si dispone di un iniettore a doppia siringa , di un bolo di soluzione fisiologica a seguire il mdc , permettono di somministrare un bolo rapido e compatto [ 11 ]  . 
le immagini , generalmente acquisite con spessori di 5 mm , per evitare artefatti da indurimento del fascio , vengono ricostruite a spessori di 10 mm , per aumentare il rapporto segnale / rumore , e a 0 , 5 s di intervallo , per incrementarne la risoluzione temporale . i dati acquisiti durante la scansione cine sono poi analizzati ad una workstation dotata di apposito software che produce ed analizza le curve tempo / densit . 
la compattezza del bolo di mezzo di contrasto determina la morfologia della curva , con ascesa ripida e discesa compresa nei 50 s di scansione . lates cbv as the area under the curve , and from these two parameters derives mean transit time ( mtt ) by solving the central volume equation . 
il modello maximum slope approssima il valore del cbf dalla pendenza ( slope ) della curva densit / tempo , calcola il cbv come larea sottesa alla stessa curva , e da questi due parametri deriva il mtt risolvendo lequazione del volume centrale ; questo modello non richiede la selezione di un arterial input function ( aif ) , ma necessita di velocit di iniezione del mdc molto alte ( > 6 ml / s ) , e fornisce solo misurazioni relative non quantitative [ 12 , 13 ] ; il modello di deconvoluzione si basa su un complesso processo matematico che richiede la scelta di un aif , ovvero di una curva densit / tempo di unarteria , con cui confrontare la curva ottenuta in corrifig . 
a maximum - slope model derives cerebral blood volume ( cbv ) values by calculating the area under the curve , cerebral blood flow ( cbf ) as the slope of the curve and mean transit time ( mtt ) by solving the central volume equation . 
again , cbv corresponds to the area under the curve , mtt to full width at half maximum ( fwhm ) and cbf is derived from the central volume equation . 
a il modello del maximum slope calcola il cbv integrando larea sotto la curva densit / tempo , il cbf secondo la tangente alla porzione ascendente della curva , e deriva il calcolo del mtt dallequazione del volume centrale . 
b il modello di deconvolution , tramite un arterial input function , trasforma la curva densit / tempo in una curva di funzione tissutale , in cui lintegrale dellarea sotto la curva esprime il cbv , il tempo alla met dellaltezza massima ( full width at half maximum fwhm ) rappresenta il mtt ed il cbf derivato dallequazione del volume centrale . 
the deconvolution method relies on a complex mathematical process that requires the use of an aif that is , the time - density curve of an artery with which to compare the curve obtained on the parenchymal pixels so as to correct for the effects of bolus dispersion and thus better reflect the postulates of the tracer kinetic model , which assumes a virtually instantaneous bolus input [ 14 ]  . 
the deconvolution model allows one to reduce the contrast injection rate ( 35 ml / s ) and provides quantitative measurements [ 1517 ] that have been validated by comparison with pet [ 18 ] , spect [ 19 ] and xe - ct [ 20 ]  . although some commercial software packages can automatically select the best aif , aif choice is one of the most controversial aspects of p - ct . 
partial - volume artefacts are practically unavoidable , as the imaging section has a thickness of 10 mm and the artery to be selected generally has a calibre of 24 mthe problem can be partially avoided by choosing an artery that runs perpendicular to the section for example , segment a2 of the anterior cerebral artery so that the roi is placed on a pixel corresponding to a voxel almost completely taken up by the vessel . 
more complex , in the case of cerebral ischaemia , is the problem of selecting an intracranial vessel that is ipsilateral or contralateral to the lesion ( generally segment m2 of the middle cerebral artery ) , or a vessel with proximal stenosis , generally at an extracranial location . 
although there is no consensus [ 21 , 22 ] and studies are underway , our advice is to choose the aif from a healthy vessel , often the anterior cerebral artery , and then compare the results with those obtained with an aif selected on the abnormal side . 
 the software then requires selection of a venous roi , which provides a reference measurement of blood density , considered free of partial - volume artefacts , such as the posterior descending portion of the superior sagittal sinus . 
this curve is used to derive mtt , identified by the width of the curve at spondenza dei pixel parenchimali , per correggere matematicamente gli effetti della dispersione del bolo e riavvicinarsi cos ai postulati teorici del kinetic tracer model , che , come detto , presuppone unimmissione pressoch istantanea del bolo [ 14 ]  . 
il modello di deconvoluzione permette di ridurre le velocit di somministrazione del bolo di mdc ( 35 ml / s ) e fornisce misurazioni quantitative [ 1517 ] , validate in studi di confronto con pet [ 18 ] , spect [ 19 ] e xetc [ 20 ]  . 
gli artefatti da volume parziale sono praticamente inevitabili , infatti , la sezione su cui si lavora ha uno spessore di 10 mm e larteria da selezionare ha generalmente un calibro di 24 mm ; per ovviare parzialmente a tale problema , si pu selezionare unarteria con decorso perpendicolare alla sezione , come nel caso del tratto a2 dellarteria cerebrale anteriore , in modo da posizionare la nostra roi su un pixel corrispondente ad un voxel quasi interamente occupato dal vaso . 
sebbene non esista definitivo consenso [ 21 , 22 ] , e siano ancora in corso studi al riguardo , il nostro consiglio di scegliere laif da un vaso ritenuto sano , spesso larteria cerebrale anteriore , e poi confrontare i risultati con quelli ottenuti con unaif scelta sul lato anormale . il software richiede poi la selezione di una roi venosa , che serve come misurazione di riferimento della densit del sangue , e viene considerata come esente da artefatti di volume parziale , come il caso del seno sagittale superiore nella sua porzione posteriore discendente . 
i parametri perfusionali sono il volume ematico cerebrale ( cerebral blood volume o cbv ) , il tempo medio di transito ( mean transit time o mtt ) ed il flusso ematico cerebrale ( cerebral blood flow o cbf ) ; alcuni software , inoltre , calcolano il tempo di picco ( time to peak o ttp )  . 
in this patient with severe stenosis of the proximal m1 segment of the right main carotid artery ( mca ) , mtt and cbf maps derived from an arterial input function ( aif ) on the anterior carotid artery ( aca ) ( healthy side ) show abnormal measurements in the right mca territory ( left column ) , whereas mtt and cbf measurements derived from an aif distal to the stenosis appear no longer abnormal ( right column )  . 
in questo paziente con stenosi serrata della porzione prossimale del tratto m1 dellacm di destra , la scelta dellaif sullaca ( lato sano ) produce mappe colore di mtt e cbf marcatamente alterate nel territorio di distribuzione dellacm destra ( colonna sinistra ) , mentre le mappe di mtt e cbf derivate da unaif sullacm destra , a valle della stenosi , appaiono sostanzialmente simmetriche ( colonna destra )  . 
5 software analysis of the arterial ( red ) and venous ( blue ) time - density curves generates the pixel - based parametric maps for mean transit time ( mtt ) , cerebral blood volume ( cbv ) and cerebral blood flow ( cbf )  . 
5 lanalisi matematica operata dal software sulla base della curva arteriosa ( in rosso ) e della curva venosa ( in blu ) genera le mappe parametriche con codice colore pixel per pixel per il mtt , il cbv ed il cbf . 
come accennato sopra , il pixel venoso , scelto da una struttura vascolare ampia , considerato essere esente da volume parziale , e quindi si assume la sua densit come unit di riferimento per il sangue ( 100 ml sangue / 100 g di tessuto ) ; la densit misurata nei singoli pixel tissutali viene poi rapportata alla misurazione venosa , ed espressa in percentuale di questa ( cbv = densit pixel parenchimale / densit pixel venoso ; es , densit nel pixel tissutale = 5% della densit nel pixel venoso cbv del pixel tissutale = 5 ml / 100 g ) ; ora pi chiaro come la sottostima della densit nella roi venosa , per artefatti di volume parziale , causi una sovrastima del cbv a livello tissutale e di riflesso , per lequazione del volume centrale cbf = cbv / mtt , anche del cbf . il cbf misura il volume di sangue distribuito a livello tissutale nellunit di tempo . 
6 in order to obtain quantitative measurements of cerebral blood volume ( cbv ) , cerebral blood flow ( cbf ) , mean transit time ( mtt ) and time to peak ( ttp ) , regions of interest ( rois ) are drawn on the different intracranial vascular territories . 
6 mediante posizionamento di apposite roi si possono ottenere misurazioni quantitative medie del cbv , cbf , mtt e ttp nei diversi territori di distribuzione delle principali arterie intra - craniche . 
it is expressed in [ s ] and is an index of the time that elapses between the beginning of contrast administration and maximum enhancement , or enhancement peak , in the roi . 
 mtt represents capillary transit time , that is , the time between arterial input in the capillary bed and venous outflow . it is expressed in [ s ] , is an sensitive index of cerebral perfusion pressure and , thanks to deconvolution , it is relatively less affected than is ttp by central haemodynamic changes such as cardiac failure or stenosis of the epiaortic arteries . mtt calculation is strictly dependent on the deconvolution process and therefore , in the event of asymmetrical perfusion as in ischaemia , on the choice of aif . 
in practice , mtt may be considered as the difference between the mean width of the time - density curve in a parenchymal pixel and the width of the curve in the reference artery ( aif ) [ 23 ]  . cbv measures the volume of cerebral blood at the capillary - tissue level , after appropriate exclusion from measurements of blood volume contained in large vessels , by means of density thresholds . 
the density measured in the single tissue pixels is then related to the venous density and expressed as a percentage of this ( cbv = parenchymal pixel density / venous pixel density ; e.g. : tissue pixel density = 5% venous pixel density = > cbv of tissue pixel = 5 ml / 100 g )  . 
it should now be clearer why an underestimation of density in the venous roi , due to partial - volume effects , leads to an overestimation of the cbv at the tissue level and consequently of cbf , given the central volume theorem cbf = cbv / mtt . cbf measures the volume of blood distributed at the tissue level in a unit of time . 
it is not measured directly but derived from the central volume theorem and is therefore dependent both on deconvolution and aif selection , which affect mtt , and on cbv measurement . 
 in brief , mtt indicates perfusion pressure , cbv reflects autoregulation mechanisms and capillary volume and cbf is the result of the above parameters . clinical applications acute ischaemic stroke thrombolysis is an approved therapy for acute ischaemic stroke [ 2 , 24 , 25 ] that aims to reperfuse the areas of ischaemic penumbra and limit the extent of the final area of infarction , thus reducing morbidity and disabling sequelae . the current indications for thrombolysis are based on a time window from symptom onset ( < 3h ) , findings of an unenhanced head ct ( no intracranial haemorrhage , no ct evidence of ischaemia of a portion of parenchyma exceeding 33% of the middle cerebral artery territory ) and absence of general contraindications for the drug [ 2 , 24 ]  . 
nevertheless , even such stringent criteria do not eliminate the significant risk of brain haemorrhage ( 15% ) [ 26 ] , and patient selection based on such criteria appears to be unsatisfactory . 
it is recognised that the time window alone is insufficient for accounting for the complex model of brain tissue viability , which in addition to a time factor includes a haemodynamic factor , a tissue factor and an intervention factor [ 27 ]  . 
as a result , individual visualisation of the extent of the infarcted and at - risk areas and of their relationship according to the pathophysiological model of infarct core and ischaemic penumbra has been proposed as a better tool for selecting patients for thrombolytic therapy . 
thrombolysis in patients with extensive cerebral infarctions and small areas of penumbra appears to be of little benefit and lead to a high risk of haemorrhage [ 25 , 28 , 29 ]  . 
on the other hand , although controversial , some studies have demonstrated that a significant proportion of patients with 1232 applicazioni cliniche ictus ischemico acuto la trombolisi una terapia approvata per lictus ischemico acuto [ 2 , 24 , 25 ] che ha lo scopo di riperfondere le aree di penombra ischemica e di ridurre lestensione dellarea finale di infarto , riducendo cos la morbilit e gli esiti invalidanti . 
le attuali indicazioni alla terapia trombolitica sono basate su una finestra temporale dallesordio clinico ( < 3 h ) , sul risultato della tc cranio in condizioni di base ( assenza di emorragia intracranica , assenza di segni tc di ischemia di una porzione di parenchima con estensione superiore al 33% del territorio vascolare dellarteria cerebrale media ) e sullassenza di controindicazioni generali al farmaco [ 2 , 24 ]  . la maggior parte dei pazienti colpiti da ictus ischemico non ricevono trattamento , poich i criteri di eligibilit sono molto restrittivi , soprattutto rispetto alla finestra temporale , anche perch in una significativa percentuale di casi il momento di insorgenza dei sintomi non determinabile con precisione ( classico il caso del cosiddetto wake - up stroke )  . 
ciononostante , perfino criteri cos restrittivi non eliminano il significativo rischio di emorragia intracranica ( 15% ) [ 26 ] e la selezione dei pazienti a cui somministrare la terapia trombolitica , basata su tali criteri , non appare soddisfacente . 
la finestra temporale infatti stata riconosciuta insufficiente a spiegare , da sola , lintricato modello della vitalit del tessuto cerebrale , che invece include , oltre al fattore tempo , un fattore emodinamico , un fattore tissutale ed un fattore terapeutico [ 27 ]  . 
perci la visualizzazione individuale dellestensione delle aree infartuate e di quelle a rischio , e del loro rapporto , secondo il modello fisiopatologico del core infartuale e della penombra ischemica , stato suggerito come un migliore strumento di selezione dei pazienti per il trattamento trombolitico . 
la trombolisi in pazienti con infarti cerebrali estesi e piccole aree di penombra , sarebbe di scarso beneficio e alto rischio emorragico [ 25 , 28 , 29 ] , mentre stato postulato e dimostrato in alcuni studi , ma ancora controverso , che una significativa percentuale di pazienti con limitate aree di infarto ed estese aree di penombra potrebbero beneficiare del trattamento di rivascolarizzazione anche oltre la classica finestra temporale delle 3 ore [ 30 , 31 ]  . il modello del core e della penombra [ 3234 ] postula che nel contesto di unarea di parenchima ipoperfusa , con elevati valori di mtt , spesso dipendente da circoli collaterali , si attuino meccanismi di autoregolazione che , inducendo vasodilatazione ed aumento del cbv , siano capaci di mantenere i valori del cbf sopra la soglia della morte cellulare neuronale ; le aree di parenchima caratterizzate da tali condizioni sono ritenute aree di penombra ischemica , ancora vitali , ad alto rischio di infarto , che potrebbero beneficiare di interventi di riperfusione . 
 limited areas of infarction and extensive areas of penumbra might benefit from revascularisation even beyond the 3 - h time window [ 30 , 31 ]  . the model of the infarct core and penumbra [ 3234 ] postulates that in the context of an area of hypoperfused parenchyma , with high mtt values , often supplied by collateral circulations , autoregulation mechanisms take place that , by inducing vasodilatation and increased cbv , are able to maintain cbf values above the threshold for neuronal death . 
where ischaemic injury is more severe and protracted , on the other hand , autoregulation is unable to maintain cbf , brain tissue sustains irreversible hypoxic damage and cbv values decrease . 
however , these pathophysiological aspects are complicated by other factors such as the duration of hypoperfusion , selective vulnerability of some neurons and physiological conditions during reperfusion [ 38 , 32 ]  . 
radiological evidence of the existence of a penumbra area has been provided by mri with combined diffusionand perfusion - weighted imaging ( dwipwi ) , the results of which have led to the pwi - dwi mismatch hypothesis [ 34 ]  . unenhanced head ct remains the first imaging modality in the assessment of patients with acute focal neurological symptoms ( stroke ) to exclude brain haemorrhage and evaluate the presence of early signs of ischaemia , such as greymatter hypoattenuation and sulcal effacement [ 35 ]  . it has been suggested that , by identifying the areas of cerebral infarction and those at risk of infarction ( ischaemic penumbra ) , the study of brain perfusion may help to select patients for thrombolytic therapy [ 15 ]  . 
other techniques for determining brain perfusion , such as xe - ct , spect and pet , although quantitative , are limited by poor availability in emergency settings and poor patient tolerance , whereas perfusion mri also suffers from an inability to provide quantitative data [ 3 ]  . 
availability in emergency settings , rapidity ( data acquisition < 5 min ) , cost and quantitative measurements probably make p - ct the technique of choice in the assessment of patients with ischaemic stroke [ 16 ]  . 
this ability of p - ct is based on the hypothesis that infarcted areas will exhibit high mtt values and low cbf and cbv values , whereas the areas at risk will show normal or increased cbv values [ 4 , 1517 , 21 , 39 , 40 ]  . 
cbv maps seem to be the least sensitive for ischaemia , with approximately 25% of acute infarctions failing to show any significant change in cbv , but are also the most specific for infarction areas [ 16 , 41 ]  . 
levidenza radiologica dellesistenza della penombra ischemica invece , stata dimostrata da studi combinati di rm con sequenze di diffusione ( dwi ) e perfusione ( pwi ) , dai cui risultati si derivato il concetto di mismatch pwi - dwi [ 34 ]  . la tc cerebrale in condizioni di base rimane la prima modalit di imaging usata per lo studio del paziente con sintomatologia neurologica focale acuta ( ictus ) , per escludere lemorragia intracranica e valutare la presenza di segni precoci di ischemia , quali lipodensit della sostanza grigia e la cancellazione dei solchi [ 35 ]  . stato suggerito che lo studio della perfusione cerebrale , identificando le aree di infarto cerebrale e quelle a rischio stage 1 stage 2 stage 3 cerebral perfusion pressure fig . 
7 the diagram illustrates the changes in mean transit time ( mtt ) , cerebral blood volume ( cbv ) , cerebral blood flow ( cbf ) and oxygen extraction fraction ( oxef ) in response to progressive reduction of cerebral perfusion pressure . 
in patients with stroke , the scan level is usually located in correspondence with the basal ganglia so as to visualise the three main supratentorial vascular territories ( fig 9 )  . 
one limitation of p - ct , especially when compared with mri , is a relative lack of sensitivity to lacunar infarctions [ 44 ] , exposure to ionising radiation and the use of iodinated contrast material . 
nonetheless , administration of iodinated nonionic di infarto ( aree di penombra ischemica ) , possa essere di ausilio nella selezione di pazienti candidati alla terapia trombolitica [ 15 ]  . 
le altre tecniche per misurare la perfusione cerebrale , quali xe - tc , spect e pet , sebbene quantitative , sono limitate dalla disponibilit in emergenza e dalla tolleranza del paziente , mentre la perfusione - rm soffre anche dellimpossibilit di fornire dati quantitativi [ 3 ]  . 
la ptc probabilmente la metodica di scelta , per disponibilit in condizioni di emergenza , per rapidit di esame ( acquisizione dati in < 5 min ) , costi contenuti , e misurazioni quantitative , nella valutazione del paziente con ictus ischemico [ 16 ]  . 
tale capacit della p - tc si basa sullipotesi che le aree infartuate presentino elevati valori di mtt , e ridotti valori di cbf e cbv , mentre nelle aree a rischio i valori di cbv siano normali o aumentati [ 4 , 1517 , 21 , 39 , 40 ]  . 
recenti studi [ 16 , 41 ] suggeriscono che le mappe di mtt siano le pi sensibili per delineare le aree ischemiche , seguite da quelle del cbf ; le mappe di cbv sembrano essere le meno sensibili allischemia , infatti , circa il 25% degli infarti acuti non mostra significative alterazioni di tale parametro , ma sono anche le pi specifiche per le aree di infarto [ 16 , 41 ]  . 
da ci deriva la raccomandazione di valutare prima le mappe di mtt e cbf alla ricerca di aree ipoperfuse , e se queste sono presenti , utilizzare le mappe di cbv per differenziare al loro interno le aree di penombra da quelle di infarto , considerando per che tali mappe possono essere normali anche in caso di infarto . 
 un importante studio prospettico [ 15 ] su pazienti affetti da ischemia cerebrale acuta , segna una pietra miliare nella validazione delle teorie su cui si basa la p - tc , mostrando significativa correlazione tra aree di ridotto cbv ( > 2 , 5 ml / 100 g ) alla p - tc di ammissione e le aree finali di infarto nei controlli a distanza , in pazienti con riperfusione , e altrettanto precisa correlazione tra aree con prolungato mtt ( > 1 , 5 volte rispetto al controlato ) e / o ridotto cbf ( riduzione > 34% rispetto al controlato ) alla p - tc di ammissione e larea finale di infarto , al controllo a distanza , in pazienti senza riperfusione . 
based on threshold values set by the operator , the software generates a colour map ( middle image in the bottom row ) where the colour green is attributed to pixels with increased mean transit time ( mtt ) , as is the case in ischaemic penumbra , and the colour red is attributed to pixels with low cerebral blood volume ( cbv ) , as is the case in the infarct core . 
sulla base di valori soglia predeterminati , il software attribuisce , nella mappa al centro nella fila in basso , il colore verde ai pixel con alterazioni perfusionali con le caratteristiche dei territori di penombra , ed il colore rosso ai pixel con caratteristiche di core infartuale . 
qualora si voglia raddoppiare la copertura anatomica , una seconda sca , nsione , ad un altro livello , pu essere effettuata osservando unattesa , rispetto alla scansione precedente , di 36 mun limite della p - tc , soprattutto in confronto alla rm , la relativa insensibilit agli infarti lacunari [ 44 ] , lesposizione alle radiazioni ionizzanti , e luso del mdc iodato . 
la somministrazione di mdc iodato non ionico in emergenza , anche senza preventiva valutazione della creatininemia , comunque risultata sicura in una larga serie di pazienti con ictus cerebrale acuto , sottoposti a p - tc , con esclusione dei pazienti con insufficienza renale nota , paraproteinemia nota , trapianto renale , assunzione di farmaci nefrotossici , e / o lunga storia di diabete mellito [ 45 ]  . in conclusione , sebbene la validit delle misurazioni quantitative della p - tc sia messa in discussione dalla loro variabilit legata alla scelta dellaif , al posizionamento delle rois ed agli artefatti da volume parziale [ 46 ] e sebbene ulteriori studi siano necessari per confrontare le misurazioni della p - tc con quelle delle altre metodiche di studio della perfusione cerebrale , i risultati sinora disponibili appaiono incoraggianti riguardo alla selezione di pazienti con estesa area di penombra ischemica , e quindi potenzialmente buoni candidati per la terapia trombolitica , ed inoltre , le misurazioni qualitative relative al controlato , offrendo dati affidabili , restano una valida alternativa , comunque indicativa dello stato della perfusionale cerebrale [ 40 ]  . infine utile qui ricordare che linterpretazione delle mappe di perfusione sicuramente facilitata e resa pi accurata dalla disponibilit di informazioni sullo stato anatomico vascolare intraed extra - cranico . 
con lavvento della tecnologia tc multidetettore , langio - tc , eseguita subito dopo la p - tc , con la somministrazione di un ulteriore bolo di 7090 ml di mdc , permette unaccurata valutazione della perviet delle arterie epiaortiche ed intra - craniche , nonch della presenza di circoli collaterali nelle patologie cerebrovascolari acute e pu avere un ruolo significativo nella selezione dei pazienti con ictus ischemico , ai fini di un corretto 1235 fig . 
9 un piano di scansione orientato lungo il tetto delle orbite , e posizionato circa 2 cm cranialmente a queste , consente la visualizzazione , nelle due fette di 10 mm , di una rappresentazione dei tre territori vascolari sopratentoriali : il territorio dellarteria cerebrale anteriore , della media e della posteriore . contrast material , even without preliminary serum creatinine assessment , proved to be safe in a large series of patients with acute cerebral stroke studied by emergency p - ct , after exclusion of those with known renal failure , known paraproteinaemia , kidney transplant , use of nephrotoxic drugs or a long - standing history of diabetes mellitus [ 45 ]  . in conclusion , although the validity of the quantitative measurements of p - ct has been questioned owing to variability related to aif choice , roi positioning and partialvolume artefacts [ 46 ] ; and although further studies are needed to compare p - ct measurements with those provided by other techniques for the study of brain perfusion , encouraging results have been obtained with regard to the selection of patients with an extensive area of ischaemic penumbra and therefore potential candidates for thrombolytic therapy . 
in addition , qualitative measurements of the contralateral side provide reliable data and offer a valuable alternative that is at any rate indicative of the state of cerebral perfusion [ 40 ]  . finally , it is useful to remember that the interpretation of perfusion maps is definitely facilitated and made more accurate by the availability of information on the state of the intraand extracranial vascular anatomy . 
with the advent of mdct technology , ct angiography , performed with the administration of an additional bolus of 7090 ml of contrast immediately after p - ct , allows accurate assessment of the patency of the epiaortic and intracranial arteries and of the presence of collateral circulations in acute cerebrovascular disease . 
in a context of acute cerebrovascular disease , p - ct combined with ct angiography therefore provides a rapid , efficient and practical tool for identifying the cause , extent and pathophysiology of ischaemia , nd for optimising treatment decisions . chronic ischaemic cerebral disease ( vascular reserve ) in patients with chronic cerebral ischaemia caused by stenosis of the intracranial arteries or epiaortic vessels , it is neca . 
nel contesto della patologia cerebro - vascolare acuta , quindi , la p - tc , combinata con langio - tc , rappresenta una soluzione rapida , efficiente e pratica per diagnosticare leziologia , lestensione e la fisiopatologia dellischemia , cos da ottimizzare la scelta terapeutica . patologia cerebrale ischemica cronica ( riserva vascolare ) nei pazienti con ischemia cerebrale cronica causata da stenosi arteriose intra - craniche o dei vasi epi - aortici , necessario distinguere le condizioni di stabile compenso emodinamico , dalle situazioni in cui il compenso instabile , ed il parenchima cerebrale sottoposto ad uno stress emodinamico e metabolico [ 48 ]  . 
tenendo presente il principio del volume centrale ( cbf = cbv / mtt ) , tipicamente , una stenosi arteriosa emodinamicamente significativa induce un prolungamento del mtt che innesca a sua volta i meccanismi dellautoregolazione cerebrale , con conseguente vasodilatazione , riflessa dallincremento del cbv , con cbf normale o lievemente diminuito . 
questo quadro perfusionale tipico per non fornisce informazioni sulla gravit dellipoperfusione , n sulle capacit residue del circolo cerebrale di adattarsi ad ulteriori modificazioni emodinamiche , ovvero sullesistenza di una riserva cerebro - vascolare . 
la valutazione della riserva cerebro - vascolare si pu ottenere osservando come la perfusione cerebrale reagisce in risposta ad uno stress emodinamico , appositamente provocato mediante somministrazione di un agente vasoattivo . 
i pazienti che hanno esaurito la riserva cerebro - vascolare presentano gi in condizioni di base vasodilatazione massima nelle aree ipoperfuse e non sono quindi in grado di rispondere allo stress emodinamico indotto dallacetazolamide . 
si ritiene che tali pazienti siano a rischio elevato di infarto cerebrale e possano beneficiare di interventi di rivascolarizzazione per aumentare il cbf nelle aree ipoperfuse [ 48 ]  . 
di 1000 mg di acetazolamide pu individuare i pazienti a rischio di infarto tra i portatori di stenosi arteriose epi - aortiche o intra - craniche [ 48 ]  . 
dopo la somministrazione di acetazolamide un incremento del cbf pari al 20%40% rispetto alle condizioni di base considerato normale , un incremento di meno del 5% considerato indicativo di insufficienza emodinamica , mentre la riduzione paradossa del cbf rispetto alla condizione di base segno di furto vascolare , indicativo di tessuto ad alto rischio di infarto [ 49 ]  . 
lacetazolamide generalmente ben tollerata ; sono stati descritti effetti collaterali lievi quali parestesie e cefalea e sebbene siano stati riportati casi di attacchi ischemici transitori , leventuale ischemia indotta sempre stata reversibile [ 48 ]  . 
10 computed tomography angiography ( cta ) , used to complement perfusion ct in this patient with acute right hemiplegia and aphasia , clearly shows left m1 occlusion ( red arrow on exam at 2.5 h ) , which is the cause of the perfusion abnormalities visible on the colour map . 
10 langio - tc cerebrale , esame complementare alla perfusione - tc , dimostra chiaramente , in questo paziente con esordio acuto di emiplegia destra ed afasia , locclusione del tratto m1 dellarteria cerebrale media di sinistra ( freccia rossa nellesame a 2 , 5 h ) , responsabile delle alterazioni perfusionali evidenziate dalla mappa colore . 
il controllo angio - tc a 24 h di distanza , dopo trattamento fibrinolitico , dimostra la ricanalizzazione del vaso ( freccia rossa )  . essary to distinguish between situations of stable haemodynamic compensation and those in which compensation is unstable and the brain parenchyma is subject to haemodynamic and metabolic stress [ 48 ]  . 
bearing in mind the central volume theorem ( cbf = cbv / mtt ) , a haemodynamically significant arterial stenosis will typically result in a prolonged mtt , which in turn will trigger cerebral autoregulation mechanisms , with consequent vasodilatation reflected by an increase in cbv , with normal or slightly decreased cbf . 
this typical perfusion pattern does not , however , give information on the severity of hypoperfusion or on the residual capacity of the cerebral circulation to adjust to further haemodynamic changes in other words , on the existence of a cerebrovascular reserve . 
patients who have exhausted the cerebrovascular reserve show maximal vasodilatation in the hypoperfused areas already at baseline and are therefore unable to respond to the haemodynamic stress induced by acetazolamide . these patients are believed to be at high risk for cerebral infarction and to benefit from revascularisation procedures to increase cbf in hypoperfused areas [ 48 ]  . 
11 perfusion - computed tomography ( p - ct ) before and after right internal carotid endarterectomy.in this patient with mild but progressive left hemisyndrome , ct angiography and p - ct ( upper row ) show severe right internal carotid stenosis causing hypoperfusion of the whole right cerebral hemisphere ( there is a foetal origin of the right posterior cerebral artery , not shown in the figure )  . 
lo studio angio - tc e p - tc ( fila superiore ) , in questo paziente con emisindrome sn lieve ma ingravescente , mostra stenosi serrata del bulbo carotideo destro , cui si associa ipoperfusione di gran parte dellemisfero cerebrale destro ( il paziente portatore di unorigine fetale dellarteria cerebrale posteriore destra , rifornita per via comunicante posteriore ; non mostrata nella figura ) , testimoniata dal prolungamento del mtt , dalla riduzione del cbf e dal lieve aumento del cbv , espressione dei meccanismi di autoregolazione . 
il controllo angio - tc e p - tc ( fila inferiore ) eseguito tre giorni dopo lintervento di endoarterectomia carotidea destra documenta la ricostituzione del lume vascolare e la risoluzione del deficit perfusionale emisferico destro . those with stenosis of the epiaortic or intracranial arteries [ 48 ]  . 
an increase less than 5% is considered indicative of haemodynamic insufficiency , whereas a paradoxical reduction of cbf relative to baseline is a sign of vascular shunting and thus of tissue at a high risk of infarction [ 49 ]  . 
acetazolamide is generally well tolerated ; the reported side effects are mild , such as paraesthesia and headache , and although there have been cases of transient ischaemic attacks , the induced ischaemia was always reversible [ 48 ]  . 
angiographic evidence of vasospasm is present in 60%80% of patients with sah , whereas 30% of patients present symptoms of vasospasm , and half of these develop cerebral infarction [ 51 ]  . 
transcranial doppler ultrasound is the most emorragia sub - aracnoidea e vasospasmo il vasospasmo una complicanza frequente dellemorragia sub - aracnoidea ( esa ) nella fase subacuta precoce [ 6 ]  . levidenza angiografica di vasospasmo presente nel 60%80% dei pazienti con esa , il 30% ne presenta i sintomi , e la met di questi sviluppa un infarto cerebrale [ 51 ]  . leco - doppler transcranico la modalit pi usata per il follow - up non invasivo in questi pazienti , per guidare le scelte terapeutiche e controllarne lefficacia , ma , oltre ad essere operatore dipendente , non offre misurazioni quantitative a livello tissutale ed gravata da scarsa specificit [ 52 ]  . 
anche se non sono ancora disponibili studi in grado di fornire valori di soglia dei parametri di perfusione , la misurazione del cbf , cbv e mtt con p - tc , associata a studi anatomici vascolari con angio - tc , potrebbe rappresentare un utile strumento diagnostico per il follow - up non invasivo dei pazienti con vasospasmo conseguente ad esa [ 6 ] , fornendo informazioni utili al trattamento medico ed anche alla scelta della tempistica chirurgica . trauma cranico la predizione della prognosi nei pazienti con trauma cranico severo rimane un compito complesso e controverso . 
however , it is operator dependent , unable to provide quantitative measurements at the tissue level and suffers from limited specificity [ 52 ]  . even though no study has yet been able to provide threshold values for the perfusion parameters , p - ct measurement of cbf , cbv and mtt , combined with vascular anatomy studies with ct angiography , might represent a valuable diagnostic tool for the noninvasive follow - up of patients with sah - related vasospasm [ 6 ] , as it provides information useful for medical management of patients and for choosing the best timing for surgery . head trauma predicting outcomes in patients with severe head trauma remains a complex and controversial issue . 
a finding of cerebral hypoperfusion , in generally hypotensive patients , indicates a loss of cerebrovascular autoregulation and is thought to be a predictor of the development of cerebral oedema , whereas normal or increased perfusion values would indicate a positive outcome [ 54 ]  . brain tumours in the study of brain tumours , there is growing interest in the noninvasive assessment of tumour vascularity [ 55 ]  . 
some brain tumours are characterised by neoangiogenesis and hypervascularity , which result in increased microvascular permeability [ measured as permeability surface area product ( ps ) or as contrast transfer coefficient ( k - trans ) , in ml / 100g per minute ] and increased cbv , due to the presence of numerous tortuous vascular structures , with immature , disrupted or absent bbb [ 56 ]  . 
contrast enhancement , the key diagnostic element in morphological imaging , is a poorly specific finding that in tumours may be caused by bbb abnormalities , hypervascularity , extent of the interstitial space or a combination of these factors . 
if the extreme disruption of microvascular permeability of some tumours causes excessive leakage of contrast material during the first pass of 1238 zionale possiede alta sensibilit nellindividuazione di lesioni intra - craniche che necessitano di intervento neurochirurgico , ma non offre validi elementi di tipo prognostico . 
il riscontro di ipoperfusione cerebrale , in pazienti generalmente ipotesi , indica la perdita dellautoregolazione cerebro - vascolare e sarebbe in grado di predire lo sviluppo di edema cerebrale , mentre valori di perfusione normali o aumentati rappresenterebbero indicatori prognostici positivi [ 54 ]  . tumori intra - cranici nello studio delle neoplasie intra - craniche crescente interesse rivolto alla valutazione non - invasiva della vascolarizzazione tumorale [ 55 ]  . 
alcuni tumori intracranici sono caratterizzati da neo - angiogenesi ed ipervascolarizzazione , che risultano in aumentata permeabilit vascolare ( misurata come permeability surface product ( ps ) o come coefficiente di trasferimento di contrasto ( k - trans ) , in [ ml / 100 g / min ] ) , e incremento del cbv , per la presenza di strutture vascolari pi numerose , tortuose , con barriera emato - encefalica immatura , danneggiata o assente [ 56 ]  . 
il contrastenhancement , elemento diagnostico chiave dellimaging morfologico , un reperto poco specifico , che nei tumori pu essere determinato dallalterazione della bee , dallipervascolarizzazione , dallampiezza dello spazio interstiziale o dalla concomitanza dei suddetti fattori . 
numerosi studi [ 55 , 56 ] dimostrano che nei gliomi , la ps ed il cbv , misurati con tecniche di rm - perfusione ( con sequenze contrast - enhanced t2 * - w , basate sulla suscettibilit magnetica , e contrast - enhanced t1 - w ) , correlano con lindice mitotico , il grading istologico e laggressivit biologica . 
se lestrema alterazione della permeabilit vascolare in alcuni tumori causa uno stravaso eccessivo del mezzo di contrasto gi durante il primo passaggio del bolo , le misurazioni del cbv , per essere accurate , necessitano di appropriate strategie di correzione ( ad esempio procedure di gamma - fitting della curva densit - tempo )  . 
in tali tumori , ipervascolarizzati e con alterata permeabilit , le misurazioni della ps o del k - trans , sembrano essere pi utili , perch correlano pi strettamente con il grado dei glomi . 
inoltre anche la risposta alle terapie anti - angiogenetiche recentemente introdotte , con riduzione della crescita tumorale , trova il suo corrispettivo in una riduzione dei valori di ps [ 57 ]  . 
in this patient with head trauma and left parietal bone fracture , p - ct shows a large area of hypoperfused brain parenchyma in the left temporoparietooccipital region , well beyond the limits of the hypodense contusion documented by enhanced ct . 
in questo paziente con trauma cranico e frattura parietale sinistra lo studio p - tc mostra una vasta area di ipoperfusione temporo - parieto - occipitale sn , caratterizzata da aumentati valori di mtt e ridotti valori di cbf e cbv , estesa ben oltre i limiti della lesione contusiva ipodensa temporo - parietale sinistra , adiacente alla frattura , visibile nellesame tc morfologico . 
in such hypervascular tumours with disrupted permeability , ps or k - trans measurements appear to be more useful , as they correlate more closely with the grade of gliomas . 
however , in the subgroup of highgrade gliomas [ world health organisation ( who ) iii - iv ] , k - trans was directly related to length of survival . 
measurement of bbb permeability by p - ct or p - mri is based on the same mathematical models , the most frequently used of which is the patlak model [ 59 ]  . 
this model describes a method to measure the transfer - rate constant of contrast material from the vessel to the interstitium , through a mathematical comparison ( deconvolution ) between the timedensity curve or a time - signal curve obtained in tumour tiscon la sopravvivenza ; questa evidenza viene spiegata dagli autori come possibile effetto dellaumentata permeabilit vascolare nel tumore , che permetterebbe una migliore penetrazione degli agenti chemioterapici ed anti - angiogenici . 
la misurazione della permeabilit della bee ottenuta con ptc o con p - rm si fonda sulluso degli stessi modelli matematici , tra i quali , uno dei pi usati il modello di patlak [ 59 ]  . 
questo modello descrive un metodo per misurare la costante di trasferimento del mezzo di contrasto dal vaso allinterstizio , operando un processo di confronto matematico ( deconvoluzione ) tra una curva densit - tempo o segnale - tempo rilevata nel tessuto tumorale ( dove presente stravaso di mdc ) ed una curva rilevata nel lume di unarteria di riferimento o aif ( dove si assume non avvenga stravaso di mdc )  . 
le tecniche di imaging di perfusione trovano utilit nello studio dei tumori intra - cranici nel tentativo di facilitare la distinzione tra tumore primitivo e metastatico , nel rendere pi accurato il grading , nella valutazione della risposta alle terapie antiangiogenetiche , e nella diagnosi differenziale tra radionecrosi e recidiva tumorale ; inoltre si sta affermando il ruolo dellimaging perfusionale per la scelta del bersaglio delle procedure bioptiche , scelta basata sulla individuazione delle aree tumorali a maggiore aggressivit 1239 a . 
perfusion - imaging techniques have been found useful in the study of brain tumours to facilitate the distinction between primary or metastatic tumour , make grading more accurate , evaluate response to antiangiogenic agents and differentiate radiation necrosis from tumour recurrence . 
in addition , the role of perfusion imaging is also gaining ground in guiding biopsy procedures , where the biopsy target is chosen based on identification of the most malignant area in a heterogeneous tumour [ 56 ]  . in contrast to p - mri , only a few small studies are available on the use of p - ct in human brain tumours [ 7 , 60 , 61 ]  . the examination is performed using a similar technique to that described for ischaemic disease , and the scan level corresponds to the tumour site . 
cbv and permeability measurements are made by positioning several small rois over the tumour mass , taking care to exclude large vascular structures from the measurements , and the measurements in the rois are compared with those of a mirror area on the contralateral side . 
the most recent scientific data , many of which are still unpublished , suggest that it is the maximum rather than the mean values of cbv and ps in the tumour that correlate with histopathological grading , as is also the case with histological assessment , which assigns a who grade on the basis of the most malignant portion of the tumour . in theory , p - ct could offer several advantages over pmri in terms of spatial resolution , insensitivity to paramagnetic susceptibility artefacts ( in cases with haemorrhage , calcifications , metallic implants ) , linear correlation between contrast concentration and density and quantitative cbv and permeability measurements , even in hypervascular tumours . in practice , however , no published study has compared the results of the two methods on representative series of patients with brain tumours . 
on the other hand , radiation dose , potential toxicity of iodinated contrast material and limited anatomical coverage of p - ct still justify the predominant use of mri , a modality that remains crucial in the morphological imaging of tumours [ 7 ]  . limitations , artefacts and controversies although the ability of p - ct to provide quantitative measurements has been confirmed by comparison with pet , spect and xe - ct , its accuracy has not yet been unquestionably demonstrated , probably as a result of heterogeneous study methodologies adopted by the various authors . 
furthermore , the quantitative measurements are dependent on several variables , such as the software used , bolus dispersion ( caused by too slow an injection rate , insufficient cardiac function , haemodynamically significant arterial stenoses ) , aif selection , partial - volume artefacts , the misleading contribution of 1240 nel contesto delleterogeneit della neoplasia [ 56 ]  . 
 contrariamente a quanto verificatosi per gli studi di rmperfusione , sono disponibili solo pochi e limitati studi sulluso della p - tc nei tumori cerebrali negli uomini [ 7 , 60 , 61 ]  . 
per la misurazione della permeabilit preferibile disporre di curve densit / tempo allequilibrio invece che limitate al primo passaggio del bolo ; quindi oltre i 50 s di scansione , si continuano ad acquisire immagini in modalit cine , per circa 3 min , al ritmo di 1 scansione ogni 15 s . 
le misurazioni del cbv e della permeabilit si effettuano posizionando multiple piccole roi nel contesto della massa tumorale , avendo cura di escludere dalla misurazione le grosse strutture vascolari , e si confrontano ad unarea speculare nel controlato . 
i pi recenti dati scientifici , molti ancora non pubblicati , suggeriscono che pi dei valori medi di cbv e ps nel contesto della lesione tumorale , siano i valori massimi a correlare con il grading istopatologico , cos come accade peraltro per lesame istologico , che assegna il grado who in base alla porzione del tumore a pi alta malignit . in teoria la p - tc potrebbe avere vantaggi rispetto alle tecniche di rm - perfusione , in termini di risoluzione spaziale , insensibilit agli artefatti di suscettibilit paramagnetica ( in casi con emorragia , calcificazioni , impianti metallici ) , correlazione lineare tra concentrazione di mdc e densit , e misurazioni quantitative di cbv e permeabilit , anche in tumori ipervascolari . 
daltra parte , la dose di radiazioni , la potenziale tossicit del mdc iodato , la limitata copertura anatomica , rappresentano fattori che ancora giustificano luso prevalente della rm , metodica comunque irrinunciabile nellimaging morfologico nei tumori [ 7 ]  . limitazioni , artefatti e controversie sebbene la capacit della p - tc di fornire misurazioni quantitative sia stata confermata , come precedentemente detto , da studi comparativi con pet , spect e xe - tc , la sua accuratezza non ancora stata provata in maniera incontrovertibile , probabilmente anche a causa di metodologie di studio non omogenee tra i diversi autori . 
le misurazioni quantitative sono inoltre dipendenti da multiple variabili , quali il software impiegato , la dispersione del bolo ( causata da uniniezione troppo lenta , uninsufficiente funzione cardiaca , stenosi arteriose emodinamicamente significative ) , la scelta dellaif , gli artefatti da volume parziale , lingannevole contributo del liquor e delle strutture vascolari alle misurazioni , il posizionamento delle roi , ed infine le significative differenze perfusionali tra sostanza grigia e sostanza bianca . 
although commercial software packages for analysis of perfusion data are relatively easy to use , operators need to master the issues inherent to the examination and postprocessing procedures and require training in using thedata derived from perfusion studies conducted by expert radiologists have been shown to have good reproducibility [ 62 ] , whereas no study has evaluated reproducibility of data among nonexpert radiologists . 
in patients with ischaemic stroke , thrombolytic therapy , when indicated , should be initiated as soon as possible , so diagnostic imaging needs to be timely , accurate and fast . 
complementation of a conventional ct study of the brain with pct and ct angiography usually requires 10 - 12 min , whereas postprocessing with automatic software takes no longer than 3 min to provide colour - coded parametric maps that can guide the clinical treatment decisions . 
the disadvantage of this small delay in imaging procedures is widely compensated for by the acquisition of diagnostic information of major clinical importance , above all in those patients in the 3to 6 - h time window and those with an unknown time interval from symptom onset of symptoms , who are currently excluded from treatment . 
 as is known , p - ct employs ionising radiation and potentially nephrotoxic , hyperosmolar and allergenic iodinated contrast material , so the use of the technique requires individual clinical assessment , appropriate indications and strategies to contain the radiation dose . 
it is therefore a valuable alternative to other modalities for the measurement of brain perfusion , compared to which it has the advantage of being readily available and accessible even in emergency settings . 
for this reason , p - ct proves most useful in acute ischaemic stroke where , used in combination with ct angiography , it offers a complete overview of the causes of hypoperfusion and its haemodynamic and pathophysiological effects on the brain in a fast , noninvasive manner . 
p - ct is also helpful in patients with other , acute or chronic , cerebrovascular diseases ; in the follow - up of patients with sah ; in preand postoperative assessment of patients undergoing cerebral revascularisation procedures ; and in brain tumours for diagnosis , grading , biopsy guidance and monitoring treatment response . further studies are required to establish the accuracy , reliability and reproducibility of the quantitative measurements , but available data appear encouraging and indicate a major clinical impact . di post - processing e di un periodo di training per il loro uso ; una buona riproducibilit stata dimostrata , nei dati estratti dagli studi di perfusione , tra radiologi esperti [ 62 ] , mentre studi di questo tipo non sono ancora stati condotti per valutare la riproducibilit tra medici non esperti . 
ulteriori svantaggi della p - tc , soprattutto nelle applicazioni che riguardano la patologia ischemica , come gi detto , sono la limitata copertura anatomica e la scarsa sensibilit verso gli infarti lacunari . 
nei pazienti colpiti da ictus ischemico di fondamentale importanza che il trattamento trombolitico sia iniziato , se indicato , il pi rapidamente possibile , ed quindi auspicabile che la fase diagnostica radiologica sia tempestiva , accurata e veloce . 
il completamento di un esame tc convenzionale dellencefalo con lo studio perfusionale e angiotc richiede solitamente 1012 minuti , mentre le procedure di post - processing , con software automatici , richiedono non pi di 3 minuti per ottenere mappe parametriche colore ai fini di decisioni clinico - terapeutiche ; solo valutazioni quantitative accurate , solitamente non necessarie in situazioni di emergenza , richiedono tempi di post - processing pi lunghi . 
lo svantaggio di questo modesto ritardo nelle procedure di imaging di solito ampiamente compensato dallacquisizione di informazioni diagnostiche di grande rilevanza clinica , soprattutto in quei pazienti nella finestra delle 36 ore ed in quelli con intervallo sconosciuto dallinsorgenza dei sintomi , attualmente esclusi dal trattamento . la p - tc , come ovvio , utilizza radiazioni ionizzanti , e mdc iodato potenzialmente nefrotossico , iperosmolare e allergenico , pertanto limpiego di tale metodica necessita di valutazione clinica individualizzata , indicazioni appropriate , e strategie di contenimento della dose di radiazioni . conclusioni in conclusione , la p - tc permette una valutazione quantitativa affidabile e veloce del cbf e del cbv , offrendo cos una visualizzazione diretta dei meccanismi di autoregolazione cerebrale , e ponendosi come valida alternativa alle altre modalit di misurazione della perfusione cerebrale , rispetto alle quali ha il maggior vantaggio di essere una tecnica prontamente disponibile ed accessibile , anche in condizioni di emergenza . 
per tale ragione la p - tc utile soprattutto nella patologia ischemica cerebrale acuta , situazione in cui , con il concomitante impiego dellangio - tc , offre in maniera rapida e non - invasiva , una completa panoramica della causa dellipoperfusione , nonch delle sue ripercussioni emodinamiche e fisiopatologiche sul parenchima cerebrale . 
krestin2 1dipartimento di radiologia e dipartimento cuore , imaging cardiovascolare non invasivo , azienda ospedaliera di parma , parma , italy 2dipartimento di radiologia e cardiologia , erasmus medical center , rotterdam , the netherlands 3dipartimento di radiologia , universit degli studi di verona , verona , italy 4dibimel , sezione di scienze radiologiche , universit di palermo , palermo , italy 5unit operativa di riabilitazione cardiovascolare , fondazione don gnocchi onlus , parma , italy correspondence to : f . 
cademartiri , imaging cardiovascolare non invasivo , dipartimento di radiologia , azienda ospedaliero - universitaria di parma , via gramsci 14 , i - 43100 parma , italy , tel . : + 39 - 052 - 1703222 , fax : + 39 - 052 - 1703630 , e - mail : filippocademartiri@hotmail.com received : 23 november 2006 / accepted : 19 march 2007 / published online : 13 december 2007 abstract purpose . 
in the 202 consecutive patients , the prevalence of anatomical variants was : left dominant circulation ( 7% ) , absent left main ( 5% ) , presence of intermediate branch ( 17% ) , aortic origin of conus branch ( 13% ) and circumflex origin of sinus node branch ( 15% )  . 
anatomical variants and anomalies of the coronary arteries are quite common and should be known and recognised promptly by the operators . key words 64 - slice ct coronary anatomy coronary artery variants coronary artery anomalies prevalence riassunto obiettivo . 
nei 202 pazienti consecutivi arruolati per lo studio la prevalenza delle varianti anatomiche risultata : dominanza sinistra ( 7% ) , tronco comune assente ( 5% ) , presenza di ramo intermedio ( 17% ) , origine aortica del ramo del cono ( 13% ) , origine dalla circonflessa dellarteria del nodo del seno ( 15% )  . 
le varianti e le anomalie coronariche sono un reperto molto comune che deve quindi essere riconosciuto agevolmente dalloperatore . parole chiave tc multistrato anatomia coronaria varianti delle arterie coronarie anomalie delle arterie coronarie prevalenza introduction introduzione effectively visualising the coronary arteries is a challenge , not only due to their rapid movement during the cardiac cycle , but also because of their small diameter , their tortuous la visualizzazione delle arterie coronarie rappresenta una sfida non solo per il loro rapido movimento durante il ciclo cardiaco , ma anche per il calibro ridotto , il decorso tortuo1117 f . 
recognising coronary artery anomalies is therefore important for correct diagnosis and treatment of patients , whether or not they are affected by atherosclerosis . the literature contains numerous descriptions of coronary artery anomalies based on conventional coronary angiography ( ca ) but still very few based on msct , and the latter are mostly review articles or case reports [ 35 ]  . the introduction of the technique allows a different anatomical approach that could change the perception of these variants . the aim of this study was to describe the prevalence of coronary artery variants and anomalies in a consecutive population of patients studied with ct coronary angiography ( ctca )  . materials and methods two hundred and two consecutive patients ( 146 men , 56 women , mean age 60 11 years , range 2183 ) with suspected coronary artery disease and already scheduled for conventional ca underwent ctca before ca in the context of a validation study of ctca . 
only patients with a sinus rhythm who had never undergone percutaneous angioplasty or bypass surgery and who were capable of holding their breath for 12 s were included in the study . 
the ethics committee in our department approved the study protocol , and all patients provided informed consent . patient preparation patients with a heart rate ( hr ) greater than 65 beats per minute ( bpm ) were administered 45 munutes before examination , in the absence of contraindications , a single oral dose of 100 mg metoprolol tartrate ( selokeen , astrazeneca pharmaceutics )  . 
apparecchiature di tomografia computerizzata multistrato ( tcms ) allo stato dellarte , ovvero a 64 strati , consentono di collocare la valutazione non invasiva delle arterie coronarie nella routine clinica in virt delle elevate prestazioni ed accuratezza della metodica [ 1 , 2 ]  . le anomalie delle arterie coronariche sono presenti alla nascita ma solo una piccola percentuale di esse si rende manifesta nei primi anni di vita . 
gran parte di esse infatti viene scoperta per caso durante langiografia coronarica o rappresenta addirittura solamente un puro reperto autoptico . ci nonostante alcune di esse possono manifestarsi con sintomi come angina pectoris , infarto miocardico , sincope , aritmie pi o meno severe o addirittura arresto cardiaco configurando il quadro di morte improvvisa . 
il riconoscimento di anomalie coronariche importante quindi per una diagnosi appropriata e per il trattamento del paziente , sia esso affetto o meno da malattia aterosclerotica . nella letteratura esistono delle descrizioni delle anomalie coronariche basate sulla ac mentre ne esistono ancora poche basate sulla tcms , peraltro per lo pi costituite da revisioni della letteratura o descrizione di singoli casi particolarmente significativi [ 35 ]  . 
lintroduzione di questa tecnologia consente un diverso approccio anatomico che pu mutare la percezione di queste varianti . pertanto lo scopo di questo lavoro quello di fornire una prevalenza di varianti ed anomalie coronariche in una popolazione consecutiva di pazienti sottoposti ad angiografia coronarica tc ( ac - tc )  . materiali e metodi duecentodue pazienti consecutivi ( 146 maschi , 56 femmine , et media 6011 anni , range 2183 anni ) con sospetta malattia coronarica e gi indirizzati a coronarografia convenzionale ( ac ) sono stati sottoposti ad angiografia coronarica tc ( ac - tc ) preliminarmente allesecuzione della ac nellambito di studi di validazione della metodica . 
solo i pazienti con ritmo sinusale , mai sottoposti ad angioplastica percutanea o ad intervento chirurgico per il posizionamento di by - pass e capaci di trattenere il respiro per almeno 12 secondi , sono stati inclusi nello studio . 
i pazienti nei quali esistevano delle controindicazioni assolute alla somministrazione endovenosa di mezzo di contrasto iodato ( ad esempio allergia nota , insufficienza renale o disordini tiroidei ) sono stati esclusi dallo studio . 
il comitato etico del nostro dipartimento ha approvato il protocollo di studio e tutti i pazienti hanno fornito un consenso informato . preparazione del paziente ai pazienti con una frequenza cardiaca ( fc ) superiore a 65 battiti per minuto ( bpm ) stata somministrata , 45 minuti prima della scansione , se non presenti delle controindicazioni , una singola dose orale di 100 mg di metoprololo tartrato ( selokeen , astrazeneca pharmaceutics )  . 
the study protocol involved a preliminary acquisition without intravenous iodinated contrast material to quantify coronary artery plaque . the parameters used for the two scans ( a ) quantification of plaque and ( b ) ctca were : 1 . 
images were reconstructed with the following parameters : effective slice thickness 3 mm , reconstruction increment 1.5 mm , field of view ( fov ) 150180 mm , dedicated convolution kernel for the calcium score . the time windows were positioned at 60% of the rr interval 2 . 
number of slices per rotation 322 , slice thickness 0.6 mm , gantry rotation time 330 ms , table feed 3.84 mm / rotation ( pitch 0.2 ) , 120 kv , 900 mas . 
prospective modulation of the x - ray tube was not used . patients were administered 100 ml of iodinated contrast material ( iomeprol , iomeron 400 , bracco , milan , italy ) with an automatic injector at 5 ml / s ( stellant , medrad , pittsburgh , pa , usa ) connected to a 20 - gauge cannula positioned in an antecubital vethe bolus tracking technique was used to optimise enhancement of the coronary arteries ( care bolus , siemens , forchheim , germany )  . 
angiography - scan data were obtained during a single breath - hold ( 912 s )  . dedicated software ( windose , institute of medical physics , erlangen , germany ) was used to calculate the radiation dose during the angiographic scan ( mean value 15.2 msv for women and 21.4 msv for men )  . 
retrospective reconstructions were performed based on the electrocardiogram ( ecg ) signal to obtain an image quality not affected by motion artefacts in the end - diastolic and end - systolic phases . coronary angiography ( ca ) ca was performed within 2 weeks of the ctca . 
cardiac catheterisation and ca were performed with standard protocols , with spider - view projections , right anterior oblique ( rao ) for the left coronary artery and left anterior oblique ( lao ) for the right coronary artery . 
a single observer unaware of the ctca results identified the coronary segments using a 17 - segment modified american heart association classification . data collection and statistical analysis all examinations judged to be of insufficient quality by two observers were excluded from the sample . 
all examinations were analysed on an off - line dedicated workstation using all the available applications ( viewing , 3d , inspace , circulation ) to achieve an accurate assessment . 
the observers obtained multiplanar reconstructions ( mpr ) , curved multiplanar reconstructions ( cmpr ) , maximum intensity projecta per via orale una dose addizionale di 1 mg di lorazepam ( temesta , wyeth - ayerst )  . protocollo di scansione e ricostruzione delle immagini per lo studio mediante tc stato utilizzato uno scanner a 64 strati ( sensation 64 , siemens , forchheim , germania )  . 
numero di strati per rotazione 322 , tempo di rotazione del gantry 330 ms , avanzamento / rotazione 6 mm ( pitch 0 , 2 ) , voltaggio del tubo radiogeno kv 120 , potenza del tubo radiogeno mas 150 . 
le immagini sono state ricostruite con i seguenti parametri : spessore di strato effettivo 3 mm , incremento di ricostruzione 1 , 5 mm , campo di vista ( fov ) 150180 mm , filtro di convoluzione dedicato per il calcium score . 
numero di strati per rotazione 322 , spessore di strato 0 , 6 mm , tempo di rotazione 330 ms , avanzamento per rotazione 3 , 84 mm ( pitch 0 , 2 ) , voltaggio del tubo radiogeno 120 kv , potenza del tubo radiogeno 900 mas , spessore di strato effettivo ricostruito 0 , 60 , 75 mm , incremento di ricostruzione 0 , 4 mm , campo di vista ( fov ) 150180 mm , filtro di convoluzione medium - smooth . 
non stata utilizzata la modulazione prospettica del tubo radiogeno . sono stati somministrati 100 ml di mezzo di contrasto iodato ( iomeprol , iomeron 400 , bracco , milano ) alla velocit di 5 ml / s mediante iniettore automatico ( stellant , medrad , pittsburgh , usa ) collegato ad unagocannula da 20 gauge preventivamente posizionata in una vena antecubitale . 
utilizzando un software dedicato ( windose , istituto di fisica medica , erlangen , germania ) stata calcolata la dose di radiazioni ionizzanti alla quale i pazienti sono stati esposti durante la scansione angiografica ( valore medio 15 , 2 msv per le donne e 21 , 4 msv per gli uomini )  . 
sono state effettuare delle ricostruzioni retrospettive basate sul segnale ecg per ottenere una qualit dellimmagine priva di artefatti da movimento in fase telediastolica e tele - sistolica . angiografia coronarica ( ac ) lac stata eseguita entro 2 settimane dallac - tc . 
la tecnica di cateterizzazione cardiaca e lesecuzione dellangiografia coronarica sono state eseguite seguendo i protocolli standardizzati , ottenendo in particolare le proiezioni spider view , oad per la arteria coronaria sinistra e oas per larteria coronaria destra . 
the same observers also assessed the presence of coronary anomalies on the basis of the type of origin and course , whether they were intrinsic to the coronary itself , or terminal . 
the proximal tract of the rca was the origin of the right conus branch in 59% of cases and the origin of the sinuatrial nodal branch in 64% of cases . 
the dellac - tc , ha identificato i segmenti coronarici utilizzando una classificazione in 17 segmenti modificata rispetto a quella fornita dallamerican heart association . raccolta dati ed analisi statistica due osservatori in consenso hanno escluso dal campione tutti gli esami la cui qualit stata giudicata insufficiente da entrambi i lettori . 
tutti gli esami sono stati analizzati su piattaforma di lavoro dedicata off - line utilizzando tutte le applicazioni disponibili ( viewing , 3d , inspace , circulation ) per raggiungere una valutazione accurata . 
gli stessi osservatori hanno infine valutato in consenso anche la presenza di anomalie coronariche sulla base della tipologia : di origine e decorso , intrinseche alla coronaria stessa , di terminazione . 
i dati sono stati espressi come prevalenze in numero assoluto e percentuale sul totale dei reperti descritti . risultati gli esami sono risultati tutti di qualit diagnostica e adatti alla valutazione . table 1 main anatomical variants of the coronary tree tabella 1 principali varianti anatomiche dellalbero coronarico main anatomical variants type principali varianti anatomiche tipo coronary dominance origin of the conus branch artery origin of the sinus node artery pda variants lm lenght intermediate branch number of diagonal branches number of marginal branches right , left , mixed rca ; rca ostium ; aorta rca ; cx ; both pathways are present early pda ; double 12 cm ; < 1cm ; > 2cm single or double 12 , > 2 12 , > 2 dominanza coronarica origine arteria del cono della polmonare origine arteria del nodo del seno varianti adp lunghezza del tcs presenza di un ramo intermedio numero di rami diagonali numero di rami marginali destra ; sinistra ; bilanciata acd ; ostio acd ; aorta acd ; acx ; entrambe le vie presenti emergenza precoce ; doppia adp 12 cm ; < 1 cm ; > 2 cm singolo o doppio 12 , > 2 12 , > 2 rca , right coronary artery ; cx , circumflex artery ; lm , left main ; pda , posterior descending artery acd , arteria coronarica destra ; acx , arteria circonflessa ; tcs , tronco comune sinistro ; adp , arteria discendente posteriore 1120 f . 
i rami settali sono stati visualizzati nell89% dei pazienti . analisi delle anomalie coronariche : le anomalie dellalbero coronarico riscontrate nella nostra popolazione comprendono 51 casi pari al 25% dei pazienti osservati . alcuni pazienti presentano multiple anomalie cos ripartite e riassunte in tabella 4 : 1 . 
anomalie di terminazione delle coronarie con decorso anomalo in 1 paziente ( 0 , 5% )  . discussione la tcms in grado di visualizzare con efficacia lanatomia complessa e variabile delle arterie coronarie sfruttando le tecfig . 
termination anomalies of the coronary arteries with an anomalous course were identified in one patient ( 0.5% ) discussion msct is able to effectively visualise the complex and variable anatomy of the coronary arteries by exploiting postprocessing techniques , thus providing valuable support to interventional cardiologists and heart surgeons [ 615 ]  . 
on the basis of the most recent published data obtained with ca , normal is defined as any morphological characteristic of the coronary circulation observed in more than 1% of a nonselected sample . a normal variant , therefore , is a characteristic observed in more than 1% of the same sample , whereas an anomaly is a condition observed in less than 1% [ 20 ]  . coronary dominance indicates which system of coronary arteries supplies the inferolateral aspect of the left ventricle [ 21 ]  . 
in 80%90% of the population the posterior descending artery ( pda ) is a branch of the rca and runs from the base of the heart to the apex in a mirror image of the lad . 
in right coronary dominance the rca gives rise to a right posterolateral ( rpl ) branch , which crosses the posterior interventricular sulcus and continues on the left side to supply the dorsal aspect of the left ventricle . 
esistono in letteratura numerosi criteri per classificare ed indicare le anomalie coronariche ; basandoci sui dati pi recenti ritrovati nella letteratura , peraltro ottenuti da casistiche di angiografia coronarica ; viene definita normale qualsiasi caratteristica morfologica del circolo coronarico osservata in pi dell1% in un campione non selezionato ; variante normale , una caratteristica osservata in pi dell1% dello stesso campione ; anomalia una condizione osservata in meno dell1% [ 20 ]  . la dominanza coronarica indica quale sistema di vasi coronarici vicaria il flusso sanguigno verso la parete infero - laterale del ventricolo sinistro [ 21 ]  . 
quando la dominanza coronarica del sistema destro la acd d origine un ramo postero - laterale destro ( pld ) , che attraversa il solco interventricolare posteriore e continua sul lato sinistro per la vascolarizzazione della parete dorsale del ventricolo sinistro . il ramo pld pu diventare di importanza notevole quando la acx occlusa , poich pu essere in grado di sostenere la vascolarizzazione della parete postero - inferiore del ventricolo sinistro . 
dai dati ottenuti nella nostra analisi emerge una stretta concordanza con quanto riportato in letteratura , ovvero una prevalenza nella dominanza destra . il primo ramo della acd larteria del cono che decorre sulla superficie antero - laterale del tratto di efflusso del ventricolo destro . 
conventional angiogram ( a ) , volume rendering ( vr ) ( b ) and maximum intensity projection ( mip ) ( c ) images of a right coronary artery ( rca ) arising from the left sinus of valsalva with an interarterial course . 
conventional angiogram ( d ) , vr ( e ) and multiplanar reconstruction ( mpr ) ( f ) images of a left coronary artery arising from the rca with a septal course . 
acc ( g ) , immagini vr ( h ) e cmpr ( i ) di acx con origine dal seno di valsalva destro e decorso retroaortico , sottoposta a stent . nance is mixed with an intermediate vascularisation pattern . data from our analysis show a strong agreement with the data reported in the literature , i.e. 
in una piccola percentuale di casi lacd pu dare origine anche ad un ampio ramo lungo il margine acuto del cuore fino a continuarsi con ladp ( emergenza precoce delladp )  . 
conventional angiogram ( a ) , volume rendering ( vr ) ( b ) and maximum intensity projection ( mip ) images ( c ) displaying an aneurysm of the circumflex artery . 
myocardial bridging of mid - left anterior descending artery displayed by conventional angiogram during systole ( d ) , vr ( e ) and mpr cross - section images ( f )  . 
sometimes , however , the rca can trifurcate at the crux , giving rise to two branches ( double pda ) for the supply of the base of the heart and an rpl branch for the supply of the dorsal aspect of the left ventricle . the length of the main lca is usually 12 cm , whereas lower values are very common and higher values more rare [ 22 ]  . 
the anomalies that prove to be haemodynamically significant , however , can lead to angina pectoris , syncope , arrhythmia , myocardial infarct or sudden death , as well as promote the onset and progression of coronary atherosclerosis or prove responsible for complications and technical difficulties during angioplasty and heart surgery . 
this anomaly could create problems during heart surgery , since the surgeon unaware of its anomalous course could mistakenly cut it . in addition , native coronary arteries are normally positioned at the origin of the aortic arch with an orientation of 120 and 150 , respectively . 
the cases we observed included 1128 zazione della base del cuore , e ad un ramo pld , per la vascolarizzazione della parete dorsale del ventricolo sinistro . la lunghezza del tcs comunemente di circa 12 cm , anche se possono essere riscontrati valori inferiori ( molto frequenti ) o superiori ( pi rari ) [ 22 ]  . 
le anomalie che risultano invece emodinamicamente significative possono indurre angina pectoris , sincope , aritmia , infarto miocardico , morte improvvisa , favorire linsorgenza e la progressione della malattia aterosclerotica coronarica o rendersi responsabili di complicanze e difficolt tecniche durante angioplastica ed interventi di cardiochirurgia . 
il chirurgo , infatti , potrebbe erroneamente reciderla non conoscendone il decorso anomalo . normalmente inoltre le arterie coronarie native sono posizionate allorigine dellarco aortico con un orientamento di 120 e 150 gradi rispettivamente . 
in caso di riposizionamento chirurgico della valvola o di una sua sostituzione importante conoscere questa condizione . alcune anomalie vengono definite anche come maligne proprio per il loro potenziale patologico e sulla base della loro rilevanza clinica ( benigne ; rilevanti correlate ad ischemia miocardia ; severe associate a morte improvvisa ; critiche associate a malattia coronarica ) , facilitandone cos la gestione ed il follow - up clinico [ 25 ]  . 
a questa classificazione si pu sovrapporre una classificazione anatomica che divide le anomalie coronariche sulla base dellorigine ( assenza del tronco comune sinistro , figura 6 , coronaria con origine dallopposto seno coronarico , figura 7 ) , del decorso ( interarterioso , settale e retroaortico , figura 7 ) , dellanatomia intrinseca ( aneurismi , ponti miocardici , figura 8 ) , e della terminazione ( fistole , figura 9 ) [ 4 , 20 ]  . 
tra i casi osservati emergono alcune di queste anomalie : la fistola coronarica una anomalia coronarica in grado di determinare o predisporre ad un evento ischemico miocardico ed a scompenso cardiaco congestizio , in quanto , drenando nelle sezioni cardiache di destra , pu determinare uno shunt sinistrof . 
coronary fistula is a anomaly capable of causing or creating the predisposition for a myocardial ischaemic event and congestive heart failure in that drainage in the right cardiac sections can lead to leftright shunt with volume overload . 
this anomaly in adults is associated with myocardial ischaemia , a higher risk of coronary disease and volume overload . coronary anomalies are a relatively frequent finding in the context of noninvasive coronary diagnostic imaging . knowledge of the characteristics and consequences of different types of anomalies allows the radiologists to judge the finding appropriately . 
nonetheless , it needs to be emphasised that the performance of ctca for suspected coronary anomaly or to exclude its presence should be carefully considered together with the cardiologist to avoid exposing a young patient to an excessive dose of ionising radiation . to date , conventional ca has been the technique of choice for the diagnosis of coronary artery anomalies . 
the diagnosis of coronary artery anomalies is very often established on the basis of the impossibility of finding the coronaries in their normal anatomical position , and therefore of characterising their ostium . in a few short years msct has modified the entire perception of coronary diagnosis by demonstrating the feasibility of a noninvasive approach . 
the complex and tortuous coronary anatomy can be easily depicted with msct . in our case series , we wanted to emphasise the importance of coronary variants and anomalies by demonstrating their prevalence in a broad population of consecutive patients treated at our tertiary referral centre . 
in our experience , the prevalence of coronary artery anomalies was 25% of cases ( 51 / 202 ) , a much higher value than reported in the literature . this can , however , be explained by the fact that the centre where the examinations were performed is a tertiary referral centre for patients with cardiovascular disease . 
in addition , it should be borne in mind that the study population was not made up of a single ethnic group but was , rather , heterogeneous , as it included patients originating from other continents and with a different and currently unknown prevalence of coronary artery anomalies . most of the coronary artery variants encountered had a minimal clinical risk , such as myocardial bridging , which was present in 12.9% of cases , a figure higher than in condestro con sovraccarico di volume . 
nelladulto questa anomalia si pu associare ad ischemia miocardica , ad un pi elevato rischio di malattia coronarica e ad un sovraccarico cardiaco . le anomalie coronariche sono un reperto relativamente frequente nel contesto dellimaging diagnostico non invasivo coronarico . 
langiografia coronarica con tcms consente grazie alle tecniche di post - processing ( mpr , cmpr , mip , vr ) una pi chiara visualizzazione delle anomalie coronariche rispetto allac . 
la ac - tcms stata recentemente considerata come test appropriato per la valutazione di una sospetta anomalia coronarica , al pari della angiografia coronarica mediante risonanza magnetica [ 2 ]  . 
occorre , tuttavia , puntualizzare che lindicazione allesecuzione di una ac - tcms per sospetta anomalia coronarica o per escluderne la presenza deve essere ben ponderata in cooperazione con il cardiologo , al fine di evitare ad un paziente di giovane et uneccessiva esposizione a radiazioni ionizzanti . sino ad oggi , la coronarografia convenzionale ha rappresentato la tecnica di riferimento per la diagnosi delle anomalie coronariche . 
la diagnosi di ac stabilita , molto spesso , sulla base dellimpossibilit di trovare la coronaria nella normale sede anatomica e , quindi , di cateterizzarne lostio . la tomografia computerizzata multistrato ( tcms ) ha modificato in pochi anni lintera percezione della diagnostica coronarica , portando in primo piano la fattibilit di un approccio non invasivo . 
questa metodica , infatti , offre uneccellente risoluzione spaziale con la possibilit di effettuare mpr ( ricostruzioni multiplanari ) , mip ( maximum intensity projections ) e vr ( volume rendering )  . 
la complessa e tortuosa anatomia coronarica pu essere agevolmente dimostrata mediante la tcms . nella casistica presentata abbiamo voluto sottolineare limportanza delle varianti e delle anomalie coronariche dimostrandone la prevalenza in una ampia popolazione di pazienti consecutivi afferenti presso un centro di iii livello . nella nostra esperienza , la prevalenza delle anomalie coronariche del 25% dei casi ( 51 / 202 ) , valore molto superiore a quanto osservato in letteratura , ma spiegabile a causa del fatto che il centro dove sono stati effettuati gli esami rappresenta centro di iii livello , di riferimento per patologie cardiovascolari . 
va tenuto conto del fatto , poi , che la popolazione di studio non rappresenta un solo gruppo etnico ma abbastanza eterogenea , poich coinvolge pazienti originanti da altri continenti e con prevalenze di anomalie coronariche quindi differenti , e attualmente non nota . delle varie varianti la maggioranza hanno scarsa rilevanza clinica , come ad esempio i ponti miocardici , la cui prevalenza risulta essere del 12 , 9% , dato anchesso superiore a 1129 f . 
in contrast , the finding of an anomalous origin or course can be clinically relevant , such as the anomalous origin of the coronary ostium from the opposite coronary sinus ( which manifests as origin in the main lca or of divided origin )  . 
in these cases , the potential compression of a single coronary vessel if it passes between the aorta and the pulmonary artery can cause ischaemia , myocardial infarct and sudden death . quanto riscontrato nelle casistiche angiografiche e pi vicino alle prevalenze evidenziate in studi autoptici [ 27 ] , mentre pu avere rilevanza clnica il reperto di anomalie di origine e decorso , quali ad esempio lanomala origine dellostio coronarico dallopposto seno coronarico ( che si estrinseca come origine in tronco comune oppure origine divisa )  . 
in questi casi la potenziale compressione di un singolo vaso coronarico se passa tra aorta ed arteria polmonare , pu provocare episodi ischemici , infarto miocardico e morte improvvisa . conclusions the anatomical complexity and variability of the coronary circulation benefits from the flexibility of the various postprocessing techniques introduced by ctca and implemented by the use of a 64 - slice scanner with isotropic voxel . 
radiologist training needs to focus on both anatomical and technical features to satisfy the criteria of professional competence and expertise required for the study of such a highly specialised area of diagnostic imaging . ctca is currently capable of providing highly accurate anatomical imaging of the cardiac vessels . 
both the arterial and the venous vessels can be visualised , although with slightly different protocols with regard to the timing of contrast administration . among all the applications requiring knowledge of the anatomy of the cardiac vessels , ctca constitutes the noninvasive diagnostic technique capable of depicting the highest number of coronary segments , particularly the distal segments . 
therefore , in patients affected by coronary artery disease , it is reasonable to expect that ctca , due to its intrinsically fast execution , noninvasiveness and low cost , will become the ideal instrument for assessing patients with a low risk of coronary artery disease and for the follow - up of percutaneous and / or surgical procedures in the coronary vascular area . conclusioni la complessit e la variabilit anatomica del circolo coronarico si giova della flessibilit delle varie metodiche di postprocessing introdotte dalla ac - tcms ed implementate dalluso di scanner a 64 strati con voxel isotropico . 
il training del medico radiologo deve necessariamente focalizzarsi su entrambi gli aspetti , anatomico e tecnico , per soddisfare quei criteri di competenza professionale ed expertise necessari per lapproccio ad un area di imaging altamente specializzata . attualmente la coronarografia tc in grado di fornire un imaging anatomico dei vasi cardiaci con elevata accuratezza . 
sia il comparto arterioso che quello venoso possono essere dimostrati , anche se con protocolli lievemente differenti legati al timing del mdc . in tutte quelle applicazioni nelle quali la conoscenza dellanatomia dei vasi del cuore importante , la coronarografia tc costituisce ad oggi la metodica diagnostica non invasiva in grado di visualizzare il maggior numero di segmenti coronarici ed in particolare anche quelli distali . 
this study was done to evaluate the diagnostic role of enteroclysis with multidetector computed tomography ( mdct ) and single - detector ct ( sdct ) in patients affected by smallbowel crohns disease . 
in comparison with double - contrast enteroclysis , sensitivity , specificity and diagnostic accuracy were 90% , 71% and 89% for sdct and 92% , 83% and 90% for mdct . 
mdct is superior to sdct because it allows a better spatial resolution and improves depiction of the pathological patterns of crohns disease . key words crohns disease ct enteroclysis multidetector ct riassunto obiettivo . 
quarantacinque pazienti sono stati sottoposti ad enteroclisi - tc a singolo detettore ( n = 20 ) o multidetettore ( n = 25 ) previa somministrazione di metilcellulosa tramite sondino naso - digiunale . 
confrontando i dati tc con quelli del clisma del tenue , abbiamo riscontrato valori di sensibilit , specificit ed accuratezza diagnostica rispettivamente del 90% , 71% e 89% con la tcsd e del 92 , 83 e 90% con la tcmd . 
la tcmd da preferire alla tcsd per la sua migliore risoluzione spaziale e perch consente una migliore rappresentazione dei segni patologici di malattia di crohn . parole chiave malattia di crohn tc enteroclisi tc multidetettore introduction introduzione crohns disease is a chronic granulomatous disease that causes transmural inflammation of the small bowel . 
complications ( abscess , fistula , stenosis ) are common and can be seen in over 40% of patients [ 1 ]  . conventional radiology techniques ( small - bowel barium examination , double - contrast enema ) visualise the small la malattia di crohn una malattia granulomatosa cronica che determina infiammazione transmurale dellintestino tenue . 
the added value of computed tomography ( ct ) is that it directly depicts the bowel wall , perienteric fat , mesentery , vessels and lymph nodes , as well as local complications of crohns disease and associated pathological conditions [ 2 ]  . 
the sensitivity of small - bowel ct is not well known and depends mainly on the quality of the study , severity of the inflammation and presence of mesenteric and intraperitoneal abnormalities . the diagnostic approach to small - bowel disease has recently been refined by the advent of spiral ct , which offers better spatial resolution and the possibility of generating multiplanar reconstructions . 
another technique proposed for the study of the small bowel is ct enteroclysis , where contrast material is administered through a nasojejunal tube , and contiguous axial images are obtained after complete distension of the small bowel [ 3 ]  . 
the function of ct enteroclysis is to overcome the individual pitfalls of ct and small - bowel enema and combine the advantages of the two techniques into a single study . 
the theoretical advantage of ct enteroclysis is the ability to simultaneously depict intraluminal , mural and extraintestinal complications of crohns disease [ 2 , 3 ]  . the aim of this study was to evaluate the diagnostic accuracy of ct enteroclysis with singleand multidetector detector techniques in symptomatic patients with a clinical suspicion or firm diagnosis of crohns disease . materials and methods we prospectively and consecutively evaluated 45 patients ( 22 men , 23 women ) aged 2570 years ( mean age 43 years ) presenting with a suspected or known diagnosis of crohns disease on the basis of clinical , laboratory and / or endoscopic findings . 
on the day before the procedure , they took a mixture of a and b sennosides with a cup of tea with sugar at 8 : 00 h , 15 mg of magnesium sulphate in three quarters of a glass of warm water at 17 : 00 h , followed by 3 l of water over the next 45 h ; at 21 : 00 h they had a cup of clear soup and fasted from 21 : 00 h onwards . all patients were intubated with a 12to 16 - fr nasojejunal tube ( create medic co . , yokohama , japan ) introduced under fluoroscopic guidance . 
patients were then taken into the ct room where contrast material ( 2 , 000 ml of 0.5% methylcellulose ) was administered manually with 60 - ml syringes , with care being taken to ensure a constant and continuous injection rate of about two syringes per minute . 
il valore aggiunto della tc quello di permettere la visualizzazione diretta della parete intestinale , del tessuto adiposo periviscerale , del mesentere , dei vasi e dei linfonodi , nonch delle complicanze locali di malattia di crohn e patologie associate [ 2 ]  . 
la sensibilit dello studio tc nelle malattie del tenue non ben conosciuta e si affida principalmente alla qualit dellindagine , alla severit dellinfiammazione e alla presenza di alterazioni mesenteriche ed intraperitoneali . 
lapproccio diagnostico allo studio del piccolo intestino stato negli ultimi anni ulteriormente modificato grazie allo sviluppo della tecnica spirale che ha consentito una migliore risoluzione spaziale e la possibilit di elaborare ricostruzioni multiplanari . 
contemporaneamente , nello studio del tenue stata proposta la tc enteroclisi in cui il mezzo di contrasto ( mdc ) viene somministrato attraverso un sondino naso - digiunale e dopo la totale distensione del piccolo intestino vengono riprese immagini assiali contigue dello stesso [ 3 ]  . 
la funzione di questa indagine quella di supplire alle carenze individuali della tc e del clisma del tenue , quindi di combinare i vantaggi di ciascuna tecnica in ununica metodica di studio . 
il teorico vantaggio della tc enteroclisi la capacit di mostrare contemporaneamente le complicanze intraluminali , murali ed extramurali della malattia di crohn [ 2 , 3 ]  . lo scopo di questo studio valutare laccuratezza diagnostica della tc enteroclisi effettuata con tecnica a singolo detettore e multidetettore in pazienti sintomatici con sospetto clinico o diagnosi certa di malattia di crohn . materiali e metodi sono stati valutati prospettivamente e consecutivamente 45 pazienti ( 22 uomini , 23 donne ) , di et compresa tra 25 e 70 anni ( et media 43 anni )  . 
il giorno prima dellesame i pazienti hanno ingerito alle ore 8 : 00 una miscela di sennosidi a e b con una tazza di t zuccherato e alle 17 : 00 15 mg di solfato di magnesio sciolti in 3 / 4 di un bicchiere di acqua tiepida , seguita dallassunzione di 3 litri di acqua nelle 45 ore successive . 
il digiuno stato rispettato delle 21 : 00 in poi . a tutti i pazienti stato introdotto sotto guida fluoroscopica un sondino naso - digiunale , del calibro 12 - 16 f ( create medic co , yokohama , giappone )  . 
the contrast - enhanced study was acquired 40 s after iv administration of 130150 ml contrast material ( ultravist 370 , schering ag , berlin , germany ) at a flow rate of 3 ml / s . axial images were processed on a computer workstation ( advantage windows , ge medical system ) to obtain 2d coronal and , if necessary , sagittal constructions . 
following ct , the nasojejunal tube was not removed , and after 68 h , all patients underwent a small - bowel barium enema according to the herlinger method [ 4 ] with digital technique . 
all ct images were assessed for the following signs : presence , site and number of abnormal bowel segments , degree of wall thickening , degree of mural enhancement [ hounsfield units ( hu ) ] , target sign , comb sign , perienteric stranding , fistulas , fibrofatty proliferation , lymphadenopathy , abscesses and other signs ( adhesions , incidental findings in the other abdominal organs , benign or malignant neoplasms of the small bowel )  . 
we evaluated small - bowel distension , which was classified according to a 4 - point scale for each segment ( 0 : no distension ; 1 : incomplete distension ; 2 : partial distension ; 3 : complete distension )  . 
the coronal and sagittal reconstructions were compared with the axial images to assess whether they provided additional information . images obtained with the small - bowel enema were checked for the following signs : oedematous folds , erosions , aphthoid and linear ulcers , fistulas , cobblestoning , micronodules , wall thickening , prestenotic dilatations , mesenteric inflammation and other signs ( adhesions , radiological findings suspicious for benign or malignant neoplasm )  . 
the ct and barium enema results were compared with those of retrograde 1190 sondino stata fissata a valle del treiz tramite linsufflazione di aria in un palloncino in modo da prevenire reflussi di mezzo di contrasto nello stomaco . 
i pazienti sono stati quindi condotti in sala tc ove il mezzo di contrasto ( 2000 ml di metilcellulosa allo 0 , 5% ) stato somministrato manualmente usando siringhe da 60 ml . 
abbiamo cercato di ottenere uniniezione costante e continua , circa 2 siringhe al minuto . al fine di evitare spasmi , ottenere unomogenea distensione del piccolo intestino , ridurre il disagio e la tensione addominale stato somministrato ai pazienti un farmaco anticolinergico ( n - butilscopolamina )  . 
abbiamo somministrato 10 mg di farmaco quando il paziente ha cominciato ad avvertire dolore addominale e 10 mg subito prima dellacquisizione delle immagini tc . venti pazienti sono stati studiati con tecnica tc spirale a singolo detettore ( tcsd ) ( prospeed , advantage sx spiral scanner , ge medical system , milwakee , usa ) con i seguenti parametri di acquisizione : collimazione 5 mm , intervallo 8 mm , ricostruzione 4 mm , kv 120 , mas 300 . 
venticinque pazienti sono stati studiati con tc multidetettore a 16 strati ( tcmd ) ( lightspeed pro16 , ge medical system , milwakee , usa ) con i seguenti parametri di scansione : collimazione 1 , 25 mm , velocit di scorrimento del tavolo 13 , 75 mm / rot , kv 120 , mas 500 , pitch 1 , 375 , tempo di rotazione 0 , 6 s . al termine dellinfusione di metilcellulosa lesame tc stato eseguito prima e durante la somministrazione di mdc iodato ev . 
nei pazienti in cui allesame preliminare senza mdc iodato veniva riscontrata una inadeguata distensione del piccolo intestino abbiamo somministrato altri 200250 ml di metilcellulosa prima di procedere allo studio tc con mdc ev . 
questultimo stato acquisito 40 secondi dopo la somministrazione ev di 130150 ml di mezzo di contrasto ( ultravist 370 , schering ag , berlino , germania ) iniettato alla velocit di 3 ml / s . con una computer workstation ( advantage windows , ge medical system ) le immagini assiali sono state rielaborate con ricostruzioni 2d , coronali ed eventualmente sagittali . 
il sondino non stato rimosso al termine dellesame tc e dopo 68 ore tutti i pazienti sono stati studiati con clisma del tenue effettuato secondo il metodo di herlinger [ 4 ] con tecnica digitale . 
sono stati somministrati 200250 ml di una sospensione di solfato di bario ( prontobario 60% , bracco , milano , italia ) seguiti dallinfusione di 10002000 ml ( in media 1200 ml ) allo 0 , 5% di metilcellulosa . 
sono stati eseguiti radiogrammi nelle proiezioni postero - anteriore , antero - posteriore , con e senza compressione delle anse . i risultati dello studio tc e del clisma del tenue sono stati confrontati e valutati da due radiologi esperti in radiologia gastroenterologica . 
abbiamo anche valutato la distensione delle anse del piccolo intestino che per ciascun segmento stata classificata con una scala di 4 punti ( 0 : distensione assente ; 1 : distensione incompleta ; 2 : distensione parziale ; 3 : distensione completa )  . 
le ricostruzioni coronali e sagittali sono state confrontate con le immagini assiali ed abbiamo valutato le eventuali informazioni aggiuntive da esse ottenibili . le immagini ottenute con clisma del tenue sono state analizzate ricercando i seguenti segni : edema delle pliche , erosioni , ulcerazioni aftoidi e lineari , fistole , aspetto ad acciottolato , micronoduli , ispessimenti parietali , dilatazioni prestenotiche , infiammazione mesenterica , altri segni ( aderenze , alterazioni radiologiche sospette per presenza di neoplasia benigna o maligna )  . 
in these loops , wall thickness ranged from 4 mm to 10 mm ( mean 6.5 mm ) , loop diameter was between 12 and 30 mm ( mean 21 mm ) and lumen diameter between 2 and 18 mm ( mean 8 mm )  . 
complete distension was observed in 27 / 45 ( 61% ) patients in the proximal jejunum , in 35 / 45 ( 77% ) in the distal jejunum , in 40 / 45 ( 88% ) in the proximal ileum , in 39 / 45 table 1 ct signs in patients with crohns disease target sign deep ulcers / sinus tracts perienteric stranding comb sign fibrofatty proliferation stenosis fistulas lymphadenopathy ( > 1 cm ) adhesions abscess segno del bersaglio ulcere profonde / sinus tract strie perienteriche segno del pettine proliferazione fibroadiposa stenosi fistole linfoadenopatie ( > 1 cm ) segni indiretti di aderenze ascesso signs sdct ; number of patients mdct ; number of patients sdct , single - detector computed tomography ; mdct , multidetector computed tomography tabella 1 segni tc nei pazienti con morbo di crohn segni tcsd ; numero pazienti tcmd ; numero pazienti tcsd , tomografia computerizzata a singolo detettore ; tcmd , tomografia computerizzata multidetettore 1191 l.m. 
i valori di densit dei segmenti coinvolti ottenuti dallo studio senza contrasto erano compresi tra 16 e 57 hu e il loro livello di enhancement dopo mdc variava tra 82 e 208 hu . 
il segno del bersaglio stato osservato in 14 pazienti , ulcere profonde / sinus tract in 4 , lo stranding perienterico in 13 , il segno del pettine in 7 . 
una completa distensione stata osservata in 27 / 45 ( 61% ) pazienti nel digiuno prossimale , in 35 / 45 ( 77% ) nel digiuno distale , in 40 / 45 ( 88% ) nellileo prossimale , in 39 / 45 ( 86% ) nellileo distale , in 41 / 45 ( 92% ) nellultima ansa ileale . 
5 tc multidetettore : due anse ileali appaiono a pareti ispessite ( frecce nere ) ; limitrofa ad esse ( freccia bianca ) presente formazione ascessuale . ( 86% ) in the distal ileum and in 41 / 45 ( 92% ) in the last ileal loop . 
the three false positive castable 2 degree of small - bowel distension on ct 0 : absent 1 : incomplete 2 : partial 3 : complete pj , proximal jejunum ; dj , distal jejunum ; pi , proximal ileum ; di , distal ileum ; lil , last ileal loop tabella 2 grado di distensione delle anse dellintestino tenue allesame tc 0 : assente 1 : incompleta 2 : parziale 3 : completa dp , digiuno prossimale ; dd , digiuno distale ; ip , ileo prossimale ; id , ileo distale ; uai , ultima ansa ileale 1193 l.m. 
abbiamo quindi riscontrato valori di sensibilit , specificit ed accuratezza diagnostica del 90% , 71% e 89% con tecnica a singolo detettore e del 92% , 83% e del 90% con tecnica multidetettore . 
essa ha confermato i reperti di normalit in 5 pazienti e un morbo di crohn in 20 , un quadro di ileite aspecifica in 1 , ileite da reflusso in 1 e ileite ischemica in 1 . 
i due terzi dei casi di leite aspecifica , diagnosticati dallenteroclisi tc , presentavano endoscopicamente un quadro compatibile con morbo di crohn . small - bowel ct allows visualisation of the entire organ and , with adequate bowel distension and elimination of respiratory and peristaltic artefacts , enables evaluation of the type and luso della tc nello studio del piccolo intestino consente la visualizzazione dellintero organo e , previa unadeguata didiscussione discussion fig . 
8a , b esempio di migliore visualizzazione dellansa patologica ( frecce ) con tecnica multidetettore ( a ) rispetto alla tecnica a singolo detettore ( b )  . extent of mural enhancement after administration of iv iodinated contrast material . 
as with conventional radiology , accurate bowel cleansing is essential for the quality of the study . because wall thickening is a characteristic feature of crohns disease , optimal bowel distension is essential . 
oral contrast agents have the disadvantage of not perstensione delle anse e leliminazione di artefatti respiratori e dei movimenti peristaltici , la valutazione del tipo e dellestensione dellenhancement parietale dopo somministrazione di mdc iodato ev . 
come nella radiologia tradizionale unaccurata pulizia intestinale il requisito fondamentale per questo tipo di studio . lispessimento parietale rappresenta una caratteristica fondamentale della malattia intestinale e per tale motivo essenziale avere la ottimale distensione delle anse . 
ct enteroclysis combines the advantages of ct with those of small - bowel enema into a single technique [ 5 ]  . in 1992 , klppel [ 6 ] conducted the first study of ct enteroclysis in small - bowel inflammatory diseases , highlighting the techniques diagnostic accuracy in evaluating mucosal abnormalities , mural thickening , fistulas and extraintestinal complications . clinical suspicion guides the choice of contrast material . it is generally preferable to use hypodense contrast agents , except in patients with intestinal obstruction , in whom positive agents are found to be more useful [ 5 , 6 ]  . 
hypodense contrast agents provide better depiction of the inner aspect of the bowel loop , allowing evaluation of the type of mural enhancement after iv administration of iodinated contrast material . 
because no infusion pump was available , we administered the contrast agent manually , trying to ensure a steady and continuous infusion . the examination was carried out after completing the methylcellulose infusion . 
moreover , to ensure uniform loop distension and reduce patient discomfort , we administered two doses of iv smooth - muscle relaxant : the first dose was given before the infusion of approximately 11.5 l of methylcellulose to reduce patient discomfort , the second just il primo requisito quindi per un ottimale studio tc ottenere unadeguata distensione del lume mediante unoculata scelta del tipo e della modalit di somministrazione del mezzo di contrasto . 
i mezzi di contrasto somministrati per via orale hanno lo svantaggio di non fare ottenere una uniforme distensione delle anse del piccolo intestino ; tale problema superato con lutilizzo dellenteroclisi tc , caratterizzata tuttavia da invasivit , tempo e costi maggiori . 
kloppel nel 1992 [ 6 ] ha realizzato il primo studio sulla tc enteroclisi nelle malattie infiammatorie dellintestino tenue mettendo in risalto laccuratezza diagnostica di questa tecnica nella valutazione delle alterazioni della mucosa , dellispessimento parietale , delle fistole e delle complicanze extra - intestinali . 
in tutti i pazienti preferibile lutilizzo di mezzi di contrasto ipodensi eccetto che per i pazienti con ostruzione intestinale nei quali un mezzo di contrasto positivo pi utile [ 5 , 6 ]  . 
un mezzo di contrasto ipodenso consente una migliore definizione dellaspetto interno dellansa intestinale , specialmente per valutare il tipo di enhancement parietale dopo la somministrazione di mdc iodato ev [ 7 ]  . 
essa ben conosciuta , sicura , non tossica , largamente utilizzata nello studio tradizionale e non viene assorbita per cui si evita il rischio di emodiluizione ; ha allincirca la stessa densit dellacqua , quindi determina un ottima differenza di contrasto tra il contenuto delle anse intestinali e lenhancement della parete intestinale . 
nei casi in cui le scansioni di base senza mdc iodato documentavano una scarsa distensione delle anse , stata somministrata unulteriore dose di metilcellulosa prima di procedere con linfusione di mdc iodato . 
inoltre , per ottenere unomogenea distensione delle anse e ridurre il disagio al paziente abbiamo somministrato ev due dosi di ipotonizzante : la prima dose stata somministrata dopo linfusione di circa 11 , 5 l di metilcellulosa in modo da ridurre il discomfort del paziente , la seconda subito prima dellesame tc . 
in tal modo abbiamo ottenuto una distensione completa del digiuno prossimale nel 61% dei pazienti , del digiuno distale nel 77% , dellileo prossimale nell88% , dellultima ansa ileale nel 92% . 
anche se la distensione delle anse migliore quando viene usata una pompa peristaltica [ 8 , 9 ] , in accordo con quanto evidenziato in letteratura [ 10 , 12 ] riteniamo possibile ottenere una sufficiente distensione delle anse anche con linfusione manuale del mezzo di contrasto . un altro elemento indispensabile per lo studio tc del morbo di crohn la somministrazione ev del mezzo di contrasto iodato . 
non tutti gli autori sono concordi sul tempo di attesa dopo la somministrazione del mezzo di contrasto iodato : alcuni ritengono utile unacquisizione arteriosa precoce ( 25 s ) [ 8 ] o arteriosa tardiva 1197 l.m. 
10a , b allesame tc a singolo detettore ( a ) evidente ispessimento parietale dellultima ansa ileale ( freccia ) ; nel clisma del tenue ( b ) essa non dissociabile dalle anse vicine . before starting the ct study . 
this enabled us to achieve complete distension of the proximal jejunum in 61% of patients , of the distal jejunum in 77% , of the proximal ileum in 88% and of the last ileal loop in 92% . 
even if loop distension is improved by the use of a peristaltic pump [ 8 , 9 ] , in agreement with the literature [ 10 , 12 ] , we believe adequate bowel distension can also be achieved by manual injection of the contrast agent . another fundamental aspect of the ct study of crohns disease is the iv administration of iodinated contrast material . 
not all authors agree on the timing of the scan after contrast administration : some prefer an early arterial acquisition ( 25 s ) [ 8 ] or late arterial acquisition ( 40 s ) [ 9 , 13 ] , whereas other prefer a portal phase ( 6070 s ) [ 14 ]  . 
in our study we decided to perform a single acquisition 40 s after iv administration of contrast material , because we preferred to study the small - bowel loops in the arterial phase . during acute inflammation , the thickened bowel wall often shows the so - called target sign , characterised by the alternation of concentric highand low - attenuation rings , on postcontrast images . 
the target sign was originally attributed to crohns disease but is now regarded as a nonspecific sign [ 14 ] ; the differential diagnosis includes crohns disease , ischaemia , infectious enteritis , actinic enteritis , vasculitis and graft - versus - host dis1198 ( 40 s ) [ 9 , 13 ] , altri prediligono una fase portale ( 6070 s ) [ 14 ]  . 
noi abbiamo deciso di realizzare una singola acquisizione a 40 s dalla somministrazione ev di mdc , poich abbiamo preferito effettuare uno studio delle anse dellintestino tenue in fase arteriosa . quando presente uninfiammazione acuta , la parete intestinale ispessita spesso mostra dopo iniezione endovena del mdc iodato il cosiddetto segno del bersaglio ( target sign ) caratterizzato dallalternanza di anelli concentrici ad alta e bassa densit . 
laspetto a bersaglio era originariamente attribuito al morbo di crohn , ma oggi si ritiene essere un segno aspecifico [ 14 ] ; la diagnosi differenziale include : malattia di crohn , ischemia , enteriti infettive , enteriti attiniche , vasculiti e grast - versushost - disease . 
il segno del pettine ( comb sign ) si evidenzia come una ipervascolarizzazione del mesentere coinvolto che si manifesta sotto forma di dilatazione arteriosa mesenterica , tortuosit , prominenza e dilatazione dei vasi retti [ 14 ]  . 
tale segno stato osservato in 7 pazienti del nostro studio . nonostante gli sviluppi tecnologici , per la nostra esperienza ancora utile la combinazione dello studio tc enteroclisi con il tradizionale clisma del tenue . 
the comb sign reflects hypervascularity of the involved mesentery , which manifests as mesenteric arterial dilatation , tortuosity , prominence and dilatation of the vasa recta [ 14 ]  . 
the comb sign was seen in seven patients in our study . despite technological advances , we find the combination of ct enteroclysis and conventional barium enema to be still useful , as it produces a better diagnostic definition , allowing identification of false positive and false negative cases on ct [ 9 , 16 , 17 ]  . 
moreover , the conventional study proved superior in characterising mucosal abnormalities , especially in the early phase , and complemented the ct findings , allowing for a diagnosis of reflux ileitis and ischaemic ileitis in two cases . 
abscesses were present in 2 / 45 patients , fistulas in 3 / 45 , perienteric stranding in 13 / 45 and fibrofatty proliferation in 5 / 45 . with regard to the comparison between the two ct techniques , it should be stressed that , as expected , the multidetector technique allowed for shorter scan times and reduced respiratory artefacts and patient discomfort while providing better spatial resolution and better depiction of pathological patterns . 
however , sensitivity , specificity and diagnostic accuracy of the two techniques are similar ( table 4 ) , as a result of the fact that both techniques are able to demonstrate the typical signs of disease , even though their visualisation is better with the multidetector technique . 
in our study , mpr did not , however , reveal any abnormalities that had not already been demonstrated in the axial scans , although they significantly increased our confidence in image interpretation and helped us better evaluate the longitudinal extent of intestinal involvement , as previously reported in the literature [ 19 , 21 ]  . 
the coronal images , especially with those obtained with the multidetector technique , were particularly well accepted by the clinicians and surgeons , given their familiarity with frontal views . even though crohns disease is the most common disease of the small bowel , we should not forget that ct enteroclysis is able to detect other pathological conditions , such as neoplasms . 
in our study , we found only two cases of carcinoid tumour , probably as a result of the preliminary clinical selection of patients with suspected or known crohns disease . 
 in conclusion , we believe that mdct should be preferred to sdct owing to its better spatial resolution and because it ne produce una migliore definizione diagnostica permettendo di identificare i casi tc falsi positivi e falsi negativi [ 9 , 16 , 17 ]  . 
nel nostro studio , infatti , 3 falsi positivi erano dovuti a scarsa distensione delle anse e 4 falsi negativi erano dovuti ad una fase iniziale di malattia e / o localizzazione prossimale di crohn . 
ascessi erano presenti in 2 / 45 pazienti , fistole in 3 / 45 , stranding perienterico in 13 / 45 , proliferazione fibroadiposa in 5 / 45 . per quanto riguarda il confronto tra le due tecniche tc vogliamo sottolineare che , come atteso , la tecnica multidetettore ha ridotto i tempi di scansione , gli artefatti respiratori e il disagio per il paziente , permettendo una migliore risoluzione spaziale e realizzando una migliore rappresentazione degli elementi patologici . 
tale dato in accordo con la letteratura [ 1517 , 19 , 20 ] .tuttavia i valori di sensibilit , specificit ed accuratezza diagnostica sono simili con le due metodiche ( tabella 4 )  . 
tuttavia le ricostruzioni multiplanari non hanno nel nostro studio mostrato alcuna alterazione che non fosse gi stata documentata nelle scansioni assiali anche se hanno migliorato significativamente la nostra confidenza nella interpretazione delle immagini e ci hanno aiutato a valutare meglio lestensione longitudinale dellinteressamento intestinale , come gi riportato in letteratura [ 19 , 21 ]  . 
le immagini coronali sono state inoltre particolarmente apprezzate dai clinici e dai chirurghi data la loro familiarit con esse , in particolare quelle ottenute con tecnica multidetettore . anche se il morbo di crohn la patologia dellintestino tenue pi frequente , non dobbiamo dimenticare che la tc enteroclisi in grado di documentare altre condizioni patologiche , come ad esempio le neoplasie . 
nel nostro studio abbiamo riscontrato solo due casi di carcinoide verosimilmente in rapporto alla preliminare selezione clinica che ci ha fatto studiare prevalentemente pazienti con sospetto o noto morbo di crohn . in conclusione noi riteniamo che la tcmd enteroclisi sia da preferire alla tcsd per la sua migliore risoluzione spaziale e perch consente una migliore rappresentazione dei segni patologici di malattia di crohn . 
in our opinion , in selected patients ( nondiagnostic ct , negative ct with a strong clinical suspicion of small bowel disease ) , ct findings still need to be combined with those from small - bowel enema to obtain all the information needed for a correct diagnosis and for planning treatment in patients with crohns disease . 
the ultimate goal is to improve visualisation of early lesions with ct in order to obtain all the necessary information for correct patient evaluation with a single examination . nione i dati tc ancora necessitano in pazienti selezionati ( tc non diagnostica , tc negativa con quadro clinico altamente suggestivo di patologia dellintestino tenue ) di essere combinati con quelli derivanti dal clisma del tenue in modo da ottenere tutte le informazioni necessarie per la corretta diagnosi e la pianificazione terapeutica dei pazienti con malattia di crohn . 
in ct - guided transthoracic needle biopsy , the final diagnosis and lesion size affect diagnostic accuracy : benign lung lesions and lesions smaller than 1.5 cm or larger than 5.0 cm in diameter provide lower diagnostic yield . key words lung biopsy computed tomography ( ct ) guidance accuracy riassunto obiettivo . 
i valori di sensibilit , specificit , valore predittivo positivo , valore predittivo negativo ed accuratezza , riferiti ad una diagnosi di malignit , sono risultati rispettivamente del 90 , 2% , 99 , 0% , 99 , 8% , 67 , 3% e 91 , 7% . 
la diagnosi finale ( benignit versus malignit ) e le dimensioni della lesione influenzano laccuratezza diagnostica della metodica : addensamenti polmonari di natura benigna e lesioni con diametro < 1 , 5 cm o > 5 cm sono caratterizzati da livelli minori di accuratezza diagnostica . parole chiave polmone biopsia tomografia vomputerizzata ( tc ) guida accuratezza diagnostica 1142 a.m. 
computed tomography ( ct ) - guided needle lung biopsy was first reported by haaga and alfidi in 1976 [ 5 ] , and since then , the techniques excellent efficacy and accuracy and acceptable morbidity and mortality rates ( 0.07% , or one death every 1 , 429 procedures ) have been widely demonstrated [ 616 ]  . 
whether carried out with fine - needle - aspiration cytology ( fnac ) or core biopsy ( cb ) , the biopsy provides adequate material for cytological or histological examination and , if necessary , for microbiological evaluation [ 1113 , 1721 ]  . 
cb is more sensitive in defining benign lesions and in characterising diffuse or lymphoproliferative diseases of the lung , where the assessment of the tissue architecture is instrumental to the diagnosis ( accuracy > 80% for cb and 50%70% for fnac ) [ 9 , 2225 ]  . 
lesions smaller than 1 cm are easier to sample with fnac [ 19 , 22 , 23 ]  . the reported sensitivity of fnac varies and is positively affected by the presence of the cytopathologist on site during the biopsy procedure [ 26 , 27 ]  . 
immediate cytological assessment increases the techniques sensitivity and reduces the number of inadequate samples and false negative results [ 2628 ] , as the on - site cytopathologist can judge the adequacy , quantity and quality of the sample , suggest the need to repeat the procedure or request cb with histological assessment [ 22 , 26 ]  . several studies have investigated the factors affecting diagnostic accuracy of ct - guided transthoracic needle biopsies . 
it is well established that the most important factor is the final diagnosis , as malignant lesions ( regardless of histological type ) are characterised by greater sensitivity compared with benign masses . 
other variables ( lesion depth , number of passes , cavitations and necrotic centre , morphology , location , radiologists experience , needle diameter and presence of pneumothorax and / or bleeding ) were not found to be significant [ 32 ]  . the purpose of this paper is to present our experience with a large cohort of patients and discuss the results achieved in relation to diagnostic accuracy . 
in particular , we extensively analyse a number of variables ( relating to the patient , the lesion or the procedure ) in an attempt to correlate them to potential influences on diagnostic accuracy and reach a conclusive judgement on the efficacy of the procedure . 
lagobiopsia polmonare tc guidata fu riportata in letteratura per la prima volta nel 1976 da haaga [ 5 ] e da allora numerosi autori ne hanno dimostrato lestrema efficacia ed accuratezza associate ad un accettabile tasso di morbidit e mortalit ( 0 , 07% , ovvero un decesso ogni 1429 manovre effettuate ) [ 616 ]  . 
il prelievo , eseguito tramite aspirazione con ago sottile ( fine needle aspiration citology , fnac ) o con ago tranciante ( core biopsy , cb ) , consente di ottenere , rispettivamente , materiale sufficiente per lesame citologico o istologico ed infine , se necessario , per una valutazione microbiologica [ 1113 , 1721 ]  . 
la cb ha maggiore sensibilit nella definizione delle lesioni benigne e nella caratterizzazione delle malattie polmonari diffuse o linfoproliferative , nelle quali la valutazione dellarchitettura tissutale importante per la diagnosi ( valori di accuratezza > 80% per la cb e tra il 50% ed il 70% per la fnac ) [ 9 , 2225 ]  . 
le lesioni di dimensioni inferiori al centimetro risultano essere pi facilmente campionabili mediante fnac [ 19 , 22 , 23 ]  . la sensibilit della fnac variabile nelle casistiche riportate in letteratura ed positivamente influenzata dalla presenza in sala radiologica del citopatologo al momento del prelievo [ 26 , 27 ]  . 
il citopatologo , infatti , in grado di dare indicazione sulladeguatezza del prelievo , sulla quantit e qualit del materiale campionato , pu suggerirne leventuale ripetizione o richiedere lintegrazione con lesame istologico dopo cb [ 22 , 26 ]  . alcuni autori hanno analizzato le variabili che influenzano laccuratezza diagnostica delle agobiopsie transtoraciche tc guidate . 
da tempo assodato come il pi importante fattore in questo senso sia la diagnosi finale corretta , dato che le lesioni maligne ( indipendentemente dallistotipo ) sono caratterizzate da livelli di sensibilit significativamente maggiori rispetto agli espansi di origine benigna . 
considerando altre variabili , tsukada stato il primo a dimostrare come la graduale riduzione delle dimensioni della lesione conducesse ad un proporzionale decremento nei valori di accuratezza diagnostica [ 13 ]  . 
i risultati di tsukada sono per stati successivamente confermati da yeow che , in una coorte di 649 soggetti , ha riscontrato come sia la diagnosi finale sia le dimensioni della lesione influenzassero il valore di accuratezza [ 20 ]  . 
the study population comprised 154 subjects who underwent surgical resection ( for whom definitive postoperative histological assessment was available ) and nonsurgical patients who had completed at least 12 months of follow - up ( 454 subjects )  . 
the population included 402 men and 206 women , with a mean age of 65.7 years [ ( median 67 ; standard deviation ( sd ) 9.4 years with 95% confidence interval ( ci ) ; range 2987 years )  . 
exclusion criteria were lesions < 5 mm in diameter ( calculated as the mean of the long and short axis ) , significant changes in blood - coagulation profile , contralateral pneumonectomy , patients inability to maintain a lying position and / or to follow verbal or visual instructions and patients refusal to accept the potential risks associated with the procedure and sign the informed consent form . all biopsies were performed according to a standard protocol by the same interventional radiologist ( ac ) under ct guidance with a spiral dual - detector - row scanner . 
after positioning the patient on the ct table in prone , supine or lateral decubitus , depending on lesion location , an initial localisation scan was obtained by acquiring the entire mass on 5mm - thick contiguous transverse sections . 
then , optimal access ( skin entry site and needle path ) was defined on ct images , taking care to sample the lesion away from low - density areas , which are often central and indicative of necrosis [ 13 , 17 , 18 ]  . 
after having established the entry site and chosen the type and length of the needle ( depending on lesion depth and , whenever possible , the shortest route [ 33 ] ) , the needle entry site was anaesthetised ( 2% lidocaine ) , the needle , or the needle guide in the case of cb , positioned and the patient instructed to hold his or her breath . at this point ct scans were obtained to visualise the needle tip in relation to the lesion . 
in particolare verranno estensivamente analizzate alcune variabili ( riferite al paziente , alla lesione od alla procedura ) nel tentativo di correlarle a potenziali influenze sui valori di accuratezza per formulare un giudizio critico conclusivo sulla efficacia della procedura interventistica . materiali e metodi popolazione di studio in un intervallo temporale compreso tra novembre 2002 ed agosto 2005 sono stati consecutivamente sottoposti a procedura interventistica transtoracica tc guidata per laccertamento della diagnosi ( agoaspirazione transtoracica con ago sottile o con ago spinale o biopsia con ago tranciante ) 608 pazienti , per un totale di 612 procedure . 
la coorte in studio ai fini della formulazione di un giudizio di accuratezza della metodica ha compreso tutti i 154 soggetti resecati ( per i quali risultata disponibile la valutazione istologica definitiva postoperatoria ) ed i pazienti non chirurgici che avessero conseguito un follow - up non inferiore a 12 mesi ( 454 soggetti )  . la popolazione ha pertanto incluso 402 soggetti di sesso maschile e 206 femminile , con unet media di 65 , 7 anni ( mediana 67 anni ; ds9 , 4 anni con ic 95% ; intervallo 2987 anni )  . 
i pazienti sono afferiti in parte con proposta di prestazione ambulatoriale ed in parte in regime di ricovero ordinario . sono state considerate tutte le lesioni intraparenchimali ( centrali e periferiche ) , ovvero gli espansi circondati da tessuto polmonare areato o a diretto contatto con le strutture ilari e / o la superficie pleurica il cui epicentro cadesse allinterno del polmone ( dimensione media delle lesioni 36 , 8 mm ; mediana 31 , 5 mm ; ds20 , 5 mm con ic 95% ; intervallo 7103 mm )  . protocollo bioptico nessun paziente risultato anamnesticamente positivo per diatesi emorragica o ha presentato alterazioni della crasi ematica e del profilo emocoagulativo . 
i soggetti in trattamento anti - aggregante e / o anticoagulante hanno sospeso tale terapia per un periodo antecedente la biopsia di 7 giorni . sono stati raccolti dal radiologo un breve raccordo anamnestico ed il consenso informato . 
al momento di ogni biopsia , risultata disponibile una precedente tc diagnostica . i criteri di esclusione da noi adottati sono stati : lesioni con diametro ( calcolato sulla media delle dimensioni degli assi maggiore e minore ) < 5 mm ; alterazioni significative dellasa.m. 
biopsies were carried out by needle aspiration with a 21 - gauge fine needle or a 20gauge spinal needle ( with the single needle technique ) , or by core biopsy with an 18 - gauge cutting needle ( and coaxial technique )  . our initial approach in pulmonary nodules always uses a chiba fine needle ( especially for small lesions ) , except in cases of a second attempt after a previous inconclusive procedure . 
this made it possible to have an evaluation of material adequacy and an extemporary general indication about the nature of the lesion within minutes of the procedure [ 2628 ]  . 
in the case of inadequate or insufficient samples for cytology or histology , the biopsy procedure was repeated immediately : in particular , 190 / 612 procedures ( 31.0% ) required more than one sampling ( mean 1.32 samples per procedure ; range 14 )  . to assess possible complications ( pneumothorax , needletrack bleeding ) , postprocedural ct scans were acquired 35 min after the procedure ( time required for the cytopathologist to prepare the slides and judge their adequacy )  . data collection : definition and selection of variables multiple discrete or continuous variables , relating to the patient , the lesion or the procedure , were considered . 
dopo aver posizionato il paziente sul lettino tc in decubito prono , supino o laterale a seconda della localizzazione della lesione , si proceduto ad una prima centratura della lesione attraverso lacquisizione dellintero espanso su sezioni trasverse contigue di 5 mm di spessore . 
successivamente , sulle immagini tc a monitor si pianificata la migliore via di accesso ( come punto di ingresso cutaneo e tragitto dellago ) al fine di campionare la lesione avendo cura di evitare regioni di ipodensit , spesso centrali ed indicative di necrosi , nel suo contesto [ 13 , 17 , 18 ]  . 
dopo aver definito il punto di ingresso dellago e scelto la tipologia e la lunghezza dellago ( a seconda della profondit della lesione e pianificando , ogni qual volta sia stato possibile , il tragitto pi breve [ 33 ] ) si effettuata unanestesia locale in corrispondenza del punto cutaneo di ingresso ( lidocaina 2% ) , quindi si posizionato lago , o lago guida per le biopsie trancianti , chiedendo al paziente di mantenere lapnea . 
per gli espansi di difficile centratura ( 162 / 612 procedure , 26 , 5% del totale ) quali piccoli noduli centrali o integralmente coperti da strutture ossee , lago bioptico stato introdotto nei tessuti molli sino alla superficie pleurica evitandone accuratamente la trasgressione . 
in alcuni casi si effettuato pi di un passaggio pleurico , dal momento che o la lesione - bersaglio non stata raggiunta dalla punta dellago al primo tentativo nonostante i riorientamenti intraparenchimali dellago o , nei prelievi senza ausilio della tecnica coassiale , il materiale risultato inadeguato e si ricorsi ad un nuovo prelievo . 
in particolare , 350 / 612 procedure ( 57 , 2% ) hanno richiesto un solo passaggio pleurico ( media 1.5 passaggi pleurici per procedura ; intervallo 14 )  . 
il prelievo stato effettuato tramite agoaspirazione con ago sottile 21 gauge ( g ) o con ago spinale 20 g ( con tecnica ad ago singolo ) , o biopsia con ago tranciante 18 g ( utilizzando la tecnica coassiale )  . 
 per i noduli polmonari sempre preferito da noi un iniziale approccio con ago sottile di chiba ( soprattutto nelle lesioni di piccole dimensioni ) , tranne nei casi di un secondo tentativo dopo un precedente prelievo non conclusivo . nellevenienza della ripetizione iterativa viene considerata la possibilit di utilizzare un ago di calibro maggiore ( spinale ) od un tranciante . 
final diagnoses were based on the surgical outcomes ( surgical patients ) or the imaging data and clinical - radiological follow - up for a period of at least 12 months ( response to first - line chemotherapy or antibiotic treatment designed on the basis of the biopsy ; unchanged imaging findings in the absence of treatment )  . 
in relation to malignant diagnoses , we considered : true positives for malignancy : all cases classified as malignant at transthoracic needle biopsy and confirmed by histology of the surgical specimen ; cytohistology of an additional biopsy from a distant metastatic lesion ; imaging evidence ( radiology or nuclear medicine ) of distant metastases ; partial or complete response to first - line chemotherapy ; clinical course . 
in surgical cases , we assessed the reliability of the needle biopsy in providing precise characterisation with specific histological type a negative finding ( benign lesion ) was classified as a true negative for malignancy at fnac or cb when its benign nature was confirmed by subsequent surgery or biopsy or when the lesion appeared stable , smaller or had totally disappeared at follow - up ct ( 12 - month follow - up ) in an untreated patient or a patient treated with appropriate antibiotic , but not antineoplastic , therapy [ 18 , 22 ] false positives for malignancy : all cases with a positive biopsy not confirmed by surgery false negatives for malignancy : all cases with a negative ( benign at fnac / cb ) or indeterminate result ( absence of cellular atypia , presence of atypical cells not otherwise specified , presence of nonspecific or granulomatous inflammatory reaction , insufficient material or blood contamination ) at transthoracic needle biopsy with positive surgical outcome or disease progression at follow - up imaging . the quality of each sample obtained by fine - needle transthoracic biopsy was scored from 0 to 3 , according to the following criteria : 0 = slides containing exclusively blood components ( blood contamination ) without epithelial cells ; 1 = finding of nonspecific benign cells ( inflammatory ; type i and ii pneumocytes ; atypical cells not otherwise specified ) ; 2 = presence of morphologically malignant cells , without characterisation of histological type or without differentiation between small - cell or non - small - cell cancer ; 3 = slides containing benign or malignant cells with definite histological characterisation . 
samples with scores 0 , 1 and 2 ( when justified by inability to differentiate between small - cell and non - small - cell cancer ) were considered nondiagnostic and therefore classed as false negatives in relation to the final diagnosis . 
samples with scores of 2 and 3 were considered adequate . size 1146 the dimensions of each lesion were measured on screen using a dedicated programme ( electronic callipers ) , in the two maximum orthogonal axial diameters , on the preliminary localisation scan reconstructed from the raw data with adequate window and level settings for lungs . 
in tutti i casi stato utilizzato una lunghezza dello scatto della pistola bioptica pari a 3 , 3 cm . la presenza del citopatologo in sala radiologica , essenziale nei prelievi citoaspirativi , ha costituito elemento caratterizzante tutte le procedure . 
in caso di materiale inadeguato od insufficiente ad unanalisi citologica od istologica si provveduto alla immediata ripetizione del prelievo : in particolare 190 / 612 procedure ( 31 , 0% ) hanno richiesto pi di un prelievo ( media 1 , 32 campioni per procedura con intervallo 14 )  . per valutare linsorgenza di complicanze ( pneumotorace , soffusione emorragica lungo il tragitto dellago ) sono state acquisite , circa 35 minuti dopo il prelievo ( nel tempo richiesto dal citopatologo per preparare gli strisci e dare giudizio di idoneit ) , scansioni tc di controllo . raccolta dei dati : definizione e selezione delle variabili sono state considerate molteplici variabili , a valore discreto o continuo , riferite al paziente , alla lesione od alla stessa manovra interventistica . 
per entrambe le categorie sono stati calcolati , attraverso la costruzione di tabelle 22 , i livelli di accuratezza ( sensibilit , specificit , valore predittivo positivo e negativo , proporzione di falsi positivi e negativi , accuratezza globale della metodica ) correlando il risultato della biopsia transtoracica con la diagnosi finale . 
la diagnosi definitiva stata ottenuta attraverso lanalisi del dato chirurgico ( per i pazienti resecati ) , dei dati di imaging e del follow - up clinico / radiologico per un periodo non inferiore a 12 mesi ( risposta a trattamenti chemioterapici di i linea o a trattamenti antibiotici impostati sulla base del risultato bioptico transtoracico ; stazionariet radiologica dei reperti in assenza di terapia )  . 
a questo proposito sono stati considerati , in rapporto alle diagnosi maligne : veri positivi per malignit i casi classificati maligni allagobiopsia transtoracica e confermati : dal dato istologico sul pezzo operatorio ; dal dato cito - istologico derivante da una ulteriore aggressione bioptica di una lesione metastatica a distanza ; dallevidenza strumentale ( indagini radiologiche e di medicina nucleare ) di metastasi a distanza ; dalla risposta parziale o completa a trattamenti chemioterapici di i linea ; dal decorso clinico . 
nei casi resecati stata valutata laffidabilit dellagobiopsia transtoracica nel fornire una precisa caratterizzazione con specificit di istotipo ; un esito negativo ( ovvero di natura benigna ) stato classificato vero negativo per malignit alla fnac o cb in caso di successiva conferma chirurgica o bioptica o se a.m. 
we recorded data on the presence of single or multiple pulmonary lesions and lobar location specifying possible contact or close proximity to an interlobar fissure , the hilum or major intrapulmonary vessels . 
the depth of the consolidation was defined as the distance between the entry site on the pleural surface and the proximal edge of the lesion , measured along the needle path on lung window settings [ 13 , 32 , 3436 ]  . 
i prelievi con punteggio 0 , 1 e 2 ( se per questi ultimi non stata possibile una differenziazione tra neoplasia a piccole cellule e non ) sono stati considerati non diagnostici e quindi classificati come falsi negativi in rispetto alla diagnosi finale . 
i prelievi con punteggio 2 e 3 sono stati invece considerati adeguati . needle type and calibre dimensioni in all procedures , we used either a single needle ( 21 - gauge chiba needle ; 20 - gauge spinal needle ; 18 - gauge cutting needle with coaxial technique ) or , in the event of multiple biopsies , a consecutive combination of different needles . 
the choice of the type of needle was based on parameters such as lesion size , clinical suspicion ( history , clinical and imaging data ) , operators preferences and the presence of the cytopathologist . 
during each anale dimensioni di ogni lesione sono state misurate a monitor mediante programma dedicato ( calibro elettronico ) , nei due diametri assiali ortogonali massimi , sulle scansioni di centratura preliminari ricostruite dai dati grezzi attraverso adeguata finestra e livello per parenchima polmonare . 
stata quindi calcolata la media aritmetica relativa alle misurazioni ottenute per ciascuna lesione in esame . morfologia e localizzazione le lesioni sono state suddivise in noduli e masse , se caratterizzate da un diametro trasverso massimo < o 30 mm rispettivamente . 
sono state considerate consolidazioni gli infiltrati cuneiformi , di natura fluida o cellulare , a distribuzione segmentaria o lobare , associati o meno a broncogramma aereo . sono stati registrati i dati relativi alla presenza di lesioni polmonari singole o multiple , la localizzazione lobare specificando leventuale contatto o la stretta vicinanza ad una scissura interlobare , allilo o a strutture vascolari intrapolmonari di rilievo . 
la profondit delladdensamento stata considerata quale distanza tra il punto di ingresso della superficie pleurica sino al margine prossimale della lesione , misurata lungo il tragitto dellago su finestra per parenchima polmonare [ 13 , 32 , 3436 ]  . 
the variables of the two groups in each category were correlated . differences between the groups were examined using the nonparametric mann - whitney u test or variance analysis according to kruskal - wallis , when indicated . 
differences between proportions were analysed with pearsons 2 test or students t test . parameters considered significant in determining diagnostic accuracy were further stratified and correlated to accuracy with the aid of contingency graphs and tables . 
a p value < 0.05 was taken as significant in all cases . results in calculating the methods diagnostic accuracy , we considered all 608 patients included in the study : there were 154 surgical patients ( for whom postoperative surgical evaluation was considered the gold standard against which the interventional technique was compared ) there were 454 nonsurgical patients who had completed , as at august 2006 , an imaging , clinical and treatment - response follow - up of at least 12 months ; in these cases the final diagnosis was inferred from the benign or malignant behaviour of the lesion we considered 612 procedures ; the quality of each of the 612 samples was assigned a discrete score from 0 to 3 at the end of each procedure in 435 / 612 procedures ( 71.1% ) , the needle biopsy recovered benign or malignant cells , allowing specific cytohistological characterisation ( score 3 )  . 
of these , 52 were benign , 382 were malignant and only one revealed the presence of a hamartochondroma associated with a primary squamous cell carcinoma of the lung there were 75 / 612 samples ( 12.2% ) that demonstrated definitely malignant cells ( score 2 ) , without providing information on histological type . 
four samples demonstrated the presence of definitely neoplastic cells ( probably epithelial ) without providing discrimination between small - cell and non - small - cell cancer , which is essential for therapy . 
in addition , the only false positive result in our cohort was encountered among these four cases : a suspected carcinoma not otherwise specified ( fnac ) was found at surgery to be an excavated granuloma characterised by severe cellular dysplasia due to previous tuberculous infection in 46 / 612 cases ( 7.5% ) , the sample was scored 1 , as 19 / 46 samples ( 41.3% ) demonstrated exclusively normal lung 1148 mento lineare di parenchima polmonare trasgredito dallago . caratteristiche interne sono state considerate laspetto solido ( se con finestra mediastinica risultava visualizzabile almeno il 50% della superficie dellespanso rispetto a quanto visibile con finestra polmonare ) , la presenza di escavazioni ( aree allinterno della lesione con valori di attenuazione propri dellaria ) , calcificazioni e broncogramma aereo . 
 stata inoltre valutata , con criterio visivo ed attraverso lausilio della misurazione digitale dei dati di attenuazione mediante roi , la densit basale della lesione per ricercare eventuali aree di disomogeneit riferibili a necrosi . tipo di ago e calibro per tutte le procedure si ricorso alluso esclusivo di un unico ago ( chiba 21 g ; spinale 20 g ; tranciante 18 g con tecnica coassiale ) o ad associazione consecutiva di diversi aghi nel caso di prelievi multipli . 
la scelta di tipologia dellago stata posta secondo alcuni parametri quali le dimensioni della lesione , il sospetto clinico ( su basi anamnestiche , obiettive e di imaging ) e le preferenze delloperatore , oltre che la presenza dellanatomopatologo in sala radiologica . 
in particolare , lago sottile di chiba ha trovato esclusivo impiego in 470 / 612 procedure ( 76 , 8% ) , lago spinale in 76 ( 12 , 4% ) , il tranciante in 35 ( 5 , 7% ) , lassociazione chibaspinale in 27 ( 4 , 4% ) e lassociazione chiba - spinale - tranciante in 4 ( 0 , 7% )  . complicanze analisi statistica la comparsa di pneumotorace o di sfumate opacit parenchimali con aspetto radiologico a vetro smerigliato ( da impegno alveolare per emorragia ) stata registrata per ogni procedura . lanalisi statistica stata condotta con lutilizzo del programma statistica 6.1 ( statsoft software , per windows ) e mirata alla valutazione dellaccuratezza della procedura . 
i pazienti sono stati , in ogni sessione analitica , suddivisi in due gruppi omogenei per variabili discrete a soluzione dicotomica ( si / no ) ed analizzati per le diverse variabili , discrete o continue , che si ipotizzava influenzare i livelli di accuratezza : i gruppi sono stati suddivisi tra le diagnosi corrette e le diagnosi non corrette o non possibili a causa dellinadeguatezza del prelievo agobioptico . 
le differenze tra i gruppi sono state esaminate utilizzando i tests non parametrici mann - whitney u - test o analisi della varianza secondo kruskal - wallis , quando indicati . 
le differenze tra le proporzioni sono state analizzate con il test 2 di pearson od il test t di student . i parametri considerati significativi , nel determinare i livelli di accuratezza diagnostica , sono stati ulteriormente stratificati e correlati allaccuratezza con lausilio di grafici a.m. 
the discordance between the biopsy result and the final diagnosis is due to sampling errors , with sampling of surrounding healthy lung parenchyma or perilesional reactive inflammatory tissue there were 56 / 612 samples ( 9.1% ) scored 0 owing to sample inadequacy ( blood contamination ) and which were therefore classed as nondiagnostic . 
in 35 / 56 samples ( 60.7% of samples with a score of 0 ) , the lesion was subsequently found to be malignant . a final diagnosis based on the criteria previously described could be obtained for all 612 procedures : 17.0% ( 104 / 612 ) of lesions were found to be benign , whereas 83.0% ( 508 / 612 ) were malignant . 
the 458 cases positive for malignancy , with subsequent confirmation ( one malignant case at biopsy was subsequently found to be a false positive ) comprised 411 primary lung cancers , seven nonepithelial neoplasms and 40 metastatic tumours with secondary localisation to the lung ( table 1 )  . of these 458 patients , 46 ( 7.6% ) had a history of neoplastic disease at another location . 
of these 46 , the metastatic origin of the lung lesion was demonstrated in 40 , whereas in the remaining six cases , the primary nature of the lung nodule was demonstrated with the aid of immunocytochemistry and comparison with cytology and / or histology of the extrapulmonary neoplasm , when available . among the 459 cases of malignancy diagnosed at biopsy , o tabelle di contingenza . 
in tutti i casi stato considerato significativo un valore di p < 0 , 05 . risultati per calcolare laccuratezza diagnostica della metodica sono stati inclusi nello studio 608 pazienti : centocinquantaquattro sono i soggetti sottoposti ad intervento chirurgico ( per i quali disponibile lesame istologico post - operatorio , considerato il gold standard di raffronto con la metodica interventistica )  . quattrocentocinquantaquattro sono , invece , i pazienti non chirurgici che presentano ad agosto 2006 un followup di imaging , clinico e di risposta terapeutica non inferiore a 12 mesi ; in base al comportamento benigno o maligno della lesione in esame stata , quindi , dedotta la diagnosi finale . complessivamente sono state considerate 612 procedure . per ciascuno dei 612 campioni ottenuti , a conclusione di ogni procedura stato attribuito un punteggio discreto da 0 a 3 per asserirne la qualit : in 435 / 612 procedure ( 71 , 1% dei casi ) si sono ottenute , dal prelievo agobioptico , cellule benigne o maligne che hanno permesso una precisa caratterizzazione citoistologica della lesione ( punteggio 3 )  . 
di queste 52 sono risultate benigne , 382 di natura maligna , mentre in 1 solo caso si registrata la presenza di un amartocondroma associato ad un carcinoma polmonare primitivo del tipo squamocellulare ; in 75 / 612 prelievi ( 12 , 2% dei casi ) sono state riscontrate cellule di sicura origine maligna ( punteggio 2 ) , ma non stato possibile trarre conclusioni sullistotipo . 
in contrast , 50 procedures were false negative for malignancy ( negative result at biopsy , with malignant cells detected at repeat biopsy , surgery or follow - up )  . 
table 3 summarises the cytological diagnoses of the 52 cases that were negative for malignancy at biopsy . if we consider only benign lesions at final diagnosis assenza di una suddivisione , critica ai fini terapeutici , in tumore a piccole cellule e non . 
questi ultimi casi pongono importanti problematiche in termini di gestione del paziente per il fatto che non si pu pianificare il corretto iter terapeutico , impedendo in questo modo allagobiopsia di porsi a termine delliter diagnostico . 
inoltre , tra questi 4 casi si riscontrato lunico risultato bioptico falso positivo dellintera coorte : un sospetto carcinoma di non pi precisa caratterizzazione ( fnac ) si poi dimostrato essere , dopo resezione chirurgica , una granulomatosi escaa final diagnoses are based on the retrospective analysis of the outcome of surgery , repeated biopsy , imaging , and clinical follow - up for at least 12 months . 
 malignant pulmonary lesions : sensitivity 90.2% ( 458 / 508 ) , specificity 99.0% ( 103 / 104 ) , positive predictive value 99.8% ( 458 / 459 ) , negative predictive value 67.3% ( 103 / 153 ) , false positive rate 0.22% ( 1 / 459 ) , false negative rate 10.9% ( 50 / 508 ) , accuracy 91.7% [ ( 458 + 103 ) / 612 ] , overall diagnostic accuracy 83.3% [ ( 458 + 52 ) / 612 ] ( for all lesions , benign and malignant ) table 2 results of 612 computed - tomography - guided transthoracic needle biopsies final diagnosisa malignant biopsy diagnosis malignant benign / inadequate total diagnosi bioptica maligno benigno - inadeguato totale 1150 tabella 2 risultati di 612 procedure agobioptiche polmonari diagnosi finalea maligno benign 103 ( 52 / 51 ) benigno 103 ( 52 / 51 ) total totale a la diagnosi finale deriva dallanalisi retrospettiva dei risultati della chirurgia , di uneventuale successiva agobiopsia transtoracica e dallesito del followup clinico / radiologico pari ad un periodo non inferiore ai 12 mesi . 
conversely , in 51 cases , the benign nature of the lesion could not be defined because of sample inadequacy ( 30 samples scored 1 and 21 scored 0 )  . with regard to malignant lesions , 99.8% of malignancies at vata caratterizzata da severa displasia cellulare in relazione alla pregressa infezione tubercolare ; in 46 / 612 casi ( 7 , 5% ) stato attribuito , al campione prelevato , un punteggio pari a 1 , dal momento che in 19 / 46 casi ( 41 , 3% ) si sono riscontrate esclusivamente cellule normali proprie del tessuto polmonare ( pneumociti di i e ii ordine ) , in 14 / 46 ( 30 , 4% ) cellule flogistiche in assenza di significative atipie ed in 13 / 46 ( 28 , 3% ) cellule atipiche non ulteriormente caratterizzabili , ma neanche classificabili come sicuramente maligne . 
in 16 di questi 46 prelievi ( 34 , 7% dei prelievi a punteggio 1 ; 2 , 6% sul totale delle procedure ) le lesioni campionate sono poi risultate di natura maligna . 
la discordanza tra risultato bioptico e diagnosi finale dovuta ad errori di campionamento , per prelievo di parenchima polmonare sano limitrofo o di tessuto flogistico reattivo perilesionale ; infine 56 / 612 prelievi ( 9 , 1% ) hanno ottenuto un punteggio pari a zero a causa dellinadeguatezza del materiale analizzato dallanatomopatologo ( campioni ematici ) e sono quindi stati classificati come non diagnostici . 
in 35 / 56 prelievi ( 60 , 7% dei campioni a punteggio 0 ) la lesione poi risultata maligna . per tutte le 612 procedure stato possibile ottenere una diagnosi finale con i criteri gi ampiamente descritti : il 17 , 0% ( 104 / 612 ) delle lesioni risultato di natura benigna , mentre lo 83 , 0% ( 508 / 612 ) maligna . 
lagobiopsia transtoracica percutanea ha conseguito un risultato diagnostico in 511 casi su 612 ( 83 , 5% , prelievi a punteggio 2 e 3 ) , mentre in 101 procedure ( 16 , 5% , prelievi a punteggio 0 e 1 ) non stato possibile fornire un giudizio affidabile . 
i 458 casi positivi per malignit , con conferma successiva ( un caso maligno alla biopsia poi risultato un falso positivo ) hanno compreso 411 carcinomi polmonari primitivi , 7 neoplasie non epiteliali e 40 tumori metastatici con localizzazione secondaria al polmone ( tabella 1 )  . di questi 458 pazienti , 46 ( 7 , 6% ) presentavano un precedente anamnestico di malattia neoplastica in altra sede . 
tra questi ultimi , in 40 si dimostrata lorigine metastatica della lesione polmonare , mentre nei restanti 6 casi stato possibile definire la natura primitiva del nodo polmonare attraverso lausilio della immunocitochimica ed il confronto , quando disponibile , con i reperti citologici e / o istologici del tumore extrapolmonare . tra i 459 riscontri risultati maligni allagobiopsia , solo in un caso la diagnosi definitiva non ha confermato il dato configurando quindi la presenza di un unico falso positivo per malignit . 
si sono invece registrate 50 procedure false negative per malignit ( risultato negativo per cellule neoplastiche con esito maligno alla ripetizione della procedura , allintervento chirurgico od al follow - up )  . 
in tabella 2 vengono riportati i risultati conseguiti , in termini di affidabilit della metodica , sulla corretta diagnosi delle lesioni maligne . la tabella 3 riassume le diagnosi citologiche nei 52 casi risultati negativi per malignit allagobiopsia . analizzando le sole lesioni benigne alla diagnosi finale ( 104 / 612 , 17% ) , lagobiopsia ha permesso di conseguire un sicuro giudizio di benignit con diagnosi specifica in soli 52 1151 a.m. 
a specific judgement of benignity of the biopsy sample could , however , be made in only 34% of samples , characterised by the definite absence of neoplastic cells ( 52 / 153 ) ( table 2 )  . 
finally , table 4 demonstrates that lesion depth ( the portion of aerated lung traversed by the needle ) , morphology and lobar location ; the presence of calcification , cavitation and necrotic centre ; and the occurrence of complications ( pneumothorax and bleeding ) have no influence on the diagnostic accuracy of transthoracic ct - guided needle biopsy . discussion with the use of percutaneous transthoracic needle biopsy , and in particular of fnac , as an initial minimally invasive procedure for evaluating the nature of pulmonary lesions , this study obtained an overall diagnostic accuracy of 84% , with levels of 92% and 67% for malignant and benign lesions , respectively . 
in 51 lesioni benigne non invece stato possibile stabilirne la natura a causa dellinadeguatezza del prelievo ( 30 prelievi a punteggio 1 e 21 prelievi a punteggio 0 )  . riferendosi agli addensamenti polmonari di natura maligna , il 99 , 8% delle lesioni considerate maligne allagobiopsia poi stata confermata ( 458 / 459 , valore predittivo positivo per malignit ) , mentre il 67 , 3% dei prelievi con assenza di cellule tumorali ( prelievi classificati come benigni , od inadeguati ) risultato benigno ( 103 / 153 , valore predittivo negativo per malignit )  . 
stato comunque possibile dare un giudizio specifico di benignit del prelievo agobioptico solo nel 34% dei campioni , caratterizzati dalla sicura assenza di cellule tumorali ( 52 / 153 ) ( tabella 2 )  . 
la sensibilit e la specificit per la diagnosi di malignit sono risultate del 91% e 99% rispettivamente , con una proporzione di falsi positivi trascurabile ( 0 , 22% ) ed una proporzione di falsi negativi dell11% . 
questo valore raggiunge l88 , 2% ( 540 / 612 ) se , tra le diagnosi agobioptiche corrette , si includono i 30 prelievi a punteggio 1 poi risultati benigni ( ovvero reperti considerati allagobiopsia negativi per cellule neoplastiche in assenza di una specifica diagnosi )  . 
in tabella 4 riassunto linsieme di variabili che si supposto esercitare una influenza sui livelli di accuratezza della metodica : le uniche variabili associate con significativit statistica ad una riduzione dei livelli di accuratezza diagnostica sono risultate la discrimante della diagnosi finale ( categoria benigno versus maligno ) e le dimensioni della lesione . 
la tabella 4 dimostra , infine , come la profondit della lesione ( ovvero la quota di parenchima polmonare aerato attraversata dallago ) , la sua morfologia e localizzazione lobare , la presenza di calcificazioni , cavitazioni e centro necrotico , oltrech loccorrenza di complicanze ( pneumotorace e sanguinamento ) non esercitino alcuna a.m. 
anumber of procedures ; bpearsons 2 test for categoric variables ; caverage lesion diameter in two axial planes ; ns , not significant tabella 4 correlazione per variabili , con la diagnosi finale , di risultati corretti ed errati od indeterminati allagobiopsia transtoracica variabile na ( 612 ) diagnosi finale errata o indeterminata n = 102 corretta n = 510 52 ( 50 , 0 ) 458 ( 90 , 2 ) 30 ( 66 , 6 ) 362 ( 87 , 3 ) 118 ( 77 , 6 ) 52 ( 50 , 0 ) 50 ( 9 , 8 ) 15 ( 33 , 3 ) 53 ( 12 , 7 ) 34 ( 22 , 4 ) i valori in parentesi sono riferiti in % . 
agrandezza del campione ; btest 2 di pearson per variabili categoriche ; cconsiderata quale dimensione media tra i due assi ( maggiore e minore ) ortogonali della lesione ; ns , non significativo . 
however , they are also dependent on the severity with which sample adequacy is judged : a sample without neoplastic cells should not be considered negative for malignancy ( consider sampling errors , for example ) unless they provide a precise and specific diagnosis of benignity ( 100% specificity is desirable in benign lesions )  . 
many studies [ 13 , 29 , 32 , 37 , 38 ] have reported substantially lower levels of diagnostic accuracy for benign lung lesions ( range 52%91% ) compared with malignant lesions influenza sui valori di accuratezza diagnostica dellagobiopsia transtoracica tc guidata . discussione attraverso lutilizzo dellagobiospia transtoracica percutanea , ed in particolar modo della fnac , quale iniziale procedura mini - invasiva nella valutazione di natura degli addensamenti polmonari si ottenuta , in questo studio , unaccuratezza diagnostica globale dell84% con valori di accu1153 a.m. 
1a - c relationship between mean lesion size and number of observations for the entire group of lesions ( a ) and in regard to the presence ( b ) or the absence ( c ) of a necrotic centre . 
1a - c correlazione tra diametro medio della lesione e numero di casi per tutte le lesioni ( a ) e suddividendo queste ultime per presenza ( b ) od assenza ( c ) di centro necrotico . 
la sensibilit della metodica per lesioni maligne risultata del 90% ( con valori di specificit del 99% ) , mentre per lesioni benigne la sensibilit risultata del 50% , ma con specificit del 100% . 
questi dati dimostrano come , nellinquadramento diagnostico delle lesioni benigne , la fnac risulti inferiore alla cb [ 20 , 24 ] , ma paiono anche giustificati dalla severit di giudizio di idoneit cui vengono sottoposti i campioni : un prelievo con assenza di cellule neoplastiche non deve essere considerato negativo per patologia tumorale ( si considerino , ad esempio , gli errori di campionamento ) , a meno che sia possibile formulare una precisa e specifica diagnosi di benignit ( sono auspicabili valori di specificit per lesioni benigne pari al 100% )  . 
molteplici studi in letteratura [ 13 , 29 , 32 , 37 , 38 ] riportano valori di accuratezza diagnostica sensibilmente inferiori per lesioni polmonari benigne ( con intervallo compreso tra il 52% ed il 91% ) se paragonate a lesioni maligne ( intervallo compreso tra l86% ed il 100% ) , sia nel prelievo aspirativo sia tranciante ; inoltre , considerando esclusivamente gli addensamenti benigni , la cb presenta valori di accuratezza e sensibilit significativamente maggiori rispetto alla fnac ( accuratezza superiore all80% versus 50%70% ) [ 9 , 2225 ]  . 
i livelli di accuratezza e sensibilit riportati dal nostro studio sono in linea con i riscontri fnac della letteratura , ma non dimensioni lesione ( cm ) lesion size ( cm ) fig . 
 ctest t di student ( per campioni indipendenti e per gruppi ) , p < 0 , 05 considerata significativa : il diametro medio delle lesioni con ( 57 , 7 mm ) e senza ( 34 , 0 mm ) necrosi differisce con significativit statistica . raggiungono i valori riportati dalla cb , elemento comunque non contraddittorio dal momento che solo nel 6 , 4% delle procedure stato utilizzato un ago tranciante ( singolarmente od in associazione con altri aghi )  . la documentazione di cellule sicuramente maligne , nella pressoch totalit dei casi , prova certa della natura maligna dellespanso . 
nella nostra coorte lunico caso classificato come maligno ( neoplasia di origine epiteliale ) e poi risultato benigno ( granulomatosi escavata ) alla diagnosi finale trova esplicazione nel fatto che la lesione benigna ha sviluppato atipie cellulari tali da farla risultare sospetta allindagine citologica per espanso di natura maligna . nonostante gli insuccessi siano sensibilmente minori per le lesioni maligne , tuttavia si evidenziato un 11% di falsi negativi nellambito di tali addensamenti ( integralmente costituiti da prelievi a punteggio 01 )  . 
in aggiunta , la nostra esperienza indica come la sola ispezione ad occhio nudo del campione ottenuto ( in termini di integrit del frustolo bioptico o di cellularit o di carattere ematico del prelievo aspirativo ) inaffidabile nel predirne ladeguatezza [ 30 , 32 , 34 , 36 , 38 ] ed quindi auspicabile la presenza dellanatomopatologo nel corso della seduta bioptica . 
la ripetizione del prelievo dovrebbe essere presa in considerazione ogni qual volta si verifichi discordanza tra risultato bioptico , presentazione clinica e caratteristiche tc ( prima di passare ad accertamenti diagnostici invasivi di ii livello ) [ 13 ]  . lanalisi della nostra coorte ha evidenziato come le uniche variabili in grado di influenzare , in termini statisticamente significativi , laccuratezza diagnostica della metodica siano la diagnosi finale ( benignit versus malignit ) e le dimensioni della lesione ( nodi con diametro medio inferiore ad 1 , 5 cm o masse di dimensioni superiori a 5 cm sono caratterizzati da livelli di minore accuratezza diagnostica )  . 
riferendosi a questultimo aspetto , gli studi presenti in letteratura esprimono giudizi discordanti in merito allinfluenza delle dimensioni della lesione sui livelli di accuratezza dia1155 dimensioni lesione ( cm ) lesion size ( cm ) fig . 
3 relazione esistente tra dimensione della lesione e presenza di necrosi : il grafico dimostra come lincidenza di necrosi si incrementi gradualmente allaumentare delle dimensioni delle lesioni ; la maggior proporzione di necrosi si riscontrata nelle lesioni di 6 cm , ovvero quelle caratterizzate da minor accuratezza diagnostica . ( from 86% to 100% ) , achieved with both fnac and cb . 
accuracy and sensitivity levels found in our study are similar to those previously reported for fnac but fail to reach those reported for cb , a finding that is not contradictory given that only 6.4% of procedures were carried out with a cutting needle ( used alone or in combination with other needles )  . demonstration of definitely malignant cells is proof of the a.m. 
4 il grafico box & whisker dimostra una differenza significativa in termini di dimensioni nel gruppo di lesioni senza ( 3 , 4 cm1 , 8 ) e con necrosi centrale intralesionale ( 5 , 7 cm1 , 8 )  . 
il diametro medio delle lesioni in assenza e presenza di necrosi differisce significativamente ( p < 0 , 001 ) ( ds , deviazione standard ; n , no ; s , si )  . malignant nature of the mass in virtually all cases . 
in our cohort , the only case classified as malignant ( epithelial neoplasm ) at biopsy and found to be benign ( excavated granuloma ) at final diagnosis is explained by the fact that the lesion had developed cellular atypia that made it suspicious for malignancy at cytological assessment . although there were substantially fewer failures in malignant lesions , the false negative rate was 11% in this group ( all for samples with a score of 01 )  . 
in addition , our experience shows that gross inspection of the sample ( in terms of integrity of the biopsy specimen , cellularity or blood contamination of the aspirate ) is unreliable in predicting adequacy [ 30 , 32 , 34 , 36 , 38 ] , making the presence of a pathologist during the biopsy procedure strongly desirable . 
found that diagnostic accuracy declined progressively with decreasing lesion size : from 100% to 93% , 87% , 79% and 67% for lesions > 5 cm , 35 cm , 23 cm , 12 cm and < 1 cm , respectively [ 13 ]  . 
reported accuracy levels of approximately 88% for lesions > 2 cm when the mean number of needle passes was 2.5 , whereas when only a single specimen was obtained , accuracy decreased to 83% [ 29 ]  . the results of our study are similar to those reported by yeow et al . 
 [ 20 ] , who found , with a mean number of passes of three ( without a pathologist present ) , lower levels of diag1156 gnostica [ 13 , 20 , 30 , 36 , 37 ]  . 
tsukada ha riscontrato un graduale e progressivo decremento dei valori di accuratezza diagnostica man mano che le dimensioni delle lesioni si riducevano : dal 100% al 93% , 87% , 79% e 67% per lesioni rispettivamente > 5 cm , tra 3 e 5 cm , tra 2 e 3 cm , tra 1 e 2 cm e < 1 cm [ 13 ]  . 
lucidarme ha riscontrato valori di accuratezza di circa l88% per lesioni > 2 cm con un numero medio di prelievi per biopsia di 2.5 , mentre laccuratezza si riduceva all83% in caso di unico prelievo per seduta bioptica [ 29 ]  . nel nostro studio si sono ottenuti risultati simili a quelli descritti da yeow [ 20 ] che riportava , con una media di 3 prelievi bioptici a seduta ( in assenza di un medico anatomopatologo in sala ) , livelli inferiori di accuratezza diagnostica per lesioni < 1 , 5 cm e > 5 cm ( dell84% e 93% rispettivamente ) quando paragonati a lesioni con diametro compreso tra 1 , 5 e 5 cm ( 96% )  . 
nel nostro lavoro vengono , invece , riportati valori di accuratezza diagnostica globale ( per lesioni maligne e benigne ) del 68% per lesioni < 1 , 5 cm , del 78% per lesioni > 5 , 0 cm e dell87% per le lesioni comprese tra 1 , 5 e 5 , 0 cm . la maggior accuratezza diagnostica riportata da yeow giustificata dal fatto che in tutti i casi stata eseguita una cb con tecnica coassiale ( caratterizzata da livelli di accuratezza diagnostica maggiori rispetto alla fnac ) , mentre nel nostro studio solo il 6% delle manovre stato eseguito con tale procedura , riservando la fnac a tutti gli altri casi . 
in aggiunta , avendo la disponibilit del citopatologo in sala radiologica , risultato sensibilmente minore il numero di prelievi per seduta ( 1 , 3 prelievi versus 3 , 0 )  . 
il numero di prelievi , essendo variabile nei diversi studi , potrebbe costituire un determinante elemento confondente in grado di influenzare laccuratezza diagnostica al pari delle dimensioni della lesione [ 20 , 29 , 37 ]  . 
con una media di 1 , 3 prelievi per procedura , il nostro studio pone come limite dimensionale critico , al di sotto del quale si verifica una sensibile riduzione di accuratezza diagnostica , il valore di 1 , 5 cquesto risultato in linea con le a.m. 
is explained by the fact that they used cb with coaxial technique ( which is more accurate than fnac ) in all cases , whereas we used cb for only 6% of procedures and fnac for the remaining cases . 
the number of needle passes should , however , be kept to a minimum , as this variable is significantly related to higher rates of pneumothorax [ 32 ] ; the presence on site of a cytopathologist during the procedure is instrumental to establishing a correct diagnosis . the substantial reduction in diagnostic accuracy in small lesions could be due to sampling errors resulting from increased technical difficulty in the localisation phase [ 11 ]  . such difficulties are further supported by the higher rate of complications within this class of lesions : yeow et al . 
 [ 32 ] reported a 17 times higher risk of pneumothorax and a six times higher risk of bleeding for lesions smaller than 2 cm . in view of the lower diagnostic accuracy for small lung nodules ( whether sampled by fnac or cb ) and the higher complication rate , fine - needle aspiration should always be preferred to cb for lesions smaller than 1 cm , as suggested by laurent et al . 
when sampling these nodules , in fact , it is impossible to avoid inclusion of portions of healthy aerated lung tissue , as the biopsy core is 1.3cm long ( shortest throw - length setting ) [ 29 , 32 , 37 ]  . 
diffuse bleeding may also occur as a result of the surrounding healthy parenchyma not being able to provide an adequate tamponade effect [ 29 , 32 , 37 ]  . 
finally , in a nonnegligible number of cases , the material recovered proves inadequate due to the abundant presence of blood . despite the fact that throw lengths of 10 mm can be used to avoid bleeding in lesions smaller than 1.5 cm , boiselle et al . reported a diagnostic accuracy of 62% with this approach [ 39 ]  . 
comunque auspicabile conseguire il minimo numero di campionamenti possibili per procedura dal momento che tale variabile significativamente correlata ad una maggior proporzione di pneumotoraci [ 32 ] ; elemento indispensabile , a garanzia dellobiettivo di ottenere una corretta diagnosi , risulta essere la presenza del citopatologo durante la seduta bioptica . la sensibile riduzione di accuratezza diagnostica per lesioni di ridotte dimensioni potrebbe trovare una valida esplicazione in errori di campionamento , dovuti a maggiori difficolt tecniche nella fase di centratura bioptica [ 11 ]  . 
le difficolt procedurali trovano ulteriore conferma nella maggior proporzione di complicanze riscontrabile in questa categoria di lesioni : yeow [ 32 ] riporta , infatti , un rischio di pneumotorace 17 volte maggiore e di sanguinamento 6 volte superiore per lesioni di diametro inferiore ai 2 cin ragione della minor accuratezza diagnostica per i piccoli noduli polmonari ( che siano sottoposti a fnac o a cb ) ed alla pi alta percentuale di complicanze riscontrate , risulta sempre opportuno preferire alla cb , come gi suggerito da laurent e yeow [ 20 , 30 ] , il prelievo aspirativo con ago sottile per le lesioni di dimensione inferiore al centimetro . 
infatti , risulta impossibile evitare , nel campionamento di questi piccoli nodi , porzioni di tessuto aerato sano , dal momento che il frustolo bioptico ha lunghezza pari ad 1 , 3 cm ( limite inferiore della lunghezza dello scatto dellago tranciante ) [ 29 , 32 , 37 ]  . 
pu inoltre verificarsi un sanguinamento diffuso dal momento che il parenchima circostante sano , interessato anchesso dal campionamento , non in grado di fornire un adeguato effetto tampone [ 29 , 32 , 37 ]  . 
nonostante si possano utilizzare lunghezze di scatto pari a 10 mm per evitare il sanguinamento in lesioni inferiori ad 1 , 5 cm , boiselle ha riportato , in tali condizioni , unaccuratezza diagnostica pari al 62% [ 39 ]  . 
questo valore risulta inferiore al 67% da noi ottenuto utilizzando esclusivamente , per tali lesioni , lagoaspirato e con un tasso di sanguinamento perilesionale di minor entit . analizzando il parametro dimensionale , quale variabile determinante laccuratezza diagnostica della procedura , si sono riscontrati decrementi di accuratezza per lesioni con dimensione maggiore ai 5 cquesta evenienza viene compresa se si considera la presenza di necrosi centrale , che risulta di frequente riscontro nelle masse polmonari > 5 cm ( nella nostra casistica percentuale tc di necrosi tumorale per lesioni sino a 5 cm compresa tra lo 0% ed il 15% , per masse 5 cm tra il 12% ed il 41% )  . 
risulta quindi evidente come , nelle grosse masse polmonari , sia necessario disporre di indagini tc acquisite con infusione di mezzo di contrasto per individuare aree lesionali necrotiche al fine di evitarle durante il campionamento . 
inoltre , in assenza di un anatomopatologo in sala , nellevenienza allispezione visiva di frustoli particolarmente fragili e frammentati o di prelievi citologici ottenuti con agoaspirato , particolarmente ematici , si dovrebbero 1157 a.m. 
moreover , when no on - site pathologist is present and the biopsy cores appear particularly fragile and fragmented or the aspirate particularly bloody on gross inspection , further samples should be obtained taking care to direct the needle to different areas of the lesion [ 32 , 40 , 41 ]  . the presence of cavitations within a lung mass does not appear to influence the diagnostic accuracy of either fnac , as demonstrated in our study , or cb , as shown by yeow et al . 
similarly , diagnostic accuracy was not significantly affected by the presence of calcification or a frankly necrotic centre ; by lesion depth ( from the pleural surface ) , morphology or location ; or by the occurrence of complications such as pneumothorax and bleeding . 
no evaluation was made with regard to needle size or the radiologists experience , variables that are ascribed different significance in the various studies [ 20 , 41 ]  . conclusions in conclusion , transthoracic ct - guided needle biopsy of pulmonary lesions is a highly accurate procedure , especially in malignant lesions . 
however , to enhance the likelihood of success and achieve a correct diagnosis , awareness of the variables affecting its effectiveness is crucial : benign lesions , nodules smaller than 1.5 cm and masses larger than 5 cm are associated with substantially lower accuracy rates . 
analogamente , la presenza di calcificazioni o di centro francamente necrotico , od ancora la profondit della lesione ( rispetto al piano pleurico ) o la morfologia o la localizzazione od infine la comparsa di complicanze , quali lo pneumotorace od il sanguinamento , non hanno significativamente alterato i valori di accuratezza diagnostica della procedura . 
non si sono , invece , formulate valutazioni relative al calibro dellago o allesperienza del radiologo prelevatore , parametri che assumono , a seconda dei diversi studi presenti in letteratura , significato diverso [ 20 , 41 ]  . conclusioni in conclusione , lagobiopsia transtoracica tc guidata di espansi polmonari metodica ad elevata accuratezza diagnostica , specie per le lesioni di natura maligna . 
risulta comunque indispensabile , al fine di implementare le probabilit di successo della procedura in termini di raggiungimento della corretta diagnosi , la conoscenza delle variabili in grado di inficiare tale efficacia : le lesioni benigne ed i nodi di dimensioni minori ad 1 , 5 cm o le masse superiori a 5 cm sono correlate a valori di accuratezza diagnostica sensibilmente inferiori . 
from january 2005 to september 2006 , 23 patients ( 10 men and 13 women ; mean age 41.38.6 years ) affected by als were evaluated with digital cineradiography to assess the grade of dysphagia . 
the cineradiographic technique enabled us to differentiate patients with disorders of the oral ( 17 / 23 ) and / or pharyngeal ( 19 / 23 ) swallowing phase from those without swallowing dysfunction ( 4 / 23 )  . 
in 14 / 23 patients , passage of contrast medium into the upper airways was observed during swallowing , whereas in 5 / 23 cases , aspiration of contrast medium into the lower airways was recorded . 
in addition , the study proved useful for planning speech therapy and for follow - up in patients with als . key words swallowing dysphagia amyotrophic lateral sclerosis cineradiography riassunto obiettivo . 
dal gennaio 2005 al settembre 2006 , 23 pazienti ( 10 uomini e 13 donne ; et media 41 , 38 , 6 anni ) affetti da sla e classificati secondo la classificazione als severity scale ( alsss ) di hillel sono stati sottoposti a studio videofluorografico della deglutizione . 
la tecnica videofluorografica utilizzata ha permesso di differenziare i pazienti con alterazioni deglutitorie , 17 / 23 con alterazioni della fase orale e 19 / 23 con alterazioni della fase faringea ( 19 / 23 pazienti ) , da quelli che non presentavano alterazioni 4 / 23 . 
lo studio videofluorografico della deglutizione una tecnica dotata di elevata capacit diagnostica per la valutazione dei deficit deglutitori , essendo in grado di identificarne il grado di compromissione e le cause determinanti . lo studio dinamico della deglutizione si inoltre dimostrato utile nellapproccio alla terapia logopedica e nel follow - up dei pazienti affetti da sla . parole chiave deglutizione disfagia sclerosi laterale amniotrofica cineradiografia introduction introduzione amyotrophic lateral sclerosis ( als ) is a progressive neurodegenerative disease that involves the upper motor neurons of the cerebral cortex and the lower motor neurons of the brainstem and spinal cord while sparing the sensory component , motor control systems and cognition . 
als is found la sclerosi laterale amiotrofica ( sla ) una malattia neurodegenerativa progressiva che coinvolge sia i motoneuroni primari della corteccia cerebrale che quelli secondari del tronco encefalico e del midollo spinale , risparmiando la componente sensitiva , i sistemi di controllo dei movimenti e 1173 g . 
pseudobulbar palsy , with exclusive involvement of the upper motor neurons . regardless of onset , as the disease progresses , both upper and lower motor neurons are involved , and the diagnosis is based on their simultaneous involvement and exclusion of all possible diagnostic alternatives [ 2 ]  . 
involvement of muscles of the oral cavity , pharynx and larynx is most frequent and earliest in patients affected by the bulbar form , and the consequent dysphagia leads to serious nutritional deficit that , over time , requires tube feeding or percutaneous gastrostomy [ 3 ]  . 
dysphagia is one of the main indicators of the clinical course of the disease , such that there are a number of classifications of its severity , some of which take into account swallowing disorders . 
the most important classification is the hillel als severity scale ( alsss ) [ 5 , 6 ] , which classifies patients according to five classes of severity : normal eating habits ( neh ) , early eating problems ( eep ) , dietary consistency changes ( dcc ) , needs tube feeding ( ntf ) and nothing by mouth ( nbm )  . the videofluoroscopic swallowing study ( vfss ) is the gold standard for assessing swallowing dysfunction , particularly in patients suffering from neurological diseases [ 714 ] , in that it provides accurate analysis of the mechanisms of the oral and pharyngeal phases of swallowing , identification of possible alterations responsible for symptoms , and planning of appropriate speech therapy . 
on the basis of the responses received , patients were divided as follows : 12 patients in the neh group ( mean age 38.4 years ) , seven patients in the eep group ( mean age 41 years ) , three patients in the dcc group ( mean age 50 years ) and one patient in the ntf group ( age 53 years ) ; there were no patients belonging to the nbm group . 
la sla diffusa in tutto il mondo ( incidenza 11 , 5 casi per 100000 pazienti ) [ 1 ] e di solito insorge dopo la seconda decade di vita ( m / f 1 , 5 : 1 ) , con frequenza progressivamente maggiore nelle decadi successive ; la prognosi infausta con attesa di vita di 35 anni dalla prima diagnosi . 
questa malattia pu presentarsi in tre forme che differiscono oltre che dal punto di vista clinico , dal punto di vista fisio - patologico e anatomo - patologico : 1 . 
paralisi pseudobulbare , con coinvolgimento esclusivo dei motoneuroni primari . indipendentemente dallesordio , nella progressione della malattia vengono coinvolti entrambi i gruppi di motoneuroni , primari e secondari e la diagnosi viene posta nel caso di un loro interessamento simultaneo con esclusione di tutte le altre possibilit diagnostiche alternative [ 2 ]  . 
il coinvolgimento dei muscoli della cavit orale , del faringe e del laringe pi frequente e pi precoce nei pazienti affetti dalla forma bulbare della malattia , e la disfagia conseguente determina gravi deficit nutrizionali che , nel tempo , rendono necessaria lalimentazione mediante luso di sondino naso - gastrico o il ricorso alla gastrostomia percutanea [ 3 ]  . 
la disfagia rappresenta uno dei principali indicatori di progressione della malattia , tanto che sono state formulate diverse classificazioni di gravit della malattia , alcune delle quali tengono in considerazione proprio la sintomatologia disfagica dei pazienti . 
fra queste classificazioni la pi importante la classificazione di severit della sla ( als severity scale , alsss ) di hillel [ 5 , 6 ] , che raggruppa i pazienti secondo cinque classi di gravit : normali abitudini alimentari ( normal eating habitus , naa ) , problemi deglutitori iniziali ( early eating problems , eep ) , cambiamenti della consistenza della dieta ( dietary consistency changes , dcc ) , alimentazione mediante sondino naso - gastrico ( needs tube feeling , ntf ) e impossibile alimentazione per via orale ( nothing by mouth , nbm )  . 
lo studio della deglutizione mediante videofluorografia ( vdfg ) rappresenta il gold standard per la valutazione dei disturbi della deglutizione , specie nei pazienti affetti da malattie neurologiche [ 714 ] , permettendo una accurata analisi dei meccanismi della fase orale e faringea della deglutizione , lindividuazione delle eventuali alterazioni responsabili della sintomatologia e la programmazione di unadeguata terapia logopedica . 
nominal abnormality : patients report slight indicators such as food lodging in the recesses of the mouth or sticking in the throat early eating problems ( eep ) dietary consistency changes ( dcc ) 7 . 
oral and pharyngeal secretions managed with aspirator and / or medications secretions cannot be managed noninvasively tabella 1 questionario per la classificazione di pazienti secondo la alsss normali abitudini alimentari ( naa ) i pazienti lamentano alcuni disturbi deglutitori lievi alterazioni : i pazienti lamentano lievi problemi come il ristagno di cibo nella bocca o in gola 10 . 
alimentazione mediante sondino naso - gastrico , con occasionale assunzione orale del cibo ; meno del 50% dellalimentazione avviene per via orale impossibile alimentazione per via orale ( nbm ) 2 . 
only in two patients ( one in the dcc group and one in the ntf group ) was the acquisition of sequences at 25 frames per second ( matrix 512512 ) necessary to adequately evaluate swallowing dysfunction . 
in 22 patients in the neh , eep and dcc groups , the study was first performed using boluses of approximately 5 ml high - density barium ( 250% weight / volume , prontobario hd , bracco , milan , italy )  . 
in patients where penetration ( the passage of the e 13 femmine , et media 41 , 38 , 6 anni ) disfagici affetti da sla sono stati sottoposti a studio videofluografico della deglutizione . 
prima dellesecuzione dellesame stato somministrato un questionario al fine di suddividere i pazienti secondo la classificazione di severit della sla di hillel ( als severity scale , alsss ) ( tabella 1 )  . 
in base alle risposte fornite questi sono stati suddivisi come segue : 12 pazienti del gruppo naa ( et media 38 , 4 anni ) , 7 del gruppo eep ( et media 41 anni ) , 3 del gruppo dcc ( et media 50 anni ) ed 1 del gruppo ntf ( et 53 anni ) ; nessun paziente apparteneva al gruppo nbm . 
lo studio della deglutizione stato eseguito con apparecchio telecomandato con arco a c digitale ( telecomandato polifunzionale eurocolumbus tr3d , milano , italia ) dotato di intensificatore di brillanza da 16 pollici , macchia focale da 0 , 6 a 1 , 2 mm e massima differenza di potenziale di 150 pkv in grafia e 120 pkv in scopia . 
sono state acquisite sequenze cineradiografiche digitali con 12 immagini / secondo e matrice 5121024 ; solo in 2 pazienti ( uno della classe dcc ed uno della classe ntf ) stata necessaria 1175 g . 
transport of the bolus into the pharynx ( swallowing with small boluses , transport of the bolus into the pharynx prior to the triggering of the oral phase of swallowing ) 6 . 
aspiration of the bolus into the airways ( transport of the bolus below the level of the vocal cords , into the trachea and / or the bronchial tree ) 11 . 
compensatory manoeuvres in the event of laryngeal penetration and / or tracheobronchial aspiration , we assessed the depth of the aspiration ( enhancement in the trachea only , in the trachea and one bronchis or in both bronchi )  . 
we assessed the statistical difference of changes in the swallowing phase of the various classes using fishers exact test ( spss 10.1 , spss , chicago , il , usa )  . 1176 lacquisizione , per una adeguata valutazione funzionale dei deficit deglutitori , di sequenze con frame - rate di 25 immagini / secondo ( matrice 512512 )  . 
in 22 pazienti appartenenti ai gruppi naa , epp e dcc lo studio stato condotto utilizzando inizialmente dei boli di 5 ml circa di bario ad alta densit ( 250% peso - volume , prontobario hd , bracco , milano , italia )  . 
per i pazienti che mostravano fenomeni di penetrazione ( passaggio del bolo nel lume laringeo al di sopra del piano delle corde vocali vere ) e / o aspirazione ( passaggio del bolo in trachea o nei bronchi ) sono state acquisite anche sequenze cineradiografiche durante la deglutizione di bario fluido ( 60% pesovolume , prontobario 60% , bracco , milano , italia ) , e di pasta baritata ( 116% peso - volume , prontobario 110% , bracco , milano , italia ) spalmata su cracker o mescolata a mollica di pane . 
al fine di individuare eventuali meccanismi di compenso si proceduto ad una ulteriore valutazione invitando questi pazienti ad assumere adeguate posture , con capo in posizione flessa ed estesa , somministrando la stessa tipologia e quantit di bario di quella utilizzata per la valutazione a capo in posizione neutra . 
al fine di ridurre il possibile rischio di complicanze gravi , come linsorgenza di fenomeni di edema polmonare , nellunico paziente del gruppo ntf stato utilizzato come mdc una soluzione al 50% di acqua e mezzo di contrasto ( mdc ) iodato non ionico idrosolubile ( iopamidolo , gastromiro bracco , milano , italia ) con le stesse modalit sopra descritte . 
passaggio del bolo in faringe ( deglutizione per piccoli boli , passaggio del bolo in faringe prima dellinnesco della fase orale della deglutizione ) ; ristagno del bolo nel faringe ( vallecule glosso - epiglottiche e / o seni piriformi ) ; escursione craniale dellosso ioide e dellepiglottide ; tempo di innesco della fase orale ; eventuale ristagno del bolo nel cavo orale ; capacit di trattenere il bolo nel cavo orale ; 8 . 
attivit contrattile dei muscoli faringei ; fenomeni di penetrazione del bolo nella via aerea ( passaggio del bolo nel lume laringeo senza superamento del piano passante per le corde vocali vere ) ; 10 . 
fenomeni di aspirazione del bolo nella via aerea ( passaggio del bolo nelle vie aeree al di sotto del piano delle corde vocali , in trachea e / o nellalbero bronchiale ) ; g . 
in five patients ( 41.6% ) , bolus stasis in the pharynx caused episodes of postdeglutitive subepiglottic penetration ( with boluses of more than 15 ml ) ; only in one case did this provoke coughing . 
none of the these patients suffered coughing fits during the examination . swallowing with the head flexed resulted in the disappearance of intradeglutitive penetration , whereas only one patient with postdeglutitive penetration benefited from the change . 
i diversi parametri presi in considerazione sono stati quindi raggruppati secondo la suddivisione in classi alsss ( tabella 2 )  . classe naa otto / 12 pazienti ( 66 , 7% ) presentavano alterazioni deglutitorie identificabili alla vdfg . 
le immagini dimostrano la presenza di fenomeni di penetrazione intra - deglutitoria ( a , freccia ) conseguente ad un ritardato movimento dellepiglottide con successiva aspirazione in trachea ( b , asterisco )  . 
nessuno di questi pazienti ha accusato accessi di tosse durante lesame . invitando i pazienti a deglutire con il capo in posizione flessa stato possibile evidenziare la scomparsa dei fenomeni di penetrazione intra - deglutitoria , mentre solo un paziente con penetrazione post - deglutitoria ha giovato del cambiamento posturale . 
in nessun paziente del gruppo naa sono state dimostrate alterazioni del movimento dellosso ioide o della contrazione del muscolo crico - faringeo , n assunzione spontanea di posture facilitanti latto deglutitorio . classe epp in 5 / 7 ( 71 , 4% ) pazienti del gruppo eep sono state riscontrate alterazioni della fase orale della deglutizione con riduzione della motilit della lingua . 
infine 3 ( 42 , 8% ) pazienti mostravano fenomeni di penetrazione post - deglutitoria , 1 paziente ( 14 , 3% ) fenomeni di penetrazione intra - deglutitoria e 1 ( 14 , 3% ) di penetrazione pre - deglutitoria . 
precoce passaggio del bolo in faringe con verniciamento dellepiglottide ( freccia bianca ) e dei seni piriformi ( punta di freccia ) e conseguente penetrazione del bolo in laringe ( freccia grigia )  . the neh group were there alterations in hyoid elevation or cricopharyngeal contraction , nor were spontaneous compensatory manoeuvres observed . eep class in 5 / 7 patients ( 71.4% ) in the eep group , alterations of the oral phase of swallowing were encountered , with a reduction in lingual motility . 
in four of these ( 57.1% ) , triggering of the oral phase of swallowing was delayed due to lingual motility alterations and stasis of the bolus in the oral cavity , whereas in one patient , nonpropulsive movements of the tongue were present , which provoked early triggering of the oral phase that , once begun , proceeded regularly . 
incontinence of the labial rhyme ( a , arrow ) , leakage from the back of the mouth in the oral phase ( b , arrow ) and postdeglutitive penetration ( c , arrow )  . 
le immagini mostrano lincapacit a contenere il bolo nel cavo orale ( a , freccia ) con precoce passaggio in faringe ( b , freccia ) e penetrazione del bolo ( c , freccia )  . 
nelle prove con diversi atteggiamenti posturali si evidenzia la riduzione dei fenomeni di penetrazione ( d , freccia ) nella posizione del capo flesso , e la loro persistenza ( e , freccia ) nella deglutizione a capo esteso . 
la deglutizione di un bolo di maggiore consistenza ( f , freccia ) determina una lieve riduzione dei fenomeni di penetrazione . in one patient ( 14.3% ) , hyoid elevation was reduced , whereas in another ( 14.3% ) , there was an associated incomplete epiglottic tilting . 
in no case were there significant alterations to cricopharyngeal contraction , nor were there cases of coughing fits due to aspiration or penetration of the bolus . dcc class all patients in the dcc class presented alterations in both phases of swallowing due to extensive changes in lingual motility and contraction and coordination of the pharyngeal muscles . 
none of these patients experienced coughing fits as a result of penetration or aspiraspontaneamente il capo durante latto deglutitorio con conseguente riduzione dei fenomeni di aspirazione e / o penetrazione . 
invitando i pazienti ad assumere adeguate posture durante la deglutizione , si evidenziato un miglioramento del quadro radiologico nel paziente con penetrazione intra - deglutitoria e in uno dei due pazienti con aspirazione del bolo . nessun cambiamento stato apprezzabile nel secondo paziente con aspirazione del bolo . 
tutti i parametri presi in esami in questo studio si presentavano alterati con aspirazione in trachea e nel bronco destro gi un bolo di 5 ml . lassunzione di posture dedicate durante la deglutizione non ha determinato sostanziali modificazioni del quadro radiologico deglutitorio . in generale , riunendo i dati di tutti i 23 pazienti dello studio sono stati individuate alterazioni della fase orale della deglutizione in 17 pazienti ( 73 , 9% ) , alterazioni della fase fa1181 g . 
patient with head flexed during swallowing of high - density contrast mediuin this position the epiglottic tilt was improved , and subsequently aspiration into airways was reduced ( a , yellow arrow )  . 
la delgutizione a capo flesso in avanti , eseguita con bario ad alta densit , mostra la riduzione dellampiezza dei fenomeni di penetrazione ( a , freccia gialla ) del bolo rispetto alla deglutizione a capo in posizione neutra ; la deglutizione a capo esteso invece determina un incremento della penetrazione del bolo in laringe ( b , freccia bianca )  . 
swallowing with the head extended brought no improvement ; on the contrary , it led to deeper aspiration in one patient . 1182 ringea in 19 ( 82 , 6% )  . 
in 14 pazienti ( 60 , 9% ) stato inoltre possibile identificare la presenza di fenomeni di penetrazione ( 1 pre - deglutitoria , 5 intra - deglutitoria e 8 post - deglutitoria ) ed in 5 ( 21 , 7% ) fenomeni di aspirazione intra - deglutitoria . 
la deglutizione a capo flesso in avanti ha determinato sostanziali miglioramenti nei pazienti con penetrazione e / o aspirazione intra - deglutitoria ed in un paziente con penetrazione post - deglutitoria . 
lassunzione della postura a capo esteso non ha determinato miglioramenti del quadro radiologico , determinando , al contrario , un maggiore approfondimento del grado di aspirazione in un paziente . analizzando la distribuzione dellet nelle quattro classi dello studio mediante il test di student non stata riscontrata alcuna differenza significativa ( p > 0 , 05 )  . 
abbiamo anche differenziato i pazienti che lamentavano lievi ed iniziali alterazioni deglutitorie ( classe naa ) dai pazienti che lamentavano disfagia di grado moderato e severo ( classi eep , dcc e nft ) , valutandone la differenza statistica con il test esatto di fisher . 
this neurodegenerative form is characterised by the presence of complex symptoms , which at times are common to different forms [ 2 ] , thus making differential diagnosis difficult [ 16 , 17 ]  . 
dysphagia is , in fact , the most common and significant aspect of the bulbar form of the disease , as this can lead to nutritional deficit and death if not adequately treated [ 18 , 19 ]  . 
 [ 13 ] formulated a classification ( alsss ) that differentiates patients on the basis of dysphagia severity and has proved useful in assessing disease progression . various studies have demonstrated the importance of vfss in the diagnosis of dysphagia in relation to a number of diseases [ 510 , 2023 ] and have obtained a good correlation between radiological findings and patients alsss class [ 24 , 25 ]  . 
in contrast , there were no statistically significant differences between groups with respect to age distribution ( p > 0.05 ) nor between the neh group and the other groups with respect to the pharyngeal phase of swallowing and the possible presence of penetration and / or aspiration of the bolus into the airways ( p = 0.59 in both cases )  . the absence of statistical significance regarding age distribution was foreseeable in that disease onset is prevalent in the second or third decade of life and has an unfavourable prognosis and limited life expectancy [ 1 ]  . 
the difference in alterations in the oral phase of swallowing between neh patients and the other groups may be explained by the fact that as long as the oral phase is normal , the patients perceive no significant disturbances , becoming accustomed to and minimising the symptoms of dysphagia . 
this process of tolerance , however , is not present in the advanced stages of the disease when dysphagia becomes so disabling that it causes prodiscussione la sla , descritta per la prima volta da charcot nel 1869 [ 15 ] una malattia neurologica a prognosi infausta causata dalla perdita progressiva delle cellule delle corna anteriori del midollo spinale e dei motoneuroni dei tratti cortico - spinali . 
questa forma neurodegenerativa si caratterizza per la presenza di sintomi complessi e a volte comuni a diverse forme [ 2 ] , rendendo difficoltosa la diagnosi differenziale [ 16 , 17 ]  . 
proprio la disfagia laspetto pi comune e rilevante della forma bulbare della malattia , potendo determinare deficit nutrizionali tali da condurre allexitus se non adeguatamente trattati [ 18 , 19 ]  . 
al fine di classificare i pazienti affetti da sla e di programmarne la terapia , hillel e collaboratori hanno formulato una classificazione ( alsss ) che distingue i pazienti in base alla gravit del grado di disfagia , dimostrandosi utile anche per la valutazione della progressione della malattia . 
 diversi autori hanno dimostrato limportante ruolo svolto dalla vdfg nella diagnosi della disfagia in vari processi patologici [ 510 , 2023 ] , ottenendo una buona correlazione fra il riscontro radiologico e la classe alsss di appartenenza dei pazienti [ 24 , 25 ]  . 
abbiamo correlato la classe di appartenenza dei singoli pazienti intervistati alle alterazioni riscontrabili allesame videofluorografico , e dai dati del nostro studio stata apprezzata una differenza statisticamente significativa tra il gruppo di pazienti naa e quello composto dai pazienti degli altri gruppi riguardo le alterazioni della fase orale ( p = 0 , 036 )  . 
viceversa non vi sono state differenze statisticamente significative fra i diversi gruppi in relazione alla distribuzione in et ( p > 0 , 05 ) e fra il gruppo naa ed i restanti gruppi in relazione alle alterazioni della fase faringea della deglutizione e alla eventuale presenza di fenomeni di penetrazione e / o aspirazione del bolo alimentare nelle vie aeree ( p = 0 , 59 in entrambi i casi )  . 
la mancata significativit statistica della distribuzione per et nelle diverse classi era prevedibile , dal momento che la malattia insorge gi dalla ii - iii decade di vita ed ha prognosi infausta con breve attesa di vita [ 1 ]  . 
la differenza nelle alterazioni della fase orale della deglutizione fra i pazienti naa e gli altri gruppi potrebbe invece essere spiegata dal fatto che fin quando lattivit orale della deglutizione regolare il paziente non avverte disturbi significativi , assuefacendosi e minimizzando i sintomi della disfagia . 
questa assuefazione , al contrario , non si realizza nelle fasi avanzate della malattia quando la disfagia diviene talmente invalidante da determinare profonde modificazioni della dieta e dello stato nutrizionale del paziente . 
si pu quindi dedurre che le alterazioni della fase orale della deglutizione sono le prime ad essere avvertite dai pazienti , anche se si manifestano tardivamente , cio quando sia la fase faringea della deglutizione sia la capacit di protezione delle vie aeree da parte delle strutture laringee sono compromesse . 
it is therefore likely that alterations to the oral phase of swallowing are the first to be noticed by patients , even if they manifest late , that is , when both the pharyngeal phase of swallowing and the ability to protect the airways with the laryngeal structures are already compromised . 
in fact , analysis of individual data from our study revealed a discrepancy between clinical symptomatology , assigned objectively on the basis of the questions put to the patients and expressed by the patient belonging to a particular class according to hillel , and the presence of swallowing alterations demonstrated with vfss . this was particularly evident in the neh and eep classes , whereas the correlation appeared greater in the higher classes . 
in particular , in the neh class , bolus penetration was present in eight patients and aspiration in one patient , and in the eep class , penetration was present in five patients and aspiration in one patient . 
only in one of these patients was the cough reflex triggered by the passage of bolus into the airways . indeed the absence of the cough reflex can lead to an increase in morbidity and mortality , thus placing the patients at greater risk of recurrent episodes of aspiration pneumonia , which in patients suffering from nutritional deficit can even lead to death [ 26 , 27 ]  . 
this can be explained by the fact that in most cases , the amount of bolus passing into the pharynx was limited , with most of the retention in the valleculae and posterior portion of the epiglottis . 
indeed , predeglutitive penetration was the result of bolus leakage into the pharynx when the laryngeal aperture was not protected by laryngeal elevation or epiglottic tilting . in contrast , in cases of intradeglutitive penetration and / or aspiration , the cause was to be found in reduced hyoid elevation and epiglottic tilting and reduction in contraction of the pharyngeal constrictor muscles . lastly , postswallowing penetration and / or aspiration was the result of bolus stasis in the valleculae and pyriform si1184 somministrate ai pazienti ed espressa dallappartenenza ad una specifica classe secondo hillel , e la presenza di alterazioni deglutitorie evidenziabili allesame vdfg . 
in particolare nella classe haa 8 pazienti mostravano penetrazione ed 1 paziente aspirazione del bolo , mentre nella classe eep la penetrazione era presente in 5 pazienti e fenomeni di aspirazione in 1 paziente . 
 proprio la mancata comparsa del riflesso tussigeno pu comportare un incremento dei tassi di morbosit e mortalit , ponendo i pazienti in una condizione di elevato rischio per lo sviluppo di episodi ricorrenti di polmonite ab ingestis che , in pazienti debilitati per il deficit nutrizionale , possono anche portare allexitus [ 26 , 27 ]  . 
 per quanto riguarda la valutazione delle singole alterazioni riscontrate nel gruppo di studio , le alterazioni della fase orale della deglutizione sono conseguenti alla comparsa di movimenti non propulsivi della lingua in 13 / 23 pazienti ( 56 , 5% ) con ritardato innesco della fase orale della deglutizione , ed al ristagno del bolo nel cavo orale in 8 / 23 pazienti ( 34 , 8% )  . 
ci spiegabile dal fatto che nella maggior parte dei casi il passaggio del bolo in faringe stato di lieve entit , raccogliendosi , per lo pi , nelle vallecule glosso - epiglottiche e nel dorso dellepiglottide . 
le alterazioni della fase faringea , invece , sono state causate prevalentemente da una riduzione dellattivit contrattile del faringe in 18 / 23 pazienti ( 78 , 3% ) e dalle alterazioni del movimento del laringe e dellepiglottide in 10 / 23 pazienti ( 43 , 5% )  . 
infatti , i fenomeni di penetrazione pre - deglutitoria sono stati conseguenti ad un precoce passaggio del bolo in faringe , quando lhaditus laringeo non viene protetto dal movimento di escursione della laringe e dellepiglottide . 
invece , nei casi di penetrazione e / o aspirazione intra - deglutitoria la causa da ricercarsi nellalterato movimento dellosso ioide e dellepiglottide e nella riduzione della capacit contrattile dei muscoli costrittori del faringe . 
in questo caso , similmente a quanto avviene nella penetrazione pre - deglutitoria , il bolo pu passare in laringe in una fase in cui lhaditus laringeo non protetto dal movimento di escursione della laringe e dellepiglottide . 
in accordo con i dati della letteratura [ 28 , 29 ] , i 19 pazienti che mostravano fenomeni di penetrazione e / o aspirazione sono stati valutati con bolo di diversa densit e consistenza al fine di riprodurre la consistenza dei cibi compresi nella normale g . 
in this case , in a similar manner to that seen in predeglutitive penetration , bolus can pass into the larynx in a phase in which the laryngeal aperture is not protected by laryngeal elevation and epiglottic tilting . 
in agreement with the literature [ 28 , 29 ] , the 19 patients who displayed penetration and / or aspiration were assessed with boluses with different densities and consistencies with the aim of reproducing the consistency of food included in the patients normal diet . 
in all cases , the patients rated the consumption of high - density barium the easiest , and this preference was justified by the greater viscosity of the bolus , which facilitates compaction in the mouth and transport to the pharynx . 
only in the patient in the ntf group was a solution of water and hydrosoluble iodinated contrast material used to reduce the risks of massive bolus aspiration into the airways . 
this approach proved just as diagnostic as the use of boluses with barium at differing densities , enabling adequate assessment of swallowing alterations . previous studies have already shown how posture changes can improve swallowing in dysphagic patients [ 30 ]  . 
flexing the head forwards lead to significant swallowing improvements in the cases of intraand postdeglutitive penetration and / or aspiration , whereas there were no changes in the case of predeglutitive penetration . 
in contrast , swallowing with the head extended increased the degree of aspiration in one patient in the dcc group and brought no changes in the radiological picture in the other patients although vfss has already been used to assess patients affected by als classified according to alsss [ 24 , 25 ] , this is the first study to our knowledge that highlights the possible compensatory mechanisms that can be implemented to improve swallowing in these patients . with regard to cricopharyngeal muscle activity , data in the literature appear to disagree in that some studies report an elevated frequency of patients with evidence of muscular hypertonia [ 31 , 32 ] , whereas others report normal contraction [ 33 ]  . 
we therefore feel that further studies are required to clarify discrepancies in the literature regarding involvement of this important muscle in the clinical course of als . there are a number of limitations to our study . 
firstly , the patient population was not evenly distributed across the various classes : however , the disease has a low incidence , and its progressive and rapid clinical course often does not allow patients belonging to the dcc and ntf classes to undergo dynamic examinations . 
in tutti i casi i pazienti hanno giudicato pi agevole lassunzione del bario ad alta densit , e questa preferenza pu essere giustificata dalla maggiore viscosit del bolo che rende pi facile il suo compattamento nel cavo orale e il successivo passaggio in faringe . 
solo nel paziente del gruppo nft si preferito , per ridurre i rischi conseguenti ad una possibile massiva aspirazione del bolo nelle vie aeree , utilizzare una soluzione di acqua e mezzo di contrasto iodato idrosolubile . 
dai dati del nostro studio si evince come alcuni pazienti tendono ad assumere spontaneamente delle posture volte alla facilitazione dellatto deglutitorio , con riduzione dei fenomeni di penetrazione e / o aspirazione . per i pazienti che non presentano spontaneamente atteggiamenti posturali di compenso si provveduto ad acquisire sequenze cineradiografiche deglutitorie invitando i pazienti a flettere ed estendere il capo . 
la flessione del capo in avanti ha determinato significativi miglioramenti dellatto deglutitorio nei casi di penetrazione e / o aspirazione intrae post - deglutitoria del bolo , mentre non ha determinato modificazioni nei casi di penetrazione pre - deglutitoria . 
viceversa , latteggiamento deglutitorio con il capo esteso ha determinato un approfondimento del grado di aspirazione in un paziente della classe dcc e nessuna modificazione del quadro radiologico negli altri pazienti . 
 sebbene in letteratura siano gi presenti degli studi di valutazione videofluorografica della deglutizione in pazienti affetti da sla classificati secondo la alsss [ 24 , 25 ] , a conoscenza dellautore questo il primo lavoro che evidenzia i possibili meccanismi di compenso posturale che possono essere intrapresi per migliorare la capacit deglutitoria di questi pazienti . 
 riguardo lattivit del muscolo crico - faringeo i dati della letteratura appaiono discordanti evidenziando in alcuni casi lelevata frequenza di pazienti con evidenza di ipertono muscolare [ 31 , 32 ] , o aspetto contrattile normale [ 33 ]  . 
solo in un paziente del nostro studio stata riscontrata unalterazione funzionale del muscolo crico - faringeo ; riteniamo che ulteriori studi vadano eseguiti per chiarire la divergenze dei dati in letteratura riguardo il coinvolgimento di questo importante muscolo nellevoluzione della sla . 
innanzi tutto il campione di pazienti incluso non omogeneamente distribuito fra le diverse classi , ma la malattia presenta bassa incidenza , inoltre la sua evoluzione rapida e progressiva spesso non consente ai pazienti appartenenti alle classi dcc e nft di sottoporsi ad esami dinamici . 
altra limitazione del nostro studio data dal fatto che i pazienti non sono stati sottoposti ad esame manometrico del faringe , per meglio correlare il riscontro sintomatologico con le alterazioni fun1185 g . 
nonetheless , as the study aimed at identifying swallowing alterations in patients affected by als in relation to their alsss classification , we feel that manometric assessment was not indispensable . zionali e con lo studio videofluorografico . 
tuttavia il presente studio finalizzato allindividuazione delle alterazioni deglutitorie in pazienti affetti da sla in relazione alla loro appartenenza alle diverse classi di hillel , rendendo , a nostro avviso , non indispensabile la valutazione manometrica . conclusions in patients affected by als , swallowing alterations are very frequent and affect both the oral and pharyngeal phases of swallowing . 
vfss therefore proved to be a technique with high diagnostic capabilities in the assessment of swallowing dysfunction of patients with als in that it is able to identify both the degree and causes of swallowing impairment . 
vfss is therefore a useful instrument in the assessment of the degree of dysphagia , both in preparation for speech therapy and in the follow - up of patients with als . conclusioni nei pazienti affetti da sla le alterazioni deglutitorie sono molto frequenti ed interessano entrambe le fasi della deglutizione , orale e faringea . 
dai dati ottenuti si pu affermare come nelle fasi iniziali la sintomatologia riferita , standardizzata dallappartenenza dei singoli pazienti ad una specifica classe di hillel , tenda a sottostimare le alterazioni deglutitorie proprie della sla . 
lo studio videofluorografico della deglutizione si quindi dimostrato una tecnica dotata di elevata capacit diagnostica per la valutazione dei deficit deglutitori dei pazienti affetti da sla , essendo in grado di identificarne il grado di compromissione e le cause determinanti . 
the purpose of this paper is to help refine the differential diagnosis among these diseases through the use of multidetector ct ( mdct ) imaging . riassunto alcune malattie di non frequente riscontro interessano localmente o diffusamente le vie aeree centrali . 
in questo gruppo di patologie sono incluse : la granulomatosi di wegener , la policondrite ricorrente , la tracheobroncopatia osteocondroplastica , lamiloidosi , la papillomatosi , il rinoscleroma , la sarcoidosi e la tubercolosi . 
lo scopo del seguente pictorial essay quello di tracciare la diagnosi differenziale fra queste malattie mediante imaging tc multidetettore ( tcmd )  . key words trachea bronchi ct stenosis parole chiave trachea bronchi ct stenosi introduction introduzione wall thickening and airways stenosis may be associated with lung diseases or with systemic diseases of varying aetiology : infectious , postinfectious , inflammatory , idiopathic and , rarely , neoplastic . 
computed tomography ( ct ) and bronchoscopy are still the techniques of choice for the study of the central airways . axial ct imaging is sufficient in most cases , as it provides information on the airway lumen and walls and on extraluminal structures . 
besides , where multidetector ct ( mdct ) scanners are available , it is easy in day - today practice to add isotropic multiplanar reconstructions ( mpr ) ( straight or curvilinear ) that are useful for demonstrating the presence of stenoses and increasing accuracy when evaluating the extent of wall abnormalities . 
volumerendering ( vr ) techniques provide images that are more immediately comprehensible to bronchoscopists than are congli ispessimenti parietali e le stenosi delle vie aeree possono essere associati a patologie polmonari o a malattie sistemiche a diversa eziologia : infettiva , post - infettiva , infiammatoria , idiopatica , e raramente neoplastica . 
inoltre , qualora si disponga di apparecchi tc multidetettore ( tcmd ) , risulta semplice nella pratica quotidiana aggiungere ricostruzioni multiplanari isotropiche ( rettilinee o curvilinee ) che sono utili nel dimostrare la presenza di stenosi e nel rendere pi accurata la valutazione 1132 a . 
although it cannot replace diagnostic sampling and does not have the interventional capabilities of bronchoscopy , 3d bronchoscopic imaging can help select candidates for bronchoscopy and may prove essential where bronchoscopy is inconclusive or unable to cross a particularly tight stenosis . the purpose of this pictorial essay is to illustrate , through the use of modern ct imaging techniques , the main characteristics of central - airway alterations that can be observed in wegeners granulomatosis , relapsing polychondritis , tracheobronchopathia osteochondroplastica , amyloidosis , papillomatosis , rhinoscleroma , sarcoidosis and tuberculosis . wegeners granulomatosis wegeners granulomatosis is a disease of unknown aetiology characterised by necrotising granulomatous vasculitis that may involve all organs and , in particular , the upper and lower airways , lungs and kidneys [ 1 ]  . 
tracheobronchial involvement occurs with a frequency between 16% and 23% [ 2 , 3 ] and is often a late complication of the disease , although rare cases of isolated involvement of the tracheobronchial tree have also been reported [ 14 ]  . 
 although chest radiography is also able to identify tracheal stenoses and / or wall thickenings ( especially in the subglottic region ) , these lesions are better characterised by ct . the most frequent ct finding is a nodule or eccentric parietal soft tissue mass associated with alterations of the tracheal rings , which may display calcifications or irregularities caused by erosion . 
further radiographic findings include parenchymal nodules generally bilateral and widely distributed cavitations , airspace consolidation , ground - glass opacities and unilateral / bilateral pleural effusion . relapsing polychondritis relapsing polychondritis is a systemic inflammatory disease , probably of autoimmune aetiology , characterised by inflammation episodes of the cartilage , with destruction and fibrosis . 
le tecniche di resa volumetrica ( volume - rendering techniques , vrt ) possono fornire immagini che sono pi immediate e comprensibili da parte dei broncoscopisti rispetto alle immagini assiali convenzionali ; queste tecniche forniscono un imaging 3d tracheobronchiale sia secondo prospettive esterne che interne ( broncoscopia virtuale )  . 
anche se non pu sostituire il campionamento diagnostico e le capacit interventistiche della broncoscopia , limaging broncoscopico 3d di aiuto nel selezionare i pazienti candidati alla broncoscopia e pu risultare fondamentale qualora la broncoscopia appaia inconcludente o non riesca a valicare una stenosi particolarmente serrata . lo scopo del presente pictorial essay quello di illustrare le caratteristiche principali , mediante moderno imaging tc , delle alterazioni delle vie aeree centrali che si osservano nella granulomatosi di wegener , policondrite ricorrente , tracheobroncopatia osteocondroplastica , amiloidosi , papillomatosi , rinoscleroma , sarcoidosi e tubercolosi . granulomatosi di wegener la granulomatosi di wegener una patologia ad eziologia sconosciuta , caratterizzata da una vasculite necrotizzante granulomatosa , che pu interessare tutti gli organi , con particolare predilezione per le alte e basse vie respiratorie , il polmone ed il rene [ 1 ]  . 
il coinvolgimento tracheale e bronchiale si presenta con una frequenza dal 16% al 23% [ 2 , 3 ] e rappresenta spesso una complicanza tardiva della malattia , sebbene siano stati riportati anche rari casi di isolato coinvolgimento dellalbero tracheo - bronchiale [ 14 ]  . 
flogosi e ulcerazioni , conseguenti allinfiammazione granulomatosa [ 5 ] , interessano la mucosa e la sottomucosa , mentre il coinvolgimento degli anelli cartilaginei meno comune , ma pu determinare deformazioni tracheali . 
il reperto tc pi frequente quello di un nodulo o massa di tessuto molle eccentrica parietale , associata ad alterazioni degli anelli tracheali che possono mostrare calcificazioni o irregolarit dovute allerosione . 
c ricostruzione in vr della stenosi tracheale . policondrite ricorrente la policondrite ricorrente una patologia infiammatoria sistemica a probabile eziologia autoimmune , caratterizzata da episodi di infiammazione del tessuto cartilagineo , con distruzione e fibrosi . 
le cartilagini delle orecchie , delle articolazioni e delle pinne nasali sono alcuni dei bersagli preferenziali della malattia ; il coinvolgimento delle vie respiratorie , pi frequentemente a carico della laringe e della parte superiore della trachea [ 7 ] , si osserva fino al 50% dei casi [ 8 ] , ed spesso associato ad una significativa morbilit e mortalit . la diagnosi di policondrite recidivante clinica e non richiede reperti istologici . 
le pareti mostrano quasi sempre the preferred sites of the disease ; the respiratory tract , more frequently the larynx and the upper trachea [ 7 ] , are involved in up to 50% of cases [ 8 ]  . 
ct is essential in establishing involvement of the respiratory tract because biopsy samples are difficult to obtain through bronchoscopy , which may encounter insurmountable obstacles and even exacerbate the inflammation . 
the use of expiratory scans is important because in most cases , they may identify areas of air trapping and bronchomalacia , which may be early , and the only , signs of disease . 
b limmagine di broncoscopia virtuale mostra chiaramente come la parete tracheale sia diffusamente tappezzata da noduli sottomucosi . mental bronchi , and rarely the larynx and subglottic area . they develop in the submucosa and often communicate with the tracheal perichondriu the nodules are generally 13 mm in size , [ 10 ] , but at advanced stages , they may coalesce to form plaque - like lesions that deform and obstruct the airways [ 11 , 12 ]  . 
i noduli hanno in genere dimensioni di 13 mm [ 10 ] , ma in fase avanzata la loro coalescenza pu dare origine a lesioni simili a placche , che provocano deformazione ed ostruzione delle vie aeree [ 11 , 12 ]  . le prime alterazioni radiologiche possono essere rappresentate da atelettasia o addensamenti su base infettiva per polmoniti ricorrenti . 
4a axial computed tomography image shows diffuse , calcified thickening of the walls of the main bronchi that does not involve the posterior wall in a patient affected by tracheobronchial amyloidosis . 
b curved coronal reconstruction shows some calcified nodules in the lower right lobe ( white arrow ) and an ipsilateral tracheal calcified wall thickening near the carina ( curved white arrow ) in a patient with systemic amyloidosis . 
4a limmagine tc assiale mette in evidenza un diffuso ispessimento calcifico delle pareti dei bronchi principali senza risparmio della parete posteriore , in un paziente affetto da amiloidosi tracheo - bronchiale . 
b in un paziente affetto da amiloidosi sistemica la ricostruzione curva sul piano coronale mostra alcuni noduli calcifici nel lobo inferiore destro ( freccia bianca ) ed un ispessimento parietale calcifico omolaterale della trachea in prossimit della carena ( freccia curva bianca )  . 
tracheobronchial involvement is the most common and is characterised by multiple submucosal and muscular deposits of amyloid plaques and nodules , which may cause wall atrophy [ 17 ]  . 
nonetheless , in 14% of patients , the amyloid deposit is isolated and may simulate a bronchial neoplasm [ 18 , 19 ]  . chest radiography may reveal a nodular and irregularly stenosing appearance of the tracheal lumen , but in 25% of cases , the study is negative [ 20 ]  . 
ct shows a nodular thickening or , more rarely , smooth , circumferential parietal thickening of the trachea and main bronchi , with luminal narrowing [ 21 , 22 ]  . 
there is also a possibility of iatrogenic transmission at the tracheal and pulmonary levels following tracheotomy and prolonged intubation with positive pressure ventilation . papillomas are benign but may undergo malignant transformation and become squamous cell carcinomas in 3%5% of cases [ 23 ]  . tracheal papilloma is usually multiple and generally caused by spread from the larynx ; the lower respiratory tract may also be affected , with cough , haemoptysis , asthma - like symptoms , recurrent pneumonia or atelectasis [ 24 , 25 ]  . 
progressive lung involvement leads to the appearance of nodules , cavitated nodules and cystic , air - filled lung lesions . rhinoscleroma rhinoscleroma is a chronic , progressive , granulomatous infection caused by klebsiella rhinoscleromatis , a gram - negative bacterium more frequently found in asia , north africa and central and south america . 
 1138 mulare una neoplasia endobronchiale [ 18 , 19 ]  . lesame radiografico del torace pu evidenziare un aspetto nodulare ed irregolarmente stenosante del lume tracheale , ma nel 25% dei casi lindagine risulta negativa [ 20 ]  . la tc mostra un ispessimento nodulare o , pi raramente , parietale circonferenziale liscio della trachea , dei bronchi , con restringimento del lume [ 21 , 22 ]  . 
a livello parenchimale si pu evidenziare una reticolazione interlobulare liscia o nodulare con opacit lineari intralobulari ( pattern interstiziale ) oppure multiple opacit rotondeggianti , ma anche uniche con diametro variabile sino ad alcuni cm ( pattern nodulare )  . papillomatosi la papillomatosi squamocellulare delle vie respiratorie una rara affezione , determinata da uninfezione da papilloma virus umano ( hpv11 - hpv6 ) , in genere acquisito alla nascita da madre infetta . 
i papillomi sono benigni , ma possono andare incontro a trasformazione maligna e diventare carcinomi squamosi nel 3%5% dei casi [ 23 ]  . il papilloma della trachea solitamente multiplo e determinato in genere da una disseminazione laringea ; si pu verificare anche un coinvolgimento del tratto respiratorio inferiore con comparsa di tosse , emottisi , sintomi simil asmatici , polmoniti ricorrenti o atelettasia [ 24 , 25 ]  . 
la progressiva estensione a livello polmonare determina la comparsa di noduli , noduli cavitati e lesioni polmonari cistiche a contenuto aereo . rinoscleroma il rinoscleroma una infezione cronica , progressiva , granulomatosa causata dal batterio gram negativo klebsiella rhinoscleromatis , di pi frequente riscontro in asia , nord africa e nellamerica centro - meridionale . 
it typically affects the hilar and mediastinal lymph nodes and lung parenchyma , but in 1%3% of cases , it may involve the larynx and trachea , mainly the upper subglottic portion . 
at a parenchymal level , millimetre - sized nodules with sharp margins and lymphatic distribution may be detected ( bronchovascular bundle , interlobular septa and subpleura )  . tuberculosis ( tb ) the prevalence of tracheobronchial stenoses in tb ranges from 40% in autoptic studies of patients with tb to 10% in patients with tb who undergo bronchoscopy [ 27 ]  . 
on gross pathology , granulomatous masses , ulcerations , necrosis of the tracheobronchial walls and , finally , fibrotic stenoses may be prevalent , although these findings may often coexist . ct allows differentiation between the active phase of disease in which the trachea is stenotic with irregular wall thickening from the fibrotic phase in which the trachea is narrowed but has a smooth and normally thick wall . 
in addition , it visualises the extent of irregular and circumferential narrowing of tracheobronchial structures [ 28 ]  . conclusions modern diagnostic imaging and in particular mdct allows identification of stenoses in the central airways with greater accuracy than with conventional diagnostics , especially in the case of extensive , not particularly tight , stenoses . 
interessa tipicamente i linfonodi ilari e mediastinici ed il parenchima polmonare , tuttavia nell1%3% dei casi pu coinvolgere la laringe e la trachea , soprattutto nella porzione superiore sub - glottica . le stenosi tracheali possono essere determinate da formazioni granulomatose della mucosa o della sottomucosa che aggettano allinterno del lume o da compressione estrinseca ( linfonodi ingranditi , fibrosi mediastinica )  . 
a livello parenchimale si possono riscontrare noduli di dimensioni millimetriche e margini netti con distribuzione linfatica ( fascio broncovascolare , setti interlobulari e subpleurica )  . tubercolosi ( tbc ) nella tbc la prevalenza di stenosi tracheo - bronchiali varia dal 40% in casistiche autoptiche in pazienti con tbc al 10% nei pazienti con tbc sottoposti a broncoscopie [ 27 ]  . 
a seconda dello stadio della malattia , da un punto di vista anatomo - patologico possono prevalere formazioni granulomatose , ulcerazioni , e necrosi delle pareti tracheo - bronchiali e infine stenosi fibrotiche , anche se questi reperti spesso possono coesistere . la tc permette di distinguere la fase attiva della malattia , in cui la trachea stenotica con un ispessimento di parete irregolare , dalla fase fibrotica in cui la trachea ristretta , ma con una parete liscia e di spessore normale ; inoltre ben visualizzabile lestensione dei restringimenti irregolari e circonferenziali delle strutture tracheo - bronchiali [ 28 ]  . conclusioni la moderna diagnostica per immagini ed in particolare la tcmd consente lindividuazione delle stenosi delle vie aeree centrali con accuratezza superiore della diagnostica convenzionale specie per quanto riguarda stenosi estese e non molto serrate ; pu fornire una diagnosi precisa o quantomeno ridurre significativamente la gamma delle diagnosi differenziali ed integrare la valutazione fibrobroncoscopica . la tabella 1 riporta schematicamente le caratteristiche principali che pi spesso sono responsabili di stenosi tra1139 a . 
zompatori1 1section of radiology , 2section of respiratory diseases , department of clinical sciences , university of parma , barbieri ospedale maggiore di parma , via gramsci 14 , i - 43100 parma , italy 3department of radiology , university of chieti correspondence to : n . 
we analysed thin - section cts and pulmonary function tests ( pfts ) of six healthy subjects and 31 patients affected by lung fibrosis , including 17 with a usual interstitial pneumonia pattern ( uip group ) , and 14 with a predominant pattern of ground - glass opacities without honeycombing ( non - uip group )  . 
together with the histogram features , fibrosis ratio ( defined as the ratio of nonfibrotic ct lung volume divided by total ct lung volume ) contributed to objectively differentiate fibrotic lungs from normal lungs . 
the visual score is still the main radiological method of quantifying the extent of abnormalities in patients with uip , whilst the range of density 700 to 400 hu can be helpfully applied in a predominant pattern of ground - glass and reticular opacities without honeycombing . key words idiomatic interstitial fibrosis high resolution computed tomography pulmonary function test riassunto obiettivo . 
abbiamo analizzato scansioni tc a strato sottile di sei soggetti sani e di 31 pazienti con fibrosi polmonare , compresi 17 con un pattern di polmonite interstiziale usuale ( gruppo di uip ) e 14 con un pattern predominante di groundglass senza honey - combing ( gruppo non - uip )  . 
insieme agli istogrammi , lindice fibrotico volumetrico ( definito come il rapporto tra il volume di polmone non fibrotico e il volume polmonare totale ) ha contribuito nel differenziare in modo obiettivo i polmoni fibrotici dai polmoni normali . 
lo score visivo ancora il metodo radiologico principale per quantificare lestensione della patologia in pazienti con uip , mentre lintervallo di densit 700400 hu pu essere vantaggiosamente applicato in un pattern predominante a groundglass e reticolare senza honey - combing . parole chiave fibrosi interstiziale idiomatica tomografia computerizzata ad alta risoluzione test di funzionalit polmonare 1160 n . 
interstitial fibrosis is a nonspecific pathological endpoint that can occur as a consequence of granulomatous diseases , collagen vascular diseases , certain drug therapies and recurring exposure to organic and inorganic antigens . 
therefore , it is crucial to develop methods for quantitative imaging of the lung that allow one to diagnose early disease , follow - up disease progression and evaluate the effects of treatment . structure - function studies have shown that high - resolution computed tomography ( hrct ) has a crucial role in diagnosis and management of patients with iips and other interstitial disorders [ 14 ]  . 
by providing images of the entire lungs , hrct ( unlike histopathological evaluation ) represents a noninvasive tool for monitoring disease extent , and it can be also correlated with pfts , which provide , instead , a global statement of lung function . 
several studies including large numbers of idiopathic pulmonary fibrosis ( ipf ) patients reported strong correlations between histological findings and functional impairment [ 5 , 6 ] ; nevertheless , these are consistently weaker than correlations observed between lung function impairment and ct scores [ 3 , 79 ]  . previous studies on ct evaluation of lung fibrosis can be categorised as visual and computer - based assessment . 
despite the relevance of a radiologists interpretation , it is increasingly accepted that the human observer , without the aid of computer - based image evaluation tools , is poorly reproducible in quantifying extent of disease [ 10 ]  . 
 computer - aided diagnosis ( cad ) has been widely used to quantify extent of emphysema ; nonetheless , quantitative cad might also be used to better evaluate the extent of lung fibrosis . 
in particular , in order to assess complex lung patterns , fractal and texture - based analyses as well as multiple neural networks have been used [ 1114 ]  . 
therefore , we still need a unique objective method for quantifying lung fibrosis and in particular the most aggressive idiopathic variants . we studied two different groups of patients with lung fibrosis to evaluate whether simple ct computer scores ( histogram features , ranges of density and one novel volumetric index ) provide a more precise discrimination between normal and pathologic areas of the lungs and a correlation between morphological and functional indices better than the visual score does . patients and methods subjects six healthy controls and 31 individuals with lung fibrosis la fibrosi polmonare pu presentarsi secondo diverse configurazioni cliniche . 
la fibrosi interstiziale uno stadio patologico terminale non specifico , che pu verificarsi come conseguenza di malattie granulomatose , connettivopatie , alcune terapie farmacologiche e ricorrente esposizione ad antigeni organici ed inorganici . 
i medici che hanno in cura i pazienti affetti da fibrosi polmonare sono frequentemente tenuti a prendere decisioni complesse riguardo al trattamento : se e quando iniziarlo , intensificarlo , o interromperlo . 
perci fondamentale sviluppare metodi di imaging quantitativo del polmone che permettano di diagnosticare precocemente la malattia , seguirne il decorso e valutare gli effetti della terapia . studi morfologici e funzionali hanno dimostrato che la tc ad alta risoluzione ( hrct ) ha un ruolo fondamentale nella diagnosi e nella gestione dei pazienti affetti da pii ed altri disordini interstiziali [ 14 ]  . 
fornendo immagini dei polmoni per intero , la hrtc ( a differenza della valutazione istopatologica ) rappresenta uno strumento non invasivo per il monitoraggio dellestensione della malattia e pu anche essere correlata con ( pfr ) , che invece forniscono solo una valutazione globale della funzionalit polmonare . 
diversi studi che includevano un numero considerevole di pazienti con fibrosi polmonare idiopatica ( ipf ) , hanno dimostrato forti correlazioni fra i reperti istologici e lalterazione funzionale [ 5 , 6 ] ; tuttavia , queste sono consistentemente pi deboli di quelle osservate tra lalterazione funzionale e gli score tc [ 3 , 79 ]  . 
nonostante limportanza dellinterpretazione del radiologo , si sempre pi concordi nel ritenere che la semplice osservazione , senza laiuto di strumenti di valutazione dellimmagine assistiti dal computer , sia scarsamente riproducibile nel quantificare lestensione della patologia [ 10 ]  . la diagnosi assistita computerizzata ( cad ) , che stata ampiamente utilizzata per quantificare lestensione dellenfisema , pu essere anche impiegata per valutare lestensione della fibrosi polmonare . 
in particolare , per analizzare i pattern pi complessi del polmone , sono stati utilizzati sistemi di analisi frattale , della texture e anche reti neurali multiple [ 1114 ]  . 
the control group included three men and three women ( mean age 46 years , range 2962 )  . all were nonsmokers , had no history of pulmonary disease , had normal lung function and were taking no medication . thirty - one patients , 19 men and 12 women ( mean age 64 years , range 3483 ) were retrospectively selected to obtain two groups of patients with usual interstitial pneumonia ( uip ) , and non - uip pattern . 
all patients were referred for thin - section ct scan of the lung on the basis of a clinicalfunctional restrictive presentation and a positive chest radiograph compatible with diffuse interstitial lung disease . uip pattern was defined according to well - established criteria and consistent mainly with reticular lines , traction bronchiectasis and honeycombing in a basal - predominant distribution [ 15 ] ; diagnosis was histological in five cases ( uip group , n = 17 )  . 
in the non - uip group ( n = 14 ) , six patients were affected by chronic extrinsic allergic alveolitis ( eaa ) and eight patients by lung fibrosis of undetermined cause despite accurate clinical , functional and radiological assessment . 
patients with asbestos exposure , sarcoidosis , connective tissue diseases and prior environmental exposure to any fibrogenic agent were not included in the study . lung - function testing , ct examination and lung biopsy had been undertaken for clinical reasons under request of patients clinicians . 
 ct and computer analysis all patients underwent thin - section ct examination of the entire thorax using a 16 - slice scanner ( sensation 16 , siemens medical solutions , forchheim , germany ) without i.v. 
scanning parameters were : 120 kvp , 100 mas , rotation time 0.5 s , feed / rotation 18 mm , the smallest possible field of view ( fov ) covering both lungs , bone filter and collimation 0.75 mm with 1 - mm reconstruction . 
the images were printed , adopting a window level of 600 hu and a window width of 1 , 600 hu for the parenchyma . to evaluate presence and extent of lung abnormalities , two chest radiologists blindly and independently evaluated all ct scans using three scans at preestablished levels : the origin of the great vessels , the carina and the right inferior pulmonary ve the radiologists were unaware of the patients lung functional data . 
the overall extent of these abnormalities was determined for each entire lung using a four - point scale ( 0 = no involvement ; 1 = 1%25% involvement ; 2 = 26%50% ; 3 = 51%75% ; and 4 = 76%100% )  . these data were used to calculate interobserver agreement . subsequently , a consensus reading was performed to obtain only one visual score for the disease extent / functional correlations . 
trentuno pazienti affetti da fibrosi polmonare , 19 maschi e 12 femmine ( et media di 64 anni , range 3483 ) , sono stati selezionati retrospettivamente in modo da ottenere due gruppi di pazienti rispettivamente con pattern di tipo polmonite interstiziale usuale ( uip ) , e pattern non uip . 
tutti i pazienti sono stati segnalati per lesecuzione di indagine hrtc sulla base di una presentazione clinico - funzionale di tipo restrittivo e di una radiografia del torace positiva , compatibile con quadro di polmonite interstiziale diffusa . il quadro di uip stato definito secondo i criteri ben consolidati letteratura che prevedono essenzialmente allhrtc la presenza di addensamenti reticolari , bronchiectasie da trazione ed honey - combing a distribuzione prevalentemente basale [ 15 ] ; la diagnosi stata supportata da dati istologici ottenuti con biopsia chirurgica in 5 casi ( gruppo uip , n = 17 )  . 
nel gruppo non uip ( n = 14 ) , 6 pazienti erano affetti da alveolite allergica estrinseca cronica ( aee ) , e 8 pazienti da fibrosi polmonare di causa indeterminata . 
pazienti con esposizione allasbesto , malattie del tessuto connettivo e precedente esposizione ambientale ad agenti fibrogenici non sono stati inclusi nel gruppo di studio . le pfr , lesame tc , e la biopsia polmonare sono stati eseguiti per ragioni cliniche previa richiesta del medico curante . 
i pazienti hanno fornito anche il loro consenso informato allo studio . tc e analisi computerizzata tutti i pazienti sono stati sottoposti a scansioni tc a strato sottile di tutto il torace usando uno scanner a 16 strati ( sensation 16 - siemens medical solutions , forchheim , germania ) , senza iniezione endovenosa del mezzo di contrasto . 
il sincronizzatore respiratorio non stato utilizzato ; gli esami sono stati eseguiti in una singola apnea ( inspiratoria completa , con decubito prono soltanto per i soggetti patologici ) , dopo aver adeguatamente istruito i pazienti . 
i parametri di scansione utilizzati erano : 120 kvp , 100 mas , tempo di rotazione 0 , 5 s , avanzamento / rotazione 18 mm , campo di vista pi piccolo possibile ( fov ) che comprendesse entrambi i polmoni , filtro di convoluzione per tessuti duri , collimazione 0 , 75 mm con ricostruzioni di 1 millimetro di spessore . 
le immagini sono state stampate applicando una finestra con livello di 600 hu e ampiezza di 1600 hu . al fine di valutare la presenza e lestensione delle lesioni , 2 radiologi toracici hanno esaminato in doppio cieco tutte le scansioni tc utilizzando tre livelli prestabiliti : lorigine dei grandi vasi , la carena tracheale ed alla vena polmonare inferiore di destra . 
b applicazione della maschera di densit con intervallo di 700400 hu . we uses 1 - mm sections for visual score , which were subsequently scored semiautomatically by the system software ( pulmo - siemens ) that allows lung - density and structure testing . 
after obtaining frequency histograms of thin - section ct scans , relative data were transferred into a different software package that calculated indices , such as mean lung attenuation ( mla ) , skewness ( representing the extent of asymmetry of histograms ) and kurtosis ( representing the peakness of histograms )  . 
finally , using the entire ct data set , we constructed a fibrosis ratio , which we define as the ratio of the non - fibrotic ct lung volume ( 1 , 024 to 500 hu ) divided by total ct lung volume ( 1 , 024 to 200 hu )  . pulmonary function tests lung function was evaluated within 1 week from the hrct exam by a flow - sensing spirometer and a body plethysmograph connected to a computer for data analysis . 
the following parameters were recorded and expressed as a percentage of the theoretical values : total lung capacity ( tlc ) , forced expiratory volume in 1 s ( fev1 ) and forced vital capacity ( fvc )  . 
diffusing capacity for carbon monoxide ( dlco ) was evaluated during a single breath - hold and considered valid only if the inspiratory volume was at least 90% of the forced vc . 
finally , a composite physiologic index ( cpi ) was derived in the uip pattern group 7 . the formula for cpi was as follows : conoscenza dei parametri funzionali dei pazienti . 
lestensione globale di queste anormalit stata determinata per ogni polmone usando una scala a 4 punti ( 0 = assenza delle lesioni ; 1 = 1%25% di estensione ; 2 = 26%50% ; 3 = 51%75% ; 4 = 76%100% )  . 
questi dati sono stati utilizzati per calcolare laccordo interosservatore ; successivamente stata fatta una lettura consensuale per ottenere un solo score visivo da porre in correlazione con i dati funzionali . 
stata inoltre valutata leventuale presenza di enfisema , di noduli , delle bronchiectasie da trazione e la distribuzione delle lesioni . le stesse sezioni da 1 mm usate per lo score visivo , sono state successivamente analizzate semi - automaticamente da un software ( pulmo - siemens ) che valuta la densit e la struttura del polmone . 
dopo aver ottenuto gli istogrammi di densit delle sezioni selezionate , i relativi dati sono stati analizzati mediante un secondo software che ha calcolato indici come la attenuazione polmonare media ( mla ) , lo skewness ( rappresentante il grado di asimmetria degli istogrammi ) e la kurtosis ( rappresentante il grado del picco degli istogrammi )  . 
three patients la funzionalit polmonare stata valutata entro una settimana dallesame hrtc mediante uno spirometro rilevatore di flusso ed un pletismografo , collegati ad un calcolatore per analizzarne i dati . 
questi sono stati espressi come percentuale dei valori teorici : capacit polmonare totale ( cpt ) , volume espiratorio forzato in 1 secondo ( fev1 ) , capacit vitale forzata ( cvf )  . 
la capacit di diffusione del monossido di carbonio ( dlco ) stata valutata durante un singolo respiro e considerata valida solo se il volume inspiratorio risultasse almeno pari al 90% della cvf . 
la formula per il cpi era la seguente : 91 ( 0 , 65 percentuale del predetto dlco ) ( 0 , 53 percentuale del predetto cvf ) + ( 0 , 34 percentuale del predetto fev1 ) analisi statistica i dati dei due gruppi sono stati comparati il t test di student . un valore di p < 0 , 05 stato considerato statisticamente significativo . 
il coefficiente di correlazione lineare di pearson stato usato per esaminare la correlazione tra ciascuno degli indici tc , gli score visivi e i test di funzionalit polmonare , ovvero il dlco e cpi . 
 uip , polmonite interstiziale usuale ; mla , densit media polmonare ; dlco , capacit di diffusione del monossido di carbonio ; cpi , indice composito funzionale also had coexisting mild to severe centrilobular emphysema predominant in the upper lobes . 
la mla , la skewness e la kurtosis degli istogrammi di densit sono risultati in media rispettivamente : 758 , 4 hu47 , 2 , 10 , 4 , 82 , 5 . 
le medie degli intervalli di densit pi basso ( 700200 hu ) , intermedio ( 700400 hu ) e pi alto ( 500200 hu ) erano rispettivamente 27 , 7%16 , 1% , 22 , 9%13 , 3% , 9 , 8%4 , 9% . 
confrontando gli istogrammi di densit del gruppo uip e quelli del gruppo non uip , non sono state osservate differenze statisticamente significative ( p > 0 , 05 )  . soggetti normali le scansioni tc dei soggetti normali non hanno mostrato alcuna alterazione polmonare . mla , skewness , kurtosis degli istogrammi di frequenza erano in media rispettivamente 837 , 5 hu29 , 4 , 3 , 50 , 5 , 18 , 84 , 5 . 
le medie degli intervalli di densit pi basso ( 700200 hu ) , intermedio ( 700400 hu ) e pi alto ( 500200 hu ) erano rispettivamente 8 , 3%2 , 4% , 6 , 5%2 , 1% , 3%0 , 8% . 
i risultati di queste analisi hanno indicato che lo score visivo era inoltre lunico strumento di predizione del cpi , con un valore di r2 di 0 , 38 ( p < 0 , 05 )  . 
although the correlation between visual score and dlco differed little from the correlation between visual score and cpi , visual score did not provide any significant predictive value for dlco . in the non - uip group , correlation between intermediate discussione nel nostro studio abbiamo cercato di verificare se i dati ottenuti col software di analisi della densit potessero distinguere soggetti normali , pazienti uip e non uip . 
 i risultati sono espressi secondo i coefficienti di correlazione di pearson ( r ) e i corrispondenti valori di r2 ranges of density ( 700 to 400 hu ) and dlco ( r = 0.71 , p < 0.05 ) was stronger than the visual score and other ct indices . 
moreover , in mild fibrotic disease , there is no definite dichotomy between the density of the normal and abnormal lung ( such as in emphysema ) ; hence , there could be disadvantages in using only ranges of density information . 
inoltre , una forma lieve della patologia fibrotica non comporta una netta dicotomia di densit tra il polmone normale e quello patologico ( come nellenfisema ) , limitando cos lutilit degli intervalli di densit . 
 al contrario si visto che gli istogrammi nei pazienti patologici erano significativamente diversi da quelli dei soggetti sani ( p < 0 , 05 ) , e tale differenza era gi evidente allispezione visiva . 
nei pazienti uip abbiamo notato una minore attenuazione media , una maggiore asimmetria , ed un picco pi acuto ( tabella 2 ) rispetto a quanto riportato negli studi di harvey et al . 
entrambi i gruppi uip e non uip avevano unestensione della patologia modicamente limitata e simile , come anche confermato dagli score visivi , dagli intervalli di densit e dal grado dinvalidazione funzionale . 
data are pearsons product moment correlation coefficients ( r ) and the corresponding r2 values mla , densit media polmonare ; dlco , capacit di diffusione del monossido di carbonio . 
i risultati sono espressi secondo i coefficienti di correlazione di pearson ( r ) skewness kurtosis 700 to 200 hu 700 to 400 hu 500 to 200 hu fibrosis ratio visual score 1168 n . 
in healthy subjects , the first - order histogram of ct attenuation is sharply peaked ( kurtotic ) and skewed to the left in comparison with the gaussian normal distribution . 
both the uip group and nonuip groups had limited and similar extent of disease , as confirmed by visual scores as well as by the range of density and functional impairment . 
indeed , analysing a wider part of the lung , the histogram features could be relatively insensitive to textural changes . to further improve the differentiation between patients and controls , we constructed a fibrosis ratio that provided information about the fibrosis fraction . 
it is well known that data derived from the density - mask technique ( histogram features and ranges of density ) are not sufficient to obtain an accurate statement of disease extent in patients with lung fibrosis . 
however , it has never been proved that these widely available tools can quantify fibrosis extent better than the visual score can , which is subjective and suffers the drawback of a moderate level of interobserver and intraobserver variation . in the uip group , visual score was the only predictor of functional impairment . 
in a study by best et al . , fvc showed the greatest magnitude of correlation with histogram features , while dlco showed the least correlation ; the correlation between kurtosis and dlco was similar to our correlation ( r = 0.37 ) [ 17 ]  . 
perhaps it is impossible to restrict the alterations of uip inside a threshold of score visivo per lestensione di malattia fosse simile sia per il gruppo uip che per il gruppo non uip ( rispettivamente 39 , 7%14 , 5% e 39 , 5%21 , 2% ) , non si sono notate differenze tra gli istogrammi dei 2 gruppi ( p > 0 , 05 )  . 
 [ 21 ] i quali riportavano che gli istogrammi di densit della polmonite interstiziale non specifica ( nsip ) erano meno deviati a sinistra ed avevano un picco pi largo di quelli uip . 
peraltro , analizzando una porzione pi ampia del polmone , le caratteristiche dellistogramma possono essere relativamente indifferenti ai cambiamenti strutturali . per migliorare ulteriormente la distinzione tra malati e sani , abbiamo formulato un indice fibrotico che fornisse informazioni riguardo il rapporto tra grado di fibrosi e polmone sano . 
il valore normale stato sempre tra 0 , 99 e 1 , 0 , ossia assenza di fibrosi , mentre un rapporto pi basso era indicativo di una quantit significativa di fibrosi ( p < 0 , 05 )  . abbiamo quindi analizzato la relazione morfologico - funzionale . 
era gi noto che i dati ottenuti dalla maschera di densit ( istogrammi ed intervalli di densit ) non fossero sufficienti per ottenere un preciso resoconto dellestensione della patologia nei pazienti con fibrosi polmonare . 
tuttavia non mai stato dimostrato come questi strumenti , largamente diffusi , potessero quantificare lestensione della fibrosi rispetto allo score visivo , un metodo soggettivo che risente dellinconveniente di una variabilit intered intra - osservatore . nel gruppo uip lo score visivo risultato essere lunico sistema per predire significativamente il danno funzionale . esso ha mostrato una buona correlazione con il cpi ( r = 0 , 60 , r2 = 0 , 38 , p < 0 , 05 )  . 
gli istogrammi sono stati insoddisfacenti , con solo la mla che mostrava una moderata correlazione con il cpi ( r = 0 , 55 , p < 0 , 05 )  . 
 [ 17 ] la cvf ha mostrato la maggiore correlazione con gli indici degli istogrammi , mentre il dlco ha mostrato la minore ; la correlazione tra kurtosis ed il dlco era simile alla nostra ( r = 0 , 37 ) [ 17 ]  . 
per esempio , i nostri intervalli di densit non tengono in considerazione lo spazio cistico dellhoneycombing ( spazi morti ) , e questo potrebbe spiegare la mancanza di correlazione con gli indici funzionali . 
le dimensioni degli spazi morti cistici ( honey - combing alla tc ) , contribuiscono alla mancanza di perfusione del polmone fibrotico e sono correlate alla riduzione del dlco e meno agli indici volumetrici pfr [ 2628 ]  . 
for example , our ranges of density did not take into account the cystic space of honeycombing ( dead spaces ) on ct , and this can explain the lack of correlation with the functional indices . 
the extent of cystic dead spaces contributes to the lack of perfusion of the fibrotic lung and is linked to the reduction of dlco and less to the volumetric pft indices [ 2628 ]  . 
fibrosis ratio in the uip group was not a predictor of dlco or cpi . in the uip group , dlco and cpi were carried out because they have the strongest correlation with the morphologic extent of disease , histologically and on the ct [ 3 , 68 ]  . 
cpi is a new powerful severity variable that accounts for coexisting emphysema , whereas in patients without emphysema on ct , cpi did not reflect the disease extent better than did dlco [ 7 ]  . 
although the non - uip group might have contained some cases of nsip , organising pneumonia ( op ) or uip with atypical imaging features , the inclusion criteria of these patients were not thought to be subject to significant selection bias . 
however , it was clearly more difficult to establish the extent of patchy ground - glass , in or out of areas of reticulation , than scoring the extent of honeycombing or reticulation alone . 
a weighting factor should be applied to the estimates of each level to correct for the differing contribution given by each level ( an upper zone section has a small cross - sectional area , and therefore contributes to the total lung volume less than a lower zone section does ) [ 26 , 30 ]  . a confounding factor in the non - uip group could be the lobular areas of air trapping , which was not taken into ac1170 gruppo uip non si correlato significativamente alla dlco o al cpi . nel gruppo uip , il dlco e il cpi sono stati ricavati poich sono gli indici a maggiore correlazione con lestensione della patologia , valutata sia istologicamente che alla tc [ 3 , 68 ]  . 
 [ 9 ] ritengono che non ci siano differenze di risultati se considerati unitamente i pazienti con e senza enfisema ; tuttavia lenfisema nei pazienti con uip un noto fattore di confondimento , potendo influenzare gli indici di funzionalit polmonare . 
la nostra analisi non stata ristretta ai soli pazienti senza enfisema a causa del numero ridotto dei soggetti dello studio e pertanto non abbiamo potuto stabilire precisamente se lenfisema fosse effettivamente un fattore fuorviante . 
 [ 7 ] , tre pazienti uip con coesistente enfisema sono stati probabilmente sufficienti per fornire una correlazione solo tra lo score visivo e il cpi ( r = 0 , 60 , r2 = 0 , 38 , p < 0 , 05 )  . le correlazioni morfologico - funzionali sono risultate significativamente diverse nel gruppo non uip . 
sebbene questo gruppo possa aver contenuto alcuni casi di nsip , o di polmonite organizzante ( op ) , o uip con caratteristiche di imaging atipiche , i criteri di selezione di questi pazienti non sono stati ritenuti essere fonte di significativo bias . 
tuttavia , chiaramente pi difficile stabilire lestensione delle irregolarit ground - glass , allinterno o allesterno delle aree di reticolazione , che misurare lestensione dellhoney - combing o la sola reticolazione . 
un fattore di ponderazione dovrebbe essere applicato per la valutazione di ogni livello , per correggerne il diverso contributo relativo ( la sezione trasversale di una zona superiore , rispetto ad una sezione basale , ha una minor area e quindi contribuisce meno al volume polmonare totale ) [ 26 , 30 ]  . un fattore fuorviante nel gruppo non uip potrebbero essere le aree lobulari di air - trapping che non erano state prese in considerazione dal nostro sistema di valutazione . 
according to hansell et al . [ 2 ] , there is no independent relationship between the extent of decreased attenuation on ct and indices of gas transfer . however , patients in that study had mainly subacute eaa , where the cellular component of bronchiolitis may be more extensive [ 2 ]  . 
despite the limited number of cases , our experience indicated a significant inverse correlation between the extent of fibrosis and expiratory air trapping , possibly because the extent of cellular bronchiolitis with heterogeneous distribution , which may persist in chronic eaa , is usually quantitatively exceeded by fibrosis [ 4 ]  . in conclusion , despite a poor correlation with functional data , fibrosis ratio together with the histogram features can differentiate fibrotic from normal lungs . 
nonostante il limitato numero di casi , la nostra esperienza indica una significativa correlazione inversa tra lestensione della fibrosi e lair - trapping espiratorio , forse perch lestensione della bronchiolite cellulata a distribuzione eterogenea che pu trovarsi nellaee cronica , di solito superata quantitativamente dalla fibrosi [ 4 ]  . in conclusione , abbiamo dimostrato che , nonostante la scarsa correlazione con i dati funzionali , lindice fibrotico assieme agli istogrammi pu distinguere i polmoni fibrotici da quelli normali . 
la stretta correlazione tra il cpi e lo score visivo nei pazienti affetti da uip suggerisce che il nostro sistema di valutazione pu essere vantaggiosamente applicato . nessun altro indice tc , inclusi gli intervalli di densit , lindice fibrotico e gli istogrammi possono correlarsi al cpi o al dlco nei pazienti uip . 
crusco , via antica vena 18 , i - 06100 - perugia , italy , tel . : + 39 - 075 - 393044 / 328 - 2896943 , fax : + 39 - 075 - 5783488 , e - mail : fcrusco@sirm.org received : 20 may 2006 / accepted : 4 april 2007 / published online : 13 december 2007 abstract the aim of this review is to provide starting from anatomical , surgical and pathophysiological data elements for evaluating the status of coronary artery bypass grafts with multidetector computed tomography ( ct ) , taking into consideration the most common conduits used ( left and right internal mammary arteries , saphenous vein , radial artery , gastroepiploic artery ) and early and late complications ( stenosis or obstruction , vasospasm , aneurysms and pseudoaneurysms , malposition )  . 
finally , we offer general guidelines for reporting the examination results . key words multidetector ct coronary artery bypass graft riassunto scopo del seguente lavoro di revisione fornire , a partire da dati di anatomia , chirurgia e fisiopatologia , gli elementi utili ai fini della valutazione con tc multidetettore dello stato di perviet dei bypass aortocoronarici , prendendo in considerazione i condotti pi comunemente utilizzati ( arteria mammaria sinistra e destra , vena safena , arteria radiale , arteria gastroepiploica ) e le eventuali complicanze precoci e tardive ( steno - ostruzione , vasospasmo , aneurismi e pseudoaneurismi , malposizionamenti ) , legate ad essi . 
infine vengono fornite alcune indicazioni di massima come guida alla refertazione dellesame . parole chiave tc multidetettore bypass aortocoronarico introduction introduzione surgical myocardial revascularisation by coronary artery bypass grafting ( cabg ) , aims at restoring and normalising coronary blood flow in hypoperfused ischaemic territories by grafting arterial and venous conduits from the aorta to the diseased coronary artery beyond the stenosis . 
surgical revascularisation is indicated in subjects with threeor two - vessel coronary disease with involvement of the proximal left anterior descending ( lad ) artery , left main coronary artery disease and depressed left ventricular function ( left ventricle ejection fraction < 45% ) [ 1 ]  . monitoring patients after cabg involves routine clinical follow - up every 46 months during the first year after surgery and where treatment has been successful , every year thereafter . 
use of electrocardiogram ( ecg ) stress testing as la terapia chirurgica di rivascolarizzazione miocardica mediante bypass aortocoronarico ( cabg ) ha come scopo il ripristino e la normalizzazione del flusso coronarico nei territori ischemici ipoperfusi , mediante limpianto a livello della porzione sottostenotica della coronaria nativa patologica , di condotti arteriosi o venosi . 
una rivascolarizzazione chirurgica indicata nei soggetti con patologia coronarica trivasale o bivasale con interessamento della lad prossimale , malattia del tronco comune , funzionalit ventricolare sinistra globale compromessa ( frazione di eiezione ventricolare sinistra < 45% ) [ 1 ]  . il monitoraggio dei pazienti sottoposti a cabg viene effettuato routinariamente mediante follow - up clinico ogni 46 mesi entro il primo anno dallintervento , mentre nei sog1087 f . 
the clinical utility of the test is , however , limited by its low sensitivity for ischaemia detection and localisation and the frequent finding , even in healthy subjects , of ecg abnormalities independent of possible ischaemia recurrence [ 2 , 3 ]  . echocardiography and myocardial scintigraphy are regarded as second - line , noninvasive imaging techniques and are used to establish the cardiac origin of angina by identifying graft stenosis or obstruction and / or coronary disease progression and to evaluate coronary functional reserve , a key element for assessing the effectiveness of the revascularisation procedure . 
these tests , performed after induction of physical or pharmacological stress ( dipyridamole or adenosine in scintigraphy ; dobutamine in echocardiography ) , reveal ischaemic territories as changes in regional wall motion , systolic function and overall end - systolic volume , or as regional perfusion defects , respectively [ 46 ]  . 
generally , decisions to use stress imaging are guided by the presence of ischaemic symptoms , with the only exception being asymptomatic highrisk patients ( with poor left ventricular function , multivessel disease , disease of the proximal left anterior descending artery , diabetes , high - risk occupations ) more than 5 years after surgery , in whom it is reasonable to perform stress single photon emission computed tomography ( spect ) imaging to stratify risk , evaluate prognosis , determine disease progression and obtain information to guide treatment [ 7 , 8 ]  . coronary angiography is currently considered the reference standard for evaluating bypass patency and planning further percutaneous or surgical revascularisation procedures [ 9 ]  . 
the main indication for coronary angiography is evidence of high - risk criteria at noninvasive stress testing ( multisegment regional wall motion abnormalities and / or reversible moderate to severe perfusion defects ) , regardless of time since surgery . 
these include age ( > 80 years ) , a functional class iv , left main disease , associated cardiac valve disease , left ventricular ejection fraction < 30% and severe noncardiac disease [ 9 , 11 ]  . 
consideration of these data related to the invasiveness of the procedure and the need to reduce the costs deriving from the substantial number of conventional coronary angiography exams not followed by percutaneous interventional procedures ( 20%27% negative , 2%58% unnecessary ) has encouraged the use of multidetector ct ( mdct ) as an alternative noninvasive cardiac imaging test for the evaluation of graft patency [ 12 ]  . 
il ricorso allecg da sforzo , quale test strumentale di primo livello , indicato in caso di comparsa di sincope , modifica della pregressa sintomatologia anginosa o comparsa di una sintomatologia suggestiva di alterazioni del ritmo e anomalie della conduzione ; il valore clinico del test tuttavia limitato a causa della bassa sensibilit nellidentificazione e nella localizzazione di ischemia e per il riscontro frequente , anche in pazienti sani , di anomalie del tracciato elettrocardiografico indipendenti dalleventuale recidiva ischemica [ 2 , 3 ]  . laccertamento della genesi cardiaca del dolore anginoso , mediante lidentificazione della steno - ostruzione del graft e / o della progressione di malattia nelle coronarie native , e la valutazione della riserva funzionale coronarica , considerata elemento fondamentale nella valutazione di efficacia della procedura di rivascolarizzazone , viene demandato allecocardiografia e alla miocardioscintigrafia , considerate tecniche di imaging non invasivo di secondo livello . 
questi test , eseguiti con induzione di stress fisico o farmacologico ( dipiridamolo o adenosina nel test scintigrafico , dobutamina in quello ecocardiografico ) , identificano i territori ischemici rispettivamente sotto forma di alterazioni della motilit parietale regionale , della funzione sistolica e del volume telesistolico globale e sotto forma di deficit perfusionali regionali [ 46 ]  . 
in linea generale , il ricorso alle metodiche di stress imaging guidato dalla positivit dei sintomi ischemici , con ununica eccezione , nei pazienti asintomatici ad alto rischio ( cio con ridotta funzione ventricolare sinistra , malattia coronarica multivasale , malattia della discendente anteriore prossimale , diabete , occupazione professionale a rischio ) , a oltre 5 anni dallintervento , nei quali pu essere considerata ragionevole leffettuazione di una stressspect per stratificare il rischio , valutare la prognosi , determinare la progressione di malattia e ottenere informazioni per approntare uneventuale terapia [ 7 , 8 ]  . la coronarografia considerata , attualmente , il gold standard diagnostico per la valutazione dello stato di perviet dei bypass e la procedura di riferimento per la pianificazione di eventuali ulteriori interventi di rivascolarizzazione , eseguibili per via percutanea o per via chirurgica [ 9 ]  . 
la principale indicazione allesecuzione dellesame rappresentata , dallevidenza con le metodiche di stress imaging non invasivo , di criteri ad alto rischio ( vale a dire anomalie della motilit parietale regionale multisegmentarie e / o difetti di perfusione moderati - severi reversibili ) qualsiasi sia il periodo postoperatorio . 
il rilievo , nellimmediato postoperatorio , di una elevazione di alcuni markers biochimici considerati indicatori in grado di predire levento ischemico da precoce ostruzione del graft ( livelli di ck - mb > 70 u / l a 6 ore dallintervento , livelli di troponina cardiospecifica i > 20 ng / ml a 1224 ore dallintervento ) , costituisce unulteriore indicazione allesecuzione dellesame , anche in assenza di dati derivati dallo stress imaging [ 10 ]  . il rischio di complicanze maggiori ( morte , infarto del miocardio , ictus ) durante cateterismo cardiaco minimo ma non trascurabile , inferiore all1% , tuttavia alcuni fattori si associano ad aumento del tasso di mortalit fino a 10 volte . tra questi let ( > 80 anni ) , una classe funzionale iv , una method has a reported sensitivity of 96% , specificity of 95% , positive predictive value of 81% , negative predictive value of 99% and interobserver agreement of 95% [ 13 , 14 ]  . mdct : general scanning principles one prerequisite for a correct study of cabgs , or the native coronaries , is that the heart rate ( hr ) must be kept < 65 bpbradycardia may be obtained by means of - blockers , administered orally 23 days before the examination or intravenously ( i.v. ) on the same day . 
 after a baseline unenhanced scan to assess calcification of the native coronary arteries and the position of the metal clips , the contrast - enhanced study is performed with i.v. 
injection of 100120 ml of nonionic iodinated contrast material ( 350400 mgi / ml ) at a rate of 34 ml / s via a dual - syringe automatic injector , followed by a 30to 50 - ml bolus of saline solution . 
contrast enhancement is optimised using the bolus tracking technique , with acquisition starting when a predefined enhancement threshold is reached ( 100150 hu in the left ventricle or ascending aorta )  . 
once the phase of the cardiac cycle with the best diagnostic quality has been identified , usually mid - to - end diastolic postprocessing is performed on the native axial images to obtain multiplanar reconstructions ( mpr ) , curved mpr ( cmpr ) , maximum intensity projections ( mip ) and three - dimensional volume rendering ( 3d - vr ) to assess the course ( proximal anastomosis , graft body , distal anastomosis ) and type of bypass gracoverage of the entire study volume [ field of view ( fov ) 150200 mm ] using 16 - slice mdct requires collimations of 0.751 mm with a 15to 20 - s inspiratory breath - hold . 
table 1 shows the technical parameters of studies carried out with 4 - , 16and 64 - slice scanners . vascular structures internal mammary artery ( ima ) better long - term patency ( 85%90% at 10 years ) currently makes the internal mammary arteries the conduits of choice in surgical revascularisation procedures [ 1517 ]  . 
lanalisi di tali dati , connessi allinvasivit della procedura , e la necessit di ridurre i costi legati allelevato numero di coronarografie convenzionali non seguite da procedure di interventistica percutanea ( il 20%27% di queste risultano negative , il 2%58% non sono necessarie ) , ha favorito il ricorso alla tc multidetettore ( tcmd ) quale metodica alternativa di imaging cardiaco non invasivo utile ai fine della valutazione dello stato di perviet dei graft [ 12 ]  . in tal senso , numerosi lavori di comparazione con la coronarografia convenzionale , hanno ormai validato lefficacia delle apparecchiature a 16 canali nella detezione di patologia dei graft ; con tale metodica sono stati riportati valori di sensibilit del 96% , specificit del 95% , valore predittivo positivo dell81% , valore predittivo negativo del 99% e accordo inter - osservatore nelle interpretazione delle immagini pari al 95% [ 13 , 14 ]  . tcmd : principi generali di scansione prerequisito fondamentale per un corretto studio dei cabg il mantenimento , analogamente a quanto avviene nello studio delle coronarie , di una frequenza cardiaca < 65 bpm . leffetto bradicardizzante pu essere ottenuto mediante il ricorso a - bloccanti , somministrabili per os nei 23 giorni precedenti lesame o endovena il giorno stesso dellesame . in tcmd il volume di studio esteso a comprendere , per i graft venosi , lanastomosi prossimale su aorta ascendente e lanastomosi distale sui diversi territori delle coronarie ; per lo studio dei graft con mammaria interna non necessario includere lorigine succlavia prossimale . dopo unacquisizione diretta senza mezzo di contrasto , utile per valutare lentit delle calcificazioni delle coronarie native e il decorso delle clips metalliche , lo studio contrastografico viene eseguito mediante somministrazione endovenosa , utilizzando un iniettore automatico a doppia siringa , di 100120 ml di mdc iodato non ionico ( 350400 mgi / ml ) , alla velocit di 34 ml / s , seguiti da un bolo di 3050 ml di soluzione salina . 
per ottimizzare il contrast enhancement viene utilizzata la tecnica del bolus tracking , e il ritardo per linizio della scansione viene selezionato a partire dal raggiungimento di una soglia predefinita ( 100150 uh in ventricolo sinistro o aorta ascendente )  . le immagini sono ricostruite con gating ecg di cardiosincronizzazione retrospettiva e finestre temporali r - r variabili ; a partire dalle immagini assiali native , identificata la fase del ciclo cardiaco con la migliore qualit diagnostica , in genere meso - telediastolica , si procede allutilizzazione nel post - processing di ricostruzioni multiplanari ( mpr ) , curve ( cmpr ) , proiezioni di massima intensit ( mip ) e volume rendering tridimensionale ( 3d - vr ) , valutando , ai fini della valutazione di perviet dei cabg decorso ( anastomosi prossimale , corpo del graft , anastomosi distale ) e tipo di bypass . con tecnica tcmd a 16 canali , al fine di coprire lintero volume di studio ( fov 150200 mm ) necessario impiegare 1089 f . 
the graft is generally used in situ , that is , with a natural proximal origin at the level of the left subclavian artery and a distal anastomosis below the stenosis or occlusion on the native coronary . 
when present , graft stenosis or occlusion generally occurs at the distal anastomosis owing to the retrograde progression of coronary disease facilitated by the small diameter and high resistance of the coronary branch [ 18 ]  . 
 the right internal mammary artery ( rima ) , rarely used as an in situ graft for the right coronary artery ( rca ) territory , is more commonly implanted as a free graft ( with proximal anastomosis on the ascending aorta and distal anastomosis on the target coronary ) or composite graft , with proximal fig . 
1 immagini 3d - vr di bypass in situ lima - lad , normalmente pervio . 1090 collimazioni di 0 , 751 mm , con risultante apnea inspiratoria di 1520 secondi . 
in tabella 1 sono riportati i parametri tecnici legati allo studio con scanner a 4 , 16 e 64 canali . strutture vasali arteria mammaria interna ( ima ) attualmente il graft arterioso con mammaria interna , grazie alla sua migliore perviet a distanza ( 85%90% a 10 anni ) , rappresenta il condotto di prima scelta nel trattamento chirurgico di rivascolarizzazione [ 1517 ]  . 
il graft generalmente utilizzato in situ , cio con origine prossimale naturale a livello dellarteria succlavia sinistra e anastomosi distale eseguita su coronaria nativa a valle della steno - ostruzione . 
la steno - ostruzione del graft , quando presente , avviene in genere in sede di anastomosi distale , per progressione retrograda della patologia coronarica nativa , favorita dal piccolo calibro e dalle elevate resistenze del ramo coronario [ 18 ]  . la mammaria interna destra ( rima ) , raramente utilizzata come graft in situ per il territorio della coronaria destra ( rca ) , pi spesso utilizzata come graft libero ( con anastomosi prossimale su aorta ascendente e anastomosi distale su coronaria target ) , o composito , con anastomosi prossimale su graft con lima e anastomosi distale sul territorio della circonflessa ( lcx ) , con configurazione risultante f . 
utilizzando entrambe le mammarie si ottiene una rivascolarizzazione miocardica totalmente arteriosa , ne consegue una riduzione delle recidive anginose e un migliore tasso di perviet a 1 anno , rispetto allutilizzo , in associazione alla lima , del graft venoso con vena safena ( sv )  . 
il limite di impiego di entrambe le mammarie interne per costituito dal rischio , nei pazienti diabetici , obesi o anziani , di infezioni e deiscenze sternali secondarie alla devascolarizzazione completa della parete toracica anteriore . 
per tali motivi la patologia del graft venoso , rispetto a quella del graft arterioso , si associa ad una maggiore instabilit di placca , ed responsabile del anastomosis on a lima graft and distal anastomosis on the left circumflex artery ( lcx ) territory , with a resulting yor t - shaped configuration [ 19 ]  . 
the use of both mammary arteries allows totally arterial myocardial revascularisation and consequently fewer angina recurrences and better 1 - year patency rates compared with the use of lima combined with a saphenous vein ( sv ) grathe limitation of using both internal mammary arteries is the risk , in diabetic , obese or elderly patients , of sternal infections or dehiscence due to complete devascularisation of the anterior chest wall . 
for these reasons , venous grafts disease is associated with greater plaque instability and is responsible for 53% of angina episodes occurring within 5 years , 76% between 5 and 10 years , and 92% beyond 10 years . 
in the same periods , progression of atherosclerosis in the native vessels is responsible for 47% , 24% and 8% of ischaemic events , respectively [ 20 , 21 ]  . 
sv grafts are normally anastomosed proximally on the anterior wall of the ascending aorta ( on the left side , with a partially protected course above the pulmonary artery for grafts to the lad territory or lcx ; on the right side for those to the rca territory ) and distally below the stenosis or obstruction of the native coronary . radial artery ( ra ) with the recent advances in surgical harvesting techniques allowing minimal endothelial damage , the characteristic features of the conduit ( relatively large diameter with well - represented muscular wall that facilitates anastomosis ) and the use of calcium antagonists postoperatively to reduce the tendency towards vasospasm , the radial artery has come to be considered a valuable alterative to ima grafts [ 22 , 23 ]  . short - term patency rates are similar to those of lima , whereas midto long - term rates are comparable with those of sv grafts ( 89% at 4 years ) [ 24 ]  . 
2 three - dimensional volume rendering images of triple coronary artery bypass grafting : free radial artery on the left circumflex artery ( lcx - mo ) , left internal mammary artery on left anterior descending artery and saphenous vein on lcx . despite limitations due to the technical difficulty of the procedure , the gastroepiploic artery has been generally been used as fig . 
 complications graft stenosis and occlusion during the immediate postoperative period , the major cause of graft stenosis or occlusion is thrombosis , a complication that arises within a month of surgery in 10%15% of cases [ 16 ]  . 
the first recognised cause is the endothelial injury sustained while harvesting the vessel , ligating the collaterals in the case of arterial grafts or as a result of mechanical trauma due to excessive pulling or stretching in the case of short grafts . 
the use of mechanical aortic connectors for creation of the proximal anastomosis with sv appears to be associated with a higher incidence of thrombosis . after the first postoperative month , stenosis or occlusion of venous grafts is due to arterialisation resulting from exposure to high - pressure levels leading to intimal hyperplasia , medial thickening and atherogenesis . 
in arterial grafts , which are more resistant and with higher patency rates than venous grafts , the most common cause is retrograde progression of coronary artery atherosclerosis from the distal anastomosis . 
occlusion of an arterial graft ( which tends to be an all - or - nothing phenomenon ) is characterised by exclusive visualisation of the metal clips without evidence of a central contrast column ( empty railway sign )  . 
specifically , the causes of elevated vasoreactivity of the radial artery lie in its wall thickness , the density and arrangement of the muscle cells in the tunica media , denervation during harvesting and competitive flow with the native coronaries involved in the distal anastomosis [ 25 ]  . 
several measures have been devised to minimise vasospasm , including preoperative prophylaxis with calcium antagonists or intraoperative treatment with topical - adrenergic antagonists . the corresponding mdct finding is diffuse , or less commonly focal , uniform narrowing of the arterial graft , with increased distance between the vascular column and the lateral metal clips , without evident intraluminal defects and thus with normal opacification . 
the narrowing , corresponding to 70%99% stenosis , is equivalent to the angiographic string 1092 53% degli episodi anginosi che si verificano entro i 5 anni , del 76% degli episodi tra i 5 e i 10 anni , del 92% di quelli oltre i 10 anni . 
normalmente i graft con sv vengono anastomizzati in sede prossimale sulla parete anteriore dellaorta ascendente ( sul lato sinistro , con un decorso parzialmente protetto al di sopra dellarteria polmonare per i graft sul territorio della lad o della lcx , sul lato destro per quelli sul territorio della rca ) , distalmente a valle della lesione stenotica od ostruttiva della coronaria nativa . arteria radiale ( ra ) grazie al recente miglioramento delle tecniche chirurgiche di isolamento che hanno portato ad una minimizzazione del danno endoteliale , alle particolari caratteristiche anatomiche del condotto ( calibro relativamente ampio con parete muscolare ben rappresentata che facilita la costruzione delle anastomosi ) e grazie allutilizzo di calcio - antagonisti nel post - operatorio in grado di ridurre la tendenza al vasospasmo , larteria radiale considerata ad oggi una valida alternativa ai graft con ima [ 22 , 23 ]  . 
il tasso di perviet equivalente a quello della lima nel breve termine mentre risulta paragonabile a quello dei graft con sv nel periodo medio - lungo ( 89% a 4 anni ) [ 24 ]  . 
il primum movens riconosciuto il danno endoteliale che pu verificarsi nella fase di isolamento del vaso , in quella di legatura dei collaterali nel caso di graft arteriosi , o come conseguenza di traumi meccanici da eccessiva trazione o stiramento del vaso nel caso di graft con lunghezza ridotta . 
lostruzione del graft arterioso ( che avviene in genere con fenomeno tutto o nulla ) si caratterizza per la sola visibilit delle clips metalliche senza evidenziazione della colonna centrale di mdc ( segno del binario vuoto , empty railway sign )  . 
specificatamente le cause dellelevata vasoreattivit dellarteria radiale sono da ricondurre allintrinseco spessore parietale , alla densit e alla particolare organizzazione delle cellule muscolari della tonaca media , alla denervazione subita durante lisolamento dallavambraccio , al flusso competitivo con le coronarie native target dellanastomosi distale [ 25 ]  . 
per minimizzare il vasospasmo sono stati considerati diversi provvedimenti , tra i quali una profilassi preoperatoria con calcio - antagonisti o un trattamento farmacologico intraoperatorio con antagonisti - adrenergici per via topica . laspetto mdct corrispondente quello del restringimento uniforme diffuso , o meno comunemente focale , del graft arterioso , con aumentata distanza tra colonna vascolare e clips metalliche laterali , in assenza di difetti endoluminali evidenti e cio con normale opacizzazione da mdc . 
tale restringimento , corrispondente ad una stenosi del 70%99% , lequivalente del segno della stringa angiografico e piuttosto che uno spasmo fisso deve essere considerato come il risultato di un rimodellamento vascolare negativo secondario ad una risposta adattativa ad una bassa richiesta di perfusione periferica per fenomeni di flusso competitivo [ 26 ]  . 
queste considerazioni sono supportate dallevidenza di una bassa incidenza di ischemia nei territori perfusi da graft con restringimento diffuso . aneurismi e pseudoaneurismi eventuali pseudoaneurismi asintomatici si formano generalmente entro i primi 6 mesi dallintervento e sono correlati 1093 fig . 
4 restringimento uniforme diffuso di bypass lima su lad , espressione di rimodellamento vascolare negativo . sign and , rather than a fixed spasm , should be considered the result of negative vascular remodelling due to an adaptive response to a low demand for peripheral perfusion because of competitive flow phenomena [ 26 ]  . 
the complications of aneurysms are mainly related to compression of adjacent structures , but thrombosis , embolisation , fistulisation and rupture with possible haemomediastinum may also occur . malposition and iatrogenic complications graft malposition may result from kinking , frequently of the proximal portion of a venous graft due to excessive length or coiling of the gra both conditions are linked to surgical technique and are associated with an increased risk of endothelial damage and subsequent thrombotic stenosis or occlusion . 
generally , however , any iatrogenic endothelial trauma affecting the graft during surgery will promote graft thrombosis . extracardiac complications within the first week after surgery , the presence of a moderate unilateral left pleural effusion , which tends to resolve spontaneously , is considered paraphysiological , and only 1%4% of patients develop a clinically relevant massive effusion requiring drainage by thoracentesis [ 28 ]  . 
likewise , the incidence of pericardial effusion during the immediate postoperative period is fairly high ( 22%85% ) ; this resolves spontaneously in most patients and becomes haemodynamically significant with cardiac tamponade in only 0.8%6% of cases [ 29 ]  . 
potentially lethal , with a mortality rate of approximately 25% , are deep sternal wound infections complicated by mediastinitis , which occur in 1%4% of patients [ 30 ]  . 
finally , pulmonary embolism , which tends to have a silent clinical course , is a further complication that arises in 0.4%9.5% of cases after cabg [ 31 ]  . 
 artefacts cardiac and respiratory motion artefacts in the presence of cardiac arrhythmias characterised by elevated or irregular hr , there is a critical reduction in the diastolic phase , the duration of which falls below the temporal resolution of the scanner . 
dilatazioni aneurismatiche vere e proprie sono complicanze rare e tardive , che si verificano ad oltre 5 anni di distanza dallintervento , pi spesso a carico dei graft venosi come effetto dellaterosclerosi [ 27 ]  . 
le complicanze degli aneurismi sono legate alla compressione delle struttura adiacenti per effetto massa ma possono essere rappresentate anche dalla trombosi , dallembolizzazione , dalla fistolizzazione , dalla rottura con potenziale emomediastino . malposizionamento e complicanze iatrogene un malposizionamento pu verificarsi per un kinking , pi frequentemente a carico della porzione prossimale di un graft venoso o per eccessiva lunghezza e tortuosit del graft stesso ( coiling )  . 
in linea generale , comunque , durante lintervento chirurgico , qualsiasi trauma endoteliale iatrogeno del graft ne favorisce la trombosi . complicanze extracardiache entro la prima settimana dallintervento la presenza di un modesto versamento pleurico unilaterale sinistro , con tendenza spontanea alla risoluzione , considerata parafisiologica solo l1%4% dei pazienti post - cabg sviluppano un versamento massivo , clinicamente rilevante , tale da richiedere una toracentesi evacuativa [ 28 ]  . 
allo stesso modo lincidenza di un versamento pericardico nellimmediato postoperatorio piuttosto elevata ( 22%85% ) ; nella maggior parte dei casi questo si risolve spontaneamente e solo nello 0 , 8%6% dei casi risulta emodinamicamente significativo appalesandosi come tamponamento cardiaco [ 29 ]  . 
potenzialmente fatali , con una mortalit di circa il 25% , sono le infezioni retrosternali profonde complicate da mediastinite , che si verificano nell1%4% dei pazienti [ 30 ]  . 
infine , lembolia polmonare , pi spesso con decorso clinico silente , un ulteriore complicanza del paziente post - cabg , manifestandosi nello 0 , 4%9 , 5% dei casi [ 31 ]  . artefatti artefatti da movimento cardiaco e respiratorio in presenza di aritmie cardiache caratterizzate da frequenza cardiaca troppo elevata o irregolare si ha una riduzione critica della fase diastolica , la cui durata risulta inferiore alla risoluzione temporale dello scanner . 
6 artefatti da indurimento del fascio con conseguente effetto blooming e mascheramento del lume vasale . tion during the last phase of an inspiratory breath - hold makes it difficult to visualise the coronary segments and distal anastomoses , especially on the right side , as , compared with the lad and lcx , the rca is affected by greater variations in velocity and thus in position during the different phases of the cardiac cycle . 
a reduction of cardiac motion artefacts can be achieved by decreasing the hr with - blocker premedication or by a more appropriate selection of the r - r interval for reconstruction . respiratory motion artefacts produce the same effects and can be differentiated from cardiac motion artefacts on the basis of the presence of linear misregistrations beyond the cardiac contours and even in the chest wall . 
cabg visualisation may also be partly affected by superimposition of adjacent anatomical structures with regular contrast enhancement , such as the left atrial auricles . la normale accelerazione cardiaca durante lultima fase dellapnea inspiratoria rende particolarmente difficoltosa la visualizzazione dei segmenti coronarici e delle anastomosi distali , specie a destra , in considerazione del fatto che la rca subisce , rispetto alla lad e alla lcx , le maggiori modifiche di velocit e quindi le maggiori escursioni di posizione durante le diverse fasi cardiache . 
la riduzione degli artefatti da movimento cardiaco pu essere ottenuta diminuendo la frequenza cardiaca mediante premedicazione con - bloccanti o mediante una selezione pi appropriata dellintervallo r - r di ricostruzione . gli artefatti da movimento respiratorio si manifestano con gli stessi effetti e la differenziazione rispetto agli artefatti da movimento cardiaco viene effettuata sulla base della presenza delle misregistrazioni lineari oltre i profili cardiaci , anche nel contesto della parete toracica . 
la riduzione di tali artefatti si ottiene riducendo i tempi dellapnea inspiratoria e istruendo il paziente a mantenere il respiro prima delleffettuazione dellesame [ 32 ]  . indurimento del fascio radiogeno clips metalliche , fili di sutura sternali , fili di pacemaker , ma anche calcificazioni aterosclerotiche nelle coronarie native , possono produrre artefatti a strisce da indurimento del fascio . 
il conseguente effetto di eccessiva luminosit di tali strutture ( blooming ) , oltre che determinare un aumento artefattuale delle dimensioni delloggetto in esame , pu contribuire , per leffetto di volume parziale , ad inficiare il corretto studio del lume vascolare , rendendo non valutabili i segmenti vasali o evidenziando pseudostenosi , specie in sede 1095 f . 
for example , poor vascular enhancement may be a direct consequence of the incorrect use of bolus tracking and inappropriate scan delay settings , of altered flow dynamics in elderly patients or of inappropriate choice of contrast volume or concentration . 
pitch selection is fundamental , above all in patients with a long r - r interval and hr < 50 bpm , in whom failure to apply a low pitch to cover the entire course of the grafts will produce blurring [ 32 ]  . 
 to avoid these technical problems , patency should always be evaluated on the combined axial , mpr , mip and vr images , as a bypass graft not visualised on vr images and erroneously thought to be obstructed may instead be patent and only detectable on mip or mpr images . reporting guide a prerequisite for the mdct study of cabg is prior knowledge of the type of surgical procedure performed on the patient . 
detailed anatomical information is required on the number and type of bypass grafts ( arterial or venous , in situ , free , composite , sequential ) and the exact location of the proximal and distal anastomoses . 
assessment of the distal anastomosis is a critical step in the examination due to relative hypermotility during the cardiac phases and the presence of more distally located metal clips , which account for a greater susceptibility to motion artefacts and beam hardening with blooming effects , respectively . 
in any case , assessment of the native coronary flow distal to the anastomosis is mandatory . the judgement of patency must be based on the degree of stenosis : patency ( complete uniform opacification of the graft without critical stenoses ) ; critical stenosis ( stenosis > 50% ) ; occlusion ( graft not visible due to lack of enhancement )  . 
in the presence of a diffuse uniform narrowing of arterial grafts , above all in ra grafts , with increased distance between vascular column and lateral metal clips and a vessel diameter1 mm , one should consider the possibility of vasospasm due to functional remodelling with preserved patency . 
la visualizzazione dei cabg pu essere inoltre parzialmente inficiata dalla sovrapposizione di strutture anatomiche adiacenti a regolare impregnazione contrastografica , quali le auricole atriali a sinistra . artefatti di ordine tecnico alcuni artefatti sono causati da errori tecnici eseguiti in fase di acquisizione dei dati , cos un enhancement vascolare insufficiente pu essere la diretta conseguenza di un errato utilizzo della temporizzazione mediante bolus tracking con inizio della scansione inappropriato , di un alterata dinamica di flusso nei pazienti anziani , di uninappropriata scelta del volume o della concentrazione del mezzo d contrasto iodato . 
la scelta del pitch di fondamentale importanza specie nei pazienti con intervallo r - r lungo e fc < 50 bpm , nei quali la mancata applicazione di un pitch basso per coprire lintero decorso dei graft causa di effetto blurring [ 32 ]  . per ovviare a tali problemi tecnici , la valutazione di perviet deve essere sempre effettuata , nel post - processing , su immagini assiali , mpr , mip e vr combinate , pu accadere infatti che un cabg non visualizzabile in vr ed erroneamente ritenuto ostruito , sia pervio e visualizzabile nelle sole immagini mip o mpr . guida alla refertazione pre - requisito essenziale per lo studio dei cabgs con mdct la conoscenza , a priori , del tipo di intervento chirurgico effettuato dal paziente . 
sono necessarie precise e dettagliate informazioni di tipo anatomico riguardanti il numero ed il tipo dei bypass confezionati ( arteriosi o venosi , in situ , liberi , compositi , sequenziali ) e lesatta collocazione delle anastomosi prossimali e distali . 
in ogni caso dobbligo la valutazione del flusso coronarico nativo distale rispetto allanastomosi . il giudizio di perviet deve essere fornito in base allentit della stenosi : perviet ( completa e uniforme opacizzazione del graft in assenza di stenosi critiche ) , stenosi critica ( stenosi > 50% ) , ostruzione ( assenza di visibilit del graft per mancata opacizzazione dello stesso )  . 
in presenza di un diffuso ed uniforme restringimento dei graft arteriosi , specie di quelli con ra , con aumento della distanza tra colonna vascolare e clips metalliche laterali e calibro vasale 1 mm , si deve prendere in considerazione un vasospasmo da rimodellamento funzionale con mantenuta perviet . dal punto di vista tecnico la valutazione dello stato di perviet dei cabg deve essere eseguita integrando le singole informazioni ottenute dalle differenti tecniche di visualizzazione delle immagini nel post - processing ( mpr , mip , vr ) e non pu prescindere in ogni caso da una valutazione iniziale delle immagini assiali native . 
le immagini vr , grazie alla visione panoramica 3d , sono molti utili nella definizione della complessa anatomia dei graft e dovrebbero essere usate , insieme alle assiali native , come base per la interpretazione dei reperti . 
gli svantaggi successiva dellanalisi singola delle mpr oblique o curve e delle mip ( rispettivamente dipendenza dallorientamento manuale del piano di vista con stenosi falsamente positive o negative nel primo caso e sovrastima delle stenosi per effetto di sovraproiezione nel secondo ) , possono essere evitati utilizzando entrambe le tecniche in maniera complementare [ 34 ]  . 
in questo modo possibile ottenere le informazioni maggiori sia sullo stato dei graft , sia su quello delle anastomosi distali . doveroso segnalare , quando presenti , tutti quei reperti cardiaci morfologici non coronarici ritenuti clinicamente rilevanti ( anomalie dello spessore parietale , delle cavit cardiache o dellorigine dei principali vasi mediastinici , masse cardiache trombotiche o non , esiti aneurismatici post - infartuali )  . infine buona regola concludere lesame contrastografico con una scansione estesa a tutto il torace , cos facendo , tra le altre cose , possibile escludere tutte quelle eventuali patologie extravascolari che si presentano clinicamente con dolore toracico tale da mimare unangina ricorrente . 
in questa classe di pazienti la diagnosi differenziale del dolore toracico include infatti il versamento pleurico , il versamento pericardico , lembolia polmonare e linfezione delle suture sternali . nei casi in cui si ha una dimostrazione di patologia si pone il problema della scelta del tipo di metodica di rivascolarizzazione da utilizzare , per tale motivo le informazioni di tipo anatomico fornite dalla tcmd sono cruciali ai fini della selezione dei pazienti da avviare a trattamento percutaneo ( angioplastica con o senza stenting ) o chirurgico . tale scelta risulta guidata dalla localizzazione e dellestensione della malattia . 
si raccomanda una rivascolarizzazione per via percutanea nei pazienti non ad alto rischio , con funzione ventricolare preservata che presentano integrit del graft con lima e stenosi focali subtotali , specialmente 1097 fig . 
7 immagini 3 dvr dellanastomosi prossimale del corpo del graft e dellanastomosi distale di triplice bypass sv - da , lima - lad d1 , ra - lcx . tion techniques ( mpr , mip , vr ) and cannot be separated from an initial assessment of the native axial images . 
vr images , providing a 3d panoramic view , are very useful to define the complex anatomy of grafts and should be used together with the native axial images as a basis for subsequent interpretation of findings . 
the drawbacks of the individual analysis of oblique or curved mpr images and mip images ( dependence on manual orientation of the plane with falsepositive or false - negative stenoses in the first case and overestimation of stenosis due to overprojection in the second ) can be overcome by using the two techniques in a complementary manner [ 34 ]  . 
 it is important to note any noncoronary morphological cardiac findings thought to be clinically relevant ( abnormalities of wall thickness , of the cardiac cavities or of the origin of the main mediastinal vessels , thrombotic or nonthrombotic cardiac masses , postinfarct aneurysm )  . finally , the contrast - enhanced study should be completed with a scan extending to the entire chest to be able to exclude , among others , the presence of possible extravascular disease manifesting with chest pain and mimicking recurrent angina . 
in this class of patients , the differential diagnosis of chest pain includes pleural effusion , pericardial effusion , pulmonary embolism and sternal wound infection . evidence of disease calls for decisions on the type of revascularisation procedure to be used , and the anatomical information provided by mdct is instrumental in selecting patients for percutaneous ( angioplasty with or without stenting ) or surgical treatment . 
repeat surgery is preferred in high - risk patients ( especially those with multivessel disease of the native arteries with left main involvement ) and those with reduced ventricular function , with diabetes , and with severe graft impairment due to chronic total occlusions [ 35 ]  . 
 in patients with severe graft impairment , the panoramic view provided by mdct allows one to provide the surgeon with all the anatomical data needed to plan the repeat operation and minimise the risks of surgery . 
for these reasons , the radiologist needs to provide detailed information on the morphology and course of the native and grafted vessels and the relationship between these and mediastinal structures , the sternum and the chest wall [ 36 ]  . 
si preferisce invece un reintervento chirurgico nei pazienti ad alto rischio ( soprattutto in presenza di malattia nativa multivasale con interessamento del tronco comune sinistro ) , con ridotta funzione ventricolare , diabetici , in presenza di grave compromissione dei graft , come nel caso di ostruzioni totali croniche degli stessi [ 35 ]  . in questa ultima classe di pazienti , la tcmd consente , grazie alla sua visione panoramica , lottenimento di tutti quei dati anatomici necessari al chirurgo per pianificare il re - intervento e minimizzare i rischi connessi a questo . 
lachman mosby elsevier , philadelphia ( 2007 ) isbn - 13 : 978 - 0 - 323 - 01931 - 6 published online : 13 december 2007 this is the fifth edition of a masterpiece book that , through the years , has been a trusty companion to paediatric radiologists and paediatricians , expanding from edition to edition to become more and more complete , being nicknamed the second bible in this field , with caffeys the first . 
the first address syndromes and metabolic disorders , now merged in a single section in contrast to the previous edition , making it easier to use and research the 860 pages , the largest section in the volume . 
these sections are followed by 46 pages listing the pertaining literature ( 22 , 500 entries and 4 , 500 literature references ! )  . as stated by the author in his short preface , this edition has been enriched by 90 new syndromes and metabolic diseases and 35 new skeletal dysplasias : whenever possible , each entity has its appropriate omim number , appropriate locus , gene name and protein involved . 
several new tables have been also prepared ( mucopolysaccharidoses / mucolipidoses entities ; general radiological features of chromosome abnormalities , for example )  . there are two appendices , as well : the first list the international nosology and classification of genetic disorders of bone 2006 ( also to be found in the american journal of medical genetics ) ; the second is a short , pithy chapter titled teaching approach to the skeletal dysplasias . 
in few pages with great clarity describe the path to be followed when one is confronted with a suspected skeletal dysplasia : how to define , describe with proper and corrects terminology ( and not jargon ) what the findings are and correctly classify them . the number of pages increased from 1 , 135 in the fourth edition to the 1 , 365 in this edition ; due to printing art miracles , volume sizes are the same but the weight is reduced ( from 3 , 600g to 3 , 290g : this matters too ! ) : font size compression and paper thickness explain these results . a small ticket , to be found at the back of the front cover , allows , by means of a personal username , free access to a web site . 
this link makes it possible and easy to expand the research or at least narrow its field , and through clinical signs or questa la quinta edizione di unopera che ha accompagnato nel corso degli anni , diventando sempre pi estesa e completa il cammino dei radiologi pediatri e dei pediatri , assumendo le caratteristiche di unaltra bibbia e di testo fondamentale in questo campo , dopo quella del caffey . il volume diviso in tre sezioni : le prima riguarda le sindromi e le malattie metaboliche , ora comprese in un unico capitolo a differenza di quanto non fosse nella quarta edizione , rendendone pi facile la consultazione ed il reperimento . 
la seconda sezione si occupa delle displasie scheletriche svolte in 243 pagine e la terza dedicate ai gamuts : 168 pagine di elenchi completate da 46 riguardanti la letteratura pertinente ( 22500 opzioni e 4500 voci bibliografiche ! )  . come precisato dallautore nella breve prefazione , ledizione attuale si arricchita di 90 nuove sindromi e malattie metaboliche e di 35 nuove displasie scheletriche : ove possibile stato riportato il numero omim , il locus genetico , il nome del gene e la proteina interessata delle affezioni trattate . 
sono state inoltre introdotte alcune nuove tabelle ( malattie da accumulo ; aspetti radiologici generali della malattie cromosomiche , per esempio )  . completano il volume due appendici , la prima , dedicata allinternational nosology and classification of genetic disorders of bone2006 ( pubblicata anche su american journal of medical genetics ) e la seconda , sotto forma di breve , ma succoso capitolo dal titolo teaching approach to the skeletal dysplasias in cui sinteticamente , ma con grande chiarezza , viene descritto il percorso da seguire quando si ha da confrontarsi con una sospetta displasia scheletrica per definirla , descriverla con termini appropriati e corretti ed eventualmente classificarla . complessivamente si passati dalle 1135 pagine della quarta edizione alle 1365 della attuale , ma miracolo dellarte tipografica le dimensioni del volume non sono diverse da quelle del precedente , ma il suo peso ridotto ( da 3600 g a 3290 : anche questo conta ! ) : compressione dei caratteri e carta pi leggera spiegano il risultato . 
 this book is of essential importance , and must absolutely be at hand in the daily practice of all of those dealing with these types of problems , be they general or paediatric radiologists , paediatricians , geneticists or orthopaedic specialists . 
the book is in good company with others on the topic , such as sprangers bone dysplasias , castriota - scanderbegs abnormal skeletal phenotypes and canepas dysmorphic syndromes and constitutional diseases of the skeleton . 
sic transit gloria mundi ! surely not the memory of a great and modest colleague depicted smiling in a photograph taken in the company of lachman that opens and illuminates the first page , followed by a moving , short memorial . 
 permette di ampliare la ricerca e di ottenere diagnosi o di restringere il campo di ricerca attraverso la diagnosi differenziale di segni clinici o di aspetti radiologici . opera fondamentale che non deve assolutamente mancare a chi si occupa di questo tipo di problemi sia radiologo , pediatra , genetista od ortopedico da consultare quotidianamente e da affiancare ad altre opere sullargomento , tipo bone dysplasias dello spranger ; abnormal skeletal phenotypes di castriotascanderbeg e sindromi dismorfiche e malattie costituzionale dello scheletro di canepa . peccato che questa nuova edizione dovr farmi mettere in pensione la precedente , in cui faceva mostra di s una bella dedica del taybi , ora da conservare come un tesoro . 
the purpose of this article is to illustrate a case where acquisition of digital imaging know - how by a modern radiotherapy division has helped to solve a technical problem while allowing substantial savings through the use of free and open - source resources . 
the system developed , called the dicom router , implemented two kinds of routing : manual and automatic , both oriented to link the images acquired with the existing electronic clinical records . 
the need to operate with more than one provider creates a series of integration issues , so that it becomes economically appealing to acquire internally the knowledge needed to interact more precisely with providers of big information technology ( it ) solutions . 
by using them , in - house it technicians are able to implement valuable technical solutions for smallto medium - sized informatization problems , which would otherwise remain unsolved except with great economic efforts . key words open source dicom low cost riassunto obiettivo . 
scopo di questo lavoro presentare un caso in cui lacquisizione in seno ad un moderno reparto di radioterapia del know how relativo allimaging digitale ha consentito di risolvere un problema con un cospicuo risparmio economico grazie allutilizzo di risorse free ed open source . 
stato realizzato un dispositivo , detto dicom router , che implementa due tipologie di instradamento : automatica e manuale , entrambe orientate allintegrazione delle immagini acquisite con il sistema di cartelle cliniche informatizzate . laspetto legato allinstabilit dellhardware stato poi risolto con una tecnica di ridondanza dei dispositivi . 
la necessit di operare con pi fornitori crea una serie di problemi di integrazione per cui spesso diventa economicamente accattivante lidea di acquisire in seno al reparto il know how per dialogare in maniera pi precisa e disambigua con i fornitori di grandi soluzioni informatiche . questa necessit ben si sposa con le possibilit offerte da tecnologie software open source ben documentate ed accessibili a tutti . 
infatti , con luso di queste , il personale tecnico informatico in seno al reparto pu implementare valide soluzioni per i piccoli / medi problemi di informatizzazione che , altrimenti , resterebbero insormontabili o economicamente molto impegnativi . parole chiave open source dicom low cost 1252 r . 
in recent years , however , new technologies have appeared in the field of information technology ( it ) , which allow substantial savings while maintaining the same level of product quality . this result is obtained thanks to the particular type of licence terms , the project conception and the developers attitude . the birth of these technologies ( the most famous of which is probably linux [ 1 ] ) is the beginning not only of a new way of thinking software , but also of a new way of working and sharing knowledge [ 2 ]  . 
this new scenario , together with increased information needs , provides a fertile environment for acquiring , directly in the clinical division , the competence and know - how necessary to manage digital imaging problems internally . here will describe a specific case that required the development of a system , called the dicom router , which is able to route in a complex manner digital diagnostic images , integrating them in the information system used in a modern radiotherapy division . 
this system allows radiographers to send images to the correct destinations with a single operation and to associate images from other divisions to the correct electronic clinical record by modifying the contents of the digital imaging and communications in medicine ( dicom ) files [ 3 ]  . materials and methods to handle both diagnostic and therapeutic information within the information system , in 2004 , our division bought a software programme for managing electronic clinical records ( normally only text data ) and a commercial dicom - compliant pacs server ( dicomed p@cs web ) for managing digital images . 
 the dicom sources initially consisted of a ge prospeed s fast computed tomography ( ct ) scanner , a vidar scanner , two precise linac systems for portal images and two different contouring stations for digital reconstructed radiograph ( drr ) images ( plato and oncentra masterplan )  . 
in addition , there is also a magnetic resonance imaging ( mri ) scanner located in the hospitals radiology division and a cdrom reader to view diagnostic images acquired at other hospitals . despite the presence of a good number of valuable , free open - source solutions [ 47 ] , the need for a flexible , simply configurable , and quickly integratable system , combined with the presence of an it technician , prompted us to develnegli ultimi anni , in molti reparti clinici che trattano limaging digitale , si osservato un aumento della necessit di informatizzare la filiera al fine di poter fruire delle potenzialit offerte dai sistemi informativi per immagini medicali ( pacs ) e dei benefici che ne derivano , soprattutto legati alla maggiore disponibilit dei dati . 
tuttavia , da qualche anno , parallelamente alle soluzioni tradizionali dei grandi gruppi produttori di software , si sono affermate sulla scena dellinformation technology ( it ) alcune tecnologie che consentono , per tipologia di licenza , per concezione progettuale , per mentalit dei realizzatori , di operare considerevoli economie mantenendo invariata la qualit di prodotto . 
la nascita di queste tecnologie ( la pi nota , probabilmente , linux [ 1 ] ) linizio non solo di un nuovo modo di pensare la realizzazione del software , ma anche di usarlo , condividendo in modo aperto le informazioni [ 2 ]  . 
questo nuovo contesto , unitamente alle sempre maggiori necessit informatiche , terreno fertile per lacquisizione in seno ai reparti clinici del know - how necessario a gestire , per quanto possibile internamente , le problematiche tecniche legate allimaging digitale . 
 in accordo con questidea mostreremo un caso specifico che ha richiesto la creazione di un sistema , detto dicom router , in grado di veicolare in maniera complessa delle immagini diagnostiche digitali integrandole con il sistema informatizzato in uso in un moderno reparto di radioterapia . 
tale sistema consente ai tecnici sanitari di radiologia ( tsr ) , con un solo invio , di veder recapitate le immagini alle corrette destinazioni e di associare le immagini acquisite anche da altri reparti alla corretta cartella clinica informatizzata tramite la modifica del contenuto dei files dicom [ 3 ]  . materiali e metodi il reparto , al fine di gestire su supporto informatico le informazioni cliniche , diagnostiche e terapeutiche di ogni paziente , si dotato nel 2004 di un sistema di cartelle cliniche informatizzate ( per la gestione dei dati testuali dei pazienti ) e di un pacs server commerciale dicom compatibile ( dicomed p@cs web ) per la gestione delle immagini digitali associate ai pazienti . 
oltre a questi da aggiungere anche una rmn di un reparto di radiologia dello stesso ospedale ed il supporto cd con il quale talvolta il paziente presenta delle immagini diagnostiche condotte presso altri ospedali . in merito alla metodologia di sviluppo , nonostante siano gi disponibili soluzioni open source e free molto valide 1253 r . 
all these technologies were adopted not only because they are free , but also because they are stable , mature and flexible and sometimes also used by pacs vendors . the hardware used is a personal computer ( pc ) with intel pentium iv processor , 256 megabyte random access memory ( ram ) , hard disk drive ( hdd ) 80 gigabyte and a pc with pentium ii processor , 128 megabyte ram and hdd 40 gigabyte . results the system developed , called the dicom router , is able to acquire images from the divisions dicom sources and to route them to the right targets . 
we thus obtained a redundant system that ensures a good level of reliability given the low likelihood of a double system crash . the two systems were realised and configured as a primary system ( the one with the better hardware ) and a secondary system ( the one with worse hardware )  . 
in particular , the estimated dos time in the case of a server crash ranges from 180 to 240 s , considered sufficient for the needs of our division . to better understand the general system architecture , two aspects need to be considered : one relating to the routing architecture ( manual and automatic ) , and one relating to the security architecture service ( fault tolerance )  . architecture for automatic routing according to the dicom standard , a node in a dicom network is identified by a triplet : < ip address , ae title ( aet ) , tcp port > this allows one to define a number of dicom nodes on the same computer by modifying the parameters of the port and / or the aet . 
in our case , the options provided by the storescp command of the dcmtk libraries made this implementation possible , allowing us to define several dicom nodes on our dicom router : node 1 : < 1.1.36.223 , dicom_router , 105 > node 2 : < 1.1.36.223 , dicom_router , 106 > node 3 : < 1.1.36.223 , dicom_router , 107 > node 4 : < 1.1.36.223 , dicom_router , 108 > node 5 : < 1.1.36.223 , dicom_router , 109 > these different nodes manage the incoming images in different ways ; thus , when a new dicom source is connected to the network and wants to send images to a destination through the dicom router , it is sufficient to configure the node corresponding to the kind of routing wanted as a target on the source . 
pi nello specifico , i due sistemi sono stati realizzati e configurati come sistema primario ( quello con hardware migliore ) e sistema secondario ( quello con hardware peggiore )  . 
il ruolo del secondario solo quello di monitorare i servizi del primario e , nel momento in cui questi non risultino correttamente funzionanti , forzarlo a spegnimento , se possibile , e prendere la sua identit diventando esso stesso primario . 
ci dovrebbe consentire al tecnico informatico di poter intervenire per risolvere i problemi che hanno afflitto il primario originale con un minimo disservizio ( dos , denial of service ) per gli utenti . 
in particolare , per il sistema proposto , si stima il dos causato da un crash del server primario allincirca in 180 , 240 secondi , il che ritenuto tollerabile per le esigenze del reparto . per meglio comprendere larchitettura del sistema generale considereremo separatamente laspetto legato allarchitettura di instradamento ( routing ) , sia manuale che automatica , e quello legato allarchitettura di messa in sicurezza del servizio ( fault tolerance )  . architettura di routing automatico secondo lo standard dicom un nodo della rete identificato da una tripletta : < indirizzo ip , ae title ( aet ) , porta tcp > ci implica la possibilit di definire pi nodi dicom su una stessa macchina modificando la porta e / o laet . 
nel nostro caso , le opzioni messe a disposizione dal comando storescp fornito dalle librerie dcmtk , hanno reso possibile questa implementazione , consentendo di realizzare un dicom router che consta di pi nodi dicom : fig . 
2 the lines labelled 1 indicate the routing of images coming from computed tomography ( ct ) : as shown , they come in the dicom router and from there are sent to the treatment planning system and picture archiving and communication systems ( pacs ) server . 
2 gli archi etichettati come 1 indicano linstradamento di alcune immagini generate dalla tac : come si pu notare queste entrano nel dicom router ed escono in direzione del treatment planning system ( tps ) e del pacs server . 
analogamente , gli archi etichettati come 2 , sempre generati dalla tac , entrano nel dicom router e , sfruttando un nodo dicom differente dal precedente , vengono instradate verso altre destinazioni ( pacs server ed una sistema di contouring contouring station )  . 
this is feasible only for images produced in our division ; for images produced in other radiology divisions ( in the same or in different hospitals ) , this is not possible . 
thus , it is not possible for images produced outside our division to implement automatic routing because the images stored on the pacs server with a wrong patient id would be matched to the wrong electronic clinical record . 
in our case , the only services requiring a database are collateral ( the viewer and the manual routing interface ) , and the data have quite a short lifespan ( no longer than 1 day )  . regarding dicom routing services , the two computers were perfectly replicated in terms of services working on diffig . 
3 sezioni per la modifica delle tag dicom e della spedizione a differenti nodi . 1256 nodo 1 : < 1.1.36.223 , dicom_router , 105 > nodo 2 : < 1.1.36.223 , dicom_router , 106 > nodo 3 : < 1.1.36.223 , dicom_router , 107 > nodo 4 : < 1.1.36.223 , dicom_router , 108 > nodo 5 : < 1.1.36.223 , dicom_router , 109 > questi differenti nodi gestiscono le immagini in ingresso instradandole in maniera differente . 
mentre ci pu essere ammissibile per le immagini prodotte allinterno del reparto , per le immagini prodotte presso altre radiologie ( anche dello stesso ospedale ) , questo non possibile . 
non possibile , quindi , per immagini di questo tipo ( come del resto anche per le immagini ottenute da cd di altri ospedali ) pensare ad un routing automatico , in quanto verrebbero spedite al server ed associate a cartelle cliniche sbagliate . 
partendo dallassunto che questa situazione si possa verificare quando per qualche motivo il server primario si sia scollegato dalla rete ( perch guasto o per motivi di crash di switch ) , la sostituzione di identit mediamente operata in 180 secondi , 240 nel caso peggiore . 
oltre a ci , sia sul primario che sul secondario ogni minuto schedulato il lancio di un agente che verifica , tramite un comando echoscu , il corretto funzionamento dei servizi dicom sulle differenti porte . 
nel momento in cui il comando non riceve loutput corretto , il sistema , primario o secondario che sia , viene forzato al riavvio ( reboot )  . nel momento in cui il server primario si accende o torna in rete dopo una sconnessione temporalmente rilevante , si affaccia alla rete con un altro indirizzo ip e manda un segnale alleventuale server che ha lindirizzo corretto del primario per forzarlo a cambiare indirizzo in quello del secondario . 
cos facendo , se anche il server primario venisse riacceso , o lipotetico switch di rete venisse riparato , non correremmo il rischio di avere conflitti di indirizzi ip nella rete . inoltre , entrambe le macchine sono sotto gruppo di continuit con spegnimento ( shutdown ) pilotato oltre i 3 minuti di mancata alimentazione ( power fault )  . il sistema cos realizzato , opera ininterrottamente da pi di un anno . discussione il sistema , nel suo complesso , ha consentito di verificare che lattuale disponibilit di software free , stabile e maturo , consente ad un qualunque reparto di attrezzarsi con del personale tecnico idoneo al fine di risolvere una serie di problematiche che , altrimenti , chiederebbero lintervento di aziende esterne . 
4 visualizzatore delle immagini dicom acquisite . ferent transmission control protocol ( tcp ) ports ( both have routing services with the same rules ) but differ in one aspect : the internet protocol ( ip ) address . 
the two pc are organised as follow : when the secondary server is not able to ping the primary server but is able to ping other servers in the network , it changes its ip address into that of the primary server , making itself identical ( for the rest of the net ) to the primary server . 
assuming that this case is compatible with a situation where the primary server is disconnected from the net ( due to damage , disc crashes or maybe a switch that has crashed somewhere ) , the identity substitution is operated in 180 s in the best case and 240 in the worst . 
furthermore , both the primary and the secondary servers have an agent , scheduled to start every minute , that checks , through an echoscu command , the state of the dicom services working on different ports . 
a bad output of this command is interpreted by the agent as a problem , so it forces the system to reboot ( primary or secondary , depending on which is giving problems )  . 
another rule requires that when the primary server is reconnected to the network after a temporally significant disconnection , it presents itself on the network with a third ip address and sends a signal to the server acting as the primary server to change its ip address and become a secondary server . 
thus , if a primary server is turned on after a system crash or the switch damage has been repaired , we should not have a conflict of ip addresses in the network . for security reasons , both computers are in a security power area driven by an uninterruptible power supply ( ups ) with controlled shutdown after 3 min of power down fault . discussion the system developed demonstrated that the availability of free and open - source software that is stable and well tested r . 
in our case , for example , the choice of operating system allowed economic savings with respect to a windows solution which , at the same stability level , usually requires an expensive server licence . 
moreover , the absence of the various viruses traditionally present on a windows platform allowed us to avoid the cost of the antivirus and the need to install the numerous windows patches . 
the advantages of this solution are not to be measured only in terms of savings but also in terms of quality : the daily presence of an in - house technician in the division increases his or her awareness of the divisions needs and refines his or her ability to produce a good analysis and develop more efficient solutions . the technical solution adopted , adequate for our aims , was evaluated concurrently with another possible routing technique that involves defining a single dicom node and searching for the right routing policy on a database using source information as a key . 
although more elegant , this method has some problems : it is more complex to implement , it allows only one possible routing for each source and is exposed to problems of concurrent access to a slow device ( the fact that image acquisition is a fast process if compared with database consultation , the system could be slowed , leading to time out )  . 
in particular , in our tests , the simultaneous transmission of images from several multislice ct scanners caused a crash of the mysqld daemon and , in a few cases , of the operating system . the architecture adopted , with several dicom nodes , was justified by its flexibility , simplicity and resulting stability . 
in fact , we think it is reasonable to suppose that the creation of many dicom nodes to be able to cope with many routing rules is not a good idea . 
for this reason , we believe to have achieved , with good margins , system stability with respect to this problem . termini di manualistica , evitando lacquisto di costose licenze per le librerie dicom . i costi nascosti dati dalladozione , in seno al reparto , di un informatico per la gestione dellinformation technology ( it ) hanno s rappresentato un costo fisso ingente ma comunque basso se valutato rispetto al costo giornaliero di quanto richiesto da aziende esterne . 
i vantaggi di questa soluzione non sono misurabili solo in quantit di denaro risparmiata ma anche in qualit di soluzione realizzata : la presenza fissa di un informatico in reparto , infatti , fa s che questi , essendo quotidianamente a contatto con il reparto , sia molto pi consapevole delle esigenze degli utenti e conseguentemente pi abile a condurre lanalisi e a fornire soluzioni efficienti . la soluzione tecnica utilizzata , pur essendo utile agli scopi preposti , stata valutata concorrentemente rispetto ad una tecnica alternativa di routing la quale prevedeva di definire un unico nodo dicom e ricavare il tipo di instradamento in funzione della diagnostica di provenienza . 
questo approccio alternativo , sicuramente pi elegante , avrebbe per sofferto di alcuni problemi : una maggiore complessit di implementazione , la possibilit per una sorgente di aver associato solo un tipo di instradamento per le immagini prodotte e lesposizione a problemi di accesso concorrente ad una risorsa lenta ( infatti dato che lacquisizione delle immagini pu essere molto veloce rispetto ai tempi di consultazione di un database il sistema , acquisita limmagine , troverebbe un collo di bottiglia su questultimo nella ricerca dellinstradamento opportuno )  . 
in particolare , il secondo problema , con carichi di lavoro particolarmente pesanti , nei nostri test ( pi tc multislice che spedivano in contemporanea ) , si tradotto in un crash del demone mysqld ed in alcuni casi in un crash del sistema operativo . in merito a questa osservazione , la scelta routing adottata che prevede pi nodi dicom , si giustificata sulla base del fatto che rappresenta una soluzione flessibile , leggera in termini di peso computazionale e semplice : da ci ne deriva una intrinseca maggiore stabilit . 
lunico elemento che potrebbe fungere da soglia decisionale sono i pesanti limiti dal punto di vista del numero di instradamenti possibili . ragionevole supporre che , nel caso in cui si debbano gestire molte sorgenti con esigenze di instradamento troppo differenti , la scelta di creare molti nodi dicom sul dicom router non sia percorribile . 
intuitivo aspettarsi che , oltre un certo numero di porte aperte , sopratutto quando il sistema sotto stress , si possano verificare dei dos dovuti al crash di qualche servizio . 
tuttavia , dati i nostri test , risultata una ottima stabilit anche per quindici porte aperte e , dato che per le esigenze del reparto ne sono apparse pi che sufficienti sei , riteniamo di essere con ottimi margini nella regione di stabilit del sistema rispetto a tale problema . conclusions conclusioni compared with a few years ago , in a technological scenario that is continuously growing in several directions , the need to operate with more than one provider creates a rispetto a pochi anni addietro , in uno scenario tecnologico in continua ed eterogenea crescita , la necessit di operare con pi aziende fornitrici crea una serie di problemi di inte1258 r . 
by using them , technical operators with no great experience are able to implement valuable technical solutions for small to medium informatization problems , which would otherwise remain unsolved except at considerable expense . 
on the basis of these two concepts , this project not only solved a specific technical problem , it also gave internal personnel hands - on experience of the possibilities that open - source technologies combined with internal know - how can offer , compared with the costly traditional solutions provided by the major it companies . grazione per cui spesso diventa economicamente accattivante lidea di acquisire in seno al reparto il know how per dialogare in maniera pi precisa e disambigua con i fornitori di grandi soluzioni informatiche . 
cos facendo personale tecnico non eccessivamente specializzato pu implementare valide soluzioni tecniche per i piccoli / medi problemi di informatizzazione che , altrimenti , resterebbero insormontabili o economicamente molto impegnativi . 
in ct - guided transthoracic needle biopsy , the final diagnosis and lesion size affect diagnostic accuracy : benign lung lesions and lesions smaller than 1.5 cm or larger than 5.0 cm in diameter provide lower diagnostic yield . key words lung biopsy computed tomography ( ct ) guidance accuracy riassunto obiettivo . 
i valori di sensibilit , specificit , valore predittivo positivo , valore predittivo negativo ed accuratezza , riferiti ad una diagnosi di malignit , sono risultati rispettivamente del 90 , 2% , 99 , 0% , 99 , 8% , 67 , 3% e 91 , 7% . 
la diagnosi finale ( benignit versus malignit ) e le dimensioni della lesione influenzano laccuratezza diagnostica della metodica : addensamenti polmonari di natura benigna e lesioni con diametro < 1 , 5 cm o > 5 cm sono caratterizzati da livelli minori di accuratezza diagnostica . parole chiave polmone biopsia tomografia vomputerizzata ( tc ) guida accuratezza diagnostica 1142 a.m. 
computed tomography ( ct ) - guided needle lung biopsy was first reported by haaga and alfidi in 1976 [ 5 ] , and since then , the techniques excellent efficacy and accuracy and acceptable morbidity and mortality rates ( 0.07% , or one death every 1 , 429 procedures ) have been widely demonstrated [ 616 ]  . 
whether carried out with fine - needle - aspiration cytology ( fnac ) or core biopsy ( cb ) , the biopsy provides adequate material for cytological or histological examination and , if necessary , for microbiological evaluation [ 1113 , 1721 ]  . 
cb is more sensitive in defining benign lesions and in characterising diffuse or lymphoproliferative diseases of the lung , where the assessment of the tissue architecture is instrumental to the diagnosis ( accuracy > 80% for cb and 50%70% for fnac ) [ 9 , 2225 ]  . 
lesions smaller than 1 cm are easier to sample with fnac [ 19 , 22 , 23 ]  . the reported sensitivity of fnac varies and is positively affected by the presence of the cytopathologist on site during the biopsy procedure [ 26 , 27 ]  . 
immediate cytological assessment increases the techniques sensitivity and reduces the number of inadequate samples and false negative results [ 2628 ] , as the on - site cytopathologist can judge the adequacy , quantity and quality of the sample , suggest the need to repeat the procedure or request cb with histological assessment [ 22 , 26 ]  . several studies have investigated the factors affecting diagnostic accuracy of ct - guided transthoracic needle biopsies . 
it is well established that the most important factor is the final diagnosis , as malignant lesions ( regardless of histological type ) are characterised by greater sensitivity compared with benign masses . 
other variables ( lesion depth , number of passes , cavitations and necrotic centre , morphology , location , radiologists experience , needle diameter and presence of pneumothorax and / or bleeding ) were not found to be significant [ 32 ]  . the purpose of this paper is to present our experience with a large cohort of patients and discuss the results achieved in relation to diagnostic accuracy . 
in particular , we extensively analyse a number of variables ( relating to the patient , the lesion or the procedure ) in an attempt to correlate them to potential influences on diagnostic accuracy and reach a conclusive judgement on the efficacy of the procedure . 
lagobiopsia polmonare tc guidata fu riportata in letteratura per la prima volta nel 1976 da haaga [ 5 ] e da allora numerosi autori ne hanno dimostrato lestrema efficacia ed accuratezza associate ad un accettabile tasso di morbidit e mortalit ( 0 , 07% , ovvero un decesso ogni 1429 manovre effettuate ) [ 616 ]  . 
il prelievo , eseguito tramite aspirazione con ago sottile ( fine needle aspiration citology , fnac ) o con ago tranciante ( core biopsy , cb ) , consente di ottenere , rispettivamente , materiale sufficiente per lesame citologico o istologico ed infine , se necessario , per una valutazione microbiologica [ 1113 , 1721 ]  . 
la cb ha maggiore sensibilit nella definizione delle lesioni benigne e nella caratterizzazione delle malattie polmonari diffuse o linfoproliferative , nelle quali la valutazione dellarchitettura tissutale importante per la diagnosi ( valori di accuratezza > 80% per la cb e tra il 50% ed il 70% per la fnac ) [ 9 , 2225 ]  . 
le lesioni di dimensioni inferiori al centimetro risultano essere pi facilmente campionabili mediante fnac [ 19 , 22 , 23 ]  . la sensibilit della fnac variabile nelle casistiche riportate in letteratura ed positivamente influenzata dalla presenza in sala radiologica del citopatologo al momento del prelievo [ 26 , 27 ]  . 
il citopatologo , infatti , in grado di dare indicazione sulladeguatezza del prelievo , sulla quantit e qualit del materiale campionato , pu suggerirne leventuale ripetizione o richiedere lintegrazione con lesame istologico dopo cb [ 22 , 26 ]  . alcuni autori hanno analizzato le variabili che influenzano laccuratezza diagnostica delle agobiopsie transtoraciche tc guidate . 
da tempo assodato come il pi importante fattore in questo senso sia la diagnosi finale corretta , dato che le lesioni maligne ( indipendentemente dallistotipo ) sono caratterizzate da livelli di sensibilit significativamente maggiori rispetto agli espansi di origine benigna . 
considerando altre variabili , tsukada stato il primo a dimostrare come la graduale riduzione delle dimensioni della lesione conducesse ad un proporzionale decremento nei valori di accuratezza diagnostica [ 13 ]  . 
i risultati di tsukada sono per stati successivamente confermati da yeow che , in una coorte di 649 soggetti , ha riscontrato come sia la diagnosi finale sia le dimensioni della lesione influenzassero il valore di accuratezza [ 20 ]  . 
the study population comprised 154 subjects who underwent surgical resection ( for whom definitive postoperative histological assessment was available ) and nonsurgical patients who had completed at least 12 months of follow - up ( 454 subjects )  . 
the population included 402 men and 206 women , with a mean age of 65.7 years [ ( median 67 ; standard deviation ( sd ) 9.4 years with 95% confidence interval ( ci ) ; range 2987 years )  . 
exclusion criteria were lesions < 5 mm in diameter ( calculated as the mean of the long and short axis ) , significant changes in blood - coagulation profile , contralateral pneumonectomy , patients inability to maintain a lying position and / or to follow verbal or visual instructions and patients refusal to accept the potential risks associated with the procedure and sign the informed consent form . all biopsies were performed according to a standard protocol by the same interventional radiologist ( ac ) under ct guidance with a spiral dual - detector - row scanner . 
after positioning the patient on the ct table in prone , supine or lateral decubitus , depending on lesion location , an initial localisation scan was obtained by acquiring the entire mass on 5mm - thick contiguous transverse sections . 
then , optimal access ( skin entry site and needle path ) was defined on ct images , taking care to sample the lesion away from low - density areas , which are often central and indicative of necrosis [ 13 , 17 , 18 ]  . 
after having established the entry site and chosen the type and length of the needle ( depending on lesion depth and , whenever possible , the shortest route [ 33 ] ) , the needle entry site was anaesthetised ( 2% lidocaine ) , the needle , or the needle guide in the case of cb , positioned and the patient instructed to hold his or her breath . at this point ct scans were obtained to visualise the needle tip in relation to the lesion . 
in particolare verranno estensivamente analizzate alcune variabili ( riferite al paziente , alla lesione od alla procedura ) nel tentativo di correlarle a potenziali influenze sui valori di accuratezza per formulare un giudizio critico conclusivo sulla efficacia della procedura interventistica . materiali e metodi popolazione di studio in un intervallo temporale compreso tra novembre 2002 ed agosto 2005 sono stati consecutivamente sottoposti a procedura interventistica transtoracica tc guidata per laccertamento della diagnosi ( agoaspirazione transtoracica con ago sottile o con ago spinale o biopsia con ago tranciante ) 608 pazienti , per un totale di 612 procedure . 
la coorte in studio ai fini della formulazione di un giudizio di accuratezza della metodica ha compreso tutti i 154 soggetti resecati ( per i quali risultata disponibile la valutazione istologica definitiva postoperatoria ) ed i pazienti non chirurgici che avessero conseguito un follow - up non inferiore a 12 mesi ( 454 soggetti )  . la popolazione ha pertanto incluso 402 soggetti di sesso maschile e 206 femminile , con unet media di 65 , 7 anni ( mediana 67 anni ; ds9 , 4 anni con ic 95% ; intervallo 2987 anni )  . 
i pazienti sono afferiti in parte con proposta di prestazione ambulatoriale ed in parte in regime di ricovero ordinario . sono state considerate tutte le lesioni intraparenchimali ( centrali e periferiche ) , ovvero gli espansi circondati da tessuto polmonare areato o a diretto contatto con le strutture ilari e / o la superficie pleurica il cui epicentro cadesse allinterno del polmone ( dimensione media delle lesioni 36 , 8 mm ; mediana 31 , 5 mm ; ds20 , 5 mm con ic 95% ; intervallo 7103 mm )  . protocollo bioptico nessun paziente risultato anamnesticamente positivo per diatesi emorragica o ha presentato alterazioni della crasi ematica e del profilo emocoagulativo . 
i soggetti in trattamento anti - aggregante e / o anticoagulante hanno sospeso tale terapia per un periodo antecedente la biopsia di 7 giorni . sono stati raccolti dal radiologo un breve raccordo anamnestico ed il consenso informato . 
al momento di ogni biopsia , risultata disponibile una precedente tc diagnostica . i criteri di esclusione da noi adottati sono stati : lesioni con diametro ( calcolato sulla media delle dimensioni degli assi maggiore e minore ) < 5 mm ; alterazioni significative dellasa.m. 
biopsies were carried out by needle aspiration with a 21 - gauge fine needle or a 20gauge spinal needle ( with the single needle technique ) , or by core biopsy with an 18 - gauge cutting needle ( and coaxial technique )  . our initial approach in pulmonary nodules always uses a chiba fine needle ( especially for small lesions ) , except in cases of a second attempt after a previous inconclusive procedure . 
this made it possible to have an evaluation of material adequacy and an extemporary general indication about the nature of the lesion within minutes of the procedure [ 2628 ]  . 
in the case of inadequate or insufficient samples for cytology or histology , the biopsy procedure was repeated immediately : in particular , 190 / 612 procedures ( 31.0% ) required more than one sampling ( mean 1.32 samples per procedure ; range 14 )  . to assess possible complications ( pneumothorax , needletrack bleeding ) , postprocedural ct scans were acquired 35 min after the procedure ( time required for the cytopathologist to prepare the slides and judge their adequacy )  . data collection : definition and selection of variables multiple discrete or continuous variables , relating to the patient , the lesion or the procedure , were considered . 
dopo aver posizionato il paziente sul lettino tc in decubito prono , supino o laterale a seconda della localizzazione della lesione , si proceduto ad una prima centratura della lesione attraverso lacquisizione dellintero espanso su sezioni trasverse contigue di 5 mm di spessore . 
successivamente , sulle immagini tc a monitor si pianificata la migliore via di accesso ( come punto di ingresso cutaneo e tragitto dellago ) al fine di campionare la lesione avendo cura di evitare regioni di ipodensit , spesso centrali ed indicative di necrosi , nel suo contesto [ 13 , 17 , 18 ]  . 
dopo aver definito il punto di ingresso dellago e scelto la tipologia e la lunghezza dellago ( a seconda della profondit della lesione e pianificando , ogni qual volta sia stato possibile , il tragitto pi breve [ 33 ] ) si effettuata unanestesia locale in corrispondenza del punto cutaneo di ingresso ( lidocaina 2% ) , quindi si posizionato lago , o lago guida per le biopsie trancianti , chiedendo al paziente di mantenere lapnea . 
per gli espansi di difficile centratura ( 162 / 612 procedure , 26 , 5% del totale ) quali piccoli noduli centrali o integralmente coperti da strutture ossee , lago bioptico stato introdotto nei tessuti molli sino alla superficie pleurica evitandone accuratamente la trasgressione . 
in alcuni casi si effettuato pi di un passaggio pleurico , dal momento che o la lesione - bersaglio non stata raggiunta dalla punta dellago al primo tentativo nonostante i riorientamenti intraparenchimali dellago o , nei prelievi senza ausilio della tecnica coassiale , il materiale risultato inadeguato e si ricorsi ad un nuovo prelievo . 
in particolare , 350 / 612 procedure ( 57 , 2% ) hanno richiesto un solo passaggio pleurico ( media 1.5 passaggi pleurici per procedura ; intervallo 14 )  . 
il prelievo stato effettuato tramite agoaspirazione con ago sottile 21 gauge ( g ) o con ago spinale 20 g ( con tecnica ad ago singolo ) , o biopsia con ago tranciante 18 g ( utilizzando la tecnica coassiale )  . 
 per i noduli polmonari sempre preferito da noi un iniziale approccio con ago sottile di chiba ( soprattutto nelle lesioni di piccole dimensioni ) , tranne nei casi di un secondo tentativo dopo un precedente prelievo non conclusivo . nellevenienza della ripetizione iterativa viene considerata la possibilit di utilizzare un ago di calibro maggiore ( spinale ) od un tranciante . 
final diagnoses were based on the surgical outcomes ( surgical patients ) or the imaging data and clinical - radiological follow - up for a period of at least 12 months ( response to first - line chemotherapy or antibiotic treatment designed on the basis of the biopsy ; unchanged imaging findings in the absence of treatment )  . 
in relation to malignant diagnoses , we considered : true positives for malignancy : all cases classified as malignant at transthoracic needle biopsy and confirmed by histology of the surgical specimen ; cytohistology of an additional biopsy from a distant metastatic lesion ; imaging evidence ( radiology or nuclear medicine ) of distant metastases ; partial or complete response to first - line chemotherapy ; clinical course . 
in surgical cases , we assessed the reliability of the needle biopsy in providing precise characterisation with specific histological type a negative finding ( benign lesion ) was classified as a true negative for malignancy at fnac or cb when its benign nature was confirmed by subsequent surgery or biopsy or when the lesion appeared stable , smaller or had totally disappeared at follow - up ct ( 12 - month follow - up ) in an untreated patient or a patient treated with appropriate antibiotic , but not antineoplastic , therapy [ 18 , 22 ] false positives for malignancy : all cases with a positive biopsy not confirmed by surgery false negatives for malignancy : all cases with a negative ( benign at fnac / cb ) or indeterminate result ( absence of cellular atypia , presence of atypical cells not otherwise specified , presence of nonspecific or granulomatous inflammatory reaction , insufficient material or blood contamination ) at transthoracic needle biopsy with positive surgical outcome or disease progression at follow - up imaging . the quality of each sample obtained by fine - needle transthoracic biopsy was scored from 0 to 3 , according to the following criteria : 0 = slides containing exclusively blood components ( blood contamination ) without epithelial cells ; 1 = finding of nonspecific benign cells ( inflammatory ; type i and ii pneumocytes ; atypical cells not otherwise specified ) ; 2 = presence of morphologically malignant cells , without characterisation of histological type or without differentiation between small - cell or non - small - cell cancer ; 3 = slides containing benign or malignant cells with definite histological characterisation . 
samples with scores 0 , 1 and 2 ( when justified by inability to differentiate between small - cell and non - small - cell cancer ) were considered nondiagnostic and therefore classed as false negatives in relation to the final diagnosis . 
samples with scores of 2 and 3 were considered adequate . size 1146 the dimensions of each lesion were measured on screen using a dedicated programme ( electronic callipers ) , in the two maximum orthogonal axial diameters , on the preliminary localisation scan reconstructed from the raw data with adequate window and level settings for lungs . 
in tutti i casi stato utilizzato una lunghezza dello scatto della pistola bioptica pari a 3 , 3 cm . la presenza del citopatologo in sala radiologica , essenziale nei prelievi citoaspirativi , ha costituito elemento caratterizzante tutte le procedure . 
in caso di materiale inadeguato od insufficiente ad unanalisi citologica od istologica si provveduto alla immediata ripetizione del prelievo : in particolare 190 / 612 procedure ( 31 , 0% ) hanno richiesto pi di un prelievo ( media 1 , 32 campioni per procedura con intervallo 14 )  . per valutare linsorgenza di complicanze ( pneumotorace , soffusione emorragica lungo il tragitto dellago ) sono state acquisite , circa 35 minuti dopo il prelievo ( nel tempo richiesto dal citopatologo per preparare gli strisci e dare giudizio di idoneit ) , scansioni tc di controllo . raccolta dei dati : definizione e selezione delle variabili sono state considerate molteplici variabili , a valore discreto o continuo , riferite al paziente , alla lesione od alla stessa manovra interventistica . 
per entrambe le categorie sono stati calcolati , attraverso la costruzione di tabelle 22 , i livelli di accuratezza ( sensibilit , specificit , valore predittivo positivo e negativo , proporzione di falsi positivi e negativi , accuratezza globale della metodica ) correlando il risultato della biopsia transtoracica con la diagnosi finale . 
la diagnosi definitiva stata ottenuta attraverso lanalisi del dato chirurgico ( per i pazienti resecati ) , dei dati di imaging e del follow - up clinico / radiologico per un periodo non inferiore a 12 mesi ( risposta a trattamenti chemioterapici di i linea o a trattamenti antibiotici impostati sulla base del risultato bioptico transtoracico ; stazionariet radiologica dei reperti in assenza di terapia )  . 
a questo proposito sono stati considerati , in rapporto alle diagnosi maligne : veri positivi per malignit i casi classificati maligni allagobiopsia transtoracica e confermati : dal dato istologico sul pezzo operatorio ; dal dato cito - istologico derivante da una ulteriore aggressione bioptica di una lesione metastatica a distanza ; dallevidenza strumentale ( indagini radiologiche e di medicina nucleare ) di metastasi a distanza ; dalla risposta parziale o completa a trattamenti chemioterapici di i linea ; dal decorso clinico . 
nei casi resecati stata valutata laffidabilit dellagobiopsia transtoracica nel fornire una precisa caratterizzazione con specificit di istotipo ; un esito negativo ( ovvero di natura benigna ) stato classificato vero negativo per malignit alla fnac o cb in caso di successiva conferma chirurgica o bioptica o se a.m. 
we recorded data on the presence of single or multiple pulmonary lesions and lobar location specifying possible contact or close proximity to an interlobar fissure , the hilum or major intrapulmonary vessels . 
the depth of the consolidation was defined as the distance between the entry site on the pleural surface and the proximal edge of the lesion , measured along the needle path on lung window settings [ 13 , 32 , 3436 ]  . 
i prelievi con punteggio 0 , 1 e 2 ( se per questi ultimi non stata possibile una differenziazione tra neoplasia a piccole cellule e non ) sono stati considerati non diagnostici e quindi classificati come falsi negativi in rispetto alla diagnosi finale . 
i prelievi con punteggio 2 e 3 sono stati invece considerati adeguati . needle type and calibre dimensioni in all procedures , we used either a single needle ( 21 - gauge chiba needle ; 20 - gauge spinal needle ; 18 - gauge cutting needle with coaxial technique ) or , in the event of multiple biopsies , a consecutive combination of different needles . 
the choice of the type of needle was based on parameters such as lesion size , clinical suspicion ( history , clinical and imaging data ) , operators preferences and the presence of the cytopathologist . 
during each anale dimensioni di ogni lesione sono state misurate a monitor mediante programma dedicato ( calibro elettronico ) , nei due diametri assiali ortogonali massimi , sulle scansioni di centratura preliminari ricostruite dai dati grezzi attraverso adeguata finestra e livello per parenchima polmonare . 
stata quindi calcolata la media aritmetica relativa alle misurazioni ottenute per ciascuna lesione in esame . morfologia e localizzazione le lesioni sono state suddivise in noduli e masse , se caratterizzate da un diametro trasverso massimo < o 30 mm rispettivamente . 
sono state considerate consolidazioni gli infiltrati cuneiformi , di natura fluida o cellulare , a distribuzione segmentaria o lobare , associati o meno a broncogramma aereo . sono stati registrati i dati relativi alla presenza di lesioni polmonari singole o multiple , la localizzazione lobare specificando leventuale contatto o la stretta vicinanza ad una scissura interlobare , allilo o a strutture vascolari intrapolmonari di rilievo . 
la profondit delladdensamento stata considerata quale distanza tra il punto di ingresso della superficie pleurica sino al margine prossimale della lesione , misurata lungo il tragitto dellago su finestra per parenchima polmonare [ 13 , 32 , 3436 ]  . 
the variables of the two groups in each category were correlated . differences between the groups were examined using the nonparametric mann - whitney u test or variance analysis according to kruskal - wallis , when indicated . 
differences between proportions were analysed with pearsons 2 test or students t test . parameters considered significant in determining diagnostic accuracy were further stratified and correlated to accuracy with the aid of contingency graphs and tables . 
a p value < 0.05 was taken as significant in all cases . results in calculating the methods diagnostic accuracy , we considered all 608 patients included in the study : there were 154 surgical patients ( for whom postoperative surgical evaluation was considered the gold standard against which the interventional technique was compared ) there were 454 nonsurgical patients who had completed , as at august 2006 , an imaging , clinical and treatment - response follow - up of at least 12 months ; in these cases the final diagnosis was inferred from the benign or malignant behaviour of the lesion we considered 612 procedures ; the quality of each of the 612 samples was assigned a discrete score from 0 to 3 at the end of each procedure in 435 / 612 procedures ( 71.1% ) , the needle biopsy recovered benign or malignant cells , allowing specific cytohistological characterisation ( score 3 )  . 
of these , 52 were benign , 382 were malignant and only one revealed the presence of a hamartochondroma associated with a primary squamous cell carcinoma of the lung there were 75 / 612 samples ( 12.2% ) that demonstrated definitely malignant cells ( score 2 ) , without providing information on histological type . 
four samples demonstrated the presence of definitely neoplastic cells ( probably epithelial ) without providing discrimination between small - cell and non - small - cell cancer , which is essential for therapy . 
in addition , the only false positive result in our cohort was encountered among these four cases : a suspected carcinoma not otherwise specified ( fnac ) was found at surgery to be an excavated granuloma characterised by severe cellular dysplasia due to previous tuberculous infection in 46 / 612 cases ( 7.5% ) , the sample was scored 1 , as 19 / 46 samples ( 41.3% ) demonstrated exclusively normal lung 1148 mento lineare di parenchima polmonare trasgredito dallago . caratteristiche interne sono state considerate laspetto solido ( se con finestra mediastinica risultava visualizzabile almeno il 50% della superficie dellespanso rispetto a quanto visibile con finestra polmonare ) , la presenza di escavazioni ( aree allinterno della lesione con valori di attenuazione propri dellaria ) , calcificazioni e broncogramma aereo . 
 stata inoltre valutata , con criterio visivo ed attraverso lausilio della misurazione digitale dei dati di attenuazione mediante roi , la densit basale della lesione per ricercare eventuali aree di disomogeneit riferibili a necrosi . tipo di ago e calibro per tutte le procedure si ricorso alluso esclusivo di un unico ago ( chiba 21 g ; spinale 20 g ; tranciante 18 g con tecnica coassiale ) o ad associazione consecutiva di diversi aghi nel caso di prelievi multipli . 
la scelta di tipologia dellago stata posta secondo alcuni parametri quali le dimensioni della lesione , il sospetto clinico ( su basi anamnestiche , obiettive e di imaging ) e le preferenze delloperatore , oltre che la presenza dellanatomopatologo in sala radiologica . 
in particolare , lago sottile di chiba ha trovato esclusivo impiego in 470 / 612 procedure ( 76 , 8% ) , lago spinale in 76 ( 12 , 4% ) , il tranciante in 35 ( 5 , 7% ) , lassociazione chibaspinale in 27 ( 4 , 4% ) e lassociazione chiba - spinale - tranciante in 4 ( 0 , 7% )  . complicanze analisi statistica la comparsa di pneumotorace o di sfumate opacit parenchimali con aspetto radiologico a vetro smerigliato ( da impegno alveolare per emorragia ) stata registrata per ogni procedura . lanalisi statistica stata condotta con lutilizzo del programma statistica 6.1 ( statsoft software , per windows ) e mirata alla valutazione dellaccuratezza della procedura . 
i pazienti sono stati , in ogni sessione analitica , suddivisi in due gruppi omogenei per variabili discrete a soluzione dicotomica ( si / no ) ed analizzati per le diverse variabili , discrete o continue , che si ipotizzava influenzare i livelli di accuratezza : i gruppi sono stati suddivisi tra le diagnosi corrette e le diagnosi non corrette o non possibili a causa dellinadeguatezza del prelievo agobioptico . 
le differenze tra i gruppi sono state esaminate utilizzando i tests non parametrici mann - whitney u - test o analisi della varianza secondo kruskal - wallis , quando indicati . 
le differenze tra le proporzioni sono state analizzate con il test 2 di pearson od il test t di student . i parametri considerati significativi , nel determinare i livelli di accuratezza diagnostica , sono stati ulteriormente stratificati e correlati allaccuratezza con lausilio di grafici a.m. 
the discordance between the biopsy result and the final diagnosis is due to sampling errors , with sampling of surrounding healthy lung parenchyma or perilesional reactive inflammatory tissue there were 56 / 612 samples ( 9.1% ) scored 0 owing to sample inadequacy ( blood contamination ) and which were therefore classed as nondiagnostic . 
in 35 / 56 samples ( 60.7% of samples with a score of 0 ) , the lesion was subsequently found to be malignant . a final diagnosis based on the criteria previously described could be obtained for all 612 procedures : 17.0% ( 104 / 612 ) of lesions were found to be benign , whereas 83.0% ( 508 / 612 ) were malignant . 
the 458 cases positive for malignancy , with subsequent confirmation ( one malignant case at biopsy was subsequently found to be a false positive ) comprised 411 primary lung cancers , seven nonepithelial neoplasms and 40 metastatic tumours with secondary localisation to the lung ( table 1 )  . of these 458 patients , 46 ( 7.6% ) had a history of neoplastic disease at another location . 
of these 46 , the metastatic origin of the lung lesion was demonstrated in 40 , whereas in the remaining six cases , the primary nature of the lung nodule was demonstrated with the aid of immunocytochemistry and comparison with cytology and / or histology of the extrapulmonary neoplasm , when available . among the 459 cases of malignancy diagnosed at biopsy , o tabelle di contingenza . 
in tutti i casi stato considerato significativo un valore di p < 0 , 05 . risultati per calcolare laccuratezza diagnostica della metodica sono stati inclusi nello studio 608 pazienti : centocinquantaquattro sono i soggetti sottoposti ad intervento chirurgico ( per i quali disponibile lesame istologico post - operatorio , considerato il gold standard di raffronto con la metodica interventistica )  . quattrocentocinquantaquattro sono , invece , i pazienti non chirurgici che presentano ad agosto 2006 un followup di imaging , clinico e di risposta terapeutica non inferiore a 12 mesi ; in base al comportamento benigno o maligno della lesione in esame stata , quindi , dedotta la diagnosi finale . complessivamente sono state considerate 612 procedure . per ciascuno dei 612 campioni ottenuti , a conclusione di ogni procedura stato attribuito un punteggio discreto da 0 a 3 per asserirne la qualit : in 435 / 612 procedure ( 71 , 1% dei casi ) si sono ottenute , dal prelievo agobioptico , cellule benigne o maligne che hanno permesso una precisa caratterizzazione citoistologica della lesione ( punteggio 3 )  . 
di queste 52 sono risultate benigne , 382 di natura maligna , mentre in 1 solo caso si registrata la presenza di un amartocondroma associato ad un carcinoma polmonare primitivo del tipo squamocellulare ; in 75 / 612 prelievi ( 12 , 2% dei casi ) sono state riscontrate cellule di sicura origine maligna ( punteggio 2 ) , ma non stato possibile trarre conclusioni sullistotipo . 
in contrast , 50 procedures were false negative for malignancy ( negative result at biopsy , with malignant cells detected at repeat biopsy , surgery or follow - up )  . 
table 3 summarises the cytological diagnoses of the 52 cases that were negative for malignancy at biopsy . if we consider only benign lesions at final diagnosis assenza di una suddivisione , critica ai fini terapeutici , in tumore a piccole cellule e non . 
questi ultimi casi pongono importanti problematiche in termini di gestione del paziente per il fatto che non si pu pianificare il corretto iter terapeutico , impedendo in questo modo allagobiopsia di porsi a termine delliter diagnostico . 
inoltre , tra questi 4 casi si riscontrato lunico risultato bioptico falso positivo dellintera coorte : un sospetto carcinoma di non pi precisa caratterizzazione ( fnac ) si poi dimostrato essere , dopo resezione chirurgica , una granulomatosi escaa final diagnoses are based on the retrospective analysis of the outcome of surgery , repeated biopsy , imaging , and clinical follow - up for at least 12 months . 
 malignant pulmonary lesions : sensitivity 90.2% ( 458 / 508 ) , specificity 99.0% ( 103 / 104 ) , positive predictive value 99.8% ( 458 / 459 ) , negative predictive value 67.3% ( 103 / 153 ) , false positive rate 0.22% ( 1 / 459 ) , false negative rate 10.9% ( 50 / 508 ) , accuracy 91.7% [ ( 458 + 103 ) / 612 ] , overall diagnostic accuracy 83.3% [ ( 458 + 52 ) / 612 ] ( for all lesions , benign and malignant ) table 2 results of 612 computed - tomography - guided transthoracic needle biopsies final diagnosisa malignant biopsy diagnosis malignant benign / inadequate total diagnosi bioptica maligno benigno - inadeguato totale 1150 tabella 2 risultati di 612 procedure agobioptiche polmonari diagnosi finalea maligno benign 103 ( 52 / 51 ) benigno 103 ( 52 / 51 ) total totale a la diagnosi finale deriva dallanalisi retrospettiva dei risultati della chirurgia , di uneventuale successiva agobiopsia transtoracica e dallesito del followup clinico / radiologico pari ad un periodo non inferiore ai 12 mesi . 
conversely , in 51 cases , the benign nature of the lesion could not be defined because of sample inadequacy ( 30 samples scored 1 and 21 scored 0 )  . with regard to malignant lesions , 99.8% of malignancies at vata caratterizzata da severa displasia cellulare in relazione alla pregressa infezione tubercolare ; in 46 / 612 casi ( 7 , 5% ) stato attribuito , al campione prelevato , un punteggio pari a 1 , dal momento che in 19 / 46 casi ( 41 , 3% ) si sono riscontrate esclusivamente cellule normali proprie del tessuto polmonare ( pneumociti di i e ii ordine ) , in 14 / 46 ( 30 , 4% ) cellule flogistiche in assenza di significative atipie ed in 13 / 46 ( 28 , 3% ) cellule atipiche non ulteriormente caratterizzabili , ma neanche classificabili come sicuramente maligne . 
in 16 di questi 46 prelievi ( 34 , 7% dei prelievi a punteggio 1 ; 2 , 6% sul totale delle procedure ) le lesioni campionate sono poi risultate di natura maligna . 
la discordanza tra risultato bioptico e diagnosi finale dovuta ad errori di campionamento , per prelievo di parenchima polmonare sano limitrofo o di tessuto flogistico reattivo perilesionale ; infine 56 / 612 prelievi ( 9 , 1% ) hanno ottenuto un punteggio pari a zero a causa dellinadeguatezza del materiale analizzato dallanatomopatologo ( campioni ematici ) e sono quindi stati classificati come non diagnostici . 
in 35 / 56 prelievi ( 60 , 7% dei campioni a punteggio 0 ) la lesione poi risultata maligna . per tutte le 612 procedure stato possibile ottenere una diagnosi finale con i criteri gi ampiamente descritti : il 17 , 0% ( 104 / 612 ) delle lesioni risultato di natura benigna , mentre lo 83 , 0% ( 508 / 612 ) maligna . 
lagobiopsia transtoracica percutanea ha conseguito un risultato diagnostico in 511 casi su 612 ( 83 , 5% , prelievi a punteggio 2 e 3 ) , mentre in 101 procedure ( 16 , 5% , prelievi a punteggio 0 e 1 ) non stato possibile fornire un giudizio affidabile . 
i 458 casi positivi per malignit , con conferma successiva ( un caso maligno alla biopsia poi risultato un falso positivo ) hanno compreso 411 carcinomi polmonari primitivi , 7 neoplasie non epiteliali e 40 tumori metastatici con localizzazione secondaria al polmone ( tabella 1 )  . di questi 458 pazienti , 46 ( 7 , 6% ) presentavano un precedente anamnestico di malattia neoplastica in altra sede . 
tra questi ultimi , in 40 si dimostrata lorigine metastatica della lesione polmonare , mentre nei restanti 6 casi stato possibile definire la natura primitiva del nodo polmonare attraverso lausilio della immunocitochimica ed il confronto , quando disponibile , con i reperti citologici e / o istologici del tumore extrapolmonare . tra i 459 riscontri risultati maligni allagobiopsia , solo in un caso la diagnosi definitiva non ha confermato il dato configurando quindi la presenza di un unico falso positivo per malignit . 
si sono invece registrate 50 procedure false negative per malignit ( risultato negativo per cellule neoplastiche con esito maligno alla ripetizione della procedura , allintervento chirurgico od al follow - up )  . 
in tabella 2 vengono riportati i risultati conseguiti , in termini di affidabilit della metodica , sulla corretta diagnosi delle lesioni maligne . la tabella 3 riassume le diagnosi citologiche nei 52 casi risultati negativi per malignit allagobiopsia . analizzando le sole lesioni benigne alla diagnosi finale ( 104 / 612 , 17% ) , lagobiopsia ha permesso di conseguire un sicuro giudizio di benignit con diagnosi specifica in soli 52 1151 a.m. 
a specific judgement of benignity of the biopsy sample could , however , be made in only 34% of samples , characterised by the definite absence of neoplastic cells ( 52 / 153 ) ( table 2 )  . 
finally , table 4 demonstrates that lesion depth ( the portion of aerated lung traversed by the needle ) , morphology and lobar location ; the presence of calcification , cavitation and necrotic centre ; and the occurrence of complications ( pneumothorax and bleeding ) have no influence on the diagnostic accuracy of transthoracic ct - guided needle biopsy . discussion with the use of percutaneous transthoracic needle biopsy , and in particular of fnac , as an initial minimally invasive procedure for evaluating the nature of pulmonary lesions , this study obtained an overall diagnostic accuracy of 84% , with levels of 92% and 67% for malignant and benign lesions , respectively . 
in 51 lesioni benigne non invece stato possibile stabilirne la natura a causa dellinadeguatezza del prelievo ( 30 prelievi a punteggio 1 e 21 prelievi a punteggio 0 )  . riferendosi agli addensamenti polmonari di natura maligna , il 99 , 8% delle lesioni considerate maligne allagobiopsia poi stata confermata ( 458 / 459 , valore predittivo positivo per malignit ) , mentre il 67 , 3% dei prelievi con assenza di cellule tumorali ( prelievi classificati come benigni , od inadeguati ) risultato benigno ( 103 / 153 , valore predittivo negativo per malignit )  . 
stato comunque possibile dare un giudizio specifico di benignit del prelievo agobioptico solo nel 34% dei campioni , caratterizzati dalla sicura assenza di cellule tumorali ( 52 / 153 ) ( tabella 2 )  . 
la sensibilit e la specificit per la diagnosi di malignit sono risultate del 91% e 99% rispettivamente , con una proporzione di falsi positivi trascurabile ( 0 , 22% ) ed una proporzione di falsi negativi dell11% . 
questo valore raggiunge l88 , 2% ( 540 / 612 ) se , tra le diagnosi agobioptiche corrette , si includono i 30 prelievi a punteggio 1 poi risultati benigni ( ovvero reperti considerati allagobiopsia negativi per cellule neoplastiche in assenza di una specifica diagnosi )  . 
in tabella 4 riassunto linsieme di variabili che si supposto esercitare una influenza sui livelli di accuratezza della metodica : le uniche variabili associate con significativit statistica ad una riduzione dei livelli di accuratezza diagnostica sono risultate la discrimante della diagnosi finale ( categoria benigno versus maligno ) e le dimensioni della lesione . 
la tabella 4 dimostra , infine , come la profondit della lesione ( ovvero la quota di parenchima polmonare aerato attraversata dallago ) , la sua morfologia e localizzazione lobare , la presenza di calcificazioni , cavitazioni e centro necrotico , oltrech loccorrenza di complicanze ( pneumotorace e sanguinamento ) non esercitino alcuna a.m. 
anumber of procedures ; bpearsons 2 test for categoric variables ; caverage lesion diameter in two axial planes ; ns , not significant tabella 4 correlazione per variabili , con la diagnosi finale , di risultati corretti ed errati od indeterminati allagobiopsia transtoracica variabile na ( 612 ) diagnosi finale errata o indeterminata n = 102 corretta n = 510 52 ( 50 , 0 ) 458 ( 90 , 2 ) 30 ( 66 , 6 ) 362 ( 87 , 3 ) 118 ( 77 , 6 ) 52 ( 50 , 0 ) 50 ( 9 , 8 ) 15 ( 33 , 3 ) 53 ( 12 , 7 ) 34 ( 22 , 4 ) i valori in parentesi sono riferiti in % . 
agrandezza del campione ; btest 2 di pearson per variabili categoriche ; cconsiderata quale dimensione media tra i due assi ( maggiore e minore ) ortogonali della lesione ; ns , non significativo . 
however , they are also dependent on the severity with which sample adequacy is judged : a sample without neoplastic cells should not be considered negative for malignancy ( consider sampling errors , for example ) unless they provide a precise and specific diagnosis of benignity ( 100% specificity is desirable in benign lesions )  . 
many studies [ 13 , 29 , 32 , 37 , 38 ] have reported substantially lower levels of diagnostic accuracy for benign lung lesions ( range 52%91% ) compared with malignant lesions influenza sui valori di accuratezza diagnostica dellagobiopsia transtoracica tc guidata . discussione attraverso lutilizzo dellagobiospia transtoracica percutanea , ed in particolar modo della fnac , quale iniziale procedura mini - invasiva nella valutazione di natura degli addensamenti polmonari si ottenuta , in questo studio , unaccuratezza diagnostica globale dell84% con valori di accu1153 a.m. 
1a - c relationship between mean lesion size and number of observations for the entire group of lesions ( a ) and in regard to the presence ( b ) or the absence ( c ) of a necrotic centre . 
1a - c correlazione tra diametro medio della lesione e numero di casi per tutte le lesioni ( a ) e suddividendo queste ultime per presenza ( b ) od assenza ( c ) di centro necrotico . 
la sensibilit della metodica per lesioni maligne risultata del 90% ( con valori di specificit del 99% ) , mentre per lesioni benigne la sensibilit risultata del 50% , ma con specificit del 100% . 
questi dati dimostrano come , nellinquadramento diagnostico delle lesioni benigne , la fnac risulti inferiore alla cb [ 20 , 24 ] , ma paiono anche giustificati dalla severit di giudizio di idoneit cui vengono sottoposti i campioni : un prelievo con assenza di cellule neoplastiche non deve essere considerato negativo per patologia tumorale ( si considerino , ad esempio , gli errori di campionamento ) , a meno che sia possibile formulare una precisa e specifica diagnosi di benignit ( sono auspicabili valori di specificit per lesioni benigne pari al 100% )  . 
molteplici studi in letteratura [ 13 , 29 , 32 , 37 , 38 ] riportano valori di accuratezza diagnostica sensibilmente inferiori per lesioni polmonari benigne ( con intervallo compreso tra il 52% ed il 91% ) se paragonate a lesioni maligne ( intervallo compreso tra l86% ed il 100% ) , sia nel prelievo aspirativo sia tranciante ; inoltre , considerando esclusivamente gli addensamenti benigni , la cb presenta valori di accuratezza e sensibilit significativamente maggiori rispetto alla fnac ( accuratezza superiore all80% versus 50%70% ) [ 9 , 2225 ]  . 
i livelli di accuratezza e sensibilit riportati dal nostro studio sono in linea con i riscontri fnac della letteratura , ma non dimensioni lesione ( cm ) lesion size ( cm ) fig . 
 ctest t di student ( per campioni indipendenti e per gruppi ) , p < 0 , 05 considerata significativa : il diametro medio delle lesioni con ( 57 , 7 mm ) e senza ( 34 , 0 mm ) necrosi differisce con significativit statistica . raggiungono i valori riportati dalla cb , elemento comunque non contraddittorio dal momento che solo nel 6 , 4% delle procedure stato utilizzato un ago tranciante ( singolarmente od in associazione con altri aghi )  . la documentazione di cellule sicuramente maligne , nella pressoch totalit dei casi , prova certa della natura maligna dellespanso . 
nella nostra coorte lunico caso classificato come maligno ( neoplasia di origine epiteliale ) e poi risultato benigno ( granulomatosi escavata ) alla diagnosi finale trova esplicazione nel fatto che la lesione benigna ha sviluppato atipie cellulari tali da farla risultare sospetta allindagine citologica per espanso di natura maligna . nonostante gli insuccessi siano sensibilmente minori per le lesioni maligne , tuttavia si evidenziato un 11% di falsi negativi nellambito di tali addensamenti ( integralmente costituiti da prelievi a punteggio 01 )  . 
in aggiunta , la nostra esperienza indica come la sola ispezione ad occhio nudo del campione ottenuto ( in termini di integrit del frustolo bioptico o di cellularit o di carattere ematico del prelievo aspirativo ) inaffidabile nel predirne ladeguatezza [ 30 , 32 , 34 , 36 , 38 ] ed quindi auspicabile la presenza dellanatomopatologo nel corso della seduta bioptica . 
la ripetizione del prelievo dovrebbe essere presa in considerazione ogni qual volta si verifichi discordanza tra risultato bioptico , presentazione clinica e caratteristiche tc ( prima di passare ad accertamenti diagnostici invasivi di ii livello ) [ 13 ]  . lanalisi della nostra coorte ha evidenziato come le uniche variabili in grado di influenzare , in termini statisticamente significativi , laccuratezza diagnostica della metodica siano la diagnosi finale ( benignit versus malignit ) e le dimensioni della lesione ( nodi con diametro medio inferiore ad 1 , 5 cm o masse di dimensioni superiori a 5 cm sono caratterizzati da livelli di minore accuratezza diagnostica )  . 
riferendosi a questultimo aspetto , gli studi presenti in letteratura esprimono giudizi discordanti in merito allinfluenza delle dimensioni della lesione sui livelli di accuratezza dia1155 dimensioni lesione ( cm ) lesion size ( cm ) fig . 
3 relazione esistente tra dimensione della lesione e presenza di necrosi : il grafico dimostra come lincidenza di necrosi si incrementi gradualmente allaumentare delle dimensioni delle lesioni ; la maggior proporzione di necrosi si riscontrata nelle lesioni di 6 cm , ovvero quelle caratterizzate da minor accuratezza diagnostica . ( from 86% to 100% ) , achieved with both fnac and cb . 
accuracy and sensitivity levels found in our study are similar to those previously reported for fnac but fail to reach those reported for cb , a finding that is not contradictory given that only 6.4% of procedures were carried out with a cutting needle ( used alone or in combination with other needles )  . demonstration of definitely malignant cells is proof of the a.m. 
4 il grafico box & whisker dimostra una differenza significativa in termini di dimensioni nel gruppo di lesioni senza ( 3 , 4 cm1 , 8 ) e con necrosi centrale intralesionale ( 5 , 7 cm1 , 8 )  . 
il diametro medio delle lesioni in assenza e presenza di necrosi differisce significativamente ( p < 0 , 001 ) ( ds , deviazione standard ; n , no ; s , si )  . malignant nature of the mass in virtually all cases . 
in our cohort , the only case classified as malignant ( epithelial neoplasm ) at biopsy and found to be benign ( excavated granuloma ) at final diagnosis is explained by the fact that the lesion had developed cellular atypia that made it suspicious for malignancy at cytological assessment . although there were substantially fewer failures in malignant lesions , the false negative rate was 11% in this group ( all for samples with a score of 01 )  . 
in addition , our experience shows that gross inspection of the sample ( in terms of integrity of the biopsy specimen , cellularity or blood contamination of the aspirate ) is unreliable in predicting adequacy [ 30 , 32 , 34 , 36 , 38 ] , making the presence of a pathologist during the biopsy procedure strongly desirable . 
found that diagnostic accuracy declined progressively with decreasing lesion size : from 100% to 93% , 87% , 79% and 67% for lesions > 5 cm , 35 cm , 23 cm , 12 cm and < 1 cm , respectively [ 13 ]  . 
reported accuracy levels of approximately 88% for lesions > 2 cm when the mean number of needle passes was 2.5 , whereas when only a single specimen was obtained , accuracy decreased to 83% [ 29 ]  . the results of our study are similar to those reported by yeow et al . 
 [ 20 ] , who found , with a mean number of passes of three ( without a pathologist present ) , lower levels of diag1156 gnostica [ 13 , 20 , 30 , 36 , 37 ]  . 
tsukada ha riscontrato un graduale e progressivo decremento dei valori di accuratezza diagnostica man mano che le dimensioni delle lesioni si riducevano : dal 100% al 93% , 87% , 79% e 67% per lesioni rispettivamente > 5 cm , tra 3 e 5 cm , tra 2 e 3 cm , tra 1 e 2 cm e < 1 cm [ 13 ]  . 
lucidarme ha riscontrato valori di accuratezza di circa l88% per lesioni > 2 cm con un numero medio di prelievi per biopsia di 2.5 , mentre laccuratezza si riduceva all83% in caso di unico prelievo per seduta bioptica [ 29 ]  . nel nostro studio si sono ottenuti risultati simili a quelli descritti da yeow [ 20 ] che riportava , con una media di 3 prelievi bioptici a seduta ( in assenza di un medico anatomopatologo in sala ) , livelli inferiori di accuratezza diagnostica per lesioni < 1 , 5 cm e > 5 cm ( dell84% e 93% rispettivamente ) quando paragonati a lesioni con diametro compreso tra 1 , 5 e 5 cm ( 96% )  . 
nel nostro lavoro vengono , invece , riportati valori di accuratezza diagnostica globale ( per lesioni maligne e benigne ) del 68% per lesioni < 1 , 5 cm , del 78% per lesioni > 5 , 0 cm e dell87% per le lesioni comprese tra 1 , 5 e 5 , 0 cm . la maggior accuratezza diagnostica riportata da yeow giustificata dal fatto che in tutti i casi stata eseguita una cb con tecnica coassiale ( caratterizzata da livelli di accuratezza diagnostica maggiori rispetto alla fnac ) , mentre nel nostro studio solo il 6% delle manovre stato eseguito con tale procedura , riservando la fnac a tutti gli altri casi . 
in aggiunta , avendo la disponibilit del citopatologo in sala radiologica , risultato sensibilmente minore il numero di prelievi per seduta ( 1 , 3 prelievi versus 3 , 0 )  . 
il numero di prelievi , essendo variabile nei diversi studi , potrebbe costituire un determinante elemento confondente in grado di influenzare laccuratezza diagnostica al pari delle dimensioni della lesione [ 20 , 29 , 37 ]  . 
con una media di 1 , 3 prelievi per procedura , il nostro studio pone come limite dimensionale critico , al di sotto del quale si verifica una sensibile riduzione di accuratezza diagnostica , il valore di 1 , 5 cquesto risultato in linea con le a.m. 
is explained by the fact that they used cb with coaxial technique ( which is more accurate than fnac ) in all cases , whereas we used cb for only 6% of procedures and fnac for the remaining cases . 
the number of needle passes should , however , be kept to a minimum , as this variable is significantly related to higher rates of pneumothorax [ 32 ] ; the presence on site of a cytopathologist during the procedure is instrumental to establishing a correct diagnosis . the substantial reduction in diagnostic accuracy in small lesions could be due to sampling errors resulting from increased technical difficulty in the localisation phase [ 11 ]  . such difficulties are further supported by the higher rate of complications within this class of lesions : yeow et al . 
 [ 32 ] reported a 17 times higher risk of pneumothorax and a six times higher risk of bleeding for lesions smaller than 2 cm . in view of the lower diagnostic accuracy for small lung nodules ( whether sampled by fnac or cb ) and the higher complication rate , fine - needle aspiration should always be preferred to cb for lesions smaller than 1 cm , as suggested by laurent et al . 
when sampling these nodules , in fact , it is impossible to avoid inclusion of portions of healthy aerated lung tissue , as the biopsy core is 1.3cm long ( shortest throw - length setting ) [ 29 , 32 , 37 ]  . 
diffuse bleeding may also occur as a result of the surrounding healthy parenchyma not being able to provide an adequate tamponade effect [ 29 , 32 , 37 ]  . 
finally , in a nonnegligible number of cases , the material recovered proves inadequate due to the abundant presence of blood . despite the fact that throw lengths of 10 mm can be used to avoid bleeding in lesions smaller than 1.5 cm , boiselle et al . reported a diagnostic accuracy of 62% with this approach [ 39 ]  . 
comunque auspicabile conseguire il minimo numero di campionamenti possibili per procedura dal momento che tale variabile significativamente correlata ad una maggior proporzione di pneumotoraci [ 32 ] ; elemento indispensabile , a garanzia dellobiettivo di ottenere una corretta diagnosi , risulta essere la presenza del citopatologo durante la seduta bioptica . la sensibile riduzione di accuratezza diagnostica per lesioni di ridotte dimensioni potrebbe trovare una valida esplicazione in errori di campionamento , dovuti a maggiori difficolt tecniche nella fase di centratura bioptica [ 11 ]  . 
le difficolt procedurali trovano ulteriore conferma nella maggior proporzione di complicanze riscontrabile in questa categoria di lesioni : yeow [ 32 ] riporta , infatti , un rischio di pneumotorace 17 volte maggiore e di sanguinamento 6 volte superiore per lesioni di diametro inferiore ai 2 cin ragione della minor accuratezza diagnostica per i piccoli noduli polmonari ( che siano sottoposti a fnac o a cb ) ed alla pi alta percentuale di complicanze riscontrate , risulta sempre opportuno preferire alla cb , come gi suggerito da laurent e yeow [ 20 , 30 ] , il prelievo aspirativo con ago sottile per le lesioni di dimensione inferiore al centimetro . 
infatti , risulta impossibile evitare , nel campionamento di questi piccoli nodi , porzioni di tessuto aerato sano , dal momento che il frustolo bioptico ha lunghezza pari ad 1 , 3 cm ( limite inferiore della lunghezza dello scatto dellago tranciante ) [ 29 , 32 , 37 ]  . 
pu inoltre verificarsi un sanguinamento diffuso dal momento che il parenchima circostante sano , interessato anchesso dal campionamento , non in grado di fornire un adeguato effetto tampone [ 29 , 32 , 37 ]  . 
nonostante si possano utilizzare lunghezze di scatto pari a 10 mm per evitare il sanguinamento in lesioni inferiori ad 1 , 5 cm , boiselle ha riportato , in tali condizioni , unaccuratezza diagnostica pari al 62% [ 39 ]  . 
questo valore risulta inferiore al 67% da noi ottenuto utilizzando esclusivamente , per tali lesioni , lagoaspirato e con un tasso di sanguinamento perilesionale di minor entit . analizzando il parametro dimensionale , quale variabile determinante laccuratezza diagnostica della procedura , si sono riscontrati decrementi di accuratezza per lesioni con dimensione maggiore ai 5 cquesta evenienza viene compresa se si considera la presenza di necrosi centrale , che risulta di frequente riscontro nelle masse polmonari > 5 cm ( nella nostra casistica percentuale tc di necrosi tumorale per lesioni sino a 5 cm compresa tra lo 0% ed il 15% , per masse 5 cm tra il 12% ed il 41% )  . 
risulta quindi evidente come , nelle grosse masse polmonari , sia necessario disporre di indagini tc acquisite con infusione di mezzo di contrasto per individuare aree lesionali necrotiche al fine di evitarle durante il campionamento . 
inoltre , in assenza di un anatomopatologo in sala , nellevenienza allispezione visiva di frustoli particolarmente fragili e frammentati o di prelievi citologici ottenuti con agoaspirato , particolarmente ematici , si dovrebbero 1157 a.m. 
moreover , when no on - site pathologist is present and the biopsy cores appear particularly fragile and fragmented or the aspirate particularly bloody on gross inspection , further samples should be obtained taking care to direct the needle to different areas of the lesion [ 32 , 40 , 41 ]  . the presence of cavitations within a lung mass does not appear to influence the diagnostic accuracy of either fnac , as demonstrated in our study , or cb , as shown by yeow et al . 
similarly , diagnostic accuracy was not significantly affected by the presence of calcification or a frankly necrotic centre ; by lesion depth ( from the pleural surface ) , morphology or location ; or by the occurrence of complications such as pneumothorax and bleeding . 
no evaluation was made with regard to needle size or the radiologists experience , variables that are ascribed different significance in the various studies [ 20 , 41 ]  . conclusions in conclusion , transthoracic ct - guided needle biopsy of pulmonary lesions is a highly accurate procedure , especially in malignant lesions . 
however , to enhance the likelihood of success and achieve a correct diagnosis , awareness of the variables affecting its effectiveness is crucial : benign lesions , nodules smaller than 1.5 cm and masses larger than 5 cm are associated with substantially lower accuracy rates . 
analogamente , la presenza di calcificazioni o di centro francamente necrotico , od ancora la profondit della lesione ( rispetto al piano pleurico ) o la morfologia o la localizzazione od infine la comparsa di complicanze , quali lo pneumotorace od il sanguinamento , non hanno significativamente alterato i valori di accuratezza diagnostica della procedura . 
non si sono , invece , formulate valutazioni relative al calibro dellago o allesperienza del radiologo prelevatore , parametri che assumono , a seconda dei diversi studi presenti in letteratura , significato diverso [ 20 , 41 ]  . conclusioni in conclusione , lagobiopsia transtoracica tc guidata di espansi polmonari metodica ad elevata accuratezza diagnostica , specie per le lesioni di natura maligna . 
risulta comunque indispensabile , al fine di implementare le probabilit di successo della procedura in termini di raggiungimento della corretta diagnosi , la conoscenza delle variabili in grado di inficiare tale efficacia : le lesioni benigne ed i nodi di dimensioni minori ad 1 , 5 cm o le masse superiori a 5 cm sono correlate a valori di accuratezza diagnostica sensibilmente inferiori . 
krestin2 1dipartimento di radiologia e dipartimento cuore , imaging cardiovascolare non invasivo , azienda ospedaliera di parma , parma , italy 2dipartimento di radiologia e cardiologia , erasmus medical center , rotterdam , the netherlands 3dipartimento di radiologia , universit degli studi di verona , verona , italy 4dibimel , sezione di scienze radiologiche , universit di palermo , palermo , italy 5unit operativa di riabilitazione cardiovascolare , fondazione don gnocchi onlus , parma , italy correspondence to : f . 
cademartiri , imaging cardiovascolare non invasivo , dipartimento di radiologia , azienda ospedaliero - universitaria di parma , via gramsci 14 , i - 43100 parma , italy , tel . : + 39 - 052 - 1703222 , fax : + 39 - 052 - 1703630 , e - mail : filippocademartiri@hotmail.com received : 23 november 2006 / accepted : 19 march 2007 / published online : 13 december 2007 abstract purpose . 
in the 202 consecutive patients , the prevalence of anatomical variants was : left dominant circulation ( 7% ) , absent left main ( 5% ) , presence of intermediate branch ( 17% ) , aortic origin of conus branch ( 13% ) and circumflex origin of sinus node branch ( 15% )  . 
anatomical variants and anomalies of the coronary arteries are quite common and should be known and recognised promptly by the operators . key words 64 - slice ct coronary anatomy coronary artery variants coronary artery anomalies prevalence riassunto obiettivo . 
nei 202 pazienti consecutivi arruolati per lo studio la prevalenza delle varianti anatomiche risultata : dominanza sinistra ( 7% ) , tronco comune assente ( 5% ) , presenza di ramo intermedio ( 17% ) , origine aortica del ramo del cono ( 13% ) , origine dalla circonflessa dellarteria del nodo del seno ( 15% )  . 
le varianti e le anomalie coronariche sono un reperto molto comune che deve quindi essere riconosciuto agevolmente dalloperatore . parole chiave tc multistrato anatomia coronaria varianti delle arterie coronarie anomalie delle arterie coronarie prevalenza introduction introduzione effectively visualising the coronary arteries is a challenge , not only due to their rapid movement during the cardiac cycle , but also because of their small diameter , their tortuous la visualizzazione delle arterie coronarie rappresenta una sfida non solo per il loro rapido movimento durante il ciclo cardiaco , ma anche per il calibro ridotto , il decorso tortuo1117 f . 
recognising coronary artery anomalies is therefore important for correct diagnosis and treatment of patients , whether or not they are affected by atherosclerosis . the literature contains numerous descriptions of coronary artery anomalies based on conventional coronary angiography ( ca ) but still very few based on msct , and the latter are mostly review articles or case reports [ 35 ]  . the introduction of the technique allows a different anatomical approach that could change the perception of these variants . the aim of this study was to describe the prevalence of coronary artery variants and anomalies in a consecutive population of patients studied with ct coronary angiography ( ctca )  . materials and methods two hundred and two consecutive patients ( 146 men , 56 women , mean age 60 11 years , range 2183 ) with suspected coronary artery disease and already scheduled for conventional ca underwent ctca before ca in the context of a validation study of ctca . 
only patients with a sinus rhythm who had never undergone percutaneous angioplasty or bypass surgery and who were capable of holding their breath for 12 s were included in the study . 
the ethics committee in our department approved the study protocol , and all patients provided informed consent . patient preparation patients with a heart rate ( hr ) greater than 65 beats per minute ( bpm ) were administered 45 munutes before examination , in the absence of contraindications , a single oral dose of 100 mg metoprolol tartrate ( selokeen , astrazeneca pharmaceutics )  . 
apparecchiature di tomografia computerizzata multistrato ( tcms ) allo stato dellarte , ovvero a 64 strati , consentono di collocare la valutazione non invasiva delle arterie coronarie nella routine clinica in virt delle elevate prestazioni ed accuratezza della metodica [ 1 , 2 ]  . le anomalie delle arterie coronariche sono presenti alla nascita ma solo una piccola percentuale di esse si rende manifesta nei primi anni di vita . 
gran parte di esse infatti viene scoperta per caso durante langiografia coronarica o rappresenta addirittura solamente un puro reperto autoptico . ci nonostante alcune di esse possono manifestarsi con sintomi come angina pectoris , infarto miocardico , sincope , aritmie pi o meno severe o addirittura arresto cardiaco configurando il quadro di morte improvvisa . 
il riconoscimento di anomalie coronariche importante quindi per una diagnosi appropriata e per il trattamento del paziente , sia esso affetto o meno da malattia aterosclerotica . nella letteratura esistono delle descrizioni delle anomalie coronariche basate sulla ac mentre ne esistono ancora poche basate sulla tcms , peraltro per lo pi costituite da revisioni della letteratura o descrizione di singoli casi particolarmente significativi [ 35 ]  . 
lintroduzione di questa tecnologia consente un diverso approccio anatomico che pu mutare la percezione di queste varianti . pertanto lo scopo di questo lavoro quello di fornire una prevalenza di varianti ed anomalie coronariche in una popolazione consecutiva di pazienti sottoposti ad angiografia coronarica tc ( ac - tc )  . materiali e metodi duecentodue pazienti consecutivi ( 146 maschi , 56 femmine , et media 6011 anni , range 2183 anni ) con sospetta malattia coronarica e gi indirizzati a coronarografia convenzionale ( ac ) sono stati sottoposti ad angiografia coronarica tc ( ac - tc ) preliminarmente allesecuzione della ac nellambito di studi di validazione della metodica . 
solo i pazienti con ritmo sinusale , mai sottoposti ad angioplastica percutanea o ad intervento chirurgico per il posizionamento di by - pass e capaci di trattenere il respiro per almeno 12 secondi , sono stati inclusi nello studio . 
i pazienti nei quali esistevano delle controindicazioni assolute alla somministrazione endovenosa di mezzo di contrasto iodato ( ad esempio allergia nota , insufficienza renale o disordini tiroidei ) sono stati esclusi dallo studio . 
il comitato etico del nostro dipartimento ha approvato il protocollo di studio e tutti i pazienti hanno fornito un consenso informato . preparazione del paziente ai pazienti con una frequenza cardiaca ( fc ) superiore a 65 battiti per minuto ( bpm ) stata somministrata , 45 minuti prima della scansione , se non presenti delle controindicazioni , una singola dose orale di 100 mg di metoprololo tartrato ( selokeen , astrazeneca pharmaceutics )  . 
the study protocol involved a preliminary acquisition without intravenous iodinated contrast material to quantify coronary artery plaque . the parameters used for the two scans ( a ) quantification of plaque and ( b ) ctca were : 1 . 
images were reconstructed with the following parameters : effective slice thickness 3 mm , reconstruction increment 1.5 mm , field of view ( fov ) 150180 mm , dedicated convolution kernel for the calcium score . the time windows were positioned at 60% of the rr interval 2 . 
number of slices per rotation 322 , slice thickness 0.6 mm , gantry rotation time 330 ms , table feed 3.84 mm / rotation ( pitch 0.2 ) , 120 kv , 900 mas . 
prospective modulation of the x - ray tube was not used . patients were administered 100 ml of iodinated contrast material ( iomeprol , iomeron 400 , bracco , milan , italy ) with an automatic injector at 5 ml / s ( stellant , medrad , pittsburgh , pa , usa ) connected to a 20 - gauge cannula positioned in an antecubital vethe bolus tracking technique was used to optimise enhancement of the coronary arteries ( care bolus , siemens , forchheim , germany )  . 
angiography - scan data were obtained during a single breath - hold ( 912 s )  . dedicated software ( windose , institute of medical physics , erlangen , germany ) was used to calculate the radiation dose during the angiographic scan ( mean value 15.2 msv for women and 21.4 msv for men )  . 
retrospective reconstructions were performed based on the electrocardiogram ( ecg ) signal to obtain an image quality not affected by motion artefacts in the end - diastolic and end - systolic phases . coronary angiography ( ca ) ca was performed within 2 weeks of the ctca . 
cardiac catheterisation and ca were performed with standard protocols , with spider - view projections , right anterior oblique ( rao ) for the left coronary artery and left anterior oblique ( lao ) for the right coronary artery . 
a single observer unaware of the ctca results identified the coronary segments using a 17 - segment modified american heart association classification . data collection and statistical analysis all examinations judged to be of insufficient quality by two observers were excluded from the sample . 
all examinations were analysed on an off - line dedicated workstation using all the available applications ( viewing , 3d , inspace , circulation ) to achieve an accurate assessment . 
the observers obtained multiplanar reconstructions ( mpr ) , curved multiplanar reconstructions ( cmpr ) , maximum intensity projecta per via orale una dose addizionale di 1 mg di lorazepam ( temesta , wyeth - ayerst )  . protocollo di scansione e ricostruzione delle immagini per lo studio mediante tc stato utilizzato uno scanner a 64 strati ( sensation 64 , siemens , forchheim , germania )  . 
numero di strati per rotazione 322 , tempo di rotazione del gantry 330 ms , avanzamento / rotazione 6 mm ( pitch 0 , 2 ) , voltaggio del tubo radiogeno kv 120 , potenza del tubo radiogeno mas 150 . 
le immagini sono state ricostruite con i seguenti parametri : spessore di strato effettivo 3 mm , incremento di ricostruzione 1 , 5 mm , campo di vista ( fov ) 150180 mm , filtro di convoluzione dedicato per il calcium score . 
numero di strati per rotazione 322 , spessore di strato 0 , 6 mm , tempo di rotazione 330 ms , avanzamento per rotazione 3 , 84 mm ( pitch 0 , 2 ) , voltaggio del tubo radiogeno 120 kv , potenza del tubo radiogeno 900 mas , spessore di strato effettivo ricostruito 0 , 60 , 75 mm , incremento di ricostruzione 0 , 4 mm , campo di vista ( fov ) 150180 mm , filtro di convoluzione medium - smooth . 
non stata utilizzata la modulazione prospettica del tubo radiogeno . sono stati somministrati 100 ml di mezzo di contrasto iodato ( iomeprol , iomeron 400 , bracco , milano ) alla velocit di 5 ml / s mediante iniettore automatico ( stellant , medrad , pittsburgh , usa ) collegato ad unagocannula da 20 gauge preventivamente posizionata in una vena antecubitale . 
utilizzando un software dedicato ( windose , istituto di fisica medica , erlangen , germania ) stata calcolata la dose di radiazioni ionizzanti alla quale i pazienti sono stati esposti durante la scansione angiografica ( valore medio 15 , 2 msv per le donne e 21 , 4 msv per gli uomini )  . 
sono state effettuare delle ricostruzioni retrospettive basate sul segnale ecg per ottenere una qualit dellimmagine priva di artefatti da movimento in fase telediastolica e tele - sistolica . angiografia coronarica ( ac ) lac stata eseguita entro 2 settimane dallac - tc . 
la tecnica di cateterizzazione cardiaca e lesecuzione dellangiografia coronarica sono state eseguite seguendo i protocolli standardizzati , ottenendo in particolare le proiezioni spider view , oad per la arteria coronaria sinistra e oas per larteria coronaria destra . 
the same observers also assessed the presence of coronary anomalies on the basis of the type of origin and course , whether they were intrinsic to the coronary itself , or terminal . 
the proximal tract of the rca was the origin of the right conus branch in 59% of cases and the origin of the sinuatrial nodal branch in 64% of cases . 
the dellac - tc , ha identificato i segmenti coronarici utilizzando una classificazione in 17 segmenti modificata rispetto a quella fornita dallamerican heart association . raccolta dati ed analisi statistica due osservatori in consenso hanno escluso dal campione tutti gli esami la cui qualit stata giudicata insufficiente da entrambi i lettori . 
tutti gli esami sono stati analizzati su piattaforma di lavoro dedicata off - line utilizzando tutte le applicazioni disponibili ( viewing , 3d , inspace , circulation ) per raggiungere una valutazione accurata . 
gli stessi osservatori hanno infine valutato in consenso anche la presenza di anomalie coronariche sulla base della tipologia : di origine e decorso , intrinseche alla coronaria stessa , di terminazione . 
i dati sono stati espressi come prevalenze in numero assoluto e percentuale sul totale dei reperti descritti . risultati gli esami sono risultati tutti di qualit diagnostica e adatti alla valutazione . table 1 main anatomical variants of the coronary tree tabella 1 principali varianti anatomiche dellalbero coronarico main anatomical variants type principali varianti anatomiche tipo coronary dominance origin of the conus branch artery origin of the sinus node artery pda variants lm lenght intermediate branch number of diagonal branches number of marginal branches right , left , mixed rca ; rca ostium ; aorta rca ; cx ; both pathways are present early pda ; double 12 cm ; < 1cm ; > 2cm single or double 12 , > 2 12 , > 2 dominanza coronarica origine arteria del cono della polmonare origine arteria del nodo del seno varianti adp lunghezza del tcs presenza di un ramo intermedio numero di rami diagonali numero di rami marginali destra ; sinistra ; bilanciata acd ; ostio acd ; aorta acd ; acx ; entrambe le vie presenti emergenza precoce ; doppia adp 12 cm ; < 1 cm ; > 2 cm singolo o doppio 12 , > 2 12 , > 2 rca , right coronary artery ; cx , circumflex artery ; lm , left main ; pda , posterior descending artery acd , arteria coronarica destra ; acx , arteria circonflessa ; tcs , tronco comune sinistro ; adp , arteria discendente posteriore 1120 f . 
i rami settali sono stati visualizzati nell89% dei pazienti . analisi delle anomalie coronariche : le anomalie dellalbero coronarico riscontrate nella nostra popolazione comprendono 51 casi pari al 25% dei pazienti osservati . alcuni pazienti presentano multiple anomalie cos ripartite e riassunte in tabella 4 : 1 . 
anomalie di terminazione delle coronarie con decorso anomalo in 1 paziente ( 0 , 5% )  . discussione la tcms in grado di visualizzare con efficacia lanatomia complessa e variabile delle arterie coronarie sfruttando le tecfig . 
termination anomalies of the coronary arteries with an anomalous course were identified in one patient ( 0.5% ) discussion msct is able to effectively visualise the complex and variable anatomy of the coronary arteries by exploiting postprocessing techniques , thus providing valuable support to interventional cardiologists and heart surgeons [ 615 ]  . 
on the basis of the most recent published data obtained with ca , normal is defined as any morphological characteristic of the coronary circulation observed in more than 1% of a nonselected sample . a normal variant , therefore , is a characteristic observed in more than 1% of the same sample , whereas an anomaly is a condition observed in less than 1% [ 20 ]  . coronary dominance indicates which system of coronary arteries supplies the inferolateral aspect of the left ventricle [ 21 ]  . 
in 80%90% of the population the posterior descending artery ( pda ) is a branch of the rca and runs from the base of the heart to the apex in a mirror image of the lad . 
in right coronary dominance the rca gives rise to a right posterolateral ( rpl ) branch , which crosses the posterior interventricular sulcus and continues on the left side to supply the dorsal aspect of the left ventricle . 
esistono in letteratura numerosi criteri per classificare ed indicare le anomalie coronariche ; basandoci sui dati pi recenti ritrovati nella letteratura , peraltro ottenuti da casistiche di angiografia coronarica ; viene definita normale qualsiasi caratteristica morfologica del circolo coronarico osservata in pi dell1% in un campione non selezionato ; variante normale , una caratteristica osservata in pi dell1% dello stesso campione ; anomalia una condizione osservata in meno dell1% [ 20 ]  . la dominanza coronarica indica quale sistema di vasi coronarici vicaria il flusso sanguigno verso la parete infero - laterale del ventricolo sinistro [ 21 ]  . 
quando la dominanza coronarica del sistema destro la acd d origine un ramo postero - laterale destro ( pld ) , che attraversa il solco interventricolare posteriore e continua sul lato sinistro per la vascolarizzazione della parete dorsale del ventricolo sinistro . il ramo pld pu diventare di importanza notevole quando la acx occlusa , poich pu essere in grado di sostenere la vascolarizzazione della parete postero - inferiore del ventricolo sinistro . 
dai dati ottenuti nella nostra analisi emerge una stretta concordanza con quanto riportato in letteratura , ovvero una prevalenza nella dominanza destra . il primo ramo della acd larteria del cono che decorre sulla superficie antero - laterale del tratto di efflusso del ventricolo destro . 
conventional angiogram ( a ) , volume rendering ( vr ) ( b ) and maximum intensity projection ( mip ) ( c ) images of a right coronary artery ( rca ) arising from the left sinus of valsalva with an interarterial course . 
conventional angiogram ( d ) , vr ( e ) and multiplanar reconstruction ( mpr ) ( f ) images of a left coronary artery arising from the rca with a septal course . 
acc ( g ) , immagini vr ( h ) e cmpr ( i ) di acx con origine dal seno di valsalva destro e decorso retroaortico , sottoposta a stent . nance is mixed with an intermediate vascularisation pattern . data from our analysis show a strong agreement with the data reported in the literature , i.e. 
in una piccola percentuale di casi lacd pu dare origine anche ad un ampio ramo lungo il margine acuto del cuore fino a continuarsi con ladp ( emergenza precoce delladp )  . 
conventional angiogram ( a ) , volume rendering ( vr ) ( b ) and maximum intensity projection ( mip ) images ( c ) displaying an aneurysm of the circumflex artery . 
myocardial bridging of mid - left anterior descending artery displayed by conventional angiogram during systole ( d ) , vr ( e ) and mpr cross - section images ( f )  . 
sometimes , however , the rca can trifurcate at the crux , giving rise to two branches ( double pda ) for the supply of the base of the heart and an rpl branch for the supply of the dorsal aspect of the left ventricle . the length of the main lca is usually 12 cm , whereas lower values are very common and higher values more rare [ 22 ]  . 
the anomalies that prove to be haemodynamically significant , however , can lead to angina pectoris , syncope , arrhythmia , myocardial infarct or sudden death , as well as promote the onset and progression of coronary atherosclerosis or prove responsible for complications and technical difficulties during angioplasty and heart surgery . 
this anomaly could create problems during heart surgery , since the surgeon unaware of its anomalous course could mistakenly cut it . in addition , native coronary arteries are normally positioned at the origin of the aortic arch with an orientation of 120 and 150 , respectively . 
the cases we observed included 1128 zazione della base del cuore , e ad un ramo pld , per la vascolarizzazione della parete dorsale del ventricolo sinistro . la lunghezza del tcs comunemente di circa 12 cm , anche se possono essere riscontrati valori inferiori ( molto frequenti ) o superiori ( pi rari ) [ 22 ]  . 
le anomalie che risultano invece emodinamicamente significative possono indurre angina pectoris , sincope , aritmia , infarto miocardico , morte improvvisa , favorire linsorgenza e la progressione della malattia aterosclerotica coronarica o rendersi responsabili di complicanze e difficolt tecniche durante angioplastica ed interventi di cardiochirurgia . 
il chirurgo , infatti , potrebbe erroneamente reciderla non conoscendone il decorso anomalo . normalmente inoltre le arterie coronarie native sono posizionate allorigine dellarco aortico con un orientamento di 120 e 150 gradi rispettivamente . 
in caso di riposizionamento chirurgico della valvola o di una sua sostituzione importante conoscere questa condizione . alcune anomalie vengono definite anche come maligne proprio per il loro potenziale patologico e sulla base della loro rilevanza clinica ( benigne ; rilevanti correlate ad ischemia miocardia ; severe associate a morte improvvisa ; critiche associate a malattia coronarica ) , facilitandone cos la gestione ed il follow - up clinico [ 25 ]  . 
a questa classificazione si pu sovrapporre una classificazione anatomica che divide le anomalie coronariche sulla base dellorigine ( assenza del tronco comune sinistro , figura 6 , coronaria con origine dallopposto seno coronarico , figura 7 ) , del decorso ( interarterioso , settale e retroaortico , figura 7 ) , dellanatomia intrinseca ( aneurismi , ponti miocardici , figura 8 ) , e della terminazione ( fistole , figura 9 ) [ 4 , 20 ]  . 
tra i casi osservati emergono alcune di queste anomalie : la fistola coronarica una anomalia coronarica in grado di determinare o predisporre ad un evento ischemico miocardico ed a scompenso cardiaco congestizio , in quanto , drenando nelle sezioni cardiache di destra , pu determinare uno shunt sinistrof . 
coronary fistula is a anomaly capable of causing or creating the predisposition for a myocardial ischaemic event and congestive heart failure in that drainage in the right cardiac sections can lead to leftright shunt with volume overload . 
this anomaly in adults is associated with myocardial ischaemia , a higher risk of coronary disease and volume overload . coronary anomalies are a relatively frequent finding in the context of noninvasive coronary diagnostic imaging . knowledge of the characteristics and consequences of different types of anomalies allows the radiologists to judge the finding appropriately . 
nonetheless , it needs to be emphasised that the performance of ctca for suspected coronary anomaly or to exclude its presence should be carefully considered together with the cardiologist to avoid exposing a young patient to an excessive dose of ionising radiation . to date , conventional ca has been the technique of choice for the diagnosis of coronary artery anomalies . 
the diagnosis of coronary artery anomalies is very often established on the basis of the impossibility of finding the coronaries in their normal anatomical position , and therefore of characterising their ostium . in a few short years msct has modified the entire perception of coronary diagnosis by demonstrating the feasibility of a noninvasive approach . 
the complex and tortuous coronary anatomy can be easily depicted with msct . in our case series , we wanted to emphasise the importance of coronary variants and anomalies by demonstrating their prevalence in a broad population of consecutive patients treated at our tertiary referral centre . 
in our experience , the prevalence of coronary artery anomalies was 25% of cases ( 51 / 202 ) , a much higher value than reported in the literature . this can , however , be explained by the fact that the centre where the examinations were performed is a tertiary referral centre for patients with cardiovascular disease . 
in addition , it should be borne in mind that the study population was not made up of a single ethnic group but was , rather , heterogeneous , as it included patients originating from other continents and with a different and currently unknown prevalence of coronary artery anomalies . most of the coronary artery variants encountered had a minimal clinical risk , such as myocardial bridging , which was present in 12.9% of cases , a figure higher than in condestro con sovraccarico di volume . 
nelladulto questa anomalia si pu associare ad ischemia miocardica , ad un pi elevato rischio di malattia coronarica e ad un sovraccarico cardiaco . le anomalie coronariche sono un reperto relativamente frequente nel contesto dellimaging diagnostico non invasivo coronarico . 
langiografia coronarica con tcms consente grazie alle tecniche di post - processing ( mpr , cmpr , mip , vr ) una pi chiara visualizzazione delle anomalie coronariche rispetto allac . 
la ac - tcms stata recentemente considerata come test appropriato per la valutazione di una sospetta anomalia coronarica , al pari della angiografia coronarica mediante risonanza magnetica [ 2 ]  . 
occorre , tuttavia , puntualizzare che lindicazione allesecuzione di una ac - tcms per sospetta anomalia coronarica o per escluderne la presenza deve essere ben ponderata in cooperazione con il cardiologo , al fine di evitare ad un paziente di giovane et uneccessiva esposizione a radiazioni ionizzanti . sino ad oggi , la coronarografia convenzionale ha rappresentato la tecnica di riferimento per la diagnosi delle anomalie coronariche . 
la diagnosi di ac stabilita , molto spesso , sulla base dellimpossibilit di trovare la coronaria nella normale sede anatomica e , quindi , di cateterizzarne lostio . la tomografia computerizzata multistrato ( tcms ) ha modificato in pochi anni lintera percezione della diagnostica coronarica , portando in primo piano la fattibilit di un approccio non invasivo . 
questa metodica , infatti , offre uneccellente risoluzione spaziale con la possibilit di effettuare mpr ( ricostruzioni multiplanari ) , mip ( maximum intensity projections ) e vr ( volume rendering )  . 
la complessa e tortuosa anatomia coronarica pu essere agevolmente dimostrata mediante la tcms . nella casistica presentata abbiamo voluto sottolineare limportanza delle varianti e delle anomalie coronariche dimostrandone la prevalenza in una ampia popolazione di pazienti consecutivi afferenti presso un centro di iii livello . nella nostra esperienza , la prevalenza delle anomalie coronariche del 25% dei casi ( 51 / 202 ) , valore molto superiore a quanto osservato in letteratura , ma spiegabile a causa del fatto che il centro dove sono stati effettuati gli esami rappresenta centro di iii livello , di riferimento per patologie cardiovascolari . 
va tenuto conto del fatto , poi , che la popolazione di studio non rappresenta un solo gruppo etnico ma abbastanza eterogenea , poich coinvolge pazienti originanti da altri continenti e con prevalenze di anomalie coronariche quindi differenti , e attualmente non nota . delle varie varianti la maggioranza hanno scarsa rilevanza clinica , come ad esempio i ponti miocardici , la cui prevalenza risulta essere del 12 , 9% , dato anchesso superiore a 1129 f . 
in contrast , the finding of an anomalous origin or course can be clinically relevant , such as the anomalous origin of the coronary ostium from the opposite coronary sinus ( which manifests as origin in the main lca or of divided origin )  . 
in these cases , the potential compression of a single coronary vessel if it passes between the aorta and the pulmonary artery can cause ischaemia , myocardial infarct and sudden death . quanto riscontrato nelle casistiche angiografiche e pi vicino alle prevalenze evidenziate in studi autoptici [ 27 ] , mentre pu avere rilevanza clnica il reperto di anomalie di origine e decorso , quali ad esempio lanomala origine dellostio coronarico dallopposto seno coronarico ( che si estrinseca come origine in tronco comune oppure origine divisa )  . 
in questi casi la potenziale compressione di un singolo vaso coronarico se passa tra aorta ed arteria polmonare , pu provocare episodi ischemici , infarto miocardico e morte improvvisa . conclusions the anatomical complexity and variability of the coronary circulation benefits from the flexibility of the various postprocessing techniques introduced by ctca and implemented by the use of a 64 - slice scanner with isotropic voxel . 
radiologist training needs to focus on both anatomical and technical features to satisfy the criteria of professional competence and expertise required for the study of such a highly specialised area of diagnostic imaging . ctca is currently capable of providing highly accurate anatomical imaging of the cardiac vessels . 
both the arterial and the venous vessels can be visualised , although with slightly different protocols with regard to the timing of contrast administration . among all the applications requiring knowledge of the anatomy of the cardiac vessels , ctca constitutes the noninvasive diagnostic technique capable of depicting the highest number of coronary segments , particularly the distal segments . 
therefore , in patients affected by coronary artery disease , it is reasonable to expect that ctca , due to its intrinsically fast execution , noninvasiveness and low cost , will become the ideal instrument for assessing patients with a low risk of coronary artery disease and for the follow - up of percutaneous and / or surgical procedures in the coronary vascular area . conclusioni la complessit e la variabilit anatomica del circolo coronarico si giova della flessibilit delle varie metodiche di postprocessing introdotte dalla ac - tcms ed implementate dalluso di scanner a 64 strati con voxel isotropico . 
il training del medico radiologo deve necessariamente focalizzarsi su entrambi gli aspetti , anatomico e tecnico , per soddisfare quei criteri di competenza professionale ed expertise necessari per lapproccio ad un area di imaging altamente specializzata . attualmente la coronarografia tc in grado di fornire un imaging anatomico dei vasi cardiaci con elevata accuratezza . 
sia il comparto arterioso che quello venoso possono essere dimostrati , anche se con protocolli lievemente differenti legati al timing del mdc . in tutte quelle applicazioni nelle quali la conoscenza dellanatomia dei vasi del cuore importante , la coronarografia tc costituisce ad oggi la metodica diagnostica non invasiva in grado di visualizzare il maggior numero di segmenti coronarici ed in particolare anche quelli distali . 
i - 00168 rome , italy 2deptartment of radiology , neuroradiology section , ucsf university of california san francisco , san francisco , ca , usa correspondence to : a . 
by providing quantitative measurements of cerebral blood flow ( cbf ) and cerebral blood volume ( cbv ) , dynamic perfusion computed tomography ( p - ct ) allows visualisation of cerebral autoregulation mechanisms and represents a fast , available and reliable imaging option for assessing cerebral perfusion . 
thanks to its feasibility in emergency settings , p - ct is considered most useful , in combination with ct angiography , in acute ischaemic patients , as it is able to provide a fast and noninvasive assessment of cerebral perfusion impairment . 
in addition , p - ct can play a diagnostic role in other types of cerebrovascular disease to assess functional reserve , and in intracranial neoplasms , where it has a role in diagnosis , grading , biopsy guidance , and follow - up during treatment . 
this article illustrates the principles , technique and clinical applications of p - ct cerebral perfusion studies . key words brain ischemia brain neoplasms brain injury hemodynamics perfusion ct riassunto lo studio dellemodinamica cerebrale , ottenuto con le metodiche di imaging , con le sue varie ed attuali applicazioni , genera interesse crescente . 
la tc perfusione dinamica ( p - tc ) permette una valutazione quantitativa del flusso cerebrale ematico ( cbf ) e del volume cerebrale ematico ( cbv ) , offrendo cos una visualizzazione diretta dei meccanismi di autoregolazione cerebrale , e si pone come una valida alternativa ad altre modalit di misurazione della perfusione cerebrale , rispetto alle quali ha il maggior vantaggio di essere una tecnica prontamente disponibile ed accessibile , in condizioni di emergenza , nella maggior parte dei centri medici . 
per tale ragione la p - tc utile soprattutto nellischemia cerebrale acuta , condizione in cui , associata allangio - tc , offre in maniera rapida e non - invasiva , la valutazione eziologica dellipoperfusione , nonch delle sue ripercussioni emodinamiche e fisiopatologiche sul parenchima cerebrale . 
inoltre la p - tc trova utile impiego in pazienti con altre patologie cerebro - vascolari e per la diagnosi , il grading , la guida alle procedure bioptiche , ed il controllo durante la terapia , dei tumori intra - cranici . 
questo articolo si propone di riassumere i principi , la tecnica e le principali applicazioni cliniche degli studi di perfusione cerebrale basati sulla metodica tc . parole chiave ischemia cerebrale neoplasie cerebrali trauma cranico perfusione tc introduction introduzione rapid technological advances in functional imaging techniques have extended the scope of radiology , and in particular of neuroradiology , beyond the boundaries of morphological imaging . 
perfusion studies can now be carried out with magnetic resonance imaging ( mri ) and computed tomography ( ct ) as well as with the gold standard modalities used until now , such as positron emission tomography ( pet ) , single - photon emission tomography ( spect ) il rapido progresso tecnologico applicato alle tecniche di imaging funzionale ha spinto la radiologia in generale , e la neuroradiologia in particolare , oltre i confini della morfologia ; in questo contesto genera crescente interesse la valutazione a scopo diagnostico dellemodinamica cerebrale ( perfusione cerebrale )  . 
gli studi di perfusione ottenuti con rm e tc si sono affiancati alle metodiche fino ad ora impiegate e considerate gold standard , quali la tomografia ad emissione di positroni ( pet ) , la tomografia ad emissione di singolo fotone ( spect ) e la xenon - tc , con 1225 a . 
this is because the introduction of thrombolytic therapy for the treatment of stroke has highlighted the need for a fast and readily available technique capable of identifying the perfusion defect and evaluating its extent and severity [ 2 , 3 ]  . 
 in patients with acute stroke undergoing emergency evaluation , dynamic perfusion ct ( p - ct ) has been proposed as a particularly useful technique after unenhanced brain ct ( which rules out intracranial haemorrhage ) on account of its ease of use , reproducibility of quantitative measurements , ready availability , limited cost and tolerability [ 3 , 4 ]  . 
moreover , the use of p - ct may be extended to the evaluation of the cerebrovascular reserve in patients with haemodynamically significant stenoses of the intraor extracranial arteries even with acetazolamide challenge in potential candidates for temporary or permanent occlusion of the internal carotid artery ( balloon occlusion test ) [ 5 ] , or in patients with subarachnoid haemorrhage at risk of vasospasm [ 6 ]  . 
in addition to acute and chronic cerebrovascular disease , a further application of p - ct is the measurement of cerebral blood volume ( cbv ) and microvascular permeability ( ps ) in brain tumours [ 7 ]  . general technique p - ct derives information on brain haemodynamics by analysing the first passage through the cerebral vessels of an intravenous contrast bolus . 
because there is a direct linear relation between the concentration of contrast material and density , passage of contrast bolus results in an increase in density of the areas being examined that is proportional to the amount of contrast material present in the blood vessels . the blood - brain barrier ( bbb ) prevents the contrast material from spreading into the interstitium , so that under normal conditions the increase in density is only transient , occurring during the first intravascular passage of the bolus . 
p - ct is based on the physical - mathematical tracer kinetic model [ 8 ] , which assumes that the contrast bolus is instantaneous , is introduced into a single vessel , passes through a capillary network , remains totally intravascular and flows out through a single venous conduit . p - ct can be performed on any spiral ct scanner capable of cine mode scans , with the aid of an automatic injector . the anatomical coverage of p - ct along the craniocaudal axis is limited , as the scan is obtained without table motion at 510 mm for single - detector devices , at 20 mm for multidetector devices ( 232 ) and at 40 mm for the recent 64 - detector - row scanners . 
for this reason , the anatomical level to be studied is selected , generally on the scout view or initial morphological scan , and the perfusion study performed at that location using a cine scan without table motion . 
one possible study protocol , applicable to both singleand multislice scanners ( 232 slices ) involves 1 rotation / s for 50 s , 1226 relativi vantaggi e limiti [ 1 ]  . 
infatti lintroduzione della terapia trombolitica nel trattamento dellictus ischemico acuto ha reso evidente il bisogno di una tecnica rapida e prontamente disponibile per lidentificazione del deficit perfusionale e la valutazione della sua estensione e della sua entit [ 2 , 3 ]  . nei pazienti con ictus , in condizioni di emergenza , dopo la tc cerebrale di base senza mdc ( che esclude lemorragia intra - cranica ) la tc perfusione dinamica ( p - tc ) , per semplicit e rapidit di esecuzione , riproducibilit di misurazioni quantitative , disponibilit sul territorio , costi contenuti e tollerabilit da parte dei pazienti , si propone come metodica di particolare interesse ed utilit [ 3 , 4 ]  . 
la p - tc pu essere effettuata velocemente con qualsiasi apparecchiatura spirale , e le mappe di perfusione possono essere ottenute in breve tempo mediante una workstation dotata di apposito software . 
limpiego della p - tc pu inoltre essere esteso alla valutazione della riserva cerebro - vascolare in pazienti con stenosi arteriose emodinamicamente significative intrao extra - craniche anche con stress - test allacetazolamide in potenziali candidati ad occlusione temporanea o definitiva dellarteria carotide interna ( balloon occlusion test ) [ 5 ] , o in pazienti con emorragia sub - aracnoidea , a rischio di vasospasmo [ 6 ]  . 
poich esiste una relazione proporzionale lineare diretta tra concentrazione del mdc e densit , il passaggio del bolo di mdc induce un aumento della densit nelle aree esaminate , proporzionale alla quantit di mdc presente nei vasi . 
la presenza della barriera emato - encefalica ( bee ) impedisce al mdc di diffondersi dai vasi allinterstizio , cosicch , in condizioni normali , laumento di densit solo transitorio , durante il suo primo passaggio intra - vascolare . 
la p - tc basata sul modello fisico - matematico del tracciante cinetico ( kinetic tracer model ) [ 8 ] , il quale assume che il bolo di mdc sia pressoch istantaneo , sia immesso in un vaso unico , passi attraverso una rete capillare , rimanga totalmente intra - vascolare , e defluisca da un singolo collettore venoso . la p - tc pu essere effettuata su qualsiasi apparecchiatura tc con tecnologia spirale che sia in grado di effettuare scansioni in modalit cine , con lausilio di un iniettore automatico . 
la copertura anatomica della p - tc lungo lasse cranio - caudale limitata , poich la scansione si effettua a tavolo fermo , a 510 mm per le apparecchiature spirali a strato singolo , a 20 mm per le apparecchiature tc multia . 
the short scan delay is aimed at the acquisition of a first baseline scan before arrival of bolus and is applied independent of the patients cardiocirculatory condition or type of ct device used . 
for a four - detector - row scanner but also possible with other multislice devices allows the effective radiation dose to be contained below that calculated for a conventional brain ct study [ 10 , 11 ]  . 
lower milliampere settings result in a reduced signal - to - noise ratio ( snr ) and poorer densitometric resolution , with consequently less precision of the parametric measurements ( expressed by increased standard deviation values ) ; nevertheless , it represents a reasonable compromise between radiation - dose containment and diagnostic quality of the examination . 
use of the automatic injector and , if a dual syringe injector is available , of a saline flush after the contrast bolus allows administration of a fast and compact bolus [ 11 ]  . 
the images , generally acquired with 5 - mm thickness to avoid beam - hardening artefacts , are reconstructed with a thicknesses of 10 mm to increase the snr , and at 0.5 - s intervals to increase temporal resolution . data acquired during the cine scan are then analysed on a workstation running dedicated postprocessing software that generates and analyses the time - density curves . 
a region of interest ( roi ) is placed on an artery in the images to view the time - density curve , which reflects the compactness of the bolus . 
it is important to note that , although ideally one might expect the arterial curve to fall back to baseline density levels after the first pass of the bolus , in practice , even when the bolus is sufficiently compact and the bbb intact , the curve tends to remain on a plateau varying height above baseline ( usually 10%30% of the peak )  . 
the maximum slope model approximates the cerebral blood flow ( cbf ) value from the slope of the time - density curve , calcustrato , dotate di multiple file di detettori ( da 2 a 32 ) , a 40 mm per le recenti tc multidetettore a 64 strati . 
per tale ragione si seleziona il livello anatomico da studiare , generalmente sulla scout view o su una scansione morfologica iniziale , ed al livello selezionato si esegue lo studio di perfusione , che consiste in una scansione cine , a tavolo fermo . 
un possibile modello di protocollo di studio , applicabile con apparecchiature tc a strato singolo o multiplo ( 232 strati ) , prevede 1 rotazione / s per 50 s , con 57 secondi di ritardo rispetto alliniezione attraverso una cannula 1820 g , in una vena antecubitale , di 4050 ml di mdc iodato non ionico ( 300370 mg% ) a 45 ml / s [ 9 ]  . 
il breve ritardo tra liniezione del mezzo di contrasto e linizio della scansione finalizzato ad ottenere lacquisizione di una linea di base , prima dellarrivo del bolo , ed applicabile indipendentemente dalla condizione cardio - circolatoria del paziente e dal tipo di apparecchiatura tc utilizzata . 
 [ 10 , 11 ] per una apparecchiatura tc multistrato con quattro file di detettori , ma applicabile anche ad altri tipi di tc multistrato , permette di contenere la dose effettiva di radiazioni al di sotto di quella calcolata per un esame tc del cranio convenzionale . 
la limitazione dellamperaggio causa una diminuzione del rapporto segnale / rumore ed una ridotta capacit di risoluzione densitometrica , cui consegue una ridotta precisione delle misurazioni parametriche ( espressa dagli aumentati valori di deviazione standard ) , ma rappresenta un ragionevole compromesso tra contenimento della dose di radiazioni e qualit diagnostica dellesame . 
luso delliniettore automatico e , se si dispone di un iniettore a doppia siringa , di un bolo di soluzione fisiologica a seguire il mdc , permettono di somministrare un bolo rapido e compatto [ 11 ]  . 
le immagini , generalmente acquisite con spessori di 5 mm , per evitare artefatti da indurimento del fascio , vengono ricostruite a spessori di 10 mm , per aumentare il rapporto segnale / rumore , e a 0 , 5 s di intervallo , per incrementarne la risoluzione temporale . i dati acquisiti durante la scansione cine sono poi analizzati ad una workstation dotata di apposito software che produce ed analizza le curve tempo / densit . 
la compattezza del bolo di mezzo di contrasto determina la morfologia della curva , con ascesa ripida e discesa compresa nei 50 s di scansione . lates cbv as the area under the curve , and from these two parameters derives mean transit time ( mtt ) by solving the central volume equation . 
il modello maximum slope approssima il valore del cbf dalla pendenza ( slope ) della curva densit / tempo , calcola il cbv come larea sottesa alla stessa curva , e da questi due parametri deriva il mtt risolvendo lequazione del volume centrale ; questo modello non richiede la selezione di un arterial input function ( aif ) , ma necessita di velocit di iniezione del mdc molto alte ( > 6 ml / s ) , e fornisce solo misurazioni relative non quantitative [ 12 , 13 ] ; il modello di deconvoluzione si basa su un complesso processo matematico che richiede la scelta di un aif , ovvero di una curva densit / tempo di unarteria , con cui confrontare la curva ottenuta in corrifig . 
a maximum - slope model derives cerebral blood volume ( cbv ) values by calculating the area under the curve , cerebral blood flow ( cbf ) as the slope of the curve and mean transit time ( mtt ) by solving the central volume equation . 
again , cbv corresponds to the area under the curve , mtt to full width at half maximum ( fwhm ) and cbf is derived from the central volume equation . 
a il modello del maximum slope calcola il cbv integrando larea sotto la curva densit / tempo , il cbf secondo la tangente alla porzione ascendente della curva , e deriva il calcolo del mtt dallequazione del volume centrale . 
b il modello di deconvolution , tramite un arterial input function , trasforma la curva densit / tempo in una curva di funzione tissutale , in cui lintegrale dellarea sotto la curva esprime il cbv , il tempo alla met dellaltezza massima ( full width at half maximum fwhm ) rappresenta il mtt ed il cbf derivato dallequazione del volume centrale . 
the deconvolution method relies on a complex mathematical process that requires the use of an aif that is , the time - density curve of an artery with which to compare the curve obtained on the parenchymal pixels so as to correct for the effects of bolus dispersion and thus better reflect the postulates of the tracer kinetic model , which assumes a virtually instantaneous bolus input [ 14 ]  . 
the deconvolution model allows one to reduce the contrast injection rate ( 35 ml / s ) and provides quantitative measurements [ 1517 ] that have been validated by comparison with pet [ 18 ] , spect [ 19 ] and xe - ct [ 20 ]  . although some commercial software packages can automatically select the best aif , aif choice is one of the most controversial aspects of p - ct . 
partial - volume artefacts are practically unavoidable , as the imaging section has a thickness of 10 mm and the artery to be selected generally has a calibre of 24 mthe problem can be partially avoided by choosing an artery that runs perpendicular to the section for example , segment a2 of the anterior cerebral artery so that the roi is placed on a pixel corresponding to a voxel almost completely taken up by the vessel . 
more complex , in the case of cerebral ischaemia , is the problem of selecting an intracranial vessel that is ipsilateral or contralateral to the lesion ( generally segment m2 of the middle cerebral artery ) , or a vessel with proximal stenosis , generally at an extracranial location . 
although there is no consensus [ 21 , 22 ] and studies are underway , our advice is to choose the aif from a healthy vessel , often the anterior cerebral artery , and then compare the results with those obtained with an aif selected on the abnormal side . 
 the software then requires selection of a venous roi , which provides a reference measurement of blood density , considered free of partial - volume artefacts , such as the posterior descending portion of the superior sagittal sinus . 
this curve is used to derive mtt , identified by the width of the curve at spondenza dei pixel parenchimali , per correggere matematicamente gli effetti della dispersione del bolo e riavvicinarsi cos ai postulati teorici del kinetic tracer model , che , come detto , presuppone unimmissione pressoch istantanea del bolo [ 14 ]  . 
il modello di deconvoluzione permette di ridurre le velocit di somministrazione del bolo di mdc ( 35 ml / s ) e fornisce misurazioni quantitative [ 1517 ] , validate in studi di confronto con pet [ 18 ] , spect [ 19 ] e xetc [ 20 ]  . 
gli artefatti da volume parziale sono praticamente inevitabili , infatti , la sezione su cui si lavora ha uno spessore di 10 mm e larteria da selezionare ha generalmente un calibro di 24 mm ; per ovviare parzialmente a tale problema , si pu selezionare unarteria con decorso perpendicolare alla sezione , come nel caso del tratto a2 dellarteria cerebrale anteriore , in modo da posizionare la nostra roi su un pixel corrispondente ad un voxel quasi interamente occupato dal vaso . 
sebbene non esista definitivo consenso [ 21 , 22 ] , e siano ancora in corso studi al riguardo , il nostro consiglio di scegliere laif da un vaso ritenuto sano , spesso larteria cerebrale anteriore , e poi confrontare i risultati con quelli ottenuti con unaif scelta sul lato anormale . il software richiede poi la selezione di una roi venosa , che serve come misurazione di riferimento della densit del sangue , e viene considerata come esente da artefatti di volume parziale , come il caso del seno sagittale superiore nella sua porzione posteriore discendente . 
i parametri perfusionali sono il volume ematico cerebrale ( cerebral blood volume o cbv ) , il tempo medio di transito ( mean transit time o mtt ) ed il flusso ematico cerebrale ( cerebral blood flow o cbf ) ; alcuni software , inoltre , calcolano il tempo di picco ( time to peak o ttp )  . 
in this patient with severe stenosis of the proximal m1 segment of the right main carotid artery ( mca ) , mtt and cbf maps derived from an arterial input function ( aif ) on the anterior carotid artery ( aca ) ( healthy side ) show abnormal measurements in the right mca territory ( left column ) , whereas mtt and cbf measurements derived from an aif distal to the stenosis appear no longer abnormal ( right column )  . 
in questo paziente con stenosi serrata della porzione prossimale del tratto m1 dellacm di destra , la scelta dellaif sullaca ( lato sano ) produce mappe colore di mtt e cbf marcatamente alterate nel territorio di distribuzione dellacm destra ( colonna sinistra ) , mentre le mappe di mtt e cbf derivate da unaif sullacm destra , a valle della stenosi , appaiono sostanzialmente simmetriche ( colonna destra )  . 
5 software analysis of the arterial ( red ) and venous ( blue ) time - density curves generates the pixel - based parametric maps for mean transit time ( mtt ) , cerebral blood volume ( cbv ) and cerebral blood flow ( cbf )  . 
5 lanalisi matematica operata dal software sulla base della curva arteriosa ( in rosso ) e della curva venosa ( in blu ) genera le mappe parametriche con codice colore pixel per pixel per il mtt , il cbv ed il cbf . 
come accennato sopra , il pixel venoso , scelto da una struttura vascolare ampia , considerato essere esente da volume parziale , e quindi si assume la sua densit come unit di riferimento per il sangue ( 100 ml sangue / 100 g di tessuto ) ; la densit misurata nei singoli pixel tissutali viene poi rapportata alla misurazione venosa , ed espressa in percentuale di questa ( cbv = densit pixel parenchimale / densit pixel venoso ; es , densit nel pixel tissutale = 5% della densit nel pixel venoso cbv del pixel tissutale = 5 ml / 100 g ) ; ora pi chiaro come la sottostima della densit nella roi venosa , per artefatti di volume parziale , causi una sovrastima del cbv a livello tissutale e di riflesso , per lequazione del volume centrale cbf = cbv / mtt , anche del cbf . il cbf misura il volume di sangue distribuito a livello tissutale nellunit di tempo . 
6 in order to obtain quantitative measurements of cerebral blood volume ( cbv ) , cerebral blood flow ( cbf ) , mean transit time ( mtt ) and time to peak ( ttp ) , regions of interest ( rois ) are drawn on the different intracranial vascular territories . 
6 mediante posizionamento di apposite roi si possono ottenere misurazioni quantitative medie del cbv , cbf , mtt e ttp nei diversi territori di distribuzione delle principali arterie intra - craniche . 
it is expressed in [ s ] and is an index of the time that elapses between the beginning of contrast administration and maximum enhancement , or enhancement peak , in the roi . 
 mtt represents capillary transit time , that is , the time between arterial input in the capillary bed and venous outflow . it is expressed in [ s ] , is an sensitive index of cerebral perfusion pressure and , thanks to deconvolution , it is relatively less affected than is ttp by central haemodynamic changes such as cardiac failure or stenosis of the epiaortic arteries . mtt calculation is strictly dependent on the deconvolution process and therefore , in the event of asymmetrical perfusion as in ischaemia , on the choice of aif . 
in practice , mtt may be considered as the difference between the mean width of the time - density curve in a parenchymal pixel and the width of the curve in the reference artery ( aif ) [ 23 ]  . cbv measures the volume of cerebral blood at the capillary - tissue level , after appropriate exclusion from measurements of blood volume contained in large vessels , by means of density thresholds . 
the density measured in the single tissue pixels is then related to the venous density and expressed as a percentage of this ( cbv = parenchymal pixel density / venous pixel density ; e.g. : tissue pixel density = 5% venous pixel density = > cbv of tissue pixel = 5 ml / 100 g )  . 
it should now be clearer why an underestimation of density in the venous roi , due to partial - volume effects , leads to an overestimation of the cbv at the tissue level and consequently of cbf , given the central volume theorem cbf = cbv / mtt . cbf measures the volume of blood distributed at the tissue level in a unit of time . 
it is not measured directly but derived from the central volume theorem and is therefore dependent both on deconvolution and aif selection , which affect mtt , and on cbv measurement . 
 in brief , mtt indicates perfusion pressure , cbv reflects autoregulation mechanisms and capillary volume and cbf is the result of the above parameters . clinical applications acute ischaemic stroke thrombolysis is an approved therapy for acute ischaemic stroke [ 2 , 24 , 25 ] that aims to reperfuse the areas of ischaemic penumbra and limit the extent of the final area of infarction , thus reducing morbidity and disabling sequelae . the current indications for thrombolysis are based on a time window from symptom onset ( < 3h ) , findings of an unenhanced head ct ( no intracranial haemorrhage , no ct evidence of ischaemia of a portion of parenchyma exceeding 33% of the middle cerebral artery territory ) and absence of general contraindications for the drug [ 2 , 24 ]  . 
nevertheless , even such stringent criteria do not eliminate the significant risk of brain haemorrhage ( 15% ) [ 26 ] , and patient selection based on such criteria appears to be unsatisfactory . 
it is recognised that the time window alone is insufficient for accounting for the complex model of brain tissue viability , which in addition to a time factor includes a haemodynamic factor , a tissue factor and an intervention factor [ 27 ]  . 
as a result , individual visualisation of the extent of the infarcted and at - risk areas and of their relationship according to the pathophysiological model of infarct core and ischaemic penumbra has been proposed as a better tool for selecting patients for thrombolytic therapy . 
thrombolysis in patients with extensive cerebral infarctions and small areas of penumbra appears to be of little benefit and lead to a high risk of haemorrhage [ 25 , 28 , 29 ]  . 
on the other hand , although controversial , some studies have demonstrated that a significant proportion of patients with 1232 applicazioni cliniche ictus ischemico acuto la trombolisi una terapia approvata per lictus ischemico acuto [ 2 , 24 , 25 ] che ha lo scopo di riperfondere le aree di penombra ischemica e di ridurre lestensione dellarea finale di infarto , riducendo cos la morbilit e gli esiti invalidanti . 
le attuali indicazioni alla terapia trombolitica sono basate su una finestra temporale dallesordio clinico ( < 3 h ) , sul risultato della tc cranio in condizioni di base ( assenza di emorragia intracranica , assenza di segni tc di ischemia di una porzione di parenchima con estensione superiore al 33% del territorio vascolare dellarteria cerebrale media ) e sullassenza di controindicazioni generali al farmaco [ 2 , 24 ]  . la maggior parte dei pazienti colpiti da ictus ischemico non ricevono trattamento , poich i criteri di eligibilit sono molto restrittivi , soprattutto rispetto alla finestra temporale , anche perch in una significativa percentuale di casi il momento di insorgenza dei sintomi non determinabile con precisione ( classico il caso del cosiddetto wake - up stroke )  . 
ciononostante , perfino criteri cos restrittivi non eliminano il significativo rischio di emorragia intracranica ( 15% ) [ 26 ] e la selezione dei pazienti a cui somministrare la terapia trombolitica , basata su tali criteri , non appare soddisfacente . 
la finestra temporale infatti stata riconosciuta insufficiente a spiegare , da sola , lintricato modello della vitalit del tessuto cerebrale , che invece include , oltre al fattore tempo , un fattore emodinamico , un fattore tissutale ed un fattore terapeutico [ 27 ]  . 
perci la visualizzazione individuale dellestensione delle aree infartuate e di quelle a rischio , e del loro rapporto , secondo il modello fisiopatologico del core infartuale e della penombra ischemica , stato suggerito come un migliore strumento di selezione dei pazienti per il trattamento trombolitico . 
la trombolisi in pazienti con infarti cerebrali estesi e piccole aree di penombra , sarebbe di scarso beneficio e alto rischio emorragico [ 25 , 28 , 29 ] , mentre stato postulato e dimostrato in alcuni studi , ma ancora controverso , che una significativa percentuale di pazienti con limitate aree di infarto ed estese aree di penombra potrebbero beneficiare del trattamento di rivascolarizzazione anche oltre la classica finestra temporale delle 3 ore [ 30 , 31 ]  . il modello del core e della penombra [ 3234 ] postula che nel contesto di unarea di parenchima ipoperfusa , con elevati valori di mtt , spesso dipendente da circoli collaterali , si attuino meccanismi di autoregolazione che , inducendo vasodilatazione ed aumento del cbv , siano capaci di mantenere i valori del cbf sopra la soglia della morte cellulare neuronale ; le aree di parenchima caratterizzate da tali condizioni sono ritenute aree di penombra ischemica , ancora vitali , ad alto rischio di infarto , che potrebbero beneficiare di interventi di riperfusione . 
 limited areas of infarction and extensive areas of penumbra might benefit from revascularisation even beyond the 3 - h time window [ 30 , 31 ]  . the model of the infarct core and penumbra [ 3234 ] postulates that in the context of an area of hypoperfused parenchyma , with high mtt values , often supplied by collateral circulations , autoregulation mechanisms take place that , by inducing vasodilatation and increased cbv , are able to maintain cbf values above the threshold for neuronal death . 
where ischaemic injury is more severe and protracted , on the other hand , autoregulation is unable to maintain cbf , brain tissue sustains irreversible hypoxic damage and cbv values decrease . 
however , these pathophysiological aspects are complicated by other factors such as the duration of hypoperfusion , selective vulnerability of some neurons and physiological conditions during reperfusion [ 38 , 32 ]  . 
radiological evidence of the existence of a penumbra area has been provided by mri with combined diffusionand perfusion - weighted imaging ( dwipwi ) , the results of which have led to the pwi - dwi mismatch hypothesis [ 34 ]  . unenhanced head ct remains the first imaging modality in the assessment of patients with acute focal neurological symptoms ( stroke ) to exclude brain haemorrhage and evaluate the presence of early signs of ischaemia , such as greymatter hypoattenuation and sulcal effacement [ 35 ]  . it has been suggested that , by identifying the areas of cerebral infarction and those at risk of infarction ( ischaemic penumbra ) , the study of brain perfusion may help to select patients for thrombolytic therapy [ 15 ]  . 
other techniques for determining brain perfusion , such as xe - ct , spect and pet , although quantitative , are limited by poor availability in emergency settings and poor patient tolerance , whereas perfusion mri also suffers from an inability to provide quantitative data [ 3 ]  . 
availability in emergency settings , rapidity ( data acquisition < 5 min ) , cost and quantitative measurements probably make p - ct the technique of choice in the assessment of patients with ischaemic stroke [ 16 ]  . 
this ability of p - ct is based on the hypothesis that infarcted areas will exhibit high mtt values and low cbf and cbv values , whereas the areas at risk will show normal or increased cbv values [ 4 , 1517 , 21 , 39 , 40 ]  . 
cbv maps seem to be the least sensitive for ischaemia , with approximately 25% of acute infarctions failing to show any significant change in cbv , but are also the most specific for infarction areas [ 16 , 41 ]  . 
levidenza radiologica dellesistenza della penombra ischemica invece , stata dimostrata da studi combinati di rm con sequenze di diffusione ( dwi ) e perfusione ( pwi ) , dai cui risultati si derivato il concetto di mismatch pwi - dwi [ 34 ]  . la tc cerebrale in condizioni di base rimane la prima modalit di imaging usata per lo studio del paziente con sintomatologia neurologica focale acuta ( ictus ) , per escludere lemorragia intracranica e valutare la presenza di segni precoci di ischemia , quali lipodensit della sostanza grigia e la cancellazione dei solchi [ 35 ]  . stato suggerito che lo studio della perfusione cerebrale , identificando le aree di infarto cerebrale e quelle a rischio stage 1 stage 2 stage 3 cerebral perfusion pressure fig . 
7 the diagram illustrates the changes in mean transit time ( mtt ) , cerebral blood volume ( cbv ) , cerebral blood flow ( cbf ) and oxygen extraction fraction ( oxef ) in response to progressive reduction of cerebral perfusion pressure . 
in patients with stroke , the scan level is usually located in correspondence with the basal ganglia so as to visualise the three main supratentorial vascular territories ( fig 9 )  . 
one limitation of p - ct , especially when compared with mri , is a relative lack of sensitivity to lacunar infarctions [ 44 ] , exposure to ionising radiation and the use of iodinated contrast material . 
nonetheless , administration of iodinated nonionic di infarto ( aree di penombra ischemica ) , possa essere di ausilio nella selezione di pazienti candidati alla terapia trombolitica [ 15 ]  . 
le altre tecniche per misurare la perfusione cerebrale , quali xe - tc , spect e pet , sebbene quantitative , sono limitate dalla disponibilit in emergenza e dalla tolleranza del paziente , mentre la perfusione - rm soffre anche dellimpossibilit di fornire dati quantitativi [ 3 ]  . 
la ptc probabilmente la metodica di scelta , per disponibilit in condizioni di emergenza , per rapidit di esame ( acquisizione dati in < 5 min ) , costi contenuti , e misurazioni quantitative , nella valutazione del paziente con ictus ischemico [ 16 ]  . 
tale capacit della p - tc si basa sullipotesi che le aree infartuate presentino elevati valori di mtt , e ridotti valori di cbf e cbv , mentre nelle aree a rischio i valori di cbv siano normali o aumentati [ 4 , 1517 , 21 , 39 , 40 ]  . 
recenti studi [ 16 , 41 ] suggeriscono che le mappe di mtt siano le pi sensibili per delineare le aree ischemiche , seguite da quelle del cbf ; le mappe di cbv sembrano essere le meno sensibili allischemia , infatti , circa il 25% degli infarti acuti non mostra significative alterazioni di tale parametro , ma sono anche le pi specifiche per le aree di infarto [ 16 , 41 ]  . 
da ci deriva la raccomandazione di valutare prima le mappe di mtt e cbf alla ricerca di aree ipoperfuse , e se queste sono presenti , utilizzare le mappe di cbv per differenziare al loro interno le aree di penombra da quelle di infarto , considerando per che tali mappe possono essere normali anche in caso di infarto . 
 un importante studio prospettico [ 15 ] su pazienti affetti da ischemia cerebrale acuta , segna una pietra miliare nella validazione delle teorie su cui si basa la p - tc , mostrando significativa correlazione tra aree di ridotto cbv ( > 2 , 5 ml / 100 g ) alla p - tc di ammissione e le aree finali di infarto nei controlli a distanza , in pazienti con riperfusione , e altrettanto precisa correlazione tra aree con prolungato mtt ( > 1 , 5 volte rispetto al controlato ) e / o ridotto cbf ( riduzione > 34% rispetto al controlato ) alla p - tc di ammissione e larea finale di infarto , al controllo a distanza , in pazienti senza riperfusione . 
based on threshold values set by the operator , the software generates a colour map ( middle image in the bottom row ) where the colour green is attributed to pixels with increased mean transit time ( mtt ) , as is the case in ischaemic penumbra , and the colour red is attributed to pixels with low cerebral blood volume ( cbv ) , as is the case in the infarct core . 
sulla base di valori soglia predeterminati , il software attribuisce , nella mappa al centro nella fila in basso , il colore verde ai pixel con alterazioni perfusionali con le caratteristiche dei territori di penombra , ed il colore rosso ai pixel con caratteristiche di core infartuale . 
qualora si voglia raddoppiare la copertura anatomica , una seconda sca , nsione , ad un altro livello , pu essere effettuata osservando unattesa , rispetto alla scansione precedente , di 36 mun limite della p - tc , soprattutto in confronto alla rm , la relativa insensibilit agli infarti lacunari [ 44 ] , lesposizione alle radiazioni ionizzanti , e luso del mdc iodato . 
la somministrazione di mdc iodato non ionico in emergenza , anche senza preventiva valutazione della creatininemia , comunque risultata sicura in una larga serie di pazienti con ictus cerebrale acuto , sottoposti a p - tc , con esclusione dei pazienti con insufficienza renale nota , paraproteinemia nota , trapianto renale , assunzione di farmaci nefrotossici , e / o lunga storia di diabete mellito [ 45 ]  . in conclusione , sebbene la validit delle misurazioni quantitative della p - tc sia messa in discussione dalla loro variabilit legata alla scelta dellaif , al posizionamento delle rois ed agli artefatti da volume parziale [ 46 ] e sebbene ulteriori studi siano necessari per confrontare le misurazioni della p - tc con quelle delle altre metodiche di studio della perfusione cerebrale , i risultati sinora disponibili appaiono incoraggianti riguardo alla selezione di pazienti con estesa area di penombra ischemica , e quindi potenzialmente buoni candidati per la terapia trombolitica , ed inoltre , le misurazioni qualitative relative al controlato , offrendo dati affidabili , restano una valida alternativa , comunque indicativa dello stato della perfusionale cerebrale [ 40 ]  . infine utile qui ricordare che linterpretazione delle mappe di perfusione sicuramente facilitata e resa pi accurata dalla disponibilit di informazioni sullo stato anatomico vascolare intraed extra - cranico . 
con lavvento della tecnologia tc multidetettore , langio - tc , eseguita subito dopo la p - tc , con la somministrazione di un ulteriore bolo di 7090 ml di mdc , permette unaccurata valutazione della perviet delle arterie epiaortiche ed intra - craniche , nonch della presenza di circoli collaterali nelle patologie cerebrovascolari acute e pu avere un ruolo significativo nella selezione dei pazienti con ictus ischemico , ai fini di un corretto 1235 fig . 
9 un piano di scansione orientato lungo il tetto delle orbite , e posizionato circa 2 cm cranialmente a queste , consente la visualizzazione , nelle due fette di 10 mm , di una rappresentazione dei tre territori vascolari sopratentoriali : il territorio dellarteria cerebrale anteriore , della media e della posteriore . contrast material , even without preliminary serum creatinine assessment , proved to be safe in a large series of patients with acute cerebral stroke studied by emergency p - ct , after exclusion of those with known renal failure , known paraproteinaemia , kidney transplant , use of nephrotoxic drugs or a long - standing history of diabetes mellitus [ 45 ]  . in conclusion , although the validity of the quantitative measurements of p - ct has been questioned owing to variability related to aif choice , roi positioning and partialvolume artefacts [ 46 ] ; and although further studies are needed to compare p - ct measurements with those provided by other techniques for the study of brain perfusion , encouraging results have been obtained with regard to the selection of patients with an extensive area of ischaemic penumbra and therefore potential candidates for thrombolytic therapy . 
in addition , qualitative measurements of the contralateral side provide reliable data and offer a valuable alternative that is at any rate indicative of the state of cerebral perfusion [ 40 ]  . finally , it is useful to remember that the interpretation of perfusion maps is definitely facilitated and made more accurate by the availability of information on the state of the intraand extracranial vascular anatomy . 
with the advent of mdct technology , ct angiography , performed with the administration of an additional bolus of 7090 ml of contrast immediately after p - ct , allows accurate assessment of the patency of the epiaortic and intracranial arteries and of the presence of collateral circulations in acute cerebrovascular disease . 
in a context of acute cerebrovascular disease , p - ct combined with ct angiography therefore provides a rapid , efficient and practical tool for identifying the cause , extent and pathophysiology of ischaemia , nd for optimising treatment decisions . chronic ischaemic cerebral disease ( vascular reserve ) in patients with chronic cerebral ischaemia caused by stenosis of the intracranial arteries or epiaortic vessels , it is neca . 
nel contesto della patologia cerebro - vascolare acuta , quindi , la p - tc , combinata con langio - tc , rappresenta una soluzione rapida , efficiente e pratica per diagnosticare leziologia , lestensione e la fisiopatologia dellischemia , cos da ottimizzare la scelta terapeutica . patologia cerebrale ischemica cronica ( riserva vascolare ) nei pazienti con ischemia cerebrale cronica causata da stenosi arteriose intra - craniche o dei vasi epi - aortici , necessario distinguere le condizioni di stabile compenso emodinamico , dalle situazioni in cui il compenso instabile , ed il parenchima cerebrale sottoposto ad uno stress emodinamico e metabolico [ 48 ]  . 
tenendo presente il principio del volume centrale ( cbf = cbv / mtt ) , tipicamente , una stenosi arteriosa emodinamicamente significativa induce un prolungamento del mtt che innesca a sua volta i meccanismi dellautoregolazione cerebrale , con conseguente vasodilatazione , riflessa dallincremento del cbv , con cbf normale o lievemente diminuito . 
questo quadro perfusionale tipico per non fornisce informazioni sulla gravit dellipoperfusione , n sulle capacit residue del circolo cerebrale di adattarsi ad ulteriori modificazioni emodinamiche , ovvero sullesistenza di una riserva cerebro - vascolare . 
la valutazione della riserva cerebro - vascolare si pu ottenere osservando come la perfusione cerebrale reagisce in risposta ad uno stress emodinamico , appositamente provocato mediante somministrazione di un agente vasoattivo . 
i pazienti che hanno esaurito la riserva cerebro - vascolare presentano gi in condizioni di base vasodilatazione massima nelle aree ipoperfuse e non sono quindi in grado di rispondere allo stress emodinamico indotto dallacetazolamide . 
si ritiene che tali pazienti siano a rischio elevato di infarto cerebrale e possano beneficiare di interventi di rivascolarizzazione per aumentare il cbf nelle aree ipoperfuse [ 48 ]  . 
di 1000 mg di acetazolamide pu individuare i pazienti a rischio di infarto tra i portatori di stenosi arteriose epi - aortiche o intra - craniche [ 48 ]  . 
dopo la somministrazione di acetazolamide un incremento del cbf pari al 20%40% rispetto alle condizioni di base considerato normale , un incremento di meno del 5% considerato indicativo di insufficienza emodinamica , mentre la riduzione paradossa del cbf rispetto alla condizione di base segno di furto vascolare , indicativo di tessuto ad alto rischio di infarto [ 49 ]  . 
lacetazolamide generalmente ben tollerata ; sono stati descritti effetti collaterali lievi quali parestesie e cefalea e sebbene siano stati riportati casi di attacchi ischemici transitori , leventuale ischemia indotta sempre stata reversibile [ 48 ]  . 
10 computed tomography angiography ( cta ) , used to complement perfusion ct in this patient with acute right hemiplegia and aphasia , clearly shows left m1 occlusion ( red arrow on exam at 2.5 h ) , which is the cause of the perfusion abnormalities visible on the colour map . 
10 langio - tc cerebrale , esame complementare alla perfusione - tc , dimostra chiaramente , in questo paziente con esordio acuto di emiplegia destra ed afasia , locclusione del tratto m1 dellarteria cerebrale media di sinistra ( freccia rossa nellesame a 2 , 5 h ) , responsabile delle alterazioni perfusionali evidenziate dalla mappa colore . 
il controllo angio - tc a 24 h di distanza , dopo trattamento fibrinolitico , dimostra la ricanalizzazione del vaso ( freccia rossa )  . essary to distinguish between situations of stable haemodynamic compensation and those in which compensation is unstable and the brain parenchyma is subject to haemodynamic and metabolic stress [ 48 ]  . 
bearing in mind the central volume theorem ( cbf = cbv / mtt ) , a haemodynamically significant arterial stenosis will typically result in a prolonged mtt , which in turn will trigger cerebral autoregulation mechanisms , with consequent vasodilatation reflected by an increase in cbv , with normal or slightly decreased cbf . 
this typical perfusion pattern does not , however , give information on the severity of hypoperfusion or on the residual capacity of the cerebral circulation to adjust to further haemodynamic changes in other words , on the existence of a cerebrovascular reserve . 
patients who have exhausted the cerebrovascular reserve show maximal vasodilatation in the hypoperfused areas already at baseline and are therefore unable to respond to the haemodynamic stress induced by acetazolamide . these patients are believed to be at high risk for cerebral infarction and to benefit from revascularisation procedures to increase cbf in hypoperfused areas [ 48 ]  . 
11 perfusion - computed tomography ( p - ct ) before and after right internal carotid endarterectomy.in this patient with mild but progressive left hemisyndrome , ct angiography and p - ct ( upper row ) show severe right internal carotid stenosis causing hypoperfusion of the whole right cerebral hemisphere ( there is a foetal origin of the right posterior cerebral artery , not shown in the figure )  . 
lo studio angio - tc e p - tc ( fila superiore ) , in questo paziente con emisindrome sn lieve ma ingravescente , mostra stenosi serrata del bulbo carotideo destro , cui si associa ipoperfusione di gran parte dellemisfero cerebrale destro ( il paziente portatore di unorigine fetale dellarteria cerebrale posteriore destra , rifornita per via comunicante posteriore ; non mostrata nella figura ) , testimoniata dal prolungamento del mtt , dalla riduzione del cbf e dal lieve aumento del cbv , espressione dei meccanismi di autoregolazione . 
il controllo angio - tc e p - tc ( fila inferiore ) eseguito tre giorni dopo lintervento di endoarterectomia carotidea destra documenta la ricostituzione del lume vascolare e la risoluzione del deficit perfusionale emisferico destro . those with stenosis of the epiaortic or intracranial arteries [ 48 ]  . 
an increase less than 5% is considered indicative of haemodynamic insufficiency , whereas a paradoxical reduction of cbf relative to baseline is a sign of vascular shunting and thus of tissue at a high risk of infarction [ 49 ]  . 
acetazolamide is generally well tolerated ; the reported side effects are mild , such as paraesthesia and headache , and although there have been cases of transient ischaemic attacks , the induced ischaemia was always reversible [ 48 ]  . 
angiographic evidence of vasospasm is present in 60%80% of patients with sah , whereas 30% of patients present symptoms of vasospasm , and half of these develop cerebral infarction [ 51 ]  . 
transcranial doppler ultrasound is the most emorragia sub - aracnoidea e vasospasmo il vasospasmo una complicanza frequente dellemorragia sub - aracnoidea ( esa ) nella fase subacuta precoce [ 6 ]  . levidenza angiografica di vasospasmo presente nel 60%80% dei pazienti con esa , il 30% ne presenta i sintomi , e la met di questi sviluppa un infarto cerebrale [ 51 ]  . leco - doppler transcranico la modalit pi usata per il follow - up non invasivo in questi pazienti , per guidare le scelte terapeutiche e controllarne lefficacia , ma , oltre ad essere operatore dipendente , non offre misurazioni quantitative a livello tissutale ed gravata da scarsa specificit [ 52 ]  . 
anche se non sono ancora disponibili studi in grado di fornire valori di soglia dei parametri di perfusione , la misurazione del cbf , cbv e mtt con p - tc , associata a studi anatomici vascolari con angio - tc , potrebbe rappresentare un utile strumento diagnostico per il follow - up non invasivo dei pazienti con vasospasmo conseguente ad esa [ 6 ] , fornendo informazioni utili al trattamento medico ed anche alla scelta della tempistica chirurgica . trauma cranico la predizione della prognosi nei pazienti con trauma cranico severo rimane un compito complesso e controverso . 
however , it is operator dependent , unable to provide quantitative measurements at the tissue level and suffers from limited specificity [ 52 ]  . even though no study has yet been able to provide threshold values for the perfusion parameters , p - ct measurement of cbf , cbv and mtt , combined with vascular anatomy studies with ct angiography , might represent a valuable diagnostic tool for the noninvasive follow - up of patients with sah - related vasospasm [ 6 ] , as it provides information useful for medical management of patients and for choosing the best timing for surgery . head trauma predicting outcomes in patients with severe head trauma remains a complex and controversial issue . 
a finding of cerebral hypoperfusion , in generally hypotensive patients , indicates a loss of cerebrovascular autoregulation and is thought to be a predictor of the development of cerebral oedema , whereas normal or increased perfusion values would indicate a positive outcome [ 54 ]  . brain tumours in the study of brain tumours , there is growing interest in the noninvasive assessment of tumour vascularity [ 55 ]  . 
some brain tumours are characterised by neoangiogenesis and hypervascularity , which result in increased microvascular permeability [ measured as permeability surface area product ( ps ) or as contrast transfer coefficient ( k - trans ) , in ml / 100g per minute ] and increased cbv , due to the presence of numerous tortuous vascular structures , with immature , disrupted or absent bbb [ 56 ]  . 
contrast enhancement , the key diagnostic element in morphological imaging , is a poorly specific finding that in tumours may be caused by bbb abnormalities , hypervascularity , extent of the interstitial space or a combination of these factors . 
if the extreme disruption of microvascular permeability of some tumours causes excessive leakage of contrast material during the first pass of 1238 zionale possiede alta sensibilit nellindividuazione di lesioni intra - craniche che necessitano di intervento neurochirurgico , ma non offre validi elementi di tipo prognostico . 
il riscontro di ipoperfusione cerebrale , in pazienti generalmente ipotesi , indica la perdita dellautoregolazione cerebro - vascolare e sarebbe in grado di predire lo sviluppo di edema cerebrale , mentre valori di perfusione normali o aumentati rappresenterebbero indicatori prognostici positivi [ 54 ]  . tumori intra - cranici nello studio delle neoplasie intra - craniche crescente interesse rivolto alla valutazione non - invasiva della vascolarizzazione tumorale [ 55 ]  . 
alcuni tumori intracranici sono caratterizzati da neo - angiogenesi ed ipervascolarizzazione , che risultano in aumentata permeabilit vascolare ( misurata come permeability surface product ( ps ) o come coefficiente di trasferimento di contrasto ( k - trans ) , in [ ml / 100 g / min ] ) , e incremento del cbv , per la presenza di strutture vascolari pi numerose , tortuose , con barriera emato - encefalica immatura , danneggiata o assente [ 56 ]  . 
il contrastenhancement , elemento diagnostico chiave dellimaging morfologico , un reperto poco specifico , che nei tumori pu essere determinato dallalterazione della bee , dallipervascolarizzazione , dallampiezza dello spazio interstiziale o dalla concomitanza dei suddetti fattori . 
numerosi studi [ 55 , 56 ] dimostrano che nei gliomi , la ps ed il cbv , misurati con tecniche di rm - perfusione ( con sequenze contrast - enhanced t2 * - w , basate sulla suscettibilit magnetica , e contrast - enhanced t1 - w ) , correlano con lindice mitotico , il grading istologico e laggressivit biologica . 
se lestrema alterazione della permeabilit vascolare in alcuni tumori causa uno stravaso eccessivo del mezzo di contrasto gi durante il primo passaggio del bolo , le misurazioni del cbv , per essere accurate , necessitano di appropriate strategie di correzione ( ad esempio procedure di gamma - fitting della curva densit - tempo )  . 
in tali tumori , ipervascolarizzati e con alterata permeabilit , le misurazioni della ps o del k - trans , sembrano essere pi utili , perch correlano pi strettamente con il grado dei glomi . 
inoltre anche la risposta alle terapie anti - angiogenetiche recentemente introdotte , con riduzione della crescita tumorale , trova il suo corrispettivo in una riduzione dei valori di ps [ 57 ]  . 
in this patient with head trauma and left parietal bone fracture , p - ct shows a large area of hypoperfused brain parenchyma in the left temporoparietooccipital region , well beyond the limits of the hypodense contusion documented by enhanced ct . 
in questo paziente con trauma cranico e frattura parietale sinistra lo studio p - tc mostra una vasta area di ipoperfusione temporo - parieto - occipitale sn , caratterizzata da aumentati valori di mtt e ridotti valori di cbf e cbv , estesa ben oltre i limiti della lesione contusiva ipodensa temporo - parietale sinistra , adiacente alla frattura , visibile nellesame tc morfologico . 
in such hypervascular tumours with disrupted permeability , ps or k - trans measurements appear to be more useful , as they correlate more closely with the grade of gliomas . 
however , in the subgroup of highgrade gliomas [ world health organisation ( who ) iii - iv ] , k - trans was directly related to length of survival . 
measurement of bbb permeability by p - ct or p - mri is based on the same mathematical models , the most frequently used of which is the patlak model [ 59 ]  . 
this model describes a method to measure the transfer - rate constant of contrast material from the vessel to the interstitium , through a mathematical comparison ( deconvolution ) between the timedensity curve or a time - signal curve obtained in tumour tiscon la sopravvivenza ; questa evidenza viene spiegata dagli autori come possibile effetto dellaumentata permeabilit vascolare nel tumore , che permetterebbe una migliore penetrazione degli agenti chemioterapici ed anti - angiogenici . 
la misurazione della permeabilit della bee ottenuta con ptc o con p - rm si fonda sulluso degli stessi modelli matematici , tra i quali , uno dei pi usati il modello di patlak [ 59 ]  . 
questo modello descrive un metodo per misurare la costante di trasferimento del mezzo di contrasto dal vaso allinterstizio , operando un processo di confronto matematico ( deconvoluzione ) tra una curva densit - tempo o segnale - tempo rilevata nel tessuto tumorale ( dove presente stravaso di mdc ) ed una curva rilevata nel lume di unarteria di riferimento o aif ( dove si assume non avvenga stravaso di mdc )  . 
le tecniche di imaging di perfusione trovano utilit nello studio dei tumori intra - cranici nel tentativo di facilitare la distinzione tra tumore primitivo e metastatico , nel rendere pi accurato il grading , nella valutazione della risposta alle terapie antiangiogenetiche , e nella diagnosi differenziale tra radionecrosi e recidiva tumorale ; inoltre si sta affermando il ruolo dellimaging perfusionale per la scelta del bersaglio delle procedure bioptiche , scelta basata sulla individuazione delle aree tumorali a maggiore aggressivit 1239 a . 
perfusion - imaging techniques have been found useful in the study of brain tumours to facilitate the distinction between primary or metastatic tumour , make grading more accurate , evaluate response to antiangiogenic agents and differentiate radiation necrosis from tumour recurrence . 
in addition , the role of perfusion imaging is also gaining ground in guiding biopsy procedures , where the biopsy target is chosen based on identification of the most malignant area in a heterogeneous tumour [ 56 ]  . in contrast to p - mri , only a few small studies are available on the use of p - ct in human brain tumours [ 7 , 60 , 61 ]  . the examination is performed using a similar technique to that described for ischaemic disease , and the scan level corresponds to the tumour site . 
cbv and permeability measurements are made by positioning several small rois over the tumour mass , taking care to exclude large vascular structures from the measurements , and the measurements in the rois are compared with those of a mirror area on the contralateral side . 
the most recent scientific data , many of which are still unpublished , suggest that it is the maximum rather than the mean values of cbv and ps in the tumour that correlate with histopathological grading , as is also the case with histological assessment , which assigns a who grade on the basis of the most malignant portion of the tumour . in theory , p - ct could offer several advantages over pmri in terms of spatial resolution , insensitivity to paramagnetic susceptibility artefacts ( in cases with haemorrhage , calcifications , metallic implants ) , linear correlation between contrast concentration and density and quantitative cbv and permeability measurements , even in hypervascular tumours . in practice , however , no published study has compared the results of the two methods on representative series of patients with brain tumours . 
on the other hand , radiation dose , potential toxicity of iodinated contrast material and limited anatomical coverage of p - ct still justify the predominant use of mri , a modality that remains crucial in the morphological imaging of tumours [ 7 ]  . limitations , artefacts and controversies although the ability of p - ct to provide quantitative measurements has been confirmed by comparison with pet , spect and xe - ct , its accuracy has not yet been unquestionably demonstrated , probably as a result of heterogeneous study methodologies adopted by the various authors . 
furthermore , the quantitative measurements are dependent on several variables , such as the software used , bolus dispersion ( caused by too slow an injection rate , insufficient cardiac function , haemodynamically significant arterial stenoses ) , aif selection , partial - volume artefacts , the misleading contribution of 1240 nel contesto delleterogeneit della neoplasia [ 56 ]  . 
 contrariamente a quanto verificatosi per gli studi di rmperfusione , sono disponibili solo pochi e limitati studi sulluso della p - tc nei tumori cerebrali negli uomini [ 7 , 60 , 61 ]  . 
per la misurazione della permeabilit preferibile disporre di curve densit / tempo allequilibrio invece che limitate al primo passaggio del bolo ; quindi oltre i 50 s di scansione , si continuano ad acquisire immagini in modalit cine , per circa 3 min , al ritmo di 1 scansione ogni 15 s . 
le misurazioni del cbv e della permeabilit si effettuano posizionando multiple piccole roi nel contesto della massa tumorale , avendo cura di escludere dalla misurazione le grosse strutture vascolari , e si confrontano ad unarea speculare nel controlato . 
i pi recenti dati scientifici , molti ancora non pubblicati , suggeriscono che pi dei valori medi di cbv e ps nel contesto della lesione tumorale , siano i valori massimi a correlare con il grading istopatologico , cos come accade peraltro per lesame istologico , che assegna il grado who in base alla porzione del tumore a pi alta malignit . in teoria la p - tc potrebbe avere vantaggi rispetto alle tecniche di rm - perfusione , in termini di risoluzione spaziale , insensibilit agli artefatti di suscettibilit paramagnetica ( in casi con emorragia , calcificazioni , impianti metallici ) , correlazione lineare tra concentrazione di mdc e densit , e misurazioni quantitative di cbv e permeabilit , anche in tumori ipervascolari . 
daltra parte , la dose di radiazioni , la potenziale tossicit del mdc iodato , la limitata copertura anatomica , rappresentano fattori che ancora giustificano luso prevalente della rm , metodica comunque irrinunciabile nellimaging morfologico nei tumori [ 7 ]  . limitazioni , artefatti e controversie sebbene la capacit della p - tc di fornire misurazioni quantitative sia stata confermata , come precedentemente detto , da studi comparativi con pet , spect e xe - tc , la sua accuratezza non ancora stata provata in maniera incontrovertibile , probabilmente anche a causa di metodologie di studio non omogenee tra i diversi autori . 
le misurazioni quantitative sono inoltre dipendenti da multiple variabili , quali il software impiegato , la dispersione del bolo ( causata da uniniezione troppo lenta , uninsufficiente funzione cardiaca , stenosi arteriose emodinamicamente significative ) , la scelta dellaif , gli artefatti da volume parziale , lingannevole contributo del liquor e delle strutture vascolari alle misurazioni , il posizionamento delle roi , ed infine le significative differenze perfusionali tra sostanza grigia e sostanza bianca . 
although commercial software packages for analysis of perfusion data are relatively easy to use , operators need to master the issues inherent to the examination and postprocessing procedures and require training in using thedata derived from perfusion studies conducted by expert radiologists have been shown to have good reproducibility [ 62 ] , whereas no study has evaluated reproducibility of data among nonexpert radiologists . 
in patients with ischaemic stroke , thrombolytic therapy , when indicated , should be initiated as soon as possible , so diagnostic imaging needs to be timely , accurate and fast . 
complementation of a conventional ct study of the brain with pct and ct angiography usually requires 10 - 12 min , whereas postprocessing with automatic software takes no longer than 3 min to provide colour - coded parametric maps that can guide the clinical treatment decisions . 
the disadvantage of this small delay in imaging procedures is widely compensated for by the acquisition of diagnostic information of major clinical importance , above all in those patients in the 3to 6 - h time window and those with an unknown time interval from symptom onset of symptoms , who are currently excluded from treatment . 
 as is known , p - ct employs ionising radiation and potentially nephrotoxic , hyperosmolar and allergenic iodinated contrast material , so the use of the technique requires individual clinical assessment , appropriate indications and strategies to contain the radiation dose . 
it is therefore a valuable alternative to other modalities for the measurement of brain perfusion , compared to which it has the advantage of being readily available and accessible even in emergency settings . 
for this reason , p - ct proves most useful in acute ischaemic stroke where , used in combination with ct angiography , it offers a complete overview of the causes of hypoperfusion and its haemodynamic and pathophysiological effects on the brain in a fast , noninvasive manner . 
p - ct is also helpful in patients with other , acute or chronic , cerebrovascular diseases ; in the follow - up of patients with sah ; in preand postoperative assessment of patients undergoing cerebral revascularisation procedures ; and in brain tumours for diagnosis , grading , biopsy guidance and monitoring treatment response . further studies are required to establish the accuracy , reliability and reproducibility of the quantitative measurements , but available data appear encouraging and indicate a major clinical impact . di post - processing e di un periodo di training per il loro uso ; una buona riproducibilit stata dimostrata , nei dati estratti dagli studi di perfusione , tra radiologi esperti [ 62 ] , mentre studi di questo tipo non sono ancora stati condotti per valutare la riproducibilit tra medici non esperti . 
ulteriori svantaggi della p - tc , soprattutto nelle applicazioni che riguardano la patologia ischemica , come gi detto , sono la limitata copertura anatomica e la scarsa sensibilit verso gli infarti lacunari . 
nei pazienti colpiti da ictus ischemico di fondamentale importanza che il trattamento trombolitico sia iniziato , se indicato , il pi rapidamente possibile , ed quindi auspicabile che la fase diagnostica radiologica sia tempestiva , accurata e veloce . 
il completamento di un esame tc convenzionale dellencefalo con lo studio perfusionale e angiotc richiede solitamente 1012 minuti , mentre le procedure di post - processing , con software automatici , richiedono non pi di 3 minuti per ottenere mappe parametriche colore ai fini di decisioni clinico - terapeutiche ; solo valutazioni quantitative accurate , solitamente non necessarie in situazioni di emergenza , richiedono tempi di post - processing pi lunghi . 
lo svantaggio di questo modesto ritardo nelle procedure di imaging di solito ampiamente compensato dallacquisizione di informazioni diagnostiche di grande rilevanza clinica , soprattutto in quei pazienti nella finestra delle 36 ore ed in quelli con intervallo sconosciuto dallinsorgenza dei sintomi , attualmente esclusi dal trattamento . la p - tc , come ovvio , utilizza radiazioni ionizzanti , e mdc iodato potenzialmente nefrotossico , iperosmolare e allergenico , pertanto limpiego di tale metodica necessita di valutazione clinica individualizzata , indicazioni appropriate , e strategie di contenimento della dose di radiazioni . conclusioni in conclusione , la p - tc permette una valutazione quantitativa affidabile e veloce del cbf e del cbv , offrendo cos una visualizzazione diretta dei meccanismi di autoregolazione cerebrale , e ponendosi come valida alternativa alle altre modalit di misurazione della perfusione cerebrale , rispetto alle quali ha il maggior vantaggio di essere una tecnica prontamente disponibile ed accessibile , anche in condizioni di emergenza . 
per tale ragione la p - tc utile soprattutto nella patologia ischemica cerebrale acuta , situazione in cui , con il concomitante impiego dellangio - tc , offre in maniera rapida e non - invasiva , una completa panoramica della causa dellipoperfusione , nonch delle sue ripercussioni emodinamiche e fisiopatologiche sul parenchima cerebrale . 
this study was done to evaluate the diagnostic role of enteroclysis with multidetector computed tomography ( mdct ) and single - detector ct ( sdct ) in patients affected by smallbowel crohns disease . 
in comparison with double - contrast enteroclysis , sensitivity , specificity and diagnostic accuracy were 90% , 71% and 89% for sdct and 92% , 83% and 90% for mdct . 
mdct is superior to sdct because it allows a better spatial resolution and improves depiction of the pathological patterns of crohns disease . key words crohns disease ct enteroclysis multidetector ct riassunto obiettivo . 
quarantacinque pazienti sono stati sottoposti ad enteroclisi - tc a singolo detettore ( n = 20 ) o multidetettore ( n = 25 ) previa somministrazione di metilcellulosa tramite sondino naso - digiunale . 
confrontando i dati tc con quelli del clisma del tenue , abbiamo riscontrato valori di sensibilit , specificit ed accuratezza diagnostica rispettivamente del 90% , 71% e 89% con la tcsd e del 92 , 83 e 90% con la tcmd . 
la tcmd da preferire alla tcsd per la sua migliore risoluzione spaziale e perch consente una migliore rappresentazione dei segni patologici di malattia di crohn . parole chiave malattia di crohn tc enteroclisi tc multidetettore introduction introduzione crohns disease is a chronic granulomatous disease that causes transmural inflammation of the small bowel . 
complications ( abscess , fistula , stenosis ) are common and can be seen in over 40% of patients [ 1 ]  . conventional radiology techniques ( small - bowel barium examination , double - contrast enema ) visualise the small la malattia di crohn una malattia granulomatosa cronica che determina infiammazione transmurale dellintestino tenue . 
the added value of computed tomography ( ct ) is that it directly depicts the bowel wall , perienteric fat , mesentery , vessels and lymph nodes , as well as local complications of crohns disease and associated pathological conditions [ 2 ]  . 
the sensitivity of small - bowel ct is not well known and depends mainly on the quality of the study , severity of the inflammation and presence of mesenteric and intraperitoneal abnormalities . the diagnostic approach to small - bowel disease has recently been refined by the advent of spiral ct , which offers better spatial resolution and the possibility of generating multiplanar reconstructions . 
another technique proposed for the study of the small bowel is ct enteroclysis , where contrast material is administered through a nasojejunal tube , and contiguous axial images are obtained after complete distension of the small bowel [ 3 ]  . 
the function of ct enteroclysis is to overcome the individual pitfalls of ct and small - bowel enema and combine the advantages of the two techniques into a single study . 
the theoretical advantage of ct enteroclysis is the ability to simultaneously depict intraluminal , mural and extraintestinal complications of crohns disease [ 2 , 3 ]  . the aim of this study was to evaluate the diagnostic accuracy of ct enteroclysis with singleand multidetector detector techniques in symptomatic patients with a clinical suspicion or firm diagnosis of crohns disease . materials and methods we prospectively and consecutively evaluated 45 patients ( 22 men , 23 women ) aged 2570 years ( mean age 43 years ) presenting with a suspected or known diagnosis of crohns disease on the basis of clinical , laboratory and / or endoscopic findings . 
on the day before the procedure , they took a mixture of a and b sennosides with a cup of tea with sugar at 8 : 00 h , 15 mg of magnesium sulphate in three quarters of a glass of warm water at 17 : 00 h , followed by 3 l of water over the next 45 h ; at 21 : 00 h they had a cup of clear soup and fasted from 21 : 00 h onwards . all patients were intubated with a 12to 16 - fr nasojejunal tube ( create medic co . , yokohama , japan ) introduced under fluoroscopic guidance . 
patients were then taken into the ct room where contrast material ( 2 , 000 ml of 0.5% methylcellulose ) was administered manually with 60 - ml syringes , with care being taken to ensure a constant and continuous injection rate of about two syringes per minute . 
il valore aggiunto della tc quello di permettere la visualizzazione diretta della parete intestinale , del tessuto adiposo periviscerale , del mesentere , dei vasi e dei linfonodi , nonch delle complicanze locali di malattia di crohn e patologie associate [ 2 ]  . 
la sensibilit dello studio tc nelle malattie del tenue non ben conosciuta e si affida principalmente alla qualit dellindagine , alla severit dellinfiammazione e alla presenza di alterazioni mesenteriche ed intraperitoneali . 
lapproccio diagnostico allo studio del piccolo intestino stato negli ultimi anni ulteriormente modificato grazie allo sviluppo della tecnica spirale che ha consentito una migliore risoluzione spaziale e la possibilit di elaborare ricostruzioni multiplanari . 
contemporaneamente , nello studio del tenue stata proposta la tc enteroclisi in cui il mezzo di contrasto ( mdc ) viene somministrato attraverso un sondino naso - digiunale e dopo la totale distensione del piccolo intestino vengono riprese immagini assiali contigue dello stesso [ 3 ]  . 
la funzione di questa indagine quella di supplire alle carenze individuali della tc e del clisma del tenue , quindi di combinare i vantaggi di ciascuna tecnica in ununica metodica di studio . 
il teorico vantaggio della tc enteroclisi la capacit di mostrare contemporaneamente le complicanze intraluminali , murali ed extramurali della malattia di crohn [ 2 , 3 ]  . lo scopo di questo studio valutare laccuratezza diagnostica della tc enteroclisi effettuata con tecnica a singolo detettore e multidetettore in pazienti sintomatici con sospetto clinico o diagnosi certa di malattia di crohn . materiali e metodi sono stati valutati prospettivamente e consecutivamente 45 pazienti ( 22 uomini , 23 donne ) , di et compresa tra 25 e 70 anni ( et media 43 anni )  . 
il giorno prima dellesame i pazienti hanno ingerito alle ore 8 : 00 una miscela di sennosidi a e b con una tazza di t zuccherato e alle 17 : 00 15 mg di solfato di magnesio sciolti in 3 / 4 di un bicchiere di acqua tiepida , seguita dallassunzione di 3 litri di acqua nelle 45 ore successive . 
il digiuno stato rispettato delle 21 : 00 in poi . a tutti i pazienti stato introdotto sotto guida fluoroscopica un sondino naso - digiunale , del calibro 12 - 16 f ( create medic co , yokohama , giappone )  . 
the contrast - enhanced study was acquired 40 s after iv administration of 130150 ml contrast material ( ultravist 370 , schering ag , berlin , germany ) at a flow rate of 3 ml / s . axial images were processed on a computer workstation ( advantage windows , ge medical system ) to obtain 2d coronal and , if necessary , sagittal constructions . 
following ct , the nasojejunal tube was not removed , and after 68 h , all patients underwent a small - bowel barium enema according to the herlinger method [ 4 ] with digital technique . 
all ct images were assessed for the following signs : presence , site and number of abnormal bowel segments , degree of wall thickening , degree of mural enhancement [ hounsfield units ( hu ) ] , target sign , comb sign , perienteric stranding , fistulas , fibrofatty proliferation , lymphadenopathy , abscesses and other signs ( adhesions , incidental findings in the other abdominal organs , benign or malignant neoplasms of the small bowel )  . 
we evaluated small - bowel distension , which was classified according to a 4 - point scale for each segment ( 0 : no distension ; 1 : incomplete distension ; 2 : partial distension ; 3 : complete distension )  . 
the coronal and sagittal reconstructions were compared with the axial images to assess whether they provided additional information . images obtained with the small - bowel enema were checked for the following signs : oedematous folds , erosions , aphthoid and linear ulcers , fistulas , cobblestoning , micronodules , wall thickening , prestenotic dilatations , mesenteric inflammation and other signs ( adhesions , radiological findings suspicious for benign or malignant neoplasm )  . 
the ct and barium enema results were compared with those of retrograde 1190 sondino stata fissata a valle del treiz tramite linsufflazione di aria in un palloncino in modo da prevenire reflussi di mezzo di contrasto nello stomaco . 
i pazienti sono stati quindi condotti in sala tc ove il mezzo di contrasto ( 2000 ml di metilcellulosa allo 0 , 5% ) stato somministrato manualmente usando siringhe da 60 ml . 
abbiamo cercato di ottenere uniniezione costante e continua , circa 2 siringhe al minuto . al fine di evitare spasmi , ottenere unomogenea distensione del piccolo intestino , ridurre il disagio e la tensione addominale stato somministrato ai pazienti un farmaco anticolinergico ( n - butilscopolamina )  . 
abbiamo somministrato 10 mg di farmaco quando il paziente ha cominciato ad avvertire dolore addominale e 10 mg subito prima dellacquisizione delle immagini tc . venti pazienti sono stati studiati con tecnica tc spirale a singolo detettore ( tcsd ) ( prospeed , advantage sx spiral scanner , ge medical system , milwakee , usa ) con i seguenti parametri di acquisizione : collimazione 5 mm , intervallo 8 mm , ricostruzione 4 mm , kv 120 , mas 300 . 
venticinque pazienti sono stati studiati con tc multidetettore a 16 strati ( tcmd ) ( lightspeed pro16 , ge medical system , milwakee , usa ) con i seguenti parametri di scansione : collimazione 1 , 25 mm , velocit di scorrimento del tavolo 13 , 75 mm / rot , kv 120 , mas 500 , pitch 1 , 375 , tempo di rotazione 0 , 6 s . al termine dellinfusione di metilcellulosa lesame tc stato eseguito prima e durante la somministrazione di mdc iodato ev . 
nei pazienti in cui allesame preliminare senza mdc iodato veniva riscontrata una inadeguata distensione del piccolo intestino abbiamo somministrato altri 200250 ml di metilcellulosa prima di procedere allo studio tc con mdc ev . 
questultimo stato acquisito 40 secondi dopo la somministrazione ev di 130150 ml di mezzo di contrasto ( ultravist 370 , schering ag , berlino , germania ) iniettato alla velocit di 3 ml / s . con una computer workstation ( advantage windows , ge medical system ) le immagini assiali sono state rielaborate con ricostruzioni 2d , coronali ed eventualmente sagittali . 
il sondino non stato rimosso al termine dellesame tc e dopo 68 ore tutti i pazienti sono stati studiati con clisma del tenue effettuato secondo il metodo di herlinger [ 4 ] con tecnica digitale . 
sono stati somministrati 200250 ml di una sospensione di solfato di bario ( prontobario 60% , bracco , milano , italia ) seguiti dallinfusione di 10002000 ml ( in media 1200 ml ) allo 0 , 5% di metilcellulosa . 
sono stati eseguiti radiogrammi nelle proiezioni postero - anteriore , antero - posteriore , con e senza compressione delle anse . i risultati dello studio tc e del clisma del tenue sono stati confrontati e valutati da due radiologi esperti in radiologia gastroenterologica . 
abbiamo anche valutato la distensione delle anse del piccolo intestino che per ciascun segmento stata classificata con una scala di 4 punti ( 0 : distensione assente ; 1 : distensione incompleta ; 2 : distensione parziale ; 3 : distensione completa )  . 
le ricostruzioni coronali e sagittali sono state confrontate con le immagini assiali ed abbiamo valutato le eventuali informazioni aggiuntive da esse ottenibili . le immagini ottenute con clisma del tenue sono state analizzate ricercando i seguenti segni : edema delle pliche , erosioni , ulcerazioni aftoidi e lineari , fistole , aspetto ad acciottolato , micronoduli , ispessimenti parietali , dilatazioni prestenotiche , infiammazione mesenterica , altri segni ( aderenze , alterazioni radiologiche sospette per presenza di neoplasia benigna o maligna )  . 
in these loops , wall thickness ranged from 4 mm to 10 mm ( mean 6.5 mm ) , loop diameter was between 12 and 30 mm ( mean 21 mm ) and lumen diameter between 2 and 18 mm ( mean 8 mm )  . 
complete distension was observed in 27 / 45 ( 61% ) patients in the proximal jejunum , in 35 / 45 ( 77% ) in the distal jejunum , in 40 / 45 ( 88% ) in the proximal ileum , in 39 / 45 table 1 ct signs in patients with crohns disease target sign deep ulcers / sinus tracts perienteric stranding comb sign fibrofatty proliferation stenosis fistulas lymphadenopathy ( > 1 cm ) adhesions abscess segno del bersaglio ulcere profonde / sinus tract strie perienteriche segno del pettine proliferazione fibroadiposa stenosi fistole linfoadenopatie ( > 1 cm ) segni indiretti di aderenze ascesso signs sdct ; number of patients mdct ; number of patients sdct , single - detector computed tomography ; mdct , multidetector computed tomography tabella 1 segni tc nei pazienti con morbo di crohn segni tcsd ; numero pazienti tcmd ; numero pazienti tcsd , tomografia computerizzata a singolo detettore ; tcmd , tomografia computerizzata multidetettore 1191 l.m. 
i valori di densit dei segmenti coinvolti ottenuti dallo studio senza contrasto erano compresi tra 16 e 57 hu e il loro livello di enhancement dopo mdc variava tra 82 e 208 hu . 
il segno del bersaglio stato osservato in 14 pazienti , ulcere profonde / sinus tract in 4 , lo stranding perienterico in 13 , il segno del pettine in 7 . 
una completa distensione stata osservata in 27 / 45 ( 61% ) pazienti nel digiuno prossimale , in 35 / 45 ( 77% ) nel digiuno distale , in 40 / 45 ( 88% ) nellileo prossimale , in 39 / 45 ( 86% ) nellileo distale , in 41 / 45 ( 92% ) nellultima ansa ileale . 
5 tc multidetettore : due anse ileali appaiono a pareti ispessite ( frecce nere ) ; limitrofa ad esse ( freccia bianca ) presente formazione ascessuale . ( 86% ) in the distal ileum and in 41 / 45 ( 92% ) in the last ileal loop . 
the three false positive castable 2 degree of small - bowel distension on ct 0 : absent 1 : incomplete 2 : partial 3 : complete pj , proximal jejunum ; dj , distal jejunum ; pi , proximal ileum ; di , distal ileum ; lil , last ileal loop tabella 2 grado di distensione delle anse dellintestino tenue allesame tc 0 : assente 1 : incompleta 2 : parziale 3 : completa dp , digiuno prossimale ; dd , digiuno distale ; ip , ileo prossimale ; id , ileo distale ; uai , ultima ansa ileale 1193 l.m. 
abbiamo quindi riscontrato valori di sensibilit , specificit ed accuratezza diagnostica del 90% , 71% e 89% con tecnica a singolo detettore e del 92% , 83% e del 90% con tecnica multidetettore . 
essa ha confermato i reperti di normalit in 5 pazienti e un morbo di crohn in 20 , un quadro di ileite aspecifica in 1 , ileite da reflusso in 1 e ileite ischemica in 1 . 
i due terzi dei casi di leite aspecifica , diagnosticati dallenteroclisi tc , presentavano endoscopicamente un quadro compatibile con morbo di crohn . small - bowel ct allows visualisation of the entire organ and , with adequate bowel distension and elimination of respiratory and peristaltic artefacts , enables evaluation of the type and luso della tc nello studio del piccolo intestino consente la visualizzazione dellintero organo e , previa unadeguata didiscussione discussion fig . 
8a , b esempio di migliore visualizzazione dellansa patologica ( frecce ) con tecnica multidetettore ( a ) rispetto alla tecnica a singolo detettore ( b )  . extent of mural enhancement after administration of iv iodinated contrast material . 
as with conventional radiology , accurate bowel cleansing is essential for the quality of the study . because wall thickening is a characteristic feature of crohns disease , optimal bowel distension is essential . 
oral contrast agents have the disadvantage of not perstensione delle anse e leliminazione di artefatti respiratori e dei movimenti peristaltici , la valutazione del tipo e dellestensione dellenhancement parietale dopo somministrazione di mdc iodato ev . 
come nella radiologia tradizionale unaccurata pulizia intestinale il requisito fondamentale per questo tipo di studio . lispessimento parietale rappresenta una caratteristica fondamentale della malattia intestinale e per tale motivo essenziale avere la ottimale distensione delle anse . 
ct enteroclysis combines the advantages of ct with those of small - bowel enema into a single technique [ 5 ]  . in 1992 , klppel [ 6 ] conducted the first study of ct enteroclysis in small - bowel inflammatory diseases , highlighting the techniques diagnostic accuracy in evaluating mucosal abnormalities , mural thickening , fistulas and extraintestinal complications . clinical suspicion guides the choice of contrast material . it is generally preferable to use hypodense contrast agents , except in patients with intestinal obstruction , in whom positive agents are found to be more useful [ 5 , 6 ]  . 
hypodense contrast agents provide better depiction of the inner aspect of the bowel loop , allowing evaluation of the type of mural enhancement after iv administration of iodinated contrast material . 
because no infusion pump was available , we administered the contrast agent manually , trying to ensure a steady and continuous infusion . the examination was carried out after completing the methylcellulose infusion . 
moreover , to ensure uniform loop distension and reduce patient discomfort , we administered two doses of iv smooth - muscle relaxant : the first dose was given before the infusion of approximately 11.5 l of methylcellulose to reduce patient discomfort , the second just il primo requisito quindi per un ottimale studio tc ottenere unadeguata distensione del lume mediante unoculata scelta del tipo e della modalit di somministrazione del mezzo di contrasto . 
i mezzi di contrasto somministrati per via orale hanno lo svantaggio di non fare ottenere una uniforme distensione delle anse del piccolo intestino ; tale problema superato con lutilizzo dellenteroclisi tc , caratterizzata tuttavia da invasivit , tempo e costi maggiori . 
kloppel nel 1992 [ 6 ] ha realizzato il primo studio sulla tc enteroclisi nelle malattie infiammatorie dellintestino tenue mettendo in risalto laccuratezza diagnostica di questa tecnica nella valutazione delle alterazioni della mucosa , dellispessimento parietale , delle fistole e delle complicanze extra - intestinali . 
in tutti i pazienti preferibile lutilizzo di mezzi di contrasto ipodensi eccetto che per i pazienti con ostruzione intestinale nei quali un mezzo di contrasto positivo pi utile [ 5 , 6 ]  . 
un mezzo di contrasto ipodenso consente una migliore definizione dellaspetto interno dellansa intestinale , specialmente per valutare il tipo di enhancement parietale dopo la somministrazione di mdc iodato ev [ 7 ]  . 
essa ben conosciuta , sicura , non tossica , largamente utilizzata nello studio tradizionale e non viene assorbita per cui si evita il rischio di emodiluizione ; ha allincirca la stessa densit dellacqua , quindi determina un ottima differenza di contrasto tra il contenuto delle anse intestinali e lenhancement della parete intestinale . 
nei casi in cui le scansioni di base senza mdc iodato documentavano una scarsa distensione delle anse , stata somministrata unulteriore dose di metilcellulosa prima di procedere con linfusione di mdc iodato . 
inoltre , per ottenere unomogenea distensione delle anse e ridurre il disagio al paziente abbiamo somministrato ev due dosi di ipotonizzante : la prima dose stata somministrata dopo linfusione di circa 11 , 5 l di metilcellulosa in modo da ridurre il discomfort del paziente , la seconda subito prima dellesame tc . 
in tal modo abbiamo ottenuto una distensione completa del digiuno prossimale nel 61% dei pazienti , del digiuno distale nel 77% , dellileo prossimale nell88% , dellultima ansa ileale nel 92% . 
anche se la distensione delle anse migliore quando viene usata una pompa peristaltica [ 8 , 9 ] , in accordo con quanto evidenziato in letteratura [ 10 , 12 ] riteniamo possibile ottenere una sufficiente distensione delle anse anche con linfusione manuale del mezzo di contrasto . un altro elemento indispensabile per lo studio tc del morbo di crohn la somministrazione ev del mezzo di contrasto iodato . 
non tutti gli autori sono concordi sul tempo di attesa dopo la somministrazione del mezzo di contrasto iodato : alcuni ritengono utile unacquisizione arteriosa precoce ( 25 s ) [ 8 ] o arteriosa tardiva 1197 l.m. 
10a , b allesame tc a singolo detettore ( a ) evidente ispessimento parietale dellultima ansa ileale ( freccia ) ; nel clisma del tenue ( b ) essa non dissociabile dalle anse vicine . before starting the ct study . 
this enabled us to achieve complete distension of the proximal jejunum in 61% of patients , of the distal jejunum in 77% , of the proximal ileum in 88% and of the last ileal loop in 92% . 
even if loop distension is improved by the use of a peristaltic pump [ 8 , 9 ] , in agreement with the literature [ 10 , 12 ] , we believe adequate bowel distension can also be achieved by manual injection of the contrast agent . another fundamental aspect of the ct study of crohns disease is the iv administration of iodinated contrast material . 
not all authors agree on the timing of the scan after contrast administration : some prefer an early arterial acquisition ( 25 s ) [ 8 ] or late arterial acquisition ( 40 s ) [ 9 , 13 ] , whereas other prefer a portal phase ( 6070 s ) [ 14 ]  . 
in our study we decided to perform a single acquisition 40 s after iv administration of contrast material , because we preferred to study the small - bowel loops in the arterial phase . during acute inflammation , the thickened bowel wall often shows the so - called target sign , characterised by the alternation of concentric highand low - attenuation rings , on postcontrast images . 
the target sign was originally attributed to crohns disease but is now regarded as a nonspecific sign [ 14 ] ; the differential diagnosis includes crohns disease , ischaemia , infectious enteritis , actinic enteritis , vasculitis and graft - versus - host dis1198 ( 40 s ) [ 9 , 13 ] , altri prediligono una fase portale ( 6070 s ) [ 14 ]  . 
noi abbiamo deciso di realizzare una singola acquisizione a 40 s dalla somministrazione ev di mdc , poich abbiamo preferito effettuare uno studio delle anse dellintestino tenue in fase arteriosa . quando presente uninfiammazione acuta , la parete intestinale ispessita spesso mostra dopo iniezione endovena del mdc iodato il cosiddetto segno del bersaglio ( target sign ) caratterizzato dallalternanza di anelli concentrici ad alta e bassa densit . 
laspetto a bersaglio era originariamente attribuito al morbo di crohn , ma oggi si ritiene essere un segno aspecifico [ 14 ] ; la diagnosi differenziale include : malattia di crohn , ischemia , enteriti infettive , enteriti attiniche , vasculiti e grast - versushost - disease . 
il segno del pettine ( comb sign ) si evidenzia come una ipervascolarizzazione del mesentere coinvolto che si manifesta sotto forma di dilatazione arteriosa mesenterica , tortuosit , prominenza e dilatazione dei vasi retti [ 14 ]  . 
tale segno stato osservato in 7 pazienti del nostro studio . nonostante gli sviluppi tecnologici , per la nostra esperienza ancora utile la combinazione dello studio tc enteroclisi con il tradizionale clisma del tenue . 
the comb sign reflects hypervascularity of the involved mesentery , which manifests as mesenteric arterial dilatation , tortuosity , prominence and dilatation of the vasa recta [ 14 ]  . 
the comb sign was seen in seven patients in our study . despite technological advances , we find the combination of ct enteroclysis and conventional barium enema to be still useful , as it produces a better diagnostic definition , allowing identification of false positive and false negative cases on ct [ 9 , 16 , 17 ]  . 
moreover , the conventional study proved superior in characterising mucosal abnormalities , especially in the early phase , and complemented the ct findings , allowing for a diagnosis of reflux ileitis and ischaemic ileitis in two cases . 
abscesses were present in 2 / 45 patients , fistulas in 3 / 45 , perienteric stranding in 13 / 45 and fibrofatty proliferation in 5 / 45 . with regard to the comparison between the two ct techniques , it should be stressed that , as expected , the multidetector technique allowed for shorter scan times and reduced respiratory artefacts and patient discomfort while providing better spatial resolution and better depiction of pathological patterns . 
however , sensitivity , specificity and diagnostic accuracy of the two techniques are similar ( table 4 ) , as a result of the fact that both techniques are able to demonstrate the typical signs of disease , even though their visualisation is better with the multidetector technique . 
in our study , mpr did not , however , reveal any abnormalities that had not already been demonstrated in the axial scans , although they significantly increased our confidence in image interpretation and helped us better evaluate the longitudinal extent of intestinal involvement , as previously reported in the literature [ 19 , 21 ]  . 
the coronal images , especially with those obtained with the multidetector technique , were particularly well accepted by the clinicians and surgeons , given their familiarity with frontal views . even though crohns disease is the most common disease of the small bowel , we should not forget that ct enteroclysis is able to detect other pathological conditions , such as neoplasms . 
in our study , we found only two cases of carcinoid tumour , probably as a result of the preliminary clinical selection of patients with suspected or known crohns disease . 
 in conclusion , we believe that mdct should be preferred to sdct owing to its better spatial resolution and because it ne produce una migliore definizione diagnostica permettendo di identificare i casi tc falsi positivi e falsi negativi [ 9 , 16 , 17 ]  . 
nel nostro studio , infatti , 3 falsi positivi erano dovuti a scarsa distensione delle anse e 4 falsi negativi erano dovuti ad una fase iniziale di malattia e / o localizzazione prossimale di crohn . 
ascessi erano presenti in 2 / 45 pazienti , fistole in 3 / 45 , stranding perienterico in 13 / 45 , proliferazione fibroadiposa in 5 / 45 . per quanto riguarda il confronto tra le due tecniche tc vogliamo sottolineare che , come atteso , la tecnica multidetettore ha ridotto i tempi di scansione , gli artefatti respiratori e il disagio per il paziente , permettendo una migliore risoluzione spaziale e realizzando una migliore rappresentazione degli elementi patologici . 
tale dato in accordo con la letteratura [ 1517 , 19 , 20 ] .tuttavia i valori di sensibilit , specificit ed accuratezza diagnostica sono simili con le due metodiche ( tabella 4 )  . 
tuttavia le ricostruzioni multiplanari non hanno nel nostro studio mostrato alcuna alterazione che non fosse gi stata documentata nelle scansioni assiali anche se hanno migliorato significativamente la nostra confidenza nella interpretazione delle immagini e ci hanno aiutato a valutare meglio lestensione longitudinale dellinteressamento intestinale , come gi riportato in letteratura [ 19 , 21 ]  . 
le immagini coronali sono state inoltre particolarmente apprezzate dai clinici e dai chirurghi data la loro familiarit con esse , in particolare quelle ottenute con tecnica multidetettore . anche se il morbo di crohn la patologia dellintestino tenue pi frequente , non dobbiamo dimenticare che la tc enteroclisi in grado di documentare altre condizioni patologiche , come ad esempio le neoplasie . 
nel nostro studio abbiamo riscontrato solo due casi di carcinoide verosimilmente in rapporto alla preliminare selezione clinica che ci ha fatto studiare prevalentemente pazienti con sospetto o noto morbo di crohn . in conclusione noi riteniamo che la tcmd enteroclisi sia da preferire alla tcsd per la sua migliore risoluzione spaziale e perch consente una migliore rappresentazione dei segni patologici di malattia di crohn . 
in our opinion , in selected patients ( nondiagnostic ct , negative ct with a strong clinical suspicion of small bowel disease ) , ct findings still need to be combined with those from small - bowel enema to obtain all the information needed for a correct diagnosis and for planning treatment in patients with crohns disease . 
the ultimate goal is to improve visualisation of early lesions with ct in order to obtain all the necessary information for correct patient evaluation with a single examination . nione i dati tc ancora necessitano in pazienti selezionati ( tc non diagnostica , tc negativa con quadro clinico altamente suggestivo di patologia dellintestino tenue ) di essere combinati con quelli derivanti dal clisma del tenue in modo da ottenere tutte le informazioni necessarie per la corretta diagnosi e la pianificazione terapeutica dei pazienti con malattia di crohn . 
fourteen patients ( nine men and five women , mean age 49 years , range 2266 years ) underwent an mr examination on a 1.5 - t systeseven patients had a history of hypertension or crf , one had takayasu disease and one had nephrovascular hypertension . 
the imaging protocol consisted of t1and t2 - weighted sequences followed by a se - epi acquisition with a diffusion gradient of 600 s / mm2 and sense factor 2 and pixel - by - pixel adc map reconstruction . 
further studies with scintigraphic correlation are needed to confirm these results and to establish reference values for this imaging technique . key words magnetic resonance imaging diffusion - weighted imaging kidney glomerular filtration rate riassunto scopo . 
quattordici pazienti ( 9 maschi e 5 femmine , et media 49 anni , range 2266 ) sono stati sottoposti ad esame rm con sistema ad alto campo 1 , 5 t ; in 5 pazienti non vi erano dati anamnestici e bioumorali di nefropatia , mentre in 7 era presente storia di ipertensione arteriosa e / o irc ingravescente , in 1 di malattia di takayasu e in 1 di ipertensione nefrovascolare . 
il protocollo prevedeva acquisizioni t1 e t2 dipendenti , nonch una sequenza seepi pesata in diffusione ( b - factor 600 s / mm2 ) con fattore sense 2 e ricostruzione di mappe delladc . 
non stata rilevata una differenza significativa delladc tra stadio i e stadio ii ( p = 0 , 27 ) e tra stadio ii e stadio iii ( p = 0 , 39 ) , mentre la differenza stata significativa tra pazienti in stadio iii e iv ( p < 0 , 01 )  . 
si rilevata una significativa correlazione tra la clearance della creatinina ( clcr ( cg ) ) e l adc parenchimale ( r = 0 , 79 ; p < 0 , 01 )  . 
la rm - dw permette di ottenere valori quantitativi del coefficiente di diffusione correlabili con la funzione renale separata , senza somministrare mezzo di contrasto e con rapidi tempi desame . ulteriori studi di correlazione con i dati scintigrafici e su pi ampie casistiche sono necessari al fine di confermare tali risultati e stabilire valori di riferimento per questa metodica . parole chiave risonanza magnetica diffusione reni filtrato glomerulare 1201 s.f. 
this impact on health and health care costs increases at least tenfold if the increased risk of cardiovascular disease among patients with kidney disease is taken into account , as this condition is the main cause of death among this population [ 2 ]  . 
whereas haemodialysis and transplant are expensive but necessary forms of therapy in the terminal phase , prevention of endstage renal failure through an attempt to slow down the progression of kidney damage is undoubtedly useful for the prognosis and management of these patients , especially considering that patients on dialysis have a mortality rate ten times greater than patients with kidney disease not undergoing dialysis [ 2 ]  . 
the therapy to adopt , therefore , needs to be based on the correct identification of patients at risk and procedures for assessing response to therapy that are minimally invasive , relatively simple and repeatable and obviously sensitive to the slightest alteration in kidney function . in addition to biochemical markers , numerous recent studies have investigated the potential of imaging techniques , and especially magnetic resonance imaging ( mri ) , in the assessment of early renal damage [ 37 ]  . 
 the technique of diffusion - weighted imaging ( dwi ) , through the use of sequences highly sensitive to proton motion such as single - shot echoplanar imaging ( ss - epi ) sequences , is capable of measuring the diffusion of water protons in the extracellular compartments and therefore proton motion in the tubular system of the kidneys [ 8 ]  . 
this measurement was made possible by using the appropriate gradient pulses ( or b factor ) [ 9 ] : the greater the motion of water molecules , the greater the decrease in signal , which can be used to quantify the apparent diffusion coefficient ( adc ) , which is measured in millimetres per second2 . 
the shortcomings of these sequences include a low signal - to - noise ratio ( snr ) , low spatial and contrast resolution and the need for relatively long echo time ( te ) [ 10 ]  . 
in addition , they are relatively sensitive to off - resonance effects , such as an inhomogeneous field , magnetic susceptibility and chemical shift , features that are often present in abdominal imaging [ 11 ]  . 
this technology , which is based on the subdivision of the signal originating from arrays of multiple receiver coils and the determination of sensitivity maps , is capable of reducing the gradient echo train and accelerating the filling of the k - space . 
the resulting increase in bandwidth per pixel in the direction of the phase encoding and reduction in the epi factor produce a net increase in image quality [ 12 ]  . with regard to the kidneys , a number of studies reported significantly reduced diffusion coefficients in the case of acute and crf ; in particular , the adc of the cortex appears to be strongly correlated with creatininaemia [ 813 ]  . the aim of the present study was to assess the adc para1202 la prevalenza dellinsufficienza renale cronica ( irc ) notevolmente aumentata negli ultimi decenni , con un significativo incremento dei costi relativi alla dialisi ed ai trapianti renali , corrispondenti secondo alcune recenti fonti a circa il 2% del budget sanitario annuo in europa [ 1 ]  . 
tale impatto sulla salute e sui costi sanitari viene poi almeno decuplicato dallaumentato rischio di patologie cardiovascolari documentato nei pazienti nefropatici , che sono le principali cause di mortalit in questa popolazione [ 2 ]  . 
se lemodialisi ed il trapianto sono le misure terapeutiche dispendiose ma necessarie nella fase terminale , per la prognosi di questi pazienti e per il loro management complessivo sicuramente utile la prevenzione dellirc terminale cercando di ridurre la velocit di progressione del danno renale , soprattutto se si tiene presente che i pazienti dializzati hanno un tasso di mortalit dieci volte superiore ai nefropatici non dializzati [ 2 ]  . 
lapproccio terapeutico deve quindi fare seguito alla corretta identificazione dei pazienti a rischio e allindividuazione di procedure di valutazione della risposta terapeutica che siano scarsamente invasive , relativamente semplici e ripetibili e , ovviamente , sensibili alle minime modificazioni della funzione renale . oltre i markers bioumorali , a tale scopo numerosi studi sono stati compiuti negli ultimi anni per indagare le potenzialit delle tecniche di imaging e soprattutto della risonanza magnetica ( rm ) nella valutazione del danno renale precoce [ 37 ]  . in particolare la tecnica di diffusione ( diffusion - weighted imaging , dwi ) consente attraverso lutilizzo di sequenze particolarmente sensibili a movimenti protonici , come le sequenze echo - planari single - shot ( ss - epi ) , di misurare la capacit di diffusione dei protoni di acqua nei compartimenti extracellulari e quindi , a livello renale , soprattutto dei movimenti protonici del sistema dei tubuli [ 8 ]  . 
tale misurazione resa possibile utilizzando impulsi di gradienti di campo appropriati ( o b - factor ) [ 9 ] : tanto pi vi movimento protonico maggiore sar il decremento di segnale , dal quale si pu ottenere una vera e propria quantificazione del coefficiente di diffusione ( apparent diffusion coefficient , adc ) , misurabile in mm2 / s . 
il limite di tali sequenze sta nel basso rapporto segnale - rumore , nella bassa risoluzione spaziale e di contrasto , nonch nella necessit di utilizzare te relativamente lunghi [ 10 ]  . 
inoltre esse presentano una elevata sensibilit agli effetti tipo off - resonance , come inomogeneit di campo , suscettibilit magnetica e chemical - shift , spesso frequenti nellimaging addominale [ 11 ]  . 
five underwent abdominal mr to characterise hepatic lesions and , as they showed no significant serum creatinine alterations and had no history of risk factors for kidney disease , were enrolled in the study as healthy controls . 
in base a questa equazione , i 9 pazienti con positiva anamnesi per patologia renale o comunque con fattori predisponenti , sono stati cos classificati : 4 in stadio 1 , 2 stadio 2 , 1 in stadio 3 e 2 in stadio 4 . 
in fase di post - processing , sono state ottenute automaticamente delle mappe di diffusione , applicando la relazione di le bihan modificata per il calcolo del coefficiente apparente di diffusione adc pixel - per - pixel : adc = ( lg si0 - lgsi1 ) / b1 - b0 [ 15 ] dove si0 e si1 sono le intensit di segnale misurate nelle immagini corrispondenti ai fattori di diffusione rispettivamente b0 e b1 . 
on the basis of this equation , the nine patients with a positive history of renal disease or predisposing factors were classified as follows : four in stage 1 , two in stage 2 , one in stage 3 and two in stage 4 . 
during the postprocessing phase , diffusion maps were automatically obtained by applying the le bihan equation modified for the calculation of the pixel - by - pixel adc : adc = ( lgsi0 - lgsi1 ) / b1 - b0 [ 15 ] where si0 and si1 are the signal intensities measured on 1204 s.f. 
nei 9 pazienti con fattori di rischio per danno renale , classificati in stadio 14 secondo lequazione di cockroft - gault , i valori di gfr erano compresi tra 215 , 23 e 19 , 46 ml / min . i dati relativi ai valori di diffusione ( adc ) nella popolazione esaminata sono riassunti nella tabella ( tabella 2 )  . 
il parametro diffusione prima e dopo somministrazione di furosemide non ha mostrato significative variazioni ( p = 0 , 7 ) ; in particolare la somministrazione di furosemide ha determinato una riduzione della correlazione lineare tra adc e clcr ( cg )  . 
a region of interest ( roi ) was then manually placed along the borders of each kidney to exclude the renal sinus in the reference se - epi images ( b = 0 )  . 
dw acquisition obtains a reference image with b = 0 s / mm2 ( a ) and an isotropic image with b = 600 s / mm2 ( b ) , based on which an apparent diffusion coefficient ( adc ) map can be calculated ( c )  . 
lacquisizione pesata in diffusione permette di ottenere unimmagine di riferimento ( a ) con b = 0 e limmagine ottenuta con b = 600 s / mm2 ( b ) , a partire dalle quali possibile calcolare una mappa delladc ( c )  . 
in patients with documented kidney damage , no significant difference was measured in terms of adc between stages 1 and 2 ( p = 0.27 ) or stages 2 and 3 ( p = 0.39 ) , whereas a marked difference in parenchymal diffusion was limpatto della terapia sulla progressione della irc fortemente influenzato dalla individuazione precoce del danno parenchimale renale e delle sue incipienti ripercussioni sulla funzione dorgano . 
tale applicazione stata resa possibile dallo sviluppo di sistemi ad elevati gradienti di campo oltre che ad alto campo magnetico stazionario , a cui si associata una significativa evoluzione tecnologica delle bobine e delle sequenze cosiddette veloci . 
tra le sequenze che sfruttano le possibilit intrinseche della risonanza di produrre contrasto tra differenti tessuti , una tecnica molto promettente in ambito addominale e di rapida acquisizione il dwi [ 1518 ]  . 
esso sfrutta la capacit della rm di produrre e misurare il movimento dei protoni liberi di acqua nei tessuti , dipendente da numerosi fattori , tra cui principalmente la presenza di barriere ( membrane cellulari , accumulo di matrice extra - cellulare , ecc )  . 
there were no significant variations in adc values before and after administration of furosemide ( p = 0.7 ) ; in fact , the administration of furosemide caused a reduction in the linear correlation between adc and clcr ( cg )  . 
this application has been made possible by the development of systems with high field gradients and high static magnetic fields , together with a significant technological development of coils and so - called fast sequences . 
the latter offer a variety of possibilities for the assessment , at least theoretically , of functional renal parameters , such as renal blood flow and gfr , by exploiting the intrinsic characteristics of mr contrast resolution or by using paramagnetic contrast agents similar to inulin [ 7 ]  . 
included among the sequences that exploit the intrinsic possibilities of resonance to produce contrast between different tissues is dwi , a fast technique that has proved highly promising in abdominal imaging [ 15 , 1618 ]  . 
6a - c bowel gas produces a magnetic susceptibility artefact in the inferior pole of the left kidney , both in the reference image with b = 600 s / mm2 diffusion - weighted and in the apparent diffusion coefficient map ( arrows )  . 
6a - c artefatto da sensibilit alla suscettibilit magnetica a livello del polo inferiore del rene sinistro , causato dalla presenza di aria colica presente sia nellimmagine di riferimento e con b = 600 s / mm2 che nella mappa delladc ( frecce )  . 
it has been shown that the b value negatively influences the snr of the image , even though it does render the sequence less sensitive to the motion of capillary flow and breathing ( or pseudodiffusion ) [ 16 ]  . 
this obtained a mean adc value in the healthy subjects and those in stage 1 ( only the presence of risk factors ) of 2.440.2410 - 3 mm2 / s , which is in line with the figures reported in the literature . 
in patients with kidney damage documented by a reduction of clcr ( cg ) , there was a progressive and significant reduction in adc ( p < 0.01 ) , with significant changes between stages 3 and 4 . 
 [ 13 ] identified a linear correlation between creatininaemia and adc ( r = 0.75 ) in the cortex and a significant but weaker correlation in the medulla ( r = 60 ) , whereas a recent preliminary study showed a significant difference between renal adc in healthy volunteers and in patients affected by crf ( p < 0.01 ) [ 8 ]  . 
though conducted on a small population , our study indicates that adc can be used as a parameter for renal damage , as it is capable of distinguishing between patients with parenchymal damage and patients with initial damage , as well as between patients in stage 3 and patients in stage 4 , although standard reference values are to be hoped for . 
in this sense , a technical standard for the acquisition appears unrealistic , given the marked 1208 diente di diffusione utilizzato ( 5001300 s / mm2 ) [ 13 , 17 , 1921 ]  . 
noto che questultimo parametro influenza negativamente il segnale / rumore dellimmagine , anche se rende meno sensibile ai movimenti da flusso capillare o respiro ( o pseudodiffusione ) la sequenza [ 16 ]  . 
in questa ottica abbiamo utilizzato un elevato fattore b di 600 s / mm2 associato ad una acquisizione in respiro sospeso , in modo da mantenere un adeguato segnale / rumore , ottenendo un valore medio di adc nei soggetti sani ed in quelli in stadio i ( presenza di soli fattori predisponenti ) di 2 , 440 , 2410 - 3 mm2 / s , in linea con quanto riportato in letteratura ; nei pazienti con danno renale documentato dalla riduzione della clcr ( cg ) si assistito ad una progressiva e significativa riduzione delladc ( p < 0 , 01 ) con modificazioni significative tra iii e iv stadio . 
 [ 13 ] hanno evidenziato una correlazione lineare tra creatininemia e adc ( r = 0 , 75 ) della corticale renale e significativa ma meno marcata con la midollare ( r = 0 , 60 ) , mentre un recente studio preliminare ha evidenziato una significativa differenza tra adc renale in volontari sani ed in pazienti affetti da irc ( p < 0 , 01 ) [ 8 ]  . 
lutilizzo della furosemide per uno studio dinamico in diffusione non ha apportato sostanziali vantaggi nei pazienti in cui stato effettuato : non abbiamo infatti riscontrato alcuna differenza in termini di adc nelle acquisizioni pree post - furosemide ( p = 0 , 7 ) , in linea con quanto peraltro gi segnalato in letteratura [ 22 ]  . 
per quanto limitata dalla esiguit della casistica , la presente esperienza indica che ladc pu essere utilizzato come parametro di danno renale , in quanto capace di distinguere i soggetti con danno parenchimale da quelli con danno iniziale e quelli in stadio 3 dai soggetti in stadio 4 , anche se valori di riferimento standardizzati sarebbero auspicabili . 
morphological findings on t1and t2 - weighted images ( a , b ) show a right renal volume reduction with lower cortical - to - medullar differentiation more evident on t1 ( b )  . 
le acquisizioni morfologiche preliminari t1 e t2 pesate ( a , b ) documentano una riduzione del volume del rene destro con minore differenziazione cortico - midollare , pi marcata in t1 ( b )  . 
the assessment of standardisation parameters in the context of the chosen technique could be the aim of future studies . the incidence of artefacts , particularly those caused by magnetic susceptibility related to the presence of the vertebral column or bowel gas , was decidedly low in our study , thanks to the sequence characteristics used and the particular application of sense technology , with a consequent reduction in off - resonance artefacts related to epi . 
this fact , which has already been emphasised in several studies of the brain [ 23 ] , was recently reported in a study of the abdomen , where parallel imaging was also associated with the use of the diffusion tensor with a significant increase in signal [ 8 ]  . overall , our data confirm the possibility of using the technique to assess renal function , above all , with reference to the functioning of a single kidney . 
questo dato , gi sottolineato in alcuni studi sul distretto encefalico [ 23 ] , stato riportato in ambito addominale in un recente lavoro , nel quale limaging parallelo peraltro associato alluso del tensore di diffusione , con un importante aumento del segnale [ 8 ]  . nel complesso i dati ottenuti confermano le possibilit di tale metodica nella valutazione della funzione renale , soprattutto in riferimento a quella del singolo rene . 
further studies are required to standardise both the technique , particularly in dwi , and the calculation , as well as to validate clinical indications on larger patient samples . i dati presenti in letteratura ed i risultati del presente studio consentono di proporre la rm in diffusione del rene come metodica di riferimento per una corretta valutazione dei parametri di funzione parenchimale in associazione ad una elevata accuratezza di valutazione morfologica sia del parenchima che dellasse vascolare . 
taouli b , martin aj , quayyum a et al ( 2004 ) parallel imaging and diffusion tensor imaging for diffusion weighted mri of the liver : preliminary experience in healthy volunteers . 
mc cullough pa ( 2003 ) beyond serum creatinine : defining the patient with renal insufficiency and why ? rev cardiovasc med 4 [ suppl 1 ] : s2s6 15 . 
bammer r , feeling sl , augustin m et al ( 2001 ) improved diffusion - weighted single - shot echo - planar imaging ( epi ) in stroke using sensitivity encoding ( sense )  . 
bartolozzi1 1division of diagnostic and interventional radiology , 2division of pathology , university of pisa , via roma 67 , i - 56100 pisa , italy 3azienda ospedaliera universitaria pisana , pisa , italy correspondence to : c . 
fifty - five patients with mammographic microcalcifications classified as bi - rads categories 3 , 4 or 5 underwent mri and biopsy with stereotactic vacuum - assisted biopsy ( vab )  . 
mri was performed with a 1.5 - tesla ( t ) unit , and t1 coronal three - dimensional ( 3d ) fast low - angle shot sequences were acquired before and after injection of paramagnetic contrast agent ( 0.1 mmol / kg )  . 
lesame rm stato eseguito con una unit da 1 , 5 tesla e sono state acquisite sequenze coronali t1 3d fast lowangle shot sequences prima e dopo liniezione di mezzo di contrasto paramagnetico ( 0 , 1 mmol / kg )  . 
lesame istologico ha rilevato 26 cancri duttali in situ ( cdis ) e cancri duttali microinvasivi ( cd mic ) , 3 iperplasie duttali atipiche ( ida ) e 26 condizioni benigne . 
la sensibilit della mammografia risultata 77% , la specificit stata 59% , il valore predittivo positivo ( vpp ) 61% , il valore predittivo negativo ( vpn ) 74% , laccuratezza diagnostica 67 , 2% . 
la sensibilit globale della rm stata 73% , la specificit 76% , il valore predittivo positivo ( vpp ) 73% , il valore predittivo negativo ( vpn ) 76% , laccuratezza diagnostica 74 , 5% . 
during the last 20 years , the prevalence of dcis has grown from less than 5% before the start of mammographic screening to 15%30% in women regularly checked with mammography [ 6 ]  . 
the 30%50% decrease in mortality is related to early diagnosis and correct management [ 7 ]  . mammography has high sensitivity and low specificity , the positive predictive value ( ppv ) being 15%30% for malignant nonpalpable lesions [ 6 ]  . 
recent studies have shown that breast magnetic resonance imaging ( mri ) with intravenous contrast injection has outstanding sensitivity for the diagnosis of invasive carcinomas ( 88%100% ) and a high negative predictive value ( npv ) [ 6 ]  . 
 considering that dcis is multifocal in more than 50% of cases and bilateral in about 30% of patients [ 1 , 15 ] , and also considering that the possible evolution into an infiltrating form depends on the histological subtype and size and adequacy of resection [ 16 , 17 ] , mri could help in early diagnosis and surgical planning , giving useful information about disease extension [ 14 ]  . 
the aim of our study was to evaluate the role of mri in patients with breast imaging reporting and data systems ( bi - rads ) 35 microcalcifications . la dimostrazione di microcalcificazioni spesso il solo segno del tumore , essendo il primo indizio riscontrato alla mammografia in circa il 70% dei carcinomi duttali in situ ( dcis ) [ 15 ]  . 
durante gli ultimi venti anni , la prevalenza del dcis cresciuta da meno del 5% prima dellinizio dello screening mammografico al 15%30% nella popolazione esaminata regolarmente con la mammografia [ 6 ]  . 
la riduzione della mortalit del 30%50% in relazione con la diagnosi precoce e con il corretto trattamento [ 7 ]  . la mammografia ha un alta sensibilit ed una bassa specificit , infatti il valore predittivo positivo ( vpp ) del 15%30% per le lesioni maligne non palpabili [ 6 ]  . 
recenti studi hanno dimostrato che la risonanza magnetica della mammella ( rm ) con somministrazione di mezzo di contrasto per via endovenosa , ha una notevole sensibilit nella diagnosi del carcinoma invasivo ( 88%100% ) ed un alto valore predittivo negativo ( vpn ) [ 6 ]  . 
invece dimostrata lutilit della rm nella pianificazione chirurgica [ 14 ]  . considerando che il dcis multifocale in pi del 50% dei casi , bilaterale in circa il 30% delle pazienti [ 1 , 15 ] e che la possibile evoluzione in una forma infiltrante dipende dal sottotipo istologico , dalle dimensioni e dalladeguatezza della exeresi chirurgica [ 16 , 17 ] , la rm potrebbe essere di aiuto nella diagnosi precoce e nella pianificazione dellintervento , dando informazioni utili sullestensione della malattia [ 14 ]  . lo scopo del nostro studio quello di valutare il ruolo della rm in pazienti con microcalcificazioni birads 35 . materials and methods we selected 55 patients ( age range 3776 years , mean 5611 years ) with bi - rads 35 microcalcifications on mammography who underwent mri and stereotactic biopsy using vacuum - assisted biopsy ( vab ) between november 2002 and november 2004 . 
mammographic magnification was performed in the view that best visualised the microcalcifications , and the microcalcification extension was evaluated in the standard views . all mammograms were evaluated in a blinded fashion by two radiologists with 15 years experience in breast imaging . materiali e metodi abbiamo selezionato 55 pazienti ( et compresa tra 37 e 76 anni , et media 5611 anni ) con microcalcificazioni birads 35 alla mammografia , che si sono sottoposte ad esame rm e a biopsia sotto guida stereotassica con tecnica di retroaspirazione ( vacuum assisted biopsy : vab ) da novembre 2002 a novembre 2004 . 
i reperti controversi sono stati ulteriormente valutati da entrambi i radiologi per raggiungere un consenso . il nostro gold standard stata la microistologia e listologia per le pazienti sottoposte ad intervento chirurgico . 
lo studio rm stato sempre eseguito prima della microistologia . la rm stata effettuata con un apparecchio da 1 , 5 t ( symphony , siemens ) in tutte le pazienti tranne in 6 in cui stata usata una macchina da 1 , 5 t ( ge , medical system )  . tutte le pazienti sono state studiate in posizione prona utilizzando una bobina dedicata per la mammella . 
il protocollo di studio rm ha incluso un sequenza trasversale t1 di localizzazione , una sequenza sagittale t2 con saturazione del grasso ed una sequenza t1 gradient eco 3d - flash ( te 2 , 5 ms , tr 12 , 7 ms , tempo di acquisizione meno di 2 minuti , fa 30 , spessore di strato 3 mm , interslice gap 0 mm , nex 1 , fov 35 , dimensione della matrice 256160 , dimensione del pixel 1 , 41 , 4 ) , con una serie di 6 acquisizione coronali . 
stato usato il gadoteridolo ( prohance , bracco , milano ) come agente di contrasto paramagnetico con boli di iniezione di 0 , 1 mmol / kg di peso corporeo . 
i processi di rielaborazione delle immagini usate sono stati : sottrazione ; ricostruzioni mip ; ricostruzioni mpr ; curve intensit / tempo nella regione di interesse . sono state identificate secondo lo score di fisher 3 tipi di curve . 
i risultati dello studio rm sono stati classificati secondo lo score di fisher in 5 categorie bi - rads : rm 12 ( fisher score 02 ) : negativo - benigno ; rm 3 ( fisher score 3 ) : probabilmente benigno ; rm 45 ( fisher score 48 ) : probabilmente maligno / maligno . sono state eseguite biopsie sotto guida stereotassica usando il mammografo giotto ims equipaggiato con tavolo prono ed un sistema di aspirazione mammotome ethicon endo - surgery ( amburgo ) con ago di 11 gauge ( g )  . 
il materiale stato inviato allesame microistologico in due differenti contenitori separando i frustoli con le microcalcificazioni da quelli senza microcalcificazioni evidenziate dalla radiografia . lintervento chirurgico stato effettuato in quelle pazienti con diagnosi microistologica di malignit o di lesione altamente sospetta . 
mammografia : le proiezioni cranio - caudale ( cc ) e medio - laterale obliqua ( mlo ) mostrano microcalcificazioni nei quadranti superoed infero - esterni birads 4 e 5 . all cases of microcalcifications were classified according to the method proposed by the american college of radiology [ 18 ] , and those classified as bi - rads 35 were selected . controversial findings were re - evaluated by the two radiologists to reach a consensus . 
mri imaging was performed with 1.5 - tesla ( t ) unit ( symphony , siemens ) in all patients but six , who were examined with a 1.5 - t unit ( ge medical system )  . 
the imaging protocol included a transverse t1 localising sequence , a sagittal fat - saturated t2 sequence and a t1 three - dimensional ( 3d ) fast low - angle shot ( flash ) gradient echo ( te 2.5 ms , tr 12.7 ms , acquisition time less than 2 min , fa 30 , slice thickness 3 mm , interslice gap 0 mm , nex 1 , fov 35 , matrix size 256160 , pixel size 1.41.4 ) , with a series of six coronal acquisitions . 
rm : le immagini mpr sagittali ( a ) ed assiali ( b ) e le mip assiali ( c ) evidenziano multiple aree di captazione contrastografica dendritica e nodulare che interessano lintera mammella sinistra . mmol / kg of body weight . 
postprocessing techniques used were : subtraction maximum intensity projection ( mip ) reconstructions multiplanar reformatting ( mpr ) reconstructions time - intensity curves in the region of interest ( roi )  . three types of curves were identified according to fisher score parameters considering enhancement morphology , we identified the following possibilities : absent enhancement , dendritic enhancement , nodular enhancement and coexistence of dendritic and nodular enhancement . 
results of the mri study were classified into five bi - rads categories according to the fisher score : mri 12 ( fisher score 02 ) negative / benign , mri 3 ( fisher score 3 ) probably benign and mri 45 ( fisher score 48 ) : probably malignant / malignant . biopsy was performed under stereotactic guidance using the giotto ims mammography system equipped with a prone table and a mammotome ethicon endo - surgery ( hamburg ) aspiration device with an 11 - gauge needle . 
considerando la difficolt di una valutazione accurata dellestensione del cdis allanatomia patologica , abbiamo diviso la mammella alla mammografia e alla rm in 5 porzioni : quadrante supero - esterno , quadrante supero - interno , quadrante infero - interno , quadrante infero - esterno , regione retroareolare e abbiamo confrontato tumorale evidenziata lestensione dallimaging con lestensione riscontrata allistologia . risultati la microistologia ha riscontrato 26 lesioni maligne , 29 lesioni benigne incluse 3 iperplasie duttali atipiche ( ida ) ( tabella 1 )  . 
la chirurgia eseguita nelle 29 pazienti ( 17 con carcinoma duttale in situ , cdis , 9 con cancro duttale microinvasivo , cdi mic , e 3 con ida ) , ha confermato in tutti i casi i dati microistologici . 
magnetic resonance imaging ( mri ) : regions of interest in the subtracted images show multiple areas of contrast enhancement with types iii and ii time - intensity curves [ breast imaging reporting and data systems ( bi - rads ) 5 ]  . 
rm : regioni di interesse nelle immagini sottratte post - contrastografiche mostrano multiple aree di captazione contrastografica con curve intensit / tempo di tipo ii e iii ( birads 5 )  . 
stata quindi eseguita una mastectomia sinistra e listologia ha rivelato multipli foci di cdis g2 e g3 . material was sent for microhistological evaluation in two separate containers , one containing the specimens with microcalcifications and the other specimens without microcalcifications . 
in consideration of the difficulties in accurately evaluating dcis extent at pathology , we decided to divide the breast at mammography and mri into five portions : upperouter quadrant , upper - inner quadrant , lower - outer quadrant , lower - inner quadrant and retroareolar region ; we compared si e g3 in 11 casi . 
surgery , performed in 29 patients [ 17 with dcis , nine with ductal microinvasive cancer ( dcmic ) and three with adh ] , confirmed the microhistological data in all cases . 
among the 55 patients studied , mammographic findings were classified as birads 3 : 23 cases , bi - rads 4 : 25 cases andbi - rads 5 : seen cases . 
of the 48 patients with mri contrast enhancement , the time - intensity curve correlated with histology was type i in ten benign lesions and one malignant lesion , type ii in nine benign lesions and nine malignant lesions and type iii in four benign lesions and 15 malignant lesions . 
in the 26 cancers , enhancement morphology logia 17 lesioni benigne e 6 maligne , nelle mammografie birads 4 abbiamo riscontrato 11 lesioni benigne e 14 maligne , nelle mammografie bi - rads 5 abbiamo riscontrato 1 lesione benigna e 6 maligne . 
nelle 48 pazienti con captazione contrastografica alla rm , la curva intensit / tempo correlata allistologia era di tipo i in 10 lesioni benigne e in 1 lesione maligna , di tipo ii in 9 lesioni benigne ed in 9 lesioni maligne e di tipo iii in 4 lesioni benigne e 15 lesioni maligne . nelle 7 pazienti che non presentavano captazione di mezzo di contrasto alla rm , 6 casi risultavano allistologia lesioni benigne e 1 caso cdis ( tabella 4 )  . 
nei 26 cancri la morfologia della captazione contrastografica risultata dendritica in 3 casi , nodulare in 8 casi , sia dendritica che nodulare in 14 casi e completamente assente in un caso ( tabella 5 )  . 
dei 9 casi di cdi mic la maggior parte dei casi avevano una captazione sia dendritica che nodulare ( n = 5 ) ; nei restanti casi la captazione era dendritica ( n = 2 ) e nodulare ( n = 2 )  . per quanto riguarda le forme maligne : nel 3 , 8% dei casi non era presente captazione contrastografica , nel 3 , 8% dei casi le curva intensit / tempo erano di tipo i , nel 34 , 6% le curve intensit / tempo erano di tipo ii e nel 57 , 7% di tipo iii . la sensibilit della mammografia stata del 77% ( 20 / 26 ) , la specificit del 59% ( 17 / 29 ) , il valore predittivo positivo ( vpp ) risultato del 63% ( 20 / 32 ) , il vpn del 74% ( 17 / 23 ) , laccuratezza diagnostica risultata del 67 , 2% ( 37 / 55 )  . 
dei 12 casi di falsi positivi in mammografia con bitable 1 histological results tabella 1 risultati istologici histology dcis / dc mic atypical ductal hyperplasia typical ductal hyperplasia fibroadipose tissue sclerosing adenosis fibroadenoma typical epithelium total istologia cdis / cd mic iperplasia duttale atipica iperplasia duttale tipica tessuto fibroadiposo adenosi sclerosante fibroadenoma epitelio tipico totale dcis , ductal carcinoma in situ ; dcmic , ductal microinvasive carcinoma cdis , carcinoma duttale in situ ; cd mic , carcinoma dentale microinvasivo a . 
as regards the nine dc mic , the majority of cases showed both nodular and dendritic enhancement ( n = 5 ) ; in the remaining cases , enhancement was dendritic ( n = 2 ) and nodular ( n = 2 )  . 
among the five false negative cases of mammographic bi - rads category 3 , mri correctly identified three cancers . of the 12 false positive cases of mammographic bi - rads category 45 , mri correctly identified nine benign lesions . however , the fisher exact test did not show a statistically rads 4 - 5 , la rm ha identificato correttamente 9 lesioni benigne . 
per quanto riguarda lestensione della neoplasia , confrontando i reperti mammografici , rm e listologia abbiamo riscontrato : una concordanza dellestensione neoplastica alla rm e allistologia in 22 / 26 casi ( 84 , 6% ) ; una sovrastima della rm in 3 / 26 casi ( 11 , 5% ) ; in 1 / 26 assenza di captazione contrastografica alla rm per cui non stato possibile valutare lestensione della malattia . abbiamo avuto una sottostima dellestensione neoplastica alla mammografia in 16 / 26 ( 73% ) e una corretta valutazioa . 
with regard to disease extension , comparison of mammographic and mri findings with histology found concordance between mri and histological disease extent in 22 / 26 cases ( 84.6% ) ; overestimation at mri in 3 / 26 cases ( 11.5% ) ; and lack of mri contrast uptake in 1 / 26 cases , preventing evaluation of disease extension . 
nellanalisi dellestensione della neoplasia la rm risultata migliore della mammografia ( test z : p < 0 , 01 )  . in tutti i casi eccetto uno ( multicentrico alla rm e multifocale alla mammografia ) lapproccio chirurgico stato deciso in base allesame rm . 
pathology mammography magnetic resonance imaging tabella 8 confrontro tra estensione neoplastica allimaging e in anatomia patologica mammografia risonanza magnetica sottostima rispetto allanatomia patologica sovrastima rispetto allanatomia patologica valutazione corretta rispetto allanatomia patologica on the basis of the mri findings . 
a mammography : craniocaudal and mediolateral oblique views of the left breast show a cluster of microcalcifications [ breast imaging reporting and data systems ( bi - rads ) 4 ] in the upper outer quadrant . 
b subtracted magnetic resonance imaging ( mri ) : dendritic and nodular irregular enhancement with types i and ii time - intensity curves with low increase of contrast enhancement [ breast imaging reporting and data systems ( bi - rads 4 ) ]  . 
a mammografia : le proiezioni cranio - caudale e medio - laterale obliqua della mammella sinistra mostrano un cluster di microcalcificazioni ( birads 4 ) nel quadrante supero - esterno . 
b immagini rm sottratte : irregolare captazione contrastografica dendritica e nodulare con curve intensit / tempo i e ii con basso incremento della captazione contrastografica ( birads 4 )  . 
a mammography : mediolateral oblique view shows microcalcifications in the retro - areolar region [ breast imaging reportingand data systems ( bi - rads ) 5 ] and other foci ( bi - rads 4 ) in the inner and outer quadrants . 
b magnetic resonance imaging ( mri ) maximum intensity projection ( mip ) : coronal and sagittal views show nodular enhancement in the upper quadrants in the para - areolar region of the right breast , associated with vascular asymmetry ipsilateral to the area of enhancement . 
c subtracted magnetic resonance imaging ( mri ) : nodular contrast enhancement with type iii time - intensity curve [ breast imaging reporting and data systems ( bi - rads ) 5 ] , vacuum - assisted biopsy of retroareolar microcalcifications : g3 dcis . 
a mammografia : la proiezione medio - laterale obliqua mostra microcalcificazioni in regione retroareolare ( birads 5 ) ed altri foci ( birads 4 ) nei quadranti infero - esterni . 
b rm : le immagini mip coronali e sagittali mostrano captazione contrastografica nodulare nei quadranti superiori in regione para - areolare nella mammella destra associata ad unasimmetria vascolare ipsilaterale allarea di captazione contrastografica . 
in one case , surgical treatment was planned on the basis of the mammographic findings rather than considering the mri finding of multifocality , and the patient had a recurrence of disease 1 year later . discussion many studies on dynamic mri [ 6 ] have reported high sensitivity values ( 90%100% ) for the diagnosis of invasive carcinoma , with a specificity of 80% [ 14 , 1923 ]  . 
concerning sensitivity and specificity of mri in the diagnosis of dcis and evaluation of isolated microcalcifications , the published values are ( sensitivity : lower and highly variable 33%100% ) [ 21 , 2426 ]  . 
the relatively low number of dcis cases included in the published studies [ 20 , 21 ] seems to be related to the reluctance of investigators to enrol patients with radiological evidence of microcalcifications . 
 the high variability of mri diagnostic accuracy in evaluating microcalcifications reported in the different studies is related to lesion size [ 6 , 27 , 28 ] , histological variability of tumours [ 20 , 24 ] , tumour angiogenesis [ 24 , 29 , 30 ] , criteria for mri evaluation such as type of enhancement , enhancement pattern , distribution in the breast ( multiple foci ) , margins ( regular , irregular ) [ 21 , 24 , 27 , 30 , 31 ] and use of different pulse sequences and scan planes [ 23 ]  . 
 [ 32 ] , for example , in an analysis of morphological and dynamic features of in situ and microinvasive breast cancer on contrast - enhanced mri , detected the typical enhancement criteria in only 50% of cases , without a significant statistical difference in enhancement pattern among the various histological grades of dcis and between pure and microinvasive dcis . 
in our study , the sensitivity of contrast - enhanced mri , according to the fisher score , was higher ( 77% vs . 50% in viehwegs study ) , with only one false negative mri result corrected by mammography . 
however , the false negative ( n = 7 ) and false positive ( n = 7 ) mris strengthen the concept that mammography is the technique of choice for detecting microcalcifications and that stereotactic biopsy is mandatory for characterising bi - rads 35 microcalcifications . 
information from conventional imaging and in particular mammography is useful to avoid mri false negative results . various studies [ 25 , 32 ] suggest a role for contrast - enhanced mri in the detection of dcis , especially for depicting further tumour foci close to or far from the main lesion that are not visible on mammography due to the absence of microcalcifications or masses or distortions [ 33 , 34 ]  . 
at the same time , it is well known that some areas of dcis can be completely noncalcified and that some sites of dcis can contain calcificadiscussione numerosi studi sulla rm dinamica [ 6 ] hanno riportato elevate valori di sensibilit ( 90% - 100% ) nella diagnosi di carcinoma invasivo , con una specificit dell80% [ 14 , 1923 ]  . riguardo alla sensibilit e specificit dellimaging rm nella diagnosi del carcinoma duttale in situ ( cdis ) e nella valutazione delle microcalcificazioni isolate , i valori pubblicati in letteratura sono pi bassi e variabili ( sensibilit : 33%100% ) [ 21 , 2426 ]  . 
il numero relativamente basso di cdis incluso negli studi pubblicati [ 20 , 21 ] sembra da correlare allavversione ad arruolare allesame rm pazienti con evidenza radiologica di microcalcificazioni . lalta variabilit dellaccuratezza diagnostica nella valutazione delle microcalcificazioni , fino ad ora pubblicata in diversi studi , correlata a : dimensioni della lesione [ 6 , 27 , 28 ] , variabilit istologica dei tumori [ 20 , 24 ] , angiogenesi tumorale [ 24 , 29 , 30 ] , criteri di valutazione della rm come tipo e morfologia della captazione contrastografica , distribuzione nella mammella ( foci multipli ) , margini ( regolari , irregolari ) [ 21 , 24 , 27 , 30 , 31 ] e tecnica di risonanza come differenti sequenze e piani di scansioni [ 23 ]  . 
nel nostro studio la sensibilit riportata per la rm dinamica , in accordo allo score di fischer , risultata pi alta ( 77% rispetto al 50% di viehweg ) con un solo caso di falso negativo corretto dalla mammografia . 
tuttavia i falsi negativi ( n = 7 ) e i falsi positivi ( n = 7 ) rafforzano il concetto che la mammografia la tecnica di scelta per individuare le microcalcificazioni e che la biopsia stereotassica obbligatoria per la caratterizzazione delle microcalcificazioni birads 35 . 
informazioni dallimaging tradizionale ed in particolare dalla mammografia sono utili per evitare i falsi negativi della rm . inoltre vari studi [ 25 , 32 ] suggeriscono un ruolo per la rm con somministrazione di mezzo di contrasto nella diagnosi di cdis specialmente per individuare ulteriori foci neoplastici vicini o lontani dalla lesione principale , che non sono visibili alla mammografia per lassenza di microcalcificazioni , masse o distorsioni [ 33 , 34 ]  . 
questi dati sono particolarmente interessanti se consideriamo levidenza di multifocalit ( 53% ) e multicentricit ( 27% ) osservate in pazienti sottoposte a mastectomia [ 30 , 35 ]  . 
la maggior prevalenza di multicentricit osservata nel cdis che nel cdi e la progressione delle forme in situ non adeguatamente trattate in forme di cancro invasivo richiede unaccurata valutazione dellestensione di malattia specialmente in donne candidate a chirurgia mammaria a . 
the higher prevalence of multicentricity observed in dcis than in dci and the progression of inadequately treated dcis into invasive forms require accurate evaluation of disease extension , especially in cases of candidates to breast - conserving surgery [ 36 , 37 ]  . 
even if the biological and clinical impact of these small foci of dcis is uncertain [ 3840 ] , according to the literature [ 41 ] , the risk of local recurrence after conservative therapy is high in patients with large multifocal tumours and low if the multifocal neoplasm is microscopic . furthermore , it is well known that histological measurement of dcis is difficult due to the difficulty in following a duct on serial sections . 
as regards histological grading , increased vascularisation was associated with more greatly differentiated dcis , which are well detected by contrast - enhanced mri , with a high positive predictive value for g3 and g2 forms . 
 even though our study has a selection bias due to the mammographic detection of microcalcifications , our data suggest that the high ppv of mri in g3 dcis can be useful mainly to depict further dcis foci not associated with mammographic signs . 
these data are in keeping with a recent publication [ 21 ] that emphasised the sensitivity of contrastenhanced mri in the detection of high - grade dcis ( g2 and g3 )  . 
however , recent studies have demonstrated that the same genetic profile is correlated with the same histological grade in both in situ and invasive cancers [ 39 , 43 ]  . 
mri could therefore be useful , especially in identifying the most aggressive forms , thereby helping in surgical planning and once the natural course of the disease and the different biological potential of dcis subtypes are better understood possibly in improving the prognosis , as early detection and correct treatment are the most important prognostic factors in breast cancer . conclusions mri cannot be considered a diagnostic tool for evaluating microcalcifications . 
anche se ad oggi limpatto clinico e biologico di questi piccoli foci di cdis incerto [ 3840 ] , in accordo ai dati di letteratura [ 41 ] il rischio di recidiva locale dopo chirurgia conservativa alto nelle pazienti con grossi tumori multifocali e basso se la neoplasia multifocale microscopica . inoltre ben noto che il cdis difficile da misurare istologicamente per la difficolt a seguire un dotto interessato in sezioni seriali . 
per quanto riguarda il grading istologico nella nostra esperienza abbiamo osservato una vascolarizzazione aumentata associata a forme di cdis pi differenziate che sono ben individuate con la rm dinamica , con elevato valore predittivo positivo per le forme g2 e g3 . anche se il nostro studio ha un bias nei criteri di inclusione rappresentato dallindividuazione mammografica delle microcalcificazioni , i nostri dati suggeriscono che lelevato valore predittivo positivo della rm nei casi di cdis g3 pu essere utile soprattutto ad individuare i foci ulteriori di cancro in situ non associati a segni mammografici . 
questi dati sono allineati con una recente pubblicazione [ 21 ] che enfatizza la sensibilit della rm dinamica nella diagnosi delle forme di cancro in situ di alto grado ( g2 e g3 )  . 
cos la rm potrebbe essere utile nell identificare specialmente le forme pi aggressive di cdis aiutando nella pianificazione dellintervento chirurgico e , una volta che siano chiariti il corso naturale della malattia e che i differenti potenziali biologici dei sottotipi di cdis , probabilmente migliorare la prognosi . infatti la diagnosi precoce del cancro mammario ed il corretto trattamento sono i fattori prognostici pi importanti . conclusioni la rm non pu essere considerata lo strumento diagnostico per valutare le microcalcificazioni . 
one hundred and seven patients with a clinical and laboratory suspicion of acute pulmonary embolism who had already undergone chest radiography were divided into four groups according to the time interval between onset of clinical suspicion and performance of the two diagnostic examinations ( 024 h , 2448 h , 048 h , 27 days )  . 
perfusion lung scintigraphy and multislice ct demonstrated elevated concordance if performed within 7 days of the onset of suspicion of acute pulmonary embolisconcordance was higher if the examinations were completed within 2448 h . 
in suspected acute pulmonary embolism , it is mandatory to reach a correct diagnosis within few hours 48 at the most . key words acute pulmonary embolism perfusion lung scintigraphy spiral computed tomography diagnostics of urgency imagings riassunto obiettivo . 
centosette pazienti con sospetto clinicolaboratoristico di epa , che avevano gi eseguito un rx torace , sono stati divisi in 4 gruppi in base allintervallo di tempo tra il sospetto clinico e lesecuzione di entrambe le indagini ( 024 ore , 2448 ore , 048 ore , 27 giorni )  . 
spp e tcsm sono risultate positive in 29 / 107 ( 27 , 1% ) e negative in 78 / 107 ( 72 , 89% ) : concordanza positiva ( cp ) in 22 casi e concordanza negativa ( cn ) in 71 . 
nel sospetto di epa importante effettuare una rapida e corretta diagnosi in poche ore , 48 al massimo . parole chiave embolia polmonare acuta scintigrafia polmonare perfusiva tc spirale multislice diagnostica per immagini durgenza introduction introduzione acute pulmonary embolism ( ape ) is the third most common acute cardiovascular disease after ischaemic heart disease and stroke [ 1 ]  . 
the yearly incidence of ape in western lembolia polmonare acuta ( epa ) rappresenta la terza pi comune patologia acuta dellapparato cardiovascolare dopo la cardiopatia ischemica e lo stroke [ 1 ]  . 
deep vein thrombosis ( dvt ) is the most common cause of pulmonary embolism ; the other principal primary and secondary risk factors are summarised in table 1 [ 2 ]  . 
in spite of the recent important advances in imaging and laboratory diagnostics , approximately 70% of cases of pulmonary embolism are identified at autopsy [ 3 ]  . in cases with an early diagnosis by multislice spiral computed tomography ( msct ) and perfusion lung scintigraphy ( pls ) , anticoagulation therapy proved to be effective in approximately 75% of patients [ 4 ]  . 
thanks to its ability to perform fast subcentimetre acquisitions with optimisation of vascular enhancement after the injection of contrast material , msct permits visualisation of the entire pulmonary artery tree down to the subsegmental level [ 5 ]  . perfusion lung scintigraphy ( pls ) plays an important role in the diagnosis of ape . 
the aim of our study was to compare and integrate the data provided by pls and by msct in cases of emergency diagnoses of ape in nuclear medicine and radiology departments operating around the clock . materials and methods from may 2002 to june 2004 , we retrospectively reviewed 107 consecutive patients ( 50 women and 57 men ) with ages ranging from 20 to 80 years ( mean age 60 + 18 ) , with a clinical and laboratory suspicion of ape . 
the first - level diagnostic approach included an assessment of predisposing factors , primary disease , symptoms ( dyspnoea , chest pain , cough , haemoptysis , syncope ) and clinical signs ( tachypnoea , tachycardia , signs of deep vein thrombosis , fever , cyanosis )  . 
furthermore , all patients had undergone chest radiography before pls and msct . pls was performed with a gamma starcam 4000 xc / t camera ( ge medical systems , west milwaukee , wi , usa ) after administration of 185 mbq of macroaggregated albumin ( number of particles between 300 , 000 and 50 , 000 and diameter from 120 to 70 m ) with 99 mtc . 
all images were acquired with the planar technique in the anterior , posterior , right and left posterior oblique and right and left anterior oblique projections using the following acquisition parameters : 128128 matrix and 500750 kcounts per projection . 
la trombosi venosa profonda ( tvp ) la pi comune causa di embolia polmonare ed i principali fattori di rischio primari e secondari sono riassunti nella tabella 1 [ 2 ]  . 
nonostante gli importanti e recenti sviluppi della diagnostica strumentale e di laboratorio , circa il 70% degli eventi embolici polmonari sono riconosciuti allesame autoptico [ 3 ]  . nei casi diagnosticati tempestivamente con la tomografia computerizzata spirale multistrato ( tcsm ) e con la scintigrafia polmonare perfusiva ( spp ) , il trattamento anticoagulante si dimostrato efficace in circa il 75% dei pazienti [ 4 ]  . pertanto la tcsm si propone come metodica utile nella valutazione del paziente con sospetta epa . 
infatti , la tcsm per la possibilit di eseguire in tempi rapidi acquisizioni subcentimetriche e di ottimizzare lopacizzazione delle strutture vascolari dopo iniezione di mdc , consente di visualizzare le diramazioni dellalbero arterioso polmonare fino al livello subsegmentario [ 5 ]  . la scintigrafia polmonare perfusiva ( spp ) ha un ruolo importante nella diagnosi di epa . 
questa tecnica non invasiva , a bassa dose di radiazioni , consente una valutazione della perfusione ematica polmonare anche in pazienti critici , non collaboranti e portatori di dispositivi metallici [ 6 ]  . 
scopo del nostro studio stato quello di comparare ed integrare i dati forniti dalla spp e dalla tcsm nella diagnosi in urgenza dellepa in centri di medicina nucleare e radiodiagnostica che svolgono indagini urgenti 24 ore su 24 . materiali e metodi nel periodo compreso tra maggio 2002 e giugno 2004 stata eseguita unanalisi retrospettiva che ha portato a selezionare 107 pazienti consecutivi ( 50 donne e 57 uomini ) di et compresa tra i 20 e gli 80 anni ( et media di 6018 ) , con sospetto clinico - laboratoristico di epa . 
per un inquadramento diagnostico in prima istanza sono stati valutati i fattori predisponenti , la patologia di base , i sintomi ( dispnea , dolore toracico , tosse , emottisi , sincope ) e i segni clinici ( tachipnea , tachicardia , segni di trombosi venosa profonda , febbre , cianosi )  . 
i criteri di inclusione sono stati : lesecuzione sia della spp che della tcsm entro 7 giorni dal sospetto clinico - laboratoristico di epa ; non aver intrapreso alcuna terapia trombolitica . 
inoltre , tutti i pazienti avevano eseguito una rx del torace prima della spp e / o della tcsm . la spp stata effettuata utilizzando una gamma camera starcam 4000 xc / t ( ge medical systems , west milwaukee , wisconsin , usa ) previa somministrazione di 185 mbq di macroaggregati di albumina ( numero di particelle variabile da 300000 a 50000 e diametro compreso tra 120 e 70 micron ) marcati con 99 mtc . 
interpretation criteria for pls were those suggested by the prospective investigative study of acute pulmonary embolism ( pisaped ) study , as reported in table 2 [ 7 ]  . msct was considered positive in the presence of eccentric filling defects or of complete occlusion of the pulmonary artery lumen , whether associated or not to the presence of areas of parenchymal hypoventilation . 
all data provided by pls and msct were independently evaluated by two nuclear physicians and two radiologists and included an assessment of which of the two studies had been ordered and performed first . the patients were grouped based on the time interval between the initial clinical suspicion and performance of the two examinations , namely , 024 h , 2448 h , 0 - 48 hours , and 27 days . 
cohens test was used for analysis of concordance between the results of the two investigations , both on the overall patient population enrolled and for the four subgroups ( 024 h , 2448 h , 048 h , 27 days )  . results pls and msct were positive in 29 / 107 ( 27.1% ) cases and negative in 78 / 107 ( 72.89% ) cases , with a positive concordance ( pc ) in 22 cases and negative concordance ( nc ) in 71 ( table 3 )  . 
in the subgroup of patients who had undergone pls and msct within 24 h of the initial clinical suspicion of ape , pls was positive in 12 / 49 ( 24.48% ) cases and negative in 37 / 49 ( 75.51% ) cases , whereas msct was positive in 17 / 49 ( 34.69% ) cases and negative in 32 / 49 ( 65.30% ) cases , with pc in 12 cases ( fig . 1 ) and nc in 32 cases ( table 4 )  . 
furthermore , 35 patients had undergone pls first , and 14 had undergone msct first . in the subgroup of patients who had undergone pls and msct from 24 to 48 h from the first clinical suspicion of ape , pls was positive in 5 / 20 ( 25% ) cases and negative in 15 / 20 ( 75% ) cases , whereas msct was positive in 3 / 20 ( 15% ) cases and negative in 17 / 20 ( 85% ) cases , with pc in three cases and nc in 15 ( table 5 )  . 
moreover , 13 patients had undergone pls first , and seven had undergone msct first . no state acquisite con tecnica planare in proiezione anteriore , posteriore , obliqua posteriore destra e sinistra , obliqua anteriore destra e sinistra utilizzando i seguenti parametri di acquisizione : matrice 128128 e conteggi per proiezione 500750 kcounts . la tc stata effettuata con apparecchiatura tcsm ( mx 8000 , philips medical system , cleveland , ohio , usa )  . 
i parametri di acquisizione utilizzati sono stati i seguenti : collimazione 2 , 5 mm , pitch 1 , 25 , incremento 1 mm , tempo di rotazione 0 , 5 s , kvp / mas - 120 / 100 . 
lesame stato eseguito con paziente in decubito supino e le scansioni sono state condotte dalle cupole diaframmatiche allo stretto toracico superioil mezzo di contrasto uroangiografico ( ultravist 370 , schering , berlin , germania ) stato iniettato nella vena cubitale attraverso un ago da 16 gauge utilizzando un iniettore automatico ( mk - iv , medrad , pittsburgh , pennsylvania , usa ) con i seguenti parametri : volume 8090 ml , flusso 33 , 5 ml / s . 
il ritardo di inizio delle scansioni stato determinato con lesecuzione di un bolus test , ed risultato variabile tra 9 e 15 s ( fase arteriosa polmonare ) , mentre la fase tardiva stata eseguita dopo 4550 s . 
i criteri dinterpretazione per la spp sono quelli proposti dallo studio prospettico pisa - ped e riportati in tabella 2 [ 7 ]  . la tcsm stata considerata positiva in presenza di difetti di riempimento eccentrici o occludenti lintero lume delle arterie polmonari , associati o meno alla presenza di aree dipoventilazione parenchimale . 
tutti i dati forniti dalla spp e dalla tcsm sono stati valutati indipendentemente da due medici nucleari e da due medici radiologi ed stato anche valutato quale dei due esami era stato richiesto ed eseguito per primo . i pazienti arruolati sono stati raggruppati in base allintervallo di tempo trascorso tra linsorgenza del sospetto clinico e lesecuzione di entrambe le indagini , ovvero 024 ore , 2448 ore , e 27 giorni . 
il test di cohen stato utilizzato per lanalisi della concordanza dei risultati delle due indagini sia per il gruppo di tutti i pazienti arruolati che per i quattro sottogruppi suddivisi in base allintervallo di tempo per lesecuzione delle due indagini 024 ore , 2448 ore , 048 ore , 48 ore7 giorni . risultati la spp e la tcsm sono risultate positive in 29 / 107 ( 27 , 1% ) e negative in 78 / 107 ( 72 , 89% ) , con una concordanza positiva in 22 casi e negativa in 71 ( tabella 3 )  . 
nel sottogruppo dei pazienti che avevano eseguito la spp e la tcsm entro 24 ore dallinsorgenza del sospetto clinico di epa , la spp risultata positiva in 12 / 49 ( 24 , 48% ) e negativa in 37 / 49 ( 75 , 51% ) , mentre la tcsm risultata positiva in 17 / 49 ( 34 , 69% ) e negativa in 32 / 49 ( 65 , 30% ) con g . 
pls ( a ) shows no perfusion in the inferior right lobe , with positive concordance with msct ( b ) , which shows an occlusive thrombus in the right inferior lobe ( white arrow )  . 
la ssp ( a ) mostra assenza di perfusione in corrispondenza del lobo inferiore destro con concordanza positiva con la tcsm ( b ) che mostra la presenza di un trombo occludente nel ramo lobare inferiore destro dellarteria polmonare ( freccia piena )  . 
la tcsm ( b ) dimostra la presenza di un trombo parzialmente occludente ( freccia )  . in the subgroup of patients who had undergone pls and msct within 48 h from the first clinical suspicion of ape , pls was positive in 17 / 69 ( 24.63% ) cases and negative in 52 / 69 ( 75.36% ) cases , while msct was positive in 20 / 69 ( 28.98% ) cases and negative in 49 / 69 ( 71% ) cases , with pc in 15 cases and nc in 47 ( table 6 )  . 
inoltre 35 pazienti avevano effettuato prima la spp e 14 prima la tcsm . nel sottogruppo dei pazienti che avevano eseguito la spp e la tcsm tra 24 e 48 ore dallinsorgenza del sospetto clinico di epa , la spp risultata positiva in 5 / 20 ( 25% ) e negativa in 15 / 20 ( 75% ) , mentre la tcsm risultata positiva in g . 
secondary antithrombin iii deficit protein c deficit factor v leiden protein s deficit plasminogen deficit factor xii deficit hyperhomocysteinaemia anti - cardiolipin antibody congenital dysfibrinogenaemia thrombomodulin tabella 1 fattori di rischio per le tromboembolie venose a . 
concordance between results 3 / 20 ( 15% ) e negativa in 17 / 20 ( 85% ) con una concordanza positiva in 3 casi e negativa in 15 ( tabella 5 )  . 
inoltre 24 pazienti avevano effettuato prima la spp e 14 prima la tcsm . nella tabella 8 riportata lanalisi della concordanza tra la spp e la tcsm nei gruppi di pazienti che hanno effettuato le due indagini entro 24 ore , tra 2448 ore , entro 48 ore , tra 27 giorni ed entro 7 giorni dal sospetto clinico di epa . 
tale concordanza stata maggiore quando le due indagini sono state eseguite entro 24 ore dal sospetto clinico , continua a mantenere valori elevati se le indagini sono eseguite entro 48 ore , mentre si riduce notevolmente se gli esami sono stati eseguiti entro 7 giorni dal sospetto clinico . inoltre fra i 22 pazienti con concordanza positiva tra la spp e la tcsm , 12 hanno eseguito entrambi gli esami entro le 24 ore , 3 entro 48 ore , 7 entro una settimana . 
tra i pazienti con concordanza positiva tra le due indagini nellintera casistica studiata , il 54 , 54% ( 12 / 22 ) apparteneva al sottogruppo studiato entro 24 ore dal sospetto clinico di epa , il 13 , 6% ( 3 / 22 ) al sottogruppo studiato entro 48 ore , il rimanente 31 , 81% ( 7 / 22 ) al sottogruppo studiato entro 7 giorni dal sospetto clinico di epa . 
tra i pazienti con concordanza negativa tra le due indagini nellintera casistica studiata , il 45 , 07% ( 32 / 71 ) apparteneva al sottogruppo studiato entro 24 ore dal sospetto clinico di epa , il 21 , 12% ( 15 / 71 ) al sottogruppo studiato entro 48 ore , il rimanente 33 , 80% ( 24 / 71 ) al sottogruppo studiato entro 7 giorni dal sospetto clinico di epa . in 11 pazienti con spp positiva in fase acuta , questo esame stato ripetuto anche dopo terapia trombolitica prima della dimissione dei pazienti ed ha dimostrato il positivo effetto del trattamento ( miglioramento in 3 , completa normalizzazione della perfusione polmonare in 8 pazienti )  . discussione la diagnosi di embolia polmonare spesso difficile e la mortalit nei casi non trattati , si aggira intorno al 30% , g . 
presence of wedgeshaped areas of hyperperfusion single or multiple wedge - shaped perfusion defects with or without corresponding radiographic parenchymal alterations . absence of wedge - shaped areas of hyperperfusion tabella 2 criteri di interpretazione della spp ( pisa - ped ) diagnosi risultati normale anormale compatibile con epa anormale non compatibile con epa difetti di perfusione non assenza di difetti perfusivi difetti di perfusione cuneiformi singoli o multipli con o senza corrispondenti alterazioni radiografiche parenchimali . 
concordance was higher when the two studies were performed within 24 h of the initial clinical suspicion and remained high when they were performed within 48 h , whereas it decreased markedly if the two studies were performed within 7 days of the initial clinical suspicion . furthermore , among the 22 patients with pc between pls and msct , 12 had undergone both exams within 24 h , three within 48 h , and seven within 1 week . 
among patients with pc between the two investigations , 54.54% ( 12 / 22 ) belonged to the subgroup studied within 24 h of the initial clinical suspicion of ape , 13.6% ( 3 / 22 ) to the subgroup studied within 48 h , and the remaining 31.81% ( 7 / 22 ) to the subgroup studied within 7 days . 
among patients with nc between the two examinations , 45.07% ( 32 / 71 ) belonged to the subgroup studied within 24 h of the initial clinical suspicion of ape , 21.12% ( 15 / 71 ) to the subgroup studied within 48 h , and the remaining 33.80% ( 24 / 71 ) to the subgroup studied within 7 days . mentre si riduce al 2%8% se viene intrapresa una adeguata terapia anticoagulante [ 4 ]  . 
nel 90% dei casi il sospetto di epa motivato dalla presenza di tosse , dispnea , dolore toracico , sincope , tachipnea , cianosi e tachicardia [ 7 , 9 ]  . 
lassenza di questi sintomi ha un valore predittivo negativo del 94% , mentre il valore predittivo positivo rimane basso , pari al 53% , anche considerando lassociazione di pi segni e sintomi riferibili ad epa . 
la radiografia del torace , il dosaggio dei d - dimeri , lemogasanalisi e lecg permettono di orientare la diagnosi e di monitorare le condizioni generali del paziente , ma non consentono una diagnosi definitiva . 
la radiografia del torace solo in rari casi permette di riconoscere lembolia polmonare tuttavia lintegrazione con la spp pu risultare utile per definire la diagnosi di epa [ 11 ]  . la spp permette la visualizzazione diretta della perfusione polmonare attraverso lidentificazione di difetti perfusivi di tipo segmentario e pertanto riveste un ruolo centrale nelliter diagnostico non invasivo di epa . 
la spp un esame semplice , rapido , economico , senza controindicazioni e anche se eseguito con tecnica planare in una sola proiezione permette di riconoscere embolie gravi , per le quali un trattamento terapeutico tempestivo necessario per salvare la vita del paziente . 
la classificazione delle immagini scintigrafiche perfusive suggerita dallo studio pisaped , da noi utilizzata , basata sullidentificazione della presenza e della morfologia di difetti perfusivi , indipendentemente dal loro numero [ 7 ]  . 
dai risultati del suddetto studio appare che la combinazione di una probabilit clinica intermedia o alta con una scintigrafia compatibile con epa ha un elevato valore predittivo positivo ( 98% ) e che una bassa probabilit clinica combinata con un spp anormale , non compatibile con epa , ha un elevato valore predittivo negativo di malattia ( 98% ) [ 7 ]  . nei centri di medicina nucleare , che eseguono indagini 24 ore su 24 in regime durgenza , come il nostro , la spp risultata lesame pi frequentemente richiesto per primo , poich fornisce i dati utili per una gestione terapeutica immediata del paziente . 
nei presidi sanitari in cui la spp non sia rapidamente disponibile la tcsm rappresenta una valida alternativa per documentare le occlusioni totali o parziali delle arterie polmonari [ 1216 ]  . 
tuttavia anche se la tcsm permette di riconoscere embolie pluri - , monoo subsegmentarie , rilevando eventuali riduzioni di densit parenchimale e di vascolarit , non in grado di valutare le modifig . 
of patients who underwent msct first : 30 nudi pazienti che hanno effettuato prima la spp : 77 nudi pazienti che hanno effettuato prima la tcsm : 30 plspls + total plspls + total sppspp + totale sppspp + totale table 4 results for patients undergoing perfusion lung scintigraphy ( pls ) and multislice computed tomography ( msct ) within 024 h tabella 4 risultati della spp e tcsm nel gruppo di pazienti studiati nellintervallo di tempo 024 ore msctmsct + total tcsmtcsm + totale no . 
of patients who underwent msct first : 14 nudi pazienti che hanno effettuato prima la spp : 35 nudi pazienti che hanno effettuato prima la tcsm : 14 in 11 patients with a positive pls in the acute phase , the examination was repeated after thrombolytic therapy and before discharge , and it confirmed the positive effect of treatment ( improvement in three , complete normalisation of lung perfusion in eight )  . discussion the diagnosis of pulmonary embolism is often difficult , and mortality in untreated cases is around 30% , whereas it drops to 2%8% if adequate anticoagulation therapy is initiated [ 4 ]  . 
in 90% of cases , the suspicion of ape is based on the presence of cough , dyspnoea , chest pain , syncope , tachypnoea , cyanosis and tachycardia [ 7 , 9 ]  . 
the absence of these symptoms has a 94% negative predictive value ( npv ) , whereas the positive predictive value ( ppv ) remains low , at 53% , even when the association of several signs and symptoms typical of ape is considered . therefore , other tests need to be used to recognise pulmonary embolism within the first hours after symptom onset . chest radiography , d - dimer , blood gas analysis and electrocardiogram ( ecg ) may suggest a diagnosis and help monitor the patients general condition , although they do not provide a definite diagnosis . 
chest radiography allows recognicazioni funzionali e quindi leffetto emodinamico di emboli o stenosi vascolari che determinano alterazioni della perfusione polmonare [ 17 , 18 ]  . nella nostra esperienza il test di cohen ha documentato una buona concordanza in tutti e 107 i pazienti selezionati . la concordanza risultata maggiore quando lintervallo temporale tra insorgenza del sospetto clinico e lesecuzione delle due indagini era minimo . 
tale non concordanza tra le due indagini spiegabile con la presenza di emboli parzialmente occludenti , che in realt non determinavano alterazioni perfusive significative e quindi di scarso o assente significato funzionale . 
tale non concordanza stata riscontrata nel 3 , 5% ( 2 su 57 ) di pazienti che avevano eseguito prima la spp e poi la tcsm studiati nellintervallo 048 ore e nel 6 , 54% ( 7 su 107 ) di pazienti dellintera casistica studiati nei 7 giorni successivi linsorgenza del sospetto clinico . 
lattivazione dei meccanismi trombolitici con frammentazione dellembolo nelle 48 ore7 giorni dopo linsorgenza del sospetto clinico pu spiegare questa non concordanza . nessun paziente che aveva eseguito entrambe le indagini entro le 24 ore dallinsorgenza del sospetto clinico ha mostrato spp positiva e tcsm negativa . 
of patients who underwent msct first : 7 nudi pazienti che hanno effettuato prima la spp : 13 nudi pazienti che hanno effettuato prima la tcsm : 7 plspls + total plspls + total sppspp + totale sppspp + totale table 6 results for patients undergoing perfusion lung scintigraphy ( pls ) and multislice computed tomography ( msct ) within 048 h tabella 6 risultati della spp e tcsm nel gruppo di pazienti studiati nellintervallo di tempo 048 ore msctmsct + total tcsmtcsm + totale no . 
of patients who underwent msct first : 12 nudi pazienti che hanno effettuato prima la spp : 57 nudi pazienti che hanno effettuato prima la tcsm : 12 tion of pulmonary embolism in sporadic cases only ; however , integration with pls may be useful to confirm the diagnosis of ape [ 11 ]  . pls allows direct visualisation of lung perfusion through the identification of perfusion defects at the segmental level , thus playing a key role in the noninvasive assessment of ape . 
pls is an easy - to - perform , fast , and inexpensive examination that does not present contraindications and , even when performed with the standard planar technique in a single projection , is able to recognise severe embolism necessitating urgent life - saving treatment . 
the classification of scintigraphic perfusion images suggested by the pisa - ped study , which we used , is based on identifying the presence and morphology of perfusion defects , independent of their number [ 7 ]  . 
the results of the above study suggest that the combination of an intermediate to high clinical probability with scintigraphic findings consistent with ape has a high ppv ( 98% ) , and that a low clinical probability combined with abnormal pls findings not consistent with ape has a high npv for this disease ( 98% ) [ 7 ]  . in nuclear medicine centres that perform emergency examinations 24 h / day , as we do , pls was found to be most frequently requested first , as it provides useful information for immediate patient management . 
la tcsm ha il grande pregio di fornire una visione panoramica di tutto il torace e di riconoscere differenti patologie ( atelettasie , focolai broncopneumonici , focolai emorragici ed enfisema )  . 
nei pazienti con spp anormale in cui la tcsm ha mostrato coesistenti patologie cardio - polmonari lintegrazione dei dati pu permettere di attribuire o meno significato embolico alle alterazioni di perfusione riscontrate alla spp [ 19 ]  . in letteratura sono stati suggeriti diversi algoritmi diagnostici sullimpiego delle differenti tecniche di diagnostica per immagini nellepa . 
i criteri di scelta delliter diagnostico devono , tuttavia , tener conto del valore clinico , della reale disponibilit delle diverse indagini , della situazione clinica del paziente ( stabile o instabile ) e della presenza di concomitanti patologie cardio - polmonari . 
this lack of concordance between the two techniques may be explained by the presence of partial occlusions causing no significant changes in perfusion and , subsequently , of little or no functional significance . 
this lack of concordance was identified in 3.5% ( 2 of 57 ) of patients who had undergone pls first and then msct , studied in the 0to 48 - h interval , and in 6.54% ( 7 of 107 ) of patients studied within 7 days of initial clinical suspicion . 
activation of thrombolytic mechanisms with embolus fragmentation during the 27 day period after the initial clinical suspicion may explain this lack of concordance . no patient who underwent both exams within 24 h of the initial clinical suspicion had a positive pls and a negative msct . 
msct has the advantage of providing a panoramic view of the entire chest and visualising different diseases ( atelectasis , bronchopulmonary foci , haemorrhagic foci , and emphysema )  . 
in patients with an abnormal pls , in which msct demonstrated concurrent cardiopulmonary diseases , integration of the data may be necessary to decide whether or not the perfusion alterations seen at pls are ascribable to embolism [ 19 ]  . several diagnostic imaging algorithms have been suggested for the detection of ape . 
other authors suggest the use of msct in cases of pls alterations not typical of pulmonary embolism , advising against its use as a stand - alone technique [ 20 , 21 ]  . however , the criteria for planning the diagnostic workup must take into account the clinical value of techniques , their availability , the patients clinical condition ( stable or unstable ) and the presence of concurrent cardiopulmonary diseases . 
on the other hand , in stable patients with complex radiologic findings , msct should be performed first , because the chances of a nondiagnostic scintigraphy are high in these cases . 
however , msct is preferred for patients with preexisting lung disease or when a differential diagnosis with mediastinal diseases is needed . table 7 results for patients undergoing perfusion lung scintigraphy ( pls ) and multislice computed tomography ( msct ) within 27 days msctmsct + total no . 
of patients with positive exams patients who underwent pls first : 15 patients who underwent msct first : 7 024 h 12 / 49 ( 24.48% ) 2448 h 3 / 20 ( 15.00% ) 27 days 7 / 38 ( 18.42% ) tabella 9 sequenza temporale degli esami spp e tcsm dei 22 pazienti con concordanza positiva raggruppati in base allintervallo di tempo tra insorgenza del sopetto clinico e completamento degli esami di diagnostica per immagini intervallo tra le due indagini numero pz con spp e tcsm positive pz che hanno effettuato prima la spp : 15 pz che hanno effettuato prima la tcsm : 7 024 ore 12 / 49 ( 24 , 48% ) 2448 ore 3 / 20 ( 15 , 00% ) 27 gg 7 / 38 ( 18 , 42% ) conclusions conclusioni in a large group of patients recruited in hospitals with round - the - clock emergency nuclear medicine and radiology services and that follow strict image interpretation criteria , pls and msct proved to have a high concordance when the exams were performed within 7 days of the initial clinical suspicion of ape . 
therefore , a clinical suspicion of ape needs to be confirmed by emergency examinations performed within hours 48 at the most . when both pls and msct are available , pls is the first exam to be ordered for a clinical suspicion of ape . 
although no diagnostic protocol has been universally adopted , pls is the first technique to identify the site and extent of the embolism , thus providing indirect evidence of pulmonary embolismsct is an accurate technique for the diagnosis of ape , and because it can provide information even on other pathological processes , it can confirm or exclude the diagnosis suspected at pls . when both diagnostic techniques are not available , either pls or msct are valuable tools for identifying the presence of severe ape necessitating urgent treatment . in unampia casistica di pazienti arruolati in grandi ospedali con centri di medicina nucleare e radiodiagnostica che effettuano indagini 24 ore su 24 , utilizzando rigorosi criteri di interpretazione delle immagini , la spp e la tcsm hanno dimostrato unelevata concordanza quando eseguite entro 7 giorni dallinsorgenza del sospetto di epa . 
la concordanza pi elevata se le due indagini sono completate entro 2448 ore . pertanto nel sospetto clinico di epa importante accertare la diagnosi con esami durgenza in poche ore , massimo 48 . la spp il primo esame richiesto nel sospetto clinico di epa quando sono disponibili entrambe le tecniche diagnostiche . 
pur non essendo unanimemente accettato un protocollo diagnostico , la spp rappresenta la tecnica di primo approccio per stabilire la sede e lestensione del processo embolico , fornendo levidenza indiretta dellembolia polmonare . 
la malformazione vascolare polmonare stata chiusa in 3 minuti e la saturazione percutanea di ossigeno passata dal 73% al 93% . la fistola da emodialisi stata chiusa con 1 dispositivo in 4 minuti . 
lavp un efficace sistema per la chiusura di grossi vasi periferici e di malformazioni vascolari , a costi contenuti , che consente unembolizzazione sicura e a basso rischio di complicanze , con risparmio in termini di tempo e di dose erogata . key words amplatzer vascular plug transcatheter embolisation vascular disease parole chiave amplatzer vascular plug embolizzazione transcatetere patologia vascolare introduction introduzione percutaneous embolisation has become a valid option and often the first - line treatment for acute bleeding , temporary or permanent devascularisation of benign or malignant hypervascular tumours [ uterine fibroids , hepatocellular carcinoma ( hcc ) ] , the closure of congenital or acquired arteriovenous lembolizzazione percutanea divenuta ormai una valida alternativa e spesso la metodica di prima istanza nel trattamento di sanguinamenti acuti , ma anche nella devascolarizzazione temporanea o permanente di tumori ipervascolarizzati benigni o maligni ( fibromi uterini , hcc ) , nella chiusura c . 
several embolisation devices are available , each with its own characteristic features : a fibrin sponge is used for temporary ( about 48 h ) embolisation , polyvinyl alcohol ( pva ) microspheres are commonly used for permanent embolisation , whereas detachable balloons , glue , metal coils and plugs are used for definitive embolisation . this paper describes our experience with a new system for definitive embolisation , the amplatzer vascular plug ( avp ) , a versatile device that was successfully used in the closure of arteries ( internal iliac and subclavian ) , a pulmonary arteriovenous malformation , a haemodialysis fistula , and a gastric varix in a subject with portal hypertension . materials and methods the avp ( aga medical corporation , golden valley , mn , usa ) is a new device approved by the u.s. 
food and drug administration for peripheral embolisation of arteries and veins and that uses the same technology as other amplatzer devices used for occluding interatrial and interventricular defects and patent foramen ovale . 
the system is preloaded inside a 100 - cm - long guiding catheter : 5 fr for plugs with a diameter of 48 mm , 6 fr for plugs with a diameter of 1012 mm and 8 fr for plugs with a diameter of 1416 mthe system ranges in size from 4 mm di malformazioni vascolari arterovenose congenite o acquisite e nella chiusura di varici . 
esistono in commercio numerosi dispositivi embolizzanti ciascuno con caratteristiche preponderanti rispetto agli altri : per lembolizzazione temporanea si utilizza la spugna di fibrina ( 48 h circa ) ; per quella permanente sono comunemente impiegate le microsfere in alcol polivinilico ( pva ) ; per lembolizzazione definitiva infine ci si avvale dei palloni staccabili , della colla , delle spirali metalliche e dei plug . gli autori descrivono la loro esperienza nellutilizzo di un nuovo sistema per lembolizzazione definitiva , lamplatzer vascular plug ( avp ) , un dispositivo dallutilizzo versatile , specie nei vasi di grosso diametro , che stato utilizzato con successo nella chiusura di arterie ( iliache interne e succlavie ) , di una malformazione arterovenosa polmonare , di una fistola da emodialisi e di una varice gastrica in soggetto con ipertensione portale . materiali e metodi lavp ( aga medical corporation , golden valley , mn , usa ) un nuovo dispositivo approvato dalla food and drug administration per lembolizzazione periferica di vasi arteriosi e venosi che utilizza la stessa tecnologia di altri device amplatzer utilizzati per occludere difetti interatriali , interventricolari e forami ovali . 
il sistema caricato allinterno di un catetere portante ( 100 cm di lunghezza ) da 5 fr per plug da 4 a 8 mm di diametro , da 6 fr per plug da 10 a 12 mm di diametro e da 8 fr per plug da 14 a 16 mm di diametro . 
il sistema disponibile in misure che vanno da 4 mm di diametro fino a 16 mm con un incremento di 2 muna volta raggiunta la sede in cui si vuole rilasciare il dispositivo basta ritirare il catetere in modo da far uscire il plug e girare in senso antiorario la guida metallica per liberare il device . 
in accordo con la casa produttrice si raccomanda di scegliere un dispositivo con un oversize almeno del 20% rispetto al diametro del vaso da trattare per evitare una possibile migrazione distale . 
le peculiarit del sistema sono la sua disponibilit in unampia gamma di misure , anche molto grandi , la possibilit di riposizionamento prima del rilascio definitivo , il minor rischio di migrazione rispetto alle spirali e un breve tempo per occludere definitivamente il vaso . presso il nostro centro sono state eseguite 11 occlusioni vascolari con lutilizzo del nuovo dispositivo avp : 5 arterie iliache interne nella prevenzione dellendoleak di tipo ii dopo posizionamento di endoprotesi aortoiliaca ; 3 arterie succlavie di sinistra per il trattamento di endoleak di ii tipo successivo al posizionamento di endoprotesi dellaorta toracica con copertura dellarteria succlavia sinistra ( ass ) ; 1 fistola arterovenosa polmonare ; 1 fistola da emodialisi e 1 grossa varice in un paziente con emorragia da varice gastrica portatore di tips . 
positive features include the availability of a wide range of sizes , even very large ; the possibility of repositioning the device before its definitive release ; lower risk of migration compared with coils ; and short occlusion times . 
 our centre performed 11 vascular occlusions using the new avp : five internal iliac arteries to prevent type ii endoleaks after aortoiliac stent - graft placement , three left subclavian arteries to treat type ii endoleaks after thoracic aorta stent - graft placement with coverage of the left subclavian artery ( lsa ) , one pulmonary arteriovenous malformation , one haemodialysis fistula , and one large gastric varix due to haemorrhage in a patient with transjugular intrahepatic portosystemic shunt ( tips )  . 
the patients were studied with a multidetector computed tomography ( mdct ) scanner ( somatom sensation 6 , siemens , germany ) , mdct workstation ( leonardo , siemens , germany ) , angiographic equipment ( axiom fa , siemens , germany ) and ultrasound ( us ) ( technos , esaote , italy )  . 
chest mdct done to assess the pulmonary arteries showed a large solitary arteriovenous fistula with two afferent arteries arising from two branches of the right lower lobar pulmonary artery and drainage into the inferior pulmonary vein of the right lower lobe . 
la mdct del torace effettuata per la valutazione delle arterie polmonari ha dimostrato una grossa e solitaria fistola arterovenosa polmonare con due arterie afferenti , provenienti da due rami dellarteria polmonare lobare inferiore di destra , e drenaggio nella vena polmonare inferiore del lobo inferiore del polmone destro . 
langiografia selettiva dellarteria polmonare destra , eseguita attraverso la vena femorale comune di destra , ha confermato la diagnosi posta con la mdct dimostrando un diametro da 10 mm e 4 mm delle due arterie afferenti . 
la chiusura del vaso di maggiori dimensioni stata eseguita utilizzando la tecnica standard gi descritta , con un avp da 12 mm ( oversize del 20% rispetto al vaso nativo ) , mentre la seconda arteria afferente , di minori dimensioni ( 4 mm di diametro ) , stata cateterizzata mediante la tecnica buddy wire : tale sistema consiste nellutilizzo di due guide allinterno dello stesso catetere , per garantire maggior portanza , facilitando il posizionamento del device in vasi a decorso pi complesso . 
 stata utilizzata una guida coronarica da 0 , 014 ( guidant spartacore middle weight ) come buddy wire che stata posizionata oltre il segmento scelto per il rilascio del dispositivo , dando stabilit al catetere , prima del passaggio del device . 
2a multidetector computed tomography ( mdct ) angiography of the abdominal aorta and iliac arteries : anterior volume - rendered ( vr ) image shows an aneurysm of the right common iliac artery ( arrowhead )  . 
c angiography 8 min after release of the avp shows the outline of the device ( arrows ) and complete absence of flow within the right internal iliac artery from the systemic blood flow . 
2a angio - mdct dellaorta addominale e delle arterie iliache : ricostruzione vrt ( volume rendering technique ) che dimostra la presenza un aneurisma dellarteria iliaca comune di destra ( testa di freccia )  . 
c controllo angiografico a 8 minuti dal rilascio dellamplatzer vascular plug , dimostra la sagoma del device rilasciato ( frecce ) e la completa assenza di flusso nellarteria iliaca interna destra dal circolo sistemico arterioso . 
3a angiography of the thoracic aorta after stent - grafting in a patient with type b dissection of the thoracic aorta demonstrates the presence of a type i + type ii endoleak ( arrowheads ) from the left subclavian artery . 
b follow - up multidetector computed tomography ( mdct ) angiography at 1 month ; sagittal maximum intensity projections ( mip ) image confirmed the type i + type ii endoleak ( arrowhead ) from the left subclavian artery and the presence of partial thrombosis of the false lumen ( * )  . 
c similar projection 5 min after release of the amplatzer vascular plug ( avp ) shows the outline of the device ( arrows ) , with the exclusion of the origin of the left subclavian artery , the patency of the left vertebral and internal mammary artery and the absence of endoleaks . 
3a angiografia dellaorta toracica dopo il posizionamento di endoprotesi toracica in un paziente con dissecazione dellaorta toracica di tipo b che dimostra un endoleak di tipo misto ( tipo i + ii ) ( teste di freccia ) dallarteria succlavia sinistra . 
b controllo angio - mdct a un mese , ricostruzione sagittale mip ( maximum intensity projections ) , che conferma la presenza di un endoleak di tipo misto ( tipo i + ii ) ( testa di freccia ) dallarteria succlavia sinistra e la presenza di trombosi parziale del falso lume ( * )  . 
c sucessivo controllo a 5 minuti dal rilascio dellamplatzer vascular plug che mette in evidenza la sagoma del device rilasciato ( frecce ) con la completa chiusura dellorigine dellarteria succlavia sinistra , la perviet dellarteria vertebrale e mammaria interna e lassenza dellendoleak . 
4a angiography shows a persistent passage of contrast material across the previously ligated haemodialysis arteriovenous anastomosis and two large aneurysms ( arrowheads ) that end in a cul - de - sac at the level of the basilic vesome collateral veins , with reverse flow , can be seen to originate from the efferent vein and course towards the hand ( arrows )  . 
b angiography performed 2 min after amplatzer vascular plug ( avp ) deployment shows a considerable reduction in the passage of contrast ( arrow ) through the device ( arrowhead ) into the basilic venote the good patency of the artery below . 
4a langiografia mette in evidenza la persistenza del passaggio di mdc attraverso lanastomosi arterovenosa da emodialisi precedentemente legata chirurgicamente e i due grossi aneurismi ( testa di frecce ) che terminano a fondo cieco a livello della vena basilica efferente : si notano inoltre alcune vene collaterali che si dipartono dalla vena efferente e si dirigono , a flusso invertito , verso la mano ( frecce )  . 
b il controllo angiografico eseguito dopo 2 minuti dallapertura del plug dimostra una marcata riduzione del passaggio di mdc ( freccia ) attraverso il dispositivo ( testa di freccia ) nella vena basilica efferente . 
c il controllo dopo altri 3 minuti evidenzia la totale chiusura della fistola , lassenza delle vene collaterali e la perviet dellasse arterioso a valle . 10 mm and 4 mm of the two afferent arteries . 
closure of the larger vessel was achieved by applying the standard technique , with a 12 - mm avp ( 20% larger than the native vessel ) , whereas the smaller afferent artery ( 4 mm ) was catheterised using the buddy wire technique , which consists of using two guidewires within the same catheter , to ensure better support and facilitate deployment of the device in more tortuous vessels . 
dopo aspirazione del trombo portale , effettuata mediante catetere tipo portante da 8 fr ( jr 4.0 , medtronic , mn , usa ) , stata eseguita una seconda angiografia che ha dimostrato la parziale riduzione del trombo e la persistenza di flusso nella varice : si quindi optato per la sua embolizzazione definitiva . 
5a portography shows , in a patient with transjugular intrahepatic portosystemic shunt ( tips ) , a filling defect ( arrowhead ) of the portal vein at the outlet of the superior mesenteric vein due to probable thrombus . 
5a portografia evidenzia , in un paziente portatore di tips , un difetto di riempimento ( testa di freccia ) della porta a livello dello sbocco della vena mesenterica superiore , da verosimile apposizione trombotica ; ben evidente inoltre la vena gastrica sinistra dilatata ( freccia ) con la presenza di spirali metalliche , da precedente embolizzazione . 
b posizionamento del plug ( testa di freccia ) allinterno della varice prima del rilascio definitivo : si riconoscono molto bene i due markers radiopachi posizionati alle due estremit del device . 
after thromboaspiration with an 8 - fr guiding catheter ( jr 4.0 , medtronic , mn , usa ) , a second angiography showed partial reduction of the thrombus and persistence of flow in the varix : we therefore opted for definitive embolisation . 
portography carried out 5 min after the procedure showed flow reduction through the device but incomplete closure ; two 12 - mm platinum coils ( nester , cook , bloomington , in , usa ) were endoprotesi aortoiliaca stato eseguito il giorno successivo per tre pazienti e due giorni dopo lembolizzazione per gli altri due . 
lembolizzazione unilaterale dellarteria iliaca interna stata tollerata da tutti e 5 i pazienti senza la comparsa di complicanze minori o maggiori quali claudicatio glutea , ischemia glutea o rettosigmoidea , disfunzioni sessuali o urologiche ; il periodo di ospedalizzazione stato in media 5 giorni dopo il posizionamento dellendoprotesi . 
il controllo angio - mdct a 1 mese ha evidenziato una buona rivascolarizzazione da parte di circoli collaterali delle due branche di divisione delliliaca interna trattata , anteriore e posteriore , senza passaggio di contrasto nel tronco comune e ha dimostrato la corretta posizione del plug con perfetta chiusura del vaso solo alla sua origine . 
 nei 3 pazienti a cui stata occlusa larteria succlavia sinistra , la mdct effettuata prima della dimissione e a 1 mese di distanza dal posizionamento dellendoprotesi aortica ha dimostrato la persistenza di endoleak di tipo ii dallarteria succlavia sinistra per due pazienti e in un paziente un endoleak misto : tipo i + tipo ii , ovvero dovuto sia ad una imperfetta sigillatura della protesi sulla parete aortica sia ad una riperfusione attraverso la succlavia : si pertanto optato per locclusione del vaso alla sua origine . 
prima di effettuare il trattamento i pazienti sono stati sottoposti a mdct e a studio eco - color doppler dei tronchi sovraortici e del circolo intracranico per escludere stenosi , ipoplasia o varianti anatomiche delle arterie carotidi , succlavie , vertebrali e del circolo del willis . 
i pazienti sono stati sottoposti quindi ad angiografia selettiva della ass che ha confermato la diagnosi di endoleak di tipo ii in due pazienti ed endoleak misto , tipo i + tipo ii , in un paziente , con perviet della vertebrale . 
tutti i pazienti sono stati dimessi due giorni dopo la procedura di occlusione : nessuno dei 3 pazienti ha avuto complicanze maggiori ( sindrome da furto della succlavia )  . 
il corretto posizionamento dellavp e lavvenuta occlusione dellarteria succlavia stato dimostrato anche mediante mdct di controllo . la fistola arterovenosa polmonare stata completamente esclusa con lutilizzo di due dispositivi avp , uno per ciascuna delle due arterie afferenti . 
il controllo angiografico ha dimostrato una chiusura totale dei due vasi embolizzati : in circa 3 minuti per il vaso maggiore e in minor tempo per quello di minori dimensioni . la saturazione percutanea di ossigeno in condizioni basali salita dal 73% al 93% subito dopo la procedura e si mantenuta stabile per i successivi 6 mesi di follow - up ; lemogasanalisi ha dimostrato normali pressioni di ossigeno e di anidride carbonica . 
il controllo clinico a 1 , 3 , 6 mesi ha dimostrato lassenza di sintomi respiratori , saturazione di ossigeno superiore al 95% in condizioni basali e un radiogramma del torace a tre mesi ha confermato una corretta e stabile posizione dei devices . nella fistola da emodialisi leco - color doppler ha dimostrato la presenza di due grosse dilatazioni vascolari anecogene , con presenza di flusso allinterno , a livello della piega del gomito . 
langiografia dellarto superiore , eseguita con accesso trans - omerale anterogrado 4 fr , ha confermato la presenza di una fistola arterovenosa in cui la legatura chirurgica della vena basilica efferente , eseguita 6 mesi prima , sia a livello dellanastomosi sia poco pi a valle , non aveva garantito una completa chiusura della connessione arterovenosa ; liperafflusso conseguente ha portato allo sviluppo di due grossi aneurismi venosi . 
al fine di escludere il flusso nei due aneurismi e nelle vene collaterali senza ricorrere ad una seconda legatura chirurgica si deciso , in accordo con il paziente , di chiudere definitivamente la fistola per via percutanea mediante avp . 
langiografia eseguita a 4 minuti dal posizionamento ha dimostrato una completa ed efficace chiusura della fistola con assenza di pasresults in the five patients undergoing closure of the common iliac artery , correct avp position was evaluated by means of a manual injection of contrast medium through the guiding catheter ; once in the correct position , the device was unscrewed and released . 
postprocedure angiography confirmed complete occlusion of the vessel within less than 8 mthe aortoiliac endografts were positioned on the following day in three patients and after 2 days in the other two . 
unilateral embolisation of the iia was tolerated by all five patients without minor or major complications , such as gluteal claudication , gluteal or rectosigmoid ischaemia and sexual or urological dysfunctions ; length of hospital stay was 5 days after endograft placement . 
mdct angiography at 1 month showed good revascularisation of the anterior and posterior branches of the treated iia by collateral circulations , without passage of contrast material in the common trunk , and demonstrated the correct position of the plug with perfect closure of the vessel origin . in the three patients who underwent occlusion of the lsa , mdct performed at discharge and 1 month after aortic endograft placement showed the persistence of type ii endoleak from the lsa in two patients and a mixed , type i and type ii , endoleak in one . 
these were both due to imperfect endograft sealing along the aortic wall and to reperfusion through the subclavian artery : we therefore decided to occlude the artery at its origbefore the embolisation procedure , the patients underwent mdct and colour - doppler us of the supraaortic trunks and intracranial vessels to exclude stenosis , hypoplasia or anatomical variants of the carotid , subclavian , vertebral arteries and the circle of willis . 
the patients were then studied by selective angiography of the lsa , which confirmed the type ii endoleak in two patients and the mixed type i and type ii endoleak in the other , with a patent vertebral artery . 
postprocedural angiography confirmed complete occlusion of the origin of the subclavian artery within less than 5 mthe patients were discharged 2 days after the procedure without developing major complications ( subclavian steal syndrome )  . 
correct avp positioning and successful subclavian artery occlusion were also demonstrated by follow - up mdct . the pulmonary arteriovenous malformation was completely excluded using two plugs , one for each of the two afferent arteries . 
baseline percutaneous oxygen saturation rose from 73% to 93% immediately after the procedure and remained stable for the following 6 months ; blood gas analysis demonstrated normal pressure of oxygen and carbon dioxide . 
arm angiography , done with 4 - fr antegrade humeral access , confirmed the presence of an arteriovenous fistula in which surgical ligation of the efferent basilic vein performed 6 months earlier at the level of the anastomosis and slightly below it had failed to guarantee complete closure of the arteriovenous communication . 
in order to exclude flow in the two aneurysms and in the collateral veins without performing a second surgical ligation , we decided in agreement with the patient to attempt definitive percutaneous closure of the fistula with an avp . 
angiography performed after 4 min demonstrated complete and effective closure of the fistula with absence of contrast in the basilic vein , two aneurysms and collateral veins and patency of the arm artery . 
colour - doppler us at 1 and 3 months confirmed the definitive closure of the fistula with complete resolution of the arm oedema and total thrombosis , with absence of flow in the two aneurysms . 
after placement of the two coils and the second plug , embolisation was achieved within about 5 mcolour - doppler us at 1 and 3 months demonstrated good patency of the tips , and angiography at 3 months confirmed varix closure . 
 discussion the avp has recently been shown to be effective in the occlusion of internal iliac arteries [ 1 ] , the treatment of pulmonary arteriovenous malformations [ 2 , 3 ] and as an occlusion system for a splenorenal shunt arising after tips [ 4 ]  . 
in our experience , avp was used to achieve vascular occlusion in five different arterial and venous settings . endograft placement in the abdominal aorta has become a valid alternative to conventional surgery and is increasingly the treatment of choice in elderly patients and subjects with high surgical risk . 
about 20% of abdominal aorta aneurysms have an iliac component [ 6 ] , and often the neck of the distal common iliac artery is insufficient to provide an adequate landing zone for the grawhen the aneurysm involves the common iliac artery as far as the origin of the iia , it is necessary to cover the iia with the graft and saggio di mdc nella vena basilica e di conseguenza nei due aneurismi e nelle vene collaterali con perviet dellasse arterioso dellarto . 
il controllo clinico ed eco - color doppler effettuato a 1 e a 3 mesi ha confermato la buona e definitiva chiusura della fistola con risoluzione completa delledema dellarto e trombosi totale , con assenza di flusso nei due aneurismi . la grossa varice gastrica stata chiusa con 2 avp e 2 spirali in platino dato il diametro particolarmente ampio e la persistenza di flusso dopo limpianto del primo plug . 
i controlli eco - color doppler a 1 e 3 mesi hanno mostrato buona perviet della tips e il controllo angiografico a 3 mesi ha confermato la chiusura della varice . discussione recentemente lefficacia dellavp stata dimostrata per locclusione di arterie iliache interne [ 1 ] , nel trattamento di fistole arterovenose polmonari [ 2 , 3 ] e come sistema per locclusione di uno shunt spleno - renale in seguito al posizionamento di tips [ 4 ]  . 
la descrizione della validit dellavp relativamente ad una casistica ampia e al trattamento di patologie vascolari pluridistrettuali , non stata ancora riportata , a nostra conoscenza , da alcun autore . nellesperienza qui riportata lavp stato utilizzato per locclusione vascolare in 5 diverse patologie , sia sul versante arterioso sia su quello venoso . il posizionamento di endoprotesi dellaorta addominale , ormai una valida alternativa alla chirurgia tradizionale e sempre pi spesso il trattamento di scelta in pazienti anziani e ad alto rischio operatorio ; i vantaggi , come noto , sono la minore invasivit , la minor durata della procedura e dellospedalizzazione , la ridotta perdita di sangue e la ridotta morbillit e mortalit rispetto alla chirurgia aperta [ 5 ]  . 
circa il 20% degli aneurismi dellaorta addominale ha una componente aneurismatica iliaca [ 6 ] e spesso il colletto dellarteria iliaca comune distale insufficiente per assicurare un adeguato atterraggio della protesi . 
quando la patologia aneurismatica coinvolge larteria iliaca comune fino allorigine dellarteria iliaca interna ( aii ) risulta necessario ricoprire con la protesi laii ed estendere la protesi fino allarteria iliaca esterna . 
nellottica quindi di prevenire un endoleak di ii tipo ( da vasi collaterali ) e quindi garantire una buona esclusione dellaneurisma raccomandata la chiusura , pre - posizionamento di endoprotesi , della aii [ 710 ]  . 
lembolizzazione prossimale della aii , utilizzando le spirali tradizionali , una tecnica relativamente sicura ed efficace ed stata riportata da molti autori [ 8 , 10 , 11 ]  . 
i maggiori difetti delle spirali sono la necessit di utilizzarne spesso un numero molto elevato , prima di riuscire a chiudere adeguatamente il vaso , lalto costo e la durata della procedura ; inoltre pu residuare un flusso anterogrado attraverso le spirali , si pu avere una dislocazione distale delle stesse o una protrusione allinterno del lume delliliaca comune o esterna [ 12 , 13 ]  . 
da marzo 2005 abbiamo iniziato ad utilizzare lavp al posto delle spirali per embolizzare preventivamente le arterie iliache interne ; questo dispositivo consente unottima occlusione prossimale del vaso allorigine , prima c . 
therefore , to prevent a type ii endoleak ( due to collaterals ) and guarantee adequate exclusion of the aneurysm , closure of the iia before endograft placement is recommended [ 710 ]  . 
in addition , there may be residual antegrade flow through the coils , distal migration , or protrusion within the lumen of the common or external iliac artery [ 12 , 13 ]  . 
in march 2005 , we started using the avp instead of coils for preventive embolisation of iias ; the device allows excellent proximal occlusion at the vessel origin , before the bifurcation , guaranteeing better revascularisation of the collateral branches of the contralateral hypogastric artery and reducing complications due to hypoperfusion . even thoracic aorta endovascular repair has been approved as a less invasive treatment compared with conventional surgery [ 14 ]  . 
the most important anatomical prerequisite for correct positioning of the stent - graft is the presence of a neck of adequate length ( at least 1520 mm ) distal to the lsa orighowever , descending thoracic aorta lesions often involve the distal aortic arch , with a proximal neck less than 15 mm : in these cases one technique enabling safe device fixation is the creation of a more proximal landing zone with deliberate overstenting of the lsa ostium [ 5 , 15 , 16 ]  . this procedure can only be done after excluding abnormalities of the vertebral and carotid arteries and the circle of willis and if the left internal mammary artery has not been used for a coronary bypass . 
all proximal endoleaks have an incidence of 0%23% for early endoleaks ( detected during stent - graft placement ) and 0%5% for late endoleaks , and they may be related to aneurysm morphology , dissection or even technical expertise [ 15 ]  . 
if this type of endoleak ( type ii from the subclavian artery ) persists , it needs to be promptly repaired because of the direct connection between the aneurysmal sac or false lumen and systemic arterial pressure . 
our three patients underwent avp embolisation of the origin of the subclavian artery because they had developed a type ii or mixed ( type i and ii ) endoleak from the subclavian artery , which was overstented at its origin due to an inadequate proximal neck . 
the use of metal coils is not advised for subclavian occlusion [ 15 ] due to the high risk of distal embolisation and the likelihood of not being able to block flow completely . 
these are abnormal , direct communications between pulmonary arteries and veins without a capillary bed , which are caused by the formation of an aneurysmal sac or a tangle of tortuous vessels [ 1820 ]  . 
treatment opdella biforcazione nei suoi due rami , garantendo una migliore rivascolarizzazione da rami collaterali dellipogastrica controlaterale riducendo in questo modo possibili complicanze da ipoperfusione . anche il trattamento endovascolare della patologia dellaorta toracica ormai stato approvato come trattamento meno invasivo rispetto alla chirurgia tradizionale [ 14 ]  . 
il pi importante requisito anatomico per il corretto posizionamento dellendoprotesi la presenza di un colletto minimo di 1520 mm distalmente allorigine dellostio dellass . tuttavia la patologia dellaorta toracica discendente spesso coinvolge larco aortico distale con un colletto prossimale inferiore a 15 mm : in tali casi una delle tecniche per un sicuro fissaggio del dispositivo un atterraggio pi prossimale con la copertura volontaria dellostio dellass [ 5 , 15 , 16 ]  . tale procedura pu essere effettuata soltanto escludendo prima patologie a carico delle arterie vertebrali , delle carotidi , del circolo del willis e se la mammaria interna sinistra non stata utilizzata per un bypass coronarico . un endoleak di tipo ii dalla ass pu insorgere dopo posizionamento di endoprotesi dellaorta discendente ma ad oggi non riportata in letteratura lesatta incidenza . 
la percentuale di tutti gli endoleak prossimali varia dallo 0% al 23% per gli endoleak precoci ( dimostrati al momento del posizionamento della protesi ) e dallo 0% al 5% per quelli tardivi e possono essere legati allanatomia dellaneurisma o dissezione o anche allesperienza delloperatore [ 15 ]  . 
questo tipo di endoleak ( tipo ii dalla succlavia ) se persiste richiede una tempestiva correzione a causa della diretta comunicazione tra la sacca aneurismatica o il falso lume e la pressione arteriosa sistemica . 
i nostri 3 pazienti sono stati sottoposti ad embolizzazione dellorigine della succlavia con avp , poich avevano sviluppato un endoleak di tipo ii o misto ( tipo i + tipo ii ) dalla succlavia che stata ricoperta dallendoprotesi alla sua origine per la presenza di un colletto prossimale insufficiente . 
lutilizzo delle spirali metalliche per locclusione della succlavia non consigliabile [ 15 ] per il forte rischio di embolizzazione distale e per lalta probabilit di non riuscire a bloccare totalmente il flusso : il nuovo dispositivo avp consente unimmediata , sicura e rapida occlusione allorigine del vaso senza complicanze emboliche distali . 
per questultima caratteristica lavp assume unimportante ruolo nel trattamento delle fistole arterovenose polmonari ( favp ) ; questultime sono delle anormali connessioni dirette tra arterie e vene polmonari con lesclusione del letto capillare dovute alla formazione di una sacca aneurismatica o ad un groviglio di canali vascolari tortuosi [ 1820 ] : estremamente eterogenea la presentazione clinica [ 18 , 21 , 22 ]  . 
le opzioni terapeutiche includono lembolizzazione percutanea o lescissione chirurgica [ 2 ]  . data la minore invasivit e lo sviluppo di nuovi materiali embolizzanti il trattamento percutaneo divenuto il trattamento di scelta per la maggior parte dei pazienti [ 2325 ]  . 
la ricanalizzazione della favp pu avvenire in una percentuale compresa tra il 5% e il 10% dei pazienti trattati [ 26 , 27 ] : pertanto , la scelta di un materiale embolizzante appropriato e laccurata localizzazione del sito dove iniziare lembolizzazione di cruciale importanza [ 23 , 24 ]  . 
recanalisation of pavf may occur in 5%10% of treated patients [ 26 , 27 ] , so the choice of appropriate embolising material and accurate localisation of the site where embolisation is to be started are crucial [ 23 , 24 ]  . 
these considerations are even more important in massive fistulas , which are more difficult to occlude with coils or detachable balloons due to the risk of distal migration [ 25 , 28 ]  . 
we believe the avp to be safer and easier to use than the above - mentioned devices owing to the simplicity and small size of the release system and , more importantly , because it was designed for vascular use and is therefore more specific and better suited to the tubular morphology of vessels seen in pavf . haemodialysis fistulas often develop complications , one of the most common being aneurysm formation [ 31 , 32 ]  . possible treatment options include surgery , endovascular treatment or both . 
we opted for the avp because its release is controlled and it can be placed precisely at the level of the neck where it readily adheres to the vessel without risking distal migration . 
to our knowledge , no author has described the use of the avp as an embolising device for the closure of a haemodialysis fistula . the efficacy of tips is well documented in the treatment of variceal bleeding refractory to endoscopic treatment [ 36 , 37 ]  . 
although tips alone is sufficient to stop variceal bleeding in many cases [ 3840 ] , in some patients with large collateral veins , it may not be enough ; embolisaton of the varix therefore becomes indispensable [ 4144 ] , as in the case treated by us . 
even very large coils ( 20 mm15 cm ) are susceptible to distal migration , and the same applies to other materials such as sclerosing agents , glue and gelatine sponge particles , which are not very effective and only increase the risk of systemic embolisation . 
in our opinion , the avp is the vascular occluder that best adapts to large vessels such as gastric varices ; in addition , it can be used is combination with coils to make vascular occlusion even more rapid . our experience indicates that the avp is an extremely versatile occlusion system that is suitable for variety of vessel to pi difficile occluderle con le spirali o i palloni staccabili a causa della possibilit di migrazione distale [ 25 , 28 ]  . 
migliori risultati sono stati ottenuti in passato con lutilizzo di vari device , ad uso cardiologico realizzati per il trattamento della perviet del dotto arterioso di botallo e dei difetti settali cardiaci , quali il gianturco - grifka , composto prevalentemente da un sacco in nylon flessibile e una guida occludente , il cardioseal , costituito da un doppio ombrellino ciascuno con quattro molle metalliche e stoffa di poliestere e lamplatzer duct occluder , realizzato con una maglia guida in nitinolo con estremit a disco [ 24 , 29 , 30 ]  . 
noi riteniamo che lavp , sia pi sicuro e facile da utilizzare rispetto ai precedenti devices menzionati , per la semplicit e le ridotte dimensioni del sistema di rilascio ma soprattutto , essendo un dispositivo dedicato allimpiego vascolare , risulta pi specifico e meglio conformabile allanatomia tubulare del vaso come nel caso delle malformazioni arterovenose polmonari . le fistole da emodialisi , sono gravate da alcune complicanze tra cui una delle pi comuni la formazione di aneurismi [ 31 , 32 ]  . 
lapproccio endovascolare pu avvalersi del posizionamento di uno stent ricoperto a livello arterioso , in modo da escludere la zona di anastomosi , oppure lembolizzazione a livello anastomotico arterioso della fistola [ 3335 ]  . 
nel nostro caso , poich la fistola era localizzata a livello del gomito , lutilizzo di uno stent ricoperto era controindicato e pertanto lunica indicazione terapeutica percutanea era lembolizzazione transcatetere . 
abbiamo preferito utilizzare un avp perch ha un rilascio controllato e pu essere posizionato esattamente a livello del colletto , aderendo prontamente al vaso senza rischio di migrazione distale sia sul versante arterioso sia su quello venoso . 
a nostra conoscenza nessun autore , prima dora , ha descritto limpiego dellavp come sistema embolizzante per la chiusura di una fistola arterovenosa da emodialisi . lefficacia della tips stata ben provata nel trattamento del sanguinamento da varici resistenti al trattamento endoscopico [ 36 , 37 ]  . 
in molti casi la tips da sola sufficiente a bloccare il sanguinamento delle varici [ 3840 ] tuttavia in alcuni pazienti con grosse vene collaterali pu non bastare ; pertanto in tali soggetti lembolizzazione della varice risulta indispensabile [ 4144 ] , come nel caso da noi trattato . 
il grande diametro di questi vasi collaterali costituisce il principale problema nella scelta del materiale embolizzante : anche spirali metalliche molto grandi ( 20 mm15 cm ) possono facilmente migrare distalmente . 
ugualmente vale per altri materiali come agenti sclerosanti , colla , particelle di spugna di gelatina che non hanno una grande efficacia e aumentano solo il rischio di embolizzazione sistemica . 
a nostro giudizio lavp il dispositivo di occlusione vascolare che meglio si adatta a vasi di grosse dimensioni come le varici gastriche : pu inoltre essere utilizzato in associazione alle spirali per rendere ancora pi rapida locclusione vascolare . in conclusione , in base alla nostra esperienza , possiamo affermare che lavp si rivelato un sistema di occlusione vascolare estremamente versatile , adatto quindi a vasi di diametro eterogeneo , facile da usare , preciso nel rilascio , c . 
ridolfi , via varthema 44 , bologna , italy , tel . : + 39 - 051 - 6235444 , fax : + 39 - 051 - 6235444 , e - mail : marcelloridolfi@yahoo.it * il lavoro spetta in parti uguali agli autori received : 21 march 2006 / accepted : 20 august 2006 / published online : 19 march 2007 abstract purpose . 
thirty - eight patients ( 15 men and 23 women ; mean age 55 years ; age range 3579 ) with neck pain were examined and divided into two groups : ( 1 ) patients already identified as rheumatic and referred for further investigation of the atlantoaxial region ; ( 2 ) patients with symptoms confined to the cervical spine , with inconclusive radiographic findings . unenhanced ct of the cervical spine ( tomoscan sr 7000 philips , eindhoven , netherlands ) was performed in all patients . 
ct demonstrated calcific deposits around the dens in 12 patients ( three men and nine women ) , in the transverse and alar ligaments , and in the anterior atlantooccipital membrane . 
crystals located in the transverse ligament of the atlas give rise to the crowned dens syndrome , usually in patients affected by severe degenerative lesions of the atlantoaxial joint and peripheral chondrocalcinosis . 
i soggetti sono stati suddivisi in due gruppi : ( 1 ) pazienti gi diagnosticati come reumatici , di cui lo specialista richiedeva un ulteriore approfondimento diagnostico della zona atlo - epistrofica ; ( 2 ) pazienti con sintomatologia desordio circoscritta al rachide cervicale , di cui la radiologia convenzionale era risultata poco significativa . 
tutti i pazienti sono stati sottoposti a tc volumetrica della colonna cervicale ( tomoscan sr 7000 philips , eindhoven , olanda ) , senza somministrazione per via endovenosa di mezzo di contrasto . 
lesame tc risultato positivo , per depositi calcifici a livello del dente , in 12 pazienti ( 3 maschi e 9 femmine ) , con calcificazioni a livello del legamento trasverso , di quelli alari e della membrana atlo - occipitale anteriore . 
radiography of other joints ( wrist , knee , pubic symphysis ) may help to ascertain whether the disease is due to calcium pyrophosphate dihydrate or hydroxyapatite crystals , and is therefore recommended for routine patient management . magnetic resonance imaging ( mri ) is indicated for the study of neurological complications . key words crowned dens syndrome cppd crystal deposition disease transverse ligament of the atlas computed tomography localizzazione , generalmente asintomatica ; quando i cristalli hanno sede nel legamento trasverso dellatlante , si realizza la sindrome del dente incoronato , abitualmente in pazienti con lesioni degenerative avanzate dellarticolazione atlo - epistrofica e con evidente condrocalcinosi periferica . 
nella sindrome del dente incoronato la tc rappresenta il gold standard diagnostico , potendo identificare la forma e la sede delle calcificazioni e le eventuali erosioni ossee . lesame radiografico di altri distretti articolari ( carpo , ginocchio , sinfisi pubica , spalla ) pu agevolare il riconoscimento eziologico della malattia , se da cppd o da ha , ed pertanto raccomandato nella gestione routinaria del paziente . 
limpiego della rm indicato nello studio della complicanze neurologiche . parole chiave sindrome del dente incoronato malattia da deposito di cristalli di cppd legamento trasverso dellatlante tomografia computerizzata introduction introduzione the crowned dens syndrome ( cds ) is a clinical - radiological entity characterised by calcification of the ligaments surrounding the odontoid process of the axis and by acute attacks of cervicooccipital pain , with fever , rigidity and general signs of inflammation lasting from days to several weeks [ 1 ]  . 
the crystal deposits , in most cases calcium pyrophosphate dihydrate ( cppd ) but also hydroxyapatite ( ha ) , may remain asymptomatic or cause chronic cervical pain and spinal cord compression [ 2 ]  . the classic presenting triad ( headache , fever and morning cervical pain ) was first described by le goff in 1980 [ 3 , 4 ] , who distinguished these acute attacks from those seen in common osteoarthritis and in crystal diseases of the distal cervical vertebrae or thoracic - lumbar spine [ 5 ]  . 
furthermore , atypical cases of cds can simulate both the painful cervicobrachial syndrome ( with shoulder weakness and stiffness ) , as well as occipitotemporal headache ( which mimics atypical rheumatic polymyalgia and / or giant cell arteritis )  . 
according to available data [ 710 ] , computed tomography ( ct ) imaging at the c1c2 level is still the reference standard and is superior to magnetic resonance imaging ( mri ) in identifying these calcifications , especially when they are small [ 11 ]  . the aim of this paper was to illustrate a series of cases of cds seen not only in crystal deposition diseases but also in other rheumatic and nonrheumatic conditions and to provide an assessment of ct imaging patterns and an analysis of the best use of current imaging techniques . 
 la sindrome del dente incoronato ( sdi ) rappresenta unentit clinico - radiologica caratterizzata dallassociazione di calcificazioni dei legamenti attorno al dente dellepistrofeo e da attacchi acuti periodici di cervicalgia occipitale con febbre , rigidit e segni generali di flogosi , di durata molto variabile , da pochi giorni ad alcune settimane [ 1 ]  . colpisce prevalentemente il sesso femminile , con et media al momento della diagnosi tra i 60 ed i 70 anni . 
i depositi di cristalli , nella maggior parte dei casi di pirofosfato di calcio diidrato ( cppd ) , ma anche di idrossiapatite ( ha ) , possono rimanere asintomatici oppure essere responsabili di dolore cronico cervicale e di compressione midollare [ 2 ]  . la tipica descrizione della sindrome , caratterizzata dalla triade cefalea , febbre e cervicalgia mattutina , stata riferita nel 1980 da le goff [ 3 , 4 ] , che distinse questi attacchi acuti da quelli osservati nelle delle malattie da microcristalli a localizzazione vertebrale cervicale distale o dorso - lombare , e da quelli della comune malattia artrosica [ 5 ]  . 
inoltre casi atipici di sdi possono simulare sia la sindrome dolorosa cervico - brachiale ( con debolezza e rigidit della spalla ) che la cefalea occipitale e temporale ( che mima la polimialgia reumatica atipica e / o larterite a cellule giganti )  . 
secondo la letteratura [ 710 ] , lesame tc focalizzato a livello di c1 - c2 rimane il gold standard della diagnostica per immagini ed superiore alla rm nellidentificare queste calcificazioni , soprattutto se minute [ 11 ]  . scopo del presente lavoro stata lillustrazione di alcuni materials and methods between 2001 and 2004 , 38 patients ( 15 men and 23 women ; age range 4572 years ; mean age 55 ) were referred by both general practitioners and specialists ( orthopaedic surgeons , rheumatologists , neurologists , and oncologists ) for investigation of neck pathe patients were divided into two groups : the first ( 16 cases , three men and fifteen women ) consisted of patients with a known clinicalrheumatologic diagnosis [ rheumatoid arthritis ( ra ) , diffuse idiopathic skeletal hyperostosis ( dish ) , etc . ] established on the basis of pain and / or dysfunction at the level of the atlantoaxial joint and alterations demonstrated by conventional radiography , and for whom the specialist had requested further diagnostic investigation the second group ( 22 cases , 12 men and fifteen women ) consisted of patients referred by rheumatologists , general practitioners and other specialists ( orthopaedic surgeon , neurologist , oncologist ) , with initial symptoms confined to the lower cervical spine , often with a history of trauma , and with inconclusive radiographic findings . data collected for each patient included signs and symptoms of the illness , social status and daily working activity , physical examination and laboratory tests , clinical diagnosis and radiographic findings . 
 the disorders affecting the patients ( table 1 ) were distributed as follows : 11 cases of rheumatoid arthritis ( ten women and one man ) , two of cppd crystal deposition disease ( both women ) , one of systemic sclerosis ( a woman ) , one of osteoarthritis ( a man ) , one of seronegative spondyloarthritis ( a man ) , four of neoplasm ( one woman and three men ) with suspected cervical metastases , one ( a man ) of malignant haematological disease ( lymphoma ) , one ( a woman ) of postmenopausal osteoporosis , ten ( five men and five women ) of recent or previous traumatic injury with suspected involvement of the skull base and first cervical vertebrae ( dens fracture , frank atlantoaxial luxation or subluxation ) and six of algo - dysfunctional syndrome of the cervical spine ( three men and three women )  . 
evaluation with ct imaging casi di sindrome del dente incoronato non solamente nellambito delle malattie da deposito di microcristalli , ma anche in altre malattie reumatiche ( i.e. , artrite reumatoide ) e non , la valutazione dei loro aspetti iconografici tc e il giudizio sul miglior impiego delle tecniche attuali di imaging . materiale e metodi nel periodo 20012004 sono stati esaminati 38 pazienti , di cui 15 maschi e 23 femmine , di et compresa fra i 45 ed i 72 anni ( media 55 ) , affetti da cervicalgia , inviati sia dal medico di base che dallo specialista ( ortopedico , reumatologo , neurologo , oncologo ) per gli accertamenti del caso . 
i pazienti sono stati divisi in due gruppi : il primo ( formato da 16 pazienti , di cui 3 maschi e 13 femmine ) , dove la diagnosi clinico - reumatologica era gi stata effettuata ( ar , dish , ecc . ) per la comparsa di sintomatologia algica e / o disfunzionale a livello della cerniera atlo - epistrofica , con alterazioni gi dimostrate in maniera inequivocabile dallesame radiografico convenzionale , e di cui lo specialista richiedeva un ulteriore approfondimento diagnostico a livello della suddetta zona ; il secondo gruppo ( formato da 22 casi , di cui 12 maschi e 10 femmine ) comprendeva pazienti inviati non dal reumatologo , ma dal medico di base e da altri specialisti ( ortopedico , neurologo , oncologo ) , la cui sintomatologia desordio era circoscritta al rachide cervicale , specie in sede prossimale , talvolta su base post - traumatica , e di cui la diagnostica per immagini convenzionale era risultata poco significativa . i dati raccolti per ogni paziente comprendevano tutti i segni ed i sintomi relativi alla malattia denunciata ; lo stato sociale e lattivit lavorativa giornaliera ; gli esami fisico e di laboratorio ; la diagnosi clinica ; i reperti ottenuti con la radiografia convenzionale . 
la visualizzazione delle immagini stata sempre effettuata in modo da avere finestra e livello di finestra sia per i tessuti molli ( i.e. , legamento trasverso ) che per le strutture ossee . 
in particolare , per lo scheletro , lampiezza della finestra stata di 1500 hu e il livello + 300 hu ; per le parti molli rispettivamente 350 hu e + 50 hu . 
non stato somministrato mezzo di contrasto . per quanto riguarda la patologia dei nostri pazienti , la cui distribuzione riportata nella tabella 1 , questa era cos rappresentata : 11 pazienti affetti da artrite reumatoide ( di cui 10 femmine e 1 maschio ) ; 2 casi portatori di malattia da p.n. 
ct demonstrated calcific deposits in the periodontoid structures ( transverse ligament of the atlas , crossed ligament , and alar ligaments ) in 12 patients , three of whom were men and nine who were women ( table 2 )  . 
ct appearance of calcifications was one of thin calcium deposits forming a cppd , entrambi di sesso femminile ; 1 caso di sclerosi sistemica ( femmina ) ; 1 caso di artrosi ( maschio ) ; 1 caso di spondiloartrite sieronegativa ( maschio ) ; 4 pazienti oncologici ( 1 femmina e 3 maschi ) con sospetta localizzazione replicativa cervicale ; 1 caso ( maschio ) affetto da patologia ematologia maligna ( linfoma ) ; 1 caso ( femmina ) con osteoporosi postmenopausale ; 10 pazienti ( 5 maschi e 5 femmine ) affetti da patologia traumatica , recente e pregressa , di cui si sospettava interessamento della base del cranio e delle prime vertebre cervicali ( frattura del dente dellepistrofeo e / o sublussazione e lussazione franca atlo - epistrofica ) e infine 6 pazienti affetti da patologia imprecisata ( 3 maschi e 3 femmine ) algico - disfunzionale cervicale . 
in essi la malattia prevalente era rappresentata dallartrite reumatoide ( ar ) 10 femmine e 1 maschio cui facevano seguito la malattia da cppd ( 2 casi ) , la sclerosi sistemica , lartrosi e la spondiloartrite sieronegativa ( 1 caso , rispettivamente )  . risultati lesame tc della giunzione atlo - epistrofica stato eseguito sulla base della sintomatologia clinica denunciata dal patable 2 patients with crowned dens syndrome ( cds ) and positive computed tomography ( ct ) findings rheumatic diseases no . 
evaluation with ct imaging women 9 rheumatoid arthritis men 3 cppd crystal deposition disease systemic sclerosis rheumatoid arthritis osteoarthritis seronegative spondyloarthritis femmine 9 artrite reumatoide maschi 3 malattia da cppd sclerosi sistemica artrite reumatoide osteoartrosi spondilite sieronegativa transverse ligament ( 4 ) alar ligaments ( 2 ) alar ligaments transverse ligament transverse ligament alar ligaments transverse ligament + atlooccipital anterior membrane legamento trasverso ( 4 ) legamenti alari ( 2 ) legamenti alari legamento trasverso legamento trasverso legamenti alari legamento trasverso + membrana atlo - occipitale anteriore tabella 2 pazienti con sindrome del dente incoronato e tc positiva malattia reumatica casi , n sede delle calcificazioni shell around the dens and located both posteriorly ( on axial images the deposits , they appeared inhomogeneous and probably located on the transverse ligament ) and at the level of the dens apex and base ( on coronal ct reformations due to involvement of the alar and apical ligaments )  . 
 [ 13 , 14 ] identified nonurate crystals in the synovial fluid of patients clinically presenting gout - like attacks ( hence the initial name pseudogout )  . these cppd crystals were characterised by a weak positive birefringence at polarised light microscopy , a finding that distinguished them from the monosodium urate crystals of gout . 
the radiographic features of the disease had , however , been already described by zitnan and sitaj in 1963 , who had called it polyarticular chondrocalcinosis [ 15 ]  . 
bennet , in particular , noted the presence of chalky , rhomboid - shaped crystals [ 16 ] during the autopsy of a patient in ireland with polyarticular chondrocalcinosis . ziente ( principalmente rappresentata da rigidit nucale e cefalea , con o senza incapacit funzionale ) o nellambito del follow - up diagnostico , soprattutto nei casi affetti da cppd dove lesame radiografico tradizionale aveva gi dimostrato la presenza di calcificazioni nelle sedi tipiche della malattia ( i.e. , ginocchio , polso )  . 
lesame tc risultato positivo per depositi calcifici in corrispondenza delle strutture viciniori al dente dellepistrofeo ( legamento trasverso dellatlante , legamento crociato , legamenti alari ) in 12 pazienti , di cui 3 maschi e 9 femmine , la cui distribuzione riportata nella tabella 2 . 
i tre soggetti erano affetti da ar ( anni 69 ) , da osteoartrosi ( anni 70 ) e da spondilite sieronegativa ( anni 48 ) ( fig . 1 ) , con calcificazioni a livello rispettivamente del legamento trasverso , di quelli alari e della membrana atlo - occipitale anteriore . 
liconografia delle calcificazioni era rappresentata da tenui depositi calcifici a guscio , attorno al dente dellepistrofeo , sia in sede posteriore ( nelle immagini tc sul piano assiale , di tipo disomogeneo , verosimilmente a carico del legamento trasverso ) , sia in corrispondenza dellapice e della base del dente stesso ( nelle immagini tc ricostruite sul piano coronale , per interessamento dei legamenti alari e del legamento apicale )  . 
the term chondrocalcinosis is very general , as it suggests apparent radiological and pathological calcification of the cartilage ( which could be due to cppd , ha , tricalcium phosphate or a combination of the three )  . 
although articular and periarticular calcifications can appear in cppd disease , similar calcifications can also be produced by other crystals [ 15 , 19 , 20 ]  . cppd crystal deposition disease is classified into sporadic , hereditary or secondary forms [ 16 ]  . 
the clinical presentation ranges from an incidental radiographic finding to discussione sebbene la malattia da deposito di pirofosfato di calcio diidrato ( cppd ) sia la pi comune artropatia da microcristalli [ 12 ] , tuttavia la scoperta di questi nelle strutture intra - articolari risale allinizio degli anni 60 , quando mccarty insieme ad hollander , kohn e faires [ 13 , 14 ] identific cristalli non uratici nel liquido sinoviale di pazienti che clinicamente presentavano attacchi simil - gottosi ( da ci il termine di pseudogotta dato inizialmente alla malattia )  . questi cristalli di cppd erano caratterizzati da una bassa birifrangenza positiva quando esaminati al microscopio a luce polarizzata , fenomeno che li distingueva da quelli di urato monosodico della gotta . 
the radiological presentation [ 2123 ] is characterised by calcifications in and around the joints and by structural articular changes ( pyrophosphate arthropathy )  . the knee , the wrist , the pubic symphysis and the hip are most commonly affected . 
the spine may be the only site of cppd disease [ 20 , 24 ] and is generally asymptomatic [ 12 ]  . crystals may be identified both in the nucleus pulposus of p.n. 
questultimo , in irlanda , durante lautopsia di un paziente affetto da condrocalcinosi poliarticolare , evidenzi la presenza di cristalli romboidali , di aspetto gessoso [ 16 ]  . i numerosi studi di resnick et al . 
 [ 12 , 17 ] hanno messo ordine nelle svariate nomenclature con cui la malattia era conosciuta , in quanto diversi sinonimi sono stati usati in maniera intercambiabile tra di loro , causando in passato una certa confusione . 
in effetti , il termine condrocalcinosi molto generico , in quanto indica una evidente calcificazione radiologica ed anatomopatologica della cartilagine ( che pu riferirsi alla presenza di cristalli di cppd , di ha , di fosfato tricalcico o alla combinazione dei tre suddetti )  . 
la dizione malattia da deposito di cristalli di cppd ( cppd crystal deposit disease ) si riferisce specificatamente a un disordine caratterizzato dalla esclusiva presenza di cristalli di cppd dentro o intorno alle articolazioni . 
le calcificazioni articolari e periarticolari possono comparire nella malattia di cppd ; tuttavia altri cristalli possono produrre calcificazioni analoghe [ 15 , 19 , 20 ]  . la malattia da cppd si classifica in forma sporadica , ereditaria o secondaria [ 16 ]  . 
infine , i cristalli di cppd possono depositarsi nel legamento trasverso dellatlante e nei legamenti alari , realizzando la c.d. sindrome del dente incoronato [ 1 , 68 ] , la cui definizione , in accordo con diversi autori [ 7 , 8 , 26 ] , dovrebbe comprendere calcificazioni di ogni componente articolare in questa sede ( membrana sinoviale , capsula articolare , legamenti )  . 
inoltre , sullapice del dente sinseriscono tre legamenti : il legamento apicale del dente e i due legamenti alari [ 6 ]  . clinicamente , la sdi dovuta a depositi di ha [ 27 ] una condizione ben definita , ancorch rara , che colpisce donne giovani e di media et , caratterizzata da intensa cervicalgia p.n. 
computed tomography ( ct ) of the atlantoaxial region : images in the axial plane ( a , b ) and reformatted in the coronal ( c , d ) and sagittal planes ( e )  . 
tc atlo - epistrofica : scansioni sul piano assiale ( a , b ) e ricostruzione elettronico sul piano coronale ( c , d ) e sagittale ( e )  . 
in some cases , there may be involvement of the yellow ligaments and posterior longitudinal ligaments , with secondary myelopathy , spinal cord compression and vertebral stenosis [ 25 ]  . 
in particular , it is important to consider the cruciform ligament , which is composed of both part of the transverse ligament of the atlas and its vertical extensions ( superior and inferior longitudinal fibres )  . 
in addition , three ligaments are inserted on the dens apex : the apical ligament and the two alar ligaments [ 6 ]  . clinically , cds due to ha crystal deposits [ 27 ] is a welldefined , though rare , condition that affects young and middle - aged women and is characterised by intense cervical pain [ 28 ] that resolves within days or weeks after treatment with nonsteroid anti - inflammatory drugs ; it does not cause spinal compression [ 4 ]  . 
the clinical picture caused by cppd crystal deposits is more complex , as it may be asymptomatic or associated with a variety of clinical manifestations , some of which are severe [ 29 , 30 ] , such as spinal cord compression [ 31 , 32 ] related in some cases to a slightly calcific mass between the dens and the transverse ligament . 
crystal deposits can be found in older patients ( 5.7% ) with evident peripheral joint chondrocalcinosis [ 11 ] and advanced degenerative lesions of the atlantoaxial joint , and they tend to worsen with age [ 9 ]  . 
 in contrast with previous studies [ 24 , 7 , 12 ] that consider cppd crystal deposition disease as the most frequent form of crowned dens syndrome , only two women in our series [ 28 ] , risolvibile in un tempo variabile da pochi giorni ad alcune settimane con trattamento anti - infiammatorio non steroideo , senza causare compressione bulbare [ 4 ]  . 
il quadro clinico sostenuto , invece , dai cristalli di cppd pi complesso , perch pu essere asintomatico o associarsi a manifestazioni cliniche varie , talora anche gravi [ 29 , 30 ] , quali la compressione midollare [ 31 , 32 ] , determinata da una massa talora finemente calcifica localizzata tra il dente dellepistrofeo e il legamento trasverso . 
i depositi cristallini si osservano in pazienti pi anziani ( 5 , 7% ) , con evidente condrocalcinosi delle articolazioni periferiche [ 11 ] e lesioni degenerative avanzate dellarticolazione atlo - epistrofica e si aggravano con lavanzare dellet [ 9 ]  . 
 nella nostra casistica , in contrasto con la letteratura [ 24 , 7 , 12 ] che indica nella malattia di cppd levenienza pi frequente di sdi , solamente due casi entrambi di sesso femminile presentavano calcificazioni peri - odontoidee . una di queste ( et 69 anni ) denunciava dolore ad entrambe le ginocchia , dove lesame radiografico dimostrava la presenza di calcificazioni moniliformi a carico di entrambi i menischi . 
analoghe calcificazioni erano presenti nella cartilagine triangolare del polso . altre malattie associate alla sdi sono lar , la ra - dish [ 33 ] , le connettiviti sistemiche , le sequele di traumi , sublussazioni e lussazioni franche . 
in particolare , nellar linteressamento dello scheletro assile [ 34 ] risulta significativo nel tratto cervicale ( 60%80% dei casi ) ; esso pu essere asintomatico oppure manifestarsi con dolore , limitazione funzionale e sindromi neurologiche anche gravi [ 20 ]  . 
qualunque sia il distretto interessato , le lesioni anatomo - patologiche sono quelle tipiche della malattia , rappresentate dalla flogosi cronica della membrana sinoviale , con formazione successiva di erosioni ossee ed indebolimento strutturale delle zone dinserzione dei legamenti . 
computed tomography ( ct ) of atlantoaxial region , images in the axial plane ( a , b ) , and reformatted images in the coronal ( c ) and sagittal ( d ) planes . 
in one ( 69 years old ) , who had pain in both knees , radiography demonstrated minute mottled calcifications in the meniscus and in the triangular cartilage of the wrist , typical of cppd crystal deposit disease . the diseases associated with cds are ra , ra - dish [ 33 ] , systemic connectivitis , trauma , frank subluxations and luxations . 
ra in particular involves the axial skeleton [ 34 ] and especially the cervical region ( in 60%80% of cases ) ; this may be asymptomatic , or present with pain , functional impairment and even severe neurological syndromes [ 20 ]  . whatever the area involved , the lesions are pathologically those of ra : chronic inflammation of the synovial membrane , bone erosions and weakening of the ligament insertions . 
laxity of the articular capsule and atlantoaxial joint ligaments causes an abnormal separation ( more than 2.5 mm ) no in sede atlo - epistrofica , con secondarie alterazioni della stabilit e della funzionalit regionale : ostacolata rotazione del capo ed instabilit articolare secondo tre direttrici fondamentali ( anteriore , laterale e verticale )  . 
la lassit delle strutture capsulo - legamentose della giunzione atlo - epistrofica determina lallontanamento anomalo ( > 2 , 5 mm ) tra arco anteriore dellatlante e dente dellepistrofeo , dando luogo alla lussazione atlo - assiale [ 20 ]  . 
nella nostra casistica sono soprattutto i pazienti affetti da ar quelli che hanno denunciato la maggiore incidenza della sdi , con prevalenza del sesso femminile ( 10 femmine e 1 maschio ) , specie in quei casi affetti da malattia reumatica inveterata . lassociazione tra ar e calcificazioni legamentose periodontoidee risulta di non facile interpretazione , dato che non ancora identificato un rapporto di causa ed effetto , tenuto between the anterior arch of the atlas and the odontoid process , resulting in atlantoaxial luxation [ 20 ]  . 
the association between ra and periodontoid calcification is not easy to interpret in that no causal relationship has yet been identified in the light of the natural evolution of ra [ 36 ]  . 
 whereas ct is indicated for the study of cortical bone and periodontoid calcifications , mri with paramagnetic contrast material is the modality of choice for the study of synovial abnormalities , as it is the only technique able to demonstrate the inflamed pannus ( hypervascular , hypovascular and fibrous ) , articular effusion and cord compression [ 37 ]  . the diagnosis of cds is not always straightforward given the broad spectrum of presentations . 
nevertheless , ct can fail to detect cds , especially if it is carried out too late after an acute attack , because the calcifications may have been reabsorbed in the meantime [ 8 ]  . 
the characteristic ct pattern is a narrow and irregular horseshoe calcification surrounding the odontoid process , at times related to osteophytes in the upper margin of the anterior arch of the atlas , as well as calcification of the transverse ligament . 
the main calcification is often surrounded by other smaller calcifications at the apex of the dens [ 10 ]  . differential diagnosis includes calcification of the tendon of the longus colli muscle , where the clinical features of cds are accompanied by pharyngeal pain and dysphagia , incorrectly suggesting meningitis , spondylodiscitis or retropharyngeal abscess . 
other radiographic appearances not to be confused with cds are disc calcifications , calcified adenopathy , sesamoid bones in the transverse ligament , ossification of the common posterior vertebral ligament , and periatlantic calcifications [ 28 , 39 , 40 ]  . 
 [ 28 ] have described 23 cases , five cases and one case of cds , respectively , with clinical presentations varying from febrile nuchal pain ( in acute forms ) to spinal compression ( in chronic forms )  . 
 se la tc indicata per lo studio della corticale ossea e delle calcificazioni periodontoidee , la rm metodica elettiva per lo studio delle lesioni sinoviali , essendo lunica tecnica capace di visualizzare il panno infiammatorio ( ipervascolarizzato , ipovascolarizzato e fibroso ) , il versamento articolare e la compressione midollare , impiegando mezzo di contrasto paramagnetico [ 37 ]  . la diagnosi della sdi non sempre facile , dato lampio spettro di presentazione dellaffezione : infatti , altre manifestazioni cliniche , come la meningite , il dolore cervico - brachiale e lemicrania occipito - temporale , possono aggiungersi al classico episodio acuto febbrile di cervicalgia [ 1 ]  . con la tc la dimostrazione delle minute calcificazioni nellarea del processo odontoideo estremamente facilitato , con sensibilit superiore alla rm [ 38 ]  . 
tuttavia la tc pu fallire nella diagnosi di sdi , specie se questa eseguita tardivamente dopo lattacco acuto , perch nel frattempo le calcificazioni possono riassorbirsi [ 8 ]  . 
laspetto tc caratteristico quello di una sottile ed irregolare calcificazione a ferro di cavallo attorno al processo odontoideo , che in alcuni casi pu essere riferito , oltre che alla calcificazione del legamento trasverso , anche a neoformazioni osteofitosiche a carico del profilo superiore dellarco anteriore dellatlante . 
questa calcificazione principale spesso circondata da altre pi piccole , situate allapice del dente [ 10 ]  . la diagnosi differenziale va posta con le calcificazioni del tendine del muscolo lungo del collo , che oltre al quadro clinico della sdi associa dolore faringeo e disfagia che evocano a torto meningite , spondilodiscite o ascesso retrofaringeo . 
altri aspetti radiografici da non confondere con la sdi sono : le calcificazioni discali , le adenopatie calcifiche , la presenza di un sesamoide nel legamento traverso , lossificazione del legamento vertebrale comune posteriore , le ossificazioni soprannumerarie peri - atlantiche [ 28 , 39 , 40 ]  . 
treves [ 39 ] , constantin [ 7 ] e denes [ 28 ] hanno descritto , rispettivamente , 23 casi , 5 casie 1 caso caratterizzati da un quadro clinico variabile da episodi di nucalgia febbrile nelle forme acute a quadri di compromissione midollare nelle forme croniche . lo studio di treves [ 39 ] ha dimostrato , con lesame tc , la presenza di calcificazioni atlo - odontoidee nel 90% dei casi di sdi : 8 pazienti su 12 di questo gruppo non avevano mai p.n. 
evaluation with ct imaging [ 39 ] demonstrated with ct the presence of atlantoaxial calcifications in 90% of cases : eight of 12 patients had never suffered from neck pa therefore , in the presence of febrile cervical pain , the discovery of periodontoid calcifications suggests cds and excludes meningitis . conclusion in cds , ct is the gold standard imaging modality in that it allows identification of the different radiographic patterns of the disease : simple band of calcification or double band of thin calcifications in the transverse ligament , irregular calcifications crowning the dens apex , and bone erosions of the dens itself . 
however , radiography of other joints ( wrist , knee , pubic symphysis and shoulder ) can help establish whether the disease is due to cppd crystal deposit disease or ha crystals and is therefore recommended for routine patient management . 
although cds is traditionally associated with crystal deposition diseases , it may be accompanied by other diseases affecting the joints , such as ra with involvement of the proximal cervical spine ( as seen in our study ) , ra - dish , and other forms of systemic connectivitis ( systemic sclerosis )  . 
pertanto , in presenza di cervicalgia febbrile , la scoperta di calcificazioni periodontoidee suggestiva di sdi , escludendo quella di meningite . conclusione nella sindrome del dente incoronato la tc rappresenta lesame gold standard , permettendo di identificare i diversi aspetti radiografici : calcificazioni semplici a banda o in doppia filiera sottile in corrispondenza del legamento trasverso ; calcificazioni irregolari a corona sopra e intorno allapice del dente ; lesioni ossee di tipo erosive del dente stesso . 
invece lesame radiografico di altri distretti articolari ( carpo , ginocchio , sinfisi pubica , spalla ) pu agevolare il riconoscimento eziologico della malattia , se da cppd o da ha , ed pertanto raccomandato nella gestione routinaria del paziente . 
la sdi , infatti , tradizionalmente associata alle malattie da microcristalli , ma altre malattie articolari si accompagnano ad essa , in primis lartrite reumatoide con compromissione della colonna cervicale prossimale ( come si evince anche dalla nostra casistica ) , seguita dalla ra - dish e da altre connettiviti sistemiche ( sclerosi sistemica )  . 
obliquecoronal 3dvr images were significantly superior to obliquecoronal sts - mip images in the evaluation of vertebrobasilar vessels ( p < 0.05 ) ; in all other cases , 3dvr images were equivalent to sts - mip images . 
le immagini sono state valutate consensualmente da 2 radiologi con lintervento di un terzo radiologo per risolvere le discordanze al fine di classificare parametri relativi alla qualit delle immagini e alladeguadezza per la definizione della patologia . 
le immagini ottenute con tecnica sts - mip sono risultate significativamene superiori alle immagini ottenute con tecnica sts - mpr nella valutazione dei diversi parametri presi in esame ( p < 0 , 05 )  . 
le immagini riformattate con tecnica sts - mip dovrebbero far parte del protocollo standard per la valutazione dei vasi epiaortici e del distretto intracranico non tralasciando di analizzare le immagini sorgenti . 
le immagini riformattate con tecnica 3dvr rivestono invece un ruolo importante in una fase successiva in cui lidentificazione della patologia stata effettuata con le immagini sts - mip e sorgenti . parole chiave tc angio - tc arterie cervico - craniali g . 
previous studies have demonstrated that cta of the intracranial circulation is equivalent if not superior to conventional angiography in demonstrating intracranial aneurysms 4 mm in size or smaller [ 36 ] and that cta of the epiaortic vessels is equivalent to colour - doppler ultrasound ( us ) in measuring the degree of carotid stenosis [ 79 ]  . however , as there are numerous reformatting techniques of cta images currently available on processing workstations , and as they influence the final quality of the examination , the radiologist needs to be aware of the advantages and limitations offered by the various reformatting techniques in order to optimise the cta examination [ 2 ]  . 
the reformatting techniques of ct images should be distinguished from image reconstruction in that they do not alter the image voxels ( i.e. , the raw data ) but use projectional techniques to visualise the image voxels in orthogonal or arbitrary planes . 
projectional include multiplanar reformatting ( mpr ) , which can be combined with the maximum intensity projection ( mip ) algorithm for visualising pixels with maximum brightness , or the sliding - thin - slab ( sts ) technique for visualising sections with variable thickness . 
although the term image reconstruction is commonly used in place of reformatting , it should be borne in mind that image reconstruction is the procedure that transforms the acquired raw data into ct images . techniques with projectional techniques , the reformatted image is what the observer would see two - dimensionally from their observation point looking through the volume being studied . with this technique , the cta images are displayed two - dimensionally in orthogonal or arbitrary planes ( mpr ) , with the possible addition of the mip algorithm to highlight opacified vascular structures that , as with bone , present the maximum brightness of the pixels . 
in addition , to improve visualisation of vascular structures , the sts technique can be used to select voxel groups of varying thickness to include or exclude them from the image and improve assessment of the structures being studied . with the 3dvr technique , density values of the ct images are assigned with properties such as visibility or nonvisibility , transparency and colour to represent the various anatomical structures on the basis of the properties assigned . in this way , the reformatted image is what the observer would see three - dimensionally from their observation point looking at the volume as if it were illuminated by an external light source , thus enabling the vessels to be visualised through structures rendered invisible or more or less transparent . because assigning the properties of visibility , colour and transparency to voxels is predefined in the various workstalutilizzo di apparecchi di tomografia computerizzata multidetettore ( tcmd ) in ambito neuroradiologico ha consentito il notevole miglioramento dellesame angio - tc per la valutazione del circolo carotideo , vertebrale e intracranico in virt dellacquisizione con voxel isotropico submillimetrico e della possibilit di ottenere riformattazioni multiplanari degli assi vascolari [ 1 , 2 ]  . 
in letteratura stato dimostrato come langio - tc del circolo intracranico sia equivalente se non superiore allangiografia convenzionale nella dimostrazione degli aneurismi intracranici di 4 mm o inferiori [ 36 ] e come lesame angio - tc dei vasi epiaortici sia equivalente alleco - color doppler per la misurazione del grado di stenosi carotidea [ 79 ]  . tuttavia , poich le tecniche di riformattazione delle immagini angio - tc oggi disponibili sulle workstation di elaborazione sono molteplici e poich esse influenzano la qualit finale dellesame , necessario che il radiologo conosca i vantaggi e i limiti offerti dalle diverse tecniche di riformattazione per ottimizzare lesame angio - tc [ 2 ]  . 
le tecniche di riformattazione delle immagini tc vanno distinte dalle ricostruzioni delle immagini in quanto non alterano i voxel dellimmagine , ovvero i dati grezzi , ma consentono una visualizzazione su piani ortogonali o arbitrari dei voxel dellimmagine utilizzando tecniche di tipo proiettivo come la riformattazione multiplanare ( mpr ) che pu avvalersi dellalgoritmo di visualizzazione dei pixel con la massima intensit luminosa denominato maximun intensity projection ( mip ) o ancora della tecnica di visualizzazione a spessore variabile denominata sliding - thin - slab ( sts )  . 
bench nelluso comune il termine ricostruzioni delle immagini viene comunque utilizzato piuttosto che riformattazione necessario ricordare che la ricostruzione delle immagini il procedimento che consente di trasformare i dati grezzi acquisiti nelle immagini tc . attraverso le tecniche di visualizzazione di tipo proiettivo limmagine riformattata ci che losservatore vedrebbe in modo bidimensionale dal suo punto di osservazione guardando attraverso il volume in esame . 
con questa tecnica le immagini angio - tc vengono rappresentate in modo bidimensionale su piani ortogonali o arbitrari ( mpr ) aggiungendo eventualmente lalgoritmo mip per enfatizzare le strutture vascolari opacizzate che come losso presentano la massima intensit luminosa dei pixel . 
inoltre , per migliorare la visualizzazione delle strutture vascolari di interesse , possibile utilizzare la tecnica sts per selezionare gruppi di voxel con spessore variabile per includere o escludere nellimmagine rappresentata gruppi di voxel e migliorare la valutazione delle strutture di interesse . con le tecniche di visualizzazione 3d di volume ( 3dvr ) ai valori di densit delle immagini tc vengono invece assegnate propriet quali se visibili o meno , la trasparenza e il colore per rappresentare le diverse strutture anatomiche in base alle propriet assegnate . 
in this study , we compared cta images of the cervicocranial arteries reformatted with sts - mpr , sts - mip and 3dvr techniques . materials and methods the cervicocranial arteries of 20 patients ( 15 men and five women , age range 5576 years , mean 66 years ) referred for cta assessment of cerebrovascular disease in the period from october 2005 to january 2006 were reviewed . 
the volume acquisition was performed during administration of 90 ml of iodinated nonionic contrast agent at a concentration of 370 mg / ml through an antecubital vein , with an 18 - gauge needle cannula using an automatic injector ( envision , medrad , pittsburgh , pa , usa ) at a flow rate of 4 ml / s . 
to obtain optimal enhancement of the arteries , the delay time between beginning the contrast agent administration and scan acquisition was calculated with the bolus test technique by measuring the enhancement curve at one of the common carotid arteries . 
the acquisition volume was set in the caudocranial direction from the aortic arch to the vertex so as to include the epiaortic vessels and the circle of willis . scan parameters were 250 mas , 120 kv , collimation 400.625 mm , pitch 0.67 , gantry rotation time 0.5 s and acquisition time 10 s . image processing and assessment the images were processed on a dedicated workstation ( extended brilliance workspace , philips , best , the netherlands ) using a standard procedure to obtain the following reformatted images : sts - mip , sts - mpr and 3dvr reformatted images in the three orthogonal planes with a slice thickness of 38 mm for visualisation of the individual arterial branches of the circle of willis , and in the obliquecoronal plane parallel to the clivus with a slice thickness of 510 mm for the visualisation in a single plane of both the basilar artery and the vertebral arteries in the intracranial tract . curvilinear reformations were not performed in that this technique is highly operator dependent and therefore cannot be standardised . 
images were transferred to a pacs mensionale dal suo punto di osservazione guardando il volume in esame come se fosse illuminato da una fonte esterna che ne consente la visione attraverso strutture rese invisibili o pi o meno trasparenti . poich lassegnazione delle propriet di visibilit , colore e trasparenza ai voxel predefinita nelle diverse workstation in base al protocollo desame e al distretto anatomico esaminato lasciando margini di modifica dei parametri alloperatore , i risultati ottenuti sono intrinsecamente soggetti a variabilit in base alla workstation in uso e al grado di addestramento delloperatore contrariamente a quanto si ottiene con le tecniche di tipo proiettivo . 
in questo lavoro vengono confrontate le immagini angio - tc del distretto arterioso cervico - craniale riformattate con tecnica sts - mpr , sts - mip e 3dvr . materiali e metodi sono stati valutati gli esami angio - tc del distretto arterioso cervico - craniale di 20 pazienti ( 15 maschi e 5 femmine di et compresa tra 55 e 76 anni , et media 66 anni ) eseguiti nel periodo ottobre 2005 - gennaio 2006 su richiesta dello specialista neurologo per patologia cerebrovascolare . 
in 1 paziente era nota la dissecazione di entrambe le arterie carotidi comuni e interne con doppio lume documentato con esame eco - color doppler . in 1 paziente era nota la stenosi dellarteria oftalmica destra documentata con esame eco doppler . gli esami sono stati effettuati utilizzando un tomografo multistrato a 40 linee di detettori ( brillance ct , philips , best , olanda )  . 
lacquisizione volumetrica stata effettuata durante infusione di 90 ml di mdc iodato non ionico alla concentrazione di 370 mg / ml attraverso accesso venoso antecubiltale con agocannula da 18 g utilizzando un iniettore automatico ( envision , medrad , pittsburgh , usa ) con flusso di 4 ml / s . 
il tempo di ritardo tra linizio dellinfusione del mdc e laquisizione delle scansioni per ottenere lopacizzazione ottimale del distretto arterioso stato calcolato con la tecnica del bolus test misurando la curva di enhancement in corrispondenza di una delle arterie carotidi comuni . 
stato impostato il volume di acquisizione delle scansioni con direzione caudo - craniale dallarco aortico fino al vertice in modo da comprendere i vasi epiaortici e il poligono di willis . 
i parametri di scansione utilizzati sono stati : 250 mas , 120 kv , collimazione 400 , 625 mm , pitch 0 , 67 , tempo di rotazione 0 , 5 s , durata delle acquisizioni 10 s . elaborazione e analisi delle immagini le immagini sono state elaborate su workstation dedicata ( extended brillance workstation , philips , best , olanda ) utilizzando una procedura standardizzata per realizzare le seguenti riformattazioni : riformattazione sts - mip , sts - mpr g . 
the following parameters were rated for each examination and for each reformation technique ( sts - mip , sts - mpr and 3dvr ) using a scale from 1 to 5 : the possibility of tracing the middle cerebral arteries e 3dvr nei 3 piani ortogonali con spessore dello strato di 38 mm per la visualizzazione dei singoli rami arteriosi del poligono di willis e in proiezione coronale obliqua , con inclinazione del piano parallelo al clivus , spessore dello strato di 510 mm , per visualizzare in un unico piano sia larteria basilare che le arterie vertebrali nel tratto intracranico . non stata presa in considerazione la riformattazione curvilinea in quanto questa tecnica fortemente operatore - dipendente e quindi non standardizzabile . 
the values were considered significant for p < 0.05. results tables 2 and 3 summarise the results of the spearman rank test for the three image reformatting techniques ( sts - mip , sts - mpr and 3dvr ) in the assessment of the various parameters studied and the significance of the test . 
le immagini sono state valutate utilizzando parametri di visualizzazione standard ( ingrandimento del 50% ; valori di finestra compresi tra 200 e 250 uh ; livello / centro compreso tra 150 e 250 uh )  . 
per ciascun esame e per ciascuna tecnica di riformattazione ( sts - mip , sts - mpr e 3dvr ) sono stati classificati , utilizzando un scala di misura ordinale con punteggio da 1 a 5 , i seguenti parametri : possibilit di delineare le arterie cerebrali medie ( acm ) , le arterie cerebrali anteriori ( aca ) , le arterie cerebrali posteriori ( acp ) , larteria basilare ( ab ) , le arterie carotidi comuni ( acc ) , la biforcazione carotidea ( bc ) , le arterie carotidi interne ( aci ) , i sifoni carotidei ( sc ) e le arterie vertebrali ( av ) ( 5 = ramo arterioso delineato in tutta la sua estensione , 1 = ramo arterioso non riconoscibile o riconoscibile solo per breve tratto ) ; omogeneit dellopacizzazione dei vasi ( 5 = eccellente , 1 = non soddisfacente ) ; sovrapposizione di strutture ossee ( 5 = nessuna influenza , 1 = influente fino a impedire la visualizzazione dei vasi ) ; identificazione di calcificazioni parietali ( 5 = eccellente , 1 = non soddisfacente ) ; adeguatezza delle ricostruzioni per delineare la patologia ( 5 = adeguato , 1 = non soddisfacente )  . analisi statistica i risultati sono stati confrontati utilizzato il test dei ranghi di spearman che ha fornito la correlazione tra i punteggi ottenuti nella valutazione dei diversi parametri presi in esame con le diverse tecniche di riformattazione ( sts - mip , stsmpr e 3dvr )  . 
lipotesi nulla del test di spearman in questo caso che non vi siano differenze significative nei punteggi ottenuti nella valutazione dei diversi parametri presi in esame con le tre tecniche di riformattazione delle immagini ( ovvero che le diverse tecniche siano equivalenti ) mentre lipotesi alternativa che vi siano differenze significative tra le diverse tecniche nella valutazione dei parametri presi in esame . 
i valori sono stati considerati significativi per p < 0 , 05 . risultati in tabella 2 e 3 sono riportati i valori della correlazione di spearman per le tre tecniche di riformattazione delle immagini ( sts - mip , sts - mpr e 3dvr ) nella valutazione dei diversi parametri presi in considerazione e la significativit del test . 
in termini di omogeneit di opacizzazione dei vasi la riformattazione sts - mip risultata superiore sia alla sts - mpr che alla 3dvr ( p < 0 , 05 )  . 
circa ladeguatezza delle immagini rispetto la definizione della patologia , in generale le immagini sts - mip si sono dimostrate pi accurate delle altre immagini ( mip vs mpr p < 0 , 05 ; mip vs 3dvr p < 0 , 05 )  . a questo proposito bisogna fare alcune considerazioni : g . 
1a - c axial sliding - thin - slab ( sts ) multiplanar reformatting ( mpr ) ( a ) , sts maximum intensity projection ( mip ) ( b ) and three - dimensional volume rendering ( 3dvr ) computed tomography angiography ( cta ) images ( c ) of circle of willis . 
2a - c sagittal sliding - thin - slab ( sts ) multiplanar reformatting ( mpr ) ( a ) , sts maximum intensity projection ( mip ) ( b ) and three - dimensional volume rendering ( 3dvr ) computed tomography angiography ( cta ) images ( c )  . 
3a - f oblique - coronal three - dimensional volume rendering ( 3dvr ) ( a - c ) and d - f sliding - thin - slab ( sts ) maximum intensity projection ( mip ) images of vertebral arteries and basilar trunk . 
3a - f immagini sul piano coronale - obliquo 3dvr ( a - c ) e sts - mip ( d - f ) della biforcazione tra arterie vertebrali e arteria basilare . 
4a - d sagittal sliding - thin - slab ( sts ) maximum intensity projection ( mip ) a and b three - dimensional volume rendering ( 3dvr ) ; c coronal sts - mip and d 3dvr images of the carotids . 
la significativa riduzione di sovrapposizione di strutture ossee sulle immagini 3dvr rende pi agevole la valutazione del decorso degli assi carotidei . discussion ct angiography with mdct of epiaortic vessels and intracranial circulation has undergone considerable improvements in recent years as a result of technological advances in mdct scanners and image processing software now available on commercial workstations . 
nonetheless , there are areas of dispute regarding the diagnostic value of the various reformation techniques and their clinical use , which is still being assessed and undergoing standardisation [ 2 , 10 ]  . 
in addition , most studies published to date have centred their attention on the assessment of quantitative parameters , such as measurement of the degree of carotid artery stenosis with comparison of findings obtained with mip and mpr reconstructions [ 1114 ]  . 
per la valutazione del doppio lume ( punta di freccia ) ci si avvalsi sia delle immagini sts - mip che delle immagini sorgenti . discussione lesame angio - tc con tcmd dei vasi epiaortici e del circolo intracranico ha subito negli ultimi anni notevoli miglioramenti in funzione degli avanzamenti tecnologici delle apparecchiature tc multidetettore e del software di elaborazione delle immagini oggi ampiamente disponibile sulle workstation commerciali . 
tuttavia in letteratura esistono controversie circa il valore diagnostico delle diverse tecniche di riformattazione delle immagini e sul loro utilizzo clinico che ancora oggetto di valutazione e standardizzazione [ 2 , 10 ]  . 
peraltro la maggior parte dei lavori pubblicati incentra la sua attenzione sulla valutazione di parametri quantitativi come la misurazione del grado di stenosi delle carotidi comparando i risultati ottenuti con le ricostruzioni mip e mpr [ 1114 ]  . 
oltre alla riformattazione mip e mpr disponibile la riformattazione 3d con algoritmo vr , notevolmente migliorata grazie allacquisizione con voxel isotropico consentita dalla tcmd a 16 strati o superiori [ 1 , 2 ]  . 
le immagini sts sono state introdotte nella pratica clinica per lo studio tc del torace [ 1618 ] e successivamente per la valutazione angio - tc della vascolarizzazione del donatore di rene [ 19 ] e di fegato [ 2022 ]  . 
le immagini sts , che possono essere visualizzate con algoritmo mip o con la tecnica mpr , presentano come caratteristica saliente un elevato contrasto e una ridotta incidenza di artefatti da volume parziale [ 19 ]  . questa caratteristica legata al fatto che , a differenza delle fig . 
7a - f basilar artery aneurysaxial a sliding - thin - slab ( sts ) multiplanar reformatting ( mpr ) and b sts maximum intensity projection ( mip ) images . 
axial a sliding - thin - slab ( sts ) multiplanar reformatting ( mpr ) , b sts maximum intensity projection ( mip ) and c three - dimensional volume rendering ( 3dvr ) images of the circle of willis . 
sagittal a sliding - thin - slab ( sts ) multiplanar reformatting ( mpr ) , b sts maximum intensity projection ( mip ) c and three - dimensional volume rendering ( 3dvr ) images . 
grossolane calcificazioni del sifone carotideo ( punta di freccia ) meglio apprezzabili nelle immagini sts - mip . provement thanks to acquisition with isotropic voxels made possible with 16 - slice or more mdct [ 1 , 2 ]  . more recently , the sts technique has been used [ 15 ]  . 
sts images were introduced into clinical practice in chest ct studies [ 1618 ] and later for cta assessment of kidney vascularisation [ 19 ] and liver donors [ 2022 ]  . 
this characteristic is related to the fact that in contrast to mip , mpr or 3dvr images , the sts technique involves selection of a very thin volume to be examined and therefore a very limited number of voxels with respect to the entire volume , thus reducing the interference of partial volume and the overlapping of other structures [ 19 , 23 , 24 ]  . 
poich lalgoritmo mip seleziona i pixel con soglia luminosa elevata , le immagini sts - mip sono pi adatte alla valutazione delle placche calcifiche rispetto le immagini sts - mpr [ 2 , 25 ]  . 
10a - i immagini riformattate sul piano assiale con algoritmo mip con spessore variabile ( sts ) da a 0 , 7 mm a i 3 , 4 mm con incremento di 0 , 3 mm ( a - i ) in corrispondenza delle arterie cerebrali medie . results of our study reveal some of the features of the reformatting planes used . 
it should be borne in mind that with the use of the oblique coronal plane adopted for this study , visualisation of the vertebrobasilar plane is easier given the possibility of displaying the vertebrobasilar axis completely and in a single plane . 
the aim of the study in fact was not comparison of diagnostic accuracy between cta and conventional angiography or colourdoppler us , which has already been established in the literature [ 39 ]  . 
this choice was prompted because by optimising arterial enhancement with the bolus test technique , we deliberately overlooked the venous district , thus avoiding possible interferences in the images , a feature not taken into consideration in other similar studies [ 23 ]  . opportuno ricordare che sono disponibili altre tecniche di analisi vascolare , denominate in modo diverso a seconda delle workstation di elaborazione in uso , quali ladvanced vessels analysis ( ava ) disponibile su workstation extended brillance philips , che permette di effettuare misurazioni quantitative del grado di stenosi e la riformattazione curvilinea . 
the aim of this study was to evaluate magnetic resonance imaging ( mri ) patterns in the affected vertebrae before and after vertebroplasty by determining changes in signal intensity and size and distribution of bone cement within the vertebra at follow - up carried out at 1 week , 6 months and 12 months . 
acrylic cement appears as an intraspongy focal area of t1 and t2 hypointensity that is mostly oval ( 34% ) or rounded ( 26.8% ) ; this appearance tends to become stable 6 months after treatment . 
la conoscenza delle modificazioni nel tempo del cemento e la reazione del tessuto osseo limitrofo fondamentale per una corretta interpretazione dellimaging post vertebroplastica . parole chiave vertebroplastica polimetilmetacrilato rm radiologia interventistica introduction introduzione first described by herv deramond in 1984 , percutaneous vertebroplasty ( pvp ) has undergone progressive technical and methodological refinement over the past few years , and now , many centres have adopted this procedure with success [ 1 , 2 ]  . 
the injection can be performed via posterior or lateral transpedicular approach in the lumbar spine , transpedicular or intercostal - transverse approach in the dorsal spine and anterolateral approach in the cervical spine [ 6 , 7 ]  . 
the aim of the procedure is to stabilise the vertebra , relieve pain , and improve patient mobility [ 8 ]  . awareness of the main imaging changes arising in treated vertebrae is therefore fundamental , and magnetic resonance imaging ( mri ) is the most widely used modality for examinations before and after treatment [ 9 , 10 ]  . 
the aim of this study was to evaluate mri patterns before and after vertebroplasty to document changes in signal intensity and size and distribution of bone cement within the treated vertebra [ 11 ]  . materials and methods between january and october 2004 , 14 nonconsecutive patients ( nine women and five men , age range 3589 years ) were examined for a total of 41 pathological vertebrae . 
candidates for the procedure were selected in accordance with eligibility criteria of the american college of radiology [ 12 ]  . to evaluate the imaging changes after pvp , all patients underwent mri at baseline and 1 week and 1 , 6 and 12 months after treatment [ 13 , 14 ]  . 
the imaging protocol used consisted of t1 - weighted spin - echo ( se ) sequences ( tr 400 , minimal te , slice thickness 4 mm , interval 0.5 mm , fov 31 cm , examination time 2 h 40 min ) , t2 - weighted se sequences ( tr 2 , 350 ms , te 103.5 ms , slice thickness 4 mm , interval 0.5 mm , fov 31 cm , examination time 4 min 04 s ) and t2weighted short tau inversion recovery ( stir ) sequences ( tr 3 , 000 ms , te 23 ms , slice thickness 4 mm , interval 0.5 mm , fov 31 cm , examination time 4 h ) ; all images were acquired in the sagittal plane . 
 mri signal intensity was evaluated in three distinct regions of the vertebra , before and after treatment ( at baseline , at 1 week and at 1 , 6 and 12 months ) by manually placing regions of interest ( rois ) within the vertebral body . 
these measurements , which expressed absolute roi values , were made at the periphery of the vertebral body , in the area adjacent to the treated area and in the treated area itself , at baseline and rela vpp consiste nelliniezione di polimetilmetacrilato ( pmm ) ( un cemento sintetico che polimerizza rapidamente , a temperatura ambiente , con una reazione esotermica ) , sotto guida tc e / o fluoroscopica , allinterno di un corpo vertebrale divenuto patologico a causa di osteoporosi [ 3 ] , metastasi , angiomi , mieloma multiplo o trauma [ 4 , 5 ]  . 
liniezione pu essere eseguita , a seconda del tratto di colonna vertebrale interessato dal processo patologico , per via trans - peduncolare posteriore o laterale nel tratto lombare , trans - peduncolare o intercosto - trasversa nel segmento dorsale e con approccio antero - laterale nel segmento cervicale [ 6 , 7 ]  . 
il fine ultimo della procedura quello di stabilizzare la vertebra , ridurre la sintomatologia dolorosa ed eventualmente di migliorare , come conseguenza , la mobilit del paziente [ 8 ]  . a tal proposito fondamentale descrivere e saper riconoscere le principali modificazioni della vertebra trattata con particolare rilievo allimaging post vpp ; tra le varie metodiche utilizzate nei controlli prima e dopo il trattamento la risonanza magnetica ( rm ) sicuramente la pi utilizzata [ 9 , 10 ]  . scopo del nostro lavoro stato quello di valutare e descrivere la semeiotica rm prima e dopo vertebroplastica , al fine di documentare le modificazioni dellintensit del segnale , delle dimensioni e della distribuzione del cemento allinterno della vertebra trattata [ 11 ]  . materiali e metodi tra gennaio e ottobre 2004 , sono stati esaminati 14 pazienti non consecutivi per un totale di 41 vertebre patologiche : i pazienti di cui 9 donne e 5 uomini , avevano unet compresa tra 35 e 89 anni . 
al fine di valutare le modificazioni dellimaging dopo vpp , tutti i pazienti sono stati studiati con esame rm prima del trattamento e seguiti nel tempo con esami di follow - up a distanza di 1 settimana , 1 , 6 e 12 mesi dal trattamento [ 13 , 14 ]  . 
i pazienti trattati erano affetti da : osteoporosi ( 8 / 14 casi , 57% ) [ 15 ] , mieloma multiplo ( 3 / 14 casi , 21 , 5% ) e fratture post - traumatiche ( 3 / 14 casi , 21 , 5% )  . le procedure di vertebroplastica sono state eseguite mediante approccio trans - peduncolare monolaterale in tutti i pazienti . 
il protocollo di studio ha previsto lutilizzo di sequenze se t1 pesate ( tr 400 , te minimo , spessore dello strato 4 mm , intervallo 0 , 5 mm , fov 31 cm , tempo di esame 2 min e 40 s ) , sequenze se t2 pesate ( tr 2350 ms , te 103 , 5 ms , spessore dello strato 4 mm , intervallo 0 , 5 , fov 31 cm , tempo esame 4 min e 4 s ) e sequenze stir t2 pesate ( tr 3000 ms , te 23 ms , spessore dello strato 4 mm , intervallo 0 , 5 , fov 31 cm , tempo di esame 4 min ) ; tutte le immagini sono state acquisite nel piano sagittale . la valutazione delle immagini rm stata eseguita , per consenso , da due radiologi e sono state prese in considerazione le caratteristiche dellintensit di segnale , le modalit r . 
intensit del segnale rm : stata valutata lintensit del segnale in tre differenti regioni della vertebra , prima e dopo il trattamento ( 1 settimana , 1 mese , 6 mesi e 12 mesi ) mediante il posizionamento manuale di aree roi nel contesto del corpo vertebrale . 
tali misurazioni , espressione di valore roi assoluto , sono state effettuate a livello della parte periferica della vertebra , della zona limitrofa allarea trattata ed a livello dellarea trattata . 
a seconda del loro aspetto morfologico , le vertebre sono state inoltre classificate , secondo la semeiotica classica prima e dopo il trattamento , in vertebra plana , vertebra con avvallamento della limitante somatica superiore , con avvallamento della limitante somatica inferiore e vertebra con aspetto a lente biconcava . 
i dati ottenuti sono stati elaborati mediante test statistico t di student , con ic del 95% . risultati la valutazione stata effettuata su un numero complessivo di 41 vertebre trattate di cui 10 dorsali ( quattro d11 e sei d12 ) , 31 lombari ( nove l1 , sei l2 , sette l3 sei l4 e tre l5 )  . relativamente alla distribuzione del cemento nel contesto del soma della vertebra trattata , la distribuzione disomogenea stata riscontrata nel 26 , 8% nel controllo dopo una settimana del trattamento e nel 12 , 2 % dopo 1 mese dal trattamento ; la distribuzione omogenea non stata mai evidenziata ; la distribuzione puntiforme del cemento stata riscontrata nel 7 , 3% dei casi dopo 1 settimana e dopo 1 mese dal trattamento ; la distribuzione a carta geografica stata evidenziata nel 14 , 6% dei casi dopo una settimana ed stata riscontrata nel 19 , 5% dei casi ad un mese dal trattamento ; la distribuzione ovalare del cemento , presente nel 21 , 9% dopo una settimana stata riscontrata nel 34 , 1% ad un mese dal trattamento mentre la distribuzione rotondeggiante , presente nel 29 , 2% dei casi ad 1 settimana , era presente nel 26 , 8% dei casi ad un mese dal trattamento ( tabella 1 )  . 
la morfologia della distribuzione del cemento osservata nel contesto del soma vertebrale a distanza di un mese dal trattamento non si in seguito modificata nei successivi controlli a sei mesi e ad un anno dal trattamento . relativamente al parametro delle altezze dei somi vertebrali , il test statistico ha evidenziato : una deviazione stanfig . 
liperintensit che circondava il cemento appare ridotta ad un esile alone . dard media di 17 , 1 nel pre - vertebroplastica e di 16 , 7 nel post - vertebroplastica in corrispondenza del muro anteriore ; una deviazione standard media di 11 , 4 pre - vertebroplastica e di 11 , 0 nel post - vertebroplastica in corrispondenza della porzione centrale del soma ; una deviazione standard media di 19 , 3 pre - vertebroplastica e di 18 , 8 nel post - vertebroplastica in corrispondenza del muro posteriore ( tabella 2 )  . 
before and after treatment , vertebrae were morphologically classified according to classic imaging patterns into vertebra plana , vertebra with concavity of the superior endplate , vertebra with concavity of the inferior endplate and biconcave vertebra . 
data obtained were processed using the students t test with 95% confidence interval ( ci )  . results a total of 41 vertebrae , ten dorsal ( four d11 and six d12 ) and 31 lumbar ( nine l1 , six l2 , seven l3 , six l4 and three l5 ) , were evaluated . 
c osteoporosi ( 12 mesi dopo vpp ; stir t2w ) l1 - l2 : lipointensit del cemento appare invariata , mentre apprezzabile una marcata riduzione dellalone di iperintensit dovuta alledema circostante . 1 month ; in no case was it homogeneous . 
la valutazione dellintensit del segnale con posizionamento di una roi perifericamente allarea trattata ha dimostrato : nelle sequenze se t2 prima del trattamento un valore medio di 80 , 658 , 30 , ad una settimana dal trattamento un valore di 101 , 678 , 34 , ad un mese dal trattamento un valore medio di 125 , 335 , 5 , a sei mesi dal trattamento un valore medio di 100 , 208 , 42 , ad un anno dal trattamento un valore medio di 98 , 518 , 91 . stata rilevata una variabilit statisticamente significativa nella modifica dellintensit del segnale esclusivamente nelle sequenze t1 ( p = 0 , 002 , < 0 , 05 ) mentre non sono emerse differ . 
nel controllo a 1 mese e a 6 mesi non sono state notate variazioni frequenza della morfologia post - vertebroplastica 1 settimana , n ( % ) post - vertebroplastica 1 , 6 , 12 mesi , n ( % ) concavity were observed in 31% and 29% of cases , respectively , whereas vertebra with inferior endplate concavity and vertebra plana were detected in 19% and 21% of cases , respectively ; these percentages remained unchanged before and after vertebroplasty . 
 results of the evaluation of signal intensity based on the roi positioned peripherally to the treated area were as follows : a mean value of 140.255.20 at baseline , 148.675.79 at 1 week , 206.59.5 at 1 month , 163.339.83 at 6 months and 163.359.88 at 12 months in t1 - weighted se sequences ; a mean value of 82.256.20 at baseline and 113.256.22 at 1 week , 134.51.5 , at 1 month 60.750.83 at 6 months and 61.510.91 at 12 months in t2 - weighted stir sequences ; and a mean value of 80.658.30 at baseline and 101.678.34 at 1 week , 125.335.5 at 1 month , 100.208.42 at 6 months and 98.518.91 at 12 months in t2 - weighted se sequences . 
levoluzione e la diffusione della vertebroplastica impone la conoscenza delle principali variazioni del soma trattato con le varie metodiche di imaging ; ci al fine di valutare e correttamente interpretare limaging dopo trattamento [ 21 ]  . 
considerata la diffusione della rm nello studio del rachide cervico - dorso - lombare e lincidenza degli esami rm abbiamo focalizzato la nostra attenzione alla descrizione delle modificazioni delle modificazioni rm del comportamento della vertebra trattata [ 22 ]  . al fine di approfondire le modificazioni del segnale rm dopo vertebroplastica abbiamo analizzato la vertebra trattata prima del trattamento ed a distanza di un mese , sei mesi ed un anno dal trattamento , valutando sia la morfologia sia le alterazioni di intensit del segnale ; tali reperti sono fondamentali in rm ai fini descrittivi e per la comprensione delle variazioni indotte dal trattamento . 
with regard to vertebral size , the material injected into the vertebra could have produced either vertebral expansion increase in size and consequent morphological changes or diffusion of the cement within the vertebra with little or no effect on size . 
this demonstrates that the acrylic cement distributes diffusively within the spongy bone trabeculae , causing no appreciable bone expansion ; it also shows that restoration of normal vertebral size and morphology should not be expected on postvertebroplasty mri . 
si evince , pertanto , che il cemento acrilico si distribuisce nelle trabecole della spongiosa ossea del soma vertebrale con un comportamento diffusivo , senza un reale e consistente effetto di espanso e che il ripristino delle dimensioni della vertebra e della sua normale morfologia non debbano essere considerati degli elementi attesi allimaging rm nel post trattamento . relativamente alla valutazione della intensit del segnale , abbiamo notato , in accordo con la letteratura [ 22 , 23 ] , una variazione di essa nelle regioni periferiche allarea trattata correlabile a presenza di edema intra - spongioso . 
lanalisi dei risultati suggerisce , quindi , che il comportamento del segnale atteso nellarea limitrofa al cemento debba presupporre un modesto incremento della intensit in t1 e in t2 dopo trattamento , che tale incremento del segnale cominci ad essere rilevato sin dalla prima settimana dopo la procedura e che divenga pi evidente nei controlli ad un mese dal trattamento per poi stabilizzarsi , con riduzione della sua intensit a sei mesi e a dodici mesi dal trattamento . 
i dati derivanti dal confronto delle dimensioni delle vertebre non sono risultati statisticamente significativi per la presenza di un p > 0 , 05 . signal intensity between t1 and t2 sequences before and after treatment were not statistically significant . 
 vertebroplasty is a minimally invasive procedure indicated for the treatment of neck and arm pain , chest pain and back pain caused by vertebral collapse due to osteoporosis [ 3 , 8 ] , metastases , invasive angiomas and myelomas . 
clinical studconclusions controllo a sei mesi dal trattamento e che il segnale rimasto invariato ad un anno . per quanto riguarda la distribuzione del cemento i reperti morfologici pi frequenti sono rappresentati dalla distribuzione ovalare e rotondeggiante ; la prima stata riscontrata nel 21 , 9% dopo una settimana e nel 34 , 1% ad un mese dal trattamento mentre la seconda presente nel 29 , 2% dei casi ad 1 settimana presente nel 26 , 8% ad un mese dal trattamento . conclusioni la vertebroplastica una metodica terapeutica mini - invasiva che ha la sua indicazione elettiva , ove la terapia medica risulti inefficace , nel trattamento delle cervico - brachialgie , delle toracoalgie , delle lombalgie dovute a crolli vertebrali da osteoporosi [ 3 , 8 ] ed indicata per altre cause di crolli vertebrali ( metastasi , angiomi invasivi e mielomi )  . 
nellimaging pree post - vertebroplastica la risonanza magnetica considerata metodica di riferimento [ 10 ] per la valutazione corretta del contenente e del contenuto ; in particolare per la capacit della metodica ies have demonstrated an immediate improvement in symptoms and quality of life [ 11 , 14 ] following treatment . 
in preand post - vertebroplasty imaging , mri is considered the reference standard [ 10 ] due to its ability to correctly evaluate both container and content and assist in patient selection by excluding those cases showing ependymal infiltration by secondary lesions . 
the information provided by mri [ 21 ] is instrumental in the evaluation of both the efficacy and possible complications of pvp [ 9 ] , and awareness of the stages influencing post - vertebroplasty mri is fundamental . 
 in our experience , post - vertebroplasty mri is characterised by three main features : the signal from the cement , the signal from the area adjacent to the cement and the effect produced on vertebral size and morphology . 
acrylic cement appeared as an oval ( 34% ) or rounded ( 26.8% ) focal area within the spongy bone , and this appearance tended to stabilise 6 months after treatment . 
 bone marrow oedema in the area adjacent to the cement [ 22 , 23 ] was also observed in our experience ; in particular , it was evident 1 week to 6 months after treatment and tended to stabilise with only moderate residual hyperintensity in t2 in the following period . 
stability of vertebral size after treatment is the result of the diffusive as opposed to expansive cement distribution pattern within the spongy bone , which means that there is no change in shape or size of the vertebra after treatment . 
le informazioni che la rm [ 21 ] in condizione di fornire sono , quindi , fondamentali sia ai fini della valutazione dellefficacia del trattamento che delle eventuali complicanze della vpp [ 9 ] ; pertanto opportuno puntualizzare le tappe che condizionano limaging rm dopo vertebroplastica . dalla nostra esperienza limaging rm dopo vertebroplastica caratterizzato da tre elementi fondamentali : il segnale del cemento , il segnale dellarea limitrofa alla localizzazione del cemento , leffetto indotto dalla procedura sulle dimensioni e sulla morfologia della vertebra . 
il cemento acrilico si presenta come una area focale intraspongiosa di forma prevalentemente ovalare ( 34% ) , rotondeggiante ( 26 , 8% ) e tale aspetto tende a stabilizzarsi dopo sei mesi dal trattamento . 
il razionale di tale comportamento del segnale del cemento indotto dalla carenza di spettro di idrogeno nel materiale amorfo e pu porre problemi di diagnosi differenziale o con residui ferromagnetici intraspongiosi da trattamento chirurgico o con presenza in sede intraspongiosa di materiali in grado di produrre artefatti di fase ( ad esempio il titanio ) , con emangiomi con componente cavernosa intraspongiosa ( in t1 ) , con fenomeni di vacuum intraspongiosi ; a tal proposito una corretta anamnesi fondamentale per linquadramento del paziente . latteso edema della spongiosa ossea nellarea limitrofa al cemento , descritto dalla letteratura [ 22 , 23 ] , stato riscontrato anche nella nostra esperienza ; in particolare abbiamo verificato come esso sia evidente soprattutto tra una settimana e sei mesi dal trattamento e tenda a stabilizzarsi nel periodo successivo con modesto residuo di iperintensit in t2 . 
simonetti universit degli studi di roma tor vergata , dipartimento di diagnostica per immagini e radiologia interventistica , policlinico universitario tor vergata , viale oxford 81 , i - 00133 roma , italy correspondence to : e . 
turbo spin echo ( tse ) t1 - , t2and t2spectral selection attenuated inversion recovery ( spair ) weighted imaging and spectroscopy for the selective evaluation of water and fat content were performed . 
the anisotropic study and the subsequent assessment of colour and vector maps can provide a noninvasive tool for assessing the risk of fracture due to osteoporosis . key words osteoporosis vertebral collapse 3 - tesla mr diffusion imaging tractography riassunto obiettivo . 
il calcolo dellanisotropia e la valutazione grafica colorimetrica e vettoriale forniscono un nuovo strumento non invasivo per valutare losteoporosi vertebrale . parole chiave osteoporosi frattura vertebrale rm a 3 tesla immagini in diffusione trattografia introduction introduzione bone mineral density ( bmd ) is perhaps the best index available for assessing the risk of osteoporotic fracture . 
these figures have been confirmed by other clinical studies that showed that the risk of a new fracture within the first year of a previous compression fracture increases from 5 to 25 times [ 2 ]  . 
hanno riportato che ad ogni riduzione di una deviazione standard della bmd corrisponde un aumento del 60% del rischio di frattura vertebrale [ 1 ]  . tali dati sono stati inoltre confermati da ulteriori studi clinici in cui si dimostrato che il rischio di una nuova frattura vertebrale entro il primo anno da una precedente frattura e . 
the aim of this study was therefore to assess the possibility of a multiparameter characterisation , and not only imaging , of vertebral osteoporosis using 3 - tesla ( t ) mr . compressiva aumenta dalle 5 alle 25 volte [ 2 ]  . 
lo scopo del nostro lavoro pertanto quello di valutare la possibilit di una caratterizzazione multiparametrica , e non solo di imaging , dellosteoporosi vertebrale mediante rm a 3 tesla . materials and methods in our study , 30 subjects underwent an mr examination of the lumbar spine . 
these included ten healthy volunteers ( three men and seven women ) aged between 31 and 68 years without metabolic bone diseases ( in particular , osteoporosis t - score - 1 ) or neoplastic disease in any district ; ten female patients aged between 55 and 78 years with osteoporosis ( t - score - 2.5 ) but without vertebral fractures identifiable with conventional radiology ; and ten female patients aged between 60 and 75 years with a single osteoporotic lumbar vertebral fracture identified in a prior radiographic examination of the spine and with dual x - ray absorptiometry ( dexa ) positive for osteoporosis , a negative history of recent traumatic events and the absence of other metabolic and neoplastic bone diseases . 
all patients were informed of the experimental nature of the examination , and they all signed informed consent before undergoing the study . the morphological , metabolic and functional images were acquired with a high - field device ( 3.0 t ) ( philips intera achieva , best , netherlands ) , with a maximum gradient strength and slew rate set to 80 mt / m and 200 mt / m / ms , respectively , using a body coil for signal transmission and a synergy spine phased - array coil for signal reception . 
 the study of the lumbar spine was performed with the following sequences : t2 - weighted turbo spin echo ( tse ) in the sagittal plane the entire including lumbarsacral tract ( tr shortest , te 120 ms , slice thickness 4 mm with an interval between the sections of 0.4 mm , fov 300 mm and a 512512 matrix ) the acquisition volume t1 - weighted tse in the sagittal plane ( tr shortest , te 7.2 ms , slice thickness 4 mm with an interval between sections of 0.4 mm , fov 300 mm and a 512512 matrix ) t2 - weighted tse with selective fat suppression [ spectral selection attenuated inversion recovery ( spair ) ] in the sagittal plane aimed at assessing the presence of bone marrow oedema , an index of recent fracture in collapsed vertebral bodies and of algodystrophic changes in response to biomechanical stress in noncollapsed vertebral bodies . on the basis of the t2 - weighted images , especially the spair images , two independent observers identified the vertebral bodies with altered signal intensity , which were selected for placement of the spectroscopy acquisition volume . spectroscopic acquisition in the healthy subjects was optimised in the mid - lumbar vertebral bodies in accordance with the literature . 
the volume of interest ( voi ) for spectroscopic analysis was chosen on the basis of the t2 and t2materiali e metodi nel nostro studio sono stati sottoposti ad esame rm del rachide lombare 30 soggetti : 10 volontari sani ( 3 maschi e 7 femmine ) , di et compresa tra 31 e 68 anni , esenti da patologie metaboliche dellosso ( in particolare osteoporosi , t - score - 1 ) e da patologie neoplastiche di qualsiasi distretto ; 10 pazienti di sesso femminile di et compresa tra 55 e 78 anni con osteoporosi ( t - score - 2 , 5 ) ma esenti da fratture vertebrali evidenziabili con esami di radiologia tradizionale ; 10 pazienti di sesso femminile di et compresa tra 60 e 75 anni con frattura vertebrale lombare singola a genesi osteoporotica , evidenziata ad un precedente esame radiografico del rachide e dexa positiva per osteoporosi , anamnesi negativa per eventi traumatici recenti , in assenza di altre patologie metaboliche e neoplastiche dellosso . 
tutti i pazienti sono stati informati sul carattere sperimentale dellesame ed hanno firmato un consenso prima di essere sottoposti allindagine . le immagini morfologiche , cos come quelle metaboliche e funzionali , sono state acquisite con unapparecchiatura ad alto campo ( 3 , 0 tesla ) ( philips intera achieva , best , netherlands ) equipaggiata con gradienti di ampiezza massima e tempo di salita di 80 mt / m e 200 mt / m / ms rispettivamente , utilizzando la bobina del corpo per la trasmissione degli impulsi di eccitazione e una bobina sinergy spine phased - array per la ricezione del segnale . lo studio del rachide lombare stato eseguito mediante le seguenti sequenze : tse t2 pesata sul piano sagittale includendo nel volume dacquisizione lintero tratto lombo - sacrale ( tr shortest , te 120 ms , spessore di sezione 4 mm con intervallo tra le sezioni di 0 , 4 mm , fov di 300 mm e una matrice di 512512 ) ; tse t1 pesata sul piano sagittale ( tr shortest , te 7 , 2 ms , spessore di sezione 4 mm con intervallo tra le sezioni di 0 , 4 mm , fov di 300 mm , matrice di 512512 ) ; tse t2 pesata con soppressione selettiva del segnale del tessuto adiposo ( spair ) sul piano sagittale , al fine di valutare leventuale presenza di edema intraspongioso , indice di frattura recente per i somi vertebrali crollati e di trasformazione da sostituzione algodistrofica per adattamenti intrinseci da sofferenza biomeccanica per quelli non crollati . sulla base delle immagini t2 pesate , soprattutto spair , sono state individuate da due osservatori indipendenti le vertebre con alterata intensit di segnale , selezionate per il successivo posizionamento del volume di acquisizione spettroscopica . 
acquisition was performed with a point - resolved spectroscopy ( press ) sequence ( double spin - echo point resolved spatially localised spectroscopic sequence ) with the two - dimensional ( 2d ) technique and echo time ( 40 ms ) optimised for quantitative assessment of water and fats , with single voxel analysis . field homogeneity was adjusted through an automatic procedure of localised three - dimensional ( 3d ) shimming . 
the following acquisition parameters were used : tr 2 , 000 ms , te 40 ms , spectral width 2.00 hz , matrix 512512 , nominal voxel resolution 25 mm25 mm15 macquisition time of the spectroscopy sequence was on average 2 min for each vertebral body examined . we performed three acquisitions with voxels positioned at the level of l2 , l3 and l4 in healthy patients and osteoporotic patients without vertebral collapse . 
the spectra obtained were processed with fourier transformation . the fat content was expressed as the percentage of the fat signal intensity in respect to total signal intensity ( fat fraction : ff )  . 
a p value lower than 0.05 was considered statistically significant . diffusion - weighted ( dw ) images were acquired with single - shot spin - echo echoplanar imaging ( se - epi ) in the sagittal plane , with a duration of 35 s ( tr 3 , 500 ms , te 62 ms , fov 200 mm , flip angle 90 , matrix 256256 , b - value set to 0 and 400 mm2 / s , three sections 5 - mm thick with a 1 - mm gap )  . 
the apparent diffusion coefficient ( adc ) was calculated by positioning regions of interest ( rois ) in the vertebral bodies examined with spectroscopy of varying size so as to almost entirely cover the body . 
the diffusion tensor images were acquired with the same technique and using the same acquisition parameters , with sampling of the random motion of free water in 32 directions in space ( directional resolution high )  . 
acquisition time was about 2 min . the fractional anisotropy ( fa ) was then calculated , a parameter that describes the degree of variation of diffusion in space ( a value between 0 = highly isotropic with the direction of water molecule diffusion being highly random , and 1 = anisotropic , with the preferential motion of water molecules being highly directional ) and the main direction of water molecule diffusion . 
this parameter , which can be derived from the tensor , is linked with the possibility of mapping the spatial orientation of tissue structures ; the technique is also known as fibre tracking or tractography [ 3 ]  . images obtained with the diffusion tensor imaging ( dti ) sequence were first analysed on the acquisition console to identify the presence of motion artefacts and image distortions related to the presence of eddy currents . 
lacquisizione stata effettuata con sequenza press ( double spin - echo point - resolved spatially localized spectroscopic sequence ) con tecnica bidimensionale ( 2dsi ) e tempo di eco ( 40 ms ) ottimizzato per la valutazione quantitativa dellacqua e dei lipidi , con analisi single voxel . lomogeneizzazione del campo magnetico stata effettuata con una procedura automatica di shimming tridimensionale localizzato . 
i parametri di acquisizione utilizzati sono stati i seguenti : tr 2000 ms , te 40 ms , ampiezza spettrale 2000 hz , matrice di 512512 , risoluzione nominale del voxel di 25 mm25 mm15 mil tempo di acquisizione della sequenza spettroscopica stato in media di 2 minuti per ogni corpo vertebrale in esame . nei pazienti sani ed in quelli osteoporotici senza crollo vertebrale sono state effettuate tre acquisizioni con voxel posizionati a livello di l2 , l3 ed l4 . 
il coefficiente di diffusione apparente ( adc ) stato calcolato posizionando delle rois nel contesto dei corpi vertebrali gi sede dellanalisi spettroscopica , di dimensioni variabili in modo da comprendere quasi completamente lintero soma . 
le immagini del tensore di diffusione sono state acquisite con la medesima tecnica , utilizzando i medesimi parametri di acquisizione , campionando i movimenti casuali dellacqua libera su 32 direzioni dello spazio ( directional resolution high )  . 
la durata dellacquisizione stata di circa 2 minuti . stato quindi calcolato il grado di anisotropia ( fractional anisotropy : fa ) , che descrive la entit di variazione della diffusione nello spazio ( valore compreso tra 0 = altamente isotropico con direzione di diffusione delle molecole di acqua assolutamente casuale , e 1 = anisotropico , con movimento preferenziale delle molecole di acqua altamente direzionato ) , e la direzione principale di diffusione delle molecole di acqua . 
data were then processed with a dedicated software package ( pride v4 fiber tracking 4.1 , idl , research systems , boulder , co , usa )  . a single rectangular roi was positioned on the images acquired with the b - value set to 0 in the same vertebral bodies assessed with spectroscopy imaging ( using the criteria for analysis of the morphological images mentioned above )  . all rois were then automatically transposed onto the corresponding image on the grey - scale maps of mean diffusivity ( md ) and fa generated by the software package . 
md values , expressed as a value of adc , and fa values were then calculated as the mean of the values derived from each pixel of the roi examined . 
extremely anisotropic structures are shown with a colour varying from black to deep blue , areas with intermediate anisotropy are shown with a colour varying from orange to purple and isotropic structures are shown in grey scale . 
the structural anatomy of the vertebral body was assessed with the representation of the preferential direction of the fibres on a directionally encoded colour ( dec ) map [ 4 ]  . this information was obtained pixel per pixel on the basis of the vector corresponding to the highest value [ 5 ] , in which red indicates the fibres that run in the left - right direction , green the fibres that run in the anterior - posterior direction , and blue the fibres that run in the superior - inferior direction . 
to this end , an roi was manually traced on the images acquired with a b - value set to 0 along the internal margin of the vertebral body cortex . 
the fibres represented in this way were superimposed on the non - dw echoplanar images ( b = 0 ) and codified by assigning them the colour corresponding to the preferential direction in accordance with the model outlined above . 
i dati sono stati poi rielaborati con software dedicato ( pride v4 , fiber tracking 4.1 , idl , research systems , boulder , co ) allanalisi del dti e al fiber tracking . una singola roi rettangolare stata posizionata sulle immagini acquisite con b factor uguale a 0 in corrispondenza degli stessi somi valutati con imaging spettroscopico ( utilizzando i criteri precedentemente visti per lanalisi delle immagini morfologiche )  . 
tutte le roi sono state poi trasposte automaticamente sullimmagine corrispondente sulle mappe in scala di grigi di diffusivit media ( md ) e di anisotropia frazionaria ( fa ) generate dal software di analisi . 
strutture estremamente anisotropiche appaiono di un colore che varia dal nero al blu ; aree con anisotropia intermedia sono rappresentate con un colore che va dallarancione al viola ; strutture isotropiche sono rappresentate in scala di grigio . 
lanatomia strutturale del corpo vertebrale stata valutata , rappresentando su di una mappa a colori ( directionally - encoded color , dec ) la direzione preferenziale delle fibre [ 4 ]  . questa informazione stata ottenuta pixel per pixel sulla base del vettore corrispondente al valore pi grande [ 5 ] nel quale il colore rosso indica le fibre che decorrono lungo la direzione destra - sinistra , il verde quelle lungo la direzione anteriore - posteriore ed il blu quelle lungo la direzione superiore - inferiore . 
il software di ricostruzione ha poi effettuato automaticamente lestrazione delle fibre concatenando , in ciascun voxel , il vettore principale con quelli dei voxel vicini . tale elaborazione risultata in una progressiva definizione della traiettoria della diffusivit preferenziale che corrisponde verosimilmente alla direzione delle trabecole dellosso spongioso del corpo vertebrale . 
le fibre cos rappresentate sono state sovrapposte alle immagini ecoplanari non pesate in diffusione ( b value = 0 ) e codificate assegnando loro il colore corrispondente alla direzione preferenziale secondo lo schema visto precedentemente . 
il tempo complessivo necessario allelaborazione dei dati del tensore di diffusione , compreso il trasferimento dei dati dalla stazione di acquisizione a quella di elaborazione e lanalisi trattografica , stato in media di circa 20 minuti . 
la valutazione qualitativa degli spettri ha evidenziato una risoluzione spettrale sufficiente a separare la risonanza dellacqua ( 4 , 7 ppm ) da quella dei lipidi ( 1 , 3 ppm )  . 
il picco dellacqua nei soggetti sani risultato significativamente pi elevato di quello dei lipidi , con andamento inverso man mano che let dei pazienti aumentava ed i valori di t - score diminuivano ( inversione del rapporto acqua / lipidi )  . 
la risoluzione spettrale della sequenza ha inoltre permesso di evidenziare un aumento degli acidi grassi saturi ( in particolare il metilene : 1 , 3 ppm ) nettamente superiore rispetto agli acidi grassi insaturi ( protoni oleofinici : 5 , 35 ppm ) [ 7 ] nei pazienti osteoporotici rispetto a quelli sani . 
il valore medio della frazione lipidica ( ff ) misurato nei corpi vertebrali dei volontari sani ottenuto dalle tre misurazioni risultato pari a 34%10 , 6% ( range compreso tra 29%9 , 6% e 56 , 3%11 , 2% ) , con incremento medio annuo di circa il 2% e valori leggermente pi alti nei maschi . 
nel gruppo dei pazienti osteoporotici la ff media calcolata stata 65 , 5%10% ( range compreso tra 55 , 2%9 , 8% e 74 , 5%11 , 2% )  . 
con questa sequenza molto meglio evidente liperintensit della spongiosa ossea di l1 , segno di edema intraspongioso e quindi di frattura recente . spectroscopy we acquired 104 spectra with the single voxel technique , excluding 12 spectra from the study that were not assessable due to motion artefacts and excessive background noise . 
the water peak in healthy subjects was significantly higher than the fat peak , with an inverse progression as patient age increased and t - score decreased ( inversion of the water / fat ratio )  . 
the spectral resolution of the sequence also revealed a notably higher increase in saturated fatty acids ( especially methylene : 1.3 ppm ) than unsaturated fatty acids ( olefinic protons : 5.25 ppm ) [ 7 ] in osteoporotic patients compared with healthy patients . 
spectroscopic analysis of vertebral bodies with a clearly hyperintense signal in the t2 - weighted sequences in osteoporotic patients , however , revealed an apparently retained water / fat ratio . 
in some cases of osteoporotic patients , there was an overlapping of ff values with those of postmenopausal patients with a normal t score . proton - diffusion and tensor - diffusion imaging we positioned 104 rois in as many vertebral bodies and calculated the adc and fa values . 
the images obtained with dti were used to process the colour and vector maps . four uncooperative patients were excluded from the study . nella tabella 1 sono riassunti i valori medi di ff correlati al t - score ed allet dei pazienti sottoposti ad esame spettroscopico . 
in alcuni casi di pazienti osteoporotici si verificata una sovrapposizione dei valori di ff con quelli di pazienti con normale t - score in et postmenopausale . diffusione protonica e tensore di diffusione sono state posizionate 104 rois in altrettanti corpi vertebrali e calcolati i relativi valori di coefficiente di diffusione apparente ( adc ) e di anisotropia frazionata ( fa )  . 
il calcolo della fa in tale gruppo ha infatti mostrato un valore medio di 0 , 29 ( valore tendente allisotropia ) con range tra 0 , 23 e 0 , 36 . 
3 analisi spettroscopica con relativo calcolo della componente lipidica ( 1 , 3 ppm ) e dellacqua in un volontario sano di sesso femminile di 39 anni che mostra normale rapporto acqua / lipidi ( ff 40 , 5% ) e . 
esiste una differenza significativa tra i valore di ff dei soggetti non osteporotici e quello dei soggetti osteoporotici indipendentemente dalla presenza di frattura vertebrale . the remaining data obtained were analysed and compared . in the group of healthy volunteers , the mean adc value obtained from measurements in the three vertebral bodies ( l2 , l3 and l4 ) was 0.480.0510 - 3 mm2 / s ( range between 0.440.0810 - 3 mm2 / s and 0.520.0710 - 3 mm2 / s )  . 
in fact , the colour maps derived from the data revealed a gradation of colours with reference to the entire vertebral body , which is compatible with a structure characterised by intermediate anisot - score - 1 ( p < 0 , 001 )  . 
il calcolo della fa nei soggetti con t - score - 2 , 5 ha mostrato un valore medio di 0 , 5 ( valore tendente allanisotropia ) con range tra 0 , 45 e 0 , 54 , ma il dato importante che tale valore aumentava proporzionalmente alla diminuzione del t - score . 
diffusion - weighted imaging ( dwi ) ( a ) and apparent diffusion coefficient ( adc ) map ( b ) with the regions of interest ( rois ) allowing quantitative analysis of the degree of both mean diffusivity and anisotropy within vertebral bodies of l2 , l3 and l4 . 
immagine pesata in diffusione ( a ) e rispettiva mappa di adc ( b ) con il posizionamento delle roi per lanalisi quantitativa del grado di diffusivit media e di anisotropia allinterno dei somi vertebrali l2 , l3 ed l4 . 
8 confronto tra valori medi di adc in soggetti sani di et giovane ( iii decade , 3040 anni ) e soggetti sani ed osteoporotici in et post - menopausa . 
quantitative histological studies performed on bone biopsies with regard to the changes in bone with age have shown the progressive , gradual and constant increase in the content of fats in bone marrow [ 9 ] , with greater evidence found in osteoporotic subjects [ 10 ]  . 
bone marrow is known to consist primarily of saturated , monounsaturated and polyunsaturated fatty acids [ 11 ] , but it is unknown if ( and how ) that composition varies in osteoporosis . 
a very recent study carried out by yeung et malmente agenti possano causare un evento fratturativo in un corpo vertebrale , n tanto meno il livello specifico dove tale rischio pi alto . 
studi istologici quantitativi eseguiti su biopsie ossee relativi alle modificazioni dellosso legate allet hanno mostrato il progressivo , graduale e costante aumento del contenuto di lipidi nel midollo osseo [ 9 ] , con maggiore evidenza nei soggetti osteoporotici [ 10 ]  . 
noto che il midollo osseo consiste primariamente di acidi grassi saturi , monoinsaturi e polinsaturi [ 11 ] , ma non noto se ( o come ) tale composizione varia nellosteporosi . 
 [ 12 ] con tomografo a risonanza magnetica da 1 , 5 t , in cui sono stati messi a confronto i risultati della spettroscopia effettuata a livello di l3 di una popolazione di donne in post - menopausa osteoporotiche con lo spettro ottenuto da un fantoccio di olio di mais , ha dimostrato che il contenuto di acidi grassi insaturi nel midollo osseo , calcolato come porzione della frazione lipidica totale ( ff ) , presentava una evidente tendenza al decremento quando si passava da soggetti sani a soggetti osteoporotici di pari et e sesso , e che contestualmente esisteva un aumento degli acidi grassi saturi . 
the findings demonstrated that the content of unsaturated fatty acids in bone marrow , calculated as a portion of the total ff , was clearly lower in osteoporotic subjects than in healthy subjects of the same age and gender , whereas the level of saturated fats was higher in the first group . 
the data obtained in our spectroscopic study fully confirm the findings of yeung , and even enable improved separation of the various molecular components of the spectrum and increased accuracy of assessing the individual fat peaks . 
this supports the fact that the techniques that benefit the most from the use of a 3 - t magnetic i dati ottenuti dal nostro studio di spettroscopia confermano pienamente i risultati riportati da yeung , ed anzi consentono di separare meglio le varie componenti molecolari dello spettro e di aumentare laccuratezza nella valutazione dei singoli picchi lipidici . 
questo a riprova del fatto che a risentire maggiormente in maniera positiva dellutilizzo di un campo magnetico a 3t dovrebbe essere proprio limaging spettroscopico ( 1h - mrsi ) , la valutazione della diffusione protonica ( dwi ) ed il calcolo del tensore di diffusione ( dti )  . la relazione esistente tra frazione di lipidi midollari e debolezza ossea pu essere spiegata da diversi fattori . 
 [ 9 ] suggested that with increasing age , the increase in bone marrow fats is even more marked than the reduction in red marrow , probably because the yellow marrow tends not only to replace the loss of bone but also the loss of red marrow . 
in fact , it is thought that the quality of bone marrow plays an important role in vertebral mechanical resistance and that an increase in fats can negatively influence this . 
our data show that postmenopausal patients with a t score - 2.5 reveal a consistent increase in ff when compared with healthy postmenopausal subjects . ff values , however , were not significantly different from those of patients with a normal t score of the same gender and age . 
this may mean that both the spectroscopic analysis and the dwi can identify early changes in the microarchitecture of the cancellous bone of the vertebral body , even when the t score is normal , and therefore before the phenomenon can be detected with dexa . currently available imaging techniques such as dexa assess the final structural result of osteoporosis , but they fail to provide information regarding the physiopathology of the disease . 
our study reveals an adc value in postmenopausal patients with a t score - 2.5 significantly lower ( p < 0.01 ) than the premenopausal group , a finding that is in agreement with the latest studies [ 17 ]  . 
 [ 18 ] were significantly different , but they were not used for comparison because the b value used in that study was below 250 mm2 / s . the lower diffusivity of water in the vertebral bodies of older patients compared with younger patients may indicate changes in the composition of bone marrow correlated with age . 
in particolare , dunnill [ 9 ] ha suggerito che , con laumentare dellet , lincremento dei lipidi midollari ancora pi marcato della riduzione del midollo rosso , presumibilmente perch il midollo giallo va a rimpiazzare tanto il midollo rosso quanto la perdita di osso . una quantit superiore di cellule adipose pertanto necessaria per rimpiazzare la perdita di osso trabecolare e riempire gli spazi prima occupati dal midollo rosso . 
i nostri dati hanno mostrato che pazienti in et postmenopausale con tscore - 2 , 5 presentavano un consistente aumento della frazione lipidica ( ff ) se comparati a soggetti sani di pari sesso in et pre - menopausa . i valori di ff non sono stati invece significativamente diversi da quelli di pazienti con t - score normale , ma di pari sesso ed et . 
tale dato stato confermato anche dal calcolo delladc effettuato negli stessi gruppi di soggetti e potrebbe significare che sia lanalisi spettroscopica che limaging di diffusione possono riflettere cambiamenti precoci nella microarchitettura della spongiosa ossea del corpo vertebrale quando ancora il t - score risulta normale e quindi prima che tale fenomeno sia rilevabile con la dexa . le attuali tecniche di imaging come la dexa valutano il risultato strutturale finale dellosteoporosi ma non danno infatti informazioni circa la fisiopatologia . 
questo studio ha evidenziato , nei soggetti in et post - menopausa con t - score - 2 , 5 , un valore di adc significativamente pi basso ( p < 0 , 01 ) rispetto al gruppo di soggetti di et pre - menopausa , in accordo con i pi recenti studi presenti in letteratura [ 17 ]  . 
 [ 18 ] sono risultati significativamente diversi , ma non sono stati confrontati perch in tale studio sono stati utilizzati valori di b inferiori a 250 mm2 / s . la minore diffusivit dellacqua nei corpi vertebrali dei soggetti pi anziani comparata con quella dei soggetti pi giovani potrebbe essere un indicatore delle alterazioni nella composizione del midollo osseo correlate allet . 
questi risultati sono in accordo con i dati del nostro studio , e suggeriscono che laumento del grasso midollare associato con losteoporosi potrebbe rendere ragione dei valori delladc significativamente pi bassi . 
the increase in fats within the trabeculae of vertebral bodies may reduce extracellular diffusion and lead to a reduction in adc values . with regard to data provided by dti , the analysis of adc and fa proved to be the most reliable . 
we retrospectively evaluated the results of percutaneous stereotactic vab with 11 - gauge needles performed over a period of 34 months on 228 nonpalpable suspicious breast lesions detectable on mammography only [ breast imaging reporting and data system ( bi - rads ) 3 : 25.9% ; bi - rads 4 : 67.1% ; bi - rads 5 : 7% ]  . 
in cases of discordance , repeat biopsy or surgical excision were recommended ; in cases of benign lesions , we urged a follow - up of at least 6 months and for borderline and malignant lesions a surgical excision . 
we obtained a suitable post - vab mammographic or histological evaluation for 78 benign lesions , 17 borderline lesions and 76 malignant lesions , with one ( 1.3% ) false negative ( fn ) case , two ( 11.8% ) underestimations of borderline lesions , 14 ( 18.4% ) underestimations of malignant lesions and no ( 0% ) false positive cases . 
percutaneous histological vab with an 11 - gauge needle proved to be , as reported in previous studies , a reliable method for diagnosing nonpalpable , mammographically detectable only breast lesions , with an underestimation rate lower than core biopsy and a fn rate similar to that of surgical biopsy , without any significant complications . key words vacuum - assisted breast biopsy breast neoplasms mammography riassunto obiettivo . 
sono stati valutati retrospettivamente i risultati delle vab eseguite sotto guida stereotassica , con ago da 11 g , nellarco di 34 mesi in corrispondenza di 228 lesioni mammarie infracliniche sospette , visibili solo mammograficamente ( bi - rads 3 : 25 , 9% ; bi - rads 4 : 67 , 1% ; birads 5 : 7% )  . 
si consigliava , per le discordanze , re - biopsia o biopsia chirurgica , per le lesioni benigne follow - up mammografico di almeno 6 mesi , per le lesioni borderline e le maligne lintervento chirurgico . 
alla vab sono state diagnosticate 123 ( 54% ) lesioni benigne ( con 6 casi di discordanza imaging - istololgia ) , 26 ( 11 , 4% ) lesioni borderline , 79 ( 34 , 6% ) lesioni maligne . 
 disponibile un adeguato riscontro mammografico o istologico successivo alla vab per 78 lesioni benigne , 17 borderline , 76 maligne , con : 1 ( 1 , 3% ) falso negativo , 2 ( 11 , 8% ) sottostime di lesioni borderline , 14 ( 18 , 4% ) sottostime di lesioni maligne , nessun ( 0% ) falso positivo . 
la vab istologica percutanea con ago da 11 g si dimostrata , in accordo con la letteratura , metodica affidabile nella diagnosi delle lesioni mammarie infracliniche visibili solo mammograficamente , con tasso di sottostima inferiore alla core biopsy e percentuale di falsi negativi sovrapponibile alla biopsia chirurgica su repere , in assenza di significative complicanze . parole chiave biopsia mammaria vacuum - assisted neoplasie mammarie mammografia c . 
surgical biopsy is considered the gold standard for this evaluation , but it is expensive , invasive , may cause intense psychological stress for the patient and can be often responsible for deep and superficial scars , possibly impairing later imaging examinations . 
the aim was to overcome the known limits of fine - needle - aspiration biopsy ( fnab ) , such as interobserver variability , the high percentage of inadequate tissue samples , the low reproducibility of results and an inability in malignant lesions to differentiate between in situ and invasive carcinomas [ 1 , 2 ]  . 
percutaneous biopsy has overcome these problems by almost constantly providing with 14gauge needles sufficient material for histological diagnosis , with results totally comparable to those of surgical excision ( sensitivity 85%97% in different reports ) and furnishing , with good accuracy , information about tumour invasiveness [ 36 ]  . 
the first experiences [ 9 , 10 ] with core biopsy used sonographic guidance , but subsequently core biopsy was applied to nonpalpable lesions detectable only by mammography [ 11 ]  . 
the vab device uses vacuum suction to pull breast tissue through the aperture of the needle while a cutter rotates at high speed , thus retrieving core specimens and acquiring multiple tissue samples with a single insertion of the probe . 
the aim of this study was to evaluate our experience with vab , focusing on its accuracy and clinical relevance in patient management decisions . materials and methods between march 2003 and december 2005 , 224 consecutive women with a total of 228 lesions underwent stereotactic vab . 
all patients had previously been studied by mammography with standard mediolateral - oblique ( mlo ) and craniocaudal ( cc ) views , supported by further views ( magnification or particular views on suspicious finding ) when necessary ( 69% of cases )  . 
mammographic findings were the folla diffusione della mammografia quale metodica di screening per il carcinoma della mammella , in grado di ridurre la mortalit per tale patologia , ha comportato un riscontro sempre pi frequente di lesioni di piccole dimensioni che richiedono , per una precisa caratterizzazione , una valutazione anatomopatologica . 
per tale inquadramento la biopsia chirurgica rappresenta sicuramente il gold standard , ma gravata da costi elevati , da una discreta invasivit e , inoltre , comporta uno stress emotivo per la paziente e pu causare frequentemente cicatrici superficiali e profonde che , a volte , compromettono la successiva valutazione strumentale . 
la necessit era quella di superare i limiti della citologia , quali la dipendenza dei risultati dallesperienza specifica degli operatori , lelevata percentuale di prelievi inadeguati , la scarsa riproducibilit e limpossibilit di diagnosi differenziale , nel caso di lesioni maligne , tra forme in situ ed infiltranti [ 1 , 2 ]  . 
il prelievo percutaneo per esame istologico superava queste difficolt , essendo in grado di fornire , nella pressoch totalit dei casi , grazie allimpiego di aghi da 14 gauge , materiale sufficiente per la diagnosi , con risultati paragonabili a quelli della biopsia chirurgica ( sensibilit 85%97% nelle diverse casistiche ) e permettendo di porre , con buona accuratezza , diagnosi di lesione in situ o infiltrante [ 36 ]  . 
sul campione istologico della biopsia percutanea inoltre possibile effettuare le valutazioni immunoistochimiche per definire , in fase preoperatoria , il profilo biologico della neoplasia e , in particolare , lo stato dei recettori ormonali [ 7 , 8 ]  . 
le prime applicazioni di tali prelievi [ 9 , 10 ] riguardarono lesioni visibili allecografia ; successivamente la tecnica venne applicata anche alle lesioni infracliniche visibili solo mammograficamente [ 11 ]  . in questo specifico settore i risultati non furono altrettanto entusiasmanti , tanto che la biopsia chirurgica su repere rimase per lungo tempo la procedura di riferimento [ 12 ]  . per tale motivo , nel 1995 venne proposta una particolare tecnica di prelievo istologico percutaneo , chiamata vacuum - assisted ( vab )  . 
tale modalit prevede linserimento dellago una sola volta e consente di prelevare successivamente numerosi frustoli tissutali , tranciando il tessuto con un mandrino tagliente e rotante e , contemporaneamente , creando una forte aspirazione . 
patients with lesions classed as bi - rads 4 and 5 underwent vab because of diagnostic suspicion , whereas those with bi - rads 3 lesions underwent vab for the following reasons : personal history of previous or synchronous cancer of the same or the contralateral breast ( 29 cases ) , scheduled liver transplantation ( one case ) , family history of breast carcinoma ( 24 cases ) and patient anxiety ( 5 cases )  . prior to biopsy , a coagulation history was obtained , and any coagulation treatment , if possible , was interrupted 48 h before and 24 h after the procedure ; any intolerance to local anaesthetics was excluded . 
we used a digital device ( giotto , ims srl , bologna , italy ) , and mammobed , with a directional vab probe ( mammotome , ethicon endo - surgery , johnson & johnson ) and an 11 - gauge automated needle . 
in cases of microcalcifications , specimen radiographs were obtained to document calcification retrieval . we obtained 12 specimens per lesion in 214 ( 93.8% ) patients and 18 in 11 ( 4.8% ) patients , due to the unsuccessful retrieval of calcifications in the first specimen radiographs . 
in two cases ( 0.9% ) , tissue sampling was interrupted ( nine and eight specimens ) because of abundant bleeding and vasovagal reaction , respectively ; in another case ( 0.4% ) , we only harvested three specimens because of patient discomfort , necessitating discontinuation of the procedure . 
in small lesions completely removed by vab , a nonmagnetic marker clip was deployed at the biopsy site to facilitate preoperative localisation in patients who would require additional surgery . histopathologic results were correlated with mammographic findings by both the radiologist and the pathologist in all cases . 
in cases of discordance , repeat biopsy or surgical excision was ordered ; patients with benign lesions were scheduled for 6 - month follow - up , in agreement with literature , whereas surgical excision was recommended for borderline or malignant lesions [ 1820 ]  . within the study period , 245 consecutive women were referred to us to under stereotactic vab , but only 224 with 228 lesions underwent vab . 
for 15 patients , the diagnostic indication was considered incorrect : in six cases , the diagnostic problem was solved through magnification or parresults materiali e metodi dal marzo 2003 al dicembre 2005 sono state sottoposte a biopsia percutanea vacuum assisted condotta sotto guida stereotassica 224 pazienti , per un totale di 228 lesioni mammarie infracliniche sospette , visibili solo mammograficamente . 
tutte le pazienti avevano eseguito una mammografia nelle due proiezioni cranio - caudale e obliqua e , quando necessario , ulteriori proiezioni e / o ingrandimenti mirati ( 69% dei casi ) sul reperto sospetto . 
la tipologia delle lesioni era cos distribuita : microcalcificazioni 194 ( 85% ) , opacit con microcalcificazioni 20 ( 8 , 8% ) , opacit 12 ( 5 , 3% ) , aree di distorsione architetturale 2 ( 0 , 9% )  . 
il diametro massimo delle lesioni era compreso tra 3 e 60 mm , con un diametro medio di 9 , 7 mm ; 176 ( 77 , 2% ) lesioni erano di dimensioni inferiori o uguali ai 10 mm , 29 ( 12 , 7% ) comprese tra 10 e 20 mm , 23 ( 10 , 1% ) superiori ai 20 mle lesioni di maggiori dimensioni corrispondevano a estesi focolai di microcalcificazioni . le lesioni venivano preliminarmente valutate , relativamente al grado di sospetto , in accordo con la classificazione bi - rads ( breast imaging reporting and data system ) dellamerican college of radiology [ 16 ]  . 
lindicazione al prelievo percutaneo mediante vab era implicita nel gruppo delle lesioni bi - rads 4 ( sospette ) e 5 ( maligne ) , mentre nel gruppo bi - rads 3 era riconducibile a problematiche particolari e , precisamente : in circa met delle pazienti ( 29 casi ) si registrava una anamnesi personale di neoplasia della mammella ( con un tumore sincrono , o precedentemente diagnosticato , nella stessa mammella o in quella controlaterale ) ; nei restanti casi , 1 paziente doveva sottoporsi ad un trapianto di fegato , 24 avevano una storia familiare di tumore della mammella e 5 avevano richiesto espressamente di essere sottoposte a biopsia , piuttosto che effettuare un followup a sei mesi . al momento in cui veniva comunicato lappuntamento per la biopsia a tutte le pazienti veniva fatta una breve anamnesi , tesa innanzitutto a stabilire se esse fossero affette da patologie della coagulazione o se assumessero terapie anticoagulanti e , qualora possibile , queste venivano sospese 48 ore prima e fino a 24 ore dopo la procedura ; venivano raccolte informazioni relativamente ad eventuali intolleranze agli anestetici locali . 
stato utilizzato un mammografo digitale giotto ( ims srl , bologna , italia ) abbinato a un letto mammobed delle stessa ditta , con dispositivo di prelievo vab ( mammotome ethicon endo - surgery , inc . johnson & johnson company )  . 
in tutti i casi sottoposti a biopsia per la presenza di microcalcificazioni , veniva effettuata la radiografia dei frustoli , per verificarne la presenza . il numero di prelievi eseguiti era in genere di 12 ; in particolare , in 214 casi ( 93 , 8% ) sono stati ottenuti 12 frustoli ; in 11 casi ( 4 , 8% ) sono stati ottenuti 18 frustoli in seguito al mancato rilievo di microcalcificazioni visibili radiologicac . 
vab could not be performed in the other six patients because of too superficial lesion location ( four cases ) or insufficient mammary thickness due to small breasts ( two cases )  . 
in six cases [ four ( 3.3% ) with a diagnosis of fat tissue , one ( 0.8% ) with normal breast tissue and one ( 0.8% ) with fcm ] , the histological results were in discordance with radiological findings . thus , two patients had repeat biopsies , which provided a diagnosis of fcm in one case but definitive diagnosis is not available in the second , as that biopsy was performed elsewhere . 
for the patient with a diagnosis of normal glandular tissue , due to the low grade of radiological suspicion ( bi - rads 3 ) , we chose radiological follow - up , at which the lesion remained unchanged . 
in the last case , surgical biopsy was performed because of radiologicalhistomente nei primi prelievi ; in 2 casi ( 0 , 9% ) sono stati ottenuti rispettivamente 9 e 8 frustoli , dal momento che la procedura stata sospesa per linsorgenza di un abbondante sanguinamento e per una reazione vaso - vagale ; in un ultimo caso ( 0 , 4% ) , in cui linterruzione della procedura stata determinata dallinsorgenza di una sintomatologia dolorosa , abbiamo prelevato 3 frustoli . 
nei casi di piccole lesioni , estesamente asportate dalla biopsia , veniva posizionata una clip metallica a - magnetica nella sede del prelievo , per consentire leventuale reperaggio della lesione in caso di successiva necessit di asportazione chirurgica . in tutti i casi stata valutata in riunione congiunta , radiologo - patologo , la congruit tra reperto anatomo - patologico e imaging . 
nei casi discordanti stata consigliata la rebiopsia , oppure la biopsia chirurgica , nelle lesioni benigne veniva consigliata la mammografia di controllo a 6 mesi , come raccomandato dalla letteratura ; lintervento chirurgico stato consigliato in tutte le lesioni maligne e borderline [ 1820 ]  . risultati nellarco di tempo considerato sono pervenute alla nostra osservazione , con indicazione alla biopsia stereotassica , 245 pazienti : 224 / 245 di esse sono state sottoposte a biopsia percutanea vacuum - assisted , per un totale di 228 lesioni . non sono state sottoposte alla procedura 15 pazienti in quanto lindicazione allesame non era corretta . 
in particolare in 6 casi il dubbio diagnostico stato risolto con lesecuzione di radiogrammi mirati e / o altre proiezioni mammografiche ( 3 casi ) , ecografia ( 2 casi ) e risonanza magnetica ( 1 caso ) ; nei restanti 9 casi stato possibile eseguire la c . 
non stato possibile procedere alla biopsia in altre 6 pazienti in quanto la lesione era localizzata vicino alla superficie cutanea ( 4 casi ) o nel contesto di mammelle di piccole dimensioni ( 2 casi )  . 
tutte le biopsie hanno fornito materiale adeguato per la diagnosi ( 100% )  . i risultati anatomopatologici sono stati i seguenti : lesioni benigne 123 ( 54% ) , lesioni borderline 26 ( 11 , 4% ) ; lesioni maligne 79 ( 34 , 6% ) , di cui 57 ( 72% ) carcinomi in situ e 22 ( 28% ) carcinomi infiltranti . 
il risultato istologico era in disaccordo con il quadro radiologico in 6 casi [ 4 ( 3 , 3% ) con diagnosi di tessuto adiposo , 1 caso ( 0 , 8% ) di tessuto ghiandolare normale e 1 caso di mfc ]  . 
nella paziente con diagnosi di tessuto ghiandolare normale , in relazione al basso grado di sospetto radiologico ( bi - rads 3 ) , si optato per un follow - up , che ha confermato la stabilit del quadro . 
in una paziente stata consigliata la biopsia chirurgica per una discrepanza tra quadro radiologico e istologico ; si trattava di un cluster di microcalcificazioni , classificato come bi - rads 4 , risultato alla vab una mfc e , alla successiva chirurgia , una iperplasia papillare epiteliale tipica . altre 2 pazienti con lesioni benigne alla biopsia percutanea sono state sottoposte a biopsia chirurgica per i seguenti motivi : una paziente stata sottoposta a quart per un carcinoma duttale infiltrante con componente in situ nello stesso quadrante e laltra paziente , che presentava una diagnosi di adenoma tubulare , stata sottoposta , per suo espresso desiderio , a escissione chirurgica . 
in 21 casi ( 29% ) disponibile un follow - up di almeno 24 mesi , considerato ottimale per confermare la benignit della lesione . sono disponibili i dati relativi a 78 lesioni benigne , con il riscontro di 1 falso negativo e 77 veri negativi . 
le lesioni borderline ( tabella 2 ) erano 26 ( 11 , 4% ) , cos distribuite : noduli scleroelastotici 8 ( 30 , 8% ) , carcinomi lobulari in situ ( clis ) 7 ( 26 , 9% ) , iperplasie lobulari atipiche ( ila ) 5 ( 19 , 2% ) , lesioni papillari 4 ( 15 , 4% ) , iperplasia duttale atipica ( ida ) 1 ( 3 , 8 % ) ; iperplasia colonnare atipica con ida 1 ( 3 , 8% )  . 
in the central , retroareolar region of the right breast , linear microcalcifications over an area of architectural distortion ( similar to a radial scar ) , with an extension of 25 mm , are shown [ breast imaging reporting and data system ( bi - rads ) 4 ]  . 
nei settori centrali retroareolari della mammella destra si apprezzano minute calcificazioni lineari sottese ad unarea di convergenza architetturale ( tipo radial scar ) , con unestensione complessiva di circa 25 mm ( bi - rads 4 )  . 
biopsia chirurgica , eseguita sei mesi dopo in relazione allincremento delle microcalcificazioni e per discordanza imaging - istologia : diagnosi istologica di cdis . logical discordance ( a cluster of microcalcifications classified as bi - rads 4 proved to be fibrocystic mastopathy at vab and usual epithelial , papillary hyperplasia at subsequent surgical biopsy )  . two more patients with benign lesions underwent surgical excision . 
in one case , at 6 - months follow - up , ultrasonography depicted a new lesion , which was located in the same quadrant as the initial finding but had no relation with it . 
in the literature , this approach is considered mandatory in the case of a percutaneous diagnosis of borderline lesions [ 2026 ]  . fifteen patients underwent surgical excision ( one was treated elsewhere , so data about her final diagnosis were lost ) , whereas 11 refused surgery and preferred to undergo followup . 
in particular , three patients have been followed up for at least 6 months ( 15 , 29 and 30 months , respectively ) , whereas six patients have been followed up only for a short period ( just recently biopsied ) ; data about the remaining two patients are not available . 
of the 14 patients who underwent surgical biopsy , percutaneous diagnosis was confirmed in 12 ( 85.7% ) , whereas two ( 14.3% ) patients were found to have a carcinoma . 
 histological diagnoses of the 79 lesions identified as malignant at vab were ( table 3 ) : 54 ( 68.3% ) dcis , three ( 3.8% ) dcis with microinfiltration , eight ( 10.1% ) invasive ductal carcinoma ( dci ) with dcis , five ( 6.3% ) dci , two ( 2.5% ) lci , one ( 1.3% ) invasive ductal - lobular carcinoma ( dlci ) , one ( 1.3% ) dlci with dcis , two ( 2.5% ) dcis in a papillary lesion , one ( 1.3% ) dci with lcis , one ( 1.3% ) dlci with lcis and one ( 1.3% ) papillary carcinoma in situ . quindici pazienti hanno effettuato la biopsia chirurgica ( una in altra sede senza riscontro istologico ) , 11 pazienti hanno rifiutato lintervento e optato per un follow - up . 
3 pazienti , in particolare , sono in follow - up da pi di 6 mesi ( una da 15 mesi , una da 29 mesi e una da 30 mesi ) mentre in altre 6 non c un sufficiente follow - up ( la biopsia ancora recente ) ; in 2 casi i dati non sono disponibili . 
si pertanto avuta una variazione diagnostica in 2 / 7 casi di clis ( 28 , 6% )  . nei 79 casi diagnosticati come lesioni maligne alla biopsia percutanea ( tabella 3 ) , le diagnosi erano cos distribuite : carcinoma duttale in situ ( cdis ) 54 ( 68 , 3% ) , cdis con microinvasione 3 ( 3 , 8% ) , carcinoma duttale infiltrante ( cdi ) con cdis 8 ( 10 , 1% ) , cdi 5 ( 6 , 3% ) , carcinoma lobulare infiltrante ( cli ) 2 ( 2 , 5% ) , carcinoma duttulo - lobulare infiltrante ( cdli ) 1 ( 1 , 3% ) , cdli con cdis 1 ( 1 , 3% ) , sospetta lesione papillare con carcinoma in situ 2 ( 2 , 5% ) , cdi con clis 1 ( 1 , 3% ) , cdli con clis 1 ( 1 , 3% ) , carcinoma papillare in situ 1 ( 1 , 3% )  . 
in 76 / 79 pazienti stato effettuato lintervento chirurgico ed disponibile la diagnosi definitiva , 3 / 79 sono state operate in altra sede e di esse non abbiamo a disposizione i dati . 
in 5 casi la lesione maligna era cos piccola da non consentire il reperimento allintervento chirurgico : si trattava di 2 casi di lesione papillare con cdis in cui allintervento stata trovata solo la lesione c . 
in tutti questi casi si trattava di lesioni inferiori ai 5 mcomplessivamente , quindi , abbiamo informazioni attendibili sul risultato istologico dellintervento chirurgico in 76 ( 96 , 2% ) lesioni maligne , 14 ( 53 , 8% ) borderline e 5 ( 4% ) benigne , sul risultato della re - biopsia in 1 ( 0 , 8% ) lesione benigna , mentre abbiamo un follow - up di almeno 6 mesi per 3 ( 11 , 5% ) lesioni borderline e 72 ( 58 , 5% ) lesioni benigne . in 45 ( 36 , 6% ) lesioni benigne , 9 ( 34 , 6% ) lesioni borderline e 3 ( 3 , 8% ) lesioni maligne non stato possibile recuperare informazioni sicure : frequentemente , infatti , le pazienti venivano inviate presso il nostro istituto per lesecuzione della biopsia e ritornavano alla sede di provenienza per il follow - up . complessivamente , abbiamo potuto valutare i dati di 78 / 123 lesioni benigne , 17 / 26 lesioni borderline e 76 / 79 lesioni maligne ( per un totale di 171 / 228 lesioni ) con i seguenti risultati : considerando borderline come negativi : 92 veri negativi ( vn ) , 3 falsi negativi ( fn ) , 76 veri positivi ( vp ) , 0 falsi positivi ( fp ) , sensibilit 96 , 2% , specificit 100% , valore predittivo negativo ( vpn ) 96 , 8% e valore predittivo positivo ( vpp ) 100% ; considerando borderline come positivi : 77 vn , 1 fn , 78 vp , 15 fp , sensibilit 98 , 7% , specificit 83 , 7% , vpn 98 , 7% e vpp 83 , 9% . per quanto riguarda lincidenza delle neoplasie maligne rispetto alla diagnosi radiologica espressa con i criteri bifig . 
a small cluster ( 10 mm ) of fine , granular microcalcifications with duct - like distribution are shown in the inferior periareolar region [ breast imaging reporting and data system ( bi - rads ) 4 ]  . 
in sede para - areolare inferiore sinistra riconoscibile un piccolo focolaio di tenui microcalcificazioni granulari con decorso simil - duttale , esteso su unarea di circa 10 mm ( birads 4 )  . 
3 vacuum - assisted biopsy ( vab ) surgical excision concordance . granular and heterogeneous ( with regard to morphology , dimensions and density ) microcalcifications , involving an area of 7 mm , are shown in upper quadrants of the left breast [ breast imaging reporting and data system ( birads ) 4 ]  . 
al passaggio tra i quadranti superiori della mammella sinistra sono riconoscibili alcune microcalcificazioni granulari , polimorfe per morfologia , dimensioni e densit , coinvolgenti unarea di circa 7 mm ( bi - rads 4 )  . 
two clusters of granular microcalcifications with segmental distribution towards the nipple are shown in the external periareolar region of the right breast . the deeper ones are projected on a small , ill - defined density [ breast imaging reporting and data system ( bi - rads ) 5 ]  . 
in sede para - areolare esterna destra si riconoscono due focolai di microcalcificazioni granulari a distribuzione segmentale dirette verso la regione del capezzolo ; in particolare le microcalcificazioni localizzate nei settori pi profondi si proiettano su di una piccola opacit a margini sfumati e mal definiti ( bi - rads 5 )  . 
biopsia chirurgica : diagnosi istologica di cdi con cdis . pathology results on the surgical specimen were available for 76 of 79 patients , whereas three patients underwent surgical intervention elsewhere . 
five very small malignancies were not retrieved in the surgical specimen : in two cases of papillary lesions with dcis , only papillary lesions were identified ; in two cases of dcis , histological evaluation of the surgical specimen revealed fcm and alh in one case and fcm and epitheliosis in the second . 
non ci sono stati casi di episodi infettivi n di ematomi che richiedessero lintervento terapeutico : gli ematomi evidenziati presentavano sempre dimensioni inferiori ai 4 cm ( in due casi il sanguinamento prolungato ha richiesto losservazione in ospedale per qualche ora )  . 
in all these cases , lesions had a diameter < 5 min summary , data about surgical excision were available for 76 ( 96.2% ) malignant lesions , 14 ( 53.8% ) borderline lesions and five ( 4% ) benign lesions ; about repeat biopsy for one ( 0.8% ) benign lesion ; whereas three ( 11.5% ) borderline and 72 ( 58.5% ) benign lesions have been followed up for at least 6 months . 
no cases of relevant scarring was observed on follow - up mammograms . discussion histological percutaneous biopsy for pathologic diagnosis of breast lesions core biopsy represents an effective alternative to surgical biopsy , which is an invasive and expensive method that may cause postsurgical scars , which can impair following diagnostic evaluation and be responsible , even though rarely , for fn results . 
compared with cytology , the complexity and costs of core biopsy are higher , but its inadequate sampling rate and fn rate are significantly lower ( less than 1%2% ) [ 14 , 19 , 23 ]  . 
its sensitivity , with 14 - gauge probes and 17 - mm tissue acquisition aperture , ranges from 85% to 97% , and its specificity is 100% according to different studies [ 27 ]  . 
moreover , its performance is similar to that of surgical biopsy , so that it can be used for decision making in patient management : follow - up for benign lesions , surgery for borderline and malignant lesions , with the possibility in the latter case of shortening the number of surgical steps compared with lesions diagnosed by surgical biopsy [ 20 , 2830 ]  . 
 discussione la biopsia istologica percutanea per la diagnosi anatomopatologica delle lesioni mammarie , detta anche core biopsy , rappresenta una valida alternativa alla biopsia chirurgica , indagine invasiva , costosa , che pu comportare cicatrici post - operatorie in grado di limitare le possibilit diagnostiche e che non scevra da una percentuale , seppur limitata , di falsi negativi . 
la complessit ed i costi della biopsia istologica percutanea sono superiori a quelli della citologia ma , rispetto a questa , la percentuale di inadeguati e falsi negativi nettamente inferiore ( meno dell1%2% ) [ 14 , 19 , 23 ]  . la sensibilit del metodo , utilizzando aghi da 14 g con finestra di prelievo di 17 mm , varia nelle diverse casistiche tra l85% ed il 97% e la specificit del 100% [ 27 ]  . 
i risultati ottenibili con biopsia istologica percutanea , sono del tutto sovrapponibili a quelli della biopsia chirurgica ed essa pu essere pertanto utilizzata per decidere il management della paziente : follow - up nel caso delle lesioni benigne , intervento chirurgico nel caso delle lesioni borderline e delle maligne , con possibilit in questultimo caso di ridurre il numero dei tempi chirurgici rispetto alle lesioni diagnosticate con biopsia chirurgica [ 20 , 2830 ]  . tuttavia , nel caso delle lesioni di piccole dimensioni , in particolare quelle visibili solo mammograficamente , quali focolai di microcalcificazioni , piccole opacit e aree di distorsione parenchimale , e quindi nelle lesioni da biopsiare sotto guida stereotassica , i risultati sono inferiori e la probabilit che la core biopsy consenta di evitare la biopsia chirurgica diagnostica minore [ 31 ]  . 
per cercare di superare tali difficolt e migliorare i risultati , stata introdotta la biopsia percutanea istologica con dispositivi vacuum - assisted che , essendo dotata della possibilit di aspirare , in grado di ridurre la contaminazione ematica della sede di biopsia e , soprattutto , aspirando il tessuto nella finestra di prelievo dellago , consente di ottenere , a parit di calibro , frustoli di maggiore volume ( circa 3 volte ) [ 32 , 33 ]  . 
questo metodo utile , in particolare , nel caso di lesioni di piccole dimensioni , nelle quali lago pu , a volte , non essere posizionato esattamente allinterno della lesione , ma in sua prossimit , per cui risulta particolarmente vantaggioso il meccanismo di aspirazione , che consente di richiamare verso lago , e conseguentemente prelevare , anche il tessuto adiacente . 
questo metodo inoltre efficace in tutti i casi di lesioni borderline e in situ , nei quali lanatomopatologo richiede una elevata quantit di tessuto per porre una diagnosi corretta . laccuratezza diagnostica di tale metodica , riportata dai dati della letteratura , del tutto confrontabile con i dati della biopsia chirurgica su repere , senza peraltro essere gravata da possibili problematiche , quali la dislocazione del repere , che riducono la sensibilit della biopsia chirurgica nelle lesioni infracliniche . 
un significativo miglioramento viene ottenuto anche nelle cosiddette lesioni borderline , anche se in queste ultime persiste , sebbene meno frequentemente , il rischio di sottostima istologica , il che non consente di evitare lintervento chirurgico [ 19 ]  . il sistema vab per il prelievo sotto guida stereotassica pi complesso e costoso di quelli utilizzati per i prelievi core biopsy sotto guida ecografica e richiede , preferibilmente , una unit per biopsia dotata di un tavolo per lesecuzione c . 
 ( % ) tabella 4 frequenza di lesioni maligne per classe breast imaging reporting and data system ( bi - rads ) categoria bi - rads lesioni totali n ( % ) lesioni maligne n ( % ) 59 ( 25.9 ) 153 ( 67.1 ) 16 ( 7 ) 59 ( 25 , 9 ) 153 ( 67 , 1 ) 16 ( 7 , 1 ) 7 ( 11.9 ) 56 ( 36.6 ) 16 ( 100 ) 7 ( 11 , 9 ) 56 ( 36 , 6 ) 16 ( 100 , 0 ) bi - rads 3 bi - rads 4 bi - rads 5 bi - rads 3 bi - rads 4 bi - rads 5 nonetheless , for small lesions and particularly for those detectable at mammography only such as microcalcification clusters , small opacities and areas of parenchymal distortion , which require biopsy under stereotactic guidance the results are poorer and the possibility of avoiding diagnostic surgical biopsy using core biopsy is lower [ 31 ]  . 
these devices , thanks to blood suctioning , can reduce hematic contamination of the sampled area and , most of all , aspirate tissue into the needle acquisition aperture , allow retrieval of larger samples ( about three times as much ) , with the needle calibre being equal [ 32 , 33 ]  . 
this is particularly useful in the case of small lesions for which the needle may be positioned not exactly within the lesion but just close to it : because of the use of vacuum , tissue can be acquired at a distance from the probe , not just within the line of fire . 
this method is also very advantageous in those scenarios , such as borderline and in situ lesions , in which pathologists need a large volume of tissue to make a correct diagnosis . the reported diagnostic accuracy of this technique is very similar to that of surgical biopsy with a localising wire , and vab is free of risks such as wire displacement that reduce the sensitivity of open surgery in nonpalpable breast lesions . a substantial improvement has also been achieved for borderline lesions , even though in these cases histological underestimation cannot be avoided and surgery excision remains mandatory [ 19 ]  . a stereotactically guided vab system is more complex and expensive than sonographically guided core biopsy and should be performed with a prone stereotactic table . 
advantages of the prone table include more working room and decreased likelihood of patient motion and vasovagal reaction ; besides , this design offers the technologist and physician a larger area in which to work and to correctly perform the procedure . 
add - on units attachable to a common mammography unit are very inexpensive but do not isolate the patient , who is more likely to move or feel discomfort as a redella biopsia con paziente prona . 
in tal modo la paziente non vede direttamente la procedura ed raro che si muova o che presenti reazioni vaso - vagali ; il tecnico ed il medico , inoltre , hanno pi spazio per lavorare e quindi effettuare correttamente la procedura . 
le cosidette add - on units , dispositivi applicati ad un comune mammografo , hanno il vantaggio di comportare costi minori ma lo svantaggio di non isolare dalla procedura la paziente cosicch questa , essendo maggiormente coinvolta , pu pi facilmente muoversi o accusare disagio . 
gli aghi utilizzati inizialmente erano da 14 g , successivamente sono stati introdotti quelli da 11 g e 8 g ; attualmente vengono considerati ottimali aghi da 11 g [ 20 , 32 , 34 ]  . la procedura prevede lidentificazione della lesione da sottoporre e biopsia sui tre radiogrammi standard ( craniocaudale , obliquo medio - laterale e medio - laterale , questultimo utile per meglio precisare la posizione della lesione in senso cranio - caudale ) ; successivamente vengono effettuati i radiogrammi di dettaglio sulla mammella compressa , a paziente posizionata sul lettino ; dopo aver identificato le coordinate x e y , vengono eseguite 2 proiezioni inclinate a + 15 e - 15 per consentire il calcolo della profondit ( z )  . 
successivamente , in anestesia locale , si esegue una piccola incisione cutanea con la lama di un bisturi , onde poter entrare nel tessuto mammario con lago , che viene poi indirizzato sul bersaglio identificato . 
in seguito , senza che lago venga pi spostato , si procede al prelievo dei frustoli che , secondo alcuni autori , devono essere in numero superiore o uguale a 20 , mentre , secondo altri , anche in numero di 12 consentono analoghi risultati [ 35 , 36 ]  . 
riteniamo che sia eccessivo ricorrere ad un numero superiore di frustoli , a meno che non sussistano indicazioni particolari , quali la mancata dimostrazione radiografica di microcalcificazioni nei frustoli [ 37 ] ( che ci ha indotto a raggiungere il numero di 18 frustoli in 11 4 , 8% casi ) o la presenza di un focolaio di microcalcificazioni particolarmente esteso . 
needles initially used were 14 gauge , then 11 - gauge and 8 - gauge needles were introduced ; at the moment , 11 - gauge probes seem to be the best choice [ 20 , 32 , 34 ]  . the first step of the procedure is lesion localisation in three standard view mammograms ( craniocaudal , mediolateraloblique and mediolateral , with the latter being useful to localise the lesion in craniocaudal direction )  . 
the highest precision in performing the procedure is needed to avoid sampling errors . after the local anaesthetic has been injected , a small skin incision is made using a bistoury , the probe is inserted , and the needle is then fired at the targeted site . 
according to some authors , at least 20 specimens are needed ; according to others , 12 samples are enough to obtain the same accuracy [ 35 , 36 ]  . 
in fact , in our opinion , a higher number of specimens is necessary in special cases only , for example , when calcifications are not demonstrated on specimen radiographs [ 37 ]  . 
the frequency of malignant lesions according to bi - rads categories was 100% in birads 5 ( 16 / 16 lesions ) , which is in agreement with published data ( 81%97% )  . 
of 153 lesions classified as birads 4 , 56 ( 36.6% ) were malignant , showing a slightly higher rate than published data , which report a proportion of 23%25% [ 28 , 38 , 39 ]  . 
finally , seven ( 11.9% ) of the 59 lesions classified as bi - rads 3 proved to be malignant , higher proportion than previously reported ( less than 2% ) [ 16 , 28 , 40 ]  . 
despite our limited sample size , it can be stated that our data , apparently in disagreement with those reported in the literature , are affected , as previously reported [ 40 , 41 ] , by difficulties in bi - rads category interpretation for microcalcifications , which suffers from subjectivity due to the lack of objective reference parameters . 
this leads to an underestimation of microcalcifications , with an incorrect tendency to classify bi - rads 3 lesions that should be included in the bi - rads 4 category . of the 123 lesion with a vab diagnosis of benignity , 78 had a follow - up of at least 6 months or a repeat biopsy or riale legata alla maggiore probabilit di identificare aree di infiltrazione neoplastica . 
in 2 ( 0 , 9% ) casi il numero contenuto a 89 prelievi stato condizionato da una reazione vaso - vagale e dallinsorgenza di unemorragia abbondante ; in 1 ( 0 , 4% ) caso la procedura stata interrotta dopo 3 prelievi per il sopraggiungere di una intensa sintomatologia dolorosa . 57 / 79 ( 72% ) lesioni maligne identificate alla biopsia percutanea corrispondevano a lesioni non invasive . 
la maggior parte delle lesioni della casistica personale era di piccole dimensioni , in particolare 176 / 228 ( 77 , 2% ) misuravano meno di 10 mm , in 194 / 228 ( 85% ) casi corrispondevano a focolai di microcalcificazioni . 
la percentuale di lesioni maligne per categoria bi - rads risultata del 100% nella categoria bi - rads 5 ( 16 / 16 lesioni maligne ) , risultato che si accorda con i dati della letteratura ( range compreso tra 81% e 97% )  . 
nella categoria bi - rads 4 sono state incluse 56 lesioni maligne su un totale di 153 lesioni , pari al 36 , 6% , con un tasso di malignit leggermente superiore rispetto ai dati della letteratura , che riportano percentuali del 23%25% [ 28 , 38 , 39 ]  . 
sette / 59 lesioni classificate come bi - rads 3 sono risultate maligne , con una percentuale dell11 , 9% , pi elevata rispetto a quella riportata in letteratura ( inferiore al 2% ) [ 16 , 28 , 40 ]  . 
con i limiti dovuti allesiguit del campione valutato , si pu affermare che i nostri dati , apparentemente in lieve disaccordo con quelli della letteratura , risentono , come gi sottolineato da altri autori [ 40 , 41 ] , della difficolt nellinterpretazione delle categorie bi - rads riguardanti le microcalcificazioni , che rimane spesso gravata da soggettivit per lassenza di parametri oggettivi di riferimento . 
si denoterebbe , quindi , un certo atteggiamento di sottostima dei casi che si presentano mammograficamente come microcalcificazioni , ovvero una erronea tendenza ad inquadrare lesioni appartenenti alla categoria bi - rads 4 in quella inferiore . tra le 123 lesioni diagnosticate benigne alla vab , in 78 si potuto avere un follow - up di almeno 6 mesi , o lesito di una re - biopsia , o di una biopsia chirurgica , che hanno consentito di classificare 77 casi come veri negativi e 1 caso falso negativo . 
dopo la diagnosi istologica su vab di tessuto adiposo , in relazione alla discrepanza radiologico - patologica , si ritenuto opportuno eseguire , in accordo con la paziente poco propensa ad essere sottoposta subito alla biopsia chirurgica , uno stretto follow - up , che , a distanza di 6 mesi , ha dimostrato un incremento delle microcalcificazioni , imponendo lesecuzione della biopsia chirurgica , che ha portato alla diagnosi di cdis . 
questo rilievo conferma come sia indispensabile un adeguato follow - up delle lesioni benigne ai fini di migliorare i risultati complessivi della metodica . relativamente alle 26 lesioni borderline , abbiamo informazioni in 14 / 15 casi sottoposti a biopsia chirurgica e in 3 / 11 in follow - up . 
i dati completi a nostra disposizione si riferiscono perci a 17 pazienti , delle quali la diagnosi stata confermata in 15 casi , mentre in 2 ( entrambi clis ) la bioc . 
after histological diagnosis of fatty tissue on vab , because of imaginghistologic discordance , a strict follow - up was advised ( as the patient refused an immediate repeat biopsy )  . 
complete data at our disposal regard 17 patients : in 15 cases , the diagnosis has been confirmed , whereas in two cases ( both lcis ) , excisional biopsy demonstrated dcis , and dcis with a microfocus of lci , respectively . 
considering fn cases of borderline lesions revealed to be underestimations at open surgery an interpretation that is still under discussion and is wekened by the general indication in these cases of performing surgical excision [ 23 ] we had three fn results , with a proportion ( 3.1% ) that is still in agreement with the published data [ 12 , 43 , 44 ]  . 
on the contrary , this underestimation rate is significantly lower than the rate reported for core biopsy , 20%50% [ 48 , 49 ] , and explains why vab is generally considered better for correctly planning the therapy . 
in two cases of papillary lesions with dcis , only papillary lesions were identified ; in two cases of dcis , histological examinations of the surgical specimens revealed fcm and alh in one case and fcm and epitheliosis in the second ; one case of dcis with suspected microinfiltration was not confirmed by surgery . 
the first four lesions were very small less than 5 mm in diameter a characteristic related to the possibility of even complete lesion removal by vab [ 19 ]  . 
in the last case , we can suppose an artefact due to epithelium displacement , which occurs when the needle , fired into the tissue , displaces ductal cells out of the duct away from the target lesion . 
this displacement can deceive the pathologist , who is , however , aware of this phenomenon , which occurs more frequently with core biopsy devices than with vab [ 19 ]  . 
for these reasons , we can consider these cases not to be fp , so we can state that , in our experience , we did not find any fp or overestimations . psia chirurgica ha reso possibile rispettivamente la diagnosi di cdis e di cdis con un microfocolaio di cli . 
2 / 7 clis diagnosticati alla vab sono risultati sottostime rispetto alla diagnosi su pezzo chirurgico , con una percentuale pari all11 , 8% delle lesioni borderline considerate , e al 28 , 6% nellambito del sottogruppo dei clis , riscontro paragonabile ai dati della letteratura [ 42 ]  . 
se si considerano falsi negativi anche le diagnosi di lesioni proliferative rivelatesi sottostime allescissione chirurgica , opinione peraltro discutibile , data la convinzione generalizzata [ 23 ] della necessit di eseguire un intervento chirurgico in questi casi , in totale si sarebbero verificati 3 casi di falsi negativi , con una percentuale ( 3 , 1% ) che rimarrebbe in accordo con i dati della letteratura [ 12 , 43 , 44 ]  . 
tale percentuale di falsi negativi , particolarmente contenuta , sarebbe sostanzialmente confrontabile anche con i falsi negativi della biopsia chirurgica su repere [ 45 ]  . per quanto riguarda le pazienti con diagnosi di malignit posta alla biopsia percutanea , tutte operate , siamo a conoscenza dei dati relativi allintervento in 76 / 79 ( 96 , 2% ) di esse . 
abbiamo riscontrato una concordanza nella diagnosi in 62 / 76 ( 81 , 6% ) pazienti , mentre nelle rimanenti 14 tutti casi di cdis alla vab stata posta diagnosi di forma infiltrante in 8 casi ( 10 , 5% ) e microinfiltrante in 6 ( 7 , 9% )  . questa variazione stata documentata nel 18 , 4% dei casi , dato che non si discosta in modo significativo da quelli della letteratura ( 5%15% ) [ 19 , 43 , 46 , 47 ]  . 
tale risultato differisce invece in maniera significativa dai valori di sottostima riportati per la core biopsy ( 20%50% ) [ 48 , 49 ] e rende ragione del maggior valore nella pianificazione terapeutica generalmente riconosciuto al prelievo vacuum - assisted . 
si trattava di : due lesioni papillari con associato un focolaio di cdis alla biopsia percutanea , nei quali la chirurgia ha riscontrato solo la lesione papillare ; due casi di cdis alla biopsia percutanea con reperto , su pezzo chirurgico , di solo mfc e ila in un caso e mfc ed epiteliosi nellaltro ; un caso di cdis con sospetta microinfiltrazione alla biopsia percutanea , non confermato alla chirurgia . 
in 4 casi si trattava di lesioni molto piccole , inferiori a 5 mm , per le quali riconosciuta la possibilit , con il prelievo vab , di asportare buona parte e talora anche la totalit della lesione [ 19 ] ; in 1 caso ipotizzabile , pi che la completa asportazione della lesione , la presenza di un artefatto da dislocazione , che consiste nel trascinamento delle cellule duttali al di fuori del dotto da parte dellago posizionato nel tessuto mammario . 
in relazione a tali considerazioni , questi casi possono non essere considerati come falsi positivi e ci ci permette di affermare di non aver riscontrato , nella nostra esperienza , casi falsi positivi , n sovrastime . conclusioni complessivamente i dati della casistica personale , seppur ancora non particolarmente numerosa , ci consentono di afc . 
we defined it as the only fn result , as we did not consider as fn the histological underestimations of borderline lesion ( vab diagnosis ) vs . malignant lesion ( surgical biopsy ) , because surgical excision is in any case planned for all of them [ 20 ]  . 
thus , we can state that this method may replace surgical biopsy for diagnosing nonpalpable lesions detectable by mammography only , allowing good patient management , with an overall reduction of costs and time for final surgery and , most probably , a positive effect on the patients outcome . fermare , in accordo con la letteratura , che la biopsia istologica percutanea vacuum - assisted si dimostrata una metodica notevolmente affidabile nella diagnostica delle piccole lesioni visibili solo mammograficamente , con un unico falso negativo e con una percentuale di sottostima in cdis del 10 , 5% nelle forme infiltranti e del 7 , 9% in quelle microinfiltranti , senza alcun caso di falsa positivit . 
tali risultati , abbinati ad una corretta gestione dei casi benigni ( valutazione della concordanza imaging / istologia nel giudicare ladeguatezza del prelievo e follow - up mammografico a 6 mesi ) , ci hanno consentito di recuperare lunico falso negativo senza significativo ritardo diagnostico , non dovendo considerare clinicamente rilevanti le sottostime istologiche di lesioni proliferative riscontrate con vab vs lesione maligna diagnosticata alla chirurgia , in quanto tali lesioni vengono sottoposte a verifica chirurgica [ 20 ]  . 
ridolfi , via varthema 44 , bologna , italy , tel . : + 39 - 051 - 6235444 , fax : + 39 - 051 - 6235444 , e - mail : marcelloridolfi@yahoo.it * il lavoro spetta in parti uguali agli autori received : 21 march 2006 / accepted : 20 august 2006 / published online : 19 march 2007 abstract purpose . 
thirty - eight patients ( 15 men and 23 women ; mean age 55 years ; age range 3579 ) with neck pain were examined and divided into two groups : ( 1 ) patients already identified as rheumatic and referred for further investigation of the atlantoaxial region ; ( 2 ) patients with symptoms confined to the cervical spine , with inconclusive radiographic findings . unenhanced ct of the cervical spine ( tomoscan sr 7000 philips , eindhoven , netherlands ) was performed in all patients . 
ct demonstrated calcific deposits around the dens in 12 patients ( three men and nine women ) , in the transverse and alar ligaments , and in the anterior atlantooccipital membrane . 
crystals located in the transverse ligament of the atlas give rise to the crowned dens syndrome , usually in patients affected by severe degenerative lesions of the atlantoaxial joint and peripheral chondrocalcinosis . 
i soggetti sono stati suddivisi in due gruppi : ( 1 ) pazienti gi diagnosticati come reumatici , di cui lo specialista richiedeva un ulteriore approfondimento diagnostico della zona atlo - epistrofica ; ( 2 ) pazienti con sintomatologia desordio circoscritta al rachide cervicale , di cui la radiologia convenzionale era risultata poco significativa . 
tutti i pazienti sono stati sottoposti a tc volumetrica della colonna cervicale ( tomoscan sr 7000 philips , eindhoven , olanda ) , senza somministrazione per via endovenosa di mezzo di contrasto . 
lesame tc risultato positivo , per depositi calcifici a livello del dente , in 12 pazienti ( 3 maschi e 9 femmine ) , con calcificazioni a livello del legamento trasverso , di quelli alari e della membrana atlo - occipitale anteriore . 
radiography of other joints ( wrist , knee , pubic symphysis ) may help to ascertain whether the disease is due to calcium pyrophosphate dihydrate or hydroxyapatite crystals , and is therefore recommended for routine patient management . magnetic resonance imaging ( mri ) is indicated for the study of neurological complications . key words crowned dens syndrome cppd crystal deposition disease transverse ligament of the atlas computed tomography localizzazione , generalmente asintomatica ; quando i cristalli hanno sede nel legamento trasverso dellatlante , si realizza la sindrome del dente incoronato , abitualmente in pazienti con lesioni degenerative avanzate dellarticolazione atlo - epistrofica e con evidente condrocalcinosi periferica . 
nella sindrome del dente incoronato la tc rappresenta il gold standard diagnostico , potendo identificare la forma e la sede delle calcificazioni e le eventuali erosioni ossee . lesame radiografico di altri distretti articolari ( carpo , ginocchio , sinfisi pubica , spalla ) pu agevolare il riconoscimento eziologico della malattia , se da cppd o da ha , ed pertanto raccomandato nella gestione routinaria del paziente . 
limpiego della rm indicato nello studio della complicanze neurologiche . parole chiave sindrome del dente incoronato malattia da deposito di cristalli di cppd legamento trasverso dellatlante tomografia computerizzata introduction introduzione the crowned dens syndrome ( cds ) is a clinical - radiological entity characterised by calcification of the ligaments surrounding the odontoid process of the axis and by acute attacks of cervicooccipital pain , with fever , rigidity and general signs of inflammation lasting from days to several weeks [ 1 ]  . 
the crystal deposits , in most cases calcium pyrophosphate dihydrate ( cppd ) but also hydroxyapatite ( ha ) , may remain asymptomatic or cause chronic cervical pain and spinal cord compression [ 2 ]  . the classic presenting triad ( headache , fever and morning cervical pain ) was first described by le goff in 1980 [ 3 , 4 ] , who distinguished these acute attacks from those seen in common osteoarthritis and in crystal diseases of the distal cervical vertebrae or thoracic - lumbar spine [ 5 ]  . 
furthermore , atypical cases of cds can simulate both the painful cervicobrachial syndrome ( with shoulder weakness and stiffness ) , as well as occipitotemporal headache ( which mimics atypical rheumatic polymyalgia and / or giant cell arteritis )  . 
according to available data [ 710 ] , computed tomography ( ct ) imaging at the c1c2 level is still the reference standard and is superior to magnetic resonance imaging ( mri ) in identifying these calcifications , especially when they are small [ 11 ]  . the aim of this paper was to illustrate a series of cases of cds seen not only in crystal deposition diseases but also in other rheumatic and nonrheumatic conditions and to provide an assessment of ct imaging patterns and an analysis of the best use of current imaging techniques . 
 la sindrome del dente incoronato ( sdi ) rappresenta unentit clinico - radiologica caratterizzata dallassociazione di calcificazioni dei legamenti attorno al dente dellepistrofeo e da attacchi acuti periodici di cervicalgia occipitale con febbre , rigidit e segni generali di flogosi , di durata molto variabile , da pochi giorni ad alcune settimane [ 1 ]  . colpisce prevalentemente il sesso femminile , con et media al momento della diagnosi tra i 60 ed i 70 anni . 
i depositi di cristalli , nella maggior parte dei casi di pirofosfato di calcio diidrato ( cppd ) , ma anche di idrossiapatite ( ha ) , possono rimanere asintomatici oppure essere responsabili di dolore cronico cervicale e di compressione midollare [ 2 ]  . la tipica descrizione della sindrome , caratterizzata dalla triade cefalea , febbre e cervicalgia mattutina , stata riferita nel 1980 da le goff [ 3 , 4 ] , che distinse questi attacchi acuti da quelli osservati nelle delle malattie da microcristalli a localizzazione vertebrale cervicale distale o dorso - lombare , e da quelli della comune malattia artrosica [ 5 ]  . 
inoltre casi atipici di sdi possono simulare sia la sindrome dolorosa cervico - brachiale ( con debolezza e rigidit della spalla ) che la cefalea occipitale e temporale ( che mima la polimialgia reumatica atipica e / o larterite a cellule giganti )  . 
secondo la letteratura [ 710 ] , lesame tc focalizzato a livello di c1 - c2 rimane il gold standard della diagnostica per immagini ed superiore alla rm nellidentificare queste calcificazioni , soprattutto se minute [ 11 ]  . scopo del presente lavoro stata lillustrazione di alcuni materials and methods between 2001 and 2004 , 38 patients ( 15 men and 23 women ; age range 4572 years ; mean age 55 ) were referred by both general practitioners and specialists ( orthopaedic surgeons , rheumatologists , neurologists , and oncologists ) for investigation of neck pathe patients were divided into two groups : the first ( 16 cases , three men and fifteen women ) consisted of patients with a known clinicalrheumatologic diagnosis [ rheumatoid arthritis ( ra ) , diffuse idiopathic skeletal hyperostosis ( dish ) , etc . ] established on the basis of pain and / or dysfunction at the level of the atlantoaxial joint and alterations demonstrated by conventional radiography , and for whom the specialist had requested further diagnostic investigation the second group ( 22 cases , 12 men and fifteen women ) consisted of patients referred by rheumatologists , general practitioners and other specialists ( orthopaedic surgeon , neurologist , oncologist ) , with initial symptoms confined to the lower cervical spine , often with a history of trauma , and with inconclusive radiographic findings . data collected for each patient included signs and symptoms of the illness , social status and daily working activity , physical examination and laboratory tests , clinical diagnosis and radiographic findings . 
 the disorders affecting the patients ( table 1 ) were distributed as follows : 11 cases of rheumatoid arthritis ( ten women and one man ) , two of cppd crystal deposition disease ( both women ) , one of systemic sclerosis ( a woman ) , one of osteoarthritis ( a man ) , one of seronegative spondyloarthritis ( a man ) , four of neoplasm ( one woman and three men ) with suspected cervical metastases , one ( a man ) of malignant haematological disease ( lymphoma ) , one ( a woman ) of postmenopausal osteoporosis , ten ( five men and five women ) of recent or previous traumatic injury with suspected involvement of the skull base and first cervical vertebrae ( dens fracture , frank atlantoaxial luxation or subluxation ) and six of algo - dysfunctional syndrome of the cervical spine ( three men and three women )  . 
evaluation with ct imaging casi di sindrome del dente incoronato non solamente nellambito delle malattie da deposito di microcristalli , ma anche in altre malattie reumatiche ( i.e. , artrite reumatoide ) e non , la valutazione dei loro aspetti iconografici tc e il giudizio sul miglior impiego delle tecniche attuali di imaging . materiale e metodi nel periodo 20012004 sono stati esaminati 38 pazienti , di cui 15 maschi e 23 femmine , di et compresa fra i 45 ed i 72 anni ( media 55 ) , affetti da cervicalgia , inviati sia dal medico di base che dallo specialista ( ortopedico , reumatologo , neurologo , oncologo ) per gli accertamenti del caso . 
i pazienti sono stati divisi in due gruppi : il primo ( formato da 16 pazienti , di cui 3 maschi e 13 femmine ) , dove la diagnosi clinico - reumatologica era gi stata effettuata ( ar , dish , ecc . ) per la comparsa di sintomatologia algica e / o disfunzionale a livello della cerniera atlo - epistrofica , con alterazioni gi dimostrate in maniera inequivocabile dallesame radiografico convenzionale , e di cui lo specialista richiedeva un ulteriore approfondimento diagnostico a livello della suddetta zona ; il secondo gruppo ( formato da 22 casi , di cui 12 maschi e 10 femmine ) comprendeva pazienti inviati non dal reumatologo , ma dal medico di base e da altri specialisti ( ortopedico , neurologo , oncologo ) , la cui sintomatologia desordio era circoscritta al rachide cervicale , specie in sede prossimale , talvolta su base post - traumatica , e di cui la diagnostica per immagini convenzionale era risultata poco significativa . i dati raccolti per ogni paziente comprendevano tutti i segni ed i sintomi relativi alla malattia denunciata ; lo stato sociale e lattivit lavorativa giornaliera ; gli esami fisico e di laboratorio ; la diagnosi clinica ; i reperti ottenuti con la radiografia convenzionale . 
la visualizzazione delle immagini stata sempre effettuata in modo da avere finestra e livello di finestra sia per i tessuti molli ( i.e. , legamento trasverso ) che per le strutture ossee . 
in particolare , per lo scheletro , lampiezza della finestra stata di 1500 hu e il livello + 300 hu ; per le parti molli rispettivamente 350 hu e + 50 hu . 
non stato somministrato mezzo di contrasto . per quanto riguarda la patologia dei nostri pazienti , la cui distribuzione riportata nella tabella 1 , questa era cos rappresentata : 11 pazienti affetti da artrite reumatoide ( di cui 10 femmine e 1 maschio ) ; 2 casi portatori di malattia da p.n. 
ct demonstrated calcific deposits in the periodontoid structures ( transverse ligament of the atlas , crossed ligament , and alar ligaments ) in 12 patients , three of whom were men and nine who were women ( table 2 )  . 
ct appearance of calcifications was one of thin calcium deposits forming a cppd , entrambi di sesso femminile ; 1 caso di sclerosi sistemica ( femmina ) ; 1 caso di artrosi ( maschio ) ; 1 caso di spondiloartrite sieronegativa ( maschio ) ; 4 pazienti oncologici ( 1 femmina e 3 maschi ) con sospetta localizzazione replicativa cervicale ; 1 caso ( maschio ) affetto da patologia ematologia maligna ( linfoma ) ; 1 caso ( femmina ) con osteoporosi postmenopausale ; 10 pazienti ( 5 maschi e 5 femmine ) affetti da patologia traumatica , recente e pregressa , di cui si sospettava interessamento della base del cranio e delle prime vertebre cervicali ( frattura del dente dellepistrofeo e / o sublussazione e lussazione franca atlo - epistrofica ) e infine 6 pazienti affetti da patologia imprecisata ( 3 maschi e 3 femmine ) algico - disfunzionale cervicale . 
in essi la malattia prevalente era rappresentata dallartrite reumatoide ( ar ) 10 femmine e 1 maschio cui facevano seguito la malattia da cppd ( 2 casi ) , la sclerosi sistemica , lartrosi e la spondiloartrite sieronegativa ( 1 caso , rispettivamente )  . risultati lesame tc della giunzione atlo - epistrofica stato eseguito sulla base della sintomatologia clinica denunciata dal patable 2 patients with crowned dens syndrome ( cds ) and positive computed tomography ( ct ) findings rheumatic diseases no . 
evaluation with ct imaging women 9 rheumatoid arthritis men 3 cppd crystal deposition disease systemic sclerosis rheumatoid arthritis osteoarthritis seronegative spondyloarthritis femmine 9 artrite reumatoide maschi 3 malattia da cppd sclerosi sistemica artrite reumatoide osteoartrosi spondilite sieronegativa transverse ligament ( 4 ) alar ligaments ( 2 ) alar ligaments transverse ligament transverse ligament alar ligaments transverse ligament + atlooccipital anterior membrane legamento trasverso ( 4 ) legamenti alari ( 2 ) legamenti alari legamento trasverso legamento trasverso legamenti alari legamento trasverso + membrana atlo - occipitale anteriore tabella 2 pazienti con sindrome del dente incoronato e tc positiva malattia reumatica casi , n sede delle calcificazioni shell around the dens and located both posteriorly ( on axial images the deposits , they appeared inhomogeneous and probably located on the transverse ligament ) and at the level of the dens apex and base ( on coronal ct reformations due to involvement of the alar and apical ligaments )  . 
 [ 13 , 14 ] identified nonurate crystals in the synovial fluid of patients clinically presenting gout - like attacks ( hence the initial name pseudogout )  . these cppd crystals were characterised by a weak positive birefringence at polarised light microscopy , a finding that distinguished them from the monosodium urate crystals of gout . 
the radiographic features of the disease had , however , been already described by zitnan and sitaj in 1963 , who had called it polyarticular chondrocalcinosis [ 15 ]  . 
bennet , in particular , noted the presence of chalky , rhomboid - shaped crystals [ 16 ] during the autopsy of a patient in ireland with polyarticular chondrocalcinosis . ziente ( principalmente rappresentata da rigidit nucale e cefalea , con o senza incapacit funzionale ) o nellambito del follow - up diagnostico , soprattutto nei casi affetti da cppd dove lesame radiografico tradizionale aveva gi dimostrato la presenza di calcificazioni nelle sedi tipiche della malattia ( i.e. , ginocchio , polso )  . 
lesame tc risultato positivo per depositi calcifici in corrispondenza delle strutture viciniori al dente dellepistrofeo ( legamento trasverso dellatlante , legamento crociato , legamenti alari ) in 12 pazienti , di cui 3 maschi e 9 femmine , la cui distribuzione riportata nella tabella 2 . 
i tre soggetti erano affetti da ar ( anni 69 ) , da osteoartrosi ( anni 70 ) e da spondilite sieronegativa ( anni 48 ) ( fig . 1 ) , con calcificazioni a livello rispettivamente del legamento trasverso , di quelli alari e della membrana atlo - occipitale anteriore . 
liconografia delle calcificazioni era rappresentata da tenui depositi calcifici a guscio , attorno al dente dellepistrofeo , sia in sede posteriore ( nelle immagini tc sul piano assiale , di tipo disomogeneo , verosimilmente a carico del legamento trasverso ) , sia in corrispondenza dellapice e della base del dente stesso ( nelle immagini tc ricostruite sul piano coronale , per interessamento dei legamenti alari e del legamento apicale )  . 
the term chondrocalcinosis is very general , as it suggests apparent radiological and pathological calcification of the cartilage ( which could be due to cppd , ha , tricalcium phosphate or a combination of the three )  . 
although articular and periarticular calcifications can appear in cppd disease , similar calcifications can also be produced by other crystals [ 15 , 19 , 20 ]  . cppd crystal deposition disease is classified into sporadic , hereditary or secondary forms [ 16 ]  . 
the clinical presentation ranges from an incidental radiographic finding to discussione sebbene la malattia da deposito di pirofosfato di calcio diidrato ( cppd ) sia la pi comune artropatia da microcristalli [ 12 ] , tuttavia la scoperta di questi nelle strutture intra - articolari risale allinizio degli anni 60 , quando mccarty insieme ad hollander , kohn e faires [ 13 , 14 ] identific cristalli non uratici nel liquido sinoviale di pazienti che clinicamente presentavano attacchi simil - gottosi ( da ci il termine di pseudogotta dato inizialmente alla malattia )  . questi cristalli di cppd erano caratterizzati da una bassa birifrangenza positiva quando esaminati al microscopio a luce polarizzata , fenomeno che li distingueva da quelli di urato monosodico della gotta . 
the radiological presentation [ 2123 ] is characterised by calcifications in and around the joints and by structural articular changes ( pyrophosphate arthropathy )  . the knee , the wrist , the pubic symphysis and the hip are most commonly affected . 
the spine may be the only site of cppd disease [ 20 , 24 ] and is generally asymptomatic [ 12 ]  . crystals may be identified both in the nucleus pulposus of p.n. 
questultimo , in irlanda , durante lautopsia di un paziente affetto da condrocalcinosi poliarticolare , evidenzi la presenza di cristalli romboidali , di aspetto gessoso [ 16 ]  . i numerosi studi di resnick et al . 
 [ 12 , 17 ] hanno messo ordine nelle svariate nomenclature con cui la malattia era conosciuta , in quanto diversi sinonimi sono stati usati in maniera intercambiabile tra di loro , causando in passato una certa confusione . 
in effetti , il termine condrocalcinosi molto generico , in quanto indica una evidente calcificazione radiologica ed anatomopatologica della cartilagine ( che pu riferirsi alla presenza di cristalli di cppd , di ha , di fosfato tricalcico o alla combinazione dei tre suddetti )  . 
la dizione malattia da deposito di cristalli di cppd ( cppd crystal deposit disease ) si riferisce specificatamente a un disordine caratterizzato dalla esclusiva presenza di cristalli di cppd dentro o intorno alle articolazioni . 
le calcificazioni articolari e periarticolari possono comparire nella malattia di cppd ; tuttavia altri cristalli possono produrre calcificazioni analoghe [ 15 , 19 , 20 ]  . la malattia da cppd si classifica in forma sporadica , ereditaria o secondaria [ 16 ]  . 
infine , i cristalli di cppd possono depositarsi nel legamento trasverso dellatlante e nei legamenti alari , realizzando la c.d. sindrome del dente incoronato [ 1 , 68 ] , la cui definizione , in accordo con diversi autori [ 7 , 8 , 26 ] , dovrebbe comprendere calcificazioni di ogni componente articolare in questa sede ( membrana sinoviale , capsula articolare , legamenti )  . 
inoltre , sullapice del dente sinseriscono tre legamenti : il legamento apicale del dente e i due legamenti alari [ 6 ]  . clinicamente , la sdi dovuta a depositi di ha [ 27 ] una condizione ben definita , ancorch rara , che colpisce donne giovani e di media et , caratterizzata da intensa cervicalgia p.n. 
computed tomography ( ct ) of the atlantoaxial region : images in the axial plane ( a , b ) and reformatted in the coronal ( c , d ) and sagittal planes ( e )  . 
tc atlo - epistrofica : scansioni sul piano assiale ( a , b ) e ricostruzione elettronico sul piano coronale ( c , d ) e sagittale ( e )  . 
in some cases , there may be involvement of the yellow ligaments and posterior longitudinal ligaments , with secondary myelopathy , spinal cord compression and vertebral stenosis [ 25 ]  . 
in particular , it is important to consider the cruciform ligament , which is composed of both part of the transverse ligament of the atlas and its vertical extensions ( superior and inferior longitudinal fibres )  . 
in addition , three ligaments are inserted on the dens apex : the apical ligament and the two alar ligaments [ 6 ]  . clinically , cds due to ha crystal deposits [ 27 ] is a welldefined , though rare , condition that affects young and middle - aged women and is characterised by intense cervical pain [ 28 ] that resolves within days or weeks after treatment with nonsteroid anti - inflammatory drugs ; it does not cause spinal compression [ 4 ]  . 
the clinical picture caused by cppd crystal deposits is more complex , as it may be asymptomatic or associated with a variety of clinical manifestations , some of which are severe [ 29 , 30 ] , such as spinal cord compression [ 31 , 32 ] related in some cases to a slightly calcific mass between the dens and the transverse ligament . 
crystal deposits can be found in older patients ( 5.7% ) with evident peripheral joint chondrocalcinosis [ 11 ] and advanced degenerative lesions of the atlantoaxial joint , and they tend to worsen with age [ 9 ]  . 
 in contrast with previous studies [ 24 , 7 , 12 ] that consider cppd crystal deposition disease as the most frequent form of crowned dens syndrome , only two women in our series [ 28 ] , risolvibile in un tempo variabile da pochi giorni ad alcune settimane con trattamento anti - infiammatorio non steroideo , senza causare compressione bulbare [ 4 ]  . 
il quadro clinico sostenuto , invece , dai cristalli di cppd pi complesso , perch pu essere asintomatico o associarsi a manifestazioni cliniche varie , talora anche gravi [ 29 , 30 ] , quali la compressione midollare [ 31 , 32 ] , determinata da una massa talora finemente calcifica localizzata tra il dente dellepistrofeo e il legamento trasverso . 
i depositi cristallini si osservano in pazienti pi anziani ( 5 , 7% ) , con evidente condrocalcinosi delle articolazioni periferiche [ 11 ] e lesioni degenerative avanzate dellarticolazione atlo - epistrofica e si aggravano con lavanzare dellet [ 9 ]  . 
 nella nostra casistica , in contrasto con la letteratura [ 24 , 7 , 12 ] che indica nella malattia di cppd levenienza pi frequente di sdi , solamente due casi entrambi di sesso femminile presentavano calcificazioni peri - odontoidee . una di queste ( et 69 anni ) denunciava dolore ad entrambe le ginocchia , dove lesame radiografico dimostrava la presenza di calcificazioni moniliformi a carico di entrambi i menischi . 
analoghe calcificazioni erano presenti nella cartilagine triangolare del polso . altre malattie associate alla sdi sono lar , la ra - dish [ 33 ] , le connettiviti sistemiche , le sequele di traumi , sublussazioni e lussazioni franche . 
in particolare , nellar linteressamento dello scheletro assile [ 34 ] risulta significativo nel tratto cervicale ( 60%80% dei casi ) ; esso pu essere asintomatico oppure manifestarsi con dolore , limitazione funzionale e sindromi neurologiche anche gravi [ 20 ]  . 
qualunque sia il distretto interessato , le lesioni anatomo - patologiche sono quelle tipiche della malattia , rappresentate dalla flogosi cronica della membrana sinoviale , con formazione successiva di erosioni ossee ed indebolimento strutturale delle zone dinserzione dei legamenti . 
computed tomography ( ct ) of atlantoaxial region , images in the axial plane ( a , b ) , and reformatted images in the coronal ( c ) and sagittal ( d ) planes . 
in one ( 69 years old ) , who had pain in both knees , radiography demonstrated minute mottled calcifications in the meniscus and in the triangular cartilage of the wrist , typical of cppd crystal deposit disease . the diseases associated with cds are ra , ra - dish [ 33 ] , systemic connectivitis , trauma , frank subluxations and luxations . 
ra in particular involves the axial skeleton [ 34 ] and especially the cervical region ( in 60%80% of cases ) ; this may be asymptomatic , or present with pain , functional impairment and even severe neurological syndromes [ 20 ]  . whatever the area involved , the lesions are pathologically those of ra : chronic inflammation of the synovial membrane , bone erosions and weakening of the ligament insertions . 
laxity of the articular capsule and atlantoaxial joint ligaments causes an abnormal separation ( more than 2.5 mm ) no in sede atlo - epistrofica , con secondarie alterazioni della stabilit e della funzionalit regionale : ostacolata rotazione del capo ed instabilit articolare secondo tre direttrici fondamentali ( anteriore , laterale e verticale )  . 
la lassit delle strutture capsulo - legamentose della giunzione atlo - epistrofica determina lallontanamento anomalo ( > 2 , 5 mm ) tra arco anteriore dellatlante e dente dellepistrofeo , dando luogo alla lussazione atlo - assiale [ 20 ]  . 
nella nostra casistica sono soprattutto i pazienti affetti da ar quelli che hanno denunciato la maggiore incidenza della sdi , con prevalenza del sesso femminile ( 10 femmine e 1 maschio ) , specie in quei casi affetti da malattia reumatica inveterata . lassociazione tra ar e calcificazioni legamentose periodontoidee risulta di non facile interpretazione , dato che non ancora identificato un rapporto di causa ed effetto , tenuto between the anterior arch of the atlas and the odontoid process , resulting in atlantoaxial luxation [ 20 ]  . 
the association between ra and periodontoid calcification is not easy to interpret in that no causal relationship has yet been identified in the light of the natural evolution of ra [ 36 ]  . 
 whereas ct is indicated for the study of cortical bone and periodontoid calcifications , mri with paramagnetic contrast material is the modality of choice for the study of synovial abnormalities , as it is the only technique able to demonstrate the inflamed pannus ( hypervascular , hypovascular and fibrous ) , articular effusion and cord compression [ 37 ]  . the diagnosis of cds is not always straightforward given the broad spectrum of presentations . 
nevertheless , ct can fail to detect cds , especially if it is carried out too late after an acute attack , because the calcifications may have been reabsorbed in the meantime [ 8 ]  . 
the characteristic ct pattern is a narrow and irregular horseshoe calcification surrounding the odontoid process , at times related to osteophytes in the upper margin of the anterior arch of the atlas , as well as calcification of the transverse ligament . 
the main calcification is often surrounded by other smaller calcifications at the apex of the dens [ 10 ]  . differential diagnosis includes calcification of the tendon of the longus colli muscle , where the clinical features of cds are accompanied by pharyngeal pain and dysphagia , incorrectly suggesting meningitis , spondylodiscitis or retropharyngeal abscess . 
other radiographic appearances not to be confused with cds are disc calcifications , calcified adenopathy , sesamoid bones in the transverse ligament , ossification of the common posterior vertebral ligament , and periatlantic calcifications [ 28 , 39 , 40 ]  . 
 [ 28 ] have described 23 cases , five cases and one case of cds , respectively , with clinical presentations varying from febrile nuchal pain ( in acute forms ) to spinal compression ( in chronic forms )  . 
 se la tc indicata per lo studio della corticale ossea e delle calcificazioni periodontoidee , la rm metodica elettiva per lo studio delle lesioni sinoviali , essendo lunica tecnica capace di visualizzare il panno infiammatorio ( ipervascolarizzato , ipovascolarizzato e fibroso ) , il versamento articolare e la compressione midollare , impiegando mezzo di contrasto paramagnetico [ 37 ]  . la diagnosi della sdi non sempre facile , dato lampio spettro di presentazione dellaffezione : infatti , altre manifestazioni cliniche , come la meningite , il dolore cervico - brachiale e lemicrania occipito - temporale , possono aggiungersi al classico episodio acuto febbrile di cervicalgia [ 1 ]  . con la tc la dimostrazione delle minute calcificazioni nellarea del processo odontoideo estremamente facilitato , con sensibilit superiore alla rm [ 38 ]  . 
tuttavia la tc pu fallire nella diagnosi di sdi , specie se questa eseguita tardivamente dopo lattacco acuto , perch nel frattempo le calcificazioni possono riassorbirsi [ 8 ]  . 
laspetto tc caratteristico quello di una sottile ed irregolare calcificazione a ferro di cavallo attorno al processo odontoideo , che in alcuni casi pu essere riferito , oltre che alla calcificazione del legamento trasverso , anche a neoformazioni osteofitosiche a carico del profilo superiore dellarco anteriore dellatlante . 
questa calcificazione principale spesso circondata da altre pi piccole , situate allapice del dente [ 10 ]  . la diagnosi differenziale va posta con le calcificazioni del tendine del muscolo lungo del collo , che oltre al quadro clinico della sdi associa dolore faringeo e disfagia che evocano a torto meningite , spondilodiscite o ascesso retrofaringeo . 
altri aspetti radiografici da non confondere con la sdi sono : le calcificazioni discali , le adenopatie calcifiche , la presenza di un sesamoide nel legamento traverso , lossificazione del legamento vertebrale comune posteriore , le ossificazioni soprannumerarie peri - atlantiche [ 28 , 39 , 40 ]  . 
treves [ 39 ] , constantin [ 7 ] e denes [ 28 ] hanno descritto , rispettivamente , 23 casi , 5 casie 1 caso caratterizzati da un quadro clinico variabile da episodi di nucalgia febbrile nelle forme acute a quadri di compromissione midollare nelle forme croniche . lo studio di treves [ 39 ] ha dimostrato , con lesame tc , la presenza di calcificazioni atlo - odontoidee nel 90% dei casi di sdi : 8 pazienti su 12 di questo gruppo non avevano mai p.n. 
evaluation with ct imaging [ 39 ] demonstrated with ct the presence of atlantoaxial calcifications in 90% of cases : eight of 12 patients had never suffered from neck pa therefore , in the presence of febrile cervical pain , the discovery of periodontoid calcifications suggests cds and excludes meningitis . conclusion in cds , ct is the gold standard imaging modality in that it allows identification of the different radiographic patterns of the disease : simple band of calcification or double band of thin calcifications in the transverse ligament , irregular calcifications crowning the dens apex , and bone erosions of the dens itself . 
however , radiography of other joints ( wrist , knee , pubic symphysis and shoulder ) can help establish whether the disease is due to cppd crystal deposit disease or ha crystals and is therefore recommended for routine patient management . 
although cds is traditionally associated with crystal deposition diseases , it may be accompanied by other diseases affecting the joints , such as ra with involvement of the proximal cervical spine ( as seen in our study ) , ra - dish , and other forms of systemic connectivitis ( systemic sclerosis )  . 
pertanto , in presenza di cervicalgia febbrile , la scoperta di calcificazioni periodontoidee suggestiva di sdi , escludendo quella di meningite . conclusione nella sindrome del dente incoronato la tc rappresenta lesame gold standard , permettendo di identificare i diversi aspetti radiografici : calcificazioni semplici a banda o in doppia filiera sottile in corrispondenza del legamento trasverso ; calcificazioni irregolari a corona sopra e intorno allapice del dente ; lesioni ossee di tipo erosive del dente stesso . 
invece lesame radiografico di altri distretti articolari ( carpo , ginocchio , sinfisi pubica , spalla ) pu agevolare il riconoscimento eziologico della malattia , se da cppd o da ha , ed pertanto raccomandato nella gestione routinaria del paziente . 
la sdi , infatti , tradizionalmente associata alle malattie da microcristalli , ma altre malattie articolari si accompagnano ad essa , in primis lartrite reumatoide con compromissione della colonna cervicale prossimale ( come si evince anche dalla nostra casistica ) , seguita dalla ra - dish e da altre connettiviti sistemiche ( sclerosi sistemica )  . 
one hundred and seven patients with a clinical and laboratory suspicion of acute pulmonary embolism who had already undergone chest radiography were divided into four groups according to the time interval between onset of clinical suspicion and performance of the two diagnostic examinations ( 024 h , 2448 h , 048 h , 27 days )  . 
perfusion lung scintigraphy and multislice ct demonstrated elevated concordance if performed within 7 days of the onset of suspicion of acute pulmonary embolisconcordance was higher if the examinations were completed within 2448 h . 
in suspected acute pulmonary embolism , it is mandatory to reach a correct diagnosis within few hours 48 at the most . key words acute pulmonary embolism perfusion lung scintigraphy spiral computed tomography diagnostics of urgency imagings riassunto obiettivo . 
centosette pazienti con sospetto clinicolaboratoristico di epa , che avevano gi eseguito un rx torace , sono stati divisi in 4 gruppi in base allintervallo di tempo tra il sospetto clinico e lesecuzione di entrambe le indagini ( 024 ore , 2448 ore , 048 ore , 27 giorni )  . 
spp e tcsm sono risultate positive in 29 / 107 ( 27 , 1% ) e negative in 78 / 107 ( 72 , 89% ) : concordanza positiva ( cp ) in 22 casi e concordanza negativa ( cn ) in 71 . 
nel sospetto di epa importante effettuare una rapida e corretta diagnosi in poche ore , 48 al massimo . parole chiave embolia polmonare acuta scintigrafia polmonare perfusiva tc spirale multislice diagnostica per immagini durgenza introduction introduzione acute pulmonary embolism ( ape ) is the third most common acute cardiovascular disease after ischaemic heart disease and stroke [ 1 ]  . 
the yearly incidence of ape in western lembolia polmonare acuta ( epa ) rappresenta la terza pi comune patologia acuta dellapparato cardiovascolare dopo la cardiopatia ischemica e lo stroke [ 1 ]  . 
deep vein thrombosis ( dvt ) is the most common cause of pulmonary embolism ; the other principal primary and secondary risk factors are summarised in table 1 [ 2 ]  . 
in spite of the recent important advances in imaging and laboratory diagnostics , approximately 70% of cases of pulmonary embolism are identified at autopsy [ 3 ]  . in cases with an early diagnosis by multislice spiral computed tomography ( msct ) and perfusion lung scintigraphy ( pls ) , anticoagulation therapy proved to be effective in approximately 75% of patients [ 4 ]  . 
thanks to its ability to perform fast subcentimetre acquisitions with optimisation of vascular enhancement after the injection of contrast material , msct permits visualisation of the entire pulmonary artery tree down to the subsegmental level [ 5 ]  . perfusion lung scintigraphy ( pls ) plays an important role in the diagnosis of ape . 
the aim of our study was to compare and integrate the data provided by pls and by msct in cases of emergency diagnoses of ape in nuclear medicine and radiology departments operating around the clock . materials and methods from may 2002 to june 2004 , we retrospectively reviewed 107 consecutive patients ( 50 women and 57 men ) with ages ranging from 20 to 80 years ( mean age 60 + 18 ) , with a clinical and laboratory suspicion of ape . 
the first - level diagnostic approach included an assessment of predisposing factors , primary disease , symptoms ( dyspnoea , chest pain , cough , haemoptysis , syncope ) and clinical signs ( tachypnoea , tachycardia , signs of deep vein thrombosis , fever , cyanosis )  . 
furthermore , all patients had undergone chest radiography before pls and msct . pls was performed with a gamma starcam 4000 xc / t camera ( ge medical systems , west milwaukee , wi , usa ) after administration of 185 mbq of macroaggregated albumin ( number of particles between 300 , 000 and 50 , 000 and diameter from 120 to 70 m ) with 99 mtc . 
all images were acquired with the planar technique in the anterior , posterior , right and left posterior oblique and right and left anterior oblique projections using the following acquisition parameters : 128128 matrix and 500750 kcounts per projection . 
la trombosi venosa profonda ( tvp ) la pi comune causa di embolia polmonare ed i principali fattori di rischio primari e secondari sono riassunti nella tabella 1 [ 2 ]  . 
nonostante gli importanti e recenti sviluppi della diagnostica strumentale e di laboratorio , circa il 70% degli eventi embolici polmonari sono riconosciuti allesame autoptico [ 3 ]  . nei casi diagnosticati tempestivamente con la tomografia computerizzata spirale multistrato ( tcsm ) e con la scintigrafia polmonare perfusiva ( spp ) , il trattamento anticoagulante si dimostrato efficace in circa il 75% dei pazienti [ 4 ]  . pertanto la tcsm si propone come metodica utile nella valutazione del paziente con sospetta epa . 
infatti , la tcsm per la possibilit di eseguire in tempi rapidi acquisizioni subcentimetriche e di ottimizzare lopacizzazione delle strutture vascolari dopo iniezione di mdc , consente di visualizzare le diramazioni dellalbero arterioso polmonare fino al livello subsegmentario [ 5 ]  . la scintigrafia polmonare perfusiva ( spp ) ha un ruolo importante nella diagnosi di epa . 
questa tecnica non invasiva , a bassa dose di radiazioni , consente una valutazione della perfusione ematica polmonare anche in pazienti critici , non collaboranti e portatori di dispositivi metallici [ 6 ]  . 
scopo del nostro studio stato quello di comparare ed integrare i dati forniti dalla spp e dalla tcsm nella diagnosi in urgenza dellepa in centri di medicina nucleare e radiodiagnostica che svolgono indagini urgenti 24 ore su 24 . materiali e metodi nel periodo compreso tra maggio 2002 e giugno 2004 stata eseguita unanalisi retrospettiva che ha portato a selezionare 107 pazienti consecutivi ( 50 donne e 57 uomini ) di et compresa tra i 20 e gli 80 anni ( et media di 6018 ) , con sospetto clinico - laboratoristico di epa . 
per un inquadramento diagnostico in prima istanza sono stati valutati i fattori predisponenti , la patologia di base , i sintomi ( dispnea , dolore toracico , tosse , emottisi , sincope ) e i segni clinici ( tachipnea , tachicardia , segni di trombosi venosa profonda , febbre , cianosi )  . 
i criteri di inclusione sono stati : lesecuzione sia della spp che della tcsm entro 7 giorni dal sospetto clinico - laboratoristico di epa ; non aver intrapreso alcuna terapia trombolitica . 
inoltre , tutti i pazienti avevano eseguito una rx del torace prima della spp e / o della tcsm . la spp stata effettuata utilizzando una gamma camera starcam 4000 xc / t ( ge medical systems , west milwaukee , wisconsin , usa ) previa somministrazione di 185 mbq di macroaggregati di albumina ( numero di particelle variabile da 300000 a 50000 e diametro compreso tra 120 e 70 micron ) marcati con 99 mtc . 
interpretation criteria for pls were those suggested by the prospective investigative study of acute pulmonary embolism ( pisaped ) study , as reported in table 2 [ 7 ]  . msct was considered positive in the presence of eccentric filling defects or of complete occlusion of the pulmonary artery lumen , whether associated or not to the presence of areas of parenchymal hypoventilation . 
all data provided by pls and msct were independently evaluated by two nuclear physicians and two radiologists and included an assessment of which of the two studies had been ordered and performed first . the patients were grouped based on the time interval between the initial clinical suspicion and performance of the two examinations , namely , 024 h , 2448 h , 0 - 48 hours , and 27 days . 
cohens test was used for analysis of concordance between the results of the two investigations , both on the overall patient population enrolled and for the four subgroups ( 024 h , 2448 h , 048 h , 27 days )  . results pls and msct were positive in 29 / 107 ( 27.1% ) cases and negative in 78 / 107 ( 72.89% ) cases , with a positive concordance ( pc ) in 22 cases and negative concordance ( nc ) in 71 ( table 3 )  . 
in the subgroup of patients who had undergone pls and msct within 24 h of the initial clinical suspicion of ape , pls was positive in 12 / 49 ( 24.48% ) cases and negative in 37 / 49 ( 75.51% ) cases , whereas msct was positive in 17 / 49 ( 34.69% ) cases and negative in 32 / 49 ( 65.30% ) cases , with pc in 12 cases ( fig . 1 ) and nc in 32 cases ( table 4 )  . 
furthermore , 35 patients had undergone pls first , and 14 had undergone msct first . in the subgroup of patients who had undergone pls and msct from 24 to 48 h from the first clinical suspicion of ape , pls was positive in 5 / 20 ( 25% ) cases and negative in 15 / 20 ( 75% ) cases , whereas msct was positive in 3 / 20 ( 15% ) cases and negative in 17 / 20 ( 85% ) cases , with pc in three cases and nc in 15 ( table 5 )  . 
moreover , 13 patients had undergone pls first , and seven had undergone msct first . no state acquisite con tecnica planare in proiezione anteriore , posteriore , obliqua posteriore destra e sinistra , obliqua anteriore destra e sinistra utilizzando i seguenti parametri di acquisizione : matrice 128128 e conteggi per proiezione 500750 kcounts . la tc stata effettuata con apparecchiatura tcsm ( mx 8000 , philips medical system , cleveland , ohio , usa )  . 
i parametri di acquisizione utilizzati sono stati i seguenti : collimazione 2 , 5 mm , pitch 1 , 25 , incremento 1 mm , tempo di rotazione 0 , 5 s , kvp / mas - 120 / 100 . 
lesame stato eseguito con paziente in decubito supino e le scansioni sono state condotte dalle cupole diaframmatiche allo stretto toracico superioil mezzo di contrasto uroangiografico ( ultravist 370 , schering , berlin , germania ) stato iniettato nella vena cubitale attraverso un ago da 16 gauge utilizzando un iniettore automatico ( mk - iv , medrad , pittsburgh , pennsylvania , usa ) con i seguenti parametri : volume 8090 ml , flusso 33 , 5 ml / s . 
il ritardo di inizio delle scansioni stato determinato con lesecuzione di un bolus test , ed risultato variabile tra 9 e 15 s ( fase arteriosa polmonare ) , mentre la fase tardiva stata eseguita dopo 4550 s . 
i criteri dinterpretazione per la spp sono quelli proposti dallo studio prospettico pisa - ped e riportati in tabella 2 [ 7 ]  . la tcsm stata considerata positiva in presenza di difetti di riempimento eccentrici o occludenti lintero lume delle arterie polmonari , associati o meno alla presenza di aree dipoventilazione parenchimale . 
tutti i dati forniti dalla spp e dalla tcsm sono stati valutati indipendentemente da due medici nucleari e da due medici radiologi ed stato anche valutato quale dei due esami era stato richiesto ed eseguito per primo . i pazienti arruolati sono stati raggruppati in base allintervallo di tempo trascorso tra linsorgenza del sospetto clinico e lesecuzione di entrambe le indagini , ovvero 024 ore , 2448 ore , e 27 giorni . 
il test di cohen stato utilizzato per lanalisi della concordanza dei risultati delle due indagini sia per il gruppo di tutti i pazienti arruolati che per i quattro sottogruppi suddivisi in base allintervallo di tempo per lesecuzione delle due indagini 024 ore , 2448 ore , 048 ore , 48 ore7 giorni . risultati la spp e la tcsm sono risultate positive in 29 / 107 ( 27 , 1% ) e negative in 78 / 107 ( 72 , 89% ) , con una concordanza positiva in 22 casi e negativa in 71 ( tabella 3 )  . 
nel sottogruppo dei pazienti che avevano eseguito la spp e la tcsm entro 24 ore dallinsorgenza del sospetto clinico di epa , la spp risultata positiva in 12 / 49 ( 24 , 48% ) e negativa in 37 / 49 ( 75 , 51% ) , mentre la tcsm risultata positiva in 17 / 49 ( 34 , 69% ) e negativa in 32 / 49 ( 65 , 30% ) con g . 
pls ( a ) shows no perfusion in the inferior right lobe , with positive concordance with msct ( b ) , which shows an occlusive thrombus in the right inferior lobe ( white arrow )  . 
la ssp ( a ) mostra assenza di perfusione in corrispondenza del lobo inferiore destro con concordanza positiva con la tcsm ( b ) che mostra la presenza di un trombo occludente nel ramo lobare inferiore destro dellarteria polmonare ( freccia piena )  . 
la tcsm ( b ) dimostra la presenza di un trombo parzialmente occludente ( freccia )  . in the subgroup of patients who had undergone pls and msct within 48 h from the first clinical suspicion of ape , pls was positive in 17 / 69 ( 24.63% ) cases and negative in 52 / 69 ( 75.36% ) cases , while msct was positive in 20 / 69 ( 28.98% ) cases and negative in 49 / 69 ( 71% ) cases , with pc in 15 cases and nc in 47 ( table 6 )  . 
inoltre 35 pazienti avevano effettuato prima la spp e 14 prima la tcsm . nel sottogruppo dei pazienti che avevano eseguito la spp e la tcsm tra 24 e 48 ore dallinsorgenza del sospetto clinico di epa , la spp risultata positiva in 5 / 20 ( 25% ) e negativa in 15 / 20 ( 75% ) , mentre la tcsm risultata positiva in g . 
secondary antithrombin iii deficit protein c deficit factor v leiden protein s deficit plasminogen deficit factor xii deficit hyperhomocysteinaemia anti - cardiolipin antibody congenital dysfibrinogenaemia thrombomodulin tabella 1 fattori di rischio per le tromboembolie venose a . 
concordance between results 3 / 20 ( 15% ) e negativa in 17 / 20 ( 85% ) con una concordanza positiva in 3 casi e negativa in 15 ( tabella 5 )  . 
inoltre 24 pazienti avevano effettuato prima la spp e 14 prima la tcsm . nella tabella 8 riportata lanalisi della concordanza tra la spp e la tcsm nei gruppi di pazienti che hanno effettuato le due indagini entro 24 ore , tra 2448 ore , entro 48 ore , tra 27 giorni ed entro 7 giorni dal sospetto clinico di epa . 
tale concordanza stata maggiore quando le due indagini sono state eseguite entro 24 ore dal sospetto clinico , continua a mantenere valori elevati se le indagini sono eseguite entro 48 ore , mentre si riduce notevolmente se gli esami sono stati eseguiti entro 7 giorni dal sospetto clinico . inoltre fra i 22 pazienti con concordanza positiva tra la spp e la tcsm , 12 hanno eseguito entrambi gli esami entro le 24 ore , 3 entro 48 ore , 7 entro una settimana . 
tra i pazienti con concordanza positiva tra le due indagini nellintera casistica studiata , il 54 , 54% ( 12 / 22 ) apparteneva al sottogruppo studiato entro 24 ore dal sospetto clinico di epa , il 13 , 6% ( 3 / 22 ) al sottogruppo studiato entro 48 ore , il rimanente 31 , 81% ( 7 / 22 ) al sottogruppo studiato entro 7 giorni dal sospetto clinico di epa . 
tra i pazienti con concordanza negativa tra le due indagini nellintera casistica studiata , il 45 , 07% ( 32 / 71 ) apparteneva al sottogruppo studiato entro 24 ore dal sospetto clinico di epa , il 21 , 12% ( 15 / 71 ) al sottogruppo studiato entro 48 ore , il rimanente 33 , 80% ( 24 / 71 ) al sottogruppo studiato entro 7 giorni dal sospetto clinico di epa . in 11 pazienti con spp positiva in fase acuta , questo esame stato ripetuto anche dopo terapia trombolitica prima della dimissione dei pazienti ed ha dimostrato il positivo effetto del trattamento ( miglioramento in 3 , completa normalizzazione della perfusione polmonare in 8 pazienti )  . discussione la diagnosi di embolia polmonare spesso difficile e la mortalit nei casi non trattati , si aggira intorno al 30% , g . 
presence of wedgeshaped areas of hyperperfusion single or multiple wedge - shaped perfusion defects with or without corresponding radiographic parenchymal alterations . absence of wedge - shaped areas of hyperperfusion tabella 2 criteri di interpretazione della spp ( pisa - ped ) diagnosi risultati normale anormale compatibile con epa anormale non compatibile con epa difetti di perfusione non assenza di difetti perfusivi difetti di perfusione cuneiformi singoli o multipli con o senza corrispondenti alterazioni radiografiche parenchimali . 
concordance was higher when the two studies were performed within 24 h of the initial clinical suspicion and remained high when they were performed within 48 h , whereas it decreased markedly if the two studies were performed within 7 days of the initial clinical suspicion . furthermore , among the 22 patients with pc between pls and msct , 12 had undergone both exams within 24 h , three within 48 h , and seven within 1 week . 
among patients with pc between the two investigations , 54.54% ( 12 / 22 ) belonged to the subgroup studied within 24 h of the initial clinical suspicion of ape , 13.6% ( 3 / 22 ) to the subgroup studied within 48 h , and the remaining 31.81% ( 7 / 22 ) to the subgroup studied within 7 days . 
among patients with nc between the two examinations , 45.07% ( 32 / 71 ) belonged to the subgroup studied within 24 h of the initial clinical suspicion of ape , 21.12% ( 15 / 71 ) to the subgroup studied within 48 h , and the remaining 33.80% ( 24 / 71 ) to the subgroup studied within 7 days . mentre si riduce al 2%8% se viene intrapresa una adeguata terapia anticoagulante [ 4 ]  . 
nel 90% dei casi il sospetto di epa motivato dalla presenza di tosse , dispnea , dolore toracico , sincope , tachipnea , cianosi e tachicardia [ 7 , 9 ]  . 
lassenza di questi sintomi ha un valore predittivo negativo del 94% , mentre il valore predittivo positivo rimane basso , pari al 53% , anche considerando lassociazione di pi segni e sintomi riferibili ad epa . 
la radiografia del torace , il dosaggio dei d - dimeri , lemogasanalisi e lecg permettono di orientare la diagnosi e di monitorare le condizioni generali del paziente , ma non consentono una diagnosi definitiva . 
la radiografia del torace solo in rari casi permette di riconoscere lembolia polmonare tuttavia lintegrazione con la spp pu risultare utile per definire la diagnosi di epa [ 11 ]  . la spp permette la visualizzazione diretta della perfusione polmonare attraverso lidentificazione di difetti perfusivi di tipo segmentario e pertanto riveste un ruolo centrale nelliter diagnostico non invasivo di epa . 
la spp un esame semplice , rapido , economico , senza controindicazioni e anche se eseguito con tecnica planare in una sola proiezione permette di riconoscere embolie gravi , per le quali un trattamento terapeutico tempestivo necessario per salvare la vita del paziente . 
la classificazione delle immagini scintigrafiche perfusive suggerita dallo studio pisaped , da noi utilizzata , basata sullidentificazione della presenza e della morfologia di difetti perfusivi , indipendentemente dal loro numero [ 7 ]  . 
dai risultati del suddetto studio appare che la combinazione di una probabilit clinica intermedia o alta con una scintigrafia compatibile con epa ha un elevato valore predittivo positivo ( 98% ) e che una bassa probabilit clinica combinata con un spp anormale , non compatibile con epa , ha un elevato valore predittivo negativo di malattia ( 98% ) [ 7 ]  . nei centri di medicina nucleare , che eseguono indagini 24 ore su 24 in regime durgenza , come il nostro , la spp risultata lesame pi frequentemente richiesto per primo , poich fornisce i dati utili per una gestione terapeutica immediata del paziente . 
nei presidi sanitari in cui la spp non sia rapidamente disponibile la tcsm rappresenta una valida alternativa per documentare le occlusioni totali o parziali delle arterie polmonari [ 1216 ]  . 
tuttavia anche se la tcsm permette di riconoscere embolie pluri - , monoo subsegmentarie , rilevando eventuali riduzioni di densit parenchimale e di vascolarit , non in grado di valutare le modifig . 
of patients who underwent msct first : 30 nudi pazienti che hanno effettuato prima la spp : 77 nudi pazienti che hanno effettuato prima la tcsm : 30 plspls + total plspls + total sppspp + totale sppspp + totale table 4 results for patients undergoing perfusion lung scintigraphy ( pls ) and multislice computed tomography ( msct ) within 024 h tabella 4 risultati della spp e tcsm nel gruppo di pazienti studiati nellintervallo di tempo 024 ore msctmsct + total tcsmtcsm + totale no . 
of patients who underwent msct first : 14 nudi pazienti che hanno effettuato prima la spp : 35 nudi pazienti che hanno effettuato prima la tcsm : 14 in 11 patients with a positive pls in the acute phase , the examination was repeated after thrombolytic therapy and before discharge , and it confirmed the positive effect of treatment ( improvement in three , complete normalisation of lung perfusion in eight )  . discussion the diagnosis of pulmonary embolism is often difficult , and mortality in untreated cases is around 30% , whereas it drops to 2%8% if adequate anticoagulation therapy is initiated [ 4 ]  . 
in 90% of cases , the suspicion of ape is based on the presence of cough , dyspnoea , chest pain , syncope , tachypnoea , cyanosis and tachycardia [ 7 , 9 ]  . 
the absence of these symptoms has a 94% negative predictive value ( npv ) , whereas the positive predictive value ( ppv ) remains low , at 53% , even when the association of several signs and symptoms typical of ape is considered . therefore , other tests need to be used to recognise pulmonary embolism within the first hours after symptom onset . chest radiography , d - dimer , blood gas analysis and electrocardiogram ( ecg ) may suggest a diagnosis and help monitor the patients general condition , although they do not provide a definite diagnosis . 
chest radiography allows recognicazioni funzionali e quindi leffetto emodinamico di emboli o stenosi vascolari che determinano alterazioni della perfusione polmonare [ 17 , 18 ]  . nella nostra esperienza il test di cohen ha documentato una buona concordanza in tutti e 107 i pazienti selezionati . la concordanza risultata maggiore quando lintervallo temporale tra insorgenza del sospetto clinico e lesecuzione delle due indagini era minimo . 
tale non concordanza tra le due indagini spiegabile con la presenza di emboli parzialmente occludenti , che in realt non determinavano alterazioni perfusive significative e quindi di scarso o assente significato funzionale . 
tale non concordanza stata riscontrata nel 3 , 5% ( 2 su 57 ) di pazienti che avevano eseguito prima la spp e poi la tcsm studiati nellintervallo 048 ore e nel 6 , 54% ( 7 su 107 ) di pazienti dellintera casistica studiati nei 7 giorni successivi linsorgenza del sospetto clinico . 
lattivazione dei meccanismi trombolitici con frammentazione dellembolo nelle 48 ore7 giorni dopo linsorgenza del sospetto clinico pu spiegare questa non concordanza . nessun paziente che aveva eseguito entrambe le indagini entro le 24 ore dallinsorgenza del sospetto clinico ha mostrato spp positiva e tcsm negativa . 
of patients who underwent msct first : 7 nudi pazienti che hanno effettuato prima la spp : 13 nudi pazienti che hanno effettuato prima la tcsm : 7 plspls + total plspls + total sppspp + totale sppspp + totale table 6 results for patients undergoing perfusion lung scintigraphy ( pls ) and multislice computed tomography ( msct ) within 048 h tabella 6 risultati della spp e tcsm nel gruppo di pazienti studiati nellintervallo di tempo 048 ore msctmsct + total tcsmtcsm + totale no . 
of patients who underwent msct first : 12 nudi pazienti che hanno effettuato prima la spp : 57 nudi pazienti che hanno effettuato prima la tcsm : 12 tion of pulmonary embolism in sporadic cases only ; however , integration with pls may be useful to confirm the diagnosis of ape [ 11 ]  . pls allows direct visualisation of lung perfusion through the identification of perfusion defects at the segmental level , thus playing a key role in the noninvasive assessment of ape . 
pls is an easy - to - perform , fast , and inexpensive examination that does not present contraindications and , even when performed with the standard planar technique in a single projection , is able to recognise severe embolism necessitating urgent life - saving treatment . 
the classification of scintigraphic perfusion images suggested by the pisa - ped study , which we used , is based on identifying the presence and morphology of perfusion defects , independent of their number [ 7 ]  . 
the results of the above study suggest that the combination of an intermediate to high clinical probability with scintigraphic findings consistent with ape has a high ppv ( 98% ) , and that a low clinical probability combined with abnormal pls findings not consistent with ape has a high npv for this disease ( 98% ) [ 7 ]  . in nuclear medicine centres that perform emergency examinations 24 h / day , as we do , pls was found to be most frequently requested first , as it provides useful information for immediate patient management . 
la tcsm ha il grande pregio di fornire una visione panoramica di tutto il torace e di riconoscere differenti patologie ( atelettasie , focolai broncopneumonici , focolai emorragici ed enfisema )  . 
nei pazienti con spp anormale in cui la tcsm ha mostrato coesistenti patologie cardio - polmonari lintegrazione dei dati pu permettere di attribuire o meno significato embolico alle alterazioni di perfusione riscontrate alla spp [ 19 ]  . in letteratura sono stati suggeriti diversi algoritmi diagnostici sullimpiego delle differenti tecniche di diagnostica per immagini nellepa . 
i criteri di scelta delliter diagnostico devono , tuttavia , tener conto del valore clinico , della reale disponibilit delle diverse indagini , della situazione clinica del paziente ( stabile o instabile ) e della presenza di concomitanti patologie cardio - polmonari . 
this lack of concordance between the two techniques may be explained by the presence of partial occlusions causing no significant changes in perfusion and , subsequently , of little or no functional significance . 
this lack of concordance was identified in 3.5% ( 2 of 57 ) of patients who had undergone pls first and then msct , studied in the 0to 48 - h interval , and in 6.54% ( 7 of 107 ) of patients studied within 7 days of initial clinical suspicion . 
activation of thrombolytic mechanisms with embolus fragmentation during the 27 day period after the initial clinical suspicion may explain this lack of concordance . no patient who underwent both exams within 24 h of the initial clinical suspicion had a positive pls and a negative msct . 
msct has the advantage of providing a panoramic view of the entire chest and visualising different diseases ( atelectasis , bronchopulmonary foci , haemorrhagic foci , and emphysema )  . 
in patients with an abnormal pls , in which msct demonstrated concurrent cardiopulmonary diseases , integration of the data may be necessary to decide whether or not the perfusion alterations seen at pls are ascribable to embolism [ 19 ]  . several diagnostic imaging algorithms have been suggested for the detection of ape . 
other authors suggest the use of msct in cases of pls alterations not typical of pulmonary embolism , advising against its use as a stand - alone technique [ 20 , 21 ]  . however , the criteria for planning the diagnostic workup must take into account the clinical value of techniques , their availability , the patients clinical condition ( stable or unstable ) and the presence of concurrent cardiopulmonary diseases . 
on the other hand , in stable patients with complex radiologic findings , msct should be performed first , because the chances of a nondiagnostic scintigraphy are high in these cases . 
however , msct is preferred for patients with preexisting lung disease or when a differential diagnosis with mediastinal diseases is needed . table 7 results for patients undergoing perfusion lung scintigraphy ( pls ) and multislice computed tomography ( msct ) within 27 days msctmsct + total no . 
of patients with positive exams patients who underwent pls first : 15 patients who underwent msct first : 7 024 h 12 / 49 ( 24.48% ) 2448 h 3 / 20 ( 15.00% ) 27 days 7 / 38 ( 18.42% ) tabella 9 sequenza temporale degli esami spp e tcsm dei 22 pazienti con concordanza positiva raggruppati in base allintervallo di tempo tra insorgenza del sopetto clinico e completamento degli esami di diagnostica per immagini intervallo tra le due indagini numero pz con spp e tcsm positive pz che hanno effettuato prima la spp : 15 pz che hanno effettuato prima la tcsm : 7 024 ore 12 / 49 ( 24 , 48% ) 2448 ore 3 / 20 ( 15 , 00% ) 27 gg 7 / 38 ( 18 , 42% ) conclusions conclusioni in a large group of patients recruited in hospitals with round - the - clock emergency nuclear medicine and radiology services and that follow strict image interpretation criteria , pls and msct proved to have a high concordance when the exams were performed within 7 days of the initial clinical suspicion of ape . 
therefore , a clinical suspicion of ape needs to be confirmed by emergency examinations performed within hours 48 at the most . when both pls and msct are available , pls is the first exam to be ordered for a clinical suspicion of ape . 
although no diagnostic protocol has been universally adopted , pls is the first technique to identify the site and extent of the embolism , thus providing indirect evidence of pulmonary embolismsct is an accurate technique for the diagnosis of ape , and because it can provide information even on other pathological processes , it can confirm or exclude the diagnosis suspected at pls . when both diagnostic techniques are not available , either pls or msct are valuable tools for identifying the presence of severe ape necessitating urgent treatment . in unampia casistica di pazienti arruolati in grandi ospedali con centri di medicina nucleare e radiodiagnostica che effettuano indagini 24 ore su 24 , utilizzando rigorosi criteri di interpretazione delle immagini , la spp e la tcsm hanno dimostrato unelevata concordanza quando eseguite entro 7 giorni dallinsorgenza del sospetto di epa . 
la concordanza pi elevata se le due indagini sono completate entro 2448 ore . pertanto nel sospetto clinico di epa importante accertare la diagnosi con esami durgenza in poche ore , massimo 48 . la spp il primo esame richiesto nel sospetto clinico di epa quando sono disponibili entrambe le tecniche diagnostiche . 
pur non essendo unanimemente accettato un protocollo diagnostico , la spp rappresenta la tecnica di primo approccio per stabilire la sede e lestensione del processo embolico , fornendo levidenza indiretta dellembolia polmonare . 
bartolozzi1 1division of diagnostic and interventional radiology , 2division of pathology , university of pisa , via roma 67 , i - 56100 pisa , italy 3azienda ospedaliera universitaria pisana , pisa , italy correspondence to : c . 
fifty - five patients with mammographic microcalcifications classified as bi - rads categories 3 , 4 or 5 underwent mri and biopsy with stereotactic vacuum - assisted biopsy ( vab )  . 
mri was performed with a 1.5 - tesla ( t ) unit , and t1 coronal three - dimensional ( 3d ) fast low - angle shot sequences were acquired before and after injection of paramagnetic contrast agent ( 0.1 mmol / kg )  . 
lesame rm stato eseguito con una unit da 1 , 5 tesla e sono state acquisite sequenze coronali t1 3d fast lowangle shot sequences prima e dopo liniezione di mezzo di contrasto paramagnetico ( 0 , 1 mmol / kg )  . 
lesame istologico ha rilevato 26 cancri duttali in situ ( cdis ) e cancri duttali microinvasivi ( cd mic ) , 3 iperplasie duttali atipiche ( ida ) e 26 condizioni benigne . 
la sensibilit della mammografia risultata 77% , la specificit stata 59% , il valore predittivo positivo ( vpp ) 61% , il valore predittivo negativo ( vpn ) 74% , laccuratezza diagnostica 67 , 2% . 
la sensibilit globale della rm stata 73% , la specificit 76% , il valore predittivo positivo ( vpp ) 73% , il valore predittivo negativo ( vpn ) 76% , laccuratezza diagnostica 74 , 5% . 
during the last 20 years , the prevalence of dcis has grown from less than 5% before the start of mammographic screening to 15%30% in women regularly checked with mammography [ 6 ]  . 
the 30%50% decrease in mortality is related to early diagnosis and correct management [ 7 ]  . mammography has high sensitivity and low specificity , the positive predictive value ( ppv ) being 15%30% for malignant nonpalpable lesions [ 6 ]  . 
recent studies have shown that breast magnetic resonance imaging ( mri ) with intravenous contrast injection has outstanding sensitivity for the diagnosis of invasive carcinomas ( 88%100% ) and a high negative predictive value ( npv ) [ 6 ]  . 
 considering that dcis is multifocal in more than 50% of cases and bilateral in about 30% of patients [ 1 , 15 ] , and also considering that the possible evolution into an infiltrating form depends on the histological subtype and size and adequacy of resection [ 16 , 17 ] , mri could help in early diagnosis and surgical planning , giving useful information about disease extension [ 14 ]  . 
the aim of our study was to evaluate the role of mri in patients with breast imaging reporting and data systems ( bi - rads ) 35 microcalcifications . la dimostrazione di microcalcificazioni spesso il solo segno del tumore , essendo il primo indizio riscontrato alla mammografia in circa il 70% dei carcinomi duttali in situ ( dcis ) [ 15 ]  . 
durante gli ultimi venti anni , la prevalenza del dcis cresciuta da meno del 5% prima dellinizio dello screening mammografico al 15%30% nella popolazione esaminata regolarmente con la mammografia [ 6 ]  . 
la riduzione della mortalit del 30%50% in relazione con la diagnosi precoce e con il corretto trattamento [ 7 ]  . la mammografia ha un alta sensibilit ed una bassa specificit , infatti il valore predittivo positivo ( vpp ) del 15%30% per le lesioni maligne non palpabili [ 6 ]  . 
recenti studi hanno dimostrato che la risonanza magnetica della mammella ( rm ) con somministrazione di mezzo di contrasto per via endovenosa , ha una notevole sensibilit nella diagnosi del carcinoma invasivo ( 88%100% ) ed un alto valore predittivo negativo ( vpn ) [ 6 ]  . 
invece dimostrata lutilit della rm nella pianificazione chirurgica [ 14 ]  . considerando che il dcis multifocale in pi del 50% dei casi , bilaterale in circa il 30% delle pazienti [ 1 , 15 ] e che la possibile evoluzione in una forma infiltrante dipende dal sottotipo istologico , dalle dimensioni e dalladeguatezza della exeresi chirurgica [ 16 , 17 ] , la rm potrebbe essere di aiuto nella diagnosi precoce e nella pianificazione dellintervento , dando informazioni utili sullestensione della malattia [ 14 ]  . lo scopo del nostro studio quello di valutare il ruolo della rm in pazienti con microcalcificazioni birads 35 . materials and methods we selected 55 patients ( age range 3776 years , mean 5611 years ) with bi - rads 35 microcalcifications on mammography who underwent mri and stereotactic biopsy using vacuum - assisted biopsy ( vab ) between november 2002 and november 2004 . 
mammographic magnification was performed in the view that best visualised the microcalcifications , and the microcalcification extension was evaluated in the standard views . all mammograms were evaluated in a blinded fashion by two radiologists with 15 years experience in breast imaging . materiali e metodi abbiamo selezionato 55 pazienti ( et compresa tra 37 e 76 anni , et media 5611 anni ) con microcalcificazioni birads 35 alla mammografia , che si sono sottoposte ad esame rm e a biopsia sotto guida stereotassica con tecnica di retroaspirazione ( vacuum assisted biopsy : vab ) da novembre 2002 a novembre 2004 . 
i reperti controversi sono stati ulteriormente valutati da entrambi i radiologi per raggiungere un consenso . il nostro gold standard stata la microistologia e listologia per le pazienti sottoposte ad intervento chirurgico . 
lo studio rm stato sempre eseguito prima della microistologia . la rm stata effettuata con un apparecchio da 1 , 5 t ( symphony , siemens ) in tutte le pazienti tranne in 6 in cui stata usata una macchina da 1 , 5 t ( ge , medical system )  . tutte le pazienti sono state studiate in posizione prona utilizzando una bobina dedicata per la mammella . 
il protocollo di studio rm ha incluso un sequenza trasversale t1 di localizzazione , una sequenza sagittale t2 con saturazione del grasso ed una sequenza t1 gradient eco 3d - flash ( te 2 , 5 ms , tr 12 , 7 ms , tempo di acquisizione meno di 2 minuti , fa 30 , spessore di strato 3 mm , interslice gap 0 mm , nex 1 , fov 35 , dimensione della matrice 256160 , dimensione del pixel 1 , 41 , 4 ) , con una serie di 6 acquisizione coronali . 
stato usato il gadoteridolo ( prohance , bracco , milano ) come agente di contrasto paramagnetico con boli di iniezione di 0 , 1 mmol / kg di peso corporeo . 
i processi di rielaborazione delle immagini usate sono stati : sottrazione ; ricostruzioni mip ; ricostruzioni mpr ; curve intensit / tempo nella regione di interesse . sono state identificate secondo lo score di fisher 3 tipi di curve . 
i risultati dello studio rm sono stati classificati secondo lo score di fisher in 5 categorie bi - rads : rm 12 ( fisher score 02 ) : negativo - benigno ; rm 3 ( fisher score 3 ) : probabilmente benigno ; rm 45 ( fisher score 48 ) : probabilmente maligno / maligno . sono state eseguite biopsie sotto guida stereotassica usando il mammografo giotto ims equipaggiato con tavolo prono ed un sistema di aspirazione mammotome ethicon endo - surgery ( amburgo ) con ago di 11 gauge ( g )  . 
il materiale stato inviato allesame microistologico in due differenti contenitori separando i frustoli con le microcalcificazioni da quelli senza microcalcificazioni evidenziate dalla radiografia . lintervento chirurgico stato effettuato in quelle pazienti con diagnosi microistologica di malignit o di lesione altamente sospetta . 
mammografia : le proiezioni cranio - caudale ( cc ) e medio - laterale obliqua ( mlo ) mostrano microcalcificazioni nei quadranti superoed infero - esterni birads 4 e 5 . all cases of microcalcifications were classified according to the method proposed by the american college of radiology [ 18 ] , and those classified as bi - rads 35 were selected . controversial findings were re - evaluated by the two radiologists to reach a consensus . 
mri imaging was performed with 1.5 - tesla ( t ) unit ( symphony , siemens ) in all patients but six , who were examined with a 1.5 - t unit ( ge medical system )  . 
the imaging protocol included a transverse t1 localising sequence , a sagittal fat - saturated t2 sequence and a t1 three - dimensional ( 3d ) fast low - angle shot ( flash ) gradient echo ( te 2.5 ms , tr 12.7 ms , acquisition time less than 2 min , fa 30 , slice thickness 3 mm , interslice gap 0 mm , nex 1 , fov 35 , matrix size 256160 , pixel size 1.41.4 ) , with a series of six coronal acquisitions . 
rm : le immagini mpr sagittali ( a ) ed assiali ( b ) e le mip assiali ( c ) evidenziano multiple aree di captazione contrastografica dendritica e nodulare che interessano lintera mammella sinistra . mmol / kg of body weight . 
postprocessing techniques used were : subtraction maximum intensity projection ( mip ) reconstructions multiplanar reformatting ( mpr ) reconstructions time - intensity curves in the region of interest ( roi )  . three types of curves were identified according to fisher score parameters considering enhancement morphology , we identified the following possibilities : absent enhancement , dendritic enhancement , nodular enhancement and coexistence of dendritic and nodular enhancement . 
results of the mri study were classified into five bi - rads categories according to the fisher score : mri 12 ( fisher score 02 ) negative / benign , mri 3 ( fisher score 3 ) probably benign and mri 45 ( fisher score 48 ) : probably malignant / malignant . biopsy was performed under stereotactic guidance using the giotto ims mammography system equipped with a prone table and a mammotome ethicon endo - surgery ( hamburg ) aspiration device with an 11 - gauge needle . 
considerando la difficolt di una valutazione accurata dellestensione del cdis allanatomia patologica , abbiamo diviso la mammella alla mammografia e alla rm in 5 porzioni : quadrante supero - esterno , quadrante supero - interno , quadrante infero - interno , quadrante infero - esterno , regione retroareolare e abbiamo confrontato tumorale evidenziata lestensione dallimaging con lestensione riscontrata allistologia . risultati la microistologia ha riscontrato 26 lesioni maligne , 29 lesioni benigne incluse 3 iperplasie duttali atipiche ( ida ) ( tabella 1 )  . 
la chirurgia eseguita nelle 29 pazienti ( 17 con carcinoma duttale in situ , cdis , 9 con cancro duttale microinvasivo , cdi mic , e 3 con ida ) , ha confermato in tutti i casi i dati microistologici . 
magnetic resonance imaging ( mri ) : regions of interest in the subtracted images show multiple areas of contrast enhancement with types iii and ii time - intensity curves [ breast imaging reporting and data systems ( bi - rads ) 5 ]  . 
rm : regioni di interesse nelle immagini sottratte post - contrastografiche mostrano multiple aree di captazione contrastografica con curve intensit / tempo di tipo ii e iii ( birads 5 )  . 
stata quindi eseguita una mastectomia sinistra e listologia ha rivelato multipli foci di cdis g2 e g3 . material was sent for microhistological evaluation in two separate containers , one containing the specimens with microcalcifications and the other specimens without microcalcifications . 
in consideration of the difficulties in accurately evaluating dcis extent at pathology , we decided to divide the breast at mammography and mri into five portions : upperouter quadrant , upper - inner quadrant , lower - outer quadrant , lower - inner quadrant and retroareolar region ; we compared si e g3 in 11 casi . 
surgery , performed in 29 patients [ 17 with dcis , nine with ductal microinvasive cancer ( dcmic ) and three with adh ] , confirmed the microhistological data in all cases . 
among the 55 patients studied , mammographic findings were classified as birads 3 : 23 cases , bi - rads 4 : 25 cases andbi - rads 5 : seen cases . 
of the 48 patients with mri contrast enhancement , the time - intensity curve correlated with histology was type i in ten benign lesions and one malignant lesion , type ii in nine benign lesions and nine malignant lesions and type iii in four benign lesions and 15 malignant lesions . 
in the 26 cancers , enhancement morphology logia 17 lesioni benigne e 6 maligne , nelle mammografie birads 4 abbiamo riscontrato 11 lesioni benigne e 14 maligne , nelle mammografie bi - rads 5 abbiamo riscontrato 1 lesione benigna e 6 maligne . 
nelle 48 pazienti con captazione contrastografica alla rm , la curva intensit / tempo correlata allistologia era di tipo i in 10 lesioni benigne e in 1 lesione maligna , di tipo ii in 9 lesioni benigne ed in 9 lesioni maligne e di tipo iii in 4 lesioni benigne e 15 lesioni maligne . nelle 7 pazienti che non presentavano captazione di mezzo di contrasto alla rm , 6 casi risultavano allistologia lesioni benigne e 1 caso cdis ( tabella 4 )  . 
nei 26 cancri la morfologia della captazione contrastografica risultata dendritica in 3 casi , nodulare in 8 casi , sia dendritica che nodulare in 14 casi e completamente assente in un caso ( tabella 5 )  . 
dei 9 casi di cdi mic la maggior parte dei casi avevano una captazione sia dendritica che nodulare ( n = 5 ) ; nei restanti casi la captazione era dendritica ( n = 2 ) e nodulare ( n = 2 )  . per quanto riguarda le forme maligne : nel 3 , 8% dei casi non era presente captazione contrastografica , nel 3 , 8% dei casi le curva intensit / tempo erano di tipo i , nel 34 , 6% le curve intensit / tempo erano di tipo ii e nel 57 , 7% di tipo iii . la sensibilit della mammografia stata del 77% ( 20 / 26 ) , la specificit del 59% ( 17 / 29 ) , il valore predittivo positivo ( vpp ) risultato del 63% ( 20 / 32 ) , il vpn del 74% ( 17 / 23 ) , laccuratezza diagnostica risultata del 67 , 2% ( 37 / 55 )  . 
dei 12 casi di falsi positivi in mammografia con bitable 1 histological results tabella 1 risultati istologici histology dcis / dc mic atypical ductal hyperplasia typical ductal hyperplasia fibroadipose tissue sclerosing adenosis fibroadenoma typical epithelium total istologia cdis / cd mic iperplasia duttale atipica iperplasia duttale tipica tessuto fibroadiposo adenosi sclerosante fibroadenoma epitelio tipico totale dcis , ductal carcinoma in situ ; dcmic , ductal microinvasive carcinoma cdis , carcinoma duttale in situ ; cd mic , carcinoma dentale microinvasivo a . 
as regards the nine dc mic , the majority of cases showed both nodular and dendritic enhancement ( n = 5 ) ; in the remaining cases , enhancement was dendritic ( n = 2 ) and nodular ( n = 2 )  . 
among the five false negative cases of mammographic bi - rads category 3 , mri correctly identified three cancers . of the 12 false positive cases of mammographic bi - rads category 45 , mri correctly identified nine benign lesions . however , the fisher exact test did not show a statistically rads 4 - 5 , la rm ha identificato correttamente 9 lesioni benigne . 
per quanto riguarda lestensione della neoplasia , confrontando i reperti mammografici , rm e listologia abbiamo riscontrato : una concordanza dellestensione neoplastica alla rm e allistologia in 22 / 26 casi ( 84 , 6% ) ; una sovrastima della rm in 3 / 26 casi ( 11 , 5% ) ; in 1 / 26 assenza di captazione contrastografica alla rm per cui non stato possibile valutare lestensione della malattia . abbiamo avuto una sottostima dellestensione neoplastica alla mammografia in 16 / 26 ( 73% ) e una corretta valutazioa . 
with regard to disease extension , comparison of mammographic and mri findings with histology found concordance between mri and histological disease extent in 22 / 26 cases ( 84.6% ) ; overestimation at mri in 3 / 26 cases ( 11.5% ) ; and lack of mri contrast uptake in 1 / 26 cases , preventing evaluation of disease extension . 
nellanalisi dellestensione della neoplasia la rm risultata migliore della mammografia ( test z : p < 0 , 01 )  . in tutti i casi eccetto uno ( multicentrico alla rm e multifocale alla mammografia ) lapproccio chirurgico stato deciso in base allesame rm . 
pathology mammography magnetic resonance imaging tabella 8 confrontro tra estensione neoplastica allimaging e in anatomia patologica mammografia risonanza magnetica sottostima rispetto allanatomia patologica sovrastima rispetto allanatomia patologica valutazione corretta rispetto allanatomia patologica on the basis of the mri findings . 
a mammography : craniocaudal and mediolateral oblique views of the left breast show a cluster of microcalcifications [ breast imaging reporting and data systems ( bi - rads ) 4 ] in the upper outer quadrant . 
b subtracted magnetic resonance imaging ( mri ) : dendritic and nodular irregular enhancement with types i and ii time - intensity curves with low increase of contrast enhancement [ breast imaging reporting and data systems ( bi - rads 4 ) ]  . 
a mammografia : le proiezioni cranio - caudale e medio - laterale obliqua della mammella sinistra mostrano un cluster di microcalcificazioni ( birads 4 ) nel quadrante supero - esterno . 
b immagini rm sottratte : irregolare captazione contrastografica dendritica e nodulare con curve intensit / tempo i e ii con basso incremento della captazione contrastografica ( birads 4 )  . 
a mammography : mediolateral oblique view shows microcalcifications in the retro - areolar region [ breast imaging reportingand data systems ( bi - rads ) 5 ] and other foci ( bi - rads 4 ) in the inner and outer quadrants . 
b magnetic resonance imaging ( mri ) maximum intensity projection ( mip ) : coronal and sagittal views show nodular enhancement in the upper quadrants in the para - areolar region of the right breast , associated with vascular asymmetry ipsilateral to the area of enhancement . 
c subtracted magnetic resonance imaging ( mri ) : nodular contrast enhancement with type iii time - intensity curve [ breast imaging reporting and data systems ( bi - rads ) 5 ] , vacuum - assisted biopsy of retroareolar microcalcifications : g3 dcis . 
a mammografia : la proiezione medio - laterale obliqua mostra microcalcificazioni in regione retroareolare ( birads 5 ) ed altri foci ( birads 4 ) nei quadranti infero - esterni . 
b rm : le immagini mip coronali e sagittali mostrano captazione contrastografica nodulare nei quadranti superiori in regione para - areolare nella mammella destra associata ad unasimmetria vascolare ipsilaterale allarea di captazione contrastografica . 
in one case , surgical treatment was planned on the basis of the mammographic findings rather than considering the mri finding of multifocality , and the patient had a recurrence of disease 1 year later . discussion many studies on dynamic mri [ 6 ] have reported high sensitivity values ( 90%100% ) for the diagnosis of invasive carcinoma , with a specificity of 80% [ 14 , 1923 ]  . 
concerning sensitivity and specificity of mri in the diagnosis of dcis and evaluation of isolated microcalcifications , the published values are ( sensitivity : lower and highly variable 33%100% ) [ 21 , 2426 ]  . 
the relatively low number of dcis cases included in the published studies [ 20 , 21 ] seems to be related to the reluctance of investigators to enrol patients with radiological evidence of microcalcifications . 
 the high variability of mri diagnostic accuracy in evaluating microcalcifications reported in the different studies is related to lesion size [ 6 , 27 , 28 ] , histological variability of tumours [ 20 , 24 ] , tumour angiogenesis [ 24 , 29 , 30 ] , criteria for mri evaluation such as type of enhancement , enhancement pattern , distribution in the breast ( multiple foci ) , margins ( regular , irregular ) [ 21 , 24 , 27 , 30 , 31 ] and use of different pulse sequences and scan planes [ 23 ]  . 
 [ 32 ] , for example , in an analysis of morphological and dynamic features of in situ and microinvasive breast cancer on contrast - enhanced mri , detected the typical enhancement criteria in only 50% of cases , without a significant statistical difference in enhancement pattern among the various histological grades of dcis and between pure and microinvasive dcis . 
in our study , the sensitivity of contrast - enhanced mri , according to the fisher score , was higher ( 77% vs . 50% in viehwegs study ) , with only one false negative mri result corrected by mammography . 
however , the false negative ( n = 7 ) and false positive ( n = 7 ) mris strengthen the concept that mammography is the technique of choice for detecting microcalcifications and that stereotactic biopsy is mandatory for characterising bi - rads 35 microcalcifications . 
information from conventional imaging and in particular mammography is useful to avoid mri false negative results . various studies [ 25 , 32 ] suggest a role for contrast - enhanced mri in the detection of dcis , especially for depicting further tumour foci close to or far from the main lesion that are not visible on mammography due to the absence of microcalcifications or masses or distortions [ 33 , 34 ]  . 
at the same time , it is well known that some areas of dcis can be completely noncalcified and that some sites of dcis can contain calcificadiscussione numerosi studi sulla rm dinamica [ 6 ] hanno riportato elevate valori di sensibilit ( 90% - 100% ) nella diagnosi di carcinoma invasivo , con una specificit dell80% [ 14 , 1923 ]  . riguardo alla sensibilit e specificit dellimaging rm nella diagnosi del carcinoma duttale in situ ( cdis ) e nella valutazione delle microcalcificazioni isolate , i valori pubblicati in letteratura sono pi bassi e variabili ( sensibilit : 33%100% ) [ 21 , 2426 ]  . 
il numero relativamente basso di cdis incluso negli studi pubblicati [ 20 , 21 ] sembra da correlare allavversione ad arruolare allesame rm pazienti con evidenza radiologica di microcalcificazioni . lalta variabilit dellaccuratezza diagnostica nella valutazione delle microcalcificazioni , fino ad ora pubblicata in diversi studi , correlata a : dimensioni della lesione [ 6 , 27 , 28 ] , variabilit istologica dei tumori [ 20 , 24 ] , angiogenesi tumorale [ 24 , 29 , 30 ] , criteri di valutazione della rm come tipo e morfologia della captazione contrastografica , distribuzione nella mammella ( foci multipli ) , margini ( regolari , irregolari ) [ 21 , 24 , 27 , 30 , 31 ] e tecnica di risonanza come differenti sequenze e piani di scansioni [ 23 ]  . 
nel nostro studio la sensibilit riportata per la rm dinamica , in accordo allo score di fischer , risultata pi alta ( 77% rispetto al 50% di viehweg ) con un solo caso di falso negativo corretto dalla mammografia . 
tuttavia i falsi negativi ( n = 7 ) e i falsi positivi ( n = 7 ) rafforzano il concetto che la mammografia la tecnica di scelta per individuare le microcalcificazioni e che la biopsia stereotassica obbligatoria per la caratterizzazione delle microcalcificazioni birads 35 . 
informazioni dallimaging tradizionale ed in particolare dalla mammografia sono utili per evitare i falsi negativi della rm . inoltre vari studi [ 25 , 32 ] suggeriscono un ruolo per la rm con somministrazione di mezzo di contrasto nella diagnosi di cdis specialmente per individuare ulteriori foci neoplastici vicini o lontani dalla lesione principale , che non sono visibili alla mammografia per lassenza di microcalcificazioni , masse o distorsioni [ 33 , 34 ]  . 
questi dati sono particolarmente interessanti se consideriamo levidenza di multifocalit ( 53% ) e multicentricit ( 27% ) osservate in pazienti sottoposte a mastectomia [ 30 , 35 ]  . 
la maggior prevalenza di multicentricit osservata nel cdis che nel cdi e la progressione delle forme in situ non adeguatamente trattate in forme di cancro invasivo richiede unaccurata valutazione dellestensione di malattia specialmente in donne candidate a chirurgia mammaria a . 
the higher prevalence of multicentricity observed in dcis than in dci and the progression of inadequately treated dcis into invasive forms require accurate evaluation of disease extension , especially in cases of candidates to breast - conserving surgery [ 36 , 37 ]  . 
even if the biological and clinical impact of these small foci of dcis is uncertain [ 3840 ] , according to the literature [ 41 ] , the risk of local recurrence after conservative therapy is high in patients with large multifocal tumours and low if the multifocal neoplasm is microscopic . furthermore , it is well known that histological measurement of dcis is difficult due to the difficulty in following a duct on serial sections . 
as regards histological grading , increased vascularisation was associated with more greatly differentiated dcis , which are well detected by contrast - enhanced mri , with a high positive predictive value for g3 and g2 forms . 
 even though our study has a selection bias due to the mammographic detection of microcalcifications , our data suggest that the high ppv of mri in g3 dcis can be useful mainly to depict further dcis foci not associated with mammographic signs . 
these data are in keeping with a recent publication [ 21 ] that emphasised the sensitivity of contrastenhanced mri in the detection of high - grade dcis ( g2 and g3 )  . 
however , recent studies have demonstrated that the same genetic profile is correlated with the same histological grade in both in situ and invasive cancers [ 39 , 43 ]  . 
mri could therefore be useful , especially in identifying the most aggressive forms , thereby helping in surgical planning and once the natural course of the disease and the different biological potential of dcis subtypes are better understood possibly in improving the prognosis , as early detection and correct treatment are the most important prognostic factors in breast cancer . conclusions mri cannot be considered a diagnostic tool for evaluating microcalcifications . 
anche se ad oggi limpatto clinico e biologico di questi piccoli foci di cdis incerto [ 3840 ] , in accordo ai dati di letteratura [ 41 ] il rischio di recidiva locale dopo chirurgia conservativa alto nelle pazienti con grossi tumori multifocali e basso se la neoplasia multifocale microscopica . inoltre ben noto che il cdis difficile da misurare istologicamente per la difficolt a seguire un dotto interessato in sezioni seriali . 
per quanto riguarda il grading istologico nella nostra esperienza abbiamo osservato una vascolarizzazione aumentata associata a forme di cdis pi differenziate che sono ben individuate con la rm dinamica , con elevato valore predittivo positivo per le forme g2 e g3 . anche se il nostro studio ha un bias nei criteri di inclusione rappresentato dallindividuazione mammografica delle microcalcificazioni , i nostri dati suggeriscono che lelevato valore predittivo positivo della rm nei casi di cdis g3 pu essere utile soprattutto ad individuare i foci ulteriori di cancro in situ non associati a segni mammografici . 
questi dati sono allineati con una recente pubblicazione [ 21 ] che enfatizza la sensibilit della rm dinamica nella diagnosi delle forme di cancro in situ di alto grado ( g2 e g3 )  . 
cos la rm potrebbe essere utile nell identificare specialmente le forme pi aggressive di cdis aiutando nella pianificazione dellintervento chirurgico e , una volta che siano chiariti il corso naturale della malattia e che i differenti potenziali biologici dei sottotipi di cdis , probabilmente migliorare la prognosi . infatti la diagnosi precoce del cancro mammario ed il corretto trattamento sono i fattori prognostici pi importanti . conclusioni la rm non pu essere considerata lo strumento diagnostico per valutare le microcalcificazioni . 
la malformazione vascolare polmonare stata chiusa in 3 minuti e la saturazione percutanea di ossigeno passata dal 73% al 93% . la fistola da emodialisi stata chiusa con 1 dispositivo in 4 minuti . 
lavp un efficace sistema per la chiusura di grossi vasi periferici e di malformazioni vascolari , a costi contenuti , che consente unembolizzazione sicura e a basso rischio di complicanze , con risparmio in termini di tempo e di dose erogata . key words amplatzer vascular plug transcatheter embolisation vascular disease parole chiave amplatzer vascular plug embolizzazione transcatetere patologia vascolare introduction introduzione percutaneous embolisation has become a valid option and often the first - line treatment for acute bleeding , temporary or permanent devascularisation of benign or malignant hypervascular tumours [ uterine fibroids , hepatocellular carcinoma ( hcc ) ] , the closure of congenital or acquired arteriovenous lembolizzazione percutanea divenuta ormai una valida alternativa e spesso la metodica di prima istanza nel trattamento di sanguinamenti acuti , ma anche nella devascolarizzazione temporanea o permanente di tumori ipervascolarizzati benigni o maligni ( fibromi uterini , hcc ) , nella chiusura c . 
several embolisation devices are available , each with its own characteristic features : a fibrin sponge is used for temporary ( about 48 h ) embolisation , polyvinyl alcohol ( pva ) microspheres are commonly used for permanent embolisation , whereas detachable balloons , glue , metal coils and plugs are used for definitive embolisation . this paper describes our experience with a new system for definitive embolisation , the amplatzer vascular plug ( avp ) , a versatile device that was successfully used in the closure of arteries ( internal iliac and subclavian ) , a pulmonary arteriovenous malformation , a haemodialysis fistula , and a gastric varix in a subject with portal hypertension . materials and methods the avp ( aga medical corporation , golden valley , mn , usa ) is a new device approved by the u.s. 
food and drug administration for peripheral embolisation of arteries and veins and that uses the same technology as other amplatzer devices used for occluding interatrial and interventricular defects and patent foramen ovale . 
the system is preloaded inside a 100 - cm - long guiding catheter : 5 fr for plugs with a diameter of 48 mm , 6 fr for plugs with a diameter of 1012 mm and 8 fr for plugs with a diameter of 1416 mthe system ranges in size from 4 mm di malformazioni vascolari arterovenose congenite o acquisite e nella chiusura di varici . 
esistono in commercio numerosi dispositivi embolizzanti ciascuno con caratteristiche preponderanti rispetto agli altri : per lembolizzazione temporanea si utilizza la spugna di fibrina ( 48 h circa ) ; per quella permanente sono comunemente impiegate le microsfere in alcol polivinilico ( pva ) ; per lembolizzazione definitiva infine ci si avvale dei palloni staccabili , della colla , delle spirali metalliche e dei plug . gli autori descrivono la loro esperienza nellutilizzo di un nuovo sistema per lembolizzazione definitiva , lamplatzer vascular plug ( avp ) , un dispositivo dallutilizzo versatile , specie nei vasi di grosso diametro , che stato utilizzato con successo nella chiusura di arterie ( iliache interne e succlavie ) , di una malformazione arterovenosa polmonare , di una fistola da emodialisi e di una varice gastrica in soggetto con ipertensione portale . materiali e metodi lavp ( aga medical corporation , golden valley , mn , usa ) un nuovo dispositivo approvato dalla food and drug administration per lembolizzazione periferica di vasi arteriosi e venosi che utilizza la stessa tecnologia di altri device amplatzer utilizzati per occludere difetti interatriali , interventricolari e forami ovali . 
il sistema caricato allinterno di un catetere portante ( 100 cm di lunghezza ) da 5 fr per plug da 4 a 8 mm di diametro , da 6 fr per plug da 10 a 12 mm di diametro e da 8 fr per plug da 14 a 16 mm di diametro . 
il sistema disponibile in misure che vanno da 4 mm di diametro fino a 16 mm con un incremento di 2 muna volta raggiunta la sede in cui si vuole rilasciare il dispositivo basta ritirare il catetere in modo da far uscire il plug e girare in senso antiorario la guida metallica per liberare il device . 
in accordo con la casa produttrice si raccomanda di scegliere un dispositivo con un oversize almeno del 20% rispetto al diametro del vaso da trattare per evitare una possibile migrazione distale . 
le peculiarit del sistema sono la sua disponibilit in unampia gamma di misure , anche molto grandi , la possibilit di riposizionamento prima del rilascio definitivo , il minor rischio di migrazione rispetto alle spirali e un breve tempo per occludere definitivamente il vaso . presso il nostro centro sono state eseguite 11 occlusioni vascolari con lutilizzo del nuovo dispositivo avp : 5 arterie iliache interne nella prevenzione dellendoleak di tipo ii dopo posizionamento di endoprotesi aortoiliaca ; 3 arterie succlavie di sinistra per il trattamento di endoleak di ii tipo successivo al posizionamento di endoprotesi dellaorta toracica con copertura dellarteria succlavia sinistra ( ass ) ; 1 fistola arterovenosa polmonare ; 1 fistola da emodialisi e 1 grossa varice in un paziente con emorragia da varice gastrica portatore di tips . 
positive features include the availability of a wide range of sizes , even very large ; the possibility of repositioning the device before its definitive release ; lower risk of migration compared with coils ; and short occlusion times . 
 our centre performed 11 vascular occlusions using the new avp : five internal iliac arteries to prevent type ii endoleaks after aortoiliac stent - graft placement , three left subclavian arteries to treat type ii endoleaks after thoracic aorta stent - graft placement with coverage of the left subclavian artery ( lsa ) , one pulmonary arteriovenous malformation , one haemodialysis fistula , and one large gastric varix due to haemorrhage in a patient with transjugular intrahepatic portosystemic shunt ( tips )  . 
the patients were studied with a multidetector computed tomography ( mdct ) scanner ( somatom sensation 6 , siemens , germany ) , mdct workstation ( leonardo , siemens , germany ) , angiographic equipment ( axiom fa , siemens , germany ) and ultrasound ( us ) ( technos , esaote , italy )  . 
chest mdct done to assess the pulmonary arteries showed a large solitary arteriovenous fistula with two afferent arteries arising from two branches of the right lower lobar pulmonary artery and drainage into the inferior pulmonary vein of the right lower lobe . 
la mdct del torace effettuata per la valutazione delle arterie polmonari ha dimostrato una grossa e solitaria fistola arterovenosa polmonare con due arterie afferenti , provenienti da due rami dellarteria polmonare lobare inferiore di destra , e drenaggio nella vena polmonare inferiore del lobo inferiore del polmone destro . 
langiografia selettiva dellarteria polmonare destra , eseguita attraverso la vena femorale comune di destra , ha confermato la diagnosi posta con la mdct dimostrando un diametro da 10 mm e 4 mm delle due arterie afferenti . 
la chiusura del vaso di maggiori dimensioni stata eseguita utilizzando la tecnica standard gi descritta , con un avp da 12 mm ( oversize del 20% rispetto al vaso nativo ) , mentre la seconda arteria afferente , di minori dimensioni ( 4 mm di diametro ) , stata cateterizzata mediante la tecnica buddy wire : tale sistema consiste nellutilizzo di due guide allinterno dello stesso catetere , per garantire maggior portanza , facilitando il posizionamento del device in vasi a decorso pi complesso . 
 stata utilizzata una guida coronarica da 0 , 014 ( guidant spartacore middle weight ) come buddy wire che stata posizionata oltre il segmento scelto per il rilascio del dispositivo , dando stabilit al catetere , prima del passaggio del device . 
2a multidetector computed tomography ( mdct ) angiography of the abdominal aorta and iliac arteries : anterior volume - rendered ( vr ) image shows an aneurysm of the right common iliac artery ( arrowhead )  . 
c angiography 8 min after release of the avp shows the outline of the device ( arrows ) and complete absence of flow within the right internal iliac artery from the systemic blood flow . 
2a angio - mdct dellaorta addominale e delle arterie iliache : ricostruzione vrt ( volume rendering technique ) che dimostra la presenza un aneurisma dellarteria iliaca comune di destra ( testa di freccia )  . 
c controllo angiografico a 8 minuti dal rilascio dellamplatzer vascular plug , dimostra la sagoma del device rilasciato ( frecce ) e la completa assenza di flusso nellarteria iliaca interna destra dal circolo sistemico arterioso . 
3a angiography of the thoracic aorta after stent - grafting in a patient with type b dissection of the thoracic aorta demonstrates the presence of a type i + type ii endoleak ( arrowheads ) from the left subclavian artery . 
b follow - up multidetector computed tomography ( mdct ) angiography at 1 month ; sagittal maximum intensity projections ( mip ) image confirmed the type i + type ii endoleak ( arrowhead ) from the left subclavian artery and the presence of partial thrombosis of the false lumen ( * )  . 
c similar projection 5 min after release of the amplatzer vascular plug ( avp ) shows the outline of the device ( arrows ) , with the exclusion of the origin of the left subclavian artery , the patency of the left vertebral and internal mammary artery and the absence of endoleaks . 
3a angiografia dellaorta toracica dopo il posizionamento di endoprotesi toracica in un paziente con dissecazione dellaorta toracica di tipo b che dimostra un endoleak di tipo misto ( tipo i + ii ) ( teste di freccia ) dallarteria succlavia sinistra . 
b controllo angio - mdct a un mese , ricostruzione sagittale mip ( maximum intensity projections ) , che conferma la presenza di un endoleak di tipo misto ( tipo i + ii ) ( testa di freccia ) dallarteria succlavia sinistra e la presenza di trombosi parziale del falso lume ( * )  . 
c sucessivo controllo a 5 minuti dal rilascio dellamplatzer vascular plug che mette in evidenza la sagoma del device rilasciato ( frecce ) con la completa chiusura dellorigine dellarteria succlavia sinistra , la perviet dellarteria vertebrale e mammaria interna e lassenza dellendoleak . 
4a angiography shows a persistent passage of contrast material across the previously ligated haemodialysis arteriovenous anastomosis and two large aneurysms ( arrowheads ) that end in a cul - de - sac at the level of the basilic vesome collateral veins , with reverse flow , can be seen to originate from the efferent vein and course towards the hand ( arrows )  . 
b angiography performed 2 min after amplatzer vascular plug ( avp ) deployment shows a considerable reduction in the passage of contrast ( arrow ) through the device ( arrowhead ) into the basilic venote the good patency of the artery below . 
4a langiografia mette in evidenza la persistenza del passaggio di mdc attraverso lanastomosi arterovenosa da emodialisi precedentemente legata chirurgicamente e i due grossi aneurismi ( testa di frecce ) che terminano a fondo cieco a livello della vena basilica efferente : si notano inoltre alcune vene collaterali che si dipartono dalla vena efferente e si dirigono , a flusso invertito , verso la mano ( frecce )  . 
b il controllo angiografico eseguito dopo 2 minuti dallapertura del plug dimostra una marcata riduzione del passaggio di mdc ( freccia ) attraverso il dispositivo ( testa di freccia ) nella vena basilica efferente . 
c il controllo dopo altri 3 minuti evidenzia la totale chiusura della fistola , lassenza delle vene collaterali e la perviet dellasse arterioso a valle . 10 mm and 4 mm of the two afferent arteries . 
closure of the larger vessel was achieved by applying the standard technique , with a 12 - mm avp ( 20% larger than the native vessel ) , whereas the smaller afferent artery ( 4 mm ) was catheterised using the buddy wire technique , which consists of using two guidewires within the same catheter , to ensure better support and facilitate deployment of the device in more tortuous vessels . 
dopo aspirazione del trombo portale , effettuata mediante catetere tipo portante da 8 fr ( jr 4.0 , medtronic , mn , usa ) , stata eseguita una seconda angiografia che ha dimostrato la parziale riduzione del trombo e la persistenza di flusso nella varice : si quindi optato per la sua embolizzazione definitiva . 
5a portography shows , in a patient with transjugular intrahepatic portosystemic shunt ( tips ) , a filling defect ( arrowhead ) of the portal vein at the outlet of the superior mesenteric vein due to probable thrombus . 
5a portografia evidenzia , in un paziente portatore di tips , un difetto di riempimento ( testa di freccia ) della porta a livello dello sbocco della vena mesenterica superiore , da verosimile apposizione trombotica ; ben evidente inoltre la vena gastrica sinistra dilatata ( freccia ) con la presenza di spirali metalliche , da precedente embolizzazione . 
b posizionamento del plug ( testa di freccia ) allinterno della varice prima del rilascio definitivo : si riconoscono molto bene i due markers radiopachi posizionati alle due estremit del device . 
after thromboaspiration with an 8 - fr guiding catheter ( jr 4.0 , medtronic , mn , usa ) , a second angiography showed partial reduction of the thrombus and persistence of flow in the varix : we therefore opted for definitive embolisation . 
portography carried out 5 min after the procedure showed flow reduction through the device but incomplete closure ; two 12 - mm platinum coils ( nester , cook , bloomington , in , usa ) were endoprotesi aortoiliaca stato eseguito il giorno successivo per tre pazienti e due giorni dopo lembolizzazione per gli altri due . 
lembolizzazione unilaterale dellarteria iliaca interna stata tollerata da tutti e 5 i pazienti senza la comparsa di complicanze minori o maggiori quali claudicatio glutea , ischemia glutea o rettosigmoidea , disfunzioni sessuali o urologiche ; il periodo di ospedalizzazione stato in media 5 giorni dopo il posizionamento dellendoprotesi . 
il controllo angio - mdct a 1 mese ha evidenziato una buona rivascolarizzazione da parte di circoli collaterali delle due branche di divisione delliliaca interna trattata , anteriore e posteriore , senza passaggio di contrasto nel tronco comune e ha dimostrato la corretta posizione del plug con perfetta chiusura del vaso solo alla sua origine . 
 nei 3 pazienti a cui stata occlusa larteria succlavia sinistra , la mdct effettuata prima della dimissione e a 1 mese di distanza dal posizionamento dellendoprotesi aortica ha dimostrato la persistenza di endoleak di tipo ii dallarteria succlavia sinistra per due pazienti e in un paziente un endoleak misto : tipo i + tipo ii , ovvero dovuto sia ad una imperfetta sigillatura della protesi sulla parete aortica sia ad una riperfusione attraverso la succlavia : si pertanto optato per locclusione del vaso alla sua origine . 
prima di effettuare il trattamento i pazienti sono stati sottoposti a mdct e a studio eco - color doppler dei tronchi sovraortici e del circolo intracranico per escludere stenosi , ipoplasia o varianti anatomiche delle arterie carotidi , succlavie , vertebrali e del circolo del willis . 
i pazienti sono stati sottoposti quindi ad angiografia selettiva della ass che ha confermato la diagnosi di endoleak di tipo ii in due pazienti ed endoleak misto , tipo i + tipo ii , in un paziente , con perviet della vertebrale . 
tutti i pazienti sono stati dimessi due giorni dopo la procedura di occlusione : nessuno dei 3 pazienti ha avuto complicanze maggiori ( sindrome da furto della succlavia )  . 
il corretto posizionamento dellavp e lavvenuta occlusione dellarteria succlavia stato dimostrato anche mediante mdct di controllo . la fistola arterovenosa polmonare stata completamente esclusa con lutilizzo di due dispositivi avp , uno per ciascuna delle due arterie afferenti . 
il controllo angiografico ha dimostrato una chiusura totale dei due vasi embolizzati : in circa 3 minuti per il vaso maggiore e in minor tempo per quello di minori dimensioni . la saturazione percutanea di ossigeno in condizioni basali salita dal 73% al 93% subito dopo la procedura e si mantenuta stabile per i successivi 6 mesi di follow - up ; lemogasanalisi ha dimostrato normali pressioni di ossigeno e di anidride carbonica . 
il controllo clinico a 1 , 3 , 6 mesi ha dimostrato lassenza di sintomi respiratori , saturazione di ossigeno superiore al 95% in condizioni basali e un radiogramma del torace a tre mesi ha confermato una corretta e stabile posizione dei devices . nella fistola da emodialisi leco - color doppler ha dimostrato la presenza di due grosse dilatazioni vascolari anecogene , con presenza di flusso allinterno , a livello della piega del gomito . 
langiografia dellarto superiore , eseguita con accesso trans - omerale anterogrado 4 fr , ha confermato la presenza di una fistola arterovenosa in cui la legatura chirurgica della vena basilica efferente , eseguita 6 mesi prima , sia a livello dellanastomosi sia poco pi a valle , non aveva garantito una completa chiusura della connessione arterovenosa ; liperafflusso conseguente ha portato allo sviluppo di due grossi aneurismi venosi . 
al fine di escludere il flusso nei due aneurismi e nelle vene collaterali senza ricorrere ad una seconda legatura chirurgica si deciso , in accordo con il paziente , di chiudere definitivamente la fistola per via percutanea mediante avp . 
langiografia eseguita a 4 minuti dal posizionamento ha dimostrato una completa ed efficace chiusura della fistola con assenza di pasresults in the five patients undergoing closure of the common iliac artery , correct avp position was evaluated by means of a manual injection of contrast medium through the guiding catheter ; once in the correct position , the device was unscrewed and released . 
postprocedure angiography confirmed complete occlusion of the vessel within less than 8 mthe aortoiliac endografts were positioned on the following day in three patients and after 2 days in the other two . 
unilateral embolisation of the iia was tolerated by all five patients without minor or major complications , such as gluteal claudication , gluteal or rectosigmoid ischaemia and sexual or urological dysfunctions ; length of hospital stay was 5 days after endograft placement . 
mdct angiography at 1 month showed good revascularisation of the anterior and posterior branches of the treated iia by collateral circulations , without passage of contrast material in the common trunk , and demonstrated the correct position of the plug with perfect closure of the vessel origin . in the three patients who underwent occlusion of the lsa , mdct performed at discharge and 1 month after aortic endograft placement showed the persistence of type ii endoleak from the lsa in two patients and a mixed , type i and type ii , endoleak in one . 
these were both due to imperfect endograft sealing along the aortic wall and to reperfusion through the subclavian artery : we therefore decided to occlude the artery at its origbefore the embolisation procedure , the patients underwent mdct and colour - doppler us of the supraaortic trunks and intracranial vessels to exclude stenosis , hypoplasia or anatomical variants of the carotid , subclavian , vertebral arteries and the circle of willis . 
the patients were then studied by selective angiography of the lsa , which confirmed the type ii endoleak in two patients and the mixed type i and type ii endoleak in the other , with a patent vertebral artery . 
postprocedural angiography confirmed complete occlusion of the origin of the subclavian artery within less than 5 mthe patients were discharged 2 days after the procedure without developing major complications ( subclavian steal syndrome )  . 
correct avp positioning and successful subclavian artery occlusion were also demonstrated by follow - up mdct . the pulmonary arteriovenous malformation was completely excluded using two plugs , one for each of the two afferent arteries . 
baseline percutaneous oxygen saturation rose from 73% to 93% immediately after the procedure and remained stable for the following 6 months ; blood gas analysis demonstrated normal pressure of oxygen and carbon dioxide . 
arm angiography , done with 4 - fr antegrade humeral access , confirmed the presence of an arteriovenous fistula in which surgical ligation of the efferent basilic vein performed 6 months earlier at the level of the anastomosis and slightly below it had failed to guarantee complete closure of the arteriovenous communication . 
in order to exclude flow in the two aneurysms and in the collateral veins without performing a second surgical ligation , we decided in agreement with the patient to attempt definitive percutaneous closure of the fistula with an avp . 
angiography performed after 4 min demonstrated complete and effective closure of the fistula with absence of contrast in the basilic vein , two aneurysms and collateral veins and patency of the arm artery . 
colour - doppler us at 1 and 3 months confirmed the definitive closure of the fistula with complete resolution of the arm oedema and total thrombosis , with absence of flow in the two aneurysms . 
after placement of the two coils and the second plug , embolisation was achieved within about 5 mcolour - doppler us at 1 and 3 months demonstrated good patency of the tips , and angiography at 3 months confirmed varix closure . 
 discussion the avp has recently been shown to be effective in the occlusion of internal iliac arteries [ 1 ] , the treatment of pulmonary arteriovenous malformations [ 2 , 3 ] and as an occlusion system for a splenorenal shunt arising after tips [ 4 ]  . 
in our experience , avp was used to achieve vascular occlusion in five different arterial and venous settings . endograft placement in the abdominal aorta has become a valid alternative to conventional surgery and is increasingly the treatment of choice in elderly patients and subjects with high surgical risk . 
about 20% of abdominal aorta aneurysms have an iliac component [ 6 ] , and often the neck of the distal common iliac artery is insufficient to provide an adequate landing zone for the grawhen the aneurysm involves the common iliac artery as far as the origin of the iia , it is necessary to cover the iia with the graft and saggio di mdc nella vena basilica e di conseguenza nei due aneurismi e nelle vene collaterali con perviet dellasse arterioso dellarto . 
il controllo clinico ed eco - color doppler effettuato a 1 e a 3 mesi ha confermato la buona e definitiva chiusura della fistola con risoluzione completa delledema dellarto e trombosi totale , con assenza di flusso nei due aneurismi . la grossa varice gastrica stata chiusa con 2 avp e 2 spirali in platino dato il diametro particolarmente ampio e la persistenza di flusso dopo limpianto del primo plug . 
i controlli eco - color doppler a 1 e 3 mesi hanno mostrato buona perviet della tips e il controllo angiografico a 3 mesi ha confermato la chiusura della varice . discussione recentemente lefficacia dellavp stata dimostrata per locclusione di arterie iliache interne [ 1 ] , nel trattamento di fistole arterovenose polmonari [ 2 , 3 ] e come sistema per locclusione di uno shunt spleno - renale in seguito al posizionamento di tips [ 4 ]  . 
la descrizione della validit dellavp relativamente ad una casistica ampia e al trattamento di patologie vascolari pluridistrettuali , non stata ancora riportata , a nostra conoscenza , da alcun autore . nellesperienza qui riportata lavp stato utilizzato per locclusione vascolare in 5 diverse patologie , sia sul versante arterioso sia su quello venoso . il posizionamento di endoprotesi dellaorta addominale , ormai una valida alternativa alla chirurgia tradizionale e sempre pi spesso il trattamento di scelta in pazienti anziani e ad alto rischio operatorio ; i vantaggi , come noto , sono la minore invasivit , la minor durata della procedura e dellospedalizzazione , la ridotta perdita di sangue e la ridotta morbillit e mortalit rispetto alla chirurgia aperta [ 5 ]  . 
circa il 20% degli aneurismi dellaorta addominale ha una componente aneurismatica iliaca [ 6 ] e spesso il colletto dellarteria iliaca comune distale insufficiente per assicurare un adeguato atterraggio della protesi . 
quando la patologia aneurismatica coinvolge larteria iliaca comune fino allorigine dellarteria iliaca interna ( aii ) risulta necessario ricoprire con la protesi laii ed estendere la protesi fino allarteria iliaca esterna . 
nellottica quindi di prevenire un endoleak di ii tipo ( da vasi collaterali ) e quindi garantire una buona esclusione dellaneurisma raccomandata la chiusura , pre - posizionamento di endoprotesi , della aii [ 710 ]  . 
lembolizzazione prossimale della aii , utilizzando le spirali tradizionali , una tecnica relativamente sicura ed efficace ed stata riportata da molti autori [ 8 , 10 , 11 ]  . 
i maggiori difetti delle spirali sono la necessit di utilizzarne spesso un numero molto elevato , prima di riuscire a chiudere adeguatamente il vaso , lalto costo e la durata della procedura ; inoltre pu residuare un flusso anterogrado attraverso le spirali , si pu avere una dislocazione distale delle stesse o una protrusione allinterno del lume delliliaca comune o esterna [ 12 , 13 ]  . 
da marzo 2005 abbiamo iniziato ad utilizzare lavp al posto delle spirali per embolizzare preventivamente le arterie iliache interne ; questo dispositivo consente unottima occlusione prossimale del vaso allorigine , prima c . 
therefore , to prevent a type ii endoleak ( due to collaterals ) and guarantee adequate exclusion of the aneurysm , closure of the iia before endograft placement is recommended [ 710 ]  . 
in addition , there may be residual antegrade flow through the coils , distal migration , or protrusion within the lumen of the common or external iliac artery [ 12 , 13 ]  . 
in march 2005 , we started using the avp instead of coils for preventive embolisation of iias ; the device allows excellent proximal occlusion at the vessel origin , before the bifurcation , guaranteeing better revascularisation of the collateral branches of the contralateral hypogastric artery and reducing complications due to hypoperfusion . even thoracic aorta endovascular repair has been approved as a less invasive treatment compared with conventional surgery [ 14 ]  . 
the most important anatomical prerequisite for correct positioning of the stent - graft is the presence of a neck of adequate length ( at least 1520 mm ) distal to the lsa orighowever , descending thoracic aorta lesions often involve the distal aortic arch , with a proximal neck less than 15 mm : in these cases one technique enabling safe device fixation is the creation of a more proximal landing zone with deliberate overstenting of the lsa ostium [ 5 , 15 , 16 ]  . this procedure can only be done after excluding abnormalities of the vertebral and carotid arteries and the circle of willis and if the left internal mammary artery has not been used for a coronary bypass . 
all proximal endoleaks have an incidence of 0%23% for early endoleaks ( detected during stent - graft placement ) and 0%5% for late endoleaks , and they may be related to aneurysm morphology , dissection or even technical expertise [ 15 ]  . 
if this type of endoleak ( type ii from the subclavian artery ) persists , it needs to be promptly repaired because of the direct connection between the aneurysmal sac or false lumen and systemic arterial pressure . 
our three patients underwent avp embolisation of the origin of the subclavian artery because they had developed a type ii or mixed ( type i and ii ) endoleak from the subclavian artery , which was overstented at its origin due to an inadequate proximal neck . 
the use of metal coils is not advised for subclavian occlusion [ 15 ] due to the high risk of distal embolisation and the likelihood of not being able to block flow completely . 
these are abnormal , direct communications between pulmonary arteries and veins without a capillary bed , which are caused by the formation of an aneurysmal sac or a tangle of tortuous vessels [ 1820 ]  . 
treatment opdella biforcazione nei suoi due rami , garantendo una migliore rivascolarizzazione da rami collaterali dellipogastrica controlaterale riducendo in questo modo possibili complicanze da ipoperfusione . anche il trattamento endovascolare della patologia dellaorta toracica ormai stato approvato come trattamento meno invasivo rispetto alla chirurgia tradizionale [ 14 ]  . 
il pi importante requisito anatomico per il corretto posizionamento dellendoprotesi la presenza di un colletto minimo di 1520 mm distalmente allorigine dellostio dellass . tuttavia la patologia dellaorta toracica discendente spesso coinvolge larco aortico distale con un colletto prossimale inferiore a 15 mm : in tali casi una delle tecniche per un sicuro fissaggio del dispositivo un atterraggio pi prossimale con la copertura volontaria dellostio dellass [ 5 , 15 , 16 ]  . tale procedura pu essere effettuata soltanto escludendo prima patologie a carico delle arterie vertebrali , delle carotidi , del circolo del willis e se la mammaria interna sinistra non stata utilizzata per un bypass coronarico . un endoleak di tipo ii dalla ass pu insorgere dopo posizionamento di endoprotesi dellaorta discendente ma ad oggi non riportata in letteratura lesatta incidenza . 
la percentuale di tutti gli endoleak prossimali varia dallo 0% al 23% per gli endoleak precoci ( dimostrati al momento del posizionamento della protesi ) e dallo 0% al 5% per quelli tardivi e possono essere legati allanatomia dellaneurisma o dissezione o anche allesperienza delloperatore [ 15 ]  . 
questo tipo di endoleak ( tipo ii dalla succlavia ) se persiste richiede una tempestiva correzione a causa della diretta comunicazione tra la sacca aneurismatica o il falso lume e la pressione arteriosa sistemica . 
i nostri 3 pazienti sono stati sottoposti ad embolizzazione dellorigine della succlavia con avp , poich avevano sviluppato un endoleak di tipo ii o misto ( tipo i + tipo ii ) dalla succlavia che stata ricoperta dallendoprotesi alla sua origine per la presenza di un colletto prossimale insufficiente . 
lutilizzo delle spirali metalliche per locclusione della succlavia non consigliabile [ 15 ] per il forte rischio di embolizzazione distale e per lalta probabilit di non riuscire a bloccare totalmente il flusso : il nuovo dispositivo avp consente unimmediata , sicura e rapida occlusione allorigine del vaso senza complicanze emboliche distali . 
per questultima caratteristica lavp assume unimportante ruolo nel trattamento delle fistole arterovenose polmonari ( favp ) ; questultime sono delle anormali connessioni dirette tra arterie e vene polmonari con lesclusione del letto capillare dovute alla formazione di una sacca aneurismatica o ad un groviglio di canali vascolari tortuosi [ 1820 ] : estremamente eterogenea la presentazione clinica [ 18 , 21 , 22 ]  . 
le opzioni terapeutiche includono lembolizzazione percutanea o lescissione chirurgica [ 2 ]  . data la minore invasivit e lo sviluppo di nuovi materiali embolizzanti il trattamento percutaneo divenuto il trattamento di scelta per la maggior parte dei pazienti [ 2325 ]  . 
la ricanalizzazione della favp pu avvenire in una percentuale compresa tra il 5% e il 10% dei pazienti trattati [ 26 , 27 ] : pertanto , la scelta di un materiale embolizzante appropriato e laccurata localizzazione del sito dove iniziare lembolizzazione di cruciale importanza [ 23 , 24 ]  . 
recanalisation of pavf may occur in 5%10% of treated patients [ 26 , 27 ] , so the choice of appropriate embolising material and accurate localisation of the site where embolisation is to be started are crucial [ 23 , 24 ]  . 
these considerations are even more important in massive fistulas , which are more difficult to occlude with coils or detachable balloons due to the risk of distal migration [ 25 , 28 ]  . 
we believe the avp to be safer and easier to use than the above - mentioned devices owing to the simplicity and small size of the release system and , more importantly , because it was designed for vascular use and is therefore more specific and better suited to the tubular morphology of vessels seen in pavf . haemodialysis fistulas often develop complications , one of the most common being aneurysm formation [ 31 , 32 ]  . possible treatment options include surgery , endovascular treatment or both . 
we opted for the avp because its release is controlled and it can be placed precisely at the level of the neck where it readily adheres to the vessel without risking distal migration . 
to our knowledge , no author has described the use of the avp as an embolising device for the closure of a haemodialysis fistula . the efficacy of tips is well documented in the treatment of variceal bleeding refractory to endoscopic treatment [ 36 , 37 ]  . 
although tips alone is sufficient to stop variceal bleeding in many cases [ 3840 ] , in some patients with large collateral veins , it may not be enough ; embolisaton of the varix therefore becomes indispensable [ 4144 ] , as in the case treated by us . 
even very large coils ( 20 mm15 cm ) are susceptible to distal migration , and the same applies to other materials such as sclerosing agents , glue and gelatine sponge particles , which are not very effective and only increase the risk of systemic embolisation . 
in our opinion , the avp is the vascular occluder that best adapts to large vessels such as gastric varices ; in addition , it can be used is combination with coils to make vascular occlusion even more rapid . our experience indicates that the avp is an extremely versatile occlusion system that is suitable for variety of vessel to pi difficile occluderle con le spirali o i palloni staccabili a causa della possibilit di migrazione distale [ 25 , 28 ]  . 
migliori risultati sono stati ottenuti in passato con lutilizzo di vari device , ad uso cardiologico realizzati per il trattamento della perviet del dotto arterioso di botallo e dei difetti settali cardiaci , quali il gianturco - grifka , composto prevalentemente da un sacco in nylon flessibile e una guida occludente , il cardioseal , costituito da un doppio ombrellino ciascuno con quattro molle metalliche e stoffa di poliestere e lamplatzer duct occluder , realizzato con una maglia guida in nitinolo con estremit a disco [ 24 , 29 , 30 ]  . 
noi riteniamo che lavp , sia pi sicuro e facile da utilizzare rispetto ai precedenti devices menzionati , per la semplicit e le ridotte dimensioni del sistema di rilascio ma soprattutto , essendo un dispositivo dedicato allimpiego vascolare , risulta pi specifico e meglio conformabile allanatomia tubulare del vaso come nel caso delle malformazioni arterovenose polmonari . le fistole da emodialisi , sono gravate da alcune complicanze tra cui una delle pi comuni la formazione di aneurismi [ 31 , 32 ]  . 
lapproccio endovascolare pu avvalersi del posizionamento di uno stent ricoperto a livello arterioso , in modo da escludere la zona di anastomosi , oppure lembolizzazione a livello anastomotico arterioso della fistola [ 3335 ]  . 
nel nostro caso , poich la fistola era localizzata a livello del gomito , lutilizzo di uno stent ricoperto era controindicato e pertanto lunica indicazione terapeutica percutanea era lembolizzazione transcatetere . 
abbiamo preferito utilizzare un avp perch ha un rilascio controllato e pu essere posizionato esattamente a livello del colletto , aderendo prontamente al vaso senza rischio di migrazione distale sia sul versante arterioso sia su quello venoso . 
a nostra conoscenza nessun autore , prima dora , ha descritto limpiego dellavp come sistema embolizzante per la chiusura di una fistola arterovenosa da emodialisi . lefficacia della tips stata ben provata nel trattamento del sanguinamento da varici resistenti al trattamento endoscopico [ 36 , 37 ]  . 
in molti casi la tips da sola sufficiente a bloccare il sanguinamento delle varici [ 3840 ] tuttavia in alcuni pazienti con grosse vene collaterali pu non bastare ; pertanto in tali soggetti lembolizzazione della varice risulta indispensabile [ 4144 ] , come nel caso da noi trattato . 
il grande diametro di questi vasi collaterali costituisce il principale problema nella scelta del materiale embolizzante : anche spirali metalliche molto grandi ( 20 mm15 cm ) possono facilmente migrare distalmente . 
ugualmente vale per altri materiali come agenti sclerosanti , colla , particelle di spugna di gelatina che non hanno una grande efficacia e aumentano solo il rischio di embolizzazione sistemica . 
a nostro giudizio lavp il dispositivo di occlusione vascolare che meglio si adatta a vasi di grosse dimensioni come le varici gastriche : pu inoltre essere utilizzato in associazione alle spirali per rendere ancora pi rapida locclusione vascolare . in conclusione , in base alla nostra esperienza , possiamo affermare che lavp si rivelato un sistema di occlusione vascolare estremamente versatile , adatto quindi a vasi di diametro eterogeneo , facile da usare , preciso nel rilascio , c . 
in our experience , the use of abdominal endografts with suprarenal fixation did not lead to any significant increase in morphological and / or functional renal complications compared with those with infrarenal fixation . key words ct angiography abdominal aortic aneurysm stent graft suprarenal fixation renal complication riassunto obiettivo . 
sono stati analizzati retrospettivamente tutti gli esami angio - tc di controllo ( follow - up a 1 , 6 , 12 mesi e quindi 1 / anno ) e i test di laboratorio eseguiti su 102 pazienti sottoposti a posizionamento di endoprotesi ad aggancio soprarenale ( 60 pazienti , gruppo a ) o sottorenale ( 42 pazienti , gruppo b )  . 
la lunghezza del colletto prossimale risultata inferiore nel gruppo a ( 2 , 14 cm0 , 84 cm ) rispetto al gruppo b ( 3 , 41 cm1 , 21 cm )  . 
nel nostro studio limpianto di endoprotesi addominali ad aggancio soprarenale non ha determinato un aumento significativo di complicanze renali morfologiche e / o funzionali rispetto ai pazienti trattati con endoprotesi ad aggancio sottorenale . parole chiave angiografia ct aneurisma aortico addominale stent - graft aggancio soprarenale complicazioni renali a.r. 
the conventional approach to aaa is surgery , which is associated with a mortality rate of 1.4%6.5% , which increases to 20% if the patients are at high surgical risk due to severe concomitant disease and reaches 50% when surgery is performed in an emergency setting due to aneurysm rupture [ 3 , 4 ]  . 
 over the last few years , a less invasive alternative to open surgery has been introduced with excellent results : the imaging - guided intraluminal positioning of a covered stent - graft . success of endovascular repair in terms of aneurysm exclusion and absence of perioperative and postoperative complications is dependent on accurate patient selection to ensure that only aneurysms with a morphology that corresponds to well - established indications are treated [ 57 ]  . 
on the other hand , the indications for endovascular repair are continuously evolving as a result of the growing experience of surgeons and the improved versatility and applicability of the new stent - grafts [ 5 ]  . 
one example is the new generation of endografts with suprarenal fixation that , by extending the proximal anchorage point cranially , have broadened the inclusion criteria to aneurysms with hostile i.e. 
however , the meshes of the uncovered metallic portion of the graft , which ensure suprarenal fixation , overlap with the ostia of the renal arteries and may cause damage to the renal arteries , the renal parenchyma and renal function . 
published results are conflicting so that the issue remains controversial [ 813 ]  . the purpose of our study was to compare the rate of morphological and functional renal complications after placement of suprarenal and infrarenal aortic stent - grafts for endovascular abdominal aortic aneurysm repair ( evar )  . materials and methods patients from january 2000 to june 2004 , we positioned aortic endografts in 140 patients with abdominal aortic aneurysms . 
indications for endovascular repair and procedure planning ( graft selection ) were defined by a team of vascular surgeons and interventional radiologists on the basis of the aneurysms clinical and morphological characteristics [ derived from preprocedural computed tomography ( ct ) angiography ] along the guidelines proposed by the italian transfemoral endovascular aneurysm management ( team ) group and by the sezione di studio di radiologia vascolare ed interventistica ( vascular and interventional radiology study section ) ( sirm ) [ 14 ]  . 
in particular , inclusion criteria for endovascular repair were : age 65 years ; aneurysm sac diameter < 7 cm ; proximal neck length 15 mm ; proximal neck diameter < 30 mm ; proximal neck angulation > 120 ; proximal laneurisma dellaorta addominale una patologia di notevole rilevanza clinica , con unincidenza stimata in 2040 casi su 100000 abitanti per anno ed responsabile dell1 , 7% dei decessi nei soggetti con et compresa tra 65 e 74 anni [ 1 , 2 ]  . 
il trattamento tradizionale dellaneurisma dellaorta addominale di tipo chirurgico ed gravato da un rischio di mortalit variabile tra l1 , 4% e il 6 , 5% , che pu aumentare sino al 20% se lintervento effettuato in pazienti ad alto rischio chirurgico per gravi patologie concomitanti e raggiunge il 50% quando lintervento viene effettuato in emergenza per rottura dellaneurisma stesso [ 3 , 4 ]  . negli ultimi anni stata proposta ed utilizzata sempre pi diffusamente una metodica alternativa alla chirurgia tradizionale , meno invasiva , che consiste nel posizionamento imaging - guidato per via endoluminale di uno stent - graft ricoperto e che ha dimostrato risultati estremamente soddisfacenti . 
il successo del trattamento endovascolare , in termini di esclusione dellaneurisma ed assenza di complicanze perie post - operatorie , dipende da unaccurata selezione dei pazienti al fine di trattare solamente quei casi nei quali le caratteristiche morfologiche dellaneurisma siano perfettamente rispondenti ad indicazioni ben codificate . 
daltra parte , le indicazioni al trattamento endovascolare sono in continua evoluzione , sia per la crescente esperienza degli operatori , che tendono ad applicare questa procedura ad una quota sempre maggiore di pazienti , sia per la disponibilit di nuovi modelli di endoprotesi con maggiori caratteristiche di versatilit ed applicabilit [ 5 ]  . 
un esempio rappresentato dalle nuove generazioni di protesi ad aggancio soprarenale che , estendendo cranialmente la sede di ancoraggio prossimale , hanno consentito di ampliare i criteri di inclusione anche ad aneurismi con colletti definiti ostili , ossia corti , tortuosi o angolati , riducendo potenzialmente il rischio di migrazione caudale dello stent - graft e di endoleak di tipo i prossimale [ 6 , 7 ]  . 
tuttavia , la porzione metallica scoperta delle protesi , deputata allaggancio soprarenale , si sovrappone con le sue maglie allostio delle arterie renali con potenziali effetti dannosi sulle arterie renali , sul parenchima renale e sulla funzionalit renale . 
gli studi presenti in letteratura a tale proposito riferiscono risultati contrastanti tali da mantenere ancora aperta tale problematica [ 813 ]  . lo scopo del nostro lavoro stato quello di confrontare il tasso di complicanze renali morfologiche e funzionali , dopo posizionamento di endoprotesi aortiche ad aggancio sopra e sottorenale per il trattamento endovascolare dellaneurisma dellaorta addominale ( evar )  . materiali e metodi pazienti da gennaio 2000 a giugno 2004 , 140 pazienti con aneurisma dellaorta addominale sono stati sottoposti presso il nostro centro a posizionamento di endoprotesi aortica . 
sixty patients ( group a ) ( 58 men ; two women ; mean age : 71.3 years , range : 6286 years ) received suprarenal endografts ( 55 talent , medtronicave ; five zenith , cook ) , whereas the remaining 42 patients ( group b ) ( 41 men ; one woman ; mean age : 69.1 years , range : 6189 years ) received infrarenal endografts ( 25 aneurx ; 17 excluder , gore )  . mean neck length was 2.140.84 cm ( range 0.85.5 cm ) in group a and 3.411.21 cm ( range 25.3 cm ) in group b , whereas the rate of hostile necks was 41.7% ( 25 / 60 patients ) in group a and 14.3% ( 6 / 42 patients ) in group b . 
in particular , in group a , the proximal neck was short ( < 1.5 cm ) in eight patients , tortuous in 12 patients , angulated ( > 120 ) in six patients , and showed significant thrombotic appositions in 15 patients and ulcerations in five patients . 
in group b , no patient had a short proximal neck ( < 1.5 cm ) ; the proximal neck was tortuous in four patients , angulated ( > 120 ) in three patients , and had significant thrombotic appositions in five patients and ulcerations in one patient ( table 1 )  . diagnostic procedures and clinical laboratory evaluation after endovascular repair , all patients underwent clinical / laboratory follow - up with creatinine clearance ( crcl ) and systemic arterial pressure ( ap ) monitoring and imaging follow - up with ct angiography at 1 , 6 and 12 months and yearly thereafter . 
renal function was evaluated by calculating crcl using the cockcraft and gault equation [ 15 ] : crcl = ( 140patients age ) patients weight ( kg ) / 72serum creatinine ( mg / 100 ml )  . 
any changes in renal function were defined on the basis of the comparison between the pretreatment crcl values and those obtained at the last follow - up test at least 1 year after the procedure . 
both the crcl and the ap values were obtained from patients medical records and reviewed by a team member not involved in the interpretation of ct images . follow - up ct angiography was done with a multidetector technique ( somatom volume zoom , siemens , medical solutions , forchheim ) before and during administration of 120 ml of contrast agent ( 300 mgi / ml ; 3 ml / s flow rate )  . 
precontrast acquisition was carried out with 42.5 - mm collimation , procedura ( scelta della protesi ) sono state definite da unequipe mista di chirurghi vascolari e radiologi interventisti sulla base di criteri clinici e caratteristiche morfologiche dellaneurisma ( queste ultime desumibili dallesame angio - tc eseguito prima del trattamento ) , tenendo conto delle linee guida proposte dal team ( transfemoral endovascular aneurysm management ) italiano , a cura della sezione di studio di radiologia vascolare ed interventistica della sirm [ 14 ]  . 
in particolare , i criteri di inclusione al trattamento endovascolare utilizzati sono stati : et del paziente 65 anni ; diametro della sacca aneurismatica < 7 cm ; lunghezza del colletto prossimale 15 mm ; diametro del colletto prossimale < 30 mm ; angolazione del colletto prossimale > 120 ; colletto prossimale con calcificazioni parietali coinvolgenti meno della met della sua circonferenza in assenza di significativa apposizione trombotica ; angolazione arterie iliache > 90 ( < 90 in assenza di diffuse calcificazioni ) ; diametro arteria iliaca esterna > 7 mm e < 14 mlindicazione al trattamento endovascolare stata inoltre posta nei pazienti giudicati ad elevato rischio chirurgico ( gravi condizioni cardiorespiratorie , addome ostile ) , indipendentemente dallet del paziente e dalle caratteristiche morfo - strutturali dellaneurisma . tutti i pazienti sottoposti ad evar e provvisti di documentazione completa riguardante i dati del follow - up strumentale e clinico - laboratoristico ad almeno 1 anno dalla procedura sono stati inclusi nel nostro studio . 
in 60 pazienti ( gruppo a ) ( maschi 58 ; femmine 2 ; et media : 71 , 3 anni ; range : 6286 anni ) stata posizionata una protesi ad aggancio soprarenale ( 55 talent , medtronic - ave ; 5 zenith , cook ) , mentre nei restanti 42 pazienti ( gruppo b ) ( maschi 41 ; femmine 1 ; et media : 69 , 1 anni ; range : 6189 anni ) stata posizionata una protesi ad aggancio sottorenale ( 25 aneurx ; 17 excluder , gore )  . la lunghezza media del colletto era di 2 , 140 , 84 cm ( range 0 , 85 , 5 cm ) nel gruppo a e 3 , 411 , 21 cm ( range 25 , 3 cm ) nel gruppo b , mentre la percentuale di colletti ostili era di 41 , 7% ( 25 / 60 pazienti ) e 14 , 3% ( 6 / 42 pazienti ) per il gruppo a e b rispettivamente . 
in particolare nel gruppo a il colletto prossimale risultato corto ( < 1 , 5 cm ) in 8 pazienti , tortuoso in 12 pazienti , angolato ( > 120 ) in 6 pazienti e ha mostrato significative apposizioni parietali trombotiche e / o ulcerazioni , in 15 e 5 pazienti rispettivamente . 
negli 8 pazienti con colletto prossimale < 1 , 5 cm , la decisione di ricorrere al trattamento endovascolare derivata dalla presenza di fattori di rischio per lintervento chirurgico tradizionale . nel gruppo b , nessun paziente presentava un colletto prossimale definito corto ( < 1 , 5 cm ) ; il colletto prossimale risultato tortuoso in 4 pazienti , angolato ( > 120 ) in 3 pazienti , con significative apposizioni parietali trombotiche ed ulcerazioni in 5 ed 1 paziente , rispettivamente ( tabella 1 )  . indagini strumentali e valutazione clinico - laboratoristica tutti i pazienti sottoposti a trattamento endovascolare sono stati controllati nel tempo con un follow - up clinico - laboratoristico , mediante il monitoraggio della clearance della creatinina ( clcr ) e della pressione arteriosa sistemica , e a.r. 
the contrast study was carried out with 41 - mm collimation , pitch = 6 , gantry rotation speed = 0.5 s , slice thickness = 1.25 mm , and reconstruction interval = 1 mthe voi extended from a plane passing through the coeliac trunk to one passing through the common femoral arteries , with an average acquisition time of 25 s . 
axial and three - dimensional ( 3d ) images were reviewed on a dedicated workstation ( leonardo , siemens , medical solutions , forchheim ) to confirm the development and / or progression of steno - occlusive lesions of the renal arteries and the presence of ischaemic parenchymal changes ( renal infarction )  . 
steno - occlusion of the renal arteries was evaluated on a 4 - point scale : grade 0 = no stenosis ; grade 1 = < 50% stenosis ; grade 2 = > 50% stenosis : grade 3 = occlusion . 
alterations of the urographic effect associated with changes in the size of the renal parenchyma were interpreted as evidence of renal infarction . renal infarctions occupying less than 20% of the entire kidney volume were considered small . strumentale , mediante esame angio - tc a 1 , 6 e 12 mesi e quindi 1 / anno dopo la procedura . 
la funzionalit renale stata valutata mediante il calcolo della clearance della creatinina ( clcr ) utilizzando la formula di cockcraft [ 15 ] : clcr = ( 140 - et del pz ) peso del pz ( kg ) / 72livello sierico della creatinina ( mg / 100 ml )  . 
la presenza / assenza di variazioni della funzionalit renale stata definita dal confronto dei valori della clcr pre - trattamento con quelli ottenuti nellultimo controllo , eseguito almeno 1 anno dalla procedura . 
in tutti i pazienti sono stati inoltre misurati e comparati i valori della pressione arteriosa sistemica pre - trattamento con quelli ottenuti nellultimo controllo , ovvero almeno 1 anno dopo evar . 
i valori della clcr e della pressione arteriosa sono stati ottenuti dalla revisione delle cartelle cliniche dei pazienti , effettuata da uno dei componenti dellequipe non coinvolto nella interpretazione delle immagini tc . gli esami angio - tc di controllo sono stati eseguiti con tecnica multistrato ( somatom volume zoom , siemens , medical solutions , forchheim ) prima e durante somministrazione di 120 ml di mdc ( 300 mgi / ml ; flusso 3 ml / s )  . 
lacquisizione senza somministrazione di mdc stata eseguita con collimazione di 42 , 5 mm , pitch 6 , velocit di rotazione del sistema tubo radiogeno - detettori di 0 , 5 s , spessore dello strato ed intervallo di ricostruzione di 5 mm , campo di vista ( fov ) , in media , di 240 mm volume di interesse ( voi ) estea.r. 
at follow - up after 1 year , there was only one case of progression from a grade 1 to a grade 2 stenosis ( 1 / 9 , 11.1% ) whereas the remaining eight cases were unchanged . 
among the 115 arteries free of stenosis before treatment , ct angiography at 1 year identified three new grade 2 stenoses ( 3 / 115 , 2.6% ) so da d12 alla sinfisi pubica . 
lacquisizione con mdc stata eseguita con collimazione di 41 mm , pitch 6 , velocit di rotazione del sistema tubo radiogeno - detettori di 0 , 5 s , spessore dello strato di 1 , 25 mm ed intervallo di ricostruzione di 1 mil voi stato esteso da un piano passante per il tripode celiaco sino ad un piano passante per le arterie femorali comuni , con un tempo di acquisizione medio di 25 s . 
il ritardo di scansione stato individuato per ogni paziente mediante un sistema automatico di bolus tracking ( care bolus , siemens ) e posizionamento di una roi ( region of interest ) nel lume dellaorta addominale soprarenale . lanalisi delle immagini tc ( esami pree post - trattamento ) stata effettuata retrospettivamente in consensus da due radiologi esperti in procedure endovascolari ed angiotc , valutando le immagini assiali e 3d su una stazione dedicata ( leonardo , siemens , medical solutions , forchheim ) al fine di verificare linsorgenza e / o la progressione di patologia steno - ostruttiva a carico delle arterie renali e la presenza di alterazioni parenchimali renali su base ischemica ( infarto renale )  . 
la valutazione della patologia steno - ostruttiva delle arterie renali stata effettuata utilizzando una scala a 4 gradi : grado 0 = assenza di stenosi ; grado 1 = stenosi < 50% ; grado 2 = stenosi > 50% : grado 3 = occlusione . 
gli infarti renali occupanti meno del 20% dellintero volume del rene sono stati considerati piccoli infarti . analisi statistica i dati clinico - laboratoristici e strumentali ottenuti in entrambi i gruppi di pazienti sono stati confrontati con il test t di student , al fine di verificare la presenza di differenze significative in termini di complicanze sul parenchima renale , sulle arterie renali e sullandamento della clearance della creatinina , considerando significativa una differenza di p < 0 , 05 . 
la valutazione statistica stata effettuata mediante lo stata statistical software ( release 8.2 , college station , tx ; stata corp lp )  . risultati in nessun paziente si sono verificati problemi tecnici al momento del posizionamento e rilascio dellendoprotesi , n la necessit di conversione chirurgica intraoperatoria e / o in fase intra / peri - procedurale . 
i due gruppi di pazienti sono risultati statisticamente simili dal punto di vista epidemiologico ( et : 71 , 3 anni versus 69 , 1 anni , p = ns ; distribuzione m / f : 58 / 2 versus 41 / 1 , p = ns )  . 
at follow - up , only one lesion was found to have progressed from a grade 1 to a grade 2 stenosis ( 1 / 8 , 12.5% ) ; the remaining seven case were unchanged . 
inoltre non si sono osservate differenze statisticamente significative n in termini di numero di pazienti con riduzione della clcr maggiore o uguale al 20% ( 11 , 86% nel gruppo a versus 7 , 1% , nel gruppo b , p > 0 , 05 ) , n in termini di decremento medio della clcr in questo gruppo selezionato di pazienti ( - 38 , 97% versus - 30% , p > 0 , 05 )  . 
multidetector computed tomography ( mdct ) angiography , three - dimensional ( 3d ) ( a , c , d ) and axial ( b ) images ; digital subtraction angiogram ( dsa ) ( e , f )  . 
lesame tc eseguito ad 1 anno dalla procedura di evar ( c , d ) documenta la comparsa di una stenosi eccentrica dellarteria renale destra ( frecce ) , non presente allesame tc pre - evar ( a , b ) ( frecce )  . 
regolare perviet dellarteria renale destra ( a ) ( freccia ) che al controllo a 12 mesi dallimpianto appare ostruita ( b ) ( freccia ) con conseguente infarto renale omolaterale ( punta di freccia )  . discussion in abdominal endografts with suprarenal fixation , the proximal anchorage point can be extended upwards to the renal arteries thanks to the presence of a portion of the prosthesis not covered by the graft ( free - flow section )  . 
nel follow - up , solo 1 stenosi progredita da grado 1 a grado 2 ( 1 / 8 , 12 , 5% ) mentre le rimanenti 7 sono rimaste immodificate . 
la valutazione delle 76 arterie renali indenni da stenosi prima del trattamento non ha documentato linsorgenza di alcuna nuova lesione steno - ostruttiva . nel gruppo a si documentata la comparsa di un rene grinzo , secondario allocclusione dellarteria renale principale omolaterale , e di un infarto polare inferiore secondario a.r. 
computed tomography ( ct ) axial images ( a , b ) show that the endograft covers one left lower polar artery , with resulting infarction of the lower kidney pole ( c ) ( arrow )  . 
 appare pertanto giustificata la tendenza a verificare leffetto nel tempo di queste protesi a livello della funzionalit renale , delle arterie renali e del parenchima renale [ 13 , 1620 ]  . in letteratura sono presenti diversi studi comparativi tra le protesi ad aggancio soprarenale e quelle ad aggancio sottorenale con risultati contrastanti . 
these figures were confirmed by bockler et al . , who reported a 19% rate of renal infarction in patients treated with suprarenal stent - grafts ( 398 patients ) compared with 3.7% in those treated with infrarenal stentgrafts ( 265 patients ) [ 21 ]  . 
studied 55 patients treated with infrarenal stent - grafts and 32 patients with suprarenal stent - grafts , finding a higher incidence of progression of steno - occlusive lesions in the group with suprarenal grafts [ 20 ]  . 
on 99 , 192 , and 113 patients , respectively [ 2426 ]  . dotto su 130 pazienti , di cui 69 trattati con protesi ad aggancio soprarenale e 61 con protesi ad aggancio sottorenale , si osservata una maggiore percentuale di piccoli infarti renali con lutilizzo di protesi ad aggancio soprarenale rispetto allaggancio sottorenale ( 5 , 8% versus 1 , 6% ) , seppure in assenza di differenze significative in termini di funzionalit renale [ 19 ]  . 
tale dato appare confermato dal lavoro di bockler che riporta una percentuale di infarti renali pari al 19% nel gruppo di pazienti trattati con protesi ad aggancio soprarenale ( 398 pazienti ) e 3 , 7% nel gruppo trattato con protesi ad aggancio sottorenale ( 265 pazienti ) [ 21 ]  . 
nello studio di lau , condotto su 55 pazienti trattati con protesi ad aggancio sottorenale e 32 pazienti con protesi aggancio soprarenale , si documentato una maggiore incidenza di progressione di patologia steno - ostruttiva nel gruppo di pazienti con protesi ad aggancio soprarenale [ 20 ]  . 
al contrario , nellesperienza di malina e marin lutilizzo di protesi ad aggancio soprarenale non ha aumentato il rischio di complicanze sulle arterie renali , sul parenchima renale e sulla funzionalit renale [ 22 , 23 ]  . 
tali risultati appaiono confermati anche dagli studi condotti da kramer , burks e surowiec rispettivamente su 99 , 192 e 113 pazienti [ 2426 ]  . nel nostro studio la valutazione retrospettiva degli esami angio - tc , pree post - evar , non ha documentato differenze statisticamente significative in termini di insorgenza / progressione delle lesioni steno - ostruttive delle arterie renali ( 11 , 1% nel gruppo a versus 12 , 5% nel gruppo b ) n in termini di alterazioni parenchimali renali . 
a significant finding is the tendency for renal function to decline in all patients undergoing evar , regardless of the type of graft employed ; as previously reported , this decline in renal function appears similar to that occurring after open surgery . 
in the case of surgery , the decline is secondary to distal embolisation phenomena connected with the operation , to the possible development of renal ischaemia after renal artery clamping or to possible renal artery injury ( dissection , occlusion ) during the operation [ 12 ]  . 
 patologia steno - ostruttiva delle arterie renali e di alterazioni parenchimali renali associata allutilizzo di protesi ad aggancio soprarenale ( 3 , 5% versus 0% ) , seppur senza differenze statisticamente significative tra i due gruppi di pazienti . la valutazione dei livelli della pressione arteriosa non ha documentato significative variazioni post - trattamento , sia in assoluto sia paragonando i due gruppi . 
tale risultato coincide con i dati riportati in letteratura secondo i quali le protesi ad aggancio soprarenale non modificano in maniera significativa i valori di pressione arteriosa del paziente [ 22 , 23 ]  . lanalisi ad un anno dalla procedura della funzionalit renale in termini di variazione della clcr registrate nei due gruppi , non ha documentato differenze statisticamente significative ( - 8 , 75 nel gruppo a versus - 6 , 40 nel gruppo b )  . 
risultati simili sono emersi anche confrontando il numero di pazienti con decremento della clcr maggiore o uguale al 20% ( 11 , 86% versus 7 , 1% ) ; in tali pazienti , inoltre , il decremento della clcr ( - 38 , 97% versus - 30% ) risultato statisticamente simile . 
un dato rilevante rappresentato dalla costante tendenza al decremento della funzionalit renale in tutti i pazienti sottoposti ad evar , indipendentemente dal tipo di protesi utilizzata ; come riportato anche in letteratura tale decremento della funzionalit renale appare del tutto simile a quanto si verifica dopo terapia chirurgica convenzionale . 
in caso di trattamento chirurgico , tale decremento secondario ai fenomeni di embolizzazione distale connessi con lintervento , alla possibile insorgenza di ischemia renale post - clampaggio delle arterie renali o in rapporto ai possibili insulti ( dissezione , occlusione ) provocati sulle arterie renali durante lintervento [ 12 ]  . conclusion conclusioni in conclusion , our study shows that the use of grafts with suprarenal fixation has broadened the indications for evar , with no significant increase in complications affecting renal function , renal arteries or renal parenchyma . 
however , we believe that further studies with longer follow - up periods and larger population samples are needed to evaluate the real risk of using suprarenal grafts as regards the development of renal artery steno - occlusive lesions and ischaemic damage to the renal parenchyma . the tendency of renal function to decline in all patients after evar should encourage us to put in place appropriate pretreatment hydration protocols and limit the use of iodinated contrast agents during the procedure and the follow - up by restricting the number of ct angiography examinations and resorting to alternative techniques such as magnetic resonance ( mr ) angiography and contrast - enhanced ultrasonography [ 27 ]  . in conclusione , dai dati del nostro studio emerge che lutilizzo di protesi ad aggancio soprarenale consente di ampliare le indicazioni allevar , in assenza di un incremento significativo di complicanze in termini di alterazioni della funzionalit renale , delle arterie renali e del parenchima renale . 
bolzano , italy , tel . : + 39 - 047 - 1908494 , fax : + 39 - 047 - 1908908 , e - mail : mosavr@yahoo.com received : 23 june 2006 / accepted : 27 july 2006 / published online : 22 february 2007 abstract purpose . 
cta is fast and relatively noninvasive , and its sensitivity appears similar to that of dsa in detecting and evaluating intracranial aneurysms , even those smaller than 3 mm . this study confirms the value of cta as the primary imaging technique in subarachnoid haemorrhage , with dsa reserved for selected patients . key words multislice ct angiography cerebral aneurysm riassunto obiettivo . 
in un periodo di 20 mesi 130 pazienti con diagnosi tc di emorragia subaracnoidea acuta non traumatica sono stati arruolati per uno studio prospettico e sottoposti ad angiotc multistrato ( 16 detettori ) ed angiografia digitale sottrattiva ( 57 maschi , 73 femmine ; et media 59 , 5 anni )  . 
langio - tc una metodica veloce e poco invasiva e la sua sensibilit nellindividuazione degli aneurismi analoga a quella dellangiografia digitale sottrattiva anche se di dimensioni inferiori ai 3 mquesto studio conferma la validit dellangiotc come tecnica di imaging di elezione nella diagnosi di emorragie subaracnoidee . 
langiografia digitale sottrattiva pu essere riservata solo a pazienti selezionati . parole chiave angio - tc multistrato aneurisma cerebrale introduction introduzione nontraumatic subarachnoid haemorrhage ( sah ) is a neurological emergency characterised by extravasation of blood into the spaces lining the central nervous system , which are normally filled with liquor . 
nonaneurysmal sah , including the perimesencephalic end systolic area ( esa ) , is in contrast characterised by a good prognosis and rare neurological complications [ 2 ]  . the global incidence of sah , which varies from region to region , is about 10.5 cases per 100 , 000 inhabitants per year and has remained stable over the last 30 years . 
the main negative prognostic factors include level of consciousness , age and the quantity of blood at initial head computed tomography ( ct ) scan [ 3 , 4 ]  . the principle variable risk factors are cigarette smoking , hypertension , use of cocaine and alcohol abuse . 
first - degree family members of patients with sah have an increased risk ; there are also hereditary diseases of the connective tissue associated with intracranial aneurysms and sah , such as polycystic kidney disease , ehlers - danlos syndrome ( type iv ) , pseudoxanthoma elasticum and fibromuscular dysplasia [ 5 ]  . on the basis of these prognostic criteria , an sah should always be suspected in patients with the typical presentation of sudden and severe headache associated with nausea , vomiting , neck stiffness , photophobia or loss of consciousness . the physical examination may reveal retinal haemorrhage , evidence of meningeal irritation , reduced level of consciousness and / or focal or unilateral neurological deficit . 
this fact supports the extensive use of ct that , if correctly performed , is capable of identifying an sah in 100% of cases within 12 h of symptom onset and in 93% of cases within 24 h [ 7 , 8 ]  . 
head ct is also able to identify the presence of intraparenchymal haematomas , hydrocephalus , cerebral oedema and , in some cases , can suggest the probable site of aneurysmal rupture [ 9 ]  . 
lumbar puncture is a valuable diagnostic examination in cases of negative ct and suspected sah . for exact localisation of the source of extravasated blood , the diagnostic protocol involves performing digital subtractive angiography ( dsa ) or ct angiography ( cta )  . 
whereas dsa is the gold standard for identifying cerebral aneurysms , cta is gaining increasing acceptance , thanks to its immediacy and noninvasiveness and a specificity and sensitivity comparable with dsa . 
cta also has a significant advantage in that it can be performed immediately following the baseline ct examination , which enables a significant shortening of examination times and the possibility of initiating treatment immediately [ 1023 ]  . the aim of this paper is to present a consecutive series of 130 patients diagnosed with sah in a single institution ( ospedale centrale regionale di bolzano ) who underwent cta and / or dsa and to analyse their sensitivity , specificity and diagnostic accuracy , comparing them with the data in the literature . le normalmente percorsi da liquido cerebrospinale . 
lesa non aneurismatica , inclusa lesa perimesencefalica , caratterizzata da buona prognosi e rare complicanze neurologiche [ 2 ]  . lincidenza mondiale aggregata dellesa , che varia da regione a regione , di circa 10 , 5 casi / 100000 abitanti per anno ed rimasta stabile negli ultimi 30 anni . 
i familiari di primo grado di pazienti con esa hanno fattore di rischio aumentato ; vi sono inoltre patologie ereditarie del tessuto connettivo associate ad aneurismi intracranici ed esa quali la malattia policistica renale , la sindrome di ehlers - danlos ( tipo iv ) , lo pseudoxantoma elastico e la displasia fibromuscolare [ 5 ]  . sulla base di tali dati prognostici unesa deve essere sempre sospettata in pazienti con presentazione tipica : cefalea violenta ed improvvisa associata a nausea , vomito , dolore cervicale , fotofobia o perdita di coscienza . 
in assenza dei classici segni e sintomi la diagnosi di esa pu essere confusa con diagnosi di emicrania e cefalea muscolo - tensiva in circa il 50% dei casi [ 6 ]  . 
tali dati supportano luso estensivo che viene fatto della tomografia assiale computerizzata ( tc ) che , se eseguita correttamente , in grado di rivelare unesa nel 100% dei casi entro 12 ore dallesordio sintomatologico e nel 93% entro 24 ore [ 7 , 8 ]  . 
la tc del cranio inoltre in grado di discriminare la presenza di ematomi intraparenchimali , idrocefalo , edema cerebrale e pu , in alcuni casi , suggerire la probabile sede della rottura aneurismatica [ 9 ]  . 
la rachicentesi mantiene una validit diagnostica nel caso di tc negativa e quadro clinico sospetto per esa . il percorso diagnostico prevede quindi , per lesatta localizzazione della sorgente del sanguinamento , lesecuzione di unangiografia digitale sottrattiva ( dsa ) oppure di un angiotc ( atc )  . 
lo studio angiografico cerebrale a sottrazione digitale rappresenta il gold standard per levidenziazione degli aneurismi cerebrali ; latc viene sempre pi utilizzata per limmediatezza e la non invasivit associate a specificit e sensibilit comparabili con la dsa . 
contrast enhancement was provided by the intravenous antecubital administration of an 80ml bolus of nonionic iodinated contrast material ( iomeron 350 mgi / ml , bracco , milan , italy ) at a 4 - ml / s flow rate followed by 50 ml saline solution at the same rate . 
the sample volume was positioned in an internal carotid artery with a threshold of + 30 hu . after acquisition data transfer to the postprocessing station ( leonardo , siemens , erlangen , germany ) , image analysis was performed interactively by the radiologist using various postprocessing techniques , including two ( 2d ) and three - dimensional ( 3d ) multiplanar reconstructions ( mpr ) , maximum intensity projections ( mip ) and volume rendering ( vr )  . each cta and dsa examination was assessed independently by the performing radiologist , and the aneurysms were classified according to the following scheme ( table 1 ) : site : aneurysms originating from the anterior circulation : internal carotid artery , ophthalmic artery , anterior choroidal artery , anterior communicating artery , anterior cerebral artery ( a1 , a2 ) , pericallosal artery , callosal - marginal artery , middle cerebral artery ( m1 , m2 , m3 ) , posterior communicating artery aneurysms originating from the posterior circulation : basilar artery , apex of the basilar artery , posteroinferior cerebellar artery , anteroinferior cerebellar artery , anterosuperior cerebellar artery , posterior cerebral artery . size : giant aneurysms : 25 mm large aneurysms : 1325 mm medium aneurysms : 513 mm small aneurysms : 35 mm very small aneurysms : < 3 mm nel periodo compreso tra giugno 2004 e febbraio 2006 ogni paziente con diagnosi tc di esa non traumatica stato sottoposto a atc ( somatom sensation 16 , siemens , erlangen , germania ) e , successivamente , a dsa ( multistar plus top , siemens , erlangen , germania )  . 
sono stati arruolati 130 pazienti consecutivi , 57 maschi e 73 femmine con et media di 59 , 515 , 2 anni ( range 2091 anni ) ; 91 pazienti provenienti dal pronto soccorso dellospedale centrale regionale di bolzano e 39 da altre strutture ospedaliere . latc stata eseguita con sistema a 16 detettori ( somaton sensation 16 , siemens , erlangen , germania ) con i seguenti parametri di acquisizione e ricostruzione : scansione sul piano assiale estesa dal soma della prima vertebra cervicale al vertice , collimazione 0 , 75 mm , tempo di rotazione 0 , 5 s , fov 230 mm , feed / rot 6 , 5 , 120 kv , 210 mas , ricostruzione in tempo reale 3 mm / 3 mm con fov 120 , ricostruzione per il postprocessing 1 mm / 0 , 5 mm con fov 80 . 
per lopacizzazione arteriosa sono stati somministrati in bolo , per via endovenosa antecubitale , 80 ml di mezzo di contrasto iodato non ionico ( iomeron 350 mgi / ml , bracco , milano , italia ) al flusso di 4 ml / s seguiti da 50 ml di soluzione salina allo stesso flusso . 
in the event of positive cta , only those patients whose cta findings were not complete for the purposes of surgical planning or were to undergo endovascular therapy were required to undergo dsa . 
cta and dsa findings were compared with regard to sensitivity , specificity and diagnostic accuracy . angioarchitettura : presenza o meno di vasi ad origine dalla sacca aneurismatica . tutti i pazienti con atc negativa sono stati sottoposti entro 48 ore a dsa . 
in caso di atc positiva sono stati inviati allo studio angiografico solamente i pazienti con quadro atc non esaustivo ai fini della programmazione chirurgica o quelli indirizzati allapproccio terapeutico endovascolare . infine , quando i reperti atc sono stati giudicati sufficienti per la pianificazione chirurgica , i pazienti sono stati sottoposti allintervento chirurgico senza ulteriori esami diagnostici . 
i dati atc ed angiografici sono stati confrontati in termini di sensibilit , specificit ed accuratezza diagnostica . all cta and dsa examinations were technically assessable . all patients who presented for the cta examination without assisted ventilation were studied without sedation ; about 60% of these required sedation at dsa . 
all cta and dsa procedures were without complications ( allergic reactions , acute renal failure or extravasation at the site of venous injection , more serious neurological complications or complications at the site of arterial catheterisation )  . all 21 patients with a negative cta underwent dsa . 
of the 109 patients with positive cta , 79 underwent emergency dsa to clarify the cta findings and plan therapy , and five underwent dsa during endovascular embolisation on average 13 days after the acute event ( 135.5 days , range 720 days )  . 
in 25 cases , the cta findings were considered complete for surgical purposes , such that these patients unrisultati tutti gli esami atc e dsa eseguiti sono risultati tecnicamente valutabili . 
tutte le procedure eseguite , atc e dsa , sono state esenti da complicazioni ( reazioni allergiche , ira o stravasi in sede di iniezione venosa , complicanze neurologiche maggiori o nella sede di cateterismo arterioso )  . tutti i 21 pazienti con atc negativa sono stati sottoposti a dsa . 
tra i 109 pazienti con atc positiva 79 sono stati sottoposti a dsa urgente per chiarire i reperti atc al fine della pianificazione terapeutica , 5 hanno eseguito lo studio angiografico durante la procedura di embolizzazione endova1325 513 total dimensioni ( mm ) 1325 513 totale results m . 
1 percorso diagnostico dei 130 pazienti arruolati nello studio . derwent dsa post - operatively only . of the 105 patients who underwent dsa , cta findings were confirmed in 104 : in 20 cases where the findings were negative and in 84 cases with the presence of one or more aneurysms . 
in 25 casi il quadro atc stato giudicato esaustivo ai fini dellintervento chirurgico , pertanto questi pazienti sono stati sottoposti a dsa solo come controllo post - operatorio . tra i 105 pazienti sottoposti a dsa il quadro atc stato confermato in 104 : in 20 casi per la negativit del reperto ed in 84 per la presenza di uno o pi aneurismi . 
solo in un caso la dsa ha dimostrato la presenza di un aneurisma del circolo anteriore delle dimensioni di 2 mm non riconosciuto allatc ; lidentificazione di questo piccolo aneurisma stata comunque possibile alla valutazione retrospettiva delle immagini . 
nei 25 pazienti sottoposti al trattamento chirurgico senza la valutazione dsa pre - operatoria stata riscontrata unottima corrispondenza tra le informazioni fornite dallatc ed il quadro chirurgico per quanto riguarda le dimensioni , la morfologia e langioarchitettura . 
2a - i a 41 - year - old woman : axial ( a ) , coronal ( b ) and sagittal ( c ) multiplanar reconstruction ( mpr ) images show a large aneurysm at the bifurcation of the left middle cerebral artery . 
axial ( d ) , coronal ( e ) and sagittal ( f ) maximum intensity projection ( mip ) images obtained with a 10 - mm - thin section more clearly show the aneurysms morphology , its multilobular profiles and relationship with surrounding vessels . 
volume rendering ( vr ) posteroanterior image ( h ) better shows vessels of cranial base and the aneurysvr and transparent vr images ( i ) : this last reconstruction shows a better three - dimensional ( 3d ) image of the aneurysmal sac and surrounding vessels . 
le ricostruzioni mpr assiale ( a ) , coronale ( b ) e sagittale ( c ) dimostrano un aneurisma di grandi dimensioni alla biforcazione dellarteria cerebrale media di sinistra . 
le ricostruzioni mip a strato sottile ( 10 mm ) assiale ( d ) , coronale ( e ) e sagittale ( f ) meglio definiscono la morfologia dellaneurisma , i profili polilobati ed i suoi rapporti con i vasi circostanti . 
3a - i a 51 - year - old woman : coronal ( a ) , axial ( b ) , sagittal ( c ) maximum intensity projection ( mip ) and volume rendering ( vr ) ( d - f ) images show a medium - sized aneurysm at the bifurcation of the right middle cerebral artery ( large arrow ) , a small one at the bifurcation of the left middle cerebral artery ( thin arrow ) and a very small aneurysm of the anterior communicating artery ( arrowhead )  . 
le ricostruzioni mip coronale ( a ) , assiale ( b ) , sagittale ( c ) e vrt ( d - f ) dimostrano un aneurisma di medie dimensioni alla biforcazione dellarteria cerebrale media destra ( freccia grande ) , un aneurisma di piccole dimensioni alla biforcazione dellarteria cerebrale media sinistra ( freccia sottile ) ed un aneurisma molto piccolo allarteria comunicante anteriore ( punta di freccia )  . 
4a - d a 38 - year - old woman : axial ( a ) , coronal ( b ) , sagittal ( c ) maximum intensity projection ( mip ) and volume rendering ( vr ) ( d ) images show a medium - sized aneurysm with a long neck of the right internal carotid artery ( arrow )  . 
ricostruzioni mip assiale ( a ) , coronale ( b ) , sagittale ( c ) e vrt ( d ) di un aneurisma di medie dimensioni dellarteria carotide interna destra con lungo colletto ( freccia )  . 
most aneurysms were of medium sized ( n = 67 ; 50% ) , followed by small ( n = 34 ; 25.4% ) , very small ( n = 16 ; 11.9% ) and large ( n = 16 ; 11.9% ) ( table 1 )  . overall , cta identified 108 of the 109 aneurysms seen at dsa and 25 aneurysms confirmed at surgery . 
6a , b a 60 - year - old man : axial ( a ) and coronal ( b ) maximum intensity projection ( mip ) images show a large , partially thrombosed , aneurysm at the bifurcation of the right middle cerebral artery ( arrow )  . 
7a - d an 81 - year - old man : axial ( a ) , coronal ( b ) paracoronal ( c ) maximum intensity projection ( mip ) and digital subtraction angiography ( dsa ) ( d ) images show a large aneurysm ( arrow ) with a wide neck of the significantly calcified left vertebral artery . 
ricostruzioni mip assiale ( a ) , coronale ( b ) , para - coronale ( c ) e corrispettivo dsa ( d ) di un aneurisma di grandi dimensioni con ampio colletto dellarteria vertebrale sinistra ( freccia ) , estesamente calcifica . 
filiforme larteria vertebrale destra ( b , punta di freccia )  . we also encountered excellent agreement between cta and dsa with regard to assessing the aneurysmal neck and angioarchitecture . 
these data were available for 92 of the 134 aneurysms in our study : we excluded the aneurysm not identified at cta , the 25 aneurysms treated surgically prior to dsa and the 16 aneurysms < 3 mm , the small size of which hindered realistic measurement of the neck . 
in 12 medium and large aneurysms prevalently located in the middle cerebral artery , the presence of a vessel originating directly from the aneurysmal sac was identified . complessivamente con atc sono stati riconosciuti 108 dei 109 aneurismi visualizzati con dsa cui vanno sommati i 25 aneurismi confermati allatto chirurgico . 
pertanto i valori di sensibilit e specificit raggiungono rispettivamente il 98 , 8% ed il 100% , la predittivit positiva del 100% e la predittivit negativa del 95 , 2% , con unaccuratezza diagnostica del 99 , 0% ( tabella 2 )  . abbiamo riscontrato unottima corrispondenza tra atc e dsa anche nella valutazione del colletto aneurismatico e dellangioarchitettura ; tali dati sono disponibili per 92 dei m . 
the technological developments of multidetector ct and especially the introduction and diffusion in clinical practice of 16 - slice scanners have led to improved spatialtemporal resolution , thus enabling an accurate and minimally invasive study of cerebral aneurysms , even in the event of acute sah . the possibility of performing cta immediately following a ct diagnosis of sah , along with the noninvasiveness of the technique , speed of execution and high diagnostic sensitivity and specificity , increasingly promote cta as a highly reliable technique in studying cerebral aneurysms . our prospective study involving 130 patients shows that in all cases , cta was technically feasible and was well tolerated by all patients . 
all examinations could be assessed , and there were no major motion artefacts . however , a limitation to our study was the absence of patients who had previously undergone neurosurgery or endovascular embolisation ; hence , we have no direct experience with artefacts caused by metal clips or coiled springs . 
in this respect , it should be noted that previous studies have demonstrated the validity of cta even in the post - operative follow - up [ 24 ]  . shorter acquisition times and increased spatial resolution are two important advantages introduced by 16 - slice scanners . 
the increased acquisition speed not only enables an exclusively arterial acquisition for the entire scan duration but also reduces the incidence of motion artefacts , which can seriously compromise image quality to the point of rendering the images nondiagnostic . 
this should not be underestimated in this type of patient , who is at times drowsy , at times suffering from psychomotor agitation and often uncooperative 134 aneurismi oggetto del nostro studio : abbiamo escluso laneurisma non riconosciuto con atc , i 25 aneurismi trattati chirurgicamente prima dellesecuzione dellangiografia ed i 16 aneurismi < 3 mm le cui piccole dimensioni non consentivano una reale definizione della base dimpianto . 
le due metodiche sono state concordi nel definire 48 aneurismi con colletto stretto e 44 aneurismi a larga base dimpianto . in 12 aneurismi di medie e grandi dimensioni , prevalentemente localizzati allarteria cerebrale media , stata identificata lemergenza di un vaso direttamente dalla sacca aneurismatica . discussione lo studio degli aneurismi cerebrali stato per lungo tempo affidato alla dsa , metodica invasiva , non priva di rischi per il paziente e caratterizzata da lunghi tempi di esecuzione . gli sviluppi tecnologici della tomografia computerizzata multidetettore ed in particolare lintroduzione e la diffusione nella pratica clinica delle apparecchiature a 16 strati , hanno portato ad un miglioramento della risoluzione spazio - temporale permettendo uno studio accurato e poco invasivo degli aneurismi cerebrali anche in caso di esa in fase acuta . la possibilit di effettuare latc subito dopo la diagnosi tc di esa , la non invasivit e la rapidit di esecuzione associate ad elevate sensibilit e specificit diagnostiche propongono sempre pi latc quale metodica altamente affidabile nello studio degli aneurismi cerebrali . questo studio prospettico relativo a 130 pazienti evidenzia che latc si dimostrata tecnicamente sempre eseguibile e ben tollerata dal paziente ; tutti gli esami sono risultati valutabili , in particolare non si sono registrati grossolani artefatti da movimento . per contro un limite di questo studio lassenza di pazienti gi precedentemente sottoposti ad interventi neurochirurgici o ad embolizzazione endovascolare : non abbiamo quindi esperienza diretta con gli artefatti da clips o spirali metalliche . 
a tale riguardo va aggiunto che precedenti studi hanno dimostrato la validit dellatc anche nei controlli post - operatori [ 24 ]  . la riduzione dei tempi di acquisizione e laumento della risoluzione spaziale sono due importanti vantaggi introdotti dagli apparecchi a 16 strati . 
la maggiore velocit di acquisizione non solo consente di ottenere unacquisizione esclusivamente arteriosa per tutta la durata della scansione , ma riduce lincidenza degli artefatti da movimento che possono inficiare la qualit delle immagini sino a renderle non diagnostiche . 
questo aspetto non va sottovalutato in questa tipologia di paziente : talvolta soporoso , talvolta in stato di agitazione psicomotoria e spesso comunque poco collaborante e poco incline a mantenere limmobilit del capo anche per pochi secondi . 
per nessuno dei pazienti sottoposti ad atc stata necessaria la sedazione , al contrario pi della met dei pazienti sottoposti a dsa ha richiesto lintervento dellanestesista al fine di ottenere immagini diagnostiche , prive di artefatti da movimento . un esame atc viene eseguito in circa 10 minuti cui va m . 
in contrast , a cerebral dsa study requires angiographic experience , and when performed by an expert has a duration of 4560 min , to which is added about 10 min for reporting the findings . the move from 4to 16 - slice devices has further increased spatial resolution . 
various studies performed with 4slice scanners have already demonstrated excellent diagnostic accuracy regarding aneurysms larger than 4 mm while underlining the limitations for smaller aneurysms [ 14 , 17 , 20 , 25 ]  . 
 mpr , which were assessed in cine mode in the three orthogonal planes in space , help confirm the presence of the aneurysm or determine whether it was not identified in the axial plane alone . 
mpr performed in various oblique planes or curved mpr along the vessel course helps define the relations with the surrounding vascular and bone structures and the size of the sac and neck , as well as parietal calcifications and partial thromboses . 
nonetheless , these easy - to - use reconstructions return a plastic , 3d - like image of the main vascular branches for most of their course , which often helps in the recognition of aneurysmal protrusions . 
these reconstructions , which are the most similar to traditional angiographic images , can be applied particularly sommato un tempo medio di elaborazione - lettura delle immagini di circa 1520 minuti : il referto quindi disponibile dopo 2530 minuti dallarrivo del paziente alla tc . 
al contrario lesecuzione di una panangiografia cerebrale richiede esperienza angiografica ed in mani esperte ha una durata media di 4560 minuti cui vanno sommati circa 10 minuti per la refertazione . il passaggio dai 4 ai 16 strati ha incrementato ulteriormente la risoluzione spaziale : questo ha permesso di aumentare le potenzialit diagnostiche dellatc soprattutto nellambito degli aneurismi di dimensioni molto piccole . 
diversi studi , condotti con apparecchi a 4 strati , hanno gi dimostrato lottima accuratezza diagnostica per gli aneurismi di dimensioni superiori ai 4 mm e sottolineato i limiti per quelli di dimensioni pi piccole [ 14 , 17 , 20 , 25 ]  . 
le ricostruzioni mpr secondo vari piani obliqui o mpr curvate lungo il decorso di un vaso aiutano a definire i rapporti con le strutture vasali ed ossee , le dimensioni della sacca e del suo colletto , calcificazioni parietali o trombosi parziali . le ricostruzioni in proiezione di massima intensit ( mip ) proiettano nel piano di ricostruzione solo i voxel pi luminosi del volume scelto : si tratta di ricostruzioni bidimensionali con perdita dellinformazione spaziale . 
tuttavia queste ricostruzioni , di facile applicazione , ci restituiscono unimmagine plastica , simil - 3d , delle principali diramazioni vasali per gran parte del loro decorso che spesso aiuta nel riconoscimento delle protrusioni aneurismatiche . 
le ricostruzioni mip , le pi vicine allimmagine angiografica tradizionale , trovano applicazione soprattutto nella valutazione delle diramazioni arteriose che si allontanano dal poligono di willis mentre non consentono una valutazione corretta delle strutture in stretto rapporto con la base cranica per lelevata attenuazione dellosso . 
it should also be borne in mind that very small aneurysms can be masked by vascular structures if the latter are projected in the same plane as the reconstruction . vr is a 3d reconstruction technique that requires definition of a threshold attenuation value ( measured in hounsfield units ) for selecting reconstruction voxels . 
these images help in interpreting the vascular relations and can be used to help in surgical planning , as they simulate the intraoperative view . postprocessing performed with a reduced fov enables improved spatial resolution . 
reconstructions in the various planes in space enable recognition and therefore sufficiently adequate characterisation of morphology , size , neck and development of an aneurysm as well as its relation to adjacent vessels and surrounding structures . 
the volumetric reconstructions , which are easy to obtain and have a high visual impact , return a plastic image that is often more useful in defining the aneurysms relations and development than in diagnosing it . 
it should also be borne in mind that only thin - section mpr , which are more laborious and less intuitive , enable correct identification of small irregularities of the sacs profile or partial thrombosis , where present . conclusions our study confirms the data in the literature . 
the ability of cta to define the relevant anatomical characteristics for surgical treatment , such as aneurysmal neck morphology , parietal calcification , sac thrombosis and relations with adjacent structures , as well as 3d reconstruction of the neurovascular angioarchitecture means that fundamental additional information can be supplied for deciding the next treatment steps . the results obtained in this study confirm the data in the literature and encourage the use of cta in routine clinical practice , with recourse to dsa only in selected cases or cases with uncertain diagnosis . 
at our institute , this study determined a change in managing patients with acute nontraumatic sah : in the event of positive cta finding for the presence of an aneurysm , in most cases , patients are scheduled for surgery without undergoing a dsa study . given the aetiology of nontraumatic sah and its highlevel of risk in the event of an incorrect diagnosis , fundamental requisites for cta study include correct technical execution and careful and interactive image assessment : where there is doubt , patients should undergo dsa . 
with regard to the future , cta is also a promising technique for follow - up to verify exclusion of the aneurysmal sac after endovascular or surgical procedures . essere mascherati dalle strutture vascolari se queste si proiettano nello stesso piano di ricostruzione . lelaborazione volume rendering technique ( vrt ) una tecnica di ricostruzione tridimensionale che richiede la definizione di un valore soglia di attenuazione ( in unit hounsfield ) per la selezione dei voxel di ricostruzione . 
queste immagini facilitano linterpretazione dei rapporti vascolari e possono essere di aiuto nella pianificazione dellapproccio chirurgico , simulando la visione intraoperatoria . le rielaborazioni con field of view ( fov ) ridotto consentono una migliore risoluzione spaziale : le ricostruzioni secondo i vari piani dello spazio permettono di riconoscere e quindi di caratterizzare in maniera adeguata morfologia , dimensioni , base dimpianto e sviluppo di un aneurisma oltre ai suoi rapporti con i vasi adiacenti e le strutture circostanti . 
le ricostruzioni volumetriche ( vrt ) facili da ottenere e di sicuro impatto visivo , restituiscono unimmagine plastica spesso pi utile nella definizione dei rapporti e dello sviluppo dellaneurisma che non nella sua diagnosi ; sono spesso richieste dai neurochirurghi perch meglio rispecchiano la spazialit e la visualizzazione intraoperatoria . 
non va dimenticato tuttavia che solo le ricostruzioni multiplanari sottili , pi laboriose e meno intuitive , ci consentono di identificare correttamente eventuali piccole irregolarit dei profili della sacca o la sua parziale trombosi . conclusioni questo studio conferma i dati della letteratura : latc una metodica poco invasiva , rapida , altamente sensibile e specifica nella diagnosi di aneurismi cerebrali nelle esa . 
la capacit dellatc di definire caratteristiche anatomiche rilevanti per il trattamento chirurgico quali la morfologia del colletto aneurismatico , le calcificazioni parietali , la trombosi della sacca ed i rapporti con le strutture adiacenti e la ricostruzione tridimensionale dellangioarchitettura neurovascolare consentono di fornire informazioni aggiuntive fondamentali per decidere il successivo trattamento dei pazienti . 
nella nostra realt questo studio ha determinato un cambiamento nella gestione del paziente con esa acuta non traumatica : in caso di reperto angio - tc positivo per la presenza di un aneurisma la maggior parte dei pazienti viene portata allintervento chirurgico senza lesecuzione della dsa . considerando leziologia dellesa non traumatica e la sua pericolosit in caso di errata diagnosi , uno studio atc non pu prescindere da unesecuzione tecnicamente corretta e da una valutazione attenta ed interattiva delle immagini : nei casi dubbi il ricorso alla dsa rappresenta tuttora lopzione di scelta . 
passariello1 1dipartimento di scienze radiologiche , universit degli studi di roma la sapienza , policlinico umberto i , v.le regina elena 324 , i - 00161 roma , italy 2servizio di fisica radiologica , dipartimento di scienze radiologiche , universit degli studi di roma la sapienza , roma , italy correspondence to : m . 
we present our initial clinical experience with a recently introduced 64 - detector computed tomography ( 64 - mdct ) scanner that makes use of a periodic motion of the focal spot in the longitudinal direction ( z - flying focal spot ) , which enables it to reach a final spatial resolution of 0.40.40.4 mm3 and a temporal resolution of 83 ms . 
a total of 114 patients ( 108 men , six women ; age range 3677 years , mean 63.1 years ) underwent retrospective electrocardiogram ( ecg ) - gated examination of the coronary arteries using a 64 - mdct scanner ( somatom sensation 64 , siemens medical solutions , germany )  . 
acquisition parameters were the following : collimation 640.6 mm , 800 quality reference milliampere second ( mas ) , 120 kvp , 0.33 - s gantry rotation time and pitch 0.2. 
administration of 80 ml of contrast agent ( iomeron 400 mgi / dl , bracco , italy ) + 30 ml of saline at 4 / s and delay time determined using a bolus triggering technique . 
oral betablockers were administered to patients with heart rate ( hr ) > 75 bpto reduce radiation exposure , an automatic exposure control system was applied in all cases to adapt tube current to patient size and anatomic shape ( care dose 4d , siemens medical solutions , germany )  . 
the optimal temporal window for raw data reconstruction was chosen from an initial preview of images reconstructed with different phase settings ( range 0%95% rr interval with 5% gap ) at a selected anatomical level in the mid part of the right coronary artery . 
in patients with hr < 60 bmp , the best reconstruction intervals were identified in the end - systolic ( 30%35% of the rr interval ) and end - diastolic ( 60%65% of the rr interval ) phases ; with faster hr ( > 80 bmp ) , high image quality was observed in end - systole ( 30%35% of the rr interval )  . 
nel presente lavoro introduciamo la nostra esperienza preliminare e lottimizzazione del protocollo di acquisizione di un apparecchio tcms in grado di acquisire 64 strati per rotazione ( tcms - 64 ) grazie allutilizzo di un movimento periodico della macchia focale lungo lasse longitudinale z che consente di raggiungere una risoluzione spaziale di 0 , 40 , 40 , 4 mm3 e temporale di 83 ms . 
114 pazienti ( 108 maschi e 6 femmine , intervallo di et 3677 anni , media 63 , 1 ) sono stati sottoposti ad esame con tcms delle arterie coronarie utilizzando uno scanner tcms - 64 ( somatom sensation 64 , siemens medical solutions , germania )  . 
i parametri di acquisizione sono stati i seguenti : collimazione 640 , 6 mm , 800 quality reference mas , 120 kvp , tempo di rotazione 0 , 33 s e pitch 0 , 2 . 
in tutti i pazienti le immagini sono state acquisite dopo somministrazione ev di 80 ml di mdc ( iomeron 400 mgi / dl , bracco , italia ) + 30 ml di soluzione fisiologica con ritardo di scansione calcolato con tecnica bolus triggering . 
in tutti i casi stato utilizzato un sistema di limitazione automatica della dose ( care dose 4d , siemens medical solutions , forchheim , germania ) in grado di modulare la corrente emessa del tubo in relazione ai valori di attenuazione della regione che si sta studiando . 
la finestra temporale ottimale per la ricostruzione dei dati grezzi stata scelta da uniniziale preview di immagini ricostruite in differenti fasi del ciclo cardiaco ( tra 0%95% dellintervallo r - r ogni 5% ) rilevato ad un livello anatomico predefinito nel tratto medio dellarteria coronaria di destra . 
durante lacquisizione tcms stata registrata una fc media di 65 , 619 , 2 bpm ( intervallo : 4496 bpm ) con bradicardizzazione effettuata in 16 / 114 pazienti ( 14 , 0% )  . 
the 64 - mdct scanner diagnostic performance for coronary ct angiography is further improved with better spatial and temporal resolution and faster scan times ; besides , initial clinical results are promising . 
nei pazienti con fc < 60 bpm , la migliore finestra temporale nella quale effettuare la ricostruzione delle immagini stata individuata rispettivamente nelle fasi telesistolica ( 35% dellintervallo r - r ) e meso - telediastolica ( 65%70% dellintervallo r - r ) ; nei casi con fc di base elevata ( > 80 bpm ) , la migliore fase per la ricostruzione delle immagini risultata la telesistole ( 35% dellintervallo r - r )  . 
il ctdi volumetrico ( ct dose index volume ) risultato in media di 36 , 538 , 30 mgy per paziente con un range compreso tra 27 , 2 e 61 , 9 mgy corrispondenti ad un eff mas . 
se confrontato con unacquisizione effettuata a amperaggio costante , lutilizzo del sistema di limitazione automatica della dose ha consentito una riduzione media del ctdi volumetrico del 33 , 3% per paziente ( p < 0 , 001 )  . 
la nostra esperienza clinica iniziale ha evidenziato come lultima generazione di tomografi tcms - 64 apra nuovi scenari per la gestione clinica del paziente coronaropatico . lutilizzo di sistemi idonei di modulazione automatica dellintensit di corrente del tubo radiogeno appare tuttavia indispensabile per limitare la dose di esposizione del paziente che rappresenta , allo stato attuale , il limite principale allutilizzo clinico della metodica . parole chiave arterie coronarie tc multidetettore coronaropatia infarto miocardico dose di esposizione in tc introduction introduzione since the introduction of multidetector computed tomography ( mdct ) into clinical practice in 1998 , diagnostic imaging of the heart and coronary arteries has been one of the main fields of interest for this technique [ 1 , 2 ]  . 
despite the major epidemiological , health and economic implications of ischaemic heart disease [ 3 ] , the study of the coronary tree using noninvasive techniques was for years a difficult problem to solve due to intrinsic technical limitations . 
the coronary artery circulatory is , in fact , a difficult vascular district to assess using the various imaging techniques available due not only to the small diameter and tortuous course of the arteries but also to the complex cardiorespiratory movements . the high spatial and temporal resolution obtainable with the most recent 16 - , 32 - , 40and 64 - detector mdct devices , coupled with the possibility of achieving adequate cardiac synchronisation , has enabled these limitations to be overcome , at least in part , thus revolutionising the management of patients with heart disease [ 7 , 8 ]  . 
the numerous clinical studies published in recent years have demonstrated the high level of diagnostic accuracy of mdct with regard to both assessment of coronary artery stenosis and follow - up of coronary artery bypass graft patency [ 916 ]  . the volume acquired for studying coronary arteries can be used for performing functional studies on volumes , calculating the ejection fraction and myocardial mass of both ventricles , and assessing regional motion and identifying the presfin dalla sua introduzione in ambito clinico nel 1998 , la diagnostica per immagini del cuore e delle arterie coronarie ha rappresentato uno dei campi di maggior interesse della tc multistrato ( tcms ) [ 1 , 2 ]  . 
malgrado le rilevanti implicazioni epidemiologiche ed economico - sanitarie della cardiopatia ischemica [ 3 ] , lo studio dellalbero coronarico con metodiche di tipo non invasivo stato per molti decenni un problema di difficile soluzione per limiti tecnici intrinseci , rimanendo dominio quasi esclusivo della coronarografia selettiva , procedura gravata da una non trascurabile mortalit e morbilit [ 46 ]  . 
il circolo arterioso coronarico rappresenta infatti un distretto vascolare di difficile valutazione per le diverse metodiche di imaging sia per il piccolo calibro ed il decorso tortuoso delle sue arterie , che per la complessit dei movimenti cardio - respiratori . lelevata risoluzione spaziale e temporale ottenibile soprattutto con i pi recenti apparecchi tcms a 16 , 32 , 40 e 64 canali e la possibilit di ottenere unadeguata cardiosincronizzazione ha consentito di superare almeno in parte tali limitazioni , dando luogo ad una rivoluzione nella gestione del paziente coronaropatico [ 7 , 8 ]  . 
i numerosi studi clinici pubblicati in questi ultimi anni hanno ormai ampiamente dimostrato come la tcms rappresenti una metodica dalla elevata accuratezza diagnostica sia per quanto riguarda la valutazione delle stenosi coronariche che il follow - up della perviet dei by - pass aorto - coronarici [ 916 ]  . il volume acquisito per studiare le arterie coronarie pu anche essere utilizzato per effettuare studi funzionali sui volumi , per calcolare la frazione di eiezione e la massa miom . 
nonetheless , clinical use of coronary artery mdct is currently hindered by several limitations [ 20 ] : imaging of coronary stents that due to their metallic struts generate beam - hardening artefacts and hinder accurate assessment of the state of the intrastent vascular lumen [ 21 , imaging of extensively calcified coronary arteries , also due to beam - hardening artefacts [ 23 ] elevated patient heart rate ( hr ) ( > 65 bmp ) , which causes progressive shortening of the electrocardiogram ( ecg ) rr interval and a consequent shortening of the end - diastolic phase in which image acquisition occurs , thus leading to motion artefacts [ 24 , 25 ] rhythm disturbances , which render cardiac synchronisation impossible [ 25 ] accuracy in assessing regional motion caused by the relatively low temporal resolution of mdct , especially when compared with other techniques such as magnetic resonance imaging ( mri ) and , above all , ecg [ 17 ]  . clearly then , most limitations of mdct depend on the resolution limitations of the technique , and therefore , as achenbach emphasised in 2004 , the key to obtaining increasingly accurate diagnostic imaging lies in increasing both spatial and temporal resolutions [ 26 ]  . in this context , we present our early clinical experience with the latest - generation 64 - mdct device in noninvasive coronary artery imaging . 
development of this latest generation of mdct in fact leads to a potential increase in radiation exposure , which could limit the clinical use of 64 - mdct , especially if proposed as a potential screening tool . materials and methods study population between january and may 2005 , 114 patients ( 108 men and six women ; age range 3677 years , mean 63.1 years ) underwent mdct angiography of the coronary arteries . 
all examinations were performed using a 64 - mdct device with a 0.33 - s gantry rotation time ( somatom sensation 64 , siemens medical solutions , forchheim , germany ) and images acquired with retrospective ecg gating . 
clinical indications for the examination were the following : typical ( n = 16 ) or atypical ( n = 7 ) chest pain in patients with a clinical indication for coronarography follow - up at 69 months of the patency of treated coronary stents ( n = 37 for a total of 63 stents )  . 
the findings of the comparison of the two techniques will therefore not be presented here , as this falls beyond the scope of the present study . follow - up of patency of aortocoronary bypass grafts ( n = 40 for a total of 48 bypasses : 32 venous grafts and 16 arterial grafts ) cardica di entrambi i ventricoli , per valutare la cinesi regionale e per identificare la presenza di aree di miocardio infartuato o non - riperfuso correlando lo stato di un vaso coronarico con quello del suo territorio di pertinenza vascolare [ 1719 ]  . 
allo stato attuale , limpiego clinico della tcms delle arterie coronarie presenta tuttavia alcune rilevanti limitazioni [ 20 ] : imaging degli stent coronarici , che a causa della loro trama metallica generano artefatti da indurimento del fascio che impediscono di valutare in modo accurato lo stato del lume vascolare intra - stent [ 21 , 22 ] ; imaging delle coronarie estesamente calcifiche , sempre per linsorgenza di artefatti da indurimento del fascio [ 23 ] ; frequenza cardiaca ( fc ) elevata del paziente ( > 65 battiti per minuto bpm ) , che determina un progressivo accorciamento dellintervallo r - r elettrocardiografico ( ecg ) e conseguentemente della durata della fase telediastolica in cui avviene lacquisizione delle immagini determinando linsorgenza di artefatti da movimento [ 24 , 25 ] ; turbe del ritmo , con impossibilit di ottenere una adeguata cardiosincronizzazione [ 25 ] ; accuratezza nella valutazione della cinesi regionale determinata dalla relativamente bassa risoluzione temporale della tcms soprattutto se confrontata con altre metodiche di riferimento quali la risonanza magnetica e soprattutto lecocardiografia [ 17 ]  . appare quindi evidente come la gran parte delle attuali limitazioni della tcms dipenda da limiti di risoluzione della metodica e pertanto , come sottolineato da achenbach nel 2004 , la chiave per ottenere prestazioni diagnostiche sempre pi accurate lincremento della risoluzione sia spaziale che temporale [ 26 ]  . in questa ottica , nel presente lavoro introduciamo la nostra esperienza clinica preliminare con apparecchiatura tcms di ultima generazione a 64 strati nellimaging non - invasivo delle arterie coronarie , analizzando anche la problematica dosimetrica . 
lo sviluppo di questa nuova generazione di tcms comporta , infatti , un potenziale incremento della dose di esposizione del paziente che rappresenta un possibile fattore limitante limpiego clinico della tcms - 64 soprattutto se si vuole proporre la metodica come potenziale strumento di screening . materiali e metodi popolazione di studio nel periodo compreso tra gennaio e maggio 2005 , 114 pazienti [ 108 maschi , 6 femmine , et compresa tra 3677 anni ( aa ) media 63 , 1 aa ] sono stati sottoposti ad unangiografia con tcms delle arterie coronarie . 
tutti gli esami sono stati effettuati utilizzando unapparecchiatura di tcms - 64 dotata di velocit di rotazione del sistema tubo radiogeno - detettori di 0 , 33 secondi ( somatom sensation 64 , siemens medical solutions , forchheim , germania ) ed acquisendo i dati con gating elettrocardiografico ( ecg ) di tipo retrospettivo . 
therefore , to obtain high - quality images , the hr was empirically slowed in all patients who at the time of the examination had a basal hr > 75 bmp . 
this was achieved using the following drug regimen : oral administration of 40 mg of beta - blockers 6090 min before the examination ( chlorhydrate , propranolol , inderal , astrazeneca ) , and monitoring arterial blood pressure and pulse rate . image acquisition protocol and automatic exposure control system for the examination , the patient was asked to lie supine on the ct table with arms behind the head . 
three electrodes were placed on the chest ( the first on right emithorax , the second 1 cm under the clavicle and the third on the left flank ) to monitor the patients ecg and obtain a tracing with a lead similar to l1 in which the p wave , the qrs complex and the t wave could be distinguished . 
in all cases , the examination was performed following iv administration of nonionic iodinated contrast material and covering a volume from the tracheal carina to the cardiac base ; for the study of coronary bypass grafts , acquisition was extended to include a volume comprising the proximal anastomosis and the cardiac base . 64 - mdct sensation 64 scanner utilizes an automatic exposure control system [ combined applications to reduce exposure ( care ) dose 4d , siemens medical solutions , forchheim , germany ] that combines both angular ( x - y axis or angular modulation system ) and z - axis ( or z - axis modulation system ) tube current modulation [ 2731 ]  . in practice , beginning with the initial topogram as a reference , the amperage is automatically modulated along the entire acquisition volume on the basis of the angular attenuation coefficient ( mean density of a tissue with respect to the tube rotation angle in the x - y axis ) of the anatomical region being studied and on the basis of the attenuation values of the different tissues along the z - axis , with data detected on the basis of the previous slice ( on - line modulation )  . 
for example , in a total - body study , lower amperages are used for rizzare , in pazienti con indicazione clinica alla coronarografia ; controllo a distanza di 69 mesi della perviet di stent coronarici medicati ( n = 37 per un totale di 63 stent )  . 
non verranno quindi riportati di seguito i risultati sul confronto tra le due metodiche poich non rappresenta lobiettivo specifico del presente lavoro ; controllo a distanza della perviet di by - pass aorto - coronarici ( n = 40 per un totale di 48 by - pass ; 32 graft venosi e 16 arteriosi ) ; pazienti sottoposti a scintigrafia miocardica perfusionale a riposo e dopo sforzo con risultati diagnostici non conclusivi ( n = 3 ) ; pazienti sottoposti ad esame ecocardiografico a riposo e dopo stress farmacologico con dobutamina con risultati diagnostici non conclusivi ( n = 11 )  . tutti i pazienti hanno firmato un consenso informato in cui veniva chiaramente spiegato il tipo di procedura a cui si sottoponevano ed i potenziali rischi . 
i criteri di esclusione per lo studio sono stati i seguenti : nota ipersensibilit ai mezzi di contrasto ( mdc ) iodati ; insufficienza renale ( creatininemia > 2 mg / dl ) ; insufficienza cardiaca conclamata ; aritmie cardiache maggiori ; insufficienza respiratoria ; asma bronchiale nei pazienti con indicazione al pre - trattamento con farmaci beta - bloccanti ( controindicazione assoluta alla somministrazione di beta - bloccanti )  . preparazione del paziente lelevata frequenza cardiaca rappresenta uno dei principali fattori in grado di influenzare negativamente la qualit diagnostica dellesame determinando un accorciamento dellintervallo r - r elettrocardiografico con insorgenza di artefatti da movimento [ 24 , 25 ]  . 
per tale motivo , al fine di ottenere massima qualit delle immagini nella nostra esperienza iniziale la bradicardizzazione stata effettuata empiricamente in tutti i pazienti che , al momento di effettuare lesame , presentavano fc basale > 75 bpm , utilizzando il seguente schema di preparazione farmacologica : somministrazione per os di 40 milligrammi ( mg ) di farmaco - bloccante 6090 minuti prima dellesame ( propranolo cloridrato , inderal , astrazeneca ) , monitorando pressione arteriosa ( pa ) e polso ( fc )  . protocollo di acquisizione delle immagini e sistema di limitazione automatica della dose al momento dellesame il paziente stato invitato a sdraiarsi in posizione supina sul lettino della tc con le braccia dietro la testa . 
posizionando tre elettrodi sul torace ( rispettivamente a livello dellemitorace destro e sinistro , un cm al di sotto della clavicola ed in corrispondenza del fianco sinistro ) stato possibile monitorare lecg del paziente , fino ad m . 
based on the initial topogram in the anteroposterior ( ap ) and lateral position ( a ) , care dose 4d automatically modulates tube current during each tube rotation according to the patients angular attenuation profile . 
1a , b sistema di modulazione automatica della dose : partendo dal topogramma iniziale ( a ) , lamperaggio viene modulato automaticamente lungo tutto il volume di acquisizione in base al coefficiente angolare di attenuazione della regione anatomica che si sta studiando . 
in tutti i casi lesame stato eseguito dopo somministrazione endovenosa ( ev ) di mdc iodato non ionico coprendo un volume cranio - caudale compreso tra la carena tracheale e la base cardiaca ; per lo studio dei bypass coronarici lacquisizione stata estesa in modo da includere un volume compreso tra lanastomosi prossimale e la base cardiaca . lo scanner di tcms - 64 utilizza un sistema di limitazione automatica della dose ( combined applications to reduce exposure , care dose 4d , siemens medical solutions , forchheim , germania ) in grado di combinare la modulazione della corrente emessa dal tubo radiogeno sia in relazione al valori di attenuazione angolare ( xy - axis o angular modulation system ) della regione in esame che attraverso un sistema di modulazione della dose lungo lasse z ( o z - axis modulation system ) [ 2731 ]  . in pratica , partendo come riferimento dal topogramma inim . 
contrast material was administered using a dual - head automatic injector ( medrad stellant dual ; medrad , palo alto , pa , usa ) in which the two syringes containing contrast material and saline solution , respectively , were connected by a y - shaped connector equipped with a one - way valve to prevent reflux . 
use of saline solution reduces the total volume of contrast material administered to the patient , thus compressing the iodine bolus and at the same time reducing the presence of beam - hardening artefacts at the level of the superior vena cava [ 32 ]  . acquisition delay was chosen using the bolus triggering technique , which involves positioning a region of interest ( roi ) in correspondence with the aortic arch and monitoring passage of the contrast material bolus with a series of low - dose dynamic scans ( 120 kvp , 30 mas ) with 1 - s intervals [ 33 ]  . 
in this way , the effective ctdi volume could be measured prior to the examination , even in conditions of constant tube - current intensity throughout the acquisition ( without the use of care dose 4d )  . an estimate of the dose absorbed [ expressed in milligray ( mgy ) ] was then made using the care dose 4d system , and this estimate was compared with the dose to which the patient would have otherwise been exposed without the use of current modulation . 
tuttavia il sistema di mudulazione della dose utilizzato in modalit cardiosincronizzata effettua una semplice riduzione del milliamperaggio in base ai valori di attenuazione individuale del paziente . i parametri tecnici utilizzati sono stati i seguenti : collimazione 3220 , 6 mm ; spessore strato 0 , 75 mm ; incremento 0 , 4 mm ; tempo di rotazione del sistema tubo radiogeno / detettori 0 , 33 s ; pitch 0 , 2 ; quality reference mas : 800 ; effective mas : variabile in relazione alle caratteristiche individuali del paziente ; 120 kvp ; filtri di convoluzione per la ricostruzione delle immagini : b20f ( tessuti molli ) e b46f ( utilizzato per la valutazione degli stent coronarici )  . somministrazione del mezzo di contrasto in tutti i pazienti stato utilizzato un mdc ad alta concentrazione di iodio ( 400 mgi / 100 ml , iomeron 400 , bracco , milano , italia ) seguito da un bolo di soluzione fisiologica . 
il mdc stato somministrato utilizzando un iniettore automatico a doppia testata ( medrad stellant dual , medrad , palo alto pa , usa ) in cui le due siringhe contenenti rispettivamente mdc e soluzione fisiologica sono state collegate tra loro tramite raccordino a y dotato di valvola unidirezionale per impedire il reflusso . 
lutilizzo della soluzione fisiologica consente di ridurre il volume totale di mdc somministrato al paziente , compattando il bolo di iodio e riducendo contemporaneamente la presenza di artefatti da indurimento del fascio a livello della vena cava superiore [ 32 ]  . per la scelta del ritardo di acquisizione stata utilizzata la tecnica del bolus triggering posizionando una regione di interesse in corrispondenza dellarco aortico e monitorizm . 
transverse images of 20 different cardiac cycle phases ( 0%95% of rr interval with 5% gap ) obtained at a predefined anatomical level in the middle part of the right coronary artery ( a )  . 
in this patient with a heart rate of 58 bmp , best image quality with minimal motion artefacts is observed in the end - systolic phase ( 20%25% of rr ) and the midto enddiastolic phase ( 70% of the rr )  . 
maximum intensity projection ( mip ) image obtained in the same patient , reconstructing images at 65% of the rr interval , allows the presence of disease throughout the course of the right coronary artery to be excluded ( b )  . 
2a , b esempio di preview series per la selezione della migliore finestra temporale ( percentuale intervallo r - r ) delle immagini utilizzando gating cardiaco di tipo retrospettivo . immagini assiali delle 20 successive serie di ricostruzioni ( dal 0% al 95% dellintervallo rr con gap del 5% ) , ottenute sulla medesima scansione , a livello del terzo medio della cdx ( a ) ; in questo paziente , con fc di 58 bpm , la minor presenza di artefatti da movimento si osserva nelle immagini ricostruite al 20%25% ( intervallo che corrisponde alla telesistole ) e tra il 60% ed il 70% dellintervallo r - r ( fase meso - telediastolica )  . 
this tends to improve temporal resolution by using the raw data derived from multiple cardiac cycles ( up to a maximum of three cycles ) to reconstruct the images [ 35 ]  . zando il passaggio del bolo di mdc attraverso una serie di scansioni dinamiche a bassa dose ( 120 kvp 30 mas ) con intervalli di 1 secondo [ 33 ]  . 
la soglia predefinita per linizio della scansione stata di 100 unit hounsfield ( hu ) con intervallo di tempo tra raggiungimento del valore soglia ed inizio dellacquisizione stessa di 7 secondi . la somministrazione del mdc stata effettuata con protocollo di tipo monofasico utilizzando i seguenti parametri [ 34 ] : 80 ml di mdc + 30 ml di fisiologica con flusso di 4 ml / s con durata totale della somministrazione di 27 , 5 secondi . image postprocessing valutazione dose di esposizione after identifying the best reconstruction interval , the images were processed using a dedicated workstation connected to tutti i pazienti sono stati studiati con utilizzo del software care dose 4d . 
volume rendering algorithms , in contrast , were used mainly in patients being studied for evaluation of aortocoronary bypasses and in all cases where the intuitive 3d approach of this algorithm enabled concise and easily recognisable representation of the state and site of coronary disease . 
this approach provides the patient with images that are easier to understand . given the large number of images obtained on average for each examination ( around 2 , 000 ) , in all cases , images were digitally stored on a compact disc equipped with software for viewing the images in dicom3 format ( digital imaging communication in medicine )  . 
all data from the mdct examination ( except the raw data ) were then archived using the picture archive and communication system ( pacs ) ( lifeweb image manager , veania imaging technologies , italy )  . results the mdct examination was carried out without complications in all patients . 
hr was slowed down in 16 / 114 patients ( 14.0% ) ; there were no cases of complications or side effects as a result of administration of oral - blockers . 
in one patient with severe chronic obstructive pulmonary disease ( copd ) and a baseline hr of 96 bmp , even though the hr could not be slowed due to contraindications for - blockers , the examination was performed nonetheless for clinical requirements . the optimal interval for image reconstruction varied in relation to patients hr during data acquisition . 
they cordi riferimento del ctdi volumetrico ( ct dose index volume ) quale parametro della dose assorbita durante lacquisizione delle immagini con gating ecg di tipo retrospettivo . il valore di ctdi viene definito automaticamente dallo scanner sulla base del topogramma in relazione alla intensit di corrente applicata durante lacquisizione . 
in tal modo , preliminarmente allesame , stato possibile registrare leffettivo valore di ctdi volumetrico anche in condizioni di costante intensit di corrente del tubo durante lintera acquisizione ( senza utilizzo del care dose 4d )  . stata quindi effettuata una stima della dose assorbita ( espressa in mgy ) utilizzando il sistema care dose 4d confrontandola con la dose a cui sarebbe stato sottoposto il paziente senza lutilizzo della tecnica di modulazione . 
in alternativa le immagini sono state ricostruite con unaltra consolle dedicata ( vitrea 2 , vital images inc , minnesota , usa )  . per standardizzare la ricostruzione 3d dei dati e fornire al medico di riferimento delle immagini il pi possibile comprensibili stato da noi utilizzato uno schema in cui sono state effettuate per ciascun paziente almeno 6 set di ricostruzioni : m . 
tale approccio consente di sfruttare al meglio lelevata risoluzione spaziale della metodica ottenendo unimmagine a spessore estremamente sottile ( 0 , 6 mm ) lungo tutto il decorso del vaso . 
gli algoritmi in volume rendering sono stati invece utilizzati prevalentemente nei pazienti inviati per studio dei by - pass aorto - coronarici e in tutti quei casi in cui lintuitivo approccio 3d di questo tipo di algoritmo ha consentito di ottenere una rappresentazione sintetica e facilmente intuibile di stato e sede della coronaropatia fornendo al paziente immagini di pi semplice comprensione . considerando il grande numero di immagini ottenute in media per ciascun esame ( circa 2000 ) , in tutti i casi stata fornita una documentazione di tipo digitale con compact disc ( cd ) su cui stato installato un software per la lettura delle immagini in formato dicom3 ( digital imaging communication in medicine )  . 
tutti i dati dellesame tcms ( esclusi i dati grezzi ) sono stati poi archiviati tramite sistema pacs ( lifeweb image manager , ferrania imaging technologies , italia )  . risultati lesame tcms stato effettuato senza alcuna complicanza in tutti i pazienti studiati . 
la bradicardizzazione stata effettuata in 16 / 114 pazienti ( 14 , 0% ) ; in nessun caso si sono avute complicanze o effetti collaterali conseguenti alla somministrazione di - bloccanti orali . 
in un paziente con bpco severa e fc basale di 96 bpm , pur non essendo stata effettuata la bradicardizzazione per la presenza di controindicazione clinica alla somministrazione di farmaco - bloccante lesame stato ugualmente eseguito per esigenze cliniche . lintervallo ottimale per la ricostruzione delle immagini variato in relazione alla fc del paziente durante lacquisizione dei dati . 
3a , b a 64 - mdct angiography performed at 6 - months follow - up after drug - eluting coronary stent implantation on the left anterior descending ( lad ) artery . 
a curved multiplanar reformation shows the presence of a metallic stent with the characteristic railway track ( arrowheads ) appearance located in the proximal part of the vessel ; the presence of contrast within and distally to the stent indirectly indicates patency ( arrows )  . 
b three - dimensional volume rendering reconstruction ; the use of dedicated cardiac software enables visualisation of the stented coronary vessel ( arrow ) along an automatically generated centreline displayed in the transverse and longitudinal axes of lad . 
a la ricostruzione multiplanare curva ( cpr ) , generata lungo lasse longitudinale dellarteria discendente anteriore , dimostra la presenza di flusso allinterno ed a valle dello stent ( frecce ) , che presenta peraltro tipico aspetto a binario ( punte di freccia )  . 
b immagine tridimensionale ottenuta con tecnica volume rendering ; utilizzando un software dedicato di campionatura selettiva del vaso in esame , possibile riprodurre , in modo pratico e veloce , il tratto di arteria selezionata ( freccia ) permettendo una visualizzazione del lume vascolare intra - stent con tecnica cpr ; in tal modo si ottiene una rappresentazione dellarteria sia secondo il suo asse trasversale che longitudinale . 
in cases with higher baseline hr ( > 80 bmp ) , the best phase for image reconstruction proved to be the end - systolic phase ( 35% rr )  . 
in patients with intermediate hr ( 60 bmp ) , no ideal reconstruction window was identified , and the rr interval used varied significantly from patient to patient . in 11 patients ( 9.6% ) , the reconstructed images were affected by artefacts due to either extrasystole during data acquisition ( n = 8 ) or inability to maintain breath - hold for the duration of the acquisition ( n = 3 )  . 
the study of only the coronary district produced the following values : ctdi volume per la ricostruzione dei dati grezzi corrispondenti alle fasi telesistolica ( 35% r - r ) e meso - telediastolica ( 65%70% rr )  . 
nei casi con fc di base elevata ( > 80 bpm ) , la migliore fase per la ricostruzione delle immagini risultata la telesistole ( 35% dellintervallo r - r )  . 
sulla base delle informazioni fornite dallesame , la paziente stata esclusa dal trattamento e dimessa evitando ulteriore accertamento diagnostico con esame coronarografico e prolungamento della ospedalizzazione . infine , nel gruppo di pazienti in cui lindicazione clinica allesame tcms - 64 era stata posta sulla base di test funzionali ecocardiografici ( n = 11 ) o scintigrafici ( n = 3 ) non conclusivi , in 6 / 14 casi sono state evidenziate immagini tcms64 compatibili con stenosi coronariche emodinamicamente significative ( mono o multivasali ) e clinicamente silenti ( fig . 7 ) ; solamente in questi 6 pazienti stato successivamente effettuato lesame coronarografico convenzionale . aspetti dosimetrici il ctdi volumetrico ( ct dose index volume ) stato in media di 36 , 538 , 30 mgy per paziente con un range compreso m . 
volume rendering reconstruction shows complete occlusion of the stent positioned in the proximal part of the vessel ( short arrow ) , with compensatory enlargement of the first diagonal branch ( long arrow ) ( a )  . thanks to the high spatial resolution achievable using a collimation of 0.6 mm , the stents metallic structure is visualised in detail ( b )  . 
4a - d coronarografia con tcms - 64 per follow - up di stent posizionato su arteria discendente anteriore 3 anni prima ; elaborazione delle immagini effettuata con workstation siemens utilizzando software syngo inspace 4d . 
le immagini ricostruite con algoritmo in volume rendering mostrano occlusione dello stent posizionato nel tratto prossimale della da ( freccia corta ) e lipertrofia compensatoria del primo ramo diagonale , che appare tortuoso ed aumentato di calibro ( freccia lunga ) ( a )  . 
nel particolare ( ingrandito ) lelevata risoluzione spaziale dellacquisizione a 0 , 6 mm di collimazione , permette di ottenere dettagli fini come la chiara definizione della trama metallica dello stent ( b )  . 
i pazienti sottoposti a studio dei by - pass coronarici , a causa del pi ampio volume cranio - caudale acquisito , presentavano valori medi di ctdi volumetrico pi elevati ( 38 , 77 , 9 mgy ) con un milliamperaggio effettivo di circa 645 , 389 , 8 . 
per il solo studio del distretto coronarico i valori dosimetrici sono risultati i seguenti : ctdi volumetrico 33 , 036 , 39 mgy , effective mas 498 , 1693 , 27 . il valore medio di ctdi volumetrico a cui sarebbe stato m . 
volume rendering image shows patency of the arterial y - shaped mammary graft connected with the left anterior descending ( lad ) artery and the first marginal branch ( arrow ) ; in the same reconstruction , venous conduit occlusion is observed at the proximal anastomosis level ( arrowhead )  . 
le immagini ricostruite con tecnica volume rendering dimostrano perviet del bypass arterioso ad y con arteria mammaria interna sinistra ( amis ) su da e primo ramo marginale ottuso ( freccia ) ; nelle stesse scansioni si evidenzia occlusione del graft aorto - coronarico venoso allorigine , con caratteristica estroflessione sulla parete aortica dellanastomosi prossimale ( punta di freccia )  . discussion patient exposure to ionising radiation is the main limitation to the clinical use of 64 - mdct , especially if it is to be used as a potential screening tool in asymptomatic patients . 
despite the patients high heart rate during image acquisition ( 96 bmp ) , maximum intensity projection ( mip ) reconstructions ( a ) show multiple stenotic lesions on the right coronary artery ( arrows ) , with subocclusive stenosis at the proximal third of the vessel . 
a 60% stenosis is also observed in the proximal portion of the left anterior descending ( lad ) artery , caused by soft tissue plaque ( arrow ) ( b )  . 
della paziente al momento dellacquisizione ( 96 bpm ) , le ricostruzioni mip ( a ) documentano la presenza di multiple alterazioni stenosanti lungo tutto il decorso della coronaria destra ( frecce ) con stenosi subocclusiva in corrispondenza del terzo prossimale del vaso . 
le ricostruzioni sono state effettuate con workstation siemens e software syngo inspace 4d . sottoposto il paziente senza lutilizzo della tecnica di modulazione risultato di 54 , 690 , 89 mgy con differenza statisticamente significativa tra ctdi volumetrico con sistema care dose e senza care dose ( p < 0 , 001 )  . 
come noto , lutilizzo del gating cardiaco retrospettivo comporta una continua e relativamente elevata esposizione del paziente ai raggi x per la necessit di effettuare lacquisizione dei dati durante lintera durata della scansione e di registrare contemporaneamente i dati dellecg per poi ricostruire le immagini in una fase definita del ciclo cardiaco [ 1 , 2 , 7 , 36 ]  . altra peculiarit tecnica dellimaging tcms delle arterie coronarie lutilizzo di bassi valori di pitch spirale ( pitch di 0 , 2 con tcms - 64 ) e notevolmente sovrapposti ( pitch < numero di strati ) che nasce dallesigenza di effettuare il campionamento dei dati per la ricostruzione delle immagini da strati sovrapposti e privi di gap nel volume acquisito , ma comporta un ulteriore significativo aumento della dose di esposizione [ 7 , 36 , 38 ]  . 
in aggiunta , rispetto alle precedenti generazioni , nella tcms - 64 lutilizzo di collimazioni submillimetriche ( 640 , 6 mm ) si associa ad un intrinseco incremento del rumore nelle immagini acquisite che pu essere limitato aumentando lamperaggio ( intensit di corrente emessa dal tubo radiogeno ) durante la scansione stessa per mantenere il rapporto segnale / rumore entro ottimali livelli diagnostici . nel nostro studio , il ctdi volume risultato in media di 36 , 538 , 30 mgy per paziente , con valori medi di 38 , 77 , 9 mgy nei pazienti con richiesta clinica per studio dei by - pass coronarici con un valore medio di mas di 567 , 83108 , 35 . rispetto ai dati disponibili in letteratura che riportano per apparecchiature a 4 e 16 canali rispettivamente ctdi volume medi di 3645 , 6 mgy e 43 , 3 mgy [ 35 ] , lutilizzo del sistema di modulazione della dose ha consentito di ottenere nella nostra popolazione valori medi di dose assorbita complessivamente inferiori rispetto alle precedenti generazioni tcms pur mantenendo una buona qualit diagnostica degli esami effettuati . 
i valori di dose efficace ottenuti sono risultati in media di 9 , 53 , 4 msv . malgrado in alcuni pazienti si siano raggiunti anche i 17 , 1 msv , questo dato preliminare , sempre se confrontato con i valori di dose efficace riportati in letteratura con apparecchiature a 4 e 16 strati ( rispettivamente 7 , 111 , 9 msv e 8 , 8 msv ) dimostra come , nella media dei pazienti , tale parametro dosimetrico non risulti significativamente superiore rispetto a quelli riportati con precedenti sistemi tcms [ 3841 ]  . 
appare tuttavia rilevante sottolineare come , a prescindere dallimpiego di sistemi di controllo dosimetrico , lesame tcms - 64 delle coronarie con cardiosincronizzazione comporti una esposizione del paziente in termini di dose efficace quasi doppia rispetto ai 57 msv di un esame tc standard del torace [ 38 , 42 ]  . nella nostra esperienza preliminare , lutilizzo del sistema care dose 4d appare quindi indispensabile per mantenere lesposizione del paziente entro livelli accettabili consentendo di ridurre lesposizione media del paziente del fig . 
7 a 64 - mdct angiography performed in a patient with atypical chest pa multiplanar reconstruction ( mpr ) of left anterior descending ( lad ) artery shows diffuse coronary artery disease with multiple vessel wall abnormalities ; note the presence of a noncalcified soft - tissue - attenuation lesion ( arrowheads ) in the proximal part of the artery , causing a > 60% narrowing of the lumen . 
la ricostruzione multiplanare ottenuta su piano curvo ( cpr ) lungo lasse longitudinale della da , documenta multiple alterazioni parietali a densit mista in corrispondenza del tratto prossimale dellarteria discendente anteriore ( da ) e la presenza di placca a densit mista , prevalentemente morbida , con riduzione significativa ( > 60% ) del lume vasale ( punte di freccia )  . 
lelaborazione delle immagini effettuata con workstation siemens utilizzando software syngo inspace 4d . to record ecg data to reconstruct images in a precise phase of the heart cycle [ 1 , 2 , 7 , 36 ]  . another technical peculiarity coronary artery mdct is the use of low spiral pitch ( 0.2 pitch with 64 - mdct ) and considerable overlap ( pitch < < number of slices ) , which arises from the need to sample data for image reconstruction with overlapping slices and no gap , but this leads to a further significant increase in radiation exposure [ 7 , 36 , 38 ]  . 
8 nella figura viene riportata la distribuzione del ctdi volumetrico ( in mgy ) utilizzando il sistema di compensazione care dose 4d , espresso in funzione del carico anodico effettivo . 
it should be borne in mind , however , that regardless of the use of an automatic dose control system , the 64 - mdct examination of the coronary arteries with cardiac gating involves exposing the patient to almost twice the effective radiation dose of a standard ct chest examination ( 57 msv ) [ 38 , 42 ]  . in our preliminary experience , the use of the care dose 4d systems appears indispensable for maintaining exposure within acceptable levels , allowing a 33.5% reduction in mean exposure compared with a similar acquisition performed without an automatic dose control syste the overall dose can be further reduced ( up to 50% compared with an acquisition without care dose 4d ) by using an ecg pulsing system , which is based on the premise that the best phase in the heart cycle for image reconstruction is the mid to end diastole , that is , the maximum immobility phase of the heart and coronary arteries [ 43 ]  . 
in this manner , during data acquisition , tube current is brought to the maximum level in the midto end - diastolic phase , where the maximum snr is required , and progressively reduced to the minimum towards the end - systolic phase . failure to use an ecg pulsing protocol in our study population derives from the evidence that the best rr interval for image reconstruction , especially in patients with elevated hr , is the end - systolic phase ( 30%35% ) , thus confirming the initial experience of wintersperger et al . 
the reduced snr of images acquired in the systolic phase resulting from an acquisition with an ecg 33 , 5% rispetto ad una analoga acquisizione senza modulazione automatica della corrente . 
la dose complessiva pu essere ulteriormente ridotta ( fino al 50% rispetto ad una acquisizione senza protocollo care dose 4d ) utilizzando un sistema di modulazione ecg - controllato dellemissione del tubo radiogeno ( sistema ecg - pulsing ) che si basa sul presupposto che la miglior fase del ciclo cardiaco nella quale effettuare la ricostruzione delle immagini sia la meso - telediastole , cio la fase di maggiore immobilit del cuore e delle arterie coronarie [ 43 ] ; in tal modo , durante lacquisizione dei dati , la corrente del tubo radiogeno viene portata a livello massimo in fase meso - telediastolica , dove necessario avere un massimo rapporto segnale / rumore e ridotta progressivamente al minimo verso la telesistole . la mancata utilizzazione di un protocollo di modulazione ecg - controllato nella nostra popolazione di pazienti deriva tuttavia dallevidenza che , particolarmente in pazienti con elevata fc , il migliore intervallo r - r per la ricostruzione delle immagini risultato essere la fase telesistolica ( 30%35% ) confermando liniziale esperienza riportata da wintersperger et al . 
although the technique needs to be validated with systematic comparisons with clinical , laboratory and coronarographic data , the latest generation of 64 - mdct scanners offers new possibilities for clinical management of patients with coronary artery disease . the increase in spatial and temporal resolution translates into improved diagnostic image quality with respect to previous generations of multidetector devices . 
thus , 64 - mdct is a noninvasive technique capable of identifying patients requiring interventional or surgical procedures such as selective coronarography with primary angioplasty or stent placement , or surgical revascularisation with bypass grafts . the use of suitable systems for the automatic control of radiation exposure seems nonetheless indispensable in order to limit patient dose , which given the current state of affairs is the main limitation to the clinical use of the technique . nella nostra esperienza clinica iniziale , lutilizzo della tcms - 64 ha fornito risultati molto promettenti . 
bonomo , i - 70031 andria ( ba ) , italy 2dipartimento di scienze radiologiche , irccs casa sollievo della sofferenza , i - 71013 san giovanni rotondo ( fg ) , italy 3dipartimento di radiologia , fondazione s . 
giglio , cefal ( pa ) , italy 4istituto di radiologia , universit di novara , italy 5istituto di radiologia , universit di ancona , italy correspondence to : t . 
scarabino , via napoli 56 , i - 70031 andria ( ba ) , italy , tel . : + 39 - 0883 - 299140 , fax : + 39 - 0883 - 299220 , e - mail : tscarabino@hotmail.com received : 19 may 2006 / accepted : 12 july 2006 / published online : 22 february 2007 abstract ever since the introduction of magnetic resonance ( mr ) , imaging with 1.5 tesla ( t ) has been considered the gold standard for the study of all areas of the body . 
indeed , their high gradient power and field intensity ( 3.0 t ) allow adjunctive and more advanced diagnostic methodologies to be performed with excellent resolution in a fraction of the acquisition time required with earlier machines . 
pi recentemente , lintroduzione di unit rm con intensit di campo di 3 , 0 t per applicazioni cliniche nuove ed avanzate ha comportato notevoli vantaggi , soprattutto in campo neuroradiologico . 
i loro alti gradienti ed intensit di campo infatti consentono di applicare metodologie diagnostiche nuove ed avanzate con eccellente risoluzione in una frazione del tempo di acquisizione richiesto dalle attrezzature precedenti . 
continuous advances in hardware ( gradients , coils ) and software ( acquisition and processing sequences ) have made magnets more compact , powerful and flexible , greatly improving patient comfort . 
higher - field units were introduced some years ago for clinical research purposes , principally in neuroradiology , and are spreading fast ( especially in the usa ) despite their dallavvento della risonanza magnetica ( rm ) le apparecchiature con intensit di campo magnetico di 1 , 5 tesla ( t ) sono considerate il gold standard per coprire le varie applicazioni cliniche in tutti i distretti corporei . 
tali apparecchiature , in virt di continui progressi che hanno interessato lhardware ( gradienti , bobine ) e il software ( sequenze di acquisizione e di elaborazione ) , sono diventate sempre pi compatte , potenti e versatili , con conseguente migliore confort del paziente . 
they are being employed not only in research and with a broader scope compared with traditional research but also in daily clinical practice to support new and sophisticated clinical applications , with considerable practical benefits . 
in the first year , the machine was employed to optimise the various morphological and functional acquisition sequences ( diffusion , perfusion , spectroscopy , cortical activation ) both in vitro ( using dedicated dummies ) and in vivo ( in healthy controls ) and the various postprocessing techniques ( with dedicated software ) with a view to creating a large database as the starting point for research on patients . 
naturally , special attention was devoted to the safety problems related to the machines use , especially issues of patient comfort in the tunnel and compatibility of various biomedical devices . after optimisation of study sequences and protocols , our research programme focused on improving morphofunctional mr imaging of the bra the higher signal - to - noise ratio ( snr ) of the 3.0 - t magnet made it possible to obtain images with greater spatial , contrast and time resolutions in shorter acquisition times compared with lower - field systems , resulting in greater patient comfort and reduction of motion artefacts . the patients studied at 3.0 t exhibited a wide and representative range of conditions that were successfully investigated using standardised dedicated sequences and protocols . 
disadvantages , which include a greater specific absorption rate ( sar ) , greater acoustic noise are not particularly severe and in newer systems are largely reduced by improved hardware and software [ 14 ]  . 
tali sistemi sono ormai diventati una realt e , nonostante lalto costo , si stanno sempre pi diffondendo ( specie in usa ) per essere utilizzati non solo nel campo della ricerca , peraltro molto pi ampia rispetto alla ricerca tradizionale , ma anche e soprattutto nella pratica clinica quotidiana per far fronte a nuove e pi sofisticate applicazioni cliniche con conseguenti importanti benefici pratici . da 5 anni il servizio di neuroradiologia del nostro istituto scientifico dotato di una apparecchiatura rm whole body ( signa horizon lx , general electric medical system , milwaukee , usa ) dotata di magnete superconduttivo , a schermatura attiva , con intensit di campo magnetico 3 , 0 t , gradienti di potenza di 50 millitesla al metro ( mt / m ) e slew rate di 150 mt / m / s . 
il sistema rm 3 , 0 t in dotazione stato utilizzato nel primo anno di lavoro , una volta acquisita lautorizzazione ministeriale alluso , per ottimizzare le diverse sequenze di acquisizione , morfologiche e funzionali ( diffusione , perfusione , spettroscopia , attivazione corticale ) , sia in vitro ( utilizzando fantocci dedicati ) che in vivo ( con controlli sani ) , e le diverse modalit di post - processing ( utilizzando software dedicati ) al fine di creare un ampio database , punto di partenza per studi di ricerca su paziente . 
naturalmente , particolare attenzione stata data alle problematiche di sicurezza inerenti luso di tale apparecchiatura ( con riferimento in particolare alla qualit di confort del paziente in esame e alla rm - compatibilit di vari dispositivi biomedicali )  . ottenuta lottimizzazione di sequenze e protocolli di studio , lobiettivo del nostro programma di ricerca stato quello di migliorare limaging morfo - funzionale cerebrale con rm . 
sfruttando il pi alto rapporto segnale / rumore ( s / r ) possibile con lapparecchiatura 3 , 0 t , stato infatti possibile ottenere immagini con pi alta risoluzione spaziale , di contrasto e temporale , in tempi peraltro ridotti rispetto ai sistemi di pi basso campo , con conseguente miglioramento del confort del paziente e riduzione degli artefatti da movimento . i pazienti sottoposti a rm 3 , 0 t hanno esibito un ampio e rappresentativo spettro di patologia che stata esaminata adeguatamente utilizzando sequenze e protocolli ben definiti e personalizzati . si quindi proceduto allo studio di diverse patologie seguendo le linee dei progetti di ricerca approvati dalla direzione scientifica del nostro istituto , previa autorizzazione del comitato etico . 
lo scopo del lavoro quello di illustrare le caratteristiche semeiologiche peculiari dellimaging cerebrale morfologico e angiografico a 3 , 0 t mettendo in risalto i relativi vantaggi e svantaggi rispetto a sistemi rm di pi basso campo . studio morfologico di base gli studi rm del cervello possono essere suddivisi in due grandi categorie , morfologici e funzionali , sulla base del tipo di informazione che si ricerca . 
lo studio morfologico di base ( detto anche convenzionale o anatomico ) riguarda limaging di tessuto , spazi periliquorali , vascolatura e fasci di fibre , mentre lo studio funzionale in senso lato comprende lo studio delle funzioni cerebrali ( fmri ) limaging di perfusione t . 
 because the user interface and sequence parameters are the same as those employed with 1.5 - t systems , at 3.0 t , in theory , the same images should be obtained with the advantage of high - field strength , i.e. 
 t1 imaging as t1 relaxation times are longer at higher magnetic fields , entailing reduction in the relative differences among different tissue types , it is generally difficult to obtain t1 - weighted images with sufficient contrast when using a 3.0 - t unit [ 6 ]  . 
for this reason , snr optimisation in the same sample using a higher - field unit requires longer time to repetition ( tr ) [ and a smaller flip angle ( fa ) ] , resulting in longer scanning times . 
t1 saturation for a given tr is greater at 3.0 t , resulting in reduced signal gain . in other words , the snr per unit of time would be optimised with the shortest possible tr / t1 . 
the possibility that the snr gain connected with the high magnetic field may substantially be offset by a longer t1 highlights the importance of optimising the imaging parameters [ 3 ]  . 
 e di diffusione , e lo studio metabolico con la spettroscopia . lo studio di rm di base si presenta a 3 , 0 t con un imaging di qualit pi alta rispetto a quello ottenuto a pi basso campo in virt di una serie di vantaggi quali un pi alto rapporto s / r , una maggiore risoluzione spaziale e temporale a fronte di alcuni svantaggi , quali lincremento del specific absorption rate ( sar ) e del rumore acustico , peraltro non particolarmente significativi e comunque in gran parte risolvibili mediante hardware e software di pi recente introduzione [ 14 ]  . 
dato che i tempi di rilassamento sono campo - dipendenti e influenzano il contrasto dellimaging , le sequenze di imaging a 1 , 5 t non possono infatti essere trasferite a 3 , 0 t . 
a ci si aggiunge la deposizione tissutale dellenergia di rf che , essendo eccessiva , va anche considerata per adattare e ottimizzare i protocolli clinici di imaging per luso a 3 , 0 t . 
 fast spgr allows thinner slices to be obtained in a shorter time and provides images suitable for reformatting in all three planes using a single volumetric acquisition of the whole brain , thus disclosing a larger number of lesions than can be depicted using se t1 - weighted sequences acquired in a single plane . 
infatti , non solo il t1 aumenta con laumentare del campo magnetico , ma addirittura converge , determinando una distribuzione meno ampia dei valori di t1 tra i diversi tipi di tessuto . 
per tale motivo per ottimizzare il rapporto s / r dello stesso campione a campo pi alto il tr deve essere pi lungo ( come anche il flip angle deve essere pi corto ) con conseguente allungamento dei tempi desame . utilizzando tr pi lunghi , il vantaggio in intensit di segnale a campo pi alto risulta essere inferiore in unit di tempo . la saturazione t1 per un dato tr infatti pi ampia a 3 , 0 t con conseguente riduzione del guadagno in segnale . 
quindi in virt del fatto che il guadagno nel rapporto s / r derivante dallalto campo pu essere sostanzialmente controbilanciato dallallungamento del t1 , risulta importante lottimizzazione dei parametri di imaging [ 3 ]  . lincremento del t1 rappresenta il pi grande problema per lo studio cerebrale a 3 , 0 t acquisito con sequenza se t1 pesata in cui il contrasto t1 appunto inaccettabile [ 1 , 2 , 4 , t . 
for instance , when studying primary and secondary brain tumours , administration of a gadolinium - based agent yields greater contrast between tumour and normal brain tissue both in single and in multiple doses . 
it also detects more metastases and demonstrates different patterns of enhancement that may be useful to assess the degree of malignancy and monitor response to therapy [ 2628 ]  . 
the greater sensitivity of 3.0 - t contrast - enhanced investigations has also been demonstrated in multiple sclerosis , with a 21% increase in the number of enhancing lesions detected , a 30% increase in enhancing lesion volume and a 10% increase in total lesion volume relative to scanning at 1.5 t [ 29 ]  . 
using a standard dose , the contrast - to - noise ratio ( cnr ) is two and a half times greater than with 1.5 - t systems [ 13 ]  . 
double or triple doses can result in meningeal contrast uptake , a common nonpathological finding at 3.0 t that at lower field strengths may , however , mimic carcinomatosis or meningitis and should thus be avoided . 
to enhance sensitivity or highlight minimal changes in the blood - brain barrier , a double dose of contrast agent may be employed in selected investigations of brain metastases or inflammatory disease [ 30 , 31 ]  . 
high spatial resolution fast spin echo ( fse ) - weighted images are especially effective at 3.0 t , as they provide more anatomical detail and better contrast due to the greater snr , which allows the use of thinner slices and broader matrices [ 1 , 2 , 4 , 32 ]  . 
tale sequenza inoltre caratterizzata da ipersegnale delle grosse strutture vascolari arteriose che comunque non inficia linterpretazione delle immagini . mentre gli studi t1 pesati sono tradizionalmente meno informativi e richiedono pi tempo , un tipico studio flair t1 pesato del cervello consente , insieme allutilizzo dellimaging parallelo , lapplicazione di protocolli a pi alta risoluzione con tempi desame pi brevi rispetto ai campi ad intensit 1 , 5 t . 
naturalmente tale aspetto diventa problematico se dobbiamo acquisire pi studi , senza e con mdc , o pi proiezioni . in rapporto al maggiore effetto inflow del mdc e alla maggiore soppressione del fondo , limaging t1 con mdc sicuramente un vantaggio del 3 , 0 t rispetto al 1 , 5 t . 
nello studio ad esempio dei tumori cerebrali primitivi e secondari la somministrazione del gadolinio produce un maggior contrasto tra lesione tumorale e parenchima normale sia a singola dose e a dose multipla , permette di rilevare un maggior numero di metastasi , pu dimostrare differenti pattern di impregnazione che risultano utili per definire il grado di malignit e per monitorare la risposta alla terapia [ 2628 ]  . 
rispetto al 1 , 5 t infatti alcuni autori hanno documentato un incremento del 21% del numero delle lesioni con impregnazione patologica , del 30% nel volume di impregnazione e del 10% del volume totale delle lesioni [ 29 ]  . 
il rapporto contrasto / rumore ( c / r ) risulta infatti maggiore rispetto allo studio con sistemi rm 1 , 5 t ( di ben 2 , 5 volte ) utilizzando una dose standard [ 13 ]  . 
da evitare la dose doppia o tripla responsabile di impregnazioni meningee , reperti comuni non patologici a 3 , 0 t ( con sistemi 1 , 5 t tali quadri possono simulare una carcinomatosi o una meningite )  . 
3 imaging fse t2 ad alta definizione della sezione mediana del cervello . infiammatoria per incrementare la sensibilit o per stressare le minime alterazioni della barriera emato - encefalica ( bee ) , una dose doppia pu essere preferita [ 30 , 31 ]  . 
di solito nello studio t1 con mdc la fast ge t1 pesata viene preferita , al posto delle tradizionali se , che presentano un contrasto minore a parit di tempo dacquisizione . 
ne consegue una migliore risoluzione spaziale ( e quindi maggiore dettaglio anatomico ) e di contrasto , possibili per effetto di un pi alto rapporto s / r che permette luso di spessore di strato inferiori e di matrici pi ampie [ 1 , 2 , 4 , 32 ]  . 
2a , b studio di microadenoma ipofisario prima ( a ) e dopo mdc endovena ( b ) con sequenza fse - xl t1 ( tr / te 600 / 12 , etl 5 , fov 180 mm , spessore di strato 2 , 5 mm / 0 , 5 , matrice 224192 , zip 512 , 29 / fase )  . creased deposition of radiofrequency ( rf ) energy , which in turn requires long tr or pulse refocalisation angles well below 180 . 
in addition , the broader bandwidth , smaller echo space , shorter minimum tr and te and shorter fse sequences employed at higher field strengths result in improved image quality , artefact reduction and enhanced diagnostic performance in much shorter examination times , which is to the patients benefit . 
these sequences allow excellent visualisation of the basal or mesencephalic nuclei by virtue of their iron content despite the intrinsically lower sensitivity of fse to magnetic susceptibility in 3.0 - t compared with 1.5 - t systems [ 5 ]  . 
4a - d high - definition ( hd ) study of the inner ear using a 3d fast imaging employing steady state acquisition ( fiesta ) sequence [ time to repetition ( tr ) 4.3 , echo time ( te ) 1.5 , flip angle ( fa ) 50 , band width ( bw ) 62 khz , field of view ( fov ) 170 , slice thickness 0.6 mm , matrix 256256 , zero fill interpolation processing ( zip ) 512 , 2 , number of excitations ( nex ) , 4 : 43 ]  . 
4a - d studio ad alta definizione dellorecchio interno con sequenza 3d fiesta ( tr / te 4 , 3 / te 1 , 5 , flip angle 50 , bw 62 khz , fov 170 , spessore di strato 0 , 6 mm , matrice 256256 , zip 512 , 2 nex , 4 : 43 ) : singole partizioni ( a ) , zoom ( b ) , mip ( c ) , elaborazione con ricostruzione di volume ( d )  . rhage , which at 3.0 t is characterised by greater hypointensity in the acute and early subacute phases [ 33 ]  . 
 flair imaging flair sequences , which are generally included in all imaging protocols , are also sharper and more sensitive at 3.0 t sono pi bassi , quindi il tempo in fse si riduce , la qualit aumenta , gli artefatti si riducono . 
con tale sequenza sono ben apprezzabili i nuclei della base o del mesencefalo che risaltano maggiormente ( per effetto del loro contenuto di ferro ) , nonostante la nota intrinseca minore sensibilit alla suscettibilit magnetica della fse , rispetto a sistemi 1 , 5 t [ 5 ]  . una variazione di semeiotica del segnale sembra interessare anche le emorragie cerebrali caratterizzate in particolare da una maggiore ipointensit a 3 , 0 t in fase acuta e subacuta precoce rispetto allintensit di segnale rilevabile a 1 , 5 t [ 33 ]  . 
a tal proposito il c / r e lintensit di segnale a 1 , 5 t e 3 , 0 t sono sostanzialmente equivalenti per cui di solito comunque non vi sono particolari problemi per interpretare correttamente let dellemorragia . 
first , it allows performance of all angiographic sequences applied routinely in clinical practice with lowerfield systems , such as 2d and 3d time of flight ( tof ) and phase contrast ( pc ) , as well as ultrafast dynamic sequences acquired after contrast agent bolus administration [ contrastenhanced mra ( ce - mra ) ]  . 
as with routine mr scanning , the technical parameters of angiographic sequences need to ge ( t2 * ) quali ad esempio la ciss ( constructive interference in the steady state ) particolarmente indicata nello studio dellorecchio interno . 
la maggiore sensibilit si ha anche grazie alluso di te pi lunghi , necessari per i tempi di rilassamento modificati a 3 , 0 t , che permettono un buon contrasto ed un rumore accettabile . 
il rilievo di piccole lesioni richiede infatti lottimizzazione del c / r piuttosto che del rapporto s / r e questo viene ottenuto appunto con te pi lunghi ( 120 ms ) anche se tale allungamento incrementa oltre il contrasto anche il rumore ma in modo accettabile . inoltre , contrariamente a quanto accade per sistemi 1 , 5 t , in virt della sostanziale mancanza di differenza del t1 del liquor ( che essenzialmente un liquido ) tra 1 , 5 t e 3 , 0 t , il tempo di inversione ( ti ) richiesto per annullare il liquor rimane dello stesso valore ( 2 , 250 millisecondi circa ) [ 5 ]  . tale caratteristica non valida in altre sequenze ir , sia in quelle in cui si cerca di massimizzare il contrasto tra differenti tessuti ( ad esempio tra sostanza bianca e sostanza grigia ) sia in quelle con soppressione del grasso , nelle quali invece il ti deve essere incrementato del 25%40% . 
le note aree di sostanza bianca soprattutto periventricolare fisiologicamente iperintense con sistemi a pi basso campo ( 1 , 5 t ) sono ancora pi accentuate a 3 , 0 t per cui c bisogno di una particolare attenzione per distinguerle da patologie reali ( ad esempio sclerosi laterale amiotrofica ) [ 1 ]  . 
in addition , the change in t1 relaxation time , namely , increased t1 , yields greater background suppression of stationary tissues due to the decrease of r1 , i.e. 
the rate of longitudinal or spin - lattice relaxation , with greater flow enhancement , as blood r1 is roughly constant thereby improving vessel - tissue contrast [ 19 , 22 , 36 ]  . 
this advantage makes magnetisation transfer application less effective than it is with 1.5 t systems [ 37 , 38 ]  . the effect is mainly evident using the 3d tof technique , as the short tr usually saturates stationary tissues but not moving blood [ 7 , 39 ] , and the tag persists for a longer time . these effects lead to greater vessel detail and conspicuity , especially in disclosing small - calibre vessels , including surface vessels not clearly depicted at 1.5 t [ 23 , 25 ]  . 
il recente sviluppo di nuovi hardware ( nuove coil ) o lutilizzo di pi recenti software ( imaging parallelo ) potranno ridurre tale inconveniente . studio angiografico con il 3 , 0 t lo studio angiografico si avvantaggia in modo significativo rispetto a sistemi rm di pi basso campo ( fig . 7 ) [ 1 , 2 , 4 , 35 ]  . 
innanizitutto un sistema rm a 3 , 0 t permette di eseguire tutte le sequenze angiografiche utilizzate di solito nella pratica clinica con sistemi a pi basso campo quali le pi comuni time of flight ( tof ) o phase contrast ( pc ) , entrambe con modalit 2d o 3d , od anche quelt . 
therefore , contrast administration further improves imaging at 3.0 t due to the high snr , which can be transformed into spatial resolution : resolution on a plane le ultrarapide utilizzate con tecnica dinamica dopo somministrazione a bolo del mdc ( contrast enhancement magnetic resonance angiography , ce - mra )  . 
tale vantaggio , che rende peraltro non particolarmente efficace ( come invece accade con sistemi 1 , 5 t ) luso della magnetization transfer ( mt ) [ 37 , 38 ] apprezzabile soprattutto quando si utilizza la tecnica angiografica 3d tof in quanto il breve tr satura di solito i tessuti stazionari ma non il sangue in movimento [ 7 , 39 ]  . 
tali effetti comportano un incremento del dettaglio e della conspicuity vascolare con in particolare evidenza di vasi di pi piccolo calibro , anche superficiali ( di iv ordine ) , non chiaramente identificabili con sistemi rm 1 , 5 t [ 23 , 25 ]  . per lo stesso motivo lutilizzo di campi magnetici elevati ( 3 , 0 t ) sembra in grado di migliorare la cospicuit dei vasi intracranici del neonato o di ridurre ulteriormente i tempi di acquisizione [ 40 ]  . 
lo studio angio - rm a 3 , 0 t in pazienti portatori di tali dispositivi biomedicali ( purch gi testati ) pu essere quindi eseguito senza particolari rischi [ 33 , 45 ]  . i benefici ottenuti a 3 , 0 t , evidenti soprattutto nella patologia vascolare malformativa ( specie aneurismatica ) e atefig . 
time - resolved imaging of contrast kinetics ( tricks ) images [ 3d time of flight spoiled gradient recalled ( tof - spgr ) , time to repetition ( tr ) 3.7 , echo time ( te ) 1.3 , matrix 224192 , slice thickness 1.8 mm , field of view ( fov ) 28 , temporal resolution 2.5 s , 8 phases ] of multiple vascular phases : contrast enhancement of arteries ( a ) , capillaries ( b ) and veins ( c )  . 
in tale modo possibile visualizzare diverse fasi vascolari : arterie ( a ) , capillari ( b ) e vene ( c )  . ments are increased at higher - field strengths resulting in artefacts caused by the susceptibility of biomedical devices such as the aneurysm clips commonly used in interventional and therapeutic neuroradiological procedures , the newer devices do not seem to present major safety or compatibility problems [ 42 , 44 ]  . 
resolution quality tends to be privileged in clinical practice , with acquisition of a larger number of slices of rerosclerotica cerebrale , rendono ancora pi competitiva langiografia con rm , nonostante una minore risoluzione spaziale rispetto allangiografia convenzionale ( dsa ) , ormai sempre pi utilizzata in campo intervenzionistico - terapeutico piuttosto che diagnostico [ 46 ]  . conclusioni grazie al suo alto rapporto s / r , la mri ad alto campo in grado , di supportare nuove applicazioni diagnostiche o di migliorare le metodiche esistenti . 
in virt dellalto rapporto s / r il neuroradiologo pu scegliere di eseguire un imaging cerebrale a 3 , 0 t in un tempo uguale a quello ottenibile con un sistema 1 , 5 t privilegiando in tal caso una pi alta qualit delle immagini ( incrementando la matrice e / o riducendo lo spessore di strato e / o il fov ) oppure di avere un imaging di qualit pari al quello di un sistema 1 , 5 t in un tempo inferiore privilegiando in tal caso il confort del paziente , od anche pu scegliere di ridurre la dose del mezzo di contrasto somministrato . 
di solito nella pratica clinica si cerca di privilegiare la risoluzione acquisendo pi fette , di spessore sottile , con campi di vista pi piccoli , con matrici pi ampie e in tempi comparabili a quelli con i sistemi 1 , 5 t . 
spesso in rapporto al quesito clinico e al tipo di paziente in esame , si cerca di trovare un compromesso tra risoluzione e velocit ( riducendo ad esempio il numero delle eccitazioni ) [ 34 ]  . lalto rapporto s / r per effetto dellalto campo pu in realt essere ottenuto anche a 1 , 5 t utilizzando i pi recenti hardware ( bobine riceventi di superficie , single o in arrays ; gradienti pi performanti capaci di ridurre il te ) e t . 
however , this cannot be done in the case of unstable or poorly co - operative patients , or it may be impossible for cost reasons ( reduced patient flow )  . 
lastly , the higher snr of high - field mr systems is a resource that neuroradiologist can exploit in clinical practice , as the higher the snr , the more the imaging protocols can be adjusted . acknowledgments to technical staff : c . 
il caso di pazienti instabili , non collaboranti , come anche ci possono essere altre motivazioni ad esempio di natura economica ( la riduzione del flusso dei pazienti )  . 
non solo , anche a 1 , 5 t possibile un incremento delle dimensioni della matrice : in tal caso per la conseguente riduzione delle dimensioni del voxel comporterebbe maggiore granulosit dellimaging . 
pertanto il maggiore rapporto s / r da alto campo sicuramente unottima arma che il neuroradiologo pu sfruttare nella pratica clinica : maggiore il rapporto s / r , maggiori sono infatti le possibilit del neuroradiologo di modificare i protocolli di imaging . rigraziamenti allo staff tecnico : c . 
 correspondence to : giuseppe caruso , via liguria , 21 , i - 90144 palermo , italy , tel . : + 39 - 91 - 6552315 , fax : 39 - 91 - 6552337 , e - mail : carusogi@libero.it received : 23 march 2006 / accepted : 18 august 2006 / published online : 22 february 2007 abstract purpose . 
a total of 1 , 729 ( 1 , 012 women and 717 men ; age range 2882 ; mean age 51 ) patients underwent contrastenhanced sonography of the liver . 
the examination was performed with a low mechanical index ( mi < 0.09 ) after injection of sulphur - hexafluoride - filled microbubbles , using different sonographic equipment and different contrast - specific algorithms . 
heterogeneous delayed liver enhancement was observed in six patients in the late phase ( 180 s ) , with the presence of multiple , partially confluent , hyperechoic areas peripheral to the portal vessels . 
the pattern appeared spontaneously between 1 and 4 h after the examination and was associated with the presence of an anomalous echogenicity in the superior mesenteric veno patient experienced adverse reactions . 
1729 pazienti ( 1012 donne e 717 uomini ; et compresa tra 2882 , media 51 ) sono stati sottoposti ad esame ecografico del fegato dopo somministrazione di ecoamplificatore esafluoruro di zolfo e basso indice meccanico ( mi < 0 , 09 )  . 
limpregnazione anomala del parenchima epatico potrebbe essere secondaria allintrappolamento nei sinusoidi epatici di gas proveniente dal microcircolo intestinale attraverso il sistema entero - portale . key words ultrasound microbubbles liver parole chiave ecografia microbolle fegato introduction introduzione sonographic contrast agents consist of microbubbles containing an inert gas or air with a diameter < 5 m surrounded by a shell or a membrane that provides transpulmonary stability and provides transpulmonary stability and increasing their persistence in the circulation . 
the total volume of mii mezzi di contrasto utilizzati in ecografia sono costituiti da microbolle ( diametro < 5 m ) contenenti gas inerte o aria , e stabilizzate da membrane a composizione differente , che ne garantiscono la stabilit attraverso il circolo transpolmonare aumentandone la persistenza in circolo . 
the second relies on detection of ultrasound ( us ) - induced physical phenomena , such as microbubble destruction , fusion or splitting [ 4 ]  . the purpose of this paper is to report a pattern of heterogeneous late - phase hepatic enhancement with anomalous duration and distribution associated with stratified hypoor hyper - echogenicity in the superior mesenteric vein , which was observed after infusion of sonovue contrast mediuan aetiological hypothesis is also presented . materials and methods from december 2001 to august 2004 , 1 , 729 patients ( 1 , 012 women and 717 men ; age range 2882 years ; mean age 51 ) underwent contrast - enhanced sonography of the liver after injection of sonovue contrast medium ( bracco , milan , italy )  . sonovue is a suspension of hexafluoride sulphur microbubbles ( mean diameter 3 m ) with a phospholipidic shell . 
the elective technique consisted of a bolus injection of 4.8 ml of contrast medium at a rate of approximately 1 ml / s , followed by a 5ml saline flush . 
sebbene si tratti essenzialmente di sostanze blood - pool , il loro comportamento nelle diverse fasi post - contrastografiche sovrapponibile a quello dei mezzi di contrasto organo - iodati , con la differenza che non si ha una vera fase interstiziale . lintroduzione del mezzo di contrasto nel circolo periferico permette di identificare tre diverse fasi della perfusione vascolare epatica , analogamente a quanto possibile ottenere allesame tc dinamico del fegato dopo somministrazione di mezzo di contrasto organo - iodato : fase arteriosa , a 2030 s : in questa fase lenhancement dovuto alla presenza del mezzo di contrasto nelle arterie di piccolo calibro ; fase portale e tardiva , a 60 e 180 s , rispettivamente : nonostante il mezzo di contrasto ecoamplificatore non passi nellinterstizio ma rimanga nei vasi , lenhancement osservato in fase portale e specialmente in fase tardiva , sembra essere dovuto alla persistenza delle microbolle nei sinusoidi o al loro up - take da parte delle cellule del kupffer e delle cellule del sistema reticolo - endoteliale [ 2 , 3 ] ; ipotesi comunque queste che devono essere avvalorate da ulteriori studi di farmacocinetica . le tecniche di imaging ecografico contrasto - specifiche sono due : la prima quella dellimaging non - distruttivo in cui limmagine viene costruita sfruttando la risposta non lineare delle microbolle , senza che queste vengano distrutte ; la seconda tecnica utilizza limaging distruttivo , che determina la distruzione , la fusione e lo splitting delle microbolle [ 4 ]  . scopo del nostro lavoro quello di riportare un fenomeno caratterizzato dalleterogeneo enhancement del parenchima epatico in fase tardiva , anomalo per durata e distribuzione , associato ad un pattern stratificato ipo - iper - ecogeno della vena mesenterica superiore dopo somministrazione di sonovue . 
sulla scorta di una serie di osservazioni abbiamo avanzato una ipotesi eziologica . materiali e metodi dal dicembre 2001 allagosto 2004 , 1729 pazienti ( 1012 donne - 717 uomini , di et compresa tra 28 e 82 anni ; et media 51 anni ) , hanno eseguito un esame ecografico del fegato con ecoamplificatore . 
il sonovue una sospensione di microbolle di esafluoruro di zolfo ( diametro medio 3 , 3 m ) , stabilizzate da una membrana fosfolipidica ; si presenta come una polvere liofilizzata in atmosfera di solfuro esafluoride posta allinterno di un flaconcino di plastica . 
una volta ricostituita , la sospensione appare come soluzione lattescente e rimane stabile per al massimo 6 ore [ 5 ]  . liniezione del mezzo di contrasto stata effettuata 510 minuti dopo la ricostituzione dello stesso . 
la tecnica consiste nelliniezione a bolo di 4 , 8 ml di mezzo di contrasto a velocit di 1 ml / s , seguita dalla infusione di 5 ml di soluzione g . 
use of these algorithms allowed real - time imaging of all vascular phases ( arterial , portal and late ) and was dependent on the availability of the us device at the time of examination ( table 1 )  . 
all examinations were performed as follows : fundamental ( b - mode ) imaging and contrast - specific algorithm without contrast agent contrast - enhanced us in the arterial phase ( 2030 s ) contrast - enhanced us in the portal and late phase ( 60 and 180 s ) contrast - enhanced us in the second late phase ( 240 s )  . patients who showed heterogeneous late - phase hepatic enhancement were examined using the b - mode technique and stimulated acoustic emission ( sae ) , both performed with a high mi ( 1.2 ) ; insonation with high mi is useful for microbubble distribution , leading to possible rapid disappearance of the heterogeneous enhancement pattern . two of the six patients underwent a second contrast - enhanced us after 24 h and 9 days , respectively , using the same us system and the same contrast agent at the same dose . 
tutti gli esami sono stati eseguiti con le stesse modalit come riportato di seguito : imaging fondamentale ( b - mode ) e algoritmo contrastospecifico senza mezzo di contrasto ; ecografia con mezzo di contrasto : fase arteriosa a 2030 s ; ecografia con mezzo di contrasto : fase portale e tardiva a 60 e 180 s rispettivamente ; ecografia con mezzo di contrasto : seconda fase tardiva a 240 s . i pazienti nei quali stato osservato , in fase tardiva , leterogeneo enhancement epatico sono stati sottoposti a esame ecografico con tecnica b - mode in e con stimulated acustic emission ( sae ) entrambe ad elevato indice meccanico ( mi = 1 , 2 ) , allo scopo di determinare la distruzione delle microbolle di mezzo di contrasto con leventuale rapida scomparsa delleterogeneo enhancement . due dei sei pazienti hanno eseguito un secondo esame ecocontrastografico a 24 ore e a 9 giorni rispettivamente , utilizzando la stessa apparecchiatura ecografica , lo stesso mezzo di contrasto , nella stessa dose . 
nei due pazienti in cui stata somministrata per la seconda volta lo stesso mezzo di contrasto nella stessa dose ( a distanza rispettivamente di 24 ore e 9 giorni ) il fenomeno non si ripresentato . 
nessuno dei sei pazienti ha presentato sintomi , n sono state osservate alterazioni della pressione arteriosa . discussione leterogeneo enhancement epatico stato descritto per la prima volta da okada [ 6 ] : egli ha osservato un fenomeno simile in sei pazienti dopo somministrazione di levovist ( 5 casi ) e di echogen ( 1 caso )  . 
1 immagine ottenuta dopo somministrazione di ecoamplificatore con coherent contrast imaging , fase tardiva ( 4 minuti ) : presenza di multiple aree iperecogene parzialmente confluenti nel contesto del parenchima epatico perifiericamente ai vasi portali ( frecce )  . 
 observation of these findings was possible thanks to the use of second - generation sonographic contrast agents , which allow real - time examination of hepatic vascular perfusion : this suggests that gas is present in the enteroportal circulation but not in the other vessels , which could account for the origin of the hepatic phenomenon . 
4 fundamental ( b - mode ) imaging , late phase ( 40 min ) : marked hyperechogenicity ( 1 ) can be seen in the superior mesenteric vein ( between the two arrows )  . 
4 immagine in basale ( b - mode ) ; fase tardiva a 40 minuti : possibile dimostrare una marcata iperecogenicit ( 1 ) nel lume della vena mesenterica superiore ( compresa tra le due frecce )  . 
liperecogenicit appare localizzata in sede ventrale mentre lipoecogenicit in sede declive come fase gassosa in fase fluida ( 2 )  . discussion heterogeneous hepatic enhancement was first described by okada [ 6 ] , who reported a similar phenomenon occurring in six patients after injection of levovist ( five cases ) and echogen ( one case )  . 
to the best of our knowledge , heterogeneous hepatic enhancement has never been observed following administration of sonovue contrast mediuunlike the agents employed by okada , sonovue relies on a low mi to provide real - time imaging of hepatic perfusion [ 7 ]  . 
il fenomeno si verificato con mezzi di contrasto diversi ( per tipo di gas e natura della membrana delle microbolle ) e con tecniche di insonazione diverse : una ad alto indice meccanico con distruzione delle microbolle ( levovist ed echogen ) ; laltra a basso indice meccanico con fenomeni di risonanza delle microbolle ( sonovue ) [ 8 ]  . il levovist un agente epato - specifico ; ha unemivita breve e il suo effetto dopo linsonazione dura solo alcuni secondi . 
moreover , has liver - specific parenchymal uptake after blood - pool clearance ; it is short - lived , and once an area has been scanned for a few seconds , the effect disappears . 
it is best seen with relatively high acoustic power settings ( although still within accepted levels for diagnostic imaging ) and shows a tendency to cluster at the level of the focal zone , where the power is maximal [ 9 , 10 ]  . 
in contrast , sonovue is purely a vascular agent , with no evidence of selective uptake ; it shows a spleen - specific enhancement that lasts longer ( 5 min ) than the blood - pool and liver enhancement phases [ 11 ]  . 
 moreover , the gas microbubbles responsible for this phenomenon are larger than the contrast agent microbubbles and are unable to cross the hepatic sinusoids , the calibre of which is 4050 in addition , this anomalous echogenicity was observed in the vena porta and superior vein only , and not in the splenic vein , hepatic vein , aorta or inferior vena cava . 
echogenicity is similar , but more intense , to the pattern seen in cases of free gas migration into the portal vessels during intestinal ischaemia , necrotic enterocolitis or in other cases of intestinal pneumatosis [ 12 , 13 ]  . 
the possibility that this phenomenon is due to free gas ( thus not incorporated in the shell ) in the superior mesenteric vein and portal vessel is confirmed by several other observations : the stratification pattern seen in the superior mesenteric vein never occurs when the gas is contained in a membrane , as in the case of contrast microbubbles , but only when the gas is free ( intestinal pneumatosis ) the phenomenon persists even after the mi has been increased , an event that should cause immediate microbubble destruction the distribution inside the periportal sinusoids is similar to that seen after injection of free co2 in the hepatic artery ( portal angioechography ) for transient obstruction in the internal sinusoids [ 14 , 15 ]  . a probable explanation for the heterogeneous hepatic enhancement is that the free gas in the superior mesenteric vein is gas emboli originating in the intestinal microcirculation and transported to the liver via enteroportal circulation . 
the pattern of heterogeneous hepatic enhancement is due to entrapment in the hepatic sinusoids of these gas emboli , which have different insonation properties from the microbubbles . several studies have reported that sonographic contrast agents may be responsible for biological damage to the cell membranes and endothelium both in vitro and in vivo [ 1720 ]  . 
di contro , il sonovue , un agente puramente vascolare , non mostra uptake selettivo epatico , mentre presenta una fase spleno - specifica , della durata di circa 5 minuti [ 11 ]  . 
il volume globale di gas contenuto nelle dosi somministrate dellordine di microlitri , invece il fenomeno osservato da noi massivo . inoltre , le microbolle di gas responsabili di questo fenomeno sono di dimensioni maggiori di quelle costituenti il mezzo di contrasto : esse non sono in grado di attraversare i sinusoidi epatici il cui calibro di 4050 abbiamo , inoltre , notato che solo la vena porta e la vena mesenterica superiore presentavano unanomala ecogenicit , non la vena splenica , n le vene sovraepatiche , laorta o la vena cava inferiore . questo anomalo aspetto ecogeno simile , ma pi intenso , al pattern che si osserva nei casi di migrazione di gas libero nei vasi portali in corso di ischemia intestinale , di enterocolite necrotizzante o in altri casi di pneumatosi intestinale [ 12 , 13 ]  . 
la possibilit che laspetto descritto sia da imputare a gas libero ( non incorporato in una membrana ) nella vena mesenterica superiore e nei vasi portali confermato da altre osservazioni : il pattern stratificato allinterno della vena mesenterica superiore non mai osservabile nel caso in cui il gas trovi allinterno di una membrana come nel caso delle microbolle di mezzo di contrasto ; si osserva solo nel caso di gas libero ( pneumatosi intestinale ) ; il fenomeno persiste anche innalzando gli indici meccanici , evento che dovrebbe determinare la distruzione immediata delle microbolle ; la distribuzione allinterno dei sinusoidi periportali sovrapponibile a quanto osservabile dopo iniezione di co2 libera nellarteria epatica ( ecocarbossigrafia ) , per transitorio ostacolo allinterno dei sinusoidi [ 14 , 15 ]  . in base a queste osservazioni si pu ipotizzare che il fenomeno del disomogeneo enhancement epatico sia il risultato di gas libero nella vena mesenterica superiore proveniente da emboli di gas formatisi a livello del microcircolo intestinale e da qui trasportati al fegato attraverso il circolo enteroportale . 
 [ 21 ] demonstrated that in mice and rats , sonographic contrast agents cause inflammation , necrosis and ulceration of the cecum and proximal colon ( cecocolonic area ) , even with a single intravenous administration of a gas - carrier contrast agent . 
 [ 16 ] reported that intestinal lesions in mice and rats after sonographic contrast agent administration are related to the formation of large intravascular gas bubbles in the cecal and colonic wall . 
another probable explanation for heterogeneous hepatic enhancement is microbubble fusion in vivo inside the sinusoids and mesenteric vessels : these microbubble macroaggregates have different insonation properties than the separate microbubbles because of their different size [ 22 ]  . our study has two limitations . 
second , we are unable to explain why this phenomenon is so rare and why it did not occur again in the same two patients after 24 h and 9 days , respectively . 
 [ 16 ] hanno osservato che le lesioni intestinali riscontrate nei topi e nei ratti dopo somministrazione di mezzo di contrasto ecografico sono correlate alla formazione di macrobolle di gas nella parete colica e cecale . 
unaltra probabile spiegazione alleterogeneo enhancement epatico la fusione delle microbolle in vivo allinterno dei sinusoidi e nei vasi mesenterici : questi macroaggregati , in ragione delle loro diverse dimensioni , presenterebbero caratteristiche di insonazione diverse da quelle delle microbolle [ 22 ]  . il nostro lavoro presenta due limiti : il primo relativo al fatto che le lesioni intestinali descritte da dirven et al . 
 [ 16 ] sono state osservate solo in animali di piccola taglia ( topi e ratti ) , non in animali di taglia maggiore ( porcellini dindia , conigli o cani )  . 
il secondo limite , legato al fatto che non siamo in grado di spiegare perch il fenomeno sia cos raro e perch non si sia ripresentato negli stessi pazienti a 24 h e 9 giorni rispettivamente . nonostante non sia stato possibile chiarire la genesi del fenomeno , le alterazioni osservate sono reversibili e non compromettono la compliance del paziente . 
caramella , radiologia diagnostica e interventistica , via roma 67 , i - 56126 pisa , italy , tel . : + 39 - 050 - 992509 , fax : + 39 - 050 - 551461 , e - mail : caramella@med.unipi.it received : 23 may 2006 / accepted : 07 august 2006 / published online : 22 february 2007 abstract purpose . 
mri exams were done with a scanner operating at 1.5 tesla ( t ) using an endorectal coil . all patients underwent radical prostatectomy within 2 weeks from the imaging assessment . 
our study suggests that by using image fusion between colour - doppler trus and endorectal mri , it is possible to improve the accuracy of pathological staging in patients who are candidates for radical prostatectomy . key words prostate cancer , imaging prostatic cancer , staging image fusion riassunto obiettivo . 
valutare il ruolo della fusione di immagini nella stadiazione del carcinoma prostatico in un campione di 32 pazienti valutati pre - operatoriamente con ecografia transrettale ( trus ) color - doppler e risonanza magnetica ( rm )  . 
il nostro studio suggerisce che la fusione di immagini tra trus color - doppler e rm permette di migliorare laccuratezza della stadiazione istopatologica in pazienti candidati a prostatectomia radicale . parole chiave carcinoma della prostata , imaging carcinoma della prostata , stadiazione fusione di immagini introduction introduzione the incidence of prostatic cancer doubles with every decade of ageing , reaching 70% over 80 years . 
although no uniform treatment regimen exists , patients with tumours confined to the prostate ( clinical stages t1 or t2 ) and with a life expectancy of 10 or more years are often considered to be good candidates for radical prostatectomy . 
those with stage t3 lesions are usually treated with radiation therapy and those with stage t4 lesions with hormonal therapy . transrectal ultrasound ( trus ) examination , either conventional or colour - doppler , must be considered as the first imaging approach , together with prostate - specific antigen ( psa ) screening . 
in the literature , some concerns have been raised concerning the ability of trus to preoperatively assess pericapsular tumour invasion [ 1 ] , although colourdoppler trus was shown to improve depiction of prostatic vasculature as well as lesion detection and targeting [ 24 ]  . magnetic resonance imaging ( mri ) has been used as a staging modality in the preoperative evaluation of patients with clinically localised prostate cancer considered to be candidates for radical prostatectomy . 
il carcinoma prostatico origina nella zona periferica della ghiandola come una lesione nodulare con la tendenza ad invadere la porzione centrale della ghiandola e a sconfinare oltre la capsula negli stadi pi avanzati . 
quando il tumore insorge nella porzione centrale , la diagnosi differenziale con un nodulo benigno risulta difficile e per tale motivo ci possono essere errori di interpretazione . la decisione terapeutica per il carcinoma della prostata dipende dalla accuratezza nella stadiazione di malattia . 
sebbene non esista un unico tipo di trattamento , pazienti con tumore localizzato alla prostata ( stadio clinico t1 o t2 ) e con aspettativa di vita superiore a 10 anni sono spesso considerati buoni canditati per un intervento di prostatectomia radicale , mentre pazienti con lesioni allo stadio t3 o t4 sono trattati rispettivamente con radioterapia e con terapia ormonale . lesame ecografico transrettale ( trus ) , convenzionale o con color - doppler , il primo approccio strumentale da utilizzare insieme alla valutazione del psa . 
sebbene in letteratura alcuni studi abbiano messo in discussione la capacit pre - operatoria della trus di valutare lestensione extra - capsulare del tumore [ 1 ] , stato ampiamente appurato che lutilizzo del color - doppler migliori notevolmente non solo la possibilit di valutare la vascolarizzazione prostatica , ma anche di rilevare la presenza delle lesioni stesse [ 24 ]  . 
la risonanza magnetica ( rm ) stata utilizzata come indagine strumentale nella stadiazione pre - operatoria dei pazienti con tumore prostatico localizzato e pertanto candidati alla prostatectomia radicale . scopo del nostro studio stato quello di valutare il possibile ruolo della fusione di immagini nella stadiazione del carcinoma prostatico e a tal fine sono stati valutati pre - oparatoriamente con trus ( convenzionale e color - doppler ) e con rm con bobina endorettale , 32 pazienti con diagnosi clinica di carcinoma della prostata . materiali e metodi pazienti in un periodo di tempo compreso tra gennaio 2004 e luglio 2005 , abbiamo valutato con trus e rm 32 pazienti ( et media 66 , 5 anni ; et compresa tra 53 e 75 anni ; psa medio 9 , 2 ; psa compreso tra 5 , 47 ng / ml e 15 , 7 ng / ml ) , successivamente sottoposti ad intervento di prostatectomia radicale . tutti i pazienti hanno firmato il consenso informato in accordo con il protocollo di studio , approvato istituzionalmente . materials and methods patient demographics a total of 32 patients ( median age 66.5 years , age range 5375 years ; median psa 9.2 , psa range 5.4715.7 ng / ml ) who underwent trus and mri followed by radical prostatectomy between january 2004 and july 2005 were included in this study . 
colour flow imaging was adjusted to the low - flow programme in every case ( threshold of velocity detection , 2.3 cm / s ) , and the colour - doppler gain was set just below the colour noise threshold . 
 vascularity of hypoechoic nodules of the peripheral zone was evaluated by comparison with that of an adjacent or contralateral peripheral zone without using a grading systea hypoechoic image or an area with a subtle decrease in echo structure was considered hypervascular if it contained more vessels than the adjacent peripheral zone . 
as benign prostatic hyperplasia has a heterogeneous pattern and commonly contains hyperplastic nodules , colour - doppler sonography was not used to detect suspicious abnormalities originating in the transition zone . magnetic resonance imaging prostate imaging was performed using a 1.5 - tesla ( t ) siemens scanner ( magnetom symphony maestro ; siemens medical solutions , erlangen , germany ) with a combined phased - array coil and endorectal coil ( mrinnervu ; medrad , indianola , ia , usa )  . 
the following pulse sequences were used : axial fast spin echo ( fse ) : turbo factor 23 , rotation time ( tr ) 4 , 400 ms , echo time ( te ) 119 ms , slice thickness 3 mm , field of view ( fov ) 180 mm , gap 0.8 mm , matrix 230256 coronal fse : turbo factor 25 , tr 3 , 070 ms , te 107 ms , slice thickness 3 mm , fov 180 mm , gap 0.8 mm , matrix 256256 sagittal fse : turbo factor 25 , tr 4 , 820 ms , te 112 ms , slice thickness 3 mm , fov 180 mm , gap 0.8 mm , matrix 224320 axial t1 fse : turbo factor 3 , tr 619 ms , te 11 ms , slice thickness 3 mm , fov 180 mm , gap 0.8 mm , matrix 230256 pathologic evaluation all patients underwent radical retropubic prostatectomy within 2 weeks from imaging assessment . 
the remainder of the gland was serially sliced from the apex to the base at 3to 4 - mm intervals , and slices were submitted for paraffin la vascolarizzazione dei noduli ipoecogeni localizzati nella porzione ghiandolare periferica stata confrontata con quella della porzione ghiandolare periferica adiacente o controlaterale , senza utilizzare nessun sistema di classificazione . unimmagine ipoecogena o unarea con ecostruttura modicamente ipoecogena veniva considerata ipervascolarizzata se presentava pi vasi rispetto alla zona periferica adiacente . al contrario un nodulo ipoecogeno era considerato ipovascolarizzato se il numero dei vasi era simile o minore a quello della zona periferica adiacente . 
inoltre , anche se lecografia transrettale convenzionale non evidenziava nessuna anomalia , il color - doppler veniva comunque utilizzato per valutare la presenza di eventuali aree della zona periferica a maggior vascolarizzazione . 
poich liperplasia prostatica benigna ha una struttura eterogenea e comunemente contiene noduli iperplastici , il color - doppler non stato utilizzato per la ricerca di zone sospette poste nella zona di transizione . gli esami di rm sono stati eseguiti con un magnete da 1 , 5 t siemens ( magnetom symphony maestro , siemens medical solutions , erlangen , germania ) , mediante uso combinato della bobina phased - array ed endorettale ( mrinnervu , medrad , indianola , usa )  . 
il palloncino della bobina endorettale stato sempre gonfiato utilizzando circa 40 cm3 di aria e successivamente tirato delicatamente indietro per garantire la completa copertura della prostata , dallapice sino alla base . 
poich il sanguinamento post - bioptico pu dunque rendere difficoltoso lo studio di rm ponendo un limite alla diagnosi di carcinoma prostatico , nei casi in cui il paziente avesse eseguito la biopsia prima dellesame rm , lindagine veniva condotta dopo un periodo di tempo minimo di almeno 4 settimane dalla esecuzione della biopsia stessa . 
per tutti i pazienti sono state sempre utilizzate le seguenti sequenze rm : fse sul piano assiale : turbo factor 23 , tr 4400 ms , te 119 ms , spessore di fetta 3 mm , fov 180 mm , gap 0 , 8 mm , matrice 230256 ; fse sul piano coronale : turbo factor 25 , tr 3070 ms , te 107 ms , spessore di fetta 3 mm , fov 180 mm , gap 0 , 8 mm , matrice 256256 ; fse sul piano sagittale : turbo factor 25 , tr 4820 ms , te 112 ms , spessore di fetta 3 mm , fov 180 mm , gap 0 , 8 mm , matrice 224320 ; fse - t1 sul piano assiale : turbo factor 3 , tr 619 ms , te 11 ms , spessore di fetta 3 mm , fov 180 mm , gap 0 , 8 mm , matrice 230256 . anatomia patologica tutti i pazienti sono stati sottoposti a prostatectomia radicale retropubica dopo circa due settimane dalla valutazione strumentale . 
la parte rimanente della ghiandola stata invece tagliata in fette da 34 mm dalla base allapice e le sezioni ottenute sono state messe in paraffina . le vescicole seminali sono state sezionate e trattate separatamente . 
after paraffin embedding , microslices were placed on glass slides and stained with hematoxylin and eos at pathologic analysis , a gleason score was assigned according to the current protocol at our institution . 
the whole mounts were then digitised using a high - resolution scanner : these constituted the tumour maps . as surgical removal and fixation can result in deformation of the gland , and as it is impossible to section identical planes with the two techniques , an exact correspondence between pathologic slices and mr sections was not expected . 
the most closely corresponding transverse t2 - weighted images and pathologic step - section slices were paired on the basis of the following anatomic landmarks : ( a ) the presence of urinary bladder and seminal vesicle tissue in superior slices , ( b ) the slice with the largest diameter and progressive changes in slice diameter , ( c ) the slice where the ejaculatory ducts enter the veru montanum , ( d ) the anteriorposterior and left - right position of the ejaculatory ducts and urethra and the shape of the urethra , ( e ) the thickness of the peripheral zone and the position of the pseudocapsule and ( f ) the presence , size , and shape of the transition zone . 
the precision of matching in the superior - inferior direction was estimated as being within one transverse slice ( 3 mm )  . data analysis trus and mr images as well as the digitised whole - mount axial pathological sections were transferred to a graphic workstation to perform image fusion . 
in three cases , image fusion was not possible , as geometrical distortion prevented reliable registration between the three data sets , and in four cases only , sagittal colour - doppler trus images were available . 
sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) were calculated by carefully analysing the fusion images in which the pathological section served as gold standard for assessing the staging obtained by trus alone , by mri alone and by the combination of mri and trus . results step - section histopathology demonstrated stage pt2b in four patients , pt2c in 14 patients , pt3a in three patients and pt3b in four patients . 
il vetrino stato successivamente digitalizzato con uno scanner ad alta risoluzione . poich la rimozione chirurgica e la fissazione possono determinare una deformazione della ghiandola e poich non possibile ottenere gli stessi piani di sezione della rm , non c stata unesatta corrispondenza tra le sezioni istologiche ed i piani acquisiti con la rm . 
la corrispondenza lungo lasse cranio - caudale delle immagini sottoposte a fusione stata stimata essere nellordine dello spessore di una fetta assiale ( 3 mm )  . analisi dei dati le immagini della trus , di rm e quelle digitalizzate dalle sezioni istopatologiche sono state successivamente inviate ad una workstation grafica al fine di eseguirne una loro fusione , tecnicamente possibile in 25 / 32 casi . 
infatti , mentre in 3 casi ci non stato possibile per la presenza di distorsioni geometriche che hanno impedito una accurata sovrapposizione delle immagini stesse , in 4 casi la sola disponibilit delle immagini sagittali di trus color - doppler ha impedito la fusione con le sezioni istopatologiche , ovviamente presenti solo sul piano assiale . 
la sensibilit , la specificit , il valore predittivo positivo ( vpp ) ed il valore predittivo negativo ( vpn ) sono stati calcolati analizzando attentamente la fusione delle immagini , nelle quali la sezione istopatologica serviva come gold standard per determinare la stadiazione ottenuta con la sola trus , con la sola rm e dalla combinazione di entrambe queste metodiche . risultati le sezioni istopatologiche hanno evidenziato uno stadio pt2b in 4 pazienti , pt2c in 14 pazienti , pt3a in 3 pazienti , pt3b in 4 pazienti . 
la correlazione tra trus e sezioni istopatologiche ha dimostrato che la trus aveva consentito una corretta stadiazione in 20 / 25 casi ( accuratezza 80% , sensibilit 29% , specificit 100% , vpp 100% , vpn 78% )  . 
la correlazione tra rm e sezioni istopatologiche ha dimostrato che la rm aveva consentito una corretta stadiazione in 22 / 25 casi ( accuratezza 88% , sensibilit 64% , specificit 100% , vpp 100% , vpn 84% )  . 
image fusion between mri and pathological sections showed that mri allowed correct staging in 22 / 25 cases ( accuracy 88% , sensitivity 64% , specificity 100% , ppv 100% , npv 84% )  . 
image fusion between trus , mri and pathological sections proved that trus and mri allowed correct staging in 23 / 25 cases ( accuracy 92% , sensitivity 71% , specificity 100% , ppv 100% , npv 90% )  . 
the three types of image fusion produced the results summarised in table 1 . discussion as nonpalpable prostatic cancers ( stage t1c ) originating in the peripheral zone have increased the use of trus - guided biopsies , we evaluated how useful it was to correlate trus ( grayscale and colour - doppler mode ) with endorectal mri , or rather , whether the fusion of images coming from these two techniques could improve diagnosis . 
unfortunately , radical prostatectomy specimens have shown that 30%51% of diazione in 23 / 25 casi ( accuratezza 92% , sensibilit 71% , specificit 100% , vpp 100% , vpn 90% )  . 
i risultati dei tre tipi di fusione sono riportati nella tabella 1 . discussione poich i tumori prostatici non palpabili della zona periferica ( stadio t1c ) hanno portato ad un uso sempre maggiore della biopsia trans - rettale eco - guidata , noi abbiamo valutato quanto possa essere utile correlare la trus ( convenzionale e color - doppler ) con la rm ed in particolare , se la fusione delle immagini appartenenti a queste due metodiche , potesse agevolare la nostra diagnosi . 
la prima la sua nota dipendenza dalloperatore , seguita dalla presenza di un limitato campo di vista , il quale oltre a non consentire una completa valutazione di tutta la struttura ghiandolare , come sua diretta conseguenza , c . 
section 20 / 25 22 / 25 23 / 25 tabella 1 confronto di tre tipi di fusione dimmagini tra color - doppler trans - rettale ( trus ) , risonanza magnetica ( rm ) e sezioni istopatologiche ( macrosezioni ) fusione di immagini pazienti , n sensibilit , % specificit , % accuratezza , % trus + macrosezioni rm + macrosezioni rm + trus + macrosezioni 20 / 25 22 / 25 23 / 25 these nonpalpable tumours display no abnormalities on trus [ 5 ]  . 
the first important and well - known limit is the strict operator dependence of trus , followed by its small fov , which does not allow depiction of nearby prostate structures , and its poor sensitivity for capsular irregularities . 
moreover , the anterior location ( transition zone ) of t1c - stage tumours reduces the diagnostic value because it is well established that trus is of limited value for revealing transitional - zone cancer [ 6 ]  . the use of colour - doppler ultrasound ( us ) does not substantially improve the accuracy of grayscale us . 
although it can improve the visibility of subtle nonpalpable grayscale abnormalities in a minority of cases , it fails to detect strictly isoechoic nonpalpable tumours , probably for reasons similar to those mentioned for grayscale trus . 
however , colourdoppler trus may add only minor information and , in particular , it seems to be superior to trus in two circumstances : the first is the significantly greater rate of high gleason grades in hypervascular tumours [ 7 ] ; the second is the better detection of tumours smaller than 3 mm , which are best resolved with colour - doppler mode . mri , performed with the combined use of phased - array and endorectal coils , provides a large fov with high spatial resolution , optimising local and distant staging of prostate carcinoma during a single mri session . 
for tumour ( t ) staging , the accuracy of endorectal coils is > 90% , also thanks to the added diagnostic value of phased - array coils that allows evaluation of nodal involvement ( n ) and detailed imaging of all anatomical structures above the pelvic floor [ 818 ]  . thus , if grayscale trus and colour - doppler trus may differentiate low - risk , nonpalpable prostate tumours which are hypovascular ( and invisible in 90% of cases ) from high - risk carcinomas ( t2 stage ) which are visible and hypervascular mri is superior because it is more sensitive and more specific in detecting all possible signal changes , ranging from small tumoural foci to coarser lesions associated or unassociated with extraprostatic tumour spread . 
inoltre , poich la trus difficilmente riesce a rilevare piccole lesioni , queste vengono spesso misconosciute ; infine la localizzazione anteriore ( zona di transizione ) dei tumori prostatici allo stadio t1c , riduce la capacit diagnostica della trus stessa , per la sua nota difficolt nellidentificare una neoplasia localizzata nella zona di transizione [ 6 ]  . luso del color - doppler non migliora laccuratezza dellecografia convenzionale in modo significativo . 
sembra comuqnue che in una minoranza di casi il suo utilizzo possa migliorare la visualizzazione di piccole alterazioni non palpabili ; tuttavia la trus color - doppler non riesce a delimitare con precisione le lesioni isoecogene non palpabili , probabilmente per le stesse ragioni descritte per la trus convenzionale . 
comunque , nonostante la trus color - doppler aggiunga solo informazioni secondarie sembra che sia comunque superiore alla trus convenzionale in due circostanze : la prima nel rilevare tumori ipervascolarizzati con gleason elevato [ 7 ] ; la seconda per la sua superiorit nellidentificare neoplasie di dimensioni inferiori ai 3 mm , meglio apprezzate grazie allausilio del color - doppler . la rm , mediante luso combinato della bobina phasedarray ed endorettale , fornisce invece una visione panoramica ad alta risoluzione spaziale , permettendo una stadiazione locale e a distanza del carcinoma . 
per la stadiazione tumorale ( t ) , laccuratezza della bobina endorettale superiore al 90% anche grazie allutilizzo combinato della bobina phased - array , che permette di valutare non solo un eventuale coinvolgimento linfonodale ( n ) ma anche tutte le restanti strutture anatomiche della pelvi [ 818 ]  . cos , anche se la trus , convenzionale e color - doppler , pu discriminare i tumori prostatici non palpabili , ipovascolarizzati e a basso rischio ( non apprezzabili nel 90% dei casi ) dai carcinomi palpabili , ipervascolarizzati e ad alto rischio ( stadio t2 ) , la rm risulta essere una metodica nettamente superiore in quanto pi sensibile e specifica nel rilevare tutte le possibili alterazioni di segnale , dai piccoli focolai tumorali alle lesioni pi grossolane associate ad eventuali sconfinamenti extraghiandolari . 
 combined analysis by using fusion images obtained from colour - doppler trus and mr images improves tumour detection and staging . in this study , we performed an additional type of analysis : the fusion between preoperative images and whole - mount radical prostatectomy specimens after having digitised the latter by means of a high - end digitiser . 
such image fusion allows immediate and intuitive assessment of the capabilities of trus , mri and fused trus and mri data in delineating local tumour extent by providing direct comparison with whole - mount prostatectomy specimens . 
tale fusione permette unimmediata ed intuitiva valutazione delle capacit della trus , della rm e delle immagini sovrapposte delle due metodiche nel delineare lestensione locale del tumore grazie al confronto diretto con le macrosezioni anatomo - patologiche ottenute dal pezzo chirurgico . conclusioni il nostro studio conferma in maniera indiscutibile lelevata attendibilit della rm come strumento diagnostico nella stadiazione del carcinoma prostatico in fase pre - operatoria . 
infatti abbiamo rilevato , non solo una buona corrispondenza tra le alterazioni evidenziate con la rm , in quanto sempre presenti nelle sezioni istopatologiche , ma anche una loro precisa corrispondenza anatomica . 
la fusione delle immagini dimostra quindi come la trus color - doppler fornisca solo miglioramenti marginali nel definire una corretta stadiazione della patologia che invece viene sicuramente ottenuta con la rm 1 , 5 t condotta con bobina endorettale . 
radiologia , ospedale di cernusco s / n , azienda ospedaliera di melegnano , milano , italy 3divisione di epidemiologia e biostatistica , istituto europeo di oncologia , milano , italy 4istituto di scienze radiologiche , facolt di medicina e chirurgia , universit degli studi di milano correspondence to : m . 
our preliminary data show that ceus can be used to obtain more accurate measurements than conventional us for the follow - up of patients with metastases in fatty liver . key words contrast - enhanced ultrasound ultrasound contrast agent breast cancer liver metastasis riassunto obiettivo . 
i nostri dati preliminari mostrano come la ceus possa essere utilizzata per ottenere parametri pi accurati di quelli della us per il follow - up delle pazienti con metastasi in fegato steatosico . parole chiave ecografia con mezzo di contrasto mezzo di contrasto ecografico cancro della mammella metastasi epatiche p . 
in italy , approximately 33 , 000 women are diagnosed with breast cancer each year , and 300 , 000 women have been diagnosed in the past , almost half of them in the past 5 years . 
most women who develop breast cancer can be treated with conservative surgery and adjuvant therapy ( chemotherapy or hormone therapy or both , depending on the biological features of the tumour ) , which substantially reduce the incidence of recurrence and metastasis or control their evolution . breast cancer patients receiving hormone therapy or chemotherapy frequently develop liver steatosis . 
in europe , it is preferred to other , more sensitive modalities , such as multidetector computed tomography ( mdct ) and magnetic resonance imaging ( mri ) because of the relative invasiveness of mdct and the high cost and limited availability of mri [ 5 , 6 ]  . 
this difference may be minimal , especially in small lesions , preventing the operator from visualising thein addition , changes in normal hepatic structure related to metabolic disorders such as diffuse or focal steatosis may make it difficult to detect lesions and determine their nature and extent [ 7 ]  . the need to improve the diagnostic accuracy of us , especially in difficult conditions , as is fatty liver disease , has recently led to the diffusion of contrast - enhanced us ( ceus )  . preliminary studies have demonstrated that ceus is more accurate than conventional us in identifying and characterising focal hepatic lesions , in part because the sonographic contrast medium appears able to reduce the parenchymal heterogeneity resulting from steatosis [ 8 , 9 ]  . correct determination of focal lesion size is fundamental because comparison between preand post - treatment dimensions is the most commonly used method to evaluate treatment response [ 10 ]  . 
 the purpose of this prospective study was to assess the accuracy of ceus in determining the diameter of liver metastases in patients operated on for breast cancer who have developed fatty liver disease and to compare it with that of us using mdct as the reference standard . materials and methods case series we enrolled 12 consecutive patients with liver metastases from resected breast cancer who had been treated with chemotherapy and / or hormone therapy and had moderate or severe fatty liver disease on us [ 11 ] that made sonographic assessment and measurement of the lesions difficult . 
we exil carcinoma della mammella il tumore pi frequente nelle donne dei paesi occidentali ; in italia si ammalano ogni anno circa 33000 donne e vivono ormai 300000 donne che hanno avuto in passato una diagnosi di cancro della mammella , di cui quasi la met negli ultimi 5 anni . 
infatti il carcinoma della mammella ha una prognosi relativamente buona : in italia , circa l80% delle donne ammalatesi nella prima met degli anni novanta era viva a 5 anni dalla diagnosi . 
la maggior parte delle donne che si ammalano di tumore della mammella oggi pu essere curata con chirurgia conservativa e ricevere trattamenti adiuvanti ( chemioterapici e / o ormonoterapia , secondo le caratteristiche biologiche del tumore ) , che riducono sensibilmente lincidenza di recidive o metastasi o ne controllano levoluzione . le pazienti con carcinoma mammario , sottoposte a ormonoterapia o chemioterapia , sviluppano con una certa frequenza una steatosi epatica . 
diversi lavori hanno dimostrato una stretta correlazione tra queste terapie e linsorgenza di una steatosi non alcolica [ 13 ] , specie nelle donne in sovrappeso [ 4 ]  . 
lecografia ( us ) la metodica di pi largo impiego per lo studio del fegato nella stadiazione e nel follow up delle pazienti con carcinoma mammario e , soprattutto in europa , la si preferisce a metodiche dotate di maggiore sensibilit quali la tomografia computerizzata multidetettore ( tcmd ) o la risonanza magnetica ( rm ) per la relativa invasivit della prima e lalto costo e la scarsa disponibilit della seconda [ 5 , 6 ]  . 
inoltre lalterazione della normale struttura epatica determinata da disordini metabolici quali la steatosi diffusa o focale , pu generare difficolt nellindividuare le lesioni e nel determinarne la natura e lestensione [ 7 ]  . la necessit di migliorare laccuratezza diagnostica dellus , particolarmente nelle condizioni difficili , come ad esempio nel fegato steatosico , ha visto di recente diffondersi lecografia con mezzo di contrasto ( ceus )  . 
studi preliminari hanno messo in evidenza una maggior accuratezza diagnostica della ceus nei confronti della us , sia nellindividuazione , sia nella caratterizzazione delle lesioni focali epatiche , anche perch il mezzo di contrasto ecografico sembra in grado di ridurre la disomogeneit parenchimale dovuta alla steatosi [ 8 , 9 ]  . la corretta determinazione delle dimensioni delle lesioni focali di fondamentale importanza se si considera che il confronto tra le dimensioni pree post - trattamento il metodo pi usato per la valutazione della risposta alla terapia farmacologia [ 10 ]  . questo studio prospettico si propone di valutare laccuratezza della ceus nel definire il diametro delle metastasi epatiche in pazienti operate per carcinoma mammario che abbiano sviluppato una steatosi epatica e di paragonarla p . 
for the purposes of the study , we focused on lesions whose topographic characteristics on initial us would ensure easy identification at the following diagnostic studies ( ceus and mdct )  . 
all patients were informed of the aims of the study and provided written informed consent for the use of contrast media . ultrasound the study was done with digital us scanners equipped with dedicated software for low - mechanical - index studies ( sequoia , siemens medical solutions , mountain view , ca , usa and logiq 500 , ge medical systems , milwaukee , wi , usa ) and 3 to 5 mhz transducers . 
the study was done at baseline and after injection of 2 ml / kg of iodinated contrast medium ( iomeron 300 , bracco imaging ) at a rate of 4 ml / s , followed by 40 ml saline solution at a rate of 4 ml / s in the arterial ( 30 s ) , portal ( 7080 s ) and late phase ( 180 s )  . scans were acquired with a collimation of 20 mm , slice thickness of 5 mm , pitch 1.75 , and table feed of 35 mm / rotation . 
 assessment criteria up to a maximum of three liver lesions per patient were measured with electronic callipers on the us , ceus and mdct images considering the largest diameter , in accordance with the response evaluation criteria in solid tumors ( recist ) criteria [ 12 ]  . 
 con quella di us , utilizzando la tcmd come metodica di riferimento . materiali e metodi casistica sono state arruolate nello studio 12 pazienti consecutive con metastasi epatiche da carcinoma mammario operato , che hanno eseguito trattamento medico sistemico con chemioterapia e / o ormonoterapia e con presenza di steatosi epatica di grado moderato o severo allus [ 11 ] , tale da rendere difficoltosa la valutazione ecografica e la misurazione delle lesioni . sono state escluse pazienti con performance status compromesso [ 45 ] , con ascite tale da ostacolare lindagine us e con controindicazioni al mdc ecografico o organoiodato . tutte le pazienti sono state sottoposte a us , ceus e tcmd . per gli scopi dello studio sono state identificate quelle lesioni che , al primo esame ecografico , presentavano caratteristiche topografiche tali da essere facilmente identificabili ai successivi esami diagnostici ( ceus e tcmd )  . 
tutte le pazienti sono state informate dello scopo dello studio e hanno sottoscritto i moduli specifici per la somministrazione dei mezzi di contrasto . ecografia lo studio si avvalso di ecografi digitali dotati di software dedicato ad esami con basso indice meccanico : sequoia ( siemens , medical solutions , view , ca , usa ) e logiq 500 ( ge , medical system , milwaukee , wi , usa ) , con sonde di frequenza variabile da 3 a 5 mhz . 
il mezzo di contrasto utilizzato stato il sonovue ( bracco imaging , milano , italia )  . la valutazione ecografica stata eseguita in condizioni basali e dopo iniezione endovenosa di mdc in bolo di 2 , 4 ml seguito da circa 5 ml di soluzione fisiologica . 
la ceus stata eseguita in imaging continuo a basso indice meccanico con scansioni panoramiche e mirate sulla lesione principale . sono state analizzate tre fasi distinte della perfusione epatica : fase arteriosa ( 1530 s dopo liniezione ) , fase portale ( 3060 s ) e fase tardiva ( da 60 s fino alla perdita delle microbolle : 240360 s )  . tc multidetettore results we studied 25 focal lesions in 12 subjects ( mean : 2.2 lesions per patient )  . 
lesame stato effettuato in condizioni basali e dopo iniezione endovenosa di 2 ml / kg di mdc organo - iodato ( iomeron300 , bracco imaging ) , con flusso di 4 ml / s seguito da 40 ml di soluzione fisiologica con flusso di 4 ml / s , nelle fasi arteriosa parenchimale ( 30 s ) , portale ( 7080 s ) e tardiva ( 180 s )  . 
comparison of us and ceus results shows that in five cases , the use of sonographic contrast medium enabled measurement of lesions that were confirmed on mdct but poorly defined on us because of marked steatosis . 
although not recommended in study protocols for evaluating results [ 12 ] , us is widely used in routine clinical practice owing to its lower economic and biological cost compared with that of mdct and mri . 
detection and accurate measurement of secondary lesions before and after treatment , which guides the clinicians decision to confirm or change treatment strategy , is therefore crucial not only for mdct and mri , but also for us . 
 use of sonovue , a second - generation contrast agent , has shown that ceus is significantly more accurate than conventional us in detection and characterisation of hepatic lesions [ 8 , 15 ]  . 
the microbubbles of second - generation contrast agents persist in the vessels up to 360 s , allowing plenty of time for a detailed study of the entire organ and depiction of secondary lesions , which are visualised in the late phase ( 60360 s ) when the surrounding parenchyma becomes homogeneous . 
sonovue proved to be well tolerated and free of side effects , in agreement with the literature [ 16 ]  . compared with conventional us , use of sonographic contrast medium allowed us to obtain measurements that were closer to those demonstrated by mdct . 
the lack of homogeneity of fatty liver also produced diagnostic doubt between a skip area and a secondary lesion ; in this case , ceus provided the correct diagnosis by demonstrating the homogeneous vascularity and echogenicity of the liver parenchyma [ 17 ]  . 
le misurazioni rilevate con us , ceus e tcmd sono state analizzate con il metodo di altmanbland [ 13 , 14 ]  . risultati sono state studiate 25 lesioni focali in 12 soggetti , con una media di 2 , 2 lesioni per paziente . 
i diametri delle lesioni misurate con us e confrontati con i valori di riferimento ottenuti con tcmd sono riportati in figura 3 . la ceus ha consentito di misurare tutte le 24 metastasi confermate dalla tcmd : i diametri misurati con ceus sono paragonati a quelli misurati con tcmd in figura 4 . 
paragonando i risultati ottenuti con us con quelli ottenuti con ceus , si osserva che in 5 casi il mezzo di contrasto ecografico ha permesso di misurare le lesioni che il grado marcato di steatosi rendeva mal definite allecografia convenzionale e confermate alla tcmd ; in un caso la lesione stata meglio definita allecografia basale ; in tre casi le misure ottenute con ecografia basale e quelle con ceus si sono equivalse . 
1a - c focal lesion in the viiviii hepatic segment , which was difficult to measure on ultrasound ( us ) images ( a ) but clearly defined by contrastenhanced us ( ceus ) ( b )  . 
1a - c lesione focale al settimo - ottavo segmento epatico , difficile da misurare con lecografia convenzionale ( a ) , ben parametrabile allecografia con mezzo di contrasto ( b )  . 
sebbene lecografia non sia un metodo consigliato per la valutazione dei risultati nei protocolli di studio [ 12 ] , di fatto ampiamente usata nella pratica clinica quotidiana per un minor costo , economico e biologico , rispetto alla mdtc o alla risonanza magnetica . 
anche con lecografia risulta pertanto determinante non solo lindividuazione , ma anche la precisa valutazione dimensionale delle lesioni secondarie pre e post trattamento che determina la decisione del clinico di confermare o cambiare la linea terapeutica . lutilizzo di un mezzo di contrasto ecografico di ii generazione , il sonovue , ha dimostrato come la ceus sia significativamente pi accurata dellecografia convenzionale nella diagnosi e nella caratterizzazione delle lesioni epatiche [ 8 , 15 ]  . 
lintroduzione dei mezzi di contrasto ecografici di seconda generazione permette una persistenza delle microbolle allinterno dei vasi fino a 360 s , e fornisce perci il tempo necessario per una valutazione approfondita di tutto lorgano , considerando che le lesioni secondarie si evidenziano meglio nella fase tardiva ( da 60 a 360 s ) con lomogeneizzazione del parenchima circostante . 
il sonovue si dimostrato ben tollerato ed , in accordo con i dati riportati in letteratura [ 16 ] , non abbiamo osservato alcun effetto collaterale . lutilizzo del mezzo di contrasto ecografico ha permesso di ottenere misure delle lesioni pi simili a quelle ottenute con la tcmd , rispetto a quelle definite con lecografia convenzionale . 
la disomogeneit del fegato interessato dalla steatosi ha creato anche un dubbio diagnostico tra una skip area ed una lesione ripetitiva ; in questo caso la ceus ha permesso di formulare la diagnosi corretta dimostrando la vascolarizzazione e lecogenicit omogenea del parenchima epatico [ 17 ]  . 
nella nostra casistica lutilizzo di mdc ecografico ha permesso di parametrare 5 lesioni ( il 20 , 8% delle metastasi considerate ) che con ecografia convenzionale erano solo intuibili a causa del marcato grado di steatosi ; inoltre ha permesso di riconoscere un falso positivo . 
le 5 lesioni non misurabili con us non avevano dimensioni inferiori alle altre ( range : 1577 mm rispetto a 1183 mm dellintera casistica ) , ma probabile che sia stata proprio la steatosi ad ostacolare lecografia convenzionale . 
the five lesions that could not be measured with us were no smaller than the others ( range : 1577 mm compared with 1183 mm ) , but it is likely that steatosis prevented detection on conventional us . comparison between us and ceus showed that by enhancing the contrast between the lesion and surrounding tissue , sonovue provides better visualisation and definition of lesion contours and consequently more accurate measurement . 
 the availability of a reliable method for measuring secondary lesions is all the more important if we consider the recent attempts made to provide oncological imaging professionals with guidelines and protocols to standardise assessment criteria across different settings [ world health organization ( who ) [ 18 ] and recist [ 11 ] ]  . 
this appears to justify endeavours to identify a modality that is not only able la possibilit di avere a disposizione una metodica affidabile nella misurazione delle lesioni secondarie appare sempre pi importante se si pensa come negli ultimi anni si sia cercato di fornire agli operatori che si occupano di imaging oncologico , linee guida e protocolli quanto pi uniformabili ai diversi ambienti ( criteri world health organization who [ 18 ] e criteri response evalutation criteria solid tumors recist [ 11 ] )  . 
sembra quindi giustificato lo sforzo di cercare una metodica che valuti la risposta alla terapia oncologica in modo accurato , ma anche quanto pi possibile accessibile ed accettabile da parte del paziente , che necessariamente dovr sottoporsi a controlli ravvicinati nel tempo . 
la tcmd conferma la morfologia e le dimensioni rilevate dallecografia con mezzo di contrasto ( c )  . to accurately evaluate treatment response but is also accessible and acceptable to patients , who will necessarily have to undergo close follow - up examinations . 
although according to protocols mdct remains the gold standard for evaluation of solid hepatic lesions , it should be noted that these guidelines do not take into account radiation protection issues or the patients age [ 19 ]  . 
despite a slight increase in costs and in view of the increasing diffusion of scanners with contrast - specific software , ceus is a safe and accurate method for evaluation of treatment response to secondary hepatic lesions , particularly in the presence of structural alterations of the liver parenchyma such as steatosis . non tengono presente alcun criterio radioprotezionistico , neppure in considerazione dellet della paziente [ 19 ]  . lutilizzo del mdc non modifica le caratteristiche dellindagine ecografica , e cio la rapidit di esecuzione , il basso costo , la non invasivit e la buona accettabilit da parte delle pazienti . 
power doppler ultrasound ( us ) with time - intensity curves was used to study renal graft function both in the absence of disease and with complications ( acute tubular necrosis and chronic rejection ) in an attempt to identify pathognomonic patterns . 
fifty - six asymptomatic renal transplant patients ( 36 men and 20 women ) , 19 of whom had altered creatinine clearance levels , were studied by power doppler us with time - intensity curves followed by biopsy . 
although confirmation by a larger series is required , our findings appear to indicate pathognomonic patterns in patients with chronic rejection and acute tubular necrosis . key words ultrasound power doppler us contrast agent kidney transplantation chronic rejection acute tubular necrosis riassunto obiettivo . 
in questo lavoro stato utilizzato il power doppler con le curve intensit / tempo per lo studio della funzionalit del rene trapiantato in assenza di malattia e con complicanze ( necrosi tubulare acuta e rigetto cronico ) , al fine di individuare pattern patognomonici . 
cinquantasei pazienti con rene trapiantato , 36 maschi e 20 femmine , con assenza di sintomi e affetti da alterati livelli di clearance della creatinina , sono stati studiati con il power doppler con le curve intensit / tempo e successivamente sottoposti ad esame bioptico . 
sono stati individuati tre gruppi di pazienti in base ai dati emersi dallo studio delle curve i / t : gruppo a : 27 pazienti presentavano curve con picco massimo di intensit raggiunto dopo un tempo compreso tra 5065 secondi dalla somministrazione ev del mdc . 
i dati ottenuti dalle curve hanno mostrato significative variazioni a seconda delle condizioni del rene ( assenza di malattia , rigetto cronico , necrosi tubulare acuta )  . i risultati ottenuti potrebbero rappresentare , a nostro giudizio , pattern patognomonici nei pazienti con rigetto cronico e necrosi tubulare acuta ; tali dati devono essere naturalmente confermati su pi ampia casistica . parole chiave ecografia power doppler mezzo di contrasto rene trapiantato rigetto cronico necrosi tubulare acuta r . 
the use of sonographic contrast material has further improved the study of graft vascularity [ 58 ] ; in particular , the study of the peripheral vasculature and intraparenchymal arteries provides important information on complications , above all in the case of chronic renal failure , chronic rejection and acute tubular necrosis [ 9 ]  . the most interesting and promising recent development in regards to us appears to be the use of sonographic contrast material to plot contrast - distribution curves known as time - intensity curves [ 1012 ]  . 
following intravenous administration of contrast material , graphs are plotted that express signal intensity values , or contrast material concentration , in the structure being examined ; this enables the study of enhancement patterns and provides important functional information for the diagnosis . 
the transplanted kidney is well suited to this type of investigation given its superficial position and the fact that it is barely affected by respiratory motion conditions that facilitate sonographic evaluation . 
the method has already been successfully used in the study of other organs [ 1618 ]  . we studied the transplanted kidney using power doppler us with time - intensity curves as well as duplex doppler us to measure the resistive index ( ri ) and correlated the results with the biopsy findings to identify pathognomonic patterns for the differential diagnosis of the complications associated with renal transplant . 
 il color e power doppler , grazie alla capacit di studiare accuratamente la vascolarizzazione renale , possono fornire numerose informazioni sul rene trapiantato [ 1 , 2 ] e le principali complicanze del trapianto [ 3 ]  . 
lutilizzo del mdc ecografico ha permesso un ulteriore progresso nello studio della vascolarizzazione del rene trapiantato [ 58 ] ; in particolare lo studio della vascolarizzazione periferica e delle arterie intra - parenchimali fornisce importanti informazioni sulle complicanze del trapianto specialmente nel caso dellinsufficienza renale cronica , rigetto cronico e necrosi tubulare acuta [ 9 ]  . recentemente lo sviluppo pi interessante e promettente dellecografia sembra essere rappresentato dallutilizzo del mdc ecografico per la determinazione delle curve di distribuzione del mdc conosciute come le curve intensit / tempo [ 1012 ]  . 
dopo aver somministrato per via ev il mdc si ottengono dei grafici che esprimono i valori di intensit di segnale provenienti dalla struttura in esame relativi alla concentrazione del mdc ; in questo modo possibile studiare la dinamica di enhancement ed avere importanti informazioni di tipo funzionale per la diagnosi . 
il rene trapiantato si presta bene a questo tipo di indagine per la sua collocazione superficiale e la scarsa mobilit con gli atti del respiro , condizioni che favoriscono lo studio ecografico . 
in dettaglio : 37 dei 56 pazienti erano asintomatici e non presentavano alterazioni degli esami di laboratorio ; 10 dei pazienti asintomatici sono stati assunti come gruppo di controllo . dopo compilazione del consenso informato tutti i pazienti sono stati sottoposti ad esame con power doppler con mdc per la determinazione delle curve intensit / tempo ( i / t ) per lo studio della dinamica di enhancement del rene trapiantato e ad esame duplex doppler per la misurazione degli indici di resistenza . 
 stato utilizzato un apparecchio ecografico idea 5 ( esaote biomedica , genova , italia ) equipaggiato con un software brevettato dallesaote per la realizzazione delle curve intensit / tempo ; stata utilizzata una sonda convex da 3 , 5 mhz con una frequenza di ripetizione degli impulsi ( prf ) di 1 , 2 khz per lesecuzione delle curve i / t e la r . 
to sample the curves time and signal - intensity values , we placed the reference line at the beginning of enhancement and at the maximal enhancement peak using the cursor . 
enhancement beginning and maximal enhancement peak values were calculated during postprocessing according to the following criteria : beginning enhancement value was sampled on the x - axis at the point where the time / intensity curve began to rise , that is , when signal intensity began to increase as a result of the arrival of the contrast material in the renal vessels the maximal enhancement peak value was sampled on the y - axis at the point where the curve reached its maximum height and which corresponded to the highest concentration of contrast material and therefore to maximum signal intensity . we therefore considered the following parameters : time to enhancement beginning time to enhancement peak . the ri was measured in the renal artery and interlobar arteries of the upper and lower pole and the middle third of the kidney . 
the same examiner performed all examinations , and all patients subsequently underwent biopsy of the renal graft . results on the basis of beginning enhancement and peak enhancement values derived from the time - intensity curves , we identified three groups of patients in addition to the control group . 
per campionare i valori di tempo e di intensit delle curve stata posizionata , con il cursore , la linea di riferimento allinizio dellenhancement e nel picco di massima intensit del segnale ; in dettaglio il valore di inizio enhancement e di raggiungimento del picco massimo di intensit sono stati calcolati in post - processing secondo i seguenti criteri : il valore di inizio enhancement stato campionato nellasse delle ascisse nel punto in cui la curva intensit / tempo si impenna e cio quando il valore dellintensit del segnale comincia ad aumentare in rapporto allingresso del mdc nei vasi renali ; il valore di picco di intensit massima stato campionato nellasse delle ordinate nel punto in cui la curva raggiunge la massima altezza e che corrisponde alla massima concentrazione e quindi al valore massimo di intensit del segnale . sono stati quindi presi in considerazione i seguenti parametri : tempo di inizio enhancement ; tempo di raggiungimento del picco massimo di intensit del segnale . gli indici di resistenza sono stati misurati nellarteria renale , nelle interlobari del polo superiore , inferiore e del terzo medio del rene . 
i valori degli indici di resistenza erano compresi tra 0 , 73 e 0 , 79 . gruppo c : tre pazienti , con valori di clearance della creatinina compresi tra 3260 ml / min , presentavano curve i / t con valori di inizio enhancement compresi tra 2530 r . 
i valori degli indici di resistenza erano compresi tra 0 , 68 e 0 , 74 . gruppo di controllo : i 10 pazienti selezionati , con valori di clearance della creatinina compresi tra 90112 ml / min , presentavano curve con inizio enhancement compreso tra 1525 secondi dalla somministrazione ev del mdc e picco massimo di intensit raggiunto dopo un tempo compreso tra 5065 secondi dalla somministrazione ev del mdc . 
i valori degli indici di resistenza erano compresi tra 0 , 62 e 0 , 68 . i dati degli ir rapportati alla clearance della creatinina sono riassunti nella tabella 1 . la misurazione degli ir , nei complessivi 56 pazienti , assumendo 0 , 70 come valore soglia ( < 0 , 70 assenza di malattia ; > 0 , 70 presenza di malattia ) , ha dimostrato 17 veri positivi , 31 veri negativi , 6 falsi positivi e 2 falsi negativi ( sensibilit 89% , specificit 83% , valore predittivo positivo 73% , valore predittivo negativo 93% )  . 
la curva intensit / tempo presenta rapido wash - in e rapido e wash - out . can be studied by diagnostic imaging techniques that provide direct or indirect information on the presence of disease . 
some routinely used methods , such as urography , computed tomography ( ct ) , magnetic resonance imaging ( mri ) and scintigraphy , have drawbacks related to the use of ionising radiation ( urography , ct ) or radioisotopes ( scintigraphy ) , and high cost ( ct , mri , scintigraphy )  . 
these changes generally translate into a reduction of renal perfusion that is directly proportional to disease severity [ 19 ]  . in our study , we used power doppler us for the advantages it offers compared with colour doppler us : less noise , and independence from the insonation angle and flow velocity [ 2022 ]  . 
the ability to demonstrate low - flow signals , such as those in the cortical region , has increased the methods potential to identify avascular areas ( infarction , acute pyelonephritis , etc . ) or areas with strongly impaired non si sono verificate reazioni dintolleranza al mdc ecografico . lesame bioptico non ha evidenziato malattia nei pazienti del gruppo a e del gruppo di controllo ; nei pazienti del gruppo b ha permesso diagnosi di rigetto cronico e nei pazienti del gruppo c diagnosi di necrosi tubulare acuta . discussione e conclusioni le patologie che possono provocare alterazioni della funzionalit e dellenhancement del parenchima renale sono numerose ; esse possono essere studiate con tecniche di diagnostica per immagini in grado di fornire informazioni dirette o indirette sullo stato della malattia . 
alcune di queste metodiche , utilizzate di routine , come lurografia , la tc , la rm e la scintigrafia , presentano alcuni svantaggi legati al rischio dellutilizzo di radiazioni ionizzanti ( urografia , tc ) o radioisotopi ( scintigrafia ) e inoltre sono gravate da costi elevati ( tc , rm , scintigrafia )  . 
 ri comparison between subjects without disease and those with disease ( acute or chronic rejection , acute tubular necrosis , cyclosporine toxicity , etc . ) generally provides an indication of disease presence but does not show significant differences among diseases and therefore does not allow a differential diagnosis [ 24 ]  . 
cut - off values between the groups were as follows : group a ( patients without disease ) : enhancement onset under 25 s and time to peak enhancement less than 65 s group b ( patients with chronic rejection ) : enhancement ai fini della diagnosi soprattutto grazie allo studio degli indici di resistenza dei vasi renali , legati ai cambiamenti che si verificano a livello della vascolarizzazione renale in corso di malattie ( vasculiti , alterazioni endoteliali , edema sub - intimale , ostruzione dei vasi ecc . ) ; tali cambiamenti si traducono generalmente in una riduzione del flusso renale , pi evidente quanto pi grave la malattia [ 19 ]  . nel nostro lavoro abbiamo utilizzato il power doppler per i vantaggi che offre rispetto al color doppler : riduzione del rumore , indipendenza dallangolo di insonazione e dalla velocit di flusso [ 2022 ]  . 
la capacit di dimostrare segnali a basso flusso come quelli della regione corticale renale ha , infatti , aumentato le potenzialit della metodica nellidentificazione daree avascolari ( infarti , pielonefriti acute ecc . ) o fortemente compromesse ( rigetto acuto , cronico , nefrotossicit da ciclosporina ecc . ) ; inoltre la correlazione con la rm ne ha evidenziato lelevata sensibilit nellidentificazione delle aree prive di flusso [ 23 ]  . 
nella nostra casistica tale metodica ha prontamente evidenziato riduzione della vascolarizzazione in quasi tutti i pazienti del gruppo b e c affetti da malattia ( fig . 4 ) , senza evidenziare , tuttavia , significative variazioni tra i due gruppi . lo studio comparativo degli indici di resistenza dei soggetti in assenza di malattia e dei soggetti con malattia ( rigetto acuto , cronico , necrosi tubulare acuta , tossicit da ciclosporina ecc . ) generalmente mette in evidenza la malattia , r . 
la curva intensit / tempo presenta lento wash - in e lento e wash - out . onset under 60 s and time to peak enhancement less than 235 s group c ( patients with acute tubular necrosis ) : enhancement onset under 30 s and time to peak enhancement less than 130 s . 
 biopsy demonstrated the presence of graft disease in all patients in group b ( n = 16 , chronic rejection ) and group c ( n = 3 , acute tubular necrosis )  . 
 on the basis of their greater sensitivity and specificity compared with ri measurements , time - intensity curves could , in our opinion , represent pathognomonic patterns in renal graft diseases and provide important information for the differential diagnosis . 
if confirmed by larger series , these data could open up new perspectives for the sonographic study of the kidney , both transplanted and native . ma non dimostra significative variazioni tra le suddette e quindi non ne permette la diagnosi differenziale [ 24 ]  . 
 curve i / t descrivono la dinamica di enhancement del rene ; infatti nei pazienti con assenza di malattia il mdc giunge rapidamente nel rene trapiantato , raggiungendo altrettanto rapidamente il picco dintensit per poi abbandonare il rene altrettanto rapidamente . 
nei reni con rc ( rigetto cronico ) e nta ( necrosi tubulare acuta ) il mdc raggiunge pi lentamente il picco di massima concentrazione e staziona pi a lungo nel rene . 
il lento raggiungimento della massima concentrazione del mdc e la ritenzione prolungata del mdc risultano quindi essere elementi legati allo stato di malattia del rene trapiantato e attribuibili quindi alla ridotta funzionalit del rene . 
 in base ai risultati ottenuti , maggiore sensibilit e specificit , rispetto alla misurazione degli indici di resistenza , le curve i / t potrebbero rappresentare , a nostro giudizio , dei pattern patognomonici nelle malattie del rene trapiantato e fornire inoltre importanti informazioni per la diagnosi differenziale delle complicanze . 
fortuna amirsys inc , salt lake city , utah ( 2005 ) isbn 1 - 4160 - 2333 - x ; isbn 0 - 8089 - 2324 - 2 ( international english edition ) in the online version of the book review unfortunately g . 
di radiodiagnostica , fondazione irccs policlinico san matteo , piazzale golgi , 19 , i - 27100 pavia radiol med ( 2007 ) 112 : 145146 doi 10.1007 / s11547 - 007 - 0129 - 5 geriatric radiology radiologia geriatrica springer - verlag italia , ( 2006 ) isbn 88 - 470 - 0485 - 3 published online : 22 february 2007 g . 
cammarota the ageing , with declining birth rates and increasing numbers of elderly individuals not only those over 65 but especially those in more advanced age groups has brought about a series of social , economic and healthcare problems . 
 with increasing frequency , doctors , whether specialists or not , are confronted with complex clinical scenarios arising in the same patient , which make management problematic and require special competences . 
over the past few years , advances in knowledge about the biology and physiology of ageing have made it possible to plan medical interventions that have the potential to slow down the functional decline that often accompanies old age and to prevent or delay the onset of disability or dependency that follow a state of disease . 
 with the use of multidimensional evaluation tools , geriatricians are able to analyse medical , physical and socioeconomic needs of elderly and especially frail elderly patients , plan personalised healthcare interventions and monitor their efficacy over time . 
multidimensional evaluation relies on the use of a variety of diagnostic methods , and healthcare professionals working as a team are able to provide the best diagnostic tests and treatment solutions for each patient and fully differentiate between physiology and pathology in the ageing process . in this context , radiology and more generally diagnostic imaging play a valuable role in the care of these patients . 
in elderly and especially geriatric patients , comorbidities , a rapidly changing clinical situation and physical and cognitive problems frequently make it difficult for radiologists to provide clinicians with the answers they expect . 
at this time of continuous technological progress , however , radiology provides not only the easy - to - perform diagnostic tests that are routinely used in the elderly , but also new imaging techniques that , if correctly applied , can be used to diagnose and monitor the evolution of disease states that are often difficult to manage clinically . 
negli ultimi anni il miglioramento delle conoscenze sulla biologia e la fisiologia dellinvecchiamento ha permesso di pianificare interventi potenzialmente in grado di rallentare il declino funzionale che spesso accompagna linvecchiamento e di prevenire o ritardare linsorgenza della disabilit e della dipendenza in seguito ad uno stato morboso . 
il geriatra , attualmente , utilizzando gli strumenti della valutazione multidimensionale , in grado di analizzare i bisogni di ordine medico , fisico , psichico e socio - economico del paziente anziano , in particolare dellanziano fragile , di programmare interventi personalizzati per il paziente e di controllarne nel tempo lefficacia . la valutazione multidimensionale si avvale di differenti metodologie diagnostiche e di numerosi operatori medici e non medici che , lavorando in maniera congiunta , riescono ad offrire le migliori soluzioni diagnostico - terapeutiche per il paziente ed a distinguere pienamente il limite tra il fisiologico e il patologico nel processo di invecchiamento . in questottica la radiologia , ed in genere la diagnostica per immagini , trova una giusta collocazione nella cura del paziente anziano . 
frequentemente in questo paziente , in particolare in quello geriatrico , a causa della comorbilit , di un quadro clinico che si modifica nel breve termine e di problemi di ordine fisico e cognitivo non facile ottenere dalla radiologia le risposte che il clinico si aspetta . 
la radiologia , oltre gli esami diagnostici di facile esecuzione utilizzati di routine anche nei pazienti anziani , in unepoca di continuo progresso tecnologico fornisce frequentemente nuove metodologie di indagine di cui anche il paziente anziano pu usufruire , se correttamente applicate , per la diagnosi e per seguire levoluzione nel tempo di un quadro morboso spesso di difficile gestione clinica . 
in molti casi una buona collaborazione tra geriatra e radiologo consente di scegliere lesame pi specifico per il singolo caso e di discutere insieme in caso di discordanze tra risultati di esami diagnostici e quadro clinico . 
 importante per il radiologo conoscere i quadri clinici pi frequenti presenti nel paziente anziano , spesso diversi da quello adulto , e per il geriatra apprendere le possibilit ed i limiti della moderna radiologia e le applicazioni nei suoi pazienti . 
ben venga dunque un testo di radiologia geriatrica scritto in prevalenza da radiologi , ma anche da alcuni geriatri nella parte introduttiva , dove book review applications in geriatric patients . 
geriatric radiology is therefore a much - need text , written for the most part by radiologists but also by some geriatricians who discuss the problems related to ageing in the introductory section . the book presents the imaging patterns of the most important body systems that undergo changes as a result of both physiological ageing processes and pathological processes that arise from or are complicated by ageing . 
besides offering in - depth descriptions with short sections on radiological anatomy and the clinical indications for the single examinations , the text is illustrated by images that make the process of learning and keeping up to date both easy and pleasant . geriatric radiology is an excellent reference book for geriatricians , radiologists , internists and for all those involved in treating the clinical problems of elderly individuals . vengono presi in esame i problemi legati allinvecchiamento . nel volume sono riportati i quadri radiologici e pi in generale di diagnostica per immagini dei pi importanti apparati che subiscono modificazioni sia per processi fisiologici di invecchiamento che a causa di patologie insorgenti o complicate in seguito allinvecchiamento . 
casella 7 , i - 90145 palermo , italy , tel . : + 39 - 091 - 6552330 , fax : + 39 - 091 - 6552337 , e - mail : sparacia@unipa.it received : 21 november 2005 / accepted : 30 march 2006 / published online : 22 february 2007 abstract purpose . 
images were continuously acquired at the basal ganglia over 40 s during injection of 90 ml of iodinated contrast medium injected at a rate of 9 ml / s with a 9 - s delay . 
z - axis coverage was 20 m all patients underwent diffusion - weighted magnetic resonance imaging ( dwi ) within 12 h of perfusion ct to define the extent of the infarct . 
perfusion ct data were analysed in regions of interests ( rois ) on regional cerebral blood volume ( rcbv ) , regional cerebral blood flow ( rcbf ) and mean transit time ( mtt ) maps placed in various parts of the perfusiondeficient territory and in the contralateral hemisphere . 
normal ct findings with abnormal ct perfusion parameters were seen in the region of infarction and in the viable tissue ( penumbra ) within a 1.5 - cm distance from the infarct margin as outlined on dwi images . 
le immagini sono state acquisite in modo continuo per una durata di 40 s attraverso la regione dei nuclei della base durante infusione di 90 ml di mdc non ionico ad una velocit di 9 ml / s con un ritardo di 9 s . 
lestensione lungo lasse z delle scansioni stata di 20 mtutti i pazienti sono stati sottoposti ad esame rm con sequenze pesate in diffusione ( dwi ) entro 12 ore dallesame tc di perfusione per delineare lestensione della lesione infartuale . 
i dati di perfusione cerebrale del flusso ematico cerebrale regionale ( rcbf ) , volume ematico cerebrale regionale ( rcbv ) e del tempo medio di transito ( mtt ) sono stati misurati con regioni di interesse ( roi ) posizionate in diverse aree di deficit perfusionale e nelle aree indenni corrispondenti dellemisfero cerebrale controlaterale . 
in tutti i pazienti lesame tc di base non dimostrava anomalie di densit da riferire a lesioni ischemiche in fase acuta mentre sono state rilevate anomalie dei parametri di perfusione cerebrale sia nelle regioni infartuate delineate nelle immagini dwi che nelle aree limitrofe ipoperfuse poste entro 1 , 5 cm di distanza dal margine del core lesionale ( dwi positiva )  . 
il prolungamento dellmtt stata lanomalia di perfusione pi frequentemente riscontrata nel tessuto ipoperfuso e nel tessuto infartuato con differenze significative ( p < 0 , 0001 ) rispetto i valori di mtt del tessuto sano . 
la media per i valori di mtt stata di 9 , 8 s nel core lesionale , 5 , 1 s nel tessuto ipoperfuso ( esterno al core lesionale ma entro 1 , 5 cm ) e 3 , 4 s nelle corrispondenti aree g . 
lanalisi delle curve roc ha dimostrato che il prolungamento dellmtt per valori superiori a 6 , 05 s consente di distinguere il tessuto infartuato con un valore predittivo positivo del 100% ( sensibilit = 84 , 6% ; specificit = 100% ; accuratezza = 92 , 3% ; p < 0 , 0001 )  . 
in selected patients , thrombolytic therapy , the intravenous administration of recombinant tissue - type plasminogen activator ( rtpa ) within 36 h of onset of neurological symptoms , is a valid treatment option to prevent disease progression and reduce the extent of disability [ 15 ]  . 
the rationale for this treatment is to restore cerebral perfusion through recanalisation in an attempt to salvage tissue in ischaemic penumbra , in other words , cerebral tissue in which perfusion values have not fallen below the viability threshold and therefore are not infarcted [ 3 ]  . 
as a consequence , discrimination of viable and ischaemic tissue using quantitative cerebral perfusion parameters measured by diagnostic imaging techniques is fundamental in selecting patients for thrombolytic therapy [ 3 , 4 , 610 ]  . 
 the cerebral perfusion threshold that defines irreversible tissue damage has been determined by positron emission tomography ( pet ) , which also provided a better understanding of the pathophysiology of brain ischaemia [ 7 , 8 ]  . 
cerebral perfusion has been assessed using nuclear medicine techniques , such as xenon - computed tomography ( ct ) , pet and single - photon emission tomography ( spect ) and diagnostic imaging methods , such as magnetic resonance imaging ( mri )  . 
however , the routine use of these techniques is hindered by the limited availability of equipment , high costs and difficulty performing the exams in uncooperative patients with acute cerebral ischaemia [ 6 , 7 , 10 ]  . in recent years , development of ct and especially multislice ct scanners has made available a study technique that nei paesi occidentali lischemia cerebrale rappresenta la terza causa di morte e la principale causa di disabilit nella popolazione adulta . 
la terapia trombolitica , basata sulla somministrazione endovena dellattivatore tissutale del plasminogeno ( recombinant tissue - type plasminogen activator : rtpa ) rappresenta , in pazienti selezionati , una opzione terapeutica valida entro le prime 36 ore dallinsorgenza dei sintomi neurologici per prevenire la progressione della malattia e ridurre il grado di disabilit [ 15 ]  . 
il ripristino dei valori di perfusione cerebrale conseguente alla ricanalizzazione rappresenta il razionale di questa terapia nel tentativo di salvare i tessuti in penombra ischemica , ovvero il tessuto cerebrale in cui i valori di perfusione cerebrale non sono scesi al disotto della soglia vitale e quindi in cui non si prodotta una lesione infartuale [ 3 ]  . 
conseguentemente , la possibilit di discriminare il tessuto cerebrale vitale da quello ischemico utilizzando parametri quantitativi di perfusione cerebrale misurati con tecniche di diagnostica per immagini fondamentale per selezionare i pazienti da sottoporre a terapia trombolitica [ 3 , 4 , 610 ]  . la soglia di perfusione cerebrale che definisce il danno tissutale irreversibile stata definita mediante studi di tomografia ad emissione di positroni ( pet ) che hanno consentito anche una migliore comprensione della fisiopatologia dellischemia cerebrale [ 7 , 8 ]  . 
tuttavia , per la sua limitata disponibilit e gli alti costi , la valutazione mediante pet non praticabile nei pazienti con ischemia cerebrale acuta , pertanto necessario identificare una tecnica di rapida esecuzione , ampiamente disponibile , che consenta di definire e quantificare il deficit di perfusione cerebrale . 
per la valutazione della perfusione cerebrale sono state utilizzate metodiche di medicina nucleare quali la xenon - tc , la pet e la tomografia ad emissione di fotone singolo ( spect ) , e metodiche di diagnostica per immagini , segnatamente la risonanza magnetica ( rm )  . 
perfusion ct , which is done during the infusion of a bolus of iodinated contrast medium , is particularly indicated in patients with acute ischaemia , as it is fast to perform and offers the possibility of ruling out haemorrhagic complications on the preliminary precontrast ct scan [ 10 , 11 ]  . 
time - concentration curves can be traced for a given region of interest ( roi ) , which reflect perfusion in that region , and the different cerebral perfusion parameters can be derived from their analysis . 
a mathematical operation , known as deconvolution of the arterial and tissue enhancement curves , allows calculation of mean transit time ( mtt ) of the contrast bolus from the arteries to the veins of the cerebral circulation . 
regional cerebral blood volume ( rcbv ) is calculated as the area under the curve ( auc ) of a parenchymal pixel divided by the auc of an arterial pixel . 
the theory underlying this technique is the central volume principle , which relates cbf , cbv and mtt in the following equation : cbf = cbv / mtt the aim of cerebral perfusion ct is therefore to identify tissue in ischaemic penumbra that is , viable tissue with reduced perfusion that can be salvaged with thrombolytic therapy initiated within the first 36 h of onset of neurological symptoms [ 1113 ]  . 
the reliability and reproducibility of perfusion parameter measurements provided by ct have been validated in the literature [ 14 , 15 ] , but controversies exist as to which parameter is best for discriminating between viable and infarcted tissue [ 16 ]  . 
 the aim of this study was to determine the haemodynamic parameters detected with perfusion ct in the infarct core and its periphery . materials and methods thirteen patients ( five men and eight women ; age range 4490 years , mean age 63.3 years ) with acute nonhaemorrhagic cerebral ischaemia in the territories of the middle ( n = 11 ) or anterior cerebral artery ( n = 2 ) were studied within 3 h of onset of neurological symptoms . 
cerebral perfusion images were obtained with a 4 - detector - row multislice spiral unit ( volume zoom ; siemens , erlangen , germany ) with scans through the basal ganglia perpendicularly to the occipital segment of the superior sagittal sinus , below the confluence of the sinuses , to visualise the lutilizzo nella pratica clinica di queste metodiche limitata dalla disponibilit delle apparecchiature , degli alti costi e dalla difficolt di eseguire lesame in pazienti non collaboranti affetti da ischemica cerebrale acuta [ 6 , 7 , 10 ]  . negli ultimi anni , grazie allevoluzione delle apparecchiature di tomografia computerizzata ( tc ) , in particolare con lintroduzione dei tomografi multistrato , stata sviluppata una tecnica di studio che consente la valutazione dei parametri di perfusione cerebrale mediante tc . 
lo studio tc di perfusione cerebrale , che si esegue durante infusione a bolo di mezzo di contrasto ( mdc ) iodato , risulta particolarmente indicato in pazienti con ischemia cerebrale acuta per la rapidit di esecuzione dellesame e per la possibilit di escludere le complicanze emorragiche allesame tc senza mdc che si esegue preliminarmente [ 10 , 11 ]  . 
la tc perfusionale una tecnica relativamente nuova che consente di rilevare le modificazioni di concentrazione del mdc iodato durante il transito del bolo attraverso le arterie cerebrali . possono quindi essere tracciate curve tempo - concentrazione per una data regione di interesse che riflettono la perfusione in quella data regione . 
attraverso un procedimento matematico noto come deconvoluzione delle curve di enhancement arterioso e tissutale si calcola il tempo medio di transito ( mtt ) del bolo di mdc nel passaggio dai vasi arteriosi del circolo cerebrale a quelli venosi . 
la teoria alla base di questa tecnica il principio del volume ematico centrale che mette in relazione il flusso ematico cerebrale ( cbf ) con il volume ematico cerebrale ( cbv ) e con il tempo medio di transito ( mtt ) nella seguente equazione : cbf = cbv / mtt lobiettivo dello studio tc di perfusione cerebrale quindi di identificare il tessuto in penombra ischemica , ovvero il tessuto vitale ma con ridotta perfusione , e dunque salvabile con terapia trombolitica attuata tempestivamente , entro le prime 36 ore dalla comparsa dei sintomi neurologici [ 1113 ]  . 
lattendibilit e la riproducibilit dei risultati relativi alla misurazione di parametri di perfusione cerebrale con tc perfusionale trova riscontro in letteratura [ 14 , 15 ] , tuttavia esistono controversie circa il parametro perfusionale da utilizzare per discriminare il tessuto vitale dal tessuto infartuato [ 16 ]  . scopo del presente lavoro di determinare i parametri emodinamici rilevati con tc perfusionale nel contesto del core lesionale ed alla sua periferia . materiali e metodi sono stati studiati 13 pazienti ( 5 maschi e 8 femmine di et compresa tra 44 e 90 anni , et media 63 , 3 anni ) affetti da ischemia cerebrale in fase acuta nel territorio di distribuzione dellarteria cerebrale media ( n = 11 ) o dellarteria cerebrale anteriore ( n = 2 ) , non complicata da emorragia , entro 3 ore dallinsorg . 
1a axial magnetic resonance ( mr ) diffusion weighted image ( dwi ) , and b calculation of perfusion computed tomography ( ct ) maps for regional cerebral blood flow ( rcbf ) , regional blood volume ( rcbv ) and mean transit time ( mtt ) in a patient with left thalamic stroke . 
1a immagine rm assiale pesata in diffusione ( dwi ) e b calcolo delle mappe di perfusione tc per i parametri rcbv , rcbf e mtt in un paziente affetto da ischemia cerebrale acuta in sede talamica sinistra . 
le immagini dwi sono state utilizzate per delineare le dimensioni finali dellarea infartuale ( core lesionale ) e tracciare sulle corrispondenti mappe di perfusione una roi entro una distanza di 1 , 5 cm dal margine del core lesionale ( per misurare i valori di perfusione nellarea peri - infartuale )  . three supratentorial vascular territories in a single scan . 
z - axis scan coverage was 20 m a 90 - ml bolus of nonionic contrast medium ( 320 mg / ml iodine concentration ) was injected into an antecubital vein at a rate of 9 ml / s via an automatic power injector ( envision ; medrad , pittsburgh , pa , usa ) and an 18 - gauge catheter . 
after a 9 - s delay from bolus administration , scans were acquired continuously for 40 s with the following parameters : 80 kv , 200 mas , 10 - mm thickness , 1 - s rotation time , 512512 matrix . 
in tutti i casi stato richiesto il consenso informato prima dellesecuzione dellesame o al paziente o al parente pi vicino in accordo con le linee guida del nostro ospedale . preliminarmente stato eseguito un esame tc dellencefalo senza mdc per escludere la presenza di emorragia cerebrale o di ischemia in fossa cranica posteriore . 
le immagini di perfusione cerebrale sono state ottenute con tomografo spirale multistrato a 4 linee di detettori ( volume zoom ; siemens , erlangen , germania ) attraverso la regione dei nuclei della base con angolazione perpendicolare al segmento occipitale del seno sagittale superiore , al disotto della confluenza dei seni , in modo da visualizzare in ununica scansione i tre territori vascolari sovratentoriali . 
lestensione delle scansioni lungo lasse z stata di 20 msono stati iniettati in una vena antecubitale 90 ml di mdc non ionico ( concentrazione di iodio pari a 320 mg / ml ) ad una velocit di 9 ml / s con catetere da 18 gauge utilizzando un iniettore aug . 
le scansioni sono state acquisite con un ritardo di 9 s dal bolo di mdc in modo continuo per una durata di 40 s con i seguenti parametri : 80 kv , 200 mas , sezioni di 10 mm , tempo di rotazione 1 s , matrice 512512 . 
le mappe di perfusione rcbf , rcbv e mtt sono state calcolate utilizzando una workstation dedicata ( vitrea ; vital images , minnetonka , usa )  . entro 12 ore dallesame tc di perfusione stato effettuato un esame rm di controllo con sequenze pesate in diffusione ( dwi ) per confermare la presenza di lesioni infartuali . 
in tabella 1 sono riportati i valori medi dei tre paramentri di perfusione considerati ( mtt , rcbf , rcbv ) nelle tre regioni di interesse ( core lesionale , tessuto ipoperfuso , regioni corrispondenti controlaterali )  . 
lunico parametro che ha mostrato una variazione significativa ( livello di significativit accettato per questo studio : p < 0 , 0001 ) stato lmtt con un valore medio di 5 , 1 s nelle aree ipoperfuse e di 9 , 8 s nel tessuto infartuato rispetto al tessuto sano che presentava valori medi di 3 , 41 s . lanalisi mediante curve roc ha consentito di individuare un valore cut - off di 6 , 05 s per lmtt come valore discriminante tra tessuto ipoperfuso ed il tessuto infartuato cos g . 
2a axial magnetic resonance ( mr ) diffusion weighted image ( dwi ) , and b perfusion computed tomography ( ct ) maps for regional cerebral blood flow ( rcbf ) , regional blood volume ( rcbv ) and mean transit time ( mtt ) in a patients with left peripheral middle cerebral artery ( mca ) stroke . 
2a immagine rm assiale pesata in diffusione ( dwi ) e b mappe tc di perfusione per i parametri rcbf , rcbv e mtt in un paziente affetto da ischemia cerebrale acuta nel territorio di distribuzione superficiale dellarteria cerebrale media si sinistra . 
ampia area di alterazione dellmtt estesa oltre 1 , 5 cm rispetto il core lesionale identificato nellimmagine dwi . the ability to discriminate infarcted and salvageable areas . this has become increasingly relevant with the development of recanalising treatment with thrombolytic agents . 
 ct during acute cerebral ischaemia may inconstantly demonstrate indirect signs , such as artery hyperattenuation due to thrombosis and a mass effect with effacement of the subarachnoid spaces due to oedema [ 1923 ]  . 
evaluation of possible discrepancies between lesion extent , as delineated by dwi , and the extent of the hypoperfused area enables definition of tissue in ischaemic penumbra , which is the target of recanalisation therapy [ 16 ]  . 
3a axial magnetic resonance ( mr ) diffusion weighted image ( dwi ) , and b perfusion computed tomography ( ct ) maps for regional cerebral blood flow ( rcbf ) , regional cerebral blood volume ( rcbv ) and mean transit time ( mtt ) in a patient with left middle cerebral artery ( mca ) stroke . 
3a immagine rm assiale pesata in diffusione ( dwi ) e b mappe tc di perfusione per i parametri rcbf , rcbv e mtt in un paziente affetto da ischemia cerebrale acuta nel territorio di distribuzione dellarteria cerebrale media si sinistra . 
ampia area di alterazione dellmtt estesa oltre 1 , 5 cm rispetto il core lesionale identificato nellimmagine dwi . the constant evolution of multidetector ct ( mdct ) scanners in recent years has led to the development of techniques for the study of cerebral perfusion , with very similar results to those obtained with perfusion mri . 
in particular , it has been demonstrated that the parameters determined by perfusion ct are reliable for defining hypoperfused cerebral tissue [ 14 , 15 ]  . to better appreciate the results of our perfusion ct study , we need to make reference to previous reports [ 24 , 25 ] that have demonstrated a significant correlation between perfusion abnormalities measured on the basis of cbf ( on both ct and mri ) and abnormalities revealed by dwi . 
this is important , as it demonstrates that a reduction in cbf greater than 66% corresponds to the infarcted tissue identified on dwi [ 26 ] , even in the early phase [ 24 , 25 ]  . 
assuming that areas with a cbf reduction > 66% are the infarcted areas [ 26 ] and that these correspond to the final infarct , as identified by diffusion abnormalities , we can hypothesise that the salvageable tissue ( ischaemic penumbra ) is situated within the difference between the area with altered mtt and the area with altered cbf ( < 66% ) , or the mismatch bete affetto da ischemia cerebrale [ 710 , 17 ]  . 
in questo settore , la ricerca stata focalizzata oltre che alla possibilit di delineare in fase precoce il focolaio ischemico , anche alla capacit di discriminare larea infartuata dalle aree salvabili . 
obiettivo della terapia ricanalizzante il ripristino dei valori di perfusione cerebrale , ovvero salvare , se presenti , i tessuti in penombra ischemica sfruttando lesigua finestra terapeutica [ 18 ]  . 
questa terapia non esente da rischi , in particolare di emorragia cerebrale , e conseguentemente necessaria una attenta selezione dei pazienti [ 35 , 18 ]  . lesame tc in corso di ischemia cerebrale acuta , pu mettere in evidenza , in maniera incostante , segni indiretti quali liperdensit delle arterie in relazione a trombosi e leffetto massa con la cancellazione degli spazi subaracnoidei per la presenza delledema [ 1923 ]  . 
tuttavia , soltanto con lesame rm con sequenze pesate in diffusione possibile individuare il focolaio ischemico in fase iperacuta sin dai primi minuti dallinsorgenza della sintomatologia clinica [ 6 , g . 
areas of perilesional oligaemia may be included within the perfusion abnormality identified by mtt , and this sensitivity in part accounts for the results of this and other studies in which mtt proved to be the most sensitive parameter for identifying penumbra . 
despite these possible limitations , it should be considered that studies correlating perfusion maps and final injury and applying the alberta stroke program early ct score ( aspects ) to perfusion maps obtained the best correlations with final injury in nonreperfused patients with analysis of cbf and mtt maps [ 27 ]  . 
 it can therefore be stated that mtt is a highly sensitive parameter for the definition of infarcted or salvageable tissue , whereas cbf has greater specificity [ 28 , 29 ]  . 
in bearing with previous reports [ 28 ] , interpretation of mtt maps in our study proved to be straightforward , as the healthy areas were homogeneously normal , and the areas with perfusion alterations were readily recognised by their inhomogeneous colour distribution . 
it should be noted , however , that progression from ischaemia to infarction depends not only on cerebral perfusion but also on duration of blood flow reduc15 , 17 ]  . 
anche con lesame rm di diffusione non tuttavia possibile discriminare il tessuto vitale , ipoperfuso , dalla lesione infartuale ed necessario ricorrere a tecniche di studio di perfusione cerebrale con rm [ 17 ]  . 
la valutazione di eventuali discrepanze tra lentit della lesione delineata in diffusione e lentit dellarea ipoperfusa consente poi di definire il tessuto in penombra ischemica , che costituisce lobiettivo della terapia ricanalizzante [ 16 ]  . negli ultimi anni , con la continua evoluzione degli apparecchi tc spirale multidetettore , stata perfezionata la tecnica di studio di perfusione cerebrale con risultati sovrapponibili a quelli ottenuti con rm di perfusione . 
 per comprendere i risultati ottenuti con lesame tc perfusionale bisogna fare riferimento ai risultati riportati in letteratura [ 24 , 25 ] che dimostrano come esista una correlazione significativa tra le anomalie di perfusione misurate attraverso il cbf ( sia con tc che con rm ) e le anomalie rilevate nelle immagini dwi . 
questo dato rilevante perch dimostra che una riduzione maggiore del 66% del cbf corrisponde al tessuto infartuato cos come identificato nelle immagni dwi [ 26 ] anche in fase precoce [ 24 , 25 ]  . 
assumendo che le aree con riduzione del cbf > 66% siano le aree infartuate [ 26 ] e che queste corrispondano allinfarto finale definito dalle anomalie di diffusione , possiamo ipotizzare che il tessuto salvabile ( in penombra ischemica ) sia compreso nella differenza tra larea con alterato mtt e larea con alterato cbf ( < 66% ) ovvero dalla discrepanza tra mtt e dwi [ 26 ]  . 
nellambito dellalterazione perfusionale identificata dallmtt possibile includere aree di oligoemia perilesionale e tale sensibilit spiega in parte i risultati ottenuti in questo ed in altri studi in cui lmtt si rivelato il parametro pi sensibile nellidentificazione del tessuto in penombra . pur con queste possibili limitazioni bisogna comunque considerare che negli studi di correlazione tra mappe di perfusione e danno finale , utilizzando gli alberta stroke program early ct score ( aspects ) per le mappe di perfusione , le migliori correlazioni con il danno finale nei pazienti senza riperfusione si ottengono mediante lanalisi delle mappe di cbf e mtt [ 27 ]  . si pu pertanto affermare che per la definizione del tessuto infartuato o salvabile lmtt un parametro altamente sensibile mentre il cbf invece dotato di maggior specificit [ 28 , 29 ]  . 
nella nostra esperienza le mappe mtt sono risultate di facile interpretazione , come peraltro riportato in letteratura [ 28 ] , in quanto le aree sane sono omogeneamente normali e le aree con alterata perfusione risultano facilmente identificabili dalla disomogenea distribuzione del colore . 
in particolare , le alterazioni di perfusione cerebrale sono state espresse da un marcato incremento dellmtt . utilizzando questo parametro stato possibile definire un valore soglia che discriminasse tra tessuto ischemico e non . lanalisi delle curve roc dimostra infatti che per valori di mtt superiori o uguali a 6 , 05 s possibile identificare il tessuto infartuato mentre con valori medi di mtt di 5 , 1 s , e quindi inferiori a 6 , 05 s , viene identificato il tessuto in penombra ( p < 0 , 0001 )  . 
4 receiver operating characteristic ( roc ) curves for perfusion computed tomography ( ct ) parameters [ regional cerebral blood volume ( rcbv ) , regional cerebral blood flow ( rcbf ) and mean transit time ( mtt ) ]  . 
4 curve roc ( receiver operator characteristic ) dei parametri di perfusione cerebrale rcbv , rcbf e mtt per identificare il valore cut - off tra infarto e tessuto sano . 
il prolungamento dellmtt per valori medi di 5 , 1 s identifica il tessuto in penombra ischemica mentre per valori uguali o superiori a 6 , 05 s identifica il tessuto infartuato con una sensibilit dell84 , 6% , specificit del 100% , valore predittivo positivo del 100% , valore predittivo negativo del 86 , 7% e accuratezza del 92 , 3% ( p < 0 , 0001 )  . 
asse x : 1specificit ( falsi positivi ) ; asse y : sensibilit ( veri positivi ) ; az , area sotto la curva . tion and the greater vulnerability of specific neuronal populations [ 30 , 31 ]  . 
 zione del flusso ematico e dalla maggiore vulnerabilit di specifiche popolazioni neuronali [ 30 , 31 ]  . conclusions conclusioni perfusion ct with latest - generation mdct scanners is a rapid technique that can be used to complete unenhanced ct , with only slightly longer examination times . 
 the disadvantages of this study protocol are increased exposure to ionising radiation and use of iodinated contrast material , which requires assessment of renal function and possible predisposition to allergies . 
in addition , cerebral perfusion ct allows limited coverage of the cerebral parenchyma , although this is destined to increase with increased detector numbers . la tc perfusionale una tecnica di studio di rapida esecuzione utilizzando tomografi multidetettore di ultima generazione e pu essere eseguita a completamento dellesame tc di base con un modesto prolungamento del tempo desame . ci particolarmente utile considerando la necessit di sfruttare la ristretta finestra terapeutica per il trattamento trombolitico . 
questa tecnica di studio quindi complementare allo studio tc di base e fornisce informazioni diagnostiche aggiuntive di grande rilievo clinico . gli svantaggi di questo protocollo desame sono correlati allaumento della dose di radiazioni ionizzanti al paziente e allutilizzo del mezzo di contrasto iodato che richiede la valutazione dei parametri di funzionalit renale e delleventuale diatesi allergica . 
eastwood jd , lev mh , azhari t et al ( 2002 ) ct perfusion imaging with deconvolution analysis : a pilot study of patients with acute middle cerebral artery stroke . 
wintermark m , fischbein nj , smith ws et al ( 2005 ) accuracy of dynamic perfusion ct with deconvolution in detecting acute hemispheric stroke . ajnr am j neuroradiol 26 : 104112 15 . 
ogasawara k , sasaki m , tomitsuka n et al ( 2005 ) early revascularization in a patient with perfusion computed tomography / diffusion - weighted magnetic resonance imaging mismatch secondary to acute vertebral artery occlusion . 
schellinger pd , chalela ja , kang dw et al ( 2005 ) diagnostic and prognostic value of early mr imaging vessel signs in hyperacute stroke patients imaged < 3 hours and treated with recombinant tissue plasminogen activator . 
eastwood jd , lev mh , wintermark m et al ( 2003 ) correlation of early dynamic ct perfusion imaging with whole - brain mr diffusion and perfusion imaging in acute hemispheric stroke . ajnr am j neuroradiol 24 : 18691875 25 . 
wintermark m , reichhart m , cuisenaire o et al ( 2002 ) comparison of admission perfusion computed tomography and qualitative diffusionand perfusionweighted magnetic resonance imaging in acute stroke patients . 
schaefer pw , roccatagliata l , ledezma c et al ( 2006 ) first - pass quantitative ct perfusion identifies thresholds for salvageable penumbra in acute stroke patients treated with intra - arterial therapy . 
grandin cb , duprez tp , smith am et al ( 2002 ) which mr - derived perfusion parameters are the best predictors of infarct growth in hyperacute stroke ? comparative study between relative and quantitative measurements . 
powers wj , grubb rl , darriet d , raichle me ( 1985 ) cerebral blood flow and cerebral metabolic rate of oxygen requirements for cerebral function and viability in humans . 
 correspondence to : giuseppe caruso , via liguria , 21 , i - 90144 palermo , italy , tel . : + 39 - 91 - 6552315 , fax : 39 - 91 - 6552337 , e - mail : carusogi@libero.it received : 23 march 2006 / accepted : 18 august 2006 / published online : 22 february 2007 abstract purpose . 
a total of 1 , 729 ( 1 , 012 women and 717 men ; age range 2882 ; mean age 51 ) patients underwent contrastenhanced sonography of the liver . 
the examination was performed with a low mechanical index ( mi < 0.09 ) after injection of sulphur - hexafluoride - filled microbubbles , using different sonographic equipment and different contrast - specific algorithms . 
heterogeneous delayed liver enhancement was observed in six patients in the late phase ( 180 s ) , with the presence of multiple , partially confluent , hyperechoic areas peripheral to the portal vessels . 
the pattern appeared spontaneously between 1 and 4 h after the examination and was associated with the presence of an anomalous echogenicity in the superior mesenteric veno patient experienced adverse reactions . 
1729 pazienti ( 1012 donne e 717 uomini ; et compresa tra 2882 , media 51 ) sono stati sottoposti ad esame ecografico del fegato dopo somministrazione di ecoamplificatore esafluoruro di zolfo e basso indice meccanico ( mi < 0 , 09 )  . 
limpregnazione anomala del parenchima epatico potrebbe essere secondaria allintrappolamento nei sinusoidi epatici di gas proveniente dal microcircolo intestinale attraverso il sistema entero - portale . key words ultrasound microbubbles liver parole chiave ecografia microbolle fegato introduction introduzione sonographic contrast agents consist of microbubbles containing an inert gas or air with a diameter < 5 m surrounded by a shell or a membrane that provides transpulmonary stability and provides transpulmonary stability and increasing their persistence in the circulation . 
the total volume of mii mezzi di contrasto utilizzati in ecografia sono costituiti da microbolle ( diametro < 5 m ) contenenti gas inerte o aria , e stabilizzate da membrane a composizione differente , che ne garantiscono la stabilit attraverso il circolo transpolmonare aumentandone la persistenza in circolo . 
the second relies on detection of ultrasound ( us ) - induced physical phenomena , such as microbubble destruction , fusion or splitting [ 4 ]  . the purpose of this paper is to report a pattern of heterogeneous late - phase hepatic enhancement with anomalous duration and distribution associated with stratified hypoor hyper - echogenicity in the superior mesenteric vein , which was observed after infusion of sonovue contrast mediuan aetiological hypothesis is also presented . materials and methods from december 2001 to august 2004 , 1 , 729 patients ( 1 , 012 women and 717 men ; age range 2882 years ; mean age 51 ) underwent contrast - enhanced sonography of the liver after injection of sonovue contrast medium ( bracco , milan , italy )  . sonovue is a suspension of hexafluoride sulphur microbubbles ( mean diameter 3 m ) with a phospholipidic shell . 
the elective technique consisted of a bolus injection of 4.8 ml of contrast medium at a rate of approximately 1 ml / s , followed by a 5ml saline flush . 
sebbene si tratti essenzialmente di sostanze blood - pool , il loro comportamento nelle diverse fasi post - contrastografiche sovrapponibile a quello dei mezzi di contrasto organo - iodati , con la differenza che non si ha una vera fase interstiziale . lintroduzione del mezzo di contrasto nel circolo periferico permette di identificare tre diverse fasi della perfusione vascolare epatica , analogamente a quanto possibile ottenere allesame tc dinamico del fegato dopo somministrazione di mezzo di contrasto organo - iodato : fase arteriosa , a 2030 s : in questa fase lenhancement dovuto alla presenza del mezzo di contrasto nelle arterie di piccolo calibro ; fase portale e tardiva , a 60 e 180 s , rispettivamente : nonostante il mezzo di contrasto ecoamplificatore non passi nellinterstizio ma rimanga nei vasi , lenhancement osservato in fase portale e specialmente in fase tardiva , sembra essere dovuto alla persistenza delle microbolle nei sinusoidi o al loro up - take da parte delle cellule del kupffer e delle cellule del sistema reticolo - endoteliale [ 2 , 3 ] ; ipotesi comunque queste che devono essere avvalorate da ulteriori studi di farmacocinetica . le tecniche di imaging ecografico contrasto - specifiche sono due : la prima quella dellimaging non - distruttivo in cui limmagine viene costruita sfruttando la risposta non lineare delle microbolle , senza che queste vengano distrutte ; la seconda tecnica utilizza limaging distruttivo , che determina la distruzione , la fusione e lo splitting delle microbolle [ 4 ]  . scopo del nostro lavoro quello di riportare un fenomeno caratterizzato dalleterogeneo enhancement del parenchima epatico in fase tardiva , anomalo per durata e distribuzione , associato ad un pattern stratificato ipo - iper - ecogeno della vena mesenterica superiore dopo somministrazione di sonovue . 
sulla scorta di una serie di osservazioni abbiamo avanzato una ipotesi eziologica . materiali e metodi dal dicembre 2001 allagosto 2004 , 1729 pazienti ( 1012 donne - 717 uomini , di et compresa tra 28 e 82 anni ; et media 51 anni ) , hanno eseguito un esame ecografico del fegato con ecoamplificatore . 
il sonovue una sospensione di microbolle di esafluoruro di zolfo ( diametro medio 3 , 3 m ) , stabilizzate da una membrana fosfolipidica ; si presenta come una polvere liofilizzata in atmosfera di solfuro esafluoride posta allinterno di un flaconcino di plastica . 
una volta ricostituita , la sospensione appare come soluzione lattescente e rimane stabile per al massimo 6 ore [ 5 ]  . liniezione del mezzo di contrasto stata effettuata 510 minuti dopo la ricostituzione dello stesso . 
la tecnica consiste nelliniezione a bolo di 4 , 8 ml di mezzo di contrasto a velocit di 1 ml / s , seguita dalla infusione di 5 ml di soluzione g . 
use of these algorithms allowed real - time imaging of all vascular phases ( arterial , portal and late ) and was dependent on the availability of the us device at the time of examination ( table 1 )  . 
all examinations were performed as follows : fundamental ( b - mode ) imaging and contrast - specific algorithm without contrast agent contrast - enhanced us in the arterial phase ( 2030 s ) contrast - enhanced us in the portal and late phase ( 60 and 180 s ) contrast - enhanced us in the second late phase ( 240 s )  . patients who showed heterogeneous late - phase hepatic enhancement were examined using the b - mode technique and stimulated acoustic emission ( sae ) , both performed with a high mi ( 1.2 ) ; insonation with high mi is useful for microbubble distribution , leading to possible rapid disappearance of the heterogeneous enhancement pattern . two of the six patients underwent a second contrast - enhanced us after 24 h and 9 days , respectively , using the same us system and the same contrast agent at the same dose . 
tutti gli esami sono stati eseguiti con le stesse modalit come riportato di seguito : imaging fondamentale ( b - mode ) e algoritmo contrastospecifico senza mezzo di contrasto ; ecografia con mezzo di contrasto : fase arteriosa a 2030 s ; ecografia con mezzo di contrasto : fase portale e tardiva a 60 e 180 s rispettivamente ; ecografia con mezzo di contrasto : seconda fase tardiva a 240 s . i pazienti nei quali stato osservato , in fase tardiva , leterogeneo enhancement epatico sono stati sottoposti a esame ecografico con tecnica b - mode in e con stimulated acustic emission ( sae ) entrambe ad elevato indice meccanico ( mi = 1 , 2 ) , allo scopo di determinare la distruzione delle microbolle di mezzo di contrasto con leventuale rapida scomparsa delleterogeneo enhancement . due dei sei pazienti hanno eseguito un secondo esame ecocontrastografico a 24 ore e a 9 giorni rispettivamente , utilizzando la stessa apparecchiatura ecografica , lo stesso mezzo di contrasto , nella stessa dose . 
nei due pazienti in cui stata somministrata per la seconda volta lo stesso mezzo di contrasto nella stessa dose ( a distanza rispettivamente di 24 ore e 9 giorni ) il fenomeno non si ripresentato . 
nessuno dei sei pazienti ha presentato sintomi , n sono state osservate alterazioni della pressione arteriosa . discussione leterogeneo enhancement epatico stato descritto per la prima volta da okada [ 6 ] : egli ha osservato un fenomeno simile in sei pazienti dopo somministrazione di levovist ( 5 casi ) e di echogen ( 1 caso )  . 
1 immagine ottenuta dopo somministrazione di ecoamplificatore con coherent contrast imaging , fase tardiva ( 4 minuti ) : presenza di multiple aree iperecogene parzialmente confluenti nel contesto del parenchima epatico perifiericamente ai vasi portali ( frecce )  . 
 observation of these findings was possible thanks to the use of second - generation sonographic contrast agents , which allow real - time examination of hepatic vascular perfusion : this suggests that gas is present in the enteroportal circulation but not in the other vessels , which could account for the origin of the hepatic phenomenon . 
4 fundamental ( b - mode ) imaging , late phase ( 40 min ) : marked hyperechogenicity ( 1 ) can be seen in the superior mesenteric vein ( between the two arrows )  . 
4 immagine in basale ( b - mode ) ; fase tardiva a 40 minuti : possibile dimostrare una marcata iperecogenicit ( 1 ) nel lume della vena mesenterica superiore ( compresa tra le due frecce )  . 
liperecogenicit appare localizzata in sede ventrale mentre lipoecogenicit in sede declive come fase gassosa in fase fluida ( 2 )  . discussion heterogeneous hepatic enhancement was first described by okada [ 6 ] , who reported a similar phenomenon occurring in six patients after injection of levovist ( five cases ) and echogen ( one case )  . 
to the best of our knowledge , heterogeneous hepatic enhancement has never been observed following administration of sonovue contrast mediuunlike the agents employed by okada , sonovue relies on a low mi to provide real - time imaging of hepatic perfusion [ 7 ]  . 
il fenomeno si verificato con mezzi di contrasto diversi ( per tipo di gas e natura della membrana delle microbolle ) e con tecniche di insonazione diverse : una ad alto indice meccanico con distruzione delle microbolle ( levovist ed echogen ) ; laltra a basso indice meccanico con fenomeni di risonanza delle microbolle ( sonovue ) [ 8 ]  . il levovist un agente epato - specifico ; ha unemivita breve e il suo effetto dopo linsonazione dura solo alcuni secondi . 
moreover , has liver - specific parenchymal uptake after blood - pool clearance ; it is short - lived , and once an area has been scanned for a few seconds , the effect disappears . 
it is best seen with relatively high acoustic power settings ( although still within accepted levels for diagnostic imaging ) and shows a tendency to cluster at the level of the focal zone , where the power is maximal [ 9 , 10 ]  . 
in contrast , sonovue is purely a vascular agent , with no evidence of selective uptake ; it shows a spleen - specific enhancement that lasts longer ( 5 min ) than the blood - pool and liver enhancement phases [ 11 ]  . 
 moreover , the gas microbubbles responsible for this phenomenon are larger than the contrast agent microbubbles and are unable to cross the hepatic sinusoids , the calibre of which is 4050 in addition , this anomalous echogenicity was observed in the vena porta and superior vein only , and not in the splenic vein , hepatic vein , aorta or inferior vena cava . 
echogenicity is similar , but more intense , to the pattern seen in cases of free gas migration into the portal vessels during intestinal ischaemia , necrotic enterocolitis or in other cases of intestinal pneumatosis [ 12 , 13 ]  . 
the possibility that this phenomenon is due to free gas ( thus not incorporated in the shell ) in the superior mesenteric vein and portal vessel is confirmed by several other observations : the stratification pattern seen in the superior mesenteric vein never occurs when the gas is contained in a membrane , as in the case of contrast microbubbles , but only when the gas is free ( intestinal pneumatosis ) the phenomenon persists even after the mi has been increased , an event that should cause immediate microbubble destruction the distribution inside the periportal sinusoids is similar to that seen after injection of free co2 in the hepatic artery ( portal angioechography ) for transient obstruction in the internal sinusoids [ 14 , 15 ]  . a probable explanation for the heterogeneous hepatic enhancement is that the free gas in the superior mesenteric vein is gas emboli originating in the intestinal microcirculation and transported to the liver via enteroportal circulation . 
the pattern of heterogeneous hepatic enhancement is due to entrapment in the hepatic sinusoids of these gas emboli , which have different insonation properties from the microbubbles . several studies have reported that sonographic contrast agents may be responsible for biological damage to the cell membranes and endothelium both in vitro and in vivo [ 1720 ]  . 
di contro , il sonovue , un agente puramente vascolare , non mostra uptake selettivo epatico , mentre presenta una fase spleno - specifica , della durata di circa 5 minuti [ 11 ]  . 
il volume globale di gas contenuto nelle dosi somministrate dellordine di microlitri , invece il fenomeno osservato da noi massivo . inoltre , le microbolle di gas responsabili di questo fenomeno sono di dimensioni maggiori di quelle costituenti il mezzo di contrasto : esse non sono in grado di attraversare i sinusoidi epatici il cui calibro di 4050 abbiamo , inoltre , notato che solo la vena porta e la vena mesenterica superiore presentavano unanomala ecogenicit , non la vena splenica , n le vene sovraepatiche , laorta o la vena cava inferiore . questo anomalo aspetto ecogeno simile , ma pi intenso , al pattern che si osserva nei casi di migrazione di gas libero nei vasi portali in corso di ischemia intestinale , di enterocolite necrotizzante o in altri casi di pneumatosi intestinale [ 12 , 13 ]  . 
la possibilit che laspetto descritto sia da imputare a gas libero ( non incorporato in una membrana ) nella vena mesenterica superiore e nei vasi portali confermato da altre osservazioni : il pattern stratificato allinterno della vena mesenterica superiore non mai osservabile nel caso in cui il gas trovi allinterno di una membrana come nel caso delle microbolle di mezzo di contrasto ; si osserva solo nel caso di gas libero ( pneumatosi intestinale ) ; il fenomeno persiste anche innalzando gli indici meccanici , evento che dovrebbe determinare la distruzione immediata delle microbolle ; la distribuzione allinterno dei sinusoidi periportali sovrapponibile a quanto osservabile dopo iniezione di co2 libera nellarteria epatica ( ecocarbossigrafia ) , per transitorio ostacolo allinterno dei sinusoidi [ 14 , 15 ]  . in base a queste osservazioni si pu ipotizzare che il fenomeno del disomogeneo enhancement epatico sia il risultato di gas libero nella vena mesenterica superiore proveniente da emboli di gas formatisi a livello del microcircolo intestinale e da qui trasportati al fegato attraverso il circolo enteroportale . 
 [ 21 ] demonstrated that in mice and rats , sonographic contrast agents cause inflammation , necrosis and ulceration of the cecum and proximal colon ( cecocolonic area ) , even with a single intravenous administration of a gas - carrier contrast agent . 
 [ 16 ] reported that intestinal lesions in mice and rats after sonographic contrast agent administration are related to the formation of large intravascular gas bubbles in the cecal and colonic wall . 
another probable explanation for heterogeneous hepatic enhancement is microbubble fusion in vivo inside the sinusoids and mesenteric vessels : these microbubble macroaggregates have different insonation properties than the separate microbubbles because of their different size [ 22 ]  . our study has two limitations . 
second , we are unable to explain why this phenomenon is so rare and why it did not occur again in the same two patients after 24 h and 9 days , respectively . 
 [ 16 ] hanno osservato che le lesioni intestinali riscontrate nei topi e nei ratti dopo somministrazione di mezzo di contrasto ecografico sono correlate alla formazione di macrobolle di gas nella parete colica e cecale . 
unaltra probabile spiegazione alleterogeneo enhancement epatico la fusione delle microbolle in vivo allinterno dei sinusoidi e nei vasi mesenterici : questi macroaggregati , in ragione delle loro diverse dimensioni , presenterebbero caratteristiche di insonazione diverse da quelle delle microbolle [ 22 ]  . il nostro lavoro presenta due limiti : il primo relativo al fatto che le lesioni intestinali descritte da dirven et al . 
 [ 16 ] sono state osservate solo in animali di piccola taglia ( topi e ratti ) , non in animali di taglia maggiore ( porcellini dindia , conigli o cani )  . 
il secondo limite , legato al fatto che non siamo in grado di spiegare perch il fenomeno sia cos raro e perch non si sia ripresentato negli stessi pazienti a 24 h e 9 giorni rispettivamente . nonostante non sia stato possibile chiarire la genesi del fenomeno , le alterazioni osservate sono reversibili e non compromettono la compliance del paziente . 
passariello1 1dipartimento di scienze radiologiche , universit degli studi di roma la sapienza , policlinico umberto i , v.le regina elena 324 , i - 00161 roma , italy 2servizio di fisica radiologica , dipartimento di scienze radiologiche , universit degli studi di roma la sapienza , roma , italy correspondence to : m . 
we present our initial clinical experience with a recently introduced 64 - detector computed tomography ( 64 - mdct ) scanner that makes use of a periodic motion of the focal spot in the longitudinal direction ( z - flying focal spot ) , which enables it to reach a final spatial resolution of 0.40.40.4 mm3 and a temporal resolution of 83 ms . 
a total of 114 patients ( 108 men , six women ; age range 3677 years , mean 63.1 years ) underwent retrospective electrocardiogram ( ecg ) - gated examination of the coronary arteries using a 64 - mdct scanner ( somatom sensation 64 , siemens medical solutions , germany )  . 
acquisition parameters were the following : collimation 640.6 mm , 800 quality reference milliampere second ( mas ) , 120 kvp , 0.33 - s gantry rotation time and pitch 0.2. 
administration of 80 ml of contrast agent ( iomeron 400 mgi / dl , bracco , italy ) + 30 ml of saline at 4 / s and delay time determined using a bolus triggering technique . 
oral betablockers were administered to patients with heart rate ( hr ) > 75 bpto reduce radiation exposure , an automatic exposure control system was applied in all cases to adapt tube current to patient size and anatomic shape ( care dose 4d , siemens medical solutions , germany )  . 
the optimal temporal window for raw data reconstruction was chosen from an initial preview of images reconstructed with different phase settings ( range 0%95% rr interval with 5% gap ) at a selected anatomical level in the mid part of the right coronary artery . 
in patients with hr < 60 bmp , the best reconstruction intervals were identified in the end - systolic ( 30%35% of the rr interval ) and end - diastolic ( 60%65% of the rr interval ) phases ; with faster hr ( > 80 bmp ) , high image quality was observed in end - systole ( 30%35% of the rr interval )  . 
nel presente lavoro introduciamo la nostra esperienza preliminare e lottimizzazione del protocollo di acquisizione di un apparecchio tcms in grado di acquisire 64 strati per rotazione ( tcms - 64 ) grazie allutilizzo di un movimento periodico della macchia focale lungo lasse longitudinale z che consente di raggiungere una risoluzione spaziale di 0 , 40 , 40 , 4 mm3 e temporale di 83 ms . 
114 pazienti ( 108 maschi e 6 femmine , intervallo di et 3677 anni , media 63 , 1 ) sono stati sottoposti ad esame con tcms delle arterie coronarie utilizzando uno scanner tcms - 64 ( somatom sensation 64 , siemens medical solutions , germania )  . 
i parametri di acquisizione sono stati i seguenti : collimazione 640 , 6 mm , 800 quality reference mas , 120 kvp , tempo di rotazione 0 , 33 s e pitch 0 , 2 . 
in tutti i pazienti le immagini sono state acquisite dopo somministrazione ev di 80 ml di mdc ( iomeron 400 mgi / dl , bracco , italia ) + 30 ml di soluzione fisiologica con ritardo di scansione calcolato con tecnica bolus triggering . 
in tutti i casi stato utilizzato un sistema di limitazione automatica della dose ( care dose 4d , siemens medical solutions , forchheim , germania ) in grado di modulare la corrente emessa del tubo in relazione ai valori di attenuazione della regione che si sta studiando . 
la finestra temporale ottimale per la ricostruzione dei dati grezzi stata scelta da uniniziale preview di immagini ricostruite in differenti fasi del ciclo cardiaco ( tra 0%95% dellintervallo r - r ogni 5% ) rilevato ad un livello anatomico predefinito nel tratto medio dellarteria coronaria di destra . 
durante lacquisizione tcms stata registrata una fc media di 65 , 619 , 2 bpm ( intervallo : 4496 bpm ) con bradicardizzazione effettuata in 16 / 114 pazienti ( 14 , 0% )  . 
the 64 - mdct scanner diagnostic performance for coronary ct angiography is further improved with better spatial and temporal resolution and faster scan times ; besides , initial clinical results are promising . 
nei pazienti con fc < 60 bpm , la migliore finestra temporale nella quale effettuare la ricostruzione delle immagini stata individuata rispettivamente nelle fasi telesistolica ( 35% dellintervallo r - r ) e meso - telediastolica ( 65%70% dellintervallo r - r ) ; nei casi con fc di base elevata ( > 80 bpm ) , la migliore fase per la ricostruzione delle immagini risultata la telesistole ( 35% dellintervallo r - r )  . 
il ctdi volumetrico ( ct dose index volume ) risultato in media di 36 , 538 , 30 mgy per paziente con un range compreso tra 27 , 2 e 61 , 9 mgy corrispondenti ad un eff mas . 
se confrontato con unacquisizione effettuata a amperaggio costante , lutilizzo del sistema di limitazione automatica della dose ha consentito una riduzione media del ctdi volumetrico del 33 , 3% per paziente ( p < 0 , 001 )  . 
la nostra esperienza clinica iniziale ha evidenziato come lultima generazione di tomografi tcms - 64 apra nuovi scenari per la gestione clinica del paziente coronaropatico . lutilizzo di sistemi idonei di modulazione automatica dellintensit di corrente del tubo radiogeno appare tuttavia indispensabile per limitare la dose di esposizione del paziente che rappresenta , allo stato attuale , il limite principale allutilizzo clinico della metodica . parole chiave arterie coronarie tc multidetettore coronaropatia infarto miocardico dose di esposizione in tc introduction introduzione since the introduction of multidetector computed tomography ( mdct ) into clinical practice in 1998 , diagnostic imaging of the heart and coronary arteries has been one of the main fields of interest for this technique [ 1 , 2 ]  . 
despite the major epidemiological , health and economic implications of ischaemic heart disease [ 3 ] , the study of the coronary tree using noninvasive techniques was for years a difficult problem to solve due to intrinsic technical limitations . 
the coronary artery circulatory is , in fact , a difficult vascular district to assess using the various imaging techniques available due not only to the small diameter and tortuous course of the arteries but also to the complex cardiorespiratory movements . the high spatial and temporal resolution obtainable with the most recent 16 - , 32 - , 40and 64 - detector mdct devices , coupled with the possibility of achieving adequate cardiac synchronisation , has enabled these limitations to be overcome , at least in part , thus revolutionising the management of patients with heart disease [ 7 , 8 ]  . 
the numerous clinical studies published in recent years have demonstrated the high level of diagnostic accuracy of mdct with regard to both assessment of coronary artery stenosis and follow - up of coronary artery bypass graft patency [ 916 ]  . the volume acquired for studying coronary arteries can be used for performing functional studies on volumes , calculating the ejection fraction and myocardial mass of both ventricles , and assessing regional motion and identifying the presfin dalla sua introduzione in ambito clinico nel 1998 , la diagnostica per immagini del cuore e delle arterie coronarie ha rappresentato uno dei campi di maggior interesse della tc multistrato ( tcms ) [ 1 , 2 ]  . 
malgrado le rilevanti implicazioni epidemiologiche ed economico - sanitarie della cardiopatia ischemica [ 3 ] , lo studio dellalbero coronarico con metodiche di tipo non invasivo stato per molti decenni un problema di difficile soluzione per limiti tecnici intrinseci , rimanendo dominio quasi esclusivo della coronarografia selettiva , procedura gravata da una non trascurabile mortalit e morbilit [ 46 ]  . 
il circolo arterioso coronarico rappresenta infatti un distretto vascolare di difficile valutazione per le diverse metodiche di imaging sia per il piccolo calibro ed il decorso tortuoso delle sue arterie , che per la complessit dei movimenti cardio - respiratori . lelevata risoluzione spaziale e temporale ottenibile soprattutto con i pi recenti apparecchi tcms a 16 , 32 , 40 e 64 canali e la possibilit di ottenere unadeguata cardiosincronizzazione ha consentito di superare almeno in parte tali limitazioni , dando luogo ad una rivoluzione nella gestione del paziente coronaropatico [ 7 , 8 ]  . 
i numerosi studi clinici pubblicati in questi ultimi anni hanno ormai ampiamente dimostrato come la tcms rappresenti una metodica dalla elevata accuratezza diagnostica sia per quanto riguarda la valutazione delle stenosi coronariche che il follow - up della perviet dei by - pass aorto - coronarici [ 916 ]  . il volume acquisito per studiare le arterie coronarie pu anche essere utilizzato per effettuare studi funzionali sui volumi , per calcolare la frazione di eiezione e la massa miom . 
nonetheless , clinical use of coronary artery mdct is currently hindered by several limitations [ 20 ] : imaging of coronary stents that due to their metallic struts generate beam - hardening artefacts and hinder accurate assessment of the state of the intrastent vascular lumen [ 21 , imaging of extensively calcified coronary arteries , also due to beam - hardening artefacts [ 23 ] elevated patient heart rate ( hr ) ( > 65 bmp ) , which causes progressive shortening of the electrocardiogram ( ecg ) rr interval and a consequent shortening of the end - diastolic phase in which image acquisition occurs , thus leading to motion artefacts [ 24 , 25 ] rhythm disturbances , which render cardiac synchronisation impossible [ 25 ] accuracy in assessing regional motion caused by the relatively low temporal resolution of mdct , especially when compared with other techniques such as magnetic resonance imaging ( mri ) and , above all , ecg [ 17 ]  . clearly then , most limitations of mdct depend on the resolution limitations of the technique , and therefore , as achenbach emphasised in 2004 , the key to obtaining increasingly accurate diagnostic imaging lies in increasing both spatial and temporal resolutions [ 26 ]  . in this context , we present our early clinical experience with the latest - generation 64 - mdct device in noninvasive coronary artery imaging . 
development of this latest generation of mdct in fact leads to a potential increase in radiation exposure , which could limit the clinical use of 64 - mdct , especially if proposed as a potential screening tool . materials and methods study population between january and may 2005 , 114 patients ( 108 men and six women ; age range 3677 years , mean 63.1 years ) underwent mdct angiography of the coronary arteries . 
all examinations were performed using a 64 - mdct device with a 0.33 - s gantry rotation time ( somatom sensation 64 , siemens medical solutions , forchheim , germany ) and images acquired with retrospective ecg gating . 
clinical indications for the examination were the following : typical ( n = 16 ) or atypical ( n = 7 ) chest pain in patients with a clinical indication for coronarography follow - up at 69 months of the patency of treated coronary stents ( n = 37 for a total of 63 stents )  . 
the findings of the comparison of the two techniques will therefore not be presented here , as this falls beyond the scope of the present study . follow - up of patency of aortocoronary bypass grafts ( n = 40 for a total of 48 bypasses : 32 venous grafts and 16 arterial grafts ) cardica di entrambi i ventricoli , per valutare la cinesi regionale e per identificare la presenza di aree di miocardio infartuato o non - riperfuso correlando lo stato di un vaso coronarico con quello del suo territorio di pertinenza vascolare [ 1719 ]  . 
allo stato attuale , limpiego clinico della tcms delle arterie coronarie presenta tuttavia alcune rilevanti limitazioni [ 20 ] : imaging degli stent coronarici , che a causa della loro trama metallica generano artefatti da indurimento del fascio che impediscono di valutare in modo accurato lo stato del lume vascolare intra - stent [ 21 , 22 ] ; imaging delle coronarie estesamente calcifiche , sempre per linsorgenza di artefatti da indurimento del fascio [ 23 ] ; frequenza cardiaca ( fc ) elevata del paziente ( > 65 battiti per minuto bpm ) , che determina un progressivo accorciamento dellintervallo r - r elettrocardiografico ( ecg ) e conseguentemente della durata della fase telediastolica in cui avviene lacquisizione delle immagini determinando linsorgenza di artefatti da movimento [ 24 , 25 ] ; turbe del ritmo , con impossibilit di ottenere una adeguata cardiosincronizzazione [ 25 ] ; accuratezza nella valutazione della cinesi regionale determinata dalla relativamente bassa risoluzione temporale della tcms soprattutto se confrontata con altre metodiche di riferimento quali la risonanza magnetica e soprattutto lecocardiografia [ 17 ]  . appare quindi evidente come la gran parte delle attuali limitazioni della tcms dipenda da limiti di risoluzione della metodica e pertanto , come sottolineato da achenbach nel 2004 , la chiave per ottenere prestazioni diagnostiche sempre pi accurate lincremento della risoluzione sia spaziale che temporale [ 26 ]  . in questa ottica , nel presente lavoro introduciamo la nostra esperienza clinica preliminare con apparecchiatura tcms di ultima generazione a 64 strati nellimaging non - invasivo delle arterie coronarie , analizzando anche la problematica dosimetrica . 
lo sviluppo di questa nuova generazione di tcms comporta , infatti , un potenziale incremento della dose di esposizione del paziente che rappresenta un possibile fattore limitante limpiego clinico della tcms - 64 soprattutto se si vuole proporre la metodica come potenziale strumento di screening . materiali e metodi popolazione di studio nel periodo compreso tra gennaio e maggio 2005 , 114 pazienti [ 108 maschi , 6 femmine , et compresa tra 3677 anni ( aa ) media 63 , 1 aa ] sono stati sottoposti ad unangiografia con tcms delle arterie coronarie . 
tutti gli esami sono stati effettuati utilizzando unapparecchiatura di tcms - 64 dotata di velocit di rotazione del sistema tubo radiogeno - detettori di 0 , 33 secondi ( somatom sensation 64 , siemens medical solutions , forchheim , germania ) ed acquisendo i dati con gating elettrocardiografico ( ecg ) di tipo retrospettivo . 
therefore , to obtain high - quality images , the hr was empirically slowed in all patients who at the time of the examination had a basal hr > 75 bmp . 
this was achieved using the following drug regimen : oral administration of 40 mg of beta - blockers 6090 min before the examination ( chlorhydrate , propranolol , inderal , astrazeneca ) , and monitoring arterial blood pressure and pulse rate . image acquisition protocol and automatic exposure control system for the examination , the patient was asked to lie supine on the ct table with arms behind the head . 
three electrodes were placed on the chest ( the first on right emithorax , the second 1 cm under the clavicle and the third on the left flank ) to monitor the patients ecg and obtain a tracing with a lead similar to l1 in which the p wave , the qrs complex and the t wave could be distinguished . 
in all cases , the examination was performed following iv administration of nonionic iodinated contrast material and covering a volume from the tracheal carina to the cardiac base ; for the study of coronary bypass grafts , acquisition was extended to include a volume comprising the proximal anastomosis and the cardiac base . 64 - mdct sensation 64 scanner utilizes an automatic exposure control system [ combined applications to reduce exposure ( care ) dose 4d , siemens medical solutions , forchheim , germany ] that combines both angular ( x - y axis or angular modulation system ) and z - axis ( or z - axis modulation system ) tube current modulation [ 2731 ]  . in practice , beginning with the initial topogram as a reference , the amperage is automatically modulated along the entire acquisition volume on the basis of the angular attenuation coefficient ( mean density of a tissue with respect to the tube rotation angle in the x - y axis ) of the anatomical region being studied and on the basis of the attenuation values of the different tissues along the z - axis , with data detected on the basis of the previous slice ( on - line modulation )  . 
for example , in a total - body study , lower amperages are used for rizzare , in pazienti con indicazione clinica alla coronarografia ; controllo a distanza di 69 mesi della perviet di stent coronarici medicati ( n = 37 per un totale di 63 stent )  . 
non verranno quindi riportati di seguito i risultati sul confronto tra le due metodiche poich non rappresenta lobiettivo specifico del presente lavoro ; controllo a distanza della perviet di by - pass aorto - coronarici ( n = 40 per un totale di 48 by - pass ; 32 graft venosi e 16 arteriosi ) ; pazienti sottoposti a scintigrafia miocardica perfusionale a riposo e dopo sforzo con risultati diagnostici non conclusivi ( n = 3 ) ; pazienti sottoposti ad esame ecocardiografico a riposo e dopo stress farmacologico con dobutamina con risultati diagnostici non conclusivi ( n = 11 )  . tutti i pazienti hanno firmato un consenso informato in cui veniva chiaramente spiegato il tipo di procedura a cui si sottoponevano ed i potenziali rischi . 
i criteri di esclusione per lo studio sono stati i seguenti : nota ipersensibilit ai mezzi di contrasto ( mdc ) iodati ; insufficienza renale ( creatininemia > 2 mg / dl ) ; insufficienza cardiaca conclamata ; aritmie cardiache maggiori ; insufficienza respiratoria ; asma bronchiale nei pazienti con indicazione al pre - trattamento con farmaci beta - bloccanti ( controindicazione assoluta alla somministrazione di beta - bloccanti )  . preparazione del paziente lelevata frequenza cardiaca rappresenta uno dei principali fattori in grado di influenzare negativamente la qualit diagnostica dellesame determinando un accorciamento dellintervallo r - r elettrocardiografico con insorgenza di artefatti da movimento [ 24 , 25 ]  . 
per tale motivo , al fine di ottenere massima qualit delle immagini nella nostra esperienza iniziale la bradicardizzazione stata effettuata empiricamente in tutti i pazienti che , al momento di effettuare lesame , presentavano fc basale > 75 bpm , utilizzando il seguente schema di preparazione farmacologica : somministrazione per os di 40 milligrammi ( mg ) di farmaco - bloccante 6090 minuti prima dellesame ( propranolo cloridrato , inderal , astrazeneca ) , monitorando pressione arteriosa ( pa ) e polso ( fc )  . protocollo di acquisizione delle immagini e sistema di limitazione automatica della dose al momento dellesame il paziente stato invitato a sdraiarsi in posizione supina sul lettino della tc con le braccia dietro la testa . 
posizionando tre elettrodi sul torace ( rispettivamente a livello dellemitorace destro e sinistro , un cm al di sotto della clavicola ed in corrispondenza del fianco sinistro ) stato possibile monitorare lecg del paziente , fino ad m . 
based on the initial topogram in the anteroposterior ( ap ) and lateral position ( a ) , care dose 4d automatically modulates tube current during each tube rotation according to the patients angular attenuation profile . 
1a , b sistema di modulazione automatica della dose : partendo dal topogramma iniziale ( a ) , lamperaggio viene modulato automaticamente lungo tutto il volume di acquisizione in base al coefficiente angolare di attenuazione della regione anatomica che si sta studiando . 
in tutti i casi lesame stato eseguito dopo somministrazione endovenosa ( ev ) di mdc iodato non ionico coprendo un volume cranio - caudale compreso tra la carena tracheale e la base cardiaca ; per lo studio dei bypass coronarici lacquisizione stata estesa in modo da includere un volume compreso tra lanastomosi prossimale e la base cardiaca . lo scanner di tcms - 64 utilizza un sistema di limitazione automatica della dose ( combined applications to reduce exposure , care dose 4d , siemens medical solutions , forchheim , germania ) in grado di combinare la modulazione della corrente emessa dal tubo radiogeno sia in relazione al valori di attenuazione angolare ( xy - axis o angular modulation system ) della regione in esame che attraverso un sistema di modulazione della dose lungo lasse z ( o z - axis modulation system ) [ 2731 ]  . in pratica , partendo come riferimento dal topogramma inim . 
contrast material was administered using a dual - head automatic injector ( medrad stellant dual ; medrad , palo alto , pa , usa ) in which the two syringes containing contrast material and saline solution , respectively , were connected by a y - shaped connector equipped with a one - way valve to prevent reflux . 
use of saline solution reduces the total volume of contrast material administered to the patient , thus compressing the iodine bolus and at the same time reducing the presence of beam - hardening artefacts at the level of the superior vena cava [ 32 ]  . acquisition delay was chosen using the bolus triggering technique , which involves positioning a region of interest ( roi ) in correspondence with the aortic arch and monitoring passage of the contrast material bolus with a series of low - dose dynamic scans ( 120 kvp , 30 mas ) with 1 - s intervals [ 33 ]  . 
in this way , the effective ctdi volume could be measured prior to the examination , even in conditions of constant tube - current intensity throughout the acquisition ( without the use of care dose 4d )  . an estimate of the dose absorbed [ expressed in milligray ( mgy ) ] was then made using the care dose 4d system , and this estimate was compared with the dose to which the patient would have otherwise been exposed without the use of current modulation . 
tuttavia il sistema di mudulazione della dose utilizzato in modalit cardiosincronizzata effettua una semplice riduzione del milliamperaggio in base ai valori di attenuazione individuale del paziente . i parametri tecnici utilizzati sono stati i seguenti : collimazione 3220 , 6 mm ; spessore strato 0 , 75 mm ; incremento 0 , 4 mm ; tempo di rotazione del sistema tubo radiogeno / detettori 0 , 33 s ; pitch 0 , 2 ; quality reference mas : 800 ; effective mas : variabile in relazione alle caratteristiche individuali del paziente ; 120 kvp ; filtri di convoluzione per la ricostruzione delle immagini : b20f ( tessuti molli ) e b46f ( utilizzato per la valutazione degli stent coronarici )  . somministrazione del mezzo di contrasto in tutti i pazienti stato utilizzato un mdc ad alta concentrazione di iodio ( 400 mgi / 100 ml , iomeron 400 , bracco , milano , italia ) seguito da un bolo di soluzione fisiologica . 
il mdc stato somministrato utilizzando un iniettore automatico a doppia testata ( medrad stellant dual , medrad , palo alto pa , usa ) in cui le due siringhe contenenti rispettivamente mdc e soluzione fisiologica sono state collegate tra loro tramite raccordino a y dotato di valvola unidirezionale per impedire il reflusso . 
lutilizzo della soluzione fisiologica consente di ridurre il volume totale di mdc somministrato al paziente , compattando il bolo di iodio e riducendo contemporaneamente la presenza di artefatti da indurimento del fascio a livello della vena cava superiore [ 32 ]  . per la scelta del ritardo di acquisizione stata utilizzata la tecnica del bolus triggering posizionando una regione di interesse in corrispondenza dellarco aortico e monitorizm . 
transverse images of 20 different cardiac cycle phases ( 0%95% of rr interval with 5% gap ) obtained at a predefined anatomical level in the middle part of the right coronary artery ( a )  . 
in this patient with a heart rate of 58 bmp , best image quality with minimal motion artefacts is observed in the end - systolic phase ( 20%25% of rr ) and the midto enddiastolic phase ( 70% of the rr )  . 
maximum intensity projection ( mip ) image obtained in the same patient , reconstructing images at 65% of the rr interval , allows the presence of disease throughout the course of the right coronary artery to be excluded ( b )  . 
2a , b esempio di preview series per la selezione della migliore finestra temporale ( percentuale intervallo r - r ) delle immagini utilizzando gating cardiaco di tipo retrospettivo . immagini assiali delle 20 successive serie di ricostruzioni ( dal 0% al 95% dellintervallo rr con gap del 5% ) , ottenute sulla medesima scansione , a livello del terzo medio della cdx ( a ) ; in questo paziente , con fc di 58 bpm , la minor presenza di artefatti da movimento si osserva nelle immagini ricostruite al 20%25% ( intervallo che corrisponde alla telesistole ) e tra il 60% ed il 70% dellintervallo r - r ( fase meso - telediastolica )  . 
this tends to improve temporal resolution by using the raw data derived from multiple cardiac cycles ( up to a maximum of three cycles ) to reconstruct the images [ 35 ]  . zando il passaggio del bolo di mdc attraverso una serie di scansioni dinamiche a bassa dose ( 120 kvp 30 mas ) con intervalli di 1 secondo [ 33 ]  . 
la soglia predefinita per linizio della scansione stata di 100 unit hounsfield ( hu ) con intervallo di tempo tra raggiungimento del valore soglia ed inizio dellacquisizione stessa di 7 secondi . la somministrazione del mdc stata effettuata con protocollo di tipo monofasico utilizzando i seguenti parametri [ 34 ] : 80 ml di mdc + 30 ml di fisiologica con flusso di 4 ml / s con durata totale della somministrazione di 27 , 5 secondi . image postprocessing valutazione dose di esposizione after identifying the best reconstruction interval , the images were processed using a dedicated workstation connected to tutti i pazienti sono stati studiati con utilizzo del software care dose 4d . 
volume rendering algorithms , in contrast , were used mainly in patients being studied for evaluation of aortocoronary bypasses and in all cases where the intuitive 3d approach of this algorithm enabled concise and easily recognisable representation of the state and site of coronary disease . 
this approach provides the patient with images that are easier to understand . given the large number of images obtained on average for each examination ( around 2 , 000 ) , in all cases , images were digitally stored on a compact disc equipped with software for viewing the images in dicom3 format ( digital imaging communication in medicine )  . 
all data from the mdct examination ( except the raw data ) were then archived using the picture archive and communication system ( pacs ) ( lifeweb image manager , veania imaging technologies , italy )  . results the mdct examination was carried out without complications in all patients . 
hr was slowed down in 16 / 114 patients ( 14.0% ) ; there were no cases of complications or side effects as a result of administration of oral - blockers . 
in one patient with severe chronic obstructive pulmonary disease ( copd ) and a baseline hr of 96 bmp , even though the hr could not be slowed due to contraindications for - blockers , the examination was performed nonetheless for clinical requirements . the optimal interval for image reconstruction varied in relation to patients hr during data acquisition . 
they cordi riferimento del ctdi volumetrico ( ct dose index volume ) quale parametro della dose assorbita durante lacquisizione delle immagini con gating ecg di tipo retrospettivo . il valore di ctdi viene definito automaticamente dallo scanner sulla base del topogramma in relazione alla intensit di corrente applicata durante lacquisizione . 
in tal modo , preliminarmente allesame , stato possibile registrare leffettivo valore di ctdi volumetrico anche in condizioni di costante intensit di corrente del tubo durante lintera acquisizione ( senza utilizzo del care dose 4d )  . stata quindi effettuata una stima della dose assorbita ( espressa in mgy ) utilizzando il sistema care dose 4d confrontandola con la dose a cui sarebbe stato sottoposto il paziente senza lutilizzo della tecnica di modulazione . 
in alternativa le immagini sono state ricostruite con unaltra consolle dedicata ( vitrea 2 , vital images inc , minnesota , usa )  . per standardizzare la ricostruzione 3d dei dati e fornire al medico di riferimento delle immagini il pi possibile comprensibili stato da noi utilizzato uno schema in cui sono state effettuate per ciascun paziente almeno 6 set di ricostruzioni : m . 
tale approccio consente di sfruttare al meglio lelevata risoluzione spaziale della metodica ottenendo unimmagine a spessore estremamente sottile ( 0 , 6 mm ) lungo tutto il decorso del vaso . 
gli algoritmi in volume rendering sono stati invece utilizzati prevalentemente nei pazienti inviati per studio dei by - pass aorto - coronarici e in tutti quei casi in cui lintuitivo approccio 3d di questo tipo di algoritmo ha consentito di ottenere una rappresentazione sintetica e facilmente intuibile di stato e sede della coronaropatia fornendo al paziente immagini di pi semplice comprensione . considerando il grande numero di immagini ottenute in media per ciascun esame ( circa 2000 ) , in tutti i casi stata fornita una documentazione di tipo digitale con compact disc ( cd ) su cui stato installato un software per la lettura delle immagini in formato dicom3 ( digital imaging communication in medicine )  . 
tutti i dati dellesame tcms ( esclusi i dati grezzi ) sono stati poi archiviati tramite sistema pacs ( lifeweb image manager , ferrania imaging technologies , italia )  . risultati lesame tcms stato effettuato senza alcuna complicanza in tutti i pazienti studiati . 
la bradicardizzazione stata effettuata in 16 / 114 pazienti ( 14 , 0% ) ; in nessun caso si sono avute complicanze o effetti collaterali conseguenti alla somministrazione di - bloccanti orali . 
in un paziente con bpco severa e fc basale di 96 bpm , pur non essendo stata effettuata la bradicardizzazione per la presenza di controindicazione clinica alla somministrazione di farmaco - bloccante lesame stato ugualmente eseguito per esigenze cliniche . lintervallo ottimale per la ricostruzione delle immagini variato in relazione alla fc del paziente durante lacquisizione dei dati . 
3a , b a 64 - mdct angiography performed at 6 - months follow - up after drug - eluting coronary stent implantation on the left anterior descending ( lad ) artery . 
a curved multiplanar reformation shows the presence of a metallic stent with the characteristic railway track ( arrowheads ) appearance located in the proximal part of the vessel ; the presence of contrast within and distally to the stent indirectly indicates patency ( arrows )  . 
b three - dimensional volume rendering reconstruction ; the use of dedicated cardiac software enables visualisation of the stented coronary vessel ( arrow ) along an automatically generated centreline displayed in the transverse and longitudinal axes of lad . 
a la ricostruzione multiplanare curva ( cpr ) , generata lungo lasse longitudinale dellarteria discendente anteriore , dimostra la presenza di flusso allinterno ed a valle dello stent ( frecce ) , che presenta peraltro tipico aspetto a binario ( punte di freccia )  . 
b immagine tridimensionale ottenuta con tecnica volume rendering ; utilizzando un software dedicato di campionatura selettiva del vaso in esame , possibile riprodurre , in modo pratico e veloce , il tratto di arteria selezionata ( freccia ) permettendo una visualizzazione del lume vascolare intra - stent con tecnica cpr ; in tal modo si ottiene una rappresentazione dellarteria sia secondo il suo asse trasversale che longitudinale . 
in cases with higher baseline hr ( > 80 bmp ) , the best phase for image reconstruction proved to be the end - systolic phase ( 35% rr )  . 
in patients with intermediate hr ( 60 bmp ) , no ideal reconstruction window was identified , and the rr interval used varied significantly from patient to patient . in 11 patients ( 9.6% ) , the reconstructed images were affected by artefacts due to either extrasystole during data acquisition ( n = 8 ) or inability to maintain breath - hold for the duration of the acquisition ( n = 3 )  . 
the study of only the coronary district produced the following values : ctdi volume per la ricostruzione dei dati grezzi corrispondenti alle fasi telesistolica ( 35% r - r ) e meso - telediastolica ( 65%70% rr )  . 
nei casi con fc di base elevata ( > 80 bpm ) , la migliore fase per la ricostruzione delle immagini risultata la telesistole ( 35% dellintervallo r - r )  . 
sulla base delle informazioni fornite dallesame , la paziente stata esclusa dal trattamento e dimessa evitando ulteriore accertamento diagnostico con esame coronarografico e prolungamento della ospedalizzazione . infine , nel gruppo di pazienti in cui lindicazione clinica allesame tcms - 64 era stata posta sulla base di test funzionali ecocardiografici ( n = 11 ) o scintigrafici ( n = 3 ) non conclusivi , in 6 / 14 casi sono state evidenziate immagini tcms64 compatibili con stenosi coronariche emodinamicamente significative ( mono o multivasali ) e clinicamente silenti ( fig . 7 ) ; solamente in questi 6 pazienti stato successivamente effettuato lesame coronarografico convenzionale . aspetti dosimetrici il ctdi volumetrico ( ct dose index volume ) stato in media di 36 , 538 , 30 mgy per paziente con un range compreso m . 
volume rendering reconstruction shows complete occlusion of the stent positioned in the proximal part of the vessel ( short arrow ) , with compensatory enlargement of the first diagonal branch ( long arrow ) ( a )  . thanks to the high spatial resolution achievable using a collimation of 0.6 mm , the stents metallic structure is visualised in detail ( b )  . 
4a - d coronarografia con tcms - 64 per follow - up di stent posizionato su arteria discendente anteriore 3 anni prima ; elaborazione delle immagini effettuata con workstation siemens utilizzando software syngo inspace 4d . 
le immagini ricostruite con algoritmo in volume rendering mostrano occlusione dello stent posizionato nel tratto prossimale della da ( freccia corta ) e lipertrofia compensatoria del primo ramo diagonale , che appare tortuoso ed aumentato di calibro ( freccia lunga ) ( a )  . 
nel particolare ( ingrandito ) lelevata risoluzione spaziale dellacquisizione a 0 , 6 mm di collimazione , permette di ottenere dettagli fini come la chiara definizione della trama metallica dello stent ( b )  . 
i pazienti sottoposti a studio dei by - pass coronarici , a causa del pi ampio volume cranio - caudale acquisito , presentavano valori medi di ctdi volumetrico pi elevati ( 38 , 77 , 9 mgy ) con un milliamperaggio effettivo di circa 645 , 389 , 8 . 
per il solo studio del distretto coronarico i valori dosimetrici sono risultati i seguenti : ctdi volumetrico 33 , 036 , 39 mgy , effective mas 498 , 1693 , 27 . il valore medio di ctdi volumetrico a cui sarebbe stato m . 
volume rendering image shows patency of the arterial y - shaped mammary graft connected with the left anterior descending ( lad ) artery and the first marginal branch ( arrow ) ; in the same reconstruction , venous conduit occlusion is observed at the proximal anastomosis level ( arrowhead )  . 
le immagini ricostruite con tecnica volume rendering dimostrano perviet del bypass arterioso ad y con arteria mammaria interna sinistra ( amis ) su da e primo ramo marginale ottuso ( freccia ) ; nelle stesse scansioni si evidenzia occlusione del graft aorto - coronarico venoso allorigine , con caratteristica estroflessione sulla parete aortica dellanastomosi prossimale ( punta di freccia )  . discussion patient exposure to ionising radiation is the main limitation to the clinical use of 64 - mdct , especially if it is to be used as a potential screening tool in asymptomatic patients . 
despite the patients high heart rate during image acquisition ( 96 bmp ) , maximum intensity projection ( mip ) reconstructions ( a ) show multiple stenotic lesions on the right coronary artery ( arrows ) , with subocclusive stenosis at the proximal third of the vessel . 
a 60% stenosis is also observed in the proximal portion of the left anterior descending ( lad ) artery , caused by soft tissue plaque ( arrow ) ( b )  . 
della paziente al momento dellacquisizione ( 96 bpm ) , le ricostruzioni mip ( a ) documentano la presenza di multiple alterazioni stenosanti lungo tutto il decorso della coronaria destra ( frecce ) con stenosi subocclusiva in corrispondenza del terzo prossimale del vaso . 
le ricostruzioni sono state effettuate con workstation siemens e software syngo inspace 4d . sottoposto il paziente senza lutilizzo della tecnica di modulazione risultato di 54 , 690 , 89 mgy con differenza statisticamente significativa tra ctdi volumetrico con sistema care dose e senza care dose ( p < 0 , 001 )  . 
come noto , lutilizzo del gating cardiaco retrospettivo comporta una continua e relativamente elevata esposizione del paziente ai raggi x per la necessit di effettuare lacquisizione dei dati durante lintera durata della scansione e di registrare contemporaneamente i dati dellecg per poi ricostruire le immagini in una fase definita del ciclo cardiaco [ 1 , 2 , 7 , 36 ]  . altra peculiarit tecnica dellimaging tcms delle arterie coronarie lutilizzo di bassi valori di pitch spirale ( pitch di 0 , 2 con tcms - 64 ) e notevolmente sovrapposti ( pitch < numero di strati ) che nasce dallesigenza di effettuare il campionamento dei dati per la ricostruzione delle immagini da strati sovrapposti e privi di gap nel volume acquisito , ma comporta un ulteriore significativo aumento della dose di esposizione [ 7 , 36 , 38 ]  . 
in aggiunta , rispetto alle precedenti generazioni , nella tcms - 64 lutilizzo di collimazioni submillimetriche ( 640 , 6 mm ) si associa ad un intrinseco incremento del rumore nelle immagini acquisite che pu essere limitato aumentando lamperaggio ( intensit di corrente emessa dal tubo radiogeno ) durante la scansione stessa per mantenere il rapporto segnale / rumore entro ottimali livelli diagnostici . nel nostro studio , il ctdi volume risultato in media di 36 , 538 , 30 mgy per paziente , con valori medi di 38 , 77 , 9 mgy nei pazienti con richiesta clinica per studio dei by - pass coronarici con un valore medio di mas di 567 , 83108 , 35 . rispetto ai dati disponibili in letteratura che riportano per apparecchiature a 4 e 16 canali rispettivamente ctdi volume medi di 3645 , 6 mgy e 43 , 3 mgy [ 35 ] , lutilizzo del sistema di modulazione della dose ha consentito di ottenere nella nostra popolazione valori medi di dose assorbita complessivamente inferiori rispetto alle precedenti generazioni tcms pur mantenendo una buona qualit diagnostica degli esami effettuati . 
i valori di dose efficace ottenuti sono risultati in media di 9 , 53 , 4 msv . malgrado in alcuni pazienti si siano raggiunti anche i 17 , 1 msv , questo dato preliminare , sempre se confrontato con i valori di dose efficace riportati in letteratura con apparecchiature a 4 e 16 strati ( rispettivamente 7 , 111 , 9 msv e 8 , 8 msv ) dimostra come , nella media dei pazienti , tale parametro dosimetrico non risulti significativamente superiore rispetto a quelli riportati con precedenti sistemi tcms [ 3841 ]  . 
appare tuttavia rilevante sottolineare come , a prescindere dallimpiego di sistemi di controllo dosimetrico , lesame tcms - 64 delle coronarie con cardiosincronizzazione comporti una esposizione del paziente in termini di dose efficace quasi doppia rispetto ai 57 msv di un esame tc standard del torace [ 38 , 42 ]  . nella nostra esperienza preliminare , lutilizzo del sistema care dose 4d appare quindi indispensabile per mantenere lesposizione del paziente entro livelli accettabili consentendo di ridurre lesposizione media del paziente del fig . 
7 a 64 - mdct angiography performed in a patient with atypical chest pa multiplanar reconstruction ( mpr ) of left anterior descending ( lad ) artery shows diffuse coronary artery disease with multiple vessel wall abnormalities ; note the presence of a noncalcified soft - tissue - attenuation lesion ( arrowheads ) in the proximal part of the artery , causing a > 60% narrowing of the lumen . 
la ricostruzione multiplanare ottenuta su piano curvo ( cpr ) lungo lasse longitudinale della da , documenta multiple alterazioni parietali a densit mista in corrispondenza del tratto prossimale dellarteria discendente anteriore ( da ) e la presenza di placca a densit mista , prevalentemente morbida , con riduzione significativa ( > 60% ) del lume vasale ( punte di freccia )  . 
lelaborazione delle immagini effettuata con workstation siemens utilizzando software syngo inspace 4d . to record ecg data to reconstruct images in a precise phase of the heart cycle [ 1 , 2 , 7 , 36 ]  . another technical peculiarity coronary artery mdct is the use of low spiral pitch ( 0.2 pitch with 64 - mdct ) and considerable overlap ( pitch < < number of slices ) , which arises from the need to sample data for image reconstruction with overlapping slices and no gap , but this leads to a further significant increase in radiation exposure [ 7 , 36 , 38 ]  . 
8 nella figura viene riportata la distribuzione del ctdi volumetrico ( in mgy ) utilizzando il sistema di compensazione care dose 4d , espresso in funzione del carico anodico effettivo . 
it should be borne in mind , however , that regardless of the use of an automatic dose control system , the 64 - mdct examination of the coronary arteries with cardiac gating involves exposing the patient to almost twice the effective radiation dose of a standard ct chest examination ( 57 msv ) [ 38 , 42 ]  . in our preliminary experience , the use of the care dose 4d systems appears indispensable for maintaining exposure within acceptable levels , allowing a 33.5% reduction in mean exposure compared with a similar acquisition performed without an automatic dose control syste the overall dose can be further reduced ( up to 50% compared with an acquisition without care dose 4d ) by using an ecg pulsing system , which is based on the premise that the best phase in the heart cycle for image reconstruction is the mid to end diastole , that is , the maximum immobility phase of the heart and coronary arteries [ 43 ]  . 
in this manner , during data acquisition , tube current is brought to the maximum level in the midto end - diastolic phase , where the maximum snr is required , and progressively reduced to the minimum towards the end - systolic phase . failure to use an ecg pulsing protocol in our study population derives from the evidence that the best rr interval for image reconstruction , especially in patients with elevated hr , is the end - systolic phase ( 30%35% ) , thus confirming the initial experience of wintersperger et al . 
the reduced snr of images acquired in the systolic phase resulting from an acquisition with an ecg 33 , 5% rispetto ad una analoga acquisizione senza modulazione automatica della corrente . 
la dose complessiva pu essere ulteriormente ridotta ( fino al 50% rispetto ad una acquisizione senza protocollo care dose 4d ) utilizzando un sistema di modulazione ecg - controllato dellemissione del tubo radiogeno ( sistema ecg - pulsing ) che si basa sul presupposto che la miglior fase del ciclo cardiaco nella quale effettuare la ricostruzione delle immagini sia la meso - telediastole , cio la fase di maggiore immobilit del cuore e delle arterie coronarie [ 43 ] ; in tal modo , durante lacquisizione dei dati , la corrente del tubo radiogeno viene portata a livello massimo in fase meso - telediastolica , dove necessario avere un massimo rapporto segnale / rumore e ridotta progressivamente al minimo verso la telesistole . la mancata utilizzazione di un protocollo di modulazione ecg - controllato nella nostra popolazione di pazienti deriva tuttavia dallevidenza che , particolarmente in pazienti con elevata fc , il migliore intervallo r - r per la ricostruzione delle immagini risultato essere la fase telesistolica ( 30%35% ) confermando liniziale esperienza riportata da wintersperger et al . 
although the technique needs to be validated with systematic comparisons with clinical , laboratory and coronarographic data , the latest generation of 64 - mdct scanners offers new possibilities for clinical management of patients with coronary artery disease . the increase in spatial and temporal resolution translates into improved diagnostic image quality with respect to previous generations of multidetector devices . 
thus , 64 - mdct is a noninvasive technique capable of identifying patients requiring interventional or surgical procedures such as selective coronarography with primary angioplasty or stent placement , or surgical revascularisation with bypass grafts . the use of suitable systems for the automatic control of radiation exposure seems nonetheless indispensable in order to limit patient dose , which given the current state of affairs is the main limitation to the clinical use of the technique . nella nostra esperienza clinica iniziale , lutilizzo della tcms - 64 ha fornito risultati molto promettenti . 
bolzano , italy , tel . : + 39 - 047 - 1908494 , fax : + 39 - 047 - 1908908 , e - mail : mosavr@yahoo.com received : 23 june 2006 / accepted : 27 july 2006 / published online : 22 february 2007 abstract purpose . 
cta is fast and relatively noninvasive , and its sensitivity appears similar to that of dsa in detecting and evaluating intracranial aneurysms , even those smaller than 3 mm . this study confirms the value of cta as the primary imaging technique in subarachnoid haemorrhage , with dsa reserved for selected patients . key words multislice ct angiography cerebral aneurysm riassunto obiettivo . 
in un periodo di 20 mesi 130 pazienti con diagnosi tc di emorragia subaracnoidea acuta non traumatica sono stati arruolati per uno studio prospettico e sottoposti ad angiotc multistrato ( 16 detettori ) ed angiografia digitale sottrattiva ( 57 maschi , 73 femmine ; et media 59 , 5 anni )  . 
langio - tc una metodica veloce e poco invasiva e la sua sensibilit nellindividuazione degli aneurismi analoga a quella dellangiografia digitale sottrattiva anche se di dimensioni inferiori ai 3 mquesto studio conferma la validit dellangiotc come tecnica di imaging di elezione nella diagnosi di emorragie subaracnoidee . 
langiografia digitale sottrattiva pu essere riservata solo a pazienti selezionati . parole chiave angio - tc multistrato aneurisma cerebrale introduction introduzione nontraumatic subarachnoid haemorrhage ( sah ) is a neurological emergency characterised by extravasation of blood into the spaces lining the central nervous system , which are normally filled with liquor . 
nonaneurysmal sah , including the perimesencephalic end systolic area ( esa ) , is in contrast characterised by a good prognosis and rare neurological complications [ 2 ]  . the global incidence of sah , which varies from region to region , is about 10.5 cases per 100 , 000 inhabitants per year and has remained stable over the last 30 years . 
the main negative prognostic factors include level of consciousness , age and the quantity of blood at initial head computed tomography ( ct ) scan [ 3 , 4 ]  . the principle variable risk factors are cigarette smoking , hypertension , use of cocaine and alcohol abuse . 
first - degree family members of patients with sah have an increased risk ; there are also hereditary diseases of the connective tissue associated with intracranial aneurysms and sah , such as polycystic kidney disease , ehlers - danlos syndrome ( type iv ) , pseudoxanthoma elasticum and fibromuscular dysplasia [ 5 ]  . on the basis of these prognostic criteria , an sah should always be suspected in patients with the typical presentation of sudden and severe headache associated with nausea , vomiting , neck stiffness , photophobia or loss of consciousness . the physical examination may reveal retinal haemorrhage , evidence of meningeal irritation , reduced level of consciousness and / or focal or unilateral neurological deficit . 
this fact supports the extensive use of ct that , if correctly performed , is capable of identifying an sah in 100% of cases within 12 h of symptom onset and in 93% of cases within 24 h [ 7 , 8 ]  . 
head ct is also able to identify the presence of intraparenchymal haematomas , hydrocephalus , cerebral oedema and , in some cases , can suggest the probable site of aneurysmal rupture [ 9 ]  . 
lumbar puncture is a valuable diagnostic examination in cases of negative ct and suspected sah . for exact localisation of the source of extravasated blood , the diagnostic protocol involves performing digital subtractive angiography ( dsa ) or ct angiography ( cta )  . 
whereas dsa is the gold standard for identifying cerebral aneurysms , cta is gaining increasing acceptance , thanks to its immediacy and noninvasiveness and a specificity and sensitivity comparable with dsa . 
cta also has a significant advantage in that it can be performed immediately following the baseline ct examination , which enables a significant shortening of examination times and the possibility of initiating treatment immediately [ 1023 ]  . the aim of this paper is to present a consecutive series of 130 patients diagnosed with sah in a single institution ( ospedale centrale regionale di bolzano ) who underwent cta and / or dsa and to analyse their sensitivity , specificity and diagnostic accuracy , comparing them with the data in the literature . le normalmente percorsi da liquido cerebrospinale . 
lesa non aneurismatica , inclusa lesa perimesencefalica , caratterizzata da buona prognosi e rare complicanze neurologiche [ 2 ]  . lincidenza mondiale aggregata dellesa , che varia da regione a regione , di circa 10 , 5 casi / 100000 abitanti per anno ed rimasta stabile negli ultimi 30 anni . 
i familiari di primo grado di pazienti con esa hanno fattore di rischio aumentato ; vi sono inoltre patologie ereditarie del tessuto connettivo associate ad aneurismi intracranici ed esa quali la malattia policistica renale , la sindrome di ehlers - danlos ( tipo iv ) , lo pseudoxantoma elastico e la displasia fibromuscolare [ 5 ]  . sulla base di tali dati prognostici unesa deve essere sempre sospettata in pazienti con presentazione tipica : cefalea violenta ed improvvisa associata a nausea , vomito , dolore cervicale , fotofobia o perdita di coscienza . 
in assenza dei classici segni e sintomi la diagnosi di esa pu essere confusa con diagnosi di emicrania e cefalea muscolo - tensiva in circa il 50% dei casi [ 6 ]  . 
tali dati supportano luso estensivo che viene fatto della tomografia assiale computerizzata ( tc ) che , se eseguita correttamente , in grado di rivelare unesa nel 100% dei casi entro 12 ore dallesordio sintomatologico e nel 93% entro 24 ore [ 7 , 8 ]  . 
la tc del cranio inoltre in grado di discriminare la presenza di ematomi intraparenchimali , idrocefalo , edema cerebrale e pu , in alcuni casi , suggerire la probabile sede della rottura aneurismatica [ 9 ]  . 
la rachicentesi mantiene una validit diagnostica nel caso di tc negativa e quadro clinico sospetto per esa . il percorso diagnostico prevede quindi , per lesatta localizzazione della sorgente del sanguinamento , lesecuzione di unangiografia digitale sottrattiva ( dsa ) oppure di un angiotc ( atc )  . 
lo studio angiografico cerebrale a sottrazione digitale rappresenta il gold standard per levidenziazione degli aneurismi cerebrali ; latc viene sempre pi utilizzata per limmediatezza e la non invasivit associate a specificit e sensibilit comparabili con la dsa . 
contrast enhancement was provided by the intravenous antecubital administration of an 80ml bolus of nonionic iodinated contrast material ( iomeron 350 mgi / ml , bracco , milan , italy ) at a 4 - ml / s flow rate followed by 50 ml saline solution at the same rate . 
the sample volume was positioned in an internal carotid artery with a threshold of + 30 hu . after acquisition data transfer to the postprocessing station ( leonardo , siemens , erlangen , germany ) , image analysis was performed interactively by the radiologist using various postprocessing techniques , including two ( 2d ) and three - dimensional ( 3d ) multiplanar reconstructions ( mpr ) , maximum intensity projections ( mip ) and volume rendering ( vr )  . each cta and dsa examination was assessed independently by the performing radiologist , and the aneurysms were classified according to the following scheme ( table 1 ) : site : aneurysms originating from the anterior circulation : internal carotid artery , ophthalmic artery , anterior choroidal artery , anterior communicating artery , anterior cerebral artery ( a1 , a2 ) , pericallosal artery , callosal - marginal artery , middle cerebral artery ( m1 , m2 , m3 ) , posterior communicating artery aneurysms originating from the posterior circulation : basilar artery , apex of the basilar artery , posteroinferior cerebellar artery , anteroinferior cerebellar artery , anterosuperior cerebellar artery , posterior cerebral artery . size : giant aneurysms : 25 mm large aneurysms : 1325 mm medium aneurysms : 513 mm small aneurysms : 35 mm very small aneurysms : < 3 mm nel periodo compreso tra giugno 2004 e febbraio 2006 ogni paziente con diagnosi tc di esa non traumatica stato sottoposto a atc ( somatom sensation 16 , siemens , erlangen , germania ) e , successivamente , a dsa ( multistar plus top , siemens , erlangen , germania )  . 
sono stati arruolati 130 pazienti consecutivi , 57 maschi e 73 femmine con et media di 59 , 515 , 2 anni ( range 2091 anni ) ; 91 pazienti provenienti dal pronto soccorso dellospedale centrale regionale di bolzano e 39 da altre strutture ospedaliere . latc stata eseguita con sistema a 16 detettori ( somaton sensation 16 , siemens , erlangen , germania ) con i seguenti parametri di acquisizione e ricostruzione : scansione sul piano assiale estesa dal soma della prima vertebra cervicale al vertice , collimazione 0 , 75 mm , tempo di rotazione 0 , 5 s , fov 230 mm , feed / rot 6 , 5 , 120 kv , 210 mas , ricostruzione in tempo reale 3 mm / 3 mm con fov 120 , ricostruzione per il postprocessing 1 mm / 0 , 5 mm con fov 80 . 
per lopacizzazione arteriosa sono stati somministrati in bolo , per via endovenosa antecubitale , 80 ml di mezzo di contrasto iodato non ionico ( iomeron 350 mgi / ml , bracco , milano , italia ) al flusso di 4 ml / s seguiti da 50 ml di soluzione salina allo stesso flusso . 
in the event of positive cta , only those patients whose cta findings were not complete for the purposes of surgical planning or were to undergo endovascular therapy were required to undergo dsa . 
cta and dsa findings were compared with regard to sensitivity , specificity and diagnostic accuracy . angioarchitettura : presenza o meno di vasi ad origine dalla sacca aneurismatica . tutti i pazienti con atc negativa sono stati sottoposti entro 48 ore a dsa . 
in caso di atc positiva sono stati inviati allo studio angiografico solamente i pazienti con quadro atc non esaustivo ai fini della programmazione chirurgica o quelli indirizzati allapproccio terapeutico endovascolare . infine , quando i reperti atc sono stati giudicati sufficienti per la pianificazione chirurgica , i pazienti sono stati sottoposti allintervento chirurgico senza ulteriori esami diagnostici . 
i dati atc ed angiografici sono stati confrontati in termini di sensibilit , specificit ed accuratezza diagnostica . all cta and dsa examinations were technically assessable . all patients who presented for the cta examination without assisted ventilation were studied without sedation ; about 60% of these required sedation at dsa . 
all cta and dsa procedures were without complications ( allergic reactions , acute renal failure or extravasation at the site of venous injection , more serious neurological complications or complications at the site of arterial catheterisation )  . all 21 patients with a negative cta underwent dsa . 
of the 109 patients with positive cta , 79 underwent emergency dsa to clarify the cta findings and plan therapy , and five underwent dsa during endovascular embolisation on average 13 days after the acute event ( 135.5 days , range 720 days )  . 
in 25 cases , the cta findings were considered complete for surgical purposes , such that these patients unrisultati tutti gli esami atc e dsa eseguiti sono risultati tecnicamente valutabili . 
tutte le procedure eseguite , atc e dsa , sono state esenti da complicazioni ( reazioni allergiche , ira o stravasi in sede di iniezione venosa , complicanze neurologiche maggiori o nella sede di cateterismo arterioso )  . tutti i 21 pazienti con atc negativa sono stati sottoposti a dsa . 
tra i 109 pazienti con atc positiva 79 sono stati sottoposti a dsa urgente per chiarire i reperti atc al fine della pianificazione terapeutica , 5 hanno eseguito lo studio angiografico durante la procedura di embolizzazione endova1325 513 total dimensioni ( mm ) 1325 513 totale results m . 
1 percorso diagnostico dei 130 pazienti arruolati nello studio . derwent dsa post - operatively only . of the 105 patients who underwent dsa , cta findings were confirmed in 104 : in 20 cases where the findings were negative and in 84 cases with the presence of one or more aneurysms . 
in 25 casi il quadro atc stato giudicato esaustivo ai fini dellintervento chirurgico , pertanto questi pazienti sono stati sottoposti a dsa solo come controllo post - operatorio . tra i 105 pazienti sottoposti a dsa il quadro atc stato confermato in 104 : in 20 casi per la negativit del reperto ed in 84 per la presenza di uno o pi aneurismi . 
solo in un caso la dsa ha dimostrato la presenza di un aneurisma del circolo anteriore delle dimensioni di 2 mm non riconosciuto allatc ; lidentificazione di questo piccolo aneurisma stata comunque possibile alla valutazione retrospettiva delle immagini . 
nei 25 pazienti sottoposti al trattamento chirurgico senza la valutazione dsa pre - operatoria stata riscontrata unottima corrispondenza tra le informazioni fornite dallatc ed il quadro chirurgico per quanto riguarda le dimensioni , la morfologia e langioarchitettura . 
2a - i a 41 - year - old woman : axial ( a ) , coronal ( b ) and sagittal ( c ) multiplanar reconstruction ( mpr ) images show a large aneurysm at the bifurcation of the left middle cerebral artery . 
axial ( d ) , coronal ( e ) and sagittal ( f ) maximum intensity projection ( mip ) images obtained with a 10 - mm - thin section more clearly show the aneurysms morphology , its multilobular profiles and relationship with surrounding vessels . 
volume rendering ( vr ) posteroanterior image ( h ) better shows vessels of cranial base and the aneurysvr and transparent vr images ( i ) : this last reconstruction shows a better three - dimensional ( 3d ) image of the aneurysmal sac and surrounding vessels . 
le ricostruzioni mpr assiale ( a ) , coronale ( b ) e sagittale ( c ) dimostrano un aneurisma di grandi dimensioni alla biforcazione dellarteria cerebrale media di sinistra . 
le ricostruzioni mip a strato sottile ( 10 mm ) assiale ( d ) , coronale ( e ) e sagittale ( f ) meglio definiscono la morfologia dellaneurisma , i profili polilobati ed i suoi rapporti con i vasi circostanti . 
3a - i a 51 - year - old woman : coronal ( a ) , axial ( b ) , sagittal ( c ) maximum intensity projection ( mip ) and volume rendering ( vr ) ( d - f ) images show a medium - sized aneurysm at the bifurcation of the right middle cerebral artery ( large arrow ) , a small one at the bifurcation of the left middle cerebral artery ( thin arrow ) and a very small aneurysm of the anterior communicating artery ( arrowhead )  . 
le ricostruzioni mip coronale ( a ) , assiale ( b ) , sagittale ( c ) e vrt ( d - f ) dimostrano un aneurisma di medie dimensioni alla biforcazione dellarteria cerebrale media destra ( freccia grande ) , un aneurisma di piccole dimensioni alla biforcazione dellarteria cerebrale media sinistra ( freccia sottile ) ed un aneurisma molto piccolo allarteria comunicante anteriore ( punta di freccia )  . 
4a - d a 38 - year - old woman : axial ( a ) , coronal ( b ) , sagittal ( c ) maximum intensity projection ( mip ) and volume rendering ( vr ) ( d ) images show a medium - sized aneurysm with a long neck of the right internal carotid artery ( arrow )  . 
ricostruzioni mip assiale ( a ) , coronale ( b ) , sagittale ( c ) e vrt ( d ) di un aneurisma di medie dimensioni dellarteria carotide interna destra con lungo colletto ( freccia )  . 
most aneurysms were of medium sized ( n = 67 ; 50% ) , followed by small ( n = 34 ; 25.4% ) , very small ( n = 16 ; 11.9% ) and large ( n = 16 ; 11.9% ) ( table 1 )  . overall , cta identified 108 of the 109 aneurysms seen at dsa and 25 aneurysms confirmed at surgery . 
6a , b a 60 - year - old man : axial ( a ) and coronal ( b ) maximum intensity projection ( mip ) images show a large , partially thrombosed , aneurysm at the bifurcation of the right middle cerebral artery ( arrow )  . 
7a - d an 81 - year - old man : axial ( a ) , coronal ( b ) paracoronal ( c ) maximum intensity projection ( mip ) and digital subtraction angiography ( dsa ) ( d ) images show a large aneurysm ( arrow ) with a wide neck of the significantly calcified left vertebral artery . 
ricostruzioni mip assiale ( a ) , coronale ( b ) , para - coronale ( c ) e corrispettivo dsa ( d ) di un aneurisma di grandi dimensioni con ampio colletto dellarteria vertebrale sinistra ( freccia ) , estesamente calcifica . 
filiforme larteria vertebrale destra ( b , punta di freccia )  . we also encountered excellent agreement between cta and dsa with regard to assessing the aneurysmal neck and angioarchitecture . 
these data were available for 92 of the 134 aneurysms in our study : we excluded the aneurysm not identified at cta , the 25 aneurysms treated surgically prior to dsa and the 16 aneurysms < 3 mm , the small size of which hindered realistic measurement of the neck . 
in 12 medium and large aneurysms prevalently located in the middle cerebral artery , the presence of a vessel originating directly from the aneurysmal sac was identified . complessivamente con atc sono stati riconosciuti 108 dei 109 aneurismi visualizzati con dsa cui vanno sommati i 25 aneurismi confermati allatto chirurgico . 
pertanto i valori di sensibilit e specificit raggiungono rispettivamente il 98 , 8% ed il 100% , la predittivit positiva del 100% e la predittivit negativa del 95 , 2% , con unaccuratezza diagnostica del 99 , 0% ( tabella 2 )  . abbiamo riscontrato unottima corrispondenza tra atc e dsa anche nella valutazione del colletto aneurismatico e dellangioarchitettura ; tali dati sono disponibili per 92 dei m . 
the technological developments of multidetector ct and especially the introduction and diffusion in clinical practice of 16 - slice scanners have led to improved spatialtemporal resolution , thus enabling an accurate and minimally invasive study of cerebral aneurysms , even in the event of acute sah . the possibility of performing cta immediately following a ct diagnosis of sah , along with the noninvasiveness of the technique , speed of execution and high diagnostic sensitivity and specificity , increasingly promote cta as a highly reliable technique in studying cerebral aneurysms . our prospective study involving 130 patients shows that in all cases , cta was technically feasible and was well tolerated by all patients . 
all examinations could be assessed , and there were no major motion artefacts . however , a limitation to our study was the absence of patients who had previously undergone neurosurgery or endovascular embolisation ; hence , we have no direct experience with artefacts caused by metal clips or coiled springs . 
in this respect , it should be noted that previous studies have demonstrated the validity of cta even in the post - operative follow - up [ 24 ]  . shorter acquisition times and increased spatial resolution are two important advantages introduced by 16 - slice scanners . 
the increased acquisition speed not only enables an exclusively arterial acquisition for the entire scan duration but also reduces the incidence of motion artefacts , which can seriously compromise image quality to the point of rendering the images nondiagnostic . 
this should not be underestimated in this type of patient , who is at times drowsy , at times suffering from psychomotor agitation and often uncooperative 134 aneurismi oggetto del nostro studio : abbiamo escluso laneurisma non riconosciuto con atc , i 25 aneurismi trattati chirurgicamente prima dellesecuzione dellangiografia ed i 16 aneurismi < 3 mm le cui piccole dimensioni non consentivano una reale definizione della base dimpianto . 
le due metodiche sono state concordi nel definire 48 aneurismi con colletto stretto e 44 aneurismi a larga base dimpianto . in 12 aneurismi di medie e grandi dimensioni , prevalentemente localizzati allarteria cerebrale media , stata identificata lemergenza di un vaso direttamente dalla sacca aneurismatica . discussione lo studio degli aneurismi cerebrali stato per lungo tempo affidato alla dsa , metodica invasiva , non priva di rischi per il paziente e caratterizzata da lunghi tempi di esecuzione . gli sviluppi tecnologici della tomografia computerizzata multidetettore ed in particolare lintroduzione e la diffusione nella pratica clinica delle apparecchiature a 16 strati , hanno portato ad un miglioramento della risoluzione spazio - temporale permettendo uno studio accurato e poco invasivo degli aneurismi cerebrali anche in caso di esa in fase acuta . la possibilit di effettuare latc subito dopo la diagnosi tc di esa , la non invasivit e la rapidit di esecuzione associate ad elevate sensibilit e specificit diagnostiche propongono sempre pi latc quale metodica altamente affidabile nello studio degli aneurismi cerebrali . questo studio prospettico relativo a 130 pazienti evidenzia che latc si dimostrata tecnicamente sempre eseguibile e ben tollerata dal paziente ; tutti gli esami sono risultati valutabili , in particolare non si sono registrati grossolani artefatti da movimento . per contro un limite di questo studio lassenza di pazienti gi precedentemente sottoposti ad interventi neurochirurgici o ad embolizzazione endovascolare : non abbiamo quindi esperienza diretta con gli artefatti da clips o spirali metalliche . 
a tale riguardo va aggiunto che precedenti studi hanno dimostrato la validit dellatc anche nei controlli post - operatori [ 24 ]  . la riduzione dei tempi di acquisizione e laumento della risoluzione spaziale sono due importanti vantaggi introdotti dagli apparecchi a 16 strati . 
la maggiore velocit di acquisizione non solo consente di ottenere unacquisizione esclusivamente arteriosa per tutta la durata della scansione , ma riduce lincidenza degli artefatti da movimento che possono inficiare la qualit delle immagini sino a renderle non diagnostiche . 
questo aspetto non va sottovalutato in questa tipologia di paziente : talvolta soporoso , talvolta in stato di agitazione psicomotoria e spesso comunque poco collaborante e poco incline a mantenere limmobilit del capo anche per pochi secondi . 
per nessuno dei pazienti sottoposti ad atc stata necessaria la sedazione , al contrario pi della met dei pazienti sottoposti a dsa ha richiesto lintervento dellanestesista al fine di ottenere immagini diagnostiche , prive di artefatti da movimento . un esame atc viene eseguito in circa 10 minuti cui va m . 
in contrast , a cerebral dsa study requires angiographic experience , and when performed by an expert has a duration of 4560 min , to which is added about 10 min for reporting the findings . the move from 4to 16 - slice devices has further increased spatial resolution . 
various studies performed with 4slice scanners have already demonstrated excellent diagnostic accuracy regarding aneurysms larger than 4 mm while underlining the limitations for smaller aneurysms [ 14 , 17 , 20 , 25 ]  . 
 mpr , which were assessed in cine mode in the three orthogonal planes in space , help confirm the presence of the aneurysm or determine whether it was not identified in the axial plane alone . 
mpr performed in various oblique planes or curved mpr along the vessel course helps define the relations with the surrounding vascular and bone structures and the size of the sac and neck , as well as parietal calcifications and partial thromboses . 
nonetheless , these easy - to - use reconstructions return a plastic , 3d - like image of the main vascular branches for most of their course , which often helps in the recognition of aneurysmal protrusions . 
these reconstructions , which are the most similar to traditional angiographic images , can be applied particularly sommato un tempo medio di elaborazione - lettura delle immagini di circa 1520 minuti : il referto quindi disponibile dopo 2530 minuti dallarrivo del paziente alla tc . 
al contrario lesecuzione di una panangiografia cerebrale richiede esperienza angiografica ed in mani esperte ha una durata media di 4560 minuti cui vanno sommati circa 10 minuti per la refertazione . il passaggio dai 4 ai 16 strati ha incrementato ulteriormente la risoluzione spaziale : questo ha permesso di aumentare le potenzialit diagnostiche dellatc soprattutto nellambito degli aneurismi di dimensioni molto piccole . 
diversi studi , condotti con apparecchi a 4 strati , hanno gi dimostrato lottima accuratezza diagnostica per gli aneurismi di dimensioni superiori ai 4 mm e sottolineato i limiti per quelli di dimensioni pi piccole [ 14 , 17 , 20 , 25 ]  . 
le ricostruzioni mpr secondo vari piani obliqui o mpr curvate lungo il decorso di un vaso aiutano a definire i rapporti con le strutture vasali ed ossee , le dimensioni della sacca e del suo colletto , calcificazioni parietali o trombosi parziali . le ricostruzioni in proiezione di massima intensit ( mip ) proiettano nel piano di ricostruzione solo i voxel pi luminosi del volume scelto : si tratta di ricostruzioni bidimensionali con perdita dellinformazione spaziale . 
tuttavia queste ricostruzioni , di facile applicazione , ci restituiscono unimmagine plastica , simil - 3d , delle principali diramazioni vasali per gran parte del loro decorso che spesso aiuta nel riconoscimento delle protrusioni aneurismatiche . 
le ricostruzioni mip , le pi vicine allimmagine angiografica tradizionale , trovano applicazione soprattutto nella valutazione delle diramazioni arteriose che si allontanano dal poligono di willis mentre non consentono una valutazione corretta delle strutture in stretto rapporto con la base cranica per lelevata attenuazione dellosso . 
it should also be borne in mind that very small aneurysms can be masked by vascular structures if the latter are projected in the same plane as the reconstruction . vr is a 3d reconstruction technique that requires definition of a threshold attenuation value ( measured in hounsfield units ) for selecting reconstruction voxels . 
these images help in interpreting the vascular relations and can be used to help in surgical planning , as they simulate the intraoperative view . postprocessing performed with a reduced fov enables improved spatial resolution . 
reconstructions in the various planes in space enable recognition and therefore sufficiently adequate characterisation of morphology , size , neck and development of an aneurysm as well as its relation to adjacent vessels and surrounding structures . 
the volumetric reconstructions , which are easy to obtain and have a high visual impact , return a plastic image that is often more useful in defining the aneurysms relations and development than in diagnosing it . 
it should also be borne in mind that only thin - section mpr , which are more laborious and less intuitive , enable correct identification of small irregularities of the sacs profile or partial thrombosis , where present . conclusions our study confirms the data in the literature . 
the ability of cta to define the relevant anatomical characteristics for surgical treatment , such as aneurysmal neck morphology , parietal calcification , sac thrombosis and relations with adjacent structures , as well as 3d reconstruction of the neurovascular angioarchitecture means that fundamental additional information can be supplied for deciding the next treatment steps . the results obtained in this study confirm the data in the literature and encourage the use of cta in routine clinical practice , with recourse to dsa only in selected cases or cases with uncertain diagnosis . 
at our institute , this study determined a change in managing patients with acute nontraumatic sah : in the event of positive cta finding for the presence of an aneurysm , in most cases , patients are scheduled for surgery without undergoing a dsa study . given the aetiology of nontraumatic sah and its highlevel of risk in the event of an incorrect diagnosis , fundamental requisites for cta study include correct technical execution and careful and interactive image assessment : where there is doubt , patients should undergo dsa . 
with regard to the future , cta is also a promising technique for follow - up to verify exclusion of the aneurysmal sac after endovascular or surgical procedures . essere mascherati dalle strutture vascolari se queste si proiettano nello stesso piano di ricostruzione . lelaborazione volume rendering technique ( vrt ) una tecnica di ricostruzione tridimensionale che richiede la definizione di un valore soglia di attenuazione ( in unit hounsfield ) per la selezione dei voxel di ricostruzione . 
queste immagini facilitano linterpretazione dei rapporti vascolari e possono essere di aiuto nella pianificazione dellapproccio chirurgico , simulando la visione intraoperatoria . le rielaborazioni con field of view ( fov ) ridotto consentono una migliore risoluzione spaziale : le ricostruzioni secondo i vari piani dello spazio permettono di riconoscere e quindi di caratterizzare in maniera adeguata morfologia , dimensioni , base dimpianto e sviluppo di un aneurisma oltre ai suoi rapporti con i vasi adiacenti e le strutture circostanti . 
le ricostruzioni volumetriche ( vrt ) facili da ottenere e di sicuro impatto visivo , restituiscono unimmagine plastica spesso pi utile nella definizione dei rapporti e dello sviluppo dellaneurisma che non nella sua diagnosi ; sono spesso richieste dai neurochirurghi perch meglio rispecchiano la spazialit e la visualizzazione intraoperatoria . 
non va dimenticato tuttavia che solo le ricostruzioni multiplanari sottili , pi laboriose e meno intuitive , ci consentono di identificare correttamente eventuali piccole irregolarit dei profili della sacca o la sua parziale trombosi . conclusioni questo studio conferma i dati della letteratura : latc una metodica poco invasiva , rapida , altamente sensibile e specifica nella diagnosi di aneurismi cerebrali nelle esa . 
la capacit dellatc di definire caratteristiche anatomiche rilevanti per il trattamento chirurgico quali la morfologia del colletto aneurismatico , le calcificazioni parietali , la trombosi della sacca ed i rapporti con le strutture adiacenti e la ricostruzione tridimensionale dellangioarchitettura neurovascolare consentono di fornire informazioni aggiuntive fondamentali per decidere il successivo trattamento dei pazienti . 
nella nostra realt questo studio ha determinato un cambiamento nella gestione del paziente con esa acuta non traumatica : in caso di reperto angio - tc positivo per la presenza di un aneurisma la maggior parte dei pazienti viene portata allintervento chirurgico senza lesecuzione della dsa . considerando leziologia dellesa non traumatica e la sua pericolosit in caso di errata diagnosi , uno studio atc non pu prescindere da unesecuzione tecnicamente corretta e da una valutazione attenta ed interattiva delle immagini : nei casi dubbi il ricorso alla dsa rappresenta tuttora lopzione di scelta . 
bonomo , i - 70031 andria ( ba ) , italy 2dipartimento di scienze radiologiche , irccs casa sollievo della sofferenza , i - 71013 san giovanni rotondo ( fg ) , italy 3dipartimento di radiologia , fondazione s . 
giglio , cefal ( pa ) , italy 4istituto di radiologia , universit di novara , italy 5istituto di radiologia , universit di ancona , italy correspondence to : t . 
scarabino , via napoli 56 , i - 70031 andria ( ba ) , italy , tel . : + 39 - 0883 - 299140 , fax : + 39 - 0883 - 299220 , e - mail : tscarabino@hotmail.com received : 19 may 2006 / accepted : 12 july 2006 / published online : 22 february 2007 abstract ever since the introduction of magnetic resonance ( mr ) , imaging with 1.5 tesla ( t ) has been considered the gold standard for the study of all areas of the body . 
indeed , their high gradient power and field intensity ( 3.0 t ) allow adjunctive and more advanced diagnostic methodologies to be performed with excellent resolution in a fraction of the acquisition time required with earlier machines . 
pi recentemente , lintroduzione di unit rm con intensit di campo di 3 , 0 t per applicazioni cliniche nuove ed avanzate ha comportato notevoli vantaggi , soprattutto in campo neuroradiologico . 
i loro alti gradienti ed intensit di campo infatti consentono di applicare metodologie diagnostiche nuove ed avanzate con eccellente risoluzione in una frazione del tempo di acquisizione richiesto dalle attrezzature precedenti . 
continuous advances in hardware ( gradients , coils ) and software ( acquisition and processing sequences ) have made magnets more compact , powerful and flexible , greatly improving patient comfort . 
higher - field units were introduced some years ago for clinical research purposes , principally in neuroradiology , and are spreading fast ( especially in the usa ) despite their dallavvento della risonanza magnetica ( rm ) le apparecchiature con intensit di campo magnetico di 1 , 5 tesla ( t ) sono considerate il gold standard per coprire le varie applicazioni cliniche in tutti i distretti corporei . 
tali apparecchiature , in virt di continui progressi che hanno interessato lhardware ( gradienti , bobine ) e il software ( sequenze di acquisizione e di elaborazione ) , sono diventate sempre pi compatte , potenti e versatili , con conseguente migliore confort del paziente . 
they are being employed not only in research and with a broader scope compared with traditional research but also in daily clinical practice to support new and sophisticated clinical applications , with considerable practical benefits . 
in the first year , the machine was employed to optimise the various morphological and functional acquisition sequences ( diffusion , perfusion , spectroscopy , cortical activation ) both in vitro ( using dedicated dummies ) and in vivo ( in healthy controls ) and the various postprocessing techniques ( with dedicated software ) with a view to creating a large database as the starting point for research on patients . 
naturally , special attention was devoted to the safety problems related to the machines use , especially issues of patient comfort in the tunnel and compatibility of various biomedical devices . after optimisation of study sequences and protocols , our research programme focused on improving morphofunctional mr imaging of the bra the higher signal - to - noise ratio ( snr ) of the 3.0 - t magnet made it possible to obtain images with greater spatial , contrast and time resolutions in shorter acquisition times compared with lower - field systems , resulting in greater patient comfort and reduction of motion artefacts . the patients studied at 3.0 t exhibited a wide and representative range of conditions that were successfully investigated using standardised dedicated sequences and protocols . 
disadvantages , which include a greater specific absorption rate ( sar ) , greater acoustic noise are not particularly severe and in newer systems are largely reduced by improved hardware and software [ 14 ]  . 
tali sistemi sono ormai diventati una realt e , nonostante lalto costo , si stanno sempre pi diffondendo ( specie in usa ) per essere utilizzati non solo nel campo della ricerca , peraltro molto pi ampia rispetto alla ricerca tradizionale , ma anche e soprattutto nella pratica clinica quotidiana per far fronte a nuove e pi sofisticate applicazioni cliniche con conseguenti importanti benefici pratici . da 5 anni il servizio di neuroradiologia del nostro istituto scientifico dotato di una apparecchiatura rm whole body ( signa horizon lx , general electric medical system , milwaukee , usa ) dotata di magnete superconduttivo , a schermatura attiva , con intensit di campo magnetico 3 , 0 t , gradienti di potenza di 50 millitesla al metro ( mt / m ) e slew rate di 150 mt / m / s . 
il sistema rm 3 , 0 t in dotazione stato utilizzato nel primo anno di lavoro , una volta acquisita lautorizzazione ministeriale alluso , per ottimizzare le diverse sequenze di acquisizione , morfologiche e funzionali ( diffusione , perfusione , spettroscopia , attivazione corticale ) , sia in vitro ( utilizzando fantocci dedicati ) che in vivo ( con controlli sani ) , e le diverse modalit di post - processing ( utilizzando software dedicati ) al fine di creare un ampio database , punto di partenza per studi di ricerca su paziente . 
naturalmente , particolare attenzione stata data alle problematiche di sicurezza inerenti luso di tale apparecchiatura ( con riferimento in particolare alla qualit di confort del paziente in esame e alla rm - compatibilit di vari dispositivi biomedicali )  . ottenuta lottimizzazione di sequenze e protocolli di studio , lobiettivo del nostro programma di ricerca stato quello di migliorare limaging morfo - funzionale cerebrale con rm . 
sfruttando il pi alto rapporto segnale / rumore ( s / r ) possibile con lapparecchiatura 3 , 0 t , stato infatti possibile ottenere immagini con pi alta risoluzione spaziale , di contrasto e temporale , in tempi peraltro ridotti rispetto ai sistemi di pi basso campo , con conseguente miglioramento del confort del paziente e riduzione degli artefatti da movimento . i pazienti sottoposti a rm 3 , 0 t hanno esibito un ampio e rappresentativo spettro di patologia che stata esaminata adeguatamente utilizzando sequenze e protocolli ben definiti e personalizzati . si quindi proceduto allo studio di diverse patologie seguendo le linee dei progetti di ricerca approvati dalla direzione scientifica del nostro istituto , previa autorizzazione del comitato etico . 
lo scopo del lavoro quello di illustrare le caratteristiche semeiologiche peculiari dellimaging cerebrale morfologico e angiografico a 3 , 0 t mettendo in risalto i relativi vantaggi e svantaggi rispetto a sistemi rm di pi basso campo . studio morfologico di base gli studi rm del cervello possono essere suddivisi in due grandi categorie , morfologici e funzionali , sulla base del tipo di informazione che si ricerca . 
lo studio morfologico di base ( detto anche convenzionale o anatomico ) riguarda limaging di tessuto , spazi periliquorali , vascolatura e fasci di fibre , mentre lo studio funzionale in senso lato comprende lo studio delle funzioni cerebrali ( fmri ) limaging di perfusione t . 
 because the user interface and sequence parameters are the same as those employed with 1.5 - t systems , at 3.0 t , in theory , the same images should be obtained with the advantage of high - field strength , i.e. 
 t1 imaging as t1 relaxation times are longer at higher magnetic fields , entailing reduction in the relative differences among different tissue types , it is generally difficult to obtain t1 - weighted images with sufficient contrast when using a 3.0 - t unit [ 6 ]  . 
for this reason , snr optimisation in the same sample using a higher - field unit requires longer time to repetition ( tr ) [ and a smaller flip angle ( fa ) ] , resulting in longer scanning times . 
t1 saturation for a given tr is greater at 3.0 t , resulting in reduced signal gain . in other words , the snr per unit of time would be optimised with the shortest possible tr / t1 . 
the possibility that the snr gain connected with the high magnetic field may substantially be offset by a longer t1 highlights the importance of optimising the imaging parameters [ 3 ]  . 
 e di diffusione , e lo studio metabolico con la spettroscopia . lo studio di rm di base si presenta a 3 , 0 t con un imaging di qualit pi alta rispetto a quello ottenuto a pi basso campo in virt di una serie di vantaggi quali un pi alto rapporto s / r , una maggiore risoluzione spaziale e temporale a fronte di alcuni svantaggi , quali lincremento del specific absorption rate ( sar ) e del rumore acustico , peraltro non particolarmente significativi e comunque in gran parte risolvibili mediante hardware e software di pi recente introduzione [ 14 ]  . 
dato che i tempi di rilassamento sono campo - dipendenti e influenzano il contrasto dellimaging , le sequenze di imaging a 1 , 5 t non possono infatti essere trasferite a 3 , 0 t . 
a ci si aggiunge la deposizione tissutale dellenergia di rf che , essendo eccessiva , va anche considerata per adattare e ottimizzare i protocolli clinici di imaging per luso a 3 , 0 t . 
 fast spgr allows thinner slices to be obtained in a shorter time and provides images suitable for reformatting in all three planes using a single volumetric acquisition of the whole brain , thus disclosing a larger number of lesions than can be depicted using se t1 - weighted sequences acquired in a single plane . 
infatti , non solo il t1 aumenta con laumentare del campo magnetico , ma addirittura converge , determinando una distribuzione meno ampia dei valori di t1 tra i diversi tipi di tessuto . 
per tale motivo per ottimizzare il rapporto s / r dello stesso campione a campo pi alto il tr deve essere pi lungo ( come anche il flip angle deve essere pi corto ) con conseguente allungamento dei tempi desame . utilizzando tr pi lunghi , il vantaggio in intensit di segnale a campo pi alto risulta essere inferiore in unit di tempo . la saturazione t1 per un dato tr infatti pi ampia a 3 , 0 t con conseguente riduzione del guadagno in segnale . 
quindi in virt del fatto che il guadagno nel rapporto s / r derivante dallalto campo pu essere sostanzialmente controbilanciato dallallungamento del t1 , risulta importante lottimizzazione dei parametri di imaging [ 3 ]  . lincremento del t1 rappresenta il pi grande problema per lo studio cerebrale a 3 , 0 t acquisito con sequenza se t1 pesata in cui il contrasto t1 appunto inaccettabile [ 1 , 2 , 4 , t . 
for instance , when studying primary and secondary brain tumours , administration of a gadolinium - based agent yields greater contrast between tumour and normal brain tissue both in single and in multiple doses . 
it also detects more metastases and demonstrates different patterns of enhancement that may be useful to assess the degree of malignancy and monitor response to therapy [ 2628 ]  . 
the greater sensitivity of 3.0 - t contrast - enhanced investigations has also been demonstrated in multiple sclerosis , with a 21% increase in the number of enhancing lesions detected , a 30% increase in enhancing lesion volume and a 10% increase in total lesion volume relative to scanning at 1.5 t [ 29 ]  . 
using a standard dose , the contrast - to - noise ratio ( cnr ) is two and a half times greater than with 1.5 - t systems [ 13 ]  . 
double or triple doses can result in meningeal contrast uptake , a common nonpathological finding at 3.0 t that at lower field strengths may , however , mimic carcinomatosis or meningitis and should thus be avoided . 
to enhance sensitivity or highlight minimal changes in the blood - brain barrier , a double dose of contrast agent may be employed in selected investigations of brain metastases or inflammatory disease [ 30 , 31 ]  . 
high spatial resolution fast spin echo ( fse ) - weighted images are especially effective at 3.0 t , as they provide more anatomical detail and better contrast due to the greater snr , which allows the use of thinner slices and broader matrices [ 1 , 2 , 4 , 32 ]  . 
tale sequenza inoltre caratterizzata da ipersegnale delle grosse strutture vascolari arteriose che comunque non inficia linterpretazione delle immagini . mentre gli studi t1 pesati sono tradizionalmente meno informativi e richiedono pi tempo , un tipico studio flair t1 pesato del cervello consente , insieme allutilizzo dellimaging parallelo , lapplicazione di protocolli a pi alta risoluzione con tempi desame pi brevi rispetto ai campi ad intensit 1 , 5 t . 
naturalmente tale aspetto diventa problematico se dobbiamo acquisire pi studi , senza e con mdc , o pi proiezioni . in rapporto al maggiore effetto inflow del mdc e alla maggiore soppressione del fondo , limaging t1 con mdc sicuramente un vantaggio del 3 , 0 t rispetto al 1 , 5 t . 
nello studio ad esempio dei tumori cerebrali primitivi e secondari la somministrazione del gadolinio produce un maggior contrasto tra lesione tumorale e parenchima normale sia a singola dose e a dose multipla , permette di rilevare un maggior numero di metastasi , pu dimostrare differenti pattern di impregnazione che risultano utili per definire il grado di malignit e per monitorare la risposta alla terapia [ 2628 ]  . 
rispetto al 1 , 5 t infatti alcuni autori hanno documentato un incremento del 21% del numero delle lesioni con impregnazione patologica , del 30% nel volume di impregnazione e del 10% del volume totale delle lesioni [ 29 ]  . 
il rapporto contrasto / rumore ( c / r ) risulta infatti maggiore rispetto allo studio con sistemi rm 1 , 5 t ( di ben 2 , 5 volte ) utilizzando una dose standard [ 13 ]  . 
da evitare la dose doppia o tripla responsabile di impregnazioni meningee , reperti comuni non patologici a 3 , 0 t ( con sistemi 1 , 5 t tali quadri possono simulare una carcinomatosi o una meningite )  . 
3 imaging fse t2 ad alta definizione della sezione mediana del cervello . infiammatoria per incrementare la sensibilit o per stressare le minime alterazioni della barriera emato - encefalica ( bee ) , una dose doppia pu essere preferita [ 30 , 31 ]  . 
di solito nello studio t1 con mdc la fast ge t1 pesata viene preferita , al posto delle tradizionali se , che presentano un contrasto minore a parit di tempo dacquisizione . 
ne consegue una migliore risoluzione spaziale ( e quindi maggiore dettaglio anatomico ) e di contrasto , possibili per effetto di un pi alto rapporto s / r che permette luso di spessore di strato inferiori e di matrici pi ampie [ 1 , 2 , 4 , 32 ]  . 
2a , b studio di microadenoma ipofisario prima ( a ) e dopo mdc endovena ( b ) con sequenza fse - xl t1 ( tr / te 600 / 12 , etl 5 , fov 180 mm , spessore di strato 2 , 5 mm / 0 , 5 , matrice 224192 , zip 512 , 29 / fase )  . creased deposition of radiofrequency ( rf ) energy , which in turn requires long tr or pulse refocalisation angles well below 180 . 
in addition , the broader bandwidth , smaller echo space , shorter minimum tr and te and shorter fse sequences employed at higher field strengths result in improved image quality , artefact reduction and enhanced diagnostic performance in much shorter examination times , which is to the patients benefit . 
these sequences allow excellent visualisation of the basal or mesencephalic nuclei by virtue of their iron content despite the intrinsically lower sensitivity of fse to magnetic susceptibility in 3.0 - t compared with 1.5 - t systems [ 5 ]  . 
4a - d high - definition ( hd ) study of the inner ear using a 3d fast imaging employing steady state acquisition ( fiesta ) sequence [ time to repetition ( tr ) 4.3 , echo time ( te ) 1.5 , flip angle ( fa ) 50 , band width ( bw ) 62 khz , field of view ( fov ) 170 , slice thickness 0.6 mm , matrix 256256 , zero fill interpolation processing ( zip ) 512 , 2 , number of excitations ( nex ) , 4 : 43 ]  . 
4a - d studio ad alta definizione dellorecchio interno con sequenza 3d fiesta ( tr / te 4 , 3 / te 1 , 5 , flip angle 50 , bw 62 khz , fov 170 , spessore di strato 0 , 6 mm , matrice 256256 , zip 512 , 2 nex , 4 : 43 ) : singole partizioni ( a ) , zoom ( b ) , mip ( c ) , elaborazione con ricostruzione di volume ( d )  . rhage , which at 3.0 t is characterised by greater hypointensity in the acute and early subacute phases [ 33 ]  . 
 flair imaging flair sequences , which are generally included in all imaging protocols , are also sharper and more sensitive at 3.0 t sono pi bassi , quindi il tempo in fse si riduce , la qualit aumenta , gli artefatti si riducono . 
con tale sequenza sono ben apprezzabili i nuclei della base o del mesencefalo che risaltano maggiormente ( per effetto del loro contenuto di ferro ) , nonostante la nota intrinseca minore sensibilit alla suscettibilit magnetica della fse , rispetto a sistemi 1 , 5 t [ 5 ]  . una variazione di semeiotica del segnale sembra interessare anche le emorragie cerebrali caratterizzate in particolare da una maggiore ipointensit a 3 , 0 t in fase acuta e subacuta precoce rispetto allintensit di segnale rilevabile a 1 , 5 t [ 33 ]  . 
a tal proposito il c / r e lintensit di segnale a 1 , 5 t e 3 , 0 t sono sostanzialmente equivalenti per cui di solito comunque non vi sono particolari problemi per interpretare correttamente let dellemorragia . 
first , it allows performance of all angiographic sequences applied routinely in clinical practice with lowerfield systems , such as 2d and 3d time of flight ( tof ) and phase contrast ( pc ) , as well as ultrafast dynamic sequences acquired after contrast agent bolus administration [ contrastenhanced mra ( ce - mra ) ]  . 
as with routine mr scanning , the technical parameters of angiographic sequences need to ge ( t2 * ) quali ad esempio la ciss ( constructive interference in the steady state ) particolarmente indicata nello studio dellorecchio interno . 
la maggiore sensibilit si ha anche grazie alluso di te pi lunghi , necessari per i tempi di rilassamento modificati a 3 , 0 t , che permettono un buon contrasto ed un rumore accettabile . 
il rilievo di piccole lesioni richiede infatti lottimizzazione del c / r piuttosto che del rapporto s / r e questo viene ottenuto appunto con te pi lunghi ( 120 ms ) anche se tale allungamento incrementa oltre il contrasto anche il rumore ma in modo accettabile . inoltre , contrariamente a quanto accade per sistemi 1 , 5 t , in virt della sostanziale mancanza di differenza del t1 del liquor ( che essenzialmente un liquido ) tra 1 , 5 t e 3 , 0 t , il tempo di inversione ( ti ) richiesto per annullare il liquor rimane dello stesso valore ( 2 , 250 millisecondi circa ) [ 5 ]  . tale caratteristica non valida in altre sequenze ir , sia in quelle in cui si cerca di massimizzare il contrasto tra differenti tessuti ( ad esempio tra sostanza bianca e sostanza grigia ) sia in quelle con soppressione del grasso , nelle quali invece il ti deve essere incrementato del 25%40% . 
le note aree di sostanza bianca soprattutto periventricolare fisiologicamente iperintense con sistemi a pi basso campo ( 1 , 5 t ) sono ancora pi accentuate a 3 , 0 t per cui c bisogno di una particolare attenzione per distinguerle da patologie reali ( ad esempio sclerosi laterale amiotrofica ) [ 1 ]  . 
in addition , the change in t1 relaxation time , namely , increased t1 , yields greater background suppression of stationary tissues due to the decrease of r1 , i.e. 
the rate of longitudinal or spin - lattice relaxation , with greater flow enhancement , as blood r1 is roughly constant thereby improving vessel - tissue contrast [ 19 , 22 , 36 ]  . 
this advantage makes magnetisation transfer application less effective than it is with 1.5 t systems [ 37 , 38 ]  . the effect is mainly evident using the 3d tof technique , as the short tr usually saturates stationary tissues but not moving blood [ 7 , 39 ] , and the tag persists for a longer time . these effects lead to greater vessel detail and conspicuity , especially in disclosing small - calibre vessels , including surface vessels not clearly depicted at 1.5 t [ 23 , 25 ]  . 
il recente sviluppo di nuovi hardware ( nuove coil ) o lutilizzo di pi recenti software ( imaging parallelo ) potranno ridurre tale inconveniente . studio angiografico con il 3 , 0 t lo studio angiografico si avvantaggia in modo significativo rispetto a sistemi rm di pi basso campo ( fig . 7 ) [ 1 , 2 , 4 , 35 ]  . 
innanizitutto un sistema rm a 3 , 0 t permette di eseguire tutte le sequenze angiografiche utilizzate di solito nella pratica clinica con sistemi a pi basso campo quali le pi comuni time of flight ( tof ) o phase contrast ( pc ) , entrambe con modalit 2d o 3d , od anche quelt . 
therefore , contrast administration further improves imaging at 3.0 t due to the high snr , which can be transformed into spatial resolution : resolution on a plane le ultrarapide utilizzate con tecnica dinamica dopo somministrazione a bolo del mdc ( contrast enhancement magnetic resonance angiography , ce - mra )  . 
tale vantaggio , che rende peraltro non particolarmente efficace ( come invece accade con sistemi 1 , 5 t ) luso della magnetization transfer ( mt ) [ 37 , 38 ] apprezzabile soprattutto quando si utilizza la tecnica angiografica 3d tof in quanto il breve tr satura di solito i tessuti stazionari ma non il sangue in movimento [ 7 , 39 ]  . 
tali effetti comportano un incremento del dettaglio e della conspicuity vascolare con in particolare evidenza di vasi di pi piccolo calibro , anche superficiali ( di iv ordine ) , non chiaramente identificabili con sistemi rm 1 , 5 t [ 23 , 25 ]  . per lo stesso motivo lutilizzo di campi magnetici elevati ( 3 , 0 t ) sembra in grado di migliorare la cospicuit dei vasi intracranici del neonato o di ridurre ulteriormente i tempi di acquisizione [ 40 ]  . 
lo studio angio - rm a 3 , 0 t in pazienti portatori di tali dispositivi biomedicali ( purch gi testati ) pu essere quindi eseguito senza particolari rischi [ 33 , 45 ]  . i benefici ottenuti a 3 , 0 t , evidenti soprattutto nella patologia vascolare malformativa ( specie aneurismatica ) e atefig . 
time - resolved imaging of contrast kinetics ( tricks ) images [ 3d time of flight spoiled gradient recalled ( tof - spgr ) , time to repetition ( tr ) 3.7 , echo time ( te ) 1.3 , matrix 224192 , slice thickness 1.8 mm , field of view ( fov ) 28 , temporal resolution 2.5 s , 8 phases ] of multiple vascular phases : contrast enhancement of arteries ( a ) , capillaries ( b ) and veins ( c )  . 
in tale modo possibile visualizzare diverse fasi vascolari : arterie ( a ) , capillari ( b ) e vene ( c )  . ments are increased at higher - field strengths resulting in artefacts caused by the susceptibility of biomedical devices such as the aneurysm clips commonly used in interventional and therapeutic neuroradiological procedures , the newer devices do not seem to present major safety or compatibility problems [ 42 , 44 ]  . 
resolution quality tends to be privileged in clinical practice , with acquisition of a larger number of slices of rerosclerotica cerebrale , rendono ancora pi competitiva langiografia con rm , nonostante una minore risoluzione spaziale rispetto allangiografia convenzionale ( dsa ) , ormai sempre pi utilizzata in campo intervenzionistico - terapeutico piuttosto che diagnostico [ 46 ]  . conclusioni grazie al suo alto rapporto s / r , la mri ad alto campo in grado , di supportare nuove applicazioni diagnostiche o di migliorare le metodiche esistenti . 
in virt dellalto rapporto s / r il neuroradiologo pu scegliere di eseguire un imaging cerebrale a 3 , 0 t in un tempo uguale a quello ottenibile con un sistema 1 , 5 t privilegiando in tal caso una pi alta qualit delle immagini ( incrementando la matrice e / o riducendo lo spessore di strato e / o il fov ) oppure di avere un imaging di qualit pari al quello di un sistema 1 , 5 t in un tempo inferiore privilegiando in tal caso il confort del paziente , od anche pu scegliere di ridurre la dose del mezzo di contrasto somministrato . 
di solito nella pratica clinica si cerca di privilegiare la risoluzione acquisendo pi fette , di spessore sottile , con campi di vista pi piccoli , con matrici pi ampie e in tempi comparabili a quelli con i sistemi 1 , 5 t . 
spesso in rapporto al quesito clinico e al tipo di paziente in esame , si cerca di trovare un compromesso tra risoluzione e velocit ( riducendo ad esempio il numero delle eccitazioni ) [ 34 ]  . lalto rapporto s / r per effetto dellalto campo pu in realt essere ottenuto anche a 1 , 5 t utilizzando i pi recenti hardware ( bobine riceventi di superficie , single o in arrays ; gradienti pi performanti capaci di ridurre il te ) e t . 
however , this cannot be done in the case of unstable or poorly co - operative patients , or it may be impossible for cost reasons ( reduced patient flow )  . 
lastly , the higher snr of high - field mr systems is a resource that neuroradiologist can exploit in clinical practice , as the higher the snr , the more the imaging protocols can be adjusted . acknowledgments to technical staff : c . 
il caso di pazienti instabili , non collaboranti , come anche ci possono essere altre motivazioni ad esempio di natura economica ( la riduzione del flusso dei pazienti )  . 
non solo , anche a 1 , 5 t possibile un incremento delle dimensioni della matrice : in tal caso per la conseguente riduzione delle dimensioni del voxel comporterebbe maggiore granulosit dellimaging . 
pertanto il maggiore rapporto s / r da alto campo sicuramente unottima arma che il neuroradiologo pu sfruttare nella pratica clinica : maggiore il rapporto s / r , maggiori sono infatti le possibilit del neuroradiologo di modificare i protocolli di imaging . rigraziamenti allo staff tecnico : c . 
bonomo , i - 70031 andria ( bat ) , italy 2dipartimento di scienze radiologiche , irccs casa sollievo della sofferenza , i - 71013 san giovanni rotondo ( fg ) , italy 3dipartimento di radiologia , fondazione s . 
giglio , cefal ( pa ) , italy 4dipartimento di fisica , irccs stella maris , pisa , italy 5istituto di radiologia , universit di napoli , italy 6istituto di radiologia , universit di pisa , italy 7istituto di neurologia , universit di campobasso , italy 8istituto di psichiatria , universit di bari , italy 9istituto di radiologia , universit politecnica delle marche , ancona , italy correspondence to : t . 
scarabino , via napoli 56 , i - 70031 andria ( bat ) , italy tel . : + 39 - 0883 - 299140 , fax : + 39 - 0883 - 299220 , e - mail : tscarabino@hotmail.com received : 19 may 2006 / accepted : 8 september 2006 / published online : 22 february 2007 abstract the aim of this paper is to illustrate the technical , methodological and diagnostic features of functional imaging ( comprising spectroscopy , diffusion , perfusion and cortical activation techniques ) and its principal neuroradiological applications on the basis of the experience gained by the authors in the 5 years since the installation of a high - field magnetic resonance ( mr ) magnet . these mr techniques are particularly effective at 3.0 tesla ( t ) owing to their high signal , resolution and sensitivity , reduced scanning times and overall improved diagnostic ability . 
the most common perfusion study ( with intravenous injection of a contrast agent ) benefits from the greater snr and higher magnetic susceptibility by achieving dramatically improved signal changes , and thus greater reliability , using smaller doses of contrast agent . functional mr imaging ( fmri ) is without doubt the modality in which high - field strength has had the greatest impact . 
images acquired with the blood - oxygen - level - dependent ( bold ) technique benefit from the greater snr afforded by 3.0 - t magnets and from their stronger magnetic susceptibility effects , providing higher signal and spatial resolution . 
this enhances reliability of the localisation of brain functions , making it possible to map additional riassunto lo scopo del lavoro quello di illustrare le caratteristiche tecnico - metodologiche e semeiologiche dellimaging funzionale ( comprendente lo studio di spettroscopia , diffusione , perfusione , attivazione corticale ) con cenni sulle principali applicazioni in campo neuroradiologico sulla base dellesperienza acquisita in 5 anni dallinstallazione di unapparecchiatura ad alto campo . 
tali modalit di studio rm risultano particolarmente migliorate a 3 , 0 t grazie ai numerosi vantaggi ( alto segnale , alta risoluzione , alta sensibilit , tempi ridotti , alta capacit diagnostica )  . 
ne consegue uno studio spettroscopico ad alta risoluzione , in tempi peraltro ridotti , non solo dei metaboliti comuni ma anche e soprattutto di quelli che , in virt della loro bassa concentrazione , difficilmente sono rilevabili con sistemi 1 , 5 t . 
lo studio di perfusione pi diffuso ( con mdc esogeno ) , in virt del maggiore s / r e della pi alta suscettibilit magnetica a 3 , 0 t , caratterizzato da variazioni di segnale drasticamente migliorate con conseguente maggiore affidabilit utilizzando peraltro minori quantit di mdc . 
le immagini ottenute con la tecnica bold infatti se ne avvantaggiano a 3 , 0 t in virt del maggiore s / r e dei maggiori effetti di suscettibilit magnetica con conseguente pi alto segnale e pi alta risoluzione spaziale . 
the data presented and results obtained to date show that 3.0 - t morphofunctional imaging can become the standard for highresolution investigation of brain disease . in definitiva sulla base dei risultati finora ottenuti , si evince che limaging morfo - funzionale ottenuto a 3 , 0 t potr in futuro diventare lo standard per lo studio ad alta definizione della patologia cerebrale . key words magnetic resonance fmri high field parole chiave risonanza magnetica imaging funzionale con rm alto campo introduction introduzione the function of the new 3.0 - tesla ( t ) magnetic resonance ( mr ) systems is to enable high spatial , contrast and time resolution morphological investigation of the brathis is essential for diagnosis but also indeed , especially to provide physiological , metabolic and functional information capable of enhancing the diagnostic power of standard mr in terms of sensitivity and specificity . 
it is well established that standard mr , the modality of choice to investigate pathological central nervous system ( cns ) conditions , is not despite its high sensitivity suitable for characterising all brain tissue abnormalities . 
hence the need for integrating morphological parameters with physiological , metabolic and functional data that may predate anatomical changes , thus promoting early diagnosis and treatment and therefore better patient outcomes . 
these data can also be obtained with mr , especially if high - field units are used . the aim of the work was to illustrate technical , methodological and diagnostic characteristics of functional mr imaging ( fmri ) [ including spectroscopy ( mrs ) , diffusionweighted imaging ( dwi ) , perfusion - weighted imaging ( pwi ) and cortical activation ] and its main neuroradiological applications based on a 5 - year experience with a high - field magnet . 
the material presented comes from work performed by the medical and technical team of the neuroradiology and physics unit of our institution in collaboration with largely italian and some foreign neuroradiologists , neuropsychiatrists , neurophysiologists , psychologists , bioengineers , physicists and other neuroimaging experts . 
the work is supplemented with literature data from other similar experiences to provide an exhaustive review of the state of the art of this field . proton spectroscopy il ruolo dei nuovi sistemi di risonanza magnetica ( rm ) 3 , 0 t deve essere quello di permettere uno studio morfologico cerebrale ad alta risoluzione spaziale , temporale e di contrasto , essenziale per la diagnosi , ma anche e soprattutto di fornire informazioni fisiologiche , metaboliche e funzionali , capaci di aumentare il potere diagnostico della rm di base in termini di sensibilit e specificit . 
a tutti noto che lo studio morfologico di base con rm , considerato lesame delezione per patologie del sistema nervoso centrale , nonostante la sua indubbia sensibilit , non sempre specifico nel caratterizzare tessuto cerebrale anomalo . 
da qui la necessaria integrazione dei parametri morfologici con quelli fisiologici , metabolici e funzio nali individuabili anche con rm , specie se eseguito con apparecchiature ad alto campo , che possono invece precedere le alterazioni anatomiche , favorendo una diagnosi e un trattamento precoci e quindi un migliore outcome del paziente . lo scopo del lavoro quello di illustrare le caratteristiche tecnico - metodologiche e semeiologiche dellimaging funzionale con rm ( comprendente lo studio di spettroscopia , diffusione , perfusione , attivazione corticale ) con cenni sulle principali applicazioni in campo neuroradiologico . 
il materiale raccolto in questo lavoro frutto dellesperienza acquisita in 5 anni dallinstallazione di tale apparecchiatura da parte dellequipe medica e tecnica delle unit operative di neuroradiologia e di fisica sanitaria del nostro centro in collaborazione con neuroradiologi , neuropsichiatri , neurofisiologi , psicologi , bioingegneri , fisici o comunque esperti di neuroimaging il cui apporto multidisciplinare essenziale per sfruttare al massimo le potenzialit di tale apparecchiatura . 
lo scritto stato completato con i risultati di altre esperienze similari raccolte dalla letteratura al fine di fornire uno stato dellarte completo sulla tematica in oggetto . the increased signal - to - noise ratio ( snr ) and greater spatial and temporal chemical shift resolution of high - field units yield improved spectroscopic imaging compared with lowerfield mr [ 17 ]  . increment of snr and chemical - shift resolution studio di spettroscopia protonica con lalto campo la spettroscopia migliora rispetto ai pi bassi campi grazie ad un incremento del rapporto segnale / rumore ( s / r ) , della risoluzione del chemical shift , spaziale e temporale [ 17 ]  . in theory , the passage from 1.5 - t to 3.0 - t systems should double the snr ; in practice , however , the improvement is only around 20%46% . 
a linear rise would only be possible if other factors , such as t1 and t2 relaxation time values or the radiofrequency ( rf ) effect did not change significantly incremento del s / r e della risoluzione del chemical shift teoricamente , il s / r dovrebbe raddoppiare passando da 1 , 5 t a 3 , 0 t , ma in pratica il miglioramento solo del 20%46% . 
to this should be added the coils lower sensitivity and poorer volume homogeneity as well as registration errors caused by the wider chemical shift , which can degrade spectra quality , thus partially offsetting the advantages of the higher field [ 46 ]  . even though the position of spectrum peaks is constant on the parts per million ( ppm ) scale [ e.g. 
the greater amplitude of the chemical shift of metabolites yields better spectral resolution and hence a more clear - cut separation between adjacent peaks , which may overlap at lower magnetic field strength . 
the increased linewidth is related to reduced t2 values and heightened local magnetic field inhomogeneity of the higher fields . field inhomogeneity can be reduced by greater and more appropriate shimming . 
the same holds for gammaaminobutyric acid ( gaba ) , usually obscured at 1.5 t by a number of other substances , including macromolecules , cr , glutamine , glutamate and extracerebral lipid contamination . two different techniques can be adopted to detect the signal from coupling protons : ( 1 ) te averaging , with acquisition of different te with subsequent sampling capable of separating selected metabolites ; and ( 2 ) editing , using additional selective rf pulses with acquisition of the sole coupled spin signal [ 9 , 10 ]  . 
the wider range of chemical - shift frequencies found at higher magnetic fields enables the use of selective pulses at lower frequency , thereby shortening the te . increment of spatial resolution in high - field magnets , voxel size can be reduced without a significant loss in snr [ 11 ]  . 
a ci vanno aggiunti la pi bassa sensibilit della bobina e la pi bassa omogeneit del suo volume , la presenza di errori di misregistrazione da pi largo chemical shift che possono degradare la qualit dello spettro vanificando in parte i vantaggi dellalto campo [ 46 ]  . anche se la posizione dei picchi dello spettro costante utilizzando la scala parti per milione ( ppm ) ( ad esempio il n - acetyl - aspartato , naa a 2 , 02 ppm ) , la frequenza di risonanza differente ( per lnaa 127 hz a 1 , 5 t , 254 hz a 3 , 0 t )  . 
questa maggiore ampiezza di frequenza del chemical shift dei metaboliti permette una migliore risoluzione spettrale e quindi una migliore separazione dei picchi vicini che possono invece sovrapporsi a pi basso campo . 
in realt questo importante vantaggio pu non essere sfruttato del tutto negli studi di spettroscopia in vivo in quanto anche lampiezza del picco ( linewidth ) ( in hz ) dei metaboliti incrementa ( almeno di un fattore due ) per cui la separazione tra i singoli picchi vicini non risulta considerevolmente migliorata a 3 , 0 t . 
lo stesso vale per il gaba di solito oscurato in uno studio condotto con rm 1 , 5 t da una serie di altre sostanze comprendenti macromolecole , creatina , glutamina , glutamato , contaminazione lipidica extracerebrale . 
1a , b magnetic resonance spectroscopy ( mrs ) glx ( glutamate and glutamine ) : spectra obtained from grey matter in a healthy subject using point - resolved spectroscopy ( press ) ( a ) and echo - time ( te ) - averaged press ( b ) sequences . 
in conventional spectroscopy sequences ( a ) , the resonances between 2 and 2.6 ppm are assigned to a mixture of glutamate ( glu ) and glutamine ( gln ) or glx . 
 incremento della risoluzione spaziale increment of time resolution one of the general limitations of lower - field spectroscopy , the long examination time , can be overcome using high - field units . 
in particular , single - volume spectra can be obtained at 3.0 t with an snr similar to that of 1.5 - t magnets in a quarter of the time , or the time for single - volume three - dimensional ( 3d ) studies can be reduced by 26% using a higher snr than that in 1.5 - t systems [ 12 ]  . 
this increases the number of protocols that can be acquired with different te and enables a larger number of brain regions and larger brain volumes to be analysed . con lalto campo possibile ridurre le dimensioni del voxel , senza una significativa perdita di s / r [ 11 ]  . 
noto che il s / r per unit di tempo della spettroscopia linearmente proporzionale al volume del voxel per cui riducendo il voxel si abbassa anche il s / r . 
a 3 , 0 t voxel al di sotto di 1 cc ( limite a 1 , 5 t ) possono essere ottenuti invece con un buon s / r in un tempo dacquisizione accettabile simile a quanto ottenuto con un 1 , 5 t . 
la pi alta risoluzione spaziale permette di distinguere differenti strutture anatomiche , normali e patologiche con conseguenti significativi vantaggi nello studio di piccole strutture quali lippocampo , i nuclei basali , le placche di sclerosi multipla , piccole lesioni con conseguenti importanti implicazioni cliniche . metabolite quantitation incremento della risoluzione temporale metabolite quantitation , another important problem in spectroscopy , benefits from the advantages offered by higher field strength [ 46 ]  . 
in addition , the relaxation times of individual brain metabolites are inversely proportional to magnetic field strength and present distinct regional variations in brain parenchyma ( especially the t2 of naa ) , probably due to local paramagnetism and different white / grey matter composition [ 14 ]  . 
these features make it possible to increase the b value to enhance the anisotropic effect , thus improving the quality of more complex examinations such as anisotropy , diffusion tensor and tractography ( dti ) , which are poorly displayed on 1.5 - t images , even with multiple excitations , due to insufficient snr [ 15 ]  . 
in fact , reconstructed dti trajectories reflect more accurately the underlying axonal fibres , particularly crossing or bifurcating bundles . uno dei limiti generali della spettroscopia a pi basso campo , e cio i lunghi tempi desame , pu essere superato con lalto campo . 
in particolare possibile ottenere con il 3 , 0 t spettri a volume singolo con s / r similari a 1 , 5 t ma in 1 / 4 di tempo oppure studi a volume singolo 3d possono essere ridotti del 26% mantenendo un pi alto s / r rispetto a 1 , 5t [ 12 ]  . 
ci permette di eseguire pi protocolli , con differenti tempi di eco ( te ) , analizzare pi sedi e pi ampi volumi cerebrali . quantizzazione dei metaboliti in virt dei vantaggi che lalto campo fornisce in spettroscopia , anche la quantizzazione dei metaboliti , altro importante problema della spettroscopia , migliora con lalto campo [ 46 ]  . 
peraltro i tempi di rilassamento dei singoli metaboliti cerebrali , oltre a presentare una proporzionalit inversa con lintensit del campo magnetico , evidenziano distinte variazioni regionali nel contesto del parenchima cerebrale ( specialmente il t2 di naa ) verosimilmente attribuibili al locale paramagnetismo e alla differente composizione sostanza bianca / sostanza grigia [ 14 ]  . 
in rapporto a questultimo parametro e al tipo di metabolita cerebrale in esame dipende il guadagno del segnale in spettroscopia a 3 , 0 t . per effetto della riduzione del t2 con lalto campo , studi di spettroscopia con te lunghi subiscono una maggiore perdita di s / r a 3 , 0 t rispetto a 1 , 5 t ed un incremento della linewidth del picco che peraltro dipende , oltre che dal t2 , dalle disomogeneit microscopiche e macroscopiche del corpo . 
in tal modo possibile incrementare il b - value per aumentare lanisotropicit e quindi migliorare la qualit di fenomeni pi complessi quali lanisotropia , il tensore di diffusione e la trattografia ( dti ) , non chiaramente rilevabili con sistemi 1 , 5 t , pur utilizzando multiple eccitazioni , in quanto il s / r non sufficiente [ 15 ]  . 
inoltre laumento lineare del segnale rm comportando una riduzione degli effetti di volume parziale particolarmente utile per ricostruire il percorso delle fibre con dti in quanto le traiettorie cos ricostruite rispecchiano pi precisamente le fibre assonali sottostanti , particolarmente i fasci che si incrociano o si biforcano . lo studio di diffusione particolarmente utilizzato nel rilievo di lesioni ischemiche iperacute con peraltro b - value t . 
4a , b multimodal magnetic resonance ( mr ) study [ contrast - enhanced spin echo ( se ) t1 , fast spin echo ( fse ) t2 , apparent diffusion coefficient ( adc ) map ] : comparison of low - grade astrocytoma ( a ) to glioblastoma ( b )  . 
4a , b studio rm multimodale ( se t1 dopo mdc , fse t2 e mappa di adc ) : confronto tra astrocitoma a basso grado ( a ) e glioblastoma ( b )  . 
in such cases , parallel imaging or new techniques such as periodically rotated overlapping parallel lines with enhanced reconstruction ( propeller ) radial k - space filling can solve this problein fact , diffusion studies adopt echoplanar techniques in which all phase - encoding steps are acquired with one rf excitation , thus accumulating phase errors responsible for susceptibility effects and image distortion . 
dti data sufficient for brain fibre tracking can be obtained in healthy subjects in < 2 min , with 2mm slice thickness , 700 s / mm2 b factor and at least six motion - probing gradient directions [ 19 ]  . 
a tal proposito usando un sistema a 3 , 0 t queste tecniche di pesatura in diffusione forniscano un maggiore contenuto di informazioni per lo studio dei tumori cerebrali , specialmente per valutare il coinvolgimento della sostanza bianca peritumorale , che pu dimostrare edema e / o infiltrazione neoplastica . 
nei gliomi a basso grado le fibre della sostanza bianca sono solo compresse e dislocate , non infiltrate o distrutte come in quelli ad alto grado : i cambiamenti rilevati alla dti possono quindi dipendere dallinfiltrazione tumorale . 
per questa ragione la dti pu essere considerata un metodo valido per rilevare uninvasione occulta della sostanza bianca da parte del glioma e quindi fornire informazioni utili per pianificare il trattamento [ 17 ]  . un grosso problema dello studio della diffusione a 3 , 0 t t . 
5a - c ischemia cerebrale iperacuta del lobulo parietale destro : dwi ( a ) , mappa di adc ( b ) , main eigenvector map area di interesse ( c )  . weighted acquisitions , avoid ambiguity in reconstructing diffusion tensor parameters , increase the snr and decrease the influence of signal distortion [ 20 ]  . a diffusion sequence based on single - shot fast spin echo ( fse ) has recently been proposed to address the problem of susceptibility , as this sequence is not affected by the spatial distortion that usually limits conventional acquisitions with echoplanar imaging ( epi ) sequences [ 21 ]  . perfusion studies perfusion mr imaging benefits from the advantages of highfield units both in examinations using exogenous contrast agents [ dynamic susceptibility contrast ( dsc ) ] and those based on endogenous contrast agents [ arterial spin labelling ( asl ) ]  . dynamic susceptibility contrast ( dsc ) dsc is without doubt the faster and more widely used technique in clinical practice . 
the higher snr and magnetic susceptibility of 3.0 - t compared with 1.5 - t units result in imrappresentato dalle pi ampie distorsioni delle immagini ( specie in aree di alta suscettibilit , quali ad esempio quelle vicino ai seni paranasali ) , conseguenza dei maggiori effetti di suscettibilit presenti a pi alto campo . 
in questi casi limaging parallelo o luso di nuove tecniche di riempimento del k - space ( cd propeller , periodically rotated overlapping parallel lines with enhanced reconstruction ) possono risolvere tale inconveniente . 
gli studi di diffusione infatti utilizzano tecniche ecoplanari in cui tutti gli step di codifica di fase sono acquisiti con ununica eccitazione di rf con quindi accumulo di errori di fase , causa di effetti di suscettibilit e di distorsione delle immagini . 
utilizzando limaging parallelo a 3 , 0 t , possibile anche ottenere dati del tensore di diffusione ( dti ) sufficienti a seguire il percorso delle fibre in soggetti sani in meno di 2 min con uno spessore di strato di 2 mm , un fattore b - value di 700 s / mm2 , almeno 6 diverse direzioni spaziali di gradienti [ 19 ]  . 
 recentemente per ovviare a tali inconvenienti stato anche proposto luso di una sequenza di diffusione basata su una fast spin echo ( fse ) single shot che non risulta appunto affetta da distorsioni spaziali che di solito invece limitano lacquisizione convenzionale con sequenze ecoplanari ( epi ) [ 21 ]  . studio di perfusione limaging mediante rm con valutazione della perfusione trae vantaggio dalluso di campi magnetici elevati sia utilizzando mezzo di contrasto ( mdc ) esogeno ( dsc , dynamic susceptibility contrast ) che mdc endogeno ( asl , arterial spin labelling )  . dsc sicuramente la pi veloce e la pi usata nella pratica clinica . 
alla potenza di campo di 1 , 5 t la misurazione dei parametri emodinamici limitata dal basso rapporto s / r dato che il passaggio del mdc cos rapido che non consente di raccogliere dati sufficienti a calcolare questi parametri con precisione . 
ci si nota in particolare in condizioni come lischemia , in cui la ipo - perfusione limita la quantit di mdc presente nei voxel . sfruttando lelevato rapporto s / r offerto dai sistemi a 3 , 0 t si possono ottenere informazioni utilizzando voxel pi piccoli ( accentuazione della risoluzione spaziale ) ad intervalli di tempo pi brevi ( incremento della risoluzione temporale )  . i gradienti rapidi inoltre consentono di usare un te breve con risoluzione temporale attorno a 1 s , il tempo necessario per seguire il passaggio del bolo di mdc attraverso lencefalo ed i suoi vasi ematici . 
questo non possibile con apparecchiature da 1 , 5 t in quanto le immagini sono in genere acquisite a bassa risoluzione spaziale per ottimizzare la velocit di acquisizione , ed anche aumentando il numero delle sezioni impossibile analizzare lintero encefalo . inoltre luso frequente nei sistemi a 3 , 0 t di sequenze t2 * pesate con valutazione della presa di contrasto e della perfusione offre alcuni potenziali vantaggi . 
in particolare , tempi di rilassamento t2 e t2 * pi brevi ed un aumentato rapporto s / r [ 24 , 25 ] possono consentire di ottenere una maggiore caduta di segnale t2 * utilizzando la stessa quantit di bolo di mdc nella fase di transito capillare [ 26 ]  . 
a tale proposito , anche se a 1 , 5 t gli studi strutturali dopo somministrazione di mdc sono solitamente effettuati utilizzando 0 , 1 mmol / kg di peso corporeo di mdc , nella maggior parte dei casi si usa 0 , 2 mmol di gadolinio chelato , per gli studi di perfusione dsc [ 23 , 2729 ]  . 
la maggiore sensibilit delle apparecchiature a campo magnetico di 3 , 0 t per la suscettibilit magnetica significa che lo stesso effetto pu essere ottenuto somministrando dosi di mdc paramagnetico molto pi piccole di quelle comunet . 
this cannot be achieved with 1.5 - t devices with which images are usually acquired at low spatial resolution to optimise acquisition speed and , even when the number of slices is increased , the whole brain cannot be covered . the frequent use of t2 * sequences with contrast - enhanced and perfusion studies also offers some potential advantages . 
in particular , shorter t2 and t2 * relaxation times and increased snr [ 24 , 25 ] may allow a greater t2 * signal drop to be obtained for a given amount of contrast bolus during capillary passage [ 26 ]  . 
in this respect , although structural contrast - enhanced brain imaging at 1.5 t is usually performed with 0.1 mmol / kg of body weight , 0.2 mmol of gadolinium chelate is the most widely used dose and is considered optimal for dsc perfusion studies [ 23 , 2729 ]  . 
the enhanced sensitivity of 3.0 - t fields to magnetic susceptibility means that the same effect can be obtained with much smaller doses of paramagnetic contrast medium than those usually injected for lower - field perfusion imaging . 
the dose can even be halved without compromising the sensitivity of intensity / time curves from which the relative cerebral blood volume ( rcbv ) and mean transit time ( mtt ) values are calculated . 
in fact , when using epi sequences , the problems of increased b0 inhomogeneity and magnetic susceptibility differences at airtissue interfaces lead to signal drop - out and geometric distortions , most notably in the phase - encode direction [ 30 ]  . 
 other drawbacks inherent in t2 * sequence susceptibility are systematic overestimation of perfusion due to the difference in relaxivity of the paramagnetic contrast agent between larger vessels ( from which the input function is obtained ) and capillaries , problems defining a good input function because of low spatial resolution on t2 * - weighted epi and , finally , problems assessing a damaged blood - brain barrier . the latter is a critical element in evaluating brain tumour perfusion because relaxivity is different when the contrast agent is confined to a small compartment as opposed to being distributed in the interstitial space [ 31 ]  . 
in particolare , gli effetti di suscettibilit pi forti determinano una completa assenza di segnale in occasione del primo passaggio di gadolinio , specialmente nella sostanza grigia , e compromettono laccuratezza dei calcoli di caduta di segnale nonch la sensibilit ad eventuali deficit di perfusione ( fig . 7 ) [ 23 ] ; la dose pu anche essere dimezzata senza compromettere la sensibilit delle curve intensit / tempo dalle quali si ottengono i valori di volume ematico cerebrale relativo ( rcbv ) e tempo di transito medio ( mtt )  . 
la dose standard di mdc non quindi necessaria per studi di perfusione effettuati a 3 , 0 t , e la dose pu essere ulteriormente ridotta somministrando concentrazioni pi elevate ( 1 molare )  . come per la diffusione anche lo studio di perfusione ad alto campo limitato dai problemi causati dagli artefatti da suscettibilit magnetica che aumentano di pari passo con laumento del campo magnetico . 
a 3 , 0 t infatti la distorsione dellimmagine pu essere critica , in particolare se si usano sequenze epi , come nella maggior parte dei protocolli per gli studi di perfusione dsc . 
infatti lutilizzo delle sequenze epi comporta gli svantaggi di un aumento della disomogeneit del b0 e differenze di suscettibilit magnetica a livello delle interfacce aria - tessuto che determinano una caduta di segnale e distorsioni geometriche evidenti maggiormente nella direzione della codifica di fase [ 30 ]  . 
a 3 , 0 t la maggiore suscettibilit magnetica richiede dosi o concentrazioni inferiori rispetto a sistemi 1 , 5 t ( per cortesia della general electric )  . sequences [ 3d fast field echo ( ffe ) t1 weighted or 3d fast low - angle shot ( flash ) t1 weighted ] [ 31 , 32 ] or selection of appropriate te has recently been proposed to minimise these negative effects [ 33 ]  . 
ne deriva un migliore rapporto s / r in quanto la marcatura decade pi lentamente e , cosa pi importante , una minore sensibilit per differenze del tempo di arrivo arterioso in quanto sono consentiti ritardi pi lunghi post - marcatura . 
tale aumentata sensibilit circa il rapporto s / r pu essere sfruttata per ridurre il tempo di scansione ( meno di 5 min rispetto agli oltre 15 di un sistema a 1 , 5 t ) , per acquisire immagini a pi alta risoluzione o per applicare tempi di ritardo post - marcatura pi lunghi . una recente applicazione della asl limaging del territorio di perfusione regionale al fine di visualizzare il territorio t . 
8a , b perfusion study of a normal brain using an endogenous contrast agent [ arterial spin labelling ( asl ) ] at 1.5 tesla ( t ) ( a ) and 3.0 t ( b )  . 
this results in a better snr because the label decays more slowly . more importantly , it results in less sensitivity to arterial arrival time differences because longer postlabelling delays are enabled . 
such increased snr sensitivity can be harnessed in order to decrease scanning time ( < 5 min compared with > 15 min at 1.5 t ) , acquire higher - resolution images , or apply longer postlabel delay times . a recent application of asl is in the study of regional perfusion territory imaging in order to visualise the vascular territory supplied by a specific labelled vessel . 
selective labelling may be useful in patients with high - grade vascular stenosis or complete vascular occlusion to guide the choice between angioplasty and stenting , evaluate cerebrovascular bypass grafts or estimate the likelihood of an embolic or atherothrombotic event in a patient with multiple bright regions on dwi [ 3742 ]  . 
 it is important to note that at one time the only way to obtain such information was by invasive catheter angiography . these data may be helpful to achieve a better understanding of vascular dynamics in patients at high risk of stroke or those being considered for corrective surgical procedures [ 43 ]  . cortical activation studies functional imaging ( fmri ) is without doubt the field in which the greater snr and magnetic susceptibility effects of 3.0 - t magnetic fields have had the strongest impact , parvascolare irrorato da uno specifico vaso marcato . 
la marcatura selettiva pu essere utile nei pazienti con stenosi vascolare elevata od occlusione completa in modo da guidare la scelta fra angioplastica e stenting , per valutare impianti di by - pass cerebrovascolare , o per stimare la probabilit di eventi aterotrombotici od embolici in pazienti con reperto allimaging pesato in diffusione di molteplici zone a segnale elevato [ 3742 ]  . importante notare che in passato lunico modo per ottenere questa informazione era langiografia per cateterismo selettivo , metodo invasivo . 
tali dati possono essere utili per ottenere una migliore comprensione delle dinamiche vascolari nei pazienti ad alto rischio di ictus o nei pazienti valutati per procedure chirurgiche correttive [ 43 ]  . studio di attivazione corticale sicuramente il settore che si maggiormente avvantaggiato con lalto campo e che , in virt del pi alto s / r e dei maggiori effetti di suscettibilit magnetica , ha avuto limpatto maggiore con in particolare pi elevata risoluzione spaziale e temporale dellimaging funzionale ( fmri )  . per ottenere vantaggi reali dallaumento di risoluzione necessario luso congiunto di strategie di acquisizione in grado di migliorare il rapporto s / r . 
infatti , oltre alla possibilit di migliorare il rapporto s / r intrinseco delle sequenze di impulsi e lutilizzo di antenne ad elevato rapporto s / r , limpiego di unit ad alto campo magnetico la soluzione che ha conseguito il maggiore successo nellaumentare il rapporto s / r per gli studi di attivazione corticale . 
indeed , besides the possibility of improving intrinsic pulse - sequence snr and the adoption of high snr coils , the most successful option for gaining snr is to use high - magnetic - field - intensity fmri units . 
in fact , the difference in magnetic susceptibility between blood which contains paramagnetic deoxyhaemoglobin and surrounding diamagnetic tissue on which difference the bold effect is based increases with the strength of the main magnetic field , determining larger mr signal changes between baseline and activated states if high - field units are used [ 45 ]  . 
as regards the magnitude of the field dependence of the bold effect , it has been considered to vary from linear to quadratic [ 4547 ] , exhibiting an inhomogeneous spatial distribution principally depending on the relationship between voxel dimension and the dimension of blood vessels contained in thein particular , studies of the field dependence of the transverse relaxation rate [ 47 ] showed that the bold effect is proportional to b0 if the voxels contain large vessels ( venules and veins ; d > 10 m ) , and that it drops to less than linear if vessels are larger than voxels . 
another , potentially more important , advantage of the higher b0 is that as the field strength increases , the field gradient around the capillaries becomes larger and extends further into the parenchyma , thus involving a greater amount of brain tissue in producing the functional signal . 
per quanto riguarda lentit della dipendenza delleffetto bold dallintensit del campo magnetico , si ritiene che essa passi da lineare a quadratica [ 4547 ] , con una distribuzione spaziale non omogenea che dipende principalmente dalla relazione fra la dimensione dei voxel e quella dei vasi contenuti al loro interno . 
in particolare studi sulla dipendenza dallintensit di campo dellentit di rilassamento trasversale [ 47 ] hanno dimostrato che leffetto bold proporzionale a b0 nei voxel che contengono vasi pi grandi ( venule e vene con diametro superiore a 10 m ) e cala a una relazione meno che lineare se i vasi sono pi grandi dei voxel . 
un altro vantaggio potenzialmente pi importante dellelevato b0 che con laumento dellintensit di campo il gradiente di campo attorno ai capillari diventa pi grande e si estende maggiormente nel parenchima , coinvolgendo pertanto una maggiore quantit di tessuto cerebrale nella produzione del segnale funzionale . 
nei gradienti di campo i vettori di magnetizzazione allinterno dei voxel possono corrispondere a fasi diverse , determinando una diminuzione del tempo effettivo di rilassamento trasversale t2 *  . contemporaneamente la diminuzione del t2 * del sangue ad elevato b0 riduce il contributo relativo delle grandi vene . cos la pesatura del contributo di segnale proveniente dai capillari diviene pi significativa ed il segnale funzionale pi strettamente correlato con lattivit neuronale [ 4852 ]  . le sequenze ge - epi sono attualmente preferite a sequenze se - epi in quanto consentono una migliore discriminazione dei vasi piccoli dai vasi grandi . 
the map obtained at 3.0 t shows more clearly the spatial extension of the activation , particularly in the supplementary motor area , and a better correspondence of the activated area with the anatomy of the motor systethe intensity of the fmri signal [ blood oxygen level dependent ( bold ) % ] doubles in the passage from the 1.5 - t to the 3.0 - t magnetic field . 
9 mappe di attivazione corticale motoria ottenute sul piano coronale a 1 , 5 t ( sinistra ) e 3 , 0 t ( destra ) durante un esperimento con schema a blocchi e pressione del pulsante alla frequenza di 0 , 5 hz . 
la mappa ottenuta a 3 , 0 t dimostra pi chiaramente lestensione spaziale dellattivazione , specie della regione motoria supplementare , ed una migliore corrispondenza dellarea attivata con lanatomia del sistema motorio . 
lintensit del segnale fmri ( bold% ) raddoppia passando da 1 , 5 t a 3 , 0 t . in synthesis , the use of high magnetic field strength fmri units considerably improves the flexibility and specificity of functional studies . 
thanks to high - field studies , fundamental neural dynamics taking place at a very small dimensional scale in specific laminar , columnar and multicolumnar domains have become directly visible , enabling more reliable localisation of brain functions , with the possibility of mapping additional areas in the millimetre or even submillimetre scale . 
these properties are especially useful when investigating minimal changes and when using paradigms requiring extra snr ( indeed , one method to measure the benefits of high field strengths is to determine the extent of activation elicited by a given paradigm )  . 
 conclusions comparison of data acquired by our team at 3.0 t and lower field strength showed that the numerous and important advantages of high - field morphofunctional imaging ( high signal , resolution and sensitivity , short acquisition times , additional and more advanced applications , enhanced diagnostic potential ) make it a promising candidate as the future standard for the study of pathological brain conditions . 
technological advances are expected to overcome some minimal limitations , such as field inhomogeneity , susceptibility and chemical - shift artefacts , high specific absorption rate ( sar ) , acoustic noise and cost , nonsignificant diagnostic differences , and scarce diffusion . 
these advances will promoting magnetico hanno consentito di visualizzare direttamente dinamiche neurali fondamentali che si verificano a scale di dimensioni molto piccole a livello di strutture laminari , colonnari e multicolonnari . 
tale beneficio particolarmente apprezzabile quando si studiano variazioni minime , o quando si utilizzano paradigmi che richiedono un s / r extra ( un modo per misurare i benefici dellalto campo appunto quello di determinare lestensione dellattivazione per un dato paradigma )  . a ci si aggiunge la possibilit di eseguire studi fmri in tempi ridotti e di seguire lattivazione corticale durante la stimolazione anche in tempo reale ( real time fmri )  . conclusioni sulla base dei nostri risultati finora ottenuti , rispetto alle precedenti esperienze con sistemi rm a pi basso campo , si evince che limaging morfo - funzionale ottenuto con un sistema rm 3 , 0 t , grazie ai suoi numerosi ed importanti vantaggi ( alto segnale , alta risoluzione , alta sensibilit , tempi ridotti , metodologie di studio aggiuntive e pi avanzate , maggiori capacit diagnostiche ) , potr in futuro diventare lo standard per lo studio della patologia cerebrale . 
il superamento di alcune minime limitazioni ( disomogeneit di campo , artefatti da suscettibilit e da chemical shift , sar elevato , rumore acustico , costi , variazioni semeiologiche non significative , scarsa diffusione ) , grazie ai futuri progressi tecnologici , sicuramente favorir sempre di pi lo studio anatomico , fisiologico , metabolico e funzionale del cervello nella pratica clifurther anatomical , physiological , metabolic and functional brain investigations in clinical practice and especially in research applied to it , as higher contrast , space and time resolution compared with the lower fields result in greater diagnostic power in terms of sensitivity and specificity . however , these systems are not to be considered the gold standard for all clinical applications and body districts . 
indeed , knowledge of technical , diagnostic and application characteristics of all field intensities is crucial for correct and optimal use of the different units based on study requirements . for all these reasons , we support the current restrictions on the use of such systems which provide excellent results , especially in neuroradiological imaging to university and hospital research centres and facilities already equipped with other mr systems employed in routine clinical practice that desire to conduct research . 
 the multidisciplinary approach adopted at our institution , with the collaboration of neuroradiologists , neuropsychiatrists , neurophysiologists , psychologists , bioengineers , physicists and other neuroimaging experts , has proved essential for optimal magnet utilisation . 
infatti essenziale conoscere le caratteristiche tecniche , semeiologiche e applicative di ogni sistema rm a diversa intensit di campo in modo da utilizzare in maniera adeguata ed ottimale le diverse apparecchiature in base alle proprie esigenze di studio . per questo motivo pensiamo sia giusto al momento confinare in centri di ricerca ( universitari o ospedalieri ) o comunque in strutture gi dotate con altri sistemi rm utilizzati per la routine clinica e che vogliano far ricerca , luso di tali sistemi che attualmente risultano ottimali soprattutto per limaging neuroradiologico . essenziale per lutilizzo ottimale di tali apparecchiature risultato lapporto multidisciplinare di diverse collaborazioni scientifiche che il nostro centro ha instaurato con neuroradiologi , neuropsichiatri , neurofisiologi , psicologi , bioingegneri , fisici o comunque esperti di neuroimaging . rigraziamenti allo staff tecnico : c . 
cova1 1uco di radiologia , 2uco di anatomia patologica , universit di trieste , ospedale di cattinara , strada di fiume 449 , i - 34149 trieste , italy correspondence to : f . 
this study was done to evaluate the effectiveness of cooperation between the radiologist and the cytopathologist in ultrasound - guided fine - needle aspiration biopsy ( fnab ) of thyroid nodules . 
la stretta collaborazione tra radiologo e citologo consente di ottimizzare i risultati . parole chiave nodulo tiroideo ecografia nodulo tiroideo , agoaspirazione introduction introduzione cytological assessment is a fundamental step in the diagnosis of nodular thyroid disease . 
fine - needle aspiration biopsy ( fnab ) of thyroid nodules is normally performed by a radiologist under ultrasound ( us ) guidance and , in some settings , by a clinician or a cytopathologist . 
le agoaspirazioni tiroidee vengono solitamente eseguite con guida ecografica dal radiologo e , in talune realt operative , dal clinico o dal citologo stesso , ma sussistono anche situazioni in cui lagoaspirazione viene effettuata dal clinico con la sola guida rappresentata dal reperto palpatorio [ 1 ]  . 
a retrospective analysis of 10 years of fnab of thyroid nodules and the operational changes that occurred during the course of these years provides an opportunity to assess the results of the cooperation between the radiologist and the cytopathologist . tuibile che una stretta collaborazione tra le figure professionali coinvolte possa migliorare lappropriatezza nella selezione dei pazienti candidati allagoaspirazione ed anche i suoi risultati [ 2 ] , ma una quantificazione di questi aspetti pu non essere immediata . 
lanalisi retrospettiva dellattivit di 10 anni di agoaspirazioni tiroidee e dei cambiamenti operativi verificatisi in questo lasso di tempo ci offre la possibilit di analizzare i risultati della collaborazione tra radiologo e citologo . materials and methods our analysis first considered the number of us - guided fnab of thyroid nodules carried out at our department between 1995 and 2005 . 
a number of operational changes occurred during these years that justify the choice of this time frame : 1994 was the last year in which fnab of thyroid nodules were performed by the radiologist alone , who was in charge of obtaining the sample and preparing the material ( smearing and fixation )  . 
as of january 1995 , a cytopathologist has worked alongside the radiologist in the sonography room ; the biopsy is still performed by the radiologist , but the cytopathologist prepares the material and possibly assesses its adequacy immediately . 
we therefore collected data for 1995 ( start of the radiologist - cytopathologist cooperation ) , 1999 ( consolidation of the cooperation ) and 2005 ( time chosen for data validation given the longterm interval )  . the peculiarity of the trieste province all fnab assessments are centralised in a single pathology department also enabled us to analyse data from other departments , where fnab procedures are carried out by clinicians using direct - palpation biopsy . 
we therefore analysed the number of fnab procedures , both us guided and palpation guided , in the trieste province during the 3 sample years ( 1995 , 1999 and 2005 )  . 
we then considered the number of fnab judged to be nondiagnostic by the cytopathologist , dividing them into us - guided and non - us - guided procedures , in the same 3 years . 
finally , we evaluated discrepancies between the cytological and histological diagnoses in the cases that underwent surgery in 1995 , 1999 , 2005 ( cytological diagnosis suggestive of benign lesion with histological evaluation of malignant lesion and vice versa )  . the us - guided fnab procedures were carried out with a variety of linear - array probes , from the initial 5 - mhz probes to the present 5to 10 - mhz multifrequency probes . 
two aspirations per nodule are considered sufficient in most cases ; additional aspirations are obtained in large or strongly suspicious nodules or when the cytopathologist judges the first specimens to be inadequate , for example , due to excess blood contamination . smearing is done on slides previously been treated with sulphochromic solution to ensure optimal cleanliness and greater adhesion of cells . 
the needle is detached and a few materiali e metodi nella nostra valutazione abbiamo inizialmente preso in esame il numero di agoaspirazioni tiroidee ecoguidate effettuate presso la nostra unit clinica operativa dal 1995 al 2005 . in questo lasso di tempo si sono verificati dei cambiamenti operativi che giustificano la scelta di questo periodo : il 1994 stato lultimo anno in cui le agoaspirazioni tiroidee sono state effettuate dal solo medico radiologo , che effettuava il prelievo e si faceva carico del trattamento del materiale ( striscio e fissazione )  . 
abbiamo quindi ritenuto che la raccolta dei dati andasse effettuata relativamente al 1995 ( inizio della attivit comune di radiologo e citologo ) , al 1999 ( momento scelto come espressione del consolidamento di questa attivit ) e al 2005 ( momento scelto per la validazione dei dati dopo un lungo intervallo temporale )  . la particolare condizione che si verifica nella nostra provincia , ove tutti gli agoaspirati afferiscono ad una sola struttura di anatomia patologica , ci ha anche permesso unanalisi dei dati relativi ad altre realt operative ove lagoaspirazione viene effettuata dal clinico sulla guida del reperto palpatorio . 
sono state inoltre valutate le discordanze tra diagnosi citologica e diagnosi istologica nei casi sottoposti ad intervento chirurgico nel 1995 , 1999 , 2005 ( diagnosi citologica orientativa per lesione benigna con riscontro istologico di lesione maligna e viceversa )  . le agoaspirazioni ecoguidate sono state effettuate avvalendosi di sonde lineari , diverse nel corso degli anni , inizialmente da 5 mhz , attualmente di frequenza variabile ( 510 mhz )  . 
la procedura stata effettuata con tecnica a mano libera utilizzando aghi non trancianti da 2527 g con lunghezza di 2 , 5 cm ai quali viene collegato un sistema di aspirazione , rappresentato da una semplice siringa . 
nella maggioranza dei casi venivano ritenute sufficienti due agoaspirazioni per nodulo , ricorrendo ad aspirazioni ulteriori in caso di noduli voluminosi , noduli fortemente sospetti o qualora il citologo non ritenesse adeguati i primi prelievi ad esempio per abbondante contaminazione ematica . la procedura di striscio utilizza preparati portaoggetti precedentemente trattati con soluzione solfocromica , al fine f . 
the needle is then reattached to the syringe and the aspirate expelled onto the slide by applying slight pressure on the plunger ; the aspirate is then quickly smeared onto the slide in an even and gentle manner , and fixed immediately . 
fixation is achieved by rapid immersion of the slides into a fixative solution , usually 95% alcohol , for 1015 mmicroscopic evaluation is normally done with papanicolaou staining . di garantire condizioni ottimali di pulizia e maggiore adesivit delle cellule al vetrino . 
estratto lago dalla siringa ed aspirati alcuni ml di aria , lago viene successivamente reinserito e il materiale agoaspirato viene distribuito sul vetrino praticando una lieve pressione sullo stantuffo della siringa e quindi rapidamente strisciato , manovra che deve essere uniforme e non traumatica , seguita da immediata fissazione . la fissazione si esegue mediante immersione rapida dei vetrini in liquido fissativo , solitamente alcool 95% , per circa 1015 minuti . 
la successiva valutazione al microscopio si avvale solitamente della colorazione secondo papanicolau . results risultati table 1 shows the number of us - guided fnab of thyroid nodules carried out at our department between 1995 and 2005 . 
that number rose steadily , both in absolute terms and relative to those performed on other body regions , from a total of 63 in 1995 ( 7% of the total ) , to 117 in 1999 ( 13% of the total ) and 395 in 2005 ( 29% of the total )  . 
table 2 shows the number of fnab of thyroid nodules ( divided into us - guided and palpation - guided ) performed in our province during the 3 sample years . 
the number of us - guided procedures , done by the radiologist with an on - site cyla tabella 1 riporta il numero di agoaspirazioni tiroidee ecoguidate effettuate nella nostra uco dal 1995 al 2005 . 
si osserva un progressivo aumento del numero delle agoaspirazioni tiroidee , sia in termini assoluti , sia in termini percentuali rispetto alle agoaspirazioni eseguite in altri distretti corporei . nel 1995 sono state infatti eseguite 63 agoaspirazioni tiroidee ecoguidate ( 7% del totale ) , salite a 117 nel 1999 ( 13% del totale ) ed a 395 nel 2005 ( 29% del totale )  . 
la tabella 2 riporta il numero di agoaspirazioni tiroidee nella nostra provincia ( distinte in ecoguidate e non ) nei tre anni campione ( 1995 , 1999 , 2005 )  . 
si osserva inoltre il progressivo incremento delle procedure ecoguidate effettuate a partire dal 1995 in collaborazione tra radiologo e citologo ( passate dal 25% al 94% del totale ) ed una progressiva riduzione dei prelievi inadeguati ai fini diagnostici ( passati dal 14% al 4% )  . 
la tabella 3 prende in esame concordanze e discordanze tra riscontro citologico dopo agoaspirazione ( nel 1995 , 1999 e 2005 ) e riscontro istologico nei pazienti successivamente sottoposti ad intervento chirurgico . 
emerge la progressiva riduzione delle discordanze che nel 1995 riguardavano il 38% dei casi , mentre nel 2005 riguardavano solamente il 13% dei casi . fnab of thyroid nodules is a nontraumatic procedure that is virtually free of complications and provides good results in terms of diagnosis [ 3 , 4 ]  . 
it is generally accepted that fnab of thyroid nodules should be performed under ultrasound guidance [ 49 ] as this makes it possible to sample lesions discussione lagoaspirazione tiroidea indagine pressoch atraumatica , sostanzialmente priva di complicanze e che fornisce buoni risultati sotto il profilo diagnostico [ 3 , 4 ]  . 
only sporadic reports have indicated higher rates of inadequate specimens with us - guided fnab procedures , probably as a result of the smaller size of nodules sampled and the greater blood contamination of aspirates from sonographically suspicious areas [ 14 ]  . 
however , the reported inadequacy rates of us - guided fnab are indeed nonnegligible and vary depending on the technique , the operators experience and , although there is no agreement on this , nodule size [ 3 , 13 , 1517 ]  . 
one recent study [ 3 ] reported a rate of inadequate specimens as high as 23% despite the fact that the procedures had been carried out by a single operator with seven to ten passes per nodule . we are convinced that through technical refinement and above all closer cooperation between the radiologist and the cytopathologist these results can be considerably improved , as has already been highlighted in the literature [ 2 , 1921 ]  . whereas very thin needles ( usually 27 gauge in our case ) and the use of forced aspiration can in part improve the results , the presence of an on - site cytopathologist is mandatory both to cooperate in collecting the patients anamnestic data and carry out the biopsy and , more importantly , to provide the expertise required in smearing the slide and assessing the adequacy of the specimen . 
this approach has markedly improved our results , as demonstrated by the reduction in the number of inadequate samples from 14% in 1995 , when cooperation was started , to 4% in 2005 , following 10 years of cooperation between the radiologist and cytopathologist . 
clearly , this set - up requires the presence of two staff physicians to perform the procedure , but we believe this to be widely compensated for by the results obtained and the minimal need to repeat the procedure due to inadequate sampling . 
 the marked increase in us - guided fnab indicates that the benefits of the approach have been readily perceived by the physicians working in this area , who have started to refer growing numbers of patients to radiology departments . moreover , besides improving adequacy rates , cooperation has also increased the expertise of the cytopathologist , as clearly demonstrated by the reduction in cytologyhistology discrepancies over the years considered . 
optimisation of results therefore depends on the close cooperation between the radiologist and the cytopathologist . ba essere ecoguidata [ 49 ] perch ci permette di effettuarla in lesioni di pochi millimetri o nei punti sospetti ( ad esempio in gettoni solidi di lesioni strutturalmente complesse ) anche se si tratta di lesioni voluminose palpatoriamente facilmente apprezzabili [ 10 , 11 ]  . 
solo lavori sporadici segnalano una maggiore percentuale di prelievi inadeguati nellagoaspirazione ecoguidata probabilmente per le minori dimensioni dei noduli e per la maggiore contaminazione ematica di agoaspirati mirati in zone ecograficamente sospette [ 14 ]  . 
tuttavia anche lagoaspirazione ecoguidata risulta gravata in letteratura da una percentuale di prelievi inadeguati non trascurabile , variabile alla luce della diversa tecnica utilizzata , dalla diversa esperienza delloperatore e delle diverse dimensioni dei noduli , anche se su questultimo aspetto non vi identit di vedute [ 3 , 13 , 1517 ]  . 
va rilevato come una pubblicazione recente [ 3 ] abbia riportato una percentuale di prelievi inadeguati del 23% pur se le aspirazioni venivano effettuate da un solo operatore e venivano eseguiti da 7 a 10 passaggi per nodulo . nostro convincimento che laffinamento tecnologico , ma soprattutto la maggior collaborazione tra radiologo e citologo , possano migliorare notevolmente questi risultati , come gi sottolineato in letteratura [ 2 , 1921 ]  . da un lato luso di aghi sottilissimi ( solitamente di 27 g nel nostro caso ) ed il ricorso allaspirazione forzata sono in grado di ottimizzare i risultati , dallaltro appare fondamentale la presenza fisica del citologo , con la possibilit di raccogliere assieme dati anamnestici e di effettuare assieme il prelievo , affidando alla sua esperienza la manovra di strisciamento sul vetrino e la valutazione delladeguatezza del campione . 
ci ha consentito un deciso miglioramento dei nostri risultati , come testimoniato dalla riduzione nel numero di agoaspirati considerati inadeguati ai fini diagnostici passati dal 14% del 1995 , allinizio della attivit in comune , al 4% del 2005 , dopo 10 anni di collaborazione . 
chiaro che questa organizzazione prevede la presenza di due dirigenti medici per leffettuazione della procedura , ma riteniamo ci sia ampiamente controbilanciato dai risultati ottenuti e dalla ridottissima necessit di effettuare eventuali nuove aspirazioni a fronte di una prima valutazione di inadeguatezza . lincremento nettissimo della quota di agoaspirazioni ecoguidate testimonia come questi vantaggi siano stati chiaramente percepiti dai medici coinvolti in questo settore , che hanno indirizzato una quota sempre pi consistente di pazienti alla struttura radiologica . 
va ancora sottolineato come la collaborazione tra radiologo e citologo abbia consentito , accanto al miglioramento in termini di adeguatezza del prelievo , anche di affinare lesperienza del citologo e ne sono chiara testimonianza il minor numero di discordanze tra citologia e istologia negli anni esaminati . 
giovagnoni3 1dipartimento diagnostica per immagini , ausl 3 umbria , ospedale foligno , perugia , italy 2dipartimento diagnostica per immagini , ausl 2 umbria , ospedale assisi , perugia , italy 3istituto di radiologia , universit politecnica delle marche , ospedale torrette , ancona , italy correspondence to : f . 
18 , i - 06087 perugia , italy , tel . : + 39 - 075 - 393044 , fax : + 39 - 075 - 5783488 , e - mail : fcrusco@sirm.org received : 19 january 2006 / accepted : 07 august 2006 / published online : 11 june 2007 abstract purpose . 
the reported sensitivity and specificity for electrocardiogram ( ecg ) testing , stress echocardiography and radionuclide myocardial perfusion imaging were 45% and 82% , 86% and 90% , and 68% and 84% , respectively . 
in our view , the completion of such randomised trials combined with the development of newgeneration scanners is required to correctly define the role of mdct in the follow - up assessment of patients who have undergone cabg . 
per ecg da sforzo , ecocardio - stress e scintigrafiastress la letteratura riporta valori di sensibilit e specificit rispettivamente del 45% e dell82% , dell86% e del 90% , del 68% e dell84% . 
gli studi tc selezionati , 15 prospettici , 1 retrospettivo , tutti di validazione mediante comparazione con la coronarografia convenzionale ed eseguiti con metodica multidetettore , hanno complessivamente coinvolto 705 pazienti per un numero totale di 1974 segmenti di cabg esaminati . 
le applicazioni della tcmd nel work - up diagnostico dei pazienti post - cabg , sono derivate da prove di tipo indiretto e lefficacia della metodica , dovrebbe essere valutata nel contesto di studi randomizzati , coinvolgenti anche le altre metodiche di stress imaging non invasivo . 
this has helped to limit inadequate healthcare behaviours and improve the health of the community , thanks to the delivery of healthcare services characterised by the best costbenefit ratio [ 1 ]  . 
the new approach is also a valuable tool for helping physicians keep abreast of new knowledge deriving from scientific advances and for continuing medical education aiming at actively impacting clinical practice . clinical reasoning based on evidence - based medicine ( ebm ) is , in other words , the application of a correct , validated and reproducible method that starts with the reading and critical appraisal of studies reported in the biomedical literature [ 2 ]  . 
a particularly challenging field for the application of ebm tools is in regard to the definition of the role of the new multidetector computed tomography ( mdct ) scanners in the diagnostic workup of patients with coronary artery disease . 
this paper , in particular , will consider the role of mdct in the follow - up of patients who have undergone revascularisation by coronary artery bypass grafting ( cabg )  . in the monitoring of post - cabg patients , stress electrocardiography ( ecg ) , which is also routinely performed in asymptomatic cases , is considered a first - line test [ 3 ]  . 
in symptomatic patients with recurrent ischaemia , however , second - line tests such as stress echocardiography and cardiac scintigraphy are required to confirm that the angina has a cardiac origin through the detection of graft stenoocclusion or disease progression in the native coronaries and to assess coronary flow reserve , considered fundamental for evaluating the efficacy of a revascularisation procedure [ 2 ]  . 
 coronary angiography is still regarded as the gold standard for assessing the patency of cabg and , in patients with known disease , for selecting the best strategy for myocardial revascularisation ( percutaneous or repeat surgery ) [ 3 , 4 ]  . 
however , the small , though not negligible , risks connected with the procedure , the high number of studies performed for diagnostic purposes only without leading to interventional treatment , and the invasiveness of the procedure have prompted the search for an equally accurate but less invasive procedure for assessing graft patency . 
recently , several reports have appeared on the diagnostic potential of latest - generation mdct scanners in the diagnosis of coronary disease and the assessment of the patency of bypass grafts and stents [ 519 ]  . 
however , owing both to the lack of relevant randomised clinical trials and the novelty of lintroduzione da parte della comunit scientifica internazionale , di un nuovo approccio metodologico basato sullutilizzazione di comportamenti pratici e linee guida basate sulle migliori prove di efficacia , ha permesso la riduzione del divario esistente tra risultati delle ricerche e pratica clinica corrente , contribuendo a diminuire i comportamenti assistenziali inadeguati e a migliorare lo stato di salute della comunit , grazie allerogazione di prestazioni sanitarie caratterizzate dal miglior rapporto costo / beneficio [ 1 ]  . 
questo approccio rappresenta anche uno dei pi validi strumenti per favorire laggiornamento del medico , inteso come acquisizione di nuove conoscenze derivanti dal progresso scientifico , e come formazione permanente tesa a modificare attivamente i comportamenti nella pratica clinica . il ragionamento clinico basato sulle evidenze in medicina ( ebm ) rappresenta , in altri termini , lapplicazione di un percorso metodologico corretto , valido , riproducibile , che ha come punto di partenza la lettura e linterpretazione critica delle esperienze presenti nella letteratura scientifica [ 2 ]  . 
da un punto di vista speculativo , un campo particolarmente stimolante dove poter utilizzare gli strumenti messi a disposizione dalla ebm , quello della reale definizione del ruolo delle nuove apparecchiature di tc multidetettore ( tcmd ) nel work up diagnostico del paziente con malattia coronarica . 
 in questo primo contributo in particolare verr considerato il ruolo della tcmd nel follow - up dei pazienti sottoposti a rivascolarizzazione mediante bypass aorto - coronarici ( cabg )  . come noto nel monitoraggio dei pazienti post - cabg , lelettrocardiogramma ( ecg ) da sforzo , eseguito di routine anche nei pazienti asintomatici , considerato il test di primo livello [ 3 ]  . 
nei pazienti sintomatici con recidiva ischemica , al contrario , va accertata la genesi cardiaca del dolore anginoso tramite lidentificazione della steno - ostruzione del graft o della progressione di malattia nelle coronarie native e va valutata la riserva coronaria , considerata elemento fondamentale nella valutazione della efficacia della procedura di rivascolarizzazione , attraverso test di secondo livello quali lecocardiografia e la miocardioscintigrafia da stress [ 2 ]  . 
 la coronarografia considerata , ad oggi , il gold standard per la valutazione dello stato di perviet dei cabg e , nei pazienti con patologia accertata , per la scelta della strategia di rivascolarizzazione miocardica ( eseguibile per via percutanea o mediante nuovo intervento chirurgico ) [ 3 , 4 ]  . 
i minimi , ma non trascurabili , rischi connessi alla procedura , lelevato numero di esami eseguiti esclusivamente con finalit diagnostica e senza successivo trattamento interventistico , linvasivit della procedura , hanno alimentato linteresf . 
the aim of this paper is to define , on the basis of the available clinical evidence , the role of mdct as a noninvasive diagnostic imaging test in the follow - up of patients after cabg . materials and methods we conducted a systematic review of the literature by searching for studies dealing with the clinical and imaging follow - up of patients after cabg , using pubmed ( medline database ) as a search engine . 
the search was done by entering multiple phrases containing the following combinations of search terms : coronary artery bypass graft , revascularisation , follow - up , outcome , management , stress testing , coronary angiography , imaging , noninvasive and computed tomography . 
all papers were printed , read and analysed , and those considered assessable for the purposes of our study were selected on the basis of language of publication ( english ) , date of publication ( after 1990 ) , imaging technique ( ecg , scintigraphy , mdct ) , level of evidence according to ebm criteria ( table 1 ) , number of patients involved and number of cabg studied . 
tuttavia , sia per la mancanza di trials clinici randomizzati sia per lancora troppo recente utilizzo , il ruolo di tale metodica nel followup dei pazienti sottoposti a rivascolarizzazione chirurgica , nella pratica clinica , non ancora definito con certezza . scopo del seguente lavoro definire , alla luce delle evidenze cliniche presenti in letteratura , il ruolo attuale della tcmd come strumento diagnostico di imaging non invasivo nel follow - up dei pazienti sottoposti a cabg . materiali e metodi stata eseguita una revisione sistematica tramite ricerca bibliografica riguardo gli studi rilevanti presenti in letteratura inerenti il follow - up clinico - strumentale dei pazienti sottoposti a cabg , utilizzando come motore di ricerca pubmed ( medline database )  . 
tale ricerca stata effettuata inserendo frasi multiple con le seguenti combinazioni di parole chiave : coronary artery bypass graft , revascularization , follow - up , outcome , management , stress testing , coronary angiography , imaging , noninvasive , computed tomography . una volta stampati , letti e analizzati , sono stati considerati valutabili , ai fini del nostro studio , quei lavori , in lingua inglese , considerati pertinenti in base a data di pubblicazione ( successiva al 1990 ) , tecnica di studio ( ecocardiografia , scintigrafia , tcmd ) , stratificazione del livello di affidabilit secondo i criteri dellebm ( tabella 1 ) , numero di pazienti coinvolti e numero di cabg studiati . 
sono stati inoltre revisionate le linee guida e le raccomandazioni redatte negli ultimi 5 anni dalle principali societ di cardiologia , medicina table 1 classification of the various types of clinical studies based on evidence - based medicine ( ebm ) level type of study meta - analysis or high - quality randomised controlled trial lower - quality randomised clinical trial nonrandomised cohort study with prospective or retrospective controls , phase iii trial study of quasi - experimental design , descriptive study , comparative study , correlation study , retrospective case - control study case series without control group tabella 1 classificazione dei diversi tipi di studi clinici in base allebm livello tipo di studio metanalisi o trials clinico controllato e randomizzato di alto potere trials clinico randomizzato di pi basso potere studio di coorte non randomizzato , con controlli concorrenti o storici , studio fase iii studio di altro disegno quasi sperimentale , studio descrittivo , studio comparativo , studio di correlazione , studio retrospettivi tipo caso controllo studio di casistica serie casi senza gruppo di controllo f . 
these were assessed in two separate groups , the first comprising studies involving conventional noninvasive imaging tests with physical or pharmacological stress ( ecg , echocardiography and scintigraphy ) and the second comprising studies performed using mdct . 
in the first group , we selected five studies on the diagnostic accuracy of scintigraphy and echocardiography ( all with a level of evidence of iii / iv ) [ 2832 ] , one meta - analysis ( level of evidence i ) on the role of testing in patients after cabg [ 33 ] , and seven studies on risk stratification , prognosis , and determination of disease progression ( all involving the use of scintigraphy ) [ 3440 ]  . the results of the single studies are shown in tables 2 , 3 and 4 . 
the sensitivity and specificity values were 45% and 82% , respectively , for stress ecg , 86% and 90% for stress echocardiography , and 68% and 84% for stress scintigraphy . 
sono stati valutati separatamente due gruppi , il primo comprendente gli studi riguardanti le metodiche tradizionali di imaging non invasivo con induzione di stress fisico o farmacologico ( ecg , ecocardiografia e scintigrafia ) , il secondo comprendente gli studi effettuati con tcmd . 
nel primo gruppo sono stati selezionati opportunamente 5 studi inerenti author level of evidence method pts time since cabg symptoms sensitivity specificity lakkis [ 28 ] deluca [ 29 ] kafka [ 30 ] crouse [ 31 ] elhendy [ 32 ] 51 months chest pain ; 40% atypical symptoms thallium - 201 spect scintigraphy , exercise stress tc - 99m - sestamibi scintigraphy , 144 non specified non specified dipyridamole stress echocardiography , exercise stress echocardiography , exercise stress echocardiography , dobutamine stress 182 2 weeks 21 years 125 6 weeks 16 years 5 years non specified chest pain chest pain pts , patients ; cabg , coronary artery bypass graft ; spect , single photon emission computed tomography 9496 3067 tabella 2 metodiche di stress imaging non invasivo : accuratezza diagnostica autore livello di evidenza metodica lakkis [ 28 ] intervallo post - cabg 51 mesi sintomatologia sensibilit specificit dolore toracico ; 80 40% sintomi atipici deluca [ 29 ] 144 non specificato non specificata 9496 3067 scintigrafia - spect con tallio - 201 , stress fisico scintigrafia con tc - 99m sestamibi , stress con dipiridamolo ecocardio , stress fisico ecocardio , stress fisico ecocardio , stress con dobutamina kafka [ 30 ] crouse [ 31 ] elhendy [ 32 ] pz , pazienti ; gabg , coronary artery bypass graft ; spect , single photon emission computed tomography 182 2 settimane 21 anni 125 6 settimane 16 anni 5 anni dolore toracico 79 dolore toracico 98 non specificata 78 f . 
tables 6 and 7 report the indications and contraindications , respectively , for the use of coronary angiography in post - cabg patients provided by the acc / aha in their 1999 report ( task force on practice guidelines committee on coronary angiography ) [ 5 , 6 ]  . 
fifteen of them were prospective and one was retrospective ; all were validation studies comparing mdct with conventional coronary angiography ( level of evidence iv ) [ 10 , 11 , 1427 ]  . 
of the 16 studies , four were performed with 4 - detector - row mdct , one with 8 - detector - row mdct , ten with 16 - detector - row mdct and one with 64 - detector - row mdct . 
overall , the studies involved 705 patients for a total of 1 , 974 cabg segments . laccuratezza diagnostica della scintigrafia e dellecocardiografia , ( tutti con livello di evidenza iii / iv ) [ 2832 ] , 1 metanalisi , ( con livello di evidenza i ) , sul ruolo dei test nel paziente post - cabg [ 33 ] , 7 studi sulla stratificazione del rischio , prognosi , determinazione della progressione di malattia ( tutti riguardanti la scintigrafia ) [ 3440 ]  . i risultati dei singoli studi sono riportati nelle tabelle 24 . 
da questi , risulta una sensibilit e una specificit per lecg da sforzo rispettivamente del 45% e dell82% , per lecocardio - stress dell86% e del 90% , per la scintigrafiastress del 68% e dell84% . 
le linee guida dellamerican college of cardiology / american hearth association ( acc / aha ) per lesecuzione di test da sforzo nel paziente postcabg sono riportate nella tabella 5 [ 3 ]  . 
class iii : conditions for which there is evidence and / or general agreement that the procedure or treatment is not useful / effective and in some cases may be harmful tabella 5 linee guida acc / aha per lesecuzione di test da sforzo nel paziente post - cabga classe i valutazione di pazienti con sintomi ricorrenti suggestivi di ischemia dopo rivascolarizzazione classe iib monitoraggio periodico di pazienti selezionati , asintomatici , ad alto rischio , ( ridotta funzione ventricolare sinistra , malattia coronaria multivasale , malattia della discendente anteriore prossimale , diabete , occupazione professionale a rischio ) al fine di escludere restenosi , ostruzione del graft o progressione di malattia coronaria nativa classe iii monitoraggio periodico , di routine , in pazienti asintomatici senza specifiche indicazioni aclasse i : test per i quali c accordo e consenso relativamente allutilit e allefficacia . 
classe ii : test per i quali si hanno evidenze conflittuali o divergenze di opinioni relativamente allutilit e allefficacia ; iia : il peso dellevidenza / opinione risulta in favore dellutilit / efficacia della metodica ; iib : lutilit / efficacia non ben stabilita . 
classe iii : test , che possono talvolta risultare dannosi , per i quali c accordo e consenso riguardo linutilit / inefficacia the percentage of assessable segments ranged from 62% to 100% . 
recurrent angina arising in the immediate follow - up period is generally related to postoperative problems , whereas angina recurring months or years after surgery is more frequently due to stenoocclusion of the graft or progression of the atherosclerotic disease of the coronary arteries [ 4345 ]  . 
gli studi tcmd selezionati , 15 prospettici , 1 retrospettivo , tutti di validazione della metodica mediante comparazione con la coronarografia convenzionale ( livello di evidenza iv ) , sono riportati in tabella 8 [ 10 , 11 , 1427 ]  . 
infine la tabella 9 riporta le principali indicazioni terapeutiche ( trattamento percutaneo versus chirurgico ) riguardo il management della sindrome coronarica acuta nel paziente post - cabg [ 41 , 42 ]  . discussione patogenesi e fisiopatologia della recidiva ischemica nei pazienti post - cabg nei pazienti sottoposti ad intervento di rivascolarizzazione f . 
recurrent angina without high - risk criteria on noninvasive testing occurring > 1 year postoperatively . asymptomatic patients in whom a deterioration in serial noninvasive testing has been documented but who are not at high risk on noninvasive stress testing class iii 1 . 
symptomatic ischaemia in patients who are not candidates for repeat revascularisation . routine angiography in asymptomatic patients , unless as part of an approved research protocol aclass iia : weight of evidence / opinion is in favour of usefulness / efficacy . 
class iii : conditions for which there is evidence and / or general agreement that the procedure or treatment is not useful / effective and in some cases may be harmful tabella 6 indicazioni dellacc / aha alla coronarografia nel paziente post - cabga classe iia 1 . 
evidenza con stress imaging non invasivo di criteri ad alto rischio ( i.e anomalie della motilit parietale regionale multisegmentarie riscontrate allecocardiografia da stress e / o difetti di perfusione moderati - severi reversibili dopo scintigrafia da stress ) qualsiasi sia il periodo postoperatorio . 
 pazienti asintomatici , nei quali stato documentato un deterioramento del bypass in test seriali non invasivi , in assenza di criteri ad alto rischio allo stress imaging classe iii 1 . 
 coronarografia di routine in pazienti asintomatici , a meno che questi non facciano parte di un protocollo di ricerca approvato aclasse i : test per i quali c accordo e consenso relativamente allutilit e allefficacia . 
classe ii : test per i quali si hanno evidenze conflittuali o divergenze di opinioni relativamente allutilit e allefficacia ; iia : il peso dellevidenza / opinione risulta in favore dellutilit / efficacia della metodica ; iib : lutilit / efficacia non ben stabilita . 
 role of conventional noninvasive stress - imaging techniques ( echocardiography and scintigraphy ) although stress ecg is the first - line investigation for the assessment of post - cabg patients , it has poor sensitivity and is unable to locate the site of ischaemia [ 28 , 30 , 33 ]  . 
stress mediante cabg , il rischio di una recidiva ischemica pi elevato in presenza di sesso maschile , et > 80 anni , presenza dei comuni fattori di rischio coronarico ( ipertensione , fumo , diabete , ipercolesterolemia )  . 
una recidiva anginosa che si verifica nellimmediato follow - up , legata , in genere , a problemi inerenti il periodo peri - operatorio , mentre , una ricorrenza anginosa a distanza di mesi o anni dallintervento pi frequentemente dovuta alla steno - occlusione del graft o alla progressione di malattia aterosclerotica nelle coronarie native [ 4345 ]  . 
in patients unable to exercise adequately , the acc / aha recommend the use of intravenous dipyridamole or adenosine for scintigraphy and dobutamine for echocardiography [ 3 , 51 ]  . 
its sensitivity for single - vessel stenosis varies dependla patologia dei graft venosi si associa ad una maggiore instabilit di placca ed responsabile del 53% degli episodi anginosi che si verificano entro i 5 anni , del 76% degli episodi tra i 5 e i 10 anni , del 92% di quelli oltre i 10 anni . 
la progressione dellaterosclerosi nei vasi nativi , causa , negli stessi periodi , rispettivamente del 47% , 24% e 8% degli eventi ischemici [ 44 , 50 ]  . ruolo delle metodiche tradizionali di stress imaging non invasivo ( ecocardiografia e scintigrafia ) lecg da sforzo rappresenta lindagine di primo livello nella valutazione dei pazienti post - cabg , tuttavia tale test dotato di bassa sensibilit ed incapace a localizzare il sito ischemico [ 28 , 30 , 33 ]  . 
lecocardiografia e la scintigrafia dopo induzione di stress sono considerate metodiche diagnostiche di secondo livello che utilizzano limaging per definire lo stato perfusionale e il recupero funzionale del miocardio rivascolarizzato . 
nei pazienti che non possono sostenere un adeguato esercizio fisico , lacc / aha raccomanda lutilizzo di dipiridamolo o adenosina ev nel test scintigrafico e di dobutamina in quello ecocardiografico [ 3 , 51 ]  . lecocardiografia identifica le aree ischemiche sotto forma di anomalie della motilit parietale regionale e come modificazione della funzione sistolica globale e del volume telesistolico . 
la sensibilit nellidentificazione di stenosi coronariche con tale metodica maggiore se la compromissione multivasale ( 23 regioni ) piuttosto che monovasale ( sensibilit 96% versus 77% ) [ 30 ]  . 
la sensibilit in caso di stenosi monovasale varia inoltre in base ai diversi territori coinvolti ( lad : 64% , clx : 50% , rca : 45% ) [ 32 ]  . 
limaging con spect incrementa laccuratezza diagnostica della metodica [ 2830 ]  . il ruolo dello stress imaging non invasivo nei pazienti sottoposti a cabg dipende da due fattori principali : 1 presenza dei sintomi ischemici , 2 tempo trascorso dallintervento . in base a quanto riportato dalle linee guida dellamerican college of cardiology / american hearth association / american society nuclear cardiology ( acc / aha / asnc ) , entro i primi 5 anni dallintervento , i pazienti asintomatici non ad alto rischio , non devono essere sottoposti a screening con stress - test non invasivi [ 4 ]  . 
per quanto riguarda i pazienti asintomatici ad alto rischio ( cio con ridotta funzione ventricolare sinistra , malattia coronaria multivasale , malattia della discendente anteriore prossimale , diabete , occupazione professionale a rischio ) c invece disaccordo tra quanto espresso nelle linee guida dellacc / aha del 2002 , e quanto riportato da quelle dellacc / aha / asnc del 2003 [ 4 , 5 ]  . 
al contrario , le seconde , concludono che il peso dellevidenza giustifica lesecuzione di test stress routinari ( livello di evidenza iia ) e raccomandano una spect dopo sforzo a 35 anni dalla rivascolarizzazione primitiva . 
the utility of scintigraphy is related to the possibility of identifying new areas of ischaemia prevalently caused by gsv stenoocclusions , which show up as areas of poor myocardial perfusion . 
trattamento percutaneo versus chirurgico pci + stent per le lesioni focali dei graft venosi , specie se subtotali e per le lesioni ostiali ( anastomosi prossimale ) dei graft arteriosi ; maggior vantaggio si ha nelle stenosi acute ( < 30 gg ) pci da sola per le lesioni anastomotiche distali , sia dei graft arteriosi sia di quelli venosi repeat - cabg per stenosi multiple e / o diffuse dei graft , specie se totali e croniche , soprattutto per graft connessi alla lad ( spect ) imaging increases the diagnostic accuracy of the method [ 2830 ]  . 
on the basis of the guidelines of the ( acc / aha / asnc ) , within the first 5 years of surgery , asymptomatic patients who are not at high risk should not be screened with noninvasive stress tests [ 4 ]  . 
as for high - risk asymptomatic patients ( with reduced left ventricular function , multivessel coronary artery disease , proximal lad artery disease , diabetes , high - risk occupation ) , there is no agreement between the acc / aha 2002 guidelines and the acc / aha / asnc 2003 guidelines [ 4 , 5 ]  . 
the acc / aha 2002 guidelines do not recommend periodic monitoring with noninvasive stress testing in this class of patients , whereas the acc / aha / asnc 2003 guidelines conclude that the weight of evidence justifies the use of routine stress tests ( level of evidence iia ) and recommend a stress spect 35 years after initial revascularisation . 
neither guideline recommends the use of stress tests for asymptomatic patients later than 5 years after revascularisation , although the fact that gsv grafts start to occlude at 5 years has led some authors to advocate a routine stress - spect exam regardless of symptoms to stratify the risk , assess the prognosis , determine disease progression and obtain information to guide possible treatment [ 36 , 39 ]  . conventional coronary angiography coronary angiography is considered the gold standard in assessing cabg patency and choosing the best strategy for cardiac revascularisation . 
tra le complicanze cosiddette minori devono essere annoverate quelle vascolari locali al sito di cateterizzazione ( ematoma retroperitoneale , fistola arterovenosa , pseudoaneurisma , trombosi arteriosa ) e quelle determinanti aritmie cardiache ( tachicardia - fibrillazione ventricolare , aritmie atriali , disturbi di conduzione )  . 
tra questi let ( < 1 anno , > 80 anni ) , una classe funzionale iv , una malattia del tronco comune della coronaria sinistra , cardiopatie valvolari associate , una frazione di eiezione ventricolare sinistra < 30% , gravi patologie di natura non cardiaca [ 6 ]  . 
nei pazienti bypassati il rischio di complicanze inoltre maggiore rispetto a quello della popolazione standard . la principale indicazione allesecuzione dellesame rappresentata , dallevidenza con stress imaging non invasivo , di criteri ad alto rischio ( ad esempio , anomalie della motilit parietale regionale multisegmentarie riscontrate allecocardiografia da stress e / o difetti di perfusione moderati - severi reversibili dopo scintigrafia da stress ) qualsiasi sia il periodo postoperatorio . 
nei pazienti in cui si ha una dimostrazione coronarografica di patologia si pone il problema della scelta del tipo di nuova rivascolarizzazione da effettuare , percutanea ( pci ) o ancora chirurgica ( repeat - cabg , r - cabg )  . 
si raccomanda lesecuzione della pci ( stent ) nelle stenosi subtotali , specie se acute , che si verificano nei pazienti con funzione ventricolare preservata , mentre si preferisce un reintervento chirurgico mediante rcabg nelle stenosi multiple , di vecchia data , in pazienti con malattia multivasale e ridotta funzione ventricolare [ 41 , 46 , 47 , 5256 ]  . 
these include age ( < 1 year , > 80 years ) , functional class iv , disease of the left coronary common trunk , associated valve disease , left ventricular ejection fraction < 30% , and severe noncardiac disease [ 6 ]  . 
in patients with bypass grafts , the risk of complications is even higher than in the standard population . the main indication for coronary angiography is evidence on noninvasive stress imaging of high - risk criteria ( multisegment regional wall motion abnormalities on stress echocardiography and / or reversible moderate to severe perfusion defects on stress scintigraphy ) whatever the postoperative period . 
patients with evidence of disease on coronary angiography pose the problem of choosing whether to perform percutaneous ( pci ) or surgical [ repeat cabg ( rcabg ) ] revascularisation . 
pci ( stent ) is recommended in subtotal , especially acute , stenoses in patients with preserved ventricular function , whereas r - cabg is preferred for multiple , long - standing stenoses in patients with multivessel disease and reduced ventricular function [ 41 , 46 , 47 , 5256 ]  . more than 40% of coronary angiography examinations conducted in haemodynamics laboratories are not followed by percutaneous or surgical revascularisation procedures but are only performed for diagnostic purposes [ 57 ]  . 
the resulting increase in costs , combined with the complications and risks connected with the method , has prompted a search for techniques with equal diagnostic yield but less invasiveness . the recent introduction of ct scanners with 4 , 16 , 32 and 64 detector rows with improved spatial and temporal resolution ( enabling the simultaneous acquisition of multiple submillimetre slices with quasi - isotropic voxels and the possibility of cardiac gating ) has revolutionised cardiovascular imaging by allowing the study of grafts with a high level of diagnostic accuracy . mdct : general scanning technique all of the studies selected were fairly homogenous in terms of scanning technique . 
in clinical practice , the study volume extends to include the proximal anastomosis on the ascending aorta for venous grafts as well as the distal anastomosis of the different territories of the coronary arteries ; for the study of lima grafts , inclusion of the origin of the proximal subclavian artery is not required . 
after an unenhanced scan to assess the extent of calcification of the native coronaries and the course of the metal clips , a contrast - enhanced study is performed after intravenous administration via a dual - syringe automatic injector of 100120 ml nonionic iodinated contrast material ( 300 400 mgi / ml ) at a flow rate of 34 ml / s , followed by a bolus of 3050 ml saline solution . 
for optimal enhancement , nearly all of the studies used the bolus tracking technique to determine scan delay , with the acquisition starting when a predefined threshold of enhancement was reached ( 100150 hu in the left ventricle or ascending aorta )  . 
images were reconstructed with retrospective ecg gating and variable r - r temporal windows and postin ogni caso pi del 40% delle coronarografie condotte nei laboratori di emodinamica non sono seguite da procedure di rivascolarizzazione percutanea o chirurgica , ma sono eseguite esclusivamente in senso diagnostico [ 57 ]  . 
il relativo aggravio dei costi , associato alle complicanze e ai non trascurabili rischi connessi alla metodica , ha spinto la ricerca verso strumenti con potenzialit diagnostiche altrettanto elevate , ma meno invasivi . 
recentemente lintroduzione dei nuovi scanner a 4 , 16 , 32 e 64 canali , grazie al miglioramento della risoluzione spaziale e temporale ( con la possibilit che ne deriva di acquisire simultaneamente slice multiple con spessore submillimetrico , voxel quasi - isotropico e con la possibilit di eseguire esami accurati con opportuna cardiosincronizzazione ) , ha rivoluzionato limaging cardiovascolare rendendo possibile lo studio dei graft con elevata accuratezza diagnostica . tcmd : principi generali di scansione per quanto riguarda i principi generali di scansione , i diversi studi selezionati sono abbastanza omogenei tra loro . 
nella pratica clinica il volume di studio esteso a comprendere lanastomosi prossimale su aorta ascendente per i graft venosi oltre che lanastomosi distale sui diversi territori delle coronarie ; per lo studio dei graft con mammaria interna non necessario includere lorigine succlavia prossimale . 
dopo unacquisizione diretta senza mezzo di contrasto , utile per valutare lentit delle calcificazioni delle coronarie native e il decorso delle clips metalliche , lo studio contrastografico viene eseguito mediante somministrazione endovenosa , utilizzando un iniettore automatico a doppia siringa , di 100120 ml di mdc iodato non ionico ( 300400 mgi / ml ) , alla velocit di 34 ml / s , seguiti da un bolo di 3050 ml di soluzione salina . 
per ottimizzare il contrast enhancement , in quasi tutti gli studi viene utilizzata la tecnica del bolus tracking , e il ritardo per linizio della scansione viene selezionato a partire dal raggiungimento di una soglia predefinita ( 100150 uh in ventricolo sinistro o aorta ascendente )  . 
le immagini sono ricostruite con gating ecg di cardiosincronizzazione retrospettiva e finestre temporali r - r variabili , a partire da quelle assiali native , nel post - processing , si procede allutilizzazione di ricostruzioni multiplanari ( mpr ) , curve ( cmpr ) , proiezioni di massima intensit ( mip ) e volume rendering tridimensionale ( 3d - vr ) , valutando , ai fini della valutazione di perviet dei cabg decorso ( anastomosi prossimale , graft body , anastomosi distale ) e tipo di bypass . 
il giudizio di perviet fornito in base allentit della stenosi : perviet ( stenosi < 50% ) , stenosi critica ( stenosi > 50% ) , ostruzione ( mancata opacizzazione del bypass )  . tcmd e cabg : applicazioni attuali della metodica e potenzialit diagnostiche in base allanalisi critica della letteratura prima dellavvento della tcmd , i primi studi di valutazione dello stato dei bypass con tomografia computerizzata , sono stati eseguiti con tecnica a fascio elettronico ( ebct )  . 
the judgement of patency was based on the degree of stenosis : patent ( stenosis < 50% ) , critical stenosis ( stenosis > 50% ) , occlusion ( no opacification of the bypass graft )  . mdct and cabg : current applications and diagnostic potential on the basis of a critical appraisal of the literature before the advent of mdct , early studies assessing the state of bypass grafts with ct used the electron beam technique ( ebct )  . 
despite its high level of diagnostic performance in identifying graft occlusion ( 95%100% sensitivity and 89%100% specificity ) , ebct never became widely used because of its poor spatial resolution and above all its limited availability [ 79 ]  . 
one of the first studies to compare the diagnostic performance of ct with that of coronary angiography was conducted in 2001 by ropers et al . , who studied 65 patients ( 182 grafts in total ) with a 4 - detector - row scanner [ 14 ]  . 
the authors reported sensitivity and specificity values of 97% and 98% , respectively , for the demonstration of complete graft occlusion and of 75% and 92% , respectively , for the demonstration of significant stenosis > 50% . 
thirty - eight percent of the grafts that were patent at coronary angiography could not be assessed with mdct owing to metal clip , respiration and motion artefacts , poor graft opacification and arrhythmias arising during the examination . 
similar results were obtained by nieman et al . , who used the same method in a sample of 24 patients and 86 grafts [ 15 ]  . the more recent studies , comparing 16and 64 - detectorrow scanners with coronary angiography , all attest to the efficacy of mdct in determining the patency or stenoocclusion of grafts [ 10 , 11 , 16 , 1827 ]  . 
 the contribution to the literature of the above studies appears , however , to be somewhat limited , and an accurate analysis shows that the studies , performed in single centres and on small patient populations , provide conflicting results that are generally of little value for establishing the true role of mdct in the diagnostic workup of this specific class of patients . 
instead , selection of the study samples were heterogeneous , not so much in terms of exclusion criteria , which are standardised ( renal failure , heart failure , respiratory failure , known allergy to iodinated contrast material , heart rate > 65 70 bpm ) as in terms of inclusion criteria . 
in at least two studies [ 16 , 19 ] by the same team , asymptomatic patients only underwent the ct examination , whereas in another , retrospective , study , out of a total of 27 patients , 16 had recurrent episodes of angina and 11 were asymptomatic and undergoing routine clinical and imaging follow - up [ 18 ]  . 
uno dei primi studi di comparazione della performance diagnostica della tc nei confronti del gold - standard coronarografico , effettuato con tcmd a 4 - canali , stato quello di ropers nel 2001 , condotto su 65 pazienti per un numero complessivo di 182 graft [ 14 ]  . 
lautore ha riportato valori di sensibilit e specificit , nella dimostrazione dellostruzione completa del graft , rispettivamente del 97% e 98% , e nella dimostrazione di stenosi significativa > 50% rispettivamente del 75% e 92% . 
il 38% dei graft che risultarono pervi alla coronarografia non poterono essere valutati con tcmd , a causa degli artefatti da clips metalliche , da respiro e da movimento , e a causa della scarsa opacizzazione del graft e delle aritmie durante lesame . 
i recenti lavori sullargomento , effettuati con scanner a 16 e 64 canali , testimoniano tutti , tramite comparazione con la coronarografia , lelevata efficacia della metodica nella valutazione di perviet o steno / ostruzione dei graft [ 10 , 11 , 16 , 1827 ]  . il contributo in letteratura apportato dai sopraccitati studi appare tuttavia non completamente esaustivo e da unanalisi accurata si scopre che gli stessi , eseguiti da singoli centri coinvolgendo un numero complessivamente esiguo di pazienti , forniscono risultati non univoci , in genere poco utili per stabilire leffettivo ruolo della tecnica nel work - up diagnostico di questa classe specifica di pazienti . 
invece la selezione del campione di studio disomogenea , non tanto per i criteri di esclusione , che sono standardizzati ( insufficienza renale , insufficienza cardiaca , insufficienza respiratoria , comprovata allergia al mezzo di contrasto iodato , frequenza cardiaca > 6570 bpm ) , quanto per i criteri di inclusione . 
in almeno due studi [ 16 , 19 ] , eseguiti peraltro dallo stesso gruppo , vengono sottoposti ad esame tc esclusivamente pazienti asintomatici , in un altro , retrospettivo , su un totale di 27 pazienti , 16 riferiscono episodi di angina ricorrente , 11 sono asintomatici in follow - up clinico - strumentale routinario [ 18 ]  . 
infine , nei rimanenti lavori , vengono studiati pazienti sintomatici , senza che venga fornita alcuna indicazione riguardo la positivit o meno dello stress imaging non invasivo ( ecocardiografia e / o scintigrafia da sforzo ) e riguardo leventuale attinenza ai criteri di accesso alla coronarografia raccomandati dallacc / aha del 1999 ( task force on practice guidelines committee on coronary angiography ) [ 5 ]  . 
nel lavoro di martuscelli uno dei criteri di esclusione dallesame la presenza di angina instabile , che per il quadro di presentazione sintomatologico del 20% del totale dei pazienti post - cabg [ 11 , 58 ]  . i dati statistici forniti dai diversi studi tendono ad essere discordanti . 
ad esempio schlosser , in uno studio condotto su 48 pazienti ( riferiti alla coronarografia e prima sottoposti a tcmd a 16 canali ) , con un totale di 131 graft valutati , rif . 
for example , in one study of 48 patients ( referred for coronary angiography and first studied by 16 - detector - row ct ) with a total of 131 grafts assessed , schlosser et al . 
reported a sensitivity of 96% , a specificity of 95% and a positive predictive value ( ppv ) of 81% and a negative predictive value ( npv ) of 99% [ 10 ]  . 
in the same study , the authors admit that as regards arterial cabg , only 76% of distal anastomoses to the anterior descending artery and 60% of distal anastomoses to diagonal branches could be visualised . 
 ( assessable segments , 95% ) [ 11 , 19 , 25 ]  . the statistical differences among the studies are also reflected in the different diagnostic evaluation of the distal anastomoses . 
distal anastomoses are notably particularly difficult to study owing to their relative mobility compared with the relatively fixed proximal anastomoses and graft body , their size ( generally < 2 mm , at the limit of mdct spatial resolution ) and the beam - hardening effect caused by metal clips . 
mdct scanners with 16 and 64 detector rows provide better results in the assessment of the proximal anastomosis , graft body and distal anastomosis compared with 4 - detector - row scanners . 
however , there are no statistically significant differences between 16 and 64 detector rows in the visualisation of the distal anastomosis ( 16 - mdct : assessability 92% , 64 - mdct : assessability 94% ) [ 12 , 24 , 26 ]  . 
 the literature data on the native coronary arteries in patients with suspected coronary disease cannot be transferred to the native coronaries of post - cabg patients , which are more difficult to assess due to severe atherosclerosis and calcification and often previous treatment with angioplasty and porta valori di sensibilit del 96% , specificit del 95% , valore predittivo positivo pari a 81% e valore predittivo negativo del 99% [ 10 ]  . 
nello stesso lavoro si ammette che , relativamente ai cabg arteriosi , stato possibile visualizzare solamente il 76% delle anastomosi distali sulla discendente anteriore e il 60% delle anastomosi distali sui rami diagonali . 
come gi detto , risultati sovrapponibili a chiurlia sono ottenuti da rossi ( sensibilit e specificit : 100% ; 91 , 4% dei segmenti di cabg valutabili ) , martuscelli ( sensibilit e specificit : 97% e 100% ; 88% dei segmenti di cabg valutabili ) e bursstahler ( 95% dei segmenti di cabg valutabili ) [ 11 , 19 , 25 ]  . 
come noto , a causa della relativa mobilit rispetto allanastomosi prossimale e al graft body , relativamente fissi , a causa delle dimensioni ( in genere , < 2 mm , ai limiti della risoluzione spaziale della tcmd ) , e per leffetto di beam hardening da clips metalliche , lo studio di tali segmenti anatomici particolarmente difficoltoso . 
le apparecchiature tcmd a 16 e 64 canali , forniscono , rispetto alla 4 canali , risultati migliori in termini di valutabilit dellanastomosi prossimale , del graft body e dellanastomosi distale . 
ai fini della visualizzazione di questultima , tuttavia , non si evidenziano , tra scanner a 16 e 64 canali , differenze statisticamente significative ( 16 - tcmd : valutabilit 92% , 64 - tcmd : valutabilit 94% ) [ 12 , 24 , 26 ]  . i dati della letteratura sulle coronarie native nei pazienti con sospetta malattia coronarica non sono trasferibili alle coronarie native dei pazienti bypassati , pi difficili da valutare in quanto severamente ateromasiche , ossia calcifiche , e spesso precedentemente trattate con angioplastica e stenting . 
a tal proposito , in questa coorte di pazienti , nieman [ 15 ] riporta una percentuale di segmenti nativi valutabili pari al 66 , 4%69 , 2% ed una sensibilit globale nella osservazione di stenosi significative , sempre nei segmenti nativi , pari al 52 , 9%69 , 6% . nonostante tali limitazioni , la tcmd , indubbiamente , presenta anche alcuni importanti vantaggi , i maggiori dei quali legati alla non invasivit e alla riduzione dei costi di esame rispetto alla coronarografia convenzionale . 
 [ 15 ] reported a percentage of assessable native segments of 66.4%69.2% and an overall sensitivity in the detection of significant stenosis in native segments of 52.9%69.6%. despite these limitations , there are undoubtedly several advantages in using mdct , the most important being related to noninvasiveness and reduced cost of the examination compared with conventional coronary angiography . 
interobserver agreement in image interpretation is high paradoxically higher for the assessment of the distal anastomosis and graft body compared with the proximal anastomosis independently of the use of thin - slab mip , mpr or vrt images [ 10 ]  . 
nonetheless , image interpretation cannot be divorced from the assessment of the native coronary arteries , which has lower reproducibility and must be performed by highly qualified staff with specific experience in cardiac imaging . 
the accf / aha / american college of physicians ( acp ) task force on clinical competence and training has recently published a statement to this effect , with the aim of identifying the most appropriate training pathways to enhance the professional competence of the physician ( radiologist or cardiologist ) interpreting the images [ 59 ]  . another advantage of mdct is its ability to detect other possible extravascular diseases that present clinically with chest pain and may mimic recurrent angina . 
in this class of patients , the differential diagnosis of chest pain includes pleural effusion , pericardial effusion , pulmonary embolism and infection of the sternal sutures [ 13 ]  . 
 in perspective , mdct could provide the information required for planning coronary revascularisation procedures . this field of application is above all justified by the need to reduce the small but nonnegligible risks and the costs related to the high number of diagnostic coronary angiograms that are not followed by percutaneous interventional procedures ( more than 40% of the total ) either because they are negative for graft disease ( or for disease progression in the native coronaries ) or because their findings warrant r - cabg . 
the unnecessary cost of invasive procedures could be reduced if mdct were used to screen symptomatic patients for focal , stenotic or substenotic acute or subacute graft disease to be referred for therapeutic coronary angiography . 
 recommendations for mdct in patients after cabg all in all , the clinical utility of mdct in the diagnostic workup of patients after cabg has not yet been correctly evaluated . 
ideally , it should be assessed in randomised trials ( involving also other noninvasive stress imaging methods , such as stress echocardiography and scintigraphy ) in which slab , mpr o vrt [ 10 ]  . 
in linea generale i valori di riproducibilit , nella valutazione dei bypass , sono elevati per il calibro maggiore e per il fatto che i cabg sono relativamente svincolati dal battito cardiaco . 
in ogni caso linterpretazione delle immagini non pu prescindere dalla valutazione delle coronarie native , che si associano a valori di riproducibilit minori , e deve essere affidata a figure professionali altamente qualificate e con esperienza specifica nel settore dellimaging cardiaco . 
a tal proposito lamerican college of cardiology foundation / american heart association / american college of physicians ( accf / aha / acp ) task force on clinical competence and training ha recentemente pubblicato uno statement su tale argomento , con lo scopo di individuare il percorso formativo pi corretto possibile , implementando lesperienza professionale del medico ( radiologo o cardiologo ? ) che interpreta le immagini [ 59 ]  . altro vantaggio della tcmd correlato alla capacit della stessa di evidenziare eventuali altre patologie extravascolari che si presentano clinicamente con dolore toracico e che possono mimare unangina ricorrente . 
in questa classe di pazienti la diagnosi differenziale del dolore toracico include il versamento pleurico , il versamento pericardico , lembolia polmonare e linfezione delle suture sternali [ 13 ]  . 
la tcmd pu fornire inoltre informazioni supplementari riguardo reperti incidentali non relati allesame coronarografico . in prospettiva , la tcmd , potrebbe offrire gli elementi necessari alla pianificazione di eventuali interventi di rivascolarizzazione coronarica . 
questo possibile campo di applicazione trova un senso soprattutto alla luce della necessit di ridurre i rischi , minimi ma non trascurabili , e i costi legati allelevato numero di coronarografie diagnostiche non seguite da procedure di interventistica percutanea ( pi del 40% del totale ) , o perch negative per patologia dei graft ( o progressione di malattia coronarica dei vasi nativi ) o perch indirizzanti verso un nuovo trattamento di tipo chirurgico mediante repeat cabg . 
la riduzione dei costi inutili legati alle procedure invasive , potrebbe essere ottenuta utilizzando la tcmd come filtro in grado di screenare quei pazienti sintomatici , con patologia dei graft focale , stenotica o substenotica , acuta o subacuta , da avviare selettivamente alla coronarografia con finalit terapeutiche . 
lidentificazione di stenosi multiple , croniche , in pazienti con malattia multivasale , potrebbe evitare il ricorso alla coronarografia e porre invece , da subito , lindicazione alla rivascolarizzazione chirurgica . raccomandazioni allesame tcmd nel paziente post - cabg in definitiva , ad oggi , lutilit clinica dellesame nel work - up diagnostico dei pazienti post - cabg , non ancora stata correttamente valutata . 
idealmente questa dovrebbe essere valutata nel contesto di studi randomizzati ( coinvolgenti anche le altre metodiche di stress imaging non invasivo , quali lecocardiografia e la scintigrafia da stress ) in cui pazienti con sospetta patologia dei graft sono assegnati a un iter diagnostico con o senza tcmd . 
in fact , no such studies , which should be conducted on an adequate number of patients , have yet been performed due to ethical as well as technical reasons . 
the evidence available on the role of mdct is therefore only indirect and derived from studies that compared its efficacy with that of coronary angiography , considered the gold standard . 
le prove disponibili sul ruolo della tcmd sono pertanto solo di tipo indiretto , derivabili da quei lavori che hanno valutato lefficacia della modalit rispetto alla coronarografia , considerata il gold standard . 
this was due to the fact that neither colour or power doppler are suitable for correct management of the signals produced by microbubble insonation , as they are limited by the heavy presence of artefacts . microbubbles may be insonated by a characteristic frequency named resonance or fundamental frequency ( f0 ) by using a high or low transmit power . 
contrast - specific us techniques have recently undergone an important technical development with the introduction of innovative algorithms able to register selectively the harmonic signals produced by microbubbles and to suppress the signal produced by stationary tissues . 
the different contrast - specific us techniques may be distinguished by their basic principle into pseudo - doppler , harmonic , phase - modulation , amplitude - modulation and phaseand amplitude - modulation techniques . 
 key words ultrasound microbubble harmonic ultrasound pulse inversion contrast - specific techniques riassunto lintroduzione dei mezzi di contrasto ecografici a base di microbolle ha determinato una importante evoluzione della tecnologia ecografica rappresentata dalla introduzione di tecniche ecografiche contrasto - specifiche . 
la ragione di questo legata al fatto che il color ed il power doppler tradizionali non sono in grado di gestire correttamente il segnale prodotto dalla insonazione delle microbolle essendo gravati dalla presenza di artefatti . 
se le microbolle vengono invece insonate a bassa potenza emettono , oltre alla frequenza di risonanza , frequenze armoniche ( 2f , 3f , 4f ) determinate dal comportamento fisico non lineare delle microbolle . 
le tecniche contrasto - specifiche hanno visto recentemente un particolare sviluppo tecnologico grazie alla introduzione di algoritmi innovativi in grado di registrare selettivamente il segnale prodotto dalle microbolle e di sopprimere il segnale prodotto dai tessuti stazionari . 
in base al loro principio di funzionamento le tecniche ecografiche contrastospecifiche si possono distingure in tecniche pseudo - doppler , armoniche , a modulazione di fase , a modulazione di ampiezza , ed a modulazione di ampiezza e di fase . parole chiave ecografia microbolle ecografia armonica pulse inversion tecniche contrasto - specifiche introduction introduzione microbubble contrast agents for ultrasound ( us ) have aroused increasing interest in recent years , and contrast - enhanced us ( ceus ) is a rapidly evolving field with many clinical applications . 
the observation that microbubbles may be employed as us contrast agents is not recent , as the first description dates back to 1968 [ 1 ] when contrast phenomena were observed in the aorta during cardiac catheterization after injection of saline solution . 
this was caused by air microbubbles that were produced by cavitation during the ini mezzi di contrasto impiegati in ecografia ( ultrasound ; us ) hanno suscitato un interesse sempre crescente negli ultimi anni , e lecografia con mezzo di contrasto divenuta una tecnica in rapida evoluzione con molte applicazioni in ambito clinico . 
la scoperta delle microbolle come mezzi di contrasto in ecografia non cos recente dato che la descrizione della loro prima applicazione risale al 1968 [ 1 ] quando si osserv la presenza di enhancement allinterno dellaorta durante la somministrazione di soluzione salina nel corso di una procee . 
in particular , innovative us techniques , known as contrast - specific techniques , were introduced to register selectively the harmonic signal produced by the nonlinear physical behaviour of microbubbles . 
this was because conventional colour and power doppler are unable to manage the harmonic signals produced by microbubble insonation , as they are limited by the presence of artefacts , including blooming and jail - bar artefacts [ 2 ]  . 
the recently introduced sulphur hexafluoride or perfluorocarbon - filled microbubbles offer both an excellent safety profile and a longer persistence in the peripheral circulation than do air - filled microbubbles . microbubble contrast agents have a shell of biocompatible material , such as proteins , lipids or biopolymers . 
they are injectable intravenously and can pass through the pulmonary capillary bed after a peripheral injection , as their diameter ( 310 m ) is below that of red blood cells . 
low - solubility low - diffusibility gases , such as perfluorocarbons and sulphur hexafluoride gas , are employed to improve the persistence of microbubbles in the peripheral bloodstrea the physical properties of microbubble contrast agents are closely related not only to their gas content and to the peripheral shell chemical composition but also to the frequency of the us beam , the pulse repetition frequency and , above all , the acoustic power employed for insonation [ 2 ]  . 
tale effetto era determinato dalle microbolle daria prodotte da un fenomeno fisico di cavitazione durante la somministrazione attraverso il catetere . da allora molti sforzi sono stati fatti per aumentare le possibili applicazioni cliniche delle microbolle . 
la ragione di questo legata al fatto che il color ed il power doppler tradizionali non sono in grado di gestire il segnale prodotto dalla insonazione delle microbolle essendo gravati dalla presenza di artefatti tra cui il blooming ed il jail bar [ 2 ]  . composizione chimica delle microbolle i mezzi di contrasto a base di microbolle sono stati approvati nella maggior parte dei paesi europei , e vengono oggi largamente impiegati anche in asia ed in canada . 
negli stati uniti la food and drug administration limita per il momento limpiego delle microbolle solo al settore ecocardiografico . le microbolle di recente introduzione a base di gas esafluoruro di zolfo o di perfluorocarburi possiedono un profilo di sicurezza elevato ed una maggiore stabilit nel circolo periferico rispetto alle microbolle a base di aria . 
le microbolle vengono somministrate per via endovenosa , e sono in grado di attraversare il letto capillare polmonare dato che presentano un diametro ( 310 m ) inferiore a quello dei globuli rossi . per migliorare la stabilit delle microbolle nel circolo periferico sono state impiegate due strategie principali : rivestire le microbolle con una capsula di composizione chimica stabile nel circolo periferico , oppure utilizzare gas a basso coefficiente di diffusione attraverso la caspula [ 3 , 4 ]  . 
la capsula periferica presenta uno spessore variabile tra 10 a 200 nm , e pu essere rigida ( ad esempio se costituita da albumina denaturata oppure biopolimeri ) , oppure pi flessibile ( ove vengano utilizzati fosfolipidi )  . 
i gas caratterizzati da una bassa solubilit e da una bassa diffusibilit in acqua come il perfluorocarburo e lesafluoruro di zolfo , determinano un aumento della stabilit delle microbolle nel circolo periferico . le propriet fisiche delle microbolle sono strettamente correlate non solo al loro contenuto gassoso ed alla composizione chimica della loro capsula periferica , ma anche alla frequenza del fascio di us impiegato , alla frequenza di ripetizione dellimpulso , e soprattutto alla potenza di insonazione impiegata [ 2 ]  . 
i diversi mezzi di contrasto a base di microbolle sono elencati nella tabella 1 . tecniche di insonazione per ottenere un segnale armonico utile per la costruzione dellimmagine le microbolle devono essere insonate mediante la loro specifica frequenza fondamentale , detta anche di table 1 microbubble ultrasound contrast agents perfluorocarbon - filled sulphur - hexafluoride - filled sonovue e . 
 tutti gli agenti possiedono una capsula a base di fosfolipidi eccetto : acaspula a base di galattosio ; bcapsula a base di albumina ; ccapsula a base di cianoacrilato ; dagente a cambiamento di fase : una emulsione di perflenapent liquido su liquido che contiene dodecafluoropentano in forma liquida in fase dispersa e che passa alla forma gassosa alla temperatura corporea formando microbolle con diametro compreso tra 38 m powee ( db ) foundamental frequency resonance frequency fig . 
1 lo spettro di intensit acustica delle microbolle a base di esafluoruro di zolfo rispettivamente per una bassa ( linea tratteggiata ) ed alta potenza di insonazione ( linea continua )  . 
i tessuti stazionari presentano un unico picco di emissione in corrispondenza della frequenza fondamentale ( risonanza ) per gentile concessione di springer science and business media , da [ 2 ]  . transmission frequency ( mhz ) ( rarefactional ) pressure that the transmitted us pulse achieves during its travel through a medium of specified attenuation coefficient , and fc is the centre frequency of the pulse . 
a more correct and reproducible manner is to express the insonation transmit power as mega pascal ( mpa ) , which is reported on the screen of some us devices . 
la formula che lo rappresenta la seguente : indice meccanico = p - / ( cid : 2 ) fc , dove pindica il picco massimo di pressione negativa ( rarefazione ) che limpulso ultrasonoro trasmesso raggiunge in un mezzo con uno specifico indice di attenuazione , mentre fc rappresenta la frequenza centrale della banda di e . 
one group of methods , commonly known as high - transmit - power insonation , uses large pulse amplitudes ( about 1 mpa ) and produces microbubble destruction with emission of an irregular wideband harmonic signal , similar to an explosion , and named stimulated acoustic emission ( sae )  . 
the threshold for destruction is variable and depends on a number of different factors , such as microbubble size , shell material and filling gas and the attenuation of the us beam power by the overlaying tissues . 
these methods are generally employed to insonate air - filled microbubble contrast agents with a soft shell , presenting a low harmonic behaviour at low - transmit - power insonation , and are limited by the transiency of the signal , which persists only for 23 frames . 
this leads to asymmetric or nonlinear oscillations , which contain both fundamental and harmonic frequencies multiple of the fundamental frequency . this insonation method is employed with sulphur hexafluoride or perfluorocarbon - filled microbubbles and allows realtime scanning given the long persistence of the signal . contrast - specific us techniques the different contrast - specific us techniques [ 6 ] may be distinguished by their basic principles into pseudo - doppler , harmonic , phase - modulation , amplitude - modulation , and phaseand amplitude - modulation techniques . 
pseudo - doppler techniques rely on the sae effect , which corresponds to the emission of a wideband signal when the microbubbles are destroyed by a high - transmit - power insonation . 
 stimulated acoustic emission ( sae ) in this mode , the microbubbles appear as a random colour mosaic extending over a limited depth range close to the focus of the transmitted us [ 7 ]  . 
un indice maggiormente ripoducibile invece rappresentato dalla potenza acustica di insonazione espressa in mpa ( mega pascal ) che viene riportata sul monitor operativo di alcuni ecografi . esistono due diverse tecniche per insonare le microbolle [ 5 ]  . 
la prima tecnica rappresentata dalla insonazione ad alta potenza acustica ( circa 1 mpa ) e determina la distruzione delle microbolle con la conseguente emissione di un segnale irregolare ad ampia banda , simile ad uno scoppio , e definito emissione acustica stimolata ( stimulated acoustic emission , sae )  . 
la soglia per raggiungere la distruzione delle microbolle variabile e dipende da numerosi fattori quali le dimensioni , la composizione chimica della capsula ed il gas presente allinterno delle microbolle , nonch dal grado di attenuazione della potenza del fascio ultrasonoro prodotto dai tessuti soprastanti . 
queste tecniche vengono generalmente impiegate per insonare le microbolle a base di aria con una capsula poco resistente e che presentano uno scarso comportamento armonico a bassa potenza di insonazione e sono limitate dalla scarsa persistenza del segnale che perdura al massimo 23 frames . 
questo limite pu essere in parte risolto impiegando una insonazione di tipo intermittente con basso frame rate ( una immagine prodotta ogni 23 secondi ) in modo da limitare la distruzione delle microbolle . la seconda tecnica rappresentata dalla insonazione delle microbolle a bassa potenza acustica ( 3070 kpa )  . 
con questa tecnica si riduce la probabilit di rottura delle microbolle , e la produzione di frequenze armoniche da parte dei tessuti stazionari , mentre si sfrutta pienamente il comportamento fisico non lineare delle microbolle . 
la tecnica a bassa potenza di insonazione viene impiegata con microbolle a base di esafluoruro di zolfo oppure di perfluorocarburo , e consente la esecuzione di una scasione ecografica in tempo reale data la lunga persistenza del segnale . tecniche ecografiche contrasto - specifiche in base al loro principio di funzionamento le tecniche ecografiche contrasto - specifiche [ 6 ] si possono distingure in tecniche pseudo - doppler , armoniche , a modulazione di fase , a modulazione di ampiezza , ed in tecniche a modulazione di ampiezza e di fase . 
il raggio delle microbolle presenta un grado di compressione durante la fase positiva dellimpulso ultrasonoro uguale al grado di espansione durante la fase negativa . le microbolle dimostrano invece un comportamento fisico non lineare se la potenza di insonazione viene progressivamente aumentata mantenendo la frequenza di risonanza ( b )  . 
questo comportamento non lineare determina la produzione di frequenze armoniche per gentile concessione di springer science and business media , da [ 2 ]  . acoustic power radius acoustic power radius struction and to the following absence of microbubbles after destruction . 
this phenomenon of loss of correlation is interpreted as a movement by the us systethe effect quickly disappears over a few frames due to the destruction of the microbubbles . cadence agent detection imaging ( adi ) this technique involves the transmission of two identical high - amplitude pulses and the temporary storage of the two resulting echo sequences before the second is subtracted from the first . 
queste tecniche sono limitate dalla forte presenza di artefatti e dalla scarsa persistenza del segnale . emissione acustica stimolata ( stimulated acoustic emission , sae ) in questa tecnica il segnale prodotto dalla insonazione delle pseudo - doppler harmonic phase modulation amplitude modulation gray - scale harmonic pulse inversion power modulation phase and amplitude modulation cadence contrast pulse sequencing e . 
quaia : contrast - specific ultrasound techniques table 2 contrast - specific ultrasound techniques stimulated acoustic emission cadence agent detection imaging advanced dynamic flow tissue signature imaging tabella 2 tecniche contrasto - specifiche pseudo - doppler imaging armonico modulazione di fase modulazione di ampiezza gray - scale harmonic pulse inversion power modulation modulazione di fase e di ampiezza cadence contrast pulse sequencing stimulated acoustic emission cadence agent detection imaging advanced dynamic flow tissue signature imaging coherent contrast imaging power pulse inversion contrast tissue discriminator vascular recognition imaging coded harmonic angio coherent contrast imaging power pulse inversion contrast tissue discriminator vascular recognition imaging coded harmonic angio flash echo imaging harmonic power doppler imaging contrast tuned imaging c cube extended pure harmonic detection ultraharmonic imaging subharmonic imaging 1.5 harmonic imaging flash echo imaging harmonic power doppler imaging contrast tuned imaging c cube extended pure harmonic detection ultraharmonic imaging subharmonic imaging 1.5 harmonic imaging bles produce strong echoes from the first pulse , but many are destroyed or substantially damaged in the process , so the echoes produced by the second pulse will be different both in intensity and morphology . 
the user can choose to display an image made up of either the subtracted echoes ( microbubble signal only ) , the echoes from the second transmission ( tissue only ) or a combination of the two , with the contrast image presented as a coloured overlay on the tissue b - mode image . microbolle appare come un mosaico di colori che si estende ad una profondit limitata e che maggiormente evidente in prossimit del punto di massima focalizzazione [ 7 ]  . 
il suo principio si basa infatti sulla iniziale identificazione delle microbolle da parte del primo impulso , e sulla loro immediata scomparsa determinata dalla loro distruzione con conseguente mancata identificazione delle microbolle da parte degli impulsi successivi ad elevata potenza . 
il fenomeno di perdita di correlazione viene interpretato come movimento da parte del sistema ecografico e advanced dynamic flow this is a wideband frequency technique that employs a lower pulse amplitude and fewer pulse transmissions per line ( from two to four ) in comparison with the pseudo - doppler techniques . 
this reduces the rate of bubble destruction , allowing real - time imaging , albeit at a lower frame rate ( four to five frames / s ) in comparison with conventional us imaging . 
this doppler threshold value is easily exceeded if microbubble contrast agents are employed resulting in the presence of artefacts such as blooming ( presence of doppler signal not determined by the mean frequency shift ) and clutter ( low frequency doppler signal produced by stationary tissues overlapping the real doppler signal )  . to limit blooming and clutter , the b - mode and doppler power for a pixel are compared , and the larger one is used to set the pixel grey level [ 6 ]  . 
the tissue and contrast information can be displayed separately ( a , b ) or simultaneously ( c ) by overlapping the colour map and the tissue image in b - mode . 
il contributo dei tessuti stazionari e del mezzo di contrasto possono essere visualizzati separatamente ( a , b ) , oppure simultaneamente mediante la sovrapposizione della mappa colore allimmagine dei tessuti in b - mode ( c )  . 
leffetto scompare rapidamente dopo alcuni frames a causa della distruzione massiva delle microbolle . cadence agent detection imaging ( adi ) questa tecnica si basa sulla trasmissione di due impulsi di uguale ampiezza , sulla registrazione temporanea dei due echi prodotti e sulla successiva sottrazione del secondo dal primo eco . 
la distruzione della microbolle produce invece due echi diversi dato che il primo impulso determina una significativa distruzione delle microbolle e , conseguentemente , il secondo impulso riscontra un numero minore di microbolle e produce un segnale di diversa intensit e morfologia . 
ci determina la formazione di un segnale complesso quando i due echi vengono sottratti che viene usato per rappresentare la distribuzione delle microbolle in ambito parenchimale per mezzo di una mappa colore . 
loperatore pu scegliere di visualizzare limmagine ottenuta solo con la sottrazione dei due echi ( esclusivo segnale delle microbolle ) , con i soli echi derivanti dal secondo impulso trasmesso ( esclusivo segnale dei tessuti ) , oppure una combinazione dei due con limmagine prodotta dalle microbolle rappresentata come mappa colore sovrapposta alla immagine dei tessuti in b - mode . advanced dynamic flow una tecnica che impiega una larga banda di frequenze in trasmissione , ed una minore potenza di insonazione ed un minor numero di impulsi ( da 2 a 4 ) per ogni linea di vista rispetto alle tecniche pseudo - doppler precedenti . 
quaia : contrast - specific ultrasound techniques a pixel will not display a power doppler signal if the b - mode signal for that pixel is large , whereas the pixels with a strong clutter will merge relatively inconspicuously with those showing strong b - mode stationary tissue signals . tissue signature imaging the tissue signature imaging mode is based on the same basic principles as the advanced dynamic flow , even though it represents the power doppler signals as grey - scale levels . this technique represents both harmonic signals produced by microbubble destruction and the nonlinear fundamental signals from stationary tissues in a gray - scale map . 
as the second harmonic ( double of the insonating frequency ) has the highest amplitude among the harmonics , it is the most relevant harmonic frequency for contrast - specific us imaging , even though the signal may be persistent or transient depending on acoustic power employed . 
le frequenze doppie rispetto alla frequenza fondamentale vengono selettivamente registrate per mezzo di un filtro passa - alto che elimina le frequenze fondamentali ( rettangolo )  . do viene ridotta la percentuale di distruzione delle microbolle permettendo quindi un imaging in tempo reale anche se con un numero di frames ridotto ( 45 frames / secondo ) rispetto allimaging ecografico convenzionale . 
questo valore soglia viene facilmente superato dopo la somministrazione delle microbolle e produce la presenza di artefatti quali il blooming ( presenza di segnale doppler non reale ) ed il clutter ( segnale doppler a bassa frequenza prodotto dai tessuti stazionari che si sovrappone al segnale doppler reale )  . 
per limitare il blooming ed il clutter la intensit del segnale b - mode e doppler vengono paragonati in ogni pixel ed il segnale a maggiore intensit viene impiegato come riferimento per regolare il livello della scala dei grigi [ 6 ]  . 
questo sistema evita la presenza del blooming e del clutter dato che un pixel non potr per principio dimostrare un segnale power doppler se il segnale b - mode per quel pixel molto intenso , mentre i pixel con forte presenza di clutter saranno soppressi dai pixel viciniori con forte segnale b - mode proveniente dai tessuti stazionari . tissue signature imaging rappresenta una tecnica che utilizza il medesimo principio delladvanced dynamic flow rappresentando per il segnale power doppler come scala di grigi . 
la seconda armonica ( frequenza doppia rispetto alla frequenza fondamentale ) la frequenza armonica di maggior importanza per limaging contrasto - specifico dato che possiede la maggiore ampiezza tra le frequenze armoniche prodotte anche se il suo segnale pu essere persistente oppure transitorio in relazione alla potenza acustica impiegata . 
moreover , if the transmit pulse bandwidth ( centred at the fundamental frequency fo ) and the receive bandwidth ( centred at 2fo ) overlap , then a portion of the echo from linear scattering will contaminate the harmonic receive signal , reducing the difference between microbubble agent and stationary tissue and , consequently , the contrast resolution of the technique . 
 flash echo imaging this technique employs intermittent high - transmit - power insonation to produce microbubble destruction , with a progressively increasing interscan delay leading to progressive microbubble accumulation in the scanning plane [ 8 ]  . 
sebbene questo sistema di filtraggio permette di eliminare gli echi che provengono dai tessuti stazionari che presentano prevalentemente un comportamento lineare , il sistema determina necessariamente una riduzione della ampiezza della banda di frequenze utilizzata determinando quindi una bassa risoluzione spaziale assiale . 
inoltre , ove la banda di frequenza del segnale trasmesso ( centrata sulla frequenza fondamentale f0 ) si sovrapponga a quella del segnale in ricezione ( centrata su 2f0 ) si riduce la differenza del segnale prodotto dalle microbolle e dai tessuti stazionari con una riduzione della risoluzione di contrasto della tecnica . harmonic power - doppler imaging flash echo imaging this technique is also known as harmonic power - doppler imaging by high - pass signal filtering . 
with conventional colour doppler , moving scatterers in a region of interest produce a low - frequency shift ( clutter signal ) , which has a doppler shift frequency nearly identical to that of the moving blood . 
 contrast - tuned imaging ( cnti ) this technique involves the transmission of the specific resonance frequency of sulphur - hexafluoride - filled microbubbles and the selective registration of the harmonic frequencies . 
this allows a significant reduction of the insonation power with reduction of the nonlinear harmonic behaviour of the stationary tissues and a selective production of harmonic frequencies from microbubbles . questa tecnica utilizza in modo intermittente una alta potenza di insonazione per distruggere le microbolle e , aumentando progressivamente il ritardo tra una insonazione e laltra , determina un progressivo accumulo delle microbolle nel piano di scansione [ 8 ]  . 
pi lungo il ritardo tra le singole scansioni e maggiore laccumulo delle microbolle pi elevata lintensit del segnale prodotto . power doppler armonico questa tecnica anche conosciuta come power doppler armonico con filtraggio passa - alto del segnale . 
lutilizzo di un filtro passa - alto consente di cancellare il segnale prodotto dai tessuti e di registrare selettivamente il segnale doppler prodotto dalle microbolle . c - cube contrast tuned imaging ( cnti ) this is a harmonic , intermittent , high - transmit - power technique that relies on the transmission of two identical us pulses down each ray line and on the comparison of returning echo signals . 
this enables the identification of nonlinear harmonic response from microbubbles from the different intensity on the two echoes due to microbubble destruction by the first us pulse [ 5 ]  . 
 extended pure harmonic detection ( ephd ) this technique consists of the transmission of a low - transmit - power us pulse characterised by a frequency equal to the resonance frequency of the microbubbles and absence of distortions or harmonic components . 
as a result , only the real harmonic signals generated by the microbubble resonance questa tecnica basata sulla trasmissione della specifica frequenza di risonanza che caratterizza le microbolle a base di esafluoruro di zolfo e sulla selettiva registrazione del segnale armonico prodotto . 
questo permette una notevole riduzione della potenza acustica di insonazione con una conseguente riduzione del comportamento armonico non lineare dei tessuti stazionari ed una selettiva produzione di frequenze armoniche da parte delle microbolle . c - cube una tecnica armonica intermittente ad alta potenza di insonazione che si basa sulla trasmissione di due impulsi identici lungo ogni linea di vista e sul confronto dei relativi echi . questo rende possibile lindividuazione della risposta non lie . 
the nonlinear nature of microbubble oscillation under the influence of sound pressure generates phase - shifted signals that identify the received harmonic signal as being originated by the contrast agent and not the surrounding tissue . 
with ultraharmonic imaging , a combination of highand low - pass filters can be used to selectively display only ultraharmonic frequencies where the signal to noise ratio ( snr ) is intrinsically greater . 
adenoma epatico ( freccia ) valutato mediante scansioni eseguite in fase arteriosa ( a ) , portale ( b ) e tardiva ( c ) dopo la somministrazione delle microbolle . 
la chiara cospicuit della lesione rispetto al background determinato dai tessuti stazionari evidente nella fase arteriosa ed determinata dalla notevole risoluzione di contrasto della tecnica . neare armonica delle microbolle che producono una differenza di intensit tra i due echi determinata dalla distruzione delle microbolle da parte del primo impulso [ 5 ]  . extended pure harmonic detection ( ephd ) questa tecnica consiste nella trasmissione di un impulso a bassa potenza acustica caratterizzato da una frequenza corrispondente alla frequenza fondamentale delle microbolle e che risulta inoltre privo di distorsioni e di qualsiasi componente armonica . 
la insonazione delle microbolle alla frequenza di risonanza determina la generazione di una variazione di fase nel segnale riflesso rispetto a quello trasmesso che caratterizza univocamente il segnale armonico generato dalle microbolle rispetto a quello generato dai tessuti stazionari . 
this , combined with the fact that nonlinear propagation of ultrasound in tissue does not generate a subharmonic component , suggests that subharmonic detection might offer an imaging method with greater contrast between microbubble and tissue echoes . 
this imaging technique reduces tissue echoes without generating motion artefacts , such as those seen in the pseudo - doppler imaging technique . contrast between tissues and bubbles is improved by 20 db compared with the second harmonic imaging technique . 
the newer modalities also share a common feature , namely , the transmission of multiple us pulses per scan line , followed by processing of the received radiofrequency signals from each line . 
le frequenze ultra - armoniche originano in minima parte dalla trasmissione non lineare degli ultrasuoni attraverso i tessuti stazionari , mentre esse vengono prodotte efficacemente dalle microbolle mediante insonazione a bassa oppure alta potenza . 
una combinazione di filtri passa - alto e passa - basso viene impiegata per rappresentare selettivamente le frequenze ultra - armoniche che producono un rapporto segnale - rumore pi elevato rispetto alle frequenze armoniche tradizionali . subharmonic imaging quando le microbolle vengono insonate vengono prodotti anche alcuni echi che presentano una frequenza che la met della frequenza di insonazione , unitamente ad echi con una frequenza pari alla frequenza di insonazione oppure alla seconda e terza armonica o anche a frequenze armoniche di ordine superiore . 
la prima certamente rappresentata dalla risposta non lineare delle microbolle ad un impulso di insonazione . la seconda causa invece rappresentata dalle oscillazioni continue delle microbolle dopo il termine della insonazione . lintensit della frequenza sub - armonica inoltre maggiore quando gli impulsi trasmessi contengono un elevato numero di cicli , e nelle microbolle che presentano una capsula meno rigida . 
questo elemento , unitamente al fatto che la propagazione non lineare degli ultrasuoni nei tessuti non genera delle frequenze subarmoniche , suggerisce che il rilevamento delle frequenza subarmoniche rappresenta una tecnica in grado potenzialmente di produrre una maggiore risoluzione di contrasto tra gli echi provenienti dalle microbolle e quelli provenienti dai tessuti . 
in particolare la banda di frequenza utilizzata presenta una frequenza centrale pari a 3 / 2 della frequenza fondamentale , e produce selettivamente echi dalle microbolle e non dai tessuti stazionari . 
per un mezzo non lineare come le microbolle , gli echi risultanti non sono copie invertite luno dellaltro e la loro somma produce un risultato diverso da zero ( c )  . better discrimination between tissue and microbubble contrast agents , and to overcome the requirement of the use of a narrow - band signal for harmonic imaging with the consequently reduced spatial axial resolution . 
in pulse inversion , two sequential us waves that are inverted replicas of each other are transmitted into tissue , and the resulting echoes are summed [ 6 ]  . 
if a high acoustic power of insonation is employed for imaging , however , tissue harmonics are not eliminated by pulse inversion and are incompletely cancelled , and tissue and microbubble second harmonics may still overlap . the advantage of the pulse inversion method over the harmonic techniques is that overlap between the fundamental and second harmonic spectra does not matter , and there is no need to restrict the transmission spectrum to the lower half of the transducer frequency range . 
the tissue - contrast discrimination that can be achieved by pulse inversion is limited by any tissue movement between the two transmissions . where there is tissue movement , the positions of the tissue interfaces in the second echo sequence will not match those fig . 
le tecniche pi recenti sono in particolare caratterizzate dalla trasmissione di multipli impulsi per ogni linea di vista , e dalla successiva elaborazione dei segnali ricevuti secondo ogni linea di vista . 
lo scopo delle tecniche a modulazione di fase di differenziare il segnale lineare prodotto dai tessuti stazionari da quello non lineare prodotto dalle microbolle con il fine di aumentare la risoluzione di contrasto ed il rapporto segnale / rumore . 
queste tecniche impiegano una bassa potenza acustica di insonazione e consentono una lunga persistenza del segnale prodotto dalle microbolle con la possibilit di eseguire le scansione in modo continuo ed in tempo reale . pulse inversion harmonic imaging ( pulse inversion a due impulsi ) questa tecnica stata introdotta per superare due importanti limiti dellimaging armonico : fornire una migliore discriminazione tra i tessuti stazionari e le microbolle , e superare il vincolo imposto dalluso di una stretta banda di frequenze con conseguente riduzione della risoluzione spaziale assiale . nella tecnica pulse inversion due impulsi ultrasonori sequenziali , di uguale ampiezza , ma di fase opposta , vengono trasmessi allinterno del tessuto e gli echi relativi vengono sommati [ 6 ]  . 
ove venga utilizzata una elevata potenza di insonazione le armoniche tissutali non vengono eliminate dato che anche i tessuti stazionari posseggono un comportamento non lineare se insonati ad alta potenza causando una sovrapposizione tra le frequenze armoniche provenienti dai tessuti e le frequenza provenienti dalle microbolle . il vantaggio della tecnica pulse inversion rispetto alla tecniche di imaging armonico costituito dal fatto che la sovrapposizione tra la frequenza fondamentale e la seconda armonica non costituisce un problema , e non quindi necessario ridurre la banda di frequenze in trasmissione . 
leffetto viene limitato se i tessuti stazionari si muovono nello spazio temporale compreso tra i due impulsi in opposizione di fase in quanto ci determina una variazione delle interfacce tissutali che producono il primo ed il secondo eco e quindi una imperfetta eliminazione del segnale lineare . microflow imaging in the first , and cancellation of the linear signal will not be perfect . microflow imaging this technique is based on the summation of multiple consecutive frames , and it represents a multi - intensity projection algorithm of pulse inversion data . 
 contrast tissue discriminator contrast tissue discriminator is a low - transmit - power multipulse mode based , like the phase inversion mode , on the pulse subtraction method using alternating phases . 
the reduction in frame rate that represents a limitation of pulse inversion can be avoided by transmitting only one pulse per scan line but by inverting the transmitted pulse on alternate scan lines [ 6 ]  . 
the sum of the two echo sequences represents an average of the two contributing lines and is therefore displayed as a synthetic line situated midway between thesumming the echo sequences from adjacent lines therefore cancels out echoes from linear scatterers , as in the two - pulse method described in the previous section . 
the avoidance of a frame - rate penalty comes at the expense of a slightly imperfect cancellation of the fundamental frequencies , as not all points in a pair of overlapping beams experience matching but inverted versions of the same pulse wavefor power pulse inversion ( pulse inversion with three transmissions ) power pulse inversion is a technique in which a series of us pulses ( 3 ) are transmitted at a high pulse repetition interval along each line [ 11 ]  . 
three interrogations of a line with alternately inverted pulses can greatly reduce the effect of tissue motion , which limits the usual pulse inversion mode . each transmitted pulse is an inverted copy of the previous pulse . 
the processing amounts to forming an average of the first and third echo sequences ( by adding them together and dividing by two ) and adding this to the second echo sequence . 
if the tissue velocity is not too great , the difference in phase between echoes from the first and third pulses ( less than a millisecond apart ) will be small , say less than 30 or so , and the average echo sequence will then be a close approximation to one with half the phase shift , corresponding to what would have been returned if an imaginary pulse had been transmitted midway in time between these two real pulses . 
as for the two - pulse method , adding the average echo sequence from the two noninverted pulses to the real sequence from the second ( inverted ) pulse will give cancellation of linear tissue echoes but not of microbubble echoes . the use of multiple pulses reduces motion artefacts while preserving true contrast signal ( noise cancels but real signals add )  . 
 vascular recognition imaging a generalisation of the pulse inversion method , combines the nonlinear detection performance of pulse inversion imaging with the motion discrimination capabilities of colour and power doppler . 
inoltre , al fine di dare continuit alla linea del vaso viene aumentata la persistenza del segnale sullo schermo . contrast tissue discriminator questa tecnica contrasto - specifica utilizza una bassa potenza di insonazione e si basa sul principio della tecnica pulse inversion consistente nella sottrazione degli echi utilizzando la modulazione della fase . 
gli echi che provengono dai tessuti stazionari vengono cancellati , mentre gli echi che derivano dalle microbolle vengono selettivamente rappresentati . coherent contrast imaging una tecnica a modulazione di fase ad alto frame rate . 
la riduzione del frame rate che rappresenta uno dei limiti della pulse inversion pu essere superata trasmettendo un singolo impulso per linea ma invertendo la fase degli impulsi a linee di vista alterne . 
viene calcolata la media del contributo di ciascuna linea di vista ed il risultato produce una ulteriore linea di vista in posizione intermedia tra le due linee di vista precedenti . 
la sommazione della sequenza degli echi derivanti da linee di vista adiacenti cancella le interfacce a comportamento lineare come avviene nelle tecniche a due impulsi in opposizione di fase in successione . 
tutto ci avviene a spese di una imperfetta cancellazione delle frequenze fondamentali , dato che due punti adiacenti sono sottoposti a due versioni coerenti ma invertite della stesso impulso ultrasonoro . power pulse inversion ( pulse inversion a tre impulsi ) la power pulse inversion una tecnica in cui una serie di impulsi ultrasonori ( 3 ) di fase invertita luno rispetto allaltro vengono trasmessi con unalta frequenza di ripetizione e ad intervalli regolari lungo ogni linea di vista [ 11 ]  . 
inviando lungo ogni linea di vista tre impulsi successivi e di fase invertita si ottiene una riduzione delleffetto derivante dal movimento dei tessuti stazionari tra 2 impulsi successivi che rappresenta uno dei limiti principali della tecnica pulse inversion . 
se la velocit del movimento tissutale non troppo elevata la differenza di fase tra gli echi che derivano dal primo e del terzo impulso ( inferiore a un ms ) sar ridotta ( < 30 ) , e la media tra il primo ed il terzo eco rappresenter quindi una approssimazione molto vicina a met della variazione di fase corrispondendo alla riflessione che si sarebbe ottenuta se un ipotetico impulso fosse stato trasmesso al centro della finestra temporale compresa e . 
come per la tecnica pulse inversion a due impulsi , aggiungendo la media degli echi ottenuta dal primo e dal terzo impulso al secondo impulso , che presenta fase opposta rispetto al primo ed al terzo , si ottiene la cancellazione degli echi prodotti dai tessuti stazionari ma non degli echi prodotti dalla insonazione delle microbolle . lutilizzo di multipli impulsi riduce quindi gli artefatti da movimento ed allo stesso tempo mantiene il contrasto tra i tessuti stazionari e le microbolle . 
lo svantaggio di questa tecnica laumento della percentuale di distruzione delle microbolle a causa dei multipli impulsi di insonazione . vascular recognition imaging questa tecnica , unisce la capacit di registrare gli echi prodotti dal comportamento non lineare delle microbolle con la capacit di discriminare la direzione del movimento tipico del color e power doppler . 
gli echi derivanti dai primi tre impulsi vengono sommati in modo ponderato ( 1 , 2 , 1 ) per produrre un segnale privo della frequenza fondamentale , e similmente gli echi che provengono dagli ultimi tre impulsi vengono sommati in modo ponderato per produrre un secondo segnale privo della frequenza fondamentale . 
la differenza di fase tra i due segnali armonici principali cos calcolati fornisce una stima della velocit delle microbolle , mentre la radice quadrata della loro ampiezza fornisce una stima della quantit di microbolle presenti . 
il fatto che vengano utilizzati un minor numero di impulsi rispetto alle tecniche color e power doppler tradizionali determina la presenza di una accuratezza minore nella misurazione del segnale doppler . coded harmonic angio questa tecnica combina la tecnica pulse inversion con la tecnica di trasmissione degli impulsi a codici con il fine di aumentare la sensibilit per il segnale prodotto dalle microbolle e sopprimere il segnale proveniente dai tessuti stazionari . essa consiste nella trasmissione di una serie di impulsi codificati e nella loro decodificazione in fase di ricezione . 
un codice consiste in una serie di variazioni predefinite della frequenza e della fase allinterno di un impulso ultrasonoro . questo codice presente negli echi utili per la formazione dellimmagine ma non nel rumore , e quindi questa tecnica determina anche un miglioramento del rapporto segnale / rumore . 
the microbubbles directed towards the transducer are represented in red ( arrow ) , those flowing away from the transducer are visualised in blue , whereas the stationary microbubbles in the liver sinusoids are visualised in green . 
le microbolle con un flusso diretto vero il trasduttore vengono rappresentate in rosso ( freccia ) , in blu se presentano un flusso in allontanamento , ed in verde se sono stazionarie a livello dei sinusoidi epatici . volves transmitting four pulses , alternately inverted , along each scan line . 
the echoes from the first three are summed with weights ( 1 , 2 , 1 ) to give one fundamental - free signal , and the echoes from the last three are summed the same way to give a second fundamental - free signal . 
a disadvantage of using only a few transmission / receive sequences for each scan line is that the accuracy of doppler frequency measurement is much less than in conventional colour flow imaging . 
 coded harmonic angio coded harmonic angio sonography combines phase - inversion harmonic imaging with a coded transmission pulse technique that boosts weak microbubble signal and suppresses tissue signals by transmitting coded pulse sequences and decoding them on receipt . 
this code is present in genuine echoes but not in noise , so by using a special matched filter , designed to respond strongly to the transmitted code , snr can be also improved by this contrast - specific technique . 
if second harmonic components are present in an echo , this quarter cycle shift at the fundamental frequency will become a half a cycle shift at the second harmonic frequency . 
thus , the code of the second harmonic component of an echo will be modified from that transmitted , whereas the fundamental component will retain the code of the transmitted pulse . 
the pulses are transmitted at two power levels , where the first and third pulses are half - height amplitude relative to the second pulse . because all pulses are transmitted at a low acoustic power , little nonlinear tissue backscatter is generated . 
subsequently , the received echoes from the half - height transmitted pulses are scaled and subtracted from the full - height signal , which results in effective removal of tissue clutter . 
amplitude and phase modulation technique cadence contrast pulse sequencing ( cps ) the contrast pulse sequencing technique works by interrogating each scan line a number of times with pulses having various amplitudes and phases . 
a processor first amplifies the echo sequence from each transmission by a particular weighting factor ( for example 2 or 1 ) and then sums all the weighted echo sequences . 
by suitable choice of transmission pulses and weighting factors , it is possible to isolate or suppress the linear fundamental echoes from stationary tissues . the amplitude and phase modulation contrast - specific techniques produce high sensitivity to the microbubble nonlinear harmonic signals and optimal suppression of the linear quando un eco codificato corrisponde perfettamente con quello di riferimento i segnali elettrici del circuito in uscita sono alti . 
una discordanza nella posizione di una sola cifra produce invece un segnale elettrico di bassa intensit . possibile sopprimere selettivamente tutti gli echi che presentano un codice uguale al codice di trasmissione , e quindi il segnale delle frequenze fondamentali prodotte dai tessuti stazionari . 
in uneco che presenti la frequenza seconda armonica , lo spostamento di fase pari ad un quarto di ciclo che si riscontra nella frequenza fondamentale corrisponde ad uno spostamento di fase pari a met ciclo per la frequenza seconda armonica . 
tecnica a modulazione di ampiezza power modulation a basse energie acustiche di insonazione il segnale tissutale pu anche essere soppresso alternando la potenza acustica di insonazione piuttosto che la fase . 
conseguentemente gli echi caratterizzati da unintensit pari alla met di quella degli impulsi trasmessi vengono sottratti dagli echi di ampiezza regolare con il risultato di rimuovere il contributo dei tessuti stazionari . 
tecnica a modulazione di fase e di ampiezza cadence contrast pulse sequencing ( cps ) la tecnica contrast pulse sequencing funziona trasmettendo multipli impulsi dotati di ampiezza e fase variabile lungo ogni linea di vista . 
10 a - d principle of cadence contrast pulse sequencing . three pulses are transmitted along each line , and the first and the third are half the amplitude of the second pulse , which presents an opposed phase with respect to the others ( a ) , and the returning echoes are summed . 
tre impulsi in successione vengono inviati ogni linea di vista , il primo e lultimo impulso possiedono la met dellampiezza del secondo impulso che presenta fase invertita rispetto agli altri ( a ) , e gli echi rispettivi vengono sommati . 
per un mezzo lineare la somma risultante zero ( b )  . per un mezzo non lineare come le microbolle la somma produce un risultato diverso da zero sia per le frequenza non lineari fondamentali ( c ) che per le frequenze non lineari armoniche ( d )  . non linear foundamental non linear harmonic fig . 
angioma epatico ( freccia ) valutato mediante scansioni eseguite in fase arteriosa ( a ) , portale ( b ) e tardiva ( c ) dopo la somministrazione delle microbolle . 
pinto 1 , i - 71100 foggia , italy 2unit sanitaria locale ba 4 , ospedale di venere , via ospedale di venere 1 , i - 70012 carbonara , bari , italy 3universit degli studi di bari , istituto di chirurgia maxillo - facciale , piazza g . 
chieffi 40 , i - 70051 barletta , italy , tel . : + 39 - 339 - 2452701 , e - mail : paomi03@libero.it received : 13 august 2006 / accepted : 26 september 2006 / published online : 11 june 2007 abstract purpose . 
this study was done to evaluate the use of multidetector computed tomography ( mdct ) in the evaluation of jaw osteonecrosis , a recently described medical entity in patients receiving long - term intravenous or oral bisphosphonates , and to help radiologists recognise it to enable early diagnosis and appropriate management . 
jaw osteonecrosis is an established medical entity discovered in patients who have undergone treatment with bisphosphonates and dental extraction or other oral surgery . mdct with multiplanar ( mpr ) , volume rendering ( vr ) and three - dimensional ( 3d ) reconstructions allows accurate assessment of affected bone structures , enabling early diagnosis and suitable treatment planning . 
radiologists should be aware of the risk of osteonecrosis in patients treated with bisphosphonates and be able to distinguish it from other bone diseases ( osteomyelitis and osteoradionecrosis ) with which it enters the differential diagnosis . key words jaw osteonecrosis bisphosphonates oral surgery mdct riassunto obiettivo . 
la tc - md con ricostruzioni multiplanari ( mpr ) , volume rendering ( vr ) e 3d consente una valutazione accurata dellosteonecrosi mascellare associata a terapia con bifosfonati fornendo informazioni utili sulle strutture ossee coinvolte per un adeguato planning terapeutico . 
i medici radiologi devono essere a conoscenza del rischio di osteonecrosi al quale vanno incontro i pazienti trattati con bifosfonati ed essere in grado di distinguerla da altri processi patologici dellosso ( osteomielite e osteoradionecrosi ) con le quale entra in diagnosi differenziale . parole chiave mascella osteonecrosi bisfosfonati chirurgia orale tc - md p . 
although the mechanism of action of bisphosphonates essentially involves a powerful inhibition of bone resorption , the manner in which they block the destruction of hydroxyapatite and interfere with osteoclast function is still unclear [ 4 ]  . 
in addition to this , they have an antiangiogenic action that inhibits proliferation of endothelial cells . at the molecular level , bisphosphonates have been shown to influence osteoclast activity by modulating a cell - surface receptor or an intracellular enzyme [ 5 , 6 ]  . a precise causal relationship between bisphosphonate therapy and jaw osteonecrosis has yet to be established . nonetheless , risk factors have been identified and guidelines published for the prevention , early diagnosis , and management of this side effect , and there are multidisciplinary recommendations for reducing the incidence of jaw osteonecrosis in patients undergoing treatment with bisphosphonates and requiring oral surgery [ 7 ]  . 
dental extraction is often the triggering event , exposing bone to changes and alterations , even though the appearance of spontaneous gingival lesions , toothache , loosening of teeth and numbness is not uncommon . 
 the aim of this study was to use multidetector computed tomography ( mdct ) to evaluate jaw - bone changes caused by bisphosphonate - related osteonecrosis to provide a radiological description of this disease entity and help radiologists differentiate it from osteomyelitis and osteoradionecrosis and enable , with the aid of an accurate history and clinical assessment , its early diagnosis and appropriate treatment . 
 materials and methods thirty - eight patients presenting with pain in the mouth and temporomandibular joints , soft tissue infection , altered taste , numbness , and lip paraesthesia were examined by mdct . all patients had a history of neoplasm or osteoporosis , bisphosphonate use and recent oral surgery . 
ten of the patients had breast cancer , 12 had multiple myeloma , five had prostate cancer , nine had osteoporosis , one had breast cancer and osteoporosis and one had multiple myeloma and pancreatic cancer . all patients with neoplastic disease at the time of the mdct scan had received 4 mg monthly of intravenous zoledronate for periods ranging from 12 to 35 months . 
the patient with breast cancer and osi bifosfonati svolgono una azione inibitrice sullattivit degli osteoclasti e sono attualmente utilizzati per la cura dellosteoporosi , dellipercalcemia associata a patologie maligne e delle lesioni metastatiche osteolitche secondarie a tumore mammario , mieloma multiplo , malattia di paget , tumore prostatico [ 13 ]  . 
sebbene il meccanismo dazione di questi farmaci sia principalmente basato sul potente effetto inibitore che esercitano sul riassorbimento osseo , resta poco chiaro come questi blocchino la distruzione dellidrossiapatite e interferiscano sulla funzione degli osteoclasti [ 4 ]  . 
a livello molecolare , stato dimostrato , che i bifosfonati influenzano lattivit degli osteoclasti attraverso la modulazione di un recettore di superficie cellulare o un enzima intracellulare [ 5 , 6 ]  . una precisa relazione causale fra la terapia con bifosfonati e losteonecrosi della mascella non stata ancora stabilita . 
esiste per una identificazione dei fattori di rischio e lo sviluppo di linee guida per la prevenzione , la diagnosi precoce , il management terapeutico ed esistono raccomandazioni multidisciplinari per ridurre lincidenza delle osteonecrosi dellosso mascellare nei pazienti sottoposti a terapia con bifosfonati e che devono far ricorso a chirurgia orale [ 7 ]  . 
una estrazione dentaria spesso il primum movens dellesposizione ossea alle modificazioni ed alterazioni ossee , anche se non insolita la comparsa di lesioni gengivali spontanee , mal di denti , mobilit degli stessi e perdita della sensibilit . 
fattori sfavorevoli sono la presenza di comorbilit dentarie , che includono carie , ascessi , periodontiti ecc , concomitanti terapie steroidee , chemio e radio - terapia [ 8 ]  . scopo del nostro lavoro stato quello di valutare con tcmd le alterazioni dellosso mascellare riferibili ad osteonecrosi da bifosfonati per una maggiore riconoscibilit radiologica di tale tipo di osteonecrosi , anche con lausilio di una corretta anamnesi e una opportuna valutazione clinica , per una diagnosi differenziale con losteomielite e le osteoradionecrosi , una diagnosi precoce di osteonecrosi ed un adeguato planning terapeutico . materiali e metodi sono stati esaminati con tc - md 38 pazienti , giunti alla nostra osservazione con algie a livello del cavo orale e delle articolazioni temporo - mandibolari , infezioni dei tessuti molli , alterazioni del gusto e della sensibilit , parestesia delle labbra . 
six of the 38 patients ( three with breast cancer , two with prostate cancer and one with multiple myeloma ) had also received radiotherapy . all patients had undergone oral radiography and orthopantomography prior to ct imaging ; some had also had a biopsy , whereas others were to undergo biopsy within days of the ct study . 
in most patients ( n = 25 ) , small areas of increased bone density , as in new bone apposition , could be seen peripherally to the lesions , forming intraoral spicules . 
i pazienti con osteoporosi erano stati sottoposti a terapia con alendronato ( 70 mg / settimana ) o alendronato e clodronato ( 10070 mg / settimana ) , somministrati per via orale . 
levento scatenante losteonecrosi era stato per tutti i pazienti unestrazione dentaria tranne che per la paziente affetta da carcinoma mammario e da osteoporosi che aveva subito invece un trattamento endodontico . 
sei dei 38 pazienti ( 3 con carcinoma mammario , 2 con carcinoma prostatico e 1 con mieloma multiplo ) erano stati sottoposti anche a trattamento radioterapico . tutti i pazienti , prima dellesame tc , avevano gi effettuato un esame radiografico endorale ed ortopantomografico ; ad alcuni di essi era gi stato effettuato anche un prelievo bioptico ; altri sarebbero stati sottoposti a biopsia nei giorni successivi lesame tc . 
perifericamente alle lesioni erano presenti , nella maggior parte dei pazienti ( 25 ) , piccole aree di addensamento osseo , come da neoapposizione , che formavano delle vere e proprie spicule endo - orali . 
lindagine tc ha mostrato una grossolana alterazione osteostrutturale di tipo necrotico del processo alveolare sinistro della mascella superiore con interessamento del processo nasale e del processo zigomatico omolaterali e del pavimento del seno mascellare corrispondente , ben visibili nelle immagini tc assiali ( a , b ) , coronali ( c ) e nelle ricostruzioni in vr ( d , e )  . schwartz in 1982 [ 9 ]  . 
other authors described patients with metastasis and jaw osteonecrosis after chemotherapy [ 2 ]  . the toxic effects of chemotherapy for head and neck cancers , especially on vascular and bony structures , have been il follow - up dopo terapia ha mostrato risultati incoraggianti che per andrebbero validati con controlli a pi ampia distanza di tempo . 
a nelle immagini tc sono ben visibili lalterazione morfostrutturale a prevalente carattere litico destruente della regione molare della mandibola di sinistra ( immagini assiale , a e b ; coronale , c ; vr , d ) , associata a moderata reazione periostale laminata del margine interno ( freccia in b ) ed esterno ( freccia in a ) dellosso mandibolare , e la disomogeneit e lispessimento delle parti molli superficiali , associate a tumefazione e perdita del profilo interno della regione tonsillare ( testa di freccia in b )  . discussed for more than 20 years ( maduro et al . , 2003 [ 10 ] )  . avascular jaw necrosis was thought to be a chemically induced disease [ 11 ] and was grouped among the jaw alterations known as phosphorus necrosis [ 12 ]  . 
 in the united states , recent reports of a characteristic jawbone alteration , which never occurs after a single treatment with pamidronate or zoledronate [ 13 ] , have attributed the origin of jaw osteonecrosis to the widespread use of bisphosphonates , which are recommended by the american society of clinical oncology ( asco ) for the treatment of bone metastasis to breast cancer [ 14 , 15 ]  . 
sugli effetti tossici , in particolar modo di quelli vascolari ed ossei , della chemioterapia nel trattamento dei tumori della testa e del collo si discute da oltre 20 anni ( maduro et al . , 2003 [ 10 ] )  . 
multidetector computed tomography ( mdct ) : an extensive morphostructural lytic - destructive alteration involves the mandibular body , well visible in axial ( a ) and coronal ( c ) projections , the palatine and alveolar maxillary processes in the right median - paramedian area ( b , c ) , the petrous apex and the basilar part of the occipital bone , and the clivus with extension to the posterior clinoid processes ( d )  . 
tc : vasta alterazione morfostrutturale a carattere litico destruente in corrispondenza del corpo della mandibola , ben visibile in proiezione assiale ( a ) , coronale ( c ) , dei processi palatino ed alveolare del mascellare in sede mediana - paramediana destra ( b , c ) , dellapice della rocca petrosa sinistra nonch della pars basilare dellosso occipitale e del clivus con interessamento dei processi clinodei posteriori ( d )  . 
e ricostruzione in volume rendering ( vr ) con software dedicato per lo studio dellosso : bene identifica e delimita alterazioni ossee dellosso mascellare superiore ed inferiore . antibiotici a lungo termine e la lunga durata della terapia agevolava ovviamente la estensione della patologia stessa . negli stati uniti , recenti descrizioni di un caratteristica alterazione dellosso mascellare , che non si verifica mai in seguito ad un solo trattamento con pamidronato o zoledronato [ 13 ] , hanno attribuito al largo uso di bifosfonati , raccomandati dallamerican society of clinical oncology ( asco ) per il trattamento di metastasi ossee secondarie a carcinoma mammario [ 1415 ] la causa delle lesioni osteonecrotiche mascellari . 
the changes in bone metabolism associated with surgical treatment and implant trauma seem to be key factors in the development of osteonecrosis . a tooth extraction is the most common trigger [ 11 , 13 , 15 , p . 
multidetector computed tomography ( ct ) examination shows the presence of a morphostructural alteration of the upper maxillary left alveolar process that is heterogeneously dense due to the presence of multiple lytic areas ( a )  . 
lesame tc dimostra la presenza di una alterazione morfostrutturale del processo alveolare sinistro dellosso mascellare superiore che presenta densitometria disomogenea per la presenza di multiple areole litiche ( a )  . 
si associano irregolare ispessimento dei tessuti gengivali corrispondenti e la presenza di sottile tramite fistoloso che mette in comunicazione la cavit orale ed il seno mascellare omolaterale ( b ) , meglio evidente nella immagine vr ( c ) , completamente occupato da essudato con ispessimento della mucosa di rivestimento . sunzione di questi farmaci e linsorgenza di osteonecrosi mascellare . 
le modificazioni del metabolismo osseo associate ad un insulto chirurgico o ad un trauma protesico sembrano essere i fattori chiave nello sviluppo delle osteonecrosi . unestrazione dentaria la pi comune causa scatenate [ 11 , 13 , 15 , 16 ] anche se esistono casi in cui si verificano lesioni spontanee ed in zone non precedentemente interessate da avulsioni dentarie . 
la localizzazione delle lesioni dai dati della letteratura risulta essere come segue : nel 38%80 , 5% nella mascella superiore , nel 14%63% nella mascella inferiore e nel 5 , 5%23% in entrambe le mascelle [ 2 , 13 , 17 ] e concorda con i risultati del nostro studio . la diagnosi di osteonecrosi da bifosfonati dovrebbe essere basata principalmente su evidenze cliniche e reperti radiografici , oltre che su una accurata anamnesi . 
culture microbiche ( aerobico ed anaerobico ) consentono la identificazione dei patogeni responsabili della secondaria infezione [ 5 ]  . le indagini radiologiche rivestono un ruolo importante nella definizione del planning terapeutico . 
la tc - md risulta per essere tecnica fondamentale per il management terapeutico del pa16 ] , even though there have been cases of lesions arising spontaneously in areas not subjected to dental extractions . the most commonly reported sites of jaw osteonecrosis are the upper jaw in 38%80.5% of cases , the lower jaw in 14%63% and both jaws in 5.5%23% [ 2 , 13 , 17 ] ; this is in agreement with the findings of our study . the diagnosis of bisphosphonate - related jaw osteonecrosis primarily relies on clinical and radiographic evidence and an accurate history . 
the intrinsic features of cr high spatial and contrast resolution and its being ideally suited for the study of bone , associated with continuous technological evolution and the possibility of multiplanar reconstructions ( mprs ) , vr and 3d imaging ensured , in our experience , a correct diagnosis and accurate staging of the osteonecrotic lesions as well as the detection of complications . currently , no established treatment exists for this type of drug - induced lesion . 
although it may seem reasonable to think that withdrawal of intravenous bisphosphonates might improve the rate of regression of the disease , this has not yet been proposed as a form of management [ 18 ] , because there is no evidence of a clinical improvement of osteonecrosis [ 17 ]  . 
this is accounted for by the fact that bisphosphonates are avidly bound to the bone matrix around the active osteoclasts and , not being metabolised , they remain there in high concentrations over long periods of time [ 4 ]  . 
there has been much discussion about the treatment strategies to be adopted : antibiotic therapy , conservative or reconstructive surgery and hyperbaric oxygen therapy [ 17 , 19 ]  . 
le caratteristiche intrinseche della tomografia computerizzata con la sua elevata risoluzione spaziale e di contrasto e la sua prerogativa di essere la tecnica gold standard per lo studio dellosso , associate al continuo progresso tecnologico e alla possibilit di effettuare ricostruzioni mpr , su tutti i piani dello spazio , vr e 3d , nella nostra esperienza , hanno garantito unaccurata diagnosi ed un adeguato bilancio destensione delle lesioni osteonecrotiche nonch la identificazione di eventuali complicanze associate . non esiste attualmente un trattamento definito per questo particolare tipo di lesioni farmaco - indotte . 
anche se pu sembrar ragionevole pensare che la sospensione della somministrazione intravenosa di bifosfonati possa migliorare la percentuale di regressione di tale fenomeno , non c nessuno che ancora lo proponga come gestione terapeutica [ 18 ]  . 
questo pu essere ben spiegato considerando che i bifosfonati restano avidamente legati alla matrice ossea che circonda gli osteoclasti attivi e , non essendo metabolizzati , ivi permangono a concentrazioni ossee elevate per periodi lunghi di tempo [ 4 ]  . 
molto si discute sulle strategie terapeutiche da adottare : terapia antibiotica , chirurgia conservativa e o ricostruttiva e terapia iperbarica [ 17 , 19 ]  . conclusions conclusioni jaw osteonecrosis is an established disease entity encountered in association with bisphosphonate use and following dental extractions or other oral surgical procedures . 
mdct with mpr , vr and 3d reconstructions helps accurate diagnosis and provides detailed information about the structures involved and is therefore useful for appropriate treatment planning and for follow - up . 
radiologists need to be aware of the risk of osteonecrosis in patients receiving bisphosphonate treatment and should be able to differentiate it from other bone diseases , such as osteomyelitis and osteoradionecrosis [ 20 ] , even with the help of an accurate history and thorough clinical assessment . losteonecrosi della mascella ormai una accertata entit nosografica che si riscontra in associazione a terapia con bifosfonati ed in seguito ad estrazione dentaria o altra chirurgia orale . 
la tc - md con ricostruzioni mpr , vr e 3d consente di farne una accurata diagnosi e di fornire precise informazioni sulle strutture coinvolte per un adeguato planning terapeutico e per il follow - up . 
adelaide di torino , via zuretti 29 , i - 10126 torino , italy correspondence to : a barile , tel . : + 39 - 086 - 2414258 , fax : + 39 - 086 - 2311277 , e - mail : antonio.barile@cc.univaq.it received : 20 september 2005 / accepted : 30 august 2006 / published online : 11 june 2007 abstract purpose . 
postprocessing was done on an independent workstation ( advantage windows , ge medical system ) , with functool ( ge ) software , which allowed a quantitative evaluation of enhancement as a function of time . 
the lesions identified in the 39 patients included 23 soft tissue tumours ( 12 benign , 11 malignant ) and 16 bone tumours ( ten benign , six malignant )  . 
comparing the time - intensity diagrams of lesions of the same histological type , we found typical enhancement patterns for some bone tumours only , especially for bone , cartilaginous , fibrohistiocytic and pseudoinflammatory lesions . 
gli esami rm dinamici sono stati eseguiti con apparecchiature rm dotate di un magnete da 1 , 5 t e da 1 , 0 t , tramite bobine di superficie dedicate scelte in funzione del distretto da studiare prima e dopo somministrazione ev di mdc utilizzando sequenze rapide ed ultrarapide in successione . 
la post - elaborazione stata eseguita in tutti i casi tramite il software functool installato su una workstation indipendente ( advantage windows , ge medical system ) , che ha permesso , attraverso il posizionamento di una roi , di effettuare una valutazione quantitativa dellenhancement in funzione del tempo . 
le lesioni dei 39 pazienti esaminati sono risultate essere : 23 tumori dei tessuti molli di cui 12 benigni e 11 maligni e 16 lesioni ossee , di cui 10 benigne e 6 maligne . 
confrontando le curve intensittempo ( tic ) delle lesioni appartenenti ad uno stesso istotipo sono state identificate curve tipiche per alcune lesioni tumorali ossee ( serie ossea , cartilaginea , fibroistiocitica ) e per le lesioni infiammatorie . non stato possibile identificare una curva tipica allinterno dello stesso istotipo per le lesioni dei tessuti molli . 
la rm perfusionale ha mostrato un discreto livello di sensibilit e specificit nella diagnosi differenziale tra lesioni ad elevata ed a bassa attivit biologica e solo in alcuni casi stato possibile riscontrare una correlazione tra pattern perfusionale e istologia della lesione . 
i valori di pendenza dovrebbero quindi essere usati insieme con le immagini spin echo convenzionali e con altri dati diagnostici e clinici per restringere le possibili diagnosi differenziali e per poter predire con una significativa attendibilit la natura della lesione . key words perfusion mri musculoskeletal tumours contrast agents tumour perfusion parole chiave risonanza magnetica perfusionale tumori muscolosheletrici mezzi di contrasto perfusione tumorale a . 
primary bone tumours account for approximately 20% of all skeletal tumours , and they frequently affect young subjects , in whom they exhibit an aggressive growth pattern and high mortality and disability rates . 
the typical presenting complaint is an asymptomatic mass , although there may be mechanical symptoms caused by compression , traction or trapping of muscles and nerves [ 3 ]  . 
 in view of the development of surgical techniques and the possibilities offered by chemotherapy , the early diagnosis and accurate staging of the disease are becoming fundamental for radical treatment . 
clinically suspected musculoskeletal tumours are usually identified by radiography and computed tomography ( ct ) in the case of bone tumours , and ultrasound ( us ) and conventional magnetic resonance imaging ( mri ) in the case of soft tissue neoplasms [ 3 ]  . 
 dynamic contrast - enhanced mri relies on dynamic imaging through the use of fast or ultrafast sequences that allow one to follow the early enhancement kinetics of the tissue being studied [ 4 ]  . 
the study was prospective and included patients aged 1386 years with a first diagnosis of musculoskeletal tumour . twenty - three patients had soft tissue tumours , and 16 had bone tumours . 
patients were excluded if they had already undergone contrast - enhanced diagnostic studies of any kind ( ultrasound , ct , mri or scintigraphy ) , if they had undergone cytological or bioptic examination , and if they had received neoadjuvant therapy ( chemotherapy or radiotherapy )  . all mri studies were performed using 1.0 - t and 1.5 - t magnets ( signa , ge medical system , milwaukee , wi , usa ) , with dedicated surface - array coils selected on the basis of the region to be studied . 
for the conventional mri studies , t1 spin - echo , t2 spin - echo , t1 sequences with fat lapparato muscoloscheletrico costituito da una componente ossea e da tessuti molli ovvero muscoli , tendini , grasso , tessuto fibroso , vasi e nervi [ 1 , 2 ]  . 
i tumori primitivi dellosso rappresentano circa il 20% della totalit delle forme neoplastiche a localizzazione scheletrica , colpiscono frequentemente let giovanile , nella quale sono caratterizzati da un atteggiamento aggressivo e da un importante indice di mortalit e di invalidit . 
il quadro di esordio pi comune rappresentato da una massa asintomatica , possono essere tuttavia presenti sintomi meccanici riferibili a compressione , alla trazione o allintrappolamento di muscoli e nervi [ 3 ]  . vista levoluzione delle tecniche chirurgiche e le possibilit offerte dalla chemioterapia , una diagnosi precoce ed una accurata stadiazione della patologia si rendono sempre pi importanti e fondamentali per raggiungere la radicalit del trattamento . 
lindividuazione di una neoplasia muscoloscheletrica clinicamente sospettabile compete di solito allesame radiografico ed alla tomografia computerizzata ( tc ) per le lesioni ossee , allecografia ed alla risonanza magnetica ( rm ) tradizionale per le lesioni dei tessuti molli [ 3 ]  . lo studio contrastografico dinamico con rm si fonda su un imaging dinamico mediante lutilizzo di sequenze rapide o ultrarapide che consentono di seguire la cinetica di impregnazione precoce con mdc del tessuto in studio [ 4 ]  . 
lo scopo di questo studio quello di valutare le potenzialit ed i limiti della rm perfusionale nella caratterizzazione delle lesioni tumorali muscoloscheletriche . materiali e metodi nel periodo compreso tra gennaio 2003 e settembre 2005 sono stati valutati gli esami rm standard e rm perfusionali di 39 pazienti . 
nello studio prospettico sono stati inclusi pazienti con patologia neoplastica muscoloscheletrica alla prima diagnosi con et compresa tra i 13 e gli 86 anni , 23 presentavano tumori dei tessuti molli e 16 tumori ossei . 
i criteri di esclusione dal reclutamento dei pazienti per questo studio sono stati : pazienti che avevano gi effettuato esami diagnostici contrastografici di qualsiasi tipologia ( ecografia , tc , rm o scintigrafia ) ; pazienti che erano gi stati sottoposti ad esame citologico o bioptico ; pazienti che erano stati sottoposti a terapie neoadiuvanti ( chemioterapia , radioterapia )  . gli esami rm sono stati eseguiti con apparecchiature rm con magneti da 1 , 0 e 1 , 5 t ( signa , ge medical system , milwakee , wi , usa )  . 
we then performed eight 19 - s scans in fast succession , with a scan delay that varied with the region being studied but that never exceeded 20 s so as to ensure good arterial - phase perfusion in all cases . 
the last scan was performed 5 min after the infusion of contrast material . postprocessing was carried out with functool software installed on an independent workstation ( advantage windows , ge medical system ) , which allows a quantitative assessment of enhancement as a function of time in a given region of interest ( roi )  . 
the rois were uniform and measured 20 mm2 in all cases , and the software - generated curves were based on graphs in which the x - axis represented a time scale reporting the progressive number of images acquired and the y - axis represented enhancement expressed as a percentage . the resulting time - intensity curves provided a graphic representation of contrast perfusion during and immediately after the administration of contrast material , from which we could derive quantitative information about the lesions : time to enhancement , wash - in ( positive slope ) , peak enhancement , and wash - out ( negative slope )  . thirty - four patients then underwent surgical excision of the tumour , and for each one , we were able to compare and confirm the imaging diagnosis , including the areas where perfusion patterns were measured , against the histological type and grade provided by gross pathology and histopathology . 
the remaining five patients underwent biopsy and histological assessment of the histological type and grade . results the lesions of the 39 patients included 23 soft tissue tumours , 12 of which were benign and 11 malignant , and 16 bone tumours , ten of which benign and six malignant . 
 una volta individuato il piano migliore per studiare la lesione , e dopo aver effettuato una prima acquisizione precontrasto , stato somministrato tramite catetere endovenoso da 20 gauge posizionato nella vena basilica o cefalica un bolo di mdc paramagnetico ( gadolinium - dtpa , 0 , 1 mmol / kg ) utilizzando un iniettore automatico , alla velocit di 2ml / s seguito da 10 ml di soluzione fisiologica . 
sono state quindi eseguite in rapida successione 8 scansioni della durata di 19 secondi ciascuna , con un ritardo di acquisizione della sequenza , dalliniezione del bolo iniziale , dipendente dal distretto in esame ma comunque non superiore ai 20 secondi per ottenere in tutti i casi una buona perfusione in fase arteriosa . 
lultima scansione stata eseguita a 5 minuti di distanza dallinfusione di mdc . la post - elaborazione stata eseguita in tutti i casi tramite il software functool installato su una workstation indipendente ( advantage windows , ge medical system , milwaukee , wi , usa ) , che ha permesso , attraverso il posizionamento di una roi , di effettuare una valutazione quantitativa dellenhancement in funzione del tempo . 
la roi in tutti i casi stata di 20 mm2 ; il software ha fornito la curva risultante rappresentata su assi cartesiani , aventi per ascissa la successione temporale delle sequenze effettuate e per ordinata lenhancement misurato in percentuale . 
questo ha permesso di ricavare informazioni quantitative sulle lesioni : tempo di inizio enhancement , frazione di wash - in ( inclinazione della curva ) , massimo enhancement e frazione di wash - out ( pendenza negativa )  . trentaquattro pazienti sono stati quindi sottoposti ad intervento chirurgico escissionale e di ognuno stato possibile confrontare e confermare la diagnosi strumentale , comprese le aree di misurazione del comportamento perfusionale della lesione , con esame macroscopico ed istologico per la valutazione dellistotipo e del grading . 
i restanti 5 pazienti sono stati invece sottoposti ad esame bioptico con successiva valutazione istologica dellistotipo e del grading . risultati le lesioni dei 39 pazienti esaminati sono risultate essere : 23 tumori dei tessuti molli di cui 12 benigni e 11 maligni e 16 lesioni ossee di cui 10 benigne e 6 maligne . 
la pendenza delle curve intensit - tempo raffrontata con i risultati del grading delle lesioni ottenuti allesame istologico ha evidenziato sensibilit e specificit rispettivamente del 64% / 58% per i tumori dei tessuti molli e del 86% / 67% per i tumori ossei . nei tumori ossei ( tabella 1 ) , lesioni ad alta ed a bassa aggressivit biologica presentavano una cinetica contrastografica perfusionale differente ed abbastanza caratteristica ; a . 
tumour growth , in fact , requires a permissive microenvironment , and the tumour itself ensures this through the induction of neoangiogenesis , without which the lesion could not grow larger than 1 or 2 mm [ 15 ]  . 
in dynamic mri , images are acquired during and immediately after bolus injection to study the dynamic , physiological phenomenon of the distribution of contrast material in the capillaries and in the interstitial spaces of the tissues ( perfusion ) [ 4 ]  . 
with the first pass of contrast material through the capillaries , a fast , unidirectional diffusion occurs in the tissue as a result of the high concentration gradient between the intravascular and interstitial space : in normal tissues , approximately 50% of the circulating contrast material diffuses from the blood into the extravascular compartment . 
infatti , affinch ci si verifichi necessaria la presenza di un microambiente permissivo ; il tumore stesso rende possibile ci grazie allinduzione di fenomeni neoangiogenetici senza i quali la lesione non potrebbe raggiungere dimensioni di pi di uno o due millimetri [ 15 ]  . 
nella rm dinamica , le immagini vengono acquisite durante e immediatamente dopo la somministrazione del bolo al fine di studiare quel fenomeno dinamico e fisiologico che la distribuzione del mdc nei capillari e negli spazi interstiziali dei tessuti ( perfusione ) [ 4 ]  . 
the interval between the end of the first pass and the equilibrium state depends on the size of the interstitial space and may vary from up to 20 s in lesions with a small interstitial space to more than 35 min in tissues with a larger interstitial ziale : nel tessuto normale circa il 50% del mdc circolante diffonde dal sangue al compartimento extravascolare . 
dopo il primo passaggio , la velocit di diffusione si riduce rapidamente perch la concentrazione media del contrasto rimesso in circolo diminuita a causa dellulteriore diluizione del sangue e del parziale accumulo nello spazio interstiziale di tutto il corpo [ 6 ]  . 
the enhancement curve of the tumour area ( a ) ( arrow a ) is characterised by a very fast and high wash - in and by a fast wash - out with a descending trend . 
a la curva di enhancement dellarea tumorale ( freccia a ) si caratterizza per un rapidissimo ed elevato wash - in ed un rapido wash - out con tendenza a scendere . 
the enhancement curve of the tumour area ( a ) ( arrow a ) is characterised by a very fast and high wash - in and high wash - out with a plateau trend . 
the enhancement curve of the tumour area ( a ) ( arrow a ) is characterised by a moderate arterial wash - in , a plateau and a moderate wash - out . 
4a , b curva intensit - tempo delle lesioni simil - infiammatorie ( osteoma osteoide della metafisi distale del femore ( b ) con segnalazione dei roi di misurazione )  . 
liposarcoma del cavo popliteo ( c ) ; d la curva di enhancement dellarea tumorale ( freccia b ) mostra un wash - in arterioso , rapido wash - out con tendenza a scendere . space [ 6 , 7 ]  . 
only in highly vascular lesions with a small interstitial space is there seen a very early wash - in with a rapidly rising slope of the time - intensity curve and an equally early wash - out within the first minutes of contrast injection [ 8 ]  . the aim of contrast - enhanced dynamic mri is to detect and depict differences in early intravascular and interstitial distribution of contrast [ 9 , 10 ]  . 
capillary perfusion , the rate of diffusion and the amount of water - soluble contrast material temporarily accumulated in the interstitial space may vary considerably from tissue to tissue : these processes are influmo di 20 secondi in lesioni con uno spazio interstiziale ridotto a pi di 35 minuti in tessuti con uno spazio interstiziale pi grande [ 6 , 7 ]  . 
solo nelle lesioni altamente vascolarizzate , con uno spazio interstiziale ridotto , abbiamo un wash - in precocissimo caratterizzato da una alta pendenza della curva intensit - tempo e un wash - out altrettanto precoce entro il primo minuto dalliniezione del bolo [ 8 ]  . lo scopo dello studio perfusionale dinamico tramite rm di individuare e descrivere le differenze nella distribuzione iniziale intravascolare e interstiziale del mdc [ 9 , 10 ]  . 
the enhancement curve of the tumour area ( b ) ( arrow a ) is characterised by a fast and early wash - in , a plateau trend and a slow , progressive and uniform washout . 
tgc delle guaine dei flessori del ginocchio ( a ) ; b la curva di enhancement dellarea tumorale ( freccia a ) presenta un rapido enhancement precoce , una tendenza al plateau ed un wash - out lento , progressivo e costante . 
as can be inferred from our results , among bone tumours , we were able to identify some typical timeintensity curves both for lesions with low and high biological aggressiveness and for some histological types of tumour . 
however , some remarks need to be made on the absolute significance of these curves : although there is a statistically significant difference in the slope values of benign and malignant lesions and in the appearance of the different curves within different histological types of tumour , these data should always be related to all the information provided by the different diagnostic modalities ( radiography , ct , mri )  . some typically benign but vascular lesions ( eosinophilic granuloma , gct , osteoid osteoma ) may , depending on the stage of lesion when the study is conducted ( active , calm etc . ) , display very similar slope values to malignant tumours . 
come si evince dai nostri risultati stato possibile , per i tumori dellosso , identificare alcune curve intensit tempo tipiche sia per lesioni a bassa che ad alta aggressivit biologica sia nel differenziare alcuni istotipi tumorali . 
 tuttavia necessario fare delle considerazioni sul significato assoluto di tali curve : infatti bench ci sia una differenza statisticamente rilevante nel valore delle pendenze delle lesioni benigne e di quelle maligne ed anche nellaspetto delle diverse curve nellambito di differenti istotipi tumorali , questi dati devono essere sempre considerati nel complesso delle informazioni ottenute dalle diverse metodiche diagnostiche ( esame radiografico , tc , rm )  . infatti alcune lesioni tipicamente benigne ma discretamente vascolarizzate e perfuse [ granuloma eosinofilo , tumore a cellule giganti del sacro ( tgc ) , osteoma osteoide ] possono avere , a seconda della fase in cui lo studio viene eseguito ( attiva , calma , ecc . ) valori di pendenza molto simili a quelli dei tumori maligni . 
al contrario , possono essere osservati tumori istologicamente maligni con valori di pendenza della curva di perfusione relativamente bassi ( tumori altamente necrotici , condrosarcomi con ridotta vascolarizzazione )  . 
although bone tumours exhibit typical curves among the different histological types , the final diagnosis and the attempt to characterise the lesion should always take into account the data provided by the different diagnostic imaging modalities . 
 soft tissue lesions do not exhibit typical curves among the different histological types , and therefore , the definitive diagnosis is dependent on all the diagnostic information provided by conventional imaging modalities ( ultrasound and mri ) and ultimately by biopsy . dal nostro studio si pu evincere che grazie ai progressi tecnologici delle apparecchiature rm e dei software ad esse collegati oggi possibile sfruttare nel migliore dei modi le informazioni relative alla fase contrastografica delle lesioni muscoloscheletriche . 
fabrizzi azienda ospedaliero - universitaria ospedali riuniti - ancona , servizio di radiologia presidio salesi , via corridoni 11 , i - 60123 ancona , italy correspondence to : c . 
the ct images were reviewed blinded to the histological findings , and attention was paid to the number and size of cysts so as to classify the lesions into the three groups described by stocker et al . 
areas with small - sized cysts ( < 2 cm ) were detected by ct in two patients ( 33.3% ) , areas with large cysts ( > 2 cm ) were seen in three cases ( 50% ) whereas in the remaining case , the diagnosis was mixed type i and type ii cam . 
ct proved to be accurate in identifying and characterising cam and provided important information on lesion site and extension . key words cystic adenomatoid malformation paediatric chest ct malformative lung lesion riassunto obiettivo . 
le immagini tc sono state rivalutate in ceco rispetto ai reperti istologici ponendo attenzione al numero e alle dimensioni delle cisti cos da classificare le lesioni nei tre tipi descritti da stocker . 
aree con cisti di piccole dimensioni ( < 2 cm ) sono state evidenziate alla tc in 2 pazienti ( 33 , 3% ) , aree con cisti voluminose ( > 2 cm ) sono state osservate in 3 casi ( 50% ) , mentre nel restante caso stata posta diagnosi di mac di tipo misto i e ii . 
la classificazione tc di stocker risultata concordante con lesame anatomo - patologico in 4 casi , mentre nei restanti 2 casi le lesioni sono state classificate come tipo i o ii alla tc e come lesioni miste ( tipo i e ii ) allistopatologia . 
lesame tc risultato essere una metodica accurata nellidentificare e caratterizzare la mac fornendo inoltre importanti informazioni sulla sede ed estensione della lesione . parole chiave malformazione adenomatoide - cistica tc del torace pediatrico lesioni malformative del polmone introduction introduzione c . 
this structural feature promotes the creation of a valve mechanism , with pulmonary hyperexpansion , compression of the normal lung and development of severe respiratory distress [ 6 , 7 ]  . 
classified cam into three types on the basis of the clinical and histological findings [ 10 ] : type i ( 50% ) : multiple large cysts , with a diameter greater than 2 cm , or a single large cyst surrounded by numerous smaller cysts type ii ( 42% ) : multiple small cysts , most smaller than 1 cm ; this type is frequently associated ( 26% of cases ) with other congenital malformations type iii ( 3% ) : malformation made up of numerous small cysts with a diameter less than 0.5 cm with cuboidal epithelium and most often collapsed walls , forming a solid mass ; it is the least common form and has the worst prognosis , with a remarkable incidence of foetal prematurity and hydrops due to cardiac compression and vena cava obstruction computed tomography ( ct ) is required for determining the type and extension of cam , information needed for surgical planning and for identifying possible coexisting malformations . 
the aim of our study was to assess the accuracy of ct in classifying the different types of cam , taking histopathology as the gold standard . materials and method we retrospectively reviewed six cases of cam ( four males and two females ; age range : 1 day to 9 years ) studied between 2000 and 2004 . 
in two cases ct , was carried out with a helical scanner ( tomoscan av e1 philips , japan ) and in four cases with a multislice device ( light speed ultra , general electric , healthcare , milwaukee , wi , usa )  . 
ct scans were carried out without contrast matela malformazione adenomatoide - cistica ( mac ) fu descritta per la prima volta come entit distinta da chin e tang nel 1949 [ 1 ]  . 
rappresenta circa il 25% di tutte le lesioni polmonari congenite ed caratterizzata da una proliferazione adenomatoide dei bronchioli terminali con formazione di masse solide , cistiche o miste allinterno del parenchima polmonare ; la vascolarizzazione ha origine dalla circolazione bronchiale , ma raramente pu dipendere da un vaso anomalo sistemico [ 25 ]  . 
le cisti , che comunicano sia tra loro sia con lalbero bronchiale , sono prive di tessuto cartilagineo di supporto a livello della connessione con i bronchi : questa caratteristica strutturale facilita linstaurarsi di un meccanismo a valvola , con iperespansione polmonare , compressione del polmone normale e comparsa di un grave distress respiratorio [ 6 , 7 ]  . 
questo rischio , associato a quello di una degenerazione maligna del tessuto adenomatoide va considerato nella decisione se trattare o meno chirurgicamente un paziente asintomatico [ 8 , 9 ]  . 
la mac stata classificata da stocker in base al quadro clinico e istologico in tre tipi [ 10 ] : i tipo ( 50% ) : cisti multiple e grandi , con un diametro superiore a 2 cm , o singola grande cisti circondata da numerose cisti pi piccole ; ii tipo ( 42% ) : cisti multiple e di piccole dimensioni , la maggior parte inferiori ad 1 cquesto tipo di lesione frequentemente associato ( 26% circa dei casi ) ad altre malformazioni congenite ; iii tipo ( 3% ) : malformazione formata da numerose piccole cisti dal diametro inferiore a 0 , 5 cm con un epitelio cuboidale e con pareti il pi delle volte collabite , cos da costituire nel suo complesso una massa solida . 
la forma pi rara e a prognosi peggiore , con notevole incidenza di prematurit e di idrope fetale per compressione cardiaca e ostruzione venocavale . la tc indispensabile nella determinazione del tipo e dellestensione della mac , informazioni necessarie per una corretta pianificazione chirurgica , e per lidentificazione di eventuali lesioni malformative coesistenti . 
scopo del nostro studio valutare laccuratezza della tc nel classificare i diversi tipi di mac assumendo come gold standard il riscontro anatomo - patologico . materiali e metodi abbiamo rivalutato in modo retrospettivo 6 casi di mac ( 4 maschi e 2 femmine ; range di et : 1 giorno9 anni ) che tra il 2000 e il 2004 sono giunti alla nostra osservazione . 
tutti i pazienti , alla luce del sospetto diagnostico ecografico prenatale o della sintomatologia clinica , sono stati sottoposti ad esame radiografico e tc del torace , e ad intervento chirurgico di lobectomia . 
patients 5 patients 1 patient symptoms asymptomatic patients symptomatic patients site < 5 months 9 years 3 asymptomatic 2 respiratory distress 1 recurrent lung infections chest x - ray : 2 negative ; 1 triangular opacity 2 opacities ; 1 hyperlucency 3 upper lobe ( 2 right - 1 left ) 1 lingula 2 left lower lobe n pazienti 5 pazienti 1 paziente sintomi pazienti asintomatici pazienti sintomatici sede < 5 mesi 9 anni 3 asintomatici 2 distress respiratorio 1 infezione polmomare ricorrente 2 negative 1 opacit triangolare 2 opacit 1 iperdiafania 3 lobo polmonare superiore ( 2 dx - 1 sn ) 1 lingula 2 lobo polmonare inferiore sn rial in four cases and with the intravenous administration of a bolus of iodinated contrast material in the remaining two cases . 
the classification of lesions obtained with ct was then correlated with the histopathological findings . results the patients age , gender , symptoms and radiographic findings are shown in table 1 . 
the lesions were located in the upper lung lobe in three cases ( two in the right lobe , one in the left lobe ) , in the lingula in one case and in the lower left lobe in the remaining two cases . 
le immagini sono state acquisite con uno spessore di strato di 5 mm , pitch 1 e ricostruite a 2 , 5 mm ( 120 kv , 50 ma , 1 s ) lesame tc stato eseguito senza mezzo di contrasto ( mdc ) in 4 casi mentre nei restanti 2 si resa necessaria la somministrazione di mdc iodato a bolo per via endovenosa . 
si proceduto poi a confrontare la classificazione delle lesioni effettuata con la tc con i corrispettivi reperti anatomo - patologici . risultati le caratteristiche dei pazienti per quanto riguarda let , il sesso , la sintomatologia e i reperti radiografici sono descritti nella tabella 1 . 
2 rx : iperdiafania rotondeggiante con orletto periferico . no in tre casi il lobo polmonare superiore ( 2 a destra , 1 a sinistra ) , in un caso la lingula e nei restanti 2 il lobo polmonare inferiore sinistro . 
le due lesioni localizzate nel lobo polmonare inferiore sinistro hanno necessitato , in aggiunta allindagine tc , di un esame di rm per meglio evidenziare il tipo di vascolarizzazione , sistemica o polmonare , cos da porre diagnosi differenziale tra mac e sequestro polmonare . il diametro massimo delle cisti stato 8 cm con un range variabile da 0 , 5 a 10 cm ( media 4 , 5 cm )  . 
la classificazione tc di stocker risultata concordante con lesame anatomo - patologico in 4 casi ( 66 , 7% ) , mentre nei restanti 2 casi ( 33 , 3% ) le lesioni sono state classificate come tipo i o ii alla tc e come lesioni miste ( tipo i e ii ) allistopatologia ( tabella 2 )  . 
in un caso la classificazione tc risultata corretta ma lesame istopatologico ha evidenziato la coesistenza di sequestro polmonare . discussione la mac una lesione malformativa dovuta ad un arresto focale dello sviluppo polmonare prima della 17a settimana di fig . 
mac di tipo misto i e ii . with histopathology in four cases ( 66.7% ) , whereas in the remaining two cases ( 33.3% ) , the lesions were classified as type i and type ii at ct and as mixed ( type i and ii ) at histopathology ( table 2 )  . 
in one case , ct classification was correct , but histopathology revealed the coexistence of pulmonary sequestration . discussion cam is a malformation due to focal arrest in foetal lung development before the 17th week of gestation and may be associated , in 4%26% of cases , with other congenital abnormalities . 
the diagnosis is generally provided by prenatal ultrasound , which has a good degree of accuracy in classifying lesions ( 77% ) but is rarely able to identify coexisting sequestration . 
when the diagnosis is prenatal , the lesion is asymptomatic at birth in 71% of cases , and in 56% it regresses spontaneously despite a possible initial enlargement [ 11 , 12 ]  . 
during the first days of life , radiography typically shows a homogeneous opacity due to the presence of fluid in the alveoli ; the appearance then becomes cystic , with multiple hyperlucent areas due to fluid reabsorption and pneumatization of the lesion . 
quando la diagnosi posta in utero la lesione asintomatica alla nascita nel 71% dei casi e nel 56% regredisce spontaneamente nonostante un possibile iniziale ingrandimento [ 11 , 12 ]  . 
nei primi giorni di vita laspetto radiografico tipico quello di una opacit omogenea per la presenza di liquido allinterno degli alveoli che poi evolve verso laspetto cistico con multiple aree radiotrasparenti dovuto al riassorbimento dei fluidi e alla pneumatizzazione della lesione . 
per un fenomeno di airtrapping le cisti possono andare incontro ad un rapido ingrandimento con shift controlaterale del mediastino e conseguente distress respiratorio [ 13 ]  . la tc la metodica pi sensibile nella diagnosi di mac . pu evidenziare il persistere di anomalie parenchimali anche a fronte di un esame eco - radiografico negativo . 
it may reveal the persistence of parenchymal abnormalities even when ultrasound and radiography are negative . ct is also necessary for determining the type and extension of cam , for identifying possible coexisting lesions and for differentiating between cam and extralobar pulmonary sequestration , which consists of a portion of lung tissue without bronchial and vascular connections . 
in congenital lobar emphysema , the hyperexpansion of a lobe , generally an upper lobe or the right middle lobe , may generate a mass effect and be confused with cam : the ct identification of hyperexpanded but normal lung parenchyma will easily lead to a diagnosis of congenital lobar emphysema . interstitial pulmonary emphysema may mimic cam when it is complicated by extensive air collections , even though these are typically associated with linear collections . 
on ct , cam may appear as an area of small - sized cysts ( < 2 cm ) associated with other lesions ( areas of consolidation or lager cysts ) , as macrocystic lesions ( > 2 cm ) or as hypodense areas made up of microcysts . 
at times , airfluid levels are visible in some cysts , above all in type i and type ii cysts [ 14 , 15 ]  . recent studies have reported that cam may be accompanied by microscopic foci of bronchioloalveolar carcinoma , mucosal hyperplasia and atypical nonmucinous adenomatous hyperplasia . 
in addition , in type iv , there is not only a risk of pleuropulmonary blastoma developing over time but also the possibility that the two entities coexist from the beginning within the same lesion [ 16 , 17 ]  . the treatment of precancerous lesions is still debated , as the risk of developing a neoplasm , generally rhabdomyosarcoma , seems to be independent of surgical resection of cam altra patologia che pu entrare in diagnosi differenziale lenfisema lobare congenito , le cisti broncogene e lernia diaframmatica . 
nellenfisema lobare congenito liperespansione di un lobo , in genere un lobo superiore o il lobo medio di destra , pu determinare un effetto massa ed essere confuso con la mac : alla tc lidentificazione di parenchima polmonare iperespanso ma normale , pu facilmente far porre la diagnosi di enfisema lobare congenito . 
lenfisema polmonare interstiziale pu mimare la mac quando complicato con ampie raccolte aeree , anche se queste si associano tipicamente a raccolte di tipo lineare e , soprattutto , preceduto da una ventilazione a pressione positiva con barotrauma . 
allesame tc la mac si pu presentare come unarea formata da cisti di piccole dimensioni ( < 2 cm ) associata ad altre lesioni ( aree di consolidazione o cisti di dimensioni maggiori ) , come lesioni macrocistiche ( > 2cm ) o come aree ipodense formate da microcisti . 
a volte visibile in alcune cisti , soprattutto nel tipo i e ii , un livello aria - fluido [ 14 , 15 ]  . recenti studi hanno messo in evidenza come la mac possa accompagnarsi a foci microscopici di carcinoma bronchioloalveolare , di iperplasia mucosa e di iperplasia adenomatosa atipica non mucinosa . 
nel tipo iv di mac inoltre c il rischio non solo di sviluppo negli anni del blastoma pleuropolmonare , ma anche la possibilit che queste due entit coesistano fin dallinizio allinterno della stessa lesione [ 16 , 17 ]  . nellottica di una lesione precancerosa la pianificazione terapeutica tuttora dibattuta in quanto il rischio di sviluppare una lesione neoplastica , in genere il rabdomiosarcoma , sembra essere indipendente dallasportazione chirurgica della mac [ 18 ]  . 
la recidiva dopo asportazione chirurgica comunque eccezionale . i risultati ottenuti con questo studio , in linea con i dati presenti in letteratura , hanno confermato come lesame radiografico sia poco sensibile nei pazienti asintomatici . 
recurrence after surgical removal is , however , an exceptionally rare occurrence . the results obtained in our study , in agreement with the literature data , confirmed that chest radiography has poor sensitivity in asymptomatic patients . 
symptoms were virtually absent in the cases of prenatal diagnosis , whereas recurrent lung infections in the eldest patient ( 9 years ) prompted the clinician to request a ct scan . 
when chest radiography is negative , ct should nonetheless be performed , as it has higher sensitivity above all in the detection of small lesions . in our study , ct correlated with histopathology in 66.7% of cases , whereas in 33.3% , histopathology revealed areas with mixed lesions ( types i and ii )  . 
mixed grading is common in cam due to the presence of different types in different areas within the same lesion . the diagnosis of cam in a context of pulmonary sequestration is not always straightforward , and in most , cases only histology is able to distinguish between the two entities . 
in particular , the differential diagnosis with extralobar sequestration proves difficult when cam is located in the lower lobes and the cystic component is poorly represented ( type iii )  . 
in addition , the use of contrast material also visualises the relationships between the lesion and the bronchial tree and large mediastinal vessels , which is essential for surgical planning . 
in two cases in our study , despite the use of an automatic system for tracking peak enhancement ( smartprep ) , the childs hyperkinetic circulation and small vessel diameter did not allow a correct vascular study of the arterial supply to the lesion . 
in both cases , we used mri , which successfully detected the bronchial origin of the anomalous vessel in one case but was unable to visualise the origin in the second case . to obtain a good ct study , thin - slice acquisitions are usually sufficient , even with low kilovoltage and amperage settings ( low dose )  . 
contrast material should always be used in doubtful cases in which the site and morphological features are not typical of cam or when the surgeon requires precise spatial visualisation of the lesion because of its closeness to mediastinal vessels . 
contrast material is also indispensable when there is a doubt between lobar sequestration and cam : in this case , use of an automatic peak enhancement tracking system can help to identify the bronchial or systemic origin of the anomalous vessel . ( 9 anni ) sono state le infezioni polmonari ricorrenti ad indurre il clinico a richiedere un esame tc . 
nel nostro studio la tc risultata essere concordante con lesame isto - patologico nel 66 , 7% dei casi mentre nel 33 , 3% listopatologia ha evidenziato aree con lesioni miste ( tipo i e ii )  . 
il grading misto frequente nella mac per la presenza di tipi differenti nelle diverse aree allinterno della stessa lesione . la diagnosi di mac su sequestro polmonare non sempre agevole e nella maggior parte dei casi solo lesame istologico riesce a differenziare le due entit . 
specialmente quando la mac si trova a livello dei lobi inferiori e la componente cistica poco rappresentata ( tipo iii ) , la diagnosi differenziale con il sequestro extralobare risulta difficoltosa . 
in questo caso solo il comportamento contrastografico della lesione alla tc consente di differenziarla dal sequestro : la mac infatti presenta una vascolarizzazione bronchiale e non sistemica come il sequestro . 
lutilizzo del mezzo di contrasto ( mdc ) inoltre permettere di studiare non solo la vascolarizzazione della lesione , ma anche il rapporto di questa con lalbero bronchiale e con i grossi vasi mediastinici , elementi indispensabili per la pianificazione chirurgica . 
nel nostro studio in due casi , nonostante lausilio del localizzatore automatico del picco massimo di enhancement ( smart - prep ) , il circolo ipercinetico del bambino e il piccolo calibro dei vasi , non ha permesso un corretto studio vascolare dellafferenza arteriosa della lesione per cui stato necessario ricorrere alla rm che ha evidenziato lorigine bronchiale del vaso anomalo in un caso mentre nel restante anche la rm non stata in grado di visualizzarne lorigine . per ottenere un buon esame tc in genere sufficiente un esame a strato sottile , anche a basso voltaggio e amperaggio ( low dose ) ; lutilizzo del mdc dovrebbe essere sempre effettuato soprattutto nei casi dubbi , in cui la sede e le caratteristiche morfologiche non sono dirimenti per mac o , qualora il chirurgo , data la stretta vicinanza con i grossi vasi mediastinici , necessiti di una precisa visualizzazione spaziale della lesione . 
il mezzo di contrasto , inoltre , indispensabile qualora sussista il dubbio tra sequestro lobare e mac : in questo caso lutilizzo del localizzatore automatico del picco massimo di enhancement utile per evidenziare lorigine sistemica o bronchiale del vaso anomalo . conclusioni conclusions given the close correlation between ct and histopathology , ct may be considered indispensable when cam is suspected , both to differentiate it from other pulmonary malformations and to study its morphological features , elements that are useful for surgery and prognosis . 
lagalla1 1dipartimento di biotecnologie mediche e medicina legale sezione di diagnostica per immagini , 2dipartimento di medicina interna , policlinico universitario , via del vespro 127 , i - 90127 palermo , italy correspondence to : t.v. 
lecografia cs in grado di individuare xta in pazienti con ife ed esame clinico negativo , offrendo una migliore qualit dellimmagine rispetto allhrus . key words achilles tendon familial hypercholesterolemia ultrasonography xanthomas parole chiave tendine dachille ipercolesterolemia familiare ecografia xantomi introduction introduzione familial hypercholesterolaemia ( fh ) is an inherited autosomal dominant disorder of lipoprotein metabolism characterised by a defect in the low - density lipoprotein ( ldl ) receptor gene [ 1 ]  . 
this leads to progressively rising ldl cholesterol levels , lipid deposition in the achilles tendon in the lipercolesterolemia familiare ( if ) una malattia ereditaria del metabolismo delle lipoproteine a trasmissione autosomica dominante , caratterizzata dal difetto di un singolo gene , quello del recettore per le lipoproteine a bassa densit ( ldl ) [ 1 ]  . 
fh may manifest in the heterozygous form ( hfh ) , with an incidence of 1 / 500 individuals in the general population , or in the homozygous form , with an incidence of 1 / 1 , 000 , 000 [ 4 ]  . 
common clinical criteria for a diagnosis of hfh include an early history of coronary atherosclerosis , increased total cholesterol levels and the presence of tendon xanthomas , usually affecting the achilles tendons , the extensor tendons of the hand and elbow , and the patellar tendons [ 6 ]  . 
ultrasonography ( us ) in particular is a reliable method for measuring the tendon thickness and identifying possible lipid deposition , both in the form of xanthomas , which appear as focal or confluent hypoechoic areas , and as a diffuse and heterogeneous tendon thickening with or without calcification , even when the physical examination is completely normal [ 7 , 8 ]  . recently , a new sonographic technique has been introduced real - time spatial compound sonography ( cs ) which relies on the possibility of electronically steering the us beam generated by the array of transducer elements in order to obtain three to nine , in part overlapping , scans of an object from multiple view angles [ 9 ]  . 
application of this technique to the sonographic study of various organs and systems , especially the musculoskeletal system , has led to improved image quality compared with conventional sonography as a result of a reduction in background noise , spurious echoes and other acoustic artefacts , such as anisotropy [ 1013 ]  . the primary aim of this study was to evaluate the incidence and sonographic patterns of achilles tendon xanthomas ( atx ) in patients with hfh and clinically normal tendons studied with high - resolution ultrasound ( hrus )  . the secondary aim was to assess the contribution of cs technology in terms of image quality . materials and methods patients after obtaining approval by our institutions ethics committee , over a period of 2 years , we recruited 40 consecutive patients with genetically proven hfh . 
all patients ( 19 men and 21 women , age range 1174 years , mean age 46.9 years ) underwent hrus and cs of both achilles tendons after providing their written informed consent . 
lif pu presentarsi in forma eterozigote ( ife ) , con unincidenza di 1 / 500 individui nella popolazione generale o in forma omozigote , con unincidenza pari a 1 / 1000000 [ 4 ]  . 
i criteri clinici usualmente presi in considerazione nella diagnosi di ife sono costituiti , soprattutto , da una storia di precoce aterosclerosi coronarica , dallaumento della concentrazione di colesterolo totale e dalla presenza di xantomi tendinei , questi ultimi pi frequentemente apprezzabili in corrispondenza dei tendini dachille , dei tendini estensori delle mani , del gomito e dei tendini patellari [ 6 ]  . 
in considerazione delle sue caratteristiche anatomo - strutturali ( sede , dimensioni ) , il tendine dachille pu essere facilmente esaminato e , in particolare , lecografia risulta un metodo affidabile per misurare il suo spessore ed individuare leventuale accumulo di lipidi , sia sotto forma di veri e propri xantomi , evidenziabili come aree ipoecogene focali o confluenti , sia come diffuso e disomogeneo ispessimento tendineo , accompagnato o meno dalla presenza di calcificazioni , anche quando lesame obiettivo locale sia del tutto negativo [ 7 , 8 ]  . recentemente , stata introdotta una nuova tecnica ecografica real time spatial compound sonography ( cs ) la quale sfrutta la possibilit di angolare elettronicamente il fascio ultrasonoro generato dalla linea di elementi trasduttori al fine di effettuare diverse ( da tre a nove ) scansioni , in parte sovrapposte , di un oggetto da diversi angoli di vista [ 9 ]  . 
lapplicazione di tale tecnica allo studio ecografico di vari organi ed apparati , con particolare riferimento al sistema muscolo - scheletrico , ha mostrato una migliore qualit dellimmagine rispetto allecografia convenzionale , principalmente grazie alla riduzione del rumore di fondo , degli echi spuri e di altri artefatti acustici , quali lanisotropia [ 1013 ]  . lobiettivo principale di questo lavoro stato quello di valutare lincidenza e gli aspetti ecografici degli xantomi del tendine dachille in pazienti con ife ed esame clinico dei tendini negativo , studiati con ecografia ad elevata risoluzione ( hrus )  . 
quale obiettivo secondario , stato valutato leventuale apporto fornito dalla tecnologia cs in termini di qualit dellimmagine . materiale e metodi pazienti durante un periodo di due anni , ottenuta lapprovazione da parte del comitato etico , 40 pazienti consecutivi ( 19 uomini e 21 donne , et 1174 anni , media 46 , 9 anni ) affetti da ife geneticamente accertata , sono stati sottoposti , previa acquisizione del consenso informato in forma scritta , ad ecografia ad alta risoluzione ( hrus ) e cs di entrambi i tendini t.v. 
once set , the technical parameters remained unchanged throughout the sonographic examination . the achilles tendons were scanned axially and longitudinally with the patients in the prone position and holding their ankles flexed over the edge of the couch with the tendon at maximum extension to facilitate contact between the probe and the tendon [ 7 ]  . 
for tendons that did not appear thickened , measurements were made 2 - cm cranially from the distal insertion . each study consisted of the acquisition of four images , two axial and two longitudinal , obtained with conventional us and cs techniques , respectively . 
overall examination time was 57 min . on - site analysis the morphological criteria used for diagnosing xanthoma were : ( 1 ) tendon thickening intended as maximum anteroposterior diameter greater than 7.1 mm , a value by at least two standard deviations ( sd ) higher than the normal sonographic values ( 5.70.7 mm ) , and ( 2 ) evidence of unior multifocal hypoechoic lesions , the largest diameter of which was also measured . 
structural abnormalities were classified as ( 1 ) no lesions , ( 2 ) unior multifocal hypoechoic lesions and ( 3 ) diffuse alterations [ 4 , 6 , 15 , 16 ]  . 
 off - site analysis the digital images stored on the workstation were retrospectively reviewed by two expert radiologists not involved in the sonographic examinations and blinded to the patients clinical data and examination technique . 
coherent wave interface or speckle , caused by excessive background noise , which gives a grainy appearance to an otherwise homogeneous region , reducing image contrast and spatial resolution 3 . 
lateral acoustic shadows , the degree of evidence of normal fibril structures and possible lesions ( visibility and sharpness in relation to the adjacent fibril structures ) [ 9 , 12 ]  . the reviewers consensually expressed their judgement on dachille . 
i tendini dachille sono stati esaminati mediante scansioni sui piani assiale e longitudinale , col paziente in posizione prona e caviglie flesse al di fuori del lettino da esame con il tendine in massima estensione per facilitare il contatto tra la sonda ed il tendine [ 7 ]  . 
ogni studio stato completato con lacquisizione di quattro immagini , due sul piano assiale e due su quello longitudinale , ottenute rispettivamente con metodica ecografica convenzionale e con tecnica compound . 
tutte le immagini sono state archiviate sul disco rigido in dotazione allunit ecografica e poi inviate , sotto forma di dati grezzi , ad un personal computer ( pc ) collegato allecografo attraverso una connessione in standard ethernet . 
definitely absent in particular , reduction of anisotropy was defined as the correct visualisation without interruptions of the tendon fibres , whereas reduction of the other artefacts was described in terms of reduced speckle and clutter . 
statistical analysis was done using the t test for paired samples , with statistical significance set at p < 0.05. results sixty - six out of 80 tendons showed no significant changes in size ( anteroposterior diameter : 4.26.9 mm , mean : 5.5 mm ) or structure . 
fourteen out of 80 ( 17.5% ) tendons in seven ( 6.2% ) patients ( four men , three women , age : 2457 years , mean : 47.3 years ) showed dimensional and / or morphostructural alterations ( table 1 )  . 
nine tendons in five patients ( two men , three women , age : 4357 years , mean : 50.8 years ) showed 12 focal hypoechoic areas classified as xanthomas ( largest diameter : 4.19.8 mm , mean : 7.1 mm ) , five of which were located on the left and seven on the right . 
the number of alterations identified by hrus and cs was equal . the degree of evidence of structural abnormalities in the 80 achilles tendons studied with hrus and cs is shown in table 2 . 
atx are extremely rare in the other forms of hyperlipidaemia , such as type iii hyperlipoproteinaemia , cerebrotendinous xanthomatosis , apolipoprotein b hyperproducnate da due esperti radiologi , non coinvolti negli esami ecografici e tenuti alloscuro dei dati clinico - anamnestici della tecnica utilizzata per ogni esame . 
le immagini sono state presentate in ordine casuale ed stata nascosta ogni informazione riguardo ai parametri tecnici e / o ai dati anagrafici . agli esaminatori stato chiesto di valutare , per ciascuna immagine , la qualit complessiva in relazione alla presenza o meno di artefatti , quali : 1 . 
coherent wave interface o speckle , riconducibile ad un eccessivo rumore di fondo che conferisce un aspetto granulare ad una regione altrimenti omogenea , riducendo il contrasto e la risoluzione spaziale dellimmagine ; 3 . 
le ombre acustiche laterali e altri artefatti acustici , nonch al grado di evidenza delle normali strutture fibrillari che di eventuali lesioni ( visibilit e chiarezza rispetto alle strutture fibrillari tendinee adiacenti ) [ 9 , 12 ]  . 
 i due radiologi hanno espresso , in consenso , la loro valutazione graduandola su una scala di cinque livelli ed il grado di evidenza delle anomalie strutturali stato conseguentemente distinto in : 1 . 
 in particolare , la riduzione dellanisotropia stata definita come la corretta rappresentazione , senza soluzione di continuit , delle fibre tendinee , mentre la riduzione degli altri artefatti stata descritta in termini , soprattutto , di riduzione dello speckle e del clutter . 
lanalisi statistica stata effettuata utilizzando il test t di student per campione doppio con livello di significativit statistica fissato a p < 0 , 05 . risultati sessantasei / 80 tendini non hanno presentato , allindagine ecografica , significative alterazioni dimensionali ( diametro antero - posteriore : 4 , 26 , 9 mm , media : 5 , 5 mm ) n morfostrutturali . 
quattordici / 80 ( 17 , 5% ) tendini in 7 ( 6 , 2% ) pazienti ( quattro maschi , tre femmine , et : 2457 anni , media 47 , 3 anni ) hanno mostrato alterazioni dimensionali e / o morfostrutturali ( tabella 1 )  . 
atx are accompanied by an enlargement of the tendon caused not only by fat deposition but also by oedema and inflammation , which may lead to achillodynia , as reported at least once in their lifetime by 30% of patients with hfh , and , rarely , to tendon rupture [ 16 , 18 ]  . 
the recent discovery of effective pharmacological treatments has placed much emphasis on the early diagnosis of hfh , above all to prevent atherosclerosis , which has a particularly poor prognosis when the coronaries are involved due to the increased risk of fatal ischaemic events [ 2 , 3 , 19 ]  . 
xanthomas , however , which are often the first clinical sign of hfh and usually manifest in the fourth decade of life , are often missed on clinical examination , whereas they are perfectly identifiable on us . 
in 9 / 80 tendini di 5 pazienti ( due maschi , tre femmine ; et : 4357 anni , media : 50 , 8 anni ) sono state identificate 12 aree focali ipoecogene classificate come xantomi ( diametro maggiore : 4 , 19 , 8 mm , media : 7 , 1 mm ) , cinque delle quali localizzate a sinistra e sette a destra . 
di contro , gli xantomi del tendine dachille sono estremamente rari nelle altre forme di iperlipidemia quali liperlipoproteinemia di tipo iii , la xantomatosi cerebrotendinea , liperproduzione di apolipoproteina b e liper - b - sitosterolemia [ 6 , 8 , 17 ] .gli xantomi del tendine dachille sono accompagnati da incremento dimensionale del tendine , causato non solo dal deposito di lipidi , ma anche da fenomeni di edema e flogosi , che possono condurre ad achillodinia , riferita almeno una volta nella vita dal 30% dei pazienti con ife , e , seppur raramente , a rottura tendinea [ 16 , 18 ]  . 
recentemente , la scoperta di terapie farmacologiche efficaci ha posto grande enfasi sulla diagnosi precoce di ife , soprattutto al fine di prevenire i fenomeni aterosclerotici , particolarmente infausti quando a localizzazione coronarica , a causa dellaumentato rischio di eventi ischemici mortali [ 2 , 3 , 19 ]  . 
tuttavia , gli xantomi , usualmente presenti a partire dalla quarta decade di vita e pur costituendo spesso la prima manifestazione clinica di ife , non di rado risultano misconosciuti allesame clinico , mentre risultano fig . 
infatti la radiologia convenzionale , storicamente impiegata come prima tecnica di studio , consente , nella proiezione laterale e con adeguati parametri di esposizione , unicamente la determinazione dello spessore tendineo e lindividuazione di eventuali calcificazioni del tendine , rimanendo pur sempre una tecnica proiettiva ed esponendo il paziente a radiazioni ionizzanti [ 20 ]  . 
questultimo limite condiviso anche dalla tomografia computerizzata ( tc ) , la quale , pur fornendo misurazioni accurate delle dimensioni del tendine achilleo grazie allintrinseco contrasto esistente tra il tendine stesso ed il circostante tessuto adiposo , non in grado di misurare valori di densit che differiscano sostanzialmente in pazienti normo o ipercolesterolemici [ 16 ]  . la risonanza magnetica , infine , non in grado di evidenziare , al di l delle alterazioni morfodimensionali e un aspecifico aspetto puntato , vere e proprie lesioni ascrivibili con certezza a xantomi tendinei [ 2022 ]  . in accordo ai dati di letteratura , la nostra esperienza conferma losservazione che lecografia , in una piccola ma non trascurabile percentuale di pazienti con tendine dachille valutato come del tutto normale allesame clinico , in grado di evidenziare significative alterazioni tendinee , quali ispessimento , calcificazioni o ancora la presenza di veri e propri xantomi [ 7 , 8 , 15 , 19 ]  . 
la minore percentuale di pazienti con ife e lesioni xantomatose achillee riscontrata nel nostro studio ( 6 , 2% ) rispetto a quanto riportato in letteratura ( 29%33 , 1% ) da mettere in relazione , almeno in parte , al gruppo di pazienti selezionato , che non presentava apprezzabili alterazioni tendinee allesame clinico [ 6 , 19 , 23 ]  . 
inoltre , la popolazione numericamente limitata e la tipologia del nostro studio non consentono di confermare i dati epidemiologici acquisiti su campioni di pi vaste proporzioni , che indicano come i soggetti con ife e con xantomi del tendine dachille tendano ad essere di sesso maschile , pi anziani e a presentare livelli di colesterolo totale e ldl pi elevati rispetto ai pazienti senza xantomi [ 19 ]  . la cs una tecnica ultrasonografica la cui applicazione ha condotto ad un miglioramento della definizione dellimmagine ecografica , che alcuni studi hanno riportato essere utile anche ai fini diagnostici , ad esempio in ambito muscoloscheletrico ed in particolare nel distretto anatomico della cuffia dei rotatori [ 9 , 12 , 13 , 24 ]  . 
questa tecnica , diversamente dallecografia b - mode convenzionale nella quale ogni immagine ottenuta da un solo angolo di insonazione , effettuando la media dei segnali ricevuti , in grado di ridurre gli artefatti , migliorando sensibilmente il rapporto segnale rumore e la risoluzione dellimfig 3 left achilles tendon in a 53 - year - old man with heterozygous familial hypercholesterolemia ( hfh )  . 
scansione sagittale che dimostra un tendine diffusamente ispessito , disomogeneo , con perdita della normale ecostruttura fibrillare ( frecce piccole ) e grossolane calcificazioni nel suo contesto ( freccia grande ) ( caso 7 della tabella 1 )  . ing modality for the assessment of atx [ 4 , 8 , 14 , 15 ]  . 
traditionally the first - line imaging technique , conventional radiography in the lateral projection and with adequate exposure settings allows determination of tendon thickness and detection of calcification only but remains a projective technique that exposes the patient to ionising radiation [ 20 ]  . 
this last limitation is shared by computed tomography ( ct ) which , while providing accurate measurement of tendon size thanks to the intrinsic contrast between achilles tendon and the surrounding fat , is unable to measure density values that differ substantially in patients with normal and elevated cholesterol levels [ 16 ]  . 
magnetic resonance imaging ( mri ) is only able to detect changes in shape and size and a nonspecific pointed appearance but cannot demonstrate lesions unequivocally referable to tendon xanthomas [ 2022 ]  . in agreement with the literature , our experience confirms that in a small but nonnegligible series of patients with clinically normal achilles tendon , us can demonstrate significant tendon alterations , such as thickening , calcification or the presence of xanthomas [ 7 , 8 , 15 , 19 ]  . 
the smaller proportion of patients with hfh and atx seen in our study ( 6.2% ) compared with previous reports ( 29%33.1% ) is in part related to our study sample not having appreciable tendon abnormalities on clinical examination [ 6 , 19 , 23 ]  . 
in addition , the limited number of patients included and the study design did not allow us to confirm or refute the epidemiological data derived from larger series , according to which subjects with hfh and atx tend to be older males with higher total and ldl cholesterol levels than those without tendon xanthomas [ 19 ]  . 
 cs is an ultrasonographic technique that has improved the definition of sonographic images and that has been reported to have diagnostic value , for example , in musculoskeletal imaging and in particular in rotator cuff studies t.v. 
4a high - resolution ultrasonography ( hrus ) sagittal image of the right achilles tendon in a 31 - year - old woman with heterozygous familial hypercholesterolemia ( hfh ) shows a normal - sized tendon ( small arrows ) ; speckle ( grainy appearance ) is clearly appreciable . 
4a immagine longitudinale del tendine di achille destro ( frecce piccole ) in una donna di 31 anni affetta da ife ottenuta mediante ecografia in scala di grigi convenzionale : notare le dimensioni normali del tendine e laspetto complessivamente granuloso dellimmagine . 
b real - time spatial compound sonography del medesimo tendine che evidenzia una migliore visibilit del nodulo stesso con riduzione della granulosit dellimmagine , permettendo cos una migliore definizione della fine struttura fibrillare del tendine . [ 9 , 12 , 13 , 24 ]  . 
cs in unlike conventional b - mode imaging in which each image is obtained from a single insonation angle ; by averaging multiple signals , cs reduces artefacts , substantially improving the signal - to - noise ratio and image resolution [ 13 ]  . 
only by analysing the tendon fibres perpendicularly is it , in fact , possible to discriminate with any certainty areas of echostructural alteration from the normal fibril network , consisting of the interfaces between the connective tissue septa of the endomysium and the parallel collagen fibre bundles [ 14 , 15 ]  . 
solo analizzando perpendicolarmente le fibre del tendine possibile , infatti , discriminare con sicurezza eventuali aree di alterazione ecostrutturale rispetto al normale disegno fibrillare , costituito dalle interfacce tra i setti connettivali dellendomisio ed i fasci di fibre collagene a decorso parallelo [ 14 , 15 ]  . in particolare , la presenza di aree ipoecogene focali o un aspetto diffusamente disomogeneo vengono considerati , in pazienti con ife ma senza storia clinica di traumi o tendinite , come altamente suggestivi di xantomi , anche in assenza di conferma istologica [ 4 ]  . 
however , although us is reliable for detecting focal hypoechoic areas on a generally hyperechoic tendon and for determining the extent of tendon thickening , it appears to be subject to a greater error margin in the evaluation of echostructural heterogeneity [ 6 ]  . 
furthermore , as already suggested for conventional us , cs could be used to monitor regression of xanthomas and / or the reduction in tendon size in patients receiving therapy [ 27 ]  . there are some limitations to our study . 
in particular , the assessment of image quality was conducted in a subjective fashion , and a double - blind review of cases would have allowed us to determine interand intraobserver variability . cremento dimensionale del tendine , la valutazione della disomogeneit ecostrutturale appare soggetta ad un pi ampio margine di errore [ 6 ]  . 
daltra parte , nella serie presa in esame , tale migliore qualit dellimmagine , per quanto utile per una maggiore confidenza diagnostica , non si tradotta in un pi elevato tasso di identificazione di xantomi achillei . 
inoltre , lecografia cs potrebbe essere utilizzata , come gi suggerito per lecografia convenzionale , per monitorare la regressione degli xantomi e / o la riduzione delle dimensioni del tendine in pazienti sottoposti a terapia [ 27 ]  . il nostro studio presenta dei limiti . 
in particolare , la valutazione della qualit dellimmagine stata condotta in maniera soggettiva e una revisione dei casi in doppio cieco avrebbe consentito il calcolo della variabilit intraed interosservatore . conclusione conclusion in conclusion , cs sonography proved useful in the evaluation of the achilles tendon in patients with hfh and normal clinical examination , as it provided early evidence of xanthomas , thereby supplying the clinician with useful information for patient management and optimisation of therapy . 
civeira f , international panel on radiol med ( 2007 ) 112 : 620 doi 10.1007 / s11547 - 007 - 0167 - z dallanatomia allimmagine silvia magnaldi luciana travan 2nd edn . 
poletto editore isbn 88 - 86786 - 94 - 8 published online : 11 june 2007 as with the first edition , and , indeed , even more so , this revised edition of dallanatomia allimmaginesucceeds in completely fulfilling its objectives . with its brilliant layout , excellent drawings and clear radiological images , the book presents radiological anatomy in a straightforward and intuitive manner . 
also excellent are the captions and cross - references in the illustrations that allow quick and easy recognition of the anatomical structures discussed in the text . anche e ancor pi nella nuova edizione il libro dallanatomia allimmagine risponde in modo completo ai suoi obiettivi . con la sua brillante impostazione editoriale , gli ottimi disegni e la chiara iconografia radiologica , il libro propone lanatomia radiologica in modo semplice e intuitivo . 
al tempo stesso il libro molto completo in tutte le sue parti . vengono illustrati ad uno ad uno gli apparati , a partire dal muscolo - scheletrico , proseguendo con il cardio - vascolare , il linfatico , il digerente , il respiratorio , luro - genitale , lendocrino , per concludere con il sistema nervoso . la trattazione prevede un testo molto ben articolato che guida liconografia e le tabelle . 
giovagnoni4 1dipartimento diagnostica per immagini , ausl 3 umbria , ospedale foligno , perugia , italy 2dipartimento diagnostica per immagini , ausl 2 umbria , ospedale assisi , perugia , italy 3sc cardiochirurgia , ospedale s . 
crusco , via antica vena 18 , i - 06087 perugia , italy , tel . : + 39 - 075 - 393044 , fax : + 39 - 075 - 5783488 , e - mail : fcrusco@sirm.org received : 17 may 2006 / accepted : 3 october 2006 / published online : 11 june 2007 abstract purpose . 
sixteen - slice mdct scanners enable accurate analysis of cabg status and are a useful noninvasive diagnostic tool for midterm clinical follow - up of patients who have undergone cabg involving the use of ra . key words radial artery coronary artery bypass graft multidetector ct riassunto obiettivo . 
cinquantuno pazienti , per un totale di 50 bypass con ami , 55 con sv e 51 con ar , sono stati sottoposti ad esame tcmd a 16 canali con intervallo temporale dallintervento chirurgico pari a 324 mesi . 
il giudizio di perviet stato valutato come grado 0 ( perviet completa ) , grado 1 ( stenosi focale , > 70% ) e grado 2 ( ostruzione del graft )  . 
la perviet dei bypass con ar stata influenzata dai diversi territori dellanastomosi distale con valori del 79 , 4% nel territorio distale della circonflessa ( acs ) , del 72 , 7% in quello della discendente anteriore ( ada ) e del 50% in quello della coronaria destra ; nel caso dei bypass con ar il tasso globale di ostruzione risultato del 20 , 0% nel caso dei graft semplici , del 18 , 2% nel caso dei graft sequenziali e del 20 , 0% nel caso dei graft compositi . 
la tcmd a 16 canali , consentendo unanalisi accurata dello stato di perviet dei graft , deve essere considerata una metodica diagnostica non - invasiva particolarmente utile nel follow - up a medio termine dei pazienti sottoposti a bypass aortocoronarico comprendente lutilizzazione dellarteria radiale . parole chiave arteria radiale intervento di bypass dellarteria coronarica tc multidetettore f . 
thus , follow - up examinations are essential . conventional coronary angiography is still considered the gold standard for the evaluation of native coronary arteries and cabg , but recent studies have shown that contrast - enhanced coronary computed tomography angiography ( cta ) , a noninvasive and less expensive diagnostic tool , enables accurate assessment of both coronary arteries [ in terms of high negative predictive value ( npv ) ] and graft patency [ 29 ]  . moreover , multidetector - row ct ( mdct ) appears to be a useful technique able to detect postoperative complications in patients presenting with chest pain resembling recurrent angina secondary to graft occlusion [ 10 ]  . 
 among cabg strategies , left internal mammary artery ( lima ) and saphenous vein ( sv ) grafts have been extensively used with good clinical results , whereas radial artery ( ra ) or gastroepiploic artery grafts are a second alternative , as are aortocoronary or composite conduits [ 1113 ]  . 
midand long - term patency rates of these grafts have been evaluated only by angiographic studies in symptomatic patients , and the findings , especially those related to ra grafts , are inconsistent and nonconclusive [ 1418 ]  . 
in asymptomatic patients , radionuclide myocardial perfusion imaging ( mpi ) seems to be a valid method for risk stratification , prognosis and determination of disease progression [ 19 , 20 ]  . to our knowledge , there are no recent midto long - term follow - up studies on cabg assessment focusing on ra patency in asymptomatic patients , performed with mdct coronary angiography . 
the purpose of our observational study was to evaluate the 3 - year outcome of patients who have undergone cabg involving the use of ra grafts in comparison with lima and sv grafts by using 16 - mdct coronary angiography . materials and methods patient population over an 8 - month period , between april 2005 and october 2005 , 51 consecutive patients with at least triple - cabgs involving ra usage were enrolled in our observational study . all of them were referred to us by a single hospital cardiothoracic department that acted as a recruitment and data control centre . 
all asymptomatic patients ( 51 ) who had undergone a routine diagnostic work - up [ including exercise electrocardiographic ( ecg ) testing ] were eligible for the ct study . 
patients with allergies to iodinated contrast media , renal ( serum creatinine level > 2.00 mg / dl ) , respiratory or cardiac failure [ new york heart association ( nyha ) iv ] , cardiac arrhythuna delle procedure pi frequentemente ed estesamente eseguita in campo cardiochirurgico la rivascolarizzazione miocardica mediante bypass aortocoronarico ( cabg ) , trattamento di scelta nei pazienti sintomatici con malattia coronarica . 
la prognosi post - chirurgica strettamente dipendente dalla perviet dei bypass , e poich entro i primi 10 anni dallintervento i bypass possono andare incontro a restenosi , con una probabilit che aumenta al passare del tempo , necessario un follow - up strumentale [ 1 ]  . langiografia coronarografica convenzionale ( ac ) considerata il gold standard diagnostico per la valutazione delle coronarie native e dei bypass , tuttavia , recentemente , alcuni studi hanno dimostrato il valore dellangio - tc multidetettore ( tcmd ) , metodica meno costosa e non - invasiva , che consente unanalisi accurata sia delle coronarie native ( specificatamente nei termini di un elevato valore predittivo negativo ) sia dello stato di perviet dei graft [ 29 ]  . 
la tcmd inoltre metodica utile nella osservazione di eventuali complicanze postoperatorie che diventano palesi clinicamente con dolore toracico tale da mimare unangina ricorrente da ostruzione del graft [ 10 ]  . nellambito delle diverse opzioni chirurgiche , i bypass pi comunemente utilizzati , associati ai risultati clinici migliori , sono quelli arteriosi con mammaria interna ( ami ) e quelli venosi con vena safena ( vs ) , mentre quelli con arteria radiale ( ar ) e gastroepiploica sono considerati condotti aortocoronarici semplici o compositi di seconda scelta [ 1113 ]  . 
il tasso di perviet a medio e lungo termine dei graft stato valutato solamente mediante studi angiografici in pazienti sintomatici con risultati , soprattutto per quanto riguarda i condotti con ar , spesso discordanti e non conclusivi [ 1418 ]  . nel sottogruppo di pazienti asintomatici , la metodica di scelta per la stratificazione dei rischi , la determinazione della prognosi e la stima della progressione di malattia , considerata la miocardioscintigrafia perfusionale [ 19 , 20 ]  . per quanto ci dato sapere non esistono in letteratura studi di follow - up a medio - lungo termine in pazienti asintomatici per la stima di perviet dei bypass con ar eseguiti con tecnica tcmd . 
obiettivo del nostro studio osservazionale perci quello di valutare , utilizzando langio - tcmd eseguita con apparecchiatura a 16 canali , lo stato di perviet a 3 anni dei bypass con ar in comparazione con quelli eseguiti con ami e sv . materiali e metodi popolazione di studio sono stati arruolati 51 pazienti , consecutivamente sottoposti , tra aprile e ottobre del 2005 , ad esame tcmd per valutare gli esiti di un intervento di triplice bypass aortocoronarico comprendente lar . 
thirty - five of the 51 ( 68.6% ) ra were free grafts ( aorta single anastomoses ) , 11 ( 21.5% ) were sequential grafts ( aorta multiple anastomoses ) and 5 ( 9.8% ) were composite grafts with the sv . 
of the 51 ra segments , 11 ( 21.5% ) were grafted onto the left anterior descending artery ( lad ) , 27 ( 52.9% ) onto the left circumflex coronary artery ( lcx ) , 6 ( 11.7% ) onto the right coronary artery ( rca ) and 7 ( 13.7% ) sequentially onto the lad - lcx territory . 
the study was approved by the local ethics committee , and informed consent was obtained from all patients . ( 51 ) in follow - up clinico - diagnostico precedentemente sottoposti ad ecg da stress . 
trentacinque / 51 segmenti ( 68 , 6% ) di bypass con ar erano condotti semplici ( aorta - aranastomosi coronarica singola ) , 11 / 51 ( 21 , 5% ) bypass di tipo sequenziale ( aortaar - anastomosi coronarica multipla ) , infine 5 ( 9 , 8% ) segmenti erano condotti compositi con origine da vs . 
intervallo di 324 mesi tra procedura chirurgica ed esame tcmd 36 ( range 5078 , et media 64 ) 15 ( range 5569 , et media 62 ) campione di studio sesso maschi femmine intevallo temporale tra procedura 324 mesi chirurgica ed esame tcmd segmenti esaminati numero numero totale dei segmenti graft venosi graft arteriosi lima segmenti con ra valore semplice ( aorta - anatomosi singola ) sequenziale ( aorta - nastomosi multiple ) composito ( sv - anastomosi singola ) distribuzione anastomosi distale sequenziale lad - lcx tipo di graft sv , vena safena ; lima , arteria mammaria sinistra ; ra , arteria radiale ; lad , discendente anteriore sinistro ; lcx , arteria coronarica circonflessa sinistra ; rca , arteria coronarica destra ct scanning protocol ct scans were obtained by using a 16 - mdct scanner ( philips brilliance 16 , philips medical systems , best , the netherlands )  . 
mean standard deviation ( sd ) interval between cardiac surgery and ct examination was 324 months . after a scout image with the patient in a supine position , scan coverage was selected from the carina to the apex of the heart in the cephalic to caudal direction to include the maximum extent of the grafts . 
in order to minimise heart rate , all patients received oral - blockers ( metoprolol 100 mg ) 23 days before the examination ; the heart rate range was 5415.3 bpafter a precontrast scan to assess the calcium burden and the course of target surgical clips , contrast - enhanced mdct was performed by administering 110 ml of low osmolar nonionic high - concentration contrast medium ( iomeprol , iomeron 400 , bracco , milan , italy ) via a dual - head anastomizzati nel territorio dellarteria discendente anteriore ( ada ) , 27 ( 52 , 9% ) in quello dellarteria circonflessa sinistra ( acs ) , 6 ( 11 , 7% ) in quello della coronaria destra , 7 ( 13 , 7% ) erano anastomizzati sequenzialmente su territorio dellada in sede prossimale e su quello della acs . 
lo studio stato approvato dal comitato etico locale previo ottenimento di un consenso informato da parte dei pazienti arruolati . protocollo di imaging la scansione stata eseguita con scanner tcmd a 16 canali ( philips brilliance 16 , philips medical systems , best , olanda ) , con un intervallo medio tra intervento chirurgico ed effettuazione dellesame di 324 mesi . 
a partire da una scout view del paziente in posizione supina , il volume di studio stato esteso in senso cranio - caudale a comprendere la massima estensione dei bypass . 
to achieve optimal contrast enhancement , the scan delay was determined by using the bolus - tracking technique after positioning a region of interest ( roi ) in the ascending aorta . 
all scans were reconstructed by using retrospective gating ( 37.5%50%62.5%75%87.5% of the r - r interval ) , with 1 - mm - thick images reconstructed every 0.5 m the cardiac phase with the least motion was chosen for further evaluation . image processing and analysis the data sets obtained were reviewed on a dedicated workstation ( mx view ; philips medical systems , best , the netherlands ) , and image interpretation was based on evaluation of axial source , multiplanar reconstructions ( mpr ) , maximum - intensity projections and volume - rendering images ( vr )  . 
advanced software tools [ arterial tree , curved multiplanar reformation along the vessel centre line , vascular two - dimensional ( 2d ) or three - dimensional ( 3d ) mapping , virtual and angioscopy ] were used in single cases . 
in the absence of clear and conclusive ct parameters to define cabg patency , we used the following three - point scale to assess the grade of single graft patency . 
concordance between readers 1 and 2 for the detection of occlusion or significant conduit stenosis by mdct was calculated by the cohen frequenza cardiaca , in tutti i pazienti si proceduto ad una premedicazione farmacologica con - bloccante ( 100 mg di metoprololo per os a partire da 23 giorni prima delleffettuazione dellesame ) : nella nostra coorte di pazienti la frequenza cardiaca media riportata stata di 5415 , 3 bpdopo unacquisizione diretta senza mezzo di contrasto , utile per quantificare lentit delle calcificazioni aterosclerotiche ed il decorso delle clips chirurgiche , si proceduto allo studio contrastografico mediante somministrazione endovenosa , con iniettore automatico a doppia siringa ( stellant , medrad , pittsburgh , usa ) e agocannula 1820 gauge posizionata in vena antecubitale di braccio , di 110 ml di mdc iodato non ionico ipo - osmolare ad alta concentrazione ( iomeprol 400 mgi / ml , iomeron , bracco , italia ) , alla velocit di 4 ml / s , seguito da un bolo di 40 ml di soluzione salina alla stessa velocit . 
per ottimizzare il contrast enhancement , lo scan delay stato selezionato in base alla tecnica del bolus tracking dopo posizionamento di una roi in aorta ascendente e scelta di una soglia di 150 uh predefinita come limite per istruire il paziente ad inspirare e trattenere lapnea . 
lacquisizione stata effettuata con i seguenti parametri : configurazione detettori 160 , 75 , spessore effettivo di strato 1 mm , indice di ricostruzione 0 , 5 mm , periodo di rotazione del tubo radiogeno 420 ms , pitch 0 , 20 , 3 , mas 500 , kvp 120 , fov 150200 mm con matrice 512512 . 
le immagini sono state ricostruite con spessore di 1 mm e indice di ricostruzione di 0 , 5 mm , gating ecg retrospettivo e finestre temporali variabili pari a 32 , 5%50%62 , 5% - 75% - 87 , 5% dellintervallo r - r : la successiva valutazione stata eseguita sulla ricostruzione giudicata migliore . analisi delle immagini il dataset di immagini assiali native stato inviato ad una stazione di lavoro dedicata ( mx view ; philips medical systems , best , olanda ) , dove si proceduto allutilizzazione di ricostruzioni multiplanari ( mpr ) , curve ( cmpr ) , proiezioni di massima intensit ( mip ) e volume rendering tridimensionale ( 3d - vr )  . 
coronary artery bypass graft ( cabg ) patency at 16 - detector - row computed tomography angiography ( cta ) : in this 60 - year - old man , three - dimensional ( 3d ) colour volume - rendered ( vr ) images ( a ) and corresponding curved multiplanar reconstruction ( cmpr ) ( b ) show perfect patency of the radial artery ( ra ) graft anastomosed to the obtuse marginal branch of the left circumflex coronary artery ( lcx ) artery ( white thick arrow ) and left internal mammary artery ( lima ) graft anastomosed via the diagonal branch to the left anterior descending ( lad ) coronary artery ( white thin arrow )  . 
in questuomo di 60 anni sottoposto ad esame tcmd a 16 canali , le immagini 3d volume rendering ( a ) e le corrispondenti ricostruzioni multiplanari curve ( b ) , dimostrano la perviet completa del bypass con arteria radiale anastomizzato distalmente sul ramo marginale ottuso della circonflessa ( freccia bianca spessa ) e di quello con mammaria sinistra anastomizzato in modo sequenziale su ramo diagonale e sulla discendente anteriore ( freccia bianca sottile )  . 
in questo caso esemplificativo la perviet di grado 0 ben dimostrabile in base allopacizzazione completa dei bypass senza evidenza di stenosi critiche nel corpo degli stessi o in corrispondenza dei siti anastomotici prossimale e distale . - value according to the formula = io - ie / 1 - ie , where io is the observed concordance and ie the expected concordance . results best image quality of all segments was obtained at a reconstruction interval of 62.5% to 75% of the cardiac cycle . 
the diagnostic quality of the images of all of the 101 arterial and 55 venous segments , all of the proximal anastomoses and 153 / 156 ( 98% ) distal anastomoses examined was sufficient to make them suitable for analysis . 
out of the 156 bypass graft segments examined , 131 ( 84.0% ) were classified as patent , 20 ( 12.8% ) as occluded and 5 ( 3.2% ) as significantly stenotic . 
among patent segments ( grade 0 ) , complete opacification with no critical stenosis was noted in 122 segments ( 78.2% ) , whereas diffuse narrowing was reported in 11 segments , which were all arterial . 
ai fini della valutazione statistica , in accordo a quanto riportato dal trial multicentrico raps ( radial artery patency study ) il diffuso restringimento del graft con ar , stato incluso in un giudizio di perviet di grado 1 [ 21 ]  . 
la concordanza interosservatore per la osservazione di stenosi del 70%99% e ostruzione del graft , stata calcolata utilizzando il statistico di cohen , in accordo alla formula = io - ie / 1 - ie dove il valore di io identifica la concordanza osservata e ie la concordanza attesa . risultati ai fini della visualizzazione dei segmenti lintervallo di ricostruzione r - r giudicato migliore per la valutazione degli stessi risultato essere quello compreso tra il 62 , 5% e il 75% del f . 
slab maximum intensity projection ( mip ) image ( a ) and corresponding volume - rendered ( vr ) image ( b ) in a 49 - year - old patient with radial artery ( ra ) bypass graft to left circumflex coronary artery ( lcx ) arterial territory demonstrate isolated surgical clips ( white thick arrow ) , appearing as bright structures without visible opacification between them at the expected site of the ra grathis finding is consistent with complete occlusion of the ra bypass granote the normal patency of the left internal mammary artery ( lima ) graft to left anterior descending ( lad ) artery and saphenous vein ( sv ) to the lcx artery . 
in questuomo di 49 anni con bypass con arteria radiale sul territorio della circonflessa , le immagini slab - mip ( a ) e le corrispondenti volume - rendering ( b ) , dimostrano la presenza delle sole clips chirurgiche ( freccia bianca spessa ) , senza visibile opacizzazione della colonna di mdc tra loro . 
la qualit diagnostica delle immagini stata giudicata idonea per la valutazione di perviet in tutti i segmenti arteriosi e venosi studiati ( rispettivamente 101 e 55 ) , in tutte le anastomosi prossimali e in 153 / 156 ( 98% ) anastomosi distali . 
su un totale di 156 segmenti esaminati , 131 ( 84 , 0% ) sono risultati pervi , 20 ( 12 , 8% ) ostruiti e 5 ( 3 , 2% ) significativamente stenotici . 
tra i segmenti giudicati pervi ( grado 0 ) , 122 ( 78 , 2% ) sono risultati completamente opacizzati in assenza di stenosi focali , mentre un restringimento diffuso del graft stato riportato in 11 segmenti , tutti arteriosi . in tabella 2 sono elencate le percentuali di ostruzione dei diversi tipi di segmenti visualizzati nellintera coorte di pazienti . 
dalla comparazione dei risultati ottenuti nei diversi gruppi , mediante applicazione del test di fischer , si evince come la percentuale di ostruzione riportata per i graft con ar sia significativamente maggiore ( p < 0 , 001 ) rispetto a quella riportata per i graft con ami e vs . 
il tasso di perviet migliore risultato essere quello per i bypass con ami , seguito da quello per i bypass con vs e infine da quello per i bypass con ar . 
la percentuale di perviet riportata per i segmenti di vs stata dell83 , 6% , infine per i segmenti di ar si sono riscontrati i valori di perviet minori , pari al 74 , 5% . nella classe di segmenti con ar un restringimento diffuso ( string sign ) era presente in 7 / 51 casi ( 13 , 7% ) ; al contrario tale aspetto semeiologico non stato riscontrato in nessun segmento confezionato con vs o ami . 
nella stessa classe una stenosi focale , in sede anastomotica distale , si evidenziata in 3 / 51 segmenti ( 5 , 9% ) ; il territorio di distribuzione dellanastomosi distale associato alla migliore percentuale di perviet risultato , sempre nel caso dei bypass con ar , quello della acs ( 79 , 4% ) seguito da quello della ada ( 72 , 7% ) e infine da quello della coronaria destra ( 50% )  . in tabella 3 sono riportate specificatamente le percentuali di ostruzione dei bypass con ar in base al sottotipo di condotto utilizzato : 20 , 0% nel caso dei bypass semplici ( aorta - radiale - anastomosi coronarica singola ) , 18 , 2% e 20 , 0% nel sottogruppo di bypass compositi ( aorta - safena - radialeanastomosi coronarica singola )  . 
stante il limitato valore numerico del campione di studio , lanalisi condotta con test di fischer non ha dimostrato alcuna correlazione tra il tipo di graft utilizzato ed il territorio di distribuzione dello stesso ( test non significativo : p > 0 , 5 )  . 
il valore del statistico per la diagnosi di perviet e stenosi significativa del dei graft arteriosi e venosi , risultato di 0 , 86 . discussione la coronarografia considerata , ad oggi , grazie allelevata risoluzione spaziale ed alla possibilit intrinseca di effettuare trattamenti di interventistica percutanea dopo la fase diagnostica , il gold standard per la valutazione dello stato di perviet dei bypass aortocoronarici . 
in the ra group , 3 / 51 ( 5.9% ) segments showed significant focal stenosis at the distal anastomosis ; the specific coronary artery receiving the di rivascolarizzazione chirurgica complessa , non sempre possibile , con tale metodica , studiare correttamente lanatomia dei condotti [ 23 ]  . 
tra le metodiche non invasive utili ai fini della valutazione dei pazienti dopo interventi di rivascolarizzazione miocadica eseguita per via chirurgica o per via percutanea , figurano i test da sforzo ( stress - test ) eseguiti mediante elettrocardiografia , miocardioscintigrafia perfusionale o ecocardiografia . 
the - value for interobserver variation in the detection of patency and significant stenoses of arterial and venous conduits was 0.86. discussion conventional coronary angiography , thanks to its high spatial resolution and the possibility of simultaneous interventional procedures , is still the gold standard for the evaluation of cabg patency . 
with the recent improvements in spatial and temporal resolution , resulting in decreased motion - related artefacts , ecg - synchronised mdct has become an emerging and useful technique for noninvasive cardiac imaging . 
initial studies with 4 - detector - row ct scanners yielded promising assessments of the native coronary arteries and graft patency , reporting 93% sensitivity and 97.8% specificity in the detection of graft occlusion and 80% sensitivity and 96% specificity in the detection of significant graft stenosis [ 24 ]  . 
sixteen - detector - row ct scanners , thanks to faster gantry rotation , decreased slice thickness , better spatial resolution , reduced respiratory and cardiac motion artefacts and nearly isotropic voxel size , reliably depict cabgs with higher diagnostic accuracy : sensitivity is 96% , specificity 95% , positive predictive value ( ppv ) 81% and npv 99% [ 4 ]  . potential advantages of mdct over conventional coronary angiography in the postoperative evaluation of patients treated with cabg surgery include the opportunity to perform a noninvasive study that does not require hospitalisation and the possibility of detecting ancillary findings ( a case of pulmonary neoplasm in our series ) and early and late exprincipali : 1 presenza dei sintomi ischemici , 2 tempo trascorso dallintervento [ 19 ]  . 
lintroduzione delle recenti apparecchiature tcmd con gating cardiaco , grazie al miglioramento della risoluzione spaziale e temporale , con la conseguente riduzione degli artefatti da movimento cardiaco e respiratorio , ha reso disponibile una metodica di imaging cardiaco non invasivo estremamente utile . 
i primi studi di valutazione delle coronarie native e dello stato di perviet dei graft , effettuati con scanner a 4 canali , hanno riportato valori di sensibilit e specificit elevati , rispettivamente del 93% e del 97 , 8% nella dimostrazione di ostruzione del graft , dell80% e del 96% nella dimostrazione di stenosi emodinamicamente significativa [ 24 ]  . 
tuttavia , con suddetta metodica , il numero dei segmenti valutabili ai fini diagnostici , pari al 62%67% , non risult essere particolarmente elevato [ 2 , 24 ]  . 
lo sviluppo della tecnologia a 16 canali , grazie alla maggiore rapidit di rotazione del sistema tubo radiogeno - detettori ed ai ridotti spessori di strato , fattori risultanti in un netto miglioramento della risoluzione spaziale , e grazie ancora alla riduzione degli artefatti da movimento respiratorio e cardiaco ed alla possibilit di sfruttare voxel quasi - isotropici , ha consentito lo studio dei bypass con elevata accuratezza diagnostica , con valori di sensibilit del 96% , specificit del 95% , valore predittivo positivo dell81% e valore predittivo negativo del 99% [ 4 ]  . 
 i vantaggi della tcmd a 16 canali , rispetto alla ac , nella valutazione dei pazienti post - cabg , sono correlati principalmente alla possibilit di eseguire uno studio non invasivo che non richiede lospedalizzazione del paziente , ed alla capacit della stessa di evidenziare eventuali patologie incidentali ( un caso di embolia polmonare nella nostra serie di studio ) o patologie extravascolari , ad esordio precoce o tardivo nel postoperatorio , che si presentano clinicamente con dolore toracico e che possono mimare unangina ricorrente . 
in questa classe di pazienti la diagnosi differenziale del dolore toracico include il versamento pleurico , il versamento pericardico , lembolia polmonare e linfezione delle suture sternali [ 10 ]  . inoltre la tcmd fornisce una pianificazione pre - operatoria particolarmente utile nei casi in cui si prospetti un re - intervento di rivascolarizzazione chirurgica , in relazione alla sua comprovata superiorit nei confronti della radiografia del torace e della ac , nel definire la corretta anatomia topografica dei graft e delle strutture mediastiniche adiacenti [ 10 , 25 ]  . nella nostra serie di studio le scansioni tc sono state acquisite con successo , senza significative complicanze per i pazienti e con buona tollerabilit dellesame da parte di questi . sono stati considerati qualitativamente valutabili ai fini diagnostici , tutti i segmenti arteriosi e tutti quelli venosi , ad eccezione di tre casi di mancata visibilit delle anastomosi distali . 
in base alla nostra esperienza , le ricostruzioni 3dvr di rendering tridimensionale , grazie alla capacit di visione panoramica , risultano particolarmente utili nella definizione della complessa anatomia dei graft e dovrebbero essere utilizzate come guida iniziale , insieme allintegrazione fornita dallo studio delle immagini assiali native , per facilitare la successiva analisi di perviet vasale , meglio eseguibile , sia per quanto riguarda il decorso del graft sia per la valutaf . 
in addition , mdct is very useful in preoperative planning before repeat cabg surgery because of its superiority over chest radiographs and conventional coronary angiography in defining the position of patent grafts and vital mediastinal structures [ 10 , 25 ]  . in our series , ct scans were acquired successfully in all patients without any complications , and the exam was well tolerated by all patients . 
based on our experience , 3d vr images , thanks to their panoramic view , are very useful in the depiction of complex graft anatomy and are used as an initial tool , together with 2d axial images , to facilitate subsequent vessel interpretation . 
by contrast , thin - slice maximum intensity projection and multiplanar reconstruction images provide the best visualisation of the graft and anastomosis sites during follow - up . regarding ra , all of the proximal and 48 / 51 ( 94.1% ) of distal anastomoses were suitable for analysis . 
in the series by khan et al . , no difference in the assessment of distal anastomoses was found between 4and 16 - detector - row ct technology [ 23 ]  . 
to maximise visualisation of distal anastomoses , we reduced motion artefacts by using recommended r - r reconstruction intervals at 62.5%75% to evaluate cabg grafted to lad or lcx arterial territory and at 50% of the cardiac cycle to visualise cabg grafted onto the rca artery [ 26 ]  . 
we also resorted to shifting the reconstruction intervals whenever necessary . because of their marked tendency to develop atherosclerosis and intimal hyperplasia , sv grafts have reduced longterm patency compared with lima grafts , with a reported 10 - year patency rate of about 60% [ 1 , 11 , 12 , 27 , 28 ]  . 
this observation has induced surgeons to prefer using other arteries as an alternative to lima arterial grafts for cabg . ra has become the most popular of these conduits , thanks to its particular anatomic features , which offer the advantages of a relatively large diameter and thick muscular walls that facilitate the construction of anastomoses and to the introduction of recent improvements in graft harvesting techniques and drug therapy to prevent spasm [ 29 , 30 ]  . 
whereas the early angiographic patency rates of ra exceed 90% , approaching those of lima [ 31 , 32 ] and higher than those of sv , the results of three recent large midto long - term studies were inconsistent [ 14 , 16 , 17 ]  . 
by contrast , zacharias et al . , in 190 symptomatic patients with zione dei siti anastomotici , mediante tecniche mip e mpr . riguardo i bypass con ar , stato possibile valutare tutte le anastomosi prossimali e 48 / 51 ( 94 , 1% ) delle anastomosi distali . 
la visualizzazione del sito anastomotico distale rappresenta il principale problema inerente lo studio dei graft , a causa degli artefatti da indurimento del fascio e delleffetto volume parziale determinati dalle clips limitrofe . 
a tal proposito , khan , nel proprio studio , non ha riscontrato differenze statisticamente significative tra scanner tcmd a 4 e 16 canali nella valutazione di perviet dellanastomosi distale [ 23 ]  . 
i dati del nostro studio sono in linea rispetto a quanto riportato da schlosser e dewey [ 4 , 5 ] , riguardo la valutabilit dellanastomosi distale , variabile nelle loro serie tra il 74% ed il 99% . 
nella nostra serie abbiamo cercato di implementare e massimizzare la visualizzazione dellanastomosi distale , minimizzando consensualmente gli artefatti da movimento , ricorrendo nella scelta della finestra temporale ottimale , allintervallo r - r 62 , 5%75% del ciclo cardiaco nel caso dei bypass anastomizzati distalmente su territorio della ada e della acs , ed allintervallo r - r 50% nel caso dei condotti anastomizzati su territorio della coronaria destra [ 26 ]  . 
 a causa della maggiore suscettibilit nello sviluppo della degenerazione aterosclerotica e delliperplasia intimale , i bypass con vs sono associati , rispetto a quelli con ami , a percentuali di perviet , nel lungo periodo , pi basse , intorno al 60% a 10 anni dallintervento [ 1 , 11 , 12 , 27 , 28 ]  . 
questa considerazione fisiopatologica ha indirizzato i chirurghi a cercare un condotto arterioso , utilizzabile come bypass , alternativo alla ami , e a tal proposito lar divenuto il condotto pi comunemente utilizzato , grazie alle sue intrinseche propriet anatomiche ( diametro e spessore parietale muscolare relativamente ben rappresentati , tali da facilitare la costruzione delle anastomosi ) , ai miglioramenti recenti nella tecnica di isolamento del vaso e alla introduzione di terapie farmacologiche ad hoc per prevenire lo spasmo arterioso [ 29 , 30 ]  . 
mentre studi angiografici hanno dimostrato che il tasso di perviet per i bypass con ar , nel breve termine , risulta superiore al 90% , valore comparabile rispetto a quello riportato per lami e superiore rispetto a quello riportato per la vs [ 31 , 32 ] , tre recenti studi angiografici hanno invece fornito dati discordanti riguardo la perviet dello stesso graft nel periodo medio - lungo [ 14 , 16 , 17 ]  . 
nella serie riportata da khot su 398 graft con ar , in un totale di 310 pazienti sintomatici in follow - up angiografico a 565511 giorni dallintervento , stata riscontrata per i graft con ar una percentuale di perviet del 51 , 3% , di ostruzione del 33 , 7% , di stenosi severa del 15 , 1% , valori peggiori rispetto a quelli riscontrati per i graft con ami e con vs [ 14 ]  . 
al contrario , dallanalisi di perviet effettuata da zacharias su 242 graft con ar , utilizzati come seconda scelta a confronto con vs rispetto a bypass dominante ami - ada , in 190 pazienti sintomatici ( follow - up a 668518 giorni dallintervento ) , si evinto un tasso di ostruzione del 31 , 8% , minore di quello riportato per vs , pari al 36 , 7 [ 16 ]  . 
on 177 ra grafts reported a 4 - year ra patency rate of 89% and a 5 - year svg patency rate of 95% [ 17 ]  . our results , with a 74.5% ra patency rate , are consistent with those reported by khot et al  . 
our patients were not matched for ra versus sv ; in fact in our cohort , ra was generally used as the third conduit of choice in cabg strategy ( after dominant lima and sv ) , with the primary objective of providing adequate global vascularisation in the perioperative period . 
considered the string sign as a sign of occlusion , diffusely narrowed grafts were considered to be patent in our study in accordance with the radial artery patency study group [ 21 ]  . 
despite the possible stenosis , this arterial conduit is still capable of responding to increased myocardial oxygen demand , and the corresponding string sign seems to be an active process of negative vascular remodelling related to an adaptive response to low flow demand rather than a fixed spasthe considerations about vascular graft remodelling are supported by the evidence that ischaemia is uncommon in myocardial territories supplied by grafts with angiographic string sign and sometimes even in regions supplied by occluded arterial ra grafts , thanks to the establishment of collateral pathways . 
this consideration explains the absence of signs or symptoms of ischaemia in the vast majority of our patients with occluded ra [ 34 ]  . a limitation of our study is the lack of validation of cabg patency with the gold standard conventional coronary angiography . 
however , considering the relatively large vessel calibre , the relative independence of grafts from heart rate compared with native coronary arteries and the high diagnostic quality of state - of - art mdct scanners , we thought that correlation with this technique was not necessary . 
thus , our study focused on the reliability of 16 - mdct in evaluating midterm status of ra used as cabg . viet a 5 anni per graft con vs pari al 95% [ 17 ]  . i risultati del nostro studio , con un tasso di perviet del 74 , 5% , sono in linea rispetto a quanto riportato da khot e tatoulis con la precisazione che nella nostra coorte di pazienti i graft con ar sono stati utilizzati , non a confronto diretto con i graft con vs , ma , generalmente , come condotti di terza scelta dopo posizionamento di due graft dominanti , il primo con ami , il secondo con vs , con lo scopo di fornire un apporto di vascolarizzazione supplementare rispetto ai due graft dominanti nel periodo peri - operatorio . 
in base alla nostra esperienza il principale segno diretto di perviet vasale dei graft con ar rappresentato dalla dimostrazione della colonna di mdc centrata tra le clips chirurgiche , presenti sui due lati . 
al contrario la sola visibilit delle clips metalliche senza evidenziazione della colonna centrale di mdc stato considerato segno evidente di ostruzione completa del graft . nonostante nei propri studi zacharias e tatoulis abbiano considerato il restringimento diffuso del graft ( string - sign ) come un segno di ostruzione , nel nostro lavoro tale reperto , in accordo a quanto espresso dal radial artery patency study group [ 21 ] , stato considerato come segno di perviet . 
infatti i graft con ar si evidenziano spesso con laspetto di restringimento diffuso in relazione alla tendenza allo spasmo dovuta allelevato spessore parietale e alla vasoreattivit intrinseca del vaso nativo [ 33 ]  . 
nonostante la presunta stenosi il condotto arterioso risulta alloccorrenza in grado rifornire con un congruo apporto ematico le diverse richieste perfusionali del miocardio ; in tal senso lo string - sign , piuttosto che uno spasmo fisso , dovrebbe essere considerato come un processo adattativo di rimodellamento vascolare negativo in risposta alle esigenze di flusso periferico . 
le considerazioni sul rimodellamento vascolare sono supportate dallevidenza che levento ischemico risulta piuttosto raro nei territori di miocardio riforniti da graft con restringimento angiografico diffuso e , talvolta , grazie allinstaurarsi di circoli collaterali , anche da graft con ar completamente ostruiti . 
in tal modo pu essere spiegata , a nostro parere , la mancanza di segni e sintomi di ischemia nella maggior parte dei nostri pazienti con graft con ar ostruiti [ 34 ]  . la principale limitazione del nostro studio stata la mancanza di validazione dei dati di perviet dei graft ottenuti con tcmd con il gold standard diagnostico , la ac . 
tuttavia , considerato il calibro vasale relativamente ampio , la relativa indipendenza dei bypass dalla frequenza cardiaca rispetto alle coronarie native , lelevata qualit diagnostica degli scanner allo stato dellarte , abbiamo ritenuto non strettamente necessaria tale correlazione . 
 conclusion conclusione in conclusion , our study shows that , by enabling accurate analysis of cabg status , 16 - mdct scanners are useful noninvasive diagnostic tools for clinical routine midterm followup of patients who have undergone cabg involving the use of ra . 
krestin2 1dipartimento di radiologia e dipartimento cuore , azienda ospedaliero - universitaria di parma , italy 2dipartimento di radiologia e cardiologia , erasmus medical center , rotterdam , the netherlands 3dibimel , sezione di scienze radiologiche , universit di palermo , italy correspondence to : f . 
cademartiri , viale rustici 2 , i - 43100 parma , italy , tel . : + 39 - 0521 - 961833 , e - mail : filippocademartiri@hotmail.com received : 20 february 2006 / accepted : 13 june 2006 / published online : 11 june 2007 abstract purpose . 
ninety - five patients ( 72 men and 23 women , mean age 588 years ) with previous percutaneous coronary intervention with stenting and suspected restenosis underwent 64 - ct ( sensation 64 , siemens )  . 
the scan parameters were : slices 322 , individual detector width 0.6 mm , rotation time 0.33 s , feed 3.84 mm / rotation , 120 kv , 900 mas . 
after the intravenous administration of iodinated contrast material ( iomeprol 400 mgi / ml , iomeron , bracco ) and a bolus chaser ( 40 ml of saline ) , the scan was completed in < 12 s . 
all coronary segments with a stent were assessed on 64 - ct by two observers in consensus and judged as : patent , with intimal hyperplasia ( lumen reduction of < 50% ) , with in - stent restenosis ( 50% ) , or with in - stent occlusion ( 100% )  . 
we found that 64 - ct has a high diagnostic accuracy for the detection of in - stent restenosis in a selected patient population . key words coronary angiography multislice computed tomography 64 - slice ct coronary stents riassunto obiettivo . 
in 95 pazienti ( 72 maschi e 23 femmine , et media 588 anni ) precedentemente sottoposti a posizionamento di stent intra - coronarico e con sospetta re - stenosi , stata effettuata una 64 - tc ( sensation 64 , siemens )  . 
i parametri di scansione erano : strati 322 ( sovracampionamento sullasse z mediante flying focal spot ) , collimazione individuale 0 , 6 mm , tempo di rotazione 0 , 33 s , avanzamento 3 , 84 mm / rot , kv 120 , ma 900 . 
dopo la somministrazione di mezzo di contrasto iodato ( iomeprolo 400 mgi / ml , iomeron ; 100 ml a 5 ml / s ) e bolus chaser ( 40 ml di soluzione fisiologica a 5 ml / s ) , la scansione stata completata in meno di 12 s . 
tutti i segmenti coronarici con uno stent sono stati valutati da due osservatori in consenso e giudicati come segue : pervi , con iperplasia intimale intra - stent ( iis ; riduzione del lume < 50% ) , con re - stenosi intra - stent ( ris ; 50% ) , o con occlusione intra - stent ( ois ; 100% )  . 
nei rimanenti 91 pazienti sono stati valutati 102 stent ( 31 in arteria coronaria destra ; 10 del tronco comune sinistro ; 54 della arteria discendente anteriore ; 7 nellarteria circonflessa )  . 
la 64 - tc fornisce una accuratezza diagnostica elevata nella rilevazione della re - stenosi intrastent in una popolazione selezionata di pazienti . parole chiave coronarografia tomografia computerizzata multistrato tc 64 strati stent coronarici introduction introduzione f . 
these scanners have proved on average more effective at assessing stenosis in native coronary arteries [ 1114 ]  . the placement of coronary artery stents has significantly reduced the development of restenosis when compared with angioplasty [ 15 , 16 ]  . 
traditionally , follow - up of stents has been performed with coronarography . studies of the diagnostic accuracy of msct using 4and 16 - slice scanners in the evaluation of restenosis of coronary arteries have demonstrated progressive improvement , although they have been hindered by significant limitations such as artefacts and small case series [ 2129 ]  . 
we report on the diagnostic accuracy of a 64 - slice scanner in the identification of in - stent restenosis of coronary artery stents . materials and methods population ninety - five patients ( 72 men and 23 women , mean age 588 years ) were enrolled in the study , all with suspected obstructive coronary artery disease who had previously undergone coronary artery angioplasty for the purposes of percutaneous stent deployment or simply for follow - up in the context of other studies being undertaken at our institute . 
exclusion criteria for the study were more than one stent per vessel in the coronary artery tree , a single stent with a diameter of less than 2.5 mm , aortocoronary bypass , atrial fibrillation , known allergy to iodinated contrast material and kidney failure ( serum creatinine > 120 mmol / l )  . 
patients with a heart rate greater than 65 beats per minute ( bpm ) received an oral dose of 100 mg metoprolol tartrate 4560 min prior to the examination . as many of the patients had been treated in peripheral hospitals , we were only able to identify the exact type of stent deployed in a limited number of patients . 
the mean time interval between 64 - ct examination and conventional coronarography ( cc ) la tomografia computerizzata multistrato ( tcms ) ha avuto nei recenti 5 anni notevoli miglioramenti tecnologici che hanno esitato in una serie di nuove applicazioni , prima fra tutte quella cardiaca e coronarica [ 13 ]  . 
questa tecnologia si dimostrata mediamente pi efficace nella valutazione delle stenosi nelle coronarie native [ 1114 ]  . il posizionamento di stent coronarici ha significativamente ridotto levenienza di re - stenosi se comparata con langioplastica [ 15 , 16 ]  . 
tradizionalmente , il follow - up degli stent ha avuto bisogno della coronarografia . le casistiche di accuratezza diagnostica per restenosi mediante tcms sugli stent coronarici con tecnologia a 4 e 16 strati hanno descritto risultati progressivamente migliorati ma sempre con significative limitazioni , artefatti e casistiche esigue [ 2129 ]  . 
riportiamo laccuratezza diagnostica di uno scanner a 64 strati nella rilevazione delle restenosi intra - stent degli stent coronarici . materiali e metodi popolazione novantacinque pazienti ( 72 maschi e 23 femmine , et media 588 anni ) con sospetta malattia coronarica ostruttiva precedentemente sottoposti ad angioplastica coronarica con posizionamento di stent per via percutanea o per semplice follow - up nel contesto di altri studi in corso nella nostra istituzione , sono stati arruolati per lo studio . 
i pazienti con pi di uno stent per vaso nellalbero coronarico , con singoli stent di diametro inferiore a 2 , 5 mm , con bypass aorto - coronarico , con fibrillazione atriale , con allergia nota al mezzo di contrasto iodato ed insufficienza renale ( creatinina sierica > 120 mmol / l ) sono stati esclusi dallo studio . 
ai pazienti con frequenza cardiaca superiore a 65 battiti per minuto ( bpm ) , 4560 minuti prima della scansione , sono stati somministrati per os 100 mg di metoprolo - tartrato . essendo molti dei pazienti seguiti in ospedali periferici , non stato possibile risalire con precisione al tipo di stent impiantato se non in un ridotto numero di pazienti . 
the study was approved by the hospital ethics committee , and all patients provided written informed consent . scan and image reconstruction the angiography scan was performed with a 64 - slice spiral ct scanner ( sensation 64 cardiac , siemens , germany ) equipped with a 32 - row detector and periodic motion of the focal spot ( z - flying focal spot ) capable of achieving an effective z - axis spatial resolution of 0.4 m contrast enhancement was achieved through the antecubital intravenous administration of 100 ml of nonionic iodinated contrast material ( iomeprol 400 mgl / ml , iomeron , bracco , italy ) at an injection rate of 5 ml / s followed by 40 ml of saline solution at the same rate . 
the region of interest ( roi ) was placed in the ascending aorta with a threshold of + 100 hu [ 31 ]  . the scan and reconstruction parameters were number of detectors / collimation 322 / 0.6 mm , gantry rotation time 330 ms , table feed / rotation 3.84 mm ( pitch 0.2 ) , 120 kvp , 900950 mas , field of view 150 mm , effective slice thickness 0.75 mm , reconstruction increment 0.4 mm , soft - tissue convolution kernel dedicated for stents ( b30fb46f )  . the images were reconstructed with a predetermined offset based on the patients electrocardiogram ( ecg ) signal [ 1 ]  . 
at the operators discretion , additional reconstructions in the end - systolic phase were performed ( + 225 / + 275 / + 325 ms from the r wave )  . image assessment the images were sent to a dedicated workstation ( leonardo , siemens , germany ) where the available reconstructions were assessed using all of the conventional postprocessing techniques [ axial images , multiplanar reconstructions ( mpr ) , curved mpr ( cmpr ) , maximum intensity projections ( mip ) and volume rendering ( vr ) ]  . 
 i parametri di scansione e ricostruzione sono : numero di detettori / collimazione 322 / 0 , 6 mm , tempo di rotazione del tubo radiogeno 330 ms , avanzamento / rotazione 3 , 84 mm ( pitch 0 , 2 ) , kvp 120 , ma 900950 , campo di vista 150 mm , spessore effettivo dello strato 0 , 75 mm , incremento di ricostruzione 0 , 4 mm , filtro di convoluzione per tessuti molli e dedicato per stent ( b30fb46f )  . le immagini sono state ricostruite con modalit retrospettiva basata sul segnale ecg [ 1 ]  . 
le finestre temporali di ricostruzione utilizzate sono state 300 ms , 350 ms , e 400 ms dalla successiva onda r ( tecnica del ritardo assoluto inverso ) [ 1 ]  . 
quando ritenuto opportuno dalloperatore sono state eseguite ulteriori ricostruzioni in fase tele - sistolica ( + 225 / + 275 / + 325 ms dallonda r )  . analisi delle immagini le immagini sono state inviate ad una stazione di lavoro dedicata ( leonardo , siemens , germania )  . 
le ricostruzioni disponibili sono state valutate con tutte le convenzionali tecniche di post processing ( immagini assiali , ricostruzioni multiplanari - mpr , riformattazione centroluminale - cmpr , proiezione di massima intensit - mip , e volume rendering - vr )  . due osservatori in consenso hanno valutato tutti gli stent dei pazienti . 
1a - d example of a patent stent with 64 - slice computed tomography ( ct ) coronary angiography in a patient who underwent percutaneous coronary intervention with stenting of the left circumflex . 
conventional coronary angiography shows a nudge of the vessel profile proximal to the stent ( a ) ( arrowhead )  . the stent appears patent with 64 - ct ( b , d )  . 
in particolare la sezione ortogonale allasse del vaso ( c ) consente di apprezzare la presenza di contrasto allinterno del lume . conventional diagnostic coronarography coronarografia convenzionale diagnostica cc was performed with a standard technique in a time interval ranging from 1 week prior to and 1 week after msct examination . 
a value 50% indicated significant stenosis . la coronarografia convenzionale ( cc ) stata effettuata con tecnica standard in un range tra una settimana prima ed una settimana dopo lac - tcms . 
un valore 50% indica una stenosi significativa . statistical analysis analisi statistica dei dati the diagnostic accuracy of 64 - ct in identifying significant restenoses ( 50% reduction of the diameter of the lumen ) was assessed using quantitative cc as a reference standard . laccuratezza diagnostica della 64 - tc per la rilevazione di re - stenosi significative ( 50% di riduzione del diametro luminale ) stata valutata utilizzando la cc quantitativa come stanf . 
2a - d example of in - stent restenosis with 64 - slice computed tomography ( ct ) coronary angiography in patient who underwent percutaneous coronary angioplasty and stenting on the left anterior descending . 
mediante 64 - tc si osserva una area ipodensa parzialmente eccentrica che inizia sul margine prossimale dello stent e si estende al suo interno determinando una restenosi significativa ( b , d )  . 
i risultati sono stati suddivisi per vaso ( arteria coronaria destra acd ; tronco comune sinistro tcs ; arteria discendente anteriore ada ; arteria circonflessa acx ) e per segmenti prossimali ( 1 , 5 , 6 , 11 ) e distali ( 24 , 710 , 1215 ) secondo la classificazione a 15 segmenti dellamerican heart association [ 32 ]  . 
tutti i valori di accuratezza diagnostica sono stati espressi mediante intervalli di confidenza del 95% . four of the 95 patients initially enrolled were excluded , two because their heart rate exceeded 65 bpm during the scan dei 95 pazienti inizialmente reclutati , 4 sono stati esclusi . due a causa di valori di frequenza cardiaca durante la scanrisultati results table 1 diagnostic accuracy of msct - ca for in - stent restenosis 50% f . 
msct - ca , multislice computed tomography coronary angiography ; no . , number of stents ; ppv , positive predictive value ; npv , negative predictive value ; rca , right coronary artery ; lm , left main coronary artery ; lad , left anterior descending coronary artery ; cx , circumflex coronary artery ; na , non - assessable . tabella 1 accuratezza diagnostica della angiografia coronarica ac - tcms per le restenosi intra - stent 50% . vessels vasi tutti n ( % ) 31 ( 30 ) 10 ( 10 ) 54 ( 53 ) 7 ( 7 ) n ( % ) 31 ( 30 ) 10 ( 10 ) 54 ( 53 ) 7 ( 7 ) sensibilit specificit 100 ( 47100 ) 85 , 7 ( 4299 ) 100 ( 15100 ) 92 , 85 ( 6699 ) 100 ( 86100 ) 30 ( 665 ) 93 , 9 ( 8398 ) 100 ( 29100 ) 88 , 63 ( 8094 ) 100 ( 47100 ) 66 , 66 ( 2992 ) 100 ( 15100 ) 56 , 52 ( 3476 ) 100 ( 86100 ) 100 ( 29100 ) 97 , 9 ( 8899 ) 100 ( 29100 ) 98 , 73 ( 9399 ) i valori di sensibilit , specificit , valore predittivo positivo e negativo sono espressi in percentuale , mentre quelli riportati tra parentesi si riferiscono ai rispettivi intervalli di confidenza al 95% . 
the study population was therefore reduced to 91 patients ( 70 men and 21 women , mean age 588 years ) , with a total of 102 stents ( mean 1.1 stent per patient )  . sixty - one patients ( 67% ) were already undergoing treatment with beta - blockers , and 33 ( 36% ) received an additional dose in accordance with the above - mentioned protocol . 
the sensitivity for the identification of restenosis by vessel was 100% for the rca , 86% for the lad and 100% for the cx . sione , superiori ai 65 bpm ed altri due per aver mostrato una qualit delle immagini a livello dello stent non compatibile con una valutazione diagnostica ( ad esempio , artefatti da movimento ed eccessivo rumore delle immagini )  . 
la sede degli stent era nel 30% ( n = 31 ) dei casi la coronaria destra , nel 10% ( n = 10 ) dei casi il tronco comune , nel 53% ( n = 54 ) dei casi la discendente anteriore , e nel 7% ( n = 7 ) la coronaria circonflessa . 
la sensibilit , specificit , valore predittivo positivo e negativo per tutti gli stent nella rilevazione delle ris + ois sono risultate 92 , 9% , 88 , 6% , 56 , 5% , e 98 , 7% , rispettivamente . 
la sensibilit per la rilevazione di restenosi per vaso risultata del 100% per lacd e del 86% per la ada e 100% per la cx . discussion studies on electron beam tomography ( ebt ) report a sensitivity of 78% and a specificity of 98% for high - grade in - stent restenosis [ 33 ]  . 
the method adopted , however , was completely different , as the study involved a multisection dynamic assessment , which measured the temporal variation of la letteratura riguardante studi con electron beam ct ( ebt ) riferisce una sensibilit del 78% ed una specificit del 98% per le stenosi intrastent di alto grado [ 33 ]  . 
the same study , building on the previous dynamic study [ 33 ] , used the degree of enhancement of the native vessel distal to the stent as a criterion for vessel patency [ 35 ]  . 
in our study , however , this proved to be a reliable measure only in one of four cases of occlusion ; in the remaining three cases , distal enhancement was normal due to collateral circulation . as with calcifications , stents are characterised by high density , which at ct leads to an enlargement of the image known as blooming [ 36 ]  . 
in small vessels such as the coronary arteries , the negative effect of the artefact is increased by the fact that its size is in the same order as the diameter of the vessel [ 36 ]  . the use of scanners with hardware and software solutions capable of improving spatial resolution promises to make the visualisation of the in - stent lumen feasible [ 9 , 37 , 38 ]  . 
the incidence of restenosis has in fact been considerably reduced , particularly following the introduction of drug - eluting stents , and this suggests the need for enrolling large patient populations to be able to achieve values of diagnostic accuracy with a sufficient level of statistical significance . 
this would , in fact , require a sensitivity of 90% or greater and an npv of 98% . the data obtained in this study on a group of 91 patients with 102 stents using a 64 - slice scanner reveal a higher diagnostic accuracy , with a sensitivity of 92% and specificity of 89% . 
the improvement with respect to earlier studies can be attributed to the technologically superior equipment used , although such direct comparisons between different devices should be made with great care given the considerable variations in the characteristics of the populations and the scan protocols . 
undoubtedly the reduction in the voxel size ( 0.4 mm3 ) linked to the z - flying focal spot plays an important role , and associated with this is the reduction in residual motion artefacts related to the improved temporal resolution ( 185 ms effective scan time ) and the use of dedicated convolution kernels ( medium - sharp b46f )  . the main limitation of the present study essentially trattandosi di una valutazione dinamica multi - sezione che misurava la variazione temporale dellattenuazione nel vaso nativo distalmente allo stent [ 33 ]  . 
questa metodologia era la sola utilizzabile stante la incapacit della ebt di visualizzare il lume del vaso allinterno dello stent [ 33 , 34 ]  . uno studio basato su msct a 4 canali ha dimostrato che impossibile in tutti gli stent visualizzare il lume , indipendentemente dal tipo di stent e dal suo diametro [ 35 ]  . 
lo stesso studio descrive come criterio per la perviet , rifacendosi allo studio dinamico precedente [ 33 ] , il grado di opacizzazione del vaso nativo distalmente allo stent [ 35 ]  . 
nella nostra esperienza , questo criterio stato affidabile in uno solo dei 4 casi di occlusione ; nei rimanenti tre casi stata osservato un grado di opacizzazione normale dovuto a circolo collaterale . gli stent cos come le calcificazioni sono caratterizzati da una densit elevata che alla tc determina un ingrandimento dellimmagine che viene definito blooming [ 36 ]  . 
questo artefatto multifattoriale deriva da effetto volume parziale , indurimento del fascio ed interpolazione e pu essere attenuato utilizzando dei filtri di convoluzione a pi alta frequenza ( fig . 3 ) [ 36 ]  . 
nei vasi di piccole dimensioni , come le coronarie , leffetto negativo dellartefatto aumentato dal fatto che la sua dimensione dello stesso ordine di grandezza del diametro del vaso [ 36 ]  . limpiego di apparecchiature con soluzioni hardware e software in grado di migliorare la risoluzione spaziale promette di rendere fattibile la visualizzazione del lume intrastent [ 9 , 37 , 38 ]  . 
infatti , soprattutto dallintroduzione degli stent medicati , lincidenza della restenosi si ridotta sensibilmente e questo presuppone larruolamento di ampie popolazioni di pazienti per raggiungere valori di accuratezza diagnostica che abbiano una sufficiente potenza statistica . 
per poterlo fare infatti sarebbe necessario avere una sensibilit del 90% o superiore ed un valore predittivo negativo superiore al 98% . la casistica presentata in questo lavoro , ottenuta su apparecchiatura a 64 strati , in un gruppo di 91 pazienti con 102 stent mostra una accuratezza diagnostica superiore con una sensibilit del 92% ed una specificit dell89% . 
il migliore risultato rispetto agli studi precedenti si spiega con la tecnologia superiore utilizzata , tuttavia ancora arduo fare paragoni diretti tra tecnologie considerando le considerevoli variazioni nelle caratteristiche delle popolazioni e nei protocolli di scansione . 
sicuramente la riduzione delle dimensioni del voxel ( 0 , 4 mm3 ) legata al sovra - campionamento sullasse z gioca un ruolo importante a questo si associano , la riduzione degli artefatti da movimento residuo legati f . 
the image at the top shows the traditional filter ( mediumsmooth ; b30f ) and the image at the bottom shows a higher dedicated filter for stents ( medium - sharp ; b46f )  . 
in alto il filtro tradizionale ( medium - smooth ; b30f ) ed in basso un filtro pi elevato ma dedicato per gli stent ( medium - sharp ; b46f )  . 
si nota come il filtro determini un aumentato rumore di fondo dellimmagine e come produca anche un incremento della attenuazione del lume coronarico allinterno dello stent . regards the inclusion criteria of the patients , which restricted the analysis to patients with a regular heart rate < 65 bp a further limitation connected with the technique is the relatively high level of exposure to ionising radiation , estimated at 15.2 msv for men and 21.4 msv for women [ 4043 ]  . although the introduction of drug - eluting stents has proved favourable for patients , it has made assessment of the diagnostic accuracy of msct in the follow - up of stents somewhat difficult . 
these new - generation stents in fact have displayed lower rates of restenosis at 6 months and lower prevalence of complete occlusions compared with earlier studies [ 1719 , 4451 ]  . future developments the recent introduction of new technologies characterised by an increased number of slices per rotation or multiple detectors will , over time , improve the visualisation of coronary artery stents but only if there is a concomitant increase in spatial resolution . 
a device capable of scanning 128 slices per rotation with an individual detector width similar to that of previous technology will only be able to increase the speed of completing the examination . 
a detector with a lower individual width will be able to achieve greater spatial resolution but only at the cost of the higher radiation dose required to obtain image quality in line with the other parameters . one simple and desirable solution lies in the development of stent materials that are less radiopaque or more radiotransparent . 
another possibility currently being tested is the use of absorbable stents made from biological materials that alla migliore risoluzione temporale ( 185 ms effettivi ) ed allutilizzo di filtri dedicati ( medium - sharp b46f )  . la limitazione principale del presente studio legata essenzialmente ai criteri di inclusione dei pazienti che hanno ristretto lanalisi ai pazienti con frequenza cardiaca regolare e < 65 bp una ulteriore limitazione , correlabile alla metodica , legata alla elevata esposizione a radiazioni ionizzanti stimata in 15 , 2 e 21 , 4 msv per maschio e femmina , rispettivamente [ 4043 ]  . lintroduzione di stent coronarici medicati ( drug - eluting stents o des ) , sebbene favorevole ai pazienti , rende difficoltosa la valutazione dellaccuratezza diagnostica della tcms nel follow - up degli stent . 
infatti , questi stent di nuova generazione hanno dimostrato delle ridotte percentuali di re - stenosi a 6 mesi ed una ridotta prevalenza di occlusioni complete se comparate con le precedenti casistiche [ 1719 , 4451 ]  . sviluppi futuri la recente introduzione di nuove tecnologie caratterizzate da un numero aumentato di strati per rotazione o di multipli sistemi tubo - detettore potranno migliorare eventualmente la visualizzazione degli stent coronarici , ma verosimilmente solo se porteranno ad un concomitante incremento della risoluzione spaziale . 
un dettetore di ampiezza individuale inferiore porter invece una maggiore risoluzione spaziale a prezzo di una dose di radiazioni superiore necessaria per ottenere una qualit delle immagini a parit degli altri parametri . una soluzione semplice ed auspicabile potrebbe essere legata allo sviluppo di materiale meno radio - opachi o ancof . 
the table shows how distal runoff was initially used for assessing patency , but with technological advances , the authors began primarily to use criteria of direct visualisation studies no . 
the projectile effect is when an object is attracted by the magnet with the risk , as reported in literature , of hitting the patient , operators and / or the instrument . objects which typically can undergo this effect are oxygen and helium cylinders , iv stands , cleaning trolleys , chairs , lamp holders , scissors , forceps , clampers , traction weights , monitoring instruments , and especially metallic splinters within the patient . twisting ( torsion ) typically occurs with cerebral vascular clamps and cochlear implants . 
burns can be caused when electrically conductive material is introduced within the magnet , for example , ecg electrodes , monitoring cables and coils which are in contact with the patients skin , as well as tattoos and eye - liners that contain iron - oxides . 
the risk for patients can be reduced by specific educational programs within individual radiology departments which include other specializations and external referring physicians with the aim of developing a standardized safety protocol . key words mr safety foreing body pacemaker pregnancy riassunto la presenza di un campo magnetico statico ( bo ) allinterno della sala rm , di un campo di radiofrequenza ( rf ) , di gradienti dinamici variabili nel tempo e di forte rumore durante lesame comportano il possibile verificarsi di eventi con implicazioni per la sicurezza del paziente . 
oggetti che tipicamente possono subire questo effetto sono : bombole di ossigeno , piantane per le fleboclisi , bombole di elio , carrelli delle pulizie , sedie , fissatori di lampadine , forbici e clamper , pesi da trazione , pulsossimetri . 
ustioni : in genere sono causate dallintroduzione di materiale elettrico conduttivo allinterno del magnete come elettrodi ecg , cavi del pulsossimetro , bobine in contatto con la superficie cutanea del paziente , ma anche tatuaggi , eye - liner che contengono ossido di ferro . 
artefatti : si possono verificare artefatti dovuti a emissione di rf da parte di dispositivi , che viene considerata rumore dalla bobina di ricezione , oppure vuoto di segnale da componente metallica , che pu mascherare o simulare una patologia . 
in 1992 , 100 , 000 , 000 mr examinations were performed in the usa alone ; today , given the increase in the number of available tomographs and routine clinical applications , it would be impossible to calculate how many are performed throughout the world . the number of serious accidents involving patients that have occurred since the introduction of mr for clinical purposes in 1984 are fortunately few , at least in part because of the growing attention being paid to safety issues [ 1 ]  . 
long - term exposure , such as that which may be experienced by an mr operator or a patient with a chronic disease requiring serial investigations over time , is still a subject of scientific debate insofar as in vitro studies have demonstrated potentially harmful effects on tissues that are apparently below the threshold of clinical significance [ 2 ]  . 
 literature concerning patient safety mainly concentrates on the two scenarios that largely reflect serious accidents that have happened in the past : interactions between the mf and ferromagnetic materials , and the malfunctioning of cardiac pacemakers ( pm )  . 
in specific terms , the presence inside the mr room of a static mf ( b0 ) , a field of radiofrequency ( rf ) , dynamic gradients varying over time and the loud noise occurring during the examination create an environment in which events may occur that threaten patient safety . 
here we review the different types of dangerous interactions in general before going into more detail about some of the situations that more frequently involve radiologists in the everyday management of patients who have to undergo mr imaging . 
we conclude with the current guidelines for performing an mr examination during pregnancy and lactation . la risonanza magnetica ( rm ) una metodica sempre pi diffusa sul territorio e sempre pi richiesta dal clinico o dal chirurgo , anche in virt della recente affermazione del body e cardiac imaging . 
nel 1992 , solo negli usa , sono stati eseguiti 100000000 di esami rm ; oggi , con lincremento del numero di tomografi disponibili e delle applicazioni entrate nella routine clinica , nel mondo vengono eseguiti un numero di esami rm difficilmente calcolabile . dallintroduzione della rm per uso clinico , nel 1984 , ad oggi , gli incidenti gravi a danno del paziente si contano per fortuna sulle dita delle mani , anche grazie ad una sempre maggiore sensibilit al tema della sicurezza [ 1 ]  . 
lesposizione a breve termine al campo magnetico di tutte le intensit di utilizzo clinico ( da 0 , 2 a 3 , 0 tesla ) e anche fino a 8 tesla ( a scopo di ricerca ) , stato approvata dalla fda americana come non dannosa per lorganismo : questa situazione - tipo quanto si verifica per il paziente in generale . 
lesposizione a lungo termine , quale potrebbe essere quella di un operatore di rm interventistica , o di un paziente con malattia cronica che esegue controlli seriati nel tempo , ancora sotto osservazione scientifica in quanto studi in vitro hanno dimostrato effetti potenzialmente dannosi per i tessuti , apparentemente sotto una soglia di significativit clinica [ 2 ]  . lattenzione sulla sicurezza del paziente si concentra in letteratura maggiormente su due tipologie di situazioni , anche sulla base degli incidenti gravi riportati dalla letteratura scientifica : linterazione del campo magnetico con materiale ferromagnetico e il malfunzionamento di pacemaker cardiaci . nello specifico , la presenza di un campo magnetico statico ( bo ) allinterno della sala rm , di un campo di radiofrequenza ( rf ) , di gradienti dinamici variabili nel tempo e di forte rumore durante lesame , creano una situazione ambientale che pu comportare il possibile verificarsi di eventi con implicazioni per la sicurezza del paziente . 
passeremo in rassegna in generale le diverse tipologie di interazioni pericolose per entrare nello specifico di alcune situazioni che pi frequentemente interessano tutti i giorni il radiologo nella gestione del paziente che deve sottoporsi ad indagine di risonanza magnetica . 
depending on the type of magnet and the intensity of the generated field , mf lines extend for a certain distance into the environment ( the fringe field ) and , to varying extents , attract objects to the centre of the magnet . 
an object in zone 2 is not only subject to a rotational interaction but also to a translational force directed towards the centre of the magnet that ceases only when it enters zone 1 . 
ferromagnetic objects near a static mf can become projectiles when they are attracted to the magnet as a result of : the strength of the mf the magnetic susceptibility of the object the mass of the object the distance of the object from the magnet the orientation of the object in relation to the mf . 
 the following is a modified list of a usa register listing the objects attracted inside the magnet during 2001 [ health devices alerts ( hda ) ] as shown in table 1 . in addition to damaging the magnet , the direct consequence of a projectile effect can be patient injury , and there are reports in the literature of injuries or fatalities affecting patients [ 3 , 4 ] , which have often involved helium or oxygen cylinders . twisting twisting refers to the deflection or torsion of magnetic objects that tend to orient themselves parallel to the direction of the b0 . 
typically , objects that can be dangerous for patients when subjected to this effect are vascular clips and cochlear implants , but there are various parts of implants or other objects that can be twisted and thus affect implant functioning or cause damage to the patient . 
at the ocular level , with the possibility of seriously damaging eyesight . vascular clips one of the reasons for screening patients is precisely that of identifying beforehand and possibly solving the impasse in which radiologists using mr often find themselves : how to manage a patient who is potentially bearing a ferromagnetic clip . 
questo fenomeno comporta un rischio di impatto sia con il paziente o loperatore che si trovino nel tragitto , sia per il magnete che ne pu risultare seriamente danneggiato . a seconda del tipo di magnete e dellintensit del campo generato , esistono linee di campo magnetico che si estendono per una certa distanza nellambiente ( campo disperso fringe field )  . 
tale distribuzione determina in varia misura lattrazione verso il centro del magnete . il campo magnetico statico suddiviso per convenzione in zona 1 ( larea circostante lisocentro magnetico , che contenuta allinterno del magnete ) e zona 2 ( larea esterna al magnete che strutturata con gradienti di intensit decrescente in senso centrifugo )  . 
oggetti ferromagnetici portati vicino al cm statico possono cio diventare proiettili per una forza di attrazione con il magnete determinata da : forza del cm ; suscettibilit magnetica delloggetto ; massa delloggetto ; distanza dal magnete ; orientamento delloggetto nei confronti del cm . esiste una registro negli usa che a tutto il 2001 riportava una lista di oggetti attirati dentro il magnete ( hda , health devices alerts ) , che riportiamo , modificata , nella tabella 1 . la conseguenza diretta del verificarsi di un effetto proiettile risiede oltre che nel possibile danneggiamento del magnete , nel possibile ferimento del paziente . 
ci sono reports in letteratura di danno o esiti fatali a carico del paziente [ 3 , 4 ] , che spesso hanno interessato bombole di elio o di ossigeno . twisting il termine twisting si riferisce alla deflessione / torsione degli oggetti magnetici che tendono a orientarsi parallelamente alla direzione del bo . 
in practice , patients often arrive for their appointment without any precise information concerning the type or model of the clip , or with a potentially unreliable certification of compatibility . in the case of endocranial clips , it is always necessary to require written certification from the neurosurgeon [ 5 ] ; if the clip is made of titanium or a titanium alloy , the examination can go ahead [ 6 , 7 ]  . 
if it is certified as being mr compatible but not made of titanium , it is possible to take 1995 as a dividing line : there are substantially no risks in the case of clips used after that date , but there is a certain risk of significant interactions with clips implanted earlier . 
of course , if the clip is declared to be ferromagnetic or otherwise incompatible with mr , the examination must be cancelled . abdominal haemostatic clips are also frequently identified during the screening procedure and , unless they are expressly declared as being compatible with mr , it is necessary to check radiologically in order to assess their radiopacity and , therefore , their metal component : if they are visible upon computed tomography ( ct ) or x - rays , they could be made of a metal alloy and will need to be certified . 
once again , mr - compatible materials have been increasingly used since the mid - 1990s , and there is likely to be an extremely low risk of incompatibility in the case of those positioned more recently . it is true that some of the sutures still used in gastroinuna delle ragioni per cui esiste il modulo di screening per il paziente proprio quella di anticipare e possibilmente risolvere limpasse nella quale si pu trovare spesso il radiologo che si occupa di rm : la gestione di un paziente potenzialmente portatore di una clip emostatica ferromagnetica . la possibilit di identificare uno di questi casi nella sede della compilazione del modulo di screening per le controindicazioni alla rm , permette da una parte di evitare grossolani errori quale potrebbe essere lintroduzione nello scanner rm di un paziente con clip non compatibile , in una sede critica quale un vaso arterioso cerebrale per esempio , e dallaltra la possibilit di richiedere al paziente di procurarsi una certificazione scritta di compatibilit della clip presso il proprio curante o il chirurgo che lha operato , e quindi di non rischiare di saltare lappuntamento per mancanza di informazione [ 5 ]  . 
in realt nella pratica si pone spesso il problema del paziente che si presenta alla data dellesame senza informazioni precise sul tipo e modello della clip , o con una non sicura certificazione di compatibilit . per quanto attiene alle clip vascolari endocraniche di fatto occorre sempre esigere una certificazione scritta da parte del neurochirurgo [ 5 ]  . 
se viene certificata come compatibile con la rm ma non costituita di titanio , si pu prendere la data del 1995 come spartiacque , oltre la quale non vi sono sostanzialmente rischi , prima della quale vi invece un certo rischio di interazione significativa . 
naturalmente se la clip dichiarata non compatibile con lambiente rm o come ferromagnetica , non si autorizzati a procedere con lesame rm . per quanto riguarda le clip emostatiche addominali , altro frequente riscontro durante la procedura di screening , salvo che non siano dichiarate espressamente come compatibili con la rm , occorre eseguire uno screening radiologico per valutarne la radiopacit e quindi la componente metallica : se visibili alla tc o in rx , potrebbero essere di lega metallica e occorre una loro certificazione . 
anche in questo ambito dalla met degli anni 90 si assistito ad un sempre maggiore uso di materiali compatibili con la rm , e il criterio di vicinanza temporale del loro posizionamento gioca a favore per un bassissimo rischio che non siano compatibili . per quanto attiene le suture per interventi gastro - intestinali , sono ancora oggi usati materiali che possono subire una certa interazione con il campo magnetico , ma senza rischi significativi per la salute del paziente , mantenendo la regola di aver permesso almeno il processo di cicatrizzazione e quindi almeno 1521 giorni dallintervento . corpi estranei la gestione di un paziente portatore di corpo estraneo endoorbitario deve prevedere unaccurata anamnesi , che non pu prescindere dallattenta analisi del modulo delle controindicazioni alla rm . 
it can be useful to repeat the questions just before entering the mr room because patients often fail to remember the presence of an eye splinter when they are first asked . 
the best strategy is to exclude the presence of a radiopaque foreign body by means of an orbital radiograph , which should always be performed in the case of a retained splinter , a history of a foreign body without precise memory of its removal , or when this was not followed by a thorough ophthalmological examination excluding the presence of other remnants . 
however , as clearly documented in the literature , radiography is not indicated as a form of blanket screening for all of the population at risk ( usually assessed on the basis of their working activities ) [ 9 ]  . 
however , not all bullets have the same ferromagnetic properties : most hunting bullets made in the usa are not ferromagnetic [ 10 ] , but those made for military purposes are and are subject to various degrees of rotation when exposed to the action of the mf . grenade fragments contain steel and are ferromagnetic [ 11 ]  . 
as bullets , pellets and shrapnel may always be contaminated by metallic material , the risk / benefit ratio of an mr examination should always be assessed in people carrying such foreign bodies . 
before undergoing an mr examination , any patient who declares himself or herself to be a bearer of metal bullets or shrapnel should have a standard radiograph of the affected body district to provide information about its position and nature [ 12 ]  . 
in a recent study , no neurological changes or painful reactions were observed in patients with spinal lesions due to the presence of bullets or metal fragments who underwent mr examinations using a 1.5 - t machine [ 13 ]  . 
none of the 13 metallic suture threads tested showed any interactions with the 1.5 - t field , whereas two ( flexon suture and steel suture , united states surgical , new haven , ct ) showed a slight deflection when exposed to a 3t field ; however , these forces were not sufficient to mobilise the stitches in situ [ 14 , 15 ]  . 
la strategia in questo caso prevede lesclusione della presenza di un corpo estraneo radiopaco con una radiografia delle orbite , da eseguirsi sempre nel caso di scheggia ritenuta o di storia clinica di pregresso corpo estraneo senza ricordo esatto di rimozione o senza che questa sia stata seguita da unaccurata visita oftalmologica che ne abbia escluse altre residue . 
la radiografia non invece indicata come screening a tappeto su tutta la popolazione a rischio ( in genere sulla base dellattivit lavorativa ) , come ben documentato anche il letteratura [ 9 ]  . la rm pu essere unindagine utile o indispensabile in pazienti giunti allattenzione del medico con ferite da arma da fuoco recenti o pregresse . 
non tutti i proiettili hanno per le stesse propriet ferromagnetiche ; la maggior parte dei proiettili da caccia sportiva fabbricati negli stati uniti non sono ferromagnetici [ 10 ] , i proiettili per uso militare sono invece ferromagnetici e sono soggetti a vari gradi di rotazione quando sottoposti allazione del campo magnetico . le schegge di granata contengono acciaio e sono ferromagnetiche [ 11 ]  . 
in ogni caso va sempre considerato che proiettili , pallottole e schegge possono essere contaminate da materiale metallico quindi bisogna sempre valutare il rapporto rischio / beneficio di unindagine di rm in pazienti portatori di tali corpi estranei . 
prima di sottoporre ad indagine rm un paziente che dichiara di essere portatore di schegge o proiettili metallici andrebbe eseguita una radiografia standard del distretto corporeo colpito per ottenere utili informazioni sulla posizione del corpo estraneo e sulla sua natura [ 12 ]  . 
la presenza di proiettili o schegge metalliche nel canale spinale di pazienti paralizzati rappresenta una controindicazione solo relativa allindagine rm ; in un recente studio infatti non si sono osservate modificazioni del quadro neurologico o reazioni dolorose in pazienti con lesioni midollari portatori di proiettili o schegge metalliche in sede endocanalare spinale sottoposti ad esame rm con apparecchiatura da 1 , 5 t [ 13 ]  . i fili di sutura possono essere fatti di diversi materiali , metallici e non . 
nessuno dei 13 fili di sutura metallici testati ha mostrato interazioni con il campo da 1 , 5 t , mentre 2 ( flexon suture e steel suture , united states surgical , north haven , ct ) hanno mostrato una lieve deflessione quando sottoposti allazione del campo da 3 t ; tali forze non erano per sufficienti a mobilizzare il filo in situ [ 14 , 15 ]  . artefatti si possono verificare artefatti dovuti a : emissione rf da parte di dispositivi , che viene considerato rumore dalla bobina di ricezione ; vuoto di segnale da componente metallica , che pu mascherare o simulare una patologia . lartefatto da clip vascolare ferromagnetica pu essere a . 
 the artefact due to a ferromagnetic vascular clip can be an aid when the findings of a previous mr examination are available , when the type of clip implanted is not known at the time of the current investigation , and its ferromagnetism needs to be assessed to decide whether or not it is safe to proceed . device malfunction ( electronic or mechanical ) such malfunctions include devices with analogic valves , electric motors , transformers , relays and switches , prostheses that use magnetisation to remain attached to a patient ( some dental devices ) , and some implants that are activated electronically , magnetically or mechanically . 
echocardiography ( ecg ) traces may be distorted , with alterations in t waves . pacemakers the presence of a cardiac pm has accompanied the development of mr over the years since the beginning as an absolute contraindication capable of threatening a patients life . 
animal and in vitro studies first documented the risk of pm malfunction in the 1980s [ 16 ] , and there were reports of at least ten deaths due to taking patients bearing pms into the mr roothe principal considerations concerning the safety of these devices were first put into a structured form in a review by mathur - de vre published in 1987 [ 17 ]  . 
on the basis of this contraindication , an increasing proportion of the population ( currently estimated at several million persons ) could not undergo mr even in the presence of one of the neurological , neurosurgical or oncological diseases , or common non - lifethreatening conditions such as musculoskeletal diseases , for which mr is the most accurate diagnostic examination . however , recent studies carried of bearers of pms undergoing mr examinations in emergency settings , together with technological advances in the pms themselves , have gradually modified these views . 
since 1996 , technological developments have led to a substantial reduction in the proportion of ferromagnetic material inside pms , and redesigns of their electronic architecture and programming , reductions in the distance between the electrodes and changes in the materials ( mp35n an alloy that is less susceptible to magnetism ) have considerably reduced their sensitivity to electromagnetic interference [ 18 ]  . the known effects of mr on cardiac pms are due to the static mf , the temporary rf field and the gradients ( additional mfs ) , and are shown in table 2 . 
il tracciato ecg pu risultare distorto con alterazioni delle onde t . pacemaker la presenza di un pacemaker ( pm ) cardiaco ha accompagnato lo sviluppo della rm negli anni fin da subito come controindicazione assoluta , in grado di porre a rischio la vita del paziente . 
negli anni 80 [ 16 ] studi su animali e studi in vitro avevano in effetti documentato il rischio di malfunzionamento del pacemaker ; a questi dati sperimentali inoltre si deve aggiungere che in letteratura sono stati riportati almeno 10 decessi dovuti a introduzione in sala rm di pazienti con pm . 
le principali considerazioni sulla sicurezza di questi dispositivi sono state elicitate per la prima volta in modo strutturato nella review di mathur - de vre , nel 1987 [ 17 ]  . 
stando a tale controindicazione , una quota sempre maggiore di popolazione , calcolato ad oggi in alcuni milioni di persone , non potrebbe essere sottoposta ad una risonanza magnetica , pur in presenza di patologia neurologica , neurochirurgica od oncologica , oppure di patologie non life - threatening ma molto diffuse nella popolazione come le patologie muscolo - scheletriche , per le quali la rm rappresenta lesame maggiormente diagnostico . tuttavia , negli ultimi anni , studi effettuati in situazioni di reale urgenza su pazienti portatori di stimolatore cardiaco sottoposti a risonanza magnetica , unitamente al progressivo sviluppo tecnologico dei pacemaker , hanno gradualmente modificato questi concetti . 
lo sviluppo tecnologico dei pm , a partire dal 1996 , porta ad una netta riduzione della quota di materiale ferro - magnetico allinterno degli stimolatori cardiaci ; la riprogettazione dellarchitettura elettronica e della programmazione , la riduzione della distanza tra gli elettrodi e il cambiamento dei materiali ( una lega con minor suscettibilit magnetica mp35n ) hanno ridotto la sensibilit alle interferenze elettromagnetiche [ 18 ]  . gli effetti noti della risonanza magnetica sui pacemaker cardiaci derivano dal campo magnetico statico ( cm ) , dal campo di radiofrequenza temporaneo ( rf ) e dai gradienti ( campi magnetici addizionali ) e sono esposti nella tabella 2 . certamente a livello clinico la complicanza potenziale pi importante da temere il decesso del paziente . 
in one in vivo study using 0.5 - t magnets , sommer describes 18 patients in whom the only recorded effect was that of the passage to the asynchronous mode [ 24 ]  . 
 in this regard , a checklist of recommendations has recently been proposed to be used when an mr examination is scheduled for patients bearing a pm [ 25 ]  . 
this was originally considered a relative contraindication but has since proved not to be insofar as studies have shown the absence of cardiac symptoms and no effects on the ecg [ 26 ]  . however , metallic endocardial guides have not yet been shown to be innocuous . furthermore , it is less dangerous to carry out the examination in a different area possibly far from the heart ( such as the head or distal joints ) , as this reduces the effects on the pm because the magnetic isocentre is shifted to the area of study . 
together with the use of coils that work as both receivers and transmitters , this certainly reduces the effect of the rf field , which is at its greatest in the isocentre . all of the above has led to the development of special low - field magnets such as those dedicated to the study of distal joints . 
in this case , the thoracic effects of the magnetic and rf fields are minimal because only the limb is inserted inside the magnet , and there is no absolute contraindication to examining patients with pms [ 27 ]  . 
 implantable cardioverters / defibrillator ( icds ) in addition to the problems raised by pms , icds also have problems of their own involving ( 1 ) batteries and ( 2 ) sensito , che si sono sottoposti a esame rm , tutti per senza monitoraggio o precauzioni allingresso [ 23 ] e soprattutto senza che fosse stata valutata la dipendenza o meno del ritmo cardiaco dal pm . in letteratura per sono presenti da tempo anche articoli riguardanti studi rm su pazienti con pacemaker , senza che vengano riportati decessi , n complicanze . 
si tratta purtroppo spesso di studi o reports abbastanza disomogenei , sia per la selezione del paziente che per il tipo di studio rm condotto . vari studi hanno dimostrato come sempre a basso campo non si osservino effetti sullattivit del pm , quando questo sia spento o posto in modalit asincrona . 
questa che era considerata una controindicazione relativa si poi rivelata non reale , in quanto vi sono studi che hanno dimostrato lassenza di sintomi cardiaci o effetti sullecg [ 26 ]  . 
non vi sono invece dimostrazioni di innocuit a carico di guide metalliche endocardiche . risulta inoltre meno pericoloso condurre lesame su una zona diversa e possibilmente lontana dal cuore , come la testa o le articolazioni distali , perch si riducono gli effetti magnetici sul pm dal momento che lisocentro magnetico risulta spostato nellarea di studio e questo , insieme allutilizzo di bobine che funzionino sia in trasmissione che in ricezione , riduce sicuramente leffetto del campo di rf , che massimo allisocentro . questo ha portato allo sviluppo di particolari magneti a basso campo come quelli dedicati allo studio delle articolazioni distali , per i quali gli effetti del cm e delle rf a livello del torace sono minimi , in quanto si inserisce solo larto nel piccolo magnete ; in tale condizione non vi una cona . 
it happens in the case of 1 - mt ( 10 gauss ) fields , which is why it is recommended not to exceed 5 gauss when examining patients bearing pms [ 19 ]  . 
the magnetic responsiveness of the latest - generation pms can be deactivated in such a way as to maintain the synchronous mode batteries and reed switches contain ferromagnetic parts and can theoretically be dislocated by the mf ( which is minimum at the isocentre and increases with the distance from it ) and the torsion ( which is maximum at the isocentre )  . 
as the direction of the b0 of closed magnets is often parallel to the axis of the patient , when a patient is lying down , the pm is also aligned with the torsion . 
however , when a patient enters the magnet room on foot , the torsion ( which tends to align paramagnetic structures with the axis of the mf ) is at its maximuin the past , patients were asked to wear an elastic pressure bandage over the pectoral pocket in order to reduce mobilisation of the device . 
the electrodes are made of an alloy ( mp35n ) that includes nickel , cobalt , chrome and molybdenum and are therefore not ferromagnetic ( c ) electrocardiogram ( ecg ) variations polarised blood flow acts as a conductor and can cause electrical alterations by superimposing electrical potentials on surface ecg measurements , thus increasing the t wave ; however , this is not very relevant insofar as the p and r waves do not seem to be affected 2 . 
temporary radiofrequency ( rf ) field ( a ) heating . the presence of instruments with electrical conduction characteristics can amplify the local electrical field and make it difficult to predict peak specific absorption rate ( sar )  . 
in this way , it is possible that thermal damage occurs at the ends of the stimulating electrodes and therefore at the interface between the myocardium and the point of the electrode . 
this phenomenon does not occur in wide and compact metal structures such as the pm generator , but it is observed in the stimulating electrodes , even when they are disconnected or insulated [ 20 ] ( b ) altered stimulation rhythm this happens because of direct interference with the pm electronics or because the induction of currents above the activation threshold of the atrial electrode activates ventricular stimulation ( c ) pm reprogramming or resetting called phantom programming , the spontaneous reprogramming of the pm during examination occurred in the past , but current pms have safety controls that prevent it from happening 3 . 
variable field gradients over time ( a ) induced voltages ( b ) reed switch these are possible insofar as the gradients are local mfs that vary rapidly over time , but the induced currents are below the threshold of excitability for cardiac fibrillation ( between 100 and 1000 a / cm2 )  . 
in 1993 , lauck postulated that it was not the stimulation capacity of the device that was affected but the detection of the autochthonous rhythm insofar as this current could be mistaken for pulsation . 
even the most recent icds can give rise to difficulties due to magnetic torsion , the forces of which have been measured in vitro [ 29 ] and found to be above the threshold for clinical detection . 
furthermore , icds are more sensitive than pms : e.g. they can be induced to recognise a ventricular tachyarrhythmia that does not exist , automatically provide the defibrillation to treat it , and thus actually cause a ventricular tachyarrhythmia which may even go unrecognised because the mfs can prevent its detection . 
effetti del cm ( a ) chiusura / apertura del reed - switch determina il passaggio da modalit sincrona , cio on demand , a modalit asincrona , con un ritmo ( b ) dislocazione del pacemaker ( c ) variazioni nellelettrocardiogramma 2 . 
effetti del campo di radiofrequenza temporaneo ( rf ) ( a ) riscaldamento pre - fissato e potenziale competizione con il ritmo di base del cuore . avviene per campi di 1 mt ( 10 gauss ) e da qui nasce la raccomandazione di non fare superare la linea dei 5 gauss ai pazienti portatori di pm [ 19 ]  . nei pm di ultima generazione possibile disattivare la responsivit magnetica e mantenere quindi la modalit sincrona . le batterie e i reed - switch contengono parti ferro - magnetiche e possono teoricamente essere dislocate dalla forza magnetica ( minima allisocentro e incrementante allontanandosi da esso ) e dalla forza di torsione ( massima a livello dellisocentro )  . dal momento che i magneti di tipi chiuso hanno spesso la direzione del b0 parallela allasse del paziente , il pm , a paziente sdraiato , si trova allineato anchesso nei confronti della forza di torsione ; quando il paziente invece entra nella sala magnete , ed in piedi , la forza di torsione che tende ad allineare le strutture paramagnetiche allasse del cm , risulta invece massima . 
questo fenomeno non avviene in strutture metalliche compatte , larghe , come il generatore del pacemaker , mentre si osserva negli elettrodi stimolatori , anche quando disconnessi o isolati [ 20 ]  . ( b ) alterazione del ritmo di stimolazione questo fenomeno avviene o per interferenza diretta con lelettronica del pacemaker , o per induzione di correnti sopra la soglia di attivazione dellelettrodo striale , attivando una stimolazione ventricolare . ( c ) riprogrammazione o resettaggio si chiama programmazione fantasma e si tratta di una riprogrammazione spontanea del dispositivo durante lesame rm . 
effetti dei gradienti di campo variabili nel tempo ( a ) voltaggi indotti sono possibili in quanto i gradienti sono campi magnetici locali rapidamente variabili nel tempo , ma le correnti indotte sono sotto la soglia di eccitabilit per la fibrillazione cardiaca ( nel range tra 100 e 1000 a / cm2 )  . lauck nel 1993 ha postulato che non ne venga affetta la capacit di stimolazione del dispositivo ma quella di riconoscimento del ritmo autoctono , in quanto tale corrente potrebbe essere scambiata per pulsazione . 
tale fenomeno sarebbe pi probabile nei pm monopolari [ 21 ]  . ( b ) reed - switch apertura e chiusura con il variare dei gradienti . rf waves the main biological effects associated with exposure to rf fields are due to their thermogenic properties because at the frequencies used for clinical purposes , some of the absorbed energy is converted into heat . 
the heat is caused by the enattualmente i pi recenti icd possono porre problematiche per le forze di torsione magnetica , che sono state misurate in vitro [ 29 ] e risultano oltre che misurabili anche sopra la soglia di riconoscimento clinico . 
the specific absorption rate ( sar , expressed in watts / kilogram ) was introduced in an attempt to limit bodily exposure to the rf field , and modern mr machinery is equipped with an sar self - limiting systeheating is less with low - field 0.5 - t magnets than with 1.5 - t magnets because the absorption is proportional to the frequency of the rf field . 
a 5 - min pause between sequences can allow total dissipation of the heat , and the use of sequences that involve a rapid switch from one gradient to another ( such as gradient recalled echo ) should be limited . automaticamente fornire il trattamento di defibrillazione , inducendo cos realmente una tachiaritmia ventricolare , che tra laltro potrebbe non essere riconosciuta in quanto i campi magnetici possono anche prevenire il suo riconoscimento . gli effetti biologici principali associati allesposizione a campi di rf sono dovuti alle caratteristiche termogeniche del campo rf . 
il riscaldamento causato dallenergia trasmessa dallimpulso rf che genera un campo elettrico che a sua volta induce correnti elettriche ; tali correnti possono determinare riscaldamento . per limitare lesposizione del corpo al campo rf stato introdotto il sar , specific absorption rate , espresso in watt per kg . 
il fenomeno del riscaldamento minore in magneti a basso campo ( 0 , 5 t ) rispetto a magneti da 1 , 5 t dal momento che lassorbimento di potenza proporzionale alla frequenza del campo rf ; il sar varia a seconda della sequenza utilizzata ; alcune sequenze , come le gradient echo ad esempio , hanno un sar minore di altre ( spin echo )  . 
una pausa di 5 minuti tra una sequenza e la successiva pu permettere la totale dissipazione del calore ; andrebbe limitato luso delle sequenze che comportino un veloce switch tra i gradienti ( come le gradient - recalled echo )  . le ustioni in genere sono causate dallintroduzione di materiale elettrico conduttivo allinterno del magnete . 
tipici oggetti che teoricamente possono scaldarsi per effetto di questi fenomeni sono gli elettrodi per ecg , i cavi del pulsossimetro , gli accessori rm ( estremi di bobine rf ) in contatto con la superficie cutanea del paziente , ma anche ( anche se con danni minori ) tatuaggi ricchi di pigmento a base di ossido ferroso . linduzione di correnti elettriche pu anche di per se stesso essere un pericolo potenziale per linterazione con altri dispositivi , ma non per impianti bio - medicali passivi . la recente diffusione del piercing a scopo decorativo o rituale un fenomeno da considerare prima di sottoporre un paziente ad esame di rm . 
il piercing pu mobilizzarsi o surriscaldarsi durante lesame rm e causare danni variabili in rapporto alla massa delloggetto , alla sua sede e alle sue propriet ferromagnetiche ; il piercing andrebbe dunque rimosso prima dellesame rm . 
se ci non possibile o se il paziente rifiuta di rimuoverlo , il paziente va informato dei potenziali rischi cui va incontro sottoponendosi allesame e vanno applicati dispositivi ( cerotti o bendaggi ) per stabilizzare loggetto . 
typical objects of this kind are ecg electrodes , the cables of pulse oximeters , and mr accessories ( such as the ends of rf coils ) , which may come into contact with a patients skhowever , such objects also ( albeit with less damaging effect ) include tattoos rich in iron - oxide - based pigments . 
the induction of electrical currents can per se be a potential danger because of their interactions with other devices , but not passive biomedical implants . the recent spread of decorative or ritual piercings is something that needs to be considered before submitting a patient to an mr examination , as the jewellery may contain nonferromagnetic metals , ferromagnetic metals or even nonmetallic materials . 
if this is not possible , or the patient refuses , he or she should be informed of the potential risks and the objects should be held firm by means of a adhesive plaster or bandage . 
to prevent burns , insulating material ( bandage or adhesive tape ) should be placed between the object and the underlying skin , and patients should also be told to inform the operator if they feel any sensation of heat at the site of the piercing or feel the object move during the examination [ 30 ]  . mr is frequently used to examine the head , neck , limbs and other anatomical regions that are common sites of tattoos . 
both permanent tattoos and cosmetics ( especially mascara and other makeup made using black pigments ) contain particles of iron oxides or other metals that , by interacting with the mf , can cause sensations of heat , pins and needles , swelling or local irritation during an examination [ 31 , 32 ]  . 
radiologists should check whether patients have any permanent tattoos before beginning the examination and , although it is not a contraindication , patients should be warned of the possible side effects and told to inform the operator if they feel any sensation of heat at the site of the tattoo . 
any cosmetic containing iron oxides or other ferromagnetic metals should be removed before starting an mr examination . permanent contraceptive devices the presence of metallic contraceptive devices could cause damage due to overheating or displacement and can generate artefacts that may reduce the diagnostic accuracy of the examination . 
the results of various studies indicate that these devices are safe when pala rm una metodica di imaging frequentemente utilizzata nello studio della testa , del collo , degli arti e di altre regioni anatomiche che sono comune sede di tatuaggi . 
sia tatuaggi permanenti che cosmetici ( in particolare mascara e altri trucchi con pigmenti neri ) contengono particelle di ossidi di ferro o di altri metalli che , interagendo con il campo magnetico , possono provocare sensazioni di calore , formicolii , gonfiore , irritazione locale durante lesecuzione dellesame rm [ 31 , 32 ]  . 
il medico radiologo deve verificare se il paziente che sta per sottoporre ad esame rm portatore di tatuaggi permanenti ; in caso affermativo ci non costituisce controindicazione allesecuzione dellindagine , tuttavia il paziente deve essere avvisato dei possibili effetti collaterali e invitato a segnalare alloperatore eventuali sensazioni di calore nella sede del tatuaggio . 
i cosmetici contenenti ossidi di ferro o altri metalli ferromagnetici andrebbero rimossi prima dellesecuzione dellesame rm . dispositivi contraccettivi permanenti la presenza di un dispositivo contraccettivo metallico potrebbe causare danni dovuti al surriscaldamento delloggetto o alla mobilizzazione dello stesso e pu generare artefatti che possono ridurre laccuratezza diagnostica dellesame di rm . gli iud ( intra - uterine devices ) sono dispositivi contraccettivi intrauterini che possono essere costituiti interamente di materiale non metallico ( plastica ) o di materiali non metallici con componenti metalliche ( tipicamente il rame il metallo utilizzato )  . 
i risultati di diversi studi indicano che questi dispositivi sono sicuri per i pazienti esaminati con magneti da 1 , 5 t o inferiori [ 35 , 36 ]  . 
le parti metalliche possono generare artefatti che sono tuttavia poco significativi considerando la bassa suscettivit magnetica del rame e le dimensioni relativamente piccole degli iud . recentemente stato sviluppato un nuovo dispositivo metallico per la contraccezione permanente chiamato essure ( conceptus , san carlos , usa ) , costituito da una microspirale posizionata nella porzione prossimale delle tube di falloppio ove induce una risposta tissutale di tipo fibrotico che determina lancoraggio del dispositivo e locclusione tubarica . 
la sicurezza dellessure stata valutata da shellock [ 37 ] utilizzando un magnete da 1 , 5 t ( general electric medical systems , milwaukee , wi ) mediante un test ex - vivo noto come test dellangolo di deflessione e misurando con un termometro rm - compatibile le variazioni di temperatura del dispositivo inserito in un fantoccio ( riempito con un gel semisolido ) forgiato in modo da simulare il tronco umano . 
essure is made of stainless steel , platinum , iridium , nickel - titanium , silver and polyethylene fibres , and its safety has been evaluated by shellock [ 37 ] , who used a 1.5 - t magnet ( general electric medical systems , milwaukee , wi , usa ) in an ex vivo deflection angle test and an mr - compatible thermometer to measure the variations in temperature of a device inserted into a dummy filled with a semisolid gel and modelled in such a way as to simulate a human trunk . 
the study revealed no interactions between essure and the mf ( deflection angle : 0 ) , and the maximum thermal variation during the examination was 0.6c , which can be sustained by human tissue and is therefore innocuous . 
the generated artefacts ( essentially a signal blank that was more extended in gradient - echo than in t1 - weighted spin - echo sequences ) would only be a problem if the area to be examined were directly adjacent to the implanted device . 
as far as is currently known , essure does not represent a risk for patients bearing it , and they can safely be examined using magnets of 1.5 t or less [ 37 ]  . most diaphragms used for contraceptive purposes contain metal rings to keep them in position , some of which are made of ferromagnetic materials and are therefore strongly attracted by the static mf ( at least of the 1.5 - t magnets used for the studies )  . 
however , it has been observed ( shellock unpublished data , 1996 ) that patients wearing them do not perceive any disturbance and , in particular , have not reported any sensation of heat or displacement during the examination . 
in brief , the presence of a diaphragm should not be considered a contraindication to an mr examination involving magnets of 1.5 t or less [ 3840 ]  . gradients gradients can cause neuromuscular stimulation , particularly in areas where soft tissue and bone are close together ( anatomical margins and angles such as the elbow , iliac crest , greater trochanter , etc . ) , because the difference in impedance leads to the accumulation of electricity and thus lowers the threshold of nervous excitability . 
the consequences are shocks and paresthesias which , however , do not leave sequelae and so do not need to be considered a real risk for patients . noise is generated by the gradients . 
in the presence of a fixed mf , rapid changes in current give rise to lorentz noise variazione termica durante lesame stata di 0 , 6c , sostenibile dai tessuti umani e quindi innocua . 
gli artefatti generati ( essenzialmente vuoto di segnale , pi esteso nelle sequenze in echo di gradiente rispetto alle spin echo t1 - pesate ) non costituiscono un problema a meno che larea oggetto dello studio rm non sia strettamente limitrofa al dispositivo impiantato . 
allo stato attuale delle conoscenze lessure non rappresenta un rischio per le pazienti ed sicuro sottoporre ad esame rm con magneti da 1 , 5 t o inferiori pazienti che ne siano portatrici [ 37 ]  . la maggior parte dei diaframmi utilizzati per la contraccezione contengono anelli metallici che mantengono in sede il dispositivo . 
alcuni di questi anelli metallici sono fatti di materiali ferromagnetici e vengono dunque fortemente attratti dal campo magnetico statico ( studi condotti su magneti da 1 , 5 t )  . stato comunque osservato ( shellock dati non pubblicati , 1996 ) che pazienti portatrici di diaframmi sottoposte ad esame rm non percepivano alcun disturbo , e in particolare non riferivano senso di calore n avvertivano sensazioni di movimento del dispositivo durante lesame . 
in definitiva , la presenza di diaframma non deve essere considerata una controindicazione allesame di rm su magneti da 1 , 5 t o inferiori [ 3840 ]  . gradienti i gradienti possono determinare stimolazione neuro - muscolare , in particolare in aree di maggior vicinanza tessuto molle tessuto osseo ( bordi e spigoli anatomici , come gomito , cresta iliaca , grande trocantere ecc ) dove si verifica per differenza di impedenza elettrica un accumulo elettrico , con conseguente abbassamento della soglia di eccitabilit nervosa e conseguente stimolazione nervosa . 
ne conseguono parestesie o scosse , che comunque sono temporanee e non lasciano reliquati ; questi effetti non sono pertanto da considerarsi un reale rischio per il paziente . rumore il rumore viene generato dai gradienti ; le rapide variazioni di corrente infatti , in presenza del campo magnetico fisso , sviluppano forze di lorentz , che , mettendo in vibrazione le strutture delle bobine stesse producono i tipici rumori di tamburellamento a bassa tonalit , knocking . 
il rumore pu causare disturbi variabili dal semplice fastidio fino alla perdita temporanea o permanente delludito ; gli effetti sono maggiormente significativi se il paziente anziano , psichiatrico o sotto terapia farmacologica ototossica , oppure nel caso del feto o del neonato , in cui limmaturit dellorecchio interno pu comportare un maggior rischio di lesione permanente . il rumore generato dipende dal tipo di sequenze ( le fast gradient , turbo spin echo e le echo planar sono le pi rumorose ) e dai parametri delle stesse ( sezioni sottili , fov piccoli e short tr and et )  . 
il potenziale rischio di noxa acustica pu essere controllato facendo indossare sempre le cuffie forces that , by making the structures of the coils vibrate , produce the typically deep noises of knocking that may even reach 115 db , whereas the limit threshold is 90 db . 
the effects are more significant if the patient is elderly , suffering from a psychiatric disorder or receiving ototoxic drug therapy , or in the case of a foetus or new born in whom the immaturity of the inner ear may lead to greater risk of a permanent lesion . the generated noise depends on the type of sequences ( the noisiest are fast gradient , turbo spin - echo and echo planar sequences ) and their parameters ( thin sections , small fov , short tr and et )  . 
the risk of hearing damage can be controlled by always having the patients wear earmuffs or earplugs ; when examining a foetus or new born , there are special sequences ( often supplied by the manufacturer of the mr equipment ) that reduce peak noise levels at the expense of greater sequence length . mr during pregnancy and postpartum technological developments over the last 20 years have made it possible to use mr to examine foetuses because the introduction of fast and ultrafast sequences has overcome the problem of artefacts due to foetal movement . 
the largest field of application for foetal mr is neurological imaging , because it is used not only to assess cerebral development [ 4348 ] but also as a means of diagnosing central nervous system diseases [ 49 ] ( agenesia of the corpus callosum [ 50 ] , hydrocephalus [ 51 ] , cerebral ischaemia [ 52 ] , neural tube defects [ 53 ] , schizencephaly [ 54 ] and abnormalities of the posterior cranial fossa [ 55 ] ) ; however , mr is now also used to study other foetal anatomical districts , such as the lungs [ 56 ] , liver [ 57 ] , neck [ 58 ] and urogenital system [ 59 ]  . foetal exposure the following are circumstances that can lead to a foetus being exposed to the biological effects of the static mfs and gradients : a pregnant patient agrees to undergo an mr examination being unaware of her condition a pregnant patient requires a diagnosis that only mr can direct mr visualisation of the foetus is necessary to clear up diagnostic doubts after ultrasound screening a pregnant woman is enrolled as a volunteer in a research protocol . it has also recently been demonstrated that mr is useful in evaluating the condition and functioning of the placenta a . 
sebbene limaging neurologico sia il pi vasto campo di applicazione della rm fetale , sia per la valutazione dello sviluppo cerebrale [ 4348 ] che per la diagnosi di patologie del sistema nervoso centrale [ 49 ] ( agenesie del corpo calloso [ 50 ] , idrocefalo [ 51 ] , ischemia cerebrale [ 52 ] , difetti del tubo neurale [ 53 ] , schizencefalia [ 54 ] , anormalit delle strutture della fossa cranica posteriore [ 55 ] ) , la rm viene oggi impiegata anche nello studio di altri distretti anatomici fetali quali i polmoni [ 56 ] , il fegato [ 57 ] , il collo [ 58 ] , il sistema uro - genitale [ 59 ]  . esposizione fetale lesposizione fetale agli effetti biologici del campo magnetico statico e di gradienti di campo pu avvenire nelle seguenti circostanze : la paziente , non consapevole di essere incinta , sottoposta ad esame rm ; la paziente , in stato di gravidanza , necessita una diagnosi che solo la rm pu fornire ; la diretta visualizzazione del feto mediante la rm pu essere necessaria per dirimere dubbi diagnostici dopo screening ecografico ; donne volontarie in gravidanza arruolate in protocolli di ricerca . stato inoltre recentemente dimostrato che la rm utile nella valutazione delle condizioni e della funzionalit della placenta [ 60 , 61 ] , del volume di liquido amniotico [ 62 ] e delle variazioni del flusso sanguigno cerebrale in gravidanza [ 63 , 64 ]  . molto difficile stabilire i rischi fetali derivanti dallesposizione di donne incinte in rapporto alla variabilit dellintensit dei campi magnetici , dei gradienti e delle sequenze di impulsi di radiofrequenza utilizzati nei diversi centri nella pratica clinica quotidiana . provide rischi per il feto a . 
however , it is still not clear what foetal damage can be caused by exposure to static mfs and field gradients , and most of the available data come from experimental animal studies . 
exposed chicken embryos to different static mfs and field gradients whose intensity varied over time and observed no effect on embryonal mortality , hatching rate or viability of the chicks [ 66 ]  . 
obtained no statistically significant data demonstrating any effects of prolonged exposure to 4 - t mfs on the foetal growth or postnatal development of exposed mice [ 68 ]  . 
found no statistically significant differences in the rate of uterine growth between a group of 74 pregnant women undergoing mr and a control group of 148 unexposed women [ 70 ]  . 
studies of humans have shown that an increase in embryonal temperature of 2c for more than 24 h can cause neural tube defects and craniofacial deformations [ 71 ]  . 
however , it has been established by law that magnet rooms must be equipped with cooling systems capable of maintaining an ambient temperature of less than 24c with less than 60% humidity . i campi magnetici statici utilizzati nella routine clinica vanno da 0 , 2 a 2 t , centri specialistici utilizzano campi a 3 t e in alcuni centri di ricerca vengono impiegati campi magnetici dellintensit di 8 t . 
tuttora non vi chiarezza sui danni fetali derivanti dallesposizione a campi magnetici statici e a gradienti di campo e i dati che possediamo derivano prevalentemente da studi sperimentali su animali . 
 [ 65 ] hanno riscontrato effetti embriotossici su topi esposti durante la fase centrale della gravidanza ad un campo magnetico dellintensit di 0 , 35 t , behr et al . 
 [ 66 ] hanno esposto embrioni di pollo a differenti campi magnetici statici e a gradienti di campo di intensit variabile nel tempo e non hanno osservato alcuna influenza sulla mortalit embrionaria , sul tasso di schiusura delle uova e sulla vitalit dei polli . 
 [ 68 ] non hanno ottenuto dati statisticamente significativi che dimostrassero effetti dellesposizione prolungata a campi magnetici di 4 t sulla crescita fetale e sullo sviluppo post - natale di topi esposti . 
 [ 69 ] in particolare non hanno riscontrato un incremento della morbilit a 3 anni in 20 bambini studiati in utero con rm a 0 , 5 t , analogamente myers et al . 
 [ 70 ] non hanno osservato differenze statisticamente significative nella velocit di crescita uterina tra un gruppo di 74 donne incinte sottoposte ad esame rm ed un gruppo di controllo di 148 donne non esposte . 
occorrono ulteriori trials clinici su pi vasta scala campionaria per determinare il reale effetto dellesposizione a campi elettromagnetici e onde di radiofrequenza sullo sviluppo endouterino . danno termico lesposizione fetale a radiofrequenze si traduce in un aumento della temperatura corporea che potenzialmente dannoso . 
studi su esseri umani hanno stabilito che un aumento della temperatura embrionale di 2c per pi di 24 ore pu determinare difetti del tubo neurale e deformazioni cranio - facciali [ 71 ]  . 
it has been demonstrated that the foetal hearing apparatus suffers from loud noises ; in particular , if pregnant women remain in very noisy environments ( > 99 db ) , this can shorten the pregnancy , lead to low birth weight and cause foetal hearing loss [ 72 ]  . 
noise intensity is proportional to mf intensity , varying from about 75 db in the case of a field of 0.2 t up to 115 db in a field of 3 t [ 73 ]  . 
some researchers have tried to establish the degree to which the maternal abdomen attenuates the noise by introducing a microphone into the stomach of a male volunteer , but this experimental model does not faithfully reproduce the foetus in all pregnancy phases [ 74 ]  . further human safety studies are needed to clarify the real risks of using mr in the early phases of pregnancy and so , as things stand , it is ethically correct to avoid undertaking mr examinations during the first trimester except in the case of an diagnostic - therapeutic urgency that cannot be delayed . given the current state of knowledge , it is deontologically and ethically correct to limit embryo / foetal exposure and therefore the indications for mr in the case of a pregnant patient to : 1 . 
the presence of neurological , neurosurgical or oncological diseases affecting the mother . use of paramagnetic contrast media during pregnancy contrast media containing gadolinium cross the placental barrier in humans and animals [ 75 ] ; however , there are no published descriptions of teratogenic or mutagenic effects on the foetus related to the administration of paramagnetic contrast media in pregnant women . 
there is , however , a reservation arising from the scientific observation that once excreted by foetal kidneys , the chelate of gadolinium remains in the amniotic fluid and is ingested through the gastrointestinal tube and re - excreted numerous times : the persistence of the molecule in a closed compartment such as the fetoplacental system could lead to the dissociation of the gadolinium ion ( which is toxic per se ) from the chelate , thus giving rise to a risk of toxicity that is still not fully known . on the basis of the above considerations , a paramagnetic contrast medium could theoretically be administered to pregnant women even though , in practical terms , it is better not to inject it unless it is absolutely necessary . 
stato dimostrato che lapparato uditivo fetale soffre il forte rumore ; in particolare la permanenza di donne in gravidanza in ambienti molto rumorosi ( > 99 db ) pu provocare accorciamento della gravidanza , basso peso alla nascita e perdita delludito nel feto [ 72 ]  . 
lintensit del rumore proporzionale allintensit del campo magnetico e in particolare varia da circa 75 db per un campo di 0 , 2 t fino 115 db in un campo dellintensit di 3 t [ 73 ]  . 
alcuni ricercatori hanno cercato di stabilire il grado di attenuazione del rumore da parte delladdome materno introducendo un microfono nello stomaco di un volontario di sesso maschile ; questo modello sperimentale non riproduceva per fedelmente il feto in tutte le fasi della gravidanza [ 74 ]  . occorrono ulteriori studi di sicurezza sulluomo per fare chiarezza sui reali rischi dellimpiego della rm nella prime fasi della gravidanza per cui , allo stato attuale delle nostre conoscenze , eticamente corretto evitare di effettuare esami di rm nel primo trimestre di gravidanza in assenza di una improcrastinabile urgenza diagnostico - terapeutica . 
a patologie della madre in ambito neurologico , neurochirurgico od oncologico . uso di mezzi di contrasto paramagnetici durante la gravidanza mezzi di contrasto contenenti gadolinio attraversano la barriera placentare sia nelluomo che nellanimale [ 75 ] ; tuttavia non sono descritti in letteratura effetti teratogeni o mutageni sul feto legati alla somministrazione di mezzi di contrasto paramagnetici a donne incinte e studi effettuati su animali non hanno documentato teratogenicit o effetti sullo sviluppo post - natale dopo somministrazione di gadopentato dimeglumina , gadoteridolo , gadobenato dimeglumina o gadoversetamide [ 7679 ]  . 
tuttavia vi una riserva che nasce dalla constatazione scientifica che il chelato di gadolinio una volta escreto dai reni fetali nel liquido amniotico , rimanendo in quel comparto viene ingerito nel tubo gastrointestinale e riescreto , numerose volte : la persistenza della molecola in un comparto chiuso come il sistema feto - placentare potrebbe portare alla dissociazione dello ione gadolinio ( di per s tossico ) dal chelato con un rischio di tossicit non del tutto noto . in base a tali considerazioni in linea teorica potrebbe essere somministrato mezzo di contrasto paramagnetico a donne incinte , anche se dal punto di vista pratico meglio mantenere un atteggiamento conservativo e quindi non iniettarlo a meno che non sia assolutamente necessario . 
these considerations initially led to the suggestion that women undergoing an mr examination with the use of a paramagnetic contrast medium should stop breast feeding during the 24 h following its intravenous injection [ 83 ]  . 
however , more recent studies have shown that the amount of medium transferred via breast milk is more than 100 times less than the maximum intravenous gadolinium dose recommended for neonatal use . 
furthermore , the intestinal absorption of ingested gadolinium is extremely small ( 0.04%0.08% of the amount ingested ) , and the absorbed amount is rapidly excreted by the kidney [ 80 , 81 ]  . 
on the basis of these findings , we believe that breast feeding can be continued without reservation after the administration of paramagnetic contrast media to the mother . lalta risoluzione di contrasto per i tessuti molli e la multiplanariet hanno condotto ad un largo impiego della rm nel post - partu molte delle nuove applicazioni della rm richiedono lutilizzo di mezzo di contrasto paramagnetico cos che la prole di donne in allattamento diviene indirettamente potenziale destinataria di tale agente . 
sebbene la farmacocinetica dei mezzi di contrasto paramagnetici extracellulari sia simile a quella dei mezzi di contrasto iodati , essi sono caratterizzati da un profilo di sicurezza di gran lunga maggiore : le reazioni anafilattiche sono molto rare e non c evidenza di nefrotossicit . i mezzi di contrasto paramagnetici vengono filtrati ed eliminati dal rene ma anche la ghiandola mammaria pu contribuire , sebbene in piccola parte , allescrezione trasferendo cos , attraverso il latte materno , una piccolissima quantit di mezzo di contrasto paramagnetico ( 0 , 011%0 , 04% della dose totale di gd - dtpa )  . 
queste considerazioni hanno condotto in passato a suggerire alle donne sottoposte ad esami rm con mezzo di contrasto paramagnetico la sospensione dellallattamento durante le 24 ore seguenti liniezione endovenosa del mdc [ 83 ]  . pi recenti studi hanno tuttavia evidenziato che la dose di mezzo di contrasto paramagnetico trasferita al feto attraverso il latte materno pi di 100 volte inferiore alla dose endovenosa massima di gadolinio raccomandata per uso neonatale ; inoltre lassorbimento intestinale di gadolinio ingerito estremamente basso ( 0 , 04%0 , 08% di quello ingerito ) , e la quota assorbita viene rapidamente escreta dallemuntore renale [ 80 , 81 ]  . 
the aims of this study were to evaluate the association of testicular microlithiasis with testicular neoplasm , to assess the accuracy of ultrasonography ( us ) in comparison with histology in detecting microlithiasis , and to identify the prevalent cytohistological features that accompany testicular cancer . 
at us examination , testicular cancer was associated with microlithiasis in seven out of 13 patients ( 53.8% ) ( the distribution pattern of microlithiasis was intranodular in two , perinodular in two and both intraand perinodular in three ) , and colour - doppler us showed perinodular and intranodular vascularity . 
dal 2004 al 2005 , sono giunti alla nostra osservazione e sottoposti ad indagine ecografica 14 pazienti , di cui 13 operati per neoplasia maligna , di et compresa tra 19 e 43 anni e solo un caso di 60 anni . 
due pazienti ( 15 , 3% ) presentavano microlitiasi testicolare in precedente indagine ecografica e due pazienti ( 15 , 3% ) presentavano alterazioni dello spermiogramma . allindagine ecografica la neoplasia testicolare maligna nel 53 , 8% ( 7 / 13 ) era associata alla presenza di microlitiasi testicolare ( in 2 / 7 in sede intranodulare , in 2 / 7 in sede perinodulare e in 3 / 7 casi in sede peri - intranodulare ) e presentava vascolarizzazione peri ed intranodulare alleco color doppler . 
le microlitiasi testicolari e le alterazioni del liquido seminale rappresentano fattore di rischio nelle neoplasie maligne del testicolo : infatti , complessivamente , il 30 , 6% ha sviluppato una eteroplasia maligna . 
some authors have indicated incidences of 0.5%0.60% [ 1 , 2 ] , 0.68% [ 3 ] , 2% [ 4 ] , 5% [ 5 ] and 9% [ 6 ] , whereas for others [ 7 ] , the incidence cannot be determined as no studies have assessed the prevalence of microlithiasis in the general population . 
this discrepancy was found to be related to an incomplete analysis of the data collected because of unavailability of histological reports for some cases [ 8 ] or the use of us probes with frequencies less than 10 mhz [ 9 ]  . 
 opinions concerning the association between microlithiasis and testicular cancer are conflicting , with no mention of microlithiasis as a risk factor in recent reviews and no levels of evidence [ 10 , 11 ]  . 
tubular hyalinisation is the end stage of tubular atrophy and includes the absence of both germ cells and sertoli cells , with alterations of the lamina propria and leydig cells ; according to some authors , these changes may be detected histologically in the parenchyma adjacent to the neoplastic lesion [ 12 ] , leading to the hypothesis that testicular cancers arise in a predisposing environment . 
 materials and methods between 2004 and 2005 , 14 patients were referred to us for us examination , 13 of whom underwent surgery for testicular neoplastheir age ranged from 19 to 43 years , except for one patient aged 60 years . 
the us examination evaluated the number of testicular lesions , their echostructure , their vascularity with colourle microlitiasi testicolari ( mt ) sono unanomalia non frequente del parenchima testicolare rappresentata della presenza di depositi calcifici nei tubuli seminiferi e / o nellinterstizio . 
lincidenza stimata in letteratura appare molto variabile : per alcuni autori , intorno allo 0 , 5%0 , 60% [ 1 , 2 ] , allo 0 , 68% [ 3 ] , al 2% [ 4 ] , al 5% [ 5 ] al 9% [ 6 ] , mentre per altri [ 7 ] , non pu essere definita , in quanto non esistono lavori che valutano la prevalenza della mt nella popolazione generale . 
infatti , la maggior parte dei lavori esamina pazienti giunti allosservazione per patologia testicolare e non risulta mai essere stata effettuata indagine di screening . in letteratura , risultano rari gli articoli che correlano il reperto ecografico di mt col rilievo istologico . 
da alcuni autori riportata la discrepanza esistente fra levidenza ecografica e il riscontro istologico di mt [ 7 ] : tale dato risult gravato da una incompleta analisi dei dati raccolti per la mancata disponibilit , in alcuni casi , del reperto istologico [ 8 ] o dallutilizzo di sonde ecografiche con frequenze inferiori a 10 mhz [ 9 ]  . 
recentemente , lutilizzo di sonde ad elevata frequenza e la maggior sensibilizzazione di radiologi ed urologi a questa problematica ha incrementato il rilievo dellincidenza della mt . allo stato attuale , le opinioni in merito allassociazione fra mt e neoplasia testicolare maligna ( ntm ) sono divergenti , in quanto le mt non vengono citate come fattore di rischio in recenti revisioni di letteratura e non vi sono livelli di evidenza riguardo queste [ 10 , 11 ]  . 
la scleroialinosi dei tuboli seminiferi ( sct ) rappresenta lo stadio terminale dellatrofia tubulare ed include lassenza sia delle cellule germinali che delle cellule di sertoli , con alterazioni della lamina propria e delle cellule di leydig ; detta alterazione , per alcuni autori , pu essere riscontrata istologicamente nel tessuto parenchimale adiacente la lesione neoplastica [ 12 ] , prospettando lipotesi che la ntm insorga su un terreno predisposto . lo scopo del lavoro stato quello di analizzare se esistono correlazioni fra la mt e le ntm , valutando , inoltre , laffidabilit dellecografia nel rilevare la microlitiasi rispetto lesame istologico . materiali e metodi dal 2004 al 2005 , sono stati sottoposti ad indagine ultrasonografica con ecografo hitachi h21 ( kashiwa , giappone ) , sonda lineare da 7 , 512 mhz , con modulo color e power doppler , 14 pazienti , di cui 13 operati per ntm , di et compresa tra 19 e 43 anni e un solo paziente di 60 anni . 
1a - c ultrasound ( us ) scan of the testis performed on 27 january 2004 shows microlithiasis in a 38 - year - old patient ( a )  . 
1a - c comparsa di neoplasia seminomatosa in pregressa microlitiasi testicolare : ( a ) il 27 / 1 / 2004 rilievo ecografico di mt in paziente di 38 anni ; ( b ) il 25 / 8 / 2005 comparsa di lesione sospetta per ntm in paziente con pregresso riscontro di mt ; ( c ) rilievo istologico di ntm e mt . per criteri di benignit della lesione , stata eseguita nodulectomia . 
allindagine ecografica si posta lattenzione nel definire il numero delle lesioni testicolari , lecostruttura , la vascolarizzazione con eco color e power doppler e la presenza di mt nella lesione eteroplastica o nel parenchima testicolare adiacente alla neoplasia . 
lesame istologico , oltre a fornire la diagnosi definitiva del tipo istologico in esame , ha ricercato la presenza di mt nel focolaio neoplastico o nel parenchima testicolare esaminato , nonch eventuali alterazioni nel parenchima circostante la ntm . risultati i sintomi di esordio che hanno indotto i pazienti ad eseguire la visita urologica e lesame ecografico sono stati : in 6 dolore scrotale , in 4 la comparsa di tumefazione intrascrotale , in 2 la comparsa di localizzazioni secondarie ( in 1 paziente adenopatia sovraclaveare e in 1 paziente lesioni polmonari ) e in 2 pazienti alterazioni dello spermiogramma ed infertilit . 
besides providing a definitive diagnosis for histological type of lesion , histology aimed to detect the presence of microlithiasis within the neoplasm or in the testicular parenchyma being examined , as well as in the parenchyma surrounding the testicular cancer . results the presenting symptoms leading to consultation of a urologist and us examination were scrotal pain in six patients , intrascrotal swelling in four , the development of secondary localisations in two ( supraclavicular adenopathy in one and pulmonary lesion in the other ) and altered sperm function and infertility in two . 
in nessuno dei pazienti erano presenti segni di ostruzione delle vie seminali e nel 53 , 8% ( 7 / 13 ) dei pazienti con ntm erano presenti mt rig.c. 
la neoplasia benigna , operata di nodulectomia , non presentava allindagine ecografica mt , dato confermato successivamente dal reperto istologico che non evidenzi neanche scleroialinosi nel tessuto adiacente alla neoplasia . allesame istologico , 9 casi erano seminomi , 2 casi forme miste , 1 caso carcinoma embrionario , 1 caso tumore del sacg.c. 
6a , b seminoma con mt in sede intraed extranodulare : a reperto us ; b reperto istologico . finding was later confirmed by histology , which , in addition , did not detect hyalinisation in the adjacent parenchyma . at histology , nine cases were seminomas , two were mixed germ - cell tumours , one was embryonal carcinoma , one was a yolk - sac tumour and one was a benign sertoli - cell tumour . 
nine out of 13 cases of testicular cancer ( 69.2% ) had microcalcifications ( in 2 / 9 cases intranodular , in 5 / 9 perinodular and in 2 / 9 periand intranodular )  . 
in 12 cases ( 92.3% ) , there was tubular hyalinisation in the parenchyma surrounding the testicular cancer . discussion the origin of testicular microlithiasis is unclear , and most authors [ 1316 ] hypothesise that the microliths are intratubular calcifications arising from calcium salt deposits in degenerated germ cells [ 13 ] or in intratubular secretions [ 14 ]  . 
according to more recent studies , the microliths may have an extratubular origin and penetrate the seminiferous tubule subsequently [ 15 ] , and some authors believe that microlithiasis may be correlated with a dysfunction of the sertoli cells , indicating anomalous gonadal embryogenesis [ 1 , 16 ]  . us examination has a decisive role in confirming a clinical suspicion or identifying a disease that requires surgical verification . 
us is also fundamental in patients with known risk factors that have either a high level of evidence ( family history , cryptorchidism , contralateral germ - cell neoplasm , gonadal dysgenesis ) or an intermediate level of evidence ( infertility , dizygous twins , testicular atrophy ) [ 10 ] , to disclose early changes in testicular parenchyma . 
risk factors with a low or controversial level of evidence are scrotal trauma , inguinal hernia , testicular ectopia , previous infections ( meningitis , pneumonia , tuberculoses ) , mumps orchitis , testicular torsion , varicocele , early puberty , down syndrome , acne , co vitellino e 1 caso tumore a cellule di sertoli variet sclerosante . 
in 9 casi su 13 ( 69 , 2% ) di ntm , sono state rilevate microcalcificazioni ( in 2 / 9 casi in sede intranodulare ; in 5 / 9 perinodulari e in 2 / 9 peri e intranodulari )  . 
in 12 casi ( 92 , 3% ) , era presente sct nel parenchima circostante la ntm . discussione lorigine della mt non chiara e la maggior parte degli autori [ 1316 ] ipotizza che queste formazioni siano calcificazioni intratubulari che originano da depositi di sali di calcio in cellule germinali degenerate [ 13 ] o in secrezioni intratubulari [ 14 ]  . 
secondo studi pi recenti , i microliti possono avere unorigine extratubulare , penetrando successivamente nel tubulo seminifero [ 15 ] e , per alcuni autori , le mt possono essere correlate ad una disfunzione delle cellule del sertoli , indicando una embriogenesi gonadica anomala [ 1 , 16 ]  . lindagine ecografica assume un ruolo decisivo nel confermare un sospetto clinico o nel rilevare una patologia che necessita di verifica chirurgica . 
inoltre , lecografia si rileva metodica insostituibile nei pazienti con fattori di rischio accertati con alto livello di evidenza ( familiarit , criptorchidismo , neoplasia germinale controlaterale , disgenesia gonadica ) o con medio livello di evidenza ( infertilit , gemelli dizigoti , atrofia testicolare ) [ 10 ] , nello svelare precoci alterazioni nel parenchima testicolare . 
vengono definiti fattori di rischio con basso o controverso livello di evidenza : il trauma scrotale , lernia inguinale , lectopia testicolare , pregresse infezioni ( meningite , polmonite , tbc ) , orchite parotitica , torsione testicolare , varicocele , pubert precoce , sindrome di down , acne , hiv , alopecia androgenica e non vi menzione alcuna in merito la mt [ 10 ]  . alcuni autori riconoscono che le mt possono essere associate a differenti patologie non neoplastiche : atrofia testig.c. 
however , the most commonly held views in the literature are those that support a correlation between microlithiasis and testicular cancer [ 1 , 3 , 4 , 6 , 1930 ]  . 
if confirmed by future prospective studies , this fact should , in our opinion , lead us to suspect the existence of an environment predisposing to the development of testicular cancer and therefore the possibility of identifying the population at risk of such cancer . 
this hypothesis is not supported by other studies [ 5 , 31 ] , and many authors question this association [ 3236 ] and do not recommend monitoring these patients [ 36 ]  . 
in our view , the prevalence of retrospective studies on microlithiasis in symptomatic patients , the ongoing technological evolution with the use of high - frequency probes and increasingly sophisticated instruments call for longitudinal prospective studies to shed light on this issue . 
our results show that testicular microliths are associated with testicular cancer in 53.8% at us and in 69.2% at histology and confirm the diagnostic accuracy of us in detecting the presence and distribution of microlithiasis . 
this discrepancy is related to the difficulty experienced by us in discriminating between healthy and neoplastic tissue in some cases of heterogeneous parenchyma with poor cleavage planes . these data , in agreement with previous studies that report a 6%46% incidence of germ - cell tumours in patients with microlithiasis [ 1 , 6 ] , support the hypothesis of a correlation between microlithiasis and the development of testicular cancer and should lead us to consider the possibility of obtaining a biopsy in all patients presenting with this type of alteration [ 30 ]  . some authors believe that the presence of unilateral microlithiasis has a high likelihood of being associated with testicular cancer , such that biopsy is justified in these cases , whereas the presence of diffuse bilateral microlithiasis , although potentially concealing a carcinoma in situ , justifies sonographic monitoring only [ 37 ]  . 
 perilesional microlithiasis , detected at us in 5 / 13 patients ( 38.4% ) and at histological examination in 7 / 13 patients ( 53.8% ) , which is external to the cancer and associated with tubular hyalinisation in the adjacent parenchyma , supports , in our opinion , the hypothesis of an environment that predisposes to cancer development and may therefore represent , as held by other authors , a premalignant condition [ 6 ]  . 
such considerations justify the view that clinical and imaging monitoring is justified in these patients . almost all of the patients examined ( 92.3% ) had tubular hyalinisation in the tissue surrounding the testicular cancer , as reported by previous studies [ 12 , 38 ] , as a result not only of autoimmune processes but also of ischaemic or obstructive events that may account for the development of a cancer in a predisposing environment . 
this is also supported by the colare , infertilit , criptorchidismo , anormalit cromosomiche ( sindromi di down o klinefelter ) ; esiti di orchite , sarcoidosi , infarto cronico [ 9 , 17 , 18 ]  . 
tuttavia , in letteratura sono prevalenti le tesi che sostengono lipotesi di correlazione fra lesistenza delle mt e la ntm [ 1 , 3 , 4 , 6 , 1930 ] ; tale rilievo , se confermato da ulteriori studi prospettici , deve far sospettare , a nostro avviso , lesistenza di un terreno favorente lo sviluppo della ntm e , quindi , la possibilit di individuare la popolazione a rischio di insorgenza della eteroplasia testicolare . 
tale ipotesi non risulta per essere sostenuta da altri studi [ 5 , 31 ] , e molti autori pongono in dubbio questa associazione [ 3236 ] e sconsigliano il monitoraggio di questi pazienti [ 36 ]  . 
a nostro avviso , la prevalenza di studi retrospettivi sulla mt in soggetti sintomatici , la continua evoluzione tecnologica con utilizzo di sonde ad elevata frequenza e strumentazioni sempre pi sofisticate , invocano studi longitudinali prospettici che possano fare chiarezza sullargomento . 
dai nostri risultati si evince che le mt sono associate alla ntm rispettivamente nel 53 , 8% allindagine ultrasonografica e nel 69 , 2% allesame istologico , confermando una buona accuratezza diagnostica dellecografia nel rilievo della presenza e della distribuzione della mt . 
in un solo paziente la mt allecografia era stata individuata nella porzione esterna della lesione eteroplastica , mentre allesame istologico questa era al di fuori della neoplasia . tale incongruenza da riferire alla difficolt in alcuni casi , in presenza di tessuto parenchimale disomogeneo e con scarsi piani di clivaggio , a discriminare ecograficamente il tessuto sano da quello neoplastico . tali dati , in accordo con altri della letteratura che riportano una incidenza del tumore a cellule germinali in pazienti con mt che varia dal 6% al 46% [ 1 , 6 ] , sono di sostegno allipotesi che vi sia una correlazione fra la mt e lo sviluppo della ntm , tale da considerare leventualit di effettuare la biopsia in tutti i pazienti che presentano questa alterazione [ 30 ]  . 
 alcuni autori sostengono che la presenza di mt raggruppate monolateralmente abbia una elevata probabilit di associarsi a ntm , tale da giustificare la biopsia , mentre la presenza di mt diffuse e bilaterali , pur potendo celare un carcinoma in situ , giustifica solo un monitoraggio ecografico [ 37 ]  . la mt perilesionale , evidenziata in 5 / 13 pazienti allecografia ( 38 , 4% ) e in 7 / 13 pazienti allesame istologico ( 53 , 8% ) , esterna alla neoplasia ed associata alla presenza di sct nel tessuto parenchimale testicolare adiacente alla neoplasia , deve far ipotizzare , a nostro avviso , la presenza di un ambiente predisponente allo sviluppo della lesione neoplastica e quindi , come gi sostenuto da alcuni autori , possa rappresentare condizione di premalignit [ 6 ]  . 
a nostro parere , tali considerazioni supportano lipotesi che sia giustificato il monitoraggio clinico - strumentale in questi pazienti . in quasi tutti i pazienti ( 92 , 3% ) da noi esaminati sono stati rilevati nel tessuto circostante la ntm fenomeni di sct , a conferma di quanto gi sostenuto da altri autori [ 12 , 38 ] , come conseguenza non solo di processi autoimmuni , ma di fenomeni ischemici ed ostruttivi che potrebbero giustificare linsorgenza della neoplasia su di un territorio predisposto . 
 two of the 13 patients with testicular cancer had poor sperm function ; this finding has already been reported in the literature and , according to some authors , 9% of patients with a diagnosis of germ - cell tumour were infertile before the diagnosis [ 39 , 40 ]  . 
furthermore , our study indicated that us with high - frequency probes is reliable in detecting and visualising microlithiasis compared with the histological examination ; in contrast to previous studies [ 7 ] , we believe this finding to be useful for identifying patients that are potentially at higher risk of developing testicular cancer . in our view , testicular microlithiasis is a risk factor for the development of testicular cancer if there are more than ten microliths per testis , or if present in a retained or atrophic testis , in a patient who has undergone surgery for retained testis , or in subjects of a critical age ( 1640 years ) without signs of seminal duct obstruction . 
 in 2 dei 13 pazienti con ntm erano presenti alterazioni dello spermiogramma ; questo reperto gi stato osservato in letteratura e secondo alcuni autori il 9% di pazienti con diagnosi di tumore germinale presentava infertilit prima della diagnosi [ 39 , 40 ]  . 
pertanto , per alcuni autori , la mt si associa ad alterazioni del liquido seminale con ipofertilit [ 41 ] e , come peraltro da noi sostenuto , tali fattori sono correlati ad un alto rischio nello sviluppo della ntm . nella nostra casistica , nel 53 , 8% dei casi , leco color doppler stato in grado di mettere in risalto in sede intrae peri - lesionale la vivace vascolarizzazione , indice , qualora presente , della neoangiogenesi tipica delle forme eteroplastiche , con la presenza , in 2 casi , di fistola artero - venosa ; questultimo dato risulta utile a nostro avviso nel rafforzare lipotesi diagnostica di eteroplasia . 
dai nostri dati emerge inoltre laffidabilit dellecografia con sonde ad elevata frequenza nel rilevare e nellevidenziare le mt testicolari rispetto al dato istologico ; tale elemento , in contrapposizione con altri autori [ 7 ] , risulta utile nellindividuare cos i pazienti che potenzialmente risultano pi a rischio della popolazione generale a sviluppare la ntm . a nostro parere le mt rappresentano fattore di rischio per lo sviluppo di ntm se sono di numero maggiore di 10 per testicolo , o se sono presenti in testicolo ritenuto , atrofico o in paziente operato per testicolo ritenuto , o in soggetti di et critica ( 1640 anni ) e che non presentano segni ostruttivi alle vie seminali . conclusions conclusioni our experience indicates that us is reliable in detecting testicular microlithiasis compared with the histological examination and suggests that patients with this alteration may be more predisposed to testicular cancer compared with the general population . 
further longitudinal prospective studies are required to validate the evidence of a parenchymal environment predisposing to the development of testicular cancer . dalla nostra esperienza emersa laffidabilit dellecografia nel rilevare le mt rispetto lesame istologico e lipotesi che pazienti con mt siano maggiormente predisposti , rispetto alla popolazione generale , allo sviluppo di un processo neoplastico . 
giovagnorio2 1dipartimento di scienze radiologiche , policlinico umberto i , universit degli studi sapienza di roma , italy 2cadi - icot latina , universit degli studi sapienza di roma polo pontino , roma , italy correspondence to : g . 
toti 6 , i - 74020 avetrana , taranto , tel . : + 39 - 347 - 7361067 , e - mail : glo.fan@gmail.com received : 16 may 2006 / accepted : 21 july 2006 / published online : 11 june 2007 abstract purpose . 
abbiamo eseguito 50 artro - tc gassose per instabilit di spalla , utilizzando scansioni a collimazione sottile ; i dati grezzi sono stati trasferiti ad una workstation e rielaborati con algoritmi di riformattazione multiplanari ( mpr ) e di rendering volumetrici ( vr )  . 
abbiamo diagnosticato 8 lesioni del cercine anterosuperiore ( i gruppo ) ; 32 lesioni del cercine antero - inferiore ( ii gruppo ) ; 2 lesioni del cercine posteriore ( iii gruppo )  . 
pathogenetically , the disorder has been divided into traumatic [ traumatic unidirectional bankart surgery ( tubs ) ] , atraumatic multidirectional bilateral rehabilitation inferior capsular shift ( ambri ) , and acquired instability overstress ( aios ) [ 5 ]  . 
a variety of diagnostic techniques have been used over the years , including conventional arthrography , ct arthrography , magnetic resonance imaging ( mri ) and mr arthrography , which provide useful information on the status of intraand periarticular structures , in particular , the fibrocartilaginous glenoid labrum , the glenohumeral ligaments , the joint capsule and the bone structures . an accurate definition of the site and extent of injury will guide the surgical strategy ( arthroscopy or arthrotomy ) , which is often necessary to restore normal shoulder articulation and function . 
the current trend is to reconstruct the labrum using arthroscopy , as it allows for less invasiveness and faster functional recovery , although the association of a labral lesion with major bone lesions ( hill - sachs or bankart lesions ) generally requires open surgery [ 610 ]  . 
the aim of this paper is to describe our experience with air - contrast 64slice ct arthrography in shoulder instability . materials and methods from november 2005 to march 2006 , we performed 50 ct arthrography examinations on 34 men and 16 women ( age range 1759 years , mean 38 ) , with a history of luxation - subluxation and / or clinical signs of acute or chronic instability . in eight patients , the instability had persisted after a previous stabilising surgical procedure . 
air - contrast ct arthrography was used to evaluate the following structures : glenoid labrum , articular capsule and recesses , glenohumeral ligaments , humeral socket , insertions of rotator cuff tendons , width of the subacromial space , and presence of effusions in the deltoid subacromial bursa and in intra - articular sites . 
in particular , glenoid labrum lesions were subdivided on the basis of their position : 123 oclock ( anterosuperior ) ( group 1 ) , 36 oclock ( anteroinferior ) ( group 2 ) and 612 oclock ( posterior ) ( group 3 ) ; superior labral anterior - posterior ( slap ) lesions were considered part of group 1 . 
the instability was secondary to the presence of one or more alterations at various levels ( labrum , capsule , socket , glenohumeral ligaments and rotator cuff )  . after obtaining the patients informed consent , 20 cc of gaseous contrast material ( air ) was injected in the articulation , via a posterior access and without fluoroscopic guidance , to obtain adequate image contrast , maximum capsule distension and differentiation of the various intra - articular components . 
the examination was conducted with a 64 - slice spiral volume ct scanner ( lightspeed vct , ge ) acquiring thin - collimation ( submillimetre ) scans in the axial planes . the data ( isotropic voxels ) were transferred to a graphics lincapacit di mantenere la testa omerale centrata nella cavit glenoidea conseguente ad uno squilibrio tra gli stabilizzatori statici ( morfologia della glena e dellapparato capsulo - labro - legamentoso ) e dinamici ( complesso muscolotendineo della cuffia dei rotatori e muscoli periscapolari ) della spalla . 
nel corso degli anni sono state messe a punto diverse tecniche diagnostiche , quali lartrografia tradizionale , lartro - tc , lrm e lartrorm , indispensabili per fornire utili informazioni sullo stato delle strutture endoe periarticolari , nel caso specifico del cercine fibrocartilagineo glenoideo , dei legamenti glenomerali , della capsula articolare e delle strutture ossee . una precisa definizione della sede e dellentit della lesione indirizza la scelta chirurgica ( artroscopica o artrotomica ) , spesso necessaria per il ripristino della normale articolarit e funzionalit della spalla . 
la tendenza attuale di ricorrere con frequenza sempre maggiore alla ricostruzione labiale con accesso artroscopico , per i vantaggi legati alla minore invasivit e al pi rapido recupero funzionale , anche se lassociazione di lesione labiale e importanti lesioni ossee ( hill - sachs o bankart ) indirizza generalmente verso un intervento chirurgico a cielo aperto [ 610 ]  . 
scopo del presente lavoro quello di esporre la nostra esperienza in materia di artro - tc gassosa con apparecchiatura a 64 strati nellinstabilit di spalla . materiali e metodi da novembre 2005 a marzo 2006 sono state eseguite 50 artro - tc in 34 maschi e 16 femmine , di et compresa tra 17 e 59 anni ( et media 38 anni ) , con anamnesi positiva per episodi di lussazione - sublussazione e / o segni clinici di instabilit acuta o cronica . 
con lartro - tc gassosa sono state valutate le seguenti strutture : il cercine glenoideo ; la capsula articolare e i recessi ; i legamenti gleno - omerali ; la glena omerale ; le inserzioni dei tendini della cuffia dei rotatori ; lampiezza dello spazio subacromiale ; la presenza di versamento nella borsa sub - acromion deltoidea ed in sede intra - articolare . 
in particolare , le lesioni del cercine glenoideo sono state suddivise in base alla posizione : da ore 12 a ore 3 ( antero - superiore ) ( i gruppo ) , da ore 3 a ore 6 ( antero - inferiore ) ( ii gruppo ) e da ore 6 a ore 12 ( posteriore ) ( iii gruppo ) ; le slap lesion sono state considerate facenti parte del i gruppo . 
il quadro di instabilit era secondario alla presenza di una o pi alterazioni a vari livelli ( cercine , capsula , glena , legamenti gleno - omerali e cuffia dei rotatori )  . previo consenso informato , senza controllo scopico , stata eseguita , attraverso un accesso posteriore , liniezione intra - articolare di circa 20 cc di mdc gassoso ( aria ) , necessaria per ottenere un sufficiente contrasto dimmagine , la distensione massima della capsula e la differenziazione delle varie componenti endoarticolari . 
using three - dimensional ( 3d ) reconstruction software ( navigator plus ) in ten cases selected randomly from group 2 , we also performed virtual arthroscopy , a perspective reconstruction of the glenohumeral joint that simulates the view of a fibreoptic endoscope . 
the acquisition parameters were as follows : 200 kv , 40 mas , slice thickness 0.6 mm , reconstruction interval 0.6 mm , fov 18 cm18 cm and matrix 512512 . 
total examination time ( including preparation and acquisition ) was around 10 mimages were analysed both in the different spatial planes ( axial , coronal , sagittal and oblique ) , and by performing perspective volume reconstructions allowing evaluation of bone structures first ( glenoid fossa and humeral head ) and then of soft tissues ( glenoid labrum and air - distended capsule )  . the patients then underwent diagnostic and / or therapeutic conventional stabilising arthroscopy , and the results were correlated with those of air - contrast ct arthrography to calculate the techniques sensitivity , specificity and diagnostic accuracy . results the diagnoses of air - contrast ct arthrography and conventional arthroscopy were identical in 42 / 50 cases of instability secondary to disorders of the capsular - labral - ligamentous and / or bone complexes . 
in 6 / 50 cases , the results were discordant , as ct arthrography failed to detect four glenoid labrum lesions from 12 to 3 oclock ( group 1 ) and two lesions from 3 to 6 oclock ( group 2 )  . 
in 2 / 50 patients , aircontrast ct arthrography proved nondiagnostic owing to failure to achieve capsular distension because of air leakage in the anteroinferior articular recess in one case and capsulitis in the other ; these patients were consequently excluded from our study . sensitivity and specificity values of mdct arthrography in the overall recognition of the various lesions of the fibrocartilaginous labrum were 88% and 100% , respectively . group 1 and group 2 labral lesions were diagnosed easily , with a calculated sensitivity of 94% ( two false negatives ) and 100% , respectively . 
i dati ottenuti ( voxel isotropici ) sono stati trasferiti ad una workstation grafica ( advantage , ge ) e rielaborati con algoritmi di riformattazione multiplanare ( mpr ) e di rendering volumetrici ( vr )  . 
utilizzando un programma di ricostruzione 3d ( navigator plus ) in 10 casi scelti in maniera random nel ii gruppo stata inoltre effettuata lartroscopia virtuale , una ricostruzione prospettica dellarticolazione gleno - omerale che simula la visione di un endoscopio a fibre ottiche . 
procedendo in senso cranio - caudale , dallarticolazione acromionclaveare alla porzione inferiore dellarticolazione glenoomerale , lindagine stata condotta con paziente supino e braccio in posizione neutrale , utilizzando i seguenti parametri : 200 kv ; 40 ma ; spessore di strato 0 , 6 mm ; intervallo di ricostruzione 0 , 6 mm ; fov 18 cm18 cm ; matrice 512512 . il tempo totale della procedura ( preparazione e acquisizione ) stato di 10 min circa . 
le immagini sono state analizzate sia nei vari piani dello spazio ( assiale , coronale , sagittale e obliquo ) , che eseguendo ricostruzioni volumetriche prospettiche che permettessero di valutare prima le strutture ossee ( fossa glenoidea e testa omerale ) e poi i tessuti molli ( labbro glenoideo e capsula distesa dallaria )  . i pazienti sono stati sottoposti , infine , ad esame artroscopico convenzionale diagnostico e / o terapeutico di stabilizzazione della spalla , e i risultati sono stati confrontati con quelli dellartro - tc gassosa , calcolandone sensibilit , specificit e accuratezza diagnostica . risultati le diagnosi dellartro - tc gassosa e dellartroscopia convenzionale sono state sovrapponibili in 42 / 50 casi di instabilit , secondaria a patologie del complesso capsulo - labro - legamentoso e / o osseo . 
in 6 / 50 casi i risultati non sono stati sovrapponibili , in quanto allartro - tc gassosa non abbiamo riconosciuto : 4 lesioni a livello del cercine glenoideo da ore 12 a ore 3 ( i gruppo ) e 2 lesioni da ore 3 a ore 6 ( ii gruppo )  . 
in 2 / 50 pazienti , lartro - tc gassosa non stata diagnostica a causa della mancata distensione capsulare secondaria rispettivamente a fuga di gas nel recesso articolare antero - inferiore e a fenomeni capsulitici ; questi pazienti sono stati pertanto esclusi dallo studio . i valori di sensibilit e specificit dellartro - tcmd nel riconoscimento complessivo delle varie lesioni del cercine fibrocartilagineo sono risultati pari rispettivamente all88% e al 100% . 
the axial section shows an abnormal configuration of the anterior labrum and of its junction with the capsule ( * ) , as well as the separation of soft tissue from the coracoid process by interposed injected air ( a )  . 
a la sezione assiale mostra anomala configurazione del labbro anteriore e della sua giunzione con la capsula ( * ) , ed interposizione di aria tra il processo coracoideo e i tessuti molli . 
2a - c la sezione assiale mostra lassit capsulare , completo scollamento del margine glenoideo libero antero - inferiore ( lesione di bankart vera ) ( a ) ; corrispettivo artroscopia convenzionale ( b ) e virtuale ( c )  . gion recorded the highest number of false negative results ( 4 ) despite the techniques ability to recognise the subtlest and most complex lesions ; sensitivity was 66% . 
5 capsula ridondante anteriormente ; avulsione completa del cercine glenoideo antero - inferiore ; legamento gleno - omerale medio ( * ) separato dalla parete capsulare , assottigliato e a densit disomogenea ( lesione di perthes )  . discussion shoulder stability depends on a complex of bone structures and soft tissues that surround and make up the glenohumeral joint . 
4 capsula ridondante con presenza di ampi recessi in sede anteriore e posteriore ; cercine glenoideo normale ; tendine del sottoscapolare ( * ) separato dalla capsula , di aspetto sinuoso , integro . diagnosticate facilmente ; stata calcolata una sensibilit rispettivamente del 94% ( 2 falsi negativi ) e del 100% . le difficolt diagnostiche maggiori si sono presentate nelle lesioni del cercine da ore 12 a ore 3 ( i gruppo ) ; a tale livello , infatti , nonostante siano state evidenziate le lesioni pi fini e complesse , si anche riscontrato il maggior numero di falsi negativi ( 4 ) ; la sensibilit stata del 66% . 
la sensibilit risultata sempre del 100% nella diagnosi delle alterazioni delle strutture ossee e / o dei tessuti molli associate alle varie lesione del cercine . discussione la stabilit della spalla dipende da un insieme di strutture ossee e di tessuti molli che circondano e formano larticolazione gleno - omerale . 
non esiste una singola anormalit che di per s responsabile dellinstabilit , ma una combinazione di differenti fattori che interagiscono fra di loro : anormalit del labbro glenoideo ; lassit o lesione capsulare ; legamenti gleno - omerali insufficienti o lesionati ; anomalie di forma e sviluppo della glenoide ; fratture di hill - sachs o bankart quali esiti di pregresse lussazioni [ 1113 ]  . lartro - tc un metodo accurato per la valutazione dellarticolazione della spalla ; fornisce infatti importanti e utili informazioni su morfologia e rapporti delle strutture endo e periarticolari [ 11 ]  . 
questa tecnica ha giocato un ruolo determinante nella diagnostica dellinstabilit di spalla fino a quando lrm basale e lartro - rm non sono state considerate il gold standard nello studio di tale patologia , in quanto dotate di multiplanariet , sensibilit e specificit intrinseca superiori [ 1 , 14 , 15 ]  . 
retrazione del tendine del bicipite ( a ) , assenza del tendine del sovraspinoso ( b )  . ct arthrography is an accurate technique for the evaluation of the shoulder joint and provides significant and useful information on the morphology and relationships of intraand periarticular structures [ 11 ]  . 
the technique played a fundamental role in the diagnosis of shoulder instability until baseline mri and mr arthrography became the gold standard in the study of this disorder in view of their multiplanar capabilities and superior intrinsic sensitivity and specificity [ 1 , 14 , 15 ]  . 
as a result , ct arthrography has taken on a secondary role , being indicated only in disorders of the glenoid labrum , glenohumeral ligaments , the long head of the biceps ruolo secondario , trovando indicazione nella patologia del labbro glenoideo , dei legamenti gleno - omerali , del capo lungo del bicipite e della cuffia dei rotatori solo nel caso di controindicazioni o di dubbi diagnostici allrm ; ha mantenuto invece un ruolo primario nello studio delle patologie ossee proprie della spalla , quali fratture , tumori , artriti , alterazioni ossee secondarie proprie dellinstabilit e dellartrosi [ 5 ]  . in uno studio prospettico di chandnani et al . 
 [ 14 ] del 1993 , stata sottolineata la maggiore sensibilit dellartrorm rispetto allrm e allartro - tc ( 96% vs 93% e 73% ) nel diagnosticare sia le alterazioni del cercine fibrocartilagineo g.p. 
it has , instead , maintained its primary role in the study of shoulder bone disorders , such as fractures , tumours , arthritis and bone alterations secondary to instability and arthrosis [ 5 ]  . a 1993 prospective study by chandnani et al . 
confirmed the better accuracy of mr arthrography in identifying the capsular - labral - ligamentous injuries responsible for shoulder instability , as the method provides panoramic and precise views of the lesions [ 6 ]  . in our opinion , ct arthrography offers several advantages in the study of the shoulder . 
it does not require arthroscopy , so the time required to introduce the gaseous contrast material into the articular cavity and perform the scan is very short , if compared with other examinations [ 15 ]  . 
the gaseous contrast agent air in this specific case has no contraindications and is free , unlike radiopaque or paramagnetic agents , which are nonetheless considered harmless [ 17 , 18 ]  . with the advent of spiral and in particular multislice technology , ct has improved considerably . 
the faster rotation speed of the x - ray tube and the use of thin collimations with larger volume acquisitions compared with conventional ct have allowed for improved temporal and spatial resolution in the z axis [ 19 , 20 ]  . 
in particular , latest - generation 64 - slice mdct scanners , as the one used by us , enable the acquisition of 64 submillimetre slices ( 0.6 mm ) per rotation and , by combining an isotropic spatial resolution ( 0.4 mm3 ) with a rotation speed of around 0.33 s , they allow optimal image quality to be routinely achieved and visualisation of the finest details . 
submillimetre and isotropic voxels were indeed particularly useful in postprocessing , with the creation of 3d images and multiplanar reconstructions in the sagittal , oblique , and coronal planes with no step - like defect in the outline , and hence with a high diagnostic quality [ 19 , 22 ]  . after these voxels have been processed with dedicated software , they can also be used to perform virtual arthroscopy of the articulation . 
not only did it provide an additional view of the joint , which confirmed our results , but it also generated more realistic , intuitive and didactic images , which are consequently more useful to orthopaedic surgeons planning surgery [ 22 , 23 ]  . a number of papers dating between 2003 and 2005 concluded that mdct arthrography has greater diagnostic accuracy than does mri / mr arthrography in the study , for example , of cartilaginous lesions of the ankle , hip , and elbow joints or in the rupture of interosseous ligaments of the wrist [ 2427 ]  . despite these results , bitzer et al . 
in a 2004 study assessing the diagnostic accuracy of mdct arthrography and mr arthrography in 29 patients affected by chronic instability reaffirmed the higher sensitivity and specificity of mr glenoideo e del legamento gleno - omerale inferiore , che la patologia della cuffia dei rotatori , utilizzando un apparecchio rm a 1 , 5 t e una tc di 3a generazione [ 14 ]  . 
 [ 6 ] , nel 2003 , hanno confermato la maggiore accuratezza dellartro - rm nellidentificare le lesioni capsulo - labro - legamentose determinanti instabilit di spalla , in quanto metodica panoramica e precisa in tali lesioni [ 6 ]  . lartro - tc , a nostro avviso , offre diversi vantaggi nello studio della spalla . 
non richiesto un controllo scopico , pertanto lintroduzione in cavit articolare del mdc gassoso e lesecuzione della scansione necessitano di un tempo minimo , se paragonato ad altre indagini [ 15 ]  . 
il mdc gassoso , nel caso specifico laria , del tutto privo di controindicazioni e di costi , a differenza dei mdc radiopachi o paramagnetici , comunque considerati innocui [ 17 , 18 ]  . con lavvento della tc spirale in particolare multistrato , la tecnica migliorata notevolmente . 
laumento della velocit di rotazione del tubo radiogeno e limpiego di collimazioni sottili con acquisizioni volumetriche pi estese rispetto alla tc convenzionale , hanno consentito di incrementare la risoluzione temporale e spaziale lungo lasse z [ 19 , 20 ]  . 
in particolare gli scanner dellultima generazione di tcms a 64 strati , da noi utilizzati , consentono lacquisizione di 64 strati sub - millimetrici ( 0 , 6 mm ) per rotazione e , combinando una risoluzione spaziale isotropica ( 0 , 4 mm3 ) e una velocit di rotazione pari a circa 0 , 33 s , permettono di raggiungere routinariamente unottima qualit di immagine e la visualizzazione dei pi fini dettagli . 
i voxel submillimetrici ed isotropici sono stati infatti particolarmente vantaggiosi nel post - processing , creando immagini 3d e ricostruzioni multiplanari , nei piani sagittale , obliquo e coronale esenti da qualsiasi difetto a gradino del contorno e dotate pertanto di alta qualit diagnostica [ 1922 ]  . questi voxel , rielaborati da software dedicati , possono inoltre essere utilizzati per eseguire lartroscopia virtuale dellarticolazione . 
nel nostro studio , la valutazione artroscopica virtuale , effettuata in maniera sperimentale in 10 artro - tc , stata particolarmente utile ; stata in grado infatti non solo di fornire un ulteriore punto di vista dellarticolazione , avvalorando i nostri risultati , ma anche di elaborare immagini pi realistiche , intuitive e didattiche , e pertanto pi utili nella pianificazione dellintervento chirurgico da parte del chirurgo ortopedico [ 22 , 23 ]  . in letteratura , dal 2003 al 2005 , sono stati pubblicati diversi lavori in cui si conclude che lartro - tcmd ha una maggiore accuratezza diagnostica a confronto con lrm / artro - rm nello studio , per esempio , delle lesioni della cartilagine articolare della caviglia , dellanca , del gomito , o nella rottura dei legamenti interossei del polso [ 2427 ]  . nonostante questi risultati , bitzer et al . 
 [ 15 ] , in uno studio del 2004 , valutando laccuratezza diagnostica dellartro - tcmd e dellartro - rm in 29 pazienti affetti da instabilit cronica , hanno ribadito una superiore sensibilit e specificit dellartro - rm rispetto allartro - tcmd nello studio delle lesioni del labbro glenoideo ( 96% vs 76% e 96% vs 92% )  . 
in a paper by waldt et al . , published in 2005 , which compared mr arthrography , ct arthrography and anatomical dissection , mdct proved to be more accurate than mri in evaluating the superior labrum insertion ( 84% vs 79% ) and the anteroposterior extension of the sublabral recess ( 81% vs 59% ) [ 28 ]  . 
the results of our study appear to be in perfect agreement with the findings of the most recent literature . in our study , the different labrum lesions ( groups 1 , 2 and 3 ) were diagnosed with mdct - arthrography with 88% sensitivity and 100% specificity . 
the best results were obtained in particular in groups 2 and 3 , with sensitivity values of 94% and 100% , respectively , and in the diagnosis of associated lesions ( capsular , hill - sachs , bankart and rotator cuff lesions ) , where sensitivity and specificity both reached 100% . 
the excellent level of diagnostic accuracy proved essential in guiding and , in some cases , modifying the surgical approach . comparavano lartro - rm , lartro - tc e la dissezione anatomica , lartro - tcmd si dimostrata pi accurata dellartrorm nel valutare linserzione del labbro superiore ( 84% vs 79% ) e lestensione antero - posteriore del recesso sottolabiale ( 81% vs 59% ) [ 29 ]  . 
i risultati del nostro studio sono apparsi perfettamente in linea con i dati della letteratura pi recente . le varie lesioni del cercine ( i , ii e iii gruppo ) allartrotcmd sono state diagnosticate con una sensibilit dell88% e una specificit del 100% . 
i migliori risultati si sono evidenziati in particolare nel ii gruppo e iii gruppo , in cui la sensibilit ha raggiunto valori del 94% e 100% , e nella diagnosi delle lesioni associate ( capsulari , di hill - sachs , di bankart e cuffia dei rotatori ) , in cui sensibilit e specificit sono state rispettivamente del 100% . 
pittore 20 , i - 00136 roma , italy , tel . : + 39 - 065 - 8703056 , fax : + 39 - 065 - 8703039 , e - mail : g.regine@sirm.org received : 4 april 2005 / accepted : 5 june 2006 / published online : 11 june 2007 abstract purpose . 
between march 2004 and april 2005 , 277 patients with blunt abdominal trauma were evaluated . twenty - eight out of 277 patients had renal lesions , the severity of which was graded according to the organ injury severity scale of the american association for the surgery of trauma ( aast )  . 
in modo particolare lecografia con mdc risulta tecnica affidabile per la valutazione del trauma renale di basso grado e nel followup dello stesso riducendo il ricorso a ripetuti esami tc con riduzione della dose radiante . 
i limiti sono dati dagli alti costi della tecnica se condotta su popolazione non selezionata . key words contrast - enhanced sonography renal trauma parole chiave ecografia con mezzo di contrasto trauma renale introduction introduzione the current diffusion of sonographic contrast agents has extended the indications of ultrasound ( us ) imaging [ 1 ]  . 
in particular , second - generation contrast agents have not only attualmente la diffusione e lutilizzo dei mezzi di contrasto ecografici ha potenziato ed arricchito di nuove indicazioni lesame ecografico [ 1 ]  . 
the study population included patients with minor blunt abdominal trauma with or without haematuria , patients who were stable but had undergone parasurgical renal procedures during the previous 12 h ( lithotripsy , renal artery stenting , postbiopsy monitoring ) and patients with acute abdomen , haematuria , and minimal but sudden drop in blood - cell count . 
for paediatric patients or patients under the age of 18 years , the parents informed consent was obtained before proceeding to contrast - enhanced us . in all cases , baseline us was integrated by the contrast study performed on a dedicated scanner ( sequoia , siemens , germany ) , with low mechanical index and contrast - specific software [ coherent contrast imaging ( cci ) 222 patients ; cadence contrast pulse sequence ( cps ) 55 patients ]  . 
patients were administered a bolus of 2.4 ml of second - generation contrast material ( sonovue ) followed by about 10 cc saline solution via peripheral vethe examination consisted of an assessment of the liver parenchyma , followed by the splenic and renal parenchyma and by the supraand submesocolic peritoneal recesses , and generated both static images and dynamic clips lasting around 20 s each taken at various times during the examination , depending on the findings . 
there were no early or late allergic reactions to the intravenous contrast material , which appeared to be very well tolerated by patients and fairly easy to use for the operators , who had received specific training in its correct preparation . patients with positive us findings underwent mdct ( light speed ultra , ge , usa ) before and after the intravenous administration of iodinated contrast material , with a specific multiphase acquisition protocol that allowed correct staging of the lesion and assessment of the integrity of the urinary tract . 
in addition , our study evaluated contrast - enhanced us in the follow - up of renal injuries using the same examination protocol as used for the initial assessment . the lesions were classified into five grades of increasing severity according to the american association for the surgery of trauma ( aast ) classification : grade 1 , subcapsular haematoma ; grade 2 , superficial renal lacerations with perirenal haematoma ; grade 3 , renal lacerations deeper than 1 cm without extension into the collecting system or evitrasto ecografici di ii generazione ha permesso non solo di incrementare le potenzialit diagnostiche dellecografia nellidentificazione e caratterizzazione delle lesioni epatiche [ 2 , 3 ] , ma anche di identificare e studiare le lesioni traumatiche di altri organi addominali : milza e fegato in modo particolare [ 4 ]  . lo scopo del presente contributo quello di definire le indicazioni , le potenzialit diagnostiche ed i limiti dellesame ecografico con mezzo di contrasto di ii generazione nel trauma renale , in base alla nostra esperienza maturata in un dea di ii livello . materiali e metodi nel periodo compreso tra marzo 2004 ed aprile 2005 sono stati valutati con esame ecografico basale e dopo somministrazione di mdc ev di ii generazione ( sonovue , bracco , italia ) 277 pazienti con trauma addominale chiuso minore , con o senza ematuria , pazienti stabili , ma sottoposti nelle 12 ore precedenti a procedure parachirugiche in ambito renale ( litotrissia , posizionamento di stent vascolari in arteria renale , monitoraggio post - bioptico ) e che avevano manifestato un quadro di addome acuto , ematuria , minima ma improvvisa riduzione dellemocromo . 
per la valutazione di pazienti pediatrici o comunque minori si procedeva allesame eco - contrastografico previo consenso dei genitori . lesame ecografico basale stato sempre integrato da quello contrastografico adoperando apparecchio ultrasonografico ( sequoia , siemens , germania ) dedicato , con tecniche a basso indice meccanico , software specifico in modalit cci ( coherent contrast imaging , 222 pazienti ) e cps ( cadence pulse sequence , 55 pazienti ) dopo somministrazione , tramite accesso venoso periferico , di una quantit di mdc di ii generazione ( sonovue , bracco , italia ) pari a 2 , 4 ml in unico bolo seguito da circa 10 cc di soluzione fisiologica con valutazione prima del parenchima epatico , poi di quello splenico ed infine di quello renale e dei recessi peritoneali sovra e sottomesocolici con documentazione iconografica sia statica che con clips della durata di circa 20 secondi ognuna , eseguite in momenti variabili , in rapporto al reperto diagnostico emerso . 
non si sono mai verificate reazioni allergiche immediate o tardive dopo somministrazione del mdc ev che apparso molto ben tollerato dai pazienti ed abbastanza maneggevole da usare dagli operatori preventivamente formati alla corretta preparazione dello stesso . i pazienti che presentavano un quadro positivo venivano sottoposti ad esame tc multidetettore ( light speed ultra , ge , usa ) prima e dopo somministrazione di mdc iodato ev , con protocollo di acquisizione specifico , multifasico , al fine di stadiare in modo corretto la lesione e di documentare lintegrit o meno della vie escretrice urinaria . 
the patient was instead monitored by contrast - enhanced us performed 12 and 48 h after the initial assessment and a further baseline examination 1 week later , with resolution of the pathological findings . the lesions identified on contrast - enhanced us were classified into three main groups based on a comparison between contrast - enhanced us , aast classification and mdct findings : ( 1 ) minor injury : presence of perirenal haematoma without evidence of renal parenchymal alteration 3 min after the i.v. 
administration of contrast ( 8 cases ) , essentially corresponding to aast grade 1 ; ( 2 ) moderate injury : perirenal haematoma with parenchymal laceration not extending to the renal hilum ( 14 cases ) , corresponding to aast grades 2 and 3 ; ( 3 ) severe injury : characterised by a deep laceration extending to all parenchymal components with involvement of the hilar region , a condition only in part corresponding to aast grades 3 and 4 , as the method does not allow us to establish whether or not the collecting system is intact ( 6 cases )  . discussion and conclusions our study shows that contrast - enhanced us has a high level of diagnostic accuracy in the assessment of renal trauma . 
it should be noted that the technique offers additional advantages over conventional us ( typically , absence of ionising radiation , fast examination times , and ready accessibility ) , in that it allows accurate evaluation of heavily vascular parenchymas such as the spleen , the liver and the kidneys . this enables the identification of lacerations - contusions and nali adoperando lo stesso protocollo desame descritto per la valutazione desordio . le lesioni identificate sono state classificate in cinque diversi gradi di gravit crescente secondo quanto definito dallaast : grado 1 , ematoma subcapsulare ; grado 2 , lacerazione superficiale renale con ematoma perirenale ; grado 3 , lacerazione renale profonda ( superiore al centimetro ) senza interessamento del sistema escretore e evidenza di stravaso urinario ; grado 4 , lacerazione interessante la struttura cortico - midollare ed il sistema escretore , coinvolgimento dellarteria renale principale , trombosi di unarteria segmentaria senza evidenti lacerazioni parenchimali ; grado 5 , grave lesione destruente il nefroparenchima che appare sovvertito con devascolarizzazione , distruzione dellarteria renale , occlusione port - traumatica dellarteria renale [ 5 ]  . risultati nei 277 pazienti valutati , di cui 45 con macroematuria posttraumatica , lecografia basale ha identificato 5 casi positivi per lesione parenchimale renale con associato ematoma perirenale . 
la tcms , integrata da ricostruzione mpvr , conferma il quadro dellesame eco - contrastografico ( c , d )  . haematomas , which appear as more or less irregularly hypoechoic and avascular areas . 
in some cases , it also visualises active bleeding as a hyperechoic blush of contrast material secondary to the interruption of a vascular structure ( fig . 3a , b ; clip 3 ) [ 4 , 69 ]  . 
a further advantage of contrast - enhanced us compared with conventional us is the better and more precise depiction of peritoneal and retroperitoneal fluid collections that results from improved definition of enterocolic wall structures , bladder and abdominal organs [ 10 ]  . nonetheless , there are also some limitations to the technique that have to be borne in mind : cost of the contrast material , need for scanners with dedicated software , need for operators familiar with this type of examination , and longer examination times . 
moreover , contrast - enhanced us does not demonstrate lesions to the urinary tract but only provides discussione e conclusioni dalla nostra esperienza emerge lelevata accuratezza diagnostica delleco - contrastografia nella valutazione del trauma renale . 
vi da precisare che tale tecnica presenta alcuni ulteriori vantaggi rispetto a quelli tipici dellesame ecografico classico ( assenza di radiazioni ionizzanti , rapidit di esecuzione , facilit di accesso ) che sono rappresentati da buona valutazione dei parenchimi fortemente vascolarizzati come milza , fegato e reni . 
2a - c baseline ultrasound ( us ) depicts the perirenal fluid collection ( a )  . contrast - enhanced us with cadence contrast pulse sequencing shows both the free fluid collection and the upper pole laceration ( arrow ) ( b )  . 
quadro tc corrispondente ( c )  . indirect evidence of injuries to the renal vascular pedicle by depicting areas of renal infarction . as regards the first point , in our experience , the use of contrast - enhanced us appears unjustified in all cases of minor injury without haematuria . 
the findings in our patients indicate that contrast - enhanced us was more likely to be positive in the following conditions , listed in order of decreasing frequency : in the presence of traumatic gross haematuria , gross haematuria or microhaematuria following renal biopsy or lithotripsy performed during the previous 12 h , traumatic micro - haematuria and normal urinalysis . 
for this reason , we have adopted the following approach in the workup of minor trauma : baseline us for stable patients with normal urinalysis , followed by contrast - enhanced us after obtaining consent to the use of the contrast agent , only in the event of a diagnostic doubt . 
in patients with a minor trauma and gross or microhaematuria or those who have undergone potentially harmful procedures within the previous 12 h , baseline us is always followed by the contrast examination . this approach allows a more accurate screening of patients not only for renal lesions but also for commonly associated injuries to other organs . 
in addition , it allows considerable savings in terms of both financial and staff resources . ct , preferably with a multidetector technique , remains the gold standard imaging test in the polytrauma patient , as it allows assessment of the renal parenchyma and vasculature and the urinary tract [ 5 , 6 , 1114 ]  . 
sonography with secondgeneration contrast material should be regarded as a technique that allows only definitely positive cases to be selected zione delle strutture parietali enterocoliche , della vescica e dei vari organi endoaddominali [ 10 ]  . accanto ai vantaggi vi da tener conto anche degli svantaggi : elevato costo del mezzo di contrasto , necessit di apparecchi ecografici con software dedicati , abitudine da parte delloperatore a questa tipologia desame , allungamento dei tempi desame . 
lecografia con mdc , inoltre , non documenta le lesioni delle vie escretrici urinarie ma indirettamente quelle del peduncolo vascolare renale identificando aree infartuali parenchimali renali . per il primo punto , dalla nostra esperienza , non sembra giustificato luso delleco - contrastografia in tutti i traumi minori senza ematuria . 
infatti come riportato nei risultati dei pazienti esaminati , la possibilit di una positivit dellesame eco - contrastografico era pi alta , con frequenza decrescente , in pazienti con macroematuria post - traumatica , macroematuria o micrometaturia in pazienti sottoposti nelle 12 ore precedenti a procedure bioptiche renali o di litotrissia , microematuria post - traumatica , pazienti con esame delle urine normale . 
evidence of active blush ( white arrow ) in the right perirenal space with free fluid collection ( asterisk ) ( a )  . multidetector computed tomography ( mdct ) confirms the findings , with optimal definition of perirenal and retroperitoneal extension . 
la clip allegata conferma il quadro ecografico ( b , c )  . delle urine nella norma , nel caso di dubbio diagnostico si procede , previo consenso alleco - contrasto . 
in pazienti con trauma minore con macroo microematuria , o sottoposti nelle 12 ore precedenti a procedure potenzialmente lesive , lecografia basale viene seguita in ogni caso dalleco - contrasto . 
questa condotta inoltre permette un notevole risparmio sia in termini di risorse economiche che gestionali del personale preposto . resta da ricordare che comunque il gold standard nel paziente traumatizzato resta sempre la tc , meglio se multidetettore , tecnica che permette di valutare sia laspetto parenchimale , vascolare ed escretore del rene [ 5 , 6 , 1114 ]  . leco - contrastografia con mdc di ii generazione deve essere considerata come metodica in grado di avviare alla valutazione tc quei pazienti sicuramente positivi per documentare lintegrit della via escretrice e del peduncolo vascolare [ 1416 ]  . 
lecografia con mdc appare metodica fondamentale nel follow - up delle lesioni riducendo cos lesposizione radiante in pazienti spesso giovani , a volte pediatrici , considerando anche che frequentemente la terapia del trauma renale di tipo conservativo [ 4 , 17 , 18 ]  . si ribadisce inoltre che la metodica non pu essere applicata nel soggetto politraumatizzato stabile in quanto questo tipo di paziente deve essere studiato con esame tc comprendente distretti corporei multipli [ 19 , 20 ]  . 
per questo motivo , nella nostra casistica , non compaiono casi di lesioni renali di alto grado in quanto si tratta di situazione critiche , spesso associate a lesioni di altri organi , che necessitano di valutazione tc che permette un adeguato bilancio del trauma . for ct assessment of the integrity of the urinary tract and vascular pedicle [ 1416 ]  . 
scuro 10 , 37134 verona , italy 2dipartimento di scienze radiologiche , policlinico umberto i , universit la sapienza , rome , italy 3reparto di chirurgia e gastroenterologia , policlinico g . 
over a period of 3 months , we prospectively studied 100 consecutive patients ( 36 men and 64 women ; age range 1587 years ; mean age 63 years ) referred to our institute for us imaging of the liver . volumetric acquisition of the liver was achieved with a 3d transducer ( 2.05.0 mhz ) and a logiq 9 us scanner . 
three categories were established : 1 = presence of focal liver lesions ; 2 = doubtful finding ; 3 = absence of focal liver lesions . concordance between volume us and conventional us was calculated by using the k statistic . 
all volume us examinations were technically adequate , allowing exploration of all hepatic sectors , except for five cases that were marred by major respiratory motion artefacts . conventional and volume us identified the same number of focal liver lesions , with the exception of four cases of doubtful findings at volume us . 
in un periodo di tre mesi , sono stati studiati consecutivamente , in modo prospettico , 100 pazienti ( 36 uomini e 64 donne ; range di et : 1587 anni ; media : 63 anni ) giunti alla sezione di ecografia del nostro istituto in regime di ricovero per eseguire un esame ecografico del fegato . 
gli esami ecografici convenzionale e volumetrico hanno identificato lo stesso numero di lesioni focali epatiche , ad eccezione di 4 casi radiol med ( 2009 ) 114 : 792801 conclusions . 
the identification of focal liver lesions on automated volume us is possible , and the examination shows a high level of concordance with conventional us . keywords ultrasound volume ultrasound liver focal liver lesions detection 3d risultati dubbi per la presenza di lesione focale epatica allesame ecografico volumetrico . 
lidentificazione delle lesioni focali epatiche su acquisizioni volumetriche automatiche del fegato possibile , risultando elevata la concordanza a tal fine dellesame volumetrico ecografico rispetto allesame ecografico convenzionale . parole chiave ecografia ecografia volumetrica fegato lesioni focali epatiche identificazione 3d introduction introduzione ultrasound ( us ) is the first - line imaging modality in the study of focal liver lesions [ 1 , 2 ] on account of its ready availability , inexpensiveness , safety and repeatability . conventional us has , however , been shown to have low sensitivity ( 40%71% ) for detecting hepatic metastases [ 35 ] and even lower levels ( 20%38.5% ) for focal liver lesions < 3 cm [ 6 , 7 ]  . 
the introduction of sonographic contrast agents has significantly improved detection and characterisation of focal liver lesions , achieving results often comparable to those of computed tomography ( ct ) and magnetic resonance ( mr ) imaging [ 3 , 810 ]  . 
conventional us is still the initial screening examination for chronic liver disease and oncological follow - up , and its role today remains firmly established in the detection of focal liver lesions [ 1 , 11 ]  . 
nonetheless , us suffers several limitations , both intrinsic and related to the patient ( obesity and bowel gas ) and / or site of the lesion under investigation , and extrinsic and related to the operators experience [ 12 , 13 ]  . 
automated 3d imaging of the liver could lead to reduced subjectivity of us examinations , allowing a reliable retrospective review of examinations following acquisition , similarly to what happens with other digital modalities such as ct and mr imaging . 
this technological application should therefore reduce operator dependency and enhance the effectiveness of cancer screening by improving the localization of lesions , an important aspect in planning conventional or minimally invasive surgical procedures . the aim of this study was to establish the reliability of automated 3d liver imaging in the detection of focal liver lesions by assessing the concordance between volume us and conventional us examinations . lecografia la metodica di imaging di primo livello nello studio delle lesioni focali epatiche [ 1 , 2 ]  . 
lesame ecografico convenzionale ha mostrato tuttavia bassa sensibilit , tra il 40% e 71% nellidentificazione di metastasi epatiche [ 35 ] raggiungendo addirittura valori ancor pi bassi , compresi tra il 20% e 38 , 5% per lesioni focali epatiche inferiori ai 3 cm [ 6 , 7 ]  . 
lintroduzione dei mezzi di contrasto ha apportato un significativo miglioramento nellidentificazione e nella caratterizzazione delle lesioni focali epatiche , con risultati paragonabili in molti casi alla tc e alla rm [ 3 , 810 ]  . 
lesame ecografico convenzionale risulta tuttora il primo esame nello screening delle epatopatie croniche e nei follow - up oncologici ed il suo ruolo , oggigiorno , rimane legato allidentificazione delle lesioni focali epatiche [ 1 , 11 ]  . nonostante ci rimangono alcuni limiti dellecografia di tipo intrinseco , derivanti dal paziente ( obesit e meteorismo ) e / o dalla sede della lesione in studio , sia estrinseci , legati allesperienza delloperatore [ 12 , 13 ]  . 
lacquisizione volumetrica automatica del fegato potrebbe portare ad una riduzione della soggettivit dellindagine ecografia consentendo una affidabile revisione dellesame retrospettiva e successiva allesecuzione tecnica dellesame stesso , analogamente a quanto avviene per altre metodiche di imaging digitali come tc ed rm . questapplicazione tecnologica dovrebbe quindi ridurre la dipendenza delloperatore e incrementare lefficienza dello screening di tumori con una pi accurata localizzazione delle lesioni , fatto questo di particolare importanza nella programmazione terapeutica chirurgica , o con tecniche mini - invasive percutanee . materials and methods patients over a period of 3 months , we prospectively studied 100 consecutive patients ( 36 men and 64 women ; age range 1587 years ; mean age 63 years ) referred to our unit to undergo us imaging of the liver . 
indications for the examination were renal colic , acute pyelonephritis , abdominal colic , arterial hypertension , fever , oncological evaluation , oncological follow - up and follow - up for chronic liver disease . 
 conventional us conventional us was carried out on a logiq 9 us scanner ( ge , healthcare , wauwatosa , wi , usa ; ge medical systems , milwaukee , wi , usa ) using a 3.55 mhz convex - array probe . 
to ensure optimal image quality , 2d b - mode imaging was optimised by appropriate setting of the speckle reduction , gain , rejection , and auto - optimisation parameters . 
conventional us was performed with scans in the epigastric , right hypochondriac and right intercostal region with the patient in the supine position , followed by right intercostal region with the patient in left lateral decubitus . 
 volume us volume us of the entire liver was performed with a volumetric convex - array 4d3c transducer ( 2.05.0 mhz ) using the same logiq 9 scanner and the volume us software volume imaging protocol ( vip )  . 
prior to acquisition , we set the spatial resolution function to maximum ( hi2 ) to improve overall quality of the 3d image and the angle of the volume window to 75 to acquire the largest amount of data possible for each single scan . 
the examination involved an initial volumetric acquisition of the left lobe of the liver , followed by an acquisition of the central sectors with the transducer placed at the level of the hepatic hilum , and finally acquisition of the paramedian and posterior segments of the right lobe with a more lateral scan . 
some patients with hepatomegaly necessitated additional scans ( up to a maximum of seven scans )  . data storage and review data were stored in digital imaging and communications in radiol med ( 2009 ) 114 : 792801 lo scopo del nostro lavoro di stabilire laffidabilit dellacquisizione volumetrica automatica del fegato nellidentificazione delle lesioni focali epatiche , valutando la concordanza tra gli esami ecografici volumetrico e convenzionale . materiali e metodi pazienti in un periodo di tre mesi , sono stati studiati consecutivamente , in modo prospettico , 100 pazienti ( 36 uomini e 64 donne ; range di et : 1587 anni , media : 63 anni ) giunti alla sezione di ecografia del nostro istituto in regime di ricovero per eseguire un esame ecografico del fegato . 
le indicazioni allesecuzione dellindagine erano : colica renale , pielonefrite acuta , coliche addominali , ipertensione arteriosa , febbre , valutazione oncologica , follow - up oncologico , follow - up per epatopatie croniche . 
 ecografia convenzionale lesame ecografico convenzionale stato eseguito con apparecchio ecografico logiq 9 ( ge , healthcare , wauwatosa , wi , usa ; ge medical systems , milwaukee , wi , usa ) , mediante sonda convex 3 , 55 mhz . 
per garantire la migliore qualit di immagine , stata effettuata una ottimizzazione dellimaging 2d b - mode mediante unadeguata impostazione dei parametri speckle reduction imaging , gain , rejection , auto optimization . 
lesame convenzionale stato condotto con scansioni in regione epigastrica , in ipocondrio di destra ed in regione intercostale destra in posizione supina , quindi in regione intercostale destra con decubito sul fianco sinistro . 
 ecografia volumetrica lacquisizione volumetrica dellintero fegato stata effettuata mediante sonda 4d3c convex volumetrica ( 2 , 05 , 0 mhz ) su medesima apparecchiatura ecografica logiq 9 con software volumetrico ecografico vip ( volume imaging protocol )  . 
prima di effettuare lacquisizione , sono stati regolati la funzione spatial resolution , impostata al massimo livello ( hi2 ) per un miglioramento della qualit dellimmagine volumetrica complessiva e langolo della finestra volume , impostato a 75 in modo da comprendere il massimo dei dati acquisibili per ogni singola scansione . 
lesame si svolto inizialmente con lacquisizione volumetrica del lobo sinistro del fegato ; quindi posizionando la sonda allaltezza dellilo epatico si radiol med ( 2009 ) 114 : 792801 medicine ( dicom ) format on a workstation equipped with logiqworks software , which was used to review the cases . volumes acquired frame by frame are displayed in a loop format ( volume review ) , which enables review of the entire volume , providing virtual scans of examined portions of the liver . 
multiplanar and volumetric reviews and reconstructions were possible on both the us system and the workstation , which offered the possibility of varying parameters such as baseline gain , time gain compensation or automatic optimisation during postprocessing . 
concordance between volume and conventional us with regard to the presence or absence of focal liver lesions was calculated by using the k statistic . results patients out of 100 patients studied , 39 were found to have focal liver lesions . 
in particular , 37 benign focal lesions were identified in 32 patients ( hepatic cysts in 23 patients for a total of 28 lesions ; hepatic angiomas in eight patients ; focal nodular hyperplasia in one patient ) , and 13 malignant focal lesions were identified in seven patients ( metastases in four patients for a total of ten lesions ; hepatocellular carcinoma in three patients )  . 
of the 23 patients with hepatic cysts , 17 had had the cysts detected at a previous us examination compared with which the cysts had remained unchanged in ottenuta lacquisizione dei settori centrali ; infine la scansione pi laterale ha acquisito i segmenti paramediani e posteriori del lobo destro . 
talora si sono rese necessarie scansioni aggiuntive ( fino ad un massimo di 7 scansioni ) in pazienti con epatomegalia . archiviazione e revisione i dati sono stati archiviati in formato dicom ( digital imaging and communication in medicine ) su una workstation fornita di software logiqworks . 
sono state possibili rivalutazioni e ricostruzioni multiplanari e volumetriche , sia sullapparecchio ecografico sulla workstation , mantenendo la possibilit di variare in post - processing i parametri quali ad esempio il guadagno basale o il tgc ( time gain compensation ) o lottimizzazione automatica . 
la concordanza dellacquisizione volumetrica con lesame ecografico convenzionale circa la presenza / assenza di lesioni focali epatiche stata calcolata con il k test . risultati pazienti su 100 pazienti studiati , in 39 sono state identificate lesioni focali epatiche . 
in particolare , in 32 pazienti sono state riconosciute 37 lesioni focali benigne ( cisti epatiche in 23 pazienti per un totale di 28 lesioni ; angiomi epatici in 8 pazienti ; iperplasia focale nodulare in 1 paziente ) , ed in 7 pazienti sono state identificate 13 lesioni focali maligne ( metastasi in 4 pazienti per un totale di 10 lesioni ; carcinoma epato - cellulare in 3 pazienti )  . 
 796 radiol med ( 2009 ) 114 : 792801 size ; in the remaining six patients , the cysts were a new finding . conventional us all examinations were technically adequate , enabling sonographic exploration of all hepatic sectors . 
examination time for exploration of the liver to identify or rule out the presence of focal liver lesions varied from 3 to 8 min in relation to the patients build and liver size , with a median time of 5 m tutte le lesioni focali maligne erano state precedentemente diagnosticate con imaging tc ed rm e giungevano alla nostra osservazione in corso di follow - up oncologico . cinque angiomi e liperplasia focale nodulare erano state precedentemente diagnosticate con esame rm ed giungevano per un follow - up ecografico . 
mentre nei restanti sei pazienti le cisti epatiche risultavano un nuovo rilievo . volume us ecografia convenzionale all 3d examinations were technically adequate , enabling sonographic exploration of all hepatic sectors , with the exception of five that were marred by severe respiratory motion artefacts . 
in view of the fact that during this time the system is unable to acquire scans , this time interval was added to each volumetric scan in calculating overall examination time . 
thus , when three volumetric scans were used to acquire the entire liver , overall examination time was approximately 133 = 39 + 503 = 189 s , whereas in the cases requiring seven scans , examination time was 137 = 91s + 503 = 241 s . 
overall time for a diagnostic volume us examination of the liver thus ranged from a minimum of 189 + 1210 = 309 s ( approximately 5 min ) to a maximum of 241 + 1210 = 361 s ( approximately 6 min )  . 
il tempo di esecuzione impiegato per il solo studio del fegato esclusivamente ai fini di identificazione / esclusione di lesioni focali epatiche risultato variabile , a seconda della corporatura del paziente e delle dimensioni del fegato , da 3 ad 8 minuti con un valore mediano pari a 5 minuti . 
 ecografia volumetrica tutti gli esami volumetrici sono risultati tecnicamente adeguati , in relazione alla esplorabilit ecografica di tutti i settori epatici , tranne 5 gravati da importanti artefatti da movimento respiratorio . 
per ottenere lacquisizione dellintero parenchima epatico sono state necessarie da 3 a 7 scansioni volumetriche automatiche della durata di 13 secondi ciascuna , con una media di 4 , 64 acquisizioni / paziente . 
in considerazione del fatto che nel corso di questo arco temporale lapparecchiatura non pu espletare scansioni , lo stesso stato sommato ad ogni scansione volumetrica ai fini del calcolo del tempo complessivo di esecuzione dellindagine . quindi nel caso di tre scansioni volumetriche per lacquisizione dellintero impiegato circa fegato , abbiamo 133 = 39 + 503 = 189 s . 
il tempo impiegato per la revisione dei volumi risultato dal numero di volte che ciascun filmato stato ripetuto per la lettura diagnostica che risultato variabile da 8 a 17 ( mediana = 12 )  . 
il tempo complessivo per un esame diagnostico volumetrico del fegato quindi risultato variabile da un minimo di 189 + 1210 = 309 s pari a circa 5 min ad un massimo di 241 + 1210 = 361 s pari a circa 6 min . gli esami ecografici convenzionale e volumetrico hanno identificato lo stesso numero di lesioni focali epatiche radiol med ( 2009 ) 114 : 792801 fig . 
istantanea ( frame ) del filmato dellacquisizione volumetrica del lobo epatico di destra visionato in modalit volume review . piccola lesione rotondeggiante solida ipoecogena ( freccia ) si identifica alla cupola epatica . table 1 concordance between volume ( vus ) and conventional ( us ) ultrasound presence doubtful absence total presence doubtful absence total kappa statistic presenza dubbio assenza totale k statistica 0.92 0 , 92 tabella 1 concordanza tra esame ecografico volumetrico ( vus ) e convenzionale ( us ) presenza assenza dubbio totale conventional us did not substantially increase the examination time to identify or rule out the presence of focal liver lesions . 
in tutti e 4 i casi dubbi si era in presenza di un fegato disomogeneamente steatosico allindagine ecografica . la concordanza tra acquisizione volumetrica automatica del fegato ed esame ecografico convenzionale ( tabella 1 ) risultata elevata ( k = 0 , 92 )  . 
non stato riscontrato un sostanziale aumento dei tempi di esecuzione dellindagine diagnostica ecografica finalizzata alla identificazione / esclusione di lesioni focali epatiche utilizzando lesame volumetrico rispetto al convenzionale . discussione le nuove tecnologie applicate agli ultrasuoni hanno consentito di ottenere un metodo che acquisisce ad ogni scansione dati volumetrici grazie ai quali possibile ricostruire lintero organo visualizzandolo nellarco di secondi . 
le misurazioni tridimensionali , originariamente , avvenivano con sistemi a mano libera ( free - hand ) e vennero utilizzati principalmente in ambito ginecologico , ostetrico e senologico [ 14 , 15 ]  . 
successivamente sono stati introdotti sistemi automatici che , modificando meccanicamente il trasduttore facendolo traslare o ruotare a predefiniti intervalli spaziali o 798 radiol med ( 2009 ) 114 : 792801 fig . 
la dinamicit dellesame ecografico convenzionale ha consentito di riferire la lesione ad area indenne da statosi . discussion the application of new technologies to us have led to the development of a technique that acquires volumetric data at each scan , thanks to which it is possible to reconstruct and display the entire organ within seconds . 
automated systems were subsequently introduced in which mechanical assemblies make the transducer translate or rotate at predefined spatial or angular intervals ( linear scanning , tilt scanning , rotational scanning )  . these automated systems have allowed organs to be imaged without requiring the usual freehand scans and thus dramatically reducing scanning time , which has always been one of the greater technical limitations of us imaging [ 16 ]  . 
the images must be acquired during breathholding to limit artefacts due to respiratory motion [ 17 , 18 ]  . the data are then sent in dicom format to a workstation where new virtual 3d scans can be obtained of the initial volume of data in any plane . 
the us system used in our study is equipped with the vip volume imaging software angolari ( linear scanning , tilt scanning , rotational scanning ) , hanno consentito di campionare un organo senza le consuete scansioni manuali da parte delloperatore , riducendo drasticamente i tempi di acquisizione dellesame , che da sempre uno dei limiti tecnici pi evidenti dellesame ecografico [ 16 ]  . 
recentemente sono state anche introdotte sonde ( 2d array ) che , attraverso lemissione di un fascio ultrasonoro piramidale che copre il volume di interesse , consentono immagini 3d real - time . 
possibile quindi acquisire ad ogni scansione dati volumetrici grazie ai quali ricostruire lintero organo , visualizzandolo nellarco di secondi . le immagini devono essere acquisite in apnea per limitare artefatti legati a movimenti respiratori [ 17 , 18 ]  . 
i dati vengono poi inviati in formato dicom ad una workstation permettendo di effettuare nuove scansioni virtuali in 3d , in qualsiasi piano di scansione , partendo dal volume dei dati acquisito inizialmente . 
lapplicazione di questo sistema ci ha permesso di ottimizzare il lavoro del medico e di renderlo pi veloce , consentendo di studiare lintero volume epatico in modo rapido e senza dover modificare il decubito del paziente . 
il tempo impiegato nelle elaborazioni in post - processing dei dati volumetrici stato tra i 5 e i 10 minuti ( per i casi dubbi ) che , se valutato nellambito di uno studio ecografico volumetrico , comporta comunque una gestione vantaggiosa dei pazienti in termini di tempi di esecuzione e risoluzione dei radiol med ( 2009 ) 114 : 792801 protocol . 
application of this system allowed us to optimise and speed up the radiologists work by enabling a rapid study of the entire liver volume without having to change the patients position . 
the time required for postprocessing of the volume data was 510 min ( for doubtful cases ) that , if evaluated within the context of volume us , entails a fast patient management in terms of examination time and diagnostic interpretation compared with conventional us . 
the data set of 3d us images can be viewed and processed using techniques common to other digital imaging modalities such as ct and mr imaging , including arbitrary - plane slicing , multiplanar reformatting , nonplanar slicing , volume rendering or surface rendering [ 19 , 20 ]  . 
volume us in fact provides greater morphological , anatomical and pathological details [ 22 ] and could improve the follow - up of focal liver lesions by providing objective and repeatable visualisation of lesions at subsequent examinations . 
 maintaining the possibility of varying the imaging parameters during postprocessing volume reviews and multiplanar and 3d reconstructions on the us system and at the workstation proved to be helpful for clearing doubts regarding the presence and site of some focal liver lesions . in our study , no major differences were found between studying the liver with the vip protocol and conventional baseline us in detecting focal liver lesions . 
 [ 3 ] , the 40% sensitivity and 63% specificity of unenhanced us in detecting metastatic liver lesions are substantially lower than the values reached after contrast administration : 83% and 84% , respectively . 
the objectivity of the volumetric study would heighten the repeatability of contrast - enhanced examinations , especially in detecting metastases in the late phase , which is useful if not indispensable for follow - up during chemotherapy or antineoangiogenic treatments . quesiti diagnostici rispetto allesame ecografico convenzionale . 
il dataset delle immagini ecografiche tridimensionali pu essere visualizzato e adoperato , utilizzando tecniche comuni alle altre metodiche di imaging digitale , come la tc e la rm , ossia secondo piani arbitrari , ricostruzioni multiplanari , non planari o basate sullanalisi della superficie o sullanalisi del volume [ 19 , 20 ]  . 
lecografia volumetrica infatti , fornisce maggiori dettagli morfologici , anatomici e patologici [ 22 ] , ma soprattutto potrebbe permettere un miglior follow - up delle lesioni focali garantendo di visualizzare in successivi controlli , in maniera oggettiva e ripetibile le formazioni segnalate . 
 grazie allapporto delle acquisizioni volumetriche si pu , in tempi brevi , avere i medesimi risultati dellesame ecografico convenzionale per quanto riguarda il numero di lesioni focali epatiche identificate secondo i risultati ottenuti . 
in particolare nel presente studio la funzione volume review ha permesso di rivedere lintero volume ottenendo scansioni virtuali delle porzioni di fegato studiate ; ogni filmato stato ripetuto il numero di volte necessario per lettura diagnostica . le rivalutazioni e ricostruzioni multiplanari e volumetriche , sia sullapparecchio ecografico che sulla workstation , mantenendo la possibilit di variare in post - processing i parametri dellimmagine si sono rivelate utili nel dirimere i dubbi sulla presenza di alcune focalit epatiche e la loro sede . 
nel nostro lavoro , in definitiva non abbiamo rilevato importanti differenze tra lo studio del fegato mediante applicazione del protocollo vip rispetto allesame ecografico basale convenzionale nella identificazione delle lesioni focali epatiche . 
 [ 3 ] la sensibilit e specificit dellesame ecografico basale nella ricerca di lesioni epatiche metastatiche del 40% e 63% rispettivamente , sono sicuramente inferiori ai valori raggiunti dopo somministrazione di mdc , vale a dire del 83% e 84% rispettivamente . 
in un lavoro pubblicato recentemente , lesame ecografico con mezzo di contrasto ( ceus ) ha consentito di identificare il 17% in pi di metastasi epatiche da carcinoma del colon - retto , rispetto allesame basale ; laccuratezza diagnostica che raggiunge la ceus , oggi , compresa tra l85%96% [ 23 , 24 ]  . 
la oggettivit dellesame volumetrico favorirebbe la ripetibilit degli esami contrastografici , specie nella identificazione delle lesioni ripetitive in fase tardiva , utile se non indispensabile nel follow - up in corso di trattamenti chemioterapici o antineoangiogenetici . moreover , by providing a 3d visualisation of lesional microand macrovascularity , the use of contrast media in inoltre lapplicazione del mezzo di contrasto allecografia 3d - 4d seppur ancora limitato , visualizzando in 3d la micro800 radiol med ( 2009 ) 114 : 792801 3d4d us , albeit still limited , could constitute a useful tool for preoperative planning before tumour resection , offering optimal information on vascular anatomy . 
giaccone , palermo , italy 4department of radiology and cardiology , university hospital , parma , italy 5department of radiology and cardiology , erasmus medical center , rotterdam , the netherlands 6cardiology unit , public hospital san gennaro , asl napoli 1 , napoli , italy 7department of diagnostic and interventional radiology , public hospital ferrarotto , catania , italy correspondence to : g . 
runza , via francesco garrano 149 , po box 96018 , pachino , italy , tel . : + 39 - 329 - 9471038 , fax : + 39 - 0931 - 594735 , e - mail : grunza@sirm.org received : 30 march 2008 / accepted : 6 october 2008 / published online : 30 may 2009 springer - verlag 2009 abstract purpose . 
from november 2006 to june 2007 , 21 patients , [ 11 men , ten women ; age standard deviation ( sd ) : 62.710.8 years ] with a history of ascending aorta replacement underwent ecg - gated mdct and were prospectively included in our study . 
ascending aorta replacement had been performed with different surgical techniques : bentall - de bono ( four patients , 19% ) , tirone - david ( five patients , 23% ) , and modified tironedavid with creation of aortic neosinuses ( 12 patients , 57% )  . 
the aortic valve area ( 3.40.2 cm2 ) , the diameter of the sinotubular junction ( 31.61.8 mm ) , the diameter of the neosinuses in the case of modified tirone - david riassunto obiettivi . 
dal novembre 2006 al giugno 2007 sono stati inclusi prospetticamente nel nostro studio 21 pazienti ( 11 maschi , 10 femmine ; et media 62 , 710 , 8 anni ) non consecutivi sottoposti ad intervento di sostituzione dellaorta ascendente . 
i pazienti sono stati sottoposti a sostituzione dellaorta ascendente mediante intervento di bentall - de bono ( 4 pazienti , 19% ) , tirone - david ( 5 pazienti , 23% ) e tirone - david modificato con ricostruzione dei neo - seni ( 12 pazienti , 57% )  . 
in tutti i pazienti la tcmd cardio - sincronizzata ha permesso una chiara valutazione dellanulus valvolare , della radice aortica e dellaorta ascendente per cui stato possibile effettuare accurate misurazioni . 
accurate clinical and therapeutic characterisation of this complex group of diseases requires complete evaluation of the aorta and its main branches and , in particular , of the coronary tree . 
an accurate imaging technique is thus needed that is capable of confirming the clinical suspicion of cardiovascular disease and providing a correct , definitive and timely diagnosis [ 1 ]  . conventional angiography has long been the method of choice for evaluating the thoracoabdominal aorta , but it presents the significant disadvantage of being invasive [ 1 ]  . noninvasive or semiinvasive imaging techniques , such as transthoracic echocardiography ( tte ) , transoesophageal echocardiography ( mr ) ( toe ) , magnetic imaging and multidetector computed tomography ( mdct ) , have all proved to be effective techniques for aortic disease imaging [ 24 ]  . resonance the most frequently used noninvasive technique in clinical practice is ct , as it is readily available and enables , thanks to the recent development of multidetector technology , the simultaneous evaluation of the aorta and coronary tree [ 511 ]  . 
conventional angiography and mr angiography are instead seldom used as initial imaging techniques [ 12 ]  . in fact , thanks to the use of cardiac synchronisation software , mdct has expanded its clinical applications in aortic pathology to become a basic step not only for preoperative nellambito delle patologie cardio - vascolari le alterazioni dellaorta toraco - addominale occupano un posto di primo piano per la necessit di un precoce riconoscimento data la rapidit della loro evoluzione verso condizioni di massima urgenza . 
si tratta di un gruppo complesso di patologie che richiedono per un corretto inquadramento clinico - terapeutico una valutazione completa non solo dellaorta , ma anche dei principali rami arteriosi che da essa prendono origine e principalmente lalbero coronarico . 
da qui la necessit di una tecnica di imaging accurata che possa confermare il sospetto clinico di una patologia cardiovascolare e fornire una diagnosi corretta , completa e quanto pi rapida possibile [ 1 ]  . langiografia convenzionale per molto tempo ha rappresentato la tecnica di scelta nella valutazione dellaorta toraco - addominale , ma presenta quale considerevole svantaggio linvasivit [ 1 ]  . 
di contro , tecniche strumentali noninvasive o semi - invasive quali lecocardiografia trans - toracica ( tte ) , lecocardiografia trans - esofagea ( tee ) , la risonanza magnetica ( rm ) e la tomografia computerizzata multidetettore ( tcmd ) si sono tutte dimostrate efficaci nellimaging della patologia aortica [ 24 ]  . nella pratica clinica , il principale strumento diagnostico non - invasivo utilizzato , poich pi facilmente disponibile nelle strutture ospedaliere , la tc , che grazie al recente sviluppo della tecnologia multidetettore consente una contemporanea valutazione non invasiva dellaorta e dellalbero coronarico [ 511 ]  . 
langio - tc rappresenta , attualmente , lindagine iniziale pi utilizzata per le patologie dellaorta , soprattutto in caso di patologia acuta ; la tte e la tee sono considerate le indagini alternative perch facilmente eseguibili anche in condizioni di emerlangiografia genza al letto del paziente instabile ; radiol med ( 2009 ) 114 : 705717 planning but also for postoperative imaging following surgical replacement of the ascending aorta [ 13 , 14 ]  . the aim of this study was to define the role of ecggated mdct in the evaluation of the postoperative ascending aorta , with special reference to the techniques contribution to the anatomical and functional evaluation of the aortic root following conservative surgery . materials and methods study population from november 2006 to june 2007 , we prospectively enrolled 21 nonconsecutive patients ( 11 men , ten women ; mean age 62.710.8 years ) who had undergone replacement of the ascending aorta . 
the study included patients treated with the bentall - de bono procedure ( n = 4 , 19% ) , tirone - david ( n = 5 , 23% ) and modified tirone - david with the creation of neosinuses ( n = 12 , 57% )  . 
inclusion criteria were heart rate < 70 bpm ( spontaneous or induced after - blocker administration ) and 15to 20 - s inspiratory breath - holding capacity to prevent respiratory motion artefacts from impairing adequate image evaluation and interpretation . 
exclusion criteria were heart rate > 70 bpm , respiratory failure ( unstable clinical condition hindering adequate breath - holding during the scan ) , severe heart failure ( patients with elevated heart rate are usually unable to maintain a supine position ) , severe renal impairment ( serum creatinine > 2 mg / dl ) , hyperkinetic arrhythmias ( e.g. , atrial fibrillation , in particular with rapid ventricular response , extrasystole , etc . ) , thyroid disorders , known allergy to iodinated contrast agents and pregnancy . mdct scan protocol the study was conducted with an mdct scanner ( brilliance 64 , philips medical systems , eindhoven , the netherlands ) with 64 detector rows , each with a minimum thickness of 0.625 m this configuration of 640.625 mm enables the study of a volume corresponding to 40 mm along the patients longitudinal axis during each gantry rotation . all mdct angiographic studies were preceded by an unenhanced baseline scan to evaluate the distribution and amount of coronary calcium ( calcium scoringtm )  . 
calcium scoring was done with the following scan protocol : prospective ecg gating , number of detectors / collimation 161.25 mm , slice thickness 2.5 mm , rotation time 4 s , voltage 120 kv , current 100 mas , craniocaudal scan direction and convenzionale e langio - rm vengono invece raramente utilizzate come modalit iniziale di imaging [ 12 ]  . 
infatti , grazie alle possibilit di utilizzare software per la cardiosincronizzazione , la tcmd ha ampliato il ventaglio delle sue applicazioni cliniche nella patologia aortica rappresentando un tappa fondamentale non solo nel planning preoperatorio , ma anche negli interventi chirurgici di sostituzione dellaorta ascendente [ 13 , 14 ]  . scopo dello studio definire il ruolo della tcmd cardiosincronizzata nella valutazione dellaorta ascendente postchirurgica , con particolare attenzione al contributo che la tecnica pu fornire nella valutazione anatomica e funzionale della radice aortica dopo chirurgia conservativa . materiali e metodi popolazione di studio dal novembre 2006 al giugno 2007 sono stati arruolati prospetticamente un totale di 21 pazienti ( 11 maschi , 10 femmine ; et media 62 , 710 , 8 anni ) non consecutivi sottoposti ad intervento di sostituzione dellaorta ascendente . sono stati inclusi nello studio pazienti sottoposti a sostituzione dellaorta ascendente mediante intervento di bentallde bono ( 4 pazienti , 19% ) , tirone - david ( 5 pazienti , 23% ) e tirone - david modificato con ricostruzione dei neo - seni ( 12 pazienti , 57% )  . 
criteri di inclusione : frequenza cardiaca < 70 bpm ( spontanea o indotta dalla somministrazione di - bloccante ) ; capacit di unapnea inspiratoria per 1520 secondi , consentendo di evitare artefatti dovuti ai movimenti respiratori che impediscono unadeguata valutazione ed interpretazione dellimmagine . 
criteri di esclusione : frequenza cardiaca > 70 bpm ; insufficienza respiratoria ; stato clinico instabile che pu impedire unadeguata apnea durante la scansione ; scompenso cardiaco severo : generalmente sono pazienti non in grado di mantenere la posizione supina con una elevata frequenza cardiaca ; grave compromissione renale ( creatinina sierica > 2 mg / dl ) ; presenza di aritmie ipercinetiche ( ad esempio : fibrillazione atriale , soprattutto con alta risposta ventricolare , extrasistolia , ecc . ) ; disordini tiroidei ; allergia nota ai mezzi di contrasto iodati ; gravidanza . protocollo di scansione tcmd lo studio stato condotto con unapparecchiatura tcmd ( brilliance 64 , philips medical systems , eindhoven , olanda ) con una matrice composta da 64 file di detettori , ciascuno dello spessore minimo di 0 , 625 m la configurazione 708 radiol med ( 2009 ) 114 : 705717 density threshold for calcium detection 130 hu . 
to monitor the arrival of the contrast material , a circular a region of interest ( roi ) was drawn over the ascending aorta , and consecutive axial scans were obtained at that level with a 10s delay from the beginning of the injection . 
scans were always triggered automatically , with a fixed delay of 8.1 s after the threshold enhancement of + 90 hu had been reached within the roi ( table 2 )  . 640 , 625 mm permette di studiare per ogni rotazione il volume corrispondente a 40 mm lungo lasse longitudinale del paziente . tutte le tcmd angiografiche sono state precedute da una scansione senza somministrazione endovenosa di mdc mirata alla valutazione della distribuzione e quantificazione del calcio coronarico ( calcium scoringtm )  . 
il protocollo di scansione per lo score del calcio stato : modalit di sincronizzazione prospettica al tracciato ecg , numero di detettori / collimazione 161 , 25 mm , spessore di strato 2 , 5 mm , tempo di rotazione 4 s , voltaggio del tubo radiogeno 120 kv , tensione del tubo radiogeno 100 mas , direzione della scansione cranio - caudale , soglia di densit per lindividuazione del calcio 130 unit hounsfield ( uh )  . 
all data sets were studied on a workstation ( extended brilliance workspace , version 3.0.1.3200 , philips medical systems , eindhoven , the netherlands ) equipped with cardiac software ( cardiac review and comprehensive cardiac )  . image analysis the evaluation included the replaced portion of the aorta ( valve , root and ascending portion ) and the remnants of the thoracic aorta ( aortic arch and descending aorta )  . 
allo scopo di ottimizzare lopacizzazione dei vasi arteriosi coronarici la sincronizzazione dellinizio della scansione con il passaggio del bolo di mezzo di contrasto stata eseguita mediante tecnica del bolus tracking . 
per monitorare larrivo del mezzo di contrasto , stata posizionata una regione circolare di campionamento ( regione dinteresse , roi ) nel tratto ascendente del lume aortico , quindi , sono state ottenute delle scansioni assiali consecutive sempre allo stesso livello , con un ritardo di 10 secondi dallinizio delliniezione . 
la scansione sempre partita automaticamente con un ritardo fisso di 8 , 1 secondi dopo il raggiungimento allinterno della roi di una soglia di + 90 uh ( tabella 2 )  . ricostruzioni dei dataset sono state effettuate delle ricostruzioni retrospettive basate sul segnale elettrocardiografico per ottenere una qualit dellimmagine priva di artefatti da movimento . 
1 immagini mpr su differenti piani per la valutazione dei diametri della giunzione sino - tubulare dell ' anulus e dei seni di valsalva . telesistolica ( 40% dellintervallo r - r ) e quella telediastolica ( 70% dellintervallo r - r )  . 
tutti i dataset sono stati valutati su una workstation ( extended brilliance workspace , version 3.0.1.3200 , philips medical systems , eindhoven , olanda ) dotata di software cardiaco dedicato ( cardiac review e comprehensive cardiac )  . analisi delle immagini oltre al tratto di aorta sostituito ( valvola , radice e ascendente ) la valutazione stata estesa anche ad i restanti tratti dellaorta toracica ( arco e discendente )  . 
2 immagine mip ( maximum intensity projection ) della radice aortica e dellaorta toracica per la misurazione dei diametri del graft in dacron sul piano sopravalvolare ( a ) ed in prossimit dellanastomosi distale ( b ) , dellarco aortico ( c ) e dellaorta discendente ( d )  . results risultati in 18 patients , ecg - gated mdct provided a clear depiction of the aortic valve annulus , aortic root and ascending aorta , enabling accurate measurements of the valve area , sinotubular junction diameter , neosinus diameter in modified in 18 pazienti la tcmd cardiosincronizzata ha permesso una chiara valutazione dellanulus valvolare , della radice aortica e dellaorta ascendente per cui stato possibile effettuare accurate misurazioni . 
grazie a questi dati , stato possibile calcolare larea valvolare , il diametro della radiol med ( 2009 ) 114 : 705717 giunzione sino - tubulare , il diametro dei neo - seni nel caso di tirone - david modificato e la distanza minima cuspidigraft in fase sistolica . 
tutti i valori ottenuti sono rientrati nel range di normalit dimostrando una buona correlazione ( r = 0 , 89 ) con quelli della tte ( tabella 3 )  . 
3 esempio dei criteri qualitativi / quantitativi utilizzati per la valutazione della distanza tra i lembi valvolari e il graft impiantato ( frecce curve )  . tirone - david procedures and the minimum distance between cusps and the graft during systole . 
4a , b a 72 - year - old man who underwent ascending aorta replacement with bentall - de bono technique for aneuryscomputed tomography ( ct ) angiography performed 6 months approximately after surgery . 
a maximum intensity projection ( mip ) reconstruction showing the mechanical aortic valve ( red curved arrows ) , the prosthetic tube ( arrowheads ) and the distal anastomosis of the graft ( arrows )  . 
b multiplanar reconstruction ( mpr ) images on the supravalvular plane illustrating the centre of the reimplanted coronary ostia and the suture material ( arrows )  . cdx , right coronary artery ; tc , left ma fig . 
a ricostruzione mip rappresentativa della valvola meccanica aortica ( frecce rosse curve ) , del tubo protesico ( punte di freccia ) e dellanastomosi distale del graft ( frecce )  . 
a three - dimensional volume - rendered ( 3d - vr ) reconstructions of the heart and thoracic aorta displaying the distal anastomosis of the dacron graft reinforced with a teflon felt strip ( arrowheads ) and the reimplanted coronary ostia ( arrows )  . 
tepi , epiaortic vessels ; arao , aortic arch ; aoa , ascending aorta ; ao , d descending aorta ; cdx , right coronary artery ; iva , left descending coronary artery ; vdx , right ventricle ; vsn , left ventricle ; tap , pulmonary artery ; adx , right atriuc mpr image in an oblique - axial plane of the native aortic valve . 
a ricostruzione 3d - vr del cuore e dellaorta toracica in cui visualizzata lanastomosi distale del tubo in dacron rinforzato con un anello in teflon ( punte di freccia ) e degli osti coronarici reimpiantati ( frecce )  . 
tepi , tronchi epiaortici ; arao , arco aortico ; aoa , aorta ascendente ; aod , aorta discendente ; cdx , arteria coronaria destra ; iva , arteria discendente anteriore ; vdx , ventricolo destro ; vsn , ventricolo sinistro ; tap , tronco arteria polmonare ; adx , atrio destro . 
una frequenza cardiaca < 70 bpm , spontanea o indotta da bloccanti considerata sufficiente per aumentare il tempo diastolico e la durata telediastolica , momento in cui il cuore , la radice aortica e le coronarie sono quasi privi di movimento [ 15 , 16 ]  . 
 la valutazione della radice aortica di fondamentale importanza per il cardiochirurgo in quanto rappresenta il tratto di aorta che subisce maggiormente lo stress meccaradiol med ( 2009 ) 114 : 705717 fig . 
electrocardiographically ( ecg ) - gated multidetector computed tomography ( mdct ) scan reduces the artefacts secondary to heart beat and allows for accurate evaluation of the dacron graft implanted onto the native aortic valve and of the neosinuses . 
maximum intensity projection ( mip ) in the oblique - sagittal plane ( panel a ) and three - dimensional volume - rendered ( 3d - vr ) ( panel b ) reconstructions of the thoracic aorta to assess the distal anastomosis of the dacron graft ( arrows ) , the neosinuses ( arrowheads ) and the centre of the reimplanted coronary ostia . 
6 uomo di 54 anni sottoposto ad intervento di sostituzione dellaorta ascendente con tecnica tirone - david e ricostruzione dei neo - seni di valsalva . lacquisizione tcmd cardiosincronizzata riduce gli artefatti da movimento pulsatile del vaso e consente unaccurata valutazione della radice aortica e dei neo - seni ricostruiti . 
ricostruzioni mip su piano parasagittale ( immagine a ) e 3d - vr ( immagine b ) dellaorta toracica per la valutazione dellanastomosi distale del graft in dacron ( frecce ) , dei neo - seni ( punte di freccia ) e della sede di impianto degli osti coronarici . 
7 a 59 - year - old man with type a aortic dissection ( according to the stanford classification ) and chest pain of recent onset who underwent replacement of the ascending aorta and aortic valve ( bentall - de bono technique )  . 
c three - dimensional volume - rendered ( 3d - vr ) reconstruction of the heart and aorta displaying the distal anastomosis of the dacron graft , the periprosthetic leakage of contrast material ( arrows ) and the intimal flap in the descending aorta lumen ( arrowheads )  . d maximum intensity projection ( mip ) reformation showing angulation of the aortic graft ( thin arrow ) and the partially calcified intimal flap involving the aortic arch , the descending aorta ( arrowheads ) and the ostium and proximal tract of the left carotid artery ( thick arrow )  . 
7 uomo di 59 anni con dissezione aortica tipo a ( secondo stanford ) sottoposto ad intervento di sostituzione dellaorta ascendente e della valvola aortica ( tecnica bentall - de bono ) e dolore toracico di recente insorgenza . 
c ricostruzione 3d - vr del cuore e dellaorta in cui possibile evidenziare lanastomosi distale della protesi , lo spandimento di mdc periprotesico ( frecce ) ed il flap intimale che coinvolge laorta discendente ( punte di freccia )  . 
d ricostruzione mip che dimostra langolazione della protesi aortica ( freccia sottile ) ed il flap intimale parzialmente calcifico che coinvolge larco , laorta discendente ( punte di freccia ) , lostio ed il tratto prossimale dellarteria carotide sinistra ( freccia spessa )  . 
e , f ricostruzioni 3d - vr del cuore e dellaorta toracica in cui si evidenzia il flap intimale ( punte di freccia )  . radiol med ( 2009 ) 114 : 705717 discussion the latest generation of mdct scanners , with their high spatial and temporal resolution , allows images to be acquired during a single short breath - hold and thus with a high level of anatomical detail [ 5 ]  . 
ecg - gated acquisitions improve the quality of the study by markedly reducing cardiac motion artefacts and providing a clear depiction of the aortic root [ 15 , 16 ]  . 
a heart rate < 70 bpm , whether spontaneous or induced by - blockers , is considered sufficient to increase diastolic time and duration of end - diastole , when the heart , aortic root and coronary arteries are nearly motionless [ 15 , 16 ]  . evaluation of the aortic root is of primary importance for the cardiac surgeon . 
this portion of the aorta is most affected by mechanical stress during surgery , influences valve dynamics and is a common site of complications , as it provides the point of proximal fixation of the gra multiplanar reconstruction ( mpr ) and volume rendering ( vr ) techniques provide the cardiac surgeon with a comprehensive depiction of the aorta , the heart and implanted structures . mdct already represents the gold standard for ruling out the possible major complications of surgery , such as periprosthetic leakage or pseudoaneurysm formation [ 17 ]  . mpr images in standard and curved planes allow for extremely accurate and reliable measurements of the graft and remaining native aorta despite intrinsic bends and possible tortuosity . 
these measurements are essential , as their comparison with the reference values provides the cardiac surgeon with the necessary elements to assess the outcome of surgery . in the case of modified tirone - david procedures with neosinus creation , the distance between the valve leaflets and the graft wall during the systolic phase can also be measured . 
this measurement is regarded as a prognostic index for neosinus creation because a sufficient distance to ensure no contact between the cusps and the graft guarantees integrity of the valve leaflets over time . conventional angiography has long represented the method of choice for evaluating the thoracoabdominal aorta , despite its considerable invasiveness [ 1 ]  . 
noninvasive imaging techniques such as toe , tte and mr angiography have also proved to be effective in imaging aortic diseases [ 24 ] , even though they present some disadvantages : semiinvasiveness and difficult visualisation of the aortic arch due to the interposition of the trachea in the case of toe , and relatively long imaging times , limited availability , and artefacts linked to the presence of the prosthesis in the case of mr angiography . 
in clinical practice , rapidity and simplicity make tte an excellent method for studying the aortic root , although the technique is limited in depicting the middledistal ascending aorta in most cases and does not permit visualisation of the descending aorta . 
additionally , the examination may be undermined by the lack of a suitable nico durante lintervento , ne influenza la dinamica valvolare e costituendo la porzione di ancoraggio prossimale della protesi , pi coinvolta nellinsorgenza di complicanze . 
grazie alle tecniche di ricostruzione mpr e 3d - vr possibile fornire al cardiochirurgo una rappresentazione completa dellaorta , del cuore e delle strutture che ha impiantato . la tcmd rappresenta gi il gold standard per escludere le possibili maggiori complicanze legate allintervento quali spandimento periprotesico o formazioni di pseudoaneurismi [ 17 ]  . 
le immagini mpr nei piani standard e curvilinei , inoltre , consentono di effettuare misurazioni altamente accurate ed attendibili della protesi e della restante aorta nativa malgrado le curve intrinseche del vaso e leventuale tortuosit di decorso . 
queste misurazioni sono di fondamentale importanza , in quanto rapportate a valori di riferimento , forniscono al cardiochirurgo degli elementi per giudicare lesito dellintervento . nel caso di interventi di tirone - david modificato con ricostruzione dei neo - seni , si pu valutare in fase sistolica anche la distanza delle cuspidi valvolari dalla parete della protesi impiantata . 
questa misurazione viene considerata come indice prognostico sulle ricostruzioni dei neoseni , dal momento che , se questa distanza tale da evitare la concussione delle cuspidi con la protesi , lintegrit dei lembi valvolari nel tempo garantita . langiografia convenzionale per molto tempo ha rappresentato la tecnica di scelta nella valutazione dellaorta toraco - addominale , ma presenta , chiaramente , quale considerevole svantaggio linvasivit [ 1 ]  . 
di contro , altre tecniche strumentali non - invasive quali la tee , la tte e langio - rm si sono dimostrate efficaci nellimaging della patologia aortica [ 24 ] , sebbene presentino alcuni svantaggi : la semi - invasivit e le difficolt nella visualizzazione dellarco aortico per linterposizione della trachea nel caso della tee ; i tempi di esecuzione dellindagine piuttosto lunghi , la limitata disponibilit sul territorio , e gli artefatti legati alla presenza delle protesi nel caso dellangio - rm . 
nella pratica clinica , la tte rappresenta , ancora , una modalit eccellente per limaging della radice aortica , in virt della rapidit e semplicit dellindagine , ma presenta limitazioni nella visualizzazione dellaorta ascendente medio - distale , nella maggior parte dei casi , e non consente la visualizzazione dellaorta discendente . 
lo strumento diagnostico non - invasivo pi utilizzato rappresentato , pertanto , dalla tc , che risulta facilmente disponibile nelle strutture ospedaliere e che , grazie al recente sviluppo della tecnologia multidetettore con la possibilit di acquisire scansioni cardiosincronizzate , consente una contemporanea 716 radiol med ( 2009 ) 114 : 705717 acoustic window due to body habitus and artefacts related to the presence of prostheses . 
it is readily available in all hospitals and , with the recent development of the ecg - gated multidetector technique , allows for the simultaneous noninvasive evaluation of the ascending aorta , aortic root and coronary artery tree , almost free of cardiac motion artefacts [ 511 ]  . 
mdct technology with ecg gating has , however , overcome these limitations , and the accurate measurements obtained with multiplanar imaging increase the cardiac surgeons confidence in formulating a judgement on the outcome of ascending aorta replacement procedures . 
speed , noninvasiveness , panoramic imaging and depiction of postoperative complications represent further advantages of mdct angiography compared with the other available imaging modalities . valutazione non invasiva e pressoch priva di artefatti da movimento cardiaco dellaorta ascendente , della radice aortica e dellalbero coronarico [ 511 ]  . 
le accurate misurazioni ottenute grazie alla multiplanarit offerta dellimaging mediante tcmd consentono , inoltre , al cardiochirurgo una migliore confidenza nel formulare un giudizio sullesito dellintervento di sostituzione dellaorta ascendente . 
la rapidit , la non invasivit , la panoramicit , la capacit di identificare le complicanze post - chirurgiche costituiscono ulteriori punti di forza dellangiografia tcmd sopra le altre metodiche disponibili . 
mdct is a reliable tool for the anatomical and functional evaluation of the aortic root , and it provides the cardiac surgeon with new and detailed information needed to formulate a prognostic judgement on the outcome of surgery . conclusioni la tcmd considerata attualmente un momento diagnostico obbligatorio nel paziente con sospetta o nota patologia aortica . 
since the 1970s , technological innovation has been accompanied by the development of methods for the multidisciplinary assessment of the technical , scientific , economic , ethical and social aspects inherent in the use of new technologies . 
because the application of hta is still in its early stages in italy , it is necessary to promote its development by drawing on consolidated international experiences . keywords health technology assessment clinical efficacy of diagnostic procedures evidence - based medicine ( ebm ) health policy riassunto obiettivo . 
dagli anni 70 ad oggi linnovazione scientifica andata di pari passo con lo sviluppo di un metodo di valutazione multidisciplinare che si occupa di analizzare gli aspetti tecnici , scientifici , economici , etici , legali e sociali conseguenti allapplicazione delle nuove tecnologie . 
dato che in italia lapplicazione dellhta ancora ad uno stadio precoce , risulta necessario incentivarne lutilizzo assumendo le gi consolidate esperienze internazionali . parole chiave health technology assessment efficacia clinica delle procedure diagnostiche evidence based medicine ( ebm ) health policy 674 introduction the incessant development and innovation of health technology over the past few decades has called for the parallel development of methods for evaluating the appropriateness of health services and the outcomes and costs of new technologies . 
this latter aspect is evaluated in terms of efficacy ( ability to improve the patients well - being in relation to a specific health problem ) , safety ( the risks inherent in the use of a new technology and assessment of their acceptability ) , cost effectiveness and ethical , social and legal implications . hta is able to provide scientific evidence to inform different decision - making levels , from agencies licensing new pharmaceuticals or medical technologies to health care institutions deciding whether to purchase new equipment or how it should be managed . 
hta therefore integrates the expertise of multiple disciplines capable of supplying the evidence base required for the optimal application of technologies , systems and procedures in medicine . materials and methods our work consisted of three phases . 
in this phase we made use of search tools such as pubmed [ 1 ] , google [ 2 ] , the cochrane central register of controlled trials [ 3 ] , the database of the cochrane collaboration reviews [ 4 ] , the health technology assessment database [ 5 ] and the national health system ( nhs ) economic evaluation database [ 6 ] of the centre for reviews and dissemination ( crd ) , the health evidence network ( hen ) web site [ 7 ] , the web sites of the online journals care [ 8 ] and point of care bulletin [ 9 , 10 ] and the data contained in the trento charter drafted by the italian network of health technology assessment in 2006 [ 11 ]  . 
following a critical analysis of the data obtained from the web site of the international network of agencies for health technology assessment ( inahta ) [ 12 ] , we defined the current composition of this network . 
using the web site of the european network of health technology assessment ( eunethta ) [ 13 ] , we identified the main collaborative projects within the european union health care policies , whereas on the basis of the information provided by the italian network for hta [ 11 ] , we systematically analysed the italian national and regional hta projects . 
the third phase allowed us to define the potential impact of hta on the decision - making radiol med ( 2009 ) 114 : 673691 introduzione lincessante sviluppo ed innovazione delle tecnologie nel campo medico sanitario che si avuto negli ultimi decenni ha richiesto una contemporanea crescita di metodologie , atte a valutare lappropriatezza delle prestazioni , degli effetti prodotti e dei costi delle nuove strumentazioni . 
lhta se da una parte quindi necessario per la valutazione di una o pi propriet tecniche , dallaltra deve considerare limpatto socio economico che la stessa tecnologia pu condizionare . questultima di conseguenza dovr essere valutata congiuntamente sul piano dellefficacia ( capacit di migliorare lo stato di salute del paziente rispetto ad una precisa problematica ) , della sicurezza ( considerazione dei rischi nellutilizzo di una nuova tecnologia e valutazione della loro accettabilit ) , del rapporto tra costi ed efficacia , nonch degli aspetti etici , sociali e medico - legali . lhta in grado di supportare scientificamente diversi livelli decisionali , dalle agenzie che autorizzano limmissione sul mercato di un farmaco o di unaltra tecnologia sanitaria , alle organizzazioni sanitarie che spesso si trovano a doverne valutare lopportunit di acquisto o la modalit di gestione . 
si tratta dunque di una tecnica che integra gli sforzi di molteplici discipline atte a produrre le informazioni necessarie alla luce delle evidenze scientifiche al fine di unottimale applicazione delle tecnologie , sistemi e procedure in medicina . materiali e metodi il lavoro si articolato in tre fasi distinte . 
la prima fase si basata sulla revisione della letteratura recente per lidentificazione dei principi , dei metodi e degli strumenti dellhta , utilizzando come motori di ricerca pubmed [ 1 ] , google [ 2 ] , il database bibliografico cochrane central register of controlled trials [ 3 ] , i database di pubblicazioni e revisioni scientifiche della cochrane collaboration [ 4 ] , lhealth technology assessment database [ 5 ] e lnhs economic evaluation database [ 6 ] del centre for reviews and dissemination ( crd ) , il sito dellhen ( health evidence network ) [ 7 ] , i siti internet delle riviste scientifiche on - line care [ 8 ] e point of care bulletin [ 9 , 10 ] , i dati ottenuti dalla carta di trento , stilata dal network italiano di health technology assessment nel 2006 [ 11 ]  . la seconda fase stata quella della valutazione dello stato attuale dellhta a livello internazionale sia extra - europeo che europeo e nazionale italiano . 
in seguito allanalisi critica dei dati ottenuti dal sito internet dellinternational network of agencies for health technology assessment [ 12 ] si definita la composizione attuale di tale esperienza radiol med ( 2009 ) 114 : 673691 process in health care institutions by analysing several successful cases of hta applied at the institutional level . results hta refers to a systematic , multidisciplinary process of evaluating health technologies and consists of the in - depth assessment of technical attributes , efficacy , clinical safety , indications for use , economic impact and ethical , social and legal implications of the technology . 
in particular , health technology refers to any intervention that may be used to promote health , to prevent , diagnose or treat disease and for patient rehabilitation and management . 
this includes medical equipment , medical devices , pharmaceuticals , diagnostic systems , medical and surgical procedures and patient care pathways , as well as the institutional and organisational settings in which health care is provided [ 11 ]  . 
utilizzando il sito del european network of health technology assessment ( eunethta ) [ 13 ] si sono identificate le principali esperienze di collaborazione dellambito delle politiche per la salute dellue , mentre sulla base delle evidenze prodotte nellambito del network italiano di hta [ 11 ] sono state presentate in maniera sistematica le esperienze di hta a livello nazionale e nelle singole regioni italiane . 
la terza fase ha permesso di definire il potenziale contributo dellhta al processo di decision making allinterno delle aziende sanitarie , analizzando alcuni casi eccellenti di applicazione di tale metodologia a livello organizzativo . risultati lacronimo ( health technology assessment ) hta definisce un processo di valutazione globale e multidisciplinare delle tecnologie utilizzate per lassistenza sanitaria e consiste in una verifica dettagliata delle caratteristiche tecniche , dellefficacia , della sicurezza clinica , delle indicazioni duso , dellimpatto economico nonch degli aspetti etici , sociali e medico - legali della tecnologia stessa . 
in particolare per tecnologia sanitaria si intendono tutti gli interventi usati nel promuovere la salute , prevenire , diagnosticare e trattare una malattia , nellattuare una riabilitazione e nel gestire i pazienti . 
in altre parole , una tecnologia sanitaria comprende le attrezzature sanitarie , i dispositivi medici , i farmaci , i sistemi diagnostici , le procedure mediche e chirurgiche ed i percorsi assistenziali , nonch gli assetti strutturali ed organizzativi nei quali viene erogata lassistenza sanitaria [ 11 ]  . 
in particolare , lhta valuta i seguenti aspetti : performance , applicabilit e indicazioni : la sensibilit e la specificit dei test diagnostici ; la conformit , affidabilit , semplicit di utilizzo , di manutenzione , di sicurezza di uno strumento o procedura sanitaria ; sicurezza clinica : considerazione dei rischi nellutilizzo di una nuova tecnologia ; efficacia : il beneficio che si ottiene sia utilizzando una tecnologia in relazione ad un preciso problema in condizioni ideali ( efficacia teorica : efficacy ) sia in condizioni generali di routine ( efficacia nella pratica : effectiveness ) ; data dal rapporto tra i risultati finali in termini di miglioramento della salute attribuibile alle prestazioni sanitarie erogate ( output ) e le prestazioni stesse ( outcome ) ; economicit ( costo , costo - efficienza , costo - beneficio ) : riguarda , a livello microeconomico , i costi , le tariffe e le modalit di rimborso ; a livello macroeconomico si riferisce , invece , alle conseguenze che nuove tecnologie possono avere sui costi della sanit a livello nazionale o agli effetti che una tecnologia pu avere sullallocazione 676 radiol med ( 2009 ) 114 : 673691 moreover , in view of the longer life expectancy of patients , a more consistent use of technologies and procedures during their lifetime will result in less discomfort , a more trusting relationship with the health care system and improved diagnostictherapeutic performance . 
 with regard to diagnostic imaging , the primary purpose of hta is to ensure the use of technologies that guarantee the most accurate and robust information from diagnostic imaging tests to optimise and increase the efficiency of both the physicians clinical and treatment decisions and the patients outcome . there are several key points to be considered to ensure that the health policy goals of an hta programme are attained : 1 . 
the evaluation of health technologies is not only a necessity for the governance of health care systems but also an opportunity that allows them to deliver qualified health services while controlling expenditure and ensuring a sustainable social and ethical impact . an hta project involves nine steps : 1 . 
in tema di implicazioni sociali particolarmente importante considerare che una strategia di valutazione multidisciplinare porta ad una migliore definizione ed applicazione di progetti che caratterizzano ed ampliano i diritti dellutente del sistema sanitario : definizione dei livelli essenziali di assistenza ; utilizzo delle migliori tecnologie / procedure in termini di costo - beneficio ; migliore informazione al paziente sulle tecnologie / procedure utilizzate . inoltre , vista laumentata aspettativa di vita del paziente , un uso pi coerente delle tecnologie e procedure a cui verr sottoposto nel corso della sua vita comporta sia una riduzione dei disagi , sia un miglior rapporto di fiducia con il sistema sanitario , nonch un miglioramento della qualit della performance diagnostico - terapeutica . 
 per quanto riguarda la diagnostica per immagini lo scopo primario quello di assicurare lutilizzo di tecnologie che garantiscano le pi accurate e solide informazioni possibili dagli esami di diagnostica per immagini per ottimizzare e rendere pi efficienti sia le decisioni cliniche e di trattamento del medico , sia loutcome del paziente . esistono dei punti fondamentali su cui un health technology assessment deve vertere affinch si possano ottenere gli obiettivi di politica sanitaria che si propone : 1 . 
prima di partire con un determinato studio deve esistere tra gli attori interessati e le autorit competenti un coordinamento in modo da ottimizzare i risultati ottenibili , la qual cosa non pu che venir favorita dallesistenza di organismi specificatamente votati allattivit di hta [ 14 ]  . 
useful sources for data and evidence include electronic databases of published literature , electronic databases of clinical and administrative data , institutional or noninstitutional reports and publications , special inventories and specialised generating evidence for an hta project may involve collecting new data . 
tale passaggio , identificato in letteratura come research question , rappresenta una specificazione , in termini empirici , della policy question : significa , in altre parole , inquadrare il problema oggetto di studio 678 radiol med ( 2009 ) 114 : 673691 5 . 
some of the possible effects of an hta project are : acquisition or adoption of a new technology changes to the frequency of use reduction or increase of a technology new allocation of resources within regional or national health care services changes to marketing plans for a given technology [ 8 ]  . hta in the field of diagnostic imaging can make use of a hierarchical model where the general rule holds true that for an imaging examination to be efficacious at a higher level , it must be efficacious at lower levels , although the reverse is not true [ 15 ]  . 
the value of the use of a hierarchical model is based on three key concepts : the concept of the global systems view of diagnosis the production and use of diagnostic information is a continuum a hierarchical model is well - suited to cost - efficiency analysis the use of this type of model to evaluate the various aspects ( technical and nontechnical ) of medical procedures is recommended , as it is simple to use , easily repeatable and helps to ensure important steps are not overlooked by sulla base dellevidenza scientifica , di ben definiti strumenti e misure dellefficacia . 
linterpretazione delle prove implica un processo di classificazione degli studi al fine di conferire a ciascuno di essi un peso e la conseguente opportunit di includerli o meno nella sintesi . 
table 1 shows a model for evaluating clinical efficacy that develops over six successive levels : in level 1 , the properties of a specific imaging technique are evaluated in traditional physical terms such as resolution , sharpness , greyscale and specific measures for digital imaging , as well as the potential for artefacts and operator error . level 2 takes into account that images must be read by a human observer in order to obtain diagnostic information and that accuracy is a function of both image technical quality and interpretation by a human observer . in level 3 , the efficacy of a diagnostic test is evaluated based on the assumption that the imaging information can affect the physicians diagnostic thinking : the more the examination influences the diagnosis , the more helpful it is to the diagnostic process . essere indirizzati ai tre livelli macro - , mesoe micro -  . 
monitoraggio dellimpatto ottenuto dalla valutazione . alcuni degli effetti a cui un hta pu condurre sono : acquisizione o adozione di una nuova tecnologia ; cambiamento della frequenza duso in termini riduttivi o di incremento di una tecnologia ; nuova allocazione di risorse nellambito sanitario regionale o nazionale ; level 4 evaluates the impact of a diagnostic test on the modificazione della pianificazione di marketing di una physicians choice of treatment . determinata tecnologia [ 8 ]  . level 5 analyses the positive impact of a diagnostic test on patient outcome in terms of reduction of morbidity , reduction in number of required health procedures , improved life expectancy and improved quality of life . level 6 addresses the cost benefit and cost - effectiveness of diagnostic imaging procedures to ascertain whether their impact on society is acceptable . table 2 describes the levels of hta of the diagnostic performance of a given technology : level a assesses whether the technology provides good - quality images that are anatomically representative ; level b evaluates whether imaging allows accurate diagnoses to be made ; level c determines the diagnostic impact of imaging ; level d considers the therapeutic impact of the technology ; level e addresses the impact of imaging on the patients health . table 3 illustrates the hta evaluation of the cost - effectiveness of a technology at each level technical and diagnostic performance , diagnostic and therapeutic impact and impact on health . 
2 shows a hierarchical model that includes implementation of a new diagnostic imaging technology in clinical practice to ensure correct use of the imaging system [ 1621 ]  . it should be noted that each hierarchical model starts from an initial assessment of technical / scientific features before passing on to aspects of efficacy , efficiency , patient outcome ( improved survival and morbidity ) and impact on society . 
in more detail , this model requires that each new technology is systematically evaluated on the basis of evidenceper quanto riguarda un esempio di valutazione che sia applicabile in modo specifico alla diagnostica per immagini , si pu utilizzare un modello gerarchico nel quale vale la regola generale che un esame per essere efficace ad un livello alto deve esserlo necessariamente ad un livello pi basso , e non viceversa [ 15 ]  . 
la validit delluso di un modello gerarchico si basa su tre concetti principali : il concetto di globalit della diagnosi ; le informazioni diagnostiche sono un processo continuo ; quello gerarchico un modello che si applica bene allanalisi costo - efficienza . lutilizzo di questo tipo di modello per valutare i vari aspetti tecnici e non delle procedure sanitarie particolarmente consigliato perch di semplice utilizzo , facilmente ripetibile ed aiuta a non trascurare nessun passaggio importante mantenendo un ordine adeguato nel considerare i vari processi costitutivi della procedura stessa . nelle tabelle 13 e fig . 
rapid advances in the field of diagnostic imaging lead the radiologist to continuously adopt new technologies to obtain more and better diagnostic information and to modify the management and ultimately the health of the patient . 
in fact , owing to the lack of clear rules , development strategies and shared and homogeneous health plans in current clinical practice , it takes less than 1 year to introduce new technologies into routine practice , and their introduction is often based on misconceptions that lead to an uneven distribution of technologies and consequently of available resources . 
new technologies and contrast media are thought to necessarily provide better , more diagnostic , images on the basis of subjective views or technical parameters , such as increased spatial or temporal resolution , new acquisition techniques and new visualisation filters . machines and software are introduced before they have been tested in the optimistic conviction that they will no doubt prove useful . 
in collaboration with the university of york , inahta has created a database of reports produced by affiliated subjects . inahta consists of 45 agencies in 23 countries worldwide . another organisation is health technology assessment international ( htai ) , an international scientific society that succeeded the international society for technology assessment in health care ( istahc ) in 2003 . 
for the first time , an italian sits on the board of directors , and the agency set up a specific interest group devoted to hospital - based hta . the cochrane collaboration is an international network of nonprofit institutions set up in 1993 with the aim of nella tabella 2 si descrivono i livelli della valutazione hta delle performance diagnostiche di una tecnologia : nel livello a necessario appurare se la tecnologia valutata garantisce una buona qualit delle immagini e se queste siano anatomicamente rappresentative ; nel livello b si valuta se le immagini ottenute garantiscano che si giunga alla formulazione di una diagnosi accurata ; nel livello c si determina limpatto diagnostico delle immagini ; nel livello d si stima limpatto terapeutico della tecnologia in discussione ed , infine , nel livello e si stima limpatto delle immagini ottenute sulla salute del paziente . 
nella tabella 3 si descrive liter nella valutazione hta del costo - beneficio di una tecnologia sia a livello tecnico , sia di performance ed impatto diagnostico , sia come impatto terapeutico e , in ultimo , come impatto sulla salute . 
2 viene descritto un modello gerarchico che comprende anche , come ultimo passaggio , limplementazione di ogni nuova tecnologia diagnostica per immagini nella pratica clinica , in modo tale da farne derivare un corretto uso dellimaging [ 1621 ]  . 682 radiol med ( 2009 ) 114 : 673691 table 2 hta of diagnostic performance of a technology [ 23 ] level a : technical performance does the technology result in good - quality images that are anatomically representative ? level b : diagnostic performance does imaging allow accurate diagnoses to be made ? level c : diagnostic impact does imaging change diagnostic confidence or the use of subsequent diagnostic tests ? level d : therapeutic impact does the imaging contribute to the management plan ? level e : impact on health does imaging lead to an improvement in patient survival or morbidity ? tabella 2 valutazione hta delle performance diagnostiche di una tecnologia [ 23 ] livello a : performance tecnica la tecnologia valutata garantisce una buona qualit delle immagini e che queste siano anatomicamente rappresentative ? livello b : performance diagnostica le immagini ottenute garantiscono che si giunga alla formulazione di una diagnosi accurata ? livello c : impatto diagnostico le immagini ottenute possono cambiare la sicurezza diagnostica o intervenire sullattitudine alluso di ulteriori esami diagnostici ? livello d : impatto terapeutico limaging contribuisce alla modifica del piano terapeutico ? livello e : impatto sulla salute le immagini ottenute possono condurre ad un miglioramento della sopravvivenza o della morbilit del paziente ? collecting , critically appraising and disseminating information about the effectiveness of health interventions from an evidence - based medicine ( ebm ) perspective through an electronic database called cochrane library that is freely accessible on the internet . 
a major goal of the cochrane collaboration is to sensitise the scientific community , the media and the general public to the risks inherent in the lack of methodological transparency in the world of research and scientific production . the earliest hta experiences developed in the united states in 1972 with the creation of the office of technology assessment ( ota )  . 
this was succeeded by similar agencies , among which is the agency for healthcare research and quality , whose mission it is to investigate health care access , health care costs and outcomes . 
the agency is active in more than 90 projects that aim to test and evaluate the si noti come ogni modello gerarchico parte dalla valutazione iniziale degli aspetti tecnico / scientifici per poi passare a considerare aspetti di efficacia , efficienza , outcome del paziente in termini di miglioramento della sopravvivenza e morbilit , impatto sulla societ ; come gi accennato i primi livelli dellorganigramma devono sempre essere verificati prima di passare al gradino successivo , in modo da scandire i corretti steps nella procedura di valutazione . 
descrivendolo in maniera pi approfondita , questo modello prevede la necessit della valutazione step - by - step di ogni nuova tecnologia sanitaria , tramite studi scientificamente basati , prima dellimplementazione di tale tecnologia . 
i rapidi progressi nel campo della diagnostica per immagini spingono infatti il radiologo alla continua implementazione di nuove tecnologie , perseguendo lobiettivo di ottenere informazioni diagnostiche rilevanti , maggiori e migliori , e modificare il comportamento gestionale del paziente , in sintesi migliorarne la salute . 
in realt nella pratica clinica attuale , in assenza di regole precise , piani di sviluppo e programmi sanitari condivisi e omogenei , si impiega meno di un anno per portare una nuova tecnologia alluso routinario , sulla base di convinzioni errate che conducono ad una distribuzione disomogenea delle tecnologie e conseguentemente delle risorse disponibili . 
si pensa infatti che le nuove tecnologie e i nuovi mezzi di contrasto debbano necessariamente fornire immagini migliori , pi diagnostiche delle vecchie , solo su base soggettiva o di parametri tecnici quali unaumentata risoluzione spaziale o temporale , nuove tecniche di acquisizione , nuovi filtri di visualizzazione e si sostituiscono macchine e software postulando ottimisticamente una loro sicura utilit prima che siano state realmente testate . 
in questo processo non da escludere la pressione del mercato sanitario che identifica la nuova macchina con un grande radiologo e un grande affare , portando allacquisto delle nuove tecnologie a seguito di logiche politico - finanziarie contingenti . stato di realizzazione attuale dellhta per quanto riguarda lo sviluppo attuale dellhta , a livello internazionale presente linahta ( international network of agencies for health technology assessment ) , istituito nel 1993 , che un network di agenzie ed istituzioni con lobiettivo di coordinare lattivit internazionale di hta e di supportare i membri nella definizione di metodiche comuni e condivise di valutazione . 
ad oggi aderiscono radiol med ( 2009 ) 114 : 673691 table 3 hta of cost - effectiveness of a technology at each level [ 23 ] level a : technical performance cost per patient examined level b : diagnostic performance cost per correct diagnosis level c : diagnostic impact cost per invasive test avoided level d : therapeutic impact cost per surgical or medical plan altered level e : impact on health cost per improvement in quality - adjusted life years ( qaly ) tabella 3 valutazione hta del costo - beneficio di una tecnologia ad ogni livello [ 23 ] livello a : performance tecnica costo per paziente esaminato . livello b : performance diagnostica costo per diagnosi corretta . livello c : impatto diagnostico costo di ogni esame invasivo evitato . livello d : impatto terapeutico costo per ogni piano terapeutico medico o chirurgico che viene cambiato in seguito all utilizzo dellesame diagnostico . livello e : impatto sulla salute costo per il miglioramento della qualit della vita c orretto sulla aspettativa di vita ( qaly ) most effective method for transferring research into practice , supporting and promoting evidence - based practice and translating recommendations into resources for providers , patients and health care systems . 
the united states is also home to the most important hta centre worldwide , the emergency care research institute ( ecri )  . canada has six agencies that deal with hta , five of which were created by provincial authorities ( british columbia , alberta , saskatchewan , ontario , quebec ) and only one that works at the national level . 
canadian universities are also particularly interested in hta and often collaborate with the agencies on hta projects . at the european level , two organisations act as coordinators of the various hta programmes . 
hta is particularly well - developed in sweden , france , the united kingdom , spain , germany , holland and denmark [ 14 ]  . allinahta 45 agenzie appartenenti a 23 paesi di tutto il mondo . 
altra organizzazione lhtai ( health technology assessment international ) , societ scientifica internazionale di valutazione delle tecnologie sanitarie , nata nel 2003 dalla chiusura dellistahc ; nellambito di tale agenzia per la prima volta un italiano siede nel board of directory ed stata promossa la costituzione di uno specifico gruppo di interesse dedicato allhospital - based hta . 
bisogna citare anche la cochrane collaboration , network internazionale di enti no - profit nato nel 1993 allo scopo di raccogliere , valutare criticamente e diffondere le informazioni relative alla efficacia degli interventi sanitari , nellottica della cosiddetta evidence based medicine ( ebm ) , attraverso un database elettronico denominato cochrane library , accessibile da tutti su internet . 
un importante compito che la cochrane collaboration cerca di svolgere quello di sensibilizzare sia il mondo scientifico , dei media ed il pubblico pi in generale sui rischi che derivano da una mancanza di trasparenza metodologica nel mondo della ricerca e della produzione scientifica . le prime esperienze di hta si sono sviluppate in america nel 1972 con la costituzione delloffice of technology assessment ( ota )  . 
in seguito alla sua chiusura sono nate altre agenzie simili fra cui la agency for healthcare research and quality la cui missione fondamentale quella di indagare laccesso ai servizi da parte dei cittadini , i costi sanitari e gli esiti ; attualmente lagenzia parte attiva in pi di 90 progetti volti a testare e valutare le metodologie pi efficaci per trasferire i risultati della ricerca nella pratica , per supportare e promuovere la pratica clinica basata sulle evidenze disponibili , per utilizzare le raccomandazioni in termini di migliore utilizzo delle risorse , per lofferta di servizi sanitari , ma anche , e soprattutto , per i pazienti e per il sistema sanitario nel suo complesso . 
 anche presente il maggiore centro di hta del mondo , quello dellecri . provinciali in canada esistono principalmente sei agenzie che si occupano di hta , cinque di queste sono state create dalle autorit ( british columbia , alberta , saskatchewan , ontario e quebec ) e solo una opera a livello nazionale . 
lattivit di produzione di hta reports molto intensa grazie al fatto che esistono dei precisi mandati a livello istituzionale per la realizzazione degli studi e anche grazie alla cultura e allesperienza ormai pluriennale in materia . 
inoltre , anche le universit sono particolarmente attente allhta e si creano spesso collaborazioni con le agenzie per la realizzazione dei lavori . a livello europeo esistono due organizzazioni che fungono da coordinatori delle varie attivit di hta . 
lhta particolarmente sviluppato in svezia , francia , regno unito , spagna , 684 radiol med ( 2009 ) 114 : 673691 germania , olanda , danimarca [ 14 ]  . la svezia da sempre uno dei paesi leader a livello europeo nellutilizzo dellhta nella programmazione di politiche sanitarie . 
oltre che a livello macro , sono poi nate molte altre iniziative che hanno coinvolto le universit , i medici e altri gruppi di professionisti sanitari ( microe meso - )  . 
laspetto molto positivo che nel corso degli anni limpatto dei report di hta via via aumentato e i decision makers hanno fatto riferimento sempre pi ai loro risultati per stabilire priorit e prendere decisioni . 
lanaes ( agenzia nazionale per laccreditamento e la valutazione in sanit ) lagenzia che si occupa pi di altre del hta con particolare riferimento a nuovi dispositivi , procedure , programmi sanitari e offerta di servizi sanitari . listituzione di riferimento in uk il national institute of clinical excellence ( nice ) [ 22 ] il quale , tecnicamente , opera solo nel territorio inglese anche se , informalmente , influenza le scelte di sanit pubblica in scozia e nellirlanda del nord . 
il ministero della salute inglese richiede valutazioni delle tecnologie al nice sulla base delleffettivo beneficio in termini sanitari di una nuova tecnologia nonch delleffettivo miglioramento nella razionalizzazione delle risorse del sistema sanitario nel suo insieme . 
importante sottolineare che in italia , dove non presente un organismo di riferimento per lhta , frequentemente si fa riferimento allattivit del nice . in spagna esiste una agenzia di hta che stata costituita a livello centrale ( aets , agencia de evaluacin de tecnologas sanitarias ) e altre tre agenzie che operano a livello locale , in particolare , in andalusia ( aetsa , agencia de evaluacin de tecnologas sanitarias de andalucia ) , in catalogna ( cahta ) e nei paesi baschi ( osteba )  . lagenzia catalana lunica che viene finanziata ( anche se in piccola parte , 20% ) dal settore privato , rimanendo comunque un ente no profit . 
 per quanto riguarda litalia , limportanza dellhta viene evidenziata dal piano sanitario nazionale 20062008 e dal riconoscimento nellambito del progetto mattoni ospedali di riferimento dellesigenza , per gli ospedali chiamati a diventare ospedali di riferimento , di adottare metodi fig . 
modificato da [ 16 ]  . sweden has always been a leader in europe in the use of hta for planning health policies , and sweden was home to the first national hta agency : the swedish council on hta ( sbu )  . 
sbu was created in 1987 and is directed by a board of representatives of the main organisations involved in the swedish health syste a scientific advisory committee is responsible for research activities that are carried out by a network of more than 300 national experts . in addition to the macro level , other initiatives have been started at the micro and meso levels ( involving universities , physicians and other groups of health professionals )  . 
a very positive aspect is that the impact of hta reports over the years has progressively increased , and policy makers increasingly make reference to them when setting priorities and making decisions . in france , hta involves numerous agencies that collaborate with one another , each one reporting to the ministry of health . 
the national agency for accreditation and assessment in health care ( anaes ) is the agency that is most closely involved in hta , with special emphasis on new devices , procedures , health plans and health services . the reference institution in the united kingdom is the national institute of clinical excellence ( nice ) [ 22 ] , which technically operates in england only , although it informally influences public health care decisions in scotland and radiol med ( 2009 ) 114 : 673691 northern ireland . 
the english department of health commissions nice to carry out technology assessments to establish the effective health benefits of a new technology and effective improvements in the rationalisation of resources of the national health systeit should be noted that in italy , which lacks a reference body for hta , nice reports are frequently referred to . spain has one central hta agency agencia de evaluacin de tecnologas sanitarias ( aets ) and three local agencies operating in andalusia : agencia de evaluacin de tecnologas sanitarias de andalucia ( aetsa ) , catalonia ( cahta ) and in the basque country ( osteba )  . 
the catalan agency receives limited funding ( 20% ) from the private sector only , but it remains a nonprofit organisation . it is also the most active of the four spanish agencies in terms of research and production of hta reports . 
 with regard to italy , the importance of hta is highlighted by the 20062008 national health plan and by the recognition that hospitals applying for benchmark hospital status according to the mattoni project on benchmark hospitals need to adopt hta methods and tools [ 9 , 10 ]  . diffusion of hta in italy has been characterised by several experiences with hospital - based hta by hospital trusts and institutions that are part of the italian hta network . 
the 2003 budget set up the national commission on medical devices ( cud ) as a consultative technical body of the ministry of health whose task it is to define and update the inventory of medical devices and to classify all products into classes and subclasses . 
among its activities , cud set up a working group to evaluate new and old medical technologies employing medical devices and to analyse their clinical benefits and costs to issue data sheets for each medical device . 
in fact , whereas no proper hta projects have been carried out at the central level , interesting studies have been completed at the local level ( veneto region , autonomous province of trento ) , even though the impact of hta in italy is still very limited . however , in 2003 , a project funded by the ministry of health led to the creation of the italian network for health technology assessment ( ni - hta ) [ 11 ] , which laid down the national reference principles for hta in the trento charter ( 2006 )  . 
members are : assessorato alla sanit della regione molise ( regional health department for molise ) agostino gemelli university hospital direzione sanitaria della azienda socio sanitaria n2 e strumenti di hta [ 9 , 10 ]  . 
nel nostro paese la diffusione dellhta stata caratterizzata in particolare dalla moltiplicazione delle esperienze di quello che viene definito hospital based hta da parte di aziende sanitarie ed organizzazioni riunite nel network italiano di hta . 
la finanziaria 2003 ha istituito la cud ( commissione unica sui dispositivi medici ) in qualit di organo consultivo tecnico del ministero della salute , che ha il compito di definire e aggiornare il repertorio dei dispositivi medici e di classificare tutti i prodotti in classi e sottoclassi specifiche . 
nellambito delle proprie attivit , ha istituito un gruppo di lavoro con il compito di valutare nuove e vecchie tecnologie mediche che richiedono luso di dispositivi medici , analizzarne i benefici clinici e i costi correlati al fine di redigere delle schede informative sui dispositivi medici valutati . 
oltre alla cud importante sottolineare che ci sono delle realt locali che fanno ben sperare per la promozione e lo sviluppo dellhta in italia . infatti , mentre a livello centrale non sono stati realizzati dei veri e propri progetti di ta , sono stati conclusi , a livello regionale , studi interessanti ( veneto , provincia autonoma di trento ) , anche se limpatto del ta in italia ancora molto esiguo . 
sempre nel 2003 , sulla base di un progetto finanziato dal ministero della salute , stato costituito il network italiano di health technology assessment ( nihta ) [ 11 ] , che ha espresso nella carta di trento ( 2006 ) i principi di riferimento nazionali per la valutazione della tecnologia . 
fanno parte del network italiano : lassessorato alla sanit della regione molise , il policlinico agostino gemelli , la direzione sanitaria della azienda socio sanitaria n 2 isontina , lirccs policlinico s . 
dannunzio di chieti , il dipartimento delle tecnologie biomediche dellistituto superiore di sanit , lagenzia per i servizi sanitari regionali sezione innovazione , sperimentazione e sviluppo , lirccs casa sollievo della sofferenza opera padre pio , la direzione generale sanit della regione lombardia . 
matteo ( san matteo research and university hospital ) of pavia azienda provinciale per i servizi sanitari di trento ( trento province health care trust ) sezione di epidemiologia e sanit pubblica dipartimento di medicina e scienze dellinvecchiamento delluniversit g . 
the aim of the project and network is to identify common minimum structural , organisational and functional requisites for the routine performance of hta projects at the regional and institutional level within the italian national health service . 
another organisation involved in hta in italy is the italian cochrane centre ( cci ) , established in 1994 with the aim of promoting the activities of the cochrane collaboration in ospedaliera di verona , listituto dermopatico dellimmacolata , lospedale bambino ges , il dipartimento di sanit pubblica e microbiologia delluniversit di torino , losservatorio tecnologie di assobiomedica , lazienda locale socio - sanitaria di rovigo , lazienda sanitaria locale 10 di firenze , il dipartimento di medicina e sanit pubblica delluniversit di verona , lazienda ospedaliera santa maria della misericordia di udine , la societ italiana dei farmacisti ospedalieri e lazienda sanitaria di bressanone . 
obiettivo del progetto , e quindi del network , di proporre una base di discussione per lidentificazione di requisiti comuni minimi strutturali , organizzativi e funzionali per lavvio routinario di attivit di hta a livello regionale e aziendale nellambito del ssn . altra organizzazione coinvolta nellhta in italia il centro cochrane italiano ( cci ) , nato nel 1994 con lo scopo di promuovere in italia ed in alcuni paesi delleuropa meridionale ( spagna , portogallo , francia , grecia ) le attivit della cochrane collaboration . 
il centro cochrane italiano ha sviluppato rapporti di collaborazione con le autorit e le agenzie regionali e nazionali incaricate di promuovere linnovazione e la qualit dellassistenza nel servizio sanitario nazionale . 
nel 2000 il cci ha collaborato formalmente alla realizzazione del piano nazionale per le linee guida ( pnlg ) , coordinato dallagenzia per i servizi sanitari regionali e dallistituto superiore di sanit . 
nel mese di gennaio 2007 stata fondata la si - hta ( societ italiana di health technology assessment ) al termine di un percorso che si sviluppato attraverso vari progetti formativi , programmi di ricerca , attivit in aziende ospedaliere ed universitarie e ha posto litalia , al pari dei paesi pi avanzati , nella condizione di avere una comunit scientifico - professionale dedicata alla valutazione delle tecnologie in sanit . 
nata per fare in modo che i professionisti che si occupano di technology assessment possano incontrarsi per far crescere la cultura del technology assessment nei nostri sistemi regionali e per promuovere la multidisciplinariet basandosi sui principi della carta di trento . 
questo programma , iniziato nel 1995 e coordinato dal centro cochrane italiano , che opera allinterno dellistituto di ricerche farmacologiche mario negri di milano , ha visto la partecipazione di 21 aziende sanitarie e ospedali dellitalia centrale e settentrionale , tra il 1995 e il 1997 , con lobiettivo di introdurre i principi e metodi dellhta nei processi decisionali a radiol med ( 2009 ) 114 : 673691 italy and other southern european countries ( spain , portugal , france and greece )  . 
in this context , the cci offers technical and methodological assistance to health professionals collaborating with the international cochrane groups and organises courses and workshops on the methods used . the cci collaborates with the regional and national authorities and agencies in charge of promoting innovation and quality of care in the national health service . 
in january 2007 , the italian society of health technology assessment ( si - hta ) was established following a series of educational projects , research programmes and activities in hospital and university institutions . 
si - hta was established to allow hta professionals to promote the culture of hta within italys regional health systems and encourage a multidisciplinary approach based on the principles contained in the trento charter . the most interesting national experience is the tripss project , an acronym that translates the english grip ( getting research into practice )  . 
this project , launched in 1995 and coordinated by the cci from the mario negri institute for pharmacological research in milan , saw the participation of 21 health care and hospital trusts of central and northern italy between 1995 and 1997 . 
its aim was to incorporate hta principles and methods in clinical and managerial decision - making processes and to introduce clinical quality of care into the appraisal systems of the health care trusts . 
these efforts have uncovered a series of problems , the most important of which is the separation between the regulations governing the national health system and the principles of efficacy and appropriateness . in this context , four projects have been developed that deal with preoperative assessment , cardiac decompensation , pregnancy and breast cancer . 
among the elements identified as relevant for the transfer of scientific knowledge to clinical practice , mention should be made of continuing education , budgeting , appropriate use of economic incentives and auditing and monitoring of clinical practice . 
projects developed in the veneto region evaluated the appropriate use of coronary angiography and angioplasty , the appropriate use of hospitals and the development of clinical guidelines in the framework of the tripss project . 
the tripss project was followed by tripss ii ( 19992003 ) that , taking advantage of the new rules that were being introduced , aimed to establish an integrated approach among health professionals and managers to define and safeguard quality of care . 
questo sforzo ha portato alla luce una serie di problemi , primo fra tutti lo scollamento tra il quadro normativo di riferimento del servizio sanitario nazionale e i principi di efficacia e appropriatezza . 
fra gli elementi identificati come rilevanti per facilitare il trasferimento delle conoscenze scientifiche nella pratica clinica vanno citati la formazione permanente , il budgeting , luso appropriato degli incentivi economici e i processi di auditing e monitoring della pratica clinica . 
nella regione veneto sono state sviluppate iniziative per valutare lutilizzo appropriato dellangiografia coronarica e dellangioplastica , luso appropriato degli ospedali e lo sviluppo di linee guida cliniche nellambito del progetto tripss . 
il progetto tripss , conclusosi nel 1997 , stato seguito dal tripss ii , lanciato nel 1999 e terminato nel 2003 che , approfittando degli strumenti normativi che si stavano configurando , si proponeva di definire un approccio integrato tra professionisti e managers per la definizione e salvaguardia della qualit dellassistenza . 
lo scopo principale di tale progetto di valutare una metodologia per limplementazione nella pratica clinica di interventi di documentata efficacia e di definire i requisiti essenziali per la realizzazione di tali programmi . 
nonostante siano progetti separati e distinti , possono essere visti come tre passi nella costruzione di un percorso per il trasferimento dei risultati che , partendo da un modello semplicistico basato principalmente sulla diffusione e divulgazione dei risultati , arriva a modelli pi articolati che hanno ridefinito il rapporto tra informazione e pratica clinica . discussione tutti i sistemi sanitari dei paesi occidentali si confrontano quotidianamente con una domanda di servizi sempre pi qualificata e , contemporaneamente , si assiste ad una rapida evoluzione sia delle tecnologie nel settore sanitario sia delle conoscenze in campo scientifico in tema di salute . 
although the tripss projects are separate and distinct , they may be seen as three steps towards the transfer of research to practice that , starting from a simple model based on the diffusion and dissemination of results , arrives at more complex models that have redefined the relationship between information and clinical practice . 
 discussion all national health systems in western countries are facing , on the one hand , an increasing demand for quality health care and , on the other hand , a rapid development of both technologies and scientific knowledge concerning health . this results in the development of new and complex procedures for prevention , diagnosis and treatment that entail high costs for the health care systems . 
in this context , health care policy making clearly needs to be guided by up - to - date scientific evidence and knowledge about the technical , economic , social , legal and ethical implications of decisions . 
such guidance can be provided by hta , which has been referred to as a bridge between scientific innovation and the technical , economic and organisational decisions of policy makers . 
it is a complex and systematic multidisciplinary assessment of the health care , economic , social and ethical impacts both direct and indirect and shortand long term of existing and new health technologies . 
the hta process involves identifying the technologies to be assessed , gathering and analysing existing evidence , synthesising data and sharing the findings and ensuring the diffusion and dissemination of resulting suggestions and recommendations . 
hta is able to support decision making at different levels from agencies authorising the marketing of a drug or health technology to health care institutions deciding whether to purchase new equipment or how it should be managed . 
an hta is principally conseguenza , nascono nuove procedure complesse in tema di prevenzione , diagnosi e trattamento in medicina che , daltro canto , comportano elevati costi economici da sostenere da parte delle strutture sanitarie . 
in questo contesto , si evidenzia chiaramente la necessit di guidare le scelte in tema di politica sanitaria in modo da tenere conto sia delle evidenze scientifiche mediche pi attuali , ma anche degli aspetti tecnici , economici e , pi in generale , delle implicazioni sociali , giuridiche , etiche di queste stesse decisioni in campo sanitario , trovando una integrazione che funga da ponte tra linnovazione scientifica e le decisioni tecniche , economiche e gestionali dei policy makers in campo sanitario . 
lhta un metodo di valutazione accurato delle nuove tecnologie sanitarie per gli aspetti sia scientifici , in termini di appropriatezza e di efficacia , sia per gli aspetti economici , sociali , giuridici ed etici ; si tratta di una valutazione multidisciplinare complessa e sistematica delle conseguenze assistenziali , economiche , sociali ed etiche provocate in modo diretto ed indiretto , nel breve e nel lungo periodo , dalle tecnologie sanitarie esistenti e da quelle di nuova introduzione . 
lhta prevede , quindi , lidentificazione delle tecnologie che necessitano di una valutazione , la raccolta delle evidenze gi esistenti e lanalisi dei dati , la sintesi delle informazioni e la distribuzione dei risultati ottenuti nonch la diffusione e la disseminazione dei conseguenti suggerimenti e raccomandazioni ; si tratta , perci , di un metodo utilizzato per promuovere la salute , prevenire e trattare una malattia , migliorare la riabilitazione o definire criteri di cura e assistenza a lungo termine . 
lhta in grado di supportare scientificamente diversi livelli decisionali , dalle agenzie che autorizzano limmissione sul mercato di un farmaco o di unaltra tecnologia sanitaria , alle organizzazioni sanitarie che spesso si trovano a doverne valutare lopportunit di acquisto o la modalit di gestione . 
un hta si basa principalmente sulla ricerca e verifica delle evidenze scientifiche secondo levidence based medicine ( fondare ogni decisione clinica , diagnostica o gestionale sulla migliore prova di efficacia disponibile nella letteratura medica , al fine di portare direttamente i risultati pi aggiornati della ricerca scientifica nella pratica quotidiana ) : ci comporta la partecipazione dei ricercatori e degli esperti nellambito di indagine , ma anche degli operatori sanitari e dei decisori che , senza la disponibilit delle migliori prove di efficacia , non sono messi nelle condizioni necessarie per decidere se adottare o meno una nuova tecnologia ; accanto agli aspetti prettamente clinici , ne vanno anche evidenziati altri come , ad esempio , quelli sociali , culturali ed etici , che coinvolgono principalmente i cittadini / utenti / pazienti del servizio sanitario pubblico , radiol med ( 2009 ) 114 : 673691 based on research and the analysis of scientific evidence according to the principles of ebm ( all decisions clinical , diagnostic or organisational must be grounded in the best available evidence to allow the transfer of the most recent scientific evidence into routine practice )  . 
this implies the participation of researchers and experts in the field but also of health professionals and decision makers who , in the absence of the current best evidence , are unable to decide whether or not to adopt a new technology . 
these mainly concern the relationship between citizens / users / patients of the public health system and the health system itself and represent an attempt to reconcile , in the decision - making process , the certainty of scientific evidence with the active participation of the effective or potential user . 
hta should therefore be considered a systemic and multidisciplinary process that is indispensable for informing decisions and ensures the optimisation of solutions pursued and the transparency of the decision - making process for all players . 
hta may represent a powerful link between the technicalscientific and the decision - making domains , provided that decision makers can guarantee the conditions for its performance and become accustomed to requesting it . 
one problem that hta has had to face since its inception is the poor communication among the various disciplines involved in the process . firstly , economic assessment is often completely unrelated to the needs of scientific knowledge and technologies . secondly , the various local hta projects often remain unknown and isolated within the international scientific setting . 
to overcome these problems , public and private hta agencies , especially international ones , have set up a wide communication and collaboration network to promote and publicise projects dealing with the assessment and implementation of scientific evidence and health technologies . this network , which is both publicly and privately funded , ensures continuity to the hta process , allowing available technologies to keep up to date with the new scientific knowledge . 
this way , hta may achieve its final goal of transferring the results of theoretical assessments to practical applications that may effectively impact the health system by informing decisions on investments and resource allocation . 
indeed , any hta project can and must act to support health care decisions at all levels [ 1113 ] : macro level : health policy decisions at the national / international level meso level : institutional decisions regarding the acquisition of new technologies or new procedures nonch il servizio stesso nel tentativo di conciliare , nelleffettuazione delle scelte , le garanzie di evidenza scientifica con la partecipazione attiva del loro destinatario effettivo o potenziale : , infatti , ormai universalmente riconosciuto che quando il cittadino partecipa al processo decisionale la sua soddisfazione maggiore e gli esiti clinici migliorano . lhta va , dunque , considerato come un processo sistemico e multidisciplinare indispensabile per la corretta istruzione delle dinamiche decisionali e garante della ottimizzazione delle soluzioni perseguite , oltre che della trasparenza di tali dinamiche nei confronti di tutti gli attori coinvolti ; lhta pu rappresentare un potente strumento di collegamento tra lambito tecnico - scientifico e quello decisionale , posto che questultimo ne garantisca i presupposti per la sua attuazione e , soprattutto , che si abitui a richiederne leffettuazione . 
un problema , non trascurabile nemmeno attualmente , con cui lhta si dovuto scontrare fin dai suoi primi passi la scarsa comunicabilit tra i vari settori che si occupano di questo tipo di disciplina : per prima cosa , la valutazione economica spesso completamente scollegata dalle esigenze delle conoscenze scientifiche e delle tecnologie ; in secondo luogo , i vari progetti di hta a livello locale spesso rimangono sconosciuti e isolati nella realt scientifica internazionale . 
per sopperire a queste problematiche , negli anni le societ pubbliche e private che si occupano di hta a livello soprattutto internazionale hanno sviluppato una fitta rete di comunicazione e collaborazione allo scopo di sviluppare e rendere pubblici i progetti che si occupano di valutazione ed implementazione delle conoscenze scientifiche e delle tecnologie sanitarie ; inoltre lesistenza di questa stessa rete , che attinge sia a finanziamenti pubblici che privati , garantisce una continuit nel tempo di lavoro , necessaria perch le tecnologie a disposizione si mantengano sempre aggiornate e allaltezza delle nuove conoscenze . 
la funzione dellhta pu cos giungere al culmine del suo scopo : portare il risultato di considerazioni valutative teoriche ad una applicazione pratica reale , che possa avere un effettivo impatto sul sistema sanitario in quanto in grado di indirizzare le scelte di investimento e di allocazione delle risorse : infatti un hta pu e deve fungere da supporto nelle decisioni sanitarie a vario livello [ 1113 ] : livello macro : decisioni di politica sanitaria a livello nazionale / internazionale ; livello meso : decisioni aziendali di investimento su nuove tecnologie o sullacquisizione di nuove procedure ; livello micro : decisioni cliniche da parte del medico o di una equipe di medici . questo riveste grande importanza nella definizione dei livelli di assistenza ottimali , nel garantire luso delle 690 radiol med ( 2009 ) 114 : 673691 micro level : clinical decisions by the individual physician or team of physicians this is crucial for defining optimal levels of care and for guaranteeing the use of the most effective technologies and procedures with a view to better coordinating the distribution and delivery of health care services . conclusioni migliori tecnologie e procedure riconosciute dalle evidenze scientifiche per poter coordinare al meglio le modalit di distribuzione e di erogazione di queste tecnologie . conclusions the recent creation of ni - hta and si - hta in italy represents a major step forward as it testifies to a growing interest in hta and marks the beginning of a new phase in the countrys health policy . 
it is increasingly accepted that decisions concerning the use of a drug , diagnostic system or health care process must be preceded by the analysis and definition of various factors including appropriateness , effectiveness , safety , and future sustainability . the lack of planning and common rules makes it necessary to encourage the use of hta to place italy on a par with countries with a longer hta tradition and to exploit the method to guide the development , assessment and use of imaging technologies , thereby enhancing the quality and cost - effectiveness of care . 
hta must therefore be seen as a multidisciplinary effort where physicians with their different clinical and organisational specialisations are often the key players who draw on the contribution of other nonmedical professionals ( physicists , economists , clinical engineers , sociologists , etc . ) to produce valid reports and inform , even at the peripheral level , research , teaching and clinical activity to enhance their effectiveness and efficiency . la recente costituzione in italia del ni - hta e della si - hta ha rappresentato un passo in avanti perch dimostra il crescente interesse sul tema e segnala lavvio di una nuova stagione per la politica sanitaria nel nostro paese . 
 progressivamente cresciuta la consapevolezza che le decisioni che riguardano luso di un farmaco , di un sistema diagnostico , di un processo assistenziale devono essere precedute da un momento di analisi e di definizione di vari fattori quali lappropriatezza , lefficacia , la sicurezza , la sostenibilit futura della stessa tecnologia sanitaria . 
la carenza di programmazione e di regole condivise rende necessario incentivare lutilizzo di tale metodologia per porre la nostra nazione al pari dei paesi pi avanzati nella gestione dellhta e per sfruttarla come strumento per guidare lo sviluppo , la valutazione e lutilizzo delle tecnologie in radiologia al fine di incrementare la qualit e il cost - effectiveness delle cure mediche . 
le complicanze pi comuni sono state la deiscenza del sistema di bendaggio ( 5 , 1% ) , lo scivolamento anteriore del bendaggio ( 3 , 5% ) e linfezione del port ( 1 , 6% )  . 
il bendaggio gastrico per via laparoscopica una procedura relativamente a basso rischio , ampiamente eseguita per trattare lobesit patologica . limaging gioca un ruolo importantissimo nella valutazione precoce e nellindividuazione delle complicanze . parole chiave bendaggio gastrico per via laparoscopica complicanze radiologiche chirurgia bariatrica obesit patologica radiol med ( 2009 ) 114 : 802810 introduction lifestyle diseases such as obesity are on the rise in singapore , similar to most nations that have undergone rapid economic growth . 
the more severe form of obesity , known as grade iii obesity or morbid obesity , is associated with the development of health problems such as dyslipidaemia , hypertension , diabetes mellitus , arthritis and obstructive sleep apnea [ 2 , 3 ]  . 
medical treatment ( conservative treatment ) has been found to be less effective in producing sustainable weight loss ( 5%8% weight loss at 2 years ) compared with bariatric surgery ( average weight loss of 16%35% in 2 years ) [ 4 ]  . surgery for morbid obesity , commonly known as bariatric surgery , is defined as gastrointestinal surgery that produces weight loss in the morbidly obese . 
another malabsorptive procedure , the biliopancreatic diversion with or without duodenal switch is associated with a high percentage of weight loss with a low risk of significant weight regain . however , the effectiveness of the malabsorption of biliopancreatic diversion with or without duodenal switch requires the patient to be supplemented with vitamins above and beyond that of the normal population , with lifelong follow - up . 
another procedure is the endoscopic insertion of an intragastric balloon , a short - term form of weight loss treatment , through the insertion of silicone balloons filled with saline and designed to occupy gastric volume and increase satiety [ 5 ]  . currently , lagb is one of the most frequently performed laparoscopic bariatric operations in many countries [ 6 , 7 ] , including in singapore . 
this study aims to review the complications of lagb , with particular emphasis on the radiological features and the role of imaging in assessing these complications . materials and methods lagb was introduced at our hospital on 30 january 2001 as part of a multidisciplinary weight management programme , and up to 3 october 2006 , 322 patients underwent this procedure . 
eligibility criteria for lagb were the asian adaptation of the criteria set by the society of american gastroenterological surgeons ( sages ) [ 21 ] and the american society of bariatric surgeons ( asbs ) [ 22 ] , namely : 1 . 
 information prospectively entered into the study database included : personal details , race , gender , presurgery polysomnography results , continuous positive airway pressure ( cpap ) requirement , preoperative very low calorie diet ( vlcd ) , age at operation , preoperative weight , height , preoperative bmi , lagb operative details including type of band used and complications , presence of comorbidities such as hypertension , diabetes mellitus , hyperlipidaemia , and postoperative follow - up information . the pars flaccida surgical technique was employed . 
in most patients , the inamed 10 ( allergan inc , irvine , ca , usa ) initially and the swedish band ( johnson & johnson , new jersey , usa ) subsequently 804 radiol med ( 2009 ) 114 : 802810 were used . 
two other bands were less frequently used , namely , the ami ( agency for medical innovations , gtzis , austria ) and the vanguard ( allergan inc , irvine , ca , usa )  . a small virtual 15 - ml gastric pouch was left above the band . 
this placement was later standardised to a site at the midline over the xiphisternu prophylactic broad - spectrum intravenous antibiotics were given at the induction of anaesthesia , and 80 mg of gentamicin was placed at the access port site before skin closure . 
measures to minimise risk of deep vein thrombosis included administering subcutaneous heparin , graduated compression stockings providing calf pumps and padded adjustable leg supports ( yellowfins , acton , ma , usa ) during the surgery , with early ambulation and physiotherapy after the operation . on the first postoperative day , the patient underwent a contrast swallow to confirm satisfactory band placement . the patient was discharged if the contrast swallow was normal and upon a successful trial of liquid ingestion . liquid diet was given for 2 weeks and pureed diet for a further 2 weeks . 
the first adjustment of the band was carried out 56 weeks after lagb , and at that time , between 1 and 3 ml of normal saline was injected into the port using the noncoring 20 - gauge deflected point huber needle ( inamed , santa barbara , ca , usa ) , depending on the band used . 
 on subsequent clinic visits , the band was tightened by incremental injections of normal saline ( ranging from 0.2 to 1 ml , depending on the band used ) into the port . 
band adjustments by percutaneous adjustment of the amount of saline in the band were generally carried out by the surgeon in his clinic except for difficult cases such as patients whose ports were located posterior to the abdominal skin folds or who could not hold their breath adequately to immobilise the port to facilitate port adjustment in the clinic . 
adjustments were aimed at : ( 1 ) sustainable , progressive small amount of weight loss , ( 2 ) sensation of early satiety after meals ( 3 ) and without severe and persistent heartburn or difficulty in swallowing . if a patient became pregnant , the band was completely emptied for the entire duration of pregnancy . 
if the patient did not lose weight despite repeated band tightening , a bandogram [ 24 ] using a water - soluble contrast agent was performed under fluoroscopic guidance to assess for leakage from the port , tubing or band . early films were taken initially with the system filled to half its capacity . 
if no leakage was demonstrated , the system was filled to its maximum capacity , and delayed films were taken after the contrast agent had been left in situ for 10 min to demonstrate small leaks . this study was planned as a study of consecutive lagb patients , and the mean and range of the patient demographic details were presented . 
lagb patients were mainly young adults with a mean age of 35.9 years and a mean bmi of 41.9. ethnic distribution differed from that of the general population in that there was a disproportionately larger number of malays and indians ( table 1 )  . of 313 patients who underwent lagb , 46 developed a total of 51 complications , giving an overall complication rate or 16.3%. 
one patient with anterior band slippage developed acute gastric volvulus and underwent emergency surgery , whereas the rest of the patients with anterior slippage underwent early elective surgery to reduce the slippage or to replace the band . 
il bario mostra un esofago distale dilatato ( frecce ) secondario a chiusura serrata del bendaggio ( punta di freccia ) radiol med ( 2009 ) 114 : 802810 fig . 
limmagine tc assiale mostra lirregolarit del tessuto molle sottocutaneo e la densit intraperitoneale ( frecce ) che rappresenta una fistola cronica a partenza dal port infetto , gi rimosso . three ports ( 1% ) had twisted around the tubing > 90 , making port adjustment even under fluoroscopic guidance impossible . 
to reduce the number of patients with rotated , displaced ports and to make needle adjustment easier , the port position was later standardised and placed anterior to the lower end of the xiphisternu since then , there were no more tubingand port - related problems . there were five ( 1.6% ) cases of port - site infection that fig . 
in one of the nondiabetic patients , the infected port was removed . however , upon complete infection resolution , the second port became infected and had to be removed . respiratory failure postoperatively in our series , one death ( 0.3% ) occurred during the early postoperative period due to aspiration pneumonia . 
the second mortality was a patient who had a perforation detected on the first postoperative day after lagb , after which the band was removed and the patient was discharged well . 
three months later , a second attempt to insert a gastric band failed due to extensive perioesophageal fibrosis . during the postoperative period , he developed a splenic abscess complicated by septic shock and succumbed to multiorgan failure . 
post - lagb patients complicated by early perforation may be asymptomatic or present with epigastric pain with / or without left shoulder - tip pathe first postoperative day contrast swallow demonstrates free extravasation at the perforation site . 
another postulated theory for early band perforation is subclinical infection around the band leading to eventual breakdown in small parts of the band [ 9 ]  . in our institution , 3.5% of lagb patients were complicated by anterior band slippage , the commonest complication in our series and also in most other series [ 27 , 28 ]  . 
oesophageal dilatation postlagb might be caused by preexisting oesophageal dysmotility [ 29 ] , overtightening or successive overzealous tightening of the band stoma , leading to oesophagus gastrification and secondary achalasia . 
 [ 30 ] found that gastric perforation may be underreported , with an incidence rate of 7.5% of gastric erosion on routine gastroscopy studies , of which all patients were asymptomatic at diagnosis . 
 [ 31 ] found that band migration through the gastric wall is a late complication , which should be suspected in patients who have late weight gain , chronic port - cutaneous fistula , neglected insidious development of peritonitis , left subphrenic abscess and protracted port - site infection . 
this typically occurs as a late complication and is postulated to be due to chronic ischaemia caused by pressure from the gastric band on the gastric walls [ 9 ] .there is no consensus regarding the mechanism of this potentially fatal complication [ 12 , 32 ]  . 
the variation in the incidence of this complication in our series compared with other series may be due to the difference in the length of follow - up in post - lagb patients , the variation in schedule of port injection , the amount of saline present in the system and the protocol for gastroscopy of post - lagb patients . 
this difference may be related to the length of follow - up , the position in which the port was placed and the expertise of the surgeons performing adjustments in the clinic . 
patients may gain weight if there is a leakage in the systeduring band tightening , if saline can be freely injected but is not aspirated in the correct amount , the surgeon should suspect a banding - system leak . 
due to the presence of a transverse skin fold of the anterior abdominal wall , the port may lie obliquely , thus causing difficulty in port adjustment in the clinic . 
under fluoroscopic control in a supine position , the omental apron can be adjusted by slight pressure on the abdomen or by the patient holding up the omental apron cranial to the transverse abdominal skin fold until the port is parallel to the fluoroscopic table top , facilitating easy assess to the port . 
in another group of patients who had achieved significant weight loss , the anterior abdominal adipose layer was reduced , and the securing suture may have come undone . during respiration , this mobile port slides on the adipose tissue , thus making the port injection difficult without fluoroscopy . 
strict aseptic technique during port adjustment can help to lower the incidence of infection . in conclusion , laparoscopic adjustable band surgery is a widely practiced method for achieving weight reduction in morbidly obese patients . 
simonetti1 1imaging diagnostics , molecular imaging , interventional radiology , and radiation therapy department , 2public health and cellular biology department , 3pathologic anatomy section , university tor vergata ( ptv ) , viale oxford 81 , 00133 rome , italy correspondence to : c.a. 
our clinical results confirm the diagnostic accuracy of both the mammotome and vacora systems , whereas our cost analysis shows that there is a considerable difference , mostly related to the initial investment . keywords breast vacuum - assisted breast biopsy mammotome vacora cost - effectiveness riassunto scopo . 
presso il nostro centro , dal gennaio a giugno 2006 sono state sottoposte a caratterizzazione istologica mediante prelievo vab 238 lesioni mammarie . di queste 5 / 238 sono state escluse dalla nostra casistica per numero insufficiente di frustoli prelevati . 
i risultati clinici confermano laccuratezza diagnostica di entrambi i sistemi mentre , ai fini della valutazione economica delle procedure , il fattore determinante linvestimento iniziale . parole chiave mammella biopsia mammaria vuotoassistita mammotome vacora costo - efficacia 744 introduction technological progress and the advent of increasingly sophisticated equipment combined with the diffusion of breast cancer screening programmes have led to the detection of a greater number of early lesions . 
in the past , surgical biopsy was considered the only technique capable of characterising these lesions , which very frequently proved benign and did not require surgery [ 14 ]  . 
major technological advances over the past few years have led to the introduction of vacuum - assisted biopsy ( vab ) systems allowing minimally invasive sampling of cores of tissue . 
the purpose of this study was to conduct a cost - effectiveness analysis on two vab systems ( mammotome and vacora ) to optimise the choice of breast biopsy systems . materials and methods two vab systems are currently available at our department : mammotome ( ethicon endo - surgery , cincinnati , oh , usa ) and vacora breast biopsy system ( bard biopsy system , tempe , az , usa )  . 
five out of 238 lesions were excluded from the series , as bleeding prevented sampling of the minimum number of tissue cores established for our study ( at least 18 cores )  . 
however , the vacora system was used for lesions visible on magnetic resonance imaging ( mri ) only , as it is the only mri - compatible device available at our department . 
 before the biopsy procedure , all patients underwent blood tests to assess coagulation status ( complete blood count , prothrombin time , thromboplastin time , antithrombin iii , fibrinogen ) and electrocardiograall patients received detailed information about the procedure , its benefits and radiol med ( 2009 ) 114 : 743756 introduzione lavanzamento tecnologico e la disponibilit di apparecchiature sempre pi sofisticate parallelamente alla maggior diffusione dei programmi di screening , ha portato negli ultimi decenni allindividuazione di un maggior numero di lesioni in fase precoce . 
in passato la biopsia chirurgica veniva considerata come unica possibilit per caratterizzare tali lesioni , che troppo spesso risultavano di natura benigna rendendo dunque inutile lintervento [ 14 ]  . 
negli ultimi anni , grazie ai notevoli progressi tecnologici , sono stati introdotti in commercio sistemi bioptici vuoto assistiti , che consentono il prelievo mini - invasivo di frustoli di tessuto . tali sistemi offrono unaccuratezza diagnostica pressoch sovrapponibile alla biopsia chirurgica , rispetto alla quale hanno tuttavia numerosi vantaggi . 
tali sistemi sono minimamente invasivi , ben tollerati , con assenti sequele cicatriziali , e conducono ad una significativa riduzione dei costi rispetto agli interventi chirurgici tradizionali [ 59 ]  . 
in una realt caratterizzata da risorse economiche sempre pi limitate , lanalisi costo - efficacia uno degli strumenti pi utili per supportare il processo decisionale nella scelta delle tecnologie da impiegare a scopo diagnostico . 
lobiettivo del lavoro quello dunque di confrontare due dispositivi bioptici vuoto assistiti ( mammotome e vacora ) in termini di analisi costo - efficacia per lottimizzazione del processo decisionale nella scelta dei sistemi bioptici in ambito senologico . materiali e metodi nel nostro dipartimento sono disponibili due sistemi di prelievo vuoto - assistito : mammotome ( biopsys ethicon endo - surgery , cincinnati , oh , usa ) ; vacora breast biopsy system ( bard biopsy system , tempe , az , usa )  . 
dal gennaio 2006 al giugno 2006 , sono state prese in considerazione 238 lesioni mammarie consecutive sottoposte a caratterizzazione istologica mediante prelievo vab in pazienti con et compresa tra i 30 ed i 74 anni ( et media 52 anni )  . 
di queste il prelievo vab stato effettuato in 108 / 233 pazienti con il sistema mammotome ed in 125 / 233 con il sistema vacora ; 5 / 238 lesioni sono state escluse dalla nostra casistica in quanto , a causa del sanguinamento , stato possibile prelevare un numero di frustoli radiol med ( 2009 ) 114 : 743756 risks and the possible diagnostic alternatives . 
lesions were located using methods previously described in the literature for each of the imaging modalities [ 1416 ]  . the breast was disinfected , and a local anaesthetic was applied ( 10 cc of 2% lidocaine hydrochloride )  . 
the mammotome system was used with us guidance in 58 / 108 lesions ( 24.9% ) ( 515 mm in diameter , mean 10 mm ) and with mammographic guidance in 50 / 108 cases ( 21.4% ) of clustered microcalcifications . 
la scelta del sistema da utilizzare non si basata sulle caratteristiche iconografiche delle lesioni , bens le pazienti sono state inviate ad uno o allaltro sistema in maniera del tutto casuale . 
 prima della procedura di biopsia percutanea mammaria tutte le pazienti sono state sottoposte ad esami ematochimici , al fine di valutare lassetto coagulativo ( emocromo con conta piastrinica , tempo di protrombina , tempo di tromboplastina , antitrombina iii , fibrinogeno ) ed elettrocardiogramma . 
 la scelta della guida strumentale si basata sullindagine diagnostica che meglio consentiva di individuare la lesione , preferendo quando possibile quella ecografica , sia per il costo minore che per la semplicit e rapidit nellesecuzione della procedura [ 10 ]  . 
la localizzazione delle lesioni avvenuta per ogni guida strumentale con modalit gi descritte in letteratura [ 1416 ]  . si proceduto dunque alla disinfezione della mammella da sottoporre a procedura ed alla somministrazione di anestetico locale ( 10 cc di lidocaina cloridrato diluita al 2% )  . 
il sistema mammotome stato utilizzato in 58 / 108 ( 24 , 9% ) lesioni sotto guida ecografica ( con diametro lesionale compreso tra 5 e 15 mm , valore medio di 10 mm ) ed in 50 / 108 ( 21 , 4% ) casi di microcalcificazioni raggruppate in cluster sotto guida stereotassica con apparecchio dedicato ( tavolo prono fischer mammotest plus / stm )  . 
il prelievo stato effettuato sotto guida ecografica in lesioni nodulari ( 47 / 58 ) , sbarramenti del fascio ultrasonoro ( 9 / 58 ) e su aree di disomogeneit ecostrutturale ( 2 / 58 ) ; sotto guida stereotassica in casi di microcalcificazioni ( 50 / 50 ) , di cui 9 ( 9 / 50 ) apprezzabili allesame mammografico in corrispondenza di aree di distorsione parenchimale da pregresso 746 radiol med ( 2009 ) 114 : 743756 after hormone replacement therapy had been discontinued for at least 2 months . 
one week after the biopsy , an excisional biopsy of all lesions diagnosed as malignant ( invasive carcinoma or carcinoma in situ ) or as atypical ductal hyperplasia was performed , and the final histological diagnoses were compared with those obtained with vab . cost analysis considered the fixed and variable costs for each procedure performed with the mammotome or vacora systems under us , mx or mri guidance . 
the precise determination of indirect costs was thought to be irrelevant to the purposes of the study , as these costs are not directly related to the procedure but to general and administrative costs common to all hospital activities . 
staff costs refer to the cost of each medical and paramedical staff unit ( radiologist , nurse , radiology technician and auxiliary nurse ) directly involved in the procedure and calculated for the mean duration of the procedures . 
for each category , we determined the cost per hour based on working hours under the contract ( radiologist 37.98 / h , nurse 24.66 / h , radiology technician 24.66 / h and auxiliary nurse 22.62 / h ) and the cost per minute , and the latter was multiplied by the mean time each staff unit was engaged in the procedure . 
this item therefore includes the cost of anaesthesia , drugs , dressing material and other disposable items ( gloves , coats , caps , sterile drapes , containers , disposable scalpel , and vab kit )  . 
in particular , with regard to the mammotome system , the items considered included the tubing set , the fluid container and the 11 - gauge probe used for the usand mx - guided procedures , which costs less than the mri - compatible probe . 
the cost of the metal clips is virtually the same with the mammotome or vacora systeour cost analysis assumed the use of metal intervento chirurgico su patologia maligna . il sistema vacora stato utilizzato per il prelievo vab in 58 / 125 ( 24 , 9% ) lesioni sotto guida ecografica ( con diametro lesionale compreso tra 5 e 15 mm , valore medio di 10 mm ) , 50 / 125 ( 21 , 4% ) lesioni sotto guida stereotassica con apparecchio dedicato ( tavolo prono fischer mammotest plus / stm )  . 
inoltre sotto guida rm sono state sottoposte a prelievo vab 17 / 125 ( 7 , 4% ) lesioni ( con diametro lesionale compreso tra 5 e 10 mm , valore medio di 7 , 5 mm )  . 
sono state caratterizzate sotto guida ecografica lesioni nodulari ( 41 / 58 ) , sbarramenti del fascio ultrasonoro ( 8 / 58 ) e aree di disomogeneit ecostrutturale ( 9 / 58 ) ; sotto guida stereotassica microcalcificazioni ( 50 / 50 ) , di cui 7 ( 7 / 50 ) su cicatrice chirurgica da pregressa patologia maligna . in pazienti con il prelievo sotto guida rm stato effettuato su lesioni non individuabili allimaging tradizionale con seguente distribuzione : 7 / 17 casi per la ricerca di foci maligni addizionali in pazienti candidate allintervento chirurgico per patologia maligna sospetta allimaging mammo - ecografico ; 5 / 17 casi per una corretta valutazione della cicatrice chirurgica in pazienti gi sottoposte ad intervento chirurgico per patologia maligna ; 3 / 17 casi per ricerca di focolai di carcinoma mammario occulto in pazienti con linfonodi positivi per metastasi da carcinoma mammario senza evidenza di lesioni primitive allesame mammografico ed ecografico ( cup syndrome ) ; in 2 / 17 casi la rm stata eseguita familiarit per carcinoma mammario . 
dopo una settimana dalla procedura bioptica stata eseguita la biopsia escissionale di tutte le lesioni diagnosticate come maligne ( carcinoma invasivo o in situ ) o come iperplasia duttale atipica ( adh ) e le diagnosi istologiche definitive sono state confrontate con quelle ottenute con il vab . 
 per quanto riguarda lanalisi dei costi relativi ad ogni singola procedura , effettuata rispettivamente con il sistema mammotome e vacora sotto guida ecografica , stereotassica e rm , sono stati presi in considerazione i costi fissi ed i costi variabili . 
to determine depreciation of the mammotome and vacora systems , handpieces and drivers we assumed a typical depreciation period of 5 years and divided the annual cost by the number of procedures per year , estimated by projecting the number performed in the 6 months considered over a 12 - month period . 
annual maintenance of the imaging devices ( us scanner , fisher table or mammography unit , mri scanner ) was instead calculated by considering the total hours of use for each device , again assuming an obsolescence period of 5 years , 45 working weeks per year , and 40 h of work per week . 
 results clinical efficacy all procedures were completed successfully , and no significant complications occurred , with the exception of five cases of bleeding that resolved after manual compression of the breast . 
in 13 / 58 mammotome procedures and in 14 / 58 vacora procedures performed under us guidance , the postprocedural mammograms and sonograms confirmed the complete excision of the nodular lesion . 
the results obtained with the mammotome system were 16 / 108 ( 14.8% ) cases of invasive carcinoma , 22 / 108 ( 20.4% ) cases of carcinoma in situ and 12 / 108 ( 11.1% ) of atypical ductal hyperplasia . 
the results obtained with the vacora system were 24 / 125 ( 19.2% ) cases of invasive carcinoma , 26 / 125 ( 20.8% ) of carcinoma in situ and 16 / 125 ( 12.8% ) of atypical ductal hyperplasia . 
discordant results at mammotome biopsy concerned five lesions diagnosed as carcinomas in situ that turned out to be invasive carcinomas and two lesions diagnosed as atypical ductal hyperplasia that were characterised as carcinomas in situ on excisional biopsy . 
as for the vacora system , six cases of carcinoma in situ turned out to be invasive carcinomas , and two cases of atypical ductal hyperplasia were characterised as carcinomas in situ at final histology ( table 2 )  . all lesions with benign histological examination of cores removed under us and mx guidance were followed up by us and mammography at 6 , 12 and 24 months and were all presi in considerazione solamente i costi diretti delle singole procedure e si ritenuta non necessaria la puntuale determinazione dei costi indiretti , in considerazione del peso marginale che questi rappresentano rispetto allobiettivo dello studio in quanto non direttamente imputabili al costo della singola procedura . 
 in particolare i costi diretti considerati sono stati quelli fissi , che consistono essenzialmente nel costo delle apparecchiature oggetto di analisi ed ausiliarie e vengono quindi indicati nella tabella 1 sotto la voce tecnologia . 
tale voce comprende il costo relativo ai due sistemi ( mammotome e vacora ) , utilizzati sotto guida ecografica ( us ) , stereotassica ( mx ) e di risonanza magnetica ( rm )  . 
per quanto riguarda i costi variabili , essi si compongono del costo del personale e materiale di consumo . la voce costo del personale comprende il costo relativo alle unit di personale sanitario e tecnico , ( medico radiologo , infermiere , tecnico di radiologia ed ausiliario ota ) , direttamente coinvolte nella procedura , per la durata media degli interventi rilevata nei casi trattati . 
il relativo costo determinato partendo dal costo annuo ( comprensivo di oneri diretti e riflessi ) di ciascuna qualifica professionale previsto nel contratto nazionale , calcolando il costo ora in base allorario settimanale previsto da contratto ( medico radiologo 37 , 98 euro / h , infermiere 24 , 66 euro / h , tecnico di radiologia 24 , 66 euro / h ed ausiliario ota 22 , 62 euro / h ) , ottenendo quindi il costo / minuto e quindi moltiplicando il costo / minuto per il tempo medio di impiego di ciascuna unit di personale nelle specifiche procedure valutato in base alla nostra esperienza . 
in tale voce quindi compreso il costo dellanestesia , dei farmaci , del materiale di medicazione e di altro materiale monouso ( guanti , camici , cuffie , telini sterili , contenitori , bisturi monouso , nonch del kit monouso )  . 
in particolare per quanto concerne il sistema mammotome sono state prese in considerazione le voci relative al set di tubi , al contenitore dei fluidi ed infine alla sonda 11 gauge , utilizzata nel nostro studio , sia per le procedure effettuate sotto guida ecografica che stereotassica , il cui costo risulta inferiore a quello della sonda rm - compatibile . 
in particular , the mammotome system had 84.4% sensitivity and 100% un periodo di ammortamento , come per tutte le alte tecnologie , di 5 anni e quindi di ripartire il costo annuo sul numero di esami annuali calcolati proiettando il numero degli esami effettuati nei sei mesi considerati sui 12 mesi . 
 il canone di manutenzione annuo per le guide strumentali ( ecografo , tavolo di fisher o mammografo , rm ) invece stato calcolato considerando le ore di utilizzo complessivo delle singole apparecchiature , ipotizzando sempre un periodo di obsolescenza di 5 anni , 45 settimane lavorative annuali e 40 ore di attivit settimanali . 
the vacora system had 86.2% sensitivity and 100% specificity , whereas the rate of underestimation was 23.1% for carcinoma in situ and 12.5% for atypical ductal hyperplasia . risultati efficacia clinica cost analysis the initial costs of the two devices are significantly different . the purchase price of the mammotome system is 42 , 217.20 inclusive of vat plus 5 , 028 for the ultrasonic handpiece , 9 , 733.20 for the stereotactic driver , and 27 , 231.60 for the upgrade required to use the system under mri guidance . 
the fixed costs are higher with the mammotome system owing to its higher purchase price and maintenance costs and the need for ultrasonic handpieces , mx drivers and dedicated programmes ( for mri guidance ) , which are not required with the vacora systethe variable costs are also higher for the mammotome system , independently of the guiding device , owing to the higher cost of supplies , with staff and procedures time being equal . 
results of the cost analysis for the two systems used under us , mx and mri guidance are shown in tables 35 , respectively . hospital reimbursement in the period considered , day surgery and outpatient procedures were reimbursed by the regional bodies according to the regional tariff nomenclature for ambulatory care specialist services approved with resolution no . 
 note that the outpatient tariffs for the us - guided procedures would not be sufficient to cover the costs for the hospital , regardless of whether the mammotome or vacora system was used . 
to date , no regional reimbursement exists for procedures performed under mri guidance , probably because they are still uncommonly performed in the lazio regional health service . discussion in recent years , several papers have widely confirmed the diagnostic value of percutaneous vab performed with con lutilizzo di entrambi i sistemi tutte le procedure sono state portate a termine con successo e non si sono verificate significative complicanze periprocedurali , ad eccezione di 5 casi di sanguinamento che si sono risolti con la compressione manuale della mammella . 
in 13 delle 58 procedure effettuate con il sistema mammotome ed in 14 delle 58 procedure eseguite con il sistema vacora sotto guida ecografica , i controlli mammo - ecografici postprocedura hanno confermato lavvenuta escissione totale della lesione nodulare . 
i risultati ottenuti al prelievo vab con il sistema mammotome sono stati rispettivamente : 16 / 108 ( 14 , 8% ) casi di carcinoma invasivo , 22 / 108 ( 20 , 4% ) casi di carcinoma in situ e 12 / 108 ( 11 , 1% ) casi di iperplasia duttale atipica . 
i risultati ottenuti al prelievo vab con il sistema vacora sono stati rispettivamente : 24 / 125 ( 19 , 2% ) casi di carcinoma invasivo , 26 / 125 ( 20 , 8% ) casi di carcinoma in situ e 16 / 125 ( 12 , 8% ) casi di iperplasia duttale atipica . 
per quanto riguarda il sistema mammotome , cinque delle lesioni diagnosticate come carcinoma in situ si sono rilevate carcinomi invasivi e due delle lesioni diagnosticate adh sono state caratterizzate come carcinoma in situ alla biopsia escissionale . 
con il sistema vacora sei casi di carcinoma in situ si sono rivelati carcinoma invasivo e due casi di adh sono stati caratterizzati come carcinoma in situ allistologico definitivo ( tabella 2 )  . tutte le lesioni diagnosticate come benigne alla diagnosi istologica dei frustoli asportati sotto guida ecografica e stereotassica sono state inserite in un programma di follow - up mammo - ecografico ogni 6 / 12 / 24 mesi e si per tutte confermata la non evolutivit allimaging . 
le lesioni caratterizzate come benigne alla diagnosi istologica su frustoli asportati sotto guida rm ( 3 / 17 ) , sono state rivalutate con esame rm dinamico dopo 1 settimana dalla procedura . 
both systems allow preoperative diagnosis and determination of the main biological prognostic markers ( receptor status , proliferation and c - erb overexpression indices ) that are essential for treatment planning [ 8 , 12 ]  . 
in particolare con il sistema mammotome i valori sono del 84 , 4% di sensibilit e 100% di specificit ; nella nostra serie la sottostima del carcinoma in situ risultata del 22 , 7% mentre per ladh pari al 16 , 7% . 
con il sistema vacora i valori di sensibilit e specificit sono stati rispettivamente del 86 , 2% e del 100% , mentre la sottostima per il carcinoma in situ pari al 23 , 1% e per ladh del 12 , 5% . 
il costo di acquisto del sistema mammotome di 42217 , 20 euro , iva inclusa , a cui si aggiungono 5028 euro per il manipolo ecografico , 9733 , 20 euro per il driver stereotassico ed infine 27231 , 60 euro per laggiornamento necessario per lutilizzo del sistema sotto guida rm . 
i costi fissi risultano maggiori nel caso di utilizzo del mammotome in quanto il prezzo di acquisto del sistema e della manutenzione maggiore ed inoltre lutilizzo di tale sistema richiede lutilizzo di manipoli ecografici , drivers stereotassici e programmi dedicati ( nel caso della guida rm ) non richiesti invece nel caso di utilizzo del vacora . 
anche i costi variabili risultano superiori nel caso di utilizzo del mammotome indipendentemente dalla guida utilizzata in quanto la voce materiali di consumo maggiore , a parit di personale impiegato e di durata delle procedure . 
vacora allows single cylindrical cores consistent in size and weight ( 150170 mg ) to be sampled during a single probe insertion [ 18 ]  . sensitivity and specificity of the two technologies observed in our study are in line with those previously reported [ 19 , 20 ] for both systems . 
the rate of underestimation of invasive cancer as carcinoma in situ was close to 23% in our series , whereas that of carcinoma in situ as atypical ductal hyperplasia was 12%16% . all tissue samples obtained with the two devices were intact and judged adequate for histological examination and , although the vacora samples were larger and heavier than the mammotome samples , there were no cases of interpretation difficulties or significant diagnostic differences in the material obtained . 
the higher fixed costs are due to the higher purchase price and a need for more accessories ; the higher variable costs are related to a greater use of supplies . 
the staff cost is equivalent , as the composition and time commitment of the medical and paramedical team are the same . with regard to the mammotome accessories , it should be regione lazio n . 
anche se linvasivit delle procedure minima , ma data la delicatezza dellintervento nonch i risvolti psicologici per la paziente , i prelievi vab vengono generalmente eseguiti , con entrambe le tecnologie , in regime di day surgery . 
 si nota che le tariffe ambulatoriali per le procedure eseguite sotto guida ecografia risulterebbero non sufficienti a coprire i costi della struttura sia nel caso che venga usato il mammotome che nel caso in cui venga usato il vacora . 
entrambe i sistemi consentono di ottenere campioni tissutali adeguati al fine di una diagnosi preoperatoria , permettendo di conoscere i principali markers biologico - prognostici ( assetto recettoriale , indici di proliferazione e sovraespressione di c - erb ) indispensabili per la pianificazione terapeutica delle lesioni mammarie maligne [ 8 , 12 ]  . 
il sistema mammotome permette tramite un unico inserimento della sonda il prelievo di multipli frustoli di tessuto la cui ispezione risulta semplice , in quanto il sistema consente un controllo visivo costante del prelievo durante la procedura . 
il sistema vacora un sistema che offre maggiore maneggevolezza , rispetto al sistema mammotome , essendo un sistema palmare ( 3640200 mm ) dotato di basso peso ( 400 g )  . 
la sottostima del carcinoma in situ rispetto al carcinoma infiltrante stata , nella nostra serie , prossima al 23% , mentre per liperplasia duttale atipica rispetto al carcinoma in situ stata variabile dal 12% al 16% circa . i campioni tissutali sono risultati integri e adeguati al fine dellesame istologico con entrambi i dispositivi utilizzati , e bench con il sistema vacora essi siano risultati di dimensioni e peso maggiori a quelli prelevati con il sistema mammotome , in nessun caso vi sono stati problemi interpretativi da parte dellanatomo patologo , n significative differenze ai fini della diagnosi sul materiale ottenuto . 
 dalla nostra analisi dei costi emerge che sia i costi fissi che i costi variabili , e quindi i costi totali , sono superiori per le procedure effettuate con mammotome piuttosto che con il vacora . 
questa differenza trova origine , per quanto concerne i costi fissi , nel maggior costo di acquisto delle apparecchiature e nella necessit di una dotazione strumentale maggiore nel caso del mammotome e nel maggior utilizzo di materiale di consumo per quanto riguarda i costi variabili . 
cost - effectiveness analysis allows the objective assessment of new technologies and the rationalisation of resources , leading to the protection of public health at affordable healthcare costs . ( 42217 , 20 euro iva compresa ) a cui devono essere aggiunti il manipolo da utilizzare sotto guida ecografia ( 5028 , 00 euro iva compresa ) , il driver necessario per la guida stereotassica ( 9733 , 20 euro iva compresa ) e laggiornamento in rm ( 27231 , 60 euro iva compresa )  . 
cornalba1 1servizio di radiologia diagnostica e interventistica , universit di milano , ospedale san paolo , milano , italy 2servizio di radiologia , istituto humanitas , bergamo , italy 3reparto di ginecologia ostetricia , clinica mangiagalli , milano , italy correspondence to : g . 
pompili , via di rudin 8 , 20142 milano , italy , tel . : + 39 - 02 - 81844798 , e - mail : gpompili@sirm.org received : 12 may 2008 / accepted : 18 july 2008 / published online : 29 may 2009 springer - verlag 2009 abstract purpose . 
fifty - eight women ( age range 1430 years , mean 20.9 ) with primary amenorrhea were studied with mri performed with a 1.0 - t superconducting magnet ( philips nt intera )  . 
t1 - weighted images were acquired in the axial and coronal planes ( tr 470 , te 15 , fov 350 , 4 - mm sections with a 0.6 - mm intersection gap , nsa 3 )  . 
valutare laccuratezza della rm in donne con amenorrea primaria , con sospetta sindrome mayerrokitansky - kuster - hauser ( mrkh ) ( assenza congenita di vagina e utero con presenza di ovaie normali )  . 
immagini turbo spin - echo t2 pesate sono state acquisite sui piani sagittale , assiale e coronale con i seguenti parametri : tr 47506686 , te 100120 , fov 350375 , sezioni di 45 mm con interspazio di 0 , 40 , 5 mm i , nsa 6 . 
immagini t1 pesate sono state acquisite sui piani assiale e coronale ( tr 470 , te 15 , fov 350 , sezioni di 4 mm di spessore con interspazio di 0 , 6 mm , nsa 3 )  . 
due radiologi esperti hanno valutato gli esami in consenso per stabilire la presenza , sede e morfologia di vagina , utero , ovaie , reni ed eventuali ulteriori malformazioni pelviche . 
la sensibilit della rm stata dell81 , 42% e c stato un buon accordo con la laparoscopia ( k cohen 0 , 55 ) e un completo accordo nella identificazione delle cavitazione dei residui tra rm e ecografia intraoperatoria . 
strong cooperation between radiologists and surgeons is highly recommended . ( 82 , 1% ) , policistica in 10 ( 17 , 8% ) ; sede pelvica in 47 ( 83 , 6% ) , extrapelvica in 8 ( 14 , 5% )  . 
una forte cooperazione tra radiologo e chirurgo fortemente raccomandata . keywords magnetic resonance ( mr ) mrkh , mayer rokitansky kuster hauser syndrome utero - vaginal agenesis mllerian anomalies primary amenorrhea parole chiave risonanza magnetica ( rm ) mrkh , sindrome di mayer rokitansky kuster hauser agenesia utero - vaginale anomalie mlleriane amenorrea primaria introduction introduzione the mayer - rokitansky - kuster - hauser ( mrkh ) syndrome is a congenital mllerian anomaly characterised by inadequate development of the mllerian ducts , leading to combined uterovaginal aplasia . 
this syndrome is considered infrequent , but with an incidence of 1 : 4 , 000 female live births , it is a significant cause of mllerian anomalies , and the second most frequent cause of primary amenorrhea , second only to gonadal dysgenesis [ 1 ]  . 
mllerian buds may be observed , which represent more or less developed uterine horns , sometimes with functioning endometriuthe ovaries and fallopian tubes are usually present and normally formed [ 4 ]  . 
the syndrome is often associated with phenotypic alterations of the urinary and skeletal systems [ 5 ]  . the aim of our study was to evaluate the possible diagnostic role of magnetic resonance imaging ( mri ) as an alternative to laparoscopy in allowing adequate anatomical study of the patient before proceeding with surgical creation of a neovagina . la sindrome di mayer - rokitansky - kuster - hauser ( mrkh ) una anomalia congenita mlleriana caratterizzata da un insufficiente sviluppo dei dotti di mller , con conseguente aplasia utero - vaginale combinata . 
questa sindrome considerata una malattia rara , ma con unincidenza di 1 : 4000 nate vive , un ' importante causa di malformazioni mlleriane , e la seconda causa pi frequente di amenorrea primaria , seconda solo alle disgenesie gonadiche [ 1 ]  . la causa dellanomalia interviene durante lembriogenesi con arresto dello sviluppo dei dotti paramesonefrici a 7 settimane dalla fertilizzazione [ 2 ]  . 
la sindrome mkrh si caratterizza con normalit dei genitali esterni , assenza o ridotto sviluppo dei due terzi superiori della vagina e dellutero in luogo del quale possono essere riconoscibili i residui mlleriani rappresentativi di abbozzi uterini pi o meno sviluppati , talora con endometrio funzionale contestuale . 
la sindrome si associa frequentemente ad alterazioni fenotipiche dellapparato urinario e scheletrico [ 5 ]  . scopo del nostro lavoro di valutare il possibile ruolo diagnostico della risonanza magnetica come strumento diagnostico alternativo alla laparoscopia , per fornire un adeguato studio anatomico della paziente prima del trattamento chirurgico volto alla creazione di una neovagina . materials and methods study population materiali e metodi popolazione esaminata our study comprised 58 consecutive patients referred to our radiology department between november 2004 and february 2008 . 
patients were adolescents and young women , with age ranging from 14 to 30 years [ mean 20.9 ; lo studio ha preso in considerazione 58 pazienti consecutive presentatesi presso il nostro servizio di radiologia nel periodo compreso tra novembre 2004 e febbraio 2008 . 
the patients , selected by means of a series of investigations to identify possible mrkh syndrome , had undergone gynaecological examination and ultrasonography , which had raised a suspicion of uterine agenesis or hypoplasia associated with unassessable vagina . mri technique patients arrived at our department with a distended bladder and without specific intestinal preparation . 
the following images were acquired : axial and coronal t1 - weighted tr / te 470 / 15 ms , flip angle 90 , fov / rfov 350 / 80% , thickness / interval 4 / 0.6 mm , nsa 3 axial and coronal t2 - weighted turbo spin - echo ( tse ) tr / te 4 , 750 / 120 ms , flip angle 90 , fov / rfov 375 / 80% , thickness / interval 5 / 0.5 mm , nsa 6 sagittal t2 - weighted turbo spin - echo ( tse ) tr / te 6 , 686 / 100 ms , flip angle 90 , fov / rfov 375 / 80% , thickness / interval 4 / 1.0 mm , nsa 6 axial images were acquired from the vaginal canal up to the iliac crests . mr image analysis a diagnosis of mrkh syndrome was established on the basis of failure to visualise the uterus and absence of a normally developed vaginal canal . 
the canal is better appreciable on sagittal and axial t2 - weighted images where it appears as a lowsignal - intensity structure between the urethra and the bladder neck anteriorly and the rectum posteriorly . 
on t2 , it is possible to evaluate potential cavitation of the popolazione presa in esame rappresentata da adolescenti e giovani donne , di et compresa tra i 14 anni e i 30 anni ( media 20 , 9 ; deviazione standard 4 , 0 ) , con storia di amenorrea primaria . 
le pazienti , selezionate attraverso una serie di indagini volte a definire uneventuale diagnosi di sindrome mrkh , erano state sottoposte ad una visita ginecologica e ad un esame ecografico con responso di sospetta agenesia o ipoplasia uterina accompagnata al riscontro di una vagina insondabile . tecnica di studio rm le pazienti sono giunte allo studio senza specifica preparazione intestinale , a vescica repleta . 
tutti gli esami del nostro studio sono stati effettuati con un magnete 1 , 0 t ( nt intera , philips medical system , best , olanda ) utilizzando una bobina multicanale a 4 elementi . 
sono state acquisite immagini : t1 pesate assiali e coronali tr / te 470 / 15 ms , flip angle 90 , fov / rfov 350 / 80% , spessore / intervallo 4 / 0 , 6 mm , nsa 3 . t2 pesate assiali e coronali turbo spin - echo ( tse ) tr / te 4750 / 120 ms , flip angle 90 , fov / rfov 375 / 80% , spessore / intervallo 5 / 0 , 5 mm , nsa 6 . t2 pesate sagittali turbo spin - echo ( tse ) tr / te 6686 / 100 ms , flip angle 90 , fov / rfov 375 / 80% , spessore / intervallo 4 / 1 , 0 mm , nsa 6 . le immagini assiali sono state acquisite dal canale vaginale fino alle creste iliache . analisi immagini rm la diagnosi di mrkh stata posta in seguito alla mancata visualizzazione dellutero e allassenza di un canale vaginale normalmente sviluppato . due radiologi esperti hanno valutato in consenso , mediante risonanza magnetica : 1 . 
presenza o assenza o incompletezza del canale vaginale . il canale meglio apprezzabile nelle immagini pesate in t2 sul piano sagittale e assiale come struttura a basso segnale tra luretra e il collo vescicale anteriormente e il retto posteriormente . 
a t2 axial turbo spin echo ( tse ) ( tr 4 , 750 ms ; te 120 ms ; thickness 4 mm ) : hypointense structure between the urethra and rectum indicating vaginal bud ( arrow )  . 
a t2 assiale tse ( tr 4750 ms ; te 120 ms ; spessore 4 mm ) : struttura a segnale ipointenso tra uretra e retto , espressione di abbozzo vaginale ( freccia )  . 
presence , morphology and location of the ovaries . ovaries have reduced signal intensity on t1 and mixed signal intensity on t2 due to the marked hyperintensity of the follicular structures and to the low signal intensity of the fibromuscular component . 
 stata infine valutata la qualit delle indagini rm con rating 0 ( esame insufficiente ) , 1 ( sufficiente ) , 2 ( buono ) , 3 ( ottimo )  . radiol med ( 2009 ) 114 : 811826 fig . 
t2 axial turbo spin echo ( tse ) ( tr 4 , 750 ms ; te 120 ms ; thickness 4 mm ) : cavitated mllerian bud with a target appearance ( arrow )  . 
41 ( tabella 3 )  . t2 assiali tse ( tr 4750 ms ; te 120 ms ; spessore 4 mm ) : strutture in sede para - annessiale , a segnale ipointenso con area iperintensa centrale , rappresentative di residui mlleriani cavitati bilaterali ( frecce )  . two different types of intervention are possible , both performed with the laparoscopic technique : the vecchietti method and the davydov procedure . 
the former is based on the constant pressure exerted on the pseudohymenal membrane by a vaginal dilator connected via two perlon wires to a traction device on the pubis , with progressive upward stretching of the vestibular dome [ 14 , 15 ]  . 
the davydov technique , by contrast , uses peritoneum from the douglas pouch to create the vaginal dome , fornices and lining of the vaginal canal [ 1619 ]  . 
the time elapsed between mri and surgery was 1180 ( mean 90 , median 60 ) days . intervento chirurgico le pazienti dello studio sono state sottoposte ad un intervento chirurgico , mirato alla creazione di un canale vaginale con asse , lunghezza e capacit secretoria normali , in grado di garantire normali rapporti sessuali [ 13 ]  . 
il primo intervento si fonda sulla pressione continua che un tutore vaginale , collegato con due fili di perlon ad un apparecchio per trazione applicato sopra al pube , esercita sulla membrana pseudo - imenale , con progressivo stiramento verso lalto della cupola vestibolare [ 14 , 15 ]  . 
lintervento secondo davydov si basa invece sullimpiego del peritoneo per la creazione della cupola vaginale , i fornici ed il rivestimento del canale vaginale ottenuto attraverso la dissezione dello spazio rettovescicale [ 1619 ]  . 
il tempo intercorso tra lesame di risonanza magnetica e lintervento chirurgico stato di 1180 giorni ( media 90 giorni , mediana 60 giorni )  . analisi statistica lanalisi dei dati forniti dallindagine rm stata condotta al fine di determinare laccuratezza diagnostica della stessa nella valutazione della sindrome verificando quanto i giudizi formulati corrispondano effettivamente alla realt fornita dal metodo di riferimento ( gold standard ) , rappresentato , in questo studio , dalla laparoscopia . 
oltre alla determinazione dellaccuratezza diagnostica della rm , mediante lutilizzo di tabelle di contingenza di tipo 33 , abbiamo valutato laffidabilit dei nostri risultati calcolando il livello di concordanza cio la misura del grado di accordo tra le due indagini , al di l del grado di concordanza tra le metodiche che ci si aspetterebbe se le due indagini fossero indipendenti , fornita dalla k di cohen . 
il grado di accordo , e quindi il valore di k ottenuto dallo studio , stato considerato facendo riferimento alla scala di valutazione impiegata per valutare la concordanza di queste stesse tecniche nello studio di reinhold et al . 
a t2 coronal turbo spin echo ( tse ) ( tr 4 , 750 ms ; te 120 ms , thickness 4 mm ) : ovaries present bilaterally in extrapelvic location , situated laterally to the psoas muscle ( arrows )  . 
a t2 coronale tse ( tr 4750 ms ; te 120 ms , spessore 4 mm ) : ovaie in sede extrapelvica bilateralmente , situate lateralmente al muscolo psoas ( frecce )  . 
rene dislocato in sede pelvica , posteriormente e lateralmente alla vescica che risulta improntata sul profilo destro . table 1 rating scale for cohens k test ( from [ 20 ] ) tabella 1 scala di valutazione dei valori di k di cohen ( da [ 20 ] ) < 0 , 4 0 , 40 , 59 0 , 60 , 74 > 0 , 75 poor agreement fair agreement good agreement excellent agreement < 0 , 4 0 , 40 , 59 0 , 60 , 74 > 0 , 75 accordo basso accordo discreto accordo buono accordo eccellente statistical analysis risultati we determined the diagnostic accuracy of mri in the evaluation of mrkh syndrome by verifying the extent to which the mri findings reflected the real situation demonstrated by the reference method ( gold standard ) in this study , laparoscopy . 
in addition to measuring diagnostic accuracy , we used 33 contingency tables to evaluate the reliability of our results by calculating the level of concordance that is , the level of agreement between the two techniques beyond that expected by mere chance with cohens k test . 
the levels of agreement ( k values ) were based on the scale adopted for evaluating the concordance between these two techniques in a previous study on uterovaginal anomalies by reinhold et al . 
in addition , we carried out a descriptive analysis of the anatomical findings of mri for patients who underwent mri only . delle 58 pazienti che hanno eseguito lesame di risonanza magnetica , 56 hanno ricevuto conferma diagnostica di mrkh , con descrizione dei riscontri pelvici correlati . 
le restanti pazienti hanno rimandato la laparoscopia al momento in cui saranno pronte ad eseguire il successivo intervento chirurgico . in 18 pazienti lintervento stato eseguito secondo vecchietti e in 23 pazienti si effettuata invece una creazione di neovagina secondo davydov . 
in questo modo stata possibile la valutazione dellaccuratezza statistica radiol med ( 2009 ) 114 : 811826 dellindagine strumentale nella diagnosi della sindrome e nella valutazione anatomica del quadro associato . 
 la tabella 2 riporta i risultati ottenuti con la sola indagine rm in tutte le 56 pazienti affette da sindrome mkrh . in 2 pazienti lesame stato viziato da artefatti di movimento ( paziente 22 e 43 ) ed pertanto risultato di scarsa qualit tecnica ma sufficiente per porre diagnosi di sindrome mrkh ( rating 1 )  . 
 stata possibile una diagnosi di mrkh in tutti i casi di malattia , la rm ha quindi dimostrato una sensibilit diagnostica pari al 100% . in nessun caso , inoltre , stata posta diagnosi di mrkh in assenza di malattia ; in 2 casi sui 58 totali stata posta diagnosi differente e , trattandosi di veri negativi , la specificit diagnostica risultata essere a sua volta del 100% . in 34 pazienti ( 60 , 7% ) stata individuata la presenza bilaterale di rudimenti . 
in 10 pazienti ( 17 , 9% ) i residui sono risultati essere monolaterali , mentre in 12 pazienti ( 21 , 4% ) si riscontrata lassenza di abbozzi uterini ( tabella 4 )  . in 54 pazienti le ovaie sono state individuate bilateralmente mentre due pazienti hanno presentato un solo ovaio monolaterale . 
la localizzazione delle ovaie risultata normalmente pelvica in 48 pazienti ( 85 , 7% ) , mentre in 8 pazienti ( 14 , 3% ) si apprezzata la presenza di annessi in sede extrapelvica , bilaterale in 6 pazienti , monolaterale in 2 . 
in un caso stata individuata la presenza di una voluminosa cisti emorragica e in un altro di una cisti semplice di discrete dimensioni ( tabella 5 )  . il canale vaginale risultato essere completamente assente in 38 pazienti ( 67 , 8% )  . 
in 15 pazienti ( 26 , 8% ) stato possibile riconoscere il terzo inferiore del canale mentre in 2 casi la maggior estensione del canale ha suggerito la presenza dei due terzi inferiori . 
in 1 paziente , invece , il canale vaginale stato classificato come normoconformato seppur di dimensioni minime poich caratterizzato da uno sviluppo comunque presente ( tabella 6 )  . la presenza di versamento liquido in cavit pelvica stata segnalata in 33 pazienti ( 58 , 9% ) localizzato pi frequentemente ( 30 / 33 ) posteriormente alla vescica dove troviamo normalmente il cavo retto - uterino del douglas . con la rm stata valutata lassociazione della sindrome con le anomalie del sistema renale . 
grosso residuo mlleriano cavitato a sede retrovescicale ( 43 cm con cavit di 7 mm ) con aspetto a bersaglio : in questa paziente il successivo intervento chirurgico render possibile la creazione di un piccolo utero funzionale . results of the 58 patients studied by mri , 56 had diagnostic confirmation of mrkh , with demonstration of characteristic pelvic findings , whereas two patients received a different diagnosis ( one morris syndrome and one isolated vaginal atresia )  . 
this allowed us to determine the accuracy of mri in diagnosing the syndrome and in evaluating the associated anatomical findings . that obtained table 2 shows the results obtained with mri alone in all 56 patients affected by mrkh syndrome . 
in no case was a diagnosis of rokitansky syndrome established in the absence of disease ; in two out of 58 cases , a different diagnosis was established and , because the results were true negative , diagnostic specificity was also 100% . bilateral buds were present in 34 patients ( 60.7% ) and unilateral buds in ten ( 17.9% ) , whereas there was no evidence of uterine buds in 12 ( 21.4% ) ( table 4 )  . the ovaries were present bilaterally in 54 patients , whereas two patients had one unilateral ovary only . 
of the latter , one patient had previously undergone adnexectomy . location of the ovaries was pelvic in 48 patients ( 85.7% ) and extrapelvic in eight ( 14.3% ) and bilateral in six and unilateral in two . 
in one case , we detected a large haemorrhagic cyst , and in another case , a fairly large simple cyst ( table 5 )  . the vaginal canal was completely absent in 38 patients ptosi e ectopia renale . 
in 2 ( 3 , 6% ) pazienti , invece , si riscontrata una regolare posizione anatomica di un rene , associata ad una localizzazione pelvica del rene controlaterale . in 1 caso ( 1 , 8% ) si riscontrata una modesta ptosi renale monolaterale ( tabella 7 )  . delle 56 pazienti con mrkh che hanno eseguito lesame di risonanza magnetica , 41 hanno poi programmato lintervento e la laparoscopia preoperatoria , permettendo il confronto dei dati anatomici ottenuti con le due indagini ( tabella 3 )  . 
in 24 pazienti sia la rm che la laparoscopia hanno condotto allindividuazione di residui bilaterali , in 6 pazienti stata concordemente riscontrata la presenza di un unico residuo monolaterale ed in 2 pazienti i residui erano , invece , assenti . 
in 15 patients ( 26.8% ) , it was possible to recognise the lower third of the cavity , whereas in two cases , the greater length of the canal suggested the presence of the lower two thirds . 
in one patient , the vaginal canal was classified as having normal morphology but very small size ( table 6 )  . fluid collection within the pelvic cavity was seen in 33 patients ( 58.9% ) , most frequently located posterior to the bladder ( 30 / 33 ) , where the rectouterine douglas pouch is normally found . 
with regard to the evaluation of associated urinary anomalies , despite the many possible alterations , we concentrated on the main ones , namely , agenesis , ptosis and renal ectopia . 
in one case ( 1.8% ) , there was moderate unilateral renal ptosis ( table 7 )  . of the 56 patients with mrkh who underwent mri , 41 laparoscopy and underwent subsequent preoperative surgery , allowing comparison of the anatomical information obtained with the two techniques ( table 3 )  . 
the information concerning the presence or absence of buds was concordant in 32 / 41 patients ( 78% )  . both techniques demonstrated bilateral buds in 24 patients , a single unilateral bud in six and the absence of buds in two . individuazione bilaterale di residui alla laparoscopia , la rm stata in grado di documentare la presenza di un solo residuo in 2 pazienti mentre , nelle restanti cinque , non sono stati individuati entrambi . 
inoltre , in 1 paziente con riscontro di struttura rudimentale monolaterale alla laparoscopia , stata posta diagnosi di mancato riscontro alla rm . in una sola paziente , delle 8 con residuo monolaterale , la rm ha posto diagnosi di residuo bilaterale . 
la sensibilit diagnostica dellindagine rm nellindividuazione dei residui 33 / 39 , 85% ; con un intervallo di confidenza , per un livello di confidenza del 95% , compreso tra 70% e 93% ( 0 , 703 < p < 0 , 927 ; 82%11 , 5% )  . 
la specificit risulta invece essere del 100% ( 2 / 2 ) poich non sono mai stati individuati residui mlleriani dove questi non erano presenti , ma ragionando anche in questo caso per reperti , la specificit scende a 11 / 12 , 91% . 
in particolare si avuto un accordo sulla presenza e localizzazione delle gonadi con localizzazione table 4 identification of mllerian buds on magnetic resonance imaging radiol med ( 2009 ) 114 : 811826 bilateral 34 / 56 ( 60.7% ) bilateral 10 / 56 ( 17.9% ) bilateral 12 / 56 ( 21.4% ) rudimentary uterus no . 
of patients patients percentage table 5 identification of the ovaries on magnetic resonance imaging regular 46 / 56 ( 82.1% ) polycistic ovaries 10 / 56 ( 17.9% ) pelvic 48 / 56 ( 85.7% ) extrapelvic 8 / 56 ( 14.3% ) conformation localization no . 
of patients patients percentage radiol med ( 2009 ) 114 : 811826 table 6 identification of the vaginal canal on magnetic resonance imaging absent 38 / 56 ( 67.8% ) 1 / 3 lower 15 / 56 ( 26.8% ) 2 / 3 lower 2 / 56 ( 3.6% ) hypoplasia 1 / 56 ( 1.8% ) portion of the vagina no . 
of patients patients percentage identified at in seven patients with bilateral buds laparoscopy , mri was able to demonstrate the presence a solitary bud in two patients , whereas neither of the buds was identified in the other five . 
specificity was 100% ( 2 / 2 ) , as mllerian buds were never detected when they were not present ; however , based on the findings , specificity falls to 11 / 12 ( 91% )  . 
the level of concordance between the two techniques was k = 0.55. nine patients also underwent intraoperative ultrasonography , which allowed us to correlate the information concerning the possible cavitation of the buds ( table 8 )  . 
in particular , an agreement was reached concerning the presence and location of the ovaries , with bilateral pelvic location in 29 patients , bilateral extrapelvic in six , unilateral extrapelvic in one and solitary pelvic ovary in two . 
in two cases , there was disagreement regarding the location of the ovaries , whereas pelvica bilaterale in 29 pazienti , extrapelvica bilaterale in 6 , extrapelvica monolaterale in una , monovaio pelvico in 2 pazienti . 
in due casi si avuta discordanza riguardo alla sede degli annessi mentre in un terzo caso una gonade riconosciuta allesame rm non sono state poi visualizzate allesame laparoscopico . discussione nonostante la sindrome mrkh sia una condizione rara , frequenti cause di essa rappresenta una delle pi amenorrea primaria [ 11 ]  . 
la popolazione del nostro studio infatti rappresentata da giovani pazienti , valutate comunque in et post - pubere , con et compresa tra i 14 e i 30 anni , con una media di 20 , 9 anni . 
a queste indagini seguono esami strumentali rappresentati , in prima istanza , dallecografia . i risultati ecografici rischiano per di essere inconclusivi o incompleti , rendendo cos necessarie ulteriori indagini diagnostiche al fine di ottenere informazioni pi dettagliate e oggettive [ 8 , 9 ]  . 
of patients patients percentage in the third , case one ovary recognised at mri was not visualised by laparoscopy . discussion although mrkh syndrome is a rare condition , it represents the second more frequent causes of primary amenorrhoea [ 11 ]  . 
ultrasonography , in fact , does not always allow the recognition of mllerian buds and of ovaries with extrapelvic location , and this information is necessary for characterising the patients anatomical situation and defining the best surgical strategy [ 23 ]  . until a few years ago , detailed anatomical definition was provided by diagnostic laparoscopy , mri having only riconoscimento dei residui mlleriani e delle ovaie a localizzazione extrapelvica , dati invece necessari per inquadrare la situazione anatomica della paziente e definire la miglior soluzione chirurgica [ 23 ]  . fino a qualche anno fa la definizione dettagliata anatomica della sindrome veniva fornita in sede laparoscopica diagnostica mentre solo in tempi pi recenti che si resa disponibile una metodica alternativa non invasiva quale la rm . 
 [ 9 ] , al preventivo studio ecografico non erano state individuate strutture anatomiche poi correttamente descritte allindagine rm . lindagine ecografica , inoltre , in quanto operatoredipendente , conduce a risultati che possono risultare divergenti se eseguita pi di una volta , senza poter garantire loggettivit necessaria alla programmazione dellintervento chirurgico . 
lo studio rm in grado di valutare in modo accurato le anomalie utero - vaginali grazie alla possibilit di uno studio multiplanare , e grazie alladozione di sequenze diversamente pesate , volte a garantire la migliore visualizzazione delle diverse strutture indagate [ 2 , 5 ]  . nel nostro studio , al fine di fornire tutte le informazioni utili alla programmazione del successivo intervento , sono state indagate le strutture dello scavo pelvico , associate allo studio delle logge renali . 
nella nostra esperienza su 58 pazienti la rm si dimostrata metodica assai efficace nella radiol med ( 2009 ) 114 : 811826 table 8 evaluation of possible cavitation of mllerian buds : comparison between magnetic resonance imaging and laparoscopy buds cavitation ( n = 9 patients ) cavitated buds not cavitated buds absent buds recently become available as a noninvasive alternative tool . in a study by fedele et al . 
 furthermore , because ultrasonography is an operatordependent technique , it may provide conflicting results when performed several times , and it fails to guarantee the neutrality required for surgical planning . 
mri can accurately evaluate uterovaginal anomalies thanks to multiplanar imaging and the use of differently weighted sequences , allowing the best visualisation of all structures being examined [ 2 , 5 ]  . in our study , to obtain all the information required for surgical planning , we studied the structures of the pelvic pouch and renal compartment . 
in our experience on 58 patients , mri proved to be an effective technique in the diagnosis of mrkh syndrome , with 100% sensitivity ( 56 / 56 patients )  . 
furthermore , it enabled correct identification of the cause of primary amenorrhoea in the two remaining patients , with 100% specificity . the search for structures consistent with mllerian buds allowed their detection in 33 / 41 patients ( 80% )  . 
inoltre stato anche possibile identificare correttamente la causa di amenorrea primaria nelle due restanti pazienti con una specificit della nostra metodica del 100% . la ricerca di strutture compatibili con residui mlleriani ha condotto a una loro identificazione in 33 / 41 pazienti ( 80% )  . 
i dati forniti dalle due indagini hanno mostrato un grado di concordanza discreto dato da k = 0 , 55 . comunque importante considerare che in 2 pazienti in cui stato effettuato lesame rm , le immagini sono risultate compromesse da artefatti di movimento . 
se la valutazione si limita a considerare gli esami tecnicamente validi , il livello di accordo nella valutazione dei residui mlleriani risulta buono ( k = 0 , 62 )  . 
poich nella nostra esperienza gli artefatti da movimento erano perlopi determinati dalla impellenza minzionale della paziente potrebbe essere utile in futuro , per quanto possibile , controllare ecograficamente lo stato di distensione vescicale prima dellinizio dellindagine rm . da ritenersi che rm e laparoscopia forniscano risultati in sostanziale accordo tra di loro nella valutazione dei residui mlleriani , soprattutto per esami tecnicamente validi . uno degli aspetti clinici pi rilevanti nelle pazienti portatrici di sindrome mkrh costituito dalla comparsa in fase post pubere di dolori pelvici ciclici . 
lo studio della causa di tali sintomi negli anni ha portato a riconoscerne come causa principale laccumulo di materiale emorragico a livello degli abbozzi uterini dotati di endometrio funzionale [ 2 ]  . 
because the motion artefacts were chiefly caused by the patients urge to urinate , it may be useful to check the level of bladder distension by ultrasound before starting the mri study . 
mri and laparoscopy provide substantially concordant results in the evaluation of mllerian buds , above all in the case of technically adequate examinations . one of the most important clinical features of mrkh syndrome is the development of cyclic pelvic pain in the postpuberal period . 
the study of these symptoms over the years has shown that their main cause is accumulation of haemorrhagic material within uterine buds with a functioning endometrium [ 2 ]  . 
however , surgical evaluation of cavitation in these studies was only indirect and based on the size of the buds and was consequently far less reliable than evaluation with mri . 
in contrast , in our study , laparoscopic ultrasonography provided direct evaluation , indicating the effective capability of mri to provide precise information . to our knowledge , no other studies have compared the two techniques in the evaluation of functioning buds in these terms , so it is reasonable to state that mri is completely reliable in the preoperative evaluation of the presence of a functioning endometriuin our experience , laparoscopic ultrasonography has become unnecessary , and we have limited its use to a few selected cases of larger buds eligible for the potential creation of a small functioning uterus . in the evaluation of the ovaries , and in particular of their location and morphology , mri and laparoscopy produced identical results in all but three patients ( 38 / 41 , 93% ) ( table 3 )  . 
as a consequence , mri proved to be a reliable technique in this evaluation . tale reperto poteva essere solo sospettato clinicamente e accertato in fase laparoscopica con uno studio ecografico intraoperatorio o con una valutazione anatomo - patologica del residuo asportato . 
purtroppo un limite nella valutazione di questo aspetto , rappresentato dal piccolo numero di pazienti in cui stato possibile il confronto coi dati laparoscopici . lavori precedenti [ 20 , 23 ] , pur sempre limitati da un basso numero di pazienti , hanno indagato questaspetto concludendo per una migliore accuratezza dellindagine rm . 
in questi studi , tuttavia , la valutazione chirurgica della cavitazione era stata solo indiretta e basata sulla dimensione del residuo , risultando pertanto assai meno affidabile della valutazione rm . 
per quanto ci dato di conoscere non esistono altri lavori in letteratura che abbiano confrontato in questi termini le due indagini nella valutazione dei residui funzionali e possiamo pertanto affermare che la rm totalmente affidabile nella valutazione prechirurgica della presenza di endometrio funzionale . 
nella nostra esperienza ci ha via via reso superfluo il ricorso allecografia intralaparoscopica limitandone luso a casi mirati di residui dimensionalmente rilevanti eleggibili per la potenziale creazione di un piccolo utero funzionante . nella valutazione delle gonadi , e in particolare della loro localizzazione e morfologia , rm e laparoscopia hanno condotto a risultati concordi in tutte le pazienti con eccezione di tre casi ( 38 / 41 , 93% ) ( tabella 3 )  . 
anche per questa valutazione la rm risultata dunque metodica affidabile . dai nostri dati quindi emersa una significativa accuratezza dellindagine rm nello studio dellanatomia pelvica ed in particolare delle gonadi e dei residui mlleriani . 
si ottenuto , infatti , un discreto grado di concordanza nellindividuazione di residui ed un ottima valutazione della cavitazione degli abbozzi stessi . il livello di accuratezza raggiunto dalla rm permette , cos , di rimandare lesplorazione laparoscopica , esame invasivo e costoso , che necessita di anestesia e ospedalizzaradiol med ( 2009 ) 114 : 811826 our data indicate that mri has a significant level of diagnostic accuracy in the study of pelvic anatomy and in particular of the ovaries and mllerian buds . 
detection of buds showed a good level of concordance , and evaluation of possible cavitation was excellent . the level of accuracy achieved by mri allows patients to postpone laparoscopic exploration , an invasive and expensive procedure requiring anaesthesia and hospitalisation , to when they feel ready to face surgery [ 2 , 5 ]  . 
in particular , mri is able to depict the vaginal canal , even when budary , which in most cases corresponds to the lower third of the canal that is , to the portion of ectodermal origin that is not systematically affected by the mllerian anomaly owing to its different embryologic orig mri identification of a vaginal bud between the rectum and bladder is surgically useful , as it may be used to guide in the creation of a vaginal canal . the ability of mri to study associated urinary anomalies is important for preoperative evaluation . 
generally , in urinary anomalies , especially in the presence of a kidney in pelvic location or of a ptosic kidney , the vecchietti technique is preferred , as it is less invasive and minimises the risk of possible renal or ureteral injury . differentiating mrkh syndrome from other mllerian anomalies and more specifically from malformations of the distal genital tract is a necessity dictated not only by the different surgical strategies required by each anomaly but also by the different reproductive potential and psychological impact . in agreement with previous studies based on smaller patient samples , we believe that mri is an essential tool in the diagnosis and anatomical evaluation of mrkh syndrome , as it provides an in - depth study of the pelvis [ 20 , 23 ] and makes it possible to postpone the more invasive and expensive laparoscopic exploration until the time of surgery , when patients are anaesthetised and hospitalised . in our opinion , cooperation between the radiologist and the surgical team is fundamental for correct preoperative management of these young patients . 
identification of cavitated mllerian buds with endometrial tissue indicates the possibility of creating a functioning uterus or , alternatively , suggests their removal . zione , al momento in cui la paziente si sentir pronta ad affrontare lintervento [ 2 , 5 ]  . 
in particolare la rm in grado di rilevare la presenza di un canale vaginale , anche se ridotto a struttura rudimentale , corrispondente , nella maggior parte dei casi , al terzo inferiore del canale di derivazione ectodermica , che non risulta sistematicamente interessato dallanomalia mlleriana , data la diversa origine embriologica . 
lidentificazione alla rm di un abbozzo vaginale tra retto e vescica chirurgicamente utile perch pu essere utilizzato come guida alla creazione del tunnel vaginale . molto importante nella valutazione prechirurgica risulta la capacit della rm di indagare anche lassociazione della sindrome con anomalie dellapparato urinario . 
generalmente in caso di anomalie del sistema renale , ed in particolare in presenza di rene pelvico o comunque ptosico , infatti preferibile il ricorso ad unoperazione secondo vecchietti che , per la minor invasivit delloperazione , riduce il rischio di uneventuale lesione renale o ureterale . 
tale necessit dovuta , non solo al diverso trattamento chirurgico che spetta a ciascuna anomalia , ma anche al diverso potenziale riproduttivo e conseguente impatto psicologico delle differenti forme di presentazione . in accordo con altri studi precedenti [ 20 , 23 ] , basati su un numero inferiore di pazienti , riteniamo che la rm sia strumento diagnostico essenziale nella diagnosi e nella valutazione anatomica della sindrome mrkh fornendo uno studio dettagliato della pelvi della paziente . 
hasbargen1 1department of obstetrics and gynecology , 2department of radiology , ludwig - maximilians - university munich , campus grosshadern , marchioninistrasse 15 , 80337 munich , germany correspondence to : m . 
on biplanar topograms , multiplanar reconstructions of 1 - mm slices , volume - rendered images of the same data and volumerendered images based on 5 - mm - thick images . interobserver agreement and variability were determined by bland - altman analysis . 
with a correlation coefficient of 0.98 , interobserver agreement was best for assessing 3d volumerendered images reconstructed from 1 - mm - thick slices . interobserver variability was very good for sagittal outlet and midpelvic diameter , transverse inlet diameter and obstetric conjugate ( correlation coefficients 0.960.99 ) but limited for intertuberous and interspinous distance . 
sono state ottenute 6 misurazioni pelvimetriche da due osservatori indipendenti in quattro data sets per ogni paziente , cio topogrammi in due proiezioni , ricostruzioni multiplanari con scansioni dello spessore di 1 mm , immagini volumetriche degli stessi dati ed immagini volumetriche basate su scansioni dello spessore di 5 mla variabilit e la concordanza interosservatore sono state determinate secondo lanalisi bland - altman . 
con un coefficiente di correlazione di 0 , 98 , la concordanza tra gli osservatori stata migliore nella valutazione delle immagini volumetriche tridimensionali ricostruite da scansioni dello spessore di 1 mla variabilit interosservatore stata molto buona per il diametro sagittale esterno e per il diametro intermedio della pelvi , per il diametro dellinserzione trasversa e per la coniugata ostetrica ( coefficiente di correlazione 0 , 960 , 99 ) , ma limitata per la distanza tra le tuberosit e tra le spine ischiatiche . 
pertanto , la pelvimetria - tc 828 radiol med ( 2009 ) 114 : 827834 keywords ct - pelvimetry cephalopelvic disproportion dystocia interobserver variability adatta ad acquisire lesatta conoscenza dellanatomia pelvica per identificare i parametri rilevanti per distocia negli studi retrospettivi . parole chiave pelvimetria - tc sproporzione cefalopelvica distocia variabilit interosservatore introduction about two thirds of all caesarean sections are performed due to dystocia or are indirectly related to cephalopelvic disproportion because they are scheduled for elective caesarean after cephalopelvic disproportion at first delivery [ 1 ]  . 
cephalopelvic disproportion should be diagnosed when there is an adequately dilated cervix and sufficient contractions of the uterus but the fetal head does not descend into the birth canal [ 2 ]  . 
the need for a reliable method to detect women at risk for dystocia has been recognised since the eighteenth century , and various approaches to pelvimetry have been used to identify small pelvis as indication for caesarean section . 
conventional x - ray and ct pelvimetry have been largely abandoned since mri pelvimetry became available , as exposure to ionising radiation , especially of the foetus , can be avoided [ 310 ]  . 
the results should be useful to identify the postprocessing method with the best reproducibility and to estimate the required radiation exposure for low - dose exams . the study can also indicate necessary slice thickness for pelvimetry on previously existing ct scans , either to perform measurements for an individual patient or to retrospectively evaluate pelvimetric measurements of large patient groups for study purposes . 
 materials and methods available ct data ct pelvimetry was performed in 25 female patients based on ct data that had been acquired in routine clinical care . the retrospective evaluation is warranted by the universitys ethics committee guidelines and adhered to the world medical association declaration of helsinki . 
ct data sets of 15 consecutive women referred for abdominal ct postpartum in 2007 had been obtained on 64 - slice ct scanners ( somatom sensation 64 or somatom definition , siemens , forchheim , germany ) with 0.6 - mm collimation at 120 kvp tube potential and 250 mas reference tube current time product with dose modulation . 
 additionally , pelvimetric measurements were acquired in ct data sets of ten women with known multiple myeloma who had undergone low - dose ct to screen for osteolysis and additionally had had a normal routine abdominal ct scan for other reasons within the last 2 years . 
the routine scans had been obtained with the routine parameters given above , whereas the low - dose scans had been acquired with a reduced tube current of 83 reference mas . 
a human cadaver skeleton was also scanned with the mentioned normal and low - dose protocols to validate ct - pelvimetric measurements , with manual measurements by ruler as external reference . 
radiation dose was calculated for each exam based on the ct dose index ( ctdi ) and the scan length , which are represented in radiol med ( 2009 ) 114 : 827834 fig . 
the equivalent dose can be calculated from the dlp with a conversion factor of 0.017 msv / mgy * cm for the pelvis [ 11 ]  . pelvimetry based on these images , pelvimetry was performed retrospectively by two independent blinded radiologists on a 3d workstation ( siemens multi - modality workplace , siemens , forchheim , germany ) using topograms , multiplanar reconstructions and volume - rendered images . 
these included the obstetric conjugate as the shortest distance between promontory and symphysis , the sagittal outlet as the distance between the inferior inner aspect of the symphysis and the distal end of the sacrum , and the midpelvic sagittal diameter from the 830 radiol med ( 2009 ) 114 : 827834 fig . 
c ricostruzione para - assiale che mostra le spine ischiatiche , la regione caudale della sinfisi e il sacro cos da poter misurare la distanza tra le spine ed il diametro intermedio della pelvi . 
as a fourth method , the same reconstructions were done based on 5 - mm - thick slices , and all measurements were obtained equally on 3d volume - rendered images . to assess clinical usability in daily routine , we clocked the times needed to measure all six pelvimetric numbers needed for pelvimetry by the different methods . 
le linee indicano la coniugata ostetrica , il diametro intermedio della pelvi e quello sagittale esterno . statistical analysis results all continuous variables are displayed as mean and standard deviation ( sd )  . 
 ex vivo ct pelvimetry agreed well with manual measurements , with a maximal deviation of 2 mm for the intertuberous distance and errors of 01 mm for the other pelvimetric distances , indicating that there is virtually no relevant systematic error . 
 regarding assessment of the different pelvimetric distances with the different methods in detail , sds of interobserver variability were generally lower for cross - sectional and 3d approaches compared with the topogram - based measurements ( table 2 )  . 
 also , variability was lower for obstetric conjugate ( m ) , transverse inlet ( c ) , sagittal midpelvic ( q ) and sagittal outlet ( s ) than for interspinal ( h ) and intertuberous ( i ) distance . regarding the different pelvimetric distances in summary , interobserver variability of 2d and 3d measurements was very low , with an sd < 2% for m , c , q and s distances , whereas variability was higher for h and i ( table 3 )  . 
 ct dose index ranged from 11 to 15 mgy at standard tube voltage of 120 kvp and a reference tube current time product of 220 mas ( ref ) with automatic exposure control , resulting in tube current values of 142180 mas . 
the impression was that a further reduction of tube current would be possible if the assessment of pelvic dimensions is the only purpose of the exa regarding reading time for measuring the various pelviradiol med ( 2009 ) 114 : 827834 metric dimensions , there were significant differences between the different postprocessing approaches . 
the topogram - based approach was performed fastest , with an average reading time of 12030 s , whereas the 3d methods required 18624 and 18015 s for 1 - mm and 5 - mm data sets , respectively . 
in comparison with the interobserver variability reported for standard mri pelvimetry in literature [ 13 ] , ct offers an at least comparable accuracy . our results indicate that a very easy , fast and accurate determination of pelvimetric dimensions can be achieved with 3d volume - rendered reconstructions , which therefore can be regarded as the method of choice . 
although the best accuracy is achieved with this approach , measurements on volume - rendered images derived from 5 - mm - thick slices achieve a comparable agreement as 2d cross - sectional reconstructions from 1 - mm data sets . 
with a standard deviation of 2% corresponding to 23 mm for most pelvimetric distances , the accuracy of these 2d and 3d measurements seems acceptable , whereas the sd of interobserver variability for the topogram - based assessment with about 5% sd may be clinically relevant . 
the round shape of the ischiadic tuber and the continuous transition between the spinous process and the ligaments inserting there with variable density and calcification can be assumed to represent the reasons for this difference [ 14 ]  . 
still , taking into account the availability of mri as an alternative method without ionising radiation , the indication for ct pelvimetry is very limited , especially in pregnant women [ 15 ]  . 
regarding the fact that dystocia can be life threatening for mother and foetus , ct may still represent an adequate diagnostic tool in critical situations if mri is not readily available or is contraindicated for other reasons , or in nonpregnant women with a history of dystocia . 
also , the results should apply similarly to mri data sets acquired with 3d pulse sequences ( e.g. vibe ) , which would release dependence on a precise primary data acquisition . 
low - dose protocols do not significantly affect accuracy and can contribute to significant dose savings , although radiation exposure may restrict the prospective application of ct pelvimetry in general . 
mazzucato piccin nuova libraria , padova , eds . isbn 978 - 88 - 299 - 1980 - 2 published online : 17 july 2009 springer - verlag 2009 the book anatomia radiologica : tecniche e metodologie in radiodiagnostica ( radiological anatomy : techniques and modalities in diagnostic imaging ) by fernando mazzucato has now reached its third edition . 
it is a pleasure to recall that many radiologists and radiographers have used and studied this textbook and that many of the teaching faculty in the postgraduate schools of radiology and undergraduate courses for radiology and radiotherapy technicians recommend it to their students . the book has always enjoyed considerable success , as testified by the numerous reprints , and its popularity is justified by its clear ability to meet the real needs of radiologists . 
the third volume is in fact entirely devoted to computed tomography ( ct ) , magnetic resonance imaging ( mri ) and positron emission tomography ( pet ) - ct . 
the approach and layout of prior editions have been maintained , with numerous images , drawings and diagrams , many of which are in colour . the three volumes offer complete coverage of technical , methodological and anatomical aspects at the level of all body organs and systems . 
in the first volume , considerable space is given to physics and technique , equipment , analogue and digital radiographic images , as well as to radiology information systems ( ris ) and picture il volume anatomia radiologica tecniche e metodologie in radiodiagnostica di fernando mazzucato giunto alla sua terza edizione . 
fa piacere sottolineare che su questo libro hanno studiato e si sono formati molti radiologi e tecnici radiologi , e molti di coloro che ricoprono incarichi di responsabilit nelle scuole di specializzazione di radiologia e nei corsi di laurea per tecnici di radiologia , per immagini e radioterapia consigliano questo libro ai propri studenti . il libro ha sempre riscosso grande successo , come testimoniato dalle numerose ristampe , perch evidentemente orientato verso le reali necessit del radiologo . 
infatti , il terzo volume interamente dedicato alla tomografia computerizzata ( tc ) , alla risonanza magnetica ( rm ) , alla tomografia ad emissione di positroni ( pet )  . inoltre , ledizione attuale comprende una nuova parte dedicata alla radiologia interventistica , a testimonianza del ruolo fondamentale di questa disciplina per il radiologo . lopera si avvale della ormai consolidata impostazione caratterizzata da una ricca iconografia , numerosi disegni e schemi didattici , molti dei quali a colori . i tre volumi presentano con grande completezza gli aspetti tecnici , metodologici e anatomici a livello di tutti gli organi e apparati . 
nel primo volume trovano ampio spazio la parte di fisica e tecnica , le apparecchiature , limmagine 836 radiol med ( 2009 ) 114 : 835836 archiving and communications systems ( pacs )  . 
 the second volume comprises most of the chapters dealing with the various organs and systems and in particular the digestive system , the liver , the biliary system , the spleen , the pancreas , the urinary system and retroperitoneum , the male and female reproductive systems , the breast and the nervous systethe volume ends with three chapters devoted to sonography in internal medicine and obstetrics and gynaecology and to doppler sonography . the third volume is the most extensive , as it deals mainly with heavy equipment , with ample space given to ct , mri and pet - ct . 
the final section is devoted to diagnostic and especially interventional angiography , with chapters also covering extravascular interventional radiology . although the book was written in collaboration with other well - known authors , it is nice to note that fernando mazzucatos work of coordinator clearly transpires through all of the chapters , such that it is easy to predict that this new edition will enjoy a success equal to that achieved by the earlier editions . radiografica analogica e digitale ; vengono inoltre presi in esame i sistemi informativi in radiologia ( ris ) e i sistemi per larchiviazione e la comunicazione delle immagini ( pacs )  . 
nel primo volume trovano ampia trattazione due capitoli fondamentali dedicati allo scheletro e articolazioni e allapparato cardiorespiratorio , con i quali deve iniziare la formazione dello specialista radiologo e del tecnico di radiologia . 
 il secondo volume contiene la maggior parte dei capitoli dedicati ai vari organi e apparati , e precisamente allapparato digerente , al fegato , alle vie biliari , alla milza , al pancreas , allapparato urinario e al retroperitoneo , allapparato genitale maschile e femminile , alla mammella e infine al sistema nervoso . 
il volume termina con tre capitoli dedicati allecografia internistica , ostetrico - ginecologica e al doppler . il terzo volume molto corposo in quanto dedicato principalmente alle macchine pesanti e pertanto trovano ampio spazio la tc , la rm e la pet . 
during a 13 - month period , 55 consecutive patients under evaluation for aortic ( 40 / 55 ) or mitral valve ( 15 / 55 ) disease before potential valve replacement underwent ctca using a 64 - detector - row scanner within 2 months of cca for comparative purposes . 
the diagnostic accuracy of 64 - row ctca for ruling out the presence of significant coronary stenoses in patients undergoing valve surgery is excellent and allows ctca to be used as a gatekeeper for invasive cca in these patients . 
mdct is a necessary preoperative examination that provides useful information for identifying potential operative complications of surgical procedures . keywords multidetector - row computed tomography conventional coronary angiography coronary artery disease valvular heart disease cardiac surgery riassunto obiettivo . 
in un periodo di 13 mesi , mediante tc a 64 detettori stata eseguita ac - tcms in 55 pazienti consecutivi affetti da patologia valvolare aortica ( 40 / 55 ) o mitralica ( 15 / 55 ) seguita da acc in un periodo minore di 2 mesi . 
laccuratezza diagnostica della ac - tcms 64 detettori per escludere la presenza di stenosi coronariche significative nei pazienti candidati allintervento valvolare eccellente , consente di utilizzarla come esame di prima istanza e di riservare la acc a pazienti selezionati . 
mdct un esame pre - operatorio che fornisce informazioni utili per identificare potenziali complicanze delle procedure chirurgiche . parole chiave tomografia computerizzato multistrato angiografia coronarica convenzionale malattia aterosclerotica coronarica malattia valvolare cardiaca chirurgia cardiaca radiol med ( 2009 ) 114 : 728742 introduction introduzione stenosis of the calcified aorta is the most common valvular heart disease in elderly patients , with a prevalence of 2%7% in the population > 65 years of age [ 1 ] and is associated with increased mortality [ 2 ]  . 
bicuspid aortic valve with fusion of the right coronary cusp and the noncoronary cusp is a common anomaly affecting 1%2% of the population and is the second most common cause of valvular disease requiring surgery [ 3 ]  . 
the surgical approach , in fact , can vary significantly according to whether the valve is bicuspid or tricuspid [ 4 ]  . valvular calcifications should be seen as a manifestation of multidistrict atherosclerotic disease , whether or not they cause valvular stenosis [ 5 ]  . 
in patients with concomitant valvular and coronary artery disease , the combined procedure of valve replacement and coronary artery bypass grafting ( cabg ) reduces perioperative and postoperative mortality and morbidity with respect to valve replacement surgery alone [ 6 ]  . 
recent guidelines recommend conventional coronary angiography ( cca ) before valvular surgery in men > 35 years of age , in premenopausal women with significant vascular risk factors and in postmenopausal women [ 7 ]  . 
as cca is a costly and invasive procedure with a mortality rate of 0.15% and morbidity rate of 1.5% [ 8 ] , an alternative , less invasive and less expensive procedure is desirable . 
thanks to its high spatial and temporal resolution , multidetectorrow computed tomography ( mdct ) with cardiac gating is able to visualise the coronary artery lumen and walls and enables accurate identification of the atherosclerotic plaque and quantification of induced stenosis [ 9 ]  . 
with multiplanar and 3d postprocessing of the data sets acquired with cardiac - gated mdct , evaluation can be made of the number of leaflets , presence of annular and valvular calcifications and valvular kinetics [ 10 ]  . 
in addition , the use of dedicated software makes possible the evaluation of ventricular function parameters , which is particularly useful in the clinical characterisation of patients referred for heart valve surgery . the aims of our study were therefore to evaluate the utility of mdct in the preoperative assessment of patients with aortic or mitral valve disease and to determine whether ct coronary angiography ( ctca ) can be used as a valid alternative to invasive cca in patients referred for heart valve surgery , so that the latter technique may be reserved for patients with significant cad . la stenosi aortica calcifica la pi comune patologia valvolare cardiaca nei pazienti anziani , con una prevalenza del 2%7% nella popolazione maggiore di 65 anni [ 1 ] ed associata ad aumentato rischio di mortalit [ 2 ]  . 
la valvola aortica di tipo bicuspide con fusione della cuspide coronarica destra e della cuspide non coronarica unanomalia comune con incidenza pari all1%2% della popolazione ed la seconda causa pi frequente di patologia valvolare che richiede intervento chirurgico [ 3 ]  . 
in fase preoperatoria , la valutazione della morfologia valvolare , il diametro dellanulus e la presenza di calcificazioni dellanulus e della valvola utile per pianificare lapproccio chirurgico . la strategia chirurgica cambia significativamente se la valvola bicuspide o tricuspide [ 4 ]  . 
nei pazienti in cui concomita malattia valvolare e coronarica , lintervento chirurgico combinato di sostituzione valvolare e bypass coronarico in unico tempo riduce la mortalit e morbilit perioperatoria e postoperatoria rispetto allesecuzione della sola sostituzione valvolare [ 6 ]  . 
le recenti linee guida raccomandano di eseguire coronarografia convenzionale ( acc ) in ogni paziente candidato allintervento valvolare se di et maggiore di 35 anni per gli uomini e nelle donne in fase post - menopausale o premenopausale se concomitano significativi fattori di rischio vascolare [ 7 ]  . 
dato che la acc una procedura invasiva che comporta un tasso di mortalit pari allo 0 , 15% e morbilit del 1 , 5% ed una metodica costosa [ 8 ] , auspicabile unindagine alternativa meno invasiva e meno costosa . 
la tc multistrato con sincronizzazione ecg essendo dotata di elevata risoluzione spaziale e temporale permette la visualizzazione del lume e della parete coronarica , inoltre consente laccurata identificazione delle placche ateromasiche e la quantificazione del grado di stenosi indotto [ 9 ]  . 
elaborando con tecnica multiplanare e 3d i dataset acquisiti con tc multidetettore con sincronizzazione ecg possibile valutare anche il numero dei lembi valvolari , lo spessore dei lembi , la presenza di calcificazioni della valvola e dellanulus e la cinetica valvolare [ 10 ]  . 
 pertanto il nostro studio ha lobiettivo di valutare lutilit della tc multistrato in fase preoperatoria nei 730 materials and methods patient population between january 2006 and january 2008 , we used ctca to examine 84 patients referred for aortic or mitral valve replacement surgery who were without contraindications to the use of nonionic iodinated contrast mediuno patients were excluded on the basis of heart rate , body mass index ( bmi ) or the presence of arrhythmias . 
the remaining 55 patients ( 29 men and 26 women , mean age 738 years ) , who underwent cca within 2 months of ctca , made up our study population . our institutional ethics committee approved the study protocol , and all patients provided informed consent . patient preparation all patients ( 35 / 55 , 64% ) with a heart rate > 65 beats per minute ( bpm ) , a left ventricular ejection fraction > 40% in the absence of arterial hypotension , asthma and obstructive respiratory disease received a 5 - mg intravenous dose of beta blockers ( propranolol , inderal )  . 
patients affected by bronchial asthma ( 4 / 55 , 7% ) with heart rate > 65 bpm received a 5 - mg intravenous dose of calcium - channel blocker ( verapamil , isoptin )  . 
in addition to the beta blockers and calcium - channel blockers , some patients ( 6 / 55 , 11% ) without contraindications received ten drops sublingually of benzodiazepine ( bromazepam , lexotan )  . 
in all patients , a preliminary unenhanced scan was performed to quantify the presence of calcium in the coronary arteries , the valves and the aorta . the parameters used for the scan , image reconstruction and data set creation are summarised in tables 13 . 
intendiamo valutare anche se la coronarografia mediante tomografia computerizzata ( ac - tcms ) pu essere una valida alternativa alla coronografia invasiva nei pazienti candidati alla sostituzione valvolare cardiaca in modo tale da riservare questultima indagine solo ai pazienti con malattia coronarica significativa . materiali e metodi popolazione esaminata da gennaio 2006 a gennaio 2008 abbiamo esaminato mediante ac - tcms 84 pazienti candidati allintervento di sostituzione valvolare aortica o mitralica , senza controindicazioni alluso del mezzo di contrasto iodato non ionico ( mdc )  . 
per il nostro studio , da questo gruppo di 84 pazienti abbiamo escluso un totale di 29 pazienti : 20 di questi perch non hanno eseguito successivamente lacc e 9 perch hanno eseguito lacc con un intervallo di tempo maggiore di 2 mesi dalla tc . 
i restanti 55 pazienti ( 29 maschi , 26 femmine ; et media 738 anni ) costituenti la nostra popolazione di studio hanno eseguito acc entro 2 mesi dalla ac - tcms . 
 il comitato etico della nostra istituzione ha approvato il protocollo di studio e tutti i pazienti hanno fornito il consenso informato . preparazione dei pazienti a tutti i pazienti ( 35 / 55 , 64% ) con frequenza maggiore di 65 battiti per minuto ( bpm ) , frazione di eiezione del ventricolo sinistro > 40% in assenza di ipotensione arteriosa , asma e malattia respiratoria ostruttiva , stata somministrata per via endovenosa una fiala di beta - bloccante da 5 mg ( propanololo , inderal )  . 
ai pazienti affetti da asma bronchiale ( 4 / 55 , 7% ) con frequenza maggiore a 65 bpm stato somministrato per via endovenosa ca - antagonista da 5 mg ( verapamil cloridrato , isoptin )  . 
oltre al beta - bloccante o ca - antagonista , in alcuni pazienti ( 6 / 55 , 11% ) privi di controindicazione ( ipersensibilit alle benzodiazepine , al principio attivo o ad uno qualsisi degli eccipienti ) , sono state somministrate 10 gocce per via sublinguale di benzodiazepina ( bromazepam , lexotan )  . 
in nessun caso sono stati somministrati nitroderivati . esame tc parametri di acquisizione tutti gli esami sono stati eseguiti mediante uno scanner a radiol med ( 2009 ) 114 : 728742 table 1 computed tomography coronary angiogram ( ctca ) scan protocol no . 
di strati spessore del detettore tempo rotazione pitch gantry volt ampere scansione senza mdc scansione con mdc 0 , 5 mm 0 , 5 mm 0 , 25 ms 0 , 25 ms variabile con i bpm variabile con i bpm 120 kv 120 kv 200 ma 500 ma table 2 image reconstruction protocol effective slice thickness reconstruction increment field of view convolution kernel unenhanced scan contrast - enhanced scan 3 mm 0.5 mm 3 mm 0.3 mm 270320 270320 calcium score coronary fc05 tabella 2 parametri di ricostruzione delle immagini spessore strato effettivo incremento di ricostruzione scansione senza mdc scansione con mdc 3 mm 0 , 5 mm 3 mm 0 , 3 mm campo di vista 270320 270320 filtro di convoluzione calcium score coronarie fc05 table 3 dataset reconstruction parameters technique using r - r interval percentage technique using absolute prospective delay technique using absolute reverse delay toshiba best phase technique ecg editing ecg , electrocardiograph tabella 3 parametri di ricostruzione dei dataset tecnica della percentuale dell ' intervallo r - r tecnica del ritardo prospettivo assoluto tecnica del ritardo assoluto inverso tecnica " best phase " toshiba ecg editing ecg , elettrocardiogramma parameters 70%75% 175 , 225 , 275 , 325 ms ( - ) 275 , ( - ) 300 , ( - ) 350 , ( - ) 400 ms automatic valori di ricostruzione 70%75% 175 , 225 , 275 , 325 ms ( - ) 275 , ( - ) 300 , ( - ) 350 , ( - ) 400 ms automatica total 100% ( 55 / 55 ) 100% ( 55 / 55 ) 100% ( 55 / 55 ) 27% ( 15 / 55 ) 38% ( 21 / 55 ) n . 
the angiographic scan was performed in 1114 s , corresponding to the length of a single breath hold . image analysis image analysis was done at a vitrea 2.0 workstation ( vital image , minnesota , mn , usa ) with axial , multiplanar ( mpr ) , curved planar ( cpr ) , 3d , maximum intensity projection ( mip ) and volume - rendered ( vr ) reconstructions . 
quantification of the aortic calcium burden by assigning a 5 - point score , corresponding to absence of aortic calcium ( 0 ) , mild ( 1 ) , moderate ( 2 ) , moderate to severe ( 3 ) and severe ( 4 ) , and location of the plaques with reference to the valvular plane 2 . 
il tempo totale medio di scansione stato di 16 secondi circa . protocollo di iniezione sono stati somministrati 90110 ml di mdc ( iomeron 400 , bracco , milano , italia ) alla velocit di 45 ml / s mediante iniettore automatico ( stellant , medrad , pittsburgh , usa ) collegato ad unagocanula da 1820 gauge precedentemente posizionata in una vena ante cubitale , seguiti da soluzione salina ( nacl 40 ml a 45 ml / s )  . 
analysis of coronary stenosis using a 17 - segment modified american heart association ( aha ) classification . each segment was classified as normal , with stenosis < 50% or with stenosis 50% . 
the segments distal to a chronic occlusion were excluded due to insufficient filling of contrast medium from collateral branches . coronary angiography examination all ccas were done within 360 days after ctca with a phillips integrix 3000 or ge innova 3100 . 
each segment was classified as normal , with stenosis < 50% or with stenosis 50% . stenoses were evaluated in two orthogonal views and classified as significant if mean luminal reduction exceeded 50% . in addition , measurements of aortic pressure , left ventricular pressure and transvalvular gradient were made for each examination , as well as an evaluation of valvular lanalisi delle immagini stata condotta mediante workstation vitrea 2.0 ( vital image , minnesota , usa ) con ricostruzioni assiali , multiplanari ( mpr ) , piani curvi ( cpr ) , 3d , massima intensit di proiezione ( mip ) , e volume rendering ( vr )  . 
i segmenti distali a unocclusione cronica sono stati esclusi per linsufficente riempimento di mdc determinato dai rami collaterali . radiol med ( 2009 ) 114 : 728742 regurgitation ( expressed with a score from 0 to 4 ) and of the number of valve cusps and their motility . 
lastly , aortography was performed in each patient . esame coronarografico statistical analysis the diagnostic accuracy of ctca was evaluated by comparing the findings with the reference standard cca and evaluating sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) and the relative 95% confidence intervals ( cis )  . 
we also calculated the positive likelihood ratio [ sensitivity / ( 1 - specificity ) ] and negative likelihood ratio [ ( 1 - sensitivity ) / specificity ]  . 
we performed a subanalysis that compared patients with aortic disease ( group a ) and patients with mitral valve disease ( group b ) and a subanalysis comparing patients with and without angina pectoris ( ap )  . results demographic characteristics of the patient population are summarised in table 4 . 
a large proportion of patients showed aortic stenosis ( 21 / 55 , 38% ) , no patient had isolated mitral stenosis and 14 patients ( 14 / 55 , 25% ) had mitral stenosis with regurgitation . 
patients with elevated heart rate were not excluded , nor were patients with arrhythmias at the time of the examination , and no selection was made on the basis of bmi . 
two examinations ( 2 / 55 , 3% ) , in patients with severe aortic stenosis , were marred by severe motion artefacts precluding ctca evaluation of the coronary arteries , so they were excluded from the analysis . 
in one patient affected by aortic stenosis and aneurysm of the ascending aorta ( 77 cm ) , the operator performing the cca failed to cannulate the ostium of the dominant left coronary artery despite numerous attempts , so on this vessel , ctca replaced cca as the reference technique . patient - based analysis ctca showed absence of significant disease in 31 patients . three cases were incorrectly evaluated as positive , thus producing a specificity of 91% . 
the operator correctly identified all 19 patients with at least one significant stenosis , tutte le acc sono state eseguite dopo ac - tcms , in un range di tempo compreso tra 3 e 60 giorni dallesecuzione della ac - tcms , con philips integrix 3000 o ge innova 3100 . 
un cardiologo con esperienza pluriennale angiografica , senza conoscere il risultato della ac - tcms , ha analizzato tutti i segmenti coronarici utilizzando la classificazione a 17 segmenti modificato dellamerican heart association ( aha )  . 
 inoltre per ogni esame stata misurata la pressione aortica , pressione ventricolare sinistra , gradiente transvalvolare , valutazione del rigurgito valvolare ( espresso con punteggio da 0 a 4 ) , valutazione del numero delle cuspidi valvolari e la loro motilit . 
infine stata eseguita laortografia in ogni paziente . analisi statistica laccuratezza diagnostica della ac - tcms stata valutata confrontando i risultati con la acc che rappresenta lo standard di riferimento e valutando la sensibilit , specificit , valore predittivo positivo ( vpp ) e negativo ( vpn ) con il relativo intervallo di confidenza al 95% . 
abbiamo eseguito una subanalisi selezionando i pazienti con patologia aortica ( gruppo a ) comparandola con patologia valvolare mitralica ( gruppo b ) e una subanalisi differenziando pazienti con e senza angina pectoris ( ap )  . risultati le caratteristiche demografiche della popolazione sono riportate in tabella 4 . 
la maggioranza dei pazienti esaminati presentava stenosi aortica ( 21 / 55 , 38% ) , nessun paziente presentava stenosi mitralica isolata e 14 pazienti ( 14 / 55 , 25% ) presentavano steno - insufficienza mitralica . non sono stati esclusi i pazienti che presentavano elevata frequenza cardiaca , n aritmie al momento dellesame , n sulla base del bmi . 
di questi 734 radiol med ( 2009 ) 114 : 728742 table 4 patient demographics tabella 4 caratteristiche della popolazione aortic stenosis , aortic regurgitation , aortic stenosis with regurgitation other valve disease male symptom angina pectoris no angina pectoris previous myocardial infarction risk factors hypertension high cholesterol level diabetes mellitus smoking family history of cad obesity ( bmi 30 kg / m2 ) coronary calcium score agatston cs volumetric cs valvular calcium score volumetric cs aortic calcium type of cardiac surgery aortic valve stenosis ( group 1 ) aortic valve regurgitation ( group 2 ) aortic valve stenosis with regurgitation ( group 3 ) mitral valve stenosis ( group 4 ) mitral valve regurgitation ( group 5 ) mitral valve stenosis with regurgitation ( group 6 ) cad absent cad not significant one vessel disease multivessel disease nyha class class i class ii class iii 749 ( 8943 ) 749 ( 8943 ) 7410 ( 4988 ) 29 ( 29 / 55 , 53% ) 16 39 25 23 11 2 , 434 0.98 21 ( 21 / 55 , 38% ) 9 ( 9 / 55 , 16% ) 10 ( 10 / 55 , 18% ) 14 ( 14 / 55 , 25% ) 1 ( 1 / 55 , 2% ) 38% ( 20 / 53 ) 26% ( 14 / 53 ) 9% ( 5 / 53 ) 26% ( 14 / 53 ) stenosi aortica , steno - insufficienza aortica , insufficienza aortica patologia di altre valvole maschi sintomi angina pectoris no angina pectoris precedente im fattori di rischio ipertensione ipercolesterolemia diabete mellito fumo familiarit di cad obesit ( body mass index 30 kg / m2 ) calcium score coronarico c.s. 
due esami ( 2 / 55 , 3% ) relativi a pazienti affetti da stenosi aortica severa presentavano artefatti da movimento del torace cos marcati da non consentire la valutazione ac - tcms dei vasi coronarici , pertanto sono stati esclusi dallanalisi . 
in un paziente affetto da stenosi aortica severa ed aneurisma dellaorta ascendente ( 77 cm ) , loperatore della coronarografia non riuscito ad incannulare lostio della coronaria sinistra dominante nonostante tentativi , pertanto nellanalisi di questo vaso la ac - tcms ha sostituito la acc come metodica di riferimento . i numerosi radiol med ( 2009 ) 114 : 728742 736 radiol med ( 2009 ) 114 : 728742 table 6 coronary artery and valvular calcium score results in patients with aortic or mitral valve disease patients with aortic valve disease patients with mitral valve disease coronary artery volumetric calcium score valvular volumetric calcium score ascending aorta calcium burden ( 04 ) 2 , 527 0.92 tabella 6 risultati del calcium score coronarico e valvolare nei pazienti con valvulopatia aortica e mitralica pazienti con patologia valvolare aortica pazienti con patologia valvolare mitralica 1 , 101 1101 c.s. 
ten patients with no aortic calcium plaques ( classified as 0 ) had aortic stenosis ( 10 / 21 , 48% ) , two had aortic regurgitation ( 2 / 9 , 22% ) , four had aortic stenosis with regurgitation ( 4 / 10 , 40% ) , eight had mitral regurgitation ( 8 / 14 , 57% ) and one has mitral stenosis with regurgitation ( 1 / 1 , 100% )  . 
the mean aortic calcium score of the patients with aortic stenosis ( n = 21 ) was 0.92 , whereas it was 0.98 for patients with aortic regurgitation ( n = 9 ) , 0.87 for patients with aortic stenosis with regurgitation ( n = 10 ) , 1.00 for patients with mitral regurgitation ( n = 14 ) and 0.00 for patients with mitral stenosis and regurgitation ( n = 1 )  . 
in the study population , ctca identified ten ( 10 / 55 , 18% ) patients with an ascending aorta > 40 mof these patients , six had aortic valve regurgitation , corresponding to 67% ( 6 / 9 ) of all patients with aortic valve regurgitation . 
the following anatomical variants were identified : four cases of common origin of the innominate artery and left common carotid artery , one case of origin of the left vertebral artery from the aortic arch , one case of arteria lusoria and two cases of separate origin of the left anterior descending and left circumflex arteries . 
in addition , the radiologist identified the presence of one saccular aneurysm ( 9 mm ) of the left anterior descending artery and left circumflex artery ( 6.8 mm ) , two patients with marked thickening of the medial mitral valve leaflet , one patient with thickening of the right aortic valve leaflet and one patient with left ventricular apical thrombus . vessel - based analysis analysis of the prevalence of cad showed that the right analisi per paziente la ac - tcms ha documentato assenza di malattia significativa in 31 pazienti , 3 casi sono stati erroneamente valutati come positivi , riportando un valore di specificit pari al 91% . 
i pazienti affetti da patologia valvolare aortica presentavano un valore di calcio valvolare medio calcolato secondo lindice volumetrico pari a 2527 ; per i pazienti affetti da patologia valvolare mitralica tale valore risultato pari a 1101 . 
dei pazienti con assenza di placche calcifiche aortiche ( classificate come 0 ) , 10 erano affetti da stenosi aortica ( 10 / 21 , 48% ) , 2 da insufficienza aortica ( 2 / 9 , 22% ) , 4 da stenoinsufficienza aortica ( 4 / 10 , 40% ) , 8 da insufficienza mitralica ( 8 / 14 , 57% ) 1 da steno - insufficienza mitralica ( 1 / 1 , 100% )  . 
i pazienti affetti da stenosi aortica ( n = 21 ) presentavano una media di calcio aortico pari a 0 , 92 ; i ( n = 9 ) pazienti con insufficienza aortica di 0 , 98 ; i pazienti ( n = 10 ) con steno - insufficienza aortica di 0 , 87 ; i pazienti ( n = 14 ) con insufficienza mitralica pari a 1 ; i pazienti ( n = 1 ) con steno - insufficienza mitralica 0 . 
nella popolazione in studio la actcms ha identificato 10 ( 10 / 55 , 18% ) pazienti con aorta ascendente maggiore di 40 mm ; di questi pazienti in 6 presentavano insufficienza aortica , che corrispondono al 67% ( 6 / 9 ) del totale dei pazienti affetti da insufficienza valvolare aortica . 
sono state individuate le seguenti variante anatomiche : 4 origine comune del tronco anonimo e della carotide comune di sinistra , 1 origine della vertebrale sinistra dallarco aortico , 1 arteria lusoria , 2 origini separate dellarteria discendente anteriore e della circonflessa . 
inoltre il radiologo ha documentato la presenza di 1 aneurisma sacciforme ( 9 mm ) dellarteria discendente anteriore e dellarteria circonflessa ( 6 , 8 mm ) , due pazienti con lembo valvolare mitralico marcato mediale , un paziente con ispessimento del lembo valvolare aortico destro e un paziente con trombo apicale del ventricolo sinistro . ispessimento del analisi per vaso dallanalisi della prevalenza di malattia dei vasi coronarici risultato che la coronaria destra il vaso con la pi elevata localizzazione di stenosi significative ( 28% )  . 
dal confronto tra ac - tcms e acc emerso che la ac - tcms raggiunge la pi alta sensibilit e specificit ( rispettivamente del 100% e 98% ) nella valutazione del tronco comune , con il riscontro di 0 falsi negativi e 1 falso positivo . la minore sensibilit e specificit ( rispettivamente del 82% e 93% ) stata raggiunta nella valutazione della circonflessa con 2 falsi negativi e 3 falsi positivi . 
1 la ricostruzione multiplanare curva ( a destra ) mostra la coronaria destra priva di lesioni , confermata dallangiografia convenzionale ( a sinistra )  . coronary artery was the vessel most commonly affected by significant stenosis ( 28% )  . 
comparison between ctca and cca revealed that ctca achieved the highest level of sensitivity and specificity ( 100% and 98% , respectively ) in the evaluation of the left main coronary artery , with no false negatives and one false positive . 
the lowest sensitivity and specificity ( 82% and 93% , respectively ) was reached in the evaluation of the left circumflex artery , with two false negatives and three false positives . 
the sensitivity and ppv of ctca were therefore 80% and 82% , respectively . subanalysis of patients with aortic valve disease versus mitral valve disease in group a patients ( aortic stenosis , aortic stenosis with regurgitation , aortic regurgitation ) , the prevalence of disease was 45% , whereas in group b ( mitral stenosis , mitral stenosis with regurgitation ) it was 13% . 
the agatston calcium score ( 731 ) and the volumetric calcium score subanalisi per pazienti con patologia valvolare aortica versus patologia mitralica nei pazienti del gruppo a ( stenosi , steno - insufficienza ed insufficienza aortica ) abbiamo riscontrato prevalenza di 738 radiol med ( 2009 ) 114 : 728742 fig . 
4 limmagine volume - rendered ac - tcms ( a sinistra ) e la ricostruzione multiplanare ( a destra ) dimostrano la perviet della coronaria sinistra confermata dalla acc ( al centro )  . ( 2577 ) in patients in group b were greater than in those in group a ( 693 and 2 , 434 , respectively )  . 
3 ricostruzione multiplanare curva e relativa sezione trasversa ( a destra ) dimostrano una stenosi > 50% allorigine del ramo interventricolare posteriore della coronaria destra ( a ) confermata dalla cag ( a sinistra ) ; b ramo postero - laterale indenne . malattia del 45% , mentre i pazienti del gruppo b ( stenosi , steno - insufficienza mitralica ) del 13% . 
il calcium score coronarico secondo agatston ( 731 ) e il calcium score valvolare volumetrico ( 2577 ) dei pazienti del gruppo b sono risultati maggiori dei pazienti del gruppo a ( rispettivamente : 693 e 2434 ) , ma in modo non significativo . 
dai risultati della diagnosi delle stenosi coronariche maggiori o uguali al 50% , emerso che nei pazienti del gruppo a la ac - tcms ha riportato sensibilit del 100% , specificit dell86% e valore predittivo positivo dell85% per la presenza di 3 falsi positivi , mentre nei pazienti del gruppo b del 100% , del 100% e del 100% rispettivamente . subanalisi dei pazienti con angina pectoris nella popolazione oggetto di studio i pazienti che riferivano angina pectoris ( ap ) sono stati 16 mentre in 39 hanno negato tale sintomo . 
escludendo i 2 pazienti con stenosi e steno - insufficienza aortica con marcati artefatti da respirazione che negavano di essere affetti da ap , i 36 pazienti rimanenti senza ap presentavano inferiore prevalenza di malattia ( 22% ) rispetto ai pazienti con ap ( 38% )  . 
laccuratezza diagnostica delle stenosi coronariche significative dei pazienti con ap stata valutata mediante sensibilit ( 100% ) , specificit ( 90% ) , valore predittivo positivo ( 86% ) e valore predittivo negativo ( 100% )  . radiol med ( 2009 ) 114 : 728742 86% and ppv of 85% , with the presence of three false positives , whereas the rates in group b were 100% , 100% and 100% , respectively . discussione subanalysis of patients with angina pectoris of the 55 patients in the study population , 16 reported ap and 39 denied having symptoms . 
with the exclusion of the two patients with stenosis and stenosis with regurgitation whose examinations were compromised by thoracic wall motion artefacts , the prevalence of disease in the remaining 36 patients without ap ( 22% ) was lower than in the group of patients with ap ( 38% )  . 
the diagnostic accuracy of significant coronary stenoses of patients with ap was evaluated with sensitivity ( 100% ) , specificity ( 90% ) , ppv ( 86% ) and npv ( 100% )  . discussion it has been demonstrated that there is a significant independent correlation between the progression of calcifications in coronary arteries and in the aortic valve , thus suggesting similar mechanisms [ 11 ]  . 
in our study , it was also seen that groups of patients with a volumetric coronary artery calcium score > 1 , 000 had similar percentages of risk factors to the group of patients with volumetric aortic valve calcium score > 1 , 000 , with a greater prevalence of hypertension and hypercholesterolaemia . 
we found no significant differences in the aortic calcium burden between the groups of patients affected by aortic and mitral valve disease . with regard to the clinical characterisation of patients with aortic valve disease , the following parameters are traditionally evaluated : measurement of aortic velocity , mean transaortic pressure gradient and continuity equation valve area [ 12 ]  . 
some authors , however , contend that percutaneous coronary intervention in patients with symptoms of angina or dyspnoea with aortic valvular stenosis and significant cad is a safe procedure able to improve symptoms in most cases and postpone heart valve surgery [ 14 ]  . there is therefore an indication for performing cca prior to valve replacement surgery in patients with severe aortic valve stenosis and angina pectoris to decide , in the case of significant cad , whether or not to perform cabg together stato dimostrato che esiste una correlazione indipendente significativa tra la progressione delle calcificazioni nelle arterie coronarie e nella valvola aortica , suggerendo simili meccanismi patogenetici [ 11 ]  . 
anche nel nostro studio , il gruppo di pazienti con calcium score coronarico volumetrico maggiore di 1000 , presentavano percentuali di fattori di rischio simili al gruppo di pazienti con calcium score volumetrico valvolare aortico maggiore di 1000 , con maggiore prevalenza di ipertensione e ipercolesterolemia . non abbiano riscontrato differenze significative del carico di calcio aortico tra i gruppi di pazienti affetti da patologia valvolare aortica e mitralica . 
 per quanto riguarda linquadramento clinico del paziente affetto da patologia valvolare aortica , tradizionalmente si valutano i seguenti parametri : velocit del sangue attraverso la stenosi valvolare , gradiente pressorio medio tra ventricolo sinistro e aorta e larea di apertura valvolare [ 12 ]  . 
tuttavia stato dimostrato che la sintomatologia del paziente il miglior indicatore della significativit emodinamica della stenosi valvolare , pertanto unaccurata anamnesi essenziale perch influenza in modo significativo la pianificazione terapeutica [ 13 ]  . 
invece secondo altri autori lintervento coronarico percutaneo ( icp ) condotto su pazienti sintomatici per angina o dispnea con stenosi valvolare aortica e mac significativa , una procedura sicura che provoca miglioramento dei sintomi nella maggior parte dei casi e permette di posporre lintervento di sostituzione valvolare [ 14 ]  . 
quindi esiste indicazione nei pazienti con stenosi valvolare aortica severa e angina pectoris , prima della sostituzione valvolare ad effettuare acc per decidere , in caso di mac significativa , se effettuare rivascolarizzazione con by - pass nello stesso tempo chirurgico dellintervento valvolare . 
nel caso di un paziente con stenosi valvolare aortica moderata e angina pectoris , se presente mac significativa , consigliabile eseguire in un primo tempo la rivascolarizzazione percutanea e quindi verificare la risoluzione del sintomo anginoso ; se tale sintomo persiste la causa potrebbe essere valvolare , pertanto opportuno procedere con la sostituzione valvolare [ 12 ]  . 
con il nostro studio abbiamo dimostrato che la ac - tcms possiede unelevata sensibilit , specificit e valore predittivo negativo ( 100% , 91% , 100% ) nella valutazione della mac rispetto alla acc nei pazienti candidati alla sostituzione valvolare . 
in the case of a patient with moderate aortic stenosis and angina pectoris , if significant cad is present , it is advisable to perform percutaneous revascularisation and then verify resolution of the angina . 
our study shows that ctca has high sensitivity , specificity and npv ( 100% , 91% , 100% , respectively ) in the evaluation of cad when compared with cca in patients referred for heart valve surgery . 
in particular , the elevated negative predictive value suggests ctca may be used in the preoperative phase to identify patients with no cad , thus ensuring that in this large group of patients ( 31 / 55 , 58% ) , no further costly and invasive examinations are performed . 
a comparison of 64slice ct in the analysis of cad with 16 - slice [ 13 , 16 ] and 40 - slice [ 17 ] scanners reveals that the diagnostic performance in our study is greater than that obtained with scanners with fewer detectors and comparable with that obtained with 64 - slice scanners on a patient population without mitral valve disease [ 18 ] , a condition known to be associated with a higher rate of ventricular arrhythmias [ 19 ] that worsen the diagnostic quality of the examination [ 20 ]  . current guidelines recommend valve replacement surgery in the presence of mitral regurgitation with significant symptoms or initial left ventricular dysfunction with ventricular dimension reaching 45 mm in the end - diastolic phase . 
surgery may also be indicated , even in the absence of symptoms or signs of left ventricular dysfunction if atrial fibrillation appears or pulmonary pressure exceeds 50 mmhg [ 7 ]  . 
in our experience , all images regarding patients with atrial fibrillation were considered of sufficiently diagnostic quality such that atrial fibrillation does not appear to be an exclusion criterion for patients referred for ctca . 
in these patients , high - quality images of the coronary tree can be obtained by positioning the time window in the end - systolic phase [ 22 ]  . 
this is because although the width of the r - r interval varies in each cardiac cycle in patients with atrial fibrillation , the difference of the width between end - diastolic intervals is greater than endsystolic intervals and between the rapid filling intervals in diastole , because their individual duration is shorter [ 22 ]  . 
in addition , images reconstructed with absolute delay tend to be of higher quality than images reconstructed with relative delay [ 22 ]  . lelevato valore predittivo negativo ci permette di proporre luso della ac - tcms in fase preoperatoria per individuare i pazienti senza mac evitando che siano condotte su questo numeroso gruppo ( 31 / 53 , 58% ) indagini pi invasive e pi costose . 
confrontando i risultati della tc 64 - strati nellanalisi della mac con studi condotti su scanner a 16 [ 3 , 16 ] e 40 - strati [ 17 ] , la performance diagnostica nel nostro studio non solo risulta migliore a quella ottenuta da scanner con minor numero di detettori ma addirittura comparabile a quella condotta con scanner a 64 - strati su una popolazione di pazienti senza patologia mitralica [ 18 ] , che presenta notoriamente maggiore incidenza di aritmie ventricolari [ 19 ] che peggiorano la qualit diagnostica dellesame [ 20 ]  . le attuali linee guida raccomandano lintervento di sostituzione valvolare mitralica per insufficienza mitralica in caso di sintomi importanti o allesordio di disfunzione ventricolare sinistra tipo dilatazione ventricolare con dimensione ventricolare di 45 mm in fase tele - diastolica . lintervento chirurgico pu inoltre essere indicato anche in assenza di sintomi o segni di disfunzione ventricolare sinistra se compare fibrillazione atriale o se la pressione polmonare supera i 50 mmhg [ 7 ]  . 
nella nostra esperienza tutte le immagini relative a pazienti con fibrillazione atriale sono state considerate di sufficente qualit diagnostica pertanto abbiamo riscontrato che la fibrillazione atriale non costituisce un fattore di esclusione della popolazione da sottoporre a ac - tcms . 
la spiegazione di questo risultato che sebbene lampiezza dellintervallo r - r varia in ogni ciclo cardiaco nei pazienti con fibrillazione atriale , la differenza di ampiezza tra gli intervalli tele - diastolici maggiore di quella tele - sistolici e tra gli intervalli di riempimento rapido nella diastole perch minore la loro singola durata [ 22 ]  . 
la presenza di severa aterosclerosi aortica responsabile dellaumento del rischio di morte radiol med ( 2009 ) 114 : 728742 cerebral ischaemic events constitute a highly relevant complication for the approximately 1 million patients who undergo heart surgery each year throughout the world [ 23 ]  . the presence of severe aortic atherosclerosis is responsible for the increased risk of intraoperative mortality due to infarction , hypoxic encephalopathy , stroke or transient ischaemic attack [ 23 ]  . 
mdct is able to identify the lesions that increase surgical risk : aortic atherosclerotic plaque , intracardiac thrombus and nodules on the valve leaflets . identification of these lesions requires modification of the surgical approach and the use of alternative techniques to minimise the risk of embolism [ 23 ]  . 
moreover , in our study , mdct proved decisive in the preoperative phase for the choice of the most appropriate surgical approach in the two patients with bicuspid aortic valve and aneurysm of the ascending aorta , in the ten patients with ascending aorta > 40 mm and in the patient with aneurysm ( 77 cm ) of the ascending aorta in whom cca was incomplete due to the failure to cannulate the left coronary artery . limitations and prospects in order to perform the evaluation of the aortic arch and coronary arteries in a single angiographic scan , we used a field of view that was larger along the z axis , such that the effective dose delivered to the patient was greater than in similar studies where the calcium burden of the aortic arch was not evaluated [ 24 ]  . our study was not done with a double - blind analysis of the ctca and cca images but by a single , highly experienced operator . 
as a result , we are unable to comment on the effective reproducibility of the excellent results achieved . a continuation of our study with a greater number of patients will allow us to verify the existence of a correlation between valvular calcium score , valvular diameter and aortic velocity measured with magnetic resonance imaging and pressure gradient measured with catheterisation and compare our findings with the data reported in the literature [ 10 ]  . 
we believe it would also be useful to investigate the existence of a correlation between aortic calcium score and intraand postoperative neurological events . conclusions ctca was demonstrated to have high sensitivity , specificity and npv in the evaluation of cad in patients referred for heart valve surgery , regardless of bmi , heart rate and arrhythmias , which tend to be particularly frequent in patients with mitral valve disease . 
inoltre la tc stata decisiva in fase preoperatoria per scegliere lapproccio chirurgico migliore nei due pazienti con valvola aortica bicuspide ed aneurisma dellaorta ascendente , nei 10 pazienti con aorta ascendente maggiore di 40 mm e nel paziente con aneurisma ( 77 cm ) dellaorta ascendente in cui la acc stata incompleta per il fallimento di incanulazione della coronaria sinistra . limiti e prospettive per eseguire in ununica scansione angiografica la valutazione dellarco aortico e delle coronarie abbiamo utilizzato un fov di dimensioni maggiori lungo lasse z , pertanto la dose effetiva del paziente risultata superiore rispetto ad altri studi analoghi in cui non si valutava il carico ateromasico dellarco aortico [ 24 ]  . 
 il nostro studio non stato condotto con analisi delle immagini della ac - tcms e della acc in doppio cieco , ma in singolo operatore con esperienza specifica pluriennale , pertanto non conosciamo riproducibilit delleccellente risultato riportato . 
 leffettiva la nostra proseguendo indagine con un numero maggiore di pazienti abbiamo intenzione di verificare lesistenza di una correlazione tra calcium score valvolare , diametro della valvola e velocit del flusso transvalvolare mediante misurazione con rm e gradiente pressorio misurato tramite cateterismo e intendiamo confrontare tale risultato con i dati rilevati in letteratura [ 10 ]  . 
riteniamo utile lesistenza di una correlazione tra calcium score aortico ed eventi neurologici intrae post - operatori . conclusioni nei pazienti candidati alla sostituzione valvolare cardiaca , senza esclusioni sulla base del valore di bmi , della frequenza cardiaca , della presenza di aritmie peraltro frequenti particolarmente nei pazienti con patologia mitralica , la ac - tcms ha dimostrato elevata sensibilit , specificit e valore predittivo negativo nella valutazione della mac . 
in particolare , lelevato valore predittivo negativo della ac - tcms consente di utilizzare tale metodica in fase preoperatoria per individuare i pazienti senza mac 742 radiol med ( 2009 ) 114 : 728742 negative predictive value of ctca makes it possible to use the technique in the preoperative phase to identify patients without cad and avoid the need for invasive and costly cca in these patients . the elevated accuracy of ctca in the evaluation of aortic atherosclerosis , an important surgical risk factor , and in the identification of aortic aneurysms is a further point in favour of the introduction of the technique in the diagnostic protocol of patients referred for heart valve surgery . evitando che siano condotte su questo gruppo indagini pi invasive e pi costose come la acc . 
 lelevata accuratezza diagnostica della tcms nella valutazione dellaterosclerosi aortica che costituisce importante fattore di rischio operatorio e nellidentificazione della patologia aneurismatica aortica rappresenta ulteriore elemento favorente lintroduzione di tale metodica nelliter diagnostico dei pazienti candidati alla sostituzione valvolare cardiaca . 
cademartiri1 , 4 1dipartimento di radiologia e cardiologia , azienda ospedaliero - universitaria di parma , parma , italy 2fondazione - irccs - sdn , napoli , italy 3dipartimento di radiologia , universit degli studi di napoli federico ii , napoli , italy 4dipartimento di radiologia e cardiologia , erasmus medical center , rotterdam , paesi bassi correspondence to : f . 
cademartiri , dipartimento di radiologia , c / o piastra tecnica piano 0 , azienda ospedaliero - universitaria , via gramsci 14 , 43100 parma , italy , tel . : + 39 - 052 - 1703516 , fax : + 39 - 052 - 1703630 , e - mail : filippocademartiri@hotmail.com received : 11 september 2008 / accepted : 18 november 2008 / published online : 23 june 2009 springer - verlag 2009 abstract purpose . 
results were compared with students t test for paired samples , pearsons r correlation coefficient and r2 coefficient of determination ; ejection fraction differences were assessed with bland - altman analysis . 
le scansioni 2d cine b - ssfp sono state utilizzate per la valutazione funzionale del ventricolo sinistro e i data - set ottenuti sono stati valutati utilizzando due software dedicati : viewforum versione 4.2 e argus versione va60c . 
nella nostra esperienza i software radiol med ( 2009 ) 114 : 718727 keywords cardiac magnetic resonance left ventricular volumes quantitative software ejection fraction . comunemente utilizzati forniscono risultati sovrapponibili in un tempo comparabile . parole chiave risonanza magnetica cardiaca volumi ventricolo sinistro software quantitativo frazione di eiezione introduction introduzione cardiac performance is key to determining disease stage and evaluating the effectiveness of treatment over time [ 1 ]  . an accurate , reproducible and quantitative evaluation of left ventricular ( lv ) function is possible , and the examination that guarantees the best results in terms of accuracy and reproducibility is magnetic resonance ( mr ) imaging [ 2 ]  . 
in particular , using two - dimensional ( 2d ) cine gradient - echo balanced steady - state free precession ( ge b - ssfp ) sequences , high - quality images are obtained that have excellent spatial , temporal and contrast resolution [ 3 , 4 ]  . these 2d gradient - echo balanced sequences are the standard of reference in the evaluation of ventricular volumes [ 5 , 6 ]  . 
the aim of our study was to compare the lv volume values obtained with two semiautomated dedicated software packages . materials and methods patients from march to august 2007 , all patients undergoing cardiac mr imaging were prospectively enrolled in the study ( 46 patients )  . 
exclusion criteria were absolute or relative contraindications to mr imaging [ 7 ] , and inclusion criteria were breath - hold capacity of at least 10 s for at least 5 consecutive times . mr imaging all images were acquired with a 1.5 - t scanner ( achieva , philips medical systems , best , the netherlands ) characterised by a maximum achievable gradient strength of 66 mt / m , and a maximum slew rate of 180 mt / m / ms . during the cine b - ssfp sequences , the maximum gradient strength was 33 mt / m and the maximum slew rate was 180 mt / m / ms . 
the 2d cine b - ssfp sequence was performed in the la performance cardiaca rappresenta attualmente un elemento cardine nel determinare lo stadio della malattia e nel valutare nel tempo lefficacia di uneventuale terapia [ 1 ]  . 
la valutazione della funzione ventricolare sinistra pu essere fatta in maniera accurata , riproducibile ed in maniera quantitativa ; lesame che garantisce migliori risultati di accuratezza e riproducibilit la risonanza magnetica ( rm ) [ 2 ]  . 
in particolare utilizzando sequenze 2d cine gradient echo balanced steady - state free precession ( ge bssfp ) si ottengono immagini di ottima qualit grazie alleccellente risoluzione spaziale , temporale e di contrasto [ 3 , 4 ]  . 
scopo del nostro lavoro stato confrontare i valori dei volumi cardiaci del ventricolo sinistro ottenuti da due differenti software semiautomatici dedicati . materiali e metodi pazienti dal marzo ad agosto 2007 , tutti i pazienti che sono stati sottoposti a rm cardiaca sono stati arruolati prospetticamente nello studio ( 46 pazienti )  . 
criteri di inclusione : capacit di mantenere unapnea di almeno 10 secondi per almeno 5 volte consecutive . imaging rm tutte le immagini sono state acquisite con uno scanner da 1 , 5 t ( achieva , philips medical systems , best , olanda ) caratterizzato da una massima forza di gradiente sviluppabile 66 mt / m e massimo slew / rate sviluppabile 180 mt / m / ms . 
durante le sequenze cine b - ssfp la massima forza di gradiente raggiungibile stata 33 mt / m e il massimo slew / rate pari a 180 mt / m / ms . 
sono stati utilizzati 720 table 1 study population patient characteristics ( n = 46 ) male / female ( no . ) mean age ( years ) mean weight ( kg ) mean heart rate ( bpm ) value range shown in parentheses caratteristiche dei pazienti ( n = 46 ) uomini / donne ( n ) et media ( anni ) peso medio ( kg ) frequenza cardiaca media ( bpm ) valore range mostrato tra parentesi 29 / 17 51 ( range 1683 ) 74.7 ( range 46113 ) 65.7 ( range 44102 ) 29 / 17 51 ( range 1683 ) 74 , 7 ( range 46113 ) 65 , 7 ( range 44102 ) tabella 1 caratteristiche generali della popolazione esaminata short axis , with coverage of the entire lv . 
evaluations were made blindly by an experienced operator who determined , for each patient , the ejection fraction ( ef ) , end - diastolic volume ( edv ) , end - systolic volume ( esv ) , stroke volume ( sv ) and cardiac output ( co ) with the two software packages , according to the criteria published in the literature [ 8 ]  . 
once the end - diastolic and end - systolic phases had been identified , the endocardial and epicardial contours were traced on the end - diastolic views . the endocardial contours were then propagated to all of the other cardiac phases , and the errors made by the software during propagation were corrected . 
the papillary muscles and the subendocardial trabeculae were included in the lv cavity [ 9 , 10 ]  . the time required by the operator to make the evaluations on the two consoles was also recorded . 
interobserver variability was assessed by having a second operator reevaluate all 46 patients with the argus software . radiol med ( 2009 ) 114 : 718727 una bobina a 5 elementi ed un elettrocardiogramma vettoriale per la ricezione del segnale e per la sincronizzazione cardiaca . 
altri parametri relativi alla sequenza 2d utilizzata : tr 3 , 1 , te 1 , 53 ; flip angle 60 ; banda 1249 , 7 hz / pixel ; risoluzione in plane 22 , 3 mm ; spessore di strato 8 mm ; intervallo tra strati 2 mm ; risoluzione temporale 31 , 525 , 93 ms ( dipendente dalla frequenza cardiaca ) ; fasi cardiache 30 ; sense off ; half scan on . 
le valutazioni sono state effettuate in cieco da un operatore esperto che ha effettuato le misurazioni volumetriche della frazione di eiezione ( fe ) , del volume tele - diastolico ( vtd ) , del volume tele - sistolico ( vts ) , della gittata sistolica ( gs ) e della gittata cardiaca ( gc ) utilizzando i due software per ciascun paziente seguendo i criteri pubblicati in letteratura [ 8 ]  . dopo aver identificato le fasi di telediastole e telesistole sono stati tracciati i contorni endocardici ed epicardici sulle immagini in telediastole ; i contorni endocardici sono stati poi propagati a tutte le altre fasi acquisite e si poi proceduto a correggere gli errori commessi dal software durante la propagazione . 
per una stima della variabilit intra - osservatore stata effettuata per due volte la valutazione di 20 pazienti utilizzando il software viewforuper una stima della variabilit interosservatore tutti i 46 pazienti sono stati valutati una seconda volta sul software argus da un secondo operatore . analisi statistica tutti i dati sono stati raggruppati e sono stati analizzati mediante test t di student per dati appaiati , test di correlazione ( r ) , di determinazione r2 e test di bland - altman . risultati la valutazione della volumetria del ventricolo sinistro effettuata con i due software non ha mostrato differenze radiol med ( 2009 ) 114 : 718727 fig . 
in a la finestra nella quale viene effettuata la segmentazione dei contorni endocardici ed epicardici . in b la finestra che mostra i risultati dellanalisi volumetrica . statistical analysis all data were collected and analysed with the student t test for paired data , the correlation coefficient ( r ) , the determination coefficient ( r2 ) and the bland - altman test . results evaluation of lv volume with the two software packages showed no significant differences ( table 2 )  . 
il tempo impiegato dalloperatore per calcolare i suddetti parametri utilizzando il software viewforum rispetto al software argus stato rispettivamente di 7 , 62 , 78 min vs 7 , 522 , 4 min ( p > 0 , 50 ; r = 0 , 85 ; r2 = 0 , 73 )  . 
the graph shows minimal dispersion of the data calculated with the two packages compared with the straight regression line for all ejection fraction values ( r2 = 0.82 ) : software performance does not decline in the six patients with depressed ef ( < 30% )  . 
il grafico dimostra una minima dispersione dei dati raccolti utilizzando i due software rispetto alla retta di regressione per tutti i valori di frazione di eiezione ( r2 = 0 , 82 ) : nei 6 pazienti con frazione deiezione depressa ( < 30% ) non si osserva un calo della performance dei due software . 
analisi di bland - altman della frazione di eiezione risultante dal calcolo dei volumi del ventricolo sinistro su workstation viewforum e argus ; sono stati utilizzati dei limiti di concordanza di 1 , 96 ds . 
to obtain more accurate and reproducible results in a la risonanza magnetica rappresenta attualmente lesame di riferimento per la valutazione volumetrica del ventricolo sinistro e lesame standard deve sempre comprendere acquisizioni dinamiche per lo studio di volumi e contrattilit . 
limplementazione degli scanner con nuovi software dedicati caratterizzati da interfacce pi intuitive e algoritmi pi sofisticati ugualmente importante rispetto allottimizzazione e / o introduzione di nuove sequenze per ottenere risultati pi accurati e riproducibili in minor tempo . lintroduzione di nuovi software dovrebbe essere preceduta da test di validazione rispetto a software conosciuti in popolazioni eterogenee di pazienti . 
i parametri volumetrici studiati dal secondo operatore non hanno mostrato differenze significative ( p > 0 , 05 ) vts ( ml ) gs ( ml ) fe ( % ) gc ( l / min ) fe , frazione di eiezione ; vtd , volume tele - diastolico ; vts , volume tele - sistolico ; gs , gittata sistolica ; gc , gittata cardiaca ; ds , deviazione standard ; min , valore minimo ; max , valore massimo fig . 
software packages may differ in several aspects : incorrect programming ( now extremely rare ) , use of different algorithms to identify enddiastolic and end - systolic phases and algorithms used for identifying the endo / epicardial contours during the propagation phase . 
error in the search for contours during the propagation phase makes the analysis time consuming , as a large number of images need to be corrected ( around 180240 images )  . in our study , we compared the performance of two common cardiac software programmes for the evaluation of ventricular function with 2d cine b - ssfp sequences . 
intraobserver and interobserver reproducibility , as measured with the viewforum and argus software , respectively , proved to be excellent in relation to the operators experience and to the intuitive display . limitations although our study sample was small , it comprised a similar number of patients to the main published series on the topic . we did not consider all of the software packages available on the market . 
nevertheless , the two packages investigated are most common and well - established platforms for the evaluation of lv volume in cardiac mr imaging . di propagazione ha conseguenze negative sul timeconsuming dellanalisi in quanto le correzioni devono essere fatte su un numero di immagini non trascurabile ( circa 180240 immagini )  . nel nostro studio stata confrontata la performance di due comuni software cardiologici dedicati alla valutazione della funzione ventricolare mediante sequenze 2d cine bssfp . 
la riproducibilit intra - osservatore dei risultati , valutata con software viewforum , e la variabilit inter - osservatore , valutata con software argus , si sono dimostrate ottime in relazione allesperienza delloperatore e alla semplicit dellinterfaccia di visualizzazione . limitazioni il numero di pazienti analizzato non stato elevato , tuttavia , le principali casistiche focalizzate su design similare fanno riferimento a numeri analoghi . 
tuttavia , si tratta di due piattaforme tra le pi diffuse e consolidate per la valutazione volumetrica del ventricolo sinistro in cardio - rm . conclusioni conclusions the software packages for the quantitative evaluation of lv volumes used in our study showed an excellent level of concordance , and all measurements were obtained within a reasonable time , compatible with average reporting times in routine clinical practic i software per valutazione volumetrica quantitativa del ventricolo sinistro utilizzati nel nostro studio hanno mostrato ottima concordanza nella valutazione dei volumi ventricolari e i risultati sono stati ottenuti in tempi ragionevoli , compatibili con un tempo medio di refertazione nel contesto clinico routinario . 
torricelli1 1dipartimento integrato dei servizi diagnostici e per immagine , cattedra e servizio di radiologia 1 , 2dipartimento integrato di emergenza - urgenza , 3dipartimento di statistica , universit degli studi di modena e reggio emilia , azienda ospedaliero universitaria policlinico , via del pozzo 71 , 41100 modena , italy correspondence to : f . 
diastolic wall thickness ( dwt ) , dobutamine - induced systolic wall thickening ( swt ) and de transmural extension were quantitatively assessed in vessel - related segments , calculating the contribution of viable tissue to swt , expressed as viability index ( vi ) = [ swt ( 100 de ) ] / 100 . 
ventitr pazienti con cto sono stati sottoposti rm con contrasto e dobutamina a bassa dose . sono stati valutati quantitativamente spessore telediastolico ( dwt ) , telesistolico ( swt ) e enhancement tardivo ( de ) nei segmenti relativi al vaso occluso , calcolando il contributo del tessuto vitale a swt , espresso come viability index ( vi ) = [ swt ( 100 - de ) ] / 100 . 
 parole chiave occlusione coronarica cronica percutaneous coronary intervention rm con dobutamina a bassa dose introduction introduzione chronic total coronary occlusions ( cto ) are observed in 35%50% of patients with significant coronary disease undergoing diagnostic angiography [ 1 , 2 ]  . 
although there have been numerous advances in the field of percutaneous coronary intervention , cto treatment remains a major challenge and is a frequent reason for referrals to cardiac surgery . 
furthermore , a large proportion of patients with cto is managed medically , the prognosis of which may vary depending on the extent of viable myocardium and ischaemia [ 4 ]  . 
the different approaches to cto are due to the fact that improvement after revascularisation is not easily predictable [ 5 ]  . in patients with cto , the vessel - related myocardium may show contractile impairment and yet be partly healthy and partly necrotic . 
the presence of severe chronic contractile dysfunction is no longer considered a contraindication to revascularisation : it has been demonstrated that patients with hibernating myocardium have a better outcome if treated invasively rather than with medical therapy [ 9 , 10 ]  . 
furthermore , retrospective observational reports [ 11 ] have documented the clinical impact of successful percutaneous cto revascularisation on long - term survival , and other studies have demonstrated statistically significant improvements in left ventricular function and regional wall motion with successful cto recanalisation [ 1219 ]  . low - dose dobutamine ( ld ) and delayed - enhancement ( de ) magnetic resonance imaging ( mri ) can evaluate contractile reserve and viability in myocardium subtended by cto in order to identify patients with potential viable myocardium that may benefit from revascularisation . 
diversi miglioramenti sono avvenuti negli ultimi anni nel campo della cardiologia interventistica , tuttavia il trattamento delle cto rimane una delle sfide maggiori per il cardiologo interventista , essendo campo di lavoro specifico per il cardiochirurgo . 
i differenti approcci alla terapia delle cto sono dovuti alla difficile valutazione del miglioramento post - rivascolarizzazione [ 5 ]  . nei pazienti con cto , il miocardio vascolarizzato dal vaso cronicamente occluso pu mostrare una alterazione contrattile ed essere contemporaneamente in parte vitale ed in parte necrotico . 
la presenza di alterazione contrattile severa e cronica non pi considerata una controindicazione alla rivascolarizzazione : stato infatti dimostrato che pazienti che presentano miocardio ibernato hanno un outcome migliore se trattati invasivamente rispetto a pazienti trattati con la sola terapia medica [ 9 , 10 ]  . 
moreover , change in left ventricular ejection fraction ( lvef ) was analysed . materials and methods study population between december 2006 and september 2007 , we enrolled 23 patients ( 21 men ; 91% ) ; mean age 65.710.6 years , with single chronic coronary occlusion . 
cto was defined as a complete occlusion of a major coronary artery for at least 3 months as obtained from previous angiogram or a clinical history of prolonged anginal chest pain or myocardial infarction . 
twochamber , four - chamber and multiple short - axis views were acquired using the fast gradient echo technique ( 8 - mm thick ; 0.8 - mm gap in multislice acquisition )  . 
acquisition parameters were te = 4.5 ms , tr = 2.2 ms , flip angle = 55 , acquired voxel = 2.034.148.0 , reconstructed voxel = 1.521.538.0 , acquisition matrix = 192256 . 
short - axis breath - hold valutare la riserva contrattile e la vitalit del miocardio sotteso alla cto , permettendo di identificare pazienti selezionati con miocardio potenzialmente vitale che potrebbero beneficiare dalla rivascolarizzazione . 
la concordanza tra rm con mezzo di contrasto e con dobutamina a bassa dose alta nei casi estremi ( infarti transmurali o subendocardici ) ma non nei segmenti con estensione intermedia del danno miocardico . 
in questo gruppo di pazienti pertanto lintegrazione della valutazione dellestensione e della transmuralit del tessuto cicatriziale esito di infarto con la valutazione quantitativa della riserva contrattile permetterebbe una identificazione ottimale dei segmenti con probabilit maggiore di ripresa funzionale post - rivascolarizzazione [ 20 ]  . lo scopo del nostro studio stato di valutare quantitativamente il valore della rm con dobutamina a bassa dose e con studio del de per predire la ripresa funzionale nei pazienti con cto da sottoporre a rivascolarizzazione percutanea con impianto di stent medicati . 
la perviet del vaso trattato e la ripresa funzionale sono stati verificati a 6 mesi mediante angiografia coronarica e rm a riposo . inoltre la modificazione della frazione di eiezione ( fe ) ventricolare sinistra stata analizzata . materiali e metodi popolazione di studio sono stati arruolati 23 pazienti ( 21 maschi ; 91% ) tra dicembre 2006 e settembre 2007 ( et media 65 , 710 , 6 anni ) con occlusione coronarica cronica di un singolo vaso . la cto stata definita come occlusione completa di un vaso coronarico maggiore per almeno tre mesi sulla base di valutazione angiografica o storia clinica di prolungato dolore toracico o infarto miocardico . 
le caratteristiche della popolazione di pazienti sono descritte nella tabella 1 . protocollo di studio rm a riposo lesame rm stato eseguito utilizzando un magnete superconduttivo a 3 tesla ( intera cv , philips medical system , best , olanda ) , con intensit di picco di 33 mt / m , tempo di picco di 0 , 2 ms e ripidit di 150 mt / m / s . 
sono state acquisite sezioni cardiache su pi piani ( piano due camere , piano quattro camere e multipli assi corti ) utilizzando sequenze veloci ad eco di gradiente ( 8 mm spessore ; 0 , 8 mm intervallo nelle acquisizioni multislice )  . 
lacquisizione in asse corto ha utilizzato un fattore sense pari a 1 , 6 ; due fette sono state acquisite in una singola apnea . rm con dobutamina a bassa dose la dobutamina stata somministrata per via endovenosa senza rimuovere il paziente dal magnete , con monitoraggio constante dellelettrocardiogramma , della pressione sanguigna e con sorveglianza visiva del paziente . 
multipli assi corti e immagini sul piano quattro camere sono stati acquisiti utilizzando la stessa sequenza e geometria dellesame a riposo . rm con studio del delayed enhancement la distribuzione tardiva del contrasto stata ottenuta 15 minuti dopo la somministrazione di gadolinio - dota ( 0 , 1 mmol / kg ) utilizzando una sequenza 3d ad eco di gradiente con modalit inversion recovery , con un pre - impulso di inversione ottimizzato per la massima soppressione del segnale del miocardio . 
i parametri della sequenza sono stati : spessore = 5 mm , te = 1 , 32 ms , tr = 4 , 4 ms , flip angle = 15 gradi , voxel acquisito = 1 , 372 , 1410 , 0 , voxel ricostruito = 1 , 371 , 375 , 0 , fattore turbo = 37 , matrice di acquisizione = 256256 . cad , coronary artery disease ; pci , percutaneous coronary intervention acquisition was completed with parallel imaging using a sense factor of 1.6. 
 follow - up low - dose dobutamine mri without removing the patient from the magnet , dobutamine was administered intravenously under continuous electrocardiography ( ecg ) , blood pressure and visual monitoring . a cardiologist was present throughout dobutamine infusion . a 6 mesi dopo la procedura di rivascolarizzazione ciascun paziente stato sottoposto a follow - up clinico , angiografico e mediante rm . 
al followup stata valutata la ripresa funzionale per definire lefficacia della procedura percutanea di rivascolarizzazione . 696 radiol med ( 2009 ) 114 : 692704 using a perfusion pump , we administered an increasing dobutamine dose of 5 and 10 mg / kg per minute for 3 min each [ 21 ]  . 
multiple short - axis and four - chamber views were acquired using the same sequence and geometry settings as the rest of the study . delayed - enhancement mri delayed - enhancement distribution was evaluated 15 min after gadolinium - dota ( 0.1 mmol / kg ) injection using a 3d inversion recovery gradient turbo field echo sequence , with prepulse delay optimised for maximum myocardial signal suppression . 
imaging parameters were thickness = 5 mm , te = 1.32 ms , tr = 4.4 ms , flip angle = 15 , acquired voxel = 1.372.1410.0 , reconstructed voxel = 1.371.37.0 , turbo factor = 37 , acquisition matrix = 256256 . follow - up six months after successful angioplasty , patients underwent clinical , angiographic and mri follow - up examinations , the latter consisting in a rest cine and delayed - enhancement study performed with the same technique as in the baseline study . 
at follow - up , functional recovery was evaluated to define a successful percutaneous coronary intervention ( pci ) procedure . data evaluation image analysis was performed on an off - line workstation ( viewforum release 4.2 ; philips medical system , best , the netherlands )  . 
global function , with end - diastolic ( edv ) and end - systolic volume ( esv ) and ejection fraction were quantified on short - axis images using the simpson rule , manually defining stolic and end - systolic borders on each tomographic slice . de quantification was performed for each segment related to the occluded vessel with a quantitative approach with a semiquantitative method , as previously validated [ 23 , 24 ]  . 
this semiquantitative analysis was based on manual contour tracing ( endocardial and epicardial borders of left ventricle ) and two regions of interest ( rois ) were manually set : one in the centre of infarction and the other in the centre of normal myocardium with normal kinesis . 
from this follows the automatic threshold delineation and valutazione dei dati lanalisi delle immagini stata effettuata su una workstation off - line ( viewforum release 4.2 ; philips medicai system , best , olanda )  . 
la funzione globale con i volumi telediastolico , telesistolico e con la frazione di eiezione sono stati quantificati sulle immagini asse corto utilizzando la regola di simpson , definendo manualmente i contorni in fase telediastolica e telesistolica su ogni fetta acquisita . la quantificazione del de stata effettuata per ogni segmento correlato al vaso cronicamente occluso con un approccio quantitativo con approccio semiquantitativo , come precedentemente validato [ 23 , 24 ]  . 
lanalisi semiquantitativa stata basata sulla delineazione manuale dei contorni endocardici ed epicardici del ventricolo sinistro e successivamente sono state posizionate due regioni di interesse ( roi ) : una nel centro del miocardio infartuato e una nel centro di miocardio sano normocinetico . 
da ci , segue la delineazione automatica del tessuto infartuato sulla base di un valore di intensit di segnale soglia derivante dalla media delle intensit del tessuto infartuato e normale . 
come riportato in letteratura se il miocardio sotteso ad una coronaria cronicamente occlusa presentava un enhancement transmurale il paziente non stato incluso nella popolazione di studio da rivascolarizzare , data la scarsa possibilit di presenza di miocardio vitale nel segmento sotteso a tale vaso . ogni esame di rm per lo studio di vitalit ( esame a riposo , sotto stress dobutaminico e post - rivascolarizzazione ) stato visionato in modalit cine per una valutazione visuale della cinesi . 
inoltre nel territorio di interesse stata calcolata la riserva contrattile espressa come lispessimento indotto dalla dobutamina ed stato calcolato sottraendo allispessimento sistolico medio indotto dallo stress dobutaminico , lispessimento sistolico medio a riposo . 
inoltre , considerando sia il de che swt , stato calcolato quanto del tessuto contribuente radiol med ( 2009 ) 114 : 692704 quantification of scar tissue , given the media of signal intensity between infarcted and normal myocardiuaccording to the literature , if the occluded coronary - related tissue presented a transmural de , the patient was not considered for revascularisation , as the possibility of having viable tissue in that segment was very scarce . 
 for viability evaluation , each mri study ( rest , during dobutamine infusion and after revascularisation ) was previewed in a cine mode to visually analyse the kinesis . considering normal coronary artery distribution territories , viability parameters were measured in the myocardial territory related to the occluded coronary . 
systolic wall thickening ( swt ) of the segments to be studied was evaluated at rest before and after revascularisation and calculated by subtracting mean dwt from mean systolic thickness . 
additionally , in the territory of interest , contractile reserve was expressed as dobutamine - induced swt and was calculated by subtracting from the mean dobutamine - induced swt the mean swt at rest . 
moreover , considering both de and swt , we calculated how much of the tissue contributing to stress wall thickening corresponded to potentially viable tissue , defined by the viability index [ ( vi ) = [ swt ( 100de ) ] / 100 ]  . 
the infarct region was defined viable , as proposed in the literature , if 50% of segments fulfilled the morphological and functional mri viability criteria [ 25 ]  . functional recovery of the infarct region after successful revascularisation was defined at follow - up mri as a rest swt 2 mm in the previous dysfunctional segments related to the occluded vessel . stenting technique all conventional angiograms before revascularisation were evaluated by two independent observers . 
all patients received a bolus of unfractionated heparin ( 70 iu / kg ) before angioplasty to obtain a timi flow of 3 and a residual stenosis of the artery [ infarct - related artery ( ira ) ] < 30% . 
after the procedure , patients were treated with aspirin and clopidogrel for 12 months and then with aspirin alone . allispessimento sotto stress corrisponde a tessuto potenzialmente vitale , definito da un indice di vitalit ( ( vi ) = [ swt ( 100de ) ] / 100 )  . 
la regione infartuata stata definita vitale , come proposto in letteratura , se 50% dei segmenti sottesi al vaso cronicamente occluso soddisfacevano i criteri rm morfologici e funzionali di vitalit [ 25 ]  . la ripresa funzionale della regione infartuata post - rivascolarizzazione stata definita alla rm di follow - up come lispessimento sistolico a riposo 2 mm nei segmenti precedentemente disfunzionali sottesi al vaso cronicamente occluso . tecnica di stenting tutti gli angiogrammi pre - rivascolarizzazione sono stati valutati da due osservatori indipendenti . 
a tutti i pazienti stata somministrata una dose eparina nonfrazionata ( 70 iu / kg ) prima dellangioplastica , per ottenere un timi flow di 3 ed una stenosi residua dellarteria ( infarct related artery , ira ) < 30% . 
dopo la procedura i pazienti sono stati trattati con aspirina e clopridogel per 12 mesi e successivamente solo con aspirina . analisi statistica i risultati sono stati espressi come valore mediodeviazione standard . 
dwt , swt , de e vi sono stati paragonati alla ripresa funzionale post - rivascolarizzazione ; lanalisi statistica stata effettuata con un modello di regressione lineare ( stata software 2006 )  . 
se un parametro funzionale variato significativamente stato comparato ai parametri rm di vitalit descritti in precedenza ( correlazione di pearson )  . risultati il cto era la coronaria destra in 13 casi , la discendente anteriore in 6 e la circonflessa in 4 . 
in tre casi ( due con malattia della coronaria destra e uno della discendente anteriore ) le immagini acquisite durante stress dobutaminico non sono state di 698 statistical analysis results are expressed as mean valuesstandard deviation ( sd )  . 
if a functional parameter varied significantly , it was related to the ld - mri viability parameters described above ( pearson correlation )  . results cto was located in the right coronary artery in 13 patients , the left descending artery in six and the circumflex artery in four . 
in three cases ( two right coronary arteries and one left descending artery ) stress - induced mri images were not suitable for diagnosis , so they were excluded from the final statistical analysis . 
four patients did not fulfil the defined criteria for myocardial viability ( one right coronary , one left and two circumflex artery ) and were therefore excluded from the revascularisation procedure . mean mri time was 4512 min . revascularisation of the occluded vessel was performed within 2 weeks ( 95 days ) after ld - mri ; revascularisation was successful in 14 / 16 cases , as in two cases the invasive procedure was complicated , with perforation and dissection of the right coronary ostium due to the highly calcified vessel . 
the overall mean procedure time was approximately 12161 min . follow - up rest , cine and de - mri were performed in the remaining 14 patients 6 months after the invasive procedure . 
mean mri time was 309 m at follow - up coronary angiography , all the treated vessels were found to be patent . regarding baseline mri , we evaluated 20 patients ( 110 segments related to the occluded vessel )  . 
purtroppo 4 pazienti non hanno soddisfatto i criteri rm richiesti per lidentificazione di vitalit e sono stati esclusi dalla procedura di rivascolarizzazione ( uno con malattia della coronaria destra , uno della discendente anteriore e due della arteria circonflessa )  . 
la durata media della rm stata di 4512 minuti . la rivascolarizzazione del vaso cronicamente occluso stata effettuata entro due settimane ( 95 giorni ) dallesame rm ; la procedura stata efficace in 14 / 16 casi , in quanto in due casi stata complicata da perforazione e dissezione dellostio del vaso a causa di severe calcificazioni ( arteria coronaria destra )  . 
il tempo medio della procedura percutanea stato circa di 12161 minuti . la rm di follow - up eseguita a riposo e con studio del de stata effettuata in 14 pazienti a 6 mesi dalla procedura di riperfusione . 
alla coronarografia di follow - up stata confermata la perviet di tutti i vasi trattati . per quanto concerne lesame rm di base sono stati valutati 20 pazienti ( 110 segmenti sottesi al vaso cronicamente occluso )  . 
per quanto concerne i 16 pazienti considerati per la rivascolarizzazione i valori medi di dwt , swt e vi presentati erano di 8 , 032 , 35 , 2 , 641 , 56 e 1 , 771 , 48 ; il valore medio di de era 41 , 9730 , 32 . 
si riscontrato una correlazione diretta statisticamente significativa tra il recupero funzionale e swt ( coefficiente 1 , 779 ; p = 0 , 015 ) e vi ( coefficiente 2 , 032 ; p = 0 , 011 )  . 
la valutazione statistica riassunta nella tabella 2 . considerando i parametri funzionali dei 14 pazienti che sono stati sottoposti a rivascolarizzazione , al follow - up si evidenziato un miglioramento significativo unicamente della frazione di eiezione del ventricolo di sinistra [ delta 95% ci 4 , 47 ( da 8 , 128 a 0 , 815 , p = 0 , 0203 ) ]  . 
follow - up rest mri shows the improvement of kinesis at the same level ( g , h ) , with no change in infarct extension ( i )  . 
revascularisation was performed , but wall motion did not improve ( c ) , and delayed enhanced images ( e , f ) show increase in infarction distribution that involves a papillary muscle . 
la rm a riposo mostra ipocinesia della parete infero - laterale del ventricolo sinistro ( a ) che migliora dopo somministrazione di dobutamina a bassa dose ( b )  . 
 stata effettuata una rivascolarizzazione del vaso occluso ma la cinesi non migliorata alla rm di follow - up a riposo ( c ) e lestensione dellinfarto appare aumentata per coinvolgimento di un muscolo papillare ( e , f )  . 
one widely accepted criterion that suggests viability in chronic infarction is considered an end - diastolic thickness > 5.5 mm , predicting vitality of the vessel - related territory with a sensitivity and specificity of 92% and 52% , respectively [ 9 , 29 ]  . in addition , de imaging has become a very accurate marker of infarct identification , with almost perfect correlation with [ 28 ]  . 
un criterio largamente accettato , ma non pi considerato sufficiente , che suggerisce la persistenza di miocardio vitale in un territorio cronicamente infartuato la presenza di spessore telediastolico > 5 , 5 mm , che ha una sensibilit e specificit rispettivamente di 92% e 52% , nel predire la vitalit del territorio relazionato ad un vaso cronicamente occluso [ 9 , 29 ]  . 
la rm mediante lo studio del delayed enhancement identifica con correlazione pressoch perfetta rispetto alla tomografia ad emissione di positroni la presenza di tessuto necrotico sia di classificarne la transmuralit [ 30 ]  . 
questo dato ritenuto essere inversamente correlato alla ripresa funzionale post - riperfusione : se linfarto si estende al 75% dello spessore di parete la ripresa funzionale avviene nel 7% dei casi , mentre se interessa il 702 radiol med ( 2009 ) 114 : 692704 positron emission tomography , thanks to the possibility of recognising the presence of necrotic tissue and classifying its transmurality [ 30 ]  . 
if infarction corresponds to 75% of wall thickness , functional recovery occurs in 7% of cases , whereas if infarction only corresponds to 25% , recovery occurs in 82% of cases [ 31 , 32 ]  . 
one functional approach to identifying hibernating myocardium is cardiac dobutamine imaging , as the response to dobutamine stress is related to the coexistence of both viable and necrotic tissue in an infarcted area and can therefore be considered superior to the sole quantification of scar tissue with de imaging [ 21 ]  . 
the clinical and prognostic importance of ld - mri has not been clearly defined , as previous studies demonstrated sensitivity and specificity of 89% and 94% , respectively , which dropped to 50% and 81% when patients with ejection fraction < 35% and multiple kinesis alterations were considered [ 25 , 33 ]  . the detection of viable tissue is important for selecting patients whose left ventricular function might benefit from revascularisation , due to the presence of potentially salvageable myocardium . in our study , we tested a comprehensive cardiac ldmri approach preinterventionally in order to identify hibernating myocardium , as under drug stress , contractility improves in dysfunctional but viable myocardium , and gadolinium storage in necrotic tissue permits evaluation of the extent of damaged tissue . 
moreover , the introduced index defined as vi , combining the two different parameters necessary for viability evaluation , is even more closely related to functional recovery than de or swt taken alone , as its coefficient and p value are more powerful than those of de and swt alone . 
 our study reflects previous findings regarding de and swt but also adds the new possibility of discriminating between patients with the same de score according to the amount of viable tissue contributing to systolic stress thickening . 
the viability index proposed can be used as a more to predict post - revascularisation accurate parameter contractile recovery , describing the healthy tissue that contributes to stress thickening in cto subtended area . this is a small step forward in the treatment of cto in patients who might benefit from the revascularisation procedure . 
a large amount of data suggests that drugeluting stents offer better long - term patency rates and freedom from restenosis and repeated revascularisation than bare - metal stents in patients with cto [ 34 , 38 ] , and in our study , all patients received drug - eluting stents . furthermore , left ventricular function is around 4% and can be considered increase observed in global the 25% dello spessore di parete , la ripresa avviene nell82% dei casi [ 31 , 32 ]  . 
un approccio funzionale allidentificazione di miocardio ibernato con limaging cardiaco con stress dobutaminico si correla alla coesistenza di miocardio vitale e necrotico allinterno di una stessa area infartuata e tale approccio pu essere considerato superiore alla sola quantificazione del tessuto cicatriziale allimaging rm di delayed enhancement [ 21 ]  . 
limportanza clinica e prognostica della rm con stress dobutaminico non stata ancora definita chiaramente anche se studi preliminari mostrano una sensibilit e specificit del 89% e 94% rispettivamente , che si abbassano per al 50% e 81% in pazienti con frazione di eiezione < 35% e / o multiple alterazioni della cinesi [ 25 , 33 ]  . 
la detection di tessuto vitale importante per selezionare pazienti in cui la frazione di eiezione potrebbe trarre beneficio da una procedura di rivascolarizzazione , in base alla presenza di miocardio potenzialmente vitale . 
 nello studio qui descritto , abbiamo testato un approccio rm comprensivo prima della procedura di rivascolarizzazione per identificare miocardio ibernato , dato che sotto stress dobutaminico a bassa dose la contrattilit migliora in caso di miocardio disfunzionale ma vitale e laccumulo di gadolinio nel tessuto necrotico permette di valutare lestensione del danno tissutale . 
inoltre lindice di vitalit introdotto , definito come vi , ottenuto da una combinazione tra i due differenti parametri necessari per lidentificazione di vitalit , ancora pi correlato al recupero funzionale rispetto al de e swt considerati singolarmente , dato che il suo coefficiente ed il suo valore p hanno una potenza statistica maggiore . 
 il nostro studio riflette quanto attualmente riportato in letteratura riguardo de ed swt ma inoltre aggiunge la possibilit di discriminare in pazienti con la medesima estensione necrotica ( simile estensione di de ) la quantit di tessuto vitale residuo . 
lindice di vitalit proposto pu essere utilizzato come parametro pi accurato per predire il recupero funzionale post - rivascolarizzazione , descrivendo il tessuto vitale che contribuisce allispessimento sotto stress in unarea sottesa ad un vaso cronicamente occluso . questo pu essere considerato un piccolo passo in avanti nel trattamento delle cto in pazienti che potrebbero beneficiare dalla procedura di rivascolarizzazione . 
molti dati attuali suggeriscono che gli stent medicati offrano percentuali maggiori di perviet a lungo termine , assenza di restenosi e , quindi , di procedure di ri - vascolarizzazione rispetto agli stent metallici in pazienti con cto [ 3438 ] e nella casistica qui riportata tutti i pazienti hanno ricevuto stent medicati . 
inoltre , il globale aumento osservato nella frazione di eiezione del ventricolo di sinistra di circa il 4% e pu essere considerato significativo anche nel follow - up a breve termine ( 6 mesi ) come riportato da un recente lavoro radiol med ( 2009 ) 114 : 692704 significant even in this short - term follow - up , as reported in a recent study where the trend in left ventricular function improvement was evaluated both in shortand long - term follow - up [ 19 ]  . 
as demonstrated , restoring blood flow to dysfunctional but viable myocardium led to a functional benefit for patients , shown by a decrease in mean esv with substantial identity of edv that caused increase in lvef . moreover the low rate of complications during intervention ( two cases out of 16 ; 12.5% ) is in line with the current literature [ 11 ] and should not prevent patients with potentially viable myocardium from being revascularised percutaneously . 
the definition of cto was more than 3 months in this study , whereas previous studies defined cto as 7 days to 3 months of occlusion time [ 38 ]  . 
 another limit is the use of a viability index that was not previously validated in the literature ; however , we decided to employ it because it enhances the contribution of viable tissue to dobutamine response in the cto vessel - related segments . 
low - dose dobutamine infusion was not preformed at follow - up mri but might provide further insight into the mechanisms underlying recovery of function of viable myocardium . dove il trend di crescita della frazione di eiezione stato valutato sia a breve che a lungo termine [ 19 ]  . 
ripristinare il flusso sanguigno in un territorio miocardico disfunzionale ma vitale migliora la funzione cardiaca dei pazienti , come dimostrato da una diminuzione del volume telesistolico con una sostanziale identit del volume telediastolico ( assenza di rimodellamento ) che porta ad un incremento della frazione di eiezione . 
inoltre la bassa frequenza di complicanze peri - procedurali ( 2 casi su 16 ; 12 , 5% ) in linea con la letteratura [ 11 ] e non dovrebbe precludere a pazienti con miocardio potenzialmente vitale di essere riperfusi in modo percutaneo . 
inoltre la definizione di cto nel nostro lavoro stata identificata come occlusione maggiore di tre mesi , mentre in altri studi stata considerate da sette giorni a tre mesi [ 38 ]  . 
 un ulteriore limite dello studio lutilizzo di un indice di vitalit non precedentemente validato in letteratura , tuttavia si voluto sottolineare il contributo del miocardio vitale allo stress dobutaminico nel territorio sotteso ad un vaso cronicamente occluso . 
lesame rm di follow - up stato condotto senza linfusione di dobutamina , anche se ci potrebbe essere utile nellidentificare meccanismi sottesi al recupero funzionale del miocardio vitale in pazienti con cto . 
twenty - seven patients with bowel infarction due to vascular obstruction were evaluated with multidetector ct ( mdct ) to establish the prognostic value of ct findings and their correlation with the origin of the ischaemia . 
bowel - wall hyperdensity ( 2 / 9 venous occlusions ) , loss of wall enhancement ( 1 / 9 venous occlusions , 2 / 18 arterial occlusions ) and wall thickening ( 8 / 9 venous obstructions , 2 / 18 arterial occlusions ) were predictive of good outcome . 
sono state valutate le immagini tc multidetettore ( tcmd ) di 27 pazienti con infarto intestinale da occlusione vascolare e sono state ricercate : significativit prognostica delle alterazioni evidenziate , correlazioni con la natura dellischemia . 
leziologia della patologia in rapporto alla prognosi risultata altamente significativa , con mortalit del 89% nelle forme arteriose e del 11% in quelle venose . liperdensit parietale ( 2 / 9 occlusioni venose ) , lassenza di enhancement ( 1 / 9 occlusioni venose , 2 / 18 occlusioni arteriose ) , lispessimento di parete ( 8 / 9 occlusioni venose , 2 / 18 occlusioni arteriose ) sono risultati significativi di evoluzione benigna , mentre la dilatazione delle anse ( 4 / 9 occlusioni venose , 13 / 18 occlusioni arteriose ) , la pneumatosi parietale ( 1 / 9 occlusioni venose , 17 / 18 occlusioni arteriose ) , laria nelle vene mesenteriche ( 2 / 9 occlusioni venose , 11 / 18 occlusioni arteriose ) , laria nei rami portali ( 1 / 9 occlusioni venose , 4 / 18 occlusioni arteriose ) , lo pneumoperitoneo ( 8 / 18 occlusioni arteriose ) e il retropneumoperitoneo ( 1 / 18 occlusioni arteriose ) sono risultati indici prognostici sfavorevoli . 
the longer life expectancy of the general population and the resulting degenerative vascular diseases have led to a steady increase in the incidence of this disease . bowel infarction is typically classified as occlusive or nonocclusive on the basis of aetiology . 
in the occlusive forms , the abnormality may involve the arterial or venous vessels ( coeliac trunk branches , superior mesenteric artery , inferior mesenteric artery and corresponding venous vessels )  . 
the nonocclusive forms are less frequent and generally caused by a rapid fall in systemic pressure or by hypovolaemia due to haemorrhagic , cardiogenic or septic shock . multidetector computed tomography ( mdct ) thanks to increased spatial resolution , high - quality multiplanar reconstructions ( mpr ) , maximum intensity projections ( mip ) , three - dimensional rendering and shorter acquisition times is now able to allow visualisation of early signs of bowel ischaemia and infarction and has become the gold standard for diagnosis [ 36 ]  . 
mdct also allows for aetiological diagnosis of the infarction which is fundamental in planning treatment in acute patients and has replaced angiography as a diagnostic study because despite having a sensitivity of 87% [ 7 ] , angiography is now exclusively reserved for therapeutic procedures . despite of our better knowledge and improved diagnostic techniques , bowel infarction is still frequently a fatal disease , with reported mortality rates between 50% and 90% [ 13 , 8 ] , depending in part on aetiology [ 9 ]  . 
 the aim of this study was to identify possible correlations between ct findings and outcomes of patients affected by occlusive bowel infarction . materials and methods we retrospectively reviewed 27 patients with bowel infarction who were examined at our department between october 2005 and january 2008 . 
patients were identified through a search of our electronic archive system using terms such as ischaemia , infarction and intestinal necrosis . thus , we obtained a list of 40 patients , 13 of whom were excluded : six , because they had circumscribed ischaemia linfarto intestinale rappresenta una causa non abituale , ma spesso sottostimata , di addome acuto non traumatico ed determinato da unalterazione dellapporto ematico alla regione mesenterica , che pu causare una necrosi della parete intestinale [ 1 , 2 ]  . 
lincremento della vita media della popolazione generale e le conseguenti patologie degenerative vascolari hanno favorito un costante aumento dellincidenza di questa patologia . linfarto intestinale viene classicamente suddiviso in occlusivo e non occlusivo in base alla differente eziologia . nelle forme occlusive , lalterazione pu interessare i vasi arteriosi o venosi ( rami di divisione del tronco celiaco , arteria mesenterica superiore , arteria mesenterica inferiore e vasi venosi corrispondenti )  . 
le forme non occlusive , meno frequenti , sono generalmente causate da un rapido decremento della pressione sistemica o dallipovolemia in rapporto a shock emorragico , cardiogeno o settico . la tomografia computerizzata multidetettore ( tcmd ) , grazie allincremento della risoluzione spaziale , allalta qualit delle ricostruzioni multiplanari mpr , mip e 3d e ai ridotti tempi di esecuzione , in grado di rilevare i segni precoci dellischemia e dellinfarto intestinale e rappresenta attualmente il gold standard per il riconoscimento di questa patologia [ 36 ]  . 
inoltre essa consente una diagnosi di tipo eziologico dellinfarto , fondamentale ai fini della pianificazione terapeutica per il paziente acuto ed ha ormai sostituito in fase diagnostica langiografia , che , pur presentando una sensibilit del 87% [ 7 ] , attualmente ha un ruolo quasi esclusivamente terapeutico . 
 nonostante lincremento delle conoscenze riguardo questa condizione clinica ed il miglioramento delle tecniche diagnostiche , linfarto intestinale risulta ancora spesso letale , con un tasso di mortalit descritto in letteratura compreso tra il 50% ed il 90% [ 13 , 8 ] , variabile anche in rapporto alla sua eziologia [ 9 ]  . 
i pazienti sono stati identificati 782 radiol med ( 2009 ) 114 : 780791 secondary to strangulation and the remaining seven because they underwent unenhanced ct only due to poor general condition . all 27 selected cases were affected by occlusive ischaemia : 18 ( 67% ) of the arterial and nine ( 33% ) of the venous type . 
only 15 patients ( 56% ) underwent surgery ; of these , nine ( 60% ) died and six ( 40% ) survived . the definite diagnosis was provided by surgery , with histology of the resected intestinal segment in 15 patients and by autopsy in five cases . 
postprocessing time was approximately 15 mct images were assessed for the following parameters : wall enhancement , considered pathological when inhomogeneous , marked and persistent , or absent wall thickness , considered normal when less than 5 mm , with distended bowel loops ; in the absence of distension , the wall was considered pathological when its thickness was greater than approximately 1 cm bowel - loop dilatation , defined as a small - bowel diameter > 2.5 cm and large - bowel diameter > 8 cm parietal pneumatosis , defined as the presence of small air bubbles within the bowel wall , irrespective of extent presence of gas within the mesenteric venules and portal venous system pneumoperitoneum / pneumoretroperitoneum ascites . finally , to define the arterial or venous nature of the vascular occlusion causing ischaemia , we evaluated the morphology and patency of the coeliac trunk , superior and inferior mesenteric arteries , superior mesenteric vein and portal trunk . ricercando nel nostro archivio computerizzato alcune parole chiave come ischemia , infarto , necrosi intestinale . 
stata in tal modo ottenuta una lista di 40 pazienti , da cui ne sono stati esclusi 13 , 6 con ischemia circoscritta secondaria a strangolamento ed i restanti 7 poich , per le precarie condizioni cliniche , erano stati valutati esclusivamente con scansioni eseguite senza iniezione di mezzo di contrasto endovenoso . 
il gruppo di pazienti considerato includeva 14 soggetti di sesso maschile ( 52% ) e 13 di sesso femminile ( 48% ) , con un intervallo di et compreso tra 42 e 92 anni ( et media 68 , 3 ; errore standard 5 , 3 )  . 
inoltre , soltanto 15 ( 56% ) sono stati trattati chirurgicamente e di questi 9 ( 60% ) sono deceduti e 6 ( 40% ) sono sopravvissuti . la diagnosi definitiva si basata sul controllo chirurgico con istologia del segmento intestinale asportato in 15 pazienti e sul controllo autoptico in 5 casi . 
 le apparecchiature utilizzate sono state : tcmd a 16 strati ( tsx - 101aaquilion 16 , toshiba medical system , tokio , giappone ) , tcmd a 4 strati ( mx 8000 , philips medical systems , royal philips electronic , best , olanda )  . in tutti i casi sono state eseguite scansioni prima e dopo iniezione endovenosa di mezzo di contrasto ( 120140 ml iniettati alla velocit di 33 , 5 ml / s ) , con acquisizione delle immagini in fase arteriolare , generalmente con un ritardo compreso tra 30 e 40 s dallinizio della somministrazione del mezzo di contrasto ed in fase venosa , con un ritardo compreso tra 60 e 90 s dallinizio della somministrazione del mezzo di contrasto . 
nel valutare le immagini tc sono stati considerati i seguenti parametri : enhancement parietale , considerato patologico quando disomogeneo , marcato e persistente , o assente ; spessore parietale , considerato normale quando inferiore a 5 mm , con anse distese ; in assenza di distensione , la parete stata considerata patologica in presenza di uno spessore superiore ad 1 cm circa ; dilatazione delle anse intestinali , diagnosticata in presenza di un diametro del tenue superiore a 2 , 5 cm e del colon superiore ad 8 cm ; radiol med ( 2009 ) 114 : 780791 statistical analysis the sample population was initially analysed using descriptive statistics and considering age , gender , aetiology of ischaemia ( arterial or venous ) , disease course ( dead or living patient ) and calculating mortality rates . 
we then examined the ability of ct to define the type of bowel ischaemia , and related the different densitometric alterations to the nature of the vascular occlusion and disease stage to determine the prognostic significance of the ct findings . 
the data obtained were considered to be statistically significant at a p value 0.05. results as shown in table 1 , disease aetiology in relation to outcome proved to be highly significant ( p = 0.0000001 ) , with 89% mortality for arterial infarctions ( 16 / 18 ) and 11% mortality for venous obstructions ( 1 / 9 )  . 
all cases of arterial obstruction regarded the main trunk of the superior mesenteric artery , which was associated with atheromatous wall alterations of the coeliac trunk in five cases and alterations of the inferior mesenteric artery in four . 
all venous obstructions involved the superior mesenteric vein , with associated involvement of the portal vein in four cases . analysis of the ct findings seen in the two types of vascular occlusion is shown in table 2 . 
wall hyperdensity was seen in 2 / 9 ( 23% ) venous occlusions and in none of the table 1 correlation between aetiology and outcome in bowel infarction pneumatosi parietale , diagnosticata in presenza di piccole bolle aeree nel contesto della parete , indipendentemente dalla entit ; presenza di aria nelle venule mesenteriche e nel sistema venoso portale ; pneumo - / retropneumoperitoneo ; ascite . 
 per definire , infine , la natura arteriosa o venosa dellocclusione vascolare responsabile dellischemia intestinale , sono state valutate la morfologia e la perviet del tronco celiaco , delle arterie mesenteriche superiore ed inferiore , della vena mesenterica superiore e del tronco portale . 
 analisi statistica lanalisi del nostro campione stata eseguita dapprima mediante statistica descrittiva , esaminando et , sesso , eziologia dellischemia ( arteriosa o venosa ) , evoluzione della patologia ( paziente deceduto o vivente ) e calcolando i valori percentuali di mortalit . 
successivamente , il campione stato suddiviso in gruppi al fine di confrontare leziologia della patologia ( gruppo degli infarti arteriosi versus quelli venosi ) , rispetto ai risultati terapeutici ( gruppo dei pazienti deceduti versus quelli viventi ) ed stata verificata la significativit statistica di tale dato , applicando il test del 2 e calcolando il valore di p . 
sono state , quindi , valutate le potenzialit della tc nel definire la natura dellischemia intestinale e le differenti alterazioni tomodensitometriche riconoscibili sono state correlate alla natura dellocclusione vascolare ed alla evoluzione della patologia , per determinare la loro significativit prognostica . 
i dati sono stati considerati statisticamente significativi per valori di p0 , 05 . risultati come riportato nella tabella 1 , leziologia della malattia , in rapporto alla prognosi , si rivelata altamente significaarterial infarctions ( n = 18 ) venous infarctions ( n = 9 ) chi - square test p value dead patients living patients dead patients living patients 16 ( 88% ) 2 ( 12% ) 1 ( 11% ) 8 ( 89% ) 0.0000001 tabella 1 correlazione tra eziologia e prognosi nellinfarto intestinale infarti arteriosi ( n = 18 ) infarti venosi ( n = 9 ) pazienti deceduti pazienti viventi pazienti deceduti pazienti viventi 16 ( 88% ) 2 ( 12% ) 1 ( 11% ) 8 ( 89% ) 119 valore p 0 , 0000001 784 radiol med ( 2009 ) 114 : 780791 fig . 
absence of wall enhancement was evident in 1 / 9 ( 12% ) venous occlusions and in 2 / 18 ( 12% ) arterial occlusions , and hence the incidence of this finding was very similar in the two forms . 
wall thickening was the most frequent abnormality in infarctions caused by venous occlusions , in which cases of thickening were marked and always > 15 mm . dilated bowel loops were present in 4 / 9 ( 45% ) venous occlusions and in 13 / 18 ( 73% ) arterial occlusions . 
lostruzione arteriosa interessava in tutti i casi il tronco principale dellarteria mesenterica superiore e si associavano alterazioni ateromasiche parietali a carico del tronco celiaco in 5 casi e dellarteria mesenterica inferiore in 4 . 
the presence of gas within the mesenteric venules was detected in 2 / 9 ( 23% ) venous occlusions and in 11 / 18 ( 62% ) arterial occlusions , with a clear prevalence in the arterial forms . 
likewise , the presence of gas within the portal branches was seen in 1 / 9 ( 12% ) venous occlusions and in 4 / 18 ( 23% ) arterial occlusions . 
in no case of venous occlusion was the presence of pneumoperitoneum / pneumoretroperitoneum detected , signs that were instead seen in 8 / 18 ( 45% ) and 1 / 18 ( 5% ) arterial occlusions , respectively . 
ascites was present in 6 / 9 ( 67% ) venous occlusions and in 12 / 18 ( 67% ) arterial occlusions , with a virtually identical incidence in the two forms . analysis of the ct findings in relation to disease course lanalisi delle alterazioni tc riscontrate in relazione alla natura dellocclusione vascolare riportata nella tabella 2 . 
uniperdensit parietale stata riscontrata in 2 / 9 ( 23% ) occlusioni venose , mai in quelle arteriose . lassenza di enhancement parietale risultata evidente in 1 / 9 ( 12% ) occlusioni venose ed in 2 / 18 ( 12% ) occlusioni arteriose , pertanto ha presentato unincidenza sovrapponibile nelle due forme . 
lispessimento parietale ha rappresentato lalterazione pi frequente negli infarti da occlusioni venose ed in tali casi , inoltre , lincremento dello spessore parietale risultato marcato e sempre superiore a 15 mla dilatazione delle anse intestinali stata riscontrata in 4 / 9 ( 45% ) occlusioni venose ed in 13 / 18 ( 73% ) occlusioni arteriose . 
la pneumatosi parietale stata riconosciuta 786 radiol med ( 2009 ) 114 : 780791 table 2 correlation between ct findings and type of vascular obstruction arterial infarctions ( n = 18 ) venous infarctions ( n = 9 ) tabella 2 alterazioni tc riscontrate in rapporto alla natura dellocclusione vascolare intramural hyperdensity loss of enhancement wall thickening loop dilation intramural pneumatosis mesenteric venous gas portal venous gas pneumoperitoneum pneumoretroperitoneum ascites 2 ( 12% ) 2 ( 12% ) 13 ( 73% ) 17 ( 95% ) 11 ( 62% ) 4 ( 23% ) 8 ( 45% ) 1 ( 5% ) 12 ( 67% ) infarti arteriosi ( n = 18 ) iperdensit parietale 2 ( 12% ) assenza di enhancement 2 ( 12% ) ispessimento parietale 13 ( 73% ) dilatazione anse pneumatosi 17 ( 95% ) aria nelle venule mesenteriche 11 ( 62% ) aria nella vena porta pneumoperitoneo retropneumoperitoneo ascite 4 ( 23% ) 8 ( 45% ) 1 ( 5% ) 12 ( 67% ) 2 ( 23% ) 1 ( 12% ) 8 ( 88% ) 4 ( 45% ) 1 ( 12% ) 2 ( 23% ) 1 ( 12% ) 6 ( 67% ) infarti venosi ( n = 9 ) 2 ( 23% ) 1 ( 12% ) 8 ( 88% ) 4 ( 45% ) 1 ( 12% ) 2 ( 23% ) 1 ( 12% ) 6 ( 67% ) is shown in table 3 . 
wall thickening was the most frequent alteration in surviving patients , with 9 / 10 ( 90% ) cases among survivors against 1 / 17 ( 6% ) among deceased patients . 
in 3 / 10 ( 30% ) surviving patients and 10 / 17 ( 59% ) deceased patients , gas was detected in the mesenteric venules , whereas in 1 / 10 ( 10% ) surviving patients and 4 / 17 ( 24% ) deceased patients , the presence of air was seen in the portal branches . 
none of the surviving patients showed pneumoperitoneum or pneumoretroperitoneum , which were instead present in 8 / 17 ( 47% ) and 1 / 17 ( 5% ) deceased patients , respectively . in 1 / 9 ( 12% ) occlusioni venose ed in 17 / 18 ( 95% ) occlusioni arteriose . 
la presenza di aria nelle venule mesenteriche stata riscontrata in 2 / 9 ( 23% ) occlusioni venose ed in 11 / 18 ( 62% ) occlusioni arteriose , con una evidente prevalenza nelle forme arteriose , come anche la presenza di aria nei rami portali , riconosciuta in 1 / 9 ( 12% ) occlusioni venose ed in 4 / 18 ( 23% ) occlusioni arteriose . 
in nessun caso di occlusione venosa stata rilevata la presenza di pneumoperitoneo o retropneumoperitoneo ; tali segni erano , invece , evidenti rispettivamente in 8 / 18 ( 45% ) e 1 / 18 ( 5% ) occlusioni arteriose . 
in 6 / 9 ( 67% ) occlusioni venose ed in 12 / 18 ( 67% ) occlusioni arteriose era presente ascite , che pertanto ha presentato una incidenza sovrapponibile nelle due forme . lanalisi delle alterazioni tc riscontrate in relazione allevoluzione della patologia riportata nella tabella 3 . uniperdensit parietale stata riscontrata in 2 / 10 ( 20% ) pazienti sopravvissuti , mai in pazienti deceduti . 
lispessimento parietale ha rappresentato lalterazione pi frequente nei pazienti sopravvissuti , con il riscontro di 9 / 10 ( 90% ) casi nel gruppo dei pazienti sopravvissuti e 1 / 17 ( 6% ) in quelli deceduti . 
la pneumatosi parietale stata riscontrata in 2 / 10 ( 20% ) pazienti sopravvissuti e 16 / 17 ( 95% ) pazienti deceduti ; lincidenza di tale alterazione ha mostrato , quindi , una netta prevalenza nel gruppo dei pazienti deceduti . 
in 3 / 10 ( 30% ) pazienti sopravvissuti e 10 / 17 ( 59% ) pazienti deceduti era evidente aria nelle venule mesenteriche , mentre in 1 / 10 ( 10% ) pazienti sopravvissuti e 4 / 17 ( 24% ) pazienti deceduti stata riscontrata la presenza di aria nei rami portali . 
in nessun paziente sopravvissuto stato rilevato pneumoperitoneo o retropneumoperitoneo , che al contrario erano evidenti rispettivamente in 8 / 17 ( 47% ) e 1 / 17 ( 5% ) pazienti deceduti . 
in 6 / 10 ( 60% ) pazienti sopravvissuti ed in 12 / 17 ( 71% ) pazienti deceduti era evidente , infine , ascite . la valutazione del valore prognostico dei reperti tc riscontrati nel nostro campione ha dimostrato una significativit statistica per tutti i segni esaminati ( p0 , 05 ) , ad eccezione della presenza di ascite ( p = 0 , 1 )  . 
in particolare , liperdensit , lassenza di enhancement e lispessimento di parete delle anse intestinali sono risultati significativi di evoluzione benigna di malattia , mentre la dilatazione delle anse , la pneumatosi parietale e porto - mesenterica e lo pneumo / retropneumoperitoneo hanno presentato una incidenza pi elevata nei pazienti deceduti . 
the presence of ascites did not provide any prognostic information , its incidence being almost identical among survivors and deceased patients . discussion thanks to its extraordinary capabilities , mdct is the most frequently used technique in the diagnostic approach to patients with suspected intestinal ischaemia , with reported sensitivity , specificity and positive and negative predictive values of 64%93% , 92%100% , 90%100% , and 94%98% , respectively [ 13 , 10 , 11 ]  . 
nonetheless , despite continuing progress in the field of abdominal imaging , the mortality rate of bowel infarction remains presentato unincidenza sovrapponibile nel gruppo dei pazienti sopravvissuti ed in quello dei pazienti deceduti . discussione nellapproccio diagnostico del paziente con sospetta ischemia intestinale la tcmd , per le sue straordinarie potenzialit , ormai divenuta lindagine pi utilizzata e presenta valori di sensibilit , specificit , valore predittivo positivo e negativo , riportati in letteratura , rispettivamente compresi tra 64%93% , 92%100% , 90%100% , 94%98% [ 13 , 10 , 11 ]  . 
nonostante i continui progressi nel campo dellimaging addominale , comunque , il tasso di mortalit in rapporto ad infarto intestinale ancora molto alto , con valori riportati in letteratura compresi tra il 50% ed il 90% [ 1 , 3 , 6 , 8 , 12 ] , e del 63% nella nostra esperienza . a tali insoddisfacenti risultati contribuiscono differenti fattori , ma certamente i pi importanti sono : clinica del paziente , eziologia dellischemia intestinale e grado del danno anatomico al momento della diagnosi . da un punto di vista clinico gli elementi pi importanti nella valutazione prognostica sono : ipertensione arteriosa , diabete mellito , et , fumo , familiarit per cardiopatia , vasculopatia periferica e / o cerebrale , coronaropatia , sintomi di ischemia mesenterica cronica , scompenso 788 radiol med ( 2009 ) 114 : 780791 very high , with reported values of 50%90% ( 63% in our experience ) [ 1 , 3 , 6 , 8 , 12 ]  . 
this unsatisfactory result is related to different factors , among which the most important are the patients clinical history , the aetiology of the intestinal ischaemia and the degree of anatomical damage at the time of diagnosis . clinically , the most important factors in evaluating prognosis are the following : arterial hypertension , diabetes mellitus , age , smoking history , family history of heart disease , peripheral and / or cerebral vascular disease , coronary disease , symptoms of chronic mesenteric ischaemia , heart failure , renal failure , previous myocardial infarction , previous surgery , chronic obstructive pulmonary disease , hypercholesterolaemia , atrial fibrillation , time elapsed between diagnosis and treatment , duration of symptoms , surgical treatment of the acute event and levels of metabolic acidosis [ 1216 ]  . as far as aetiology is concerned , the classification of bowel infarction into nonocclusive and occlusive forms , in relation to arterial or venous obstructions , is now universally recognised in the literature [ 24 , 9 , 17 ]  . 
nonocclusive forms account for 20%30% of cases of acute mesenteric ischaemia , are associated with often adverse outcomes with mortality rates of 58%70% [ 18 , 19 ] , and occur in conditions of systemic hypoperfusion characterised by low infarction , cardiac output : heart failure , myocardial arrhythmias , aortic failure , traumatic , haemorrhagic or septic shock [ 2 , 3 , 11 , 20 ]  . 
in particular , occlusions of the mesenteric arteries are more frequent than those of the corresponding veins [ 3 , 9 , 18 , 2123 ] , as seen in our patient population in which 67% of cases were arterial occlusions against 33% venous occlusions . 
among occlusive forms , the reported mortality rates of forms caused by arterial obstruction range from 17% to 90% , whereas those of lesions secondary to venous obstruction range from 0% to 30% [ 18 , 24 , 25 ]  . 
hence , the aetiology of ischaemia is an important prognostic factor , and its definition should be a fundamental goal of diagnostic imaging . until recently , the use of conventional ct scanners in the study of bowel ischaemia allowed detection of wall alterations , but only rarely was it possible to define disease aetiology [ 26 ]  . 
nowadays , diffusion of scanners with multislice acquisition systems that provide excellent vascular studies has increasingly facilitated this evaluation , allowing both detection and aetiological definition of the disease , as seen in our series [ 4 , 11 , 2729 ]  . another fundamental parameter for ct evaluation is the type of alterations , which have a different incidence in arterial or venous vascular obstructions and express the extent of anatomical damage . 
in this regard , as previously stated cardiaco , insufficienza renale , precedente infarto miocardico , precedente intervento chirurgico , broncopatia cronica ostruttiva , ipercolesterolemia , fibrillazione atriale , intervallo di tempo intercorso tra diagnosi e terapia , durata dei sintomi , trattamento di tipo chirurgico dellevento acuto e livelli di acidosi metabolica [ 1216 ]  . per quanto riguarda leziologia , ormai universalmente riconosciuta in letteratura [ 24 , 9 , 17 ] la suddivisione dellinfarto intestinale in forme non occlusive ed occlusive , in rapporto ad ostruzioni arteriose o venose . 
le forme non occlusive rappresentano il 20%30% dei casi di ischemia mesenterica acuta , si associano ad una prognosi pi spesso sfavorevole , con tasso di mortalit compreso tra il 58% ed il 70% [ 18 , 19 ] e si verificano nelle condizioni di ipoperfusione sistemica caratterizzate da una bassa gittata cardiaca : scompenso cardiaco , infarto del miocardio , aritmie , insufficienza aortica , shock traumatico , emorragico o settico [ 2 , 3 , 11 , 20 ]  . le forme occlusive risultano molto pi frequenti di quelle non occlusive , ed in particolare le occlusioni delle arterie mesenteriche sono pi frequenti di quelle dei vasi venosi corrispondenti [ 3 , 9 , 18 , 2123 ] , come anche riscontrato nel nostro campione in cui le occlusioni arteriose incidono nel 67% dei casi , mentre quelle venose nel 33% . 
nellambito delle forme occlusive , sono stati riportati valori di mortalit nelle forme secondarie ad ostruzione arteriosa compresi tra 17% e 90% , nelle forme secondarie ad ostruzione venosa , valori compresi tra 0% e 30% [ 18 , 24 , 25 ] ; nel gruppo di pazienti da noi studiati , la mortalit stata del 89% nelle forme arteriose e soltanto del 11% in quelle venose . 
 in passato , utilizzando le apparecchiature tradizionali , nello studio dellischemia intestinale era possibile riconoscere le alterazioni parietali , ma solo raramente era possibile una definizione eziologica della patologia [ 26 ]  . 
attualmente , al contrario , la diffusione di apparecchiature dotate di sistemi di acquisizione multistrato , che consentono eccellenti studi vascolari , ha reso agevole tale valutazione , per cui possibile non soltanto il riconoscimento della patologia , ma anche la sua definizione eziologica , cos come anche nella casistica personale [ 4 , 11 , 2729 ]  . 
in fact , parietal hyperdensity , the absence of enhancement and bowel - wall thickening all indicate a favourable outcome , whereas loop dilatation , parietal and portomesenteric pneumatosis and pneumoperitoneum / pneumoretroperitoneum are all indicators of unfavourable outcome . these findings are readily explained if we consider the correlation between progression of intestinal ischaemic damage and abnormalities visible on ct [ 2 , 4 , 17 , 28 , 29 ]  . bowel - wall hyperdensity reflects vasodilation , which is the first consequence of hypoxic damage . 
the absence of wall enhancement corresponds to the ensuing vasoconstriction ; bowel - loop thickening and dilatation are related to the increased capillary permeability , pneumatosis and the presence of air within the mesentericportal system are an expression of necrosis of the intestinal mucosa , whereas pneumoperitoneum / pneumoretroperitoneum corresponds to transmural extension of the necrosis [ 3 , 4 , 3032 ]  . 
therefore , wall hyperdensity , the absence of enhancement and wall thickening represent an early stage of the disease , in contrast to loop dilation , parietal and portomesenteric pneumatosis and pneumoperitoneum / pneumoretroperitoneum , which reflect an advanced stage of disease characterised by high mortality rates . furthermore , our experience suggests that hyperdensity and bowel - wall thickening are significantly correlated to forms of venous infarction , whereas loop dilatation , parietal pneumatosis , the presence of gas within the mesenteric venules and portal branches , as well as pneumoperitoneum / pneumoretroperitoneum are characteristic of forms related to arterial occlusions . 
on the other hand , the absence of wall enhancement and the presence of ascites are nonspecific ct findings with respect to the nature of ischaemia , as their incidence is almost the same in arterial and venous obstructions . these findings may have a temporal justification in that , in arterial obstructions , the pathophysiological events leading to wall necrosis occur in rapid succession , whereas a longer time interval is required for the initial vascular damage of venous occlusions to develop into frank anatomical damage . 
infatti , liperdensit parietale , lassenza di enhancement e lispessimento delle anse indicano una prognosi pi favorevole , mentre la dilatazione delle anse , la pneumatosi parietale e porto - mesenterica e lo pneumo - / retropneumoperitoneo rappresentano indici prognostici sfavorevoli . tali risultati appaiono agevolmente giustificabili , considerando la correlazione tra la progressione del danno ischemico intestinale e le alterazioni riscontrabili allesame tc [ 2 , 4 , 17 , 28 , 29 ]  . 
liperdensit parietale intestinale riflette il meccanismo di vasodilatazione , che rappresenta la prima conseguenza del danno ipossico ; lassenza di enhancement di parete corrisponde alla successiva vasocostrizione che rapidamente si instaura ; lispessimento e la dilatazione delle anse sono dovuti allincremento della permeabilit capillare ; la pneumatosi e la presenza di aria nel sistema mesenterico - portale sono espressione della necrosi della mucosa intestinale ed infine lo pneumo - / retropneumoperitoneo corrispondono allevoluzione transmurale della necrosi [ 3 , 4 , 3032 ]  . 
pertanto , liperdensit parietale , lassenza di enhancement e lispessimento parietale rappresentano una fase precoce della patologia , al contrario la dilatazione delle anse , la pneumatosi parietale e porto - mesenterica e lo pneumo - / retropneumoperitoneo rappresentano una patologia avanzata , gravata da un alto tasso di mortalit . dalla nostra esperienza emerge , inoltre , che liperdensit e lispessimento di parete delle anse intestinali si associano con elevata significativit a forme di infarto venoso , mentre la dilatazione delle anse , la pneumatosi parietale , la presenza di aria nelle venule mesenteriche e nei rami portali e lo pneumo - / retropneumoperitoneo presentano una maggiore incidenza nelle forme secondarie ad occlusioni arteriose . 
lassenza di enhancement di parete e la presenza di ascite hanno invece rappresentato alterazioni tc aspecifiche in relazione alla natura dellischemia , poich la loro incidenza risultata sovrapponibile nelle ostruzioni arteriose ed in quelle venose . la giustificazione di tali risultati probabilmente riconducibile ad un fattore temporale , poich nelle ostruzioni arteriose gli eventi fisiopatologici , che esitano nella necrosi parietale , si susseguono rapidamente ; al contrario , nelle forme venose , occorre un maggior intervallo di tempo affinch dal danno vascolare iniziale si arrivi ad una fase pi conclamata del danno anatomico . 
pertanto la possibilit di riconoscere in corso di esami tc alterazioni parietali riconducibili ad una fase pi precoce della patologia maggiore negli infarti da occlusione venosa , piuttosto che in quelli secondari ad ostruzione arteriosa . 
 [ 3 ] with regard to wall thickening and the target appearance of intestinal loops , which represents a favourable prognostic indicator and a sign more frequently associated with venous infarction owing to a larger intramural haemorrhagic component and superinfection . 
 [ 33 ] , who reported that portomesenteric pneumatosis is indicative of advanced bowel necrosis and correlates with unfavourable outcomes , above all when associated with other ischaemic wall abnormalities . by contrast , our results conflict with those obtained in other series [ 34 , 35 ] and those reported by wiesner [ 36 ] , who states that the outcome of patients with bowel ischaemia depends on severity and extent of the initial disease and that bowel wall and portomesenteric pneumatosis have no prognostic significance , as they are not generally indicative of transmural infarction and may also be seen in partial ischaemia . i nostri risultati concordano con quanto riportato da wiesner et al . 
 [ 3 ] per quanto riguarda lispessimento parietale , con aspetto a bersaglio delle anse intestinali , che rappresenta un indice prognostico favorevole e si associa pi frequentemente ad infarto di tipo venoso , in rapporto ad una maggiore componente emorragica intraparietale e superinfezione . 
 [ 33 ] , secondo i quali la pneumatosi porto - mesenterica indicativa di necrosi intestinale avanzata con prognosi sfavorevole , soprattutto quando associata ad altre alterazioni ischemiche parietali . tuttavia , i nostri risultati si differenziano da quelli ottenuti in altre casistiche [ 34 , 35 ] e dallo stesso wiesner [ 36 ] , i quali sostengono che la prognosi dei pazienti affetti da ischemia intestinale dipende dalla gravit e dalla estensione della patologia di base e che la pneumatosi parietale intestinale e porto - mesenterica causate da ischemia generalmente non indicano un infarto transmurale e pertanto non hanno significato prognostico , in quanto tali segni possono essere osservati anche in pazienti con ischemia solo parziale . 
mdct allows for correct diagnosis , contributes to appropriate treatment planning and provides important prognostic information thanks to its ability to define the nature of the disease and the extent of anatomical damage . la tcmd fondamentale e deve essere tempestivamente eseguita in tutti i pazienti con addome acuto da sospetta ischemia intestinale . 
lindagine , infatti , consente di diagnosticare tale situazione patologica , contribuisce ad una corretta impostazione terapeutica e fornisce importanti valutazioni prognostiche , essendo in grado di definire la natura della patologia e lentit del danno anatomico . 
pinto 187 , 71100 foggia , italy , e - mail : drfperfetto@libero.it received : 20 june 2007 / accepted : 26 march 2008 / published online : 30 may 2009 springer - verlag 2009 abstract purpose . 
we sought to identify breast magnetic resonance imaging ( mri ) criteria capable of influencing the differential diagnosis between radial scars related to benign proliferative disease and those associated with breast cancer with a view to proposing breast mri as a promising and cost - effective modality to be carried out between mammography and surgical biopsy . 
sulle immagini di risonanza magnetica , sono state identificate 27 lesioni di dimensioni comprese tra 7 mm e 30 mdelle 27 lesioni mammarie , 15 mostravano caratteristiche di lesioni benigne , 8 apparivano come lesioni maligne e 4 erano classificate come lesioni sospette alla rm . 
la valutazione della risonanza magnetica della mammella dimostrava che solo la percentuale di enhancement e lanalisi della curva intesit di 758 radiol med ( 2009 ) 114 : 757770 be considered a useful diagnostic tool that can guide the choice between follow - up or surgical excision of radial scars . keywords breast magnetic resonance radial scar breast cancer segnale / tempo sono realmente utili nella diagnosi differenziale tra lesioni mammarie benigne e maligne . 
riteniamo che la rm possa sicuramente costituire un valido ausilio diagnostico nella scelta decisionale per il follow - up o per lescissione chirurgica di una radial scar mammaria . parole chiave mammella risonanza magnetica radial scar tumore mammario introduction introduzione radial scar ( rs ) is one of the most frequent impalpable benign proliferative lesions of the breast ( 32% ) [ 1 ]  . 
rs is bilateral or multicentric in 43% [ 2 , 3 ] and 67% [ 4 , 5 ] of cases , respectively . according to the royal college of pathologists working group , breast lesions < 10 mm are defined as rs , whereas those > 10 mm are complex sclerosing lesions [ 6 , 7 ]  . mammography depicts these lesions as dendritic alterations of the breast parenchyma caused by trabecular distortion , with thin , long , radiating spicules against a central radiolucent fibroelastotic core [ 8 ]  . 
additionally , it may occur in association with breast cancer ( ductal carcinoma in situ , invasive ductal and tubular carcinoma ) in 29%35% of cases [ 912 ]  . 
the radiological differential diagnosis of rs thus constitutes a challenge that often results in surgical excision to obtain final histological characterisation [ 1315 ]  . breast magnetic resonance imaging ( mri ) has recently been shown to play an important role in the differential diagnosis between benign and malignant lesions . 
on the basis of the time - intensity curve , it has a reported sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and diagnostic accuracy of 91% , 83% , 77% , 94% and 86% , respectively , whereas on the basis of lesion enhancement rate within the first minute of the examination , the values are 91% , 37% , 47% , 87% and 58% , respectively , [ 8 , 1618 ]  . 
 the purpose of our study was to identify possible breast mri criteria helping to differentiate between rs related to benign proliferative disease and those associated with breast cancer , with the aim of proposing breast mri as a promising and cost - effective modality to be carried out between mammography and surgical biopsy . 
secondo il royal college of pathologist working group le lesioni inferiori ai 10 mm vengono definite rs mentre quelle superiori ai 10 mm vengono definite lesioni sclerosanti complesse ( csl ) [ 6 , 7 ]  . 
laspetto mammografico tipico quello di unimmagine stellata , per la distorsione trabecolare , con tralci lungi e sottili convergenti verso un nucleo centrale radiotrasparente ( di natura sclero - elastosica ) [ 8 ]  . 
questa lesione , purtroppo , spesso non chiaramente distinguibile dal punto di vista morfologico da una lesione maligna ; frequente inoltre lassociazione tra rs e carcinoma ( carcinoma intraduttale in situ dcsi , carcinoma invasivo intraduttale e tubulare ) riportata in letteratura tra il 29% ed il 35% [ 912 ]  . 
pertanto la diagnosi mammografica di rs gravata da un importante problema diagnostico differenziale che spesso induce allescissione chirurgica della lesione per la sua tipizzazione istologica definitiva [ 1315 ]  . la risonanza magnetica ( rm ) della mammella , negli ultimi anni ha assunto un ruolo importante nella diagnosi differenziale tra lesioni benigne e carcinomi riportando in letteratura una sensibilit del 91% , una specificit del 83% ( vpp 77% , vpn 94% ) ed unaccuratezza diagnostica del 86% considerando come parametro discriminate solo la curva intensit - tempo ; considerando invece fattore discriminate la percentuale di contrast enhancement al primo minuto in letteratura si riporta una sensibilit del 91% , una specificit del 37 % ( vpp 47% , vpn 87% ) ed unaccuratezza diagnostica del 58% [ 8 , 1618 ]  . radiol med ( 2009 ) 114 : 757770 materials and methods from 1998 to june 2006 , we studied 20 patients with a mammographically detected focal architectural distortion . on mammography , eight lesions appeared probably benign [ breast imaging reporting and data system ( bi - rads ) category 3 ] ; eight were suspicious ( bi - rads category 4 ) and eight were highly suspicious for breast cancer ( birads category 5 )  . 
postprocessing , carried out on an easy vision philips console , allowed us to obtain subtraction images , detect lesion enhancement kinetics and evaluate the time - intensity curve within a region of interest ( roi ) ( no larger than nine pixels ) placed over the area of maximum enhancement . 
after acquisition of the native images , a 0.2 - ml / kg bolus of paramagnetic contrast medium was administered at a flow rate of 2 ml / s , followed by a 10 - cc saline flush . dynamic imaging was started after administration of contrast material . la finalit del nostro studio stata pertanto la ricerca , mediante rm , di criteri diagnostici differenziali tra rs determinate esclusivamente da fenomeni proliferativi di tipo benigno e rs associate ad una componente carcinomatosa , con lobiettivo di proporre la rm come test intermedio tra la mammografia e la biopsia chirurgica cost - effective . materiali e metodi dal 1998 al primo semestre del 2006 sono state reclutate 20 pazienti sulla base di un riscontro mammografico di disordine architetturale orientativo in 8 casi di lesione probabilmente benigna ( bi - rads 3 ) , in 8 casi per lesione sospetta ( bi - rads 4 ) ed in altri 8 casi con elevato sospetto di neoplasia ( bi - rads 5 )  . 
le lesioni sono state classificate in considerazione dellentit della distorsione architetturale , della presenza o meno di un nucleo denso centrale , delle sue dimensioni nonch della presenza di microcalcificazioni contestuali . 
tutte le 8 pazienti inquadrate nella categoria mammografica bi - rads 3 , come lesioni probabilmente benigne , hanno preferito volontariamente sottoporsi allintervento chirurgico , piuttosto che affrontare lo stress dei successivi controlli ; 3 di esse peraltro condizionate dalla familiarit materna per carcinoma mammario . 
 rm mammella tecnica desame gli esami sono stati eseguiti con apparecchio philips gyroscan da 1t , dotato di gradienti da 16 mts , con bobina dedicata , previo posizionamento di accesso venoso mediante ago - cannulla collegato ad iniettore automatico . 
il post - processing , oltre alla sottrazione delle immagini , ha previsto lo studio dellenhancement delle lesioni e la valutazione della curva intensit / tempo previo posizionamento di una roi non superiore a 9 pixel nel punto di massima intensit del segnale della lesione ; la ricostruzione tridimensionale stata ottenuta con tecnica mip ed mpr , mediante elaborazione della iii e vi fase dinamica . 
la scelta del piano assiale invece consente , a 760 diagnostic criteria all lesions were retrospectively evaluated on the basis of the morphological and dynamic criteria proposed by fischer et al . 
the enhancement rate is calculated with the following formula : enh = si postcontrast ( within the first minute ) si precontrast 100 si precontrast the enhancement rate is regarded as an important criterion for differentiating benign from malignant breast lesions . 
in particular , we considered : enhancement rate < 50% : score 0 enhancement rate between 50% and 100% : score 1 enhancement rate > 100% : score 2 [ 1924 ] radiol med ( 2009 ) 114 : 757770 nostro avviso , una migliore visualizzazione delle regioni retro - areolari e pre - pettorali che , spesso sul piano coronale , sono sedi di fenomeni artefattuali , specie nelle immagini sottratte . 
allacquisizione delle immagini in condizioni basali seguiva liniezione rapida di mdc paramagnetico in ragione di 0 , 2 ml / kg alla velocit di 2 ml / s con successiva iniezione di 10 cc di soluzione fisiologica . 
in questo gruppo abbiamo compreso le curve inizialmente classificate come ia e ib secondo il criterio di kuhl [ 22 ] ; incremento rapido dellintensit del segnale entro il primo minuto con successivo plateau ( + 10% , 10% ) : score 1 ; incremento rapido con massima intensit ( wash - in ) entro il primo minuto ed altrettanto rapido decremento del segnale ( wash - out > 10% ) : score 2 . radiol med ( 2009 ) 114 : 757770 all patients underwent excision biopsy with final pathological diagnosis . 
lesions with fischer scores between 0 and 2 were classified as benign , those with a score of 3 as probably benign , those with scores of 45 as suspicious and those with scores between 6 and 8 as malignant . results ten of the 20 patients examined had a family history of breast cancer . 
in three cases , mri revealed a double lesion where mammography had depicted one lesion only ( pathology diagnosed rs in two cases and rs with ductal epithelial hyperplasia in one )  . 
histopathological examination confirmed all of the 27 lesions detected on breast mri and revealed 16 benign proliferative lesions and three ductal hyperplasias ( in two cases associated with rs ) , one fat necrosis with inflammatory change and three invasive ductal carcinomas ( two tubular and one cribriform ) associated with rs and four ductal carcinomas in situ . 
eight lesions had a fischer score of valutazione della percentuale di impregnazione valutazione della percentuale di impregnazione della lesione entro il primo minuto dalliniezione del mdc calcolata mediante la seguente formula : enh = si post ( entro il primo minuto dopo mdc ) si pre100 si pre tale percentuale considerata un fattore discriminate nella tipizzazione delle lesioni . 
abbiamo considerato : percentuali inferiori al 50% : score 0 ; percentuali comprese tra 50% e 100% : score 1 ; percentuali superiori al 100% : score 2 [ 1924 ]  . tutte le pazienti esaminate sono state sottoposte a biopsia escissionale con esame istologico definitivo . abbiamo considerato benigne le lesioni che presentavano un fischer score compreso tra 0 e 2 ; probabilmente benigne le lesioni con uno score di 3 ; sospette quelle con uno score di 45 ed infine maligne quelle con uno score compreso tra 6 ed 8 . 
delle 24 lesioni individuate alla mammografia , la diagnosi istologica ha confermato 17 lesioni benigne e 7 maligne . table 1 correlation between mammographic features and histological examination mammography : bi - rads classification benign breast lesionsa malignant breast lesionsa bi - rads , breast imaging and reporting data system a histological diagnosis tabella 1 correlazione tra reperti mammografici e diagnosi istologica classificazione bi - rads mammografica lesioni benignea lesioni malignea bi - rads 3 bi - rads 4 bi - rads 5 total bi - rads 3 bi - rads 4 bi - rads 5 totale bi - rads , breast imaging and reporting data system a diagnosi istologica 762 radiol med ( 2009 ) 114 : 757770 fig . 
histology confirmed all 15 benign lesions seen at mri and revealed the benign nature la successiva rm ha rilevato 27 lesioni di dimensioni comprese tra 7 mm e 30 mm ; in 3 casi stata rilevata una doppia lesione a fronte della mammografia che evidenziava ununica lesione ( di esse allesame istologico sono risultate essere in 2 casi entrambe rs ed in 1 caso rs con associata iperplasia duttale )  . 
allesame istologico sono state confermate tutte le 27 lesioni rilevate con rm , di cui 16 erano lbp , 3 iperplasie duttali ( 2 associate a rs ) , 1 steatonecrosi radiol med ( 2009 ) 114 : 757770 fig . 
in 4 / 20 cases , ( 20% ) there was either no focal enhancement or enhancement that was not significantly different from the normal breast parenchyma around the roi . 
several authors have investigated the association between enhancement pattern and tissue characteristics such as microvessel density [ 25 ] in an attempt to differentiate between benign and malignant breast lesions and predict aggressiveness and prognosis of malignancies [ 2730 ]  . 
although the results have not been univocal , there is general agreement that rim type iii type ii type iii type iii type ii total tipo iii tipo ii tipo iii tipo iii tipo ii totale 3 ( 40% ) 1 ( 15% ) 1 ( 15% ) 2 ( 30% ) 1 ( 15% ) 7 / 7 ( 100% ) a malignant malignant malignant malignant malignant 3 ( 40% ) 1 ( 15% ) 1 ( 15% ) 2 ( 30% ) 1 ( 15% ) 7 / 7 ( 100% ) a maligna maligna maligna maligna maligna dendritica anche se lo stesso aspetto era ugualmente presente in 4 lesioni benigne . 
non abbiamo mai rilevato lesioni rotondeggianti od ovali a margini lisci . il pattern del contrast enhancement si mostrato omogeneo in 19 delle 20 lesioni benigne , ma anche in 3 delle 7 lesioni maligne ; il pattern centripeto ( rim enhancement ) si evidenziato in 3 delle 7 lesioni maligne ed in 1 lesione benigna . la percentuale di contrast enhancement , entro il primo minuto dalliniezione del mdc , nelle lesioni benigne confermate istologicamente stata : inferiore al 50% in 11 / 20 casi ( 55% ) ; con valore compreso tra il 50%100% in 5 / 20 casi ( 25% ) e senza enhacement di tipo focale o comunque con contrast enhancement non significativamente differente da quello del parenchima ghiandolare adiacente la regione dinteresse ( roi ) in 4 / 20 casi ( 20% )  . 
homogeneous contrast enhancement , a characteristic of benign lesions , can also be found in some malignant lesions , such as tubular carcinoma , which is sometimes associated with rs , probably as a result of its mainly fibrotic component [ 26 , 31 ]  . 
 enhancement rate and time - signal intensity curve were mx , mammografico ; rm , risonanza magnetica alesioni non rilevate allesame mammografico discussione la rm , a differenza della mammografia , fornisce importanti indicazioni non solo sulla morfologia , ma anche su alcuni aspetti bio - funzionali delle lesioni , come ad esempio la vascolarizzazione ( neoangiogenesi ) e la permeabilit capillare , che influenzano il pattern e lentit dellenhancement della lesione stessa [ 2527 ]  . 
molti autori hanno riportato in letteratura varie esperienze sulla correlazione tra modalit di enhancement e la presenza di alcune caratteristiche tissutali , come la densit microvascolare [ 25 ] , nel tentativo di differenziare le lesioni benigne da quelle maligne , e tra queste ultime di predirne il grado di malignit nonch la prognosi [ 2730 ]  . 
i risultati tuttavia non sono univoci , pur concordando che il cosiddetto rim enhancement ritenuto essere attualmente lunico aspetto pi indicativo di lesione maligna , con fattori istomorfologici e prognostici pi sfavorevoli , mentre lassenza di un significativo enhancement nel contesto di una lesione , comunque rilevabile morfologicamente , fortemente indicativo di lesione benigna [ 25 ]  . nella nostra esperienza , la morfologia ed il pattern di impregnazione di mdc non sono elementi discriminanti tra table 6 statistical evaluation histology diseased healthy total test + test total test + test totale tabella 6 valutazione statistica istologia malati sani totale found to be more helpful in that they reflected the neoangiogenesis that characterises the presence of breast cancer . our experience showed plateau and wash - out time - intensity curves in all malignant lesions , and an enhancement rate > 100% in 6 / 7 malignant lesions . 
it is well established that vascular endothelial growth factors and vessel permeability factors may be present in rs , even in the absence of neoplastic proliferation , a fact that may lead to false positive findings [ 3235 ]  . 
this may explain our single false positive case and the four breast lesions classified as suspicious at mri . therefore , if we take into account enhancement rate and time - intensity curve and we consider the four suspicious lesions as false positive results , breast mri had a sensitivity of 100% , a specificity of 75% , an accuracy of 81% , a ppv of 58% and a npv of 100% ( table 6 )  . conclusions our experience confirmed that breast mri has very high sensitivity ( 100% ) but lower specificity ( 75% ) , even in the study of rs . 
because morphological assessment , with either breast mri or conventional imaging , proved unreliable for characterising radiol med ( 2009 ) 114 : 757770 rs benigne e lesioni maligne ; tale dato era prevedibile poich si tratta di lesioni che determinano macroscopicamente unalterazione morfologica abbastanza simile . infatti , 16 lesioni benigne mostravano aspetto amorfo irregolare mentre non abbiamo mai rilevato lesioni maligne di forma rotondeggiante od ovalare , a margini lisci . 
lomogeneit o meno del contrast enhancement non discriminante nella diagnosi differenziale tra benignit e malignit . lomogeneit del contrast enhancement , caratteristica delle lesioni benigne , si ritrova infatti anche in alcune lesioni maligne , come ad esempio nel carcinoma tubulare , talvolta associato alle rs , probabilmente in ragione della sua prevalente componente fibrotica [ 26 , 31 ]  . 
la nostra esperienza ha dimostrato curve intensit / tempo del tipo plateau e wash - out in tutte le lesioni maligne , con una percentuale di enhancement > 100% in 6 delle 7 lesioni maligne . 
tuttavia in letteratura riportata nelle rs la presenza di fattori di crescita vasculo - endoteliali e di aumento della permeabilit vascolare , anche in assenza di proliferazione neoplastica , entrambe probabile causa di false positivit [ 3235 ] , come rilevato anche nella nostra esperienza con la presenza di 1 caso falso positivo e di 4 lesioni benigne con caratteristiche rm sospette . 
considerando quindi la percentuale del contrast enhancement e la curva intensit / tempo ed includendo tra i falsi positivi anche le 4 lesioni classificate come sospette alla rm , la sensibilit della metodica si rilevata essere del 100% , la specificit del 75% , laccuratezza dell81% , il vpp del 58% ed il vpn del 100% ( tabella 6 )  . conclusioni i risultati del nostro studio confermano lelevata sensibilit ( 100% ) della rm anche nello studio della radial scar mammaria , sebbene la specificit ( 75% ) non sia entusiasmante . 
tuttavia il dato di maggior interesse si rivelato il vpn ( 100% ) , basato essenzialmente sulla valutazione dellintensit del segnale nel tempo , determinata dalle modalit di impregnazione di mdc delle lesioni . 
poich lanalisi morfo - strutturale si dimostrata essere non affidabile al fine della tipizzazione di queste lesioni , sia mediante rm e maggiormente con le metodiche tradizionali , riteniamo che la rm costituisca un ausilio per il radio - senologo nella scelta radiol med ( 2009 ) 114 : 757770 rs , we believe that dynamic breast mri might be considered a useful diagnostic tool in guiding decisions towards follow - up care or surgical excision of rs . 
 decisionale per il follow - up o per lescissione chirurgica di una radial scar mammaria e quindi anche una indagine intermedia cost - effective , in considerazione della futuribile riduzione del numero delle biopsie chirurgiche . 
between april 2006 and february 2008 , 18 patients ( 13 males and five females , age range 842 years ) with blunt abdominal trauma were evaluated with computed tomography ( ct ) and ceus at the emergency department of our institution . 
as a noninvasive , versatile , easy to perform and repeatable technique with a low rate of adverse reactions , ceus is ideally suited for this purpose and allowed us to reduce the number of ct scans , especially in the follow - up of young patients . keywords contrast - enhanced ultrasound liver spleen trauma follow - up riassunto obiettivo . 
diciotto pazienti ( 13 maschi e 5 femmine ) di et compresa fra gli 8 e i 42 anni e con trauma addominale chiuso sono giunti alla nostra osservazione da aprile 2006 a febbraio 2008 . 
a tal proposito , lecografia con mdc , in virt della sua non invasivit , facilit di esecuzione , ripetibilit del mdc e assenza di documentate reazioni avverse allo stesso , la metodica ideale per la gestione del follow - up , riducendo il numero degli esami tc ai quali i pazienti , anche di giovane et , sono sistematicamente sottoposti . 
 parole chiave ecografia con mezzo di contrasto fegato milza trauma follow - up 772 introduction ultrasound ( us ) is one of the most common techniques performed in the initial evaluation of patients with abdominal trauma [ 1 ] , as it is a noninvasive , versatile and repeatable technique . 
however , it is limited by a low accuracy in detecting and determining the extent of hepatic injuries if compared with contrast - enhanced computed tomography ( ct ) , which is still considered the gold standard [ 2 ]  . 
us in abdominal trauma has been used to detect peritoneal effusion as an indirect sign of lesions of hollow or parenchymatous organs , an application known as focused assessment sonography for trauma ( fast ) among anglo - america authors [ 3 , 4 ]  . 
us can be considered extremely valid , especially in the follow - up of known hepatic or splenic lesions in patients treated conservatively . the recent introduction of contrast - enhanced us ( ceus ) based on sulphur - hexafluoride - filled microbubbles ( sonovue ) has improved the overall accuracy of us , thanks to the possibility of carrying out a real - time study of the region of interest [ 5 , 6 ]  . 
although the high diagnostic accuracy of ceus in the detection of both the presence and extension of lesions has been widely demonstrated , little is known about its value in the long - term follow - up of patients with liver and / or splenic trauma . 
the aim of our study was to assess the role of ceus in the follow - up of patients treated conservatively after minor abdominal trauma ( hepatic and / or splenic ) diagnosed in the emergency room with ceus and ct scans . materials and methods patients between april 2006 and february 2008 , we studied 18 patients ( 13 males and five females ; age range 842 years ; mean age 29 ) who arrived in our emergency room with blunt abdominal trauma . 
the remaining 11 patients ( enrolled in the study ) were haemodynamically stable and were studied with standard us , ceus and contrast - enhanced ct to exclude haemoperitoneum within 13 h after the trauma . 
lesion type and the patients clinical conditions justified an initial conservative treatment , and all patients were admitted to the intensive care unit where they underwent close clinical and laboratory monitoring . 
as radiol med ( 2009 ) 114 : 771779 introduzione lecografia per le sue caratteristiche di semplicit di esecuzione , ripetibilit e non invasivit , rappresenta la metodica di primo approccio nella valutazione dei pazienti che giungono con riferito trauma addominale [ 1 ]  . 
tuttavia ormai noto che lecografia ha una bassa accuratezza nella identificazione diretta della lesione dorgano e nella definizione dellestensione della stessa , lasciando pertanto alla tc con mdc il ruolo di tecnica di riferimento [ 2 ]  . 
lutilizzo dellecografia nei traumi addominali stato finalizzato per molto tempo , infatti , alla identificazione di versamenti peritoneali come segno indiretto di lesione dorgano cavo o parenchimatoso , ricordando pertanto la tecnica fast ( focused assessment sonography for trauma ) degli anglosassoni [ 3 , 4 ]  . 
la metodica ecografica pu essere ormai considerata come estremamente valida soprattutto nei follow - up dei pazienti con lesione traumatica a livello epatico e / o splenico ormai accertata e che sono stati sottoposti a trattamento conservativo . la recente introduzione dellecografia con mezzo di contrasto ( contrast - enhanced ultrasound , ceus ) a base di microbolle di esafluoruro di zolfo ( sonovue ) con uno studio real - time della regione dinteresse , ha fornito numerose informazioni supplementari rispetto a quelle date dalla sola ecografia di base , fornendo pertanto allecografia unaccuratezza complessiva maggiore [ 5 , 6 ]  . 
tuttavia esistono ancora poche evidenze scientifiche riguardanti laccuratezza diagnostica della ceus nel follow - up a lungo termine di pazienti con trauma epatico e / o splenico minore monitorati esclusivamente mediante ceus . 
scopo del nostro lavoro stato , pertanto , valutare il ruolo dellecografia con mdc nel follow - up di pazienti con trauma addominale minore ( splenico e / o epatico ) sottoposti a trattamento conservativo e valutati in acuto con lecocontrastografia e a tc con mdc . materiali e metodi pazienti nel periodo compreso fra aprile 2006 e febbraio 2008 sono giunti nel pronto soccorso del nostro ospedale 18 pazienti , 13 maschi e 5 femmine , di et compresa fra gli 8 e i 42 anni , et media 29 anni , che avevano riportato un trauma addominale chiuso . 
sette di questi ( 2 femmine e 5 maschi ) sono radiol med ( 2009 ) 114 : 771779 this finding was confirmed by the laboratory parameters , after 48 h of observation in the intensive care unit , the patients were moved to a surgical ward and discharged after 79 days . 
subsequently , when standard us showed a slightly inhomogeneous area at the level of the liver or spleen parenchyma with or without effusion , ceus was performed with the intravenous administration of the contrast agent sonovue ( bracco , milan , italy )  . 
this contrast agent consists of microbubbles containing an inert gas , sulphur hexafluoride , which resonates at low acoustic pressure ranges ( mechanical index < 0.2 ) , emitting a specific signal . 
by using a specific lowmechanical - index software programme ( contrast tuned imaging , cnti , esaote ) , it is possible to detect exclusively the echoes from the microbubbles and identify both the macroand microcirculation to obtain a dynamic exploration of organ perfusion [ 7 , 8 ]  . all patients were studied with a technos mpx unit equipped with cnti software , which enables selective tuning of the unit to the signal generated by the contrast microbubbles resonating in the sonicated tissue . 
we used convex - array 3.5 mhz variable - band probes ( ca 430 ) and low us beam energy with a 4050 kpa derated pressure and a mechanical index of 0.060.08. 
then , depending on the case and the size of the patients parenchymatous organs , the operator decided whether or not to use a complete volume of contrast agent , 4.8 cc , or half the dose . 
the injection of contrast material was continuous and immediately followed by an infusion of saline solution to prevent any contrast material remaining in the cannula and to flush the contrast material into the peripheral circulation . 
exploration lasted at least 56 min to estimate changes in vascularity during the different dynamic phases , taking care not to confuse areas of altered vascularity due to the trauma with the physiological inhomogeneous enhancement of the liver and spleen during the arterial phase . 
i restanti 11 pazienti allingresso si presentavano emodinamicamente stabili e pertanto venivano sottoposti in urgenza , in un arco di tempo variabile da unora a tre ore dal trauma , ad esame ecografico standard eseguito per la ricerca di emoperitoneo , ad ecografia con mdc e a tc addominale senza e con mdc e.v. 
per il tipo di lesione e per le condizioni cliniche stato deciso un iniziale trattamento conservativo ed i pazienti sono stati ricoverati in terapia intensiva per un sistematico monitoraggio clinico - laboratoristico . 
tale dato veniva anche confermato dai parametri laboratoristici per cui , dopo 48 ore di osservazione in terapia intensiva , i pazienti sono stati trasferiti nel reparto di chirurgia e successivamente dimessi in viiix giornata . il follow - up ecografico stato eseguito a 30 giorni e nei casi di persistenza di alterazioni strutturali parenchimali relative al trauma , veniva effettuato un ulteriore controllo a 90 giorni . tecnica desame tutti i pazienti sono stati sottoposti ad ecografia di base addominale per la valutazione degli organi parenchimatosi e dei recessi peritoneali addomino - pelvici . 
successivamente di fronte ad aree di fine disomogeneit strutturale a livello dei parenchimi epatico o splenico con o senza la presenza di versamento periviscerale , si procedeva alla somministrazione e.v. 
il sonovue ( bracco , milano , italia ) costituito da microbolle di un gas inerte , lesafluoruro di zolfo , dotate della capacit di risuonare a basse pressioni acustiche ( indice meccanico inferiore a 0 , 2 ) e di emettere uno specifico segnale . 
tramite un software specifico ( cnti ) a basso indice meccanico , possibile rilevare esclusivamente gli echi provenienti dalle microbolle riuscendo cos a visualizzare sia il macroche il microcircolo in esame fornendo pertanto una valutazione dinamica della perfusione dorgano [ 7 , 8 ]  . tutti i pazienti sono stati valutati con un ecografo technos ( mpx , esaote ) dotato di software contrasto - specifico contrast tuned imaging ( cnti , esaote ) che permette una sintonizzazione selettiva dellapparecchio con il segnale proveniente dalla risonanza delle microbolle del mezzo di contrasto presenti nel tessuto insonato . 
nel presente studio stata impiegata una potenza bassa del fascio us , con derated pressure di 4050 kpa cui corrisponde un indice meccanico basso di 0 , 060 , 08 . 
where standard us showed a slightly inhomogeneous area at the level of the spleen and segment viii of the liver , the administration of contrast material showed the presence of well - defined lacerated and contused foci appearing as hypoechoic streaks , bands or areas . 
the findings included mild hepatic lacerations without evidence of perihepatic effusion in three patients ; one liver laceration with perihepatic effusion showing a maximum thickness of 2 mm , more evident in morrisons pouch and at the subdiaphragmatic level in one patient ; and splenic lesions , two of which were associated with perisplenic effusion measuring 1.5 and 3 mm maximum thickness , respectively , in six patients . 
in no case was effusion observed along the paracolic gutters or in the pelvic fossa . no change was observed at follow - up performed after 12 , 24 and 48 h . 
the hepatic or splenic trauma was thus considered healed when it showed an echogenic signal homogeneous to the remaining parenchyma at standard us and regular distribution of contrast medium during the contrast - enhanced examination . discussion ceus has taken on an increasingly important role in the study of parenchymatous organs , in particular , the liver and spleen , thanks to the advent of contrast material based on sulphur - hexafluoride - filled microbubbles that permit visualisation of hypovascular lesions in a hypervascular background . 
splenic and hepatic perfusion is 150 ml / min , and this has always limited the usefulness of color doppler imaging , which is unable to evaluate hypovascular lesions in a hypervascular background [ 9 , 10 ]  . 
lesaminatore , in base alla costituzione del paziente e alla grandezza degli organi parenchimatosi da visualizzare , ha scelto se iniettare lintera dose di sonovue , pari a 4 , 8 cc , o met dose . 
liniezione di mezzo di contrasto era continua ed immediatamente seguita da una rapida infusione di soluzione fisiologica al fine di evitare che il mezzo di contrasto residuasse , seppur in misura minima , nellago - cannula e allo stesso tempo venisse vigorosamente spinto nel circolo periferico . 
subito dopo liniezione del mezzo di contrasto iniziato lo studio dinamico dei parenchimi ; losservazione durava almeno 56 minuti per valutare le modificazioni della vascolarizzazione nelle varie fasi dinamiche , tenendo presente che in fase arteriosa il fegato e ancora di pi la milza presentano fisiologicamente molte disomogeneit parenchimali da non confondere con aree di alterata vascolarizzazione indotte dal trauma . 
 importante considerare che al momento delliniezione veniva fatto partire il cronometro dellapparecchio che ci indicava le varie fasi dellesame ; sono state fatte delle registrazioni di alcuni secondi sulla lesione e sul restante parenchima in esame , che sono state archiviate e revisionate in un secondo tempo anche insieme al medico chirurgo . 
dopo circa dieci minuti il mezzo di contrasto stato eliminato per via respiratoria in modo pressoch completo ; in alcuni casi stata iniettata una ulteriore dose di sonovue ed stata eseguita una seconda valutazione ecocontrascografica . 
nessuno dei pazienti osservati ha presentato ipersensibilit precoce o ritardata al mezzo di contrasto . risultati nella nostra esperienza luso dellecografia con mdc ha evidenziato un netto miglioramento nella identificazione e nella valutazione della reale estensione della lesione dorgano sospettata o evidenziata allesame ecografico standard . 
in tutti i casi abbiamo evidenziato una buona corrispondenza tra tc e ceus per sede , numero abbiamo evidenziato : in 3 pazienti lacerazioni epatiche senza segni di versamento periepatico , in 1 paziente lacerazione visualizzati estensione pertanto lesioni . 
lecografia con mezzo di contrasto , a due ore dal trauma , mostra area lamellare ipovascolare che si estende a tutto spessore senza , tuttavia , coinvolgimento capsulare ( b )  . la tc con mezzo di contrasto conferma la sede e lestensione della lacerazione splenica ( c )  . 
il controllo a 30 giorni con ecografia con mezzo di contrasto mostra risoluzione completa della zona di lacerazione splenica ( d )  . hypervascular parenchyma , thus revealing minimal alterations of the microcirculation as well [ 11 , 12 ]  . 
 many authors have investigated the beneficial role of ceus for blunt abdominal trauma in the acute phase and demonstrated its extraordinary capabilities for detecting major hepatic , splenic and renal traumas . 
however , the usefulness of ceus in these cases is limited by the need to epatica con versamento periepatico di circa 2 mm di spessore massimo , pi evidente nello spazio del morrison e in sede sottodiaframmatica e in 6 pazienti lacerazioni spleniche di cui 2 accompagnate da versamento perisplenico rispettivamente di 1 , 5 e 3 mm di spessore massimo . 
2a - c lecografia epatica standard in paziente politraumatizzato mostra area di alterazione strutturale disomogeneamente iperecogena ( a )  . lecografia con mezzo di contrasto , a 3 ore dal trauma , documenta area caratterizzata da scarso enhancement in relazione a lesione epatica lacero - contusiva ( b )  . 
il controllo a 30 giorni con ecografia con mezzo di contrasto mostra risoluzione pressoch completa della lesione , residua piccola zona avascolare ( c )  . radiol med ( 2009 ) 114 : 771779 nei controlli a 12 , 24 e 48 ore non vi sono state modificazioni . 
pertanto abbiamo considerato risolto il trauma epatico o splenico quando la lesione traumatica mostrava ecogenicit omogenea al restante parenchima allesame standard e presentava regolare distribuzione del mezzo di contrasto durante la fase contrastografica dellesame . discussione negli ultimi anni lecocontrastografia ha acquisito una importanza via via maggiore nello studio degli organi parenchimatosi , in particolare del fegato e della milza , grazie allavvento della ceus che permette di visualizzare aree di ipovascolarizzazione in tessuti estremamente irrorati . 
il fegato e la milza infatti ricevono una frazione della gittata cardiaca pari a 150 ml di sangue ad ogni pulsazione ; tale caratteristica ha sempre limitato lutilit del color - doppler non in grado di valutare alterazioni ipovascolari in un contesto fortemente vascolarizzato [ 9 , 10 ]  . 
il sonovue al contrario permette di identificare perfettamente aree ipovascolari nel contesto di parenchimi ipervascolari svelando cos alterazioni del microcircolo anche di dimensioni estremamente limitate [ 11 , 12 ]  . molti autori hanno valutato quale sia il potenziale della ceus nella valutazione dei traumi addominali chiusi in fase acuta mettendo alla luce straordinarie capacit di individuazione di traumi epatici , splenici e renali maggiori . in questi casi tuttavia lutilit dellecocontrastografia limitata dalla necessit di effettuare una tc total body per poter valutare non solo la condizione addominale ma anche quella toracica ed encefalica nel minor tempo possibile [ 13 , 14 ]  . per quanto riguarda i traumi minori , i dati in letteratura sono altamente discordanti in quanto si passa da livelli di sensibilit pari al 90% a livelli pari al 41% . 
queste notevoli differenze nelle varie casistiche dipendono non solo dalle capacit delloperatore e dalla costituzione fisica del paziente ma anche dalle condizioni cliniche del traumatizzato che possono limitarne la collaborazione , la grandezza e la sede della lesione e leventuale assenza di versamento intraperitoneale [ 15 , 16 ]  . radiol med ( 2009 ) 114 : 771779 fig . 
3a - c contrast - enhanced ultrasound ( ceus ) performed 3 h after the trauma shows multiple confluent areas of altered enhancement at the right centrilobular level due to posttraumatic contusion in a patient with abdominal trauma ( a )  . 
3a - c lecografia epatica con mezzo di contrasto a 3 ore dal trauma in paziente con trauma addominale documenta multiple aree confluenti di alterato enhancement in sede centrolobare destra riferibili a contusioni post - traumatiche ( a )  . 
il controllo a 30 giorni con ecografia con mezzo di contrasto mostra marcata riduzione delle aree di contusione epatica ( c )  . perform whole - body ct for the purposes of neurological , thoracic and abdominal evaluation [ 13 , 14 ]  . with regard to less severe traumas , the literature data are controversial , with sensitivities of 90% and 41% both being reported . 
this remarkable variability depends not only on the operators skill and patients build but also on the patients clinical status , which may limit cooperation ; on the size and site of the lesion ; and on the possible absence of intraperitoneal effusion [ 15 , 16 ]  . during the follow - up phase , these problems are in part overcome , as the lack of urgency and knowledge of the lesion site allows for a more detailed baseline us and selection of the best acoustic window for visualising the region of interest . 
if used in these conditions , ceus achieves a detection power similar to that of ct or magnetic resonance ( mr ) imaging while allowing real - time study of lesions . in all cases in our series , on ceus , the lesions initially appeared as clearly hypoechoic streaks , bands or areas , with nella fase di follow - up una parte di queste problematiche risulta ridotta in quanto , non essendo pi in regime di urgenza e conoscendo il sito della lesione si potr tranquillamente effettuare una ecografia di base pi dettagliata al fine di utilizzare la finestra acustica migliore per visualizzare la regione di interesse e , solo successivamente , effettuare una ecografia con mdc [ 1719 ]  . 
se utilizzata in queste condizioni lecocontrastografia acquisisce un potere di identificazione assolutamente sovrapponibile a quello di una tc o di una rm con mdc offrendo inoltre lopportunit di uno studio dinamico real - time delle lesioni . nella nostra casistica infatti , in tutti i pazienti osservati , le lacerazioni sono state identificate come strie o bande o aree ipoecogene dopo iniezione e.v. 
con morfologia e dimensioni inizialmente sovrapponibili a quelle della tc e poi via via minori fino ad una pressoch completa restitutio ad integruil fatto di sostituire in maniera efficace la tc con una metodica non invasiva particolarmente importante in considerazione della giovane et dei pazienti , sia 778 radiol med ( 2009 ) 114 : 771779 similar shape and size to those seen on ct . 
the use of ceus guarantees the absence of problems arising from both ionising radiation and the nephrotoxic effects of ct contrast agents . conclusioni per la dose elevata di radiazioni ionizzanti sia per la possibilit di evitare la nefrotossicit del mezzo di contrasto iodato impiegato in tc . 
 conclusions given the high levels of sensitivity and specificity shown by ceus in the follow - up of minor traumatic liver and spleen lesions , one might consider the possibility of replacing ct with ceus in these cases . 
moreover , ceus shows a very low rate of adverse reactions and allows one to reduce the number of ct scans , which is particularly important in the follow - up of young patients . lecografia con mezzo di contrasto ha mostrato una specificit ed una sensibilit estremamente elevate nel follow - up delle lesioni traumatiche minori epatiche e spleniche tanto da poter essere completamente sostituita alla tc in questa fase diagnostica . 
pertanto lecografia con mdc grazie ad una serie di vantaggi quali la non invasivit , la facilit di esecuzione , la ripetibilit del mdc , lassenza di documentate reazioni avverse allo stesso , risulta essere la metodica ideale nel monitoraggio delle lesioni suddette , riducendo pertanto il numero degli esami tc di follow - up in pazienti peraltro di giovane et . 
simonetti department of diagnostic and molecular imaging , interventional radiology and radiation therapy , university of rome tor vergata , 81 oxford street , 00133 rome , italy correspondence to : s . 
vertebral fractures usually become evident because of pain of varying intensity that reduces the patients quality of life , producing functional limitations , depression , disability , height loss , spinal instability and kyphotic deformity associated with impaired lung capacity . 
vertebroplasty , as derived from our experience and a review of the literature data , has more than 70%90% effectiveness for short - term pain reduction and return to activity . 
the aim of this paper was to describe the state of the art of this spinal interventional radiology procedure and to examine the future directions of percutaneous vertebroplasty . keywords vertebroplasty cryoablation radiofrequency ablation spinal interventional radiology bone cement vertebral fracture riassunto la prima vertebroplastica percutanea stata condotta da un gruppo francese nel 1984 , e riportata in letteratura nel 1987 , per il trattamento di un emangioma doloroso cervicale . 
le fratture vertebrali vengono generalmente diagnosticate in relazione alla marcata algia , di vario grado , lamentata dal paziente , che determina riduzione della qualit della vita , limitazione funzionale , depressione , disabilit , riduzione in altezza , instabilit spinale e deformit cifotica associata a compromissione respiratoria . 
la vertebroplastica , in relazione a quanto derivato dalla nostra esperienza e dai dati presenti in letteratura , presenta una efficacia di oltre il 70%90% nella riduzione del dolore a breve termine con pronta ripresa delle attivit quotidiane . 
lo scopo di questo lavoro descrivere lo stato dellarte di questa procedura di radiologia interventistica spinale e di esaminare le future direzioni della vertebroplastica percutanea . parole chiave vertebroplastica crioablazione ablazione a radiofrequenza radiologia interventistica spinale cemento osseo frattura vertebrale background and state of the art background e stato dellarte a vertebral compression fracture ( vcf ) is defined as a decrease in vertebral height by 20% or more of its initial dimensions [ 1 , 2 ]  . 
reduction in vertebral body strength may result from infiltrative processes produced by benign or la frattura vertebrale compressiva ( vcf ) definita come una riduzione in altezza del soma vertebrale di almeno il 20% rispetto alle dimensioni iniziali [ 1 , 2 ]  . 
una riduzione della resistenza offerta dal corpo vertebrale pu essere radiol med ( 2009 ) 114 : 976983 malignant neoplasms or , more commonly , from bone mineral loss precipitated by osteoporosis . 
with regard to bone neoplasms , the skeletal system is the most common localisation of metastatic deposits , and in particular , the spinal column is frequently affected by metastatic dissemination [ 3 , 4 ]  . 
in particular , painful bone metastases commonly occur in advanced cancer patients , whom are difficult to manage because of pain , reduced mobility and performance status . in osteoporotic vertebral collapses , a conservative therapy is generally achieved with bed rest , bracing , analgesics and physical therapy [ 5 , 6 ]  . 
instead , current conventional treatments for patients with painful bone metastases are primarily palliative and include localised therapies ( fractionated external beam radiation therapy and surgery ) , systemic therapies ( chemotherapy , hormonal therapy , radiopharmaceuticals , and bisphosphonates ) and analgesics ( opioids and nonsteroidal anti - inflammatory drugs ) [ 3 , 4 ]  . 
however , in secondary bone tumours , few patients are surgical candidates . all painful vertebral collapses , regardless of the underlying disease , can be adequately treated through percutaneous injection of polymethylmethacrylate ( pmma ) bone cement into the vertebral body . 
first used by galibert et al . in 1984 as vertebroplasty , the procedure was successfully applied to the treatment of c2 aggressive haemangiomas [ 7 ] and subsequently used on vcf secondary to osteoporosis and osteolytic tumours [ 811 ]  . 
over the past year , much debate and controversy has surrounded the issue of the complications of vertebroplasty ( 22 papers ) and the comparison between vertebroplasty and kyphoplasty ( 21 papers ) ; many manuscripts concerned case reports ( 16 papers ) , technical aspects ( 26 papers ) , experimental tests ( 15 papers ) , cement assessments ( 19 papers ) , reviews ( 34 papers ) , preoperative magnetic resonance imaging ( mri ) evaluation ( 4 papers ) and unspecific discussion ( 41 papers )  . causata da processi infiltrativi , prodotti da neoplasie benigne o maligne , o pi comunemente , dalla perdita della componente minerale ossea dovuta allosteoporosi . losteoporosi la principale causa di vcf in europa e negli usa . 
per quanto concerne invece le neoplasie va ricordato come il sistema scheletrico sia la pi frequente sede di localizzazione metastatica ed in particolare il rachide rappresenti uno dei segmenti maggiormente colpiti [ 3 , 4 ]  . il sintomo pi frequentemente associato alle vcf rappresentato da un intenso dolore cronico altamente debilitante in grado di compromette in modo considerevole la qualit di vita dei pazienti . 
in particolare le metastasi ossee , che solitamente si presentano in pazienti oncologici in stadio avanzato , presentano una complessa gestione a causa dellintenso dolore e della severa compromissione psico - fisica che determinano nei pazienti . nei crolli vertebrali di natura osteoporotica un iniziale trattamento conservativo generalmente condotto attraverso lallettamento , corsetti ortopedici , terapia medica ( analgesici ) e riabilitativa [ 5 , 6 ]  . 
di contro nei pazienti con localizzazioni metastatiche spinali dolorose i trattamenti convenzionali hanno prevalentemente un intento palliativo ed includono : terapie localizzate ( radioterapia stereotassica e resezione chirurgica ) , terapie sistemiche ( chemioterapia , terapia ormonale , trattamento con radiofarmaci e bifosfonati ) e terapia antidolorifica ( oppioidi e fans ) [ 3 , 4 ]  . 
in alcuni casi la rt non pu essere utilizzata per insensibilit della neoplasia o per il raggiungimento della soglia di tollerabilit di tessuti precedentemente irradiati mentre la chemioterapia pu non essere indicata a causa della sua tossicit . 
dosi elevate di antidolorifici possono produrre effetti collaterali non tollerabili . la sola resezione chirurgica pu essere considerata potenzialmente curativa , tuttavia solo pochi pazienti con metastasi ossee rientrano tra i candidati a questo tipo di trattamento . tutte le vcf , indipendentemente dalla patologia sottostante , sono adeguatamente trattate attraverso liniezione percutanea di cemento osseo del tipo polimetilmetacrilato ( pmma ) allinterno del corpo vertebrale . 
nel 1984 per il trattamento di un angioma aggressivo di c2 [ 7 ] e denominata vertebroplastica , attualmente utilizzata con successo nel trattamento delle vcf secondarie ad osteoporosi o tumori osteolitici [ 811 ]  . ad oggi sono stati pubblicati 1208 lavori sulla vertebroplastica , con 224 review su questo argomento . 
solo nel 978 radiol med ( 2009 ) 114 : 976983 published results [ 1214 ] support the view that percutaneous vertebroplasty is the treatment of choice in painful vertebral fractures refractory to medical management . 
this approach involves specifically a strict assessment of patients based on accurate clinical and imaging examinations , assessment by a dedicated team of highly specialised operators and the use of high - quality imaging guidance . new technical aspects and future directions review of the literature shows that new tendencies are essentially directed towards the development of a new generation of injectable bone cements and the use of hybrid treatments obtained by combining vertebroplasty with other minimally invasive procedures [ radiofrequency ablation ( rfa ) , cryoablation , microwave ablation ( mwa ) ] [ 15 ]  . conventional pmma , with its inherent shortcomings including highly exothermic polymerisation , incorporation of toxic components in the formulation , and , critically , the exhibition of poor integration between cement and bone , has been compared with more recent alternative materials such as bisphenol - a - glycidyl dimethacrylate ( bis - gma ) , aluminium - free zinc - based glass polyalkenoate cement ( zngpc ) , bioactive and bioresorbable calcium sulphate cement ( csc ) and calcium phosphate cement ( cpc )  . recently , in the management of vertebral neoplasms , minimally invasive surgical techniques ( miss ) have been added to the therapeutic armamentarium as an alternative option to conventional treatments in order to improve the patients quality of life and duration of survival . 
to qualify for these treatments , patients must complain of unremitting spinal pain in the absence of symptomatic spinal cord or nerve root compression and refractory to conventional therapeutic options such as radiation therapy , chemotherapy , surgery and analgesics . radiofrequency ( rf ) is one of the most promising thermal ablation techniques for the treatment of nonresectable tumors , related to multifocality or poor clinical condition of the patient [ 16 ]  . 
the tip of shielded electrode needle placed in osteolytic lesions is used to concentrate the energy produced by alternating electric current operating in the range of rf ( 2001200 khz ) in order to produce local ionic agitation and subsequent frictional heat that leads to focal coagulation necrosis in living tissue [ 17 , 18 ]  . 
 the aim of performing rf heat ablation before vertebroplasty is to obtain thermal destruction of tumour tissue and thrombose the paravertebral and intravertebral venous plexus , thereby minimising procedure - related complications , before vertebral stabilisation through intrasomatic cement injection . 
nello scorso anno dibattiti e controversie sono state incentrate principalmente sulle complicanze procedurali ( 22 articoli ) , confronto tra vertebroplastica e chifoplastica ( 21 articoli ) , case report ( 16 articoli ) , aspetti tecnici ( 26 articoli ) , test sperimentali ( 15 articoli ) , valutazione dei cementi ( 19 articoli ) , review ( 34 articoli ) valutazione preoperatoria con mri ( 4 articoli ) ed argomentazione generiche ( 41 articoli )  . 
questa affermazione contempla specificatamente una ortodossa valutazione del paziente , attraverso una accurata valutazione clinico - strumentale , equipe dedicate super - specialistiche ed un imaging procedurale di elevata qualit . 
 nuovi aspetti tecnici e future direzioni le nuove tendenze sono essenzialmente rivolte , in accordo a quanto riportato in letteratura , allo sviluppo di nuove generazioni di cementi ossei iniettabili ed allutilizzo di trattamenti ibridi ottenuti combinando la vertebroplastica con altre procedure mini - invasive ( ablazione a radiofrequenza , rfa , crioablazione ed ablazione con microonde , mwa ) [ 15 ]  . il convenzionale polimetilmetacrilato con i suoi intrinseci difetti , quali la solidificazione ad elevata esotermia , la presenza di componenti tossici nella sua formulazione e soprattutto la dimostrazione di una scarsa integrazione tra cemento e tessuto osseo , stato confrontato con nuovi recenti materiali alternativi quali il bisfenol - a - glicidil dimetacrilato ( bis - gma ) , alluminio free , zinco based glass polialkenoato cemento ( zn - gpc ) , calcio - solfato cemento bioattivo e bioriassorbibile ( csc ) e calcio - fosfato cemento ( cpc )  . recentemente nella gestione delle neoplasie vertebrali , nuove tecniche mini - invasive sono state aggiunte allarmamentario terapeutico come alternativa ai trattamenti convenzionali con il fine di implementare la qualit di vita e la sopravvivenza dei pazienti . 
per essere candidato allesecuzione di queste procedure il paziente deve lamentare un dolore spinale non remittente , in assenza di compressioni midollari o radicolari sintomatiche , refrattario alle comuni terapie convenzionali , quali radioterapia , chemioterapia , chirurgia e terapia antidolorifica . la radiofrequenza ( rf ) rappresenta una delle pi promettenti tecniche di ablazione per il trattamento dei tumori non resecabili , in relazione alla multifocalit o alle radiol med ( 2009 ) 114 : 976983 immediate cell death and preliminary assessment of lesion size . 
la punta dellago - elettrodo , posta allinterno della lesione osteolitica , utilizzata per concentrare lenergia prodotta da una corrente alternata operante nel range della radiofrequenza ( 2001200 khz ) , in modo da ottenere una agitazione ionica localizzata ed un conseguente riscaldamento frizionale in grado di produrre una necrosi coagulativa tissutale [ 17 , 18 ]  . lablazione a radiofrequenza prima della vertebroplastica ha lobiettivo di ottenere la necrosi termo - indotta del tessuto tumorale e la trombosi del plesso venoso intra e paravertebrale , cos da ridurre le complicanze peri - procedurali , prima di stabilizzare il corpo vertebrale attraverso la somministrazione intraspongiosa di cemento osseo . 
il pmma viene quindi caricato allinterno di un sistema di iniezione dedicato e somministrato attraverso lago da vertebroplastica allinterno del corpo vertebrale sotto guida fluoroscopica continua [ 19 ]  . 
inoltre il congelamento , riducendo lattivit dei nocicettori e le trasmissioni sinaptiche , produce una parziale analgesia diminuendo cos il dolore intra - procedurale avvertito dal paziente . la crioablazione attualmente estesamente utilizzata con successo nel trattamento di molteplici neoplasie quali il tumore prostatico , renale , epatico , polmonare , mammario ed uterino . 
il funzionamento degli aghi da crioablazione ( 17 gauge = 1 , 47 mm ) basato sul rilascio e la rapida espansione di gas argon attraverso una porzione isolata della sonda che determina il congelamento con il raggiungimento di temperature inferiori ai 180c nellarco di pochi secondi . 
ai diversi cicli di congelamento / riscaldamento , il cui numero e durata dipende dalle caratteristiche della lesione e delle strutture circostanti , viene fatta seguire la somministrazione del pmma allinterno della lesione osteolitica somatica attraverso gli aghi da vertebroplastica . 
2 immagine tc assiale a livello di l5 documenta la presenza delliceball allinterno della lesione osteolitica vertebrale rappresentata da unarea di ridotta densit . and synaptic transmission so that patients do not experience pain during the procedure . cryoablation has been extensively used to successfully treat neoplasms in different organs , including the prostate , kidney , liver , lung , breast and uterus . 
after multiple freeze - thaw - freeze cycles performed for each lesion , the number and duration of which depend on the features of the lesion itself and the proximity of critical structures , the procedure is concluded by injecting pmma in the vertebral body osteolytic lesion through the vertebroplasty needle . 
different combined transpedicularly approaches to metastasis are aimed at freezing the entire circumference of the lesion ; 17 - gauge cryoprobes ( icerod , galil medical , yokneam , israel ; iceseed , galil medical , yokneam , israel ) are then introduced coaxially , up to the distant border of the lesion , immediately behind the vertebral cortical wall . 
the biopsy needles are then retracted slightly to obtain a better exposure of the tip of the freezing radiol med ( 2009 ) 114 : 976983 crioablazione da 17 g ( icerod o iceseed , galil medical , yokneam , israele ) sono quindi introdotti coassialmente sino al margine distale della lesione , subito al di sotto della superficie corticale vertebrale . 
sono attualmente disponibili in commercio due differenti tipi di sonde da crioablazione , licerod ( 16 mm41 mm ellissoide a 40c ) con una doppia camera per lespansione dei gas che permette di ottenere un ice - ball di maggiori dimensioni e liceseed ( 10 , 5 mm19 mm ellissoide a 40c ) con una sola camera despansione per i gas [ 20 , 21 ]  . il gas argon compresso attraversando lago da crioablazione ne determina il raffreddamento della punta e la formazione dellice - ball ( effetto joule - thomson )  . 
 intervallati da una i cicli di congelamento della durata di 10 min sono generalmente fase di riscaldamento passivo di 5 min in modo da permettere un adeguato scongelamento del tessuto lesionale circostante alla probe e permetterne la rimozione , prima della successiva somministrazione del pmma . 
le cellule che sopravvivono al primo insulto da congelamento muoiono successivamente per i danni strutturali ed ischemici subiti . dopo ogni ciclo di congelamento un controllo tc rileva la presenza dellice - ball intralesionale visualizzato come unarea di ipodensit che dalla punta dellago si diparte a coinvolgere la lesione neoplastica e parte del tessuto limitrofo , risparmiando la corticale vertebrale . 
entrambe le procedure combinate di ablazione a radiofrequenza o crioablazione e vertebroplastica sono eseguite sotto guida fluoroscopica e dopo somministrazione di anestesia locale . conclusioni attualmente la somministrazione percutanea di cemento osseo allinterno del soma vertebrale rappresenta un fig . 
the icerod ( 16 mm41 mm ellipsoid at 40c ) , with its double - chambered gas expansion , allows wider ice - ball formation in comparison with the iceseed ( 10.5 mm19 mm ellipsoid at 40c ) , which has a single gas chamber on the exposed tip [ 20 , 21 ]  . 
 generally , freezing cycles ( 10 min each ) are interspersed with a 5 - min passive thaw in order to defrost surrounding tissue and release probes for subsequent pmma injection . a double freeze / thaw cycle increases the statistical probability of intracellular ice formation and cell damage . 
after each freezing step , a ct scan reveals the presence of ice balls within the lesion , visualised as hypodense areas arising from the probe tips enveloping the greater part of the lesion and a small surrounding area , sparing the wall of the vertebral bone . during the last thaw phase , the pmma is prepared by combining the monomer and powder polymer and then delivering it into the vertebral body . 
according to published reports , new tendencies , in particular in interventional oncology , are essentially directed towards the development of hybrid treatments , obtained by combining vertebroplasty with other minimally invasive procedures . 
rf heat ablation or cryoablation and vertebroplasty have shown to be effective , simple and safe in treating vertebral collapse consequent to metastases and to provide early and long - lasting pain relief with evident increase in vertebral weight - bearing resistance . over the next few years , we will probably see the development and validation of other new procedures to be combined with vertebroplasty . approccio mini - invasivo per il trattamento delle fratture vertebrali compressive amieliche . 
in accordo con quanto riportato in letteratura le nuove tendenze soprattutto nellambito dellinterventistica oncologica mirano allo sviluppo di nuovi trattamenti ibridi ottenuti combinando la vertebroplastica ad altre procedure mini - invasive . 
la rfa o la crioablazione associate alla vertebroplastica si sono dimostrate procedure efficaci , semplici e sicure nel trattamento delle vcf conseguenti a localizzazione metastatica , in grado di garantire una repentina e duratura riduzione del dolore associata ad un significativo incremento della resistenza vertebrale . 
ct helps to detect a growing number of increasingly small lesions , but , as with chest radiography , the primary goal in the evaluation of small pulmonary nodules is to exclude malignancy . 
the role of the radiologist is therefore to help the clinician determine the most appropriate management strategy by using all available modalities [ ct , magnetic resonance ( mr ) imaging , positron emission tomography ( pet ) ] and evaluating the patients clinical history and the imaging features leading to a diagnosis of benignity or malignancy . 
imaging features include nodule size , margins , calcifications and fatty component , internal features ( cavitations , pseudocavitations , air bronchogram , halo sign ) , as well as advanced techniques for characterisation ( growth rate , contrast enhancement ) and management ( computer - aided diagnosis , bayesian analysis , neural networks )  . 
the aim of this paper is to summarise the approach to pulmonary nodules from the point of view of the radiologist , oncologist and thoracic surgeon . riassunto il ruolo della tc nella diagnostica dei noduli polmonari in continua espansione : con tale metodica , infatti , si individua un numero crescente di lesioni e di dimensioni sempre minori , ma come avveniva con le radiografie , il primo obiettivo nel valutare questi piccoli noduli escluderne la natura maligna . 
il ruolo del radiologo consiste quindi nellaiutare il clinico a scegliere la strategia diagnostica pi appropriata , adottando tutte le metodiche a sua disposizione ( tc , rm , pet ) e valutando la storia clinica e quegli aspetti radiologici che lo indirizzino verso una diagnosi di benignit o di malignit : dimensioni , margini , presenza di calcificazioni o di una componente adiposa , caratteristiche proprie del nodulo ( cavitazioni , pseudocavitazioni , broncogramma aereo , segno dellalone ) , integrati a tecniche avanzate di caratterizzazione ( tasso di crescita , contrast enhancement ) e di gestione ( diagnosi computer - assistita , analisi bayesiana , reti neurali )  . 
obiettivo di questo lavoro fare il punto della situazione sullapproccio al nodulo polmonare dal punto di vista del radiologo e delloncologo da un lato e del chirurgo toracico dallaltro . keywords pulmonary nodule computed tomography lung screening parole chiave nodulo polmonare tomografia computerizzata polmone diagnosi precoce 872 introduction the term solitary pulmonary nodule ( spn ) traditionally refers to a round or oval lesion 30 mm or less in diameter surrounded on at least two thirds of its perimeter by lung parenchyma and not associated with atelectasis or hilar adenopathy [ 1 , 2 ]  . 
this definition is predominantly based on information obtained from the standard chest radiograph . the expanding role of computed tomography ( ct ) in medical imaging has offered additional important insights , including that of focal opacity , a term encompassing solid , semisolid and nonsolid lesions of variable shape . 
 the incidence of spns , based on standard chest radiography , was believed to be approximately 150 , 000 new cases per year in the united states [ 3 ]  . 
however , the incidence has increased dramatically with the diffusion of ct equipment and especially of new - generation scanners that allow thin - slice imaging of the entire chest in a few seconds . 
 as with radiographically detected nodules , the first concern in evaluating an spn is to exclude malignancy . despite the availability of many diagnostic tests of variable invasiveness , it should be noted that diagnostic accuracy tends to decline as nodule size decreases . 
the role of the radiologist is therefore to help the clinician select the most appropriate management strategy . clinical presentation and differential diagnosis the majority of neoplastic spns are lung carcinomas , whereas metastases from extrapulmonary tumours account for approximately one third of all cancerous nodular lesions . our experience with a lung cancer screening programme has shown that approximately 97% of screen - detected pulmonary lesions are benign and likely to be related to granulomatous infectious diseases ( tuberculosis , histoplasmosis , coccidioidomycosis ) , which account for 80% of benign diseases , followed by hamartomas ( 11% of lung resections for benign pulmonary nodules ) ( unpublished personal data )  . 
the imaging features of an spn that is subsequently characterised as bronchogenic carcinoma are of a mass of variable diameter , with poorly defined or spiculated margins , commonly located in the upper lobes . 
the corona radiata sign is a common finding that may be ascribed surrounding tissues and obliteration of the small vessels and bronchioles . infiltration , oedema of to neoplastic although the presence and site of the primary tumour are generally known in the case of lung metastases from an extrapulmonary tumour , metastases may occasionally arise before the signs and symptoms of the primary tumour become manifest or many years after apparently radical surgery . 
in this case , the differential diagnosis must include radiol med ( 2009 ) 114 : 871889 introduzione con il termine nodulo polmonare solitario ( nps ) si indica tradizionalmente una lesione tondeggiante o ovalare di diametro non superiore ai 30 mm , circondata per almeno i due terzi da parenchima polmonare , in assenza di atelettasia o adenopatie ilari [ 1 , 2 ]  . 
questa definizione si basa soprattutto su informazioni ottenute dal radiogramma standard ; con la tc ( tomografia computerizzata ) , il cui ruolo nella diagnostica medica in continua espansione , sono emersi ulteriori aspetti che non si possono trascurare nel definire un nps . 
 lincidenza dei nps , calcolata sui radiogrammi standard del torace , stata stimata in passato intorno ai 150000 nuovi casi / anno negli usa [ 3 ] , ma lincidenza aumenta drasticamente considerando la diffusione sul territorio delle apparecchiature tc e soprattutto quelle di ultima generazione le quali consentono di acquisire immagini dellintero torace in pochi secondi utilizzando collimazioni sottili . cos come avveniva con le classiche radiografie , il primo obiettivo che ci si pone nel valutare un nps quello di escluderne la natura maligna . 
a tale fine sono disponibili numerosi tests diagnostici , pi o meno invasivi , ma occorre notare come la loro accuratezza diagnostica diminuisca con la riduzione delle dimensioni del nps . 
il ruolo del radiologo consiste quindi nellaiutare il clinico a scegliere la strategia diagnostica pi appropriata . clinica e diagnosi differenziale la maggior parte dei nps neoplastici costituita da carcinomi polmonari , mentre le metastasi da tumori extrapolmonari rappresentano allincirca un terzo di tutte le lesioni nodulari neoplastiche . 
dalla nostra esperienza , derivata da un programma di screening , emerso che allincirca il 97% delle lesioni polmonari riscontrate con questi programmi sono di natura benigna e le cause pi frequenti sono da ricercarsi nella patologia infettiva granulomatosa ( tubercolosi , istoplasmosi , coccidiomicosi ) , rappresentante l80% circa della patologia benigna , seguita dagli amartomi ( 11% circa delle resezioni polmonari per noduli benigni polmonari ) ( dati personali , non pubblicati )  . 
laspetto di un nps , che risulti poi essere un carcinoma broncogeno quello di una formazione , pi frequentemente localizzata a carico dei lobi superiori , di diametro variabile , a margini scarsamente definiti , o frastagliati . 
frequente il segno radiologico della corona raggiata , da attribuirsi ai fenomeni infiltrativi della neoplasia , alledema dei tessuti circostanti e a fenomeni obliterativi a carico di piccoli vasi e bronchioli . nel caso di metastasi polmonari da tumore primario extrapolmonare , in genere la presenza e la sede del tumore radiol med ( 2009 ) 114 : 871889 the possibility of a new primary bronchopulmonary malignancy or a benign lesion . metastases , which tend to occur in the subpleural regions of the lower lobes , may originate from cancers affecting any organ with venous drainage to the right heart and hence to the pulmonary vascular bed : head and neck carcinomas , renal carcinoma , testicular carcinoma , choriocarcinoma , osteosarcoma and soft - tissue sarcomas , melanoma and endocrine tumours ( thyroid and adrenal glands ) , in addition to breast and prostate cancer . 
most paediatric cancers , with the exception of neuroblastoma , tend to metastasise to the lungs . the following abnormalities may also appear as spns : pulmonary abscess ( in the initial phase of circumscribed parenchymal consolidation ) , tuberculoma , colonisation by ( especially aspergillus ) , amoebiasis , fungal hyphae echinococcosis , rheumatoid arthritis , wegeners granulomatosis , bronchogenic cyst , lung sequestration and arteriovenous fistula ( associated with rendu - oslers disease in 30%60% of cases )  . 
determining whether an spn is benign or malignant therefore requires the interpretation and integration of several clinical and radiological features by using the techniques described below . basic radiological evaluation criteria a small spn is more likely to be benign [ 46 ]  . 
similarly , lobulated contours , a sign of uneven growth of the cellular components , are often associated with malignant spns [ 11 ] , even though 25% of benign spns exhibit lobulated appearance [ 6 ]  . 
 ( 4 ) popcorn and primitivo sono note , ma occasionalmente possono manifestarsi prima che insorgano segni e sintomi della neoplasia primaria o molti anni dopo una resezione chirurgica apparentemente radicale . 
in questo caso la diagnostica differenziale deve considerare anche lipotesi di una nuova neoplasia maligna broncopolmonare primitiva o di una lesione benigna . le metastasi , la cui localizzazione preferenziale a carico dei lobi inferiori , in regione mantellare sottopleurica , possono derivare da tutti gli organi con drenaggio venoso diretto al cuore destro e quindi al letto vascolare polmonare : carcinomi della testa e del collo , del rene , del testicolo , corioncarcinomi , osteosarcomi e sarcomi delle parti molli , melanomi e tumori endocrini ( tiroide e ghiandola surrenale ) , oltre a carcinomi mammario e prostatico . anche la maggioranza dei tumori pediatrici , con leccezione del neuroblastoma , metastatizzano preferibilmente in ambito polmonare . possono apparire come nps anche lascesso polmonare ( nelliniziale fase di condensazione parenchimale circoscritta ) , il tubercoloma , la colonizzazione di cavit polmonari da parte di ife fungine ( per lo pi quelle dellaspergillo ) , lamebiasi , lechinococcosi , lartrite reumatoide , la granulomatosi di wegener , la cisti broncogena , il sequestro polmonare e la fistola artero - venosa ( nel 30%60% dei casi associata alla malattia di rendu - osler )  . 
stabilire , quindi , se un nps sia benigno o maligno richiede linterpretazione e lintegrazione di molteplici elementi clinici e radiologici con le tecniche che saranno ora esaminate . criteri radiologici elementari di valutazione dimensioni generalmente un nps di piccole dimensioni ha buone probabilit di essere benigno [ 46 ]  . 
tuttavia , le dimensioni ridotte non escludono con certezza un potenziale di malignit , poich il 15% delle lesioni maligne risultano di diametro inferiore a 10 mm [ 79 ]  . 
sebbene la maggior parte dei nps con margini lisci e regolari possa essere definita benigna , necessario ricordare che tale aspetto presente anche nel 21% delle lesioni maligne [ 10 ] ; analogamente i contorni lobulati , sinonimo di crescita non sincronizzata delle componenti cellulari , con zone a maggior tasso di crescita ed altre nelle quali la proliferazione minore , sono spesso associati alla 874 radiol med ( 2009 ) 114 : 871889 fig . 
2 nodulo completamente calcifico in sede apicale destra , visualizzato con la finestra del parenchima e del mediastino ( pregressa tbc )  . ( 5 ) punctate [ 15 ]  . 
the first four are typical of benign lesions ( in particular , the first three are typical of granulomas or infectious lesions such as histoplasmosis or tuberculosis , whereas popcorn calcifications are characteristic of hamartomas )  . 
pseudocavitation is not related to tissue necrosis but to lepidic growth , that is , to the proliferation of tumour cells around the alveolar wall , with sparing of air - filled areas . these features , when associated with air bronchogram , will lead to a diagnosis of a bronchioloalveolar adenocarcinoma [ 6 , 19 ]  . 
la cavitazione un fenomeno pi frequente in noduli di dimensioni superiori a 3 cm , ma stata osservata anche in lesioni di diametro di 7 mm [ 13 ]  . 
la pseudocavitazione deve il suo aspetto non a fenomeni di necrosi tissutale , ma alla crescita lepidica , ovvero alla proliferazione delle cellule tumorali intorno alla parete alveolare , con risparmio di aree a contenuto aereo . 
5a noduli a " vetro smerigliato " periferici , risultati essere all ' esame istologico definitivo un ' area di adenomatosi bronchiolo - alveolare e b un carcinoma bronchiolo - alveolare . haemorrhage , although it has also been seen in conjunction with other infections , vasculitis and neoplasms [ 20 ]  . nodules with ground - glass appearance are increasingly frequently detected at high - resolution ct ( hrct )  . 
recently , a close correlation was established between ct diagnoses of ground - glass spns and the histological findings of adenocarcinoma : the ground - glass component in adenocarcinomas is associated with lepidic growth or mucin production [ 19 , 22 ] , and its possible predominance over the solid component is related to prognosis . 
il segno dellalone , costituito da unarea di ground - glass che circonda una nodularit [ 1 ] , fu descritto per la prima volta da kuhlman in pazienti immunocompromessi con aspergillosi polmonare angioinvasiva precoce , correlandosi ad aree di emorragia polmonare , ma stato osservato anche in presenza di altre infezioni , vasculiti e neoplasie [ 20 ]  . 
recentemente si giunti a definire una stretta corrispondenza tra le diagnosi tc di nps a vetro smerigliato ed il corrispettivo istologico di adenocarcinoma : la componente a vetro smerigliato negli adenocarcinomi associata alla crescita lepidica o alla produzione di mucina [ 19 , 22 ] e la sua preponderanza o meno rispetto alla componente solida correlata alla prognosi . 
 [ 23 ] hanno evidenziato che la maggior presenza della componente solida nel contesto di nps ground - glass associata ad un comportamento maggiormente invasivo ( tipi c , d , e , f della classificazione noguchi , ovvero , rispettivamente , carcinomi bronchioloalveolari con presenza di proliferazione fibroblastica , carcinomi poco differenziati , tubulari e papillari , con un pattern di crescita di tipo compressivo ) [ 23 , 24 ]  . 
 [ 25 ] hanno dimostrato che la maggior presenza della componente ground - glass inversamente correlata , in adenocarcinomi periferici inferiori a 30 mm , alla probabilit di sviluppare recidive [ 25 ]  . 
il tempo di raddoppiamento ( ossia il tempo necessario affinch un nps raddoppi il suo volume ) per la maggior parte dei nps maligni si colloca tra i 30 ed i 400 giorni e corrisponde ad un aumento del diametro del nps del 26% [ 26 ]  . 
la stabilit dimensionale per un periodo superiore a due anni , ai controlli radiografici o tc , implica un tempo di radiol med ( 2009 ) 114 : 871889 component within ground - glass spns is associated with a more invasive behaviour ( noguchi type c , d , e , f ; or , respectively , bronchioloalveolar carcinoma with fibroblastic proliferation , poorly differentiated adenocarcinoma , tubular adenocarcinoma and papillary adenocarcinoma with a compressive growth pattern ) [ 23 , 24 ]  . 
for most malignant spns , doubling time ( the time required for a nodule to double in volume ) is between 30 and 400 days and corresponds to a 26% increase in diameter [ 26 ]  . 
6 evoluzione nell ' arco di 12 mesi di un nodulo ( carcinoma broncogeno )  . raddoppiamento di almeno 730 giorni ed generalmente considerata un affidabile indicatore di benignit [ 3 , 27 ] , sebbene recentemente anche questa assunzione sia stata messa in dubbio . 
infatti una rivalutazione dei dati in letteratura dimostra che il valore predittivo di benignit correlato alla stabilit dimensionale per una finestra di due anni solo del 65% [ 2830 ]  . 
per esempio un nps di 5 mm pu raddoppiare di volume in 6 mesi ( tasso di crescita indicante malignit ) , ma il diametro aumenter solamente di 1 , 25 mm , divenendo di 6 , 25 m questa minima crescita di diametro assai difficile da apprezzare , cosicch i nps maligni di piccole dimensioni possono raddoppiare di volume apparendo stabili radiologicamente , con un conseguente ritardo nella diagnosi . 
per superare questo problema stato proposto di misurare il tasso di crescita dei nps di piccole dimensioni con misurazioni seriali non tanto del diametro quanto del volume [ 31 ]  . 
a tale scopo esistono dei software che , attraverso algoritmi matematici acquisiti per mezzo di una roi applicata al nps stesso , in maniera pi o meno automatica , calcolano volume , dimensioni e massa . 
questi software volumetrici 3d hanno il limite di non permettere la corretta valutazione dei nps non solidi , mentre la loro affidabilit sembra essere buona nella valutazione dei nps parzialmente solidi , nel cui caso , essendo ad alto rischio di malignit , lo studio deve essere pi accurato e pi rapido [ 21 ]  . 
sono necessari ulteriori studi per determinare precisamente lefficacia di questi software nel calcolare lintervallo di raddoppiamento e decidere se integrarli nella pratica quotidiana , al fine di diminuire lintervallo di follow - up . contrast enhancement ( impregnazione contrastografica ) nella caratterizzazione del nps importante anche valutare il contrast - enhancement . 
alcuni studi presenti in letteratura hanno dimostrato che il flusso di sangue nei nps maligni quantitativamente e qualitativamente diverso rispetto ai nps benigni [ 32 , 33 ] ed il grado di enhancement direttamente correlato con la vascolarizzazione del nps e con la probabilit di malignit [ 34 ]  . 
con la tecnica definita come quantitative contrast - enhanced ct ( qect ) , la valutazione dellenhancement pu essere ottenuta per mezzo di scansioni sottili contigue ( 13 mm ) prima e dopo la somministrazione di mezzo di contrasto per via endovenosa con flusso di 2 ml / s ( dose totale di 420 mg / kg )  . 
le scansioni vengono acquisite ogni 30 secondi per 5 minuti e lindice di attenuazione viene misurato allinterno del nps stesso prima della somministrazione del mdc ed al raggiungimento del picco massimo di contrast - ehnancement . 
un enhancement inferiore a 15 uh fortemente indicativo di lesione benigna , mentre se 878 radiol med ( 2009 ) 114 : 871889 exhibiting faster or slower doubling times tend to be benign . 
dimensional stability for > 2 years on chest radiography or ct implies a doubling time of at least 730 days and is generally considered a reliable indicator of benignity [ 3 , 27 ]  . 
this assumption has , however , recently been challenged , with a review of the literature showing that the predictive value for benignity of 2 - year stability was only 65% [ 2830 ]  . 
software has been developed that provides semiautomated calculations of volume , size and mass by applying mathematical algorithms to data acquired in a region of interest ( roi ) placed over the spn . 
although this 3d volumetric software has limits in the evaluation of nonsolid spns , it appears to have a good level of reliability in semisolid spns , whose higher risk of malignancy warrants a more accurate and faster study [ 21 ]  . 
further research is needed to establish the true effectiveness of these software packages in calculating doubling time and decide whether they should be used in clinical practice to reduce the interval between follow - up examinations . contrast enhancement contrast enhancement is another factor to be assessed when evaluating an spn . 
some studies have shown that blood flow in malignant spns is quantitatively and qualitatively different from that in benign spns [ 32 , 33 ] and that the degree of enhancement is directly related to spn vascularity and the likelihood of malignancy [ 34 ]  . 
with quantitative contrast - enhanced ct ( qect ) , enhancement is evaluated on contiguous thin sections ( 13 mm ) acquired before and after intravenous administration of contrast material at a rate of 2 ml / s ( total dose 420 mg / kg )  . 
the scans are acquired every 30 s for 5 min , and the attenuation index is measured in the spn before contrast administration and once the maximum enhancement peak has been reached . 
per migliorare ulteriormente lattendibilit del contrast - enhancement , recentemente si provato ad operare una segmentazione automatica computerizzata dei nps ed una determinazione del valore medio di densit di tutti i voxel della lesione segmentata [ 29 ]  . 
 [ 32 ] , nel loro studio per lidentificazione precoce del cancro polmonare con tc a bassa dose , hanno utilizzato il contrast - enhancement in caso di nps inferiori a 8 mm . rmn ( risonanza magnetica nucleare ) il principio di base che consiste nel confermare o escludere la presenza di vascolarizzazione mediante esami dinamici applicabile anche alla risonanza magnetica ( rm ) [ 21 , 31 ]  . la rm dinamica offre il vantaggio potenziale di permettere lacquisizione di pi misure nel tempo e di conseguenza consente la valutazione dellandamento della curva di enhancement . 
 [ 36 ] hanno evidenziato un picco di aumento di contrasto pi alto nei nps maligni rispetto a quelli benigni e in aggiunta , valutando la pendenza della curva di enhancement ( che non pu essere derivata dalla qect utilizzando la tecnica usuale ) , hanno dimostrato che le lesioni maligne avevano una pendenza pi pronunciata rispetto alle lesioni benigne [ 36 ]  . 
questo studio , inoltre , differenziava le lesioni benigne ( ad esempio il granuloma ) dalle infezioni attive : la maggior parte delle lesioni benigne aveva un picco di aumento di contrasto molto basso , mentre le infezioni attive mostravano in media un aumento di contrasto persino maggiore rispetto ai tumori maligni , come confermato da kono et al . 
di conseguenza , il picco di aumento di contrasto e la pendenza della curva potrebbero consentire di distinguere tra lesioni benigne e tutte le altre ( lesioni maligne e infezioni attive ) , esistendo una sostanziale sovrapposizione tra lesioni maligne e infezione attiva per entrambi i parametri . 
la variazione di densit ( 94 , 6 hu ) del nodulo suggestiva per una lesione evolutiva maligna ( diagnosi post - chirurgica di carcinoma broncogeno )  . rispetto alla tc limita ancor di pi lutilizzo della rm per la valutazione dei nps di piccole dimensioni . 
pertanto , il valore della rm dinamica , rispetto alla qect , che disponibile ovunque ed a costi ridotti ( con valore predittivo negativo pari al 96% ) , deve ancora essere accertato . 
 pet ( tomografia ad emissione di positroni ) lutilit della pet , metodica in grado di fornire informazioni complementari a quella morfologica di tc e rm nella valutazione dei nps , stata oggetto di numerosi studi , molti dei quali hanno evidenziato una sensibilit ed una specificit del 90% circa [ 3840 ]  . 
tuttavia , a quanto ci risulta , non esistono studi che comparino laccuratezza della pet e della pet - tc nella valutazione del nps . esistono , inoltre , alcune potenziali limitazioni allutilizzo della pet nella pratica clinica che devono essere prese in considerazione . 
per esempio , numerosi processi infiammatori ed infettivi , come la tbc o le infezioni fungine , possono captare il fluorodesossiglucosio ( fdg ) e simulare la presenza di un nodulo maligno . 
quindi necessario conoscere in dettaglio lanamnesi patologica prossima e remota del paziente [ 41 , 44 ] e sfruttare le informazioni ottenute dalla sinergia di una metodica morfologica quale la tc con una funzionale quale la pet . 
to further improve the reliability of contrast enhancement , researchers have recently attempted to perform automated segmentation of spns and determination of the mean density value of all voxels in the segmented lesion [ 29 ]  . 
used contrast enhancement for spns < 8 mm [ 32 ]  . mri ( magnetic resonance imaging ) the primary goal of confirming or excluding lesion vascularity by means of a dynamic study also applies to magnetic resonance ( mr ) imaging [ 21 , 31 ]  . 
in agreement with the ct findings , ohno et al . observed a higher enhancement peak in malignant spns than in benign lesions and , by assessing the slope of the enhancement curve ( which cannot be derived from qect using the normal technique ) , demonstrated that malignant lesions had more pronounced slopes compared with benign lesions [ 36 ]  . the authors also distinguished between benign lesions ( e.g. granulomas ) and active infections : most benign lesions had a very low enhancement peak , whereas active infections generally presented enhancement peaks that were even higher than those of malignant tumours , a finding confirmed by kono et al . 
consequently , the enhancement peak and the slope of the enhancement curve might enable benign lesions to be differentiated from all other lesions ( malignant lesions and active infections ) , given the substantial similarity of both parameters in nonbenign lesions . 
thus , the value of dynamic mr imaging compared with qect , a widely available and inexpensive technique with 96% negative predictive value , has yet to be established . positron emission tomography ( pet ) the utility of pet , a method providing complementary information to the morphological data offered by ct and mr imaging , has been investigated in several studies , most of which have reported sensitivity and specificity values around 90% [ 3840 ]  . 
however , to our knowledge , no study has compared the accuracy of pet and pet - ct in the evaluation of spns . there are potential limitations to the use of pet in clinical practice . 
for example , numerous inflammatory and infectious processes , such as tuberculosis or fungal infections , may take up fluorodeoxyglucose ( fdg ) and mimic a malignancy ; similar findings have been observed in the presence of sarcoidosis , silicosis and other granulomatous processes [ 38 , 4143 ]  . 
differentiation thus requires a thorough knowledge of the patients recent and past medical history [ 41 , 44 ] and integration of information resulting from the combination of morphological ct imaging and functional pet imaging . 
 to distinguish malignant spns from inflammatory and maligni da processi infiammatori ed infettivi , alcuni autori sostengono lutilit di unacquisizione pet o pet - tc ad unora dalla somministrazione del fdg ( come avviene comunemente ) e di una seconda acquisizione dopo 24 ore circa [ 41 , 45 , 46 ]  . 
la ragione che sottende a questa seconda scansione che le lesioni maligne generalmente tendono a continuare ad accumulare il fdg col passare del tempo , mentre i processi infiammatori ed infettivi diminuiscono la captazione di fdg nel tempo . 
per quanto concerne le dimensioni , la maggior parte degli scanner pet in uso attualmente ha una risoluzione spaziale di circa 6 mm , a patto che la lesione sia metabolicamente molto attiva . 
tuttavia , falsi negativi possono aversi in caso di noduli primitivi o secondari con minor attivit metabolica , come nel caso del carcinoma bronchiolo - alveolare o dei carcinoidi [ 4749 ]  . 
il valore soglia di suv ( standardized up - take value ) minimo riportato in letteratura per definire come maligno un nodulo solitario di 2 , 5 [ 50 , 51 ]  . 
in realt dalle meta - analisi questa valutazione quantitativa non sembra migliorare i dati di efficacia rispetto alla sola valutazione qualitativa . futuri sviluppi : tc perfusionale , diagnosi computerassistita ( cad ) , analisi bayesiana e reti neurali la neoangiogenesi promuove la crescita del tumore assicurando il necessario nutrimento alle cellule neoplastiche e gioca un ruolo importante nella metastatizzazione , grazie alla mancanza di integrit della parete vasale [ 5254 ]  . 
i cambiamenti nel volume dei vasi , nella perfusione e nella permeabilit vascolare sono valutati mediante limaging di perfusione , che si basa sullanalisi di curve tempo - densit ( tdc ) calcolate per ogni pixel della sezione acquisita con scansioni consecutive , dopo iniezione a bolo di mezzo di contrasto iodato [ 55 ]  . 
lavvento delle tc 16 banchi ha consentito , grazie ai brevi tempi di acquisizione e alla migliore risoluzione temporale e spaziale sullasse z , di campionare i parametri fisiologici su un maggiore volume di tessuto ( 68 sezioni )  . alcuni autori hanno evidenziato come lenhancement di un tumore polmonare periferico e quello dellaorta avvengano con la stessa tempistica , a causa dellabbondante apporto vascolare derivante dalle arterie bronchiali [ 5658 ]  . 
inoltre , questo tipo di lesione mostra un abbondante interstizio ed una rete linfatica ridotta o assente , entrambi responsabili della ritenzione del mezzo di contrasto nel nodulo con caratteristico ritardato wash - out . le dimensioni del tumore nelle mappe parametriche ottenute con limaging di perfusione sono generalmente pi piccole di quelle valutabili nelle immagini originali per la presenza di processi essudativi , ma le mappe parametriche esprimono in maniera essenziale le differenze di perfusione radiol med ( 2009 ) 114 : 871889 infectious processes , some authors advocate performing a pet or pet - ct scan 1 h after administration of fdg ( as is usually the case ) and a second scan some 24 h later [ 41 , 45 , 46 ]  . 
the rationale for the second scan is the general tendency of malignant lesions to continue accumulating fdg over time , unlike inflammatory and infectious processes that show reduced fdg uptake with time . 
with regard to nodule size , most modern pet scanners have a spatial resolution of around 6 mm , provided that the lesion is metabolically active . false negative results may nonetheless occur in the event of primary or secondary nodules with lower metabolic activity , as in the case of bronchioloalveolar carcinoma and carcinoid tumours [ 4749 ]  . 
however , meta - analyses have shown that this quantitative assessment does not improve the techniques effectiveness compared with qualitative evaluation alone . future developments : perfusion ct , computer - aided diagnosis ( cad ) , bayesian analysis and neural networks neoangiogenesis promotes tumour growth by supplying neoplastic cells with the required nutrients , and it plays a major role in metastasisation as a result of the limited integrity of the vessel wall [ 5254 ]  . 
changes in vessel volume , perfusion and vascular permeability are assessed by means of perfusion imaging , which is based on analysis of time - density curves calculated for each pixel of the section acquired with consecutive scans after injection of a bolus of iodinated contrast material [ 55 ]  . 
the introduction of 16 - detector - row ct with its fast acquisition times and improved temporal and spatial resolution in the z - axis has allowed physiological parameters to be sampled over a larger volume of tissue ( six to sections )  . some authors have observed that enhancement of peripheral lung tumours and that of the aorta occur with the same timing owing to abundant vascular supply from the bronchial arteries [ 5658 ]  . 
furthermore , lung tumours show a large interstitial space and a reduced or absent lymphatic flow , both responsible for the retention of contrast material in malignant nodules and the characteristic delay in wash - out . on parametric maps obtained with perfusion imaging , tumour size is generally smaller than assessed on the original images owing to exudation processes . 
perfusion parametric maps , however , essentially express the perfusion differences between the different portions of the nodule , providing valuable guidance for ct - guided biopsies [ 55 ]  . computer - aided diagnosis ( cad ) can be understood as an integrated computerised system capable of locating an spn , acquiring information on its dimensions , calculating its volume and mass and comparing it with a database to enable differentiation of significant nodules from artefacfra le diverse parti del nodulo , fornendo una valida guida per le biopsie tc - guidate [ 55 ]  . il concetto di diagnosi computer - assistita ( cad ) pu essere tradotto come sistema informatico integrato in grado di localizzare un nps , acquisirne informazioni dimensionali , calcolarne volume e massa e paragonarlo ad un database che permette di discriminare una nodularit significativa da unimmagine costruita / fittizia . 
questa tecnica , utilizzata inizialmente come secondo lettore per lo screening mammografico , chiaramente necessita di una sorta di autoapprendimento che la porti ad una riduzione progressiva dei falsi positivi [ 5962 ]  . 
il cad pu assumere un ruolo cruciale per eliminare numerosi fattori ostacolanti una diagnosi efficace dei nps , come la variabilit interosservatore , le caratteristiche del nps , la tecnica di acquisizione e i metodi di visualizzazione [ 63 ]  . 
dato il suo potenziale nella localizzazione delle nodularit polmonari e nel successivo follow - up , con metodi di comparazione computerizzata delle dimensioni , tale tecnica permette di migliorare la sensibilit della tc , aumentando il numero di nps diagnosticati , e conseguentemente di migliorare lefficienza clinica [ 64 ]  . 
per mezzo del cad possibile analizzare larchitettura interna del nps per differenziare nps con attenuazione uniforme da altri con attenuazione non omogenea , ma lo stesso principio potrebbe essere applicato anche dopo somministrazione di mdc per la valutazione dellenhancement nei nps stessi [ 65 , 66 ]  . 
lintegrazione dei risultati di analisi cad con sistemi di database potrebbe essere utile per assistere le attivit cliniche , nonch per fornire dati indispensabili alla ricerca ( ad esempio per studiare lassociazione tra alcune caratteristiche dei nps ed il tasso di malignit )  . la probabilit di malignit di un nps pu essere determinata utilizzando lanalisi bayesiana , sistema complesso che somma tra loro segni radiologici ( margini , dimensioni , densit , ecc . ) e clinici ( et , abitudine tabagica ) , integrandoli ed assegnando un punteggio finale [ 67 ]  . 
viene valutato il likelihood ratio ( lr ) , definito come il rapporto tra il numero di lesioni maligne che presentano una determinata caratteristica ed il numero di lesioni benigne con la stessa caratteristica [ 68 ]  . 
per esempio , un lr di 5 , 54 per quanto concerne i margini spiculati significa che su 13 nps con questo aspetto , 11 risulteranno maligni e 2 benigni . inoltre , per migliorare le capacit diagnostiche della tc nella diagnosi differenziale tra nps benigni e maligni si prevede in futuro lutilizzo di reti neurali artificiali le quali , mediante lanalisi computerizzata ( cad ) delle caratteristiche morfologiche del nps , saranno in grado di esprimere una stima delle probabilit di malignit della lesione in esame [ 69 ]  . 882 radiol med ( 2009 ) 114 : 871889 tual structures . 
this technique , initially used as a second reader for mammographic screening , clearly requires a selflearning feature to progressively reduce the rate of false positive results [ 5962 ]  . 
cad may play a crucial role in eliminating some of the variables hampering the effective diagnosis of spns , such as interobserver variability , spn characteristics , acquisition technique and viewing methods [ 63 ]  . 
given the potential of cad for detecting and following up pulmonary nodules with computerised size comparison , it can improve the sensitivity of ct by increasing the number of detected spns and consequently maximising clinical efficiency [ 64 ]  . cad can analyse the internal architecture of spns to differentiate spns with homogeneous attenuation from those with heterogeneous attenuation , but the same principle could also be applied to assess enhancement of spns after contrast medium administration [ 65 , 66 ]  . 
integration of the results from cad analyses with database systems might assist in clinical work , as well as provide indispensable data for research ( for example , to study the association between certain features of spns and malignancy rates )  . the likelihood of spn malignancy can be determined using bayesian analysis , a complex system that combines imaging ( margins , size , density , etc . ) and clinical findings ( age , smoking history ) to assign a final score [ 67 ]  . 
the system calculates the likelihood ratio ( lr ) , defined as the ratio between the number of malignant and benign lesions exhibiting a certain feature [ 68 ]  . 
for example , an lr of 5.54 for spiculated margins means that out of 13 spns exhibiting this feature , 11 are malignant and two are benign . in the future , artificial neural networks will be used to improve the diagnostic capabilities of ct in differentiating between benign and malignant spns : computerised analysis of spn morphology will provide an estimation of the likelihood of malignancy [ 69 ]  . management of the spn the pulmonary oncologists opinion the management of spns remains a subject of debate . new - generation ct scanners are able to detect increasingly small spns and the numerous lung - cancer screening programmes using low - dose ct throughout the world require the development of effective diagnostic and therapeutic protocols for managing spns . 
data from one of the main low - dose ct screening programmes , in the united states , showed that none of the detected malignancies was < 5 mm [ 70 ] , and similar results were obtained in an analogous 5 - year research project conducted at our institute [ 71 ]  . these data , which await validation by studies that are still approccio al nodulo polmonare : lopinione dello pneumo - oncologo il management del nps un argomento assai dibattuto e tuttora non completamente definito in quanto le nuove apparecchiature tc consentono di riconoscere nps di dimensioni sempre pi piccole ed i numerosi programmi di screening con tc a bassa dose , applicati ormai in tutto il mondo , richiedono lo sviluppo di percorsi diagnostico - terapeutici efficaci per la gestione del nps . 
da uno dei principali programmi di screening con tc a bassa dose , condotto negli stati uniti , emerso che nessuna delle lesioni maligne diagnosticate presentava dimensioni inferiori a 5 mm [ 70 ] e risultati sovrapponibili sono stati conseguiti al termine di 5 anni di un lavoro di ricerca analogo , condotto nel nostro istituto [ 71 ]  . 
questi dati , che peraltro andranno consolidati e supportati da studi ancora in corso , suggeriscono limprobabilit che un nps di dimensioni inferiori a 5 mm possa essere maligno : al momento vanno quindi considerati inadeguati latteggiamento aggressivo o ricontrolli troppo ravvicinati . limpatto economico e limpegno di risorse necessari per gestire un numero cospicuo di soggetti che dovrebbero essere sottoposti ad ulteriori indagini ha reso necessaria lelaborazione di linee guida , applicabili al di fuori dei progetti di screening . 
tra queste , nella tabella 1 riportiamo le raccomandazioni proposte da macmahon che , basandosi sul solo criterio dimensionale , identifica percorsi differenziati in base al rischio clinico del soggetto , da seguire in caso di riscontro occasionale di nps [ 72 ]  . il singolo criterio dimensionale non tuttavia sufficiente e deve essere associato ad altri , quale quello morfologico . 
lidentificazione di caratteristiche radiologiche tipiche di un nps benigno consente di soprassedere ad ulteriori indagini ; al contrario , laspetto francamente maligno di una lesione pu indirizzare verso un atteggiamento meno conservativo ( per esempio , fnab al posto del follow - up )  . sempre da dati riportati in programmi di screening con lutilizzo della tac spirale , si evince che la presenza di nps multipli ( > 7 ) , di piccole dimensioni , raggruppati , fortemente indicativa di un processo flogistico , piuttosto che di forme maligne . 
table 1 summarises the recommendations of macmahon et al . , who identified , on the basis of nodule size alone , different management strategies for incidentally detected spns based on the subjects clinical risk [ 72 ]  . nodule size alone is , however , insufficient and should be combined with other criteria , such as morphological appearance . 
the detection of radiological features typical of soprattutto in soggetti anziani , risultando nella quasi totalit dei casi di natura post - tubercolare . il riscontro di un nps va inoltre inserito in un contesto globale , che prevede una attenta valutazione anamnestica e clinica . 
la scoperta occasionale di un nps con diametro massimo pari a 56 mm , in un soggetto di et avanzata ed in presenza di importanti co - morbilit , essendo assai ridotta la probabilit che leventuale malattia diventi sintomatica nellarco della vita futura , giustifica lastenersi da ulteriori accertamenti . 
thus , in the presence of multiple small parenchymal nodules , follow - up at 1 month after adequate antibiotic therapy is recommended , as this will facilitate differentiation between inflammation and a lung cancer manifesting as a dominant spn surrounded by small satellite nodules [ 73 , 74 ]  . lesion site is another major criterion . 
although there is a higher incidence of lung cancer in the upper lobes , subpleural spns with scar - like appearance are often seen at the apical level , especially in elderly subjects . 
an spn with a maximum diameter of 56 mm detected incidentally in an elderly person with significant comorbid conditions is unlikely to develop into a symptomatic cancer during the subjects lifetime and therefore does not require further investigation . 
by contrast , a patient without comorbid conditions and with a solid spn > 8 mm and a high risk of malignancy requires a more aggressive diagnostic approach . management of the spn the surgeons opinion the aim of spn diagnosis and management is to promptly operate on all patients with operable malignant nodules while avoiding unnecessary thoracotomy in patients with benign disease [ 75 ]  . 
in the absence of previous radiographs demonstrating the stability of an spn , three possible decision alternatives exist : ( 1 ) serial follow - up examinations , ( 2 ) further diagnostic investigations and ( 3 ) surgery for diagnostic / therapeutic purposes . 
a rational choice among these alternatives is based on the application of bayess theorem and decision analysis principles , which in turn are based on the estimation of the likelihood of malignancy of the spn [ 76 ]  . 
clinical risk factors include the patients age , smoking history , occupational exposure to known carcinogens and history of cancer . radiological signs include nodule size , margins and site . schematically , patients with a low risk of malignancy and ct findings consistent with a malignant lesion should undergo pet - ct and / or fine - needle aspiration biopsy , whereas those with a low risk of malignancy and ct findings consistent with a benign lesion may undergo serial ct scans [ 77 ]  . 
in patients with an intermediate risk of approccio al nodulo polmonare : lopinione del chirurgo lassunto alla base dellapproccio diagnostico - terapeutico al nps rimane quello di sottoporre senza indugio a intervento chirurgico i pazienti portatori di lesioni maligne resecabili , evitando al contempo di esporre ai rischi di una toracotomia non necessaria quelli portatori di lesioni benigne [ 75 ]  . 
in assenza di dati radiografici che ci consentano di affermare con certezza la natura non evolutiva di un nps , le possibili alternative a nostra disposizione comprendono : ( 1 ) lesecuzione di controlli seriati nel tempo ; ( 2 ) lesecuzione di ulteriori indagini diagnostiche ; ( 3 ) lintervento chirurgico con finalit diagnostico - terapeutiche . 
la scelta razionale fra queste alternative basata sullapplicazione dei principi del teorema di bayes e dellanalisi decisionale , che fanno a loro volta riferimento alla stima delle probabilit che il nps abbia natura maligna [ 76 ]  . 
schematicamente , nei pazienti con basso rischio di cancro e reperto tc suggestivo di lesione maligna , appare preferibile eseguire una pet / tc e / o una fna , mentre di fronte a un reperto tc suggestivo di lesione benigna consentita lesecuzione di controlli tc seriati [ 77 ]  . 
nei pazienti con rischio intermedio di cancro e reperto tc suggestivo di lesione maligna , appare indicato eseguire una fna oppure procedere demble allintervento chirurgico , mentre nei casi di lesione tc benigna pu ancora essere consentita lesecuzione di controlli periodici . 
infine , nei pazienti con rischio elevato di cancro e reperto tc di lesione maligna , in presenza di un rischio operatorio limitato ( vedi pi avanti ) , la scelta pi adeguata appare quella di procedere allintervento chirurgico . lultimo elemento indispensabile per una scelta razionale fra le possibili alternative a nostra disposizione nellapproccio al singolo paziente portatore di un nps costituito dalla valutazione del rischio operatorio cui questi sarebbe esposto nel caso di intervento chirurgico . 
 la valutazione del rischio operatorio tiene conto di numerosi fattori , fra i quali et e condizioni generali del paziente , eventuali comorbidit ( in primo luogo , cardiovascolari ) , riserva funzionale respiratoria e tipo di exeresi che si presume necessaria per la rimozione radicale della lesione . 
poich sia la stima delle probabilit di cancro che la valutazione del rischio operatorio costituiscono , al meglio , delle scienze inesatte , ai fini di una scelta radiol med ( 2009 ) 114 : 871889 malignancy and ct findings suggestive of malignancy , fineneedle aspiration ( fna ) or surgery are warranted , whereas those with benign ct results may undergo serial ct scans . finally , in patients with a high risk of cancer and a malignant ct finding , surgery is the preferred strategy provided that the patient is not at high surgical risk ( see below )  . the last important factor to be considered for a rational choice of diagnostic strategy in the individual patient with an spn is the patients surgical risk . 
evaluation of surgical risk includes several factors , such as age and general condition , possible comorbidities ( mainly cardiovascular ) , respiratory functional reserve and the type of resection necessary to radically remove the lesion . 
as estimation of the likelihood of malignancy and assessment of surgical risk are at best both inexact sciences , the patients preferences and attitude must also be taken into account to make a clinically oriented decision . 
nor should we neglect the inevitable impact of new imaging technologies on our diagnostic and therapeutic decisions [ 78 ]  . concluding remarks although the clinician and surgeon have a different initial approach to the patient with an spn , the key points in managing these patients are nonetheless shared by a multidisciplinary team [ 78 ]  . 
there are two main points : on the one hand , a watchfulwaiting approach through follow - up after 1 year in the case of small nodules , and on the other , a standard diagnostic workup jointly established by the radiologist , pulmonary oncologist and surgeon and that may be adjusted to the individual patient . 
benign features are lesion stability for at least 2 years ( with the exception of groundglass lesions , which require longer follow - up ) and the presence of fat or benign calcification patterns . on the basis of the experience acquired during a lung cancer screening programme started in 2000 , we have devised a protocol for managing patients at intermediate to high risk ( table 2 )  . 
this protocol , which we have strongly supported , has enabled us to compromise between a watchfulwaiting clinical approach and a more aggressive surgical approach . all the above considerations should be viewed in the light of new models describing the natural history of lung cancer [ 79 ]  . 
this is based on the classical model , which considers advanced lung cancer as the more - or - less rapid evolution of an early , limited and localised but at any rate curable precursor lesion . 
 n va dimenticata linfluenza che la continua evoluzione delle tecniche radiografiche necessariamente esercita sulle nostre indicazioni diagnostico - terapeutiche [ 78 ]  . commento conclusivo da quanto emerso nei due paragrafi precedenti si evince un approccio iniziale diverso tra clinico e chirurgo nellaffrontare i problemi del paziente con nodulo polmonare . 
tuttavia i punti chiave nella gestione di questi pazienti sono comunque condivisi essendo essa multidisciplinare ad opera di una equipe da tempo costituita [ 78 ]  . due sono i punti fondamentali : da una parte un atteggiamento attendistico mediante un follow - up annuale in caso di noduli di piccole dimensioni , dallaltra lutilizzo di un percorso diagnostico standardizzato condiviso da radiologo , pneumo - oncologo e chirurgo , che possa per anche adattarsi al singolo caso . 
indipendentemente da qualsiasi considerazione clinica , il primo passo che il radiologo deve effettuare consiste nel riconoscimento dei caratteri di benignit della lesione , grazie ai quali si pu evitare di proseguire nelliter diagnostico . 
tali criteri consistono nella stabilit della lesione per almeno 2 anni ( eccetto per le lesioni a vetro smerigliato ove consigliabile un pi lungo monitoraggio ) e la presenza di grasso o di calcificazioni di tipo benigno . basandoci sullesperienza acquisita grazie al progetto uno screening iniziato nel 2000 , abbiamo elaborato un nostro protocollo che utilizziamo nei pazienti con rischio intermedio o elevato ( tabella 2 )  . 
questo percorso , da noi fortemente voluto , ha consentito di fare da trait dunion tra lapproccio clinico pi di attesa e quello chirurgico pi aggressivo . tutte queste considerazioni devono essere interpretate anche alla luce dei nuovi modelli che descrivono la storia naturale del tumore polmonare [ 79 ]  . 
si evince , infatti , che il tumore polmonare identificato allo screening in stadio precoce non sia in realt precursore dello stadio avanzato , ma un lato di una stessa medaglia ( modello bipartito )  . 
should this hypothesis be confirmed , new scenarios might emerge with regard to both screening and management of pulmonary nodules . finally , by allowing detection of increasingly small nodules , modern technology has produced excellent results , and the use of neural networks and metabolic and perfusion imaging will enhance performance with regard to the characterisation of these lesions . 
guidelines will no doubt be of assistance in managing pulmonary nodules , but they should also be flexible in that , in the opinion of clinicians , surgeons and radiologists alike , each patient requires a different approach given the numerous variables affecting the choice of diagnostic and therapeutic management strategies . mata si aprirebbero nuovi scenari sia per quanto concerne lo screening sia per lapproccio al nodulo polmonare . in conclusione , possiamo affermare che la moderna tecnologia permette di ottenere grandi risultati consentendo di identificare noduli di dimensioni sempre pi piccole ed inoltre lutilizzo di reti neurali , dellimaging metabolico e perfusionale aumenteranno le performance per quanto riguarda la tipizzazione di queste lesioni . 
fugazzola1 1department of radiology , university of insubria , ospedale di circolo , varese , italy 2department of cardiology , university of insubria , ospedale di circolo , varese , italy 3department of cardiology , ospedale di circolo , varese , italy 4department of medical physics , ospedale di circolo , varese , italy correspondence to : d . 
lumia , university of insubria , ospedale di circolo , viale borri 57 , 21100 varese , italy , tel : + 39 - 0332 - 278763 , fax : + 39 - 0332 - 393535 , e - mail : domenicolumia@gmail.com received : 23 september 2008 / accepted : 12 december 2008 / published online : 1 july 2009 springer - verlag 2009 abstract purpose . 
we retrospectively examined the coronary mdct studies of 89 patients ( 73 males , 16 females , average age 62.5 years , range 3179 ) referred for suspected coronary artery disease . 
the lateral cardiac vein was visualised in 56 / 84 patients ( 67% ) and the posterior cardiac vein in 63 / 84 patients ( 75% ) , never both missing . 
 along the postero - lateral wall of the left ventricle , only one branch was present in 44 cases , two branches in 21 cases and three or more branches in 19 / 84 cases ( 22% )  . evaluation of the maximum diameter revealed that the lateral vein was dominant over the posterior vein in 20 / 40 cases . 
sono stati valutati retrospettivamente 89 pazienti consecutivi ( 73 maschi e 16 femmine , et media 62 , 5 anni , range di et 3179 ) , sottoposti ad angiotcms per sospetta patologia coronarica . 
la vena cardiaca laterale stata visualizzata in 56 / 84 casi ( 67% ) , mentre la vena cardiaca posteriore in 63 / 84 casi ( 75% ) ; in nessun caso mancavano entrambe . 
complessivamente in prossimit della parete postero - laterale del ventricolo sinistro stato possibile visualizzare un solo ramo in 44 casi , due rami in 21 casi , tre o pi rami in 19 casi . 
nei pazienti che presentavano due rami per la parete posterolaterale del ventricolo sinistro , valutando il calibro massimo della vena , la vena laterale stata considerata dominante sulla posteriore in 20 / 40 casi . 
pertanto , questa tecnica di imaging potrebbe essere proposta nel planning dei pazienti da sottoporre alla crt . parole chiave scompenso cardiaco terapia di resincronizzazione cardiaca tcms vene cardiache seno coronarico introduction introduzione since the launch of the first single - slice scanner , computed tomography ( ct ) has undergone incessant technological advances that have rapidly broadened its fields of use [ 1 , 2 ]  . the advent of multidetector - row systems has made ct study of the heart possible , despite the hearts involuntary motion and complex anatomy , which have been the main hurdles for noninvasive cardiac imaging [ 35 ]  . 
the use of cardiac synchronisation techniques and pharmacological agents for slowing the heart rate have in fact enabled the reduction of pulsatility artefacts and the attainment of submillimetre volumetric data sets of good diagnostic quality in any phase of the cardiac cycle [ 2 , 4 ]  . 
over the years , a number of non - coronary applications of cardiac ct have been developed , such as the study of cardiac masses ( thrombotic or neoplastic ) , evaluation of the left atrium and pulmonary veins , the study of myocardial function and imaging of cardiac veins [ 3 , 4 , 68 ]  . interest in the study of the coronary venous system deals with the diffusion of an innovative approach to the treatment of heart failure : cardiac resynchronisation therapy ( crt ) [ 9 , 10 ]  . 
the use of crt is based on clinical evidence that the deterioration of ventricular function in patients with heart failure is to a large extent related to the onset of an intramyocardial conduction disorder [ 11 , 12 ]  . 
crt aims to reduce symptoms and slow the progression of heart failure by resynchronising the activity of the left ventricle with those of the right ventricle and atriuin order to do so , a pacing lead for the left ventricle , in addition to leads for the right atrium and ventricle , needs to be implanted in a vein of the coronary venous system , preferably on the posterolateral wall of the left ventricle [ 913 ]  . the use of intravascular venous access offers a number of advantages over the minimally invasive thoracotomy approach [ 1214 ] because it eliminates the need for surgery and general anaesthesia in patients who already present haemodynamic instability . 
crt can nonetheless fail due to the marked anatomical variability ( number and course ) of the cardiac veins and the intrinsic difficulty in catheterising the coronary sinus [ 9 , 1214 ]  . the aim of our study was to evaluate the use of multidetector - row computed tomography ( mdct ) in assessing the sin dallintroduzione sul mercato del primo scanner a singolo strato , la tomografia computerizzata ( tc ) ha mostrato un rapido ed incessante sviluppo tecnologico che ne ha ampliato rapidamente i campi di utilizzo [ 1 , 2 ]  . 
la comparsa dei sistemi multidetettore ha consentito di estendere lo studio tc al cuore , nonostante da sempre la sua motilit involontaria e la sua complessa anatomia abbiano rappresentato i principali ostacoli allimaging non invasivo [ 35 ]  . 
lutilizzo delle tecniche di cardiosincronizzazione e limpiego della bradicardizzazione farmacologica hanno reso possibile infatti , la riduzione degli artefatti da pulsatilit , consentendo di ottenere dataset volumetrici submillimetrici di buona qualit diagnostica in qualsiasi fase del ciclo cardiaco [ 2 , 4 ]  . 
negli anni si sono sviluppate diverse applicazioni non coronariche della cardio - tc , come lo studio delle masse cardiache ( trombotiche o neoplastiche ) , la valutazione dellatrio sinistro e delle vene polmonari , lo studio della funzionalit miocardica ed infine limaging delle vene cardiache [ 3 , 4 , 68 ]  . linteresse per lo studio del sistema venoso coronarico legato al diffondersi di un approccio innovativo al trattamento dello scompenso cardiaco : la terapia di resincronizzazione cardiaca ( crt ) [ 9 , 10 ]  . 
il presupposto alluso della crt si basa sullevidenza clinica che il deterioramento della funzione ventricolare in pazienti con scompenso cardiaco in gran parte legato allinstaurarsi di disturbi della conduzione intramiocardica [ 11 , 12 ]  . 
la crt si pone quindi lobiettivo di migliorare i sintomi e rallentare la progressione dello scompenso attraverso la resincronizzazione dellattivit del ventricolo sinistro con quella del ventricolo e dellatrio destro ; per ottenere questo risultato necessario posizionare un elettrodo stimolatore per il ventricolo sinistro in aggiunta a quelli per latrio ed il ventricolo destro , in una vena del sistema venoso coronarico , preferibilmente in corrispondenza della parete postero - laterale del ventricolo sinistro [ 913 ]  . lutilizzo di un accesso endovascolare per via venosa presenta numerosi vantaggi rispetto a quello mediante mini - toracotomia [ 1214 ] , perch elimina la necessit di ricorrere ad un intervento chirurgico , con limpiego dellanestesia generale , in pazienti che gi presentano un radiol med ( 2009 ) 114 : 837851 anatomical variants of the coronary venous system in order to identify the most appropriate veins for placement of the pacing lead required for crt . materials and methods we retrospectively evaluated 89 consecutive patients ( 73 males and 16 females , mean age 62.5 years , range 3179 years ) who underwent mdct angiography for suspected coronary artery disease ( cad ) from september 2007 to may 2008 ( table 1 )  . 
patients with a heart rate > 70 bpm received a 100 mg oral dose of metoprolol 6090 min before the examination in addition to 0.6 mg of nitroglycerine administered sublingually 5 min prior to the beginning of the scan . 
exclusion criteria included a heart rate > 70 bpm even after pharmacological treatment or in patients with contraindications to beta - blocker administration , an inability to maintain the breath - hold during the scan , kidney failure ( creatinine > 2 mg / dl ) , heart and / or respiratory failure , refractory arrhythmia , pregnancy or a known hypersensitivity to iodinated contrast agents . all examinations were performed with a 64 - detectorrow multislice scanner ( aquilion 64 , toshiba medical systems corporation , otawara , japan ) with retrospective cardiac gating . 
among selected , two were excluded because of inadequate electrocardiography ( ecg ) gating due to an increase in the heart rate after contrast agent administration , one because of the patients precario equilibrio emodinamico . 
la crt comunque pu risultare talvolta inefficace a causa dellampia variabilit anatomica ( numero e decorso ) delle vene cardiache e delle difficolt intrinseche nelle procedure di cateterizzazione del seno coronarico [ 9 , 1214 ]  . scopo del nostro lavoro valutare lutilit della tomografia computerizzata multistrato ( tcms ) nellidentificazione delle varianti anatomiche del sistema venoso coronarico al fine di riconoscere quelle ritenute pi idonee per limpianto dellelettrocatetere necessario per la crt . materiali e metodi sono stati valutati retrospettivamente 89 pazienti consecutivi ( 73 maschi e 16 femmine , et media 62 , 5 anni , range di et 3179 ) , sottoposti ad angio - tcms per sospetta patologia coronarica ( cad ) nel periodo compreso tra settembre 2007 e maggio 2008 ( tabella 1 )  . 
nei pazienti con frequenza cardiaca > 70 bpm stata praticata una bradicardizzazione farmacologica con 100 mg di metoprololo per os , 6090 min prima dellesecuzione dellesame , in aggiunta a 0 , 6 mg di nitroglicerina per via sublinguale somministrata 5 minuti prima dellinizio della scansione . 
i criteri di esclusione dallo studio comprendevano una frequenza cardiaca superiore ai 70 bpm anche dopo trattamento farmacologico o in concomitanza di controindicazioni alla somministrazione di beta - bloccanti , lincapacit di mantenere unadeguata apnea inspiratoria durante la scansione , la presenza di insufficienza renale ( creatinina > 2 mg / dl ) , insufficienza cardiaca e / o respiratoria , aritmie cardiache non controllate dalla terapia , stato di gravidanza , nota ipersensibilit ai mezzi di contrasto iodati . tutti gli esami sono stati eseguiti con apparecchiatura multi - strato a 64 file di detettori ( aquilion 64 , toshiba medical systems corporation , otawara , giappone ) , mediante limpiego di sincronizzazione cardiaca con tecnica retrospettiva . 
lenhancement vascolare stato ottenuto mediante linfusione di un bolo di 90100 ml di mezzo di contrasto ( mdc ) iodato non ionico ( iomeprolo , iomeron 400 mgi / ml , bracco , milano , italia ) con velocit di flusso di 45 ml / s , seguito da bolo di 40 ml di soluzione fisiologica a 45 ml / s , utilizzando unagocannula da 18 gauge inserita in una vena antecubitale del braccio ed un iniettore automatico a doppia testa ( stellant d , medrad , pa , usa )  . 
the lateral cardiac vein ( lcv ) was visualised in 56 / 84 cases ( 67% ) and the posterior cardiac vein ( pcv ) in 63 / 84 cases ( 75% ) ; failure to visualise both veins occurred in no cases . 
the overall vein visualization along the postero - lateral wall of the left ventricle resulted as follows : one branch only in 44 / 84 cases ( 52% ) , two branches in 21 / 84 cases ( 25% ) and three or more branches in 19 / 84 cases ( 22% )  . 
near the lm bifurcation , it opens into the gcv , which lies in the left atrioventricular groove close to the left circumflex artery and in turn opens into the cs . 
 cs , coronary sinus ; gcv , great cardiac vein ; aiv , anterior interventricular vein ; mcv , middle cardiac vein ; lcv , lateral ( also marginal ) cardiac vein ; pcv , posterior cardiac vein ; scv , small cardiac vein ; lm , left main coronary artery ; d1 , first diagonal ; mo , obtuse marginal . 
 cs , seno coronario ; gcv , grande vena cardiaca ; aiv , vena inter - ventricolare anteriore ; mcv , vena cardiaca media ; lcv , vena cardiaca laterale ; pcv , vena cardiaca posteriore ; scv , piccola vena cardiaca ; lm , tronco comune ; d1 , primo diagonale ; mo , marginale ottuso . the distance between the postero - lateral veins and the cs was 62.5132.5 mm for the pcv and 58.6632.0 mm for the lcv . 
the mean diameters of the other tributaries of the cs were as follows : aiv 3.2 mm , mcv 4.6 mm , lcv 2.6 mm , pcv 3.4 mm and scv 2.2 mm ( table 3 )  . therefore , in 79 of the 84 patients ( 94% ) who underwent mdct coronary angiography for suspected cad , at least one venous branch with good diameter and regular course was identified along the postero - lateral wall of the left ventricle . 
in 5 / 84 patients ( 6% ) , the postero - lateral branch had a short course ( < 12 mm )  . una buona visualizzazione del circolo venoso coronarico . 
la vena cardiaca laterale ( lcv ) stata visualizzata in 56 / 84 casi ( 67% ) mentre la vena cardiaca posteriore ( pcv ) in 63 / 84 casi ( 75% ) ; in nessun caso mancavano entrambe . 
complessivamente in prossimit della parete postero - laterale del ventricolo sinistro stato possibile visualizzare un solo ramo in 44 / 84 casi ( 52% ) , due rami in 21 / 84 casi ( 25% ) , tre o pi rami in 19 / 84 casi ( 22% ) ; nei pazienti che presentavano due rami per la parete posterolaterale del ventricolo sinistro , valutando il calibro massimo della vena , la lcv stata considerata dominante sulla vena posteriore in 20 / 40 casi . 
2 multiplanar reconstruction ( mpr ) of the coronary venous systethe image displays the coronary sinus ( cs ) , the great cardiac vein ( gcv ) , the anterior interventricular vein ( aiv ) and the middle cardiac vein ( mcv )  . 
la figura mostra il seno coronarico ( cs ) , la grande vena cardiaca ( gcv ) , la vena interventricolare anteriore ( aiv ) e la vena cardiaca media ( mcv )  . 
cs , seno coronario ; gcv , grande vena cardiaca ; aiv , vena interventricolare anteriore ; mcv , vena cardiaca media ; lcv , vena cardiaca laterale ; pcv , vena cardiaca posteriore . 844 radiol med ( 2009 ) 114 : 837851 fig . 
angolo di confluenza inferiore a 90 , con possibilit di un facile cateterismo ( a ) ; angolo di confluenza superiore a 90 , che potrebbe comportare delle difficolt durante limpianto di elettrodo stimolatore per crt ( b )  . discussion heart failure is one of the major health problems of industrialised nations , partly as a result of the ageing population . pharmacological therapy alone is unable to produce la distanza delle vene della parete postero - laterale dal cs risultata 62 , 5132 , 5 cm per la pcv e di 58 , 6632 cm per la lcv . 
langolo di raccordo dei rami venosi della parete postero - laterale apparso inferiore a 90 in 59 pazienti ( 93 , 7% ) in corrispondenza della pcv e in 53 pazienti ( 94 , 6% ) in corrispondenza della lcv , superiore a 90 in 4 pazienti ( 6 , 3% ) in corrispondenza della pcv e in 3 pazienti ( 5 , 4% ) in corrispondenza della lcv . 
quindi in 79 degli 84 pazienti ( 94% ) sottoposti ad angio - tcms coronarica per sospetta cad , stato possibile visualizzare almeno un ramo venoso di buon calibro e decorso regolare in corrispondenza della parete posterolaterale del ventricolo sinistro ; in 5 / 84 pazienti ( 6% ) il ramo venoso postero - laterale visualizzato presentava breve decorso ( < 12 mm )  . discussione lo scompenso cardiaco costituisce uno dei maggiori problemi sanitari dei paesi industrializzati , anche in relazione al progressivo aumento dellet anagrafica della popolazione . 
la sola terapia farmacologica non sempre consente di raggiungere risultati soddisfacenti , mentre lassociazione di pi farmaci pu risultare mal tollerata dai pazienti pi anziani ; per tale motivo sono state sviluppate negli anni , terapie alternative [ 11 , 14 ]  . la crt rappresenta un approccio innovativo al paziente affetto da insufficienza cardiaca cronica [ 9 , 10 , 12 ]  . 
la crt agendo sulla meccanica cardiaca mediante limpianto di un pacemaker biventricolare , migliora lo stato clinico , la qualit della vita e la prognosi dei pazienti affetti da insufficienza cardiaca cronica avanzata associata ad incremento della durata del qrs , ripristinando una corretta sincronizzazione dellattivit di atri e ventricoli . 
 per il posizionamento di tali elettrodi , un approccio endovascolare per via venosa da preferire a quello chirurgico mediante mini - toracotomia , per gli ovvi vantaggi derivanti dallevitare ad un paziente in precarie condizioni cardiocircolatorie sia lanestesia generale che il trauma chirurgico . 
this has led to the development of alternative treatments [ 11 , 14 ]  . crt is an innovative approach to the patient affected by chronic heart failure [ 9 , 10 , 12 ]  . 
the technique involves implantation of three pacing leads to stimulate the right atrium , the right ventricle and the left ventricle [ 9 , 1518 ]  . for lead implantation , an intravascular approach with venous access is preferable to minimally invasive thoracic surgery because of the obvious advantage of sparing general anaesthesia and surgical trauma for patients with unstable haemodynamic status . 
the pacing leads destined for the right heart chambers can be positioned through a central venous catheter , generally with access from the left subclavian vestimulation of the left ventricle is performed with a usually bipolar pacing lead specially developed for placement in a cardiac vein , which is reached through catheterisation of the cs . the cs is a large vein that runs across the diaphragmatic sviluppato per limpianto in una vena cardiaca , che viene raggiunta mediante cateterizzazione del cs . 
il cs riceve inferiormente la pcv , che origina dalla parete posteriore del ventricolo stesso , e superiormente la vena obliqua dellatrio sinistro ( o di marshall ) , la quale discende medialmente dalla parete posteriore di tale atrio [ 1517 ]  . 
a complete study requires at least two fluoroscopic views in order to clearly visualise the anatomy , the angulation and the course of each vein [ 16 ]  . the limitations of this technique include invasiveness , examination duration , a not always optimal depiction of the cardiac veins , an inability to visualise both the vessels and the cardiac wall at the same time , a need for large amounts of contrast agent ( in some cases up to 500 ml per examination ) and the impossibility to selectively catheterise the cs and its tributaries in 4%5% of patients [ 10 , 16 , 2022 ]  . in recent years , mdct has been taking on an increasingly important role as a noninvasive modality for studying the cardiac veins , thanks to its high spatial and temporal resolution , cardiac gating and sophisticated image postprocessing and reconstruction techniques , which are able to produce detailed visualisation of the cardiac venous system [ 2027 ]  . trans - venoso ed impianto dellelettrocatetere in corrispondenza della regione mediale o basale della parete libera del ventricolo di sinistra , in una vena laterale o postero - laterale , zona del ventricolo sinistro che risulta essere quella maggiormente colpita dal processo di desincronizzazione [ 13 ]  . non tutti i pazienti risultano candidabili alla crt , a causa dellassenza di una vena cardiaca di calibro o lunghezza adeguata ad alloggiare lelettrocatetere o per la presenza di ostacoli alla procedura di cateterizzazione . 
la possibilit di ottenere una visualizzazione dettagliata del sistema venoso cardiaco risulta perci preziosa nella pianificazione della procedura di impianto dei pacemaker biventricolari [ 19 ]  . langiografia venosa retrograda con pallone occludente , attualmente rappresenta la metodica convenzionale di studio delle vene coronariche [ 9 , 20 ] : viene ottenuta mediante iniezione di mdc durante cateterizzazione selettiva del seno coronarico e delle vene tributarie . 
i limiti di questa metodica sono da ricercare nellinvasivit della procedura , nella durata dellesame , nella visualizzazione non sempre ottimale delle vene cardiache , nellimpossibilit di visualizzare contemporaneamente i vasi e la parete cardiaca , nella necessit di impiegare grandi quantit di mdc ( in alcuni casi fino a 500 ml per esame ) ed infine nellimpossibilit , riscontrata nel 4%5% dei pazienti , di procedere al cateterismo selettivo del seno coronarico e delle vene tributarie [ 10 , 16 , 2022 ]  . in questi ultimi anni la tcms , grazie allelevata risoluzione spaziale e temporale , alle tecniche di gating cardiaco , alle sofisticate capacit di processazione e ricostruzione delle immagini , che consentono di ottenere una visualizzazione dettagliata dellanatomia del sistema venoso coronarico , si sta proponendo come metodica non invasiva per lo studio delle vene cardiache [ 2027 ]  . 
5a - f anatomia normale e varianti del sistema venoso del seno coronarico ( tcms ricostruzioni 3dvr ) : anatomia normale ( a ) , sc bifido ( b ) , sc high riding ( c ) , sc varicoide ( d ) , sc filiforme ( e ) , sc windsock o ectasico ( f )  . 
the presence of anatomical conditions unsuitable for correct placement of the bipolar lead needs to be ruled out , such as accessory valve leaflets ( thebesian or vieussens ) in the cs , certain anatomical configurations of the cs ( bifid , high riding , varicoid , filiform , windsock or ectatic , diverticular , atresic ) ( fig . 5af ) , a left circumflex artery crossing the gcv and causing external compression , and unfavourable angles between the gcv non favorevoli ( 90 ) ad unagevole cateterizzazione . 
 lintento del nostro lavoro non quello di analizzare laccuratezza diagnostica della tcms nei confronti della venografia , ma di far risaltare i vantaggi che possono derivare dallutilizzo routinario della tcms nella valutazione del sistema venoso coronarico . 
in letteratura sono riportate percentuali di visualizzazione dei diversi segmenti venosi coronarici , variabili in relazione al calibro dei vasi considerati : il cs stato visualizzato nel 98% dei casi , la gcv nel 99% dei casi , la aiv nel 99% dei casi , la mcv nel 98% dei casi , la pvc nel 89% dei casi , la lcv nel 73% dei casi , la pvc nel 14% dei casi . 
il diametro medio del cs 848 radiol med ( 2009 ) 114 : 837851 postero - lateral veins and the gcv ( 90 )  . the aim of our study was not to analyse the diagnostic accuracy of mdct compared with venography but , rather , to highlight the advantages of the routine use of mdct in the evaluation of the coronary venous syste the literature reports detection rates of the various venous segments that vary in relation to the diameter of the vessels considered . 
possible explanations for this discrepancy include nonoptimal timing of contrast enhancement , much faster scan times with respect to the earlier multislice scanners ( 48and 16 slices ) and interobserver variability in the linear measurements of vessel diameter . even though postprocessing of new - generation angiographic images makes possible the creation of 3d volume rendering ( vr ) reconstructions immediately after retrograde venography [ 28 ] and linear measurements on 2d images , the vr and mpr images created from mdct acquisitions with isotropic voxels may provide additional advantages . 
in addition to clearly depicting the course of the vessel in relation to the left ventricular free wall and the left circumflex artery [ 28 ] , these reconstructions offer the possibility of evaluating the anatomy of the mitral valve and enable precise measurement of the diameter , or better of the section , of the cs and its branches . 
 the presence of reduced diameters , short vessels or sharp vascular angles can in fact make difficult or even impossible the insertion of the bipolar pacing [ 10 , 16 , 2123 ]  . normally the anatomy of the cardiac veins is not optimally visualised with the contrast - enhancement protocol used for the coronary arteries , with the exception of the juxta - atrial portions ( cs and mcv )  . 
this occurs because the veins situated more cranially , when the acquisitions are performed in the cranio - caudal direction , show valid contrast enhancement towards the end of the scan [ 1 ]  . undoubtedly , the visibility of the cardiac veins on the postero - lateral wall of the left ventricle could be improved by performing the scan in the caudo - cranial direction or by using an additional delay of 1215 s after reaching the enhancement threshold in the ascending aorta ( baseline hu + 100 hu ) [ 1 , 2 , 17 ]  . 
althrough our experience is still limited , this bolus - tracking method , which does not appear to be reported in the literature , risultato 13 , 2 mm , quello della gcv 6 , 8 m i diametri medi delle altre vene tributarie del seno coronarico sono risultati rispettivamente : aiv 3 , 9 mm , mcv 5 , 2 mm , pvc 3 , 9 mm e lcv 3 , 7 mm [ 2027 ]  . 
i dati riguardanti la percentuale di visualizzazione delle vene cardiache riportati in letteratura sono in buona parte sovrapponibili ai dati estrapolati dalla nostra esperienza fatta eccezione per la visualizzazione della lcv ( 73% vs 67% letteratura vs nostra casistica ) ed in maniera ancora pi significativa della pvc ( 89% vs 75% )  . 
possibile che il motivo di tali discrepanze risieda in un timing contrastografico non ottimizzato , in tempi di scansione estremamente pi veloci rispetto ai primi scanner multi - slice ( 4 - 8 - 16 strati ) ed infine nella variabilit inter - osservatore delle misure lineari del diametro vasale . 
 nonostante le implementazioni degli angiografi di nuova generazione permettano di creare in post - processing immagini 3d volume rendering ( vr ) subito dopo lesecuzione della venografia retrograda [ 28 ] e di eseguire misurazioni lineari su immagini bidimensionali , le ricostruzioni vr e mpr ricavate da acquisizioni tcms con voxel isotropico , possono fornire ulteriori vantaggi . 
queste ricostruzioni , oltre che mettere bene in evidenza il decorso dei vasi in relazione alla parete libera del ventricolo di sinistra e allarteria circonflessa [ 28 ] offrono la possibilit di valutare lanatomia dellanello mitralico e consentono di misurare in maniera precisa il diametro , o ancor meglio la sezione , del seno coronarico e dei suoi rami ; la presenza di calibri ridotti , di vasi a breve decorso o di brusche angolazioni vasali , possono infatti rendere difficoltoso , o addirittura impossibile , linserimento dellelettrocatetere bipolare durante la procedura di crt [ 10 , 16 , 2123 ]  . 
 normalmente lanatomia delle vene cardiache non viene visualizzata in maniera ottimale applicando il protocollo di studio contrastografico utilizzato per le arterie coronarie , ad eccezione delle porzioni iuxta - atriali ( seno coronarico e vena cardiaca media )  . 
questo avviene perch le vene situate pi cranialmente , quando lacquisizione viene effettuata in senso cranio - caudale , presentano un valido contrast enhancement verso il termine della scansione [ 1 ]  . 
 sicuramente effettuare lacquisizione della scansione in direzione caudo - craniale o lutilizzo di un ritardo aggiuntivo di 1215 secondi dal raggiungimento della soglia di attenuazione in aorta ascendente [ hu basale + 100 hu ] , consente di migliorare la visibilit delle vene cardiache lungo la parete postero - laterale del ventricolo sinistro [ 1 , 2 , 17 ]  . 
un ulteriore accorgimento , attualmente oggetto di studio presso il nostro centro , lutilizzo contemporaneo di due regioni di interesse ( roi ) in corrispondenza del seno coronarico e dellaorta discendente ; questa modalit di bolus tracking , che non ci risulta essere mai stata descritta , nella nostra casistica seppure ancor limitata , permetterebbe di ridurre al minimo la criticit del timing contrastografico del sistema venoso coronarico , specie in pazienti che a causa della radiol med ( 2009 ) 114 : 837851 could minimise the problem of contrast enhancement timing of the coronary venous system , particularly in patients presenting a severely depressed ejection fraction ( < 35% , nyha 34 ) due to desynchronicastion of the left ventricle . the limitations of our study include the use of a nonoptimal protocol for cardiac veins ( retrospective evaluation in patients with suspected cad ) , clinical heterogeneity of patients undergoing mdct with respect to those referred for crt and absence of a direct assessment of the effective clinical advantages of the use of mdct ( reduced procedure times , fewer unsuccessful attempts and lower overall dose delivered to the patient )  . finally , it should be noted that the study of the coronary veins with ecg - gated mdct angiography in the planning of crt cannot be correctly performed in the presence of a high heart rate ( > 70 bpm ) , frank arrhythmias and / or inability to maintain the breath - hold for an adequate duration ( around 10 s )  . 
in fact patients with severe heart failure could not tolerate supine position or present an absolute contraindication to pharmacological control of the heart rate [ 1 , 2 ]  . increased exposure to ionising radiation of the patient referred for crt should not be overlooked , as in addition to undergoing venography , the patient needs to undergo an mdct examination of the coronary venous systems for crt planning ( effective dose varying from 14 to 24 msv ) [ 1 , 16 ]  . 
of course the use and further development of dose modulation systems , such as ecg pulsing , to prospectively adjust the tube current intensity in order to deliver the maximum current only during the midto end - diastolic window will help reduce the dose delivered during this examination ( 1014 msv ) [ 2932 ]  . desincronizzazione del ventricolo sinistro presentano una frazione di eiezione gravemente despressa < 35% ( nyha 3 - 4 )  . 
 non inoltre possibile tralasciare lincremento di esposizione alle radiazioni ionizzanti del paziente candidato a crt , che oltre a doversi sottoporre a venografia , debba eseguire una tcms del sistema venoso coronarico durante il planning ( dose efficace variabile tra 1424 msv ) [ 1 , 16 ]  . certo che lutilizzo e lulteriore sviluppo di sistemi di modulazione , come lecg - pulsing , in grado di regolare lintensit di corrente del tubo radiogeno in modalit prospettica , sfruttando la massima corrente solo in corrispondenza della finestra temporale mesoe tele - diastolica , consentiranno di ridurre la dose erogata durante lesame ( 1014 msv ) [ 2932 ]  . conclusions introduction of the mdct study of cardiac veins as a preliminary examination in patients referred for crt may reduce procedure times , the number of unsuccessful procedures , the amount of contrast agent used and the overall dose delivered to the patient and operators during the venographic study . 
the use of a dedicated contrast enhancement protocol could further improve the diagnostic confidence of mdct , which already today as our study shows provides good visualisation of the coronary veins and can accurately define anatomical variants , as well as the course and angulation of the vessel in relation to the free wall of the left ventricle . 
 conclusioni lintroduzione dello studio delle vene cardiache mediante tcms come indagine preliminare nei pazienti da sottoporre a crt , potrebbe consentire di ridurre i tempi della procedura , il numero degli insuccessi , la quantit di mdc impiegata e la dose complessiva erogata a paziente ed operatori durante lo studio venografico . 
algarotti 8 , 00137 rome , italy , tel . : + 39 - 349 - 1946942 , fax : + 39 - 06 - 8276895 , e - mail : stepieri@excite.it received : 4 september 2008 / accepted : 12 november 2008 / published online : 25 june 2009 springer - verlag 2009 abstract purpose . 
between january 1997 and december 2005 , 23 patients ( 14 men and nine women ; age range 2148 years ) , after a previous study with computed tomography ( ct ) and / or magnetic resonance ( mr ) imaging , underwent percutaneous drainage of a tuberculous fluid collection in the psoas muscles . 
tra gennaio 1997 e dicembre 2005 , 23 pazienti ( 14 uomini e 9 donne ) , di et compresa tra i 21 ed i 48 anni , precedentemente studiati con tc e / o rm , sono stati sottoposti a drenaggio percutaneo di una raccolta fluida , di origine tubercolare , localizzata nei muscoli psoas . 
il massimo diametro trasverso stato di 7 cm , mentre quello longitudinale stato di 14 cil posizionamento del catetere di drenaggio sempre avvenuto con successo , con una permanenza del catetere di 536 giorni ( media 18 , 4 giorni ) ; la regressione della sintomatologia avvenuta gi allatto dellevacuazione della raccolta fluida . 
gli ascessi dei muscoli psoas rappresentano una seria complicanza nella localizzazione ossea della radiol med ( 2009 ) 114 : 984995 drainage catheter requires daily monitoring to identify when it can be safely removed without risk of recurrence . keywords tuberculosis psoas abscess percutaneous drainage tubercolosi , trattabili efficacemente con il drenaggio percutaneo , capace di risolvere subito la sintomatologia dolorosa . 
il catetere di drenaggio necessita di unassistenza giornaliera , per poter decidere il momento pi idoneo alla sua rimozione , senza andare incontro a recidive . parole chiave tubercolosi ascesso muscolo psoas drenaggio percutaneo introduction introduzione despite the systematic use of targeted therapy and serial laboratory testing , tuberculosis continues to be a major cause of morbidity and mortality , affecting more than 30 million people worldwide , especially in developing countries where hygienic conditions are poor [ 13 ]  . 
although past prevention and treatment strategies decreased its frequency in western countries [ 2 ] , recent trends in alcohol and drug abuse , the rising numbers of hiv and aids cases [ 4 ] and waves of migration from the third world have led to a resurgence of the disease in developed countries as well [ 5 ]  . in addition to primary and secondary pulmonary manifestations , tuberculosis may also affect the skeletal system , which is the most frequent extrapulmonary localisation [ 3 ]  . 
in particular , the thoracic and lumbar vertebral bodies and intervertebral discs are common sites of involvement , and the formation of paraspinal tuberculous abscesses is seen in approximately 50%75% of cases of bone tuberculosis [ 1 , 3 ]  . the clinical diagnosis of tuberculous abscesses is not easy , as the symptoms have indolent onset and gradual progression owing to the slow course of tuberculosis . 
diagnostic suspicion can be confirmed with computed tomography ( ct ) [ 6 ] or magnetic resonance ( mr ) imaging [ 7 , 8 ] , as both diagnostic modalities are able to document changes in the margins , size and density of the psoas muscles before and after contrast administration . whereas conservative management ( analgesics , immobilisation , external orthoses ) was the mainstay of treatment in the past , with surgery reserved for patients not responding to conservative treatment [ 9 ] and with disease in other anatomical regions [ 10 ] , today , percutaneous drainage is increasingly being used to treat the fluid collections [ 4 , 9 , 11 ]  . 
 la tubercolosi , nonostante limpiego sistematico di una terapia mirata e i controlli seriati di laboratorio , continua ad essere una importante causa di morbilit e mortalit e a colpire oltre 30 milioni dindividui nel mondo , specie nelle nazioni in via di sviluppo , caratterizzate da scarse condizioni igieniche [ 13 ]  . 
pur essendo pi rara nelle nazioni occidentali , grazie a politiche di prevenzione e cura , attuate in passato [ 2 ] , recentemente , labuso di alcool e droga , laumento dei casi di hiv e aids [ 4 ] e le notevoli ondate migratorie dal terzo mondo , hanno fatto registrare un aumento del numero di casi annui anche nelle nazioni pi progredite [ 5 ]  . oltre alle diverse manifestazioni polmonari , espressione della localizzazione primaria e secondaria della malattia , la tubercolosi in grado di coinvolgere anche il sistema scheletrico : questo infatti rappresenta la localizzazione extrapolmonare pi frequente [ 3 ]  . 
in particolare risultano colpiti i corpi vertebrali dorso lombari e i dischi intervertebrali ; inoltre , la formazione degli ascessi tubercolari paraspinali osservata in circa il 50%75% dei casi di tubercolosi ossea [ 1 , 3 ]  . la diagnosi clinica degli ascessi non agevole : i sintomi sono subdoli , ad esordio ritardato rispetto allinsorgenza della malattia : ci per la lenta evoluzione della tubercolosi ; molto spesso a richiamare lattenzione possono essere un dolore lombare inspiegabile o la comparsa di disturbi motori degli arti inferiori [ 5 ]  . 
al giorno doggi , la conferma del sospetto diagnostico avviene sia con la tc [ 6 ] , che con la rm [ 7 , 8 ] : entrambe le metodiche diagnostiche sono in grado di documentare le variazioni di margini , dimensioni e densit dei muscoli psoas , prima e dopo somministrazione di mezzo di contrasto . se le modalit conservative ( farmacoterapia basata sugli antidolorifici , immobilizzazione , applicazione esterna di tutori ) rappresentavano , nel recente passato , i capisaldi delle opzioni terapeutiche a disposizione del clinico , riservando alla chirurgia quei pazienti dove non era stata raggiunta una risoluzione del quadro clinico [ 9 ] , come per altre regioni anatomiche [ 10 ] , attualmente si fa sempre pi 986 materials and methods over a period of 7 years , 62 patients with psoas - muscle fluid collections were treated with percutaneous drainage at our centre . 
the patients , 14 men and nine women , age 2148 ( mean 39 ) years , had been admitted to different respiratory disease divisions of our hospital because of the gradual onset of nonspecific symptoms such as worsening fatigue , persistent mild evening fever , productive cough at times with blood - tinged sputum , and pain in the thoracic spine in nine patients and in the lumbar spine in 14 . 
pain had been partially controlled with analgesics and in three cases even with the occasional addition of opiates . diagnostic tests revealed the presence of a lung lesion on plain chest radiography or on subsequent ct scan ( 16 active lesions , seven sequelae ) , as well as associated findings at the level of the spine and psoas muscles . 
ct was performed on 16 patients with a conventional ct scanner ( rhota ctw 950 sr , esaote biomedica , genoa , italy ) and a protocol consisting of a study of the spine with a slice thickness of 5 mm before and after contrast administration ( optiray 300 , tyco healthcare spa , milan , italy )  . 
abscesses were considered appropriate for percutaneous drainage if they met the following criteria [ 7 ] : abscesses associated with spondylodiscitis , larger than 3 cm in diameter , causing pain refractory to systemic antituberculous chemotherapy , and associated with altered laboratory parameters ( neutrophil leukocytosis , elevated erythrocyte sedimentation rate )  . 
 in letteratura sono presenti pochi articoli che descrivono lapplicazione sistematica di questa procedura , tra laltro su un numero limitato di pazienti ; riportiamo la nostra esperienza nella gestione percutanea di tali quadri clinici , nel breve e medio termine , maturata su un discreto gruppo di pazienti . 
 materiali e metodi nellarco di 7 anni , nel nostro servizio sono stati complessivamente sottoposti a trattamento percutaneo 62 pazienti che presentavano una raccolta fluida allinterno dei muscoli psoas : solo 23 sono risultati di natura tubercolare e rappresentano il gruppo di popolazione oggetto del nostro articolo . 
questi pazienti , 14 uomini e 9 donne , di et compresa tra i 21 e i 48 anni ( et media 39 anni ) , erano stati ricoverati nelle varie unit pneumologiche della nostra azienda ospedaliera , per la progressiva comparsa di una sintomatologia aspecifica , caratterizzata da astenia ingravescente , febbricola serotina persistente , tosse produttiva , talvolta con striature ematiche , da dolori prevalentemente toracici in 9 pazienti e lombari in 14 . 
solo quattro pazienti presentavano anche disturbi alla deambulazione e incapacit a tenere la stazione eretta ; in due casi tale sintomatologia era insorta durante la gravidanza e si era aggravata durante lallattamento . 
la sintomatologia dolorosa era stata controllata in modo incompleto con antidolorifici , in tre casi anche con laggiunta saltuaria di oppiacei . nel corso dei vari accertamenti eseguiti in ambito ospedaliero , in tutti i pazienti stata riscontrata la presenza di una lesione polmonare alla radiografia diretta del torace o allapprofondimento diagnostico con la tc ( 16 in fase attiva , mentre in 7 erano presenti esiti ) e di una contemporanea alterazione a livello della colonna e dei muscoli psoas . 
lo studio tc stato eseguito su 16 pazienti , utilizzando una tc tradizionale ( rhota ctw 950 sr , esaote biomedica , genova , italia ) e un protocollo che prevedeva dopo liniziale scansione lo studio della colonna con uno spessore di sezione di 5 mm , prima e dopo la somministrazione di mezzo di contrasto ( optiray 300 , tyco healthcare spa , milano , italia ) ; lo studio rm stato eseguito su 7 pazienti , utilizzando lapparecchiatura da 1 , 0 t ( siemens , erlangen , germania ) , con scansioni sagittali e longitudinali t1 , prima e dopo somministrazione di mezzo di contrasto ( magnevist , schering , berlino , germania ) , e t2 spin echo ( tempo di ripetizione ms / tempo di echo ms , 500600 / 1520 nelle prime e 2 , 000 / 90 nel secondo caso )  . raccolta fluida , o ascesso , stata definita quella radiol med ( 2009 ) 114 : 984995 fig . 
gli ascessi venivano considerati appropriati per un drenaggio percutaneo secondo indicazioni gi riportate da altri autori [ 7 ] , che brevemente riassumiamo : dovevano essere associati ad una spondilodiscite , dovevano avere dimensioni superiori ai 3 cm di diametro , con una sintomatologia dolorosa che non aveva tratto beneficio dalla chemioterapia antitubercolare sistemica , con la presenza di unalterazione dei parametri di laboratorio ( leucocitosi neutrofila , aumento della ves )  . 
i criteri di esclusione sono stati il riconoscimento di un tessuto di granulazione paravertebrale o paraspinale associato con una raccolta fluida ipodensa , o la localizzazione epidurale della raccolta , con compressione midollare . 
coronal mr imaging confirms the presence of an abscess originating from the vertebral body of t11 - t12 . altered signal intensity can be seen in the vertebral bodies and intervertebral disc , along with a left - sided swelling with fluid characteristics involving the cranial portion of the ipsilateral psoas muscle . 
la rm nella scansione coronale conferma lesistenza di una formazione ascessuale a partenza dal corpo di d11 - d12 ; inoltre presente unalterazione di segnale nei corpi vertebrali e nel disco intersomatico , con presenza , a sinistra , di una tumefazione con caratteristiche liquide , che coinvolge la porzione craniale del muscolo psoas omolaterale . 
3a , b sonography prior to the interventional procedure with transducer oriented towards the lower pole of the ipsilateral kidney shows a 2.85 - cm hypoechoic mass with homogeneous and well - defined margins in the left paravertebral region ( a )  . 
3a , b lindagine ecografica propedeutica alla procedura interventistica , in sede paravertebrale sinistra , con sonda orientata verso il polo inferiore del rene omolaterale , mostra la presenza di una formazione ipoecogena , a margini omogenei e bene definiti , delle dimensioni di cm 2 , 8 per 5 circa ( a )  . 
grazie alla buona visibilit della lesione viene eseguito il drenaggio ecoguidato , con posizionamento di catetere pig tail 8 fr , a fori multipli terminali ( b )  . modality because it is faster and provides real - time monitoring throughout the drainage procedure . 
the initial puncture was performed under sonographic guidance with a 15 - cm - long 18 - gauge needle ( hs spa , aprilia , rome , italy ) inserted into the most distal portion of the fluid collection . 
the fluid was aspirated with a 10 - ml luer - lock syringe to confirm the position of the needle and guide the choice of drainage catheter based on the texture of the caseous material . 
con il paziente in posizione prona , dopo aver disinfettato larea cutanea dinteresse ed aver effettuato lanestesia locale , stata effettuata una piccola incisione cutanea con una punta di bisturi ( retto , n11 )  . 
successivamente , dopo iniezione di mezzo di contrasto ( optiray 240 , tyco healthcare spa , milano , italia ) per dimostrare lestensione della raccolta ed escludere eventuali comunicazioni fistolose , si proceduto al posizionamento di un filo guida idrofilico 0 , 038 , a punta j , lungo 80 cm , molto rigido ( terumo corporation , gamma international , roma , italia )  . 
si preferito impiegare la pi indaginosa tecnica di seldinger , e non la trocar , per evitare ulteriori traumatismi e dolori al paziente , visto lampio calibro del catetere necessario ad eseguire il drenaggio in prima battuta . 
tutto il materiale purulento stato aspirato ; parte stato inviato per le ricerche batteriologiche . tutti i cateteri sono stati lasciati drenare per gravit ; sono stati lavati quotidianamente con 100 ml di soluzione fisiologica , almeno tre volte al giorno , impiegando siringhe luer lock da 20 ml ; inoltre sono stati utilizzati anche per liniezione di farmaci anti - tubercolari in loco ( rifampicina 600 mg )  . 
il follow - up consistito in una ricerca mensile del mycobacterium tubercolaris , attraverso lesame dellespettorato ed accertamenti di laboratorio , e in una valutazione radiologica semestrale con la tc della colonna e la radiografia diretta del torace . 
la distruzione localizzata di uno o pi corpi vertebrali era visibile in tutti i pazienti : 3 / 23 avevano il coinvolgimento di un solo corpo vertebrale , 16 / 23 di due corpi vertebrali e 4 di tre corpi vertebrali . 
 tutti hanno fatto registrare la contemporanea presenza di un ascesso del muscolo psoas o di entrambi : 6 erano unicamente a destra , 3 a sinistra e 14 erano bilaterali : di cui 10 comunicanti . 
le dimensioni degli ascessi erano variabili : il massimo diametro trasverso era a livello lombare , probabilmente per la maggiore gravit , e raggiungeva i 7 cm , mentre il minimo era a livello dorsale e raggiungeva i 3 , 7 cm ( media 5 , 3 cm )  . 
la massima estensione longitudinale stata di 14 cm , mentre la minima stata di 6 , 3 cm ( media 9 , 8 cm ) tutti i pazienti sono stati sottoposti al drenaggio percutaneo in associazione con la chemioterapia sistemica ( rifampicina 600 mg , isoniazide 300 mg , etambutolo 1250 mg , pirazinamide 1500 mg ) e loco - regionale ( rifampicina 600 mg )  . 
il controllo radiografico , al termine della procedura di posizionamento del catetere di drenaggio , evidenzia le caratteristiche del catetere ( posto allinterno della porzione pi ampia della raccolta ascessuale , in grado di raccogliere per gravit anche la produzione pi craniale , grazie ai multipli fori laterali presenti , allinterno della porzione arrotolata del catetere tale conformazione garantisce latraumaticit del catetere ) e della raccolta appena drenata . the one - step trocar technique . 
all purulent material was aspirated , and part of it was sent for bacteriological testing . all catheters were left to drain by gravity and were flushed at least three times daily with 100 ml of saline solution injected through 20 - ml luer - lock syringes . 
after an initial finding of bilateral and confluent psoas muscle abscesses ( a ) , the first follow - up study 7 days after the procedure shows a significant bilateral reduction in the size of the collections ( b ) , more evident after 15 days ( c )  . 
da uniniziale reperto di ascessi dei muscoli psoas , bilaterali e confluenti ( a ) , il primo controllo radiologico del drenaggio , dopo 7 giorni dalla procedura , evidenzia una notevole riduzione bilaterale delle dimensioni delle raccolte ( b ) , pi evidenti dopo 15 giorni ( c )  . 
dopo 21 giorni , la raccolta sinistra praticamente guarita , mentre a destra permangono ancora dei residui , anche se la produzione giornaliera di essudato inferiore a 10 ml ( d )  . results all patients but one were immigrants . 
sono stati posizionati sempre , in prima istanza , cateteri di drenaggio 8 fr ; si fatto ricorso sistematicamente alla tecnica di seldinger , per evitare il traumatismo diretto di un catetere con calibro radiol med ( 2009 ) 114 : 984995 fig . 
confronto tra la originaria raccolta ascessuale nella porzione pi declive del muscolo psoas di sinistra ( a ) e controllo effettuato dopo 8 giorni di permanenza del catetere di drenaggio ( b )  . bodies was seen in all patients , with involvement of only one vertebra in 3 / 23 patients , two vertebrae in 16 / 23 and three vertebrae in 4 / 23 . 
 all patients had an associated abscess in either one or both psoas muscles : six were unilateral on the right side , three unilateral on the left and 14 bilateral ; ten were the communicating abscesses . 
the largest longitudinal extension was 14 cm , whereas the smallest was 6.3 cm ( mean 9.8 cm ) all patients underwent percutaneous drainage combined with systemic chemotherapy ( rifampicin 600 mg , isoniazid 300 mg , ethambutol 1 , 250 mg , pyrazinamide 1 , 500 mg ) and locoregional chemotherapy ( rifampicin 600 mg )  . 
all procedures were carried out with 8 - fr drainage catheters as a first choice , and the seldinger technique was used systematically to avoid any direct trauma being caused by the large - bore catheters used with trocar technique . 
 we observed no major complications and only two minor complications : in two patients , the catheter became dislodged due to excessive traction during daily catheter care and had to be replaced with a larger ( 10 - fr ) drainage catheter inserted via the existing pathway . 
 non si sono avute complicanze maggiori , ma solo 2 minori : in due pazienti si resa necessaria la sostituzione dei cateteri per una loro dislocazione a seguito di eccessiva trazione durante leffettuazione di una medicazione giornaliera : in tali pazienti stato posizionato un nuovo catetere di drenaggio , di dimensioni maggiori ( 10 fr ) , utilizzando il tramite preesistente . 
in nessuno dei pazienti , compresi quelli con recidiva polmonare , si dovuto ricorrere a drenaggio chirurgico per inefficacia dellatto interventistico , per cui il drenaggio risultato curativo nel 100% dei casi . 
 il periodo di drenaggio stato 536 giorni ( media 18 , 4 giorni ) , con un sensibile miglioramento della sintomatologia gi nelle prime fasi di posizionamento del catetere ed proseguito nei giorni successivi . 
il batterio tubercolare stato isolato in 18 dei 23 ascessi dei muscoli psoas , mentre negli altri la diagnosi stata indiretta , basata cio sui reperti della contemporanea localizzazione polmonare e sui reperti radiologici della colonna vertebrale . 
 durante i primi 12 mesi del periodo di controllo , ben 19 pazienti si sono presentati regolarmente , sottoponendosi sia agli esami di laboratorio sia ai controlli radiografici , mentre 4 non sono risultati pi reperibili . 
mentre tutti erano guariti per la spondilodiscite e le raccolte ascessuali , solo un paziente ha fatto registrare la ripresa della malattia polmonare per una resistenza allisoniazide , per cui si resa necessaria la somministrazione di una differente associazione di farmaci antitubercolari . 
the tuberculosis bacterium was isolated in 18 of the 23 psoas abscesses , whereas in the remainder , the diagnosis was reached indirectly on the basis of findings of the simultaneous involvement of the lung and spine . 
whereas the spondylodiscitis and abscesses had resolved in all cases , one patient had recurrence of pulmonary tuberculosis due to isoniazid resistance , leading to a treatment change . discussion the skeleton , and particularly the spine , is involved in 3% of cases of extrapulmonary tuberculosis . 
tubercularis can reach the spine through haematogenous spread , with embolisation to a site distant from the primary focus , or through direct extension from contiguous foci of infection ( rarely ) ; no cases of venous dissemination or direct seeding during surgery or interventional radiology procedures have been reported [ 12 , 13 ]  . spondylodiscitis starts in the inferior and anterior portion of the vertebral body , at the level of the metaphysis , which has a rich arterial supply . 
the inflammation spreads to the entire vertebral body along the medullary canals and to adjacent vertebrae along the longitudinal anterior ligament , with delayed disc involvement due to the lack of specific cartilage proteolytic enzyme in m . 
disc involvement leads to destruction and collapse of the vertebral body , resulting in severe spinal deformity . abscess formation is caused by the spread of the vertebral disease to neighbouring areas . 
however , because many of its compartments are not completely covered by septations , they may potentially become open and act as a route for disease dissemination , allowing spinal infection to spread . 
the size of the abscess appears to depend on the amount of fat displaced by the inflammatory process or fluid collection [ 14 ]  . although a final diagnosis of tuberculous abscess requires specimen cultures to confirm the presence of the bacterium , cultures often prove positive in only della malattia tubercolare . 
il mycobacterium tubercolaris pu raggiungere la colonna vertebrale attraverso una disseminazione ematogena , con una embolizzazione distante dalla sede di localizzazione dei germi , oppure con una propagazione da sedi dinfezione limitrofe ( rara ) , mentre non sono state segnalate disseminazioni per via venosa o per impianto diretto durante procedure chirurgiche o di radiologia interventistica [ 12 , 13 ]  . linizio della spondilodiscite si verifica nella porzione inferiore e anteriore del corpo vertebrale , a livello della metafisi , dove esiste una ricca vascolarizzazione arteriosa . si verifica un infarto osseo , causato dallembolizzazione settica , a cui fa seguito una reazione infiammatoria granulomatosa . 
dalla primitiva sede , linfiammazione si propaga a tutto il corpo vertebrale , lungo i canali midollari , e a quelli adiacenti lungo il legamento longitudinale anteriore , con coinvolgimento tardivo del disco o dei dischi interposti , grazie allassenza dellenzima proteolitico cartilagineo [ 6 , 13 ]  . 
il risultato di tale coinvolgimento la distruzione e il collasso del corpo vertebrale , con severa deformit della colonna . la formazione degli ascessi il risultato della progressione della malattia vertebrale nei territori circostanti : non trovando ostacoli , pu colpire il canale midollare , posteriormente e / o il retroperitoneo e i muscoli psoas , anteriormente e lateralmente . 
il retroperitoneo diviso in compartimenti da multiple fasce ; molti dei compartimenti non sono completamente ricoperti da tali sepimenti , per cui possono divenire potenzialmente aperti , costituire una rotta di propagazione , attraverso i quali la malattia pu diffondersi . linfezione della colonna pu quindi diffondersi ; le raccolte ascessuali possono comportare spesso una sintomatologia aspecifica , fino alla claudicatio , oppure mimare una lesione osteolitica . 
lestensione delle raccolte sembra dipendere dal grado di spostamento del grasso da parte del processo infiammatorio o del fluido accumulato [ 14 ]  . pur se per una diagnosi definitiva di ascesso tubercolare necessario un esame colturale per confermare la presenza del batterio , tuttavia molto spesso le colture sono positive solo nel 50%60% dei casi , per cui , come segnalato da altri autori [ 15 ] , la diagnosi di tubercolosi talvolta pu essere anche presuntiva , basata sulle caratteristiche di presentazione clinica , sui reperti radiologici allesordio ed a seguito della risposta alla terapia antitubercolare . 
 per quanto riguarda la terapia , il solo impiego dei farmaci antitubercolari associati al riposo a letto e alla terapia fisica hanno notevolmente migliorato i risultati clinici , ma necessitano di tempi molto lunghi per una piena ripresa funzionale . 
in passato , il ricorso alla chirurgia stato legato principalmente allesito non soddisfacente o non risolutivo della terapia conservativa , alla deformit spinale o allinstabilit della colonna , secondaria alle fratture patologiche ed alla presenza di una sintomatologia neurologica : la radiol med ( 2009 ) 114 : 984995 50%60% of cases . 
consequently , as reported by other authors [ 15 ] , the diagnosis of tuberculosis is at times presumptive and based on clinical presentation and imaging findings at onset and following response to antituberculous treatment . with regard to therapy , the use of antituberculous drugs combined with bed rest and physical therapy have significantly improved clinical outcomes , although a full functional recovery will take a long time . 
in the past , surgery was principally used in the case of unsatisfactory results of conservative management , spinal deformity or spinal instability secondary to pathological fractures , and neurological symptoms : anterior decompression , drainage , and debridement with posterior fusion have represented the mainstay of the surgical approach , with mortality rates ranging from 2% to 11% and recurrence rates between 10% and 20% [ 16 , 20 ]  . 
widely and successfully employed in other anatomical regions [ 11 ] , percutaneous drainage under sonographic or ct guidance may also be used in tuberculous abscesses of the psoas muscles to help accelerate healing with full remission of symptoms . 
whereas ct is superior to sonography in depicting the location and extent of the abscesses and their relations with neighbouring organs and in suggesting the most appropriate route for catheter insertion [ 6 ] , sonography has the advantage of being faster and enabling real - time monitoring of the entire procedure . 
 percutaneous drainage of a fluid collection includes various phases : locating the fluid collection , choosing the path to follow and placing one or more catheters [ 23 ]  . 
in consideration of the clinical situation , the imaging findings of extensive vertebral destruction and of abscess formation in the psoas muscles , all patients in our series underwent complete evacuation of the infected collection and never diagnostic aspiration only . 
the final drainage catheter was chosen according to the exudate characteristics , with 8 - fr catheters being used in all cases given that the exudate appeared moderately dense at initial needle puncture [ 9 ]  . 
in addition , our decision took into account the traumatic nature of the procedure in subjects already suffering severely and unable to tolerate placement of larger - bore catheters under local anaesthesia alone . 
the greater laboriousness of the seldinger technique is offset by the lower level of pain endured by the patient during the procedure . the choice of percutaneous drainage catheter proved optimal in terms of resolution of the clinical symptoms in decompressione anteriore , il drenaggio , lo sbrigliamento con la fusione posteriore hanno rappresentato i capisaldi dellopzione chirurgica , con percentuali di mortalit tra il 2% e l11% , e di recidive tra il 10% e il 20% [ 16 , 20 ]  . 
 ad affiancare tali opzioni terapeutiche , negli anni 8090 comparso il drenaggio percutaneo , effettuato in anestesia locale , sotto lassistenza di una tecnica per immagini , il cui obiettivo quello di favorire la risoluzione della raccolta fluida senza dover sottoporre il paziente ad un intervento chirurgico [ 21 ]  . 
gi ampiamente impiegato con successo in altre regioni anatomiche [ 11 ] , anche gli ascessi dei muscoli psoas di natura tubercolare possono giovarsi di tale procedura percutanea , con lausilio della guida ecografica o tc , contribuendo cos ad accelerare il processo di guarigione di queste complicanze della malattia tubercolare , con piena risoluzione della sintomatologia . mentre la tc pi sensibile degli ultrasuoni nel mostrare lesatta localizzazione e lestensione degli ascessi , la loro relazione con gli organi vicini e nel consigliare leventuale tragitto da seguire per posizionare il catetere di drenaggio , evitando il percorso organi e strutture [ 6 ] , lecografia ha il vantaggio di essere pi rapida e di seguire in tempo reale le varie fasi della procedura . 
 lungo la procedura di drenaggio percutaneo di una raccolta fluida comprende le varie fasi di localizzazione della raccolta stessa , la scelta della traiettoria da seguire , il posizionamento di uno o pi cateteri [ 23 ]  . 
visti il contesto clinico , i reperti radiologici di ampia distruzione dei corpi vertebrali e di formazione di ascessi nei muscoli psoas , nella nostra esperienza non abbiamo mai eseguito la sola aspirazione diagnostica , ma sempre il drenaggio completo della raccolta infetta . 
il definitivo catetere di drenaggio stato scelto sulla base delle caratteristiche dellessudato : stato sempre impiegato un catetere 8 fr , in relazione ad una non eccessiva densit della raccolta fluida , verificato gi al momento della iniziale puntura della raccolta con lago [ 9 ]  . 
anche per questi motivi abbiamo preferito non ricorrere alla tecnica trocar ; il maggior numero di passaggi della tecnica di seldinger compensato da una quota minore di dolore che il paziente deve sostenere durante la procedura . la scelta del catetere di drenaggio percutaneo si dimostrata ottimale per la risoluzione del quadro clinico , sia negli ascessi uniloculati , che in quelli pluriconcamerati . 
 la sintomatologia dolorosa notevolmente regredita , in maniera quasi istantanea , gi al momento delliniziale evacuazione della raccolta fluida , dopo la puntura il successivo con lago , rendendo meno doloroso 994 radiol med ( 2009 ) 114 : 984995 both unilocular and multilocular abscesses . 
bilateral and communicating abscesses were treated with the double drainage technique to allow the passage of the smaller fluid collection to the larger one and help close the communication between the two abscess cavities . 
these results are in line with other authors experiences [ 1719 ]  . there were no major complications ( septic shock , bacteremia , haemorrhage , bowel transgression , pleural transgression ) or minor complications ( except for catheter dislodgement during dressing removal )  . 
we had no cases of abscess recurrence , which may occur in the event of resistance to antituberculous medication ( in endemic areas , resistance to isoniazid is 4% ) , incorrect diagnosis ( tuberculous abscess misdiagnosed as bacterial abscess ) , early cessation of systemic antituberculous medication ( therapy should continue for at least 12 months ) and presence of large abscesses extending to different areas , not all drained by the catheter [ 15 , 17 ]  . 
furthermore , the chronic nature of tuberculous spondylitis and the persistence of a active tuberculous focus in the spine after catheter removal may also be responsible for recurrence [ 5 ]  . 
in genere , stato sufficiente posizionare un solo catetere di drenaggio per ogni raccolta fluida ; per gli ascessi bilaterali e comunicanti , stata scelta la tecnica del doppio drenaggio , per favorire dapprima il passaggio della raccolta fluida minore verso la maggiore e poi la chiusura della comunicazione tra le due cavit . 
non abbiamo avuto casi clinici con drenaggi curativi parziali , cio che hanno implicato il ricorso ad un intervento chirurgico per risolvere problemi sottostanti o eventuali fistole , a dimostrazione dellefficacia di questa procedura . 
questi risultati sono in linea con le esperienze di altri autori [ 1719 ]  . non abbiamo avuto complicanze maggiori ( shock settico , batteriemia , emorragia , lesione di anse intestinali , lesioni pleuriche ) , n minori ( se si eccettua la dislocazione del catetere di drenaggio avvenuta durante la rimozione energica della medicazione )  . 
nella nostra esperienza , non abbiamo avuto episodi di recidiva degli ascessi : il motivo di un tale evento da ascrivere alla resistenza ai farmaci antitubercolari ( nelle aree endemiche la resistenza allisoniazide del 4% ) , ad unerrata diagnosi ( ascesso tubercolare scambiato per batterico ) , ad una precoce interruzione della terapia antitubercolare sistemica ( la terapia dovrebbe essere continuata per almeno 12 mesi ) , alla presenza di larghi ascessi che si estendono verso aree differenti , non tutte drenate dal catetere [ 15 , 17 ]  . 
inoltre , la natura cronica della spondilite tubercolare e la persistenza di un focolaio tubercolare attivo nella colonna vertebrale dopo la rimozione del catetere possono essere responsabili della recidiva [ 5 ] : per questi motivi abbiamo voluto associare alla terapia sistemica anche quella loco - regionale , durante il periodo di permanenza del catetere di drenaggio , in modo da aumentare la concentrazione del farmaco nella sede dinfezione tubercolare ; cos in parte possibile spiegare lassenza di recidive nella nostra pur limitata esperienza . 
percutaneous drainage of these fluid collections , if performed by experienced operators , carries a low risk of morbidity and is much less traumatic than the surgical approach . percutaneous drainage requires daily care of the drainage catheter ( flushing with saline solution ) and monitoring over time to ascertain complete abscess resolution and patient recovery . 
percutaneous drainage is curative in 100% of cases . gli ascessi dei muscoli psoas risultano essere unevenienza non rara nella odierna pratica clinica , viste le continue ondate migratorie a cui litalia sottoposta . 
necessita di una gestione quotidiana del catetere di drenaggio ( lavaggio con soluzione fisiologica ) , di un monitoraggio nel tempo del paziente , per garantire la risoluzione completa delle raccolte ascessuali e la guarigione del paziente . 
muzzio1 , 2 1department of medical diagnostic sciences and special therapies , university of padua , via giustiniani 2 , padua , italy 2the veneto institute of oncology ( iov - irccs ) , via gattamelata 64 , padua , italy 3department of oncologic and surgical sciences , university of padua , via giustiniani 2 , padua , italy correspondence to : f . 
institutional review board approval and informed patient consent were obtained . colonic segmental radiopaque markers were counted and transit times calculated in 36 healthy subjects studied using a technique involving daily radiopaque marker ingestion and single radiological visit , with oral administration of 810 ml of a thick barium paste as a colonic trace for the marker count . 
the upper limit for colonic transit times was : 45.6 h in the colon as a whole , 31.2 h in the right colon , 19.2 h in the left colon and 16.8 h in the rectosigmoid . 
in healthy subjects , a barium trace affords optimal visibility of the different colonic segments , enabling accurate location of all markers and thus providing an anatomy - related , repeatable and reproducible fluoroscopic segmental marker count . 
trentasei adulti sani vengono studiati con tecnica di ingestione giornaliera di indicatori radiopachi , ingestione di 810 ml di bario - solfato denso per tracciare il colon e singolo controllo radiologico . 
limite superiore dei tempi di transito in ore : 45 , 6 nellintero colon , 31 , 2 nel colon destro , 19 , 2 nel colon sinistro e di 16 , 8 nel sigma - retto . 
nei soggetti sani , la traccia colica di bario fornisce visibilit ottimale dei segmenti colici , consentendo la localizzazione di tutti gli indicatori ed un conteggio fluoroscopico basato sullanatomia , ripetibile e riproducibile . 
suggeriamo di utilizzare i medesimi valori di riferimento per i tempi di transito colico normali negli italiani di entrambi i sessi . keywords colonic transit radiopaque markers normal value gender difference observer agreement parole chiave transito colico indicatori radiopachi valori normali differenza fra sessi accordo tra osservatori 926 introduction following clinical assessment based on medical history and physical examination , various function tests are used to assess functional constipation in adults , including evacuation proctography ( defaecography ) [ 1 , 2 ] , measurement of colonic transit time ( ctt ) by radiology or scintigraphy [ 3 , 4 ] , magnetic resonance imaging ( mri ) [ 5 , 6 ] , ultrasound us [ 7 , 8 ] , and anorectal and colonic manometry [ 9 , 10 ]  . 
in times of cross - sectional imaging , gastroenterologists seem to have lost interest in using radiopaque markers and scintigraphy to study transit in the assessment of chronic constipation [ 11 , 12 ]  . 
however , radiopaque marker transit studies can reveal slow transit constipation or normal colorectal transit times in patients complaining of constipation , enabling assessment of colonic transit segment by segment . 
the validity of the test has been questioned , particularly because of discrepancies in published colonic segmental transit time studies in distinguishing between different subtypes of constipation [ 9 ] , i.e. colonic dysfunction ( of the proximal or transverse colon ) or outlet obstruction ( involving the sigmoid colon and rectum ) [ 13 ]  . 
individual diversity [ 14 ] , different study methods [ 9 ] , difficulties in defining the different colonic segments when locating and counting retained markers on abdominal x - rays [ 15 ] and differences in the way transit times are defined and calculated might also help to explain discrepancies in published data and result in variations in the numerical reference values adopted for both constipated and normal subjects . 
 for instance , normal reference values for ctts have yet to be established for italian adults , and observer agreement in marker counts in healthy subjects has not been tested . 
the purposes of this study were to measure total and segmental ctt in 36 healthy italian adults with no symptoms of constipation and to estimate intraobserver repeatability and interobserver reproducibility for marker counts using a technique based on daily marker ingestion and single radiological visit . materials and methods subjects radiol med ( 2009 ) 114 : 925934 introduzione dopo laccertamento clinico basato sullanamnesi e lesame obiettivo , vari test funzionali vengono utilizzati per la valutazione della stipsi cronica negli adulti : la proctografia evacuativa ( defecografia ) [ 1 , 2 ] , la misura dei tempi di transito colico ( ttc ) con tecniche radiologiche o scintigrafiche [ 3 , 4 ] , la risonanza magnetica [ 5 , 6 ] , gli ultrasuoni [ 7 , 8 ] , la manometria anorettale e colica [ 9 , 10 ]  . 
per la disponibilit attuale delle tecniche di immagini tomografiche , sembra che i gastroenterologi non siano pi interessati alla misura dei ttc con indicatori radiopachi e con scintigrafia nella valutazione della stipsi cronica [ 11 , 12 ]  . comunque , lo studio del transito con indicatori radiopachi nei pazienti che lamentano stipsi distingue quelli con transito colico rallentato da quelli con transito colico normale e valuta il transito nei vari segmenti colici . 
la validit del test stata criticata soprattutto per la discrepanza dei risultati pubblicati negli studi dei tempi di transito colico segmentali per la differenziazione di sottotipi di stipsi [ 9 ] : inerzia colica ( disfunzione del colon prossimale o trasverso ) oppure stipsi per blocco alluscita ( disfunzione di sigma e retto ) [ 13 ]  . 
differenze individuali [ 14 ] , diversit delle tecniche radiologiche [ 9 ] , difficolt nella identificazione dei segmenti colici per la localizzazione ed il conteggio degli indicatori non espulsi visibili nei radiogrammi delladdome [ 15 ] e differenze nel modo in cui viene definito e calcolato il tempo di transito possono giustificare le discrepanze tra i dati pubblicati , con conseguente variabilit dei parametri di riferimento per gli asintomatici di controllo ed i pazienti con stipsi . 
finora , ad esempio , i valori normali di riferimento dei ttc ed il grado di accordo tra osservatori nel conteggio degli indicatori non sono stati misurati in adulti italiani sani . 
gli scopi di questo studio sono : misurare i ttc totale e segmentali in 36 adulti italiani che non avevano sintomi di stipsi e stimare la ripetibilit intraosservatore e la riproducibilit inter - osservatore del conteggio degli indicatori , utilizzando una tecnica che prevede lingestione quotidiana di indicatori ed un unico controllo radiologico . 
 thirty - six healthy volunteers ( 19 women , 17 men ; median age 60 years , range 4172 years ; median body mass index 22 kg / m2 , range 1625 kg / m2 ) were recruited from among subjects presenting for standard chest radiography at our radiology unit . 
individuals complaining of gastrointestinal symptoms specified in the rome iii criteria for functional bowel disorders [ 13 ] or with a history of gastrointestinal disease or abdominal surgery were excluded , as were women pregnant within the last year . 
all included subjects were negative on physical examination and had always been in good health , with no history of constipation or use of laxatives or other drugs known to affect gastrointestinal motility . 
on the days before and during the test period , volunteers kept to their usual dietary and social habits , as our aim was to conduct a real - life study . imaging technique ctts were measured using a technique described elsewhere [ 15 ]  . 
key steps in the technique included : ( 1 ) a brief preliminary fluoroscopy to ensure that supine fluoroscopic images focused on the colon and rectum for the count of all retained markers ; ( 2 ) saving fluoroscopic abdominal views . 
the right colon ( the caecum and ascending and right transverse colon ) , the left colon ( left transverse and descending colon ) and the rectosigmoid , based on the barium colonic trace , as previously reported [ 15 ]  . 
the markers were counted independently by two radiologists : one ( observer a ) an experienced gastrointestinal radiologist ; the other ( observer b ) with no specific training on marker counting and with 3 years of experience in radiology . 
total and segmental ctts were calculated from the distribution of the markers throughout the colon and in the above - specified three separate segments of the colon using the classic equation : t = tn / n , where t is the alla nostra radiologia per un esame radiografico standard del torace . 
tutti avevano frequenza dellalvo di almeno tre evacuazioni alla settimana ( media 1 , 380 , 69 [ ds ] evacuazioni al giorno ; range 0 , 433 , 14 evacuazioni al giorno )  . 
sono stati esclusi coloro che avevano sintomi gastrointestinali specificati nei criteri rome iii per le alterazioni intestinali funzionali [ 13 ] o anamnesi positiva per malattie gastrointestinali oppure per chirurgia addominale . sono state escluse anche le donne che avevano avuto una gravidanza entro il periodo di un anno dallindagine . 
inoltre , tutti i soggetti arruolati nello studio avevano un esame clinico negativo , avevano sempre goduto di un buono stato di salute , avevano anamnesi negativa per stipsi e non avevano mai assunto lassativi o farmaci che possono alterare la motilit gastrointestinale . 
tutte le donne in et fertile che hanno partecipato allo studio dovevano produrre un test di gravidanza ( gonadotropina corionica umana urinaria ) negativo , eseguito non oltre 48 ore prima dello studio . 
nei giorni precedenti e durante lesame , tutti i volontari dovevano continuare la loro alimentazione consueta e le loro abitudini di vita perch il nostro proposito stato di realizzare uno studio rispettoso della realt quotidiana . 
 calcolo dei dati gli indicatori presenti sono stati contati in tutti i soggetti e localizzati in tre segmenti colici tracciati dal bario : colon destro ( cieco , colon ascendente e met destra del colon 928 radiol med ( 2009 ) 114 : 925934 trasverso ) , colon sinistro ( met sinistra del colon trasverso e colon discendente ) e sigma - retto , come gi descritto [ 15 ]  . gli indicatori sono stati contati indipendentemente da due radiologi : uno ( osservatore a ) era un esperto radiologo gastrointestinale , laltro ( osservatore b ) aveva unesperienza radiologica di tre anni , ma non aveva una pratica specifica nel conteggio degli indicatori . 
non ci sono stati limiti di tempo per linterpretazione delle immagini fluroscopiche memorizzate il cui ordine di analisi stato casuale . ciascun osservatore ha ripetuto il conteggio degli indicatori dopo un intervallo di sei settimane e non conosceva i risultati ottenuti dal collega fino al completamento dello studio . 
pertanto , per calcolare il tempo di transito segmentale in ore il numero di indicatori contati in un dato segmento colico stato moltiplicato per 2 , 4 perch sono stati ingeriti quotidianamente dieci indicatori e lintervallo tra due ingestioni stato di 24 ore . 
la ripetibilit intra - osservatore dei conteggi per ciascun segmento colico , stata stimata con il coefficiente di ripetibilit ( cr ) che considera le variazioni intra - osservatore dei conteggi ( secondo conteggio verso primo conteggio , sia dellosservatore a che b )  . 
il cr stato calcolato con la formula : 1 , 96 ( cid : 2 ) ( cid : 3 ) 2sw , dove sw la deviazione standard intra - soggetto . il cr definisce i valori estremi delle differenze fra due conteggi fatti dallo stesso osservatore entro cui cade il 95% dei soggetti e pu essere rappresentato graficamente considerando le differenze tra i due conteggi effettuati da ogni osservatore e le loro medie . 
la riproducibilit inter - osservatore dei conteggi per ciascun segmento colico stata stimata con i limiti di accordo ( la ) per il 95% dei casi , valutando le variazioni inter - osservatore ( primo conteggio dellosservatore a verso il primo di b )  . 
the barium trace enables clear identification of the different anatomical segments of the colon and an easy segmental marker count , thus overcoming the potential drawback of the weak fluoroscopic signal . 
la traccia di bario consente lidentificazione sicura dei diversi segmenti anatomici del colon ed il facile conteggio segmentale degli indicatori , superando lo svantaggio potenziale della debolezza del segnale fluoroscopico . 
 transit time in a given colonic segment , t is the time between two marker ingestions , n is the number of markers counted in a given colonic segment , and n is the number of markers ingested daily [ 16 ]  . 
as ten markers were ingested daily and the interval between two ingestions was 24 h , the number of markers counted in a given colonic segment was multiplied by 2.4 to calculate the segmental transit time in hours . 
intraobserver repeatability for the marker counts in each colonic segment was assessed using the repeatability coefficient ( rc ) , considering the intraobserver variations ( second count vs first count for both observers )  . 
the rc was calculated using the formula : 1.96 ( cid : 2 ) ( cid : 3 ) 2sw , where sw is the standard within - subject deviation . the rc defines the range of the differences between two counts made by the same observer for 95% of the subjects , and it was analysed by plotting the differences between the counts repeated by each observer and their means . 
interobserver agreement on the marker counts for each colonic segment was assessed using 95% limits of agreement ( la ) , radiol med ( 2009 ) 114 : 925934 considering interobserver variations ( observer b versus a ; first count used )  . 
the la were calculated using the formula : is the mean difference and sd the standard 1.96sd , where d deviation of the differences , considering the two observers as two counting methods . 
the la define the range expected to include 95% of the differences between the two observers marker counts , and it was analysed by plotting the difference between the counts obtained by the two observers against the mean of the two values . 
8.2 ( sas institute inc . , cary , nc , usa )  . results total and segmental ctts were evaluated in 36 healthy adults using daily marker ingestion and a single radiological examination . 
the rc ( intraobserver repeatability ) showed that individual differences between repeat counts relating to the right colon , left colon and rectosigmoid were always less than two markers ( table 1 ) , meaning a strong repeatability of the counts . 
the p values for total and segmental ctts according to the two observers radiographic measurements ( a vs b , considering their first count ) ranged from 0.92 for the rectosigmoid to 0.97 for the whole colon , meaning that their measurements were statistically identical . table 2 summarises previously published values for total la mediads per poter confrontare i nostri risultati con quelli di altri autori . 
il cr ( ripetibilit intra - osservatore ) ha dimostrato differenze individuali tra i conteggi replicati sempre inferiori a 2 indicatori sia nel colon di destra che di sinistra e sigma - retto ( tabella 1 ) , a testimonianza di unottima ripetibilit del conteggio . 
infine , i valori di p relativi alle misure radiologiche dei ttc totale e segmentali ottenute dai due osservatori ( primo conteggio di a verso il primo di b ) erano compresi fra 0 , 92 per il sigma - retto e 0 , 97 per lintero colon , a dimostrazione che le misure del transito erano statisticamente identiche . 
la tabella 2 riassume i valori dei ttc totale e segmentali riportati in pubblicazioni precedenti dove stata utilizzata la tecnica di ingestione quotidiana degli indicatori con singolo controllo radiografico , in pi di 30 soggetti [ 1822 ] e fornisce la mediads ed il limite superiore della norma ottenuti nel presente studio ( primo conteggio dellosservatore a )  . 
la mediana dei tempi di transito colico totale , destro , sinistro e nel sigma - retto era rispettivamente di 45 , 6 ore , 31 , 2 ore , 19 , 2 ore e 16 , 8 ore . 
2a - c accordo inter - osservatore per il conteggio fluoroscopico degli indicatori in 36 soggetti adulti sani : nei grafici sono segnati i valori delle differenze individuali e delle medie dei conteggi effettuati da due osservatori e i la . 
esiste una notevole concordanza tra i conteggi dei due osservatori nel colon di destra ( a ) , nel colon di sinistra ( b ) e nel sigma - retto ( c )  . si notino i numerosi punti che cadono vicino alla linea dello zero . and segmental ctts in studies using daily marker ingestion and a single x - ray in series of more than 30 subjects [ 1822 ] , showing the total and segmental ctts expressed as the meansd and upper limits of normal for the present radiological marker study ( observer a , first count )  . 
table 3 shows the median total , right , left and rectosigmoid ctts for women and men and the correspondent upper limits of normal ( observer a , first count )  . tutti gli indicatori presenti nel colon era di 61 , 21%15 , 56% , 18 , 05%12 , 82% e 20 , 72%15 , 39% rispettivamente nel colon destro , sinistro e sigma - retto . 
la tabella 3 riporta la mediana dei tempi di transito colico totale , destro , sinistro e nel sigma - retto per le donne e gli uomini ed i limiti superiori della norma corrispondenti ( primo conteggio dellosservatore a )  . discussione stato proposto che i ttc segmentali possano essere utilizzati per classificare i pazienti stitici in sottogruppi fisiolopatologici [ 23 ] , ma altri autori non considerano validi gli studi sul transito colico segmentale con ingestione radiol med ( 2009 ) 114 : 925934 table 2 total and segmental colonic transit times in healthy adults expressed as the meansd and upper limit of normala author [ ref ] country / year no . 
such a technique only monitors the markers progress through the colon for 72 h , which is not long enough for patients with functional constipation because it has been demonstrated that the upper limit ( 95th percentile ) of the total ctt may be more than 220 h in constipated patients [ 3 ]  . 
an evidence - based review of the utility of physiological and imaging tests in evaluating chronic constipation identified valid evidence to support colonic transit study with radiopaque markers [ 24 ]  . 
other common physiological tests , such as colonic transit study using scintigraphy , anorectal manometry , the balloon expulsion quotidiana di indicatori per tre giorni e controllo radiologico in quarta giornata [ 11 ]  . 
questa tecnica monitora la progressione degli indicatori nel colon solo per 72 ore che un periodo insufficiente nei pazienti con stipsi funzionale perch stato dimostrato che negli stitici il limite superiore ( 95 percentile ) del ttc totale pari a pi di 220 ore [ 3 ]  . tuttavia uno studio di revisione sullutilit dei test fisiologici e con immagini , basata sullevidenza clinica , riconosce validit alla misurazione del transito colico con indicatori radiopachi nella valutazione della stipsi cronica [ 24 ]  . 
ad altri test fisiologici usuali , come la scintigrafia , la manometria anorettale , il test dellespulsione del palloncino , la manometria colica , viene attribuito lo stesso grado o un grado inferiore di utilit clinica basata sullevidenza [ 24 ]  . lo studio radiologico dei ttc fornisce unidea obiettiva dei disturbi dei pazienti con stipsi ed utile criterio nella selezione dei pazienti destinati alla terapia chirurgia [ 2527 ] o alle terapie con farmaci enterocinetici [ 2830 ]  . 
ctts give an objective idea of patients complaints of constipation and are useful in selecting patients for surgery [ 2527 ] and evaluating the effects of enterokinetic drugs [ 2830 ]  . 
we opted to use fluoroscopic abdominal views because it has been demonstrated that fluoroscopy exposes individuals to a median skin dose of 0.239 cgy , which is approximately a third of the radiation exposure of a classic abdominal x - ray obtained using a digital recording plate [ 15 ]  . 
i diversi segmenti colici sono identificati facilmente con scintigrafia [ 31 ] che ha il vantaggio di una dose di esposizione alle radiazioni minima : tra 1 , 1 e 9 , 0 msv allintero corpo [ 12 , 32 ] , a seconda del tempo impiegato dal radioisotopo nellattraversamento del colon . 
da parte nostra abbiamo scelto la fluoroscopia delladdome perch stato provato che lesposizione fluoroscopica dei soggetti , con una dose mediana alla cute di 0 , 239 cgy , circa un terzo dellesposizione per una radiografia standard delladdome , ottenuta usando una piastra digitale [ 15 ]  . 
gli autori hanno somministrato per bocca grosse capsule contenenti una soluzione salina di gadolinio ed hanno calcolato un ttc medio simile a quello calcolato con lesame radiologico [ 33 ]  . 
poich prevedibile che gli asintomatici di controllo abbiano un tempo di transito colico inferiore alle 72 ore , il calcolo del ttc sarebbe stato accurato con una tecnica di monitoraggio dei sani per 72 ore , ovvero con assunzione degli indicatori per tre giorni consecutivi [ 18 ]  . 
comunque abbiamo optato per la stessa tecnica usata negli stitici , ovvero gli indicatori sono assunti per dieci giorni consecutivi , per un confronto diretto delle radiol med ( 2009 ) 114 : 925934 as for constipated patients to enable a stricter comparison of the measurements obtained . 
the total ctt calculated in our study comes within the range shown in the series in table 2 , but our measurements tend to reveal longer right ctt and shorter rectosigmoid ctt than other studies ( table 2 )  . 
this was probably partly because any overlap of the pelvic caecum with the rectosigmoid may negatively affect marker count accuracy if no colonic barium trace is used to enhance the anatomical boundaries between the different colonic segments . 
some authors found that women had a slower ctt than men [ 18 , 34 ] , but we could find no significant ctt differences between women and men , in agreement with other reports [ 21 , 30 ]  . in conclusion , this study produced a set of normal ctts in healthy italian individuals . 
nel nostro studio , il ttc totale rientra nel range di quelli riportati in tabella 2 ; invece il tempo di transito nel colon destro e nel sigma - retto tende ad essere rispettivamente pi lungo e pi breve del corrispondente riportato in altri studi ( tabella 2 )  . 
probabilmente ci in parte dovuto al fatto che leventuale sovrapposizione di un cieco pelvico e del sigma - retto riduce laccuratezza del conteggio degli indicatori , in assenza di una traccia di bario che evidenzi i contorni dei diversi segmenti colici . alcuni autori hanno misurato un ttc pi lungo nelle donne che negli uomini [ 18 , 34 ] ; la nostra esperienza non dimostra significative differenze del ttc nei due sessi , in accordo con altri studi [ 21 , 30 ]  . in conclusione , questo studio fornisce una serie di misure dei ttc normali in adulti italiani sani . 
these were accounted for by the presence of a major lesion distracting the examiners attention from a less important associated lesion in one case , a false negative result in a patient examined on a incompletely radiolucent spinal board and underexposure of the coccyx region in an obese patient . six cases were related to an interpretation error by the radiologist . 
di queste , in una il rilievo di una lesione maggiore ha distolto lattenzione da una lesione associata di minore importanza ; un falso negativo stato osservato in paziente esaminato su barella spinale , non completamente radiotrasparente ; uno era per sottoesposizione della regione coccigea in soggetto obeso . in 6 casi vi stato errore interpretativo del radiologo . 
abbiamo individuato strumenti che speriamo atti a ridurre il livello di contenzioso . keywords radiology audit civil liability litigation clinical risk management parole chiave radiologia audit responsabilit civile contenzioso management del rischio clinico introduction introduzione claims for compensation and legal action against physicians for presumed error are an increasingly serious problem in all countries and in all medical specialties . 
litigations arising from these claims have led to a constant increase in insurance premiums paid by healthcare institutions and individual professionals , and these costs have become an increasingly important item in the expenditure of hospitals and their personnel . there are numerous strategies for reducing litigation that tackle different aspects of the probleone of them is based on the retrospective review of legal proceedings already underway to analyse the causes of the different litigation episodes and the possible personal or organisational factors responsible for the error with a view to planning corrective measures and drawing up guidelines to improve patient management in healthcare institutions [ 1 , 2 ]  . 
from a technical point of view , this approach follows the methods used in the clinical audit [ 38 ]  . the accident and emergency department is the healthcare facility with the highest healthcare expectations by the general public and is therefore the most common site of claims for damages due to inappropriate treatment , whether real or presumed . 
radiology is not the most frequently affected specialty among those operating in accident and emergency departments , but it does particularly lend itself to critical analysis and review of the contested episodes because of the existence of objective documents ( radiographs ) that testify to the patients clinical situation at the time of evaluation . 
interpretation errors of radiographs performed for a suspected fracture represent the most frequent cause of claims for damages against radiologists in italy [ 9 ]  . le richieste di risarcimento e le denunce contro i medici per presunti errori sono un problema di crescente gravit in tutti i paesi e per tutte le specialit mediche . 
il contenzioso risultante da queste richieste causa un continuo incremento delle tariffe assicurative a carico delle aziende sanitarie e dei singoli professionisti e queste sono diventate un importante capitolo di spesa aziendale e personale . 
tra queste , una si basa sullanalisi retrospettiva del contenzioso gi instauratosi analizzando le cause dei diversi episodi e i possibili fattori predisponenti allerrore , personali o organizzativi al fine di pianificare cambiamenti atti a evitare gli stessi e a stendere linee guida per migliorare il percorso dei pazienti nelle strutture sanitarie [ 1 , 2 ]  . 
da un punto di vista tecnico , questo approccio segue le modalit utilizzate nel corso degli audit clinici [ 38 ]  . il pronto soccorso la struttura sanitaria verso cui sono pi alte le aspettative di salute dei cittadini e , conseguentemente , pi frequenti le richieste di risarcimento danni per inappropriatezza delle cure , vera o presunta che sia . 
tra le varie unit operative del pronto soccorso , la radiologia non quella pi frequentemente chiamata in causa , ma si presta particolarmente ad una analisi critica degli episodi contestati perch esistono documenti oggettivi ( i radiogrammi ) a testimonianza della situazione clinica del paziente al momento della valutazione che possono essere rivalutati criticamente . 
una errata interpretazione degli esami radiografici eseguiti per sospetta frattura la prima causa , in ordine di frequenza , delle richieste di risarcimento danni nei confronti dei radiologi in italia [ 9 ]  . 998 radiol med ( 2009 ) 114 : 9961008 in this study , we reviewed the documentation regarding episodes that involved radiological services provided by radiologists in our hospitals accident and emergency radiology department over the period 20042006 to verify the causes of these episodes , identify the predisposing factors and plan the necessary corrective measures . nel nostro lavoro abbiamo rivalutato la documentazione relativa ai sinistri verificatisi nel corso delle prestazioni sanitarie fornite dai radiologi del dipartimento emergenza e accettazione ( dea ) della nostra azienda nel triennio 20042006 al fine di verificare le cause degli stessi , riconoscerne i fattori predisponesti e pianificare i necessari interventi correttivi . materials and methods we analysed the litigation database held by our institutions general and legal affairs office regarding the period 20042006 to extract episodes in which there was a failure to diagnose a fracture at the radiology unit of the accident and emergency department . 
for each patient , the radiology report and relevant images were collected from the radiology information and picture archiving system , as well as the data regarding admission , treatment and discharge from the accident and emergency department . each episode was studied using clinical audit techniques [ 5 , 6 ] , with an analysis of administrative aspects followed by clinical aspects . 
 with regard to the first component , we analysed all procedures relating to the collection and processing of data for the litigation episode against the radiology service of the accident and emergency department and the insurance outcome of each episode . 
type of event ( classified into five types : misdiagnosis or failure to diagnose , inappropriate treatment or failure to treat , onset of complications , falls , other ) 10 . 
di ciascun paziente sono stati tratti , dal sistema informativo e dallarchivio radiologico , i referti e le immagini radiografiche pertinenti e i dati relativi alla scheda di accettazione , trattamento e dimissione dal pronto soccorso . ciascun episodio stato studiato seguendo le tecniche utilizzate nel corso degli audit clinici [ 5 , 6 ] , analizzando in primo luogo la parte amministrativa e , quindi , la parte clinica per quanto riguarda la prima componente sono state analizzate le modalit globali di raccolta e trattamento del contenzioso riguardante la radiologia del dea e lesito assicurativo di ciascun episodio . 
list of documents contained in each file , in chronological order with regard to the clinical component , the images of each patient were evaluated by two radiologists in musculoskeletal radiology expert . 
the evaluation took place without the radiologists being aware that the images regarded cases of litigation and was performed separately . in the event of disagreement between the two experts , no attempt was made to reach a consensus . after the blinded review of the radiographs , a final judgement of each case was formulated by two radiologists , a forensic pathologist and the head of the general and legal affairs office . 
it aimed to understand whether the compensation claim was truly due to a diagnostic error arising from the performance or interpretation of the radiographic examination and , if so , to analyse the events that led to the error . analysis of overall results was then used to implement corrective measures to improve the organisational and technical aspects of patient management and to produce patient information sheets that we trust will influence patient expectations in relation to the service provided . results the number of compensation claims regarding accident and emergency services at our hospital in the 3 - year period was 84 ( 35 in 2004 , 29 in 2005 and 20 in 2006 )  . 
for those occurring in 2006 ( 20 cases ) , 14 claims were filed in 2006 and 6 in 2007 . with regard to the claimants names , several transcription errors were identified that were corrected to avoid association with different cases due to coincidence of names . transcription errors were also identified in the dates of some events , which had , as a result , been catalogued incorrectly . 
in addition , in some cases , even the compensation claims were unable to shed light on precisely what the presumed error was that had motivated the claim . the analysis then identified the service within the accident and emergency department that was involved in each 13 . 
elenco dei documenti presenti in ciascun fascicolo secondo un ordine cronologico . per quanto riguarda la parte clinica , le immagini di ogni paziente sono state sottoposte a valutazione da parte di due radiologi esperti in radiologia muscoloscheletrica . lanalisi ha avuto luogo senza che gli stessi fossero a conoscenza che le immagini riguardavano episodi di contenzioso ed stata svolta in modo disgiunto . 
questa ha considerato congiuntamente i documenti del file amministrativo , i referti , le immagini radiologiche , le cartelle cliniche del pronto soccorso e i risultati dellanalisi da parte degli esperti e ha cercato di comprendere se la richiesta risarcitoria era realmente dovuta ad un errore diagnostico avvenuto durante leffettuazione o linterpretazione dellindagine radiografica e , in caso positivo , di analizzare le modalit con cui si giunti allerrore . lanalisi dei risultati complessivi stata quindi utilizzata per costruire azioni correttive per migliorare le modalit organizzative e tecniche delle attivit e stilare informazioni da fornire ai pazienti che , speriamo , incidano sulle loro aspettative nei confronti del servizio fornito . 
 risultati i casi di richiesta risarcitoria relativi alle attivit svolte nel dea della nostra azienda ospedaliera sono stati , nel triennio analizzato , in numero di 84 ( 35 con evento verificatosi nel 2004 , 29 nel 2005 e 20 nel 2006 )  . 
per il 2006 ( 20 casi ) , 14 sono stati denunciati nello stesso anno e 6 nel 2007 . per quanto riguarda i nominativi dei richiedenti , sono stati identificati alcuni errori di trascrizione degli stessi che sono stati corretti in quanto associati a casi differenti per riguardavano anche la data di alcuni eventi che erano stati precedentemente catalogati , di conseguenza , in modo erroneo . 
errori trascrizione 1000 radiol med ( 2009 ) 114 : 9961008 claithe radiology service was involved in 22 events over the 3 - year period , subdivided as follows : in 2004 , seven events ; in 2005 , eight events ; in 2006 , seven events . tables 13 summarise the overall results ( subdivided by year )  . 
in another case ( fracture of the coccyx ) , the final judgement established that stata precisata , in quasi tutti i casi la descrizione dellevento con una terminologia pi opportuna ed accurata dal punto di vista medico - specialistico . 
inoltre , anche le richieste da cui muovevano le pretese risarcitorie non hanno permesso , in qualche caso , di capire a quali presunti errori fosse ascrivibile la motivazione della richiesta . si individuato quindi in ciascun fascicolo il reparto interessato allinterno del dea . 
radiologia risultava coinvolta in 22 eventi complessivi che , suddivisi nei tre anni , risultavano : nel 2004 : 7 eventi ; nel 2005 : 8 eventi ; nel 2006 : 7 eventi . 
no dislocation of third finger fracture of radius fracture of d12 no fracture no fracture a & e , accident and emergency department tabella 1 contenzioso relativo allanno 2004 frattura falange iv dito mano no frattura frattura capitello radiale frattura riscontrata frattura ginocchio frattura riscontrata sospetta frattura gomito dopo lussazione no frattura frattura iv dito lussazione iii dito frattura radio frattura d12 no frattura iv lussazione iii no frattura no frattura ps , pronto soccorso fracture visible with difficulty litigation not related to radiology litigation not related to radiology fracture identified on radiograph obtained 15 days after episode . 
 litigation closed without further consequences minimal fracture , not identified because attention distracted by dislocation fracture not seen fracture not seen , patient on spinal board frattura visibile con difficolt contenzioso non inerente la radiologia contenzioso non inerente la radiologia riscontrata frattura in esame rx 15 gg dopo lepisodio . 
yes / yes no fracture of radius yes / yes a & e , accident and emergency department tabella 2 contenzioso relativo allanno 2005 paziente lesione diagnosi ps esperto 1 esperto 2 epicrisi frattura d12 frattura piede no frattura no frattura fracture visible with difficulty . 
manca nella richiesta se frattura ossea o cartilaginea frattura non vista frattura non vista frattura radio frattura iv metacarpo frattura piede frattura riscontrata frattura riscontrata no frattura si , malleolo si , cuboide non valutabile . 
manca specifica ( dubbia ) ( dubbia ) frattura naso no frattura frattura rotula frattura polso no frattura frattura stiloide ulna no frattura radio si / si si / si ps , pronto soccorso the radiographic image was of suboptimal quality ( obese patient incorrectly imaged by the available equipment )  . yet another case of failure to diagnose ( vertebral fracture ) due to technical problems was seen in a patient examined on a spinal board . 
in addition , the different segments of the vertebral column were represented on different radiographs , with the fractured vertebra visualised at the periphery of only one of the images obtained . 
un ulteriore episodio , in cui il paziente reclamava un danno per omessa diagnosi di frattura associata a lussazione del gomito ( lussazione correttamente identificata e trattata ) , stato chiuso dallassicurazione aziendale senza seguito . 
 stata stilata una istruzione operativa riguardante lo studio dei pazienti su barella spinale che prevede una attenta centratura dei radiogrammi e un consulto con il radiologo prima di allontanare il paziente dalla sala desame . 
questi dovr porre particolare attenzione a che tutti i segmenti interessati dal trauma siano ben visualizzati , sottolineare sul referto le problematiche tecniche incorse ed eventualmente richiedere una ulteriore indagine dopo stabilizzazione del paziente . 
si raccomandato al radiologo di mantenere viva lattenzione su tutto quanto presente sul radiogramma anche dopo aver identificato una lesione , in quanto non infrequente il riscontro di lesioni multiple . 
si predisposto un foglio informativo per il paziente in cui , a fronte della possibilit di falsi negativi per lesioni impercettibili , si invita lo stesso a ripresentarsi presso il reparto in caso di persistenza della sintomatologia . 
nellambito delle programmazioni di rinnovo tecnologico in azienda , si messo in programma lacquisto di un apparecchio pi performante per il pronto soccorso , in grado di esaminare correttamente anche soggetti obesi . discussione le strategie per ridurre il contenzioso in medicina sono numerose e affrontano aspetti diversi del problema . 
da pi parti , anche a livello istituzionale , si sta cercando di sviluppare e promuovere la cultura di imparare dagli eventi avversi attraverso linee guida sul come trattare gli errori e gli episodi problematici [ 6 , 7 ]  . 
a further case , in which the patient was claiming damages for failure to diagnose a fracture associated with a dislocation of the elbow ( dislocation correctly identified and treated ) was closed by the insurance company without further consequences . 
on this 1004 radiol med ( 2009 ) 114 : 9961008 basis , the fracture was not considered due to an error by the radiologist . table 4 shows the cases divided into five groups . 
cases type specifications interpretation error of the radiologist litigation not related to radiology imperceptible fracture nonassessable episode litigation closed without compensation fracture of patella ( 1 ) , radius ( 1 ) , wrist ( 1 ) , lumbar vertebra ( 1 ) , femur ( 2 ) lesions correctly identified ; claim probably due to dissatisfaction with treatment 3 fractures not identified by one reviewer ; 1 fracture of finger not seen ( associated with dislocation ) ; 1 fracture not seen on spinal board ; 1 fracture of coccyx not seen in obese patient lacking clear description of lesion for which compensation was being claimed lesion not identifiable even retrospectively , visualised some time after trauma tabella 4 suddivisione degli episodi per tipologia n casi tipologia specificazioni errore interpretativo del radiologo contenzioso non ascrivibile alla radiologia fratture impercettibili episodi non valutabili contenzioso chiuso senza risarcimento frattura rotula ( 1 ) , radio ( 1 ) , polso ( 1 ) , vertebra lombare ( 1 ) , femore ( 2 ) lesioni correttamente identificate , contenzioso verosimilmente dovuto a insoddisfazione per la cura 3 fratture non riconosciute da uno dei revisori ; 1 frattura dito non vista ( associata a lussazione ) ; 1 frattura non vista su barella spinale ; 1 frattura coccige non vista in paziente obeso mancava chiara descrizione della lesione per cui si richiedeva il risarcimento lesione non riconoscibile anche in retrospettiva , visualizzata a distanza di tempo dal trauma radiol med ( 2009 ) 114 : 9961008 1005 device for the accident and emergency radiology department that is capable of correctly examining even obese patients . discussion there are numerous strategies for reducing litigation in medicine that tackle different aspects of the proble at various levels , including the institutional level , efforts are being made to promote a culture of learn from your mistakes with the use of guidelines on how to deal with errors and problem cases [ 6 , 7 ]  . 
numerous healthcare institutions have followed this type of approach , creating specific departments dedicated to clinical risk management [ 6 , 8 ]  . the philosophy characterising the function of these departments consists of a complex system of culture , policies , objectives , individuals , resources and procedures . 
at the same time , however , the system needs to be able to act immediately by identifying problems at an early stage , analysing them , recognising the mechanisms responsible for the error or mismanagement and correcting them [ 3 , 4 ]  . 
in addition , it needs to be able to perform the task of conciliation , following the principle whereby most cases of litigation arise from poor communication between the healthcare institution and the user . the approach adopted in our case series followed , from a technical point of view , the method used in the clinical audit [ 3 , 4 ] , with a retrospective analysis of administrative and clinical factors for each episode . 
the administrative analysis revealed that the claims for compensation did not immediately follow the event , with claims being filed in some cases as late as 3 years after the event . 
for classification purposes , therefore , the claims need to be ordered both by the date of the event and the date of the arrival of the claithis is able to produce useful and significant data regarding annual incidence . 
contemporaneamente , per , il sistema deve mettersi immediatamente in grado di agire identificando precocemente i problemi , analizzandoli , riconoscendo i meccanismi attraverso i quali si giunti allerrore o al disservizio e correggendo gli stessi [ 3 , 4 ]  . 
inoltre , deve essere capace di svolgere opera di conciliazione seguendo il principio per cui la gran parte del contenzioso nasce da cattiva comunicazione tra le strutture del sistema sanitario e il cittadino . 
 lapproccio seguito nella nostra casistica segue , da un punto di vista tecnico , le modalit utilizzate nel corso degli audit clinici [ 3 , 4 ] analizzando retrospettivamente fattori amministrativi e clinici di ciascun episodio . allanalisi amministrativa , la valutazione ha mostrato che le richieste danni non sono immediate rispetto allevento . 
peraltro , la progressiva riduzione numerica delle richieste negli anni fa ipotizzare che leventuale variazione possa essere solo di poche unit . sono state anche individuate difficolt nella attribuzione delle richieste . 
anche in questo caso , appare necessaria particolare attenzione alla catalogazione degli episodi perch le lettere di richiesta di risarcimento sono spesso oltremodo succinte e prive di elementi che consentono di ricostruire correttamente levento . per quanto riguarda la componente clinica , da sottolineare che lincidenza del problema delle diagnosi radiologiche errate in pronto soccorso stato esaminato estesamente in letteratura . 
la percentuale di diagnosi errate varia , a seconda degli articoli considerati , dal 1% al 1006 radiol med ( 2009 ) 114 : 9961008 possible difference is in the order of several units only . difficulties were also identified in the attribution of the claims . 
in this case , too , there appears to be a need for special care in classifying episodes , because the letters claiming compensation tend to be brief and without elements enabling a precise reconstruction of the event . with regard to the clinical component , the incidence of radiological misdiagnoses in the accident and emergency department has been widely discussed in the literature . 
there are , however , no studies that demonstrate a difference in the quality of radiological reports performed at the beginning or the end of a shiin our series , this factor may be taken into consideration to explain the episodes in which the radiologists error could not be attributed to anything other than a failure to identify a lesion . a further cause ( that explains one of the episodes ) is the presence of two associated lesions , whereby one is readily identifiable and the other is less evident . 
in the first instance , a patient was examined on an incompletely radiolucent spinal board , and the radiographs failed to guarantee an optimal evaluation of all of the segments requested by the study . 
we feel that it is useful to recommend that the radiologist note in the report any technical difficulties encountered during the examination , emphasising how the resulting radiographs cannot be used to rule out the presence of anything but patently evident lesions and inviting the requesting physician to have the patient undergo the examination again once stabilised , if necessary . with regard to the second episode , availability of a more appropriate device for the study of obese subjects might have 6% [ 1012 ]  . 
per quanto riguarda le fratture , due lavori affrontano specificatamente il problema riportando valori che vanno dal 2 , 8% al 3 , 7% [ 13 , 14 ]  . 
nella nostra casistica , questo fattore pu forse essere preso in considerazione per spiegare gli episodi in cui lerrore del radiologo non poteva essere riferito ad altro se non ad un deficit identificativo . una causa ulteriore ( spiega uno degli episodi ) la presenza di due lesioni associate , di cui una facilmente riconoscibile e la seconda di difficile identificazione . 
nel primo , un paziente stato esaminato su barella non completamente radiotrasparente e linsieme dei radiogrammi eseguiti non garantiva valutazione ottimale di tutti i segmenti di cui era richiesto lo studio . 
riteniamo sia utile raccomandare al radiologo segnalare nel referto la difficolt tecnica di esecuzione , sottolineando come i radiogrammi risultanti non consentano di escludere la presenza di lesioni men che grossolane e invitando il richiedente ad inviare nuovamente il paziente dopo stabilizzazione , se necessario . 
necessario tenere in considerazione questo fatto nelle programmazioni di sostituzione delle attrezzature radiografiche . un lavoro della letteratura riporta che il 33% dei casi di mancata diagnosi di frattura ha lesioni impercettibili al momento dellesame [ 14 ]  . 
this fact should be borne in mind when planning of the upgrade of radiological equipment . one study reported that 33% of cases of failure to diagnose a fracture were related to imperceptible lesions at the time of the examination [ 14 ]  . 
informing the radiologist of a specific clinical suspicion or an anatomical site requiring greater attention because it is particularly symptomatic can in fact influence their interpretation of dubious images or findings at the limit between normal and pathological , thus prompting additional views being taken that are better able to reveal an abnormality . 
 we believe it is useful to provide the patient with an information sheet reminding them the radiographic examination can return false - negative errors in the presence of small fractures and that such fractures are often identifiable several days later . 
the patient is therefore invited to return for a further evaluation in the event that symptoms persist . a correct characterisation was not possible in three cases . here , the radiographs showed no fractures and the administrative documentation gave no description of the episode and / or lesion able to reveal its nature . lastly , in six out of 22 cases , analysis of the episode demonstrated that the problem giving rise to the litigation did not originate from the radiological service ( the radiologist had correctly identified the lesion ) but was probably due to the overall treatment of the trauma or the aftereffects thereof . it should be noted that our series refers to litigation regarding the accident and emergency radiology department and does not enable an analysis of the rate of error in the diagnoses performed by radiologists who work in the department . 
the discrepancy between the rate of error reported in the literature and the litigation rate in our study is probably due to the fact that in clinical practice , very few cases can present evidence of harm due to error , and therefore the claim for compensation . however , we believe that the study we performed is still useful . 
una stretta collaborazione con il medico che ha visitato il paziente pu , in taluni casi , rivelarsi utile : la segnalazione di un sospetto clinico specifico o di sedi anatomiche su cui necessaria maggiore attenzione perch particolarmente sintomatiche pu infatti influire sullinterpretazione di immagini dubbie o di reperti al limite tra normale e patologico , inducendo talora lesecuzioni di proiezioni aggiuntive in grado di meglio svelare unalterazione . 
riteniamo utile fornire ai pazienti un foglio informativo su cui si ricorda che le indagini radiografiche possono presentare errori falso - negativi a fronte di piccole fratture e che queste possono essere spesso riconoscibili a distanza di pochi giorni . 
il paziente viene quindi invitato a ripresentarsi per una nuova valutazione qualora la sintomatologia persista . non stato possibile inquadrare correttamente tre casi . in essi i radiogrammi non mostravano fratture e la documentazione amministrativa non presentava una descrizione dellepisodio e / o della lesione sufficiente a comprenderne la natura . 
radiologia ( il radiologo aveva identificato correttamente la lesione ) ma , verosimilmente , riguardava il trattamento complessivo del trauma o i postumi dello stesso . importante sottolineare che la nostra casistica si riferisce al contenzioso relativo alla radiologia del dea e non consente di analizzare la percentuale di errore nelle diagnosi effettuate dai radiologi che lavorano nel reparto . in considerazione del fatto che , mediamente , si effettuano circa 50000 esami radiologici per anno in questa struttura , la percentuale di contenzioso si aggira intorno allo 0 , 0016% . 
la discrepanza tra le percentuali di errore riportate in letteratura e la nostra incidenza di contenzioso verosimilmente ascrivibile al fatto che , nella pratica clinica , solo in pochissimi casi si arriva allevidenza del danno da errore e , quindi , alla richiesta risarcitoria . riteniamo comunque che rimanga lutilit dellanalisi da noi effettuata . 
questa infatti ha consentito di rivisitare alcune prassi amministrative e riconoscere alcune criticit organizzative , tecniche o interpretative che ci hanno portato a individuare strumenti che speriamo atti a ridurre ulteriormente il gi basso livello di contenzioso ed a permettere la creazione di una pi dettagliata mappa del rischio , con la conseguente rivalutazione di premi e franchigie , al fine di favorire il recupero di risorse per lassistenza . 
802810 published online : 18 september 2009 springer - verlag 2009 radiological features of complications of laparoscopic adjustable gastric banding caratteristiche radiologiche delle complicanze del bendaggio gastrico per via laparoscopica t . 
bellomi1 , 2 1department of radiology , european institute of oncology , via ripamonti 435 , 20141 milan , italy 2school of medicine , university of milan , milan , italy 3department of pathology and laboratory medicine , european institute of oncology , milan , italy 4department of clinical haemato - oncology , european institute of oncology , milan , italy 5breast imaging unit , department of radiology , european institute of oncology , milan , italy correspondence to : s . 
magnetic resonance imaging ( mri ) features of these malignancies can be relevant in establishing the extent of disease and planning the appropriate therapeutic strategy , usually represented by chemoand radiotherapy , rather than surgery . 
for mass - like lesions , kinetic curve assessment of initial rise showed slow enhancement in one lesion , rapid enhancement in four lesions and medium enhancement in one lesion . 
mri features of primary breast lymphomas in this study cohort suggest that the occurrence of a pbnhl should be considered in the presence of large enhancing lesions of the breast , especially if associated with skin thickening . 
le caratteristiche alla risonanza magnetica ( rm ) di queste neoplasie possono essere importanti nello stabilire lestensione della malattia e nel pianificare la pi efficace strategia terapeutica , che di solito rappresentata da chemio - radioterapia , piuttosto che dallescissione chirurgica . 
il diametro principale medio delle lesioni alla rm risultato di 44 mm ( range 1269 ) ; 6 lesioni hanno mostrato un enhancement di tipo nodulare ; 1 lesione ha mostrato un enhancement di tipo non - nodulare . 
per le lesioni nodulari , la fase iniziale della curva contrastografica dellintensit del segnale nel tempo ha mostrato un enhancement lento in 1 lesione , intermedio in 1 lesione , rapido in 4 lesioni . 
le caratteristiche rm dei linfomi primitivi mammari riscontrate nella nostra casistica non si sono 916 radiol med ( 2009 ) 114 : 915924 in the assessment of response to therapy and diagnosis of recurrence . keywords primary breast lymphoma breast mri dimostrate patognomoniche della patologia , tuttavia la presenza di un linfoma primitivo mammario dovrebbe essere presa in considerazione in caso di lesioni mammarie di grandi dimensioni , dotate di contrastenhancement , specie se associate ad ispessimento cutaneo . la rm ha inoltre un ruolo importante nella valutazione della risposta alla terapia e nella diagnosi di recidiva . parole chiave linfoma mammella rm introduction introduzione primary lymphomas of the breast ( pbnhl ) are uncommon , accounting for 0.04%0.05% of all breast malignancies and < 1% of all non - hodgkins lymphomas . 
diagnostic imaging of these malignancies is particularly relevant in establishing disease extent and planning the most effective therapeutic strategy , which relies on chemotherapy plus radiotherapy , sparing patients unnecessary and potentially harmful surgical procedures . 
mammographic and ultrasonographic imaging findings of pbnhl have been reported to be nonspecific [ 2 , 3 ] , whereas mri findings have been described in single case reports [ 48 ] or in a study analysing multiple diagnostic modalities , where mri was performed in one case only [ 9 ]  . 
 the aim of this study was to ascertain the occurrence of specific mri features in pbnhl and assess the importance of mri in studying this rare pathology . i linfomi primitivi della mammella sono rari , costituendo lo 0 , 04%0 , 05% di tutte le neoplasie maligne della mammella e meno dell1% di tutti i linfomi non - hodgksolo l1% dei pazienti che si presentano con malattia extranodale ha un coinvolgimento mammario [ 1 ]  . 
limaging diagnostico di queste lesioni pu essere particolarmente rilevante nello stabilire lestensione della malattia e nel pianificare la strategia terapeutica pi efficace , che si fonda su chemioterapia e / o radioterapia , piuttosto che su procedure chirurgiche , non necessarie e potenzialmente dannose . 
limaging pu avere inoltre un ruolo importante nella valutazione della risposta alle terapie e nella diagnosi di recidiva . gli esami diagnostici oggi a disposizione per i tumori mammari sono rappresentati da mammografia , ecografia e risonanza magnetica ( rm )  . 
laspetto mammografico ed ecografico dei linfomi primitivi mammari risultato non specifico [ 2 , 3 ] , mentre laspetto rm stato descritto solo in singoli case report [ 48 ] e in uno studio che ha valutato le altre modalit diagnostiche ma la rm in un unico caso [ 9 ]  . 
ci si propone inoltre di valutare limportanza del contributo della rm nello studio di tale patologia rara . materials and methods patients materiali e metodi pazienti all patients provided signed informed consent , including the possibility of use of anonymous data for scientific purposes . 
seven patients with pbnhl who underwent a breast mri examination during the diagnostic pretreatment workup were retrospectively retrieved from the november 2001 to november 2007 database of our radiology department . 
the diagnosis of p bnhl was obtained by biopsy ( n = 4 ) or by surgery ( n = 3 ) according to : ( 1 ) specific morphological features , such as size , nuclear features , diffuse or follicular architectural pattern of proliferation ; ( 2 ) immunophenotypical features , such as the use of antibodies recognising the cd3 , cd5 , cd10 , cd20 , cd23 and tutte le pazienti hanno sottoscritto il consenso informato per lesame rm con contrasto e un consenso informato generico comprensivo della possibilit di utilizzo dei dati acquisiti e resi anonimi per scopi scientifici . 
dai database disponibili presso la divisione di radiologia relativi alle rm mammarie eseguite tra novembre 2001 e novembre 2007 , sono state selezionate retrospettivamente sette pazienti con linfoma mammario primitivo , sottoposte a rm durante liter diagnostico pree post - trattamento . 
la diagnosi di linfoma mammario primitivo era stata ottenuta su prelievi bioptici in 4 casi e sul pezzo chirurgico in 3 casi , sulla base di specifiche analisi basate sullo studio della morfologia cellulare ( dimensione , caratteristiche dei nuclei e crescita follicolare o diffusa ) ; sulle caratteristiche radiol med ( 2009 ) 114 : 915924 ki - 67 antigens ; and ( 3 ) according to molecular analyses , such as evaluation of the ig heavy chain gene rearrangement by polymerase chain reaction analysis at fr2 and fr3a . 
 breast mri technique all mri examinations were performed on a 1 - t signa horizon lx ( ge medical systems , milwaukee , wi , usa ) in use during the period considered ( 20012007 )  . 
the imaging sequence included a localising sequence followed by coronal t1 - weighted fatsuppressed fast spoiled gradient - echo sequences without breast compression with the following parameters : tr = 7 ms , te = 1.6 ms , flip angle = 10 , ti = 25 ms , receiver bandwidth = 32 khz , nex = 1 , matrix = 320320 , fov = 3618 cm . slice thickness ranged between 2 mm and 3 mm according to the volume of the entire breast in order to maintain the single volume acquisition time within 2 m the same sequence was acquired once before and five consecutive times starting 10 s after rapid intravenous contrast injection of 0.1 mmol / kg gadolinium diethylenetriamine pentaacetic acid ( gd - dtpa ) ( magnevist bayer schering , germany ) at a flow rate of 2 ml / s , followed by a 20 - ml saline flush at the same flow rate . 
relative enhancement ( percentage changes of signal intensity course ) was calculated according to the following formula : sicsi / si100 , where sic and si represent signal intensities after and before contrast administration , respectively . interpretation of mri findings and lesion classification two radiologists retrospectively reviewed each preand post - treatment breast mri examination in consensus on a digital picture - archiving and communication system diagnostic workstation ( dynacad , invivo , orlando fl , usa ) also evaluating the maximum intensity projection ( mip ) images generated from the subtraction images . 
for each lesion , maximum diameter and longest orthogonal diameter in the same plane and distances of lesion to skin , to nipple and to chest wall were measured on the screen by using electronic callipers . 
 immunofenotipiche ( usando anticorpi specifici per gli antigeni cd3 , cd5 , cd10 , cd20 , cd23 e ki67 ) e sulle caratteristiche molecolari ( valutazione del riarrangiamento del gene codificante per le catene pesanti delle immunoglobuline tramite analisi pcr [ polymerase chain reaction ] delle regioni fr2 e fr3a )  . 
 risonanza magnetica tutti gli esami sono stati acquisiti con lapparecchio rm horizon lx 1t ( ge medical systems , milwaukee , wi , usa ) , in uso durante il periodo in esame ( 20012007 )  . 
dopo la sequenza di localizzazione sono state acquisite sezioni coronali fspgr t1 pesate con soppressione del grasso , senza compressione delle mammelle , secondo i seguenti parametri : tr = 7 ms , te = 1 , 6 ms , flip angle = 10 , ti = 25 ms , banda di ricezione = 32 khz , nex = 1 , matrice = 320320 , fov = 3618 c lo spessore delle sezioni variato da 2 a 3 mm , a seconda dello spessore complessivo della mammella , per mantenere il tempo di acquisizione totale inferiore ai 2 minuti per ciascuna acquisizione di volume . 
la stessa sequenza stata acquisita nelle condizioni basali e cinque volte dopo iniezione endovenosa rapida di mezzo di gadopentetato dimeglumina ( magnevist , bayer schering , germania ) 0 , 1 mmol / kg , velocit di flusso = 2 ml / s , seguita da un bolo di 20 ml di soluzione salina alla stessa velocit di flusso . 
lenhancement relativo ( percentuale di variazione dellintensit del segnale iniziale e tardivo ) stato calcolato sulla base della seguente formula : sic - si / si100 , dove sic e si rappresentano le intensit di contrasto del segnale rispettivamente dopo e prima la somministrazione di contrasto . interpretazione delle immagini rm e classificazione delle lesioni due radiologi hanno rivalutato retrospettivamente ciascun esame rm della mammella pree post - trattamento su una workstation di archiviazione digitale delle immagini ( dynacad , invivo corp . , orlando fl , usa ) , dove sono state valutate anche le ricostruzioni mip ( massima intensit di proiezione ) generate dalle immagini di sottrazione . 
per ogni lesione sono stati misurati a monitor : il diametro maggiore e il diametro massimo ad esso ortogonale nello stesso piano ; le distanze della lesione dalla cute , dal capezzolo e dalla parete toracica . 
 the noncontrast t1 appearance of lesions was described ogni lesione stata valutata secondo i criteri rm di 918 radiol med ( 2009 ) 114 : 915924 iso - , hyperor hypointense relative to breast parenchyma . 
the presence or absence of skin thickening in the same quadrant as the lesion was recorded and , if present , its distance from the lesion was also recorded . each lesion was evaluated according to the mr lexicon for enhancing breast lesions [ 10 , 11 ]  . 
for mass - like enhancement , descriptors used were shape ( round , oval , lobular , irregular or spiculated ) , margins ( smooth , lobular , irregular or spiculated ) and internal enhancement ( homogeneous , heterogeneous , rim enhancement , bright internal enhancing septations , dark septations ) [ 10 ]  . 
for nonmass enhancement , shape and borders are not assessable , and descriptors used were : distribution of contrast enhancement ( linear , linear branching , segmental , regional , patchy or diffuse enhancement ) [ 10 , 12 ] and internal contrast enhancement ( for linear or linear branching duct margins defined as smooth , undulating , or irregular borders , for segmental , regional , patchy or diffuse enhancement defined as homogenous , heterogeneous , clumped and stippled ) [ 10 , 11 ]  . kinetic curve assessment was performed by placing a freehand region of interest ( roi ) including the entire lesion in case of homogeneous internal pattern of contrast enhancement and on the most enhancing area for all other types of internal enhancement . 
delayed - phase enhancement was rated as persistent ( when curves exhibited steady signal intensity increase ) , plateau ( when curves exhibited fast early upstroke followed by plateau after the third postcontrast minute ) and washout ( when curves exhibited fast early upstroke followed by a loss of signal intensity after the third postcontrast minute )  . 
 an overall assessment category was eventually attributed to each lesion according to the mri breast imaging reporting and data system ( bi - rads ) categories introduced by the american college of radiology [ 11 , 1315 ]  . assessment categories included six classes : i = negative findings , ii = benign finding , iii = probably benign finding , iv = suspicious abnormality , v = highly suggestive of malignancy and vi = proven malignancy [ 14 ]  . 
 results the study population included seven women ( mean age 63 , range 4477 years ) with a histological diagnosis of pbnhl classified as follows : five diffuse large b - cell lymphoma ( dlbcl ) , one dlbcl with a focal ( accounting for 5% of neoplastic cells ) marginal zone [ mucosa - associated classificazione delle lesioni dotate di contrast - enhancement riportati in letteratura e internazionalmente accettati [ 10 , 11 ]  . 
secondo tali criteri le lesioni dotate di contrast - enhancement vengono classificate come : lesioni con enhancement nodulare ( mass - like enhancement ) , e lesioni con enhancement non nodulare ( non - mass like enhancement ) [ 10 , 11 ]  . per lenhancement nodulare , vengono comunemente valutate le seguenti caratteristiche : forma ( definita come rotonda , ovale , lobulare , irregolare , spiculata ) ; margini ( definiti come regolari , lobulari , irregolari o spiculati ) ; enhancement interno ( definito come omogeneo , eterogeneo , ad anello , con setti intralesionali contrastati , con setti intralesionali non contrastati ) [ 10 ]  . 
per le lesioni con enhancement non nodulare , forma e margini non sono valutabili , e pertanto in tali lesioni vengono comunemente valutate le seguenti caratteristiche : distribuzione del contrast enhancement ( definita come lineare , ramificata , segmentario , regionale , in regioni multiple , diffusa ) [ 10 , 12 ] ; enhancement interno ( definito a margini lisci , ondulati o irregolari per le lesioni lineari e ramificate ; definito come omogeneo , eterogeneo , addensato o puntinato per le lesioni segmentarie , regionali , in regioni multiple o diffuse ) [ 10 , 11 ]  . 
 lanalisi dinamica stata effettuata nelle lesioni nodulari posizionando una roi a mano libera comprendente tutta la lesione in caso di enhancement interno omogeneo , e nellarea dotata di maggior contrast - enhancement per tutti gli altri tipi di contrast - enhancement interno . 
lenhancement tardivo stato classificato come : persistente quando la curva mostrava un incremento dellintensit del segnale costante ; con plateau quando la curva mostrava una fase iniziale di incremento rapido seguita da una stabilit della curva anche dopo il terzo minuto dalliniezione del mezzo di contrasto ; con wash out quando la curva mostrava una fase iniziale di incremento rapido seguita da una diminuzione dellintensit del segnale dopo il terzo minuto dalliniezione del mezzo di contrasto . 
tale classificazione include 6 categorie : i = reperto negativo , ii = reperto francamente benigno , iii = reperto probabilmente benigno , iv = reperto sospetto per malignit , v = reperto altamente suggestivo di malignit , vi = reperto con diagnosi istologica di malignit [ 14 ]  . risultati lo studio ha incluso 7 donne ( et media 63 , range di et 4477 ) con diagnosi istologica di linfoma mammario primitivo , classificato come segue : 5 linfomi a grandi cellule b , 1 linfoma a grandi cellule b con una piccola componente della zona marginale ( che ammontava a meno del 10% delle cellule neoplastiche ) , 1 linfoma extranodale della zona marginale ( malt - linfoma )  . 
tutte le lesioni erano clinicamente palpabili prima del trattamento e localizzate come segue : 2 supero - esterne a destra , 1 supero - esterna sinistra , 1 radiol med ( 2009 ) 114 : 915924 lymphoid tissue ( malt ) ] component and one extranodal marginal zone ( malt ) lymphoma . 
all lesions were clinically palpable before treatment and were located as follows : two right upper - outer , one left upper - outer , one left upper - internal , one right lower - outer , one left lowerinternal and one right upper - internal . 
all patients with a diffuse large b - cell lymphoma received acod ( adriamicin - ciclophosphamide - vincristine - deltacortene ) [ chop - like chemotherapy ( ciclophosphamide - hydroxydaunorubicina - onsovin - prednisone ) ] for four to six cycles , followed by 4446 gy radiotherapy of the breast and positive nodes if present . 
 posttreatment mri examinations showed complete remission of local disease at the end of therapy for all seven patients , whereas for two of them ( one large b - cell lymphoma , one malt lymphoma ) , mri detected recurrence in the contralateral breast . 
the mean mri longest diameter was 44 mm ( range 1269 ) , and the mean longest orthogonal diameter measured in the same plane was 32 mm ( range 943 )  . 
the mean distance from the skin , the nipple and the chest wall was 15 mm ( range 423 ) , 43 mm ( range 670 ) and 21 mm ( range 549 ) , respectively . 
tutte le pazienti con linfoma diffuso a grandi cellule b hanno ricevuto chemioterapia acod ( adriamicin - ciclophosphamide - vincristinedeltacortene ) [ chop - like ( ciclophosphamide - hydroxydaunorubicina - onsovin - prednisone ) ] , per 4 o 6 cicli , seguita da 4446 gy di radioterapia della mammella e di eventuali linfonodi positivi , quando presenti . 
 gli esami rm post - trattamento hanno mostrato remissione completa della malattia locale alla fine delle terapie per le 7 pazienti , ma per 2 di loro ( 1 con linfoma a grandi cellule b , 1 con malt - linfoma ) la rm di follow - up ha dimostrato una recidiva nella mammella controlaterale . queste due pazienti sono state pertanto nuovamente sottoposte a chemioterapia , dopo la quale hanno mostrato remissione completa . 
le distanze medie dalla cute , dal capezzolo e dalla parete toracica sono risultate rispettivamente di 15 mm ( range 423 ) , 43 mm ( range 670 ) e 21 mm ( range 549 )  . 
laspetto t1 pre - contrasto risultato iso - intenso rispetto al parenchima mammario in cinque lesioni ( 4 linfomi a grandi cellule b , 1 maltlinfoma ) e iper - intenso in due lesioni ( entrambe linfomi a grandi cellule b )  . 
in 3 / 7 casi ( due linfomi diffusi a grandi cellule b e un lnh con una piccola componente della zona marginale ) , si osservato ispessimento cutaneo , la cui distanza dalla lesione era di 10 , 16 e 17 mper le caratteristiche rm di contrast - enhancement , sei lesioni sono state classificate come nodulari ( 5 linfomi a grandi cellule b , 1 malt - linfoma ) ; e una lesione come non - nodulare ( linfoma a grandi cellule b )  . 
per quanto riguarda le lesioni nodulari , la forma stata classificata come segue : rotonda n = 2 , lobulare n = 2 , ovale n = 1 , irregolare n = 1 . 
i margini sono stati valutati come : regolari n = 2 , irregolari n = 3 , spiculati n = 1 . lenhancement delle lesioni nodulari stato classificato come : omogeneo n = 2 , eterogeneo n = 2 , ad anello n = 2 . landamento iniziale della curva dinamica del segnale ha mostrato : enhancement lento n = 1 , enhancement intermedio n = 1 , enhancement rapido n = 4 . 
il picco medio di enhancement risultato del 110% ( range 78162 ) , dove il picco maggiore ( 162% ) apparteneva allunico linfoma a grandi cellule b che ha mostrato anche wash out tardivo . per lunica lesione con enhancement non nodulare , che era un linfoma a grandi cellule b , la distribuzione dellenhancement stata definita come enhancement in regioni multiple , con caratteristiche interne di tipo puntinato . 
1a - c post - gadolinium t1 - weighted breast magnetic resonance imaging ( mri ) ( a ) of a 69 - mm b - cell primary right breast lymphoma showing a masslike enhancement area , with lobular shape , irregular margins and heterogeneous contrast enhancement . 
kinetic curve assessment based on a freehand region of interest ( roi ) placed on the entire area ( b ) shows a 128% initial rise and a delayed plateau ( c )  . 
1a - c immagine rm mammaria t1 pesata post - gadolinio ( a ) di linfoma primitivo a grandi cellule b della mammella destra di 69 mm , che mostra un area solida , con forma loblulare , margini irregolari , ed eterogeneo contrastenhancement . 
la valutazione della curva dinamica basata sul posizionamento manuale di una roi nellintera area ( b ) mostra una crescita iniziale del 128% e un plateau tardivo ( c )  . 
in a recent study performed at stanford university and encompassing 10 , 125 lymphoma patients admitted from 1981 to 2005 , only 51 cases had lymphoma involving the breast [ 17 ]  . 
as a consequence , there are few data concerning the radiological features of breast lymphoma , which mainly arise from mammographic and ultrasonographic examinations [ 2 , 3 , 6 , 7 , 9 , 18 , 19 ] , which led to the conclusion that pbnhl does not show specific radiological features [ 2 , 3 ]  . 
described one or more than one primitivo della mammella , con o senza diffusione ai linfonodi regionali , in assenza di linfoma pre - esistente o simultaneo in altre sedi [ 2 , 4 ]  . 
il linfoma mammario , sia come sede primitiva che come sede secondaria di malattia , raro , rappresentando lo 0 , 04%0 , 53% di tutti i tumori maligni della mammella [ 5 , 16 ]  . 
la maggior parte di tali lavori ha valutato solo esami mammografici e ecografici [ 2 , 3 , 6 , 7 , 9 , 18 , 19 ] , e sostanzialmente tutti concordano sul fatto che i linfomi primitivi della mammella non hanno aspetti radiologici specifici [ 2 , 3 ]  . 
per quanto riguarda gli aspetti mammografici , i risultati descritti non sono omogenei : alcuni autori hanno descritto una o pi lesioni ben circoscritte senza microcalcificazioni e con reazione desmoplastica [ 2 ] ; con densit parenchimale diffusamente aumentata , anche associata ad ispessimento cutaneo [ 2 , 5 ] ; altri radiol med ( 2009 ) 114 : 915924 relatively well - circumscribed lesion without calcifications or desmoplastic reaction [ 2 ] ; soyupak et al . 
described bilateral miliary densities , especially during secondary involvement [ 20 ]  . at ultrasonography , primary and secondary breast lymphomas have been described as well or as poorly defined masses with a hypoto hyperechoic appearance , focal or diffuse heterogeneous parenchymal involvement [ 21 ] and variable attenuation [ 7 ]  . 
due to the rarity of this condition , mri findings of pbnhl have been previously described only as single case reports [ 48 ] or as single cases included in a larger series [ 9 ]  . 
in our study cohort , 2 / 7 lesions ( both dlbcl ) were hyperintense relative to breast parenchyma . a recent study has emphasised the importance of mri lesion size , suggesting that it may be helpful in predicting the likelihood of malignancy . 
this finding was not compared with mammographic and ultrasonographic images of the same time period because they were not available , but it was not mentioned in the radiological report and therefore probably not apparent . 
 in agreement with the above - mentioned studies [ 48 ] showing a rapid and strong contrast enhancement with an increase in signal intensity of > 80% in the first part of the curve , in our series , the mean peak of enhancement was 110% . 
unfortunately , this finding is generically expected for all malignant lesions , regardless of histological type , autori hanno descritto lesioni con margini netti o lievemente irregolari [ 8 ] ; altri ancora hanno descritto un coinvolgimento linfomatoso secondario con densit bilaterali miliariformi [ 20 ]  . 
 laspetto ecografico dei linfomi mammari primitivi o secondari stato variamente descritto come masse da ipoa iperecogene , ben defnite o poco definite , con coinvolgimento parenchimale eterogeneo focale o diffuso [ 21 ] , e con attenuazione variabile del fascio ultrasonoro [ 7 ]  . 
a causa della relativa rarit di questa patologia , gli aspetti rm dei linfomi primitivi sono stati finora descritti solo come report di singoli casi [ 48 ] o come singoli casi inclusi in casistiche pi numerose [ 9 ]  . 
nella nostra casistica , 2 / 7 lesioni ( entrambe linfomi a grandi cellule b ) hanno mostrato iperintensit rispetto al parenchima mammario , mentre le rimanenti 5 hanno mostrato sostanziale isointensit . 
tale reperto rm non stato da noi confrontato con gli esami ecografico e mammografico dello stesso periodo in quanto non disponibile , tuttavia esso non era riportato nel referto e pertanto verosimilmente non evidenziabile . 
 in accordo coi suddetti case report [ 48 ] che descrivevano un contrast - enhancement rapido e marcato dei linfomi mammari primitivi , con crescita iniziale dellintensit del segnale maggiore dell80% , nella nostra casistica il picco medio di enhancement stato del 110% . 
2a , b postgadolinium t1 - weighted breast mri of a b - cell primary left breast lymphoma showing a 12 - mm mass - like enhancing lesion , with round shape , smooth margins and homogenous contrast enhancement ( a )  . 
kinetics of the lesion , based on a freehand region of interest ( roi ) ( b ) showed a 92% initial rise and a postinitial plateau ( c )  . 
2a , b imamgine rm mammaria t1 post - gadolinio di un linfoma primitivo a cellule b della mammella sinistra , che mostra una lesione solida di 12 mm , con forma rotonda , margini regolari e omogeneo contrast - enhancement ( a )  . la cinetica della lesione , basata sul posizionamento manuale di una roi ( b ) , mostrava una crescita iniziale del 92% ed un plateau tardivo ( c )  . 
 at variance with a previous study [ 9 ] where the only pbnhl analysed by mri showed a washout , in this series , only one lesion showed a washout ( dlbcl ) , whereas the remaining lesions showed a plateau in five cases . 
in our series , we did not find multifocality or multicentricity at the time of diagnosis , but the diagnosis of multifocality or multicentricity of breast lymphomas may be less important than for other breast malignancies in that surgery is not the most appropriate therapy . 
however , the possibility of a differenza di uno studio precedente [ 9 ] , in cui il solo linfoma mammario analizzato con rm mostrava un wash out nella fase tardiva della curva contrastografica , nella nostra casistica soltanto 1 lesione ( linfoma a grandi cellule b ) ha mostrato un wash out , mentre le altre 5 lesioni con contrast - enhancement di tipo nodulare hanno mostrato un plateau . 
 [ 9 ] potrebbe dipendere dalle grandi dimensioni della lesione da lui analizzata ( 11 , 6 cm ) , che sostituiva quasi lintera mammella mentre le nostre lesioni , pur avendo un diametro mediamente grande ( 44 mm ) comprendevano un range tra 12 e 69 mm . relativamente al noto importante contributo della rm nella valutazione di multicentricit e multifocalit dei tumori mammari , kiresi et al . 
nella nostra casistica non erano presenti tumori multifocali alla diagnosi e comunque la diagnosi di multifocalit o multicentricit dei linfomi rispetto ad altri tumori mammari pu radiol med ( 2009 ) 114 : 915924 demonstrating multifocal and multicentric lesions by mri may be helpful during staging in order to eventually correctly evaluate response to treatment . 
indeed , the examination performed after treatment showed complete remission in all patients and , during follow - up , showed the occurrence of a contralateral relapse in two patients , both of whom underwent additional treatments , achieving a complete final remission . 
nevertheless , this technique may hamper immunophenotyping and molecular analyses , which are mandatory to correctly diagnose and subclassify nonhodgkins lymphoma lesions [ 2426 ] and to plan subsequent radiotherapy and chemotherapy instead of surgery . 
thus , when mri interpretation of enhancing lesions of the breast includes pbnhl in the differential diagnosis , the patient should undergo a core biopsy rather than an fnac . treatments , which include a possible limitation of our study is the lack of data relative to the t2 appearance of lesions . 
 another limitation of this study is the lack of data relative to the mammographic and ultrasonographic appearance of the lymphoma lesions and to the consequent lack of a comparison among different diagnostic methods . 
unfortunately , the cases included in this study had been evaluated by mammography and ultrasound in other hospitals , and only the written reports without images were available . however , this limitation may lead to a possible future study comparing mammographic , sonographic and mri appearance of breast lymphomas . 
 in conclusion , our data suggest that the occurrence of a pbnhl should be kept in mind , especially for large enhancing lesions and in the presence of skin thickening , in order to plan the most accurate diagnostic procedures , which should include core biopsy rather than fine - needle aspiration . 
furthermore , mri may also have an important role in monitoring the response to therapy and during follow - up in the diagnosis of recurrence . essere meno importante , in quanto la terapia pi appropriata non chirurgica . 
tuttavia la possibilit della rm di dimostrare eventuali lesioni multifocali e multicentriche pu essere importante nella valutazione basale per la valutazione della risposta alle terapie . in accordo a quanto affermato da espinosa et al . 
infatti , gli esami rm eseguiti dopo trattamento hanno mostrato una completa remissione di malattia in tutte le pazienti e , durante il follow - up , lesame rm ha mostrato la presenza di recidiva controlaterale in due pazienti , che sono state sottoposte ad ulteriori cicli di terapia medica , con successiva remissione completa . la diagnosi di una massa tumorale mammaria spesso basata sullanalisi citologica da ago - aspirato . 
tuttavia , lago - aspirato pu non consentire lesecuzione di analisi immunofenotipiche e molecolari , che sono fondamentali per la corretta diagnosi e classificazione dei linfomi non - hodgkin [ 2426 ] e per la conseguente pianificazione terapeutica che per i linfomi si basa su chemioterapia e radioterapia e non sulla chirurgia . 
pertanto , quando linterpretazione rm di lesioni mammarie include i linfomi nella diagnosi differenziale , potrebbe essere opportuno eseguire una biopsia piuttosto che un ago - aspirato . un limite del presente studio la mancanza di dati relativi allaspetto dei linfomi mammari nelle sequenze t2 . questo dovuto al fatto che la selezione delle pazienti con diagnosi istologica di linfoma mammario primitivo stata fatta retrospettivamente . 
tale limite rappresenta un possibile spunto di studio futuro che potrebbe confrontare i reperti mammografici , ecografici e rm dei linfomi primitivi della mammella . in conclusione , le caratteristiche rm dimostrate dai linfomi primitivi mammari nella nostra casistica non sono patognomoniche della patologia . 
tuttavia i nostri dati suggeriscono che la presenza di un linfoma mammario primitivo dovrebbe essere presa in considerazione in presenza di lesioni di grandi dimensioni dotate di contrastenhancement , specie se associate ad ispessimento cutaneo . tale sospetto dovrebbe suggerire lesecuzione di una biopsia invece che di un ago - aspirato per unaccurata diagnosi e pianificazione terapeutica . 
sartor. springer - verlag berlin heidelberg , 2009 isbn 978 - 3 - 540 - 32983 - 1 published online : 15 september 2009 springer - verlag 2009 the monumental and revolutionary steps in the development of technical procedures in coronary radiology that have taken place in the past few years are properly and clearly depicted in the second edition of this book . 
 to study and evaluate coronary events nowadays there is not only the old - fashioned invasive and in some respects dangerous arteriography ; but also non invasive techniques ( multidetector ct angiography and dual source tomography , 2d and 3d visualization techniques , mri coronary angiography ) that quikly provide and with no danger to the patient ( beware in any case high dose multidetector ct ) all possible information for the clinician and radiologist . 
all these modalities may in any case be implemented by conventional catheterization , quantitative angiography as well as intravascular ultrasound . following the introductory chapter on coronary anatomy , the results that can be obtained with all the above techniques are well described in two chapters ( invasive coronary catheterization ; non - invasive coronary imaging ) subdivided in ten sections . 
one longer chapter ( non - invasive measurements of coronary atherosclerosis ) which is divided in 6 sections , deals with the problem of coronary atherosclerosis and calcifications ( pathophysiology , detection , measure , epidemiology , calcium as indicator of disease , screening ) and how to evaluate and treat these according to the recommendations of the us and european societies of cardiac imaging and radiology as well as the cardiology societies on both sides of the ocean . the last chapter provides an overview of the problems with image visualization , how to obtain , store , retrieve and use these in the daily practice in the ever increasing and demanding world of hospital generated images ( dicom , pacs etc . ) many beautiful black and white and colour images are to i progressi giganteschi ed in un certo modo rivoluzionari nello sviluppo delle tecniche di indagine per lo studio delle coronarie che si avuto negli ultimi anni messo ben in evidenza e spiegato in modo chiaro nella seconda edizione di questo volume . 
montemezzi6 1centro di prevenzione senologica ( cps ) , po marzana , ulss 20 , verona , italy 2istituto di radiologia , universit degli studi di verona , policlinico g.b. 
di senologia clinica e screening mammografico , dipartimento di radiodiagnostica , trento , italy 4registro tumori , istituto oncologico veneto ( iov ) , padova , italy 5istituto per lo studio e la prevenzione oncologica ( ispo ) , firenze , italy 6dipartimento interaziendale di radiologia , verona , italy correspondence to : s . 
volta 171 , 50131 firenze , italy , tel . : + 39 - 055 - 5012275 , fax : + 39 - 055 - 5001623 , e - mail : stefano - ciatto@gmail.com received : 17 october 2008 / accepted : 13 january 2009 / published online : 23 june 2009 springer - verlag 2009 abstract purpose . 
le mammografie refertate come negative precedenti i ci sono state sottoposte a revisione con modalit cieca ( mescolate casualmente con controlli negativi in ragione 2 : 1 ) da tre radiologi esperti e classificati in base ai criteri delle linee guida europee ( oc = occulto , sm = segni minimi , es = errore di screening ) in base al giudizio di maggioranza . 
il tasso di incidenza proporzionale stato del 10 , 8% nel primo e del 40 , 0% nel secondo anno dellintervallo ( standard europeo rispettivamente < 30% o < 50% , )  . 
alla revisione i ci sono stati classificati come es , sm , o oc rispettivamente nel 15 , 0% , 14 , 0% , o 71 , 0% dei casi . 
la verifica degli indicatori precoci di efficacia basati sui ci fattibile in un programma corrente di screening e dovrebbe divenire una procedura di routine . parole chiave carcinoma mammario screening mammografia carcinomi di intervallo introduction introduzione interval cancers ( ics ) account for a relatively infrequent event in mammography screening practice , their frequency being reported to be lower than 0.1% of screened subjects [ 13 ]  . 
ics are categorised according to the year of diagnosis in the interval ( first or second ) and classified according to the presence of different degrees of interpretation error at review . 
ics , however , are the most reliable indicators of screening programme sensitivity . monitoring ic is recommended as a standard procedure by several institutions [ 13 ] , and ic surveys have been reported by some italian screening programmes [ 46 ]  . 
 ic survey is based on the assessment or their frequency ( proportional incidence : ratio of observed ic to cancers expected according to underlying baseline incidence , in absence of screening ) and on the review of screening mammograms preceding ic , originally reported as negative , and their classification according to categories predefined by the european guidelines [ 3 ]  . 
 our study aimed to report the proportional incidence and review of ic observed in an italian screening programme during 20002006 . materials and methods i carcinomi intervallo ( ci ) costituiscono un evento relativamente raro nella pratica dello screening mammografico , con un tasso inferiore allo 0 , 1% soggetti esaminati [ 13 ]  . definiti come tutti i carcinomi invasivi insorti a seguito di episodio negativo di screening e prima della successiva mammografia di screening , vengono categorizzati in base allanno di comparsa ( primo o secondo ) e classificati in base alla presenza o meno di un errore diagnostico alla revisione . 
il monitoraggio dei ci raccomandato come procedura standard da diverse istituzioni [ 13 ] ed stato oggetto di pubblicazione da parte di alcuni programmi di screening italiani [ 46 ]  . 
 lanalisi dei ci si basa classicamente sia sulla verifica della loro frequenza ( incidenza proporzionale , o rapporto tra ci osservati e carcinomi attesi in assenza di screening , questi ultimi pari alla incidenza di base ) , che sulla revisione dei radiogrammi di screening precedenti il ci , refertati come negativi , e classificati in base a categorie predeterminate , definite nelle linee guida europee [ 3 ]  . il presente studio si ripropone di presentare i dati relativi alla incidenza proporzionale e alla revisione dei ci osservati in un programma di screening italiano dal 2000 al 2006 . a population - based mammography screening programme , inviting resident women aged 5059 years , has been ongoing in verona since 1999 and is run by the centre for breast prevention of marzana hospital , local health unit 20 . 
the main characteristics of the programme have been already materiali e metodi un programma di screening mammografico biennale di popolazione rivolto alle donne residenti , dai 50 ai 69 anni di et , in corso a verona dal 1999 , gestito dal centro di radiol med ( 2009 ) 114 : 907914 reported : the most original feature is real - time diagnostic assessment of screen positives , a practice associated with higher recall rates [ 7 ] but also with good sensitivity levels , as recently reported for some programmes of the veneto region [ 6 ]  . 
this study refers to a period when single reading was in use , whereas conventional double reading has been recently adopted , according to european guidelines [ 3 ]  . the studied series was identified in the computerised database of the screening programme , and consists of 85 , 380 negative screening episodes during 20002006 . 
in order to identify ic occurring within 2 years of a negative screening episode , the screening database was linked to different sources , namely : ( a ) screening programme archives of detected cancers ( the screening unit also runs a breast clinic for symptomatic or self - referring subjects from the same population area ) ; ( b ) cancer registry archives of the veneto oncological institute ; and ( c ) hospital discharge records ( hdr ) made available from the veneto regional health authorities . 
the latter hdr archive , originally created for administrative purpose , is increasingly used as a support for clinical studies ( and for cancer registries ) , as it provides the final diagnosis and is rapidly available ( within a few months ) after discharge . 
hdr use to identify ic has been recently recommended in a ministry of health position paper [ 2 ] and has been employed with success in some italian screening experiences [ 5 , 6 ]  . ic proportional incidence was thus calculated as the ratio of observed ic ( identified according to the above - mentioned linkage process ) to cancers expected ( obtained by multiplying the age - specific underlying incidence rate by the number of women - year at risk )  . 
ic proportional incidence is a direct indicator of sensitivity according to the formula : sensitivity = 1 ( observed ic / expected cancers ) this formula is the most reliable to estimate screening sensitivity and is recommended by the european guidelines , which also provide reference standards for the maximum acceptable proportional incidence in the first ( reference < 30% ) or second ( reference < 50% ) year of the interval [ 3 ]  . finally , mammograms preceding ic were reviewed to assess radiologists responsibility . 
this number is different from the total of observed ic ( n = 95 ) considered to calculate proportional ic incidence as : ( a ) the review process also reviewed carcinomas in situ ( 7 of 100 cases ) , as recommended by european guidelines , which are excluded when assessing proportional incidence ; ( b ) some cases were not reviewed , as mammograms were not retrieved or had indelible marks that would have influenced review ; and ( c ) some reviewed cases were not included when assessing proportional incidence as they were too recent , occurring later than the considered period . 
le principali caratteristiche del programma sono gi state oggetto di pubblicazione : la pi peculiare certamente lesecuzione immediata degli approfondimenti diagnostici delle alterazioni rilevate alla mammografia , pratica che se risultata essere associata a un tasso di richiamo piuttosto elevato [ 7 ] consente comunque una buona sensibilit , come stimato in una recente valutazione di alcuni programmi della regione veneto [ 6 ]  . 
lo studio fa riferimento a un periodo in cui era in uso la lettura singola , recentemente implementata dalla doppia lettura in ottemperanza alle raccomandazioni europee [ 3 ]  . la casistica mammografica oggetto di studio stata identificata dallarchivio computerizzato del programma di screening ed pari a 85380 mammografie refertate come negative dal 2000 al 2006 . 
questultimo strumento , nato principalmente per uso amministrativo , viene sempre pi usato come supporto a studi clinici ( e a registri tumori ) , in quanto riporta la diagnosi di dimissione ed disponibile con breve latenza ( pochi mesi ) rispetto al ricovero . 
luso delle sdo per lidentificazione dei ci stato recentemente raccomandato da una position paper del ministero della salute [ 2 ] , ed stato gi usato con successo in esperienze di screening italiane [ 5 , 6 ]  . identificati in tal modo i ci nel biennio successivo allesame mammografico , si proceduto al calcolo dellincidenza proporzionale di ci : tale misura fa riferimento al rapporto tra ci effettivamente osservati e carcinomi attesi in assenza di screening ( questi ultimi calcolati in base agli anni - persona e ai tassi incidenza et specifici forniti dal registro tumori locale ) , corrisponde al reciproco della sensibilit , che viene calcolata secondo la formula : sensibilit = 1 ( ci osservati / carcinomi attesi ) tale formula quella pi affidabile per il calcolo della sensibilit ed raccomandata dalle linee guida europee che forniscono anche standard di riferimento per il rapporto osservati / attesi , pari ad un massimo del 30% per il primo anno , o del 50% per il secondo anno dellintervallo [ 3 ]  . infine sono stati rivalutati i radiogrammi di screening precedenti ai ci per verificare la presenza o meno di un 910 radiol med ( 2009 ) 114 : 907914 reviewed as : ( a ) screening error ( se : abnormality worthy of further diagnostic assessment ) ; ( b ) minimal sign ( ms : minimal changes , not enough to prompt diagnostic assessment , perceived by the reviewer only as a consequence of adopting a lower threshold for suspicion [ the attempt to maximise sensitivity is common in a test setting ] or with knowledge of ic side , site and features at diagnosis ) ; or ( c ) occult ( oc : no abnormality worthy of diagnostic assessment )  . 
as three reviewers were involved , review classification category was determined according to majority report : lesions identified by at least two reviewers were classified as se , lesions identified by one reviewer only were classified as ms and lesions not identified by any reviewer were classified as oc . 
involved reviewers ( a , b , c ) are expert radiologists currently involved in screening programmes and with at least 5 years of mammography practice , blinded to which were cases and controls in the set . 
table 1 shows observed ics and expected cancers , overall and by age group . observed / expected ratio ( o / e ) was 10.8% in the first and 40.0% in the second year of the interval . 
lo standard di buona pratica indicato nelle linee guida europee di una proporzione non superiore a 20 es ogni 100 ci sottoposti a revisione . essendo stato impiegato un numero dispari di revisori , la classificazione stata definita a maggioranza : sono stati classificati es le lesioni identificate da almeno 2 revisori , sm le lesioni identificate da un solo revisore , e oc le lesioni non identificate da alcun revisore . 
i tre revisori ( a , b , c ) sono radiologi esperti ( con almeno 5 anni di esperienza mammografica ) , coinvolti in programmi di screening di popolazione , e che non avevano visto in precedenza i casi in revisione . 
al fine di evitare una sovrastima dellerrore di interpretazione a maggior tutela del radiologo lettore , come raccomandato dal ministero della salute [ 2 ] , stata applicata la procedura della revisione cieca . tale procedura , che vede i casi di ci ( n = 100 ) mescolati a un elevato numero di controlli negativi verificati ( negativi anche allo screening successivo , n = 200 , in rapporto 2 : 1 ) , scelti a caso dalla stessa casistica di screening , risulta assai pi garantista per il radiologo lettore rispetto alla revisione semi - informata ( del solo esame di screening precedente il ci ) o informata ( del solo esame di screening precedente il ci , con disponibile anche la mammografia diagnostica che rivela lato , sede e morfologia del ci )  . 
positive predictive value for ic was 51.7% for se lincrocio con i diversi archivi ha portato allidentificazione per il periodo 20002006 di 95 ci , 28 comparsi nel primo anno dellintervallo e 67 nel secondo anno . 
la tabella 1 presenta i ci osservati e i carcinomi attesi , complessivi e per classe di et . il rapporto osservati / attesi risultato essere 10 , 8% per il primo e 40 , 0% per il secondo anno . 
assumendo lo stesso numero di anni - paziente nel primo e secondo anno , il rapporto osservati / attesi e la sensibilit nellintervallo biennale risultano rispettivamente 25 , 4% e 74 , 6% . i 100 casi e i 200 controlli selezionati per la revisione non differiscono significativamente per et ( et media casi = 59 , 7 , controlli = 57 , 0 anni , p = 0 , 58 ) , mentre differiscono significativamente per densit radiologica ( bi - rads casi : d1 = 41% , d2 = 32% , d3 = 17% , d4 = 10% ; controlli : d1 = 58% , d2 = 25% , d3 = 12 , 5% , d4 = 4 , 5% ) quando si considerino quattro categorie di densit ( d1 / 2 / 3 / 4 ; p = 0 , 02 ) e con significativit marginale quando se ne considerino due ( d1 - 2 / 3 - 4 ; p = 0 , 07 )  . 
i risultati della revisione dei radiogrammi di screening negativi sono riportai nella tabella 2 . la frazione di ci classificati come es stata del 15 , 0% , mentre la stessa categoria stata attribuita al 7 , 0% dei controlli negativi ( p = 0 , 04 )  . 
blind review of ic is the most correct methodology to assess potential reading errors [ 8 , 9 ] , as it tends to reproduce the current screening scenario , where radiologists are not aware of which cases will develop ic , among a large majority of true negative cases . 
blind review , recommended by the italian study group for mammography screening ( gisma ) [ 1 ] , has been recently suggested as a standard procedure by the italian ministry of health [ 2 ]  . 
 the choice of a 2 : 1 control / case ratio was motivated by the large number of cases to be reviewed and by the need to use a set of cases of a proper size to allow a relatively easy review process . 
the latter is no doubt higher than currently recommended at first screening ( 7% ) , although in the range of what is reported in similar studies [ 5 , 6 , 8 ]  . 
 la casistica in oggetto e sufficientemente ampia e studiata con una metodologia affidabile per la valutazione della performance del programma di screening in oggetto . lanalisi del tasso proporzionale dei ci nel primo e secondo anno successivo allo screening raccomandata dalle linee guida europee [ 3 ] , e lidentificazione dei ci mediante le sdo , raccomandata da autorit scientifiche e sanitarie nazionali [ 1 , 2 ] , risulta sufficientemente affidabile , dato il basso tasso di migrazione terapeutica extraregionale , ed stata gi usata con successo in altri studi analoghi [ 5 , 6 ]  . 
i tassi proporzionali di incidenza di ci osservati nel primo e secondo anno rientrano ampiamente nei limiti di sufficienza raccomandati ( primo anno 10 , 8% vs 30% raccomandato , secondo anno 40 , 0% vs 50% raccomandato )  . 
complessivamente la sensibilit del programma rispetto allintervallo biennale risulta essere del 74 , 6% , nella media rispetto a quanto osservato in centri di eccellenza [ 10 ]  . la revisione cieca dei ci risulta la modalit pi garantista rispetto al possibile errore diagnostico del radiologo [ 8 , 9 ] , in quanto si sforza di riprodurre la condizione corrente di screening ove il lettore ignaro di quali casi manifesteranno un ci tra una maggioranza di casi negativi . tale modalit di revisione , caldeggiata dal gruppo italiano di studio per lo screening mammografico ( gisma ) [ 1 ] , stata recentemente suggerita come quella ideale dal ministero della salute [ 2 ]  . 
 la scelta di accoppiare ad ogni caso solo due controlli deriva dallelevato numero di ci in studio e dalla necessit di costruire un set di dimensioni non eccessive per rendere agevole la revisione . 
 la scelta di definire le categorie di revisione ( ms o es ) non in base al giudizio di un solo revisore , ma in base alla maggioranza delle identificazioni della lesione da parte di un panel di revisori , appare pi affidabile , per le inevitabili differenze soggettive di giudizio delle anomalie radiologiche : che tali differenze di giudizio esistano ben evidente radiol med ( 2009 ) 114 : 907914 the choice of defining review categories ( ms or se ) according to majority detection rate of a reviewer panel rather than to a single reviewers judgement appears more reliable for the unavoidable interobserver variation in judging the degree of suspicion of perceived abnormalities . such a low reproducibility was confirmed in our study by the wide variation of recall rates of negative controls among reviewers . 
even when determined according to majority , se and ms categories maintained a substantially different positive predictive value for ic ( se = 51.7% , ms = 21.8% ) , as recommended in european guidelines . comparison between reviewers suggests that adopting significantly higher recall rates does not necessarily imply higher detection rates . 
as a collateral finding , we observed that ics are significantly associated with denser breast compared with a random sample of negative controls , thus confirming previous reports on the subject [ 11 ]  . overall , the study results suggest that the considered screening programme has a good performance as to sensitivity , as it fulfilled the standards recommended by european guidelines , a goal that is not frequently achieved by service screening programmes [ 5 , 6 , 9 ]  . 
further improvement is expected in ic rate thanks to the recent adoption of routine double reading , and a study is ongoing on this aspect , which will be the object of a future report . 
the experience reported here , together with a relatively limited number of italian studies on this subject to date [ 46 ] , confirms that the assessment of ic proportional incidence and blind review of ic are fairly easy and feasible procedures , which should not remain episodic but should be performed routinely , as they are probably the most reliable among the available recommended early indicators of screening efficacy [ 10 ]  . dal diverso tasso di richiamo osservato per i diversi revisori . 
la differenza di significato tra le categorie ms e es , enunciata nelle line guida europee , chiaramente rispettata , come dimostrato dal diverso valore predittivo positivo osservato per le due categorie ( es = 51 , 7% , sm = 21 , 8% )  . il confronto tra diversi revisori suggerisce che ladozione di tassi di richiamo eccessivamente elevati non ripaghi in termini di tasso di identificazione di ci : in questo studio infatti , differenze statisticamente molto significative del tasso di richiamo non si traducono in differenze significative nel tasso diagnostico di ci . 
come rilievo collaterale abbiamo osservato che i ci , rispetto a un campione random di controlli negativi , sono associati a una maggiore probabilit di seno denso , confermando cos quanto gi riportato in letteratura [ 11 ]  . la revisione ha fornito un risultato anchesso indicativo di buona pratica , con un tasso di ci classificato come es ben inferiore al massimo raccomandato dalle linee guida europee ( 15% vs 20% raccomandato )  . 
complessivamente i risultati di questo studio dimostrano che il programma di screening valutato ha una buona performance , in linea con quanto raccomandato dalle linee guida europee , risultato non sempre facilmente ottenuto dai correnti programmi di screening [ 5 , 6 , 9 ]  . 
un ulteriore miglioramento nella frequenza dei ci atteso a seguito delladozione della doppia lettura quale pratica routinaria , e uno studio attualmente in corso su questo aspetto sar oggetto di futura pubblicazione . 
lo studio dimostra anche che la valutazione del tasso proporzionale dei ci e la loro revisione fattibile con una ragionevole facilit , come dimostrato anche da un numero a tuttoggi assai limitato di studi italiani [ 46 ]  . 
mazzini 121 , 00195 rome , italy , tel . : + 39 - 06 - 20902419 , fax : + 39 - 06 - 20902469 , e - mail : orazio.schillaci@uniroma2.it received : 12 june 2008 / accepted : 5 november 2008 / published online : 4 july 2009 springer - verlag 2009 abstract purpose . 
the aim of our prospective study was to compare the diagnostic accuracy of early , delayed and dual - timepoint positron emission tomography ( pet ) acquisition with contrast enhanced computed tomography ( ct ) within a pet - ct examination in the evaluation of pulmonary solitary nodules ( spns )  . 
whole - body pet images were acquired at 50 min after fluorine18 - fluorodeoxyglucose ( 18f - fdg ) administration ( early ) , followed by a chest acquisition ( delayed )  . 
presso il nostro dipartimento sono stati sottoposti a valutazione pet - tc dual - time con mezzo di contrasto 30 pazienti con nodulo polmonare solitario . le immagini pet total - body sono state acquisite 50 minuti dopo la somministrazione di 18f - fdg ( fase precoce ) seguite da unacquisizione del torace ( fase tardiva )  . 
stato calcolato il valore di suvmax sulle immagini precoci e tardive : lincremento ( suvmax ) del suvmax tra fase precoce e fase tardiva 10% stato considerato suggestivo di malignit ; in valutazione tc invece stata considerato suggestivo di benignit lassenza di un significativo incremento di densit ( < 15 hu ) tra lacquisizione basale e quella postcontrastografica . 
la sensibilit , la radiol med ( 2009 ) 114 : 890906 dual - time - point suvmax values were 83% , 67% , 75% and 74% ; hu values were 94% , 34% , 67% , 96% ; ct morphologic evaluation values were 61% , 46% , 60% , 47% . 
area under the curve ( auc ) for early suvmax was 0.79 , for delayed suvmax 0.80 , for dual - time - point suvmax 0.85 , for hu 0.63 and for ct morphologic assessment 0.58. 
however , dual - time - point suvmax was most sensitive , whereas single - time - point suvmax was most specific . keywords dual - time - point suv single - time - point suv contrast - enhanced ct lung nodules specificit , il vpp e il vpn del suvmax ottenuto in fase precoce sono state del 77% , 91% , 79 , 5% e 66 , 7% rispettivamente ; in fase tardiva abbiamo ottenuto valori di 83% , 67% , 75% e 74% rispettivamente . 
la differenza del valore in hu ( hu ) tra acquisizione tc basale e quella post - contrastografica ha mostrato valori di sensibilit del 94% , di specificit del 34% , vpp del 67% e vpn del 96% . la valutazione tc morfologica ha riportato valori di sensibilit , specificit , di vpp e di vpn di 61% , 46% , 60% , 47% . 
nel nostro piccolo gruppo di pazienti il valore di suvmax sia in fase precoce che tardiva ha mostrato accuratezza sovrapponibile , le valutazioni tc morfologica e contrastografica hanno mostrato laccuratezza pi bassa . il valore di suvmax calcolato in dual - time ( suvmax ) ha mostrato laccuratezza diagnostica maggiore . 
about one third of spns in patients older than 35 years are malignant [ 2 ] and are often discovered accidentally during a routine chest x - ray , with radiological features such as size , location or calcification [ 2 , 3 ] helping to discriminate between benign and malignant nodularities . 
furthermore , the recent use of computed tomography ( ct ) as a screening technique for lung cancer has further increased the number of incidentally discovered pulmonary nodules in asymptomatic patients . 
the reported incidence of malignant lesions i noduli polmonari solitari ( nps ) sono definiti come lesioni polmonari ovalari o rotondeggianti con dimensioni fino a 3 cm di diametro , circondate da parenchima polmonare , non associate ad atelettasia o ispessimento parenchimale infiammatorio [ 1 ] ; le lesioni di dimensioni maggiori di 3 cm sono invece definite come masse e possono essere pi frequentemente di natura maligna . 
circa un terzo dei nps nei pazienti con et maggiore di 35 anni sono maligni [ 2 ] e spesso vengono rinvenuti incidentalmente durante lesecuzione di una radiografia ( rx ) del torace di routine , con caratteristiche radiografiche come dimensioni , localizzazione o calcificazioni [ 2 , 3 ] che aiutano a discriminare fra benignit o malignit . 
il recente impiego della tomografia computerizzata ( tc ) come tecnica di screening per il carcinoma del polmone ha repentinamente aumentato il numero di noduli polmonari scoperti in pazienti asintomatici . 
molti di 892 radiol med ( 2009 ) 114 : 890906 among solitary pulmonary nodules ranges from 5% to 69% [ 79 ] , the variability essentially depending on the imaging technique used and the population examined . 
on account of its ability to scan the whole lung in a few seconds using thin collimations , msct plays a crucial role in the noninvasive detection , characterisation and follow - up of pulmonary nodules . 
the increasing importance of thin - section ct , with or without intravenous ( i.v. ) contrast enhancement , in the study of spns is due to its capability of assessing the pattern of calcification within a nodule and precisely assessing its doubling time , margins , density , attenuation and contrast enhancement , therefore defining potential benignity or malignancy , e.g. 
popcorn or diffuse calcifications within a nodule suggest benignity , whereas eccentric calcifications are more frequently found in malignant lesions [ 2 , 3 ]  . however , the above - mentioned features may fail to confidently discriminate between benign and malignant spns , making management decision difficult . 
in fact , the use of msct in combination with 99mtc sestamibi single photon emission tomography ( spect - ct ) in spns has been proposed , with interesting future perspectives in differential diagnosis [ 10 ]  . 
over the last decade , diagnostic imaging of spns and staging of lung cancers have rapidly changed with the increasing use of pet scan , which measures the degree of fluorine18 - fluorodeoxyglucose ( 18f - fdg ) uptake or c11 - choline [ 11 ] within a lesion . 
in fact , it is possible to calculate standardised uptake value ( suv ) , that is , the ratio between the average radioactivity registered in a lesion and the administered amount of the same tracer . 
rationale for the use of 18f - fdg is the higher glucose metabolism displayed by malignant cells compared with benign cells , probably due to up - regulation of glucose transporters ( glut ) and hexokinase , with down - regulated synthesis of glucose - 6phosphatase [ 13 , 14 ]  . 
actually , whereas pet sensitivity and specificity are high for hypermetabolic solid spns 13 cm in size , false negatives are described for low metabolic neoplasms , such as bronchioloalveolar cell carcinoma and carcinoids or in solid nodules < 1 cm [ 15 ]  . 
false - positive results of pet examination include , on the other hand , some benign pulmonary conditions such as tuberculosis and sarcoidosis due to monocytes increased turnover [ 16 ] , which lowers pet positive predictive value ( ppv )  . 
dual - time - point pet acquisition has been proposed to discriminate between benign and malignant processes [ 16 ] , in the evaluation of suspected secondary liver lesions [ 17 ] and in distinguishing between benign and malignant lung nodules [ 18 , 19 ]  . 
a lesion is likely to be malignant if the suv increases over time , whereas it is likely to be benign if the suv is stable or decreases 10% [ 19 ]  . 
lincidenza riportata di lesioni maligne fra i nps varia dal 5% al 69% [ 79 ] : tale variabilit dipende essenzialmente dalla tecnica di imaging utilizzata e dalla popolazione esaminata . 
in virt della capacit di esaminare lintero torace con acquisizione di pochi secondi , la tcms gioca un ruolo cruciale nella individuazione non - invasiva , nella caratterizzazione e nel monitoraggio dei noduli polmonari . 
la crescente importanza della tc a strato sottile nello studio dei nps , con o senza somministrazione di mezzo di contrasto endovena ( mdc e.v. ) , dovuta alla sua capacit di descrivere pattern di calcificazione nellambito nodulare di determinare il tempo di raddoppiamento delle dimensioni , i margini , la densit , lattenuazione , limpregnazione dopo mdc e pertanto nel definirne la potenziale benignit o malignit ; ad esempio calcificazioni a pop - corn o diffuse sono suggestive di benignit mentre quelle eccentriche sono riscontrate pi spesso nei noduli maligni [ 2 , 3 ]  . 
 nella differenziale luso di metodiche ibride che permettano studi morfofunzionali pu agevolare la diagnosi e la gestione dei nps . a tale proposito stata recentemente proposta lintegrazione della tcms con la spect - tc con 99mtc - sestamibi nella valutazione dei noduli polmonari con interessanti prospettive [ 10 ]  . diagnostica nellultima decade limaging dei nps e la stadiazione del carcinoma polmonare sono stati velocemente modificati con lavvento della tomografia ad emissione di positroni ( pet ) che misura lintensit della captazione del 18f - fluorodeossiglucosio ( 18f - fdg ) o della 11c - colina [ 11 ] nellambito di una lesione . 
infatti possibile misurare lo standardised uptake value ( suv ) , valore di captazione standard , che il rapporto fra la radioattivit media misurata nella lesione e la dose di tracciante somministrata . nei nps un valore di suv di 2 , 5 viene impiegato come cutoff per differenziare lesioni benigne ( valore di suv inferiore ) da quelle maligne ( valore di suv maggiore ) [ 12 ]  . 
il razionale per lutilizzo del 18f - fdg laumentato metabolismo glucidico espresso dalle cellule maligne rispetto a quelle benigne , dovuto probabilmente alla maggiore espressione di trasportatori di glucosio ( glut ) e di esochinasi , con minor espressione della glucosio - 6 - fosfatasi [ 13 , 14 ]  . 
altro punto da considerare che sensibilit e specificit della pet sono state dimostrate elevate per le lesioni ad aumentato metabolismo glucidico con dimensioni da 1 a 3 cm , mentre falsi negativi sono descritti per quanto riguarda neoplasie a basso metabolismo , quali il carcinoma bronchiolo - alveolare e il carcinoide o in caso di noduli solidi con dimensioni inferiori al centimetro [ 15 ]  . radiol med ( 2009 ) 114 : 890906 vivo show that malignant cells tend to accumulate 18f - fdg over time , with an increase of suv > 10% , whereas mononucleate cells of the monocyte - macrophage system show stable or decreasing ( 10% ) suv [ 16 ]  . 
recently , 18f - fdg in - line pet - ct have demonstrated an emerging role in the evaluation of spns [ 20 ] and in lung carcinoma staging , in the assessment of response to chemo / radiation therapy and risk stratification of lung carcinoma [ 21 ]  . 
in previous studies , dual - time - point and single - time - point fdg - pet accuracies were calculated as a percentage of correct diagnoses in the entire sample . 
moreover , no studies compared dual - timepoint pet accuracy with contrast - enhanced ct diagnostic accuracy in a single clinical setting . the aim of the present study was to compare single - timepoint maximum suv max , dual - time - point suvmax and contrast - enhanced ct diagnostic accuracy by means of area under the receiver - operating characteristic ( roc ) curves in the evaluation of solitary pulmonary nodules . materials and methods patients from november 2006 to january 2007 , 35 patients underwent pet - ct examination for metabolic evaluation of spns after giving written informed consent to the examination . 
patients had either a known lung cancer history , or an oncological history with a newly discovered lung nodule suspicious for metastasis , or were referred for metabolic evaluation of an spn . 
finally , 30 patients ( 17 men and 13 women , mean age 59 , range 3680 ) were included in the study ( table 1 )  . twenty - four patients with lung nodules incidentally discovered at chest radiography or at high - resolution ct ( hrct ) performed for other reasons ( cough , chest pain ) and six oncology patients ( two with suspected recurrent lung cancer , two with colorectal carcinoma , one with liposarcoma , one with ovarian carcinoma ) were prospectively enrolled . dual - time - point pet and contrast - enhanced ct all patients fasted for at least 5 h before f18 - fdg i.v. 
this daltro canto esiste la possibilit di falsi positivi in pet per reperti polmonari benigni , come tubercolosi e sarcoidosi , a causa dellaumentato turn - over monocitario espresso [ 16 ] , che possono inficiare il valore predittivo positivo della metodica . 
 stata dunque proposta la pet con metodica di acquisizione dual - time per migliorare la performance nella diagnosi differenziale fra processi benigni e maligni [ 16 ] , ad esempio in caso di sospette lesioni secondarie epatiche [ 17 ] e dei noduli polmonari benigni e maligni [ 18 , 19 ] : una lesione ha maggiore probabilit di essere maligna se il valore di suv aumenta nel tempo mentre tendenzialmente benigna se tale valore rimane stabile o si riduce al di sotto del 10% [ 19 ]  . 
studi eseguiti sia in vitro che in vivo mostrano che le cellule maligne tendono ad accumulare il 18f - fdg nel tempo con incremento di suv fino al 10% , mentre le cellule del sistema monocito - macrofagico mostrano suv stabile o decrescente ( 10% ) [ 16 ]  . 
ultimamente la pet - tc con 18f - fdg ha dimostrato un ruolo emergente nella valutazione dei nps [ 20 ] , nella stadiazione , nella valutazione della risposta alla chemioterapia / radioterapia e nella stratificazione del rischio del carcinoma polmonare [ 21 ]  . 
in studi precedenti stata calcolata laccuratezza della pet con 18f - fdg in dual - time e in single - time come percentuale di diagnosi corretta nellintero campione ; tuttavia non ci sono studi che confrontino in ununica sessione laccuratezza diagnostica della pet dual - time con la tc con mdc . scopo del nostro studio confrontare la performance diagnostica del valore della pet con valore di suvmax in singola misurazione ( precoce o tardiva ) , della pet con valore di suvmax in dual - time e della tc con mdc con lutilizzo di curva roc nella valutazione del nodulo polmonare solitario ( nps )  . materiali e metodi pazienti da novembre 2006 a gennaio 2007 , 35 pazienti hanno eseguito lesame pet - tc per la caratterizzazione metabolica di nps , dopo consenso informato . 
i pazienti , sono stati inviati presso il nostro dipartimento per la caratterizzazione di nodulo polmonare evidenziato come reperto occasionale , per la stadiazione di carcinoma polmonare primitivo o per la valutazione di sospetto nodulo polmonare secondario ad altra patologia oncologica . 
sono stati inclusi nello studio pazienti con singolo nodulo polmonare . i noduli con dimensioni < 9 mm , dati i limiti del potere di risoluzione della metodica pet , sono stati esclusi dallo studio . 
infine il numero totale di pazienti arruolati patients table 1 patients gender clinical query tabella 1 pazienti pazienti 17 m , 13 f 59.3 years ( range 3680 ) 24 metabolic characterisation of spn 6 oncological history sesso quesito clinico 17 m , 13 f 59 , 3 anni ( range 3680 ) 24 caratterizzaione metabolica nps 6 storia oncologica system combines a high - speed ultra 16 - detector - row ( 912 detectors per row ) ct unit and a pet scanner with 10080 bismuth germanate ( bgo ) crystals in 24 rings . 
after nonenhanced ct , pet images were acquired 50 min after 18f - fdg i.v. administration , with a chest scan and early suv calculation ( two bed positions , 4 min per bed ) , followed by whole - body acquisition ( five to seven bed position , depending on the patient , 4 min per bed ) for delayed suv calculation 90 min after the i.v. 
total - body pet examination in the caudocranial direction , from upper thighs to vertex , was performed after 40 mimages were reconstructed using a standard iterative algorithm [ ordered subsets expectation maximisation ( osem ) ] after 2530 min of acquisition . 
administration of nonionic iodinated contrast material ( 100120 ml , 370 mgi / ml , 420 mgi / kg at 3 ml / s ) , obtaining two successive stacks of scans . 
the first comprised the upper abdomen with a 30 - s delay from the injection onset ; the second extended from the neck to the pelvis with a 60 - s delay . 
change of spn enhancement was assessed 2 , 3 and 4 min after contrast administration , using 3.75 - mm - thick volumetric scans . data analysis images were evaluated on a dedicated workstation by a nuclear physician and a radiologist , both aware of the clinical history of the patient . 
lung nodules were evaluated by radiol med ( 2009 ) 114 : 890906 risultato essere di 30 soggetti ( 17 uomini e 13 donne ; et media 59 anni ; range 3680 anni ) ( tabella 1 )  . 
ventiquattro pazienti presentavano noduli polmonari evidenziati come reperto occasionale in un esame rx del torace o tc ad alta risoluzione ( hrct ) precedentemente eseguiti per altri motivi ( tosse , dolore toracico , esami preoperatori di routine ) ; 6 pazienti presentavano una precedente storia clinica oncologica ( 2 pazienti con sospetta recidiva di carcinoma polmonare , 2 pazienti con carcinoma colorettale , 1 paziente con liposarcoma , 1 paziente con carcinoma ovarico )  . 
 tutti i pazienti sono stati mantenuti a digiuno per almeno 5 ore prima della somministrazione del 18f - fdg e in tutti il livello ematico di glucosio era nella norma . 
con adeguata idratazione ( 500 ml di soluzione salina di nacl 0 , 9% ) per ridurre la presenza di radiofarmaco nei reni . inoltre circa 600 cc di soluzione di contrasto sono stati somministrati per os , al fine di opacizzare le anse intestinali . 
questo sistema associa uno scanner tc di tipo high speed ultra con 16 file di detettori ( 912 detettori per fila ) con tomografo pet con 10080 cristalli di germinato di bismuto ( bgo ) disposti in 24 anelli . 
prima dellacquisizione stata eseguita una tc a basso amperaggio per la correzione dellattenuazione delle immagini pet ( 80 ma , 140 kv , campo visivo da 420 a 500 mm , spessore della sezione tc di 3 , 75 mm )  . 
del radiofarmaco con unacquisizione del torace per la misura del suv in fase precoce ( 2 lettini della durata di 4 minuti ciascuno ) seguite dallacquisizione whole body ( da 5 a 7 lettini , della durata di 4 minuti ciascuno ) per la misurazione del suv in fase tardiva , a distanza di 90 minuti dalla somministrazione del 18f - fdg . 
le immagini pet whole body sono state acquisite in direzione caudo - craniale dal terzo prossimale dei femori fino al cranio , e di seguito ricostruite utilizzando un algoritmo iterativo standard ( osem , ordered subsets expectation maximization ) dopo 2530 minuti di acquisizione . 
la tc diagnostica stata eseguita con il seguente protocollo : 120140 kv , amperaggio automatico ( 330350 ma ) , modalit di acquisizione 27 , 50 / 1 , 375 : 1 , velocit di rotazione del gantry 0 , 6 secondi , fov largo matrice di 512512 . 
liniezione di mezzo di contrasto iodato ( 100120 ml , 370 mgi / kg , 3 ml / s ) stata effettuata in due pacchetti consecutivi di scansioni : il primo comprendente laddome radiol med ( 2009 ) 114 : 890906 means of semiquantitative analysis ( suvmax ) , and suv > 2.5 was considered malignant [ 12 ]  . 
in lesions with low 18f - fdg uptake , the rois were extrapolated from the axial chest ct tomograms by placing it at the nodule equator . dual - time - point suv was calculated on early and delayed images according to the following formula : suvmax ( % ) = ( suvdelayed suvearly ) / suvearly100 . 
nodule density changes , between nonenhanced and contrast - enhanced ct scan , after the emission scan , were quantified to determine the significance of lesion enhancement using circular standardised rois ( 10 mm2 ) manually placed on the axial tomograms within the lesion . 
ct examination data sets were reprocessed and reconstructed according to the ala software instructions : 1.25 - mm slice thickness , 3.27mm interval to ensure correspondence with the spacing of the pet sections , and bone filter , applied to the nonenhanced acquisition . 
minimum follow - up was 10 months . statistical analysis the meanstandard error ( sem ) of the different variables ( early suvmax , delayed suvmax , dual - time - point suvmax , hu ) was calculated . 
in order to test variables normal distribution , the shapiro - wilk test was performed . according to the test results , mean values of the variables were compared using the nonparametric mann - whitney u test . 
statistical analysis was performed using a standard commercially available software package ( ncss 2007 )  . con un ritardo di 30 secondi rispetto alliniezione del mdc e il secondo esteso dal collo alla pelvi con un ritardo di 60 secondi . 
lenhancement post - contrastografico dei nps stato misurato al 2 , 3 e 4 minuto dopo la somministrazione del contrasto , utilizzando spessore di scansione di 3 , 75 m analisi dei dati le immagini sono state esaminate su consolle dedicata da un medico nucleare e da un radiologo , entrambi consapevoli della storia clinica del paziente . 
regioni di interesse ( roi , region of interest ) sferiche sono state utilizzate sulle immagini mip ( maximum intensity projection ) per calcolare il valore di suvmax ; nelle lesioni con scarsa captazione del 18f - fdg le roi sono state estrapolate da immagini assiali tc del torace posizionando il cursore al centro del nodulo . 
la variazione di suv ( suv ) sulle immagini precoci e tardive stato calcolata seguendo questa formula : suvmax ( % ) = ( suvtardivosuvprecoce ) / suvprecoce100 . lincremento di suv > 10% stato considerato suggestivo di malignit mentre un valore di suv stabile o decrescente stato considerato suggestivo di benignit [ 19 ]  . 
i cambiamenti nella densit del nodulo tra tc basale e tc post - contrasto sono stati misurati usando roi circolari standardizzate ( 10 mm2 ) posizionate manualmente sui tomogrammi assiali delle lesioni . 
i dati tc sono stati processati e ricostruiti secondo le istruzioni del software ala : 1 , 25 mm di spessore delle slices , intervallo fra le slices di 3 , 27 mm per assicurare la coincidenza tra sezioni pet e filtro per losso applicati allacquisizione basale . lenhancement post - contrastografico stato definito come differenza fra la densit della lesione misurata in unit hounsfield prima e dopo la somministrazione di mdc ( hu )  . 
eighteen of 30 nodules ( 60% ) were malignant , and 12 / 30 were benign ( 40% ) , as confirmed by pathological assays or clinical follow - up . 
among malignant lesions ( table 2 ) , seven were adenocarcinoma of the lung , two squamous cell carcinoma , one large - cell carcinoma , one carcinoid , one liposarcoma metastasis that responded to chemotherapy , two colorectal carcinoma that responded to chemotherapy , three lung cancer recurrence ( after chemoor radiation therapy ) and one ovarian carcinoma metastasis . 
 risultati caratteristiche dei noduli sono stati valutati complessivamente 30 noduli polmonari : il diametro massimo medio dei noduli maligni risultato di 22 , 136 , 8 mm ( range 9 , 5030 mm ) mentre quello dei noduli benigni risultato di 14 , 817 , 38 ( range 3 , 528 mm )  . 
le tabelle 2 e 3 mostrano le caratteristiche della popolazione in esame divisa in noduli maligni e benigni . diciotto / 30 noduli ( 60% ) sono risultati maligni , 12 / 30 ( 40% ) benigni , come confermato dai campioni istologici o dal monitoraggio clinico - strumentale . 
tra le lesioni maligne ( tabella 2 ) , sette si sono dimostrate adenocarcinoma del polmone , due carcinoma squamocellulare , uno carcinoma a grandi cellule , un carcinoide , una metastasi da liposarcoma ( risposta alla chemioterapia ) , due carcinoma colo - rettale ( risposta alla chemioterapia ) , tre recidive di carcinoma polmonare ( dopo chemioterapia o radioterapia ) , una metastasi da carcinoma ovarico . 
1a , b distinction between benign and malignant nodules obtained with the early and delayed maximum standardised uptake value ( suvmax ) value ( a ) and the suvmax ( b )  . 
1a , b distinzione tra noduli benigni e maligni ottenuta sia con il valore di suvmax precoce e tardivo ( a ) sia con il suvmax ( b )  . 
although 30% of the nonselected population with spn identified at first chest radiography has a 20%40% likelihood of being malignant [ 2 , 24 ] , radiol med ( 2009 ) 114 : 890906 differenza statistica significativa tra il valore di suv precoce e suv tardivo quando i pazienti sono stati classificati in riferimento a patologia benigna o maligna ( p = 0 , 93 e p = 0 , 090 , rispettivamente )  . 
analogamente non stata osservata differenza statistica significativa tra enhancement postcontrastografico dei noduli maligni rispetto a quello dei noduli benigni ( 36 , 2213 , 24 vs 23 , 8333 , 15 , p = 0 , 17 )  . applicato il test di fisher non stata osservata significativa correlazione fra i criteri morfologici documentati in tc e la malignit ( p = 0 , 58 )  . la sensibilit , la specificit , il valore predittivo positivo ( vpp ) e il valore predittivo negativo ( vpn ) del hu hanno presentato rispettivamente valori percentuali di : 94% , 34% , 67% e 96% . 
i valori massimi corrispondenti riscontrati sulla performance diagnostica del valore di suv tardivo sono stati : 77% , 66% , 74% , 66% ; i valori riscontrati per il valore di suv precoce : 77% , 91% , 79 , 5% , 66 , 7% ; infine i valori corrispondenti alla performance del valore di suvmax in dualtime hanno dimostrato : sensibilit 83% , specificit 70% , vpp 75% , vpn 74% . 
in quasi il 30% dei soggetti in popolazioni non selezionate con nps identificato al primo radiogramma del torace sussiste la probabilit tra il 20% e il 40 % che questo nodulo diventi maligno [ 2 , 24 ] ; nei due terzi di casi inoltre la tc o le radiografie standard non riescono a discriminare fra lesioni benigne e maligne sulla base della presenza di calcificazioni o degli aspetti tomodensitometrici [ 25 , 26 ]  . 
2a , b dual - time - point maximum standardised uptake value ( suvmax ) values ( a ) and early suvmax values ( b ) in the receiver - operating characteristic ( roc ) curves . 
larea roc del suvmax in dualtime pi alta di quella del suvmax in singola acquisizione ; comunque , per un tasso di falsi positivi minore di 0 , 20 , preferibile utilizzare il suvmax in singola acquisizione rispetto al suvmax in dual - time . in two thirds of cases , both ct and chest x - rays are unable to differentiate between benign and malignant lesions on the basis of calcifications or density features alone [ 25 , 26 ] moreover , 5 - year survival after resection of bronchogenic carcinoma ranges from 40% to 80% depending on the timing of diagnosis . 
recently , dual - time - point suv has been evaluated in lung carcinoma [ 16 , 19 , 29 ] , showing a better accuracy than single - time - point evaluation . 
morphologic evaluations of spns can help differentiate benign and malignant spns when they have typical benign or malignant features , but there may be overlap between nodule types in terms of morphologic presentations [ 22 , 30 ]  . 
in our series , no intralesional calcifications were evidenced , and the fisher exact test indicated no significant association between lesion 40%80% ci dipende dalla timing della diagnosi iniziale ; pertanto la diagnosi precoce delle lesioni maligne fondamentale al fine di iniziare un trattamento subitaneo e adeguato [ 27 , 28 ]  . 
ultimamente stata studiata lefficacia del valore diagnostico del suvmax in dual - time nella valutazione del carcinoma polmonare [ 16 , 19 , 29 ] , che ha dimostrato una maggiore accuratezza della valutazione in singola acquisizione . 
nel nostro studio stata osservata una differenza significativa tra noduli benigni e maligni per quanto riguarda i valori suvmax precoce , suvmax tardivo e suvmax in dual - time . secondo tali risultati il valore di suv con cut - off 2 , 5 rappresenta un affidabile criterio per differenziare i noduli benigni da quelli maligni ; tale dato stato riscontrato in uno studio analogo eseguito sulla performance della pettc con somministrazione di mezzo di contrasto nella valutazione dei nps . 
in questo studio stato valutato peraltro il valore di suv in singola acquisizione tardiva associato ai criteri morfologici tc [ 30 ]  . per quanto riguarda il valore hu densitometrico o per quanto concerne gli aspetti morfologici tc nel nostro studio non si sono osservate differenze significative . proprio la valutazione morfologica del nodulo polmonare solitario ( nps ) pu aiutare nella differenziazione di lesioni benigne e maligne quando queste presentino aspetti tipici di benignit o malignit . 
inoltre lefficacia diagnostica valutata misurando larea sotto la curva roc dellanalisi dellenhancement ( hu ) , risultata maggiore dellarea roc dellanalisi morfologica . probabilmente ci dovuto ai limiti connessi al numero di pazienti inclusi nel nostro studio . 
analogamente ad altri dati precedentemente pubblicati [ 19 , 31 ] , i nostri risultati hanno inoltre dimostrato che il suvmax in dual - time possiede maggiore accuratezza del suvmax in single - time ( suvmax dual - time : auc , 0 , 84 ; suvmax precoce : auc , 0 , 80 ; suvmax tardivo 0 , 79 )  . 
di conseguenza i valori di suvmax in dual - time hanno dimostrato sensibilit e vpn pi elevati se confrontati al valore di suvmax in singola acquisizione ( dual - time suvmax : 83% , 74% ; suvmax precoce : 77% , 66% ; suvmax tardivo : 77% , 66% )  . 
moreover , the area under the roc curve reflecting assessment of the enhancement of each lesion was greater than the area under the roc curves reflecting the irregularity of the shape . 
indeed , dual - timepoint suvmax values showed higher sensitivity and npv compared with single - time - point suv ( dual - time - point suv 83% , 74% ; early suvmax 77% , 66% ; delayed suvmax 77% , 66% )  . 
roc curve for dual - time - point suv is better than early suvmax curve in the high false positive range ( high sensitivity range ) , whereas early suvmax is better than dual - time - point suvmax in the false positive rate of < 0.20. noteworthy is that a diagnostic test should be judged relative to the clinical setting to which the test is applied . for the evaluation of a lung nodule , in low - risk populations ( nonsmokers , no history of neoplasm ) where the prevalence of lung cancer is very low , a high specificity and low false positive test such as early suvmax evaluation would be of preference . 
however , when screening for lung cancer in a high - risk population ( smokers , history of lung neoplasm , etc . ) , the diagnostic test should provide the best sensitivity , such as dual - time - point suvmax , even if the false positive rate is higher , as false negative test results may have serious consequences on patient survival in high - risk patients . figures 3 and 4 show two false negatives for semiquantitative suv evaluation but true positive for dual - time - point suv and hu evaluation . 
in both cases , nodule size was > 9 mm but < 1 cm , and single - time - point suv evaluation was probably affected by the suv threshold commonly used [ 12 ]  . 
nodules < 9 mm were excluded from this study , as our aim was to evaluate an suv with 2.5 cutoff , which was demonstrated to be not efficient in discriminating benign lung micro - nodules [ 32 ]  . 
daltro canto larea sotto la curva per il suvmax precoce risultata maggiore di quella del suvmax dual - time , utilizzando un cut - off di falsi positivi < 0 , 20 . 
per la valutazione di un nodulo polmonare in popolazioni a basso rischio ( non fumatori , non precedenti neoplasie ) , nei quali la prevalenza del carcinoma polmonare molto bassa , sarebbe preferibile ottenere un valore di specificit elevato associato a un basso tasso di falsi positivi , come verificatosi nella nostra valutazione del valore precoce di suvmax . daltro canto , quando si attui screening per carcinoma polmonare in popolazione ad alto rischio ( fumatori , storia di precedente neoplasia polmonare etc . ) il test diagnostico dovrebbe provvedere la maggiore sensibilit possibile , come nel caso del valore di suvmax in dual - time , anche se possibile che si verifichi un elevato tasso di falsi positivi ; si consideri infatti che un risultato di un test falso negativo avrebbe serie conseguenze sulla sopravvivenza del paziente nelle popolazioni ad alto rischio . 
le figure 3 e 4 mostrano 2 risultati falsi negativi nella valutazione semiquantitativa suv che invece si sono dimostrati veri positivi nelle valutazioni del suv in dual - time e della variazione di hu . 
in entrambi i casi le dimensioni del nodulo erano > 9 mm ma < 1 cm e la valutazione del suvmax in dual - time stata probabilmente influenzata dal valore soglia suv utilizzato [ 12 ]  . 
i noduli con dimensioni < 9 mm sono stati esclusi dal nostro studio , in quanto lo scopo era di valutare il valore di suv con cut - off di 2 , 5 , che stato dimostrato non affidabile nel discriminare fra micronoduli benigni e maligni [ 32 ]  . 
in ogni caso le lesioni polmonari 1 cm con assente o trascurabile captazione di 18f - fdg hanno una bassa probabilit di malignit ( < 5% ) [ 3 ]  . 
 la nostra analisi potrebbe anche essere limitata dal fatto che la metodologia roc utilizzata per attribuire laccuratezza diagnostica inficiata dallesperienza degli esaminatori e dal tasso di falsi negativi e falsi positivi nella popolazione arruolata . 
daltro canto lanalisi roc la misura pi accurata di una performance diagnostica rispetto alla valutazione percentuale di corretta diagnosi nellintero campione , mentre la curva roc mostra la sensibilit e i tassi di falsi positivi a tutti i valori di cut - off radiol med ( 2009 ) 114 : 890906 fig . 
3a immagini assiale precoce e tardiva coronale del paziente numero 2 in tabella 2 ( noduli maligni ) , con suvmax di 1 , 4 e 2 , 1 rispettivamente . 
questo paziente si dimostrato un falso negativo per lanalisi semiquantitativa suv ma vero positivo sia per lanalisi del suvmax in dual - time sia per la valutazione densitometrica in hu . 
 racy was affected by the readers experience and by false negative and false positive rates in the population enrolled . on the contrary , roc analysis is a more accurate measure of diagnostic performance than evaluation of the percentage of correct diagnoses in the entire sample , as roc curve displays sensitivities and false positive rates at all possible cutoff levels , thus allowing assessment of the performance of a test independently of the decision threshold . conclusions in our small series of patients , malignant and benign nodules showed significantly different early suvmax , delay suvmax and dual - time - point suvmax values , disponibili , valutando la performance del test indipendentemente dalla scelta del valore soglia . 
 conclusioni nella nostra piccola casistica di pazienti i noduli benigni e quelli maligni hanno dimostrato valori di suvmax precoce , suvmax tardivo e suvmax in dual - time significativamente differenti , mentre non sono state osservate differenze per quanto riguarda il hu o gli aspetti morfologici . 
questo paziente si dimostrato falso negative per lanalisi del suvmax in singola acquisizione e vero positivo per lacquisizione suvmax in dual - time e per la misura densitometrica in hu . whereas no differences were observed in terms of hu or morphologic features . 
di conseguenza , per la valutazione di un nodulo polmonare solitario in ambito clinico , in popolazione a basso rischio ( ad esempio in non - fumatori o con anamnesi negativa per precedenti neoplasie maligne ) , dove la prevalenza del carcinoma polmonare bassa , dovrebbe essere eseguito preferenzialmente un test ad alta specificit come il suvmax precoce . 
quando daltro canto si applichi screening per carcinoma polmonare in popolazione ad alto rischio ( ad esempio in soggetti fumatori o pazienti con precedenti oncologici ) il test diagnostico dovrebbe garantire la migliore sensibilit , come il valore di suvmax in dual - time , anche tenuto conto dellelevato tasso di falsi radiol med ( 2009 ) 114 : 890906 fig . 
pathological examination of the surgical specimen revealed the presence of atypical mycobacteriosis : single - time - point pet , dual - time - point suvmax and contrast - enhanced ct were all false positive for malignant pulmonary disease . 
la prima riga mostra visione assiale in tc e ricostruzione ala del nodulo polmonare ; la seconda riga mostra immagini pet assiali , precoce e tardiva , dello stesso nodulo ( suvmax 3 , 7 e 5 , 2 rispettivamente )  . 
il reperto anatomo - patologico dopo la resezione chirurgica ha dimostrato presenza di micobatteriosi atipica : la singola acquisizione pet , il suvmax in dual - time e la tc con contrasto si sono dimostrate falsamente positive per patologia polmonare maligna . 
 the false positive rate is higher , as false negative test results may have serious consequences on patient survival in a high - risk population . positivi , in quanto il risultato negativo di tale test potrebbe avere serie ripercussioni sulla sopravvivenza dei pazienti in popolazioni ad alto rischio . 
ventriglia2 1dipartimento di scienze radiologiche , 2dipartimento di pediatria , servizio di cardiologia pediatrica , sapienza universit degli studi di roma , policlinico umberto i , viale del policlinico 155 , 00161 rome , italy correspondence to : s . 
this is a prospective observational study of 83 pregnant women who underwent fetal cardiac mri : 43 patients ( cases ) had echocardiographic suspicion of congenital heart disease ; 40 patients ( controls ) did not . 
si stratta di uno studio osservazionale prospettico di 83 donne in gravidanza sottoposte ad esami di rm cardiaca fetale : 43 con il sospetto ecocardiografico di patologia cardiovascolare ( casi ) e 40 senza ( controlli )  . lo studio rm cardiaco fetale consiste in una fase statica con sequenze multiplanari ssfp ed in una fase dinamica con sequenze ssfp in tempo reale . 
le sequenze ssfp permettono uno studio rm morfologico del distretto cardiovascolare mentre radiol med ( 2009 ) 114 : 852870 sequences may provide adjunctive albeit suboptimal functional information . attualmente riescono ad aggiungere dati funzionali solamente in maniera subottimale . 
 keywords fetal heart fetal mri congenital heart disease cardiac malformations prenatal diagnosis parole chiave cuore fetale rm fetale malattie congenite cardiache malformazioni cardiache diagnosi prenatale introduction introduzione magnetic resonance imaging ( mri ) has taken on an increasingly important role in the evaluation of high - risk pregnancy . 
fetal mri is , in fact , considered a third - line modality for the study of complex fetal disease after prenatal screening ultrasound and specialist ultrasound in secondary care centres [ 1 , 2 ]  . 
currently , the main fetal indications for performing mri in pregnancy are central nervous system ( cns ) malformations , disease of the chest and neck region , some abdominal abnormalities and urinary tract malformations [ 3 , 4 ]  . up until now , however , the study of the heart and great vessels has been hindered by the intrinsic characteristics of the organ and the technological limitations of the current mri sequences . 
the high fetal heart rate requires elevated temporal resolution , the small size of the heart and the vessels requires elevated spatial resolution and the rapid blood flow requires sequences that are not sensitive to fluids in motion . ultrafast t2 - weighted sequences , which are commonly used for fetal mri , are instead highly sensitive to flow and visualise the blood - filled heart and vessels as structures characterised by a lack of signal [ 5 ]  . 
 the heart has therefore been considered the black hole of fetal mri , and the aorta and great vessels were only studied by evaluating the image negatives , as they were depicted as structures without signal . steady - state free precession ( ssfp ) sequences use the alternating application , between two successive radiofrequency pulses , of balanced gradients that cause each stationary magnetic spin to return to the phase prior to the application of the gradient . 
therefore , whether stationary or in motion , fluids are visualised as structures with elevated signal ( just like stationary fluids in t2 - weighted sequences ) and at the same time the elevated contrast resolution of the t1weighted sequences is maintained [ 6 , 7 ]  . in addition , thanks to the acquisition speed of the individual slices , these sequences also enable dynamic visualisation ( cine mri ) of the heart with the possibility of evaluating the contractility of the cardiac chambers [ 8 ]  . la risonanza magnetica ( rm ) ha acquisito un ruolo progressivamente importante nella valutazione delle gravidanze a rischio . 
la rm fetale infatti considerata metodica di terzo livello per lo studio delle patologie fetali complesse dopo lecografia ostetrica di screening e quella specialistica eseguita in centri di secondo livello [ 1 , 2 ]  . attualmente le principali indicazioni fetali per lesecuzione della rm in gravidanza sono le malformazioni del sistema nervoso centrale ( snc ) , le patologie del distretto cervicotoracico , alcune anomalie addominali e le malformazioni urinarie [ 3 , 4 ]  . 
 fino ad oggi tuttavia lo studio del cuore e dei grossi vasi stato ostacolato dalla presenza di peculiari caratteristiche di tali organi e dalla limitazione tecnologica delle attuali sequenze rm : lelevata frequenza cardiaca fetale richiede infatti unelevata risoluzione temporale ; le piccole dimensioni del cuore e dei vasi rendono necessario lutilizzo di unelevata risoluzione spaziale ; il rapido flusso sanguigno necessita di sequenze non sensibili ai fluidi in movimento . le sequenze ultraveloci t2 pesate , comunemente utilizzate per lo studio rm fetale , sono invece molto sensibili ai flussi e visualizzano il cuore ed i vasi , percorsi da sangue in movimento , come strutture caratterizzate da mancanza di segnale [ 5 ]  . 
 il cuore stato pertanto considerato il buco nero della rm fetale e laorta ed i grossi vasi sono stati studiati solo attraverso la valutazione in negativo delle immagini , essendo rappresentate come strutture senza segnale . 
 le sequenze steady state free precession ( ssfp ) utilizzano lapplicazione alternata , tra due impulsi di radiofrequenza successivi , di gradienti bilanciati che agiscono su ogni spin magnetico stazionario facendolo tornare alla fase precedente allapplicazione del gradiente . 
in tal modo i gradienti bilanciati mantengono sia la magnetizzazione longitudinale che trasversale e nellimmagine rappresentato sia il contrasto t1 che t2 : pertanto i fluidi , sia stazionari che in movimento , vengono visualizzati come strutture ad elevato segnale ( proprio come i fluidi stazionari nelle sequenze t2 pesate ) e , allo stesso tempo , viene mantenuta lelevata risoluzione di contrasto delle sequenze t1 pesate [ 6 , 7 ]  . inoltre tali sequenze grazie alla rapidit di acquisizione delle singole fette permettono anche la visualizzazione dinamica ( cine - rm ) del distretto cardiaco con la possibilit della valutazione della contrattilit delle camere cardiache [ 8 ]  . the aim of this study was to demonstrate the feasibility scopo di questo lavoro quello di dimostrare la 854 radiol med ( 2009 ) 114 : 852870 of identifying the main anatomical and pathological appearances of the fetal cardiovascular system by using fetal mri with ssfp . possibilit di riconoscere i principali aspetti anatomici e patologici del distretto cardiovascolare fetale attraverso lo studio rm fetale con sequenze ssfp . materials and methods between september 2007 and august 2008 , we performed a prospective observational study based on fetal mri of the cardiovascular region . 
for this study , we enrolled 43 cases represented by 43 pregnant women with echocardiographic suspicion of fetal congenital heart disease referred to our service from the paediatric cardiology division and 40 controls represented by 40 pregnant women without known cardiovascular or thoracic disease referred from the prenatal diagnosis centre with a suspicion of cns ( n = 27 ) or non - cns disease ( n = 13 )  . 
the group of cases included one twin pregnancy and 42 singleton pregnancies , whereas the control group consisted of 40 singleton pregnancies . mean maternal age was 30 ( range 2342 ) years among cases and 32 ( range 1844 ) years among controls , whereas mean gestational age was 29 weeks in both groups ( range 2038 and 2237 weeks in cases and controls , respectively )  . all women underwent fetal mri without sedation of mother or fetus after giving informed consent and filling in a questionnaire regarding their pregnancy and family history of congenital disease . 
fetal mri was performed in both cases and controls with a 1.5 - t system ( siemens magnetom avanto , erlangen , germany ) with one or two multichannel phased - array surface coils and a standard protocol . 
in each case , a localisation sequence was acquired to enable placement of the volumes investigated as well as a t2 - weighted half - fourier single - shot turbo spin - echo ( haste ) sequence with a thick slice ( 6 mm ) in the coronal plane to evaluate fetal lie ( longitudinal , transverse , oblique ) and presentation ( cephalic , breech , shoulder )  . 
a study with thin - slice t2 - weighted haste sequences was then performed ( tr 1 , 000 , te 118 / 151 , matrix 256134 , slice thickness 34 mm , fov 2720 cm ) with multiplanar orientation on the organ of interest and lastly on the cardiovascular region . the study of the heart involved two phases . 
t1 and t2 weighting was intermediate , and sequences were oriented in the three planes in materiali e metodi tra settembre 2007 ed agosto 2008 abbiamo eseguito uno studio osservazionale prospettico , basato sullesecuzione di esami rm fetali mirati allo studio del distretto cardio - vascolare . 
per questo studio abbiamo arruolato 43 casi comprendenti 43 donne in gravidanza con il sospetto ecocardiografico di patologia fetale cardio - vascolare , inviate presso il nostro servizio dal servizio di ecocardiografia del reparto di cardiologia pediatrica e 40 controlli comprendenti 40 donne in gravidanza senza patologia fetale cardiovascolare n toracica nota , inviate dal servizio di diagnosi prenatale del nostro policlinico per il sospetto di patologie del snc ( 27 ) o non - snc ( 13 )  . 
let media materna era di 30 anni ( range 2342 anni ) nel gruppo dei casi e 32 anni ( range 1844 anni ) nel gruppo dei controlli e let gestazionale media era di 29 settimane in entrambi i gruppi ( range 2038 e 2237 rispettivamente nei casi e nei controlli )  . tutte le donne sono state sottoposte ad esame di rm fetale senza sedazione n materna n fetale e previa raccolta del consenso scritto allesecuzione dellesame rm preceduto dalla compilazione di un questionario anamnestico sulla gravidanza in corso e sulla storia familiare per patologie congenite . 
lo studio rm fetale stato effettuato sia nei casi che nei controlli con un magnete da 1 , 5 tesla ( siemens magnetom avanto , erlangen , germania ) con una o due bobine di superficie multicanale phased array attraverso un protocollo standard comune . 
in ogni caso stata acquisita una sequenza di localizzazione che consente il posizionamento dei volumi indagati e una sequenza t2 half - fourier acquisition singleshot turbo spin echo ( haste ) a strato spesso ( 6 mm ) sul piano coronale per la valutazione della situazione ( longitudinale , trasversale , obliqua ) e della presentazione ( cefalica , podalica , di spalla ) fetale : lorientamento dei successivi pacchetti stato effettuato su quelli precedentemente acquisiti per ridurre al minimo i problemi dovuti al cambiamento della posizione fetale per il movimento spontaneo del feto . quindi stato effettuato uno studio con sequenze t2 haste a strato sottile ( tr 1000 , te 118 / 151 , matrice 256134 , spessore dello strato 34 mm , fov 27 20 cm ) con orientamento multiplanare sullorgano anatomico di interesse ed infine lo studio del distretto cardiovascolare . 
la fase statica prevede lacquisizione di sequenze a gradiente di eco ultraveloci con steady state free precession chiamate truefisp ( true fast imaging with steady state free precession ) sulla apparecchiatura rm da noi utilizzata ( tr / te 3 , 5 / 1 , 5 , matrice 256144 , spessore dello strato 34 mm , fov 40 30 cm ) , radiol med ( 2009 ) 114 : 852870 space orthogonal to the cardiac axis to identify the main cardiovascular structures and measure the cardiac axis . 
the entire study was performed and evaluated in consensus by two radiologists with longstanding experience in obstetric radiology . with regard to image interpretation in controls , the anatomical study of the heart involved evaluation of cardiac orientation , cardiac volume , morphology and volume of the cardiac chambers and structure of the myocardial walls , as well as a study of the integrity of the interventricular septum ( ivs ) and the interatrial septum ( ias ) with the oval foramen ( of )  . 
the anatomical study of the great vessels , instead , involved evaluation of the origin , diameter and course of the aorta ( ao ) , pulmonary artery ( pa ) , ductus arteriosus ( da ) , superior vena cava ( svc ) , inferior vena cava ( ivc ) and pulmonary veins ( pv )  . with regard to interpretation of images in the cases , the radiologists were always aware of the suspicion of cardiovascular disease based on echocardiography , and the examination was targeted towards evaluating mri signs of the disease being studied . 
direct signs were defined as morphological and volumetric abnormalities of the cardiac chambers and the myocardium , malrotations , septal defects and abnormalities of origin , course and diameter of the great vessels . 
in the cases , mri findings were compared with echocardiography performed after mri or at birth or with the postmortem findings , which were considered the standard of reference . con pesatura intermedia t1 e t2 , orientate sui tre piani dello spazio ortogonali allasse cardiaco per lidentificazione delle la misura principali strutture cardiovascolari e per dellangolazione dellasse cardiaco . 
la fase dinamica prevede lacquisizione di sequenze real - time cine - rm truefisp 2d chiamate tf2d sulla apparecchiatura da noi utilizzata ( tr / te 248 , 5 / 1 , 1 , matrice 19299 , spessore dello strato 34 mm , fov 3826 cm , fa 77 , risoluzione temporale 2 fette / secondo ) , ripetute in maniera dinamica ( per 40 volte ) ed orientate sulle precedenti scansioni statiche per ottenere piani rm simili ai principali piani di scansione ecocardiografici ( assiali , sagittali ed obliqui ) ed inclinati lungo i piani anatomici cardiaci fetali dellafflusso ed efflusso vascolare . lintero esame stato eseguito e valutato da due radiologi esperti in radiologia in ostetricia in consensus . 
 per quanto riguarda linterpretazione delle immagini nei controlli lo studio anatomico del cuore ha previsto la valutazione dellorientamento cardiaco , della volumetria cardiaca , della morfologia e volumetria delle camere cardiache e della struttura delle pareti miocardiche , nonch lo studio dellintegrit del setto interventricolare ( siv ) e di quello interatriale ( sia ) con il forame ovale ( fo )  . 
lo studio anatomico dei grossi vasi ha invece previsto la valutazione dellorigine , del calibro e del decorso dei vasi di efflusso cardiaco ( aorta , ao , arteria polmonare , ap ) e del dotto arterioso ( da ) e la valutazione del calibro e decorso dei vasi di afflusso ( vena cava superiore , vcs , vena cava inferiore , vci , vene polmonari , vp )  . per quanto riguarda linterpretazione delle immagini nei casi , i radiologi erano sempre a conoscenza della patologia cardiovascolare sospettata dallecocardiografia e lesame stato mirato alla valutazione degli aspetti di semeiotica rm della patologia oggetto di studio . sono stati individuati segni diretti ed indiretti di patologia cardiovascolare considerando come segni diretti le anomalie morfovolumetriche delle camere cardiache e del miocardio , le malrotazioni , i difetti settali e le anomalie di origine , decorso e calibro dei grossi vasi e come segni indiretti lassenza delle strutture anatomiche nella scansione di riferimento , laumento di calibro a monte di una stenosi vascolare , la presenza di cardiomegalia e versamento pericardico . 
the walls of the myocardium were instead visualised as hypointense structures with optimal contrast resolution le sequenze ssfp truefisp statiche e tf2d dinamiche hanno permesso una visualizzazione ottimale del cuore e dei grossi vasi in tutti i feti ( 40 / 40 ) : in particolare le camere cardiache ed il lume vascolare sono stati visualizzate come strutture ad elevata intensit di segnale per la presenza nel loro contesto di sangue in movimento ; le pareti del miocardio 856 radiol med ( 2009 ) 114 : 852870 fig . 
a four - chamber view with visualisation of the ventricular ( vs , left ventricle ; vd , right ventricle ) and atrial chambers ( as left atrium , ad right atrium ) , the ventricular ( siv ) and atrial septum ( sia ) and the atrioventricular plane . 
c three - vessel view with the origin of the pulmonary artery ( ap ) and the ductus arteriosus ( da ) continuing with the descending aorta ( aod ) , the aorta and superior vena cava ( vcs )  . 
a scansione 4 camere con visualizzazione delle cavit ventricolari ( vs , ventricolo sinistro e vd , ventricolo destro ) , delle cavit atriali ( as , atrio sinistro e ad , atrio destro ) , del setto interventricolare ( siv ) e del setto interatriale ( sia )  . 
c scansione tre vasi con la visualizzazione dellarteria ( ap ) e del dotto arterioso ( da ) che si continua nellaorta discendente ( aod ) , dellao e della vena cava superiore ( vcs )  . 
the static true - fisp sequences in planes orthogonal to the cardiac axis made possible the visualisation of all of the main cardiovascular structures of interest covered by echocardiographic screening during the second trimester morphological examination ( four - chamber views and views of the outflow tracts of the aorta and pulmonary trunk ) in 28 / 40 fetuses ( 70% )  . 
le sequenze statiche truefisp , orientate su piani ortogonali allasse cardiaco , hanno consentito di ottenere la visualizzazione di tutte le principali strutture cardiovascolari di interesse previste dallo screening ecografico durante lesame morfologico del ii trimestre ( 4 camere cardiache e tratto di efflusso dellaorta e del tronco polmonare ) in 28 / 40 feti ( 70% ) ed in particolare le camere cardiache sono state visualizzate in tutti i feti ( 100% ) , il tratto di efflusso dellaorta in 28 feti ( 70% ) e il tratto di efflusso del tronco polmonare in 33 feti ( 82 , 5% ) : i rimanenti 12 feti nei quali uno o entrambi i tratti di efflusso cardiaci non erano visualizzabili con le sequenze statiche presentavano rispettivamente unepoca gestazionale compresa tra 22 e 25 settimane in 9 casi e 26 e 30 settimane nei restanti 3 casi . 
 the dynamic tf2d sequences oriented according to le sequenze dinamiche tf2d , orientate secondo le radiol med ( 2009 ) 114 : 852870 table 1 frequency of projections obtained with fetal mri in normal fetuses planes views mri frequency transverse views sagittal views angulated views scansioni assiali scansioni sagittali scansioni oblique 4 - chamber 5 - chamber ( aortic origin ) 3 vessels ( pulmonary outflow tract ) aortic arch short axis of the left ventricle tricuspid - aortic cut long axis of the ductus arteriosus long axis of the aortic arch long axis of the left ventricle aortic arch and ductus arteriosus simultaneously 4 camere 5 camere ( origine dellaorta ) 3 vasi ( tratto di efflusso polmonare ) scansione assiale dellarco aortico asse corto del ventricolo sinistro scansione tricuspide - aorta asse lungo del dotto arterioso asse lungo dellarco aortico asse lungo del ventricolo sinistro scansione simultanea dellarco aortico e del dotto arterioso 40 / 40 21 / 40 17 / 40 6 / 40 38 / 40 11 / 40 22 / 40 33 / 40 18 / 40 11 / 40 40 / 40 21 / 40 17 / 40 6 / 40 38 / 40 11 / 40 22 / 40 33 / 40 18 / 40 11 / 40 tabella 1 frequenza delle proiezioni ottenute con rm cardiaca fetale nei controlli normali piano di scansione scansione frequenza visualizzazione rm standard echocardiographic scans produced mri planes that were comparable to the main echocardiographic scans with the frequency reported in table 1 . 
the five - chamber and long - axis views of the lv , which enable evaluation of the aorta origin , were obtained in 21 / 40 fetuses ( 52.5% ) and 18 / 40 fetuses ( 45% ) , respectively , with overall visualisation of the rv outflow tract in 38 / 40 fetuses ( 95% , in 17 with double scan ) and the lv outflow tract in 31 / 40 fetuses ( 77.5% , in six with double scan )  . the distinction between right and left cardiac chambers was possible in 32 / 40 fetuses ( 80% )  . 
in 12 fetuses , it was based on direct evidence of the presence of the moderator band at the rv apex , which was represented by a thin band projecting into the ventricular lumen in the fourand fivechamber views . 
in the remaining eight fetuses , the distinction was not possible because the moderator band scansioni ecocardiografiche standard , hanno permesso di ottenere piani rm comparabili alle principali scansioni ecocardiografiche con la frequenza riportata in tabella 1 . in particolare i piani previsti dallo screening ecografico durante lesame morfologico del ii trimestre ( scansione 4 camere , scansione tratti di efflusso ) sono stati riprodotti secondo tale frequenza : la scansione 4 camere stata ottenuta in tutti i controlli ( 40 / 40 feti , 100% ) ; la scansione asse corto del ventricolo sinistro ( vs ) e la scansione tre vasi che permettono la visualizzazione del tratto di efflusso del ventricolo destro ( vd ) rispettivamente in 38 / 40 feti ( 95% ) e 17 / 40 feti ( 42 , 5% ) ; la scansione 5 camere e la scansione asse la valutazione dellorigine dellaorta rispettivamente in 21 / 40 ( 52 , 5% ) ed in 18 / 40 feti ( 45% ) , con una complessiva visualizzazione del tratto di efflusso del vd in 38 / 40 feti ( 95% , in 17 con doppia scansione ) e del tratto di efflusso del vs in 31 / 40 ( 77 , 5% , in 6 con doppia scansione )  . lungo del vs che permettono la distinzione tra camere cardiache di destra e di sinistra stata possibile in 32 / 40 feti ( 80% ) : in 12 feti in base allevidenza diretta della presenza della banda moderatrice allapice del vd rappresentata da una tenue e sottile banda che aggetta nel lume ventricolare nelle scansioni 4 e 5 camere ; in 20 feti in base alla morfologia del vs che risultava pi profondo nella porzione che costituisce lapice rispetto al vd ; nei rimanenti 8 feti tale distinzione non stata possibile poich la banda moderatrice non era 858 radiol med ( 2009 ) 114 : 852870 was not recognisable and the morphology of the cardiac chambers appeared similar . 
the volume measurements of the cardiac chambers showed that the rv in the fetus is larger than the lv up until the 32nd week of gestation , whereas the atria are similar in size even at term . the ivs was well depicted in all fetuses and characterised by low signal intensity in the ssfp sequences . 
the ias was visualised in only 12 / 40 fetuses , where it appeared as a hypointense , slender and mildly fragmented structure . lastly , the flap of the oval foramen from right to left was only rarely visualised ( 3 / 40 fetuses , at weeks 32 , 34 and 38 of gestation , respectively ) in the cine mri tf2d views oriented in the short axis of the heart . 
the cardiac apex appeared directed towards the left , and the angle of the ivs on the midsagittal plane was a mean 455 in all fetuses . the atrioventricular plane was visualised in all fetuses in the fourand five - chamber views , although the opening and closing of the valves was perceived in the dynamic sequences in six fetuses only . 
the analysis of signal intensity of the myocardial wall also revealed a difference between the walls of the lv and rv , with a more marked signal hypointensity of the lv than the rv in 38 / 40 fetuses regardless of gestational age . with regard to the study of the cardiac outflow vessels , the origin of the ao and the pa were well visualised in 31 / 40 and 38 / 40 fetuses , respectively . 
measuring the diameter of those vessels revealed that the pa was on average slightly larger than the ao , which in turn was slightly larger than the svc and similar in size to the da . 
la volumetria delle camere cardiache ha mostrato che nel feto il vd risulta di dimensioni maggiori rispetto al vs fino alla 32 settimana di gestazione , mentre gli atri risultano di dimensioni comparabili anche a termine . il siv risultato ben visualizzabile in tutti i feti e caratterizzato da bassa intensit di segnale nelle sequenze ssfp ; il sia stato visualizzato solo in 12 / 40 feti nei quali appariva comunque come una struttura sottile e frammentata lievemente ipointensa ; il flap del forame ovale da destra verso sinistra stato infine visualizzato raramente ( 3 / 40 feti , rispettivamente alla 32 , 34 e 38 settimana di gestazione ) attraverso le scansioni cine - rm tf2d orientate sullasse corto del cuore . 
il piano atrioventricolare stato visualizzato in tutti i feti nelle scansioni 4 e 5 camere , tuttavia lapertura e la chiusura delle valvole stata percepita nelle sequenze dinamiche solamente in 6 feti . 
lanalisi dellintensit di segnale del miocardio parietale ha mostrato inoltre una differenza tra le pareti del vs e quelle del vd con una pi marcata ipointensit di segnale del vs rispetto al vd in 38 / 40 feti indipendentemente dallepoca gestazionale . per quanto riguarda lo studio dei vasi di efflusso cardiaco , lorigine dellao e dellap sono risultati ben visualizzabili rispettivamente in 31 / 40 feti e in 38 / 40 feti : la misura del calibro di tali vasi ha dimostrato che lap risulta in media di dimensioni poco maggiori dellao , la quale , a sua volta , mostra dimensioni poco maggiori della vcs e simili al da ; il decorso dei grossi vasi stato valutato attraverso varie scansioni assiali a diverse altezze e attraverso le scansioni asse lungo dellarco aortico e del fig . 
b long - axis view of the aortic arch with the origin of the aorta ( origine ao ) , the ascending tract , the arch and the descending tract ( aod ) and some of the head and neck vessels ( left carotid artery , csn ; left subclavian artery , ssn )  . 
ben documentabile il ventricolo destro ( vd ) e lorigine dellaorta ( ao )  . radiol med ( 2009 ) 114 : 852870 the pulmonary arteries were visualised in 10 / 40 fetuses as very small structures at the limit of the techniques spatial resolution arising from the pa with asymmetrical course from the hylar region to the periphery . 
the da , which in the fetus is a fundamental structure of the systemic circulation , was well visualised in 32 fetuses in transverse section in the three - vessel scan ( n = 17 ) and / or in the longitudinal section in the long - axis scan of the da ( n = 22 )  . the svc and the ivc were visualised with the tricuspidaorta scan in 11 fetuses . 
il da che nel feto costituisce una struttura fondamentale per la circolazione sistemica stato ben visualizzato in 32 feti , in sezione trasversale nella scansione 3 vasi ( 17 ) e / o in sezione longitudinale nella scansione asse lungo del dotto arterioso ( 22 )  . 
 per quanto riguarda i vasi di afflusso cardiaco la vcs e la vci sono state visualizzate con la scansione tricuspideaorta in 11 feti ; con maggiore difficolt in soli 5 feti stato possibile visualizzare anche le vene polmonari con una scansione paracoronale allaltezza degli atri . pathological appearance aspetti patologici at least one congenital heart disease was diagnosed with postnatal echocardiography or postmortem examination in 42 / 43 pathological fetuses , whereas the condition was ruled out in the remaining fetus ( table 2 )  . 
fetal mri with static ssfp true - fisp and dynamic tf2d sequences was able to visualise the pathological fetal heart in all 42 fetuses with congenital heart disease ( 42 true positives ) and successfully rule out hypoplastic left - heart syndrome in the normal fetus ( one true negative )  . 
mri was therefore diagnostic in 43 / 43 cases . however , even though mri identified the major disease in all cases , it also raised a suspicion of an ivs defect , which was ruled out at birth in two fetuses ( one fetus with cardiomegaly and one with cardiac rhabdomyomas ) ( two false positives ) and visualised a significant ivs defect corresponding at birth to multiple interventricular defects in a fetus with truncus arteriosus ( one diagnostic inaccuracy )  . congenital heart disease was diagnosed on the basis of the mri depiction of direct pathological signs in 21 fetuses , indirect pathological signs in six fetuses and both in 15 cases . 
however , as stated above , in the fetus with truncus arteriosus , this defect in fact corresponded to multiple adjacent interventricular defects at birth , whereas in two other fetuses , interventricular defect was ruled out at birth . 
 almeno una patologia congenita cardiaca stata diagnosticata attraverso lecocardiografia postnatale o lesame autoptico in 42 / 43 feti patologici mentre stata esclusa nel rimanente feto ( tabella 2 )  . 
lo studio rm fetale con le sequenze ssfp truefisp statiche e tf2d dinamiche ha permesso la visualizzazione del cuore fetale patologico in tutti i 42 feti portatori di patologia cardiaca ( 42 veri positivi ) , mentre nel feto normale alla nascita aveva escluso la sindrome del cuore sinistro ipoplastico ( 1 vero negativo ) : la rm risultata pertanto complessivamente diagnostica in 43 / 43 casi . 
 tuttavia lesame rm , pur diagnosticando la patologia maggiore in tutti i casi , ha anche sospettato la presenza di un difetto del siv escluso alla nascita in 2 feti ( 1 feto con cardiomegalia e 1 feto con rabdomiomi cardiaci ) ( 2 falsi positivi ) ed ha visualizzato un ampio difetto del siv corrispondente alla nascita a multipli difetti interventricolari in un feto con tronco arterioso ( 1 imprecisione diagnostica )  . 
 la patologia cardiaca stata diagnosticata attraverso la visualizzazione in rm dei segni patologici diretti in 21 feti , dei segni indiretti in 6 feti e attraverso entrambi in 15 casi . 
3a , b feto di 34 settimane con sindrome del cuore sinistro ipoplasico : a nella scansione 4 camere il ventricolo sinistro ( vsx ) appare molto ridotto in volume con lume quasi virtuale ; a , b il ventricolo destro ( vdx ) appare aumentato di volume con pareti di spessore maggiore che di norma e marcatamente ipointense in relazione ad ipertrofia del miocardio . 
4a - d feto di 35 settimane con miocardiopatia spongiosa : le pareti del miocardio ( frecce ) appaiono aumentate di spessore , con intensit di segnale ridotta e con struttura compatta . radiol med ( 2009 ) 114 : 852870 fig . 
7a - e feto di 27 settimane con trasposizione completa dei grossi vasi : nelle immagini in successione si evidenzia lorigine dellaorta ( ao ) dal ventricolo destro ( vdx ) e dellarteria polmonare ( ap ) dal ventricolo sinistro ( vsx ) in cuore con regolare connessione atrio - ventricolare . table 4 mri direct signs of abnormalities of the origin and course of the great vessels abnormalities of origin and course of the great vessels mri direct sign 1 truncus arteriosus 2 complete transposition of the great arteries 7 overriding aorta ( 6 tetralogy of fallot , 1 vsd ) 3 right - sided aortic arch vsd , ventricular septal defect ; ivs , interventricular sept a single great artery exiting heart overriding the ventricles the aorta arises from the right ventricle and the pulmonary artery arises from the left ventricle in a heart with normal atrioventricular connections the aorta arises in the middle of the heart and overrides both the ventricles right - to - right course of the aorta tabella 4 segni rm diretti delle anomalie di origine e decorso dei grossi vasi anomalie di origine e decorso dei grossi vasi segno rm diretto 1 tronco arterioso bilanciato 2 trasposizione completa dei grossi vasi 7 aorta a cavaliere ( 6 tetralogie di fallot , 1 div ) 3 arco aortico destroposto div , difetto interventricolare ; siv , setto interventricolare un unico vaso di efflusso arterioso che origina a cavaliere del siv origine dellartao dal ventricolo destro e della arteria polmonare dal ventricolo sinistro in un cuore con normale connessione atrioventricolare origine dellaorta in sede mediana a cavallo del siv e dei due ventricoli un arco aortico che mostra decorso da destra verso destra radiol med ( 2009 ) 114 : 852870 fig . 
magnetic resonance images show a small right ventricle ( vdx in b , * in a , c ) , a large right atrium ( adx in b ) and a ventricular septal defect ( arrow in c )  . 
8a - c feto di 32 settimane con atresia della tricuspide : si visualizza marcata riduzione volumetrica del ventricolo destro ( vdx in b , * in a e c ) , aumento volumetrico dellatrio destro ( adx in b ) , il difetto interventricolare ( freccia in c )  . 
as , atrio sinistro ; vs , ventricolo sinistro ; ao , aorta ; vcs , vena cava superiore . table 5 mri in direct signs of abnormalities of vessel size or valve anomalies abnormalities of the size of the vessels / valve anomalies mri indirect sign 2 pulmonary stenosis 6 fallot , 1 hypoplastic pa 1 coarctation of the aorta 1 tricuspid atresia 1 mitral - aortic disease increased size and hypertrophy of the rv increased size of the right ventricle increased size of the right ventricle and nonvisualisation of the ascending aorta and aortic arch increased size of the ra decreased size of the lv with myocardial hypertrophy and low - flow phenomena pa , pulmonary artery ; rv , right ventricle ; ra , right atrium ; lv , left ventricle tabella 5 segni rm indiretti delle anomalie di calibro dei vasi e delle anomalie valvolari anomalia di calibro dei vasi / anomalia valvolare segno rm indiretto 2 stenosi polmonari 6 tetralogie di fallot , 1 ipoplasia ap 1 coartazione dellaorta 1 atresia della tricuspide 1 patologia mitro - aortica dilatazione ed ipertrofia del vd dilatazione del vd dilatazione del vd e la scarsa visualizzazione dellaorta ascendente e dellarco dilatazione dellad riduzione volumetrica del vs con lipertrofia del miocardio e fenomeni di flusso lento endocavitari ap , arteria polmonare ; vd , ventricolo destro ; ad , atrio destro ; vs , ventricolo sinistro sequences ascribable to the presence of slow or turbulent flows were signs suggestive of incipient heart failure in three fetuses , one of which had a vein of galen aneurysm , one a malformation of the dural sinuses and one tuberous sclerosis . cardiache nelle sequenze t2 haste riconducibile alla presenza di flussi lenti o turbolenti sono segni che hanno fatto ipotizzare un incipiente scompenso cardiaco in 3 feti di cui 1 con aneurisma della vena di galeno , 1 con una malformazione dei seni durali e 1 con una sclerosi tuberosa . 
 discussion abnormalities of the cardiovascular system are the most common congenital diseases in the fetus , they can lead to intrauterine death and are often the cause of premature birth le anomalie congenite del distretto cardiovascolare sono le pi frequenti patologie congenite nel feto , possono portare ad discussione 866 radiol med ( 2009 ) 114 : 852870 fig . 
magnetic resonance images show a large heart with a cardiothoracic index of 0.41 , a pericardial effusion larger than 2 mm ( * in b - d ) and a large ductus arteriosus ( da ) ( a )  . 
9a - d feto di 34 settimane con cardiomegalia e versamento pericardico : il cuore appare aumentato di volume in toto con rapporto cardiotoracico di 0 , 41 ; si evidenzia la presenza di versamento pericardico > 2 mm ( * in b - d ) ; il dotto arterioso lievemente aumentato di dimensioni ( a )  . 
screening for fetal cardiovascular malformations is performed with obstetric ultrasound with four - chamber scans and outflow - vessel scans , although the reference standard for disease diagnosis is echocardiography [ 10 ]  . 
fetal echocardiography is performed in tertiary referral centres by paediatric cardiologists or obstetrician / gynaecologists and is a costly procedure reserved for women with specific risk factors for congenital cardiac diseases arising from family , personal or fetal indications [ 1113 ]  . fetal mri has been taking on an increasingly important role as a support modality to obstetric ultrasound in prenatal diagnosis of fetal malformations [ 14 ]  . 
to date , no negative effects have been demonstrated for the fetus exposed to magnetic fields less than or equal to 1.5 t , and fetal mri is advisable from the second trimester of pregnancy , when a preliminary ultrasound examination proves inadequate or diagnostically [ 1517 ]  . 
however , until recently , the cardiovascular system was difficult to evaluate with mri due to the techniques limitation in evaluating fluid in motion and the organs through which it travels and the need to adjust sequence parameters to the elevated fetal heart rate and the small size of the heart and vessels in the fetus [ 18 ]  . 
lo screening della patologia cardiovascolare fetale affidata allecografia ostetrica attraverso la scansione 4 camere e la scansione per i vasi di efflusso , tuttavia il gold standard per la diagnosi di tale patologia lecocardiografia [ 10 ]  . 
lecocardiografia fetale viene eseguita in centri ultraspecialistici da cardiologi pediatri o da ginecologi ostetrici ed un esame dispendioso riservato a donne con fattori di rischio specifici per le patologie cardiache congenite derivanti da indicazioni familiari , personali o da indicazioni fetali [ 1113 ]  . la rm fetale ha acquisito sempre maggior importanza come metodica di supporto allecografia ostetrica nella diagnosi prenatale delle malformazioni fetali [ 14 ]  . 
allo stato attuale non sono dimostrati effetti dannosi per il feto con campi di esposizione uguali o inferiori a 1 , 5 tesla e lesame rm fetale consigliato a partire dal secondo trimestre di gravidanza quando un preliminare esame ecografico risulti inadeguato o in conclusivo nella diagnosi [ 1517 ]  . tuttavia il distretto cardio - vascolare sembrava fino a poco tempo fa essere malvalutabile con la rm a causa dellostacolo tecnico della metodica nella valutazione dei fluidi in movimento e degli organi da essi attraversati come il sangue e il distretto cardio - vascolare e della necessit di adeguare i parametri tecnici delle sequenze allelevata radiol med ( 2009 ) 114 : 852870 with progressive technological improvement , sequences have been developed that are less sensitive to flow , faster and less subject to fetal movement , and thus produce better temporal and spatial resolution . 
in particular , ssfp sequences have been shown to be excellent for anatomical and functional study of the heart and great vessels in many studies of both adults and neonates [ 19 , 20 ]  . 
recently , these sequences have also been applied to mri study of the fetus and have proven able to identify the heart and great vessels in the uterus [ 21 , 22 ]  . our study confirms the possibility of visualising the fetal heart anatomy and great vessels with static ssfp true - fisp and dynamic tf2d sequences , with good contrast resolution between the blood contained in the cardiac chambers and the myocardial wall as well as the blood contained in the vessels and the vessel walls . in our study all the main anatomical structures were identified and followed in their course in all cases , with progressively reduced difficulty identifying the structures as the cardiac examinations were performed and familiarity with fetal images was acquired . 
in addition , cardiac and vascular structures of interest were visualised not only in the mri scans corresponding to the echocardiographic scans ( obtained with tf2d cine mri ) but also in contiguous scans oriented in planes orthogonal to the fetal cardiac axis ( obtained with static true - fisp sequences )  . this enabled anatomical continuity of the vascular structures to be followed , even in those cases where mri scans corresponding to the echocardiography could not be obtained . the greater difficulty visualising cardiac outflow vascular structures in the earlier weeks of gestation was most likely due to the smaller size of the vessels combined with greater fetal motion during mri acquisitions in this gestational period . 
indeed , the technical impossibility of triggering the fetal heart rate and the still limited temporal resolution of the current cine mri sequences ( two frames per second ) do not allow cardiac functionality to be tracked in real time . instead , cardiac motion is acquired in an artificial manner with its speed being dependent on the rapidity of slice acquisition and not on the real speed of atrioventricular contraction . 
it is therefore fundamental that whoever wishes to perform mri examinations of the fetal heart undergo training in the execution and reporting of fetal cardiac mri . the imaging technique should seek to maximise spatial resolution in both static and dynamic acquisitions by using a small fov , a sufficiently large matrix and thin slices . dynamic scans also require sequences with higher temporal resolution . 
lastly , orienting the data sets in the correct direction orthogonal to the heart and great frequenza cardiaca fetale e alle piccole dimensioni del cuore e dei vasi nel feto [ 18 ]  . con il progressivo miglioramento tecnologico sono state ideate sequenze meno sensibili ai flussi e sempre pi veloci e meno influenzabili dal movimento del feto , con risoluzioni sia temporale che spaziale pi performanti . 
in particolare le sequenze ssfp si sono dimostrate ottime sequenze per lo studio anatomico e funzionale del cuore e dei grossi vasi in numerosi lavori in letteratura sulladulto e sul neonato [ 19 , 20 ]  . 
recentemente queste sequenze sono state applicate anche nello studio rm del feto ed stata dimostrata in tal modo la possibilit di evidenziare il cuore e dei grossi vasi anche in utero [ 21 , 22 ]  . il nostro studio ha confermato la possibilit di visualizzare lanatomia del cuore e dei grossi vasi fetali con le sequenze ssfp truefisp statiche e tf2d dinamiche , con una buona risoluzione di contrasto tra il sangue contenuto nelle camere cardiache e il miocardio parietale cos come tra il sangue contenuto nei vasi e la parete vasale . nel nostro studio in ogni caso tutte le principali strutture anatomiche sono state riconosciute e seguite nel loro decorso , pur dovendo sottolineare una difficolt progressivamente minore in tale riconoscimento man mano che si eseguivano gli esami rm cardiaci e si acquisiva una maggiore familiarit con le immagini fetali . 
inoltre le strutture di interesse cardiache e vascolari sono state visualizzate non solo nelle scansioni rm corrispondenti alle scansioni ecocardiografiche ( ottenute con le cine - rm tf2d ) ma anche nelle scansioni contigue orientate sui piani ortogonali allasse cardiaco fetale ( ottenute con le sequenze statiche truefisp ) consentendo in ultima analisi di seguire la continuit anatomica delle strutture vascolari anche in quei casi in cui non si riuscivano ad ottenere scansioni rm corrispondenti allecocardiografia . da notare una maggiore difficolt di visualizzazione delle strutture vascolari di efflusso cardiaco nelle settimane di gestazione pi precoci probabilmente da metter in relazione alle minori dimensioni dei vasi ed al contemporaneo maggiore movimento del feto durante lacquisizione rm riscontrati in tali epoche gestazionali . 
la contrattilit cardiaca e la funzionalit valvolare sono invece attualmente non valutabili con la rm in maniera attendibile ; infatti limpossibilit tecnica di effettuare il trigger del battito cardiaco fetale e lancora limitata risoluzione temporale delle attuali sequenze cine rm ( 2 frame al secondo ) non permettono di seguire la funzionalit cardiaca in real time ma acquisiscono il movimento cardiaco in maniera artificiale , con velocit dipendente dalla rapidit di acquisizione delle fette e non dalla reale velocit di contrazione atrioventricolare . 
 per quanto riguarda infatti lesecuzione dellesame fondamentale sia nelle acquisizioni statiche che dinamiche ricercare la maggiore risoluzione spaziale possibile utilizzando piccoli fov , matrici sufficientemente grandi e 868 radiol med ( 2009 ) 114 : 852870 vessels is also fundamental in order to obtain images corresponding to those we are used to seeing in echocardiography . reporting of the examination requires recognition of the different anatomical structures and evaluation of their course in subsequent scans . 
in this sense , we believe that graphics software can be particularly useful for rotating and manipulating images to obtain the standard visualisation we are used to seeing on mri or ct studies of the adult , which depict the right cardiac chambers on the left of the image and the left cardiac chambers on the right of the image . in contrast , very few studies with significantly large patient populations have been published on the applicability of fetal mri study for evaluating congenital heart disease [ 23 ]  . 
our experience shows that fetal mri is able to visualise all the main congenital morphological abnormalities , whereas it cannot be used to evaluate cardiac rhythm or atrioventricular valvular abnormalities . in our study , fetal cardiac mri proved to be diagnostic in all 42 cases found to be truly pathological at postnatal echocardiography . 
in addition , in the only case of suspected cardiac disease at prenatal echocardiography that was excluded at the postnatal study , mri ruled out the disease ( one true negative )  . 
however , in the strictest sense of diagnostic accuracy , we need to emphasise the presence of two additional false positives in our series corresponding to two interventricular defects in a fetus with cardiomegaly and another with multiple rhabdomyomas that were ruled out at birth , as well as one diagnostic inaccuracy by depicting multiple interventricular defects as a single , large interventricular defect in a fetus with truncus arteriosus . 
these diagnostic findings , in fetuses at weeks 26 , 33 and 26 of gestation , respectively , seem to depend more on the still limited spatial resolution of the sequences rather than on the early gestational age . fetal cardiac mri relies on both direct and indirect signs that are sought after in all cases of suspected cardiovascular disease . 
marked malrotation can , for example , alter the anatomical planes we are used to seeing in the normal fetus , thus requiring sequence orientation in new planes obtained during the examination itself , leading to longer examination times and occasionally the need to repeat the sequences until the correct standard anatomical scan is obtained . even with these drawbacks , the study of fetal cardiac malformations is possible with mri and has the significant advantage that even the numerous extracardiac abnormalities , which are often associated with cardiac malformations , can be evaluated with a single examination and with the accuracy of mri , especially with regard to the fetal piccoli spessori di strato ; per le scansioni dinamiche inoltre altrettanto importante avvalersi di sequenze con la pi elevata risoluzione temporale . 
infine fondamentale orientare i pacchetti nella giusta direzione ortogonale al cuore e ai grossi vasi per ottenere immagini corrispondenti a quelle che siamo abituati a vedere in ecocardiografia . per quanto riguarda la refertazione , importante riconoscere le diverse strutture anatomiche e seguirne il decorso attraverso la valutazione delle scansioni successive ; a tale proposito risulta in nostra opinione molto utile lutilizzo di software grafici che permettono la rotazione ed il ribaltamento delle immagini per ottenere la visualizzazione standard come siamo abituati ad avere negli esami rm o tc delladulto delle camere cardiache di destra alla sinistra dellimmagine e di quelle di sinistra alla destra dellimmagine . 
 pochissimi lavori con casistiche di rilievo sono stati pubblicati invece sullapplicabilit dello studio rm fetale per la valutazione delle patologie congenite cardiache [ 23 ]  . secondo la nostra esperienza la rm fetale permette la visualizzazione delle principali patologie congenite morfologiche del distretto cardiaco mentre non pu essere utile per la valutazione delle anomalie del ritmo cardiaco e delle anomalie delle valvole atrio - ventricolari . nel nostro studio in tutti i 42 casi realmente patologici al controllo ecocardiografico postnatale la rm cardiaca fetale risultata diagnostica . 
se consideriamo tuttavia laccuratezza diagnostica pi fine dobbiamo sottolineare nella nostra casistica la presenza di 2 falsi positivi aggiuntivi corrispondenti a 2 div in un feto con cardiomegalia e uno con rabdomiomi poi esclusi alla nascita e di una imprecisione diagnostica nella descrizione di multipli div come un unico ampio div in un feto con tronco arterioso . 
tali dati peraltro diagnosticati rispettivamente in un feto a 26 , 33 e 26 settimane di gestazione sembrano dipendere maggiormente dallattuale ancora limitata risoluzione spaziale della sequenza piuttosto che dallepoca gestazionale precoce . 
certamente anche lo studio rm nel caso di anomalie cardiache complesse risulta pi difficile sia da impostare che da interpretare : le marcate anomalie di rotazione alterano per esempio i piani anatomici cui siamo normalmente abituati nel feto normale richiedendo lorientamento delle sequenze su nuovi piani ricavati durante lesame stesso con un prolungamento del tempo di esame e la necessit talvolta di ripetere le sequenze fino allottenimento della corretta scansione anatomica standard . pur con queste puntualizzazioni lo studio del cuore patologico nel feto possibile anche con la rm con il notevole vantaggio che in questo caso anche le numerose patologie extracardiache che spesso sono associate alle patologie radiol med ( 2009 ) 114 : 852870 bra of course , much still needs to be done from the technological perspective to obtain sequences with an optimal compromise between elevated spatial and contrast resolution and enabling real - time acquisition of mri images of the fetal heart that are capable of evaluating cardiac dyskinesia . the main limitation of our study is the knowledge on the part of the radiologists who performed and interpreted the fetal cardiac mri examinations of the presence or absence of a congenital heart disease suspected at fetal echocardiography and the type of congenital cardiac disease suspected in all cases . 
however , although this knowledge may have influenced the search for specific signs of the disease , it is typical of current clinical practice where mri is performed as a third - line investigation in the prenatal diagnosis of congenital disease and operators are inevitably aware of the findings of other previous investigations . in conclusion , fetal mri with ssfp sequences in static and dynamic acquisition enables evaluation of the normal fetal heart and identification of the main morphological alterations of the pathological fetal heart . cardiache possono essere valutate con un unico esame e con laccuratezza della metodica di rm soprattutto per quanto riguarda lencefalo fetale . 
certamente ancora molto deve essere fatto dal punto di vista tecnologico per ottenere sequenze con un ottimale compromesso tra elevata risoluzione spaziale e di contrasto che permettano di avere immagini rm in tempo reale del cuore fetale in grado di valutare anche le discinesie cardiache . 
 il principale limite del nostro studio la conoscenza da parte dei radiologi che hanno effettuato ed interpretato gli esami rm cardiaci fetali della presenza o assenza di una patologia congenita cardiaca sospettata allecocardiografia fetale e del tipo di cardiopatia congenita sospettata in tutti i casi . 
tuttavia tale fatto che potrebbe avere influenzato la ricerca di segni specifici della patologia rispecchia la corrente pratica clinica che prevede lesecuzione dellesame rm come esame di terzo livello nella diagnosi prenatale delle patologie congenite e pertanto impone inevitabilmente ed insostituibilmente la conoscenza dei rilievi delle altre metodiche precedentemente eseguite . in conclusione allo stato attuale la rm fetale con le sequenze ssfp in acquisizione statica e dinamica permette di valutare il cuore fetale normale e di riconoscere le principali alterazioni morfologiche del cuore fetale patologico . acknowledgements the authors thank the engineers at siemens , and particularly leonardo boccaccini , for their constant and fruitful cooperation in implementing the static true - fisp and cine mri sequences for studying the fetus . ringraziamenti gli autori ringraziano gli ingegneri della siemens in particolare ling . 
 + 39 - 045 - 8124301 , fax : + 39 - 045 - 8124775 , e - mail : riccardo.manfredi@univr.it received : 25 september 2008 / accepted : 23 october 2008 / published online : 14 may 2009 springer - verlag 2009 abstract purpose . 
images were assessed for tumour site and size , infiltration of the cervical stroma , infiltration of vaginal fornices and relationship between the tumour and the internal os of the endocervical canal and the presence and dimensions of pelvic and lumboaortic lymph nodes ( cutoff values 1 cm and 0.5 cm minimum axial diameter )  . 
the endocervix was the site of origin of 25% ( 13 / 53 ) of the cervical tumours and the exocervix the site of origin of 75% ( 40 / 53 )  . 
in the assessment of the lymph nodes , when using a cutoff value of 1 cm , mr imaging had a sensitivity , specificity and diagnostic accuracy of 28% , 100% and 89% , respectively . 
sono state sottoposte ad esame rm 53 pazienti con carcinoma della cervice uterina confermato istologicamente , candidate a chirurgia conservativa . lanalisi delle immagini ha compreso : localizzazione e dimensioni del tumore , infiltrazione dellanello stromale , infiltrazione dei fornici vaginali ed estensione oltre lorifizio uterino interno , presenza e dimensioni di linfonodi pelvici o lombo - aortici ( valore soglia 1 cm e 0 , 5 cm di diametro assiale minimo )  . 
nella valutazione dellinfiltrazione dei fornici vaginali la rm ha riportato una sensibilit del 67% , una specificit del 92% ed unaccuratezza diagnostica del 91% ; nella valutazione dellestensione oltre lorifizio uterino interno dell 86% , 93% e 92% , rispettivamente . 
la rm ha dimostrato unelevata accuratezza nella valutazione pre - operatoria dellestensione ( t ) radiol med ( 2009 ) 114 : 960975 imaging had a sensitivity , specificity and diagnostic accuracy of 33% , 92% and 83% , respectively . 
mr imaging had a high level of accuracy in the preoperative assessment of the extent of cervical tumour in patients eligible for conservative surgery . accuracy is lower in the evaluation of the pelvic and lumboaortic lymph nodes . keywords uterine neoplasms cervical cancer magnetic resonance imaging staging tumorale in pazienti candidate ad intervento chirurgico conservativo . 
laccuratezza minore nella valutazione dei linfonodi pelvici e lombo - aortici . parole chiave neoplasie dellutero carcinoma della cervice risonanza magnetica stadiazione introduction introduzione localized uterine cervical carcinoma [ stage iia , with tumour diameter < 4 cm , according to the classification of the international federation of gynaecologists and obstetricians ( figo ) ] may be treated with surgery or exclusive radiation therapy , with similar 5 - year survival rates [ 1 , 2 ]  . nonetheless , in many centres , surgery remains the treatment of choice [ 3 ]  . 
in patients with localized cervical carcinoma , radical surgery consisting of hysterectomy with parametrectomy extended to the pelvic wall , and pelvic and lumboaortic lymphadenectomy may represent overtreatment that exposes the patient to an increased risk of postoperative complications and morbidity [ 47 ]  . 
attempts to modulate the extent of surgical treatment while preserving the degree of oncological disease control may be justified both by the reduction of periand postoperative complications and by the lesser impact on the patients quality of life and sexual functioning . in this context , accurate preoperative clinical and imaging staging of local tumour extension and pelvic and lumboaortic lymph node involvement represent a primary medical need for the planning of surgical treatment . 
 magnetic resonance ( mr ) imaging has shown to be a valuable noninvasive diagnostic modality for evaluating parametrial invasion , which is key in distinguishing localised from advanced disease , leading to different treatment strategies [ 1315 ]  . 
to be able to plan a less aggressive surgical procedure , gynaecological oncologists need to be il carcinoma della cervice uterina localizzato allutero ( stadioiia , con diametro tumorale < 4 cm , secondo la classificazione della federazione internazionale di ginecologia e ostetricia figo ) , pu essere trattato mediante chirurgia o radioterapia esclusiva , con risultati a distanza simili in termini di sopravvivenza [ 1 , 2 ] ; tuttavia , la chirurgia il trattamento di scelta in molti centri [ 3 ]  . 
in pazienti con carcinoma della cervice localizzato allutero , un trattamento chirurgico radicale , che consiste in isterectomia con parametrectomia estesa alla parete pelvica e linfoadenectomia pelvica e lombo - aortica , pu risultare in un sovra - trattamento , esponendo la paziente ad un aumentato rischio di complicanze post - operatorie e di morbilit [ 47 ]  . 
ad eccezione delle neoplasie di grosse dimensioni ( bulky disease ) , le pazienti con carcinoma cervicale localizzato allutero sono infatti potenziali candidate ad una chirurgia meno aggressiva [ 811 ]  . 
un tentativo di modulare lestensione del trattamento chirurgico , preservandone comunque la radicalit oncologica , potrebbe essere giustificato sia dalla riduzione delle complicanze perie postoperatorie , che dal miglior impatto sulla qualit della vita delle pazienti e sulla loro vita sessuale . in questa ottica , unaccurata stadiazione pre - operatoria clinica e strumentale della diffusione locale del tumore e del coinvolgimento dei linfonodi pelvici e lombo - aortici , rappresenta una necessit medica di primaria importanza nella pianificazione del trattamento chirurgico . 
la stadiazione del carcinoma della cervice si basa su criteri clinici , codificati dalla figo ; la stadiazione clinica , tuttavia , comparata con quella chirurgica , ha mostrato un range di errore che va dal 20% al 39% , a seconda dello stadio della malattia [ 12 ]  . 
five were found to have locally advanced disease at mr imaging and were consequently treated with neoadjuvant therapy following a multidisciplinary assessment . the other two refused to undergo mr imaging . 
at histopathology , 39 / 53 ( 73% ) patients had squamous cell carcinoma , 9 / 53 ( 18% ) had adenocarcinoma and 5 / 53 ( 9% ) had adenosquamous cell carcinoma . 
 magnetic resonance imaging mr images were obtained with a 1.5 - t machine ( echospeed , ge medical system , milwaukee , wi , usa ) using a multichannel pelvic coil . 
to limit intestinal peristalsis , patients were instructed to fast for at least 4 h prior to the examination and received an intramuscular injection of 1 mg of butyl scopolamine ( buscopan , schering , germany ) 10 min before the examination . axial t1 - weighted spin - echo ( se ) images were acquired with the following parameters : tr / te 500 / 14 ms , slice thickness 4 mm , interval 1 mm , matrix 256256 . 
axial t2weighted rapid acquisition with relaxation enhancement ( rare ) fast spin - echo ( fse ) images were acquired with tr / te 4 , 000 / 85 ms , echo train length ( etl ) 12 , slice thickness 4 mm , interval 1 mm , matrix 256256 . 
 lo scopo di questo studio quello di determinare laccuratezza della rm nella stadiazione pre - operatoria di pazienti con carcinoma della cervice clinicamente localizzato , candidate ad un intervento chirurgico meno esteso . 
in accordo con i criteri di stadiazione della figo , tutte le pazienti presentavano una neoplasia della cervice uterina localizzata ( stadio < iib ) , alla visita ginecologica in narcosi , e sono state sottoposte a risonanza magnetica ( rm )  . 
sette pazienti sono state escluse dallo studio : 5 di queste avevano una malattia localmente avanzata allindagine rm e , di conseguenza , sono state sottoposte a trattamento neoadiuvante , a seguito di una valutazione multidisciplinare . 
allesame istopatologico 39 / 53 ( 73% ) pazienti avevano un carcinoma squamoso , 9 / 53 ( 18% ) pazienti un adenocarcinoma e 5 / 53 ( 9% ) pazienti un carcinoma adeno - squamoso . 
 risonanza magnetica le immagini rm sono state ottenute utilizzando un magnete da 1 , 5 t ( echospeed , ge medical system , milwaukee , wis ) , usando una bobina pelvica multicanale . 
interpretation differences were resolved by consensus ; evaluation was concordant in 92% of cases and discordant in 8% . in each patient , tumour extent and nodal involvement was assessed on the mr images . 
evaluation of the tumour , performed on t2 - weighted images , comprised site of origin , size , infiltration of the cervical stroma and vaginal fornices and relationships between tumour and internal os of the endocervical canal . 
tumour size was determined by measuring its maximum diameters in three planes and calculating its volume by multiplying the three diameters by 0.52 , according to the ellipsoid formula . tumour infiltration of the cervical stroma was assessed based on the interruption of stromal hypointensity on t2weighted images and classified as < 50% or > 50% of total stromal thickness . 
likewise , the vaginal fornices ( anterior , posterior , left and right ) were considered infiltrated in the presence of an interruption of the hypointensity of the vaginal wall on t2 - weighted images . 
the relationship between the tumour and the internal os of the endocervical canal allowed for the distinction between tumours confined to the cervix from those extending to the uterine body . 
the internal os was recognised as the point of constriction of the uterine contours and the point of evidence of the uterine arteries and , on t2 - weighted images , as the point at which the low signal intensity of the cervical stroma changed into the intermediate signal intensity of the myometrium . the presence , size and site of the pelvic and lumboaortic lymph nodes were also recorded . 
lymph nodes were classified as external iliac , internal iliac , common iliac , internal obturator , bilaterally and lumboaortic in agreement with the surgeons and pathologists [ 17 ]  . 
le immagini assiali t2 - dipendenti rare - fse ( rapid acquisition with relaxation enhancement - fast spin echo ) sono state ottenute con tr / te 4000 / 85 ms , lunghezza del treno di echi ( etl ) 12 , spessore di scansione 4 mm , intervallo 1 mm , matrice 256256 . 
le immagini sagittali e coronali oblique ( parallele allasse longitudinale del canale endocervicale ) t2 - dipendenti rare - fse sono state ottenute con i seguenti parametri : tr / te 35004000 / 90 ms , etl 12 , spessore di scansione 3 mm , intervallo 1 mm , matrice 256256 . sono state inoltre acquisite immagini assiali t2 - dipendenti rare - fse , usando una bobina per il corpo , fino agli ili renali , per valutare leventuale presenza di linfoadenomegalie della catena lombo - aortica . 
 analisi delle immagini lanalisi dellimmagine stata effettuata da due radiologi ( rm , bg ) con esperienza in diagnostica per immagini in ginecologia oncologica ( > 10 anni ) , e la valutazione stata eseguita senza conoscere i dati clinici . 
le discrepanze interpretative sono state risolte per consenso ; le valutazioni sono state concordi tra i due osservatori nel 92% dei casi e discordanti nel 8% . per ogni paziente sono stati analizzati mediante rm lestensione tumorale ed il coinvolgimento linfonodale . lanalisi del tumore , eseguita nelle immagini t2 - dipendenti , ha compreso : la sede di insorgenza , le dimensioni del tumore , linfiltrazione dello stroma cervicale e dei fornici vaginali , i rapporti tra la neoplasia e lorifizio interno del canale endocervicale . 
le dimensioni del tumore sono state determinate misurando i diametri massimi sui tre piani , e calcolandone il volume 3d moltiplicando i tre valori per 0 , 52 , secondo la formula dellellissoide . linfiltrazione tumorale dello stroma cervicale stata valutata in base allinterruzione dellipointensit stromale nelle immagini t2 - dipendenti e classificata come < 50% o > 50% del suo spessore totale . 
analogamente , i fornici vaginali ( anteriore , posteriore , sinistro e destro ) sono stati considerati infiltrati quando presente uninterruzione dell ipointensit , nelle immagini t2 - dipendenti , della parete vaginale . 
twenty - three out of 53 patients ( 43% ) underwent piver type 3 hysterectomies [ 14 ] , 17 / 53 ( 32% ) underwent type 2 , 9 / 53 ( 17% ) underwent type 4 and 4 / 53 ( 8% ) underwent piver type 1 . 
the cervix was separated from the uterine body , completely sectioned along the longitudinal plane ( 12 sections on average ) and stained with haematoxylin and eos the sections were analysed by a single pathologist ( gz ) according to the following criteria : presence and degree of infiltration of the cervical stroma measured from the basal membrane to the maximum penetration depth , involvement of the surgical margins ( positive or negative margins ) and infiltration of the vaginal fornices . 
subsequently , the pathologist documented the total number of lymph nodes , their position , presence / absence of metastatic foci , size of lymph nodes along the short axis and any metastatic foci detected in the lymph nodes . 
 finally , mr findings in regard to infiltration of the cervical stroma and vaginal fornices , relationship of the tumour with the internal os of the endocervical canal and the presence of pelvic or lumboaortic nodal metastases were consensually compared with histological findings by two radiologists ( rm , bg ) , a pathologist ( gz ) and a stroma cervicale cambia nellintermedia intensit di segnale del miometrio . sono stati inoltre rilevati la presenza , le dimensioni e la sede dei linfonodi pelvici e lombo - aortici . 
i linfonodi sono stati classificati in : iliaci esterni , iliaci interni , iliaci comuni , otturatori interni , bilateralmente e lombo - aortici , in accordo con i chirurghi ed i patologi [ 17 ]  . 
sono stati impiegati due valori soglia per distinguere i linfonodi metastatici da quelli infiammatori : 1 cm ( criterio 1 ) e 0 , 5 cm ( criterio 2 )  . infine laccuratezza della rm stata valutata in ciascuna paziente combinando tutti i parametri sopra menzionati , in modo da considerare la reale utilit dellindagine rm per il ginecologo oncologo al fine di pianificare la terapia pi appropriata per ciascuna paziente . tutte le pazienti sono state sottoposte ad un programma di follow - up che consiste in : esame clinico e test di papanicolau ( pap - test ) ogni 3 mesi per i primi 3 anni ed ogni 6 mesi per i seguenti 2 anni ; indagine rm ogni 6 mesi per i primi 2 anni e annuale per i seguenti 3 . 
durante il follow - up 49 / 53 pazienti ( 93% ) non hanno mostrato nessuna evidenza di recidiva di malattia , mentre 4 / 53 ( 7% ) hanno avuto una recidiva a diversa distanza di tempo : 1 paziente dopo 3 mesi dallintervento chirurgico , 1 paziente dopo 5 mesi , 1 paziente dopo 16 mesi ed 1 paziente dopo 20 mesi . 
 chirurgia / istopatologia linfoadenectomia tutte le pazienti sono state sottoposte ad intervento chirurgico entro un intervallo di tempo medio dalla rm di 18 , 8 giorni ( 155 giorni )  . 
ventitre / 53 pazienti ( 43% ) hanno subito unisterectomia di tipo 3 secondo piver [ 14 ] , 17 / 53 ( 32% ) di tipo 2 , 9 / 53 pazienti ( 17% ) di tipo 4 e 4 / 53 pazienti ( 8% ) sono state sottoposte ad unisterectomia di tipo 1 secondo piver . 
nei casi in cui il risultato della palpazione chirurgica e dellesame istologico intraoperatorio stato negativo , il chirurgo ha eseguito unisterectomia radicale . quando il risultato stato positivo , il chirurgo ha completato la procedura tramite isterectomia radicale e radiol med ( 2009 ) 114 : 960975 gynaecologist ( mds ) to determine the diagnostic accuracy of mr imaging . linfoadenectomia lombo - aortica fino allorigine dellarteria mesenterica inferiore . 
 statistical analysis sensitivity , specificity , positive and negative predictive values and diagnostic accuracy were calculated for infiltration of the vaginal fornices , tumour extension to the internal os of the endocervical canal and nodal involvement . 
with regard to nodal involvement , two series of values were calculated based on two cutoff values 1 cm and 0.5 cm measured along the short axis of the lymph node . 
interobserver variability was calculated using the k statistic . concordance between mr imaging and histopathological findings was calculated for each patient , taking into account all parameters described above . results at surgery / histopathology , 45 / 53 ( 85% ) patients had stage ib cervical carcinoma , 5 / 53 ( 9% ) stage ia and 3 / 53 ( 6% ) stage iia . 
le sezioni sono state analizzate da un solo anatomo - patologo ( gfz ) , secondo i seguenti criteri : presenza e grado di infiltrazione dello stroma cervicale , misurata dalla membrana basale fino alla massima profondit di infiltrazione ; coinvolgimento dei margini chirurgici da parte del tumore ( margini positivi o negativi ) ; infiltrazione dei fornici vaginali . 
successivamente , il patologo ha documentato il numero totale di linfonodi , la loro posizione , la presenza / assenza di focolai metastatici , le dimensioni dei linfonodi lungo lasse minore e dei focolai metastatici eventualmente rilevati nei linfonodi . 
 infine i rilievi della rm sullinfiltrazione dello stroma cervicale , sui fornici vaginali , sui rapporti della neoplasia con lorifizio interno del canale endocervicale , cos come la presenza di linfonodi metastatici pelvici o lombo - aortici , sono stati confrontati con i risultati dellistologia collegialmente da due radiologi ( rm , bg ) , un anatomo - patologo ( gfz ) ed un ginecologo ( mds ) , ed stata quindi determinata laccuratezza diagnostica della rm . analisi statistica fornici vaginali , la sensibilit , la specificit , i valori predittivi positivi e negativi , laccuratezza diagnostica sono stati calcolati per linfiltrazione dei tumorale allorifizio interno del canale endocervicale ed il coinvolgimento linfonodale ; per questo ultimo , sono state calcolate due diverse serie di valori , in base ai due valori soglia rispettivamente di 1 cm e 0 , 5 cm , misurati lungo lasse minimo del lestensione fig.1a , b cervical carcinoma : site of origin . sagittal t2 - weighted rapid acquisition with relaxation enhancement ( rare ) image ( tr / te 4 , 000 / 90 ms ) shows site of origin of the cervical neoplasa cervical carcinoma originating from the endocervix in a 54 - year - old woman . 
a sagittal t2 - weighted rapid acquisition with relaxation enhancement ( rare ) image ( tr / te 4 , 000 / 90 ms ) shows cervical carcinoma ( arrowhead ) in a 61 - year - old woman , which is infiltrating the underlying cervical stroma for < 50% of its thickness ( arrow )  . 
a immagine sagittale t2 - dipendente rare ( tr / te 4000 / 90 ms ) , che mostra il carcinoma della cervice uterina ( testa di freccia ) in una donna di 61 anni , che infiltra lo stroma cervicale sottostante per meno della met del suo spessore ( freccia )  . 
b immagine assiale t2 - dipendente rare ( tr / te 4000 / 90 ms ) della stessa paziente di a , che conferma linfiltrazione dello stroma cervicale da parte della neoplasia . 
c immagine assiale t2 - dipendente rare ( tr / te 4000 / 90 ms ) , che mostra infiltrazione dello stroma cervicale ( punta di freccia ) per pi della met del suo spessore da parte del carcinoma cervicale ( freccia curva ) , in una donna di 37 anni . ( 4% ) true positive results , 4 / 53 ( 7% ) false positive results and 1 / 53 ( 2% ) false negative result . 
la concordanza tra immagini di rm ed i reperti istopatologici stata calcolata per ciascuna paziente considerando tutti i parametri sopra menzionati . risultati alla chirurgia / istopatologia , 45 / 53 ( 85% ) pazienti avevano un carcinoma della cervice con stadio ib , 5 / 53 ( 9% ) con stadio ia e 3 / 53 ( 6% ) presentavano una malattia con stadio iia . 
neoplastic infiltration was present in 61 / 2 , 266 ( 3% ) lymph nodes , 44 / 61 ( 72% ) of which were pelvic and 17 / 61 ( 28% ) lumboaortic . 
positive pelvic nodes were located in the following sites : left common iliac in 14 / 44 ( 32% ) cases , left internal obturator in 15 / 44 ( 34% ) , right internal obturator in 6 / 44 ( 14% ) , left external iliac in 4 / 44 ( 9% ) , right external iliac in 4 / 44 ( 9% ) and left internal iliac in 1 / 44 ( 2% )  . 
la sensibilit , la specificit , il valore predittivo positivo e negativo e laccuratezza diagnostica delle immagini rm t2 - dipendenti sagittali , per infiltrazione tumorale attraverso lorifizio uterino interno sono stati 86% , 93% , radiol med ( 2009 ) 114 : 960975 fig . 
a coronal t2 - weighted rapid acquisition with relaxation enhancement ( rare ) image ( tr / te 4 , 200 / 90 ms ) shows infiltration of the right and the left vaginal fornices ( large arrows ) with obliteration of the hypointense vaginal wall ( small arrow ) in a 41year - old woman , the infiltration was confirmed at surgery . 
b the coronal t2 - weighted rare image ( tr / te 4 , 200 / 90 ms ) shows infiltration of the left vaginal fornix in a 27 - year - old woman . 
a immagine coronale t2dipendente rare ( tr / te 4200 / 90 ms ) , che mostra linfiltrazione dei fornici vaginali destro e sinistro ( freccia grande ) , caratterizzata dallobliterazione della parete vaginale ipointensa ( freccia piccola ) in una donna di 41 anni . 
b limmagine coronale t2 - dipendente rare ( tr / te 4200 / 90 ms ) , mostra linfiltrazione del fornice vaginale di sinistra in una donna di 27 anni . 
c nella stessa paziente , il prelievo istologico ( fissato con emotossilina ed eosina , 4 ) nel fornice vaginale di sinistra non ha confermato linfiltrazione , ma ha dimostrato solo la presenza di reazione infiammatoria iperplastica . 
conversely , with the cutoff value of 0.5 cm along the minimum axial diameter ( criterion 2 ) , its sensitivity , specificity , diagnostic accuracy and positive and negative predictive value were 33% , 92% , 83% , 43% and 89% , respectively ( table 1 )  . 
61 / 2266 ( 3% ) linfonodi mostravano infiltrazione neoplastica : tra questi ultimi 44 / 61 ( 72% ) erano pelvici e 17 / 61 ( 28% ) erano lombo - aortici ; tutte le pazienti con linfonodi neoplastici lombo - aortici avevano anche linfonodi pelvici neoplastici . 
i linfonodi pelvici positivi erano localizzati nelle seguenti sedi : iliaca comune di sinistra in 14 / 44 ( 32% ) casi , otturatoria interna sinistra in 15 / 44 ( 34% ) casi , otturatoria interna destra in 6 / 44 ( 14% ) casi , iliaca esterna sinistra in 4 / 44 ( 9% ) , iliaca esterna destra in 4 / 44 ( 9% ) ed iliaca interna sinistra in 1 / 44 ( 2% ) caso . 
allesame istologico , il diametro medio dei linfonodi metastatici , misurato lungo lasse minore , era di 0 , 5 cm ( intervallo 0 , 22 cm ) , mentre era di 0 , 8 cm ( intervallo 0 , 22 cm ) quando misurato lungo lasse maggiore ; il diametro radiol med ( 2009 ) 114 : 960975 medio dei focolai metastatici era di 0 , 5 cm ( intervallo 0 , 11 , 5 cm )  . 
 alla rm erano risultati positivi 80 linfonodi ; 29 / 80 ( 36% ) erano localizzati in sede otturatoria interna di sinistra , 22 / 80 ( 27% ) in sede otturatoria interna di destra , 14 / 80 ( 18% ) in sede iliaca esterna di destra , 8 / 80 ( 10% ) in sede iliaca esterna di sinistra , 3 / 80 ( 4% ) in sede iliaca comune di sinistra , 1 / 80 ( 1% ) era localizzato in sede iliaca comune destra e 3 / 80 ( 4% ) in sede lombo - aortica . 
in 9 / 12 pazienti ( 75% ) lerrore riguardava un solo parametro : 3 / 12 pazienti ( 25% ) mostravano linfonodi falsi negativi , 3 / 12 pazienti ( 25% ) mostravano fornici vaginali falsi positivi , ( 8% ) presentava un coinvolgimento 1 / 12 paziente dellorifizio uterino interno falsamente negativo , 1 / 12 paziente ( 8% ) presentava un coinvolgimento dellorifizio uterino interno falsamente positivo , e 1 / 12 paziente ( 8% ) mostrava fornici vaginali falsamente negativi . 
a sagittal t2 - weighted rapid acquisition with relaxation enhancement ( rare ) image ( tr / te 4 , 000 / 90 ms ) shows infiltration of the myometrium ( curved arrow ) by cervical cancer ( arrow ) beyond the internal os of the endocervical canal ( confirmed at surgery ) in a 62 - year - old woman . 
a immagine sagittale t2 - dipendente rare ( tr / te 4000 / 90 ms ) , che mostra linfiltrazione del miometrio ( freccia curva ) da parte del carcinoma cervicale ( freccia ) , attraverso lorifizio interno del canale endocervicale ( confermata alla chirurgia ) , in una donna di 62 anni . 
in 9 / 12 patients ( 75% ) , the error concerned one parameter only : 3 / 12 patients ( 25% ) showed false negative lymph nodes , 3 / 12 ( 25% ) had false positive vaginal fornices , 1 / 12 ( 8% ) had false negative extension to the internal os , 1 / 12 ( 8% ) had false positive extension to the internal os and 1 / 12 ( 8% ) had false negative vaginal discussione nelle pazienti con carcinoma della cervice uterina in stadio iniziale ( stadio figo < iia ) , la chirurgia il trattamento di scelta nella maggior parte dei centri , con leccezione delle neoplasie di diametro > 4 cm ( ib bulky ) [ 1 , 2 , 13 ]  . 
b in the same patient , the histopathology specimen ( hematoxylin and eosin stain , 10 ) confirmed the nodal metastases with complete invasion by neoplastic tissue . c in this other 35 - year - old patient , normal - size lymph nodes ( minimum diameter < 1 cm ) were detected on t2 - weighted axial images in the left external iliac space . 
b nella stessa paziente il prelievo istologico ( fissato con emotossilina e cosina , 10 ) , conferma le metastasi linfonodali , con invasione completa da parte di tessuto neoplastico . 
c in questaltra paziente di 35 anni , le immagini assiali t2 - dipendenti dello spazio iliaco esterno di sinistra , mostrano linfonodi con dimensioni nei limiti della norma ( diametro minimo < 1 cm )  . 
patients with early cervical carcinoma are potential candidates for a less extensive , but nonetheless oncologically radical , surgical operation that aims to reduce periand postoperative complications ( haemorrhage , fistula , bladder lymphocele , urinary lymphoedema , dysfunction ) and limit the heavy psychological impact of surgery on the patients quality of life and sexual functioning [ 8 , 18 ]  . 
this information regards , on the one hand , the tumour and specifically its size , location and infiltration of the internal os and vaginal fornices and on the other , the number , size and location of lymph nodes . 
 tumour size has prognostic implications , as it correlates with locoregional infiltration and nodal metastases [ 16 , 19 ]  . mr imaging allows direct measurement of tumour size , especially as regards its endocervical portion . 
nonetheless , the accuracy of mr imaging in determining tumour size has been widely demonstrated in previous studies [ 13 , 16 ]  . the depth of stromal infiltration is a major prognostic factor , as it is associated with the risk of nodal involvement [ 16 ]  . 
in our study , there was agreement between mr imaging and histopathology for parametrial infiltration in 40 / 53 ( 75% ) patients , with a tendency towards underestimation in 11 / 53 ( 21% )  . 
this is easily explained by the failure to visualize microscopic foci of infiltration . the fornices represent the upper third of the vagina , where the vaginal wall reflects back to merge with the cervical stroma . 
the presence of infiltration of the fornices indicates figo stage iia cervical carcinoma and implies a worse prognosis relative to tumour confined to the cervix ( stage ib )  . 
from the surgical point of view , infiltration of the vaginal fornices involves a more aggressive hysterectomy extending the upper vagina , consequently increasing the piver class [ 15 ]  . 
our study demonstrates a high specificity and accuracy , 92% and 91% , respectively , of mr imaging in evaluating infiltration of the vaginal fornices , but a low sensitivity ( 67% )  . 
given that the presence of false negative results for vaginal infiltration leads to undertreatment , it may be useful to introduce gadolinium chelates or air into the vagina to facilitate this evaluation [ 18 ] , or to perform a gynaecological examination under anaesthesia , as this allows direct visual inspection and biopsy of the fornices [ 13 , 20 ]  . 
in addition , in our study , there were four per la pianificazione del trattamento riguardano da una parte il tumore ed in particolare , le dimensioni , la localizzazione e linfiltrazione dellorifizio uterino interno e dei fornici vaginali , dallaltra i linfonodi , con il loro numero , le dimensioni e la localizzazione . 
un limite del nostro studio stato il mancato riscontro di una correlazione diretta tra la misurazione rm e quella istopatologica ; comunque , laccuratezza della rm nel determinare le dimensioni tumorali stata ampiamente dimostrata in precedenti studi [ 13 , 16 ]  . la profondit dellinfiltrazione stromale un importante fattore prognostico , in quanto si associa al rischio di diffusione linfonodale di malattia [ 16 ] ; inoltre , la sua integrit comporta un alto valore predittivo negativo per infiltrazione parametriale ( 96%99% ) [ 14 , 15 , 20 , 21 ]  . 
nel nostro studio c stata una concordanza rm / istopatologia per infiltrazione parametriale in 40 / 53 ( 75% ) pazienti , con una tendenza alla sottostima in 11 / 53 ( 21% ) , che pu essere facilmente spiegata dalla mancata visualizzazione dei focolai di infiltrazione microscopici . i fornici rappresentano il terzo superiore della vagina , dove la parete vaginale si ripiega per unirsi con lo stroma cervicale . 
la presenza di infiltrazione dei fornici , indica un carcinoma cervicale allo stadio iia , secondo la classificazione figo , ed implica una prognosi peggiore rispetto al tumore confinato alla cervice ( stadio ib )  . 
dal punto di vista chirurgico , linfiltrazione dei fornici vaginali comporta lesecuzione di unisterectomia pi estesa , che comprende anche la parte superiore della vagina , aumentando , di conseguenza , la classe nello schema secondo piver [ 15 ]  . 
il nostro studio dimostra unelevata specificit ed accuratezza , 92% e 91% rispettivamente , della rm nel valutare linfiltrazione dei fornici vaginali , ma una bassa sensibilit ( 67% )  . 
abbiamo riportato 1 falso negativo , in cui non siamo stati in grado di distinguere il tumore dalla parete vaginale . dato che la presenza di falsi negativi per infiltrazione vaginale comporta un sotto - trattamento , pu essere daiuto lintroduzione nel lume vaginale di chelati del gadolinio o di aria , per una loro pi agevole valutazione [ 18 ] , oppure affidarsi alla visita ginecologica in narcosi , in quanto questa in grado di visualizzare direttamente i fornici vaginali e permette di eseguire prelievi bioptici [ 13 , 20 ]  . 
nel nostro studio sono stati inoltre riportati 4 falsi positivi , due dei quali dovuti alla presenza di una reazione flogistica aberrante , che molto difficile da distinguere dallinfiltrazione neoplastica con la rm . 
questi falsi positivi hanno portato ad un sovra - trattamento , che comunque non ha interferito con la prognosi delle pazienti . 972 radiol med ( 2009 ) 114 : 960975 false positive results , two due to the presence of an aberrant inflammatory reaction , which is very difficult to distinguish from neoplastic infiltration on mr imaging . 
these false positive results led to overtreatment , which did not , however , interfere with the patients prognosis . the relationship between tumour and internal os of the endocervical canal and the presence of tumour extension to the uterine body constitute an important prognostic factor and are correlated with the risk of nodal involvement [ 1 ]  . moreover , this prognostic factor may be important in young patients ( < 35 years ) with localised cervical cancer who wish to have a pregnancy . 
this group of patients requires a surgical approach that combines radical excision of the tumour with conservation of the uterus to preserve fertility ( trachelectomy ) [ 9 , 2022 ]  . 
in our study , we achieved a high level of diagnostic accuracy ( 92% ) in determining the relationship between the tumour and the internal os of the endocervical canal , with a specificity of 93% and a negative predictive value of 98% . in women with cervical carcinoma , the presence of nodal metastasis is an important risk factor that has a radical impact on prognosis and treatment planning and is associated with parametrial involvement in nonadvanced stages of disease [ 10 , 11 ]  . 
to differentiate normal from metastatic lymph nodes on mr imaging , a dimensional criterion is used that involves a cutoff value of 1 cm measured along the short axis of the node [ 2329 ]  . 
in our study , mr accuracy in evaluating nodal metastasis was 89% , considering a cutoff value of 1 cm , and 83% with a cutoff value of 0.5 ctherefore , lowering the cutoff value from 1 cm to 0.5 cm leads to a higher false positive rate , with consequent reduction in specificity , accuracy and positive predictive value and a slight increase in sensitivity ( one patient ) ( table 1 )  . 
all lymph nodes that were found to be metastatic at histology and were false negative at mr imaging had a minimum axial diameter < 1cm and were thus not classified as pathological ( table 2 )  . 
in our study , surgically excised lymph nodes had a mean minimum diameter of 0.5 cm and maximum diameter of 0.8 cthe mean maximum diameter of small intraparenchymal metastases was 0.5 c the main limitation of diagnostic imaging lies in identifying small nodal metastases , as seen in our study . 
the presence of internal necrosis , which appears as an area of signal hyperintensity on t2 - weighted mr images , was of no assistance in our study : none of the 61 neoplastic lymph nodes showed this feature , which is more commonly found in large lymph nodes , as reported by other studies ( mean diameter 2.3 cm ) [ 30 ]  . il rapporto del tumore con lorifizio interno del canale endocervicale e la presenza di estensione del tumore al corpo dellutero sono un importante fattore prognostico e sono correlati al rischio di coinvolgimento linfonodale [ 1 ]  . inoltre , questo fattore prognostico pu essere importante nelle pazienti giovani ( < 35 anni ) con carcinoma cervicale localizzato che desiderano una gravidanza . 
in questo gruppo di pazienti richiesto un approccio chirurgico che combini lescissione radicale del tumore e la conservazione dellutero per preservare la fertilit ( trachelectomia ) [ 9 , 2022 ]  . 
nel nostro studio stato raggiunta unalta accuratezza diagnostica ( 92% ) nel determinare il rapporto tra tumore e orifizio interno del canale endocervicale , con una specificit del 93% ed un valore predittivo negativo del 98% . nelle donne con carcinoma della cervice la presenza di linfonodi metastatici un importante fattore di rischio che cambia radicalmente la prognosi e la pianificazione terapeutica ed associato a coinvolgimento del parametrio negli stadi non avanzati [ 10 , 11 ]  . 
nellimaging rm per differenziare i linfonodi normali da quelli metastatici viene utilizzato un criterio dimensionale che prevede un valore soglia di 1 cm misurato lungo il diametro minore [ 2329 ]  . laccuratezza della rm nellidentificare linfonodi metastatici varia dal 75% al 100% , con un valore medio del 86% [ 2329 ]  . 
nel nostro studio laccuratezza della rm nella valutazione dei linfonodi metastatici stata del 89% , considerando come valore soglia 1 cm , e del 83% considerando come valore soglia 0 , 5 cm ; quindi , abbassando il limite del valore soglia da 1 cm a 0 , 5 cm , aumentano i falsi positivi , con conseguente riduzione della specificit , dellaccuratezza e del valore predittivo positivo e con lieve aumento della sensibilit ( 1 paziente ) ( tabella 1 )  . 
tutti i linfonodi metastatici allistologia e falsi negativi alla rm , presentavano un diametro assiale minimo < 1 cm e , pertanto , non sono stati classificati come patologici ( tabella 2 )  . 
il diametro medio dei linfonodi asportati chirurgicamente nel nostro studio stato , infatti , di 0 , 5 cm il minimo e di 0 , 8 il massimo ; il diametro massimo medio delle piccole metastasi intraparenchimali stato di 0 , 5 c il limite pi grande della diagnostica per immagini stato quello di individuare le piccole metastasi linfonodali , come emerso dai dati del nostro studio . 
una possibile soluzione potrebbe essere lutilizzo di mezzo di contrasto ultrasmall superparamagnetic iron oxide ( uspio ) [ 30 ] , o la tomografia ad emissione di positroni [ 29 ]  . 
la presenza di necrosi interna , che si visualizza tramite rm come unarea di iperintensit nelle sequenze t2 - dipendenti , non ci stata daiuto nel nostro studio : dei 61 linfonodi neoplastici , nessuno mostrava questo reperto , che invece pi facile ritrovare in linfonodi di grandi dimensioni , come riportato in altri studi in cui il diametro medio era di 2 , 3 cm [ 30 ]  . nel nostro studio stata riportata unimportante discrepanza tra il numero di linfonodi asportati e quelli osservati radiol med ( 2009 ) 114 : 960975 table 2 evaluation of false negative lymph nodes on magnetic resonance ( mr ) imaging ( using a cutoff value of 1 cm ) compared with histopathology histopathology mr imaging total no . 
totale dei sede dei linfonodi diametro linfonodi metastatici metastatici massimo del linfonodo pi voluminoso ( cm ) dimensioni dei n totale di foci metastatici ( cm ) linfonodi visualizzati diametro minimo del linfonodo pi voluminoso ( cm ) istopatologia para - aortici para - aortici 10 , 4 0 , 80 , 4 10 , 4 10 , 8a 10 , 3 0 , 90 , 4 10 , 6 10 , 5a 0 , 70 , 5 0 , 50 , 5 0 , 90 , 8 10 , 5 1 , 20 , 7 0 , 50 , 2 0 , 70 , 4 1 , 20 , 5 1 , 2a 1 , 1a sede dei linfonodi non visualizzati non visualizzati non visualizzati non visualizzati non visualizzati non visualizzati para - aortici d , destra ; s , sinistra ; oi , otturatori interni ; ie , iliaci esterni ; ic , iliaci comuni aestensione extracapsulare 974 radiol med ( 2009 ) 114 : 960975 in our study , there was a major discrepancy between the number of excised lymph nodes and the number of nodes observed on mr imaging . 
most probably this was due to two factors : some lymph nodes were counted more than once at histology owing to sectioning , and others were too small to be visualised by mr imaging . in conclusion , mr imaging had relatively good accuracy ( 77% ) in evaluating all surgical factors that the gynaecological oncologist needs for planning treatment . 
molto probabilmente questo dovuto a due fattori : alcuni linfonodi sono stati contati pi di una volta allistologia a causa dei tagli ed altri linfonodi erano troppo piccoli per essere visualizzati con la rm . in conclusione , la rm ha dimostrato una discreta accuratezza ( 77% ) nel valutare tutti i fattori chirurgici di cui ha bisogno il ginecologo oncologo per pianificare il trattamento . 
magrassi , ii universit di napoli , azienda ospedaliera universitaria , 80100 napoli , italy 4dipartimento di scienze chirurgiche , radiologiche e odontostomatologiche , unit di oncologia chirurgica universit di perugia , ospedale s . 
andrea delle fratte , 06134 perugia , italy 5dipartimento di radiologia , ospedale di budrio , azienda sanitaria locale bologna , 40054 budrio , italy 6dipartimento di scienze della salute , sezione di diagnostica per immagini , universit del molise , c.da tappino , 86100 campobasso , italy correspondence to : m . 
we retrospectively reviewed the hepatic arterial - dominant phase ( hap ) , portal venous phase ( pvp ) and delayed - phase ( dp ) helical ct images of 47 patients with 52 atypical small ( 2cm ) hhs associated with 34 typical small hhs . 
sono stati valutati retrospettivamente gli esami tc trifasici in fase dominante - arteriosa ( fa ) , fase venosa portale ( fvp ) e fase tardiva ( ft ) , di 47 pazienti con 52 emangiomi epatici ( ee ) atipici di piccole dimensioni ( 2 cm ) associati a 34 ee tipici piccoli . 
on dp images , the appearance of atypical small hhs was identical to that of typical small hhs in all cases ( p < 0.0001 ) , with lesions showing homogeneous isoattenuation to the aorta or liver parenchyma without peripheral capsule . 
in ft laspetto degli ee atipici di piccole dimensioni ( isodensit rispetto allaorta o al parenchima epatico con assenza di capsula periferica ) risultato sovrapponibile a quello degli ee tipici di piccole dimensioni ( p < 0 , 0001 )  . 
unlike typical hhs , these lesions do not show globular or nodular peripheral enhancement similar to the attenuation of blood vessels at rapid ct with bolus administration of contrast material . 
to our knowledge , there have been only few reports investigating three - phase on hepatic arterialdominant phase ( hap ) , portal venous phase ( pvp ) , and delayed phase ( dp ) helical ct imaging in characterising hhs [ 7 ]  . 
the rationale for using three - phase helical ct is that the pvp is the most sensitive phase for lesion detection , whereas the hap and dp can supply additional information on the vascularity of the lesions that may help to clarify their nature [ 7 ]  . 
the purpose of this paper is to describe the findings of atypical small ( 2 cm ) hhs in non - cirrhotic liver to verify the usefulness of triple - phase helical ct . 
 materials and methods patients we retrospectively reviewed the ct images of 52 small ( < 2 cm ) hhs in 47 patients studied over the past 4 years lemangioma epatico ( ee ) rappresenta la neoplasia benigna pi frequente del fegato . 
lintroduzione nella pratica clinica della tomografia computerizzata ( tc ) spirale ha determinato un ulteriore aumento del riscontro di ee di piccole dimensioni ( 2 cm ) e di forme atipiche [ 2 ]  . queste ultime sono lesioni con aspetto inusuale che , diversamente dagli ee classici , non esibiscono , dopo iniezione a bolo di mezzo di contrasto ( mdc ) per vena , enhancement globulare e periferico , ad andamento centripeto . 
il rilievo di un ee atipico di piccole dimensioni , in particolare nel paziente oncologico , pone spesso problemi di diagnosi differenziale , poich tali lesioni possono simulare neoplasie epatiche benigne e maligne sia ipoche ipervascolari o metastasi . 
rari lavori , invece , su casistiche limitate , hanno valutato mediante tc trifasica in fase dominante - arteriosa ( fa ) , fase venosa portale ( fvp ) e fase tardiva ( ft ) laspetto degli ee atipici [ 7 ]  . 
il razionale della tc trifasica , nella caratterizzazione delle lesioni focali epatiche consiste in una maggiore sensibilit della fvp nellidentificazione delle lesioni , mentre la fa e la ft consentono di ottenere informazioni sulla loro vascolarizzazione e sulla loro possibile natura [ 7 ]  . 
scopo del presente studio quello di riportare i diversi aspetti degli ee atipici di piccole dimensioni ( 2 cm ) con tc spirale a singolo e multistrato trifasica nel paziente non - cirrotico , al fine di verificare laffidabilit diagnostica della metodica . 
patients ( 16 women and 31 men ; age range 2584 years , mean age 56.8 years ) underwent triple - phase ct to further investigate atypical small hhs classified as indeterminate lesions at ultrasonography ( us ) or single - phase or biphasic ct , or as part of preoperative staging or follow - up of known extrahepatic primary malignancies . exclusion criteria included steatosis , cirrhosis , elevated - fetoprotein levels , concurrent benign adenoma , focal nodular hyperplasia ( fnh ) or malignant hepatic tumours , or use of oral contraceptives in the 6 months prior to the study . lesions excluding the large ( > 2 cm ) hhs associated with atypical small hhs , the 52 atypical small hhs were solitary in 19 patients , ranging from one to three and / or coexisting with 34 typical small hhs in 28 patients . 
in the remaining patients , the final diagnosis was based on the clinical course or lack of change in lesion appearance or size for at least 25 ( range 648 ) months at follow - up with us , ct and magnetic resonance imaging ( mri )  . ct technique ct scans were obtained by using a single - detector - row helical ct scanner ( advantage ave 1 , philips medical systems , best , the netherlands ) in 26 patients and a 16detector - row helical ct scanner ( lightspeed plus and lightspeed plus 16 pro , ge healthcare , milwaukee , wi , usa ) in 21 patients . 
 materiali e metodi pazienti sono state analizzate retrospettivamente le immagini tc di 52 ee atipici di piccole dimensioni ( 2 cm ) , osservati nellarco degli ultimi quattro anni in 47 pazienti sottoposti al medesimo protocollo di studio con tecnica tc spirale trifasica . 
i pazienti ( 16 femmine e 31 maschi di et compresa tra 25 e 84 anni , et media 56 , 8 anni ) , sono stati sottoposti a tc trifasica in seguito al rilievo di ee atipici piccoli identificati come lesioni indeterminate riscontrate occasionalmente in corso di una precedente ecografia ( us ) , tc monoo bifasica oppure per una stadiazione preoperatoria o follow - up per neoplasia extraepatica primitiva maligna nota . 
 nessuno dei pazienti inclusi nello studio presentava steatosi epatica , cirrosi , elevati livelli di - fetoproteina , lesioni epatiche benigne adenoma , iperplasia nodulare focale ( inf ) o maligne , n assunzione di contraccettivi orali da almeno 6 mesi . 
 lesioni con esclusione degli ee epatici > 2 cm associati agli ee atipici di piccole dimensioni , i 52 ee epatici piccoli atipici in 19 pazienti erano unici ed in 28 pazienti in numero variabile da uno a tre e / o coesistenti con 34 ee tipici piccoli . 
la diagnosi di ee atipico in 7 dei pazienti con lesione unica stata confermata mediante esame istologico ( n = 2 ) e citologia aspirativa sotto guida ecografica ( n = 5 ) ; nei rimanenti pazienti la diagnosi radiologica stata confermata in base al decorso clinico ed allimmodificazione dellaspetto e delle dimensioni delle lesioni ad un follow - up radiologico con us , tc e / o risonanza magnetica ( rm ) variabile da 6 mesi a 48 mesi ( media , 25 mesi )  . 
 tecnica tc gli esami tc sono stati eseguiti con apparecchiatura tc spirale singolo strato ( advantage ave 1 ; philips medical systems , best , olanda ) in 26 pazienti e con tc 16 - strati / rotazione ( light speed plus and light speed pro 16 , ge healthcare , milwaukee , usa ) in 21 pazienti . 
il tempo di ritardo dallinizio delliniezione del mdc per vena , stato registrato a 30 secondi , 70 secondi e 5 minuti per la tc spirale a singolo strato ed a 35 secondi , 80 secondi e 5 minuti per la tc 16 - strati . 
 stata utilizzata una quantit media di 150 ml di mdc organo - iodato per paziente alla concentrazione di 300370 mgi / ml ( iopromide , ultravist schering , berlino , germania ) , 938 radiol med ( 2009 ) 114 : 935947 scanning parameters were : section thickness 2.5 mm and 5 mm and table feed 18.75 mm / s and 5 mm / s for the 16 - detector - row scanner and single - detector - row scanner , respectively . 
hap , pvp and dp images were reviewed for the presence of hhs . according to the literature [ 7 ] , the appearance of each lesion in each phase was described on the basis of the attenuation and homogeneity of the lesion in comparison with the aorta and recorded as follows : 1 . 
isoattenuating relative to the arterial syste atypical small hhs were defined as lesions with diameter < 2 cm and with unusual appearance on hap or pvp images with respect to classic forms . 
patterns of enhancement of atypical hhs were classified into three types : type 1 : hyperattenuating hh ( but less attenuating than the aorta ) either without ( type 1a ) or with transient hepatic attenuation difference ( thad ) around the lesion on hap images type 2 : homogeneously hypoattenuating hh in the ha or pvp ( type 2a ) and hypoattenuating area with brightdot sign in the hap or pvp ( type 2b )  . 
the bright - dot sign was defined as a tiny enhancing dot within an hh that did not progress to globular enhancement [ 4 ] type 3 : hypoattenuating hh with central enhancing area ( centrifugal enhancement pattern ) in the pvp . 
in accordo con i dati della letteratura [ 7 ] le caratteristiche di enhancement delle lesioni in ciascuna fase sono state classificate come segue , utilizzando come riferimento la densit dellaorta : 1 . 
 nel nostro studio sono stati definiti ee atipici di piccole dimensioni , lesioni con diametro 2 cm , che in corso di tc trifasica mostravano aspetti inusuali non riscontrabili nelle forme classiche in fa e fvp . 
tali lesioni , in base al pattern di enhancement , sono state distinte in tre tipi : tipo 1 , ee iperdenso ( ma con densit pi bassa rispetto allaorta ) in fa senza alterazione transitoria della densit epatica o thad ( transient hepatic attenuation difference ) ( tipo 1a ) o con associata thad ( tipo 1b ) ; tipo 2 , ee ipodenso , omogeneo in fa o fvp ( tipo 2a ) e ipodenso con puntiforme iperdensit intralesionale periferica in fa o fvp ( tipo 2b ) che non realizza un enhancement globulare [ 4 ] ; tipo 3 , ee ipodenso con iperdensit centrale in fvp e ad enhancement centrifugo . per ogni ee atipico di piccole dimensioni sono stati valutati : radiol med ( 2009 ) 114 : 935947 categorized as rapid if > 75% of the tumour volume enhanced ; intermediate if approximately 25%75% of the tumour volume enhanced and slow if < 25% of the tumour volume enhanced the qualitative ( visual ) analysis of the attenuation of atypical and typical small hhs during the dp . statistical analysis all the lesions were reviewed by three radiologists . 
statistical analysis was carried out using spss software for windows ( release 13.0 ; spss chicago , il , usa )  . results the results of triple - phase helical ct in atypical and typical small hhs are summarised in tables 1 and 2 , respectively . 
in fa stata correlata la presenza di thad con il tipo di enhancement delle lesioni ed in ft stato correlato laspetto degli ee atipici ( n = 52 ) di piccole dimensioni con quello delle forme tipiche ( n = 34 ) di piccole dimensioni . le differenze statistiche sono state calcolate mediante il test esatto di fisher . 
one type 1a lesion was resected , and pathology revealed the typical vascular pattern of cavernous hh consisting of multiple vascular spaces filled with blood , areas of fibrosis and organised thrombi . 
type 2a lesions showed hypoattenuation ( n = 2 ) risultati i risultati della tc trifasica relativi agli ee atipici e tipici di piccole dimensioni sono riportati rispettivamente nelle tabelle 1 e 2 . 
un ee atipico di tipo 1a stato asportato ; lesame istologico ha evidenziato la presenza di pattern vascolare tipico dellee cavernoso con ampie lacune vascolari ripiene di eritrociti e zone fibrotiche con piccoli vasi . 
1a - d triphasic helical ct of an atypical small hepatic haemangioma in the right liver lobe ( segment vi ) : type 1a , hyperattenuating area with rapid fill - in ( a - c ) and corresponding histological examination ( d )  . 
ct scan shows a homogeneous hyperattenuating lesion in the arterial phase ( a ) , substantially isoattenuating in the portal venous phase ( b ) and isoattenuating in the delayed phase at 5 minutes ( c ) after administration of contrast material . 
1a - d tc trifasica di un emangioma epatico atipico di piccole dimensioni nel lobo destro epatico ( segmento vi ) : tipo 1a , aspetto iperdenso a riempimento rapido ( a - c ) e corrispettivo esame istologico ( d )  . 
la lesione esibisce intenso omogeneo enhancement in fase arteriosa ( a ) ed aspetto sostanzialmente isodenso in fase venosa portale ( b ) e tardiva a 5 minuti ( c )  . 
2a - c triphasic helical ct of an atypical small hepatic haemangioma in the right liver lobe ( segment v ) : type 1b , hyperattenuating area with rapid fillin associated with transient hepatic attenuation difference ( thad )  . 
ct scans show a homogeneous hyperattenuating lesion with thad during the arterial phase ( a ) , which appears isoattenuating during the portal venous phase ( b ) and delayed phase at 5 minutes ( c ) after administration of contrast material . 
2a - c tc trifasica di un emangioma epatico atipico di piccole dimensioni nel lobo destro epatico ( segmento v ) : tipo 1b , aspetto iperdenso a riempimento rapido con thad . 
la lesione mostra intenso omogeneo enhancement e thad adiacente in fase arteriosa ( a ) ed aspetto isodenso ai vasi in fase venosa portale ( b ) e tardiva a 5 minuti ( c )  . 
six out of nine type 2a lesions underwent histological examination ( n = 1 ) or us - guided aspiration cytology completa di enhancement ( tipo 2a ) e forme ipodense con minimo puntiforme enhancement periferico ( bright - dot sign ) ( tipo 2b )  . 
il tipo 2a stato riscontrato in 9 dei 52 ee atipici ; tali lesioni in fa sono apparse ipodense ( n = 2 ) ed radiol med ( 2009 ) 114 : 935947 fig . 
3a - d triphasic helical ct of an atypical small hepatic haemangioma in the left liver lobe ( segment ii ) : type 2a , homogeneously hypoattenuating area during the portal - venous phase ( a - c ) and corresponding histological examination ( d )  . 
ct scans show an isoattenuating lesion in the arterial phase ( a ) , hypoattenuating in the portal venous phase ( b ) and isoattenuating in the delayed phase at 5 minutes ( d ) after administration of contrast material . 
3a - d tc trifasica di un emangioma epatico atipico di piccole dimensioni nel lobo sinistro epatico ( segmento ii ) : tipo 2b , aspetto ipodenso con completa assenza di enhancement in fase venosa portale e corrispettivo esame istologico ( d )  . 
la lesione isodensa in fase arteriosa ( a ) appare ipodensa in fase venosa portale ( b ) ed isodensa ai vasi in fase tardiva a 5 minuti ( c )  . 
 ( n = 5 ) : histology revealed extensive fibrosis with reduction of vascular channels , whereas aspiration cytology revealed few erythrocytes with predominant spindle cells , suggestive of fibrosis . 
in 6 delle 9 forme di tipo 2a la diagnosi stata ottenuta mediante esame istologico ( n = 1 ) e citologia aspirativa sotto guida ecografica ( n = 5 )  . 
lesame istologico ha dimostrato estesa fibrosi con riduzione dei vasi ; le 5 lesioni aspirate sotto guida ecografica hanno mostrato scarsi eritrociti con predominanza di cellule connettivali fusate espressione di fibrosi . 
 in ft stata osservata completa concordanza tra i tre osservatori in tutti i casi relativamente allaspetto degli ee atipici piccoli rispetto a quelli piccoli tipici ( p < 0 , 0001 ) con rilievo di lesioni isodense rispetto allaorta o al parenchima epatico ed assenza di capsula periferica . 
univoca concordanza tra i tre osservatori stata osservata in tutti i casi di ee atipico di piccole dimensioni di tipo 1a , 1b , 2a e 3 . concordanza elevata stata riscontrata , invece , nei casi di radiol med ( 2009 ) 114 : 935947 fig . 
4a - c triphasic helical ct of an atypical small hepatic haemangioma in the left liver lobe ( segment iv ) : type 2b , hypoattenuating area with slow fill - in and bright - dot sign . 
ct scan show isoattenuating lesion in the arterial phase ( a ) , hypoattenuating with peripheral bright dot in the portal venous phase ( b ) and isoattenuating in the delayed phase at 5 minutes ( c ) after administration of contrast material . 
4a - c tc trifasica di un emangioma epatico atipico di piccole dimensioni nel lobo sinistro epatico ( segmento iv ) : tipo 2b , aspetto ipodenso a riempimento lento con puntiforme iperdensit periferica ( bright - dot sign )  . 
la lesione isodensa in fa ( a ) appare ipodensa con puntiforme iperdensit periferica in fase venosa portale ( b ) e sostanzialmente isodensa ai vasi in fase tardiva a 5 minuti ( c )  . 
la lesione isodensa in fase arteriosa ( a ) , mostra aspetto ipodenso con puntiforme enhancement centrale in fase venosa portale ( b ) ed aspetto isodenso allaorta in fase tardiva a 5 minuti ( c )  . 
because the high incidence of atypical hh in radiological practice makes their surgical resection unfeasible , the accuracy of a radiological diagnosis of hh is based on follow - up imaging and changes in appearance and size over a long period of time [ 13 ]  . 
 the rationale for evaluating small ( 2 cm ) hhs by triplephase helical ct scans [ hepatic arterial - dominant phase ( hap ) , portal venous phase ( pvp ) , and delayed phase ( dp ) ] was that each phase of contrast enhancement provides useful information about the enhancement of lesions and thus helps to determine their vascularity . 
 [ 16 ] reported that thad is present in the 21% of atypical hh 2 cthad is thought to be related to the hyperdynamic status of rapidly enhancing small hhs where the large arterial inflow and consequently the large and rapid outflow results in early opacification of the draining vein and peritumoural enhancement [ 17 ]  . the incidence of atypical hypoattenuating or slow hhs ranges from 8% to 16% [ 4 , 17 ]  . 
such lesions include those with complete absence of enhancement ( that pose a significant diagnostic challenge and cannot be characterised by dual - phase ct ) [ 4 ] and hypoattenuating lesions with minimal enhancement or bright - dot sign , which has a considerable diagnostic value [ 4 , 18 ]  . 
 [ 19 ] reported that the histological examination of atypical hhs with inside - out pattern revealed fibrosis in variable degrees and a central area composed of numerous vascular spaces , which showed early and prolonged enhancement on dynamic bolus ct . 
 the aim of our study was to verify the usefulness of triple - phase helical ct in the diagnosis and to determine the enhancement pattern of small atypical hhs in noncirrhotic patient . 
la dizione radiologica ee atipico non quindi riconducibile ad una lesione vascolare di tipo capillare , ma esclusivamente allee cavernoso , eventualmente con alterazioni regressive ( infarto , necrosi , fibrosi , forme peduncolate , trombi , calcificazioni distrofiche )  . 
poich non proponibile lasportazione chirurgica per il numero elevato di casi riscontrati nella pratica radiologica , al fine di documentare con appropriatezza la diagnosi radiologica di ee , possiamo basarci sul follow - up ed evidenziare eventuali modificazioni del loro aspetto e delle dimensioni [ 13 ]  . 
a tal proposito va ricordato che una lesione che rimane stabile nel follow - up oltre ad un ee pu essere riferibile , sebbene pi raramente , ad un adenoma o a uniperplasia nodulare focale ( inf )  . nella valutazione degli ee atipici di piccole dimensioni ( 2 cm ) la tecnica tc trifasica , in fase dominante arteriosa ( fa ) , venosa - portale ( fvp ) e tardiva ( ft ) , trova il suo razionale nellidentificazione e nella valutazione del comportamento dellenhancement delle lesioni , elemento fondamentale per determinare la natura vascolare di una lesione in presenza di reperti atipici . 
 [ 16 ] , riportano che un differenza transitoria della densit del parenchima epatico o thad si riscontra nel 21% dei casi di ee atipici 2 csi tratta di unalterazione emodinamica dovuta al prevalente flusso arterioso degli ee atipici iperdensi di piccole dimensioni laddove un cospicuo afflusso arterioso ed il conseguente deflusso determinano una precoce opacizzazione della vena di drenaggio ed un enhancement peritumorale [ 17 ]  . relativamente agli ee atipici ipodensi lincidenza riportata in letteratura varia dal 8% al 16% [ 4 , 17 ] ; tali lesioni comprendono forme con completa assenza di enhancement ( per le quali si pongono le maggiori problematiche diagnostiche e che non possono essere caratterizzate con tecnica tc bifasica ) [ 4 ] , e forme ipodense con puntiforme minimo enhancement intralesionale periferico o segno del brightdot [ 4 , 18 ]  . 
in fact , 6 / 9 of type 2a hhs underwent histological examination ( n = 1 ) or us - guided aspiration cytology ( n = 5 )  . thirteen hypoattenuating hhs showed the bright - dot sign ( type 2b )  . 
 all atypical and typical small hhs exhibited isoattenuation or slight hyperattenuation relative to the aorta and no peripheral capsule on ct scan during dp ( 5 minutes after endovenous contrast material injection )  . 
hypervascular metastases , compared with hhs , have rapid wash - out and low attenuation on pvp and dp images [ 27 ] ; however , some metastases will show peripheral areas of low attenuation surrounding an enhanced centre on dp images [ 26 ]  . 
hypovascular metastases exhibit an enhancing rim ( probably due to well - perfused tumour tissue ) in the hap and fibrous tissue or central necrosis at histology [ 7 ]  . our study emphasises the role of adding dp acquisitions to the biphasic study for the diagnosis of atypical small hhs . 
in atypical hhs , the enhancement on dp is probably caused to longer retention of contrast material in large intravascular spaces , atipici di piccole dimensioni in pazienti non - cirrotici . 
nella serie esaminata , le forme con pattern iperdenso sono state le pi frequenti , riscontrate in 29 dei 52 ( 55% ) ee atipici di piccole dimensioni ; di queste 17 senza thad ( tipo 1a ) e 12 associate a thad ( tipo 1b )  . 
in un ee atipico di tipo 1a , asportato chirurgicamente , lesame istologico ha evidenziato aspetto tipico dellee cavernoso con ampie lacune vascolari ripiene di eritrociti e zone regressive fibrotiche con vasi di piccolo calibro . 
in 13 casi stato rilevato aspetto ipodenso con puntiforme iperdensit decentrata intralesionale o segno del bright - dot ( tipo 2b ) che , quando riconoscibile , assume particolare valore diagnostico . 
 in ft a 5 minuti dallinizio delliniezione del mdc per vena , tutti gli ee atipici , come quelli tipici di piccole dimensioni , sono apparsi isodensi o lievemente iperdensi allaorta e senza capsula periferica . 
tale aspetto risulta particolarmente valido nella diagnosi differenziale tra ee atipico 2 cm , lesioni ipervascolari [ adenoma , inf , carcinoma fibrolamellare epatocellulare , e metastasi ] e lesioni ipovascolari ( cisti < 1 , 5 cm , noduli di steatosi focale e metastasi ) [ 2026 ]  . 
 in tc multifasica gli ee atipici piccoli possono presentare aspetti sovrapponibili sia ai piccoli adenomi epatocelullari che ai noduli di inf senza cicatrice centrale ; appare in questi casi giustificato il ricorso alla risonanza magnetica ( rm ) con mdc epatotospecifico [ 23 , 24 ]  . 
le metastasi ipovascolari esibiscono orletto periferico di impregnazione ( tessuto neoplastico perfuso ) in fa e tessuto fibrotico o necrosi nella porzione centrale quando esaminate istologicamente [ 7 ]  . il nostro studio sottolinea limportanza della ft in associazione alla tecnica bifasica nella diagnosi di ee atipici di piccole dimensioni . 
negli ee atipici infatti , in ft lenhancement determinato probabilmente da una pi 946 radiol med ( 2009 ) 114 : 935947 resulting from slow flow , puddling and partial thrombosis [ 28 ]  . our study has several limitations . 
in agreement with the literature [ 29 , 30 ] , our study included patients who received a similar , if not identical , volume of contrast material with the same dose of iodine per body weight . 
particularly no change in the size or appearance of the lesion at six months as the main diagnostic criterion was used [ 3 , 4 , 6 , 13 , 15 , 18 ]  . 
however , in clinical practice , these disadvantages are offset by the benefits derived from the additional diagnostic information provided by the dp acquisition , which allows a confident diagnosis of hh and a significant reduction of further diagnostic investigations such as mri or biopsy . in summary , triple - phase helical ct can distinguish several types of atypical small hhs . 
dp acquisition ( after 5 minutes ) in association with biphasic ct technique further increases its value in the diagnosis of atypical hhs . the demonstration of patterns similar to typical forms is fundamental in establishing the vascular nature of the lesions and excluding nonvascular benign or malignant lesions . 
in accordo con la letteratura [ 29 , 30 ] , nella casistica sono stati inclusi pazienti in cui era stato somministrato un volume effettivo di mdc simile se non identico ovvero una quantit di contrasto con la stessa dose di iodio per peso corporeo . 
tuttavia , nella pratica clinica , linterpretazione delle immagini in ft , in associazione a quelle acquisite in fa e fvp , consente di ottenere informazioni utili ai fini della diagnosi di ee atipico determinando una sensibile riduzione di ulteriori indagini come la rm e la biopsia . in conclusione , la tc trifasica consente di distinguere vari tipi di ee atipici di piccole dimensioni . 
lacquisizione della ft a 5 minuti , in associazione ad un esame tc con tecnica bifasica , rafforza ulteriormente il ruolo di questa metodica nella diagnosi di ee atipico mostrando aspetti sovrapponibili a quelli delle forme tipiche ; tale elemento risulta essenziale nella conferma della natura vascolare delle lesioni , consentendo di escludere lesioni benigne e maligne di natura non vascolare . 
over a 23 - month period , 45 women ( mean age 58 years , range 2776 years ) with a known diagnosis of systemic sclerosis and a history of dysphagia underwent a dynamic and morphological study of the oral , pharyngeal and oesophageal phases of swallowing with videofluorography . 
the vfg swallow study identified alterations of epiglottal tilting associated with intraswallowing laryngeal penetration in 26 patients ( 57.8% ) , pooling of contrast agent in the valleculae and pyriform sinuses in 23 ( 51.1% ) and radiographic signs of nonspecific hypertrophy of the riassunto obiettivo . 
in un periodo di 23 mesi 45 pazienti di sesso femminile ( et media 58 anni ; range 2776 anni ) , con diagnosi certa di sclerodermia , che riferivano anamnesticamente disfagia sono stati sottoposti a studio dinamico e morfologico del tratto oro - faringeo della deglutizione e del tratto faringo - esofageo - cardiale mediante videofluorografia . 
tutti gli esami sono stati eseguiti con apparecchio telecomandato con arco a c digitale dotato di intensificatore di brillanza da 16 pollici , macchia focale da 0 , 6 a 1 , 2 mm e massima differenza di potenziale di 150 pkv in grafia e 120 pkv in scopia . 
per lo studio della deglutizione sono state acquisite sequenze cineradiografiche digitali con 12 immagini / secondo con matrice 512512 previa ingestione di boli progressivi di mdc baritato ad alta densit ( 250% p / v )  . 
la valutazione della peristalsi esofagea stata documentata con sequenze cineradiografiche digitali con 6 immagini / secondo , in ortostasi e clinostasi durante deglutizione di boli di bario ( 60% p / v ) ed , infine , stato effettuto , in clinostasi , il test del sifone dacqua ( wst ) per la valutazione del reflusso gastroesofageo ( gerd )  . 
lo studio della funzione deglutitoria , effettuato mediante vfg , ha evidenziato alterazioni del tilting epiglottico associato a fenomeni di penetrazione subepiglottica intradeglutitoria in 26 pazienti ( 57 , 8% ) , ristagno del mdc in corrispondenza delle vallecule glosso - epiglottiche e dei radiol med ( 2009 ) 114 : 948959 lingual and / or palatine tonsils in 18 ( 40% )  . 
indeed , in 40% of patients , radiographic signs were found that indicated nonspecific hypertrophy of the lingual tonsil and / or palatine tonsils and nonspecific signs of chronic pharyngeal inflammation , and gord was identified in 93% of patients , which in 40% of cases extended to the proximal third of the oesophagus . 
the data obtained were confirmed in 85% of cases with ph monitoring and in all cases with laryngoscopy . keywords scleroderma dysphagia gastro - oesophageal reflux disease videofluorography ( vfg ) seni piriformi in 23 ( 51 , 1% ) e segni radiografici di ipertrofia aspecifica della tonsilla linguale e / o delle tonsille palatine in 18 ( 40% )  . 
lo studio del viscere esofageo ha evidenziato la presenza di alterazioni della peristalsi nella totalit della popolazione ed in particolare 36 pazienti ( 80% ) manifestavano evidenti segni di atonia . 
infatti nel 40% dei pazienti , sono stati evidenziati segni radiografici riferibili ad unipertrofia aspecifica della tonsilla linguale e / o delle tonsille palatine segni aspecifici di flogosi cronica faringea ed stato , inoltre , identificato gerd , nel 93% dei pazienti che , nel 40% dei casi , si estendeva sino al iii prossimale del viscere . i dati ottenuti sono stati confermati nell85% anche da valutazione ph - metrica e nella totalit dei casi da valutazione laringoscopica . 
 parole chiave sclerodermia disfagia reflusso gastroesofageo videofluorografia ( vfg ) introduction introduzione scleroderma or systemic sclerosis ( ssc ) is a chronic disease of the connective tissue of unknown but probably autoimmune aetiology characterised by idiopathic fibrosis in multiple anatomical regions with involvement of the blood vessels , the skin and the internal organs [ 1 ]  . 
not always , however , is there a direct correlation between the clinical symptoms and la sclerodermia o sclerosi sistemica ( ssc ) una malattia cronica del tessuto connettivo ad eziologia sconosciuta , verosimilmente autoimmune , caratterizzata da fibrosi idiopatica pluridistrettuale con coinvolgimento dei vasi sanguigni , della cute e degli organi interni [ 1 ]  . 
i dati epidemiologici indicano una prevalenza di 12 , 625 casi per 100000 , incidenza di 0 , 06 e 1 , 9 / 100000 , et media 4565 anni con rapporto maschi / femmine di 1 / 4 [ 2 ]  . la patologia interessa il tratto gastroenterico , precocemente e severamente , con disordini della motilit conseguenti ad atrofia e fibrosi della muscolatura liscia , con prevalente coinvolgimento dellesofago , 75%90% dei casi [ 36 ] , specie nel tratto tratto medio e distale [ 7 ]  . 
le disfunzioni motorie stanno alla base delle alterazioni manometriche dello sfintere esofageo inferiore con conseguente insorgenza di reflusso acido gastro - esofageo ( gerd ) ed irritazione della mucosa esofagea , determinando una triade sintomatologica tipicamente caratterizzata da disfagia , generalmente descritta per i cibi solidi , epigastralgia e pirosi retro - sternale ( 50%90% dei casi ) [ 5 ]  . 
non sempre , per , vi una diretta correlazione tra la 950 radiol med ( 2009 ) 114 : 948959 the alterations recorded with manometry , ph monitoring and endoscopy [ 5 ]  . 
although these techniques are currently the reference methods for identifying motor alterations , gord , lesions of the mucosa and hiatus hernia [ 8 , 9 ] , none of them is able to provide direct information regarding the possible presence of primary alterations of swallowing of the food bolus . 
videofluorography ( vfg ) is the reference examination for the morphodynamic study of swallowing and is also a reliable technique for identifying oesophageal motor alterations , gord and hiatus hernia [ 1018 ]  . the aim of our study was to evaluate the role of vfg in the study of swallowing and oesophageal peristalsis in a group of patients with ssc . materials and methods from june 2005 to april 2007 , 45 women ( mean age 58 years , range 2776 years ) with ssc underwent a vfg study . the only inclusion criterion was a known diagnosis of ssc with a positive history of dysphagia . 
dynamic acquisitions were done with patients in the upright and supine positions during swallowing of progressive boluses ( 5 / 10 / 20 ml ) of barium ( 60% weight / volume ) ( prontobario bracco ) , and the water siphon test ( wst ) was done with the patient supine to evaluate the presence of gord . 
the following parameters were evaluated for each patient and each type of contrast agent with a morphological and functional study : vocal cord motility , soft palate motility , oral phase of swallowing , pharyngeal phase of swallowing , pooling of contrast agent in the valleculae and / or pyriform sinuses , oesophageal sintomatologia clinica e le alterazioni manometriche , phmetriche ed endoscopiche [ 5 ]  . 
le suddette metodiche rappresentano , oggi , le tecniche di riferimento per lidentificazione di alterazioni motorie , gerd , lesioni della mucosa ed ernia jatale [ 8 , 9 ]  . 
lo studio videofluorografico ( vfg ) lindagine di riferimento nello studio morfodinamico della deglutizione ed affidabile metodica anche nellidentificazione delle alterazioni motorie esofagee , del gerd e dellernia jatale [ 1018 ]  . 
 materiali e metodi da giugno 2005 ad aprile 2007 , 45 pazienti di sesso femminile ( et media 58 anni ; range 2776 anni ) affetti da sclerodermia sistemica sono stati sottoposti a studio videofluorografico . 
tutti gli esami sono stati eseguiti con apparecchio telecomandato con arco a c digitale ( telecomandato polifunzionale eurocolumbus tr3d , milano , italia ) dotato di intensificatore di brillanza da 16 pollici , macchia focale da 0 , 6 a 1 , 2 mm e massima differenza di potenziale di 150 pkv in grafia e 120 pkv in scopia . 
per lo studio della deglutizione sono state acquisite sequenze cineradiografiche digitali con 12 immagini / secondo con matrice 512512 , con paziente in ortostasi , mediante acquisizioni dinamiche durante la fonazione e durante deglutizione di boli progressivi ( 5 / 10 / 20 ml ) di bario ad alta densit ( 250% p / v ) ( bracco , milano , italia )  . 
la valutazione della peristalsi esofagea stata documentata con sequenze cineradiografiche digitali con 6 immagini / secondo con matrice 10241024 , con paziente in ortostasi e clinostasi mediante acquisizioni dinamiche durante deglutizione di boli progressivi ( 5 / 10 / 20 ml ) di bario ( 60% p / v ) ( prontobario , bracco ) ed , infine , stato effettuato , in clinostasi , il test del sifone dacqua ( wst ) per la valutazione del gerd . 
the study of the oesophageal phase instead showed the presence of generally hypotonic oesophageal dyskinesia in almost the entire patient population , with prevalent involvement of the middle and distal tract . 
per ogni paziente e per ogni tipo di mdc utilizzato sono stati valutati , mediante studio morfologico e funzionale , i seguenti parametri : motilit delle corde vocali ; motilit del palato molle ; fase orale della deglutizione ; fase faringea della deglutizione ; ristagno di mdc baritato nel contesto delle vallecule glosso - epiglottiche e / o dei seni piriformi , discinesia esofagea ; dilatazione esofagea ; deficit del clearing esofageo ; ernia jatale ; reflusso gastro - esofageo . tutti i pazienti sono stati sottoposti successivamente a valutazione laringoscopica , endoscopica e ph - metrica . 
1a , b fenomeni di penetrazione subepiglottica intradeglutitoria ( freccia ) ( a ) ; asimmetrico ribaltamento dellepiglottide per deficit a destra ( freccia ) da alterata motilit ( b )  . 952 radiol med ( 2009 ) 114 : 948959 fig . 
2a - d multiple hypopharyngeal findings due to hypertrophy of the lingual tonsil ( arrows ) , evaluated in the anteroposterior ( a , b ) and laterolateral ( c ) views . 
laryngoscopy confirmed the presence of laryngeal inflammation , with signs of arytenoid and interarytenoid oedema and reactive hypertrophy of the palatine tonsils and / or lingual tonsil in all patients . 
3a - d oesophageal motility disorder in upright ( a , b ) and supine ( c , d ) positions , with multiple antiperistaltic waves ( arrows )  . 
si apprezza inoltre formazione linfonodale calcifica ed ernia jatale da scivolamento ( punte di freccia )  . reflux oesophagitis in 34 ( 75% ) , the coexistence of sliding hiatus hernia and reflux oesophagitis in 34 ( 75% ) and the presence of sliding hiatus hernia alone in four ( 9% )  . 
the data obtained with the swallow study in patients with scleroderma were compared with those from 45 nonsclerodermic controls with a known history of modiche quote di mdc adese alla parete . 
5a - c impaired oesophageal clearing ( upright position ) with small amounts of contrast agent adhering to the walls ( arrow ) ( a ) ; sliding hiatus hernia with return to normal in the upright position ( arrows ) associated with multiple antiperistaltic waves ( arrowheads ) ( b , c )  . 
5a - c deficit di clearing esofageo ( decubito ortostatico ) con lieve verniciatura della parete viscerale ( frecce ) ( a ) ; ernia jatale da scivolamento riponibile in ortostasi ( frecce ) associata a multiple onde antiperistaltiche ( punte di freccia ) ( b , c )  . radiol med ( 2009 ) 114 : 948959 fig . 
6a , b reflusso gastro - esofageo con ricanalizzazione della colonna baritata sino al tratto medio - prossimale del viscere ( a ) e sino alla giunzione faringo - esofagea ( frecce ) ( b )  . gord and similar characteristics of age , gender and symptoms . 
the comparison revealed that in the controls , there was a substantially similar incidence of altered epiglottal motility ( n = 25 , 55.6% ) , of contrast agent pooling in the valleculae and pyriform sinuses ( n = 21 , 46.7% ) and of radiological signs attributable to hypertrophy of the lingual and / or palatine tonsils ( n = 16 , 35.6% ) ( table 1 )  . confermato la presenza di segni di flogosi laringea , con segni di edema aritenoideo , iteraritenoideo ed ipertrofia reattiva delle tonsille palatine e / o della tonsilla linguale , nella totalit dei pazienti esaminati . 
lindagine endoscopica ha confermato la presenza di ernia jatale da scivolamento in 38 pazienti ( 85% ) ed ha , inoltre , evidenziato segni di esofagite da reflusso in 34 di essi ( 75% ) , in 34 ( 75% ) la coesistenza di ernia jatale da scivolamento e segni di esofagite da reflusso ed in 4 ( 9% ) la presenza esclusivamente di ernia jatale da scivolamento . 
in rapporto allo scopo del lavoro , i dati ottenuti dallo studio della deglutizione dei paziente sclerodermici sono stati confrontati con quelli provenienti da una popolazione di controllo formata da 45 pazienti , non sclerodermici , con storia nota di gerd ed analoghe caratteristiche di et , sesso e sintomatologia . 
il confronto ha evidenziato come , nella popolazione di controllo , vi sia unincidenza sostanzialmente sovrapponibile , rispetto alla popolazione di studio , dei fenomeni di alterata motilit epiglottica presente in 25 pazienti ( 55 , 6% ) , del ristagno di mdc baritato in corrispondenza delle vallecule glossoepiglottiche e dei seni piriformi , presente in 21 di essi ( 46 , 7% ) ed , infine , dei segni radiologici riferibili ad ipertrofia della tonsilla linguale e / o palatine presente in 16 pazienti ( 35 , 6% ) ( tabella 1 )  . 
in esso si assiste , infatti , allinsorgenza di una progressiva atrofia e fibrosi della muscolatura liscia , conseguente ad un danno microvascolare su base ischemica da correlarsi ad abnorme deposito di collagene [ 36 ]  . 
tale evento , la cui causa primitiva risulta non essere nota , appare maggiormente evidente in corrispondenza della porzione medio - distale con progressiva compromissione della motilit e quindi della funzionalit globale del viscere [ 7 ]  . 
inoltre lesofago pu essere considerato un affidabile indicatore dellavanzamento globale della patologia mediante lincremento delleterogenea sinomatologia clinica , caratterizzata con maggiore frequenza da disfagia , epigastarlgia e pirosi retrosternale , correlata alle suddette progressive alterazioni . 
la severa compromissione esofagea , nei pazienti con ssc , da collegare a molteplici e coesistenti fattori come riduzione o assenza delle onde peristaltiche primarie e secondarie , ridotta pressione del les , associazione con ernia jatale , ritardato vuotamento gastrico e gerd . 
questultimo appare correlato sia ad una riduzione dellonda 956 radiol med ( 2009 ) 114 : 948959 table 1 oropharyngeal alterations in patients with and without scleroderma patients with scleroderma patients without scleroderma altered epiglottal motility contrast agent pooling in valleculae and / or pyriform sinuses radiological signs ascribable to hypertrophy of the lingual and / or palatine tonsil 26 ( 57.8% ) 23 ( 51.1% ) 18 ( 40% ) tabella 1 rilievo delle alterazioni oro - faringee in pazienti sclerodermici e non sclerodermici alterata motilit epiglottica ristagno mdc nelle vallecule glosso - epiglottiche e / o nei seni piriformi segni radiologici riferibili ad ipertrofia della tonsilla linguale e / o palatine 26 ( 57 , 8% ) 23 ( 51 , 1% ) 18 ( 40% ) 25 ( 55.6% ) 21 ( 46.7% ) 16 ( 35.6% ) 25 ( 55 , 6% ) 21 ( 46 , 7% ) 16 ( 35 , 6% ) pazienti sclerodermici pazienti non sclerodermici discussion the oesophagus is the gastrointestinal organ with the earliest and most frequent involvement in ssc . 
this event , the primary cause of which is unknown , appears particularly evident in the middledistal portion , with progressive compromise of motility and therefore overall function of the organ [ 7 ]  . 
in addition , the oesophagus can be considered a reliable indicator of the overall progression of disease , with the increase of the clinical symptoms more commonly dysphagia , chronic gastritis and retrosternal pyrosis being correlated with the above - mentioned alterations . 
severe oesophageal impairment in patients with ssc is linked to multiple coexisting factors such as the reduction or absence of primary and secondary peristaltic waves , reduced pressure of the lower oesophageal sphincter , association with hiatus hernia , delayed gastric emptying and gord . 
gord seems to be correlated with a reduction in the secondary peristaltic wave in the distal segment , a reduction in the physiological mechanism of oesophageal clearing and pressure changes of the lower oesophageal sphincter [ 5 , 7 ]  . 
in addition , the excessive exposure of various oesophageal segments to gastric acid leads to the onset of chronic inflammatory processes ( 33%66% ) , which in some cases can progress into a frank barretts oesophagus ( 13%16% ) and subsequent adenocarcinomatous degeneration ( 0.5% ) [ 9 ]  . the techniques most commonly used in the diagnostic workup of patients with ssc are manometry , endoscopy and ph monitoring . 
in particular , oesophageal manometry is considered the reference technique in the evaluation of motor alteration , given its ability in general to record reduced contraction in the distal 2 / 3 of the oesophagus and reduced pressure of the lower oesophageal sphincter . 
inoltre leccessiva esposizione dei diversi segmenti viscerali al succo acido gastrico permette linsorgenza di processi flogistici ( 33%66% ) cronici che , in alcuni casi , possono evolvere sino a configurare chiari quadri di metaplasia di barrett ( 13%16% ) e successivamente di degenerazione neoplastica adenocarcinomatosa ( 0 , 5% ) [ 9 ]  . 
in particolare , la manometria esofagea considerata la metodica di riferimento nella valutazione delle alterazioni motorie , documentando , in genere , la ridotta contrazione nei 2 / 3 distali del viscere ed una ridotta pressione del les , segni che , seppur presenti in corso di ssc , non sono specifici poich frequenti anche in altre patologie del tessuto connettivo come lamiloidosi , lalcolismo cronico , ecc [ 5 , 7 ]  . 
la ph - metria , oggi considerata metodica di riferimento nella diagnosi di gerd , permette lidentificazione qualitativa e quantitativa , delle alterazioni del ph intraesofageo presenti nel 54%90% dei pazienti affetti da ssc [ 8 ]  . 
lendoscopia , in ultimo , metodica di riferimento nella valutazione diretta della mucosa esofagea e , quindi , nellidentificazione sia dei quadri di esofagite da reflusso ( 60%75% ) sia dellernia jatale da scivolamento ( 85% ) [ 10 ]  . 
tuttavia nessuna delle suddette metodiche permette unaffidabile e sensibile valutazione , morfologica e funzionale , della fase orale e faringea della deglutizione , inoltre , non vi sono , nella recente letteratura , specifici studi che possano escludere la presenza di alterazioni primitive della deglutizione in corso di sclerosi sistemica [ 17 ]  . 
in questo contesto si colloca la vfg che si identifica come affidabile metodica radiologica nello studio della ssc , essendo in grado di radiol med ( 2009 ) 114 : 948959 signs are present in ssc , they are nonspecific and frequently seen in other connective tissue diseases such as amyloidosis , chronic alcoholism , etc . 
monitoring ph , which is currently considered the reference technique in the diagnosis of gord , is able to qualitatively and quantitatively identify the intraoesophageal ph alternations present in 54%90% of patients with ssc [ 8 ]  . 
finally , endoscopy is the reference technique for the direct evaluation of the oesophageal mucosa and therefore for identifying reflux oesophagitis ( 60%75% ) and sliding hiatus hernia ( 85% ) [ 10 ]  . 
vfg is in fact able to ensure detection and evaluation of changes not only to the oral and pharyngeal phases of swallowing , for which many authors suggest it is the reference standard [ 1216 ] , but also to the oesophageal phase , showing good sensitivity ( 70%90% ) in the identification of oesophageal motility disorders and gord and in the overall evaluation of the lower oesophageal sphincter [ 8 , 10 , 11 ]  . nonetheless , vfg is unable to identify the early signs of oesophagitis , which instead can be detected with endoscopy in 33%66% of patients [ 36 , 9 ]  . 
our study , therefore , was aimed at identifying primary alterations of the oral and pharyngeal phases and the overall evaluation of the oesophageal phase with the use of a variety of techniques . thanks to vfg , in all patients , we observed no signs indicating primary alterations of the swallowing mechanism and noted that both the oral and pharyngeal phases appeared morphologically and dynamically valid . 
this consideration was confirmed by direct radiological findings , such as positive wst , with identification of gord extending to the pharyngealoesophageal junction , and indirect signs such as hypertrophy of the lingual tonsil and the palatine tonsils [ 18 , 19 ]  . further confirmation was provided by laryngoscopy , ph monitoring and endoscopy . 
in addition , the vfg swallow study with direct fluorographic acquisitions investigated the morphology and function of the oesophageal phase , making possible the reliable evaluation of kinetic alterations and therefore the transit time of the barium bolus within the oesophagus in the different patient positions . 
the latter is crucial , because a progressive increase in transit time is directly correlated with a loss of physiological oesophageal kinesis and therefore with the progression of fibrosis , which is pathognomonic of the disease . 
in addition , vfg enabled evaluation of the incidence of concomitant diseases associated with ssc , such as hiatus hernia and gord , thus garantire lidentificazione e la valutazione delle alterazioni sia del tratto oro - farigeo , per il quale metodica di riferimento secondo molti autori [ 1216 ] , sia per il tratto faringo - esofageo - cardiale , con buona sensibilit , che si attesta tra 70%90% , nellidentificazione delle dismotilit esofagee e del gerd e nella valutazione complessiva della giunzione esofago gastrica [ 8 , 10 , 11 ]  . 
la vfg , tuttavia , non consente di evidenziare i segni precoci di esofagite , invece riscontrabili con lesame endoscopico nel 33%66% dei pazienti [ 36 , 9 ]  . 
il nostro studio , pertanto , stato mirato allidentificazione delle alterazioni primitive del tratto oro - faringeo ed alla valutazione complessiva del tratto farigo - esofageo - cardiale mediante lutilizzo di diverse metodiche . 
grazie alla vfg abbiamo costatato come in nessuno dei pazienti presi in esame vi sono segni riconducibili ad alterazioni primitive del meccanismo deglutitorio e come , sia la fase orale sia la fase faringea , nei loro diversi momenti , si presentino morfologicamente e dinamicamente valide . 
lidentificazione di un deficit del ribaltamento dellepiglottide , unica alterazione repertata , nel 57 , 8% dei pazienti , con conseguente penetrazione del mdc in sede subepiglottica risulta essere secondaria , infatti , ad insulto cronico da gerd . tale considerazione confermata da rilievi radiografici diretti , quali la positivit al wst con identificazione di gerd ad estensione sino alla giunzione faringo - esofagea , ed indiretti , lipertrofia della tonsilla linguale e delle tonsille palatine [ 18 , 19 ]  . 
inoltre lindagine videfluorografica , grazie allultilizzo di acquisizioni dirette in grafia ha indagato la morfologia e la funzionalit del tratto faringo - esofageo - cardiale permettendo cos la corretta ed affidabile valutazione delle alterazioni della cinetica e , quindi anche del tempo di transito del bolo baritato nel contesto del viscere esofageo nei diversi decubiti . 
inoltre la vfg ha permesso di valutare lincidenza di patologie concomitanti , associate al quadro di sclerosi sistemica , quali ernia jatale e gerd , costatando come vi sia unassociazione tra ipoe aperistalsi esofagea ed aumento del numero degli episodi di reflusso , che , secondo le recenti evidenze della letteratura , potrebbero non essere direttamente correlati alla pressione vigente in corrispondenza les [ 7 ]  . 
le verifiche gastroscopiche e phmetriche , come indagini di riferimento non radiologiche , hanno confermato ulteriormente lelevata affidabilit ( > 80% ) della metodica nellidentificazione e valutazione 958 radiol med ( 2009 ) 114 : 948959 that there demonstrating is an association between oesophageal hypo / aperistalsis and an increase in the number of reflux episodes , which may be directly correlated with the pressure of the lower oesophageal sphincter [ 7 ]  . 
this suggests that reflux is due to a reduction in clearing mechanisms more than the pressure of the lower oesophageal sphincter . gastroscopy and ph monitoring , as nonradiological reference techniques , further confirmed the high reliability ( > 80% ) of the technique in the identification and evaluation of both gord and hiatus hernia , reliability that has been emphasised several times in the recent literature . 
those values , however , are significantly lower in the identification of oesophagitis , the diagnosis of which is the exclusive realm of endoscopy . conclusions our study has shown that in patients affected by scleroderma with a subjective history of dysphagia , there is no primary deficit in the oral or pharyngeal phase of swallowing , in that no significant morphological and / or functional alterations can be appreciated , in either the preparatory or the transit phase of swallowing . 
in addition , our study population showed no compensatory mechanisms or manoeuvres , either in the oral or pharyngeal phase , events that instead are very common in the setting of primary swallowing alterations [ 20 ]  . 
therefore , our findings show that the alterations of epiglottal tilting associated with intraswallowing laryngeal penetration of high - density barium are secondary to chronic gord extending to the pharyngealoesophageal junction [ 18 ]  . 
this event is confirmed , on the one hand , by the identification in 40% of the study population of nonspecific hypertrophy of the lingual tonsil and / or palatine tonsils , which in adult subjects is an indirect sign of chronic pharyngeal inflammation [ 19 ] and , on the other , by the visualisation of gord in 97% of patients , which in 40% extended to the pharyngeal - oesophageal junction . 
our study also confirmed that vfg is a reliable modality for dynamic evaluation of both dynamic and morphological evaluation of the mechanisms and different phases underlying the swallowing reflex and is therefore able to demonstrate the presence of pathological conditions that can cause dysphagia . 
we can therefore conclude by emphasising that in patients with scleroderma , a systematic study of the oropharyngeal phase of swallowing with vfg is useful given the broad range of information that can be easily obtained with only a slight increase in radiation dose . 
moreover , we confirmed that vfg is a valid modality in the overall study of the oesophagus , although limitations to the technique remain , especially with regard to identification of superficial lesions of the oesophageal mucosa , the evaluation of which is the exclusive realm of endoscopy . sia del reflusso gastro - esofageo sia dellernia jatale , pi volte sottolineata nella recente letteratura , mentre tali valori si riducono drasticamente nellidentificazione delle esofagiti essendo la diagnosi di pertinenza esclusivamente endoscopica . 
 conclusioni il nostro studio ha evidenziato come , in pazienti affetti da sclerodermia che riferiscono anamnesticamente disfagia soggettiva , non vi alcun deficit primitivo della fase orale o faringea della deglutizione , in quanto non sono apprezzabili significative alterazioni morfologiche e / o funzionali sia nella fase preparatoria sia nella fase esecutiva del suddetto meccanismo . 
inoltre , nella nostra popolazione di studio , non stato osservato alcun meccanismo o atteggiamento compensatorio sia durante la fase orale si durante la fase faringea , eventi invece molto frequenti in corso di alterazioni primitive della deglutizione [ 20 ]  . quindi dai nostri dati si evince come le alterazioni del ribaltamento dellepiglottide ( tilting ) associate a fenomeni di penetrazione subepiglottica intradeglutitoria , del mdc baritato ad alta densit , sono secondarie ad insulto cronico da gerd ad estensione faringo - esofagea [ 18 ]  . tale evento confermato dallidentificazione , nel 40% dei pazienti in esame , di unipertrofia aspecifica della tonsilla linguale e / o delle tonsille palatine che , in soggetti adulti , segno indiretto di flogosi cronica faringea [ 19 ] e dalla visualizzazione nel 97% dei pazienti di gerd che nel 40% presentava unestensione sino alla giunzione faringoesofagea . 
il nostro studio , ha confermato , inoltre , come la videofluorografia rappresenta una metodica affidabile sia nella valutazione dinamica sia morfologica dei meccanismi e delle diverse fasi che sottendono al riflesso deglutitorio , permettendo leventuale dimostrazione dei quadri patologici che possono causare disfagia . 
inoltre lo studio da noi effettuato ha ulteriormente confermato come la metodica si dimostri altamente attendibile nella valutazione ed identificazione dei quadri patologici coinvolgenti anche il tratto faringo - esofageo - cardiale . 
quindi possiamo concludere sottolineando come , nei pazienti affetti da sclerodermia , sia utile effettuare , sistematicamente , anche lo studio della fase oro - faringea della deglutitizione , mediante vfg , in relazione alle molteplici informazioni e rilievi facilmente ottenibili , a fronte di un modesto incremento della dose radiogena somministrata al paziente . 
inoltre si conferma come la vfg rappresenti una valida metodica nello studio globale dellesofago pur permanendo i limiti della stessa , soprattutto , nellidentificazione delle lesioni superficiali della mucosa esofagea la cui valutazione permane di esclusiva pertinenza endoscopica . 
giaccone , via del vespro 127 , 90127 palermo , italy 2department of radiology , university of verona , italy 3department of cardiology , san gennaro hospital , naples , italy 4department of radiology and cardiology , university hospital , parma , italy 5department of radiology and cardiology , erasmus medical center , rotterdam , the netherlands correspondence to : l . 
a total of 277 patients ( mean age 6011 years ) we consecutively examined with 64 - slice msct - ca for suspected or known coronary atherosclerosis were retrospectively reviewed for myocardial bridging . 
bridges were of variable length ( < 1 cm 58% ; 12 cm 32% ; > 2 cm 10% ) and depth ( superficial 69% , intramyocardial 31% ) and frequently localised in the mid - distal segment of the left anterior descending artery ( 95% )  . 
coronary segments proximal to the bridge showed no atherosclerotic disease ( 33% ) , positive remodelling ( 27% ) , < 50% stenosis ( 20% ) or > 50% stenosis ( 20% )  . 
in una popolazione di 277 pazienti ( et media 6011 ) , sottoposti consecutivamente ad ac - tcms con scanner a 64 - strati per malattia aterosclerotica coronarica sospetta o nota , stata ricercata la presenza di decorsi miocardici . 
ottantadue pazienti ( 30% , et media 5912 ) presentano decorso miocardico superficiale ( 69% ) o intramiocardico ( 31% ) , con lunghezza variabile ( < 1 cm : 58% ; 12 cm : 32% ; > 2 cm : 10% ) , frequentemente localizzato nel tratto medio - distale dellarteria coronaria discendente anteriore ( 95% )  . 
msct - ca is a reliable , noninvasive method that is able to depict myocardial bridging and associated atherosclerotic plaque in the proximal segments . keywords myocardial bridging coronary artery disease coronary angiography multislice computed tomography . > 50% ( 20% )  . 
la ac - tcms una metodica non invasiva efficace nel dimostrare i ponti miocardici e le placche aterosclerotiche associate presenti nei segmenti prossimali . parole chiave ponte miocardico coronaropatia aterosclerotica angiografia coronarica tomografia computerizzata multistrato introduction introduzione myocardial bridging is considered a congenital anomaly of the coronary arteries in which a segment of a major epicardial coronary artery courses intramurally through the myocardium beneath a muscle bridge [ 13 ]  . 
it is characterised by systolic compression of the intramyocardial segment and may cause angina , myocardial infarction , left ventricular dysfunction , paroxysmal atrioventricular block ( pavb ) , ischaemia during exertion and sudden death by cardiac arrest [ 13 ]  . 
the segment proximal to the bridge is more prone to formation of atherosclerotic plaque , whereas the intramyocardial segment is typically spared [ 3 ]  . the gold standard for diagnosing myocardial bridging is conventional coronary angiography ( cca ) , which shows characteristic compression of the intramyocardial segment on systolic angiograms . 
new imaging techniques have recently been used , such as intravascular ultrasound ( ivus ) , intracoronary doppler ( icd ) and noninvasive multislice computed tomography coronary angiography ( msct - ca ) [ 413 ]  . 
recent studies with 16and 64 - slice msct - ca have shown that myocardial bridging can be identified in a noninvasive manner , thanks to the techniques multiplanar capabilities and the flexibility of postprocessing tools , demonstrating a higher prevalence than reported in cca series [ 614 ]  . the purpose of our study was to retrospectively assess the prevalence of myocardial bridging and its association with coronary atherosclerosis in a large population of consecutive patients examined with 64 - slice msct - ca . il ponte miocardico considerato unanomalia congenita delle arterie coronarie nella quale un tratto di coronaria epicardica maggiore assume un decorso intramurale allinterno del miocardio al di sotto del ponte muscolare [ 13 ]  . 
 caratterizzato dalla compressione sistolica del segmento intramiocardico e pu causare angina , infarto del miocardio , disfunzione del ventricolo sinistro , blocco atrioventricolare ( bav ) parossistico , oltre che ischemia da sforzo e morte cardiaca improvvisa [ 13 ]  . 
tuttavia , il ponte miocardico asintomatico nella maggior parte dei casi . laterosclerosi coronarica associata con la presenza di ponti miocardici stata studiata principalmente nellarteria discendente anteriore ( ada )  . 
il segmento prossimale al ponte predisposto alla formazione di placche aterosclerotiche , mentre il segmento intramiocardico tipicamente risparmiato [ 3 ]  . il gold standard attuale per la diagnosi dei ponti miocardici langiografia coronarica convenzionale ( acc ) , che mostra la caratteristica compressione del tratto intramiocardico nellangiogramma sistolico . 
nuove tecniche di imaging sono state recentemente impiegate , quali lecografia intravascolare ( ivus ) , il doppler intracoronarico ( icd ) , e langiografia coronarica non invasiva mediante tomografia computerizzata ( tc ) multistrato ( ac - tcms ) [ 413 ]  . 
studi recenti con ac - tcms a 16 e 64 strati hanno dimostrato che i ponti miocardici possono essere non invasivamente individuati in virt della multiplanariet della metodica e della flessibilit dei mezzi di post - processing , con prevalenza pi elevata rispetto alle casistiche di acc [ 614 ]  . scopo del nostro studio quello di valutare retrospettivamente in una ampia popolazione consecutiva di pazienti sottoposti ad ac - tcms a 64 strati la prevalenza dei ponti miocardici e la loro associazione con laterosclerosi coronarica . 1026 materials and methods population radiol med ( 2009 ) 114 : 10241036 materiali e metodi popolazione a total of 277 consecutive patients ( 209 men , mean age 6011 years ) who had undergone 64 - slice msct - ca between 27 february 2007 and 30 march 2008 at our department were retrospectively reviewed to determine the prevalence of myocardial bridging . 
msct - ca had been performed for the following indications : atypical angina ( n = 107 ) , typical angina with a nonconclusive stress test ( n = 29 ) , high risk of major coronary events ( n = 63 ) , followup of proximal stents ( n = 36 ) and aortocoronary bypass grafts ( n = 25 ) , and evaluation of aneurysms of the ascending aorta ( n = 17 ) ( table 1 )  . 
the study protocol received the approval of the ethics committee . duecentosettantasette pazienti consecutivi ( 209 uomini , et media 6011 ) , sottoposti ad ac - tcms a 64 strati tra il 27 / 02 / 2007 ed il 30 / 03 / 2008 nel nostro dipartimento , sono stati valutati retrospettivamente per determinare la prevalenza dei ponti miocardici . 
i pazienti erano stati sottoposti ad ac - tcms con le seguenti indicazioni : angina atipica ( n = 107 ) , angina tipica con stress test non conclusivo ( n = 29 ) , alto rischio di eventi coronarici maggiori ( n = 63 ) , follow - up di stent prossimali ( n = 36 ) e by - pass aorto - coronarici ( n = 25 ) , e valutazione di aneurismi dellaorta ascendente ( n = 17 ) ( tabella 1 )  . 
il comitato etico ha approvato il protocollo di studio . scanning and reconstruction parameters parametri di scansione e ricostruzione all studies were carried out using a 64 - slice ct scanner ( brilliance 64 , philips medical systems , cleveland , oh , usa ) with the following parameters : slices / collimation 64 / 0.6 mm , rotation time 420 ms , effective temporal resolution ( with 180 algorithm ) 210 ms , 120 kv , 8001 , 040 mas , table speed 11.9 mm / s , effective slice thickness 0.8 mm , reconstruction increment 0.4 mm , submillimeter isotropic voxel , field of view 140240 mm ( extended cranially for evaluation of aortocoronary bypass grafts and ascending aorta aneurysms only )  . patients with heart rate > 65 bpm received 2040 mg of propranolol hydrochloride per os ( inderal , astrazeneca reims , reims , cedex , france ) 1 h before the scan . 
the patients information sheets recommended treatment with 2040 mg twice daily under medical supervision during the 3 days preceding the examination to reduce and regulate heart rate in patients with anxiety - related tachycardia . 
all patients were also administered 2.5 mg of diazepam ( tranquirit , aventis pharma , waterford , ireland ) 1 h prior to the scan . a 100to 120 - ml bolus of nonionic iodinated contrast agent ( iomeprol , iomeron 400 , bracco , milan , italy ) , depending on scan range , was injected at a flow rate of 5 ml / s using an automatic injector ( stellant , medrad , pittsburgh , pa , usa ) connected to an 18 - gauge needle - cannula inserted into a right antecubital ve in order to optimise intraluminal enhancement of the coronary arteries , the beginning of the scan was synchronised with the passage of the contrast bolus by using the bolus tracking technique with a region of interest ( roi ) in the ascending aorta . 
nella nota informativa preliminare di preparazione allesame era stato consigliato un trattamento con 2040 mg due volte al giorno sotto controllo medico nei tre giorni precedenti lindagine per ridurre e regolarizzare la frequenza cardiaca nei pazienti con tachicardia su base ansiosa . 
allintera popolazione stata , altres , somministrata una dose di 2 , 5 mg di diazepam ( tranquirit , aventis pharma , waterford , irlanda ) unora prima della scansione . un bolo di 100120 ml di contrasto iodato non - ionico ( iomeprol , iomeron 400 , bracco , milano , italia ) , a seconda del range di scansione , stato iniettato con un flusso di 5 ml / s mediante iniettore automatico ( stellant , medrad , pittsburgh , usa ) collegato ad unagocannula da 18 gauge , posizionata in una vena antecubitale destra . 
all data were analysed with postprocessing tools such as multiplanar reconstructions ( mpr ) , curved mpr ( cmpr ) , maximum intensity projection ( mip ) and volume rendering ( vr ) to visualise possible myocardial 1027 regione di interesse ( roi ) posizionata al livello dellaorta ascendente . 
il tempo di scansione risultante stato di 13 , 21 , 8 s ( range 11 , 720 , 1 s )  . i dati sono stati ricostruiti con tecnica retrospettiva alla fine della fase diastolica ( dal 65% all80% dellintervallo rr ) o alla fine della fase sistolica ( 40%45% ) per una migliore visualizzazione dellarteria coronaria destra ( acd )  . analisi delle immagini e dei dati gli esami ac - tcms sono stati revisionati da due radiologi con esperienza in tc coronarica [ 15 ] , che hanno caricato i set di dati in una workstation dedicata ( extended brilliance tm workspace , version 3.0.1.3200 , philips medical systems , cleveland , ohio , usa )  . 
tutti i dati sono stati analizzati mediante mezzi di post - processing quali ricostruzioni multiplanari ( mpr ) , mpr curve ( cmpr ) , proiezioni di massima intensit ( mip ) , e volume rendering ( vr ) per visualizzare i ponti miocardici e le placche aterosclerotiche eventualmente presenti . 
i segmenti sono stati classificati secondo lo schema dellamerican heart association ( aha )  . la prevalenza , la lunghezza ( < 1 cm , 12 cm , > 2 cm ) , e la localizzazione dei ponti miocardici sono state determinate . la profondit stata definita come superficiale o intramiocardica in base alla deviazione dei vasi nel miocardio . 
 stata , altres , valutata la presenza di placche aterosclerotiche ( no aterosclerosi , rimodellamento positivo , stenosi < 50% , stenosi > 50% ) nei segmenti prossimali e distali al ponte [ 16 ]  . 
sono state valutate anche eventuali lesioni aterosclerotiche nel tronco comune ( tcm ) , nella ada , nei rami diagonali , nella arteria circonflessa ( cx ) , nei rami marginali , nella acd e nel ramo intermedio ( ri ) , quando presente . la valutazione statistica stata eseguita con software dedicato ( spss 10 : 1 ; spss , chicago , iii )  . 
il test del chi - quadrato stato eseguito per verificare la differenza statistica riguardo alla presenza della aterosclerosi coronarica rispetto ai decorsi miocardici in rapporto alla sintomatologia ( dolore tipico o atipico )  . 
il test del chi - quadrato stato impiegato , anche , per analizzare la differenza statistica riguardo alla distribuzione dellaterosclerosi tra i segmenti prossimali e distali dei ponti miocardici , ed i restanti vasi coronarici . 
una p < 0 , 0001 stata considerata statisticamente significativa . il cramers v test stato impiegato per valutare la correlazione tra i segmenti prossimale e distale al ponte rispetto al grado di aterosclerosi . 
the prevalence , length ( < 1 cm , 12 cm , > 2 cm ) and location of myocardial bridges were determined . depth was defined as superficial or intramyocardial based on the vessels deviation in the myocardiuthe presence of atherosclerotic plaque was also assessed ( absence of atherosclerosis , positive remodelling , < 50% stenosis , > 50% stenosis ) in the segments proximal and distal to the bridging [ 16 ]  . 
any atherosclerotic lesions in the left main trunk ( lm ) , lad , diagonal branches , circumflex artery ( cx ) , marginal branches , rca and in the intermediate branch ( ib ) , if present , were also assessed . statistical analysis was performed using dedicated software ( spss 10 : 1 ; spss , chicago , il , usa )  . 
the chi - square test was used to verify statistical differences between the presence of coronary atherosclerosis and myocardial bridging in relation to symptoms ( typical or atypical pain )  . 
the chi - square test was also used to analyse the statistical difference , with reference to the distribution of atherosclerosis , between the proximal and distal segments of myocardial bridging and the remaining coronary vessels . 
the test was also used to assess the potential relationship between the segment proximal to the bridge and the other coronary vessels in relation to the degree of atherosclerosis . results single ( 90% ) or multiple ( 10% ) myocardial bridges of variable length ( < 1 cm in 58% , 12 cm in 32% and > 2 cm in 10% of cases ) were identified in 82 patients ( 30% , 56 men , mean age 6010 years , average heart rate 629 bpm ; table 1 )  . 
bridges were either superficial ( n = 63 , 69% ) without vessel deviation inside the myocardium or intramyocardial ( n = 28 , 31% ) with u - shaped deviation myocardiu bridges were frequently localised in the middistal segment of the lad ( n = 86 , 95% )  . 
sono stati individuati ponti superficiali ( n = 63 , 69% ) , senza deviazioni del vaso allinterno del miocardio , o intramiocardici ( n = 28 , 31% ) , con deviazioni a u nel miocardio . 
i ponti risultano frequentemente localizzati nel segmento medio - distale della ada ( n = 86 , 95% )  . conformemente allo schema aha , i ponti presentano la seguente distribuzione : segmento 7 , 40 casi ; segmento 8 , 32 casi ; segmenti 78 , 13 casi ; segmenti 67 , 1 caso ; segmento 9 , 1 caso ; segmento 11 , 1 casi ; segmento 14 , 1 caso ; segmento 16 , 2 casi . il ponte miocardico non rappresenta un fattore di rischio rilevante per laterosclerosi coronarica nella nostra popolazione ( or 0 , 49 per aterosclerosi coronarica ; or 0 , 47 per presenza di stenosi > 50% ) rispetto ai tradizionali fattori di rischio cardiovascolari ( colesterolemia : or 2 , 04 per aterosclerosi coronarica , or 1 , 25 per presenza di stenosi > 50% ; diabete : or 1 , 18 per aterosclerosi coronarica , or 1 , 17 per presenza di stenosi > 50% ; ipertensione : or 2 , 51 per aterosclerosi coronarica , or 0 , 64 per presenza di stenosi > 50% ; storia familiare : or 1 , 06 per aterosclerosi coronarica , or 1 , 17 per presenza di stenosi > 50% ; fumo : or 1 , 39 per aterosclerosi coronarica , or 1 , 4 per presenza di stenosi > 50% ; obesit : or 1 , 12 per aterosclerosi coronarica , or 0 , 79 per presenza di stenosi > 50% )  . 
tuttavia , solamente nel 29% dei pazienti con dolore atipico ( n = 11 ) o tipico ( n = 2 ) la ac - tcms ha identificato stenosi > 50% ( p < 0 , 05 rispetto alla popolazione che non presenta ponti miocardici )  . 
in seven patients with atypical ( n = 6 ) or typical ( n = 1 ) pain , msct - ca evidence of myocardial bridging without > 50% stenosis and stress electrocardiogram ( ecg ) evidence of nonspecific signs of ischaemia or rhythm alterations , myocardial bridging was considered clinically symptomatic , and drug therapy with - blockers was initiated . considering subjects with myocardial bridging , the coronary artery segments proximal to the bridge showed the following pattern of atherosclerosis : no sign of disease ( n = 30 , 33% ) , positive remodelling ( n = 25 , 27% ) , < 50% stenosis ( n = 18 , 20% ) and > 50% stenosis ( n = 18 , 20% ) ( table 2 )  . 
six noncalcified ( 46% ) , six mixed ( 46% ) and one calcified ( 8% ) atherosclerotic plaque were identified in the distal segments . the degree of atherosclerosis was also determined in the main coronary vessels : rca , lm , lad , diagonal branches , cx , marginal branches and ib ( found in 29% of the population )  . 
a moderate correlation ( cramers v = 0.52 ) was identified between segments proximal or distal to the bridge and the degree of atherosclerosis ( table 2 )  . discussion in 1737 , reyman first described a peculiar congenital coronary anomaly in which myocardial fibres are arranged to cover segments of a major coronary artery . 
myocardial bridging may be characterised by systolic compression of the intramymiocardico , i segmenti di coronaria prossimale al ponte presentano il seguente pattern aterosclerotico : nessun segno di malattia ( n = 30 , 33% ) , rimodellamento positivo ( n = 25 , 27% ) , stenosi < 50% ( n = 18 , 20% ) , stenosi > 50% ( n = 18 , 20% ) ( tabella 2 )  . 
nei segmenti distali sono state individuate 6 placche aterosclerotiche non calcifiche ( 46% ) , 6 miste ( 46% ) , e 1 calcifica ( 8% )  . lestensione dellaterosclerosi stata , anche , determinata nei principali vasi coronarici : acd , tcm , ada , rami diagonali , cx , rami marginali e ri ( presente nel 29% della popolazione )  . 
 stata riscontrata una discreta correlazione ( cramers v = 0 , 52 ) tra segmento prossimale o distale al ponte e grado di malattia aterosclerotica presente ( tabella 2 )  . discussione reyman descrisse nel 1737 per la prima volta una particolare anomalia congenita coronarica , nella quale le fibre miocardiche si dispongono a ricoprire tratti di unarteria coronaria maggiore . 
in un ponte superficiale ( o incompleto ) , il segmento non completamente ricoperto dalle fibre miocardiche , ma da un sottile strato di tessuto connettivo , nervi e tessuto adiposo [ 17 ]  . esiste una discrepanza tra la bassa prevalenza ( < 5% ) riportata dopo acc e la elevata prevalenza riportata negli studi autoptici ( 5%86% )  . 
in soggetti con coronarie angiograficamente normali , luso di test di provocazione pu aumentare la compressione sistolica del miocardio nel 40% dei casi [ 1 , 3 ]  . inoltre , la presenza di una lesione stenotica prossimale pu impedire la visualizzazione del successivo ponte . 
in a superficial ( or incomplete ) bridge , the segment is not completely covered by myocardial fibres but by a thin layer of connective tissue , nerves and fat tissue [ 17 ]  . there is some discrepancy between the low prevalence ( < 5% ) reported after cca and the high prevalence reported in post - mortem studies ( 5%86% )  . 
the lower depiction rate of angiography may , instead , be attributed to the prevalence of superficial bridges causing minimal compression undetectable by cca [ 1 , 3 ]  . 
a high prevalence of bridging has been reported in patients with hypertrophic obstructive cardiomyopathy [ 18 ]  . myocardial bridges may cause angina , myocardial infarction , left ventricular dysfunction , paroxysmal avb , prevalenza riportata nei pazienti con cardiopatia ipertrofica ostruttiva [ 18 ]  . i ponti miocardici possono causare angina , infarto del miocardio , disfunzione ventricolare sinistra , bav parossistico , ischemia da sforzo e morte cardiaca improvvisa . 
i pazienti possono presentarsi con dolore toracico atipico o equivalenti anginosi , senza una consistente associazione tra severit dei sintomi e lunghezza , profondit o grado di compressione sistolica del ponte [ 17 ]  . mentre i ponti miocardici brevi e superficiali sono sovente asintomatici e non richiedono intervento di alcun tipo , le opzioni di gestione per i ponti miocardici sintomatici possono essere farmacologiche ( - bloccanti e calcio - antagonisti ) o interventistiche ( stenting , bypass grafting e miotomia chirurgica ) in relazione alla profondit ed alla lunghezza del decorso [ 20 ]  . i ponti miocardici sono ritenuti una delle principali cause di infarto miocardico in circa il 6% dei pazienti che non presentano evidenza di aterosclerosi coronarica n alla acc , n alla autopsia [ 21 ]  . 
il ponte miocardico , invece , un evento sistolico alla acc , per cui la sua rilevanza radiol med ( 2009 ) 114 : 10241036 1031 ischaemia during exertion and sudden death by cardiac arrest . 
patients may present with atypical chest pain or anginal equivalents without any consistent association between the severity of symptoms and the length , depth or degree of systolic compression of the bridge [ 17 ]  . whereas short and superficial myocardial bridges are often asymptomatic and require no intervention , management options for symptomatic myocardial bridges may be pharmacological ( - blockers and calcium antagonists ) or interventional ( stenting , bypass grafting and surgical myotomy ) , depending on the depth and length of the bridge [ 20 ]  . myocardial bridging is considered one of the main causes of myocardial infarction in approximately 6% of patients with no evidence of coronary atherosclerosis on either cca or post - mortem examinations [ 21 ]  . 
ischaemia can be attributed to a combination of the following factors : sudden tachycardia ( compromising diastolic filling of coronary arteries ) , coronary spasm and systolic kinking of the coronaries ( leading to endothelial damage with resulting platelet activation and thrombosis ) [ 2 , 22 , 23 ]  . 
haemodynamic forces may explain the formation of atherosclerotic plaque close to the intramyocardial segment where the endothelium is flat , polygonal and polymorphic , indicating low shear stress [ 24 , 25 ]  . 
furthermore , local increase in wall tension may cause damage to the endothelium and fissuring of the plaque , resulting in the formation of proximal thrombi [ 27 ]  . cca is the imaging gold standard in diagnosing myocardial bridging . 
new invasive imaging techniques , such as ivus and icd , can be used to demonstrate the morphological and functional features of myocardial bridging [ 4 , 5 ]  . 
ivus studies support the absence of atherosclerosis in intramyocardial vessels , although at least 90% of patients show formation of atheromatous plaque in proximal tracts [ 4 ]  . few studies have investigated the feasibility of msct - ca for visualising myocardial bridging [ 614 ]  . 
the incidence ed il suo significato clinico sono stati messi in dubbio . lischemia pu essere attribuita alla combinazione dei seguenti fattori : improvvisa tachicardia ( tale da compromettere il riempimento diastolico delle coronarie ) , spasmo coronarico , kinking sistolico delle coronarie ( tale da determinare un danno endoteliale con conseguente attivazione piastrinica e trombosi ) [ 2 , 22 , 23 ]  . 
le forze emodinamiche possono spiegare la formazione di placche aterosclerotiche in prossimit del segmento intramiocardico , dove lendotelio piatto , poligonale e polimorfo , indicando la presenza di un ridotto shear stress [ 24 , 25 ]  . 
inoltre , un aumento locale della tensione di parete pu causare un danno endoteliale e la fissurazione della placca con conseguente formazione di trombi prossimali [ 27 ]  . il gold standard attuale di imaging , per la diagnosi di ponte miocardico la acc . 
nuove tecniche di imaging invasive , quali la ivus ed il icd , possono essere utilizzate per mostrare le caratteristiche morfologiche e funzionali del bridging miocardico [ 4 , 5 ]  . studi lassenza di aterosclerosi allinterno dei decorsi miocardici , sebbene almeno il 90% dei pazienti mostri la formazione di placche ateromasiche nei tratti prossimali [ 4 ]  . ivus supportano pochi studi hanno descritto la fattibilit della visualizzazione dei ponti miocardici mediante ac - tcms [ 614 ]  . lincidenza dei ponti miocardici diagnosticati con ac - tcms a 16 strati compresa tra il 3 , 5% ed il 5 , 7% [ 8 , 9 ]  . 
gli studi effettuati con tc a 64 strati riportano una prevalenza pi elevata , compresa tra il 10 , 9% ed il 44% , superiore alle casistiche angiografiche , ma non dissimile dai tassi riportati negli studi autoptici [ 1014 ]  . 
studies conducted with 64 - slice ct report a prevalence of bridging ( 10.9%44% ) that is higher than that reported for angiographic series but similar to postmortem studies [ 1014 ]  . 
the incidence of myocardial bridging ( 30% ) is consistent with that reported by post - mortem studies and higher than that found in most angiographic studies in which the low depiction rate can be attributed to superficial bridging and minimal compression . 
lincidenza dei ponti miocardici ( 30% ) in accordo con quella degli studi autoptici e pi elevata di quella riportata nella maggior parte degli studi angiografici , nei quali il basso tasso di riscontri attribuibile a decorsi superficiali responsabili di modeste compressioni . la ac - tcms offre , inoltre , diversi potenziali vantaggi . 
la prevalenza di malattia inferiore nella popolazione con ponte miocardico rispetto a quella che non presenta tale condizione anatomica ( aterosclerosi coronarica rilevata 78% vs 87% , stenosi > 50% 41% vs 60% , p < 0 , 05 )  . 
1a - d superficial myocardial bridging ( arrow ) of the middistal tract of the left anterior descending artery on vr ( a ) , mpr ( b ) and stretched - vessel mpr images ( c )  . 
1a - d ponte miocardico superficiale ( freccia ) del tratto medio - distale della arteria discendente anteriore mediante immagini vr ( a ) , mpr ( b ) , e stretch - vessel mpr ( c )  . 
decorso miocardico ( testa di freccia ) del tratto prossimale del primo ramo marginale mediante stretch - vessel mpr ( d )  . radiol med ( 2009 ) 114 : 10241036 1033 fig . 
2a - c superficial myocardial bridging ( white arrow ) of the mid - distal tract of the left anterior descending artery and intramyocardial course ( arrowhead ) of the first diagonal branch ( d1 ) : vr ( a ) and mpr ( b , c ) images . 
2a - c ponte miocardico superficiale ( freccia bianca ) del tratto medio - distale dellarteria discendente anteriore e decorso intramiocardico ( testa di freccia ) del primo ramo diagonale ( d1 ) : immagini vr ( a ) ed mpr ( b , c )  . 
with regard to the association with coronary atherosclerosis , and in agreement with the literature [ 1 , 35 , 9 , 2427 ] , the segments proximal to myocardial bridges show a distinctly different atherosclerotic pattern from that of the distal segments . 
in riferimento alla associazione con la aterosclerosi coronarica ed in accordo con la letteratura [ 1 , 35 , 9 , 2427 ] , i segmenti prossimali ai ponti miocardici presentano un pattern aterosclerotico significativamente differente rispetto ai segmenti distali . 
la actcms non invasiva stata , infatti , spesso eseguita nel tentativo di non sottoporre i pazienti alla diagnostica invasiva con acc , in virt dellalto valore predittivo della metodica [ 28 ]  . 
a tal proposito , scanner con pi elevata risoluzione temporale potrebbero fornire precise indicazioni circa la compressione sistolica [ 29 ]  . 1034 radiol med ( 2009 ) 114 : 10241036 fig . 
3a - f myocardial bridging ( arrow ) of the middle tract of the left anterior descending artery shown on vr ( a , d ) , mpr ( b ) and stretched - vessel mpr images ( c ) in a patient with atypical chest panonsignificant noncalcified plaque can be seen proximal to the myocardial bridging ( arrowhead )  . 
3a - f ponte miocardico ( freccia ) del tratto medio della arteria discendente anteriore visualizzato mediante immagini vr ( a , d ) , mpr ( b ) e stretch - vessel mpr ( c ) in paziente con dolore toracico atipico . 
angiografia coronarica convenzionale del ponte miocardico in fase telediastolica ( e ) e telesistolica ( f )  . diastolic images may enable evaluation of temporary luminal restriction in the myocardial segments [ 13 ]  . 
in this respect , scanners allowing higher temporal resolution may provide an accurate indication of systolic compression [ 29 ]  . finally , our study population reflects a selection bias , as a suspicion of coronary atherosclerosis and related clinical symptoms were the main reasons for the msct - ca examinations . 
despite the above limitations , msct - ca is defined as a reliable method for coronary imaging and an excellent tool for describing detected anomalies [ 30 ]  . infine , la popolazione del nostro studio presenta un bias di selezione poich il sospetto di aterosclerosi coronarica e la correlata sintomatologia clinica rappresentano il motivo principale dellesecuzione dellindagine ac - tcms . 
nonostante tali limitazioni , la ac - tcms definita attualmente come una metodica affidabile per limaging coronarico ed uno strumento eccellente per la descrizione di eventuali anomalie [ 30 ]  . conclusioni conclusions radiologists have the opportunity to assess myocardial i radiologi hanno la concreta opportunit di valutare la presenza dei ponti miocardici in vivo , grazie ai vantaggi della tecnologia tcms , quali lalta risoluzione spaziale e radiol med ( 2009 ) 114 : 10241036 1035 fig . 
4a - f decorso intramiocardico ( freccia ) del tratto medio della arteria discendente anteriore con associata placca non calcifica eccentrica prossimale che determina stenosi significativa ( testa di freccia ) : immagini vr ( a ) , mpr ( b ) , stretch - vessel mpr ( c ) , mip ( d ) , e sezioni mpr in corrispondenza della stenosi significativa ( e ) e del decorso intramiocardico ( f )  . bridges in vivo , thanks to the advantages offered by msct technology such as high spatial resolution and postprocessing flexibility . 
a 64 - slice msct - ca can provide exhaustive information on myocardial bridging and associated proximal atherosclerotic lesions , which may support clinical management and , where necessary , interventional planning . 
la ac - tcms a 64 strati pu fornire informazioni esaurienti riguardo i ponti miocardici e le associate lesioni aterosclerotiche prossimali , che possono supportare la gestione clinica ed , eventualmente , il planning interventistico dei cardiologi . 
di rudin 8 , 20142 milano , italy 3unit di radiologia , dipartimento di scienze medico - chirurgiche , universit degli studi di milano , irccs policlinico san donato , san donato milanese , italy correspondence to : s . 
the locations of the parathyroid glands were typical cervical ( n = 77 ) , thyrothymic ligament ( n = 3 ) , carotid sheath ( n = 2 ) , and mediastinum ( n = 3 )  . 
per - patient sensitivity and positive predictive values , respectively , were 84% ( 64 / 76 ) and 99% ( 64 / 65 ) for us , 68% ( 44 / 65 ) and 100% ( 44 / 44 ) for scintigraphy and 91% ( 59 / 65 ) and 98% ( 59 / 60 ) for both techniques combined . 
la sede di tali paratiroidi ingrandite , correttamente identificata dallecografia per 74 paratiroidi in 64 pazienti , era : cervicale tipica ( n = 77 ) , legamento tireotimico ( n = 3 ) , ricorrenziale ( n = 2 ) , mediastino ( n = 3 )  . 
in due pazienti stato identificato un microadenoma intratiroideo allesame istologico . sensibilit e valore predittivo positivo , per paziente , sono risultati 84% ( 64 / 76 ) e 99% ( 64 / 65 ) per lecografia , 68% ( 44 / 65 ) e 100% ( 44 / 44 ) per la scintigrafia e 91% ( 59 / 65 ) e 98% ( 59 / 60 ) per le due metodiche combinate , rispettivamente . 
lidentificazione e la localizzazione 1160 radiol med ( 2009 ) 114 : 11591172 inexpensive and widely available method , limiting the use of scintigraphy to those cases with negative and / or doubtful findings on us . keywords parathyroid glands primary hyperparathyroidism parathyroidectomy ultrasonography preoperatoria di paratiroidi ingrandite possono basarsi sullecografia , metodica assai diffusa e con costi contenuti , limitando lutilizzo della scintigrafia ai casi negativi e / o dubbi allecografia . parole chiave paratiroidi iperparatiroidismo primario paratiroidectomia ecografia introduction introduzione primary hyperparathyroidism is a disorder characterised by an overproduction of parathormone independently of the feedback of calcium homeostasis . 
the estimated incidence of this disorder in the general population is 40 / 100 , 000 cases per year and is four to five times higher among postmenopausal women [ 13 ]  . 
however , thanks to the possibility of localising the enlarged parathyroid glands preoperatively , the use of minimally invasive procedures with shorter procedure times is becoming increasingly frequent [ 1 , 4 ]  . 
 the parathyroid glands can be localised preoperatively by using different imaging techniques , including ultrasound ( us ) , scintigraphy , computed tomography ( ct ) and magnetic resonance ( mr ) imaging . 
although scintigraphy was once the most widely used preoperative localising technique , recent advances in us , including high - frequency probes and colour doppler and power doppler , have increased the diagnostic performance of us to levels comparable with those of scintigraphy , with clear benefits in terms of greater speed and reduced cost and dose [ 1 , 57 ]  . the purpose of our study was to retrospectively evaluate the diagnostic capabilities of preoperative us in patients with primary hyperparathyroidism by correlating and comparing the results with the surgical and histological findings . materials and methods this retrospective study received the approval of our ethics committee , which authorised the anonymous publication of the data even in the absence of individual patients informed consent . 
attualmente , grazie anche alla possibilit di identificare preoperatoriamente la sede delle paratiroidi ingrandite , sempre pi frequente lesecuzione di interventi mini - invasivi , con riduzione dei tempi dintervento [ 1 , 4 ]  . 
pi di recente i progressi della tecnica ecografica , con lutilizzo di sonde ad alta frequenza e la possibilit di avvalersi di color doppler e power doppler , la capacit diagnostica dellecografia equiparabile a quella della scintigrafia con indubbi vantaggi in termini di rapidit di esecuzione , e risparmio economico e dosimetrico [ 1 , 57 ]  . rendono scopo del nostro studio stato valutare retrospettivamente la capacit diagnostica dellecografia preoperatoria in pazienti affetti da iperparatiroidismo primario , rispetto ai reperti chirurgici e allesame istologico . materiali e metodi il presente studio retrospettivo stato autorizzato dal comitato etico , consentendo la pubblicazione dei dati in forma anonima anche senza disponibilit di specifico consenso informato da parte dei singoli pazienti . 
 popolazione we retrospectively reviewed patients with primary hyperparathyroidism [ increased serum parathormone ( pth ) with consequent hypercalcaemia and hypophosphataemia ] who had been operated on at the endocrine surgery department of our hospital between september 1999 and october 2006 . abbiamo valutato retrospettivamente i pazienti con iperparatiroidismo primario ( livelli ematici aumentati di paratormone [ pth ] con conseguente ipercalcemia ed ipoil reparto di chirurgia fosfatemia ) operati presso radiol med ( 2009 ) 114 : 11591172 1161 patients in whom preoperative us had been performed at another centre or not performed at all were excluded from the study . 
thus , the finally study population included 77 patients . imaging methods all 77 patients had undergone preoperative us of the neck , and 65 had also been studied by scintigraphy . 
where possible , in relation to the patients somatic structure and to the diagnostic requirements , the upper mediastinal region immediately below the jugulum was evaluated using a micro convex high - frequency transducer ( 48 mhz )  . 
the examination was completed with an evaluation of the lateral neck regions and with colour doppler and power doppler of identified expansive masses . predominantly perilesional vascularity was considered indicative of thyroid nodules , whereas predominantly intralesional vascularity indicated enlarged parathyroid glands . 
in particular , we did not evaluate the lower diagnostic capabilities of us in patients with thyroid goitre [ 3 ]  . subtraction scintigraphy was performed on 84% ( 65 / 77 ) of patients with 99mtc - sestamibi ( mibi ) and 99mtc - pertechnetate . 
scintigraphic studies performed at other centres had in some cases preceded the us scan , whereas at our hospital , scintigraphy was always performed to supplement doubtful or inconclusive us scans . 
the longest time interval between us and scintigraphy was approximately 3 months , and the maximum time between diagnostic investigations and surgery was 6 months . surgery three different kinds of surgical procedures were performed : neck dissection with either unilateral ( n = 54 ) or bilateral ( n = 23 ) neck exploration and minimally invasive video - assisted neck dissection ( localised parathyroid , absence of goitre or previous neck dissection ) ( n = 1 )  . 
la popolazione in esame risultata di 77 pazienti . metodiche dimaging tutti i 77 pazienti , prima dellintervento , sono stati sottoposti ad indagine ecografica del collo , 65 anche ad indagine scintigrafica . 
lesame ecografico , per cui stata utilizzata una sonda lineare ad alta frequenza ( 7 , 510 mhz ) , stato eseguito da un operatore dedicato ( 3 radiologi con pi di 10 anni di esperienza )  . 
per quanto stato possibile , in relazione alla struttura somatica del soggetto ed alla necessit diagnostica , stata valutata la regione del mediastino superiore immediatamente al di sotto del giugulo mediante lutilizzo di trasduttore microconvex ad alta frequenza ( 48 mhz )  . 
le paratiroidi ingrandite ( diametro > 4 mm ) sono state descritte come formazioni rotondeggianti ipoecogene rispetto al parenchima tiroideo , con ricca vascolarizzazione alla valutazione power doppler ; di ciascuna sono state registrate e classificate informazioni sulle dimensioni ( diametro massimo ) , il lato ( destro o sinistro ) e la sede ( superiore od inferiore )  . 
in particolare non abbiamo valutato linferiore capacit diagnostica in pazienti portatori di struma tiroideo [ 3 ]  . lesame scintigrafico , effettuato nell84% ( 65 / 77 ) dei pazienti , stato eseguito mediante tecnica di sottrazione digitale con il 99mtc - sestamibi ( mibi ) e 99mtc pertecnetato . in alcuni casi lesame scintigrafico , eseguito presso altre sedi , aveva preceduto lesame ecografico mentre presso il nostro centro la scintigrafia stata eseguita ad integrazione di ecografie dubbie o inconcludenti . 
il periodo massimo intercorso tra ecografia e scintigrafia stato di circa 3 mesi ; il periodo massimo intercorso tra indagini diagnostiche e successivo intervento chirurgico stato di 6 mesi . intervento chirurgico sono stati eseguiti tre diversi tipi di intervento : cervicotomia 1162 radiol med ( 2009 ) 114 : 11591172 according to the literature [ 2 , 8 ] , a fall in serum pth of more than 50% 10 min after parathyroidectomy compared with the level at anaesthesia induction confirms the complete removal of all hyperfunctioning parathyroid tissue and justifies the end of the surgical procedure . 
if there is no fall in serum pth , a histological examination is carried out to confirm the parathyroid nature of the resected tissue , and a further pth assay is performed . 
where the removal of multiple parathyroid glands does not lead to a fall in pth , the thyroid is partially or completely removed as a possible site of intrathyroid microadenoma . 
we considered as true positives all patients in whom us correctly detected at least one abnormal parathyroid gland ( on the right or left side of the neck )  . 
all patients with negative preoperative us and at least one pathological parathyroid at surgery and those with negative us and a surgical diagnosis of intrathyroid microadenoma were considered false negatives . 
false positives were defined as patients with a us finding of enlarged parathyroid glands not confirmed on surgery and those with incorrect us localisation of the enlarged parathyroid gland ( enlarged parathyroid gland on the right side of the neck described by us as being located on the left side or vice versa )  . 
 sensitivity and positive predictive value were then calculated for the two modalities separately and in combination and for the us and scintigraphy detection of parathyroid glands in relation to weight ( mann - whitney u test )  . comparison between the sensitivity of us and scintigraphy was based on the mcnemar test . results parathyroidectomy was performed in 77 patients 60 women ( 78% ) and 17 men ( 22% ) aged 2279 years ( table 1 )  . 
in two patients ( 3% ) , the procedure revealed no grossly enlarged parathyroid glands , and the histological diagnosis ( partial right lobectomy in one case , total thyroidectomy in the other ) was thyroid microadenoma . 
at surgery , 77 enlarged parathyroid glands were identified in the classic lateral neck position , three in the con esplorazione cervicale monolaterale ( n = 54 ) o bilaterale ( n = 23 ) e minicervicotomia videoassistita ( paratiroide localizzata , assenza di gozzo o di pregressa cervicotomia ) ( n = 1 )  . 
 in accordo con la letteratura [ 2 , 8 ] una caduta del livello serico di pth di oltre il 50% a 10 minuti dalla paratiroidectomia rispetto al valore allinduzione dellanestesia fornisce la conferma della rimozione di tutto il tessuto paratiroideo iperfunzionante e giustifica la fine dellintervento . 
in assenza di caduta del livello serico di pth si procede ad esame istologico per confermare la natura paratiroidea del tessuto asportato e ad ulteriore prelievo per il dosaggio del pth . 
se la natura paratiroidea confermata e non si ha riduzione tardiva del pth , si procede ad unulteriore paratiroidectomia ; in caso la rimozione di pi paratiroidi non si associ a discesa del pth si procede ad asportazione parziale o totale della tiroide possibile sede di microadenoma intratiroideo . in tutti i casi la natura della lesione asportata stata valutata con esame istologico . 
abbiamo considerato veri positivi tutti i pazienti con almeno una paratiroide patologica correttamente localizzata ( sede laterocervicale destra o sinistra ) allecografia ; falsi negativi i pazienti con ecografia preoperatoria negativa ed almeno una paratiroide patologica al riscontro operatorio ed i pazienti con ecografia negativa e diagnosi di microadenoma intratiroideo al riscontro operatorio . 
abbiamo considerato falsi positivi i pazienti con riscontro ecografico di paratiroidi ingrandite non confermate allintervento e quelli con errata localizzazione ecografica di paratiroide ingrandita ( paratiroide ingrandita in sede laterocervicale destra descritta a sinistra allesame ecografico o viceversa )  . 
 sono stati quindi calcolati sensibilit e valore predittivo positivo di ciascuna delle due metodiche e della loro combinazione come pure lidentificazione ecografica e scintigrafica delle paratiroidi in relazione al loro peso , per entrambe le tecniche ( test u di mann - whitney )  . 
il confronto tra la sensibilit dellecografia e quella della scintigrafia stato effettuato mediante test di mcnemar . risultati sono stati sottoposti a paratiroidectomia 77 pazienti , 60 radiol med ( 2009 ) 114 : 11591172 1163 table 1 population with primary hyperparathyroidism included in the study no . 
of patients percent of patients mean age ( years ) women total maschi femmine totale tabella 1 popolazione con iperparatiroidismo primario inclusa nello studio numero pazienti percentuale pazienti et media ( anni ) table 2 histological type and site of the removed parathyroid glands no . 
it produced 12 false negatives and incorrectly located the side of the lesion in one patient only ( correctly diagnosed by scintigraphy )  . femmine ( 78% ) e 17 maschi ( 22% ) , di et compresa tra 22 e 79 anni ( tabella 1 )  . 
in due pazienti ( 3% ) lintervento non ha evidenziato alcuna parala diagnosi tiroide macroscopicamente ingrandita e 1164 radiol med ( 2009 ) 114 : 11591172 table 3 histological characteristics of removed parathyroid glands . 
the diameter of abnormal parathyroid glands identified on us was 19.714.4 mm ; 16 mm ; 475 mm [ meanstandard deviation ( sd ) ; median ; range ]  . 
the us and scintigraphy results were concordant in 54% of patients who underwent both examinations ( 35 / 65 ) , with sensitivity and positive predictive value of 81% ( 52 / 64 ) and istologica ( lobectomia destra parziale in un caso , tiroidectomia totale nellaltro ) stata di microadenoma intratiroideo . 
allintervento chirurgico 77 paratiroidi ingrandite sono state identificate nella classica sede laterocervicale , 3 nel legamento tireotimico , 2 in sede ricorrenziale , 3 ectopiche nel mediastino superiore ( tabelle 2 e 3 )  . lecografia preoperatoria ha identificato correttamente fig . 
1a , b a 52 - year - old man with a 10 - year history of kidney stones and a 2 - year history of skeletal paus detected an enlarged left lower parathyroid gland that after removal was found to weigh 1 , 780 mg . 
il diametro delle paratiroidi anormali identificate allecografia risultato : 19 , 714 , 4 mm ; 16 mm ; 475 mm ( mediadeviazione standard ; mediana ; range )  . 
neither us nor scintigraphy detected the hypertrophic parathyroid gland at the level of the carotid sheath . we obtained the weight of 63 ( 74% ) of the 85 resected parathyroid glands . 
no weight was recorded for the following : 12 adenomatous parathyroid glands , five hyperplastic parathyroid glands , two intrathyroid microadenomas and three cases in which only fragments of the parathyroid gland were available . 
whereas in the past hyperparathyroidism tended to be detected in symptomatic patients with disorders due to hypercalcaemia ( kidney stones , fractures , neuromuscular syndrome ) , nowadays , it is increasingly discovered in asymptomatic patients . 
although medical treatment to control disease progression is an option in some cases , the only chance for cure remains surgery , which leads to a dramatic reduction of symptoms , a substantial fall in serum pth levels and normalisation of serum calcium levels [ 2 , 810 ]  . 
il confronto tra la sensibilit dellecografia e quella della scintigrafia ha mostrato un trend in favore dellecografia ( p = 0 , 134 , test di mcnemar )  . i casi in cui lecografia risultata falsamente negativa erano 9 pazienti con adenoma singolo , compresi una paratiroide in sede mediastinica e una in sede ricorrenziale ( in 6 di questi compresa la paratiroide ectopica in sede mediastinica la paratiroide patologica stata identificata con la scintigrafia ) , un paziente con adenoma duplice ( negativo anche alla scintigrafia ) e i due pazienti con microadenoma intratiroideo . delle 3 paratiroidi ectopiche intramediastiniche , tutte correttamente identificate alla scintigrafia , 2 , grazie alle dimensioni ed alla posizione subito sotto al giugulo , erano state sospettate anche allecografia . 
tutte le paratiroidi ingrandite situate nel legamento tireotimico sono state correttamente identificate , una di esse sulla base della sola ecografia in quanto il paziente non stato sottoposto ad esame scintigrafico . 
a ultrasound , axial view : symmetrical , regularly echoic thyroid gland ; on the right side , a hypoechoic intrathyroidal nodule ( arrowheads ) and an expansive hypoechoic mass ( arrow ) posterior to the thyroid lobe ( in the upper parathyroid gland site )  . 
b on the same side , a hypoechoic thyroidal nodule with peripheral vascularisation can be seen inside the thyroid gland , probably the reason why scintigraphy failed to detect the parathyroid lesion . 
a ecografia , scansione trasversale : la tiroide appare simmetrica con echi regolari ; a destra , oltre a nodulo ipoecogeno intraghiandolare ( punte di freccia ) , posteriormente al lobo ( nella sede della paratiroide superiore ) si rileva neoformazione espansiva ipoecogena ( freccia )  . 
la paziente esegue intervento chirurgico mini - invasivo con visualizzazione di ghiandola paratiroidea superiore destra aumentata di volume del peso di 300 mg ( diagnosi istologica di adenoma )  . 
3a , b a 29 - year - old woman with recurring bilateral renal colic and sudden growth of an anterior cervical mass on the left side of the neck . both scintigraphy and us confirmed the parathyroid nature of the mass . 
la diagnosi istologica stata di adenoma paratiroideo atipico del peso di 2100 mg . roidism , with hyperplasia ( 15%20% ) , multiple adenomas ( 4%5% ) and parathyroid carcinoma ( 1% ) being less frequent causes [ 2 , 9 , 11 ]  . 
the us examination does not enable even with the aid of doppler imaging the differential diagnosis between these disease entities , but it does allow recognition of the abnormal gland to be dissected [ 5 ]  . radiol med ( 2009 ) 114 : 11591172 frequente nelle donne ( 1 / 500 vs 1 / 2000 negli uomini ) , in particolare tra la quinta e la settima decade di vita [ 1 , 2 , 8 ]  . 
bench in alcuni casi si possa optare per la terapia medica e tenere sotto controllo la progressione di malattia , lunica possibilit di guarigione resta lintervento chirurgico che riduce drasticamente i sintomi e induce una caduta sostanziale dei livelli di pth e la normalizzazione della calcemia [ 2 , 810 ]  . 
 la maggior parte dei lavori in letteratura riporta come causa principale di iperparatiroidismo primario ladenoma singolo ( 80%90% dei casi ) , sono pi rari i casi di iperplasia ( 15%20% ) , di adenomi multipli ( 4%5% ) e di carcinoma paratiroideo ( 1% ) [ 2 , 9 , 11 ]  . 
la popolazione del nostro studio appare del tutto sovrapponibile con valori di circa 86% , 9% , 3% , 0% , rispettivamente ( tabella 3 )  . lesame ecografico non permette , neanche con lausilio del doppler , una diagnosi differenziale tra queste entit nosologiche , ma consente di riconoscere la ghiandola anormale su cui mirare lintervento chirurgico [ 5 ]  . lesecuzione dellintervento chirurgico in pazienti spesso asintomatici e , in cos larga misura , con lesione unica , ha posto lesigenza di interventi sempre meno invasivi . 
nel nostro studio , grazie allidentificazione della ghiandola interessata , circa il 70% dei pazienti ( 54 / 77 ) stato sottoposto ad esplorazione cervicale monolaterale , circa il 30 % ( 23 / 77 ) ad esplorazione cervicale bilaterale . 
i tempi medi di degenza e , di conseguenza , i costi sono ridotti [ 4 , 9 , 1116 ]  . in questo contesto ha assunto unimportanza sempre maggiore la corretta identificazione preoperatoria delle ghiandole patologiche . 
varie sono le tecniche di imaging capaci di identificare le paratiroidi ipertrofiche : ecografia , scintigrafia , tc , rm , tomografia computerizzata ad emissione di fotone singolo ( single photon emission computed tomography , spect )  . 
pi recentemente ad essa si affiancata lecografia , pi conveniente in termini di costi , disponibilit sul territorio , ripetitivit e dose per il paziente [ 1 , 79 , 17 , 18 ]  . 
nel nostro studio sono state prese in considerazione radiol med ( 2009 ) 114 : 11591172 1169 the use of surgery in often asymptomatic patients , most of whom have a single lesion only , has called for increasingly less invasive procedures . 
in our study , preoperative identification of the pathological gland led to unilateral neck exploration in approximately 70% of patients ( 54 / 77 ) and bilateral exploration in 30% ( 23 / 77 )  . 
unilateral transcervical access reduces operative time and morbidity by lowering transient and permanent postoperative hypocalcaemia and reducing the risk of compromising correctly functioning parathyroid glands . given that the contralateral glands are spared , the risk of permanent hypoparathyroidism is very low . 
mean hospitalisation stay is shorter and costs are reduced [ 4 , 9 , 1116 ]  . in this context , correct preoperative identification of abnormal glands has become increasingly important . several imaging techniques are able to detect hypertrophic parathyroid glands : us , scintigraphy , ct , mr imaging and single photon emission ct ( spect )  . 
more recently , us has taken on a significant role , as it is less expensive , readily available , repeatable and non - radiating [ 1 , 79 , 17 , 18 ]  . 
our study took into consideration us and scintigraphy only , as these are the techniques most commonly used by surgeons ( who consider ct , mri and spect third - line techniques to be reserved for exceptional cases )  . 
 [ 1 ] have emphasised that us sensitivity for detecting parathyroid glands , which was 34%82% in the 1980s , greatly improved beginning in the mid - 1990s , in parallel with technological developments ( highfrequency probes , colour and power doppler ) , reaching the current levels of 83%93% [ 1 , 17 , 1921 ]  . 
 the further advantages of us in addition to the abovementioned practical aspects are the possibility of detecting , measuring and characterising parathyroid lesions , which are usually solid , hypoechoic masses with welldefined margins and parenchymal vascularity . 
in cases of large neck nodules , where intraand extrathyroid formations are difficult to distinguish , the support of colour and power doppler proved useful in our experience [ 6 ]  . in our study , there was a trend in favour of us as opposed to scintigraphy in the identification of enlarged parathyroid glands and an impact of the weight of parathyroid glands on scintigraphic detection rates , confirming the usefulness of us as an initial diagnostic approach in primary hyperparathyroidis in agreement with published experiences , the sensitivity of scintigraphy alone ( 68% ) was lower than solo lecografia e la scintigrafia , essendo le tecniche pi richieste dai chirurghi ( i quali considerano tc , rm e spect tecniche di terzo livello e ne limitano la richiesta a casi eccezionali )  . 
 [ 1 ] si sottolinea come la sensibilit dellesame ecografico nella localizzazione delle paratiroidi , variabile dal 34% all82% negli anni ottanta , sia di gran lunga migliorata , di pari passo con i miglioramenti tecnologici ( sonde ad alta frequenza , utilizzo di color e power doppler ) , dalla met degli anni novanta , raggiungendo valori attorno all83%93% [ 1 , 17 , 1921 ]  . 
 vantaggi dellecografia sono sicuramente , oltre agli aspetti pratici gi elencati , la possibilit di localizzare , misurare e caratterizzare le lesioni paratiroidee ; solitamente , lesioni solide a margini ben definiti , ipoecogene , con vascolarizzazione di tipo parenchimale . 
lecografia , a differenza della scintigrafia , inoltre , consente unadeguata diagnosi differenziale con noduli tiroidei , linfonodi , residui timici , strutture vascolari [ 16 , 22 , 23 ]  . 
nel caso di lesioni nodulari voluminose a livello del collo , in cui era difficile distinguere tra formazioni intrao extratiroidee risultato utile , nella nostra esperienza , il supporto di color e power doppler [ 6 ]  . 
 nel presente studio abbiamo rilevato un trend in favore dellecografia nellidentificazione delle paratiroidi ingrandite in confronto alla scintigrafia ed uninfluenza del peso delle paratiroidi nella loro identificazione scintigrafica confermando lutilit dellecografia come prima indagine diagnostica nei casi di iperparatiroidismo primario . 
nel nostro studio , in accordo con numerosi lavori in letteratura , i valori di sensibilit del solo esame scintigrafico ( 68% ) sono inferiori a quelli della sola ecografia ( sensibilit dellesame ecografico pari a 84% considerando tutti i 77 pazienti ; 81% considerando solo i 65 pazienti sottoposti ad entrambi gli esami ) , ma raggiungono una significativit decisamente maggiore in caso di esecuzione combinata ( sensibilit di ecografia pi scintigrafia pari a 91% )  . 
il relativamente basso valore di concordanza ( 54% ) tra le due tecniche ottenuto nel nostro studio sicuramente limitato dal fatto che la maggior parte delle scintigrafie sono state eseguite su pazienti con ecografia dubbia o non conclusiva . 
the relatively low concordance ( 54% ) between the two techniques in our study is certainly dependent on the fact that most scintigraphy examinations were carried out on patients with inconclusive or doubtful us results . 
because of embryonic migration defects , in 25% of the population , the inferior parathyroid glands are located in the superior mediastinum [ 1 , 19 , 24 ]  . however , us succeeded in correctly identifying two ectopic glands ( located immediately below the jugulum ) out of three in our study . 
we believe the combination of us and scintigraphy is useful in cases with a clinical suspicion and negative us , given that the detection of abnormal parathyroid glands in ectopic locations is not an uncommon event [ 1 , 7 , 22 , 2426 ]  . a number of studies have reported a significant reduction in us sensitivity in detecting glands with lower weight compared with heavier ones ; a similar , though not statistically significant , trend is reported for scintigraphy [ 7 , 15 , 17 , 27 ]  . 
in our study , considering the difference between identified and missed parathyroid glands , the greatest significance was recorded in the evaluation of the two numerically balanced subgroups of scintigraphy ( 33 identified and 23 missed ) , confirming that limited spatial resolution plays a significant role in the us detection rates . 
the mcnemar test ( table 7 ) revealed only a trend in favour of us ( p = 0.1340 ) , not a statistically significant difference , a result most likely due to small sample size . 
however , in the choice of diagnostic tool , lack of invasiveness , wider availability and lower cost weigh heavily in favour of us , which should be the technique of choice , even in the vent of equivalent diagnostic performance . 
riteniamo comunque utile nei casi con sospetto clinico ed ecografia negativa , data la possibilit non infrequente di riscontrare paratiroidi anormali in sede ectopica , affiancare allecografia la scintigrafia [ 1 , 7 , 22 , 2426 ]  . in alcuni lavori si riporta una significativa riduzione di sensibilit dellecografia nellidentificazione di ghiandole di basso peso rispetto a quelle di peso maggiore , simile andamento , ma non statisticamente significativo , riguarda la sensibilit della scintigrafia [ 7 , 15 , 17 , 27 ]  . nel nostro studio , considerando la differenza tra le paratiroidi identificate e quelle non identificate , la maggiore significativit si ha nella valutazione dei due sottogruppi , numericamente bilanciati ( 33 identificate e 23 non identificate ) della scintigrafia , che conferma come la limitata risoluzione spaziale abbia un ruolo importante nellidentificazione delle ghiandole con questa tecnica . tuttavia la larga deviazione standard dei pesi ghiandolari indica che sono sicuramente coinvolti altri fattori , per esempio una concomitante patologia tiroidea . 
lo stesso si potrebbe dire per lecografia , anche se il non bilanciato rapporto tra i due sottogruppi ( 55 identificate versus 9 non identificate ) crea un limite statistico dovuto alle piccole dimensioni del campione ; inoltre , sempre considerando lecografia , la notevole differenza tra le mediane del peso ( 470 mg per le paratiroidi identificate , 180 mg per quelle non identificate ) si contrappone a valori simili delle medie ( 1022 , 5 mg versus 921 , 8 mg ) , a causa della presenza di singoli casi con peso di molto superiore alla media ( come nel caso di un adenoma paratiroideo ectopico con peso superiore alla media di pi di una ds )  . la minore influenza del peso delle paratiroidi nella sensibilit dellecografia rispetto a quella della scintigrafia il verosimile motivo dei maggiori livelli di sensibilit della stessa ecografia . 
il test di mcnemar ( tabella 7 ) ha mostrato solo un favore dellecografia ( p = 0 , 1340 ) , e non una significativit statistica , verosimilmente a causa delle ridotte dimensioni del campione . tuttavia , nella scelta tra i due mezzi diagnostici , la non invasivit , la maggior disponibilit e i minori costi giocano pesantemente a favore dellecografia che dovrebbe rappresentare comunque la tecnica di prima istanza anche nel caso di uguali performance diagnostiche . 
essi hanno anche realizzato unanalisi dei costi e concludono dicendo che il ricorso ad una metodica di iii livello come la spect solo nei casi di ecografia dubbia o negativa pu ridurre considerevolmente i costi dellimaging prima della paratiroidectomia . trend radiol med ( 2009 ) 114 : 11591172 1171 reported by tublin et al . 
their cost analysis showed that limiting the use of a third - line modality such as spect to cases of doubtful or negative us only can considerably reduce preoperative imaging costs . a few limitations of this study need to be acknowledged . firstly , its design was retrospective , even though the analysis was based on us diagnosis produced prospectively before surgery . 
the sensitivity of scintigraphy , ct and mr imaging is , however , even more affected in these cases [ 1 , 3 , 7 , 11 , 16 , 18 , 20 , 22 ]  . finally , us being an operator - dependent technique , our results are likely to be affected by the radiologists experience and by their long cooperation with the surgeons [ 1 , 9 , 10 ]  . 
different clinical settings and low levels of experience might undermine the reproducibility of our results . in conclusion , our study has shown that us represents the technique of choice for the detection of pathological parathyroid glands in patients with primary hyperparathyroidism and a high likelihood of single adenoma . 
un secondo limite quello relativo alla dimensione campionaria , non sufficiente a determinare una significativit statistica in favore della sensibilit dellecografia rispetto alla scintigrafia , ma solo un trend ( p = 0 , 1340 )  . 
i nostri risultati , infine , sono probabilmente dovuti anche allesperienza degli ecografisti , come ovvio per una tecnica operatore dipendente , e alla prolungata collaborazione con i chirurghi [ 1 , 9 , 10 ]  . 
contesti clinici differenti e minore esperienza specifica da parte dei colleghi radiologi potrebbero rendere non riproducibili i risultati del presente studio . in conclusione , il nostro studio ha mostrato che lesame ecografico rappresenta la tecnica di prima istanza nellidentificazione delle paratiroidi patologiche in soggetti con iperparatiroidismo primario e alta probabilit di adenoma singolo . 
42 chapters divided into 7 sections : technique ; neuro / ear - nose - throat ; cardiovascular imaging ; lung ; oncology ; intervention ; trauma imaging / acute care are intended to increase the readers confidence regarding the enormous and ever - increasing progress in multidetector imaging that has taken place since the publication of the second edition in 2002 . 
 much of what is described has its roots in the results of the 5th international symposium on multi - detector row ct . the aim and desire of the editors was to provide fundamental knowledge on this field , coupled with the latest ct scanner technology , updating the important clinical scenario . great importance is given to technical and also practical details , which are essential when using these ultrafast machines in order to fully exploit their enormous potential . 
chapters are devoted to indepth evaluation of the head and neck ( 6 chapters ) , cardiovascular system ( 11 chapters ) and lungs ( 3 chapters ) as well as oncology and intervention . 
importance is also given to multi - slice ct in the field of trauma . the book can be considered a salute to the mnich school of radiology , since most of the authors belong to this group , in conjunction with internationally known coauthors from europe and the usa . 
 in some of the chapters , the reader will need a pico della mirandolas memory due to the robust use of acronyms or short - listings , some of which are not defined . 
in some ways the subject index tries to obviate this phenomenon , yet one cannot always peruse the index in order to find ( not always ) what he is searching . 
in the text , unfortunately , some of the references to figures are incorrect as well as figure captions il lettore trover 614 pagine in questa terza edizione di multislice ct dal peso di 1.900 grammi , divisa in 42 capitali raggruppati in 7 sezioni dal titolo : technique ; neuro / ear - nose - throat ; cardiovascular imaging ; lung ; oncology ; intervention ; trauma imaging / acute care . 
scopo del volume di rendere edotto il lettore degli enormi e continui progressi nello studio per immagini con attrezzature multidetettore avvenuti nel corso di questi ultimi anni dalla data della seconda edizione nel 2002 . molto di quanto descritto trae le sue origini dalla presentazioni fatte al 5 simposio internazionale sulla tc con multidetettore : scopo e desiderio espresso dagli editori del volume stato di voler diffondere quanto pi possibile una conoscenza di base fondamentale in questo campi associata ai pi recenti progressi tecnologici nei riguardi anche delle ricadute nello scenario clinico da parte degli stessi . nel volume viene data grande importanza non solo ai dettagli tecnici ma anche a quelli pratici , che sono di assoluta e necessaria conoscenza nelluso di queste macchine ultra veloci in modo tale da poterne sfruttare al meglio le loro enormi potenzialit . 
vengono descritti nel dettaglio la preparazione del paziente e la radioprotezione dello stesso , luso dei mezzi di contrasto , langio - tc , lo studio con tomografia a doppio sorgente e linterpretazione delle immagini ottenute . 
vari capitoli sono dedicati ad una valutazione e studio in profondit della testa e del collo ( 6 capitoli ) del sistema cardiovascolare ( 11 capitoli ) , dei polmoni ( 3 capitoli ) come anche alloncologia ed allinterventistica . 
 il volume pu essere considerato il trionfo della scuola di radiologia di monaco di baviera dal momento che buona parte degli autori ne fa parte , in collaborazione con un gruppo di ben conosciuti co - autori europei ed americani . in alcuni dei capitoli chi legge dovr mettere in campo una memoria da pico della mirandola dato leccessivo , esorbitante uso di acronimi ed abbreviazioni , di cui alcune neppure spiegate . 
ballesio1 1dipartimento di scienze radiologiche , policlinico umberto i , universit sapienza di roma , roma , italy 2dipartimento di ginecologia e ostetricia , universit del sacro cuore di roma , roma , italy 3dipartimento di scienze ginecologiche , perinatologia e puericultura , policlinico umberto i , universit sapienza di roma , roma , italy correspondence to : s . 
savelli , via del risaro 81 , 00127 roma , italy , tel . : + 39 - 348 - 6709705 , fax : + 39 - 06 - 52372327 , e - mail : sarasavelli@hotmail.it received : 12 november 2008 / accepted : 7 january 2009 / published online : 5 september 2009 springer - verlag 2009 abstract purpose . 
to evaluate the additional diagnostic value of fetal mri to evaluate cerebral ventriculomegaly assessed by ultrasonography ( us ) for the possibility to change the diagnosis , the counseling and the management of pregnancy . 
from february 2006 to october 2008 , we studied 55 pregnant women by fetal mri ( mean age 28 years ) , 4 with twin pregnancy , for a total of 59 fetuses with mean gestational age of 27 weeks . 
the findings showed that the two techniques are substantially in agreement in defining the degree of vm , with the exception of some cases in which the disagreement could be attributed to the possible progression of the dilatation between the us and mri examinations , which sway between two days and two weeks . 
we proved a low correlation between us and mri in the evaluation of ventriculomegaly associated either with cns or non - cns anomalies : in fact while fetal mri detected 26 / 55 ( 47 , 3% ) vm associated with cns anomalies , us demonstrated only 18 / 55 ( 32 , 7% )  . 
nel periodo compreso fra febbraio 2006 ed ottobre 2008 , abbiamo sottoposto a risonanza magnetica 55 donne in stato di gravidanza ( et media 28 aa ) , 4 delle quali con gravidanze gemellari , per un totale di 59 feti di et gestazionale con et gestazionale media di 27 settimane . 
tutti i feti avevano diagnosi ecografica di ventricolomegalia : 29 feti con ventricolomegalia isolata e 26 feti con ventricolomegalia associata ; in particolare in questultimo gruppo 18 feti riportavano associazioni con anomalie del snc ed 8 feti con anomalie di altri distretti . 
emerso cos che le due metodiche sono sostanzialmente in accordo nel definire lentit della ventricolomegalia , fatta eccezione per alcuni casi nei quali la discordanza potrebbe attribuirsi alla possibile progressione della dilatazione nellarco di tempo intercorso fra ecografia e risonanza magnetica , variabile fra 2 giorni e 2 settimane . 
our experience demonstrated that fetal mri has an important role as adjunctive tool to sonography in the evaluation of cerebral ventriculomegaly for the additional informations given to parents and for the possibility to change the diagnosis , the counseling and the management of pregnancy . 
the condition may present in an isolated form or in association with other abnormalities of the central nervous system ( cns ) and / or other regions , and often represents the tip of the iceberg of a much more complex underlying pathological condition . 
vm is also known to be associated with chromosome disorders and intrauterine infectious diseases , especially cytomegalovirus . ultrasonography ( us ) is the first - line modality for the morphological study of the foetal brain and can easily identify the presence of vm . 
however , the technique is not free from the difficulty of studying the structure of the posterior cranial fossa in foetuses of advanced gestational age , which already show cranial ossification or critical conditions such as oligohydramnios , and maternal obesity , which can hamper the examination . 
there are also abnormalities of brain parenchyma that appear as findings at the limits of the resolution power of us and brain diseases with nonspecific characteristics [ 35 ]  . the aim of our study was to evaluate the role of magnetic resonance imaging ( mri ) in the diagnostic characterisation of vm for the consequences that a change in diagnosis can have on maternal counselling , pregnancy management and the planning of possible preand postnatal procedures in the setting of multidisciplinary patient management . le ventricolomegalie ( vm ) rappresentano lanomalia encefalica fetale di pi frequente riscontro con una prevalenza variabile fra 0 , 3 e 1 , 5 / 1000 nati [ 1 ] fino a valori riportati di circa 22 / 1000 nati [ 2 ]  . 
queste possono presentarsi in forma isolata o in associazione ad ulteriori anomalie del sistema nervoso centrale ( snc ) e / o di altri distretti , rappresentando spesso la punta delliceberg di un quadro patologico sottostante ben pi complesso . 
nota inoltre lassociazione delle ventricolomegalie con cromosomopatie e con patologie infettive intrauterine , prima fra tutte linfezione da citomegalovirus . lecografia rappresenta lindagine di prima scelta per lo studio morfologico dellencefalo fetale ed in grado di individuare facilmente la presenza di una ventricolomegalia . tuttavia essa non scevra da limiti , come la difficolt di studiare le strutture della fossa cranica posteriore in feti di et gestazionale avanzata che mostrano gi ossificazione cranica o in condizioni critiche quali loligo - anidramnios e lobesit materna , che possono ostacolare lesecuzione dellesame . 
esistono inoltre anomalie del parenchima cerebrale che si manifestano con reperti ai limiti del potere risolutivo dellecografia e patologie encefaliche con caratteristiche non specifiche [ 35 ]  . lo scopo del nostro studio valutare il ruolo della risonanza magnetica ( rm ) nellinquadramento diagnostico delle ventricolomegalie per le conseguenze che un cambiamento della diagnosi pu avere sul counselling materno , sul radiol med ( 2009 ) 114 : 10131023 materials and methods between february 2006 and october 2008 , we performed an mri evaluation of 55 pregnant women ( maternal age range 1839 years , mean 28 years ) , four with twin pregnancies , for a total of 59 foetuses with a gestational age range of 1938 weeks ( mean gestational age 27 weeks )  . 
the number of foetuses with vm identified at us was 55 , and these made up our study population . all foetuses were referred to our mri service with a us diagnosis of vm : 29 with isolated vm and 26 with associated vm . 
in the latter group , 18 showed associations with cns abnormalities and eight with abnormalities in other regions , one of these with a diagnosis of trisomy 21 and one with known cytomegalovirus infection . examinations were performed with a 1.5 - tesla scanner ( siemens magnetom avanto , siemens medical systems , erhlangen , germany ) using a phased - array coil positioned on the maternal pelvis . 
the study protocol of the foetal brain included the use of the following sequences : t2 - weighted half fourier acquisition single - shot turbo spin - echo ( haste ) : tr 1 , 500 ms ; te 151 ms ; slice thickness 4 mm ; fov 260350 mm ; matrix 192256 ; ta 20 s , in axial , coronal and sagittal planes of the foetal brain t1 - weighted 2d fast low - angle shot ( flash ) : tr 362 ms ; te 4.8 ms ; slice thickness 4 mm ; flip angle 70 ; fov 350300 mm , matrix 256192 ; ta 20 s , in axial or coronal planes . in some cases , when considered appropriate , the examination was performed with the following sequences : true fast imaging with steady - state precession ( fisp ) : tr 4.8 ms ; te 2.3 ; slice thickness 4 mm ; fov 400300 mm ; matrix 256144 ; ta 14 s , repeated in the same planes as the t2 - weighted haste sequences diffusion weighted imaging ( dwi ) : tr 8 , 000 ms , te 90 ms ; ti 185 ms ; slice thickness 5 mm ; fov 420300 mm ; matrix 192192 ; ta 45 s ; three b - factors ( diffusion gradients ) per plane : 0 , 200 and 700 s / mm2 , in the x , y , z orthogonal axes , acquired in axial or coronal planes of the foetal brain fluid - attenuated inversion recovery ( flair ) : tr 10 , 000 ms ; te 102 ; slice thickness 4 mm ; fov 300300 mm ; matrix 128128 ; acquisition time 40 s , in axial or coronal planes the measurement of ventricular diameters was performed in the axial and / or coronal planes , at the atrial level , in correspondence of the choroids plexuses . 
the management della gravidanza e sulla pianificazione di eventuali interventi pree post - natali , nellottica di una gestione multidisciplinare . 1015 materiali e metodi nel periodo compreso fra febbraio 2006 ed ottobre 2008 , abbiamo sottoposto a rm 55 donne in stato di gravidanza ( et materna compresa fra 18 e 39 anni , et media 28 anni ) , quattro delle quali con gravidanze gemellari , per un totale di 59 feti di et gestazionale ( eg ) compresa fra 19 e 38 settimane ( eg media 27 settimane )  . 
 tutti i feti venivano mandati al nostro servizio di rm con diagnosi ecografica di ventricolomegalia : 29 feti con ventricolomegalia isolata e 26 feti con ventricolomegalia associata ; in particolare in questultimo gruppo 18 feti riportavano associazioni con anomalie del snc ed 8 feti con anomalie di altri distretti , uno dei quali con diagnosi posta di trisomia 21 ed uno con infezione nota da citomegalovirus ( cmv )  . gli esami sono stati eseguiti con un magnete superconduttore da 1 , 5 tesla ( siemens magnetom avanto , siemens medical systems , erhlangen , germania ) , utilizzando una bobina phased array , posizionata sulla pelvi materna . 
le pazienti sono state esaminate in posizione supina o in decubito laterale qualora ne riportassero la necessit e non stata effettuata alcuna sedazione , n sulle donne n sul feto . per la localizzazione del feto sono state ottenute in primo luogo immagini di scanogramma multiplanare , poi , durante lesame , ciascuna sequenza servita come scanogramma per la successiva . 
where possible , we compared the findings with postnatal imaging findings or biopsy examinations , and only in five cases do we have follow - up information of the neonates from patient history data gathered directly from the mother or paediatrician . results we compared the diagnoses of the two modalities in terms of two parameters , i.e. 
the degree of dilatation of the lateral ventricles ( table 1 ) and on the basis of the presence / absence of abnormalities associated with vm ( table 2 )  . 
the findings showed that the two techniques are substantially in agreement in defining the degree of vm , with the exception of some cases in which the disagreement could be attributed to the possible progression of the dilatation in the interval between the us and mri examinations , which varied from 2 days to 2 weeks . s / mm2 , sugli assi ortogonali x , y , z , acquisite sui piani assiale o coronale dellencefalo fetale ; fluid - attenuation invertion recovery ( flair ) : tr 10000 ms ; te 102 ms ; spessore strato 4 mm ; fov 300300 mm ; matrice 128128 ; tempo di acquisizione 40 s , sui piani assiale o coronale . 
la soglia considerata di normalit era un diametro atriale inferiore a 10 mla classificazione adottata per definire il grado di dilatazione stata la seguente [ 6 , 7 ] : vm borderline : 10 mmda12 mm ; vm lieve : 13 mmda15 mm ; vm severa : da > 15 mm . i reperti rm sono stati confrontati con le relative indicazioni ecografiche . 
quando possibile abbiamo avuto il riscontro con esami strumentali post - natali o esami bioptici , ed in soli 5 casi disponiamo del follow - up dei piccoli pazienti attraverso dati anamnestici raccolti direttamente dalle madri o dai pediatri di riferimento . risultati a lesser degree of agreement was found with regard to the association of the cases of vm with further abnormalities abbiamo confrontato le diagnosi delle due metodiche rispetto a due parametri , ossia rispetto al grado di dilatazione table 1 degree of ventriculomegaly : us vs . 
in fact , mri revealed 26 / 55 ( 47.3% ) cases of vm associated with abnormalities of the cns against 18 / 55 ( 32.7% ) cases of diagnosis of association with other brain abnormalities identified with us . 
table 4 reports the diagnoses of associated abnormalities made at us and later not identified at mri . discussion the impact of vm on foetalneonatal morbidity and mortality is influenced by different factors , including : degree of dilatation unilateral or bilateral presentation symmetry / asymmetry of the dilatation regression , stability or progression of the condition association with additional abnormalities of the brain or other organs dei ventricoli laterali ( tabella 1 ) ed in base alla presenza / assenza di anomalie associate alla ventricolomegalia ( tabella 2 )  . 
emerso cos che le due metodiche sono sostanzialmente in accordo nel definire lentit della ventricolomegalia , fatta eccezione per alcuni casi nei quali la discordanza potrebbe attribuirsi alla possibile progressione della dilatazione nellarco di tempo intercorso fra ecografia e risonanza magnetica , variabile fra 2 giorni e 2 settimane . minor grado di accordo invece emerso dal confronto rispetto alla associazione delle ventricolomegalie con ulteriori anomalie sia del snc fetale che di altri organi e apparati ( tabella 2 )  . 
la rm ha infatti evidenziato 26 / 55 ( 47 , 3% ) vm associate ad anomalie del snc , versus 18 / 55 ( 32 , 7% ) diagnosi di associazione con ulteriori anomalie encefaliche poste con lecografia ; quanto alle associazioni con anomalie di altri distretti , la rm ne ha posto diagnosi in 10 / 55 casi ( 18 , 2% ) e lecografia in 8 / 55 feti ( 14 , 5% )  . 
la tabella 3 mostra tutte le anomalie del snc associate , sia quelle gi diagnosticate allesame ecografico morfologico , sia quelle inizialmente non riconosciute dallecografia e poi diagnosticate con rm . 
 [ 10 ] riportano che l86% delle ventricolomegalie isolate stabili o progressive presentano una morbilit / mortalit in percentuali molto elevate mentre altri autori riportano un normale sviluppo psicomotorio nel 90% dei casi di vm che si risolvono prima della nascita [ 11 ]  . studiare questi dati limportanza di sottolineano radiol med ( 2009 ) 114 : 10131023 1019 normal psychomotor development in 90% of cases of vm that resolve prior to birth [ 11 ]  . 
these figures emphasise the importance of thoroughly studying cases of vm , and some authors believe that the diagnosis of isolated vm should be a diagnosis of exclusion , thus justifying the use of mri despite the high costs and complexities of the examination . mri in fact provides a detailed study of the brain parenchyma , which makes possible not only the measurement of biometric values but also the evaluation of neuronal migration , gyration and sulcation and myelination . 
it also can identify haemorrhagic foci , porencephaly , cortical and subependymal tubers , abnormalities of the structures of the midline and partial and complete agenesis of the corpus callosum , and abnormalities of the posterior cranial fossa ( cysts , hypoplasia and agenesis of the cerebellar vermis ) [ 5 ]  . some studies in the literature [ 12 ] report in their own series a us classification error of isolated / associated vm in 37% of cases in which associated diseases are incorrectly identified , even by expert operators , with relative consequences on prognosis and management . 
indeed , whereas the failure to identify an agenesis of the corpus callosum can have mild consequences ( favourable prognosis in 85% of cases ) , this is not the case for other conditions such as abnormalities of neuronal migration , which significantly influence prognosis and often only appear as an alteration in mri signal [ 13 ] , and therefore difficult to identify with us . in a study on 214 foetuses with vm , levine et al . 
these were six cases ( 10.9% ) of cns abnormalities and two cases of abnormalities of other regions ( two cases of pulmonary hypoplasia with retarded parenchymal maturation )  . 
in another four cases , mri negated the us diagnosis of agenesis of the corpus callosum ( two cases ) , hypoplasia of the cerebellar vermis and cysts of the posterior cranial fossa . it should be noted that in our series , in agreement with the literature [ 15 ] , associated diseases identified with mri and not detected with us are for the most part abnormalities of the cerebral / cerebellar parenchyma , such as neuronal approfonditamente i casi di ventricolomegalia ed alcuni autori ritengono che la diagnosi di vm isolata debba essere una diagnosi di esclusione giustificando limpiego della risonanza magnetica a dispetto degli elevati costi e della complessit dellesame . 
la rm consente infatti un dettagliato studio del parenchima cerebrale permettendo , oltre alla misurazione dei valori biometrici , anche la valutazione della migrazione neuronale , della girazione e solcazione , e della mielinizzazione ; permette inoltre di identificare foci emorragici , cavit porencefaliche , tuberi subependimali e corticali , anomalie delle strutture della linea mediana ed agenesie parziali e complete del corpo calloso , anomalie delle strutture della fossa cranica posteriore ( cisti vermiane , ipoplasia ed agenesia del verme cerebellare ) [ 5 ]  . alcuni studi in letteratura [ 12 ] riportano nella propria casistica un errore ecografico di classificazione delle vm isolate / associate nel 37% dei casi in cui patologie associate non venivano riconosciute anche in mani esperte , con relative conseguenze sulla prognosi e sul management . 
infatti se il mancato riconoscimento di unagenesia del corpo calloso pu avere conseguenze talvolta modeste ( prognosi favorevole nell85% dei casi ) , ci non vero per altre condizioni come le anomalie di migrazione neuronale che influiscono in maniera rilevante sulla prognosi e che si manifestano spesso solo come alterazioni di segnale in rm [ 13 ] , pertanto difficilmente rilevabili ecograficamente . 
 [ 14 ] , in uno studio su 214 feti con ventricolomegalia , hanno notato che la rm comportava : cambiamento della diagnosi nel 23% circa dei casi gi anomali allecografia ; cambiamento del counselling nel 41% circa dei casi ; variazione del management nel 13 , 5% dei casi . 
nel nostro studio , la rm ha diagnosticato la presenza di anomalie associate alla ventricolomegalia in ulteriori 8 / 55 casi ( 14 , 5% ) rispetto allecografia , trattandosi in 2 casi di anomalie di altri distretti ( due ipoplasie polmonari con ritardo di maturazione parenchimale ) ed in 6 casi ( 10 , 9% ) di anomalie del snc . 
si evince pertanto che le vm possono rappresentare la punta delliceberg di un quadro patologico pi complesso caratterizzato dalla presenza di ulteriori anomalie , ed pertanto necessario approfondirne lo studio e seguirne levoluzione sia per monitorare la stabilit sia per diagnosticare anomalie che possono manifestarsi in et gestazionale pi avanzata . la rm diventa inoltre di cruciale importanza quando si debba individuare la causa determinante la ventricolomegalia , radiol med ( 2009 ) 114 : 10131023 1021 fig . 
3 feto alla 35a settimana di gestazione : vm severa monolaterale con parenchima cerebrale assottigliato e di alterato segnale ; le immagini mostrano inoltre ritardo della girazione e solcazione omolateralmente alla dilatazione . 
5 feto alla 31a settimana di gestazione : vm severa bilaterale , dilatazione del iii ventricolo , parenchima cerebrale assottigliato , ipoplasia cerebellare con polimicrogiria , dilatazione della cisterna magna . 
the correct characterisation can in fact have significant ramifications on the one hand for counselling and consequently for the management of the pregnancy in the legal context with regard to abortion , and on the other hand for the type of treatment in terms of the planning of a possible neurosurgical procedure [ 16 ]  . the literature does not provide specific indications regarding when further study of a diagnosis of vm is indicated with mri . 
cova1 1uco di radiologia , universit di trieste , ospedale di cattinara , strada di fiume 449 , 34149 trieste , italy 2uo di urologia , ospedale fondazione san raffaele giglio , contrada pietrapollastra , 90015 cefal , italy correspondence to : m . 
phosphodiesterase - 5 ( pde - 5 ) inhibitors have an established role in the treatment of erectile dysfunction , but there is increasing evidence that these drugs are effective also for the treatment of lower urinary tract symptoms and benign prostatic hyperplasia ( bph )  . 
the aim of this study was to investigate whether administration of tadalafil , a pde - 5 inhibitor , in patients with bph produces haemodynamic changes in the prostate that can be assessed using contrast - enhanced us ( ceus )  . 
changes in enhancement peak , time to peak ( ttp ) , sharpness of the bolus transit and area under the curve ( auc ) were considered for further analysis . 
gli inibitori della 5 - fosfodiesterasi ( pde - 5 ) hanno un ruolo ben definito nella terapia della disfunzione erettile , ma vi una crescente evidenza che questi farmaci siano efficaci anche per il trattamento dei disturbi delle basse vie urinarie e delliperplasia prostatica benigna ( ipb )  . 
dodici pazienti consecutivi con ipb sono stati studiati con ecocontrastografia prima e dopo la somministrazione di 20 mg di tadalafil . lecocontrastografia stata eseguita con un software contrastospecifico non distruttivo dopo somministrazione in bolo di sonovue ( 4 , 8 ml )  . 
le variazioni del picco di enhancement , del tempo di picco ( ttp ) , della ripidezza del transito del bolo e dellarea sotto la curva ( auc ) sono state valutate statisticamente . radiol med ( 2009 ) 114 : 11061114 1107 observed in the prostate after tadalafil administration , which can be detected with ceus . keywords prostate phosphodiesterase - 5 contrast enhanced ultrasound benign prostatic hyperplasia ( bph ) tadalafil risultati . 
nei pazienti con ipb dopo somministrazione di tadalafil si osservano variazioni della vascolarizzazione prostatica rilevabili con lecocontrastografia . parole chiave prostata 5 - fosfodiesterasi ecocontrastografia iperplasia prostatica benigna tadalafil introduction introduzione phosphodiesterases ( pde ) are intracellular enzymes that catalyse the hydrolysis of cyclic adenosine monophosphate ( camp ) and cyclic guanosine monophosphate ( cgmp )  . 
 several pde - 5 inhibitors are available , including sildenafil ( viagra , pfizer , italy ) , vardenafil ( levitra , bayer , italy ) and tadalafil ( cialis , lilly , italy )  . 
by inhibiting the enzyme reactions catalysed by pde - 5 , these drugs increase intracellular cgmp and reduce intracellular calcium , leading to an alteration of intracellular calcium - dependent processes , including relaxation of the smooth muscles of parenchyma and vessels [ 8 ]  . 
the therapeutic success achieved in this field recently prompted clinicians to search for new applications in treating lower urinary tract disorders in patients with benign prostatic hyperplasia ( bph ) and / or chronic prostatitis [ 1013 ]  . 
the most widely accepted hypothesis , based on studies conducted on prostate tissue specimens [ 15 , 16 ] , is that these agents act to relax prostatic smooth muscle , thereby increasing the washout of secretions [ 12 ]  . 
it is also likely that these agents relax the prostatic parenchymal vasculature , as demonstrated at the systemic and penile level [ 4 , 9 ] , which translates into changes in blood flow . the aim of this study was to establish whether the signal le fosfodiesterasi ( pde ) sono enzimi intracellulari che catalizzano lidrolisi delladenosin - monofosfato ciclica ( camp ) e della guanosin - monofosfato ciclica ( cgmp )  . esistono almeno 11 isoforme di tali enzimi , con varia distribuzione a carico dei diversi tessuti . 
la 5 - pde , in particolare , oltre che a livello penieno , espressa nei tessuti encefalici [ 1 ] , nel cuore [ 2 , 3 ] , nella muscolatura liscia dei vasi [ 4 ] , nella vescica [ 5 ] e nella prostata [ 6 , 7 ]  . 
 sono disponibili diversi farmaci inibitori della 5 - pde quali sildenafil ( viagra , pfizer italia ) , vardenafil ( levitra , bayer italia ) e tadalafil ( cialis , lilly italia )  . 
questi farmaci , inibendo le reazioni enzimatiche catalizzate dalla 5 - pde , producono un aumento del cgmp intracellulare ed una riduzione del calcio intracellulare , ci comporta unalterazione dei processi intracellulari calcio - dipendenti , tra cui il rilasciamento della muscolatura liscia dei parenchimi e dei vasi [ 8 ]  . 
il successo terapeutico in questo campo ha recentemente spinto i clinici a ricercare nuove applicazioni nel trattamento dei disturbi delle basse vie urinarie in pazienti con iperplasia prostatica benigna ( ipb ) e / o con prostatiti croniche [ 1013 ]  . 
lipotesi pi accreditata , basata su studi condotti su campioni di tessuto prostatico [ 15 , 16 ] , teorizza che tali farmaci producano essenzialmente un rilasciamento della muscolatura liscia prostatica che favorisce il drenaggio delle secrezioni [ 12 ]  . peraltro verosimile che questi farmaci producano il rilasciamento dei vasi parenchimali anche a livello prostatico , come stato dimostrato a livello sistemico e penieno [ 4 , 9 ] , evento che si traduce in variazioni dellapporto ematico . scopo di questo lavoro stabilire se nei pazienti con iperplasia prostatica benigna possibile misurare in 1108 radiol med ( 2009 ) 114 : 11061114 intensity of sonovue microbubbles can be determined in a reproducible manner in patients with bph and to assess whether tadalafil administration induces changes in prostate vascularity that can be detected with contrastenhanced ultrasound ( ceus )  . 
 materials and methods materiali e metodi twelve consecutive patients ( age range 3686 years ; mean age 66 years ) with bph underwent us examination of the prostate with the transrectal approach . 
a new - generation us unit ( iu22 , philips medical systems , bothell , wa , usa ) equipped with an end - fire broadband endocavitary transducer supporting nondestructive ceus imaging ( c9 - 5 , pulse inversion harmonic imaging , pihi ) was used . after a preliminary colour doppler us examination , an obliquetransverse scan plane was selected for the ceus study . 
 baseline ceus was conducted during the bolus injection of 4.8 ml sonovue , and digital video recordings were obtained for at least 2 min from the start of the injection . after baseline ceus , the patient was administered 20 mg oral tadalafil and was examined again 90 min after receiving the drug , with the same parameters as used for the baseline examinations . the digital clips obtained before and after tadalafil administration were retrospectively reviewed with dedicated software ( qontraxt v.360 , amid , rome , italy ) by two blinded independent readers who were not involved in scanning and were unaware of whether the scans had been obtained before or after tadalafil administration . 
the following changes in the enhancement curve parameters supplied by the software and indicative of enhancement kinetics before and after tadalafil administration were considered : enhancement peak ( expressed as a percentage , maximum intensity = 100% ) ; time to peak ( expressed in seconds ) ; sharpness of the bolus transit ( expressed in seconds - 1 ) ; area under the curve ( auc ; expressed in seconds - 1 )  . 
 stato utilizzato un ecografo di ultima generazione ( iu22 , philips medical systems , bothell , wa ) dotato di un trasduttore endocavitario endfire a larga banda abilitato allimaging ecografico non distruttivo con mezzo di contrasto ( c9 - 5 , pulse inversion harmonic imaging , pihi )  . dopo un preliminare esame eco - color doppler stato scelto un piano di scansione trasversale obliquo per lo studio ecocontrastografico della prostata . 
per ridurre al minimo la rottura delle microbolle la potenza acustica del trasduttore stata regolata su bassi livelli di indice meccanico ( im = 0 , 07 ) e mantenuta invariata per tutti i pazienti . per limitare le cause di variabilit nella valutazione dellenhancement , il guadagno stato regolato per ogni paziente prima delliniezione delle microbolle e mantenuto costante per tutta la durata dellesame . 
il paziente stato quindi esaminato nuovamente dopo 90 minuti dalla somministrazione del farmaco utilizzando gli stessi parametri dellesame di base . i filmati digitali ottenuti prima e dopo la somministrazione del farmaco sono stati analizzati retrospettivamente in forma anonima utilizzando un software dedicato ( qontraxt v.360 , amid , roma , italia ) da due lettori indipendenti non coinvolti nellesecuzione dellesame e che non sapevano se le scansioni erano state ottenute prima o dopo la somministrazione di tadalafil . 
sono state considerate le variazioni prima e dopo la somministrazione di tadalafil dei seguenti parametri della curva di enhancement , forniti automaticamente dal software , descrittivi della cinetica del contrasto : picco di enhacement ( espresso in percentuale , massima intensit = 100% ) ; tempo di picco ( espresso in secondi ) ; ripidezza del transito del bolo ( espresso in secondi - 1 ) ; area sotto la curva di enhancement ( espresso in secondi - 1 )  . 
le variazioni di questi parametri prima e dopo la somministrazione di tadalafil sono state valutate statisticamente utilizzando il test dei ranghi con segno di wilcoxon ( wilcoxon signed - rank test )  . 
i valori del picco di enhancement , del tempo di picco , della ripidezza del transito del bolo e dellarea sotto la curva ottenuti prima e dopo lassunzione di tadalafil sono riportati in tabella 1 . 
sotto leffetto del farmaco si osservato un aumento statisticamente significativo del picco di enhancement e dellarea sotto la curva ( p < 0 , 002 ) , mentre non sono state rilevate variazioni statisticamente significative dei restanti parametri . i diagrammi di bland - altman relativi alla variabilit interosservatore per i diversi parametri sono riportati in figura 3 . 
gli intervalli di confidenza ( ic ) al 95% per il picco di enhancement , per larea sotto la curva , per il 1110 radiol med ( 2009 ) 114 : 11061114 fig . 
immagini ottenute 10 s , 20 s , 37 s , 60 s dopo liniezione delle microbolle con un software contrastospecifico non distruttivo ( im = 0 , 07 )  . 
3a - d bland - altman plots show interobserver variability ( with two observers ) for the measurement of enhancement peak ( a in per cent of maximum enhancement ) ; time to peak ( b in seconds ) ; sharpness of the bolus transit ( c in s - 1 ) ; and area under the curve ( d in s - 1 ) in a randomly selected group of ten perfusion curves . 
3a - d diagrammi di bland - altman relativi alla variabilit interosservatore ( con 2 osservatori ) per le misure del picco di enhancement ( a , in percentuale del massimo enhancement ) ; del tempo di picco ( b , in secondi ) ; ripidezza del transito del bolo ( c , in secondi - 1 ) ; e dellarea sotto la curva ( d , in secondi1 ) in un gruppo di 10 curve di perfusione scelte a caso . 
questa variabilit accettabile se rapportata alle differenze medie tra le misure dei parametri ottenute dai due osservatori che erano rispettivamente di 31 , 62% , 4 , 41 s - 1 , 35 , 36 s e 0 , 146 s - 1 . discussion discussione studies conducted on the heart [ 17 ] , liver [ 18 ] , kidney [ 19 ] and other organs [ 2023 ] have demonstrated that ceus provides a useful and noninvasive tool for evaluating changes in the vascularity of the parenchyma and lesions . 
in studi condotti sul cuore [ 17 ] , sul fegato [ 18 ] , sul rene [ 19 ] e su altri organi [ 2023 ] hanno dimostrato che lecocontrastografia fornisce un utile strumento non invasivo per valutare variazioni della vascolarizzazione dei parenchimi e delle lesioni . 
in ambito prostatico lecocontrastografia 1112 radiol med ( 2009 ) 114 : 11061114 prostatic imaging , ceus has been proposed as a helpful technique for the detection and targeted biopsy of isoechoic neoplasms [ 2427 ]  . 
in fact , it is only in recent years that highly performing endocavitary transducers have become clinically available for the realtime , nondestructive , contrast - specific imaging of the prostate . 
by using this approach , we were able to demonstrate that prostatic blood flow was altered following tadalafil administration and that this change could be detected in a reproducible and noninvasive manner with ceus . 
moreover , we were unable to determine whether the changes observed in prostate vascularity were due to a local effect of tadalafil or to systemic effects resulting from a generalised relaxation of the smooth muscles of arteries . 
another limitation is related to the fact that enhancement of the prostatic parenchyma was evaluated by measuring variations in the intensity of the video signal on the sonographic images . more accurate measurements can be achieved by using specific software that quantifies the signal on a linear scale before logarithmic compression for video display [ 18 , 28 ]  . another limitation lies in the fact that the microbubbles were administered as a bolus and the enhancement measurements were fitted to a gamma - variate curve . 
with this method , the parameters reflecting parenchymal perfusion ( blood velocity , blood volume and blood flow ) can be extrapolated on condition that one can correct for microbubble recirculation [ 29 ]  . 
to perform this correction , one needs to know the concentration of microbubbles in blood and acquire enhancement curves both in the arteries supplying the organ and in the parenchyma . 
analysis of the enhancement curves of the prostatic parenchyma after bolus administration of microbubbles thus allows one to recognise changes in organ vascularity but not to obtain absolute measurements of flow parameters . 
other methodological approaches , such as evaluation of reperfusion kinetics after microbubble rupture during the continuous administration of contrast at a constant rate , allow for a more direct correlation between enhancement curve characteristics and perfusion parameters [ 19 ] , but they require administration of a larger dose of contrast material . 
one limitation that is common to all sonographic perfusion techniques is that any accurate and rigorous quantitative analysis of enhancement characteristics is made extremely stata proposta da alcuni autori come uno strumento utile per lindividuazione e leffettuazione di una biopsia mirata di neoplasie isoecogene [ 2427 ]  . 
utilizzando questo approccio il nostro studio dimostra che dopo somministrazione di tadalafil si verifica unalterazione del flusso ematico nel parenchima prostatico e che questa alterazione rilevabile in maniera riproducibile e non invasiva con lecocontrastografia . 
inoltre , non possibile stabilire se le variazioni osservate nella vascolarizzazione del parenchima prostatico siano dovute ad un effetto locale del tadalafil o ad effetti sistemici dovuti , in particolare , ad un rilasciamento generalizzato della muscolatura liscia delle arterie . un altro limite legato al fatto che lenhancement del parenchima prostatico stato valutato misurando le variazioni dellintensit del segnale video sulle immagini ecografiche . 
misurazioni pi accurate possono essere ottenute utilizzando software dedicati progettati per quantificare il segnale in scala lineare , prima della compressione logaritmica necessaria per la presentazione a video delle immagini [ 18 , 28 ]  . 
utilizzando questo approccio i parametri che descrivono la perfusione parenchimale ( velocit del sangue , frazione del volume ematico e flusso ematico ) possono essere estrapolati solo quando possibile effettuare una correzione per leffetto del ricircolo delle microbolle [ 29 ]  . 
per eseguire questa correzione necessario conoscere la concentrazione delle microbolle nel sangue e acquisire la curva di enhancement sia nelle arterie afferenti allorgano , sia nel parenchima . nel nostro caso non possibile misurare la curva di enhancement nelle arterie afferenti alla prostata per la disposizione anatomica sfavorevole ed il piccolo calibro . 
lanalisi delle curve di enhancement del parenchima prostatico dopo somministrazione in bolo delle microbolle permette pertanto di riconoscere variazioni della vascolarizzazione dellorgano , ma non di ottenere misurazioni assolute sulle caratteristiche del flusso . 
altri approcci metodologici , quali la valutazione della cinetica di riperfusione dopo rottura delle microbolle durante somministrazione continua del contrasto a velocit costante , permettono una correlazione pi diretta tra le caratteristiche delle curve di enhancement e i parametri di perfusione [ 19 ] , ma richiedono la somministrazione di una maggiore quantit di contrasto . 
anzidei , tel : + 39 - 06 - 4455602 , fax : + 39 - 06 - 490243 , e - mail : michele.anzidei@gmail.com received : 3 november 2008 / accepted : 24 february 2009 / published online : 22 september 2009 springer - verlag 2009 abstract purpose . 
in the mr imaging protocol , we acquired t2weighted turbo spin - echo ( tse ) sequences , true fast imaging steady - state free precession ( true - fisp ) and gadolinium - enhanced t1 - weighted volumetric interpolated breath - hold examination ( vibe ) 3d sequences . 
le immagini rm sono state acquisite con sequenze turbo spin echo ( tse ) t2 pesate ad alta risoluzione , true fast imaging with steady state precession ( truefisp ) e volumetric interpolated breath hold examination ( vibe ) 3d t1 pesate dopo somministrazione ev di chelati del gadolinio ( gdbopta )  . 
la rm si rivelata pi accurata della tc nella valutazione delle lesioni t1 ( 50% vs 37 , 5% ) , mentre la tc risultata pi affidabile nella valutazione delle lesioni t2 ( 81 , 2% vs 68 , 7% )  . 
patients with locally advanced lesions ( t3t4 ) are , instead , normally treated with neoadjuvant chemoradiotherapy with the aim of shrinking the tumour to make resection possible [ 3 , 4 ]  . stage t3t4 is , however , associated with worse outcomes [ 5 , 6 ]  . 
however , these methods suffer several limitations that preclude their use in routine practice : eus is invasive , of limited use in severely debilitated patients , highly operator dependent and with a field of view restricted to the gastric wall ; laparoscopy is considered a surgical and hence highly invasive technique that requires anaesthesia and hospitalisation . 
over the past 10 years , the widespread diffusion of multislice computed tomography ( ct ) scanners and implementation of volumetric imaging have enabled the noninvasive staging of gastric cancers , with a sensitivity of 85%90% in t staging [ 1316 ]  . 
although magnetic resonance ( mr ) imaging provides higher contrast resolution in the study of soft tissues and particularly the walls of hollow organs , it has never been considered a real alternative to ct [ 17 ] owing to its long acquisition times and susceptibility to motion artefacts ( breathing , peristalsis and cardiovascular pulsation )  . 
these limitations have recently been partially overcome with technological developments and the introduction of fast imaging , leading to a significant increase in the accuracy of mr imaging [ 18 , 19 ]  . 
 [ 20 ] compared the diagnostic performance of ct and mr imaging in the local staging of gastric tumours , reporting encouraging results with the use of old - generation scanners . 
the aim of our study was to prospectively evaluate the diagnostic accuracy of ct and mr imaging in the locoregional staging of gastric carcinei pazienti affetti da carcinoma gastrico la stadiazione dellestensione locale di malattia rappresenta il parametro principale nella pianificazione terapeutica ed determinante nella prognosi . 
pazienti con lesioni localizzate ( t1t2 ) possono essere trattati chirurgicamente con intento radicale , garantendo ottima sopravvivenza a lungo termine [ 1 , 2 ] ; pazienti con lesioni localmente avanzate ( t3t4 ) sono invece generalmente trattati con chemio - radioterapia neoadiuvante con lobiettivo di ridurre le dimensioni del tumore per renderne possibile la resezione [ 3 , 4 ] ; lo stadio t3t4 tuttavia correlato con una prognosi peggiore [ 5 , 6 ]  . lecoendoscopia ( eus ) [ 79 ] e la laparoscopia esplorativa [ 1012 ] sono tecniche affidabili nella stadiazione locale del carcinoma gastrico . 
tuttavia queste metodiche presentano limitazioni che ne impediscono lutilizzo nella routine diagnostica : leus una metodica invasiva , limitata nei pazienti defedati , fortemente operatore dipendente e con un campo di vista ristretto alla parete gastrica ; la laparoscopia invece considerata metodica del tutto chirurgica , altamente invasiva , che necessita di anestesia e di alti costi di ospedalizzazione . 
negli ultimi dieci anni lampia diffusione delle apparecchiature di tomografia computerizzata ( tc ) multistrato e limplementazione dellimaging volumetrico hanno reso fattibile la stadiazione non - invasiva del tumore gastrico , con una sensibilit dell85%90% [ 1316 ] nella stadiazione del parametro t . 
sebbene la risonanza magnetica ( rm ) permetta una risoluzione di contrasto superiore nello studio dei tessuti molli , ed in particolare della parete degli organi cavi , il suo utilizzo non stato finora considerato quale reale alternativa alla tc [ 17 ] , soprattutto per la lunga durata delle acquisizioni e per la suscettibilit agli artefatti da movimento ( respiro , peristalsi e pulsazioni cardiovascolari )  . 
attualmente queste limitazioni sono state almeno parzialmente superate : i recenti sviluppi tecnologici e lintroduzione del fast - imaging hanno apportato un significativo incremento dellaccuratezza diagnostica della rm [ 18 , 19 ]  . 
lo scopo del nostro studio stato di valutare prospetticamente radiol med ( 2009 ) 114 : 10651079 1067 noma using state - of - the - art imaging protocols and technology . laccuratezza diagnostica della tc e della rm nella stadiazione loco - regionale del carcinoma gastrico utilizzando protocolli di imaging e tecnologie allo stato dellarte . materials and methods materiali e metodi from january to october 2008 , we enrolled 40 patients ( 26 men , 14 women ; mean age 53.6 years ) with an endoscopic diagnosis of gastric carcinoma . 
scopolamine ( buscopan , boehringer international , ingelheim , germany ) to relax the stomach musculature and reduce peristalsis , and 1 l of water orally to distend the stomach . 
 a first scan of the upper abdomen was obtained without contrast agent administration ( 120 kv , slice thickness 3 mm , reconstruction increment 1.5 mm ) , and later scans ( 250 mas , 120 kv , slice thickness 1.5 mm and reconstruction increment 1.0 mm ) were acquired in the arterial ( upper abdomen ) and venous ( chestabdomenpelvis ) phases after contrast administration . 
optimal delay between contrast administration ( 0.5 ml / kg iomeron 350 bracco spa , milan , italy , at a flow rate of 4 ml / s ) , and the beginning of the arterial - phase scan was calculated using the bolus - tracking technique , with a region of interest ( roi ) placed at the level of the coeliac trunk , threshold set at 150 hu and delay set at 15 s after reaching the threshold . mr examinations were performed with a 1.5 - t scanner ( magnetom avanto , siemens medical solutions , erlangen , germany ; gradient strength 45 mt / m , slew rate 346 t / m / s , rise time 400 ms )  . 
precontrast images were obtained with t2 - weighted turbo spin echo ( tse ) and true fast imaging with steady - state precession ( truefisp ) sequences in the axial and coronal planes ( table 1 )  . after the intravenous administration of 15 ml gadolinium benzyloxyproprionic - tetraacetic acid ( gd - bopta ) ( multihance , bracco spa , milan , italy ) , images were acquired in the arterial and venous phases using t1 - weighted 3d volumetric interpolated breath - hold examination ( vibe ) sequences ( table 1 )  . 
both the ct and mr imaging studies were evaluated preoperatively by two groups of tra gennaio ed ottobre 2008 , sono stati arruolati 40 pazienti ( 26 uomini , 14 donne ; et media di 53 , 6 anni ) con diagnosi endoscopica di carcinoma gastrico . 
per 7 pazienti ( 17 , 5% ) stato programmata una chemioterapia neoadiuvante prima di una eventuale resezione e 3 ( 7 , 5% ) pazienti hanno subito interventi di chirurgia palliativa a causa dellestensione locale della malattia . 
circa 10 minuti prima dellesame sono stati iniettati 20 ml di scopolamina ( buscopan , boehringer international , ingelheim , germania ) per rilassare la muscolatura viscerale e ridurre la peristalsi ed stato somministrato 1 l di acqua per os per la distensione gastrica . 
una prima scansione delladdome superiore stata ottenuta senza mezzo di contrasto ( 120 kv , slice thickness 3 mm , recon increment 1 , 5 mm ) e successivamente sono state acquisite scansioni ( 250 mas , 120 kv , slice thickness 1 , 5 mm e recon increment 1 , 0 mm ) in fase postcontrastografica artero - portale ( addome superiore ) e venosa ( torace - addome - pelvi ) ; il ritardo ideale tra la somministrazione del mezzo di contrasto ( mdc ; 0 , 5 ml / kg di iomeron 350 bracco spa , milano , italia , ad una velocit di flusso di 4 ml / s ) e linizio della scansione artero - portale stato calcolato tramite tecnica di bolus - tracking , con una regione di interesse ( roi ) posizionata a livello del tripode celiaco , soglia a 150 uh e ritardo a 15 secondi dal raggiungimento della soglia . 
gli esami rm sono stati eseguiti con unapparecchiatura a 1 , 5 t ( magnetom avanto , siemens medical solutions , erlangen , germania ; intensit dei gradienti 45 mt / m , slew rate 346 t / m / s , tempo di risalita 400 msec )  . 
le immagini precontrasto sono state ottenute con sequenze turbo spin echo ( tse ) e true fast imaging with steady state precession ( truefisp ) t2 pesate acquisite su piani assiali e coronali ( tabella 1 )  . 
the t parameter was staged according to the tumour node metastasis ( tnm ) classification established by the american joint committee on cancer and by the international union against cancer [ 21 ]  . 
progression of tumour invasion on ct and mr images was classified as follows : t1 : slight enhancement and focal thickening of the inner layer of the gastric wall t2 : diffuse enhancement and thickening of the entire gastric wall without extramural spread t3 : diffuse enhancement and thickening of the entire gastric wall , with irregularities and invasion of the perivisceral fat t4 : infiltration of adjacent organs , including vascular structures the ct studies were evaluated using 1.5 - mm - thick multiplanar reconstructions ( mpr ) parallel and perpendicular to the lesion . 
mr studies were evaluated using both the precontrast sequences ( tse and true - fisp ) and the sequences obtained after gd - bopta administration ( vibe 3d )  . 
la progressione dellinvasione tumorale nelle immagini tc ed rm stata classificata come segue : t1 : sottile enhancement ed ispessimento focale dello strato interno della parete gastrica ; t2 : diffuso enhancement ed ispessimento dellintera parete gastrica senza estensione extramurale ; t3 : diffuso enhancement ed ispessimento dellintera parete gastrica con irregolarit e invasione del grasso periviscerale ; t4 : infiltrazioni degli organi adiacenti , incluse le strutture vascolari . gli esami tc sono stati valutati utilizzando delle ricostruzioni multiplanari ( mpr ) parallele ed ortogonali alla lesione con spessore di 1 , 5 mm ottenute dai dati grezzi . 
gli esami rm sono stati valutati utilizzando sia le sequenze pre - constrastografiche ( tse e truefisp ) che le sequenze ottenute dopo somministrazione gd - bopta ( vibe 3d )  . 
relativamente alla rm , la stadiazione del parametro t stata effettuata con una valutazione combinata , ma le tre sequenze sono state visualizzate indipendentemente per consentire anche un confronto intra - metodica . 
lagreement tra i due gruppi stato misurato con il radiol med ( 2009 ) 114 : 10651079 1069 technique were compared with statistical package ( spss , chicago , il , usa )  . 
the percentage of detected lesions and stage attributed based on each diagnostic histopathological findings for lesions staged t1t3 and with exploratory laparotomy for lesions staged t4 ; mcnemar test was used to evaluate the accuracy of each technique in lesion detection and staging . 
compared with the histological and laparoscopic findings , ct correctly staged 33 / 40 lesions with a sensitivity of 82.5% , and mr imaging staged 32 / 40 lesions with a sensitivity of 80% ( table 2 )  . 
there were no statistically significant differences between mr imaging and ct in either sensitivity or accuracy ( mcnemar test p > 0.05 ) : the two techniques were concordant in 35 ( 87.5% ) cases with regard to the presence or absence of disease and in 37 ( 92.5% ) cases with regard to disease stage . 
evaluation performed on t1 - weighted contrast - enhanced 3d vibe sequences alone resulted in nine ( 22.5% ) lesions being understaged , whereas evaluation of t2 - weighted tse sequences led to the understaging of ten ( 25% ) lesions ( table 4 )  . test di cohen ( scarso : = 0 , 210 , 40 , moderato : = 0 , 410 , 69 , buono : = 0 , 610 , 80 , eccellente = 0 , 811 , 00 )  . la percentuale di lesioni individuate e lo stadio attribuito da ciascuna metodica diagnostica sono stati confrontati con il risultato isto - patologico nelle lesioni stadiate come t1t3 e con la laparotomia esplorativa nelle lesioni stadiate come t4 ; il test di mcnemar stato utilizzato per valutare laccuratezza di ogni metodica sia nellindividuazione che nella stadiazione delle lesioni . 
 risultati lagreement tra i due gruppi di osservatori stato eccellente , sia per la tc ( = 0 , 957 ) che per la rm ( = 0 , 933 )  . rispetto ai reperti istologici e laparoscopici , la tc ha correttamente stadiato 33 / 40 lesioni neoplastiche con una sensibilit dell82 , 5% e la rm ha stadiato 32 / 40 lesioni con una sensibilit dell80% ( tabella 2 )  . 
non stata rilevata nessuna differenza statisticamente significativa tra rm e tc n per la sensibilit , n per laccuratezza ( test di mcnemar , p > 0 , 05 ) : le due metodiche si sono rivelate concordi in 35 ( 87 , 5% ) casi per la presenza o lassenza di malattia ed in 37 ( 92 , 5% ) casi per la stadiazione . 
la valutazione con le sole sequenze t1 pesate contrast enhanced ( ce ) - vibe 3d ha invece portato a sottostadiarne 9 ( 22 , 5% ) mentre valutando le sequenze t2 pesate tse erano state sottostadiate 10 lesioni ( 25% ) ( tabella 4 )  . discussione nello studio del carcinoma gastrico la scelta diagnostica per lidentificazione e la stadiazione delle lesioni ampia ed 1070 table 2 t staging at histopathologic examination , ct and mr imaging . 
all values are expressed as numbers and percentages histopathological examination radiol med ( 2009 ) 114 : 10651079 ct , computed tomography ; mr , magnetic resonance tabella 2 grado di stadiazione delle lesioni attribuito da ciascuna metodica . 
moreover , the possibility of routinely using multiplanar reconstruction and volume - rendering techniques represents an additional advantage for the accurate evaluation of the gastric wall and vascular infiltration . mr imaging , if performed with state - of - the - art technique and technology , is a potentially innovative modality for the study of gastric lesions . 
la tc e , pi recentemente , la rm sono state proposte quali alternative non invasive e con elevata accuratezza diagnostica per stadiazione e follow - up [ 13 , 2527 ]  . 
le pi recenti apparecchiature tc permettono di acquisire immagini con voxel isotropici e spessore di strato sub - millimetrico in tempi brevissimi [ 28 ] ; inoltre la possibilit di utilizzare di routine le tecniche di ricostruzione multiplanare e di volume rendering costituisce un ulteriore vantaggio per unaccurata valutazione della parete gastrica e dellinfiltrazione vascolare . 
la rm , se effettuata con criteri tecnici e tecnologie allo stato dellarte , una metodica potenzialmente innovativa per lo studio delle lesioni gastriche : rispetto alla tc , la superiore risoluzione di contrasto ottenibile sulle apparecchiature di ultima generazione pu consentire una miglior differenziazione dei diversi strati della parete incrementando laccuratezza nella stadiazione delle lesioni in stadio precoce . 
the extent of wall infiltration is better evaluated on true - fisp ( c ) and tse ( d ) sequences rather than on contrast - enhanced vibe sequences ( e ) and ct images . 
non si osservano fenomeni infiltrativi a carico degli strati profondi della parete ( stadio t1 ) , n nelle immagini tc ( a , b ) , n nelle sequenze rm ( c - e )  . 
lestensione della lesione appare meglio valutabile nelle sequenze truefisp ( c ) e tse ( d ) piuttosto che nelle sequenze vibe ( e ) acquisite dopo somministrazione di mdc e nelle immagini tc . 
i reperti descritti sono stati confermati allesame istologico macro ( f ) e microscopico ( g )  . contrast resolution obtained with the latest - generation scanners may improve discrimination of the various wall layers , thus increasing staging accuracy in early lesions . 
the new devices have almost completely overcome the limitations due to long examination times , allowing for significant improvements in the study of abdominal organs and a diagnostic performance almost identical to that of ct in many gastrointestinal imaging applications [ 7 , 2932 ]  . 
furtheri limiti dovuti alla durata dellesame , consentendo un significativo miglioramento nello studio degli organi addominali e garantendo performance diagnostiche sostanzialmente sovrapponibili a quelle della tc in molte applicazioni di imaging gastrointestinale [ 7 , 2932 ]  . 
inoltre la diffusione delle tecniche di imaging parallelo [ 33 , 34 ] ha ulteriormente colmato il gap esistente tra le due metodiche riguardo alla risoluzione spaziale / temporale ed al rapporto segnale rumore delle immagini rm . 
in this case , the involvement of the deep layers of the gastric wall is better seen on ct images ( a , b ) : on mr images ( c - e ) the lesion appears to be involving the superficial layer only . infiltration of the perigastric fat tissue can be excluded with both techniques . 
2a - g lesione a tutto spessore della grande curvatura gastrica ( punta di freccia ) , senza coinvolgimento del tessuto adiposo periviscerale ( stadio t2 )  . in questo caso le immagini tc ( a , b ) dimostrano meglio il coinvolgimento degli strati profondi di parete : nelle acquisizioni rm ( c - e ) la lesione sembra infatti limitata ai soli strati superficiali . 
i reperti sono stati confermati allesame istologico macro ( f ) e microscopico ( g )  . more , diffusion of parallel imaging techniques [ 33 , 34 ] has bridged the gap between the two modalities with regard to spatial / temporal resolution and signal - to - noise ratio of mr images . 
relativamente ai risultati del nostro studio , necessario notare che , anche se sono state utilizzate apparecchiature allo stato dellarte , linclusione di numerose lesioni in stadio t1 particolarmente impegnative da identificare e differenziare ha radiol med ( 2009 ) 114 : 10651079 1073 fig . 
the extension of the tumour and the infiltration of both gastric wall and extraparietal fat are clearly evaluated on both ct ( a , b ) and mr ( c - e ) images . 
with regard to the results of our study , it should be noted that despite the use of state - of - theart devices , the inclusion of several t1 lesions which are particularly difficult to detect and discriminate led to very similar diagnostic values as those obtained in previous reports . 
this difference was determinato valori diagnostici sovrapponibili a quelli delle precedenti esperienze : valutando globalmente la performance delle due metodiche , la tc risultata leggermente superiore alla rm con una sensibilit dell82 , 5% ed una accuratezza diagnostica del 76 , 7% contro l80% ed il 74 , 4% . 
tuttavia , tale differenza non si rivelata statisticamente significativa : il dato dimostra come entrambe le metodiche , se adeguatamente utilizzate , permettano di ottenere risultati non solo adeguati alla richiesta clinica in termini di accuratezza diagnostica , ma anche altamente riproducibili . 
4a - d extensive tumour of the antropyloric passage ( arrowhead ) with diffuse infiltration into the perivisceral fat tissue and involvement of the superoanterior aspect of the pancreatic head ( arrow ) ( stage t4 )  . 
4a - d avanzata neoplasia della regione antro - pilorica ( punta di freccia ) con diffusa infiltrazione extraparietale e coinvolgimento della porzione superiore della regione cefalo - pancreatica ( freccia ) ( stadio t4 )  . 
sia nelle immagini tc ( a , b ) che nelle sequenze rm ( c , d ) la lesione appare chiaramente visualizzabile , con perdita del piano di clivaggio adiposo rispetto alla testa del pancreas . 
 not , however , statistically significant , implying that when used correctly , either technique is capable of providing results that are not only sufficiently accurate for the clinical purpose but also highly reproducible . 
in routine practice , accurate staging is the cornerstone of treatment planning . thus , in terms of clinical relevance , the significance of the ppv and accuracy of the two modalities in our study is noteworthy : both mr imaging and ct reached 100% ppv for t1 , t3 and t4 lesions ; in t2 lesions , instead , ct had 81.2% ppv , whereas mr imaging reached 78.5% , with no statistically significant difference between the two modalities . 
there were very small differences in favour of mr imaging in stage t1 and of ct in stage t2 , but the low prevalence of these cases in our study population and the small number of patients studied ( not sufficient for statistically significant evaluations ) do not allow for any scientifically sound conclusions to be drawn on this topic . 
however , on an observational basis , only valid within our small patient population , the limitation of ct partially consisted of suboptimal contrast resolution for the detection of focal wall alterations ( t1 )  . 
in such cases , mr quotidiana , laffidabilit nella stadiazione il presupposto fondamentale per la scelta terapeutica ; al fine della rilevanza clinica pertanto interessante notare la significativit del vpp e dellaccuratezza delle due metodiche evidenziate dal nostro studio : sia la rm che la tc hanno ottenuto un vpp del 100% per le lesioni di tipo t1 , t3 e t4 ; per le lesioni in stadio t2 invece la tc ha ottenuto un vpp dell81 , 2% mentre la rm del 78 , 5% , senza evidenza di differenze statisticamente significative tra le due metodiche . 
laccuratezza ha mostrato un costante incremento dallo stadio t1 allo stadio t4 per entrambe le metodiche ; si sono evidenziate delle minime differenze a favore della rm nello stadio t1 e della tc nello stadio t2 , ma la ridotta percentuale di tali casi sul totale della popolazione e lesiguit del campione stesso ( non sufficiente per valutazioni statisticamente significative ) non consentono di estrapolare considerazioni scientificamente valide a tale riguardo . 
su una base osservazionale , e pertanto valida unicamente nellambito della nostra ristretta popolazione , il limite della tc si parzialmente manifestato nella risoluzione di contrasto non ottimale per la discriminazione di focali alterazioni parietali ( t1 ) ; a tale riguardo la rm ha mostrato una miglior capacit di identificare il tessuto patologico , anche se questo beneficio stato ottenibile in un unico caso , in cui peraltro alla tc la scarsa distensione dellantro gastrico ha radiol med ( 2009 ) 114 : 10651079 1075 table 3 positive predictive value , diagnostic accuracy and sensitivity of computed tomography ( ct ) and magnetic resonance ( mr ) imaging in the evaluation of the t parameter . 
studies using in vivo [ 38 ] and , with more interesting results , in vitro experimental technologies [ 18 , 29 ] have shown mr imaging to have excellent potential for accurately depicting the gastric wall layers , thus facilitating the diagnosis and differentiation of superficial lesions . 
in some cases of t2 lesions understaged as t1 on mr imaging , the parietal signal alteration due to neoplastic infiltration had wrongly been attributed to perilesional oedema / inflammatory reaction , leading to incorrect staging . 
on the subject of the diagnostic performance of mr imaging , it is interesting to note that the shorter examination times made possible by state - of - theart equipment allowed lesions to be accurately staged , even in patients with locally advanced disease who were potentially unable to sustain long breath - holds or prolonged immobility . 
finally , both techniques showed a moderate tendency to understage t3 lesions : in three cases at ct and in two cases at mr imaging , infiltration into the perivisceral fat , later confirmed at histology , could not be demonstrated . 
on this subject , in addition to the diagnostic limitations of each technique , we also need to consider the psychological influence exerted by the risk of precluding surgical treatment in patients with cancer stages within the ulteriormente ridotto la possibilit di identificare la lesione . studi con tecnologie sperimentali in vivo [ 38 ] e , con risultati pi interessanti , in vitro [ 18 , 29 ] hanno evidenziato come la rm abbia uneccellente potenzialit di visualizzare accuratamente gli strati della parete gastrica , potendo rendere pi facile la diagnosi e la differenziazione delle lesioni superficiali . 
tale capacit deve tuttavia essere attentamente considerata in sede di refertazione anche quale possibile fonte di pitfalls : in alcuni casi di lesioni t2 sottostadiate alla rm come t1 , lalterazione di segnale della parete dovuta allinfiltrazione neoplastica era stata erroneamente attribuita ad edema / reazione flogistica perilesionale , determinando unerrata determinazione dellestensione della patologia . ancora riguardo alla performance diagnostica della rm , interessante notare come la contenuta durata degli esami , ottenuta grazie allimpiego di apparecchiature allo stato dellarte , abbia permesso di ottenere unaccurata stadiazione anche nei pazienti con patologia localmente avanzata e pertanto potenzialmente poco cooperanti nel mantenimento di lunghe apnee o prolungata immobilit . 
entrambe le metodiche hanno mostrato infine una modesta tendenza a sottostimare le lesioni t3 : in 3 casi alla tc ed in 2 casi alla rm non stato possibile dimostrare linfiltrazione del tessuto adiposo periviscerale successivamente confermato allesame istologico . 
relativamente a tale dato , oltre ai limiti diagnostici di ciascuna metodica , senza dubbio da tenere in conto linfluenza psicologica esercitata dal rischio di poter precludere trattamenti chirurgici in pazienti con stadiazioni nei cutoff del protocollo terapeutico t2 / t3 e dunque la potenziale tendenza a dichiarare come immediatamente operabili lesioni 1076 radiol med ( 2009 ) 114 : 10651079 fig . 
on ct images ( a , b ) , mainly due to incomplete distension of the stomach , the lesion was wrongly interpreted as a nonspecific scarring of the gastric wall . 
at mr imaging ( c - e ) , adequate distension of the stomach and particularly of the antrum allows identification of the lesion that seems confined to the superficial wall layer , especially on the contrast - enhanced vibe ( e ) images . 
allesame rm ( c - e ) , unadeguata distensione dello stomaco e soprattutto della regione antrale consente di evidenziare chiaramente la formazione polipoide che appare limitata allo strato superficiale della parete , soprattutto nelle sequenze vibe ottenute dopo somministrazione di mdc ( e )  . 
i reperti sono stati confermati tramite resezione endoscopica ( f ) ed esame istologico microscopico ( g )  . t2 / t3 cutoff for treatment , which could lead one to report as immediately operable locally advanced lesions that are instead eligible for neoadjuvant therapy . 
tuttavia , il ridotto numero di lesioni osservabili per ogni stadio patologico , e maggiormente in relazione allo stadio t1 ( 10% dei casi ) ed allo stadio t4 ( 10% dei casi ) , comporta una riduradiol med ( 2009 ) 114 : 10651079 1077 table 4 mr sequences vs . 
concordance in the evaluation of the t parameter histopathological examination w , pesata ; tse , turbo spin echo ; truefisp , true fast imaging with steady state precession ; ce - vibe , contrast enhanced volumetric interpolated breath hold examination tabella 4 sequenze rm vs reperti istopatologici . 
clearly , further studies on larger patient populations are required for a more reliable comparison of the roles of ct and mr imaging . in conclusion , our results confirm that ct is the modality of choice in gastric carcinoma staging . 
from a purely clinical point zione dellattendibilit delle misurazioni statistiche sui nostri dati : saranno ovviamente necessari ulteriori studi che coinvolgano un maggior numero di pazienti per poter confrontare con maggior attendibilit i rispettivi ruoli di tc ed rm . 
 in conclusione , i risultati del nostro studio confermano la tc come metodica di elezione per la stadiazione del carcinoma gastrico ; la rm pu tuttavia essere considerata unalternativa non solo fattibile , ma anche affidabile ed in grado di garantire una performance diagnostica soddisfacente nella stadiazione locale . 
da un punto di vista strettamente clinico , attualmente appare ancora per difficile 1078 radiol med ( 2009 ) 114 : 10651079 of view , mr imaging cannot , however , compete with ct : the higher costs , the greater difficulty achieving a technically adequate examination and , more importantly , the inability to perform comprehensive staging with evaluation of extra - abdominal lymph nodes and metastases are all very real limitations , well beyond possible considerations on the ability to detect and characterise local extent of disease . poter proporre la rm come metodica in competizione con la tc : il costo , la maggior difficolt nellesecuzione di un esame tecnicamente valido e , soprattutto , la sostanziale impossibilit di effettuare una stadiazione completa , comprensiva della valutazione linfonodale e metastatica in sede extra - addominale rappresentano limiti rigidamente realistici , ben aldil delle possibili considerazioni sulla capacit di poter identificare e caratterizzare la semplice estensione locale della patologia . 
fugazzola1 1vascular and interventional radiology , department of radiology , university of insubria , 21100 varese , italy 2department of vascular surgery , university of insubria , 21100 varese , italy correspondence to : d . 
all patients underwent intentional exclusion of the left subclavian artery and placement of a straight graft ; four patients underwent supra - aortic vessel transposition and four underwent carotid - carotid bypass in one case combined with carotid - subclavian bypass . 
in tutti i pazienti stata esclusa intenzionalmente la succlavia sinistra e posizionata unendoprotesi retta ; in 4 pazienti stata effettuata una trasposizione completa dei tronchi epiaortici mentre in altri 4 pazienti stato effettuato un by - pass carotidocarotideo , in un caso associato a by - pass carotidosucclavio . 
sono necessari ulteriori studi per verificare i risultati a medio e lungo termine . keywords aortic arch aneurysm aortic aneurysms endovascular treatment hybrid treatment parole chiave aneurisma arco aortico aneurismi aortici trattamento endovascolare trattamento ibrido radiol med ( 2009 ) 114 : 11301140 introduction introduzione 1131 open surgery is considered the treatment of choice for isolated aortic arch aneurysms , as it involves prosthetic replacement of the arch and thus represents a definitive treatment [ 1 ]  . 
the recently proposed endovascular approach [ 2 , 3 ] excludes the aneurysm from the systemic circulation by using an endograft but does not prevent disease progression . endovascular treatment of descending thoracic aortic aneurysms with endograft placement in patients at high surgical risk has become a well - established approach , thanks to its lower invasiveness [ 47 ]  . 
similarly , in selected patients at high surgical risk it is now possible to adopt a hybrid approach in isolated aortic arch aneurysms with inadequate neck [ 8 ]  . 
treatment of zone 0 , the most proximal , involves complete transposition of the supra - aortic vessels by means of an aortoinattualmente il trattamento chirurgico convenzionale la terapia di scelta per gli aneurismi isolati dellarco aortico , in quanto prevede la sostituzione protesica dellarco e quindi la definitiva risoluzione della malattia [ 1 ] ; il trattamento endovascolare , proposto recentemente [ 2 , 3 ] , esclude dal circolo sistemico la dilatazione aneurismatica mediante unendoprotesi , ma non impedisce la progressione della malattia . ormai ben consolidato lapproccio solo endovascolare mediante stent - graft degli aneurismi dellaorta toracica discendente in pazienti ad alto rischio chirurgico in relazione alla minore invasivit [ 47 ]  . 
il trattamento prevede dapprima un intervento chirurgico di trasposizione dei vasi epiaortici e successivamente , realizzandosi delle zone nuove di atterraggio , si procede al posizionamento di stent - graft con esclusione della sacca aneurismatica dal circolo sistemico [ 3 ]  . in relazione alla classificazione di criado - ishimaru ( fig . 1 ) [ 9 ] , larco dellaorta viene suddiviso in 3 zone di atterraggio a cui corrispondono interventi chirurgici differenti . il trattamento della zona 0 , la pi prossimale , prevede la trasposizione completa dei vasi epiaortici mediante un bypass aorto - anonima e aorto - carotideo sinistro con o senza by - pass carotido - succlavio . 
la zona 2 , pi distale , pu essere trattata anche solo per via endovascolare con esclusione intenzionale dellarteria succlavia di sinistra nei casi in cui ci siano gli estremi affinch si realizzi il furto della succlavia ; negli altri casi viene effettuato un by - pass carotido - succlavio . il radiologo gioca un ruolo essenziale nel planning della procedura in quanto identifica mediante imaging la zona sana di atterraggio prossimale che prevede approcci chirurgici differenti e inoltre valuta lindicazione alla esclusione intenzionale della succlavia in relazione alla possibilit di documentare lintegrit dei vasi epiaortici e del circolo intracranico . 
zone 2 , the most distal , may be treated with endovascular treatment alone , with intentional exclusion of the left subclavian artery in cases likely to develop subclavian steal syndrome . 
imaging is used to identify the healthy proximal landing zone , and thus determine the surgical approach , and to document the integrity of the supra - aortic vessels and intracranial circulation , thus providing the indication for intentional exclusion of the subclavian artery . 
in our experience , the radiologist also plays a fundamental role in endograft placement and in preventing and treating endoleaks . the aim of our study was to evaluate the results of the hybrid treatment and to highlight the role of the radiologist in treatment planning and follow - up . materials and methods from december 2000 to december 2006 , we selected 14 patients ( nine men and five women ; mean age 66.2 years , range 5685 ) with isolated aortic arch aneurysms ( mean diameter 62 mm ; range 5572 mm ) located in zone 0 ( n = 4 ) , zone 1 [ n = 6 , two with bovine aortic arch configuration ( fig . 2 ) ] and zone 2 ( n = 4 )  . 
complete transposition of the supraaortic trunks was performed in four patients , whereas a carotidcarotid bypass was done in another four , in one case combined with a carotidsubclavian bypass . 
planning involved assessment of the integrity and diameter of the landing zones and patency of the supra - aortic vessels and willis circulation ( to identify stenosis of the vertebral arteries or carotids or abnormalities of the intracranial circulation ) , a search for possible areas of cerebral ischaemia and evaluation of the peripheral vascular point of access . 
stata effettuata una trasposizione completa dei tronchi epiaortici in 4 pazienti , mentre in altri 4 pazienti stato confezionato un by - pass carotido - carotideo , in un caso associato a bypass carotido - succlavio . 
i pazienti presentavano le seguenti comorbilit : ipertensione ( 8 casi ) ; cardiomiopatia ischemica ( 8 casi ) ; broncopneumopatia cronica ostruttiva ( 6 casi ) ; insufficienza renale cronica ( 4 casi ) e diabete ( 5 casi )  . il planning pre - procedura stato effettuato mediante angio - tomografia computerizzata multistrato ( angio - tcms ) ( lightspeedplus , ge , milwaukee , usa ; aquilion 64 , toshiba , tokyo , giappone ) con ricostruzioni multiplanari ( mpr ) e 3d per la valutazione dellintegrit e dei diametri della zona di atterraggio , della perviet dei vasi epiaortici e del circolo di willis ( per lidentificazione di stenosi a carico delle arterie vertebrali , carotidi o anomalie del circolo intra - cranico ) e per la ricerca di eventuali aree ischemiche cerebrali , nonch valutazione del sito daccesso vascolare periferico . 
solo in casi selezionati ( placche calcifiche in arterie vertebrali con impossibilit di valutazione della riduzione in calibro ) stata effettuata langiografia per lo studio in particolare dei vasi epiaortici e del circolo intracranico e successivamente , mediante simultaneo approccio trans - omerale , con pallone occlusore allorigine dellarteria succlavia di sinistra , stato effettuato un test con cateterismo selettivo trans - femorale dellarteria vertebrale di destra per verificare linversione di flusso dellarteria vertebrale sinistra in succlavia [ 10 ]  . sono stati considerati criteri di inclusione : pazienti senza pregresse aree ischemiche cerebrali e con regolare perviet del circolo di willis ; eventuali stenosi delle arterie carotidi sono state corrette prima del posizionamento della protesi ( 1 caso )  . 
nella selezione dei pazienti sono stati considerati criteri di esclusione locclusione intenzionale dellarteria succlavia di sinistra , i pazienti con arteria lusoria , con fistole dialitiche , quelli in cui larteria mammaria interna era stata utilizzata per un by - pass coronarico e in tutti i casi di steno - occlusione delle arterie vertebrali in cui non vi era indicazione alla ricanalizzazione endovascolare . 
a tutti i pazienti stata somministrata una profilassi antibiotica a breve termine ( vancomicina 2 g ) , associata alla somministrazione di 5000 ui di eparina , e quindi stato realizzato il cateterismo trans - omerale sinistro per la valutazione angiografica dellarco nelle prime fasi della procedura endovascolare . successivamente stata posizionata lendoprotesi radiol med ( 2009 ) 114 : 11301140 1133 fig 2a - c aortic arch aneurysm in a patient with bovine aortic arch configuration ( type iii )  . 
a intraoperative digital subtraction angiography ( dsa ) : large aortic arch aneurysm in a patient with left common carotid artery originating from the innominate artery ( type iii aortic arch )  . 
il controllo agiografico al termine della procedura dimostra il furto dellarteria succlavia , sinistra in assenza di endoleak di tipo ii ad origine dallarteria succlavia che pertanto non viene occlusa . intracranial circulation and subsequently , with simultaneous transhumeral approach and selective transfemoral catheterisation of the right vertebral artery , a balloon occlusion test with balloon positioned at the origin of the left subclavian artery to verify flow inversion from the left vertebral artery into the subclavian artery [ 10 ]  . inclusion criteria were patients without previous areas of cerebral ischaemia and with patenty of willis circulation . any carotid artery stenoses were repaired before endograft placement ( n = 1 )  . 
exclusion criteria included intentional occlusion of the left subclavian artery , patients with lusory arteries , dialysis fistulas , coronary bypass with internal mammary artery and all cases of stenosis / occlusion of the vertebral arteries without an indication for endovascular ( excluder , gore , flagstaff , az , usa , in 5 casi ; talent , medtronic , santa rosa , ca , usa , in 5 casi ; zenith , cook , bloomington , in , usa , in 4 casi ) mediante monitoraggio fluoroscopico in proiezione obliqua anteriore sinistra , con un oversizing del 15%20% . 
in 6 casi stata coperta intenzionalmente larteria succlavia di sinistra e solo in 3 casi stata embolizzata mediante posizionamento , per via transomerale , di vascular plug ( amplatzer , aga medical corporation , plymounth , mn , usa ) ( fig . 3d ) , per la presenza di un endoleak di tipo ii documentato al controllo angiografico post - procedura . 
all patients underwent short - term antibiotic prophylaxis ( vancomycin 2 g ) combined with 5 , 000 iu heparin , followed by left transhumeral catheterisation for angiographic assessment of the aortic arch during the initial phases of the endovascular procedure . the endograft [ excluder , gore , flagstaff , az , usa ( n = 5 ) ; talent , medtronic , santa rosa , ca , usa ( n = 5 ) ; zenith , cook , bloomington , in , usa ( n = 4 ) ] was subsequently placed under fluoroscopic guidance in the left anterior oblique projection , with 15%20% oversizing . 
follow - up was performed by msct angiography at 1 , 3 , 6 and 12 months , and yearly thereafter . results immediate technical success ( exclusion of the sac from the systemic circulation ) was achieved in all cases . 
three patients remained in the intensive care unit for 2 , 15 and 33 days , respectively . we had four cases of cerebral ischaemia ( three transient ischaemic attacks and one stroke ) related to zone - 0 procedures in two cases and zone - 1 procedures in the other two . postprocedural complications included one case of pneumonia and one of atrial fibrillation , both of which were treated medically . 
mean hospital stay was 12 days ( 10 days for zone 0 and 6.5 days for zone 2 )  . during a mean follow - up of 21 months ( range 1248 months ) , two patients died ( one of myocardial infarction and one of respiratory failure ) 26 and 36 months after the procedure , respectively . 
il followup stato espletato mediante angio - tcms a 1 , 3 , 6 e 12 mesi e successivamente annualmente . risultati stato ottenuto un successo tecnico immediato ( esclusione della sacca dalla circolazione sistemica ) in tutti i casi . 
tre pazienti sono stati ricoverati in terapia intensiva rispettivamente per 2 , 15 , 33 giorni . abbiamo osservato 4 ischemie cerebrali ( 3 ischemie cerebrali transitorie [ tia ] e 1 stroke ) , in 2 casi di ancoraggio della protesi in zona 0 e in 2 casi di ancoraggio nella zona 1 . 
lospedalizzazione media stata di 12 giorni ( 10 giorni per zona 0 e 6 , 5 per zona 2 )  . durante un follow - up medio di 21 mesi ( range 1248 mesi ) 2 pazienti sono deceduti ( 1 per infarto miocardico e 1 per insufficienza respiratoria , rispettivamente 26 e 36 mesi dopo le procedure )  . 
 [ 11 ] riportarono la prima esperienza di esclusione endovascolare di un aneurisma dellaorta addominale ( aaa ) ; sfortunatamente questo lavoro rimase a lungo sconosciuto in quanto pubblicato in lingua russa . 
3a - f posttraumatic chronic aortic arch pseudoaneurysa , b computed tomography angiography ( cta ) [ a : sagittal maximum intensity projection ( mip ) ; b : 3d volume rendering ( vr ) ] : chronic bilobate posttraumatic pseudoaneurysm with marginal calcified cap proximal to the origin of the left subclavian artery . 
d intraoperative dsa : endograft placement and occlusion of the origin of the left subclavian artery with vascular plug due to type - ii endoleak , with initial reverse flow of the vertebral artery ( black arrows ) to the mediodistal subclavian artery ( white arrows )  . 
d angiografia intra - operatoria : posizionamento dellendoprotesi e occlusione dellemergenza dellarteria succlavia mediante vascular plug per endoleak di tipo ii , con iniziale inversione di flusso dellarteria vertebrale ( frecce nere ) in arteria succlavia medio - distale ( freccie bianche )  . 
visibile un guscio calcifico lungo la convessit ( teste di frecce ) e la concavit ( freccia ) dellarco aortico . the other , from the innominate artery , by coil embolisation . in the other cases , msct angiography demonstrated the exclusion of the aneurysmal sac with no migration of the endografts from the landing zone . stanford [ 13 ] tratt 103 pazienti con aneurismi dellaorta toracica ; in 8 di questi pazienti lendoprotesi fu posizionata a valle dellarteria succlavia sinistra . 
the 3d vr reconstruction well depicts the short aneurysmal neck and the bovine aortic arch configuration ( type ii : arrows ) , an indication for transposition of the supra - aortic vessels . 
la ricostruzione 3d vr ben evidenza la presenza di breve colletto aneurismatico , la configurazione bovina dellarco ( tipo ii , frecce ) quindi vi indicazione alla trasposizione dei vasi epiaortici . 
trasposizione dei vasi epiaortici con by - pass aorto - anonima , aorto - carotideo ( freccia ) con legatura dei vasi a valle , del posizionamento di cuffia protesica ( testa di frecce ) e del furto dellarteria succlavia ( frecce )  . discussion just over 20 years have passed since balko et al . 
 [ 11 ] first described the endovascular exclusion of an abdominal aortic aneurysm ( aaa ) in a report that remained largely unknown , as it was written in russian . 
the need to develop an endograft large enough for the ascending aorta decidedly larger than the abdominal aorta that would not give rise to problems at the transfemoral access point [ 14 ]  . 
another hindrance to the use of this approach in aortic arch and ascending aortic aneurysms was the increased risk of endograft migration due to high blood pressure , mechanical stress produced by the heart and possible progression of aneurysmal disease . further problems may arise in relation to the different landing zones on the aorta , the angle between the aortic arch and the descending aorta , which is not well suited to the stiffness of endografts , and the anatomical peculiarity of the unendoprotesi di calibro maggiore , adeguata alle dimensioni dellaorta ascendente , decisamente maggiore di quelle dellaorta addominale , ma allo stesso tempo che non creasse difficolt sul sito di accesso trans - femorale [ 14 ] ; importante stata la creazione di un puntale protesico di ridotte dimensioni , atraumatico , che non danneggiasse la valvola aortica durante il posizionamento della protesi . 
un ulteriore freno allutilizzo di tale approccio nel trattamento degli aneurismi dellarco e dellaorta ascendente stato il maggiore rischio di sposizionamento dellendoprotesi a causa dellelevata pressione del flusso sanguigno , delle sollecitazioni meccaniche del cuore e delleventuale progressione della malattia aneurismatica . 
infine , altre difficolt possono derivare dalle diverse zone di atterraggio sullaorta , dallangolazione tra larco aortico e laorta discendente che mal si adatta alla rigidit delle protesi impiegate , e dallanatomia peculiare dellarco aortico dalla cui convessit nascono , a poca distanza luno dallaltro , i vasi epiaortici [ 14 ]  . 
queste difficolt di carattere tecnico , sono state in parte superate mediante lintroduzione di stent - graft di seconda generazione , ben pi flessibili di quelli fino ad allora impiegati , in grado di adattarsi meglio alla curvatura dellarco e dotati inoltre di efficaci sistemi di fissaggio ed ancoraggio [ 14 ]  . radiol med ( 2009 ) 114 : 11301140 1137 fig . 
b , c superselective dsa of the thyrocervical trunk : leakage of contrast material into the aneurysmal sac ( b : arrow ) and treatment with glue embolisation ( c )  . 
perviet dellendoprotesi in assenza di endoleak e riduzione dimensionale della sacca aneurismatica . 1138 radiol med ( 2009 ) 114 : 11301140 aortic arch , with the supra - aortic vessels arising at short intervals along its convex surface [ 14 ]  . 
the concavity of the aortic arch and emergence of the supra - aortic vessels from its convex surface make it difficult to seal this portion of the aorta with an endogra these technical difficulties have been partially overcome by second - generation endografts , which are much more flexible than the earlier devices and thus able to adapt better to the curvature of the arch , as well as being equipped with effective fixing and landing systems [ 14 ]  . in addition , during the planning phase , the use of msct angiography with its high spatial and temporal resolution and the possibility of performing electrocardiography - gated examinations can increase the diagnostic accuracy of msct by enabling the attenuation or elimination of cardiac motion artefacts , especially at the level of the ascending aorta and supra - aortic vessels , with 3d volume rendering ( vr ) , multiplanar reconstructions ( mpr ) and 3d virtual endoscopy ( ve )  . the combination of technical advances and technological developments in thoracic endografting allows endovascular treatment in a large number of patients affected by major aortic lesions and several comorbidities who would otherwise have no other alternative [ 8 ]  . 
according to the criado / ishimaru classification [ 9 ] , zone 0 of the aortic arch requires complete transposition of the supra - aortic vessels through aortoinnominate and left aortocarotid bypass , whereas zone 1 requires carotid - carotid and , if necessary , carotidsubclavian bypass [ 18 ]  . 
zone 2 may undergo endovascular treatment alone , with intentional exclusion of the left subclavian artery in cases likely to develop subclavian steal syndrome [ 18 ]  . hybrid treatment potentially reduces procedural morbidity and mortality and may thus represent the only therapeutic choice for a subset of patients at high surgical risk . 
however , manipulation of supra - aortic and aortic arch vessels , particularly in the presence of multiple atheromatous plaques , is not devoid of complications such as embolism and consequent cerebral ischaemia . 
in order to avoid ischaemic events , doubtful cases require a detailed angiographic study of the supra - aortic vessels followed by an occlusion test of the left subclavian artery and in aggiunta , lesecuzione nel planning di unangiotcms , grazie allelevata risoluzione spaziale e temporale , nonch alla possibilit in casi selezionati , di esecuzione di esami con sincronizzazione cardiaca ( ecg - gated ) permette di incrementare mediante ricostruzioni 3d volume rendering ( vr ) , ricostruzioni multiplanari ( mpr ) e 3d virtual endoscopy ( ve ) , le potenzialit diagnostiche della tcms , attenuando o eliminando i possibili artefatti da pulsatilit cardiaca , specie in corrispondenza dellaorta ascendente e sui tronchi sovra - aortici . quindi la combinazione tra avanzamenti tecnici e sviluppi tecnologici nellendo - grafting toracico permette attualmente di trattare per via endovascolare un numero di pazienti con importati lesioni aortiche e numerose comorbilit che altrimenti non avrebbero nessuna altra alternativa [ 8 ]  . 
il trattamento chirurgico convenzionale , prevedendo infatti larresto di circolo e la circolazione extracorporea , presenta degli indici di mortalit compresa tra 8 , 5% e 25 , 2% [ 1517 ] con danno neurologico permanente tra 4 , 2% e 12 , 8% [ 15 ]  . la suddivisione in zone di atterraggio in relazione alla necessit di interventi chirurgici differenti stata confermata nel corso del primo summit internazionale di thoracic aortic endografting a tokio nel 2001 [ 9 ]  . 
in accordo con la classificazione di criado - ishimaru [ 9 ] la zona 0 dellarco prevede la trasposizione completa dei vasi epiaortici mediante un by - pass aorto - anonima e aorto - carotideo sinistro ; la zona 1 prevede il confezionamento di un by - pass carotido - carotideo ed eventuale carotido - succlavio [ 18 ]  . 
la zona 2 pu essere tratta anche solo per via endovascolare con esclusione intenzionale dellarteria succlavia di sinistra nei casi in cui si possa realizzare il furto della succlavia [ 18 ]  . il trattamento ibrido potenzialmente riduce la morbilit e la mortalit costituendo , in alcuni pazienti ad alto rischio chirurgico , lunica possibilit terapeutica . 
al fine di evitare eventi ischemici , nei casi dubbi necessario eseguire uno studio di particolare angiografico dei vasi epiaortici con test di occlusione dellarteria succlavia di sinistra e valutazione dellinversione del flusso . 
 [ 19 ] dopo il posizionamento di endoprotesi tipo talent in zona 2 . il follow - up a lungo termine di queste procedure radiol med ( 2009 ) 114 : 11301140 1139 assessment of flow inversion . 
 [ 19 ] after positioning a talent endograft in zone 2 . long - term follow - up of these procedures is still inadequate , and published studies have reported on small patient populations [ 8 ]  . 
for this reason , some authors [ 20 ] have suggested surgical bending of the ascending aorta near landing zone 0 to ensure lasting fixation and sealing of the endogra although bending the proximal neck clearly increases the invasiveness of the procedure , it is thought to prevent endograft migration . 
in fact , in the case of complete transposition of the supra - aortic vessels , endoleak prevention requires either surgical ligation of the origin of the vessels [ 21 ] or subsequent embolisation in the event that a type - ii endoleak occurs [ 8 ]  . in our experience , all type - ii endoleaks were treated with endovascular embolisation owing to our conviction that we should limit overall procedure time in consideration of the type of patients selected . 
furthermore , with regard to the intentional exclusion of the left subclavian artery , our experience shows occlusion at the origin is only indicated when postprocedural dsa demonstrates a type - ii endoleak , a rare occurrence when the subclavian artery is not involved by the aneurys it is , however , possible to perform preliminary endovascular occlusion , although this might turn out to be useless due to increased flow towards the upper limb . limitato , sono state pubblicate solo casistiche numericamente ridotte [ 8 ] ; inoltre la perviet a lungo termine di questi bypass non stata ancora verificata . 
da qui alcuni autori [ 20 ] hanno proposto il bending chirurgico dellaorta ascendente in corrispondenza dellatterraggio in zona 0 , per garantire un fissaggio duraturo e per poter sigillare in maniera ermetica lendoprotesi . 
infatti , nei casi di trasposizione completa dei tronchi epiaortici la prevenzione dellendoleak richiede o la legatura chirurgica dellorigine degli stessi [ 21 ] o lembolizzazione in un secondo tempo se si realizza lendoleak di tipo ii [ 8 ]  . nella nostra esperienza gli endoleak di tipo ii sono stati trattati tutti per via endovascolare mediante embolizzazione per convinzione , anche chirurgica , di non eccedere sul tempo operatorio complessivo in relazione al tipo di pazienti selezionati , quindi di concentrare un primo tempo solo dedicato alla esclusione della sacca dalla circolazione sistemica e il successivo trattamento dei rifornimenti della sacca in ambiente radiologico mediante angiografo fisso . inoltre nella esclusione intenzionale dellarteria succlavia sinistra , nella nostra esperienza indicata locclusione allorigine solo nei casi in cui il controllo angiografico post - procedura dimostri lendoleak di tipo ii : fatto poco frequente se non direttamente coinvolta dallaneurisma . 
endovascular exclusion of isolated aortic arch aneurysms , in the small number of cases treated , allowed us to control the in conclusione , il trattamento ibrido una valida alternativa alla chirurgia convenzionale in pazienti ad alto rischio chirurgico per la minore invasivit con tassi di mortalit inferiori . 
questa tecnica ha un basso tasso di mortalit peri - operatoria , ma un tasso di complicanze non indifferente da insulto ischemico cerebrale , anche se pu essere nella maggior parte dei casi di tipo transitorio . 
 di fondamentale importanza il planning della procedura dove gioca un ruolo determinante il radiologo per la valutazione del calibro vascolare del sito daccesso , per la scelta del device pi idoneo allesclusione della sacca aneurismatica , per stabilire il tipo di intervento chirurgico in relazione alla scelta della zona di atterraggio e per dare gli estremi per la realizzazione dellinversione del flusso in arteria vertebrale di sinistra in quanto questo evita il bypass carotido - succlavio . 
renieri piccin nuova libraria s.p.a. , padova 2009 isbn 978 - 88 - 299 - 2002 - 0 published online : 9 october 2009 springer - verlag 2009 these soft - bound books complement one another and can be considered a short and simple crib for the much larger work , summa teologica ( deformit congenite dello scheletro ) published some years ago by canepa . their aim is to present , most of all to clinical genetists , paediatricans and orthopaedics , clues and bases for observing and diagnosing congenital bone diseases and congenital deformities of the skeleton . atlante disegnato lists 193 dysplasias , syndromes and chromosomal diseases . 
each page describes two conditions . at glance , in half a page each , one will find drawings extracted by deformit congenite dello scheletro , plus in the shortest possible text a listing ( a check - list one could call it ) of the main and most important diagnostic features of the described condition and type of genetic transmission , viability and main phenotypic expression . 
at the end of the book one will find some pages listing genetic features and loci of the single conditions ( heredity , mim , locus and gene involved )  . atlante elementare in 95 pages and 10 sub - headings addresses what one should know about the genetics clinical genetics of congenital deformities of the skeleton . 
as stressed in their short introduction by the authors , the book addresses geneticists and biologists in order to remind the firsts that clinical genetics is not an out - dated subject overrun by molecular genetics ; and to remind the second group that biology prevails over medicine in their study curricula . 
at the end of this book the listing of loci and genes is shorter than in the previous one ; actually here one will find a subject index . once one has read or perused the atlante elementare he / she will better understand the atlante disegnato and appreciate the research and effort put into the preparation and lay - out of both books . 
 scopo degli autori e dei volumi , di presentare a genetisti clinici , pediatri , ed ortopedici quali sono le tracce e le basi sulle quali sospettare e diagnosticare le malattie congenite dellosso e le deformit congenite dello scheletro . 
in mezza pagina , con un solo sguardo , si ritovano disegni con i tratti caratteristici dellaffezione , ed un elenco sintetico ( tipo check - list dei piloti ) delle pi importanti e significative caratteristiche diagnostiche della stessa , le modalit di trasmissione genetica , le possibilit di vita o non nonch il tipo di genotipo . 
alla fine del volume alcune pagine elencano le caratterisctiche genetiche ed i loci delle single affezioni ( ereditariet , mim , locus e gene interessato )  . latlante elementare descrive in 95 pagine e 10 capitoli ci che si dovrebbe sapere sulla genetica genetica clinica delle deformit congenite dello scheletro . 
come anche ben specificato , nella breve introduzione , da parte degli autori il volume ha come obiettivo di sensibilizzare i genetisti ed anche i biologi : i primi perch ricordino che la genetica clinica non qualche cosa da dimenticare a fronte della genetica molecolare ; i secondi perch ricordino che la biologia prevale sulla medicina nel loro corso di studio . 
 dopo avere letto o sfogliato latlante elementare il lettore potr meglio comprendere latlante disegnato ed apprezzare quanto di sforzo e ricerca stato profuso nella preparazione ed impostazione di entrambi i volumi . 
dato anche il prezzo modesto ( importante di questi tempi ) questi volumi dovrebbero avere una ampia diffusione nel campo di coloro che si occupano di bambini portatori di displasie o radiol med ( 2009 ) 114 : 11841185 1185 even though these conditions may be deemed to be in a world apart , both for diagnosis and therapy , due to their rarity they should not be overlooked or archived in some obscure corner of the mind . 
sensibilit , specificit , valore predittivo positivo e negativo per lus sono stati rispettivamente 70 , 2% , 59 , 2% , 74 , 7% e 53 , 7% ; per la ceus sono stati 96 , 4% , 98% , 98 , 8% e 94 , 1% . 
la metodica riconosce il sanguinamento attivo e le lesioni vascolari , evita lesposizione a radiazioni ionizzanti ed utile nel monitoraggio dei pazienti con trattamento conservativo . keywords ultrasonography contrast agent abdominal trauma parole chiave ecografia mezzo di contrasto trauma addominale radiol med ( 2009 ) 114 : 10801093 introduction introduzione 1081 trauma represents the most frequent cause of death in the population younger than 40 years [ 1 ] , and there is evidence that a prompt diagnosis can prevent severe consequences for the patient . 
because of this , in europe and in many centres in the usa , ultrasonography ( us ) , a modality that can be performed at the bedside without suspending first - aid procedures , has become the first - line investigation in evaluating abdominal trauma [ 510 ]  . 
emergency us performed to detect haemoperitoneum is termed focused assessment with sonography for trauma ( fast ) and has 81%94% sensitivity , 88%100% specificity and 86%98% diagnostic accuracy [ 1116 ]  . 
whereas it is generally recognised that us has a key role in unstable patients [ 20 ] , its use in stable patients remains controversial owing to its inability to exclude abdominal organ lesions , even in the presence of negative findings , with the result that patients need to undergo contrast - enhanced computed tomography ( ct )  . 
 contrast - enhanced us ( ceus ) relies on the use of contrast agents containing microbubbles filled with a gas other than air , which resonate at a low acoustic pressure emitting a specific signal . 
these real - time images allow identification of any solid organ lesion in a manner similar to ct but with the advantage of continuous exploration . initially used in the study of focal hepatic lesions , ceus was later successfully used to investigate a variety of conditions in other anatomical regions . 
one application is evaluating patients with blunt abdominal trauma , a field in which its success in recognising traumatic solid organ lesions has suggested its use in the triage of abdominal trauma [ 2125 ]  . the aim of this paper is to present our experience with the use of ceus in patients with blunt abdominal trauma and to assess its impact on clinical practice in the emergency department . materials and methods the study was conducted on a cohort of patients with blunt abdominal trauma in a university hospital featuring a level ii emergency department but with no trauma centre . between 2004 and 2008 , 1 , 462 patients underwent fast examination according to a shared protocol to detect il trauma costituisce la causa pi frequente di morte nella popolazione di et inferiore ai 40 anni [ 1 ]  . 
per tale motivo , in europa e in molti centri degli usa , lecografia ( us ) , eseguibile immediatamente al letto del paziente senza interrompere le manovre di soccorso , costituisce la metodica di primo approccio nella valutazione del trauma addominale [ 510 ]  . 
lus in urgenza , finalizzata alla ricerca dellemoperitoneo , chiamata focused assessment with sonography for trauma ( fast ) e ha una sensibilit dell81%94% , una specificit dell88%100% e unaccuratezza diagnostica dell86%98% [ 1116 ]  . 
il suo limite pi importante rappresentato dalla scarsa sensibilit , che varia tra il 41% e il 44% [ 1719 ] , nel riconoscimento diretto delle lesioni degli organi solidi delladdome . mentre lus ha un ruolo sicuramente riconosciuto nel paziente instabile [ 20 ] , la sua applicazione resta a tuttoggi pi discutibile nel paziente stabile , nel quale anche se negativa non in grado di escludere la presenza di lesioni degli organi addominali e quindi non permette di evitare lesecuzione di una tc con mezzo di contrasto ( mdc )  . lecografia con mezzo di contrasto , definita ceus ( dallinglese contrast - enhanced ultrasononography ) , si basa sullimpiego di mezzi di contrasto costituiti da microbolle ripiene di un gas diverso dallaria capaci di risuonare a bassa pressione acustica e di emettere un segnale specifico . 
con la tecnica a basso indice meccanico possibile ottenere in real time immagini quasi esclusivamente dipendenti dal segnale delle microbolle , identificando le eventuali lesioni degli organi solidi , in un modo del tutto simile a quello della tc , ma con il vantaggio dellesplorazione continua . 
tra queste la valutazione dei pazienti con trauma chiuso delladdome , campo in cui si dimostrata molto efficace nel riconoscimento delle lesioni traumatiche degli organi solidi al punto da venire proposta nel triage del trauma addominale [ 2125 ]  . 
 lo scopo del presente studio riportare la nostra esperienza nellimpiego della ceus nel paziente con trauma chiuso delladdome e di valutarne leffetto sulla pratica clinica in emergenza . 1082 radiol med ( 2009 ) 114 : 10801093 table 1 causes of traumatic lesion in 1462 patients including in the study materiali e metodi cause n . 
of this cohort , 133 consecutive patients ( 99 male and 34 female , mean age 40.2 years ) with a suspicion of abdominal lesion justified by accident dynamics ( direct impact , deceleration impact or impact with blunt object with a small surface ) or positive fast for free abdominal fluid and in stable haemodynamic conditions ( pulse pressure 90 mmhg , heart rate < 100 bpm , respiratory rate < 20 respirations per minute ) underwent ceus followed by contrast - enhanced ct . us scans were performed by experienced physicians ( radiologists or emergency physicians ) , whereas ct was performed by a second radiologist within 1 h after the us and ceus exams . 
the study was approved by the hospital ethics committee ( clinical study n1 / 2004 / o )  . patients were informed of the type of examination and study protocol , and consent was obtained from either the patients , or from their relatives in the case of minors . ultrasonography and contrast - enhanced ultrasonography examinations were performed with an atl 5000 hdi scanner , version 10.4 equipped with a 2 - 5 - mhz convexarray probe with a dedicated low mechanical index software for second - generation contrast agents . 
us was performed to search for haemoperitoneum with scans of the right upper quadrant , including the hepatorenal fossa and pleural space ; of the left upper quadrant , including the perisplenic region and pleural space ; of the epigastrium , including the pericardiac space ; of the right and left paracolic gutters ; and of the pelvis . solid abdominal organs were assessed for the presence of lesions in all patients . 
us was considered positive in the lo studio stato condotto in un ospedale universitario sede di un dipartimento di emergenza di 2 livello , ma non trauma center , su una coorte di pazienti con trauma chiuso delladdome . 
nel periodo 2004 - 2008 sono stati valutati 1462 pazienti , che sono stati sottoposti , secondo un protocollo condiviso , ad esame fast nel corso della valutazione primaria allinterno della sala urgenze . 
di questa coorte , 133 pazienti consecutivi ( 99 maschi e 34 femmine , di et media di 40 , 2 anni ) , con sospetta lesione addominale per la dinamica dellevento ( impatto diretto , da decelerazione o da corpo contundente di piccola superficie ) o con fast positiva per versamento addominale , in condizioni di stabilit emodinamica ( pressione sistolica 90 mmhg , frequenza cardiaca < 100 bpm , frequenza respiratoria > 20 respirazioni al minuto ) , sono stati sottoposti a ceus seguita da tc con mdc . gli esami ecografici sono stati eseguiti da medici dedicati ( radiologo o medico di pronto soccorso ) , mentre la tc stata eseguita da un secondo radiologo entro 1 ora dallesame us e ceus . 
i pazienti sono stati informati del tipo di indagine e del protocollo di studio , e il consenso stato ottenuto dal paziente o dai familiari presenti in caso di minori . ecografia ed ecografia con mezzo di contrasto gli esami sono stati eseguiti con un ecografo atl 5000 hdi , versione 10.4 , equipaggiato con sonda convex da 25 mhz e dotato di specifico software a basso indice meccanico per limpiego dei mezzi di contrasto di 2 generazione . stato utilizzato limaging armonico per massimizzare le performance diagnostiche dellus basale . 
lus stata effettuata per la ricerca dellemoperitoneo con scansioni del quadrante superiore destro , inclusa la fossa epatorenale e lo spazio pleurico ; del quadrante superiore sinistro , inclusa la regione perisplenica e lo spazio pleurico ; dellepigastrio , incluso lo spazio pericardico ; delle docce paracoliche destra e sinistra ; della pelvi . 
lus stata considerata positiva in presenza di versamento peritoneale o alterazione dellecostruttura parenchimale compatibile con una lesione traumatica . la ceus stata eseguita immediatamente dopo lus basale secondo un protocollo standardizzato nel quale lagente ecocontrastografico stato iniettato in due dosi di 2 , 4 ml per studiare gli organi solidi del quadrante superiore destro ( rene destro , fegato e pancreas ) e superiore sinistro ( rene sinistro e milza )  . 
tale procedura consente la radiol med ( 2009 ) 114 : 10801093 1083 presence of peritoneal effusion or changes to parenchymal echostructure consistent with a traumatic lesion . ceus was performed immediately after baseline us . 
a standard protocol was used consisting of the injection of two 2.4 - ml doses of contrast agent to study the solid organs in the right upper quadrant ( right kidney , liver and pancreas ) and left upper quadrant ( left kidney and spleen )  . 
this procedure allows evaluation of solid abdominal organs during the most favourable vascular phase for the detection of traumatic lesions . on us , lesions were recognised as a hypoor hyperechoic alteration within the organ being studied . 
passage of microbubbles outside a lacerated organ was defined as active bleeding . ct was performed within 1 h after ceus with and without nonionic contrast agent during the venous phase . 
in the presence of collections , a late - phase study at 315 min was performed to identify any active bleeding or urinoma . examination results were stored in digital form and subsequently assessed by two radiologists who graded the lesions on the basis of the organ injury scale of the american association for the surgery of trauma ( aast ) [ 26 ]  . statistical analysis for the purposes of statistical analysis , us and ceus were considered positive in the presence of a parenchymal change related to a traumatic lesion . 
the positive and negative lr were subsequently entered into the fagan nomogram in order to calculate , starting from the pre - test probability ( 63.2% prevalence in this study ) , the positive and negative post - test probability of disease . us and ceus exams were evaluated by two operators blinded to the final result , who defined the concordance of the two techniques compared with the standard of reference ( ct )  . valutazione degli organi solidi delladdome nella fase vascolare pi favorevole per lidentificazione delle possibili lesioni traumatiche . 
alla ceus la lesione traumatica stata descritta come un difetto di perfusione nel contesto dellorgano caratterizzato da una ipoecogenicit con limiti sfumati o ben definiti , con o senza interruzione del profilo dellorgano . 
in presenza di raccolte stato effettuato uno studio in fase tardiva , a 315 minuti , per evidenziare un eventuale sanguinamento attivo o un urinoma . gli esami , archiviati su supporto digitale , sono stati successivamente valutati da due radiologi e le lesioni graduate sulla base dellorgan injury scale of the american association for the surgery of trauma ( aast ) [ 26 ]  . analisi statistica per lanalisi statistica , us e ceus sono state considerate positive in presenza di unalterazione parenchimale riferibile ad una lesione traumatica . 
i valori di sensibilit , specificit , valore predittivo positivo e negativo , il rapporto di verosimiglianza ( likehood ratio , lr ) positivo e negativo , ed il numero necessario per la diagnosi ( number needed to diagnosis , nnd , cio il numero medio di pazienti da sottoporre al test in esame per diagnosticare correttamente la patologia in oggetto ) , tutti con il loro intervallo di confidenza al 95% ( ci 95% ) per us e ceus rispetto alla tc sono stati calcolati utilizzando i seguenti calcolatori on line [ 27 , 28 ]  . 
 gli esami us e ceus sono stati successivamente valutati da due operatori ciechi sul risultato finale , che hanno definito la concordanza tra le due metodiche rispetto alla metodica di riferimento ( tc )  . 
us identified free fluid or altered parenchymal echostructure in 59 of the 84 patients with lesions on ct and free fluid in 20 of the 49 patients without lesions on ct . 
la diagnosi definitiva di sede della lesione traumatica stata di frattura splenica ( 48 pazienti ) , epatica ( 21 pazienti ) , rene o surrene ( 13 pazienti ) e pancreas ( 2 pazienti ) ( tabella 2 )  . 
lus ha identificato versamento libero o alterazione dellecostruttura parenchimale in 59 degli 84 pazienti con lesioni alla tc , e versamento libero in 20 dei 49 pazienti senza alterazioni post1084 site fig . 
7 and 8 , respectively . discussion us is widely utilised in blunt abdominal trauma mainly as a result of its ease of use , which allows examinations to be il numero di pazienti veri positivi , falsi positivi , veri negativi e falsi negativi per us e ceus rispetto alla tc con mdc sono riportati rispettivamente in tabella 3 e tabella 4 . i valori di sensibilit , specificit , valore predittivo positivo e negativo per lus sono stati rispettivamente 70 , 2% , 59 , 2% , 74 , 7% e 53 , 7% ; per la ceus sono stati 96 , 4% , 98% , 98 , 8% e 94 , 1% . 
la ceus ha dimostrato un numero significativamente pi elevato di lesioni correttamente identificate rispetto alla us , fornendo una stretta correlazione tra le dimensioni delle lesioni misurate con la gradazione ottenuta con la tc ( tabella 6 )  . 
 in stable patients , on the other hand , organ damage needs to be precisely evaluated to initiate the most appropriate treatment , whether conservative , surgical or interventional [ 29 ]  . 
the use of contrast material significant improves the diagnostic discussione lus ampiamente utilizzata nel trauma chiuso delladdome soprattutto per le sue caratteristiche di maneggevolezza che ne consente lutilizzo allinterno dellarea di emergenza , al letto del paziente , senza interrompere le manovre di rianimazione . 
la scansione ecocontrastografica rileva un focolaio lacerativo del terzo medio della milza con fuoriuscita ( freccia ) di mdc ecografico dallorgano . radiol med ( 2009 ) 114 : 10801093 fig . 
c la tc dimostra la presenza di una piccola lesione del labbro mediale del rene ( freccia ) con raccolta perirenale . con il trattamento pi idoneo , conservativo , chirurgico o di radiologia interventistica [ 29 ]  . 
limpiego del mezzo di contrasto ecografico determina un significativo miglioramento delle performance diagnostiche dellus , che raggiunge una sensibilit quasi uguale a quella della tc . la societ europea di ultrasuoni in medicina e biologia ( efsumb ) ha recentemente riportato limpiego della ceus nelle sue linee guida in caso di tc non disponibile o contro - indicata per precedenti reazioni allergiche al mdc iodato ; tc non conclusiva per artefatti ; monitoraggio di lesioni traumatiche note ; trauma chiuso minore , soprattutto in et pediatrica [ 30 ]  . nella nostra esperienza , la ceus ha consentito di rilevare un maggior numero di lesioni rispetto allus e di definirne le dimensioni , i rapporti con la capsula e le eventuali situazioni di sanguinamento attivo . 
infatti , nella gestione del paziente traumatizzato , tale reperto , associato alla presenza di emoperitoneo , suggerisce unelevata probabilit di fallimento del trattamento conservativo ed orienta ad un rapido trattamento chirurgico o di embolizzazione [ 31 ]  . la mancanza di contrast - enhancement in parte o in tutto lorgano studiato , consente di porre la diagnosi di lesione vascolare . 
with improved detection and precise localisation of organ injuries , the emergency surgeon can receive in real time accurate lesion grading in accordance with the reference standard ct . also in our experience , we found the possibility of identifying areas of active bleeding as contrast extravasation from the lesion to be particularly useful . 
in fact , in managing trauma patients , this finding if associated with the presence of haemoperitoneum strongly predicts the failure of conservative treatment , indicating a need for a prompt surgical or embolisation procedure [ 31 ]  . the lack of contrast enhancement in part of or in the entire organ allowed us to establish a diagnosis of vascular lesion . 
in one of our cases , lack of perfusion of the left kidney in a young patient with a complex splenic laceration led to detection of an injury to the renal vascular pedicle , which was treated with simultaneous nephrectomy and splenectomy . in patients undergoing conservative treatment , ceus allows effective monitoring , as it does not require transferring the patient to the ct room and avoids any exposure to radiation and iodinated contrast agents . 
in our department , ceus has become the most frequent procedure performed in children , a setting in which it has recorded its major sione del rene sinistro , in un giovane traumatizzato con frattura complessa di milza , ha permesso di identificare lavulsione del peduncolo vascolare del rene , trattata con nefrectomia simultaneamente alla splenectomia . 
 nel paziente avviato al trattamento conservativo , la ceus rappresenta un efficace sistema di monitoraggio , evitando lo spostamento del paziente verso la sala tc e lesposizione a radiazioni ionizzanti ed al mdc iodato . tale procedura rappresenta , nella nostra realt , la maggioranza dei trattamenti in ambito pediatrico e proprio in tale ambito la metodica ha un importante impatto sulla gestione del paziente traumatizzato [ 31 , 32 ]  . 
questo aspetto pu essere di particolare impatto soprattutto nelle donne in et fertile , nelle quali frequente il riscontro di una piccola falda fluida nello scavo di douglas , il cui significato pu essere ambiguo . 
uno studio di brown et al , a questo proposito , concludeva che la presenza di una raccolta fluida isolata nel cul - di - sac in donne in et fertile poteva essere considerata fisiologica e che un follow - up clinico poteva essere sufficiente [ 33 ]  . 
probabilit pre - test e post - test di us ( linea rossa ) e ceus ( linea blu ) per pazienti veri negativi . impact on trauma evaluation [ 31 , 32 ]  . 
on this subject concluded that fluid collections isolated to the pouch of douglas in women of reproductive age could be considered physiological and requiring clinical follow - up only [ 33 ]  . 
in clinical practice , the finding of small peritoneal fluid collections in trauma patients is an indication for an immediate ct scan without and with contrast agent to exclude organ lesions . 
ceus allows for a rapid assessment of solid organs , potentially avoiding the need for ct and thus reducing exposure to radiation and iodinated contrast agents . ceus errors included one false splenic lesion and failure to detect one renal lesion and one adrenal haematoma . 
the false splenic lesion on ceus was correctly evaluated as an in un paziente traumatizzato determina limmediata esecuzione di una tc senza e con mdc per escludere eventuali lesioni dorgano . 
la ceus permette di valutare rapidamente gli organi solidi evitando potenzialmente lesecuzione della tc , e quindi riducendo lesposizione a radiazioni ionizzanti e al mdc iodato . gli errori della ceus sono stati rappresentati da 1 falsa lesione splenica e dalla mancata identificazione di 1 lesione renale e di un ematoma del surrene . 
i 2 pazienti con lesione del rene e del surrene , identificati solo con tc , sono stati entrambi casi di lesioni non chirurgiche , trattate conserva1092 radiol med ( 2009 ) 114 : 10801093 ischaemic lesion on ct . 
the two patients with renal and adrenal lesions , identified by ct only , were both cases of nonsurgical lesions , the missed detection of which did not modify patient outcome . clearly , ceus is burdened by the typical limitations of sonography , such as the less panoramic view in comparison with ct and the difficulty in examining large patients , even though the new us scanners accommodate this class of patients as well . as our study was conducted in a single centre , it considered a relatively small number of patients compared with the large series in the literature regarding us and ct . 
a multicentre study could allow us to investigate a statistically significant number of patients and better define the real advantages of this new technique . conclusions our prospective study , in line with other experiences reported in the literature , has confirmed that ceus is an accurate diagnostic tool that may find an indication for evaluating stable patients with abdominal trauma , in that it provides performance values approaching those of ct . 
in particular , we believe ceus to be useful in patients with minor abdominal trauma and in the presence of free peritoneal fluid , as it enables identification of the injured organ and definition of the severity of the injury without causing delays in the diagnostic workup . 
 conclusioni il nostro studio prospettico , in linea con altre esperienze della letteratura , ha confermato che la ceus uno strumento diagnostico accurato e che pu trovare indicazione trauma nella valutazione del paziente stabile con delladdome , in quanto consente di ottenere valori di performance che si avvicinano molto a quelli della tc . 
in particolare riteniamo che limpiego del mdc in ecografia sia particolarmente utile nel paziente con trauma minore delladdome , quando sia presente un versamento peritoneale , per identificare lorgano sede di lesione e graduare lentit del danno senza alcun ritardo nelliter diagnostico . la ceus , inoltre , dovrebbe essere eseguita anche in assenza di versamento libero , quando vi siano alterazioni degli esami di laboratorio ( ad esempio aumento delle transaminasi o delle amilasi , microematuria , anemizzazione ) o fratture ossee che pongano il sospetto di lesioni intra - addominali ( ultime coste , apofisi trasverse delle vertebre lombari ) e in tutti i casi in cui la dinamica del trauma sia definibile come maggiore [ 20 ]  . 
cova unit clinico operativa di radiologia , universit degli studi di trieste , ospedale di cattinara , strada di fiume 447 , 34149 trieste , italy correspondence to : s . 
three readers independently reviewed the axial ct scans and multiplanar oblique and twoand three - dimensional reconstructions ( maximum intensity projection and volume rendering ) images to assess degree of stenosis according to four categories : 1 ( 0%49% stenosis ) ; 2 ( 50%99% stenosis ) ; 3 ( occluded ) ; 4 ( not evaluable )  . 
in 53 patients , 1 , 440 segments were evaluated ( infrarenal aorta and 16 arterial segments for each leg ; 42 bilateral studies , 11 unilateral studies )  . 
sixty - four - row multislice ct proved to be helpful in detecting haemodynamically significant lesions in peripheral arterial occlusive disease and improved the results obtained with 4and 16 - slice multidetector ct . 
tre radiologi hanno valutato le immagini assiali e le ricostruzioni multiplanari oblique e bi - tridimensionali ( maximum intensity projection [ mip ] , volume rendering [ vr ] ) , e hanno classificato le lesioni in 4 gradi : 1 = stenosi 0%49% , 2 = stenosi 50%99% , 3 = occlusione , 4 = non valutabile . 
langio - tc a 64 strati si dimostrata affidabile nella valutazione delle lesioni steno - ostruttive dellaoai ed stata in grado di migliorare ulteriormente i risultati ottenuti con tc a 416 strati . 
inoltre , grazie allelevata risoluzione spaziale e ad una tecnica rigorosa , non sono stati rilevate variazioni nei dati ottenuti al di sotto del ginocchio , superando un limite delle tc delle generazioni precedenti . 1116 radiol med ( 2009 ) 114 : 11151129 keywords 64 - row multislice computed tomography digital angiography peripheral arterial occlusive disease parole chiave tomografia computerizzata a 64 strati angiografia digitale arteriopatia ostruttiva arti inferiori introduction introduzione lifestyles occlusive arterial disease ( oad ) of the lower extremities is a disorder of leg arteries associated with a clinical situation resulting from altered tissue perfusion , which may evolve into chronic or acute ischaemia . 
in 95% of cases , it is caused by atherosclerosis [ 1 ] and manifests as a diffuse disease with constant involvement of other regions , such as the coronary and carotid arteries [ 2 ]  . 
in the remaining 5% of cases , oad has inflammatory causes ( buergers disease , takayasus arteritis , hortons arteritis ) or iatrogenic causes ( ergotamine , beta - blockers , etc )  . 
the main risk factors of oad of the lower extremities are the same as those identified for oad in other vascular districts , namely , age , smoking , diabetes , hypertension , hyperlipidaemia , hyperhomocysteinaemia , and male sex [ 35 ]  . for many years , angiography with arterial catheterisation , initially with conventional acquisition then with digital subtraction technique , was the mainstay diagnostic technique for planning surgical and endovascular treatment . however , it is an invasive technique that requires hospitalisation and is burdened by a small risk of immediate ( dissection , mobilisation of atheromatous plaques or microemboli ) and late ( pseudoaneurysms ) complications [ 6 , 7 ]  . 
over the last few years , other less invasive techniques have appeared alongside intra - arterial catheterisation angiography , such as colour - doppler ultrasound ( us ) and magnetic resonance ( mr ) imaging or computed tomography ( ct ) angiography . ct angiography performed with multislice scanners and a large number of detectors is a rapid , noninvasive and welltolerated technique that is less costly than digital angiography as a result of less utilisation of personnel and supplies , the outpatient nature of the procedure and smaller amount of contrast material required [ 8 ]  . the purpose of this study was to evaluate the diagnostic capabilities of 64 - slice ct angiography in the evaluation of haemodynamically significant lesions in lower - extremity arteries using digital subtraction angiography ( dsa ) as the gold standard . larteriopatia ostruttiva ( ao ) degli arti inferiori una patologia che colpisce le arterie degli arti inferiori , associata ad una condizione clinica conseguente allalterata perfusione ematica dei tessuti con evoluzione verso unischemia cronica e talvolta acuta . 
nel 95% dei casi si riconosce come causa laterosclerosi [ 1 ] e si manifesta come una malattia diffusa che coinvolge costantemente anche altri distretti quali le arterie coronarie e carotidi [ 2 ]  . 
i principali fattori di rischio di ao degli arti inferiori sono gli stessi riconosciuti per altre aree vascolari , vale a dire et , fumo , diabete , ipertensione , iperlipidemia , iperomocisteinemia , sesso maschile [ 35 ]  . per molti anni langiografia mediante cateterismo arterioso , dapprima con tecnica di acquisizione convenzionale e quindi mediante sottrazione digitale , ha rappresentato lindagine diagnostica fondamentale al fine della programmazione del trattamento terapeutico sia chirurgico che endovascolare . 
tuttavia tale tecnica risulta essere invasiva e necessita il ricovero del paziente e , seppur in basse percentuali , gravata da alcune complicanze sia immediate ( dissezione , mobilizzazione di placche ateromasiche o di microemboli ) che tardive ( pseudoaneurismi ) [ 6 , 7 ]  . negli ultimi anni , allangiografia mediante cateterismo arterioso si sono affiancate tecniche sicuramente meno invasive quali leco - color doppler e langiografia mediante risonanza magnetica ( rm ) o mediante tomografia computerizzata ( tc )  . 
questultima grazie alla tecnologia multistrato ad elevato numero di detettori , rappresenta una tecnica rapida e priva di invasivit , con unottima compliance da parte dei pazienti ed una significativa riduzione dei costi rispetto allangiografia digitale per minor impegno di personale e consumo di materiali , per il radiol med ( 2009 ) 114 : 11151129 materials and methods we retrospectively reviewed 53 patients who underwent 64slice ct angiography of the lower extremities followed by dsa within 36 days ( range 1966 days ) at our institute between january 2006 and april 2008 . 
indications for ct angiography included specific clinical symptoms ( intermittent claudication , leg pain , trophic changes to the skin , nail dystrophy , etc ) and findings on colour - doppler us . 
all patients gave their informed consent to both procedures . ct angiography ct angiography was performed with a ct scanner featuring 64 rows of solid - state detectors ( toshiba aquilion , toshiba medical systems , tokyo , japan ) , a toshiba megacool tube and a 60 - kw high - frequency xray generator , multiple kilovolt settings ( 80100120135 kv ) , and milliampere range of 50500 ma with steps of 10 ma [ 9 ]  . 
contrast material was injected in an antecubital vein via an 18 - 20 - gauge needle cannula connected to an automated power injector ( stellant sct 211 , medrad , indianola , ia , usa )  . 
the scan protocol included a double orthogonal scout view from the diaphragm to the feet , followed by a 30 - ml saline test injection to establish the venous access and injection of a single 80to 120 - ml contrast bolus , depending on patients height and build , at a flow rate of 4 ml / s . 
the entire scan volume was divided into two parts to facilitate postprocessing : the first , from the renal arteries to the knee , was reconstructed with a 40 - cm field of view ( fov ) , 512512 pixel matrix , 1 - mm slice thickness at an interval of 0.5 mm ; the second , from the knee to the foot , was 1117 carattere ambulatoriale della prestazione e per la minor quantit di mezzo di contrasto ( mdc ) necessaria per lesecuzione dellesame tc [ 8 ]  . lo scopo di questo studio quello di valutare le reali capacit diagnostiche dellangio - tc a 64 strati nella valutazione delle lesioni emodinamicamente significative delle arterie degli arti inferiori , utilizzando come gold standard langiografia digitale ( ad ) a sottrazione dimmagine . materiali e metodi nel nostro istituto , nel periodo tra gennaio 2006 e aprile 2008 , sono stati valutati retrospettivamente 53 pazienti , sottoposti , in seguito a sintomatologia clinica specifica ( claudicatio intermittens , sintomatologia dolorosa agli arti inferiori , alterazioni del trofismo cutaneo , distrofie ungueali , ecc . ) e sulla base di rilievi eco - color doppler , ad angio - tc a 64 strati delle arterie degli arti inferiori e successivamente , in un arco di tempo medio di 36 giorni ( min , 19 giorni ; max , 66 giorni ) , ad ad dello stesso distretto . 
tutti i pazienti hanno fornito un consenso informato ad entrambe le procedure . angio - tc gli esami di angio - tc sono stati eseguiti utilizzando un tomografo computerizzato a 64 strati di detettori allo stato solido ( toshiba aquilion , toshiba medical systems , tokyo , giappone ) composto da un tubo toshiba megacool e da un generatore di raggi x ad alta frequenza con una potenza di 60 kw , un range di kv disponibili tra 80 e 135 ( 80 / 100 / 120 / 135 ) ed un range di ma tra 50 e 500 con step di 10 ma [ 9 ]  . 
i parametri per lesecuzione dellangio - tc degli arti inferiori sono stati : uno spessore nominale di strato pari a 0 , 5 mm per 64 strati , un movimento del tavolo di 26 , 5 mm per rotazione , un pitch factor di 0 , 828 ( helical pitch : 53 ) ed un tempo di rotazione di 0 , 4 secondi ; sono stati utilizzati 120 kv e 230 ma con esposizione modulata per ridurre la dose ( sure exposure : standard )  . 
il paziente stato posto in posizione supina , con le caviglie dentro al poggiatesta e ginocchia e bacino in posizione neutrale , al fine di limitare gli artefatti da movimento durante lacquisizione delle immagini . 
il mdc stato iniettato attraverso unagocannula da 1820 g inserita in una vena a livello 1118 radiol med ( 2009 ) 114 : 11151129 reconstructed with 25 - cm fov , 512512 matrix , and 0.5mm slice thickness at an interval of 0.3 mm to minimise voxel size and maintain maximal spatial resolution in the most distal sectors where the arteries have smaller diameter . 
to optimise scan timing , we used the sure - start function , with a low - dose pulsed scan at the level of the infrarenal abdominal aorta 10 s after the beginning of the injection and placement of a region of interest ( roi ) over the aorta lumen to calculate the density increase inside the aorta . 
adequate opacification of the entire vascular axis of interest was thus achieved in all cases . software ( vitrea , vital images were automatically transferred to a workstation with dedicated images , minnetonka , mn , usa )  . 
in the presence of significant stenoses or heavily calcified arteries , further reconstructions such as curved multiplanar ( cmpr ) and vessel probe ( vp ) reconstructions were performed to improve depiction of the course of the diseased arteries and provide panoramic support for the information obtained from axial images . 
under angiographic guidance , a straight or pigtail 5 - french diagnostic catheter was placed in the infrarenal abdominal aorta . in the humeral approach , a hydrophilic guidewire and pigtail catheter were preferred . 
image acquisition was carried out with the successive fields technique : five separate adjacent fields were acquired from the aortoiliac to the tibioperoneal area , with a slight overlap to ensure inclusion of all segments of the arterial tree . 
il protocollo di acquisizione prevede , dopo lesecuzione di una doppia scout - view ortogonale dal diaframma ai piedi , uniniezione test di 30 ml di soluzione fisiologica per verificare laccesso venoso , e liniezione di un unico bolo di mdc pari a 80120 ml , a seconda dellaltezza e della costituzione fisica del paziente , ad un flusso di 4 ml al secondo ; liniezione del mdc seguita da un piccolo bolo ( 30 ml ) di soluzione fisiologica sempre ad un flusso di 4 ml / s . 
lintero volume stato suddiviso in due parti per agevolare il postprocessing : la prima , dalle arterie renali al ginocchio , ricostruita con campo di vista ( fov ) pari a 40 cm , matrice di 512512 pixel , spessore di strato pari 1 mm ad un intervallo di 0 , 5 mm ; la seconda , dal ginocchio al piede , ricostruita con fov di 25 cm , matrice di 512512 e spessore di strato pari a 0 , 5 mm ad un intervallo di 0 , 3 mm , per ridurre al minimo le dimensioni del voxel al fine di mantenere la massima risoluzione spaziale nei settori pi distali dove i vasi arteriosi presentano un calibro minore . 
to improve opacification of the leg arteries , some cases required injection of 40 ml instead of 30 ml of contrast agent during the acquisition of the most distal field . 
tutti i pazienti sono stati sottoposti a un cateterismo arterioso del tratto distale dellaorta addominale dopo puntura dellarteria femorale comune o dellarteria omerale con tecnica di seldinger : attraverso la guida angiografica stato posizionato un catetere diagnostico da 5 french a radiol med ( 2009 ) 114 : 11151129 1120 fig . 
ct angiography and dsa images were first examined separately by three radiologists with different levels of experience with angiography ( sc , fpm , ap ) and then , in the case of discordant interpretations , they were discussed jointly to reach a consensus . 
the arteries were evaluated by region and rated according to a previously reported scoring system [ 10 ] : 1 punta dritta o pig - tail a livello del tratto sottorenale dellaorta addominale . 
lacquisizione delle immagini stata poi effettuata con la modalit a campi successivi : sono stati acquisiti 5 campi distinti contigui a partire dal distretto aorto - iliaco sino a quello tibio - peroneale , in radiol med ( 2009 ) 114 : 11151129 1121 table 1 scoring system grade degree of stenosis tabella 1 punteggio di valutazione grado grado di stenosi < 50% 50%99% occlusion not evaluable < 50% 50%99% occlusione non valutabile minima parte sovrapposti per avere la certezza di non escludere dallo studio nessun tratto dellalbero arterioso in esame . 
per ciascun campo stato iniettato un bolo di mdc non ionico con concentrazione di iodio compresa tra 320 e 370 mgi / ml ( visipaque 320 , amersham health , princeton , new jersey , usa ; ultravist 370 , bayer - schering pharma ag , berlin , germania ) mediante iniettore automatico ( medrad mark v , indianola , usa ) di 2530 ml con un flusso di 15 ml / s . 
per ottenere un maggiore tenore di opacizzazione delle arterie della gamba , talvolta stato necessario iniettare 40 ml di mdc , anzich 30 ml , durante lacquisizione del campo pi distale . 
le immagini dellangio - tc e dellangiografia digitale sono state esaminate dapprima separatamente da tre radiologi con esperienza angiografica di diverso livello ( sc , fpm , ap ) e poi , nel caso di eventuali discordanze , ridiscusse insieme fino ad ottenere una concordanza di giudizio . 
le arterie sono state valutate distretto per distretto , assegnando uno score ricavato dalla letteratura [ 10 ] , che prevede lassegnazione di un punteggio pari a 1 se larteria risulta sana o con una stenosi minore del 50% , a 2 se presenta una stenosi tra il 50% ed il 99% , a 3 se occlusa , a 4 se non valutabile ( tabella 1 )  . 
3 la suddivisione dellalbero arterioso oggetto dello studio . for healthy arteries or arteries with < 50% stenosis , 2 for 50%99% stenosis , 3 for occlusion , and 4 for not evaluable arteries ( table 1 )  . 
in the event of multiple lesions in the same region , the lesion with the highest degree of stenosis was considered . separate evaluations were made for lesions of the following arteries : common iliac ( ci ) , internal iliac ( ii ) , external iliac ( ei ) , common femoral ( cf ) , deep femoral ( df ) , proximal ( sf1 ) and distal superficial femoral ( sf2 ) , supra - articular ( p1 ) and subarticular popliteal ( p2 ) , proximal ( at1 ) and distal ( at2 ) anterior tibial , tibioperoneal ( tr ) , proximal ( pe1 ) and distal peroneal ( pe2 ) , and proximal ( tp1 ) and distal ( tp2 ) posterior tibial . 
sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) , diagnostic accuracy and concordance in degree of stenosis between the two modalities were calculated for all segments , taking dsa as the gold standard [ 8 , 10 , 11 ]  . 
patients with a score of 1 were considered negative ( healthy ) , whereas those with scores of 2 and 3 that is , 1122 table 2 summary of statistical results dsa + dsa 64 - slice ct + 64 - slice ct total 1 , 017 1 , 048 tabella 2 riepilogo statistico dei nostri risultati atcms64 + atcms64 totale 1017 1048 total 1 , 028 1 , 440 totale 1028 1440 dsa , digital subtraction angiography ; ct , computed tomography atcms64 , angio - tomografia computerizzata a 64 strati ; ad , angiografia digitale radiol med ( 2009 ) 114 : 11151129 sullarteria iliaca comune ( ic ) , iliaca interna ( ii ) , iliaca esterna ( ie ) , femorale comune ( fc ) , femorale profonda ( fp ) , femorale superficiale prossimale ( fs1 ) e distale ( fs2 ) , poplitea sopra ( p1 ) e sottoarticolare ( p2 ) , tibiale anteriore prossimale ( ta1 ) e distale ( ta2 ) , tronco tibioperoneale ( tr ) , peroneale prossimale ( pe1 ) e distale ( pe2 ) e tibiale posteriore prossimale ( tp1 ) e distale ( tp2 )  . 
sul totale dei segmenti sono stati calcolati sensibilit , specificit , valore predittivo positivo ( vpp ) , valore predittivo negativo ( vpn ) , accuratezza diagnostica e la concordanza del grado di stenosi tra le due metodiche , ponendo come gold standard langiografia digitale [ 8 , 10 , 11 ]  . 
sono stati considerati negativi al test ( sani ) i pazienti con uno score di 1 e positivi al test ( malati ) i pazienti con uno score 2 e 3 ovvero i pazienti che presentavano un quadro di stenosi superiore al 50% oppure unocclusione completa del vaso [ 10 , 12 ]  . with > 50% stenosis or complete occlusion were considered positive ( diseased ) [ 10 , 12 ]  . risultati results the total number of segments considered for which comparisons were possible in the 53 patients was 1 , 440 ( table 2 )  . we excluded from the analysis segments that were not assessed with both methods , that is , a total of 309 segments in 11 patients for whom dsa was performed on one extremity only . 
with regard to overand underestimation , there were 31 cases ( 2.1% ) of significant overestimation with positive ct angiography ( > 50% stenosis or obstruction ) but negative dsa ( < 50% stenosis ) , 10 cases ( 0.7% ) of slight overestimation with occlusion on ct angiography but > 50% stenosis on dsa , 11 cases ( 0.8% ) of significant underestimation with negative ct angiography but positive dsa ( > 50% stenosis or occlusion ) , and 14 cases ( 1% ) of slight underestimation where ct angiography was positive for > 50% stenosis whereas dsa showed occlusion . 
concordance in the degree of stenosis was 95.4% ( table 3 )  . in addition , we analysed separately the data regarding the vascular segments below the knee , as the small size of the vessels and frequent calcifications make this area particularly critical . 
per quanto riguarda i casi di sovrae sottostime , sono stati evidenziati 31 casi ( 2 , 1% ) di sovrastima significativa con paziente positivo allangio - tc ( stenosi > 50% o ostruzione ) , ma negativo alla ad ( stenosi < 50% ) , 10 casi ( 0 , 7% ) di sovrastima lieve , con paziente occluso allangio - tc , ma con stenosi > 50% alla ad , 11 casi ( 0 , 8% ) di sottostima significativa , con paziente negativo allangio - tc , ma positivo alla ad ( stenosi > 50% o occlusione ) e 14 casi ( 1% ) di sottostima lieve con paziente positivo in angio - tc per una stenosi > 50% , in realt occluso alla ad . 
considerando i dati complessivi , stata riscontrata una sensibilit del 97 , 2% , specificit del 97 , 0% , vpp al 92 , 5% , vpn al 98 , 9% e accuratezza pari al 97 , 1% . 
la concordanza del grado di stenosi risultata pari a 95 , 4% ( tabella 3 )  . oltre ai dati complessivi , sono stati valutati separatamente i dati relativi ai segmenti dellalbero vascolare al di sotto del ginocchio , distretto questo particolarmente critico per le esigue dimensioni dei vasi e la frequente presenza di calcificazioni ; in questo caso il totale dei segmenti valutati corrisponde a 604 ( tabella 4 )  . 
in questo distretto sono stati riscontrati i valori di sensibilit pari a 97 , 5% , specificit 96 , 9% , vpp 95 , 5% , vpn 98 , 3% , accuratezza 97 , 2% , concordanza del grado di stenosi 93 , 5% ( tabella 5 )  . radiol med ( 2009 ) 114 : 11151129 1123 fig . 
sixty - four - slice ct angiography with its near - isotropic voxels produces very high - quality reconstructions in the three spatial planes , thereby modifying the diagnostic approach to a technique that formerly relied on evaluation of axial images only . multislice ct has overcome the problems of the early spiral ct devices ( limited craniocaudal coverage during a single contrast injection ) by enabling fast acquisition times , reduced contrast agent doses and increased spatial resolution , with the possibility of studying even the smallest vessels [ 18 , 19 ]  . 
recent studies report that the introduction of multislice ct scanners have increased the sensitivity and specificity of ct to levels very close to those of dsa , with the exception of small vessels [ 13 , 2022 ]  . 
the purpose of our study was to ascertain whether the latest - generation equipment can further improve sensitivity and specificity compared with the earlier 4and 16 - slice scanners , particularly in the study of small - calibre vessels . 
langio - tc a 64 strati grazie alla sostanziale isotropicit del voxel permette di ottenere ricostruzioni di qualit altissima nei tre piani dello spazio , modificando di fatto lapproccio diagnostico di tale metodica , un tempo legata alla sola valutazione delle immagini assiali . 
la tc multistrato ha ridimensionato le problematiche delle prime apparecchiature spirali ( limitata copertura cranio - caudale durante una singola iniezione del mdc ) , offrendo tempi di acquisizione brevi , una riduzione della dose di mdc ed un aumento della risoluzione spaziale che ha permesso lo studio anche dei vasi di minor calibro [ 18 , 19 ]  . 
le pubblicazioni pi recenti riportano che , con lintroduzione delle apparecchiature tc multistrato , la sensibilit e la specificit di questindagine risultano molto vicine a quella dellangiografia digitale , fatto salvo , anche in questo caso , per i vasi di calibro minore [ 13 , 2022 ]  . lobiettivo del nostro lavoro stato quello di verificare se le apparecchiature di ultima generazione siano in grado di apportare un ulteriore miglioramento dei valori di sensibiradiol med ( 2009 ) 114 : 11151129 1124 fig . 
the 3d mip ( a - c ) reformation apparently shows patency of the proximal and middle segments , of the peroneal ( arrows ) and of the left posterior tibial artery , respectively . 
the use of high - iodine - concentration contrast material has led to greater opacification of the arterial tree , with clear attenuation differences from the surrounding tissues , thus reducing postprocessing time [ 23 ]  . however , in our experience , postprocessing amounts to 20 min on average for each study for an experienced operator . the speed of the equipment significantly increases patient compliance , and images are virtually free from motion artefacts . 
langio - tc a 64 strati degli arti inferiori si rivelata , nel complesso , una tecnica di semplice esecuzione , rapida , con un tempo desame brevissimo , un tempo di preparazione del paziente molto contenuto , legato alla sola repertazione di un accesso venoso , ma con un notevole impegno di tempo per il post - processing . 
lutilizzo di mdc ad alta concentrazione di iodio ha permesso di ottenere una maggior opacizzazione dellalbero arterioso in modo da realizzare un netto distacco in termini di uh dai tessuti circostanti per ridurre il dispendio di tempo nel post - processing [ 23 ] , comunque quantificabile nella nostra esperienza in 20 minuti in media ad esame , per un operatore esperto . favorisce molto la compliance del paziente , risultando le immagini velocit dellapparecchiatura radiol med ( 2009 ) 114 : 11151129 1125 fig . 
b , d limmagine angiografica mostra una stenosi di modesta entit , inferiore al 50% , pertanto non significativa . overall analysis of our results indicates that in most cases ( 95.4% ) , there is excellent concordance between the results of 64 - slice ct angiography and dsa , with a further slight improvement over published data for older - generation ct scanners ( table 6 )  . 
our data are fully concordant with those reported in the literature , with slightly higher values for sensitivity , ppv and npv ; the very high npv of ct angiography emphasises its well - known reliability in demonstrating the real absence of disease in healthy patients . 
sixty - four - slice equipment has also overcome , through optimisation of the examination technique and very high spatial resolution , the problems reported in previous studies , allowing almost identical results in the leg vessels to those in the upper regions , thus overcoming the limitations due to the vessel diameter and the close relation with bone structures . 
all this would not , however , be possible without the necessary parallel evaluation of the reconstructed and axial images , which basically eliminates diagnostic errors due to incorrect reconstructions . praticamente esenti da artefatti da movimento . 
riportavano invece nel 2004 [ 21 ] i dati della tc spirale a 4 strati con valori di sensibilit del 93% , una specificit del 95% , un valore predittivo positivo del 90% ed un valore predittivo negativo del 97% ; nel 2005 willmann et al . 
 analizzando globalmente i risultati da noi ottenuti si evince che nella maggior parte dei casi ( 95 , 4% ) esiste una ottima concordanza tra i risultati forniti dallangio - tc a 64 strati e dallad con un ulteriore lieve miglioramento dei dati riportati in letteratura per apparecchiature tc delle precedenti generazioni ( tabella 6 )  . 
underestimations can be in part explained by heavily calcified vessels simulating vascular patency ; overestimates , instead , often result from inadequate opacification of a portion of vessel owing to slower blood circulation tiva dellesame angio - tc sottolinea lormai nota affidabilit nel dimostrare la reale assenza di patologia nei pazienti sani . 
lapparecchiatura a 64 strati ha inoltre permesso , con lottimizzazione della tecnica di esame e con laltissima risoluzione spaziale , di superare le problematiche emerse in tutti i lavori precedenti in letteratura e di ottenere quindi a livello dei vasi della gamba risultati pressoch identici a quelli del distretto superiore , superando di fatto i limiti dovuti al calibro vasale ed al rapporto stretto con le strutture ossee , comunque mai prescindendo dallindispensabile valutazione parallela delle immagini ricostruite ed assiali , che sostanzialmente azzera gli errori diagnostici da errate ricostruzioni . importante evidenziare anche il numero estremamente basso di sovrastime e sottostime che abbiamo rilevato , dato questo che rende lesame angio - tc a 64 strati , in termini di accuratezza diagnostica , sicuramente superiore alla angiorm , molto influenzata dal ritorno venoso e da stent che determinano artefatti da suscettibilit magnetica , e molto vicina al gold standard angiografico . 
le sottostime sono in parte spiegabili da vasi estremamente calcifici che simulano la perviet vascolare ; le sovrastime invece spesso sono derivate da uninsufficiente opacizzazione di un tratto vasale per un circolo pi rallentato anche monolateralmente . 
risulta chiaro come tali limiti , intrinseci alla tecnica tc , siano stati minimizzati migliorando la risoluzione spaziale lungo lasse z , mantenuta da noi volutamente il pi possibile elevata proprio a questo livello ed ottimizzando la tecnica dindagine . 
 [ 22 ] : 16 strati casistica personale : 64 strati 90 , 9 97 , 2 vpp , valore predittivo positivo ; vpn , valore predittivo negativo 92.4 92 , 4 92.5 92 , 5 98.9 98 , 9 97.1 97 , 1 studio sensibilit ( % ) specificit ( % ) vpp ( % ) vpn ( % ) accuratezza ( % ) unilaterally . 
these limitations inherent in the ct technique were clearly minimised by improving z - axis spatial resolution , which was intentionally kept as high as possible at this level , and by optimising the study technique . 
in diagnostic terms , compared with dsa , multislice ct angiography has two additional advantages : first , it is very accurate in evaluating eccentric stenoses , thanks to axial imaging of the vessel lumen ; for dsa to obtain the same information in those locations requires further oblique or lateral projections with increased patient exposure and contrast material dose . second , it visualises the segments located immediately after an obstruction , which angiography fails to depict [ 14 ]  . 
the same accuracy of information means ct angiography could replace dsa as an initial investigation in the preoperative evaluation of patients [ 14 ]  . langio - tc multistrato presenta due ulteriori vantaggi : essa risulta estremamente accurata nella valutazione delle stenosi eccentriche grazie alle immagini assiali del lume dei vasi , l dove per ottenere le stesse informazioni , la ad necessit di ulteriori proiezioni oblique o laterali con aumento di dose e di somministrazione del mezzo di contrasto al paziente ; in secondo luogo rende possibile la visualizzazione dei segmenti immediatamente successivi ad unostruzione che non risultano visibili in angiografia [ 14 ]  . in particolare langio - tc pu fornire un quadro globale dellalbero arterioso del paziente e pu servire a mirare lintervento del radiologo interventista velocizzando tale procedura , risparmiando mdc e dose al paziente grazie alla possibilit di scegliere il materiale pi adatto e lapproccio pi idoneo ( orientandosi magari verso un approccio omerale piuttosto che femorale , o una puntura in senso anterogrado omolaterale piuttosto che retrogrado controlaterale ) come gi suggerito da kock et al . 
 [ 8 ] ; le stesse informazioni rendono lindagine fruibile in prima istanza anche dal chirurgo vascolare al posto dellangiografia diagnostica , in previsione di un intervento chirurgico [ 14 ]  . conclusions conclusioni the study of oad of the lower extremities with 64 - slice ct angiography cannot be considered a screening method for asymptomatic patients but rather a valid alternative to mr angiography , especially in patients with absolute lo studio dellao degli arti inferiori con angio - tc a 64 strati non pu essere considerata una metodica di screening di pazienti asintomatici , ma si pone come valida alternativa allangio - rm , in primis nei pazienti con controindicazioni 1128 radiol med ( 2009 ) 114 : 11151129 contraindications to mr imaging . 
it should be proposed in patients with steno - obstructive disease diagnosed clinically and / or with colour doppler us or in patients with clear signs and symptoms and risk factors for obliterative arteriopathy such as hypertension , diabetes , hyperlipoproteinaemia and smoking , especially if combined . 
at our institute , diagnostic angiography has been virtually replaced by mr and ct angiography , its use being reserved for interventional procedures or in targeted studies on the basis of results of the diagnostic investigations . 
 assolute alla rm ; essa deve essere proposta a pazienti con patologia steno - ostruttiva gi inquadrata allesame obiettivo e / o strumentale mediante eco - color doppler , anche in evoluzione , oppure in pazienti con una chiara sintomatologia ed un corollario di fattori di rischio per arteriopatia obliterante quali ipertensione , diabete , dislipidemie , fumo , specialmente se in associazione , che abbisognino di una tecnica rapidamente disponibile , panoramica e molto affidabile . 
nel nostro istituto gli esami di angiografia diagnostica sono stati praticamente azzerati da angio - rm ed angio - tc , riservando interventista , allangiografia un ruolo esclusivamente comunque mirato in base ai risultati degli esami diagnostici . 
pedicelli , via trionfale 7130 , 00135 rome , italy , tel . : + 39 - 06 - 30156054 , fax : + 39 - 06 - 35501928 , e - mail : apedicelli@rm.unicatt.it received : 19 october 2008 / accepted : 21 november 2008 / published online : 22 september 2009 springer - verlag 2009 abstract purpose . 
clinically relevant improved mobility and reduction of pain and analgesics were observed , with overall significant results ( p < 0.0001 ) in all patients at 24 h after pvp and in 83 available patients at 6 months . 
pvp is a safe , rapid , effective and costeffective therapy for vcfs , requiring only brief hospital admission and with long - lasting clinical results , when performed under good - quality radiological guidance , when correct indications are respected and when it is associated with rehabilitation therapy in the follow - up . 
la vpp costituisce un trattamento sicuro , rapido , efficace , poco costoso , che richiede solo un ricovero breve e con risultati clinici duraturi qualora sia impiegato un sistema di guida radiologica di buona qualit , siano rispettate le corrette indicazioni e venga associata una terapia riabilitativa nel follow - up . rappresenta una valida alternativa alla terapia 1142 radiol med ( 2009 ) 114 : 11411158 immobilisation of frail and elderly patients at risk of clinical complications . keywords treatment efficacy osteoporosis fluoroscopy spinal fractures vertebroplasty conservativa , questultima gravata da elevati costi sanitari e che richiede spesso limmobilizzazione prolungata di pazienti fragili e anziani , a rischio di complicanze cliniche . parole chiave efficacia terapeutica osteoporosi fluoroscopia fratture vertebrali vertebroplastica introduction introduzione percutaneous vertebroplasty ( pvp ) involves the injection of polymethylmethacrylate ( pmma ) through a special needle into the vertebral body under radiological guidance . the main indication for the procedure is back pain associated with osteoporotic or osteolytic vertebral compression fracture that is refractory to conservative medical treatment [ 1 , 2 ]  . 
total direct costs were calculated at around 31.7 billion euro , which is forecast to increase to 76.7 billion in 2050 given the expected demographic changes across the continent [ 3 ]  . 
approximately 25%30% of the population affected by vcfs has such severe symptoms as to require medical attention [ 5 ] , with serious consequences for both patients and society at large , which is burdened by the significant healthcare costs for treatment and disability arising from this type of fracture . 
clinical consequences of vcfs such as acute and chronic pain , kyphosis , reduced mobility , respiratory difficulties , gastro - oesophageal reflux , bedridden status and depression can lead to reduced quality of life and are associated with an increase in mortality in proportion with the number of vcfs [ 6 ]  . traditional conservative therapy of symptomatic vcfs is often ineffective in the short term and can necessitate prolonged immobility and administration of high doses of analgesics with their associated side effects and a possible increase in risk and poor tolerance by this fragile patient population [ 7 ]  . 
while back pain may persist for 612 weeks , other complications can arise , such as respiratory infections , bedsores , venous thromboembolism and even death [ 8 ]  . 
currently , pvp and kyphoplasty are becoming valuable procedures for treating symptomatic vcfs , with the aim of obtaining pain relief and stabilisation of the fracture over the short term [ 1 , 2 , 9 ]  . in this study , we report our 3 - year experience with pvp for treating vcfs refractory to conservative therapy or in patients unable to tolerate prolonged immobility or orthopaedic braces because of associated reduction in quality of life , with special attention paid to optimization criteria of the procedure and overall patient management . la vertebroplastica percutanea ( vpp ) consiste nelliniezione di polimetilmetacrilato ( pmma ) attraverso uno speciale ago allinterno del corpo vertebrale sotto la guida radiologica . 
la principale indicazione alla procedura costituita dal dolore rachideo associato a frattura vertebrale su base osteoporotica o tumorale , refrattario al trattamento medico conservativo [ 1 , 2 ]  . 
i costi diretti totali sono stati calcolati intorno ai 31 , 7 miliardi di euro e dovrebbero aumentare a 76 , 7 miliardi nel 2050 in base ai cambiamenti attesi nella demografia europea [ 3 ]  . 
circa il 25%30% della popolazione colpita da fvc ha sintomi di tale gravit da richiedere lattenzione medica [ 5 ] , con serie conseguenze sia sui pazienti che sulla societ la quale deve caricarsi di onerosi costi sanitari per la terapia e la disabilit che consegue a questo tipo di frattura . 
conseguenze cliniche delle fvc quali dolore acuto e cronico , cifosi , ridotta mobilit , difficolt respiratorie , reflusso gastroesofageo , allettamento e depressione psicologica possono portare ad una ridotta qualit di vita e sono associate ad un incremento della mortalit a seconda della prevalenza del numero di fvc [ 6 ]  . 
la terapia conservativa tradizionale delle fvc sintomatiche spesso inefficace nel breve periodo e pu dover necessitare di una prolungata immobilizzazione e della somministrazione di elevate dosi di analgesici gravate da effetti collaterali , con un possibile aumento di pericoli e scarsa tolleranza da parte di una popolazione di pazienti fragili [ 7 ]  . 
mentre il dolore rachideo pu perdurare per 612 settimane , possono insorgere complicanze quali : polmonite , ulcere da decubito , tromboembolia venosa e persino il decesso [ 8 ]  . 
attualmente , la vpp e la cifoplastica stanno diventando validi strumenti per il trattamento delle fvc sintomatiche , con il fine di ottenere sollievo dal dolore e stabilizzazione della frattura in breve tempo [ 1 , 2 , 9 ]  . in questo lavoro riportiamo la nostra esperienza triennale con la vpp per il trattamento delle fratture vertebrali refrattarie alla terapia conservativa o in pazienti non in grado di sopportare lallettamento prolungato o il busto radiol med ( 2009 ) 114 : 11411158 materials and methods following the activation of pvp procedures in our radiology department , between september 2005 and september 2008 , we treated 120 consecutive patients with symptomatic vertebral fractures after obtaining informed consent . 
preliminary workup was based on a clinical and radiological examination [ magnetic resonance ( mr ) and / or computed tomography ( ct ) ] to establish the differential diagnosis of the back pain and to evaluate the correct indication for pvp ( table 1 )  . 
the inclusion criteria were : ( 1 ) focal pain on the median or paravertebral line , accentuated by digital pressure on the vertebral spinous process at the same level and associated with the radiological finding of one or multiple vertebral fractures ; ( 2 ) when an mr examination was available , signal hyperintensity of the fractured vertebral body on t2weighted and short - tau ( stir ) sequences ; ( 3 ) no radiological evidence of other spinal disorders that could account for the symptoms at the same level ; ( 4 ) absence of neurological signs ; and ( 5 ) ineffective medical treatment after at least 3 weeks . 
upon request by the referring physician due to marked intolerance of the patients to conservative treatment , 12 vcfs in ten patients were treated early , on average at 1 week from symptom onset . 
another 21 of the 120 treated patients instead suffered from old vcfs with little or no signal hyperintensity on t2 - weighted or stir sequences but with associated chronic pain persisting inversion recovery ortopedico perch causa di riduzione della qualit di vita , con particolare attenzione ai criteri di ottimizzazione della procedura e della gestione complessiva del paziente . 1143 materiali e metodi dopo lattivazione delle procedure di vpp nel nostro dipartimento di radiologia , da settembre 2005 a settembre 2008 abbiamo trattato 120 pazienti consecutivi con fratture vertebrali sintomatiche dopo consenso informato . 
la causa di frattura era losteoporosi in 102 casi , malattie linfoproliferative in 4 casi e metastasi ossee in 12 casi ; sono stati trattati anche 2 casi di angiomi vertebrali aggressivi . 
il work - up preliminare stato basato su un esame clinico e radiologico ( risonanza magnetica [ rm ] e / o tomografia computerizzata [ tc ] ) ai fini di una diagnosi differenziale del dolore rachideo e per valutare la corretta indicazione alla vpp ( tabella 1 )  . 
of treated vertebrae gender osteoporotic compression fracture primary or secondary tumour haemangioma m , male ; f , female ; mri , magnetic resonance imaging ; ct , computed tomography tabella 1 caratteristiche generali della popolazione trattata e tipologia degli esami radiologici preliminari patologia vertebrale numero pazienti numero vertebre trattate sesso preliminary radiological examinations x - ray esami radiologici preliminari frattura da osteoporosi neoplasia primaria o secondaria angioma m , maschio ; f , femmina ; rm , risonanza magnetica ; tc , tomografia computerizzata 1144 radiol med ( 2009 ) 114 : 11411158 fig . 
1 numero e distribuzione delle fvc trattate con vpp . listogramma mostra il numero ( asse verticale ) e la distribuzione ( asse orizzontale ) dei corpi vertebrali trattati con vpp nella nostra serie . for at least 1 year from symptom onset . 
the exclusion criteria were : ( 1 ) noncorrectable blood dyscrasias ; ( 2 ) effective medical therapy ; ( 3 ) stable and asymptomatic vertebral fracture ; and ( 4 ) local or systemic infection . 
pvp was performed with injection of pmma ( volume range 25 ml ) within the vertebral body with the use of a screw - type ( leveen ) or pressure syringe after transpedicular access . 
in all cases , the procedure was guided and monitored with low - dose digital fluoroscopy alone ( single plane tube , allura xper fd 20 with flat - panel detector , philips medical systems , the netherlands ) and performed with a 13 or 1011 - gauge vertebroplasty needle according to the pedicle diameter and the degree of access difficulty , as in the case of severe vertebral body collapse or vertebrae affected by heteroplastic lesions where a smaller bore needle was generally preferred . 
the vertebroplasty needle was introduced with the assistance of an orthopaedic hammer , although after our initial experience with the procedure , the hammer was in most cases only used to introduce and direct the needle through the pedicle , with further advancement within the vertebral body being easily performed with manual screwsettimana dallinsorgenza dei sintomi , su richiesta del medico curante a causa della marcata intolleranza da parte dei pazienti a sostenere la terapia conservativa . 
tra i 120 pazienti trattati , altri 21 soffrivano invece di vecchie fratture vertebrali con scarsa o assente iperintensit di segnale nelle sequenze rm t2w o stir , ma associate a dolore cronico perdurante da almeno 1 anno dallinsorgenza dei sintomi . 
tutti i trattamenti sono stati effettuati in condizioni di sterilit , dopo anestesia locale ( 78 ml di mepivacaina 2% per ogni singolo accesso percutaneo con infiltrazione anche del periostio peduncolare ) , il paziente in posizione prona , e hanno necessitato solamente di una blanda sedazione ( midazolam e / o fentanyl ) in 56 pazienti . 
la vpp stata effettuata con iniezione di pmma ( range volumetrico : 25 ml ) allinterno del corpo vertebrale per mezzo di siringa a vite ( di leveen ) o pistola a pressione e dopo accesso trans - peduncolare . 
la procedura sempre stata guidata e controllata esclusivamente da fluoroscopia digitale a bassa dose ( tubo monoplano , allura xper fd 20 con detettore flat - panel , philips medical systems , olanda ) ed eseguita con ago per vertebroplastica a becco di flauto di 13 o 1011 gauge , a seconda del diametro peduncolare e del grado di difficolt daccesso , come in caso di severo radiol med ( 2009 ) 114 : 11411158 1145 like manoeuvres without the need for any orthopaedic device . 
in the event of particular fluidity of the cement or the detection of cement in the perivertebral veins , the infusion of the pmma was immediately halted and between 30 and 45 s were allowed for polymerisation to reduce the fluidity and therefore the risk of cement leakage outside of the vertebral body . 
on the other hand , in the presence of particularly compact bone , as is often seen in severe vertebral collapse , the high resistance to penetration and diffusion of the pmma was almost always overcome by reinserting the stylet into the cement - filled needle . 
by manually pushing the stylet with constant force within the needle , the right pressure was obtained for the outflow and appropriate penetration of the pmma , even in cases where this could not be achieved with the leveen syringe or pressure needle alone , not even when exerting maximum pressure . 
after treatment , all patients were prescribed bedrest in the supine position for 23 h and then mobilised with discharge on the same day or after a short stay of 12 days , depending on the clinical conditions of the patient following the procedure . 
prior to discharge , each patient was advised about simple precautions to consider in their daily routine regarding the risk of further vertebral fractures , which persists in the presence of osteoporosis , and encouraged not only to regularly take medication for osteoporosis prescribed by their specialist but also to undergo rehabilitation therapy as soon as possible . in order to evaluate the results of the procedure , we measured pain [ visual analogue scale ( vas ) ] and the degree of mobility before treatment , 24 h after treatment and during long - term follow - up by means of a telephone interview . after the first evaluation of all patients at 24 h , subsequently , only patients with at least 6 months of follow - up were included in the study . 
the vas score for pain was evaluated in a range from 0 to 10 , where 0 represents the absence of pain and 10 the worst and intolerable pain , on the basis of the pain intensity numeric rating scale . 
the degree of mobility was calculated in a range from 0 to 5 , where 0 = normal mobility , 1 = mobility with assistance , 2 = mobility only for brief periods with assistance , 3 = mobility only for vital daily activities or for appointments and with assistance , 4 = need for a wheelchair and 5 = bedridden status . 
the use of analgesics was classified in a range from 0 to 4 , where 0 = no use of medication , 1 = use of oral nonsteroid anti - inflammatory drugs , 2 = oral opioids according to need , 3 = programmed use of opioids and collasso somatico vertebrale o su metameri sovvertiti da patologia eteroplasica ove generalmente si preferito luso dellago di minor calibro . 
lago da vpp stato introdotto con laiuto di un martello ortopedico , ma dopo le fasi iniziali di esperienza con la procedura , nella gran parte dei casi il martello stato utilizzato solo per introdurre e indirizzare lago attraverso il peduncolo , mentre il successivo avanzamento allinterno del corpo vertebrale stato sempre effettuato agevolmente con manovre di avvitamento manuale senza alcun ricorso al dispositivo ortopedico . 
in caso di particolare fluidit del cemento o al momento in cui si rilevato limpegno di vene perivertebrali da parte di questo , si sempre interrotta immediatamente linfusione del pmma e atteso tra i 30 e i 45 secondi di polimerizzazione per ridurne la fluidit e pertanto il rischio di stravaso extrasomatico . 
in caso , invece , di elevata compattezza ossea , come spesso osservato nei crolli vertebrali severi , lelevata resistenza alla penetrazione e diffusione del pmma stata pressoch sempre vinta grazie al reinserimento del mandrino nellago riempito di cemento : spingendo infatti il mandrino manualmente e con forza costante allinterno dellago si ottenuta la giusta pressione con fuoriuscita e penetrazione adeguata del pmma anche in questi casi ove con la sola siringa di leveen o pistola a pressione ci non era ottenibile neanche esercitando la massima pressione . 
dopo il trattamento tutti i pazienti sono stati messi a riposo a letto in decubito supino per 23 ore e quindi mobilizzati con dimissione nella stessa giornata o dopo un breve ricovero di 12 giorni a seconda delle condizioni cliniche successive allintervento . 
prima della dimissione stato effettuato un colloquio con ogni paziente al fine di indicare alcune semplici precauzioni da osservare nellattivit quotidiana in considerazione del rischio di ulteriori fratture vertebrali che persiste in presenza dellosteoporosi e per incoraggiare , oltre alla regolare assunzione di eventuale terapia per losteoporosi prescritta dallo specialista , una fisioterapia riabilitativa pi precoce possibile . per lanalisi dei risultati sono stati misurati degli score per il dolore ( con una visual analogue scale [ vas ] ) e il grado di mobilit prima , a 24 ore dal trattamento e durante un follow - up a lungo termine per mezzo di intervista telefonica . 
i pazienti colpiti da nuove fratture vertebrali sintomatiche prima dei 6 mesi , a livelli differenti da quelli trattati , non sono stati inclusi nelle misurazioni del follow - up . 
sono state adottate scale numeriche ( per il dolore , la mobilit e luso di analgesici ) 1146 radiol med ( 2009 ) 114 : 11411158 4 = opioids with infusion pump . 
the effectiveness of pvp was determined on the basis of comparative results of paired data using a nonparametric test ( wilcoxon signed - rank test )  . statistical significance was established for a value of p < 0.05. 
no major complications were recorded at treatment or during follow - up that could be associated with the procedure , not even in patients suffering from cardiopulmonary disease such as severe chronic obstructive pulmonary disease ( ten cases )  . 
the examination performed during and immediately after treatment revealed a total of 82 mild cases of cement leakage gi usate in lavori con ampie popolazioni di pazienti [ 1 , 10 ]  . 
il vas score per il dolore stato valutato in un range da 0 a 10 , dove 0 rappresenta lassenza di dolore e 10 il dolore peggiore e insopportabile , in base alla scala pain intensity numeric rating scale ( pi - nrs ) ; il grado di mobilit stato calcolato in un range da 0 a 5 ( 0 = mobilit normale ; 1 = mobilit con assistenza ; 2 = mobilit solo per brevi periodi e con assistenza ; 3 = mobilit solo per attivit vitali quotidiane o per appuntamenti e con assistenza ; 4 = necessit della sedia a rotelle ; 5 = allettamento ) ; luso di analgesici stato classificato in un range scalare da 0 a 4 ( 0 = nessun uso di farmaci ; 1 = uso di antinfiammatori nonsteroidei orali ; 2 = oppiacei orali alla bisogna ; 3 = uso programmato di oppiacei ; 4 = oppiacei tramite pompa di infusione )  . 
2a , b trattamento di vertebra dorsale medio - alta ( d5 ) con approccio transpeduncolare ( a ) e controllo post - vpp tramite immagini di ricostruzione assiali mpr thick slab da acquisizione radiografica rotazionale ( b ) , ove si nota distribuzione del pmma anche lungo il tragitto transpeduncolare dellago . radiol med ( 2009 ) 114 : 11411158 1147 fig . 
in this case a bipedicular approach is needed because the narrow pedicles do not allow the direct insertion of the needle towards the midline of the vertebral body ( c )  . 
in questo caso necessario un approccio bi - peduncolare poich i peduncoli stretti non permettono di orientare lintroduzione dellago in direzione della linea mediana del corpo vertebrale ( c )  . 
dopo iniezione del pmma si ottiene una buona e simmetrica distribuzione intravertebrale del cemento ( d )  . outside the vertebral body , of which 80 cases were completely asymptomatic ( 39 cases of perivertebral leakage within the segmental veins ; 23 cases of intradiscal leakage ; 18 cases of posterior epidural leakage )  . 
non si sono registrate complicanze maggiori al momento del trattamento o nel follow - up che potessero essere attribuibili alla procedura , neanche in pazienti sofferenti di diverse patologie cardio - polmonari , quali bronco - pneumopatie ostruttive 1148 radiol med ( 2009 ) 114 : 11411158 fig . 
dopo vpp , la radiografia nella stessa proiezione mostra una distribuzione omogenea del cemento allinterno del corpo vertebrale e della fissurazione con ripristino in altezza di 2 mm ( b )  . radiol med ( 2009 ) 114 : 11411158 1149 table 2 technical results and complications of percutaneous vertebroplasty ( pvp ) site of pvp no . 
of treated vertebrae perivertebral leak posterior / foraminal leak intradiscal leak partial somatic height restoration complications thoracic lumbar - sacral dorsale lombare - sacrale tabella 2 risultati tecnici e complicanze della vertebroplastica percutanea ( vpp ) sede vpp numero vertebre leak trattate perivertebrale leak posteriore / foraminale leak intradiscale parziale ripristino complicanze somatico in altezza 1 radiculopathy 1 pulmonary microembolism ; 1 radiculopathy 1 radiculopatia 1 microembolia polmonare ; 1 radiculopatia fig . 
in one case ( a 67 - year - old woman with bone metastases from breast cancer ) , an asymptomatic pulmonary microembolism was observed , which caused no clinical symptoms at 20 months . 
in another two cases ( 64 - and 76 - year - old women with osteoporotic vcf ) , posterior intraforaminal leakage of pmma was croniche di grado severo ( 10 casi )  . 
il controllo durante e immediatamente dopo il trattamento ha mostrato un totale di 82 modesti stravasi extra - vertebrali di cemento , risultati del tutto asintomatici in 80 casi ( 39 perivertebrali allinterno delle vene segmentarie ; 23 intra - discali ; 18 posteriori epidurali )  . 
nei casi in cui si riscontrata la coesistenza di una fissurazione da osteonecrosi in una vertebra fratturata , il pmma si distribuito sempre in modo omogeneo e compatto allinterno della fissurazione ( 72 livelli ) , senza che si siano mai verificati stravasi posteriori e 1150 radiol med ( 2009 ) 114 : 11411158 observed that caused transient radiculopathy , which was resolved with oral nonsteroid anti - inflammatory drugs for 1 month in both patients ( table 2 )  . 
the mean mobility score prior to treatment was 2.1 ( sd = 1.6 ) , whereas at 24 h after treatment , it had fallen to 0.2 ( sd = 0.5 ) , which was also highly statistically significant ( p < 0.0001 ) in terms of improvement of the degree of mobility after comparison of the data . 
at the time of writing , a revision of our data suggested a total of 95 patients had undergone pvp at least 6 months earlier . of these , two patients could not be contacted for follow - up , two had died of cancer and eight had been affected by 14 new symptomatic vcfs on different vertebrae from those treated and arising between 1 and 10 months after the procedure . 
it should be noted that the lowest overall scores over time were reported by patients who during the follow - up period had undergone rehabilitation , and specifically hydrotherapy , proprioceptive and postural training and / or magnetic laser therapy . 
in 1 caso ( femmina , 67 anni , con metastasi scheletriche da carcinoma mammario ) si osservata una microembolia polmonare di pmma asintomatica che non ha determinato manifestazioni cliniche neanche nel follow - up fino a 20 mesi . 
in altri 2 casi ( femmine , rispettivamente di 64 e 76 anni , con fvc osteoporotiche ) si documentato uno stravaso di pmma posteriore intra - foraminale che ha causato una transitoria radiculopatia risolta per mezzo di antinfiammatori non - steroidei orali per un mese in entrambe le pazienti ( tabella 2 )  . 
stato possibile dimettere i pazienti nella stessa giornata in 28 / 120 casi mentre negli altri casi , la maggior parte dei quali con et superiore a 70 anni , si preferito un ricovero con osservazione clinica per ulteriori 24 ore a causa della particolare fragilit dei pazienti , per moderato dolore nella sede di accesso percutaneo ( al fine di escludere lo sviluppo di eventuali ematomi ) e / o per il recupero funzionale dopo la somministrazione di farmaci sedativi durante il trattamento . nella nostra popolazione di pazienti , il vas score medio misurato per il dolore prima del trattamento stato di 6 , 8 ( deviazione standard [ ds ] = 1 , 8 ) e di 1 , 7 ( ds = 1 , 5 ) a 24 ore dal trattamento ; il confronto dei due gruppi di dati appaiati ha prodotto come risultato un valore statistico estremamente significativo in termini di riduzione del dolore ( p < 0 , 0001 )  . 
lo score medio di mobilit prima del trattamento risultava di 2 , 1 ( ds = 1 , 6 ) , mentre a 24 ore dal trattamento sceso a 0 , 2 ( ds = 0 , 5 ) , anchesso risultando estremamente significativo ( p < 0 , 0001 ) in termini di miglioramento del grado di mobilit dopo comparazione dei dati . 
di questi , 2 pazienti non erano pi reperibili per il controllo di follow - up , 2 pazienti erano deceduti per cancro , 8 pazienti erano stati colpiti da 14 nuove fvc sintomatiche , incorse tra 1 e 10 mesi dopo la procedura , su metameri diversi da quelli precedentemente trattati . 
la valutazione del grado di mobilit ha documentato un significativo miglioramento in tutti i pazienti poich solamente 14 casi avevano ancora necessit costante di supporto con busto ortopedico mentre tutti gli altri a quel tempo avevano una mobilit normale o necessitavano del supporto ortopedico solo occasionalmente . 
lo score medio delluso di farmaci analgesici prima del trattamento era di 1 , 9 ( ds = 0 , 8 ) , mentre era di 0 , 6 ( ds = 0 , 7 ) a 6 mesi di distanza negli 83 pazienti reperibili , risultando in una significativa riduzione delluso di farmaci ( p < 0 , 0001 )  . importante notare come gli score complessivamente pi bassi a distanza di tempo siano stati riportati dai pazienti che nel corso del follow - up si siano sottoposti a terapia riabilitativa e nello specifico : idrochinesiterapia , ginnastica propriocettiva e posturale e / o magneto - laser - terapia . 
i singoli gruppi di dati utilizzati per valutare lefficacia della vpp per tutti i pazienti sono riportati nelle tabelle 35 . allinterno del gruppo controllato nel follow - up , 50 pazienti erano stati sottoposti a esami radiologici della colonna vertebrale ( rx , rm o tc ) in diverse epoche dopo il trattamento percutaneo e in gran parte nel nostro istituto ove stato possibile revisionare le immagini per mezzo dellarchivio informatico . 
after percutaneous treatment of l2 and l3 , a new symptomatic fracture of l1 occurred , and pvp was performed at this level ( a , arrow ) after 3 days from symptom onset . 
dopo il trattamento percutaneo di l2 e l3 si verificata una nuova frattura sintomatica di l1 ed stata eseguita vpp su questo livello ( a , freccia ) dopo 3 giorni dalla comparsa dei sintomi . 
una radiografia di controllo a 6 mesi rileva un nuovo ulteriore cedimento , stavolta asintomatico , di l1 precedentemente trattata ( b , freccia ) con severa riduzione in altezza del soma e segni di cedimento sulla limitante somatica inferiore di d12 . communication system ( pacs )  . 
la vpp oggigiorno considerata una tecnica mininvasiva sicura ed efficace per il trattamento delle fratture vertebrali sintomatiche non responsive alla terapia medica conservativa ed caratterizzata da un rischio di complicanze estremamente basso . 
complicanze gravi sono riportate [ 12 , 13 ] , ma la maggior parte degli studi concorde sul fatto che tali evenienze possono essere ridotte al minimo utilizzando sistemi radiologici di guida di elevata qualit ed eseguendo le procedure in una sala angiografica , dove lapparecchiatura fluoroscopica migliore , piuttosto che in una sala operatoria dove sono riportate le complicanze pi gravi a causa della qualit inferiore del tubo a c [ 1 , 2 , 9 , 1316 ]  . 
la vpp pu essere eseguita in regime di day - surgery con significativo contenimento dei costi ospedalieri , e si dimostra essere una tecnica rapida e facile in mani esperte . 
pvp is today considered to be a safe and effective minimally invasive technique for treating symptomatic vertebral fractures refractory to conservative medical treatment and is characterised by an extremely low risk of complications . 
severe complications have been reported [ 12 , 13 ] , but most studies agree that these outcomes can be minimised with the use of high - quality radiological guidance systems and by performing the procedure in an angiography suite where the fluoroscopy equipment is better , rather than in an operating theatre where the most severe complications have been reported due to the inferior quality of the c - arm device [ 1 , 2 , 9 , 1316 ]  . 
 pvp can be performed as a day - surgery procedure with significant reductions in hospital costs , and it has proved to be a rapid and easily performed technique in expert hands . the calculation of the mean cost of materials we used for each procedure falls within the range estimated by the single commission for medical equipment of the italian ministry of health [ 17 ]  . 
in most cases , because all that was required was a vertebroplasty needle , a maximum dose of 5 ml of pmma and a leveen syringe for the injection , with no need for complex and expensive sets , costs were able to be kept within the minimum of the estimated range . percutaneous access and the absence of general anaesthesia , together with the possibility of treating elderly patients with no apparent age limit , are some of the main advantages of the technique . 
severe vertebral collapse ( < 1 / 3 of the normal vertebral height ) is considered by some authors to be a relative contraindication to the procedure due to technical difficulties regarding the approach [ 9 , 18 ] , as is the high risk in treating patients with severe cardiopulmonary disease due to the possibility of fat embolism and the cardiotoxic effect of pmma [ 14 , 19 , 20 ]  . 
in our series , we believe that the use of high - resolution digital fluoroscopy as the guidance system , the complete and continuous monitoring of vital signs , the possibility of a unilateral approach in most cases and the injection of cement limited to a maximum volume of 5 ml in a single vertebral body are decisive features for minimising risks and adverse effects . this , therefore , allowed us to treat even fragile patients with chronic obstructive pulmonary disease and cases of severe vertebral collapse without the onset of severe complications . our findings show that the use of the orthopaedic hammer , which is often poorly tolerated by patients from a psychological point of view , can be limited to introducing the needle . 
this favours greater tolerance of the procedure and as a consequence improved patient compliance , and 1153 medio dei materiali da noi utilizzati per singola procedura rientra nel range stimato dalla commissione unica dei dispositivi medici del ministero della salute ( sito web , sul portale del ministero della salute : lute.it / dispositivi / paginainterna.jsp ? id = 88&menu = commissione )  . 
nella maggior parte dei casi , essendo sufficiente lutilizzo dellago per vertebroplastica , una dose massima di 5 ml di pmma e la siringa di leveen per liniezione senza necessario ricorso a set pi complessi , articolati e dispendiosi , tali costi possono essere contenuti entro il minimo del range stimato . laccesso percutaneo e nessuna necessit di anestesia generale , insieme alla possibilit di trattare pazienti anziani senza apparente limite det , rappresentano alcuni dei principali vantaggi della tecnica . 
il collasso vertebrale severo ( < 1 / 3 della normale altezza somatica ) considerato da alcuni autori una relativa controindicazione alla procedura a causa delle difficolt tecniche di approccio [ 9 , 18 ] , cos come da considerarsi ad alto rischio il trattamento di pazienti con gravi malattie cardio - polmonari a causa della possibile embolia grassosa e delleffetto cardiotossico del pmma [ 14 , 19 , 20 ]  . 
nella nostra serie , riteniamo che lassistenza della fluoroscopica digitale ad alta risoluzione come sistema di guida , il completo e continuo monitoraggio dei parametri vitali , linutilit di un approccio bilaterale nella maggior parte dei casi e liniezione di cemento limitata ad un volume massimo di 45 ml in un singolo corpo vertebrale , siano stati decisivi a minimizzare i rischi e gli effetti avversi , consentendoci pertanto di trattare anche pazienti fragili con bronco - pneumopatie ostruttive croniche e casi di vertebra plana senza insorgenza di complicanze gravi . 
lintroduzione manuale pressoch sempre agevole e meno dolorosa e favorisce pertanto una maggiore tolleranza alla procedura a cui consegue una migliore collaborazione da parte del paziente , permettendo di ridurre i tempi di esecuzione e il ricorso a farmaci sedativi . 
 in caso di peduncoli vertebrali molto stretti , come a livello di vertebre toraciche alte , pu essere necessario un approccio paravertebrale o intercosto - vertebrale , ma ci comporta un maggiore rischio di pneumotorace o ematoma paraspinale [ 9 , 16 ]  . 
la buona visualizzazione fluoroscopica della struttura anatomica vertebrale ci ha permesso di trattare ogni paziente con un sicuro accesso trans - peduncolare anche sulle vertebre toraciche pi alte , che nella nostra serie stato a livello di d4 in 2 casi . dibattuto se debba essere somministrato un antibiotico al paziente che si sottopone a vpp e alcuni autori sostengono che la profilassi sia un imperativo solo per pazienti immunocompromessi [ 9 ]  . 
in accordo con i lavori maggiori 1154 radiol med ( 2009 ) 114 : 11411158 therefore reduces procedure times and the need for sedatives . in the presence of very narrow vertebral pedicles , as for example at the level of the high thoracic vertebrae , a paravertebral or intercostal - vertebral approach may be required , although this increases the risk of pneumothorax or paraspinal haematoma [ 9 , 16 ]  . 
good fluoroscopic visualisation of the vertebral structures allowed us to treat each patient with safe transpedicular access even in high thoracic vertebrae , which in our series was at the level of t4 in two cases . it is debatable as to whether antibiotics should be administered to patients undergoing pvp , and some authors suggest prophylaxis is a requirement only in immunocompromised patients [ 9 ]  . 
in agreement with the major studies to date [ 1 , 20 ] , we prefer to administer antibiotics to all patients prior to the procedure to avoid infections , a rare complication of pvp but one of the most feared , which may necessitate neurosurgery . our findings regarding the effectiveness of pvp in producing long - term pain relief from vcfs are in accordance with the literature [ 1 , 1416 , 1820 ]  . 
a significant improvement in mobility and reduction in the use of analgesics have also been reported by most authors , with an overall improvement of the patients quality of life . 
although some studies report better results associated with fewer complications in osteoporotic fractures compared with osteolytic fractures [ 16 ] , we found no significant differences in our series in terms of pain reduction and complications after pvp , regardless of the underlying disease . 
our data are therefore similar to most of the other studies in the literature , which report favourable outcomes in a variety of patient subgroups [ 1 , 15 ]  . 
this finding was supported by 21 cases in our series that showed a significant reduction in pain after treating an old vcf that had been symptomatic for more than 1 year . as stated in other studies [ 1 , 14 , 16 ] , we believe that the effectiveness and favourable clinical outcome of pvp are closely related to careful patient selection established by preliminary clinical and radiological workup . 
in particular , where available , spinal mr was considered the modality of choice for establishing the correct indication for the procedure on the basis of the society of interventional radiology guidelines [ 2 ]  . 
in agreement with most authors , we believe that the presence of signal hyperintensity due to bone marrow oedema on t2 - weighted and stir sequences is a determining sign for establishing the indication for pvp and predicting the positive outcome of the procedure . the reason pvp is able to produce its analgesic effect on [ 1 , 20 ] noi preferiamo la somministrazione dellantibiotico prima della procedura in tutti i casi , al fine di prevenire infezioni , una complicanza rara , ma tra le pi temibili della vpp , che pu richiedere un intervento neurochirurgico . i nostri risultati riguardanti lefficacia della vpp nel produrre un prolungato sollievo dal dolore per le fvc sono in linea con la letteratura [ 1 , 1416 , 1820 ]  . 
anche il significativo miglioramento della mobilit e la riduzione delluso di farmaci analgesici sono riportati dalla maggior parte degli autori con un complessivo miglioramento della qualit di vita dei pazienti . sebbene alcuni studi mostrino migliori risultati associati a minori complicanze nelle fratture osteoporotiche piuttosto che in quelle legate a patologie tumorali [ 16 ] , nella nostra serie non abbiamo trovato significative differenze in termini di riduzione del dolore e complicanze dopo vpp qualsiasi fosse la patologia di base . 
lo stesso risultato stato ottenuto in 21 dei nostri casi che hanno mostrato una significativa riduzione del dolore dopo trattamento di vecchie fvc con sintomatologia che perdurava da almeno 1 anno . come gi espresso in altri lavori [ 1 , 14 , 16 ] , riteniamo che lefficacia della vpp e lesito clinico favorevole siano strettamente dipendenti da unaccurata selezione dei pazienti stabilita dal work - up clinico e radiologico preliminare . 
in particolare , quando disponibile , la rm spinale da considerarsi la metodica di scelta al fine di stabilire la corretta indicazione alla procedura sulla base delle linee guida della society of interventional radiology [ 2 ]  . 
in accordo con la maggior parte degli autori , riteniamo che la presenza delliperintensit di segnale dovuta ad edema intraspongioso sulle sequenze t2w e stir , rappresenti un segno determinante per stabilire lindicazione alla vpp e predittivo dellesito positivo della procedura . non ancora chiara la ragione per cui la vpp sia una tecnica in grado di produrre un effetto antalgico nelle fvc . sono stati proposti diversi meccanismi che includono la stabilizzazione della frattura , la necrosi termica e la chemiotossicit tissutale , delle terminazioni nervose e dei recettori intraossei del dolore [ 14 , 16 , 20 ]  . un problema emergente da numerosi studi riguarda il rischio di sviluppare nuove fratture vertebrali dopo trattamento con vpp . 
nella nostra casistica , dopo il follow - up di 6 mesi , la percentuale di pazienti nei quali si sono verificate nuove fratture vertebrali dopo la procedura a livelli diversi da quelli trattati stata del 6 , 7%  . 
 [ 1 ] hanno riportato l8% di nuove fratture dopo un follow - up medio di 7 mesi ; la percentuale di nuove fratture riportate radiol med ( 2009 ) 114 : 11411158 1155 vcfs is still not clear . 
a number of mechanisms have been proposed , including fracture stabilisation , thermal necrosis and tissue , nerve endings and intraosseous pain receptors chemiotoxicity [ 14 , 16 , 20 ]  . a problem that has emerged from numerous studies regards the risk of developing new vertebral fractures after pvp treatment . 
in our series , after 6 months of follow - up , the percentage of patients in whom new vertebral fractures occurred after the procedure at levels other than those treated was 6.7%. 
recent studies suggest that increased rigidity of the treated vertebra , leakage of intradiscal cement , an elevated degree of restoration of the height of the vertebra or treating vertebral bodies at the thoracolumbar junction [ 27 , 28 ] may be risk factors for new vertebral fractures at levels adjacent to those treated and that a significant restoration of the anterior vertebral body height increases the risk of further fractures in the treated vertebrae [ 29 ]  . 
in our series we noted that all new fractures in previously treated vertebrae ( five cases ) occurred in the group of ten patients who underwent early pvp at around 1 week from symptom onset , but we were unable to establish whether this coincidence can be considered a causative factor of new fractures due to the small number of cases . with regard to the problems of new fractures , we agree with baroud et al . 
as pvp is a relatively new technique , prospective studies are required to conclusively define the cause ( s ) of new fractures at different levels and on previously treated vertebrae . 
on the basis of these preliminary studies , and given the known analgesic and stabilising effect of pvp regardless of the volume of pmma injected [ 14 , 16 , 31 ] , we believe that the volume of cement injected should be limited to a precautionary 23 ml , particularly during the treatment of vertebrae at the thoracolumbar junction , to reduce the risk of new fractures on adjacent vertebral bodies . 
this conservative approach also reduces the risk of extravertebral leakage of cement and fat embolism [ 1 , 14 , 31 ]  . in our study , we observed the best long - term results in patients who underwent rehabilitation therapy during the follow - up period . 
infatti , dimostrato che i pazienti che sviluppano una fvc da osteoporosi sono a rischio di nuove fratture entro un anno , con unincidenza del 19 , 2% [ 26 ]  . 
recenti studi suggeriscono che unaumentata rigidit della vertebra trattata , uno stravaso di cemento intradiscale , un elevato grado di ripristino in altezza del soma o il trattamento di metameri alla giunzione toracolombare [ 27 , 28 ] possano costituire dei fattori di rischio per nuove fratture vertebrali a livelli adiacenti a quelli trattati e che un significativo recupero in altezza del muro somatico anteriore aumenti il rischio di successive fratture in vertebre gi trattate [ 29 ]  . 
nella nostra serie abbiamo notato che tutti i nuovi cedimenti somatici in vertebre gi trattate ( 5 casi ) si sono verificati nel gruppo di 10 pazienti sottoposti a vpp precocemente a circa una settimana dallinsorgenza dei sintomi , ma non siamo in grado di stabilire se questa coincidenza possa ritenersi una possibile condizione causativa di nuova frattura in relazione allesiguo numero di casi . riguardo alla problematica delle nuove fratture concordiamo con lasserzione di baroud et al . 
 [ 30 ] per cui sebbene siano in aumento prove a supporto di alcune ipotesi riguardanti i fattori di rischio , bisogna essere cauti : la vpp una tecnica relativamente nuova e sono necessari studi prospettici per definire in modo conclusivo la / e causa / e di nuove fratture a livelli differenti e sulle stesse vertebre gi trattate . 
sulla base di questi studi preliminari ed essendo noto che leffetto antalgico e la stabilizzazione possono ottenersi dopo vpp indipendentemente dal volume di pmma iniettato [ 14 , 16 , 31 ] , la nostra opinione che si debba considerare in modo precauzionale di iniettare un volume di cemento limitato a 23 ml , in particolare durante il trattamento di vertebre al passaggio toraco - lombare , al fine di ridurre il rischio di nuove fratture su metameri adiacenti . 
questo approccio cauto riduce anche il rischio di stravasi di cemento extra - vertebrali e di embolia grassosa [ 1 , 14 , 31 ]  . nel nostro studio abbiamo osservato i migliori risultati a distanza nei pazienti che nel corso del follow - up avevano eseguito dei cicli di fisioterapia riabilitativa . 
il paziente colpito da fratture che hanno richiesto limmobilizzazione , lallettamento o comunque luso del busto ortopedico ha infatti necessit di ristabilire un buon tono ed equilibrio muscolare e la ripresa di una normale attivit fisica mirando a riacquistare lindipendenza e 1156 radiol med ( 2009 ) 114 : 11411158 an integral part of the postoperative management protocol for pvp . 
the patient suffering from fractures who required immobilisation , bedrest or the use of an orthopaedic brace in fact needed to reestablish good muscular tone and equilibrium and the recovery of normal physical activity and reacquire the independence and autonomy that the condition had limited . 
the probability of this being achieved was greater thanks to this therapy , which above all reduces the risk of new vertebral fractures over time [ 32 , 33 ]  . 
by relieving pain and stabilising the fracture , pvp in turn enabled the patient to undertake physiotherapy in a reduced time frame and without limitations . there are a number of limitations to our study . 
firstly , with regard to the technique , we were unable to make a comparison with a group of patients treated under ct guidance , because all of our cases were treated under fluoroscopic guidance . 
ct is considered the best guidance system for precise introduction of the pvp needle [ 9 , 34 ] , but in our opinion , and in agreement with most authors , most interventional radiologists are well versed with fluoroscopy , and this technique is fast , inexpensive and reliable as a guidance system for pvp if the operator has a good understanding of the radiographic anatomical landmarks . 
although both techniques are valid , the preference is obviously also influenced by the possibility of access and use of one or the other in accordance with type of activity and diagnostic workload of the individual centres and should be subject to the good quality of the equipment . the second limitation of our study was the absence of a comparison with a population treated with conservative therapy in order to establish whether pvp is more effective with respect to traditional medical treatment . 
during conservative therapy , complications and worsening of vertebral collapse may arise , which suggest early referral for pvp in patients at risk of prolonged immobilisation and who need high doses of analgesics [ 2 , 16 ]  . 
in addition , as occurred in our series , the patients are generally satisfied with the procedure , and they are the ones to request new treatment in the event of new symptomatic fractures . lastly , we were unable to determine the clinical importance of changes in the degree of pain after pvp because the pain intensity numerical rating scale does not provide information regarding the subjective nature of the paas with anselmetti et al . 
on a large patient population that puts forward a possible standard definition of major clinical improvement in chronic pain by establishing that a mean reduction of lautonomia che la patologia aveva limitato . 
la vpp , dal canto suo , alleviando il dolore e stabilizzando la frattura , permette al paziente di avviare in breve tempo la fisioterapia e affrontarla senza limitazioni . analizziamo alcuni limiti del nostro studio . 
innanzitutto , riguardo alla tecnica , non abbiamo termini di comparazione con un gruppo di pazienti trattati sotto guida tc giacch tutti i nostri casi sono stati trattati sotto guida fluoroscopica . 
la tc considerata il sistema di guida migliore per una esatta introduzione dellago da vpp [ 9 , 34 ] , ma nella nostra opinione e in accordo con la maggior parte degli autori , la gran parte dei radiologi interventisti ha unelevata dimestichezza con la fluoroscopia e questa metodica veloce , poco costosa e affidabile come sistema di guida per la vpp se loperatore ha una buona conoscenza dei reperi anatomici radiografici . 
se pur valide entrambe le metodiche , la preferenza ovviamente determinata anche dalla possibilit di accesso e utilizzo delluna o dellaltra a seconda del tipo di attivit e carico diagnostico dei singoli centri , e deve comunque essere subordinata alla buona qualit dellapparecchiatura . il secondo limite del nostro lavoro consiste nel mancato confronto con una popolazione trattata con terapia conservativa al fine di stabilire se la vpp sia pi efficace rispetto al trattamento medico tradizionale . 
tuttavia , alcuni studi prospettici preliminari , randomizzati e non - randomizzati [ 3537 ] , hanno comparato gruppi di pazienti sottoposti a vpp con pazienti trattati per mezzo della terapia conservativa . 
durante la terapia conservativa possono verificarsi complicanze e peggioramento del collasso vertebrale che suggeriscono lindicazione alla vpp precoce in pazienti a rischio di prolungata immobilizzazione e che necessitano di elevate dosi di farmaci analgesici [ 2 , 16 ]  . 
inoltre , come accaduto nella nostra casistica , i pazienti sono generalmente soddisfatti della procedura e richiedono essi stessi il nuovo trattamento in caso di nuove fratture sintomatiche . infine , non abbiamo potuto determinare limportanza clinica delle modificazioni del grado di dolore dopo vpp giacch la pi - nrs non fornisce informazioni sulla soggettiva natura del dolore . 
 [ 38 ] su di unampia popolazione , il quale propone una possibile definizione standard di importante miglioramento clinico del dolore cronico stabilendo che una riduzione media di circa il 30% nella pi - nrs rappresenta una differenza clinicamente importante . in conclusione , la vpp si dimostra essere una terapia radiol med ( 2009 ) 114 : 11411158 1157 approximately 30% in the pain intensity numerical rating scale is a clinically important difference [ 38 ]  . in conclusion , pvp has been shown to be an effective , safe , relatively inexpensive and minimally invasive procedure for symptomatic vcfs , with an extremely low rate of complications when performed under the guidance of goodquality equipment , such as low - dose digital fluoroscopy , and by experienced operators . 
the technique may be indicated as a day - surgery procedure when the clinical conditions of the patient allow it and are thoroughly evaluated and when vital signs are monitored before , during and after the procedure . 
careful selection of patients , evaluation of the correct indications and postoperative management that includes rehabilitation therapy are crucial for increasing the probability of a favourable outcome for the patient in both the short and long term . efficace , sicura , poco costosa e mininvasiva per le fvc sintomatiche , con una percentuale di complicanze estremamente bassa qualora eseguita sotto la guida di apparecchiature di buona qualit , quale la fluoroscopia digitale a bassa dose , e da parte di operatori esperti . 
la procedura effettuabile in regime di day - surgery qualora le condizioni cliniche del paziente lo consentano e siano valutate in modo accurato e quando i parametri vitali sono monitorati prima , durante e dopo la procedura . 
the choice made by radiologists in moral dilemmas is inspired by an adherence to moral principles , which in italy and elsewhere refer to the judaeo - christian tradition or to neo - darwinian relativism . whatever the choice , the radiologist is bound to adhere to that choice and to provide the patient with all the relevant information regarding his or her state of health . riassunto il continuo aumento della richiesta di esami radiologici che si verifica in tutti i paesi economicamente sviluppati riconosce tra le sue cause lintervento attivo dei pazienticonsumatori . 
il consumismo pone i radiologi di fronte ad un dilemma etico , tra il principio di autonomia e gli altri principi etici di beneficenza , non maleficenza e giustizia . la scelta operata da ciascun radiologo nei dilemmi morali sar ispirata dalladesione a principi morali , che nella nostra societ si riferiscono alla tradizione giudaicocristiana , o al relativismo neo - darwiniano . 
qualunque sia la scelta , il radiologo tenuto a rendere conto di essa e a comunicare al paziente tutte le informazioni rilevanti per il suo stato di salute . keywords ethics radiology medical consumerism autonomy accountability parole chiave etica radiologia consumismo medico autonomia accountability is there a problem of ethics in radiology ? esiste un problema etico in radiologia ? like all medical practitioners , the radiologist is faced with numerous ethical problems on a daily basis . 
this rather selfevident statement should not overlook the fact that often the healthcare professional , pressed by the needs of providing the service , has little time to dedicate to an ethical assessment and is forced to act upon impulse , following habitual actions or going along with the prevailing pressures . 
in the ordered sequence of operations that a modern radiological unit performs , it may appear that there is no margin for doubt , or even that it does not exist . 
of course , that is not so . the reason we are interested in this situation is that it il medico radiologo , come ogni medico , chiamato a misurarsi quotidianamente con numerosi problemi etici . 
questa affermazione banale non deve far dimenticare che spesso il sanitario , pressato dalle esigenze del servizio , ha ben poco tempo da dedicare allanalisi etica ed costretto ad agire dimpulso , seguendo labitudine o assecondando le pressioni prevalenti . 
nella sequenza ordinata di operazioni che una moderna sala radiologica esegue , pu cos sembrare che non vi sia alcuno spazio per il dubbio , o addirittura che esso non esista . 
la ragione per cui , come medici del lavoro , ci interessiamo di tale situazione che essa costituisce una 1174 radiol med ( 2009 ) 114 : 11731181 constitutes an effective threat to the health of healthcare professionals . moral conflicts can lead to stress and burnout in physicians , a stress - response syndrome characterised by emotional exhaustion , depersonalisation and reduced personal realisation . 
a recent norwegian study reported that more than 50% of medical practitioners are aware of having to professionally act in a manner contrary to their conscience , and that this is a cause of tension [ 1 ]  . 
the prospect of revealing the error , or even having to report behaviour of a colleague who is noncompliant with professional standards , is a serious decision that may compromise ones career or working conditions and is therefore highly stressful . 
specific research indicates that healthcare professionals are relatively intolerant of criticism of their own work and that in some cases medical practitioners avoid turning to ethics committees precisely because they fear criticism or hostility from their colleagues [ 2 ]  . 
in contrast , the less tolerant environments , in which discussion of ethical problems is more likely to be stifled both before relevant decisions are made and after the controversy has arisen , are a greater threat for the psychophysical well - being of those working in them . on a more general note , healthcare professionals complain of the lack of time available for caring for patients as they would like to , and they lament the commitments in contrast with their professional obligations . 
the time dedicated to administrative and bureaucratic activities , which is constantly on the increase in part due to the growing need for transparency limits the time available for caring for patients . 
for all physicians , ideal ethics constitute an important part of ones professional image , and being constrained to working in a manner out of line with these ideals can significantly reduce job satisfaction and induce stress [ 1 ]  . 
it can be said , therefore , that quality in medicine does not only depend on professional attitude but also on the ability to effettiva minaccia per la salute degli operatori . 
 i conflitti morali possono determinare nei medici uno stato di stress e la comparsa del burnout , una sindrome di risposta allo stress caratterizzata da esaurimento emotivo , depersonalizzazione e ridotta realizzazione personale . 
una recente indagine sui medici norvegesi ha documentato che oltre la met di essi consapevole di aver dovuto agire professionalmente in modo contrario alla propria coscienza , e che tale fatto motivo di tensione [ 1 ]  . 
tra i momenti di conflitto pi frequentemente indicati vi la necessit di affrontare errori clinici commessi da colleghi . lipotesi di rivelare lerrore , o addirittura di dover segnalare la condotta non conforme agli standard o alle norme deontologiche di un collega , ritenuta una decisione grave , suscettibile di compromettere la propria carriera o la condizione lavorativa , e quindi fortemente stressante . ricerche specifiche indicano che i medici hanno pochissima tolleranza per le critiche al proprio operato , e che in alcuni casi i medici evitano di rivolgersi ai comitati etici proprio perch temono critiche o ostilit da parte dei colleghi [ 2 ]  . daltro canto , gli ambienti di lavoro nei quali si soliti formulare e ricevere critiche sono anche quelli caratterizzati da maggiore collegialit e sostegno per gli errori , e quindi i meno stressanti . 
al contrario , gli ambienti meno tolleranti , nei quali si soliti sopprimere la discussione sui problemi etici sia prima di prendere decisioni rilevanti che dopo che la controversia si presentata , risultano maggiormente rischiosi per il benessere psicofisico di chi lavora . pi in generale , i medici lamentano la mancanza di tempo per prendersi cura come dovrebbero dei pazienti e gli impegni contrastanti con i propri doveri professionali . 
il tempo dedicato ad attivit amministrative e burocratiche , in continuo aumento anche per le accresciute esigenze di trasparenza , riduce la possibilit di occuparsi dei pazienti . inoltre i medici sentono come un problema rilevante dal punto di vista etico lesistenza di lunghe liste di attesa e il fatto che pazienti pi forti ( ad esempio , solventi ) hanno spesso la priorit rispetto ai pi bisognosi . le condizioni di lavoro di molti medici , caratterizzate da carichi di lavoro eccessivi e ristrettezza di tempo , possono ostacolare il raggiungimento dei desiderati livelli professionali ed etici nellattivit lavorativa . 
per tutti i medici , gli ideali etici costituiscono una parte importante della propria immagine professionale , ed essere costretti a lavorare in modo da non aderire a questi ideali pu significativamente ridurre la soddisfazione tratta dal lavoro ed indurre uno stato di stress [ 1 ]  . 
si pu ben dire quindi che la qualit in medicina non dipende solo dalle attitudini radiol med ( 2009 ) 114 : 11731181 1175 identify and resolve ethical conflicts [ 1 ]  . a recent study of ours on italian radiologists and radiation therapists reported an increase in work - related stress . this has been due on the one hand to an increasing workload , often in the presence of limited resources , and on the other to the introduction of increasingly sophisticated and invasive techniques that require greater professional commitment , and has increasingly penalised the time available to the physician to evaluate the examination or the treatment plan , and definitively to treat the patient [ 3 ]  . 
this involves issues , such as allocation of healthcare resources , which regard all aspects of medicine and especially involves radiology in relation to the high cost of some examinations and their limited availability . 
an ethical conflict can arise between the principle of autonomy of the patient requesting an inappropriate examination on the one hand , and the principles of nonmaleficence towards the patient , who would be unnecessarily exposed to the undesired effects of the radiological technique , and justice and beneficence towards other patients , who may have a greater need for the examination than the other . 
similar considerations can be made with regard to defensive radiology , which is responsible for a squandering of resources motivated by the sole intent of protecting oneself from possible accusations of malpractice . professionali , ma anche dalla capacit di identificare e risolvere i conflitti etici [ 1 ]  . una nostra recente indagine sui medici radiologi e radioterapisti italiani ha evidenziato lemergere di un accentuato stress da lavoro , riconducibile da un lato alle crescenti esigenze produttive , spesso in limitatezza di risorse , dallaltro allintroduzione di tecniche sempre pi sofisticate e di metodiche invasive , che richiedono un impegno professionale crescente , penalizzando sempre di pi il tempo a disposizione del medico per valutare lesame o il piano terapeutico , e in definitiva per la cura del malato [ 3 ]  . 
si tratta spesso di argomenti , come quelli relativi allallocazione delle risorse sanitarie , che riguardano tutta la medicina nel suo insieme , e coinvolgono la radiologia soprattutto in relazione allelevato costo di taluni esami ed alla loro limitata disponibilit . 
un conflitto etico pu sorgere tra il principio di autonomia del paziente che richiede un esame non appropriato ed i principi di non maleficenza verso lo stesso paziente , che sarebbe esposto indebitamente agli effetti indesiderati della tecnica radiologica , e i principi di giustizia e beneficenza verso altri pazienti che potrebbero avere maggiore bisogno dellesame . 
analoghe considerazioni possono essere fatte per la radiologia difensiva , che responsabile di una dissipazione di risorse motivata solo dallintento di difendersi da possibili accuse di malpractice . healthcare consumerism in radiology il consumismo sanitario in radiologia in radiology , the problem of healthcare consumerism is particularly evident . 
the number of examinations is on the increase at an annual rate of 5%11% in all developed countries , and the request appears to be infinite [ 4 ]  . 
multiple factors are responsible for this trend , the main ones being the development of new technology , the use of reimbursement systems per service , the endorsement of self - prescription by general practitioners , defensive medicine and defensive radiology . 
undeniably , the autonomy of the patient , with the abandonment of the traditional paternalism that left the power to decide to the physician , has led to an acceleration of the phenomenon . 
an increasing number of patients see the acquisition of computed tomography ( ct ) or magnetic resonance ( mr ) images a compulsory act and they feel disregarded if the physician fails to prescribe thein addition , many patients self - prescribe examinations on the basis of the conviction in radiologia il problema del consumismo sanitario particolarmente presente . 
molteplici fattori sono responsabili di questa tendenza : lo sviluppo di nuove tecnologie , luso di sistemi di rimborso a prestazione , lavallo alle auto - prescrizioni da parte dei medici di medicina generale , la medicina difensiva e radiologia difensiva sono le principali . 
sempre pi numerosi sono i pazienti che percepiscono lesecuzione di immagini di tomografia computerizzata ( tc ) o risonanza magnetica ( rm ) come un atto dovuto , e che si sentono trascurati se il medico rifiuta di prescriverli . 
molti pazienti , inoltre , si auto - prescrivono gli esami , sulla base del concetto che 1176 radiol med ( 2009 ) 114 : 11731181 that one examination too many is better than one too few . moreover , although the harmful effects of ionizing radiation have been known for a long time , and provisions have been made in italy that proscribe the unjustified use of diagnostic imaging , there appears to be no evidence of the application of professional or legal sanctions for unnecessary examinations . 
as a result , radiological examinations of the chest are indiscriminately performed as a matter of routine , even prior to a clinical examination . a number of competing factors are at play that lead to the failure of physicians to oppose performing clinically useless examinations . 
traditionally , radiologists have felt that concerns over patient autonomy are a useless waste of time [ 4 ] and that informing patients about the risks of a radiological examination is a way of making them unnecessarily apprehensive or even inducing them to refuse the examination . the individualistic interpretation of the principle of autonomy ends up downgrading the relationship between patient and physician to one between patient and technician , whereby the sole task of the radiologist is to satisfy the request . 
the systems of reimbursement for services encourage consumerism and competition between radiologists , with the effect of further downgrading the doctorpatient relationship to one of trade . on the other hand , it has been observed that patients are considered to be free to autonomously decide about much more relevant healthcare matters , such as abortion , so that there appears little ground for limiting them with regard to radiological examinations [ 5 ]  . standing against patient autonomy is the fact that patient knowledge is never neutral , balanced or based on evidence . often , radiology services do not provide adequate information in that the information is aimed at reassuring the patient or it does not accompany a verification of what the patient has understood . the prerequisite for an effectively autonomous decision is a state of psychophysical balance by the person making it . even healthy and asymptomatic subjects can exhibit a state of anxiety or fear of disease that may influence choice . patients may be subject to pressure from family members or the mass media , through which interest groups spread the notion that benefits may be obtained from new technology , particularly if this can be obtained without an economic cost , within the national health service . of course , patient autonomy offers important benefits for the healthcare profession . 
leaving the patient free to choose is a way of transferring professional responinduced meglio effettuare un controllo in pi che uno di meno di quello che in un secondo tempo si rivela necessario . 
per di pi , nonostante gli effetti nocivi delle radiazioni ionizzanti siano note da tempo , e siano in vigore nel nostro paese provvedimenti che proscrivono il ricorso ingiustificato a diagnostica radiologica , non si ha notizia dellapplicazione di alcuna sanzione professionale o legale per esami non necessari . 
si assiste cos allesecuzione di esami radiologici del torace come parte di routine indiscriminate , addirittura prima di una visita medica con finalit di certificazione . numerosi fattori concorrono perch i medici non si oppongano allesecuzione di esami clinicamente inutili . tradizionalmente i radiologi hanno sempre ritenuto che le preoccupazioni per lautonomia del paziente siano una inutile perdita di tempo [ 4 ] e che informare i pazienti sui rischi degli esami radiologici sia il modo di renderli inutilmente apprensivi o addirittura indurli a rifiutare lesame . linterpretazione individualistica del principio di autonomia finisce per ridurre il rapporto tra paziente e medico ad un rapporto tra paziente e tecnico , nel quale al radiologo spetta solo il compito di soddisfare la richiesta . 
i sistemi di rimborso a prestazione incoraggiano il consumismo e la competizione tra radiologi , con leffetto di appiattire ulteriormente la relazione medico - paziente ai soli aspetti mercantili . daltro canto stato osservato che i pazienti sono ritenuti liberi di decidere autonomamente su questioni sanitarie molto pi rilevanti , come ad esempio laborto , e non si vede perch debbano essere limitati nelle indagini radiologiche [ 5 ]  . contro lautonomia sta il fatto che le conoscenze del paziente non sono mai neutrali , bilanciate o basate sullevidenza . 
i servizi di radiologia spesso non forniscono informazioni adeguate , in quanto spesso le informazioni sono rivolte a rassicurare il paziente , o non si accompagnano ad una verifica di ci che il paziente ha compreso . 
 il pre - requisito perch una decisione sia effettivamente autonoma lo stato di equilibrio psicofisico di chi la adotta . anche i soggetti sani e asintomatici possono presentare uno stato dansia o di patofobia che pu influenzare la scelta . 
i malati , a maggior ragione , possono essere soggetti a pressioni da parte di familiari o mass - media , tramite i quali gruppi di interesse propalano lidea che si possa trarre vantaggio da ogni nuova tecnologia , soprattutto se questa ottenibile senza esborso economico , in regime di servizio sanitario nazionale . lautonomia del paziente offre naturalmente importanti vantaggi al medico . 
in this way , moreover , the declaimed autonomy of the patient ends up becoming an insidious form of paternalism aimed at protecting the radiologist . the increasing use of radiology leads to an inevitable reduction in resources earmarked for other healthcare activities . 
in this way , the autonomy of some patients ends up limiting the autonomy of others and violating the principle of equity . consumerism does not only concern radiology but all branches of medicine . 
the diagnosis of spastic colitis , which regards one third of all gastroenterological disease [ 7 ] , binds the physician to prescribing antispastic medication , which has not proven to be more effective than a placebo and which may have side effects . 
the failure to prescribe , in addition to being unacceptable for the patient , could expose the physician to legal consequences . how can ethical problems be resolved ? it seems clear that the problem of allowing access to diagnostic technology or treatment of unproven effectiveness or uncertain benefit essentially derives from the fact that resources are not infinite . 
on the other hand , it is known that 10% of patients are responsible for some 70% of healthcare costs [ 9 ]  . given the premise that all are agreed upon the need to allocate healthcare resources to obtain the greatest benefit , the question of who shall be responsible for making this allocation becomes crucial . 
should it be the patients themselves who go without a certain type of service to avoid taking healthcare resources away from others ? should it be physicians who balance treatment among their patients , withholding it from one in favour of another , with the aim of obtaining the greatest benefit for society as a whole ? or should it be a collective decision that establishes a system that binds medical practitioners to using resources only up to a certain level and not beyond ? clearly , the solution should be sought in a balance between the different individual , technical and collective aspects . patients expect that the physician acts in their interest to guarantee the best treatment possible . 
however , they should be aware that physicians are constrained to rationing their services for financial reasons that are beyond their dere un modo di trasferire a questultimo la responsabilit professionale . 
in questo modo , peraltro , la declamata autonomia del paziente finisce per diventare una forma subdola di paternalismo , a protezione del radiologo . lincremento delluso della radiologia determina inevitabilmente una contrazione delle risorse destinate ad altre attivit sanitarie . 
in questo modo , lautonomia di alcuni pazienti finisce per limitare quella di altri e violare anche il principio di equit . il consumismo non riguarda solo la radiologia , ma tutta la medicina . 
principi etici mal applicati sono responsabili di altre , anche pi gravi , distorsioni della spesa sanitaria . un esempio quello fornito da evans e hungin [ 6 ] , riguardo la sindrome del colon irritabile , patologia comune quanto priva di trattamenti di provata efficacia . 
la diagnosi di colite spastica , che riguarda un terzo delle patologie gastroenterologiche [ 7 ] vincola il medico a prescrivere un farmaco antispastico che non si rivelato pi efficace del placebo , e che pu presentare effetti collaterali . 
la mancata prescrizione , oltre ad essere inaccettabile per il paziente , potrebbe esporre il medico a conseguenze legali . come risolvere i problemi etici ? evidente che il problema di concedere laccesso a tecnologie diagnostiche o trattamenti di efficacia non documentata o di incerto beneficio deriva essenzialmente dal fatto che le risorse non sono infinite . 
 posto che tutti sono daccordo sulla necessit di allocare le risorse sanitarie in modo da ottenere il maggior beneficio , la questione di chi debba essere responsabile di questa allocazione diventa centrale . 
deve essere il paziente stesso a rinunciare a qualche tipo di servizi , per non sottrarre ad altri risorse sanitarie ? deve essere il medico a bilanciare i trattamenti tra i propri pazienti , negandoli ad uno a favore di un altro , nellottica di ricavare il maggiore beneficio per la popolazione ? o deve essere la collettivit a stabilire un sistema che vincoli i medici ad usare le risorse solo fino a un certo livello e non oltre ? la soluzione , evidentemente , va ricercata in un equilibrio delle diverse istanze individuali , tecniche e collettive . i pazienti si attendono che il medico agisca nel loro interesse , per assicurare loro le migliori cure possibili . 
the shortage of beds or the long waiting lists for performing a mr may induce the physician to putting off the referral or even cancelling it totally , thus playing an active role in the implicit limitation of resources . 
unfortunately , this does not always occur solely on the basis of clinical needs , in that more demanding patients end up obtaining the referral to the detriment of those with the same clinical needs but who are less insistent . 
these patients , in fact , limit their own access to medical treatment , due to time constraints or fear of disease , with possible clinically manifest consequences . taxpayers ( who coincide with the patients , before they show signs of the disease ) have a very different attitude to patients . 
overall , as a society , they demand that physicians make optimal use of healthcare resources , without waste or incongruities , and obtain the maximum possible improvement in the health of the population . 
certain highly advantageous procedures , such as screening for colon cancer and influenza vaccines , have limited success , whereas certain radiological examinations are overutilized in terms of real clinical needs . in addition to the healthcare ramifications of their decisions , physicians are also called on to consider the economic and psychological costs for patients . 
unfortunately , they are not always able to act in the exclusive interest of the patient . long waiting lists and reduced accessibility to those healthcare resources can induce them to modify their decisions to favour healthcare services that are more available than useful . 
in addition , they can find themselves in an ambiguous role , because society requires that they ration healthcare resources among their patients in order to provide the greatest collective benefit possible , even to the detriment of the individual case . 
there is the expectation that they propose treatment plans to the individual patient that may not be the most useful , for the purposes of making savings . clearly , this condition compromises the trust in the doctorpatient relationship . the need to distribute healthcare resources to all , and therefore the need to ration them , has no evident ethical solutions [ 10 ]  . 
by way of extreme simplification , these are the system of judaeo - christian values and the scientific relativist system that goes by the name of social darwinism . possono indurre il medico a rinviare la prescrizione o a volte di cancellarla del tutto , in questo espletando un ruolo attivo di limitazione implicita delle risorse . 
sfortunatamente , ci non sempre avviene solo sulla base delle esigenze cliniche , in quanto i pazienti pi esigenti finiscono per ottenere comunque la prescrizione , a scapito di coloro che , a parit di necessit clinica , pongono richieste meno insistenti . 
taluni pazienti , di fatto , auto - limitano il proprio accesso alle cure mediche , per esigenze di tempo o patofobia , talora con conseguenze clinicamente manifeste . i contribuenti ( che coincidono con gli stessi pazienti , prima che si sia manifestato levento morboso ) hanno un atteggiamento molto diverso da quello dei pazienti . 
essi chiedono in primo luogo di non dover sopportare personalmente i costi dellaumentato ricorso alle cure mediche . nellinsieme , come societ , essi chiedono che i medici facciano un uso ottimale delle risorse sanitarie , senza sprechi e incongruenze , ottenendo il massimo miglioramento della salute della popolazione . 
noto che procedure molto vantaggiose , come lo screening dei tumori del colon e la profilassi vaccinale anti - influenzale , hanno scarso successo , mentre alcuni esami radiologici sono sovra - utilizzati rispetto alle reali necessit cliniche . i medici sono chiamati a considerare , oltre alle conseguenze sanitarie delle loro decisioni , anche i costi economici e psicologici per i pazienti . 
inoltre essi si trovano in un ruolo ambiguo , perch la societ chiede che essi razionino i beni sanitari tra i pazienti , ricavando il maggior beneficio collettivo , anche se a discapito del singolo caso . 
evidentemente , questa condizione compromette il rapporto fiduciario tra paziente e medico . la necessit di distribuire a tutti , e quindi di razionare , le risorse sanitarie , non ha soluzioni eticamente ovvie [ 10 ]  . il dissidio etico pu essere risolto in modi diametralmente opposti , a seconda di quello che il sistema di valori di riferimento . 
in estrema semplificazione , essi sono il sistema di valori giudaico - cristiano e quello scientifico - relativista che va sotto il nome di darwinismo sociale . judaeo - christian tradition the holy bible ( i kings 17 : 12 ) narrates how the prophet nella sacra bibbia ( 1re 17 : 12 ) si narra che il profeta elia , inviato dal signore in un luogo di preghiera , durante una la morale giudaico - cristiana radiol med ( 2009 ) 114 : 11731181 1179 elijah was sent by the lord to a place of prayer during a period of high drought . 
he turned to a widow ( in jewish tradition representative of the most abject poverty and social isolation ) for some food and water , and against all logic , the woman agreed to give him her final reserves , which miraculously were not used up . this episode in the old testament has a corresponding one in the gospels with the story of the multiplication of the loaves ( matthew 14 : 1321 ) , although it may be more correct to speak of the distribution of the loaves . in contrast to what the term multiplication may lead one to believe , the episode in the new testament , like that in the old testament , bears no suggestion of the magical creation of the food . 
on the contrary , magic , even if beneficial as for example the transformation of stones into bread is proffered by satan as a temptation and explicitly rejected by jesus ( luke 4 : 34 )  . 
the apparently impromptu distribution of goods that are essential for survival is unexpectedly followed not by them being used up , but by the miraculous discovery of unexpected resources : the flour and oil were not used up for the entire famine , the loaves and the fishes were distributed among all and a large quantity remained , probably because by following jesus teachings they had brought food with them and spontaneously accepted to share it . 
in line with these teachings , the judaeochristian tradition calls for giving each person all that they need without reservations . social darwinism the judaeo - christian tradition is decisively countered in our society by a set of moral principles that goes by the name of social darwinissocial darwinism is a naturalistic form of ethics based on the theory of evolution proposed by charles darwin ( 18091882 ) , which aims at replacing former dominant ethical principles , or those transcendent principles of the judaeo - christian tradition or the philosophical principles of kantian teachings . 
the principles of natural selection proposed by darwin in his origins of species were applied to human beings under the influence of the principles of malthusianism , which point to the need to combat excessive population , and the thinking of spencer , who rejected the idea of treating the sick on the basis of the principle of survival of the fittest . 
this led to the work by ernst haeckel , who rejected the sanctity of human life and invoked infanticide , abortion and the elimination of the mentally ill while exalting the model of ancient sparta . the step towards mein kampf by hitler is intuitive and immediate . 
less well known , perhaps , is the fact that tremenda siccit , si rivolgesse ad una vedova ( cio alla figura che rappresentava nel mondo ebraico la pi estrema miseria ed isolamento sociale ) per avere da lei cibo e acqua , e che avendo la donna , contro ogni logica , accettato di dare fondo alle ultime sue riserve , queste miracolosamente non si esaurirono . lepisodio biblico ha una corrispondenza con quello riportato nei vangeli con il nome di moltiplicazione dei pani ( matteo 14 : 1321 ) , ma che sarebbe forse pi corretto indicare come distribuzione dei pani . 
 contrariamente a quello che il termine moltiplicazione potrebbe far intendere , nellepisodio del nuovo testamento , cos come in quello della bibbia , non c alcun cenno alla creazione magica di un alimento . 
al contrario , la magia anche benefica , ad esempio la trasformazione delle pietre in pane , proposta dal maligno come tentazione , ed esplicitamente respinta da ges ( luca 4 : 34 )  . 
la distribuzione , apparentemente improvvida , di beni indispensabili per la sopravvivenza seguita inaspettatamente non dal loro esaurimento , ma dal reperimento , questo s miracoloso , di risorse impreviste : la farina e lolio non si esauriscono per tutta la carestia , i pani e i pesci vengono distribuiti tra tutti e ne avanza una gran quantit : probabilmente perch quanti , seguendo la predicazione di ges , avevano portato con s risorse alimentari , accettano spontaneamente di condividerle . 
in linea con questi insegnamenti , la morale giudaico - cristiana impone di dare a ciascuno , senza riserve , tutto ci di cui ha bisogno . il darwinismo sociale alla morale giudaico - cristiana si contrappone decisamente nella nostra societ una morale che si pu indicare con il nome di darwinismo sociale . 
il darwinismo sociale una forma naturalistica di etica basata sulla teoria dellevoluzione di charles darwin ( 18091882 ) , che mira a sostituire i principi etici prima dominanti , quelli trascendenti della cultura giudaico - cristiana o quelli filosofici della deontologia kantiana . 
lapplicazione agli esseri umani dei principi di selezione naturale proposti da darwin nella sua opera fondamentale lorigine della specie , sotto linfluenza dei principi del malthusianesimo , secondo i quali occorre combattere leccesso della popolazione , e del pensiero di spencer , che rigettava lidea di occuparsi dei malati in ragione del principio di sopravvivenza del migliore , ha prodotto lopera di ernst haeckel , che rigetta la santit della vita umana e invoca linfanticidio , laborto e leliminazione dei malati di mente , esaltando il modello della civilt spartana . 
il passaggio al mein kampf di hitler 1180 radiol med ( 2009 ) 114 : 11731181 concepts in line with these ideologies have been expressed by modern scientists such as watson , who was awarded the nobel prize in 1962 for the discovery of the dna double helix and who felt that abortion was justified in the event it was demonstrated that the fetus was genetically predisposed towards homosexuality ; or contemporary bioethicists such as peter singer , who justify euthanasia if the quality of life is not satisfactory . reflecting upon the links between darwinism and nazism , the irish bioethicist omathuna noted in a recent work that the vitality of the principles of social darwinism more than 60 years after the fall of the nazi dominion bears witness to the persistence of some principles that significantly undermine the concept of human dignity [ 11 ]  . the first of these principles is that ethics have a relative and not absolute value . 
if ethics are modified with changing environmental conditions , then even the evaluation of human dignity is relative . the second principle is evolutionis if humans have gradually evolved from animals , then there is no reason for treating humans differently from animals . 
this does not necessarily translate into an increased level of attention for animals , but rather into attributing to humans a value lower than that of animals . the third principle refers to the observation that inequality exists in nature and leads to different degrees of ability to adapt . 
meno noto , forse , il fatto che concetti in linea con tali ideologie siano stati espressi da scienziati moderni come watson , lo scopritore della struttura ad elica del dna , premio nobel 1962 , che riteneva giustificato laborto nel caso si fosse dimostrato che il nascituro fosse geneticamente predisposto allomosessualit ; o da bioeticisti contemporanei come peter singer , che giustifica leutanasia se la qualit della vita non soddisfacente . in un recente lavoro , omathuna , bioeticista irlandese , riflettendo sui legami tra darwinismo e nazismo , osserva come la vitalit dei principi del darwinismo sociale , a pi di sessantanni dalla caduta del dominio nazista , sia testimoniata dalla persistenza di alcuni principi che mettono in crisi il concetto di dignit umana [ 11 ]  . il primo di tali principi che letica ha un valore relativo , non assoluto . 
ci non si traduce necessariamente in un aumento del livello di attenzione verso gli animali , ma pu al contrario consistere nellattribuzione ad esseri umani di un valore inferiore a quello degli animali . il terzo principio si riferisce allosservazione che lineguaglianza esiste in natura e conduce a diversi gradi di capacit di adattamento . 
per analogia , la razza e le abilit mentali sono considerate determinanti la dignit umana . from this follows the fourth principle , which states that some existences have such limited value as to be considered unworthy of being lived . dai precedenti , consegue il quarto principio , che alcune esistenze hanno cos scarso valore , da essere ritenute indegne di essere vissute . last is the fifth principle , whereby natural selection is based on survival of the fittest . 
as a consequence , helping disadvantaged humans corresponds to opposing the laws of nature and can compromise the well - being and , in the end analysis , the survival of the human species . it is no hard task to realise which of these principles has survived the fall of nazism in modern capitalist society . 
the growing economic pressure on healthcare systems throughout the world and the economic and social inequalities between countries could , as occurred in germany prior to the rise in nazism , lead to a recovery of the principles of relativism and social darwinis any decision regarding the criteria of healthcare management in particular and society in general must take into account the reference system of values , whether they be judaeo - christian or darwinian . 
di conseguenza , aiutare gli esseri umani svantaggiati corrisponde ad opporsi alle leggi della natura e pu compromettere il benessere e in ultima analisi la sopravvivenza della specie umana . non difficile rilevare quanto di questi principi sia sopravvissuto alla caduta del nazismo nelle moderne societ capitalistiche . 
le pressioni economiche crescenti esercitate sui sistemi sanitari di tutto il mondo e le ineguaglianze economiche e sociali tra diversi paesi , potrebbero , come gi accadde in germania prima dellavvento del nazismo , agire per una ripresa dei principi del relativismo e del darwinismo sociale . 
qualunque decisione sui criteri di gestione della sanit , e della societ pi in generale , non pu prescindere dal confronto con i sistemi di valori di riferimento , siano essi giudaico - cristiani o darwinisti . 
in particular , the physician should evaluate and correctly and comprehensibly explain the possible risks of radiation exposure . in general , the radiologist who aspires to judaeo - christian values should seek to protect the patient from their own choices if these are not sufficiently valid . 
by performing the requested examinations without regard to their appropriateness , the radiologist will obtain evident and immediate advantages by increasing his or her own personal case series and professional experience , avoiding an undermining of relations with patients , and increasing their own fame and earnings . in this case , as in other situations , the choice based on ethical or religious principles is neither painless nor without consequences . ciascun radiologo potr compiere dipende direttamente dalladesione ad un preciso sistema di valori . 
per evitare di incorrere nel paternalismo , il medico dovr informare i pazienti in modo completo ed esaustivo non solo sulle procedure possibili , ma anche sulle controindicazioni e rischi di ciascuna procedura . 
in particolare , egli tenuto a valutare ed esporre correttamente e in modo comprensibile al paziente il rischio stocastico da radiazioni . in generale , il radiologo che si ispira ai principi giudaico - cristiani dovr cercare di proteggere i pazienti dalle loro stesse scelte , se non sufficientemente valide . 
eseguendo gli esami richiesti senza preoccuparsi della loro appropriatezza il radiologo otterr evidenti e immediati vantaggi : potr aumentare la propria casistica ed esperienza professionale , eviter di mettere in crisi il rapporto con i pazienti , accrescer la propria fama e i propri guadagni . 
cademartiri1 , 2 1dipartimento di radiologia e cardiologia , azienda ospedaliero - universitaria , parma , italy 2dipartimento di radiologia e cardiologia , erasmus medical center , rotterdam , the netherlands correspondence to : f . 
cademartiri , dipartimento di radiologia e diagnostica per immagine , c / o piastra tecnica piano 0 , azienda ospedaliero - universitaria di parma , via gramsci 14 , 43100 parma , italy , tel . : + 39 - 0521 - 703756 , fax : + 39 - 0521 - 704838 , e - mail : filippocademartiri@hotmail.com received : 13 november 2008 / accepted : 7 january 2009 / published online : 7 august 2009 springer - verlag 2009 abstract purpose . 
weighted ct dose index ( ctdi ) was measured by using an anthropomorphic phantom in spiral cardiac mode ( retrospective ecg gating ) at five pitch values adapted with two heart - rate - adaptive ecg pulsing windows using four algorithms : narrow pulsing window , with tube current reduction to 20% ( a ) and 4% ( b ) of peak current outside the pulsing window ; wide pulsing window , with tube current reduction to 20% ( c ) and 4% ( d )  . 
this feature is a potential limitation to its clinical use . as a result , algorithms have been developed that are able to modulate the radiation exposure without significantly deteriorating image quality at mdct coronary angiography ( mdct - ca ) [ 710 ]  . recently , it has been introduced dual - source ct ( dsct ) equipped with two x - ray tubes and two detectors mounted on the rotating gantry with an angular offset of 90 . 
single - source mdct [ 1013 ]  . in single - source scanners , the modulation modality allows a progressive reduction in radiation dose with a reduction in hr ( fixed - pitch protocol ) [ 1416 ]  . 
the scanners also have other special attributes : the collimation system is equipped with an additional bow - tie filter to limit the field of view ( fov ) to the cardiac region ; the dose can be adapted to the patients build [ combined applications to reduce exposure ( care ) dose 4d ] ; and as with single - source systems , an echocardiography ( ecg ) pulsing system can be used to vary the width of the application window by 100% of ma , as well la tomografia computerizzata multistrato ( tcms ) diventata un importante strumento diagnostico non invasivo per la patologia coronarica ( cad ) e cardiaca . 
tuttavia , lo sviluppo di apparecchiature sempre pi performanti ( per esempio tcms a 64 strati ) , comporta di regola un incremento della dose di esposizione alle radiazioni ionizzanti [ 46 ]  . 
questo fattore rappresenta una potenziale limitazione allimpiego clinico . sono stati pertanto sviluppati degli algoritmi capaci di modulare lesposizione alle radiazioni senza deteriorare significativamente la qualit dellimmagine mediante angiografia coronarica tcms ( ac - tcms ) [ 710 ]  . di recente stata introdotta sul mercato lapparecchiatura tcms a doppia sorgente ( dsct ) dotata di due tubi radiogeni e due detettori montati sulla pista di rotazione del gantry ad una compensazione angolare di 90 . 
questa configurazione determina una maggiore risoluzione temporale ( 83 ms ) e quindi una conseguente maggiore flessibilit di utilizzo a frequenza cardiaca elevata ( > 65 bpm ) rispetto alla tcms a singola sorgente [ 1013 ]  . nelle apparecchiature a singola sorgente la modalit di modulazione consentiva una progressiva riduzione della dose di radiazioni con la riduzione della frequenza cardiaca ( protocollo a pitch fisso ) [ 1416 ]  . 
inoltre , il sistema di collimazione equipaggiato con un addizionale cardiac bow - tie filter per limitare il field of view ( fov ) alla regione cardiaca ; la dose pu essere adattata al paziente in base alla radiol med ( 2009 ) 114 : 10371052 1039 as a system of ecg - controlled tube current modulation [ 13 ]  . 
with the combination of these techniques , dedicated dose - reduction algorithms can be applied according to the situation and the quantity of information that needs to be extrapolated from the examination . the aim of this study was to compare the difference of total radiation dose between two different pulsing windows and two different tube current modulation modalities at different hrs . materials and methods for this study , performed on a phantom , we used a firstgeneration dsct system ( siemens medical solutions , forchheim , germany )  . 
with a gantry rotation time of 330 ms and peak power of 80 kw / tube ( straton , siemens medical solution ) , a constant temporal resolution was obtained equal to a quarter of a gantry rotation ( 83 ms ) at the centre of the scan fov . 
this is one of the key differences with the conventional mdct systems , which can theoretically provide the same temporal resolution with multisegment reconstruction algorithms and a rhythmical and regular hr . the conventional indices of ct dose are also applicable to dsct because the doses regarding a single tube or both tubes simultaneously are the same value of combined doses . in dsct , the tube current value is calculated as the current time product per gantry rotation ( mas / rot ) with the sum of the mas of both x - ray tubes [ 13 , 1719 ]  . dose - reduction methods in cardiac dsct adaptive pitch - heart rate the pitch can be increased proportionally with increasing hr . 
the slowest heart beat during prescan monitoring is used for calculating the pitch . cardiac bow - tie filter conformazione corporea ( combined applications to reduce exposure [ care ] dose 4d ) ; pu essere utilizzato ( come per i sistemi a singola sorgente ) un sistema di modulazione cardio - sincronizzata dellemissione del tubo radiogeno ( electrocardiographic [ ecg ] - pulsing ) per variare larghezza della finestra di applicazione del 100% di amperaggio e un sistema di riduzione cardio - sincronizzata della corrente del tubo al di fuori della finestra di acquisizione ( ecg - controlled tube current modulation ) [ 13 ]  . 
 lo scopo di questo studio di confrontare la differenza di dose totale di radiazione tra due diverse finestre di pulsing e due diverse modalit di modulazione della corrente del tubo , a diversi valori di frequenza cardiaca . 
 materiali e metodi per questo studio su fantoccio stato utilizzato il sistema dual - source tc ( siemens medical solutions , forchheim , germania ) di prima generazione . principi di tcms a doppia sorgente il sistema dsct valutato dotato di due tubi radiogeni e due detettori montati ad una compensazione angolare di 90 . con un tempo di rotazione del gantry di 330 ms ed una potenza di picco di 80 kw / tubo ( straton , siemens medical solution ) , si ottenuta una costante risoluzione temporale pari ad un quarto del tempo di rotazione del gantry ( 83 ms ) , al centro del fov di scansione . 
per un imaging cardiaco dsct , sono necessari esclusivamente i dati provenienti da un solo ciclo cardiaco , quindi la risoluzione temporale indipendente dalla frequenza cardiaca ( fc ) del paziente e la modalit base di un sistema dsct corrisponde alla ricostruzione a singolo segmento . 
questa una delle maggiori differenze con i sistemi tcms convenzionali , che teoricamente possono fornire la stessa risoluzione temporale con algoritmi di ricostruzione multi - segmentari e con un battito cardiaco ritmico e regolare . 
i convenzionali indici di dose nella tomografia computerizzata ( tc ) sono applicabili anche in dsct , poich le dosi relative ad un singolo tubo o su entrambi i tubi radiogeni simultaneamente risultano nello stesso valore di dose combinate . 
in dsct , il valore della corrente del tubo viene calcolato come il prodotto della corrente al secondo per il tempo di rotazione del gantry ( mas / rot ) sommando i mas di entrambi i tubi [ 13 , 1719 ]  . 
 metodi di riduzione della dose in dsct cardiaco pitch heart rate - adattativo as the region of interest ( roi ) is the heart , the collimation il pitch pu aumentare parallelamente allaumento della frequenza cardiaca . 
questo adattamento porta ad una 1040 radiol med ( 2009 ) 114 : 10371052 system of the dsct is equipped with two bow - tie filters . the first is defined cardiac and is used to limit the fov to the cardiac region , whereas the second is the body filter , which is also present in 64 - slice mdct . riduzione dellesposizione alle radiazioni corrispondente ad una dose media direttamente proporzionale a 1 / pitch . 
il pi basso ritmo cardiaco rilevato durante il monitoraggio prescansione determina il valore del pitch . care dose 4d filtro bow - tie cardiaco this is an automatic exposure control system ( care dose 4d , siemens medical solution , forchheim , germany ) , which can combine automatic modulation of the tube current along the acquisition volume in relation to the angular attenuation coefficient of the examined region and to attenuation values of different tissues on the basis of data acquired from a single localiser radiograph ( on - line modulation ) [ 15 ]  . ecg - controlled tube current modulation tube current modulation during the entire cardiac cycle was introduced in cardiac ct to help reduce exposure by ~50% [ 1416 ]  . 
prior to the scan , the operator can manually select the width of the image acquisition window ( pulsing window = window of max ma , or 100% of ma ) in the r - r interval corresponding to the phase of greatest immobility of the heart and coronary arteries . 
thanks to a system of ecg - controlled tube current modulation , the radiation delivered can be reduced outside the pulsing window to 20% ( functional imaging ) or 4% ( mindose ) of the peak tube current , thus markedly reducing radiation exposure . 
modulation to 4% is applied to minimise the total time of radiation exposure [ 13 , 17 , 20 ]  . in mdct equipment , ecg - controlled tube current modulation requires a rhythmical and regular heart beat . when a patients heart beat is not regular , the optimal reconstruction interval of the raw data may occur during the window of reduced tube current , thus requiring an interpolation of the data of two adjacent heart beats . 
in dsct equipment , the algorithm that analyses the heart beat is able to recognise an r - r interval significantly different from the expected r - r interval and automatically widen the pulsing window ( ecg editing )  . 
this adaptation tends to increase the dose delivered to the patient but at the same time automatically optimises the acquisition data set , thus overcoming one of the main limitations of previous mdct equipment [ 10 , 11 , 13 ]  . three - dimensional adaptive noise reduction algorithm a further system of dose optimisation in dsct systems is the 3d reconstruction algorithm , which employs special adaptive convolution kernels ( b26 ) that are able to reduce poich la regione dinteresse quella cardiaca , il sistema di collimazione della dsct dotato di due bow - tie filters . 
il primo definito cardiac , per limitare il fov alla regione cardiaca ; il secondo il filtro body , presente anche in tc a 64 strati . care dose 4d il care dose 4d un sistema di limitazione automatico della dose ( siemens medical solution ) in grado di combinare la modulazione automatica della corrente del tubo radiogeno lungo il volume di acquisizione in relazione al coefficiente di attenuazione angolare della regione in esame e ai valori di attenuazione dei diversi tessuti , sulla base dei dati rilevati dallo strato precedente durante il topogramma ( on - line modulation ) [ 15 ]  . modulazione cardio - sincronizzata della corrente del tubo la modulazione dellemissione del tubo radiogeno durante lintera durata del ciclo cardiaco stata introdotta in cardio - tc , come aiuto per ridurre la dose di ~50% [ 1416 ]  . prima della scansione , loperatore pu selezionare manualmente la larghezza della finestra di acquisizione delle immagini ( finestra di pulsazione , o pulsing window vale a dire la finestra di massimo amperaggio [ max ma o 100% di ma ] ) nellintervallo r - r in corrispondenza della fase di maggior immobilit del cuore e delle arterie coronariche . 
grazie ad un sistema di modulazione cardio - sincronizzato della corrente del tubo , o ecg - controlled tube current modulation , lerogazione raggi pu essere ridotta al di fuori della finestra di pulsing al 20% ( functional imaging ) o al 4% ( mindose ) del valore nominale della corrente del tubo , per ridurre fortemente lesposizione alle radiazioni . 
la qualit delle immagini acquisite al 20% di ma permette lo studio cinetico - morfologico del ventricolo sinistro ; la modulazione al 4% viene applicata per ridurre al minimo i tempi totali di esposizione raggi [ 13 , 17 , 20 ]  . 
quando il ritmo cardiaco del paziente non regolare , lintervallo di ricostruzione ottimale dei dati grezzi potrebbe verificarsi durante la finestra di riduzione della corrente del tubo richiedendo un interpolazione dei dati dei due battiti cardiaci adiacenti . 
nellapparecchiatura dsct , lalgoritmo di analisi dellandamento del ritmo cardiaco permette di riconoscere un intervallo r - r significativamente differente da quello previsto e allargare automaticamente la finestra di pulsing ( ecg - editing )  . 
the common scan parameters were the following : two x - ray sources , 380 mas / rot with 120 kv , detector collimation 320.6 mm , slice collimation 640.6 mm ( zaxis flying focal spot ) ; gantry rotation time 330 ms . 
the pitch varied between 0.2 ( 45 bpm ) and 0.5 ( 120 bpm ) at low and high hrs , being automatically adapted by the device to the hr utilised : 45 , 60 , 75 , 90 and 120 bpthe craniocaudal range of the scan was 90 m images were reconstructed in single - segment modality with 0.75 - mm slice thickness , reconstruction increment 0.7 mm and mediumsmooth convolution kernel ( b31f )  . 
for each hr value , four series of reconstructions were done from 0% to 95% of the r - r interval with 5% steps for a total of 20 data sets . in dsct - ca a narrow pulsing window can be used in patients with low ( 65 bpm ) and hig ( 80 bpm ) hr without significant deterioration in image quality . 
however , in most patients with an intermediate hr ( 7075 bpm ) , a wider data acquisition window is required to obtain the best - quality data sets [ 2123 ]  . 
1 fantoccio tc cardiaco antropomorfico . mento determina un aumento della dose di esposizione del paziente , ma allo stesso tempo unottimizzazione automatica del data set di acquisizione , superando uno dei principali limiti delle apparecchiature tcms precedenti [ 10 , 11 , 13 ]  . algoritmo 3d adattativo di riduzione dal rumore come ulteriore sistema di ottimizzazione della dose , nel sistema dsct stato implementato un algoritmo di convoluzione adattativo di ricostruzione 3d ( kernel b26 ) , in grado di ridurre il rumore dellimmagine a parit di dose . 
il kernel b25 di un sistema 64 tc usa un algoritmo simile , ma implementato solo in 2 dimensioni : allinterno del piano dellimmagine ( x , y ) , ma non lungo lasse z . 
i parametri di scansione comuni sono : due sorgenti a raggi x ; 380 mas / rot ad una tensione di 120 kv , collimazione del detettore 320 , 6 mm , collimazione della slice 640 , 6 mm ( z - axis flying focal spot ) ; tempo di rotazione del gantry 330 ms . 
il pitch variava tra 0 , 2 ( 45 bpm ) e 0 , 5 ( 120 bpm ) , a basse ed elevate frequenze cardiache , adattato automaticamente dalla macchina alle frequenze cardiache utilizzate : 45 , 60 , 75 , 90 e 120 bpil range cranio - caudale di scansione era 90 mle immagini sono state ricostruite in modalit a singolo - segmento con spessore della slice 0 , 75 mm ; incremento 0 , 7 mm ; kernel di convoluzione medium - smooth ( b31f )  . 
per ogni valore di frequenza cardiaca sono state ricostruite 4 serie di ricostruzioni , dallo 0% al 95% dellintervallo r - r con passo del 5% , per un totale di 20 data set . in dsct - ca , nei pazienti con bassa ( 65 bpm ) ed alta frequenza cardiaca ( 80 bpm ) pu essere utilizzata una finestra di pulsazione stretta , senza significativo deterioramento della qualit dellimmagine . 
invece , nella maggioranza dei pazienti con frequenza cardiaca intermedia ( 7075 bpm ) necessaria una finestra di acquisizione dati ampia per ottenere la miglior qualit del data set [ 2123 ]  . lottimale finestra di pulsing a basse , intermedie , alte frequenze cardiache 65%76% ( fase telediastolica ) , 30%77% ( telediastole + telesistole ) , 31%47% ( fase telesistolica )  . 
 in questo studio stata valutata la dose di radiazione relativa allutilizzo degli algoritmi dsct di risparmio della dose , alla valutazione morfo - funzionale del ventricolo sinistro e alla frequenza cardiaca . 
the use of the optimal window produces a 64% reduction in exposure at both low and high hr [ 2123 ]  . our study evaluated radiation exposure in terms of hr , the morphofunctional evaluation of the left ventricle , and the use of the dsct algorithms for dose reduction . 
the constant hr produced by the ecg simulator did not allow evaluation of the ecg editing system . dose exposure evaluation we calculated the ct dose index volume [ ctdivol ( mgy ) , where ( j ) ( kg - 1 ) = ( gy ) ] and the dose - length product [ dlp ( mgy cm ) ] as parameters of absorbed dose ( table 2 )  . ctdivol considers the scan parameters ( kv , mas / rot , scan time ) and describes the mean dose relative to the scan volume , the type of equipment and the pitch . 
for the scan length used in our study ( 90 mm ) , the mean number of cardiac cycles required for to reconstruct the total scan volume varied between seven and six beats . 
il ctdivol mette in relazione i parametri di scansione ( kv , mas / rot , tempo di scansione ) e descrive la dose media relativa al volume di scansione , al tipo di apparecchiatura e al pitch . 
per la lunghezza di scansione utilizzata in questo studio ( 90 mm ) , il numero medio di cicli cardiaci necessari alla ricostruzione del volume totale di scansione variava tra 78 battiti . in unapparecchiatura dsct , lutilizzo dellalgoritmo di modulazione cardio - sincronizzato della corrente del tubo comporta una dose di radiazione proporzionale al valore medio della corrente applicata durante la scansione [ 13 ] : [ ( tminmamin ) + ( tmaxmamax ) ] / ( tmin + tmax ) dove tmin e tmax sono i tempi alla minima ( mamin ) e alla massima corrente del tubo ( mamax ) , rispettivamente , allinterno di un ciclo cardiaco . 
 [ ( tminmamin ) + ( tmaxmamax ) ] / ( tmin + tmax ) statistica where tmin and tmax are the times at the minimum ( mamin ) and maximum tube current ( mamax ) , respectively , within a cardiac cycle . 
in addition , we calculated the effective dose by using the conversion coefficient for the chest ( dpl0.017 ) ( table 2 )  . tutti i dati sono presentati come mediadeviazione standard ( ds )  . 
il confronto tra i valori di dlp e dlp effettivo stato effettuato mediante test t di student per dati appaiati e una p < 0 , 05 stata considerata significativa . 
the correlations were evaluated with the pearson r coefficient . risultati statistics results the scan time was ~810 s at low hr ( 60 bpm ) , 6 s at intermediate hr ( 75 bpm ) and ~45 s at high hr ( 90 bpm )  . 
con una finestra di acquisizione stretta , la modulazione della corrente al 4% di ma riduce la dose del 35 , 15% ( 2 , 51 msv ; p < 0 , 005 )  . 
pitch ( table feedgantry rotation time ) / slice collimation ; fc , heart rate , bpm beats per minute . pitch / fc ( 2 ) = pitch / hr ( 2 ) fig . 
il valore del pitch aumenta parallelamente allaumentare della frequenza cardiaca , sulla base del pi basso battito rilevato durante il monitoraggio pre - scansione . pitch = ( table feedtempo di rotazione ) / collimazione slice ; fc , frequenza cardiaca ; bpm , battiti per minuto . pitch / fc ( 2 ) = pitch / hr ( 2 ) dose exposure evaluation table 2 shows estimates of the ctdivol and dlp dose indices . 
the dose delivered beyond the scan range corresponds to the difference between dlp and effective dlp ( dlpe ) calculated for the scan length used in this study ( mgy 90 mm )  . 
la dose erogata oltre il range di scansione corrisponde alla differenza tra il dlp ed il dlp effettivo ( dlpe ) , calcolato per la lunghezza di scansione utilizzata in questo studio ( mgy90 mm )  . 
il dlpe risultato 5 , 651 , 48 msv , 3 , 660 , 86 msv , 7 , 62 , 48 msv , 5 , 91 , 87 msv , nel protocollo a , b , c e d , rispettivamente ( tabella 2 )  . il decreto legge 230 / 95 relativo alle norme radio - protezionistiche emanate dallinternational commission on radiological protection ( icrp ) , sancisce che il personale classificato non esposto alle radiazioni ionizzanti ( per esempio i pazienti ) suscettibile ad unesposizione globale annua < 1 msv . 
in patients with low , intermediate and high heart rates , the use of a narrow pulsing window produces a significant reduction in dose , with image quality remaining unchanged . 
nei pazienti con bassa , intermedia ed alta frequenza cardiaca , lutilizzo di una finestra di pulsazione stretta determina una significativa riduzione di dose , a parit di qualit di immagine . 
therefore , all the available dose reduction algorithms should be implemented . discussione lesposizione alle radiazioni ionizzanti rappresenta uno dei limiti allimpiego clinico delle apparecchiature tc multistrato nellimaging cardiaco non invasivo [ 13 ]  . 
come noto , in modalit spirale i dati sono continuamente raccolti attraverso il ciclo cardiaco per essere successivamente ricostruiti in una fase del ciclo cardiaco definita arbitrariamente [ 710 , 24 ]  . 
the graphs show the difference between the radiation dose delivered during the scan ( dlp ; a ) and the dose calculated for effective scan length of 90 mm ( dlpe ; b )  . 
i grafici mostrano la differenza tra la dose di esposizione erogata durante la scansione ( dlp ; a ) e la dose relativa leffettiva lunghezza di scansione di 90 mm ( dlpe ; b )  . 
therefore , on the basis of the data obtained , the choice of the optimal ecg pulsing window seems to be indispensable for achieving a significant reduction in exposure at low hr [ 710 , 22 , 23 ]  . dependence on minimum pulsing value on the basis of the scan parameters used , ( 120 kv , 380 mas / rot , slice thickness 0.75 , pitch - hr adaptive protocol ) , the dose delivered with 20% modulation to the maximum ma with narrow and wide pulsing window ( r = 0.99 ; p < 0.005 ) can be significantly reduced with modulation to 4% . 
 the results obtained suggest that : the use of the pitchhr adaptive protocol produces a reduction in dose proportional to the increase in hr in dsct - ca examinations with retrospective ecg gating , the use of dose - reduction algorithms , ecg pulsazione , narrow e wide pulsing window , modulate al 20% e al 4% del valore nominale della corrente del tubo . dipendenza dalla frequenza cardiaca nelle acquisizioni pulsate , ladattamento del valore di pitch alla frequenza cardiaca risulta in una significativa riduzione del ctdivol allaumentare della frequenza cardiaca ( 45120 bpm )  . 
determina una significativa riduzione di dose con e senza valutazione dei volumi del ventricolo sinistro ( r = 0 , 97 ; p < 0 , 05 ) , se confrontato con il protocollo wide pulsing window . 
quindi in base ai dati ottenuti , lottimale finestra ecg - pulsing sembra essere indispensabile per una significativa riduzione della dose a bassa ed elevata frequenza cardiaca [ 710 , 22 , 23 ]  . 
 dipendenza dal valore minimo di pulsazione sulla base dei parametri di scansione utilizzati ( 120 kv , 380 mas / rot , slice thickness 0 , 75 , pitch heart rate - adattativo ) , la dose erogata con modulazione al 20% di max ma mediante narrow e wide pulsing window ( r = 0 , 99 ; p < 0 , 005 ) pu essere significativamente ridotta con modulazione al 4% . 
 dai risultati ottenuti possibile dedurre che : lutilizzo del pitch heart rate - adattativo permette una riduzione di dose proporzionale allaumento della frequenza cardiaca ; nelle indagini dsct - ca eseguite mediante ecg - gated retrospettivo , lutilizzo degli algoritmi di risparmio della dose , ecg - pulsing e ecg - controlled tube current modulation , risultano in una riduzione di dose a tutte le frequenze cardiache , con i migliori risultati a basse ed alte frequenze cardiache ; la modulazione della corrente del tubo al 4% del valore nominale di ma al di fuori della finestra di 1050 radiol med ( 2009 ) 114 : 10371052 pulsing and ecg - controlled tube current modulation produces a reduction in dose at all hrs , with the best results at low and high hr tube current modulation to 4% of the peak tube current outside the pulsing window is recommended in cases where a cardiac motion - morphological evaluation of the left ventricle is not required , with a dose reduction proportional to the increase in hr . with respect to data in the literature that report exposure values for dsct systems of 12 / 9 msv ( hr 65 bpm ) , 15 / 12 msv ( hr 6679 bpm ) and 7 / 4 msv ( hr 80 bpm ) with the use of optimal pulsing window and modulation to 20% / 4% [ 22 , 23 ] , application of ecg pulsing and ecgcontrolled tube current modulation allowed us to achieve overall lower exposure values at all hrs [ 28 ]  . a recent study comparing prospective ecg triggering and retrospective ecg gating in dsct - ca [ 29 ] reported significantly lower values of effective dose with the use of prospective scan algorithms . 
dsct systems have not yet been equipped with a similar mobile system . limitations the optimisation of scan protocols in cardiac ct is based solely on the calculation of the dose of ionising radiation delivered derived from measurement on a phantom and on the basis of the algorithms developed for this study . 
in addition , not all dose - reduction algorithms available with this technique were evaluated , such as reduction and / or increase in tube voltage , the use of double voltage , the b26 convolution kernel , the care dose 4d system and ecg editing . 
however , the considerations formulated appear reasonable and take into account the technical features of dsct systems . recommendations for use results from our study suggest the use of tube current acquisizione - dati raccomandabile nei casi in cui non sia richiesta una valutazione cinetico - morfologica del ventricolo sinistro , per una riduzione di dose proporzionale allaumento della frequenza cardiaca . rispetto ai dati disponibili in letteratura che riportano per apparecchiature dsct valori di dose di 12 / 9 msv ( fc65 bpm ) , 15 / 12 msv ( fc 6679 bpm ) e 7 / 4 msv ( fc80 bpm ) con lutilizzo dellottimale finestra di pulsing e modulazione al 20% / 4% [ 22 , 23 ] , lapplicazione del sistema di modulazione ecg - pulsing ed ecg - controlled tube current modulation ha consentito di ottenere nel nostro studio valori di dose complessivamente inferiori , a tutti i valori di frequenza cardiaca [ 28 ]  . un altro recente studio eseguito mediante confronto ecg - prospettico vs ecg - retrospettivo in dsct - ca [ 29 ] , mostra valori di dose efficace significativamente inferiori mediante lapplicazione di algoritmi di scansione prospettici . 
questi algoritmi sembrano ridurre maggiormente la dose ed appaiono pi efficaci a bassa frequenza cardiaca . tuttavia , il principio su cui si basano non garantisce una perfetta coerenza tra la fase del ciclo cardiaca identificata e la fase effettiva . nel 2008 , sono stati introdotti ulteriori metodi di limitazione della dose su apparecchiature tc multistrato ( definition as + , siemens medical solutions ) che consistono in un nuovo sistema di collimazione mobile che si posiziona davanti alla finestra di uscita dei raggi x prima e dopo leffettiva lunghezza di scansione , formando uno scudo che frena le radiazioni erogate oltre il range di scansione . lapparecchiatura dsct non ancora dotata di un simile sistema mobile . 
 limitazioni lottimizzazione dei protocolli di scansione in tc del cuore si basa solo sul calcolo della dose di esposizione alle radiazioni ionizzanti risultata da misurazioni su fantoccio e in base agli algoritmi elaborati per questo studio . 
inoltre non sono stati valutati tutti gli algoritmi di risparmio della dose disponibili con questa metodica , quali : la riduzione e / o laumento del voltaggio del tubo radiogeno , lutilizzo del doppio voltaggio , il kernel di convoluzione b26 , il sistema care dose 4d ed ecg - editing . 
 raccomandazioni di utilizzo in generale , ad ogni valore di frequenza cardiaca , dovrebbe essere utilizzata una modulazione della corrente al 20% del valore nominale della corrente del tubo per una valutazione radiol med ( 2009 ) 114 : 10371052 1051 modulation to 20% of the peak tube current for a kineticfunctional evaluation of the left ventricle and in all other cases the minimisation of the pulsing value to spare patients unnecessary radiation exposure : low hr ( 65 bpm ) : use a narrow pulsing window corresponding to the end - diastolic phase of the cardiac cycle ( 65%76% of the r - r interval ) intermediate hr ( 65 bpm < hr < 80 bpm ) : use a wide pulsing window that includes end diastole and end systole ( 31%76% of the r - r interval ) high hr ( hr > 80 bpm ) : use a narrow pulsing window corresponding to the end - systolic phase ( 31%47% of the r - r interval )  . cinetico - funzionale del ventricolo sinistro ; in tutti gli altri casi , si dovrebbe prediligere una riduzione al minimo valore di pulsazione per preservare il paziente da uninutile esposizione alle radiazioni ionizzanti : frequenza cardiaca bassa ( fc65 bpm ) : utilizzare una finestra di pulsazione stretta in corrispondenza della fase telediastolica del ciclo cardiaco ( 65%76% dellintervallo r - r ) ; frequenza cardiaca intermedia ( 65 bpm < fc < 80 bpm ) : utilizzare una finestra di pulsing ampia che comprenda telediastole e telesistole ( 31%76% dellintervallo r - r ) ; frequenza cardiaca elevata ( fc > 80 bpm ) : utilizzare una finestra di pulsazione stretta in corrispondenza della fase telesistolica del ciclo cardiaco ( 31%47% dellintervallo r - r )  . conclusions conclusioni the dose - reduction algorithms available on dsct systems enable the effective dose to be kept below 10 msv in most cases , with the exception of low hr ( 12.68 msv at 4560 bpm ) with imaging protocol . 
when the mindose protocol is routinely applied ( foregoing the possibility of performing an evaluation of left ventricular volumes ) , the dose delivered on average is below 8 msv . the wide functional gli algoritmi di riduzione della dose disponibili su apparecchiatura dsct consentono di mantenere la dose efficacie al di sotto dei 10 msv nella maggior parte dei casi , ad eccezione delle basse frequenze cardiache ( 12 , 68 msv a 4560 bpm ) con protocollo wide functional imaging . 
this study evaluated the effectiveness of contrast - enhanced ultrasound ( ceus ) , performed immediately after percutaneous ethanol injection ( pei ) or radiofrequency thermal ablation ( rfta ) , by comparing results with the computed tomography ( ct ) follow - up . 
in the first group , ct identified complete necrosis in 28 / 54 ( 52% ) lesions , 21 ( 75% ) of which had avascular , one ( 4% ) isovascular and six ( 21% ) hypovascular patterns at ceus . 
in the second group , ct showed complete necrosis in 31 / 36 ( 86% ) lesions , all ( 100% ) of which had a corresponding avascular pattern at ceus . 
sensitivity , specificity , positive predictive value , negative predictive value and accuracy of the avascular pattern at ceus compared with ct findings were 75% , 69% , 72% , 72% and 72% for pei and 100% , 20% , 89% , 100% and 89% , for rfta , respectively . 
sono stati descritti tre pattern di enhancement alla ceus ( 1 , isovascolare ; 2 , ipovascolare ; 3 , avascolare ) e quindi confrontati con la tc di follow - up . 
nel primo gruppo , la tc ha evidenziato necrosi completa in 28 / 54 ( 52% ) lesioni , di cui 21 ( 75% ) con pattern ceus avascolare , 1 ( 4% ) isovascolare e 6 ( 21% ) ipovascolare . 
sensibilit , specificit , valore predittivo positivo e negativo ed accuratezza diagnostica per necrosi completa alla tc del pattern ceus avascolare sono 75% , 69% , 72% , 72% e 72% dopo pei , 100% , 20% , 89% , 100% e 89% dopo rfta . 
ceus performed immediately after percutaneous ablation of hepatocellular carcinoma to evaluate treatment efficacy is compulsory in the case of rfta but not for pei . keywords contrast - enhanced ultrasound hepatocellular carcinoma ablation therapy radiofrequency ethanol injection computed tomography conclusioni . 
la ceus al termine della procedura ablativa dellepatocarcinoma ai fini della valutazione del successo terapeutico mandatoria dopo rfta , ma non dopo pei . parole chiave ecografia con mdc carcinoma epatocellulare terapie ablative radiofrequenza alcolizzazione tomografia computerizzata introduction introduzione hepatocellular carcinoma ( hcc ) is one of the most common cancers in the world , representing about 85% of primary liver tumours , characterised by an incidence of about 500 , 000 new cases per year [ 1 , 2 ]  . 
based upon the high positive predictive value ( ppv ) and a good cost - effectiveness ratio [ 3 ] , the guidelines published by the american association for the study of liver diseases ( aasld ) recommend ultrasonography as the most useful imaging method for screening and surveillance . surgical resection is still considered to be the treatment of choice for curative therapy for hcc in patients with good hepatic functional reserve ( child - pugh class a and minimal portal hypertension : platelet count > 100 , 000 / mm3 and / or hepatic venous pressure gradient < 10 mmhg )  . however , patients in this category represent only about 15% [ 4 , 5 ]  . 
thus , in recent years , some nonsurgical treatments such as percutaneous ethanol injection ( pei ) [ 6 , 7 ] and radiofrequency thermal ablation ( rfta ) [ 8 , 9 ] were developed . 
the aim of these therapies is to treat patients who are not candidates for resection due to advanced age , underlying diseases , multinodular hccs or unfavorable locations , such as tumours too close to vascular and biliary structures [ 6 , 812 ] , by means of a complete coagulation tumor necrosis . 
usually , the response is monitored with computed tomography ( ct ) , magnetic resonance ( mr ) [ 1517 ] and , more recently , with contrast - enhanced ultrasonography ( ceus ) [ 1722 ]  . 
in most published studies , these examinations are not performed immediately lepatocarcinoma ( hcc ) una delle neoplasie pi comuni al mondo e costituisce l85% dei tumori primitivi del fegato , con incidenza pari a circa 500000 nuovi casi / anno [ 1 , 2 ]  . 
il principale fattore di rischio la cirrosi epatica , in partico ( virus lare quando secondaria ad dellepatite b , hbv , o virus dellepatite c , hcv ) o ad abuso di alcool . 
come raccomandato dalle linee guida pubblicate dallamerican association for the study of liver diseases ( aasld ) , lecografia addominale la tecnica dimmagine pi utilizzata per lo screening e la sorveglianza , avendo un alto valore predittivo positivo [ 3 ] e buon rapporto costoeffacacia . infezioni virali la resezione chirurgica tuttora considerata il gold standard per la terapia curativa dellhcc nei pazienti con buona riserva funzionale epatica ( child - pugh a e minima ipertensione portale : piastrine > 100000 / mm3 e / o gradiente pressorio venoso epatico < 10 mmhg )  . 
pertanto nel corso degli ultimi anni sono stati proposti alcuni tipi di trattamento percutaneo non chirurgico , come liniezione percutanea di etanolo ( pei ) [ 6 , 7 ] e la termoablazione con radiofrequenza ( rfta ) [ 8 , 9 ]  . 
lo scopo di tali trattamenti di poter trattare , mediante il raggiungimento di una completa necrosi coagulativa del tessuto tumorale , i pazienti considerati inoperabili per let avanzata , per comorbilit , per caratteristiche proprie del tumore , come la stretta adiacenza a strutture vascolari e biliari o per la multifocalit delle lesioni [ 6 , 812 ]  . 
inoltre , la resezione nei pazienti cirrotici con ridotta funzionalit epatica conduce ad un significativo rischio di insufficienza epatica post - chirurgica e di recidive post - chirurgiche per foci tumorali non diagnosticati allimaging pre - operatorio . 
 tali trattamenti mini - invasivi consentono peraltro di poter trattare pi volte il paziente , senza ridurre in maniera significativa la funzionalit epatica , con indici di sopravvivenza paragonabili a quelli del trattamento chirurgico [ 7 ]  . una precoce e corretta valutazione della risposta al trattamento rappresenta un importante fattore prognostico della sopravvivenza a lungo termine [ 13 , 14 ]  . 
generalmente , la risposta ai trattamenti viene valutata mediante tomografia computerizzata ( tc ) , risonanza magnetica ( rm ) 1096 radiol med ( 2009 ) 114 : 10941105 but 13 months after the procedure [ 16 , 1821 ]  . 
ceus has been proposed in the early posttreatment phase at 2448 h after the procedure [ 17 , 21 , 22 ]  . the aim of our study was to evaluate the efficacy of ceus , performed immediately after pei and / or rfta treatments , by comparing our results with the ct follow - up . materials and methods from december 2005 to december 2007 , 69 consecutive patients ( 55 men and 14 women ; mean age 69 years ; age range 2889 years ) with a proven diagnosis of hcc and candidates to percutaneous procedures ( pei or rfta ) were included in this prospective study , which was approved by the ethics committee of our institute . 
before all examinations and procedures , all patients gave written informed consent . the inclusion criteria were : ( a ) proven diagnosis of hcc by means of histology or derived from the agreement of two tumour main diagnostic modalities [ 23 ] ; ( b ) good detectability at us ; ( c ) cirrhosis with child - pugh risk class b7 or less ; ( d ) single hcc with main diameter < 5 cm ; ( e ) multinodular hcc ( up to three lesions ) with main diameter < 3 cm per lesion ; ( f ) absence of thrombosis of portal vein or distant metastases ; ( g ) prothrombin time > 50% and platelet count > 60109 / l ; ( h ) refused surgery . the exclusion criteria were : ( a ) cirrhosis with childpugh risk class b8 or more ; ( b ) single hcc with main diameter > 5 cm ; ( c ) multinodular disease with more than three lesions with main diameter > 3 cm per lesion ; ( d ) thrombosis of portal vein ; ( e ) distant metastases ; ( f ) refractory ascites ; ( g ) severe coagulopathy ; ( h ) underlying diseases with a poor life expectancy . 
 all patients , divided into two homogeneous groups , underwent conventional us and ceus before treatment . during the same interventional session , immediately after each single percutaneous procedure , ceus was performed to evaluate residual perfusion within the treated lesion . after 1 month , a contrast - enhanced ct as gold standard was performed to assess the degree of tumour necrosis . 
sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and accuracy of ceus after pei or rfta were evaluated and compared with ct findings . 
a p value < 0.05 was considered statistically significant . conventional ultrasonography and contrast - enhanced ultrasonograph the evaluation and precise localisation of the lesions to be [ 1517 ] e , pi di recente , con ecografia con mezzo di contrasto ( ceus ) [ 1722 ]  . 
alcuni autori hanno proposto lapplicazione della ceus nella precoce valutazione dei trattamenti , a 2448 ore dal termine della procedura [ 17 , 21 , 22 ]  . lo scopo del nostro studio stimare la reale efficacia dellecografia con mezzo di contrasto eseguita nellimmediato periodo post - pei e / o rfta , nel corso della stessa sessione interventistica , confrontando i risultati con la tc di follow - up . materiali e metodi da dicembre 2005 a dicembre 2007 sono stati inseriti prospetticamente nello studio 69 pazienti consecutivi ( 55 maschi e 14 femmine ; et media di 69 anni , range : 2889 anni ) con diagnosi certa di epatocarcinoma , candidati a procedure percutanee ( pei o rfta )  . 
sono stati quindi valutati sensibilit , specificit , valore predittivo positivo ( vpp ) e negativo ( vpn ) ed accuratezza diagnostica della ceus post - pei o rfta rispetto ai reperti riscontrati in tc . 
il confronto statistico stato effettuato utilizzando il test del 2e valori di p < 0 , 05 sono stati considerati statisticamente significativi . radiol med ( 2009 ) 114 : 10941105 1097 treated was performed by using a sequoia 512 ( acusonsiemens , erlangen , germany )  . 
a nonlinear imaging method that detects the reflected harmonics ( usually the second harmonic ) of a transmitted pulse or a phase inversion method can be used . when a suspected lesion was identified , it was measured routinely . then , the study protocol involved ceus before and immediately after the ablation therapy and performed by the same operator . 
harmonic microbubble - specific imaging with low acoustic us pressure ( 24 mhz transducer ; mechanical index < 0.2 ; 1213 frame rate / s ) was performed utilising the same machine . 
all ceus examinations were recorded on a magneto - optical disk syste a standard real - time dynamic study , in the arterial phase ( hyperechogenicity of the intrahepatic arterial branches ) about 1525 s after contrast injection , and in the portal venous phase ( hyperechogenicity of the portal vein and its intrahepatic branches ) about 4060 s after contrast injection and finally in the late sinusoidal phase ( hyperechogenicity of the hepatic veins ) from 120 s after contrast injection up to 5 min , was performed in all cases . successful treatment was defined when no focal and / or irregular enhancement within the treated lesion during the dynamic study was detected , whereas treatment failure was defined by the presence of residual enhancement within the treated lesion . three enhancement patterns were observed on the treated lesions compared with the liver parenchyma : ( 1 ) isovascular residual lesion perfusion ; ( 2 ) hypovascular residual lesion perfusion ; ( 3 ) avascular lesion perfusion . computed tomography ct was performed at 1 month after the procedure using 6or 64 - row multidetector scanners ( philips , eindhoven , the netherlands )  . 
a nonenhanced ct and a multiphase study after administration of 120140 ml of nonionic contrast agent ( ultravist 370 , schering , berlin , germany ) through an antecubital vein at a 3to 4 - ml / s flow rate were performed . 
scan parameters for all phases were a collimation of 5 mm , table feed / rotation 7.5 / 12 mm and a reconstruction interval of 5 / 3 m utilising a test bolus , the contrast - enhanced ct images were usually acquired with a delay time after contrast injection set at 2530 s for the arterial phase , at 6070 s for the portal venous phase and at > 23 min for the delayed interstitial phase . ecografia basale ed ecografia con mezzo di contrasto la valutazione e la precisa localizzazione delle lesioni da trattare stata eseguita utilizzando un ecografo sequoia 512 ( acuson - siemens , erlangen , germania )  . 
utilizzando una sonda convex multi - frequenza 4c stata eseguita una ecografia convenzionale b - mode del fegato , con scala di grigi , per identificare la lesione focale epatica . 
sono state utilizzate metodiche di imaging non - lineari in grado di identificare le armoniche riflesse ( solitamente la seconda armonica ) di un impulso trasmesso o metodiche di inversione di fase . 
quando una lesione focale sospetta veniva identificata , si procedeva alla sua misurazione . il protocollo di studio prevede quindi lesecuzione , da parte dello stesso operatore , di un esame ceus prima ed immediatamente al termine della procedura percutanea . viene iniettato esafluoruro di zolfo ( dosi di 2 , 4 ml ) in forma di microbolle ( sonovue , bracco , milano , italia ) in una vena antecubitale , con ago - cannula a 20 gauge , in bolo rapido , seguito da 5 ml di soluzione salina . 
 stato utilizzato un imaging armonico specifico per le microbolle , con bassa pressione acustica ( 24 mhz coherent contrast imaging ; indice meccanico < 0 , 2 ; 1213 frame rate / s ) sullo stesso ecografo . 
tutti gli esami ecografici con contrasto sono stati registrati su sistemi magneto - ottici . dopo liniezione del mezzo di contrasto , in tutti i casi stata effettuata unosservazione dinamica in real - time delle fasi contrastografiche : fase arteriosa ( definita dalliperecogenicit dei rami arteriosi intraepatici ) dopo circa 1525 secondi dalliniezione ; fase venosa portale ( definita dalliperecogenicit della vena porta e delle sue diramazioni intraepatiche ) dopo circa 4060 secondi dalliniezione ; fase sinusoidale tardiva ( definita dalliperecogenicit delle vene epatiche , per il progressivo passaggio delle microbolle dopo il loro accumulo a livello sinusoidale ) da 120 secondi a 5 minuti dalliniezione . 
 il successo terapeutico viene definito dallassenza di focale e / o irregolare impregnazione contrastografica nella lesione trattata durante lo studio dinamico , mentre il fallimento terapeutico definito dalla presenza di impregnazione residua nella lesione trattata . 
 sono stati pertanto descritti tre differenti pattern di enhancement a carico delle lesioni trattate , valutate a confronto con il parenchima epatico circostante : ( 1 ) perfusione lesionale residua isovascolare ; ( 2 ) perfusione lesionale residua ipovascolare ; ( 3 ) perfusione lesionale avascolare . tomografia computerizzata un mese dopo la procedura percutanea stata eseguita una tc di controllo , utilizzando macchine a 6 o 64 detettori ( philips , eindhoven , olanda )  . 
sono state effettuate scansioni sia prima che dopo la somministrazione endovenosa di 120140 m di mezzo di contrasto non ionico ( ultravist 370 , schering , berlino , germania ) , con velocit di flusso di 34 ml / s . 
per tutte le scansioni sono stati applicati i seguenti parametri : collimazione di 5 mm , rapporto tra la 1098 percutaneous ethanol injection patients lay down in supine or left lateral position , and after local anaesthesia ( lidocaine s.c. ) , a 20to 22 - gauge needle was percutaneously inserted under us guidance . 
 radiofrequency thermal ablation after total anaesthesia , the active needle electrode ( 1419 gauge ) was inserted percutaneously into the lesion under us guidance , inducing coagulation necrosis through interstitial reactive hyperthermia , without any systemic damage . 
the rf delivery system uses active - needle cooled and expandable electrodes with nine hooks , connected to a high frequency ( 150 - w ) generator ( rita model 1500 , rita medical system , ca , usa )  . 
finally , the needle was removed [ 2527 ]  . data analysis sensitivity , specificity , ppv , npv and accuracy of ceus after pei or rfta compared with ct in terms of presence / absence of residual perfusion remnant / recurrence were evaluated . 
al termine della procedura , la lesione trattata appare come unarea iperecogena [ 24 ]  . termoablazione con radiofrequenza previa anestesia generale , sotto guida ecografica viene inserito nel tessuto neoplastico da trattare un ago - elettrodo attivo ( 1419 g ) per via percutanea per indurre necrosi coagulativa mediante ipertermia reattiva interstiziale , senza alcun danno sistemico . 
il sistema per la termoablazione con radiofrequenza prevede lutilizzo di aghi - elettrodi espandibili a 9 uncini , con punta autoraffreddata , connessi ad un generatore ad alta frequenza a 150 watt ( rita modello 1500 , rita medical system , ca , usa )  . 
sotto guida ecografica stata eseguita una valutazione in tempo reale della procedura termoablativa , con evidenza di un alone iperecogeno . al termine della procedura , lago stato rimosso [ 2527 ]  . analisi dei dati la concordanza tra i risultati ottenuti con la ceus post - trattamento e con la tc eseguita ad un mese stata valutata calcolando sensibilit , specificit , vpp , vpn ed accuratezza diagnostica , in termini di presenza / assenza di perfusione residua sospetta per residuo di malattia a carico delle lesioni trattate . 
le sensibilit del pattern ceus avascolare dopo pei e dopo rfta e lefficacia delle due procedure ablative , in termini di percentuale di necrosi completa ottenuta , sono state valutate e confrontate tra loro con il test del 2 . 
valori di p < 0 , 05 sono stati considerati statisticamente significativi . risultati sono stati reclutati 69 pazienti per un totale di 90 lesioni tutte ipervascolarizzate in fase arteriosa , con numero medio radiol med ( 2009 ) 114 : 10941105 1099 fig . 
d alla tc di follow - up si osserva necrosi completa della lesione trattata ( freccia )  . suggestive for residual tumour in 25 / 54 ( 46% ) lesions , two of which ( 4% ) were characterised by an isovascular and 23 / 54 ( 42% ) by a hypovascular pattern . 
tutti i pazienti sono stati sottoposti a pei o rfta e quindi suddivisi in due gruppi : nel primo gruppo tutti i pazienti trattati con pei e nel secondo gruppo quelli trattati con rfta . la pei stata eseguita in 43 pazienti per un totale di 54 / 90 ( 60% ) noduli con diametro medio di 2 cm ( range : 0 , 54 , 8 cm )  . 
d alla tc di follow - up si osserva necrosi completa della lesione trattata ( freccia )  . thus , in 32 / 36 ( 89% ) cases ( 31 / 31 = 100% cases characterised by complete necrosis and 1 / 5 = 20% cases with residual tumour ) , ct confirmed ceus imaging . 
in 4 / 36 ( 11% ) cases , the agreement between ceus and ct was not satisfied due to 4 / 5 ( 80% ) false positives at ceus . 
 sensitivity , specificity , ppv , npv and accuracy of the avascular pattern at ceus immediately after pei and after rfta , respectively , compared with ct assessment of necrosis performed after 1 month were 75% , 69% , 72% , 72% and 72% for pei and 100% , 20% , 89% , 100% and 89% , for rfta ( table 2 )  . 
in nessun caso alla ceus post - rfta stato evidenziato pattern isovascolare ( tabella 1 )  . pertanto , in 32 / 36 ( 89% ) casi ( 31 / 31 = 100% dei casi con necrosi completa e 1 / 5 = 20% dei casi con residuo tumorale ) la tc ha confermato il reperto ceus ; in 4 / 36 ( 11% ) casi non c stata concordanza tra le due tecniche di immagine per il riscontro ceus di 4 / 5 ( 80% ) falsi positivi . 
 la sensibilit , la specificit , il vpp , il vpn e laccuratezza diagnostica del pattern avascolare alla ceus eseguito immediatamente dopo la procedura di pei rispetto alla necrosi completa dimostrata alla tc ad un mese erano rispettivamente di 75% , 69% , 72% , 72% e 72% . 
il pattern avascolare alla ceus eseguito immediatamente dopo la procedura di rfta rispetto alla tc eseguita un mese dopo ha mostrato invece valori di sensibilit , specificit , vpp , vpn ed accuratezza diagnostica pari a 100% , 20% , 89% , 100% e 89% ( tabella 2 )  . 
infine , la differenza tra lefficacia del trattamento mediante ablazione con radiofrequenza e con alcolizzazione risultata statisticamente significativa ( p < 0 , 05 )  . discussione nel corso degli ultimi anni sono state proposte diverse tecniche per il trattamento ablativo percutaneo dellepatocarcinoma in grado di offrire ottime possibilit terapeutiche ai pazienti non candidabili alla chirurgia . 
tra queste un posto di radiol med ( 2009 ) 114 : 10941105 1103 sensitivity after pei and after rfta showed a p value < 0.05 ( statistically significant difference )  . 
a statistically significant difference ( p < 0.05 ) between the necrosis obtained by rfta and pei was also demonstrated . discussion during recent years , several percutaneous ablation techniques were proposed to treat hcc , offering good therapeutic possibilities for patients who are not candidates for surgery . 
for lesions with a mean diameter from 2 to 4 cm , rfta achieved complete necrosis with fewer sessions [ 29 ]  . evaluation of the efficacy of percutaneous treatments is monitored by imaging techniques , such as contrastenhanced ct or mr [ 1517 ] and recently also by means of ceus [ 1722 ]  . 
high concordance between all these methods has been demonstrated , leading to an important role of ceus for the characterisation and follow - up for hccs [ 20 , 30 , 31 ]  . 
in these studies , ceus was not performed immediately but at least 2448 h or more after treatment [ 17 , 2122 ]  . although ceus is characterised by intrinsic limitations , it is more accessible than other methods and leads to a realtime evaluation of the dynamic vascularity of tissue , which is able to identify vascular structures around and within the lesion [ 1820 , 30 , 31 ]  . 
the correct timing to perform the evaluations is still under discussion , with authors performing the follow - up 1 month [ 21 ] or 2448 h after treatment [ 17 , 21 , 22 ]  . the most important question is the differential diagnosis at ct , the gold standard , between residual tumour and reactive hyperaemia , generally characterised by focal irregular enhancement and regular ring enhancement , respectively . some authors have suggested that a correct follow - up should be performed 3 months after treatment [ 16 ] , when reactive hyperaemia cannot be present , leading to less false positive diagnoses but with a later detection of residual tumour . 
regarding pei , quite a good agreement ( 72% ) between ceus and ct was shown , without any significant difference between cases with residual tumour ( 69% ) and with complete necrosis ( 75% )  . 
in other terms , false positives ( 31% ) and rilievo occupato dallablazione con etanolo ( pei ) e con radiofrequenza ( rfta ) [ 68 , 13 , 14 ]  . 
i risultati ottenuti dalle due tecniche in esame sono apparsi pressoch paragonabili per il trattamento di hcc ipervascolarizzati con diametro2 cm , ma con dimostrato minor rischio di complicanze per la prima [ 28 ] ; per lesioni con diametro compreso tra 2 e 4 cm , la rfta si dimostrata pi efficace nel raggiungimento di necrosi completa in un minor numero di sedute [ 29 ]  . 
 la valutazione dellefficacia dei trattamenti percutanei affidata allimaging radiologico , sostenuto da metodiche consolidate , quali tc e rm con mezzo di contrasto [ 1517 ] , e negli ultimi anni anche dalla ceus [ 1722 ]  . 
numerosi studi hanno dimostrato lelevata concordanza tra le suddette metodiche , decretando pertanto allecografia con mezzo di contrasto un ruolo chiave non solo per la caratterizzazione ed il follow - up degli hcc [ 20 , 30 , 31 ] , ma anche per la valutazione della vascolarizzazione residua nel contesto della lesione trattata [ 17 , 18 , 21 , 22 ]  . 
tuttavia , in questi studi la ceus non viene eseguita immediatamente al termine della procedura interventistica , ma ad almeno 2448 ore o pi dal trattamento [ 17 , 21 , 22 ]  . pur essendo gravato dai noti limiti intriseci della metodica , la ceus rimane senza dubbio di pi facile accesso e rapida esecuzione , consentendo una valutazione dinamica in tempo reale della lesione trattata dopo la somministrazione del mezzo di contrasto , con capacit di identificare vasi perio intra - lesionali [ 1820 , 30 , 31 ]  . 
alcuni autori hanno considerato lipotesi di un corretto follow - up ad un mese [ 21 ] o a 2448 ore dalla procedura percutanea [ 17 , 21 , 22 ]  . il principale limite sembra rappresentato dalla diagnosi differenziale tra residuo ed iperemia reattiva , valutati alla tc di controllo considerata come gold standard di riferimento , e caratterizzati generalmente il primo da impregnazione contrastografica focale , irregolare e la seconda da impregnazione a cercine perilesionale , regolare . 
 col nostro studio ci siamo proposti di valutare lefficacia della ceus effettuata immediatamente al termine della procedura percutanea , pei o rfta , durante la stessa seduta interventistica , in termini di affidabilit nel predire il successo terapeutico . 
per quanto concerne la pei , i risultati hanno dimostrato discreta concordanza ( 72% ) tra le due tecniche di immagine , senza differenza significativa tra il gruppo di casi con residuo tumorale ( 69% ) e quelli con necrosi completa ( 75% )  . 
in altre parole , i falsi positivi ( 31% ) ed i falsi negativi ( 25% ) sono confrontabili e questo esprime una moderata sensibilit dellindagine ( 75% )  . 
per quanto riguarda la rfta , abbiamo documentato una concordanza molto buona ( 89% ) tra i rilievi ceus e tc , in 1104 radiol med ( 2009 ) 114 : 10941105 false negatives ( 25% ) were comparable , resulting in a moderate sensitivity ( 75% )  . 
regarding rfta , a very good agreement ( 89% ) between ceus and ct results was obtained , particularly when the avascular pattern , suggestive for complete necrosis [ 68 , 13 , 14 ] , was detected within the treated lesion . 
furthermore , the high true positive rate ( 100% ) , the few true negative diagnoses ( 20% ) and the absence of false negative diagnoses indicate the greater efficacy of rfta in comparison with pei . finding an avascular pattern at ceus immediately after rfta is highly predictive of a therapeutic success in term of complete necrosis ( sensitivity 100% and ppv 89% ) than after pei ( sensitivity 75% and ppv 72% )  . particolare con assoluta affinit in caso di pattern avascolare ( 100% ) , espresso alle metodiche di imaging con mezzo di contrasto come assenza di flusso nel contesto della lesione trattata ed espressione pertanto di necrosi coagulativa completa [ 68 , 13 , 14 ]  . 
inoltre , considerando lelevata percentuale di veri positivi ( 100% ) , lesigua percentuale di veri negativi ( 20% ) e lassenza di falsi negativi , dal nostro studio risulta chiaramente evidente la maggior efficacia della rfta rispetto alla pei . 
 per concludere , il riscontro di pattern avascolare alla ceus eseguita immediatamente dopo la procedura percutanea fortemente predittivo per successo terapeutico ( sensibilit 100% e vpp 89% ) dopo rfta , ma non dopo pei ( sensibilit 75% e vpp 72% )  . 
this is made possible by using midto low - end scanners and simple acquisition techniques accessible to both radiologists and clinicians . major advantages include ready availability at the bedside , the absence of ionising radiation , high reproducibility and cost efficiency . 
when such artefacts are widely detected on anterolateral transthoracic lung scans , diffuse alveolar - interstitial syndrome can be diagnosed , which is often a sign of acute pulmonary oedema . 
this condition rules out exacerbation of copd as the main cause of acute dyspnoea . keywords lung ultrasound dyspnea pulmonary oedema alveolar - interstitial syndrome riassunto in questo lavoro viene discussa lutilit dellecografia polmonare nella diagnosi delle diverse cause di dispnea acuta in emergenza , in particolare focalizzando lattenzione sulla diagnosi differenziale tra ledema polmonare e la riacutizzazione della broncopneumopatia cronica ostruttiva ( bpco )  . 
quando tali artefatti sono diffusamente visualizzati nelle scansioni trans - toraciche antero - laterali , possibile diagnosticare la sindrome alveolo - interstiziale diffusa , che spesso un segno di edema polmonare acuto . 
questa condizione esclude la riacutizzazione di bpco quale causa di dispnea acuta . parole chiave ecografia polmonare dispnea edema polmonare sindrome alveolo - interstiziale 1054 introduction the new millennium , diagnostic imaging has with continued to advance rapidly on all fronts as a result of advances in technology and information systems . 
its application to chest diagnostics has always been considered impossible given the poor accessibility of the lung to ultrasound ( us ) , as opposed to the study of more superficial structures , both muscular and subcutaneous , where us has always been widely used and accepted . 
obstacles to the sonographic study of deep chest structures are related to both the air content of the lung and the bone component of the rib cage , which prevent penetration of the us beam and lead to the development of artefacts . 
this , combined with the widespread use of conventional radiology and the development of multidetector computed tomography ( mdct ) , has hindered the application of us imaging to the lung . 
in recent years , the use of us as a clinical tool in the emergency setting where the patients critical condition limits any clinical evaluation and impairs the quality of chest radiographs , as well as preventing access to more complex modalities such as mdct has become increasingly popular , leading to the development of new applications . 
evaluation of the lung is among the most innovative . the latest developments in lung us are based on the interpretation of a few signs ( mostly artefacts ) rather than on technological advances . 
analysis of the correlations of artefact patterns with clinical and radiological diagnoses in intensive care unit ( icu ) patients led to the important insights of lichtenstein et al . , the first to describe the potential of us in the diagnosis of alveolarinterstitial syndrome [ 1 ]  . 
growing interest in the practical applications of lung us bodes well for its future as an increasingly indispensable tool for intensivists , emergency medicine physicians and radiologists . this paper describes the clinical application of lung us in the recognition of alveolarinterstitial syndrome , focusing in particular on the differential diagnosis between pulmonary oedema and exacerbation of chronic obstructive pulmonary disease ( copd ) as causes of acute dyspnoea . the first part examines the reasons for the complexity of the differential diagnosis of the possible causes of acute dyspnoea ( in particular , cardiogenic and respiratory causes ) at the patients bedside in the emergency department and analyses the potential benefits of lung us compared with radiological techniques . 
the second part describes the us radiol med ( 2009 ) 114 : 10531064 introduzione nel nuovo millennio la diagnostica per immagini ha continuato a progredire a ritmi elevati su tutti i fronti , spinta dallo sviluppo tecnologico e dei sistemi informatici . lecografia b - mode non una nuova tecnologia , in quanto venne proposta per la prima volta per scopi medici pi di cinquanta anni fa . 
la sua applicazione nelle procedure diagnostiche delle malattie del torace sempre stata considerata impossibile per la scarsa accessibilit del polmone agli ultrasuoni , al contrario dello studio delle strutture pi superficiali , sia muscolari che sottocutanee , dove lecografia ha sempre mantenuto un ruolo diffusamente accettato . 
gli ostacoli allo studio ecografico delle strutture toraciche profonde sono dovuti sia al contenuto aereo polmonare sia alla componente ossea della gabbia toracica , che impediscono la progressione del fascio ultrasonoro e causano lo sviluppo di artefatti . 
questa considerazione , insieme al diffuso utilizzo della radiologia tradizionale ed al progressivo sviluppo della tomografia computerizzata multidetetettore ( tcmd ) , ha impedito per diversi anni lo sviluppo di metodiche ecografiche di imaging del polmone . negli ultimi anni , lutilizzo degli ultrasuoni come strumento clinico da applicare nel paziente in emergenza , laddove la criticit del malato limiti sia la valutazione clinica sia la qualit della radiografia del torace , precludendo anche laccesso a metodiche complesse quali la tcmd , sta ottenendo consensi crescenti che hanno portato allo sviluppo di nuove applicazioni . 
piuttosto che sul progresso tecnologico , i pi recenti sviluppi dellecografia polmonare si sono basati sulla scoperta del significato di alcuni segni ( per lo pi veri e propri artefatti )  . 
le analisi delle correlazioni di alcuni patterns di artefatti con le diagnosi cliniche e radiologiche in pazienti in unit di terapia intensiva ( uti ) , hanno condotto alle importanti intuizioni di lichtenstein et al . 
 [ 1 ] , i primi a descrivere le possibilit della diagnosi mediante ecografia della sindrome alveolo - interstiziale . questa condizione morbosa caratterizza molte patologie eterogenee che hanno in comune un diffuso coinvolgimento dellinterstizio polmonare con conseguente riduzione della capacit di scambio alveolo - capillare . 
il crescente interesse nelle sue applicazioni pratiche , fa prevedere per lecografia polmonare un futuro come strumento sempre pi indispensabile per il medico dellunit di terapia intensiva , per i medici di medicina durgenza ed anche per il radiologo . 
in lapplicazione clinica questo articolo descriveremo dellecografia polmonare nel riconoscimento della sindrome alveolo - interstiziale , focalizzando lattenzione tra edema soprattutto sulla diagnosi differenziale radiol med ( 2009 ) 114 : 10531064 1055 technique used for the diagnosis of alveolarinterstitial syndrome , providing the reader with the latest data published in the literature . differential diagnosis of acute dyspnoea the emergency diagnosis and management of adults with acute dyspnoea remains a major challenge , often with many pitfalls . 
in some cases , this problem cannot be readily solved , especially in emergency settings , and in elderly patients who often have associated disorders and atypical clinical presentations [ 2 , 3 ]  . 
the literature indicates that the diagnosis of each lung abnormality whether restrictive , obstructive , or due to increased ventricular filling pressure traditionally relies on the integration of different diagnostic information . 
this includes physical signs , case history and results of conventional diagnostic tests such as chest radiography , electrocardiogram and laboratory investigations [ 4 , 5 ]  . when taken individually , both the physical examination and the history have insufficient specificity [ 68 ]  . 
radiographic signs such as redistribution of blood flow to the apices , blurring of the pulmonary vessels , and cardiomegaly have good predictive value but depend on the quality of the examination and on the radiologists skill and experience [ 5 , 9 ]  . 
recent studies show that chest radiography is often misleading in the evaluation of patients with decompensated heart failure in emergency settings [ 10 ] and may lead to diagnostic errors and inappropriate treatment . 
blood natriuretic peptide ( bnp and nt - pro bnp ) assays have recently been described as accurate tests for diagnosing heart failure . however , the precise role of these peptides in the emergency room has still to be clarified and validated [ 1114 ]  . electrocardiography is not sufficiently accurate for a correct diagnosis [ 15 ]  . 
transthoracic echocardiography allows exclusion of left ventricular dysfunction or right heart overload but fails to identify diastolic dysfunction , which may be the main cause of cardiac failure in approximately 50% of cases [ 16 ]  . 
pulsed doppler and tissue doppler are recent additions that allow estimation of left ventricle filling pressures and , as a result , identification of diastolic dysfunction [ 17 , 18 ]  . 
however , although relatively easy to apply , these methods require complex and expensive equipment that is not always available to emergency physicians and radiologists . lung us has been shown to have greater diagnostic accuracy in differentiating the causes of acute dyspnoea in emergency settings compared with the traditional methods commonly employed in emergency departments ( ed )  . 
its polmonare e riacutizzazione della broncopneumopatia cronica ostruttiva ( bpco ) quali cause di dispnea acuta . nella prima parte esamineremo le motivazioni per cui la diagnosi differenziale tra le diverse cause di dispnea acuta ( in particolare cause cardiogene e cause respiratorie ) al letto del paziente in urgenza pu essere complessa ed analizzeremo i potenziali benefici dellecografia polmonare rispetto alle tecniche radiologiche . 
la seconda parte si propone di descrivere la tecnica ecografica utilizzata per la diagnosi della sindrome alveolo - interstiziale , fornendo un aggiornamento sui dati pi recenti pubblicati in letteratura . diagnosi differenziale della dispnea acuta la diagnosi e la gestione in urgenza dei pazienti adulti con dispnea acuta ancora oggi da considerare una grande sfida , spesso con molte insidie . 
alcune volte questo problema non facile da risolvere , in particolare in emergenza e nei pazienti anziani che spesso hanno patologie associate e presentazioni cliniche atipiche [ 2 , 3 ]  . 
noi sappiamo dalla letteratura che la diagnosi di ogni alterazione polmonare , sia essa restrittiva , da ostruzione al flusso aereo , da aumento della pressione di riempimento ventricolare cardiaco , tradizionalmente si basa sulla integrazione di vari dati diagnostici . 
tali sono i segni fisici , i dati anamnestici ed i risultati dei test diagnostici tradizionali quali radiografia del torace , elettrocardiogramma e dati di laboratorio [ 4 , 5 ]  . quando considerati singolarmente , sia lesame fisico che i dati anamnestici non sono dotati di sufficiente specificit [ 68 ]  . 
i segni radiografici , quali la redistribuzione del flusso verso gli apici , la sfumatura dei vasi polmonari e la cardiomegalia , dimostrano buoni valori predittivi , ma sono dipendenti dalla qualit dellesame e dallabilit ed esperienza dello specialista radiologo [ 5 , 9 ]  . 
inoltre , recenti studi riportano che la radiografia del torace spesso fuorviante nella valutazione dei pazienti con insufficienza cardiaca scompensata in emergenza [ 10 ] , potendo condurre ad errori diagnostici e conseguenti terapie inappropriate . 
lecocardiografia transtoracica permette di escludere la disfunzione del ventricolo sinistro o il sovraccarico cardiaco destro , ma non consente di identificare la disfunzione diastolica che in circa il 50% dei casi pu essere la causa principale della 1056 radiol med ( 2009 ) 114 : 10531064 major advantages , particularly over radiographic techniques , are the absence of ionising radiation , speed and the fact that it is unaffected by the patients breath - hold limitations or agitation [ 19 ]  . 
the required information can be obtained by using linear , abdominal and microconvex probes , but to analyse comet - tail artefacts , low frequencies have to be used to allow the us beam to reach the deeper structures . 
a review of the literature shows that there is disagreement as to the most effective probe , although the earliest published experience on us diagnosis of alveolarinterstitial syndrome was performed with a 5mhz microconvex probe [ 1 ]  . 
the most influential opinion on this subject is that of lichtensteins group , who consider the microconvex probe to be most effective , as it combines sufficient penetration depth to enable analysis of vertical artefacts with sufficient compactness to allow satisfactory analysis of the intercostal spaces [ 19 ]  . 
in our studies and daily experience , we have used a 3.5 - mhz convex abdominal probe that offers the advantage of combining a wider and more effective depiction of the pleural surfaces with the ability to examine deeper structures [ 20 ]  . 
this type of probe is also preferred by other authors because of the characteristics described above [ 21 ]  . sonographic signs the lung is characterised by a mixture of water and air , and disruption of their balance underlies the pathophysiology of lung diseases . 
the sonographic probe can detect this imbalance as a result of the very different acoustic impedance values of air and water , and the contrast between these two elements generates pulmonary artefacts that enable diagnosis . 
solo recentemente stato introdotto luso del doppler pulsato e del doppler tissutale che permettono la stima delle pressioni di riempimento del ventricolo sinistro , e quindi lidentificazione della disfunzione diastolica [ 17 , 18 ]  . 
si tratta per di metodiche che , pur essendo di applicazione relativamente facile , necessitano di sistemi complessi e costosi non sempre a disposizione del medico durgenza e del radiologo . lecografia polmonare ha dimostrato valori di accuratezza diagnostica pi elevati nella diagnosi differenziale delle cause di dispnea acuta in urgenza rispetto alle comuni metodiche che tradizionalmente vengono utilizzate nel dipartimento di emergenza ( dea )  . 
essa presenta alcuni sicuri vantaggi in particolare rispetto alle tecniche radiografiche , in quanto priva di radiazioni ionizzanti , una metodica di rapida ed immediata esecuzione , e non influenzata da due fattori quali limpossibilit a mantenere una sufficiente apnea inspiratoria o lo stato di agitazione del paziente [ 19 ]  . 
inoltre , consente di distinguere fra lesioni solide o liquide ( consolidamenti vs versamento ) e fornisce informazioni dinamiche , permettendo di osservare le strutture in movimento in tempo reale . apparecchiatura la visualizzazione delle patologie della parete toracica , della pleura e del polmone richiede una apparecchiatura ecografica b - mode di tipo convenzionale senza la necessit di ricorrere a tecniche e tecnologie pi sofisticate , quali lutilizzo del mezzo di contrasto o il color doppler . 
le informazioni necessarie possono essere ottenute utilizzando sonde lineari , addominali e microconvex , ma per lanalisi degli artefatti a coda di cometa dobbiamo avvalerci di basse frequenze in modo da permettere al fascio di ultrasuoni di estendersi alle strutture pi profonde . 
da una analisi della letteratura , vi discordanza di vedute su quale sia il tipo di sonda pi efficace , ma la prima esperienza pubblicata sulla diagnosi ecografica della sindrome alveolo - interstiziale stata eseguita utilizzando la sonda di tipo microconvex da 5 mhz [ 1 ]  . 
lopinione pi autorevole su questo argomento viene dal gruppo di lichtenstein , il quale considera la sonda microconvex come la pi indicata , in quanto unirebbe il vantaggio di penetrare sufficientemente in profondit per permettere lanalisi degli artefatti verticali con quello di essere abbastanza compatta da inserirsi negli spazi intercostali del torace [ 19 ]  . 
nei nostri studi ed esperienza quotidiana utilizziamo una sonda addominale convex da 3.5 mhz , la quale presenta il vantaggio di unire la possibilit di una pi ampia ed efficace visualizzazione dei foglietti pleurici con la capacit di esaminare le strutture pi profonde [ 20 ]  . altri autori concordano sulla preferenza di questo tipo di sonda per le caratteristiche descritte [ 21 ]  . radiol med ( 2009 ) 114 : 10531064 1057 between fig . 
the typical sonographic sign is a vertical artefact originally called comet tail or b - line [ 1 , 22 , 23 ] , or also lung comet by some authors [ 24 , 25 ]  . 
we know from the literature how to define b - lines , which exhibit the characteristics described above and should be distinguished from other similar artefacts that have different properties and meanings , such as eand z - lines . 
e - lines are vertical , similar to b - lines , but originate from the chest wall and not from the pleural line , as they segni ecografici il polmone caratterizzato da una commistione di aria e acqua , e il turbamento del loro equilibrio il principio che sta alla base della fisiopatologia delle malattie polmonari . la sonda ecografica in grado di rilevare questo squilibrio , in quanto aria ed acqua hanno valori di impedenza acustica opposta . 
3 scansione ecografica intercostale obliqua che dimostra la presenza di numerosi artefatti a coda di cometa o linee b , con una distanza tra di loro inferiore ai 7 mm ( quadro definito anche lung rockets )  . 
in the sonographic images on either side of the radiogram , the presence of multiple adjacent comet - tail artefacts ( at least three per scan and in all chest areas examined ) can be easily distinguished . 
4 tipico pattern ecografico della sindrome interstizio - alveolare diffusa ( ai lati ) e corrispondente radiografia del torace ( al centro ) in un paziente affetto da edema polmonare acuto cardiogeno . 
nelle immagini ecografiche ai lati della radiografia , facilmente distinguibile la presenza di multipli e ravvicinati artefatti a coda di cometa ( almeno 3 per scansione , presenti in tutti i settori toracici esaminati )  . 
z - lines are also vertical lines that , like b - lines , originate from the pleural line but end before the bottom of the screen , fading out within a few centimetres [ 23 ]  . 
their significance is still uncertahow many b - lines have to be identified in a lung scan to be considered pathological ? isolated b - lines may be detected in any chest scan , even in normal lungs . 
dal confronto con le immagini tc si evince come le linee b corrispondano ai setti interlobulari edematosi [ 1 ]  . tali strutture hanno uno spessore non superiore ai 700 radiol med ( 2009 ) 114 : 10531064 1059 pathological pattern is instead characterised by multiple blines seen in a single scan ( b + pattern )  . 
it has been demonstrated that the separation between individual lines should never exceed 7 mm , as this distance corresponds to that existing between two subpleural interlobular septa [ 1 ]  . these artefacts thus have the same clinical relevance as kerley b - lines , which can often be seen on chest radiographs of patients with interstitial oedema . 
the discrepancy between these criteria is due to the greater extension of the pleural line , which can be depicted with a linear probe rather than with a microconvex probe . 
our criterion for defining a b + scan with an abdominal convex probe is based on recognition of at least three artefacts per scan , with a maximum distance between adjacent b - lines of 7 mm [ 20 ]  . 
anterior areas extend from the sternal border line to the anterior axillary line and are divided into an upper and a lower half , located between the clavicle and the second / third intercostal spaces and between the third intercostal space and the diaphragm , respectively . 
once the sonographic image of the pleural line has been obtained by identifying the ribs and the so - called bat micrometri , quindi al di sotto della risoluzione degli ultrasuoni . 
sappiamo dalla letteratura come definire le linee b , che presentano le caratteristiche appena descritte e che devono essere distinte da altri artefatti simili , ma con propriet e significato differente , come le linee e e z . 
le linee e sono linee verticali simili alle linee b ma originano dalla parete toracica e non dalla linea pleurica , essendo presenti in caso di enfisema sottocutaneo [ 23 ]  . 
le linee z sono anchesse linee verticali che , come le linee b , originano dalla linea pleurica , estinguendosi per prima del fondo dello schermo e svanendo in pochi centimetri [ 23 ]  . 
quante linee b si devono individuare in una scansione polmonare per essere considerate patologiche ? linee b isolate possono essere rilevate in qualunque scansione toracica del polmone anche in polmoni normali . 
stato dimostrato che la separazione tra le singole linee non deve mai essere superiore a 7 mm , in quanto questa distanza riflette quella tra 2 setti interlobulari a localizzazione subpleurica [ 1 ]  . 
in alcuni casi gli artefatti ecografici a coda di cometa possono essere pi vicini , con distanza tra di loro fino a 3 mci corrisponderebbe al coinvolgimento dellinterstizio intralobulare , che si traduce nelle tecniche radiologiche con il classico pattern a vetro smerigliato oppure con pattern di microreticolazione se viene eseguita la tc ad alta risoluzione . 
per convenzione viene comunemente accettato che , utilizzando la sonda microconvex , sia sufficiente lindividuazione di tre linee b per considerare positiva una scansione polmonare . in uno studio eseguito con sonda lineare , reissig et al . 
il nostro criterio per definire una scansione con un pattern b + utilizzando una sonda convex addominale si basa sul riconoscimento di almeno tre artefatti per scansione , con una distanza tra le linee b adiacenti non superiore ai 7 mm [ 20 ]  . 
le aree anteriori sono comprese tra la linea margino - sternale e la linea ascellare anteriore , e sono suddivise in una met superiore ed una inferiore , rispettivamente comprese tra la clavicola e il secondo - terzo spazio intercostale e dal terzo spazio intercostale al diaframma . le aree laterali sono comprese tra la linea ascellare anteriore e quella posteriore e sono suddivise in una met superiore ed una inferiore o basale . 
una volta ottenuta limmagine ecografica della linea pleurica attraverso lindividuazione delle coste e la visualizzazione del cosiddetto segno del pipistrello ( bat sign , vedere la figura 6 e la didascalia per la spiegazione ) [ 23 ] , ruotiamo la sonda per ottenere una scansione intercostale che consenta di visualizzare la pi ampia estensione possibile della porzione di pleura ( scansione obliqua )  . 
il tempo necessario per eseguire lesame varia da pochi secondi a 12 minuti [ 1 , 20 ]  . la definizione di un esame ecografico polmonare positivo richiede alcune attente considerazioni . 
 [ 1 ] si espressero in modo generico , asserendo semplicemente che il riscontro di un pattern b + da ambo i lati nelle aree anteriori del torace sufficiente per la diagnosi di sindrome alveolointerstiziale [ 1 ]  . 
in una recentissima pubblicazione , gli stessi autori hanno proposto il protocollo blue ( bedside lung ultrasound examination ) nella insufficienza respiratoria acuta , che prevede la esclusione della diagnosi di edema polmonare acuto quando le scansioni anteriori non presentino linee b predominanti bilateralmente [ 27 ]  . 
5 schematic subdivision of the anterolateral chest wall into eight areas . areas 1 , 2 , 5 and 6 correspond to the upper and lower right and left anterior quadrants , respectively . 
6 a longitudinal sonographic lung scan displaying the typical bat sign . this pattern appears when the pleural line , in the middle , is surmounted by the two adjacent lateral ribs , resembling a flying bat . 
questa immagine si evidenzia quando la linea pleurica , disposta al centro , sovrastata dalle due coste , disposte ai lati , in maniera tale da simulare un pipistrello in volo . 
the time required to carry out the examination varies from a few seconds to 12 min [ 1 , 20 ]  . defining a lung us scan as positive requires careful consideration . 
made the general statement that the finding of a b + pattern on both sides in the anterior areas was sufficient to establish a diagnosis of alveolarinterstitial syndrome [ 1 ]  . 
in a recent publication , the same authors suggested employing the bedside lung ultrasound examination ( blue ) protocol for acute respiratory failure , a protocol that excludes a diagnosis of acute pulmonary oedema when the anterior scans show no predominant b - lines bilaterally [ 27 ]  . 
when applying lung us to cardiological settings , other authors have defined a positive examination as one characterised by multiple bilateral comet - tail artefacts disseminated along the anterolateral surface or restricted to the lateral surface of the lung [ 28 ]  . 
in our opinion , the routine practice of an ed , in particular , requires a more precise definition of a positive ( or pathological ) examination , as the diseases seen in the ed are more diverse and often less severe than those seen in the icu and cardiology departments . 
furthermore , that many b - lines may also be detected when performing lateralbasal scans in patients with normal lungs , with rates ranging from 14% to 28% [ 1 , 26 , 29 ]  . 
in our experience , multiple b - lines ( b + ) have been identified in transthoracic us scans in patients presenting to the ed with different diagnoses , even in the presence of normal lungs . 
another aspect to be emphasised is that b - lines may also be found in areas surrounding parenchymal consolidation of various origins [ 1 , 20 , 30 , 31 ]  . 
these findings may therefore be confusing when lung us is used to exclude alveolarinterstitial syndrome . this is why we believe that recognition of the b + pattern in at least two scans per hemithorax should be adopted as a criterion for diagnosing diffuse alveolarinterstitial syndrome when four anterolateral scans per hemithorax are performed [ 20 , 30 ]  . 
an probabilmente il criterio di limitare lesame polmonare alle scansioni toraciche anteriori sufficiente solo nei pazienti critici con insufficienza respiratoria severa , ricoverati in uti . applicando lecografia polmonare in ambiente cardiologico , altri autori hanno definito come positivo un esame caratterizzato dalla presenza di multipli artefatti a coda di cometa bilaterali e disseminati lungo la superficie antero - laterale o limitati alla superficie laterale del polmone [ 28 ]  . 
nella nostra opinione , specialmente nella pratica quotidiana di un dea , occorre una definizione pi precisa di ci che dovrebbe essere considerato positivo ( o patologico ) , in quanto le malattie sono pi varie e spesso meno gravi rispetto alle patologie presenti nelle uti ed in ambiente cardiologico . inoltre , i dati provenienti dalla letteratura dimostrano che molte linee b possono essere riconosciute anche nelle scansioni latero - basali di pazienti con polmoni normali . 
nella nostra esperienza su pazienti presentatisi in dea con diagnosi diverse , linee b multiple ( b + ) sono state evidenziate in scansioni ecografiche trans - toraciche anche in caso di polmoni normali . 
un altro punto da sottolineare che le linee b possono anche essere ritrovate in aree circostanti addensamenti parenchimali di varia origine [ 1 , 20 , 30 , 31 ]  . 
di conseguenza , la rilevazione di linee b in alcune aree del torace non necessariamente correlata con una patologia interstiziale diffusa del polmone . tali reperti possono quindi essere confondenti nel momento in cui venga applicata lecografia polmonare per escludere la sindrome alveolo - interstiziale . 
questo il motivo per cui secondo noi dovrebbe essere adottato il criterio del riconoscimento del pattern b + in almeno 2 scansioni per ciascun emitorace per porre diagnosi di sindrome alveolo - interstiziale diffusa , allorquando vengano effettuate 4 scansioni antero - laterali per emitorace [ 20 , 30 ]  . 
uno scorretto inquadramento diagnostico tra cause cardiogene o polmonari di dispnea acuta pu tradursi in un pericoloso ritardo terapeutico oppure nellimpiego di farmaci inutili . linterpretazione dei segni radiologici tradizionali di sovraccarico del piccolo circolo , quali la redistribuzione di flusso e ledema interstiziale , sono spesso discutibili e soggettivi specialmente nel caso di radiografie del torace di scarsa qualit tecnica . 
inoltre linterpretazione di un radiogramma del torace a paziente supino e poco collaborante piena di insidie e la corretta interpretazione delle immagini necessita 1062 radiol med ( 2009 ) 114 : 10531064 table 1 distribution of 85 positive scans ( b + lines ) recorded in the 8 individualized areas of transthoracic lung ultrasound in a group of 145 patients with normal lungs at chest radiography and clinical final diagnosis ( 30 ) tabella 1 distribuzione delle 85 scansioni positive ( linee b + ) ottenute nelle 8 aree esaminate in un gruppo di 145 pazienti con polmoni normali al radiogramma del torace e con normale diagnosi clinica finale ( 30 ) areas of thoracic ultrasound positive scans settori esaminati scansioni positive upper anterior right lower anterior right upper lateral right latero - basal right upper anterior left lower anterior left upper lateral left latero - basal left 2 , 8 18 , 6 22 , 8 antero - superiore destro anterinferiore destro latero - superiore destro latero - basale destro antero - superiore sinistro antero - inferiore sinistro latero - superiore sinistro latero - basale sinistro 2 , 8 18 , 6 22 , 8 cardiogenic or pulmonary causes of acute dyspnoea may lead to dangerous delays in treatment or use of unnecessary drugs . interpretation of the conventional radiology signs of overload of the pulmonary circulation , such as blood flow redistribution and interstitial oedema , are often questionable and subjective , especially in the case of poor - quality chest radiographs . 
interpretation of a chest radiogram of a supine and poorly cooperating patient is full of pitfalls , and correct interpretation may call for specific competences . the availability of a us scanner 24 h a day in the ed allows emergency physicians , intensivists or radiologists to confirm or exclude alveolar - interstitial syndrome and settle any interpretation doubts regarding the chest radiogram . two published studies have assessed the accuracy of lung us for recognising alveolarinterstitial syndrome , comparing us with chest radiography and the final clinical diagnosis . 
another study performed by intensivists in the ed concluded that us recognition of diffuse interstitial through b - line detection allows some involvement pulmonary diseases to be rapidly ruled out , in particular , copd exacerbation , which is one of the most common causes of acute dyspnoea [ 32 ]  . 
the same paper demonstrated that us signs of diffuse alveolarinterstitial syndrome were seen in all patients with pulmonary oedema but were absent in a vast majority of patients with copd exacerbation ( 100% sensitivity and 92% specificity ) [ 32 ]  . recently , the same group published the results of a thorough assessment of the effectiveness of lung us in the differential diagnosis of the main causes of respiratory failure , reporting excellent accuracy values [ 27 ]  . 
la disponibilit di un ecografo 24 ore su 24 in dea consente al medico durgenza , rianimatore o radiologo di confermare od escludere in qualsiasi momento la sindrome alveolo - interstiziale e dirimere alcuni dubbi interpretativi sul radiogramma del torace . ci sono due studi pubblicati che hanno valutato laccuratezza dellecografia polmonare nel riconoscimento della sindrome alveolo - interstiziale , ponendola a confronto con la radiografia del torace e la diagnosi clinica finale . 
il secondo studio stato eseguito in un dea , ed ha dimostrato risultati simili al precedente ( sensibilit 85 , 7% , specificit 97 , 7% , fattibilit 98 , 3% , variabilit inter - osservatori 4 , 9% ) [ 20 ]  . 
in un altro lavoro eseguito da rianimatori in dea , gli autori hanno concluso che il riconoscimento con esame ecografico di un coinvolgimento interstiziale diffuso grazie alla visualizzazione delle linee b , permette di escludere rapidamente alcune patologie polmonari ma specialmente la riacutizzazione di bpco , una delle cause di dispnea acuta di pi frequente riscontro [ 32 ]  . 
nello stesso lavoro stato dimostrato che i segni ecografici della sindrome alveolo - interstiziale diffusa erano presenti in tutti i pazienti con edema polmonare , ma assenti nella grande maggioranza dei pazienti con riacutizzazione di bpco ( sensibilit del 100% e specificit del 92% ) [ 32 ]  . 
recentemente lo stesso gruppo ha prodotto i risultati di una attenta valutazione della efficacia della tecnica ecografica polmonare nella diagnosi differenziale di tutte le principali cause di insufficienza respiratoria , dimostrando ottimi valori di accuratezza [ 27 ]  . 
l archiviazione dei dati permette di monitorare levoluzione della congestione polmonare durante il trattamento medico di pazienti affetti da scompenso cardiaco radiol med ( 2009 ) 114 : 10531064 1063 known that us allows acquisition and storage of digital films , and this is a further , undoubted advantage over the plain evaluation of clinical signs during a physical examination . 
data storage allows one to monitor the evolution of pulmonary congestion during medical management of acute cardiac decompensation and to observe the gradual resolution of the b + pattern [ 3335 ]  . 
a number of studies have documented that lung us targeted to detection of b + lines can be easily performed by general radiologists and even by clinicians , such as cardiologists , intensivists and emergency physicians [ 1 , 20 , 24 , 26 , 36 , 37 ]  . a further aspect to be emphasized is that the thickening of interlobular septa , as seen on high - resolution ct , is not a highly specific sign , as it can manifest in different interstitial diseases of various aetiology . 
the b + pattern cannot distinguish between the various acute and chronic diseases underlying the interstitial syndrome , such as cardiac decompensation , acute respiratory distress syndrome ( ards ) , interstitial pneumonia and pulmonary fibrosis . 
we are convinced that integration of us data with those from other emergency bedside investigations , patient history and physical examination can better guide the diagnosis , even in dubious cases . 
moreover , observation of changes in the lung us pattern during medical treatment can facilitate diagnosis for example , in the event of a failure to induce any reduction or disappearance of b - lines after diuretic therapy or continuous positive airway pressure ( c - pap ) , an event that would exclude the cardiogenic origin of alveolarinterstitial syndrome [ 33 ]  . finally , some theories exist concerning the possibility of differentiating between hydrostatic ( cardiac decompensation ) and ards by analysing the distribution of b - lines , pleural sliding and subpleural alveolar consolidation [ 21 ]  . these theories are still to be validated . 
these are readily identifiable us signs that represent the indirect visualisation of interlobular septal thickening due to oedema or fibrosis and intra - alveolar fluid and allow a bedside differential diagnosis between oedema and copd exacerbation as causes of acute dyspnoea in the emergency setting . we believe that lung us , in its new role as a visual stethoscope , will be rapidly taken up in the daily practice of radiologists , emergency physicians , intensivists , cardiologists and pneumologists . 
diversi studi hanno documentato come lecografia polmonare dedicata al riconoscimento delle linee b + possa essere facilmente eseguita da radiologi generali ed anche da clinici come i cardiologi , i rianimatori e i medici durgenza [ 1 , 20 , 24 , 26 , 36 , 37 ]  . a completamento delle precedenti osservazioni occorre sottolineare che lispessimento dei setti interlobulari , come accade per la tcar , non un segno molto specifico potendo essere presente in diverse patologie dellinterstizio polmonare con diversa eziologia . 
il pattern b + non in grado di distinguere tra le diverse malattie acute e croniche causa della sindrome interstiziale , come lo scompenso cardiaco , la sindrome da distress respiratorio acuto ( ards ) , la polmonite interstiziale e le fibrosi polmonari . 
siamo convinti che lintegrazione dellecografia con altri esami eseguibili in urgenza al letto del paziente e con i dati anamnestici e dellesame fisico , permetta un pi efficace orientamento diagnostico anche nei casi dubbi . 
inoltre , losservazione delle modifiche del pattern ecografico polmonare durante il trattamento medico pu facilitare lorientamento , come per esempio nel caso di una mancata riduzione o scomparsa delle linee b dopo terapia diuretica o ventilazione a pressione positiva continua ( c - pap ) , fenomeno che negherebbe lorigine cardiogena della sindrome alveolointerstiziale [ 33 ]  . 
infine , esistono alcune teorie ancora da validare che prevederebbero per esempio la diagnosi differenziale ecografica tra edema polmonare idrostatico ( scompenso cardiaco ) e lesionale ( ards ) attraverso lanalisi della distribuzione delle linee b , dello scorrimento pleurico e dei consolidamenti alveolari sub - pleurici [ 21 ]  . 
il riconoscimento ecografico al letto del paziente di una sindrome alveolo - interstiziale diffusa si basa sulla ricerca e riconoscimento degli artefatti verticali iperecogeni detti a coda di cometa o linee b . 
questi sono segni ecografici semplici da riconoscere che rappresentano la visualizzazione indiretta dei setti interlobulari ispessiti da edema o fibrosi e dei fluidi intralveolari , e permettono la diagnosi differenziale al letto del paziente tra edema o riacutizzazione di bpco come cause di dispnea acuta in emergenza . 
di fisica sanitaria , irccs casa sollievo della sofferenza , san giovanni rotondo , italy 6facolt di medicina , universit di bari , bari , italy 7dipartimento di radiologia , universit novara , novara , italy 8dipartimento di scienze neurologiche e psichiatriche dellet evolutiva , universit la sapienza , roma , italy 9dipartimento di radiologia , azienda ospedaliera universitaria , umberto i , torrette , ancona , italy correspondence to : t . 
scarabino , via napoli 56 , 70031 andria , italy , tel . : + 39 - 088 - 3299140 , fax : + 39 - 088 - 3299220 , e - mail : tscarabino@hotmail.com received : 10 september 2008 / accepted : 6 october 2008 / published online : 10 march 2009 springer - verlag 2009 abstract purpose . 
this study evaluated the sensitivity of a 3.0 - tesla ( t ) magnetic resonance imaging ( mri ) in measuring cerebral phenylalanine using proton magnetic resonance spectroscopy and in assessing mr - documented whitematter changes by means of diffusion studies ( diffusionweighted imaging , apparent diffusion coefficient map ; diffusion tensor imaging ) in patients with phenylketonuria . 
lampiezza del segnale della fenilalanina relativo al rapporto creatina / fosfocreatina incrementa linearmente con la fenilalanina ematica ( r = 0 , 7067 ; p < 0 , 001 )  . 
lo studio di diffusione ha 462 radiol med ( 2009 ) 114 : 461474 exhibiting mri changes as well as decreased apparent diffusion coefficient values , but fractional anisotropy indices were normal . 
in particular , the multimodal approach with mri , proton magnetic resonance spectroscopy and diffusion magnetic resonance imaging can provide more information than previous studies performed with low - field systems . 
in particolare lapproccio multimodale con imaging rm di base , di spettroscopia e di diffusione pu fornire maggiori informazioni rispetto ai precedenti studi acquisiti con sistemi a pi basso campo . keywords phenylketonuria magnetic resonance imaging ( mri ) proton mr spectroscopic imaging diffusion mri parole chiave fenilchetonuria risonanza magnetica studio di spettroscopia protonica con risonanza magnetica studio di diffusione con risonanza magnetica introduction introduzione phenylketonuria ( pku ) is a disorder of amino acid metabolism caused by an inborn error in the phenylalanine hydroxylase gene ( pha ) , which strongly reduces hepatic hydroxylation of phenylalanine ( phe ) to tyrosine [ 1 ]  . 
previous morphological mri studies have documented focal symmetrical lesions in periventricular and , especially , parieto - occipital white matter ( wm ) in pku patients with phe blood levels exceeding 10 mg / dl . these lesions , the significance of which is as yet unclear , seem to be due to a reversible structural myelin alteration , as they regress when blood phe levels fall [ 2 ]  . proton magnetic resonance spectroscopy ( 1h - mrs ) measures noninvasively the concentration of several brain metabolites in vivo and can help to further elucidate the cerebral metabolic characteristics of pku . 
it allows evaluation of several aspects of brain biochemistry that could shed light on the nature of wm lesions , including measurement of n - acetyl - containing moieties [ mainly n - acetylaspartate ( naa ) ] , choline - containing compounds ( cho ) , creatine ( cr ) and phosphocreatine ( pcr ) , and myo - inositol ( mi ) [ 3 ]  . 
in particular , changes in naa ( a marker of neuronal integrity ) la fenilchetonuria ( pku ) un disordine del metabolismo degli amminoacidi causata da una alterazione congenita del gene della fenilalanina - idrossilasi che determina una significativa riduzione , a livello epatico , della idrossilazione della fenilalanina ( phe ) in tirosina [ 1 ]  . 
i pazienti non diagnosticati e quindi non trattati possono sviluppare diversi segni e sintomi : in particolare ritardo mentale e nella crescita , crisi epilettiche , manifestazioni cutanee ( eczemi e ipopigmentazioni )  . 
la diagnosi neonatale ha radicalmente cambiato la prognosi della pku anche se in realt possiamo riscontrare alcuni sintomi e alterazioni neuro - radiologiche anche in pazienti trattati precocemente . la pku stata studiata sotto diversi aspetti . 
essa documenta la presenza di focali e simmetriche lesioni a livello della sostanza bianca periventricolare , specie a livello parieto - occipitale , evidenti in tutti i pazienti in cui il livello ematico di phe supera il valore di 10 mg / dl . 
tali lesioni , il cui significato non ancora chiaro , sembra siano dovute a una alterazione strutturale e reversibile della mielina : infatti esse regrediscono nel momento in cui il livello di phe decresce verso valori normali [ 2 ]  . la spettroscopia protonica con rm ( 1h - mrs ) misura in maniera non invasiva e in vivo la concentrazione a livello cerebrale di alcuni metaboliti fornendo ulteriori informazioni sulle caratteristiche metaboliche cerebrali della pku e soprattutto permettendo una pi chiara analisi biochimica delle alterazioni della sostanza bianca tipiche di questa radiol med ( 2009 ) 114 : 461474 or in mi ( a potential astrocytic marker ) suggest a possible neurochemical dysfunction , whereas cho is one measure of the degree of tissue myelination [ 4 , 5 ]  . 
despite continuing progress in 1h - mrs acquisition methods and the development of increasingly sophisticated quantification routines , its determination is affected by the detection threshold at 1.5 t ( > 200 mmol / kg ) [ 3 ]  . 
furthermore , the very large volume of interest ( voi ) applied at 1.5 t ( > 30 cc ) makes phe signal detection unreliable due both to concurrent line - broadening effects on the resonance and the presence of macromolecular contributions at 7.3 ppm [ 36 ]  . in recent years , diffusion - weighted mri ( dwi - mri ) has been introduced in clinical practice . 
however , these studies have failed to clarify the significance of dwi changes in relation to clinical findings . the aim of this work was to analyse the potential and advantages of a 3.0 - t system in the multimodal study of brain alterations of patients with pku . 
they were 17 male and 15 female subjects aged from 7 to 34 ( mean 18.9 , standard deviation 6.0 ) years , 21 with early and 11 with late diagnosis . 
in particolare andiamo a misurare la concentrazione di molecole contenenti il gruppo n - acetile ( in prevalenza ln - acetil - aspartato , naa ) , le macro - molecole contenenti colina ( cho ) , la creatina ( cr ) , la fosfocreatina ( pcr ) e il mio - inositolo ( mi ) [ 3 ]  . 
le variazioni di naa ( marker di integrit neuronale ) e di mi ( potenziale marker astrocitario ) suggeriscono una possibile disfunzione neurochimica mentre mediante la quantificazione della cho abbiamo un indice del grado della mielinizzazione tissutale [ 4 , 5 ]  . 
lo studio di 1h - mrs con unapparecchiatura a 1 , 5 tesla ( t ) ha permesso di quantificare i livelli cerebrali di phe in pazienti con pku : tali misurazioni hanno fornito dati interessanti sulla biochimica delle alterazioni della sostanza bianca in tale patologia [ 35 ]  . 
la valutazione dei livelli di phe , ottenuta utilizzando sistemi a 1 , 5 t , per ancora soggetta a delle limitazioni : nonostante i miglioramenti dei protocolli di acquisizione 1h - mrs e di quantificazione dei metaboliti c un limite intrinseco dovuto alla soglia di detezione del metabolita ( > 200 mmol / kg ) legato direttamente alla intensit del campo magnetico ( 1 , 5 t ) [ 3 ]  . 
inoltre la notevole ampiezza del volume di interesse ( voi ) in sistemi a 1 , 5 t ( > 30 cc ) rende il valore di phe non attendibile e ci dovuto sia a effetti concomitanti di allargamento sulla risonanza che allinterferenza delle altri componenti macromolecolari presenti a 7 , 3 ppm [ 36 ]  . negli ultimi anni sono state introdotte di routine nella pratica clinica le sequenze rm pesate in diffusione ( dwmri )  . 
 [ 8 ] hanno dimostrato in pazienti con pku la presenza di alterazioni di segnale in dwi che si sovrappongono sostanzialmente alle alterazioni rilevate in condizioni rm di base e che sono caratterizzate da iperintensit in t2 . 
le mappe del coefficiente di diffusione apparente ( adc ) invece documentano valori compatibili con una diffusione ristretta dei protoni . tali studi comunque non hanno ancora chiarito lorigine di tali alterazioni e una chiara correlazione col quadro clinico . lo scopo del nostro lavoro di analizzare le possibilit e i vantaggi della rm 3 , 0 t nello studio multimodale delle alterazioni cerebrali di pazienti con pku ed in particolare di valutare la sensibilit di un sistema rm ad alto campo nel misurare la phe cerebrale con la 1h - mrs e nel documentare le alterazioni della sostanza bianca rilevate con lo studio rm di base utilizzando gli studi di dw - mri . magnetic resonance technique materiali e metodi mr studies were performed with a 3.0 - t scanner ( signa horizon lx , general electric medical system , milwaukee , wi , usa ) with a standard quadrature transmit / receive birdcage head coil . 
wm involvement was assessed by morphological mr with sagittal fast spoiled gradient recalled ( fspgr ) t1 - weighted [ repetition time ( tr ) / echo time sono stati inclusi nello studio 32 pazienti con un tipico quadro biochimico - clinico della pku : 17 maschi e 15 femmine , et compresa tra 7 e 34 anni ( media 18 , 9 , deviazione standard 6 , 0 ) : 21 con diagnosi precoce e 11 con diagnosi tardiva di malattia . 
in tutti i pazienti stato valutato il valore ematico di phe ed stata effettuata una 464 radiol med ( 2009 ) 114 : 461474 ( te ) / flip angle ( fa ) 225 / minimum / 75 ms , acquisition time 1 min 57 s ] sequences ; axial fluid - attenuated inversion recovery ( flair ) ( tr / te / ti , inversion time , 11 , 000 / 130 / 2 , 250 ms , acquisition time 2 min 26 s ) and spin echo ( se ) t1 - weighted ( tr / te 500 / 10 ms , acquisition time 2 min 21 s ) sequences , and coronal fast spin echo ( fse ) t2weighted ( tr / te 5 , 000 / 55 / 20 ms , acquisition time 1 min 05 s ) sequences . 
standard mr scans were acquired using 5mm - thick slices , 1 - mm gap , matrix size 256192 , and field of view ( fov ) 22 cm . single - voxel 1h - mrs was performed using a short te point - resolved spectroscopy ( press ) sequence ( tr / te 2 , 000 / 35 ms , total averages 128 , spectral width 2 , 500 hz , 1 , 024 complex data points , acquisition time 4 min 56 s )  . 
a voxel volume of just 8 cc was placed in the altered periventricular deep wm . the diffusion studies [ dwi , adc maps and diffusion tensor imaging ( dti ) ] were performed in the axial plane using a pulsed gradient se echo - planar sequence ( tr / te 8 , 000 / minimum ms ; dwi acquisition time 44 s and for dti 6 min 57 s ) without ( b - value = 0 ) and with ( b - value = 1 , 000 s / mm2 ) diffusion gradients applied along 25 different directions to cover the three - dimensional space uniformly . 
 additional dwis were acquired in the axial plane , with identical parameters except for the b - value , which was set at values from 500 to 2 , 500 s / mm2 ( 500 , 1 , 000 , 1 , 500 , 2 , 000 and 2 , 500 ) for five different acquisitions . data analysis all postprocessing was performed on a unix workstation . wm changes were analysed on mri both qualitatively , with reference to signal intensity in the different pulse sequences , and semiquantitatively , with reference to their anatomical location ( frontal , temporal , occipital , cerebellar and in brainstem ) and extension ( deep and / or subcortical wm )  . 
wm involvement was scored on t2 - weighted images by raters who were unaware of patients clinical history , as follows : 1 for deep wm involvement only ( limited involvement ) ; 2 for involvement of deep and subcortical wm ( intermediate involvement ) ; 3 for greater involvement of brainstem and / or cerebellum ( severe involvement )  . 
 brain phe was quantified by computing the amplitude of the phe signal at 7.36 ppm relative to the cr / pcr peak at 3.05 ppm , considered as an internal reference , using the sage / idl software . 
the other metabolites ( naa , cho , cr / pcr , mi ) were analysed by means of lcmodel routines . the diffusion images were analysed assuming a monoexponential relation between signal intensity and the product of the b - matrix ( a 33 matrix expressing the relationship between signal attenuation and elements of the diffusion tensor matrix )  . 
trenta soggetti sani ( 9 maschi e 21 femmine ; et compresa tra 12 e 58 anni ; media 32 , 9 ) sono stati arruolati nello studio come gruppo di controllo per confrontare i dati ottenuti dallimaging rm . 
il consenso informato stato firmato da tutti i pazienti o da loro parenti prossimi . tecnica rm lo studio rm stato effettuato con unapparecchiatura rm 3 , 0 t ( signa horizon lx , general electric medical system , milwaukee , wi , usa ) dotata di bobina della testa standard in quadratura trasmittente / ricevente . 
le alterazioni della sostanza bianca sono state valutate con la rm di base utilizzando le sequenze : fast spoiled gradient recalled ( fspgr ) t1 - pesata ( tr , tempo di ripetizione , / te , tempo di echo , / fa , flip angle , 225 / minimum / 75 ms , tempo di acquisizione 1 min 57 s ) acquisita in proiezione sagittale ; fluid - attenuated inversion recovery ( flair ) ( tr / te / ti , tempo di inversione , 11000 / 130 / 2250 ms , tempo di acquisizione 2 min 26 s ) e spin echo ( se ) t1 - pesata ( tr / te 500 / 10 ms , tempo di acquisizione 2 min 21 s ) in proiezione sagittale ; fast spin echo ( fse ) t2 - pesata ( tr / te 5000 / 55 / 20 ms , tempo di acquisizione 1 min 05 s ) in proiezione coronale . 
il volume di interesse di 8 cc stato posizionato nelle aree di alterazione della sostanza bianca periventricolare profonda . lo studio in diffusione ( dwi , mappe adc e diffusion tensor imaging , dti ) stato ottenuto sul piano assiale utilizzando sequenze pulsed - gradient se eco - planari ( tr / te 8000 / minimum ms , tempo di acquisizione dwi 44 s e 6 min 57 s per il dti ) senza ( b - value = 0 ) e con gradienti di diffusione ( b - value = 1000 s / mm2 ) applicati lungo 25 direzioni per coprire uniformemente e tridimensionalmente il volume di interesse . ulteriori immagini pesate in diffusione sono state acquisite sul piano assiale con gli stessi parametri fatta eccezione per il b - value che stato impostato in valori variabili compresi tra 500 e 2500 s / mm2 ( 500 , 1000 , 1500 , 2000 e 2500 ) per un totale di cinque diverse acquisizioni . analisi dei dati la successiva elaborazione dei dati stata effettuata utilizzando una workstation unix . 
le alterazioni della sostanza bianca sono state analizzate sia qualitativamente , con riferimento alle variazioni di intensit di segnale nelle varie sequenze , che semi - quantitativamente considerando la loro radiol med ( 2009 ) 114 : 461474 tensor diagonalisation , of the main eigenvalues min , med , and max , were generated and the fractional anisotropy index ( fai ) , was derived from the eigenvalues for each pixel . 
 the analysis was performed on parieto - occipital wm , as all patients exhibited dwi changes in this region ; a spherical region of interest ( roi ) of uniform size ( ~45 mm2 ) was placed on the dwis and adc maps , and eigenvalues and fai values were then calculated . 
these data were confirmed by dti quantitative analysis , as decreased adc values but normal fai indices were found in the rois located on the hyperintense parieto - occipital wm ( table 1 )  . 
qualitative analysis of anisotropy images acquired with b - values > 1 , 000 s / mm2 showed an increased contrast between normal tissue and t2 / flair / dwi - mri hyperintense areas . 
il punteggio utilizzato stato calcolato come segue : 1 punto per lesioni interessanti solo la sostanza bianca profonda ( coinvolgimento limitato ) , 2 punti per lesioni interessanti anche la sostanza bianca sottocorticale ( coinvolgimento intermedio ) , 3 punti per leventuale coinvolgimento del tronco e / o del cervelletto ( coinvolgimento severo )  . il valore di phe cerebrale stato quantificato valutando lampiezza del picco di phe a 7 , 36 ppm in relazione al picco di cr / pcr a 3 , 05 ppm ( considerato come un valore interno costante e quindi di riferimento , usando il software sage / idl )  . 
gli altri metaboliti ( naa , cho , cr / pcr e mi ) sono stati analizzati utilizzando lcmodel software . le immagini in diffusione sono state analizzate utilizzando una relazione mono - esponenziale tra lintensit di segnale e il segnale prodotto della matrice b ( matrice 33 che esprime la relazione tra lattenuazione di segnale e gli elementi della matrice del tensore di diffusione )  . 
 lanalisi stata effettuata sulla sostanza bianca parietooccipitale poich tutti i pazienti presentavano alterazioni di segnale in dwi in questa regione ; una regione di interesse ( roi ) sferica e di misura uniforme ( ~45 mm2 ) stata posizionata sulle immagini pesate in diffusione , quindi mappe di adc , autovalori e valori di fai sono stati calcolati . 
the advantages of a higher mr field , with respect to the low - field system used to date , is a larger diagnostic ability in the management of patients with this pathology . 
resolution quality tends to be privileged in clinical practice , with acquisition of a larger number of slices of reduced thickness , use of smaller fov and broader matrices , and acquisition times similar to those of 1.5 - t ai soggetti normali . 
questi dati sono stati confermati dalla analisi quantitativa del dti : nelle regioni di interesse localizzate nelle aree iperintense della sostanza bianca parieto - occipitale stato trovato un valore di adc ridotto ma un normale valore di fai ( tabella 1 )  . 
lanalisi qualitativa delle immagini dellanisotropia , acquisite con b - value pi elevato di 1000 s / mm2 , mostra un aumento del contrasto tra il tessuto normale e le aree di iperintensit in t2 / flair / dw - mri . 
2a - e proton magnetic resonance spectroscopy allows detection of an abnormal phenylalanine ( phe ) signal at 7.36 pp values of the main metabolites [ n - acetylaspartate ( naa ) , choline - containing compounds ( cho ) , creatine ( cr ) / phosphocreatine ( pcr ) ] were normal ( a , arrow )  . 
all patients exhibit variable white - matter hyperintensities on t2 ( c ) and fast low - angle inversion recovery ( flair ) ( d ) scans , mainly in temporal , occipital and periatrial regions . 
in our work , instead , we found the optimum acquisition time as a tradeoff between a time - consuming multimodal acquisition and the patients comfort during the exafurther technological advances are expected to overcome some minor limitations of this system , such as field discussione in tale studio nei pazienti con pku si confermano i dati dei precedenti studi circa la frequenza e le caratteristiche delle alterazioni della sostanza bianca e le correlazioni tra il radiol med ( 2009 ) 114 : 461474 fig . 
a questo valore il confronto dellintensit di segnale sullasse x ( a ) e y ( b ) permette di rilevare una sottile variazione di segnale nella sostanza bianca alterata in relazione ad una parziale conservata anisotropia ( freccia gialla )  . inhomogeneity , susceptibility and chemical - shift artefacts , high specific absorption rate ( sar ) and acoustic noise . in detail , the use of higher fields for spectroscopy allows reduced acquisition time ( higher s / r ) and improved spectral resolution [ 18 , 19 ]  . 
as a result of such improvements ( increased snr and greater spectral resolution compared with 1.5 - t studies ) , the phe signal was easily detected and was measurable despite the low phe concentrations . 
the relative amplitude of the phe signal increased linearly with blood phe up to 1 , 200 micromoles , where growing dispersion of brain phe values was noted , suggesting a more marked interindividual variability , probably due to saturation of the specific phe transport system at the level of the bloodbrain barrier ( bbb )  . a short te ( 35 ms ) was applied better to detect the phe signal as well as those of the principal metabolites . 
furthermore , spectroscopic studies with longer te are subject to greater snr loss at 3.0 t than at 1.5 t due to t2 reduction at higher field strengths and increased peak line width in relation to reduced t2 and microscopic and macroscopic body inhomogeneities . 
these features allow an increased b - value to enhance the anisotropic effect , thus improving the quality of more complex examinations such as fai , diffusion tensor and tractography , which are poorly displayed on 1.5 - t images , even with multiple grado di severit e parametri clinici e laboratoristici . 
per ottenere maggiori informazioni sulla patogenesi di tali alterazioni abbiamo condotto con apparecchiatura a 3 , 0 t uno studio rm multimodale che ha compreso nel protocollo uno studio di base , di spettroscopia e di diffusione al fine di migliorare , sfruttando i vantaggi derivanti dalluso dellalto campo rispetto a quello dei tradizionali sistemi a 1 , 5 t , la capacit diagnostica che pu risultare utile nel management di tali pazienti [ 1517 ]  . in generale lintroduzione di magneti ad alto campo ha infatti portato dei benefici in campo neuroradiologico . 
il confronto delle immagini acquisite con sistemi a 3 , 0 t rispetto a quelle ottenute con 1 , 5 t ha mostrato degli indubbi vantaggi quali : aumento della risoluzione spaziale , temporale e di contrasto , intensit di segnale pi elevato , ridotto tempo di acquisizione , maggiori e pi avanzate applicazioni , elevata sensibilit ed eccellente potere diagnostico nelle sequenze con mezzo di contrasto . 
 il neuroradiologo quindi , grazie allalto s / r dellalto campo , utilizzando lo stesso tempo di un esame con sistemi a 1 , 5 t , pu ottenere immagini di qualit superiore ( aumentando la matrice o riducendo lo spessore di strato o il fov ) oppure immagini della stessa qualit di un basso campo ma in un tempo ridotto : ci si traduce in un maggior confort per il paziente e in una riduzione degli artefatti da movimento . 
la qualit della risoluzione tende ad essere privilegiata nella pratica clinica , grazie allacquisizione di un maggior numero di slices con uno spessore di strato ridotto tramite lutilizzo di un fov pi piccolo , di matrici pi ampie con un tempo di acquisizione risultante simile a quello speso se si utilizzano scanner a 1 , 5 t . 
4a , b comparison of proton magnetic resonance spectroscopy brain phenylalanine ( phe ) detection at 1.5 t ( a ) and 3.0 t ( b ) at 7.36 ppdetection of phe signal in the 1.5 - t study requires a 32 - cc volume of interest ( voi )  . 
4a , b confronto della fenilalanina cerebrale rilevata con la spettroscopia protonica a 1 , 5 t ( a ) e 3 , 0 t ( b ) a 7 , 36 ppla documentazione del segnale della fenilalanina a 1 , 5 t necessita di un voi di 32 cc . 
luso di un pi piccolo voi ( 8 cc ) riduce gli effetti da sovrapposizione con altri metaboliti quali istidina , omocarnosina , macromolecole presenti a 7 , 3 ppm . radiol med ( 2009 ) 114 : 461474 quale compromesso tra il tempo necessario per uno studio multimodale e che garantisse un adeguato confort per il paziente durante lesame . nello specifico in spettroscopia luso dellalto campo permette di ridurre i tempi di acquisizione ( in virt del pi alto s / r ) ma anche migliora la risoluzione spettrale [ 18 , 19 ] la maggior ampiezza del chemical shift dei metaboliti in esame permette infatti di avere una migliore risoluzione spettrale che si traduce in una pi definita separazione dei picchi adiacenti che invece con un sistema a pi basso campo andrebbero a sovrapporsi . 
in virt di tali miglioramenti ( aumento del s / r e migliore risoluzione spettrale ) possibile individuare e misurare pi facilmente il picco della phe , persino a basse concentrazioni . 
lampiezza relativa del picco di phe aumenta linearmente con la concentrazione ematica della stessa sino a un valore di 1200 micromoli oltre il quale si osserva una dispersione dei valori cerebrali di phe dovuta probabilmente a una variabilit individuale derivante dalla saturazione dei sistemi di trasporto della phe localizzati a livello della barriera emato - encefalica ( bee )  . stato utilizzato un te ridotto ( 35 ms ) per avere una migliore individuazione del segnale ( picco ) della phe e degli altri principali metaboliti in esame . 
utilizzando un te pi lungo si avrebbe una riduzione del rapporto s / r pi marcata con lutilizzo di sistemi a 3 , 0 t che con quelli a 1 , 5 t , per effetto della riduzione del segnale in t2 che si ha ad alto campo , dellaumento dellampiezza della linewidth causata dalla riduzione del segnale sempre in t2 e per le disomogeneit a livello microa macro - scopico . anche limaging pesato in diffusione beneficia dellaumento del rapporto s / r , della risoluzione spaziale e della accuratezza delle immagini [ 1517 ]  . 
si verifica in tal modo un miglioramento della qualit di alcune misurazioni come la fractional anisotropy ( fai ) , il tensore di diffusione ( dti ) e di alcune tecniche di ricostruzione come la trattografia . questi valori non sono ben rappresentati a causa del basso s / r utilizzando un sistema a 1 , 5 t anche se si dovesse aumentare il numero delle eccitazioni . 
infatti le ricostruzioni dei tratti assonali riflettono con maggior accuratezza la realt anatomica , specialmente a livello delle biforcazioni dei fasci . radiol med ( 2009 ) 114 : 461474 excitations , owing to the low snr . 
in fact , the reconstructed dti trajectories reflect more accurately the underlying axonal fibres , particularly crossing or bifurcating bundles . for some years , it has been possible to reconstruct threedimensional pathways of wm tracts by combining anisotropy characteristics and directionality , applying the technique known as fibre tracking or tractography . 
the method allows reconstruction of continuous fibre trajectories by sequentially piecing together discrete and shortly spaced estimates of fibre orientation to form continuous fibre trajectories [ 2022 ]  . the colour map is an easy method to show dti values , as it displays the main direction of wm fibres in three fundamental colours : green , blue and red . 
in particular , ontogenetically older fibre systems , such as the optic radiation , had a normal appearance . wm changes depicted in pku patients do not therefore necessarily reflect an altered organisation of cerebral myelin pathways detectable on dti . 
some mr findings in pku appear to be more consistent with reversible dysmyelination associated with an increased myelin turnover than with a demyelination process [ 2 , 15 , 25 , 26 , 29 , 30 ]  . our latest report of decreased adc values with preserved anisotropic diffusivity on dwi - mri and abnormal elevation of brain phe levels on 1h - mrs [ 31 ] seems to be in line with the hypothesis of water accumulation in myelin sheaths [ 7 ] due to an abnormally increased myelin turnover with consequent preservation of anisotropy , which can occur only in structures whose microscopic barriers have not been either destroyed or partially vacuolated . however , a third hypothesis consistent with these findings is that of intracellular accumulation of a hydrophilic metabolite produced by the phe hydroxylase defect , leading da alcuni anni possibile effettuare una ricostruzione tridimensionale del decorso delle fibre mieliniche . 
utilizzando le caratteristiche di anisotropia e tecniche note come il fiber tracking o trattografia si ricostruisce la direzione delle fibre nervose interpolando in modo sequenziale , stime discrete e poco spaziate dellorientamento della fibra per ottenere la sua traiettoria continua [ 2022 ]  . 
 ne derivano mappe a colori che costituiscono un metodo semplice per mostrare i valori del tensore di diffusione attraverso la visualizzazione delle principali direzioni delle fibre nervose in tre diversi colori ( grigio , blu e rosso )  . lapplicazione di tale tecnica nei pazienti con pku ci consente di poter valutare lintegrit delle fibre nervose nelle aree caratterizzate da riduzione dei valori di adc . 
al momento attuale il principale problema nella tecnica del fiber tracking e nella rappresentazione delle mappe a colori dovuto alla difficolt di ottenere una buona delineazione delle fibre mieliniche , senza una parziale contaminazione da parte dei tratti nervosi adiacenti [ 24 ]  . lanalisi del dti ha mostrato una riduzione dei valori di adc nelle aree iperintense nellimaging di base , in linea con i precedenti lavori in letteratura [ 7 , 8 ]  . 
in particolare le fibre ontogeneticamente pi vecchie come i tratti ottici appaiono normali . le alterazioni della sostanza bianca , viste nella pku , non necessariamente riflettono una alterazione architetturale del decorso delle fibre mieliniche , valutato con dti . 
le alterazioni della sostanza bianca nei pazienti con pku sono state documentate in molti studi effettuati con sistemi rm a pi basso campo , ma il loro significato non ancora stato chiarito [ 315 , 2528 ]  . 
alcuni pattern rm sembrano essere correlati pi con un reversibile processo di dismielinizzazione che con uno di demielinizzazione [ 2 , 15 , 25 , 26 , 29 , 30 ]  . in un precedente nostro lavoro la riduzione dei valori di adc con una preservazione dei valori di fa in diffusione e lanormale incremento dei livelli di phe cerebrale in spettroscopia protonica [ 31 ] potrebbero suggerire un accumulo di acqua nelle guaine mieliniche [ 7 ] dovuto a un incremento anomalo del turn - over mielinico stesso che si ha in strutture in cui le barriere microscopiche non sono state ancora del tutto degradate o vacuolizzate . 
 una terza ipotesi in linea con le nostre valutazioni che laccumulo di una metabolita idrofilico intracelllulare prodotto dalla phe - idrossilasi porta a un aumento del contenuto di acqua intracellulare . 
5a - d imaging di diffusione : mappa di adc ( a ) con selezione di una roi , mappa di fai ( b ) e a colori ( c ) ; i valori delle roi di adc e fa ( d ) misurati nelle aree di ciascun paziente ( punti ) e in range normali ( barra : meansd )  . to increased intracellular water content [ 31 ]  . 
the latter data require further examination given that no other abnormalities were detected by in vivo mrs examination , apart from the phe increase . pattern di dwi sia le anormalit dei valori ottenuti con la spettroscopia ( accumulo di phe con valori normali degli altri metaboliti )  . 
questi ultimi dati richiedono ulteriori studi dato che nessuna altra anomalia stata rilevata nellesame di spettroscopia in vivo a parte lincremento della phe . conclusions conclusioni the higher snr of high - field mr systems is a resource that neuroradiologists must exploit in clinical practice , as the higher the snr , the more the imaging protocols can be adjusted . 
i sistemi a 3 , 0 t permettono infatti di effettuare oltre a un accurato studio di base , anche delle valutazioni di carattere fisiologico , metabolico e funzionale che consentono di aumentare la sensibilit e la specificit del potere diagnostico della rm . sono comunque necessari ulteriori progressi tecnologici che permettano di superare alcune piccole limitazioni di tali sistemi rm come la disomogeneit del campo magnetico , radiol med ( 2009 ) 114 : 461474 inhomogeneity , susceptibility and chemical - shift artefacts , high sar and acoustic noise . the growing diffusion of high - field mr systems over the next few years will help a clearer understanding of the pathogenic role of signal abnormalities of the cerebral parenchyma in patients having several kinds of neurological diseases , such as pku . 
in addition , measurement of brain phe levels with 1h - mrs and dwi - mr parameters could become the technique of choice to monitor the influx of blood phe into brain tissue , especially during therapy in follow - up studies . gli artefatti da suscettibilit magnetica e da chemical shift , lelevato sar ( specific absorption rate ) e lalto rumore acustico . la crescente diffusione di tali sistemi nei prossimi anni permetter una migliore comprensione delle cause di alterazione del segnale in pazienti con patologie come la pku . 
guerci , radiologia padiglione barbieri , ospedale maggiore di parma , via gramsci 14 , 43100 parma , italy , tel . : + 39 - 052 - 1703660 , fax : + 39 - 052 - 1986352 , e - mail : leonardo.guerci@libero.it received : 12 august 2008 / accepted : 16 september 2008 / published online : 12 march 2009 springer - verlag 2009 abstract two women with profoundly different backgrounds were brought together in a destiny that saw their paths cross and join in the discovery of radioactivity : marie curie and blanche wittman . 
the discovery of radioactivity was the common denominator underlying the vicissitudes of their lives , the same radioactivity that was so acclaimed and of such incredible diagnostic and therapeutic potential while at the same time so underrated in the everyday life of the time that disregarded , almost disparagingly , the deleterious biologic effects it was capable of provoking . 
at the beginning of the twentieth century , those effects were in fact often underestimated or scarcely considered , and it was only after world war ii that there came an awareness of the ambiguous properties of ionising radiation . 
after numerous studies on radiation exposure , much of the current debate concerns the possible effects of exposure to small doses , such as those delivered in most radiological examinations . 
the theories proposed include the unorthodox theory of hormesis , which requires careful riassunto due donne , due storie profondamente diverse , unite da un destino che le incrocia e le unisce nella scoperta della radioattivit : marie curie e blanche wittman . 
la prima , una delle pi grandi donne e scienziate di tutti i tempi , unica nella storia ad aver vinto due premi nobel per la scienza , dotata di uno spirito umanitario fuori dal comune , dedic , nonostante lo scandalo sentimentale che la vide al centro della cronaca , la maggior parte della sua vita alla ricerca scientifica . 
la seconda , passata alla storia come la regina delle isteriche durante il suo ricovero nel famigerato manicomio della piti salpetrire di parigi , divenne alla sua guarigione una delle pi strette collaboratrici di m.me curie nellestrazione del radium dai fanghi della boemia , per ben 16 anni di duro lavoro , fino al momento della sua morte . 
ed proprio la scoperta della radioattivit il comun denominatore che sta alla base delle loro vicende umane , quella stessa radioattivit cos celebrata e dalle incredibili potenzialit diagnostiche e terapeutiche , ma allo stesso tempo cos svilita nella quotidianit della vita di quel tempo , che ignorava , talora quasi a sprezzo , gli effetti nocivi biologici in grado di provocare . 
nei primi anni del 900 tali effetti furono infatti spesso sottovalutati o scarsamente considerati , e fu solo dal secondo dopoguerra in poi che si manifest una forte presa di coscienza sulle ambigue propriet delle radiazioni ionizzanti . 
dopo numerosi studi condotti sullesposizione alle radiazioni ionizzanti , ci di cui molto si discute oggi riguarda principalmente i possibili effetti provocati dallesposizione a piccole dosi , come quelle erogate nella 348 radiol med ( 2009 ) 114 : 347357 reevaluation . 
much light has been shed on radiology since the time of blanche and marie , but there still remain many shadows to dispel , and this can only be done by serious and constant scientific commitment . keywords curie wittman ionizing radiation biological risk radiobiology maggior parte degli esami radiologici : tre le teorie proposte , una delle quali ( quella eretica della ormesi ) richiede di essere rivalutata con maggiore attenzione . molte luci si sono accese dai tempi della radiologia di blanche e marie , tuttavia al giorno doggi rimangono da chiarire ancora molte ombre , che solo un serio e costante impegno scientifico potr dissipare in futuro . parole chiave curie wittman radiazioni ionizzanti rischio biologico radiobiologia introduction introduzione a few years ago , the swedish writer p.o. 
enquist [ 1 ] wrote a charming book about two very different women brought together by the vagaries of life to work side by side and share hardships , danger and emotions linked with the early study of radioactivity : marie curie and blanche wittman . the clearly fictionalised vicissitudes narrated by enquist were the starting point for research that led us to analyse several aspects of the history of radiology . qualche anno fa , lo scrittore svedese p.o. 
enquist [ 1 ] ha dedicato un bel libro alla storia di due donne , molto diverse tra loro ma che i casi della vita hanno portato a lavorare fianco a fianco e a condividere fatiche , pericoli ed emozioni legate ai primi studi sulla radioattivit : marie curie e blanche wittman . 
a few days before she was awarded the prize in stockholm , a scandal over her affair with another famous physicist , paul langevin ( 18721946 ) broke out in paris . 
 this event was considered of such importance that the marie curie marie sklodowska , nata a varsavia , in polonia , nel 1867 , era figlia di un libero pensatore e di una cattolica praticante . 
pochi giorni prima della consegna del secondo nobel a stoccolma , a parigi scoppi lo scandalo per la relazione che la ancor giovane vedova aveva iniziato con un altro fisico famoso , paul langevin ( 18721946 )  . 
 radiol med ( 2009 ) 114 : 347357 questo evento ebbe una rilevanza tale che il fisico svante arrhenius , membro del comitato per il nobel , giunse a consigliare a marie di rifiutare il premio . 
lei stessa e la figlia irne ( allora diciassettenne ) , impararono a guidare questi automezzi , studiarono anatomia e , in compagnia di personale medico , prestarono a lungo la propria opera in zona di guerra . madre e figlia si sottoposero cos a forti dosi di raggi x senza alcuna protezione . 
negli stessi anni , marie fond un servizio di radioterapia militare , isolando personalmente il gas radioattivo ( radon ) in tubi di vetro che venivano spediti a diversi ospedali francesi . 
marie vers anche alla patria acquisita la maggior parte delle somme ricevute dalla fondazione nobel [ 3 ]  . dopo la fine della guerra , negli anni dal 1919 al 1934 , marie ormai una celebrit mondiale viaggi molto , recandosi anche pi volte negli stati uniti , dove incontr i presidenti harding e hoover , lanci raccolte di fondi per la ricerca e ottenne notevoli finanziamenti , nonostante la timidezza le impedisse di parlare in pubblico con facilit . 
during her stay in england , marie , deeply shocked by the parisian scandal , came into contact with the movement for womens suffrage , the leading member of which was emmeline pankhurst ( 18581928 ) , who was arrested on several occasions . back in paris after a break of 14 months , marie returned to her laboratory , as the scandal began to die down with the separation by mutual agreement of the langevins . 
marie and paul did not resume their affair , although their relationship remained friendly and fruitful on the professional level . at the outbreak of world war i , only two people remained on the premises of the radium institute , which was planned by marie and built in the latin quarter in the street renamed for the occasion in honour of pierre curie : marie , and a mechanic who was exempt from the draft due to a serious heart condition . although until then , maries main interest had been the radioactive phenomena , she realised the enormous diagnostic potential of artificial x - rays ( roentgen rays ) in saving the lives of wounded soldiers at the front . 
she set up 350 radiol med ( 2009 ) 114 : 347357 marie curie cominci a soffrire di disturbi fisici connessi alle radiazioni intorno al 1920 , con una doppia cataratta che richiese quattro dolorosi e , per lepoca , complicati interventi . 
negli ultimi anni , manifest una progressiva debolezza , dapprima attribuita erroneamente alla tubercolosi , finch uno specialista di ginevra cap che poteva trattarsi di una forma simil - tumorale di anemia aplastica , dovuta al danno midollare da radiazioni [ 4 ]  . quando mor , nel 1934 , 28 anni dopo il marito pierre , listituto da lei diretto era una delle massime realt scientifiche del tempo e molte applicazioni mediche e industriali delle scoperte dei curie testimoniavano gi delle vaste potenzialit applicative della radioattivit . 
negli anni compresi tra il 1919 ed il 1934 , listituto diretto da marie curie produsse 483 lavori scientifici o libri , 31 dei quali scritti da marie [ 3 ]  . marie venne sepolta due volte ; la prima a sceaux , con il marito pierre . 
il castello , in quegli anni , attirava la curiosit morbosa di frotte di visitatori , da sarah bernhardt ad axel munte , a numerosi giovani studiosi di neuropsichiatria , da broca a gilles de la tourette , al giovane freud . 
in sostanza , alla fine dell800 , non molto era cambiato nella vita degli internati da quando , un secolo prima , il castello era diretto dall illuminista dottor pinel , il quale aveva tentato con scarso successo , nel 1795 , loperazione insieme politica ed umanitaria di liberare i folli dalle catene . 
foucault ha scritto , a proposito del tentativo di pinel : le catene cadono , il folle si ritrova libero e in quel momento recupera la ragione la brutalit che appare nei dementi non che il riflesso della bestialit dei guardiani e ancora : a partire da pinel si vedr nella follia uno slancio venuto dal profondo , che oltrepassa i limiti dellindividuo e tende ad una apoteosi di se stesso [ 5 ]  . blanche , ricoverata dal 1878 al 1893 , divenne presto famosa come la regina delle isteriche , protagonista delle esibizioni organizzate e dirette settimanalmente dal dottor fig . 
marie and her daughter , irne ( aged 17 at the time ) , learned how to drive these vehicles , studied anatomy and , in the company of medical personnel , gave long - term service in the war zone . 
during the same period , marie established a military radiotherapy service and personally isolated the radioactive gas ( radon ) in glass tubes that were sent to various french hospitals . 
she also donated most of the money received from the nobel foundation to her adopted homeland [ 3 ]  . after the war , in the years between 1919 and 1934 , marie , who had become an international celebrity , travelled widely . 
the frail figure of this now elderly woman dressed constantly in black became legendary , and due to her austere appearance and moral force , marie was compared with a buddhist monk . launched very successful marie began to suffer from physical problems connected with radiation around 1920 , with the appearance of a double cataract that required four painful and for the times complicated operations . 
in her later years , she developed progressive weakness , which was at first erroneously attributed to tuberculosis until a specialist from geneva realised it could have been caused by aplastic anaemia , a tumour - like condition due to radiation - induced bone marrow damage [ 4 ]  . between 1919 and 1934 , the institute directed by marie produced 483 scientific papers or books , 31 of which were written by her [ 3 ]  . 
in those years , the piti salptrire attracted the morbid curiosity of a stream of visitors , from sarah bernhardt to axel munte and numerous neuropsychiatrists , from paul broca to georges gilles de la tourette and the young sigmund freud . 
in essence , at the end of the nineteenth century , little had changed in the lives of the patients from when a century earlier the hospital was directed by the enlightened physician philippe pinel , who , in 1795 , had attempted without success to perform the political and humanitarian task of freeing the inmates from their shackles . 
and at that moment they recovered their reasonthe brutality which appears in the demented is nought else than the reflection of the animality of the guardians and from pinel onwards , the classical mind recognised in madness an impulse from the depths which exceeded the juridical limits of the individual , ignoring fixed moral limits and tending towards an apotheosis of the self [ 5 ]  . blanche , who was hospitalised between 1879 and 1893 , soon became famous as the queen of hysterics , being the protagonist of the spectacular weekly lectures organised and directed by jean - martin charcot . 
the cures without support became real cures of false diseases , as in fact hysteria proved to be , which failed to outlive charcot . hysterical madness was a mixture of persuasion and mystification , even though , many years later , blanche stated that charcots weekly performances were not based on feigning . although charcot has been accused , and probably correctly , of misogyny [ 6 ] , in 1885 , he accepted into his group a brilliant young medical graduate , blanche edwards [ 7 ]  . after the death of charcot , blanche wittman , having been declared clinically cured , found temporary work as a technical assistant in the recently instituted radiology department of salptrire . 
indeed , roentgens discovery ( 1895 ) was made only 2 years after the death of charcot and was contemporary to freuds studies of hysteria . in 1898 , at the time of the dreyfus affair , blanche moved to the laboratory of the curies , with the thankless task of isolating radium from the pitchblende from sankt joachimsthal in bohemia and in which she remained for hours and hours each day immersed up to her knees in pitchblende . this was the beginning of the collaboration and cohabitation of blanche and marie , which lasted 16 years . 
after 4 years of work , the curies , with the help of several assistants , including blanche , managed to isolate 0.1 g of radium from tonnes of pitchblende . the lesions caused by the radioactive pitchblende caused blanche to have first her legs and then her left arm amputated . 
reduced virtually to a torso and forced to move about in a wooden cart , blanche had to stop working in the laboratory but remained until her death , in 1913 , within the curies family circle and their closest friends and collaborators , such as becquerel [ 8 ]  . ha potuto compiersi se non con la complicit del malato stesso . 
la follia isterica era un insieme di persuasione e di mistificazione anche se , parecchi anni dopo , blanche ebbe a dichiarare che gli spettacoli settimanali di charcot non erano basati sulla simulazione . 
nonostante charcot sia stato accusato , probabilmente con ragione , di misoginia [ 6 ] , nella sua quipe egli accett anche , nel 1885 , una giovane e brillante laureata in medicina , blanche edwards [ 7 ]  . 
 nel 1898 , allepoca dellaffare dreyfus , blanche si trasfer nel laboratorio dei coniugi curie , con lingrato compito di isolare il radium dai fanghi della pechblenda provenienti dalle cave di sankt joachimsthal in boemia e nei quali restava immersa fino alle ginocchia per ore ed ore , ogni giorno . 
da una tonnellata di fanghi e terriccio , dopo quattro anni di duro lavoro , i curie , con laiuto di alcuni aiutanti , tra cui blanche wittman , riuscirono ad isolare 0 , 1 grammi di radiu le lesioni provocate dai fanghi radioattivi portarono la povera blanche a subire progressivamente lamputazione delle gambe e del braccio sinistro . 
ridotta ad un torso e costretta a spostarsi su un carretto di legno , blanche cess ovviamente di lavorare nel laboratorio , ma rest fino alla morte , avvenuta nel 1913 , nella cerchia familiare dei curie e dei loro pi stretti amici e collaboratori , come il grande fisico henri becquerel [ 8 ]  . popularity and demonisation of ionising radiation at the beginning of the twentieth century , artificially produced x - rays and , to a lesser extent , the radiation emitted by newly discovered radioactive elements , had become quite fashionable . 
this popularity can be seen not only in the press of the time [ 9 ] but also in the numerous handmade articles and industrial products whose labels referred to the mysterious rays for publicity purposes , such as household products , detergents , postcards , razors , citrus squeezers , prophylactics , soap , spirits and a variety of other items [ 10 ]  . 
questa popolarit testimoniata , oltre che dalla stampa dellepoca [ 9 ] , anche da numerosi reperti , manufatti o prodotti industriali , che nelletichetta richiamavano i raggi misteriosi a scopo pubblicitario , come prodotti per la casa , detergenti , cartoline , rasoi , spremiagrumi , profilattici , sapone , alcolici ed apparecchi di ogni tipo [ 10 ]  . 
at circuses and trade fairs , as well as elegant salons , x - ray demonstrations were performed , mainly for the macabre fancy of observing the skeleton of friends and relatives in the penumbra of the fluoroscopic screen , illuminated by a weak green glow . 
to have her hand x - rayed , as roentgens wife berta had done several years earlier , the celebrated actress sarah bernhardt remained motionless on her knees for 5 min in a fashionable parisian salon [ 4 ]  . the new rays captured the imagination of the public , more so than the cinmotagraphe invented by the lumire brothers , auguste and louis , in the same year as roentgens discovery , for they embodied the guarantee of a better future , in a positivist vision of the world characterised by endless faith in progress . 
photographs of the time bear witness to the very slow growth in the awareness of the ambiguous properties of radiation , which can be a powerful weapon against disease in both diagnosis and treatment but also highly pathogenetic and therefore should be dominated and controlled , rather than used indiscriminately . 
nei circhi e nelle fiere di paese , come nei salotti eleganti , venivano eseguite dimostrazioni dei raggi x , principalmente per il gusto un po macabro di osservare lo scheletro di amici e parenti nella penombra dello schermo fluoroscopico , illuminata da una debole luminescenza verdastra . 
per farsi radiografare la mano , come aveva fatto pochi anni prima la moglie di roentgen , berta , la grande attrice sarah berhardt rimase immobile in ginocchio per 5 minuti in un salon parigino alla moda [ 4 ]  . i nuovi raggi catturavano limmaginazione del pubblico con grande intensit , pi dellinvenzione del cinematografo , che i fratelli lumire realizzarono nello stesso anno della scoperta di roentgen e rappresentavano la garanzia per un futuro migliore , in una visione del mondo ancora positivista , caratterizzata da grande fiducia nel progresso . liconografia del tempo testimonia di una presa di coscienza collettiva molto lenta delle ambigue propriet delle radiazioni , che possono costituire unarma potente contro le malattie nella diagnosi e nella terapia , ma anche risultare altamente patogene e dunque devono essere dominate e controllate , piuttosto che utilizzate indiscriminatamente . 
la invisibilit delle radiazioni portava ad una sottovalutazione degli effetti biologici . quando , un anno dopo la scoperta di roentgen , 354 radiol med ( 2009 ) 114 : 347357 could have ( unpredictable ) effects on the body . 
in 1903 , the first rudimentary forms of lead or leadrubber shields appeared , but they were seldom used , as their rigidity hampered free movement . the radiologists of the time performed hours of radioscopy every day without any protection whatsoever , and after several years of work , they developed paleness , weakening and severe asthenia . 
in 1925 , she was part of a commission of the french academy of medicine , which recommended the use of lead screens and periodic blood tests in subjects exposed to radiation in the workplace . 
hair and fingernails tended to fall out and not grow back . a list of radiation victims compiled in hamburg in 1936 and clearly incomplete bore the names of 220 physicians , physicists , chemists , technicians , nurses and religious workers . 
gendreau described the first case of occupational disease associated with radiation [ 4 ]  . workers in a watch factory established in 1914 ( radium corporation , newark , nj , usa ) used fine brushes to spread radioactive material ( known as glow in the dark ) on the watch faces to render them fluorescent in the dark . many of these watches were supplied to troops on the european fronts during world war i . 
the workers in the factory , who tended to moisten the brushes on their tongues , suffered severe ulcerations , jaw necrosis , tooth loss and various types of tumours . 
in 1929 , legal action was taken against the company , which defended itself by attributing the workers illnesses to symptoms of hysteria . the use of radiation for diagnostic and therapeutic purposes , particularly in the fight against cancer , was actively promoted as early as the latter phase of maries life after world war i . 
the biological effects of radiation became effectively controlled , and devices capable of delivering very low doses were developed without sacrificing diagnostic accuracy ( such as those quipped with image intensifiers and television chains )  . 
this growing sullamerican journal of the medical association , comparve il primo articolo sulle applicazioni mediche dei raggi x , il direttore della rivista ipotizz che la forma di energia appena scoperta potesse esercitare effetti ( imprevedibili ) sullorganismo ma , a parte il grande clinico inglese lister , nessuno raccolse queste osservazioni [ 4 ]  . 
nel 1903 comparvero le prime , rudimentali protezioni in piombo o in gomma piombifera ma pochi le utilizzarono , perch la loro rigidit ostacolava i movimenti . i radiologi praticavano ore di radioscopia ogni giorno , senza alcuna protezione e , dopo pochi anni di lavoro , accusavano pallore , deperimento e grave astenia . 
tiraboschi , da 15 anni in attivit come radiologo presso lospedale cittadino . lautopsia dimostr gravi lesioni a carico del midollo osseo e della milza ed il patologo ipotizz un ruolo eziologico delle radiazioni . 
nel 1925 marie fece parte di una commissione dellaccademia francese di medicina , che raccomand luso di schermi protettivi piombati e di test periodici del sangue in soggetti esposti professionalmente . 
i capelli e le unghie cadevano , per non ricrescere pi . una lista di vittime delle radiazioni , compilata ad amburgo nel 1936 e chiaramente incompleta , riporta 220 nomi di medici , fisici , chimici , tecnici , infermieri e religiosi . gendreau descrisse il primo caso di patologia lavorativa industriale legata alle radiazioni [ 4 ]  . 
 le operaie di una fabbrica di orologi fondata nel 1914 ( radium corporation , newark , new jersey ) utilizzavano sottili pennelli per spalmare materiale radioattivo ( detto : glow in the dark ) sui quadranti , allo scopo di renderli fluorescenti e consentire di leggere lora al buio . 
nel 29 venne istruito un processo e la ditta si difese attribuendo i disturbi delle operaie a sintomi isterici . limpiego delle radiazioni a scopo diagnostico e terapeutico , specie nella lotta contro i tumori , venne promosso attivamente gi nellultima fase della vita di marie curie , nel primo dopoguerra , ma solo nel secondo dopoguerra le conoscenze di radiobiologia raggiunsero un livello soddisfacente ; il controllo degli effetti biologici delle radiazioni divenne efficace e furono proposte apparecchiature in grado di erogare dosi molto basse , senza sacrificare radiol med ( 2009 ) 114 : 347357 awareness was certainly encouraged by the devastating effects of the atomic bombs dropped on hiroshima and nagasaki in japan in 1945 . 
it has been estimated that to obtain the same quantity of ( gamma ) radiation unleashed by the hiroshima bomb alone , millions of tonnes of the curies radium would be required . 
the nuclear proliferation in the cold war years and the chernobyl nuclear disaster in russia further contributed to the growing alarm that pervaded public opinion and the mass media in the second half of the twentieth century . the american philosopher john dewey wrote : the destructive use made of the fission of the nucleus of an atom has become the stock - in - trade of the assault upon science . what is so ignored as to be denied is that this destructive consequence occurred not only in a war but because of the existence of war , and that war as an institution antedates by unknown millennia the appearance on the human scene of anything remotely resembling scientific enquiry [ 11 ]  . today , the sustainability of medical imaging is being questioned [ 12 ]  . 
the so - called deterministic effects , which occur only in the presence of very high doses and coincide with cell death ( apoptosis ) , have a threshold effect and are not ( fortunately ) seen in individuals who undergo irradiation for diagnostic purposes or in personnel exposed to radiation in the workplace ( of course , radiotherapy is a case apart )  . the stochastic or random effects on the body are due to dna alterations produced by radiation , with consequent hereditary effects if these occur in germ cells or radioinduced tumours if they occur in somatic cells . 
particular concern regards computed tomography examinations , the number of which has increased 12 - fold over the last 20 years in europe and more than 20 - fold in the united states , with an increase of at least seven times the average dose per examination delivered to the population [ 15 ]  . laccuratezza diagnostica ( come gli impianti dotati di amplificatore di brillanza e di catene televisive )  . 
si pensi che , per ottenere la stessa quantit di radiazioni ( gamma ) prodotte dalla sola bomba di hiroshima , sarebbero stati necessari milioni di tonnellate del radium dei curie . 
la proliferazione nucleare negli anni della guerra fredda e la recente catastrofe nucleare di chernobyl hanno ulteriormente contribuito al crescente allarme , che ha pervaso lopinione pubblica ed i grandi media nella seconda met del novecento . 
si ignora che tale conseguenza distruttiva avvenuta non solo durante una guerra , ma a causa dellesperienza stessa della guerra , una istituzione nata parecchi millenni prima che comparisse sulla scena umana alcunch di lontanamente somigliante allindagine scientifica [ 11 ]  . oggi ci si interroga sulla sostenibilit dellimaging medico [ 12 ]  . 
per comprendere leffetto delle radiazioni bisogna tener conto del tipo di radiazioni coinvolte , della dose , cio della quantit di energia che esse trasferiscono nei tessuti e della variabile radiosensibilit dei tessuti stessi [ 13 ]  . 
i cosiddetti effetti deterministici , che si verificano solo per dosi molto elevate e coincidono con la morte delle cellule ( apoptosi ) , hanno un effetto - soglia e non si osservano ormai pi ( fortunatamente ) nelle persone sottoposte ad irradiazione per scopi diagnostici o in personale esposto lavorativamente ( diverso , ovviamente , il caso della radioterapia )  . 
 gli effetti stocastici o casuali sono dovuti ad alterazioni del dna prodotte dalle radiazioni , con conseguenti effetti ereditari se avvengono nelle cellule germinali o tumori radioindotti , se avvengono nelle cellule somatiche . 
le preoccupazioni riguardano soprattutto gli esami di tomografia computerizzata ( tc ) , il cui numero aumentato negli ultimi venti anni di 12 volte in europa e di oltre venti volte negli usa , con incremento di almeno 7 volte della dose media erogata per indagini radiologiche alla popolazione [ 15 ]  . state of the art and conclusions stato dellarte e conclusioni there are three theories with regard to the effects of low doses of ionising radiation . 
the best known and official doctrine , which has inspired international radiation protection laws , states that there is no threshold for stochastic effects and that even a single event or a very low dose can cause damage , the probability of which is linearly correlated with the dose ( linear nonthreshold [ lnt ] model )  . 
it has been estimated that one third of all computed tomography examinations could thus be avoided without sacrificing diagnostic accuracy . a second theory , which is not without biological and epidemiological evidence , suggests that the stochastic effects are in proportion to the dose , but in the case of low doses , there is a sort of practical threshold below which cell repair mechanisms manage to avoid damage . 
it may even be possible that the stochastic effects are not completely random , but at low doses only regard genetically predisposed individuals ( who may be identified with appropriate tests )  . the third theory , which goes contrary to accepted belief and is politically incorrect but supported by significant proof , some of which is experimental , is hormesis ( from the greek meaning to excite ) [ 16 ]  . 
moreover , life on earth evolved in conditions of environmental radioactivity much greater than the current average annual background level of around 3 msv , and the longevity of individuals exposed for professional or environmental reasons to small additional doses ( contemporary radiologists , aeroplane pilots or flight attendants , etc . ) is not lower but higher than the average , with a lower incidence of cancer . 
la lnt , estrapolata dagli effetti delle bombe atomiche sui sopravvissuti di hiroshima e nagasaki , considerata vera precauzionalmente , ma non vi sono prove certe che per dosi molto basse , come quelle coinvolte in quasi tutti gli esami di radiodiagnostica in et adulta , essa sia valida . 
la lnt ha ispirato il principio conosciuto con lacronimo alara ( as low as reasonably available ) , secondo il quale la dose erogata dovrebbe essere sempre la pi bassa ragionevolmente possibile in quelle condizioni cliniche [ 15 ]  . 
bisogna dunque mettere in atto ogni presidio tecnico per ridurre la dose erogata , senza che ci vada a discapito della accuratezza diagnostica e bisogna cercare di evitare esami inutili , di efficacia non dimostrata ( esempio : screening del tumore polmonare ) o sostituibili con altre metodiche di analoga efficacia diagnostica ( esempio : ecografia nella diagnosi della appendicite acuta )  . 
si calcola che almeno un terzo degli esami tc potrebbe cos essere evitato , senza pregiudizio per laccuratezza diagnostica . una seconda teoria , non priva di evidenze biologiche ed epidemiologiche , sostiene che gli effetti stocastici sono s proporzionali alla dose , ma che , quando si tratta di dosi piccole , esiste una sorta di soglia pratica , al di sotto della quale i meccanismi riparativi delle cellule e dei tessuti riescono ad evitare danni . 
anche possibile che gli effetti stocastici non siano poi del tutto casuali , ma che riguardino , per dosi basse , solo individui geneticamente predisposti ( ed individuabili con appositi test )  . la terza teoria , eretica e politicamente scorretta , ma sostenuta da notevoli elementi di prova anche sperimentali , lormesi ( dal termine greco che significa stimolazione ) [ 16 ]  . 
del resto , la vita sulla terra si evoluta in condizioni di radioattivit ambientale ben superiori allattuale fondo annuale medio di circa 3 millisievert e la longevit delle persone sottoposte professionalmente o per ragioni ambientali a piccole dosi aggiuntive ( i radiologi contemporanei , i piloti di aereo o le hostess ecc . ) non minore , ma maggiore della media , con minore incidenza di patologia neoplastica , il che sembra convalidare tale teoria . 
lormesi stata fortemente contrastata nei decenni scorsi , anche perch uno dei suoi ideatori ( arndt ) , oltre che psichiatra , era anche omeopata , ma le notevoli evidenze sembrano richiedere una pi seria valutazione di questa teoria , anche in campo radiologico [ 18 ]  . it is surprising that despite the widespread diffusion of clinical radiology and the advances in scientific knowledge that such an important question has still not received certain pu stupire che , nonostante la grande diffusione della radiologia medica e levoluzione delle conoscenze scientifiche , un problema di questa importanza non abbia ancora radiol med ( 2009 ) 114 : 347357 answers . 
maria delle croci , v.le randi 5 , 48100 ravenna , italy 2scuola di specializzazione in radiodiagnostica , universit degli studi di ferrara , corso giovecca 203 , 44100 ferrara , italy 3servizio di anatomia patologica , ospedale civile s.maria delle croci , v.le randi 5 , 48100 ravenna , italy 4istituto di radiodiagnostica , universit degli studi di ferrara , corso giovecca 203 , 44100 , ferrara , italy correspondence to : g . 
cdus revealed an inflammatory process in 277 patients ( 34.58% ) , testicular trauma in 112 ( 13.9% ) , funicular torsion or torsion of the vestigial remnant in 44 ( 5.4% ) , findings suggestive of testicular neoplasm in 35 ( 4.3% ) and no abnormality in 41.5%. 
mri , used to further investigate the cdus findings in 46 cases , showed three cases of intraparenchymal haematoma , one of intrascrotal cavernous body rupture , one of testicular abscess with intrascrotal fistula , two of testicular infarction and 15 of neoplasmri allowed the exclusion of focal abnormalities in ten patients with testicular microlithiasis , in three with chronic orchitis and in four with atrophic involution . 
on the basis of our experience , cdus is irreplaceable as an initial approach to patients affected by scrotal disease , whereas mri is an ideal second - line investigation . 
lecd ha rilevato : in 277 pazienti ( 34 , 58% ) processo flogistico , in 112 ( 13 , 9% ) trauma del testicolo , in 44 ( 5 , 4% ) torsione del funicolo o di un residuo embrionario , in 35 ( 4 , 3% ) alterazioni riconducibili ad eteroplasia testicolare mentre nel 41 , 5% non erano presenti alterazioni patologiche . 
in 46 casi si ricorsi alla rm per chiarimento o miglior definizione diagnostica del reperto ecd rilevando : 3 casi di ematoma intraparenchimale , 1 caso di rottura intrascrotale del corpo cavernoso , 1 caso di ascesso testicolare con fistola intrascrotale , 2 casi di infarto , 15 casi di eteroplasia . 
la rm utile in casi selezionati , potendo rilevare situazioni complesse e precedentemente insospettabili , risultando in un miglioramento della gestione del paziente e in un contenimento globale dei costi . 
 radiol med ( 2009 ) 114 : 414424 keywords scrotum mri color doppler ultrasound scrotal disorders ultrasonography parole chiave scroto rm eco - color doppler patologia scrotale ultrasonografia introduction introduzione colour doppler ultrasonography ( cdus ) plays a key role as a first - line approach to scrotal disease , as it can help solve diagnostic dilemmas and establish a precise diagnosis , thus leading to appropriate clinical and therapeutic decisions . nevertheless , when the cdus findings are difficult to interpret in relation to the patients clinical data and medical history , or when the clinical and sonographic findings are unjustified , magnetic resonance imaging ( mri ) may be used to elucidate or reveal unusual features [ 1 ]  . the value of mri as a second - line technique after inconclusive sonography [ 2 , 3 ] has rarely been addressed in the literature , and the few existing studies are dated and involve small patient populations . 
the aim of this study was to review our experience of the past 7 years in an attempt to validate the usefulness of cdus in the study of scrotal disease and evaluate the role of mri in selected cases . materials and methods from 2000 to 2007 , 801 male subjects aged from 0 to 87 years were referred to our department to undergo cdus of the scrotuexaminations were carried out on a ge logic 7 sonographic unit with a 715 mhz linear - array probe and colour doppler , power doppler , and b - flow imaging . patients included urgent cases referred from the emergency department and high - priority outpatients referred by the urologist to investigate suspicion of acute or subacute scrotal disease . transverse planes panoramic grey - scale scans were obtained in the longituto compare size and dinal and echogenicity on the two sides . 
colour doppler imaging was then performed , with optimisation of the scanner to obtain the highest sensitivity to slow flow , with low pulse repetition frequency ( prf ) , low wall filter and appropriate colour ga in patients with suspected torsion or infarction , the asymptomatic side was evaluated first to ensure that the parameters for flow evaluation had been set appropriately . mri was performed in 46 patients to confirm or elucidate the cdus findings . 
the criteria adopted to define inconclusive sonographic evaluation were unclear nature of the lesion , inability to delineate the lesion spatially in terms of site and / or extension , conflicting clinical and sonographic diagnosis and marked structural heterogeneity associated with conditions predisposing to malignancy , such as testicular leco - color doppler ( ecd ) riveste un ruolo di primo approccio nella patologia dello scroto risolvendo nella maggior parte dei casi lenigma diagnostico oppure delineando in modo esaustivo il quadro patologico , permettendo cos corrette decisioni e idonei approcci clinico - terapeutici . 
pur tuttavia , in presenza di quadri ecd di dubbia interpretazione alla luce del reperto clinico - anamnestico o in presenza di reperti clinico - ecografici ingiustificabili , la risonanza magnetica ( rm ) pu trovare applicazione per risolvere , chiarire o svelare aspetti a volte inusuali [ 1 ]  . solo pochi autori hanno esaminato il ruolo della rm come metodica di secondo livello in caso di reperti ecografici non esaustivi [ 2 , 3 ]  . 
finalit del lavoro stata quella di effettuare una revisione della nostra casistica degli ultimi 7 anni allo scopo di riconfermare lutilit dellecd nellapproccio alle patologie dello scroto e far emergere il ruolo della rm in casi selezionati . materiali e metodi dal 2000 al 2007 sono stati inviati al nostro servizio 801 pazienti maschi di et compresa tra 0 e 87 anni , per essere sottoposti ad indagine ecd dello scroto con ecografo ge logic 7 con sonda lineare 715 mhz con modulo color doppler , power doppler e b flow . 
i pazienti erano giunti alla nostra osservazione in regime di urgenza , in quanto inviati dal pronto soccorso , oppure in regime prioritario ambulatoriale su consiglio dello specialista urologo , nel sospetto di una patologia acuta o subacuta . sono state eseguite scansioni in scala di grigi quanto pi possibile panoramiche , sui piani longitudinale e trasversale , per confrontare le dimensioni e lecogenicit dei due lati . 
lo studio color doppler stato condotto successivamente , ottimizzando lapparecchiatura per ottenere la massima sensibilit ai flussi lenti , con una bassa pulse repetition frequency ( prf ) , bassi filtri di parete e un appropriato guadagno di colore . 
nei casi di sospetta torsione o infarto , il lato asintomatico stato valutato per primo , per assicurarsi che i parametri di valutazione del flusso fossero settati in modo opportuno . in 46 pazienti si ritenuto di procedere ad indagine rm , per confermare o chiarire il dato ecd , rilevando in alcuni 416 radiol med ( 2009 ) 114 : 414424 microlithiasis , testicular retention and chronic orchitis . mri was performed on a 1.5 - tesla philips achieva unit with a 14 - cm circular surface coil . 
for studying the scrotum in the three orthogonal planes , a t2 - weighted fast spin - echo sequence with a 1012 - cm field of view and a 256256 matrix was used . 
axial t1 - weighted spoiled gradient - echo images were also obtained to detect haemorrhage . only selected cases were studied with mri following intravenous administration of gadolinium with diethylenetriamine penta - acetic acid ( gd - dtpa ) ; in these cases , axial and coronal t1 - weighted spoiled gradient - echo sequences with fat saturation were obtained after contrast injection . definitive diagnoses were confirmed at surgery in the cases of funicular torsion , morgagnis hydatid torsion and inguinoscrotal hernia . 
in the case of inflammatory processes , thrombophlebitis of the pampiniform plexus , complicated cysts and scrotal trauma with intact tunica vasculosa , the diagnosis was declared ex adjuvantibus after successful medical treatment , as confirmed by lesion regression at serial us follow - up and resolution of clinical symptoms . results among the 801 patients examined , medical history , clinical data and cdus findings led to a definite diagnosis of inflammatory process in 277 patients , traumatic lesion in 112 , funicular torsion in 33 ( three cases appearing at birth , with intrauterine torsion ) and torsion of the hydatid of morgagni [ 4 , 5 ] in 11 . 
i criteri utilizzati per definire la valutazione ecografia non esaustiva sono stati : incerta natura della lesione , incapacit dellecografia di definire spazialmente la lesione in termini di sede e / o di estensione della stessa , la discrepanza tra la clinica e la diagnosi ecografia , la presenza di una marcata disomogeneit strutturale in presenza di condizioni predisponesti linsorgenza di neoplasie quali microlitiasi testicolare ( mt ) , ritenzione testicolare , orchite cronica . abbiamo impiegato unapparecchiatura philips achieva 1 , 5 tesla con una bobina di superficie , circolare da 14 cm . per la patologia del canale inguinale si studiata la pelvi mediante scansioni assiali usando un campo di vista pi ampio . 
per lo studio dello scroto sui 3 piani ortogonali sono stati utilizzati : una sequenza fast spin - eco pesata in t2 , un campo di vista 1012 cm e una matrice 256256 . 
sono anche state acquisite immagini assiali spoiled - gradient - eco t1 pesate , molto utili per identificare lemorragia . lacido dietilen - triamino - penta - acetico chelato con gadolinio ( gd - dtpa ) , per via endovenosa , stato somministrato solo in casi selezionati ; sono state condotte acquisizioni assiali e coronali dopo somministrazione del mezzo di contrasto , sfruttando sequenze spoiled - gradient - eco con saturazione per il grasso t1 pesate . 
per le lesioni di sospetta origine neoplastica si proceduto con esame citologico estemporaneo intraoperatorio seguito in caso di reperto dubbio o positivo da orchiectomia e in caso di negativit da nodulectomia . 
nei processi flogistici , nelle tromboflebiti del plesso pampiniforme , nelle cisti complicate e nei traumi del testicolo con conservazione dellintegrit della tunica vasculosa , la diagnosi stata confermata ex adjuvantibus dal successo della terapia medica , confermato dalla regressione delle lesioni nel corso di controlli ecografici seriati e dalla regressione del quadro clinico . 
 risultati tra gli 801 pazienti esaminati lanamnesi , i dati clinici e i reperti ecd hanno permesso la diagnosi definitiva di : processo flogistico in 277 pazienti , lesione traumatica in 112 , torsione del funicolo in 33 casi ( in 3 con comparsa alla nascita e torsione intrauterina ) e torsione dellidatide del morgagni [ 4 , 5 ] in 11 pazienti . 
in 15 patients , mri confirmed the suspicion of neoplastic lesion by showing t1 isohyperintensity and clear t2 hypointensity relative to the surrounding parenchyma . the use of contrast material allowed us to evaluate lesion vascularity ( with findings virtually coincident with cdus and b - flow imaging )  . 
in ten patients with testicular microlithiasis on cdus , mri enabled us to rule out alterations in signal intensity or enhancement that might indicate ecografica , sottoposti a rm come indagine di secondo livello , sono stati 46 pari al 5 , 74% . 
in 15 pazienti la rm ha confermato il sospetto di lesione neoplastica in base allaspetto isoiperintenso in t1 e francamente ipointenso in t2 rispetto al parenchima circostante . lutilizzo del mdc ha permesso di valutare la vascolarizzazione delle lesioni ( con reperto sostanzialmente sovrapponibile al dato ecd e b flow )  . 
in 10 pazienti con reperto ecd di mt la rm ha permesso di escludere con sicurezza alterazioni di segnale o di contrast - enhancement ( c.e. ) riferibili a lesioni parenchimali ( in 1 caso ha consentito di rilevare accumulo focale di calcio - pirofosfatodiidratato )  . 
in 4 pazienti affetti da ritenzione testicolare grazie alla rm stato possibile identificare la sede del testicolo atrofico non visibile ecograficamente , differeziando tale condizione dallagenesia testicolare , ed escludere una patologia eteroproduttiva a cui tali soggetti sono predisposti . 
3a - c cisti intrascotale con contenuto infiammatorio : a aspetto ecd ; b rm : sequenza pesata in t1 ; c rm sequenza pesata in t2 . parenchymal lesions ( in one case , it allowed detection of a focal accumulation of calcium - pyrophosphate - dihydrate )  . mri was also used to confirm the sonographic diagnosis of inguinoscrotal hernia in three cases , in which it showed the presence of intestinal loops in the scrotal region . 
in four patients with cryptorchidism , mri helped to locate the undescended testis , which was not visible at us , to differentiate this condition from testicular agenesis and to exclude possible malignancy . 
likewise , it enabled us to exclude nella nostra casistica i casi ritenuti dubbi e sottoposti a rm come indagine di secondo livello sono stati 46 pari al 5 , 74% . 
 radiol med ( 2009 ) 114 : 414424 areas of signal alteration or increased contrast enhancement in three patients with chronic orchitis in whom cdus had shown marked echostructural inhomogeneity . 
mri was also used to follow - up conservative treatment in two patients with scrotal trauma and cdus findings of discontinuity of the tunica albuginea and intact capsular vascularity . discussion in our series , mri was performed as a second - line investigation in 46 patients ( 5.74% ) with inconclusive cdus findings . 
this pattern , which has been previously described in the literature [ 6 ] , is particularly useful for solving interpretation doubts : it should be noted that 10%15% of testicular neoplasms are detected as a consequence of traumas [ 79 ]  . in one patient with a large intrascrotal collection , mri enabled a diagnosis of epididymo - orchitis with abscess formation and intrascrotal fistula , which could not be detected by cdus . 
on the basis of the literature and patients clinical data , we interpreted the findings as complicated testicular cysts , an interpretation that was confirmed ex adjuvantibus after symptom resolution with appropriate medical treatment . 
si tratta di un reperto , gi descritto in letteratura [ 6 ] , utile laddove sussistano dubbi interpretativi : si tenga conto che il 10%15% delle lesioni eteroplasiche testicolari vengono scoperte in conseguenza di un trauma [ 79 ]  . in un pazienze con voluminosa raccolta intrascrotale la rm ha permesso di rilevare orchiepididimite ascessualizzata con tramite fistolizzato in sede intrascrotale , difficilmente rilevabile allindagine ecd . 
analogamente basandoci su tali osservazioni e sulla clinica abbiamo interpretato i nostri reperti come cisti del testicolo complicate , confermate poi ex juvantibus dalla risoluzione del quadro dopo adeguata terapia medica . 
contrast - enhanced t1 sequences demonstrated the characteristic rim of enhancement delimiting the lesion [ 12 , 13 ]  . in 15 patients with cdus findings suggestive of testicular neoplasm , mri confirmed the diagnosis ( definitive histology revealed 11 cases of germ - cell tumour and four benign neoplasms , two of which were adenomatoid tumours and two sertoli - cell tumours )  . 
in these patients , mri also enabled preoperative staging by depicting possible infiltration of the tunica albuginea , tunica vaginalis , epididymis and / or of the para - epididymal tissues . 
in questi pazienti la rm ha inoltre permesso la stadiazione pre - operatoria , potendo definire tunica albuginea , della tunica vaginale , dellepididimo e / o dei tessuti paraepididimari . 
in letteratura si trovano opinioni discordanti sul significato della mt , tuttavia prevalgono le tesi che attribuiscono alla mt il ruolo di condizione precancerosa [ 1418 ]  . in un paziente che presentava formazione dubbia del testicolo , del diametro di circa 1 cm , in sede parailare , iperecogena e senza significativo flusso intralesione all ecd , la rm ha evidenziato alta intensit del segnale nelle sequenze pesate in t1 e basso segnale in t2 . 
tale reperto risulta invariato da 3 anni e , alla luce di quanto riferito da alcuni aa [ 1921 ] , pu essere attribuito ad accumulo di calcio - pirofosfato - diidratato . la rm non in grado di identificare la mt [ 2224 ] : questo aspetto pu facilitare il rilievo di aree di alterato segnale nel parenchima testicolare in pazienti affetti da questa condizione . 
perci , ove la disomogeneit ecostrutturale del parenchima associata alla mt non ha permesso lassoluta esclusione con lecd di lesioni eteroproduttive , siamo ricorsi alla rm che ha escluso alterazioni di segnale radiol med ( 2009 ) 114 : 414424 fig . 
5a - d infarto testicolare ; a immagine ecd ; b immagine rm pesata in t1 ; c immagine rm pesata in t2 ; d immagine rm con mdc . low signal intensity on t2 . 
this finding has remained unchanged for 3 years and , based on previous reports [ 1921 ] , it may be due to an accumulation of calciumpyrophosphate - dihydrate . mri is unable to depict testicular microlithiasis [ 2224 ] , and this may facilitate the detection of parenchymal areas with pathological signal in affected patients . 
therefore , when the parenchymal echostructural inhomogeneity associated with testicular microlithiasis did not allow malignant lesions to be confidently excluded with cdus , the patient was further studied with mri , which revealed no signal alterations or areas of contrast enhancement in the testis . similarly , mri proved useful in three patients with marked parenchymal echostructural alteration due to chronic inflammation , in whom it confirmed the cdus findings and revealed no focal alteration . four patients with atrophic testicular involution secondary to cryptorchidism and / or funicular torsion and who presented diffuse parenchymal echostructural alterations on cdus were also studied with mri to exclude focal parenchymal lesions . 
cryptorchidism is a well - known risk factor for nel testicolo , ma soprattutto non ha rilevato aree di potenziamento dopo mezzo di contrasto . con analogo significato la rm risultata utile in 3 pazienti con marcata alterazione ecostrutturale del parenchima per flogosi cronica , nei quali ha confermato il reperto ecd non rilevando alterazioni focali . sempre per escludere lesioni focali parenchimali sono stati sottoposti a rm 4 casi di involuzione atrofica del testicolo , secondaria a ritenzione testicolare e / o a torsione del funicolo , che presentavano sovvertimento ecostrutturale diffuso del parenchima allecd . 
la ritenzione testicolare rappresenta un noto fattore di rischio per linsorgenza di neoplasie testicolari [ 2527 ] e in questi pazienti consigliato il monitoraggio ecografico : a volte la marcata alterazione ecostrutturale del parenchima e / o la scarsit del flusso parenchimale all ecd nel testicolo atrofico , possono creare dubbi allecografista . 
6a - c trauma dello scroto con rottura della tunica albuginea e integrit della tunica vascolosa : a quadro ecd ; b controllo ecd ; c quadro rm in esiti di rottura della tunica albuginea post - traumatica . testicular neoplasm [ 2527 ] , and sonographic surveillance is recommended in all patients with this condition . 
our experience confirms the usefulness of mri to exclude cancerous lesions . in three cases , mri confirmed the cdus suspicion of omental herniation within the inguinal canal , thus proving its extreme utility in the study of this structure [ 7 ]  . a ruptured tunica albuginea is an indication for exploratory surgery [ 2832 ]  . 
our relatively limited experience demonstrates that surgery is required only in patients with compromised capsular vascularity , that is , of the tunica vasculosa that runs below the tunica albuginea . as is known , on reaching the lower pole of the testis , the testicular artery divides into branches that pierce the tunica albuginea and run along the periphery of the testis in a layer known as the tunica vasculosa . 
dalla nostra seppur limitata esperienza si evince che i pazienti da sottoporre a revisione chirurgica , sarebbero solo quelli con compromissione della vascolarizzazione capsulare e cio della tunica vasculosa che decorre sotto la tunica albuginea  . 
come noto larteria testicolare giunta in prossimit del polo inferiore del testicolo si suddivide in diversi rami , che decorrono alla periferia del testicolo al di sotto della tunica albuginea , formando la cos detta tunica vascolosa . 
da queste arterie capsulari si sviluppano branche centripete che penetrano nel parenchima testicolare decorrendo verso il mediastinum testis ove si arborizzano in rami ricorrenti [ 10 , 33 ]  . 
si pu radiol med ( 2009 ) 114 : 414424 parenchyma and run towards the testicular mediastinum , where they arborise into recurrent rami [ 10 , 33 ]  . 
consequently , it appears reasonable to believe that discontinuity of the tunica albuginea with normal capsular vascularity is an indication for conservative treatment and close clinical and imaging follow - up . conclusions in our experience , cdus is irreplaceable as a first - line approach to patients with scrotal disease . 
we have proposed cdus evaluation of tunica vasculosa integrity in cases of testicular rupture to determine the feasibility of a conservative approach , even in the presence of discontinuity of the tunica albuginea . testicular infarction ; mri is highly valuable in the diagnosis of intratesticular haematoma ; cysts with atypical , presumably inflammatory content ; testicular abscess with intrascrotal fistula ; and in the diagnosis and preoperative staging of testicular neoplasms . 
mri proved to be an ideal second - line modality to be used in selected complex cases , as it improved patient management , leading to fewer unnecessary surgical operations and lower overall costs . quindi sostenere lipotesi che la soluzione di continuit della tunica albuginea con integrit della vascolarizzazione capsulare rappresenti indicazione alla terapia conservativa , con stretto monitoraggio clinico - strumentale . conclusioni dalla nostra esperienza lindagine ecd appare di insostituibile utilit nel primo approccio al paziente con patologia scrotale . 
abbiamo proposto la valutazione con ecd dellintegrit della tunica vasculosa , nei casi di rottura del testicolo , al fine di valutare lipotesi di un approccio terapeutico conservativo , anche in presenza di soluzione di continuit della tunica albuginea . la rm si offre come metodica di insostituibile utilit nella diagnosi di ematoma intratesticolare , di cisti a contenuto atipico verosimilmente infiammatorio , di infarto testicolare , di ascesso testicolare con fistolizzazione intrascrotale nonch nella diagnosi e stadiazione preoperatoria delle eteroplasie testicolari . 
salvatore dipartimento di scienze biomorfologiche e funzionali ( dsbmf ) , universit degli studi di napoli federico ii , istituto di biostrutture e bioimmagini , consiglio nazionale delle ricerche ( cnr ) ; fondazione sdn ( irccs ) , napoli , italy correspondence to : s . 
the aim of this study was to directly compare the results of magnetic resonance cholangiopancreatography ( mrcp ) with those of ultrasonography ( us ) and multislice computed tomography ( msct ) in the diagnosis of pancreaticobiliary diseases . 
a total of 70 patients ( 41 men , 29 women ) aged 2289 years were studied either before ( n = 59 ) or after cholecystectomy ( n = 11 ) for biliary lithiasis . clinical signs and symptoms were jaundice ( n = 15 ) , abdominal pain ( n = 37 ) and proven biliary lithiasis ( n = 18 )  . mrcp was performed in all patients , whereas abdominal us was performed in 55 ( group 1 ) and msct in 37 ( group 2 ) patients . 
histology ( n = 27 ) , biopsy ( n = 5 ) , endoscopic retrograde cholangiopancreatography ( ercp ) ( n = 28 ) and / or clinicalimaging follow - up ( n = 10 ) were considered standards of reference . 
in particular , patients were classified as showing benign ( n = 47 ) or malignant ( n = 12 ) lesions or normal biliary anatomy ( n = 11 )  . 
in group 1 , the results of mrcp and us were concordant in the majority ( 92% ) of cases ; however , statistically significant discordance ( p < 0.01 ) was found in the evaluation of the extrahepatic ducts , with nine cases riassunto obiettivo . 
sono stati arruolati 70 pazienti ( 41 maschi , 29 femmine ) , di et compresa tra 22 e 89 anni , in fase diagnostica pre - chirurgica ( n = 59 ) o dopo colecistectomia per litiasi biliare ( n = 11 ) ; i pazienti presentavano ittero ( n = 15 ) , sintomatologia dolorosa addominale ( n = 37 ) e litiasi biliare documentata ( n = 18 )  . 
in tutti i pazienti stata eseguita la cprm ; in 55 casi stata effettuata us addominale ( gruppo 1 ) , mentre 37 pazienti sono stati sottoposti ad esame tcms ( gruppo 2 )  . 
stata eseguita unanalisi regionale nei principali distretti del sistema bilio - pancreatico : colecisti e dotto cistico , vie biliari intraepatiche ( vbi ) , via biliare principale ( vbp ) e dotto pancreatico principale . 
per linterpretazione dei risultati degli esami diagnostici , i dati istologici post - chirurgici ( n = 27 ) , bioptici ( n = 5 ) , della colangio - pancreatografia retrograda endoscopica ( cpre ) ( n = 28 ) o del follow - up ( 36 mesi ) clinico - strumentale ( n = 10 ) sono stati considerati gli standard di riferimento ; in particolare sono stati osservati 47 casi di lesioni biliari benigne , 12 lesioni maligne e 11 casi con normale anatomia delle vie biliari . radiol med ( 2009 ) 114 : 390402 ( 16% ) of middle - distal common bile duct stones being detected on mrcp only . 
the results of our study confirm the diagnostic potential of mrcp in the study of the pancreaticobiliary duct systein particular , the comparison between mrcp and us and msct indicates the superiority of mrcp in evaluating bile ducts and detecting stones in the common bile duct . keywords ultrasound computed tomography magnetic resonance biliary lithiasis risultati . 
nel gruppo 1 , stata osservata una concordanza globale dei risultati della cprm e dellecografia nel 92% dei casi ; tuttavia , stata riscontrata una discordanza del 16% ( n = 9 ) statisticamente significativa ( p < 0 , 01 ) tra i risultati delle due metodiche nella valutazione della vbp ove la cprm mostrava lesioni litiasiche in sede medio distale non evidenti allecografia . 
i risultati della nostra esperienza confermano le potenzialit diagnostiche della cprm nello studio del sistema duttale bilio - pancreatico ; in particolare , i dati di confronto con lecografia e la tcms dimostrano i vantaggi della cprm nella valutazione distrettuale dei dotti biliari con particolare riferimento alla coledocolitiasi . parole chiave ecografia tomografia computerizzata risonanza magnetica litiasi biliare introduction introduzione diagnostic imaging of pancreaticobiliary disease includes numerous techniques that are selected on the basis on the patients signs and symptoms , the diagnostic information sought , the techniques invasiveness and the therapeutic needs of each individual case . 
in particular , ultrasonography ( us ) and computed tomography ( ct ) , the most commonly used noninvasive modalities in the study of the pancreaticobiliary system , appear to have high specificity but lower sensitivity , especially in patients with common bile duct stones that require alternative techniques such as endoscopic retrograde cholangiopancreatography ( ercp ) [ 14 ]  . 
however , despite its high sensitivity , specificity and diagnostic accuracy and its inherent therapeutic possibilities , ercp is invasive and thus has limited use for diagnostic purposes alone [ 47 ]  . 
this has led to the use of other techniques , the most important of which is magnetic resonance cholangiopancreatography ( mrcp ) that allows for correct depiction of the pancreaticobiliary ducts without being invasive for the patient . 
the availability of multiple imaging modalities , each with different levels of invasiveness and accuracy , calls for protocols or guidelines to ensure that patients with pancreaticobiliary disease are managed correctly . comparative studies on the modalities used for diagnosing pancreaticobiliary disease indicate a fundamental la diagnostica per immagini delle malattie del sistema bilio - pancreatico rappresentata attualmente da numerose tecniche la cui scelta funzione di diversi fattori , quali la sintomatologia e le condizioni del paziente , linformazione diagnostica richiesta , linvasivit della metodica e le necessit terapeutiche di ogni singolo caso ; in particolare , lecografia ( us ) e la tomografia computerizzata ( tc ) , le pi comuni metodiche di imaging non invasivo utilizzate nello studio del sistema bilio - pancreatico , mostrano unelevata specificit ma una minore sensibilit diagnostica particolarmente nei pazienti affetti da calcolosi del coledoco , tanto che in tali casi si ricorre frequentemente a tecniche alternative quali ad esempio la colangio - pancreatografia retrograda endoscopica ( cpre ) [ 14 ]  . 
la cpre , nonostante lelevata sensibilit , specificit e accuratezza diagnostica e le possibilit terapeutiche intrinseche alla metodica stessa , mostra tuttavia uninvasivit che ne rende limitato il suo solo utilizzo diagnostico [ 47 ]  . 
pertanto , in base a tali considerazioni stato necessario ricorrere ad altre tecniche tra cui principalmente la colangio - pancreatografia con risonanza magnetica ( cprm ) che fornisce una corretta rappresentazione del sistema duttale bilio - pancreatico senza alcuna invasivit per il paziente ; dunque , la disponibilit di molteplici metodiche di diagnostica per immagini , 392 radiol med ( 2009 ) 114 : 390402 role for mrcp , which has high sensitivity , specificity and accuracy [ 3 , 4 , 811 ]  . 
the literature indicates that us has a good level of accuracy in the study of the extrahepatic common hepatic duct and of the intrahepatic bile ducts when dilated but performs less well in the assessment of the middle - distal portion of the extrahepatic ducts [ 10 , 1214 ]  . 
multislice ct technology allows reconstruction of three - dimensional volumetric images , with significant advantages in the evaluation of bile duct anatomy . however , given that ct cannot always visualise the cause of the obstruction because of the frequent isodensity of stones relative to bile , detection is based on the same indirect criteria as used at us , such as dilatation of the bile ducts above the stenosis . 
for this reason , it has been suggested that ct with biliary contrast material may help identify filling defects caused by the presence of radiolucent stones within the opacified bile duct lumen , especially in patients with contraindications to mrcp [ 15 ]  . the purpose of our study was to evaluate the role of mrcp compared with us and msct in diagnosing pancreaticobiliary disease . 
to this end , we performed a comparative analysis of the results of imaging in the main regions of the pancreaticobiliary system ( gallbladder , intrahepatic bile ducts , extrahepatic bile ducts , main pancreatic duct )  . materials and methods population a total of 70 patients ( 41 men , 29 women ) aged 2289 ( mean 58 ) years were retrospectively reviewed . 
patients were studied before ( n = 59 ) or after cholecystectomy for lithiasis ( n = 11 ) and were referred because of jaundice ( mean total bilirubin = 4.9 mg / ml ; mean direct bilirubin = 4.0 mg / ml ) ( n = 15 , 21% ) , abdominal pain ( n = 37 , 53% ) or bile stones detected on us ( n = 18 , 26% )  . 
for 55 patients , the results were compared with abdominal us scans ( group 1 ) and for 37 patients with abdominal ct scans ( group 2 )  . ultrasonography patients were imaged with an atl 5500 hdi unit after a 6h fast and in various positions ( supine , left lateral , upright )  . examinations were carried out with a 3.54 mhz probe initially placed subcostally during deep breath - holding or between the 10th and 11th rib , without breath - holding . 
transverse , longitudinal and oblique scans of the upper abdomen were performed , as reported in the literature [ 16 ]  . pi o meno invasive ed accurate , richiede la definizione di specifici protocolli diagnostici o linee guida clinicamente fondamentali per la corretta gestione dei pazienti affetti da malattie del sistema bilio - pancreatico . gli studi di confronto attualmente disponibili tra le varie metodiche di imaging impiegate nella valutazione diagnostica del sistema bilio - pancreatico dimostrano un ruolo fondamentale della cprm che ha mostrato elevati valori di sensibilit , specificit e accuratezza [ 3 , 4 , 811 ]  . 
in particolare , nei pazienti con calcolosi biliare , che rappresenta la condizione morbosa pi frequente , laccuratezza diagnostica delle varie tecniche nellidentificazione dei calcoli biliari varia in funzione della sede , delle dimensioni e della struttura delle lesioni litiasiche ; i dati riportati in letteratura mostrano una buona accuratezza dellecografia nello studio del dotto epatico comune nel suo tratto extra - epatico e dei dotti biliari intra - epatici se dilatati , ma una pi difficile valutazione del tratto medio - distale della via biliare principale [ 10 , 1214 ]  . 
levoluzione tecnologica in tc con la metodica multi - strato consente la ricostruzione di immagini tridimensionali volumetriche determinando un significativo vantaggio nella valutazione dellanatomia delle vie biliari ; tuttavia , la tc non potendo sempre identificare la causa dellostruzione per lisodensit che i calcoli possono mostrare rispetto alla bile , utilizza i medesimi criteri indiretti usati in us quali la dilatazione delle vie biliari a monte della lesione stenosante ; per tale motivo stato proposto , in particolare nei pazienti che non possono essere sottoposti alla cprm per controindicazioni tecniche , lutilizzo in tc di un mezzo di contrasto ad escrezione biliare al fine di identificare difetti di riempimento da formazioni litiasiche radiotrasparenti allinterno del lume biliare opacizzato dal mezzo di contrasto [ 15 ]  . lo scopo del nostro studio stato la valutazione del ruolo specifico della cprm nella diagnostica del sistema bilio - pancreatico in confronto ad altre tecniche per immagini quali lus e la tc ; a tale proposito , stata effettuata unanalisi regionale comparativa del risultato delle immagini nei principali distretti biliari ( colecisti , dotti intraed extra - epatici , dotto pancreatico principale )  . materiali e metodi popolazione sono stati valutati in una analisi retrospettiva 70 pazienti ( 41 maschi , 29 femmine ) di et compresa tra 22 e 89 anni ( et media 58 anni )  . 
i pazienti sono stati studiati in fase diagnostica pre - chirurgica ( n = 59 ) o dopo colecistectomia per litiasi ( n = 11 ) ; i pazienti sono stati valutati per ittero ( valore medio di bilirubina totale = 4 , 9 mg / ml ; valore medio di bilirubina diretta = 4 , 0 mg / ml ) ( n = 15 , 21% ) , dolore addoradiol med ( 2009 ) 114 : 390402 computed tomography the ct study was carried out with the multislice technique ( toshiba aquilion , with 4 detector rows ) after administration of both oral and intravenous contrast material . 
oral contrast material ( barium sulphate , prontobario tac , bracco , italy ) was administered at a dose of 10 ml per 200 ml of water approximately half an hour before the examination . 
early scans in the angiographic , arterial ( 30 s ) and portal ( 60 s ) phase permitted differentiation between portal vessels and bile ducts , whereas the late scans in the equilibrium phase ( 180300 s ) produced hepatobiliary parenchymal enhancement . 
 mr cholangiography all mrcp examinations were carried out after administration of 900 ml oral superparamagnetic contrast agent [ a suspension of silicon - coated iron oxide crystals ( ferumoxsil ) , lumirem , guebert ]  . 
patients were imaged on a high - field device ( intera 1.5 t , philips ) using a phased - array synergy body coil and dedicated breath - hold t2 - weighted sequences ( mrcp - rad - bh )  . 
the sequence was a 40 - mm - thick singleslab half - fourier sequence , with acquisition matrix 256 , reconstruction matrix 256512 , fov 250 mm , effective echo time ( teeff ) 1 , 050 ms , and repetition time ( tr ) 2 , 600 ms . 
in all patients , the cholangiographic acquisitions were complemented with axial and coronal sequences of the upper abdomen , t1 [ fastfield echo breath - hold ( ffe - bh ) ] sequences without fat suppression , tr / te 214 / 46 ms , fa 80 , matrix 192512 , slice thickness 5 mm , conventional t2 turbo spin echo ( tse ) ( with and without fat suppression ) , tr / te 831 / 80 ms , fa 80 , matrix 192512 , slice thickness 5 mm , and fatsuppressed t1 ffe - bh using a multiphase dynamic technique after intravenous administration of 0.1 mmol / kg body weight paramagnetic contrast material ( gadopentetic acid , magnevist , schering )  . 
 data analysis in the two patient groups , a regional analysis of the concordance or discordance of the results obtained with the two imaging techniques was carried out by comparing the main regions of the pancreaticobiliary system : gallbladder and cystic duct , intrahepatic bile ducts , extrahepatic bile ducts ( divided into common hepatic duct and common bile duct ) minale ( n = 37 , 53% ) o litiasi biliare documentata allecografia ( n = 18 , 26% )  . 
tutti i pazienti sono stati sottoposti ad cprm ; in 55 pazienti stato disponibile il confronto con lesame ecografico delladdome ( gruppo 1 ) , mentre in 37 pazienti stato disponibile il confronto con lesame tc delladdome ( gruppo 2 )  . ecografia lesame ecografico ( atl 5500 hdi ) stato eseguito con paziente a digiuno assoluto da almeno 6 ore , in vari decubiti ( supino , fianco sinistro ed ortostasi ) , in apnea inspiratoria profonda con sonda da 3 , 54 mhz posta inizialmente in sede sottocostale oppure , senza apnea respiratoria , a livello intercostale tra la x e la xi costa . 
in tutti gli studi ecografici , limpiego dellcolor doppler ha consentito di differenziare le immagini dei rami portali da quelle dovute a dilatazione delle vie biliari ; sono state eseguite scansioni trasversali , longitudinali ed oblique delladdome superiore , come riportato in letteratura [ 16 ]  . tomografia computerizzata lo studio tc stato eseguito con tecnica multistrato ( aquilion toshiba 4 file di detettori ) dopo somministrazione di mezzo di contrasto sia per via orale che endovenosa . 
il contrasto orale , prontobario tac ( solfato di bario , bracco ) , stato somministrato circa mezzora prima dellesame al dosaggio di 10 ml ogni 200 ml dacqua ; il mezzo di contrasto endovenoso organoiodato ( ultravist , schering 370 mg / i / kg ) stato iniettato ad un dosaggio di 120150 ml ( 3 ml / s per 25 secondi ) con tecnica dinamica multifasica standardizzata ( collimazione 3 mm ; pitch 0 , 871 , 25 ) ; le scansioni precoci in fase angiografica , arteriosa ( 30 s ) e portale ( 60 s ) hanno consentito la differenziazione tra i vasi portali e i dotti biliari , mentre le scansioni tardive allequilibrio ( 180300 s ) hanno offerto leffetto parenchimografico epato - biliare . 
 colangiografia rm tutti i pazienti , previa assunzione orale di mezzo di contrasto super - paramagnetico [ sospensione di cristalli di ossido di ferro rivestiti di silicone ( ferumoxsil ) lumirem , guebert ] alla dose di 900 ml , sono stati studiati con cprm utilizzando unapparecchiatura ad alto campo ( intera 1 , 5 t , philips ) e sequenze dedicate in apnea espiratoria t2 pesate ( cprm - rad - bh ) , usando una bobina phased array synergy body coil ; come sequenza stata usata la single slab half fourier , a singola fetta di 40 mm di spessore con matrice di acquisizione 256 e matrice di ricostruzione 256512 , un fov di 250 mm , con tempo di eco effettivo ( teeff ) di 1050 millisecondi , tempo di ripetizione ( tr ) di 394 radiol med ( 2009 ) 114 : 390402 and main pancreatic duct . 
the assessment criteria have been previously reported [ 19 , 20 ] and include : gallbladder ( degree of distension , dimorphism , heterogeneity of content and / or presence of filling defects , wall thickness and wall irregularity ) cystic duct ( calibre , normal tortuosity of course and filling defects ) , intrahepatic bile ducts ( increase in calibre > 2 mm , heterogeneity of content and / or presence of filling defects ) , common hepatic duct ( increase in calibre > 5 mm , heterogeneity of content and / or presence of filling defects ) , common bile duct ( increase in calibre > 7 mm , heterogeneity of content and / or presence of filling defects and regular course ) , and main pancreatic duct ( according to recently proposed criteria ) [ 21 ]  . 
 in the interpretation of results , postoperative histological ( n = 27 ) , bioptic ( n = 5 ) , ercp ( n = 28 ) findings and / or those of the clinicalimaging ( 36 months ) follow - up ( n = 10 ) were considered standards of reference . 
in particular , we tested the hypothesis that there is significant discordance between results observed in the two patient groups , as confirmed by the reference standards . results group 1 figure 1 shows the results of the comparative regional analysis of mrcp and us . 
in particular , 212 biliary regions were analysed : gallbladder and cystic duct in 47 cases , intrahepatic bile ducts in 55 , extrahepatic ducts in 55 and main pancreatic duct in 55 . 
in the evaluation of the gallbladder region , there was concordance between mrcp and us in 45 cases ( 96% ) , which included negative findings ( n = 17 ) , lithiasis ( n = 20 ) and inflammation ( n = 8 ) , as confirmed by postoperative histology ( n = 24 ) or follow - up ( n = 4 )  . 
le acquisizioni colangiografiche sono state integrate in tutti i pazienti da sequenze secondo piani di scansione assiali e coronali delladdome superiore , t1 ( ffe - bh ) senza soppressione del segnale del tessuto adiposo , tr / te pari a 214 / 46 ms , fa di 80 , matrice 192512 , spessore di scansione di 5 mm , t2 convenzionali tse ( con e senza soppressione del tessuto adiposo ) , tr / te pari a 831 / 80 ms , fa di 80 , matrice 192512 , spessore di scansione di 5 mm , e t1 ffe - bh con soppressione del segnale del tessuto adiposo con tecnica dinamica multi - fasica dopo somministrazione endovena di mdc paramagnetico ( acido gadopentetico , magnevist , schering ) al dosaggio di 0 , 1 mmol / kg peso corporeo ; la tecnica dinamica multi - fasica prevedeva la fase arteriosa a 30 secondi dalla somministrazione , la fase portale a 60 secondi e la fase allequilibrio a 180300 secondi . 
 analisi dei dati nei due gruppi in cui stata suddivisa la popolazione stata eseguita unanalisi regionale relativa alla concordanza o alla discordanza dei risultati ottenuti con le due metodiche poste a confronto nella valutazione dei principali distretti del sistema bilio - pancreatico : colecisti e dotto cistico , vie biliari intra - epatiche ( vbi ) , via biliare principale ( vbp ) , suddivisa in dotto epatico comune e dotto coledoco , e dotto pancreatico principale . 
due osservatori indipendenti non a conoscenza dei dati clinici hanno valutato gli esami di diagnostica per immagini ; i criteri di valutazione utilizzati per lanalisi degli esami diagnostici sono stati i seguenti come riportato in letteratura [ 19 , 20 ] : colecisti ( grado di distensione , dimorfismo , disomogeneit del contenuto e / o presenza di difetti di riempimento e spessore ed irregolarit della parete ) ; dotto cistico ( calibro , regolare tortuosit del decorso e difetti di riempimento ) ; vbi ( incremento di calibro > 2 mm , disomogeneit del contenuto e / o presenza di difetti di riempimento ) ; dotto epatico comune ( incremento di calibro > 5 mm , disomogeneit del contenuto e / o presenza di difetti di riempimento ) , coledoco ( incremento di calibro > 7 mm , disomogeneit del contenuto e / o presenza di difetti di riempimento e regolarit del decorso ) e dotto pancreatico principale secondo i criteri recentemente proposti [ 21 ]  . 
in ascissa sono indicati i distretti bilio - pancreatici : colecisti , vie biliari intraepatiche ( vbi ) , via biliare principale ( vbp ) e dotto pancreatico principale ( wirsung ) ; in ordinata stato riportato il valore percentuale di concordanza e discordanza dei risultati delle due metodiche . 
in the evaluation of the main pancreatic duct , there was concordance between mrcp and us in 52 cases ( 95% ) and , in particular , in cystic lesions of the main pancreatic duct ( n = 2 ) and pancreatic tumour ( n = 4 ) , as confirmed by ercp ( n = 2 ) or postoperative histology ( n = 4 ) ; mrcp and us were also concordant in yielding negative findings in the remaining 46 cases . 
 in the study of the extrahepatic bile ducts , there was concordance between mrcp and us in 46 cases ( 84% ) , which included negative findings ( n = 34 ) , lithiasis ( n = 7 ) and inflammation ( n = 5 ) , as demonstrated by ercp . 
based on statistical analysis , the 16% discordance between mrcp and us in the evaluation of the extrahepatic bile ducts was statistically significant ( p = 0.003 , mcnemar test )  . group 2 results of the comparative regional analysis between mrcp and ct are shown in fig . 
in particular , 143 biliary regions were analysed : gallbladder and cystic duct in 32 cases , intrahepatic bile ducts in 37 , extrahepatic bile ducts in 37 and main pancreatic duct in 37 . 
five patients were come affetti da lesioni benigne ( n = 47 ; 29 litiasi , 13 flogosi , 2 cirrosi biliari primitive , 2 cisti del dotto pancreatico principale e 1 lesione iatrogena ) o maligne ( n = 12 ; 4 colangiocarcinoma e 8 tumori del pancreas ) del sistema biliopancreatico ; nei restanti 11 pazienti non stata riscontrata evidenza strumentale di lesioni patologiche delle vie biliari e / o del pancreas per cui sono stati considerati liberi da malattia . al fine di valutare la significativit statistica dei risultati delle tecniche per immagini osservati nellanalisi regionale del sistema bilio - pancreatico eseguita nei due gruppi di pazienti in cui stata suddivisa la popolazione stato applicato in test di mc nemar , considerando un valore significativo della p < 0 , 05 [ 22 ] ; in particolare , lipotesi da dimostrare stata la significativit statistica della discordanza dei risultati osservati nei due gruppi di pazienti , supportata dai dati degli standard di riferimento . risultati gruppo 1 i risultati della analisi regionale comparativa lecografia e la cprm sono riportati nella fig . 
in particolare , sono stati analizzati 212 distretti biliari , di cui 47 relativi alla colecisti ed al dotto cistico , 55 corrispondenti alla vbi , 55 relativi alla vbp e 55 riguardo il dotto pancreatico principale ; in particolare , 8 pazienti sono stati studiati in follow - up post - colecistectomia per cui non stato valutato il distretto colecistico . 
2a - c gallbladder and common bile duct stones : ultrasonography ( us ) and magnetic resonance cholangiopancreatography ( mrcp ) concordance for the gallbladder and discordance for the common bile duct . 
a us : the image shows ectasia of the intrahepatic bile ducts caused by the presence of stones located near the hilub mrcp reveals signs of intrahepatic bile stones consisting of diffuse intrahepatic duct dilatation and filling defects ( arrow ) near the hilum ; a large stone can also be appreciated in the infundibulum of the gallbladder . 
in ascissa sono indicati i distretti bilio - pancreatici : colecisti , vie biliari intra - epatiche ( vbi ) , via biliare principale ( vbp ) e dotto pancreatico principale ( wirsung ) ; in ordinata riportato il valore percentuale di concordanza e discordanza dei risultati delle due metodiche . 
in the evaluation of the gallbladder region , there was concordance between mrcp and ct in 29 cases ( 91% ) , which included lithiasis ( n = 9 ) , as confirmed by postoperative data , and negative findings ( n = 20 )  . 
similarly , in the evaluation of the main pancreatic duct , there was concordance between mrcp and ct in most cases ( 92% ) , both with ( n = 11 ) and without ( n = 23 ) disease . 
among the cases with positive findings on both methods , ercp confirmed the presence of cystic lesions of the main pancreatic duct ( n = 2 ) , whereas postoperative histology confirmed the presence of a tumour of the head of the pancreas ( n = 8 ) or cholangiocarcinoma of the terminal common bile duct ( n = 1 )  . in contrast , in the study of the intrahepatic and extrahepatic ducts , the two methods yielded a rate of discordance of 19% ( n = 7 ) and 16% ( n = 6 ) , with positive findings on mrcp compared with ct in all cases . 
la valutazione del dotto pancreatico principale ha mostrato in 52 casi ( 95% ) risultati concordanti tra la rm e lecografia ; in particolare , il risultato stato concordante in termini di positivit degli esami in caso di lesioni cistiche del dotto pancreatico principale ( n = 2 ) o di tumori pancreatici ( n = 4 ) , come confermato dai dati della cpre ( n = 2 ) o dellistologia postchirurgica ( n = 4 ) ; al contrario , il risultato delle due tecniche stato concordante in termini di negativit nei rimanenti 46 casi . 
5a , b intrahepatic bile stones : discordance of computed tomography ( ct ) and magnetic resonance cholangiopancreatography ( mrcp ) findings . a ct shows intrahepatic bile duct dilatation , without stones inside . 
b la cprm in immagine assiale t2 pesata convenzionale conferma la dilatazione delle vie biliari intra - epatiche , ma identifica inoltre la presenza di due difetti di riempimento focali ( freccia ) a livello dei dotti biliari principali da lesioni litiasiche . discussion the availability of several imaging techniques , such as us , ct , mri and ercp , for evaluating patients with bile duct diseases requires selecting the most accurate and less invasive modality for each clinical problethis is also a prerequisite for defining the most appropriate diagnostic protocol according to the clinical presentation of each patient [ 23 ]  . in this study , we report the results of a direct comparison of mrcp , us and ct in a group of patients with clinicallaboratory evidence of biliary diseases , 47 of whom had benign lesions , 12 had malignant lesions and 11 were without evidence of organic lesions . 
in particolare , sono stati analizzati 143 distretti biliari , di cui 32 relativi alla colecisti ed al dotto cistico , 37 corrispondenti alla vbi , 37 relativi alla vbp e 37 riguardo il dotto pancreatico principale ; in particolare , 5 pazienti sono stati studiati in followup post - colecistectomia per cui non stato valutato il distretto colecistico . 
in base allanalisi statistica , la percentuale di discordanza ( 19% ) tra la rm e la tc nella valutazione della vbi ha mostrato una significativit al test di mc nemar ( p = 0 , 008 ) ; analogamente la percentuale di discordanza ( 16% ) tra la rm e la tc nella valutazione della vbp ha mostrato una significativit al test di mc nemar ( p = 0 , 01 )  . radiol med ( 2009 ) 114 : 390402 fig . 
6a - c extrahepatic bile duct lithiasis in a patient who underwent cholecystectomy for gallstones : discordant results between computed tomography ( ct ) and magnetic resonance cholangiopancreatography ( mrcp ) at the level of the common bile duct . 
a mrcp indicates dilatation of the segmental intrahepatic bile ducts and of the common hepatic duct that shows a saccular dilatation ; faint and inhomogeneous magnetic resonance imaging ( mri ) signal in the distal bile duct with evidence of multiple fine filling defects , with evidence of residual biliary flow ( arrow )  . 
a la cprm mostra lectasia delle vie biliari intra - epatiche segmentarie , dei dotti biliari principali e del dotto epatico che mostra una dilatazione sacciforme ; il segnale rm appare scarso e disomogeneo a livello del coledoco con evidenza di multipli e fini difetti di riempimento da litiasi , pur tuttavia con segni di flusso biliare residuo ( freccia )  . 
c la tc non mostra definiti segni di litiasi biliare a livello del coledoco intra - pancreatico ( freccia )  . discussione la disponibilit di varie tecniche di diagnostica per immagini , quali lecografia , la tc , la rm e la cpre , per la valutazione dei pazienti affetti da malattie delle vie biliari comporta necessariamente la scelta della metodica pi accurata e meno invasiva in relazione alla specifica problematica clinica ; tale considerazione rappresenta tra laltro il presupposto per la corretta definizione dei protocolli diagnostici in tali pazienti per le diverse patologie a seconda dellesordio clinico [ 23 ]  . in questo studio , riportiamo la nostra esperienza relativa al confronto diretto dei risultati della cprm con quelli dellecografia ( us ) e della tc in un gruppo di pazienti con evidenza clinico - laboratoristica di patologia delle vie biliari , di cui 47 con lesioni di tipo benigno , 12 con lesioni maligne e 11 senza evidenza di lesioni organiche delle vie biliari agli esami di diagnostica per immagini . 
in particolare , il confronto diretto delle tre tecniche per immagini ( cprm , us e tc ) stato eseguito mediante unanalisi regionale dei diversi distretti del sistema bilio - pancreatico quali la colecisti , le vbi , la vbp e il dotto pancreatico principale allo scopo di definire la tecnica di diagnostica pi idonea e valida nella valutazione del singolo distretto biliare in relazione al tipo di patologia ; in particolare , la valutazione dei risultati stata suddivisa in un primo gruppo di pazienti in cui stata eseguito un confronto tra cprm ed us ( gruppo 1 ) ed un secondo gruppo in cui stato effettuato un confronto tra cprm e tc ( gruppo 2 )  . 
 nel gruppo 1 , il confronto diretto tra cprm ed us ha dimostrato una validit diagnostica di entrambe le metodiche di imaging per la valutazione del distretto colecistico , delle vbi e del dotto pancreatico principale sia in caso di patologia benigna che maligna , oltre che nei casi di assenza di lesioni organiche del sistema duttale biliopancreatico . 
al contrario , i risultati comparativi relativi alla valutazione della vbp hanno dimostrato una differenza statisticamente significativa a vantaggio della cprm rispetto allus in particolare nellidentificazione di lesioni litiasiche localizzate a livello del tratto medio e distale della vbp ; tale risultato appare ragionevole in quanto il tratto medio o distale della vbp pu non essere ben studiato dallus per linterferenza del meteorismo intestinale o nei soggetti obesi . 
 [ 8 ] hanno riportato valori di sensibilit ( 91% ) ed accuratezza diagnostica ( 97% ) della cprm superiori a quelli dellus ( 38% e 89% ) ; analogamente , ferrari et al . 
 [ 10 ] nella loro casistica hanno riscontrato una percentuale di risultati falsi negativi allus significativamente superiore a quello della 400 radiol med ( 2009 ) 114 : 390402 and main pancreatic duct with the aim of establishing the most appropriate and reliable diagnostic technique for each region based on the type of disease . 
evaluation of results was divided into two groups : in one group , mrcp was compared with us ( group 1 ) , whereas in the second group , mrcp was compared with ct ( group 2 )  . 
 in group 1 , the comparison between mrcp and us demonstrated that both imaging methods are of value in evaluating the gallbladder region , intrahepatic bile ducts and main pancreatic duct in both benign and malignant diseases , as well as in cases without organic lesions of the pancreatobiliary systeby contrast , results of the evaluation of the extrahepatic bile ducts showed a statistically significant superiority of mrcp , especially in detecting stones in the middle - distal tract of the extrahepatic bile ducts . 
this result is reasonable , given that the middle or distal tract of the extrahepatic bile ducts is not well visualised on us owing to interference from intestinal gas or in obese subjects . 
 [ 8 ] reported high levels of sensitivity ( 91% ) and diagnostic accuracy ( 97% ) for mrcp compared with us ( 38% and 89% ) , and ferrari et al . 
on the other hand , before conventional or laparoscopic cholecystectomy for gallstones , it is crucial to ensure that there are no migrating stones in the common bile duct , as these can cause colic and jaundice in the postoperative period . 
in such cases , mrcp allows the degree of ductal dilatation , the location of the stone visualised as a biliary filling defect , the degree of stenosis caused by the lesion and any residual bile flow to be determined . 
in the diagnostic evaluation of patients with biliary malignancies , although our study showed no statistically significant difference in the analysis of the various biliary regions , mrcp with conventional t1and t2 - weighted sequences and intravenous paramagnetic contrast material has a higher contrast resolution than us and allows a detailed study of biliary tree anatomy ( site , degree , extent and cause of the stenosis , vascular and / or lymph node involvement ) [ 2 ]  . in group 2 , direct comparison between mrcp and ct demonstrated the diagnostic value of both techniques in evaluating the gallbladder and main pancreatic duct , both in cprm nellidentificazione della coledoco - litiasi a sede distale . 
per ovviare a tali limitazioni dellus convenzionale nello studio del tratto distale della vbp , lecografia endoscopica e le pi recenti tecniche ecografiche tridimensionali consentono uno studio pi dettagliato dellalbero biliare [ 11 , 24 , 25 ] ; in particolare , solo lapproccio usendoscopico mostra una sensibilit diagnostica superiore ( 100% ) rispetto a quello della cprm ( 88% ) [ 11 ]  . 
pertanto la cprm certamente la tecnica di diagnostica per immagini di scelta per la valutazione del tratto medio - distale della vbp , sede frequente di lesioni litiasiche ; del resto , prima di una colecistectomia per calcolosi con approccio laparoscopico o tradizionale , fondamentale accertarsi che non vi siano calcoli migranti nel coledoco che determinano coliche ed ittero nel post - intervento o , comunque , in tutti i casi di ittero da stasi necessario una studio accurato delle vie biliari extra - epatiche ; in particolare , la cprm consente di valutare con precisione il grado di dilatazione delle vie biliari , la sede della lesione litiasica identificata semeiologicamente come difetto di riempimento biliare , il grado di stenosi determinato dalla lesione stessa ed , eventualmente , il flusso biliare residuo . 
certamente per quanto riguarda la valutazione diagnostica dei pazienti con lesioni maligne delle vie biliari , nonostante i risultati del nostro studio non hanno mostrato differenze statisticamente significative nellanalisi regionale dei diversi distretti biliari , la cprm offre rispetto allus una maggiore risoluzione di contrasto in quanto , integrata dallimpiego delle sequenza t1 e t2 pesate convenzionali associate alla somministrazione endovenosa di mezzo di contrasto paramagnetico , consente di valutare dettagliatamente le condizioni anatomiche dellalbero biliare ( sede della stenosi , grado della stenosi , estensione della stenosi , causa stenosante , impegno vascolare e / o linfoghiandolare ) [ 2 ]  . 
 nel gruppo 2 , il confronto diretto tra cprm e tc ha dimostrato una validit diagnostica di entrambe le metodiche dimaging per la valutazione del distretto colecistico e del dotto pancreatico principale sia in caso di patologia benigna che maligna , oltre che nei casi di assenza di lesioni organiche del sistema duttale bilio - pancreatico . 
al contrario , i risultati comparativi relativi alla valutazione delle vbi e della vbp hanno dimostrato una differenza statisticamente significativa a vantaggio della cprm rispetto alla tc in particolare nel riconoscimento di lesioni litiasiche nella maggioranza dei casi ; solo in un caso la maggiore risoluzione di contrasto della cprm ha consentito di identificare correttamente una stenosi maligna da colangiocarcinoma della confluenza dei dotti biliari principali nel dotto epatico comune ( tumore di klatskin tipo iv )  . 
 [ 9 ] che dimostrano una maggiore sensibilit diagnostica della cprm ( 100% ) rispetto a quella della radiol med ( 2009 ) 114 : 390402 benign and malignant diseases and in the case of no organic lesions affecting the pancreaticobiliary ducts . 
conversely , results of the evaluation of the extrahepatic bile ducts demonstrated a statistically significant superiority of mrcp over ct , particularly for detecting bile stones of the intraor extrahepatic bile ducts . 
in one case only did the higher contrast resolution of mrcp allow correct identification of a malignant stenosis due to cholangiocarcinoma at the confluence of the main bile ducts into the common hepatic duct ( klatskin type iv tumour )  . 
indeed , despite the improved spatial and temporal resolution brought about by volumetric multislice ct and multiplanar reconstructions , it is well known that ct cannot detect stones that do not have calcium density . 
 [ 11 ] reported similar sensitivity ( 88% ) for mrcp and ct in detecting choledocholithiasis , with both techniques yielding the same number of false negatives due to stones smaller than 5 mdespite having similar accuracy to mrcp , ct cholangiography with biliary contrast agents should be considered only in patients who cannot undergo mrcp or in the case of diagnostic doubts on us and / or mri [ 17 ] owing to the risk of hypersensitivity reactions . 
with regard to malignant lesions , the high contrast resolution of mrcp with the different t1and t2 - weighted sequences enabled correct identification of a klatskin type iv tumour and accurate definition of its hilar and perihilar extension . 
the role of mrcp in patients with cholangiocarcinoma has previously been well documented by others [ 2628 ]  . in conclusion , results of our study suggest that compared with us and ct , mrcp provides greater diagnostic accuracy in evaluating the intrahepatic and extrahepatic bile ducts because it identifies a larger number of strictures secondary to gallstones and their impact on biliary dynamics ( prestenotic dilatation , poststenotic bile flow )  . 
the noninvasiveness , lack of ionising radiation , panoramic capabilities and high spatial and contrast resolution of mrcp in depicting the pancreatobiliary system are significant technical advantages that justify its routine clinical use as a supplement to us , with ct being restricted to patients with contraindications to mrcp or to settings where mri is not available . tc multi - strato ( 82% ) nella coledocolitiasi ; in questo studio , infatti , due casi con lesioni litiasiche del coledoco non risultavano adeguatamente riconosciute dalla tc che inoltre non identificava unostruzione del dotto epatico comune . 
infatti , nonostante i progressi tecnologici della tc , quali la metodica multistrato volumetrica e le ricostruzioni multiplanari che offrono vantaggi in termini di risoluzione spaziale e temporale , noto che lesioni litiasiche non a densit calcica possono non essere identificate dalla tc . 
 [ 11 ] hanno mostrato unanaloga sensibilit diagnostica ( 88% ) della cprm e della tc nel riconoscimento della coledocolitiasi , riportando le stesse false negativit dovute a lesioni litiasiche inferiori ai 5 millimetri . 
la tc con mezzo di contrasto ad escrezione biliare , pur avendo dei valori di accuratezza diagnostica paragonabili a quelli della cprm , a causa del rischio di reazioni da ipersensibilit va considerata come tecnica alternativa solo nei pazienti che non possono essere sottoposti alla cprm o in caso di dubbi diagnostici allesame ecografico e / o di risonanza magnetica [ 25 ]  . 
levent1 1department of radiology , medical faculty , atatrk university , erzurum , turkey 2department of radiology , bilim university , florence nightingale hospital , istanbul , turkey 3department of radiology , numune hospital , erzurum , turkey 4department of pediatric cardiology , medical faculty , atatrk university , erzurum , turkey correspondence to : m . 
sok no 5 , dadaskent , erzurum , turkey , tel . : + 90 - 442 - 2361212 / 1521 , fax : + 90 - 442 - 2361301 , e - mail : akkanrad@hotmail.com received : 28 september 2008 / accepted : 31 october 2008 / published online : 14 april 2009 springer - verlag 2009 abstract purpose . 
these 18 patients were initially referred due to indications such as chest pain ( n = 11 ) , risk - factor assessment ( n = 3 ) , coronary artery anomaly ( n = 1 ) , suspected aberrant right subclavian artery due to dysphagia ( n = 1 ) and coronary artery bypass graft ( n = 2 )  . 
diciotto pazienti presentavano una tasca o una sacca a livello del setto interventricolare , verosimile espressione di chiusura spontanea del difetto interventricolare ( prevalenza 0 , 66% )  . i reperti della tcms di questi 18 pazienti sono stati correlati con i reperti ecocardiografici . 
il nostro studio dimostra che lincidenza di chiusura spontanea del difetto del setto interventricolare , radiol med ( 2009 ) 114 : 370375 patients initially diagnosed with ventricular diverticula with an apical and marginal septum location may actually have spontaneously closed muscular vsd . 
 keywords cardiac ct heart mdct angiography closed muscular ventricular septal defect introduction ventricular septal defects ( vsds ) are the second most common congenital heart defect after bicuspid aortic valves , but their true rate of incidence is , surprisingly , unknown [ 1 ]  . the incidence rate of vsds among live births is 1.53.5 per 1 , 000 term infants and 4.57.0 per 1 , 000 premature infants [ 2 ]  . the natural history of vsd , including incidence of spontaneous closure ( sc ) , is also uncerta however , some studies have reported the sc rate of vsd to be 48% by 6.9 years ( the mean age ) and 72% in patients without congestive heart failure ( chf ) [ 13 ]  . 
radiologists have begun to interpret coronary artery diseases , and in reporting the results of images obtained for coronary artery disease , they have begun to evaluate nonvascular cardiac structures as well . one of these is sc of muscular vsd observed in the interventricular septu in this study , we describe the experience of our institutions ( two centres ) in using cardiac multidetector ct angiography ( mdcta ) to diagnose sc of muscular vsd . materials and methods the study included 2 , 725 consecutive patients referred to atatrk university hospital and florence nightingale hospital for mdcta . 
in questo modo , alcuni pazienti , ai quali inizialmente era stata fatta diagnosi di diverticoli ventricolari a livello apicale e marginale del setto , in realt possono aver avuto una chiusura spontanea del difetto del setto interventricolare . 
questo risultato pu incidere sulle percentuali precedentemente riportate sia per quanto riguarda i diverticoli ventricolari sia per quanto riguarda la chiusura spontanea del difetto del setto interventricolare . parole chiave tc cardiaca cuore angiografia tcms chiusura del difetto del setto interventricolare disease , congenital malformation , atypical chest pain , screening for coronary artery disease and inconclusive conventional angiography . 
images were reviewed by a radiologist and a cardiologist in consensus , and the following data were recorded : gender , age , degree of coronary artery disease and presence of any other cardiac abnormality . 
similar records for images of the central muscular region to those of the earlier cases ( sc of muscular vsd ) were noted in the apical and marginal areas of the interventricular septuthese cases , with no history of surgery , were evaluated for sc of vsd . however , in the literature , ventricular diverticulum has been reported in all locations of the ventricle wall except 372 radiol med ( 2009 ) 114 : 370375 the interventricular septu in our study , patients with similar lesions in the basal inferoseptal , apical septum and midinferoseptal locations were not included because these lesions could not be differentiated from ventricular diverticula [ 5 , 6 ]  . 
 the procedures used were in accordance with the recommendations found in the declaration of helsinki , and approval of the local ethics committee was obtained . results retrospective electrocardiogram ( ecg ) - gated reconstructions were generated every 10% during the 50%90% of the r - r interval . 
 mdct scan protocol mdct was performed using two different 16 - detector - row ct scanners ( sensation16 , siemens medical systems , forchheim , germany , and aquillon , toshiba medical systems , tokyo , japan ) during one breath - hold ( 1624 s )  . with the first scanner , the following parameters were used : 160.75 - mm collimation , 1 - mm slice thickness and 0.6 - mm reconstruction interval . 
the indications for mdcta were suspected coronary artery disease ( n = 2 , 310 ) , coronary artery anomaly ( n = 22 ) , coronary artery stent patency ( n = 220 ) and coronary artery bypass graft assessment ( n = 173 )  . 
these 18 patients were referred due to indications such as chest pain ( n = 11 ) , risk factor assessment ( n = 3 ) , coronary artery anomaly ( n = 1 ) , suspected aberrant right subclavian artery ( n = 1 ) and coronary artery bypass graft ( n = 2 )  . 
1a - c a 46 - year - old man with spontaneous closure of muscular ventricular septal defect in the interventricular septu multiplanar reformatted multidetector computed tomography angiography ( mdcta ) images from 16 - mdct single - source scanner show long - axis ( a , c ) and short - axis ( b ) views of vsd ( ventricular septal defect ) ( arrows )  . 
le immagini angiografiche con tc a 16 strati mostrano lasse lungo ( a , c ) e lasse corto ( b ) del difetto del setto ventricolare ( frecce )  . 
of vsd location of vsd other abnormalities degree of coronary artery disease radiol med ( 2009 ) 114 : 370375 table 1 patient characteristics . tabella 1 caratteristiche dei pazienti . patient vsd , ventricular septal defect ; lad , left arterial dilatation central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular none moderate none mild none severe moderate none none none mild none mild severe none severe mild none myocardial bridging at lad middle segment aberrant right subclavian artery prior coronary artery bypass grafting aortic valve disease origin of the left circumflex coronary artery from the right sinus of valsalva myocardial bridging at lad middle segment prior percutaneous coronary intervention prior coronary artery bypass grafting ventricular function with no evidence of regional wallmotion abnormalities . 
vsds create a volume load on the left side of the heart secondary to the left - to - right shunt at the ventricular level , causing left atrial and left ventricular dilatation . 
the inlet defect makes up 5%8% of all vsds and is posterior and inferior to the perimembranous defect . muscular vsds make up 5%20% of all vsds [ 7 ]  . 
2a , b immagini assiale ( a ) e sagittale ( b ) del cuore di una donna di 39 anni con chiusura spontanea del difetto del setto interventricolare , che non si pu distinguere dai diverticoli settali a localizzazione apicale . laboratory tests for the diagnosis are not available . 
in the literature , ventricular diverticula have been reported to be located on all the ventricular walls ( apical and marginal septum ) except on the interventricular septum [ 5 ]  . 
nevertheless , similar appearances were observed in the apical and marginal septum as well . because they could not be differentiated from ventricular diverticula , these patients were excluded from the study . 
naturally , this finding may affect previously reported rates of both ventricular diverticula and the incidence of sc of muscular vsd . this will negatively affect the incidence rates of ventricular diverticula reported in earlier studies . 
it appears that there is a very high incidence of asymptomatic small muscular vsds that close spontaneously . isolated ventricular noncompaction is another entity that should be considered in the differential diagnosis . 
diagnosis of ventricular noncompaction usually requires the finding of more than three deep intertrabecular recesses [ 11 , 12 ]  . other pathologies that should be differentiated from sc muscular vsd are other causes of acquired ventricular aneurysms : those that occur after myocardial infarction , myocardial inflammatory disease such as sarcoidosis or myocarditis , infectious endocarditis or trauma [ 13 ]  . 
additional causes of ventricular aneurysm include connective tissue disease and ventricular dysplasia , as seen in patients with arrhythmogenic right ventricular cardiomyopathy . often , the distinction between fibrous ventricular diverticula and these entities can be made only with knowledge of cardiac history and visualisation of an associated abnormalitye.g. 
the true incidence of muscular vsd may have been underreported in the literature because of limitations inherent in the previously available imaging techniques and differences in the patient populations studied . 
because cardiac mdcta is frequently performed in patients with symptoms of chest discomfort , and because chest pain may be a signal of a cardiac abnormality , the incidence of sc muscular vsd is probably higher in this population than in the general asymptomatic population . however , in our study , a higher incidence of sc muscular vsd was shown in a large patient population . 
at our institutions , almost all of the cardiac mdcta studies are prospectively interpreted by two reviewers in consensus to generate an official report , and most reports are generated by two of the most senior reviewers . 
second , although a strict criterion consistent with the pathologic description of muscular vsd was chosen for the identification of this entity , pathologic proof of diagnosis was not obtained in any of our patients . 
however , given that the true prognostic implication of muscular vsd is unknown and that treatment of this entity is controversial , there is no indication to pursue an invasive procedure to confirm the diagnosis in these patients . in summary , this study shows that the incidence of sc muscular vsd with central septum location is probably higher than expected . 
3 immagine assiale del cuore di un uomo di 52 anni con chiusura spontanea del difetto del setto interventricolare , che non si pu distinguere dai diverticoli settali a localizzazione basale . this study has some limitations . 
dwi with adc mapping provides functional information on fetal renal parenchyma development and may thus become a useful tool in the management of pregnancy and treatment of the newborn child . 
abbiamo studiato con esame rm 88 feti ( et gestazionale 1740 settimane ) , gi valutati con esame ecografico che escludeva malformazioni del tratto urinario . lapparato urinario fetale stato studiato con sequenze t2 pesate ultraveloci ; successivamente sono state acquisite sui reni fetali sequenze dwi ( con calcolo automatico delle mappe di adc ) ed i valori di adc messi in relazione con let gestazionale , suddividendo i feti in sei gruppi in base allet gestazionale . 
abbiamo riscontrato che i valori di adc , compresi fra 0 , 99 e 1 , 6210 - 3 mm2 / s ( media 1 , 22 ; 95% ci 1 , 191 , 25 ; deviazione standard 0 , 147 ) tendono a decrescere con laumentare delle settimane di gestazione . 
le sequenze dwi ed le mappe di adc , fornendo informazioni funzionali sullo sviluppo del parenchima renale fetale , risultano di grande utilit soprattutto nella gestione della gravidanza e nel management post - natale . 404 radiol med ( 2009 ) 114 : 403413 keywords fetus mri kidney diffusion adc parole chiave feto risonanza magnetica rene diffusione adc introduction introduzione prenatal ultrasound is the imaging modality of choice in the study of fetal anatomy , although its accuracy may be limited by pathological fetal conditions such as anhydramnios , oligohydramnios and complex malformations as well as maternal conditions such as obesity [ 17 ]  . 
magnetic resonance ( mr ) imaging provides a detailed morphological study of the fetal renal system anatomy as well as allowing assessment of possible urinary tract disorders , which may be isolated or associated with abnormalities of other organs or systems , especially of the gastrointestinal tract or lungs [ 810 ]  . prior to the introduction of ultrafast sequences [ 11 , 12 ] , the use of diffusion - weighted imaging ( dwi ) , which is extremely sensitive to motion , was confined to neuroradiology [ 13 ]  . 
in recent years , researchers have suggested the possibility of using dwi sequences with calculation of the apparent diffusion coefficient ( adc ) of water in the human kidney for the study of renal function in adults [ 16 , 17 ]  . the aim of our study was to apply dwi sequences to the study of the fetal kidney in order to verify the existence of a correlation between adc maps and gestational age in normal fetuses , as such additional functional information would be useful in the event of suspected urinary system abnormalities . materials and methods patients between june 2005 and april 2008 , 85 pregnant women with 88 fetuses ( gestational age 1740 weeks , mean 28 weeks ; maternal age 1844 years , mean 29 years ) were consecutively evaluated with mr imaging . 
patients had previously undergone obstetric ultrasound examination performed with aloka prosound ssd - 5500 equipment by one of three gynaecologists with > 10 years experience at the department of gynaecology and obstetrics of our teaching hospital . the clinical indications for the mr study were diverse , but in no case was there a suspicion of urinary tract malformations . 
in particular , the fetal organs with suspected malformations to be confirmed by mr imaging were the lultrasonografia pre - natale rappresenta limaging di prima scelta nello studio dellanatomia fetale , ma la sua accuratezza pu essere limitata da condizioni patologiche fetali , quali anidramnios , oligoidramnios , sindromi malformative complesse e condizioni di pertinenza materna , come lobesit [ 17 ]  . 
la rm consente un dettagliato studio morfologico dellanatomia fetale , con riguardo al tratto urinario , ma anche una valutazione delle eventuali patologie del tratto urinario stesso che possono presentarsi in forma isolata o associate ad anomalie di altri organi o apparati , soprattutto del tratto gastrointestinale e dei polmoni [ 810 ]  . prima del perfezionamento delle sequenze ultraveloci [ 11 , 12 ] , le sequenze pesate in diffusione , che sono estremamente sensibili al movimento , venivano utilizzate esclusivamente lavvento in neuroradiologia [ 13 ] ; con dellimaging ultrafast invece , la loro applicazione stata di molto ampliata [ 14 , 15 ]  . 
negli ultimi anni alcuni autori hanno prospettato la possibilit di utilizzare le sequenze rm pesate in diffusione ( dwi ) nello studio della funzionalit renale delladulto , calcolando il coefficiente di diffusione apparente dellacqua ( adc ) nel rene umano [ 16 , 17 ]  . il nostro scopo quello di applicare le sequenze dwi allo studio del rene fetale , per valutare lesistenza di una correlazione fra coefficiente di diffusione apparente ( mappe di adc ) ed et gestazionale nei feti sani ; tale dato rappresenta infatti unulteriore informazione , di tipo funzionale , utile soprattutto nel sospetto di patologia del tratto urinario . materiali e metodi pazienti da giugno 2005 ad aprile 2008 , abbiamo valutato in successione , mediante esame rm , 85 donne in gravidanza , con 88 feti ( et gestazionale 1740 settimane di amenorrea , media 28 settimane ; et materna range 1844 anni , media 29 anni )  . 
le pazienti erano gi state sottoposte ad esame ecografico ostetrico eseguito con apparecchio aloka prosound ssd - 5500 , da uno di tre ginecologi con esperienza decennale , del dipartimento di ginecologia ed ostetricia del nostro policlinico . 
in particolare gli organi sui quali pendeva il sospetto di malformazioni , da confermare con rm , erano : encefalo , cuore , distretto toracoaddominale per la presenza di ernie intestinali transdiaframmatiche , oppure vi era evidenza clinica di infezione materna da cmv o , ancora , esisteva il dato anamnestico di precedenti radiol med ( 2009 ) 114 : 403413 brain , the heart and the thoracoabdominal regions due to the presence transdiaphragm intestinal herniations . 
other included clinical evidence of maternal indications cytomegalovirus ( cmv ) infection or a history of previous pathological pregnancies ( spina bifida , genetic syndrome )  . patients were divided into six groups according to gestational age at the time of the mr examination . 
in particular , the following groups were defined : group a : 8 fetuses of 1720 weeks group b : 19 fetuses of 2124 weeks group c : 21 fetuses of 2528 weeks group d : 20 fetuses of 2932 weeks group e : 17 fetuses of 3336 weeks group f : 3 fetuses of 3740 weeks informed consent was obtained from the all patients prior to the examination , and the study was approved by the hospitals ethics committee . mr imaging mr imaging was performed using a superconducting 1.5tesla magnet ( siemens magnetom avanto ) with a phasedarray coil placed over the mothers pelvis . 
the multiplanar study of the urinary system was acquired by using the following : t2 - weighted halffourier single - shot turbo spin - echo ( haste ) sequences ( tr 1 , 900 , te 111 , fov 350300 mm , matrix 256256 , slice thickness 4 mm , acquisition time 20 s ) in the fetal axial , coronal and sagittal planes t2 - weighted thick - slab ( cholangiographic ) sequences ( tr 1 , 900 , te 111 , fov 350300 mm , matrix 256256 , slice thickness 4 mm , acquisition time 14 s ) with maximum intensity projection ( mip ) reconstructions , obtained in the fetal coronal plane t1 - weighted gradient - echo fast low - angle shot ( flash ) two - dimensional sequences ( tr / te = 362 / 4.8 , flip angle 70 , fov 350300 mm , matrix 256192 , slice thickness 4 mm , acquisition time 20 s ) were also acquired in the axial and coronal plane to rule out haemorrhage . axial or coronal single - shot echoplanar dwi sequences of the renal kidneys were subsequently acquired in all cases ( tr 8 , 000 , te 90 , ti 185 ms , fov 420300 mm , matrix 192192 , slice thickness 5 mm , total acquisition time 90 s ) with diffusion gradients applied along the three orthogonal directions ( x , y , z )  . 
three b - factor values were applied for each plane ( 0 , 200 and 700 s / mm2 ) ; the number of images acquired with the dwi sequences was 168 , that is , the sum of the images acquired with the three b factors applied in the three directions . 
in particolare si sono definiti i seguenti gruppi : gruppo a : 8 feti fra la 17 e la 20 settimana ; gruppo b : 19 feti fra la 21 e la 24 settimana ; gruppo c : 21 feti fra la 25 e la 28 settimana ; gruppo d : 20 feti fra la 29 e la 32 settimana ; gruppo e : 17 feti fra la 33 e la 36 settimana ; gruppo f : 3 feti fra la 37 e la 40 settimana . ogni esame stato effettuato previo consenso scritto delle donne ; il nostro studio stato approvato da una commissione etica di studi clinici del nostro ospedale . risonanza magnetica lesame rm stato condotto con magnete superconduttore da 1 , 5 tesla ( siemens magnetom avanto ) , utilizzando una bobina phased array , posizionata sulla pelvi materna . 
le pazienti sono state esaminate in posizione supina e non stata effettuata alcuna sedazione , n sulle donne n sul feto . per la localizzazione del feto sono state ottenute in primo luogo immagini di scanogramma multiplanare , poi , durante lesame , ciascuna sequenza servita come scanogramma per la successiva . 
lo studio multiplanare del sistema urinario stato ottenuto utilizzando : sequenze half - fourier single - shot turbo spin - echo ( haste ) t2 pesate ( tr 1900 , te 111 , fov 350300 mm , matrice 256256 , spessore di strato 4 mm , tempo di acquisizione 20 s ) , eseguite sui piani assiale , coronale e sagittale fetali ; sequenze thick slab ( colangiografiche ) t2 pesate ( tr 1900 , te 111 , fov 350300 mm , matrice 256256 , spessore di strato 4 mm , tempo di acquisizione 14 s ) , con successive ricostruzioni mip , eseguite sul piano coronale fetale ; sequenze flash ( fast low angle shot ) 2d gradient - echo t1 pesate ( tr / te = 362 / 4 , 8 , flip angle 70 , fov 350300 mm , matrice 256192 , spessore di strato 4 mm , tempo di acquisizione 20 s ) sui piani assiale o coronale , per escludere la presenza di eventuali emorragie . successivamente , in tutti i casi , sono state acquisite sui piani assiale o coronale dei reni fetali sequenze in diffusione single - shot echo - planare ( tr 8000 , te 90 , ti 185 ms , fov 420300 mm , matrice 192192 , spessore 5 mm , tempo di acquisizione totale 90 s ) con gradienti di diffusione applicati sui tre assi ortogonali ( x , y , z )  . 
sono stati applicati tre b - factor per piano ( 0 , 200 e 700 s / mm2 ) ; il numero di immagini acquisite con le sequenze pesate in dwi in totale di 168 , risultando tale cifra dalla somma delle immagini ottenute con i tre valori del b - factor applicati sui tre assi . 
il tempo di acquisizione totale dellesame rm fetale stato mediamente di 20 minuti ( 1645 min ) ; il tempo di elaborazione totale stato di circa 7 minuti ( 412 min )  . 
evaluation of fetal kidney adc was obtained by placing a region of interest ( roi ) over the renal parenchyma on all of the slices containing renal tissue and then averaging the results . 
normal renal function was confirmed after birth by means of studio dei reni stato effettuato dunque con acquisizione sui tre piani coronale , assiale e sagittale . per la misurazione dei valori di adc sono state selezionate le immagini non alterate da artefatti da movimento ; in 4 casi le sequenze pesate in diffusione risultavano alterate per artefatti da movimento in corso di acquisizione e sono state ripetute per la seconda volta al fine di ottenere immagini ottimali per il calcolo del valore di adc ; in un solo caso , non preso in considerazione nel nostro studio , non siamo riusciti ad ottenere immagini utili per calcolare il valore di adc . 
coronal t2 - weighted image ( a ) , diffusion - weighted image ( b ) and corresponding apparent diffusion coefficient ( adc ) map ( c ) , with regions of interest covering the entire area of the renal parenchyma . 
la figura mostra acquisizioni coronali sui reni fetali , sui quali sono state posizionate le roi : immagine t2 pesata antomica ( a ) , immagine pesata in diffusione ( b ) e relativa mappa di adc ( c )  . 
il valore medio di adc del parenchima renale in questo caso era di 1 , 5610 - 3 mm2 / s . radiol med ( 2009 ) 114 : 403413 fig . 
axial t2 - weighted image ( a ) , diffusion - weighted image ( b ) , and corresponding apparent diffusion coefficient ( adc ) map ( c )  . 
il miglior risultato stato espresso dallequazione di regressione : adc ( mm2 / s ) = 1 , 690 , 0169 ( eg ) ( r2 = 37 , 7% , r2 adj = 37 , 0% , p < 0 , 005 , correlazione di pearson = 0 , 614 )  . inoltre bisogna precisare che la correlazione pi significativa fra valore medio di adc ed et gestazionale stata riscontrata nei gruppi b , c , d ed e ( p < 0 , 005 ) ; nel gruppo a non si evidenziata correlazione adc / et gestazionale ( p = 0 , 321 ) , ma questo nel nostro studio pu dipendere dal numero inferiore di feti esaminati in tale epoca gestazionale . 410 radiol med ( 2009 ) 114 : 403413 fig . 
in group a , no correlation between adc and gestational age was identified ( p = 0.321 ) , but this finding may well depend on the small number of fetuses in this group . 
with regard to group f ( 3740 weeks ) , the mean adc values , considered individually , reflect the decreasing trend , but in consideration of the small number of fetuses in the group , this finding lacks statistical significance [ 8 ]  . 
dwi and adc determinations can also be performed in fetuses by using dw echoplanar sequences [ 2022 ] , and they can be used for the study of the urinary system [ 23 , 24 ]  . 
 clearly , fetal studies entail more difficulties than postnatal studies owing to possible artefacts due to maternal or fetal movement , aorta pulsations , lower signal - to - noise ratio and reduced spatial resolution [ 24 ]  . 
in a 3 - year follow - up of fetuses studied with mr imaging with echoplanar sequences , they found no abnormality ( including hearing deficit ) arising as a result of per quanto riguarda il gruppo f ( 3740 settimane ) i valori medi di adc riscontrati , considerati singolarmente sono in accordo allandamento decrescente , ma dato il numero esiguo di feti esaminati non hanno un valore statisticamente significativo [ 8 ]  . 
 discussione la rm considerata la sola metodica valida per calcolare la diffusione molecolare in vivo attraverso la misura del movimento browniano delle molecole dacqua in un tessuto . i valori di adc del parenchima renale , sono condizionati sia dal suddetto movimento browniano dellacqua , ma anche da altri fattori , quali la perfusione nella rete capillare e il flusso tubulare . 
le immagini pesate in diffusione e le determinazioni dei valori di adc sono realizzabili anche nei feti , utilizzando sequenze ecoplanari pesate in diffusione [ 2022 ] e possono essere sfruttate per lo studio del tratto urinario [ 23 , 24 ]  . certamente lo studio fetale comporta maggiori difficolt rispetto a quello post - natale , per i possibili artefatti da movimento materni e fetali , per la pulsatilit dellaorta e per il pi basso rapporto segnale - rumore e la minore risoluzione spaziale [ 24 ]  . 
 [ 26 ] noted that dwi sequences have considerably ( approximately six times ) lower specific absorption rates ( sar ) when compared with the t2 sequences used in fetal studies and may therefore be considered harmless for the fetus . 
in 2005 , gubhaju and black [ 27 ] proposed a functional estimate of fetal renal parenchyma on the basis of the morphological information provided by t2 - weighted sequences . 
they noted the existence of a positive linear correlation between fetal renal diameters and gestational age and suggested that this information may be useful not only for monitoring normal renal development but also for predicting possible renal function renal size being correlated with the number of nephrons . 
quantification of the mean adc value , in relation to gestational age , is a truly functional parameter that can help define normal renal function . in agreement with the recent literature [ 23 , 24 ] , in our series of fetuses without urinary system abnormalities , we observed a change in adc values in relation to gestational age and , in particular , the tendency of adc to decrease with increasing gestational age . 
this has been related to the development of the renal parenchyma ( increase in the number and size of nephrons , the functional unit of the kidney ) and hence to other contributing age - related factors , such as blood flow , tubular flow , water content or cell density [ 24 ]  . 
renal blood flow amounts to 2%4% of cardiac output during fetal life as against 20% in adult life [ 28 ]  . we must acknowledge some limitations to our study . 
in addition , it is worth noting that dwi sequences and related adc values have the sole aim of providing an indication of the correct development and normal functioning of the fetal kidney . 
in the event of abnormal findings , dwi with adc is unable to guide the diagnosis with regard to the nature of the kidney disease . this is , instead , achieved with ultrafast t2 sequences , which definitely have better spatial and contrast resolution and consequently excellent anatomical definition [ 29 ]  . however , the anatomical information offered by t2weighted sequences is complemented by the functional information provided by dwi , which allows identification of residual renal parenchyma [ 30 ] and assessment of whether its development , in relation to the adc values , reflects that expected for the fetuss gestational age . sequenze pesate in diffusione presentano un tasso di assorbimento specifico ( sar : specific absorption rates ) molto pi basso ( circa sei volte inferiore ) se comparato alle sequenze t2 pesate utilizzate nello studio fetale e dunque queste possono essere considerate innocue per i feti . 
nel 2005 , gubhaju e black [ 27 ] hanno proposto una stima di tipo funzionale del parenchima renale fetale in base al dato morfologico emergente dalle sequenze t2 pesate . 
costoro infatti hanno notato lesistenza di una correlazione positiva di tipo lineare fra diametri renali fetali ed et gestazionale ipotizzando che tale dato sia utile non solo per monitorare il normale sviluppo renale , ma anche per prevedere la possibile funzionalit renale , essendo le dimensioni dei reni correlate al numero di nefroni . 
la misura quantitativa del valore medio di adc , riferito a ciascuna et gestazionale , rappresenta un reale parametro funzionale utile a definire la normale funzionalit dei reni . in accordo con la recente letteratura [ 23 , 24 ] , nella nostra serie di feti , tutti senza anomalie del tratto urinario , abbiamo notato lesistenza di una variazione dei valori di adc con let gestazionale , in particolare emersa la tendenza di questi a decrescere con lavanzare delle settimane di gestazione : ci stato posto in relazione allo sviluppo parenchimale renale ( aumento del numero e delle dimensioni dei nefroni che rappresentano lunit funzionale del rene ) , e quindi a fattori concorrenti anchessi variabili con let gestazionale , quali il flusso ematico , il flusso tubulare , il contenuto dacqua o la densit cellulare [ 24 ]  . 
inoltre va ricordato che le sequenze pesate in dwi e i relativi valori di adc hanno il solo scopo di orientare circa il corretto sviluppo e dunque il normale funzionamento dei reni fetali , ma nelleventualit di un dato anomalo non consentirebbero di orientare la diagnosi di natura della nefropatia , che invece affidata alle sequenze t2 pesate ultraveloci , le quali sono sicuramente caratterizzate da una migliore risoluzione spaziale e di contrasto e dunque da uneccellente definizione anatomica [ 29 ]  . 
accanto al dato anatomico delle sequenze t2 pesate tuttavia le dwi forniscono il dato funzionale consentendo innanzitutto di identificare la presenza di parenchima renale residuo [ 30 ] e di valutare se il suo sviluppo , in rapporto ai valori di adc , in accordo con i valori attesi per let gestazionale . 412 conclusions conclusioni radiol med ( 2009 ) 114 : 403413 although our study is preliminary and further research on larger populations is needed , we believe that dwi sequences should be considered a useful adjunct to conventional t2 - weighted ultrafast imaging in the study of the fetal kidneys . 
midiri3 1dipartimento di radiologia e dipartimento cardio - polmonare , imaging cardiovascolare non invasivo , azienda ospedaliera di parma , parma , italy 2dipartimento di radiologia e cardiologia , erasmus medical center , rotterdam , the netherlands 3dibimel , sezione di scienze radiologiche , universit di palermo , palermo , italy 4dipartimento di radiologia , universit degli studi di verona , verona , italy 5dipartimento di radiologia , universit degli studi di trieste , trieste , italy correspondence to : f . 
cademartiri , viale rustici 2 , 43100 parma , italy , tel . : + 39 - 052 - 1961833 , e - mail : filippocademartiri@hotmail.com received : 8 august 2007 / accepted : 25 october 2007 / published online : 14 april 2009 springer - verlag 2009 abstract purpose . 
lm dimensions ( length , ostial and bifurcation diameters ) , quantitative [ location , hounsfield unit ( hu ) attenuation ] and qualitative ( composition , shape ) analysis of plaques within the lm were performed . 
stato esaminato un gruppo di 62 pazienti consecutivi ( 41 uomini , et media 6011 ) sottoposti ad ac - tcms con scanner a 64 strati per sospetta malattia aterosclerotica coronarica . 
sono state misurate le dimensioni del tcs ( lunghezza , diametri allostio ed alla biforcazione ) , nonch stata effettuata unanalisi quantitativa ( sede , densit hu ) e qualitativa ( composizione , forma ) delle placche in esso presenti . 
la ac - tcms mette in evidenza aspetti fondamentali del carico aterosclerotico del tcs dal momento che la rottura e la conseguente trombosi delle placche vulnerabili possono svilupparsi a partire da lesioni non significative alla acc . parole chiave coronaropatia aterosclerotica tronco comune sinistro tomografia computerizzata multistrato tc 64 strati placca vulnerabile introduction introduzione the distribution of fissured or rupture - prone plaques is known to be nonuniform throughout the coronary tree . histopathological and angiographic studies suggest that atheromas with a thin fibrous cap and necrotic lipid core at a high risk of rupture are not frequently encountered in the left main ( lm ) branch and the distal tracts of the coronary arteries [ 13 ]  . 
intravascular ultrasonography ( ivus ) has in fact shown that plaque fissuring rarely occurs in the lm or the distal tracts of the coronary arteries , whereas it is much more common in the proximal tract [ 4 ] , particularly the left anterior descending ( lad ) coronary artery [ 5 ]  . 
the nonuniform distribution of coronary atherosclerosis , with emphasis on the varying lipid content of the plaques , has recently been confirmed by studies with ivus [ 8 , 9 ]  . a number of studies have highlighted the ability of multidetector - row computed tomography ( mdct ) coronary angiography ( ca ) to distinguish between calcified , noncalcified and mixed plaques on the basis of the different attenuation values of each structure [ 10 , 11 ]  . 
the aim of our study was to quantify the coronary atherosclerotic burden of the lm and its bifurcation in a consecutive series of patients la distribuzione delle placche fissurate o prone alla rottura conosciuta non essere uniforme in tutto lalbero coronarico . 
studi istopatologici ed angiografici suggeriscono che gli ateromi con cappuccio sottile e core lipidico necrotico ad alto rischio di rottura non sono frequenti nel tronco comune sinistro ( tcs ) e nei tratti distali delle arterie coronarie [ 13 ]  . 
lecografia intravascolare ( ivus ) ha , infatti , mostrato che la fissurazione delle placche avviene raramente nel tcs o nel tratto distale delle arterie coronarie , mentre molto pi comune riscontrarla nel tratto prossimale [ 4 ] , specialmente nellinterventricolare anteriore ( iva ) [ 5 ]  . tuttavia , la ragione del perch placche vulnerabili o fissurate tendano a risparmiare il tcs ed i segmenti distali delle arterie coronarie sinistre non ancora nota . 
la distribuzione non uniforme dellaterosclerosi coronarica , con attenzione al differente contenuto lipidico delle placche , stata gi confermata da recenti studi condotti con ivus [ 8 , 9 ]  . diversi lavori mettono in evidenza le capacit della angiografia coronarica con tc multistrato ( ac - tcms ) nel distinguere tra placche calcifiche , non calcifiche e di aspetto misto , basandosi sulla diversa attenuazione dei raggi x di ciascuna struttura [ 10 , 11 ]  . 
the scan and reconstruction parameters were : number of detectors / collimation 322 / 0.6 mm , gantry rotation time 330 ms , feed / rotation 3.84 mm ( pitch 0.2 ) , kvp 120 , mas 900950 , field of view 150 mm , effective slice thickness 0.75 mm , reconstruction increment 0.4 mm and convolution kernels b30f and b46f . 
a 100 - ml bolus of nonionic iodinated contrast material ( iomeprol 400 mgi / ml , iomeron , bracco , italy ) was intravenously administered with antecubital access at a flow rate of 5 ml / s , followed by a bolus chaser of 40 ml of physiological solution at the same injection rate . 
the available reconstructions were examined with all the conventional postprocessing techniques [ native axial images , multiplanar reconstructions ( mpr ) , curved multiplanar reconstructions ( cmpr ) , maximum intensity projections ( mi ) p and volume rendering ( vr ) ]  . 
a semiquantitative analysis of the following lm characteristics was performed : length and diameter at the ostium and bifurcation , presence of atherosclerotic plaques ( at least 1 - mm thick ) , their position and involvement of the bifurcation , extension , eccentric development ( up to two thirds of the vessel circumference ) or concentric development ( covering 360 of the vessel circumference ) , composition ( calcified , noncalcified or mixed ) and density in hounsfield units ( hu )  . 
a quantitative analysis of the thickness ( vessel diameter vessel lumen ) and the area ( vessel area lumen area ) of the plaques visualised in the axial sections of the vessel was performed . 
in questo contesto , i dati sono stati correlati con le caratteristiche dimensionali del tcs . materiali e metodi un gruppo di 62 pazienti consecutivi ( 41 uomini , 21 donne , et media 6011 anni ) sottoposti ad ac - tcms per patologia coronarica nota o sospetta stato retrospettivamente esaminato per la valutazione del carico aterosclerotico nel tcs ed alla sua biforcazione . 
i parametri di scansione e ricostruzione sono : numero di detettori / collimazione 322 / 0 , 6 mm , tempo di rotazione del tubo radiogeno 330 ms , avanzamento / rotazione 3 , 84 mm ( pitch 0 , 2 ) , kvp 120 , mas 900950 , campo di vista 150 mm , spessore effettivo dello strato 0 , 75 mm , incremento di ricostruzione 0 , 4 mm , filtri di convoluzione b30f e b46f . 
stato somministrato per via endovenosa antecubitale un bolo di mezzo di contrasto iodato non ionico ( iomeprol 400mgi / ml , iomeron , bracco , italia ) da 100 ml ad una velocit di 5 ml / s seguito da un bolo da 40 ml di soluzione salina alla stessa velocit . 
per la somministrazione del mezzo di contrasto stato utilizzato un iniettore automatico a doppia testa ( stellant , medrad , usa )  . due radiologi con elevata esperienza in ac - tcms ( livello 3 ) [ 12 ] hanno esaminato su una stazione di lavoro dedicata ( leonardo , siemens , germania ) le caratteristiche del tcs . 
le ricostruzioni disponibili sono state valutate con tutte le convenzionali tecniche di post processing ( immagini assiali , ricostruzioni multiplanari - mpr , ricostruzioni multiplanari curve - cmpr , proiezioni di massima intensit - mip , e volume rendering - vr )  . 
stata effettuata unanalisi semiquantitativa delle caratteristiche seguenti del tcs : lunghezza e diametri allostio ed alla biforcazione , eventuale presenza di placche aterosclerotiche ( con almeno 1 mm di spessore ) , loro posizione e coinvolgimento della biforcazione , estensione , sviluppo eccentrico ( fino a 2 / 3 della circonferenza vaso ) o concentrico ( sui 360 della circonferenza del vaso ) , composizione ( calcifiche , non calcifiche o miste ) e densit in unit hounsfield ( hu )  . 
stata , altres , effettuata unanalisi quantitativa dello spessore ( diametro vasodiametro lume ) e dellarea ( area vaso - area lume ) delle placche presenti visualizzate nella sezione assiale del vaso . tutti i pazienti sono stati sottoposti ad angiografia coronarica convenzionale ( acc )  . 
i filmati sono stati poi analizzati da un operatore in cieco con un software radiol med ( 2009 ) 113 : 358369 the study population was divided into two groups on the basis of the presence ( n = 30 ) or absence ( n = 32 ) of plaques in the lm . 
vessel dimensions ( length and diameter at the ostium and bifurcation ) and plaques were correlated with the pearson test . quantitativo ( caas , pie medical , olanda )  . 
un valore 50% indica una stenosi significativa . la nostra popolazione stata suddivisa in due gruppi di pazienti sulla base della presenza ( n = 30 ) o meno di placche ( n = 32 ) nel tcs . 
the plaques prevalently involved the distal tracts of the lm ( 69% ) , the bifurcation ( 71% ) and the ostial tracts of the lad ( 57% ) and the left circumflex coronary artery ( lcx ) ( 31% )  . 
mpr in sezione che evidenziano una placca non calcifica non significativa e ne consentono un analisi semiquantitativa ( c , d )  . radiol med ( 2009 ) 113 : 358369 fig . 
3a - d conventional coronary angiography ( ca ) displays a significant lesion ( arrowhead ) at the ostium ( a )  . multiplanar reconstruction ( mpr ) using multidetector - row computed tomography ca : visualisation and length measurement of the left main ( lm ) coronary artery ( b )  . 
a moderate correlation ( r = 0.56 ) was found between lm diameter at the bifurcation ( mean diameter 6.31.3 mm ) and the corresponding plaque area ( mean area 20.8 mm2 ) in patients with plaques at the bifurcation ( n = 25 )  . 
most of these plaques were eccentric ( n = 20 , 80% ) , with a variable composition ( eight calcified , 12 mixed , five noncalcified ) , frequently ( 80% ) involving the proximal tracts of the lad or lcx . 
in ten patients ( 16% ) with nonsignificant atherosclerotic plaques ( < 50% of the lumen ) at mdct , conventional ca identified the presence of vessel wall irregularities of the lm . 
in the remaining 49 patients ( 79% ) , conventional ca identified a healthy lm , whereas mdct detected nonsignificant atherosclerotic lm plaques in 19 of these patients ( 31% )  . ( 71% ) ed i tratti ostiali dellarteria interventricolare anteriore ( 57% ) e della circonflessa ( 31% )  . 
le placche riscontrate presentavano estensione media di 54 mm , spessore medio di 20 , 8 mm , ed area media 1 , 80 , 9 mm2 . i diametri misurati allostio ed alla biforcazione del tcs erano significativamente aumentati nei pazienti con placche aterosclerotiche ( p < 0 , 05 )  . 
una correlazione moderata ( r = 0 , 56 ) stata riscontrata tra il diametro del tcs alla biforcazione ( diametro medio 6 , 31 , 3 mm ) e la corrispondente area della placca ( area media 20 , 8 mm2 ) dei pazienti con placche alla biforcazione ( n = 25 )  . 
tali placche si presentano nella maggior parte dei casi eccentriche ( n = 20 , 80% ) , con composizione variabile ( 8 calcifiche , 12 miste , 5 non calcifiche ) , coinvolgendo frequentemente ( 80% ) i tratti prossimali della iva o della circonflessa . 
visualisation of noncalcified plaque is limited to the size of the plaque and the size of the vessel , with greater accuracy when the analysis is limited to the proximal segments [ 10 ]  . 
noncalcified plaques were more frequently encountered in the first group , and as such , the severity of atherosclerosis is underestimated if calcified plaques only are taken into consideration [ 15 ]  . 
nei rimanenti 49 pazienti ( 79% ) la acc ha evidenziato un tcs indenne , mentre la ac - tcms ha dimostrato in 19 di questi ( 31% ) placche aterosclerotiche non significative del tcs . discussione la visualizzazione delle placche aterosclerotiche coronariche rappresenta una delle sfide pi recenti per la ac - tcms , nel tentativo di giungere a quella caratterizzazione tessutale che la acc non in grado di effettuare . 
la malattia aterosclerotica progredisce verso la stenosi clinicamente significativa con un carico crescente di placche , in principio non calcifiche , che scatenerebbero gli eventi acuti [ 6 , 7 , 13 ]  . 
la tc in virt della capacit di attribuire valori numerici di densit in hu alle strutture esaminate sembrerebbe il metodo di imaging ideale per visualizzare e quantificare i componenti della placca aterosclerotica che langiografia non riesce ad apprezzare [ 14 ]  . 
la visualizzazione delle placche non calcifiche limitata dalle dimensioni della placca e del vaso , con una migliore accuratezza quando si restringe lanalisi ai segmenti prossimali [ 10 ]  . 
 [ 15 ] hanno dimostrato una sostanziale differenza di composizione tra le placche dei pazienti con ima e quelle dei pazienti radiol med ( 2009 ) 113 : 358369 plaques in patients with acute ischemic events have a lower mean density than those in patients with stable angina [ 16 ]  . 
nonetheless , according to the most recent evidence , the attenuation values measured in hu within the plaque seem to be influenced by the intravascular attenuation and the convolution kernel used . 
used ivus to verify whether the distance of the plaque from the ostium influences plaque composition in terms of necrotic lipid content and therefore whether this is relevant to plaque fissuring [ 8 ]  . 
a later study by the same group clarified how the composition of plaques in the lm and the distal tracts is similar , whereas plaques in the proximal tracts of the coronary arteries are characteristically more vulnerable , with a necrotic lipid core . 
this core is greater in patients with acute coronary syndromes and is weakly associated with high levels of high - density lipoprotein ( hdl ) cholesterol , although there was no correlation with inflammatory markers . 
in patients with a short lm , the lipid cores tend to be concentrated between the bifurcation and the proximal tracts of the coronary arteries , whereas in patients with a long lm , they tend to appear more proximally , prior to the proximal coronary artery tracts [ 9 ]  . in light of these considerations , in our study , we described the presence of atherosclerosis in the lm correlated with vessel dimensions ( length , diameter ) using 64 - mdct and evaluated the characteristics of the atherosclerotic plaques present with a semiquantitative method . the length of the lm is commonly about 12 cm , even though it is frequently shorter and more rarely longer . 
a recent study demonstrated that measurements of the proximal diameters of the proximal coronary arteries at mdct are well correlated with measurements made with ivus and conventional ca [ 20 ]  . 
plaque distribution may be determined by anatomical factors that influence the shear stress ( length of the lm , diameters of the ostium and the bifurcation ) , by the lipid profile and by inflammatory con angina stabile . 
al riguardo i dati presenti in letteratura concordano : le placche dei pazienti con evento ischemico acuto hanno densit media minore di quella dei pazienti con angina stabile [ 16 ]  . 
 [ 8 ] hanno inizialmente verificato mediante icus come la distanza della placca dallostio sia un fattore determinante sulla composizione della stessa , in termini di contenuto lipidico necrotico , e , quindi , rilevante sulla propensione della stessa alla fissurazione . 
uno studio successivo dello stesso gruppo ha meglio chiarito come la composizione delle placche nel tcs e nei tratti distali sia simile , mentre nei tratti prossimali delle coronarie assuma pi spiccati caratteri di vulnerabilit , caratterizzati da un nucleo lipidico necrotico . 
di converso , un lungo tcs si associa a nuclei lipidici precoci immediatamente entro i tratti prossimali delle coronarie [ 9 ]  . alla luce di queste considerazioni , nel nostro studio abbiamo descritto landamento dellaterosclerosi nel tcs correlata alle dimensioni del vaso ( lunghezza , diametri ) mediante angiografia coronarica con tc a 64 strati e valutato le caratteristiche delle placche aterosclerotiche presenti con un metodo semiquantitativo . 
un recente studio ha dimostrato che le misure dei diametri prossimali delle arterie coronarie prossimali in tcms mostrano unottima correlazione con le misurazioni condotte durante icus ed acc [ 20 ]  . 
 [ 21 ] hanno recentemente confermato che la ac - tcms con 64 strati raggiunge risultati incoraggianti nellidentificazione e caratterizzazione di placche posizionate nei segmenti prossimali [ 21 ]  . 
tale distribuzione potrebbe 366 radiol med ( 2009 ) 113 : 358369 markers ( c - reactive protein , fibrinogen , interleukin - 6 , tumour necrosis factor - alpha )  . 
clearly , with hindsight , the clinical presentation mirrors the plaque typology present [ 9 , 15 ]  . one study reported that the rupture of coronary plaques occurs most frequently in the distal tract of the lm near the bifurcation [ 22 ]  . 
low - oscillation shear stress is known to induce a loss of flow - dependent endothelial dilation , an increase in the expression of adhesion molecules and a weakening of intercellular junctions , which in the end cause an increase in permeability to lipids and macrophages [ 23 ]  . 
due to the turbulence of highvelocity blood flow , which impacts the anatomical essere determinata da fattori anatomici ( lunghezza del tcs , diametri allostio ed alla biforcazione ) , che influenzano lo shear stress , dal profilo lipidico e probabilmente dai marker dellinfiammazione ( proteina c reattiva , fibrinogeno , interleuchina 6 , tnf - )  . 
certamente , a posteriori , la presentazione clinica riflette la tipologia di placche presenti [ 9 , 15 ]  . stato riportato che la rottura delle placche coronariche avviene pi frequentemente nel tratto distale del tcs in prossimit della biforcazione [ 22 ] nella nostra popolazione la maggior parte dei pazienti mostrava placche in questa area , con composizione non calcifica ed eccentricit prevalente . 
lo shear stress a bassa oscillazione conosciuto indurre una perdita del fisiologico allineamento flusso - orientato delle cellule endoteliali , un aumento dellespressione di molecole di adesione ed un indebolimento delle giunzioni intercellulari , che determinano , fig . 
4a - d forma e composizione di placche aterosclerotiche del tcs : placca eccentrica mista ( a ) ; placca non calcifica eccentrica alla biforcazione ( b ) ; placca ostiale calcifica ( c ) ; placca non calcifica alla biforcazione ( d )  . radiol med ( 2009 ) 113 : 358369 structures [ 24 ] , the proximal segments could be more exposed to shear stress [ 25 ]  . 
clearly , a parallel measurement of the shear stress and plaque composition along the vessels needs to be carried out in vivo to confirm these hypotheses . in our study , the plaques proved to be prevalently eccentric , particularly at the bifurcation , confirming the data in the literature [ 9 ]  . although coronary artery bypass grafting surgery is still the recommended first - choice treatment for significant lm stenosis ( 50% of the lumen ) , the introduction of drug - eluting stents with relatively low rates of in - stent restenosis has encouraged the alternative choice of angioplasty with stenting of the lm [ 26 ]  . 
the description of the progression of atherosclerosis in the lm and at the bifurcation is therefore of primary importance in the planning of stent deployment in that it can provide information on the type of stent to be used as well as the precise extent and the placement technique [ 27 ]  . 
i segmenti prossimali potrebbero a causa della turbolenza del flusso di sangue ad alta velocit che impatta contro le strutture anatomiche [ 24 ] essere pi esposti allo shear stress [ 25 ]  . 
nel nostro lavoro le placche risultano prevalentemente eccentriche , in particolare alla biforcazione , confermando il dato presente in letteratura [ 9 ]  . nonostante gli interventi di by - pass aorto - coronarico siano ancora lopzione terapeutica raccomandata per le stenosi significative del tcs ( 50% del lume ) , lintroduzione degli stent medicati ( drug - eluting stents - des ) con tassi di restenosi piuttosto bassi ha incoraggiato la scelta alternativa dellangioplastica del tcs con posizionamento di stent [ 26 ]  . la descrizione dellandamento dellaterosclerosi nel tcs ed alla biforcazione risulta , quindi , di primaria importanza nel planning di uno stent , nella misura in cui fornisce indicazioni sul tipo di stent da utilizzare , sulla sua precisa estensione e sulla tecnica di posizionamento [ 27 ]  . 
la ac - tcms ha raggiunto , allo stesso tempo , notevoli risultati nella valutazione degli stent del tcs nellescludere la restenosi intra - stent , come alternativa di primo livello alla cca [ 28 ]  . esistono , tuttavia , alcune limitazioni nel nostro studio . in primo luogo non stata effettuata una correlazione icus , ma solamente un riscontro acc , che conferma la bassa prevalenza di malattia significativa nel tcs [ 29 ]  . 
allo stato attuale , non vi ancora accordo circa le reali potenzialit della ac - tcms nello stimare le dimensioni delle placche , dal momento che le misurazioni sono senza dubbio influenzate da artefatti da volume parziale e movimento [ 10 ]  . 
la ricerca di un protocollo desame ottimizzato per la valutazione della placca e lo sviluppo di un software , che consenta alloperatore unaffidabile e riproducibile caratterizzazione , potrebbero essere valide soluzioni a queste problematiche . 
una questione da sollevare relativa a tutti gli attuali studi ac - tcms la significativa esposizione del paziente a radiazioni ionizzanti ( 914 msv ) , che ne deve obbligatoriamente limitare luso nei pazienti asintomatici ed in particolare nelle donne al di sotto dei 40 anni [ 30 ]  . conclusions conclusioni plaques in the lm have a specific appearance according to their anatomical location . 
in particolare , la ac - tcms fornisce dettagli anatomici del tcs ed informazioni morfologiche rilevanti circa lestensione dellaterosclerosi coronarica , dal momento che la rottura e 368 radiol med ( 2009 ) 113 : 358369 and consequent thrombosis of vulnerable plaques can develop from lesions characterised as nonsignificant at conventional ca . 
 + 39 - 081 - 5665201 , fax : + 39 - 081 - 5665201 , e - mail : salvatore.cappabianca@unina2.it received : 11 april 2008 / accepted : 28 july 2008 / published online : 11 march 2009 springer - verlag 2009 abstract purpose . 
plain chest radiographs and hrct scans of 42 consecutive patients with ntm pulmonary infection ( m / f 26 / 16 ; mean age 57 , range 4183 ) were retrospectively reviewed . 
abbiamo valutato gli esami rx / tc a strato sottile ( hrct spessore 1 mm / interslice 10 mm ) relativi a 42 casi consecutivi di infezione polmonare da ntmb , insorta in pazienti immunocompetenti ( m / f , 26 / 16 ; et media 57 anni ; range 4183 ) ; abbiamo eseguito il follow - up in 10 casi entro 18 mesi dalla diagnosi . 
la micronodulia ( < 10 mm ) pi frequente della macronodulia , la consolidazione segmentaria della lobare ; le cavitazioni , il tree - in - bud , le bronchiectasie sono pi frequenti nei lobi superiori . 
lhrct consente la corretta identificazione ed il monitoraggio a distanza del pattern che pi frequentemente si riscontra nella localizzazione polmonare dellinfezione : micronodulia diffusa , bronchiectasie , consolidazione segmentaria lobare superiore , cavit . 
although humans are exposed daily to the microorganisms , the incidence of ntm pulmonary infection is low , as the bacteria tend to colonise rather than infect their host [ 7 ]  . 
it is not completely clear whether ntm infection is responsible for the clinical presentation at disease onset , or whether this is a late manifestation of the infection . the most common pathogens causing ntm pulmonary infection are intracellular mycobacterium avium [ no longer distinguished from m . 
predisposing factors for ntm infection include interferon - gamma - deficiency cystic fibrosis , alcoholism , chronic obstructive pulmonary disease ( copd ) , alpha - 1 antitproteinase gene defects , pathological thinness , prior surgery ( especially gastrectomy ) , oesophageal disease and rib - cage anomalies ( pectus excavatum and kyphoscoliosis ) [ 13 ]  . 
the recent increase in the prevalence of ntm infections may therefore be correlated with the reduced incidence of tuberculosis ( tb ) in the general population in industrialised areas [ 9 ]  . 
fever , night sweats , weight loss , lethargy and symptoms related to chronic bronchopulmonary disease are more common in advanced stages of disease , thus making the recognition of the symptoms difficult in patients with underlying pulmonary disease [ 911 ]  . i micobatteri atipici ( ntmb ) sono microrganismi ubiquitari comunemente rinvenuti nellambiente e riconosciuti sempre pi frequentemente come causa di infezione cronica polmonare in soggetti immunocompetenti [ 15 ]  . 
linfezione da ntmb rappresenta lo 0 , 5%30% di tutte le infezioni micobatteriacee e , sebbene luomo sia quotidianamente esposto a tali microrganismi , lincidenza di infezioni polmonari da ntmb bassa perch essi colonizzano pi che infettare lospite [ 7 ]  . linfezione pu essere acquisita per inalazione , ingestione ed inoculazione diretta ; la trasmissione interumana rara [ 8 ]  . 
non del tutto chiaro se linfezione da ntmb sia responsabile di quadri patologici respiratori ab initio , ovvero se questi rappresentino una manifestazione tardiva della stessa . i patogeni pi frequentemente riscontrati come causa di infezioni polmonari micobatteriacee non tubercolari sono m . 
deficit di inf , fibrosi cistica , alcolismo , patologie croniche ostruttive polmonari , difetti genici dell1 - antiproteinasi , magrezza patologica , interventi chirurgici ( in particolare gastrectomia ) , patologie esofagee ed anomalie della gabbia toracica ( pectus excavatum e cifoscoliosi ) possono costituire fattori predisponenti alle infezioni da ntmb [ 13 ]  . 
lesposizione al bacillo tubercolare ed il vaccino antitubercolare con bcg possono dare unimmunit crociata con i micobatteri ; il recente incremento della prevalenza di tali infezioni potrebbe correlare , dunque , con la riduzione dellincidenza di tubercolosi nella popolazione generale delle aree industrializzate [ 9 ]  . 
febbre , sudorazione notturna , perdita di peso , letargia , sintomi spesso legati a broncopneumopatie croniche , sono pi comuni nelle fasi avanzate di malattia rendendo difficile , pertanto , il riconoscimento dei sintomi legati alle micobatteriosi in pazienti con patologia polmonare soggiacente [ 911 ]  . immunocompetent patients affected by mycobacterial i pazienti immunocompetenti affetti dalle micobatteriosi 378 radiol med ( 2009 ) 114 : 376389 infection can be divided into two groups : ( 1 ) adult males with preexisting chronic pulmonary disease such as copd and fibrosis [ 12 , 1416 ] and ( 2 ) young females without evident pulmonary disease or concomitant systemic disease [ 1416 ]  . 
nel primo gruppo la patologia tende a presentarsi in forma classica , ovvero in maniera quasi indistinguibile dalla forma attiva della tubercolosi ; nel secondo , invece , si riscontra , nella maggior parte dei casi , una forma non classica . 
 scopo del nostro lavoro stato quello di definire , analizzandoli retrospettivamente , i reperti hrct pi frequenti e significativi per la diagnosi di micobatteriosi polmonare atipica , correlando il dato tc con i reperti radiografici e levoluzione clinica della malattia . materials and methods we retrospectively reviewed the radiographic and hrct examinations performed between january 2004 and december 2007 on 42 immunocompetent patients ( m / f 26 / 16 , age range 4183 years ) with a clinical and radiographic suspicion of pulmonary ntm infection that was subsequently confirmed by using the american thoracic society 1997 criteria ( table 1 ) and 2007 criteria for the patients examined in the final year ( table 2 ) [ 11 , 19 ]  . the study included patients who underwent not only clinical and laboratory diagnosis of ntm infection but also radiography and hrct examinations to locate the pulmonary infection . 
nine additional patients were excluded due to bronchogenic carcinoma ( n = 6 ) , sarcoidosis ( n = 1 ) and diffuse interstitial pneumonia ( n = 2 ) because the hrct findings associated with these diseases may coincide and mask the signs of ntm pulmonary infection . 
the terminology recommended by the fleischner society [ 20 , 21 ] was used to define the hrct findings . radiographic and hrct examinations were separately evaluated by two groups of radiologists with equal experience and blinded to each others conclusions . 
radiographic findings correlated with the clinical findings leading to a suspicion of ntm infection included : cavitations , areas of consolidation , diffuse nodular opacities , fibrosis associated materiali e metodi abbiamo valutato retrospettivamente gli esami radiografici e di tc ad alta risoluzione ( hrct ) eseguiti nel periodo gennaio 2004dicembre 2007 relativi a 42 pazienti immunocompetenti ( m / f : 26 / 16 ; range di et 4183 anni ) con sospetto diagnostico clinico e radiografico di micobatteriosi atipica polmonare , successivamente accertato mediante i criteri dellamerican thoracic society del 1997 ( tabella 1 ) e del 2007 per i pazienti esaminati nellultimo anno ( tabella 2 ) [ 11 , 19 ]  . 
 nel nostro studio sono stati inclusi pazienti in cui oltre alla diagnosi clinico - laboratoristica di infezione da ntmb erano stati eseguiti , per dimostrare la localizzazione polmonare dellinfezione , esami radiografici del torace e studi hrct condotti con lutilizzo dei seguenti parametri : slice thickness 1 mm , avanzamento 10 mm e filtro di visualizzazione per osso . 
abbiamo escluso dal protocollo di valutazione tutti i pazienti con hiv , ipogammaglobulinemia , leucemia e quelli in terapia steroidea , in quanto deficit immunitari possono modificare notevolmente limaging delle infezioni polmonari da ntmb ; abbiamo inoltre escluso 9 pazienti perch affetti da carcinoma broncogeno ( n = 6 ) , sarcoidosi ( n = 1 ) , polmonite interstiziale diffusa ( n = 2 ) , poich i segni tc associati a tali malattie possono sovrapporsi ed in parte mascherare i reperti legati a polmonite da ntmb . 
nella definizione dei reperti tc stata impiegata la terminologia raccomandata dalla fleischner society [ 20 , 21 ]  . gli esami radiografici e di hrct sono stati valutati separatamente in cieco da due gruppi di radiologi di pari esperienza . 
allanalisi degli esami rx gli elementi semeiologici , che uniti al quadro clinico , hanno indotto al sospetto di infezione da ntmb sono stati : cavitazioni , aree consolidative , opacit nodulari diffuse , fibrosi associata a perdita di radiol med ( 2009 ) 114 : 376389 table 1 diagnostic criteria 1997 modified for immunocompetent patients only american thoracic society a . 
compatible signs / symptoms ( cough ; fatigue most common ; fever ; weight loss ; hemoptysis ; dyspnea may be present , particularly in advanced disease ) with documented deterioration in clinical status if an underlying condition is present b . 
at least three available sputum / bronchial wash samples within 1 year three positive cultures with negative afb smears , or two positive cultures and one positive afb smear b . 
single available bronchial wash and inability to obtain sputum samples positive culture with 2 + , 3 + , or 4 + growth , or positive culture with a 2 + , 3 + , or 4 + afb smear c . 
tissue biopsy any growth bronchopulmonary tissue biopsy granuloma and / or afb on lung biopsy with one or more positive cultures from sputum / bronchial wash any growth from usually sterile extrapulmonary site for a diagnosis of pulmonary disease , all three criteria : ( 1 ) clinical , ( 2 ) radiographic and ( 3 ) bacteriologic must be satisfied hrct , high - resolution computed tomography ; afb , acid - fast bacillus with volume loss , focal pleural thickening and pleural effusion . 
 hrct findings suggestive of ntm infection included : small nodules ( < 10 mm ) , nodules ( 1030 mm ) , tree - in - bud sign , parenchymal consolidation , cavitation and bronchiectasis . 
 alla tc sono stati ricercati , quali segni suggestivi di infezione da ntmb : micronoduli ( meno di 10 mm di diametro ) ; macronoduli ( diametro compreso tra 1030 mm ) ; tree - in - bud pattern ; consolidazioni parenchimali ; cavitazioni ; bronchiectasie . 
tutti i reperti patologici sono stati categorizzati impiegando precostituiti modelli sinottici al fine di ottenere la maggiore sovrapponibilit delle interpretazioni dei due gruppi di osservatori ; le differenze interpretative sono state risolte in consenso . 
nella definizione di sede delle lesioni abbiamo tenuto in considerazione la suddivisione in lobi dei polmoni , assumendo la lingula come 380 radiol med ( 2009 ) 114 : 376389 tabella 1 criteri diagnostici sec . 
segni / sintomi compatibili ( tosse , astenia , febbre , perdita di peso , emottisi , dispnea possono essere presenti , specie nella malattia in fase avanzata ) con un documentato deterioramento dello stato clinico se presente una condizione sottostante b . 
qualunque dei seguenti reperti allrx del torace ; se precedenti ad un anno , dovrebbe esserci evidenza di progressione infiltrati con o senza noduli ( persistent da pi di 2 mesi o progressivi ) cavitazioni solo noduli ( multipli ) b . 
almeno 3 campioni di espettorato disponibili / campioni di lavaggio bronchiale in un anno tre colture positive con striscio afb negativo o due colture positive ed uno striscio afb positivo oppure b . 
singolo lavaggio bronchiale disponibile ed impossibilit di ottenere campioni di espettorato coltura positiva con crescita 2 + , 3 + , o 4 + oppure coltura positive con striscio afb 2 + , 3 + , o 4 + oppure c . 
biopsia tissutale crescita dal tessuto bioptico broncopolmonare granuloma e / o afb su biopsia polmonare con uno o pi colture positive su espettorato / lavaggio bronchiale crescita da sedi extrapolmonari di solito sterili per la diagnosi di malattia polmonare , tutti e tre i criteri ( 1 ) clinico , ( 2 ) radiografico e ( 3 ) batteriologico devono essere soddisfatti hrtc , tc ad alta risoluzione ; afb , acid - fast bacillus months from diagnosis was possible in ten of the 42 patients after adequate medical treatment . 
in 2 dei 10 pazienti seguiti nel tempo , stato possibile correlare il dato radiologico con quello anatomo - patologico su campioni di lobectomia . among the 42 patients , 23 were affected by m . 
the most common clinical presentations were dyspnoea ( 50% ) , cough ( 46.4% ) , fever ( 42.8% ) risultati dei 42 pazienti inclusi nel nostro studio , 23 erano affetti da infezione da m . 
transbronchial or other lung biopsy with mycobacterial histopathologic features ( granulomatous inflammation or afb ) and positive culture for ntm or biopsy showing mycobacterial histopathologic features ( granulomatous inflammation or afb ) and one or more sputum or bronchial washings that are culture positive for ntm 4 . 
making the diagnosis of ntm lung disease does not , per se , necessitate the institution of therapy , which is a decision based on environmental contamination is firmly established or excluded potential risks and benefits of therapy for individual patients afb , acid - fast bacillus ; ntm , nontuberculous mycobacteria 1 . 
sintomi polmonari , opacit nodular o cavitazioni allrx del torace , o una hrct che mostri bronchiectasie multifocali con multipli tabella 2 criteri diagnostici ats 2007 clinici ( entrambi richiesti ) micronoduli 2 . 
biopsia transbronchiale o polmonare con caratteristiche istologiche da micobatteriosi ( infiammazione granulomata o afb ) e colture positive per ntmb o biopsie che mostrino caratteristiche istologiche da micobatteriosi ( infiammazione granulomata o afb ) ed uno o pi colture di espettorato o lavaggio bronchiale positive per ntmb 4 . 
il quadro clinico di presentazione pi frequentemente riscontrato era costituito da dispnea ( 50% ) , tosse ( 46 , 4% ) , febbre ( 42 , 8% ) ed espettorato ( 39 , 3% ) ; meno spesso emoftoe ( 28 , 6% ) , astenia ( 17 , 8% ) , dimagrimento ( 17 , 8% ) e dolore toracico ( 10 , 7% )  . 
the tree - inbud sign was present in 27 patients ( 40.5% ) , with location in the upper right lobe in seven patients ( 41.2% ) and in the middle lobe and the upper left lobe in five patients ( 29.4% ) ( table 3 )  . all patients were treated with combination therapy consisting of daily isoniazid , rifampin and ethambutol in the m . 
kansasii and 12 months of mac onset . conventional radiology was performed every 6 months , whereas hrct was carried out only after completion of ( 21 , 4% ) , gastroresezione ( 23 , 8% ) e patologia esofagea ( 11 , 9% ) erano quelle maggiormente riscontrate nel nostro gruppo di pazienti . 
3 parenchymal consolidation of the posterior segment of the right upper lobe with thick - walled cavitation associated with extensive bronchiectasis and scattered nodulation in both upper lobes in a patient with m . 
3 consolidazione parenchimale del segmento posteriore del lobo superiore destro con cavitazione a pareti spesse associati ad estesi fenomeni bronchectasici e nodulazioni sparse in entrambi i lobi superiori in paziente con infezione da m . 
due pazienti mostravano un quadro hrct sostanzialmente invariato ed in altri 2 si avuta resistenza alla terapia medica con progressione della patologia dimostrata dallaumento di estensione delle lesioni pre - esistenti e / o dalla comparsa di nuove lesioni cavitate , bronchiectasie , tree - in - bud e micronoduli , reperti che hanno reso necessario il ricorso alla terapia chirurgica . 
in two others , the infection was refractory to medical treatment , with disease progression demonstrated by the increase in the extension of preexisting lesions and / or the appearance of new cavitations , bronchial dilatation , tree - in - bud and small nodules . 
these findings led to surgical treatment , and gross pathology on the resected specimens showed a close correlation with the radiological findings . discussion the insidious nature of ntm pulmonary infections has been underlined in many studies . 
the symptoms can be underestimated for months or even years prior to diagnosis , especially in patients with preexisting pulmonary disease [ 9 ]  . dyspnoea , cough , expectoration and fever were the most common signs and symptoms in our patient population . 
the nonspecific nature of these signs and symptoms , which are often present in advanced chronic bronchopulmonary disease , confirm the diagnostic difficulties that can be nei pazienti con patologie polmonari preesistenti [ 9 ]  . dispnea , tosse , espettorato e febbre , sintomi e segni aspecifici , sono stati i pi frequenti nellambito del gruppo di pazienti selezionati per il nostro studio ; la aspecificit di tali sintomi e segni , che spesso sono presenti nelle broncopneumopatie croniche avanzate , confermano le difficolt di inquadramento diagnostico che possono verificarsi in pazienti affetti da polmoniti da ntmb . 
alcuni pazienti mantengono un quadro clinico e radiografico stabile per lungo tempo , mentre altri hanno una rapida progressione della malattia , che senza terapia opportuna , tende a risultare fatale . nella nostra casistica le micobatteriosi atipiche di pi frequente riscontro sono state quelle sostenute da m . kansasii e da mac e , nellambito della coorte di pazienti esaminati , in accordo con i dati presenti in letteratura , abbiamo potuto osservare che il m . 
kansasii colpisce prevalentemente uomini ( m / f = 20 / 3 ) con et media di circa 70 anni , mentre le infezioni da mac si riscontrano prevalentemente in donne ( m / f = 7 / 12 ) con et media di circa 57 anni . i pazienti immunocompetenti possono essere sostanzialmente ricondotti a due grossi gruppi : quello dei maschi anziani con patologia polmonare cronica e quello delle donne di et media senza storia di patologia polmonare . radiol med ( 2009 ) 114 : 376389 fig . 
b al controllo hrct a distanza di un anno , dopo terapia medica mirata , le bronchiectasie risultano aumentate di numero e dimensioni . encountered in patients with ntm pulmonary infections . the natural history of the infection is unpredictable in immunocompetent patients . 
some patients maintain stable clinical and radiological presentation over a long period , whereas others face rapid clinical progression , which may be fatal without appropriate treatment . the most frequent ntm in our patient population were m . 
in agreement with the literature , m . kansasii infections were prevalent among males ( m / f 20 / 3 ) with a mean age approximately 70 years , whereas mac infections were found mostly among women ( m / f 7 / 12 ) with a mean age of approximately 57 years . 
immunocompetent patients can be divided into two broad groups : elderly men with chronic pulmonary disease , and middle - aged women with no history of pulmonary disease . radiological patterns of ntm infection are not mycobacterium specific , and our findings , in agreement with the recent literature , demonstrated two different radiological presentations : a classic and a nonclassic for the classic form typical of m . 
kansasii infection and less commonly associated with mac infections manifests with areas of lobar or segmental consolidation , commonly situated in the upper lobes , whereas involvement of the i quadri radiologici in caso di micobatteriosi atipica non sono micobatterio specifici e la nostra esperienza , in accordo a quanto riportato nella letteratura recente , ci ha consentito di verificare che esistono due differenti tipi di presentazione radiologica dinfezione : una forma classica ed una non classica . 
kansasii , meno spesso legata ad infezione da mac , si manifesta di solito con aree di consolidazione lobare o segmentaria , pi frequentemente localizzate ai lobi superiori , mentre il coinvolgimento dei lobi inferiori pi raro . 
le caverne , reperto comune alla tubercolosi , sono eterogenee per dimensioni e spessore di parete ; queste caratteristiche non permettono di differenziare una tbc da uninfezione da ntmb , ma correlano semplicemente con i vari stadi evolutivi di malattia . 
la forma non classica , conosciuta anche come lady windermere syndrome , ha delle caratteristiche peculiari consistenti in bronchiectasie cilindriche 386 radiol med ( 2009 ) 114 : 376389 lower lobes is rarer . 
these features do not allow differentiation between tb and ntm infection ; rather , they are simply correlated with the various clinical stages of the disease . apical pleural thickening is another relatively common finding in ntm infections with a classic presentation and is often associated with progressive fibrosis and loss of parenchymal volume . 
the nonclassic form , also known as lady windermere syndrome , has atypical features consisting of mild to moderate cylindrical bronchiectasis and small nodules situated in the middle lobe and the lingula [ 22 , 23 ] , i.e. 
in our patient population , we included these two forms in the two broad categories described above [ 8 ]  . chest radiography of patients with ntm infections only provided information on the presence or absence of cavitations , consolidation , nodules , pleural thickening and volume loss . 
kansasii ( 100% ) predominantly in the upper lobes ( 30%39% ) and in patients with mac infection ( 52.6% ) predominantly in the middle lobe ( 70% ) and the lingula ( 50% ) [ 2325 ]  . 
a similar distribution was also observed for segmental consolidation . nodules the least common finding ( 45.2% ) were encountered more frequently in the upper lobes in m . kansasii infections ( 60% ) and in the middle lobe and lingula in mac infections ( 40% )  . 
lastly , the most common hrct findings in patients with mac infection were bronchiectasis involving the middle lobe and lingula , nodules no larger than 1 cm and focal consolidation [ 26 , 27 ]  . 
patients with preexisting pulmonary disease and mac infection very often present cavitary lesions . medio - moderate e micronoduli localizzati al lobo medio ed alla lingula [ 22 , 23 ] , sedi nelle quali le caverne , le consolidazioni e la perdita di volume sono molto meno comuni [ 23 ]  . 
in letteratura sono state descritte anche altre due forme di infezione polmonare , quella nodulare in pazienti asintomatici e quella consolidativa in pazienti con acalasia , che , nei nostri raggruppamenti , abbiamo fatto rientrare nelle due grandi categorie di pazienti appena descritte [ 8 ]  . lesame radiografico standard del torace in pazienti con infezioni da ntmb ci ha consentito solo di ottenere informazioni circa la presenza o meno di caverne , consolidazioni , macronoduli , ispessimenti pleurici e perdita di volume ; il ricorso alla hrct si reso necessario per una corretta valutazione dellinteressamento polmonare sia prima che dopo terapia . 
allesame tc la micronodulia ( 78 , 6% ) , le cavit ( 71 , 4% ) , le bronchiectasie ( 66 , 6% ) e la consolidazione segmentaria ( 66 , 6% ) sono stati gli aspetti maggiormente riscontrati alla diagnosi . 
kansasii ( 100% ) maggiormente ai lobi superiori ( 30%39% ) e nei pazienti con infezione da mac ( 52 , 6% ) maggiormente a livello del lobo medio ( 70% ) e della lingula ( 50% ) [ 2325 ]  . 
kansasii a livello del lobo superiore destro ( 43% ) , si localizzavano , nellinfezione da mac , al lobo superiore destro ( 33 , 3% ) , al lobo medio ( 33 , 3% ) nonch ai lobi inferiori ( 16 , 7% )  . 
in ultima analisi , nellinfezione da mac i reperti pi frequentemente riscontrati alla tc consistevano in bronchiectasie estese pi spesso al lobo medio ed alla lingula , noduli di diametro massimo di 1 cm e pochi piccoli foci di consolidazione [ 26 , 27 ]  . 
 in letteratura si tentato in numerose serie di trovare una correlazione tra tipo di micobatterio e spessore delle pareti delle caverne ; le caverne correlate ad infezione da mac tendono ad essere pi sottili e con meno opacizzazione parenchimale circostante [ 11 ]  . 
kansasii , sebbene il radiol med ( 2009 ) 114 : 376389 numerous studies in the literature have attempted to find a correlation between type of mycobacteria and the thickness of the cavitation walls . 
kansasii infections , although the most common radiological presentation is cavitations and small nodules in the upper lobes . the use of hrct , with its ability to demonstrate bronchiectasis and small nodules , and the knowledge that the infection tends to have a chronic course and slow progression have shown the importance of treating these patients with medical therapy [ 9 ] and performing adequate follow - up of the parenchymal lesions . 
over the years , there have been various studies aimed at experimenting new and prevalently multidrug protocols , which have had varying success ( 60%80% ) in the different series [ 28 ]  . our follow - up data show that bronchi affected by preexisting bronchiectasis tend to increase in size , and new areas of dilatation can form over time . 
some nodules can undergo cavitation , whereas other smaller nodules may disappear with appropriate therapy . surgical excision should be taken into consideration in patients with localised disease , especially if refractory to medical therapy [ 9 ]  . 
the histological findings , correlated with the corresponding radiological findings , showed that the centrilobular nodules correspond to granulomas and caseous material at the level of the terminal or respiratory bronchioles . 
nodules > 10 mm and lobar consolidation correspond to areas of central caseous granulomas , associated with nonspecific marginal inflammation and lymphocyte infiltration , which completely replaces the alveolar spaces . 
the wall of the cavitations consists of caseous material , epithelioid cells , multinucleated giant cells , granulomatous tissue and a fibrotic capsule . conclusions in agreement with the literature , our findings demonstrate that chest radiography exclusively has a first - line role in the quadro di presentazione pi frequentemente evidenziato consista in cavit e micronoduli ai lobi superiori . 
 con lutilizzo dellhrct , capace di dimostrare bronchiectasie e micronodulazioni , e con la consapevolezza che linfezione tende ad avere decorso cronico e lenta progressione , si dimostrata limportanza di trattare con terapia medica questi pazienti [ 9 ] e di seguire con adeguati followup levoluzione delle lesioni parenchimali . 
negli anni si sono susseguiti studi volti alla sperimentazione di nuovi protocolli , prevalentemente di tipo multifarmaco , che hanno portato al successo terapeutico in percentuali variabili ( 60%80% ) nelle differenti serie [ 28 ]  . i dati in nostro possesso , relativi ai controlli a distanza , hanno permesso di dimostrare che le bronchiectasie preesistenti tendono ad aumentare di dimensioni , mentre nuove aree bronchiectasiche possono formarsi nel tempo . 
la tc dimostra anche che , con il passare del tempo , la diffusione dei noduli si verifica in altri segmenti ; alcuni macronoduli possono escavarsi , mentre alcuni noduli di piccole dimensioni possono , con una valida terapia , scomparire . lexeresi chirurgica deve essere presa in considerazione in pazienti con patologia localizzata , specie se resistenti alla terapia medica [ 9 ] ; tuttavia localizzazioni bilaterali , et avanzata e concomitanti patologie croniche limitano il numero di candidati alla chirurgia [ 11 , 2830 ]  . 
dei nostri pazienti , infatti , soltanto 2 sono stati sottoposti ad intervento chirurgico di lobectomia ed i dati istologici , correlati con i corrispondenti reperti radiologici , hanno dimostrato che i noduli centrolobulari corrispondono a granulomi e materiale caseoso a livello dei bronchioli terminali o respiratori ; i noduli con diametro maggiore di 10 mm e la consolidazione lobare corrispondono ad aree di granulomi caseosi centrali , associati ad infiammazione aspecifica marginale ed infiltrato linfocitario che rimpiazza completamente gli spazi alveolari ; la parete delle caverne costituita da materiale caseoso , cellule epitelioidi , cellule giganti multinucleate , tessuto di granulazione e capsula fibrotica . 
 conclusioni i nostri dati , in accordo con quanto riportato dalla letteratura , dimostrano ampiamente come allo stato attuale lesame radiografico del torace abbia un ruolo esclusivamente di primo livello nelliter diagnostico delle infezioni polmonari da ntmb . 
sicuramente la tc ed in particolare la hrct hanno aperto nuovi scenari nella diagnostica per immagini delle infezioni polmonari da ntmb , permettendo lidentificazione di specifici pattern che spesso , ma non costantemente , 388 radiol med ( 2009 ) 114 : 376389 diagnostic work - up of ntm pulmonary infections . 
ct , and especially hrct , have opened up new horizons in the diagnostic imaging of ntm pulmonary infections by allowing the detection of specific patterns that are often though not always correlated with the different stages of disease and reliable indicators of the efficiency and effectiveness of treatment . 
only with the correct use of hrct can the sign be identified and correlated with other equally important findings to define the stage of the disease . currently , there is limited knowledge and awareness of this diagnostic possibility . 
the incidence of disease might therefore be much higher than reported in the literature , given the nonspecific nature of the clinical symptoms and the similar presentation of preexisting pulmonary disease and ntm infection at conventional radiography . possono essere correlabili alle differenti fasi evolutive della malattia ed essere indicatori attendibili della efficacia / efficienza delle terapie poste in atto . 
scarabino , via napoli 56 , 70031 andria ( ba ) , tel . : + 39 - 088 - 3299140 , fax : + 39 - 088 - 3299220 , e - mail : tscarabino@hotmail.com received : 29 february 2008 / accepted : 8 april 2008 / published online : 12 december 2008 springer - verlag 2009 abstract the objective of this study was to evaluate the potential role of newly developed , advanced magnetic resonance ( mr ) imaging techniques ( spectroscopy , diffusion and perfusion imaging ) in diagnosing brain gliomas , with special reference to histological typing and grading , treatment planning and posttreatment follow - up . 
these limitations can be overcome by supplementing the morphological data obtained with conventional mr imaging with the metabolic , structural and perfusional information provided by new mr techniques that are increasingly becoming an integral part of routine mr studies . 
in addition , the recent development of new , more effective , treatments for cerebral glioma strongly relies on morphofunctional mr imaging with its ability to provide a biological interpretation of these characteristically heterogeneous tumours . riassunto lo scopo del lavoro di illustrare le potenzialit delle nuove e pi avanzate modalit di studio rm ( spettroscopia , diffusione , perfusione ) nella diagnostica dei gliomi cerebrali , con particolare riferimento alla definizione dellistotipo e del grading , alla pianificazione del trattamento e al follow - up post - trattamento . 
necessario pertanto integrare le informazioni fornite dalla rm di base con le informazioni di carattere metabolico , strutturale ed emodinamico fornite dalle pi recenti tecniche rm , oramai parte integrante di uno studio di routine . 
the gold standard for establishing brain tumour histological type and grade is histopathology of tissue specimens obtained with stereotactic or open biopsy , both of which are invasive procedures [ 2 ]  . 
there is thus a need to use noninvasive modalities capable of providing a diagnosis closely corresponding to the histological diagnosis but without risks for the patient and affording a more global view not restricted to the surgical specimen . in this context , a key role may be played by magnetic resonance ( mr ) imaging supplemented by techniques that provide anatomicalpathological information of considerable diagnostic and clinical utility . 
under ideal conditions , conventional mr imaging with its multiplanar capabilities , ability to evaluate multiple parameters , excellent contrast resolution and lack of invasiveness helps to detect the lesion , identify its intra - axial site , define its precise location and hypothesise its nature . 
however , it has limited sensitivity and specificity in defining glioma type and grade [ 4 ] and does not always permit precise delineation of tumour margins or tumour differentiation from oedema and / or treatment effects ( recurrence vs radionecrosis ) [ 5 , 6 ]  . 
 the diagnostic limitations of conventional mr imaging can be overcome by new , advanced mr techniques that give metabolic , structural and perfusional information , thereby providing the surgeon with increasingly precise and comprehensive diagnoses . 
the aim of this paper is to illustrate the potential of the new mr techniques , such as spectroscopy , diffusion and perfusion imaging , in diagnosing brain gliomas , with special reference to histological typing and grading , treatment planning and posttreatment monitoring . 
the paper reviews data from the literature and the authors personal experience . i gliomi costituiscono circa il 70% dei tumori primitivi maligni cerebrali , e sono caratterizzati da una notevole eterogeneit nellaspetto neuropatologico , nellespressione genica e nella prognosi [ 1 ]  . 
una diagnosi precoce e soprattutto una approfondita valutazione della reale estensione del tumore e dei rapporti con le strutture anatomiche circostanti sono elementi importanti ai fini della prognosi e dellimpostazione delle diverse strategie terapeutiche . 
lesame gold standard per la diagnosi di tipo e grado di tumore cerebrale lo studio isto - patologico su tessuto prelevato attraverso stereotassi o resezione chirurgica , metodi entrambi invasivi [ 2 ]  . 
da qui la necessit di utilizzare modalit di studio non invasive che possano condurre ad una diagnosi il pi vicino possibile a quella istologica , senza rischi per il paziente e con un punto di vista pi ampio , non limitato al solo pezzo anatomico prelevato . in questa prospettiva si inserisce perfettamente la risonanza magnetica ( rm ) , corredata da una serie di metodiche complementari allimaging di base che , nellinsieme , offrono informazioni anatomo - patologiche di grandissima utilit diagnostica e clinica . 
in condizioni ideali , infatti , in virt di una serie di vantaggi , quali la multiplanariet , la multiparametricit , lottima risoluzione di contrasto , lassenza di invasivit , la rm di base consente di identificare la lesione , stabilirne la sede intrassiale , definirne una precisa localizzazione e proporre unipotesi di natura . 
inoltre la rm di base non sempre permette una precisa definizione dei margini della neoplasia n consente di distinguere con certezza il tumore dalledema e / o dagli effetti del trattamento ( recidiva vs radionecrosi ) [ 5 , 6 ]  . 
 le nuove ed avanzate modalit di studio rm , grazie alle informazioni metaboliche , strutturali ed emodinamiche che riescono a fornire , riducono i limiti diagnostici della sola rm di base permettendo diagnosi sempre pi precise ed esaustive per il chirurgo . 
lo scopo del lavoro di illustrare le potenzialit delle nuove e pi avanzate modalit di studio rm , quali la spettroscopia , la diffusione e la perfusione , nella diagnostica dei gliomi cerebrali con particolare riferimento alla definizione dellistotipo e del grading , alla pianificazione del trattamento e al follow - up post - trattamento . 
verranno presi in considerazione i dati della letteratura integrati con i risultati della nostra esperienza . 450 spectroscopy proton mr spectroscopy ( 1h - mrs ) is a noninvasive technique that provides metabolic information about normal and pathological tissue components [ 79 ]  . 
the physical principle underlying 1h - mrs is the chemical shift that varies in relation to differences in precession frequency of atoms belonging to different molecules ( metabolites )  . 
 the most commonly used metabolites in diagnosing gliomas are choline ( cho ) , creatine ( cr ) , n - acetyl - aspartate ( naa ) , lactate ( lac ) and lipid ( lip )  . 
il principio fisico sfruttato dalla 1h - mrs il chemical shift che varia in rapporto alle differenze di frequenza di precessione di atomi appartenenti a singole molecole ( metaboliti )  . 
 i metaboliti maggiormente coinvolti nella diagnostica dei gliomi sono la colina ( cho ) , la creatinina ( cr ) , ln - acetilaspartato ( naa ) , il lattato ( lac ) e i lipidi ( lip )  . 
combining this information with the morphostructural data from conventional mr imaging leads to a 15% increase in the number of correct diagnoses compared with conventional mr imaging alone , with no increase in incorrect diagnoses [ 10 ]  . 
the major challenge in interpreting the metabolic information of gliomas is related to the extreme heterogeneity among tumours of different grades , tumours of the same grade and even within individual tumours [ 11 ]  . 
lip is also essential for tumour grading , as it reflects tissue necrosis , which in turn is related to tumour grade , thereby permitting differentiation of grade iv from grades ii and iii lesions [ 7 , 9 ]  . 
 from a methodological point of view , several authors have demonstrated the usefulness of employing short echo time ( te ) ( 35 ms ) rather than intermediate te ( 144 ms ) sequences on high - field mr spectroscopy in grading gliomas , despite the different cho / cr and laclip ( ll ) / cr ratios obtained . 
in particular , if a single sequence must be chosen , the short te sequence is preferable because of the poor lactate inversion at intermediate te [ 12 ]  . in a recent study , by exploiting the potential of a highfield magnet , we were able to analyse the spatial distribution ormai noto che la 1h - mrs in grado di fornire informazioni sulle caratteristiche metaboliche dei tumori , ed in particolare dei gliomi , che complementariamente a quelle prettamente morfo - strutturali fornite dal solo imaging conducono ad un incremento di diagnosi corrette pari al 15% rispetto a quanto accade con la sola rm di base , senza alcun incremento di diagnosi non corrette [ 10 ]  . 
la difficolt maggiore nellinterpretazione delle informazioni metaboliche fornite dai glomi legata alla estrema eterogeneit tumorale tra differenti gradi , allinterno dello stesso grado ed anche allinterno del singolo tumore [ 11 ]  . 
la cho ha un ruolo importante in virt della diretta correlazione con la proliferazione cellulare tumorale che comunque non sempre correlata direttamente con il grading ( il caso del meningioma )  . 
anche i lip sono essenziali ai fini delle differenziazione tumorale : essi infatti esprimono la necrosi tissutale che , a sua volta , correlata al grado tumorale , permettendo cos di differenziare lesioni di grado iv da lesioni di ii e iii grado essendo correlati con la necrosi [ 7 , 9 ]  . 
in particular , in high - grade gliomas , cho is reduced in the central necrotic areas , elevated in the tumour mass and margins , reduced in peritumoural oedema and increased to normal levels in the surrounding healthy tissue ; naa is reduced in the central areas , increased in the periphery and normal in the surrounding healthy tissue ; lac - lip are substantially increased in the central necrotic areas and reduced in the tumour margins . 
in low - grade gliomas , cho is usually increased in the central areas , reduced in the margins and slightly increased in the surrounding healthy tissue ; naa is reduced in the mass and tumour margins and normal in the surrounding healthy tissue ; lac - lip are not detectable ( personal communication )  . 
 treatment planning supplementing conventional mr imaging with metabolic imaging improves the definition of tumour margins . conventional mr imaging alone does not allow distinction between infiltrated and noninfiltrated peritumoural oedema . this distinction is made possible by spectral analysis , as infiltrated oedema shows an abnormal cho / naa ratio , whereas the ratio is normal in pure vasogenic oedema . tumour extent can thus be defined beyond the margins detected by conventional imaging ( beyond the borders of contrast enhancement or peritumoural t2 hyperintensity ) , allowing differentiation of active tumour from normal oedematous tissue [ 1317 ]  . 
it has been reported that when radiosurgery includes peritumoural parenchymal areas with high cho and abnormal cho / naa ratio and hence infiltrated areas patient survival is prolonged [ 15 ]  . 
 posttreatment follow - up 1h - mrs has higher sensitivity than does conventional mr imaging in detecting tumour progression , as it can demonstrate spectral abnormalities beyond the limits of the radiation target , even in the absence of contrast enhancement or before an increase in contrast enhancement is detected [ 16 ]  . in particular , changes in cho and the magnitude of the cho / cr and cho / naa ratios are predictive of tumour progression . 
in particolare nei gliomi di alto grado la cho risulta ridotta nelle aree centrali necrotiche , aumentata nella massa e nei margini tumorali , ridotta nelle aree edematose e di nuovo incrementata fino ai livelli normali nel tessuto sano circostante ; lnaa invece ridotto nelle aree centrali , aumentato verso la periferia e raggiunge valori normali nel tessuto sano circostante ; i lac - lip risultano invece notevolmente aumentati nelle aree centrali necrotiche e ridotti ai margini della neoplasia . 
nei gliomi di basso grado , di solito , la cho elevata nelle aree centrali , si riduce verso i margini e persiste leggermente alta nel tessuto sano circostante ; lnaa ridotto nella massa e nei margini tumorali , raggiunge valori normali nel tessuto sano circostante ; i lac - lip non risultano per nulla rilevabili ( comunicazione personale )  . 
con la valutazione degli spettri ci invece possibile , dato che nelle aree edematose infiltrate da neoplasia presente uno spettro con anomalo rapporto cho / naa contrariamente a quello che accade in presenza di edema vasogenico puro caratterizzato dalla normalit di tale rapporto . 
in tal modo possibile delineare lestensione del tumore oltre i margini rilevati con limaging di base ( al di fuori dei bordi del contrast enhancement , ce , o delliperintensit peritumorale in t2 ) distinguendo il tumore attivo dal tessuto normale ed edematoso [ 1317 ]  . 
tali indicazioni sono utili al fine di effettuare una biopsia pi mirata e una terapia ( chirurgica o radiante ) maggiormente differenziata , in modo da risparmiare la maggior quota di tessuto sano . 
peraltro alcuni autori hanno evidenziato che quando la radiochirurgia include le aree parenchimali peritumorali con alta cho e alterato rapporto cho / naa e quindi infiltrate , vi un allungamento della sopravvivenza del paziente [ 15 ]  . 
 controllo post - trattamento la 1h - mrs presenta una sensibilit maggiore rispetto allo studio rm convenzionale nel rilevare una progressione neoplastica , evidenziando anomalie spettrali al di fuori della sede del letto chirurgico , anche in assenza di ce e comunque prima di un incremento dello stesso [ 16 ]  . 
per la cho , laumento oltre il 45% del valore normale misurato controlateralmente indice di progressione di malattia ; mentre un valore uguale o inferiore al 35% indicativo di stabilit [ 17 ]  . 
 tumour response to surgical and / or radiation therapy usually involves an initial reduction of cho , with possibly an increase in lac / lip , indicating radiation necrosis , which is characterised by the absence of any metabolic signals for up to several months [ 10 ]  . 
 in the follow - up after chemotherapy , the cho / cr and lac / cr ratios are the most reliable markers for assessing the response of low - grade gliomas . 
in the enhancing tumour regions , a significant association was detected between increased lac / cr during treatment and decreased progression - free survival time , whereas a low naa / cr in normalappearing brain tissue adjacent to nonenhancing tumour at baseline was associated with decreased progression - free survival time . 
an increase in cho / cr and lac / cr in normalappearing brain regions next to the tumour may be noted several months before mr imaging reveals disease progression [ 24 ]  . 
the result may be visualised in different , increasingly complex , forms , each of which information : ( 1 ) diffusion - weighted provides unique raggiunge una sensibilit del 93 , 8% e una specificit del 85 , 7% [ 18 ]  . 
essi sono significativamente pi alti nelle recidive rispetto al danno radiante e ancor pi alti rispetto alla sostanza bianca apparentemente sana ; naa / cr ha un andamento opposto [ 21 ]  . 
sensibilit , specificit ed accuratezza diagnostica dei rapporti cho / cr e cho / naa sono risultati rispettivamente del 94 , 1% , 100% e 96 , 2% [ 22 ]  . 
 la risposta di un tumore alla terapia chirurgica e / o radiante consiste , di solito , nella iniziale riduzione della cho e nella possibile comparsa del picco di lac / lip per la necrosi da irradiazione caratterizzata soprattutto dalla assenza di segnale metabolico che persiste per diversi mesi [ 10 ]  . 
 nel follow - up post - chemioterapia i rapporti cho / cr e lac / cr risultano essere i marker pi utili per il follow - up dei gliomi a basso grado . 
nelle aree con ce stata individuata una significativa associazione tra lincremento del lac / cr durante il trattamento e la riduzione della progressione / sopravvivenza , mentre nellimaging di base un basso naa / cr nel tessuto cerebrale apparentemente normale adiacente al tumore senza ce associato ad una ridotta progressione / sopravvivenza . 
un aumento di cho / cr e lac / cr nel tessuto cerebrale apparentemente normale adiacente al tumore pu evidenziarsi qualche mese prima della progressione di malattia in rm [ 24 ]  . 
the different cellularity of tissues corresponds to changes in the diffusivity of water molecules : the greater the cellularity and consequently the number of cell membrane interfaces , the lower the diffusivity . histological typing and grading besides the inverse relationship between the adc and cellularity ( an important parameter in grading ) , data on the possibility of discriminating histological type and grade are conflicting because of tissue heterogeneity [ 25 , 26 ]  . normally , it is not possible to distinguish low - grade from high - grade gliomas on the basis of dwi and adc values alone [ 27 ]  . 
dwi is , instead , far more useful in differentiating cystic from solid components of a mass and the tumour mass from necrosis or surrounding healthy tissue [ 28 , 29 ]  . 
 treatment planning dwi is not generally accepted as a useful parameter for determining the extent of tumour infiltration and differentiating infiltrated from vasogenic oedema owing to its intrinsic low spatial resolution [ 25 ]  . 
moreover , there is some overlap of the adc and fractional anisotropy ( fa ) values in peritumoural regions with high signal intensity on t2 and in the adjacent normal - appearing white matter between various types of brain tumour [ 30 ]  . 
dti is no doubt more useful for planning neurosurgery or radiotherapy in that it visualises white - matter tracts that may be displaced and oedematous ( as in low - grade gliomas , meningiomas or metastases ) , or infiltrated or destroyed ( as in high - grade tumours ) [ 31 ]  . information about the integrity of structures inside or outside the tumour obtained by visualising its relationships with critical cortical areas and white - matter tracts may guide treatment decisions ( radical or partial ) so as to optimise tumour resection and minimise permanent posttreatment neurological morbidity [ 32 ]  . 
the combination of dti and cortical activation maps ( fmri ) improves the accuracy of presurgical mapping by delineating the relationship between the tumour and white - matter tracts ( with dti ) and eloquent cortical areas ( with fmri )  . 
in the posttreatment follow - up of a high - grade glioma , it helps to differentiate the effects of radiotherapy from disease recurrence or progression [ 34 ]  . 
con il variare della cellularit di un tessuto varia infatti la diffusivit delle molecole dacqua : maggiore la cellularit , e quindi il numero delle interfacce di membrane cellulari , minore la diffusivit . istotipo e grading nonostante la presenza di una correlazione inversa tra adc e cellularit ( parametro importante per definire il grading ) , i dati riguardanti la possibilit di discriminare istotipo e grading tumorale sono contrastanti in virt della eterogeneit tissutale [ 25 , 26 ]  . 
di solito non possibile distinguere un glioma di basso grado da uno di alto grado utilizzando solo le immagini pesate in diffusione e i valori di adc [ 27 ]  . 
la dwi invece molto pi utile nel differenziare le componenti cistiche e solide di una massa , la massa tumorale dalla necrosi o dal tessuto sano circostante [ 28 , 29 ]  . 
 pianificazione del trattamento la dwi non da tutti considerato un parametro utile per determinare lestensione della infiltrazione tumorale e per differenziare linfiltrazione neoplastica dalledema vasogenico per lintrinseca limitazione della risoluzione spaziale [ 25 ]  . 
inoltre , vi sovrapposizione dei valori di adc e fa ( fractional anisotropy ) nelle aree peritumorali iperintense in t2 e nelladiacente sostanza bianca apparentemente normale tra tumori cerebrali di vario tipo [ 30 ]  . 
sicuramente pi importante il dti che fornisce informazioni utili per il planning neurochirurgico / radioterapeutico visualizzando i fasci di sostanza bianca che possono essere dislocati ed edematosi ( in gliomi di basso grado , meningiomi o metastasi ) , oppure infiltrati o distrutti ( in tumori di alto grado ) [ 31 ]  . 
le informazioni circa la integrit o meno delle strutture poste allinterno e al di fuori del tumore , infatti , evidenziando i rapporti della lesione con le aree corticali critiche e con i fasci di sostanza bianca , possono guidare la scelta del tipo di trattamento ( radicale o parziale ) , ottimizzando la resezione tumorale e riducendo al minimo il danno cerebrale e quindi la morbilit neurologica permanente post - trattamento [ 32 ]  . 
la combinazione del mapping dti con quello di attivazione corticale ( fmri ) fornisce un mapping prechirurgico ancora pi accurato , delineando i rapporti del tumore con i fasci di sostanza bianca ( con il dti ) e le aree corticali eloquenti ( con lfmri )  . 
 controllo post - trattamento ladc nellimmediato periodo post - operatorio pi utile del ce nel definire la persistenza o meno di patologia [ 33 ]  . nel follow - up di un glioma di alto grado trattato , esso permette di differenziare gli effetti della radioterapia dalla recidiva o progressione tumorale [ 34 ]  . 
in particular , the adc is significantly higher in radionecrosis than in tumour recurrence , even though there is some overlap [ 34 ]  . in the presence of an enhancing lesion , the adc is significantly higher in tumour recurrence than in radiation injury , whereas in white - matter tracts in peritumoural oedema , the adc is higher in radiation injury than in tumour recurrence . 
fa is significantly higher in the normalappearing white matter adjacent to the peritumoural oedema in radiation injury compared with tumour recurrence [ 35 ]  . perfusion imaging mr perfusion - weighted imaging ( pwi ) is a useful , noninvasive , and rapid technique that is increasingly being used in the characterisation of brain tumours [ 36 , 37 ]  . 
numerous techniques have been proposed for measuring brain perfusion parameters : cerebral blood volume , transit time , clearance , extraction fraction , cerebral blood flow and the permeability - surface area product [ 38 ]  . 
the most commonly used technique in clinical practice is dynamic susceptibilityweighted bolus - tracking mr imaging , which is based on the susceptibility effects obtained after intravenous injection of a bolus of paramagnetic contrast material and first - pass acquisition of t2 - weighted echoplanar ( epi ) sequences of the region of interest . 
 histological typing and grading when a tumour is present , there is increased microvessel density and neoangiogenesis , both of which are crucial histological criteria for determining glioma aggressiveness . this leads to increased cbv , which is considered one of the main predictors of tumour aggressiveness and survival , even though it is not synonymous for malignancy ( e.g. highly vascular benign tumours such as oligodendroglioma and meningioma ) [ 40 , 41 ]  . 
in particolare significativamente pi alto nella radionecrosi che nella recidiva tumorale anche se esiste un certo grado di sovrapposizione [ 34 ]  . in presenza di lesione con ce , ladc significativamente pi alto nella recidiva rispetto al danno radiante , mentre nei fasci di sostanza bianca nelledema perilesionale ladc risultato pi alto nel danno da irradiazione rispetto alla recidiva . 
la fa invece significativamente pi alto nella sostanza bianca apparentemente normale adiacente alledema perilesionale in presenza di danno radiante rispetto alla recidiva [ 35 ]  . perfusione limaging di perfusione ( pwi ) con rm una tecnica non invasiva , di rapida esecuzione e di indubbia utilit che viene sempre pi spesso utilizzata nella caratterizzazione delle neoformazioni cerebrali [ 36 , 37 ]  . 
numerose tecniche sono state proposte per misurare la perfusione a livello cerebrale , ed altrettanto numerosi sono i parametri che possono essere misurati : il volume ematico cerebrale , il tempo di transito , la clearance , la frazione di estrazione , il flusso ematico cerebrale e il prodotto tra superficie di permeabilit e flusso ematico [ 38 ]  . 
nella pratica clinica di solito si utilizza il metodo che si basa sulle caratteristiche di suscettibilit che si ottengono dopo somministrazione endovenosa a bolo di un mdc paramagnetico e nelle acquisizioni , nellarea di interesse , al primo passaggio del bolo , di sequenze ecoplanari ( epi ) pesate in t2 . 
ci comporta laumento del cbv considerato uno dei maggiori fattori predittivi di aggressivit neoplastica e di sopravvivenza in vari istotipi anche se non sinonimo di malignit ( il caso di tumori benigni molto vascolarizzati quali loligodendroglioma e il meningioma ) [ 40 , 41 ]  . 
 studies investigating the possibility of delimiting the true tumour margins with pwi have yielded conflicting results . in a recent study involving 36 patients with gliomas of different grades , we performed pwi with a 3 - tesla magnet , which allowed us to distinguish not only necrosis from tumour but also oedema mixed with tumour ( with high cbv ) from pure oedema ( with low cbv ) [ 45 ]  . 
there is a good correlation between cbv and 1h - mrs in delineating tumour margins . posttreatment follow - up cbv is significantly higher in tumour recurrence than in radionecrosis , although there is some overlap [ 27 ]  . 
given the recent development of new treatments aimed at reducing neoangiogenesis ( both pharmacological and targeted radiation ) , pwi may take on a primary role as a means to monitor their efficacy in a simple and rapid fashion . multimodal mr evaluations use of a single conventional or advanced mr imaging pattern for diagnosing gliomas may prove insufficient for histological typing and grading , treatment planning and posttreatment follow - up . 
hence the need for combined studies capable of enhancing the techniques diagnostic accuracy [ 4 , 4750 ]  . we recently conducted a multimodal study involving the combination of conventional mr imaging with spectroscopy , diffusion and perfusion imaging on 31 patients with cerebral gliomas of different grades . 
in una nostra recente esperienza che ha coinvolto 36 pazienti con glioma di diverso grado lo studio di perfusione effettuato con apparecchiatura a 3 tesla , oltre che permettere la distinzione tra necrosi e massa tumorale , ha permesso di differenziare ledema misto a tumore ( con alto cbv ) dalledema puro ( con basso cbv ) [ 45 ]  . 
esiste , comunque , una buona correlazione tra cbv e 1h - mrs nella delineazione dei margini tumorali . controllo post - trattamento il cbv significativamente pi alto nella recidiva tumorale rispetto alla radionecrosi , ma esiste un certo grado di sovrapposizione [ 27 ]  . 
con il recente interesse e sviluppo di nuove tecniche terapeutiche mirate a ridurre la neo - angiogenesi ( farmacologiche e radianti mirate ) la pwi pu assumere ruolo primario per valutare in maniera semplice ed immediata la loro efficacia . valutazioni rm multimodali luso di un solo pattern rm di base o avanzato per la diagnosi dei gliomi pu non bastare ai fini soprattutto di definire istotipo e grading , pianificazione del trattamento , follow - up up post - trattamento . 
da qui la necessit spesso di studi combinati capaci di aumentare laccuratezza diagnostica [ 4 , 4750 ]  . in una recente esperienza personale , abbiamo condotto uno studio multimodale abbinando alla rm di base una valutazione di spettroscopia , diffusione e perfusione . 
in tale studio che ha coinvolto 31 pazienti con glioma cerebrale di diverso grado , tutti i parametri utilizzati , ad eccezione delladc , hanno permesso di differenziare i gliomi di basso grado da quelli di alto grado . 
e infatti la ricerca dei dati semeiologici basilari sede di origine , modalit di crescita , caratterizzazione tissutale ed il confronto con let e le informazioni cliniche dei pazienti pu condurre alla corretta diagnosi pre - trattamento . 
1a - f contrast - enhanced t1 - weighted image ( a ) and fluid - attenuated inversion recovery ( flair ) image ( b ) , apparent diffusion coefficient ( c ) and cerebral blood volume ( d ) maps , localising flair image ( e ) and proton mr spectroscopy ( 1h - mrs ) spectra in regions of interest ( roi ) ( f ) in a 49 - yearold patient with a grade ii frontoparietal fibrillar astrocytoma . 
spectra 1 , 2 and 3 show the typical pattern of low - grade gliomas , characterised by elevated choline ( cho ) , abnormal cho / n - acetyl - aspartate ( naa ) ratio ( > 1 ) and absence of lactate and lipid ( lac - lip ) peak . 
1a - f immagine rm pesata in t1 dopo mezzo di contrasto ( a ) e flair ( b ) , mappe di coefficiente di diffusione apparente ( adc ) ( c ) e di volume ematico cerebrale ( cbv ) ( d ) , immagine localizzatrice flair ( e ) e spettri 1h - mrsi in regioni di interesse ( roi ) ( f ) , in un paziente di 49 anni con astrocitoma fibrillare di ii grado in sede fronto - parietale sinistra . 
gli spettri 1 , 2 e 3 presentano un pattern tumorale tipico dei glomi di basso grado , caratterizzato da elevata cho , anomalo rapporto cho / naa ( > 1 )  . 
2a - f contrast - enhanced t1 - weighted image ( a ) and fluid - attenuated inversion recovery ( flair ) image ( b ) , apparent diffusion coefficient ( adc ) ( c ) and cerebral blood volume ( cbv ) ( d ) maps localising t2 - weighted image ( e ) and proton mr spectroscopy ( 1h - mrs ) spectra in regions of interest ( roi ) ( f ) in a 56 - year - old patient with right temporooccipital glioblastoma multiforme . 
spectra 1 , 3 and 4 at the lesion margins show the typical tumoural pattern , characterised by elevated choline ( cho ) , abnormal cho / nacetyl - aspartate ( naa ) ratio ( > 1 ) and lactate and lipid ( lac / lip ) peak . 
spectra 5 and 6 , in a region of apparent oedema beyond the enhancing margins , show reduced levels of all metabolites ( relative to the normal spectra 7 , 8 and 9 ) but different patterns : spectrum 5 has a normal cho / naa ratio ( < 1 ) , suggesting noninfiltrated oedema ; spectrum 6 has a tumoural pattern suggesting infiltrating tumour cells . 
2a - f immagine rm pesata in t1 dopo mezzo di contrasto ( a ) e flair ( b ) , mappe di coefficiente di diffusione apparente ( adc ) ( c ) e di volume ematico cerebrale ( cbv ) ( d ) , immagine localizzatrice pesata in t2 ( e ) e spettri 1h - mrsi in regioni di interesse ( roi ) ( f ) , in una paziente di 56 anni con glioblastoma temporo - occipitale destro . 
gli spettri 1 , 3 e 4 , sui margini del glioblastoma , presentano il tipico pattern tumorale , caratterizzato da elevata cho , anomalo rapporto cho / naa ( > 1 ) e presenza di lac / lip ( ll )  . 
lo spettro 2 , allinterno dei margini impregnati , presenta una riduzione dei livelli di tutti i metabolici ad eccezione di ll che risulta ulteriormente incrementato ; in tale sede , ladc tende ad essere aumentato e il cbv ridotto . gli spettri 5 e 6 , in unarea apparentemente edematosa esterna ai margini impregnati , presentano nel complesso livelli ridotti di tutti i metaboliti ( rispetto agli spettri normali 7 , 8 e 9 ) , ma pattern differenti : lo spettro 5 ha un normale rapporto cho / naa ( < 1 ) e suggerisce la presenza di edema vasogenico non infiltrato ; lo spettro 6 ha un pattern tumorale e suggerisce la presenza di cellule tumorali infiltranti . 
in queste sedi , adc e cbv hanno un comportamento opposto , essendo il primo elevato e il secondo ridotto nellarea di edema puro e viceversa nellarea di edema infiltrato . 
the extreme heterogeneity of brain tumours and the limited specificity of mr imaging may make it difficult to arrive at a clear histological diagnosis , which is straightforward in typical cases only . 
furthermore , the wider availability of 3 - tesla mr equipment and awareness of its numerous advantages ( high signal , high resolution , high sensitivity , better quality , reduced examination times , additional and more advanced imaging techniques ) is stimulating the development and application of morphofunctional evaluation of brain tumours in routine neuroradiological practice [ 5658 ]  . 
this becomes all the more important if we consider the recent development of new and more effective antitumoural treatments that rely on a complete morphofunctional mr study to obtain a noninvasive in vivo neuropathological characterisation and thus a biological interpretation of the typical heterogeneity of such tumours . interpretazione non solo per il neuroradiologo , ma anche per il neuropatologo . 
tali tecniche , fornendo dati relativi al metabolismo cellulare , alla diffusione e allemodinamica , migliorano laccuratezza diagnostica , la sensibilit e la specificit della metodica rm [ 5355 ]  . 
inoltre , la maggiore diffusione sul territorio di apparecchiature con intensit di campo a 3 tesla , in virt dei numerosi vantaggi ( alto segnale , alta risoluzione , alta sensibilit , migliore qualit , tempi desame ridotti , metodologie di studio aggiuntive e pi avanzate ) sta favorendo lo sviluppo e lapplicazione nella pratica neuroradiologica di routine della valutazione morfo - funzionale della patologia cerebrale [ 5658 ]  . 
baffoni2 1uos neuroradiologia , 2uoc radiologia , 3uos di staff programmazione sanitaria e miglioramento qualit , 4direzione medica ospedaliera , 5uoc nefrologia e dialisi , 6uoc anestesia e rianimazione , 7uos di staff ufficio relazioni con il pubblico , 8uos di staff formazione ed aggiornamento , 9uos medicina legale , 10uos diabetologia e malattie metaboliche , 11uoc patologia clinica , 12uos allergologia , asur marche zona territoriale 11 , via zeppilli 18 , 63023 fermo , italy correspondence to : m.g. 
138 , 63023 fermo , italy , tel . : + 39 - 0734 - 6252246 , fax : + 39 - 0734 - 6253200 , e - mail : massimogiuliano.bonetti@tin.it received : 10 january 2008 / accepted : 9 may 2008 / published online : 25 march 2009 springer - verlag 2009 abstract purpose . 
the new documentation ( patient history / screening form and informed consent form ) is available on the hospital web site and requests serum creatinine determination with estimation of glomerular filtration rate ( gfr ) for every patient . 
un evento formativo di rinforzo per i medici invianti e la verifica del fg al momento della prenotazione dellesame sono giudicati indispensabili per la corretta programmazione delle indagini nei pazienti a rischio . radiol med ( 2009 ) 114 : 496508 keywords diagnostic imaging iodinated contrast media guidelines parole chiave diagnostica per immagini mezzi di contrasto organo - iodati linee guida introduction introduzione stimulated by the sirm emilia romagna and marche regional chapters and the experience of the modena university hospital and hospital trust ( protocollo sulluso dei mezzi di contrasto 26 / 9 / 2001 ) , the authors sought to implement the new national guidelines on the use of injectable iodinated contrast media ( icm ) issued by sirm ( italian society of medical radiology ) [ 1 , 2 ] to prevent icm toxicity at their institution . the project was presented and informally discussed with the institutions radiologists at two departmental meetings held in 2004 and 2005 , where the new documentation ( patient history / screening form and informed consent form ) was presented through a powerpoint slide show ( mgb , personal communication )  . 
one point that emerged from these preliminary meetings was the need to adopt a systematic approach to the problem of icm , as its use is still governed by consolidated practice and there is some resistance to change . 
critically review the list of laboratory tests ordered before icm administration ( for all patients : blood urea nitrogen , glucose , serum creatinine , serum protein and protein electrophoresis , urinalysis ) 3 . 
provide information about the comorbidities capable of increasing the risk of reaction to icm and to devise specific protocols for preventing organ damage and , where needed , for patient preparation 4 . 
create a tool capable of promoting the sharing of responsibility between the referring physician and the radiologist , as indicated in the directive euratom 43 / 97 and incorporated in our legislation [ 3 ] , by reviewing the patient history / screening form and informed consent form 5 . 
train radiology personnel in the first - line techniques and treatment to be used in the event of adverse reactions to alla luce dei recenti orientamenti sulluso del mezzo di contrasto organo iodato per via iniettiva ( mdc ) espressi dalla sirm ( societ italiana di radiologia medica ) [ 1 , 2 ] e stimolati in ambito regionale sirm emilia romagna e marche dallesperienza gi attuata nelle aziende ospedaliere policlinico e usl di modena ( protocollo sulluso dei mezzi di contrasto 26 / 9 / 2001 ) , abbiamo voluto implementare anche nella nostra realt operativa delle linee guida aggiornate sulla profilassi della tossicit da mdc . la preliminare presentazione e discussione informale della problematica con i colleghi radiologi nellambito di due riunioni di reparto negli anni 2004 e 2005 , tramite presentazione power - point ( m.g.b. , comunicazione personale ) comprensiva di una nuova modulistica per linformativa ed il consenso informato , ha indicato la necessit di un approccio organico alla problematica dei mdc , molto influenzata dalla prassi consolidata e poco incline alle novit . 
during a series of meetings held in the first semester of 2006 , the group jointly prepared the draft documentation the patient history / screening form ( to be filled in by the referring physician and by the patient ) and the informed consent form ( to be filled in by the radiologist and patient during the routine interview before the examination )  . 
the group devised specific protocols for patients at risk of adverse reactions to icm and / or for those requiring special preparation and identified the relevant contact telephone numbers to be given to the service users . in collaboration with the hospitals public relations department ( mfs ) , the new forms for radiological investigations , including those employing icm , were posted on the hospitals web site ( asp ? co_id = 6493 ) , together with links to sirm web pages providing practical information on patient preparation for imaging studies ( esami / ) and the national guidelines [ 5 ]  . in collaboration with the hospitals education and training department ( sm ) , two training courses were developed to illustrate the draft document on icm use , the national guidelines ( with special reference to the appropriateness of imaging procedures , harmful effects of radiation exposure and icm administration ) and the patient history / screening and consent forms posted on the hospital web site . 
all materials were supplied to the course participants as hypertexts on cd - rom . the first course , titled diagnostic imaging : national guidelines [ two afternoons for a total of 10 h , 10 continuing medical education ( cme ) credits ] was repeated in two sessions ( january and february 2007 ) and was addressed to medical , nursing and technical personnel of the radiology department . 
the same personnel were also enrolled in inhouse training sessions on first - aid techniques [ bls ( basic life support ) or blsd ( basic life support and defibrillation ) ]  . 
in addition to hospital trolleys with emergency medications , diagnostic imaging rooms were also provided with kits containing specific drugs for adverse reactions to contrast media [ chlorphenamine maleate ( trimeton 10 mg / ml vials , schering - plough ) ; ranitidine chloride ( ranitidine angenerico 50 mg / 5 ml , angelici ) ; hydrocortisone ( flebocortid 1g / 10 ml , sanofi aventis ) ; methylprednisolone hemisuccinate ( urbason 40 mg / ml , aventis ) ; adrenaline im ( 0.33 mg fastjekt adults , merck ; 0.165 mg fastjekt junior , merck ) ; and the telephone numbers of the anaesthetistresuscitator for emergencies . 
in una serie di riunioni nel primo semestre del 2006 stata quindi preparata una bozza di documento condivisa , comprensiva della nota informativa ( da compilarsi a cura del medico inviante e del paziente ) e del consenso informato ( da compilarsi a cura del medico radiologo e del paziente nel consueto colloquio pre - esame )  . 
sono stati previsti i percorsi per i pazienti a rischio per la somministrazione del mdc e / o con necessit di particolare preparazione e sono stati identificati i numeri telefonici di riferimento da fornire agli utenti . in collaborazione con la uos di staff ufficio relazioni con il pubblico [ m.f.s. ] , la modulistica relativa alle indagini radiologiche , comprese quelle con mdc , stata resa disponibile sul sito aziendale ( viewdoc.asp ? co_id = 6493 ) , assieme ad un link sulle informazioni pratiche per la preparazione dei pazienti alle indagini di imaging ( esami / ) ed alle linee guida nazionali di riferimemento [ 5 ]  . in collaborazione con la nostra uos di staff formazione ed aggiornamento [ s.m. ] sono stati preparati due progetti formativi per illustrare : la bozza del documento condiviso sui mdc , le linee guida nazionali di riferimento ( con particolare riguardo allappropriatezza delle indagini di imaging , agli effetti dannosi associati allesposizione alle radiazioni ionizzanti ed alla somministrazione dei mdc ) , fornite ai corsisti in versione ipertestuale di facile consultazione su cd , e la modulistica disponibile sul sito aziendale di cui sopra . 
 il primo progetto formativo , dal titolo la diagnostica per immagini : linee guida nazionali di riferimento ( due pomeriggi per un totale di 10 ore , 10 crediti ecm ) stato presentato in due edizioni ( gennaio e febbraio 2007 ) al personale medico , infermieristico e tecnico della nostra radiologia . 
at the same time , the report forms for serum creatinine used by the hospitals clinical laboratory services were integrated , with an estimation of glomerular filtration rate ( gfr ) to be calculated automatically by applying the simplified modification of diet in renal disease ( mdrd ) formula [ 6 ] , namely : gfr ( ml / min per 1.73 m2 ) = 186 ( serum creatinine ) 1.154 ( age ) 0.2030.742 ( if patient is female ) 1.210 ( if patient is african american )  . on 10 may 2007 , initial feedback was obtained at a meeting between the hospital director staff ( av ) , the radiologist ( mgb ) and several clinical directors ( surgery , urology , oncology ) who had experienced difficulties in applying the protocol , and the forms were reviewed . 
on 22 and 30 may 2007 , two meetings were held between the hospital director staff , the radiologist , the reference nephrologist ( mc ) and endocrinologist ( pp ) , the clinical director of the clinical laboratory services ( cc ) and the union and scientific representatives of the general practitioners and community paediatricians of our area . 
proposals to improve the clarity and usability of the forms were incorporated , and procedures were established for arranging a nephrological consultation in selected cases and for admitting outpatients to the nephrology and dialysis unit for treatment before and after contrast injection . 
the radiology department purchased an oven to heat the contrast medium to body temperature , weighing scales with a heightmeasuring rod , and a timer for timing the observation of patients after the examination with icm ( from a minimum of 30 min to a maximum of 60 min in high - risk patients )  . during the first week of june , the radiologist ( mgb ) incorporated all revisions to produce a final draft of the patient history / screening form and informed consent form . on 12 june 2007 , the final document was posted on the hospital trust web site and transmitted to the hospital clinical directors , to the district directors and to the head of primary care services ( general practitioners and specialist services )  . anestesia , analgesia , rianimazione e terapia intensiva ( siaarti ) [ 1 ]  . il secondo progetto formativo , dal titolo nuovi strumenti di lavoro per la diagnostica per immagini ( una mattina per un totale di 4 ore , 2 crediti ecm ) stato presentato in quattro edizioni ( febbraio e marzo 2007 ) al personale medico ( ospedalieri , medici di medicina generale , pediatri di libera scelta , emergenza territoriale , distretti , guardia medica , etc . ) della nostra zona territoriale . dopo la conclusione dei progetti formativi , il 23 marzo 2007 stata trasmessa nota ai responsabili delle uu.oo. ospedaliere ed ai responsabili dei due distretti della nostra zona territoriale con le informazioni relative allutilizzo del nuovo modello per gli esami di imaging con mdc da impiegare in via sperimentale , affinch fosse portato a conoscenza di tutto il personale interessato . 
contemporaneamente il laboratorio di analisi dellospedale ha aggiunto nei suoi referti della creatininemia la stima del filtrato glomerulare ( fg ) calcolata automaticamente secondo la formula mdrd semplificata [ 6 ] , che si ottiene come segue : fg ( ml / min / 1 , 73 m2 ) = 186 ( creatininemia ) 1 , 154 ( et ) 0 , 2030 , 742 ( se di sesso femminile ) 1 , 210 ( se di razza afroamericana )  . il 10 maggio 2007 si svolta in direzione sanitaria [ a.v. ] una riunione preliminare tra il radiologo del gruppo [ m.g.b. ] ed alcuni primari ospedalieri ( chirurgia , urologia , oncologia ) che avevano riscontrato problemi nellapplicazione del modello , con rivalutazione del modello stesso ed acquisizione di suggerimenti . 
il 22 e 30 maggio 2007 sono state svolte in direzione sanitaria [ a.v. ] due riunioni con il radiologo del gruppo [ m.g.b. ] , convocando il nefrologo [ m.c. ] e lendocrinologo di riferimento [ p.p. ] , il primario del laboratorio di analisi [ c.c. ] , i rappresentanti sindacali e scientifici dei medici di medicina generale e dei pediatri di libera scelta della nostra zona territoriale . 
il modello stato rivalutato a seguito della sperimentazione e di nuove acquisizioni della letteratura [ 7 ] e sono stati recepiti suggerimenti finalizzati alla sua chiarezza dei contenuti ed alla fattibilit di utilizzo . stata codificata la procedura per la consulenza del nefrologo nei casi indicati e per la presa in carico dei pazienti ambulatoriali da parte della uoc di nefrologia e dialisi per la somministrazione della terapia indicata pree post - somministrazione di mdc . 
in radiologia sono stati acquisiti un fornetto per scaldare a temperatura corporea il mdc , una bilancia con asta graduata per la misura dellaltezza e dei timer per la misurazione del periodo di osservazione ( minimo 30 minuti , fino a 60 minuti nei pazienti a rischio ) del paziente dopo lesame contrastografico . 500 results the training course diagnostic imaging : national guidelines was successfully attended by 74.5% of the medical , nursing and technical staff of the radiology department ( 38 of 51 invited )  . 
the bls or blsd courses were successfully attended by 80% of the same personnel ( 44 of 55 invited )  . the training course new working tools for diagnostic imaging was successfully attended by 57.2% of doctors of our area ( 206 of 360 invited )  . two - sided page , one side containing the final version of the document ( available on our hospital trusts web site asp ? co_id = 6493 > modulo mezzo di contrasto ) consists of the patient history / screening form for elective examinations with injectable icm and the informed consent form , and the other providing guidance notes for completion and useful telephone numbers . 
the form contains sections on indications for the examination and the patients clinical data ( inclusive of possible neoplasms and surgical operations ) , and any risk factors for the use of icm . 
if the gfr is less than 60 ml / min per 1.73 m2 of body surface , a nephrological consultation must be arranged before the examination to prevent possible nephrotoxic damage . 
for adults , among the various recommended methods [ 11 , 12 ] , we decided to continue using the classic cockcroftgault formula [ 13 ] , with which we are familiar . 
the formula was modified by replacing body weight in numerator position with ideal body weight ( useful also in obese or pathologically underweight patients ) : gfr = [ 140 - age ( in years ) ] ideal body weight ( in kg ) / [ 72 serum creatinine ( in mg / dl ) ]  . 
ospedaliere , ai direttori di distretto ed al responsabile delluos medicina di base e specialistica per lopportuna diffusione . risultati il progetto formativo la diagnostica per immagini : linee guida nazionali di riferimento stato seguito con profitto dal 74 , 5% del personale medico , infermieristico e tecnico della radiologia ( 38 persone su 51 convocati )  . 
il progetto formativo nuovi strumenti di lavoro per la diagnostica per immagini stato seguito con profitto dal 57 , 2% dei medici della nostra zona territoriale ( 206 persone su 360 convocati )  . il modello definitivo ( disponibile sul nostro sito aziendale allindirizzo co_id = 6493 > modulo mezzo di contrasto ) in due facciate , comprende un fronte ( informativa per gli esami di diagnostica per immagini programmati con mezzi di contrasto organo - iodati per via iniettiva , consenso informato ) ed un retro ( note , numeri di telefono utili )  . 
linformativa , necessaria per procedere allesecuzione dellesame , deve essere compilata e firmata dal medico inviante e dal paziente . dopo le notizie relative allindicazione allesame ed alla sintesi clinico - anamnestica del paziente ( comprensiva di eventuali neoplasie ed interventi chirurgici ) , vengono riportati gli eventuali fattori di rischio alluso del mdc . 
la valutazione di laboratorio della sola creatininemia ci ha fatto abbassare la spesa da 11 , 40 euro a 1 , 50 euro a paziente ( risparmio del 86 , 8% ) rispetto al passato . dalla cratininemia viene richiesto , nella nota informativa , di calcolare il fg stimato , usando le formule riportate nel retro del modulo . 
se il filtrato glomerulare risulta inferiore a 60 ml / min / 1 , 73 m2 di superficie corporea necessario ricorrere ad una consulenza nefrologica prima dellesecuzione dellesame con mezzo di contrasto organo - iodato per prevenire leventuale danno nefrotossico . 
per let pediatrica stata considerata la formula di schwartz [ 810 ] : fg = k altezza ( in cm ) / creatininemia ( in mg / dl ) , dove k uguale a 0 , 33 nel pretermine , 0 , 45 nel bambino nato a termine e fino a 12 mesi di et , 0 , 55 nel bambino dal compimento del primo anno fino a 13 anni , successivamente e fino a 18 anni 0 , 55 nelle femmine e 0 , 65 nei maschi . 
as a result , in consideration of the number of examinations with icm performed in patients older than 60 years at our centre ( 71% , and specifically , 57% of patients aged 6180 years and 14% of patients aged over 80 years ) , the clinical laboratory services have restricted the use of automatic gfr calculations from serum creatinine to patients aged 1860 years only until a large enough case series has been collected to enable comparison of the two formulas across our patient population . to date , at least 80% of patients have presented to the radiology department on the day of the examination without any indication of estimated gfr noted on the patient history / screening form by the referring physician , with no substantial difference between inpatients and outpatients . 
in all patients , the radiology department nursing and medical personnel independently evaluate this parameter , even when already calculated , by using the formulas reported on the back of the patient history / screening form . on average 20% of patients scheduled for an examination with icm have abnormal estimated gfr on the day of the examination ( < 60 ml / min per 1.73 m2 , significant renal insufficiency )  . 
the nephrologist assesses the patient for risk factors , quantifies the risk ( table 1 ) and prescribes the prophylactic treatment to be administered in connection with the examination , hydration with isotonic saline solution and administration of n - acetylcysteine . 
table 1 is derived from a study on the intra - arterial administration of contrast medium [ 14 ] , which used a semiquantitative method for calculating contrast - induced risk . the fact that the risk of contrast - induced nephropathy may be higher with intra - arterial contrast administration [ 15 ] allows for a greater safety margin in our patients , who receive intravenous icm . 
in particular , with regard to hydration , our policy is to administer 0.9% saline solution intravenously at a dose of 1.01.5 ml / kg per hour , as suggested by the recent literature [ 16 ]  . 
whereas there is no problem starting the infusion 12 h before the examination and afterwards continuing it for at least 12 h in inpatients , in outpatients we are obliged to reduce administration times to 3 h before and 6 h after the examination . 
in addition to hydration , patients receive 600 mg oral n - acetylcysteine twice daily the day before and the day of sica formula di cockcroft - gault [ 13 ] , modificata mettendo al numeratore , al posto del peso corporeo , il peso corporeo ideale ( utile anche nei pazienti obesi o patologicamente magri ) : fg = [ 140 - et ( in anni ) ] peso corporeo ideale ( in kg ) / [ 72 creatininemia ( in mg / dl ) ]  . 
a titolo esemplificativo , nel normotipo , i valori limite di creatininemia sono 40 anni : uomo > 1 , 67 ; donna > 1 , 42 ; 65 anni : uomo > 1 , 25 ; donna > 1 , 06 ; 80 anni : uomo > 1 , 00 ; donna > 0 , 85 . per quanto riguarda la formula mdrd semplificata , nella nostra preliminare esperienza ( periodo 1 aprile15 maggio 2007 ) abbiamo rilevato in alcuni pazienti anziani un valore di fg stimato maggiore della soglia di 60 a fronte di un valore inferiore alla medesima soglia nel calcolo eseguito con la formula di cockcroft - gault . 
di conseguenza , alla luce dellalta prevalenza di esami con mdc in pazienti di et superiore a 60 anni nella nostra realt operativa ( 71% , ed in particolare 57% dei pazienti con et compresa tra 61 e 80 anni e 14% dei pazienti con et superiore a 80 anni ) , il laboratorio di analisi ospedaliero dal giugno 2007 ha eseguito il calcolo automatico del fg dalla creatininemia solo nella fascia di et 1860 anni , in attesa di poter raccogliere una ampia casistica di confronto tra le due formule nella nostra popolazione . fino al momento attuale almeno l80% dei pazienti giunge in radiologia il giorno dellesame senza che sia riportato nella nota informativa il calcolo del fg da parte del medico inviante . 
in ogni paziente il personale infermieristico e medico della radiologia valuta indipendentemente questo parametro , anche se gi calcolato , impiegando le formule riportate nel retro del modulo . in media il 20% dei pazienti prenotati per un esame con mdc mostra il giorno dellesame un fg stimato patologico ( < 60 , insufficienza renale significativa )  . 
il paziente con insufficienza renale viene riprenotato nellagenda degli appuntamenti della radiologia e contemporaneamente indirizzato allambulatorio nefrologico per lopportuna valutazione e preparazione allesame con mdc ( consulenza nefrologica e profilassi dei danni da somministrazione di mezzo di contrasto )  . 
il nefrologo valuta il paziente per i fattori di rischio alla somministrazione del mdc , quantizza il rischio stesso ( tabella 1 ) e prescrive la profilassi , da eseguirsi in connessione allesame con mdc mediante idratazione con soluzione fisiologica isotonica e somministrazione di acetilcisteina . 
la tabella 1 stata derivata da uno studio eseguito per la somministrazione intra - arteriosa di mdc [ 14 ] , in cui si usata una metodica di calcolo semiquantitativo del rischio da mdc . 
the usefulness of n - acetylcysteine in preventing contrast - induced nephropathy [ 19 ] is still being debated in the literature ; however , until conclusive evidence becomes available , we believe it reasonable to continue its use , as it is inexpensive , simple to administer and free of side effects . 
in particolare per lidratazione del paziente abbiamo seguito i consigli della recente letteratura [ 16 ] con la somministrazione di soluzione fisiologica 0 , 9% per via venosa alla dose di 1 , 01 , 5 ml / kg / ora . mentre nel paziente ricoverato non abbiamo problemi ad iniziare linfusione 12 ore prima dellesame con mdc ed a continuarla per almeno 12 ore dopo lindagine , nel paziente ambulatoriale siamo obbligati a ridurre la tempistica a 3 ore prima ed a 6 ore dopo lindagine . 
allidratazione abbiamo aggiunto la somministrazione di acetilcisteina per os secondo queste dosi : 600 mg due volte al giorno , per due giorni a partire dal giorno precedente allesame con mdc [ 18 ]  . 
in letteratura lutilit dellacetilcisteina per la prevenzione della nefropatia da mdc assai controverso [ 19 ] , ma in attesa di conoscere dati conclusivi sullargomento , noi labbiamo impiegata perch poco costosa , somministrabile facilmente e priva di effetti collaterali . 
se ritenuto necessario il nefrologo prescrive un dosaggio della creatininemia dopo lesame con mdc ( a 12 , 24 , 36 o 48 ore a seconda del caso ) , considerando come nefropatia da mezzo di contrasto [ 20 ] un aumento del 25% della creatininemia entro 3 giorni dalla somministrazione del mdc . 
inoltre per prevenire leventuale acidosi lattica [ 21 ] , ai pazienti diabetici in terapia con metformina viene prescritta la sospensione del farmaco 48 ore prima dellesame con mdc , con ripresa della terapia antidiabetica orale 48 ore dopo , previa verifica della funzionalit renale . 
per gli esami non programmabili stato istituito un protocollo di profilassi rapido ( soluzione di bicarbonato di sodio 1 , 4% : 3 ml / kg in unora prima della somministrazione del mdc , seguiti da una dose di 1 ml / kg / ora per 6 ore dopo linfusione del mdc [ 22 ] )  . 
 in addition , to prevent possible lactic acidosis [ 21 ] , diabetic patients receiving metformin are instructed to discontinue the medication 48 h before and not resume it until 48 h after the examination , after renal function has been evaluated . 
a quick prophylactic protocol has been developed for nonelective examinations ( 1.4% sodium bicarbonate : 3 ml / kg 1 h before icm injection , followed by 1 ml / kg per hour for 6 h after icm injection [ 22 ] )  . 
these include hyperthyroidism ( table 2 ) , previous allergic reactions to iodinated substances and / or previous adverse reaction to icm and severe asthma or allergy ( evaluation by allergologist and anaesthesiologist before the day of the examination , with eventual desensitisation therapy prior to examination ) , need for sedation / general anaesthesia ( preliminary evaluation by anaesthesiologist in uncooperative patients )  . 
 the patient history / screening form then reminds the referring physician and the patient of the need to bring along to the examination any previous relevant documentation and requests information on the patients weight and height to dose the amount of icm required . 
with regard to the maximum dose of contrast medium , it was reported almost 20 years ago that in the case of renal insufficiency , the maximum dose should be 5 ml of icm per kilogram of body weight divided by the creatinine value [ 23 ]  . 
per quanto riguarda la dose massima di mdc da somministrare al paziente , quasi venti anni fa stato segnalato in letteratura che in caso di insufficienza renale bisogna considerare una dose limite di 5 ml di mdc per kg di peso corporeo diviso il valore della creatininemia [ 23 ]  . 
per i mdc attualmente in uso lesur [ 24 ] ha segnalato che nei pazienti adulti con normale funzionalit renale non si dovrebbero superare i 400 ml di mdc ( alla concentrazione di 300 mg di iodio per ml ) , mentre la dose andrebbe drasticamente ridotta nei pazienti con compromissione della funzionalit renale ( 150 ml se linsufficienza renale modesta , 100 ml se linsufficienza renale grave )  . 
anche il cin consensus working panel ha raccomandato di mantenersi ad una dose minore di 100 ml di mdc nei pazienti con fg < 60 ml [ 15 ]  . 
infine il paziente viene informato che sconsigliato ripetere un esame con mdc prima che sia trascorsa almeno una settimana da una precedente analoga somministrazione . segue la parte del consenso allatto radiologico , da compilare e firmare nel corso del colloquio preliminare tra il medico radiologo ed il paziente . 504 table 2 thyroid function radiol med ( 2009 ) 114 : 496508 evidence of manifest hyperthyroidism is a contraindication to the administration of iodinated contrast media . subjects at risk of thyrotoxicosis after iodinated contrast media ( icm ) administration are patients with basedow disease and multinodular toxic goitre , especially those living in areas with dietary deficiency of iodine and / or elderly patients . patients at risk should be monitored after icm administration by means of thyroid - stimulating hormone ( tsh ) and ft4 testing , preferably by an endocrinologist . in selected high - risk patients , tsh and ft4 testing may be performed , and prophylactic pharmacological treatment may be given prior to icm administration . 
 tabella 2 funzionalit tiroidea levidenza di franco ipertiroidismo una controindicazione alla somministrazione di mezzi di contrasto organo - iodati . sono a rischio di tireotossicosi dopo somministrazione di mezzi di contrasto organo - iodati i pazienti con morbo di basedow e gozzo multinodulare tossico , specie se residenti in aree geografiche a carenza iodica e / o anziani . questi pazienti a rischio dovrebbero essere monitorati dopo lesame con mezzi di contrasto organo - iodati mediante il dosaggio di tsh e ft4 , preferibilmente dallendocrinologo . in pazienti ad alto rischio selezionati possono essere indicati il dosaggio di tsh e ft4 ed una eventuale profilassi farmacologica tireostatica prima dellesame con mezzi di contrasto organo - iodati . used icm , the european society of urogenital radiology ( esur ) [ 24 ] stated that in adults with normal renal function , the maximum recommended dose is 400 ml icm ( at a concentration of 300 mg of iodine per millilitre ) , whereas this amount should be drastically reduced in patients with renal insufficiency ( 150 ml if moderate , 100 ml if severe )  . even the contrast - induced nephropathy consensus working panel recommended a dose of icm less than 100 ml in patients with gfr < 60 ml [ 15 ]  . 
finally , the patient is informed that it is inadvisable to repeat an examination with icm before at least 1 week has passed after a previous icm administration . the patient history / screening form is followed by the informed consent form , to be filled in and signed during the verbal interview between the radiologist and patient prior to the examination . discussion the aim of this paper is to share our experience in addressing the problem of icm use so as to assist others seeking to tackle the same problem in their setting . 
the excellent and exhaustive material made available by sirm [ 1 , 2 ] regarding the new guidelines for the use of injectable contrast media had had insufficient uptake among both discussione lo scopo del presente lavoro stato quello di voler condividere la nostra esperienza di fronte al problema dellimpiego dei mdc , perch possa essere di aiuto a chi si trovi nella propria realt operativa a dover affrontare unanaloga situazione . 
lottimo ed esaustivo materiale pubblicato in ambito societario [ 1 , 2 ] sui nuovi orientamenti nelluso del mdc per via iniettiva non aveva avuto nella nostra zona una sufficiente divulgazione , sia tra gli specialisti in radiologia che tra i medici invianti , per far sentire come necessario un cambiamento radicale nellapproccio al problema ( preparazione del paziente , nota informativa , consenso informato , addestramento del personale alla gestione delle reazioni avverse al mdc )  . 
di qui la necessit di una rivisitazione locale della problematica con i referenti non radiologi interessati ( nefrologo , anestesista rianimatore , endocrinologo , allergologo , medico legale , direzione medica ospedaliera ) , affrontata con entusiasmo da tutti i colleghi coinvolti . 
molto pi difficile nella nostra esperienza stata ( ed tuttora ) la condivisione della problematica con gli altri . per quanto riguarda il personale della radiologia , dal punto di vista concettuale , le perplessit ed i dubbi rilevati nelle riunioni di reparto del 2004 e del 2005 sono stati chiariti con la buona partecipazione del personale ai corsi bls o d - bls ed al progetto formativo la diagnostica per immagini : linee guida nazionali di riferimento , utile radiol med ( 2009 ) 114 : 496508 radiologists and referring physicians in our area , calling for a radical change in the approach to the problem ( patient preparation , patient history / screening , informed consent , training of personnel in the management of adverse reactions to icm )  . 
the issue of icm use was thus revisited locally with all the professionals , other than radiologists , involved in the process ( nephrologist , anaesthetistresuscitator , endocrinologist , allergologist , legal medicine specialist , hospital management ) , an opportunity that was met with enthusiasm by all concerned . 
 we experienced ( and still experience ) far more difficulty in sharing the project with other professionals . with regard to radiology department personnel , conceptually , the concerns and doubts aired at the meetings held in 2004 and 2005 were solved , with a good level of participation in the bls and blsd courses and the training course diagnostic imaging : national guidelines , which also served as a refresher course on indications for radiological procedures and the use of the various imaging modalities in individual clinical cases . in practice , whereas gfr calculation has become routine practice at our radiology department , there are still problems with the 20% of patients who are found to have a pathological gfr on the day of the examination , causing major logistic difficulties in rescheduling the examinations to a date compatible with management of the patients condition . the reason for this is , we believe , the fact that despite their moderately good level of participation in the course new working tools for diagnostic imaging and their extensive involvement in the project ( through meetings with their scientific and union representatives ) , referring physicians still do not feel the problem of icm use in a homogeneous manner . 
preliminary evaluation of the gfr appears to be the cornerstone of the entire process of preventing icm - induced toxicity , and advance gfr evaluation by the referring physician allows proper patient preparation to be planned . 
failure to evaluate the gfr exposes the patient to the risk of not being able to undergo contrast administration on the day of the examination ( scheduled with great difficulty given the normal waiting lists ) , with obvious consequences on patient care ; on the other hand , it is difficult for the radiology department to reschedule examinations for which there is no alternative and that become increasingly urgent over time . we therefore believe that serum creatinine determination with gfr calculation needs to be done at the time of referral , whether examinations are performed on inpatients , urgent outpatients or elective outpatients . 
furthermore , we believe that another refresher course on the subject , scheduled for 2009 for both physicians and nurses in our area , may be more effective than sending out formal and informal reminders for them to fill in the patient history / inoltre per aggiornare chi ne avesse bisogno anche sullindicazione agli esami e sulluso delle varie metodiche di imaging nel singolo caso clinico . dal punto di vista pratico , mentre ormai entrato nella routine lavorativa della nostra radiologia il calcolo del fg , grandi disagi ancora derivano da quel 20% di pazienti che vengono quotidianamente rinviati il giorno dellesame per il riscontro in diagnostica di un fg patologico , con grosse difficolt logistiche nel riprogrammare tali esami in tempi utili per la gestione della malattia del paziente . 
ci dovuto a nostro avviso al fatto che i medici invianti , nonostante la discreta partecipazione al progetto formativo nuovi strumenti di lavoro per la diagnostica per immagini ed al loro ampio e prezioso coinvolgimento ( attraverso gli incontri con le loro rappresentanze scientifiche e sindacali ) , non sentono ancora in maniera omogenea la problematica . e , fra tutti i fattori di rischio , la valutazione preliminare del fg appare nella nostra esperienza come il punto cardine di tutto il processo . 
la mancata valutazione preliminare del fg invece espone il paziente al rischio di non poter effettuare lesame con mdc il giorno dellappuntamento ( programmato con fatica viste le normali liste di attesa ) , con ovvie conseguenze sulla gestione della sua malattia , e la radiologia a non sapere quanto riprogrammare esami che bisogna in ogni caso fare e che pi tempo passa pi diventano urgenti . 
pensiamo quindi che sia necessario verificare il valore della creatininemia con il calcolo del fg gi al momento della prenotazione degli esami dei pazienti ricoverati ed ambulatoriali urgenti , nonch degli ambulatoriali programmati nel tempo . 
per questi ultimi , se a rischio per nefropatia da mdc , tale verifica andr ripetuta anche in stretta prossimit della data di esecuzione dellindagine con mdc . inoltre al proposito pensiamo che un nuovo progetto formativo di rinforzo sullargomento , gi in programma per lanno prossimo ( sia per i medici che per gli infermieri professionali della nostra zona territoriale ) , possa essere pi utile dei richiami informali e formali gi effettuati per una compilazione completa ( comprensiva del calcolo del fg ) della nota informativa . infatti utile ribadire come la prevalenza di insufficienza renale non nota nella popolazione elevata ( fg < 60 = 7 , 4% [ 25 ] )  . 
questo ovviamente risulta tanto pi vero quanto pi il soggetto anziano ( fisiologico calo della funzionalit renale di 1 ml / min per anno dopo i 2030 anni di et [ 26 ] ) , vasculopatico o sottoposto a terapia con farmaci nefrotossici ( tipo la chemioterapia )  . 
daltra parte essenziale cercare di evitare il pi possibile il danno della funzionalit renale dovuto alluso non prudente del mdc ( linsufficienza renale da mdc stata stimata come la terza causa di insufficienza renale acuta in ospedale [ 27 ] )  . 
a tal proposito 506 radiol med ( 2009 ) 114 : 496508 screening sheet completely ( including gfr calculation )  . it should be emphasised that the prevalence of undetected renal insufficiency in the general population is high ( gfr < 60 ml = 7.4% [ 25 ] )  . 
this is particularly true in the elderly ( renal function decreases physiologically by 1 ml / min per year after the age of 2030 years [ 26 ] ) and in subjects with vascular disease or undergoing treatment with nephrotoxic agents ( such as chemotherapy )  . 
on the other hand , it is important to minimise the risk of renal impairment due to the imprudent use of contrast media ( icm - induced nephrotoxicity is reported to be the third leading cause of acute renal failure in a hospital setting [ 27 ] )  . 
the possibility of a logistic support system in charge of prophylactic treatment before and after icm administration , such as the one activated at our nephrology and dialysis department , is thus essential . 
 owing to the lack of agreement in the literature on the subject of nephrological consultation ( only with gfr < 30 ml [ 28 ] ; in all patients with gfr < 60 ml [ 29 , 30 ] ) , we initially opted for nephrological consultation in all patients with gfr < 60 ml . 
the gfr threshold value required for nephrological consultation may be lowered in the future should it be demonstrated that this is not detrimental to the patient . because it is impossible to routinely request determination of actual gfr ( measured as creatinine clearance = urine creatinineurine volume in 1 min / serum creatinine ) , which requires 24 - h urine collection , we decided to use the cockcroftgault formula for gfr estimation [ 13 ] , which we have used successfully for many years . 
the method is also inexpensive ( it requires only serum creatinine determination : cost = 1.50 euro ) and is thus suitable for screening purposes . its main disadvantage , in our experience , is that it does not allow for the automatic calculation of gfr , as the hospital clinical laboratory does not record patients weight and height . 
 another recently proposed formula is the mdrd [ 31 ] : gfr = 170 ( serum creatinine ) 0.999 ( age ) 0.176 ( blood urea nitrogen ) 0.170 ( serum albumin ) 0.3180.762 ( if patient is female ) / 1.73 m2 ( of body surface )  . 
this led us to perform an in - depth comparison of the cockcroftgault and simplified mdrd formula in each patient in quindi riteniamo indispensabile lattivazione di un percorso che coinvolga il nefrologo per i pazienti a rischio . 
la possibilit di un supporto logistico competente per il paziente ambulatoriale per la profilassi pree post - somministrazione di mdc , come quello da noi attivato presso la nefrologia e dialisi ospedaliera , risulta quindi essenziale . avendo ritrovato in letteratura dati discordanti sullargomento ( consulenza nefrologica solo con fg < 30 [ 28 ] ; consulenza nefrologica in tutti i pazienti con fg < 60 [ 29 , 30 ] ) , abbiamo optato inizialmente per la consulenza nefrologica in tutti i pazienti con fg < 60 . 
anche se ci comporta un notevole carico di lavoro , vogliamo poter verificare in tutti questi pazienti eventuali modificazioni della funzionalit renale indotte dalluso del mdc , riservandoci in futuro di ridurre il valore del fg necessario per la consulenza nefrologica se dimostreremo nella nostra popolazione che ci possa essere fatto senza detrimento per il paziente . non essendo possibile nella routine pratica richiedere la valutazione reale del fg ( misurato come clearance della creatinina = creatinina urinaria volume urinario al minuto / creatinina plasmatica ) , che necessita della raccolta completa delle urine delle 24 ore , tra le formule a disposizione per la stima del fg , abbiamo scelto quella di cockcroft - gault [ 13 ] perch la usiamo con soddisfazione clinica da molti anni . 
il suo svantaggio principale nella nostra esperienza quello di non consentire un calcolo automatico del fg , dal momento che non vengono registrati al laboratorio di analisi il peso e laltezza dei pazienti . 
tale formula , validata , utile soprattutto nei pazienti con insufficienza renale avanzata , si presterebbe ad una valutazione automatica del fg da parte del laboratorio di analisi , ma necessit di pi dosaggi ( costo = 4 , 5 euro ) rispetto alla cockcroft - gault e quindi ha un costo maggiore . 
nella letteratura inoltre il suo confronto con la formula di cockcroft - gault non ne ha ancora dimostrato una definitiva maggiore affidabilit , in particolare nel paziente anziano [ 32 ]  . 
i dati in proposito sono in corso di raccolta e quando avranno raggiungo un congruo numero saranno oggetto di un successivo lavoro . per quanto riguarda i tempi tra il dosaggio della creatininemia e lespletamento dellesame con mdc , nel modello radiol med ( 2009 ) 114 : 496508 our population . 
on this subject , the esur guidelines [ 35 ] state that , even in areas with dietary iodine deficiency , as is ours , there is no need to evaluate thyroid function before icm administration in patients without clinical evidence of thyroid disease . 
moreover , because the bottles of contrast medium always contain amounts of free iodine ( much higher than the daily requirements ) , the esur considers patients with basedows disease and toxic nodular goitre as being at risk of developing thyrotoxicosis after icm administration , especially those living in areas with dietary iodine deficiency and / or the elderly . 
 finally , with regard to collaboration with the anaesthetistresuscitator , the ministerial circular of 17 / 09 / 97 [ 1 ] recommends only preliminary consultation with the anaesthesiologist and his / her presence in the hospital during the performance of examinations with icm on patients at risk for adverse reactions . 
however , in consideration of the distance between the radiology department and the operating theatre suite , we request that the anaesthesiologist is physically present in the radiology department during the examination . non si impone un tempo definito ( per esempio un massimo di un mese ) , ma si richiede lindicazione della data del dosaggio di laboratorio , per potersi regolare caso per caso , dal momento che nel paziente con buona funzionalit renale viene accettato anche un tempo di sei mesi [ 34 ] , mentre nel paziente con insufficienza renale pu essere necessario un dosaggio anche pi recente di una settimana . per quanto riguarda altri fattori di rischio alluso del mdc , ci sembra utile segnalare che , limitatamente al periodo sperimentale di 1 , 5 mesi di impiego del modello , i problemi riscontrati hanno riguardato anche la funzionalit tiroidea , con una transitoria esplosione di richieste di dosaggio di ft3 , ft4 e tsh , non giustificata dalla situazione clinica dei pazienti . 
al proposito le linee guida dellesur [ 35 ] chiariscono che , anche in aree geografiche con carenza di iodio come la nostra , non vi alcuna necessit di un dosaggio di laboratorio della funzione tiroidea prima di esami con mdc in pazienti clinicamente normali da questo punto di vista . 
inoltre , dal momento che i flaconi di mdc contengono sempre quantit di iodio libero ( di molto superiori al fabbisogno quotidiano ) , lesur cataloga i malati con morbo di basedow e gozzo multinodulare tossico come pazienti a rischio di sviluppare una tireotossicosi dopo la somministrazione di mdc , specie se residenti in aree geografiche a carenza iodica e / o anziani . 
the authors provide a simple yet exhaustive introduction to the concept of file format and describe the algorithms and main features of the most common formats ( bmp , jpeg , gif , dicom , tif , png ) and portable network graphics ( png )  . 
 the different formats are compared in terms of dimension , quality , portability and with reference to the following specific needs : electronic communications , publication on the world wide web , presentation of electronic posters , video presentations for teaching and manuscript publishing . 
we also illustrate how to handle the various formats with the programmes supplied with standard software installations . the large number of digital applications of image file formats calls for a simplification in daily radiological practice . 
we recommend the use of jpeg and png for electronic communications ; png and gif for publication on the worldwide web ; jpeg and png for electronic poster presentations ; dicom , png and jpeg for teaching presentations ; tif and png for printing on paper . riassunto evidenziare le differenze tra i pi comuni formati di salvataggio delle immagini radiologiche digitali . suggerire un uso appropriato e garantire la facilit di manipolazione nella gestione delle pi tipiche applicazioni pratiche nel lavoro quotidiano in radiologia . vengono presentati in modo semplice ma esaustivo il concetto di formato file , gli algoritmi dei formati pi comuni ( bmp , jpeg , gif , dicom , tif , png ) e le loro caratteristiche principali . vengono messi a confronto i differenti formati in base a dimensioni , qualit , portabilit ed in relazione alle seguenti situazioni specifiche : comunicazione elettronica , pubblicazione su pagine internet , presentazione di poster elettronici , comunicazioni didattiche , stampa di pubblicazioni . 
viene inoltre illustrato come gestire tali formati con i programmi che sono distribuiti assieme alle installazioni software standard . le numerose applicazioni digitali dei file immagine rendono dobbligo una semplificazione nella pratica radiologica quotidiana . 
in this context , it is very difficult to obtain that broad overview of the subject that is essential for selecting the most appropriate image file format for specific situations . 
 moreover , there is a widespread casualness in handling images , in part due to indifference in the choices made when sending e - mails , displaying images on screen or organising an institutional policy for image storage . 
such inappropriate decisions can result in minor or major mishaps that may be more or less strongly perceived ; for example , obliging a colleague to wait an additional 35 min at the monitor to download an attachment or , far worse , being obliged to continuously purchase supplementary hard disks because the storage server has reached full capacity . as it is not necessary to go into extreme detail to master the competences required to handle these tasks , we felt it might be useful to provide an overview of common resources and give a few simple recommendations , in plain language and using concrete examples . introduzione la gestione delle immagini digitali nella quotidiana pratica radiologica pu essere molto complicata . 
 assistiamo inoltre ad una diffusa leggerezza nella gestione delle immagini , in parte sostenuta da una certa indifferenza , nelle scelte che si operano quando si decide di inviare una email , di presentare a video delle immagini , o di organizzare una politica di archiviazione di istituto . 
si passa dallobbligare un collega ad attendere tre / cinque minuti in pi davanti ad un monitor per scaricare un allegato , a quello ben pi costoso di costringerci continuamente allacquisto di unit disco supplementari perch il nostro server di archiviazione gi pieno . dal momento che non necessario scendere nel dettaglio per impadronirsi delle competenze utili in tale direzione , ci parso appropriato stendere una panoramica delle risorse di uso comune e qualche semplice linea guida , senza mai sacrificare la semplicit del linguaggio , attraverso esempi concreti . image formats i formati immagine digital images are handled by computers as a grid of rows and columns of coloured dots , something like a battleship grid . 
each square corresponds to a dot on the screen a pixel which represents the elementary unit of every image . each pixel is characterised not only by its colour but by its coordinates that is , by its position within the image . 
otherwise , we could describe the content of each square in order , by announcing first row : water , water , water , ship , water second row : water , ship , ship and so on . 
in computing , these different techniques for describing the same image are known as formats . a format is a standardised method , that is , one that can be reproduced on any computer , for reading and arranging the le immagini digitali sono gestite da un computer come una griglia di righe e colonne di punti colorati , simili ad una tavola da battaglia navale . 
immaginiamo il seguente esempio : una griglia da battaglia navale di dieci righe per dieci colonne . essa contiene cento caselle , di cui la maggior parte sono azzurre ( lacqua appunto ) , ed una minore parte sono grigie , poich occupate dalle navi . 
oppure si potrebbe descrivere il contenuto di ogni casella ordinatamente , dichiarando prima riga : acqua , acqua , acqua , nave , acqua , seconda riga : acqua , nave , nave , e cos via . 
o ancora si potrebbe dire prima riga : tre caselle di acqua , una occupata da una nave , poi acqua fino a fine 486 radiol med ( 2009 ) 114 : 484495 fig . 
however , all formats can be classified according to three main features : the size of the image file , the maximum number of colours and possible loss of detail . 
 in particular , the size of the image file refers to the amount of physical memory ( in kilobytes or megabytes ) that a file takes up on a storage device . 
compression algorithms are extremely important , as the amount of memory needed to store an image can be reduced to the point that the image is several times smaller than the original . 
queste differenti tecniche di descrivere la stessa immagine sono note in informatica con il termine di formati . un formato un metodo standardizzato , ovvero riproducibile su qualsiasi pc , per leggere ed organizzare le informazioni contenute in un file . 
una volta selezionato un file immagine , il computer ne riconosce lestensione , abbina il formato corretto , e attua una precisa sequenza di operazioni per manipolare i dati e presentarli a schermo , chiamato algoritmo . 
ogni formato nasce per soddisfare particolari esigenze , ma tutti sono classificabili in base a tre caratteristiche principali : la dimensione del file immagine , il numero massimo di colori e leventuale perdita di dettaglio . 
 in particolare , con il termine dimensione del file immagine intendiamo la quantit di memoria fisica ( in unit kilobyte o megabyte ) che una immagine occupa su un supporto per memorizzarla . 
2 la stessa immagine , rappresentata con due profondit di grigi molto diverse . a destra , con una bassa profondit di colore , limmagine perde tutti i dettagli . whose size has been reduced by 90% will require only a tenth of time to be transferred from one peripheral device to another . 
some formats are very effective in compressing images ; others less so . the maximum number of colours refers to the colour depth , or the number of different colour nuances that the image can encode . 
this happens because each colour in the image will be specified much more accurately , with a larger series of digits . absence of loss of detail refers to the formats ability to preserve the individual details of the original image . 
as even minimal loss of data allows for very effective compression , some formats have been developed that expressly eliminate some barely perceivable details in favour of an excellent reduction in volume . 
alcuni formati sono eccellenti nel comprimere immagini , altri meno . con il termine numero massimo di colori , intendiamo la cosiddetta profondit del colore , ovvero il numero di differenti sfumature di colore che limmagine pu codificare . per fare un esempio pratico si pensi ad una proiezione pa di un torace . 
con una profondit del colore molto alta , ovvero con la possibilit di rappresentare milioni di tinte diverse , otteniamo una rappresentazione a video dellimmagine cos come siamo soliti pensarla , cio ricca di dettagli e di sottili differenze di sfumatura tra una struttura e laltra . allestremo opposto immaginiamo una profondit di colore di sole poche tinte . 
dal momento che anche piccole perdite di dati permettono una compressione molto efficace , si sono sviluppati alcuni formati che prevedono esplicitamente lo scarto di alcuni dettagli difficilmente percepibili in nome di una eccellente riduzione di volume . 
la conversione verso formati lossy accumula perdita di dati , che irreversibile . is thus a dot - by - dot description of the image , and for this reason , it is regarded as the standard of reference . 
the jpeg format has excellent colour depth , as it can have up to several million colours . the strength of this type of algorithm lies in its ability to reduce the size of images on the disk . 
essi sono i formati bitmap , jpeg , gif , dicom , tiff e png [ 18 ]  . formato bitmap un file immagine in formato bitmap costituito dalla serie di coordinate e colori di ogni singolo punto dellimmagine . si tratta quindi di una descrizione punto a punto dellimmagine , e per tale motivo limmagine bitmap considerata una misura di riferimento . 
il formato jpeg ha il grande pregio di permettere una compressione regolabile dallutente , che pu quindi scegliere quanto efficace essa debba essere . purtroppo ogni singolo programma di fotoritocco gestisce lefficacia della compressione jpeg in modo diverso . alcuni programmi , come ms paint , non danno affatto la possibilit di scegliere lefficacia della compressione , e ne utilizzano una pre - impostata , a basso rendimento . 
noi abbiamo usato come riferimento paint.net , poich si tratta di una alternativa gratuita a ms paint , semplice da usare e molto completa . va inoltre sottolineato come il formato jpeg sfrutti un algoritmo lossy , ovvero con una perdita di dati . 
il formato jpeg viene oggi impiegato soprattutto nel salvataggio e nella memorizzazione di immagini fotografiche , dove permette una minima perdita di dettaglio percepibile contro un grande vantaggio in termini di spazio di archiviazione . formato gif il formato gif un po obsoleto e molto sottodimensionato in quanto a caratteristiche . 
si tratta di un formato con forti limiti nella profondit del colore , e un algoritmo di compressione non particolarmente efficace su immagini fotografiche , che perdono notevolmente di dettaglio nella riduzione dei colori disponibili . 
sebbene infatti lalgoritmo gif sia teoricamente loss - less , nella pratica la conversione in questo formato di immagini di uso comune , come radiografie o fotografie , produce immagini assolutamente incomparabili alloriginale , a causa della sopracitata importante perdita di profondit di colore . 
the jpeg format is especially used for storing photographic images , where it allows a minimal loss of perceivable detail against major advantages in terms of storage space . gif format formato dicom the gif format is rather obsolete , and its features have been heavily reconsidered . 
the format has strong limitations in colour depth and employs a compression algorithm that is not very effective on photographic images , which lose considerable detail in the colour reduction process . 
in fact , although the gif algorithm is theoretically loss - less , in practice , the conversion of commonly il formato file dicom uno standard industriale [ 68 ] tuttora in sviluppo . 
ci vuol dire che un file dicom pu contenere una immagine a pieno dettaglio in formato bitmap , oppure compressa con algoritmo jpeg , 490 radiol med ( 2009 ) 114 : 484495 used images such as radiographs or photographs into gif produces images that are absolutely unlike the originals , owing to the substantial loss of colour depth . 
for this reason , the gif format is preferred for web pages , which tend to contain many of these elements . dicom format the dicom format is an industrial standard [ 68 ] that is still being developed . 
it is an anomalous format that does not introduce any new compression algorith instead , it merely encapsulates , or contains , an image in the format in which it was produced . 
however , the main difference between the two formats is that dicom is able to memorise a series of data not necessarily linked to the image , such as the patients demographic information , type of examination , date and time of the examination and so on . 
for this reason , dicom images are particularly suited for use in integrated reporting workstations or software - based reconstructions , but there is no difference in the quality of the image itself . also , as with the bmp format , dicom often generates very large images that are difficult to store . 
tipicamente le immagini dicom prodotte dalle apparecchiature radiologiche sono riproduzioni non compresse dellimmagine originale , e per questo motivo sono comparabili a delle immagini bitmap , di cui ereditano le caratteristiche . 
tuttavia la grande differenza tra questi due formati che il dicom in grado di memorizzare una serie di dati non necessariamente legati allimmagine visiva , come i dati anagrafici del soggetto , il tipo di esame , la data e lora di esecuzione , e cos via . 
per questo motivo esse sono molto pratiche nelle console di refertazione integrate o nelle ricostruzioni tramite software , ma non si distinguono dagli altri formati per nessuna vera qualit riferibile allimmagine stessa , anzi , parimenti al formato bitmap spesso producono immagini di grandi dimensioni , poco pratiche da archiviare . 
purtroppo i file tiff sono molto voluminosi , e per questo motivo non sono molto utilizzati , se non per scopi di archiviazione di immagini molto importanti , campo in cui si impongono come prima scelta . 
esso stato sviluppato appositamente per concentrare i punti di forza di molti formati in uno solo . le principali qualit delle immagini png risiedono in una buona compressione senza perdita di dati ( loss - less ) , nella capacit di supportare profondit di colore molto alte , e nella gestione delle trasparenze . 
sempre pi programmi supportano questo formato , e probabilmente il formato png si imporr a breve termine come unalternativa estremamente valida nei compiti di archiviazione e stampa [ 4 ]  . png format discussione png ( pronounced ping ) is the newest among the image file formats discussed here . 
it was specifically developed to combine the strengths of several formats in a single format . the main qualities of png images include efficient lossallo scopo di illustrare pi chiaramente le differenze tra i diversi formati che abbiamo discusso , e prefigurarne un utilizzo pratico , abbiamo allestito una semplice tavola sinottica che ne riassuma le caratteristiche essenziali radiol med ( 2009 ) 114 : 484495 less compression , very high colour depths and ability to handle transparencies . 
a growing number of programmes support this format , and it is likely that png will soon impose itself as an extremely valuable option for storage and print publishing [ 4 ]  . discussion the differences and practical uses of the various formats discussed in this paper are summarised in the synoptic table ( table 1 )  . 
per rendere lidea della capacit di compressione dei vari formati abbiamo ideato un semplice test . abbiamo convertito una immagine di riferimento bitmap di 466382 pixel raffigurante una proiezione pa di un torace , nei formati jpeg , gif , tiff e png . 
abbiamo quindi indicato nella colonna efficacia compressione torace la dimensione finale del file , dove a percentuali pi basse corrisponde una dimensione file minore ( e quindi una compressione pi alta ed una relativa perdita di dettaglio ) ( tabella 1 )  . 
 per quanto riguarda lutilizzo pratico delle immagini , nella routine radiologica le situazioni che ne richiedono un uso accorto sono essenzialmente : linvio tramite posta elettronica , la pubblicazione di immagini su pagine internet , la preparazione di poster elettronici illustrati , la presentazione a video di diapositive e limpaginazione di elaborati per la stampa cartacea . table 1 synoptic table of the main features of the various file formats . 
a questo punto nel menu a tendina salva come : possibile selezionare il formato di destinazione . of the compressed file and indicated the output file size in the column titled compression effectiveness chest , where low percentages correspond to smaller file sizes ( and to a higher compression ratio and relative loss of detail ) ( table 1 )  . 
in such situations , the png format can be used , as it offers good compression performance without sacrificing the information that the image must convey . web publishing when publishing images on the internet , special attention should be paid to the size of image files because in that context , it is more important to provide users with a smooth comunicazione elettronica ( e - mail ) nellinvio di immagini attraverso la rete internet , e in particolar modo quando si tratta di pi immagini , fondamentale considerare la dimensione dei file che vogliamo inviare . 
dal momento che il formato jpeg un formato lossy , in alcune occasioni consigliabile optare per una soluzione che preservi tutti i dettagli dellimmagine di partenza , a scapito dellefficacia di compressione . 
in tali situazioni pu essere consigliabile adoperare il formato png , che presenta una buona compressione senza sacrificare linformazione che deve veicolare limmagine . pubblicazione su internet quando si desidera pubblicare immagini su una pagina web bisogna prestare particolare attenzione alle dimensioni dei file immagine . 
per tali motivi le indicazioni che si pongono per le comunicazioni tramite email sono particolarmente adatte anche in questo caso . poich tuttavia la velocit di caricamento delle pagine dipende in larghissima parte dalle dimensioni delle immagini ( in rapporto inversamente proporzionale ) [ 9 ] , potrebbe essere consigliabile utilizzare rapporti di compressione pi spinti nelle immagini jpeg . 
however , because download speed is highly dependent on image size ( the larger the image , the slower the download ) [ 9 ] , it may be useful to use higher compression ratios in jpeg images . 
in addition , web pages are often rich in animations , arrows and graphs with few colours and diagrams , so one should also consider using a format , such as the gif format , capable of handling simple animations and characterised by excellent compression capabilities for simple graphics . preparation of electronic posters all images included in an electronic poster should , in our opinion , be of excellent quality , because individual slides often remain on screen for several seconds , giving viewers the time to notice even subtle details such as discolouration or border imperfection . 
even in this situation , the jpeg format ( possibly at low - compression ratio ) may be best for images in which some detail can be sacrificed , whereas the png format may be best for images that should be graphically perfect , as in the magnification of a detail . 
to better appreciate these points , it may be useful to view the many electronic posters posted at news / eposternews and presented at the sirm 2006 and 2008 congresses . slide shows the video presentation of a series of slides is largely similar to an electronic poster , and the same principles apply . 
this also provides the possibility of manipulating the images in real time for example , with multiplanar reconstructions or renderings which will enhance the interactiveness of the presentation . print publishing the most important aspects in printing images on paper are the quality of the image and its size in pixels , as these will directly affect the quality of the printed image . 
in fact , in the process of transforming a digital image into a printed image , one needs to consider the physical dimension of the internet sono molto ricche di animazioni , frecce , grafici con pochi colori e schemi , e quindi non va ignorata la possibilit di utilizzare un formato in grado di gestire semplici animazioni e caratterizzato da una ottima capacit di compressione nella grafica semplice come il formato gif . preparazione di poster elettronici liconografia di un poster elettronico dovrebbe , a nostro parere , essere particolarmente curata nei dettagli , anche nelle immagini meno importanti . 
ci giustificato dal fatto che spesso le singole diapositive rimangono a schermo a lungo , dando il tempo di notare anche sottili dettagli come le discromie o limperfezione dei bordi . 
ecco perch anche in questa situazione raccomandabile optare per il formato jpeg ( possibilmente a basso rapporto di compressione ) nelle immagini in cui si pu sacrificare un po di dettaglio , mentre il formato png in quelle che devono essere graficamente ineccepibili , come in un ingrandimento di particolare . 
a tale proposito risulta utile prendere visione dei numerosi poster elettronici pubblicati allindirizzo news , riguardanti il congresso sirm 2006 e 2008 . presentazione a video la presentazione a video di una serie di diapositive per molti aspetti una situazione simile a quella di un poster elettronico , ed infatti si applicano gli stessi principi . 
questo fornisce anche la possibilit di manipolare in tempo reale le immagini , per esempio con ricostruzioni multiplanari o rendering , che innalzano linterattivit della presentazione . stampa su carta per quanto riguarda la stampa su supporto cartaceo ci che maggiormente conta sono la qualit e le dimensioni in pixel dellimmagine , poich esse incideranno direttamente sulla bont dellimmagine nella pagina . 
per quanto riguarda le dimensioni delle immagini , la stampa su carta assume alcune caratteristiche singolari rispetto ad altre situazioni . infatti nel processo che porta dallimmagine digitale a quella stampata si introduce il concetto della dimensione fisica dellimmagine , ovvero della dimensione tangibile delloggetto ( carta , o altro supporto ) che contiene limmagine nella realt . 
per effettuare tale passaggio necessario specificare le dimensioni reali di un pixel , in modo che una stampante sappia , per usare un esempio limite , che un pixel 494 radiol med ( 2009 ) 114 : 484495 image , that is , the tangible dimension of the object ( paper or other medium ) containing the image . 
when transforming the image , one needs to specify the true dimensions of a pixel , so as to inform the printer that to use an extreme example a pixel on screen corresponds to a dot of 1 mm2 on paper . 
in practice , one specifies how many pixels should be contained in an inch ( 2.54 cm ) , the so - called dots per inch ( dpi )  . 
the smaller the pixels , the better the quality of the printed images , as more pixels will be printed per inch of paper , with a more natural - looking result . 
on the other hand , it should be noted that the value of the attribute dots per inch is an extrinsic value that bears no relation to the image displayed on screen . 
finally , although the tiff format is considered the reference standard , it is not necessarily superior to png for conventional printing purposes , and in some cases , it may be much easier to manipulate and send images at the more effective compression rates provided by the png format . conclusions each image file format was developed for a specific purpose , whether to minimise the size of the file or to preserve a faithful image . 
the tiff is the de facto standard when the maximum quality of the image data is needed . whenever possible , however , we recommend the use of the png format , which combines good compression effectiveness with preservation of image quality . sullo schermo corrisponde ad un punto di 1 mm2 sulla carta . nella pratica si preferisce specificare quanti pixel andranno contenuti in un pollice ( 2 , 54 cm ) , i cosiddetti punti per pollice , sintetizzati con lacronimo inglese dpi ( dots per inch )  . 
la qualit delle immagini su carta sar migliore se ci saranno pixel di minori dimensioni , poich in un pollice di carta ne verranno stampati un numero maggiore , dando allocchio una impressione pi naturale . 
per esempio , per stampare una immagine di 1010 pollici ad una risoluzione di 300 dpi ( la stessa risoluzione necessaria per la pubblicazione di immagini su la radiologia medica e su molte altre riviste ) necessaria una immagine di 30003000 pixel ( 300 dpi10 pollici )  . 
lo stesso risultato si pu ottenere stampando una immagine di 60006000 pixel a 600 dpi . daltro canto si vuole sottolineare come il valore dellattributo punti per pollice sia una caratteristica estrinseca dellimmagine , del tutto svincolata dalla sua rappresentazione a monitor . 
infatti possibile specificare tale valore a posteriori , e modificarlo a piacimento prima della stampa . a questo proposito si raccomanda lutilizzo di formati in grado di non perdere dettaglio e di poter gestire agevolmente tale attributo , come il formato tiff ed il formato png . 
a volte infatti pu essere di gran lunga pi agevole manipolare e spedire immagini alle compressioni pi efficaci garantite dal formato png . conclusioni ogni formato di file immagine nasce con uno scopo preciso , sia esso quello di minimizzare le dimensioni dei file , o quello di preservare una immagine fedele . 
levent1 1department of radiology , medical faculty , atatrk university , erzurum , turkey 2department of radiology , bilim university , florence nightingale hospital , istanbul , turkey 3department of radiology , numune hospital , erzurum , turkey 4department of pediatric cardiology , medical faculty , atatrk university , erzurum , turkey correspondence to : m . 
sok no 5 , dadaskent , erzurum , turkey , tel . : + 90 - 442 - 2361212 / 1521 , fax : + 90 - 442 - 2361301 , e - mail : akkanrad@hotmail.com received : 28 september 2008 / accepted : 31 october 2008 / published online : 14 april 2009 springer - verlag 2009 abstract purpose . 
these 18 patients were initially referred due to indications such as chest pain ( n = 11 ) , risk - factor assessment ( n = 3 ) , coronary artery anomaly ( n = 1 ) , suspected aberrant right subclavian artery due to dysphagia ( n = 1 ) and coronary artery bypass graft ( n = 2 )  . 
diciotto pazienti presentavano una tasca o una sacca a livello del setto interventricolare , verosimile espressione di chiusura spontanea del difetto interventricolare ( prevalenza 0 , 66% )  . i reperti della tcms di questi 18 pazienti sono stati correlati con i reperti ecocardiografici . 
il nostro studio dimostra che lincidenza di chiusura spontanea del difetto del setto interventricolare , radiol med ( 2009 ) 114 : 370375 patients initially diagnosed with ventricular diverticula with an apical and marginal septum location may actually have spontaneously closed muscular vsd . 
 keywords cardiac ct heart mdct angiography closed muscular ventricular septal defect introduction ventricular septal defects ( vsds ) are the second most common congenital heart defect after bicuspid aortic valves , but their true rate of incidence is , surprisingly , unknown [ 1 ]  . the incidence rate of vsds among live births is 1.53.5 per 1 , 000 term infants and 4.57.0 per 1 , 000 premature infants [ 2 ]  . the natural history of vsd , including incidence of spontaneous closure ( sc ) , is also uncerta however , some studies have reported the sc rate of vsd to be 48% by 6.9 years ( the mean age ) and 72% in patients without congestive heart failure ( chf ) [ 13 ]  . 
radiologists have begun to interpret coronary artery diseases , and in reporting the results of images obtained for coronary artery disease , they have begun to evaluate nonvascular cardiac structures as well . one of these is sc of muscular vsd observed in the interventricular septu in this study , we describe the experience of our institutions ( two centres ) in using cardiac multidetector ct angiography ( mdcta ) to diagnose sc of muscular vsd . materials and methods the study included 2 , 725 consecutive patients referred to atatrk university hospital and florence nightingale hospital for mdcta . 
in questo modo , alcuni pazienti , ai quali inizialmente era stata fatta diagnosi di diverticoli ventricolari a livello apicale e marginale del setto , in realt possono aver avuto una chiusura spontanea del difetto del setto interventricolare . 
questo risultato pu incidere sulle percentuali precedentemente riportate sia per quanto riguarda i diverticoli ventricolari sia per quanto riguarda la chiusura spontanea del difetto del setto interventricolare . parole chiave tc cardiaca cuore angiografia tcms chiusura del difetto del setto interventricolare disease , congenital malformation , atypical chest pain , screening for coronary artery disease and inconclusive conventional angiography . 
images were reviewed by a radiologist and a cardiologist in consensus , and the following data were recorded : gender , age , degree of coronary artery disease and presence of any other cardiac abnormality . 
similar records for images of the central muscular region to those of the earlier cases ( sc of muscular vsd ) were noted in the apical and marginal areas of the interventricular septuthese cases , with no history of surgery , were evaluated for sc of vsd . however , in the literature , ventricular diverticulum has been reported in all locations of the ventricle wall except 372 radiol med ( 2009 ) 114 : 370375 the interventricular septu in our study , patients with similar lesions in the basal inferoseptal , apical septum and midinferoseptal locations were not included because these lesions could not be differentiated from ventricular diverticula [ 5 , 6 ]  . 
 the procedures used were in accordance with the recommendations found in the declaration of helsinki , and approval of the local ethics committee was obtained . results retrospective electrocardiogram ( ecg ) - gated reconstructions were generated every 10% during the 50%90% of the r - r interval . 
 mdct scan protocol mdct was performed using two different 16 - detector - row ct scanners ( sensation16 , siemens medical systems , forchheim , germany , and aquillon , toshiba medical systems , tokyo , japan ) during one breath - hold ( 1624 s )  . with the first scanner , the following parameters were used : 160.75 - mm collimation , 1 - mm slice thickness and 0.6 - mm reconstruction interval . 
the indications for mdcta were suspected coronary artery disease ( n = 2 , 310 ) , coronary artery anomaly ( n = 22 ) , coronary artery stent patency ( n = 220 ) and coronary artery bypass graft assessment ( n = 173 )  . 
these 18 patients were referred due to indications such as chest pain ( n = 11 ) , risk factor assessment ( n = 3 ) , coronary artery anomaly ( n = 1 ) , suspected aberrant right subclavian artery ( n = 1 ) and coronary artery bypass graft ( n = 2 )  . 
1a - c a 46 - year - old man with spontaneous closure of muscular ventricular septal defect in the interventricular septu multiplanar reformatted multidetector computed tomography angiography ( mdcta ) images from 16 - mdct single - source scanner show long - axis ( a , c ) and short - axis ( b ) views of vsd ( ventricular septal defect ) ( arrows )  . 
le immagini angiografiche con tc a 16 strati mostrano lasse lungo ( a , c ) e lasse corto ( b ) del difetto del setto ventricolare ( frecce )  . 
of vsd location of vsd other abnormalities degree of coronary artery disease radiol med ( 2009 ) 114 : 370375 table 1 patient characteristics . tabella 1 caratteristiche dei pazienti . patient vsd , ventricular septal defect ; lad , left arterial dilatation central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular central muscular none moderate none mild none severe moderate none none none mild none mild severe none severe mild none myocardial bridging at lad middle segment aberrant right subclavian artery prior coronary artery bypass grafting aortic valve disease origin of the left circumflex coronary artery from the right sinus of valsalva myocardial bridging at lad middle segment prior percutaneous coronary intervention prior coronary artery bypass grafting ventricular function with no evidence of regional wallmotion abnormalities . 
vsds create a volume load on the left side of the heart secondary to the left - to - right shunt at the ventricular level , causing left atrial and left ventricular dilatation . 
the inlet defect makes up 5%8% of all vsds and is posterior and inferior to the perimembranous defect . muscular vsds make up 5%20% of all vsds [ 7 ]  . 
2a , b immagini assiale ( a ) e sagittale ( b ) del cuore di una donna di 39 anni con chiusura spontanea del difetto del setto interventricolare , che non si pu distinguere dai diverticoli settali a localizzazione apicale . laboratory tests for the diagnosis are not available . 
in the literature , ventricular diverticula have been reported to be located on all the ventricular walls ( apical and marginal septum ) except on the interventricular septum [ 5 ]  . 
nevertheless , similar appearances were observed in the apical and marginal septum as well . because they could not be differentiated from ventricular diverticula , these patients were excluded from the study . 
naturally , this finding may affect previously reported rates of both ventricular diverticula and the incidence of sc of muscular vsd . this will negatively affect the incidence rates of ventricular diverticula reported in earlier studies . 
it appears that there is a very high incidence of asymptomatic small muscular vsds that close spontaneously . isolated ventricular noncompaction is another entity that should be considered in the differential diagnosis . 
diagnosis of ventricular noncompaction usually requires the finding of more than three deep intertrabecular recesses [ 11 , 12 ]  . other pathologies that should be differentiated from sc muscular vsd are other causes of acquired ventricular aneurysms : those that occur after myocardial infarction , myocardial inflammatory disease such as sarcoidosis or myocarditis , infectious endocarditis or trauma [ 13 ]  . 
additional causes of ventricular aneurysm include connective tissue disease and ventricular dysplasia , as seen in patients with arrhythmogenic right ventricular cardiomyopathy . often , the distinction between fibrous ventricular diverticula and these entities can be made only with knowledge of cardiac history and visualisation of an associated abnormalitye.g. 
the true incidence of muscular vsd may have been underreported in the literature because of limitations inherent in the previously available imaging techniques and differences in the patient populations studied . 
because cardiac mdcta is frequently performed in patients with symptoms of chest discomfort , and because chest pain may be a signal of a cardiac abnormality , the incidence of sc muscular vsd is probably higher in this population than in the general asymptomatic population . however , in our study , a higher incidence of sc muscular vsd was shown in a large patient population . 
at our institutions , almost all of the cardiac mdcta studies are prospectively interpreted by two reviewers in consensus to generate an official report , and most reports are generated by two of the most senior reviewers . 
second , although a strict criterion consistent with the pathologic description of muscular vsd was chosen for the identification of this entity , pathologic proof of diagnosis was not obtained in any of our patients . 
however , given that the true prognostic implication of muscular vsd is unknown and that treatment of this entity is controversial , there is no indication to pursue an invasive procedure to confirm the diagnosis in these patients . in summary , this study shows that the incidence of sc muscular vsd with central septum location is probably higher than expected . 
3 immagine assiale del cuore di un uomo di 52 anni con chiusura spontanea del difetto del setto interventricolare , che non si pu distinguere dai diverticoli settali a localizzazione basale . this study has some limitations . 
guerci , radiologia padiglione barbieri , ospedale maggiore di parma , via gramsci 14 , 43100 parma , italy , tel . : + 39 - 052 - 1703660 , fax : + 39 - 052 - 1986352 , e - mail : leonardo.guerci@libero.it received : 12 august 2008 / accepted : 16 september 2008 / published online : 12 march 2009 springer - verlag 2009 abstract two women with profoundly different backgrounds were brought together in a destiny that saw their paths cross and join in the discovery of radioactivity : marie curie and blanche wittman . 
the discovery of radioactivity was the common denominator underlying the vicissitudes of their lives , the same radioactivity that was so acclaimed and of such incredible diagnostic and therapeutic potential while at the same time so underrated in the everyday life of the time that disregarded , almost disparagingly , the deleterious biologic effects it was capable of provoking . 
at the beginning of the twentieth century , those effects were in fact often underestimated or scarcely considered , and it was only after world war ii that there came an awareness of the ambiguous properties of ionising radiation . 
after numerous studies on radiation exposure , much of the current debate concerns the possible effects of exposure to small doses , such as those delivered in most radiological examinations . 
the theories proposed include the unorthodox theory of hormesis , which requires careful riassunto due donne , due storie profondamente diverse , unite da un destino che le incrocia e le unisce nella scoperta della radioattivit : marie curie e blanche wittman . 
la prima , una delle pi grandi donne e scienziate di tutti i tempi , unica nella storia ad aver vinto due premi nobel per la scienza , dotata di uno spirito umanitario fuori dal comune , dedic , nonostante lo scandalo sentimentale che la vide al centro della cronaca , la maggior parte della sua vita alla ricerca scientifica . 
la seconda , passata alla storia come la regina delle isteriche durante il suo ricovero nel famigerato manicomio della piti salpetrire di parigi , divenne alla sua guarigione una delle pi strette collaboratrici di m.me curie nellestrazione del radium dai fanghi della boemia , per ben 16 anni di duro lavoro , fino al momento della sua morte . 
ed proprio la scoperta della radioattivit il comun denominatore che sta alla base delle loro vicende umane , quella stessa radioattivit cos celebrata e dalle incredibili potenzialit diagnostiche e terapeutiche , ma allo stesso tempo cos svilita nella quotidianit della vita di quel tempo , che ignorava , talora quasi a sprezzo , gli effetti nocivi biologici in grado di provocare . 
nei primi anni del 900 tali effetti furono infatti spesso sottovalutati o scarsamente considerati , e fu solo dal secondo dopoguerra in poi che si manifest una forte presa di coscienza sulle ambigue propriet delle radiazioni ionizzanti . 
dopo numerosi studi condotti sullesposizione alle radiazioni ionizzanti , ci di cui molto si discute oggi riguarda principalmente i possibili effetti provocati dallesposizione a piccole dosi , come quelle erogate nella 348 radiol med ( 2009 ) 114 : 347357 reevaluation . 
much light has been shed on radiology since the time of blanche and marie , but there still remain many shadows to dispel , and this can only be done by serious and constant scientific commitment . keywords curie wittman ionizing radiation biological risk radiobiology maggior parte degli esami radiologici : tre le teorie proposte , una delle quali ( quella eretica della ormesi ) richiede di essere rivalutata con maggiore attenzione . molte luci si sono accese dai tempi della radiologia di blanche e marie , tuttavia al giorno doggi rimangono da chiarire ancora molte ombre , che solo un serio e costante impegno scientifico potr dissipare in futuro . parole chiave curie wittman radiazioni ionizzanti rischio biologico radiobiologia introduction introduzione a few years ago , the swedish writer p.o. 
enquist [ 1 ] wrote a charming book about two very different women brought together by the vagaries of life to work side by side and share hardships , danger and emotions linked with the early study of radioactivity : marie curie and blanche wittman . the clearly fictionalised vicissitudes narrated by enquist were the starting point for research that led us to analyse several aspects of the history of radiology . qualche anno fa , lo scrittore svedese p.o. 
enquist [ 1 ] ha dedicato un bel libro alla storia di due donne , molto diverse tra loro ma che i casi della vita hanno portato a lavorare fianco a fianco e a condividere fatiche , pericoli ed emozioni legate ai primi studi sulla radioattivit : marie curie e blanche wittman . 
a few days before she was awarded the prize in stockholm , a scandal over her affair with another famous physicist , paul langevin ( 18721946 ) broke out in paris . 
 this event was considered of such importance that the marie curie marie sklodowska , nata a varsavia , in polonia , nel 1867 , era figlia di un libero pensatore e di una cattolica praticante . 
pochi giorni prima della consegna del secondo nobel a stoccolma , a parigi scoppi lo scandalo per la relazione che la ancor giovane vedova aveva iniziato con un altro fisico famoso , paul langevin ( 18721946 )  . 
 radiol med ( 2009 ) 114 : 347357 questo evento ebbe una rilevanza tale che il fisico svante arrhenius , membro del comitato per il nobel , giunse a consigliare a marie di rifiutare il premio . 
lei stessa e la figlia irne ( allora diciassettenne ) , impararono a guidare questi automezzi , studiarono anatomia e , in compagnia di personale medico , prestarono a lungo la propria opera in zona di guerra . madre e figlia si sottoposero cos a forti dosi di raggi x senza alcuna protezione . 
negli stessi anni , marie fond un servizio di radioterapia militare , isolando personalmente il gas radioattivo ( radon ) in tubi di vetro che venivano spediti a diversi ospedali francesi . 
marie vers anche alla patria acquisita la maggior parte delle somme ricevute dalla fondazione nobel [ 3 ]  . dopo la fine della guerra , negli anni dal 1919 al 1934 , marie ormai una celebrit mondiale viaggi molto , recandosi anche pi volte negli stati uniti , dove incontr i presidenti harding e hoover , lanci raccolte di fondi per la ricerca e ottenne notevoli finanziamenti , nonostante la timidezza le impedisse di parlare in pubblico con facilit . 
during her stay in england , marie , deeply shocked by the parisian scandal , came into contact with the movement for womens suffrage , the leading member of which was emmeline pankhurst ( 18581928 ) , who was arrested on several occasions . back in paris after a break of 14 months , marie returned to her laboratory , as the scandal began to die down with the separation by mutual agreement of the langevins . 
marie and paul did not resume their affair , although their relationship remained friendly and fruitful on the professional level . at the outbreak of world war i , only two people remained on the premises of the radium institute , which was planned by marie and built in the latin quarter in the street renamed for the occasion in honour of pierre curie : marie , and a mechanic who was exempt from the draft due to a serious heart condition . although until then , maries main interest had been the radioactive phenomena , she realised the enormous diagnostic potential of artificial x - rays ( roentgen rays ) in saving the lives of wounded soldiers at the front . 
she set up 350 radiol med ( 2009 ) 114 : 347357 marie curie cominci a soffrire di disturbi fisici connessi alle radiazioni intorno al 1920 , con una doppia cataratta che richiese quattro dolorosi e , per lepoca , complicati interventi . 
negli ultimi anni , manifest una progressiva debolezza , dapprima attribuita erroneamente alla tubercolosi , finch uno specialista di ginevra cap che poteva trattarsi di una forma simil - tumorale di anemia aplastica , dovuta al danno midollare da radiazioni [ 4 ]  . quando mor , nel 1934 , 28 anni dopo il marito pierre , listituto da lei diretto era una delle massime realt scientifiche del tempo e molte applicazioni mediche e industriali delle scoperte dei curie testimoniavano gi delle vaste potenzialit applicative della radioattivit . 
negli anni compresi tra il 1919 ed il 1934 , listituto diretto da marie curie produsse 483 lavori scientifici o libri , 31 dei quali scritti da marie [ 3 ]  . marie venne sepolta due volte ; la prima a sceaux , con il marito pierre . 
il castello , in quegli anni , attirava la curiosit morbosa di frotte di visitatori , da sarah bernhardt ad axel munte , a numerosi giovani studiosi di neuropsichiatria , da broca a gilles de la tourette , al giovane freud . 
in sostanza , alla fine dell800 , non molto era cambiato nella vita degli internati da quando , un secolo prima , il castello era diretto dall illuminista dottor pinel , il quale aveva tentato con scarso successo , nel 1795 , loperazione insieme politica ed umanitaria di liberare i folli dalle catene . 
foucault ha scritto , a proposito del tentativo di pinel : le catene cadono , il folle si ritrova libero e in quel momento recupera la ragione la brutalit che appare nei dementi non che il riflesso della bestialit dei guardiani e ancora : a partire da pinel si vedr nella follia uno slancio venuto dal profondo , che oltrepassa i limiti dellindividuo e tende ad una apoteosi di se stesso [ 5 ]  . blanche , ricoverata dal 1878 al 1893 , divenne presto famosa come la regina delle isteriche , protagonista delle esibizioni organizzate e dirette settimanalmente dal dottor fig . 
marie and her daughter , irne ( aged 17 at the time ) , learned how to drive these vehicles , studied anatomy and , in the company of medical personnel , gave long - term service in the war zone . 
during the same period , marie established a military radiotherapy service and personally isolated the radioactive gas ( radon ) in glass tubes that were sent to various french hospitals . 
she also donated most of the money received from the nobel foundation to her adopted homeland [ 3 ]  . after the war , in the years between 1919 and 1934 , marie , who had become an international celebrity , travelled widely . 
the frail figure of this now elderly woman dressed constantly in black became legendary , and due to her austere appearance and moral force , marie was compared with a buddhist monk . launched very successful marie began to suffer from physical problems connected with radiation around 1920 , with the appearance of a double cataract that required four painful and for the times complicated operations . 
in her later years , she developed progressive weakness , which was at first erroneously attributed to tuberculosis until a specialist from geneva realised it could have been caused by aplastic anaemia , a tumour - like condition due to radiation - induced bone marrow damage [ 4 ]  . between 1919 and 1934 , the institute directed by marie produced 483 scientific papers or books , 31 of which were written by her [ 3 ]  . 
in those years , the piti salptrire attracted the morbid curiosity of a stream of visitors , from sarah bernhardt to axel munte and numerous neuropsychiatrists , from paul broca to georges gilles de la tourette and the young sigmund freud . 
in essence , at the end of the nineteenth century , little had changed in the lives of the patients from when a century earlier the hospital was directed by the enlightened physician philippe pinel , who , in 1795 , had attempted without success to perform the political and humanitarian task of freeing the inmates from their shackles . 
and at that moment they recovered their reasonthe brutality which appears in the demented is nought else than the reflection of the animality of the guardians and from pinel onwards , the classical mind recognised in madness an impulse from the depths which exceeded the juridical limits of the individual , ignoring fixed moral limits and tending towards an apotheosis of the self [ 5 ]  . blanche , who was hospitalised between 1879 and 1893 , soon became famous as the queen of hysterics , being the protagonist of the spectacular weekly lectures organised and directed by jean - martin charcot . 
the cures without support became real cures of false diseases , as in fact hysteria proved to be , which failed to outlive charcot . hysterical madness was a mixture of persuasion and mystification , even though , many years later , blanche stated that charcots weekly performances were not based on feigning . although charcot has been accused , and probably correctly , of misogyny [ 6 ] , in 1885 , he accepted into his group a brilliant young medical graduate , blanche edwards [ 7 ]  . after the death of charcot , blanche wittman , having been declared clinically cured , found temporary work as a technical assistant in the recently instituted radiology department of salptrire . 
indeed , roentgens discovery ( 1895 ) was made only 2 years after the death of charcot and was contemporary to freuds studies of hysteria . in 1898 , at the time of the dreyfus affair , blanche moved to the laboratory of the curies , with the thankless task of isolating radium from the pitchblende from sankt joachimsthal in bohemia and in which she remained for hours and hours each day immersed up to her knees in pitchblende . this was the beginning of the collaboration and cohabitation of blanche and marie , which lasted 16 years . 
after 4 years of work , the curies , with the help of several assistants , including blanche , managed to isolate 0.1 g of radium from tonnes of pitchblende . the lesions caused by the radioactive pitchblende caused blanche to have first her legs and then her left arm amputated . 
reduced virtually to a torso and forced to move about in a wooden cart , blanche had to stop working in the laboratory but remained until her death , in 1913 , within the curies family circle and their closest friends and collaborators , such as becquerel [ 8 ]  . ha potuto compiersi se non con la complicit del malato stesso . 
la follia isterica era un insieme di persuasione e di mistificazione anche se , parecchi anni dopo , blanche ebbe a dichiarare che gli spettacoli settimanali di charcot non erano basati sulla simulazione . 
nonostante charcot sia stato accusato , probabilmente con ragione , di misoginia [ 6 ] , nella sua quipe egli accett anche , nel 1885 , una giovane e brillante laureata in medicina , blanche edwards [ 7 ]  . 
 nel 1898 , allepoca dellaffare dreyfus , blanche si trasfer nel laboratorio dei coniugi curie , con lingrato compito di isolare il radium dai fanghi della pechblenda provenienti dalle cave di sankt joachimsthal in boemia e nei quali restava immersa fino alle ginocchia per ore ed ore , ogni giorno . 
da una tonnellata di fanghi e terriccio , dopo quattro anni di duro lavoro , i curie , con laiuto di alcuni aiutanti , tra cui blanche wittman , riuscirono ad isolare 0 , 1 grammi di radiu le lesioni provocate dai fanghi radioattivi portarono la povera blanche a subire progressivamente lamputazione delle gambe e del braccio sinistro . 
ridotta ad un torso e costretta a spostarsi su un carretto di legno , blanche cess ovviamente di lavorare nel laboratorio , ma rest fino alla morte , avvenuta nel 1913 , nella cerchia familiare dei curie e dei loro pi stretti amici e collaboratori , come il grande fisico henri becquerel [ 8 ]  . popularity and demonisation of ionising radiation at the beginning of the twentieth century , artificially produced x - rays and , to a lesser extent , the radiation emitted by newly discovered radioactive elements , had become quite fashionable . 
this popularity can be seen not only in the press of the time [ 9 ] but also in the numerous handmade articles and industrial products whose labels referred to the mysterious rays for publicity purposes , such as household products , detergents , postcards , razors , citrus squeezers , prophylactics , soap , spirits and a variety of other items [ 10 ]  . 
questa popolarit testimoniata , oltre che dalla stampa dellepoca [ 9 ] , anche da numerosi reperti , manufatti o prodotti industriali , che nelletichetta richiamavano i raggi misteriosi a scopo pubblicitario , come prodotti per la casa , detergenti , cartoline , rasoi , spremiagrumi , profilattici , sapone , alcolici ed apparecchi di ogni tipo [ 10 ]  . 
at circuses and trade fairs , as well as elegant salons , x - ray demonstrations were performed , mainly for the macabre fancy of observing the skeleton of friends and relatives in the penumbra of the fluoroscopic screen , illuminated by a weak green glow . 
to have her hand x - rayed , as roentgens wife berta had done several years earlier , the celebrated actress sarah bernhardt remained motionless on her knees for 5 min in a fashionable parisian salon [ 4 ]  . the new rays captured the imagination of the public , more so than the cinmotagraphe invented by the lumire brothers , auguste and louis , in the same year as roentgens discovery , for they embodied the guarantee of a better future , in a positivist vision of the world characterised by endless faith in progress . 
photographs of the time bear witness to the very slow growth in the awareness of the ambiguous properties of radiation , which can be a powerful weapon against disease in both diagnosis and treatment but also highly pathogenetic and therefore should be dominated and controlled , rather than used indiscriminately . 
nei circhi e nelle fiere di paese , come nei salotti eleganti , venivano eseguite dimostrazioni dei raggi x , principalmente per il gusto un po macabro di osservare lo scheletro di amici e parenti nella penombra dello schermo fluoroscopico , illuminata da una debole luminescenza verdastra . 
per farsi radiografare la mano , come aveva fatto pochi anni prima la moglie di roentgen , berta , la grande attrice sarah berhardt rimase immobile in ginocchio per 5 minuti in un salon parigino alla moda [ 4 ]  . i nuovi raggi catturavano limmaginazione del pubblico con grande intensit , pi dellinvenzione del cinematografo , che i fratelli lumire realizzarono nello stesso anno della scoperta di roentgen e rappresentavano la garanzia per un futuro migliore , in una visione del mondo ancora positivista , caratterizzata da grande fiducia nel progresso . liconografia del tempo testimonia di una presa di coscienza collettiva molto lenta delle ambigue propriet delle radiazioni , che possono costituire unarma potente contro le malattie nella diagnosi e nella terapia , ma anche risultare altamente patogene e dunque devono essere dominate e controllate , piuttosto che utilizzate indiscriminatamente . 
la invisibilit delle radiazioni portava ad una sottovalutazione degli effetti biologici . quando , un anno dopo la scoperta di roentgen , 354 radiol med ( 2009 ) 114 : 347357 could have ( unpredictable ) effects on the body . 
in 1903 , the first rudimentary forms of lead or leadrubber shields appeared , but they were seldom used , as their rigidity hampered free movement . the radiologists of the time performed hours of radioscopy every day without any protection whatsoever , and after several years of work , they developed paleness , weakening and severe asthenia . 
in 1925 , she was part of a commission of the french academy of medicine , which recommended the use of lead screens and periodic blood tests in subjects exposed to radiation in the workplace . 
hair and fingernails tended to fall out and not grow back . a list of radiation victims compiled in hamburg in 1936 and clearly incomplete bore the names of 220 physicians , physicists , chemists , technicians , nurses and religious workers . 
gendreau described the first case of occupational disease associated with radiation [ 4 ]  . workers in a watch factory established in 1914 ( radium corporation , newark , nj , usa ) used fine brushes to spread radioactive material ( known as glow in the dark ) on the watch faces to render them fluorescent in the dark . many of these watches were supplied to troops on the european fronts during world war i . 
the workers in the factory , who tended to moisten the brushes on their tongues , suffered severe ulcerations , jaw necrosis , tooth loss and various types of tumours . 
in 1929 , legal action was taken against the company , which defended itself by attributing the workers illnesses to symptoms of hysteria . the use of radiation for diagnostic and therapeutic purposes , particularly in the fight against cancer , was actively promoted as early as the latter phase of maries life after world war i . 
the biological effects of radiation became effectively controlled , and devices capable of delivering very low doses were developed without sacrificing diagnostic accuracy ( such as those quipped with image intensifiers and television chains )  . 
this growing sullamerican journal of the medical association , comparve il primo articolo sulle applicazioni mediche dei raggi x , il direttore della rivista ipotizz che la forma di energia appena scoperta potesse esercitare effetti ( imprevedibili ) sullorganismo ma , a parte il grande clinico inglese lister , nessuno raccolse queste osservazioni [ 4 ]  . 
nel 1903 comparvero le prime , rudimentali protezioni in piombo o in gomma piombifera ma pochi le utilizzarono , perch la loro rigidit ostacolava i movimenti . i radiologi praticavano ore di radioscopia ogni giorno , senza alcuna protezione e , dopo pochi anni di lavoro , accusavano pallore , deperimento e grave astenia . 
tiraboschi , da 15 anni in attivit come radiologo presso lospedale cittadino . lautopsia dimostr gravi lesioni a carico del midollo osseo e della milza ed il patologo ipotizz un ruolo eziologico delle radiazioni . 
nel 1925 marie fece parte di una commissione dellaccademia francese di medicina , che raccomand luso di schermi protettivi piombati e di test periodici del sangue in soggetti esposti professionalmente . 
i capelli e le unghie cadevano , per non ricrescere pi . una lista di vittime delle radiazioni , compilata ad amburgo nel 1936 e chiaramente incompleta , riporta 220 nomi di medici , fisici , chimici , tecnici , infermieri e religiosi . gendreau descrisse il primo caso di patologia lavorativa industriale legata alle radiazioni [ 4 ]  . 
 le operaie di una fabbrica di orologi fondata nel 1914 ( radium corporation , newark , new jersey ) utilizzavano sottili pennelli per spalmare materiale radioattivo ( detto : glow in the dark ) sui quadranti , allo scopo di renderli fluorescenti e consentire di leggere lora al buio . 
nel 29 venne istruito un processo e la ditta si difese attribuendo i disturbi delle operaie a sintomi isterici . limpiego delle radiazioni a scopo diagnostico e terapeutico , specie nella lotta contro i tumori , venne promosso attivamente gi nellultima fase della vita di marie curie , nel primo dopoguerra , ma solo nel secondo dopoguerra le conoscenze di radiobiologia raggiunsero un livello soddisfacente ; il controllo degli effetti biologici delle radiazioni divenne efficace e furono proposte apparecchiature in grado di erogare dosi molto basse , senza sacrificare radiol med ( 2009 ) 114 : 347357 awareness was certainly encouraged by the devastating effects of the atomic bombs dropped on hiroshima and nagasaki in japan in 1945 . 
it has been estimated that to obtain the same quantity of ( gamma ) radiation unleashed by the hiroshima bomb alone , millions of tonnes of the curies radium would be required . 
the nuclear proliferation in the cold war years and the chernobyl nuclear disaster in russia further contributed to the growing alarm that pervaded public opinion and the mass media in the second half of the twentieth century . the american philosopher john dewey wrote : the destructive use made of the fission of the nucleus of an atom has become the stock - in - trade of the assault upon science . what is so ignored as to be denied is that this destructive consequence occurred not only in a war but because of the existence of war , and that war as an institution antedates by unknown millennia the appearance on the human scene of anything remotely resembling scientific enquiry [ 11 ]  . today , the sustainability of medical imaging is being questioned [ 12 ]  . 
the so - called deterministic effects , which occur only in the presence of very high doses and coincide with cell death ( apoptosis ) , have a threshold effect and are not ( fortunately ) seen in individuals who undergo irradiation for diagnostic purposes or in personnel exposed to radiation in the workplace ( of course , radiotherapy is a case apart )  . the stochastic or random effects on the body are due to dna alterations produced by radiation , with consequent hereditary effects if these occur in germ cells or radioinduced tumours if they occur in somatic cells . 
particular concern regards computed tomography examinations , the number of which has increased 12 - fold over the last 20 years in europe and more than 20 - fold in the united states , with an increase of at least seven times the average dose per examination delivered to the population [ 15 ]  . laccuratezza diagnostica ( come gli impianti dotati di amplificatore di brillanza e di catene televisive )  . 
si pensi che , per ottenere la stessa quantit di radiazioni ( gamma ) prodotte dalla sola bomba di hiroshima , sarebbero stati necessari milioni di tonnellate del radium dei curie . 
la proliferazione nucleare negli anni della guerra fredda e la recente catastrofe nucleare di chernobyl hanno ulteriormente contribuito al crescente allarme , che ha pervaso lopinione pubblica ed i grandi media nella seconda met del novecento . 
si ignora che tale conseguenza distruttiva avvenuta non solo durante una guerra , ma a causa dellesperienza stessa della guerra , una istituzione nata parecchi millenni prima che comparisse sulla scena umana alcunch di lontanamente somigliante allindagine scientifica [ 11 ]  . oggi ci si interroga sulla sostenibilit dellimaging medico [ 12 ]  . 
per comprendere leffetto delle radiazioni bisogna tener conto del tipo di radiazioni coinvolte , della dose , cio della quantit di energia che esse trasferiscono nei tessuti e della variabile radiosensibilit dei tessuti stessi [ 13 ]  . 
i cosiddetti effetti deterministici , che si verificano solo per dosi molto elevate e coincidono con la morte delle cellule ( apoptosi ) , hanno un effetto - soglia e non si osservano ormai pi ( fortunatamente ) nelle persone sottoposte ad irradiazione per scopi diagnostici o in personale esposto lavorativamente ( diverso , ovviamente , il caso della radioterapia )  . 
 gli effetti stocastici o casuali sono dovuti ad alterazioni del dna prodotte dalle radiazioni , con conseguenti effetti ereditari se avvengono nelle cellule germinali o tumori radioindotti , se avvengono nelle cellule somatiche . 
le preoccupazioni riguardano soprattutto gli esami di tomografia computerizzata ( tc ) , il cui numero aumentato negli ultimi venti anni di 12 volte in europa e di oltre venti volte negli usa , con incremento di almeno 7 volte della dose media erogata per indagini radiologiche alla popolazione [ 15 ]  . state of the art and conclusions stato dellarte e conclusioni there are three theories with regard to the effects of low doses of ionising radiation . 
the best known and official doctrine , which has inspired international radiation protection laws , states that there is no threshold for stochastic effects and that even a single event or a very low dose can cause damage , the probability of which is linearly correlated with the dose ( linear nonthreshold [ lnt ] model )  . 
it has been estimated that one third of all computed tomography examinations could thus be avoided without sacrificing diagnostic accuracy . a second theory , which is not without biological and epidemiological evidence , suggests that the stochastic effects are in proportion to the dose , but in the case of low doses , there is a sort of practical threshold below which cell repair mechanisms manage to avoid damage . 
it may even be possible that the stochastic effects are not completely random , but at low doses only regard genetically predisposed individuals ( who may be identified with appropriate tests )  . the third theory , which goes contrary to accepted belief and is politically incorrect but supported by significant proof , some of which is experimental , is hormesis ( from the greek meaning to excite ) [ 16 ]  . 
moreover , life on earth evolved in conditions of environmental radioactivity much greater than the current average annual background level of around 3 msv , and the longevity of individuals exposed for professional or environmental reasons to small additional doses ( contemporary radiologists , aeroplane pilots or flight attendants , etc . ) is not lower but higher than the average , with a lower incidence of cancer . 
la lnt , estrapolata dagli effetti delle bombe atomiche sui sopravvissuti di hiroshima e nagasaki , considerata vera precauzionalmente , ma non vi sono prove certe che per dosi molto basse , come quelle coinvolte in quasi tutti gli esami di radiodiagnostica in et adulta , essa sia valida . 
la lnt ha ispirato il principio conosciuto con lacronimo alara ( as low as reasonably available ) , secondo il quale la dose erogata dovrebbe essere sempre la pi bassa ragionevolmente possibile in quelle condizioni cliniche [ 15 ]  . 
bisogna dunque mettere in atto ogni presidio tecnico per ridurre la dose erogata , senza che ci vada a discapito della accuratezza diagnostica e bisogna cercare di evitare esami inutili , di efficacia non dimostrata ( esempio : screening del tumore polmonare ) o sostituibili con altre metodiche di analoga efficacia diagnostica ( esempio : ecografia nella diagnosi della appendicite acuta )  . 
si calcola che almeno un terzo degli esami tc potrebbe cos essere evitato , senza pregiudizio per laccuratezza diagnostica . una seconda teoria , non priva di evidenze biologiche ed epidemiologiche , sostiene che gli effetti stocastici sono s proporzionali alla dose , ma che , quando si tratta di dosi piccole , esiste una sorta di soglia pratica , al di sotto della quale i meccanismi riparativi delle cellule e dei tessuti riescono ad evitare danni . 
anche possibile che gli effetti stocastici non siano poi del tutto casuali , ma che riguardino , per dosi basse , solo individui geneticamente predisposti ( ed individuabili con appositi test )  . la terza teoria , eretica e politicamente scorretta , ma sostenuta da notevoli elementi di prova anche sperimentali , lormesi ( dal termine greco che significa stimolazione ) [ 16 ]  . 
del resto , la vita sulla terra si evoluta in condizioni di radioattivit ambientale ben superiori allattuale fondo annuale medio di circa 3 millisievert e la longevit delle persone sottoposte professionalmente o per ragioni ambientali a piccole dosi aggiuntive ( i radiologi contemporanei , i piloti di aereo o le hostess ecc . ) non minore , ma maggiore della media , con minore incidenza di patologia neoplastica , il che sembra convalidare tale teoria . 
lormesi stata fortemente contrastata nei decenni scorsi , anche perch uno dei suoi ideatori ( arndt ) , oltre che psichiatra , era anche omeopata , ma le notevoli evidenze sembrano richiedere una pi seria valutazione di questa teoria , anche in campo radiologico [ 18 ]  . it is surprising that despite the widespread diffusion of clinical radiology and the advances in scientific knowledge that such an important question has still not received certain pu stupire che , nonostante la grande diffusione della radiologia medica e levoluzione delle conoscenze scientifiche , un problema di questa importanza non abbia ancora radiol med ( 2009 ) 114 : 347357 answers . 
di fisica sanitaria , irccs casa sollievo della sofferenza , san giovanni rotondo , italy 6facolt di medicina , universit di bari , bari , italy 7dipartimento di radiologia , universit novara , novara , italy 8dipartimento di scienze neurologiche e psichiatriche dellet evolutiva , universit la sapienza , roma , italy 9dipartimento di radiologia , azienda ospedaliera universitaria , umberto i , torrette , ancona , italy correspondence to : t . 
scarabino , via napoli 56 , 70031 andria , italy , tel . : + 39 - 088 - 3299140 , fax : + 39 - 088 - 3299220 , e - mail : tscarabino@hotmail.com received : 10 september 2008 / accepted : 6 october 2008 / published online : 10 march 2009 springer - verlag 2009 abstract purpose . 
this study evaluated the sensitivity of a 3.0 - tesla ( t ) magnetic resonance imaging ( mri ) in measuring cerebral phenylalanine using proton magnetic resonance spectroscopy and in assessing mr - documented whitematter changes by means of diffusion studies ( diffusionweighted imaging , apparent diffusion coefficient map ; diffusion tensor imaging ) in patients with phenylketonuria . 
lampiezza del segnale della fenilalanina relativo al rapporto creatina / fosfocreatina incrementa linearmente con la fenilalanina ematica ( r = 0 , 7067 ; p < 0 , 001 )  . 
lo studio di diffusione ha 462 radiol med ( 2009 ) 114 : 461474 exhibiting mri changes as well as decreased apparent diffusion coefficient values , but fractional anisotropy indices were normal . 
in particular , the multimodal approach with mri , proton magnetic resonance spectroscopy and diffusion magnetic resonance imaging can provide more information than previous studies performed with low - field systems . 
in particolare lapproccio multimodale con imaging rm di base , di spettroscopia e di diffusione pu fornire maggiori informazioni rispetto ai precedenti studi acquisiti con sistemi a pi basso campo . keywords phenylketonuria magnetic resonance imaging ( mri ) proton mr spectroscopic imaging diffusion mri parole chiave fenilchetonuria risonanza magnetica studio di spettroscopia protonica con risonanza magnetica studio di diffusione con risonanza magnetica introduction introduzione phenylketonuria ( pku ) is a disorder of amino acid metabolism caused by an inborn error in the phenylalanine hydroxylase gene ( pha ) , which strongly reduces hepatic hydroxylation of phenylalanine ( phe ) to tyrosine [ 1 ]  . 
previous morphological mri studies have documented focal symmetrical lesions in periventricular and , especially , parieto - occipital white matter ( wm ) in pku patients with phe blood levels exceeding 10 mg / dl . these lesions , the significance of which is as yet unclear , seem to be due to a reversible structural myelin alteration , as they regress when blood phe levels fall [ 2 ]  . proton magnetic resonance spectroscopy ( 1h - mrs ) measures noninvasively the concentration of several brain metabolites in vivo and can help to further elucidate the cerebral metabolic characteristics of pku . 
it allows evaluation of several aspects of brain biochemistry that could shed light on the nature of wm lesions , including measurement of n - acetyl - containing moieties [ mainly n - acetylaspartate ( naa ) ] , choline - containing compounds ( cho ) , creatine ( cr ) and phosphocreatine ( pcr ) , and myo - inositol ( mi ) [ 3 ]  . 
in particular , changes in naa ( a marker of neuronal integrity ) la fenilchetonuria ( pku ) un disordine del metabolismo degli amminoacidi causata da una alterazione congenita del gene della fenilalanina - idrossilasi che determina una significativa riduzione , a livello epatico , della idrossilazione della fenilalanina ( phe ) in tirosina [ 1 ]  . 
i pazienti non diagnosticati e quindi non trattati possono sviluppare diversi segni e sintomi : in particolare ritardo mentale e nella crescita , crisi epilettiche , manifestazioni cutanee ( eczemi e ipopigmentazioni )  . 
la diagnosi neonatale ha radicalmente cambiato la prognosi della pku anche se in realt possiamo riscontrare alcuni sintomi e alterazioni neuro - radiologiche anche in pazienti trattati precocemente . la pku stata studiata sotto diversi aspetti . 
essa documenta la presenza di focali e simmetriche lesioni a livello della sostanza bianca periventricolare , specie a livello parieto - occipitale , evidenti in tutti i pazienti in cui il livello ematico di phe supera il valore di 10 mg / dl . 
tali lesioni , il cui significato non ancora chiaro , sembra siano dovute a una alterazione strutturale e reversibile della mielina : infatti esse regrediscono nel momento in cui il livello di phe decresce verso valori normali [ 2 ]  . la spettroscopia protonica con rm ( 1h - mrs ) misura in maniera non invasiva e in vivo la concentrazione a livello cerebrale di alcuni metaboliti fornendo ulteriori informazioni sulle caratteristiche metaboliche cerebrali della pku e soprattutto permettendo una pi chiara analisi biochimica delle alterazioni della sostanza bianca tipiche di questa radiol med ( 2009 ) 114 : 461474 or in mi ( a potential astrocytic marker ) suggest a possible neurochemical dysfunction , whereas cho is one measure of the degree of tissue myelination [ 4 , 5 ]  . 
despite continuing progress in 1h - mrs acquisition methods and the development of increasingly sophisticated quantification routines , its determination is affected by the detection threshold at 1.5 t ( > 200 mmol / kg ) [ 3 ]  . 
furthermore , the very large volume of interest ( voi ) applied at 1.5 t ( > 30 cc ) makes phe signal detection unreliable due both to concurrent line - broadening effects on the resonance and the presence of macromolecular contributions at 7.3 ppm [ 36 ]  . in recent years , diffusion - weighted mri ( dwi - mri ) has been introduced in clinical practice . 
however , these studies have failed to clarify the significance of dwi changes in relation to clinical findings . the aim of this work was to analyse the potential and advantages of a 3.0 - t system in the multimodal study of brain alterations of patients with pku . 
they were 17 male and 15 female subjects aged from 7 to 34 ( mean 18.9 , standard deviation 6.0 ) years , 21 with early and 11 with late diagnosis . 
in particolare andiamo a misurare la concentrazione di molecole contenenti il gruppo n - acetile ( in prevalenza ln - acetil - aspartato , naa ) , le macro - molecole contenenti colina ( cho ) , la creatina ( cr ) , la fosfocreatina ( pcr ) e il mio - inositolo ( mi ) [ 3 ]  . 
le variazioni di naa ( marker di integrit neuronale ) e di mi ( potenziale marker astrocitario ) suggeriscono una possibile disfunzione neurochimica mentre mediante la quantificazione della cho abbiamo un indice del grado della mielinizzazione tissutale [ 4 , 5 ]  . 
lo studio di 1h - mrs con unapparecchiatura a 1 , 5 tesla ( t ) ha permesso di quantificare i livelli cerebrali di phe in pazienti con pku : tali misurazioni hanno fornito dati interessanti sulla biochimica delle alterazioni della sostanza bianca in tale patologia [ 35 ]  . 
la valutazione dei livelli di phe , ottenuta utilizzando sistemi a 1 , 5 t , per ancora soggetta a delle limitazioni : nonostante i miglioramenti dei protocolli di acquisizione 1h - mrs e di quantificazione dei metaboliti c un limite intrinseco dovuto alla soglia di detezione del metabolita ( > 200 mmol / kg ) legato direttamente alla intensit del campo magnetico ( 1 , 5 t ) [ 3 ]  . 
inoltre la notevole ampiezza del volume di interesse ( voi ) in sistemi a 1 , 5 t ( > 30 cc ) rende il valore di phe non attendibile e ci dovuto sia a effetti concomitanti di allargamento sulla risonanza che allinterferenza delle altri componenti macromolecolari presenti a 7 , 3 ppm [ 36 ]  . negli ultimi anni sono state introdotte di routine nella pratica clinica le sequenze rm pesate in diffusione ( dwmri )  . 
 [ 8 ] hanno dimostrato in pazienti con pku la presenza di alterazioni di segnale in dwi che si sovrappongono sostanzialmente alle alterazioni rilevate in condizioni rm di base e che sono caratterizzate da iperintensit in t2 . 
le mappe del coefficiente di diffusione apparente ( adc ) invece documentano valori compatibili con una diffusione ristretta dei protoni . tali studi comunque non hanno ancora chiarito lorigine di tali alterazioni e una chiara correlazione col quadro clinico . lo scopo del nostro lavoro di analizzare le possibilit e i vantaggi della rm 3 , 0 t nello studio multimodale delle alterazioni cerebrali di pazienti con pku ed in particolare di valutare la sensibilit di un sistema rm ad alto campo nel misurare la phe cerebrale con la 1h - mrs e nel documentare le alterazioni della sostanza bianca rilevate con lo studio rm di base utilizzando gli studi di dw - mri . magnetic resonance technique materiali e metodi mr studies were performed with a 3.0 - t scanner ( signa horizon lx , general electric medical system , milwaukee , wi , usa ) with a standard quadrature transmit / receive birdcage head coil . 
wm involvement was assessed by morphological mr with sagittal fast spoiled gradient recalled ( fspgr ) t1 - weighted [ repetition time ( tr ) / echo time sono stati inclusi nello studio 32 pazienti con un tipico quadro biochimico - clinico della pku : 17 maschi e 15 femmine , et compresa tra 7 e 34 anni ( media 18 , 9 , deviazione standard 6 , 0 ) : 21 con diagnosi precoce e 11 con diagnosi tardiva di malattia . 
in tutti i pazienti stato valutato il valore ematico di phe ed stata effettuata una 464 radiol med ( 2009 ) 114 : 461474 ( te ) / flip angle ( fa ) 225 / minimum / 75 ms , acquisition time 1 min 57 s ] sequences ; axial fluid - attenuated inversion recovery ( flair ) ( tr / te / ti , inversion time , 11 , 000 / 130 / 2 , 250 ms , acquisition time 2 min 26 s ) and spin echo ( se ) t1 - weighted ( tr / te 500 / 10 ms , acquisition time 2 min 21 s ) sequences , and coronal fast spin echo ( fse ) t2weighted ( tr / te 5 , 000 / 55 / 20 ms , acquisition time 1 min 05 s ) sequences . 
standard mr scans were acquired using 5mm - thick slices , 1 - mm gap , matrix size 256192 , and field of view ( fov ) 22 cm . single - voxel 1h - mrs was performed using a short te point - resolved spectroscopy ( press ) sequence ( tr / te 2 , 000 / 35 ms , total averages 128 , spectral width 2 , 500 hz , 1 , 024 complex data points , acquisition time 4 min 56 s )  . 
a voxel volume of just 8 cc was placed in the altered periventricular deep wm . the diffusion studies [ dwi , adc maps and diffusion tensor imaging ( dti ) ] were performed in the axial plane using a pulsed gradient se echo - planar sequence ( tr / te 8 , 000 / minimum ms ; dwi acquisition time 44 s and for dti 6 min 57 s ) without ( b - value = 0 ) and with ( b - value = 1 , 000 s / mm2 ) diffusion gradients applied along 25 different directions to cover the three - dimensional space uniformly . 
 additional dwis were acquired in the axial plane , with identical parameters except for the b - value , which was set at values from 500 to 2 , 500 s / mm2 ( 500 , 1 , 000 , 1 , 500 , 2 , 000 and 2 , 500 ) for five different acquisitions . data analysis all postprocessing was performed on a unix workstation . wm changes were analysed on mri both qualitatively , with reference to signal intensity in the different pulse sequences , and semiquantitatively , with reference to their anatomical location ( frontal , temporal , occipital , cerebellar and in brainstem ) and extension ( deep and / or subcortical wm )  . 
wm involvement was scored on t2 - weighted images by raters who were unaware of patients clinical history , as follows : 1 for deep wm involvement only ( limited involvement ) ; 2 for involvement of deep and subcortical wm ( intermediate involvement ) ; 3 for greater involvement of brainstem and / or cerebellum ( severe involvement )  . 
 brain phe was quantified by computing the amplitude of the phe signal at 7.36 ppm relative to the cr / pcr peak at 3.05 ppm , considered as an internal reference , using the sage / idl software . 
the other metabolites ( naa , cho , cr / pcr , mi ) were analysed by means of lcmodel routines . the diffusion images were analysed assuming a monoexponential relation between signal intensity and the product of the b - matrix ( a 33 matrix expressing the relationship between signal attenuation and elements of the diffusion tensor matrix )  . 
trenta soggetti sani ( 9 maschi e 21 femmine ; et compresa tra 12 e 58 anni ; media 32 , 9 ) sono stati arruolati nello studio come gruppo di controllo per confrontare i dati ottenuti dallimaging rm . 
il consenso informato stato firmato da tutti i pazienti o da loro parenti prossimi . tecnica rm lo studio rm stato effettuato con unapparecchiatura rm 3 , 0 t ( signa horizon lx , general electric medical system , milwaukee , wi , usa ) dotata di bobina della testa standard in quadratura trasmittente / ricevente . 
le alterazioni della sostanza bianca sono state valutate con la rm di base utilizzando le sequenze : fast spoiled gradient recalled ( fspgr ) t1 - pesata ( tr , tempo di ripetizione , / te , tempo di echo , / fa , flip angle , 225 / minimum / 75 ms , tempo di acquisizione 1 min 57 s ) acquisita in proiezione sagittale ; fluid - attenuated inversion recovery ( flair ) ( tr / te / ti , tempo di inversione , 11000 / 130 / 2250 ms , tempo di acquisizione 2 min 26 s ) e spin echo ( se ) t1 - pesata ( tr / te 500 / 10 ms , tempo di acquisizione 2 min 21 s ) in proiezione sagittale ; fast spin echo ( fse ) t2 - pesata ( tr / te 5000 / 55 / 20 ms , tempo di acquisizione 1 min 05 s ) in proiezione coronale . 
il volume di interesse di 8 cc stato posizionato nelle aree di alterazione della sostanza bianca periventricolare profonda . lo studio in diffusione ( dwi , mappe adc e diffusion tensor imaging , dti ) stato ottenuto sul piano assiale utilizzando sequenze pulsed - gradient se eco - planari ( tr / te 8000 / minimum ms , tempo di acquisizione dwi 44 s e 6 min 57 s per il dti ) senza ( b - value = 0 ) e con gradienti di diffusione ( b - value = 1000 s / mm2 ) applicati lungo 25 direzioni per coprire uniformemente e tridimensionalmente il volume di interesse . ulteriori immagini pesate in diffusione sono state acquisite sul piano assiale con gli stessi parametri fatta eccezione per il b - value che stato impostato in valori variabili compresi tra 500 e 2500 s / mm2 ( 500 , 1000 , 1500 , 2000 e 2500 ) per un totale di cinque diverse acquisizioni . analisi dei dati la successiva elaborazione dei dati stata effettuata utilizzando una workstation unix . 
le alterazioni della sostanza bianca sono state analizzate sia qualitativamente , con riferimento alle variazioni di intensit di segnale nelle varie sequenze , che semi - quantitativamente considerando la loro radiol med ( 2009 ) 114 : 461474 tensor diagonalisation , of the main eigenvalues min , med , and max , were generated and the fractional anisotropy index ( fai ) , was derived from the eigenvalues for each pixel . 
 the analysis was performed on parieto - occipital wm , as all patients exhibited dwi changes in this region ; a spherical region of interest ( roi ) of uniform size ( ~45 mm2 ) was placed on the dwis and adc maps , and eigenvalues and fai values were then calculated . 
these data were confirmed by dti quantitative analysis , as decreased adc values but normal fai indices were found in the rois located on the hyperintense parieto - occipital wm ( table 1 )  . 
qualitative analysis of anisotropy images acquired with b - values > 1 , 000 s / mm2 showed an increased contrast between normal tissue and t2 / flair / dwi - mri hyperintense areas . 
il punteggio utilizzato stato calcolato come segue : 1 punto per lesioni interessanti solo la sostanza bianca profonda ( coinvolgimento limitato ) , 2 punti per lesioni interessanti anche la sostanza bianca sottocorticale ( coinvolgimento intermedio ) , 3 punti per leventuale coinvolgimento del tronco e / o del cervelletto ( coinvolgimento severo )  . il valore di phe cerebrale stato quantificato valutando lampiezza del picco di phe a 7 , 36 ppm in relazione al picco di cr / pcr a 3 , 05 ppm ( considerato come un valore interno costante e quindi di riferimento , usando il software sage / idl )  . 
gli altri metaboliti ( naa , cho , cr / pcr e mi ) sono stati analizzati utilizzando lcmodel software . le immagini in diffusione sono state analizzate utilizzando una relazione mono - esponenziale tra lintensit di segnale e il segnale prodotto della matrice b ( matrice 33 che esprime la relazione tra lattenuazione di segnale e gli elementi della matrice del tensore di diffusione )  . 
 lanalisi stata effettuata sulla sostanza bianca parietooccipitale poich tutti i pazienti presentavano alterazioni di segnale in dwi in questa regione ; una regione di interesse ( roi ) sferica e di misura uniforme ( ~45 mm2 ) stata posizionata sulle immagini pesate in diffusione , quindi mappe di adc , autovalori e valori di fai sono stati calcolati . 
the advantages of a higher mr field , with respect to the low - field system used to date , is a larger diagnostic ability in the management of patients with this pathology . 
resolution quality tends to be privileged in clinical practice , with acquisition of a larger number of slices of reduced thickness , use of smaller fov and broader matrices , and acquisition times similar to those of 1.5 - t ai soggetti normali . 
questi dati sono stati confermati dalla analisi quantitativa del dti : nelle regioni di interesse localizzate nelle aree iperintense della sostanza bianca parieto - occipitale stato trovato un valore di adc ridotto ma un normale valore di fai ( tabella 1 )  . 
lanalisi qualitativa delle immagini dellanisotropia , acquisite con b - value pi elevato di 1000 s / mm2 , mostra un aumento del contrasto tra il tessuto normale e le aree di iperintensit in t2 / flair / dw - mri . 
2a - e proton magnetic resonance spectroscopy allows detection of an abnormal phenylalanine ( phe ) signal at 7.36 pp values of the main metabolites [ n - acetylaspartate ( naa ) , choline - containing compounds ( cho ) , creatine ( cr ) / phosphocreatine ( pcr ) ] were normal ( a , arrow )  . 
all patients exhibit variable white - matter hyperintensities on t2 ( c ) and fast low - angle inversion recovery ( flair ) ( d ) scans , mainly in temporal , occipital and periatrial regions . 
in our work , instead , we found the optimum acquisition time as a tradeoff between a time - consuming multimodal acquisition and the patients comfort during the exafurther technological advances are expected to overcome some minor limitations of this system , such as field discussione in tale studio nei pazienti con pku si confermano i dati dei precedenti studi circa la frequenza e le caratteristiche delle alterazioni della sostanza bianca e le correlazioni tra il radiol med ( 2009 ) 114 : 461474 fig . 
a questo valore il confronto dellintensit di segnale sullasse x ( a ) e y ( b ) permette di rilevare una sottile variazione di segnale nella sostanza bianca alterata in relazione ad una parziale conservata anisotropia ( freccia gialla )  . inhomogeneity , susceptibility and chemical - shift artefacts , high specific absorption rate ( sar ) and acoustic noise . in detail , the use of higher fields for spectroscopy allows reduced acquisition time ( higher s / r ) and improved spectral resolution [ 18 , 19 ]  . 
as a result of such improvements ( increased snr and greater spectral resolution compared with 1.5 - t studies ) , the phe signal was easily detected and was measurable despite the low phe concentrations . 
the relative amplitude of the phe signal increased linearly with blood phe up to 1 , 200 micromoles , where growing dispersion of brain phe values was noted , suggesting a more marked interindividual variability , probably due to saturation of the specific phe transport system at the level of the bloodbrain barrier ( bbb )  . a short te ( 35 ms ) was applied better to detect the phe signal as well as those of the principal metabolites . 
furthermore , spectroscopic studies with longer te are subject to greater snr loss at 3.0 t than at 1.5 t due to t2 reduction at higher field strengths and increased peak line width in relation to reduced t2 and microscopic and macroscopic body inhomogeneities . 
these features allow an increased b - value to enhance the anisotropic effect , thus improving the quality of more complex examinations such as fai , diffusion tensor and tractography , which are poorly displayed on 1.5 - t images , even with multiple grado di severit e parametri clinici e laboratoristici . 
per ottenere maggiori informazioni sulla patogenesi di tali alterazioni abbiamo condotto con apparecchiatura a 3 , 0 t uno studio rm multimodale che ha compreso nel protocollo uno studio di base , di spettroscopia e di diffusione al fine di migliorare , sfruttando i vantaggi derivanti dalluso dellalto campo rispetto a quello dei tradizionali sistemi a 1 , 5 t , la capacit diagnostica che pu risultare utile nel management di tali pazienti [ 1517 ]  . in generale lintroduzione di magneti ad alto campo ha infatti portato dei benefici in campo neuroradiologico . 
il confronto delle immagini acquisite con sistemi a 3 , 0 t rispetto a quelle ottenute con 1 , 5 t ha mostrato degli indubbi vantaggi quali : aumento della risoluzione spaziale , temporale e di contrasto , intensit di segnale pi elevato , ridotto tempo di acquisizione , maggiori e pi avanzate applicazioni , elevata sensibilit ed eccellente potere diagnostico nelle sequenze con mezzo di contrasto . 
 il neuroradiologo quindi , grazie allalto s / r dellalto campo , utilizzando lo stesso tempo di un esame con sistemi a 1 , 5 t , pu ottenere immagini di qualit superiore ( aumentando la matrice o riducendo lo spessore di strato o il fov ) oppure immagini della stessa qualit di un basso campo ma in un tempo ridotto : ci si traduce in un maggior confort per il paziente e in una riduzione degli artefatti da movimento . 
la qualit della risoluzione tende ad essere privilegiata nella pratica clinica , grazie allacquisizione di un maggior numero di slices con uno spessore di strato ridotto tramite lutilizzo di un fov pi piccolo , di matrici pi ampie con un tempo di acquisizione risultante simile a quello speso se si utilizzano scanner a 1 , 5 t . 
4a , b comparison of proton magnetic resonance spectroscopy brain phenylalanine ( phe ) detection at 1.5 t ( a ) and 3.0 t ( b ) at 7.36 ppdetection of phe signal in the 1.5 - t study requires a 32 - cc volume of interest ( voi )  . 
4a , b confronto della fenilalanina cerebrale rilevata con la spettroscopia protonica a 1 , 5 t ( a ) e 3 , 0 t ( b ) a 7 , 36 ppla documentazione del segnale della fenilalanina a 1 , 5 t necessita di un voi di 32 cc . 
luso di un pi piccolo voi ( 8 cc ) riduce gli effetti da sovrapposizione con altri metaboliti quali istidina , omocarnosina , macromolecole presenti a 7 , 3 ppm . radiol med ( 2009 ) 114 : 461474 quale compromesso tra il tempo necessario per uno studio multimodale e che garantisse un adeguato confort per il paziente durante lesame . nello specifico in spettroscopia luso dellalto campo permette di ridurre i tempi di acquisizione ( in virt del pi alto s / r ) ma anche migliora la risoluzione spettrale [ 18 , 19 ] la maggior ampiezza del chemical shift dei metaboliti in esame permette infatti di avere una migliore risoluzione spettrale che si traduce in una pi definita separazione dei picchi adiacenti che invece con un sistema a pi basso campo andrebbero a sovrapporsi . 
in virt di tali miglioramenti ( aumento del s / r e migliore risoluzione spettrale ) possibile individuare e misurare pi facilmente il picco della phe , persino a basse concentrazioni . 
lampiezza relativa del picco di phe aumenta linearmente con la concentrazione ematica della stessa sino a un valore di 1200 micromoli oltre il quale si osserva una dispersione dei valori cerebrali di phe dovuta probabilmente a una variabilit individuale derivante dalla saturazione dei sistemi di trasporto della phe localizzati a livello della barriera emato - encefalica ( bee )  . stato utilizzato un te ridotto ( 35 ms ) per avere una migliore individuazione del segnale ( picco ) della phe e degli altri principali metaboliti in esame . 
utilizzando un te pi lungo si avrebbe una riduzione del rapporto s / r pi marcata con lutilizzo di sistemi a 3 , 0 t che con quelli a 1 , 5 t , per effetto della riduzione del segnale in t2 che si ha ad alto campo , dellaumento dellampiezza della linewidth causata dalla riduzione del segnale sempre in t2 e per le disomogeneit a livello microa macro - scopico . anche limaging pesato in diffusione beneficia dellaumento del rapporto s / r , della risoluzione spaziale e della accuratezza delle immagini [ 1517 ]  . 
si verifica in tal modo un miglioramento della qualit di alcune misurazioni come la fractional anisotropy ( fai ) , il tensore di diffusione ( dti ) e di alcune tecniche di ricostruzione come la trattografia . questi valori non sono ben rappresentati a causa del basso s / r utilizzando un sistema a 1 , 5 t anche se si dovesse aumentare il numero delle eccitazioni . 
infatti le ricostruzioni dei tratti assonali riflettono con maggior accuratezza la realt anatomica , specialmente a livello delle biforcazioni dei fasci . radiol med ( 2009 ) 114 : 461474 excitations , owing to the low snr . 
in fact , the reconstructed dti trajectories reflect more accurately the underlying axonal fibres , particularly crossing or bifurcating bundles . for some years , it has been possible to reconstruct threedimensional pathways of wm tracts by combining anisotropy characteristics and directionality , applying the technique known as fibre tracking or tractography . 
the method allows reconstruction of continuous fibre trajectories by sequentially piecing together discrete and shortly spaced estimates of fibre orientation to form continuous fibre trajectories [ 2022 ]  . the colour map is an easy method to show dti values , as it displays the main direction of wm fibres in three fundamental colours : green , blue and red . 
in particular , ontogenetically older fibre systems , such as the optic radiation , had a normal appearance . wm changes depicted in pku patients do not therefore necessarily reflect an altered organisation of cerebral myelin pathways detectable on dti . 
some mr findings in pku appear to be more consistent with reversible dysmyelination associated with an increased myelin turnover than with a demyelination process [ 2 , 15 , 25 , 26 , 29 , 30 ]  . our latest report of decreased adc values with preserved anisotropic diffusivity on dwi - mri and abnormal elevation of brain phe levels on 1h - mrs [ 31 ] seems to be in line with the hypothesis of water accumulation in myelin sheaths [ 7 ] due to an abnormally increased myelin turnover with consequent preservation of anisotropy , which can occur only in structures whose microscopic barriers have not been either destroyed or partially vacuolated . however , a third hypothesis consistent with these findings is that of intracellular accumulation of a hydrophilic metabolite produced by the phe hydroxylase defect , leading da alcuni anni possibile effettuare una ricostruzione tridimensionale del decorso delle fibre mieliniche . 
utilizzando le caratteristiche di anisotropia e tecniche note come il fiber tracking o trattografia si ricostruisce la direzione delle fibre nervose interpolando in modo sequenziale , stime discrete e poco spaziate dellorientamento della fibra per ottenere la sua traiettoria continua [ 2022 ]  . 
 ne derivano mappe a colori che costituiscono un metodo semplice per mostrare i valori del tensore di diffusione attraverso la visualizzazione delle principali direzioni delle fibre nervose in tre diversi colori ( grigio , blu e rosso )  . lapplicazione di tale tecnica nei pazienti con pku ci consente di poter valutare lintegrit delle fibre nervose nelle aree caratterizzate da riduzione dei valori di adc . 
al momento attuale il principale problema nella tecnica del fiber tracking e nella rappresentazione delle mappe a colori dovuto alla difficolt di ottenere una buona delineazione delle fibre mieliniche , senza una parziale contaminazione da parte dei tratti nervosi adiacenti [ 24 ]  . lanalisi del dti ha mostrato una riduzione dei valori di adc nelle aree iperintense nellimaging di base , in linea con i precedenti lavori in letteratura [ 7 , 8 ]  . 
in particolare le fibre ontogeneticamente pi vecchie come i tratti ottici appaiono normali . le alterazioni della sostanza bianca , viste nella pku , non necessariamente riflettono una alterazione architetturale del decorso delle fibre mieliniche , valutato con dti . 
le alterazioni della sostanza bianca nei pazienti con pku sono state documentate in molti studi effettuati con sistemi rm a pi basso campo , ma il loro significato non ancora stato chiarito [ 315 , 2528 ]  . 
alcuni pattern rm sembrano essere correlati pi con un reversibile processo di dismielinizzazione che con uno di demielinizzazione [ 2 , 15 , 25 , 26 , 29 , 30 ]  . in un precedente nostro lavoro la riduzione dei valori di adc con una preservazione dei valori di fa in diffusione e lanormale incremento dei livelli di phe cerebrale in spettroscopia protonica [ 31 ] potrebbero suggerire un accumulo di acqua nelle guaine mieliniche [ 7 ] dovuto a un incremento anomalo del turn - over mielinico stesso che si ha in strutture in cui le barriere microscopiche non sono state ancora del tutto degradate o vacuolizzate . 
 una terza ipotesi in linea con le nostre valutazioni che laccumulo di una metabolita idrofilico intracelllulare prodotto dalla phe - idrossilasi porta a un aumento del contenuto di acqua intracellulare . 
5a - d imaging di diffusione : mappa di adc ( a ) con selezione di una roi , mappa di fai ( b ) e a colori ( c ) ; i valori delle roi di adc e fa ( d ) misurati nelle aree di ciascun paziente ( punti ) e in range normali ( barra : meansd )  . to increased intracellular water content [ 31 ]  . 
the latter data require further examination given that no other abnormalities were detected by in vivo mrs examination , apart from the phe increase . pattern di dwi sia le anormalit dei valori ottenuti con la spettroscopia ( accumulo di phe con valori normali degli altri metaboliti )  . 
questi ultimi dati richiedono ulteriori studi dato che nessuna altra anomalia stata rilevata nellesame di spettroscopia in vivo a parte lincremento della phe . conclusions conclusioni the higher snr of high - field mr systems is a resource that neuroradiologists must exploit in clinical practice , as the higher the snr , the more the imaging protocols can be adjusted . 
i sistemi a 3 , 0 t permettono infatti di effettuare oltre a un accurato studio di base , anche delle valutazioni di carattere fisiologico , metabolico e funzionale che consentono di aumentare la sensibilit e la specificit del potere diagnostico della rm . sono comunque necessari ulteriori progressi tecnologici che permettano di superare alcune piccole limitazioni di tali sistemi rm come la disomogeneit del campo magnetico , radiol med ( 2009 ) 114 : 461474 inhomogeneity , susceptibility and chemical - shift artefacts , high sar and acoustic noise . the growing diffusion of high - field mr systems over the next few years will help a clearer understanding of the pathogenic role of signal abnormalities of the cerebral parenchyma in patients having several kinds of neurological diseases , such as pku . 
in addition , measurement of brain phe levels with 1h - mrs and dwi - mr parameters could become the technique of choice to monitor the influx of blood phe into brain tissue , especially during therapy in follow - up studies . gli artefatti da suscettibilit magnetica e da chemical shift , lelevato sar ( specific absorption rate ) e lalto rumore acustico . la crescente diffusione di tali sistemi nei prossimi anni permetter una migliore comprensione delle cause di alterazione del segnale in pazienti con patologie come la pku . 
maria delle croci , v.le randi 5 , 48100 ravenna , italy 2scuola di specializzazione in radiodiagnostica , universit degli studi di ferrara , corso giovecca 203 , 44100 ferrara , italy 3servizio di anatomia patologica , ospedale civile s.maria delle croci , v.le randi 5 , 48100 ravenna , italy 4istituto di radiodiagnostica , universit degli studi di ferrara , corso giovecca 203 , 44100 , ferrara , italy correspondence to : g . 
cdus revealed an inflammatory process in 277 patients ( 34.58% ) , testicular trauma in 112 ( 13.9% ) , funicular torsion or torsion of the vestigial remnant in 44 ( 5.4% ) , findings suggestive of testicular neoplasm in 35 ( 4.3% ) and no abnormality in 41.5%. 
mri , used to further investigate the cdus findings in 46 cases , showed three cases of intraparenchymal haematoma , one of intrascrotal cavernous body rupture , one of testicular abscess with intrascrotal fistula , two of testicular infarction and 15 of neoplasmri allowed the exclusion of focal abnormalities in ten patients with testicular microlithiasis , in three with chronic orchitis and in four with atrophic involution . 
on the basis of our experience , cdus is irreplaceable as an initial approach to patients affected by scrotal disease , whereas mri is an ideal second - line investigation . 
lecd ha rilevato : in 277 pazienti ( 34 , 58% ) processo flogistico , in 112 ( 13 , 9% ) trauma del testicolo , in 44 ( 5 , 4% ) torsione del funicolo o di un residuo embrionario , in 35 ( 4 , 3% ) alterazioni riconducibili ad eteroplasia testicolare mentre nel 41 , 5% non erano presenti alterazioni patologiche . 
in 46 casi si ricorsi alla rm per chiarimento o miglior definizione diagnostica del reperto ecd rilevando : 3 casi di ematoma intraparenchimale , 1 caso di rottura intrascrotale del corpo cavernoso , 1 caso di ascesso testicolare con fistola intrascrotale , 2 casi di infarto , 15 casi di eteroplasia . 
la rm utile in casi selezionati , potendo rilevare situazioni complesse e precedentemente insospettabili , risultando in un miglioramento della gestione del paziente e in un contenimento globale dei costi . 
 radiol med ( 2009 ) 114 : 414424 keywords scrotum mri color doppler ultrasound scrotal disorders ultrasonography parole chiave scroto rm eco - color doppler patologia scrotale ultrasonografia introduction introduzione colour doppler ultrasonography ( cdus ) plays a key role as a first - line approach to scrotal disease , as it can help solve diagnostic dilemmas and establish a precise diagnosis , thus leading to appropriate clinical and therapeutic decisions . nevertheless , when the cdus findings are difficult to interpret in relation to the patients clinical data and medical history , or when the clinical and sonographic findings are unjustified , magnetic resonance imaging ( mri ) may be used to elucidate or reveal unusual features [ 1 ]  . the value of mri as a second - line technique after inconclusive sonography [ 2 , 3 ] has rarely been addressed in the literature , and the few existing studies are dated and involve small patient populations . 
the aim of this study was to review our experience of the past 7 years in an attempt to validate the usefulness of cdus in the study of scrotal disease and evaluate the role of mri in selected cases . materials and methods from 2000 to 2007 , 801 male subjects aged from 0 to 87 years were referred to our department to undergo cdus of the scrotuexaminations were carried out on a ge logic 7 sonographic unit with a 715 mhz linear - array probe and colour doppler , power doppler , and b - flow imaging . patients included urgent cases referred from the emergency department and high - priority outpatients referred by the urologist to investigate suspicion of acute or subacute scrotal disease . transverse planes panoramic grey - scale scans were obtained in the longituto compare size and dinal and echogenicity on the two sides . 
colour doppler imaging was then performed , with optimisation of the scanner to obtain the highest sensitivity to slow flow , with low pulse repetition frequency ( prf ) , low wall filter and appropriate colour ga in patients with suspected torsion or infarction , the asymptomatic side was evaluated first to ensure that the parameters for flow evaluation had been set appropriately . mri was performed in 46 patients to confirm or elucidate the cdus findings . 
the criteria adopted to define inconclusive sonographic evaluation were unclear nature of the lesion , inability to delineate the lesion spatially in terms of site and / or extension , conflicting clinical and sonographic diagnosis and marked structural heterogeneity associated with conditions predisposing to malignancy , such as testicular leco - color doppler ( ecd ) riveste un ruolo di primo approccio nella patologia dello scroto risolvendo nella maggior parte dei casi lenigma diagnostico oppure delineando in modo esaustivo il quadro patologico , permettendo cos corrette decisioni e idonei approcci clinico - terapeutici . 
pur tuttavia , in presenza di quadri ecd di dubbia interpretazione alla luce del reperto clinico - anamnestico o in presenza di reperti clinico - ecografici ingiustificabili , la risonanza magnetica ( rm ) pu trovare applicazione per risolvere , chiarire o svelare aspetti a volte inusuali [ 1 ]  . solo pochi autori hanno esaminato il ruolo della rm come metodica di secondo livello in caso di reperti ecografici non esaustivi [ 2 , 3 ]  . 
finalit del lavoro stata quella di effettuare una revisione della nostra casistica degli ultimi 7 anni allo scopo di riconfermare lutilit dellecd nellapproccio alle patologie dello scroto e far emergere il ruolo della rm in casi selezionati . materiali e metodi dal 2000 al 2007 sono stati inviati al nostro servizio 801 pazienti maschi di et compresa tra 0 e 87 anni , per essere sottoposti ad indagine ecd dello scroto con ecografo ge logic 7 con sonda lineare 715 mhz con modulo color doppler , power doppler e b flow . 
i pazienti erano giunti alla nostra osservazione in regime di urgenza , in quanto inviati dal pronto soccorso , oppure in regime prioritario ambulatoriale su consiglio dello specialista urologo , nel sospetto di una patologia acuta o subacuta . sono state eseguite scansioni in scala di grigi quanto pi possibile panoramiche , sui piani longitudinale e trasversale , per confrontare le dimensioni e lecogenicit dei due lati . 
lo studio color doppler stato condotto successivamente , ottimizzando lapparecchiatura per ottenere la massima sensibilit ai flussi lenti , con una bassa pulse repetition frequency ( prf ) , bassi filtri di parete e un appropriato guadagno di colore . 
nei casi di sospetta torsione o infarto , il lato asintomatico stato valutato per primo , per assicurarsi che i parametri di valutazione del flusso fossero settati in modo opportuno . in 46 pazienti si ritenuto di procedere ad indagine rm , per confermare o chiarire il dato ecd , rilevando in alcuni 416 radiol med ( 2009 ) 114 : 414424 microlithiasis , testicular retention and chronic orchitis . mri was performed on a 1.5 - tesla philips achieva unit with a 14 - cm circular surface coil . 
for studying the scrotum in the three orthogonal planes , a t2 - weighted fast spin - echo sequence with a 1012 - cm field of view and a 256256 matrix was used . 
axial t1 - weighted spoiled gradient - echo images were also obtained to detect haemorrhage . only selected cases were studied with mri following intravenous administration of gadolinium with diethylenetriamine penta - acetic acid ( gd - dtpa ) ; in these cases , axial and coronal t1 - weighted spoiled gradient - echo sequences with fat saturation were obtained after contrast injection . definitive diagnoses were confirmed at surgery in the cases of funicular torsion , morgagnis hydatid torsion and inguinoscrotal hernia . 
in the case of inflammatory processes , thrombophlebitis of the pampiniform plexus , complicated cysts and scrotal trauma with intact tunica vasculosa , the diagnosis was declared ex adjuvantibus after successful medical treatment , as confirmed by lesion regression at serial us follow - up and resolution of clinical symptoms . results among the 801 patients examined , medical history , clinical data and cdus findings led to a definite diagnosis of inflammatory process in 277 patients , traumatic lesion in 112 , funicular torsion in 33 ( three cases appearing at birth , with intrauterine torsion ) and torsion of the hydatid of morgagni [ 4 , 5 ] in 11 . 
i criteri utilizzati per definire la valutazione ecografia non esaustiva sono stati : incerta natura della lesione , incapacit dellecografia di definire spazialmente la lesione in termini di sede e / o di estensione della stessa , la discrepanza tra la clinica e la diagnosi ecografia , la presenza di una marcata disomogeneit strutturale in presenza di condizioni predisponesti linsorgenza di neoplasie quali microlitiasi testicolare ( mt ) , ritenzione testicolare , orchite cronica . abbiamo impiegato unapparecchiatura philips achieva 1 , 5 tesla con una bobina di superficie , circolare da 14 cm . per la patologia del canale inguinale si studiata la pelvi mediante scansioni assiali usando un campo di vista pi ampio . 
per lo studio dello scroto sui 3 piani ortogonali sono stati utilizzati : una sequenza fast spin - eco pesata in t2 , un campo di vista 1012 cm e una matrice 256256 . 
sono anche state acquisite immagini assiali spoiled - gradient - eco t1 pesate , molto utili per identificare lemorragia . lacido dietilen - triamino - penta - acetico chelato con gadolinio ( gd - dtpa ) , per via endovenosa , stato somministrato solo in casi selezionati ; sono state condotte acquisizioni assiali e coronali dopo somministrazione del mezzo di contrasto , sfruttando sequenze spoiled - gradient - eco con saturazione per il grasso t1 pesate . 
per le lesioni di sospetta origine neoplastica si proceduto con esame citologico estemporaneo intraoperatorio seguito in caso di reperto dubbio o positivo da orchiectomia e in caso di negativit da nodulectomia . 
nei processi flogistici , nelle tromboflebiti del plesso pampiniforme , nelle cisti complicate e nei traumi del testicolo con conservazione dellintegrit della tunica vasculosa , la diagnosi stata confermata ex adjuvantibus dal successo della terapia medica , confermato dalla regressione delle lesioni nel corso di controlli ecografici seriati e dalla regressione del quadro clinico . 
 risultati tra gli 801 pazienti esaminati lanamnesi , i dati clinici e i reperti ecd hanno permesso la diagnosi definitiva di : processo flogistico in 277 pazienti , lesione traumatica in 112 , torsione del funicolo in 33 casi ( in 3 con comparsa alla nascita e torsione intrauterina ) e torsione dellidatide del morgagni [ 4 , 5 ] in 11 pazienti . 
in 15 patients , mri confirmed the suspicion of neoplastic lesion by showing t1 isohyperintensity and clear t2 hypointensity relative to the surrounding parenchyma . the use of contrast material allowed us to evaluate lesion vascularity ( with findings virtually coincident with cdus and b - flow imaging )  . 
in ten patients with testicular microlithiasis on cdus , mri enabled us to rule out alterations in signal intensity or enhancement that might indicate ecografica , sottoposti a rm come indagine di secondo livello , sono stati 46 pari al 5 , 74% . 
in 15 pazienti la rm ha confermato il sospetto di lesione neoplastica in base allaspetto isoiperintenso in t1 e francamente ipointenso in t2 rispetto al parenchima circostante . lutilizzo del mdc ha permesso di valutare la vascolarizzazione delle lesioni ( con reperto sostanzialmente sovrapponibile al dato ecd e b flow )  . 
in 10 pazienti con reperto ecd di mt la rm ha permesso di escludere con sicurezza alterazioni di segnale o di contrast - enhancement ( c.e. ) riferibili a lesioni parenchimali ( in 1 caso ha consentito di rilevare accumulo focale di calcio - pirofosfatodiidratato )  . 
in 4 pazienti affetti da ritenzione testicolare grazie alla rm stato possibile identificare la sede del testicolo atrofico non visibile ecograficamente , differeziando tale condizione dallagenesia testicolare , ed escludere una patologia eteroproduttiva a cui tali soggetti sono predisposti . 
3a - c cisti intrascotale con contenuto infiammatorio : a aspetto ecd ; b rm : sequenza pesata in t1 ; c rm sequenza pesata in t2 . parenchymal lesions ( in one case , it allowed detection of a focal accumulation of calcium - pyrophosphate - dihydrate )  . mri was also used to confirm the sonographic diagnosis of inguinoscrotal hernia in three cases , in which it showed the presence of intestinal loops in the scrotal region . 
in four patients with cryptorchidism , mri helped to locate the undescended testis , which was not visible at us , to differentiate this condition from testicular agenesis and to exclude possible malignancy . 
likewise , it enabled us to exclude nella nostra casistica i casi ritenuti dubbi e sottoposti a rm come indagine di secondo livello sono stati 46 pari al 5 , 74% . 
 radiol med ( 2009 ) 114 : 414424 areas of signal alteration or increased contrast enhancement in three patients with chronic orchitis in whom cdus had shown marked echostructural inhomogeneity . 
mri was also used to follow - up conservative treatment in two patients with scrotal trauma and cdus findings of discontinuity of the tunica albuginea and intact capsular vascularity . discussion in our series , mri was performed as a second - line investigation in 46 patients ( 5.74% ) with inconclusive cdus findings . 
this pattern , which has been previously described in the literature [ 6 ] , is particularly useful for solving interpretation doubts : it should be noted that 10%15% of testicular neoplasms are detected as a consequence of traumas [ 79 ]  . in one patient with a large intrascrotal collection , mri enabled a diagnosis of epididymo - orchitis with abscess formation and intrascrotal fistula , which could not be detected by cdus . 
on the basis of the literature and patients clinical data , we interpreted the findings as complicated testicular cysts , an interpretation that was confirmed ex adjuvantibus after symptom resolution with appropriate medical treatment . 
si tratta di un reperto , gi descritto in letteratura [ 6 ] , utile laddove sussistano dubbi interpretativi : si tenga conto che il 10%15% delle lesioni eteroplasiche testicolari vengono scoperte in conseguenza di un trauma [ 79 ]  . in un pazienze con voluminosa raccolta intrascrotale la rm ha permesso di rilevare orchiepididimite ascessualizzata con tramite fistolizzato in sede intrascrotale , difficilmente rilevabile allindagine ecd . 
analogamente basandoci su tali osservazioni e sulla clinica abbiamo interpretato i nostri reperti come cisti del testicolo complicate , confermate poi ex juvantibus dalla risoluzione del quadro dopo adeguata terapia medica . 
contrast - enhanced t1 sequences demonstrated the characteristic rim of enhancement delimiting the lesion [ 12 , 13 ]  . in 15 patients with cdus findings suggestive of testicular neoplasm , mri confirmed the diagnosis ( definitive histology revealed 11 cases of germ - cell tumour and four benign neoplasms , two of which were adenomatoid tumours and two sertoli - cell tumours )  . 
in these patients , mri also enabled preoperative staging by depicting possible infiltration of the tunica albuginea , tunica vaginalis , epididymis and / or of the para - epididymal tissues . 
in questi pazienti la rm ha inoltre permesso la stadiazione pre - operatoria , potendo definire tunica albuginea , della tunica vaginale , dellepididimo e / o dei tessuti paraepididimari . 
in letteratura si trovano opinioni discordanti sul significato della mt , tuttavia prevalgono le tesi che attribuiscono alla mt il ruolo di condizione precancerosa [ 1418 ]  . in un paziente che presentava formazione dubbia del testicolo , del diametro di circa 1 cm , in sede parailare , iperecogena e senza significativo flusso intralesione all ecd , la rm ha evidenziato alta intensit del segnale nelle sequenze pesate in t1 e basso segnale in t2 . 
tale reperto risulta invariato da 3 anni e , alla luce di quanto riferito da alcuni aa [ 1921 ] , pu essere attribuito ad accumulo di calcio - pirofosfato - diidratato . la rm non in grado di identificare la mt [ 2224 ] : questo aspetto pu facilitare il rilievo di aree di alterato segnale nel parenchima testicolare in pazienti affetti da questa condizione . 
perci , ove la disomogeneit ecostrutturale del parenchima associata alla mt non ha permesso lassoluta esclusione con lecd di lesioni eteroproduttive , siamo ricorsi alla rm che ha escluso alterazioni di segnale radiol med ( 2009 ) 114 : 414424 fig . 
5a - d infarto testicolare ; a immagine ecd ; b immagine rm pesata in t1 ; c immagine rm pesata in t2 ; d immagine rm con mdc . low signal intensity on t2 . 
this finding has remained unchanged for 3 years and , based on previous reports [ 1921 ] , it may be due to an accumulation of calciumpyrophosphate - dihydrate . mri is unable to depict testicular microlithiasis [ 2224 ] , and this may facilitate the detection of parenchymal areas with pathological signal in affected patients . 
therefore , when the parenchymal echostructural inhomogeneity associated with testicular microlithiasis did not allow malignant lesions to be confidently excluded with cdus , the patient was further studied with mri , which revealed no signal alterations or areas of contrast enhancement in the testis . similarly , mri proved useful in three patients with marked parenchymal echostructural alteration due to chronic inflammation , in whom it confirmed the cdus findings and revealed no focal alteration . four patients with atrophic testicular involution secondary to cryptorchidism and / or funicular torsion and who presented diffuse parenchymal echostructural alterations on cdus were also studied with mri to exclude focal parenchymal lesions . 
cryptorchidism is a well - known risk factor for nel testicolo , ma soprattutto non ha rilevato aree di potenziamento dopo mezzo di contrasto . con analogo significato la rm risultata utile in 3 pazienti con marcata alterazione ecostrutturale del parenchima per flogosi cronica , nei quali ha confermato il reperto ecd non rilevando alterazioni focali . sempre per escludere lesioni focali parenchimali sono stati sottoposti a rm 4 casi di involuzione atrofica del testicolo , secondaria a ritenzione testicolare e / o a torsione del funicolo , che presentavano sovvertimento ecostrutturale diffuso del parenchima allecd . 
la ritenzione testicolare rappresenta un noto fattore di rischio per linsorgenza di neoplasie testicolari [ 2527 ] e in questi pazienti consigliato il monitoraggio ecografico : a volte la marcata alterazione ecostrutturale del parenchima e / o la scarsit del flusso parenchimale all ecd nel testicolo atrofico , possono creare dubbi allecografista . 
6a - c trauma dello scroto con rottura della tunica albuginea e integrit della tunica vascolosa : a quadro ecd ; b controllo ecd ; c quadro rm in esiti di rottura della tunica albuginea post - traumatica . testicular neoplasm [ 2527 ] , and sonographic surveillance is recommended in all patients with this condition . 
our experience confirms the usefulness of mri to exclude cancerous lesions . in three cases , mri confirmed the cdus suspicion of omental herniation within the inguinal canal , thus proving its extreme utility in the study of this structure [ 7 ]  . a ruptured tunica albuginea is an indication for exploratory surgery [ 2832 ]  . 
our relatively limited experience demonstrates that surgery is required only in patients with compromised capsular vascularity , that is , of the tunica vasculosa that runs below the tunica albuginea . as is known , on reaching the lower pole of the testis , the testicular artery divides into branches that pierce the tunica albuginea and run along the periphery of the testis in a layer known as the tunica vasculosa . 
dalla nostra seppur limitata esperienza si evince che i pazienti da sottoporre a revisione chirurgica , sarebbero solo quelli con compromissione della vascolarizzazione capsulare e cio della tunica vasculosa che decorre sotto la tunica albuginea  . 
come noto larteria testicolare giunta in prossimit del polo inferiore del testicolo si suddivide in diversi rami , che decorrono alla periferia del testicolo al di sotto della tunica albuginea , formando la cos detta tunica vascolosa . 
da queste arterie capsulari si sviluppano branche centripete che penetrano nel parenchima testicolare decorrendo verso il mediastinum testis ove si arborizzano in rami ricorrenti [ 10 , 33 ]  . 
si pu radiol med ( 2009 ) 114 : 414424 parenchyma and run towards the testicular mediastinum , where they arborise into recurrent rami [ 10 , 33 ]  . 
consequently , it appears reasonable to believe that discontinuity of the tunica albuginea with normal capsular vascularity is an indication for conservative treatment and close clinical and imaging follow - up . conclusions in our experience , cdus is irreplaceable as a first - line approach to patients with scrotal disease . 
we have proposed cdus evaluation of tunica vasculosa integrity in cases of testicular rupture to determine the feasibility of a conservative approach , even in the presence of discontinuity of the tunica albuginea . testicular infarction ; mri is highly valuable in the diagnosis of intratesticular haematoma ; cysts with atypical , presumably inflammatory content ; testicular abscess with intrascrotal fistula ; and in the diagnosis and preoperative staging of testicular neoplasms . 
mri proved to be an ideal second - line modality to be used in selected complex cases , as it improved patient management , leading to fewer unnecessary surgical operations and lower overall costs . quindi sostenere lipotesi che la soluzione di continuit della tunica albuginea con integrit della vascolarizzazione capsulare rappresenti indicazione alla terapia conservativa , con stretto monitoraggio clinico - strumentale . conclusioni dalla nostra esperienza lindagine ecd appare di insostituibile utilit nel primo approccio al paziente con patologia scrotale . 
abbiamo proposto la valutazione con ecd dellintegrit della tunica vasculosa , nei casi di rottura del testicolo , al fine di valutare lipotesi di un approccio terapeutico conservativo , anche in presenza di soluzione di continuit della tunica albuginea . la rm si offre come metodica di insostituibile utilit nella diagnosi di ematoma intratesticolare , di cisti a contenuto atipico verosimilmente infiammatorio , di infarto testicolare , di ascesso testicolare con fistolizzazione intrascrotale nonch nella diagnosi e stadiazione preoperatoria delle eteroplasie testicolari . 
simonetti department of imaging diagnostics , molecular imaging , interventional radiology and radiotherapy , policlinico universitario tor vergata , viale oxford 81 , 00133 rome , italy correspondence to : e . 
twenty - eight women , mean age 50 years , and with a diagnosis of pudendal neuralgia on the basis of clinical and electromyographic criteria were enrolled in the study . 
in pudendal nerve entrapment , ct - guided perineural injection in the anatomical sites of nerve impingement is a safe and reproducible treatment with a clinical efficacy of 92% at 12 months . 
nella valutazione a ventiquattrore post - trattamento stato riferito un miglioramento qualitativo significativo della sintomatologia dolorosa in 21 casi su 27 ; in 24 su 27 casi stato riportato un miglioramento 20% dellindice di qualit della vita ( qol index ) nel follow - up clinico a 3 , 6 , 9 e 12 mesi . 
nella sindrome da intrappolamento del nervo pudendo , liniezione perineurale tc guidata delle sedi anatomiche di conflitto nervoso risulta una procedura sicura e ripetibile con una efficacia clinica nel 92% dei pazienti trattati nel follow - up a 12 mesi . parole chiave nevralgia blocco del nervo pudendo intrappolamento nel canale di alcock tomografia computerizzata guidata 426 introduction chronic pelvic pain can be defined as noncyclic pain in the pelvis , anterior abdominal wall below the umbilical line and lumbosacral region and / or glutei that persists continuously for at least 6 months and is sufficiently severe to cause functional disability and / or require nonspecific pharmacotherapy . 
although the exact prevalence of chronic pelvic pain in the two genders is unknown , 15%20% of women aged 1850 years are estimated to be affected [ 1 ]  . 
chronic pelvic pain may occur in a variety of clinical syndromes [ 4 ]  . the diagnostic and therapeutic roles of interventional radiology have increasingly gained importance in three specific chronic pelvic pain syndromes : pudendal neuralgia , piriformis syndrome syndrome and border nerve ( ilioinguinal , iliohypogastric and genitofemoral nerve neuropathy )  . pudendal neuralgia is a clinical entity that was underrecognised and poorly understood for many years [ 3 , 4 ]  . the pudendal nerve is a mixed sensory and motor nerve that arises from the somatic component of sacral nerve roots s2 and s4 on the ventral aspect of the piriformis muscle in the pelvic cavity and crosses the gluteal region , passing through the greater ischiatic foramen into the infrapiriformis canal . it supplies the anus , the urethral sphincters , the pelvic floor and the perineum and is responsible for genital sensitivity . the nerve is accompanied by the internal pudendal artery and is surrounded by a venous plexus ( pudendal neurovascular bundle )  . 
if thickened , duplication of the obturator fascia may also contribute to the entrapment [ 6 , 7 ]  . radiol med ( 2009 ) 114 : 425436 introduzione il dolore pelvico cronico si pu definire come dolore nonciclico localizzato alla regione pelvica , alla parete addominale anteriore inferiormente alla linea ombelicale e alla regione lombo - sacrale e / o ai glutei , che persiste continuativamente per almeno 6 mesi , di entit sufficiente da causare disabilit funzionale e / o richiedere cure farmacologiche generiche . 
sebbene la prevalenza del dolore pelvico cronico nella popolazione generale in entrambe i sessi non sia ancora completamente definita , si stima che ne siano affette circa il 15%20% delle donne di et compresa tra i 18 e i 50 anni [ 1 ]  . 
 il ruolo diagnostico e terapeutico della radiologia interventistica appare di crescente importanza in tre quadri sindromici di dolore pelvico cronico : nevralgia del pudendo , sindrome del piriforme e sindrome border nerve ( neuropatia iliaco - inguinale , iliaco - ipogastrica e del nervo genito - femorale )  . 
 la nevralgia del nervo pudendo stata unentit clinica a lungo non riconosciuta o scarsamente identificata [ 3 , 4 ]  . il nervo pudendo un nervo misto sensitivo e motorio , derivante dalla componente somatica delle radici del sacro s2 ed s4 dalla parte ventrale del muscolo piriforme nella cavit pelvica , attraversa la regione glutea passando attraverso il pi grande forame ischiatico nel canale infrapiriforme . 
il nervo predisposto allintrappolamento a livello della spina ischiatica ed allinterno del canale pudendo . alla spina ischiatica , il nervo pu essere compresso di frequente tra i legamenti sacrospinoso e sacrotuberoso . 
a livello del canale di alcock , il nervo pu essere compresso radiol med ( 2009 ) 114 : 425436 pudendal nerve sacrospinosus legament sacrotuberosus legament ischiatic spine pudendal canal fig . 
enmg is indicated for determining pudendal motor innervation prior to surgical decompression but not for locating the site of compression or monitoring the surgical procedure [ 12 ]  . just as there are no definitive clinical or imaging criteria for diagnosing pudendal neuralgia , neither is there a standard therapeutic approach , with conservative treatment , nerve block , decompressive surgery and neuromodulation all having shown to be useful [ 13 ]  . 
se ispessita , la duplicazione della fascia otturatoria pu anche contribuire essa stessa allintrappolamento [ 6 , 7 ]  . boisson [ 8 ] sugger per primo , nel 1966 , che la causa del dolore anoperineale cronico potesse essere una neuropatia ; altri autori hanno successivamente confermato tale ipotesi , definendo la nevralgia del pudendo come una neuropatia da entrapment [ 6 , 9 , 10 ]  . 
tuttavia se lintrappolamento senza dubbio la causa pi frequente di nevralgia del pudendo , altre eziologie sono state descritte [ 11 ] : polineuropatia periferica , neuropatia post - erpetica , neuropatia post - attinica , compressione neoplastica . anche se gli studi neurofisiologici [ 12 ] hanno contribuito alla conoscenza della malattia , non possono essere riconosciuti come indagine diagnostica di prima istanza . gli studi elettroneuromiografici ( enmg ) presentano limitate sensibilit e specificit nella diagnosi differenziale tra lesione nervosa da conflitto e sindrome da dolore perineale cronico . 
lesame enmg indicato nella definizione della innervazione motoria del territorio di innervazione del pudendo prima della decompressione chirurgica ma non nella localizzazione della sede di compressione o nel monitoraggio intra - operatorio [ 12 ]  . se la diagnosi di nevralgia del pudendo non ha criteri diagnostici clinico - strumentali definitivi , altrettanto valido per il suo trattamento . 
la radiologia interventistica si propone in modo efficace come terapia infiltrativa percutanea del nervo pudendo sotto guida tc , ecografica o fluoroscopica . lo scopo del presente lavoro di descrivere i vantaggi della tecnica di blocco tc guidata del nervo pudendo e confrontarla con altre tecniche di radiologia interventistica imaging - guidate recentemente proposte in letteratura . 
end point primario dello studio la risoluzione persistente della sintomatologia algica nel periodo di follow - up individuale . materiali e metodi nel periodo compreso tra maggio 2006 e gennaio 2008 sono state incluse nel nostro studio prospettico 28 donne con et media 50 anni e diagnosi di nevralgia del pudendo , posta sulla base di criteri clinici ed elettromiografici , con algia significativa e invariata / persistente dopo 6 settimane di terapia conservativa ( tabella 1 )  . 
lintensit minima del dolore > 5 / 10 vas score ( visual analog pain scale livello di dolore riferito a intensit , distress muscolare , spasmo muscolare , interferenza con normali attivit quotidiane ) per tre giorni consecutivi e dolore posturale intermittente valutato durante test biomeccanico . 428 radiol med ( 2009 ) 114 : 425436 this paper describes the advantages of ct - guided pudendal nerve block and compares it with other imagingrecently guided proposed in the literature . 
the primary end - point of the study was persistent pain relief in the follow - up period . radiology procedures interventional materials and methods between may 2006 and january 2008 , we recruited 28 women ( mean age 50 years ) with a diagnosis of pudendal neuralgia based on clinical and electromyographic ( emg ) criteria and suffering from persistent significant pain after 6 weeks of conservative therapy ( table 1 )  . 
minimum pain intensity was a score of > 5 / 10 on the visual analogue scale ( vas ) : pain level related to intensity , muscular distress , muscular spasm and interference with normal daily activities for 3 consecutive days and intermittent postural pain assessed with a biomechanical test . i pazienti ( n = 28 ) hanno firmato un consenso informato allo studio precedentemente approvato dalla commissione etica del nostro policlinico . 
tutte le pazienti , con evidenza clinica di dolore cronico inabilitante gravativo non urente e non - ciclico della regione ano - rettale , labbra e perineo sono state sottoposte a rm del rachide lombo - sacrale per escludere la presenza di ernia discale e / o impingement radicolare . 
 tutte le pazienti erano maggiorenni , con un test di gravidanza negativo ( 6 in et riproduttiva ) , con un peso corporeo 50 kg ed erano stabili da un punto di vista medico sistemico . 
il protocollo di trattamento prevede che le inoculazioni siano eseguite in successione a distanza di 15 giorni una dallaltra per 3 volte ; in ogni seduta la prima sessione effettuata a livello della spina ischiatica e la seconda a livello del canale pudendo [ 14 ]  . 
patients with clinical evidence of chronic , disabling , nonburning , noncyclic aching pain in the anorectal , labial and perineal regions underwent magnetic resonance imaging ( mri ) of the lumbosacral spine to rule out intervertebral disk herniation and / or radicular impingement . 
the scans should allow clear la paziente posta in decubito prono ; il protocollo impiegato si avvale della acquisizione di scansioni da 2 , 5 mm , con pitch 1 , estese dal tetto dellacetabolo alla sinfisi pubica . 
il nervo pudendo sovrasta la spina ischiatica esattamente a livello della inserzione del legamento sacrospinoso , ed rivestito a questo livello dalla superficie interna del legamento sacrotuberoso , visibile come una linea iperdensa sovrapposta alla parte ventrale del muscolo grande gluteo . 
previa disinfezione della cute della regione glutea , viene inserito un ago spinale da 22 g per via trans - glutea , verso il fascio pudendo con direzione verticale in corrispondenza della parte caudale della spina ischiatica , evitando il nervo sciatico . 
the pudendal nerve crosses the ischial spine exactly at the point where the sacrospinous ligament attaches to the spine , and at this level , it is covered by the inner surface of the sacrotuberous ligament , which appears as a dense linear structure over the ventral portion of the gluteus maximus muscle . 
after the skin of the gluteal region has been disinfected , a 22 - gauge spinal needle is inserted transgluteally and advanced towards the pudendal bundle following a vertical direction at the caudal extremity of the ischial spine , with care being taken to avoid the sciatic nerve . 
once the needle tip is correctly placed in the interligamentous space ( between the sacrospinous and sacrotuberous ligaments , as close as possible to the caudal portion of the ischial spine ) , 0.75 ml of diluted contrast agent ( 5% iomeron 300 mg / ml , bracco , milan , italy ) is injected , which should appear to surround the pudendal bundle in the interligamentous space . this is followed by an injection of a mixture of methylprednisolone ( 1 ml depo - medrol 40 mg / ml solution , pfizer , new york , ny , usa ) and lidocaine ( 1 ml 2% 200 mg / 10 ml solution , bioindustria lim , novi ligure , italy )  . 
si inietta quindi una miscela di metilprednisolone acetato ( 1 ml di soluzione di depo - medrol 40 mg / ml , pfizer , new york , usa ) e di lidocaina cloridrato ( 1 ml di soluzione al 2% 200 mg / 10 ml , bioindustria lim , novi ligure , italia )  . la lidocaina , anestetico long - acting , produce unanestesia cutanea del territorio del nervo pudendo e fa da indicatore dellubicazione esatta del blocco del nervo . 
il cortisonico provvede alla risposta a lungo termine sia per gli effetti anti - infiammatori degli steroidi che per la necrosi adiposa indotta dagli steroidi con riduzione dellazione compressiva peri - neurale . 
dopo aver introdotto un ago da 22 - gauge con decorso verticale tramite un approccio transgluteo , si dispone lago a livello del tessuto adiposo posto subito lateralmente alla fascia otturatoria . 
una volta valutata la corretta posizione della punta dellago mediante iniezione di una soluzione di 0 , 75 ml di mezzo di contrasto diluito , che dimostra il nervo pudendo allingresso del canale di alcock , somministriamo localmente la miscela di anestetico e di steroide nelle stesse quantit e proporzioni della prima iniezione . 
 per una valutazione dellesito del trattamento dal punto di vista della valutazione soggettiva del paziente , a ciascuna delle donne inserite al protocollo di studio stato radiol med ( 2009 ) 114 : 425436 fig . 
 to obtain patients subjective evaluation of outcome , each woman was given a questionnaire ( including the vas ; pain score referred to intensity , muscular distress , muscular spasm , interference with normal daily activities ) to be filled in once a week at the same hour throughout the follow - up period ( from 3 to 12 months ) and handed in at the monthly hospital visits . 
patients were examined and interviewed every month to rate their perceived quality of life ( qol index ) after treatment on a five - point scale ( 1 = qol worsened ; 2 = qol unchanged ; 3 = qol improved with occasional symptom remission ; 4 = qol improved with persistent symptom remission ; 5 = qol optimal , with disappearance of pain )  . results a total of 82 procedures were completed over 164 sessions ( table 1 )  . 
in one case , the procedure had to be discontinued because the patient had respiratory insufficiency and could sottoposto un questionario relativo al vas score ( visual analog pain scale livello di dolore riferito a intensit , distress muscolare , spasmo muscolare , interferenza con normali attivit quotidiane ) da compilare settimanalmente a casa , allo stesso orario del giorno per la durata del followup ( variabile da 3 a 12 mesi ) e riconsegnato mensilmente in ospedale . 
con cadenza mensile i pazienti trattati sono stati visitati ed intervistati per ottenere un valore individuale di percezione della qualit della vita post - trattamento ( qol index ) in una scala variabile da 1 ( peggioramento della qualit della vita dopo il trattamento ) a 5 ( qualit della vita ottimale con scomparsa della sintomatologia dolorosa ) , con valori intermedi di 2 ( qualit della vita invariata rispetto al pre - trattamento ) 3 ( qualit della vita migliorata con remissione saltuaria dei sintomi ) e 4 ( qualit della vita migliorata con remissione persistente dei sintomi )  . risultati sono state completate 82 procedure per 164 sessioni ( tabella 1 )  . 
in un caso la procedura stata sospesa per impossibilit della paziente a mantenere la posizione prona in relazione ad una condizione di insufficienza respiratoria ; la paziente stata pertanto esclusa dalla successiva valutazione . 
nei restanti 27 casi sono state effettuate tutte le procedure ( 81 ) , nei tempi e nelle modalit sopradescritte . la procedura stata sempre eseguita senza complicanze in un periodo di tempo variabile da 20 a 30 minuti . 
in the remaining 27 patients , all procedures ( 81 ) were successfully completed within the timeframe and in the manner described above . all procedures were completed without complications in 2030 m infiltrations were unilateral on the symptomatic side in 26 / 27 cases and bilateral in one case . 
 minor treatment complications included a burning sensation at the injection site in 12 / 27 patients ( 44% ) , a temporary ( < 12 - h ) worsening of pain in 3 / 27 patients ( 11% ) and transient ( > 12 - h ) block of the sciatic nerve in 11 / 27 patients ( 40% )  . 
 evaluation of pain after treatment compared with perceived pain before treatment showed a significant improvement in 25 / 27 cases , with a vas ( meanstandard deviation ) of 2.120.41 , with a score of 5 in six patients ( 22% ) , a score of 4 in five ( 18% ) and a score of 3 in 14 ( 52% )  . 
 complicanze minori del trattamento sono state il bruciore a livello del sito di iniezione ( 12 / 27 pazienti , 44% ) , temporaneo ( < 12 ore ) peggioramento della sintomatologia algica ( 3 / 27 pazienti , 11% ) , blocco transitorio ( > 12 ore ) del nervo sciatico ( 11 / 27 pazienti , 40% )  . 
una dolenzia nelle sedi della infiltrazione , una volta scomparso leffetto dellanestetico , stata riscontrata in 26 casi su 27 ( 96% )  . il questionario a cui sono state sottoposte le pazienti ha fornito le seguenti valutazioni . 
a 24 ore dalla procedura in 21 su 27 casi ( 77% ) stata riferita una riduzione significativa del dolore rispetto alla baseline ( tabella 2 ) , mentre in 6 su 27 casi il dolore si ridotto in modo non significativo intensit 0 , 230 , 68 ; spasmo ( attivit 0 , 0590 , 49 ; 0 , 160 , 58 ; distress 1 , 680 , 58 )  . 
un miglioramento del 20% o pi del qol index stato riferito in 24 su 27 casi ( 88% ) rispetto al relativo follow - up ( 48% a 3 mesi ; 4% a 6 mesi ; 15% a 9 mesi ; 33% a 12 mesi ) mentre in 3 casi su 27 stato inferiore al 20% . 
 la valutazione del dolore al termine del trattamento rispetto alla percezione del dolore prima dellesecuzione del trattamento , ha riportato in 25 casi su 27 un miglioramento significativo con un vas ( mediadeviazione standard ) di 2 , 120 , 41 con score di 5 in 6 pazienti ( 22% ) , score 4 in 5 pazienti ( 18% ) e score 3 in 14 ( 52% )  . 
in 2 casi su 27 non stato riferito alcun beneficio a distanza dal trattamento eseguito ( vas 0 , 780 , 11 )  . discussione leziologia del dolore ano - perineale cronico in assenza di una identificabile causa urologica , ginecologica o proctologica frequente e di difficile individuazione . 
numerose sindromi sono state messe in causa per spiegare questo tipo di dolore , come ad esempio la coccigodinia , la proctalgia fugace o la sindrome del muscolo elevatore dellano . in assenza di imaging patognomonico , reperti laboratoristici ed elettrofisiologici , la diagnosi di nevralgia del pudendo rimane clinica . 
semplici recenti criteri utili alla identificazione clinica della nevralgia del pudendo dovuta ad intrappolamento del nervo sono i cinque criteri di nantes [ 9 ] : ( 1 ) dolore nel territorio del nervo pudendo : dallano verso il pene o il clitoride ; ( 2 ) dolore accentuato dalla posizione seduta ; ( 3 ) dolore che non risveglia il paziente di notte ; ( 4 ) sintomatologia algica in assenza di perdita di sensibilit ; ( 5 ) miglioramento del dolore dopo blocco anestetico del nervo . criteri di esclusione della neuropatia sono dolore puramente coccigeo , gluteo o ipogastrico , dolore esclusivamente parossistico , prurito , reperti di imaging che spiegano la sintomatologia . 
tuttavia la presenza di una sintomatologia radiol med ( 2009 ) 114 : 425436 discussion chronic anoperineal pain in the absence of any identifiable urological , gynaecologic or proctological cause is common and its aetiology difficult to ascertanumerous syndromes have been cited to explain this type of pain , such as coccygodynia , proctalgia fugax or levator ani syndrome . in the absence of pathognomonic imaging , laboratory and electrophysiological findings , the diagnosis of pudendal neuralgia remains clinical . 
simple criteria for the clinical demonstration of pudendal neuralgia due to nerve entrapment are the following ( nantes criteria ) [ 9 ] : ( 1 ) pain in the anatomical area supplied by the pudendal nerve ( from the anus to the penis or clitoris ) ; ( 2 ) pain worsens when patient is sitting ; ( 3 ) pain never causes the patient to wake in the night ; ( 4 ) no sensory loss is found on clinical examination ; ( 5 ) pain improves after anaesthetic nerve block . exclusion criteria for neuropathy are purely coccygeal , gluteal or hypogastric pain ; exclusively paroxysmal pain ; pruritus ; and imaging findings accounting for the symptoms . 
however , the presence of pain in the area supplied by the pudendal nerve should always arouse suspicion of pudendal neuralgia related to strain and compression . compression is more likely if the pain worsens upon sitting and is relieved upon standing or lying . 
long - term effectiveness is , however , undermined by late recurrence due to irreversible nerve damage ; incomplete release of the nerve , especially in the case of entrapment in the distal alcocks canal ; and the appearance of postsurgical perineural fibrosis . diagnostic imaging plays an important role in excluding other conditions that may simulate anoperineal pain and in providing guidance during nerve infiltration . 
 as in other entrapment syndromes , nerve infiltration at the site of compression has a dual role : diagnostic when performed with an anaesthetic , and therapeutic when performed with corticosteroids . 
treatment of pudendal neuropathy with corticosteroid and anaesthetic infiltration is not new , with pudendal nerve block via a transperineal approach having been first described by muelener in 1908 [ 14 ]  . 
pudendal nerve block with conventional fluoroscopic guidance has become popular in clinical practice owing to the easy identification of anatomical landmarks [ 18 , 19 ] , with placement of the needle tip near the apex of the iliac crest . 
nonetheless , the pudendal nerve courses in an anatomical plane formed by the sacrospinous and sacrotuberous ligament dolorosa nel territorio di innervazione del nervo pudendo deve sempre far considerare la presenza di una nevralgia dello stesso nervo , sia da allungamento sia da compressione ; la compressione pi probabile come causa del dolore quando questo peggiora con la posizione seduta mentre descresce in ortostatismo e nella posizione sdraiata . 
lefficacia a lungo termine risulta tuttavia inficiata dalla ricomparsa tardiva del dolore a causa di danneggiamento irreversibile del nervo , incompleto rilascio dello stesso soprattutto in caso entrapment a livello della parte distale del canale di alcock e dalla comparsa di fibrosi peri - neurale post - chirurgica . 
la diagnostica per immagini riveste un ruolo importante non nellaccertamento della patologia in se stessa quanto nella esclusione delle condizioni che possono simulare il dolore ano - perineale e come guida nellinfiltrazione del nervo . 
 come nelle altre sindromi da intrappolamento linfiltrazione del nervo nella sede della compressione ha un duplice effetto : diagnostico quando essa viene effettuata con anestetico e terapeutico quando vengono iniettati nella sede dei cortisonici . 
il trattamento della neuropatia del pudendo mediante infiltrazione con cortisonici ed anestetici non una terapia nuova ; il blocco del nervo pudendo con approccio transperineale stato descritto per la prima volta da muelener nel 1908 [ 14 ]  . 
 la terapia percutanea pu essere eseguita sotto guida tc [ 14 , 16 , 17 ] , fluoroscopica [ 18 ] oppure impiegando una guida elettromiografica [ 13 ]  . 
la tecnica di blocco del nervo pudendo con guida fluoroscopica convenzionale ha guadagnato popolarit nella pratica clinica per il facile riconoscimento dei reperi anatomici [ 18 , 19 ] , posizionando la punta dellago nella adiacenza dellapice della cresta iliaca . 
tuttavia il nervo pudendo decorre in un piano anatomico tra il legamento sacrospinoso e sacrotuberoso ( piano interligamentoso ) che non visualizzabile in fluoroscopia . la guida ecotomografica per il blocco percutaneo del nervo pudendo a livello della spina ischiatica una tecnica semplice e ripetibile [ 20 , 21 ]  . 
lecografia garantisce una diretta visualizzazione dei reperi anatomici in diretta contiguit con il nervo pudendo quali la spina ischiatica , larteria pudenda interna , ed i legamenti sacrospinoso e sacrotuberoso . 
us ensures direct visualisation of anatomical landmarks immediately contiguous to the pudendal nerve , such as the ischial spine , the internal pudendal artery and the sacrospinous and sacrotuberous ligaments . 
the disadvantages of us are related to the difficulty in visualising the pudendal nerve ( 12% in a recent series [ 22 ] ) , which measures 46 mm at the ischial spine [ 23 ] , the sacrotuberous ligament and the pudendal artery , which may be confused with the inferior gluteal artery , with consequent frequent sciatic nerve block . 
however , at the level of the ischial spine , in 30%40% of cases , the pudendal nerve shows anatomical variants , with two or three nerve trunks [ 7 ] covered by dense connective and / or fat tissue [ 22 ] , resulting in poor sonographic conspicuity and limited response to nerve stimulation . 
the depth of the ischial spine from the cutaneous plane is usually > 7 c this requires intraoperative fluoroscopy to confirm correct needle placement and solution delivery , with a us / fluoroscopy concordance of 82% . 
nerve stimulation ( graded electrical stimulation ) to confirm correct placement of the us - guided needle does not appear to be an absolute technique because of the size of the nerve at the level of the interligamentous space [ 24 , 25 ]  . 
 ct - guided infiltration is a problem - solving technique that ensures correct needle placement at the site of passage of the pudendal nerve close to the ischial spine and at the entrance to alcocks canal . 
 [ 19 ] reported on 200 infiltrations , with two or three injections of 5 ml of lidocaine and 1.5 ml of corticosteroids , but they failed to describe the final outcome in terms of pain relief . 
benson and griffis [ 14 ] obtained an improvement of symptoms in 31% of cases ( 12 / 38 ) by injecting 1 cc of triamcinolone and 9 cc of lidocaine and performing up to three injections 6 weeks apart . 
furthermore , they reported exacerbation of symptoms for 710 injection , which was , however , days following performed under emg guidance . the assessment of outcomes may prove difficult owing to the subjective nature of the evaluation of symptom improvement . the scale adopted in our study allows for a more systematic evaluation of the evolution of symptoms , as it distinguishes between the benefits deriving from the individual injections from the final overall benefits and includes an assessment dimensioni a livello della spina ischiatica sono di 46 mm [ 23 ] , del legamento sacrotuberoso , della arteria pudenda , facilmente confusa con la glutea inferiore con conseguente frequente blocco del nervo sciatico . 
sempre a livello della spina ischiatica , il nervo pudendo nel 30%40% dei casi presenta varianti anatomiche con 2 o 3 tronchi nervosi [ 7 ] , rivestiti da connettivo denso e / o da tessuto adiposo [ 22 ] , con conseguente ridotta visibilit ecografia del nervo nonch scarsa risposta alla stimolazione nervosa . 
la profondit della spina ischiatica dal piano cutaneo comunemente > 7 cm , con conseguente necessit intra - procedurale di ricorrere alla fluoroscopia per confermare il centraggio dellago e la somministrazione della soluzione , con una concordanza ecografia / fluoroscopia dell82% . 
la stimolazione nervosa ( stimolazione elettrica graduata ) per confermare il posizionamento dellago eco - guidato non appare una tecnica assoluta , a causa delle dimensioni del nervo a livello dello spazio interlegamentoso [ 24 , 25 ]  . liniezione perineurale pu determinare la comparsa di alterazioni enmg a breve termine , anche quando la sintomatologia dolorosa sia risolta . 
 linfiltrazione tc guidata una tecnica problem solving garantendo il corretto posizionamento dellago nella sede di passaggio del nervo pudendo in prossimit della spina ischiatica e a livello dellingresso nel canale di alcock . 
 [ 19 ] eseguendo 2 o 3 iniezioni di 5 ml di lidocaina e di 1 , 5 ml di cortisonico riporta una casistica di 200 infiltrazioni , ma non specifica il risultato finale delle stesse riguardo il sollievo dalla sintomatologia . 
 [ 16 ] riporta su di una casistica di 36 pazienti un miglioramento della sintomatologia nei 3 / 4 dei casi ; la sua tecnica consiste in 5 infiltrazioni ripetute a distanza di 30 giorni luna dallaltra . 
benson e griffis [ 14 ] riferisce un miglioramento dei sintomi nel 31% dei casi ( 12 / 38 ) iniettando 1 cc di triamcinolone e 9 cc di lidocaina ed eseguendo un massimo di 3 iniezioni a distanza di 6 settimane ; inoltre riporta unesacerbazione dei sintomi per i successivi 710 giorni a seguito delliniezione , eseguita per sotto guida emg e non mediante imaging . la valutazione dei risultati appare senza dubbio difficile , essendo molto soggettiva la valutazione del miglioramento sintomatologico . 
la scala da noi adottata permette una valutazione pi sistematica dellevoluzione dei sintomi , distinguendo il beneficio di ciascuna iniezione da quello finale complessivo e valutando anche la capacit del soggetto di svolgere normalmente la sua attivit quotidiana . in tutte le casistiche riportate non sono mai riferiti insuccessi tecnici della procedura ; nelliniezione in corrispondenza della spina ischiatica occorre tuttavia prestare attenzione a non spostare lago troppo lateralmente , pena una radiol med ( 2009 ) 114 : 425436 of the subjects ability to carry out daily activities . none of the previous series reported on any technical failures . 
however , with regard to injections at the ischial spine level , it is important to avoid moving the needle too laterally so as not to cause sciatic nerve block . 
secondary complications include haematoma at the injection site , pain worsening for a few days and transient nerve blocks lasting hours . as shown in our experience , perineural injection of the pudendal nerve is to be considered a safe procedure that is free of significant complications . 
in the case of suspected pudendal nerve entrapment syndromes , we believe that ctguided infiltration of impingement sites can and should be performed , partly in consideration of the fact that other approaches conservative therapy , decompressive surgery and neuromodulation [ 13 ] fail to provide consistent and long - lasting results . conseguente analgesia del nervo sciatico . 
le complicanze secondarie possono lematoma nella sede includere delliniezione , il peggioramento del dolore della durata di alcuni giorni ed il blocco transitorio del nervo per alcune ore . i risultati da noi presentati sono indicativi comunque della efficacia terapeutica e della mancanza di complicanze . 
giacomo , unit operativa di radiodiagnostica , monopoli , bari , italy 3universit degli studi di catania , cattedra di radiodiagnostica , azienda policlinico di catania gaspare rodolico , catania , italy correspondence to : p . 
chieffi 40 , 70051 barletta , italy , tel . : + - 39 - 340 - 0781993 , fax : + 39 - 088 - 1733866 , e - mail : paomi03@libero.it received : 11 october 2008 / accepted : 5 march 2009 / published online : 9 november 2009 springer - verlag 2009 abstract purpose . 
of these 31 patients , we included in the study 22 patients ( mean age 53 years ; range 4657 years ) who underwent liver transplantation within 1224 h after mr examination . 
dei 31 pazienti selezionati per il trapianto dorgano , sono stati inclusi nello studio 22 pazienti , di et media di 53 anni ( range 4657 anni ) , sottoposti a trapianto di fegato a distanza di circa 1224 ore dallesecuzione dellesame rm . i pazienti inclusi nello studio sono stati sottoposti ad esame rm con un apparecchio da 1 , 5 t . 
unenhanced baseline mr imaging correctly identified and characterised 20 lesions , equal to 33.90% of all lesions : 6 hcc , 12 dn and 2 dn with a subfocus of hcc . 
spio - enhanced mr imaging showed greater sensitivity detecting and characterising 45 lesions , equal to 76.27% of all lesions identified at histology : 14 hcc , 27 dn and 4 dn with subfocus of hcc . 
spio - enhanced mr imaging proved to be of value in detecting and characterising lesions in the cirrhotic liver , allowing differentiation of dn from hcc and providing an early diagnosis of neoplastic degeneration of dn . keywords spio dysplastic nodules hepatocellular carcinoma magnetic resonance imaging 14 hcc , 3 sono risultati ben differenziati , 8 moderatamente differenziati , 3 scarsamente differenziati . dei 39 nd , 28 erano a basso grado di malignit e 11 ad alto grado di malignit . 
con lesame rm di base abbiamo identificato e caratterizzato correttamente 20 lesioni , pari al 33 , 90% delle lesioni complessivamente individuate , di cui 6 hcc , 12 nd e 2 nd con foci di hcc . 
lesame rm , eseguito dopo somministrazione di mdc spio , ha mostrato una maggiore sensibilit identificando e caratterizzando complessivamente 45 lesioni , pari al 76 , 27% delle lesioni totali individuate con lesame istologico : 14 hcc , 27 nd e 4 nd con foci di hcc . 
i falsi negativi con la rm - spio sono stati 12 noduli displasici , pari al 31% , che allesame istologico sono risultati essere nd a basso grado di malignit e con diametro inferiore al c conclusioni . 
la rm con utilizzo di mdc spio , nella nostra esperienza risultata utile nella identificazione e caratterizzazione delle lesioni epatiche nel fegato cirrotico mostrandosi in grado di differenziare i noduli displasici dagli hcc e ha consentito una diagnosi precoce della trasformazione carcinomatosa dei noduli displasici . parole chiave spio noduli displasici carcinoma epatocellulare risonanza magnetica introduction introduzione cirrhosis is a progressive , diffuse disease of the liver characterised by hepatocyte necrosis , fibrosis , distortion of the normal hepatic architecture and a spectrum of nodular lesions that includes regenerative nodules ( rn ) , dysplastic nodules ( dn ) and hepatocellular carcinomas ( hcc ) [ 1 ]  . 
in italy , the likelihood of developing hcc secondary to cirrhosis ranges from 2% to 5% [ 3 ]  . in recent decades , the survival of hcc patients has been significantly increased by improvements and refinements in both nonsurgical treatments , such as percutaneous ethanol injection ( pei ) , thermoablation and lipiodol computed tomography ( ct ) ; and surgical treatments , such as liver resection and transplantation . 
the results of these treatments are , however , heavily dependent on accurate disease staging , so that the early diagnosis of hcc and its precursor , the dn , is mandatory [ 4 , 5 ]  . the screening protocol for high - risk cirrhotic patients entails ( ultrasonographic ) imaging and alpha - fetoprotein testing at 3to 6 - month intervals . 
unfortunately , an la cirrosi un progressivo e diffuso processo patologico del fegato caratterizzato da necrosi epatocitaria , fibrosi , sovvertimento della normale architettura epatica e dalla presenza di uno spettro di lesioni nodulari che include noduli di rigenerazione ( nr ) , noduli displasici ( nd ) ed epatocarcinomi ( hcc ) [ 1 ]  . 
in italia la probabilit annuale di sviluppare hcc su cirrosi epatica varia dal 2% al 5% [ 3 ]  . negli ultimi decenni si verificato un significativo incremento della sopravvivenza dei pazienti affetti da tale patologia per il miglioramento e laffinamento sia delle terapie che si basano sui trattamenti locali , come liniezione percutanea di etanolo ( pei ) , la termoablazione e la lipiodoltomografia computerizzata , sia di quelle che si basano invece su tecniche chirurgiche , quali la resezione o il trapianto . 
i risultati di queste terapie sono tuttavia fortemente condizionati dallo staging della malattia ed , quindi , estremamente importante effettuare una diagnosi precoce ed accurata dellhcc e del suo precursore , il nd [ 4 , 5 ]  . il protocollo di screening , nei pazienti cirrotici ad radiol med ( 2009 ) 114 : 12671282 1269 elevation of alpha - fetoprotein levels is neither sensitive nor specific enough to be used to screen for hcc . 
this test may , in fact , fail to detect small tumours ( < 2 cm ) , and high alpha - fetoprotein levels are seen in only 50% of patients with hcc and 26% of those with dn [ 6 , 7 ]  . imaging techniques therefore play a primary role in the diagnosis of focal lesions in the cirrhotic liver , even though there is no general agreement as to the most appropriate modality for the study of this disease . 
the literature reports conflicting data regarding not only the diagnostic capabilities of the different imaging modalities ultrasonographic ( us ) , ct and magnetic resonance ( mr ) imaging in the detection and characterisation of hcc and dn , but also the possibility of arriving at a differential diagnosis among the different lesions . 
histological analysis of the entire cirrhotic liver and a short interval between mr imaging and transplantation are factors that , if neglected , may lead to considerable variability among results . 
indeed , a long interval between mr examination and transplantation could mean the transformation of a dn into an hcc , whereas a histological examination limited to biopsies or specimens from partial liver resection could preclude identification of other nodular lesions in the liver parenchyma [ 812 ]  . in recent years , investigators have emphasised the value of hepatospecific superparamagnetic contrast agents based on iron oxide particles ( spio ) in the detection and characterisation of focal lesions with mr imaging , and their use has been advocated for staging patients scheduled for surgical or locoregional treatment of malignant focal hepatic lesions [ 13 , 14 ] and for monitoring patients with cirrhosis [ 1517 ]  . the rationale for using spio contrast agents to differentiate among the wide range of nodular lesions seen in cirrhosis is not based on vascular changes but on the cellular changes rn undergo as they develop into dn and then into hcc . 
in fact , during this process , the number and function of kupffer cells , the cells that accumulate spio , decrease [ 10 , 1820 ] , and some investigators have reported that hcc and dn show different enhancement patterns after spio administration . 
dn , on the other hand , have preserved cellular composition and function and hence greater spio accumulation , with the result that they appear markedly hypointense even relative to the surrounding liver . 
these different enhancement patterns led researchers to hypothesise that it was possible to differentiate between these two lesions and identify the transformation of a dn into an hcc at an early stage . the aim of this study was to evaluate the potential of spio - mr imaging in the detection and characterisation of elevato rischio , prevede lesecuzione di esame ecografico e dei dosaggi dellalfa - fetoproteina ( afp ) ad intervalli di 36 mesi . 
tale test , infatti , non sempre in grado di rilevare piccoli tumori ( < 2 cm ) ed elevati livelli di afp sono presenti solo nel 50% di pazienti con hcc e nel 26% di pazienti con nd [ 6 , 7 ]  . le tecniche dimaging svolgono pertanto un ruolo fondamentale nella diagnostica delle lesioni focali nella cirrosi epatica ; tuttavia non vi uniformit di vedute su quale sia la metodica pi idonea per lo studio di tale patologia . 
in letteratura sono piuttosto controversi i dati circa le reali potenzialit diagnostiche delle diverse tecniche dimaging ecografia ( us ) , tomografia computerizzata ( tc ) e risonanza magnetica ( rm ) nella identificazione e nella caratterizzazione degli hcc e dei nd , nonch riguardo la possibilit di effettuare una diagnosi differenziale tra le diverse lesioni . questo si verifica per mancanza , in tutti i lavori finora pubblicati , di uniformit dei parametri di valutazione attendibili ; infatti , lanalisi istologica di tutto il fegato cirrotico ed il breve timing tra lesecuzione dellesame rm e il trapianto sono fattori che se non presi in considerazione possono portare una notevole variabilit nei risultati . 
infatti , un lungo tempo intercorso tra lesame rm e lesecuzione del trapianto potrebbe portare alla trasformazione di un nd in hcc , mentre uno studio anatomo - patologico limitato alle sole lesioni sottoposte a biopsia o a resezione parziale dellorgano potrebbe escludere lidentificazione di altre lesioni nodulari nel restante parenchima epatico [ 812 ]  . negli ultimi anni stata sottolineata lutilit dei mezzi di contrasto ( mdc ) superparamagnetici epatospecifici , a base di particelle di ossido di ferro ( spio ) , per lidentificazione e la caratterizzazione delle lesioni focali epatiche con rm ed il loro uso stato proposto per lo staging di pazienti da sottoporre a trattamenti chirurgici o loco - regionali per lesioni focali epatiche maligne [ 13 , 14 ] e nel follow - up dei pazienti portatori di cirrosi epatica [ 1517 ]  . il razionale di impiego dei mdc spio per la diagnosi differenziale tra il vasto spettro di lesioni nodulari presenti nella cirrosi epatica , si basa , pi che sulle modificazioni vascolari , sulle alterazioni cellulari che i nr subiscono nella loro trasformazione in nd prima e in hcc poi ; infatti , in rapporto a tale evoluzione , decrescono progressivamente il numero e la funzionalit delle cellule del kupffer , deputate alla captazione del mdc spio [ 10 , 1820 ]  . 
in relazione a tali riscontri , alcuni autori hanno rilevato un differente comportamento contrastografico con i mdc spio tra gli hcc e i nd , in quanto i primi , a causa delle alterazioni cellulari subite , non presenterebbero captazione di mdc spio , risultando marcatamente iperintensi rispetto al parenchima epatico contiguo ; i nd invece , preservando composizione e funzionalit cellulari , presenterebbero una maggiore captazione di mdc spio , apparendo francamente ipointensi anche rispetto al fegato sano limitrofo . 
tali comportamenti contrastografici hanno indotto a considerare 1270 radiol med ( 2009 ) 114 : 12671282 dn and hcc in the cirrhotic liver by correlating the findings on mr imaging performed 1224 h before liver transplantation with histopathology of the entire explanted liver . materials and methods in the setting of a screening programme for cirrhotic patients awaiting liver transplantation , we performed mr studies on 400 patients , 31 of whom had been selected for transplantation . 
prior to the mr examination , all patients received full explanation of the aims and design of the study and gave their informed consent . all patients were studied with a 1.5 - tesla unit ( magnetom 63p , siemens , germany ) using baseline proton density ( pd ) / t2 - weighted spin echo ( se ) sequences ( tr / te 2 , 0002 , 300 / 2080 ms ) , and t1 - weighted fast low - angle shot ( flash ) gradient echo ( ge ) sequences ( tr / flip angle / te 153 ms / 70 / 6 ms ) and t2 * - weighted fast imaging with steady - state precession ( fisp ) ge sequences ( tr / flip angle / te 130 ms / 40 / 10 ms )  . 
for the pd / t2weighted sequences obtained under free breathing , two acquisition and presaturation bands were positioned above and below the imaging volume so as to reduce flow artefacts . 
the t1weighted ge sequences provided a stack of 15 slices that was sufficient to study the entire liver in most patients . with the t2 * - weighted ge sequences , a stack of five slices was used so that three to four stacks were required to study the entire liver . 
in all sequences , we used a slice thickness of 8 mm with 1 - mm gap , 128256 matrix with 3 / 4 reconstruction algorithm , and 380to 450 - mm field of view . 
 following unenhanced baseline imaging , spio ( ferumoxide , endorem , guerbet , villepinte , france ) was administered by slow infusion ( 820 drops / min ) over 3045 min at a dose of 23.6 md iron per gram ( fe / g ) diluted in a 100cc flask of 5% glucose solution . 
approximately 30 - 60 min after the end of spio administration , t2 * - weighted ge sequences with the same parameters as used at baseline were obtained in all patients . the baseline and spio - enhanced images were evaluated singly and then in combination by observers blinded to the histopathological results who recorded the number and site of lesions and expressed their judgement as to the dysplastic , neoplastic or other nature of the lesions . 
for each nodule , the observers recorded signal intensity on t1and t2 - weighted images and enhancement pattern after spio administration and , based on these parameters , expressed a la possibilit di una diagnosi differenziale tra queste due lesioni , nonch di una precoce identificazione della trasformazione del nd in hcc . scopo del nostro lavoro stato quello di valutare le possibilit della rm con mdc spio nella identificazione , caratterizzazione di nd e hcc nel fegato cirrotico , confrontando i risultati della rm , eseguita nelle 1224 ore precedenti il trapianto di fegato , con lesame anatomopatologico dellintero fegato espiantato . materiali e metodi nellambito di un programma di screening di pazienti cirrotici in lista dattesa di trapianto epatico , abbiamo studiato con rm 400 pazienti , 31 dei quali sono stati selezionati in seguito al reperimento di un fegato da trapiantare e di questi solo 22 sono stati inclusi nello studio , in quanto sottoposti a trapianto di fegato a distanza di circa 1224 ore dallesecuzione dellesame rm . 
per le sequenze se dpt2w ottenute durante la respirazione libera del paziente , abbiamo utilizzato due acquisizioni e bande di pre - saturazione , al di sopra ed al di sotto del campo di studio , al fine di ridurre gli artefatti vascolari . le sequenze ge t1w e ge t2 w sono state eseguite durante lapnea respiratoria dei pazienti , con ununica acquisizione e senza bande di pre - saturazione . 
in tutte le sequenze stato utilizzato spessore di strato di 8 mm con un gap di 1 mm , matrice di 128256 con algoritmo di ricostruzione 3 / 4 , campo di vista ( fov ) di 380450 mm . dopo le sequenze di base stato somministrato mdc spio ( ferumoxide , endorem , guerbert , francia ) , in infusione lenta ( 820 gocce / min ) alla dose di 23 , 6 micromol fe / g , diluito in un flacone da 100 cc di soluzione glucosata al 5% , con un tempo di infusione di circa 3045 mdopo circa 3060 min dalla fine della somministrazione del mdc spio in tutti i pazienti sono state ripetute le sequenze ge t2 * w con gli stessi parametri utilizzati nelle sequenze di base , eseguite prima dellinfusione del mdc spio . le immagini di base e quelle dopo spio sono state valutate separatamente e comparativamente , alloscuro dei radiol med ( 2009 ) 114 : 12671282 1271 diagnostic hypothesis as to histological type . 
on baseline images , in agreement with the reported mr criteria ( table 1 ) , dn were defined as nodular lesions that appeared hyperintense on t1 - weighted images and hypointense on t2weighted images or isohyperintense on t1 - weighted images and isointense on t2 - weighted images . 
hcc were defined as lesions appearing isohyperintense on t1 - weighted images , hyperintense in t2 or hyperintense in t1 , and isointense in t2 or hypointense in t1 , and hyperintense in t2 . hcc subfoci in dn ( nodule - in - nodule ) were defined as nodular areas of heterogeneous signal intensity seen within a dn with typical signal intensity . on spio - enhanced images , the diagnosis was based on the pattern of spio uptake . 
dn were defined as nodules presenting variable uptake depending on the degree of dysplasia , appearing in some cases strongly hypointense , often even more hypointense than the surrounding liver . hcc were defined as nodules with no spio uptake and hence markedly hyperintense . 
hcc subfoci in dn were defined as nodular lesions with hyperintense appearance due to failure to accumulate spio in the context of a larger , hypointense dn due to spio uptake . 
the definitive diagnosis was reached with the histopathological examination performed within 1 week of surgery on the entire explanted liver . dn were classified according to the international working party terminology of hepatic lesions into : lowgrade dn , high - grade dn and dn containing a subfocus of hcc [ 21 ]  . 
hcc were classified based on histology into well differentiated , moderately differentiated and poorly risultati dellanatomia patologica , riportando il numero e la sede delle lesioni ed esprimendosi sulla natura displasica , neoplastica o di altro tipo delle lesioni stesse . 
per ogni nodulo stata indicata lintensit di segnale nelle sequenze di base pesate in t1 e in t2 , il comportamento dopo somministrazione di mdc spio e , in base a tali parametri , si avanzata una ipotesi diagnostica sul tipo istologico . 
nelle sequenze di base , in accordo con la semeiotica rm descritta in letteratura ( tabella 1 ) abbiamo considerato nd le lesioni nodulari che apparivano iperintense in t1w ed ipointense in t2w o iso - / iperintense in t1w e isointense in t2w ; hcc le lesioni che apparivano iso - / iperintense in t1w e iperintense in t2w o iperintense in t1w e isointense in t2w o ipointense in t1w ed iperintense in t2w ; foci di hcc in nd ( nodulonel - nodulo ) le aree nodulari di disomogenea intensit di segnale riscontrate allinterno di un nodulo displasico con le caratteristiche intensit di segnale descritte . dopo iniezione di mdc spio , la diagnosi si basata sul tipo di captazione dello stesso e sono stati considerati nd i noduli che presentavano una captazione di mdc variabile in relazione al grado di displasia , apparendo in alcuni casi fortemente ipointensi , spesso anche in maggior grado rispetto al parenchima epatico limitrofo . 
lidentificazione di foci di hcc in nd si invece basata sul riscontro , dopo mdc spio , di una lesione nodulare iperintensa per mancata captazione di mdc nel contesto di un nodulo displasico di maggiori dimensioni , ipointenso per lup - take di mdc . 
nine patients were excluded because of mr or clinicallaboratory evidence of abnormalities that constituted contraindications or indications to defer surgery , findings later confirmed by clinical - imaging follow - up and biopsy . 
three of these nine patients had suspicious focal liver lesions that were > 5 cm and involved both hepatic lobes ; another two patients had cancercirrhosis , with extensive involvement of the right lobe in one case and left lobe in the other . 
in one patient , mr imaging demonstrated a large confluent fibrosis that made it impossible to confidently rule out the presence of cancer - cirrhosis , which was later excluded by biopsy . 
 risultati i risultati del nostro studio sono riportati nella tabella 2 . dei 31 pazienti selezionati per il trapianto dorgano , sono stati inclusi nello studio 22 pazienti , sottoposti a trapianto fegato a distanza di 1224 ore dallesecuzione dellesame rm . 
nove pazienti sono stati eliminati dallo studio in quanto allesame rm e agli esami clinico - laboratoristici presentavano alterazioni che contravvenivano o consigliavano il posticipo dellesecuzione del trapianto , reperti poi supportati dal follow - up clinico - radiologico e dallesame bioptico . 
in 3 , di questi 9 casi , erano presenti lesioni focali epatiche sospette per hcc che avevano dimensioni complessivamente superiori a 5 cm ed interessavano entrambi i lobi epatici ; in altri 2 casi si trattava di cancro - cirrosi con esteso interessamento del lobo destro in un caso e del sinistro nellaltro ; in 1 caso il paziente era portatore di tips che si estendeva nellatrio destro e per il quale si preferito posticipare lintervento ai fini di una migliore pianificazione chirurgica ; in 1 caso la rm evidenziava una estesa fibrosi confluente che non consentiva di escludere con certezza una eventuale cancro - cirrosi , successivamente non confermata dallesame bioptico ; in 2 casi le scadenti condizioni cliniche dei pazienti hanno imposto di soprassedere al trapianto e di proseguire la terapia medica . 
sono stati quindi inclusi nel nostro studio 22 pazienti , di et media di 53 anni ( range 4657 anni ) , successivamente sottoposti a trapianto dorgano entro 1224 ore dallesecuzione dellesame rm . la cirrosi stata classificata utilizzando il child - pugh syste quindici erano pazienti con cirrosi child - pugh classe a , 7 con cirrosi child - pugh classe b . 
unidici avevano cirrosi correlata con il virus dellepatite b ( hbv ) , 7 cirrosi correlata con il virus dellepatite c ( hcv ) , 4 cirrosi alcool correlata . risultati anatomo - patologici lesame anatomo - patologico dei 22 fegati espiantati ha dimostrato in tutti i casi un aspetto di tipo nodulare : macronodulare in 9 casi , micronodulare in 3 casi e medio - macronodulare in 10 casi . 
sono state identificate , inoltre , 59 lesioni focali epatiche , di cui 14 hcc , 4 noduli displasici con allinterno foci di hcc , 39 noduli displasici e 2 radiol med ( 2009 ) 114 : 12671282 1273 seven were child - pugh class b . 
at baseline mr imaging , four of the 12 dn exhibited the typical pattern of t1 hyperintensity relative to liver and t2 hypointensity ; eight dn were isointense on t1and t2weighted images . 
the two hcc subfoci within dn showed the classic nodule - in - nodule appearance , with isohyperintense signal on t1 - weighted images and heterogeneous hyperintensity on t2 - weighted images , contained within a larger dn that appeared isointense on t1weighted images and isohypointense on t2 - weighted images . 
the signal characteristics of the remaining 39 undetected lesions were such that precise identification and characterisation was precluded . spio administration increased mri sensitivity by 42.37% relative to the baseline study . 
il diametro medio delle lesioni stato per gli hcc 1 , 7 cm ( range 12 , 5 cm ) , per noduli displasici 0 , 8 cm ( range 0 , 52 ) , per i noduli displasici con allinterno foci di hcc 2 , 1 cm ( range 0 , 82 , 8 ) e per i cistoadenomi 1 , 2 cm ( range 11 , 4 )  . risultati rm gli esami rm effettuati senza mdc ( rm di base ) e con ferumoxide hanno mostrato una differente sensibilit nellidentificazione delle lesioni focali epatiche . 
lesame rm di base ha consentito di diagnosticare correttamente 20 lesioni , pari al 33 , 90% di quelle identificate con lesame anatomopatologico , di cui , allesame anatomopatologico , 12 sono risultate nd e 8 hcc . 
nelle sequenze di base , dei 12 nd identificati , 4 hanno mostrato comportamento caratteristico con intensit di segnale aumentata rispetto al parenchima epatico circostante nelle immagini t1w e ridotta in quelle t2w ; 8 nd sono risultati isointensi in t1w e isointensi in t2w . 
per quanto riguarda gli hcc , allesame di base in 3 casi hanno mostrato iperintensit di segnale nelle sequenze t1w ; in 2 casi isointensit di segnale ed in 1 caso ipointensit di segnale . 
i due foci di hcc in nodulo displasico hanno presentato il classico aspetto rm di nodulo - nel - nodulo mostrando segnale iper - / isointenso nelle sequenze t1w , e disomogeneamente iperintenso nelle sequenze t2w allinterno di un nodulo displasico di pi grandi dimensioni isointenso nelle sequenze t1w ed iso / ipointenso nelle sequenze t2w . 
le rimanenti 39 lesioni non diagnosticate presentavano caratteristiche di segnale tali da non consentire una precisa identificazione ed una adeguata caratterizzazione . dopo somministrazione di mdc spio , la rm ha mostrato una maggiore sensibilit diagnosticando complessivamente 45 lesioni , pari al 76 , 27% delle lesioni totali individuate con lesame istologico , di cui , allesame anatomo - patologico , 27 sono risultate nd e 18 hcc , con un incremento del 42 , 37% rispetto alle sequenze rm di base . 
i falsi negativi con la rm - spio sono stati 12 noduli displasici , pari al 31% , che allesame istologico sono risultati essere nd a basso grado di malignit e con diametro inferiore al centimetro . 
due lesioni , caratterizzate istologicamente come cistoadenomi , non sono state identificate in prima istanza allesame rm di base n dopo mdc spio , ma solo valutando retrospettivamente le immagini , sulla scorta dei risultati dellesame istologico . 
baseline protondensity - weighted spin echo ( a ) , t2 - weighted spin echo ( b ) , fisp ( c ) and t1 - weighted flash ( d ) magnetic resonance ( mr ) sequences : the nodule seems slightly hyperintense in all baseline sequences ( a - d )  . 
il reperto anatomo - patologico corrispondente conferma la presenza di multipli e diffusi noduli di rigenerazione con nodulo di hcc bianco - verdastro ( f )  . radiol med ( 2009 ) 114 : 12671282 1275 fig . 
3a - c nodulo di hcc e nodulo displasico con focus di hcc allviii segmento epatico . rm di base , sequenza flash t1w ( a ) e fisp t2w ( b ) : sono ben evidenti due formazioni nodulari iperintense . 
il nodulo di minori dimensioni mostra , nella sequenza fisp t2w , disomogenea intensit di segnale con evidenza di piccolo nodulo centrale contestuale . tale reperto configura il tipico pattern di nodulo - nel - nodulo . 
il reperto anatomopatologico ( c ) dimostra il caratteristico aspetto di nodulo nel nodulo della lesione . in 12 dn , equal to 31% , which at histology proved to be low - grade dn < 1 cm in diameter . 
two lesions that were histologically characterised as cystoadenomas went unrecognised on initial viewing of the baseline and spio - enhanced mr images but were later identified on retrospective review after the results of the histological examination . 
in fact , according to several authors , the transition from a dn to an hcc occurs over a period of mancata identificazione stata probabilmente dovuta alle ridotte dimensioni , alla localizzazione in sede sottoglissoniana e allaspetto simil - cistico . 
infatti lo sviluppo di un hcc a partire da un nd avviene , secondo molti autori , in un tempo variabile di circa 46 mesi [ 12 , 23 ] e laspettativa di vita media in un paziente non trattato di soli 13 mesi , con una sopravvivenza a 2 anni inferiore al 33% [ 24 ]  . 
il reperto anatomo - patologico ( d ) ha dimostrato la presenza di area di fibrosi e lassenza di lesioni focali . approximately 46 months [ 12 , 23 ] , and mean life expectancy of untreated patients is only 13 months , with a 2 - year survival rate < 33% [ 24 ]  . 
this pathogenic theory is supported by other studies and confirmed by the international working party on terminology of hepatocellular lesions , which stated that carcinogenesis in the cirrhotic liver is a multistep process that involves a transition from large rn to lowand high - grade dn to dn with subfocus of hcc and eventually to hcc [ 21 ]  . identification of dn and hcc in the cirrhotic liver is difficult because of the marked architectural distortion of the parenchyma due to areas of fibrosis , steatosis , parenchymal necrosis and regenerative nodules [ 10 , 25 , 26 ]  . 
found that us had a sensitivity of 20.5% for the detection of hcc and of 1.6% for detection of dn , emphasising the difficulty in differentiating hcc and dn with us , as both manifest as hypoechoic nodular lesions [ 8 ]  . 
although the use of sonographic contrast agents has improved the techniques epatiche , in cui si affermato che la carcinogenesi nel fegato cirrotico avviene con un meccanismo multi - step che include la transizione da grossi noduli rigenerativi a noduli displasici a basso ed alto grado di malignit , quindi a noduli displasici con foci di hcc sino agli hcc [ 21 ]  . lidentificazione di nd e di hcc nel fegato cirrotico per difficoltosa per la notevole alterazione strutturale del parenchima epatico a causa della presenza di aree di fibrosi , steatosi , necrosi parenchimale e noduli di rigenerazione [ 10 , 25 , 26 ]  . 
 [ 8 ] , in uno studio su 200 pazienti con cirrosi , hanno rilevato una sensibilit dellecografia nella identificazione degli hcc del 20 , 5% e dei nd del 1 , 6% , sottolineando la difficolt di differenziare con lecografia gli hcc dai nd , poich entrambi apparivano come lesioni nodulari ipoecogene [ 8 ]  . 
lutilizzo dei mezzi di contrasto ecografici ha migliorato laccuratezza diagnostica nella identificazione di hcc e nd rispetto alla ecografia convenzionale ; tuttavia esistono ancora difficolt nella diagnosi differenziale tra le due lesioni con tale metodica [ 27 ]  . 1278 radiol med ( 2009 ) 114 : 12671282 accuracy in identifying hcc and dn , the difficulties in differentiating the two lesions with this modality remain [ 27 ]  . spiral ct is currently considered amongst the most reliable modalities for detecting hcc . 
reported a mean sensitivity of dynamic spiral ct in identifying hcc of 76% and mean sensitivity values of 39% in the detection of dn depending on lesion size [ 10 ]  . the lower sensitivity in dn detection is most likely due to the fact that some dn are hypoattenuating in the portal venous and delayed phases , and few dn show hyperattenuation in the hepatic arterial phase . 
in the early stages of carcinogenesis , this is similar to that of healthy liver parenchyma , whereas in the subsequent hcc stage , it is characterised by a predominance of abnormal arteries ( not accompanied by bile ducts and for this reason known as unpaired or nonportal arteries ) and arteriovenous shunts livers undergoing neoplastic degeneration [ 19 , 20 , 29 ]  . 
thus , the arterial and portal supply to lowand high - grade dn is variable and inconsistent , making difficult the detection and characterisation of dn on the basis of their vascular characteristics alone [ 10 ]  . 
 typical of cirrhotic unlike ct , mr imaging often proves capable of differentiating hcc from dn based on signal characteristics alone as a result of its multiparametric properties and highcontrast resolution [ 23 ]  . 
on unenhanced baseline mr imaging , dn normally have isohyperintense signal on t1weigthed images in relation to their glycoprotein content and degree of dysplasia ; the signal becomes hypointense to surrounding parenchyma on t2 - weighted images . 
unfortunately , however , the signal intensity of dn may be identical to that of well - differentiated hcc , especially of small hcc ( < 2cm )  . the signal intensity of hcc on unenhanced t1weighted mr images varies widely and hcc may appear as hypointense masses as a result of greater water content or fibrous tissue or poor cellular differentiation or alternatively as hyperintense due to steatosis , accumulation of glycoproteic material or areas of intralesional haemorrhage . in some cases , however , moderately and well - differentiated hcc may also appear isointense compared with the healthy hepatic parenchyma . 
conversely , on t2 - weighted images , hcc tend to appear as generally ill - defined and hyperintense lesions [ 12 , 30 ] with the exception of well - differentiated hcc , which are isointense relative to the healthy parenchyma . 
in letteratura riportato che la sensibilit della tc multifasica nella identificazione di hcc di diametro superiore a 2 cm del 93 , 6% , percentuale che si riduce al 61% nella valutazione di lesioni di minori dimensioni [ 28 ]  . 
 [ 10 ] hanno riportato una sensibilit media della tc spirale dinamica nellidentificazione dellhcc del 76% e valori medi di sensibilit del 39% nellidentificazione dei nd in relazione alle dimensioni delle lesioni [ 10 ]  . 
questi minori valori di sensibilit nella identificazione dei nd sono verosimilmente dovuti al fatto che solo alcuni nd sono ipodensi in fase venosa portale e in fase tardiva , cos come sono pochi i nd che mostrano iperdensit nella fase arteriosa epatica . 
questo fenomeno correlato al tipo di apporto ematico arterioso e venoso che , nelle prime fasi della carcinogenesi , simile a quello del tessuto epatico sano ; successivamente , nello stadio di hcc , prevalgono vasi arteriosi anomali , non accompagnati dai canalicoli biliari , e per tale motivo denominati unpaired o non portal arteries , e shunt artero - venosi tipici dei fegati cirrotici ed in degenerazione neoplastica [ 19 , 20 , 29 ]  . 
pertanto lapporto arterioso e portale ai nd a basso ed alto grado di malignit variabile ed incostante ; , dunque , difficile identificare e caratterizzare i noduli displasici soltanto sulla base delle caratteristiche vascolari [ 10 ]  . la rm , grazie alle sue intrinseche multiparametricit ed alta risoluzione di contrasto , rispetto alla tc si rivela spesso in grado , di differenziare gli hcc dai nd sulla base delle sole caratteristiche di segnale [ 23 ]  . 
nelle immagini rm di base , il nd presenta solitamente , nelle sequenze t1w , un segnale che , in relazione al contenuto glicoproteico ed al grado di displasia , pu essere iso - / iperintenso ; appare invece iso - / ipointenso rispetto al parenchima circostante nelle sequenze t2w . 
purtroppo per lintensit di segnale dei nd pu essere sovrapponibile a quella degli hcc ben differenziati , soprattutto se di piccole dimensioni ( hcc < 2 cm )  . le caratteristiche di segnale degli hcc nelle immagini rm di base t1w variano molto e possono apparire come formazioni ipointense , per il maggior contenuto di acqua o per la componente di tessuto fibroso e per la scarsa differenziazione cellulare ; o possono presentare elevata intensit di segnale per la presenza di steatosi , accumulo di materiale glicoproteico o aree di emorragia intratumorale . 
nelle immagini t2w , invece , gli hcc appaiono comunemente come lesioni non sempre ben definite , iperintense rispetto al parenchima circostante [ 12 , 30 ] ad eccezione degli hcc ben differenziati che mostrano isointensit di segnale rispetto al parenchima sano . la capacit di identificare e caratterizzare le lesioni nodulari nel fegato cirrotico aumenta con lausilio dei mdc siano essi paramagnetici o superparamagnetici . 
la rm con mdc paramagnetico sfrutta , come la tc , le caratteristiche vascolari delle lesioni focali epatiche sia per la loro radiol med ( 2009 ) 114 : 12671282 1279 imaging with paramagnetic contrast medium relies on the vascular characteristics of focal liver lesions for their detection and characterisation . 
gadolinium has shown a good level of accuracy , even in the study of small hcc ( < 1.52 cm ) and in cirrhotic livers , with a sensitivity of 84% [ 11 , 31 , 32 ]  . 
sensitivity in the detection of dn is instead only 15% , with low specificity values since the signal intensity of dn on mr imaging with paramagnetic contrast medium is very similar to that of many hcc [ 33 ]  . the introduction of spio contrast agents , which provide information about the composition and function of cells forming the nodular lesions and which overcome the limitations of relying on vascularisation changes , has made it possible to detect even small hcc , which in the early stage may preserve normal vascularity and thus be difficult to differentiate from other nodular lesions [ 34 , 35 ]  . 
 the mechanism of action of spio contrast agents is based on their uptake by kupffer cells or cells of the hepatic reticuloendothelial syste spio uptake is reflected in a marked reduction in signal intensity of the tissues containing kupffer cells . 
conversely , any malignant lesions , which do not contain reticuloendothelial cells , do not accumulate the contrast medium and therefore appear as areas of hyperintensity [ 36 , 37 ]  . 
the use of these contrast agents has in part solved the problem of false positive results seen with paramagnetic contrast agents injected as a bolus , a problem related to the presence of small arteriovenous shunts which , appearing as small areas of vascular enhancement during the arterial phase , may mimic the enhancement pattern of small hcc . 
these false positive results are similar to those seen during contrast - enhanced dynamic ct imaging . on this subject , the literature also contains studies on the combined use of gadolinium diethylenetriamine pentaacetic acid ( gd - dtpa ) and spio , which , by fulfilling the criteria of vascularity as well as cellular composition and function [ 11 , 38 ] , reduces both the false positive results of paramagnetic contrast agents and the false negative results of spio agents the latter related to the presence , in moderately and well - differentiated hcc , of functioning kupffer cells that accumulate the contrast agent . 
as a result , the t2 - weighted signal intensity is higher in low - grade tumours , in which the number and function of kupffer cells is decreased [ 40 ]  . 
il gadolinio in molti studi ha mostrato una buona accuratezza anche nello studio dei piccoli hcc ( < 1 , 52 cm ) e nei fegati cirrotici mostrando una sensibilit dell84% [ 11 , 31 , 32 ]  . la sensibilit nellidentificazione dei nd invece , solo del 15% con bassi valori di specificit in quanto lintensit di segnale dei nd ottenuta dopo mdc paramagnetico paragonabile a quella di molti hcc [ 33 ]  . lintroduzione dei mdc spio fornendo informazioni sulla composizione e sulla funzione delle cellule che costituiscono le lesioni nodulari e , superando i limiti diagnostici legati alle alterazioni della vascolarizzazione , consente di identificare anche hcc di piccole dimensioni che nello stadio precoce possono in parte conservare una vascolarizzazione normale ed essere quindi difficilmente differenziabili dalle altre lesioni nodulari [ 34 , 35 ]  . 
il meccanismo dazione dei mdc spio si basa sulla loro captazione da parte delle cellule del sistema reticolo - endoteliale epatico , le cellule di kupffer , che si traduce in una marcata riduzione dellintensit di segnale dei tessuti in cui sono presenti tali cellule . pertanto , leffetto prodotto nelle immagini pesate in t2 fa s che alcune lesioni benigne captano il mezzo di contrasto come il parenchima epatico sano ; di contro , le lesioni focali maligne eventualmente presenti , sprovviste di cellule del sistema reticolo - endoteliale , non captano il mdc e appaiono come aree iperintense [ 36 , 37 ]  . 
lutilizzo di questi mdc ha risolto in parte il problema dei falsi positivi che si osservano con i mezzi di contrasto paramagnetici iniettati a bolo , legato alla presenza di piccoli shunt artero - venosi che , manifestandosi come piccole aree di enhancement vascolare in fase arteriosa , possono simulare il pattern contrastografico di piccoli hcc . 
 a tale proposito , in letteratura sono stati anche riportati studi sulluso combinato di gadolinio legato ad acido acetico dietilenetriaminepenta ( gd - dtpa ) e spio che , soddisfacendo sia i criteri di vascolarizzazione sia quelli di composizione e funzionalit cellulare [ 11 , 38 ] , riduce il problema dei falsi positivi dei mezzi di contrasto paramagnetici e dei falsi negativi con mdc spio , questi ultimi legati alla presenza , in hcc ben differenziati o moderatamente differenziati , di cellule di kupffer sane che di conseguenza captano il mdc . infatti , uno studio anatomo - patologico sulla carcinogenesi multi - step ha dimostrato che cellule del kupffer sono ancora presenti nei noduli epatici durante il processo di degenerazione ; ci che varia il numero di tali cellule che si riduce durante le fasi della malattia cirrotica e nellevoluzione dellhcc da uno stadio di alta differenziazione ad uno di moderata o scarsa differenziazione [ 35 , 39 ]  . 
lintensit di segnale nelle sequenze t2w , quindi , sar tanto pi elevato quanto meno differenziato sar il tumore , essendo inferiori il numero e la funzionalit delle cellule del kupffer presenti [ 40 ] ; questo offre la possibilit di effettuare una diagnosi differenziale tra hcc e noduli displasici . sebbene la rm con i mezzi di contrasto spio sia una 1280 radiol med ( 2009 ) 114 : 12671282 differentiating between hcc and dn . although spio - enhanced mr imaging is an accurate technique in the cirrhotic liver , the heterogeneous uptake of spio agents may affect the interpretation of findings . 
in liver cirrhosis , the marked subversion of hepatic structure and function , intrahepatic vascular changes , or the presence of inflammatory processes , thromboses , hepatitis or steatosis , may affect the intracellular uptake and distribution of spio agents in the liver , thereby reducing their effectiveness in the detection and characterisation of small focal lesions [ 43 , 44 ]  . 
such alterations , known as spio liver uptake and distribution alterations ( spio - luda ) lead to the formation of hyperintense areas due to reduced uptake or hypointense areas , which is due to increased uptake . although the incidence of such alterations is relatively low , in some cases , they give rise to diagnostic problems on mr imaging [ 26 ]  . we observed no significant spio uptake and distribution alterations ( table 1 ) , and the signal intensity of the nodular lesions seen in our study ( dn and hcc ) corresponded to the patterns reported in the literature . 
there were no cases of well - differentiated hcc showing spio uptake , as reported by other authors [ 45 ] , and hence no cases of false negative hcc diagnoses . 
 on the basis of our results and in agreement with the literature , spio - enhanced mr imaging proved to be a valuable tool for the detection and characterisation of focal lesions in the cirrhotic liver . 
in particular , spio - enhanced mr imaging is currently the only imaging modality capable of distinguishing between dn and hcc and thus providing the opportunity for an accurate and timely diagnosis of malignant change of dn , a crucial aspect for prognosis . tecnica dimaging accurata nello studio del fegato cirrotico , leterogeneo up - take degli spio pu compromettere linterpretazione dei reperti . 
nella cirrosi epaticail notevole sovvertimento della struttura e delle funzioni epatiche , le alterazioni vascolari intraepatiche o la presenza di processi flogistici , trombosi vascolari , di epatiti , steatosi , possono modificare la captazione intra - cellulare e la distribuzione dei mdc spio nel fegato riducendone lefficacia , con una minore utilit degli stessi nella identificazione e caratterizzazione di piccole lesioni focali [ 43 , 44 ]  . 
tali alterazioni , denominate alterazioni di captazione e distribuzione epatiche del mdc spio ( spio - acde ) , provocano la formazione di aree di iperintensit da ridotta captazione o di ipointensit da aumentata captazione . 
lincidenza di queste alterazioni relativamente bassa ma in alcuni casi determina difficolt diagnostiche allesame rm [ 26 ]  . in questa esperienza non si sono dimostrate significative alterazioni della captazione e della distribuzione dei mezzi di contrasto ( tabella 1 ) e lintensit di segnale delle lesioni nodulari epatiche considerate nel nostro studio ( nd e hcc ) stata corrispondente ai possibili pattern di segnale rm riportati in letteratura . 
non stato riscontrato nessun caso di hcc ben differenziato che captava mdc spio , come descritto da alcuni autori in letteratura [ 45 ] ; non sono stati pertanto riscontrati falsi negativi nella diagnosi di hcc . sono stati riscontrati , invece , falsi negativi per la diagnosi di nd . 
tali risultati sono probabilmente legati al fatto che il nostro campione di studio era relativamente limitato , quindi ridotta stata la possibilit di riscontro di hcc con comportamento contrastografico simile a quello dei nd . 
rossi , universit di verona , verona , italy 2istituto di radiologia , ospedale cafoncello , treviso , italy 3istituto di radiologia , ospedale san bassiano , bassano del grappa , vicenza , italy correspondence to : n . 
thirteen of 15 patients underwent mr examination for clinical suspicion of a lesion located in the periscapular region , whereas in 2 / 15 cases it was an incidental finding during mr examination performed for other diseases . 
mr imaging with its multiplanar capabilities and high - contrast resolution has a high level of accuracy in characterising elastofibroma dorsi and may avoid the need for biopsy or surgical operation . keywords elastofibroma dorsi magnetic resonance riassunto obiettivo . 
sono stati analizzati gli esami rm del distretto toracico presenti nellarchivio di tre istituti di radiologia , effettuati con magnete superconduttivo da 1 , 5 t su 1233 pazienti in un arco di nove anni , al fine di identificare retrospettivamente la presenza di elastofibromi del dorso . 
sono stati identificati 17 elastofibromi del dorso in 15 pazienti ( 12 femmine , 3 maschi ; et media 58 anni , range 2882 anni ) ; due pazienti presentavano lesioni bilaterali . 
la rm presenta elevata affidabilit nella caratterizzazione dellelastofibroma del dorso e consente di evitare il ricorso alla biopsia ed al trattamento chirurgico , qualora non indicati . parole chiave elastofibroma del dorso risonanza magnetica 1284 introduction elastofibroma dorsi is a pseudotumoral lesion of mesenchy mal origin , presumably of a reactive nature , which develops electively in the soft tissues of the periscapular region between the scapula and the chest wall . 
it is observed in elderly subjects , mostly women , with a marked correlation with manual work carried out over long periods of time [ 1 ]  . microscopically , elastofibromas are characterised by fibroelastic connective tissue with poorly organised , thick and elastic fibres and rare collagen fibres , with varying amounts of intermingled fatty strands [ 2 , 3 ]  . 
considering the dramatic intensification of computed tomography ( ct ) and magnetic resonance ( mr ) examinations , a progressive increase in the incidental detection of scapulohumeral elastofibromas is likely to occur . 
there is therefore a possibility that these lesions are misinterpreted as suspected neoplasms by radiologists unaware of the features of this disease . mr imaging allows characterisation of the site and morphostructural features of typical elastofibroma dorsi , in particular , with t2 - weighted sequences , thanks to the possibility of differentiating it from other soft tissue lesions of the infrascapular region [ 58 ]  . 
therefore , the aim of our study was to analyse the mr features of elastofibroma dorsi by correlating imaging and pathologic findings to help decrease the number of unjustified surgical biopsies and resections . radiol med ( 2009 ) 114 : 12831291 introduzione lelastofibroma del dorso una lesione pseudotumorale ad origine mesenchimale di verosimile natura reattiva che si sviluppa elettivamente nei tessuti molli della regione periscapolare , fra la scapola e la parete toracica . 
colpisce una popolazione di et media - avanzata , in prevalenza di sesso femminile , mostrando inoltre una correlazione positiva con lespletamento di lavori manuali per lunghi periodi di tempo [ 1 ]  . 
dato il vertiginoso aumento di indagini di tomografia computerizzata ( tc ) e risonanza magnetica ( rm ) , verosimile che si andr incontro ad un aumento progressivo dei riscontri incidentali di elastofibromi scapolo - omerali . 
esiste pertanto la possibilit che tali lesioni vengano erroneamente considerate come sospette neoplasie dal radiologo non a conoscenza delle caratteristiche di queste entit nosologiche . la rm consente la caratterizzazione , in particolare mediante sequenze t2 pesate , degli elastofibromi del dorso tipici , per sede e caratteristiche morfo - strutturali , grazie alla possibilit di differenziare altre lesioni dei tessuti molli della regione infra - scapolare [ 58 ]  . 
lo scopo del nostro lavoro quindi quello di analizzare le caratteristiche rm degli elastofibromi del dorso , correlando i reperti imaging con quelli anatomo - patologici , allo scopo di promuovere la riduzione del numero di biopsie ed escissioni chirurgiche ingiustificate . materials and methods materiali e metodi we retrospectively evaluated chest mr imaging examinations performed on 1 , 233 patients in our institute ( n = 365 ) and at two other hospitals ( n = 444 and n = 424 ) from january 2000 to october 2008 to look for the presence of elastofibroma dorsi . 
sono state individuate 17 lesioni in 15 pazienti ( 12 femmine , 3 maschi , range det 2882 anni , media 58 anni ) , con due pazienti con lesioni bilaterali simmetriche . 
 in tutti i pazienti sono state ottenute sequenze multiplanari spin - echo pesate in t1 ( te 9 , tr 550 , flip angle 90 ) e fast spin echo t2 ( te 90 , tr 2000 , flip angle 90 ) , sequenze short tau inversion recovery ( stir ) ( te 70 , tr 4800 , flip radiol med ( 2009 ) 114 : 12831291 1285 90 ) and short - tau inversion recovery ( stir ) sequences ( te 70 , tr 4 , 800 , flip angle 90 ) with slice thickness 4 mlesion enhancement patterns were studied in 12 / 17 lesions . 
the contrast - enhanced examination was carried out with gadolinium chelates ( dotarem , guerbet , france ) injected at 0.2 mg / kg using an automatic power injector ( medrad , spectris ) to administer a single bolus at a rate of 1 ml / s , followed by acquisition of t1 - dependent sequences with and without fat saturation ( te 2 , tr 5 , flip angle 10 )  . 
we used high - resolution sequences with 256256 matrix and variable field of view ( fov )  . the mr images were retrospectively analysed to evaluate site ( infrascapular or subscapular ) ; shape ( elliptical or rounded ) ; size ; margins ( regular or irregular ) ; structure ( homogeneous or heterogeneous ) ; signal intensity on t1weighted , t1 with fat suppression and t2 - weighted sequences ; subjective evaluation of enhancement after contrast administration ( poor , fair , high ) ; and any associated finding ( bone erosion , thickening or infiltration of the surrounding tissues )  . 
in the six lesions without histological confirmation , we evaluated stability over time ( up to 18 months follow - up ) of size , margins and signal to confirm the benign nature of the lesion . all examinations were interpreted by radiologists with 5 , 10 and 12 years experience with mr imaging in each of the three hospitals participating in this study . 
in all cases , the presence of thick , disorganised elastic fibres confirmed by a positive verhoeff - van gieson test and of collagen fibres interspersed with more or less thick , fatty tissue septations confirmed the diagnosis of elastofibroma . results results are summarised in table 1 . 
thirteen of 17 lesions had infrascapular location , and only 4 / 17 were subscapular . all lesions had an elliptical shape , with the longest axis in the craniocaudal direction . 
the lesion margins were regular in 14 / 17 cases ; only three lesions had margins indistinguishable from the surrounding structures , in particular , from the adjacent muscles . all lesions were characterised by a heterogeneous structure . 
lesame con mezzo di contrasto ( mdc ) paramagnetico stato eseguito mediante iniezione di chelati del gadolinio ( dotarem , guerbet , francia ) , alla dose di 0 , 2 mg / kg . 
il mdc stato iniettato mediante pompa automatica ( medrad , spectris , usa ) , in un bolo unico con velocit di iniezione di 1 ml / s , e successiva acquisizione di sequenze t1 - dipendenti con e senza saturazione del grasso ( te 2 , tr 5 , flip angle 10 )  . 
sono state utilizzate sequenze ad elevata risoluzione con matrice di 256256 ed un campo di vista ( fov ) variabile . le indagini rm sono state analizzate retrospettivamente per valutare : sede ( infra - scapolare o sotto - scapolare ) , forma ( ellittica o rotondeggiante ) , dimensioni , margini ( regolari o irregolari ) , struttura ( omogenea o disomogenea ) , intensit di segnale nelle sequenze pesate in t1 , t1 con soppressione del grasso , t2 , valutazione soggettiva dellenhancement dopo somministrazione di contrasto ( scarsa , buona , intensa ) , ed eventuali reperti associati ( erosione ossea , ispessimento o infiltrazione tessuti circostanti )  . 
nel caso delle 6 lesioni di cui non stata ottenuta una conferma istologica si ricorso alla valutazione della stabilit dimensionale nel tempo ( follow - up fino a 18 mesi ) , dei margini e delle caratteristiche di segnale per confermare la benignit della lesione . tutti gli esami sono stati interpretati da medici radiologi con esperienza di 5 , 10 e 12 anni in risonanza magnetica in ognuna delle 3 sedi coinvolte in questo studio . 
in tutti i casi la presenza di spesse fibre elastiche disorganizzate , confermate dalla positivit al test di verhoeff - van gieson , e di fibre collagene , alternate a setti pi o meno spessi di tessuto adiposo , ha confermato la diagnosi di elastofibroma . risultati la tabella 1 mostra schematicamente i nostri risultati . tredici lesioni su 17 avevano sede infra - scapolare e solo 4 / 17 sede sotto - scapolare . 
on t1 - weighted sequences , 14 / 17 lesions showed intermediate signal intensity , mostly isointense to muscle , whereas three were predominantly hyperintense due to the presence of a moderate amount of fatty tissue . 
1a , b axial t1 - weighted spin - echo image ( a ) and t2 - weighted image ( b ) show an elastofibroma dorsi in its usual location in the periscapular region in a 62 - year - old woman with clinically evident mass . 
1a , b immagine assiale spin - echo t1 - dipendente ( a ) e t2 - dipendente ( b ) che mostra un elastofibroma del dorso nella sua tipica sede infrascapolare in una donna di 62 anni con tumefazione clinicamente evidente . 
2a - d axial t1 - weighted se ( a ) , t1weighted ( b ) and t1weighted se images acquired after intravenous administration of gadolinium chelates in the axial ( c ) and coronal ( d ) planes show an elliptical lesion located in the left infrascapular region in a 66 - year - old woman with clinically evident swelling of the periscapular region and limited shoulder motion . 
2a - d le immagini assiali spin - echo t1dipendente ( a ) , t1 - dipendenti fs ( b ) , e le immagini spin - echo t1 - dipendenti dopo somministrazione endovena di chelati del gadolinio acquisite sul piano assiale ( c ) e coronale ( d ) , mostrano una lesione della regione infrascapolare sinistra a morfologia ellittica , prevalentemente ipointensa in t1 ( a ) , isointensa al muscolo in t1 fs ( b ) , con discreto e disomogeneo rinforzo contrastografico ( c , d ) , in una donna di 66 anni con tumefazione periscapolare e limitazione funzionale . 
 conversely , the 3 / 17 lesions rich in lipids showed intermediate signal intensity , being mildly hyperintense to muscle due to the presence of large amounts of intralesional fat . 
on t1 - weighted sequences with selective fat suppression , all lesions were markedly hypointense , with loss of signal of grasso intra - lesionale rispetto al muscolo ; nelle sequenze pesate in t1 con soppressione selettiva del grasso tutte le lesioni si presentavano marcatamente ipointense , con caduta di segnale delle componenti adipose . 
3a - d axial ( a ) and coronal ( b ) t1 - weighted se , axial t2 - weighted stir ( c ) and t1 - weighted fat - suppressed ( d ) images show a bilateral symmetrical elliptical lesion in the infrascapular region in a 58 - year - old woman with clinically evident bilateral symmetrical swelling . 
3a - d le immagini assiali spin - echo t1 - dipendente sul piano assiale ( a ) e coronale ( b ) , e assiali t2 - dipendente stir ( c ) e spin - echo t1 - dipendenti con soppressione del grasso ( d ) mostrano due lesioni simmetriche della regione infrascapolare a morfologia ellittica , disomogeneamente iperintense in t1 per la presenza di spesse bande di tessuto adiposo ( a , b ) , tendenzialmente isointense al muscolo in t2 stir ( c ) e dopo soppressione del grasso ( d ) , mantenendo tuttavia un segnale pi intenso del grasso sottocutaneo , in una donna di 58 anni con tumefazione periscapolare bilaterale simmetrica . 
by contrast , 2 / 12 lesions showed less enhancement , in part due to the presence of abundant fat inside thein none of the cases did we note alterations in the surrounding bone and muscular structures , except for the mass effect produced by the larger masses on the belly of adjacent muscles . ma disomogenea in 10 / 12 lesioni , verosimilmente per il progressivo accumulo di chelati del gadolinio nel contesto del tessuto fibroso . 
in nessun caso sono state riscontrate alterazioni delle strutture ossee e muscolari circostanti , fatta eccezione per limpronta dovuta alleffetto massa sui ventri muscolari adiacenti ad opera delle masse di maggiori dimensioni . 
 discussion elastofibroma dorsi is a pseudotumoral lesion characteristically located in the periscapular soft tissues between the scapula and the chest wall or caudally to the lower pole of the scapula [ 1 , 4 , 9 , 10 ]  . 
in approximately 10% of cases , elastofibroma dorsi is bilateral and symmetrical , whereas other locations are extremely rare and include the extremities , ischial tuberosities , small intestine , colon and tracheodiscussione lelastofibroma del dorso una lesione pseudotumorale che si localizza elettivamente nei tessuti molli della regione peri - scapolare , tra la scapola e la parete toracica o caudalmente al polo inferiore della scapola stessa [ 1 , 4 , 9 , 10 ]  . 
in circa il 10% dei casi lelastofibroma del dorso bilaterale e simmetrico , mentre rarissime sono le altre sedi di riscontro come in corrispondenza delle estremit , delle tuberosit ischiatiche , del piccolo intestino , del colon , e dellalbero tracheobronchiale [ 1 , 912 ]  . 
come dimostrato da alcuni autori , queste lesioni non sono affatto rare ai controlli radiol med ( 2009 ) 114 : 12831291 1289 bronchial tree [ 1 , 9 , 1012 ]  . 
as shown by some authors , these lesions are not infrequent findings at autopsy , and they affect 24% of women and 11% of men older than 55 years of age [ 9 , 10 ]  . 
the snapping scapula sign , almost pathognomonic , calls for diagnostic confirmation with either ct or mr imaging [ 2 , 4 , 13 ]  . the imaging appearance of these lesions is closely related to their histopathological features : composed of elastic fibres , collagen and fat [ 3 , 57 , 14 , 15 ] , elastofibromas may grow slowly , possibly as a result of repeated microtrauma between the scapula and the chest wall during heavy physical work [ 1 , 16 ]  . 
considering the increase in chest ct and mr examinations , the number of elastofibroma dorsi diagnoses is likely to rise . hence , awareness of the imaging findings may help to reduce the number of unnecessary surgical biopsies and resections . 
the presence of an elliptical lesion with more or less thick , fatty septations in the subscapular region , even though not pathognomonic , should suggest a diagnosis of elastofibroma in all typical settings . 
mr imaging is the modality of choice for characterising expansile infrascapular lesions , leading in most cases to a definite diagnosis [ 4 , 68 , 11 , 14 ]  . 
the mr appearance of elastofibroma dorsi is of a solid , low - signal - intensity mass that may be moderately heterogeneous due to the presence of a fibroelastic component , almost isointense to skeletal muscle both on t1and on t2 - weighted sequences , with an internal fatty component arranged into septations of variable size and number . 
the internal fatty component arranged in linear or curvilinear streaks parallel to the longest axis of the lesion has high signal intensity on t1 - weighted sequences , intermediate on t2 - weighted sequences and shows the typical signal loss on sequences with fat suppression [ 7 , 8 , 14 ]  . lesion margins may be more or less defined , although infiltration into surrounding structures has never been documented . the enhancement pattern of elastofibroma varies , with mild to marked enhancement being described [ 5 ]  . 
in our experience , most lesions showed intense and heterogeneous enhancement after contrast administration . elastofibroma develops deep to the serratus anterior muscle , rhomboid muscle , and latissimus dorsi at the level of the lower scapular angle and over the ribcage from the autoptici , arrivando ad interessare il 24% dei soggetti di sesso femminile e l11% di sesso maschile sopra i 55 anni [ 9 , 10 ]  . 
il segno dello schiocco scapolare , pressoch patognomonico , necessita di conferma diagnostica mediante tc o rm [ 2 , 4 , 13 ]  . laspetto imaging di queste lesioni strettamente correlato alle loro caratteristiche anatomopatologhe : essendo costituiti da fibre elastiche , collagene e grasso [ 3 , 57 , 14 , 15 ] , gli elastofibromi possono crescere lentamente in verosimile rapporto a microtraumatismi fra la scapola e la parete toracica durante lespletamento di lavori manuali pesanti [ 1 , 16 ]  . 
dato il continuo aumento delle indagini tc ed rm del distretto toracico verosimile attendersi un progressivo incremento delle diagnosi di elastofibroma del dorso : la conoscenza dei reperti imaging da parte del radiologo pu pertanto contribuire a ridurre il numero delle biopsie e delle escissioni chirurgiche non necessarie . 
la presenza di una lesione a morfologia ellittica , contenente setti adiposi pi o meno spessi nella tipica sede sotto - scapolare , anche se non patognomonica , deve indirizzare alla diagnosi in tutti i casi tipici di elastofibroma . 
la rm la metodica di scelta per la caratterizzazione delle lesioni espansive del distretto infra - scapolare consentendo nella maggior parte dei casi di indirizzare ad una diagnosi definitiva [ 4 , 68 , 11 , 14 ]  . 
laspetto rm dellelastofibroma del dorso quello di una massa solida a bassa intensit di segnale , che pu essere moderatamente disomogenea , dovuta alla compresenza di una componente fibro - elastica , quasi isointensa al tessuto muscolare scheletrico sia nelle sequenze t1che t2 - dipendenti , nel cui contesto si pu identificare talvolta una componente adiposa distribuita a setti di entit e numero variabile . lipointensit nelle sequenze t2 - dipendenti ascrivibile alla scarsa cellularit del tessuto fibroso e delle fibre elastiche [ 6 ]  . 
la componente adiposa , frammista al tessuto fibroso in fasci lineari o curvilinei disposti parallelamente allasse maggiore della lesione , presenta unintensit di segnale elevata nelle sequenze t1 - pesate , intermedia nelle sequenze t2 - pesate , e presenta il tipico abbattimento del segnale nelle sequenze con soppressione selettiva di tipo spettrale del grasso [ 7 , 8 , 14 ]  . 
i margini lesionali possono essere pi o meno definiti , ma in nessun caso stata documentata linfiltrazione delle strutture vicine . il comportamento contrastografico dellelastofibroma variabile , essendo stati descritti casi da lieve a marcata impregnazione [ 5 ]  . 
 lelastofibroma si sviluppa profondamente al muscolo serrato anteriore , al romboide ed al latissimus dorsi a livello dellangolo scapolare inferiore ed a ridosso della 1290 radiol med ( 2009 ) 114 : 12831291 sixth to the eighth rib [ 1 , 2 , 4 , 7 ]  . 
the differential diagnosis , limited to atypical lesions ( intralesional haemorrhage , abundance or scarcity of fat , marked irregularity of margins ) must include desmoid tumour , neurofibroma , fibroma and malignant fibrous histiocytoma . 
all these lesions have poor cellularity and predominant fibrous tissue , and all appear hypointense on t2 - weighted sequences , although generally without hyperintense fatty septations on t1 [ 6 , 17 ]  . 
on the other hand , lipoma , well - differentiated liposarcoma , and haemangioma , which appear hyperintense on t1 - weighted sequences , do not show the typical hypoisointensity on t2 nor the streaked pattern typical of the fatty septations [ 5 ]  . the high frequency of bilateral findings ( 10% ) , in addition to reinforcing a possible diagnostic suspicion , must prompt investigation of the contralateral region , even when clinically asymptomatic [ 2 , 4 , 11 , 12 ]  . on the basis of our experience , biopsy should be limited to atypical cases when radiological examinations , especially mr imaging , do not allow for a confident diagnosis . surgical treatment is only necessary in symptomatic cases with functional limitations and / or pain , or for masses > 5 c alternatively , a large asymptomatic mass may be monitored over time , after biopsy , to rule out a malignant nature [ 2 ]  . analysis of the mr findings that we retrospectively evaluated proved that in 13 / 17 cases the diagnostic hypothesis was correct . 
the four noncharacterised lesions included two cases of incidental findings , subjected to biopsy , and two atypical lesions subsequently surgically resected . we believe that contrast administration is not necessary for characterising lesions with typical clinical and radiological findings and that its use could be limited to clarifying the nature of the lesion and its relation to neighbouring structures in atypical cases . 
among all symptomatic patients , only three , showing abundant fatty component arranged in thick and irregular septa , had an uncertain interpretation . gabbia toracica a livello delle coste , dalla sesta allottava [ 1 , 2 , 4 , 7 ]  . 
la diagnosi differenziale , limitata alle lesioni atipiche ( emorragia intra - lesionale , abbondanza o scarsit di adipe , marcata irregolarit dei margini ) deve includere il tumore desmoide , il neurofibroma , il fibroma e listiocitoma fibroso maligno , tutti caratterizzati da scarsa cellularit e prevalenza fibrosa e che quindi appaiono prevalente ipointensi nelle sequenze t2 - dipendenti , non mostrando di regola setti adiposi iperintensi in t1 [ 6 , 17 ] ; il lipoma , il liposarcoma ben differenziato e lemangioma , che appaiono invece iperintensi nelle sequenze t1 - dipendenti , non mostrano la tipica ipo - isointensit in t2 n il pattern a bande dei setti adiposi [ 5 ]  . 
lelevata frequenza di bilateralit ( 10% ) di queste formazioni espansive , oltre che rafforzare lipotesi diagnostica quando presente , deve indurre ad indagare il distretto controlaterale , anche se clinicamente silente [ 2 , 4 , 11 , 12 ]  . 
 secondo la nostra esperienza , la biopsia dovrebbe essere attualmente limitata ai casi atipici , qualora le indagini radiologiche , ed in particolare la rm , non consentano una diagnosi sicura ; il trattamento chirurgico si rende necessario solo nei casi sintomatici con limitazione funzionale e / o dolore , o per masse superiori ai 5 cm di maggior asse . in alternativa una massa di cospicue dimensioni , ma clinicamente silente , pu essere semplicemente monitorata nel tempo dopo averne escluso la malignit con prelievo bioptico [ 2 ]  . lanalisi dei referti delle indagini rm da noi retrospettivamente valutate ha mostrato che in 13 / 17 casi lipotesi diagnostica formulata era corretta . 
le 4 lesioni non caratterizzate comprendevano 2 casi di riscontro incidentale , sottoposte a biopsia , e 2 lesioni atipiche successivamente sottoposte ad escissione chirurgica . riteniamo che la somministrazione di mdc non sia indispensabile alla caratterizzazione delle lesioni con tipici reperti clinici e radiologici , e che potrebbe essere limitata ai casi atipici per meglio chiarire la natura della lesione ed i rapporti con le strutture circostanti . 
in realt non da escludere che questa nostra ipotesi sia stata condizionata dal fatto che la nostra serie comprendeva in prevalenza pazienti sintomatici con lesioni tipiche sia per caratteristiche morfo - strutturali che per sede . 
knauth springer - verlag berlin heidelberg , 2009 isbn 978 - 3 - 540 - 72784 - 5 published online : 20 november 2009 springer - verlag 2009 this is an easy to read and very informative book on the use of contrast media in daily diagnostic practice . 
going through its 29 chapters , plus the appendix containing the european society of urogenital radiology ( esur ) guidelines on contrast media ( version 7.0 ) and one page dedicated to the official publications from the same society , provides the reader with all possible information on the use of different contrast media . the second edition of the book is the result of in depth study and research by the academic members of the esur contrast medium safety committee updating in more than one chapter , information on the use of gadolinium - based contrast media and their possible complications , in particular those related to the alarming nefrogenic systemic fibrosis ( nsf )  . 
 nsf , whose real cause is yet unknown , has come slowly to the attention of the medical community in the past few years rendering what was thought ( gadolinium contrast agents ) to be a very safe diagnostic tool as a possible , harmful instrument in the radiologists hands . since in the daily practice there is not only gadoliniumbased contrast media in use , much discussion is devoted also to the other different types of contrast media and their use . 
one will find information on ionic and non - ionic as well as barium and ultrasound contrast media , detailed information on meta - analysis in research , and information on the rules that must be followed in contrast - media research , approval , manufacturing , and clinical application . great attention is given to the discussion of contrast - media related complications : this issue is thoroughly discussed in the relevant section of many chapters and thoroughly reviewed in the esur appendix guidelines . 
one should realize that each time a contrast medium is used , however safe it might be considered , it can given the different individual conditions of the patient create adverse reactions . 
scorrendo i 29 capitoli che lo compongono , pi lappendice che elenca le linee guida della european society of urogenital radiology ( esur ) sulluso dei mezzi di contrasto nella sua pi recente versione ( 7.0 ) e la pagina dedicata allelenco delle pubblicazioni ufficiali della stessa societ , il lettore trover tutte le informazioni possibili circa luso dei diversi mezzi di contrasto . la seconda edizione il risultato di una gran lavoro di ricerca e studio da parte dei membri accademici del comitato esur sulla sicurezza dei mezzi di contrasto , lavoro che aggiorna , in particolare in pi di un capitolo , le informazioni circa limpiego dei mezzi di contrasto a base di gadolinio e le loro possibili complicanze , in particolare quelle riferibili alla pi temuta e cio la fibrosi nefrogenica sistemica ( nsf )  . la nsf , le cui cause sono ancora sconosciute , stata riconosciuta con lentezza e ritardo dalla comunit medica negli ultimi anni , rendendo quelli che erano pensati come mezzi di contrasto sicuri ( cio a base di gadolinio ) , quali possibili strumento di danno nelle mani del radiologo . dal momento per che nella pratica quotidiana non esiste solo limpiego dei mezzi di contrasto a base di gadolinio , una parte consistente del volume e di discussione viene riservata a tutti i restanti tipi di mezzo di contrasto . 
il lettore trover informazioni sui mezzi di contrasto ionici e non ionici , su quelli baritati e per impiego in ecografia : il tutto correlato da informazioni precise sulla meta - analisi nella loro ricerca ed informazioni importanti circa le regole che debbono essere impiegate nella ricerca , approvazione , preparazione ed applicazione clinica dei mezzi di contrasto . 
 grande attenzione viene dedicata alla discussione delle complicanze provocate dallimpiego dei mezzi di contrasto : questo tema viene discusso con precisione nella parti relative di molti capitoli e ben riassunta nellappendice sulle linee guida dellesur . 
ci si deve rendere conto che tutte le volte che viene utilizzato un mezzo di contrasto , per quanto sicuro lo si possa ritenere , questi pu , in funzione anche della differenti condizioni cliniche di ciascun paziente in cui viene impiegato , provocare delle reazioni , reazioni che possono essere assai lievi ed a risoluzione rapida e spontanea ma anche , 1384 radiol med ( 2009 ) 114 : 13831384 resolving over a very short period to very severe and unexpected , requiring immediate and correct treatment . how many people know the correct doses of adrenaline or atropine to be used in an emergency situation ? are there any emergency procedure - charts in your diagnostic radiology suites ? in this respect some of the pages are unnerving and hair - raising . 
 in summary , this is a very useful book and a highly recommended resource text for every radiology department , it should be easily accessible not only for the old staff radiologists but most importantly for those who are in training . al contrario , essere piuttosto evidenti ed impreviste , necessitanti un intervento terapeutico immediato e corretto . quante persone conoscono la dose corretta di adrenalina o di atropina da utilizzare in situazioni di emergenza ? nelle sale di diagnostica radiologica vi sono cartelli riportanti le procedure da seguire nelleventuale emergenza ? da questo punto di vista alcune della pagine del volume sono piuttosto preoccupanti , tali da far venire i brividi . 
between april and august 2008 , we examined 65 patients with 64 - slice mdct - ca : 6 / 65 using the step - and - shoot dose - saving protocol , 45 / 65 the cardiac dose right protocol and 14 / 65 using a standard protocol . 
in the per - patient analysis , image quality was excellent in 100% of the step - and - shoot protocols , in 91.1% of the cardiac dose right protocols and in 85.8% of the standard protocols . 
langiografia coronarica con tomografia computerizzata multistrato ( ac - tcms ) comporta una dose elevata a causa di spessori submillimetrici e ridotti tempi di acquisizione ; le case costruttrici quindi hanno prodotto protocolli di risparmio di dose che per possono ridurre la qualit delle immagini e laccuratezza diagnostica . 
tra aprile e agosto 2008 , 65 pazienti sono stati sottoposti ad ac - tcms a 64 detettori ; 6 / 65 mediante protocollo step and shoot , 45 / 65 con protocollo cardiac dose right , 14 / 65 con protocollo standard . 
nelle analisi per paziente la qualit delle immagini risultata ottimale nel 100% dei casi per il protocollo step and shoot , nel 91 , 1% dei casi per il protocollo cardiac dose right e nell85 , 8% dei casi per il protocollo standard . 
il radiologo deve utilizzare il protocollo di scansione pi adatto a seconda dellindicazione clinica , del paziente e del tipo di informazioni necessarie per liter diagnostico . radiol med ( 2009 ) 114 : 11961213 1197 keywords mdct coronary angiography dosimetry dose saving parole chiave angiografia coronarica tcms dosimetria risparmio di dose introduction introduzione the rapid technological advancements of multidetector - row computed tomography ( mdct ) have increased the diagnostic accuracy of the noninvasive imaging of the coronary arteries . 
this has led to an increase in the number of diagnostic examinations being performed and has placed the technique within the diagnostic workup of the patient with coronary artery disease [ 15 ]  . 
the increasingly widespread use of the technique has emphasised the need to monitor and limit radiation exposure to the patient during these procedures . the use of mdct coronary angiography ( mdct - ca ) in the study of the heart does , in fact , involve high dose levels owing to the special operating conditions with which these examinations are performed ( submillimetre thickness of the elements being studied ; reduced acquisition times ) and the reconstruction methods used . 
the studies performed to date are in agreement on attributing a dose of 13 msv for calcium scoring and 1020 msv for mdct - ca [ 6 , 7 ]  . 
focusing on issues of dosimetry therefore becomes crucial . this is even more so with regard to mdct - ca when used as an elective noninvasive technique for diagnostic and not therapeutic purposes in patients with a low - medium risk of disease . 
the aim of this study was to evaluate diagnostic quality in assessing the coronary arteries with a number of dose - saving protocols . incrementato i rapidi avanzamenti tecnici della tomografia computerizzata multistrato ( tcms ) hanno laccuratezza diagnostica dellimaging non invasivo delle arterie coronarie portando conseguentemente ad un incremento delle indagini diagnostiche effettuate e ponendo la tecnica allinterno del workup diagnositco del paziente coronaropatico [ 15 ]  . 
 limpiego della angiografia coronarica mediante tcms ( ac - tcms ) nello studio del cuore comporta infatti una dose elevata in considerazione delle particolari condizioni operative con cui vengono eseguite queste indagini ( spessori submillimetrici degli elementi di rivelazione , ridotti tempi di acquisizione ) e delle modalit di ricostruzione . 
gli studi finora eseguiti concordano nellattribuire una dose di 13 msv al calcium score ( ca - score ) e di 1020 msv alla ac - tcms [ 6 , 7 ]  . 
 questo ancora pi vero in caso di ac - tcms che rappresenta una metodica non invasiva in elezione per pazienti con basso - medio rischio e che ha finalit diagnostica ma non terapeutica come la angiografia coronarica ( acc ) che in corso della stessa procedura effettua la diagnosi e la eventuale terapia con valori di esposizione relativamente bassi , mediamente pari a 57 msv se confrontati con lac - tcms [ 8 , 9 ]  . 
per fare fronte al problema dosimetrico le case costruttrici si sono mosse per la produzione di protocolli di risparmio di dose : tale risparmio di dose pu per comportare una riduzione della qualit delle immagini andando quindi a discapito quindi dellaccuratezza diagnostica [ 1015 ]  . 
an additional inclusion criterion was the presence of a heart beat with sinus rhyth patients with chronic atrial fibrillation with a mediumlow ventricular response ( inducible < 65 bpm ) were also admitted . exclusion criteria the following patients were excluded from the mdct - ca study : symptomatic patients with acute chest pain and an electrocardiogram ( ecg ) positive for ischaemia , patients with a heart rate 65 bpm , patients with a known allergy to iodinated contrast material , pregnant patients , patients with respiratory failure , patients with unstable clinical conditions and severe heart failure . 
patients unable to maintain breathhold for at least 810 s and with an unstable heart beat were also excluded . patient preparation the day before the examination , ecg monitoring was performed . 
at the time of the examination , the heart rate was monitored again , and when required , was lowered further with a 2.5 - mg intravenous dose of atenolol . the patient was positioned on the patient tray and connected to the electrodes monitoring a single ecg lead corresponding to either lead i or lead iii . 
the electrodes were positioned above the left and right clavicles with a ground electrode placed on the abdominal surface of the left tutti i pazienti sottoposti allindagine sono stati ammessi allo studio non invasivo delle coronarie se la loro frequenza cardiaca era < 65 bpm ( spontanea o indotta dalla somministrazione di - bloccanti ) e se avevano capacit di mantenere una apnea sufficiente per il periodo di acquisizione del volume di dati , in media pari a 1012 secondi . 
sono stati accettati allo studio anche i pazienti con fibrillazione atriale cronica con risposta ventricolare medio bassa , inducibile < 65 bp criteri di esclusione i pazienti sintomatici con dolore toracico acuto e elettrocardiogramma ( ecg ) positivo per ischemia , con frequenza cardiaca 65 bpm , allergia nota al mezzo di contrasto iodato , gravidanza , insufficienza respiratoria , stato clinico instabile e scompenso cardiaco di grado severo vengono esclusi dallo studio mediante ac - tcms . 
al momento dellesame stata controllata le frequenza cardiaca e se necessario ulteriormente abbassata per via endovenosa dal personale medico dedicato ( atenololo 2 , 5 mg )  . il paziente stato posizionato sul tavolo porta - paziente e collegato agli elettrodi dellecg monitorando una sola derivazione ecg corrispondente a di o diii . 
heart rate variability was observed , and a test was performed to assess whether the duration of the patients breath - hold was compatible with the scan time . the iodinated contrast material with an iodine concentration of 400 mgi / ml ( iomeron 400 , bracco , milan , italy ) was administered intravenously into an antecubital vein of the left arm with the use of an 18to 20 - gauge needle cannula and an injection rate of 5 ml / s . 
the standard dose of 80 ml was followed by a 20 - ml bolus of saline solution . acquisition synchronisation was achieved with the bolus tracking technique , which involves positioning a region of interest ( roi ) in the descending aorta . 
when the attenuation value within the roi reached the preset value of 120 hu , the scan was begun . protocols standard sono stati posizionati in sede sovraclaveare destra e sinistra con elettrodo terra sulla superficie addominale del fianco sinistro . 
 stata osservata la variabilit della frequenza cardiaca ed , inoltre , stato eseguito un test per valutare se la durata dellapnea del paziente era compatibile con il tempo di scansione . 
 il mezzo di contrasto iodato alla concentrazione iodica di 400 mgi / ml ( iomeron 400 , bracco , milano , italia ) stato somministrato per via endovenosa attraverso una vena antecubitale del braccio sinistro mediante unagocannula da 1820 gauge somministrato al flusso di 5 ml / s , alla dose standard di 80 ml seguito da un bolo di soluzione fisiologica di 20 ml . 
 la sincronizzazione dellacquisizione stata ottenuta mediante tecnica di bolus tracking che consiste nel posizionare una regione dinteresse ( roi ) nellaorta discendente ; quando il valore di attenuazione allinterno della roi ha raggiunto la soglia prestabilita , pari a 120 hu , stato dato il via alla scansione . all patients with a body mass index ( bmi ) > 27 , with early ectopic heart beats , extrasystoles , uncertain breath - hold or unstable heart rate , underwent a standard acquisition protocol . 
the protocol parameters included 120 kv , 800970 mas with a maximum intensity at 75% of the r - r interval and decreasing ( minimum 20% ) in the other phases of the cardiac cycle . 
in tutte le fasi del ciclo cardiaco viene somministrata dose piena , essendo quindi possibile una ricostruzione retrospettiva sia in fase telediastolica ( 75% ) che telesistolica ( 40% )  . 1200 step and shoot all patients with a stable heart rate < 65 bpm and bmi < 23 underwent the step - and - shoot dose - saving protocol . 
the protocol parameters included 120 kv and 210 mas , with the dose being delivered only at 75% of the r - r interval and nothing in the other phases of the cardiac cycle . 
the examination protocols are summarised in table 1 . a precontrast enhancement phase was obtained for all patients to evaluate the level of calciu the quantity of calcium in the coronary artery walls was evaluated with the calcium score technique following the agatston protocol . the ed was judged to be the best parameter for the evaluation and comparison of radiation exposure . 
tale protocollo prevede scansioni a 120 kv e 800970 mas con intensit massima al 75% del ciclo r - r , e decrescente ( min 20% ) nelle altre fasi del ciclo . 
only in the end - diastolic ( 75% ) phase is standard dose applied to the patient , and a lower dose is given in the rest of the cardiac cycle . 
the exposure is expressed as the dose length product ( dlp ) obtained in milligray per centimetre ( mgy / cm ) and was multiplied by a dose absorption conversion factor for the chest equivalent to 0.017. 
the patient characteristics subdivided by dose - saving protocol are summarised in table 2 . image analysis two expert observers , a radiologist and a cardiologist , performed a qualitative evaluation in consensus , which considered all segments of the coronary artery tree following the 17 - segment classification system of the american heart association [ 18 ]  . 
evaluation of the diagnostic quality of the images was performed by giving a score per segment and per examination of 0 in the event the examination or segment could not be evaluated and was therefore of insufficient quality , 1 for moderate quality and 2 for excellent quality . statistical analysis we used the spearman test to correlate image quality with the protocol used . 
tale dose , denominata anche dose length product ( dlp ) ed espressa in mgy / cm , stata moltiplicata per un fattore di conversione di assorbimento dose per il torace pari a 0 , 017 . 
le caratteristiche riassuntive dei pazienti suddivise per protocollo di modulazione di dose sono riassunte nella tabella 2 . analisi delle immagini due osservatori esperti specialisti in radiologia e cardiologia hanno effettuato in consenso una valutazione qualitativa considerando tutti i segmenti dellalbero coronarico , seguendo il sistema di classificazione della american heart association , diviso in 17 segmenti [ 18 ]  . 
al fine di giudicare la qualit diagnostica delle immagini stato conferito per segmento e per esame un punteggio pari a 0 in caso di esame o segmento non valutabile , di qualit insufficiente , 1 in caso di qualit discreta , 2 in caso di qualit ottimale . 
la distribuzione dei fattori di rischio risultata omogenea non mostrando significativa prevalenza di un fattore di rischio rispetto ad un altro nelle tre differenti categorie di protocolli di modulazione di dose . 
lanalisi delle caratteristiche dei pazienti suddivise per protocollo di modulazione di dose riassunta nella tabella 3 . radiol med ( 2009 ) 114 : 11961213 1203 mgy / cm for the cardiac dose right protocol and 290 mgy / cm for the step - and - shoot protocol . 
in total , 780 segments were identified ( mean 1314 per patient )  . with regard to the per - patient analysis , an image quality rating of 2 ( excellent ) was achieved in 6 / 6 ( 100% ) patients for the step - and - shoot protocol , 41 / 45 ( 91.1% ) patients for the cardiac dose right protocol and 12 / 14 ( 85.8% ) for the standard protocol . 
with regard to the per - segment analysis , an image quality rating of 2 was achieved in 31 / 31 ( 100% ) segments for the step - and - shoot protocol , 483 / 547 ( 82% ) segments for the cardiac dose right protocol and 165 / 202 ( 81.6% ) segments for the standard protocol . 
the results of the per - patient and per - segment analyses are summarised in table 4 . discussion mdct - ca has become a part of routine clinical practice only recently . 
its use in the past was mostly limited to validation studies in comparison with cca , with extensive use only in a few centres of applied clinical research [ 1921 ]  . the increasing use of the technique has focused attention on the need to maintain radiation exposure to the patient as low as possible to conform to the alara criteria for the optimisation of the examination [ 22 ]  . 
the term optimisation here suggests maintaining the dose at the lowest possible level achievable that is compatible with the image quality required for obtaining the sought - after diagnostic information [ 12 , 22 ]  . our findings show that the use of the appropriate acquisition technique makes possible a significant saving in radiation exposure of around 70% with the use of the cardiac dose right protocol and 30% with the step - and - shoot protocol when compared with the standard protocol . 
il bmi medio per i pazienti sottoposti a protocollo standard risultato essere pari a 26 , 6 , per il protocollo cardiac dose right di 22 , 6 e per il protocollo step and shoot di 21 , 3 . la dose registrata in termini di dlp comprensiva di acquisizione contrastografica e ca - score stata pari a 1205 , 58 mgy / cm in caso di protocollo standard , 872 , 07 mgy / cm in caso di protocollo care dose right e 390 mgy / cm in caso di protocollo step and shoot . 
la dose assorbita per ogni indagine di ca - score in termini di ctdivol era pari a 7 e , considerando che la lunghezza delle scansioni era mediamente pari a 13 cm ( range 1015 ) , ne risultata una dose assorbita in termini di dlp pari a 91 mgy / cm che , espresso in msv e rapportato al distretto anatomico in esame ha portato ad una dose effettiva per il solo ca - score pari a 1 , 547 . la dose effettiva al paziente dellintero esame comprendente scout , ca - score , premonitoring , monitoring e actcms in termini di msv risultata essere pari a 20 , 49 msv in caso di protocollo standard , 14 , 8 msv in caso di protocollo cardiac dose right e 6 , 63 msv in caso di protocollo step and shoot . 
sono stati analizzati in totale 780 segmenti ( media 1314 segmenti per paziente )  . nel contesto di una analisi per paziente la qualit delle immagini in caso di protocollo step and shoot risultata essere pari a 6 / 6 ( 100% ) pazienti valore 2 ( qualit ottimale ) , in caso di protocollo cardiac dose right risultata essere pari a 41 / 45 ( 91 , 1% ) pazienti valore 2 , in caso di protocollo standard risultata essere pari a 12 / 14 ( 85 , 8% ) pazienti valore 2 . 
nel contesto di una analisi per segmento la qualit delle immagini in caso di protocollo step and shoot risultata essere pari a 31 / 31 segmenti valore 2 ( 100 % ) , in caso di protocollo cardiac dose right risultata essere pari a 483 / 547 segmenti valore 2 ( 82% ) , in caso di protocollo standard risultata essere pari a 165 / 202 segmenti valore 2 ( 81 , 6% )  . 
i risultati delle analisi per paziente e per segmento sono riassunti in tabella 4 discussione lac - tcms una metodica entrata nella routine clinica solo molto recentemente , per lo pi utilizzata nel passato per validazione della metodica a paragone con lacc con un uso estensivo della metodica riservato solamente ad alcuni centri di ricerca clinica applicata [ 1921 ]  . 
volume - rendered and axial native scan obtained from retrospectively gated reconstructions at 40% ( a , b ) and 75% ( c , d ) of the r - r interval . 
la qualit delle immagini risulta pi scarsa nella finestra temporale al 40% con risparmio di dose . to perform a correct evaluation , the dose values must be correlated with the operating parameters used ( slice thickness , mas , kv , volume covered ) and the image quality parameters ( spatial resolution , noise , contrast ) [ 22 ]  . 
new ct scanners are equipped with dose - modulation systems that vary the tube current so as to achieve the highest possible image quality by optimising the use of x - rays and thus reducing radiation exposure to the patient . 
to obtain the same quantity of radiation and therefore the same level of noise , modulation of the tube current is crucial . ( as low as reasonably achievable ) di ottimizzazione dellindagine [ 22 ]  . 
con il temine ottimizzazione si intende il mantenimento della dose al livello pi basso ottenibile , compatibilmente con la qualit dellimmagine necessaria ad ottenere linformazione diagnostica ricercata [ 12 , 22 ]  . i nostri risultati dimostrano come con lutilizzo della tecnica di acquisizione appropriata si riesca ad ottenere un notevole risparmio di dose pari mediamente al 70% in caso di utilizzo di protocollo cardiac dose right e 30% in caso di protocollo step and shoot rispetto al protocollo standard . nonostante la riduzione di dose , la qualit complessiva dellesame rimasta soddisfacente , confermando che non 1208 radiol med ( 2009 ) 114 : 11961213 fig . 
volume - rendered and axial native scan obtained from retrospectively gated reconstructions at 40% ( a , b ) and 75% ( c , d ) of the rr interval . 
la qualit delle immagini risulta pi ottimale in tutte le finestre di acquisizione . scanner manufacturers adopt a variety of approaches to achieve this aim , ranging from the choice of the value of the current , the reference noise or the reference image . 
in addition to the normal dose - reduction techniques available on all scanners , techniques dedicated to the study of the heart have been implemented with two different modalities . the first ecg - gated dose modulation reduces the delivery of the radiation beam during the cardiac cycle ( e.g. systolic phase ) , which can be excluded a priori from the retrospective reconstruction phases [ 12 ]  . 
with this approach , the peak tube current ( mas ) is applied only during the diastolic phase of the cardiac cycle , necessario sempre utilizzare il protocollo standard di acquisizione . 
 per effettuare una corretta valutazione essenziale correlare i valori di dose ai parametri operativi utilizzati ( spessore di strato , mas , kv , volume coperto ) e ai parametri di qualit dellimmagine ( risoluzione spaziale , rumore , contrasto ) [ 22 ]  . 
i nuovi sistemi tc sono dotati di controllo della dose che agiscono variando il valore dei mas con lo scopo di raggiungere un migliore livello di qualit di immagine , ottimizzando limpiego di radiazione x e riducendo di conseguenza la dose radiante al paziente . 
where available , this technique can be used in patients without arrhythmias , with a constant heart rate and in whom the interval of the diastolic phase can be readily calculated , as the low level of radiation in the remainder of the cardiac cycle can compromise image quality [ 25 ]  . technique the second dose optimisation involves prospective triggering whereby the reconstruction window is preset by the radiologist before the contrast - enhanced study [ 15 ]  . 
the dose delivered to the patient is significantly reduced in that there is a sequential and nonspiral acquisition , which therefore reduces oversampling of the data acquired [ 13 ]  . 
because a rising bmi negatively influences image quality , which appear markedly affected by noise , this low - dose protocol can only be used for subjects who weigh < 70 kg and have a bmi < 20 . 
it should nonetheless be noted that mdct - ca is only rarely used to evaluate ventricular radiazione e quindi lo stesso livello di rumore , indispensabile agire sul valore dei mas . le ditte costruttrici utilizzano approcci diversi che variano tra la scelta di un valore di corrente , di rumore di riferimento o di immagine di riferimento . 
 la prima ( ecg - gated dose modulation ) riduce lerogazione del fascio di radiazione durante fasi come la sistole e che , a priori , si possono escludere dalle fasi di ricostruzione retrospettiva [ 12 ]  . 
tramite questo approccio loutput nominale massimo impostato ( mas ) viene applicato solo durante la fase diastolica del ciclo cardiaco , ovvero nella fase in cui i dati possono essere utilizzati per la ricostruzione . 
con questo metodo il valore della coronaro - tc pu dare valori di dose efficace dellordine di 57 msv , in funzione della frequenza cardiaca del paziente . dove disponibile questa tecnologia pu essere utilizzata per tutti i pazienti in assenza di aritmia , per pazienti con battito costante e in cui facilmente calcolabile lintervallo della fase diastolica su cui andare a ricostruire i dati , in quanto il 1210 radiol med ( 2009 ) 114 : 11961213 function , because echocardiography is in widespread use and because cardiac mr is the reference standard for this analysis . the data from our patient series show that the correct selection of the dose - saving protocol within subcategories of the patient population can provide a good compromise between the dose delivered and image quality and therefore overall diagnostic value . on the basis of the characteristics of the sample population , it is apparent that the most significant variable that determines the choice of one protocol over another is bmi . heart rate may play a significant role only in the choice between the step - and - shoot protocol and the other two protocols , whereas it has no significant effect on the choice between the standard protocol and the cardiac dose right protocol . at our centre , we make regular and widespread use of dose optimisation protocols with the following strategy . 
if the patient weighs < 70 kg , has a bmi < 20 and a stable heart rate < 65 bpm , then they undergo the mdct - ca study with the step - and - shoot protocol . 
if the patient weighs > 70 kg , has a bmi between 20 and 25 but a stable heart rate < 65 bpm , then they undergo mdct - ca with the cardiac dose right protocol . 
if , instead , the patient weighs > 70 kg , has a bmi > 25 and an unstable heart rate and we foresee the need for ecg editing or the evaluation of images reconstructed from different time windows , then we apply the standard protocol . 
in all cases where a functional evaluation is requested , we do not apply the step - and - shoot protocol but rather use the cardiac dose right protocol when possible . 
7 dose effettiva al paziente media nei differenti protocolli di modulazione di dose . basso livello di radiazioni del resto del ciclo cardiaco possono compromettere la qualit delle immagini [ 25 ]  . la seconda tecnica di ottimizzazione della dose prevede un triggering prospettico nel quale viene preimpostata dal radiologo prima dellesame contrastografico lunica finestra di ricostruzione [ 15 ]  . 
la dose al paziente decresce in modo importante in quanto vi una acquisizione sequenziale e non spirale che porta quindi a una riduzione del sovracampionamento dei dati acquisiti [ 13 ]  . 
per il fatto che lindice di massa corporea ( bmi ) influenza negativamente la qualit delle immagini che appaiono pi rumorose , tale protocollo a basso dosaggio pu essere utilizzato al fine di ottenere immagini diagnostiche solamente per soggetti con peso inferiore ai 70 kg e bmi inferiore a 20 . 
va detto per che la ac - tcms solo raramente viene utilizzata come metodica per la valutazione della funzione ventricolare essendo largamente in uso lecocardiografia ed essendo la risonanza magnetica cardiaca ( cardio - rm ) lo standard di riferimento per tale analisi . 
 dai dati ricavati dalla nostra casistica abbiamo dimostrato come una corretta selezione del protocollo di risparmio di dose in subpopolazioni campione da sottoporre allindagine porti a un buon rapporto tra la dose erogata , la qualit dellimmagine ottenuta e quindi il valore diagnostico complessivo . 
 sulla base dellanalisi delle caratteristiche della popolazione campione emerge come la variante pi significativa che determina la scelta di un protocollo rispetto ad un altro sia lindice di massa corporeo . 
la frequenza cardiaca poi varia in maniera pi significativa solo tra il protocollo step and shoot e gli altri 2 , mentre non varia significativamente tra il protocollo standard e cardiac dose right . nel nostro istituto utilizziamo regolarmente i protocolli di ottimizzazione della dose seguendo la seguente strategia di impostazione : se il paziente ha un peso inferiore ai 70 kg e bmi inferiore a 20 e mantiene una frequenza cardiaca stabilmente sotto i 65 bpm viene sottoposto ad indagine actcms mediante protocollo step and shoot . 
se il paziente ha un peso superiore ai 70 kg e bmi compreso tra 20 e 25 ma con frequenza cardiaca stabilmente sotto i 65 bpm viene sottoposto ad indagine ac - tcms mediante protocollo di modulazione della dose cardiac dose right . 
nel caso in cui il paziente sia sopra i 70 kg e con bmi superiore a 25 e con frequenza cardiaca instabile e prevediamo la necessit di un editing dellecg o la valutazione delle immagini secondo pi finestre temporali di ricostruzione applichiamo il protocollo standard . 
utilizziamo questa strategia di risparmio di dose in quanto lindagine , esponendo il paziente ad una dose considerevole , non pu andare incontro a un esame di qualit generalmente insoddisfacente e deve permettere una diagnosi nella stragrande maggioranza dei casi . 
 va infine ricordato come lac - tcms ormai nelluso clinico si proponga come metodica valida in grado spesso di modificare liter diagnostico del paziente cardiopatico evitando in molti casi lesecuzione di indagini invasive ed in alcuni casi inutili come lacc ( nel caso in cui non si dimostrino stenosi significative e quindi non si proceda a rivascolarizzazione ) oppure diagnostiche , ma con accuratezza 1212 radiol med ( 2009 ) 114 : 11961213 myocardial scintigraphy , which generally involves a dose to the patient per examination of around 2023 msv [ 2528 ]  . inferiore se paragonate allac - tcms come ad esempio la scintigrafia miocardica che generalmente prevede una dose al paziente per esame media di circa 2023 msv [ 2528 ]  . 
 conclusions conclusioni given that the latest generation of scanners has led to an increase in the dose delivered to patients , it is of crucial importance that dose - saving systems are adopted whenever possible . 
the radiologist must be aware of acquisition techniques with dose - saving protocols to avoid exposing the patient to an unnecessary and dangerous dose and must propose the most appropriate protocol in relation to the clinical indication , the type of patient and the information required for the diagnostic process . dato che con le ultime generazioni di scanner vi un incremento di dose al paziente di primaria importanza lutilizzo di sistemi di risparmio di dose , ove possibile . 
de filippo , scienze radiologiche , azienda ospedaliero - universitaria di parma , via gramsci 14 padiglione barbieri , 43100 parma , italy , tel . : + 39 - 052 - 1703660 , e - mail : massimo.defilippo@unipr.it received : 20 march 2008 / accepted : 13 october 2008 / published online : 19 november 2009 springer - verlag 2009 abstract purpose . 
through a search of the pathology databases of four italian hospitals , we identified six men ( mean age , 56 years ) with a histological diagnosis of ecd . histology was performed on retroperitoneal or pulmonary biopsy , depending on disease involvement on imaging . patients underwent plain radiography of the lower limbs and chest , total - body computed tomography ( ct ) and bone scintigraphy . 
magnetic resonance ( mr ) imaging was performed in two patients to evaluate the lower limbs and in one patient to study the brain , the chest and the abdomen . 
imaging studies revealed extraskeletal manifestations in all patients , including involvement of the retroperitoneal space ( n = 4 ) , the lung ( n = 4 ) and the heart ( n = 2 )  . 
sono stati identificati nel data base dellunit operativa di anatomia patologica di quattro ospedali italiani , sei pazienti con ecd , di sesso maschile , con et media di 56 anni . 
la diagnosi di natura dellecd istologica , 1320 radiol med ( 2009 ) 114 : 13191329 keywords erdheim - chester disease retroperitoneal fibrosis histiocytosis tuttavia il sospetto diagnostico pu essere agevolmente posto con limaging radiologico . parole chiave malattia di erdheim - chester fibrosi retroperitoneale istiocitosi introduction introduzione erdheim - chester disease ( ecd ) is a rare , systemic xanthogranulomatous infiltrative disease characterised by deposition of lipid - laden histiocytes in various organs , with the constant involvement of bone [ 16 ]  . 
ecd was first described by the american physician william chester and the viennese pathologist jacob erdheim in 1930 , who described two cases of a peculiar form of histiocytosis that they distinguished from other disorders such as handschller - christian disease and niemann - pick disease [ 7 ]  . the eponym erdheim - chester disease was coined by jaffe in 1972 [ 8 ]  . 
 the disease affects both genders , though with a slight predilection for men , and has a peak incidence between the fifth and sixth decade of life , although it may manifest at any age [ 4 ]  . 
skeletal involvement is most common in the long bones of the lower limbs . approximately 50% of patients present with extraskeletal manifestations , the most commonly involved sites being the heart , the lungs , the kidneys , the retroperitoneal space , the central nervous system and the skthe clinical course of ecd ranges from the absence of symptoms to a very poor outcome [ 9 ]  . 
 materials and methods we retrospectively searched the pathology databases of four italian hospitals for cases of ecd entered between january 2002 and january 2008 to identify organ involvement and clinical and radiological features . 
the diagnostic criteria for ecd are histopathological and based on the presence of lipid - laden histiocytes immunopositive for cd68 and immunonegative for s - 100 , cd1a and birbeck granules . 
 imaging studies included plain radiography of the lower limbs and chest , bone scintigraphy with technetium 99 ( 99mtc ) and chest and abdominal multidetector computed tomography ( mdct )  . 
patient 1 , who had a history of la malattia di erdheim - chester ( ecd ) una rara patologia sistemica di tipo infiltrativo xantogranulomatoso , caratterizzata dalla presenza di depositi di istiociti contenenti materiale lipoideo in vari organi , con coinvolgimento osseo pressoch costante [ 16 ]  . 
lecd fu descritta per la prima volta nel 1930 da un medico nordamericano , william chester e da un patologo viennese , jacob erdheim , che identificarono in due pazienti una particolare forma di istiocitosi , distinguendola da altri disordini , come la malattia di hand - schllerchristian e di niemann - pick [ 7 ]  . 
 la malattia presente in entrambi i sessi , con una lieve prevalenza nel sesso maschile ed un picco di incidenza tra la 5 e la 6 decade di vita , sebbene possa manifestarsi a qualsiasi et [ 4 ]  . 
linteressamento scheletrico dellecd riguarda prevalentemente le ossa lunghe degli arti inferiori ; circa la met dei pazienti presentano manifestazioni extra - scheletriche , le sedi maggiormente coinvolte sono : cuore , polmoni , reni , retroperitoneo , sistema nervoso centrale e cute . 
 materiali e metodi abbiano ricercato retrospettivamente nel database delle unit operative di anatomia patologica di quattro ospedali italiani la presenza di casi di malattia di erdheim - chester ( ecd ) , nel periodo compreso tra il gennaio 2002 e il gennaio 2008 , al fine di valutarne limpegno nei differenti organi e gli aspetti clinico - radiologici . 
i criteri diagnostici di ecd , sono istopatologici , basati sulla presenza di istiociti ricchi di lipidi , positivi allantigene per il cd68 e prive degli antigeni della proteina s - 100 , del cd1a e dei granuli di birbeck . 
il campionamento tissutale era stato ottenuto mediante biopsia retroperitoneale in tre casi e polmonare nei rimanenti tre casi . i pazienti erano stati sottoposti a radiografia del torace e degli arti inferiori , scintigrafia ossea con tecnezio 99 ( 99mtc ) , tomografia computerizzata multidetettore ( tcmd ) toracica e addominale . 
il paziente 1 , con anamnesi positiva per diabete insipido e microadenoma ipofisario , fu studiato radiol med ( 2009 ) 114 : 13191329 1321 diabetes insipidus and pituitary microadenoma , was studied with high - field magnetic resonance ( mr ) imaging to evaluate the brain , the chest , the abdomen and the lower limbs . patient 2 underwent mr imaging of the lower limbs to investigate diffuse bone changes identified on plain radiography . 
le indagini di imaging sono state valutate da due radiologi . results patients 1 and 2 had cardiac involvement , which in one patient manifested clinically as chest tightness and in the other as dyspnoea on exertion . 
in one case , the retroperitoneal solid tissue caused renal artery stenosis with resulting arterial hypertension , whereas in the other three cases , it surrounded the aorta and the iliac arteries , causing risultati nei pazienti 1 e 2 si evidenziava coinvolgimento cardiaco dellecd . 
lesordio clinico di ecd nei pazienti 3 e 4 era caratterizzato da alterazione degli indici di funzionalit renale , associato a ripetuti episodi di infezioni delle vie urinarie nel caso 3 . 
nei primi quattro pazienti la tc addome evidenziava infiltrazione retroperitoneale da parte di tessuto solido , prevalentemente perirenale e simmetrico , ipodenso e dotato di lieve contrast enhancement dopo somministrazione di mezzo di contrasto ( mdc )  . 
il tessuto solido retroperitoneale determinava nel caso 1 stenosi dellarteria renale con conseguente ipertensione arteriosa mentre negli altri tre casi circondava laorta e le arterie iliache , con fenomeni di incarceramento della fig . 
magnetic resonance image of the chest ( balanced sequence ) in the coronal plane ( a ) and contrast - enhanced computed tomography of the chest with four - chamber multiplanar reconstruction ( b )  . 
rm del torace ( balance w ) in sezione coronale ( a ) e tc con mdc del torace in sezione assiale obliqua four chambers mpr ( b )  . 
contrast - enhanced computed tomography of the upper abdomen ( a , b ) : the coated aorta , a specific finding in erdheim - chester disease , is well - depicted in the magnified image ( arrows in b )  . fig 2a , b ecd in fase avanzata . 
tc con mdc delladdome superiore ( a , b ) : aorta rivestita da tessuto solido , aspetto peculiare dellecd , ben evidente nellimmagine ingrandita ( frecce in b )  . 
renal pyelectasia , more evident on the left side , is due to compression by the fibrous tissue , which is also present at the level of the proximal periureteral spaces  . fig 3a , b ecd in fase iniziale . 
nel caso 5 lecd si manifestava con dolori al rachide in assenza di alterazioni allrx tradizionale , mentre la scintigrafia con 99mtc mostrava iperfissazione del radiol med ( 2009 ) 114 : 13191329 1323 fig . 
magnetic resonance images of the upper abdomen : axial t2 - weighted turbo spin - echo ( a ) and t1 - weighted fat - saturated fast - field echo sequence after the intravenous administration of paramagnetic contrast material . 
note the homogeneous enhancement of the fibrous tissue during the venous phase ( b ) and the sparing of the periaortic and pericaval regions ( a , b )  . fig 4a , b ecd in fase avanzata . 
il sottoslivellamento laterale sn della sella turcica ( freccia ) segno indiretto della presenza di un microadenoma ipofisario . scintigraphy showed increased tracer uptake at the level of a thoracic vertebra . four patients ( 2 , 3 , 5 and 6 ) had involvement of the pulmonary interstitiuchest radiography showed pleural and interstitial thickening ( kerley b lines )  . 
chest ct demonstrated a reticular interstitial pattern , smooth septal and fissural thickening , diffuse ground - glass opacities and centrilobular nodules with subpleural , peribronchovascular radiofarmaco in corrispondenza di una vertebrale dorsale . in quattro pazienti ( caso 2 , 3 , 5 e 6 ) vi era coinvolgimento dellinterstizio polmonare . 
i radiogrammi in proiezione antero - posteriore di gamba , mettono in evidenza bilateralmente , in corrispondenza della spongiosa tibiale e peroneale , diffuse e sfumate zone di radio - opacit , cui si associano ispessimenti focali delle corticali scheletriche , in assenza di reazioni periostali . radiol med ( 2009 ) 114 : 13191329 1325 fig . 
the clinical suspicion was confirmed by a biopsy of the retroperitoneal tissue in three patients ( 1 , 3 , and 4 ) and of pulmonary tissue in the remaining three patients ( 2 , 5 , 6 )  . 
il sospetto clinico stato confermato dallindagine bioptica su tessuto retroperitoneale in 3 pazienti ( casi 1 , 3 , 4 ) e polmonare nei rimanenti 3 casi ( 2 , 5 , 6 )  . 
 discussion discussione ecd is a rare form of non - langerhans histiocytosis , the reactive or neoplastic nature of which is still being debated [ 10 , 11 ]  . 
axial ( a ) and sagittal ct ( b ; multiplanar reconstruction ) demonstrates extensive erosion of t6 vertebral body with interruption of the posterior end - plate and initial involvement of the left vertebral pedicle . 
dimostrazione tc sul piano assiale ( a ) e sagittale ( b ; mpr ) di voluminosa erosione del soma di t6 con interruzione della limitante somatica posteriore ed inizile impegno del peduncolo vertebrale di sn . 
la rm ( c ) in se t1 w dimostra lindennit del canale vertebrale . in our experience , there was a frequent correlation between a histological diagnosis of ecd and skeletal involvement . 
bone involvement is an almost constant feature of ecd , there being only one report of a case without bone lesions [ 13 ]  . ecd generally affects the long bones of the lower limbs ( tibia and fibula )  . 
the related radiographic findings are diffuse or patchy opacities of the tibia and / or fibula , medullary sclerosis and cortical thickening of the metaphyseal and diaphyseal portions without epiphyseal changes . on 99mtc scintigraphy , intense , bilateral and symmetrical tracer uptake by the long - bone diaphyses and metaphyses is revealed , reflecting increased osteoblastic activity in those regions [ 1521 ]  . 
in our series , osteosclerotic changes were seen in the metaphyseal and diaphyseal portions of the tibia and fibula in three patients ( 1 , 3 and 4 )  . 
le lesioni ossee rappresentano un aspetto pressoch costante dellecd ; viene riportato in letteratura un solo caso di ecd in cui non erano state dimostrate lesioni ossee [ 13 ]  . lecd generalmente interessa le ossa lunghe degli arti inferiori ( in particolar modo tibia e perone ) e le alterazioni radiografiche si configurano come radio - opacit diffuse o a chiazze della tibia e / o del perone , la sclerosi midollare e lispessimento corticale delle regioni metafisarie e diafisaria ; scarse invece sono le alterazioni epifisiarie [ 13 ]  . 
la scintigrafia 99mtc evidenzia intensa iperfissazione , bilaterale , simmetrica a carico delle diafisi e metafisi delle ossa lunghe , conseguente ad aumento dellattivit osteoblastica in tali sedi [ 1521 ]  . 
nei casi 2 e 6 si osservano le caratteristiche radiol med ( 2009 ) 114 : 13191329 1327 the osteosclerotic changes were seen at the femoral level . bone involvement in these patients was asymptomatic , in contrast to previous reports where > 50% of patients have mild but persistent pain of the affected bone segment , most commonly the hips and knees [ 9 ]  . only patient 4 had symptomatic bone involvement , reporting with spinal pain despite the absence of significant abnormalities on plain radiography of the spine and lower limbs . 
are the only authors to have reported cases of lytic vertebral lesions ( two cases ) , and indeed , the spine is typically spared in ecd [ 22 ]  . 
otomastoiditis , spleen and bone marrow involvement are also more frequent in lch , whereas retroperitoneal involvement of the perirenal spaces and circumferential involvement of the aorta are typical features of ecd [ 23 ]  . 
in support of the latter hypothesis , cases of ecd coexisting with lch have been reported , suggesting the possibility that the two diseases are linked by a common precursor , cd34 + [ 24 , 25 ]  . 
il coinvolgimento osseo in questi pazienti era asintomatico , a differenza dei dati riportati in letteratura , secondo i quali oltre la met dei pazienti presentano dolore lieve ma persistente , localizzato al segmento osseo colpito , prevalentemente alle anche e alle ginocchia [ 9 ]  . solo il quarto paziente presentava sintomatologia dolorosa al rachide , in assenza di significative alterazioni allindagine radiografica del rachide e degli arti inferiori ; lesame scintigrafico evidenziava iperfissazione del radiofarmaco in corrispondenza di una vertebra dorsale ; la biopsia in tale sede non risultava diagnostica per campionamento inadeguato ; il prelievo bioptico a livello polmonare consentiva la diagnosi di ecd . 
 [ 22 ] sono gli unici autori ad avere riportato casi di lesioni osteolitiche vertebrali ( 2 pazienti ) ; infatti caratteristicamente nellecd si osserva risparmio del rachide . le lesioni osteolitiche dello scheletro assiale sono pi frequenti nellistiocitosi a cellule di langerhans ( lch ) , ma generalmente , queste tendono a risparmiare le ossa lunghe . inoltre i pazienti affetti da lch sono pi giovani rispetto a quelli con ecd ed hanno una prognosi migliore , con tassi di mortalit del 30% ( versus il 57% dellecd ) [ 9 ]  . 
a favore di tale ipotesi sono stati riportati alcuni casi in cui ecd ed lch coesistono , suggerendo la possibilit che le due patologie siano legate da un comune precursore cd34 + [ 24 , 25 ]  . 
in nessuno dei nostri pazienti stato osservato esoftalmo , sebbene rappresenti una frequente presentazione neurologica di ecd , conseguente ad una infiltrazione del tessuto adiposo retroconale e della guaina del nervo ottico . 
diabete insipido ed esoftalmo sono presenti in entrambe le forme di istiocitosi , in particolare nellistiocitosi a cellule del langerhans ( lch ) rappresentano in associazione ad osteolisi la classica triade di presentazione . 
tali reperti associati a diabete insipido sono rilevabili anche nello xantoma disseminato ( xd ) che pu simulare clinicamente lecd ; tuttavia nellxd il diabete insipido di grado lieve e transitorio ( nellecd persistente e progressivo ) , i pazienti hanno et inferiore a quelli con ecd , presentano diffuse lesioni cutanee e mucose e , raramente , possono presentare lesioni osteolitiche periarticolari [ 25 ]  . nei casi 1 e 2 stato riscontrato coinvolgimento cardiaco 1328 radiol med ( 2009 ) 114 : 13191329 among the typical manifestations of ecd [ 27 ]  . 
these same patients also exhibited thoracoabdominal periaortic infiltration , which is the most common cardiovascular manifestation of ecd [ 27 ] and is referred to as a coated aorta when the entire course of the vessel aorta is surrounded by fibrosis . 
at ct , care should be taken not to confuse periaortic infiltration with the aortic wall thickening seen in takayasu arteritis , a condition characterised by involvement of the full thickness of the aortic wall and sparing of the periaortic spaces . 
the use of contrast - enhanced ct is decisive for identifying this feature at the early stages of perirenal fibrosis . displacement of the aorta and ureters by the fibrous tissue may also be seen in forms secondary to malignant diseases , as in some histological types of non - hodgkins lymphomas . these , however , will rarely show the typical bilateral and symmetrical distribution of ecd [ 28 ]  . 
the differential diagnosis of pulmonary involvement by ecd prevalently includes cardiogenic interstitial oedema , where centrilobular nodules are absent , and carcinomatous lymphangitis and sarcoidosis , where septal thickening is typically nodular [ 29 , 30 ]  . our experience suggests that a clinical presentation of bilateral hydronephrosis , persistent bone pain , diabetes insipidus and exophthalmus should raise suspicion of ecd and prompt investigation with plain radiography of the lower limbs and ct of the chest and abdomen . 
if the examination reveals the characteristic diaphyseal and metaphyseal osteosclerosis , brain mr imaging should be used to search for any pituitary microadenomas ( at times hyposecreting )  . in conclusion , ecd is a rare multiorgan disease that has heterogeneous clinical presentations and a prevalence that appears to be underestimated . 
among the possible organs con riscontro di versamento pericardico , che recentemente stato incluso tra le manifestazioni tipiche dellecd [ 27 ]  . negli stessi pazienti stata anche evidenziata la presenza di infiltrato periaortico toraco - addominale ; tra le manifestazioni cardiovascolari questa la pi frequente in corso di ecd [ 27 ] e viene indicata anche con il termine di aorta rivestita , quando lintero decorso aortico circondato da fibrosi . 
allesame tc linfiltrato periaortico non deve essere confuso con lispessimento parietale aortico presente nellarterite di takayasu ; questultima condizione infatti si caratterizza per linteressamento a tutto spessore della parete aortica con risparmio degli spazi periaortici . 
la dislocazione dellaorta e degli ureteri da parte del tessuto fibrotico pu essere riscontrata anche nelle forme secondarie a patologie maligne , come nel caso di alcuni istotipi di linfomi nonhodgkin ; tuttavia rara la distribuzione bilaterale e simmetrica propria dellecd [ 28 ]  . 
la tc torace evidenziava un pattern interstiziale di tipo reticolare con ispessimento liscio dei setti e delle scissure , diffuse aree a densit ground glass e noduli centrolobulari con distribuzione subpleurica , peribroncovasale e interlobulare . 
la diagnosi differenziale in caso di coinvolgimento polmonare include principalmente ledema interstiziale cardiogenico in cui sono assenti i noduli centrolobulari , la linfangite carcinomatosa e la sarcoidosi ove lispessimento dei setti tipicamente nodulare [ 29 , 30 ]  . la nostra esperienza suggerisce che un quadro clinico caratterizzato da idronefrosi bilaterale , dolore osseo persistente , diabete insipido , esoftalmo , pu orientare il sospetto diagnostico di ecd e giustificare lesecuzione di un esame radiografico degli arti inferiori e di una tc toracica ed addominale . 
il riscontro occasionale in ecografia , tc o rm , di fibrosi perirenale giustificano un approfondimento con rx standard degli arti inferiori ; quando a tali livelli , si documenti la caratteristica osteosclerosi diafisaria e metafisaria dellecd , consigliabile ricercare la possibile presenza di microadenomi ipofisari ( talvolta iposecernenti ) con rm dellencefalo . in conclusione , lecd una patologia multiorgano rara , con manifestazioni cliniche estremamente eterogenee , la cui radiol med ( 2009 ) 114 : 13191329 1329 involved by the disease , skeletal tissue , the retroperitoneum and , in particular , perirenal spaces , were found to be constantly involved . 
hence they require a multidisciplinary and multispecialty approach , which must begin with an accurate medical examination , conducted in compliance with the lege artis principles and with respect for the victims dignity . 
this paper describes the radiologists key role in identifying physical injuries due to child abuse , in accordance with current regulations . keywords child abuse forensic radiology battered child syndrome riassunto labuso ed il maltrattamento sui minori una tematica di grande attualit con notevoli risvolti di carattere sociosanitario che interessano il medico sia come cittadino che in qualit di esercente una professione sanitaria . 
la molteplicit e la peculiarit di alcune lesivit di abuso , siano queste di tipo fisico , psichico o sessuale necessitano di un approccio multidisciplinare e polispecialistico che non pu prescindere tuttavia da una visita medica che , nel rispetto della dignit della presunta vittima , sia effettuata secondo i criteri propri della lege artis . 
il presente articolo descrive il ruolo del radiologo nella valutazione degli abusi fisici perpetrati a danno di minori alla luce della normativa vigente . parole chiave abuso sui minori radiologia forense sindrome del bambino battuto introduction introduzione in 1946 , radiologist john caffey following a . 
this was a concise , pragmatic definition of what is not so much a clinical syndrome as a sociocultural phenomenon that had strong repercussions on trauma medicine and on physicians themselves [ 1 ]  . 
tardieu del 1860 definiva in maniera concisa e pragmatica pi che una sindrome clinica un fenomeno socio - culturale che aveva pesanti riflessi sulla medicina traumatologica e notevoli ricadute sui medici [ 1 ]  . radiol med ( 2009 ) 114 : 13561366 1357 an accurate definition of child abuse is given in the world health report 2002 : child abuse or maltreatment constitutes all forms of physical and / or emotional illtreatment , sexual abuse , neglect or negligent treatment or commercial or other exploitation , resulting in actual or potential harm to the childs health , survival , development or dignity in the context of a relationship of responsibility , trust or power [ 2 ]  . 
therefore , child abuse includes the results not only of actions ( sexual or other kinds of violence ) but also of neglect or deprivation ( lack of care , attention , affection )  . 
in both cases , the physical examination alone , for legal purposes as well as for diagnosis and therapy , is unable to confirm or exclude suspected abuse ( at least from a medical point of view ) without the use of radiological tools . 
thus , radiology is an essential tool in medicolegal investigations of physical child abuse , which gives the specialist a proper understanding of type , extent and age of the injury , and the dynamics behind it ( aetiopathogenesis )  . extent of child abuse child abuse has become a very worrying issue with an impact on the whole of society . 
introna states : the abused child is the expression of multiple problems of a legal , cultural and economic nature involving his / her family at a given moment in time , and therefore he / she is the clue to pain that goes beyond his / her own person [ 3 , 4 ] as requested . child abuse is often rejected or negated , and it is always underreported . 
this is especially true in southern italy , where it often occurs within a complex family setting , which makes it difficult to evaluate the victim and type of abuse suffered . 
the child presents distinctive injuries , often inflicted with bare hands or by using various objects ( sticks , chains , strings , cigarettes or other sources of heat )  . 
this form of child abuse results from indeuna precisa e completa definizione pu essere tradotta dalla world health organization ( who ) che nel 2002 cos recitava : per abuso allinfanzia si intendono tutte le forme di cattiva salute fisica e / o emozionale , abuso sessuale , trascuratezza o negligenza o altro che comportino un pregiudizio reale o potenziale per la salute del bambino , per la sua sopravvivenza , per il suo sviluppo o per la sua dignit nellambito di una relazione caratterizzata da responsabilit , fiducia o potere [ 2 ]  . 
in relazione a tanto , labuso sui minori deve intendersi comprensivo sia degli atti ( violenze vere e proprie , non necessariamente sessuali ) che delle carenze e le deprivazioni ( sottrazione delle cure , dellattenzione , dellaffetto )  . 
in entrambe le ipotesi laccertamento medico , effettuato a fini giuridici lindagine oltrech diagnostico - terapeutici , utilizzer radiologica quale importantissimo strumento diagnostico per fondare ovvero escludere , quanto meno da un punto di vista medico , il sospetto di un abuso . 
lutilizzo di immagini digitali , utile ad una pi fine valutazione della lesione fisica osservata e correlato ad una specifica competenza del radiologo riguardo allepoca di mineralizzazione ossea trauma ed dei segmenti scheletrici allidoneit lesiva del trauma stesso ( eziopatogenesi ) , rende la valutazione radiologica strumento fondamentale nella valutazione medico - legale degli abusi fisici a carico di minori . interessati dal dimensioni del problema il maltrattamento dei bambini ha assunto dimensioni tali da costituire un serio motivo di preoccupazione da parte di tutta la societ civile . 
introna in un suo felicissimo intervento recita : il bambino maltrattato testimone di problemi multipli che investono il nucleo familiare in quel determinato momento storico , giuridico , culturale ed economico e quindi egli la spia di una sofferenza che va oltre la sua persona [ 3 , 4 ]  . 
questo vero soprattutto nelle regioni del sud dove la complessit dellambiente familiare , in cui , nella maggior parte dei casi , avviene tale tipologia di reati , rende estremamente difficile unesatta valutazione delle vittime e del tipo di abuso subito . 
these injuries are a sign of neglect by those responsible for the childs care because the injuries were not treated promptly and eventually became chronic . this paper focuses only on the battered child syndrome because this feature directly concern radiologists . radiological features of child abuse the radiologist is consulted during the investigation of battered child injuries , especially those related to cranial and musculoskeletal injuries , and he or she must be cognizant of the often critical features of such injuries . 
later , in 1962 , kempe coined the expression battered child syndrome [ 6 ] to group together the clinical and radiological signs of beaten children and to draw attention of the medical community to the problem , which , although steadily escalating , had been totally disregarded . incomplete or complete fractures are generally observed in the upper and lower limbs and in the ribs directly struck by blows , punches or kicks whilst the child is hunched up for self protection . 
it must be pointed out that the radiologist is always able to identify an injury but can never be absolutely sure of its mechanis nevertheless , some fractures are highly specific of the battered child syndrome , i.e. 
the knee ( proximal tibia ) , the wrist , the femur and the humerus are the most frequent sites of such fractures , which are rarely accompanied by a periosteal reaction and seldom cause growth disturbances or bending deformities [ 10 , 11 ]  . 
con questo termine si vogliono comprendere tutti quei maltrattamenti occulti costituiti da mancanza di protezione , di educazione , di amore e di accettazione che si estrinsecano con una serie di trascuratezze , rifiuti e sfruttamenti che sfociano nellabbandono psicologico ma , talvolta , possono determinare il decesso della vittima per inanizione . 
oltre alle sequele psicologiche , si possono riscontrare anche dermatosi comuni croniche da scarsa igiene , ripetute infezioni , ascessi e fistole che sono indicative della trascuratezza di chi si dovrebbe prendere cura del minore ed indicare affezioni perduranti nel tempo e non curate tempestivamente . in questo articolo tratteremo esclusivamente la valutazione specialistica degli abusi fisici sui minori ( battered child syndrome ) , i cui aspetti coinvolgono in maniera diretta e pressante lo specialista radiologo . aspetti radiologici degli abusi fisici sui minori lo specialista radiologo consultato in corso di accertamenti sul bambino battuto ( abusato da un punto di vista fisico ) , prevalentemente nei casi di violenza da trauma sullapparato cranico - muscolo - scheletrico e deve conoscerne gli aspetti , a volte del tutto peculiari , con cui questi possono palesarsi . 
 gi caffey nel 1957 aveva messo in evidenza ed illustrato alcuni nuovi e particolari tipi di frattura e di lesioni ossee che si possono riscontrare nei bambini battuti , specie nei pi piccoli [ 5 ]  . 
kempe successivamente , nel 1962 , allo scopo di puntare lattenzione del mondo scientifico su un problema sociale assolutamente trascurato , ma in fase di importante espansione , coni il termine di battered child radiol med ( 2009 ) 114 : 13561366 1359 fig . 
b frattura a manico di secchio . table 1 differential diagnosis between accidental and nonaccidental injuries abusive fractures metaphysis proximal tibia femur accidental fractures epiphysis distal tibia other sites with high energy mechanism tabella 1 diagnosi differenziale tra fratture accidentali e da abuso fratture da abuso sede metafisaria tibia prossimale femore fratture accidentali epifisi tibia distale altri siti con meccanismo elevata energia the lower limbs of children who are not ambulatory ( table 1 )  . 
 the so - called toddlers fracture , namely , the spiroidal fracture of the distal portion of the tibia , is a very common accidental injury in the paediatric age . 
on the other hand , if the same fracture is detected with identical radiological features in children not yet ambulatory , it should undeniably be considered as resulting from abuse [ 1214 ]  . 
multiple syndrome per indicare tutto il corteo di sintomi clinici e radiologici che caratterizzano gli aspetti traumatici del bambino battuto [ 6 ]  . come aspetto generale le fratture incomplete o complete interessano lo scheletro appendicolare , specie gli arti superiori ed inferiori e le coste che sono oggetto della violenza soprattutto a seguito di percosse , pugni e calci a bambini rannicchiati in atteggiamento di autoprotezione . 
raro che le lesioni scheletriche siano fatali e rare sono anche le lussazioni e le lesioni vertebrali , in ogni caso opportuno ricordare che il radiologo pu identificare la lesione , ma mai determinarne con certezza le modalit . 
tuttavia esistono alcune particolarit del tipo di fratture che possono indirizzare o far propendere il radiologo verso un trauma non accidentale e quindi da riferire a sindrome da bambino battuto . 
these are caused by traumatic anteroposterior compression of the rib cage , and the radiologist must bear in mind the fact that they cannot be caused even by the use of improper resuscitation procedures [ 1518 ]  . 
as they are extremely serious injuries , they may appear to indicate abuse , but , at the same time , they are very often associated with accidents such as tumbling out of bed , falling off the changing table or down the stairs . 
8a , b frattura lineare del parietale destro nella battered child syndrome . in the case of indirect head injuries , guthkelchi [ 24 ] made a further distinction by introducing shaken baby syndrome , in which lesions are secondary to the attendant axonal damage . when the infant is shaken violently and head movements are not synchronised with the rest of the body , in 90% of cases , this produces retinal haemorrhage , but it may also produce subdural haematoma ( related to vascular tears ) and osseous injuries ( detectable only by means of magnetic resonance imaging )  . 
 we must remember the possibility , even if remote , of finding damage to parenchymal organs , which is regularly accompanied by other types of lesions ( haematoma , pseudocyst , etc . ) that will be seen only through ultrasound or computed tomography [ 26 ]  . current regulations and forensic medicine considerations the entire medical profession , including radiologists , is bound by italian law to cooperate with police authorities to prevent and combat crime . 
evidenti le lesioni ipodense cerebrali , esiti di trauma da scuotimento . deontology code ( 2006 ) states that the doctor must protect children in cases of physical or psychological maltreatment or sexual abuse , and in the event of opposition from the legal guardians , the doctor must report to the appropriate legal authorities . 
361medici specialisti in radiodiagnostica , sono previsti , dalla legislazione attualmente vigente , obblighi giuridici finalizzati a forme di collaborazione con lautorit giudiziaria , al fine di prevenire e contrastare la criminalit . 
32 del codice di deontologia medica ( 2006 ) che prevede , oltrech una specifica tutela sui minori in casi di maltrattamenti fisici o psichici , violenze o abusi sessuali anche la possibilit in caso di opposizione dei legali rappresentanti alla necessaria cura dei minori di ricorrere alla competente autorit giudiziaria . 
 doveroso ricordare che la violazione del dovere o omissione di inoltrare il referto / rapporto costituisce un delitto contro lamministrazione della giustizia ; la mancata ottemperanza di tale dovere prevede automaticamente il reato di omissione datti dufficio qualora si accerti che il sanitario abbia , con coscienza e volont , voluto omettere o ritardare la presentazione del referto [ 28 ]  . 
failure to comply with this duty is malfeasance in office when it is ascertained that the health professional wilfully and knowingly failed to report or delayed reporting the crime [ 28 ]  . 
 conclusions too often do we hear about presumed physical , psychological or sexual child abuse based on psychological evaluations without any reference to the medical follow - up or specialistic investigations that could unequivocally confirm or exclude this suspicion . 
in fact , they have the knowledge to determine the time of mineralisation of skeletal segments , reconstruct the mechanisms of injuries to the osteoarticular system and interpret imaging findings . reporting child abuse is always a very delicate question , especially because it always involves the families . 
although a mere suspicion of abuse is sufficient for current regulations , the professional must be aware of the consequences of a misdiagnosis on the family and on the victitherefore , the radiologist is required to make both a cultural effort in understanding the problem and a methodological effort in evaluating the case in terms of collecting a complete clinical history and carefully interpreting the imaging findings . implementation of guidelines and protocols such as those proposed by the american college of radiology [ 29 ] , together with cooperation between several specialists , will help produce a methodologically flawless evaluation of the true extent of this serious problem , which has been neglected and underreported for too long . 
la particolare complessit della fenomenologia di abuso sui minori necessita , ai fini di un corretto inquadramento del fenomeno stesso , di un approccio multidisciplinare da parte di una equipe di specialisti , laddove di fondamentale importanza appare il contributo del radiologo pediatrico . 
linterpretazione delle lesioni eventualmente riscontrate con limaging non pu essere di pertinenza del pediatra ovvero dellortopedico ; solo il radiologo ha le competenze per discernere laddove possibile caratteristiche proprie di un trauma accidentale oppure di un abuso . 
infatti , la conoscenza della mineralizzazione ( epoca di ossificazione ) dei segmenti scheletrici , dei meccanismi traumatici che sottendono la genesi di lesivit soprattutto dellapparato osteo - articolare nonch la loro interpretazione diagnostica , di pertinenza del radiologo che ne certamente lunico e il migliore conoscitore . 
se da un lato la normativa vigente prevede che sia sufficiente il sospetto e non la certezza , per denunciare siffatto reato altres chiaro che una erronea diagnosi di abuso pu sconvolgere un nucleo familiare , ivi inclusa la vittima . 
allo specialista in radiodiagnostica richiesto pertanto uno sforzo culturale nei termini di comprensione / conoscenza del problema e metodologico nella valutazione di tale fenomeno che non potr prescindere dalla raccolta di un completo raccordo anamnestico , oltrech dalla successiva analisi dellimaging . 
bergamaschi1 1istituto di medicina del lavoro delluniversit cattolica del sacro cuore , largo gemelli 8 , 00168 roma , italy 2istituto nazionale di riposo e cura dellanziano ( inrca ) , u.o. 
le donne radiologo sono meno soddisfatte degli uomini per quanto riguarda i riconoscimenti per il lavoro ben fatto , la variet del lavoro , la distribuzione dei carichi di lavoro . 
appare realizzabile una indagine trasversale a campione multistratificato . parole chiave soddisfazione lavoro radiologo stress radiol med ( 2009 ) 114 : 13301344 introduction introduzione 1331 the scientific literature has reported increasing numbers of studies on job satisfaction among physicians [ 16 ] in that this measure it closely related to physical and psychological well - being , work - related choices and the quality of care delivered [ 7 , 8 ]  . 
 this pilot study aimed to investigate the professional satisfaction of italian radiologists and evaluate the feasibility of extending the investigation to the national level . materials and methods physicians attending two congresses of radiology and radiation oncology were invited to complete an anonymous questionnaire exploring the level of work stress , physical and psychological well - being and other factors potentially associated with medical error . 
in consideration of the findings of previous studies [ 11 , 12 ] , in the italian version , we included an additional item on equity of workload distribution , which was not present in the original version . 
surveys of physicians working in private practice have generally used a short form of the questionnaire , that is , excluding items concerning satisfaction with relationships with superiors or management . 
solo pochi studi hanno affrontato il tema della soddisfazione professionale dei radiologi . il presente studio - pilota il primo condotto nel nostro paese sulla soddisfazione professionale dei medici radiologi e mira , tra laltro , a valutare la fattibilit di una indagine estesa a tutto lambito nazionale . materiali e metodi i medici specialisti partecipanti a due congressi di radiologia e di radioterapia oncologica sono stati invitati a compilare un questionario anonimo , che indagava il livello di tensione lavorativa , il benessere psicofisico ed altri fattori potenzialmente associati allerrore medico . 
la scala di warr consta di 15 domande , a ciascuna delle quali si pu rispondere , secondo una scala likert in 7 punti , che va da estremamente insoddisfatto ( 1 punto ) a estremamente soddisfatto ( 7 punti )  . 
sulla base di studi precedenti [ 11 , 12 ] nella versione italiana stata aggiunta una domanda , relativa allequit nella ripartizione dei compiti di lavoro , che non figura nella versione originale . 
nelle ricerche condotte su medici liberi professionisti il questionario stato generalmente impiegato in forma breve , escludendo le domande riguardanti la 1332 radiol med ( 2009 ) 114 : 13301344 fig . 
job satisfaction scale ( jss ) [ 10 ] , italian version . what is your level of satisfaction with your job ? to complete the questionnaire , mark the number corresponding to one of the following levels of satisfaction in the box next to each question : 1 im extremely dissatisfied 2 im very dissatisfied 3 im moderately dissatisfied 4 im not sure 5 im moderately satisfied 6 im very satisfied 7 im extremely satisfied ( - ) satisfaction how satisfied are you with... w1 the physical work conditions w2 the freedom to choose your own method of working w3 your fellow workers w4 the recognition you get for good work w5 your immediate boss w6 the amount of responsibility you are given w7 your rate of pay w8 your opportunity to use your abilities w9 relations between management and workers w10 your chance of promotion w11 the way your company is managed w12 the attention paid to suggestions you make w13 your hours of work w14 the amount of variety in your job w15 your job security w16 distribution of workloads w17 now , taking everything into consideration , how do you feel about your job as a whole ? fig . 
1 questionario jss ( job satisfaction scale ) di warr et al [ 10 ] , versione italiana . quale il tuo livello di soddisfazione dal lavoro ? per rispondere al questionario , occorre segnare accanto a ciascuna domanda , nellapposita casella , il numero che corrisponde ad uno dei seguenti livelli di soddisfazione : 1 ono estremamente insoddisfatto 2 sono molto insoddisfatto 3 sono moderatamente insoddisfatto 4 sono incerto 5 sono moderatamente soddisfatto 6 sono molto soddisfatto 7 sono estremamente soddisfatto ( - ) soddisfazione quanto ti soddisfa... w1 lambiente di lavoro w3 i compagni di lavoro w5 il superiore w2 la libert di scegliere da s il proprio metodo di lavoro w4 i riconoscimenti che si ricevono per il lavoro ben fatto w6 linsieme delle responsabilit che ti vengono date w7 il guadagno economico w8 la possibilit di usare le proprie capacit professionali w9 le relazioni tra lamministrazione ed i lavoratori w10 le tue possibilit di promozione e carriera w11 il modo in cui lazienda in cui lavori diretta w12 lattenzione rivolta ai suggerimenti che fai w13 lorario di lavoro w14 la variet nel lavoro w15 la sicurezza dellimpiego w16 la ripartizione dei carichi di lavoro w17 ora , prendendo in considerazione tutte queste cose , quale il livello di soddisfazione che ricavi dal lavoro nel suo insieme ? radiol med ( 2009 ) 114 : 13301344 1333 a total of 314 physicians were interviewed , of whom 108 were radiotherapists and 206 radiologists . 
because the group of radiotherapists did not differ significantly from that of radiologists in terms of demographics ( sex , age ) or work - related variables ( experience , position , public or private sector ) , all data were pooled for the analysis . 
the subjects were predominantly men [ 251 men , ( 79.9% ) and 63 women ] , and nearly all of them worked in the public sector [ 289 physicians ( > 92% ) ]  . 
among those employed by the italian national health service ( nhs ) , 20% were heads of department ( n = 59 ) , > 19% were division heads ( n = 56 ) and the remainder were staff - grade consultants ( n = 174 ; 61% )  . 
as controls , we used a group of 34 specialists in infectious diseases , all of whom were hospital employees , who were invited to complete the same questionnaire on the occasion of their periodical medical check - up . 
the control group did not differ significantly from the group of radiologists in terms of age and sex . data were analysed using the statistical package for social science , version 11.5. 
this test measures the correlation between variables of single items in the scale , and the closer it is to a value of 1.0 , the higher the scales internal consistency . 
binomial logistic regression with backward stepwise selection was used to evaluate which of the many variables investigated were more directly correlated to physician satisfaction or dissatisfaction . results questionnaire validation the italian version of the satisfaction scale maintains the unitary nature of the original version by warr et al . 
this component correlated with all of the 17 items , with greater weight ( 0.810 ) attributed to the last item , which elicits a judgement on satisfaction derived from the job as a whole considering all the soddisfazione dei rapporti con i superiori o il management . la versione breve formata dalle domande 1 , 2 , 3 , 4 , 6 , 7 , 8 , 13 , 14 e dellultima , riassuntiva . 
nel nostro studio abbiamo preferito impiegare la versione estesa , in considerazione del fatto che gli intervistati operavano prevalentemente in condizioni di lavoro subordinato . sono stati intervistati 314 medici , di cui 108 specialisti in radioterapia e 206 specialisti in radiologia . 
il gruppo dei radioterapisti non presentava differenze significative rispetto a quello dei radiologi per nessuna delle variabili anagrafiche ( genere , et ) n per quelle legate al lavoro ( esperienza , ruolo , attivit nel settore pubblico o privato )  . 
i soggetti esaminati risultavano prevalentemente di sesso maschile ( 251 maschi , pari al 79 , 9% , 63 femmine ) , e quasi tutti operanti nel settore pubblico ( 289 , oltre il 92% )  . 
i medici dipendenti del sistema sanitario nazionale ( ssn ) erano nel 20% dei casi direttori di unit operativa ( 59 unit ) , in oltre il 19% dei casi responsabili di struttura semplice ( 56 unit ) , per il rimanente dirigenti medici ( 174 unit , 61% )  . 
la classe di et pi frequente risultata quella compresa tra 46 e 50 anni ( 27 , 7% ) , la categoria di esperienza lavorativa pi frequente quella superiore a 26 anni ( 22 , 6% )  . 
come controllo esterno stato impiegato un gruppo di 34 specialisti in malattie infettive , dipendenti ospedalieri , che sono stati invitati a compilare lo stesso questionario in occasione della visita medica periodica . 
il gruppo di controllo non differisce significativamente per et e sesso dai radiologi . i dati sono stati analizzati mediante il software statistico statistical package for social science ( spss ) , versione 11.5. 
convenzionalmente un valore di alfa pari a 0 , 60 ritenuto accettabile , un valore di 0 , 70 adeguato , un valore di 0 , 80 buono . lanalisi fattoriale stata impiegata per conoscere la struttura latente del set di variabili composto dalle domande del jss , riducendo linsieme delle variabili ad un ristretto gruppo di fattori omogenei e dotati di coerenza logica . 
le tabelle di contingenza sono state testate con il test del chi quadro di pearson , per verificare lipotesi zero di mancanza di associazione tra righe e colonne nei dati tabulati . 
per valutare quali , tra le numerose variabili indagate , risultassero pi direttamente legate alla soddisfazione o insoddisfazione dei medici , stata impiegata la regressione logistica binomiale , con selezione condizionale a ritroso ( backward stepwise )  . table 1 factor analysis : component matrix obtained using the extraction method . 
cronbachs coefficient is based on the correlation between the items in the scale and may be interpreted as the percentage of variance accounted for by the scale with respect to universal variance ; in other words , the correlation between the scale used and all possible other scales in the entire romain made up of the same number of items . 
the value found indicates that the jss is highly reliable for evaluating satisfaction . job satisfaction responses to the last item on the jss indicate that 49% of radiologists were satisfied to varying extents with their job . the proportion of satisfied radiotherapists was significantly higher ( 64% ) , as was that of infectious disease specialists ( 62% ) ( table 3 )  . 
analysis of responses to each item on the questionnaire better illustrates the differences among the groups of professionals ( table 4 )  . the differences between radiologists and radiotherapists in the mean scores for each item were statistically risultati validazione del questionario la scala di soddisfazione conserva , anche nella versione italiana , il carattere unitario della versione originale di warr et al . 
questa componente correlata a tutte le 17 domande , con il maggiore peso ( 0 , 810 ) attribuito allultima domanda , che appunto chiede di specificare la soddisfazione complessivamente tratta dal lavoro , tenendo conto di tutti i fattori indagati dalle domande precedenti . 
applicando nellanalisi fattoriale la rotazione obliqua varimax in modo da massimizzare la differenza tra le due componenti ( tabella 2 ) , si osserva che la prima componente riguarda soprattutto motivi di soddisfazione legati al soggetto e allambiente in cui lavora , mentre la seconda , correlata alle domande 9 , 10 , 11 , 12 e , in minor misura , 4 , 5 , 7 , 16 , si riferisce ai rapporti con la struttura in cui lavora . la consistenza interna del questionario jss , misurata mediante il coefficiente alfa di cronbach , molto elevata ( 0 , 9457 )  . 
il coefficiente di cronbach , basato sulla correlazione tra le domande , pu essere interpretato come la percentuale di varianza che la scala usata pu spiegare rispetto alla varianza universale , ovvero la correlazione tra la scala usata e tutte le possibili altre scale delluniverso composte da un analogo numero di domande . 
differences in satisfaction with physical working conditions , freedom to choose work method , lultima domanda del jss indica che il 49% dei radiologi soddisfatto , in misura maggiore o minore , del proprio lavoro ; tra i radioterapisti la percentuale di soddisfatti significativamente pi alta ( 64% ) e cos tra gli infettivologi ( 62% ) ( tabella 3 )  . 
se si analizzano le risposte fornite in corrispondenza di ciascuna delle domande del questionario , la differenza fra le categorie professionali viene meglio precisata ( tabella 4 )  . le differenze tra radiodiagnosti e radioterapisti nei punteggi medi riportati in ciascuna delle domande sono statisticamente significative ( p < 0 , 05 mediante il test t di student ) per quasi tutte le domande ; risultano sovrapponibili solo le risposte relative ai rapporti con colleghi e collaboratori , mentre approssimano la significativit senza raggiungerla le domande sulle possibilit di carriera e sulla variet del lavoro . 
risulta altamente significativa ( p < 0 , 01 ) la differenza di soddisfazione per le caratteristiche dellambiente di lavoro , per la discrezionalit nei metodi di lavoro , per le responsabilit affidate , per i rapporti con il direttore e con il management , per la stabilit del posto di lavoro . 
altamente significativa la differenza di punteggio per ( misurata dallultima domanda ) e per le scale di soddisfazione intrinseca , estrinseca e globale che si ottengono sommando i punteggi delle singole domande . 
 la soddisfazione globale nel confronto con gli infettivologi ospedalieri , i radiologi addetti alla diagnostica per immagini esprimono livelli sovrapponibili di soddisfazione ( o di insoddisfazione ) solo per quanto riguarda la libert di scegliere da s il metodo di lavoro , i riconoscimenti per il lavoro ben fatto , le possibilit di carriera . 
essi sono significativamente meno soddisfatti per tutte le altre variabili , e la differenza risulta altamente significativa ( p < 0 , 01 ) per lambiente di lavoro , le responsabilit , il guadagno economico , il modo in cui gestita lazienda , lattenzione rivolta ai propri suggerimenti , gli orari di lavoro , la variet dei compiti professionali e la stabilit del posto di lavoro , oltre che per la soddisfazione complessiva indicata dallultima domanda e per le scale combinate di soddisfazione intrinseca , estrinseca e totale . tra i medici radiologi e radioterapisti di genere femminile si osserva una maggiore prevalenza di insoddisfatti che tra i maschi ( tabella 5 ) , ma la differenza non raggiunge significativit statistica ( chi quadro = 0 , 073 )  . 
se per si effettua il confronto dei punteggi medi di ciascuna domanda si evidenziano alcune significative differenze , con le donne meno soddisfatte dei colleghi maschi per quanto riguarda i riconoscimenti per il lavoro compiuto ( p = 0 , 034 ) , la ripartizione dei carichi di lavoro ( p = 0 , 011 ) e la variet del lavoro ( p = 0 , 047 )  . 
likewise , the difference in scores for overall satisfaction ( measured by the last question ) and the scales of intrinsic , extrinsic and total satisfaction obtained by adding up the scores on the single items were also statistically significant . 
 compared with infectious disease specialists , diagnostic radiologists reported similar levels of satisfaction ( or dissatisfaction ) only as regards freedom to choose work method , recognition for good work and career prospects . 
 the prevalence of subjects satisfied with their job did not vary consistently across the various age groups ; the greatest frequency of dissatisfied professionals was recorded among pi basso che negli uomini ( p = 0 , 028 ) , anche se le risposte allultima domanda , sulla soddisfazione complessiva , non differiscono nei due sessi ( p = 0 , 110 )  . 
 la prevalenza di soggetti soddisfatti dal lavoro non varia nelle diverse classi di et con un andamento univoco ; la maggiore frequenza di soggetti insoddisfatti si registra nelle et di mezzo , tra 41 e 50 anni ( tabella 6 )  . 
appare invece chiara la maggiore soddisfazione del lavoro man mano che si procede dalle categorie di lavoro precario ai dirigenti medici , ai responsabili di struttura semplice , alla figure apicali ( tabella 7 )  . 
non si osserva alcuna differenza nei livelli medi di soddisfazione in funzione del tipo di struttura pubblica o privata in cui lattivit viene esercitata ( p = 0 , 427 )  . applicando il metodo della regressione logistica con selezione condizionale a ritroso ai dati relativi a tutti i medici intervistati ( tabella 8 ) si osserva che le variabili maggiormente associate con la soddisfazione sono : lambiente di lavoro , il superiore , lattenzione rivolta ai propri suggerimenti , la libert di scegliere da s il proprio metodo di lavoro , la variet del lavoro . 
il metodo di selezione stepwise finalizzato ad escludere le variabili meno significative , fino a determinare il set di variabili che pi efficacemente determinano leffetto finale , in questo caso la soddisfazione professionale . 
on the other hand , there was a clear trend towards greater satisfaction among physicians in the higher ranks of the hierarchy from temporary staff , to staff grade , to division heads and heads of department ( table 7 )  . 
numerosi studi hanno valutato i livelli di soddisfazione nei medici di medicina generale o di varie specialit e le relazioni con il contesto organizzativo , sociale e culturale della professione [ 16 ]  . 
the position held within the organisation was the last variable to be excluded by the statistical process and was thus the variable that correlated most closely among those excluded . risultati di tali studi non possono essere estrapolati direttamente alla radiologia , in quanto questa differisce sostanzialmente dalle altre specialit mediche , in primo luogo per le modalit di interazione con i pazienti , poi per il contesto organizzativo e relazionale , infine per la rapidit dellevoluzione tecnologica . 
 stata proprio lintroduzione di nuove tecnologie a motivare recentemente la pubblicazione di una serie di lavori , secondo i quali la modificazione dellambiente e dellorganizzazione di lavoro indurrebbero un aumento della soddisfazione professionale dei radiologi [ 1316 ]  . curiosamente , i livelli di soddisfazione dei radiologi non sono stati misurati a priori in nessuno di tali studi , ma sono stati indagati anamnesticamente solo dopo lintroduzione del miglioramento tecnologico . radiol med ( 2009 ) 114 : 13301344 1339 table 7 rate of professional satisfaction as a function of role ( excluding subject reporting uncertainty ) tabella 7 andamento della soddisfazione professionale in funzione del ruolo ( esclusi gli incerti )  . 
the results of these studies cannot be directly extrapolated to radiology , as the discipline differs sono pochi gli studi che affrontano specificamente il tema della soddisfazione professionale dei radiologi , e i risultati paiono talora contraddittori . 
nel regno unito unindagine trasversale condotta nei primi anni 90 su 214 radiologi e altri tre gruppi di consulenti ospedalieri , al fine di valutare lincidenza dello stress e dei disturbi ad esso correlato , ha stimato che il 27% dei consulenti presenta affezioni psichiatriche , senza significative differenze tra le varie specialit , ed ha dimostrato che la soddisfazione professionale ha un effetto protettivo sullo stress e 1340 radiol med ( 2009 ) 114 : 13301344 substantially from other medical specialties in terms of interaction with patients , organisational and relational context and the speed of technological evolution . 
 indeed , it was the introduction of new technologies that recently prompted the publication of a series of papers that maintained that the changes to the physical work setting and organisation would increase job satisfaction among radiologists [ 1316 ]  . 
oddly , the level of satisfaction of radiologists was not measured a priori in any of these studies , but only investigated historically after the introduction of the technological improvements . few studies have specifically addressed the issue of job satisfaction among radiologists , and the results appear somewhat conflicting . 
a cross - sectional survey conducted in the uk in the early 1990s to investigate stress and associated disturbances in 214 radiologists and three other groups of hospital consultants estimated that 27% of consultants had some psychiatric morbidity , without significant differences between the various specialist groups , and demonstrated that job satisfaction has some protective effect against stress and burnout [ 7 ]  . 
 the alarming tone of this study is apparently countered by the findings of a survey conducted in poland , where 80% of radiologists reported being satisfied with the clinical relevance of their specialty , meaningfulness of their job and appreciation shown by superiors and colleagues [ 18 ]  . however , the same 70 radiologists also reported suffering from work - related stress due to low salaries , haste , lack of clinical data about patients and fear of misdiagnoses , leading the authors to conclude that radiologists are at a high risk of burnout . 
the lack of homogeneity between the questionnaires , as well as methodology and objectives of the two studies , make it impossible to establish whether the inconsistent findings are the reflection of completely different situations or the result of different interpretations of a complex and multifaceted data set . 
 this difficulty in correctly interpreting results because of methodological differences is present in the series of north american papers by sunshines team [ 1923 ] who interpreted the data provided by the periodical stratified sample surveys conducted by the american college of radiology ( acr ) on a large number of specialists . 
these surveys , in fact , do not investigate professional satisfaction explicitly , so the authors have to estimate current satisfaction in two ways : ( a ) as a positive reply as to a question on whether sullesaurimento emozionale [ 7 ]  . 
il principale fattore di insoddisfazione per i radiologi risultato leccessivo carico di lavoro , seguito dalla carenza di personale e di fondi nel sistema sanitario , e dalle richieste incongrue poste dai clinici al radiologo . 
 al tono allarmante di questo studio si contrappone apparentemente il risultato dellindagine condotta in polonia , dove l80% dei radiologi si dichiara soddisfatto del lavoro , a causa del rilievo clinico della disciplina , della significativit del compito professionale , dellapprezzamento da parte di superiori e colleghi [ 18 ]  . 
da notare per che lo stesso gruppo di 70 radiologi polacchi segnala di subire stress professionale per le retribuzioni basse , i ritmi eccessivi , la mancanza di dati clinici relativi ai pazienti e la paura di denunce per gli errori diagnostici , per cui gli autori concludono che i radiologi sono ad elevato rischio di burn - out . 
la disomogeneit degli strumenti usati , della metodologia e delle finalit strategiche dei due lavori , rende impossibile stabilire se i quadri descritti corrispondano a situazioni diametralmente opposte o se derivino da differenti interpretazioni di un insieme complesso e variegato . 
 la difficolt di prescindere da vincoli metodologici che spesso limitano la possibilit di interpretare correttamente i risultati presente nella serie di pubblicazioni statunitensi del gruppo di sunshine [ 1923 ] che interpretano i dati forniti dalle periodiche indagini a campione multi - strato condotte dallamerican college of radiology ( acr ) su un consistente numero di specialisti . 
a prescindere da una valutazione del costrutto , che soprattutto per il secondo criterio questionabile ( si pu avere unottima opinione della disciplina e del lavoro in genere , pur avendo una pessima esperienza personale ) , si deve notare che entrambe le domande sono molto indirette , il che pone dubbi sulla loro sensibilit e specificit . 
di fatto , emerge dal confronto delle successive pubblicazioni che le percentuali di soggetti classificabili come soddisfatti professionalmente divergono sostanzialmente , a seconda che si adotti luno o laltro criterio . 
il primo criterio ha radiol med ( 2009 ) 114 : 13301344 1341 respondents would recommend a career in medicine to a college - age adult , combined with a positive reply as to whether they would recommend a specialisation in radiology ; ( b ) as a positive reply to the question bearing in mind all aspects of your work , what do you think of radiology as a profession ? apart from evaluating the validity of the construct , which is questionable especially with regard to the second criterion ( one may have an excellent opinion of the discipline and job in general but a very bad personal experience ) , it should be noted that both questions are extremely indirect , which raises doubts as to their sensitivity and specificity . 
using both criteria , the level of satisfaction of north american radiologists appears to be declining . a second measure was derived from the acr survey , called dynamic satisfaction and was based on a single question with five possible replies , which measured respondents level of satisfaction relative to the past ( compared to 5 years ago , what is your feeling about working as a radiologist ? )  . 
the studies conducted on the basis of the 1995 survey identified professional qualification and improved physical working conditions as causes of increased satisfaction , whereas the main source of decreased satisfaction , which mainly referred to men and the 4554 age group , was uncertainty due to reorganisation of the health system [ 21 , 22 ]  . 
the study based on the 2003 data , as well as confirming that satisfaction was increasing among women and young individuals and decreasing among men aged 4554 years , associated reduced satisfaction with excessive workloads [ 23 ]  . 
surprisingly , this last survey revealed no correlation between radiological subspecialty and level of satisfaction [ 23 ] , whereas previous surveys found satisfaction to be significantly higher among nuclear medicine physicians and radiation oncologists than among general diagnostic radiologists [ 20 ]  . as the authors admit , the question exploring dynamic satisfaction contains an evident recall bias , as it is imposto objectively evaluate ones current feelings sible compared to ones feelings five years ago . 
when reading the numerous studies published by north american authors one cannot but admire the expertise in statistical analysis , fornito tassi di soddisfazione che oscillano dal 42% della prima indagine del 1988 , al 65% del 1990 , al 51% del 1995 ; sulla base del secondo criterio sono stati descritti livelli di soddisfazione che vanno dal 97% dei primi sondaggi al 93% del 2003 [ 1923 ]  . 
 dallinchiesta dellacr stata ricavata una seconda grandezza , denominata soddisfazione dinamica , sulla base di una singola domanda con risposta prefissata in 5 punti , che pi propriamente misura il gradimento dellattivit di radiologo rispetto al passato ( compared to 5 years ago , what is your feeling about working as a radiologist ? )  . da questa variabile , ricavato il maggior numero di informazioni . 
gli studi condotti sulla base dellinchiesta del 95 identificano come cause di aumentata soddisfazione la qualificazione professionale e il miglioramento dellambiente di lavoro , mentre la ridotta soddisfazione , che riguarda soprattutto i maschi e la fascia di et tra 45 e 54 anni , sarebbe da ricondurre alle incertezze per la riorganizzazione del sistema sanitario [ 21 , 22 ]  . 
lo studio dei dati raccolti nel 2003 , oltre a confermare che la soddisfazione in aumento nelle donne e nei giovani , mentre in diminuzione per i maschi tra 45 e 54 anni , associa la riduzione della soddisfazione con leccessivo carico di lavoro [ 23 ]  . 
sorprendentemente , in questultima inchiesta non si rileva alcuna relazione tra la specialit radiologica praticata e il livello di soddisfazione [ 23 ] , mentre nelle analisi precedenti la soddisfazione negli specialisti in medicina nucleare e radioterapia era significativamente maggiore che nei radiodiagnosti generalisti [ 20 ]  . come riconoscono gli stessi ricercatori , la domanda su cui si basa la valutazione della soddisfazione dinamica negli studi statunitensi contiene un evidente recall bias , errore sistematico di richiamo , perch impossibile valutare oggettivamente le proprie sensazioni attuali a confronto con quelle che erano le proprie sensazioni cinque anni addietro . 
leggendo i numerosi studi pubblicati dagli autori statunitensi non si pu che rimanere ammirati dalla perizia di analisi statistica , mortificata purtroppo dallinconsistenza dei dati su cui poggia lanalisi . come riconoscono gli stessi autori , misurare il concetto di soddisfazione tratta dal lavoro intrinsecamente difficile , cos come lo cercare di stabilire un preciso nesso tra la soddisfazione da lavoro e levolversi del contesto professionale [ 22 ]  . 
le percezioni sono molto pi 1342 radiol med ( 2009 ) 114 : 13301344 which is , however , undermined by the quality of the data used for the analysis . as acknowledged by the authors , it is intrinsically difficult to measure the concept of job satisfaction , just as it is to try to establish a clear link between job satisfaction and a changing professional context [ 22 ]  . 
the fact that data are based on radiologists perceptions , memory of perceptions and willingness to report them is clearly a limitation . perceptions are far more variable than factual data , and they may change rapidly and be reported inconsistently by the same radiologist at two different times . 
the concept of satisfaction , derived from a single question , oversimplifies a very complex issue and excludes , in the north american surveys , other important factors such as pay , family environment and relationships with fellow workers [ 22 ]  . professional satisfaction has been investigated by clinical psychologists since the 1930 using different methods based on one question or an aggregate of questions . 
the many questionnaires proposed over the years tended to contain redundant and overlapping items , were often long and cumbersome and tended to confuse descriptive and evaluative judgements [ 10 ]  . 
 [ 10 ] designed the jss , which rapidly imposed itself as the most reliable instrument for measuring job satisfaction . our study demonstrates that a nonnegligible proportion of specialists in our sample was not satisfied with their job : one radiologist in four and one radiotherapist in five were professionally dissatisfied . 
less than half of those who worked in diagnostic imaging were satisfied with their job . this finding , which is important in itself , is even more striking if we consider that professional satisfaction has a strong negative correlation with occupational stress and a positive correlation with psychological well - being and healthy life style [ 8 ]  . the least satisfied professionals were the general diagnostic radiologists , especially if they were on the lower rungs of the hierarchical ladder , middle aged or women . even with the difficulties in comparing our data with the situation in north america because of methodological differences , italian radiologists appear to have lower levels of professional satisfaction compared with their north american colleagues . 
several factors relating to work organisation ; quality of interpersonal relations with peers , superiors and subordinates ; material and especially immaterial rewards for work done ; and fairness in workload distribution all affect the level of job satisfaction of italian radiologists significantly and in many cases negatively . 
if the differences in satisfaction among the various age groups and hierarchical levels can be ascribed to career progression and its consequences ; the differences between the sexes are explained by persisting discrimination towards female radiologists , who feel they receive less recognition for their variabili dei dati fattuali , possono cambiare rapidamente nel tempo ed essere riportate in modo molto diverso dallo stesso radiologo in due momenti diversi . 
il concetto di soddisfazione , ricavato da una sola domanda , semplifica eccessivamente un concetto molto complesso escludendo , nelle indagini americane , altri importanti concetti come la retribuzione , linterazione con il contesto familiare , le relazioni con i colleghi [ 22 ]  . la soddisfazione professionale stata indagata dagli psicologi clinici fin dagli anni 30 con diversi metodi , basati su una singola domanda , ma pi spesso su un aggregato di pi domande . 
i numerosi questionari proposti negli anni tendevano a contenere domande ridondanti e parzialmente sovrapponibili , erano spesso lunghi e complessi , tendevano a confondere giudizi descrittivi e valutativi [ 10 ] ; per ovviare a tali carenze il gruppo di warr et al . 
ha elaborato il jss , che si rapidamente imposto come lo strumento pi affidabile per misurare la soddisfazione da lavoro . la nostra indagine dimostra che una quota non trascurabile dei medici intervistati non soddisfatta del proprio lavoro : un radiodiagnosta su quattro e un radioterapista su cinque sono professionalmente insoddisfatti . 
questo risultato , di per s non trascurabile , assume maggiore rilievo se si considera che la soddisfazione professionale risulta significativamente associata , in senso inverso , con lo stress da lavoro , e si correla positivamente al livello di benessere psicologico e ad uno stile di vita sano [ 8 ]  . allinterno della categoria , i pi insoddisfatti risultano coloro che si dedicano alla diagnostica per immagini , soprattutto se occupano un livello basso nella scala gerarchica , sono di mezza et e di sesso femminile . 
pur considerando la difficolt di confrontare i dati da noi raccolti con la situazione statunitense , per le evidenti differenze metodologiche , sembra che i radiologi italiani presentino un livello di soddisfazione professionale inferiore rispetto ai colleghi doltre oceano . 
numerosi fattori legati allorganizzazione del lavoro , alla qualit dei rapporti interpersonali in senso orizzontale e verticale , alle ricompense materiali e soprattutto immateriali per il lavoro compiuto , allequit organizzativa del lavoro , influenzano significativamente , e spesso in senso negativo , il livello di soddisfazione professionale dei radiologi italiani . 
se le differenze nei livelli di soddisfazione tra le varie classi di et e tra diversi livelli gerarchici sembrano da attribuire in primo luogo allevoluzione della carriera e a tutte le sue conseguenze , le differenze tra i due generi sono spiegabili dal persistere di condizioni discriminatorie per le dottoresse , che sentono di ottenere meno riconoscimenti per il lavoro compiuto e di subire una ripartizione ingiusta dei carichi di lavoro , con una maggior quota di lavori ripetitivi . i fattori che maggiormente risultano capaci di determiradiol med ( 2009 ) 114 : 13301344 1343 work and suffer an unfair distribution of workloads , with a greater share of repetitive work . the factors that most affect the professional satisfaction of physicians are in part related to the physical working conditions and in part to human aspects , such as relationship with ones superior , freedom to choose ones work method , attention to ones suggestions and amount of job variety . our analysis , conducted with a sensitive instrument ( the jss ) , confirmed the role of organisational factors and work climate as the main determinants of professional satisfaction , along with improvements in physical working conditions , machines and diagnostic and care facilities . 
even with the difficulties in comparing our findings with the north american studies , it is interesting to note that environmental and organisational factors are closely related to professional satisfaction in the north american surveys , as well . our data inevitably suffer from a lack of sampling , which makes it impossible to extrapolate results to all italian radiologists . 
the interest of the findings of this initial analysis and the samples willingness to participate in the survey lead us to believe that extension of the study to the whole professional category would be well received and would provide highly relevant data for planning the development of the profession . 
increasing the data set of information would enable us to identify corrective actions capable of improving the professional satisfaction of radiologists . nare la soddisfazione professionale nei medici sono in parte collegati allambiente di lavoro fisico , in parte invece derivano da aspetti umani , come il rapporto con il superiore , la libert di scegliere da s il proprio metodo di lavoro , laccoglienza dei suggerimenti avanzati , la variet dei compiti . 
la nostra analisi , condotta con uno strumento sensibile come il jss , conferma il ruolo dei fattori organizzativi e del clima lavorativo come principali determinanti della soddisfazione professionale , accanto al miglioramento dellambiente fisico , delle macchine e delle strutture diagnostiche e terapeutiche . 
pur ribadendo la difficolt di effettuare confronti tra le nostre osservazioni e le ricerche statunitensi , interessante rilevare che una stretta relazione tra fattori ambientali e organizzativi e la soddisfazione professionale emerge anche dalle indagini americane . i dati da noi raccolti soffrono inevitabilmente della mancanza di un campionamento , il che impedisce di estrapolare i risultati alluniverso dei radiologi italiani . 
linteresse dei dati emersi da questa prima analisi e la disponibilit manifestata dai partecipanti allindagine ci inducono a ritenere che una estensione della ricerca a tutta la categoria sarebbe bene accetta e fornirebbe dati di sicuro interesse ai fini della programmazione dellevoluzione della professione . 
guglielmi2 , 3 1sezione dipartimentale di medicina legale , universit degli studi di foggia , ospedale colonnello davanzo , via degli aviatori 1 , 71100 foggia , italy 2dipartimento di diagnostica per immagini , universit degli studi di foggia , azienda ospedaliero - universitaria ospedali riuniti , viale luigi pinto 1 , 71100 foggia , italy 3dipartimento di radiologia , ospedale irccs casa sollievo della sofferenza , viale cappuccini 1 , san giovanni rotondo , 71013 foggia , italy correspondence to : g . 
mri , in addition , is also well suited to the examination of surviving victims of assault , especially choking , and helps visualise internal injuries sometimes not seen on external examination of the victivarious postprocessing techniques can provide strong forensic evidence for use in legal proceedings . 
the documentation and analysis of postmortem findings with msct and mri and postprocessing techniques ( virtopsy ) is investigator independent , objective and noninvasive and will lead to qualitative improvements in forensic pathologic investigation . 
apart from the accuracy and three dimensionality that conventional documentations lack , these techniques allow for the re - examination of the corpse and the crime scene even decades later , after burial of the corpse and liberation of the crime scene . 
 keywords virtopsy postmortem imaging multislice computed tomography ( msct ) magnetic resonance imaging ( mri ) noninvasive autopsy forensic radiology digital autopsy riassunto la tomografia computerizzata multistrato ( tcms ) e la risonanza magnetica ( rm ) sono metodiche dimaging sempre pi richieste in patologia forense . 
tali tecniche possono essere di grande ausilio alle classiche autopsie medico - legali . la visualizzazione degli organi interni del cadavere pu essere eseguita preventivamente utilizzando la tcms e / o la rm . 
la rm , in aggiunta , indicata anche per la valutazione delle vittime superstiti di azioni violente , in particolare lo strangolamento , in quanto aiuta ad individuare lesioni interne talvolta non apprezzabili neanche ad unattenta osservazione esterna della vittima . 
la documentazione e lanalisi dei reperti post - mortem individuati mediante tcms e rm come pure alcune tecniche di post - elaborazione ( virtopsy ) sono esecutore - indipendenti , oggettive , non invasive e porteranno nel tempo a miglioramenti qualitativi nelle indagini patologiche forensi . 
a parte laccuratezza e la tridimensionalit di cui mancano le documentazioni convenzionali , queste tecniche consentono di riesaminare sia il cadavere che il luogo del delitto anche decenni pi tardi , dopo la sepoltura della salma e lalterazione del luogo del reato . 
noi crediamo che questo approccio virtuale , noninvasivo o minimamente invasivo permetter di migliorare la medicina forense nel prossimo futuro . parole chiave virtopsy post - mortem imaging tomografia computerizzata multistrato ( tcms ) risonanza magnetica ( rm ) autopsia non - invasiva radiologia forense autopsia digitale 1368 introduction how relevant it is to deal with modern postmortem techniques in medicine is witnessed by the need for a modern and shared reflection about the contribution of radiologic science in necroscopic practice for forensic purposes . the progress and evolution of radiological sciences towards an increasingly improved definition of technical parameters enabling study of the details of the human body and its components , both in an anatomical - structural and a physiologicalpathophysiological sense , has brought closer two disciplines that only appear to be so different . 
in fact , both disciplines are based on the study and interpretation of anatomical findings , with radiology aiming to provide rapid responses with implications , forensic medicine to shed light on legal issues and both being carried out for research purposes . 
the methodological and operational approach between the two disciplines is recent , dating back to the early uses of conventional radiology in the study and detection of foreign bodies retained in the corpse . therapeutic - operational in the 1970s , the american college of pathologists reported on the importance of the correct use of preventive radiography in some cases of death and , at the time of its introduction , carried out studies on the use of ultrasoundguided techniques of anatomical sampling [ 1 ]  . 
in 1984 , di maio in italy published a paper instructing on the correct use of radiography in the study of firearm deaths , which immediately assumed the value of a protocol for the discipline . 
over the past 20 years , radiologic examinations have become increasingly available and accessible in both practical and economic terms , leading to a closer collaboration between the two disciplines , with a move from a needed sensitivity to performing radiographic examination in special cases to the almost routine and objective experience of the postmortem radiologic investigation , universally named virtopsy [ 36 ]  . the term virtopsy derives from the terms virtual and autopsy . 
merged the terms virtual and autopsy deleting autos to create the term virtopsy [ 7 ]  . virtopsy basically consists of documenting and analysing a body volume using multislice computed tomography ( msct ) , magnetic resonance imaging ( mri ) and 3d surface reconstructions by processing raw data sets radiol med ( 2009 ) 114 : 13671382 introduzione quanto attuale possa essere il discutere di moderne tecniche autoptiche allinterno del sistema medicina , testimoniato dalla necessit di unodierna , condivisa riflessione , sullapporto della scienza eidologica nella pratica autoptica a fini forensi . il progredire e levolversi delle scienze radiologiche , verso la sempre maggiore definizione di parametri tecnici atti allo studio dei dettagli del corpo umano e delle sue componenti , tanto in senso anatomo - strutturale , quanto in senso fisiologico / fisiopatologico hanno , forse come non mai , avvicinato due discipline solo apparentemente cos diverse . 
entrambe le discipline , infatti , a ben riflettere , basano il loro humus sullo studio e linterpretazione dei reperti anatomici , volti luna , la radiologia , allesigenza di una sempre pi rapida risposta dai risvolti terapeuticointerventistici , laltra , la medicina legale , allacclaramento dei fatti di giustizia , oltre che , naturalmente , per entrambe , ai fini di ricerca . 
laccostamento metodologico operativo tra le due discipline , invero , ha origini assai recenti , che risalgono ai primi impieghi della radiologia tradizionale per lo studio e la repertazione di corpi estranei ritenuti nellorganismo . gi negli anni 70 , negli stati uniti , lamerican college of pathologists segnalava limportanza del corretto utilizzo dellindagine radiografica preventiva in alcuni casi mortali , cos come , coeva alla sua introduzione , portava avanti studi sullutilizzo delle tecniche di prelievo anatomico eco - guidate [ 1 ]  . 
in italia , nel 1984 , di maio pubblicava un decalogo del corretto impiego della radiografia nello studio delle morti da armi da fuoco che , da subito , assumeva senso e valore protocollare per la disciplina . 
si riteneva cio indispensabile , per un corretto esperimento settorio nei casi di morti da armi da fuoco , la preventiva individuazione dei proiettili ritenuti integri o in frammenti cos come indispensabile era la loro consequenziale rimozione e repertazione [ 2 ]  . 
da allora passato oltre un ventennio , nel quale la sempre maggiore disponibilit degli esami eidologici , vieppi in termini di accessibilit , non solo di fruibilit pratica , ma anche economica , hanno , negli ultimi anni , posto in collaborazione le due discipline affinando metodiche e facendo crescere esperienze . 
si passati dallauspicata sensibilit alleffettuazione dellesame radiografico in particolari casi , alla pressoch routinaria ed oggettiva esperienza della sofiindagine eidologica pre - autoptica denominata sticata universalmente virtopsy [ 36 ]  . 
il termine autopsia una combinazione dei vocaboli classici greci autos ( auto , propri ) e opsomei ( io vedr )  . cos , autopsia significa vedere con i propri occhi . 
 la virtopsy consiste fondamentalmente nella documentazione ed analisi di un volume corporeo mediante tomografia radiol med ( 2009 ) 114 : 13671382 1369 with volume - rendering ( vr ) tools at a workstation in such a way that a virtual autopsy can be performed at any time or place . 
in deceased persons , the main goals are to determine the cause and manner of death , to evaluate the vitality of the sustained injuries and to develop a forensic reconstruction based on the findings ( table 1 )  . although autopsies are still recognised as important medical table 1 main objectives of virtopsy identification gender , age , body length , organ weight estimation , individual features ( dental , intracorporeal ) documentation foreign material ( bullets , metal fragments , inserted foreign bodies ) cause and manner of death as the result of different manners : accident , suicide , homicide , natural or iatrogenic causes possible causes : polytrauma , drowning , shooting , burn injuries , hanging , etc . vitality of sustained injuries forensic reconstruction education research tabella 1 principali obiettivi della virtopsy identificazione sesso , et , lunghezza corporea , stima del peso degli organi , caratteristiche individuali ( dentarie , intracorporee ) documentazione corpi estranei ( proiettili , frammenti metallici , corpi estranei impiantati ) causa e modalit di decesso quale esito di differenti modalit : incidente , suicidio , omicidio , cause naturali o iatrogene possibili cause : politrauma , annegamento , lesioni da arma da fuoco , ustioni , impiccamento , ecc . ricerca di segni vitali a carico delle strutture anatomiche lesionate ricostruzione forense istruzione ricerca computerizzata multistrato ( tcms ) , risonanza magnetica ( rm ) e ricostruzioni 3d di superficie , elaborando il set dei dati acquisiti con strumenti di volume rendering ( vr ) mediante una workstation dedicata , permettendo quindi di eseguire unautopsia virtuale in qualsiasi momento e in qualsiasi luogo , il tutto non alterando alcun reperto utile ai fini medico - legali a differenza di quanto avviene con le tecniche invasive utilizzate nelle autopsie tradizionali [ 8 ]  . 
 i principali obiettivi della medicina forense sono documentare , analizzare e chiarire le prove medico - scientifiche , sia che si tratti di soggetti viventi che di persone decedute , presentandole in un modo facilmente comprensibile in unaula giudiziaria . 
in persone decedute , gli obiettivi principali sono determinare la causa e le modalit di morte , valutare la presenza di segni vitali a carico delle strutture anatomiche lesionate e sviluppare una ricostruzione forense sulla base dei diversi reperti ( tabella 1 )  . 
tale fase comporta la scansione dellintero cadavere , al fine di acquisire mediante tcms o rm un insieme di dati da rielaborare poi con tecniche 3d volume - rendering [ 9 ]  . 
diversamente dalla genetica forense ( in cui viene utilizzato il dna ) e dalla tossicologia forense , la documentazione dei reperti patologici utili ai fini medico - legali ancora basata prevalentemente sulluso degli stessi protocolli e tecniche autoptiche utilizzate da secoli . 
lebraismo ortodosso , per esempio , vieta la manomissione dei corpi esanimi , tranne quando tali azioni possono salvare la vita ad altri o in situazioni di emergenza per la rimozione di organi ai fini di donazione . 
 pertanto da considerare che lautopsia convenzionale , al giorno doggi spesso respinta da parte di familiari o non tollerata per tali motivi religiosi nella nostra societ multietnica , potrebbe essere in parte sostituita da una documentazione non - invasiva di immagini e , se necessario , da tecniche di campionamento tissutale mini - invasive imagingguidate e dallangiografia per la valutazione delle alterazioni vascolari . 
i dati acquisiti in maniera digitale potrebbero invece essere consultati ogni qual volta emergono nuove problematiche medico - legali o essere inviati ad altri esperti esterni per un secondo parere . 
this involves cadaver scanning to acquire msct or mri data sets and the use of 3d direct volume - rendering techniques [ 9 ]  . autopsies are known for their importance in establishing the cause of death . 
other than in forensic genetics ( in which dna is used ) and forensic toxicology , the documentation of forensic pathologic findings is still predominantly based on the same autopsy techniques and protocols that have been used for centuries . 
religious prohibitions are one reason for this trend . orthodox judaism , for example , prohibits disturbing dead bodies except when such action may save others and urges avoidance of organ removal . 
followers of islam are opposed to any desecration or exposure of the body of a deceased believer . conventional autopsy , nowadays often refused by the family or not tolerated for religious reasons in our multiethnic societies , may thus be replaced by noninvasive imaging and , when required , by minimally invasive imaging - guided tissue sampling and by angiography to address vascular questions . 
in addition , autopsy specimens such as tissue sections are difficult to store indefinitely . digitally acquired data could instead be consulted whenever new issues arise , or they could be sent to other experts for a second opinion . 
several studies have shown that virtual autopsy holds great potential to aid forensic investigations [ 5 , 7 , 10 , 11 ]  . virtual autopsies have several other advantages over traditional methods of investigation . 
assessing the entire bone structure or searching for air , for example , is otherwise difficult and time - consuming . virtual autopsies also have a number of disadvantages . postmortem imaging with msct and mri provides no colour documentation of the body . 
mri , on the other hand , is better suited for the lautopsia virtuale ha la grande potenzialit di supportare le indagini forensi [ 5 , 7 , 10 , 11 ]  . 
infatti , la tcms ha dimostrato di essere molto utile nella repertazione di alterazioni ossee , dei corpi estranei , di embolia gassosa , e di grossolane alterazioni dei tessuti molli [ 7 , 12 ]  . 
la visualizzazione di strutture vascolari e la possibile emorragia allo stesso modo difficile da rilevare , ma risultati promettenti sono stati raggiunti dallangiografia post - mortem [ 13 , 14 ]  . 
 tecniche di imaging sia la tcms che la rm possono essere utilizzate per uno studio di imaging post - morte necessario illustrare agli specialisti del settore le principali applicazioni di ogni metodica come pure i loro limiti per trarre i maggiori vantaggi sfruttando le capacit insite in ciascuna tecnica . 
 gli strumenti a disposizione per facilitare la visualizzazione dei reperti dimaging post - mortem comprendono software che rendono pi facile e veloce la valutazione e linterpretazione di un grande volume di dati . 
msct has shown to be very useful in detecting bone lesions , foreign bodies , air embolisms and gross abnormalities of the soft tissue [ 7 , 12 ]  . 
visualising the circulation and possible bleeding is also difficult , but promising results have been achieved with postmortem angiography [ 13 , 14 ]  . postmortem information on imaging provides no histology and chemistry . 
this can be solved to a certain extent with msct - guided biopsies or mr spectroscopy . postmortal gas can be difficult to distinguish from bowel gas or gas in wound channels . 
it is thus important to perform postmortal imaging as soon as possible after death . imaging techniques both msct and mri can be used for postmortem imaging . practitioners need to appreciate the principal applications of each technique as well as its limitations to make the most of each modalitys capabilities . 
it is relatively easy to visualise bone , gas and metal with msct , for instance , but discrimination of soft tissue is limited . tools available to facilitate the visual presentation of postmortem imaging data include software packages that make it easier and quicker to view and interpret largevolume data sets . 
it is theoretically possible to reconstruct an infinite number of images from data acquired for a typical whole - body msct virtual autopsy , leading to data sets several gigabytes in size . 
this technique makes it possible to obtain not only tissue specimens but also samples of urine , bile or blood for toxicological or dna rielaborate dovrebbero fornire agli esaminatori una buona comprensione della anatomia 3d post - mortem in questione come pure orientare sulla probabile causa di morte . una peculiare caratteristica dellautopsia virtuale rappresentata dalla stragrande quantit di dati ottenuti . 
 teoricamente possibile ricostruire un infinito numero di immagini a partire dai dati acquisiti per una autopsia virtuale whole - body mediante tcms , comportando quindi pacchetti di dati delle dimensioni di diversi gigabyte . 
la tecnica di scansione della superficie corporea mediante scanner con ottica di superficie 3d fotogrammetrica pu documentare e confrontare gli oggetti e le ferite sul corpo . inoltre possibile utilizzare uno scanner di superficie dotato di un proiettore a fasci di luce e due fotocamere combinate che misurano la deformazione di tali fasci in relazione alla superficie corporea . 
successivamente il software in grado di elaborare un modello 3d basato sulla deformazione di tali strie luminose [ 1517 ]  . stata inoltre perfezionata per scopi forensi la tecnica mini - invasiva di biopsia imaging - guidata ( per esempio sotto guida tc ) su cadaveri per consentire il prelievo di campioni tissutali per lanalisi istologica . 
in tal modo possibile , in casi selezionati , eseguire una completa indagine autoptica in maniera mini - invasiva , specie ove sia impraticabile per qualsivoglia motivo la tradizionale autopsia . uno dei problemi cruciali in medicina - legale la valutazione del tempo trascorso dal momento del decesso . 
la spettroscopia mediante rm in grado di fornire una stima di tale tempo partendo da informazioni metaboliche ottenute dallanalisi di predefinite regioni encefaliche [ 18 ]  . la possibilit di ricavare multiple informazioni mediante tutte queste metodiche consente quindi lesecuzione in ultima analisi di una autopsia completa , minimamente invasiva , dalla cui fusione dei dati raggiunti possibile ottenere uno strumento importante da utilizzare nelle aule giudiziarie del futuro . principali applicazioni lautopsia virtuale in grado di fornire informazioni in merito alle possibili cause di morte . politrauma le fratture possono essere pi facilmente individuate utilizzando le ricostruzioni 3d . 
thus , a complete , minimally invasive autopsy examination is possible in selected cases in which traditional autopsy is impracticable for whatever reason . assessing the time since death is a fundamental problem . mr spectroscopy can provide an estimation of this time from metabolic information collected in a predefined region of the brain [ 18 ]  . 
data fusion may also be an important tool in the courtroom of tomorrow . main applications polytrauma virtual autopsy can provide information for many possible causes of death . fractures can be visualised using 3d reconstruction . 
the direction of the force causing bone fracture can be assessed by analysing the fracture systethus , the base of a wedge - shaped fracture piece indicates the direction of the force . 
in injuries to the head , determining whether the injury was caused by a fall to the ground or a blow to the head is an important forensic assessment when the deceased person was initially found lying injured on the ground . 
this determination can be made on the basis of typical skull fracture systems , the morphologic features of the crush wound and contrecoup lesions within the brain [ 20 ]  . as it does not use ionising radiation , and as it is well suited for the depiction of soft - tissue pathology , mri is also regularly utilised within the project in the assessment of surviving victims of blunt trauma , especially of manual strangulation [ 21 ]  . 
the problem in such cases is that the superficial findings may be innocuous , but the underlying soft tissuesespecially around the reflectogenic structures of the throatmay be severely traumatised , thus leading to a potentially life - threatening situation . identification prior to any postmortem investigation , the identity of the body must be clarified and proved ; otherwise , the postmortem investigation has as one of its goals the re - estabtura . 
in caso di lesioni alla testa , determinare se il danno stato causato da una caduta al suolo o da un colpo alla testa un importante elemento di valutazione forense specie quando la persona deceduta viene inizialmente ritrovata gi ferita per terra . 
questa valutazione pu essere effettuata sulla base del tipico aspetto di frattura delle ossa craniche , sulle caratteristiche morfologiche della ferita da impatto e in base alle lesioni da contraccolpo rinvenute allinterno del cranio [ 20 ]  . dal momento che non utilizza radiazioni ionizzanti e poich risulta essere particolarmente adatta alla valutazione della patologia dei tessuti molli , la rm regolarmente utilizzata anche nellambito del protocollo di valutazione delle vittime superstiti di traumi contusivi , in particolare di strangolamento manuale [ 21 ]  . 
il problema in questi casi che i reperti superficiali a volte possono anche essere di lieve entit e quindi non facilmente apprezzabili , mentre i tessuti molli sottostanti , specialmente quelli che circondano le strutture laringee , possono essere gravemente traumatizzati , determinando in tal modo una situazione di potenziale pericolo di vita . identificazione prima di qualsiasi tipo di indagine post - mortem , deve essere chiarita e dimostrata lidentit del cadavere ; se ci non possibile , uno dei principali obiettivi dellindagine post - mortem risulta essere la ricerca dellidentit del corpo non identificato . 
poich lesame del dna la tecnica pi costosa e che richiede maggior tempo , si utilizzano pi comunemente a tale scopo il rilevamento delle impronte digitali e lidentificazione e dello status dentario . 
i dati ottenuti dalla tcms di un cranio di un cadavere , possono essere utilizzati per ricostruire qualsivoglia proiezione radiografica utile al confronto con lo status ante - mortem [ 23 ]  . 
inoltre , il materiale utilizzato in trattamenti ortodontici pu essere accertato sulla base di dati tc postmortem e confrontato con i reperti dentari ante - mortem nel caso si sospetti di persone scomparse [ 25 ]  . 
in aggiunta a grossolani reperti morfologici come endoprotesi di spalla , anca , ginocchio o segni di percutaneo - vertebroplastica , reperti peraltro individuabili sulla base di un efficiente isperadiol med ( 2009 ) 114 : 13671382 1373 lishment of the identity of the unidentified body . 
the human or animal remains of past cultures can be investigated without disturbing them ( as would traditional autopsy ) , thereby providing information regarding the age and gender of , as well as any injuries or diseases suffered by , the deceased animal or person , as recently performed on the newly discovered egyptian mummies [ 2830 ]  . drowning traditional autopsies provide no unambiguous indications of drowning . 
drowning has especially been related to young adults who are under the influence of alcohol and other drugs whilst being near water [ 34 , 35 ]  . diagnosis relies primarily on the subjects individual characteristics and circumstances , macroand micropathological postmortem changes and studies of the lungs , zione esterna , una tcms whole - body di un cadavere rivela ulteriori reperti utilizzabili positivamente per lidentificazione , nonch per lesclusione di una presunta identit , conclusioni in alcuni casi non raggiungibili mediante lautopsia di routine come nel caso , ad esempio , di un cadavere completamente bruciato [ 26 , 27 ]  . unaltra disciplina in cui lodierno imaging di inestimabile valore paleoradiologia . 
i resti umani o animali , appartenenti ad epoche passate , possono essere studiati senza alterare minimamente il loro stato ( come avverrebbe invece con lautopsia tradizionale ) , mettendo cos a disposizione degli esperti informazioni riguardanti let e il sesso , cos come qualsiasi lesione o malattia subita dal defunto animale o persona , come ultimamente effettuato sulle mummie egiziane recentemente scoperte [ 2830 ]  . annegamento le autopsie tradizionali non forniscono indicazioni inequilorganizzazione vocabili di annegamento . 
attualmente lannegamento si riscontra soprattutto nei giovani adulti che abusano di alcol e altre droghe [ 34 , 35 ]  . la diagnosi si basa principalmente sulle caratteristiche individuali del soggetto e sulle circostanze , sulle alterazioni post - mortem macroe micro - patologiche , e sugli studi dei polmoni , tra cui il volume polmonare . 
gli specialisti del settore possono studiare il volume polmonare post - mortem , lattenuazione media , le differenze di attenuazione antero - posteriore , il profilo di densit polmonare , e lammontare di acqua presente nei polmoni . 
 [ 36 ] , analizzando i reperti tcms post - mortem preautoptici di dieci casi di annegamento e confrontandoli con 20 casi di non - annegamento sottoposti ugualmente ad autopsia e tcms post - mortem , avevano riscontrato la presenza di fluido nelle vie aeree in tutti i casi di annegamento . 
tutti questi reperti , tipici dellannegamento , ad eccezione degli spots di paltau ( depositi pleurici di emoglobina da emolisi ) , sono stati rilevati utilizzando la tcms post - mortem , ed stata trovata una buona correlazione tra la tcms e lautopsia tradizionale . 
 [ 36 ] , analysing the postmortem preautopsy msct findings of ten drowning cases in comparison with autopsy and postmortem msct of 20 nondrowning cases observed that fluid in the airways was present in all drowning cases . 
all the typical findings of drowning , except paltaus spots ( pleural haemoglobin deposits from haemolysis ) , were detected using postmortem msct , and a good correlation between msct and autopsy was found . 
knowing the bullets location before commencing physical autopsy will save the pathologist time , especially if the bullet has actually left the body . bullets frequently lodge far from their entry points , particularly if they are deflected by curved bones such as the ribs or skull . postmortem radiology serves to locate the projectile , entry and exit wounds , depict the bullet course and may help identify the ammunition and the weapon type utilised [ 37 ]  . 
to assess the direction from which gunshot wounds were created , different characteristics are visualizzazione diretta del broncospasmo , dellemodiluizione e dellacqua in tutti i seni paranasali , reperto piuttosto complicato o impossibile da rilevare con la classica autopsia . 
la conoscenza dellesatta posizione di un proiettile allinterno del corpo prima dellinizio di una autopsia tradizionale consentir al medico - legale di risparmiare il tempo necessario allindagine autoptica , specialmente se il proiettile ha in realt abbandonato il corpo . 
i proiettili infatti sono spesso rinvenuti anche in sedi distanti dai loro punti di ingresso , particolarmente se deviati da ossa curve come le coste o il cranio . la radiologia post - mortem utile quindi a individuare il proiettile , i fori dingresso e di uscita , indica il percorso compiuto dal proiettile , e pu aiutare a identificare le munizioni e il tipo di arma utilizzata [ 37 ]  . 
anche la precisa valutazione del percorso compiuto dal proiettile attraverso il corpo di estrema importanza , specie poich aiuta a ricostruire il luogo del delitto , la posizione della vittima e dellomicida [ 38 ]  . 
inoltre , la regola di puppe pu contribuire a valutare lordine cronologico in cui si sono formate le fratture , in quanto , secondo tale regola , le fratture pi recenti si interromperanno in corrispondenza di quelle precedentemente formate [ 39 ]  . 
 [ 40 ] dallanalisi forense di 57 casi mediante tecniche di neuroimaging , basate sulluso della tcms e della rm e dal confronto dei risultati ottenuti con i reperti autoptici , hanno osservato che abrasioni , contusioni e lacerazioni del cuoio capelluto sono state rilevate dalla rm piuttosto che dalla tc ; tuttavia , e non sorprendentemente , per quanto riguarda tutti i tipi di lesioni al cuoio capelluto , lautopsia era di gran lunga superiore alle tecniche di imaging . 
la tc e la rm sono state quasi equivalenti nella valutazione delle emorragie extra - assiali che , nel complesso , hanno trovato una soddisfacente correlazione con lautopsia . nei casi in cui la tc e la rm non hanno identificato radiol med ( 2009 ) 114 : 13671382 1375 fig . 
1 examination of a gunshot victim by msct : msct , 2d and 3d reconstructions of the body of a homicide victim . forensic radiology can be of great help in detecting entrance and exit wounds , bullet and bullet fragments within the body , bullet track and inflicted injuries , as well as in identifying the ammunition and the weapon type utilised . 
la radiologia forense pu essere di grande aiuto nellindividuazione del foro di ingresso e di uscita , del proiettile , dei suoi frammenti , del suo percorso e delle lesioni arrecate allinterno del corpo , oltre ad identificare il tipo di munizione e di arma utilizzata . 
 [ 40 ] , analysing 57 forensic cases by neuroimaging techniques such as msct and mri and comparing the results to the autopsy findings , observed that abrasions , contusions and lacerations of the scalp were detected by mri rather than ct . 
ct and mri were the evaluation of extra - axial almost equivalent emorragie subdurali , i reperti autoptici indicavano un sanguinamento con spessore inferiore a 3 mil 53% delle contusioni a carico della sostanza grigia sono state osservate mediante tc , di cui la rm ne ha rilevato circa il 41% ; le tecniche radiologiche sono state sufficienti per la valutazione di reperti definiti come lesioni da colpo o da contraccolpo allautopsia . 
la rm e la tcms si sono dimostrate superiori allautopsia in merito alla valutazione dellemorragia ventricolare , nonch dello pneumocefalo che non era stato osservato allindagine autoptica , anche se il cranio era stato parzialmente distrutto . 
la rm stata superiore alla tc nellindividuazione delle lesioni del parenchima cerebrale ( ad esempio nel mostrare chiaramente il percorso compiuto dal proiettile ) e a carico del troncoencefalo . lesioni da ustioni lautopsia virtuale pu apportare un contributo significativo in diversi casi che coinvolgono corpi gravemente bruciati , fornendo informazioni su eventuali fratture , distribuzione di gas , ed altri reperti . 
2 examination of a gunshot victim by msct : msct , 2d and 3d reconstructions of the skull of a gunshot victiknowing the bullets location in gunshot injuries before starting physical autopsy will save the pathologist time . 
in this case , postmortem radiology serves to locate the projectile and entry and exit wounds , depict the bullet course and may help in identifying the ammunition and the weapon type utilised . 
tcms e ricostruzioni 2d e 3d del cranio di una vittima darma da fuoco . la conoscenza preliminare dellesatta posizione della pallottola nelle lesioni da arma da fuoco ridurr al medico - legale il tempo necessario per lindagine autoptica . 
in questo caso la radiologia forense serve a individuare il proiettile , il foro di ingresso e di uscita , identificare il percorso compiuto dal proiettile agevolando lindividuazione del tipo di munizione e di arma adoperata . 
questo facilita il recupero del proiettile e di frammenti potenzialmente importanti nel rivelare informazioni circa langolazione e la direzione seguita dal proiettile stesso . hemorrhage that overall was found in a satisfactory correlation with the autopsy . 
mri and msct were superior to autopsy concerning evaluatius of ventricular hemorrhage , as well as pneumencephalus that had not been noted in the autopsy protocols , even if the head had been partially destroyed . 
quindi , il heat epidural , spesso riscontrato in cadaveri carbonizzati , non rappresenta una lesione antemortem e non dovrebbe essere erroneamente interpretata come tale [ 41 ]  . impiccamento per escludere un simulato suicidio in caso di impiccamento messo in atto per mascherare un qualche tipo di omicidio , i medici legali ricercano sulla vittima segni di reazioni vitali . le ricostruzioni 3d dei dati acquisiti con la tcms possono radiol med ( 2009 ) 114 : 13671382 burn injuries virtual autopsy can make a significant contribution to several cases involving severely burned bodies , providing information on fractures , gas distribution and other areas . 
thus , heat epidural is often seen in burned corpses but does not indicate an antemortem injury and should not be misinterpreted as such [ 41 ]  . hanging to exclude simulated suicidal hanging covering some kind of homicide , forensics experts search for vital reactions . three dimensional reconstruction of msct data can verify vital signs that are caused by the hanging procedure to exclude postmortem simulated hanging . 
bleeding into the insertions of the sternocleidomastoid muscle or softtissue structures of the neck prove that circulation was ongoing at the onset of strangulation , and strong breathing attempts against the occluded airways cause alveolar ruptures with subsequent pneumomediastinum and softtissue emphysema ascending into the neck [ 43 ]  . vitality of sustained injuries cutaneous vital reactions elucidate the sequence of injury and death in forensic pathologic investigations . 
the answer is provided by forensic findings that occur only with intact circulation ( e.g. fatal haemorrhage , air and fat embolism ) , respiration ( e.g. aspiration , emphysema ) , metabolism or consciousness . 
 cardiac applications the leading cause of natural death is cardiac insufficiency . postmortem imaging must be able to separate these natural deaths from unnatural deaths because the heart is often the target of injury in suicides as well as homicides . 
 1377 verificare la presenza di segni vitali causati dalla procedura di impiccamento per escludere che si tratti di una simulazione post - mortele immagini 3d possono inoltre visualizzare i segni della legatura aiutando a ricostruire il processo di strangolamento [ 19 , 21 , 42 ]  . 
il sanguinamento in corrispondenza delle inserzioni dei muscoli sternocleidomastoidei o a carico dei tessuti molli del collo dimostrano che la vittima era in vita al momento dello strangolamento , come pure i tentativi di respirazione forzata , per vincere locclusione delle vie aeree , possono causare rotture alveolari con conseguente pneumomediastino ed enfisema dei tessuti molli con carattere ascendente nel collo [ 43 ]  . 
la risposta fornita dal riscontro di reperti medico - legali identificabili solo quando la circolazione sanguigna ancora presente ( ad esempio , emorragia mortale , embolia adiposa e gassosa ) , in presenza di respirazione ( ad esempio , aspirazione , enfisema sottocutaneo ) , con metabolismo attivo e in stato di coscienza . 
la presenza di materiale aspirato , sia esso sangue , contenuto gastrico , o fuliggine , dimostra una ventilazione ancora in corso dopo il trauma , cos come un diffuso enfisema dei tessuti molli dopo un trauma contusivo . 
anche se lenfisema sottocutaneo apprezzabile alla palpazione dei tessuti molli , difficilmente tale reperto resta permanentemente tale allindagine autoptica in quanto laria tende a scomparire al momento dellincisione della cute sovrastante . 
limaging post - mortem deve essere in grado di discriminare questi decessi naturali dalle morti innaturale in quanto il cuore rappresenta il principale obiettivo da lesionare in suicidi e omicidi . 
acute bleeds tend to have higher hounsfield unit values than older bleeds on msct [ 4447 ]  . forensic outlook today we could translate virtopsy with digital autopsy so as to fit the expression to the method . 
indeed , a careful review of scientific papers published to date clearly shows that digital autopsy is particularly well suited to the basic objective of forensic medicine , that is , the investigation of scientific evidence . however , before moving towards the future , we should consider the present situation : the autopsy . 
judicial autopsy , far from exhausting the investigations on the corpse , is just one step in the process : not the first , much less the last [ 52 , 53 ]  . autopsy , as external inspection of the corpse and internal examination , is just one necessary step in the approach to the corpse . 
in the range of such ancillary examinations to the main investigation ( autopsy ) , the radiologic - forensic investigation rises to a new dimension and becomes rightfully placed among the instruments enabling the search for scientific ( validated and repeatable ) evidence . 
 awareness of the need for a guarantee of validity ( the best in a technical sense ) and reliance on increasingly objective technicalscientific aids has permeated the coroners causato da lesioni del miocardio o dei vasi epicardici pu essere documentato sia con la tcms che mediante rm . alla tcms le emorragie acute tendono a presentare pi alti valori densitometrici , in termini di unit hounsfield , rispetto ai sanguinamenti cronici [ 4447 ]  . 
invero , a ben scandagliare il contenuto della produzione scientifica sin qui prodotta , ben si comprende come , a fronte delleccezionale lavoro esperito , in realt lautopsia digitale ben si incardina su un concetto basilare della medicina legale quale quello della ricerca di evidenze scientifiche quale mezzi di prova . in realt , ancor prima di fare un passo verso il futuro , appare opportuno interrogarsi sul suo presente : the autopsy , do we still need it ? piace riportare il quesito in inglese poich esso parafrasa per intero il titolo di un editoriale edito nel 1970 sulla prestigiosa rivista chest [ 48 ]  . 
ed invero , ad una scorsa della letteratura nazionale ed internazionale pi recente , la ovvia risposta che lautopsia non solo necessaria ma da dire assolutamente irrinunciabile [ 4951 ]  . 
ed infatti , come abbiamo gi avuto modo di esprimere , lautopsia giudiziaria , lungi dallesaurire gli accertamenti sul cadavere , non che un momento di essi : non il primo , n tanto meno lultimo [ 52 , 53 ]  . lautopsia intesa come esame esterno cadaverico ed esperimento settorio , solo uno ( ineluttabile ) dei momenti di approccio al cadavere , forse neanche il pi importante , ma certamente propedeutico al corretto prelievo , campionamento e studio istologico di ogni singolo organo o frammento di esso . 
lindagine tossicologica , genetico - forense , e cos via , sino ad arrivare ai pi specialistici esami di metabolomica e / o proteonomica costituiscono oggi il corretto corollario ad una moderna indagine autoptica . 
nellambito di tali innesti ancillari alla indagine principe ( la autopsia ) , assurge a nuova dimensione lindagine eidologico - forense in grado di essere annoverata a pieno titolo tra i mezzi di ricerca di evidenze scientifiche ( come tali validate e ripetibili )  . 
 la consapevolezza della necessit di una forte garanzia di validit ( la migliore possibile in senso tecnico ) , della riferibilit a supporti tecnico - scientifici sempre pi oggettivi , ha permeato negli ultimi anni la sensibilit del medicolegale [ 54 ]  . 
ben si comprende , allora , come e perch alcuni autori siano giunti ad auspicare lo studio tc del cadavere nello specifico settore delle morti da arma da fuoco , alla stregua di una autopsia virtuale ( virtopsy ) da configurarsi come uno strumento observer - indipendent , che non altera la realt del corpo e poich digitalizzato ed archiviato , anche a distanza di tempo , sempre ripetibile ed oggettivabile [ 5 , 5557 ]  . indubbiamente , lo studio tridimensionale del corpo umano permette una esatta localizzazione dei corpi estranei di natura metallica eventualmente ritenuti ed offre un radiol med ( 2009 ) 114 : 13671382 1379 sensitivity in recent years [ 54 ]  . 
it is not surprising , then , that some authors advocated ct study of corpse in firearms deaths to be used as a virtual autopsy ( virtopsy ) and thus an observer - independent instrument that does not alter the corpse and that is repeatable and objectifiable , even over time [ 5 , 5557 ]  . undoubtedly , the 3d study of the human body allows correct localisation of any metallic foreign bodies and helps greatly in identifying the exact intracorporeal channels with a precision very similar to that of autopsy studies : very similar , but not replaceable , as findings without the physical detection and accurate description of gross features such as margins , rims and halos can be appreciated , even in intracorporeal parenchymas , and are indispensable for the correct description of the death event ( not merely the cause , but also with information regarding the manner and time )  . such a diagnostic aid is clearly of great benefit to the practice of forensic medicine : the possibility of new evaluations , subsequent verification and multidisciplinary consultation in complex investigations , even after the end of the classic postmortem procedures , is a positive opportunity . 
 digital autopsy can disclose findings that might escape even the most careful observation : while a minute bone splinter can easily escape an accurate direct inspection , it will not escape an aseptic database that will return this piece of information at a later evaluation allowing , for instance , confirmation of the differential diagnosis between the entry and the exit wounds . 
however , at this particular stage of the cultural debate , the above must lead to the correct interpretation and methodologicaloperational collocation of digital autopsy as an asset to two disciplines that depend on each other for the purposes of reaching specific diagnostic conclusions . 
 to conclude , it should be noted that although crucial to the autopsic investigation as a whole , these advanced studies will remain ancillary and supplemental to the classic postmortem investigation , without replacing it . 
hence the need to use semantic caution and steer the meaning of virtopsy toward the notion of digital autopsy that better reflects the nature of the technique and the intrinsic meaning of an ancillary method rather than a possible replacement , contributo eccellente nella esatta individuazione di tramiti intracorporei , con una precisione che raggiunge livelli di sovrapponibilit allosservazione settoria . 
sovrapponibile , ma non per questo sostituibile , poich reperti privi della materiale individuazione e dellesatta descrizione di caratteristiche macroscopiche , quali margini , orletti ed aloni , invero sono ben apprezzabili anche nei parenchimi intracorporei ed indispensabili per il corretto inquadramento dellevento morte ( inteso non solo come momento causativo ma corredato di informazioni preziose relative al modus ed al tempo della morte )  . evidente che lavere a disposizione un simile supporto diagnostico nella pratica di medicina forense in occasione delle ricognizioni autoptiche sia di straordinario vantaggio : la possibilit di procedere a nuove valutazioni , a riscontri successivi nel tempo , a consulti multidisciplinari per indagini complesse , anche dopo che si sono concluse le procedure autoptiche classiche , rappresenta una vantaggiosa opportunit . lautopsia digitale in grado di fornire rilievi che possono sfuggire anche alla pi attenta e sperimentata osservazione medico - legale : una minuscola scheggia ossea , ad esempio , pu assai facilmente sfuggire allosservazione diretta , pur accuratissima , ma non ad un asettico data - base che restituir questa informazione ad una valutazione successiva consentendo , ad esempio , di arricchire di ulteriori conferme la diagnosi differenziale tra fori di ingresso e fori di uscita . 
tutto questo , per , proprio in questa attuale fase del vivo dibattito culturale ci deve condurre alla corretta interpretazione e collocazione metodologico - operativa dellautopsia digitale quale patrimonio di due discipline non autonome luna dallaltra ai fini delle specifiche conclusioni diagnostiche , ma interdisciplinari . 
 peraltro ci sentiamo , in conclusione , di ammonire come tali indagini siano sovente di fondamentale importanza nellambito dellindagine autoptica intesa in maniera omnicomprensiva , ma con intendimento che tali sofisticate metodiche siano di accompagnamento ed approfondimento , ma non certo sostitutive , neppure parzialmente , dellindagine autoptica classicamente intesa . 
da qui la necessit di un pi prudente viraggio semantico dalla virtopsy al concetto di autopsia digitale pi rispondente tanto ad un corretto inquadramento della tecnica quanto allintrinseco significato di un affiancamento e non mai dellavvicendamento , che porti quindi non ad una virtopsy , ma semmai verso una virtuous autopsy o autopsia virtuosa . 1380 radiol med ( 2009 ) 114 : 13671382 leading not to a virtopsy but , if anything , to a virtuous autopsy . conclusioni conclusions in some countries , virtual autopsy is now used routinely for forensic work . 
findings identified during physical autopsy often raise additional questions that can be answered by virtual autopsy . the pathologist can also re - examine the cadaver at any point during the investigation and search for additional information . 
 the applications of msct technology in the forensic domain suggest that a mobile scanner could be used for postmortem data acquisition in cases of mass casualties such as airplane crashes or natural disasters . 
future applications of this approach include assessment of morbidity and mortality in the general population and , perhaps , routine screening of bodies prior to burial . several forces must work in unison if a new era of digital autopsies is to emerge . 
however , digital autopsy is and will remain merely an aid in the practice of forensic medicine and one that is not always available and that cannot be considered an alternative to conventional postmortem procedures . in alcuni paesi lautopsia virtuale attualmente utilizzata di routine per scopi forensi . 
i reperti identificati allindagine autoptica talvolta sollevano ulteriori problematiche cui lautopsia virtuale in alcuni casi in grado di rispondere . pertanto lo specialista del settore pu anche riesaminare virtualmente il cadavere in qualsiasi momento durante linchiesta giudiziaria alla ricerca di ulteriori utili informazioni . 
nuove scoperte emerse dalle indagini compiute sul luogo del reato possono comportare il riesame di altre ipotesi mediante imaging . lacquisizione delle immagini con gli attuali scanner tcms e la successiva sessione di elaborazione richiedono peraltro molto meno tempo rispetto a quello necessario per eseguire un esame autoptico completo . 
lindagine virtuale pu anche ridurre il tempo necessario per lautopsia tradizionale essendo il medico - legale preventivamente informato dei reperti pi significativi da valutare . le applicazioni della tecnologia tcms in ambito forense hanno suggerito luso di apparecchiature mobili per lacquisizione di informazioni nel post - mortem in caso di incidenti di massa , come incidenti aerei o calamit naturali . 
future applicazioni di questo approccio comprendono la valutazione della morbilit e della mortalit nella popolazione generale e , forse , lo screening di routine dei corpi prima della loro sepoltura . 
 diversi organi competenti devono lavorare allunisono , se una nuova era delle autopsie digitali dovr emergere . medici specializzati e autorit giudiziarie devono stabilire protocolli standard per la scansione e la memorizzazione dei dati . 
i radiologi avranno anche bisogno di una formazione specifica nel settore dellimaging post - mortem in quanto limaging del cadavere si presenta spesso in maniera molto diversa da quanto routinariamente osservato in pazienti viventi . 
non si pu , tuttavia , nascondere che quello dellautopsia virtuale , e rimane , soltanto un supporto alla pratica di medicina legale , non sempre disponibile e che , non pu certamente , costituirsi come alternativa alle procedure autoptiche usuali . acknowledgments the authors thank giampaolo grilli ( dipartimento di radiologia ospedaliera , azienda ospedalierouniversitaria di foggia ) for his contribute in the aquisition and post - elaboration of the images . ringraziamenti gli autori ringraziano il dott . 
the solution to this dilemma is particularly complex in an environment in which the tendency to sue physicians for civil liability or incriminate them for criminal liability appears to be particularly high . 
there does not appear to be any evidence to suggest disclosure modifies the probability of litigation against the physician . keywords ethics medical error disclosure malpractice apology riassunto nella pratica radiologica , come in qualsiasi attivit medica , lerrore inevitabile , anche se prevedibile e prevenibile . 
la soluzione di tale dilemma particolarmente complessa in un ambiente nel quale la tendenza a citare il medico per responsabilit civile o a incriminarlo penalmente appare oggettivamente elevata . la rivelazione dellerrore certamente utile ad aumentare la consapevolezza del paziente , ridurne le sofferenze , migliorare la qualit dellassistenza e limitare le conseguenze del danno . 
non vi evidenza che tale atto modifichi la probabilit che il medico sia sottoposto ad una azione giudiziaria . parole chiave etica errore medico rivelazione dellerrore mala pratica richiesta di scuse medical error lerrore medico a few years ago under the title to err is human , a book by the united states institute of medicine [ 1 ] dramatically drew attention to the issue of medical errors . 
figures published in the report that stated that between 44 , 000 and 98 , 000 patients die in us hospitals each year due to medical errors were greeted with alarm by the media : medical errors were said to be responsible for twice the number of deaths as the vietnam war or equivalent to a 9 / 11 every day . 
 pochi anni fa , sotto il titolo errare umano un saggio dellistituto di medicina statunitense ( iom ) [ 1 ] ha posto drammaticamente lattenzione sul tema degli errori medici . le cifre del rapporto , secondo il quale negli ospedali statunitensi morirebbero ogni anno da 44000 a 98000 pazienti a seguito di errori medici , sono state riprese con toni allarmanti dalla stampa : gli errori medici causerebbero ogni anno negli stati uniti il doppio di morti della guerra del 1346 radiol med ( 2009 ) 114 : 13451355 the tone was no different in the scientific press , according to which medical errors are the fifth cause of death in the us and cost some us $29 billion each year [ 2 ]  . 
it was rightly noted that if these statements were correct , the us healthcare system was a threat to public health of pandemic proportion [ 3 ]  . the estimate of errors and adverse events is generally based on the review of medical charts or analysis of litigation claims , error reporting systems or other methods of investigation , none of which is without limitations or pitfalls [ 4 ]  . 
a literature review taking into consideration this and seven other studies conducted in the united states , canada , the united kingdom , australia and new zealand estimated that adverse events during hospitalisation occur in one in every six patients [ 6 ]  . 
in italy there are no reliable statistics available , and estimates oscillate between 15 , 000 and 50 , 000 deaths per year as a result of intrahospital errors and adverse events . 
the figure of 30 , 00035 , 000 preventable deaths reported by many press agencies corresponds to 5.5% of total deaths , more than the number of deaths on the road or due to heart attack or many cancers . 
the associated costs are said to be in the order of 10 billion euro , or 1% of the gross domestic product . although the interpretation of the statistics by the media is undoubtedly distorted , the institute of medicine report gives voice to a real problethe deaths occur above all in the terminal phase of critical patients . 
the evaluation conducted a priori , which leads to the extrapolation of the number of preventable deaths , is fraught with limitations , is particularly influenced by the operator who performs it and is unable to make any inference regarding the possible survival that would have occurred had the error not been made [ 3 ]  . 
the phenomenon therefore tends to be overestimated , because the evaluation fails to take into consideration the risk of death in the absence of medical error [ 7 ]  . one study that considered the prognosis of patients who were the victims of medical error came to the conclusion that only 0.5% would have enjoyed at least 3 months of life if the care had been optimal [ 3 ]  . 
nonetheless , it is clear that there is a broad margin for the improvement of medical procedures . even irrespective of the drama of the preventible loss of human life , it should be borne in mind that a medical error can have significant psychological repercussions for both the family of the victim and the physician responsible for the error . 
il tono non diverso nei commenti della stampa scientifica , secondo la quale gli errori medici rappresentano per la popolazione degli usa la quinta causa di morte , e costano 29 miliardi di dollari lanno [ 2 ]  . 
 stato giustamente osservato che se queste affermazioni fossero corrette , il sistema sanitario rappresenterebbe una minaccia per la salute pubblica di proporzioni epidemiche [ 3 ]  . le stime degli errori e degli eventi avversi sono generalmente basate su metodi di revisione delle cartelle cliniche , o su analisi delle denunce , dei sistemi di segnalazione degli errori o su altri metodi di indagine , nessuno dei quali esente da limiti e punti deboli [ 4 ]  . 
una delle indagini pi accurate , lo harvard medical practice study , ha rilevato che eventi avversi occorrono nel 3 , 7% dei ricoveri ospedalieri , e che in pi di un quarto di questi casi ( quindi , nell1% di tutti i ricoveri ) rilevabile negligenza [ 5 ]  . 
una revisione della letteratura , che tiene conto di questo e di altri sette studi condotti in usa , canada , regno unito , australia e nuova zelanda , stima che gli eventi avversi durante lospedalizzazione riguardino quasi un paziente su dieci [ 6 ]  . 
la cifra di 3035 mila morti evitabili , che riportata da molte agenzie giornalistiche , corrisponde al 5 , 5% dei decessi totali , pi degli incidenti stradali , dellinfarto e di molti tumori . 
la valutazione che porta a determinare il numero di morti evitabili , condotta a posteriori presenta numerose limitazioni , risentendo spiccatamente delloperatore che la effettua , e non potendo inferire in alcun modo sulleventuale sopravvivenza che ci sarebbe stata se lerrore non si fosse verificato [ 3 ]  . 
uno studio che tiene conto della prognosi dei pazienti vittime di errori medici giunto alla conclusione che solo lo 0 , 5% di essi avrebbe goduto di almeno tre mesi di vita se le cure fossero state ottimali [ 3 ]  . tuttavia evidente che esiste un largo margine di miglioramento nelle procedure mediche . anche se si volesse prescindere dal dramma della perdita evitabile di una vita umana , occorre tenere presente che lerrore medico pu avere gravi conseguenze psicologiche sia per i familiari della vittima che per lo stesso medico che ne autore . indeed , once the patients family members have been victims of and witness to a fatal error , they may feel a real sense of guilt for not having safeguarded the health of their infatti , una volta che i familiari dei pazienti siano stati vittime e testimoni di un errore fatale , possono sentire un vivo senso di colpa per non avere vigilato sufficientemente radiol med ( 2009 ) 114 : 13451355 1347 loved one sufficiently well or for not having obtained the best available care or the most reliable physician . 
this conflict can increase the sense of isolation and abandonment prompted by the loss of their family member [ 8 ]  . at the same time , physicians , too , may feel a sense of guilt resulting from the error and the fear of suffering professional and economic consequences and of being isolated by their own colleagues and clients [ 8 ]  . 
added to this is the consideration that medical professionals who have committed a severe error are open to a reduction in quality of life and an increase in the frequency of burnout . 
perceived stress is associated with an increase in the number of errors committed in the subsequent period , thus creating a vicious circle whereby errors lead to stress and stress leads to new errors [ 9 ]  . in a pilot study conducted among a group of italian radiologists and radiation therapists , we observed that those who had been subject to litigation for presumed malpractice display a high rate ( > 60% of cases ) of reactions of anxiety and anger , and in one third of cases , even feelings of helplessness , disappointment , anguish and humiliation . 
one in 20 radiologists have reported physical disturbances following a malpractice suit , and almost 40% have admitted to having changed their own practice to adopt strategies of defensive radiology [ 10 ]  . error in radiology medical errors in radiology can be mainly classified in two categories : procedural errors , which arise from incorrectly performing radiological procedures such as the injection of iodinated contrast medium , the barium enema and , more generally , interventional radiology procedures ( biopsy , angioplasty , stenting , vascular embolisation , drainage , ablation , etc . ) , and diagnostic errors . 
procedural errors , which in italy are at the basis of 10%15% of malpractice suits against radiologists [ 11 ] , have the same clinical and medicolegal characteristics as those that occur in surgical specialisations and therefore have no specific features . 
in contrast , diagnostic errors , which are at the basis of 60% of claims against radiologists [ 11 ] , have a profoundly different nature in radiology from that in other medical and surgical specialisations . diagnostic imaging differs from clinical diagnostics in that in the latter there is direct contact between physician and patient . 
in radiology , instead , due to historical and practical reasons that are still prevalent in italy , the patient does not self - refer to the radiologist but is referred by a clinician . sulla salute del proprio caro e per non aver procurato le cure migliori o il medico pi affidabile . 
 di contro , anche i medici possono avvertire il senso di colpa per lerrore , la paura di subirne le conseguenze professionali ed economiche e di essere isolati dai propri colleghi e clienti [ 8 ]  . 
il distress percepito associato con laumento della commissione di errori nel periodo seguente : si crea cos un circolo vizioso , per cui gli errori portano a stress e questo a nuovi errori [ 9 ]  . in uno studio - pilota condotto su un gruppo di radiologi e radioterapisti italiani abbiamo osservato che coloro che hanno subito una denuncia per presunta malpractice manifestano con elevata frequenza ( oltre il 60% dei casi ) reazioni di ansia e rabbia e , in circa un terzo dei casi , anche sensazione di essere indifeso , e sensazioni di delusione , angoscia e umiliazione . 
un radiologo su venti ha riferito di avere riportato in seguito alla denuncia danni fisici obiettivabili , e quasi il 40% ha ammesso di avere cambiato la propria condotta professionale , per adottare strategie di radiologia difensiva [ 10 ]  . lerrore in radiologia gli errori medici in radiologia appartengono principalmente a due categorie : gli errori procedurali , che derivano dallo svolgimento non corretto di tecniche radiologiche , come liniezione di mezzo di contrasto iodato , il clisma opaco , e pi in generale tutte le tecniche proprie della radiologia interventistica ( biopsia , angioplastica , stenting , embolizzazione vascolare , drenaggio , ablazione ecc . ) , e gli errori diagnostici . 
gli errori procedurali , che danno origine nel nostro paese al 10%15% delle richieste di risarcimento contro i radiologi [ 11 ] , hanno le stesse caratteristiche cliniche e medico - legali di quelli che occorrono nelle specialit chirurgiche e non presentano quindi particolari specificit . 
al contrario gli errori diagnostici , che sono allorigine di oltre il 60% delle denunce contro i radiologi [ 11 ] , riconoscono in radiologia un profilo profondamente diverso da quello di altre discipline mediche e chirurgiche . la diagnostica per immagini differisce dalla diagnostica clinica per il fatto che in questultima c un contatto diretto tra medico e paziente , mentre in radiologia , per ragioni 1348 radiol med ( 2009 ) 114 : 13451355 communication with the referring clinician and the patient are two critical and distinguishing features of the radiologist that we will discuss below . the work of the radiologist consists of two distinct but integrated phases : the complete identification of all abnormalities in the radiological image and their correct interpretation . 
the inability to detect an abnormality in a radiological examination , are the leading cause of litigation against radiologists , particularly in the case of breast [ 12 ] or chest cancer [ 11 ]  . 
they appear to be significantly correlated with a number of psychophysiological factors , such as the level of alertness of the radiologists , which in turn depends on physical and mental fatigue and therefore on work - related stress , and on their state of visual tiredness , which depends on ergonomic conditions of the work environment , the duration of the visual task , and therefore on the pauses and workload . 
such a complex pattern explains why this type of error cannot be completely eliminated , even though specific techniques have been developed and taught that make up good radiological practices and are aimed at reducing the rate of perception errors [ 13 ]  . the reading of radiographs by a second radiologist would undoubtedly reduce ( although not completely eliminate ) the number of perception errors . 
however , this practice is possible only in some cases where a second radiologist is available , and it is in fact limited to cases of doubtful diagnosis and not adopted routinely for the reading of images except in some screening programmes . perception errors are committed even by expert radiologists operating in ideal conditions . 
the frequency and inevitability of perception errors and the availability to the patient of his or her radiological images explain why radiologists are more exposed to medicolegal risk than are other medical specialties . 
whereas in the traditional working model storiche e pratiche tuttora prevalenti nel nostro paese , il paziente non si presenta direttamente al radiologo , ma viene inviato da un clinico . 
la comunicazione con il clinico curante e quella con il paziente sono due aspetti critici peculiari della radiologia , dei quali ci occuperemo in seguito . il lavoro del radiologo consiste di due momenti separati , ancorch fra loro integrati : lidentificazione completa di tutte le anomalie dellimmagine radiologica e la loro corretta interpretazione . 
il secondo momento , lerrore di interpretazione , si pu ritenere analogo a quello di altre branche della medicina , nelle quali possibile interpretare in modo errato un rilievo obiettivo , ad esempio un soffio cardiaco o un altro segno clinico . lerrore percettivo , cio lincapacit a rilevare una anomalia in un esame radiografico , la principale causa di contenzioso contro i radiologi , soprattutto nel caso del tumore della mammella [ 12 ] o del torace [ 11 ]  . 
esso appare significativamente correlato con numerosi fattori psicofisiologici , come il livello di allerta del radiologo , che a sua volta dipende dalla fatica fisica e mentale e quindi dallo stress da lavoro , e il suo stato di affaticamento visivo , che dipende dalle condizioni ergonomiche dellambiente di lavoro , dalla durata dellimpegno visivo , e quindi dalle pause e dal carico di lavoro . 
un quadro cos complesso rende ragione del perch non sia possibile eliminare del tutto questo tipo di errore , nonostante siano state messe a punto ed insegnate tecniche specifiche , che rientrano nelle buone pratiche radiologiche , finalizzate proprio a ridurre il tasso di errori percettivi [ 13 ]  . la lettura dei radiogrammi da parte di un secondo radiologo potrebbe indubbiamente ridurre ( non eliminare in assoluto ) gli errori percettivi , ma tale pratica , possibile solo nei casi in cui vi sia la disponibilit di un secondo radiologo , di fatto limitata ai casi di dubbio diagnostico e non adottata routinariamente per la lettura delle immagini se non in alcuni programmi di screening . gli errori percettivi sono commessi anche da radiologi esperti , che operano in condizioni ideali . 
la frequenza e inevitabilit degli errori percettivi , e la disponibilit piena da parte del paziente della documentazione radiologica che lo riguarda , rendono ragione del fatto radiol med ( 2009 ) 114 : 13451355 1349 the presence of the prescribing physician is an implicit guarantee against the failure to transmit , or delayed transmission of , the report by the radiologist , this cannot be said to be the case in screening programmes . 
it therefore comes as no surprise that in the united states there has been a significant rise in the number of malpractice suits related to delayed or incomplete communication of diagnostic findings by the radiologist . 
in italy , communication problems are encountered in a part of criminal proceedings ( ex art . 589 or 590 of the penal code ) , which involve the radiologist and other specialists and account for > 12% of malpractice litigation against radiologists [ 11 ]  . in the united states , in cases where a communication deficit is dependent on a radiologist , the courts have established damages on average twice that awarded in cases where only a diagnostic error was found [ 16 ]  . 
it has in fact been noted that the marked development of radiological techniques has not been matched by a similar improvement in reporting and communication [ 17 ]  . it has therefore been suggested that perhaps radiologists should communicate the findings of examinations directly to patients to at least partially avoid errors in communication [ 18 ]  . in the united states it has been estimated that 40% of radiologists are sued for malpractice on average once every 5 years [ 19 ]  . 
in that country , litigation against radiologists accounts for 10%15% of total malpractice claims , even though radiologists in the united states are less exposed to the risk of litigation than are surgeons , but they are much more exposed than internists and other specialists [ 20 ]  . 
the average compensation payouts agreed by the courts have tripled over the last 15 years , and approximately one third of cases conclude with conviction of the radiologist [ 15 ]  . in the same period , italy has gone from a situation of low - level medicolegal conflict [ 21 , 22 ] to one similar to that in the united states . 
with regard to practising radiologists , approximately half have received or will receive a claim for compensation or a summons related to their professional practice over the last 10 years . 
with regard to young radiologists who are beginning their career in italy , it can be stated with statistical certainty that each and every one will receive a claim for compensation at some point during their working life [ 11 ]  . the contrast is even more striking when compared with countries with a particularly low level of judicial conflict , such as those in northern europe . 
in the united kingdom che i radiologi siano esposti ad un rischio medico - legale pi elevato rispetto ad altre specialit mediche . un altro aspetto peculiare dellerrore in radiologia la comunicazione . 
se nel modello di lavoro tradizionale la presenza di un medico prescrittore rappresenta una implicita garanzia contro la mancata o ritardata trasmissione del referto da parte del radiologo , ci non pu ritenersi valido nel caso dei programmi di screening , nel quale i pazienti presumono di essere sani , ed i loro medici curanti , che si attendono un risultato negativo , non hanno alcun motivo di sollecitare una risposta . 
 negli usa nei casi in cui sia stato riscontrato un difetto di comunicazione a carico del radiologo le corti hanno stabilito un risarcimento di entit mediamente doppia rispetto ai casi in cui fosse stato accertato solo un errore diagnostico [ 16 ]  . 
ci si interroga pertanto se non sia il caso che i radiologi comunichino direttamente ai pazienti i risultati degli esami , cos da evitare almeno in parte gli errori di comunicazione [ 18 ]  . negli stati uniti si stima che il 40% dei radiologi sia citato in giudizio mediamente una volta ogni 5 anni [ 19 ]  . sempre negli usa le denunce contro i radiologi rappresentano il 10%15% del totale delle denunce contro i medici per imperizia o negligenza anche se , in quel paese , i radiologi risultano nel complesso meno esposti al rischio di citazioni rispetto ai chirurghi , ma molto pi esposti dei medici internisti e specialisti [ 20 ]  . 
per i radiologi gi in attivit si pu dire che , mediamente , circa la met di essi ha ricevuto o ricever una richiesta di risarcimento o una citazione per la propria attivit professionale degli ultimi dieci anni . 
per i giovani radiologi che iniziano oggi la professione in italia , si pu dire che ciascuno di essi ha la certezza statistica di ricevere almeno una richiesta di risarcimento nel corso delle propria vita professionale [ 11 ]  . 1350 radiol med ( 2009 ) 114 : 13451355 over the past two centuries , from 1795 to today , only 85 physicians have been taken to court with the charge of manslaughter , and only 22 of those have been convicted ; none of them were radiologists [ 23 ]  . 
in italy , however , more than 100 radiologists have been charged with manslaughter in the last decade [ 11 ]  . in sweden , where the malpractice claims are dealt with through administrative channels and compensated by a national insurance system if an independent medical committee confirms the claim , litigation against physicians is decidedly low . 
this principle , which is included in the hippocratic oath , is older than radiology , but there is no doubt that it should also be applied to this branch of medicine . 
it places a number of specific obligations on both the physician who commits it and on their colleagues and the healthcare centre they work in . on an individual basis , the physician who commits an error has a responsibility towards the patient . 
if the error causes bodily harm , informing the patient of the error is a moral obligation , which in the united states is also a legal obligation [ 25 ]  . 
this act of conscience , this awareness of ones own error and the harm caused , leads to contrition and therefore repentance and the resolution not to repeat the error , together with satisfaction or reparation of the harm done . 
even though the expectation of the penitent is forgiveness , there is no guarantee that the mistreated patient is willing to forgive . the fact that the indications regarding disclosing medical error is based on religious values whereas we live in a secular society explains why they are only partially applied in medical practice . 
in gran bretagna negli ultimi due secoli , dal 1795 ad oggi , solo 85 medici hanno dovuto subire un processo per omicidio colposo , e soltanto 22 sono stati condannati ; nessuno di essi era radiologo [ 23 ]  . 
in italia , viceversa , oltre cento radiologi sono stati indiziati per omicidio colposo nellultima decade [ 11 ]  . in svezia , dove i reclami dei pazienti sono trattati per via amministrativa ed indennizzati da un sistema assicurativo nazionale se un collegio medico indipendente conferma la congruit della richiesta , la conflittualit legale per i medici decisamente ridotta . 
il tasso delle richieste di indennizzo per malpractice medica dopo un ricovero ospedaliero fermo da anni allo 0 , 2% dei ricoveri [ 24 ]  . responsabilit del medico e rivelazione degli errori i medici hanno sempre avuto il dovere di non causare alcun danno ai pazienti ( principio di non maleficenza )  . 
lerrore rappresenta una deviazione dallo standard della disciplina ; esso pone diversi e specifici obblighi sia al medico che lo commette , che ai suoi colleghi e allazienda sanitaria in cui operano . sul piano individuale il medico che ha commesso un errore responsabile verso il paziente . 
latto di coscienza , la consapevolezza del proprio errore e del danno causato determinano contrizione , pentimento , quindi il ravvedimento e il proponimento di non ripetere lerrore , uniti alla soddisfazione , riparazione del danno . 
anche se lattesa del penitente nel perdono , non vi alcuna garanzia che il paziente offeso sia disposto a perdonare . il fatto che le indicazioni sulla rivelazione degli errori medici siano basate sui valori religiosi , ma che la nostra sia una societ secolare , rende ragione del fatto che esse siano applicate solo parzialmente nella pratica medica . 
indagini condotte sui medici statunitensi indicano che il 95% di essi ha assistito ad almeno un errore ; oltre l80% ritiene che rivelare gli errori migliori la sicurezza dei futuri pazienti , ma la radiol med ( 2009 ) 114 : 13451355 1351 improves the safety of future patients , in 1991 , some 76% stated they failed to report a significant error . 
today , only one in 30 inform patients of serious errors , and one in five states that they have not reported errors they have committed [ 27 ]  . 
the opinions regarding appropriate behaviour in the event of error , the type of error that should be reported and the manner of doing so are radically different in patients and physicians [ 28 ]  . experience indicates that in some cases , the disclosure of an error by a physician can be used by the patient to launch litigation against the same physician [ 29 ]  . 
even though laws prompted by the intervention of senators hillary clinton in new york state and barack obama in illinois rule out the possibility of using as circumstantial evidence the apology of a physician to a patient , these laws do not rule out the possibility of the physicians being sentenced if they have admitted their own guilt . 
indeed , lawyers advise physicians never to apologise , or at least never to make a complete apology , but rather to display an attitude limited to expressing empathy and understanding without ever referring to ones own errors or those of other physicians and repeating the causal chain of events ( apologia or botched apology )  . 
an authentic apology requires recognition that a procedure has been violated , the consequent admission of blame for that violation , the expression of genuine remorse , regret for the harm caused , an explicit offer of reparation or the commitment to modify procedures such that the violation never occurs again [ 30 ]  . as can be easily imagined , patients have a rather different opinion from physicians and their legal representatives regarding the information that should be provided in the event of error . 
they expect complete admission of responsibility and are unwilling to accept partial admissions , even though in most cases they state that error disclosure by a physician would not deter them from seeking legal action [ 31 ]  . 
it has nonetheless been noted that the discovery of an error not disclosed by a radiologist or clinician tends to sharpen a patients determination to undertake legal proceedings and prompts the courts to set higher compensation than would be applied in the event the error was immediately disclosed [ 32 ]  . all the techniques of clinical risk management are based on the principle that the search for errors is made to identify the cause and not the perpetrator , and they guarantee that error reporting is anonymous . 
nonetheless , there is a fine line between analysis carried out for preventative purposes percentuale di quanti dichiarano di non avere riportato gli errori gravi commessi era del 76% nel 1991 . 
le opinioni sui comportamenti da tenere in caso di errore , su quali errori segnalare ed in che modo farlo , sono radicalmente diverse nei pazienti e nei medici [ 28 ]  . lesperienza indica che in alcuni casi lammissione di errore da parte del medico pu essere usata dal paziente per promuovere una azione giudiziaria contro il medico stesso [ 29 ]  . 
anche se , per intervento dei senatori hillary clinton nello stato di new york e barack obama nellillinois , sono state approvate leggi che escludono la possibilit di usare come prova indiziaria il fatto che il medico abbia offerto le proprie scuse al paziente , queste leggi non escludono che il medico possa essere condannato se nel farlo ha ammesso la propria colpa . 
di fatto , gli avvocati consigliano ai medici di non offrire mai le proprie scuse , o al massimo , di non fare una piena e completa offerta di scuse ( apology ) , ma una esposizione limitata ad esprimere empatia e comprensione , senza mai fare riferimento a errori propri o di altri medici e ribadendo la causalit degli accadimenti ( apology o botched apology )  . questa forma di comunicazione strategicamente diretta non a fornire informazioni , ma a giustificare la propria azione e neutralizzare potenziali effetti negativi [ 30 ]  . 
una autentica apology richiede invece un riconoscimento che una procedura stata violata , la conseguente ammissione di colpa per tale violazione , lespressione di un genuino rimorso , il dispiacere per il danno causato , una esplicita offerta di riparazione e limpegno a modificare le cose in modo che la violazione non debba pi accadere [ 30 ]  . i pazienti hanno , come facile prevedere , una opinione piuttosto diversa da quella dei medici e dei loro legali su quali debbano essere le informazioni fornite in caso di errore . 
essi pretendono una completa ammissione di responsabilit e non sono disposti ad accettare ammissioni parziali , anche se , nella maggioranza dei casi , dichiarano che la rivelazione di un errore da parte del medico non li distoglierebbe dallintraprendere una azione legale [ 31 ]  . 
 stato per osservato che la scoperta di errori non rivelati dal radiologo o dal medico rende i pazienti pi determinati a procedere giudizialmente e spinge le corti a fissare risarcimenti pi elevati di quelli che vengono determinati nei casi in cui vi sia stata una tempestiva rivelazione [ 32 ]  . anche se tutte le tecniche di clinical risk management partono dal principio che la ricerca degli errori si fa per identificare la causa e non il colpevole , e garantiscono lanonimato delle segnalazioni , la distinzione tra indagini a fini preventivi e a fini ispettivi molto sottile e pu essere 1352 radiol med ( 2009 ) 114 : 13451355 and investigative purposes , a line that can be easily overstepped . 
in the united states , consumer organisations demand that data regarding complications recorded in hospitals be available on line so that patients may make an informed decision [ 33 ]  . 
such a move would create the real possibility of triggering an uncontrollable spiral of alarm , with an increase in malpractice claims and therefore of insurance costs and defensive medical practices ( which already account for 8%10% of unjustified costs for various healthcare organisations )  . with regard to the issue of clinical risk , the attitude of healthcare organisations in italy and the english - speaking world is markedly different . 
in english - speaking countries , the principle has always been in force that the healthcare system is accountable in the event of errors committed by healthcare personnel when the latter are unable to demonstrate that they acted specifically to prevent the errors . roman law , in contrast , rules out criminal liability of the system ( societas delinquere non potest ) , and in this respect , art . 
this orientation has been a real hindrance to the principle of social accountability of the healthcare system and in the specific sense has led to an underestimation of the problem and a general omission of prevention measures , unjustly considered the exclusive responsibility of individuals . 
only recently has legislative decree 231 / 01 introduced into the italian judicial system the principle of institutional liability for administrative offences on the basis of failure to act and negligence in the management of the institutional body . 
the recent legislative decree 81 / 08 ( known as the consolidation act in matters of health and workplace safety ) extended this form of liability to offences in the workplace . these differences in legislative frameworks and traditions explain the difficulty in comparing the experiences and choices made in english - speaking countries with those in italy . 
in the united states , a number of healthcare organisations have managed to reduce expenses due to medical malpractice suits by applying a policy whereby the physicians and hospital managerial staff not only disclose errors but admit blame and offer compensation [ 29 ]  . 
some healthcare organisations have adopted a policy of absolute honesty , according to which the patient who has suffered the error is fully informed , receives care and is compensated . 
this system seems to have led to a reduction in medicolegal costs for healthcare organisations [ 34 ] , even though a reduction in the number of compensation claims has not been shown . 
negli stati uniti le organizzazioni dei consumatori chiedono che siano resi accessibili in rete i dati sulle complicazioni registrate negli ospedali , cos che sia possibile una scelta informata da parte dei pazienti [ 33 ]  . in italia , forse a solo scopo propagandistico , stato proposto di rendere disponibili on - line gli errori di ciascun medico . 
la possibilit di innescare in questo modo una spirale incontrollabile di allarme , con incremento delle denunce di malpractice e quindi dei costi assicurativi e delle pratiche di medicina difensiva ( che gi oggi rappresentano l8%10% di spese ingiustificate a carico dei diversi sistemi sanitari ) , concreta . riguardo al tema del rischio clinico latteggiamento delle aziende sanitarie anglosassoni e di quelle italiane estremamente diverso . 
nei paesi anglosassoni , difatti , stato sempre vigente il principio della responsabilit delle aziende sanitarie , che possono essere chiamate a rispondere degli eventuali errori dei propri dipendenti , qualora non siano in grado di dimostrare di avere attivamente operato per prevenire tali errori . 
il diritto romano , al contrario , escludeva la responsabilit penale delle aziende ( societas delinquere non potest ) e su questa linea la nostra costituzione ha stabilito , allart . 27 , che la responsabilit penale personale . 
tale orientamento ha costituito un reale ostacolo al principio della responsabilit sociale delle aziende e , nel campo specifico , ha indotto la sottovalutazione del problema e la generale omissione delle misure di prevenzione , a torto ritenute di esclusiva competenza dei singoli . 
solo di recente con il d . lgs 231 / 01 stato introdotto anche nel nostro sistema giudiziario il principio della responsabilit dellente per i delitti amministrativi , sulla base del comportamento omissivo e negligente nella gestione della propria struttura organizzativa . 
lgs 81 / 08 ( cosiddetto testo unico sulla salute e sicurezza sul lavoro ) ha esteso tale responsabilit anche ai delitti in campo lavorativo . le differenze del quadro normativo e delle tradizioni rendono ragione della difficolt di confrontare le esperienze e le scelte anglosassoni con quelle nostrane . 
negli usa alcune aziende sanitarie hanno ridotto le spese derivanti dal contenzioso medico - legale applicando una politica secondo la quale i medici e dirigenti dellospedale non solo rivelano gli errori , ma ammettono la colpa ed offrono un risarcimento [ 29 ]  . 
alcune organizzazioni sanitarie hanno adottato una politica di estrema onest secondo la quale il paziente che ha subito un errore viene informato in modo completo , ottiene assistenza e viene indennizzato . 
questo sistema sembra avere determinato una riduzione delle spese medico - legali da parte delle aziende sanitarie [ 34 ] , anche se non dimostrato che esso abbia ridotto il numero di richieste di risarcimento . 
 stato osservato infatti che il paziente o i suoi familiari hanno una risposta positiva verso radiol med ( 2009 ) 114 : 13451355 1353 lies respond positively towards the physician who openly and fully acknowledges having made a clinical error , thus maintaining their trust and satisfaction with the care provided , but it is unclear whether this dissuades them from taking legal action . 
in cases where the error causes damage of little count , an offer by the physician to compensate costs could have a negative effect [ 35 ]  . all this , however , is not to suggest that the disclosure of errors is a universal and painless practice in englishspeaking countries . 
many healthcare workers are loathe to reveal their own errors for fear of possible repercussions . nonetheless , there is strong cultural pressure in favour of such behaviour , right from the early stages of medical training . 
medical students are , in fact , instructed on how to behave in the event of errors committed by their tutor [ 36 , 37 ]  . the way to behave in the event of errors committed by colleagues in fact reveals the greatest distance between the ethical principles taught and applied in english - speaking countries and those of latin origin ( mind your own business )  . 
in the former , the physician is obliged to intervene should they note or receive an anonymous report of a colleague not adhering to standards : the failure to do so can lead to disciplinary action . 
the intervention should , of course , be graded according to the situation and may range from a simple discussion with the colleague to clarify matters and possibly involve advice to avoid the problem being repeated , to a report to a superior , right up to a report to professional bodies . il medico che riveli in modo aperto e completo di avere commesso un errore clinico , conservando la loro fiducia e la soddisfazione per loperato del medico , ma non chiaro se ci li distolga dallintraprendere una azione giudiziaria . nei casi in cui lerrore abbia prodotto danni poco rilevanti , il fatto che il medico si offra di rimborsare i costi potrebbe avere effetti negativi [ 35 ]  . non bisogna credere che la divulgazione degli errori sia una pratica universale e indolore nei paesi anglosassoni : molti sanitari sono riluttanti a rivelare i propri errori per il timore delle possibili ripercussioni . 
tuttavia presente una forte pressione culturale per favorire tale comportamento , sin dalla prime fasi delleducazione medica : gli studenti sono istruiti sul comportamento da tenere nel caso di errori compiuti dal loro tutore [ 36 , 37 ]  . 
 proprio sullatteggiamento da tenere nel caso di errori commessi dai colleghi che si rileva la maggiore distanza tra i principi etici insegnati e applicati nei paesi anglosassoni e la pratica popolare dei latini ( fatti i fatti tuoi )  . 
il medico obbligato ad intervenire nel caso che abbia notato , o abbia avuto segnalazione anche anonima , del fatto che un suo collega ha operato in modo difforme dagli standard ; nel caso che ometta di farlo , pu essere egli stesso sottoposto ad azione disciplinare . 
lintervento da compiere , naturalmente , graduato in funzione della necessit , e pu andare da un semplice colloquio con il collega , per chiarire i fatti ed eventualmente elargire consigli per evitare il ripetersi del problema , ad una segnalazione per via gerarchica , fino alla segnalazione agli organismi professionali . conclusions conclusioni as with all ethical problems , there are no simple and painless solutions that can be immediately applied . 
they may inform the patient that there has been a clinical problem , without accepting liability and possibly illustrating the factors that may cause undesirable effects or diagnostic difficulty in radiology . 
they may inform the patient that there has been a clinical problem , adding comforting statements such as : i am really sorry that this had to happen to you . 
they may inform the patient of the error , accept liability and offer compensation . in appears clear that in the setting of personal ethics , the correct choice can only be the latter . 
this is by no means an easy choice to make , and the physician should not expect that the patient sees things in the same light and offers come in ogni altro problema etico , non esistono ricette semplici e indolori da applicare automaticamente . 
il medico radiologo che si accorga di avere commesso un errore grave , che ha causato un danno ad un paziente , ha la possibilit di scegliere una delle cinque possibili alternative . 
pu comunicare al paziente che ci sia stato un problema clinico , ma senza assumersene la responsabilit , ed eventualmente illustrare i motivi che determinano effetti indesiderati o difficolt diagnostiche in radiologia . 
pu comunicare il fatto al paziente , dichiarando che si trattato di un errore ed assumendosene la responsabilit . infine , pu comunicare lerrore al paziente , assumersene la responsabilit ed offrire un risarcimento . appare evidente che , nellambito di unetica personalistica , la scelta corretta sar solo questultima . 
pozzi mucelli1 1department of radiology , 2department of biomedical and surgical science , university of verona , p.le scuro 10 , 37134 verona , italy 3erasmus university , medical center rotterdam , 3000 ca rotterdam , the netherlands correspondence to : v . 
the observers evaluated pancreatic parenchymal enlargement , signal intensity abnormalities , enhancement , vascular involvement , bile - duct diameter and main pancreatic duct ( mpd ) narrowing ( diffuse / focal / segmental )  . 
mr imaging showed diffuse pancreatic enlargement in 8 / 28 ( 29% ) cases , focal pancreatic enlargement in 16 / 28 ( 57% ) cases and no enlargement in 4 / 28 ( 14% ) cases . 
the alteration of pancreatic signal intensity was diffuse in 8 / 28 ( 29% ) cases ( 8 diffuse aip ) and focal in 20 / 28 ( 71% ) cases ( 20 focal aip )  . delayed pancreatic enhancement was present in all aip , with peripheral rim of enhancement in 8 / 28 ( 29% ) aip ( 1 / 8 diffuse , 7 / 20 focal ) ; vascular encasement was present in 7 / 28 ( 25% ) aip ( 1 / 8 diffuse , 6 / 20 focal ) ; distal common bile duct narrowing was present in 12 / 28 ( 43% ) aip ( 5 / 8 diffuse , 7 / 20 focal )  . 
scopo di questo lavoro stato valutare i rilievi di pancreatite autoimmune ( aip ) nella revisione retrospettiva delle immagini di risonanza magnetica ( rm ) , colangiopancreatografia - rm ( cprm ) e cprm con secretina . 
le immagini sono state analizzate considerando i seguenti parametri : volume del pancreas , alterazioni dellintensit di segnale del parenchima , impregnazione del pancreas , coinvolgimento vascolare , calibro delle vie biliari e restringimento ( diffuso / focale / segmentale ) del dotto pancreatico principale ( dpp )  . 
lesame rm ha evidenziato un incremento dimensionale diffuso del pancreas in 8 / 28 ( 29% ) casi , un ingrandimento di una parte del pancreas focale in 16 / 28 ( 57% ) casi e pancreas di regolare dimensione in 4 / 28 ( 14% ) casi . 
lalterazione dellintensit di segnale del pancreas era diffusa in 8 / 28 ( 29% ) casi ( 8 aip di tipo diffuso ) e focale in 20 / 28 ( 71% ) casi ( 20 aip di tipo focale )  . 
secretinenhanced mrcp is a problem - solving tool in the differential diagnosis between focal aip and ductal adenocarcinoma . keywords autoimmune pancreatitis mrcp secretin stimulation pancreas inflammatory diseases ( 5 / 8 aip di tipo diffuso , 7 / 20 aip di tipo focale )  . 
lesame cprm con secretina ha fatto rilevare il segno del dotto penetrante in 6 / 14 ( 43% ) aip ( 1 caso di aip diffusa con restringimento segmentale del dpp , 5 casi di aip focale con restringimento focale del dpp ) dimostrano lintegrit del dpp . 
 parole chiave pancreatite autoimmune cprm somministrazione di secretina pancreas malattie infiammatorie introduction introduzione autoimmune pancreatitis ( aip ) is defined as an autoimmune reaction around the pancreatic ducts resulting in inflammatory and fibrotic reaction with obliteration of the pancreatic ductal system [ 1 ]  . 
according to the different classification systems , the diagnosis of aip is based on the combination of four main criteria : imaging findings , laboratory data , histology and response to corticosteroid therapy , as assessed by both clinical and imaging findings [ 14 ]  . 
as a localised enlargement of the gland , which typically involves the head of the pancreas and the main pancreatic duct ( mpd ) upstream [ 5 , 6 ]  . 
differential diagnosis between focal aip and adenocarcinoma is extremely challenging but of paramount importance because of the different prognosis and because steroid therapy is effective in reversing clinical and morphological changes of aip [ 712 ]  . 
 most literature reports to date have concentrated on endoscopic retrograde cholangiopancreatography ( ercp ) and computed tomography ( ct ) features of aip [ 4 , 5 , 710 , 13 ]  . 
magnetic resonance ( mr ) imaging has a growing role in imaging the pancreas and biliary ducts , mainly related to visualisation of the ducts by means of mr cholangiopancreatography ( mrcp )  . 
few reports have evaluated the contribution of mr imaging in aip . furthermore , to our knowledge , the utility of secretin administration has not been evaluated , and only few studies la pancreatite autoimmune ( aip ) definita come una reazione autoimmunitaria attorno ai dotti pancreatici associata a reazione infiammatoria e fibrotica che risulta nella obliterazione del sistema duttale pancreatico [ 1 ]  . 
secondo i diversi sistemi di classificazione , la diagnosi di aip si basa sulla combinazione di 4 criteri principali : rilievi di imaging , dati di laboratorio , quadro istologico e risoluzione delle alterazioni cliniche e strumentali dopo terapia con corticosteroidi [ 14 ]  . 
 i reperti macroscopici di aip sono la diffusa ipertrofia del pancreas o pi raramente , la presenza di una alterazione espansiva formante massa , vale a dire un allargamento focale della ghiandola , che generalmente interessa la porzione della testa del pancreas e dilata il decorso del dotto pancreatico principiale ( dpp ) a monte [ 5 , 6 ]  . 
la diagnosi differenziale tra questultima , la forma focale di aip , e ladenocarcinoma pancreatico estremamente impegnativa , ma di fondamentale importanza , a causa della diversa prognosi e terapia ; il trattamento steroideo infatti efficace in caso di aip per risolvere le alterazioni cliniche e le modificazioni morfologiche [ 712 ]  . 
 fino ad oggi la maggior parte dei lavori riportati in letteratura si sono concentrati sugli aspetti delle aip rilevabili con colangio - pancreatografia retrograda endoscopica ( ercp ) e tomografia computerizzata ( tc ) [ 4 , 5 , 710 , 13 ]  . 
lindagine di risonanza magnetica ( rm ) ha un ruolo sempre pi importante nellimaging del pancreas e delle vie biliari , grazie alla capacit di visualizzazione dei dotti 1216 radiol med ( 2009 ) 114 : 12141231 have described mrcp features in aip [ 6 , 14 ]  . 
 the purpose of our study was to retrospectively review the imaging findings of mr imaging in general , and mrcp in particular , with and without administration of secretin , in patients with aip . materials and methods patients this study was approved by our investigator review board , and informed consent requirement was waived . 
a search of our institutions pathology , radiology and medical records from february 2001 to january 2006 revealed 55 patients with aip whose data were considered for inclusion in this retrospective study . 
patients were included if they fulfilled the histological criteria for aip ( fibrotic pancreatic change with lymphocyte and plasma - cell infiltration ) and had a complete mr and mrcp examination at the time of diagnosis . 
exclusion criteria were as follows : lack of histopathological diagnosis ( n = 16 ) , poor image quality ( n = 6 ) and lack of mr imaging before steroid treatment ( n = 5 )  . 
thus , the study population consisted of 28 patients ( 18 men , ten women ) , with a mean age of 45 ( range 2179 ) years at clinical presentation . 
 the frequency of symptoms at presentation was as follows : jaundice in 14 / 28 ( 50% ) , abdominal pain in 4 / 28 ( 14% ) , pancreatic - like abdominal pain ( with posterior irradiation ) in 8 / 28 ( 28% ) , weight loss in 2 / 28 ( 7% ) and diabetes in 2 / 28 ( 7% )  . 
eleven out of 28 ( 39% ) patients showed association with other autoimmune disorders : ulcerative colitis in 6 / 28 ( 21% ) , sjgren syndrome in 1 / 28 ( 3% ) , hashimotos thyroiditis in 1 / 28 ( 3% ) , sclerosing cholangitis in 2 / 28 ( 7% ) and retroperitoneal fibrosis in 1 / 28 ( 3% )  . 
the clinical and laboratory data are summarised in table 1 . the results of laboratory tests showed increased serum levels of bilirubin and cholestatic liver function tests in 10 / 28 ( 36% ) patients , increased serum amylase and lipase in 4 / 28 ( 12% ) , serum antibodies in 2 / 28 ( 6% ) patients , increased immunoglobulin ( ig ) g ( > 2.0 g / dl ) in 1 / 28 ( 3% ) patients and increased carbohydrate antigen ( ca ) 19 - 9 in 6 / 28 ( 18% ) patients ( table 1 )  . 
a history of habitual ( < 80 g / day ) alcohol consumption and cigarette smoking was present in 7 / 28 patients ; there were no patients with history of alcohol abuse ( > 80 g / day for at least 5 years )  . 
the pathological diagnosis was obtained by percutaneous ultrasonography ( us ) - guided biopsy in 23 / 28 ( 82% ) patients and by core biopsy during mediante colangiopancreatografia con rm ( cprm )  . 
un ridotto numero di studi ha valutato il contributo della rm nei casi di aip ; inoltre lutilit della somministrazione di secretina non stato ancora analizzata e solo pochi lavori descrivono le caratteristiche delle aip rilevabili allindagine cprm [ 6 , 14 ]  . 
 lo scopo del presente studio quello di valutare retrospettivamente i rilievi rm , cprm , in particolare , con e senza la somministrazione di secretina , nei pazienti con aip . 
 materiali e metodi pazienti lo studio stato approvato dal comitato etico della struttura e si soprasseduti al rilascio del consenso informato . dallanalisi degli archivi patologici , radiologici e clinici riguardanti il periodo compreso fra febbraio 2001 e gennaio 2006 sono stati evidenziati 55 pazienti con aip i cui dati sono stati presi in considerazione per linclusione in questo studio . 
i criteri di inclusione sono stati : presenza dei criteri istologici di aip ( fibrosi pancreatica con infiltrazione di linfociti e plasmacellule ) e di documentazione rm e cprm completa al momento della diagnosi . 
i criteri di esclusione sono stati i seguenti : mancanza di diagnosi istopatologica di aip ( n = 16 ) , cattiva qualit delle immagini ( n = 6 ) e mancato espletamento di indagine rm prima del trattamento steroideo ( n = 5 )  . 
la popolazione in studio , quindi , formata da 28 pazienti ( 18 uomini , 10 donne ) , con et media alla presentazione clinica di 45 anni ( range 2179 anni )  . 
 la frequenza dei sintomi di presentazione stata la seguente : ittero in 14 / 28 ( 50% ) , dolore addominale in 4 / 28 ( 14% ) , dolore addominale tipo pancreatite ( con irradiazione posteriore ) in 8 / 28 ( 28% ) , perdita di peso in 2 / 28 ( 7% ) e diabete di 2 / 28 ( 7% )  . 
altre malattie autoimmuni erano presenti in 11 / 28 ( 39% ) pazienti ; in particolare , colite ulcerosa in 6 / 28 ( 21% ) , sindrome di sjogren in 1 / 28 ( 3% ) , tiroidite di hashimoto in 1 / 28 ( 3% ) , colangite sclerosante in 2 / 28 ( 7% ) e fibrosi retroperitoneale in 1 / 28 ( 3% )  . 
 i risultati degli esami di laboratorio hanno dimostrato un aumento dei livelli sierici di bilirubina colestatica e dei test di funzionalit epatica in 10 / 28 ( 36% ) pazienti , aumento della lipasi e amilasi sierica in 4 / 28 ( 12% ) , innalzamento degli anticorpi in 2 / 28 ( 6% ) pazienti , aumento delle igg radiol med ( 2009 ) 114 : 12141231 1217 1218 radiol med ( 2009 ) 114 : 12141231 radiol med ( 2009 ) 114 : 12141231 1219 surgery in the remaining 5 / 28 ( 18% ) patients ( duodenocephalopancreasectomy : 3 / 5 patients ; bilioenteric anastomosis and cholecystectomy : 2 / 5 patients )  . 
in patients with jaundice associated with increased ca 19 - 9 ( n = 6 ) , pancreatic - like abdominal pain and jaundice ( n = 2 ) , diabetes ( n = 2 ) or those with suspected pancreatic tumour ( based on clinical signs and other imaging examinations such as ercp , us and ct performed before mr examination ) , dynamic mrcp after secretin administration was indicated . 
thus , secretin - enhanced mrcp was performed in 14 / 28 patients ( 50% ) : ten with symptoms suggestive of pancreatic disease and four with suspected diagnosis of pancreatic tumour . mr and mrcp protocols the conventional mr examination was performed with a 1.5 - t magnet ( symphony ; siemens , erlangen , germany )  . 
 the non - contrast - enhanced mr examination consisted of t1and t2 - weighted images acquired with gradient - echo ( ge ) fat - saturated ( fs ) and fast spin - echo ( fse ) sequences , with repetition time ( tr ) / echo time ( te ) ( ms / ms ) equal to 107 / 4.8 and 4950 / 102 , respectively . 
the additional delayed acquisition was performed in all cases at 5 min from the injection of the contrast mediu the mrcp examination was performed after oral administration of 100 ml superparamagnetic agent ( ferumoxsil , lumirem , guerbet , france ) with t2 - weighted rapid acquisition with relaxation enhancement turbo spin - echo ( raretse ) sequence in the paracoronal and axial planes . 
the imaging parameters were as follows : tr / te 4 , 500 / 991 ; turbo factor 410 ; field of view ( fov ) 350 mm ; matrix 410512 ; 64 - mm - thick slab . 
considering its higher spatial resolution , mrcp was also performed using a 3d t2weighted half - fourier single - shot turbo se ( haste ) sequence with respiratory triggering in the coronal oblique plane ( tr / te 4 , 450 / 585 )  . 
the imaging parameters were as follows : fov ( > 2 , 0 g / dl ) in 1 / 28 ( 3% ) pazienti , e incremento del marcatore tumorale ca - 19.9 in 6 / 28 ( 18% ) pazienti ( tabella 1 )  . una storia di consumo abituale di alcol ( < 80 g / giorno ) e fumo di sigaretta era presente in 7 / 28 pazienti ; nessuno riportava storia di abuso di alcool ( > 80 g / giorno per almeno 5 anni )  . 
la diagnosi patologica stata ottenuta con biopsia percutanea sotto guida ecografica in 23 / 28 ( 82% ) pazienti e con core - biopsy durante intervento chirurgico nei restanti 5 / 28 ( 18% ) pazienti ( duodeno - cefalopancreasectomia in 3 / 5 pazienti ; anastomosi bilio - enterica e colecistectomia negli altri 2 / 5 pazienti )  . 
nei pazienti con ittero associato ad aumento del ca - 19.9 ( 6 casi ) , con dolore addominale tipo pancreatite e ittero ( 2 casi ) , con diabete ( 2 casi ) o con sospetta diagnosi di tumore ( sulla base dei segni clinici e degli altri esami strumentali come ercp , ecografia e tc eseguita prima dellesame rm ) , stata espletata la valutazione cprm dopo secretina . 
quindi , lo studio cprm dopo secretina stato effettuato in 14 / 28 pazienti ( 50% ) , 10 pazienti con sintomi suggestivi di malattie del pancreas , 4 pazienti con sospetta diagnosi di tumore pancreatico . protocolli rm e cprm lesame rm stato eseguito con magnete 1 , 5 t ( symphonia , siemens , erlangen , germania ) in condizioni basali e con somministrazione di mezzo di contrasto in fase arteriosa , venosa e tardiva . 
gadoterate meglumina ( gd - dota , dotarem , guerbet , aulnay - sous - bois , francia ) stato iniettato per via endovenosa ad una dose di 0 , 2 mmol / kg . 
 nella fase pre - contrasto , sono state espletate sequenze t1 e t2 - dipendenti , acquisite con gradiente - echo ( gre ) fat saturation ( fs ) e fast spin - echo , e tr / te ( ms / ms ) , pari rispettivamente a 107 / 4 , 8 e 4950 / 102 . 
lo spessore di fetta stato 5 , 5 mm per le immagini t1 - dipendenti e 7 mm per quelle t2 - dipendenti , con slice - gap variabile ( 0 , 5 e 0 , 7 , rispettivamente )  . 
in fase arteriosa , come stabilito dalla tecnica timing run , stata utilizzata una sequenza t1 ( vibe ) dipendente volume ( tr / te : 107 / 4 , 8 ms ) ; in fase venosa le immagini , ottenute con la stessa sequenza , sono state acquisite con un ritardo di 65 secondi dallinizio delliniezione . 
 interpolated breath - hold lesame cprm stato condotto dopo somministrazione orale di 100 ml di agente superparamagnetico ( ferumoxsil , lumirem , guerbet , francia ) , con sequenze t2 - dipendenti rapid acquisition with relaxation enhancement - turbo spin echo ( rare - tse ) nei piani para - assiali e coronali con i seguenti parametri : tr / te : 4500 / 991 ; turbo factor : 410 ; campo di vista , 350 mm ; matrice , 410512 ; 64 mm di spessore di fetta . 
considerando la pi alta risoluzione spaziale , lo studio cprm stato effettuato utilizzando una sequenza 3d half - fourier acquisition single - shot turbo spin - echo 1220 radiol med ( 2009 ) 114 : 12141231 300 mm ; matrix 256256 interpolated ; 64 - mm - thick slab ; 0.95 - mm - thick partitions . a dynamic mrcp examination during secretin ( secrelux , goldham - bioglan , zusmarhausen , germany ) intravenous administration at 1 iu / kg was performed . 
after secretin stimulation , a 64 - mm t2 - weighted thick - slab rare sequence was repeated every 30 s for 5 min and every 60 s for other 5 min , reaching a total of 15 images . at the end of the dynamic phase , the same 3d t2 - weighted haste sequence with respiratory triggering , described above , was acquired in the paracoronal plane . image analysis two radiologists ( gc , rm ) skilled in abdominal imaging with 11 and 15 years of experience , respectively , in abdominal radiology , and blinded to the laboratory and histological data , independently reviewed all the examinations at the workstation in separate sessions . 
firstly , they evaluated the conventional mr images for parenchymal abnormalities , such as focal / diffuse enlargement of the pancreatic gland and / or signal intensity alterations of pancreatic parenchyma compared with those of the liver and spleen at the same level , peripancreatic oedema , early enhancement after contrast - medium administration and presence of delayed enhancement or peripheral rim and vascular involvement ( narrowingcompression of the vessels shown by decrease in signal intensity of perivascular fat )  . 
in particular , the imaging studies were analysed for the following features : dilation of common bile duct ( > 5 mm in normal fasting patients ; > 11 mm in patients with previous surgery ) focal , segmental or diffuse narrowing ( < 1 mm ) of the mpd , focal or diffuse dilation ( > 4 mm ) of the mpd and of the branch ducts . 
i parametri utilizzati sono stati i seguenti : campo di vista 300 mm ; matrice 256256 ; 64 mm di spessore di fetta , 0 , 95 mm di spessore partizioni . 
 lo studio cprm dinamico stato condotto prima e dopo somministrazione endovenosa di 1 ui / kg di secretina ( secrelux , goldham - bioglan , zusmarhausen , germania ) con sequenza thick slab rare t2 - dipendente ( 64 mm ) , ripetuta dopo iniezione di secretina ogni 30 secondi per i primi cinque minuti e ogni 60 secondi per i successivi cinque minuti , ottenendo un numero complessivo di 15 immagini . alla fine della fase dinamica stata ripetuta la stessa sequenza 3d haste t2w nel piano para - coronale descritta in precedenza . 
 analisi delle immagini due radiologi ( gc , rm ) , con rispettivamente 11 e 15 anni di esperienza in imaging addominale , alloscuro della documentazione clinica e istologica , hanno valutato indipendentemente e separatamente tutti gli esami alla work - station ; in caso di discrepanza si raggiunto il consenso . 
le immagini sono state analizzate considerando i seguenti rilievi : ingrandimento focale / diffuso del pancreas , alterazioni focali / diffuse dellintensit di segnale del pancreas rispetto allintensit del fegato e della milza rilevabili nella stessa scansione , edema peri - pancreatico , impregnazione parenchimale dopo somministrazione di mezzo di contrasto e comparsa di impregnazione periferica con aspetto di cercine iperintenso in fase tardiva , interessamento vascolare ( riduzione dellintensit di segnale del tessuto adiposo perivascolare suggestivo di restringimento o compressione esterna del vaso ) , e infine eventuali linfonodi peri - pancreatici ( tabella 2 )  . 
 lindagine cprm stata valutata analizzando le seguenti alterazioni duttali : dilatazione del dotto biliare comune ( > 5 mm in pazienti a digiuno ; > 11 mm in pazienti con precedente intervento chirurgico ) , assottigliamento ( < 1 mm ) del dpp , focale , segmentale o diffusa , dilatazione ( > 4 mm ) del dpp e dei dotti collaterali , focale , segmentale o diffusa . 
il restringimento del calibro del dpp stato definito focale quando lassotigliamento del dotto coinvolgeva un breve tratto del dpp , segmentale quando interessava il dotto per un tratto pi ampio rispetto a quello segmentale , corrispondente ad una regione del pancreas ( vale a dire la porzione nella testa o nel corpo , coda ) , ed infine diffuso quando lintero dpp appariva assotigliato [ 4 ]  . 
the cases in which secretin administration showed neither reduction of mpd stenosis nor the duct - penetrating sign were considered unresponsive to secretin cases in which secretin administration reduced mpd stenosis , the length and number of the decreased strictures were considered . 
other parameters assessed after secretin administration were visualisation of side branches , and impairment of the pancreatic function ( based on timing and degree of duodenal filling ) according to criteria reported in the literature ( table 4 ) [ 16 ]  . 
 histopathological diagnosis histopathologic evaluation was performed by one pathologist with > 10 years of experience in pancreatic pathology . the histopathologic criteria used to diagnose aip were as follows : infiltration around small pancreatic ducts , swirling fibrosis centred around ducts and veins , and obliterative phlebitis [ 10 ]  . lymphoplasmacytic statistical analysis interessava una regione del pancreas e diffusa quando coinvolgeva lintero decorso del dpp ( tabella 3 ) [ 4 ]  . 
 nei 14 casi in cui stata somministrata secretina , gli osservatori hanno analizzato leventuale miglioramento della visualizzazione del dpp in base alla presenza , al numero e alla estensione del restringimento del dpp e la positivit del segno del dotto penetrante [ 4 , 15 ]  . 
i casi in cui dopo secretina il tratto di dpp assotigliato e stenotico diventava meglio definito , irregolare ma non ostruito , riacquistando la continuit nel suo decorso che prima non era visualizzable , sono stati considerati positivi per la presenza del segno del dotto penetrante [ 15 ]  . 
i casi in cui dopo somministrazione di secretina non si rilevato n la riduzione della stenosi del dpp , n il segno del dotto penetrante sono stati considerati non responsivi alla secretina . 
gli altri parametri di valutazione dopo somministrazione di secretina sono stati : visualizzazione dei dotti collaterali , alterazioni della funzionalit esocrina pancreatica ( in base al tempo e grado di riempimento duodenale ) secondo i criteri riportati in letteratura ( tabella 4 ) [ 16 ]  . interobserver variability in determining the extent of parenchyma involvement as modification of normal signal intensity was assessed with - statistics . 
the strength of agreement was assessed as follows : values of 0.20 represented poor agreement ; 0.20.40 fair agreement ; 0.410.60 diagnosi patologica lesame patologico stato effettuato da un patologo con pi di 10 anni di esperienza in patologia pancreatica . 
axial t1 - weighted ge image ( a ) shows diffuse enlargement of the pancreas ; axial t2 - weighted image ( b ) shows increased signal intensity of peripancreatic fat tissue ( peripancreatic oedema ; arrows in b )  . 
immagine gre t1 - dipendente nel piano assiale ( a ) : diffuso allargamento del pancreas . immagine t2 - dipendente nel piano assiale ( b ) : aumentata intensit di segnale del tessuto adiposo peripancreatico ( edema peripancreatico : frecce in b )  . dopo somministrazione di mezzo di contrasto ( mdc ) , in fase tardiva , limmagine gre t1 - dipendente fs nel piano assiale ( c ) mostra impregnazione pancreatica periferica ( cercine iperintenso di impregnazione : frecce in c ) , reperto tipico nella pancreatite autoimmune . 
immagine gre t1 - dipendente fs nel piano assiale ( e ) , espletata 28 giorni dopo lassunzione di terapia steroidea , evidenzia una riduzione del volume pancreatico ; il dotto pancreatico principale visualizzabile lungo lintero decorso ( cprm , f )  . 1224 radiol med ( 2009 ) 114 : 12141231 fig . 
mrcp examination ( d ) shows stenosis of cbd in the intrapancreatic portion , with marked dilation of the bile duct upstream , and mpd narrowing at the head with irregular dilation at the body - tail associated with dilation of collateral branches . 
immagine gre t1 - dipendente fs nel piano assiale ( a ) : allargamento focale della ghiandola pancreatica localizzato alla testa ove lintensit del segnale diminuita rispetto a quella del parenchima epatico e splenico . 
dopo somministrazione di mdc , in fase tardiva , limmagine gre t1 - dipendente fs nel piano assiale ( c ) mostra un restringimento della confluenza portomesenterica ( freccia in c )  . 
lindagine cprm ( d ) , evidenzia una riduzione del calibro del dotto biliare comune nella porzione intrapancreatica con marcata dilatazione dei dotti biliari a monte e un restringimento del dpp nel tratto della testa con irregolare dilatazione al corpo - coda associata a estasia dei rami collaterali . 
la forza di accordo stata valutata come segue : scarso accordo per valori di minori o uguale a 0 , 20 ; accordo equo tra 0 , 200 , 40 ; accordo moderato tra radiol med ( 2009 ) 114 : 12141231 1225 fig . 
immagine gre t1 - dipendente fs nel piano assiale ( a ) : diminuzione dellintensit di segnale proveniente dal corpo - coda del pancreas con solo lieve allargamento del volume della ghiandola pancreatica . 
dopo somministrazione di mdc , immagine gre t1 - dipendente fs nel pianio assiale ( b ) : linfonodo globoso adiacente alla vena porta ( freccia in b )  . 
il calibro a livello della confluenza portomesenterica ridotto a causa dello stretto rapporto con la lesione pancreatica , meglio visualizzabile nella immagine coronale gre t1 - dipendente fs ( freccia in c )  . 
the features of the mpd are shown in table 3 . secretin - enhanced mrcp was performed in 4 / 8 patients with diffuse - type aip ( 50% ) ( table 4 )  . 
in the remaining 3 / 4 patients ( 75% ) , two with segmental irregular narrowing of the mpd and one with diffuse mpd dilation , secretin administration did not change the mpd calibre as depicted on presecretin mrcp ( table 4 )  . 
after contrast medium administration , enhancement of the pancreas was homogeneously reduced compared with the spleen and adjacent unaffected parenchyma in all patients ( 20 / 20 ; 100% )  . 
in 1 / 8 pazienti ( 12% ) era presente compressione della confluenza portomesenterica e in un altro paziente ( 1 / 8 , 12% ) linfonodi aumentati di volume ( tabella 2 )  . 
 lo studio cprm con secretina stato eseguito in 4 / 8 pazienti con aip di tipo diffuso ( 50% ) ( tabella 4 ) e ha rilevato le seguenti modificazioni . 
in un caso ( 1 / 4 , 25% ) , che aveva un irregolare assottigliamento di un segmento del dpp , dopo somministrazione di secretina era apprezzabile il segno del dotto penetrante . 
nei restanti 3 / 4 pazienti ( 75% ) , 2 dei quali con irregolare restringimento segmentale del dpp e laltro con diffusa dilatazione del dpp , la somministrazione di secretina non ha avuto alcun effetto sullaspetto morfologico del dpp ( tabella 4 )  . 
 aip di tipo focale nei casi di aip focale ( 20 / 28 casi : 71% ) nelle immagini t1dipendenti fs il parenchima pancreatico presentava una focale diminuzione dellintensit del segnale e appariva ipointenso rispetto alla milza e al parenchima pancreatico adiacente . 
lalterazione dellintensit del segnale era localizzata nella porzione testa in 14 / 20 pazienti ( 70% ) ( fig . 2a ) e nel corpo - coda in 6 / 20 pazienti ( 30% ) ( tabella 2 )  . nelle immagini t2 - dipendenti , un alone periferico iperintenso stata osservato in 4 / 20 pazienti ( 20% )  . 
dopo iniezione di mezzo di contrasto , limpregnazione della parte di pancreas coinvolta risultata diminuita rispetto al parenchima sano adiacente e alla milza in tutti i pazienti ( 20 / 20 , 100% )  . 
sia nei pazienti con dilatazione delle vie biliari che in quelli con stent biliare , la lesione era localizzata nella testa pancreatica ; nei pazienti con lesione localizzata al corpocoda le via biliare intra ed extra - epatica era normorappresentata ( tabella 3 )  . 
in un altro caso ( 10% ) con restringimento multifocale , in pi punti della porzione del corpo - coda , dopo somministrazione di secretina si rilevato la diminuzione del numero di stenosi . 
in 1 / 10 pazienti ( 10% ) con focale restringimento del dpp nella coda , la somministrazione di secretina non ha avuto alcun effetto sul calibro del dotto e la morfologia del dpp rimasta invariata . 
nei rimanenti 2 / 10 pazienti ( 20% ) che avevano un dpp dilatato rispettivamente nella porzione della testa e del corpo , dopo somministrazione di secretina si resa evidente la estasia dei dotti pancreatici collaterali ( tabella 4 )  . 
dopo iniezione di secretina ( b ) , il dotto pancreatico principale dimostrabile lungo tutto il suo decorso con visualizzazione del segmento della testa , non identificabile pre - secretina , grazie alla riduzione della stenosi ( frecce in b , segno del dotto penetrante )  . discussione the body , after secretin administration , there was a decrease in length of the stenosis . 
in 1 / 10 ( 10% ) patients who had mpd segmental irregular narrowing with multiple stenosis in the body - tail , secretin administration showed a reduction in number of the stenoses . 
in 1 / 10 patients ( 10% ) who had focal narrowing of mpd in the tail , administration of secretin did not change the mpd calibre as depicted on presecretin mrcp . 
in the remaining 2 / 10 patients ( 20% ) who had mpd dilation in the head ( n = 1 ) and in the body la pancreatite autoimmune una malattia infiammatoria del pancreas , non comune ( 4 , 6%6% delle pancreatiti croniche ) , con eziopatogenesi autoimmunitaria , di recente scoperta e in via di definizione [ 2 , 17 ]  . 
nella presente serie sono stati retrospettivamente analizzati i rilievi rm , cprm , e cprm con secretina in ragione del fatto che il comportamento delle pancreatiti autoimmuni allindagine cprm con secretina non stato ancora documentato e potrebbe fornire 1228 radiol med ( 2009 ) 114 : 12141231 ( n = 1 ) , dilation of collateral branches was appreciable after administration of secretin ( table 4 )  . in all patients , secretin administration showed preserved exocrine pancreatic function . 
as demonstrated by normal duodenal filling ( table 4 )  . discussion inflammatory involvement of the pancreas caused by autoimmune reaction is a recent pathological entity that is still incompletely understood [ 2 , 17 ]  . 
in this study , we performed a retrospective determination of mr , mrcp and secretin - mrcp findings . the use of mrcp after secretin administration for aip is a new method that has not been previously reported , and evaluation of aip by this method may provide important information . 
according to the literature , aip predominantly affects the elderly male population , with relatively mild symptoms compared with the acute abdominal pain accompanying forms of acute pancreatitis [ 3 , 4 ]  . 
the incidence of associated autoimmune diseases and the detection rate of autoantibodies show variable values ranging from 12% to 50% [ 24 ] and from 10% to 100% [ 1820 ] , respectively . 
we found associated autoimmune disease in 39% of cases and autoantibodies in 6% of cases only . in agreement with the literature [ 3 , 4 , 10 , 1922 ] , in our series we noted a male predominance ( 64% )  . 
although the diffuse form is the most frequently reported in the literature [ 6 , 10 , 11 , 21 , 23 ] , in our series the focal form of aip was more common . 
this may be due in part to the fact that we are a referral centre for pancreatic surgery , but it also reflects the fact that the focal form of aip still represents a major diagnostic challenge . 
 [ 11 ] : once again , we believe that being a referral centre for pancreatic surgery contributes to the selection of younger patients with suspected pancreatic tumour who could successfully undergo radical intervention . 
on the other hand , the younger age of the patients with focal aip is consistent with some reports of progression from segmental to diffuse disease observed on serial ercp without steroid importanti elementi diagnostici . 
in letteratura riportato che le pancreatiti autoimuni colpiscono prevalentemente le persone anziane di sesso maschile , con sintomi lievi in confronto agli attacchi di dolore addominale che si accompagnano alle forme di pancreatite acuta [ 3 , 4 ]  . 
la pancreatite autoimmune si associa ad altre malattie autoimmuni e allinnalzamento del tasso sierico di auto - anticorpi in un numero variabile di casi con incidenza rispettivamente del 12%50% [ 24 ] , e del 10%100% [ 1820 ]  . 
 in accordo con la letteratura [ 3 , 4 , 10 , 1922 ] , la popolazione colpita stata in prevalenza maschile ( 64% ) ; tuttavia , altri dati sono diversi da quanto rilevato nei precedenti lavori . 
let media era di 45 anni , quindi pi giovane rispetto a quella di 5568 anni riportata in altre esperienze [ 3 , 4 , 6 , 1922 ]  . 
inoltre , nonostante la forma diffusa di aip venga riportata come quella pi comune [ 6 , 10 , 11 , 21 , 23 ] , nella serie esaminata la forma focale di aip risultata pi numerosa . 
la preponderanza della forma focale di aip imputabile in parte al fatto che la casistica stata raccolta in centro di riferimento per la chirurgia del pancreas , ma dallaltra al fatto che le aip focali rappresentano ancora un importante problema diagnostico . 
 [ 11 ] probabilmente perch un centro specializzato in chirurgia pancreatica contribuisce alla selezione di giovani pazienti con sospetto tumore del pancreas , che potrebbero essere sottoposti con successo a intervento radicale . 
daltro canto , per , la giovane et dei pazienti con aip di tipo focale in linea con la teoria della progressione temporale della malattia , il cui esordio sarebbe localizzato e in assenza di trattamento medico diffonderebbe al resto del pancreas [ 4 , 22 , 2426 ] , suggerendo quindi che la forma focale e quella diffusa di aip siano spettro della stessa malattia piuttosto che di entit distinte [ 4 ]  . 
 nella presente serie , non tutti i pazienti con aip mostravano ipertrofia pancreatica ; in 4 casi vi era una modificazione dellintensit di segnale del parenchima non accompagnata da un aumento dimensionale della porzione di pancreas corrispondente . 
 dopo iniezione di mezzo di contrasto , limpregnazione radiol med ( 2009 ) 114 : 12141231 1229 treatment [ 4 , 22 , 2426 ] , suggesting than the different types are a spectrum of the same disease rather than separate entities [ 4 ]  . in our study , not all patients with aip had enlargement of the pancreas . 
these findings probably suggest earlier onset of signal intensity alteration compared with glandular enlargement in focal aip . after contrast medium administration , pancreatic enhancement was delayed in all cases , and a capsule - like smooth rim was seen in eight patients ( 28% )  . 
in our study , mr imaging identified the peripheral rim in a higher number of cases ( n = 8 ; 28% ) compared with other ct series [ 6 , 24 , 27 ] and more frequently in focal aip compared with diffuse aip ( seven focal aip vs one diffuse aip ) [ 13 ]  . 
moreover , the review of ercp features from the literature [ 22 ] of aip suggests that its typical appearance is not only diffuse irregular narrowing of the mpd but also segmental irregular narrowing that may advance to diffuse irregular narrowing . 
even if the role of mrcp may appear to be limited in diagnosing aip ( the entity being a narrow - duct disease ) [ 4 ] and mrcp cannot differentiate mpd irregular narrowing of aip from the stenosis of pancreatic carcinoma [ 22 ] , the administration of secretin opens new perspectives in the utilisation of mrcp as a diagnostic tool for the differential diagnosis of aip and inflammatory pancreatic disease versus pancreatic cancer [ 4 , 15 ]  . the focal type of aip is difficult to differentiate from pancreatic carcinoma on the basis of morphological features and early enhancement pattern , and due to these difficulties , most of these forms undergo surgery [ 28 ]  . 
the associated findings such as elevation of tumour markers , focal mass forming , dilatation of mpd and common bile duct upstream ( the double - duct sign ) , together with vascular encasement , can raise further concern for malignancy [ 12 , 29 , 30 ]  . 
also in our study , 56% of focal aip of the head had the diagnosis of pancreatic suspicious findings for del pancreas risultata ritardata in tutti i casi e il cercine periferico di enhancement stato osservato in 8 pazienti ( 28% )  . 
limpregnazione periferica tardiva un reperto distintivo della pancreatite autoimmune e utile nella diagnosi differenziale con il carcinoma ; in questa casistica studiata con indagine rm , il cercine di impregnazione periferica stato individuato in un numero pi elevato di casi ( 8 casi , 28% ) rispetto ad altre serie esaminate con indagine tc [ 6 , 24 , 27 ] , e pi frequentemente nelle aip focali rispetto alla forma diffusa ( in particolare in 7 casi di aip focale vs 1 caso di aip diffusa ) [ 13 ]  . 
il restringimento del tratto distale del dotto biliare comune una evenienza frequentemente descritta nelle pancreatiti autoimmuni [ 21 ]  . in modo analogo , la dilatazione delle vie biliari stata osservata in 5 pazienti con aip diffusa ( 63% ) e in 7 pazienti con aip focale della testa ( 50% ) mentre nessuna anomalia biliare stata rinvenuta nei casi di aip del corpo e della coda del pancreas . 
dalla disamina della letteratura [ 22 ] emerge che laspetto tipico del dpp nelle aip non solo quello di diffuso ed irregolare restringimento , ma anche quello di assottigliamento segmentale , cio pi breve e sfumato , che pu progredire fino alla stenosi diffusa . 
il contributo della indagine rm nellinquadramento delle malattie del pancreas deriva dalla possibilit di ottenere uno studio parenchimale e dei vasi sanguigni in modo simile alla tc e , con lo studio cprm , una rappresentazione dei dotti biliari e del pancreas . 
anche se lapporto dellindagine cprm pu apparire limitato nella diagnosi di aip essendo questa una patologia che si manifesta prevalentemente con un restringimento dei dotti [ 4 ] e non potendo differenziare il restringimento provocato da aip dalle stenosi neoplastiche [ 22 ] , luso dello stimolo secretinico apre nuove prospettive di utilizzo della cprm come strumento di diagnosi differenziale tra le aip e il carcinoma pancreatico [ 4 , 15 ]  . 
 la pancreatite autoimmune di tipo focale infatti difficile da distinguere dal carcinoma pancreatico sulla base dei reperti morfologici e del pattern di impregnazione in fase precoce , tanto che , a causa di queste difficolt , la maggior parte delle aip focali sottoposta a intervento chirurgico [ 28 ]  . 
la combinazione di rilievi quali aumento dei marcatori tumorali , alterazione espansiva del pancreas formante massa , dilatazione del dpp e del dotto biliare comune a monte del restringimento ( segno del doppio dotto ) , insieme con la compressione dei vasi peri - pancreatici possono destare il sospetto di malignit [ 12 , 29 , 30 ]  . anche nella presente casistica , il 56% delle aip focali della testa aveva caratteri macroscopici sospetti per carcinoma del pancreas ; in particolare , 2 pazienti con aip focale della testa ( 14% ) mostravano un innalzamento del ca - 19.9 e una 1230 radiol med ( 2009 ) 114 : 12141231 carcinoma . 
the other three patients with focal aip of the head ( 21% ) had increased ca 19 - 9 alone , and another three patients with focal aip of the head ( 21% ) had stenosis of peripancreatic vessels alone . the role of mrcp in the morphological evaluation of the pancreatic duct has been extensively demonstrated in the literature [ 14 , 30 , 3134 ]  . 
mr imaging with mrcp is superior to both mr imaging alone and ct in differentiating focal aip from carcinomas , considering that focal aip tends to have an irregular , but not obstructed , mpd , which tends to penetrate the mass after secretin administration , the socalled duct - penetrating sign [ 15 , 30 ]  . 
consequently , it increases fluid in the pancreatic duct and its delineation is improved at mrcp . the use of dynamic mrcp with secretin has already proven useful to visualise the pancreatic duct and evaluate the behaviour of the mpd [ 15 , 3134 ]  . 
in six cases in particular , in one case of diffuse aip and in five cases of focal aip of the head ( 43% of all aip and 50% of focal aip ) the mpd segmental or focal stenosis resolved during the dynamic study showing the duct - penetrating sign . 
in those cases , especially in the focal aip of the head with no typical sausage - like pancreatic enlargement , the administration of secretin was of paramount importance in excluding a ductal adenocarcinoma and avoiding unnecessary surgical intervention . 
moreover , considering the relatively small number of patients , it is difficult to assess the statistical significance of the imaging findings . in conclusion , in the right clinical scenario , conventional mr imaging with mrcp allows precise classification of the aip in most cases , thanks to the demonstration of the late rim enhancement and the behaviour of the stenotic biliary and pancreatic ducts , particularly after secretin stimulation . for these reason , we suggest mr imaging with mrcp in suspected aip , and especially in the presence of a focal pancreatic lesion mimicking a ductal adenocarcinoma . 
the administration of secretin represents a problem - solving tool capable of demonstrating the integrity of the mpd along with preserved pancreatic function , thus enabling a correct diagnosis . stenosi dei vasi peripancreatici , altri 3 pazienti con aip focale della testa ( 21% ) presentavano solo ca - 19.9 aumentato e gli altri 3 pazienti con aip focale della testa ( 21% ) avevano una stenosi dei vasi peripancreatici isolata . 
lo studio rm con cprm superiore allindagine rm e tc nella differenziazione delle forme di aip focale dai carcinomi visto che la aip focale si caratterizza per un dpp irregolare , assotigliato , ma non ostruita che tende a penetrare la massa , ridisegnandone la continuit dopo stimolo secretinico , il cosiddetto segno del dotto penetrante [ 15 , 30 ]  . 
lo stimolo secretinico aumenta la secrezione di fluidi nel sistema duttale e contemporaneamente aumenta il tono sfinteriale delloddi incrementando quindi la quantit di liquido nel dotto pancreatico e migliorandone la visualizzazione con cprm . 
lo studio cprm con secretina si gi dimostrato utile per la delineazione del dotto pancreatico e per la valutazione del comportamento dinamico del dpp [ 15 , 3134 ]  . 
in 6 casi , nel dettaglio , in 1 caso di aip diffusa e in 5 casi di aip focale della testa ( ovvero nel 43% della casistica globale e nel 50% delle aip focali ) , il restringimento segmentale focale del dpp si risolto dopo lo stimolo secretinico , indicando che il dotto era penetrante . 
in questi casi , soprattutto nelle aip focali della testa , la somministrazione di secretina stata di fondamentale importanza per escludere un adenocarcinoma duttale , e per eludere la necessit di intervento chirurgico . 
 tuttavia , questo studio presenta inevitabili limitazioni . in primo luogo , la popolazione esaminata comprende solo casi di aip e , in secondo luogo , essendo una valutazione retrospettiva , non tutti i pazienti sono stati sottoposti alla prova con secretina . 
 in conclusione , in presenza di un appropriato contesto clinico , le indagini rm e cprm consentono lesatta classificazione della pancreatite autoimmune nella maggior parte dei casi , grazie alla dimostrazione del cercine di impregnazione periferica e al pattern di restringimento duttale del sistema biliare e pancreatico e alle modificazioni indotte dalla stimolazione con secretina . 
i valori di attenuazione medi degli insulinomi e del pancreas normale nelle fasi precontrastografiche arteriosa e portale erano , rispettivamente , pari a 40 , 58 , 75 uh , 114 , 4827 , 30 uh , 112 , 1919 , 52 uh e 44 , 566 , 48 uh , 81 , 1615.22 uh , 90 , 5413 , 80 uh , con differenze statisticamente significative in tutti i casi ( p < 0 , 000 )  . 
lenhancement degli insulinomi nelle fasi arteriosa e portale era pari a 74 , 0329 , 51 uh e 70 , 9021 , 93 uh , rispettivamente , senza differenze statisticamente significative ( p = 0 , 499 )  . 
lacquisizione delle immagini nella fase arteriosa la pi utile nel riconoscimento di questi tumori . radiol med ( 2009 ) 114 : 12321238 1233 keywords pancreas pancreas neoplasms insulinomas computed x - ray tomography parole chiave pancreas tumori pancreatici insulinomi tomografia computerizzata introduction insulinoma is the most common syndromic pancreatic endocrine tumour . 
in addition , patients may report symptoms such as paroxysmal headache , tiredness , palpitations , sweating , fainting and morning unconsciousness [ 1 ]  . surgery is the preferred treatment , and patients may have a long survival without symptoms . 
some experts believe that because an insulinoma has been confirmed biochemically , preoperative imaging is not necessary , as nearly all insulinomas are located in the pancreas and can be detected with intraoperative inspection , palpation and sonography . 
for example , insulinomas located near the surface of the gland may undergo simple enucleation ; however , if tumours are located deep in the gland , partial pancreatectomy may be required . 
thus , if the sensitivity of noninvasive preoperative imaging improves , preoperative imaging evaluation is essential to insulinomas [ 3 ]  . along with the development of multidetector - row computed tomography ( ct ) and improvement and optimisation of scanning techniques , we are now able to rapidly image the pancreas during multiple phases of contrast enhancement . 
some experts have shown improved conspicuity of islet - cell tumours during the arterial phase of enhancement , whereas others have shown portal - venous - phase imaging to be more beneficial [ 35 ]  . 
 the aims of this study were to review our experience in the preoperative detection of insulinomas by using pancreatic thin - section dual - phase helical ct technology , quantitatively determine the enhancement characteristics and relative conspicuity of insulinomas in the different phases of pancreatic enhancement , establish the ability of multidetector - row ct to localise these tumours , and analyse the cause of false negative findings on multidetector helical ct . 
 materials and methods patients during the 6 - year period from 2002 to 2007 , 56 patients suspected of having insulinoma on the basis of clinical symptoms and laboratory data underwent surgery , and in 52 cases the insulinoma was proved by pathology . 
all patients in our study group presented with classic whipples triad , and most of them suffered from paroxysmal headache , weakness , palpitations , sweating , fainting , blurring of vision or morning unconsciousness . 
 ct acquisition all 46 patients underwent contrast - enhanced scanning with a 4or 16 - detector helical ct scanner ( light speed , ge healthcare ) ; ten patients ( group 1 ) only underwent portal - phase ct scanning , and the remaining 36 patients ( group 2 ) underwent dual - phase ct scanning . 
all patients were administered 500800 ml of water 30 min prior to the study and an additional 250300 ml of water immediately prior to the study to achieve adequate distension of the stomach and duodenum . the unenhanced ct acquisition was obtained with slice thickness and slice intervals of 7.5 mm ( 250 ma , 120 kv ) , and the enhanced ct acquisition was obtained with slice thickness and slice intervals of 3.755 mm ( 280300 ma , 120 kv ) covering the liver and pancreas . 
 all patients were given nonionic iodinated contrast material ( omnipaque 300 mg i / ml , ge healthcare ) administered with a power injector ( ulrich medical , germany ) at a rate of 34 ml / s through a 21 - gauge angiocatheter placed in an antecubital vethe volume of contrast material delivered was 90120 ml . dual - phase ct acquisition used a bolus - tracking 1234 radiol med ( 2009 ) 114 : 12321238 program ( smartprep , ge healthcare ) that monitored contrast enhancement after injection of contrast medium before initiation of the diagnostic scans . 
real - time lowdose ( 120 kvp , 50 ma ) serial monitoring scanning was initiated 1012 s after the start of the contrast injection , and the scanning threshold value was set at 80 hu . 
when the ct value of the aorta was > 80 hu , the arterial - phase acquisition was triggered , and the portal - phase acquisition was started 30 s after arterial phase acquisition . surgical and pathologic analysis we reviewed the surgical and pathology reports providing the diagnosis based on the gross , microscopic and immunohistochemical examination of surgical tissues . 
one roi was obtained from the lesion on corresponding unenhanced , arterial and portal - venous phases , and three rois that excluded lesions and edges of the organ , and vascular or ductal structures were obtained from homogeneous areas of the head , body and tail of the pancreas for a same section . 
then , the attenuation values were averaged , and lesion contrast enhancement was obtained by subtracting the average attenuation value on unenhanced images from the average attenuation value on contrast - enhanced images . 
students t test was used for qualitative analysis , and p0.05 were considered statistically significant . results surgical and pathology findings thirty - six patients were treated with pancreatic insulinoma enucleation or resection , two patients with pancreatic insulinoma enucleation under celioscopy , six patients with partial pancreatectomy and two patients with the child procedure . all patients were affected by insulinoma , as proved by the pathology reports describing the gross , microscopic and immunohistochemical examination of surgical tissues . imaging studies tumour detection sensitivities of ct for detecting insulinomas are listed in table 1 . 
dual - phase helical ct ( group 2 ) was significantly superior to single - phase helical ct ( group 1 ) ( 2 = 4.064 ; p = 0.044 ) with regard to sensitivity for depicting pancreatic insulinoma . 
1 caso di insulinomi multipli , due dei quali localizzati nella testa e uno nella coda del pancreas . radiol med ( 2009 ) 114 : 12321238 1235 pancreas : 15 ( 32.6% ) were located in the head of the pancreas , seven ( 15.2% ) in the neck , 13 ( 28.3% ) in the body and 11 ( 19.6% ) in the tail in 44 patients ; another two ( 4.3% ) tumours were ectopic in two patients and located near the left and right kidney , respectively . 
la massa della coda del pancreas appare isodensa nellesame senza mezzo di contrasto . essa presenta enhancement marcato nella fase arteriosa e lieve nella fase venosa portale . defined edges ( n = 31 / 86.1% ) and only five ( 13.9% ) had illdefined edges . 
contrast enhancement differences between the normal pancreas and the lesions were significantly higher during the arterial phase than the portal - venous phase ( p = 0.011 ) , resulting in greater tumour conspicuity . most insulinomas were hyperenhancing on at least one of the postcontrast phases ( n = 36 ) ; 28 ( 77.8% ) of them enhanced homogeneously , three ( 8.3% ) enhanced inhomogeneously and five ( 13.9% ) showed circular enhancement with filling in the portal venous phase . 
5 il grafico evidenzia l ' incremento medio di densit degli insulinomi e del pancreas normale e della differenza di contrasto tumore / pancreas nelle fasi precontrastografica , arteriosa e venosa portale . unenhanced phase portal investigated the enhancement characteristics of pancreatic insulinomas . 
 [ 5 ] reported that portal - venous - phase images were more helpful than arterial - phase images for detecting pancreatic islet - cell tumour on the basis of receiver operating curve analysis of qualitative data . 
 [ 8 ] reported that the sensitivity for detecting pancreatic adenocarcinoma was significantly greater during the pancreatic than the arterial phase and that the mean attenuation of the pancreas in all patients was greatest in the pancreatic phase at 107 hu versus 65 hu in the arterial phase . 
further studies will be necessary to assess this issue for detecting insulinomas . other investigators have shown good sensitivities with other modalities , such as magnetic resonance imaging ( mri ) and endoscopic sonography [ 5 , 6 , 1016 ]  . 
 [ 5 ] found that mri and biphasic ct had similar effectiveness in detecting islet - cell tumours . 1238 radiol med ( 2009 ) 114 : 12321238 gouya et al . 
 [ 6 ] found that the diagnostic sensitivity for dual - phase helical ct and endoscopic sonography was > 90% and for combined biphasic thin - section helical ct and endoscopic sonography was 100% . 
however , in our experience , pancreatic biphasic helical ct with thin sections is the preferred modality for suspected insulinomas . if there is a contraindication to ct or negative findings on ct , we will perform mri examination . 
in addition , the study may have had a selection bias , as some patients may not undergo ct examination if other modalities were performed , such as mri or endoscopic sonography as the initial modality and had positive results . 
therefore , our results cannot indicate the absolute sensitivity of ct in the prospective diagnosis of pancreatic islet - cell tumours . we can draw several conclusions from our evaluation of a biphasic multidetector - row ct protocol for detecting pancreatic insulinomas . 
first , dual - phase ct has a promising sensitivity in detecting pancreatic insulinomas . second , the acquisition of images in the arterial phase is helpful to detect insulinomas because tumour - to - gland attenuation differences are significantly conspicuous . finally , using a standard and uniform ct scanning technique and being aware of atypical appearances such as isoattenuating , extruded , pedunculated or ectopic insulinomas will help obtain the highest possible sensitivities . 
hence they require a multidisciplinary and multispecialty approach , which must begin with an accurate medical examination , conducted in compliance with the lege artis principles and with respect for the victims dignity . 
this paper describes the radiologists key role in identifying physical injuries due to child abuse , in accordance with current regulations . keywords child abuse forensic radiology battered child syndrome riassunto labuso ed il maltrattamento sui minori una tematica di grande attualit con notevoli risvolti di carattere sociosanitario che interessano il medico sia come cittadino che in qualit di esercente una professione sanitaria . 
la molteplicit e la peculiarit di alcune lesivit di abuso , siano queste di tipo fisico , psichico o sessuale necessitano di un approccio multidisciplinare e polispecialistico che non pu prescindere tuttavia da una visita medica che , nel rispetto della dignit della presunta vittima , sia effettuata secondo i criteri propri della lege artis . 
il presente articolo descrive il ruolo del radiologo nella valutazione degli abusi fisici perpetrati a danno di minori alla luce della normativa vigente . parole chiave abuso sui minori radiologia forense sindrome del bambino battuto introduction introduzione in 1946 , radiologist john caffey following a . 
this was a concise , pragmatic definition of what is not so much a clinical syndrome as a sociocultural phenomenon that had strong repercussions on trauma medicine and on physicians themselves [ 1 ]  . 
tardieu del 1860 definiva in maniera concisa e pragmatica pi che una sindrome clinica un fenomeno socio - culturale che aveva pesanti riflessi sulla medicina traumatologica e notevoli ricadute sui medici [ 1 ]  . radiol med ( 2009 ) 114 : 13561366 1357 an accurate definition of child abuse is given in the world health report 2002 : child abuse or maltreatment constitutes all forms of physical and / or emotional illtreatment , sexual abuse , neglect or negligent treatment or commercial or other exploitation , resulting in actual or potential harm to the childs health , survival , development or dignity in the context of a relationship of responsibility , trust or power [ 2 ]  . 
therefore , child abuse includes the results not only of actions ( sexual or other kinds of violence ) but also of neglect or deprivation ( lack of care , attention , affection )  . 
in both cases , the physical examination alone , for legal purposes as well as for diagnosis and therapy , is unable to confirm or exclude suspected abuse ( at least from a medical point of view ) without the use of radiological tools . 
thus , radiology is an essential tool in medicolegal investigations of physical child abuse , which gives the specialist a proper understanding of type , extent and age of the injury , and the dynamics behind it ( aetiopathogenesis )  . extent of child abuse child abuse has become a very worrying issue with an impact on the whole of society . 
introna states : the abused child is the expression of multiple problems of a legal , cultural and economic nature involving his / her family at a given moment in time , and therefore he / she is the clue to pain that goes beyond his / her own person [ 3 , 4 ] as requested . child abuse is often rejected or negated , and it is always underreported . 
this is especially true in southern italy , where it often occurs within a complex family setting , which makes it difficult to evaluate the victim and type of abuse suffered . 
the child presents distinctive injuries , often inflicted with bare hands or by using various objects ( sticks , chains , strings , cigarettes or other sources of heat )  . 
this form of child abuse results from indeuna precisa e completa definizione pu essere tradotta dalla world health organization ( who ) che nel 2002 cos recitava : per abuso allinfanzia si intendono tutte le forme di cattiva salute fisica e / o emozionale , abuso sessuale , trascuratezza o negligenza o altro che comportino un pregiudizio reale o potenziale per la salute del bambino , per la sua sopravvivenza , per il suo sviluppo o per la sua dignit nellambito di una relazione caratterizzata da responsabilit , fiducia o potere [ 2 ]  . 
in relazione a tanto , labuso sui minori deve intendersi comprensivo sia degli atti ( violenze vere e proprie , non necessariamente sessuali ) che delle carenze e le deprivazioni ( sottrazione delle cure , dellattenzione , dellaffetto )  . 
in entrambe le ipotesi laccertamento medico , effettuato a fini giuridici lindagine oltrech diagnostico - terapeutici , utilizzer radiologica quale importantissimo strumento diagnostico per fondare ovvero escludere , quanto meno da un punto di vista medico , il sospetto di un abuso . 
lutilizzo di immagini digitali , utile ad una pi fine valutazione della lesione fisica osservata e correlato ad una specifica competenza del radiologo riguardo allepoca di mineralizzazione ossea trauma ed dei segmenti scheletrici allidoneit lesiva del trauma stesso ( eziopatogenesi ) , rende la valutazione radiologica strumento fondamentale nella valutazione medico - legale degli abusi fisici a carico di minori . interessati dal dimensioni del problema il maltrattamento dei bambini ha assunto dimensioni tali da costituire un serio motivo di preoccupazione da parte di tutta la societ civile . 
introna in un suo felicissimo intervento recita : il bambino maltrattato testimone di problemi multipli che investono il nucleo familiare in quel determinato momento storico , giuridico , culturale ed economico e quindi egli la spia di una sofferenza che va oltre la sua persona [ 3 , 4 ]  . 
questo vero soprattutto nelle regioni del sud dove la complessit dellambiente familiare , in cui , nella maggior parte dei casi , avviene tale tipologia di reati , rende estremamente difficile unesatta valutazione delle vittime e del tipo di abuso subito . 
these injuries are a sign of neglect by those responsible for the childs care because the injuries were not treated promptly and eventually became chronic . this paper focuses only on the battered child syndrome because this feature directly concern radiologists . radiological features of child abuse the radiologist is consulted during the investigation of battered child injuries , especially those related to cranial and musculoskeletal injuries , and he or she must be cognizant of the often critical features of such injuries . 
later , in 1962 , kempe coined the expression battered child syndrome [ 6 ] to group together the clinical and radiological signs of beaten children and to draw attention of the medical community to the problem , which , although steadily escalating , had been totally disregarded . incomplete or complete fractures are generally observed in the upper and lower limbs and in the ribs directly struck by blows , punches or kicks whilst the child is hunched up for self protection . 
it must be pointed out that the radiologist is always able to identify an injury but can never be absolutely sure of its mechanis nevertheless , some fractures are highly specific of the battered child syndrome , i.e. 
the knee ( proximal tibia ) , the wrist , the femur and the humerus are the most frequent sites of such fractures , which are rarely accompanied by a periosteal reaction and seldom cause growth disturbances or bending deformities [ 10 , 11 ]  . 
con questo termine si vogliono comprendere tutti quei maltrattamenti occulti costituiti da mancanza di protezione , di educazione , di amore e di accettazione che si estrinsecano con una serie di trascuratezze , rifiuti e sfruttamenti che sfociano nellabbandono psicologico ma , talvolta , possono determinare il decesso della vittima per inanizione . 
oltre alle sequele psicologiche , si possono riscontrare anche dermatosi comuni croniche da scarsa igiene , ripetute infezioni , ascessi e fistole che sono indicative della trascuratezza di chi si dovrebbe prendere cura del minore ed indicare affezioni perduranti nel tempo e non curate tempestivamente . in questo articolo tratteremo esclusivamente la valutazione specialistica degli abusi fisici sui minori ( battered child syndrome ) , i cui aspetti coinvolgono in maniera diretta e pressante lo specialista radiologo . aspetti radiologici degli abusi fisici sui minori lo specialista radiologo consultato in corso di accertamenti sul bambino battuto ( abusato da un punto di vista fisico ) , prevalentemente nei casi di violenza da trauma sullapparato cranico - muscolo - scheletrico e deve conoscerne gli aspetti , a volte del tutto peculiari , con cui questi possono palesarsi . 
 gi caffey nel 1957 aveva messo in evidenza ed illustrato alcuni nuovi e particolari tipi di frattura e di lesioni ossee che si possono riscontrare nei bambini battuti , specie nei pi piccoli [ 5 ]  . 
kempe successivamente , nel 1962 , allo scopo di puntare lattenzione del mondo scientifico su un problema sociale assolutamente trascurato , ma in fase di importante espansione , coni il termine di battered child radiol med ( 2009 ) 114 : 13561366 1359 fig . 
b frattura a manico di secchio . table 1 differential diagnosis between accidental and nonaccidental injuries abusive fractures metaphysis proximal tibia femur accidental fractures epiphysis distal tibia other sites with high energy mechanism tabella 1 diagnosi differenziale tra fratture accidentali e da abuso fratture da abuso sede metafisaria tibia prossimale femore fratture accidentali epifisi tibia distale altri siti con meccanismo elevata energia the lower limbs of children who are not ambulatory ( table 1 )  . 
 the so - called toddlers fracture , namely , the spiroidal fracture of the distal portion of the tibia , is a very common accidental injury in the paediatric age . 
on the other hand , if the same fracture is detected with identical radiological features in children not yet ambulatory , it should undeniably be considered as resulting from abuse [ 1214 ]  . 
multiple syndrome per indicare tutto il corteo di sintomi clinici e radiologici che caratterizzano gli aspetti traumatici del bambino battuto [ 6 ]  . come aspetto generale le fratture incomplete o complete interessano lo scheletro appendicolare , specie gli arti superiori ed inferiori e le coste che sono oggetto della violenza soprattutto a seguito di percosse , pugni e calci a bambini rannicchiati in atteggiamento di autoprotezione . 
raro che le lesioni scheletriche siano fatali e rare sono anche le lussazioni e le lesioni vertebrali , in ogni caso opportuno ricordare che il radiologo pu identificare la lesione , ma mai determinarne con certezza le modalit . 
tuttavia esistono alcune particolarit del tipo di fratture che possono indirizzare o far propendere il radiologo verso un trauma non accidentale e quindi da riferire a sindrome da bambino battuto . 
these are caused by traumatic anteroposterior compression of the rib cage , and the radiologist must bear in mind the fact that they cannot be caused even by the use of improper resuscitation procedures [ 1518 ]  . 
as they are extremely serious injuries , they may appear to indicate abuse , but , at the same time , they are very often associated with accidents such as tumbling out of bed , falling off the changing table or down the stairs . 
8a , b frattura lineare del parietale destro nella battered child syndrome . in the case of indirect head injuries , guthkelchi [ 24 ] made a further distinction by introducing shaken baby syndrome , in which lesions are secondary to the attendant axonal damage . when the infant is shaken violently and head movements are not synchronised with the rest of the body , in 90% of cases , this produces retinal haemorrhage , but it may also produce subdural haematoma ( related to vascular tears ) and osseous injuries ( detectable only by means of magnetic resonance imaging )  . 
 we must remember the possibility , even if remote , of finding damage to parenchymal organs , which is regularly accompanied by other types of lesions ( haematoma , pseudocyst , etc . ) that will be seen only through ultrasound or computed tomography [ 26 ]  . current regulations and forensic medicine considerations the entire medical profession , including radiologists , is bound by italian law to cooperate with police authorities to prevent and combat crime . 
evidenti le lesioni ipodense cerebrali , esiti di trauma da scuotimento . deontology code ( 2006 ) states that the doctor must protect children in cases of physical or psychological maltreatment or sexual abuse , and in the event of opposition from the legal guardians , the doctor must report to the appropriate legal authorities . 
361medici specialisti in radiodiagnostica , sono previsti , dalla legislazione attualmente vigente , obblighi giuridici finalizzati a forme di collaborazione con lautorit giudiziaria , al fine di prevenire e contrastare la criminalit . 
32 del codice di deontologia medica ( 2006 ) che prevede , oltrech una specifica tutela sui minori in casi di maltrattamenti fisici o psichici , violenze o abusi sessuali anche la possibilit in caso di opposizione dei legali rappresentanti alla necessaria cura dei minori di ricorrere alla competente autorit giudiziaria . 
 doveroso ricordare che la violazione del dovere o omissione di inoltrare il referto / rapporto costituisce un delitto contro lamministrazione della giustizia ; la mancata ottemperanza di tale dovere prevede automaticamente il reato di omissione datti dufficio qualora si accerti che il sanitario abbia , con coscienza e volont , voluto omettere o ritardare la presentazione del referto [ 28 ]  . 
failure to comply with this duty is malfeasance in office when it is ascertained that the health professional wilfully and knowingly failed to report or delayed reporting the crime [ 28 ]  . 
 conclusions too often do we hear about presumed physical , psychological or sexual child abuse based on psychological evaluations without any reference to the medical follow - up or specialistic investigations that could unequivocally confirm or exclude this suspicion . 
in fact , they have the knowledge to determine the time of mineralisation of skeletal segments , reconstruct the mechanisms of injuries to the osteoarticular system and interpret imaging findings . reporting child abuse is always a very delicate question , especially because it always involves the families . 
although a mere suspicion of abuse is sufficient for current regulations , the professional must be aware of the consequences of a misdiagnosis on the family and on the victitherefore , the radiologist is required to make both a cultural effort in understanding the problem and a methodological effort in evaluating the case in terms of collecting a complete clinical history and carefully interpreting the imaging findings . implementation of guidelines and protocols such as those proposed by the american college of radiology [ 29 ] , together with cooperation between several specialists , will help produce a methodologically flawless evaluation of the true extent of this serious problem , which has been neglected and underreported for too long . 
la particolare complessit della fenomenologia di abuso sui minori necessita , ai fini di un corretto inquadramento del fenomeno stesso , di un approccio multidisciplinare da parte di una equipe di specialisti , laddove di fondamentale importanza appare il contributo del radiologo pediatrico . 
linterpretazione delle lesioni eventualmente riscontrate con limaging non pu essere di pertinenza del pediatra ovvero dellortopedico ; solo il radiologo ha le competenze per discernere laddove possibile caratteristiche proprie di un trauma accidentale oppure di un abuso . 
infatti , la conoscenza della mineralizzazione ( epoca di ossificazione ) dei segmenti scheletrici , dei meccanismi traumatici che sottendono la genesi di lesivit soprattutto dellapparato osteo - articolare nonch la loro interpretazione diagnostica , di pertinenza del radiologo che ne certamente lunico e il migliore conoscitore . 
se da un lato la normativa vigente prevede che sia sufficiente il sospetto e non la certezza , per denunciare siffatto reato altres chiaro che una erronea diagnosi di abuso pu sconvolgere un nucleo familiare , ivi inclusa la vittima . 
allo specialista in radiodiagnostica richiesto pertanto uno sforzo culturale nei termini di comprensione / conoscenza del problema e metodologico nella valutazione di tale fenomeno che non potr prescindere dalla raccolta di un completo raccordo anamnestico , oltrech dalla successiva analisi dellimaging . 
knauth springer - verlag berlin heidelberg , 2009 isbn 978 - 3 - 540 - 72784 - 5 published online : 20 november 2009 springer - verlag 2009 this is an easy to read and very informative book on the use of contrast media in daily diagnostic practice . 
going through its 29 chapters , plus the appendix containing the european society of urogenital radiology ( esur ) guidelines on contrast media ( version 7.0 ) and one page dedicated to the official publications from the same society , provides the reader with all possible information on the use of different contrast media . the second edition of the book is the result of in depth study and research by the academic members of the esur contrast medium safety committee updating in more than one chapter , information on the use of gadolinium - based contrast media and their possible complications , in particular those related to the alarming nefrogenic systemic fibrosis ( nsf )  . 
 nsf , whose real cause is yet unknown , has come slowly to the attention of the medical community in the past few years rendering what was thought ( gadolinium contrast agents ) to be a very safe diagnostic tool as a possible , harmful instrument in the radiologists hands . since in the daily practice there is not only gadoliniumbased contrast media in use , much discussion is devoted also to the other different types of contrast media and their use . 
one will find information on ionic and non - ionic as well as barium and ultrasound contrast media , detailed information on meta - analysis in research , and information on the rules that must be followed in contrast - media research , approval , manufacturing , and clinical application . great attention is given to the discussion of contrast - media related complications : this issue is thoroughly discussed in the relevant section of many chapters and thoroughly reviewed in the esur appendix guidelines . 
one should realize that each time a contrast medium is used , however safe it might be considered , it can given the different individual conditions of the patient create adverse reactions . 
scorrendo i 29 capitoli che lo compongono , pi lappendice che elenca le linee guida della european society of urogenital radiology ( esur ) sulluso dei mezzi di contrasto nella sua pi recente versione ( 7.0 ) e la pagina dedicata allelenco delle pubblicazioni ufficiali della stessa societ , il lettore trover tutte le informazioni possibili circa luso dei diversi mezzi di contrasto . la seconda edizione il risultato di una gran lavoro di ricerca e studio da parte dei membri accademici del comitato esur sulla sicurezza dei mezzi di contrasto , lavoro che aggiorna , in particolare in pi di un capitolo , le informazioni circa limpiego dei mezzi di contrasto a base di gadolinio e le loro possibili complicanze , in particolare quelle riferibili alla pi temuta e cio la fibrosi nefrogenica sistemica ( nsf )  . la nsf , le cui cause sono ancora sconosciute , stata riconosciuta con lentezza e ritardo dalla comunit medica negli ultimi anni , rendendo quelli che erano pensati come mezzi di contrasto sicuri ( cio a base di gadolinio ) , quali possibili strumento di danno nelle mani del radiologo . dal momento per che nella pratica quotidiana non esiste solo limpiego dei mezzi di contrasto a base di gadolinio , una parte consistente del volume e di discussione viene riservata a tutti i restanti tipi di mezzo di contrasto . 
il lettore trover informazioni sui mezzi di contrasto ionici e non ionici , su quelli baritati e per impiego in ecografia : il tutto correlato da informazioni precise sulla meta - analisi nella loro ricerca ed informazioni importanti circa le regole che debbono essere impiegate nella ricerca , approvazione , preparazione ed applicazione clinica dei mezzi di contrasto . 
 grande attenzione viene dedicata alla discussione delle complicanze provocate dallimpiego dei mezzi di contrasto : questo tema viene discusso con precisione nella parti relative di molti capitoli e ben riassunta nellappendice sulle linee guida dellesur . 
ci si deve rendere conto che tutte le volte che viene utilizzato un mezzo di contrasto , per quanto sicuro lo si possa ritenere , questi pu , in funzione anche della differenti condizioni cliniche di ciascun paziente in cui viene impiegato , provocare delle reazioni , reazioni che possono essere assai lievi ed a risoluzione rapida e spontanea ma anche , 1384 radiol med ( 2009 ) 114 : 13831384 resolving over a very short period to very severe and unexpected , requiring immediate and correct treatment . how many people know the correct doses of adrenaline or atropine to be used in an emergency situation ? are there any emergency procedure - charts in your diagnostic radiology suites ? in this respect some of the pages are unnerving and hair - raising . 
 in summary , this is a very useful book and a highly recommended resource text for every radiology department , it should be easily accessible not only for the old staff radiologists but most importantly for those who are in training . al contrario , essere piuttosto evidenti ed impreviste , necessitanti un intervento terapeutico immediato e corretto . quante persone conoscono la dose corretta di adrenalina o di atropina da utilizzare in situazioni di emergenza ? nelle sale di diagnostica radiologica vi sono cartelli riportanti le procedure da seguire nelleventuale emergenza ? da questo punto di vista alcune della pagine del volume sono piuttosto preoccupanti , tali da far venire i brividi . 
giacomo , unit operativa di radiodiagnostica , monopoli , bari , italy 3universit degli studi di catania , cattedra di radiodiagnostica , azienda policlinico di catania gaspare rodolico , catania , italy correspondence to : p . 
chieffi 40 , 70051 barletta , italy , tel . : + - 39 - 340 - 0781993 , fax : + 39 - 088 - 1733866 , e - mail : paomi03@libero.it received : 11 october 2008 / accepted : 5 march 2009 / published online : 9 november 2009 springer - verlag 2009 abstract purpose . 
of these 31 patients , we included in the study 22 patients ( mean age 53 years ; range 4657 years ) who underwent liver transplantation within 1224 h after mr examination . 
dei 31 pazienti selezionati per il trapianto dorgano , sono stati inclusi nello studio 22 pazienti , di et media di 53 anni ( range 4657 anni ) , sottoposti a trapianto di fegato a distanza di circa 1224 ore dallesecuzione dellesame rm . i pazienti inclusi nello studio sono stati sottoposti ad esame rm con un apparecchio da 1 , 5 t . 
unenhanced baseline mr imaging correctly identified and characterised 20 lesions , equal to 33.90% of all lesions : 6 hcc , 12 dn and 2 dn with a subfocus of hcc . 
spio - enhanced mr imaging showed greater sensitivity detecting and characterising 45 lesions , equal to 76.27% of all lesions identified at histology : 14 hcc , 27 dn and 4 dn with subfocus of hcc . 
spio - enhanced mr imaging proved to be of value in detecting and characterising lesions in the cirrhotic liver , allowing differentiation of dn from hcc and providing an early diagnosis of neoplastic degeneration of dn . keywords spio dysplastic nodules hepatocellular carcinoma magnetic resonance imaging 14 hcc , 3 sono risultati ben differenziati , 8 moderatamente differenziati , 3 scarsamente differenziati . dei 39 nd , 28 erano a basso grado di malignit e 11 ad alto grado di malignit . 
con lesame rm di base abbiamo identificato e caratterizzato correttamente 20 lesioni , pari al 33 , 90% delle lesioni complessivamente individuate , di cui 6 hcc , 12 nd e 2 nd con foci di hcc . 
lesame rm , eseguito dopo somministrazione di mdc spio , ha mostrato una maggiore sensibilit identificando e caratterizzando complessivamente 45 lesioni , pari al 76 , 27% delle lesioni totali individuate con lesame istologico : 14 hcc , 27 nd e 4 nd con foci di hcc . 
i falsi negativi con la rm - spio sono stati 12 noduli displasici , pari al 31% , che allesame istologico sono risultati essere nd a basso grado di malignit e con diametro inferiore al c conclusioni . 
la rm con utilizzo di mdc spio , nella nostra esperienza risultata utile nella identificazione e caratterizzazione delle lesioni epatiche nel fegato cirrotico mostrandosi in grado di differenziare i noduli displasici dagli hcc e ha consentito una diagnosi precoce della trasformazione carcinomatosa dei noduli displasici . parole chiave spio noduli displasici carcinoma epatocellulare risonanza magnetica introduction introduzione cirrhosis is a progressive , diffuse disease of the liver characterised by hepatocyte necrosis , fibrosis , distortion of the normal hepatic architecture and a spectrum of nodular lesions that includes regenerative nodules ( rn ) , dysplastic nodules ( dn ) and hepatocellular carcinomas ( hcc ) [ 1 ]  . 
in italy , the likelihood of developing hcc secondary to cirrhosis ranges from 2% to 5% [ 3 ]  . in recent decades , the survival of hcc patients has been significantly increased by improvements and refinements in both nonsurgical treatments , such as percutaneous ethanol injection ( pei ) , thermoablation and lipiodol computed tomography ( ct ) ; and surgical treatments , such as liver resection and transplantation . 
the results of these treatments are , however , heavily dependent on accurate disease staging , so that the early diagnosis of hcc and its precursor , the dn , is mandatory [ 4 , 5 ]  . the screening protocol for high - risk cirrhotic patients entails ( ultrasonographic ) imaging and alpha - fetoprotein testing at 3to 6 - month intervals . 
unfortunately , an la cirrosi un progressivo e diffuso processo patologico del fegato caratterizzato da necrosi epatocitaria , fibrosi , sovvertimento della normale architettura epatica e dalla presenza di uno spettro di lesioni nodulari che include noduli di rigenerazione ( nr ) , noduli displasici ( nd ) ed epatocarcinomi ( hcc ) [ 1 ]  . 
in italia la probabilit annuale di sviluppare hcc su cirrosi epatica varia dal 2% al 5% [ 3 ]  . negli ultimi decenni si verificato un significativo incremento della sopravvivenza dei pazienti affetti da tale patologia per il miglioramento e laffinamento sia delle terapie che si basano sui trattamenti locali , come liniezione percutanea di etanolo ( pei ) , la termoablazione e la lipiodoltomografia computerizzata , sia di quelle che si basano invece su tecniche chirurgiche , quali la resezione o il trapianto . 
i risultati di queste terapie sono tuttavia fortemente condizionati dallo staging della malattia ed , quindi , estremamente importante effettuare una diagnosi precoce ed accurata dellhcc e del suo precursore , il nd [ 4 , 5 ]  . il protocollo di screening , nei pazienti cirrotici ad radiol med ( 2009 ) 114 : 12671282 1269 elevation of alpha - fetoprotein levels is neither sensitive nor specific enough to be used to screen for hcc . 
this test may , in fact , fail to detect small tumours ( < 2 cm ) , and high alpha - fetoprotein levels are seen in only 50% of patients with hcc and 26% of those with dn [ 6 , 7 ]  . imaging techniques therefore play a primary role in the diagnosis of focal lesions in the cirrhotic liver , even though there is no general agreement as to the most appropriate modality for the study of this disease . 
the literature reports conflicting data regarding not only the diagnostic capabilities of the different imaging modalities ultrasonographic ( us ) , ct and magnetic resonance ( mr ) imaging in the detection and characterisation of hcc and dn , but also the possibility of arriving at a differential diagnosis among the different lesions . 
histological analysis of the entire cirrhotic liver and a short interval between mr imaging and transplantation are factors that , if neglected , may lead to considerable variability among results . 
indeed , a long interval between mr examination and transplantation could mean the transformation of a dn into an hcc , whereas a histological examination limited to biopsies or specimens from partial liver resection could preclude identification of other nodular lesions in the liver parenchyma [ 812 ]  . in recent years , investigators have emphasised the value of hepatospecific superparamagnetic contrast agents based on iron oxide particles ( spio ) in the detection and characterisation of focal lesions with mr imaging , and their use has been advocated for staging patients scheduled for surgical or locoregional treatment of malignant focal hepatic lesions [ 13 , 14 ] and for monitoring patients with cirrhosis [ 1517 ]  . the rationale for using spio contrast agents to differentiate among the wide range of nodular lesions seen in cirrhosis is not based on vascular changes but on the cellular changes rn undergo as they develop into dn and then into hcc . 
in fact , during this process , the number and function of kupffer cells , the cells that accumulate spio , decrease [ 10 , 1820 ] , and some investigators have reported that hcc and dn show different enhancement patterns after spio administration . 
dn , on the other hand , have preserved cellular composition and function and hence greater spio accumulation , with the result that they appear markedly hypointense even relative to the surrounding liver . 
these different enhancement patterns led researchers to hypothesise that it was possible to differentiate between these two lesions and identify the transformation of a dn into an hcc at an early stage . the aim of this study was to evaluate the potential of spio - mr imaging in the detection and characterisation of elevato rischio , prevede lesecuzione di esame ecografico e dei dosaggi dellalfa - fetoproteina ( afp ) ad intervalli di 36 mesi . 
tale test , infatti , non sempre in grado di rilevare piccoli tumori ( < 2 cm ) ed elevati livelli di afp sono presenti solo nel 50% di pazienti con hcc e nel 26% di pazienti con nd [ 6 , 7 ]  . le tecniche dimaging svolgono pertanto un ruolo fondamentale nella diagnostica delle lesioni focali nella cirrosi epatica ; tuttavia non vi uniformit di vedute su quale sia la metodica pi idonea per lo studio di tale patologia . 
in letteratura sono piuttosto controversi i dati circa le reali potenzialit diagnostiche delle diverse tecniche dimaging ecografia ( us ) , tomografia computerizzata ( tc ) e risonanza magnetica ( rm ) nella identificazione e nella caratterizzazione degli hcc e dei nd , nonch riguardo la possibilit di effettuare una diagnosi differenziale tra le diverse lesioni . questo si verifica per mancanza , in tutti i lavori finora pubblicati , di uniformit dei parametri di valutazione attendibili ; infatti , lanalisi istologica di tutto il fegato cirrotico ed il breve timing tra lesecuzione dellesame rm e il trapianto sono fattori che se non presi in considerazione possono portare una notevole variabilit nei risultati . 
infatti , un lungo tempo intercorso tra lesame rm e lesecuzione del trapianto potrebbe portare alla trasformazione di un nd in hcc , mentre uno studio anatomo - patologico limitato alle sole lesioni sottoposte a biopsia o a resezione parziale dellorgano potrebbe escludere lidentificazione di altre lesioni nodulari nel restante parenchima epatico [ 812 ]  . negli ultimi anni stata sottolineata lutilit dei mezzi di contrasto ( mdc ) superparamagnetici epatospecifici , a base di particelle di ossido di ferro ( spio ) , per lidentificazione e la caratterizzazione delle lesioni focali epatiche con rm ed il loro uso stato proposto per lo staging di pazienti da sottoporre a trattamenti chirurgici o loco - regionali per lesioni focali epatiche maligne [ 13 , 14 ] e nel follow - up dei pazienti portatori di cirrosi epatica [ 1517 ]  . il razionale di impiego dei mdc spio per la diagnosi differenziale tra il vasto spettro di lesioni nodulari presenti nella cirrosi epatica , si basa , pi che sulle modificazioni vascolari , sulle alterazioni cellulari che i nr subiscono nella loro trasformazione in nd prima e in hcc poi ; infatti , in rapporto a tale evoluzione , decrescono progressivamente il numero e la funzionalit delle cellule del kupffer , deputate alla captazione del mdc spio [ 10 , 1820 ]  . 
in relazione a tali riscontri , alcuni autori hanno rilevato un differente comportamento contrastografico con i mdc spio tra gli hcc e i nd , in quanto i primi , a causa delle alterazioni cellulari subite , non presenterebbero captazione di mdc spio , risultando marcatamente iperintensi rispetto al parenchima epatico contiguo ; i nd invece , preservando composizione e funzionalit cellulari , presenterebbero una maggiore captazione di mdc spio , apparendo francamente ipointensi anche rispetto al fegato sano limitrofo . 
tali comportamenti contrastografici hanno indotto a considerare 1270 radiol med ( 2009 ) 114 : 12671282 dn and hcc in the cirrhotic liver by correlating the findings on mr imaging performed 1224 h before liver transplantation with histopathology of the entire explanted liver . materials and methods in the setting of a screening programme for cirrhotic patients awaiting liver transplantation , we performed mr studies on 400 patients , 31 of whom had been selected for transplantation . 
prior to the mr examination , all patients received full explanation of the aims and design of the study and gave their informed consent . all patients were studied with a 1.5 - tesla unit ( magnetom 63p , siemens , germany ) using baseline proton density ( pd ) / t2 - weighted spin echo ( se ) sequences ( tr / te 2 , 0002 , 300 / 2080 ms ) , and t1 - weighted fast low - angle shot ( flash ) gradient echo ( ge ) sequences ( tr / flip angle / te 153 ms / 70 / 6 ms ) and t2 * - weighted fast imaging with steady - state precession ( fisp ) ge sequences ( tr / flip angle / te 130 ms / 40 / 10 ms )  . 
for the pd / t2weighted sequences obtained under free breathing , two acquisition and presaturation bands were positioned above and below the imaging volume so as to reduce flow artefacts . 
the t1weighted ge sequences provided a stack of 15 slices that was sufficient to study the entire liver in most patients . with the t2 * - weighted ge sequences , a stack of five slices was used so that three to four stacks were required to study the entire liver . 
in all sequences , we used a slice thickness of 8 mm with 1 - mm gap , 128256 matrix with 3 / 4 reconstruction algorithm , and 380to 450 - mm field of view . 
 following unenhanced baseline imaging , spio ( ferumoxide , endorem , guerbet , villepinte , france ) was administered by slow infusion ( 820 drops / min ) over 3045 min at a dose of 23.6 md iron per gram ( fe / g ) diluted in a 100cc flask of 5% glucose solution . 
approximately 30 - 60 min after the end of spio administration , t2 * - weighted ge sequences with the same parameters as used at baseline were obtained in all patients . the baseline and spio - enhanced images were evaluated singly and then in combination by observers blinded to the histopathological results who recorded the number and site of lesions and expressed their judgement as to the dysplastic , neoplastic or other nature of the lesions . 
for each nodule , the observers recorded signal intensity on t1and t2 - weighted images and enhancement pattern after spio administration and , based on these parameters , expressed a la possibilit di una diagnosi differenziale tra queste due lesioni , nonch di una precoce identificazione della trasformazione del nd in hcc . scopo del nostro lavoro stato quello di valutare le possibilit della rm con mdc spio nella identificazione , caratterizzazione di nd e hcc nel fegato cirrotico , confrontando i risultati della rm , eseguita nelle 1224 ore precedenti il trapianto di fegato , con lesame anatomopatologico dellintero fegato espiantato . materiali e metodi nellambito di un programma di screening di pazienti cirrotici in lista dattesa di trapianto epatico , abbiamo studiato con rm 400 pazienti , 31 dei quali sono stati selezionati in seguito al reperimento di un fegato da trapiantare e di questi solo 22 sono stati inclusi nello studio , in quanto sottoposti a trapianto di fegato a distanza di circa 1224 ore dallesecuzione dellesame rm . 
per le sequenze se dpt2w ottenute durante la respirazione libera del paziente , abbiamo utilizzato due acquisizioni e bande di pre - saturazione , al di sopra ed al di sotto del campo di studio , al fine di ridurre gli artefatti vascolari . le sequenze ge t1w e ge t2 w sono state eseguite durante lapnea respiratoria dei pazienti , con ununica acquisizione e senza bande di pre - saturazione . 
in tutte le sequenze stato utilizzato spessore di strato di 8 mm con un gap di 1 mm , matrice di 128256 con algoritmo di ricostruzione 3 / 4 , campo di vista ( fov ) di 380450 mm . dopo le sequenze di base stato somministrato mdc spio ( ferumoxide , endorem , guerbert , francia ) , in infusione lenta ( 820 gocce / min ) alla dose di 23 , 6 micromol fe / g , diluito in un flacone da 100 cc di soluzione glucosata al 5% , con un tempo di infusione di circa 3045 mdopo circa 3060 min dalla fine della somministrazione del mdc spio in tutti i pazienti sono state ripetute le sequenze ge t2 * w con gli stessi parametri utilizzati nelle sequenze di base , eseguite prima dellinfusione del mdc spio . le immagini di base e quelle dopo spio sono state valutate separatamente e comparativamente , alloscuro dei radiol med ( 2009 ) 114 : 12671282 1271 diagnostic hypothesis as to histological type . 
on baseline images , in agreement with the reported mr criteria ( table 1 ) , dn were defined as nodular lesions that appeared hyperintense on t1 - weighted images and hypointense on t2weighted images or isohyperintense on t1 - weighted images and isointense on t2 - weighted images . 
hcc were defined as lesions appearing isohyperintense on t1 - weighted images , hyperintense in t2 or hyperintense in t1 , and isointense in t2 or hypointense in t1 , and hyperintense in t2 . hcc subfoci in dn ( nodule - in - nodule ) were defined as nodular areas of heterogeneous signal intensity seen within a dn with typical signal intensity . on spio - enhanced images , the diagnosis was based on the pattern of spio uptake . 
dn were defined as nodules presenting variable uptake depending on the degree of dysplasia , appearing in some cases strongly hypointense , often even more hypointense than the surrounding liver . hcc were defined as nodules with no spio uptake and hence markedly hyperintense . 
hcc subfoci in dn were defined as nodular lesions with hyperintense appearance due to failure to accumulate spio in the context of a larger , hypointense dn due to spio uptake . 
the definitive diagnosis was reached with the histopathological examination performed within 1 week of surgery on the entire explanted liver . dn were classified according to the international working party terminology of hepatic lesions into : lowgrade dn , high - grade dn and dn containing a subfocus of hcc [ 21 ]  . 
hcc were classified based on histology into well differentiated , moderately differentiated and poorly risultati dellanatomia patologica , riportando il numero e la sede delle lesioni ed esprimendosi sulla natura displasica , neoplastica o di altro tipo delle lesioni stesse . 
per ogni nodulo stata indicata lintensit di segnale nelle sequenze di base pesate in t1 e in t2 , il comportamento dopo somministrazione di mdc spio e , in base a tali parametri , si avanzata una ipotesi diagnostica sul tipo istologico . 
nelle sequenze di base , in accordo con la semeiotica rm descritta in letteratura ( tabella 1 ) abbiamo considerato nd le lesioni nodulari che apparivano iperintense in t1w ed ipointense in t2w o iso - / iperintense in t1w e isointense in t2w ; hcc le lesioni che apparivano iso - / iperintense in t1w e iperintense in t2w o iperintense in t1w e isointense in t2w o ipointense in t1w ed iperintense in t2w ; foci di hcc in nd ( nodulonel - nodulo ) le aree nodulari di disomogenea intensit di segnale riscontrate allinterno di un nodulo displasico con le caratteristiche intensit di segnale descritte . dopo iniezione di mdc spio , la diagnosi si basata sul tipo di captazione dello stesso e sono stati considerati nd i noduli che presentavano una captazione di mdc variabile in relazione al grado di displasia , apparendo in alcuni casi fortemente ipointensi , spesso anche in maggior grado rispetto al parenchima epatico limitrofo . 
lidentificazione di foci di hcc in nd si invece basata sul riscontro , dopo mdc spio , di una lesione nodulare iperintensa per mancata captazione di mdc nel contesto di un nodulo displasico di maggiori dimensioni , ipointenso per lup - take di mdc . 
nine patients were excluded because of mr or clinicallaboratory evidence of abnormalities that constituted contraindications or indications to defer surgery , findings later confirmed by clinical - imaging follow - up and biopsy . 
three of these nine patients had suspicious focal liver lesions that were > 5 cm and involved both hepatic lobes ; another two patients had cancercirrhosis , with extensive involvement of the right lobe in one case and left lobe in the other . 
in one patient , mr imaging demonstrated a large confluent fibrosis that made it impossible to confidently rule out the presence of cancer - cirrhosis , which was later excluded by biopsy . 
 risultati i risultati del nostro studio sono riportati nella tabella 2 . dei 31 pazienti selezionati per il trapianto dorgano , sono stati inclusi nello studio 22 pazienti , sottoposti a trapianto fegato a distanza di 1224 ore dallesecuzione dellesame rm . 
nove pazienti sono stati eliminati dallo studio in quanto allesame rm e agli esami clinico - laboratoristici presentavano alterazioni che contravvenivano o consigliavano il posticipo dellesecuzione del trapianto , reperti poi supportati dal follow - up clinico - radiologico e dallesame bioptico . 
in 3 , di questi 9 casi , erano presenti lesioni focali epatiche sospette per hcc che avevano dimensioni complessivamente superiori a 5 cm ed interessavano entrambi i lobi epatici ; in altri 2 casi si trattava di cancro - cirrosi con esteso interessamento del lobo destro in un caso e del sinistro nellaltro ; in 1 caso il paziente era portatore di tips che si estendeva nellatrio destro e per il quale si preferito posticipare lintervento ai fini di una migliore pianificazione chirurgica ; in 1 caso la rm evidenziava una estesa fibrosi confluente che non consentiva di escludere con certezza una eventuale cancro - cirrosi , successivamente non confermata dallesame bioptico ; in 2 casi le scadenti condizioni cliniche dei pazienti hanno imposto di soprassedere al trapianto e di proseguire la terapia medica . 
sono stati quindi inclusi nel nostro studio 22 pazienti , di et media di 53 anni ( range 4657 anni ) , successivamente sottoposti a trapianto dorgano entro 1224 ore dallesecuzione dellesame rm . la cirrosi stata classificata utilizzando il child - pugh syste quindici erano pazienti con cirrosi child - pugh classe a , 7 con cirrosi child - pugh classe b . 
unidici avevano cirrosi correlata con il virus dellepatite b ( hbv ) , 7 cirrosi correlata con il virus dellepatite c ( hcv ) , 4 cirrosi alcool correlata . risultati anatomo - patologici lesame anatomo - patologico dei 22 fegati espiantati ha dimostrato in tutti i casi un aspetto di tipo nodulare : macronodulare in 9 casi , micronodulare in 3 casi e medio - macronodulare in 10 casi . 
sono state identificate , inoltre , 59 lesioni focali epatiche , di cui 14 hcc , 4 noduli displasici con allinterno foci di hcc , 39 noduli displasici e 2 radiol med ( 2009 ) 114 : 12671282 1273 seven were child - pugh class b . 
at baseline mr imaging , four of the 12 dn exhibited the typical pattern of t1 hyperintensity relative to liver and t2 hypointensity ; eight dn were isointense on t1and t2weighted images . 
the two hcc subfoci within dn showed the classic nodule - in - nodule appearance , with isohyperintense signal on t1 - weighted images and heterogeneous hyperintensity on t2 - weighted images , contained within a larger dn that appeared isointense on t1weighted images and isohypointense on t2 - weighted images . 
the signal characteristics of the remaining 39 undetected lesions were such that precise identification and characterisation was precluded . spio administration increased mri sensitivity by 42.37% relative to the baseline study . 
il diametro medio delle lesioni stato per gli hcc 1 , 7 cm ( range 12 , 5 cm ) , per noduli displasici 0 , 8 cm ( range 0 , 52 ) , per i noduli displasici con allinterno foci di hcc 2 , 1 cm ( range 0 , 82 , 8 ) e per i cistoadenomi 1 , 2 cm ( range 11 , 4 )  . risultati rm gli esami rm effettuati senza mdc ( rm di base ) e con ferumoxide hanno mostrato una differente sensibilit nellidentificazione delle lesioni focali epatiche . 
lesame rm di base ha consentito di diagnosticare correttamente 20 lesioni , pari al 33 , 90% di quelle identificate con lesame anatomopatologico , di cui , allesame anatomopatologico , 12 sono risultate nd e 8 hcc . 
nelle sequenze di base , dei 12 nd identificati , 4 hanno mostrato comportamento caratteristico con intensit di segnale aumentata rispetto al parenchima epatico circostante nelle immagini t1w e ridotta in quelle t2w ; 8 nd sono risultati isointensi in t1w e isointensi in t2w . 
per quanto riguarda gli hcc , allesame di base in 3 casi hanno mostrato iperintensit di segnale nelle sequenze t1w ; in 2 casi isointensit di segnale ed in 1 caso ipointensit di segnale . 
i due foci di hcc in nodulo displasico hanno presentato il classico aspetto rm di nodulo - nel - nodulo mostrando segnale iper - / isointenso nelle sequenze t1w , e disomogeneamente iperintenso nelle sequenze t2w allinterno di un nodulo displasico di pi grandi dimensioni isointenso nelle sequenze t1w ed iso / ipointenso nelle sequenze t2w . 
le rimanenti 39 lesioni non diagnosticate presentavano caratteristiche di segnale tali da non consentire una precisa identificazione ed una adeguata caratterizzazione . dopo somministrazione di mdc spio , la rm ha mostrato una maggiore sensibilit diagnosticando complessivamente 45 lesioni , pari al 76 , 27% delle lesioni totali individuate con lesame istologico , di cui , allesame anatomo - patologico , 27 sono risultate nd e 18 hcc , con un incremento del 42 , 37% rispetto alle sequenze rm di base . 
i falsi negativi con la rm - spio sono stati 12 noduli displasici , pari al 31% , che allesame istologico sono risultati essere nd a basso grado di malignit e con diametro inferiore al centimetro . 
due lesioni , caratterizzate istologicamente come cistoadenomi , non sono state identificate in prima istanza allesame rm di base n dopo mdc spio , ma solo valutando retrospettivamente le immagini , sulla scorta dei risultati dellesame istologico . 
baseline protondensity - weighted spin echo ( a ) , t2 - weighted spin echo ( b ) , fisp ( c ) and t1 - weighted flash ( d ) magnetic resonance ( mr ) sequences : the nodule seems slightly hyperintense in all baseline sequences ( a - d )  . 
il reperto anatomo - patologico corrispondente conferma la presenza di multipli e diffusi noduli di rigenerazione con nodulo di hcc bianco - verdastro ( f )  . radiol med ( 2009 ) 114 : 12671282 1275 fig . 
3a - c nodulo di hcc e nodulo displasico con focus di hcc allviii segmento epatico . rm di base , sequenza flash t1w ( a ) e fisp t2w ( b ) : sono ben evidenti due formazioni nodulari iperintense . 
il nodulo di minori dimensioni mostra , nella sequenza fisp t2w , disomogenea intensit di segnale con evidenza di piccolo nodulo centrale contestuale . tale reperto configura il tipico pattern di nodulo - nel - nodulo . 
il reperto anatomopatologico ( c ) dimostra il caratteristico aspetto di nodulo nel nodulo della lesione . in 12 dn , equal to 31% , which at histology proved to be low - grade dn < 1 cm in diameter . 
two lesions that were histologically characterised as cystoadenomas went unrecognised on initial viewing of the baseline and spio - enhanced mr images but were later identified on retrospective review after the results of the histological examination . 
in fact , according to several authors , the transition from a dn to an hcc occurs over a period of mancata identificazione stata probabilmente dovuta alle ridotte dimensioni , alla localizzazione in sede sottoglissoniana e allaspetto simil - cistico . 
infatti lo sviluppo di un hcc a partire da un nd avviene , secondo molti autori , in un tempo variabile di circa 46 mesi [ 12 , 23 ] e laspettativa di vita media in un paziente non trattato di soli 13 mesi , con una sopravvivenza a 2 anni inferiore al 33% [ 24 ]  . 
il reperto anatomo - patologico ( d ) ha dimostrato la presenza di area di fibrosi e lassenza di lesioni focali . approximately 46 months [ 12 , 23 ] , and mean life expectancy of untreated patients is only 13 months , with a 2 - year survival rate < 33% [ 24 ]  . 
this pathogenic theory is supported by other studies and confirmed by the international working party on terminology of hepatocellular lesions , which stated that carcinogenesis in the cirrhotic liver is a multistep process that involves a transition from large rn to lowand high - grade dn to dn with subfocus of hcc and eventually to hcc [ 21 ]  . identification of dn and hcc in the cirrhotic liver is difficult because of the marked architectural distortion of the parenchyma due to areas of fibrosis , steatosis , parenchymal necrosis and regenerative nodules [ 10 , 25 , 26 ]  . 
found that us had a sensitivity of 20.5% for the detection of hcc and of 1.6% for detection of dn , emphasising the difficulty in differentiating hcc and dn with us , as both manifest as hypoechoic nodular lesions [ 8 ]  . 
although the use of sonographic contrast agents has improved the techniques epatiche , in cui si affermato che la carcinogenesi nel fegato cirrotico avviene con un meccanismo multi - step che include la transizione da grossi noduli rigenerativi a noduli displasici a basso ed alto grado di malignit , quindi a noduli displasici con foci di hcc sino agli hcc [ 21 ]  . lidentificazione di nd e di hcc nel fegato cirrotico per difficoltosa per la notevole alterazione strutturale del parenchima epatico a causa della presenza di aree di fibrosi , steatosi , necrosi parenchimale e noduli di rigenerazione [ 10 , 25 , 26 ]  . 
 [ 8 ] , in uno studio su 200 pazienti con cirrosi , hanno rilevato una sensibilit dellecografia nella identificazione degli hcc del 20 , 5% e dei nd del 1 , 6% , sottolineando la difficolt di differenziare con lecografia gli hcc dai nd , poich entrambi apparivano come lesioni nodulari ipoecogene [ 8 ]  . 
lutilizzo dei mezzi di contrasto ecografici ha migliorato laccuratezza diagnostica nella identificazione di hcc e nd rispetto alla ecografia convenzionale ; tuttavia esistono ancora difficolt nella diagnosi differenziale tra le due lesioni con tale metodica [ 27 ]  . 1278 radiol med ( 2009 ) 114 : 12671282 accuracy in identifying hcc and dn , the difficulties in differentiating the two lesions with this modality remain [ 27 ]  . spiral ct is currently considered amongst the most reliable modalities for detecting hcc . 
reported a mean sensitivity of dynamic spiral ct in identifying hcc of 76% and mean sensitivity values of 39% in the detection of dn depending on lesion size [ 10 ]  . the lower sensitivity in dn detection is most likely due to the fact that some dn are hypoattenuating in the portal venous and delayed phases , and few dn show hyperattenuation in the hepatic arterial phase . 
in the early stages of carcinogenesis , this is similar to that of healthy liver parenchyma , whereas in the subsequent hcc stage , it is characterised by a predominance of abnormal arteries ( not accompanied by bile ducts and for this reason known as unpaired or nonportal arteries ) and arteriovenous shunts livers undergoing neoplastic degeneration [ 19 , 20 , 29 ]  . 
thus , the arterial and portal supply to lowand high - grade dn is variable and inconsistent , making difficult the detection and characterisation of dn on the basis of their vascular characteristics alone [ 10 ]  . 
 typical of cirrhotic unlike ct , mr imaging often proves capable of differentiating hcc from dn based on signal characteristics alone as a result of its multiparametric properties and highcontrast resolution [ 23 ]  . 
on unenhanced baseline mr imaging , dn normally have isohyperintense signal on t1weigthed images in relation to their glycoprotein content and degree of dysplasia ; the signal becomes hypointense to surrounding parenchyma on t2 - weighted images . 
unfortunately , however , the signal intensity of dn may be identical to that of well - differentiated hcc , especially of small hcc ( < 2cm )  . the signal intensity of hcc on unenhanced t1weighted mr images varies widely and hcc may appear as hypointense masses as a result of greater water content or fibrous tissue or poor cellular differentiation or alternatively as hyperintense due to steatosis , accumulation of glycoproteic material or areas of intralesional haemorrhage . in some cases , however , moderately and well - differentiated hcc may also appear isointense compared with the healthy hepatic parenchyma . 
conversely , on t2 - weighted images , hcc tend to appear as generally ill - defined and hyperintense lesions [ 12 , 30 ] with the exception of well - differentiated hcc , which are isointense relative to the healthy parenchyma . 
in letteratura riportato che la sensibilit della tc multifasica nella identificazione di hcc di diametro superiore a 2 cm del 93 , 6% , percentuale che si riduce al 61% nella valutazione di lesioni di minori dimensioni [ 28 ]  . 
 [ 10 ] hanno riportato una sensibilit media della tc spirale dinamica nellidentificazione dellhcc del 76% e valori medi di sensibilit del 39% nellidentificazione dei nd in relazione alle dimensioni delle lesioni [ 10 ]  . 
questi minori valori di sensibilit nella identificazione dei nd sono verosimilmente dovuti al fatto che solo alcuni nd sono ipodensi in fase venosa portale e in fase tardiva , cos come sono pochi i nd che mostrano iperdensit nella fase arteriosa epatica . 
questo fenomeno correlato al tipo di apporto ematico arterioso e venoso che , nelle prime fasi della carcinogenesi , simile a quello del tessuto epatico sano ; successivamente , nello stadio di hcc , prevalgono vasi arteriosi anomali , non accompagnati dai canalicoli biliari , e per tale motivo denominati unpaired o non portal arteries , e shunt artero - venosi tipici dei fegati cirrotici ed in degenerazione neoplastica [ 19 , 20 , 29 ]  . 
pertanto lapporto arterioso e portale ai nd a basso ed alto grado di malignit variabile ed incostante ; , dunque , difficile identificare e caratterizzare i noduli displasici soltanto sulla base delle caratteristiche vascolari [ 10 ]  . la rm , grazie alle sue intrinseche multiparametricit ed alta risoluzione di contrasto , rispetto alla tc si rivela spesso in grado , di differenziare gli hcc dai nd sulla base delle sole caratteristiche di segnale [ 23 ]  . 
nelle immagini rm di base , il nd presenta solitamente , nelle sequenze t1w , un segnale che , in relazione al contenuto glicoproteico ed al grado di displasia , pu essere iso - / iperintenso ; appare invece iso - / ipointenso rispetto al parenchima circostante nelle sequenze t2w . 
purtroppo per lintensit di segnale dei nd pu essere sovrapponibile a quella degli hcc ben differenziati , soprattutto se di piccole dimensioni ( hcc < 2 cm )  . le caratteristiche di segnale degli hcc nelle immagini rm di base t1w variano molto e possono apparire come formazioni ipointense , per il maggior contenuto di acqua o per la componente di tessuto fibroso e per la scarsa differenziazione cellulare ; o possono presentare elevata intensit di segnale per la presenza di steatosi , accumulo di materiale glicoproteico o aree di emorragia intratumorale . 
nelle immagini t2w , invece , gli hcc appaiono comunemente come lesioni non sempre ben definite , iperintense rispetto al parenchima circostante [ 12 , 30 ] ad eccezione degli hcc ben differenziati che mostrano isointensit di segnale rispetto al parenchima sano . la capacit di identificare e caratterizzare le lesioni nodulari nel fegato cirrotico aumenta con lausilio dei mdc siano essi paramagnetici o superparamagnetici . 
la rm con mdc paramagnetico sfrutta , come la tc , le caratteristiche vascolari delle lesioni focali epatiche sia per la loro radiol med ( 2009 ) 114 : 12671282 1279 imaging with paramagnetic contrast medium relies on the vascular characteristics of focal liver lesions for their detection and characterisation . 
gadolinium has shown a good level of accuracy , even in the study of small hcc ( < 1.52 cm ) and in cirrhotic livers , with a sensitivity of 84% [ 11 , 31 , 32 ]  . 
sensitivity in the detection of dn is instead only 15% , with low specificity values since the signal intensity of dn on mr imaging with paramagnetic contrast medium is very similar to that of many hcc [ 33 ]  . the introduction of spio contrast agents , which provide information about the composition and function of cells forming the nodular lesions and which overcome the limitations of relying on vascularisation changes , has made it possible to detect even small hcc , which in the early stage may preserve normal vascularity and thus be difficult to differentiate from other nodular lesions [ 34 , 35 ]  . 
 the mechanism of action of spio contrast agents is based on their uptake by kupffer cells or cells of the hepatic reticuloendothelial syste spio uptake is reflected in a marked reduction in signal intensity of the tissues containing kupffer cells . 
conversely , any malignant lesions , which do not contain reticuloendothelial cells , do not accumulate the contrast medium and therefore appear as areas of hyperintensity [ 36 , 37 ]  . 
the use of these contrast agents has in part solved the problem of false positive results seen with paramagnetic contrast agents injected as a bolus , a problem related to the presence of small arteriovenous shunts which , appearing as small areas of vascular enhancement during the arterial phase , may mimic the enhancement pattern of small hcc . 
these false positive results are similar to those seen during contrast - enhanced dynamic ct imaging . on this subject , the literature also contains studies on the combined use of gadolinium diethylenetriamine pentaacetic acid ( gd - dtpa ) and spio , which , by fulfilling the criteria of vascularity as well as cellular composition and function [ 11 , 38 ] , reduces both the false positive results of paramagnetic contrast agents and the false negative results of spio agents the latter related to the presence , in moderately and well - differentiated hcc , of functioning kupffer cells that accumulate the contrast agent . 
as a result , the t2 - weighted signal intensity is higher in low - grade tumours , in which the number and function of kupffer cells is decreased [ 40 ]  . 
il gadolinio in molti studi ha mostrato una buona accuratezza anche nello studio dei piccoli hcc ( < 1 , 52 cm ) e nei fegati cirrotici mostrando una sensibilit dell84% [ 11 , 31 , 32 ]  . la sensibilit nellidentificazione dei nd invece , solo del 15% con bassi valori di specificit in quanto lintensit di segnale dei nd ottenuta dopo mdc paramagnetico paragonabile a quella di molti hcc [ 33 ]  . lintroduzione dei mdc spio fornendo informazioni sulla composizione e sulla funzione delle cellule che costituiscono le lesioni nodulari e , superando i limiti diagnostici legati alle alterazioni della vascolarizzazione , consente di identificare anche hcc di piccole dimensioni che nello stadio precoce possono in parte conservare una vascolarizzazione normale ed essere quindi difficilmente differenziabili dalle altre lesioni nodulari [ 34 , 35 ]  . 
il meccanismo dazione dei mdc spio si basa sulla loro captazione da parte delle cellule del sistema reticolo - endoteliale epatico , le cellule di kupffer , che si traduce in una marcata riduzione dellintensit di segnale dei tessuti in cui sono presenti tali cellule . pertanto , leffetto prodotto nelle immagini pesate in t2 fa s che alcune lesioni benigne captano il mezzo di contrasto come il parenchima epatico sano ; di contro , le lesioni focali maligne eventualmente presenti , sprovviste di cellule del sistema reticolo - endoteliale , non captano il mdc e appaiono come aree iperintense [ 36 , 37 ]  . 
lutilizzo di questi mdc ha risolto in parte il problema dei falsi positivi che si osservano con i mezzi di contrasto paramagnetici iniettati a bolo , legato alla presenza di piccoli shunt artero - venosi che , manifestandosi come piccole aree di enhancement vascolare in fase arteriosa , possono simulare il pattern contrastografico di piccoli hcc . 
 a tale proposito , in letteratura sono stati anche riportati studi sulluso combinato di gadolinio legato ad acido acetico dietilenetriaminepenta ( gd - dtpa ) e spio che , soddisfacendo sia i criteri di vascolarizzazione sia quelli di composizione e funzionalit cellulare [ 11 , 38 ] , riduce il problema dei falsi positivi dei mezzi di contrasto paramagnetici e dei falsi negativi con mdc spio , questi ultimi legati alla presenza , in hcc ben differenziati o moderatamente differenziati , di cellule di kupffer sane che di conseguenza captano il mdc . infatti , uno studio anatomo - patologico sulla carcinogenesi multi - step ha dimostrato che cellule del kupffer sono ancora presenti nei noduli epatici durante il processo di degenerazione ; ci che varia il numero di tali cellule che si riduce durante le fasi della malattia cirrotica e nellevoluzione dellhcc da uno stadio di alta differenziazione ad uno di moderata o scarsa differenziazione [ 35 , 39 ]  . 
lintensit di segnale nelle sequenze t2w , quindi , sar tanto pi elevato quanto meno differenziato sar il tumore , essendo inferiori il numero e la funzionalit delle cellule del kupffer presenti [ 40 ] ; questo offre la possibilit di effettuare una diagnosi differenziale tra hcc e noduli displasici . sebbene la rm con i mezzi di contrasto spio sia una 1280 radiol med ( 2009 ) 114 : 12671282 differentiating between hcc and dn . although spio - enhanced mr imaging is an accurate technique in the cirrhotic liver , the heterogeneous uptake of spio agents may affect the interpretation of findings . 
in liver cirrhosis , the marked subversion of hepatic structure and function , intrahepatic vascular changes , or the presence of inflammatory processes , thromboses , hepatitis or steatosis , may affect the intracellular uptake and distribution of spio agents in the liver , thereby reducing their effectiveness in the detection and characterisation of small focal lesions [ 43 , 44 ]  . 
such alterations , known as spio liver uptake and distribution alterations ( spio - luda ) lead to the formation of hyperintense areas due to reduced uptake or hypointense areas , which is due to increased uptake . although the incidence of such alterations is relatively low , in some cases , they give rise to diagnostic problems on mr imaging [ 26 ]  . we observed no significant spio uptake and distribution alterations ( table 1 ) , and the signal intensity of the nodular lesions seen in our study ( dn and hcc ) corresponded to the patterns reported in the literature . 
there were no cases of well - differentiated hcc showing spio uptake , as reported by other authors [ 45 ] , and hence no cases of false negative hcc diagnoses . 
 on the basis of our results and in agreement with the literature , spio - enhanced mr imaging proved to be a valuable tool for the detection and characterisation of focal lesions in the cirrhotic liver . 
in particular , spio - enhanced mr imaging is currently the only imaging modality capable of distinguishing between dn and hcc and thus providing the opportunity for an accurate and timely diagnosis of malignant change of dn , a crucial aspect for prognosis . tecnica dimaging accurata nello studio del fegato cirrotico , leterogeneo up - take degli spio pu compromettere linterpretazione dei reperti . 
nella cirrosi epaticail notevole sovvertimento della struttura e delle funzioni epatiche , le alterazioni vascolari intraepatiche o la presenza di processi flogistici , trombosi vascolari , di epatiti , steatosi , possono modificare la captazione intra - cellulare e la distribuzione dei mdc spio nel fegato riducendone lefficacia , con una minore utilit degli stessi nella identificazione e caratterizzazione di piccole lesioni focali [ 43 , 44 ]  . 
tali alterazioni , denominate alterazioni di captazione e distribuzione epatiche del mdc spio ( spio - acde ) , provocano la formazione di aree di iperintensit da ridotta captazione o di ipointensit da aumentata captazione . 
lincidenza di queste alterazioni relativamente bassa ma in alcuni casi determina difficolt diagnostiche allesame rm [ 26 ]  . in questa esperienza non si sono dimostrate significative alterazioni della captazione e della distribuzione dei mezzi di contrasto ( tabella 1 ) e lintensit di segnale delle lesioni nodulari epatiche considerate nel nostro studio ( nd e hcc ) stata corrispondente ai possibili pattern di segnale rm riportati in letteratura . 
non stato riscontrato nessun caso di hcc ben differenziato che captava mdc spio , come descritto da alcuni autori in letteratura [ 45 ] ; non sono stati pertanto riscontrati falsi negativi nella diagnosi di hcc . sono stati riscontrati , invece , falsi negativi per la diagnosi di nd . 
tali risultati sono probabilmente legati al fatto che il nostro campione di studio era relativamente limitato , quindi ridotta stata la possibilit di riscontro di hcc con comportamento contrastografico simile a quello dei nd . 
de filippo , scienze radiologiche , azienda ospedaliero - universitaria di parma , via gramsci 14 padiglione barbieri , 43100 parma , italy , tel . : + 39 - 052 - 1703660 , e - mail : massimo.defilippo@unipr.it received : 20 march 2008 / accepted : 13 october 2008 / published online : 19 november 2009 springer - verlag 2009 abstract purpose . 
through a search of the pathology databases of four italian hospitals , we identified six men ( mean age , 56 years ) with a histological diagnosis of ecd . histology was performed on retroperitoneal or pulmonary biopsy , depending on disease involvement on imaging . patients underwent plain radiography of the lower limbs and chest , total - body computed tomography ( ct ) and bone scintigraphy . 
magnetic resonance ( mr ) imaging was performed in two patients to evaluate the lower limbs and in one patient to study the brain , the chest and the abdomen . 
imaging studies revealed extraskeletal manifestations in all patients , including involvement of the retroperitoneal space ( n = 4 ) , the lung ( n = 4 ) and the heart ( n = 2 )  . 
sono stati identificati nel data base dellunit operativa di anatomia patologica di quattro ospedali italiani , sei pazienti con ecd , di sesso maschile , con et media di 56 anni . 
la diagnosi di natura dellecd istologica , 1320 radiol med ( 2009 ) 114 : 13191329 keywords erdheim - chester disease retroperitoneal fibrosis histiocytosis tuttavia il sospetto diagnostico pu essere agevolmente posto con limaging radiologico . parole chiave malattia di erdheim - chester fibrosi retroperitoneale istiocitosi introduction introduzione erdheim - chester disease ( ecd ) is a rare , systemic xanthogranulomatous infiltrative disease characterised by deposition of lipid - laden histiocytes in various organs , with the constant involvement of bone [ 16 ]  . 
ecd was first described by the american physician william chester and the viennese pathologist jacob erdheim in 1930 , who described two cases of a peculiar form of histiocytosis that they distinguished from other disorders such as handschller - christian disease and niemann - pick disease [ 7 ]  . the eponym erdheim - chester disease was coined by jaffe in 1972 [ 8 ]  . 
 the disease affects both genders , though with a slight predilection for men , and has a peak incidence between the fifth and sixth decade of life , although it may manifest at any age [ 4 ]  . 
skeletal involvement is most common in the long bones of the lower limbs . approximately 50% of patients present with extraskeletal manifestations , the most commonly involved sites being the heart , the lungs , the kidneys , the retroperitoneal space , the central nervous system and the skthe clinical course of ecd ranges from the absence of symptoms to a very poor outcome [ 9 ]  . 
 materials and methods we retrospectively searched the pathology databases of four italian hospitals for cases of ecd entered between january 2002 and january 2008 to identify organ involvement and clinical and radiological features . 
the diagnostic criteria for ecd are histopathological and based on the presence of lipid - laden histiocytes immunopositive for cd68 and immunonegative for s - 100 , cd1a and birbeck granules . 
 imaging studies included plain radiography of the lower limbs and chest , bone scintigraphy with technetium 99 ( 99mtc ) and chest and abdominal multidetector computed tomography ( mdct )  . 
patient 1 , who had a history of la malattia di erdheim - chester ( ecd ) una rara patologia sistemica di tipo infiltrativo xantogranulomatoso , caratterizzata dalla presenza di depositi di istiociti contenenti materiale lipoideo in vari organi , con coinvolgimento osseo pressoch costante [ 16 ]  . 
lecd fu descritta per la prima volta nel 1930 da un medico nordamericano , william chester e da un patologo viennese , jacob erdheim , che identificarono in due pazienti una particolare forma di istiocitosi , distinguendola da altri disordini , come la malattia di hand - schllerchristian e di niemann - pick [ 7 ]  . 
 la malattia presente in entrambi i sessi , con una lieve prevalenza nel sesso maschile ed un picco di incidenza tra la 5 e la 6 decade di vita , sebbene possa manifestarsi a qualsiasi et [ 4 ]  . 
linteressamento scheletrico dellecd riguarda prevalentemente le ossa lunghe degli arti inferiori ; circa la met dei pazienti presentano manifestazioni extra - scheletriche , le sedi maggiormente coinvolte sono : cuore , polmoni , reni , retroperitoneo , sistema nervoso centrale e cute . 
 materiali e metodi abbiano ricercato retrospettivamente nel database delle unit operative di anatomia patologica di quattro ospedali italiani la presenza di casi di malattia di erdheim - chester ( ecd ) , nel periodo compreso tra il gennaio 2002 e il gennaio 2008 , al fine di valutarne limpegno nei differenti organi e gli aspetti clinico - radiologici . 
i criteri diagnostici di ecd , sono istopatologici , basati sulla presenza di istiociti ricchi di lipidi , positivi allantigene per il cd68 e prive degli antigeni della proteina s - 100 , del cd1a e dei granuli di birbeck . 
il campionamento tissutale era stato ottenuto mediante biopsia retroperitoneale in tre casi e polmonare nei rimanenti tre casi . i pazienti erano stati sottoposti a radiografia del torace e degli arti inferiori , scintigrafia ossea con tecnezio 99 ( 99mtc ) , tomografia computerizzata multidetettore ( tcmd ) toracica e addominale . 
il paziente 1 , con anamnesi positiva per diabete insipido e microadenoma ipofisario , fu studiato radiol med ( 2009 ) 114 : 13191329 1321 diabetes insipidus and pituitary microadenoma , was studied with high - field magnetic resonance ( mr ) imaging to evaluate the brain , the chest , the abdomen and the lower limbs . patient 2 underwent mr imaging of the lower limbs to investigate diffuse bone changes identified on plain radiography . 
le indagini di imaging sono state valutate da due radiologi . results patients 1 and 2 had cardiac involvement , which in one patient manifested clinically as chest tightness and in the other as dyspnoea on exertion . 
in one case , the retroperitoneal solid tissue caused renal artery stenosis with resulting arterial hypertension , whereas in the other three cases , it surrounded the aorta and the iliac arteries , causing risultati nei pazienti 1 e 2 si evidenziava coinvolgimento cardiaco dellecd . 
lesordio clinico di ecd nei pazienti 3 e 4 era caratterizzato da alterazione degli indici di funzionalit renale , associato a ripetuti episodi di infezioni delle vie urinarie nel caso 3 . 
nei primi quattro pazienti la tc addome evidenziava infiltrazione retroperitoneale da parte di tessuto solido , prevalentemente perirenale e simmetrico , ipodenso e dotato di lieve contrast enhancement dopo somministrazione di mezzo di contrasto ( mdc )  . 
il tessuto solido retroperitoneale determinava nel caso 1 stenosi dellarteria renale con conseguente ipertensione arteriosa mentre negli altri tre casi circondava laorta e le arterie iliache , con fenomeni di incarceramento della fig . 
magnetic resonance image of the chest ( balanced sequence ) in the coronal plane ( a ) and contrast - enhanced computed tomography of the chest with four - chamber multiplanar reconstruction ( b )  . 
rm del torace ( balance w ) in sezione coronale ( a ) e tc con mdc del torace in sezione assiale obliqua four chambers mpr ( b )  . 
contrast - enhanced computed tomography of the upper abdomen ( a , b ) : the coated aorta , a specific finding in erdheim - chester disease , is well - depicted in the magnified image ( arrows in b )  . fig 2a , b ecd in fase avanzata . 
tc con mdc delladdome superiore ( a , b ) : aorta rivestita da tessuto solido , aspetto peculiare dellecd , ben evidente nellimmagine ingrandita ( frecce in b )  . 
renal pyelectasia , more evident on the left side , is due to compression by the fibrous tissue , which is also present at the level of the proximal periureteral spaces  . fig 3a , b ecd in fase iniziale . 
nel caso 5 lecd si manifestava con dolori al rachide in assenza di alterazioni allrx tradizionale , mentre la scintigrafia con 99mtc mostrava iperfissazione del radiol med ( 2009 ) 114 : 13191329 1323 fig . 
magnetic resonance images of the upper abdomen : axial t2 - weighted turbo spin - echo ( a ) and t1 - weighted fat - saturated fast - field echo sequence after the intravenous administration of paramagnetic contrast material . 
note the homogeneous enhancement of the fibrous tissue during the venous phase ( b ) and the sparing of the periaortic and pericaval regions ( a , b )  . fig 4a , b ecd in fase avanzata . 
il sottoslivellamento laterale sn della sella turcica ( freccia ) segno indiretto della presenza di un microadenoma ipofisario . scintigraphy showed increased tracer uptake at the level of a thoracic vertebra . four patients ( 2 , 3 , 5 and 6 ) had involvement of the pulmonary interstitiuchest radiography showed pleural and interstitial thickening ( kerley b lines )  . 
chest ct demonstrated a reticular interstitial pattern , smooth septal and fissural thickening , diffuse ground - glass opacities and centrilobular nodules with subpleural , peribronchovascular radiofarmaco in corrispondenza di una vertebrale dorsale . in quattro pazienti ( caso 2 , 3 , 5 e 6 ) vi era coinvolgimento dellinterstizio polmonare . 
i radiogrammi in proiezione antero - posteriore di gamba , mettono in evidenza bilateralmente , in corrispondenza della spongiosa tibiale e peroneale , diffuse e sfumate zone di radio - opacit , cui si associano ispessimenti focali delle corticali scheletriche , in assenza di reazioni periostali . radiol med ( 2009 ) 114 : 13191329 1325 fig . 
the clinical suspicion was confirmed by a biopsy of the retroperitoneal tissue in three patients ( 1 , 3 , and 4 ) and of pulmonary tissue in the remaining three patients ( 2 , 5 , 6 )  . 
il sospetto clinico stato confermato dallindagine bioptica su tessuto retroperitoneale in 3 pazienti ( casi 1 , 3 , 4 ) e polmonare nei rimanenti 3 casi ( 2 , 5 , 6 )  . 
 discussion discussione ecd is a rare form of non - langerhans histiocytosis , the reactive or neoplastic nature of which is still being debated [ 10 , 11 ]  . 
axial ( a ) and sagittal ct ( b ; multiplanar reconstruction ) demonstrates extensive erosion of t6 vertebral body with interruption of the posterior end - plate and initial involvement of the left vertebral pedicle . 
dimostrazione tc sul piano assiale ( a ) e sagittale ( b ; mpr ) di voluminosa erosione del soma di t6 con interruzione della limitante somatica posteriore ed inizile impegno del peduncolo vertebrale di sn . 
la rm ( c ) in se t1 w dimostra lindennit del canale vertebrale . in our experience , there was a frequent correlation between a histological diagnosis of ecd and skeletal involvement . 
bone involvement is an almost constant feature of ecd , there being only one report of a case without bone lesions [ 13 ]  . ecd generally affects the long bones of the lower limbs ( tibia and fibula )  . 
the related radiographic findings are diffuse or patchy opacities of the tibia and / or fibula , medullary sclerosis and cortical thickening of the metaphyseal and diaphyseal portions without epiphyseal changes . on 99mtc scintigraphy , intense , bilateral and symmetrical tracer uptake by the long - bone diaphyses and metaphyses is revealed , reflecting increased osteoblastic activity in those regions [ 1521 ]  . 
in our series , osteosclerotic changes were seen in the metaphyseal and diaphyseal portions of the tibia and fibula in three patients ( 1 , 3 and 4 )  . 
le lesioni ossee rappresentano un aspetto pressoch costante dellecd ; viene riportato in letteratura un solo caso di ecd in cui non erano state dimostrate lesioni ossee [ 13 ]  . lecd generalmente interessa le ossa lunghe degli arti inferiori ( in particolar modo tibia e perone ) e le alterazioni radiografiche si configurano come radio - opacit diffuse o a chiazze della tibia e / o del perone , la sclerosi midollare e lispessimento corticale delle regioni metafisarie e diafisaria ; scarse invece sono le alterazioni epifisiarie [ 13 ]  . 
la scintigrafia 99mtc evidenzia intensa iperfissazione , bilaterale , simmetrica a carico delle diafisi e metafisi delle ossa lunghe , conseguente ad aumento dellattivit osteoblastica in tali sedi [ 1521 ]  . 
nei casi 2 e 6 si osservano le caratteristiche radiol med ( 2009 ) 114 : 13191329 1327 the osteosclerotic changes were seen at the femoral level . bone involvement in these patients was asymptomatic , in contrast to previous reports where > 50% of patients have mild but persistent pain of the affected bone segment , most commonly the hips and knees [ 9 ]  . only patient 4 had symptomatic bone involvement , reporting with spinal pain despite the absence of significant abnormalities on plain radiography of the spine and lower limbs . 
are the only authors to have reported cases of lytic vertebral lesions ( two cases ) , and indeed , the spine is typically spared in ecd [ 22 ]  . 
otomastoiditis , spleen and bone marrow involvement are also more frequent in lch , whereas retroperitoneal involvement of the perirenal spaces and circumferential involvement of the aorta are typical features of ecd [ 23 ]  . 
in support of the latter hypothesis , cases of ecd coexisting with lch have been reported , suggesting the possibility that the two diseases are linked by a common precursor , cd34 + [ 24 , 25 ]  . 
il coinvolgimento osseo in questi pazienti era asintomatico , a differenza dei dati riportati in letteratura , secondo i quali oltre la met dei pazienti presentano dolore lieve ma persistente , localizzato al segmento osseo colpito , prevalentemente alle anche e alle ginocchia [ 9 ]  . solo il quarto paziente presentava sintomatologia dolorosa al rachide , in assenza di significative alterazioni allindagine radiografica del rachide e degli arti inferiori ; lesame scintigrafico evidenziava iperfissazione del radiofarmaco in corrispondenza di una vertebra dorsale ; la biopsia in tale sede non risultava diagnostica per campionamento inadeguato ; il prelievo bioptico a livello polmonare consentiva la diagnosi di ecd . 
 [ 22 ] sono gli unici autori ad avere riportato casi di lesioni osteolitiche vertebrali ( 2 pazienti ) ; infatti caratteristicamente nellecd si osserva risparmio del rachide . le lesioni osteolitiche dello scheletro assiale sono pi frequenti nellistiocitosi a cellule di langerhans ( lch ) , ma generalmente , queste tendono a risparmiare le ossa lunghe . inoltre i pazienti affetti da lch sono pi giovani rispetto a quelli con ecd ed hanno una prognosi migliore , con tassi di mortalit del 30% ( versus il 57% dellecd ) [ 9 ]  . 
a favore di tale ipotesi sono stati riportati alcuni casi in cui ecd ed lch coesistono , suggerendo la possibilit che le due patologie siano legate da un comune precursore cd34 + [ 24 , 25 ]  . 
in nessuno dei nostri pazienti stato osservato esoftalmo , sebbene rappresenti una frequente presentazione neurologica di ecd , conseguente ad una infiltrazione del tessuto adiposo retroconale e della guaina del nervo ottico . 
diabete insipido ed esoftalmo sono presenti in entrambe le forme di istiocitosi , in particolare nellistiocitosi a cellule del langerhans ( lch ) rappresentano in associazione ad osteolisi la classica triade di presentazione . 
tali reperti associati a diabete insipido sono rilevabili anche nello xantoma disseminato ( xd ) che pu simulare clinicamente lecd ; tuttavia nellxd il diabete insipido di grado lieve e transitorio ( nellecd persistente e progressivo ) , i pazienti hanno et inferiore a quelli con ecd , presentano diffuse lesioni cutanee e mucose e , raramente , possono presentare lesioni osteolitiche periarticolari [ 25 ]  . nei casi 1 e 2 stato riscontrato coinvolgimento cardiaco 1328 radiol med ( 2009 ) 114 : 13191329 among the typical manifestations of ecd [ 27 ]  . 
these same patients also exhibited thoracoabdominal periaortic infiltration , which is the most common cardiovascular manifestation of ecd [ 27 ] and is referred to as a coated aorta when the entire course of the vessel aorta is surrounded by fibrosis . 
at ct , care should be taken not to confuse periaortic infiltration with the aortic wall thickening seen in takayasu arteritis , a condition characterised by involvement of the full thickness of the aortic wall and sparing of the periaortic spaces . 
the use of contrast - enhanced ct is decisive for identifying this feature at the early stages of perirenal fibrosis . displacement of the aorta and ureters by the fibrous tissue may also be seen in forms secondary to malignant diseases , as in some histological types of non - hodgkins lymphomas . these , however , will rarely show the typical bilateral and symmetrical distribution of ecd [ 28 ]  . 
the differential diagnosis of pulmonary involvement by ecd prevalently includes cardiogenic interstitial oedema , where centrilobular nodules are absent , and carcinomatous lymphangitis and sarcoidosis , where septal thickening is typically nodular [ 29 , 30 ]  . our experience suggests that a clinical presentation of bilateral hydronephrosis , persistent bone pain , diabetes insipidus and exophthalmus should raise suspicion of ecd and prompt investigation with plain radiography of the lower limbs and ct of the chest and abdomen . 
if the examination reveals the characteristic diaphyseal and metaphyseal osteosclerosis , brain mr imaging should be used to search for any pituitary microadenomas ( at times hyposecreting )  . in conclusion , ecd is a rare multiorgan disease that has heterogeneous clinical presentations and a prevalence that appears to be underestimated . 
among the possible organs con riscontro di versamento pericardico , che recentemente stato incluso tra le manifestazioni tipiche dellecd [ 27 ]  . negli stessi pazienti stata anche evidenziata la presenza di infiltrato periaortico toraco - addominale ; tra le manifestazioni cardiovascolari questa la pi frequente in corso di ecd [ 27 ] e viene indicata anche con il termine di aorta rivestita , quando lintero decorso aortico circondato da fibrosi . 
allesame tc linfiltrato periaortico non deve essere confuso con lispessimento parietale aortico presente nellarterite di takayasu ; questultima condizione infatti si caratterizza per linteressamento a tutto spessore della parete aortica con risparmio degli spazi periaortici . 
la dislocazione dellaorta e degli ureteri da parte del tessuto fibrotico pu essere riscontrata anche nelle forme secondarie a patologie maligne , come nel caso di alcuni istotipi di linfomi nonhodgkin ; tuttavia rara la distribuzione bilaterale e simmetrica propria dellecd [ 28 ]  . 
la tc torace evidenziava un pattern interstiziale di tipo reticolare con ispessimento liscio dei setti e delle scissure , diffuse aree a densit ground glass e noduli centrolobulari con distribuzione subpleurica , peribroncovasale e interlobulare . 
la diagnosi differenziale in caso di coinvolgimento polmonare include principalmente ledema interstiziale cardiogenico in cui sono assenti i noduli centrolobulari , la linfangite carcinomatosa e la sarcoidosi ove lispessimento dei setti tipicamente nodulare [ 29 , 30 ]  . la nostra esperienza suggerisce che un quadro clinico caratterizzato da idronefrosi bilaterale , dolore osseo persistente , diabete insipido , esoftalmo , pu orientare il sospetto diagnostico di ecd e giustificare lesecuzione di un esame radiografico degli arti inferiori e di una tc toracica ed addominale . 
il riscontro occasionale in ecografia , tc o rm , di fibrosi perirenale giustificano un approfondimento con rx standard degli arti inferiori ; quando a tali livelli , si documenti la caratteristica osteosclerosi diafisaria e metafisaria dellecd , consigliabile ricercare la possibile presenza di microadenomi ipofisari ( talvolta iposecernenti ) con rm dellencefalo . in conclusione , lecd una patologia multiorgano rara , con manifestazioni cliniche estremamente eterogenee , la cui radiol med ( 2009 ) 114 : 13191329 1329 involved by the disease , skeletal tissue , the retroperitoneum and , in particular , perirenal spaces , were found to be constantly involved . 
patients treated with these hearing devices are often children who require close follow - up with frequent functional and radiological examinations ; in particular , multislice computed tomography ( msct )  . 
dental volumetric cone - beam ct ( cbct ) has been reported as a reliable technique for acquiring images of the temporal bone while delivering low radiation doses and containing costs . 
one hundred patients ( mean age 26 years , range 743 ) with vibrant soundbridge implants on the round window underwent follow - up : 85 with cbct and 15 with msct . 
i pazienti trattati con questi tipi di ausili , spesso bambini , necessitano di un follow - up di esami sia funzionali che radiologici , in particolare mediante tomografia computerizzata multistrato ( mstc )  . la tc volumetrica dentale a fascio conico ( cbct ) una metodica utile allo studio dellosso temporale , con il vantaggio di erogare basse dosi di radiazioni ionizzanti e di avere costi di gestione contenuti . 
sono stati inclusi 100 pazienti ipoacusici ( et media 26 anni , range 743 ) , trattati mediante protesi vibrant soundbridge alla finestra rotonda , 15 studiati in follow - up mediante tc e 85 mediante cbct , calcolando le dosi medie assorbite per tessuto sia durante esame mstc che cbct : ogni studio era focalizzato sullosso temporale utilizzando il pi piccolo campo di vista ed il protocollo a bassa dose radiante . 
le immagini ottenute con la cbct sono di qualit inferiore rispetto alla mstc , ma sufficientemente diagnostiche , permettendo di valutare con sicurezza la radiol med ( 2009 ) 114 : 13081318 1309 technique for postoperative imaging and follow - up of patients with bionic ear implants . 
la cbct , grazie alla bassa dose impartita e alla sufficiente qualit delle immagini , pu essere considerata tecnica radiologica adeguata per i controlli postoperatori ed il follow - up dei pazienti con orecchio bionico . parole chiave orecchio bionico , impianti acustici tomografia computerizzata a fascio conico dose radiante introduction introduzione cone - beam computed tomography ( cbct ) is an evolution of conventional computed tomography ( ct ) from which it has inherited some physical features , such as the use of ionising radiation , the ability to provide high - quality images and the possibility of imaging anatomical structures with multiplanar and three - dimensional ( 3d ) reconstructions . 
the first applications of cbct were in the fields of dental and maxillofacial imaging , where it replaced in part orthopantomography and multislice ct ( mstc ) , which were limited by insufficient spatial information and high radiation doses , respectively . 
cbct has played a primary role in dental implantology owing to its ability to provide an estimate of the thickness and depth of residual alveolar bone and visualise anatomical structures at risk during surgery , such as the mandibular canal , the mental foramen , the nasopalatine duct and the maxillary sinus . 
cbct has also been employed to study the temporomandibular joints , bone neoplasms and single dental elements [ 57 ]  . the use of cbct is establishing itself in the otological field , where msct is the gold standard [ 8 , 9 ]  . 
the first clinical indications were middle - ear diseases causing conductive hearing impairment , in which it was used to identify the damaged conduction structure or to follow - up patients after ossiculoplasty [ 10 ]  . 
cbct was subsequently used for postoperative imaging and follow - up of patients with vibrant soundbridge ( vsb ) electromagnetic implants , both on the incus and on the round window [ 11 , 12 ]  . 
cbct may offer a valuable alternative to msct in vsb hearing implants , as the patients most of whom are children require periodic follow - up , and cbct delivers lower radiation doses . 
 the purpose of this study was to evaluate a cohort of hearing - impaired patients with middle ear implants to determine how much dose could be saved by studying the la cone beam computed tomography ( cbct ) o tomografia a fascio conico rappresenta una evoluzione della tomografia computerizzata convenzionale ( tc ) , condividendone caratteristiche di natura fisica , come lutilizzo di radiazioni ionizzanti , la capacit di ottenere immagini di elevata qualit , e la possibilit di studiare le varie strutture anatomiche con ricostruzioni multiplanari e tridimensionali ; si differenzia dalla progenitrice tc per le dimensioni ridotte , i costi contenuti , il limitato campo di vista e la minore dose radiante somministrata al paziente [ 14 ]  . 
le prime applicazioni della cbct sono state realizzate in campo odontoiatrico e maxillo - facciale , in parte sostituendo lortopantomografia e la tc multistrato ( mstc ) , per le quali linsufficiente informazione spaziale per la prima , e la dose radiante per la seconda rappresentavano dei limiti . 
la cbct ha rivestito un ruolo primario nellimplantologia dentale , per la capacit di fornire una stima dello spessore e della profondit dellosso alveolare residuo e nellevidenziare strutture anatomiche a rischio durante le procedure chirurgiche , come il canale mandibolare , il foro mentoniero , il dotto naso - palatino ed il seno mascellare ; inoltre , la cbct trova impiego anche nella valutazione delle articolazioni temporo - mandibolari , delle lesioni neoplastiche dellosso , e dei singoli elementi dentari [ 57 ]  . lutilizzo della cbct si sta imponendo in campo otologico , dove la mstc rappresenta il gold standard [ 8 , 9 ]  . 
le prime indicazioni cliniche hanno riguardato patologie dellorecchio medio che determinavano ipoacusia di tipo trasmissivo , al fine di comprendere quale struttura di conduzione fosse lesa , o per il follow - up dei pazienti sottoposti ad intervento di ossiculoplastica [ 10 ]  . 
successivamente la cbct stata impiegata per limaging postoperatorio ed il follow - up dei pazienti impiantati con protesi elettromagnetiche vibrant soundbridge ( vsb ) sia allincudine che alla finestra rotonda [ 11 , 12 ]  . 
la cbct pu rappresentare una valida alternativa allo studio mstc in questo tipo di 1310 radiol med ( 2009 ) 114 : 13081318 petrous bones with cbct rather than with mstc , as well as to evaluate image quality obtained with the two techniques . materials and methods patients the study was conducted on 100 hearing - impaired patients ( mean age 26 years , range 743 ) who , after initial clinical and aetiological assessment at our hospitals ear , nose and throat department , were treated with vsb implants on the round window . 
the time period considered spanned from may 2004 to november 2007 . postoperative follow - up included periodic functional tests to demonstrate restoration of hearing and radiological examinations to define the position of the implant and identify possible dislocations where clinically suspected . patients underwent conventional radiology ( anteroposterior and lateral skull radiography ) and / or computed tomography ( msct / cbct )  . equipment cbct images were obtained with maxiscan equipment ( qr - dvt 9000 , verona , italy ) , which generates a pulsed emission of a fixed 110 - kvp conical beam of x - rays rotating around the patients head and is capable of reconstructing a cylindrical volume with a diameter and height of 15 cthe 400 - cm2 - wide detector is made up of an image intensifier coupled with a ccd camera [ 1 , 2 , 13 , 14 ]  . 
raw data are processed with primary reconstruction to obtain sections in the axial plane or other planes specified by the operator of a nominal thickness of 1 mm ( in the protocol used )  . 
on the basis of the anteroposterior and lateral projection tomograms , cbct calculates the milliampere per second ( mas ) values for the axial scan ( generally between 100 and 150 mas )  . msct images of the petrous bones were obtained with a 120 - kvp , 60 - mas , low - exposure protocol in the transverse plane with electronic reconstruction in the coronal plane ( thickness 1 mm ; spiral technique ; pitch 0.4 ) using a brilliance ct 6 msct device ( philips , eindhoven , the netherlands )  . measurement of values of exposure to ionising radiation a comparison between the doses delivered to the patients and the associated radiological risk was performed with both devices . 
 lo scopo del presente lavoro quello di valutare una coorte di pazienti ipoacusici impiantati allorecchio medio per definire il risparmio di dose ottenibile eseguendo lo studio delle rocche petrose mediante cbct rispetto allesame mstc e di valutare la qualit delle immagini ottenute con le due diverse tecniche . materiali e metodi pazienti sono stati inclusi 100 pazienti ipoacusici ( et media 26 anni , range 743 ) , inizialmente inquadrati dal punto di vista clinico ed eziologico presso listituto di otorinolaringoiatria della nostra struttura e successivamente trattati mediante posizionamento di protesi vsb alla finestra rotonda . 
lintervallo di tempo considerato stato tra il maggio 2004 ed il novembre 2007 . il follow - up post - operatorio di questi pazienti includeva lesecuzione periodica di test funzionali , in grado di provare la ripresa della capacit uditiva , nonch di esami radiologici , per la valutazione della posizione della protesi e la sua eventuale dislocazione , qualora clinicamente sospettata . 
questi pazienti sono stati sottoposti ad indagini di radiologia convenzionale ( rx cranio in antero - posteriore e latero - laterale ) e / o tecniche tomografiche computerizzate ( mstc / cbct )  . apparecchiature le immagini cbct sono state ottenute utilizzando lapparecchiatura maxiscan ( qr - dvt 9000 , verona , italia ) che genera una emissione pulsata di un fascio conico di raggi x , con tensione di 110 kvp , non modificabile , che ruota attorno alla testa del paziente , ed in grado di ricostruire un volume cilindrico con diametro e altezza di 15 c il rivelatore , con estensione di 400 cm2 , costituito da un intensificatore di brillanza accoppiato ad una videocamera con schermo ccd [ 1 , 2 , 13 , 14 ]  . 
i dati grezzi ottenuti sono rielaborati mediante ricostruzione primaria , ottenendo sezioni assiali o su altri piani indicati dalloperatore , con spessore nominale di 1 mm ( nel protocollo da noi utilizzato )  . 
the tlds were wrapped in polyethylene and inserted into the holes in the phantom at the sites of interest ( crystalline lens , thyroid , cerebrum , hypophysis and marrow of the skull and of c1 , c2 and c7 vertebrae and nuchal skin ) and at other significant sites to determine the dose distribution . the tlds , gr200a lithium fluorides ( central research laboratory , beijing , china ) , were read with a pcl3 reader ( fimel , vlizy , france ) while the annealing process occurred inside an ett oven ( fimel , vlizy , france )  . 
the dosimeters were calibrated in terms of air kerma for doses between 5 mgy and 30 mgy using an x - ray tube with 80 kvp and 2.8 mm al filtration , through comparison with an ionisation chamber . 
the phantom was examined at different times with cbct and msct , following the manufacturers protocols for the specific examination ( table 1 )  . the tlds readings were used to derive the air kerma values . 
le misure sono state effettuate posizionando 46 dosimetri termoluminescenti ( tlds ) allinterno della testa di un fantoccio antropomorfo rando ( alderson research laboratories , stanford , cn , usa )  . 
i tlds sono stati avvolti nel polietilene e quindi inseriti negli appositi fori del fantoccio sia in siti di interesse ( cristallino , tiroide , cervello , ipofisi , midollo osseo delle ossa della teca e delle vertebre cervicali c1 , c2 e c7 , cute nucale ) che in altre posizioni significative per la determinazione della distribuzione di dose . i tlds , fluoruri di litio del tipo gr200a ( central research laboratory , pechino , cina ) , sono stati letti con lettore pcl3 ( fimel , vlizy , francia ) mentre il processo di annealing ( temperaggio ) stato eseguito con un forno ett ( fimel , vlizy , francia )  . 
i dosimetri sono tarati in termini di kerma in aria per dosi comprese tra 5 mgy e 30 mgy , utilizzando un tubo radiologico con 80 kvp e filtrazione di 2 , 8 mm al , mediante confronto con camera a ionizzazione . tre tlds non sono stati irradiati in modo da poter sottrarre il segnale di fondo . 
il fantoccio stato esaminato in momenti diversi alla cbct e alla msct seguendo i protocolli predisposti dalle rispettive ditte fornitrici per lesame in oggetto ( tabella 1 )  . dalla lettura dei tlds sono stati ricavati i valori di kerma in aria ; moltiplicando questi ultimi per il rapporto dei coefficienti di assorbimento di energia massici ariamuscolo [ 15 ] si ottenuta la dose in tessuto . 
le dosi equivalenti impartite ai diversi organi ( ht , misurato in msv ) sono state ricavate moltiplicando la dose ottenuta mediante tlds per la frazione del volume dellorgano irradiato ( dt , misurato in mgy ) e per il coefficiente di peso della radiazione ionizzante ( wr ) : il coefficiente di peso per la radiazione considerato uguale a 1 . il contributo di un singolo organo / tessuto nel determinare il rischio globale del paziente durante lesposizione radiante viene ottenuto moltiplicando la dose equivalente ( ht ) per il coefficiente di peso del tessuto ( wt )  . 
the image obtained with cbct measures 15 cm in diameter and therefore does not cover the whole phantom section . the slice thickness of the reconstructed image was determined by measuring its width at half the height of the profile of the lamellae fitted into the appropriate insert . 
when measuring uniformity , the value considered was the maximum ratio between the average pixel values in the regions of interest ( roi ) placed at the centre and in four peripheral locations of an image of the homogeneous insert of the phanto finally , we assessed whether it was possible to visualise the various anatomical components of the tympanic cavity and specifically of the vsb implants to determine the position of the distal end at the level of the round window or of the long apophysis of the incus . results all 100 patients underwent msct within 6 h of surgery , according to the protocol . 
per le scansioni con la cbct limmagine ottenuta ha diametro di 15 cm , quindi non comprende lintera sezione del fantoccio . lo spessore di strato dellimmagine ricostruita stato determinato misurando la larghezza a met altezza del profilo delle lamelle inserite nellapposito inserto ; per evitare lerrore dovuto al campionamento stata determinata la media delle dimensioni in diverse posizioni dellimmagine . per la determinazione della risoluzione spaziale ad alto contrasto , nel fantoccio in plexiglas sono presenti otto serie di cinque fori pieni daria a sezione circolare con diametro da 2 , 5 mm a 0 , 75 mm distanziati fra loro in modo tale che la distanza fra i centri sia doppia del diametro del foro . 
per valutare la visibilit dei particolari stata selezionata la scala di grigi con finestra ridotta al minimo scegliendo il livello che meglio permette di distinguerli ; questi vengono considerati visibili se risultano tutti distinti . nel fantoccio sono inseriti dei cilindri con diametro di un pollice ( 25 , 4 mm ) immersi in acqua con le seguenti densit : policarbonato = 1 , 20 g / cm3 , acrilico = 1 , 19 g / cm3 , nylon = 1 , 10 g / cm3 , polistirene = 1 , 05 g / cm3 , polietilene = 0 , 95 g / cm3 . questi inserti possono fornire una indicazione della risoluzione a basso contrasto . 
per la misura delluniformit viene considerato il massimo rapporto fra i valori medi dei pixel nelle regioni di interesse ( roi ) posizionate al centro ed in quattro posizioni alla periferia di una immagine acquisita in corrispondenza dellinserto omogeneo del fantoccio . 
 stata infine valutata la possibilit di evidenziare le diverse componenti anatomiche presenti allinterno del cavo timpanico , e nel caso specifico delle protesi vsb , in particolare per definire con certezza la posizione dellestremo distale a livello della finestra rotonda o allapofisi lunga dellincudine . risultati tutti i 100 pazienti sono stati sottoposti a esame mstc entro le 6 ore dallintervento , come da protocollo postchirurgico . 
successivamente , in corso di follow - up , i pazienti che hanno eseguito rx cranio nelle proiezioni ortogonali sono stati 2 / 100 ; i pazienti studiati mediante mstc delle rocche petrose sono stati 15 / 100 , e 85 / 100 mediante cbct . 
1a , b paziente portatore di impianto vibrant soundbridge alla finestra rotonda . sezione assiale msct ( a ) e corrispondente immagine cbct ( b ) che mostra il corretto posizionamento della massa vibrante . 85 / 100 were studied with cbct . 
with regard to the crystalline lens , the measured dose was 4.5 mgy for cbct and 15.5 mgy for msct , values that are substantially lower than those likely to cause deterministic effects , even after repeated exams . an overall analysis of the single results shows that the effective dose delivered to the individual patient during cbct of the ear was equal to 39.2% of the dose imparted during msct . 
per quanto riguarda il cristallino la dose risultata pari a 4 , 5 mgy per la cbct ed a 15 , 5 mgy per la mstc , valori decisamente inferiori a quelli che possono provocare effetti deterministici anche per esami ripetuti . 
 da una analisi globale dei singoli risultati si evince come la dose efficace somministrata ad un paziente durante esame cbct dellorecchio sia il 39 , 2% rispetto a quella di unindagine mstc : nello specifico la dose efficace della mstc stata di 0 , 28 msv , mentre quella della cbct stata di 0 , 11 msv . results of image quality are summarised in table 3 . 
con queste premesse , abbiamo considerato leventualit di sottoporre i pazienti operati ad un follow - up radiologico che sfruttasse tecniche alternative alla mstc : poich metodiche non ionizzanti non erano usufruibili , la radiografia standard del cranio e la cbct rappresentavano le uniche opzioni . 
 tutti i 100 pazienti della nostra casistica sono stati valutati nellimmediato post - operatorio come da protocollo ( entro le 6 ore ) mediante esame mstc al fine di escludere focolai emorragici in sede di intervento o un eventuale mal posizionamento della protesi . 
il follow - up standard includeva lesecuzione di una indagine mstc a 6 settimane , ossia allattivazione dellimpianto elettromagnetico , ed a 1 radiol med ( 2009 ) 114 : 13081318 1315 fig . 
4a , b low - contrast resolution on msct ( a ) and cbct ( b ) refers to the systems ability to distinguish small - sized objects against a low - contrast background . 
4a , b la risoluzione a basso contrasto per la mstc ( a ) e per la cbct ( b ) intesa come la capacit del sistema di distinguere oggetti di piccole dimensioni in un contesto di basso contrasto . 
 evidente la migliore capacit discriminativa della mstc rispetto alla cbct . discussion this study sought to identify the technique providing the best ratio between diagnostic quality and patient dose in the evaluation of the correct position of vsb ear implants . 
quindici / 100 pazienti hanno seguito liter tradizionale , e quindi sono stati studiati mediante mstc . il follow - up da noi proposto implicava lesecuzione di un rx cranio e / o di una cbct rispetto alla mstc : i primi 1316 radiol med ( 2009 ) 114 : 13081318 fig . 
5a , b uniformity is defined as the maximum ratio between the average values of pixels in different regions of interest , with one placed at the centre of the image and four in peripheral locations . 
5a , b luniformit definita come il massimo rapporto fra i valori medi dei pixel di regioni di interesse poste una al centro e quattro in periferia , con la mstc ( a ) e con cbct ( b )  . 
fifteen out of 100 patients were followed up with the standard protocol ( msct )  . the experimental follow - up protocol involved performing skull radiography and / or cbct instead of msct . 
finally , cbct was considered sufficient so that msct could be reserved for cases suspected to have more severe complications than implant dislocation ( no case in the period in question )  . analysis of dosimetric data , in agreement with the literature [ 1822 ] , showed that the use of cbct permits considerable dose saving compared with msct . 
in addition , although cbct provides lower - quality images , these are nonetheless adequate for evaluating the position of the hearing implant . in contrast to msct , cbct scans and reconstructions are not affected by artefacts related to the metallic components of the vibrating mass of the implant , which often limit visualisation of the structures being investigated . 
these two 2 / 100 pazienti hanno espletato indagine rx del cranio nelle proiezioni ortogonali , dalla quale non si riuscivano ad estrapolare sufficienti reperi , e quindi stata abbandonata e non riproposta nei successivi pazienti . 
si infine ritenuto sufficiente lesecuzione di questa sola metodica , riservando la mstc a quelle circostanze in cui il sospetto fosse di complicazioni pi gravi della semplice dislocazione della protesi ( nessun caso nella nostra casistica nel periodo in esame )  . lanalisi dei dati dosimetrici , in accordo con la letteratura [ 1822 ] , dimostra come lutilizzo della cbct comporti una considerevole riduzione di dose alla mstc , determinata in particolare dalla geometria del fascio , dal tipo di emissione ( pulsata , rispetto ad unemissione continua su tutto langolo di rotazione del tubo ) e da una maggiore efficienza di rilevazione . 
a differenza della mstc , la cbct non presenta nelle scansioni e nelle successive ricostruzioni artefatti derivanti dalle componenti metalliche della massa vibrante dellimpianto acustico , procuranti spesso limitazioni nella visualizzazione delle strutture dinteresse . 
la cbct conferma di avere una buona risoluzione ad alto contrasto ma a differenza della mstc risulta deficitaria nel basso contrasto : queste due caratteristiche conferiscono alla cbct la capacit di visualizzare con buona risoluzione di contrasto le strutture ossee , mentre con difficolt i tessuti molli . 
lorecchio medio una struttura anatomica , inserita in un contesto osseo , la rocca petrosa dellosso temporale , e riempita di aria attraverso la tuba di eustachio ; al suo interno la trasmissione del suono garantita dalla catena ossiculare , formata da singoli ossicini . 
la cbct riesce le diverse componenti a evidenziare correttamente radiol med ( 2009 ) 114 : 13081318 1317 characteristics mean that cbct visualises bone structures with good contrast resolution , whereas it is somewhat limited in depicting soft tissues . 
cbct correctly depicts the various anatomical components within the tympanic cavity and , in the case of vsb implants , allows accurate definition of the position of the distal end at the level of the round window or of the long apophysis of the incus . 
 because cbct has been reported to impart smaller radiation doses compared with msct , we attempted to determine the difference in dosimetric performance between the devices available at our department . 
data analysis showed that cbct delivers a patient dose equal to 39% of that imparted by msct with a low - dose protocol . according to the directive of the dimond iii european research group , when choosing between two techniques capable of solving a diagnostic problem , one needs to take into account both its usefulness in depicting the object being studied and the patient dose [ 23 ]  . 
in the case of vsb devices , it is important to consider that the majority of patients are children , and they have to undergo a intensive postoperative follow - up protocol involving repeated radiological examinations . 
such patients are those most likely to be affected by the cumulative effects of ionising radiation . cbct meets the european criteria for diagnostic capability , and its place in otology where msct is still the gold standard should be reassessed . 
 the limitations of this study , in which the comparison was based on the standard protocols in use at our hospital and on the best costbenefit ratio for each of the ct scanners available at the time of assessment , concern the unexpressed potential for reducing the msct dose by refining the protocol . 
however , there remains an objective difference between the doses delivered to individual patients , even after attempting to stress the protocols at a dosimetric level , which is due to the intrinsic characteristics of the two ct applications . 
we therefore believe that our study may encourage a more widespread use of cbct in other fields as well , given that while yielding the same diagnostic information , cbct allows lower patient doses and lower costs per examination . anatomiche presenti allinterno del cavo timpanico e , nel caso specifico delle protesi vsb , a definire con certezza la posizione dellestremo distale a livello della finestra rotonda o allapofisi lunga dellincudine . 
 dalla letteratura si conferma come la cbct somministri una quantit di radiazioni ionizzanti minore rispetto alla mstc ; si quindi voluto ottenere un riscontro oggettivo della differenza di prestazioni dosimetriche fra le apparecchiature a disposizione presso il nostro dipartimento . 
a conclusione dei rilievi e dellelaborazione dei dati si rileva come la cbct eroghi una dose radiante pari al 39% di quella della mstc con protocollo a bassa esposizione . secondo la raccomandazione del gruppo di ricerca europeo dimond iii nella scelta fra due tecniche bisogna considerare sia lutilit della stessa nel mettere in evidenza loggetto di studio , sia la dose radiante , se il quesito diagnostico viene soddisfatto [ 23 ]  . 
nel caso delle protesi vsb si deve tenere conto che spesso i pazienti sono in et pediatrica e che dal momento dellintervento entrano a far parte di un protocollo di follow - up radiologico intenso , composto da controlli ripetuti ; sono proprio questi pazienti che pi potrebbero risentire degli effetti cumulativi delle radiazioni ionizzanti . 
 i limiti di questo lavoro , nel quale il confronto stato compiuto secondo i protocolli di comune uso nella pratica clinica nella nostra struttura , e secondo il miglior rapporto costo - beneficio per ognuna delle macchine a tecnologia tc possedute al momento della valutazione dei pazienti , riguardano la potenzialit non espressa di diminuire la dose in msct , affinando il protocollo : resta comunque oggettivo il divario tra le dose impartite ai singoli pazienti , anche cercando di stressare dosimetricamente i protocolli , proprio per le caratteristiche intrinseche delle due diverse applicazioni di tecnologia tc , e per questo noi riteniamo che questo lavoro possa comunque essere di stimolo per cercare di diffondere luso della cbct anche in altre applicazioni , in modo da ridurre la dose al paziente , poich in caso di parit di informazione diagnostica si ha una minor dose al paziente e un minor costo per esame . conclusioni conclusions during cbct of the ear , the radiation dose delivered to the patient is lower than that of msct performed with a lowdose protocol . 
in comparison with msct , cbct provides an image quality that is lower in terms of low - contrast resolution and uniformity and slightly lower in terms of highdurante un esame cbct dellorecchio la dose radiante somministrata al paziente inferiore a quella erogata dalla tc con protocollo a bassa esposizione . 
la qualit dellimmagine ottenuta con cbct , rispetto alla mstc , minore in termini di risoluzione a basso contrasto e uniformit , leggermente inferiore per risoluzione ad alto contrasto , mentre equivalente per quanto riguarda lo 1318 radiol med ( 2009 ) 114 : 13081318 contrast resolution , whereas slice thickness is equal . 
the images are sufficiently diagnostic to identify the correct placement of vsb , both on the round window and on the incus , with the advantage of lower radiation exposure for the patient . 
sorrentino , via san lorenzo 291 / a , 90146 palermo , italy , tel . : + 39 - 091 - 6552335 , fax : + 39 - 091 - 6552337 , e - mail : drdiving@yahoo.it received : 05 junuary 2007 / accepted : 22 april 2008 / published online : 30 january 2009 springer - verlag 2009 abstract purpose . 
thirteen patients with a magnetic resonance imaging ( mri ) diagnosis of pcl lesions ( ten acute and three chronic ) and 20 healthy controls underwent conventional and compound hrus performed by the same radiologist who was blinded to the subjects case - control status . 
tredici pazienti con diagnosi rm di lesione del lcp , 10 acuta e 3 cronica , sono stati sottoposti ad hrus convenzionale e compound eseguita in cieco dallo stesso radiologo . 
in consenso ed in cieco altri due radiologi hanno misurato nelle immagini hrus lo spessore degli lcp , quindi i casi sono stati classificati in normali / patologici e questi ultimi in acuti / cronici . 
nelle immagini hrus convenzionali e compound lo spessore del lcp era rispettivamente nel normale 4 , 50 , 7 mm e 4 , 60 , 7 mm , nelle lesioni acute 9 , 11 , 5 mm e 9 , 21 , 7 mm e nelle croniche 70 , 9 mm e 70 , 8 mla riproducibilit intermetodo nella misurazione dello spessore del lcp nelle immagini convenzionali versus compound era del 98 , 6% . 
lhrus consente lo studio del lcp con riconoscimento incidentale delle alterazioni traumatiche . radiol med ( 2009 ) 114 : 312320 keywords posterior cruciate ligament knee sonography real - time spatial compound sonography magnetic resonance imaging parole chiave legamento crociato posteriore ginocchio ecografia real - time spatial compound risonanza magnetica introduction introduzione posterior cruciate ligament ( pcl ) lesions are a relatively uncommon consequence of traumatic knee injuries , and the reported incidence of pcl rupture ranges between 1% and 44% of all acute injuries of the knee ligaments [ 1 ]  . 
in addition , in acute settings , many of the manoeuvres used for diagnosis , among which the most important is the posterior drawer test , may give false - negative results [ 2 , 3 ]  . arthroscopy permits direct visualisation of pcl lesions and is considered the diagnostic standard . 
however , it is an invasive procedure characterised by postprocedural discomfort and a small but finite complication rate ( 1.4% of cases ) and should preferably be performed for therapeutic purposes [ 4 ]  . 
diagnostic imaging modalities such as computed tomography ( ct ) and magnetic resonance imaging ( mri ) allow noninvasive visualisation of the pcl and hence play a key role in detecting and evaluating traumatic pcl lesions . 
mri is a reliable noninvasive technique , but its use is limited by high costs though lower than those of arthroscopy and reduced availability , especially of high - field - strength units . in contrast , high - resolution ultrasonography ( hrus ) is a inexpensive , noninvasive and widely available technique . 
in studying the knee joint , hrus has proved to be highly reliable for evaluating extra - articular disease [ 6 ] but has yielded conflicting results in evaluating cruciate ligament and meniscal injuries [ 710 ]  . 
by reducing the negative effect of acoustic artefacts ( speckle and clutter ) and increasing the definition of tissue planes , real - time spatial compounding allows better spatial and contrast resolution . 
la diagnosi clinica allesame obiettivo , soprattutto in fase acuta , pu essere molto difficoltosa e / o pu sottostimare lentit della lesione . in queste condizioni molte delle manovre cliniche , e tra queste la pi rilevante il test del cassetto posteriore , possono risultare falsamente negative [ 2 , 3 ]  . 
 anche se lartroscopia rappresenta lo standard diagnostico , consentendo la visualizzazione diretta delle lesioni del lcp , una procedura invasiva eseguita preferibilmente a scopi terapeutici , caratterizzata da discomfort post - procedurale e con bassa , seppur presente , incidenza di complicanze ( 1 , 4% dei casi ) [ 4 ]  . 
la diagnostica per immagini , mediante la tomografia computerizzata ( tc ) e la risonanza magnetica ( rm ) , consente la visualizzazione non invasiva del lcp e pertanto riveste un ruolo fondamentale nellidentificazione e valutazione delle alterazioni traumatiche del lcp . 
la rm una metodica affidabile e non invasiva , ma condizionata dallalto costo , comunque inferiore a quello dellartroscopia , e dalla ridotta diffusione sul territorio , specialmente se ci riferiamo alle apparecchiature ad alta intensit di campo . 
nello studio dellarticolazione del ginocchio lhrus si dimostrata molto affidabile nella valutazione delle alterazioni delle strutture extra - articolari [ 6 ] mostrando risultati controversi nella valutazione della alterazioni dei legamenti crociati e dei menischi [ 710 ]  . 
il real - time spatial compound riducendo linterferenza negativa degli artefatti acustici ( speckle and clutter ) consente una migliore risoluzione spaziale e di contrasto incrementando la tissue - plane definition . 
mri was performed with both low - field - strength ( artoscan c , esaote biomedica , genoa , italy ) and high - field - strength ( signa excite hd , ge medical systems , milwaukee , wi , usa ) units . 
the acute or chronic nature of lesions was established on the basis of previously published mri criteria [ 12 , 13 ]  . to assess the reliability of hrus with and without spatial compounding and determine the normal appearance of the pcl , a group of 20 healthy volunteers ( 12 men and eight women ; age range 2036 years ; mean age 24.6 ) with no history of traumatic or degenerative knee lesions and who had not undergone mri evaluation were enrolled as controls . 
in accordance with the declaration of helsinki [ 14 ] , approval by the ethics committee was not required . hrus technique conventional and real - time spatial compound hrus imaging was performed by the same radiologist , who was blinded to the subjects case - control status . 
patients underwent hrus of the affected knee only , and controls underwent hrus of one knee , which was chosen by tossing a co in consideration of the results obtained by miller in patients and controls , pcl thickness of the contralateral knee was not measured [ 7 ]  . 
hrus examinations were carried out on an hdi 5000 scanner ( atl - philips , bothell , wa , usa ) with a high - resolution multifrequency lineararray transducer ( 712 mhz )  . 
the proximal end of the probe was pushed down as much as possible with the heel - toe manoeuvre [ 8 ] and tilted slightly to achieve a plane parallel to the long pcl axis . 
hrus criteria used to define the presence of an acute pcl lesion were : ( 1 ) marked hypoechogenicity with nonvisualisation of the normal fibrillar pattern , ( 2 ) thickening , ( 3 ) ill - defined irregmateriali e metodi popolazione in un periodo di 15 mesi , 13 pazienti con diagnosi rm di lesione del lcp , 8 uomini e 5 donne ( et compresa tra 19 e 36 anni ; et media 24 , 5 anni ) , 10 ( 73 , 3% ) con lesione acuta e 3 ( 26 , 7% ) con lesione cronica , sono stati sottoposti ad hrus con tecnica convenzionale e con lapplicazione del real - time spatial compound . 
le indagini rm erano state eseguite sia con apparecchiature a basso campo ( artoscan c ; esaote biomedica , genova , italia ) che ad alto campo ( signa excite hd , ge medical systems , milwakee , wisconsin , usa )  . 
 per valutare laffidabilit dellhrus con entrambe le tecniche e dimostrare il normale aspetto del lcp stato reclutato un gruppo controllo non sottoposto a valutazione rm composto da 20 volontari sani , 12 uomini e 8 donne ( et compresa tra 20 e 36 anni ; et media 24 , 6 anni ) , con anamnesi negativa per patologia traumatica o degenerativa del ginocchio . 
i pazienti e i controlli informati degli scopi dello studio e delle modalit operative delle varie procedure hanno fornito in forma scritta il proprio consenso informato a partecipare allo studio . 
in considerazione del rispetto delle norme della dichiarazione di helsinki [ 14 ] non stato richiesto il parere del comitato etico . tecnica hrus le indagini hrus ( convenzionale e con real - time spatial compound imaging ) sono state eseguite in cieco dallo stesso radiologo non a conoscenza dei pazienti e dei controlli . 
nei pazienti il ginocchio sottoposto a valutazione hrus stato quello affetto dalla patologia del lcp ; nei volontari stato valutato un solo ginocchio ed il lato da valutare stato scelto in modo random con luso di una moneta prima dellindagine . 
per lesecuzione delle indagini hrus stata utilizzata unapparecchiatura hdi 5000 ( atl - philips , bothell , wa , usa ) utilizzando una sonda lineare multifrequenza ( 712 mhz ) ad alta risoluzione . 
each conventional and compound hrus image was rated for echostructure and margins using a fourpoint scale : ( 1 ) unacceptable , ( 2 ) poor quality but marginally acceptable , ( 3 ) good quality and ( 4 ) excellent quality . analysis of variance to quantify differences in pcl thickness measured with conventional and compound hrus was carried out by another author who pooled the results of the measurements obtained . 
measurements were reported as averagestandard deviation ( sd )  . results as no patient enrolled in the study was obese , the results of the hrus examinations were unaffected by this potential limitation . 
these patients underwent arthroscopy , which confirmed the pcl injury . the remaining three patients with pcl injury underwent physiotherapy , and the injury was confirmed by follow - up mri at 2 months . 
lanalisi della varianza dello spessore per quantizzare le differenze della misurazione legate alla metodica hrus ( convenzionale versus compound ) stata eseguita da un altro autore che ha combinato i risultati delle misurazioni ottenute . 
 risultati nessuno dei pazienti pervenuti alla nostra osservazione e reclutati nello studio era obeso ; pertanto il risultato delle indagini ecografiche non stato inficiato da questo 316 radiol med ( 2009 ) 114 : 312320 possibile limite . 
nei pazienti con lesione del lcp abbiamo evidenziato in tutti i casi nella classificazione della lesione come acuta o cronica una completa concordanza tra la classificazione rm e quelle hrus . 
 in 10 / 13 pazienti ( 76 , 92% ) , 8 con lesione acuta e 2 con lesione cronica del lcp , stata evidenziata alla rm unalterazione associata : al legamento crociato anteriore nel 70% e ai menischi nel 60% . 
lo spessore del lcp nelle lesioni acute era 9 , 11 , 5 mm nelle immagini hrus convenzionali e 9 , 21 , 7 mm in quelle con lapplicazione real - time spatial compound . 
mentre lo spessore del lcp nelle lesioni croniche era 70 , 9 mm nelle immagini hrus convenzionali e 70 , 8 mm in quelle con lapplicazione real - time spatial compound . 
confrontando lo spessore nelle immagini hrus convenzionali versus quello nelle compound stata rilevata una riproducibilit inter - metodo del 98 , 6% . le immagini hrus convenzionali e compound nella valutazione dellecostruttura degli lcp patologici hanno ricevuto un punteggio rispettivamente di 2 , 70 , 6 e 3 , 30 , 3 , in quella del margine superficiale un punteggio rispettivamente di 2 , 90 , 3 e 4 e in quella del margine profondo un punteggio rispettivamente di 2 , 40 , 7 e 3 , 50 , 5 . 
 le lesioni del lcp sono poco comuni essendo il risultato di traumi di notevole intensit e le cause di maggiore incidenza sono i traumi sportivi e della strada [ 15 ]  . 
in precedenza alcuni autori [ 79 ] hanno eseguito una valutazione ecografica non in cieco del lcp in volontari sani e in soggetti con lesioni acute o croniche ma nessuno ha mai effettuato valutazioni hrus in cieco . 
le scansioni hrus convenzionale ( a ) e real - time spatial compound ( b ) mostrano una formazione semilunare ipoecogena a struttura fibrillare , con margini regolari , ben distinguibile dal tessuto adiposo limitrofo iperecogeno . discussione results , with 100% sensitivity , specificity and positive predictive value . 
le scansioni hrus convenzionale ( a ) e real - time spatial compound ( b ) mostrano ispessimento e marcata ipoecogenicit del lcp con mancata identificazione del disegno fibrillare , irregolarit e sfumatura del margine superficiale , e scarsa identificazione del margine profondo . 
3a - c chronic injury of the posterior cruciate ligament ( pcl ) on conventional ( a ) and real - time spatial compound high - resolution ultrasonography ( hrus ) images ( b ) : thick and inhomogeneously hyperechoic structure with nonvisualisation of normal fibrillar pattern , irregular and smooth margins and inhomogeneity of the surrounding fat . 
le scansioni hrus convenzionale ( a ) e real - time spatial compound ( b ) mostrano aumentato di spessore e disomogenea iperecogenicit del lcp con mancata identificazione del disegno fibrillare , irregolarit e sfumatura dei margini , e disomogeneit del tessuto adiposo limitrofo . 
as reported by schulz et al . , knowledge of the biomechanics of the injury is crucial , as it can prompt the search for multiple ligament injuries or meniscal lesions [ 1 ]  . 
although there are previous reports [ 79 ] of unblinded sonographic evaluations of the pcl in healthy volunteers and in patients with acute or chronic injuries , ours is the first study to have performed blinded hrus evaluations . 
the evaluation of conventional and compound hrus images revealed that both techniques allow identification of diagnostic criteria , enabling detection of normal or injured pcls and acute or chronic injuries . 
the wider variability of the thickness values found in these studies may be explained by : ( 1 ) interindividual and interracial variations in the populations studied , for which no anthropometric correlation was determined in either our study or previous studies , ( 2 ) the level of measurement of pcl thickness differed between our study and previous studies ; in particular , cho et al . 
 [ 8 ] measured pcl thickness at the level of the tibial spine , whereas miller did not specify the level of measurement [ 7 ]  . pcl thickness in acute lesions was 9.11.6 mm on conventional hrus images and 9.21.7 mm on compound valutazione comparativa tra i due lcp normali nei volontari sani . 
la valutazione delle immagini hrus ottenute con la tecnica convenzionale e con quella compound ha messo in evidenza come con entrambe stata possibile lidentificazione dei criteri diagnostici per la classificazione degli lcp in normali / patologici e in questi ultimi in acuti e cronici . 
la nostra esperienza pertanto come confermato dalla variabilit intermetodo , con un valore del 98 , 6% , non ha mostrato differenze significative tra lhrus convenzionale e quella con lapplicazione compound nella capacit di caratterizzazione del lcp . 
pertanto entrambe hanno permesso in tutti i casi la corretta classificazione del lcp come normale o patologico con completa concordanza tra la classificazione sulle immagini rm e quella eseguita sulle immagini hrus ottenute con la tecnica convenzionale e con lapplicazione compound . nei volontari sani lo spessore del lcp misurato nelle immagini hrus eseguite senza e con compound risultato rispettivamente di 4 , 50 , 7 mm e 4 , 60 , 7 mnon stata evidenziata una differenza statisticamente significativa nello spessore del lcp misurato con entrambe le tecniche . 
 lo spessore del lcp nelle lesioni acute era 9 , 11 , 6 mm nelle immagini hrus convenzionali e 9 , 21 , 7 mm in quelle con lapplicazione compound . 
 [ 8 ] si rileva , come gi evidenziato in precedenza nella comparazione dello spessore dei lcp normali , una maggiore dispersione dei valori di spessore rispetto a quelli del nostro studio e le possibili giustificazioni sono le medesime prima menzionate per gli lcp normali . 
in our study , we classified pcl injuries as acute or chronic and separately evaluated the diagnostic role of the two hrus techniques in each injury . a comparison of our results with those of miller [ 7 ] and cho et al . 
although not an objective of this study , the use of pcl thickness alone as a diagnostic criterion proved unhelpful for classifying chronic injuries but relatively useful in acute injuries , as statistical analysis revealed a significant difference between the thickness of acutely injured and normal pcls . as with other muscles , tendons and ligaments , the pcl is mainly composed of specular reflectors . 
when these structures are insonated , the amplitude of the echo strongly depends on the angle of incidence the greatest amplitude being obtained when the ultrasound beam is perpendicular to the structure [ 11 ]  . 
averaging the images obtained from different angles of insonation results in suppression of artefacts , with improved image quality and a better signal - to - noise ratio [ 17 ]  . our comparison of conventional and compound hrus showed that the latter provides better visualisation of anatomical details , and in particular , improves fibrillar echostructure definition and pcl margins . 
in our experience , while not providing a diagnostic gain , compound imaging nevertheless improved depiction of the pcl and its pathological changes . one of the main limitations of spatial compounding is mostrato una certa utilit nella valutazione di quelli con lesioni acute mostrando rispetto ai normali una differenza statisticamente significativa . 
 llcp come altre strutture muscolo - tendineo - legamentoso costituito principalmente da reflettori speculari ; quando queste strutture vengono insonate lampiezza delleco di risposta dipende dallangolo di incidenza del fascio ultrasonoro e la maggiore ampiezza si ottiene quando questo perfettamente perpendicolare alla struttura in esame [ 11 ]  . 
le immagini hrus convenzionali sono spesso soggette allinterferenza degli artefatti a specchio e del rumore , i quali riducono la qualit dellimmagine non consentendone a volte una corretta valutazione [ 16 ]  . 
tutti gli artefatti si combinano riducendo la risoluzione di contrasto con degradazione dellecostruttura e riduzione della definizione dei dettagli , ed in particolare dei margini ; inoltre si assiste alla riduzione della cospicuit delle piccole strutture a basso contrasto e alla possibile illusione di discontinuit delle strutture continue . 
il real - time spatial compound imaging acquisisce diverse immagini sovrapposte delloggetto da vari punti di vista ; queste immagini sono quindi processate con una media digitale e combinate in una singola immagine in tempo reale . dalla media delle immagini acquisite dai differenti punti di vista si ottiene la soppressione degli artefatti con incremento della qualit dellimmagine e ne risulta un migliore rapporto segnale - rumore [ 17 ]  . 
 nel nostro studio confrontando lhrus convenzionale con quella con lapplicazione compound abbiamo osservato nel secondo caso una pi accurata visualizzazione dei dettagli anatomici , ed in particolare una migliore identificazione dellecostruttura fibrillare e della definizione dei margini del lcp . 
le immagini hrus convenzionali e compound nella valutazione dellecostruttura degli lcp patologici hanno ricevuto un punteggio rispettivamente di 2 , 70 , 6 e 3 , 30 , 3 , con migliore visualizzazione del disegno fibrillare nelle immagini hrus compound . 
nella valutazione dei margini superficiale e profondo degli lcp patologici le immagini hrus convenzionali e compound hanno ricevuto per il margine superficiale un punteggio rispettivamente di 2 , 90 , 3 e 4 e in quella del margine profondo un punteggio rispettivamente di 2 , 40 , 7 e 3 , 50 , 5 . 
nonostante i punteggi ottenuti sulle immagini hrus con lapplicazione del compound siano migliori rispetto a quelli ottenuti dalle immagini hrus convenzionale nel nostro studio non sono state rilevate differenze statisticamente significative tra i punteggi ottenuti . 
 una delle maggiori limitazioni del compound la riduzione del frame rate con riduzione della risoluzione temporale 320 radiol med ( 2009 ) 114 : 312320 the decreased frame rate that occurs with lower temporal resolution and a resulting persistence effect . 
the main limitations of our study were : ( 1 ) small case series , which precluded stringent statistical validation , and ( 2 ) body habitus , in that our series did not include obese patients , in whom spatial resolution is reduced by the greater distance between the transducer and the pcl resulting from increased thickness of popliteal fossa fat ( even though this shortcoming may generally be overcome by using multifrequency transducers )  . 
hrus is not recommended in the diagnostic workup of patients with knee trauma , as adequate information is obtained about superficial structures only ( such as collateral ligaments ) , whereas the information on inner structures ( menisci and anterior cruciate ligaments ) is partial and often incomplete and unreliable . traumatic knee injuries may result in multiple lesions affecting the menisci , ligaments and bones , as seen in our population . 
given the high level of accuracy of hrus in detecting pcl lesions , the technique could be used for the follow - up of isolated chronic lesions , making the use of mri unnecessary and thus providing considerable cost savings . che risulta in effetti di persistenza . 
lhrus non raccomandata nel corretto iter diagnostico dei pazienti con patologia traumatica del ginocchio consentendo di ottenere adeguate informazioni solo sulle strutture superficiali , come i legamenti collaterali , ma parziali e spesso incomplete e inaffidabili su quelle profonde , come i menischi e il legamento crociato anteriore . 
lhrus come evidenziato nel nostro studio consente con buona accuratezza il riconoscimento delle alterazioni del lcp e potrebbe essere impiegata nel follow - up delle lesioni croniche isolate non rendendo necessario lutilizzo della rm , con marcata riduzione dei costi . 
clinical , histological and atypical radiological patterns selected among our cases il tumore fibroso solitario della pleura ( tfsp ) : patologia dai mille volti . clinica , istologia e rappresentazione degli aspetti radiologici insoliti selezionati dalla nostra casistica l . 
fava1 1istituto di radiologia , universit degli studi di torino , ospedale san luigi gonzaga , regione gonzole 10 , 10143 orbassano , italy 2reparto di radiologia , ospedale degli infermi , biella , italy 3s.c.d.u. 
sftp usually presents as a peripheral mass abutting the pleural surface , to which it is attached by a broad base or , more frequently , by a pedicle that allows it to be mobile within the pleural cavity . 
a precise preoperative diagnosis can be arrived at with a cutting - needle biopsy , although most cases are diagnosed with postoperative histology and immunohistochemical analysis of the dissected sample . 
sftp , owing to its large size or unusual locations ( paraspinal , paramediastinal , intrafissural and intraparenchymal ) , can pose interpretation problems or , indeed , point towards a diagnosis of diseases of a totally different nature . 
we present some unusual radiographic and computed tomography ( ct ) images of large sftp or sftp located in atypical thoracic locations in patients who underwent surgical resection . keywords mesenchymal neoplasms pleura computed tomography riassunto il tumore fibroso solitario della pleura ( tfsp ) , descritto per la prima volta da klemperer e rabin nel 1931 , una neoplasia mesenchimale che solitamente coinvolge la pleura , ma che viene descritta anche in altre sedi sia toraciche , quali il mediastino , il pericardio ed il parenchima polmonare , sia extratoraciche , quali le meningi , lepiglottide , le ghiandole salivari , la tiroide , i reni e la mammella . 
di solito si presenta come massa periferica a contatto con la superficie pleurica alla quale unito da una larga base dimpianto o pi spesso da un peduncolo che lo pu rendere mobile allinterno del cavo pleurico . 
la diagnosi preoperatoria di certezza pu essere ottenuta mediante biopsia con ago tranciante , ma nella maggior parte dei casi post - chirurgica , frutto dellesame istologico e dellanalisi immuno - istochimica sul pezzo operatorio . 
il tumore fibroso solitario della pleura tuttavia , spesso a causa delle notevoli dimensioni o dello sviluppo in sedi inconsuete ( paraspinali , paramediastiniche , intrascissurali ed intraparenchimali ) pu creare numerose difficolt di interpretazione o addirittura orientare la diagnosi verso patologie di tuttaltra natura . 
in questo lavoro presentiamo gli aspetti radiologici inusuali di tfsp di grosse dimensioni o localizzati in sedi toraciche atipiche in pazienti sottoposti ad intervento chirurgico di exeresi . parole chiave tumori mesenchimali pleura tomografia computerizzata radiol med ( 2009 ) 114 : 204215 introduction solitary fibrous tumour of the pleura ( sftp ) is a mesenchymal tumour that tends to involve the pleura , although it has also been described in other thoracic areas ( mediastinum , pericardium and pulmonary parenchyma ) and in extrathoracic sites ( meninges , epiglottis , salivary glands , thyroid , kidneys and breast ) [ 12 ]  . 
sftp usually presents as a peripheral mass abutting the pleural surface , to which it is attached by a broad base or , more frequently , by a pedicle that allows it to be mobile within the pleural cavity [ 24 ]  . 
unlike mesothelioma , sftp is not asbestos - related and is usually a benign , rarely aggressive , tumour , although a small percentage of patients may develop locoregional recurrence [ 2 , 5 , 6 ]  . 
 the most accredited hypothesis is that these tumours originate from submesothelial stromal cells with fibroblastic or myofibroblastic phenotype , whose growth is promoted by an aberrant reaction to inflammatory or hormonal stimuli [ 7 ]  . 
most patients are asymptomatic at the time of the diagnosis , with the lesion being discovered during a chest x - ray performed for other reasons [ 4 , 8 ]  . 
in the remaining patients , particularly those with large and aggressive masses , the most common clinical manifestations are chest pain , cough and dyspnoea [ 4 , 6 , 9 ]  . 
the rate of incidental diagnoses is increasing , as demonstrated by our series in which symptomatic patients did not exceed 30% and confirmed by a comparison of studies published between 1942 and 1972 and between 1973 and 1980 in which symptomatic patients decreased from 72% to 54% [ 4 ]  . 
sftp , owing to its large size or unusual locations ( paraspinal , paramediastinal , intrafissural and intraparenchymal ) , can pose interpretation problems or , indeed , point towards a diagnosis of diseases of a totally different nature . 
 we present some unusual radiographic and computed tomographic ( ct ) images selected from our case history of sftp located in atypical thoracic locations differential diagnosis . histology and immunohistochemistry introduzione il tumore fibroso solitario della pleura ( tfsp ) una neoplasia mesenchimale che solitamente coinvolge la pleura , ma che viene descritta anche in altre sedi sia toraciche , quali il mediastino , il pericardio ed il parenchima polmonare , sia extratoraciche , quali le meningi , lepiglottide , le ghiandole salivari , la tiroide , i reni e la mammella [ 1 , 2 ]  . 
generalmente si presenta come massa periferica a contatto con la superficie pleurica alla quale unito da una larga base dimpianto o pi spesso da un peduncolo che lo pu rendere mobile allinterno del cavo pleurico [ 24 ]  . il tfsp , a differenza del mesotelioma , non una malattia asbesto - correlata e di solito non presenta un comportamento aggressivo , sebbene una ridotta percentuale di pazienti possa andare incontro a recidive loco - regionali [ 2 , 5 , 6 ]  . 
lipotesi pi accreditata sostiene che questi tumori possano originare delle cellule stromali sottomesoteliali con fenotipo fibroblastico o miofibroblastico , alla cui crescita contribuisce unaberrante reattivit a stimoli infiammatori od ormonali [ 7 ]  . 
la maggior parte dei pazienti asintomatico al momento della diagnosi essendo la lesione riscontrata in occasione di un radiogramma del torace eseguito per altri motivi [ 4 , 8 ] ; nei restanti casi , soprattutto in presenza di lesioni di grandi dimensioni e con comportamento aggressivo , le pi comuni manifestazioni cliniche sono dolore toracico , tosse e dispnea [ 4 , 6 , 9 ]  . 
la percentuale di casi riscontrati occasionalmente in continuo aumento , come testimoniato dalla nostra casistica in cui la percentuale di pazienti sintomatici non supera il 30% e dal confronto fra studi pubblicati nel periodo 1942 1972 e nella decade successiva , in cui la percentuale di pazienti sintomatici si ridotta dal 72% al 54% [ 4 ]  . 
questi dati sono da attribuire ad una sempre maggiore ( e talora inappropriata ) diffusione delle metodiche di imaging , con un incremento del numero di casi scoperti in fase iniziale ed in modo del tutto occasionale . tali lesioni tuttavia , spesso a causa delle cospicue dimensioni o dello sviluppo in sedi inusuali ( paraspinali , paramediastiniche , intrascissurali ed intraparenchimali ) possono creare numerose difficolt di interpretazione o addirittura orientare la diagnosi verso patologie di tuttaltra natura . in questo lavoro , abbiamo selezionato dalla nostra casistica alcuni dei casi pi interessanti caratterizzati da aspetti radiologici inconsueti sia per sede che per dimensioni per i quali si dovuto sensibilmente ampliare il ventaglio delle possibili diagnosi differenziali . 
 sftp is a rare mesenchymal neoplasm , previously defined as benign localised mesothelioma or fibrous benign mesothelioma , although the use of these terms is discouraged owing to the potential confusion with mesothelioma . as the name suggests , it is usually a single , occasionally large , pleural neoplasm , although multiple forms have been reported in the literature [ 4 , 8 , 10 ]  . 
macroscopically , it istologia ed immuno - istochimica il tfsp una rara neoplasia mesenchimale , in passato definita mesotelioma benigno localizzato o mesotelioma fibroso benigno , termini il cui uso deve essere scoraggiato per la potenziale confusione con il mesotelioma . 
come il termine suggerisce una neoplasia pleurica singola , 206 radiol med ( 2009 ) 114 : 204215 originates from the visceral pleura [ 6 ] , to which it is often connected by a short pedicle [ 2 , 3 , 11 ]  . 
microscopically , the typical finding is that of a patternless pattern consisting of spindle cells with round nuclei with scarce cytoplasm arranged in bundles or in a fishbone configuration , in close contiguity with small vessels , resulting in a haemangiopericytoma - like appearance . 
sftp is usually histologically benign , but the presence of cellular atypia and a high mitotic index [ defined as being in excess of 4 / 10 high power field ( hpf ) ] is strongly indicative of malignancy [ 10 ]  . 
the differential diagnosis of sftp includes sarcomatoid mesothelioma , primary pleural synovial sarcoma and haemangiopericytoma [ 1 , 1921 ]  . talvolta di grosse dimensioni , anche se in letteratura ne sono state descritte forme multiple [ 4 , 8 , 10 ]  . 
solitamente i tfsp sono istologicamente benigni , ma la presenza di atipie cellulari e di un alto indice mitotico ( definito come maggiore di 4 / 10 hpf ) fortemente indicativa di malignit [ 10 ]  . 
la diagnosi differenziale dei tfsp include il mesotelioma sarcomatoide , il sarcoma sinoviale pleurico primitivo e lemangiopericitoma [ 1 , 1921 ]  . radiological aspects chest film sftp usually appears on chest x - ray as a peripheral opacity with well - defined borders and homogeneous density [ 8 , 9 , 2224 ]  . 
il versamento pleurico di natura reattiva ( sieroso o siero - emorragico ) concomitante in una proporzione di casi che varia in letteratura fra il 6% e il 17% [ 3 , 4 , 12 , 27 , 28 ] , soprattutto in presenza di forme maligne [ 3 , 8 , 29 ]  . la valutazione dellangolo di raccordo con la parete toracica permette la distinzione fra masse di pertinenza pleurica e lesioni parenchimali , risultando ottuso nei piccoli tumori a genesi pleurica , acuto nelle neoplasie intrapolmonari e nei tfsp di maggior volume e / o peduncolati [ 30 , 31 ]  . la mobilit e le modificazioni di morfologia , imposte dalle diverse fasi della dinamica respiratoria e / o dai diversi decubiti del paziente , accertate fluoroscopicamente , costituiscono i segni patognomonici di una lesione peduncolata di pertinenza pleurica [ 3 , 9 , 32 , 33 ]  . radiol med ( 2009 ) 114 : 204215 fig . 
f immunoistochimica20 : bcl2 +  . pathognomonic signs of a pleural pedunculated lesion and can be verified with fluoroscopic examination [ 3 , 9 , 32 , 33 ]  . tomografia computerizzata ( tc ) computed tomography ( ct ) the size of the lesion will strongly affect its appearance on computed tomography ( ct )  . 
smaller fibromas usually appear as homogeneous , well - defined , rarely lobulated masses forming obtuse angles against the pleural surface [ 3 , 22 , 34 , 35 ]  . 
i fibromi di piccole dimensioni si presentano di solito come masse ben delimitate , omogenee , solo occasionalmente lobulate , a contatto con la superficie pleurica e con angolo di raccordo ottuso [ 3 , 22 , 34 , 35 ]  . quando di cospicue dimensioni sono invece piuttosto disomogenee e con angoli di raccordo acuti . 
il riscontro di un angolo di raccordo a detta degli autori anglosassoni , smooth tapering [ 22 , 34 ] un altro segno tc affidabile di neoplasia a partenza pleurica . 
nei tumori intrascissurali esso bilaterale e ne facilita la distinzione da una massa 208 radiol med ( 2009 ) 114 : 204215 junction of the mass with the pleural surface [ 22 , 34 ] is another reliable ct sign of a neoplasm of pleural origin intrafissural tumours , the finding is bilateral , facilitating differentiation from an intraparenchymal mass ( fig 3 c , d ) [ 22 ]  . 
intralesional calcifications ( punctate , linear or coarse ) constantly associated with areas of necrosis may be recognised in larger lesions [ 3 , 4 , 8 , 22 ]  . 
the presence of the pedicle is rarely identified on ct but can be inferred by evaluating lesion mobility [ 4 , 36 ]  . sftp exhibits medium to high attenuation on baseline scans owing to the high physical density of the collagen fibres and the abundant vascular network of the lesions ( fig . 4c ) [ 4 , 36 , 37 ]  . 
con laumentare delle dimensioni , dopo somministrazione di mezzo di contrasto costante il riscontro di lesioni piuttosto disomogenee caratterizzate da un pattern tomodensitometrico definito a carta geografica oppure da aree serpiginose iperdense , espressione della ricca rete vascolare , cui possono associarsi aree ipodense , riferibili a fenomeni di degenerazione cistica fig . 
3a - d a 35 - year - old man with incidental finding of intrafissural lung opacity on chest x - ray ( a , b ) confirmed on axial computed tomography ( c ) and parenchymal ( d ) windows . 
involvement of hilar - mediastinal lymph nodes enables a diagnosis of sftp to be definitely excluded . differential diagnosis of sftp in atypical locations the differential diagnosis of primary pleural tumours is relatively narrow and includes , in addition to pleural fibromas and malignant mesothelioma , the rarer lipomas , synovial sarcomas and epithelioid haemangioendothelioma [ 3941 ]  . 
linteressamento linfonodale ilo - mediastinico esclude con certezza la diagnosi di tfsp . diagnosi differenziale delle localizzazioni insolite il ventaglio diagnostico differenziale dei tumori primitivi della pleura relativamente contenuto , coinvolgendo oltre ai fibromi pleurici solitari ed al mesotelioma pleurico maligno anche i pi rari lipomi , sarcomi e lemangioendotelioma epitelioide [ 3941 ]  . 
il problema della diagnosi differenziale si rende ancora pi complesso quando queste lesioni si sviluppano in sedi inconsuete , rendendo ancora pi ampio il numero di patologie che entrano in diagnosi differenziale . 
in these cases , it is important to evaluate the ribs : chest wall involvement by sftp is rare [ 4 , 42 ] and usually manifests as sclerosis or cortical erosion at the costal level , a feature more typical of tumours of neurogenic origin [ 4 ]  . sftp that have a mediastinal pleural origin can mimic a thymic neoplasm or dysembryogenetic tumour . 
in questi casi , la valutazione della parete toracica importante ; infatti , in letteratura , la presenza di segni di estensione parietale riportata molto raramente [ 4 , 42 ] e si manifesta generalmente come sclerosi o erosione costale , un aspetto pi caratteristico delle neoplasie di origine neurogena [ 4 ]  . se invece si sviluppano dalla pleura mediastinica possono simulare una neoplasia timica oppure un tumore disembriogenetico . 
5a - d a 74 - year - old woman with a mediastinal mass discovered incidentally on chest x - ray ( a , b ) and confirmed at computed tomography ( ct ) ( c , d )  . 
5a - d donna di 74 anni con riscontro occasionale ( a , b ) di lesione a partenza mediastinica , sospetto confermato alla tc ( c , d )  . 
intraparenchymal and inverted sftp present as pulmonary nodules or rounded or oval masses with well - defined margins and enter the differential diagnosis with some benign diseases ( hamartochondromatosis ) or with less aggressive lesions such as carcinoids [ 44 ]  . two reports in the literature address ct imaging of intrapulmonary pleural fibromas [ 44 , 45 ] , and only one of them describes ct features of intense , predominantly peripheral , heterogeneous enhancement [ 45 ]  . 
attualmente la tc multidetettore con le ricostruzioni multiplanari ottenute da acquisizioni sottili permettono una migliore visualizzazione della scissura ed i suoi rapporti con il tumore . 212 radiol med ( 2009 ) 114 : 204215 fig . 
6a , b solitary fibrous tumour of the pleura ( sftp ) in a 63 - year - old patient with an incidental finding of left paraspinal opacity on chest x - ray ( a , b ) , which was confirmed at computed tomography ( ct ) ( c ) and initially diagnosed as a neurogenic tumour . 
7a - c a 44 - year - old man with atypical precordial pain who underwent chest x - ray in the lateral view ( a ) and computed tomography ( ct ) with sagittal maximum intensity projection ( mip ) reformations ( b ) and transverse lung windowing ( c )  . 
a radiogramma mirato in proiezione laterale , b tc con ricostruzione mip in sezione sagittale e c tc in sezione assiale con finestra per il parenchima dimostrano la presenza di una lesione nodulare localizzata nel lobo superiore sinistro che data la stretta contiguit con le strutture bronchiali e vascolari stata sospettata la diagnosi di carcinoide . 
8a - d a 47 - year - old man with incidental finding of a 3 - cm opacity on chest x - ray . computed tomography ( ct ) ( a , b ) confirmed the presence of a well - marginated parenchymal mass within the left lower lobe ( c , d )  . 
la tc prima e dopo somministrazione di mezzo di contrasto ( a , b ) mostra un nodulo intraparenchimale a margini ben definiti nel contesto del lobo inferiore sinistro ( c , d ) , in stretta contiguit con un vaso polmonare ( d )  . 
la diagnosi istopatologica definitiva stata di tfsp . conclusion conclusione in conclusion , sftp is a rare mesenchymal neoplasm that is relatively easy to diagnose when located in typical sites , as the diseases entering the differential diagnosis are few and rare . 
cova1 1unit clinica operativa di radiologia , 2unit clinica operativa di anatomia ed istologia patologica , ospedale di cattinara , universit di trieste , strada di fiume 447 , 34149 trieste , italy correspondence to : e . 
seven intratumoural tissue components can be identified both at ct and at pathology : ( 1 ) solid component , ( 2 ) haemorrhagic component , ( 3 ) coagulation necrosis , ( 4 ) liquefactive necrosis , ( 5 ) parenchymal consolidation , ( 6 ) diffuse peripheral component and ( 7 ) fibrotic component . 
necrotic and haemorrhagic components are typically observed in malignant lesions , whereas solid and fibrotic components may be seen both in solid lung malignancies and in benign lesions . keywords lung tumours ct radiological - pathological correlation riassunto lo scopo di questa presentazione descrivere le componenti tissutali intratumorali presenti nei tumori polmonari solidi che risultino evidenti allanalisi macroscopica e / o microscopica del campione patologico , autoptico oppure chirurgico , e che siano identificabili anche allindagine tomografia computerizzata ( tc ) eseguita mediante metodica diretta e dopo la somministrazione di mezzo di contrasto . 
le componenti a carattere necrotico ed emorragico vengono osservate tipicamente nelle neoplasie maligne , mentre le componenti solide ed a carattere fibrotico possono essere osservate sia nei processi polmonari solidi a carattere maligno sia nelle lesioni benigne . 
malignant tumours are the most common , with bronchogenic carcinoma being the leading cause of death from cancer in many countries i tumori solidi del polmone rappresentano un reperto frequente al radiogramma diretto del torace oppure alla tomografia computerizzata ( tc )  . 
although surgery has a high success rate in the initial stages of disease [ 2 , 3 ] , survival and general outcomes are closely related to the histological type of the tumour . 
in particular , histological typing of primary lung tumours is based on their most differentiated components , whereas grading is based on the cell features of their least differentiated components [ 13 ]  . 
four histological types account for more than 95% of primary lung cancers : squamous cell carcinoma , adenocarcinoma , undifferentiated large cell carcinoma and small cell carcinoma ( microcytoma )  . 
squamous cell carcinoma is characterised by the presence of intercellular bridging ( thin parallel strands connecting adjacent cells ) , abundant keratin production by the individual cells ( intense eosinophilia of the single cells ) and formation of keratin pearls in the well - differentiated forms . adenocarcinoma is the most common histological type of lung cancer and represents almost 50% of all bronchogenic carcinomas . 
the tumour cells are large , with abundant cytoplasm , large nuclei , prominent nucleoli and a growth pattern of cords of uniform cells . giant cell carcinomas are a subtype of undifferentiated large cell carcinomas and are characterised by giant pleomorphic cells with bizarre morphology . 
anche se la chirurgia raggiunge un elevato livello di successo negli stadi iniziali della patologia [ 2 , 3 ] , la sopravvivenza ed il risultato generale della terapia sono strettamente correlati allistotipo tumorale . 
in particolare , listotipo dei tumori polmonari primitivi viene classificato sulla base delle aree tumorali maggiormente differenziate mentre il grading tumorale basato sulle caratteristiche cellulari delle aree tumorali meno differenziate [ 13 ]  . 
quattro istotipi corrispondono ad oltre il 95% delle neoplasie primitive polmonari : tumore a cellule squamose , adenocarcinoma , carcinoma indifferenziato a grandi cellule , e tumore a piccole cellule ( microcitoma )  . 
il tumore a cellule squamose caratterizzato dalla presenza di ponti intercellulari ( sottili linee parallele tra i bordi di cellule contigue ) , abbondante produzione di cheratina da parte delle single cellule ( intensa eosinofilia delle singole cellule ) , e della formazione di perle di cheratina nelle forme ben differenziate . 
il carcinoma del polmone a cellule giganti rappresenta un sottotipo del carcinoma indifferenziato ed caratterizzato da cellule giganti pleomorfe ed a morfologia radiol med ( 2009 ) 114 : 173189 fig . 
adenocarcinoma del polmone con pattern bronchioloalveolare con massiva diffusione lobare ( frecce ) ( d )  . lung cancers show several macroscopic patterns in relation to the type of growth : 1 . 
peripheral single - nodule or multiple - nodule lung carcinoma : in this macroscopic pattern , single or multiple peripheral nodules are seen within the lung , resembling a secondary lesion . 
lung carcinoma with subpleural diffusion : this growth pattern is distinctive of adenocarcinoma . the aim of this paper is to describe the intratumoural tissue components of solid lung cancers evidenced by macroscopic and / or microscopic analysis of autopsic or surgical specimens and are also visible on nonenhanced and contrast - enhanced ct . 
the purpose is to illustrate some characteristic patterns of lung cancers that can facilitate their characterisation by ct on the basis of the morphological evaluation of the lesion . intratumoural tissue components seven distinct intratumoural tissue components may be described that have correlates on both nonenhanced and contrast - enhanced ct and gross and / or microscopic / histological examination of the pathology specimen : 1 . 
fibrotic component these tissue components may be encountered separately or in various combinations within the same tumour . carcinoma bronchiale in ordine di frequenza , ed tipico del carcinoma a cellule squamose e del tumore a piccole cellule . 
carcinoma del polmone con diffusione subpleurica . questo pattern di crescita caratteristico delladenocarcinoma . lo scopo di questa presentazione descrivere le componenti tissutali intratumorali presenti nei tumori polmonari solidi che risultino evidenti allanalisi macroscopica e / o microscopica del campione autoptico oppure chirurgico , e che siano anche identificabili allindagine tc diretta ed eseguita dopo la somministrazione di mezzo di contrasto . questo al fine di descrivere alcuni patterns caratteristici delle lesioni polmonari maligne che ne facilitino la caratterizzazione alla tc sulla base della valutazione morfologica della lesione . 
 radiol med ( 2009 ) 114 : 173189 solid component this tissue component consists of the simultaneous presence , though in varying proportions , of tumour cells and fibrovascular stroma . 
the solid component may have exclusive endobronchial growth , infiltrating growth within the bronchial wall and the mediastinal structures and peribronchial growth with progressive invasion or compression of the adjacent parenchyma , or again it may cause bronchial obstruction with atelectasis and postobstructive pneumonia . the peribronchial solid component may also manifest as multiple nodules in the lung parenchyma . 
bronchial solid component with invasion of adjacent structures ( bronchial wall and mediastinum ) : lung malignancies presenting with this type of solid component produce focal stenosis or complete occlusion of the affected bronchus . 
this pattern represents the evolution of early endobronchial solid lesions with subsequent invasion of the adjacent lung parenchyma or mediastinum . 3 peribronchial solid component : lung cancers with this type of solid component have solid density and welldefined margins or show infiltration or compression of the adjacent lung parenchyma . 
la componente solida pu presentare una crescita esclusivamente endobronchiale , una crescita infiltrante nei confronti della parete bronchiale e delle strutture mediastiniche , una crescita peribronchiale con progressiva infiltrazione ovvero compressione del parenchima polmonare adiacente , ovvero ancora pu causare ostruzione bronchiale con atelettasia e polmonite post - ostruttiva . 
 haemorrhagic component this intratumoural component may be observed at the level of the central or peripheral region of the solid component in squamous cell carcinomas in undifferentiated large cell carcinomas and in small cell tumours . 
at nonenhanced ct , this radiol med ( 2009 ) 114 : 173189 sostanzialmente , questo pattern rappresenta levoluzione di lesioni tumorali precoci endobronchiali con invasione successiva del parenchima polmonare adiacente oppure del mediastino . 
 componente emorragica questa componente intratumorale pu essere osservata a livello della regione centrale oppure periferica della componente solida nel carcinoma a cellule squamose , nel carcinoma indifferenziato a grandi cellule , e nel tumore a piccole cellule . 
the cut surface of the surgical specimen shows a bronchial lesion ( long arrow ) partially obstructing the lower bronchial lumen and infiltrating the adjacent lung parenchyma ( short arrow ) ( c )  . 
c la superficie di sezione del campione chirurgico del medesimo tumore dimostra un tumore bronchiale ( freccia lunga ) che ostruisce parzialmente il lume del bronco inferiore ed infiltra il parenchima polmonare adiacente ( freccia corta )  . 
lanalisi microscopica rivela il tipico aspetto istologico del carcinoma a cellule squamose con evidenza di ponti intercellulari , abbondante produzione di cheratina da parte delle single cellule con intensa eosinofilia delle singole cellule , e formazione di perle di cheratina . 
frequentemente , questa componente necrotica non chiaramente identificabile allanalisi delle immagini tc dato che risulta commista alla componente solida , nel qual caso determina radiol med ( 2009 ) 114 : 173189 fig . 
lanalisi microscopica rivela il tipico aspetto istologico del carcinoma a cellule squamose con evidenza di ponti intercellulari , abbondante produzione di cheratina da parte delle single cellule con intensa eosinofilia delle singole cellule , e formazione di perle di cheratina . 
the tumour appears heterogeneous , with an intratumoural area of low density that does not enhance after contrast material administration , whereas the solid viable peripheral component shows evident enhancement . 
the section surface of the specimen shows a mass with well - defined lobulated margins , with a brownish central component ( arrow ) that corresponds to the area of intratumoural low density identified at ct and reflecting central coagulation necrosis within the neoplasm ( c )  . 
b la superficie di sezione del campione macroscopico dimostra una massa a margini distinti e lobulati con una componente centrale brunastra ( freccia ) che corrisponde alla regione intratumorale ipodensa identificata alla tc e che riflette la necrosi centrale a carattere coagulativo nel contesto della neoplasia . 
questo pattern tipicamente osservato nel mesotelioma pleurico con coinvolgimento del parenchima polmonare e nelladenocarcinoma periferico con infiltrazione pleurica . parenchymal consolidation this component manifests as a process with massive lobar spread or parenchymal consolidation at gross pathology and exhibits air bronchogram at ct . 
 diffuse peripheral component on gross pathology , this solid component shows a subpleural growth pattern , and it partially or completely involves the peripheral lung region and the visceral and / or parietal pleura , with frequent simultaneous involvement of the lung fissures . 
this pattern is typically observed in pleural mesothelioma with parenchymal involvement and in peripheral adenocarcinoma with pleural infiltration . nente si presenta nella forma di noduli periferici , singoli oppure multipli , a volte coalescenti , oppure si pu manifestare come lesione localizzata a livello dellapice polmonare . 
on gross pathology , this component presents in the form of peripheral single or multiple at times coalescing nodules or as a lesion located at the level of the lung apices . 
extensive parenchymal consolidation at the level of the right lung . contrast - enhanced computed tomography ( b , c ) ( b mediastinal window ; c lung window )  . diffuse parenchymal consolidation involving the upper lobe of the right lung is visualised ( arrow )  . 
 discussion benign lung lesions must be differentiated from lung malignancies , in which infiltration of the adjacent parenchyma , invasion of mediastinal structures or bronchial obstruction are typically present [ 47 ]  . 
anche se lindagine tc standard ( non - dinamica ) eseguita con metodica diretta e con mezzo di contrasto non fornisce ovviamente la medesima risoluzione spaziale della microscopia ottica , alcune distinte componenti tissutali intratumorali possono essere riconosciute mediante una attenta analisi morfologica delle lesioni polmonari . 
in questa presentazione abbiamo descritto le componenti tissutali intratumorali presenti nei tumori polmonari solidi che risultano evidenti alla valutazione macroscopica e / o microscopica dei campioni patologici autoptici oppure chirurgici , e che risultano anche evidenti alla valutazione delle immagini tc dirette e con mezzo di contrasto ottenute 186 radiol med ( 2009 ) 114 : 173189 fig . 
d la microscopia ad alto ingrandimento ( 200 ; colorazione con ematossilina - eosina ) dimostra un adenocarcinoma ben differenziato con estesa reazione desmoplastica periferica e fibrosi ( frecce )  . 
 of contrast material clearly do not provide the same spatial resolution as optical microscopy , some distinct intratumoural tissue components may be recognised by a careful morphological analysis of the lung lesions . 
in this paper , we described the intratumoural tissue components seen in solid lung tumours at gross pathology and / or microscopic analysis of autopsic or surgical specimens , which are also appreciable on examination of nonenhanced and contrastenhanced ct images obtained with the standard nondynamic technique . the solid component related to the presence of proliferating neoplastic cells is common to all lung cancers . 
in particular , squamous cell carcinomas and small cell lung secondo la tecnica standard ( non - dinamica )  . la componente solida determinata dalla presenza di cellule neoplastiche in proliferazione comune a tutte le neoplasie polmonari maligne . 
in particolare , il carcinoma a cellule squamose ed il tumore polmonare a piccole cellule si manifestano tipicamente come lesioni centrali a livello del bronco principale , del bronco lobare , ovvero a livello dei bronchi segmentari in circa i due terzi dei casi e si possono quindi manifestare come masse polmonari ilari con invasione delle strutture mediastiniche adiacenti , oppure anche come lesioni bronchiali ostruttive con conseguente atelettasia ovvero polmonite post - ostruttiva . 
c la superficie di sezione del campione autoptico dimostra una lesione biancastra disomogenea ( frecce ) che rivela una prevalente componente fibrotica alla istologia ( d ) e caratteristiche tipiche della reazione tubercolare granulomatosa ( freccia )  . 
on gross pathology , these endobronchial tumours range from an irregular focal growth in the bronchial mucosa to a polypoid endobronchial mass with diffuse wall infiltration leading to stenosis and / or bronchial occlusion . 
circa un terzo dei carcinomi a cellule squamose presentano una localizzazione periferica a causa della prevalente crescita peribronchiale e si manifestano come noduli polmonari solitari periferici ( 3 cm in diametro ) ovvero come voluminose masse polmonari [ 5 ]  . 
 la componente emorragica e coagulativa pu essere riscontrata in tutti gli istotipi maligni mentre la componente necrotica colliquativa si riscontra tipicamente nel carcinoma a cellule squamose e nel carcinoma bronchioloalveolare [ 13 ]  . 
squamous cell carcinomas constitute the histological type that most commonly produces cavitation , owing to the presence of liquefactive central necrosis in approximately 10% of cases , leaving a cavity with a clear airfluid level with thick , irregular walls [ 5 , 6 ]  . 
the cystic or cavitated pattern , rarely seen in adenocarcinomas [ 11 , 12 ] , may be found in bronchioloalveolar carcinomas [ 13 , 14 ] , and it is generally suggestive of liquefactive intratumoural necrosis . intratumoural areas of coagulation or liquefactive necrosis are frequently observed in undifferentiated large cell lung carcinomas because of the large size of these tumours . 
they cause central hypoxia of the mass and appear as areas of brownish discolouration on examination of the cut surface of the specimen . parenchymal consolidation may be considered a solid tumoural component with diffuse alveolar involvement , similar to a pulmonary infection , and it is typically observed in bronchioloalveolar carcinoma . 
the diffuse peripheral component , which is found both in pleural mesothelioma and in lung adenocarcinoma , is related to the tumoural growth that involves the lung parenchyma starting from the pleural region , or to the neoplastic peribronchial growth with subsequent pleural infiltration . 
the fibrotic component is observed both in lung cancers , in particular adenocarcinomas , and in chronic lung infections . in fact , many benign lesions may mimic malignancies , and the most common of these are solid fibrotic areas , chronic pseudotumoural inflammatory processes [ 15 ] and tubercular lesions [ 16 ]  . 
in particular , active postprimary pulmonary tuberculosis may appear as a cavitation or bronchial wall thickening or in the form of miliary nodules , whereas chronic tuberculosis may appear in the form of calcified nodules , parenchymal consolidation or a spiculated peripheral lung mass [ 1517 ]  . conclusions several intratumoural tissue components may be identified in solid lung tumours , both on gross pathology and / or microscopic analysis of the specimen and on standard ct . 
the necrotic and haemorrhagic components are typically observed in malignant lung lesions , whereas the solid and fibrotic components may be observed in both malignant and benign solid lung lesions . listotipo che pi comunemente produce cavitazione per la presenza di necrosi centrale a carattere colliquativo in circa il 10% dei casi , residuando una cavit con netto livello idroaereo con pareti spesse ed irregolari [ 5 , 6 ]  . laspetto cistico ovvero cavitato , di raro riscontro nelladenocarcinoma [ 11 , 12 ] , pu invece essere riscontrato nel carcinoma bronchioloalveolare [ 13 , 14 ] ed generalmente suggestivo per necrosi intratumorale di tipo colliquativo . 
le aree intratumorali di necrosi coagulativa ovvero colliquativa sono frequentemente osservate nei carcinomi polmonari indifferenziati a grandi cellule a causa delle grandi dimensioni raggiunte spesso dal tumore con conseguente ipossia centrale della massa , ed appaiono come aree di discolorazione brunastra alla valutazione della superficie della sezione del reperto patologico . laddensamento parenchimale pu essere considerato come una componente tumorale solida con diffuso coinvolgimento alveolare , simile ad una infezione polmonare , e viene tipicamente riscontrato nel carcinoma bronchioloalveolare . la componente diffusa periferica , che viene osservata sia nel mesotelioma pleurico come anche nelladenocarcinoma polmonare , determinata dalla crescita tumorale che coinvolge il parenchima polmonare a partire dalla regione pleurica , oppure dalla crescita neoplastica peribronchiale con successiva infiltrazione della pleura . 
di fatto molte lesioni polmonari di tipo benigno possono mimare le neoplasie maligne , e tra queste le pi comuni sono le aree solide da esiti a carattere fibrotico , i processi infiammatori di tipo cronico a carattere pseudotumorale [ 15 ] e le lesioni tubercolari [ 16 ]  . 
in particolare , la tubercolosi polmonare attiva post - primaria si pu manifestare come cavitazione oppure come ispessimento della parete bronchiale ovvero anche come noduli miliari , mentre il processo tubercolare cronico pu manifestarsi come noduli calcifici ovvero anche come addensamento parenchimale oppure ancora come massa polmonare periferica a margini spiculati [ 1517 ]  . 
le componenti a carattere necrotico ed emorragico sono osservate tipicamente nelle neoplasie polmonari maligne , mentre le componenti solide ed a carattere fibrotico possono essere osservate nelle lesioni polmonari solide sia a carattere maligno che benigno . 
thirty - two chest radiographs and 32 chest us examinations were performed in 28 consecutive patients ( aged 4 months to 17 years ) with a clinical suspicion of pneumonia . 
chest us examinations were carried out with a convex - array broadband probe ( 25 mhz ) and a high - frequency linear - array broadband probe ( 512 mhz )  . 
for this reason , chest us may be a valuable aid and possible alternative to standard chest radiography in the evaluation and follow - up of children with suspected pneumonia . keywords thoracic sonography lung consolidation pleural effusion pneumonia riassunto obiettivo . 
in 28 pazienti consecutivi , con esame obiettivo sospetto per polmonite ( di et tra 4 mesi e 17 anni ) sono stati eseguiti , considerando sia gli esami allesordio che i controlli , 32 radiogrammi del torace ed altrettante ecografie della parete toracica . 
lecografia permette di identificare gli addensamenti parenchimali polmonari , qualora siano situati in sede subpleurica , quantomeno con la stessa sensibilit dellrx e come noto valuta molto bene il versamento pleurico . 
pertanto lecografia si pone come un valido supporto ed eventuale alternativa agli esami radiologici tradizionali nel monitoraggio dei pazienti pediatrici . parole chiave ecografia toracica addensamenti polmonari versamento pleurico polmonite 322 introduction chest radiography is considered the reference examination in the diagnosis of pneumonia in children , whereas chest ultrasound ( us ) is not typically used for this purpose [ 1 ]  . chest radiography presents , however , some important disadvantages , including exposure to ionising radiation and a high degree of interand intraobserver variability , not only in typical parenchymal consolidation but especially in consolidations affecting areas that are difficult to explore with radiography , such as retrocardiac locations or the lung bases [ 2 ]  . 
given the smaller volume of lung parenchyma in children , it is likely that the pleura becomes involved more rapidly and to a greater extent than in adults and that chest us may provide results at least comparable with those obtained in adults . 
in many cases , chest us may provide equally useful clinical information and be used as an alternative to standard chest radiography . the aim of this study was to evaluate the sensitivity of chest us compared with chest radiography in identifying parenchymal consolidation and pleural effusion in children with suspected pneumonia . the normal lung is poorly accessible to the us beam because the great difference in impedance between the pleura and the alveolar air causes us waves to be completely reflected . 
sometimes , when the bronchioles are traversed by the us beam , the air bronchogram may also appear as hyperechoic rounded images produced by the air trapped within the bronchial structures [ 5 , 6 ]  . 
us may also identify fluid bronchograms , which correspond to fluid - filled bronchial structures ( typically mucus and / or inflammatory exudate ) that assume a tubular , binary appearradiol med ( 2009 ) 114 : 321330 introduzione la radiografia del torace viene attualmente considerato lesame di riferimento per la diagnosi di polmonite nellet pediatrica , mentre lecografia della parete toracica , da alcuni definita anche ecografia polmonare , non risulta fra le diagnostiche per immagini utilizzate a tal fine [ 1 ]  . 
innanzitutto lesposizione alle radiazioni ionizzanti , secondariamente la variabilit dellinterpretazione intered intra - osservatore , non tanto in un classico quadro di consolidamento polmonare , quanto soprattutto in presenza di addensamenti polmonari in sedi mal esplorabili col radiogramma , ad esempio retrocardiaca o a livello delle basi [ 2 ]  . 
 ragionevole ipotizzare che nei bambini la polmonite possa estendersi alla pleura pi rapidamente ed in percentuali maggiori , in relazione al minor volume del parenchima polmonare , e quindi che con lecografia polmonare si possano ottenere risultati perlomeno sovrapponibili a quelli degli adulti . 
nei pazienti pediatrici , spesso affetti da flogosi delle vie respiratorie , a volte anche ripetutamente nel giro di pochi mesi , nei casi in cui non si sia un netto miglioramento allesame obiettivo nonostante la terapia ci si trova davanti alla necessit di eseguire un monitoraggio nel tempo con radiogramma del torace [ 1 ]  . 
scopo di questo lavoro valutare la sensibilit dellecografia della parete toracica , rispetto al radiogramma del torace nei pazienti pediatrici con sospetta polmonite , nellidentificazione degli addensamenti polmonari e del versamento pleurico . il polmone normale non raggiungibile , quindi non esplorabile con gli ultrasuoni , poich lelevata differenza dimpedenza acustica nel passaggio tra la pleura e laria contenuta allinterno degli alveoli polmonari determina una riflessione completa degli ultrasuoni . 
essi tuttavia possiedono ancora molta energia , per cui vengono riflessi pi volte tra la superficie impedente ed il trasduttore , generando artefatti costituiti da multipli echi trasversali , che vengono rappresentati sul monitor ad intervalli equidistanti tra loro e profondit via via crescenti ; essi sono definiti riverberi . quando invece gli alveoli in prossimit della pleura contengono materiale liquido , semiliquido o solido ( nel nostro caso essudato flogistico ) , gli ultrasuoni possono oltrepassare la pleura e raggiungere la zona di parenchima polmonare addensato [ 4 ]  . 
lecostruttura degli addensamenti spesso disomogenea in quanto possono coesistere zone con diversa radiol med ( 2009 ) 114 : 321330 ance , with hypoor anechoic fluid content and no doppler flow signals [ 7 ]  . 
anechoic tubular structures may also be seen that correspond to the vessels and are easily distinguished from a fluid bronchogram in that they show colour signals on colour doppler us [ 8 ]  . materials and methods between october 2006 and july 2007 , we performed a total of 32 chest us examinations and 32 chest radiographs ( including diagnostic and follow - up examinations ) on 28 consecutive children ( 17 boys , 11 girls ; mean age 4.5 years ; age range 4 months to 17 years ) admitted to the paediatric emergency ward for respiratory symptoms and a clinical suspicion of pulmonary disease . 
 before the us examination , the childrens parents or guardians were informed about the limitations inherent in the small series reported in the literature and thus the need for further investigation by chest radiography , even if the us examination provided an apparently diagnostic result . the parents and guardians were also informed that the data would be stored and used in accordance with the law in force , and their informed consent was obtained . patients were divided into three age groups : 024 months , 24 years and 4 years or older . 
patients from 0 to 24 months of age underwent ventrodorsal chest radiography in the supine position , 2to 4 - year - olds underwent only posteroanterior radiography in the standing position and patients aged 4 years or older underwent posteroanterior and laterolateral radiography in the standing position . 
pleural effusion was systematically looked for at the level of the anterior , lateral and posterior costophrenic angles . where pleural effusion was detected on us but not on chest radiography , the patient underwent serial us monitoring until complete reabsorption of the effusion . 
questo visibile come stria ecogena e riverberante , reperto simile ai rami biliari epatici in caso di aerobilia , e sarebbe presente in circa l80%90% dei casi di addensamenti polmonari secondo i dati presenti in letteratura . talvolta , quando i bronchioli vengono attraversati dagli ultrasuoni determinandone una sezione assiale , il broncogramma aereo pu apparire anche come immagini rotondeggianti iperecogene generate appunto dallaria intrappolata nelle strutture bronchiali [ 5 , 6 ] con lecografia si possono identificare anche i broncogrammi fluidi , cio strutture bronchiali a contenuto liquido ( generalmente muco e / o essudato flogistico ) che quindi assumono morfologia tubulare , a binario , con contenuto liquido ipo o anecogeno , prive di flusso al campionamento doppler [ 7 ]  . 
 materiali e metodi in un periodo di 9 mesi ( ottobre 2006luglio 2007 ) , considerando sia gli esami allesordio che i controlli seriati , sono state eseguite 32 ecografie della parete toracica ed altrettanti radiogrammi del torace in un totale di 28 pazienti consecutivi ( 17 maschi , 11 femmine ) afferiti al pronto soccorso pediatrico per sintomatologia respiratoria . tutti avevano un esame obiettivo sospetto per coinvolgimento polmonare , cio presenza di rantoli , ridotto murmure vescicolare , tachipnea , dispnea e / o tosse febbre > 37 , 5c . let media stata di 4 , 5 anni con un intervallo compreso tra 4 mesi e 17 anni . 
 i tutori legali dei pazienti , prima che questi ultimi fossero sottoposti allecografia , sono stati informati sui limiti derivanti dalle esigue casistiche a disposizione e pertanto della necessit di eseguire ulteriori approfondimenti ( in particolare il radiogramma del torace ) anche qualora lesame fosse apparentemente risultato diagnostico . 
ai tutori legali dei pazienti stato reso noto che i dati sarebbero stati conservati ed utilizzati in ottemperanza alle leggi ed alle norme vigenti ed stato ottenuto il loro consenso informato . 
ai pazienti nella fascia det 024 mesi stato eseguito il radiogramma del torace in proiezione ventro - dorsale in posizione supina , nella fascia 24 anni solo in proiezione postero - anteriore in stazione eretta ; infine nella fascia det superiore ai 4 anni sono strate eseguite le due proiezioni ortogonali postero - anteriore e latero - laterale in stazione eretta . 
si preso come standard di riferimento il solo radiogramma del torace . i pazienti sono stati sottoposti ad ecografia della parete 324 radiol med ( 2009 ) 114 : 321330 pendent radiologists who were blinded to each others findings . 
the data obtained were processed to calculate sensitivity , specificity and positive and negative predictive values of chest us in the identification of lung consolidation and pleural effusion . results the radiographic and us examinations were successfully performed in all patients . 
chest us depicted 22 cases of subpleural consolidation , none of which extending exclusively to the perihilar area ; pleural effusion was identified in 15 cases and air bronchogram in eight cases . 
the results are summarised in table 1 . among the examinations positive for consolidation , air bronchogram was seen on chest radiography in 29.1% ( 7 / 24 ) of cases and on us in 36.3% ( 8 / 22 ) of cases . 
pleural effusion was detected in 33.3% ( 8 / 24 ) of the cases that were positive for consolidation on chest radiography and in 68.1% ( 15 / 22 ) of those positive for consolidation on us . 
in our series , chest us compared with chest radiography had 91.67% sensitivity and 100% specificity for lung consolidation ; its negative predictive value was 84.6% and positive predictive value 100% . 
sono state eseguite scansioni longitudinali ed assiali ( intercostali ) lungo quelle che sono i tradizionali punti di repere : la linea emiclaveare , parasternale , ascellare in modo da coprire tutta la gabbia toracica . 
stato sistematicamente ricercato versamento pleurico a livello dei seni costofrenici anteriori , laterali e posteriori . nei casi di versamento pleurico con riscontro solo ecografico senza corrispettivo al radiogramma del torace si optato per un monitoraggio ecografico seriato nel tempo fino al completo riassorbimento dello stesso . 
con i dati ottenuti sono stati calcolati i valori di sensibilit , specificit , valore predittivo positivo e negativo dellecografia toracica nellidentificazione degli addensamenti polmonari e del versamento pleurico . risultati in tutti i pazienti stato possibile eseguire esami radiografici ed ecografici diagnostici . 
lecografia ha riscontrato in 22 casi addensamenti subpleurici , nessuno che si estendesse esclusivamente in sede perilare , in 15 casi stato segnalato il versamento pleurico ed in 8 casi il broncogramma . 
i dati sono riassunti anche nella tabella 1 . il broncogramma aereo , in rapporto ai soli esami positivi per addensamento , al radiogramma del torace stato riscontrato nel 29 , 1% ( 7 / 24 ) dei casi , mentre in ecografia nel 36 , 3% ( 8 / 22 ) dei casi . 
il versamento pleurico stato riscontrato nel 33 , 3% ( 8 / 24 ) dei casi nei quali i radiogrammi del torace avessero riscontrato un addensamento e nel 68 , 1% ( 15 / 22 ) delle ecografie positive per addensamento . 
nella nostra casistica lecografia della parete toracica , quando confrontata con il radiogramma del torace , ha riportato una sensibilit del 91 , 67% nellindividuare gli radiol med ( 2009 ) 114 : 321330 the statistical analysis because of the lack of a reference standard better than chest radiography . 
however , modalities employing ionising radiation must be used very sparingly in children for two reasons : their higher tissue sensitivity to ionising radiation ( as tissues are rich in proliferating cells ) , and their longer life expectancy , during which radiation - induced damage may emerge [ 9 ]  . 
once the clinical need for the examination has been ascertained , appropriate measures and technical parameters ( depending on the patients weight and / or age ) should always be implemented to achieve a significant reduction of exposure [ 11 ] while providing sufficient diagnostic quality ( following the principle of keeping radiation doses as low as reasonably achievable alara )  . 
the aim , it should be remembered , is not to produce a beautiful radiological image but to obtain an image allowing a diagnosis [ 9 , 12 ]  . 
chest radiography is thus only indicated in a restricted number of patients admitted to hospital for suspected pneumonia , that is , those who present severe symptoms and signs of pneumonia [ 1 ]  . 
in a small minority of patients with a suspicion of severe complications , with worsening signs and symptoms , discrepancy between imaging and clinical findings or with particularly severe and extensive lesions on radiography , computed tomography may also be used . although us has long been used to identify pleural effusion , only recently [ 13 , 14 ] has the evaluation of lung parenchyma become possible , partly as a result of technological advances and multifrequency transducers ( which have improved the techniques spatial resolution ) [ 15 ]  . 
 however , in the case of perihilar consolidation not extending to the subpleura and thus inaccessible to us , sonography is unable to visualise the diseased parenchyma addensamenti polmonari ed una specificit del 100% . 
in tutti i casi in cui era stato segnalato il versamento pleurico questo stato identificato anche con lecografia ; in pi sono stati identificati ulteriori 7 pazienti in cui il versamento era visibile solo ecograficamente ; in questi casi si monitorato il paziente fino al completo riassorbimento del versamento , eseguendo quindi in totale ulteriori 11 ecografie , i cui dati per , mancando uno standard di riferimento migliore del radiogramma del torace , non sono stati utilizzati al fine della valutazione statistica . 
per quanto concerne i versamenti pleurici , con i limiti sopra esposti , lecografia ha dimostrato dei valori di sensibilit e specificit nellordine del 100% . discussione il radiogramma del torace viene classicamente considerato la metodica di primo livello nella valutazione degli addensamenti polmonari . 
tuttavia in et pediatrica lutilizzo di metodiche che utilizzino radiazioni ionizzanti deve essere molto oculato , per due ragioni : la maggiore sensibilit dei tessuti agli effetti delle radiazioni ionizzanti ( perch pi ricchi di cellule in proliferazione ) e la maggiore aspettativa di vita durante la quale possono manifestarsi i danni radioindotti [ 9 ]  . 
la pi importante misura per ridurre la dose di radiazioni erogata alla popolazione per scopi medici si attua in primo luogo eseguendo lindagine ( sia essa un radiogramma , piuttosto che un esame di tomografia computerizzata ) solamente quando questa sia realmente giustificata . 
una volta assodata la correttezza dellindicazione allesame radiologico bisogna ricordare che tale tipo di indagini vanno sempre eseguite utilizzando adeguati accorgimenti e parametri tecnici ( variabili in funzione del peso e / o dellet del paziente ) che consentano di limitare al massimo lesposizione alle radiazioni ionizzanti [ 11 ] , pur ottenendo unindagine di sufficiente qualit diagnostica ( secondo il criterio del mantenere la dose il pi possibile bassa per quanto ragionevolmente fattibile alara , as low as reasonably achievable )  . 
pertanto lesecuzione del radiogramma del torace indicato solamente in una limitata parte dei pazienti che giungono allosservazione medica per sospetta polmonite , cio in caso di polmoniti con un quadro clinico severo [ 1 ]  . 
in casi ancora pi selezionati in cui si sospettino gravi complicanze , vi sia un importante peggioramento della sintomatologia , reperti 326 fig 1a axial image ( left ) showing pulmonary consolidation ( arrows ) with tiny hyperechoic spots ( air bronchogram , arrowhead )  . 
longitudinal image ( right ) with hypoechoic pneumonic lesion ( arrows ) above the left hemidiaphragm ( d , diaphragm , c , costal arch ) and small anechoic pleural effusion . 
1a a sinistra scansione assiale che dimostra laddensamento ( frecce ) ed alcuni minuti spot iperecogeni ( broncogramma aereo , testa di freccia )  . a destra scansione longitudinale ( d , diaframma , c , costa ) con addensamento ipoecogeno ( frecce ) in sede basale sinistra con sottile falda di versamento pleurico anecogeno . 
b allrx torace tenue e sfumato addensamento basale sinistro in sede paracardiaca , con velatura del seno costofrenico omolaterale . directly because of the interposition of the aerated lung . another reason for the failure of us to detect disease may be lesions located in areas difficult to reach with us , such as the posterior apical regions that are covered by the scapulae and supraclavicular fossa , and the axillary region . 
in such cases , however , us may depict indirect signs of radiol med ( 2009 ) 114 : 321330 strumentali discordanti con la clinica , o quadri particolarmente gravi ed estesi al radiogramma , pu essere utilizzata la tomografia computerizzata . da tempo nota la possibilit di valutare ecograficamente il versamento pleurico , ma solo recentemente [ 13 , 14 ] , grazie anche allevoluzione tecnologica degli ecografi e delle sonde multifrequenza ( che consentono di avere maggiore risoluzione spaziale ) si pensato di utilizzare questa metodica anche per la valutazione del parenchima polmonare [ 15 ]  . 
un ulteriore motivo di mancato riconoscimento delle lesioni pu essere rappresentato da sedi difficilmente esplorabili con lecografia quali le apicali posteriori , coperte dalle scapole e dalla fossa sovraclaveare , nonch la regione ascellare . lecografia in questi casi potrebbe tuttavia rilevare dei segni indiretti di coinvolgimento parenchimale , per esempio un versamento pleurico che , in base ai dati presenti in letteratura pu coesistere nei pazienti con polmonite , a seconda dellet e delle casistiche tra il 20% ed il 61% dei casi [ 16 ]  . nella nostra casistica il versamento stato riscontrato nel 33 , 3% dei casi nei quali i radiogrammi del torace riscontrassero un addensamento e nel 68 , 1% delle ecografie positive per addensamento . 
questi pazienti , come gi anticipato nei risultati , sono stati comunque sottoposti a controllo ecografico fino al completo riassorbimento del versamento , eseguendo quindi ulteriori 11 ecografie toraciche ottenendo dati concordanti con la letteratura , la quale riporta la migliore sensibilit dellecografia nella valutazione del versamento pleurico rispetto al radiogramma [ 17 ]  . 
 un aspetto peculiare dellecografia inoltre quello di poter anche caratterizzare , entro certi limiti , il tipo di versamento , quanto meno facendoci orientare verso un versamento non organizzato , con aspetto anecogeno o , viceversa , organizzato nel caso sia ad ecostruttura disomogenea , corpuscolata , con presenza di setti iperecogeni nel cui caso pu rendersi necessaria lesecuzione di toracentesi [ 18 , 19 ]  . in un caso il monitoraggio seriato nel tempo mediante ecografia , dimostrando la graduale riduzione e riassorbimento spontaneo del versamento pleurico ha permesso di evitare una toracentesi per il posizionamento del drenaggio pleurico . 
viceversa in un ulteriore caso , bench il contenuto del versamento non apparisse ecograficamente corpuscolato , al momento del drenaggio pleurico ( eseguito per la mancata risoluzione del quadro clinico ) stato evacuato materiale di aspetto lattescente , francamente purulento . 
in questo caso quindi lecografia avrebbe potuto fuorviare , portando ad un atteggiamento pi attendista ; naturalmente per il quadro radiologico , cos come quello laboratoristico , sono solamente degli aspetti di cui bisogna tenere conto alla luce della clinica nella gestione globale del paziente . i vantaggi del radiogramma rispetto allecografia della parete toracica , a fronte di unesposizione a radiazioni ionizzanti , sono quelli di permettere una maggiore panoramicit , facilit di esecuzione e refertazione , oltre a permettere una misurazione oggettiva del diametro della lesione . lecografia tra i suoi limiti ha limpossibilit ad esplorare alcune zone ( ad esempio la superficie pleurica al di sotto della scapola ) , lelevata operatore - dipendenza e tra laltro anche quando ripetuta sulla stessa apparecchiatura dallo stesso operatore non sempre pu essere facile valutare la riduzione dimensionale di un addensamento , specie se questo si estende molto in profondit rispetto alla superficie pleurica . 
bisogna inoltre ricordare che dovendo esplorare ecograficamente due emitoraci su tutta la loro estensione superficiale ( anteriore , posteriore , laterale ) , questo esame richiede sicuramente una quantit di tempo maggiore rispetto alla lettura di un radiogramma del torace , pertanto non facilmente applicabile nel contesto di un servizio di radiologia durgenza . daltro canto lecografia , soprattutto una volta che sia stata individuata la zona patologica , davvero molto rapida nelleffettuare una valutazione mirata relativa alla presenza o meno di un addensamento e / o di un versamento di precefig . 
on the left , an unevenly hypoechoic pulmonary consolidation ( arrows ) near the chondrocostal cartilage ( c ) , with tiny hyperechoic spots corresponding to an air bronchogram ( arrowhead )  . 
a sinistra addensamento disomogeneamente ipoecogeno ( frecce ) in sede paracardiaca subito posteriormente alle cartilagini condrocostali ( c ) , con evidenza di alcuni spot iperecogeni , tipo broncogramma aereo ( testa di freccia )  . 
nellimmagine a destra posteriormente alle cartilagini condrocostali ( c ) presente un addensamento parenchimale ( freccia ) ; nel suo contesto immagine lineare iperecogena ( testa di freccia ) tipo broncogramma aereo . 
b al radiogramma si apprezza uno sfumato addensamento in sede parailare inferiore - paracardiaca destra in corrispondenza del lobo medio . parenchymal involvement , such as pleural effusion that has been reported to coexist with pneumonia in 20%61% of cases , depending on age and patient series [ 16 ]  . 
the greater number of cases of pleural effusion detected with us may be explained by the fact that , especially in children , us is able to clearly depict even small effusions at the costophrenic angles , thanks to the 328 radiol med ( 2009 ) 114 : 321330 high spatial resolution afforded by high - frequency probes . additionally , it is always able to depict the region of interest directly without the interposition of other structures . in the case of very compact consolidations , colour doppler us also plays an important role , as it allows one to visualise vessels within the lung parenchyma . 
we thus performed 11 chest us scans achieving results comparable with those in the literature , which reports that us is more sensitive than chest radiography in the evaluation of pleural effusion [ 17 ]  . another peculiarity of us is its ability to give some indication of the type of effusion by indicating whether it is an unorganised anechoic effusion or , conversely , an organised effusion with an inhomogeneous corpuscular echostructure and hyperechoic septations , which might require a thoracentesis [ 18 , 19 ]  . 
in one case , serial us monitoring demonstrated the gradual reduction and spontaneous reabsorption of the pleural effusion , thus allowing thoracentesis to be avoided by placing a pleural drainage catheter . 
in another case , instead , despite the noncorpuscular appearance of the effusion on us , the pleural drainage catheter ( performed for persisting clinical signs and symptoms ) yielded milky , clearly purulent material . 
in this case , us could have misled us into adopting a more expectant approach , even though patient management is clearly guided not only by the radiological findings but also by the laboratory data and overall clinical condition . the advantages of radiography over chest us , despite the exposure to ionising radiation , include a more panoramic view , easier performance and reporting and objective determination of lesion diameter . 
in addition , because the entire surface of the two hemithoraces ( anterior , posterior , lateral ) need to be explored , us takes longer compared with chest radiography and is therefore poorly suited to emergency radiology settings . on the other hand , once the diseased area has been identified , us provides a very rapid and targeted evaluation of the presence of a previously detected consolidation and / or effusion , allowing serial monitoring over time , even in response to clinical changes indicating improvement or fig 3a on the left , axial intercostal image of a inhomogeneous consolidation with prevalently hypoechoic texture ( arrows ) and air bronchogram ( arrowheads )  . 
b al radiogramma si conferma una sfumata opacit a carico del campo polmonare superiore - medio di destra che si estende fino alla pleura . dente riscontro , potendo fare pi monitoraggi seriati nel tempo , anche in relazione a momenti in cui ci siano delle variazioni della sintomatologia clinica e quindi si sospetti un miglioramento o , viceversa , un peggioramento del quadro . lecografia permette inoltre una precisa e rapida valutazione anche dellevoluzione del versamento pleurico [ 20 ]  . nel caso di pazienti molto piccoli talvolta pu esser pi semplice eseguire lecografia , in cui si possono assecondare piccoli movimenti del paziente , esplorabile anche mentre dorme . 
in very young patients , it may at times be easier to perform us , as small movements can be followed and the patient can be studied even while asleep . 
paradoxically , a crying infant does not represent a major problem as occurs with ct or chest radiography , as crying enables the optimal evaluation of diaphragmatic motion with good visibility of the costophrenic angles during deep breathing . there are some limitations to our study . 
first , it included a small number of patients , and it is likely that we would have obtained lower sensitivity values if we had analysed a larger patient series with more cases of perihilar consolidation not extending to the pleura . 
likewise , had we used a more sensitive reference standard than chest radiography , our results with chest us would no doubt have been less encouraging . conclusions in consideration of the good sensitivity and positive and negative predictive values in identifying both parenchymal consolidation and pleural effusion , chest us proved to be a promising technique for the detection and follow - up of lung infections and associated pleural effusion . 
patients could be spared further radiological examinations that expose them to ionising radiation and which do not provide additional relevant clinical information . chest us also provides information about the need for further procedures , such as thoracentesis . 
us may also be used to guide thoracentesis to reduce the risks associated with the procedure , thereby decreasing morbidity and recovery time [ 19 ]  . the ease , speed and availability of us could also allow a higher frequency of follow - up examinations during treatment and an earlier detection of disease exacerbations , for example due to drug resistance . 
this would be beneficial in terms of both a more rational use of economic resources and a more appropriate management of the patient . del torace , in quanto ci permette di avere anche unottima valutazione dellescursione diaframmatica con una buona visibilit dei seni costofrenici durante i profondi respiri . il nostro studio presenta alcuni limiti . 
in primo luogo la bassa numerosit , per cui analizzando casistiche pi ampie , in cui siano compresi pi pazienti con addensamenti periilari senza estensione alla pleura , sarebbe plausibile ottenere valori di sensibilit peggiore . 
cova1 1unit clinica operativa di radiologia , ospedale di cattinara , universit degli studi , strada di fiume 447 , 34149 trieste , italy 2dipartimento di economia aziendale , universit degli studi , trieste , italy 3struttura complessa di cardiologia , ospedale di cattinara , trieste , italy correspondence to : f . 
sono stati calcolati i costi differenziali ( costo apparecchiature , costi variabili , costo del personale medico , tecnico e infermieristico ) , i costi comuni e i costi esterni . 
la coronaro - tc ha fatto registrare : costo differenziale 222 , 23 , costo comune 5 , 50 , costo esterno 2 , 30 , costo totale 230 , 03 . 
per la coronarografia si verificato : costo differenziale 366 , 18 , costo comune 0 , 50 , costo esterno 9 , 20 , costo degenza 1652 , costo totale 2027 , 88 . 
la coronaro - tc ha costi notevolmente inferiori alla coronarografia e un rapporto costo - efficacia migliore nella larga maggioranza dei pazienti . 240 radiol med ( 2009 ) 114 : 239252 keywords cost analysis multidetector computed tomography ct coronary angiography coronary angiography cost - effectiveness parole chiave analisi dei costi tomografia computerizzata multistrato coronaro - tc coronarografia costo efficacia coronaro - tc introduction introduzione coronary angiography with computed tomography ( ctca ) is rapidly establishing itself as an accurate technique for studying coronary arteries . 
in segmental analysis , the literature reports 72%95% sensitivity and 86%98% specificity for 16 - slice ct [ 1 , 2 ] and 95%99% sensitivity and 95%97% specificity , with a negative predictive value ( npv ) of 99%100% for 64 - slice ct [ 35 ]  . 
 the main strength of the technique lies in its high npv , which makes it a valuable diagnostic alternative for the noninvasive evaluation of the coronary circulation , allowing screening of patients with equivocal symptoms for significant coronary artery disease ( cad )  . 
the costs of ctca in particular have received sparse attention in the literature . the objective of our study was to analyse the costs of ctca performed at a department of radiology and compare them with those of conventional coronary angiography performed at the department of cardiology of the same hospital . 
a further aim was to compare the cost - effectiveness of the two modalities . materials and methods examination technique ctca la coronaro - tc si sta rapidamente imponendo come indagine accurata nella valutazione delle arterie coronarie . 
in letteratura , nellanalisi per segmento , vengono riportati valori di sensibilit tra il 72% e il 95% e di specificit del 86%98% mediante tc a 16 strati [ 1 , 2 ] , mentre gli studi relativi alla tecnologia a 64 strati riportano sensibilit pari al 95%99% e specificit pari al 95%97% , con un valore predittivo negativo del 99%100% [ 35 ]  . 
 il suo elevato valore predittivo negativo ne fa il reale punto di forza , proponendola come valido ed alternativo strumento diagnostico per la valutazione del circolo coronarico in modo non invasivo , al fine di gestire una porzione della popolazione con un corteo sintomatologico dubbio , in modo da evitare unindagine invasiva , e allo stesso tempo escludendo la presenza di una patologia ateromasica significativa . 
ulteriore obiettivo quello di confrontare ulteriormente le due metodiche con unanalisi di costo - efficacia . materiali e metodi tecnica desame coronaro - tc the ct scan was performed with an aquilion 64 multislice scanner ( toshiba medical systems , tokyo , japan ) with the following scanning parameters : slice thickness 640.5 mm , variable pitch and rotation time determined by the cardiac software on the basis of heart rate and adjusted to obtain maximum temporal resolution , 120 kv and variable ma . 
in particular , tube current was tailored to each patients weight and ranged from 300 ma ( for patients < 60 kg ) to 450 ma ( for those > 100 kg )  . ct scans were performed during the infusion of iomeprol , ( iomeron , a high - iodine - concentration contrast agent bracco , milan , 400 mgi / ml )  . 
a dose of 80 ml of contrast medium was la scansione tc stata eseguita con apparecchiatura multistrato aquilion 64 ( toshiba medical systems , tokyo ) con i seguenti parametri di scansione : spessore di strato 640 , 5 mm , pitch e tempo di rotazione variabili stabiliti dal software cardiologico dedicato in base alla frequenza cardiaca e regolati per ottenere la massima risoluzione temporale possibile , 120 kv e ma variabili . 
in particolare , la corrente del tubo radiogeno stata personalizzata in radiol med ( 2009 ) 114 : 239252 injected at a flow rate of 4 ml / s followed by a 40 - ml bolus of saline at the same flow rate . 
the contrast medium was injected through an 18 - gauge needle cannula placed in an antecubital vein of the right ara stellant dual - head automatic power injector ( medrad , indianola , ia , usa ) was used . 
the scan was synchronised with the contrast bolus by using the systems sure start triggering protocol , which involves placing a region of interest ( roi ) in the descending aorta and setting the enhancement threshold at 100 hu above baseline . the electrocardiographic trace was recorded throughout the scan for the retrospective reconstruction of isocardiophasic images . 
images were reconstructed by retrospective gating and unisegmental ( where possible ) or multisegmental technique , with initial placement of the temporal window in the end - diastolic phase at 75% of the r - r interval . 
where the end - diastolic phase proved inadequate , the dedicated software imagexact was used to identify the correct phase . axial images were processed on a dedicated workstation for 3d processing ( vitrea version 3.8.1 , vital images inc . minnetonka , mn , usa )  . 
reconstructions included 2d multiplanar reformations ( mpr ) , curved mpr ( cmpr ) along the course of the coronaries , maximum intensity projection ( mip ) and 3d volume rendering . 
whenever possible , we used vessel probe software to study the course of the coronaries and analyse any stenotic lesions in two orthogonal planes . conventional coronary angiography conventional coronary angiography was performed at the department of cardiology by selective catheterisation of the right and left coronary district . 
images were acquired with a dedicated c - arm innova 2100 ( general electric medical systems , chicago , il , usa ) and an additional ge innova 2000 c - arm equipped with high - definition flatpanel systems ( 1 , 0241 , 024 pixels12 bits )  . cost analysis we calculated the differential cost ( defined as the sum of the variable costs , equipment costs and personnel costs ) of the two procedures . 
we then calculated the common cost , which includes cost components that are the same for any type of procedure carried out at the departments of relazione al peso del paziente con un valore compreso fra 300 ma ( per pazienti di peso inferiore ai 60 kg ) e 450 ma ( per pazienti di peso superiore a 100 kg )  . lesame stato eseguito durante la somministrazione di iomeprolo , mezzo di contrasto ( mdc ) organoiodato ad alta concentrazione ( iomeron 400 mgi / ml )  . 
in pazienti portatori di bypass confezionati con arterie mammarie interne stato iniettato un volume di mdc di 100 ml a causa della maggior ampiezza del volume di acquisizione e quindi del maggior tempo di scansione . 
la scansione stata sincronizzata al bolo di contrasto mediante protocollo di triggering sure start fornito dalla apparecchiatura che prevede il posizionamento di una roi in aorta discendente ed un valore soglia regolato in 100 uh sopra il valore basale . durante la scansione stato acquisito il tracciato elettrocardiografico per la ricostruzione retrospettiva delle immagini isocardiofasiche . 
le immagini isocardiofasiche sono state ricostruite mediante gating retrospettivo e tecnica mono - segmentaria ( quando possibile ) o multisegmentaria con posizionamento iniziale della finestra temporale nella fase telediastolica al 75% dellintervallo rr . 
quando la qualit delle immagini lo ha reso possibile stato utilizzato il pacchetto software dedicato allo studio vascolare vessel probe per lo sviluppo del percorso coronarico e per lanalisi delle eventuali lesioni stenosanti effettuando lo studio del vaso su due piani ortogonali . coronarografia langiografia tradizionale stata eseguita presso la s.c. 
di cardiologia del nostro ospedale mediante cateterismo arterioso selettivo del distretto coronarico destro e sinistro . durante liniezione di iodixanolo ( visipaque 320 mgi / ml ) sono state ottenute serie di immagini ad elevato frame rate ( 15 immagini / s )  . 
solitamente vengono iniettati complessivamente 120140 ml di mdc per uno studio diagnostico . durante liniezione a bolo di mdc si acquisiscono le 242 radiol med ( 2009 ) 114 : 239252 radiology and cardiology . 
we then evaluated the variable costs , related to the type and amount of supplies used and the services connected to their use . physician , technologist and nursing personnel costs were also determined . 
examination duration was estimated on the basis of the time allocated by the hospitals computerised systein evaluating the differential costs , we only considered the technical act and consequently the costs incurred by the departments of radiology and cardiology , disregarding any relationship between these two departments and our hospitals other cost centres and the impact of the procedures on the patient - care process . every patient undergoing ctca and conventional coronary angiography requires a blood test to evaluate serum creatinine . 
patients undergoing coronary angiography are also screened for coagulation disorders [ platelets , international normalised ratio ( inr ) , thromboplastin time ]  . patients with prior allergic reactions to various substances approximately 1% require pharmacological preparation ( corticosteroids and anti - h1 and anti - h2 agents )  . 
although the costs for these procedures are not incurred by the department of radiology , they are considered , as they are essential for the performance of ctca and conventional coronary angiography and have a certain economic impact . 
 [ 9 , 10 ] to compare ctca and conventional coronary angiography . ctca cost = ne ( fe + rec ) + na ( fa + rac ) + nf ( rfc ) effectiveness = nex ( 1 - ndxe ) xpxsne + nexpxndxe . conventional coronary angiography cost = naxfa effectiveness = naxp test ; where ne = number of patients having na = number of patients having coronary angiography because of positive or nondiagnostic ctca = nex ( 1 - ndxe ) x [ pxsne + ( 1 - p ) x ( 1 - spe ) ] + nexndxe ; nf = number initial immagini in diverse proiezioni , usualmente quattro per la coronaria sinistra e due per la coronaria destra . 
sono stati poi valutati i costi variabili , relativi a tipologia e quantit dei materiali impiegati e ai servizi legati a tale impiego . successivamente sono stati valutati anche i costi del medico , del tecnico e del personale infermieristico . 
di cardiologia , ignorando i rapporti tra queste strutture e gli altri centri di costo dellazienda ospedaliero - universitaria e il ruolo che tali indagini hanno avuto sul processo di cura . va ricordato che ogni paziente che deve eseguire un esame di coronaro - tc e di coronarografia viene sottoposto a prelievo ematico per valutare i livelli sierici della creatinina . 
nell1% dei casi poi , essendoci unanamnesi positiva per allergia a diverse sostanze , si esegue anche una preparazione farmacologica ( basata sullimpiego di cortisonici associati ad anti - h1 ed anti - h2 )  . i costi legati a tali procedure non sono di pertinenza dellu.c.o. 
di radiologia ; tuttavia queste sono fondamentali ai fini dellesecuzione dellesame di coronaro - tc e della coronarografia ed avendo comunque un certo rilievo economico , verranno brevemente presi in considerazione . 
si ricorda ancora che i costi di materiali ed apparecchiature sono comprensivi di iva al 20% . stata inoltre effettuata unanalisi di costo - efficacia . radiol med ( 2009 ) 114 : 239252 of patients with false negative results who do not have coronary angiography = nex ( 1 - ndxe ) xpx ( 1 - sne ) ; fe = cost of ctca ; fa = cost of conventional coronary angiography ; re = rate of complications of ctca ; ra = rate of complications of coronary angiography ; rf = rate of complications over a 10year follow - up period in patients with cad and false negative tests ; c = mean cost of complications ; ndxe = number of nondiagnostic examinations at ctca ; sne = ctca sensitivity ; spe = ctca specificity ; p = pre - test likelihood the mathematical model adopted in our study evaluates a hypothetical cohort of patients with different pretest likelihoods who are studied with the two diagnostic tests . 
in particular , calculation of ctca costs included the rate and cost of complications as well as the equipment costs ; to this we added the cost of patients requiring coronary angiography ( because ctca is either nondiagnostic or positive for lesions eligible for percutaneous treatment ) and the cost of false negative patients who do not undergo coronary angiography . 
 [ 13 ] , who estimated an average overall cost for myocardial infarction of 11 , 742 ( hospitalisation , rehabilitation , nonproductive time ) , assuming this to be the most serious complication and considering the same cost for both procedures . 
by applying a complex mathematical model , this set of data can be used to compare the cost and cost - effectiveness of the two modalities based on the different pretest likelihoods . results ctca differential costs 1 . 
the lanalisi di costo - efficacia una forma di piena valutazione economica dove sia i costi che le conseguenze di un programma diagnostico o terapeutico vengono contemporaneamente esaminati [ 8 ]  . 
una riduzione dei costi con diagnosi corretta indica miglioramento del rapporto costo - efficacia . per la valutazione del rapporto costo - efficacia ci siamo basati sul modello decisionale sviluppato da patterson et al . 
 [ 9 , 10 ] per confrontare la coronaro - tc e la coronarografia . coronaro - tc costo = ne ( fe + rec ) + na ( fa + rac ) + nf ( rfc ) efficacia = ne ( 1nde ) psne + nepnde . coronarografia costo = nafa efficacia = nap ( ne = numero di pazienti con test iniziale ; na = numero di pazienti che eseguono la coronarografia perch la coronaro - tc positiva o non diagnostica = ne ( 1nde ) [ psne + ( 1 - p ) ( 1 - spe ) ] + nende ; nf = numero di pazienti falsi negativi che non eseguono coronarografia = ne ( 1nde ) p ( 1sne ) ; fe = costo coronaro - tc ; fa = costo coronarografia ; re = percentuale complicanze coronaro - tc ; ra = percentuale complicanze coronarografia ; rf = percentuale complicanze in un periodo di 10 anni di follow - up nei pazienti con cad e test falsi negativi ; c = costo medio complicanze ; nde = numero di esami non diagnostici alla coronaro - tc ; sne = sensibilit coronaro - tc ; spe = specificit coronaro - tc ; p = probabilit pre - test ) nel modello matematico che abbiamo adottato viene valutata una coorte ipotetica di pazienti con diverse probabilit pre - test che vengono sottoposti alle due indagini diagnostiche . 
in particolare , nel computo del costo della coronaro - tc si considera oltre al costo dellapparecchiatura , la percentuale delle complicanze e il loro costo ; a ci va sommato il costo dei pazienti che devono eseguire la coronarografia ( perch la coronaro - tc risulta positiva per lesioni da sottoporre a trattamento percutaneo o non diagnostica ) e il costo dei falsi negativi che non eseguono la coronarografia . 
nel 2006 , che dimostrano valori di sensibilit del 98 , 4% , di specificit del 90 , 7% ed una quota di esami non diagnostici del 3 , 6% . 
la coronarografia , che rappre244 radiol med ( 2009 ) 114 : 239252 maintenance and service costs were 91 , 800 annually . to this we added the cost of an electrocardiographic monitor ( hme lifepulse 10 tg ) compatible with the toshiba mdct scanner , which has a purchase price of 3 , 500 ( equal to 437 annually )  . 
the picture archiving and communication system ( pacs ) had a purchase price of 2 , 500 , 000 , corresponding to 312 , 500 yearly for 8 years of amortisation . 
additional materials include an 18 - gauge abbocath needle cannula ( 0.44 ) , a small amount of disinfectant , cotton wool and an adhesive plaster ( the cost of these is negligible )  . 
ctca protocols recommend the oral administration of 100 mg of - blocker ( metoprolol ) at least 1 h before the examination in subjects with a heart rate > 65 beats per minute . 
la quota di complicanze , ricavata dalla letteratura , risulta del 0 , 004% per la coronaro - tc e del 1 , 5% per la coronarografia [ 11 , 12 ]  . 
 [ 13 ] , che stima in circa 11742 il costo medio di un infarto miocardico ( ospedalizzazione , riabilitazione , non produttivit ) , valutando questa come la complicanza pi grave e considerando di utilizzare tale valore per entrambe le metodiche . 
dallinsieme di questi dati , attraverso un complesso modello matematico , si ottiene prima una comparazione dei costi e successivamente del rapporto costo - efficacia delle due apparecchiature in base alla diverse probabilit pre - test . risultati costi differenziali coronaro - tc 1 . 
a ci va ancora aggiunto il costo dellelettrocardiografo ecg monitor , hme lifepulse 10 tg compatibile con la mdtc toshiba che ha un costo dacquisto di 3500 ( pari a 437 annui )  . 
va ancora considerato il sistema pacs : il suo costo dacquisto stato pari a 2500000 , a cui corrispondono 312500 di costo annuo per 8 anni radiol med ( 2009 ) 114 : 239252 3 . 
we calculated the personnel costs ( radiologist , technologist and nurse ) by considering their yearly hours and subtracting time off for ordinary paid leave , extraordinary paid leave and sick leave ( 30% on average )  . 
radiologist costs were 120 / examination based on an annual cost of 101 , 040 , a daily cost of 577 , an hourly cost of 80 and an estimated commitment of 90 min ( preliminary evaluation of the patient , attendance during acquisition , image postprocessing , reporting )  . 
we considered the price of the dedicated haemodynamic unit ge innova 2000 ( general electric medical systems )  . the cost to be amortised ( purchase price ) is 1 , 004 , 611 , leading to a yearly cost of 125 , 576 for 8 years of amortisation . 
materials used during the procedure : sterile gauze ( 0.01 ) , four 10 - cc luerlock syringes ( 0.68 ) , four 20 - cc syringes ( 0.72 ) , arterial puncture needle ( 1.25 ) , two vials of lidocaine ( 0.11 ) , one 0.38 175 - cm , j - tip cordis emerald guidewire ( 6.78 ) , one 6 - fr st . 
lesecuzione di una coronaro - tc prevede lutilizzo di 80100 ml di mezzo di contrasto iomeprolo 400 ( iomeron , bracco , milano ) in flaconi da 250 ml ( 116 , 66 )  . 
viene inoltre utilizzato un ago cannula abbocath da 18 g ( 0 , 44 ) con una piccola quantit di disinfettante , un batuffolo di cotone ed un cerotto ( il costo di questo materiale trascurabile )  . 
nei protocolli di coronaro - tc prevista la somministrazione di 100 mg di - bloccante ( metoprololo ) per os almeno unora prima dellindagine nei soggetti che presentano una frequenza cardiaca superiore ai 65 battiti al minuto . 
si utilizza inoltre un cd rom ( costo unitario di 0 , 33 ) che viene fornito ai pazienti ambulatoriali , non provenienti dal reparto di cardiologia , che rappresentano il 28% delle coronaro - tc eseguite presso la nostra u.c.o. 
poich il costo dacquisto del tubo radiogeno di 91800 e la sua durata prevista pari a 300000 colpi , con un costo per ogni colpo di 0 , 30 emerge un costo per indagine di 6 , 12 . 
abbiamo calcolato il costo del personale ( medico , tecnico e infermieristico ) considerando il suo debito orario annuale , sottraendo da esso le assenze per congedo ordinario , straordinario e malattia ( in media 30% ) e stimando quindi in 35 settimane / anno il tempo lavorativo effettivo . 
ne consegue un costo medico di 120 / esame in base ad un costo annuo di 101040 , giornaliero di 577 e orario di 80 avendo stimato il suo impegno temporale per tale metodica in 90 minuti ( valutazione preliminare del paziente , presenza in fase di acquisizione dellesame , elaborazione delle immagini , stesura del referto )  . 
il costo del tecnico radiologo ( tsrm ) risulta di 15 , 5 in base ad un costo annuo di 39679 , giornaliero di 226 ed orario di 31 , stimando un suo impiego per 30 minuti . 
we calculated , using the same method as used for ctca , a cardiologist cost of 86.60 on the basis of a yearly cost of 101 , 040 , a daily cost of 577 and hourly cost of 80 and estimating a time commitment of approximately 65 min per examination . 
technologist cost was 31 based on a yearly cost of 39 , 679 , a daily cost of 226 and hourly cost of 31 , with a commitment of 60 min per examination . 
based on the data provided by our hospitals planning and control office , the cost of 1 day of hospitalisation at the department of cardiology is 1 , 652  . 
all patients undergoing conventional coronary angiography are hospitalised for at least 1 day , whether the puncture site is closed with a sealing device ( angioseal ) or by application of manual compression . 
the patient is discharged on the following day . common costs common costs are those related to support staff and support materials ( computers , printers , recording equipment , electrical and network materials , stationery , cleaning supplies )  . in calculating the cost of support staff , we considered all those professionals not discussed so far : one chief consultant radiologist , two chief technologists , three technologists , four auxiliary staff , four file clerks , six assistants and these professionals administrative assistants . 
materiale utilizzato dopo la procedura : dispositivo angio - seal ( 138 ) ; questo dispositivo stato utilizzato in 129 pazienti su 821 eseguiti nel periodo preso in considerazione per tale valutazione ovvero nel 15 , 71% dei casi . 
quindi , il materiale preparatorio ha un costo totale pari a 10 , 41 , il materiale utilizzato radiol med ( 2009 ) 114 : 239252 common cost of 5 per examination . 
the lack of support staff at the department of cardiology results in different common costs for the two procedures . external costs blood tests performed before the two procedures involve an external cost of 1.90 per examination for ctca and 8.80 for digital coronary angiography . 
this depends on the fact that a correct diagnosis is considered a positive factor in any cost - effectiveness analysis , and correct diagnoses will be all the more frequent the higher the pretest likelihood . 
in particular , the data demonstrate that ctca is more costeffective in patients with a pretest likelihood up to 86% , with a cost per correct diagnosis of cad ranging from 7 , 295.10 ( 10% pretest likelihood ) to 2 , 593.9 ( 86% pretest likelihood )  . 
by contrast , coronary angiography shows better cost - effectiveness for pretest likelihoods of 87%100% , with a cost per correct diagnosis of cad between 2 , 563.59 ( 87% pretest likelihood ) and 2 , 207.60 ( 100% pretest likelihood )  . 
new durante la procedura ha un costo totale pari a 112 , 9 e il materiale utilizzato dopo la procedura ha un costo di 21 , 68 , per un costo complessivo di 144 , 99 . 
abbiamo calcolato , con analoga modalit rispetto alla coronaro - tc , un costo medico di 86 , 6 in base ad un costo annuo di 101040 , giornaliero di 577 e orario di 80 stimando un impegno per tale metodica di circa 65 minuti . 
il costo del personale tecnico di 31 in base ad un costo annuo di 39679 , giornaliero di 226 e orario di 31 con un impegno di 60 minuti / esame . 
il costo del personale infermieristico di 29 in base ad un costo annuo di 39174 , giornaliero di 223 e orario di 29 con un impegno di 60 minuti / esame . 
viene effettuata nei pazienti sottoposti a coronarografia almeno una giornata di degenza , sia che venga posizionato il dispositivo di chiusura del foro dentrata ( angio - seal ) , sia che si applichi una compressione manuale . 
il paziente viene dimesso nella giornata successiva . costi comuni i costi comuni sono rappresentati dai costi relativi al personale di supporto e da quelli dei materiali di supporto ( computer , stampanti , registratori , materiale elettrico e di rete , materiale di cancelleria , materiale di pulizia )  . 
si tratta di 1 primario , 2 capotecnici , 3 tecnici , 4 ausiliari , 4 archivisti , 6 tra coadiutori e assistenti amministrativi . complessivamente questinsieme di unit lavora per il funzionamento di tutta lu.c.o. ; non riteniamo che il loro lavoro sia influenzato dalla tipologia di esami e quindi il loro costo complessivo stato ripartito proporzionalmente sullinsieme di tutte le indagini eseguite presso lu.c.o. 
nel 2006 il costo totale di tale pool di operatori risultato di 557646 ; il numero complessivo di esami stato di 111367 ; risulta un costo comune aggiuntivo per esame di 5 , 00 . 
the impact of new technology on health care expenditure depends exclusively on its appropriate use , even relative to other available diagnostic and therapeutic tools , to optimise resource utilisation . 
this is especially true in the field of diagnostic radiology , as technology has a major impact on overall hospital costs , and the clinical impact of the radiological process heavily affects how the patient is managed following diagnosis , with effects on treatment decisions and consequently on the overall cost of the disease . the main point to recall when dealing with an economic analysis is that the costs refer to the investment made when a resource is consumed during a therapeutic or diagnostic programme . 
one of the main aspects to be faced when costi esterni per il dosaggio dei diversi parametri ematochimici utili preliminarmente alleffettuazione delle due metodiche vi un costo esterno per esame che di 1 , 90 per la coronaro - tc e di 8 , 8 per la coronarografia digitale . 
inoltre nell1% dei pazienti si esegue una profilassi farmacologica ( basata sullimpiego di cortisonici associati ad anti - h1 ed anti - h2 ) , per un costo di 4 , in media 0 , 04 in pi per ogni esame . 
1 valutazione grafica dellanalisi di costo - efficacia delle due metodiche in relazione alle diverse probabilit pre - test di malattia coronarica . coronaro - tc : 222 , 23 + 5 , 50 + 2 , 3 = 230 , 03 coronarografia : 366 , 18 + 0 , 50 + 9 , 2 + 1652 = 2027 , 88 . le tabelle 2 e la fig . 
ci dipende dal fatto che una diagnosi corretta viene considerata un fattore positivo in unanalisi di costo - efficacia e una diagnosi corretta sar tanto pi frequente quanto pi elevata la probabilit pre - test . 
in particolare si dimostra come la coronaro - tc sia la metodica con il migliore rapporto di costo - efficacia nei pazienti con probabilit pre - test fino all86% con un costo per identificare correttamente la malattia coronaria che varia tra 7295 , 10 ( 10% probabilit pre - test ) e 2593 , 9 ( 86% probabilit pre - test )  . 
mentre dall87% al 100% di probabilit pre - test la metodica con il migliore rapporto costo - efficacia la coronarografia che presenta un costo per identificare correttamente la malattia coronarica che varia tra 2563 , 59 ( 87% probabilit pre - test ) e 2207 , 60 ( 100% probabilit pre - test )  . 
le nuove tecnologie , soprattutto al momento della loro introduzione nella pratica clinica , presentano costi elevati , che risultano giustificabili solo attraverso la dimostrazione di un reale impatto diagnostico - terapeutico . 
gli effetti sulla spesa sanitaria di un innovazione tecnologica dipendono esclusivamente da un suo appropriato utilizzo , anche in relazione ai mezzi diagnostici e terapeutici gi disponibili , in modo da ottimizzare luso delle risorse . 
in ambito radiologico queste considerazioni sono ancor pi valide in quanto le tecnologie utilizzate hanno un grosso peso sul totale dei costi aziendali , ed inoltre limpatto clinico fornito dal radiologo influenza notevolmente la storia del paziente successiva alla diagnosi , condizionando le scelte terapeutiche e di conseguenza i costi complessivi della patologia . il punto principale da ricordare quando si affronta unanalisi economica che i costi si riferiscono allinvestimento fatto quando una risorsa viene consumata in un programma di trattamento o di diagnosi . 
the only significant cost factor that is higher for ctca is medical personnel cost owing to the long postprocessing time , a factor that is likely to decrease with future software improvements . 
if , on the other hand , we correctly consider hospitalisation costs , the cost of coronary angiography exceeds that of ctca by a factor of 9 . this form of cost accounting employs a method that is frequently used in industrial organisations and that ensures a rigour that reduces the approximations of traditional costcalculation methods [ 14 ]  . 
our analysis illustrates the situation at the departments of radiology and cardiology at a given point in time , and as such cannot be extrapolated directly to other centres , where costs may differ considerably because of different equipment or materials used or different organisational strategies that affect , for example , personnel utilisation and cost . 
this analysis may thus serve as a methodological model to be applied to other settings . given that coronary angiography is performed on inpatients , any cost analysis should be compared against the reimbursement rates based on the diagnosis - related groups ( drg )  . 
in the friuli venezia giulia region , coronary angiography is listed under drg 124 ( cardiovascular disease except acute myocardial infarction , with cardiac catheterisation ) , which has a reimbursement rate of 4 , 876.00 , regardless of the actual number of days in hospital . ctca , instead , is normally performed on outpatients , so it is not allocated a reimbursement based on the drg . 
if the examination was to be performed on inpatients , drg 140 ( angina pectoris ) would probably apply , which has a reimbursement rate of 332.00 for up to 3 days in hospital and of 2 , 929.00 for hospitalisations exceeding 3 days . when considering cost - effectiveness , the least costly technique should ideally also be the most sensitive . although the sensitivity of ctca has been substantially improved by the advent of the new 64 - slice ct scanners , it nonetheless remains lower than that of coronary angiography ( the gold standard )  . 
in evaluating a patient at high risk of cad , the decision to perform ctca as a first - line study is not justified in either clinically or economically , as it is likely that the patient will subsequently have to undergo coronary angiography , at least for therapeutic purposes . 
this finding , affrontare per intraprendere questo tipo di analisi sono in primis il periodo di tempo nel quale valutare il costo poich una procedura bench pi conveniente pu richiedere nel tempo unulteriore procedura , per un risultato falsamente negativo o per linsorgenza di una complicanza , vanificando cos il vantaggio economico [ 8 ]  . lanalisi dei costi da noi effettuata , qualora non si consideri il costo della degenza , ha fatto rilevare un costo superiore del 38 , 8% della coronarografia rispetto alla coronaro - tc . 
lunico fattore di costo significativo in cui la coronaro - tc ha un valore superiore rappresentato dal costo del personale medico , dovuto al tempo di elaborazione tuttora elevato che questi esami richiedono , fattore che del tutto verosimilmente si ridimensioner negli anni a venire grazie ai miglioramenti dei software dedicati . qualora si prenda correttamente in considerazione anche il costo della degenza , il costo della coronarografia sopravanza quello della coronaro - tc di un fattore nove . questa analisi dei costi utilizza una tecnica cui si fa ricorso frequentemente in ambito industriale e che garantisce un rigore che riduce le approssimazioni dei metodi tradizionali di calcolo dei costi [ 14 ]  . 
di cardiologia , in un determinato momento storico , e i dati non possono essere esportati acriticamente , in quanto in altre strutture i costi possono essere estremamente diversi , vuoi per diverse apparecchiature e diversi materiali utilizzati , vuoi per diverse scelte organizzative che possono incidere ad esempio sullimpiego e quindi sui costi del personale . questa analisi assume quindi rilievo soprattutto quale modello metodologico utilizzabile in altre realt . dal momento che la coronarografia viene effettuata su pazienti ospedalizzati , unanalisi dei costi opportuno si raffronti con i rimborsi basati sul drg . 
nella nostra regione il drg 124 che richiama la coronarografia ( malattia cardiovascolare eccetto infarto miocardio acuto , con cateterismo cardiaco ) prevede un rimborso di 4876 , 00 , indipendentemente dalla durata della degenza . la coronaro - tc viene invece effettuata nella larghissima maggioranza dei casi in pazienti ambulatoriali e quindi ad essa non si applica il rimborso sulla base del drg . qualora venisse effettuata in paziente ospedalizzato , si applicherebbe verosimilmente il drg 140 ( angina pectoris ) che prevede un rimborso di 332 , 00 se la degenza inferiore ai 3 giorni e di 2929 , 00 se invece superiore ai 3 giorni . quando si consideri il rapporto costo - efficacia , va anzitutto rilevato come idealmente sia auspicabile che la metodica meno costosa sia anche la pi sensibile . 
however , because the pretest likelihood of disease is the decisive factor in establishing which of the two techniques ctca or coronary angiography is more cost effective , determining the pretest likelihood is an important step in selecting the most appropriate diagnostic strategy [ 15 , 16 ]  . it is also important to consider the impact of the different diagnostic approaches on patient survival and quality of life . this aspect could be addressed by further studies . our results can be compared with a recently published cost - effectiveness analysis carried out by dewey et al . 
 [ 13 ] , which sought to determine the cost - effectiveness of ctca and magnetic resonance imaging ( mri ) according to the pretest likelihood of cad compared with the gold standard digital coronary angiography . 
our findings differ from those reported by dewey et al . , in particular as regards their statement that ctca has good cost - effectiveness up to a pretest likelihood of 60%70% , whereas coronary angiography is more cost - effective in patients with higher pretest likelihoods . 
 [ 13 ] 230 , 03 2027 , 88 costo coronaro - tc costo coronarografia sensibilit della coronaro - tc 98 , 4% specificit della coronaro - tc 90% coronaro - tc : esami non diagnostici 3 , 6% 175 , 69 630 , 99 95 , 6% 78 , 8% 1 , 15% grafia ( considerata come gold standard ) e comunque bisogna ricordare come nella scelta della metodica diagnostica ci non sia sufficiente . 
infatti nella valutazione di un paziente ad alto rischio coronarico la scelta di effettuare come prima indagine la coronaro - tc non giustificata n dal punto di vista clinico n da quello economico poich verosimile che successivamente tale paziente dovr eseguire una coronarografia quantomeno ai fini terapeutici . 
 in base ai nostri dati , nella nostra realt operativa , abbiamo dimostrato come la coronaro - tc sia la metodica con il migliore rapporto costo - efficacia nella valutazione di pazienti che presentano una probabilit pre - test inferiore all87% . 
variazioni dei costi e variazioni dellaccuratezza diagnostica ( ad esempio ulteriore incremento della sensibilit con i progressi tecnologici delle apparecchiature tc ) potranno modificare il quadro anche in un prossimo futuro . 
va comunque sottolineato che la probabilit pre - test il fattore determinante nello stabilire quale tra la coronaro - tc e la coronarografia sia lindagine con il migliore rapporto di costo - efficacia , per cui stabilire il valore di probabilit pre - test un importante criterio nel selezionare la strategia diagnostica pi appropriata [ 15 , 16 ]  . 
ci potrebbe rappresentare loggetto di studi futuri . i nostri risultati possono essere confrontati con una valutazione di costo - efficacia comparsa recentemente in letteratura ed effettuata da dewey et al . 
 [ 13 ] che si proponeva di verificare il valore di costo - efficacia , in base alla probabilit pre - test di malattia coronarica di due indagini diagnostiche , la coronaro - tc e la risonanza magnetica , rispetto al gold standard rappresentato dalla coronarografia digitale . nella valutazione dei costi vi sono alcune differenze significative tra il lavoro di dewey et al . 
infatti per questa analisi noi ci siamo basati su costi ottenuti da una scrupolosa analisi dei costi differenziali , comuni ed esterni , mentre il lavoro di dewey utilizza dei dati di costo apparecchiature che derivano dai drg . 
questo deriva dal fatto che noi abbiamo eseguito unanalisi dei costi per ottenere il nostro dato , 252 radiol med ( 2009 ) 114 : 239252 besides , there was an important difference in the cost of coronary angiography , which was derived from the german equivalent of the drg system in dewey et al.s study but was actually computed by means of cost analysis in our study . consequently , dewey et al . 
found coronary angiography to be most cost effective for a pretest likelihood of at least 70% , with costs per correctly identified cad patient ranging from 1 , 153 ( 70% likelihood ) to 807 ( 100% likelihood ) , whereas we found coronary angiography to be most cost effective only for a pretest likelihood > 86% . 
 [ 13 ] riportano come la coronarografia sia la metodica con migliore rapporto costo - efficacia al di sopra del 70% di probabilit pre - test con un costo per identificare correttamente un paziente con malattia coronarica che varia tra 1153 ( 70% probabilit pre test ) e 807 ( 100% probabilit pre test ) , mentre dai nostri dati ci risulta vero solo con una probabilit pre - test di malattia superiore all86% . 
radiodiagnostica , fondazione irccs policlinico san matteo , v.le golgi 19 , 27100 pavia , italy 2specializzanda di radiologia , istituto di radiologia delluniversit di pavia , pavia , italy correspondence to : g . 
we retrospectively reviewed the radiographs taken during the past 26 years in children ( age 018 years ) undergoing imaging of the spine or of other body segments in which the spine was adequately depicted , to determine possible intervertebral disc calcifications . 
the following clinical evaluation was extrapolated from the patients charts : presence of spinal symptoms , history of trauma , suspected or clinically evident scoliosis , suspected or clinically evident syndromes , bone dysplasias , and preor postoperative chest or abdominal x - rays . 
five calcifications were asymptomatic ( one newborn baby with patau syndrome ; three patients studied to rule out scoliosis , hypochondroplasia and syndromic traits ; one for dyspnoea due to sunflower seeds inhalation )  . 
apart from the calcification in the patient with cervical pain , all calcifications were asymptomatic and constituted an incidental finding ( particularly those detected at the thoracic level in the patient studied for sunflower - seed inhalation )  . 
sono stati valutati retrospettivamente tutti i radiogrammi eseguiti nel corso di 26 anni in bambini ( di et compresa tra 0 e 18 anni ) sottoposti a studio del rachide o di altri distretti corporei in cui esso era compreso e ne era possibile la valutazione , alla ricerca di eventuali calcificazioni . 
abbiamo rilevato la presenza di calcificazioni dei dischi intervertebrali in 6 pazienti ; per 5 dei quali non vi era in anamnesi sintomatologia dolorosa ( un neonato affetto da sindrome di patau ; tre pazienti erano stati sottoposti ad esame radiologico per diagnosi clinica di scoliosi , ipocondroplasia e stato sindromico , rispettivamente ; uno perch aveva inalato semi di girasole ) , mentre uno solo era sintomatico , con anamnesi di dolore acuto al rachide cervicale . 
con la sola eccezione del paziente con algia cervicale , tutte le calcificazioni erano asintomatiche , il loro riscontro risultando del tutto fortuito ( in particolare quelle a livello toracico nel paziente sottoposto a radiografia del torace per la inalazione di semi di girasole ) , avevano di forma variabile , da lineari a rotondeggianti . 
la casistica esaminata conferma che il riscontro di calcificazioni dei dischi intervertebrali evento raro nellinfanzia e che non deve destare 332 radiol med ( 2009 ) 114 : 331341 only very few cases , such as those of medullary compression or severe dysphagia due to anterior herniation of cervical discs , may require surgical procedures . keywords spine childhood calcifications intervertebral disc calcifications preoccupazione , anche in presenza di sintomatologia dolorosa , dato che in questo caso la calcificazione tende a regredire spontaneamente ed in breve tempo ( contrariamente a quelle indolenti che possono perdurare per anni , per sparire entro i 20 ) , con o senza terapia antidolorifica ed immobilizzazione , richiedendo solo in rarissimi casi ( compressioni midollari o gravi fatti disfagici , in caso di erniazione anteriore nelle localizzazioni cervicali ) la necessit di interventi chirurgici . parole chiave rachide bambini calcificazioni calcificazioni dischi intervertebrali introduction introduzione intervertebral disc calcification is a chance finding in both childhood and the adult population . 
since the first case reported by bron [ 1 ] in 1924 of a 12 - year - old boy presenting with thoracic spine pain , fever and scoliosis , no more than 400 cases have been reported in the 0to 20 - year age group [ 225 ]  . 
the finding may be entirely serendipitous during x - ray studies performed for other clinical problems or may be detected in cases of a typical clinical syndrome characterised by acute pain , mostly affecting the cervical spine , accompanied by fever , raised erythrocyte sedimentation rate ( esr ) and sometimes leucocytosis [ 2 , 3 , 5 , 710 , 1619 , 2123 ]  . 
the time of appearance of calcifications is uncertain , although it certainly precedes by far the time of discovery , as shown by the many cases in which a careful re - evaluation of previous x - rays following the detection of calcifications showed that the lesions were indeed present even in childhood but had gone undetected and unreported [ 2 , 7 , 16 , 17 , 23 ]  . 
 we retrospectively reviewed x - rays taken during the past 26 years in children ( age 018 years ) undergoing imaging of the spine or other body segments in which the spine adequately visualised to discover possible intervertebral disc calcifications . 
the following clinical data were extrapolated from the patients charts : presence of spinal symptoms , history of trauma , suspected or clinically evident scoliosis , suspected or clinically evident syndromes , bone dysplasias and preor postoperative chest or abdominal x - rays . 
dalla prima segnalazione , nel lontano 1924 da parte di bron ( un ragazzo di 12 anni con dolori dorsali , febbre e scoliosi ) [ 1 ] , si sono avuti nel corso degli anni segnalazioni che hanno interessato non pi di 400 casi nel gruppo di et neonato fino a 20 anni [ 225 ]  . 
il reperto pu essere del tutto occasionale , nel corso di indagini radiologiche per altro motivo , o di riscontro in presenza di una sindrome clinica piuttosto tipica , caratterizzata da sintomatologia dolorosa acuta , con localizzazione quasi sempre a livello del rachide cervicale , accompagnata da rialzo febbrile , aumento della ves e saltuaria leucocitosi [ 2 , 3 , 5 , 710 , 1619 , 2123 ]  . 
incerta let in cui compaiono le calcificazioni : certo che essa deve essere situata a distanza anche di molti anni dalla loro effettiva scoperta , come dimostrato da casi in cui , dopo la scoperta di calcificazioni , lattenta rivalutazione , quando possibile , di indagini radiologiche eseguite in precedenza , ne ha dimostrato la presenza , allora per non segnalata , anche in bambini tenera et , e ci anche a distanza di anni dal successivo riscontro [ 2 , 7 , 16 , 17 , 23 ]  . 
dallesame delle richieste sono state registrate le seguenti informazioni : presenza di sintomatologia riferibile al rachide , anamnesi di trauma , sospetto o diagnosi clinica di scoliosi , di sindromi , di displasia ossea , richiesta di radiografia di addome e / o torace preo postoperatori . radiol med ( 2009 ) 114 : 331341 performed during her first day of life to evaluate bone changes related to the syndrome showed a differentiation defect at the thoracolumbar passage . 
eleven pairs of ribs were detected ( hypoplasia of the t11 right rib , no left rib ) and intervertebral disc calcifications at t10 - t11 and l1 - l2 levels . 
the first one was thin , oval in shape and slightly dense ; the second was round , large , fairly opaque and caused a small increase of the intervertebral space . 
this patient was a 121 / 2 - year - old girl who underwent x - ray study of the spine to rule out scoliosis in the absence of pa intervertebral disc calcifications were detected at t5 - t6 ( round and located in the posterior portion of the intervertebral space ) , t6 - t7 ( linear , central ) and possibly at t7 - t8 levels . 
lo studio radiologico dello scheletro per la determinazione di eventuali alterazioni ossee correlabili alla sindrome da cui affetta , eseguito nella prima giornata di vita , ha evidenziato vizio di differenziazione al passaggio dorso - lombare con presenza di 11 paia di coste ( ipoplasica la destra di d11 ed assente la sinistra ) , calcificazioni dei dischi corrispondenza di d10 - d11 e di l1 - l2 . 
i radiogrammi del torace evidenziano , oltre ad area di opacit disventilatoria in corrispondenza del lobo inferiore destro , calcificazioni dei dischi intervertebrali in corrispondenza di d2 - d3 e d5 - d8 , sotto forma di immagini radiopache di diversa densit , e di forma ovalare allungata . 
vizio di differenziazione al passaggio dorso - lombare con presenza di 11 paia di coste ( ipoplasica la destra di d11 ed assente la sinistra ) , calcificazioni dei dischi intervertebrali in corrispondenza di d10 - d11 e di l1 - l2 . 
a chest x - ray taken to determine the location of the foreign body showed right lower - lobe parenchymal opacity and intervertebral disc calcifications at t2t4 and t5t8 levels . 
according to the boys parents , the boy had never complained of spinal pa patient 4 caso 6 radiol med ( 2009 ) 114 : 331341 mai avvertito sintomatologia dolorosa in corrispondenza del rachide . 
vengono rilevate calcificazioni degli spazi intersomatici d4 - d5 , d9 - d10 , d12 - l1 , questultima di ampie dimensioni , anche se non omogenea , di forma ovalare , localizzata nei due terzi posteriori dello spazio intersomatico . 
3a il radiogramma del torace evidenzia opacit del lobo inferiore destro dovuta allaspirazione di semi di girasole nonch presenza di calcificazioni sottili , quasi lineari degli spazi intersomatici d2 - d4 e d5 - d8 , ben visibili anche b nella proiezione ll . radiol med ( 2009 ) 114 : 331341 fig . 
4a grossolana calcificazione dello spazio intersomatico posteriore d11 - d12 che impronta le limitanti somatiche , in particolare b la superiore di d11 . hospital to assess her dysmorphic features clinically unrelated to any syndrome . 
a very large , coarse , intervertebral disc calcification was detected in the posterior third of the t11 - t12 intersomatic space , with indentation of the vertebral margins , especially of t12 . 
this patient was an 11 - year - old boy examined to rule our hypochondroplasia in the absence of back paintervertebral disc calcifications were found at t4 - t5 , t9 - t10 , t12 - l1 levels . 
this patient was a 101 / 2 - year - old boy admitted to hospital due to sudden onset of progressively worsening neck pain unresponsive to analgesic therapy , with impairment of neck movement and severe neck , arm and back pa cervical spine x - ray showed a tiny wedge - shaped calcification posteriorly located at the c2 - c3 intervertebral mezzo , causa linsorgenza improvvisa di dolenzia in regione sterno - cleido - mastoidea che non recede con terapia antalgica , in aumento progressivo , con limitazione marcata della mobilit del collo associata ad estrema sensazione dolorosa al collo , braccio , schiena . 
a distanza di 6 mesi le calcificazioni erano ancora evidenti in assenza di altri episodi dolorosi . nessuno di questi soggetti stato sottoposto a studio tc od rm . quadro clinico , frequenza e sintomatologia le calcificazioni del disco intervertebrale nel bambino sono sempre osservate in corrispondenza del nucleo polposo , in particolare quello dei somi del tratto cervicale . hanno aspetto di solito ovalare , rotondeggiante , appiattito , raramente sono frammentate o puntiformi e sono localizzate quasi sempre in sede centrale [ 2 ]  . 
sono possibili minime alterazioni della morfologia dei somi adiacenti alla / e calcificazione / i , reperto presente nel 90% dei casi in pazienti sintomatici [ 2 , 6 , 9 , 10 , 1517 , 22 ] , sotto forma di riduzione in altezza , deformazione a cuneo anteriore o posteriore , irregolarit o sclerosi marginale , scomparsa del nucleo di ossificazione delle limitanti somatiche angolari anteriori [ 6 , 10 , 16 , 17 ] ed impronte da parte delle calcificazioni sulle limitanti somatiche adiacenti , reperti cui si pu associare riduzione od allargamento degli spazi intersomatici corrispondenti [ 3 , 6 , 8 , 10 , 18 , 19 ]  . 
 anche se le calcificazioni possono essere riscontrate in ogni tratto del rachide , quelle cervicali sono le pi frequenti ( con prevalente localizzazione in corrispondenza dello spazio intersomatico c5 - c6 e c6 - c7 , mentre molto rara la localizzazione in corrispondenza di c7 - d1 ) , con associata erniazione anteriore o posteriore nel 38% dei casi [ 4 , 6 , 10 , 12 , 1517 , 25 ] , seguite dalle toraciche , mentre le lombari sono di riscontro , molto raro [ 7 , 23 ]  . 
 possibile linteressamento multiplo nel 30%40% dei casi [ 2 , 16 , 19 ] , potendosi arrivare al numero di 8 osservate in un paziente asintomatico [ 2 ] e ad uno medio di 1 , 9 calcificazioni per paziente secondo girodias et al . 
il maggior riscontro nel gruppo di et 610 anni [ 7 , 8 , 16 , 19 , 20 , 22 , 23 , 25 ] con casi osservati anche nelle prime ore o giorni di vita [ 2 , 11 , 16 ] , con una certa maggior frequenza nei maschi delle calcificazioni a livello cervicale mentre nelle femmine la distribuzione variabile nei vari tratti ( 7 : 5 ) e con netta 336 radiol med ( 2009 ) 114 : 331341 fig . 
5a - d calcificazioni intersomatiche in corrispondenza di d4 - d5 , d9 - d10 , d - 12 - l1 di cui grossolana quella d12 - l1 , di forma quasi ovalare , al terzo medio dello spazio intersomatico . 
a distanza di un anno c , d , aspetto frammentato in due parti della calcificazione d12 - l1 ; meno densa e riconoscibile solo in parte quella tra d9 - d10 . space and a larger , inhomogeneous round calcification at the c5 - c6 central intervertebral space . 
six months later , the patient was free of symptoms , intervertebral disc calcifications were still the and detectable . none of the patients in this case series underwent computed tomography ( ct ) or magnetic resonance imaging ( mri )  . clinical findings , frequency and signs and symptoms intervertebral disc calcifications in children are always found in the nucleus pulposus , in particular those of the cervical spine . 
calcifications are usually oval or round , flat , rarely fragmented or punctiform , and located centrally , without reduction of the affected intervertebral disc space , which may be increased [ 2 ]  . 
slight changes may be detected in the adjacent vertebral bodies in nearly 90% of symptomatic cases [ 2 , 6 , 9 , 10 , 1517 , 22 ]  . 
these include flattening , anterior or posterior wedging , irregularities or marginal sclerosis , disappearance of anterior ring apophysis [ 6 , 10 , 16 , 17 ] and impression due to the calcification on the fig . 
noteworthy is the absence of swelling of the perivertebral soft tissue [ 17 ]  . even though calcifications may be found in any part of the spine , cervical calcifications are the most frequent ( especially at c5 - c6 and c6 - c7 intersomatic space , whereas the c7 - t1 localisation is rare ) , associated in 38% of cases with anterior or posterior herniation [ 4 , 6 , 10 , 12 , 1517 , 25 ]  . second most common are thoracic calcifications , whereas lumbar ones are very rare . 
multiple localisation is possible in 30%40% of cases [ 2 , 16 , 19 ] , with up to eight disc calcifications having been found in one asymptomatic patient [ 2 ] and a median of 1.9 calcifications according to girodias et al . 
there is a slight predominance among males ( 7 : 5 male - to - female ratio ) , in whom they more commonly affect the cervical spine , whereas distribution is uneven at the cervical and dorsal spine in females ; there is a clear - cut prevalence in caucasians patients [ 3 , 7 , 8 , 16 , 21 , 23 , 25 ]  . 
 radiological findings are completely different from those of spondylodiscitis , which is usually confined to a single disc lumbar spine involvement being more frequent followed by reduction of the intervertebral space , irregularities of vertebral - body plates , perivertebral soft tissue swelling , possible vertebral - body fusion and never disc calcification [ 6 , 7 , 16 , 17 , 22 ]  . in those cases in which neurological symptoms are present in addition to pain , mri will accurately demonstrate the possible compression of the medulla and nerve roots [ 6 , 17 , 2022 , 25 , 26 ] , diminished calcification signal intensity [ 12 , 23 ] and initial involvement of other discs , especially at the cervical level [ 26 ]  . 
mri may also show lower signal intensity of the vertebral body below the affected disc [ 12 ]  . ct will better define the site of calcified and probably herniated discs [ 16 , 25 ]  . for radiation protection reasons , mri should be the preferred imaging modality in paediatric patients , even though the procedure is longer and sometimes more poorly tolerated by children compared with the faster spiral ct , which has been advocated by some authors [ 16 , 25 ]  . 
mri also has the advantage of being able to adequately demonstrate spinal - cord compression in the case of disc herniation [ 6 , 17 , 2022 , 25 , 26 ] and revealing further calcifications undetectable on plain x - ray in cases of calcified discitis [ 26 ]  . 
 il quadro radiologico ben diverso da quello della spondilo - discite , di regola localizzata ad un solo disco spesso a livello lombare e che determina riduzione dello spazio intersomatico , irregolarit delle limitanti somatiche corrispondenti , manicotti perisomatici , possibile fusione tra i somi interessati ma mai calcificazione del disco [ 6 , 7 , 16 , 17 , 22 ]  . in casi in cui la sintomatologia oltre che dolorosa abbia anche una componente neurologica , lo studio con risonanza magnetica nucleare definir con precisione la eventuale compressione sul midollo spinale e le radici nervose [ 16 , 17 , 2022 , 25 , 26 ] , la ridotta intensit di segnale della calcificazione [ 12 , 23 ] , potendo evidenziare segni di iniziale interessamento anche di altri dischi , soprattutto a livello cervicale [ 26 ] ed una ridotta intensit del segnale del corpo vertebrale sottostante al disco interessato [ 12 ] mentre la tomografia computerizzata meglio evidenzier la posizione del nucleo calcificato ed eventualmente erniato [ 16 , 25 ]  . nei soggetti in et pediatrica , per motivi protezionistici dovrebbe essere preferito limpiego della rm , anche se lindagine pi lunga e spesso mal sopportata dai pazienti , a confronto della pi rapida tc spirale come suggerito in letteratura [ 16 , 25 ]  . 
la rm ha anche il vantaggio oltre al protezionistico di ben dimostrare non solo le compressioni sul midollo in caso di disco erniato [ 16 , 17 , 2022 , 25 , 26 ] , ma anche la possibile esistenza di ulteriori calcificazioni , non dimostrabili sui radiogrammi diretti , in caso di discite calcifica [ 26 ]  . clinicamente , le calcificazioni nel bambino possono essere divise in due gruppi , quelle sintomatiche , associate a un quadro clinico particolare , e quelle asintomatiche . 
le calcificazioni sintomatiche sono soprattutto localizzate a livello cervicale , in particolare in corrispondenza di c5 - c6 e c6 - c7 e sono pi frequenti nei maschi con et media di presentazione sui 7 , 1 anni [ 6 , 8 , 15 , 17 , 20 ]  . 
la comparsa dei sintomi improvvisa , nel giro di 12 / 24 ore , accompagnata da un quadro clinico tipico , caratterizzato da rialzo febbrile , torcicollo , aumento della ves , modica leucocitosi . 
il dolore quasi sempre il motivo causale dello studio del rachide e della conseguente scoperta delle calcificazioni , in particolare a livello cervicale , ove la loro presenza associata nell85%90% dei casi a spasmi muscolari peri - vertebrali , riduzione della mobilit e dolorabilit alla palpazione e da torcicollo nel 40% dei casi [ 4 , 7 , 13 , 16 , 17 , 19 , 20 , 25 ] , mal di schiena e talora a scoliosi [ 7 ] , mentre il dolore assai meno frequente , se non assente , nelle localizzazioni multiple , in particolare dorsali e lombari . 
la risoluzione della sintomatologia rapida ( 2 / 3 settimane ) [ 8 , 13 , 16 , 17 , 21 ] , salvo nei rari casi di pazienti con compressione midollare [ 8 ] , in cui a fronte di sofferenza radicolare resistente alla terapia 338 radiol med ( 2009 ) 114 : 331341 symptomatic calcifications are found at the cervical level , especially at c5 - c6 and c6 - c7 . 
presentation is usually abrupt over a 12to 24hour time span and usually found in connection with a typical clinical syndrome of low - grade fever , tenderness , neck pain , slight increase in esr and leucocytosis . 
pain is usually the reason for the radiological study and consequent discovery of calcifications , especially at the cervical level , where calcifications are associated with cervical pain , perivertebral muscle spasms , reduction in motility and tenderness in 85%90% of cases , torticollis in 40% of cases [ 4 , 7 , 13 , 16 , 17 , 19 , 20 , 25 ] , back pain and , sometimes , scoliosis [ 7 ]  . 
symptom resolution is fast ( 23 weeks ) [ 8 , 13 , 16 , 17 , 21 ] , except in rare cases of patients with medullary compression [ 8 ] in whom , due to compression radiculopathy resistant to conservative therapy , a surgical procedure is sometimes necessary [ 5 , 8 , 13 , 20 , 24 ]  . 
in the case of anterior herniation , there may be dysphagia due more to perioesophageal inflammatory swelling than to direct oesophageal compression by the displaced disc [ 4 , 8 , 1416 ]  . 
in cases of posterior herniation , neurological symptoms from simple paraesthesia to more severe radiculopathy due to cord compression by the extruded disc fragments [ 5 , 8 , 16 , 26 ] are rather infrequent and not the rule [ 17 , 23 , 24 ]  . 
they are followed in most cases by spontaneous resolution after noninvasive treatment , even in the case of severe and sometimes dramatic radiological findings [ 2 , 7 , 17 , 23 ]  . 
whereas it is uncertain whether trauma can trigger symptoms related to the presence of intervertebral disc calcifications , it is certainly true that a radiological survey of the spine will show the presence of calcifications or herniated disc fragments , but in only symptomatic patients [ 2 , 5 , 16 , 19 , 25 ]  . 
moreover , x - ray is unable to establish the time of appearance [ 1719 ]  . asymptomatic disc calcifications are more frequent in females , at an average presentation age of 4.1 years [ 8 , 17 ]  . they are usually found at the thoracic level and contrary to cervical calcifications that disappear in a relatively short time after discovery tend to persist for a long time before disappearing within the 20th year of life [ 2 , 8 , 16 , 17 , 23 ]  . the real frequency of disc calcifications in children is unknown . 
 [ 7 ] reported 15 cases of which 11 were symptomatic and four were studied for other reasons discovered over a 14 - year time span in the swedish town of ume ( 100 , 000 inhabitants but with no details on the paediatric population )  . 
in caso di erniazione anteriore possibile lassociazione con fenomeni disfagici , dovuti per pi a fatti infiammatori peri - esofagei che non a diretta compressione sullesofago da parte del nucleo erniato [ 4 , 8 , 1416 ]  . 
nelle erniazioni posteriori i sintomi neurologici , dalle semplici parestesie a quadri pi severi [ 5 , 8 , 16 , 26 ] di radicolopatia da compressione midollare per estrusione di frammenti del disco , con compressione sulle radici nervose , sono piuttosto infrequenti e non la regola [ 17 , 23 , 24 ] , avendo quasi sempre risoluzione spontanea con misure terapeutiche non invasive , anche a fronte di quadri radiologici talvolta drammatici [ 2 , 7 , 17 , 23 ]  . 
se non certo che fatti traumatici possano scatenare la sintomatologia legata alla presenza delle calcificazioni dei dischi intervertebrali certo invece che lo studio radiologico del rachide , in occasione di tali evenienze , evidenzier le calcificazioni eventualmente presenti , come certo il possibile riscontro di frammenti erniati nei soli pazienti sintomatici [ 2 , 5 , 16 , 19 , 25 ] non potendosi per stabilire da quanto essi siano presenti [ 1719 ]  . le calcificazioni asintomatiche sono invece localizzate quasi sempre a livello toracico , sono pi frequenti nelle femmine , con et mediana di presentazione sui 4 , 4 anni [ 8 , 17 ] e , a differenza delle cervicali , che tendono a scomparire in tempi relativamente brevi dopo la loro scoperta , persistono per lunghi periodi di tempo , prima di scomparire entro i 20 anni di vita [ 2 , 8 , 16 , 17 , 23 ]  . 
 [ 7 ] segnalano 15 pazienti , di cui 11 con sintomatologia dolorosa e 4 studiati per altri motivi , scoperti in un arco di tempo di 14 anni nella popolazione della citt di ume ( 100000 abitanti , senza precisazione per della componente pediatrica )  . 
nelladulto invece le calcificazioni intervertebrali , localizzate allannulus fibroso , sono di frequente riscontro dopo la quinta decade di vita , con un periodo libero in corrispondenza della terza e quarta e localizzazione in pi spazi intervertebrali nei vari segmenti del rachide , in particolare dorsale inferiore e lombare [ 27 ]  . 
queste calcificazioni , accompagnate da irregolarit delle limitanti , con riduzione degli spazi intersomatici , cui possono corrispondere associate fusioni intersomatiche , sono di solito asintomatiche , permanenti e di scoperta casuale nel corso di indagini radiologiche eseguite per altri motivi e da considerare un fisiologico fenomeno legato allinvecchiamento [ 23 , 27 , 28 ]  . 
 radiol med ( 2009 ) 114 : 331341 intervertebral calcifications at the annulus fibrosus are frequently discovered after the fifth decade of life , with a free period during the third and fourth decades , and are localised at several intervertebral spaces in different parts of the spine , especially at the lower thoracic and lumbar levels [ 27 ]  . 
these calcifications , usually accompanied by vertebral - plate irregularities and possible intervertebral body fusion , are usually asymptomatic , permanent and an incidental finding during radiological procedures : these are thought to represent a physiological consequence of aging [ 23 , 27 , 28 ]  . conclusions it is difficult to evaluate how long calcifications have been present before their incidental discovery or the appearance of pain , and for how long they will remain detectable after their discovery , given that follow - up over time shows their reduction and disappearance [ 2 , 13 , 16 , 17 , 23 ] without being able to demonstrate the time of disappearance . 
disc calcifications tend to disappear faster than the associated vertebral - body changes [ 10 , 17 ]  . acute cases of symptomatic , painful disc calcification , cervical in particular , are followed by disappearance within weeks or even days ( 9 days in one case ) [ 3 ] through a process of fragmentation and progressive reduction in opacity [ 2 , 5 , 6 , 13 , 16 ]  . 
at the thoracic and lumbar levels , calcifications tend to last unchanged for up to 10 years before they resolve spontaneously [ 2 , 7 , 16 ]  . in patients with disc calcifications , especially when symptomatic , it is possible to detect a reduction in intervertebral disc space and a slight flattening of the affected vertebral bodies a long time after their disappearance [ 17 ]  . 
metabolic causes , such as hypervitaminosis d , alcaptonuric hyperparathyroidism , haemochromatosis , ochronosis ( typical of adults , as a long time is necessary before calcified deposits appear ) , are not justified owing to the absence of analogous findings in other organs , whereas inflammatory , microtraumatic or vascular causes are more likely [ 2 , 7 , 21 , 22 ]  . 
 the large blood supply to the intervertebral disc in children and its more active metabolism which is increased in case of inflammation could explain the sudden appearance and disappearance or enlargement of the intervertebral disc calcification in comparison with that seen in adult patients [ 16 , 28 ]  . 
traumatic events may lead to the discovery conclusioni risulta difficile valutare da quanto tempo , prima della scoperta causale o della comparsa della sintomatologia dolorosa , siano gi presenti le calcificazioni e per quanto tempo rimangano poi visibili , in quanto controlli a distanza di tempo ne evidenziano la riduzione e la completa scomparsa [ 2 , 13 , 16 , 17 , 23 ] senza per poterne stabilire con esattezza il momento . 
da tenere ben presente che le calcificazioni scompaiono assai pi rapidamente delle alterazioni dei corpi vertebrali [ 10 , 17 ]  . certo che i casi acuti di calcificazione sintomatica dolorosa , in particolare cervicale , sono seguiti da scomparsa in tempi relativamente rapidi , di poche settimane se non di giorni ( 9 in un caso ) [ 3 ] , attraverso un processo di frammentazione e progressiva riduzione della loro radiopacit [ 2 , 5 , 6 , 13 , 16 ] , mentre a livello dorsale le calcificazioni tendono a permanere immodificate anche per lunghi periodi di tempo ( fino a 10 anni ) prima della scomparsa [ 2 , 7 , 16 ]  . nei soggetti in cui sono presenti le calcificazioni discali , in particolare quelle sintomatiche , sono osservabili a distanza di tempo dalla loro scomparsa fenomeni di riduzione dello spazio discale e relativo appiattimento dei corpi vertebrali interessati [ 17 ]  . 
cause metaboliche come lipervitaminosi d , liperparatiroidismo , la pseudogotta , lemocromatosi , locronosi alcaptonurica ( nelladulto in quanto richiede tempi molto lunghi prima che si giunga allosservazione dei depositi calcifici ) non sono giustificate per lassenza di analoghe localizzazioni in altre sedi , mentre fatti infiammatori , microtraumatici o vascolari sono stati chiamati in causa con forse maggior veridicit [ 2 , 7 , 21 , 22 ]  . sono state anche segnalate associazioni con anomalie congenite , in particolare cardiopatie , cataratte bilaterali , calasia [ 2 ] e sindrome di patau [ 11 ]  . la maggior vascolarizzazione dei dischi intervertebrali nel bambino e di conseguenza il suo pi attivo metabolismo , aumentato in caso di flogosi , potrebbe essere una giustificazione della rapida comparsa e successiva scomparsa od aumento nelle dimensioni delle calcificazioni del disco intervertebrale rispetto a quanto osservabile nelladulto [ 16 , 28 ]  . 
traumatismi sono stati chiamati in causa ( anche se assai difficili da valutare in et pediatrica ) non tanto come evento scatenante il meccanismo che sta alla base della formazione delle calcificazioni , quanto come elemento che permette la dimostrazione delle stesse . attualmente lipotesi della natura vascolare della lesione sembra essere la pi seguita , in particolare se in 340 radiol med ( 2009 ) 114 : 331341 of calcifications , but they are very difficult to evaluate in paediatric patients . 
 the most popular hypothesis is that the lesion has a vascular nature , particularly when in association with perinatal lesions , as shown by the rare cases of disc calcification found at birth [ 2 , 11 , 16 , 22 ]  . 
none of these hypotheses is in any case supported by real evidence . with the exception of the rare cases of anterior disc protrusion with associated dysphagia [ 8 , 13 , 1517 ] or posterior protrusion with slight [ 22 ] or evident spinal cord compression necessitating a surgical procedure [ 8 , 13 , 16 , 20 ] , intervertebral disc calcification is a benign and selflimiting disorder , which resolves with bed rest , analgesics , muscle - relaxing and anti - inflammatory medication without any need for invasive surgical procedures [ 2 , 16 , 17 , 2224 ]  . it is noteworthy that even in our small patient series ( three females and three males ) , there was only one case of symptomatic painful disc localised at the cervical level and that single or multiple , mostly thoracic , disc calcifications were detected during x - ray procedures performed for different reasons in asymptomatic patients . 
it is also interesting that calcifications were discovered in a newborn patient ( one of the few in the literature [ 2 , 11 ] ) affected by patau syndrome . we conclude with a citation from melnick that intervertebral disc calcification in children appears to be a selflimited disease and requires only symptomatic treatment and as stressed also by dias et al . 
seldom requires an operation [ 17 ]  . associazione con lesioni perinatali , come dimostrato dai rari casi di riscontro delle calcificazioni alla nascita [ 2 , 11 , 16 , 22 ]  . 
nessuna per di queste ipotesi corroborata da riscontri certi per la corretta spiegazione delle cause di questa affezione che rimane perci misteriosa riguardo ai suoi meccanismi scatenanti . salvo i rari casi di protrusione anteriore con associata disfagia [ 8 , 13 , 1517 ] o posteriore con compressione lieve [ 22 ] o significativa del midollo in cui stato necessario lintervento chirurgico [ 8 , 13 , 16 , 20 ] , la prognosi della affezione , che da considerarsi autolimitante , benigna con ritorno a condizioni di normalit con il riposo , farmaci miorilassanti ed antiflogistici senza che sia necessario il ricorso ad interventi pi cruenti [ 2 , 16 , 17 , 2224 ]  . significativa nella nostra casistica , anche se basata su soli 6 casi ( 3 femmine e 3 maschi ) , la presenza in un solo caso dei sintomi acuti tipici della localizzazione cervicale ed invece il riscontro di localizzazioni dorsali , singole od interessanti pi dischi intervertebrali , avvenuto in modo del tutto fortuito , in occasione di indagini radiologiche eseguite per altri motivi , in assenza di sintomatologia dolorosa . significativo invece il riscontro delle calcificazioni nel paziente in et neonatale , affetto da sindrome di patau , uno dei pochi casi segnalati in tale et [ 2 , 11 ]  . 
 possiamo concludere con laffermazione di melnick che nei bambini la calcificazione del disco intervertebrale pu essere considerata come una malattia autolimitantesi e che richiede solo terapia sintomatica e che , come sottolineato da dias et al . 
catalano , corso vittorio emanuele 89 bis , 80121 napoli , italy , tel . : + 39 - 081 - 7612417 , fax : + 39 - 081 - 5903689 , e - mail : orlandcat@tin.it received : 2 february 2008 / accepted : 6 march 2008 / published online : 12 december 2008 springer - verlag 2009 abstract purpose . 
il contenuto cistico appariva limpido in 6 papillomi e nelliperplasia duttale atipica e a fini echi in 2 papillomi ; nei carcinomi il contenuto era sotto forma di grossolani echi in 2 casi e di fini echi in 11 . 
calcificazioni intralesionali erano rilevabili 254 radiol med ( 2009 ) 114 : 253266 coarse echoes in two cases and fine echoes in 11 . intralesional calcifications were seen in three carcinomas . posterior enhancement was present in all carcinomas , whereas none showed posterior shadowing . 
leco - color doppler dimostrava assenza di flusso in 4 papillomi e segnali modesti in 4 papillomi e nelliperplasia duttale atipica ; i 13 carcinomi mostravano tutti segnali vascolari diffusi , con multipli vasi sparsi e da multipli poli vascolari . 
gli indici resistivi , misurabili in 4 / 8 papillomi , erano in media di 0 , 43 ; nei carcinomi lo studio flussimetrico era sempre possibile , con indice resistivo medio di 0 , 71 . 
i tumori mammari intracistici presentano aspetti ecostrutturali ed eco - color doppler peculiari , tali da consentire unefficace differenziazione rispetto alle cisti non tumorali ; spesso ipotizzabile una differenziazione tra papillomi e carcinomi . 
la presenza di vascolarizzazione pone invece lindicazione allescissione oppure alla biopsia dei gettoni solidi , anche quando lanalisi citologica risultata negativa . parole chiave mammella ecografia tumore intracistico carcinoma papillare introduction introduzione cystic lesions of the breast are a very common finding , especially in middle - aged women , being detected in one in four women aged 3550 years [ 1 ]  . 
a variety of breast disorders may exhibit a cystic appearance , at least in a number of cases , in a portion of the lesion and / or in a specific stage of their evolution : haematoma , abscess , plasma - cell mastitis , seroma , galactocele , lymphocele , fat necrosis ( lipid cyst ) , angioma , lymphangioma , aneurysm , arteriovenous malformation , fibroadenoma ( 3% of cases ) , phyllodes tumour , fibrocystic change , atypical ductal hyperplasia , papilloma ( with or without atypia ) and carcinoma [ 24 ]  . 
possible malignancies with cystic appearance include ductal carcinoma in situ and infiltrating ductal carcinoma , papillary carcinoma in situ and infiltrating papillary carcinoma , invasive lobular carcinoma , medullary carcinoma , squamous cell carcinoma and hypersecreting ductal adenocarcinoma [ 3 , 5 ]  . 
 on ultrasound ( us ) imaging , complicated or dirty cysts are defined as cystic masses characterised by mild posterior enhancement or multiple homogeneous internal low - level echoes but are otherwise identical to simple cysts . 
numerose lesioni possono avere , almeno in una parte dei casi , in maniera parziale e / o in una determinata fase della loro evoluzione , un aspetto similcistico , ematoma , ascesso , mastite plasmacellulare , sieroma , galattocele , linfocele , steatonecrosi ( cisti oleosa ) , angioma , linfangioma , aneurisma , malformazione artero - venosa , fibroadenoma ( 3% dei casi ) , tumore filloide , modifiche fibrocistiche , iperplasia duttale atipica , papilloma ( con o senza atipia ) , carcinoma [ 24 ]  . 
nellambito dei possibili carcinomi cistici , bisogna ricordare innanzitutto quello duttale in situ e infiltrante e quello papillare in situ e infiltrante , poi quelli lobulare infiltrante , midollare , squamoso nonch ladenocarcinoma duttale ipersecernente [ 3 , 5 ]  . 
 dal punto di vista ecografico , vengono definite complicate o sporche quelle formazioni cistiche , altrimenti semplici , ma caratterizzate da un rinforzo di parete posteriore poco evidente oppure da multipli e omogenei echi interni di basso livello , eventualmente mobili ( come dimostrabile anche con lm - mode , con leco - color doppler radiol med ( 2009 ) 114 : 253266 demonstrated by m - mode us , colour doppler us or vocal fremitus combined with doppler techniques )  . 
complex or partially solid cysts , in contrast , show some evidence of echostructural atypia , such as mural projections , thick septa ( > 0.4 mm ) or thick ( > 0.4 mm ) and irregular walls . 
 [ 2 ] distinguished among four types of complex cysts : cysts with thick walls ; cysts with one or more intracystic solid masses ; cysts with mixed solid and cystic components ( > 50% cystic ) ; cysts with mixed solid and cystic components ( > 50% solid with eccentric cystic foci )  . 
 [ 6 ] recently developed a broader classification covering all forms of cystic breast lesions , whihs was also based on sonographic morphostructural features : type i ( simple cyst ) , anechoic mass with imperceptible circumscribed border and posterior acoustic enhancement ; type ii ( clustered cysts ) , cluster of anechoic formations with no discrete solid component ; type iii ( septated cyst ) , cyst with thin septa ( < 0.5 - mm thick ) ; type iv ( complicated cyst ) , mass with homogeneous low - level echoes or floating debris or with fluid - debris levels ; type v ( complex fluid cyst ) , mass with thick walls or septa ( > 0.5 mm ) or nodules with > 50% fluid component ; type vi ( complex solid cyst ) , mass with < 50% fluid component , essentially solid , with eccentric cystic foci corresponding to dilated ducts , acini or necrosis . 
types v and vi are malignant in 26% and 62% of cases , respectively , being possibly related to fibroadenomas , phyllodes tumours or abscesses [ 6 ]  . although the classification systems of berg et al . 
in contrast , we believe angioarchitectural features to be an integral part of any sonographic assessment of breast masses and as such very useful for differentiating between true ( vascular ) intracystic solid projections and their avascular mimickers , and for distinguishing between intracystic papillomas and carcinomas . 
le cisti complesse , anche definite come parzialmente solide , sono invece quelle che mostrano qualche segno di atipia ecostrutturale , come gettoni murali , setti spessi ( > 0 , 4 mm ) o pareti spesse ( > 0 , 4 mm ) e irregolari ; queste cisti , che vanno inquadrate come bi - rads 4 , sono maligne nel 23%31% dei casi [ 2 , 3 ]  . 
 [ 2 ] hanno distinto le cisti complesse in quattro tipologie : cisti a parete spessa ; cisti con una o pi formazioni solide endocistiche ; cisti con quote solide e cistiche miste ( con queste ultime > 50% ) ; cisti con quote solide e cistiche miste ( con le prime > 50% e le seconde riconoscibili sottoforma di foci eccentrici )  . 
 [ 6 ] hanno recentemente pubblicato una pi ampia classificazione comprensiva di tutte le formazioni cistiche mammarie , sempre basata sui soli aspetti ecografici morfostrutturali : tipo i ( cisti semplice ) , formazione anecogena con bordo impercettibile e circoscritto e con rinforzo posteriore ; tipo ii ( cisti a grappolo ) , formazione anecogena a grappolo senza componenti solide ; tipo iii ( cisti settata ) , formazione con setti sottili ( < 0 , 5 mm di spessore ) ; tipo iv ( cisti complicata ) , formazione con echi omogenei di basso livello o con detriti galleggianti o con livello liquido - detriti ; tipo v ( cisti complessa liquida ) , formazione con pareti o setti spessi ( > 0 , 5 mm ) o con noduli ma comunque con componente liquida > 50% ; tipo vi ( cisti complessa solida ) , formazione con componente liquida < 50% , essenzialmente solida con foci cistici eccentrici , corrispondenti a dotti dilatati , acini o necrosi . 
i tipi da i a iv sono risultati sempre benigni , con il pattern iv spesso corrisponde ad ascessi ; i tipi v e vi sono maligni rispettivamente nel 26% e 62% dei casi , potendo anche essere espressione di fibroadenomi , tumori filloidi , ascessi [ 6 ]  . sebbene i sistemi classificativi di berg et al . 
nostro giudizio invece che i reperti angioarchitettonici , parte integrante di qualsiasi valutazione ecografica di una formazione mammaria , siano molto utili , sia per unimmediata distinzione tra i gettoni solidi endocistici , con segnali di flusso , ed i loro simulatori , avascolari , che per una distinzione , entro determinati limiti , tra papillomi e carcinomi intracistici . 
lo scopo del presente lavoro stato pertanto di valutare lapporto delle tecniche doppler nella diagnostica differenziale e nellinquadramento dei tumori intracistici della mammella . 256 radiol med ( 2009 ) 114 : 253266 cystic breast masses corresponding to type iv , v or vi cysts according to the chang classification [ 6 ] , with the exclusion of type vi predominantly solid cysts with eccentric cystic foci . 
selected patients were 45 women ( one case of male breast cancer was excluded ) aged 3187 years ( mean 59 ) , each with a single complicated / complex cyst . 
 us examinations were conducted on a technos mpx unit ( esaote , genoa , italy ) equipped with a broadband linear - array transducer ( 5.512.5 mhz , generally used at 10 mhz )  . 
a single focus was placed at the level of the deep aspect of the mass being examined . the gain curve was adjusted to the depth of the lesion , attempting to avoid intracystic artefactual images . 
during the scan , the cystic mass was compressed with the transducer and explored with partial changes in patient position . very superficial masses were scanned with the aid of a gel standoff pad . angioarchitecture was studied with colour doppler , in particular , power doppler ( with directional coding ) , with settings allowing maximal sensitivity to slow flows : pulse repetition frequency 750 hz ; low wall filter ( 50 hz ) ; colour gain just below the threshold for artefacts ; colour box restricted to the region of interest . 
emission frequency for colour and power doppler was adjusted to lesion depth : 7.1 mhz for superficial lesions ; 6.3 mhz for intermediate lesions ; 5.6 mhz for deep lesions . 
care was taken not to apply excessive compression with the transducer during the doppler study . evaluation of the us examinations was retrospective and based on video clips acquired with the us unit and transferred via a local network to a personal computer . maximum diameter and maximum diameter perpendicular to the first diameter of both the cystic lesion and any major intracystic projections were measured : the ratio of solid ( or pseudosolid ) to fluid component was defined as < 50% , 50% and > 50% . 
orientation of the mass relative to the cutaneous plane was classified as parallel ( if the long axis was parallel to the skin ) or antiparallel ( if the short axis was parallel to the skin )  . 
echogenicity of the fluid materiali e metodi tra gennaio e dicembre 2006 abbiamo selezionato prospetticamente le pazienti con riscontro ecografico di una o pi formazioni cistiche della mammella assimilabili ai tipi iv , v o vi secondo chang et al . 
i casi selezionati erano costituiti da 45 donne ( un caso di carcinoma maschile veniva escluso ) , di 3187 anni ( media 59 ) , ciascuna con una singola cisti complicata / complessa . 
 gli esami venivano condotti con ecografo technos mpx ( esaote , genova , italia ) equipaggiato con sonda lineare a larga banda ( escursione da 5 , 5 a 12 , 5 mhz , con impiego generalmente a 10 mhz )  . 
per formazioni particolarmente superficiali si ricorreva anche allimpiego di un cuscinetto distanziatore in gelatina . lo studio angio - architettonico veniva praticato con ecocolor doppler e soprattutto con power doppler ( in modalit con codifica direzionale ) , utilizzando dei parametri di studio tali da massimizzare la sensibilit ai flussi lenti : frequenza di ripetizione dellimpulso di 750 hz , filtro di parete al minimo ( low , pari a 50 hz ) , guadagno del colore subito al di sotto della soglia degli artefatti , box del colore ristretto sullarea di interesse . 
la frequenza di emissione per il color e power doppler veniva correlata alla profondit della lesione : 7 , 1 mhz per lesioni superficiali , 6 , 3 mhz per quelle a media profondit e 5 , 6 mhz per quelle pi profonde . 
quando possibile si campionava lo spettro flussimetrico doppler , ottenuto a livello del vaso intralesionale di calibro maggiore e con il flusso pi vivace allosservazione color doppler ; in caso di campionamenti su pi poli vascolari si prendeva in considerazione lo spettro pi pulito e che mostrasse i valori pi estremi , in senso benigno o maligno , dellindice resistivo . 
loperatore aveva cura di evitare una compressione eccessiva con la sonda durante lo studio doppler . la valutazione degli esami ecografici stata retrospettiva , basata sui filmati acquisiti sullecografo ed inviati via rete ad un personal computer . 
veniva misurato il diametro massimo ed il massimo diametro ad esso perpendicolare , sia della lesione cistica che delle eventuali proiezioni solide radiol med ( 2009 ) 114 : 253266 component was classified as homogenously anechoic , inhomogeneously anechoic with fine internal echoes , inhomogeneously anechoic with coarse internal echoes and hypoechoic . 
note was taken of the presence of thick walls ( > 0.5 mm ) , thick septa ( > 0.5 mm ) or solid papillary projections . solid projections were defined as single or multiple and hypoechoic or hyperechoic . 
finally , cysts were assessed for the presence of calcification in the solid component , posterior acoustic enhancement , posterior shadowing and solid satellite nodules . assessment parameters on colour doppler us were presence or absence of flow signal in the wall / septa or solid projections , number of vascular poles ( single or multiple ) , extent of flow ( few isolated signals , moderate diffuse vascularity or abundant diffuse vascularity )  . 
an on - site cytologist provided immediate evaluation , and only in equivocal cases was a core biopsy of the solid component obtained with an automated biopsy gun and 14 - gauge needles . results twenty - three nontumoural masses were treated conservatively , and the diagnosis was confirmed by follow - up ; three symptomatic large masses were aspirated for therapeutic purposes . 
lorientamento della formazione rispetto al piano cutaneo veniva catalogato come parallelo ( se il maggior asse lesionale parallelo alla cute ) o antiparallelo ( se il minior asse lesionale parallelo alla cute )  . 
la reflessivit della quota liquida veniva distinta in anecogena omogenea , anecogena disomogenea con echi interni fini , anecogena disomogenea con echi interni grossolani , presenza di pareti spesse ( > 0 , 5 mm ) , setti spessi ( > 0 , 5 mm ) o proiezioni papillari solide ; questi gettoni solidi venivano distinti in singoli e multipli ed in ipoecogeni e iperecogeni . veniva infine valutata la presenza di calcificazioni nelle quote solide , di rinforzo di parete posteriore e di attenuazione posteriore del fascio , nonch di noduli solidi satelliti . come parametri color - power doppler venivano definiti : presenza / assenza di segnali di flusso in pareti / setti o gettoni solidi , numero di poli vascolari ( singolo o multipli ) , entit ipoecogena . 
assenza di segnali di flusso sia a livello delle pareti che della quota ecogena luminale . 258 radiol med ( 2009 ) 114 : 253266 table 1 morphostructural , angioarchitectural and spectral findings in 45 patients with complicated / complex breast cyst findings features nontumoural lesions ( n = 23 ) papilloma ( n = 8 ) atypical ductal hyperplasia ( n = 1 ) carcinoma ( n = 13 ) maximum diameter ( mm ) 931 ( mean 18 ) 413 ( mean 8 ) 922 ( mean 13 ) orientation solid / fluid component shape margins fluid portion walls septa parallel antiparallel < 50% > 50% round oval oblong lobulated irregular welldefined illdefined homogeneously anechoic anechoic with fine echoes anechoic with coarse echoes hypoechoic thin ( 5 mm ) thick ( > 0.5 mm ) absent thin ( 5 mm ) thick ( > 0.5 mm ) absent present and hypoechoic present and hyperechoic papillary projections calcifications posterior enhancement absent present shadowing satellite solid nodules absent present absent present absent present septal or nodular signals absent present no . 
in the papillomas , del flusso ( qualche segnale isolato , discreta vascolarizzazione diffusa oppure cospicua vascolarizzazione diffusa )  . lanalisi spettrale , quando ottenibile , veniva utilizzata per calcolare lindice resistivo . 
 i prelievi citologici di conferma venivano effettuati con radiol med ( 2009 ) 114 : 253266 tabella 1 reperti morfo - strutturali , angio - architettonici e flussimetrici in 45 pazienti con cisti mammaria complicata / complessa reperti possibilit lesione non tumorale ( n = 23 ) papilloma ( n = 8 ) iperplasia duttale atipica ( n = 1 ) carcinoma ( n = 13 ) diametro massimo in mm 931 ( media 18 ) 413 ( media 8 ) 922 ( media 13 ) orientamento quota solida / liquida forma margini quota liquida pareti setti proiezioni papillari parallelo antiparallelo < 50% > 50% rotondeggiante ovalare allungata polilobulati irregolare netti e regolari mal definiti anecogena omogenea anecogena con echi fini anecogena con echi grossolani ipoecogena sottili ( 5 mm ) spesse ( > 0 , 5 mm ) assenti sottili ( 5 mm ) spessi ( > 0 , 5 mm ) assenti presenti e ipoecogene presenti e iperecogene calcificazioni rinforzo posteriore attenuazione del fascio noduli solidi satelliti assenti presenti assente presente assente presente assenti presenti segnali in setti o gettoni assenti presenti numero di poli vascolari singolo vascolarizzazione indice resistivo mmultipli qualche segnale isolato discreta e diffusa cospicua e diffusa 0 , 330 , 58 ( media 0 , 43 ) 0 , 550 , 77 ( media 0 , 71 ) colour doppler us detected no flow signals in four cases and moderate flow signals in the remaining four cases , as well as in the atypical ductal hyperplasia . 
il materiale aspirato veniva poi strisciato su vetrino , lasciato asciugare allaria e sottoposto a colorazione rapida secondo may - grnwald - giemsa 260 radiol med ( 2009 ) 114 : 253266 flow , with diffuse vascularity characterised by multiple scattered vessels or multiple vascular poles emerging from the base of the papillary projections . 
2 papilloma intracistico , immagine con power doppler direzionale ( modalit dual )  . piccola formazione ( freccia ) aggettante nel lume di una cisti uniloculata , per il resto a contenuto omogeneo . 
assenza di segnali di flusso sia nelle pareti cistiche che nellaggetto . risultati ventitre formazioni cistiche non tumorali venivano trattate conservativamente e la diagnosi veniva confermata dal follow - up ; 3 venivano aspirate a scopo soprattutto terapeutico , perch voluminose o sintomatiche . 
il contenuto cistico appariva limpido in 6 papillomi e nelliperplasia duttale atipica ed a fini echi sparsi in 2 papillomi ; nei carcinomi il contenuto era sotto forma di grossolani echi di basso livello in 2 casi e di fini echi sparsi nei restanti 11 casi . 
nei papillomi leco - color doppler dimostrava assenza di segnali di flusso in 4 casi e modesti segnali vascolari nei rimanenti 4 papillomi nonch nelliperplasia duttale atipica , sottoforma di un singolo peduncolo vascolare centrale o di qualche segnale sparso . 
tutti i 13 carcinomi mostravano invece segni di flusso intralesionale , con una vascolarizzazione diffusa , caratterizzata da multipli vasi sparsi e da multipli poli vascolari emergenti alla base delle proiezioni papillari . 
the smaller one is anterior and with a single flow signal internally ( single arrow in a ) , whereas the other is deeper and larger ( arrows in a )  . 
5a - c carcinoma papillare intracistico , immagini con power doppler direzionale ( a ) , con ago per biopsia ( b ) e dopo svuotamento ( c )  . 
al suo interno aggettano due formazioni solide , una pi piccola e anteriore con singolo segnale di flusso allinterno ( freccia singola in a ) ed una pi profonda e voluminosa ( frecce in a )  . 
in una serie di 1805 pazienti sottoposte ad esame ecografico , questa patologia veniva rilevata in 20 casi ( 1 , 1% ) tra cui solo 6 di tipo maligno [ 10 ]  . 
i carcinomi intracistici ( 0 , 3%2% dei cancri mammari nel sesso femminile ) sono generalmente del tipo papillare ed in questo caso insorgono in donne di et medio - avanzata ( media 65 anni ) , presentando una crescita lenta e una bassa frequenza di metastasi linfonodali alla presentazione , con buona prognosi ( in assenza di chiara diffusione extracistica ) [ 4 , 5 , 11 , 12 ]  . data lorigine duttale , i carcinomi papillari si localizzano soprattutto nelle regioni retroareolari ( almeno nel 50% dei casi ) : essi si formano infatti per dilatazione cistica di un assessment was possible in all carcinomas , where it yielded a mean resistive index of 0.71. 
owing to their ductal origin , papillary carcinomas tend to develop in the retroareolar regions ( in at least 50% of cases ) , where they form as a result of cystic dilatation of a duct around a solid vegetation that obstructs it . 
alternatively , they may present with bloody nipple discharge ( 22%34% of cases ) [ 14 ] or , in the case of internal haemorrhage , with rapid enlargement of the mass that may become tender . 
bleeding may be spontaneous and essentially related to torsion and infarction of the papillary projections or it may be induced by the diagnostic needle puncture itself [ 13 , 14 ]  . 
solid papillary tumours are rare ( four invasive papillary carcinomas in a series of 940 invasive breast carcinomas studied with mammography [ 15 ] ) and generally invasive and have an nonspecific sonographic pattern consisting of single or multiple lobular nodules with welldefined margins , relatively homogeneous hypoechoic echostructure and possible posterior enhancement [ 13 , 14 , 16 , 17 ]  . 
possible papillary projections tend radiol med ( 2009 ) 114 : 253266 dotto intorno ad una vegetazione solida che ne determina lostruzione ; in genere la vegetazione nasce come papilloma , per poi trasformarsi [ 4 , 13 ]  . quando voluminosi , i tumori intracistici possono manifestarsi come tumefazioni palpabili indolenti . 
in alternativa si pu avere una secrezione ematica dal capezzolo ( 22%34% dei casi [ 14 ] ) oppure , per unemorragia interna , un rapido ingrandimento della formazione che diviene anche dolente ; in un caso [ 5 ] la presentazione era sottoforma di cisti emorragiche multiple , insorte dopo un trauma , ma ricorrenti . 
il sanguinamento pu essere spontaneo , determinato essenzialmente dalla torsione e infarto dei gettoni papillari , o pu essere indotto dalla stessa puntura diagnostica [ 13 , 14 ]  . 
 lelemento istologico distintivo di tutti i tumori papillari dato dalla presenza di uno stroma fibrovascolare arborizzante , che supporta la componente epiteliale ; nelle forme carcinomatose si rileva una scarsit o una virtuale assenza dello strato mioepiteliale [ 13 , 14 ]  . 
quelli solidi , rari ( 4 carcinomi papillari invasivi su una casistica di 940 carcinomi mammari invasivi studiati con xeromammografia [ 15 ] ) e generalmente invasivi , hanno un aspetto ecografico aspecifico , sottoforma di noduli singoli o multipli , di forma lobulata , con margini definiti , a struttura ipoecogena relativamente omogenea , con eventuale rinforzo posteriore [ 13 , 14 , 16 , 17 ]  . 
i tumori papillari intraduttali e soprattutto intracistici sono di solito in situ , anche se la quota solida spesso cospicua al momento della diagnosi [ 6 ] ; la diffusione degli eventuali elementi invasivi viene in parte ostacolata dallo strato fibroso parietale [ 14 ]  . 
queste lesioni presentano un aspetto ecografico caratteristico , come formazioni cistiche ovalari , a margini netti , con rinforzo posteriore generalmente presente ( di solito da lieve a moderato )  . 
le cisti mostrano allinterno un contenuto liquido leggermente disomogeneo , con echi spesso di variabile diametro , e dei setti pi o meno polimorfi ; le eventuali proiezioni papillari hanno generalmente una morfologia semilunare , con possibili calcificazioni interne e con base di impianto sessile [ 14 , 18 , 19 ]  . 
in alcuni dei casi personali si osservavano una o pi piccole nodulazioni solide satelliti al carcinoma intracistico e ci conferma il dato della letteratura di un possibile riscontro di foci di carcinoma intraduttale adiacenti ai carcinomi papillari intracistici [ 14 ]  . i carcinomi intracistici vanno quindi distinti dai semplici papillomi , dai tumori solidi di alto grado con ampia necrosi , emorragia e colliquazione centrale , dai tumori maligni che abbiano infiltrato una cisti pre - esistente e dai coaguli ematici adesi alla parete delle cisti ( anche dopo cisticentesi ma in questultimo caso lanamnesi fondamentale ) [ 4 , 6 , 20 ]  . 
per dimostrare leffettiva natura della proiezione papillare , e distinguerla da semplice materiale ecogeno adeso alla parete cistica , possibile ricorrente alla radiol med ( 2009 ) 114 : 253266 to be crescent - shaped with internal calcifications and a sessile base [ 14 , 18 , 19 ]  . 
several cases in our series showed one or more small solid satellite nodules , which confirms previous reports on the possibility of identifying foci of intraductal carcinoma adjacent to intracystic papillary carcinomas [ 14 ]  . intracystic carcinomas thus need to be distinguished from simple papillomas ; from high - grade solid tumours with extensive central necrosis , haemorrhage and colliquation ; from malignant tumours invading a preexisting cyst and from blood clots adhering to the cyst walls ( even after cyst aspiration , although in this case , the patients history is fundamental ) [ 4 , 6 , 20 ]  . 
techniques that help to demonstrate the true nature of the papillary projection and distinguish it from echoic material adhering to the cyst wall include graded compression , changes in patient position , vocal fremitus on power doppler or flow signal detection with doppler techniques [ 3 , 7 ]  . 
detection of nonartefactual colour signals provides a simple and immediate means to demonstrate the solid nature of echoic luminal projections . however , even on the basis of our experience , whereas the presence of colour signals demonstrates the solid nature of the projecting structure , its absence does not exclude , it as it is impossible to adequately differentiate between adhering debris and solid projections [ 3 ]  . 
similarly , the presence of flow on doppler analysis is alone insufficient to discriminate papillomas from carcinomas , even though the signals are few or absent in papillomas and substantially more intense and diffuse in carcinomas . 
 [ 18 ] , power doppler demonstrated regular , nonanarchic vascularity with a single afferent vessel , suggesting a presumptive diagnosis of benignity , whereas histological examination subsequently revealed a papillary carcinoma . in early studies on intracystic tumours , us was reported to have had limited accuracy in diagnosing malignancy [ 10 ]  . in reality , even with modern sonographic equipment , the malignant nature of an intracystic projection cannot be definitely excluded or demonstrated . 
although the presence of vascular signals in an intracystic projection increases the level of suspicion of malignancy ( though benign intracystic projections may also demonstrate internal vessels , generally with low resistive index ) , in fact , all complex cysts should be aspirated and evaluated by cytology . 
it should also be noted that particularly large and / or fast - growing carcinomas may be massively necrotic and thus mimic a dirty cyst or an abscess : demonstration of flow signals at colour doppler us may be diagnostic in such cases [ 1 , 21 ]  . the classic elements for the differential diagnosis of breast nodules were not applicable in our series because all nontumoural lesions , papillomas and carcinomas constantly compressione dosata , alla variazione di decubito , al fremito vocale con power doppler oppure alla dimostrazione di segnali di flusso con tecniche doppler [ 3 , 7 ]  . 
quindi chiaro che , anche in base alla nostra esperienza , la presenza di segnali di colore dimostra la natura solida dellaggetto mentre la loro assenza non la esclude , non potendosi in questultimo caso porre un adeguata differenziazione tra detriti adesi e gettoni solidi [ 3 ]  . 
 inoltre chiaro che la sola presenza di flussi allanalisi doppler non sufficiente da sola per discriminare con certezza papilloma e carcinoma , anche se nel primo caso si osservano segnali scarsi , quando non assenti , mentre nel carcinoma la vascolarizzazione molto pi intensa e diffusa . 
 [ 18 ] il power doppler dimostrava una vascolarizzazione regolare , non anarchica , con singolo polo afferente , reperti tali da suggerire unipotesi di benignit , laddove poi lesame istologico avrebbe dimostrato un carcinoma papillare . nelle prime casistiche sui tumori intracistici lecografia presentava una limitata accuratezza nella diagnosi di malignit [ 10 ]  . 
sebbene la presenza di segnali vascolari in un gettone intracistico renda maggiore il sospetto di malignit ( quantunque anche le vegetazioni intracistiche benigne possono mostrare vasi interni , generalmente con spettro a basso indice resistivo ) , in realt tutte le cisti complesse andrebbero aspirate e sottoposte a valutazione citologica ; in caso di svuotamento incompleto dopo aspirazione poi spesso indicata una biopsia microistologica del residuo . 
bisogna anche ricordare come alcuni carcinomi , particolarmente voluminosi e / o a rapida crescita , possano essere massicciamente necrotici e simulare pertanto una cisti sporca o un ascesso : la dimostrazione di qualche segnale di flusso alleco - color doppler pu essere dirimente [ 1 , 21 ]  . gli elementi classici della diagnostica - differenziale del nodulo mammario sono poco applicabili , poich sia le lesioni non tumorali , che i papillomi che i carcinomi presentavano nella nostra casistica , in maniera costante o pressoch costante , la riconoscibilit di un rinforzo posteriore ( e lassenza di unattenuazione del fascio ) e la presenza di forma rotondeggiante - ovalare , di margini netti e regolari e di disposizione parallela rispetto al piano cutaneo . 
la natura invasiva del tumore intracistico pu essere sospettata soprattutto quando la base dei gettoni endocistici appare irregolare con strie ipoecogene penetranti nel parenchima adiacente , quando si osservano piccoli noduli solidi satelliti e , chiaramente , in presenza di localizzazioni linfonodali ascellari . 
a questo proposito , bisogna segnalare come la biopsia sia spesso limitata , 264 radiol med ( 2009 ) 114 : 253266 or almost constantly showed posterior enhancement ( and absent posterior shadowing ) , a rounded - oval shape , sharp regular margins and orientation parallel to the cutaneous plane . 
the invasive nature of an intracystic tumour may be suspected , particularly when the base of the intracystic projections appears irregular and with hypoechoic bands penetrating the adjacent parenchyma , when small satellite nodules are seen and , clearly , in the presence of axillary nodal metastases . 
on this subject , it should be noted that biopsy is often limited , as it is often performed on the largest portion of the intracystic solid component and not at the periphery where the invasive elements would be detected [ 5 , 13 ]  . 
in many cases , only evaluation of the entire surgical specimen enables a definitive distinction between benign and malignant papillary forms , as the foci of malignant degeneration are interspersed with benign papillary elements [ 18 ]  . high - resolution us , now also with harmonic imaging [ 22 ] and spatial compounding technology [ 20 ] , no doubt allows effective morphostructural assessment of breast cysts , enabling differentiation of the various types . 
the septa and solid projections of intracystic tumours show fairly large vessels at histological examination [ 14 ] , which explains the significant flow signals detected in the majority of lesions in our patients . 
if our experience is confirmed on larger series , diagnostic sampling of type iv and v cysts according to chang can be foregone and the lesion merely followed up whenever colour doppler us does not depict flow signals ( which is always the case in type iv cysts )  . 
in type vi cysts , however , where flow signals are constantly present , a pattern of diffuse hypervascularity on colour doppler us should constitute an indication for surgical excision or core biopsy ( also vacuum - assisted ) , even in the case of negative cytological examination . 
this finding does not , however , necessarily indicate malignancy , nor does a different appearance allow carcinoma to be ruled out : sonographic suspicion must lead to excision , even in the presence of negative cytological findings [ 5 , 11 ]  . 
aspiration of bloody fluid from a cyst dictates cytological evaluation of the aspirate [ 9 ] , which some authors reserve for cysts with haemorrhagic content only [ 2 ]  . 
further investigation may also be advisable for cysts , including those with simple appearance , which are not completely emptied with aspiration or which form again rapidly [ 5 ]  . 
in the case of complex cysts , however , cytological evaluation of the aspirate is considered to be poorly accurate : normally , it is recommended to perform a core biopsy , or vacuum - assisted biopsy , of the residual solid material , as only biopsy is considered capable of consistently excluding the risk of malignancy [ 3 , 4 , 6 ]  . poich viene condotta verso la parte pi ampia della quota solida endocistica e non verso la periferia ove si rileverebbe il carattere invasivo [ 5 , 13 ]  . 
in molti casi solo la valutazione dellintero pezzo operatorio asportato consente una distinzione finale tra forme papillari benigne e maligne , poich non di rado i foci degenerativi sono frammisti agli aspetti papillari benigni [ 18 ]  . lo studio ecografico ad alta risoluzione , anche grazie alle attuali possibilit tecnologiche derivanti dallimpiego dellarmonica tissutale [ 22 ] e dal compound spaziale [ 20 ] , consente sicuramente una valida valutazione morfo - strutturale delle cisti mammarie , distinguendone le diverse tipologie . 
i setti ed i gettoni solidi dei tumori intracistici presentano allesame istologico dei vasi di discreto calibro [ 14 ] , cosa che spiega i significativi segnali di flusso rilevati nella nostra casistica nella maggioranza delle lesioni . 
in base allesperienza personale , se confermata anche da casistiche pi ampie , si pu soprassedere dalla puntura diagnostica delle cisti tipo iv e v di chang et al . , limitandosi ad un controllo a distanza di tempo , se lo studio eco - color doppler non dimostra segnali di flusso ( cosa peraltro costante nelle cisti di tipo iv )  . 
nella tipologia vi secondo chang et al . , invece , ove peraltro i segnali di flusso sono risultati sempre presenti , un aspetto di ipervascolarizzazione diffusa allecocolor doppler deve porre unindicazione allescissione chirurgica oppure alla biopsia microistologica ( anche come vacuum - assisted ) , anche quando un eventuale esame citologico sia risultato negativo per cellule maligne . 
 il materiale aspirato dalle cisti complesse generalmente di aspetto ematico , pi o meno scuro , sebbene tale reperto non indichi necessariamente la natura maligna della cisti n , allopposto , un contenuto di aspetto differente permette di escludere il carcinoma : un sospetto ecografico deve condurre allescissione anche dinanzi ad un reperto citologico negativo [ 5 , 11 ]  . 
dinanzi allaspirazione di materiale ematico da una cisti si impone la valutazione citologica dello stesso [ 9 ] , da alcuni limitata proprio alle cisti che dimostrano un contenuto emorragico [ 2 ]  . 
una valutazione pi approfondita pu essere poi consigliabile nel caso di cisti , anche di aspetto semplice , che dopo una cisticentesi non si vuotano completamente o che si riformano rapidamente [ 5 ]  . nel caso delle cisti complesse la citologia su liquido aspirato considerata tuttavia poco accurata : generalmente viene invece consigliata la puntura delle quote solide residue dal prelievo del materiale liquido , con biopsia microistologica o anche con biopsia vacuum - assisted , poich si ritiene che solo la biopsia infatti ritenuta in grado di escludere consistentemente il rischio di malignit [ 3 , 4 , 6 ]  . 
riteniamo radiol med ( 2009 ) 114 : 253266 nonetheless , we prefer to carry out extemporaneous cytological analysis of the material obtained from the solid component and proceed to core biopsy only in doubtful cases . 
indeed , we believe that aspiration of the fluid component for cytological analysis causes decompression of the cyst and possibly extracystic leakage of the fluid component , which may make it more difficult to identify and sample the solid component [ 3 ]  . 
we thus prefer to sample the solid component directly with needle aspiration and only later to sample the fluid portion : if extemporaneous cytological analysis of the solid portion shows no signs of atypia or malignancy , we proceed with core biopsy of the remaining solid portion . 
because needle aspiration of the solid components has generally proved to be sufficient for the diagnosis in our experience , we prefer not to perform a core biopsy first , in contrast to what has been suggested in the literature [ 3 ]  . infatti che , se si esegue in prima istanza laspirazione della quota liquida ai fini dellanalisi citologica , si determina anche una decompressione della formazione cistica , con eventuale diffusione extracistica di liquido , e quindi vi possono essere maggiori difficolt ad identificare ed a campionare adeguatamente le porzioni solide [ 3 ]  . 
noi preferiamo quindi prelevare direttamente , mediante ago - aspirazione , la quota solida e solo in un secondo momento prelevare la parte liquida : qualora lanalisi citologica estemporanea della parte solida non dimostri segni di atipia o malignit , si proceder alla biopsia microistologica della parte solida residuata dalla cisticentesi . 
poich nellesperienza personale lago - aspirato dei gettoni solidi si generalmente dimostrato sufficiente per la diagnosi , preferiamo non eseguire la biopsia microistologica in prima istanza , a differenza di quanto consigliato in letteratura [ 3 ]  . conclusions intracystic breast tumours show distinctive echostructural and colour doppler us features that allow effective differentiation , not only from nontumoural complex cysts but also in many cases between intracystic papillomas and carcinomas . 
the main signs of malignancy detected in our series were elevated solid - to - cystic ratio , multiple solid projections , intralesional calcifications , intense vascularity of the papillary projections , multiple vascular poles and high resistive index of the intralesional vessels . 
the presence of neovascularity represents an indication for surgical excision of the lesion or biopsy of the solid projections , even in the case of negative cytological analysis . conclusioni i tumori mammari intracistici presentano aspetti eco - strutturali ed eco - color doppler peculiari , tali da consentire unefficace differenziazione non solo rispetto alle cisti complesse non tumorali , ma anche , in molti casi , tra papillomi e carcinomi intracistici . 
i maggiori indicatori di malignit sono risultati : elevato rapporto tra quota solida e cistica , multipli gettoni solidi , calcificazioni intralesionali , intensa vascolarizzazione delle proiezioni papillari , multipli poli vascolari , elevate resistenze al flusso in corrispondenza dei vasi intralesionali . 
sartor springer - verlag , berlin heidelberg , 2008 isbn 978 - 3 - 540 - 33004 - 2 this second edition of pediatric uroradiology , edited by fotter of gratz university with the help of a distinguished team , updates all 29 chapters for technical and theoretical changes in paediatric uroradiology that have occurred over the last few years . 
for instance , what was considered dogma in the management of urinary tract infections and vesicoureteral reflux is amply discussed and debated after presenting new sometimes contradictory knowledge and different opinions by leaders in the field . due to these new clinical findings , it will be necessary to re - evaluate procedures and conduct and implement the suggestions and recommendations found throughout the book . in particular , careful attention should be given to the chapter clinical management of common nephrourologic disorders ( guidelines and beyond )  . 
new insights are provided on how to manage prenatally diagnosed congenital uropathies , how to deal with the case of renal hypertension , how to use nuclear medicine and be spering in voiding cystourethrography , how to study renal tumours and how to say goodbye to intravenous urography . 
as in the first edition , emphasis is placed on voiding dysfunction due to either nonneurogenic or neurogenic bladder sphincter dysfunction . the number of pages has been increased from 431 in the first to 533 in the second edition and the number of chapters from 26 to 29 . 
fotter delluniversit di graz che ha coordinato un gruppo di esperti nella materia , porta alla luce nei suoi 29 capitoli tutti i cambiamenti , non solo tecnici , ma soprattutto teorici , avvenuti nel campo delluroradiologia pediatrica nel corso degli ultimi anni . 
quanto ritenuto per esempio un dogma nel trattamento delle infezioni delle vie urinarie e del reflusso vescico - ureterale viene ampiamente dibattuto alla luce delle recenti e talvolta contraddittorie acquisizioni , esponendo differenti opinioni e effettuando una discussione in merito , da parte di importanti cultori della materia . 
a causa di queste recenti acquisizioni dal punto di vista clinico sar necessario prendere in considerazione la rivalutazione di condotte e procedure ed adeguarsi ai suggerimenti presenti nel volume , soprattutto meditando con attenzione quanto scritto nel capitolo dal titolo : clinical management of common nephrourologic disorders ( guidelines and beyond )  . 
nuove indicazioni e suggerimenti vengono dati su come comportarsi in caso di uropatie riscontrate in utero ; come comportarsi in caso di ipertensione ; come utilizzare la medicina nucleare ed essere avari nellesecuzione di cistouretrografie ; come studiare i tumori renali ecc . 
viene data importanza , come nel volume della prima edizione alle disfunzioni della minzione sia non - neurogene che da disfunzioni neurogene dello sfintere vescicale . le pagine sono aumentate da 431 a 533 e con esse il numero delle figure , delle immagini e delle tabelle ed il numero dei capitoli da 26 a 29 . 
in alcuni capitoli mr urography oppure urinary tract trauma sono state effettuate modificazioni sostanziali mentre in altri solo delle aggiunte ( come langioplastica renale percutanea nel capitolo riguardante pediatric genitourinary intervention )  . 
 according to the new springer style , in this series , all boxes and relevant information sections of the book are highlighted in two shades of blue , which immediately catches the readers attention and makes them easier to understand . 
i do , however , regret the absence of contributions from italian colleagues , which were included in the first edition . in summary , this is an important and useful text . 
i believe this edition will become and important reference text for paediatric and adult radiologists , general paediatric practitioners , paediatric surgeons , urologists and paediatric nephrologists and will hopefully reach the largest possible circulation . apportata una modifica nella scaletta dei capitoli , ora pi consequenziali che non nella precedente edizione . secondo la nuova impostazione grafica della casa editrice , tutte le tabelle ed i quadri riassuntivi sono ben evidenziati in blu e blu pallido , cosa che richiama subito lattenzione rendendole pi facili da usare e memorizzare . 
purtroppo non si ha pi traccia dei contributi dei colleghi italiani presenti nella prima edizione . in conclusione , questo un libro di grande importanza ed utilit : solleva anche problematiche da risolvere , si spera , nel futuro con ulteriori studi dellargomento nellottica della miglior cura dei bambini . 
between 1 january 2006 and 30 june 2007 , 23 consecutive patients ( 21 men and 2 women ) presenting with electrocardiographic abnormalities mimicking acute myocardial infarction and a clinical suspicion of acute myocarditis ( fever , chest pain and elevated troponin levels ) underwent contrast - enhanced cardiac mri within a week of admission . 
dal 1 gennaio 2006 al 30 giugno 2007 , ventitre pazienti consecutivi ricoverati con alterazioni elettrocardiografiche di tipo simil - infartuale ( stemi ) e sospetto clinico di miocardite acuta ( febbre , dolore toracico , positivit dei valori di troponina ) sono stati sottoposti , entro 6 giorni dallesordio , a risonanza magnetica ( rm ) cardiaca con mdc . 
la rm cardiaca pu avere ruolo fondamentale nella diagnosi precoce di miocardite acuta , documentando un quadro di alterazioni di segnale del miocardio che esclude la genesi ischemica . 230 radiol med ( 2009 ) 114 : 229238 keywords magnetic resonance imaging myocarditis myocardial infarction parole chiave risonanza magnetica miocardite infarto del miocardio introduction introduzione myocarditis is an inflammation of the myocardium associated with myocyte necrosis and is usually of viral origin [ 1 , 2 ]  . 
persistence of viraemia associated with an autoimmune response can cause chronic inflammation that may progress to dilated cardiomyopathy [ 4 ]  . because noninvasive imaging modalities are unable to provide a definitive diagnosis , invasive procedures are used , such as endomyocardial biopsy , which is considered the reference standard for the diagnosis . 
in addition , there is some variability in the interpretation of specimens , and the standard histological criteria for inflammation and myocytolysis tend to underestimate the true incidence of myocarditis , requiring integration with immunohistochemical analysis [ 5 ]  . 
endomyocardial biopsy is indicated in patients with rapidly progressive cardiomyopathy not responding to conventional treatment or in cases of cardiomyopathy associated with severe arrhythmias . there is thus a need for imaging modalities able to assist in the early diagnosis and follow - up of myocarditis [ 6 ]  . magnetic resonance imaging ( mri ) has been shown to be effective not only for the diagnosis [ 7 ] , where evidence of late gadolinium enhancement reflects irreversible myocardial damage , but also for quantifying disease extent and thus allowing lesion monitoring [ 8 , 9 ]  . 
these findings have also been validated by histopathology , as reported in the literature [ 8 ]  . the late enhancement obtained with mri permits visualisation of small myocardial lesions that in patients with myocarditis tend to be located in the subepicardial or intramyocardial regions with sparing of the subendocardium , conversely to what occurs in ischemic necrosis where the damage is subendocardial or transmural [ 9 , 10 ]  . the aims of our study were to confirm the diagnostic reliability of mri in acute myocarditis in patients with infarction - like clinical findings and to evaluate the possibility of la miocardite una infiammazione del miocardio associata a necrosi , nella maggior parte dei casi di origine virale [ 1 , 2 ]  . 
il persistere della viremia associato alla risposta auto - immunitaria pu causare infiammazioni croniche che possono evolvere in cardiomiopatia dilatativa [ 4 ]  . allo stato attuale le metodiche non invasive non permettono di ottenere la diagnosi definitiva di miocardite cosicch necessario il ricorso a procedure diagnostiche invasive , come la biopsia endomiocardica , considerata la metodica di riferimento nella diagnosi di miocardite . 
in primo luogo la sensibilit della metodica strettamente correlata alla qualit dei frustoli ed inoltre si possono avere falsi - negativi quando il prelievo sia stato effettuato in unarea non coinvolta dal processo infiammatorio ; inoltre esiste variabilit nella interpretazione dei campioni ed i semplici criteri istologici di infiammazione e miocitolisi spesso sottostimano la reale incidenza di miocardite tant che debbono essere integrati con una analisi immunoistochimica [ 5 ]  . 
in secondo luogo la biopsia endomiocardica una procedura che comporta dei rischi per cui opinabile lutilizzo di questa metodica , specie nei casi in cui la patologia ha andamento benigno e regredisce spontaneamente in breve tempo . 
 stato dimostrato che la risonanza magnetica pu essere di aiuto non solo per la diagnosi [ 7 ] , identificando il late gadolinium enhacement ( le ) espressione di danno miocardico irreversibile ma in grado anche di quantificare lestensione della patologia , consentendone il monitoraggio [ 8 , 9 ]  . 
 il le ottenuto con rm permette di visualizzare piccoli danni miocardici che , in pazienti affetti da miocardite , sono localizzati usualmente nella porzione subepicardica o intramiocardica senza interessamento del subendocardio contrariamente a quanto avviene nella necrosi a genesi ischemica , radiol med ( 2009 ) 114 : 229238 monitoring disease evolution during the follow - up by correlating mri findings with cardiac function . materials and methods patient inclusion criteria patient inclusion criteria in our study were the following : 1 . 
absence of significant coronary artery stenosis on coronary angiography . twenty - three consecutive patients meeting the above criteria were enrolled in the study between 1 january 2006 and 30 june 2007 . 
two patients were excluded due to claustrophobia . magnetic resonance imaging mri examinations were performed on a superconductive 1.5 - t magnet ( intera , philips medical systems , best , the netherlands ) within 6 days of patient admission to hospital . 
all patients were imaged during a breath - hold . localisation was achieved with electrocardiograph ( ecg ) gated balanced fast - field echo ( ffe ) steady - state freeprecession sequences in planes corresponding to the cardiac axes . 
ecg - gated t1and t2 - weighted black - blood sequences were acquired in the short axis ( base , mid ventricle and apex ) , with tr dependent on the trigger ( t1 with tr 1 rr , te 25 ms , flip angle 90 , t2 with tr 2 rr , te 80 ms , flip angle 90 )  . 
due pazienti tuttavia sono stati esclusi in quanto non hanno potuto espletare lesame di rm per manifesta claustrofobia . risonanza magnetica gli esami di rm sono stati acquisiti utilizzando un magnete superconduttivo da 1.5 t ( intera , philips medical systems , best , olanda ) entro 6 giorni dallingresso in ospedale . 
le sequenze t1 e t2 pesate black blood sono state acquisite su asse corto ( basale , medio - ventricolare e apicale ) , cardiosincronizzate con tr dipendente dal trigger ( t1 con tr 1 rr , te 25 ms , flip angle 90 ; t2 con tr 2 rr , te 80 ms , flip angle 90 )  . 
 le immagini post - contrastografiche t1 pesate sono state acquisite con un ritardo di 1015 min dopo la 232 radiol med ( 2009 ) 114 : 229238 short - axis and twoand four - chamber long - axis views . 
areas of late enhancement were defined as those exhibiting a signal intensity 2 standard deviations ( sd ) above the mean signal intensity of the remote myocardiuthe portion of the heart exhibiting late enhancement was classified according to the segmentation established by the american heart association , which divides the left ventricle into 17 segments [ 11 ]  . 
standard coronary angiography using a philips integris allura unit was performed within 3 h of patient admission to exclude the presence of coronary vascular abnormalities . results the number of patients included in the study was 21 ( 20 men and one woman ; mean age 30 years ) ( table 1 )  . 
systolic function was preserved in most patients ( ef > 50% ) , with a mild dysfunction being encountered three patients only ( ef 45%48% ) ( table 1 )  . 
only mild segmental wall motion abnormalities were encountered , with no cases of akinesia . t2 - weighted sequences demonstrated increased signal intensity corresponding to the areas of late enhancement , a possible expression of oedema . 
la risoluzione spaziale stata sempre di 1 , 21 , 8 mla dose di contrasto utilizzata ( gadoteridol , magnevist , shering ag , berlin , germania ) stata di 0 , 1 mmol / kg . lestensione dellenhancement post - contrastografico stata misurata sulle immagini ad asse corto ed asse lungo due camere e quattro camere . 
sono state considerate come aree di le solo quelle con un livello di intensit 2 sd ( deviazioni standard ) rispetto alla media delle intensit di segnale di tutte le pareti del miocardio . 
la porzione di cuore interessata dal le stata classificata in accordo con la segmentazione dellamerican heart association , che suddivide il ventricolo sinistro in 17 segmenti [ 11 ]  . 
la coronarografia convenzionale stata effettuata con tecnica standard per escludere la presenza di alterazioni vascolari coronariche con apparecchio philips integris allura nellintervallo di tempo massimo di 3 ore dal momento dellarrivo del paziente nel nostro ospedale . risultati i pazienti inclusi nello studio sono stati 21 di cui 20 uomini e 1 donna . 
in questultimo paziente il test virologico ha rilevato una infezione da coxsackievirus . dal punto di vista clinico nel follow - up nessun paziente ha riportato eventi aritmici e / o ulteriori complicanze cardiologiche ; la frazione di eiezione risultata stabile o migliorata in tutti i pazienti . 
nel follow - up con le sequenze t2 non stata riscontrata presenza di edema ; anche paziente , che aveva presentato al controllo una nuova area di le , non si sono riscontrate aree edemigene . discussione il nostro studio , in accordo con quanto riportato in letteratura , conferma laffidabilit della rm nella diagnosi del coinvolgimento miocardico , definendo estensione e localizzazione del le durante la fase acuta di una miocardite attiva [ 13 , 14 ] , ed in grado di valutare la progressione o regressione dellaffezione cardiaca nel follow - up [ 9 ]  . 
inoltre non stata ancora chiarita la correlazione tra i pattern miocardici della rm e la riduzione della funzione sistolica . nel nostro studio stata analizzata la localizzazione del le e la sua correlazione con la severit della disfunzione miocardica in 21 pazienti con quadro clinico simil - infartuale , affetti da miocardite acuta , con arterie coronarie indenni . 
2 distribuzione delle aree di late enhancement della popolazione studiata in accordo con la segmentazione dellamerican heart association . discussion in agreement with previous reports , our study confirms the effectiveness of mri in diagnosing myocardial damage , as it is able to delineate the extent and location of late enhancement during the acute phase of active myocarditis [ 13 , 14 ] and evaluate disease progression or regression during the follow - up [ 9 ]  . 
nonetheless , mri is not regarded as the reference standard for diagnosing myocarditis , nor has the correlation between mri myocardial patterns and reduced systolic function been clarified to date . radiol med ( 2009 ) 114 : 229238 fig . 
piccola area di late enhancement intramiocardico in sede laterale medio - distale . our study analysed the location of the late enhancement and its correlation with myocardial dysfunction severity in 21 patients affected by acute myocarditis mimicking infarction and with healthy coronary arteries . 
we observed a peculiar distribution of the myocardial lesions , which involved the epicardial and / or intramyocardial region of the left ventricular wall , with prevalent involvement of the inferiore rispetto alla parete laterale . 
la distribuzione delle lesioni miocardiche da noi riscontrata , con il frequente coinvolgimento infiammatorio della parete laterale , ampiamente confermata da quanto riportato in letteratura , che inoltre , nei pi recenti contributi [ 15 ] ipotizza persino una stretta correlazione fra agente patogeno e sede della lesione miocardica [ 16 , 17 ]  . 
6a , b inversion - recovery gradient recalled echo ( ir - gre ) long - axis four - chamber view showing myocardial damage in the intermediate and distal segments of the lateral wall during acute myocarditis ( a )  . 
6a , b sequenze ir - gre post - contrastografiche : acquisizione in asse lungo 4 camere allesordio con aree di late enhancement localizzate a livello della parete laterale nei segmenti medi e distali ( a )  . 
the pattern of myocardial damage seen in our study with frequent involvement of the lateral wall has been widely reported in the literature , and recent contributions [ 15 ] have even suggested the possibility of a close relationship between the nature of the pathogen and the site of myocardial damage [ 16 , 17 ]  . durante il follow - up le aree di le si sono progressivamente ridotte nel tempo ad eccezione di un unico caso in cui sono comparse nuove aree di le . 
in tutti i pazienti non si mai rilevata impregnazione post - contrastografica tardiva ( le ) a partenza dal subendocardio , reperto tipico e , quadro rm ormai consolidato , di coinvolgimento miocardico su base ischemica [ 5 , 18 , 19 ]  . il meccanismo responsabile dellimpregnazione postcontrastografica tardiva non stato ancora completamente radiol med ( 2009 ) 114 : 229238 during the follow - up , the areas of late enhancement decreased progressively , with the exception of one patient who developed new areas of late enhancement . 
none of the patients exhibited late enhancement originating in the subendocardial portion , a typical and consolidated mri pattern of ischaemic myocardial damage [ 5 , 18 , 19 ]  . the mechanism responsible for late enhancement has not been elucidated , and various pathophysiological hypotheses have been put forward . 
during healing of the inflammatory process , contrast enhancement is progressively reduced , and in some cases , only a microscar can be identified , which reflects fibrous replacement of the necrotic myocytes . during the follow - up , in some of our patients , the late enhancement albeit less intense persisted in the same area for more than 4 months . 
this finding confirms the need to further investigate the long - term prognostic significance of late enhancement . finally , mri can provide information about the precise location of the myocardial damage induced by myocarditis and may serve as a guide for the execution of endomyocardial biopsy [ 20 ]  . left ventricular ef and the extent of late enhancement were not found to be closely correlated . 
a further explanation that may account for normal ef lies in the fact that the damage identified by contrast enhancement is overestimated during the acute phase , in part because not all the cells contained in the area of enhancement are totally damaged , as opposed to what happens in myocardial infarction . chiarito ed in merito sono state formulate varie ipotesi fisiopatologiche . 
fra queste quella che gode di maggior credito quella che identifica nella rottura della membrana dei miociti la causa della diffusione del mezzo di contrasto non solo nello spazio extracellulare ma anche in quello intracellulare [ 10 ]  . 
questa ipotesi supportata dai riscontri isto - patologici che identificano nelle fasi precoci della miocardite la presenza di edema interstiziale con infiltrato linfocitario e necrosi dei miociti ; il successivo danno cellulare si instaura nelle ore e / o giorni successivi al momento iniziale dellinfezione . 
peraltro il miocardio in condizioni di normalit non presenta impregnazione post - contrastografica perch i miociti , con membrane sarcolemmatiche integre , non permettono la diffusione del mezzo di contrasto al loro interno e quindi risulta impregnarsi solo lo spazio extra - cellulare , peraltro esiguo . 
quando il processo infiammatorio in fase di guarigione limpregnazione post - contrastografica si riduce progressivamente ed in alcuni casi pu essere identificata solamente una micro - cicatrice , espressione di sostituzione fibrotica delle aree di necrosi dei miociti . 
 nel follow - up della nostra serie si riscontrata , in alcuni casi , persistenza del le nella stessa sede , seppur ridotta in intensit , anche dopo 4 mesi . 
tale osservazione conferma quanto finora riportato in letteratura vale a dire che il significato del le , come valore prognostico clinico a lungo termine , deve essere ancora validato . infine da ricordare che la rm pu fornire informazioni sulla esatta localizzazione del danno miocardico causato dalla miocardite e pu pertanto essere utilizzata quale eventuale guida per la biopsia endomiocardica [ 20 ]  . la frazione di eiezione ventricolare sinistra ( lvef ) e lestensione del le non sono risultate strettamente correlate . 
una ulteriore spiegazione , a giustificazione del normale lvef , risiede nel fatto che il danno identificato dallimpregnazione post - contrastografica sovrastimato nella fase acuta , anche perch non tutte le cellule ubicate nellarea di enhancement sono totalmente danneggiate , contrariamente a ci che avviene nei casi di infarto del miocardio . 238 conclusions conclusioni radiol med ( 2009 ) 114 : 229238 late contrast enhancement at mri is a constant finding in the diagnosis of myocarditis and is directly correlated to myocardial damage . 
this late enhancement manifests with a characteristic pattern in that it originates from the epicardial and / or intramyocardial surface , and this distinguishes it from ischaemic necrosis that shows subendocardial and / or transmural involvement . 
finally , mri proved to be very useful in evaluating and monitoring the progression of inflammatory damage over time . limpregnazione post - contrastografica tardiva ( le ) ottenuta con rm un reperto costante nella diagnosi delle miocarditi ed direttamente correlata al danno miocardico . il pattern con cui si manifesta caratteristico originando dalla superficie epicardica e / o intramiocardica , il che lo distingue dalla necrosi di origine ischemica che ha una interessamento subendocardico e / o transmurale . 
trentatre pazienti ( 22 maschi , 11 femmine , di et media di 62 , 4 ) sono stati sottoposti a risonanza magnetica ( rm ) utilizzando un magnete da 1 , 5 tesla ( general electric , hd )  . 
a riposo sono state impiegate sequenze rm cine di tipo steady - state con imaging parallelo in 4 - camere , per il tratto di efflusso del ventricolo sinistro ed in asse corto . 
dobutamine stress cardiac mri is an accurate method for assessing myocardial ischaemia in patients with cad , and it could be useful as a noninvasive tool for excluding the disease . 
the increase in signal intensity and acquisition speed obtained by using steadystate free precession with parallel imaging proved useful in increasing test specificity with respect to previous similar studies . keywords magnetic resonance imaging dobutamine stress testing heart ischemia analizzato in totale 960 segmenti ed abbiamo osservato comparsa o peggioramento della cinesi in sette pazienti ed in 29 differenti segmenti . 
lincremento dellintensit del segnale e della velocit di acquisizione ottenuta utilizzando le sequenze steady - state free precession con imaging parallelo risultata utile nellincrementare la specificit del test rispetto a precedenti simili studi . parole chiave risonanza magnetica test da sforzo con dobutamina cuore ischemia introduction introduzione previous studies have demonstrated that cardiac magnetic resonance imaging ( mri ) with dobutamine stress has a clinically useful accuracy for detecting myocardial ischemia [ 16 ]  . 
 [ 6 ] demonstrated that stress cardiac mri provided better sensitivity ( 86% vs 74% ) and specificity ( 86% vs 70% ) than stress echocardiography for detecting new regional kinetic alterations in 172 patients compared with coronary angiography . 
 the fast - gradient - echo technique has been used to evaluate a group of patients referred for diagnosis of ischaemia . the technique provided useful data for diagnosing inducible ischaemia in patients unable to undergo stress echocardiography [ 6 , 7 ]  . 
acquisition time has been reduced to 67 s or less for each slice , with 30 phases per cardiac cycle , thus improving regional kinetic analysis and ventricular exploration during pharmacological stress studies [ 8 ]  . the purpose of this study was to assess the clinical utility of dobutamine stress mri in demonstrating inducible ischaemia in patients with atypical chest pain or equivocal studi precedenti hanno dimostrato che la risonanza magnetica cardiaca ( rmc ) da stress con dobutamina possiede unaccuratezza clinica utile alla rilevazione di ischemia miocardica [ 16 ]  . 
nonostante ci lecocardiografia ( eco ) da stress resta nella pratica clinica il metodo preferito per lanalisi della funzione regionale ventricolare in virt dellelevata accuratezza , versatilit , disponibilit e del costo relativamente contenuto . 
 [ 6 ] hanno dimostrato che la risonanza magnetica cardiaca da stress forniva una migliore sensibilit ( 86% vs 74% ) e specificit ( 86% vs 70% ) rispetto alleco - stress per la rilevazione di nuove alterazioni cinetiche regionali in 172 pazienti confrontati con angiografia coronarica . 
inoltre , la superiorit della rmc rispetto allecografia risultava pi evidente in pazienti che presentavano una qualit ecografica moderata . utilizzando la tecnica fast gradient echo , sono stati analizzati un gruppo di pazienti con sospetto di malattia ischemica ; questa tecnica ha fornito dati utili per la diagnosi di ischemia inducibile in pazienti non sottoponibili a eco - stress [ 6 , 7 ]  . 
il tempo di acquisizione stato ridotto a 67 secondi o meno per ogni sezione con 30 fasi acquisite nel singolo ciclo cardiaco , migliorando cos lanalisi cinetica regionale e 218 radiol med ( 2009 ) 114 : 216228 stress electrocardiography ( ecg ) not suitable for stress echocardiography using steady - state parallel imaging sequences . materials and methods the study population consisted of 33 patients ( 22 men and 11 women ; age range 5268 years , mean age 62.4 years ) with a suspicion of ischaemia due to atypical chest pain or equivocal stress ecg not suitable for stress echocardiography because of inadequate endocardial visualization despite the use of second harmonic imaging at the evaluation at rest . 
patients were excluded if they had known general contraindications to mri or dobutamine , a recent history of myocardial infarction , significant q waves and prior coronary surgery ( table 1 )  . 
 mri technique mri was performed using a 1.5 - tesla superconducting whole - body system ( general electric medical system , milwaukee , wi , usa ) equipped with 33 mt / m gradients ( slew rate 120 mt / m / s )  . 
in - plane resolution was 1.4 mm , with a slice thickness of 8 mm . first - pass perfusion was performed using t1 - weighted saturation recovery ( sr ) sequences repeated for 2530 s for each r - r or 2r - r interval , depending on heart rate , to obtain at least three slices for each acquisition at the apical , papillary and basal level . 
 study protocol intravenous access was gained through the right antecubital ve mri was performed with the patient in the supine position on the scanning table with an eight - channel phasedarray surface coil . 
a three - channel ecg monitor was lesplorazione del ventricolo durante lo stress farmacologico [ 8 ]  . lo scopo del presente studio stato di stabilire lutilit clinica della rmc da stress con dobutamina nella diagnosi di ischemia inducibile mediante lutilizzo di sequenze ssfp e di tecniche di imaging parallelo , in pazienti con dolore toracico atipico o ecg da sforzo dubbio non sottoponibili a eco - stress . materiali e metodi abbiamo studiato 33 pazienti ( 22 uomini e 11 donne di et compresa da 52 a 68 anni , et media 62 , 4 anni ) con il sospetto di ischemia cardiaca per la presenza dolore toracico atipico o ecg da sforzo dubbio , non idonei ad essere sottoposti a eco - stress a causa di inadeguata visualizzazione dei contorni endocardiaci , nonostante limpiego della seconda armonica , gi nella valutazione a riposo . lecocardiografia stata eseguita con un apparecchio recente ( philips 5000 ) da due diversi operatori con 9 e 7 anni di esperienza . 
i pazienti non erano arruolabili se presentavano controindicazioni generiche alla rm o alla dobutamina o se affetti da infarto miocardio di recente insorgenza , onde q significative e se precedentemente sottoposti a chirurgia coronaria ( tabella 1 )  . 
a tutti i partecipanti allo studio stato richiesto di sospendere lassunzione di - bloccanti e farmaci antiangina , rispettivamente 72 e 24 ore prima dellanalisi rmc . tecnica rm per limaging a risonanza magnetica stato usato uno scanner whole - body con magnete superconduttivo a 1 , 5 tesla ( general electric medical system , milwaukee usa ) con gradienti da 33 mt / m e tempo di risalita da 120 mt / m / s . 
 stato collegato un monitor ecg 3 canali e contemporaneamente misurati saturazione di o2 e pressione sanguigna brachiale . a riposo , abbiamo eseguito una valutazione funzionale standard che includeva una acquisizione in asse lungo verticale , acquisizioni multiple in asse corto al livello del ventricolo sinistro dallapice alla base senza alcun intervallo tra le sequenze ( 89 sequenze ) e acquisizione in 4 camere ( 46 sequenze senza intervallo ) e 34 sequenze nel tratto di deflusso del ventricolo sinistro ( lvot )  . 
lanalisi della perfusione al primo passaggio stata ottenuta in asse corto dopo iniezione di 0 , 1 m / kg di gadolinio alla velocit di infusione di 5 ml / s . 
lo stress test stato eseguito tramite lacquisizione di 6 sezioni ssfp dalla base allapice del ventricolo sinistro con un intervallo tra le sezioni di 46 msono state acquisite fino a 2 sequenze durante una singola apnea per una durata massima di 1315 secondi . 
le immagini sono state acquisite dopo 3 minuti dalliniezione di dosi crescenti di dobutamina endovena pari a 510203040 g / kg / min fino a raggiungere l85% della frequenza cardiaca massimale teorica calcolata dalla formula ( 220 et ) 0 , 85 per gli uomini e ( 200 et ) 0 , 85 per le donne . 
 stata monitorata costantemente la frequenza ed il ritmo cardiaco , ogni due minuti la pressione sistemica , sulle immagini acquisite la funzione regionale ; laumento della pressione sistolica oltre i 40 mmhg , la presenza di frequente extrasistolia o la comparsa di nuove alterazioni cinetiche parietali sono state considerate motivo di sospensione del test . analisi delle immagini la cinesi regionale stata analizzata da 2 osservatori non a conoscenza dei risultati degli altri esami . 
first - pass perfusion analysis was obtained on short - axis views after injection of 0.1 m / kg of gadolinium at a flow rate of 5 ml / s . 
stress evaluation was performed using six ssfp acquisitions obtained from the left - ventricle base to apex with an interslice gap of 46 mm . up to two acquisitions were acquired during a single breathhold lasting 1315 s . 
images were acquired starting 3 min after the intravenous injection of 5 , 10 , 20 , 30 or 40 g / kg / min dobutamine to reach 85% of the theoretical maximal heart rate predicted by the following formula : ( 220age ) 0.85 for men and ( 200age ) 0.85 for women [ 1 ]  . 
the test was discontinued in the event of an increase of systolic pressure > 40 mmhg or a drop in blood pressure , frequent arrhythmia or the appearance of new wall motion abnormalities . 
each of the six slices was divided into five segments : anteroseptal , anterior , lateral , posterior and posteroseptal from the basal level ( i ) to the apex ( vi )  . 
the test was considered positive for ischaemia if the score increased by at least 1 point during the infusion or indicative of viability if a hypokinetic segment became normal at low dobutamine dose and worsened at high dose . cases of disagreement were resolved by reviewing the images and reaching a consensus . 
coronary angiography was considered the standard of reference and was performed in 22 out of 32 patients included in the study within 3 months from the mri examination in the absence of cardiac events . at least two orthogonal planes were obtained , and standard analysis was used for each vessel . 
il test stato considerato positivo per ischemia se vi stato un aumento del punteggio di almeno un livello durante linfusione o indicativo di vitalit se un segmento diventava normale a basso dosaggio di dobutamine per poi peggiorare di nuovo allaumentare del dosaggio del farmaco . 
in caso di discordanza tra i due osservatori stato effettuato una rivalutazione per ottenere laccordo . lapporto arterioso coronarico stato definito secondo la nomenclatura e segmentazione della american heart association ( aha ) [ 9 ]  . 
la gravit della stenosi , durante lesame coronarografico , stata considerata significativa se superiore al 50% in coronarie 2 mm . analisi statistica tutti i parametri continui sono stati espressi come mediadeviazione standard ; stato considerato significativo un valore di p < 0 , 05 . 
sensibilit e specificit sono stati calcolati per stenosi superiori al 50% . risultati una paziente presentava a riposo grave ipertensione ed stata esclusa ; pertanto stata effettuata analisi cinetica e perfusionale a riposo e stress con dipiridamolo e non risultato evidente di difetto di perfusione o disfunzione cinetica dovuta a ischemia inducibile . 
i restanti presentavano cinesi normale in tutti i segmenti del ventricolo sinistro . tutti i pazienti sono stati in grado di raggiungere l85% della frequenza cardiaca massima teorica prevista dallet . per raggiungere tale valore , stata necessaria in 6 pazienti liniezione di atropina alla fine della somministrazione incrementale di dobutamina . 
in 4 pazienti stato sufficiente 1 mg di atropina , nei restanti due si dovuto ricorrere ad un dosaggio incrementale di rispettivamente 1 , 5 mg e 2 mg . radiol med ( 2009 ) 114 : 216228 rest 5 microg / kg / min 10 microg / kg / min 20 microg / kg / min 30 microg / kg / min 40 microg / kg / min fig . 
all patients were able to reach 85% of the theoretical maximum heart rate la frequenza cardiaca basale stata di 6918 battiti al minuto ( 5081 ) , la frequenza cardiaca massima di 145 battiti al minuto ( media 13411 )  . 
la pressione sanguigna di base stata tra 150 / 95 e 110 / 70 mmhg , con picco di pressionedurante il test tra 185 / 110 e 160 / 93 mmhg . 
 durante linfusione di dobutamina 12 pazienti hanno mostrato segni di ansia , ma solo in 4 casi questa stata responsabile di una cooperazione incompleta nellapnea e di conseguenza ha prodotto artefatti nelle immagini . 
atropine injection after incremental dobutamine administration was necessary in six patients to achieve at least 85% of the theoretical maximum heart rate . a dose of 1 mg of atropine was sufficient in four patients , whereas incremental doses of 1.5 and 2 mg were necessary in the remaining two patients . 
baseline blood pressure was between 150 / 95 mmhg and 110 / 70 mmhg , and peak blood pressure during the test was between 185 / 110 mmhg and 160 / 93 mmhg . during dobutamine infusion , 12 patients developed anxiety , but only in four of them did this result in incomplete cooperation in the breath - hold sequences , with consequent image artefacts . 
i dieci pazienti che non hanno eseguito la coronarografia e che non presentavano evidenza di ischemia inducibile alla rm da stress ( tre pazienti hanno rifiutato lesame coronarografico mentre nei restanti 7 casi , sulla scorta dei dati clinici e strumentali ottenuti abbiamo ritenuto inadeguato ed eccessivo il rischio di unangiografia coronarica ) sono stati ricontattati 912 mesi dopo ( media 10 , 2 mesi )  . 
parziale recupero funzionale stato osservato alla bassa dose di dobutamina nel segmento anterosettale ( c ) con peggioramento della cinesi alle alte dosi di dobutamina . examination and coronary angiography was between 1 week and 3 months ( mean 26 days )  . 
ten patients , who had no evidence of inducible ischaemia at mri , did not undergo coronary angiography ( three patients due to refusal and seven because review of the clinical and instrumental data indicated high risk )  . 
the reasons are sono presenti ad un esame basale cos come durante i test sotto sforzo e sono frequentemente correlate ad enfisema polmonare e conseguente bassa velocit di propagazione degli ultrasuoni . 
in definitiva , la penetrabilit acustica resta un aspetto determinante per la qualit dellimmagine [ 13 ]  . tutti i pazienti inclusi nel nostro studio presentavano una riduzione evidente della qualit ecocardiografica , responsabile di delineazione difficoltosa dei bordi endocardici . lapplicazione di imaging con seconda armonica non stata in grado di superare le difficolt nellanalisi cinetica . per questi pazienti leco - stress con dobutamina sarebbe rest 5 microg / kg / min 10 microg / kg / min radiol med ( 2009 ) 114 : 216228 69 hb / min 70 hb / min 73 hb / min 20 microg / kg / min 30 microg / kg / min 40 microg / kg / min 79 hb / min 112 hb / min 139 hb / min fig . 
4a - g acinesia del segmento postero - settale evidente allo stress massimale nelle immagini tele - sistoliche ( f ) , correlato alla presenza di stenosi significativa della coronaria destra ( g )  . related to the high accuracy achievable with this modality , its availability , versatility and relatively low costs . 
considerando tutti questi aspetti , la possibilit di valutare la cinesi del ventricolo sinistro tramite rm potrebbe rivelarsi unalternativa efficace sia per la valutazione basale sia durante il test da stress farmacologico [ 7 ]  . 
inoltre la risoluzione ad alto contrasto della rm molto utile per delineare in modo corretto non solo lendocardio ma anche i bordi epicardici in modo da ottenere una stima corretta dellispessimento della parete [ 14 , 15 ]  . 
lunica limitazione tecnica relativa alla rm dovuta alla sensibilit ad artefatti da movimento che possono essere presenti in modo particolare durante lo radiol med ( 2009 ) 114 : 216228 all the patients in our series had an evident reduction of echocardiographic quality that was considered responsible for difficulties in delineating the endocardial borders . 
in consideration of these aspects , the possibility of evaluating left ventricle kinesis by mri could be a useful alternative for evaluation both at baseline and during pharmacological stress test [ 7 ]  . 
moreover , the high - contrast resolution of mri is useful for correctly delineating not only the endocardium but also the epicardial borders and thus obtaining an accurate estimation of wall thickening [ 14 , 15 ]  . 
the only technical limitation of mri is related to its sensitivity to movement artefacts , which may be particularly present during stress imaging as a result of anxiety or pain affecting certain patients . previous studies have reported the feasibility of mri combined with pharmacological stress testing . 
the first investigation was performed in 1992 by pennel et al . , who used an intermediate dose of dobutamine ( up to 20 g / kg / min ) to examine 25 patients with exertional chest pa the study achieved a 90% agreement with dobutamine thallium - 201 single - photon - emission computed tomography [ 16 ]  . 
a few years later , two studies reported a good level of accuracy using a similar dose of dobutamine in a small group of patients [ 17 , 18 ]  . 
it was not until 1999 that nagel et al . reported the efficacy of mri with high doses of dobutamine ( up to 40 g / kg per minute ) followed by atropine as needed to reach the maximal theoretical heart rate for detecting inducible ischaemia . 
on the other hand , the quality of the images obtained with dobutamine stress echocardiography has been reported to be good or very good in 50% of cases with the standard modality and up to 70% using harmonic imaging [ 7 ]  . 
reported an 83% sensitivity and specificity of dobutamine mri in detecting noninducible ischaemia in patients not suitable for second harmonic imaging , particularly those with a large body , severe obstructive airway disease or prior cardiothoracic surgery [ 7 ]  . 
pennel , il quale nel 1992 ha esaminato 25 pazienti con dolore toracico da sforzo usando una dose intermedia di dobutamina ( fino a 20 g / kg / min )  . 
 [ 6 ] hanno riportato lefficacia del rm nellindividuare ischemia inducibile con alte dosi di dobutamina , fino a 40 g / kg / min , seguita , quando necessario , da atropina se per raggiungere la frequenza cardiaca massimale teorica ; loro hanno inoltre comparato i risultati con unampia popolazione sottoposta ad angiografia coronarica . 
gli autori hanno riscontrato una migliore attendibilit rispetto all eco - stress con dobutamina sia in termini di sensibilit ( 88 , 7% contro 74 , 3% ; p < 0 , 05 ) che di specificit per ischemia inducibile ( 85 , 7% contro 69 , 8% ; p < 0 , 05 ) comparata con angiografia coronarica [ 6 ]  . 
dallaltro lato , la qualit dellimmagine ottenibile tramite eco - stress con dobutamina stata considerata da buona a molto buona in solo il 50% dei casi con la modalit standard , e fino al 70% tramite imaging con armonica [ 7 ]  . 
 [ 7 ] hanno riferito lutilit della rm con dobutamina nel rilevare ischemia non inducibile in pazienti non sottoponibili ad imaging con seconda armonica , in particolare quelli sovrappeso , con gravi malattie ostruttive delle vie respiratorie o sottoposti a precedente chirurgia cardiotoracica , riportando una sensibilit e specificit dell83% [ 7 ]  . 
nel 2003 hanno riscontrato una sensibilit del 96% e una specificit del 95% usando un apparato rm a 1.0 tesla su 211 pazienti [ 19 ]  . tutti questi dati sperimentali sono stati ottenuti usando sequenze fast gradient - echo . 
solo nel 2004 patesch et al . [ 4 ] hanno riportato uno studio su stress con dobutamina combinata con adenosina condotto su 79 pazienti , fornendo rispettivamente una sensibilit dell89% e una specificit del 96% , usando sequenze ssfp [ 4 ]  . 
utilizzando queste sequenze essi sono stati in grado di ottenere un aumento nella risoluzione temporale fino a 25 fasi per ciclo cardiaco e una migliore delineazione dei bordi endocardici dovuta 226 radiol med ( 2009 ) 114 : 216228 reported a sensitivity of 96% and a specificity of 95% using a 1.0 - tesla mr apparatus on 211 consecutive patients [ 19 ]  . 
report on a study investigating combined adenosine and dobutamine stress in 79 consecutive patients , providing a sensitivity in comparison with coronary angiography of 89% and a specificity of 96% obtained using the steady - state free - precession sequences [ 4 ]  . 
use of these sequences enabled them to increase temporal resolution up to 25 phases per cardiac cycle and to achieve a better delineation of the endocardial borders due to an almost complete reduction of the signal void during the systolic phase that generally affects fast - gradient - echo sequences . 
nevertheless , the level of accuracy obtained was similar to that of the other previous experiences . in our study , we employed an ssfp sequence with parallel imaging that allowed us to obtain 2530 cardiac phases per cycle in half the time required for the previously reported sequences , making it possible to simultaneously acquire two different slices with a higher contrast - to - noise ratio compared with gradient - echo acquisition . 
a five - segment segmentation was performed in accordance with the american heart association [ 9 ]  . ventricular regional analysis was performed on a 30segment model , producing a definitely greater segmentation than reported by previous dobutamine stress mri studies . 
this could be related to the limited number of cases enrolled in our study or , alternatively , it could mean that increased regional segmentation does not offer an increase in sensitivity and specificity . 
the latter hypothesis , however , appears unrealistic if one considers that an increase in spatial coverage may increase the detection of inducible ischaemia , which may be evident only in small areas of the left ventricle due to stenosis of the distal coronary arteries [ 2023 ]  . 
the specificity obtained in our study was at the maximum level achievable , and this could , again , be due to the small number of patients examined or to the better border delineation obtained with the steady - state sequences and the multichannel phased - array coil [ 24 ]  . 
in agreement with other authors , we found the procedure to be safe , with no side effects being observed in our series [ 3 , 2729 ]  . the use of ssfp sequences is helpful for obtaining higher signal intensity and thus providing greater contrast alla riduzione quasi totale del vuoto di segnale durante la fase sistolica , che invece ostacolava le sequenze fast gradient - echo . 
 nel nostro studio abbiamo impiegato una sequenza ssfp con imaging parallelo con cui stato possibile ottenere 2530 fasi cardiache per ciclo in met del tempo richiesto per le sequenze precedentemente riportate rendendo possibile lacquisizione simultanea di due diverse sezioni con un rapporto contrasto / rumore superiore rispetto allacquisizione gradient - echo . 
lanalisi regionale ventricolare stata effettuata su un modello di 30 segmenti , producendo un segmentazione decisamente maggiore rispetto agli studi riportati finora . tuttavia , la sensibilit non risultata superiore rispetto ai dati precedentemente riportati . 
nel nostro studio abbiamo riportato una sensibilit dell85% , anche inferiore rispetto alla sensibilit riportata da kuijpers et al . [ 19 ] nel 2003 con limpiego di una rm ad 1 , 0 tesla e sequenze fast gradient - echo . 
questultima ipotesi sembra tuttavia poco realistica se si considera che lincremento della copertura spaziale pu incrementare la detezione di ischemia inducibile che pu essere evidente anche solo in piccoli territori del ventricolo sinistro a causa di stenosi di arterie coronarie distali [ 2023 ]  . 
dallaltro lato , la specificit da noi ottenuta stata pari al massimo livello raggiungibile e questo pu riflettere il limitato campione da noi studiato cosi come descritto per lanalisi dei dati sulla sensibilit , ma pu anche essere collegato alla migliore della delineazione dei bordi ottenuta usando le sequenze ssfp e la bobina multicanale phased array [ 24 ]  . 
la procedura si rivelata sicura anche nella nostra esperienza , cos come negli studi precedenti , infatti non sono stati riscontrati effetti collaterali nella nostra serie [ 3 , 2729 ]  . limpiego di sequenze ssfp utile per ottenere unintensit del segnale maggiore nelle immagini , fornendo un contrasto pi elevato tra sangue e miocardio , determinato principalmente dal loro differente rapporto t1 / t2 . 
lelevato rapporto segnale / rumore efficace anche per unacquisizione radiol med ( 2009 ) 114 : 216228 between blood and myocardium , which is principally determined by their different t1 / t2 ratios . 
these sequences are able to generate a significant increase in the delineation of endocardial borders , papillary muscles and trabeculations . the high signal - to - noise ratio is also useful for rapid acquisition , thanks to the possible use of very short tr values in the range of 35 ms [ 8 , 14 , 30 ]  . 
this is extremely useful when the heart rate increases during the dobutamine test , as it allows one to obtain a high frame rate even at maximal stress that is , when the r - r is reduced [ 3133 ]  . parallel imaging may represent a key factor in cardiac stress mri , as it reduces acquisition time by half . 
 the combination of the high signal intensity of ssfp sequences and parallel imaging , where signal intensity is inversely proportional to the square root of the acceleration factor , is ideal for dobutamine stress imaging [ 1 , 4 , 34 ]  . in conclusion , mri with ssfp and parallel imaging appears to be an advantageous technique for dobutamine stress mri , as it theoretically increases the high potential of the modality , which is considered the gold standard for evaluating inducible ischaemia . 
nevertheless , studies on larger populations are required to better evaluate the effects of technical improvements on the accuracy and especially the sensitivity of dobutamine stress testing . rapida con la possibilit di usare valori tr molto piccoli , di 35 ms [ 8 , 14 , 30 ]  . 
laumento nella risoluzione temporale rende possibile inoltre di aumentare i frames per ciclo cardiaco , cosa estremamente utile quando la frequenza cardiaca aumenta durante la somministrazione di dobutamina , cos da ottenere un alto rapporto frame / ciclo cardiaco anche a livello massimale di stress cio quando lr - r ridotto [ 3133 ]  . 
la combinazione tra lelevato segnale delle sequenze steady - state e limaging parallelo , nel quale lintensit del segnale inversamente proporzionale alla radice quadrata del fattore di accelerazione , si rivela ideale nel campo dellimaging da stress con dobutamina [ 1 , 4 , 34 ]  . in conclusione , limpiego di sequenze ssfp e imaging parallelo sembra una tecnica vantaggiosa per la rm da stress con dobutamina , aumentando teoricamente le potenzialit di questa metodica considerata la migliore per valutare lischemia inducibile . 
lagalla1 1dipartimento di biotecnologie e medicina legale , sezione di scienze radiologiche , 2dipartimento di medicina , pneumologia , fisiologia e nutrizione umana , sezione di pneumologia e medicina , 3dipartimento biomedico di medicina interna e specialistica , sezione di reumatologia , universit di palermo , palermo , italy correspondence to : m . 
bellia , via del vespro 127 , palermo , italy , tel . : + 39 - 091 - 6802652 , fax : + 39 - 091 - 6891857 , e - mail : maria.bellia@libero.it received : 10 december 2007 / accepted : 9 may 2008 / published online : 12 march 2009 springer - verlag 2009 abstract purpose . 
thirty - one patients with ssc ( 16 with the diffuse form and 15 with the limited form ) underwent functional and hrct evaluations of the lung . the semiquantitative evaluation of radiological involvement , as proposed by warrick , provides a score for each lesion based on the severity and the extent of the pulmonary damage . 
scopo del lavoro stato contribuire alla validazione del metodo del punteggio ( score ) radiologico di warrick nella valutazione del danno polmonare nella sclerosi sistemica ( scs ) e di correlare i risultati con le alterazioni della funzionalit respiratoria . 
trentuno pazienti affetti da scs , 16 da forma diffusa ( dscs ) , 15 da forma limitata ( lscs ) , sono stati sottoposti a valutazione respiratoria funzionale e radiologica tramite hrct . 
la valutazione semiquantitativa della compromissione radiologica stata effettuata mediante il metodo di warrick , che attribuisce un punteggio alle varie lesioni secondo un criterio di gravit ( score di severit del danno ) e di estensione delle stesse alterazioni allinterno del parenchima polmonare ( score di estensione del danno )  . 
i risultati del presente studio confermano il valore diagnostico dellhrct nel rappresentare il tipo e lestensione del danno polmonare determinato dalla scs . inoltre , la suddivisione in punteggi parziali ( alveolite e fibrosi ) apre una ulteriore prospettiva di valutazione dei rapporti tra interessamento polmonare e compromissione sistemica . parole chiave tomografia computerizzata ad alta risoluzione sclerosi sistemica alveolite fibrosi polmonare introduction introduzione systemic sclerosis ( ssc ) , or scleroderma , is a connectivetissue disease of unknown orig it is typically characterised by fibrosis of the skin , which may be diffuse or limited . 
the disease may also involve the blood vessels and internal organs , and the lung , the kidneys and the cardiovascular system are often affected simultaneously [ 1 ]  . 
clinical and radiological involvement of the lung is seen in 70%90% of cases , although autopsic studies have reported it to occur in 74% [ 3 ] to 100% of cases [ 4 ]  . 
the natural history of interstitial lung disease in ssc includes an initial phase of predominant alveolitis that is potentially reversible if treated immediately and adequately , and a progressive evolution with a prevalence of irreversible fibrosis leading to endstage lung disease . to substantially there is a growing interest in techniques capable of defining the stage of organ damage in its progression from inflammation irreversible structural damage . 
in this context , although chest radiography remains irreplaceable as a first - line imaging technique for detecting interstitial involvement , it is insufficient for discriminating between irreversible fibrosis and active alveolitis . 
considerable progress has been made with highresolution computed tomography ( hrct ) , which compared with conventional chest radiography offers high sensitivity even in the early phase of the disease , high la sclerosi sistemica ( scs ) o sclerodermia una malattia del tessuto connettivo ad eziologia sconosciuta , caratterizzata pi spesso da fibrosi della cute : questa pu essere colpita in forma diffusa o limitata . 
la malattia pu determinare inoltre un coinvolgimento dei vasi sanguigni e di organi interni : polmone , reni e apparato cardiovascolare tendono spesso ad essere interessati contemporaneamente [ 1 ]  . 
la patologia dellinterstizio polmonare costituisce limpegno viscerale pi frequente nella scs [ 2 ] e rappresenta una delle principali cause di morte in questi pazienti . linteressamento clinico - radiologico polmonare presente nel 70%90% dei casi , mentre studi basati sui rilievi autoptici hanno evidenziato dati di prevalenza del coinvolgimento polmonare posti tra il 74% [ 3 ] ed il 100% [ 4 ]  . 
la storia naturale dellinterstiziopatia da scs riconosce una fase iniziale con prevalenza di fenomeni alveolitici potenzialmente reversibili , se tempestivamente ed opportunamente trattati , ed una evoluzione progressiva con prevalenza di fenomeni fibrotici non suscettibili di intervento terapeutico fino alla condizione di end - stage lung . 
 un argomento di crescente interesse costituito dalle metodologie dirette a definire lo stadio evolutivo delle lesioni dorgano , lungo litinerario che dalla flogosi conduce al danno strutturale anatomico sostanzialmente irreversibile . 
a tal proposito , lesame radiologico del torace , pur conservando linsostituibile valore dellindagine di primo approccio che segnala il coinvolgimento dellinterstizio , non sufficiente per distinguere la fibrosi irreversibile 192 radiol med ( 2009 ) 114 : 190203 levels of accuracy in discriminating between idiopathic lung fibrosis and lung disease secondary to rheumatic disorders , and good capabilities in quantifying disease extent . 
for ssc in particular , warrick et al . [ 5 ] devised a semiquantitative method for evaluating radiological abnormalities whereby the different types of lesions are rated according to severity and extent of lung damage , leading to an overall hrct score . the goal of this study was to contribute to validating the warrick method by : ( 1 ) evaluating correlations with the main indices of lung function abnormalities in ssc with diffuse skin involvement ( dssc ) and ssc with limited skin involvement ( lssc ) ; ( 2 ) distinguishing between the fibrotic and alveolitic components of the warrick index to establish whether any of the radiological abnormalities reflect different stages of lung damage and correlate with specific lung - function abnormalities . materials and methods this study was conducted on 31 patients ( males : females 1 : 30 ; mean age 5410.4 years ) affected by ssc diagnosed on the basis of the criteria established in 1980 by the american college of rheumatology [ 6 ] and admitted to the department of rheumatology of the university of palermo . disease duration ranged from 5 months to 21 years . 
on the basis of the extent of skin involvement , 16 patients were classified as affected by dssc and the remaining 15 by lssc . all subjects tested positive for antinuclear antibody ( ana ) , 16 tested positive for anti - scl 70 , and 15 tested positive for anticentromere . 
patients with a history of allergy to organic or inorganic substances were excluded . functional evaluation each subject underwent respiratory function testing , which included spirometry , residual volume measurement with the helium dilution technique , and single - breath carbon monoxide ( co ) diffusion test . 
un significativo progresso stato realizzato con lintroduzione della tomografia computerizzata ad alta risoluzione ( hrct ) del torace che rispetto alla radiologia tradizionale si dimostrata tecnica dotata di alta sensibilit , sin dalle fasi precoci della malattia , elevata accuratezza nella discriminazione tra fibrosi idiopatica polmonare e patologia polmonare secondaria a malattie reumatiche nonch di elevata capacit nel quantificare lestensione della malattia . 
 [ 5 ] hanno elaborato un metodo di valutazione semiquantitativa delle alterazioni radiologiche che attribuisce un punteggio alle varie lesioni secondo criteri di gravit e di estensione del danno del parenchima polmonare , dalla cui somma si ottiene uno score hrct globale . 
la durata di malattia variava da 5 mesi a 21 anni . in base allestensione cutanea della malattia , 16 pazienti risultavano affetti dalla dscs e i restanti 15 dalla lscs . 
una storia di allergie a sostanze organiche o inorganiche costituiva un criterio di esclusione dallo studio . valutazione funzionale ciascun soggetto stato sottoposto ad una valutazione funzionale respiratoria comprendente la spirometria , la misura del volume residuo mediante tecnica di diluizione dellelio e test di diffusione del monossido di carbonio ( co ) radiol med ( 2009 ) 114 : 190203 hrct study all patients gave informed consent for the hrct study . scans were performed with a 4 - detector philips mx 8000 quad ( eindhoven , the netherlands ) ct scanner . 
scanning parameters were as follows : 120 kv , 170 mas , slice thickness 1 mm , interval 10 mm , acquisition time 0.8 s , smallest field of view ( fov ) providing coverage of both lungs and convolution filter for hard tissue . in the presence of mild ground - glass opacities in the posterior basal subpleural zone , a limited number of prone scans were added to exclude the possibility of gravitational effects causing increased parenchymal density . 
capacit polmonare totale ( tlc ) e la capacit di diffusione del co ( dlco ) sono state misurate ed espresse in percentuale del valore teorico di riferimento . valori inferiori all80% del teorico sono stati considerati anormali . 
per lo studio stato utilizzato uno spirometro a campana dotato di moduli per la diluizione dellelio e la misura del transfer gassoso per il co ( baires system ; biomedin , padova , italia )  . 
 studio hrct lesame hrct , previo consenso informato , stato eseguito con apparecchio tc multistrato a 4 detettori philips mx 8000 quad ( einthoven , olanda ) e lacquisizione stata realizzata con tecnica assiale , con scansioni dagli apici alle basi con paziente in posizione supina , in apnea inspiratoria , senza somministrazione di mezzo di contrasto . 
per la realizzazione dellindagine radiologica stato utilizzato un protocollo che prevede i seguenti parametri di scansione : 120 kv , 170 mas , spessore di strato 1 mm , intervallo di scansione 10 mm , tempo di scansione 0 , 8 s , campo di vista pi piccolo possibile ( fov ) che comprendesse entrambi i polmoni , filtro di convoluzione per tessuti duri . in presenza di opacit con aspetto a vetro smerigliato di lieve entit in sede mantellare basale posteriore , stato aggiunto un numero limitato di scansioni in decubito prono , allo scopo di escludere che laumentata densit parenchimale osservata non fosse dovuta a fenomeni di natura gravitazionale . 
le lesioni elementari considerate ai fini del calcolo del punteggio , cui viene attribuito uno valore crescente da 1 a 5 in relazione alla gravit del danno , sono le seguenti : 1 . 
1 areas of lung hyperintensity with ground - glass attenuation and prevalent distribution in the lower lobe ; a subpleural line is present on the right in the posterior subpleural zone . 
 [ 5 ] attribuisce inoltre un punteggio da 1 a 3 in relazione allaumentare del numero dei segmenti in cui diviso topograficamente il polmone , 194 radiol med ( 2009 ) 114 : 190203 interessati da ciascun tipo di alterazione parenchimale , ovvero punteggio 1 se la lesione in esame presente in un numero di segmenti compreso tra 1 e 3 , punteggio 2 se il numero di segmenti interessati compreso tra 4 e 9 e punteggio 3 se il numero di segmenti maggiore di 9 . 
tale metodo di valutazione semiquantitativa della malattia polmonare allhrct stato applicato anche nella valutazione delle alveoliti fibrosanti e in altre malattie interstiziali del polmone , dimostrando una bassa variabilit inter - osservatori [ 7 ]  . 
 oltre al punteggio hrct totale , abbiamo inoltre arbitrariamente distinto un indice di alveolite , che tenesse cio conto della presenza ed estensione delle opacit a vetro smerigliato , e un indice di fibrosi , che comprendesse invece alterazioni radiologiche quali gli ispessimenti settali , il polmone ad alveare e le cisti subpleuriche . 
il calcolo dellindice di alveolite stato ottenuto estrapolando il subtotale relativo alla presenza ( score di severit = 1 ) o assenza ( score di severit = 0 ) di ground glass e alla sua estensione , ovvero al numero di segmenti ove la lesione era riscontrabile ( valore di estensione da 1 a 3 ) ; ne deriva che il range di valori dellindice di alveolite compreso tra 0 e 4 . 
il calcolo dellindice di fibrosi stato ottenuto prendendo in esame le irregolarit dellinterfaccia tra pleura periferica e parenchima ( score di severit = 2 ) , le linee settali da ispessimento dei setti interlobulari e le linee subpleuriche ( score di severit = 3 ) , il polmone ad alveare ( score di severit = 4 ) e le cisti subpleuriche ( score di severit = 5 ) , e la loro estensione ( valore di estensione compreso tra 1 e 3 per ciascuna delle quattro alterazioni )  . 
 elaborazione statistica i dati sono espressi come medie e deviazione standard . lanalisi di regressione semplice stata eseguita per valutare la correlazione tra gli indici radiologici e i parametri respiratori funzionali . 
thin - walled subpleural cystic areas . furthermore , the warrick score [ 5 ] assigns a score from 1 to 3 according to the number of segments involved by each type of lesion : a score of 1 indicates that the lesion is present in from one to three segments , a score of 2 that it is present in from four to nine segments and a score of 3 that it is present in more than nine segments . 
this semiquantitative technique for hrct evaluation of lung disease has also been applied to the evaluation of fibrosing alveolitis and other interstitial lung diseases , showing a low interobserver variability [ 7 ]  . in addition to the total hrct score , we arbitrarily distinguished between an alveolitis index , based on the presence and extent of ground - glass opacities , and a fibrosis index , based on septal thickening , honeycombing and subpleural cysts . 
the alveolitis index was calculated by isolating the subtotal scores for the presence ( severity score = 1 ) or absence ( severity score = 0 ) of ground - glass attenuation and for its extent , that is , the number of lung segments exhibiting the lesion ( extent score = 13 ) ; hence , the alveolitis index score ranges from 0 to 4 . 
the fibrosis index was calculated based on irregularities in pleural margins ( severity score = 2 ) , septal lines due to thickening of the interlobular septa and subpleural lines ( severity score = 3 ) , honeycombing ( severity score = 4 ) , subpleural cysts ( severity radiol med ( 2009 ) 114 : 190203 table 1 criteria used in the warrick score tabella 1 criteri utilizzati per la valutazione del punteggio radiologico di warrick lesions and lung segments score lesions and lung segments score parenchymal abnormalities ground - glass opacities irregularities in the pleural margins septal / subpleural lines honeycomb lung subpleural cysts number of lung segments disease severity score disease extent score alterazioni parenchimali opacit a vetro smerigliato score di severit del danno ( ground glass ) irregolarit dei margini pleurici 2 linee settali / subpleuriche polmone a favo dapi cisti subpleuriche score di estensione del danno n di segmenti polmonari score = 5 ) and the extent of each of the four abnormalities ( extent score = 13 ) ; the fibrosis index score ranges from 0 to 26 . 
the alveolitis and fibrosis indices were applied to all patients , and correlations were assessed between the specific radiological abnormalities and the different forms of disease . statistical analysis senso restrittivo con alterazioni del transfer gassoso per il co in 23 soggetti ( 75% del campione ) ( tabella 2 )  . 
il confronto dei due gruppi clinici ( dssc vs lssc ) non ha mostrato differenze nei test funzionali polmonari n per quanto riguarda la tlc% del teorico ( dssc vs lssc : 89%16% vs 82%19% , p = 0 , 31 ) , n per quanto riguarda la dlco% del teorico ( dssc vs lssc : 70%21% vs 55%18% , p = 0 , 06 )  . 
 hrct results functional tests in all patients , lung - function testing showed a moderately restrictive pattern with abnormal gas transfer for co in 23 subjects ( 75% ) ( table 2 )  . 
seventeen patients lo score totale hrct secondo warrick risultato pari a 167 , 7 nel campione complessivo , senza differenze significative tra il gruppo di pazienti con forma diffusa e limitata di malattia ( dssc vs lssc : 168 vs 178 , p = 0 , 71 )  . 
 linee settali e linee subpleuriche erano lunica alterazione presente in tutti i pazienti ( 31 / 31 ) , mentre lopacit a vetro smerigliato era la seconda pi frequente alterazione riscontrata ( 18 / 31 )  . 
la correlazione tra le alterazioni radiologiche e il grado di compromissione della diffusione del co si confermata quando i punteggi hrct di estensione e di severit delle lesioni radiologiche sono stati valutati separatamente ( punteggio di estensione : r = 0 , 41 , p = 0 , 02 ; punteggio di severit : r = 0 , 39 , p = 0 , 03 )  . 
viceversa , il punteggio di estensione correlava con la tlc% del teorico 196 radiol med ( 2009 ) 114 : 190203 showed all of the abnormalities considered in the warrick index . 
septal and subpleural lines were the only abnormalities seen in all patients ( 31 / 31 ) , whereas ground - glass appearance was the second most common abnormality ( 18 / 31 )  . 
on the basis of these observations , we propose to extend the warrick method by analysing the indices separately in order to improve the characterisation of patients with scleroderma in clinical practice . hrct offers undisputed advantages in defining severity and extent of lesions in scleroderma [ 8 ]  . 
 al fine di valutare quale tipo di danno strutturale ( ground glass vs manifestazioni fibrotiche ) influenzasse specificamente le alterazioni funzionali respiratorie , sono stati utilizzati i punteggi radiologici relativi di alveolite e fibrosi . lindice di alveolite e lindice di fibrosi sono risultati rispettivamente pari a 1 , 91 , 1 e 14 , 27 , 4 . 
sulla base di queste osservazioni , lestensione del metodo di warrick attraverso lanalisi separata degli specifici indici da esso derivati pu essere proposta per una migliore caratterizzazione del paziente sclerodermico nella pratica clinica . lhrct offre vantaggi innegabili per quanto riguarda la definizione della qualit delle lesioni e della loro estensione nel paziente affetto da tale patologia [ 8 ]  . 
numerosi studi [ 9 , 10 ] hanno affrontato il tema delle correlazioni tra alterazioni morfologiche osservabili allhrct e modificazioni funzionali respiratorie nellintento di definire i rapporti patogenetici ed il valore clinico di tali alterazioni . 
come era lecito attendersi , i risultati del nostro studio confermano il rapporto tra entit del danno strutturale parenchimale , evidenziato alla hrct e alterazioni funzionali in senso restrittivo , caratterizzate da una riduzione dei volumi polmonari . 
as expected , the results of our study confirm the correlation between severity of structural parenchymal damage seen on hrct and restrictive functional abnormalities , characterised by reduced pulmonary volumes . 
this was thought to reflect the fact that evidence of fibrosis or ground - glass pattern corresponds to parenchymal lesions responsible for abnormalities in alveolocapillary diffusion , whereas other phenomena , not bili , per motivi diversi , delle alterazioni della diffusione alveolo - capillare , laddove alla base delle alterazioni della cvf si pongono altri fenomeni ( per esempio , fatica o debolezza muscolare o le alterazioni della cute del tronco ) non descritti dallhrct [ 5 ]  . 
 [ 11 ] , che hanno osservato come nei pazienti senza honeycomb lung la capacit di diffusione fosse pi bassa nei pazienti con ground glass rispetto a quelli che non presentavano tale segno , mentre una tale differenza non era riscontrabile per i volumi polmonari ; per contro tra quelli caratterizzati dal polmone ad alveare gli autori hanno osservato come valori spirometrici normali possano ancora essere rilevabili in soggetti con limitata diffusione dellhoneycomb ( < 15% ) , mentre tutti i pazienti con honeycomb diffuso ( in media 32% ) presentavano una sindrome restrittiva alla spirometria . 
 [ 11 ] , who observed that among patients without honeycomb lung , those with ground - glass opacities had lower dlco compared with those without , whereas no such difference was found for lung volumes . 
among patients with honeycomb lung , instead , the authors observed that normal spirometry values may still be detected in subjects with limited honeycombing ( < 15% ) , whereas all patients with diffuse honeycombing ( mean 32% ) showed a restrictive pattern at spirometry . thus , the available data confirm the role of the warrick index in the early stages of disease . 
 our proposal to subdivide the warrick score into two separate indices one for alveolitis and one for fibrosis dati disponibili confermano pertanto il ruolo dellindice di warrick nelle fasi precoci di malattia . 
 la nostra proposta di distinguere lo score di warrick rispettivamente in due indici , di alveolite e di fibrosi , nasce dal tentativo di introdurre degli indici parziali , che riflettano diverse fasi evolutive della malattia , in relazione alla prevalenza di peculiari alterazioni radiologiche . 
infatti , i punteggi relativi di alveolite e di fibrosi vengono ottenuti scorporando lindice di warrick , e mantenendo la divisione in estensione e gravit delle alterazioni : obiettivo quello di analizzare quali caratteristiche dellindice di warrick influenzino le alterazioni della diffusione alveolo - capillare e quali invece le alterazioni dei volumi polmonari . 
i punteggi subtotali hanno dimostrato che lindice di alveolite correla con il grado di compromissione della diffusione dei gas ma non con le alterazioni dei volumi polmonari ; al radiol med ( 2009 ) 114 : 190203 table 3 distribution of high - resolution computed tomography ( hrct ) abnormalities for each patient patients ground - glass attenuation irregularities in the pleural margins septal / subpleural lines honeycombing subpleural cysts stems from an attempt to introduce partial indices reflecting the prevalence of specific radiological abnormalities and thus the different stages of disease . 
in fact , the scores for alveolitis and fibrosis are obtained by isolating the components of the warrick index and maintaining the division into lesion severity and extent : the goal is to determine which items of the warrick index affect alveolocapillary diffusion and which affect pulmonary volumes . 
the subtotal scores showed that the alveolitis index correlates with the degree of impairment of gas diffusion but not with changes in lung volumes ; on the other hand , the fibrosis index correlates significantly with both of these functional abnormalities . 
da ci si pu dedurre che lindice di alveolite , espressione di un danno acuto e , verosimilmente , iniziale , contribuisce ad alterare la diffusione dei gas , mantenendo inalterati i volumi polmonari ; solo quando il danno si cronicizza e si estende ( aumento dellindice di fibrosi ) si assiste anche ad una compromissione dei volumi polmonari in senso restrittivo . 
tale pattern determinato da unopacit parenchimale tenue , una velatura della trasparenza che non cancella il sottostante disegno bronco - vasale . il segno del ground glass viene spesso considerato tra i reperti che si manifestano pi precocemente e rappresenta un indicatore di potenziale trattabilit e reversibilit del coinvolgimento polmonare , in quanto si assume che identifichi tabella 3 distribuzione delle alterazioni hrct per ciascun paziente pazienti aspetto a vetro smerigliato irregolarit dellinterfaccia linee settali e pleura / parenchima linee subpleuriche aspetto a favo dapi cisti subpleuriche radiol med ( 2009 ) 114 : 190203 the alveolitis index chiefly reflects changes affecting subpleural zones of the lower lobes , in which a pattern of interstitial thickening with ground - glass appearance may also be seen . 
the ground - glass pattern is often considered to be one of the earliest pulmonary manifestations and an indicator of potential treatability and reversibility of pulmonary involvement in that it is thought to represent areas of inflammation , where oedema and cells ( especially lymphocytes and / or neutrophils ) accumulate denoting the stage of active alveolitis . 
 however , a ground - glass pattern may also reflect other anatomical abnormalities : it may correspond to a thickening of the small airspaces and thus to fibrosis at a microscopic stage . 
finally , both inflammation le aree di flogosi , in cui edema e cellule ( in particolare linfociti e / o neutrofili ) si accumulano , connotando in tal modo la fase dellalveolite attiva . 
questa potenziale correlazione radiologico - funzionale potrebbe rivestire una grande importanza clinica in quanto nella scs lalveolite non trattata si accompagna ad una pi elevata mortalit [ 12 ]  . 
secondo uno studio su 15 pazienti con sclerodermia [ 14 ] , la correlazione anatomoradiologica del ground glass potrebbe essere diversa in relazione alla topografia delle lesioni : gli autori hanno correlato i risultati dellhrct con quelli della citologia del radiol med ( 2009 ) 114 : 190203 tlc % predicted dlco % predicted fig . 
4 correlazione tra lindice di alveolite e a la dlco% del teorico , b tlc% del teorico . dlco % predicted tlc % predicted and vascular abnormalities may lead to increased capillary blood volume , which contributes to forming the typical ground - glass opacity . 
according to a study performed on 15 patients with scleroderma [ 14 ] , the anatomicalradiological correlations of ground - glass opacities may differ depending on the location of the lesions : the authors correlated the findings of hrct and bronchoalveolar lavage ( bal ) repeated in two different sites either the lower lobe and lingual or the lower lobe and middle lobe . 
in the lower lobe , instead , bal ripetuto in due sedi diverse , rispettivamente nel lobo inferiore e , nei diversi casi , o nella lingula o nel lobo medio . i risultati dello studio segnalano uneccellente correlazione tra il rilievo di una alveolite e la presenza di ground glass a livello del medio o della lingula , ma non per quanto riguarda il lobo inferiore ; in questa ultima sede era invece riconoscibile una correlazione tra levidenza citologica di alveolite ed i segni hrct di franca fibrosi polmonare . 
 nellevoluzione della malattia la comparsa di un pattern reticolare o reticolo - nodulare segnala lispessimento del connettivo peribronchiale , dei setti interlobulari , dellinterstizio subpleurico oltre che dei setti intralobulari e della 202 radiol med ( 2009 ) 114 : 190203 fig . 
5 correlazione tra lindice di fibrosi e a la dlco% del teorico , b tlc% del teorico . dlco % predicted tlc % predicted there was a significant correlation between fibrosis on hrct and evidence of alveolitis on bal . 
 lung , which in the history of the disease , the appearance of a reticular or reticulonodular pattern indicates thickening of the peribronchial connective tissue , interlobular septa , subpleural airspaces and centrilobular bronchiolar walls changes that combine to create the fibrosis index . 
it is characterised by the presence of rounded cystic spaces ( 210 mm ) that are thick - walled ( 13 mm ) and arranged over several concentric layers at the subpleural level , with interposed fibrosis and consequent parenchymal architectural distortion . 
even though these lesions are typically found in peripheral locations [ 3 ] , they have also been reported in central lung regions in a non - negligible number of cases [ 15 ]  . 
as a result of the structural and functional derangements caused by the airspace disease and the traction and distortion effects on the airways , the lung pattern in ssc may be characterised by a high prevalence of bronchiectasis , as demonstrated by hrct . 
in a study of 21 patients with diffuse and limited disease , bronchiectasis was present in 59% of cases , apparently with no correlation with the diffuse or limited nature of disease or the ground - glass or fibrotic features of lesions [ 16 ]  . 
 in conclusion , the advent of new imaging modalities such as hrct has helped to expand our knowledge about this disease , allowing us to accurately define the severity and distribution of diffuse interstitial pulmonary disease and detect the presence of subclinical alveolitis . 
lapprodo del percorso evolutivo rappresentato dal polmone ad alveare , che costituisce evidenza patognomonica dellend - stage lung : esso si caratterizza per la presenza di lacune cistiche rotondeggianti ( 210 mm ) che condividono pareti spesse ( 13 mm di spessore ) e si dispongono su pi strati concentrici a livello subpleurico , con tessuto fibroso interposto e distorsione consequenziale della normale architettura parenchimale . 
anche se la disposizione periferica di queste lesioni caratteristica [ 3 ] , stato segnalato come esse possano non risparmiare le zone centrali del polmone in un numero non trascurabile di casi [ 15 ]  . 
in relazione al disordine strutturale e funzionale determinato dallinterstiziopatia e dagli effetti di trazione e di distorsione esercitati dal parenchima alterato sulle vie aeree , il quadro del polmone nella scs pu essere caratterizzato da una elevata prevalenza di bronchiectasie , come dimostrato dallhrct : in uno studio condotto su 21 pazienti con malattia estesa e limitata le bronchiectasie erano presenti nel 59% dei casi , senza apparente relazione col carattere esteso o limitato della malattia o con le caratteristiche di ground glass o fibrosi presentate dalle lesioni [ 16 ]  . 
 in conclusione , lavvento di nuove tecniche di diagnostica per immagini come lhrct ha contribuito negli anni ad ampliare le conoscenze su questo tema , consentendo di definire con accuratezza lentit e la distribuzione della pneumopatia interstiziale diffusa , evidenziando precocemente la presenza di alveolite subclinica . 
pertanto , la disponibilit di uno score per la valutazione semiquantitativa della radiol med ( 2009 ) 114 : 190203 radiological impairment has a major impact on clinical practice , as it allows the clinician to make appropriate treatment decisions and provides an adequate tool for the follow - up . however , the clinical significance of findings of early and ill - defined impairment remains to be investigated , and further studies that include patients with more severe structural pulmonary impairment are required to define the diagnostic and prognostic roles of the partial radiological indices . compromissione radiologica riveste una notevole importanza pratica , consentendo al clinico le opportune valutazioni ai fini terapeutici , e fornendo un mezzo adeguato per un accurato follow up . 
sardanelli1 1servizio di radiologia , dipartimento di scienze medico - chirurgiche , universit degli studi di milano , irccs policlinico san donato , via morandi 30 , 20097 san donato milanese , italy 2servizio di radiologia , irccs istituto ortopedico galeazzi , via r . 
galeazzi 4 , 20161 milano , italy 3unit di ortopedia , dipartimento di scienze medico - chirurgiche , universit degli studi di milano , irccs policlinico san donato , via morandi 30 , 20097 san donato milanese , italy correspondence to : a . 
for each sequence , we evaluated the visibility of the anatomical structures of the central pivot , lateral compartment , and anterior compartment using a semiquantitative score ( 0 = total masking ; 1 = insufficient visibility ; 2 = sufficient visibility ; 3 = optimal visibility )  . 
the visibility index ranged between 83% and 89% for the first four sequences without significant differences among them , 58% for stir and 11%36% for the last five sequences . significant differences were found between each of the four first sequences and the remaining sequences ( p < 0.004 ) and riassunto obiettivo . 
quattro pazienti portatori di protesi monocompartimentale mediale oxford iii sono stati sottoposti a rm a 1 , 5 t mediante scansioni coronali : spinecho ( se ) pesata in t1 ; turbo - se ( tse ) pesata in t1 ; tse pesata in densit protonica ( dp ) e t2 ; gradient - echo ( ge ) pesata in t1 ; short tau inversion recovery ( stir ) ; multi echo data image combination ( medic ) ; ge pesata in t2 * ; volumetric interpolated breath - hold examination ( vibe ) ; dual echo steady state ( dess )  . 
per ciascuna sequenza stata valutata la visibilit delle strutture anatomiche del pivot centrale , del compartimento laterale e del compartimento anteriore mediante un punteggio semiquantitativo ( 0 = totale mascheramento ; 1 = insufficiente riconoscibilit ; 2 = sufficiente riconoscibilit ; 3 = ottimale riconoscibilit )  . 
lindice di riconoscibilit risultato tra l83% e l89% per le prime quattro sequenze , senza differenze significative tra loro , 58% per la stir e tra l11% e il 36% per le ultime cinque sequenze . 
le sequenze se , tse e stir potrebbero essere utilmente incluse in un protocollo per lo studio del ginocchio con protesi monocompartimentale mediale . parole chiave ginocchio protesi monocompartimentali risonanza magnetica introduction introduzione medial unicompartmental knee replacement is considered a valid surgical option for patients affected by medial arthritis [ 14 ]  . 
long - term clinical results are optimal , with reported rates of implant survival equal to 97% at 10 years [ 6 ] and 94% at 15 years [ 7 ]  . middleand long - term evaluation of the remaining anatomical structures of the knee , in particular progression of arthritis of the untreated compartment [ 8 , 9 ] , should be performed with dedicated imaging techniques . 
whereas bone segments can be studied using radiographic examinations [ 8 , 10 , 11 ] , synovial and capsular structures and collateral ligaments can be investigated with ultrasound , an approach already proposed for evaluating soft tissues after total knee arthroplasty [ 12 ]  . 
no real solution , however , exists for evaluating the lateral and anterior compartments ( in particular , bone , cartilage and lateral meniscus ) or structures of the central pivot of knees treated with medial unicompartmental arthroplasty . magnetic resonance ( mr ) imaging is considered an optimal technique for imaging the knee and evaluating new therapeutic approaches [ 13 , 14 ]  . 
until now , mr imaging of the replaced knee has been practically considered to be contraindicated , even though pilot experiences of mr imaging of the knee after total arthroplasty have been reported [ 15 , 16 ]  . 
in fact , metallic objects inside the human body generate artefacts on mr images , and such artefacts vary in size and type according the radiofrequency sequence used and its technical parameters [ 17 ]  . 
gradient - echo ( ge ) sequences are more sensitive ( and thus produce larger artefacts ) when compared with spin - echo ( se ) sequences , and the size of the artefact increases as t2 * weighting increases [ 1820 ]  . 
therefore , orthopaedic metallic devices are generally considered a possible limitation for mr imaging , being a possible source of masking the anatomical districts to be studied [ 20 ]  . 
in particular , the current opinion is that in the limpianto di una protesi monocompartimentale mediale ( pmcm ) del ginocchio da tempo considerato una valida opzione chirurgica per il trattamento dellartrosi del compartimento mediale [ 14 ]  . 
la prognosi a lungo termine ottimale : sono state riportate sopravvivenze medie delle pmcm a 10 anni del 97% [ 6 ] , a 15 anni del 94% [ 7 ]  . la valutazione a medio e lungo termine delle strutture anatomiche residue , in particolare la progressione artrosica del compartimento laterale [ 8 , 9 ] , necessita di un utilizzo appropriato di tecniche di imaging . 
mentre lo studio delle strutture ossee agevolmente condotto mediante indagini radiografiche [ 8 , 10 , 11 ] , quello della sinovia , delle strutture capsulari e dei legamenti collaterali si giova dellapproccio ecografico , gi proposto per il follow - up delle protesi totali [ 12 ]  . 
resta aperto , nei pazienti con pmcm , il problema della valutazione dei compartimenti laterale e anteriore ( con particolare riferimento alle strutture osteocartilaginee e al menisco laterale ) , oltre che delle strutture del pivot centrale . la risonanza magnetica ( rm ) oggi considerata tecnica di imaging ottimale nello studio del ginocchio , anche relativamente alla valutazione di approcci terapeutici innovativi [ 13 , 14 ]  . 
fino ad oggi , nei pazienti con protesi del ginocchio la rm stata ritenuta sostanzialmente controindicata ai fini della valutazione del ginocchio operato , anche se sono state riportate alcune esperienze pilota nello studio rm di pazienti con protesi totali [ 15 , 16 ]  . 
infatti , la presenza di oggetti metallici intracorporei genera nelle immagini rm artefatti che variano a seconda del tipo di sequenza di impulsi a radiofrequenza e dei suoi parametri tecnici [ 17 ]  . 
in generale le sequenze gradient - echo ( ge ) sono pi sensibili ( e quindi producono artefatti di maggiori dimensioni ) rispetto a quelle spinradiol med ( 2009 ) 114 : 301311 presence of total arthroplasty of the knee , mr imaging does not allow a reliable evaluation of even superficial structures such as the quadriceps tendon , patellar tendon and collateral ligaments , which are better investigated with ultrasound [ 12 ]  . 
 the aim of our study was to test a series of mr sequences for evaluating the knee after medial unilateral arthroplasty in order to identify sequences that provide images affected by smaller artefacts and thus of potential use for assessing the anterior and lateral compartments and the central pivot . materials and methods patients between june and september 2005 , four symptomatic patients ( two women aged 60 and 65 years and two men aged 67 and 79 years ) who had undergone medial unicompartmental arthroplasty ( oxford iii , biomet , london , uk ) underwent mr imaging of the treated knee . 
this implant is composed of two metallic devices ( cobaltchromemolybdenum alloy ) implanted into the medial femoral condyle and into the corresponding portion of the tibial plate , which can be easily recognised at radiography , and of a radiolucent polymer meniscus [ 10 ]  . 
 mr protocol examinations were performed with a 1.5 - t superconductive magnet , 40 - mt / m gradient power and a 20 - cm flexible coil surrounding the knee ( sonata maestro class , siemens , erlangen , germany )  . 
in fact , whereas the sagittal scan plane would have prevented direct evaluation of mediolateral extent of artefacts , the axial scan plane would have allowed the direct evaluation of mediolateral and anteroposterior extent of artefacts ( though not the craniocaudal extent ) , but only with a far higher number of slices compared with coronal scanning and , as a consequence , longer scan time . 
 we tested seven sequences commonly used in musculoskeletal mr imaging : t1 - weighted se , t1 - weighted echo ( se ) e lentit dellartefatto aumenta col crescere della pesatura in t2 * [ 1820 ]  . 
per tale motivo , le protesi metalliche ortopediche sono in generale considerate condizioni potenzialmente limitanti lindagine rm in quanto potenziale fonte di mascheramento delle regioni anatomiche oggetto di studio [ 20 ]  . 
in particolare , in presenza di protesi totali del ginocchio , secondo lopinione corrente la rm non consente una valutazione affidabile neppure di strutture superficiali quali il tendine del quadricipite , i legamenti collaterali e il tendine rotuleo , per le quali appare nel complesso superiore il contributo diagnostico dellecografia [ 12 ]  . 
 scopo dello studio stato testare una serie di sequenze rm per lo studio del ginocchio trattato con pmcm al fine di identificare quelle che consentono di ottenere immagini gravate da minori artefatti , quindi potenzialmente utili per lo studio del pivot centrale e dei compartimenti anteriore e laterale . materiali e metodi pazienti tra giugno e settembre 2005 , 4 pazienti sintomatici ( 2 femmine di 60 e 65 anni e 2 maschi di 67 e 79 anni ) portatori di pmcm oxford iii ( biomet , london , uk ) sono stati sottoposti a rm del ginocchio protesizzato . 
tale protesi costituita da due componenti metalliche in lega al cobaltocromo - molibdeno impiantate sul condilo femorale mediale e sul corrispondente emipiatto tibiale , ben riconoscibili allesame radiografico , e da un menisco in polimero radiotrasparente [ 10 ]  . 
 protocollo rm le indagini sono state eseguite con apparecchiatura a magnete superconduttivo operante a 1 , 5 t , gradienti con potenza di 40 - mt / m ( sonata maestro class , siemens , erlangen , germania ) e bobina flessibile ( 20 cm ) posta a circondare larticolazione . 
infatti , mentre il piano sagittale non avrebbe permesso la valutazione diretta dellestensione medio - laterale degli artefatti , il piano assiale avrebbe s 304 radiol med ( 2009 ) 114 : 301311 turbo - se ( tse ) , proton - density ( pd ) and t2weighted tse , t1 - weighted spoiled ge ( fast low - angle shot ) , shorttau inversion recovery ( stir ) and multi - echo data image combination ( medic )  . 
moreover , to investigate artefact extent on images obtained with sequences highly sensitive to magnetic inhomogeneity , we tested a t2 * - weighted spoiled ge sequence and two three - dimensional sequences : the volumetric interpolated breath - hold examination ( vibe ) and the dual - echo steady - state ( dess ) sequences . 
each of the four patients remained in our department about 30 min after the examination and was asked if she or he had any discomfort during or after the exa image analysis for each of the ten sequences , two radiologists with about consentito la valutazione diretta dellestensione mediolaterale e antero - posteriore degli artefatti ( rinunciando a quella cranio - caudale ) ma con un numero di strati molto pi elevato rispetto al piano coronale e , quindi , tempi di scansione prolungati . 
 sono state testate sette sequenze tra quelle pi utilizzate in rm muscolo - scheletrica : se pesata in t1 ; turbo - se ( tse ) pesata in t1 ; tse pesata in densit protonica ( dp ) e t2 ; ge con spoiler ( fast low angle shot ) pesata in t1 ; short tau inversion recovery ( stir ) ; multi echo data image combination ( medic )  . 
allo scopo di verificare lestensione degli artefatti in sequenze ad alta sensibilit alla disomogeneit magnetica , sono state altres testate una sequenza ge con spoiler pesata in t2 * e due sequenze con acquisizione table 1 technical parameters of ten mr sequences used in four patients for the 1.5 - t study of the knee with unicompartmental oxford iii biomet arthroplasty sequences tr ( ms ) te ( ms ) flip angle thickness ( mm ) number of slices acquisition time ( min : sec ) tr , time of repetition ; te , time of echo ; pd , proton density ; tse , turbo spin - echo ; se , spin - echo ; stir , short tau inversion - recovery ; ge , gradient - echo ; dess , double echo in the steady state ; vibe , volumetric interpolated breath - hold examination ; medic , multi echo data image combination . 
for all sequences : matrix 256256 ; field of view 200200 mm ; number of excitations , nex = 1 , with the exception of t1and t2 * - weighted ge sequences ( nex = 2 )  . 
per tutte le sequenze : matrice 256256 ; campo di vista 200200 mm ; numero di eccitazioni , nex = 1 , ad eccezione delle sequenze ge t1e t2 * - pesate ( nex = 2 )  . 
 a tempo di interpulso = 130 ms radiol med ( 2009 ) 114 : 301311 10 and 4 years , respectively , of experience in musculoskeletal mr imaging rated in consensus the visibility of the anatomical structures of the central pivot ( cruciate ligaments ) , lateral compartment ( femoral and tibial bone and cartilages ; lateral meniscus ; capsule and lateral collateral ligament ) and anterior compartment ( femoral and tibial bone and cartilages ; hoffa fat pad , patellar tendon )  . we used a semiquantitative scoring system for the visibility of anatomical structures related either to the presence of image artefacts or to the contrast resolution for each sequence ( 0 = total masking ; 1 = insufficient visibility ; 2 = sufficient visibility ; 3 = optimal visibility )  . 
 statistical analysis differences among the visibility indexes of the ten sequences were globally evaluated with the friedman test . post hoc analysis for direct comparison between pairs of sequences was performed using the sign test . 
none of the four patients reported pain , heat or other local discomfort either during the examination or during the following 30 m visibility indexes of anatomical structures obtained with each of the ten sequences are reported in table 2 . 
nonsignificant differences ( p = 0.733 , friedman test ) were observed among the visibility indexes of the first four sequences ( pdand t2weighted tse , t1weighted se , and t1 - weighted tse )  . the visibility indexes of the first four sequences were significantly higher ( p < 0.004 , sign test ) than those of the remaining six sequences . 
visibility index of the stir sequence ( fifth , decreasing order ) was significantly higher ( p < 0.008 , sign test ) than that of the last five sequences ( t1and t2 * - weighted ge , dess , vibe , medic )  . 
the difference between the visibility index of the t1 - weighted ge tridimensionale , volumetric interpolated breath - hold sequence ( vibe ) e dual echo steady state ( dess )  . 
ciascuno dei quattro pazienti rimasto in reparto in osservazione per circa 30 minuti dopo lesecuzione dellindagine ed stato interrogato sulleventuale insorgenza di qualsiasi disturbo durante e dopo lindagine . analisi delle immagini due radiologi con circa 10 e 4 anni di esperienza in rm muscolo - scheletrica hanno valutato in consenso , per ciascuna sequenza , la visibilit delle strutture anatomiche del pivot centrale ( legamenti crociati ) , del compartimento laterale ( osso e cartilagine femorale e tibiale ; menisco laterale ; capsula e legamento collaterale laterale ) , del compartimento anteriore ( osso e cartilagine femorale e tibiale ; corpo di hoffa ; tendine rotuleo )  . 
 stato utilizzato un punteggio semiquantitativo di riconoscibilit delle strutture anatomiche in relazione alla presenza di artefatti e alla risoluzione di contrasto della sequenza ( 0 = totale mascheramento ; 1 = insufficiente riconoscibilit ; 2 = sufficiente riconoscibilit ; 3 = ottimale riconoscibilit )  . 
la somma dei punteggi riportati da ciascuna sequenza in ciascuno dei tre compartimenti e per tutti e quattro i pazienti stata divisa per il valore massimo ottenibile ( score = 3 per i tre compartimenti e per ognuno dei quattro pazienti , cio 334 = 36 ) , ottenendo per ciascuna sequenza un indice percentuale di riconoscibilit . 
 analisi statistica le differenze tra gli indici di riconoscibilit delle dieci sequenze sono stati globalmente valutati con il test di friedman ; lanalisi post - hoc per il confronto delle differenze tra gli indici di riconoscibilit di coppie di sequenze stata eseguita con il test dei segni . 
valori di p inferiori a 0 , 05 sono stati considerati significativi . risultati lindagine rm durata circa 3032 minuti , 24 dei quali per il tempo di acquisizione delle dieci sequenze , i rimanenti 68 minuti per il posizionamento del paziente , le sequenze di centratura e la discesa del paziente dal lettino . nessuno dei quattro pazienti ha lamentato dolore , riscaldamento o altre sensazioni locali durante lindagine o nei 30 minuti successivi . 
 arapporto percentuale tra la somma dei punteggi e il valore massimo ottenibile ( vedi testo ) and that of the t2 * - weighted ge was not significant ( p = 0.453 , sign test )  . 
this result was expected , given that orthopaedic implants are generally defined not as real contraindications to the study but as patient conditions possibly limiting the diagnostic effectiveness of the examination ; that is , as a possible source of image artefacts in the body region being investigated [ 22 ]  . secondly , comparison among sequences confirmed the in ordine decrescente in tabella 2 . 
non sono state osservate differenze significative ( p = 0 , 733 , test di friedman ) tra gli indici di riconoscibilit delle prime quattro sequenze ( tse dpe t2 - pesata , se t1 - pesata e tse t1 - pesata )  . 
lindice di riconoscibilit della sequenza stir ( quinta in ordine decrescente ) risultato significativamente maggiore ( p < 0 , 008 , test dei segni ) di quello delle ultime cinque sequenze ( ge t1e t2 * - pesata , dess , vibe , medic )  . 
1a - h comparison among magnetic resonance ( mr ) sequences used at 1.5 t for studying the knee after unicompartmental arthroplasty ( coronal plane , anterior compartment ) : a t1 - weighted turbo spin - echo ( tse ) ; b proton - density - weighted tse ; c t2 - weighted tse ; d short - tau inversion recovery ( stir ) ; e gradient - echo ( ge ) t2 * - weighted ; f dual - echo steady state ( dess ) ; g multi - echo data image combination ( medic ) ; h volumetric interpolated breath - hold examination ( vibe )  . 
note the visibility of the patellar tendon only in a , b , c , and d ( full arrow ) , whereas in the remaining images , the artefact entirely masks the image below the patellar apex . 
1a - h comparazione tra sequenze rm ( 1 , 5 t ) per lo studio del ginocchio con protesi monocompartimentale mediale ( piano coronale , compartimento anteriore ) : a turbo spin - echo ( tse ) pesata in t1 ; b tse pesata in densit protonica ; c tse pesata in t2 ; d short tau inversion - recovery ( stir ) ; e gradientecho ( ge ) pesata in t2 ; f dual echo steady - state ( dess ) ; g multi echo data image combination ( medic ) ; h volumetric interpolated breath - hold examination ( vibe )  . 
si osservi come il tendine rotuleo sia riconoscibile solo in a , b , c e d ( freccia piena ) mentre lartefatto metallico maschera completamente limmagine caudalmente allapice rotuleo . 
in b , c e d la sottile linea arcuata artefattuale non compromette la riconoscibilit del tendine rotuleo ( freccia vuota )  . higher sensitivity of the ge sequences to magnetic susceptibility artefacts when compared with se sequences [ 18 , 19 , 22 ]  . 
moreover , t1weighted ge sequences obtained a visibility index ( 36% ) that was slightly higher , though not significant ( p = 0.453 ) , than that of t2 * - weighted ge sequences ( 31% ) due to the effect of t2 * weighting , which emphasises image artefacts [ 18 , 19 ]  . 
however , neither of these sequences appeared to be diagnostic . in our study , the spatial relationship between the coronal scans and the course of the patellar tendon limited its evaluation . 
sagittal or axial scans allowed a better evaluation of due sequenze ge t1e t2 * - pesate risultata non significativa ( p = 0 , 453 , test dei segni )  . 
4. discussione i risultati del presente studio indicano in primo luogo che lindagine rm del ginocchio a 1 , 5 t in presenza di pmcm non comporta linsorgenza di eventi avversi durante o dopo lesecuzione della stessa . 
tale risultato era atteso in ragione della generale definizione delle protesi ortopediche non come controindicazioni ma come condizioni potenzialmente limitanti lindagine , ovvero fonte potenziale di artefatti nellambito della regione studiata [ 22 ]  . 308 radiol med ( 2009 ) 114 : 301311 fig . 
2a - h comparison among magnetic resonance ( mr ) sequences used at 1.5 t for studying the knee after unicompartmental arthroplasty ( coronal plane , central pivot ) : a t1 - weighted turbo spin - echo ( tse ) ; b proton - density - weighted tse ; c t2 - weighted tse ; d short - tau inversion recovery ( stir ) ; e gradient - echo ( ge ) t2 * - weighted ; f dual - echo steady state ( dess ) ; g multi - echo data image combination ( medic ) ; h volumetric interpolated breath - hold examination ( vibe )  . 
note that the anterior cruciate ligament is visualised only in a , b , c and d ( arrow ) , whereas in the remaining images , the artefact masks entirely the central pivot and partially the lateral condyle , allowing limited visibility of the lateral meniscus . 
2a - h comparazione tra sequenze rm ( 1 , 5 t ) per lo studio del ginocchio con protesi monocompartimentale mediale ( piano coronale per il pivot centrale ) : a turbo spin - echo ( tse ) pesata in t1 ; b tse pesata in densit protonica ; c tse pesata in t2 ; d short tau inversion - recovery ( stir ) ; e gradientecho ( ge ) pesata in t1 ; f ge pesata in t2 * ; g dual echo steady - state ( dess ) ; h multi echo data image combination ( medic )  . 
si osservi come il legamento crociato anteriore sia riconoscibile solo in a , b , c e d ( freccia ) mentre nelle restanti immagini lartefatto maschera interamente il pivot centrale e parzialmente il condilo laterale consentendo una limitata riconoscibilit del menisco laterale . 
 the stir sequence , even though limited by an overall visibility index ( 58% ) significantly lower than those of the first four se and tse sequences , must be regarded as a useful approach due to its high sensitivity for fluid collections and soft tissue and spongy bone oedema . 
the structure of the stir sequence used by us is that of an inversion pulse ( ti = 130 ms ) lowering the fat signal , in secondo luogo , il confronto tra sequenze ha confermato la maggiore sensibilit agli artefatti da suscettivit magnetica delle sequenze ge rispetto a quelle se [ 18 , 19 , 22 ]  . 
3a - h comparison among magnetic resonance ( mr ) sequences used at 1.5 t for studying the knee after unicompartmental arthroplasty ( coronal plane , lateral compartment ) : a t1 - weighted turbo spin - echo ( tse ) ; b proton - density - weighted tse ; c t2 - weighted tse ; d short - tau inversion recovery ( stir ) ; e gradient - echo ( ge ) t2 * weighted ; f dual - echo steady state ( dess ) ; g multi - echo data image combination ( medic ) ; h volumetric interpolated breath - hold examination ( vibe )  . 
in the area indicated by the oval in a , b , c and d , all anatomical structures can be recognised , in particular , the lateral portion of the capsule , lateral collateral ligament , external femorotibial joint space and lateral meniscus . 
3a - h comparazione tra sequenze rm ( 1 , 5 t ) per lo studio del ginocchio con protesi monocompartimentale mediale ( piano coronale per il compartimento laterale ) : a turbo spin - echo ( tse ) pesata in t1 ; b tse pesata in densit protonica ; c tse pesata in t2 ; d short tau inversion - recovery ( stir ) ; e gradient - echo ( ge ) pesata in t1 ; f ge pesata in t2 * ; g dual echo steady - state ( dess ) ; h volumetric interpolated breath - hold examination ( vibe )  . nellarea compresa dallovale in a , b , c e d si riconoscono tutte le strutture , in particolare il compartimento capsulare laterale , il legamento collaterale laterale , la rima articolare femoro - tibiale esterna e il menisco esterno . 
we did not perform sequences with spectral fat saturation due to their high sensitivity to the magnetic field distortion determined by the presence of ferromagnetic material [ 24 , 25 ] and to avoid a longer total examination time . 
on the basis of these results , an mr imaging protocol including sagittal t1weighted se , coronal pd - weighted tse , axial t2 - weighted tse and sagittal and coronal stir sequences could be used to study the knee with unicompartmental arthroplasty . our experience confirms the absence of contraindications to mr imaging for patients with joint replacement . 
in particular , for unicompartmental knee arthroplasty , mr imaging could be a diagnostic tool for evaluating the progression of arthritis of the lateral compartment and associated cartilage and meniscal changes and assessing the fini diagnostici : la pesatura in t2 * enfatizza gli artefatti [ 18 , 19 ]  . nelle sequenze coronali da noi utilizzate langolazione del piano di scansione rispetto al decorso del tendine rotuleo ne ha limitato la valutazione diagnostica che avrebbe potuto essere ottimizzata da piani di scansione sagittali o assiali . 
4 visibility index of the anatomical structures of the central pivot , anterior compartment , and lateral compartment of ten magnetic resonance ( mr ) sequences ( 1.5 t ) used to study the knee after medial unicompartmental arthroplasty . 
4 grafico a barre dellindice di riconoscibilit delle strutture anatomiche del pivot centrale , del compartimento anteriore del compartimento laterale per dieci diverse sequenze utilizzate per lo studio rm del ginocchio con protesi monocompartimentale a 1 , 5 t . 
the diagnostic value and clinical efficacy of this information require investigation with a specifically designed prospective study . radiol med ( 2009 ) 114 : 301311 prime quattro sequenze ( se e tse ) , rappresenta un approccio utile in virt dellelevata sensibilit alle raccolte liquide e alledema sia dei tessuti molli che , in particolare , dellosso spongioso . 
la struttura delle sequenza stir da noi utilizzata , del resto , quella di una sequenza tse t2pesata ( te = 54 ms ) preceduta da un impulso di inversione ( ti = 130 ms ) che abbatte il segnale del grasso . 
si deciso di non eseguire sequenze con saturazione spettrale del grasso in quanto marcatamente sensibili alla distorsione del campo magnetico determinata dalla presenza di materiale ferromagnetico [ 24 , 25 ] e per non prolungare la durata dellesame . 
in particolare , per le pmcm di ginocchio , indica nella rm una tecnica diagnostica che potrebbe fornire informazioni circa la progressione dellartrosi del compartimento laterale e delle associate degenerazioni cartilaginee e meniscali , nonch sullarticolazione femoro - rotulea e delle sue superfici cartilaginee , non valutabili con tecniche di imaging alternative . 
sardanelli2 1unit of breast imaging , university of genoa and san martino hospital , largo rosanna benzi 10 , 16132 genoa , italy 2department of medical and surgical sciences , university of milan school of medicine , unit of radiology , irccs policlinico san donato , via morandi 30 , 20097 san donato milanese , milan , italy 3unit of cytopathology , university of genoa and san martino hospital , largo rosanna benzi 10 , 16132 genoa , italy 4department of worldwide medical & regulatory affairs , bracco imaging spa , via e . 
a number of women who should undergo magnetic resonance ( mr ) imaging of the breast cannot use this diagnostic tool due to claustrophobia or excessive body size for the restricted confines of standard closed mr systems . 
the standard of reference was pathological examination for 16 lesions classified with a maximal breast imaging reporting and data system ( bi - rads ) score from 3 to 5 , fine - needle aspiration cytology and ( cid : 2 ) 2 - year follow - up for two lesions classified as bi - rads 3 , and ( cid : 2 ) 2 - years follow - up for five lesions classified as bi - rads 2 . 
le donne claustrofobiche o obese non possono eseguire la rm mammaria nei comuni magneti chiusi . nostro obiettivo stato valutare la performance diagnostica di un sistema aperto a basso campo per lo studio rm mammario dinamico in queste pazienti , utilizzando un mezzo di contrasto paramagnetico ad alta relassivit . 
stata eseguita sequenza gradient - echo dinamica 3d t1 - pesata ( risoluzione temporale = 94 secondi ) con gadobenato dimeglumina ( 0 , 1 mmol / kg )  . 
tre lesioni ( due tumori invasivi e una cisti ) non erano valutabili per la presenza di artefatti da movimento e sono state considerate come due falsi negativi e un falso positivo . performance diagnostica : sensibilit 86% ( 13 / 15 ; ic [ intervallo di confidenza ] 95% 70% , 100% ) ; specificit 87% ( 7 / 8 ; ic 95% 65% , 100% ) ; accuratezza 87% ( 20 / 23 ; 268 radiol med ( 2009 ) 114 : 267285 95% ci : 73%100% ) were obtained . 
in pazienti claustrofobiche o obese la rm mammaria a basso campo e magnete aperto con gadobenato dimeglumina consente una buona perfomance diagnostica . parole chiave mammella , tumori risonanza magnetica magneti aperti a basso campo gadobenato dimeglumina ( gd - bopta ) introduction introduzione magnetic resonance ( mr ) imaging of the breast is typically performed using 1.5 - t closed magnets [ 19 ]  . 
unfortunately , a number of women who should undergo mr imaging of the breast cannot use this diagnostic tool due to claustrophobia [ 10 ] or a body size that is too large for the restricted confines of closed systems [ 11 ]  . 
furthermore , the studies conducted at low field strength have been performed only with conventional contrast agents with standard r1 relaxivities of approximately 3.74.6 l mmol1 s1 ( as measured at 1.5 t ) [ 17 , 18 ]  . an inherent limitation of low - field magnets for diagnostic mr imaging of the breast is the reduced signal - tonoise ratio attainable compared with that attainable at higher field strengths . 
numerous studies have shown not only that gadobenate dimeglumine is effective for breast mr imaging [ 1922 ] but also that it offers improved diagnostic performance compared with gadopentetate dimeglumine administered at equivalent doses [ 23 ]  . our purpose was to evaluate the diagnostic performance of breast mr imaging on an open low - field magnet in la risonanza magnetica ( rm ) mammaria tipicamente eseguita con magneti chiusi a 1 , 5 t [ 19 ]  . 
un numero limitato di studi ha valutato la performance diagnostica delle indagini rm a contrasto dinamico a basso campo , per lo pi a fini interventistici ( biopsie e centraggi rm - guidati ) [ 1215 ] , e la stragrande maggioranza degli studi di rm mammaria allo stato dellarte sono realizzati con campi magnetici pi elevati che consentono migliori performance tecniche ( superiore risoluzione spaziale e temporale ) [ 1 , 16 ]  . 
occorre inoltre considerare che tutti gli studi a basso campo sono stati condotti con gli usuali mezzi di contrasto caratterizzati da relassivit r1 nellintervallo tra 3 , 7 e 4 , 6 l mmol1 s1 ( misurate a 1 , 5 t ) [ 17 , 18 ]  . unintrinseca limitazione allutilizzo di magneti a bassa intensit di campo per limaging mammario a rm il ridotto rapporto segnale / rumore rispetto a quello ottenibile con intensit di campo pi elevate . 
una possibilit di superare questa limitazione lutilizzo di mezzi di contrasto con relassivit r1 maggiore di quella dei mezzi di contrasto usuali . tale potenziale vantaggio offerto dal gadobenato dimeglumina ( multihance , gd - bopta ; bracco imaging spa , milano , italia ) la cui relassivit r1 nel plasma umano a 0 , 2 t ( 10 , 9 l mmol1 s1 ) circa doppia rispetto a quella del gadopentetato dimeglumina ( gd - dtpa ) ( 5 , 7 l mmol1 s1 ) [ 18 ]  . molteplici studi hanno dimostrato lefficacia del gadobenato dimeglumina per la rm mammaria [ 1922 ] , con migliore performance diagnostica rispetto al gadopentetato dimeglumina somministrato alla stessa dose [ 23 ]  . il nostro obiettivo stato valutare la performance diagnostica della rm mammaria a basso campo con magnete aperto in pazienti che non potevano essere sottoposte a indagine standard con magnete chiuso ad alto campo per ragioni psicologiche o fisiche , utilizzando come mezzo di contrasto il gadobenato dimeglumina . 
 radiol med ( 2009 ) 114 : 267285 patients who were precluded from undergoing standard mr imaging with a closed higher - field magnet for psychological or physical reasons using gadobenate dimeglumine as a contrast agent . materiali e metodi disegno dello studio e pazienti materials and methods study design and patient population from march 2002 to septemper 2004 , a total of 397 consecutive breast mr examinations were performed in genoa at the san martino university hospital . 
the low - field examinations were performed because of claustrophobia ( the patients refused the examination on the closed magnet , even with medication ) in 15 cases or large body size in three cases ( the patients were unable to enter the closed magnet ) ( table 1 )  . 
all 18 patients [ age 4816 years ; meanstandard deviation ( sd ) ] were specifically informed about the potential limitations of the mr exam performed at low field with a body coil , including the possibility of missing malignant lesions . 
the clinical indications for breast mr in these 18 patients were presurgical cancer staging ( n = 12 ) , high familial risk of breast cancer ( n = 4 ) and suspected local cancer recurrence ( n = 2 )  . 
 clinical breast examination and conventional imaging clinical breast examination ( cbe ) and mammography and / or ultrasound ( us ) were performed on the 18 patients as part of their clinical care ( table 1 )  . 
fourteen patients also underwent bilateral two - view screen - film mammography with additional projections as necessary ( mammomat 2000 , siemens medical solutions , erlangen , germany )  . 
because a dedicated breast coil tra marzo 2002 e settembre 2004 presso lazienda ospedale san martino e cliniche universitarie convenzionate ( genova ) sono state eseguite 397 indagini rm mammarie consecutive . 
di queste 397 indagini , 379 ( 95 , 5% ) sono state eseguite a 1 , 5 t ( vision , siemens , erlangen , germania ) con bobina dedicata bilaterale . 
si trattava di 15 pazienti claustrofobiche ( che avevano rifiutato di eseguire lindagine con magnete chiuso , anche con sedazione ) e di tre pazienti obese ( che non riuscivano ad entrare nel magnete chiuso ) ( tabella 1 )  . 
tutte le 18 pazienti ( et 4816 anni [ mediadeviazione standard ] ) sono state specificamente informate circa i potenziali limiti dellindagine rm eseguita a basso campo con bobina body , inclusa la possibilit di mancata diagnosi di lesioni maligne . 
le indicazioni allindagine erano : stadiazione prechirurgica ( n = 12 ) ; alto rischio familiare di tumore mammario ( n = 4 ) ; sospetta recidiva locale ( n = 2 )  . lo studio stato approvato dal comitato etico e tutte le 18 pazienti hanno fornito consenso informato scritto . 
 visita senologica , mammografia ed ecografia in tutte le 18 pazienti sono state eseguite visita senologica ( vs ) e mammografia e / o ecografia nel contesto dellusuale iter diagnostico ( tabella 1 )  . 
 risonanza magnetica stato utilizzato un sistema aperto operante a 0 , 2 t ( concerto , siemens medical solutions , erlangen , germania ) con gradienti da 20 mt / m e slew rate di 40 t / m / s . 
 le prime due pazienti della serie sono state studiate in posizione prona ( con le braccia posizionate in avanti , anteriormente alla testa ) utilizzando alcuni cuscini e supporti in 270 radiol med ( 2009 ) 114 : 267285 radiol med ( 2009 ) 114 : 267285 272 radiol med ( 2009 ) 114 : 267285 fig . 
1a - d benign breast lesions detected at gadobenate - dimeglumine - enhanced ( 0.1 mmol / kg ) dynamic breast magnetic resonance imaging with a 0.2 - t open magnet ( patients studied in the supine position ) : coronal first postcontrast subtracted images ( a , c ) and enhancement - to - time curves ( b , d )  . 
a small , round , homogenously enhancing mass ( arrow in a ) in a 28 - year - old woman and a large inhomogenously enhancing lobulated mass ( arrow in c ) in a 24year - old woman , both at the upper outer quadrant of the left breast . 
integrating morphology and dynamics , we obtained a breast imaging reporting and data system ( bi - rads ) 3 ( probably benign ) score , in agreement with the lesion features observed at ultrasound . 
1a - d lesioni benigne alla rm mammaria a basso campo ( 0 , 2 t ) con 0 , 1 mmol / kg di gadobenato dimeglumina ( pazienti in posizione supina ) : immagini coronali sottratte della prima fase dinamica ( a , c ) e curve dinamiche ( b , d )  . 
una piccola formazione rotondeggiante con enhancement omogeneo ( freccia in a ) in una donna di 28 anni e grande formazione lobulata con enhancement disomogeneo ( freccia in c ) in una donna di 24 anni , entrambe al quadrante supero - esterno della mammella sinistra . 
in b possiamo apprezzare il modesto enhancement precoce ( 49% ; valori assoluti da 286 , 2 a 425 , 3 unit arbitrarie ) e la dinamica incrementale ( curva di tipo 1 ) della lesione di piccole dimensioni , che depongono a favore della benignit , confermata dallassenza di modificazioni al follow - up . 
la curva dinamica della lesione maggiore ( d ) presenta invece un enhancement iniziale del 104% ( da 328 , 3 a 670 , 4 unit arbitrare ) con un andamento postiniziale con wash - out non significativo ( 17 , 1 unit arbitrare , pari al 5 , 0% dellenhancement iniziale ) , ovvero una curva a plateau ( tipo 2 )  . 
a causa delle difficolt incontrate durante queste indagini ( tempo di posizionamento prolungato e relativa instabilit della posizione della paziente ) , le rimanenti 16 pazienti sono stati studiate in posizione supina con mammelle libere ( senza uso di fasce o altri mezzi di contenzione ) e con le braccia distese lungo il corpo . 
2a , b breast cancer detected at gadobenate - dimeglumine - enhanced ( 0.1 mmol / kg ) dynamic breast magnetic resonance with a 0.2 - t open magnet in a 42 - year - old patient studied in the supine position : coronal first postcontrast subtracted images ( arrow in a ) and enhancement - to - time curve ( b )  . 
2a , b carcinoma mammario alla rm mammaria a basso campo ( 0 , 2 t ) con 0 , 1 mmol / kg di gadobenato dimeglumina in paziente di 42 anni studiata in posizione supina : immagini coronali sottratte della prima fase dinamica ( freccia in a ) e curva dinamica ( b )  . 
la curva dinamica ( b ) mostra un enhancement precoce del 74% ( da 206 , 6 a 359 , 1 unit arbitrarie ) e wash - out postiniziale ( curva di tipo 3 )  . 
3a , b breast cancer detected at gadobenate - dimeglumine - enhanced ( 0.1 mmol / kg ) dynamic breast magnetic resonance with a 0.2 - t open magnet in a 82 - year - old patient studied in the supine position : coronal first postcontrast subtracted image ( arrows in a ) and percent enhancement - to - time curve ( b )  . 
3a , b carcinoma mammario alla rm mammaria a basso campo ( 0 , 2 t ) con 0 , 1 mmol / kg di gadobenato dimeglumina in paziente di 82 anni studiata in posizione supina : immagini coronali sottratte della prima fase dinamica ( frecce in a ) e curva dinamica ( b )  . 
however , because of technical difficulties encountered during these examinations ( long positioning time and relative instability of the patients position ) , the remaining 16 patients were placed in the informate dellimportanza di evitare di muoversi durante lindagine , in particolare durante lacquisizione dinamica che avrebbe seguito le sequenze di centratura . 
stato altres richiesto alle pazienti di respirare con regolarit . 274 radiol med ( 2009 ) 114 : 267285 supine position with free breasts ( no use of bands or strips ) and with the arms placed along the body . 
all patients were carefully informed about the importance of avoiding any movement during the examination , in particular during the dynamic acquisition following the initial scout sequence . finally , the patients were instructed to breathe quietly . in order to reduce overall imaging time , the examination was limited to the dynamic study following the preliminary three - plane localising sequence . 
a three - dimensional axial ( eight patients ) or coronal ( ten patients ) gradient - echo ( fast low - angle shot ) sequence was used with the following common technical parameters : te 10.6 ms ; flip angle 30 ; one excitation ; partition thickness 3 mm without interslice gap ; acquisition time 94 s . 
 the dynamic study was acquired continuously without any time interval between the preand post - contrast phases . at the end of the first ( precontrast ) scan , the same operator manually injected a single 0.1 mmol / kg of body weight dose of gadobenate dimeglumine at a rate of approximately 2 ml / s , followed by a 20 - ml saline flush at a rate of approximately 1 ml / s . 
 mr postprocessing and image evaluation postprocessing involved image subtraction ( postcontrast minus precontrast ) of all dynamic phases , generation of maximum intensity projection ( mip ) reconstructions for bilateral axial and coronal views and right and left lateral views . 
moreover , we dotained enhancement - to - time curves for one or more regions of interest positioned within the lesion on the area subjectively recognised as showing maximal enhancement on the subtracted images of the dynamic phase judged as the best phase for lesion conspicuity [ 25 ]  . all mr examinations were prospectively evaluated in consensus by a senior radiologist and a resident in radiology with 12 and 2 years experience in breast mr , respectively . they were blinded to the results of cbe , mammography and us . 
 [ 4 ] , taking into account , for the levels of initial enhancement , the higher r1 relaxivity al fine di ridurre il tempo - esame , lindagine stata limitata allo studio dinamico dopo la preliminare sequenza triplanare di centratura . 
stata utilizzata una sequenza tridimensionale gradient - echo ( fast low angle shot ) assiale ( 8 pazienti ) o coronale ( 10 pazienti ) con i seguenti parametri tecnici comuni : te = 10 , 6 ms ; flip angle = 30 ; una eccitazione ; spessore di partizione = 3 mm , senza interspazio ; tempo di acquisizione per fase dinamica time = 94 secondi . 
la sequenza assiale stata acquisita con un tr di 29 ms , 44 partizioni , campo di vista rettangolare 380285 mm e matrice dimmagine 256192 ( pixel di 1 , 481 , 48 mm )  . 
la sequenza coronale stata acquisita con un tr 23 ms , 32 partizioni , campo di vista rettangolare 380309 mm e matrice dimmagine 256192 ( pixel di 1 , 481 , 61 mm )  . 
alla fine della prima scansione ( precontrasto ) , sempre il medesimo operatore ha somministrato manualmente 0 , 1 mmol / kg di gadobenato dimeglumina ( multihance ; bracco imaging spa , milano ) a circa 2 ml / s e 20 ml di soluzione fisiologica a circa 1 ml / s . 
 post - processing e valutazione delle immagini il post - processing stato effettuato mediante sottrazione dimmagine ( post - contrasto meno pre - contrasto ) per tutte le fasi dinamiche , ricostruzioni con algoritmo di massima intensit bilaterali ( assiali e coronali ) e laterali ( destra e sinistra ) e calcolo delle curve dinamiche dellenhancement in funzione del tempo per una o pi regioni di interesse posizionate nel contesto della lesione sullarea soggettivamente riconosciuta come maggiormente contrastata alle immagini sottratte della fase dinamica giudicata ottimale per la cospicuit della stessa lesione [ 25 ]  . tutte le indagini rm sono state valutate prospetticamente , in consenso , da un radiologo con 12 anni di esperienza in rm mammaria e da uno specializzando con due anni di esperienza in rm mammaria , in cieco rispetto ai risultati di vs , mammografia ed ecografia . 
le indagini sono state inizialmente valutate per il livello di qualit e la presenza di artefatti sulle immagini native ( da movimento ) e sottratte ( insufficiente coregistrazione )  . 
 [ 4 ] , tenendo conto , per il livello di enhancement iniziale , della pi elevata relassivit r1 del mezzo di contrasto utilizzato in questo studio , ovvero adottando le soglie modificate < 100% , 100% 240% e > 240% invece delle soglie classiche < 50% , 50% 100% e > 100% , come descritto in letteratura [ 27 ]  . 
ne conseguito il radiol med ( 2009 ) 114 : 267285 of the contrast agent used in this study , that is , adopting the modified thresholds of < 100% , 100% 240% and > 240% instead of the classic thresholds < 50% , 50% 100% and > 100% , as previously described [ 27 ]  . 
this score was translated into the 15 bi - rads score , as previously described [ 9 ] , as follows : no detectable enhancement = no enhancing focus = bi - rads 1 ; score 02 = benign = bi - rads 2 ; score 3 = probably benign = bi - rads 3 ; score 45 = suspicious = bi - rads 4 ; score 68 = highly suggestive of malignancy = bi - rads 5 . 
lesions with a maximal bi - rads score of 3 ( probably benign ) at one or more imaging modalities were also examined histopathologically after surgical excision ( n = 1 ) or by us - guided fine needle aspiration cytology ( n = 2 )  . 
the patients with lesions assigned a maximal bi - rads score of 2 ( benign ) underwent annual follow - up for 24 years , with cbe and us until 39 years of age , also with mammography for older women . 
annual follow - up was also performed for the two bi - rads 3 lesions evaluated with us - guided fine - needle aspiration cytology . all surgical samples were evaluated according to the guidelines of the association of directors of anatomic and surgical pathology [ 28 ]  . 
all tumours were classified according to the world health organization ( who ) system [ 29 ]  . statistical analysis sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) and overall accuracy were calculated against a standard of reference comprising seguente sistema di valutazione di lesioni mammarie alla rm dinamica : forma ( rotondeggiante / ovale / lobulata = 0 ; lineare / dendritica / stellata = 1 ) ; margini ( definiti = 0 ; indefiniti = 1 ) ; pattern dellenhancement ( omogeneo = 0 ; disomogeneo = 1 ; anulare = 2 ) ; enhancement iniziale ( < 100% = 0 ; 100%240% = 1 ; > 240% = 2 ) ; enhancement postiniziale ( continuo = 0 ; plateau = 1 ; washout = 2 )  . 
questo punteggio stato tradotto in un punteggio bi - rads da 1 a 5 , come precedentemente descritto [ 9 ] , nel modo seguente : nessun enhancement identificabile = nessuna lesione con enhancement = bi - rads 1 ; punteggio 02 = lesione benigna = birads 2 ; punteggio 3 = lesione probabilmente benigna = birads 3 ; punteggio 45 = lesione sospetta per malignit = birads 4 ; punteggio 68 = lesione altamente sospetta per malignit = bi - rads 5 . 
 il diametro massimo di ciascuna lesione stato misurato elettronicamente dal primo autore sulle ricostruzioni mip ottenute dalle immagini sottratte assiali o coronali della fase dinamica considerata ottimale per la cospicuit della lesione . 
 correlazione radiologica - patologica tutte le lesioni sospette per malignit alla vs o classificate bi - rads 4 o 5 alla mammografia , allecografia o alla rm ( n = 13 ) sono state valutate istologicamente dopo asportazione chirurgica ( 4 lesioni non palpabili sono state centrate per mezzo di iniezione di sospensione di carbone sotto guida ecografica )  . 
lesioni con massimo punteggio birads 3 ( probabilmente benigno ) a una o pi delle modalit di imaging sono state valutate istologicamente dopo asportazione chirurgica ( n = 1 ) o mediante citologia su agoaspirato ecoguidato ( n = 2 )  . 
le pazienti che presentavano lesioni con massimo punteggio bi - rads 2 ( reperto benigno ) sono state sottoposte a follow - up da 2 a 4 anni con vs ed ecografia fino a 39 anni di et , anche con mammografia dopo i 39 anni di et . 
tutte le lesioni sono state valutate secondo la classificazione oms [ 29 ]  . analisi statistica sensibilit , specificit , valore predittivo positivo ( vpp ) , valore predittivo negativo ( vpn ) e accuratezza diagnostica sono stati calcolati rispetto allo standard di riferimento dato dalla combinazione tra esame istologico , citologia su agoaspirato e follow - up . 
sono state realizzate determinazioni separate per tutte e 23 le lesioni nelle 18 pazienti e 276 radiol med ( 2009 ) 114 : 267285 pathological examination , cytological examination and follow - up . 
 maximal diameters of the lesions measured on the mips were compared with those obtained on pathological specimens ; the comparison was presented using a bland - altman plot [ 30 ] , whereas the global agreement between the two measurements was calculated using the intraclass correlation coefficient [ 31 ]  . results diagnostic image quality was achieved for 15 / 18 ( 83% ) patients overall . 
images for the remaining 3 / 18 ( 17% ) patients ( all with claustrophobia ) were considered nondiagnostic due to patient movement and resulting misregistration artefacts during the dynamic phase of the mr examination . 
one of these was a 10 - mm invasive ductal carcinoma ( idc ) ( patient number 5 , lesion number 6 ) and another was a 20 - mm invasive tubular carcinoma ( patient 14 , lesion 19 )  . 
the third lesion ( patient 11 , lesion 16 ) was a 10 - mm cyst , as confirmed at us - guided fine - needle - aspiration cytology and negative follow - up . the final diagnosis of all lesions is listed in table 1 . 
one of these lesions ( patient 11 , lesion 16 ) was the 10 - mm cyst described above , whereas the others comprised a giant 45 - mm fibroadenoma ( patient 8 , lesion 11 ) that was surgically excised and a 20 - mm fibroadenoma ( patient 10 , lesion 15 ) characterised as benign on fineneedle - aspiration cytology and confirmed as benign at 2 - year follow - up with mammography and us . the morphologic and kinetic features of the lesions on low - field mr imaging are listed in table 2 . 
whereas all eight confirmed idcs showed morphological and kinetic parameters indicative of a malignant lesion , only three of five invasive lobular carcinomas ( ilcs ) , all in one patient ( patient 9 , lesions 12 , 13 and 14 ) showed morphological and kinetic parameters suspicious of malignancy . 
the other two ilcs demonstrated benign morphology but inhomogeneous enhancement with rapid wash - in followed by washout ( patient 2 , lesion 3 ) and suspicious morphology , together with inhomogeneous enhancement and intermediate washin followed by plateau ( patient 4 , lesion 5 )  . 
examples of dopo esclusione delle indagini di qualit non diagnostica . per gli indici di performance diagnostica sono stati calcolati gli intervalli di confidenza al 95% ( 95%ci ) secondo lapprossimazione della distribuzione binomiale alla distribuzione normale . il diametro massimale delle lesioni misurato alla rm sulle ricostruzioni mip stato comparato con quello istopatologico ( analisi di bland - altman [ 30 ] ) mentre laccordo globale tra le due misurazioni stato valutato mediante il coefficiente di correlazione intraclasse [ 31 ]  . risultati sul totale delle 18 indagini , 15 ( 83% ) sono state giudicate con qualit di immagine di livello diagnostico e vi sono state identificate 20 lesioni . 
la qualit di immagine ottenuta per le tre rimanenti 3 indagini ( 17% ) in 3 pazienti ( tutte claustrofobiche ) stata considerata non diagnostica a causa di artefatti da insufficiente coregistrazione dovuti a movimento della paziente durante lacquisizione dinamica . in queste tre pazienti mammografia e / o ecografia avevano identificato una lesione per ciascuna paziente ( tabella 1 ) : un carcinoma duttale invasivo ( cdi ) di 10 mm ( paziente numero 5 , lesione numero 6 ) ; un carcinoma tubulare invasivo di 20 mm ( paziente 14 , lesione 19 ) ; una cisti di 10 mm ( paziente 11 , lesione 16 ) confermata alla citologia su agoaspirato ecoguidato e follow - up negativo . la diagnosi conclusiva di tutte le lesioni elencata in tabella 1 . 
tre lesioni considerate bi - rads 3 sono state valutate mediante citologia su agoaspirato o istologia del pezzo operatorio : la cisti di 10 mm ( paziente 11 , lesione 16 ) descritta poco sopra , un fibroadenoma gigante di 45 mm ( paziente 8 , lesione 11 ) asportato chirurgicamente ; un fibroadenoma di 20 mm ( paziente 10 , lesione 15 ) risultato benigno alla citologia su agoaspirato e confermato come benigno al follow - up a due anni con mammografia ed ecografia . le caratteristiche morfologiche e dinamiche delle lesioni alla rm sono elencate in tabella 2 . 
mentre tutti gli otto cdi hanno mostrato caratteristiche morfologiche e dinamiche indicative di malignit , solo tre dei cinque carcinomi lobulari invasivi ( cli ) , tutti nella stessa paziente ( paziente 9 , lesioni 12 , 13 e 14 ) hanno mostrato caratteristiche morfologiche e dinamiche indicative di malignit . 
gli altri due cli hanno mostrato : uno morfologia di tipo benigno con enhancement disomogeneo , rapido wash - in seguito da wash - out ( paziente 2 , lesione 3 ) ; laltro morfologia sospetta con enhancement disomogeneo , wash - in intermedio seguito da plateau ( paziente 4 , lesione 5 )  . 
2 e 3 ( lesioni maligne )  . il confronto del diametro delle lesioni alla rm e allesame istologico ha mostrato un completo accordo per 9 / 14 lesioni asportate chirurgicamente ( 64% ) e una sottoradiol med ( 2009 ) 114 : 267285 mr diagnoses are shown in fig . 
2 and 3 ( malignant lesions )  . comparison between lesion diameter at mr imaging and that at pathological examination revealed full agreement for 9 / 14 ( 64% ) surgically excised lesions and an underestimation at mr imaging for 5 / 14 ( 36% ) lesions . 
for this latter determination , the two nonassessable malignant lesions on mr imaging ( one idc , one invasive tubular carcinoma ) were considered false negatives , whereas the nonassessable cyst was considered a false positive . 
however , many studies focused on the role of lowfield systems for preoperative localisation of breast lesions and for interventional procedures [ 1214 , 35 ] rather than for lesion detection and diagnosis . 
the long acquisition times ( primarily due to low - power gradient systems ) and low signal - to - noise ratios associated with these systems precluded their use for clinical dynamic imaging of the breast . more recently , pkk et al . 
investigated the potential clinical value of low - field breast mr imaging using a dynamic contrast - enhanced mr sequence in conjunction with an open 0.23 - t permanent magnet [ 15 ]  . 
in their study , they evaluated 16 malignant and 11 benign lesions with a mean lesion size of 2 cm and obtained sensitivity , specificity and accuracy values of 100% , 82% and 92% , respectively . 
interestingly , the corresponding specificity and accuracy values for the same patients examined using a 1.5t closed magnet were slightly worse ( 73% and 89% , respectively ) , even with an equal 100% sensitivity [ 15 ]  . however , a relatively high dose ( > 0.2 mmol / kg ) of a conventional contrast agent ( gadopentetate dimeglumine ) was considered necessary to achieve sufficient enhancement in this study . stima alla rm per le rimanenti 5 lesioni ( 36% )  . 
il diametro , misurato per 20 lesioni alla rm , risultato di 28 , 810 , 3 mm ( mediadeviazione standard ) con mediana di 20 m gli indici di performance diagnostica della rm mammaria a basso campo con gadobenato dimeglumina sono presentati in tabella 3 . 
se consideriamo solo le indagini di qualit diagnostica ( ovvero valutiamo 20 lesioni in 15 pazienti ) , sensibilit , specificit , vpp , vpn e accuratezza sono risultati tutti pari al 100% . 
se invece includiamo nellanalisi anche le tre indagini di qualit non diagnostica ( ovvero valutiamo 23 lesioni in 18 pazienti ) , gli indici di performance diagnostica sono risultati 86% , 87% , 93% , 78% e 87% , rispettivamente . 
in questultimo calcolo le due lesioni maligne non valutabili ( un cdi e un carcinoma tubulare invasivo ) sono considerate falsi negativi mentre la cisti non valutabile considerata come falso positivo . 
tuttavia , linteresse clinico si per lo pi focalizzato sul ruolo dei sistemi a basso campo per le biopsie e i centraggi prechirurgici rm - guidati [ 1214 , 35 ] piuttosto che sullidentificazione e caratterizzazione lesionale . 
i tempi di acquisizione prolungati ( dovuti innanzitutto alla scarsa potenza dei gradienti di campo ) e il ridotto rapporto segnale / rumore tipici di questi sistemi hanno precluso il loro utilizzo clinico per lo studio delle mammelle a contrasto dinamico . pi recentemente , pkk et al . 
 [ 15 ] hanno investigato il potenziale valore clinico della rm mammaria a contrasto dinamico a basso campo con un magnete aperto permanente operante a 0 , 23 t [ 15 ]  . 
 interessante notare come lo studio delle stesse pazienti con magnete chiuso operante a 1 , 5 t avesse dato una sensibilit egualmente elevata ( 100% ) ma livelli inferiori di specificit ( 73% ) e accuratezza ( 89% ) [ 15 ]  . 
tuttavia , in questo studio , gli autori hanno ritenuto necessaria una dose relativamente elevata ( maggiore di 0 , 2 mmol / kg ) di un mezzo di contrasto a bassa r1 relassivit ( gadopentetato dimeglumina )  . le potenzialit cliniche della rm mammaria a basso campo vanno correlate alle crescenti indicazioni , agli avanzamenti tecnologici dellhardware rm e , in particolare , alla rilevanza epidemiologica del numero di pazienti claustrofobiche o obese che necessiterebbero di unindagine da eseguirsi su sistemi a magnete chiuso . 
la claustrofobia da tempo un problema per alcuni dei pazienti che necessitano 278 radiol med ( 2009 ) 114 : 267285 radiol med ( 2009 ) 114 : 267285 280 radiol med ( 2009 ) 114 : 267285 fig . 
4 bland - altman comparison between magnetic resonance ( mr ) and pathological maximal diameters for 14 breast lesions . differences are obtained for each of the 14 lesions by subtracting the diameter measured at pathology from that measured at mr imaging . 
le differenze sono ottenute per ciascuna lesione sottraendo il diametro misurato allistopatologia a quello misurato alla rm . la visualizzazione di soli 11 punti dovuta alla perfetta sovrapposizione della differenza e dei valori medi per tre lesioni ( tabella 1 )  . mean ( mm ) table 3 diagnostic performance of breast magnetic resonance ( mr ) imaging performed at 0.2 t using a body coil and 0.1 mmol / kg of gadobenate dimeglumine in 18 claustrophobic or oversized patients tabella 3 performance diagnostica della rm mammaria eseguita a 0 , 2 t con bobina body e 0 , 1 mmol / kg di gadobenato dimeglumina in 18 pazienti claustrofobiche o obese index value 95% ci indice valore ic 95% sensitivity specificity positive predictive value negative predictive value overall accuracy 86% ( 13 / 15 ) 87% ( 7 / 8 ) 93% ( 13 / 14 ) 78% ( 7 / 9 ) 87% ( 20 / 23 ) 70%100% 65%100% 79%100% 51%100% 73%100% sensitibilit specificit valore predittivo positivo valore predittivo negativo accuratezza 86% ( 13 / 15 ) 87% ( 7 / 8 ) 93% ( 13 / 14 ) 78% ( 7 / 9 ) 87% ( 20 / 23 ) 70%100% 65%100% 79%100% 51%100% 73%100% numbers in parentheses were used to calculate the percentages . 
gli indici di performance diagnostica sono statti calcolati per tutte le 23 lesioni nelle 18 pazienti , considerando le tre indagini rm di qualit non diagnostica come due falsi negativi e un falso positivo ( tabella 1 : pazienti 5 , 11 e 14 ; lesioni numero 6 , 16 e 19 )  . ic , intervallo di confidenza the potential for low - field clinical mr imaging of the breast is related to the increasing indications for breast mr , to the technological advances in mr hardware and , importantly , to the number of claustrophobic or obese patients who require breast mr imaging . 
the increasing prevalence of obesity is an additional problem , not only given the practical problems associated with imaging oversized patients but also because obesity is an associated risk factor for breast cancer [ 36 ]  . 
in the usa , roughly 34% of the female population aged 2074 years were considered obese ( body mass index 30 ) in the period between 2001 and 2004 , whereas the prevalence of extreme obesity ( body mass index 40 ) among women in the same period was 6.9% [ 37 , 38 ]  . 
on this subject , it should be noted that our study was performed in italy , where the overwhelming majority of the population adopt a mediterranean lifestyle and diet and where the incidence of obesity is comparatively lower . 
lobesit un ulteriore problema , in notevole incremento , da valutare non solo in s ( per le difficolt tecniche che pu determinare per lesecuzione della rm mammaria su sistemi a magnete chiuso ) ma anche per il noto ruolo dellobesit quale fattore di rischio per il carcinoma mammario [ 36 ]  . 
negli usa , tra il 2001 e il 2004 , circa il 34% della popolazione femminile tra 20 e 74 anni era in condizioni di obesit ( body mass index maggiore o uguale a 30 ) e il 6.9% era in condizioni di estrema obesit ( body mass index maggiore o uguale a 40 ) [ 37 , 38 ]  . 
va notato che il nostro studio stato realizzato in italia , laddove la stragrande maggioranza della popolazione adotta stili di vita e dieta di tipo mediterraneo cui conseguono minori tassi di incidenza dellobesit . 
lincidenza di obesit grave cui consegue lesclusione dallaccesso alla rm su sistemi a magnete chiuso quindi verosimilmente maggiore negli usa laddove lobesit rappresenta un problema nazionale di dimensioni rilevanti [ 38 ]  . 
 radiol med ( 2009 ) 114 : 267285 closed magnets is likely to be much greater in the usa , where obesity is a national concern [ 38 ]  . 
 in terms of incidence , among the 397 consecutive patients evaluated over a 30 - month period at our institution , 18 patients ( approximately 5% ) of whom 15 ( 3.7% ) were claustrophobic and three ( 0.8% ) obese would otherwise have been excluded from evaluation with mr imaging had an open low - field mr system not been available . 
this percentage is not negligible ( it is equal to 1 in 20 ) , which may even be an underestimation given the likelihood of self - exclusion by claustrophobic or obese patients who know that breast mr is usually performed on closed magnets . 
although patient motion precluded the acquisition of diagnostic images in three women , overall diagnostic performance was good , with an overall accuracy of 87% for the 18 patients combined and an excellent correlation between the maximal lesion diameters measured on mr images and pathological specimens . 
 indications for breast mr imaging are rapidly expanding , even when filtered by critical analysis and consensus conferences among radiologists , surgeons , oncologists and other colleagues involved in breast diagnosis and care , as happened recently in italy [ 2 ]  . 
in addition to screening high - risk women [ 9 , 39 ] , breast mr is also used in clinical practice for presurgical staging [ 40 , 41 ] , for evaluating the effect of neoadjuvant chemotherapy on locally advanced cancers [ 42 , 43 ] , and for assessing equivocal breast findings on mammography and us [ 24 ] , even if this indication is under discussion and criticised [ 2 ]  . 
 our diagnostic performance values obtained using wellestablished morphological and kinetic assessment criteria are comparable with those determined elsewhere using conventional closed magnets and dedicated breast coils [ 4547 ]  . 
sebbene gli artefatti da movimento abbiano reso non diagnostica la qualit di immagine di tre indagini , la performance diagnostica complessiva stata buona , con unaccuratezza diagnostica globale dell87% su tutte e 18 le pazienti e uneccellente correlazione tra rm e istopatologia relativamente alle dimensioni lesionali . 
le recenti le indicazioni alla rm mammaria sono in rapida espansione , anche qualora sottoposte ad analisi critica e consensus conference tra radiologi , chirurghi , oncologi e altri specialisti del mondo senologico , com accaduto recentemente linee - guida dellamerican cancer society raccomandano la rm mammaria come modalit di screening per le donne con un rischio di tumore mammario nellarco di vita maggiore o uguale al 20%25% ( donne ad alto rischio genetico - familiare o esposte a radioterapia toracica tipicamente per il trattamento di linfomi tra 10 e 30 anni di et ) [ 39 ]  . 
oltre alla sorveglianza delle donne ad alto rischio [ 9 , 39 ] , la rm mammaria sempre pi utilizzata per la stadiazione prechirurgica [ 40 , 41 ] , per la valutazione delleffetto della chemioterapia neoadiuvante dei tumori localmente avanzati [ 42 , 43 ] e anche per la valutazione di reperti dubbi alla mammografia e allecografia [ 24 ] , anche se questultima indicazione oggetto di discussione e non esente da critiche [ 2 ]  . 
considerata la crescente importanza della rm mammaria e i limiti delle tecniche convenzionali in alcune situazioni ( per esempio lo studio mammografico delle mammelle ad elevata densit [ 8 , 44 ] ) , la rm mammaria dovrebbe essere accessibile a tutte le pazienti che non abbiano controindicazioni assolute allindagine . 
 i livelli di performance diagnostica da noi ottenuti utilizzando criteri standardizzati per la valutazione morfologica e dinamica sono comparabili a quelli altrimenti ottenuti con gli usuali magneti chiusi e bobine dedicate [ 4547 ]  . 
con leccezione delle tre indagini di qualit non diagnostica , tutte le lesioni maligne , inclusi cinque cli , sono stati correttamente diagnosticate utilizzando un mezzo di contrasto ad alta relassivit . 
the value of this agent lies in its increased r1 relaxivity in blood compared with other standard gadolinium agents [ 17 , 18 , 50 ] derived from weak and transient interaction of the gdbopta contrast - effective moiety of this agent with serum albumin [ 51 ]  . 
this results in markedly increased enhancement compared with that achievable with standard agents at an equivalent dose of 0.1 mmol / kg body weight and thus seems to improve breast lesion detection and visualisation [ 19 , 23 ]  . 
an association between one - sided increased breast vascularity and ipsilateral invasive cancer has been shown with gadobenate dimeglumine [ 20 , 52 ]  . the greater enhancement achievable with gadobenate dimeglumine could be important when using low - field magnets with inherently reduced signal - to - noise ratio [ 53 ]  . an alternative approach to achieving adequate enhancement would be to use a high dose of conventional gadolinium agent . 
this approach was recently adopted by pkk et al . , who administered 30 ml of gadopentetate dimeglumine ( corresponding to a dose of 0.21 mmol / kg body weight for a 70 - kg subject ) to women undergoing low - field breast mr imaging [ 15 ]  . 
however , the use of high doses is currently a matter of debate among the radiological community given the recently reported association between the administration of gd - chelates and the development of nephrogenic systemic fibrosis in patients with severe renal insufficiency [ 54 , 55 ]  . 
 our study suffered a series of important limitations concerning both the study design and the mr technique . these include the lack of a direct ( intraindividual ) comparison with data deriving from mr examinations conducted at higher field strength and the small number of patients and lesions evaluated . 
sebbene alcuni studi abbiano gi mostrato una superiore performance diagnostica della rm mammaria con gadobenato a 1 , 0 e 1 , 5 t [ 19 , 20 , 23 ] , per quanto a nostra conoscenza , questa la prima esperienza di rm mammaria con questo mezzo di contrasto a basso campo . 
il vantaggio fondamentale del gadobenato sta nella sua maggiore relassivit r1 nel sangue rispetto agli usuali mezzi di contrasto a base di gadolinio [ 17 , 18 , 50 ]  . 
ci determina un marcato aumento dellenhancement rispetto a quello ottenibile con gli usuali mezzi di contrasto a dosi equivalenti ( 0 , 1 mmol / kg ) cui sembra conseguire una superiore capacit di identificazione e visualizzazione delle lesioni mammarie [ 19 , 23 ]  . 
laumentata relassivit r1 consente altres un maggiore enhancement della vascolarizzazione mammaria ; lassociazione tra incremento asimmetrico della vascolarizzazione mammaria e tumore invasivo ipsilaterale stata infatti dimostrata con gadobenato [ 20 , 52 ]  . la possibilit di ottenere un maggiore enhancement con gadobenato potrebbe giocare un ruolo rilevante quando la rm mammaria realizzata con magneti a basso campo che producono immagini con rapporto segnale / rumore intrinsecamente ridotto [ 53 ]  . 
un approccio alternativo per ottenere un enhancement adeguato potrebbe essere luso di alte dosi di mezzi di contrasto paramagnetici a bassa relassivit . tale approccio stato recentemente utilizzato da pkk et al . 
 [ 15 ] che hanno somministrato 30 ml di gadopentetato dimeglumina ( corrispondenti a una dose di 0 , 21 mmol / kg per un peso corporeo di 70 kg ) a pazienti che eseguivano rm mammaria a basso campo [ 15 ]  . 
tuttavia , luso di alte dosi oggi molto discusso nella comunit radiologica in considerazione dellassociazione recentemente riportata tra la somministrazione di chelati del gadolinio e linsorgenza di fibrosi nefrogenica sistemica in pazienti con insufficienza renale grave [ 54 , 55 ]  . 
nonetheless , despite these limitations , our study shows that breast mr imaging on low - field open systems can be considered a possibility for claustrophobic or obese women who cannot undergo mr study at higher field strength on closed magnets . 
the availability of a dedicated breast coil should , however , be considered a requirement for future studies exploring the clinical use of lowfield open - unit breast mr imaging . given the potential for worldwide dissemination of lowfield magnets for breast mr screening programmes ( at least for high - risk women ) , further studies are needed to fully evaluate the potential of low - field open magnets with dedicated breast coils , possibly by means of direct ( intraindividual ) comparison with findings on higher field closed systems . 
moreover , low - field open breast mr imaging offers interesting possibilities for mr - guided breast biopsy and presurgical localisation [ 12 , 5659 ] as well as for reducing overall costs and possibly making breast mr imaging accessible to more women and more imaging centres . 
these include the recently available closed wide - bore or open high - field scanners [ 60 , 61 ] and mr examination under sedation or anaesthesia for claustrophobic women . 
 in conclusion , our preliminary experience showed that dynamic breast mr imaging of claustrophobic or oversized patients can be performed successfully on a low - field 0.2 - t open magnet using a single 0.1 mmol / kg body weight dose of gadobenate dimeglumine as a contrast agent . 
it is important to note that this technical combination can be used for claustrophobic or oversized patients only and not as a substitute for standard 1.5 - t breast mr imaging in normal subjects . 
further larger studies using dedicated breast coils are required to validate our general hypothesis regarding the value of breast mr imaging using open low - field magnets . insufficiente rappresentazione dello spettro di malattia . 
dal punto vista tecnico , il limite maggiore stato luso di una bobina body non dedicata ( la bobina dedicata per lo studio delle mammelle non era commercialmente disponibile per il nostro sistema aperto a basso campo )  . 
la risoluzione spaziale relativamente bassa ( dimensioni del pixel da 2 , 2 a 2 , 4 mm2 ) potrebbe avere limitato la valutazione della morfologia delle lesioni , limite che potrebbe essere superato disponendo di una bobina dedicata e utilizzando matrici dimmagine maggiori e spessori di partizione ridotti ( anche prolungando il tempo di acquisizione fino a 120 secondi )  . 
comunque , anche considerando questi limiti , il nostro studio mostra che la rm mammaria con magneti aperti a basso campo pu essere considerata una possibilit per le donne claustrofobiche o obese che non possono eseguire lindagine a pi alto campo con magneti chiusi . tuttavia , noi riteniamo che la disponibilit della bobina dedicata debba essere considerata necessaria per futuri studi sulluso clinico dei sistemi aperti a basso campo per la rm mammaria . date le potenzialit che i magneti a basso campo potrebbero offrire per la diffusione di programmi di screening mammario con rm ( almeno per le donne ad alto rischio ) , occorrono studi con disegno comparativo intraindividuale che mettano a confronto indagini a basso campo con indagini ad alto campo con magneti chiusi . 
inoltre , magneti aperti a basso campo offrono interessanti potenzialit per lesecuzione di biopsie e centraggi rm - guidati [ 12 , 5659 ] e per una sensibile riduzione dei costi economici che renderebbe la rm mammaria disponibile in un maggiore numero di centri e per un maggiore numero di donne . 
 infine opportuno tenere presente anche che possibili alternative per le donne claustrofobiche o obese possono essere date dalla recente disponibilit di sistemi chiusi con ampie dimensioni di accesso o sistemi aperti a campo relativamente alto [ 60 , 61 ]  . 
 in conclusione , la nostra esperienza preliminare ha mostrato che la rm mammaria in donne claustrofobiche o obese pu essere eseguita con successo utilizzando un sistema a magnete aperto a basso campo ( 0 , 2 t ) e 0 , 1 mmol / kg di gadobenato dimeglumina . 
importante sottolineare che questa combinazione tecnica pu essere utilizzata solo per pazienti claustrofobiche o obese e non pu in alcun modo essere proposta come alternativa allindagine standard a 1 , 5 t in soggetti normali . 
guglielmi2 , 5 1department of human physiology , university of rome tor vergata , via montpellier 1 , 00173 rome , italy . 2department of radiology , university of foggia , viale luigi pinto 1 , 71100 foggia , italy 3department of radiology , university of rome tor vergata , via montpellier 1 , 00173 rome , italy 4department of orthopaedics and traumatology , university hospital policlinico tor vergata rome , italy 5department of radiology , scientific institute hospital css , viale cappuccini 1 , san giovanni rotondo , 71013 foggia , italy correspondence to : g . 
this review discusses the application of dual - energy x - ray absorptiometry ( dxa ) on bc determination , given that dxa has the potential to provide overall and regional assessment of bc in terms of fat , lean mass and bone . 
dxa is widely used in many clinical settings primarily diagnosis osteoporosis . this article describes the use of whole - body dxa in assessing bc in patients with chronic diseases ( e.g. metabolic syndrome ) as well as in different sport activities to evaluate the effects of exercise . keywords total - body dxa body composition body fat lean body mass bone mineral density riassunto la valutazione della composizione corporea , da qui in avanti denominata con il termine anglosassone di body composition ( bc ) , un metodo essenziale per analizzare lo stato di salute in termini nutrizionali , sia a livello di popolazione sia a livello individuale , come pure per indagare lefficacia delle strategie nutrizionali preventive primarie e secondarie . 
questi tuttavia possono manifestarsi anche nel soggetto anziano , per il quale il trascorrere degli anni pu causare significativi cambiamenti nel rapporto tra grasso corporeo e muscolo . per misurare la composizione corporea esistono numerose tecniche ; alcune semplici , ma con margini di errore ancora elevati , altre invece costose e sofisticate ( come per esempio la valutazione a livello atomico e molecolare della bc ) ma pi affidabili nel rilevamento dei risultati a vari livelli . 
questa revisione della letteratura riguarda lapplicazione della tecnica dual - energy x - ray absorptiometry ( dxa ) per la determinazione della composizione corporea , in quanto tale metodica permette di misurare sia in maniera totale che segmentale , per singoli distretti , i vari componenti quali : massa grassa , massa magra e tessuto osseo . 
in questo articolo verranno descritte le altre possibili radiol med ( 2009 ) 114 : 286300 indicazioni , tra cui lo studio della bc nei pazienti con sindromi metaboliche croniche e lapplicazione in condizioni fisiologiche , quali per esempio nello sportivo , per valutare leffetto dellesercizio fisico sulla massa corporea . parole chiave dxa composizione corporea massa grassa massa magra densit minerale ossea introduction introduzione two parallel trends exist in the developed world in an increasing percentage of the population : ageing and obesity . debilitating illnesses such as cardiovascular disease and diabetes are common to both obesity and ageing , and osteoporosis , in particular , is the most common metabolic bone disease and indeed one of the most prevalent disorders associated with ageing [ 1 , 2 ]  . because obesity is becoming a worldwide epidemic , there is an increasing interest in the study of body composition ( bc ) both overall and regional to monitor the condition and delay the development of obesity - related diseases such as cardiovascular disease and type 2 diabetes [ 3 ]  . frailty and sarcopoenia may represent the advanced stages of age - related bc changes . 
it has been reported that loss of skeletal muscle mass occurs with advancing age in elderly men and women , even in independently living healthy subjects , and that men lose significantly more leg skeletal muscle mass than do women [ 4 ]  . furthermore , skeletal muscle mass loss in men is masked by weight stability resulting from a corresponding increase in total body fat mass ( fm )  . 
age - related changes in bc , including the increase and redistribution of fat tissue and the decrease in skeletal muscle and bone mass , begin as early as the fourth decade of life . 
preventive and therapeutic options for optimising bone mass and bc in old age is central to the care of patients in midlife and beyond . because of the high impact of osteoporotic fractures on morbidity and mortality and indirectly on health care costs , early and accurate diagnosis of these bc changes is of major importance [ 1 , 6 , 7 ]  . 
there is general agreement that lifestyle changes focused on improving dietary intake and increased daily physical activities are the cornerstones in both preventing and treating obesity and the metabolic syndrome [ 8 ]  . 
authors have shown a positive effect of exercise on bc , especially in reducing menopausal risk factors in women [ 9 ] and enhancing treatment efficacy in older nei paesi industrializzati si sta assistendo allo sviluppo di due tendenze che aumentano parallelamente nella popolazione , ossia obesit e invecchiamento . 
in particolare , losteoporosi risulta la patologia metabolica dellosso associata con maggiore prevalenza allavanzare dellet [ 1 , 2 ]  . per quanto riguarda lobesit , poich sta diventando una patologia a diffusione quasi epidemica , diventa necessario nei pazienti coinvolti effettuare una valutazione della composizione corporea ( bc ) anche distrettuale , per monitorare landamento dellaffezione e cercare di ritardare lespressione di patologie associate quali diabete e disturbi cardiovascolari [ 3 ]  . 
laffaticamento muscolare , il rischio di caduta , le limitazioni funzionali , limmobilit e di conseguenza le fratture da fragilit , sono tutte condizioni connesse alla perdita di massa muscolare . 
come noto , dalla letteratura si evince che la riduzione della massa muscolare scheletrica un processo fisiologico legato al progredire dellet che coinvolge entrambi i sessi indipendentemente dallo stato di salute . 
bisogna sottolineare inoltre che anche la bc soggetta a cambiamenti che iniziano allincirca dalla quarta decade di vita , nel senso di un incremento e ridistribuzione del tessuto adiposo e di una riduzione della massa muscolare e ossea . 
 necessario perci mettere in campo misure preventive e attuare trattamenti terapeutici mirati per fasce det . in considerazione dellalto impatto che le fratture da osteoporosi hanno sulla morbilit , sulla mortalit e di riflesso sui costi sanitari correlati , risulta necessario attuare una diagnosi precoce e attendibile dei suddetti 288 radiol med ( 2009 ) 114 : 286300 total 1 . , 0 - 2 , 5 fracture risk increased non increased high t - score vs . 
for this reason , and because bc is known to have a significant effect on athletic performance and because exercise has the potential to alter bc , there is considerable interest in the evaluation of bc in exercise and sport [ 11 , 12 ]  . 
nonetheless , fluctuations in body weight alone cannot be adequately understood unless they are interpreted as quantitative variations of the fat - free mass ( ffm ) , fm and total body water ( tbw ) components , as each component varies independently . 
most centres use axial dual - energy x - ray absorptiometry ( dxa ) of the spine , lumbar spine and nondominant proximal hip , as the hip is considered the most suitable site for predicting fracture risk [ 7 ] and the spine the most suitable for monitoring treatment effects [ 14 ] , as indicated by the world health organization , which also established that the results should be reported as a t - score for osteoporosis . 
esiste comunque un consenso comune sul fatto che un corretto stile di vita abbinato a una regolare attivit fisica e ad unalimentazione corretta e bilanciata , si configurano quali fattori fondamentali nella prevenzione delle sindromi metaboliche [ 8 ]  . 
alcuni autori hanno dimostrato leffetto benefico dellesercizio fisico svolto in modo regolare sulla bc soprattutto per quanto concerne la riduzione dei fattori di rischio legati alla menopausa nelle donne [ 9 ] cos come nei superobesi con diabete alimentare nellaumentare gli effetti terapeutici [ 10 ]  . 
per questo motivo , permane un considerevole interesse per la valutazione della bc associata allattivit fisica agonistica e non , in quanto noto che la bc ha un effetto significativo sulla prestazione atletica . 
non di meno , le fluttuazioni del peso corporeo non possono essere adeguatamente interpretate se non come variazioni quantitative delle seguenti componenti : massa magra ( fat free mass , ffm ) , massa grassa ( fat mass , fm ) e acqua corporea totale ( total body water , tbw )  . 
ci accade perch il femore risulta essere il migliore sito predittivo di rischio di frattura [ 7 ] mentre la colonna il miglior sito per esaminare landamento della terapia [ 14 ] , come da indicazione della who che si riferisce al valore espresso in t - score per effettuare la diagnosi di osteoporosi . 
nella scelta del metodo bisogna considerare , oltre ai costi legati allo strumento stesso e al personale deputato allutilizzo , leventuale esposizione del soggetto a radiazioni , il tempo necessario a ottenere linformazione richiesta e , non ultimo , laccuratezza dellinformazione ( tabella 1 )  . radiol med ( 2009 ) 114 : 286300 fig . 
il distretto addominale e le regioni prossimali degli arti inferiori possono essere definite manualmente , mentre altre sedi sono determinate automaticamente dal software , sebbene possano essere successivamente modificate . methods of assessing body composition depending on the information needed , several methods can be used to measure bc , each with some advantages and limitations [ 16 , 17 ]  . 
in selecting the method , one has to take into account cost ( equipment and personnel ) , possible radiation exposure , time required to obtain the information and accuracy of the information obtained ( table 1 )  . bc measurement is of interest to clinicians , nutritionists and physiologists . 
the most frequently used methods are based on a two - component model comprising fm and ffm , because the amount of fat in the body is of special nutritional interest . 
the three most common methods used to calculate bc based on the two - component model are underwater weighing ( uww ) , bioelectrical impedance le figure cliniche maggiormente interessate ai risultati della valutazione della bc sono linternista , il nutrizionista e il fisiologo . 
i metodi pi comunemente utilizzati si basano su modelli a due compartimenti che comprendono la massa grassa ( fat mass , fm ) e la massa magra ( fat free mass , ffm ) in quanto linteresse principale naturalmente la misurazione del grasso corporeo . 
i tre pi comuni metodi usati per calcolare la bc sul modello a due compartimenti sono la pesata idrostatica ( uww ) , lesame impedenziometrico ( bia ) e la plicometria ( sk )  . questo modello presuppone che nella ffm sia compresa una quantit fissa di acqua , proteine e minerali . 
tuttavia , bisogna tenere in considerazione il fatto che la massa ossea , lacqua e le proteine variano per ciascun individuo e sono influenzati dallet , dalletnia , dal sesso , dai fattori genetici , cos come da elementi modificabili quali la dieta e 290 radiol med ( 2009 ) 114 : 286300 analysis ( bia ) and skinfold thickness measurements . 
gli studi effettuati in passato sulla valutazione della bc si basavano per la maggior parte su misurazioni antropometriche indirette come lindice di massa corporea ( bmi ) e le circonferenze corporee . 
il bmi , la circonferenza vita e fianchi , con relativo rapporto ( whr ) , sono dati antropometrici indiretti , comunemente utilizzati come indice di obesit nelladulto . queste misure , essendo semplici da eseguire e standardizzate , si configurano come ideali per la compilazione di studi epidemiologici . 
 [ 19 ] hanno evidenziato che la dxa e due modelli di bia davano risultati simili di massa grassa in uomini e donne table 1 methods for assessing body composition techniques pros cons body mass index ( bmi ) free ; fast ; predictor of life expectancy skinfold thickness measurements convenient ; fast ; portable ; useful for detecting changes in percentage of body fat waist - hip ratio ( whr ) fast ; free with tape measure bioelectrical impedance analysis ( bia ) inexpensive ; portable ; fast ; accurate for estimating total body water in populations if height , weight and other variables are added underwater weighing ( uww ) relatively accurate ; very consistent ( good for measuring changes in percentage of body fat ) dual - energy x - ray absorptiometry ( dxa ) most precise ; most accurate ; fast ; gives information on total and regional percentage of fat does not give precise assessment of percentage of body fat for athletes . 
possible causes of error are posture ( height ) , hydration and bowel status ( weight ) requires precise and consistent technique ; not accurate for very thin or very fat people ; only measures fat under the skin ; not an accurate estimate of actual percentage of body fat . 
 possible sources of error are dehydration , bladder status , temperature , fat asymmetry , arm position , etc . uncomfortable ; wet ; bulky apparatus ; skimpy outfit required ; time consuming ; expensive ; hard to find ; bone density assumption causes error . 
possibile fonte di errore sono : deidratazione , stato di replezione vescicale , temperatura , asimmetria del tessuto adiposo , posizione degli arti , ecc . poco confortevole perch in acqua ; attrezzatura voluminosa ; tempi lunghi desame ; costosa ; difficile da trovare ; non calcola la densit dellosso . 
however , the differences depend on gender and body - weight status , indicating the importance of considering these factors when identifying people with excess fm [ 19 ]  . in addition to total body measurements , the interest of clinicians and researchers has also focused on regional assessment of bc . 
the application of techniques such as ct and mri to bc analysis over the last two decades has enabled the anatomical delineation and quantification of several types of adipose tissue depots [ 20 ]  . unfortunately , the routine use of ct and mri in bc con diverso grado di obesit e di attivit fisica . 
poich le differenze erano correlate al sesso e al peso corporeo , gli autori hanno sottolineato lincidenza di tali fattori nella valutazione di persone con eccesso di massa grassa [ 19 ]  . oltre alle valutazioni corporee totali , lattenzione del clinico particolarmente rivolta ai distretti corporei : i metodi di riferimento utilizzabili sono la risonanza magnetica ( rm ) e la tomografia computerizzata ( tc )  . 
sfortunatamente , lutilizzo di routine della tc e rm nella valutazione della bc ostacolato dai costi elevati , inoltre , la tc richiede una significativa esposizione a radiazioni 292 radiol med ( 2009 ) 114 : 286300 fig . 
results of the total - body scan are calculated automatically for bone mineral density ( bmd ) , fat tissue , and lean tissue for the total body and subregions . 
b evaluation of total bmd and bone mineral content ( bmc ) ( left ) , soft tissue ( middle ) and fat - mass distribution expressed as a percentage with a colour map ( right )  . 
b valutazione della bmd totale e del bmc ( sulla sinistra ) , dei tessuti molli ( al centro ) e della distribuzione del tessuto adiposo espressa in percentuale con una mappa a colori ( sulla destra )  . analysis is hampered by access and cost . 
 [ 23 ] compared abdominal adipose tissue measured by mri and dxa in nonobese men and defined dxa regions of interest ( rois ) in two different ways ( between the second and fourth lumbar vertebra or the iliac crest ) and found both of these regions comparable with mri total abdominal adiposity and mri - derived narrow abdominal slices . 
 [ 24 ] reported that dxa yielded accurate measurements of abdominal adipose tissue compared with ct in overweight / obese individuals weighing 66134 kg . dxa divides the body into three components : bone , fatfree and bone - free tissue , and fat [ 14 , 25 ]  . 
 [ 23 ] hanno misurato il tessuto adiposo addominale in uomini non obesi con la dxa e con la rm , definendo le regioni di interesse della dxa in due differenti modi ( tra la seconda e la quarta vertebra lombare e a livello delle creste iliache )  . 
quindi , per la dxa , che attualmente si configura come il metodo pi usato per misurare la densit minerale ossea , si prevede un utilizzo su larga scala anche per analisi distrettuali e totali della bc , in virt della sua accuratezza diagnostica ed economicit . 
la dxa ancora oggi una delle tecniche pi utilizzate per la valutazione della bc come risulta dallincremento della disponiradiol med ( 2009 ) 114 : 286300 result of the increasing worldwide availability of these scanners . 
inoltre , la metodica suscita interesse in quanto non invasiva , a basso rischio di radiazione e relativamente facile da eseguire sia per loperatore che per il soggetto che vi si sottopone . 
bene sottolineare che gli scanner di seconda generazione consentono una maggiore velocit di scansione e una minore esposizione alle radiazioni ionizzanti [ 15 , 19 , 26 ]  . principles of dxa , advantages and limitations principi della dxa , vantaggi e limitazioni the major commercial manufacturers of bone densitometers employ a subtly different technology ; however , our further discussion does not address the particulars of each technology and / or manufacturer . 
each system uses a source that generates x - rays at two energies : a detector , and an interface with a computer system for imaging the scanned areas of interest . 
dxa employs an x - ray tube and filtres to produce one low and one high pseudomonoenergetic beathe x - ray tube may be operated at constant potential or switched between two potentials . 
soft tissue , consisting largely of water and organic compounds , reduces photon flux to a much lesser extent than does bone mineral , and pixels containing bone are relatively easily distinguished from those with no bone present . 
however , hydration can vary from 67% to attualmente le principali ditte costruttrici di densitometri impiegano nella realizzazione degli scanner una tecnologia molto simile , in ogni caso la seguente discussione non riguarda nessuna delle diverse tecnologie in particolare . ciascun sistema utilizza una sorgente che genera raggi x a doppio livello energetico , un detettore , e uninterfaccia con un sistema computerizzato per la visualizzazione delle aree di interesse . 
viene effettuata una scansione rettilinea del corpo in posizione supina che divide il corpo in una serie di pixel , in cui ciascuno delle attenuazioni fotoniche misurata ai due livelli energetici . 
lapproccio della dxa per la misurazione della bc assume che il corpo consiste di tre componenti che sono distinguibili in base alle loro propriet di attenuazione : massa grassa , tessuto osseo e massa magra . 
allinterno di ciascun pixel le proporzioni di soli due componenti possono essere ottenute dal differente grado di assorbimento della doppia energia fotonica . i tessuti molli , composti principalmente da acqua e da componenti organici , riducono il flusso di fotoni molto meno del tessuto osseo , ed relativamente facile distinguere i pixel contenenti osso da quelli che non lo contengono . nelle zone dove losso non presente sono necessarie ulteriori calibrazioni per distinguere la componente grassa da quella magra appartenente ai tessuti molli . 
4 il software per lanalisi della composizione corporea ( body composition analysis , bca ) permette una valutazione totale e distrettuale della distribuzione della massa grassa , massa magra , massa totale e del bmc . 
 dxa software enables one to determine bone mineral and soft - tissue composition in different regions of the body . thus , bone mineral can be determined by scanning the spine , hip , forearm or whole body . 
these whole - body scans can then be analysed to measure the bc in defined regions of these scans , for example , in the arms , legs and trunk . 
thus , it is becoming increasingly clear that different skeletal regions , even those that have similar quantities of trabecular or cortical bone , may respond variably in different physiological situations [ 3135 ]  . the precision of a particular dxa device for assessing whole bc is generally good , with coefficients of variation of about 1% for bmc and 2%3% for total body fat [ 36 ]  . for this reason , dxa has been used to determine both longterm change in bc with ageing [ 4 ] and short - term change in bc as a result of intervention [ 3739 ]  . 
however , in regions such as the thorax , arm and head , the percentage of bone - free pixels is lidratazione tuttavia pu invece variare dal 67% al 85% . se un soggetto contiene pi della met della quantit di acqua , alcune scansioni dxa sovrastimeranno il contenuto di grasso [ 28 ]  . 
sebbene una variazione dellidratazione dal 68 , 2% al 78 , 2% non alteri significativamente la percentuale di grasso [ 29 ] una severa iperidratazione , cos come nellascite o edema , pu inficiare il risultato della percentuale di grasso [ 30 ]  . il software dellapparecchiatura dxa in grado di determinare la densit minerale ossea e la valutazione dei tessuti molli in differenti regioni del corpo umano . 
infatti , comincia ad essere evidente che , in differenti situazioni fisiologiche , si possono ottenere risposte non uguali nelle diverse regioni scheletriche comprese quelle che hanno la medesima distribuzione di trabecole [ 3135 ]  . la dxa possiede una precisione di scansione per la valutazione della bc con un coefficiente di variazione di circa 1% per il contenuto minerale osseo e del 2%3% per il contenuto totale di grasso corporeo [ 36 ]  . 
per questo motivo , la dxa stata usata per determinare cambiamenti a lungo termine della bc con let [ 4 ] e a breve termine come risposta al trattamento farmacologico [ 3739 ]  . in tutte le misure dxa si assume che la quantit di grasso sullosso lo stesso di quello intorno allosso . 
tuttavia , nelle regioni come il tronco , le braccia e la testa , la percentuale radiol med ( 2009 ) 114 : 286300 much lower and may be insufficient for accurate soft - tissue and hence bone determinations to be made [ 40 ]  . during whole - body scans , measurements are made over a very wide range of tissue depths . 
during the last few years , manufacturers have introduced major software upgrades as they have become aware of inaccuracies in their systems . physiological and clinical applications with ageing , changes in bc have an adverse impact on mortality and morbidity , as well as on quality of life . 
the study of the effects of ageing on bc measurements will enable a better understanding of the relative contribution of obesity and sarcopoenia morbidity and mortality in older adults [ 5 , 18 ]  . 
 several changes in bc may occur with ageing , including a reduction in fat - free tissue mass and an increase in fm , leading to a higher percentage of body fat at any given body weight in older adults . 
similarly , repositioning of fat to a more central distribution and loss of muscle bulk in the gluteal region will increase waist circumference and whr without an increase in fm . 
these and other age - related changes ( such as loss of skin elasticity , body cell mass and abdominal muscle bulk , and an increase in thoracic kyphosis ) may affect the validity of indirect anthropometric measures in determining bc [ 4 , 5 , 37 , 43 ]  . loss of skeletal muscle mass with ageing increases an individuals risk for sarcopoenia , which is characterised by low skeletal muscle mass , reduced muscle strength and increased risk for adverse health outcomes such as falls , fractures , impaired physical functioning , disability and frailty in older populations [ 22 ]  . 
 [ 18 ] studying 351 men ( mean age 63.5 years ) and 380 women ( mean age 62.1 years ) observed a trend towards sarcopoenia with dei pixel privi di osso sar molto bassa e quindi insufficiente per unaccurata analisi del tessuto molle [ 40 ]  . durante la scansione total body , le misure sono effettuate in un ampio spettro di spessore del tessuto . 
lo studio degli effetti dellet su ciascuna misura della composizione corporea sar capace di offrire in futuro una migliore conoscenza del contributo relativo dellobesit e sarcopenia sulla morbilit e mortalit dei soggetti in et adulta [ 5 , 18 ]  . tra i diversi cambiamenti che avvengono nella bc con lavanzare dellet sono inclusi una riduzione della massa magra ( ffm ) e un incremento della massa grassa che comporta una maggiore incidenza della percentuale di grasso corporeo sul peso totale delladulto . 
similmente , la ridistribuzione centrale del grasso e la riduzione della massa muscolare in regione glutea saranno responsabili dellaumento della circonferenza e whr senza un corrispondente incremento del tessuto adiposo . 
tutti i cambiamenti finora elencati ( quali la perdita di elasticit cutanea , della massa cellulare e della massa muscolare e unaccentuazione della cifosi dorsale ) possono alterare la validit di misure antropometriche indirette associate alla bc [ 4 , 5 , 37 , 43 ]  . la perdita di massa muscolare con lavanzare dellet incrementa il rischio di sarcopenia cui consegue la perdita della forza muscolare e di seguito il rischio di caduta , quindi di frattura , disabilit e fragilit nella popolazione anziana [ 22 ]  . 
 [ 18 ] hanno condotto uno studio su 351 uomini ( di et media 63 , 5 anni ) e 380 donne ( di et media 62 , 1 anni ) e hanno osservato , soprattutto negli uomini , un trend in ascesa della sarcopenia con let . 
such changes include agerelated loss of gluteal muscle bulk , kyphotic posture and loss of abdominal muscle tone , and shift of fm from a peripheral to a more central distribution . 
these changes may lead to an increase in whr in the absence of an increase in total body fat [ 4 , 18 ]  . it is well known that excess of intra - abdominal or visceral adipose tissue ( iaat or vat ) is related to the risk of developing coronary heart disease and type - 2 diabetes . researchers have suggested that iaat , independent of obesity severity , is a good predictor of the associated abnormalities of metabolic syndrome . 
their use could help in the early detection of abdominal / visceral obesity [ 23 , 44 , 45 ]  . most recent studies using new types of scanners and updated software [ 38 , 40 ] support the capability of dxa to measure small changes in bc . 
furthermore , a recent study in 76 postmenopausal women followed for 1 year showed that dxa was a more sensitive method for assessing small changes in bc compared with uww and a multicomponent model [ 38 , 47 ]  . 
they also provide the android / gynoid fat ratio and colour maps of the percentage of fat , thus helping to determine body fat distribution an important risk factor in a variety of serious diseases . in clinics it is important to know individuals bc , because in addition to the concept that bc naturally changes with age , physical activity and disease , fat is associated with health , and lean body mass is a better indicator of fitness and health than is weight , and increased fitness may reduce body fat but not reduce weight . a decrease in physical activity may lead to an increased loss of bone mineral and an increase in the incidence of osteoporotic fractures . 
questi cambiamenti possono portare ad un incremento di whr in assenza di un aumento del grasso corporeo totale [ 4 , 18 ]  . gi noto che leccesso di tessuto adiposo intra - addominale o tessuto viscerale ( iaat or vat ) correlato al rischio di sviluppare patologie cardiovascolari e diabete mellito di tipo 2 . 
alcuni studi hanno identificato per le scansioni dxa alcune regioni di interesse ( roi ) che possono essere validamente misurate usando siti anatomici specifici per la valutazione della distribuzione del tessuto adiposo . 
il loro utilizzo faciliterebbe la diagnosi precoce di obesit addominale / viscerale [ 23 , 44 , 45 ]  . studi recenti hanno evidenziato che limpiego di nuovi tipi di scanner e software aggiornati , supportano la capacit della dxa di misurare anche piccoli cambiamenti nella bc [ 38 , 40 ]  . 
inoltre , uno studio recente effettuato per un anno su 76 donne in stato post - menopausale , ha dimostrato che la dxa il metodo pi sensibile per valutare piccoli cambiamenti nella bc confrontato con la pesata idrostatica e con il modello multicompartimentale [ 38 , 47 ]  . 
 particolari dxa scanner sono inoltre in grado di esaminare soggetti gravemente obesi e di calcolare automaticamente i risultati raddoppiando i valori ottenuti da una semivalutazione total body , cos come la percentuale di grasso androide / ginoide e la mappa a colori della percentuale di grasso . 
ci pu aiutare a determinare con accuratezza la distribuzione di questultimo , acquisendo informazioni aggiuntive su quello che rimane un importante fattore di rischio per unampia variet di patologie . in clinica importante conoscere la composizione corporea perch , oltre al concetto che la bc si modifica con let , lattivit fisica e in condizioni patologiche , la quantit di grasso associata allo stato di salute e la distribuzione della massa magra un migliore indice predittivo rispetto al peso ; lincremento di massa magra pu infatti ridurre la quantit di grasso , ma non il peso corporeo totale . 
 una diminuzione dellattivit fisica pu condurre ad un aumento della perdita di massa ossea e un incremento radiol med ( 2009 ) 114 : 286300 athletes , especially those who are strength - trained , have greater bmd than do nonathletes and that strength , muscle mass and maximal oxygen uptake correlate with bone density [ 47 ]  . the factors associated with attaining optimal bmd and bmc have not been clearly identified . 
furthermore , physical activity has an effect on bc , especially skeletal muscle , which is the largest nonadipose tissue component and which plays an important role in physical activity and many biochemical processes . 
recent studies have confirmed that dxa can be used as a method for estimating total body muscle mass in healthy men and women of different age groups and that dxa can be used for estimating appendicular muscle mass ( amm ) [ 11 ]  . 
 [ 11 ] comparing three groups of athletes judo , karate and water polo with an ageand gendermatched control group , have shown that the athletes had significantly greater bmd and amm than the did nonathletes of similar age . 
in particular , weightbearing activities , such as walking , have a greater effect than nonweightbearing activities , such as cycling and swimming , whereas a reduction in mechanical loading , e.g. , bed rest or space flight , leads to bone loss . 
it has been previously suggested that the type of physical activity necessary to build and maintain bone density must be weightbearing , in part , because the loss of ambulation or weightlessness results in marked skeletal atrophy . 
within the athletic groups , however , water polo players had the highest amm but a lower total body bmd and an intermediate level of bmd for the arms , legs and trunk compared with the judo and karate groups , suggesting that the amm alone is not entirely responsible for the increased bmd in the athletic individuals . 
 [ 12 ] found significant regional tissue differences , measured by dxa , between soccer forwards and backs , which may be related to playing function , as well as differences between rugby players and controls . 
studi crosssectional mostrano che gli atleti , specialmente quelli che praticano allenamenti di tipo agonistico , hanno maggiore bmd rispetto ai sedentari ; la forza muscolare , la massa e il massimo consumo di ossigeno sono correlati alla bmd [ 47 ]  . i fattori associati al conseguimento di una bmd e bmc ottimale non sono stati chiaramente identificati . 
lattivit fisica ha un effetto sulla composizione corporea ( bc ) , specialmente sulla muscolatura scheletrica , che il pi vasto componente tissutale non - adiposo e riveste un importante ruolo nellattivit fisica e in alcuni processi biochimici . 
 [ 11 ] hanno valutato e messo a confronto tre gruppi di atleti praticanti judo , karate e water polo , con un gruppo controllo avente le medesime caratteristiche antropometriche . 
lo studio ha mostrato che gli atleti presentavano un valore maggiormente significativo di bmd e amm rispetto ai controlli di stessa et ; in particolare stato notato che le attivit svolte sotto carico , come per esempio camminare , hanno un maggiore effetto rispetto a quelle svolte fuori carico , come il ciclismo e il nuoto , mentre una riduzione nel carico meccanico , per esempio nellimmobilit a letto o nellattivit in assenza di gravit , conducono alla perdita di massa ossea . 
in precedenza stato suggerito che il tipo di attivit fisica relativo alla stimolazione della formazione ossea e al mantenimento dovrebbe essere svolta in condizione di carico , perch in condizioni opposte si arriva allatrofia scheletrica . 
considerando il gruppo di atleti , i praticanti water polo avevano un maggiore valore di amm ma un pi basso valore di bmd in total body , un livello intermedio di bmd per gli arti superiori , inferiori e al tronco comparato con i gruppi praticanti judo e karate , suggerendo che il valore di amm da solo non interamente responsabile dellincremento della bmd negli atleti . 
una variet di altri fattori pu essere responsabile delle differenze osservate , per esempio , variazioni nellintroito alimentare potrebbero determinare differenze nel valore energetico totale , del calcio e vitamina d [ 11 ]  . le informazioni fornite dallanalisi della bc su gruppi di atleti pu essere un importante indicatore dello stato nutrizionale e della prestazione fisica . 
 [ 12 ] hanno riscontrato differenze significative nella valutazione dei tessuti per distretti corporei , misurati con esame dxa , tra giocatori di calcio che ricoprivano in campo ruoli diversi . tali difformit possono essere correlate alla funzione di gioco cos come appare tra i giocatori di rugby e i controlli , 298 radiol med ( 2009 ) 114 : 286300 players , the correlation between age and fm was significant , whereas there was no correlation between fat and age in control subjects . 
in addition , soccer athletes who begin a season with a high level of fitness can maintain , and in some cases improve , bc and physical performance from before to after a competitive season . 
a correct combination of soccer - specific practices and strength and conditioning programmes can maintain and develop physical performance , allowing a soccer athlete to perform optimally throughout pre - , in - , and postseason play [ 51 ]  . 
data in the literature indicate that caution is necessary when dxa is used to compare patients with control subjects or to assess changes in bc in subjects whose relative weight changes significantly between measurements . 
it is often used in clinical settings , as well as to compare various bc methods for assessing body fat in overweight and obese children . in conclusion , bc assessments by dxa are readily available , less expensive and less invasive compared with other diagnostic imaging techniques , such as mri and ct . 
dxa is a simple , safe and precise technique that can measure bmd and soft - tissue composition not only in the whole body but also in specific regions of the body . this makes it an important tool not only for assessing and managing osteoporosis , but also for studying how softtissue composition changes in healthy and disease conditions . 
in aggiunta , i calciatori che cominciano una stagione con un alto livello di allenamento possono mantenere , e in alcuni casi migliorare , attraverso una valutazione della bc , la prestazione fisica prima e dopo la stagione calcistica . 
 necessaria una combinazione tra lattivit pratica calcistica specifica e un programma che mantenga e sviluppi la prestazione fisica [ 51 ]  . conclusioni la dxa stata designata per effettuare le misurazioni sulla massa ossea ed stato ampiamente dimostrato come questa sia una metodica accurata e precisa per tale utilizzo . inoltre , questa tecnica inizia ad essere utilizzata anche per la valutazione della bc . 
i dati presenti in letteratura suggeriscono una certa cautela nellimpiego di questa metodica nei confronti tra pazienti affetti da patologie e i controlli , come anche nella valutazione dei cambiamenti della bc in soggetti che presentano significative variazioni del peso tra le valutazioni . 
 la dxa offre facili e veloci informazioni circa il grasso corporeo , la massa magra e la massa ossea e , per la sua accuratezza , considerata un metodo di indagine superiore ad altri . 
non a caso utilizzata spesso nelle valutazioni cliniche , nella comparazione della bc con gli altri metodi per la misurazione del grasso corporeo , anche quello dei bambini obesi e sovrappeso . 
 per concludere , la valutazione della composizione corporea con metodica dxa prontamente disponibile , poco costosa e meno invasiva se confrontata con le altre metodiche diagnostiche come rm e tc . 
caudana1 1diagnostic imaging unit , 2vascular surgery unit , 3orthopaedics and traumatology unit , carlo poma hospital , viale albertoni 1 , 46100 , mantua , italy correspondence to : r . 
if diagnostic uncertainty persists , a bone biopsy is indicated . the inflammatory hyperaemia caused by the ulcer deteriorates the diagnostic quality of 40%50% of mr angiography studies in the infrapopliteal region . 
sedici pazienti diabetici sono stati sottoposti da gennaio 2006 a settembre 2007 a rm del piede per sospetta osteomielite monolaterale ; in 3 / 16 vi erano alterazioni radiografiche da osteo - artropatia neuropatica di charcot . 
la rm ha elevata sensibilit per losteomielite nel piede diabetico , ma pi bassa specificit dovuta soprattutto allosteo - artropatia neuropatica di charcot ; se permane dubbio diagnostico indicata la biopsia ossea . 
liperemia flogistica causata dallulcera deteriora il 40%50% degli studi angio - rm a livello sottopopliteo : in tali casi appropriata larteriografia selettiva per la possibilit di effettuare angioplastica nella stessa seduta . 122 radiol med ( 2009 ) 114 : 121132 keywords diabetic foot osteomyelitis foot mri peripheral vascular disease mr angiography parole chiave piede diabetico osteomielite rm piede vasculopatia periferica angio - rm introduction introduzione diabetic foot complications caused by neuropathy , ischaemia and resulting infection often lead to lower limb amputation , with severe disability and a significant impact on mortality . 
early recognition of osteomyelitis is essential for guiding medical and surgical treatment , which are usually implemented in an inpatient hospital setting [ 1 ]  . ( 89% ) the diagnosis of the clinical diagnosis of the infected foot is problematic in diabetic patients : up to two - thirds of patients may not present with the classic symptoms of deep infection owing to immunodeficiency related to diabetes mellitus . even laboratory examinations are often of little help , as inflammation indices may be absent [ 2 ]  . 
bone probing through skin ulceration is a clinical test with high positive predictive value foot osteomyelitis , although its low sensitivity ( 66% ) means that negative findings do not exclude bone infections [ 3 ]  . 
as a result , diabetic patients with suspected foot osteomyelitis require musculoskeletal imaging , both for the differential diagnosis particularly with neuropathic osteoarthropathy ( charcot foot ) and for an accurate evaluation of the extension of the infection , so as to plan surgery and avoid recurrences . 
moreover , diabetologists recommend that diabetic patients with foot ulcers should always undergo a vascular study to exclude the possibility that healing is prevented by peripheral arterial disease , which often has an insidious clinical onset due to the absence of neuropathic pain [ 4 ]  . our retrospective study presents the modern imaging approach to the infected diabetic foot , emphasizing the important role of magnetic resonance imaging ( mri ) in musculoskeletal and vascular assessment performed with the objective of selecting the most appropriate treatment . le complicanze del piede diabetico causate da neuropatia , ischemia e conseguente infezione portano spesso ad amputazione darto inferiore con grave disabilit e rilevante impatto anche sulla mortalit . 
lulcera del piede rappresenta in genere il primo segnale di allarme nel paziente diabetico e tende a progredire ( 15% dei casi ) verso losteomielite , in prevalenza con meccanismo di diffusione per contiguit . 
il riconoscimento precoce dellinsorgenza di osteomielite essenziale per indirizzare il trattamento sia medico che chirurgico , entrambi in genere attuati in ambiente ospedaliero [ 1 ]  . linquadramento clinico del piede infetto risulta difficile nel paziente diabetico : infatti fino a 2 / 3 dei pazienti possono non presentare i sintomi classici di infezione profonda a causa del deficit immunitario legato al diabete mellito ; anche gli esami di laboratorio spesso non sono di aiuto in quanto gli indici di flogosi possono essere assenti [ 2 ]  . 
il sondaggio osseo con specillo attraverso lulcera cutanea rappresenta un test clinico con elevato valore predittivo positivo ( 89% ) per la diagnosi di osteomielite del piede , ma la sua negativit non esclude linfezione ossea a causa della limitata sensibilit ( 66% ) [ 3 ]  . 
dunque nei pazienti diabetici con sospetta osteomielite del piede le metodiche di imaging muscolo - scheletrico sono necessarie sia per la diagnosi differenziale in particolare con losteo - artropatia neuropatica del piede di charcot sia per laccurato bilancio di estensione dellinfezione , cos da pianificare lintervento chirurgico ed evitare le recidive . 
inoltre , i diabetologi raccomandano di sottoporre sempre a studio vascolare il piede diabetico ulcerato , per escludere che larteriopatia periferica con esordio clinico spesso insidioso per lassenza di dolore dovuta alla neuropatia possa impedire la guarigione dellinfezione [ 4 ]  . lo scopo del nostro studio retrospettivo quello di presentare il moderno approccio imaging al piede diabetico infetto , sottolineando limportanza del ruolo che la risonanza magnetica ( rm ) ha avuto nella nostra esperienza nella valutazione muscolo - scheletrica e vascolare ai fini della scelta terapeutica ottimale . materials and methods materiali e metodi between january 2006 and september 2007 , 16 diabetic patients ( 11 men and five women , mean age 58 years , range 4278 ) with unilateral infected ulcer affecting the forefoot in ten cases , the midfoot in two and the hindfoot in four ( table 1 ) underwent mri of the foot . 
all patients underwent plain radiography of the foot , which was positive for osteomyelitis in 7 / 16 patients owing to the presence of corticoperiosteal thickening and / or of osteolytic foci in the cortical spongiosa close to the skin ulcer . 
tutti i pazienti sono stati sottoposti ad esame radiografico del piede , ritenuto positivo per osteomielite in 7 / 16 pazienti in base alla presenza di ispessimento cortico - periosteale e / o di focolai di osteolisi cortico - spongiosa in adiacenza allulcera cutanea . 
t1 - weighted spin - echo all musculoskeletal mri examinations were conducted with a 1.5 - tesla superconductive unit ( magnetom avanto : siemens ag medical solutions , erlangen , germany ) and an extremity coil . 
the mri protocol , selected on the basis of clinical suspicion and ulcer site , was different for studies of the forefoot compared with those of the midfoot and hindfoot and used a flex surface coil , a smaller field of view ( fov ) and predominantly coronal scans perpendicular to the metatarsal bones as recommended in the literature ( se : tr / te / fa 775 / 10 / 90 ) sequences and short - tau inversion - recovery sequences ( stir : tr / te / ti 5160 - 5760 / 53 / 150 ) were obtained in at least two planes , one of which was always sagittal , for selective fat suppression . 
in a few cases , a double t2and proton - density - weighted turbo - spin - echo sequence ( tse : tr / te / fa 3380 / 11103 / 150 ) was also obtained in the sagittal plane . 
parameters included 3 - mm slice thickness , 0.3 - mm interval for the t1 - weighted se sequences and 0.9 - mm interval for the stir sequences , and 16to 20 - cm fov . 
enhancement was assessed with a t1 - weighted tse sequence with selective ( tse - fs : tr / te / fa 791 / 10 / 150 ) with the same localization parameters as the stir sequences . fat suppression based on the suspicion of peripheral arteriopathy , 12 / 16 patients with infected diabetic foot also underwent mr angiography of the lower limbs with paramagnetic contrast agent ( table 1 ) 3 days to 6 weeks after mri of the foot , using the same scanner . 
this technique , through parallel imaging with dedicated phasedarray coils and high performing gradients , allows acquisithree peripheral arterial regions ( aortoiliac , tion of femoropopliteal , infrapopliteal ) within 42 s only ( excluding table motion )  . 
t1 - weighted 3d fast gradient - recalled - echo ( 3d fast - gre ) sequences were acquired in the coronal plane before and after administration of contrast material to allow subsequent subtraction . 
gadoliniumbenzyloxyproprionic - tetraacetic acid ( gd - bopta ) ( multihance , bracco , milan , italy ) at a dose of 0.15 mmol / kg , equivalent to 0.3 ( 3 / 9 oss . ) erano prevalenti le alterazioni dellosteo - artropatia neuropatica di charcot ( osteorarefazione , fratture , frammentazione ossea , lussazione , reazione osteoblastica riparativa )  . 
un paziente con ulcera del retropiede ed esame radiografico negativo stato sottoposto a biopsia ossea tcguidata del calcagno per interpretare il quadro rm dubbio . la diagnosi finale di osteomielite o la sua esclusione stata ottenuta integrando i rilievi clinico - laboratoristici con quelli imaging in tutti casi ; in 11 / 16 pazienti la diagnosi stata completata con esame batteriologico e / o istologico . tutti gli esami rm muscolo - scheletrici sono stati effettuati con apparecchiatura superconduttiva da 1 , 5 tesla ( magnetom avanto , siemens ag medical solutions , erlangen , germania ) e bobina da estremit . 
il protocollo rm , scelto sulla base del sospetto clinico e della sede dellulcera , stato diverso per lo studio dellavampiede rispetto a quello del medio e retropiede , impiegando bobina flex di superficie , fov pi piccolo e acquisizioni prevalenti sul piano coronale ovvero perpendicolare ai metatarsi come raccomandato in letteratura [ 5 ]  . 
sono state acquisite almeno in due piani , di cui uno sempre sagittale sequenze spin - echo t1 - pesate ( se : tr / te / fa 775 / 10 / 90 ) , sequenze short - inversion - time inversion - recovery per la selettiva soppressione del segnale adiposo ( stir : tr / te / ti 51605760 / 53 / 150 )  . 
in alcuni casi stata acquisita anche la sequenza turbo - spin - echo a doppia pesatura t2 e densit protonica ( tse : tr / te / fa 3380 / 11103 / 150 ) sul piano sagittale . 
sono stati impiegati spessore di fetta 3 mm , intervallo di 0 , 3 mm per le se t1 - pesate e di 0 , 9 mm per le stir , fov compreso tra i 16 cm ed i 20 cin tutti i pazienti lo studio rm stato completato con somministrazione di mezzo di contrasto ( mdc ) paramagnetico per via endovenosa ( dotarem , guerbet , aulnay - sous - bois , francia ) alla dose di 0 , 1 mmol / kg equivalente a 0 , 2 ml / kg ; lenhancement stato valutato con sequenza turbo - spin - echo t1 - pesata a selettiva soppressione del segnale adiposo ( tse - fs : tr / te / fa 791 / 10 / 150 ) con gli stessi parametri di centratura delle sequenze stir . nel sospetto di arteriopatia periferica 12 / 16 pazienti con piede diabetico infetto hanno effettuato anche lo studio angio - rm degli arti inferiori con mdc paramagnetico ( tabella 1 ) , a distanza di tempo variabile ( range : 3 giorni6 settimane ) dallesame rm del piede , utilizzando la stessa apparecchiatura rm . 
langio - rm degli arti inferiori stata effettuata con tecnica bolus - chase a tavolo mobile [ 6 ] : tale tecnica , grazie alla disponibilit dellimaging parallelo con bobine phased - array dedicate e di gradienti pi performanti , consente lacquisizione di 3 distretti arteriosi periferici ( aorto - iliaco , femoro - popliteo , infra - popliteo ) nellarco di soli 42 secondi ( escluso il movimento del tavolo )  . 
le sequenze fast - gradient - recalled - echo tridimensionali ( 3d fast - gre ) t1 - pesate sono state acquisite sul piano coronale prima e dopo somministrazione di mdc per consentire la successiva sottrazione . 
la somministrazione per via endovenosa di gadolinio - bopta ( multihance , bracco , milano , italia ) alla dose di 0 , 15 mmol / kg , equivalente a 0 , 3 ml / kg , radiol med ( 2009 ) 114 : 121132 ml / kg , was administered as an intravenous bolus ( 20 ml on average ) using a two - phase flow injector ( the first half at 1.2 ml / s , the second half at 0.6 ml / s ) , followed by the injection of 20 ml of saline at a flow rate of 1 ml / s . 
acquisition of the 3d gre sequences started when the care bolus technique detected the arrival of the contrast bolus in the aorta . during the postprocessing stage , after the subtraction process , panoramic coronal maximum intensity projection ( mip ) reconstructions were obtained from the aorta to the foot arteries by creating a composite image of the three regions . three radiologists retrospectively reviewed the mri images from the musculoskeletal study of the foot ( ur , ev , rc ) and the vascular study ( ur , at , rc )  . 
the most experienced radiologist ( rc > 15 years ) was considered the reference standard in the event of disagreement among less experienced radiologists ( ur , ev , at < 5 years )  . 
a primary sign of osteomyelitis on mri is evidence of low - signal - intensity areas in the bone marrow on t1 - weighted se images , with higher signal intensity on stir images and enhancement after contrast administration . 
secondary signs are identified close to the altered bone marrow signal and include oedema caused by septic inflammation ( cellulitis or phlegmon ) , soft - tissue abscess , skin ulcer and fistula , with possible interruption of the cortical bone . 
mr angiography of the lower limbs assessed the patency or stenosis - occlusion of the peripheral arteries and the presence of venous contamination artifacts superimposed enhancement of the venous circulation which is one of the main limitations of the technique , as it deteriorates the diagnostic quality of the images . 
with this assessment , the images of each of the three vascular regions were judged as either adequate or inadequate for peripheral revascularization . avvenuta in bolo ( in media 20 ml ) tramite iniettore con velocit di flusso bifasica ( la prima met a 1 , 2 ml / s , la seconda met a 0 , 6 ml / s ) , seguita dalla iniezione di 20 ml di soluzione salina con flusso di 1 ml / s . 
la diagnosi rm di osteomielite stata formulata sulla base della concordanza di segni primari e secondari [ 7 ] : la semeiotica rm della osteomielite considera come segno primario la presenza a livello del tessuto osteo - midollare di aree di ridotta intensit di segnale nelle immagini se t1pesate , con incremento dellintensit di segnale in quelle stir ed enhancement dopo somministrazione di mdc paramagnetico , mentre i segni secondari sono quelli rilevabili in adiacenza allalterazione del segnale osteo - midollare e comprendono ledema dovuto alla flogosi settica ( cellulite o flemmone ) , lascesso dei tessuti molli , lulcera cutanea e la fistola con leventuale interruzione della corticale ossea . nelle indagini angio - rm degli arti inferiori sono state valutate la perviet o la steno - occlusione delle arterie periferiche e la presenza di artefatti da contaminazione venosa ovvero lenhancement sovrapposto del circolo venoso uno dei principali limiti in quanto riduce la qualit diagnostica dellesame . 
i reperti angio - rm evidenziati sulle ricostruzioni mip sono stati analizzati per conferma anche nelle sequenze sottratte con software 3d ; cos valutate , le immagini di ciascuno dei tre distretti vascolari esaminati sono state giudicate sufficienti o insufficienti per la scelta del trattamento di rivascolarizzazione periferica . 
 results risultati in 13 / 16 patients with infected diabetic foot ulcers , the final diagnosis was osteomyelitis , whereas in 3 / 16 patients it was cellulitis ( table 1 )  . 
a the sagittal short - tau inversion - recovery sequence ( stir ) image shows a pathological fracture of the calcaneus and dislocation of the proximal fragment due to retraction of the achilles tendon . 
b magnetic resonance angiography ( mra ) of the right lower limb is hindered by venous contamination in the distal third , whereas the left infrapopliteal arteries are clearly visible up to the plantar arch . 
the mri diagnosis of exclusion of osteomyelitis proved correct in two patients with infected ulcer and cellulitis ( true negatives ) in whom conventional radiology had identified alterations due to neuropathic osteoarthropathy . 
overall , foot mri permitted correct identification or exclusion of osteomyelitis in 15 / 16 patients , with a high accuracy and sensitivity , even in the presence of radiographic evidence of neuropathic osteoarthropathy ( three cases )  . 
mri also proved useful for assessing the extent of osteomyelitis for surgical planning purposes ( four minor and one major amputation ) , for identifying soft - tissue abscesses requiring drainage ( three cases ) as well as other infectious complications including septic arthritis ( four cases ) , pathological fracture ( one case , fig . 1a ) and septic tenosynovitis ( one case )  . non da flogosi settica del tessuto osteo - midollare . 
2a - c reactive bone marrow oedema of the talus and calcaneus due to neuropathy in a diabetic patient with mal perforans and normal radiographic findings [ false - positive magnetic resonance imaging ( mri ) diagnosis of osteomyelitis ]  . 
a the coronal t1 - weighted turbo - spin - echo ( tse ) image with fat suppression after administration of paramagnetic contrast material reveals diffuse changes in signal intensity of bone marrow and soft tissues , as well as moderate contrast enhancement in the talus and in the subtalar portion of the calcaneus , wrongly interpreted as osteomyelitis associated with cellulitis . 
b in the axial short - tau inversion recovery ( stir ) image , the area of increased signal intensity in the calcaneus is identified to perform a bone biopsy . 
2a - c edema osteo - midollare reattivo talo - calcaneare da neuropatia in paziente diabetico con mal perforante e quadro radiografico normale ( errore rm di falsa positivit per la diagnosi di osteomielite )  . 
a nellimmagine coronale tse t1 - pesata con soppressione del segnale adiposo dopo somministrazione di mdc paramagnetico diffuse alterazioni del segnale osteo - midollare e dei tessuti molli in sede talare e nella porzione subtalare del calcagno caratterizzate da discreto enhancement , erroneamente interpretate come osteomielite associata a cellulite . 
c la biopsia ossea sotto guida tc ha escluso linfezione ossea . regions , the diagnostic patients with infected diabetic foot allowed evaluation of the diagnostic quality of the images obtained in the three vascular regions . 
these five patients later underwent selective arteriography of the ulcerated lower limb with an anterograde approach , owing to a suspicion of concomitant distal diabetic arterial disease ( table 1 )  . 
in 4 / 5 patients , arteriosclerotic occlusive lesions of the crural arteries were identified , and percutaneous transluminal angioplasty ( pta ) was performed during the same session . after the mr angiography , 9 / 12 patients underwent three of whom had a surgical vascular surgery , effettuati in 12 pazienti con piede diabetico infetto ha permesso di valutare la qualit diagnostica delle immagini ottenute in ciascuno dei tre distretti vascolari esaminati . 
questi 5 pazienti sono stati successivamente sottoposti ad arteriografia selettiva con approccio anterogrado dellarto inferiore affetto dallulcera , nel sospetto di concomitante arteriopatia diabetica distale ( tabella 1 )  . 
in 4 / 5 pazienti sono state riscontrate lesioni arteriosclerotiche steno - occlusive delle arterie crurali e si proceduto ad angioplastica ( pta ) nella stessa seduta . complessivamente 9 / 12 pazienti sono stati sottoposti dopo lo studio angio - rm a intervento vascolare , di cui 3 mediante by - pass chirurgico femoro - popliteo e 6 con tratta128 radiol med ( 2009 ) 114 : 121132 fig . 
3a - c severe charcot neuropathic osteoarthropathy , infected plantar ulcer and bone infarcts due to peripheral arterial disease [ osteomyelitis excluded by magnetic resonance imaging ( mri ) ]  . 
3a - c grave osteo - artropatia neuropatica di charcot , ulcera infetta plantare e infarti ossei da arteriopatia periferica ( rm negativa per osteomielite )  . a nel radiogramma in incidenza antero - posteriore frammentazione ossea del medio - piede in fase cronica con tipica frattura - dislocazione di lisfranc . 
liperintensit di segnale a livello del primo e secondo dito dovuta ad un artefatto ( insufficiente soppressione del segnale adiposo alla periferia della bobina da estremit )  . femoropopliteal bypass , and six underwent endovascular angioplasty ( three the femoropopliteal and infrapopliteal regions , three in the infrapopliteal region only ) with immediate technical success in 5 / 6 cases . in both mento endovascolare di angioplastica ( 3 a livello sia femoro - popliteo che infra - popliteo , 3 solo in sede infrapoplitea ) con successo tecnico immediato in 5 / 6 casi . discussion conventional radiology is still the first - line modality in the study of the diabetic foot owing to its rapidity , ease of use and inexpensiveness . 
it also allows the diagnosis of clinically overt osteomyelitis , reveals alterations due to charcots neuroarthropathy and is useful for the follow - up . known limits of conventional radiology , especially if performed in the early stage of bone infection , are its low sensitivity ( 60% ) and specificity ( 66% ) [ 8 ]  . 
the former is due to a need for 30%50% bone reabsorption , a process taking up to 23 weeks from the onset of infection , whereas the latter depends on the wide spectrum of diseases entering the differential diagnosis , first of all neuropathic discussione lindagine radiologica convenzionale rimane la metodica di primo livello per lo studio del piede diabetico per rapidit , facilit di esecuzione e bassi costi : oltre a permettere la diagnosi di osteomielite nella fase conclamata rileva le alterazioni da osteo - artropatia neuropatica di charcot ed utile per il follow - up . 
i limiti noti dellindagine radiologica , specie se espletata nelle prime fasi dellinfezione ossea , sono la bassa sensibilit ( 60% ) e specificit ( 66% ) [ 8 ] ; la prima dovuta alla necessit di un riassorbimento osseo pari al 30%50% , processo che impiega fino a 23 settimane dallinizio dellinfezione , mentre la seconda dipende dallampio spettro di patologie coinvolte nella diagnosi differenziale , prima fra tutte losteo - artropatia neuropatica che colpisce fino al 3% dei pazienti diabetici . 
the midfoot and hindfoot bones are the most commonly involved by this disease , which presents with demineralization , fractures , fragmentation , joint dislocation and collapse of the plantar arch , with the classic charcot deformity . 
the use of more sophisticated imaging modalities ( either mri or scintigraphy ) is indicated when radiography demonstrates alterations due to charcot neuroarthropathy or whenever the clinicallaboratory suspicion of osteomyelitis is high , or finally to demonstrate complications . 
bone biopsy , although not routinely performed because of its invasiveness , is considered the gold standard in the diagnosis of osteomyelitis and is still recommended to confirm an uncertain diagnosis and isolate the pathogen [ 9 ]  . mri has come to replace ct , as it is better able to identify bone marrow changes and evaluate septic involvement of the soft tissues , which allows surgery to be planned in such a way as to prevent recurrence of infection after amputation or abscess drainage [ 10 ]  . 
it is thus reserved for patients with contraindications to mri or with doubtful mri findings , or to assess response to antibiotic therapy during the follow - up [ 9 ]  . 
when there is topographic concordance between the primary and secondary mri signs , the mean reported sensitivity and mediopiede e del retropiede sono quelle pi spesso coinvolte da questa patologia che si manifesta con demineralizzazione , fratture , frammentazione , lussazione articolare fino al collasso dellarcata plantare , con la classica deformit del piede di charcot . 
il ricorso a metodiche di imaging pi sofisticate ( rm o scintigrafia ) indicato quando lesame radiologico riscontri alterazioni da osteo - artropatia neuropatica di charcot oppure qualora il sospetto di osteomielite sia elevato dal punto di vista clinico - laboratoristico o , infine , per evidenziare le complicanze dellinfezione . 
la biopsia ossea , bench non praticata di routine a causa dellinvasivit , tuttoggi considerata il gold standard per la diagnosi di osteomielite e viene dunque ancora raccomandata per confermare una diagnosi dubbia e per isolare lagente patogeno [ 9 ]  . la rm oggi ha sostituito la tc per la migliore capacit di identificare le alterazioni osteo - midollari e di valutare il coinvolgimento settico dei tessuti molli , ci che consente di pianificare gli interventi chirurgici al fine di evitare recidive infettive dopo amputazione o drenaggio di ascessi [ 10 ]  . la scintigrafia con granulociti tecneziati presenta accuratezza lievemente inferiore a quella della rm nella diagnosi di osteomielite [ 11 ] e risulta pi indaginosa e costosa ; viene attualmente pertanto riservata ai pazienti che hanno controindicazioni alla rm oppure in quelli con quadro rm dubbio o , infine , nel follow - up per valutare la risposta alla terapia antibiotica [ 9 ]  . 
la rm rappresenta pertanto lindagine preferenziale di secondo livello per la diagnosi di osteomielite : qualora vi sia concordanza topografica tra segni rm primari e secondari i valori medi di sensibilit e 130 radiol med ( 2009 ) 114 : 121132 specificity are 94% ( range 88%99% ) and 83% ( range 78%89% ) , respectively [ 2 , 7 ]  . 
on the other hand , a positive mri study is not always sufficient to establish a diagnosis of osteomyelitis , even when there is topographic concordance between primary and secondary signs . 
administration of paramagnetic contrast material increases specificity because enhancement of bone marrow , as assessed in the t1weighted sequences with selective fat suppression , is regarded as typical of osteomyelitis rather than of neuropathic osteoarthropathy [ 12 ]  . 
nonetheless , some authors have found that neuropathy - related reactive bone marrow oedema often associated with trabecular microfractures and joint effusion may also enhance , thus becoming indistinguishable from osteomyelitis [ 13 ]  . 
 the differential diagnosis between osteomyelitis and neuropathic osteoarthropathy is not a problem when the skin is intact but only when stress - related biomechanical alterations promote skin ulceration and the foot appears swollen , warm and erythematous . 
 in the chronic phase of neuropathic osteoarthropathy , subchondral cysts , osteoporosis and bone fragmentation may appear , with loss of articular relationships and subsequent endosteal and periosteal reparative bone proliferation , which are easily detected even on conventional radiology . in these cases , mri can usually be interpreted correctly if compared with the radiographic pattern [ 1214 ]  . 
the ankle - arm index ( aai ) may overestimate systolic pressure owing to calcified sclerosis of the tunica media hindering compression of the arteries , transcutaneous oximetry may be misleadingly low due to the presence of oedema and , finally , colour doppler ultrasound may not allow adequate evaluation of the infrapopliteal arterial region , the most commonly affected by diabetic arterial disease [ 4 ]  . 
 in genere la rm permette di escludere con affidabilit losteomielite se negativa o se lalterazione del segnale osteo - midollare lontana dallulcera , ma non sempre sufficiente a formulare diagnosi di osteomielite quando positiva , anche se vi concordanza topografica tra i segni rm primari e quelli secondari . 
la somministrazione di mdc paramagnetico risulta utile per aumentare la specificit dellesame dato che lenhancement del tessuto osteo - midollare , valutato nelle sequenze t1 - pesate con selettiva soppressione del segnale adiposo , considerato tipico dellosteomielite pi che delle alterazioni dovute a osteo - artropatia neuropatica [ 12 ]  . 
tuttavia , alcuni autori hanno riscontrato la possibilit che in alcuni casi ledema midollare reattivo da neuropatia spesso associato a micro - fratture trabecolari e versamento articolare possa presentare enhancement , risultando indistinguibile dallosteomielite [ 13 ]  . 
 il problema della diagnosi differenziale tra osteomielite e osteo - artropatia neuropatica non si pone quando la cute integra , ma solo quando le alterazioni bio - meccaniche dovute al carico favoriscono lulcerazione della cute ed il piede appare tumefatto , caldo , eritematoso ; in tale fase acuta la neuro - artropatia pu simulare losteomielite sia dal punto di vista clinico che radiologico . 
pertanto , in tutti i casi con rm positiva , ma discordante dai dati clinici , colturali , laboratoristici e radiologici , necessario ricorrere alla biopsia ossea per conferma , utilizzando la guida fluoroscopica o tc per raggiungere la sede della alterazione del segnale rm , scegliendo un tragitto che attraversi la cute integra , al fine di evitare la possibile contaminazione ossea [ 9 ]  . 
 nellosteo - artropatia neuropatica fase cronica possono comparire cisti subcondrali , osteoporosi e frammentazione ossea con perdita dei rapporti articolari e successiva proliferazione ossea riparativa endo e periosteale , facilmente rilevabili gi allesame radiologico convenzionale . 
 le indagini strumentali vascolari pi comunemente utilizzate per lo screening dellarteriopatia periferica presentano alcuni limiti nel paziente diabetico con osteomielite del piede : lindice caviglia - braccio ( abi ) pu sovrastimare la pressione sistolica per la sclerosi calcifica della tunica media che rende le arterie non comprimibili , lossimetria trans - cutanea pu essere falsamente bassa per la presenza di edema in corso di infezione e , infine , leco - color doppler radiol med ( 2009 ) 114 : 121132 quently , mr angiography plays a crucial role in these highrisk patients , allowing a noninvasive panoramic study of the lower - limb arteries [ 15 ]  . 
the advantages of mr angiography over ct angiography are the low nephrotoxicity of paramagnetic contrast agents , the absence of exposure to ionizing radiation and easier and more accurate postprocessing , especially in patients with diffuse wall calcifications [ 16 ]  . 
 until recently before the implementation of parallel imaging that significantly reduces acquisition times one of the major limits of lower - limb mr angiography was the filling of the venous circulation at the infrapopliteal level , which rendered up to 43% of mr angiography studies nondiagnostic [ 17 ]  . 
there is evidence that cellulitis and ulcers are the only two circumstances capable of significantly accelerating the transit time of contrast material through the peripheral arteries [ 18 ]  . 
in our experience , within the limits shown in the infrapopliteal region , mr angiography proved adequate for planning surgical or endovascular treatment , allowing healing of the ulcer and infection or identification of the level of amputation for critical limb ischaemia . conclusions pu non consentire una adeguata valutazione del distretto arterioso infra - popliteo , il pi frequentemente colpito dallarteriopatia diabetica [ 4 ]  . 
dunque , langio - rm ha assunto un ruolo cruciale in questi pazienti ad alto rischio , consentendo la valutazione panoramica arteriosa degli arti inferiori in maniera non invasiva [ 15 ]  . 
i vantaggi dellangiorm rispetto allangio - tc sono rappresentati dalla bassa nefrotossicit dei mdc paramagnetici , dallassenza di esposizione radiante e dal post - processing pi facile ed accurato , specie nei pazienti con estese calcificazioni parietali [ 16 ]  . 
 fino a pochi anni fa , prima dellapplicazione dellimaging parallelo che permette di ridurre sensibilmente i tempi di acquisizione , uno dei limiti maggiori dello studio angio - rm degli arti inferiori era rappresentato dal riempimento del circolo venoso a livello infra - popliteo : fino al 43% degli esami angio - rm risultavano non diagnostici [ 17 ]  . 
nella nostra esperienza langio - rm , pur con i limiti evidenziati a livello del distretto infrapopliteo , risultata idonea a pianificare la strategia di trattamento chirurgico o endovascolare per permettere la guarigione dellulcera e dellinfezione o comunque a individuare il livello di amputazione nellischemia critica di arto . mri of the diabetic foot is highly sensitive in the study of osteomyelitis and infectious complications . 
bolus - chase mr angiography of the lower limbs is the preferred vascular study in diabetic patients for planning peripheral revascularization . inflammatory hyperaemia of the diabetic foot , however , affects visualization of the infrapopliteal arteries in around 40%50% of limbs with infected ulcers . 
in these cases , in the presence of known patency of the femoropopliteal axis , the use of selective arteriography is justified by the opportunity to perform angioplasty during the same session . conclusioni la rm del piede diabetico ha elevata sensibilit per losteomielite e per le complicanze infettive . 
nei casi dubbi la biopsia ossea con guida imaging resta lunica indagine in grado di discriminare i casi di falsa positivit alla rm dovuti a osteo - artropatia neuropatica di charcot . 
we initiated a prospective , blinded investigation on 29 patients affected by oncological diseases ( n = 14 ) or lymphoma ( n = 15 ) , who underwent fluorodeoxyglucose ( fdg ) - based pet - ct and whole - body dwibs for restaging purposes . 
reader 1 had a sensitivity of 89.07% , a specificity of 98.5% , and an accuracy of 97.65% , with a positive predictive value ( ppv ) of 85.48% and a negative predictive value ( npv ) of 98.91%. 
abbiamo avviato uno studio prospettico , in cieco , su 29 pazienti oncologici ( 15 con linfoma e 14 con altre neoplasie ) , che si sono sottoposte nel loro normale percorso diagnostico a tac - pet con fluro - desossi - glucosio ( fdg ) e anche ad unindagine con wb - dwibs , per restaging di malattia . 
lagreement tra i due radiologi risultata quasi perfetta ( = 0 , 93 )  . rispettivamente i due radiologi hanno avuto una sensibilit del 89 , 07% e 87 , 39% , specificit del 98 , 5% e del 98 , 39% , accuratezza diagnostica del 97 , 65% e 97 , 8% per i due lettori , ppv del 85 , 48% e 88 , 13% e npv 98 , 91 e 98 , 75% . radiol med ( 2009 ) 114 : 117 conclusions . 
un difetto dello studio sicuramente larruolamento di pazienti con lesioni multiple disseminate , leterogeneit della casistica . keywords diffusion weighted imaging whole - body pet - ct parole chiave diffusion weighted imaging whole body tc - pet introduction introduzione in oncology , tumour therapy and follow - up are strongly based on reliable diagnostic imaging techniques enabling evaluation of the entire body for disease detection and staging . 
whole - body imaging is mainly applied to screening and staging , and the role of magnetic resonance imaging ( mri ) and positron emission tomography computed tomography ( pet - ct ) among whole - body imaging techniques has been well assessed in the literature [ 1 ]  . whole - body mri , intended as a conventional mri technique , has excellent tissue contrast and high spatial resolution and allows detailed morphological evaluation . 
these properties make it particularly suited to the study of bone marrow , very sensitive in detecting and locating skeletal metastasis and superior to ct in depicting bone and parenchymal infiltration [ 2 ] in almost all organs ( though not yet for lung parenchyma )  . since the introduction of pet - ct in 2001 , anatomicmetabolic studies have been found to be extremely sensitive in characterising lymph nodes and detecting metastasis . however , they also tend to be affected by false - positive results mainly related to brown fat , muscle tissue or inflammatory processes given that fluorodeoxyglucose ( fdg ) is not entirely a cancer - specific tracer . 
in addition , bowel muscular activity and urinary tract excretion hinder the interpretation of studies of the gastrointestinal and urinary system [ 3 ]  . the other whole - body technique , 99mtc - phosphonatebased skeletal scintigraphy ( or bone scan ) , is the standard nuclear - medicine method for the initial staging of primary or secondary bone tumours . 
however , it provides limited anatomical detail , has low sensitivity and specificity and only depicts bone metastasis at a relatively late stage when osteoblastic reaction to tumour deposits has already occurred [ 49 ]  . diffusion - weighted imaging ( dwi ) , a validated technique for mr evaluation of the brain , has recently been in oncologia , la terapia ed il controllo della risposta ad essa ( follow - up ) sono strettamente legati alla disponibilit di metodiche di imaging diagnostico , che permettano lanalisi di tutti i distretti corporei potenzialmente interessati dalla patologia e dalla sua diffusione ( whole - body ) , e che siano affidabili nel riconoscere e stadiare la patologia . 
per essere appropriato , lo screening di secondo e terzo livello deve disporre di una metodica caratterizzata da elevata specificit , applicata su una patologia ad elevata prevalenza nella popolazione . 
limaging whole - body ( whole body imaging , wbi ) orientato pertanto principalmente a scopi di screening e staging ; in letteratura [ 1 ] , ben assodato il ruolo dellimaging con risonanza magnetica ( whole body magnetic resonance imaging , wb - mri ) e della tomografia computerizzata abbinata alla tomografia ad emissione di positroni ( tc - pet )  . 
 per wb - mri , si intende lapplicazione total - body della tecnica rm convenzionale ; tale tecnica caratterizzata da eccellente risoluzione di contrasto ed alta risoluzione spaziale , permette una valutazione morfologica dettagliata ; queste caratteristiche rendono la metodica particolarmente idonea allo studio del midollo osseo , molto sensibile nella individuazione e localizzazione delle metastasi scheletriche , superiore alla tc nella definizione dellinfiltrazione ossea e parenchimatosa [ 2 ] , per quasi tutti gli organi ( allo stato attuale , non per il parenchima polmonare )  . dallintroduzione della tc - pet nel 2001 , lo studio anatomo - funzionale risultato particolarmente sensibile per la caratterizzazione dei linfonodi e delle metastasi , sebbene limitato da false positivit . 
queste ultime sono generate prevalentemente dal grasso bruno , da processi infiammatori o da tessuti muscolari metabolicamente attivi , dal momento che il marcatore metabolico il fluorodesossi - glucosio ( fdg ) , marker non specifico per lattivit cellulare neoplastica ; inoltre lattivit muscolare intestinale e lescrezione urinaria del metabolita rendono di difficile interpretazione questi due apparati [ 3 ]  . laltra principale metodica wb disponibile in medicina nucleare la scintigrafia ossea con tecnezio ( bone scan , bs ) , che rappresenta la metodica standard di staging dei radiol med ( 2009 ) 114 : 117 extended to extracranial districts [ 10 , 11 ] , although with several technical difficulties due to hardware limitations and the mobile nature of the organs to be studied . 
this is an echo planar imaging pulse sequence combined with short tau inversion recovery ( epi - stir ) suitable for evaluating extracranial districts and for whole - body imaging , partly owing to the excellent images obtained with postprocessing algorithms . apart from one congress abstract [ 13 ] , no papers have been published on the subject since takaharas report [ 12 ] , so the accuracy of whole - body dwibs in comparison with the pet - ct is as yet unknown . 
 the aim of this study was to assess the overall diagnostic accuracy of whole - body dwibs in comparison with petct , which is considered the standard of reference among whole - body imaging modalities . materials and methods patients from february 2007 to august 2007 , we enrolled 29 consecutive patients undergoing pet - ct for oncological and oncohaematological diseases ( table 1 ) and fulfilling the requisites for the study . inclusion criteria patients were eligible if they met the following criteria : 1 . 
they volunteered for the mr scan , were able to understand and sign an informed consent form informing them that : ( a ) the examination was not necessary but might tumori ossei primitivi o secondari . 
con lo sviluppo di sequenze pi veloci , insieme al potenziamento dei gradienti variabili e allintroduzione della tecnica del tavolo mobile , limaging rm pesato in diffusione , esteso allo studio di tutto il corpo diventato tecnicamente realizzabile . 
 [ 12 ] hanno messo a punto una sequenza per limaging rm pesato in diffusione , denominata diffusion weighted body imaging with background body signal suppression ( dwibs ) , una sequenza echo - planar abbinata alla soppressione del segnale del grasso con tecnica stir , ottimizzata per la valutazione dei distretti extracranici e particolarmente adatta ad un impiego whole - body , grazie anche agli ottimi risultati iconografici ottenuti con opportuni algoritmi di post - processing . 
pertanto , fino ad oggi , laccuratezza diagnostica della tecnica wb - dwibs in confronto con la tc - pet non nota . scopo del presente studio di valutare laccuratezza diagnostica della wb - dwibs in comparazione con il gold standard nellambito delle metodiche di imaging wholebody , la tc - pet . table 1 patient series grouped by type of neoplasm tabella 1 casistica raggruppata per tipologia di neoplasia primary tumour number of patients sedi del tumore primitivo numero di pazienti lymphoma lung breast kidney sarcoma primitive neuroectodermal tumour prostate linfoma polmone mammella intestino rene sarcoma pnet prostata radiol med ( 2009 ) 114 : 117 help to understand or stage their disease in the future , ( b ) the study required no contrast medium injection , ( c ) the study took 20 min , and ( d ) no ionising radiation would be used 2 . 
a q - body coil was used , with the patient positioned feet first on an extended anatomical coverage table , based on rolling - table technology ( mobitrak , philips )  . 
after a scout pulse sequence , we acquired a dwibs pulse sequence and an epi - stir single - shot pulse ( table 2 ) in the axial plane , repeated for up to four stacks to encompass all anatomical districts from the head to at least the distal thigh , similarly to the pet - ct acquisition protocol . 
we adopted two different b - factor values in the diffusion pulse sequence , b = 500 and b = 1000 s / mm2 , but only the latter was used for reading purposes , as suggested by takahara et al . 
the acquisition time for the protocol was 18 min which , summed to the scout sequence time , implied a total room occupation time of 20 min . nuclear medicine pet - ct scans were acquired on a hybrid siemens ( siemens , erlangen , germany ) system consisting of an lutetium oxyorthosilicate ( lso ) pet scanner ( hi - rez ) with pico - 3d electronics and a 16 - row ct device ( somatom sensation 16 )  . 
 image reformatting and analysis after acquisition and the choice of the higher b value , the materiali e metodi pazienti da febbraio 2007 fino ad agosto 2007 abbiamo arruolato 29 pazienti consecutivi , in previsione di eseguire una valutazione tc - pet , per patologie oncologiche , anche del distretto emolinfopoietico ( tabella 1 ) , che soddisfacevano i criteri di inclusione . criteri di inclusione i pazienti sono stati considerati reclutabili in relazione ai seguenti criteri : 1 . 
abbiamo utilizzato la bobina q - body , posizionando il paziente ( in modo che entrasse nel gantry caudocranialmente , feet first ) , su un tavolo porta - paziente a copertura anatomica estesa , basata sulla tecnologia del tavolo mobile ( mobitrak , philips )  . 
dopo una sequenza di centratura , abbiamo acquisito una sequenza dwibs ( stirecho planar , single shot ) ( tabella 2 ) sul piano assiale , ripetuta 4 volte per coprire tutti i distretti anatomici dalla testa alla coscia distale , in modo da replicare la copertura anatomica dellesame tc - pet . 
la sequenza stata pesata con un doppio valore di b ( b = 500 e b = 1000 s / mm2 ) , utilizzando solo il secondo per la refertazione , come suggerito da takahara et al . 
al termine , si provveduto a invertire la scala dei grigi , in modo che su sfondo bianco ( le aree prive di segnale ) si potesse riconoscere la patologia come immagini con diverse intensit di grigio ( le aree con segnale accentuato ) , con tutte le gradazioni intermedie . 
le immagini whole - body mip e mpr cos elaborate sono state poi salvate nel database dello scanner . anche le immagini native assiali sono state separate sulla base del parametro b , e quelle con b = 1000 s / mm2 , dopo aver preparato la finestra di visualizzazione in modo ottimale , sono state salvate nel database delle immagini . 
tutte le immagini , elaborate da un medico specializzando esperto , sono state private delle informazioni anagrafiche e salvate su una memoria di massa mobile ( cd ) , in formato dicom , registrando ogni supporto con il numero progressivo del paziente . 
le sessioni di refertazione sono state condotte , indipendentemente , da due radiologi esperti in rm ( a.s , lettore 1 e a.c. lettore 2 ) , rispettimente con 8 e 18 anni di pratica professionale in rm , reciprocamente inconsapevoli del risultato della refertazione dellaltro , e del risultato della lettura della tc - pet . i due radiologi hanno riempito una scheda di lettura predefinita , costituita da un foglio elettronico ( excel , microsoft , redmond , usa ) , nel quale sono state individuate 40 sedi anatomiche , scheletriche e viscerali , basandosi su unanaloga schematizzazione elaborata da lauenstein et al . radiol med ( 2009 ) 114 : 117 4 - mm - thick multiplanar reformations ( mpr ) in the coronal plane , and ( c ) multiple 4 - mm - thick mpr in the sagittal plane oriented to include the midline as well as the spine and paraspinal regions . 
all reformatted images were then fused by means of mobiview software ( philips ) with the smooth fusion algorithm to obtain whole - body mip and mpr images . the grey scale was subsequently inverted to allow visualisation of abnormalities ( increased signal ) as grey areas of varying intensity against a white background ( without signal )  . all of the whole - body mip and mpr images were saved in the scanners patient - image database . 
the native axial slices were separated on the basis of the b value , and those acquired with b = 1 , 000 s / mm2 were also saved in the image database after optimal window setting . 
all image sets , processed by an experienced trainee radiologist , were rendered anonymous and stored in digital imaging and communications in medicine ( dicom ) format on an optical disc marked with the patients progressive number for subsequent reading . 
images were read independently by two experienced mri radiologists ( reader 1 : as , with 8 years experience ; reader 2 : ac , with 18 years experience ) who were unaware of each others mri report and of the pet - ct results . the two readers noted their findings on a predefined reading grid consisting of a spreadsheet ( excel , microsoft , redmond , usa ) listing 40 skeletal and visceral sites and subsites , modified from the classification proposed by lauenstein et al . 
radiological reading time for each patient was also recorded . the pet - ct study was reported by the nuclear physician on a similar reading grid as that used by the radiologists . 
the nuclear physician first read the source axial pet images separately and then overlaid on axial ct images and next reviewed the coronal and sagittal images reconstructed with the mpr algorith image analysis included assessment of metabolic uptake of the tracer in terms of standardised uptake value ( suv )  . postprocessing and mean reading times were calculated . the cohen constant was calculated to determine the grade of interobserver agreement . 
for each reader , we calculated diagnostic accuracy , sensitivity and specificity of the wholebody dwibs readings together with negative and positive predictive values ( npv and ppv ) and compared the readings with those performed by the nuclear physician on the pet - ct studies . 
 lo specialista medico nucleare ha refertato lesame tcpet , su unanaloga scheda di lettura , valutando le immagini assiali pet , poi sovrapposte alle immagini tc e infine ricostruite nei piani coronale e sagittale tramite algoritmo mpr , utilizzando anche la valutazione dellintensit della captazione metabolica ( suv ) come criterio per definire patologico un reperto . statistica sono stati calcolati il tempo di post - processing e di refertazione . 
per ogni lettore , stata calcolata laccuratezza diagnostica , la sensibilit e la specificit delle letture wb - dwibs , insieme al valore predittivo negativo e positivo ( npv e ppv ) , comparando le letture dei radiologi con quella del medico nucleare eseguita sulle immagini tc - pet . risultati la tecnica wb - dwibs ha comportato un tempo di occupazione della sala rm di circa 20 minuti . 
il tempo di refertazione medio risultato di 20 minuti ( range 1525 minuti ) per il primo lettore e 18 minuti per il secondo ( range 1322 minuti )  . 
il tempo di refertazione ha avuto un trend decrescente per ognuno dei radiologi dal primo paziente fino al ventinovesimo . laccordo tra i due lettori stato quasi perfetto , con un valore di di 0 , 93 . 
i due radiologi , su 29 pazienti ( ognuno analizzato nelle 40 sedi anatomiche ) hanno considerato positive ( affette ) rispettivamente 124 e 128 sedi , delle quali 106 e 104 veri positivi ( vp ) , 18 e 14 falsi positivi ( fp ) , contro il rilievo ct - pet di positivit in 119 sedi . sono state considerate negative dai due radiologi 1196 e 1202 sedi , delle quali rispettivamente 1183 e 1187 veri negativi ( vn ) e 13 e 15 falsi negativi ( fn ) , rispetto alle 1121 sedi considerate negative dalla metodica di riferimento ( tabella 4 )  . 
quattordici dei 29 pazienti hanno avuto un responso esattamente speculare a quello della tc - pet . negli altri 15 pazienti si verificato un mismatch di lettura ( fp e fn ) da 1 a massimo 5 sedi . 
i due radiologi hanno avuto rispettivamente : il primo radiologo una sensibilit del 89 , 07% , specificit del 98 , 5% e accuratezza del 97 , 65% , con vpp e vpn di 85 , 48% e 98 , 91% ; il secondo radiologo una sensibilit del 87 , 39% , specificit del 98 , 39% e accuratezza diagnostica del 97 , 8% , con vpp del 88 , 13% e vpn del 98 , 75% . analizzando le discrepanze tra tc - pet e wb - dwibs , abbiamo rilevato rispettivamente per i due radiologi 18 e 14 falsi positivi , 13 e 15 falsi negativi , distribuiti come evidente nella tabella 5 , dove stata riportata la media tra i due radiol med ( 2009 ) 114 : 117 table 3 categorisation of skeletal and visceral sites used in the radiology report skeletal sites visceral sites skull , orbit and maxillary bones cervical spine thoracic spine lumbar spine sacrum coccyx sternum clavicle scapula humerus forearm ilium - ischium femur lower leg cranio , orbite e massiccio colonna cervicale colonna toracica colonna lombare sacro coccige sterno coste clavicola scapola omero avambraccio ilio - ischio femore arto inferiore neck neck nodes lung mediastinum pleura thoracic nodes breast axillary nodes liver adrenal glands kidneys pancreas spleen gallbladder bladder abdominal nodes retrocrural nodes female pelvis reproductive apparatus male pelvis reproductive apparatus omental / peritoneal pelvic nodes inguinal nodes soft tissues collo linfonodi del collo polmone mediastino pleura linfonodi toracici mammella linfonodi ascellari fegato surreni reni pancreas milza intestino colecisti vescica linfonodi addominali linfonodi retrocrurali pelvi femminile apparato riproduttivo pelvi maschile apparato riproduttivo omento peritoneo linfonodi pelvici linfonodi inguinali tessuti molli tabella 3 elenco delle sedi scheletriche e viscerali adottato per la scheda di lettura sedi scheletriche sedi parenchimali time of approximately 20 mpostprocessing had a mean duration of 15 min ( range 1017 min )  . 
of these sites , 106 and 104 were true positive ( tp ) and 18 and 14 were false positive ( fp ) compared with positive pet - ct findings in 119 sites . 
readers rated 1 , 196 and 1 , 202 sites , respectively , as negative , of which 1 , 183 and 1 , 187 were true negatives ( tn ) and 13 and 15 were false negatives ( fn ) compared with negative pet - ct findings in 1 , 121 sites ( table 4 )  . dallaltra parte , nelle sedi addominali e pelviche , appaiono pi concentrati i reperti di falsa positivit . 
un caso di non completa corrispondenza tra le tc - pet e wb - dwibs , quello di un paziente ( p.l. ) , affetto da sarcoma della coscia , trattato chirurgicamente e poi con chemioterapia . 
i numeri rappresntano la media tra i due lettori distribution of results distribuzione dei risultati false negative falsi negativi skull , orbit and maxillary bones sacrum sternum scapula ilium - ischium lung mediastinum thoracic nodes pancreas retrocrural nodes total thoracic spine scapula femur neck neck nodes breast axillary nodes liver adrenal glands kidneys omental / peritoneal pelvic nodes inguinal nodes soft tissues total cranio , orbite e massiccio sacro sterno scapola ileo - ischio polmone mediastino linfonodi toracici pancreas linfonodi retrocrurali totale colonna dorsale scapola femore collo linfonodi del collo mammella linfonodi ascellari fegato surreni reni omento / peritoneo linfonodi pelvici linfonodi inguinali tessuti molli totale false positive falsi positivi there was a perfect match with the pet - ct findings in 14 / 29 patients ; there was some mismatch , ranging from one site ( fp or fn ) to five sites in the same patient in the remaining 15 / 29 patients . 
 with regard to the sites of disagreement between petct and whole - body dwibs , the two readers provided 18 and 14 false - positive results and 13 and 15 false - negative results , distributed as seen in table 5 , which shows the mean between the two readers . 
table 5 shows that the spine was almost free of fp and fn results , whereas the lung - mediastinum and the skull - maxillary bones recorded more fn results than all the other areas taken as a whole . 
colour multiplanar reformation ( mpr ) fusion of pet and ct data ( a , b )  . single pet and ct axial images before coregistration and fusion ( c , d )  . 
1a - d paziente affetto da sarcoma alla coscia destra , trattato con chirurgia e chemioterapia , attualmente con riscontro di metastasi allilo sinistro e ai polmoni ( frecce ) , oltre a recidiva alla coscia controlaterale ( punta di freccia ) ; a , b fusione a colori mediante algoritmo mpr tra pet e tc ; c , d immagini assiali singole di pet e tc prima della co - registrazione e fusione . 
si notino le calcificazioni lesionali a livello dellilo polmonare ( c ) e del nodulo polmonare ( d )  . thigh sarcoma mismatch between pet - ct findings and dwibs refers to a previous treated with surgery and chemotherapy . 
nella stessa figura , si pu osservare un fp ( a livello renale ) con un mancato riconoscimento della lesione peri - cefalopancreatica , verosimilmente linfonodale ( falso negativo )  . discussione limaging rm a diffusione ( dwi ) , stato inizialmente introdotto nella pratica clinica in ambito neuroradiologico , principalmente per la valutazione dello stroke ischemico . solo recentemente il dwi stato esteso ad applicazioni rm in ambito body [ 11 ] anche per le problematiche tecniche intrinseche al distretto toraco - addominale : movimento , numerosit di interfacce tissutali , contenuto intestinale , contenuto gassoso . 
con il miglioramento tecnologico delle apparecchiature rm , con sequenze sempre pi veloci che hanno permesso lacquisizione delle immagini in respiro libero , la rm body e la diffusione a rm nello stesso ambito , sono divenute tecnicamente fattibili . 
alcuni autori [ 15 ] , inoltre , hanno dimostrato la possibilit di caratterizzare le radiol med ( 2009 ) 114 : 117 b = 500 b = 1000 fig . 
diffusion - weighted imaging with optimised background suppression pulse sequence ( dwibs ) coronal ( a ) , sagittal ( b ) and axial multiplanar reformation ( mpr ) images ( c ) at b = 1 , 000 did not confirm the pet - ct findings ( dotted circles )  . 
dwibs elaborata con mpr in coronale ( a ) , sagittale ( b ) e mpr ( c ) a b = 1000 , senza conferma dei reperti osservati in tac - pet ( circoli punteggiati )  . 
6. the same figure shows an fp result ( kidney ) and a missed , probably nodal , pericephalopancreatic lesion . discussion dwi was originally introduced into clinical practice for neuroimaging purposes and mainly for assessing stroke . lesioni maligne , in ambito body , mediante lo studio della diffusione e la misurazione quantitativa con roi posizionate su mappe adc . 
 [ 12 ] che hanno utilizzato una sequenza echo - planar single - shot accoppiata ad un impulso stir , denominata dwibs : questa sequenza ha permesso una maggiore e pi omogenea soppressione del segnale di fondo e lacquisizione con la tecnica del respiro libero , garantendo cos la possibilit di un tempo di acquisizione pi lungo e per questo con un numero maggiore di radiol med ( 2009 ) 114 : 117 fig . 
3 tac - pet : metastasi ossee diffuse ( frecce ) da primitivo ignoto . only recently has its use been extended to whole - body mr applications [ 11 ] owing to technical problems intrinsic to the thoracoabdominal area , including movement , number of tissue interfaces , bowel content and air content . 
dwi with quantitative measurement of apparent diffusion coefficient ( adc ) has been shown to be able to characterise malignant lesions in whole - body imaging [ 15 ]  . 
more recently , dwi has been improved by the introduction of dwibs , a diffusionweighted pulse sequence paired with a stir sequence [ 11 ]  . this sequence allowed greater homogeneous suppression of the background signal as well as acquisition during free breathing , thus permitting more time for the acquisition , a higher number of signal averages , a better signal - to - noise ratio ( snr ) and acquisition of thinner slices . 
the better fat suppression at the edges of the field of view ( fov ) provided by the stir sequence and the acquisition of thinner slices also allow for better mpr and mip reformamedie del campionamento del segnale , con conseguente incremento del snr e con la possibilit di acquisire sezioni pi sottili . 
in questo modo possibile ottenere migliori immagini mip e mpr , sia per le sezioni pi sottili , sia per la migliore soppressione del grasso ai margini del campo di vista indotta dallimpulso stir . 
in questi ultimi anni , la rm whole body diventata pi accessibile e tecnicamente realizzabile grazie allintroduzione commerciale della tecnologia del tavolo mobile , che insieme anche alla disponibilit del software di fusione di pi pacchetti di acquisizione in ununica immagine , ha permesso lacquisizione e la ricostruzione di immagini whole body . 
questi progressi tecnici sono stati preliminari allintroduzione della tecnica wb - dwibs , nel 2004 . ad oggi , nessuno ha comparato i risultati di questa tecnica con le metodiche di medicina nucleare come la tcpet e la scintigrafia ossea , in pazienti oncologici . 
abbiamo condotto questo studio per comparare la wb - dwibs con la tc - pet , attualmente considerata il gold standard nello staging tnm della patologia neoplastica maligna [ 16 ]  . alcune problematiche relative allimaging wb - dwi , radiol med ( 2009 ) 114 : 117 fig . 
in recent years , whole - body mri has become more accessible and easier to perform , thanks to the advent of rolling or moving table technology that , together with the availability of software allowing the fusion of multiple stacks , has made possible the acquisition and reconstruction of whole - body images . 
all these technical advances paved the way for the introduction of the whole - body dwibs technique in 2004 . to our knowledge , no published study has compared the results of whole - body dw - mri with whole - body nuclear imaging techniques ( pet - ct and bone scan ) in cancer patients . 
this study was thus conducted to compare wholebody dwibs and pet - ct , which is considered the standard of reference in the tumour - node - metastasis ( tnm ) staging system for malignancies [ 16 ]  . limitations of whole - body dwi are the huge amount of data to be postprocessed and interpreted . 
in this study , we had a mean postprocessing time of 15 min , mainly spent for window adjustments before completing image fusion and possono essere sicuramente considerate la notevole quantit di immagini da ri - elaborare nel post - processing e da interpretare . 
nel nostro studio , abbiamo riscontrato un tempo di ri - elaborazione delle immagini piuttosto lungo , circa 15 minuti , prevalentemente spesi nellottimizzazione delle finestre di visualizzazione , prima della fusione dei pacchetti e nella generazione delle ricostruzioni mip e mpr . 
il tempo di lettura medio risultato di 19 minuti , come media tra i due lettori ; le letture sono state molto concordanti e questo garantisce la riproducibilit del risultato , in quanto sembra che le immagini siano facilmente interpretate nello stesso modo da differenti lettori . i nostri risultati dimostrano che la wb - dwibs ha alta specificit ed accuratezza diagnostica ed un elevato valore predittivo negativo ( npv ) , tutti vicino al 98% , quando paragonata alla tc - pet . 
analizzando i falsi positivi e negativi della nostra casistica , si evidenziano tre macroaree critiche , che necessitano ulteriore approfondimento : la regione cranio - mascellare , la regione toracomediastinica e la regione addomino - pelvica . 
in addition , there was high interobserver agreement between the two readers . our results show that whole - body dwibs has high specificity , high accuracy and high npv ( all close to 98% ) when compared with pet - ct . 
analysing the fp and fn results of whole - body dwibs in our series , we observed three critical sites that require further investigation : the skull - maxillary bones , the lung - mediastinum and the abdominopelvic site . 
in particular , the thorax , one of the most investigated districts in cancer screening or staging , requires further investigation by means of dwi with dedicated multichannel coils and a specific pulse protocol . 
specifically , the spine was well assessed , with no fn findings , and only one fp result out of 18 tp results ( table 5 )  . sono responsabili della maggioranza dei fn e fp della nostra casistica . 
in particolare , il torace , che uno dei distretti pi esplorati per screening o staging oncologico , necessita un approfondimento ulteriore , con imaging di diffusione mediante bobine multicanale specifiche ed un protocollo con sequenza ottimizzata per la sua valutazione . invece la tecnica appare estremamente accurata nel riconoscimento di lesioni ossee del rachide e delle ossa lunghe , del cranio e del bacino , e per il rilievo di patologia linfonodale . 
nello specifico , la valutazione della colonna vertebrale appare beneficiare , senza falsi negativi e con un solo falso positivo ( tabella 5 ) su 18 rilievi patologici , delle potenzialit diagnostiche di questa nuova tecnica . 
in a e b si osservano multipli foci di alterazione del segnale a livello latero - cervicale bilaterale , a livello splenico , lomboaortico , pelvico e inguinale sinistro ( freccie nere )  . 
con le punte di freccia si osservano due foci segnalati da entrambi i radiologi a livello renale , dei quali ( a destra , punta di freccia rossa ) uno una localizzazione peri - cefalopancreatica ( errore di sede ) e uno ( a sinistra , punta di freccia verde ) un falso positivo non corrispondente a patologia . 
conferma nelle immagini tc - pet in assiale delle lesioni osservate sia in wb - dwibs che nella ricostruzione tc - pet con mpr in coronale , delle lesioni laterocervicali ( e - g , frecce tratteggiate ) , spleniche ( h - l , frecce intere ) , lombo - aortiche ( m - o , punte di freccia )  . radiol med ( 2009 ) 114 : 117 these preliminary data show that the two major clinical applications could be ( a ) bone lesion detection , and specifically screening or staging of the spine - bony pelvis , and ( b ) lymphoma staging , due to the accurate delineation of nodal disease . 
we will direct our future scientific efforts in these two directions . these data , to be confirmed on a larger number of patients , appear to point to a role for whole - body dwibs in cancer screening , staging , restaging and follow - up , given that the higher the specificity and npv , the better the technique as a screening tool . 
whole - body mri has been shown to be more accurate than pet - ct in evaluating the liver , bone and central nervous system [ 17 , 18 ]  . 
from this point of view , a comparison with pet - ct cannot substitute a comparison based on clinical and pathological follow - up or autopsic confirmation ( the true gold standard ) and , in this sense , our study is limited by a certain degree of bias . 
thus , in presenting our preliminary data , we refer to previous studies in which a direct comparison was made between mri and nuclear medicine . the main limitations of the present study inherent to its pilot nature are a heterogeneous case series , the presence of diffuse disease and the small sample size . 
even though wholebody dwibs is beginning to be used in clinical practice , the standard diagnostic workup of patients enrolled in the study was not modified on the basis of the findings of whole - body dwibs because of the lack of scientific validation . direzioni condurremo i nostri prossimi studi . 
 questi risultati , da confermare su una popolazione di pazienti pi ampia , sembrano indicare un ruolo della wbdwibs nello screening , stadiazione , ristadiazione e followup della malattia tumorale ; infatti , pi alta la specificit e il npv , migliore la tecnica per lo screening . 
osserviamo inoltre che la sensibilit non cos elevata ( < 90% ) come la specificit , sempre in riferimento alla tc - pet . la tc - pet ha comunque le sue limitazioni specifiche , in particolare riguardo ai falsi positivi [ 17 ] , che possono influenzare anche il calcolo dellaccuratezza diagnostica delle tecniche di imaging messe a confronto , e in particolare la sensibilit della wb - dwibs nel nostro studio . 
in molte forme tumorali , la tc - pet pi adatta nella individuazione del tumore ( parametro t ) e nello staging linfonodale ( parametro n ) , ma ci sono alcune forme neoplastiche che hanno scarsa captazione di fdg , come il carcinoma renale o i tumori con frequente diffusione alle ossa , al fegato e al cervello ( come il carcinoma della mammella ) , che sono pi idonei a essere studiati con metodiche whole body alternative : infatti gi stato dimostrato che la rm whole body convenzionale pi accurata della tc - pet nella valutazione del fegato , dellosso e del cervello [ 17 , 18 ]  . 
da questo punto di vista , un confronto con la tc - pet , non pu sostituire un confronto tra una metodica di imaging da validare e il follow - up clinico e patologico o autoptico , che rappresenta il vero gold standard ; pertanto i nostri risultati sono gravati da un certo margine di errore . 
gli autori fanno pertanto riferimento alla letteratura , dove il confronto tra rm e tecniche di medicina nucleare stato pi volte descritto , nel presentare questi dati preliminari . nel nostro studio , anche per le sue caratteristiche di studio pilota , principali bias possono essere considerati la casistica eterogenea , la presenza di patologia neoplastica diffusa e il campione di piccole dimensioni : attualmente stiamo estendendo lo studio ad un campione pi omogeneo , introducendo anche il follow - up clinico - patologico come standard di riferimento . 
parisi1 1department of radiation oncology , scientific institute hospital casa sollievo della sofferenza , san giovanni rotondo , 71013 foggia , italy 2university of bari , department of radiology , bari , italy 3university of foggia , department of radiology , scientific institute hospital casa sollievo della sofferenza , san giovanni rotondo , 71013 foggia , italy correspondence to : g . 
a systematic review of external radiation therapy studies in urinary cancers was performed . this synthesis of the literature is based on data from metaanalyses , randomised and prospective trials and retrospective studies . 
there are several reports on multimodality treatment in invasive bladder cancer : intravesical surgery and neoadjuvant chemotherapy to radiotherapy or concomitant radiochemotherapy with organ preservation . the conclusions reached for renal cancer are controversial , and data on cancers of the urethra and ureter are few and inconclusive . 
sufficient data now exist in the literature to demonstrate that conservative management with organ preservation is a valuable alternative to radical cystectomy , the traditional gold standard , in invasive bladder cancer , keywords urinary cancers treatment radiotherapy ( rt ) chemotherapy ( ct ) organ preservation overall survival ( os ) riassunto i tumori invasivi del tratto urinario sono relativamente rari ed il loro trattamento pu causare importanti cambiamenti nelle funzioni sessuali , sociali ed urinarie . 
ci sono pochi studi clinici controllati che hanno utilizzato la radioterapia adiuvante o radicalechemioterapia nei tumori del rene , degli ureteri e delluretra ; ci sono diversi studi sul trattamento multimodale del carcinoma invasivo della vescica : chirurgia intravescicale e chemioterapia neoadiuvante alla radioterapia o radio - chemioterapia concomitante con preservazione dellorgano . 
le conclusioni raggiunte sul carcinoma renale sono controverse ; i dati sul carcinoma degli ureteri e delluretra sono pochi e inconclusivi ; invece , vi sono ora in letteratura dati sufficienti che dimostrano come lapproccio con preservazione dellorgano , per il carcinoma vescicale invasivo rappresenta una valida alternativa alla cistectomia radicale , considerata per lungo tempo il gold standard . parole chiave tumore urinario trattamento radioterapia ( rt ) chemioterapia ( ct ) preservazione dellorgano sopravvivenza globale ( sg ) radiol med ( 2009 ) 114 : 7082 introduction surgery is the primary therapeutic option in the curative treatment of clinically localized urinary cancer . 
preoperative irradiation with doses of 2040 gy has been used to facilitate resection , to sterilise well - oxygenated peripheral extensions of the tumour that might be transected during nephrectomy , and to decrease the chance of dissemination at the time of nephrectomy . 
no advantage has been demonstrated in patients receiving preoperative irradiation with doses of 2040 gy with respect to overall survival ( os ) or survival free from distant metastases ( ffdm ) , although improvement in resectability has been noted [ 16 ]  . 
two studies [ 10 , 12 ] have reported excessive bowel and hepatic complications and an irradiation - related mortality rate of 13%19% , explained in part by the fractionation scheme . 
doses of 4550 gy can be given , with acceptable bowel and hepatic complications ; however , a dose of 50 gy is suggested as being the tolerance limit for the upper abdominal gastrointestinal tract [ 13 ]  . 
a recent study [ 15 ] has shown that 5year disease - free survival ( dfs ) is 66% in the radiotherapy introduzione la chirurgia la principale opzione terapeutica nella cura del tumore dellapparato urinario clinicamente localizzato . ad ogno modo , sebbene questappoccio sia associato ad un eccellente controllo locale , non scevro dal rischio di complicanze e , a volte , di una scarsa qualit di vita . 
nel trattamento del cancro invasivo della vescica come alternativa alla cistectomia radicale immediata si pu utilizzare la resezione la resezione transuretrale , la chemioterapia e la radioterapia con cistectomia di salvataggio . 
la maggior parte dei pazienti trattati con terapia trimodale conserva una buona funzione vescicale . rene la nefrectomia radicale resta la sola terapia efficace per il carcinoma renale ( cr ) clinicamente localizzato . 
la rt pre - operatoria con dosi da 20 a 40 gy stata utilizzata per facilitare la resezione , per sterilizzare i margini periferici ben ossigenati del tumore che potrebbero essere sezionati durante la nefrectomia , e per ridurre la possibilit di disseminazione al momento della nefrectomia . 
nessun vantaggio stato dimostrato nei pazienti sottoposti a rt pre - operatoria con dosi da 20 a 40 gy per ci che riguarda la sopravvivenza globale ( sg ) o sopravvivenza libera da metastasi a distanza ( slmd ) , sebbene sia stato notato un miglioramento della resecabilit [ 16 ]  . 
in studi datati , la radioterapia postoperatoria ha dimostrato un vantaggio di circa il 15% sul controllo locale ( cl ) rispetto alla nefrectomia da sola [ 711 ] , ma con risultati insoddisfacenti sulla sopravvivenza . 
due studi [ 10 , 12 ] hanno riportato eccessive complicanze intestinali ed epatiche , ed una percentuale del 13%19% di mortalit legata alla rt , spiegata in parte dallo schema di frazionamento . 
dosi di 4550 gy possono essere erogate con complicanze intestinali ed epatiche accettabili ; comunque suggerita una dose di 50 gy come limite di tolleranza per il tratto gastrointestinale superiore [ 13 ]  . 
 [ 11 ] lirradiazione post nefrectomia con dose mediana di 46 gy ha ridotto la percentuale di recidive locali nei pazienti con tumore in stadio pt3 dal 37% ( nei pazienti non irradiati ) al 10 % ( nei pazienti irradiati ) ( p = 0 , 05 ) ; ma , in un aggiornamento del radiol med ( 2009 ) 114 : 7082 group and 16% in the no - treatment group and has shown reduction of local recurrence , with a significant difference in both univariate and multivariate analyses , although no benefit was noted in patients with earlier - stage disease . another recent study [ 16 ] concluded that adjuvant rt is unlikely to improve treatment outcome . 
however , these series suggest a benefit in patients with a high risk of local recurrence ( incomplete resection t3 / t4 stage , nodal metastases ) , but there is no advantage for os . 
the authors achieved an overall partial / complete response ( cr ) of 90%98% . this new radiation technique is a big challenge , because the side effects are few , and local control is high due to easy dose escalation . 
complete resection is the only known cure for renal cancer , and the value of rt is controversial . results are awaited of multi - institutional prospective randomised trials using modern rt techniques to evaluate the role of rt and its effect on survival , especially in selected patients with a high risk of local or regional failure . ureter and urethra carcinomas of the ureter and urethra are unusual diseases with insufficient clinical experience , and the role of rt in situ not well defined . 
 [ 20 ] reviewed the records of 31 patients with locally advanced transitional cell carcinoma of the renal pelvis and ureter who underwent surgery followed by adjuvant rt with or without concurrent chemotherapy ( ct ) [ methotrexate , cisplatin ( cddp ) and vinblastine ( mcv ) ]  . five - year os and disease - specific survival ( dss ) with adjuvant concurrent chemoradiotherapy ( ccrt ) ( cddp ) improved in patients with resected , locally advanced uppertract urothelial malignancies . 
 the results of 34 women with primary urethral carcinoma were retrospectively reviewed at princess margaret hospital [ 23 ] .these patients were treated with rt with or lavoro , la rt post - operatoria non ha migliorato la sopravvivenza ed ha mostrato tossicit [ 14 ]  . 
un recente studio [ 15 ] ha mostrato che la sopravvivenza libera da malattia ( slm ) a 5 anni del 66% nel gruppo sottoposto a rt e del 16% nel gruppo non sottoposto a rt ed ha mostrato una riduzione della recidiva locale , con una differenza significativa sia allanalisi univariata che multivariata ; ma non stato notato nessun beneficio nei pazienti in stadio iniziale di malattia . 
comunque , tali lavori hanno suggerito un beneficio nei pazienti ad alto rischio di recidiva ( resezione incompleta negli stadi t3 / t4 , metastasi linfonodali ) , ma nessun vantaggio riguardo alla sg . 
 [ 18 ] riguardo lutilizzo della rt stereotassica extracranica nei tumori renali primitivi e metastatici : gli autori hanno riportato il 90%98% di risposte parziali / complete ; questa nuova tecnica di irradiazione una grande sfida perch gli effetti collaterali sono pochi ed il cl alto , dovuto alla facilit di aumentare la dose erogata . 
attendiamo i risultati di studi prospettici randomizzati multi - istituzionali che utilizzano tecniche moderne di rt per valutare il ruolo ed i suoi effetti sulla sopravvivenza , specialmente in pazienti selezionati con alto rischio di recidiva locale o regionale . 
la sg e la ssm a 5 anni con chemio - rt concomitante e adiuvante ( ccrt ) sono migliorate nei pazienti con neoplasie resecate e localmente avanzate del tratto urinario inferiore . 
i pazienti con malattia avanzata trattati con chirurgia da sola hanno avuto una slm pi breve ( 23 , 3 mesi ) rispetto quelli trattati con combinazione di rt - ct ( 45 , 2 mesi )  . grigsby [ 22 ] ha analizzato i dati di 44 donne con carcinoma delluretra . 
brachytherapy reduced the risk of local recurrence , and the beneficial effect was most prominently seen in patients with bulky primary disease . in the conservative management of these tumours , the combination of external beam brachytherapy with or without ct play a significant role , and the multimodality approach appears to be the optimal way to treat these patients , with surgery to be used for biopsy - proven persistent tumours or recurrence [ 24 ]  . radiotherapy bladder radical cystectomy with bilateral pelvic lymph node dissection followed by urinary diversion remains the gold standard for treating muscle - invading bladder cancer and is the single most successful modality , reporting long - term os in the range of 40%60% [ 2528 ]  . 
rt is utilised as single modality treatment , as neoadjuvant or adjuvant to surgery , or combined with ct with or without conservative local surgery . i risultati di 34 pazienti con carcinoma uretrale sono stati analizzati in modo retrospettivo al princess margaret hospital [ 23 ] , trattati con rtbrachiterapia . 
 nella strategia terapeutica conservativa di questi tumori , la combinazione di rt e brachiterapia con o senza ct , riveste un ruolo significativo e , lapproccio multimodale sembra essere il modo ottimale per trattare questi pazienti , riservando la chirurgia per laccertamento istologico della persistenza o recidiva tumorale [ 24 ]  . vescica la cistectomia radicale con linfadenectomia pelvica bilaterale seguita da diversione urinaria rimane il gold standard per il trattamento del carcinoma della vescica invasivo ed la singola modalit pi vantaggiosa che ha riportato una sg a lungo termine del 40%60% [ 2528 ]  . 
such comparisons were very difficult , because patients selected for radical cystectomy had less advanced tumours at diagnosis , were younger and in a better general condition than patients selected for definitive irradiation . 
results reported in the literature are not significant : the 5 - year survival rate for patients treated with rt alone ( dose of 6066 gy at a 1.8to 2 - gy daily fraction ) is 32% ( range 22%50% ) [ 29 , 30 ]  . 
a recent review of treatment outcomes in rt - treated patients showed the following 5 - year survival rates : t1 , 35%71% ; t2 , 10%59% ; t3 , 10%38% ; and t4 , 0%16% [ 31 ]  . 
tali confronti sono molto difficoltosi perch i pazienti selezionati per la cistectomia radicale avevano tumori in stadio meno avanzato alla diagnosi , erano pi giovani ed in migliori condizioni generali rispetto ai pazienti selezionati per la rt radicale . 
in letteratura i risultati non sono significativi : la sopravvivenza a 5 anni per i pazienti trattati con rt da sola ( dosi di 6066 gy , con 1 , 82 gy come frazione giornaliera ) del 32% ( 22%50% ) [ 29 , 30 ]  . 
una review recente riguardo i risultati della terapia nei pazienti sottoposti a rt , ha mostrato la seguente incidenza di sg a 5 anni : t1 , da 35% a 71% ; t2 , da 10% a 59% ; t3 , da 10% a 38% e t4 da 0% a 16% [ 31 ]  . radioterapia pre - operatoria la pi ampia esperienza ( 338 pazienti ) con rt pre - operatoria ha ottenuto una sotto - stadiazione patologica nel 65% dei pazienti ed una risposta patologica completa nel 42% dei casi . 
la sopravvivenza a 5 anni dei pazienti che hanno ricevuto la rt seguita da cistectomia stata 38% vs radiol med ( 2009 ) 114 : 7082 followed by cystectomy was 38% vs 29% for those treated with rt alone ( not statistically significant )  . 
 strong support for the use of preoperative irradiation in patients with t3b bladder cancer was reported by cole et al . [ 34 ] : the 5 - year local control in the preoperative group was 91% compared with 72% for those treated with radical cystectomy alone . 
however , a meta - analysis of five randomised trials showed no statistical difference in treatment outcomes between patients treated with preoperative rt and cystectomy alone [ 35 ]  . 
 preoperative ccrt showed better results than rt alone . in fact , in the nordic cystectomy trial [ 37 ] , the combination of ct ( adriamycin + cddp for two cycles ) plus rt ( 20 gy in four fractions ) followed by cystectomy showed a 5 - year os improvement of 15% only for t3 - t4 disease compared with rt or surgery alone ( p = 0.03 ) , whereas no survival benefit was found for early stages of disease ( t1t2 )  . 
in the canadian randomised study [ 38 ] , concurrent cisplatin improved pelvic disease control with preoperative 29% per quelli trattati con rt da sola ( non statisticamente significativo , nss )  . 
 [ 34 ] : il cl a 5 anni nel gruppo sottoposto a rt preoperatoria stato del 91% rispetto al 72% per quelli trattati con cistectomia radicale e c stato anche un beneficio in termini di sg e slm ( nss )  . 
comunque , una metanalisi di 5 studi randomizzati non ha mostrato differenze statisticamente significative tra i pazienti trattati con rt pre - operatoria e quelli trattati con cistectomia radicale [ 35 ]  . 
una conclusione simile stata raggiunta in uno studio di fase iii su 140 pazienti , che confronta la chirurgia da sola con chirurgia e rt preoperatoria [ 36 ]  . la ccrt pre - operatoria ha mostrato migliori risultati rispetto alla rt da sola . 
infatti , nello studio nordico sulla cistectomia [ 37 ] la combinazione di ct ( adriamicina + cisplatino per 2 cicli ) pi rt ( 20 gy in 4 frazioni ) seguita da cistectomia ha mostrato un incremento della sg a 5 anni del 15% solo per i t3 - t4 rispetto alla rt o chirurgia da sola ( p = 0 , 03 ) , mentre nessun beneficio di sopravvivenza stato trovato negli stadi iniziali di malattia ( t1t2 )  . 
only one randomised study of radical cystectomy alone vs cystectomy and postoperative rt [ 41 ] reported a 5 - year pelvic recurrence of 5% ( 50% in the cystectomy arm ) and an improvement in dfs in patients with schistosomiasis . 
the main disadvantage of postoperative rt is the high rate ( 20%40% ) of serious late gastrointestinal complications [ 39 , 40 , 4244 ] because of the larger volume of small bowel occupying the pelvis after cystectomy . 
patients at high risk of recurrence are probably better treated with ct , which may prevent local and distant relapse and is usually well tolerated . bladder - preservation therapy the relative negative impact on quality of life for urinary diversion and the trend towards organ preservation led many authors to explore the role of conservative management . 
neoadjuvant ct compared with rt or surgery was investigated in an attempt to improve the results of surgery alone or definitive rt maintaining a functioning bladder . in the european organisation for research and treatment of cancer ( eortc ) / medical research council [ 45 ] trial , 976 patients undergoing curative cystectomy or radical rt were randomised to receive three cycles of neoadjuvant ct ( mcv ) in 491 patients or no ct in 485 . 
a recent meta - analysis [ 46 ] confirmed that neoadjuvant platinum - based combination ct reduces mortality risk of 13% , with moderate improvement ( 5% , p = 0.016 ) of 5 - year os , irrespective of the type of local treatment ( surgery or rt alone or rt and cystectomy ) and without differences between subgroups of patients . 
there was no evidence to support the use of single - agent platinum . these studies showed that patients treated with rt ( conservative approach ) have preserved bladder function . over the past 1520 years , several prospective trials have investigated conservative therapy in which all patients were treated with transurethral resection of bladder tumours ( turbt ) with or without ct followed by ccrt . 
alcuni studi hanno riportato una riduzione del rischio di recidive pelviche dal 30% al 50% al 10% al 20% [ 3941 ] , ma alcuni pazienti hanno anche ricevuto rt pre - operatoria . 
solo uno studio randomizzato sulla cistectomia radicale da sola verso cistectomia e rt postoperatoria [ 41 ] , ha riportato recidive pelviche a 5 anni del 5% ( 50% nel braccio della cistectomia ) ed un incremento della slm nei pazienti con schistosomiasi . 
il principale svantaggio della rt post - operatoria lalta percentuale ( 20%40% ) di gravi complicanze gastrointestinali tardive [ 39 , 40 , 4244 ] , poich una gran parte di volume dellintestino tenue occupa la pelvi dopo cistectomia . 
i pazienti ad alto rischio di recidiva sono probabilmente trattati in modo migliore con la ct , che pu prevenire recidive locali e a distanza ed di solito ben tollerata . 
 terapia di preservazione della vescica il relativo impatto negativo sulla qualit della vita per la diversione urinaria e la tendenza verso la preservazione dellorgano , ha permesso a molti autori di esplorare il ruolo di un trattamento conservativo . 
 nello studio delleortc ( european organisation for research and treatment of cancer ) / medical research council [ 45 ] , 976 pazienti sottoposti a cistectomia curativa o rt radicale sono stati randomizzati a ricevere 3 cicli di ct neoadiuvante ( mcv , metotressato , cddp , vinblastina ) in 491 pazienti , oppure nessuna ct in 485 pazienti . 
la differenza assoluta tra i gruppi per quanto riguarda la sg a 3 anni del 5 , 5% ( 55 , 5% per ct vs 50% nessuna ct ) non risultata statisticamente significativa ( p = 0 , 075 ) : la ct neoadiuvante stata associata con una patologica completa pi alta , ma non c stato nessuna chiara evidenza sulla sopravvivenza . 
una recente metanalisi [ 46 ] ha confermato che la combinazione di ct neoadiuvante a base di platino riduce il rischio di mortalit del 13% , con un incremento moderato ( 5% , p = 0 , 016 ) della sg a 5 anni , indipendentemente dal tipo di trattamento locale ( chirurgia o rt da sola o rt e cistectomia ) , e senza differenze tra i sottogruppi di pazienti , non vi stata alcuna evidenza a favore di platino da solo . 
 negli ultimi 1520 anni , diversi studi prospettici hanno analizzato terapie conservative in tutti quei pazienti trattati radiol med ( 2009 ) 114 : 7082 patients with cr , a consolidation rt or ccrt regimen was administered . 
 since 1985 , radiation therapy oncology group ( rtog ) trials have used a trimodality conservative treatment ( turbt , rt , ct ) in patients with t2t4a muscleinvasive bladder cancer who were candidates for cystectomy . 
in three rtog trials [ 4749 ] , patients were treated with neoadjuvant turbt and ccrt ( conventional or altered fractionation ) , whereas in two trials [ 50 , 51 ] , patients were treated with induction by turbt with or without ct and ccrt . 
cystectomy was reserved for incompletely responders or relapse . in the rtog 89 - 03 phase iii study , the combination of neoadjuvant ct ( mcv2 cycles ) with ccrt with cddp failed to show significant benefits in terms of 5 - year local control ( 61% vs 49% ) , os ( 48% vs 49% ) and ffdm ( 33% vs 39% ) compared with ccrt alone , with more toxicity in the mcv arm [ 51 ]  . 
in fact , subsequent studies by the rtog emphasised the impact of adjuvant ct [ mcv or cddp + 5fluorouracil ( fu ) ] following rt ( hypoor hyperfractionated ) [ 49 ] , with good results at 3 years . 
from paris began a prospective trial of preoperative ct using 5 - fu and cddp with concomitant rt ( 3 gy b.i.d. , total dose 24 gy ) , followed by either cystectomy or additional ccrt . 
this treatment strategy resulted in a pathological cr of 77% and may be proposed as conservative treatment in patients with a cr to the initial course of chemoradiation [ 54 ]  . 
globally , os at 5 years was 51% . similar results were obtained in further series of combined modality treatment for bladder preservation with excellent cr ( ~70% ) , and 5 - year os ( ~50% ) , with bladder con resezione transuretrale della vescica ( turbt ) ct seguita da ccrt , e per i pazienti con risposta completa stata somministrata una rt di consolidamento o ccrt ; la cistectomia stata riservata per le risposte incomplete o per le recidive di malattia . 
 dal 1985 , gli studi dellrtog ( radiation therapy oncology group ) hanno utilizzato un trattamento conservativo trimodale ( turbt , rt , ct ) nei pazienti con carcinoma della vescica t2t4a , che erano candidati alla cistectomia . in tre studi dellrtog [ 4749 ] i pazienti sono stati trattati con turbt neoadiuvante e ccrt ( convenzionale o frazionamenti alterati ) ; mentre in 2 studi [ 50 , 51 ] i pazienti sono stati trattati con induzione mediante turbtct e ccrt ; la cistectomia stata riservata per le risposte incomplete o in caso di recidive . 
 nello studio di fase iii dellrtog 89 - 03 , la combinazione di ct neoadiuvante ( mcv2 cicli ) con ccrt mediante cddp non ha mostrato alcun beneficio significativo in termini di cl a 5 anni ( 61% vs 49% ) , sg ( 48% vs 49% ) e slm ( 33% vs 39% ) , rispetto alla ccrt da sola , con una maggiore tossicit nel braccio che ha usato lmcv [ 51 ]  . 
infatti gli studi successivi dellrtog hanno puntualizzato limpatto della ct adiuvante ( mcv o cddp + 5 - fu ) alla rt ( ipoo iper - frazionamenti ) [ 49 ] , con buoni risultati a 3 anni . 
 [ 54 ] di parigi , hanno iniziato uno studio prospettico di ct preoperatoria usando 5 - fu e cddp con rt concomitante ( 3 gy bid , dose totale di 24 gy ) , seguita sia da cistectomia o ccrt addizionale . 
questa strategia di trattamento ha determinato una risposta patologica completa nel 77% dei casi , e pu essere proposta come terapia conservativa nei pazienti con risposta completa al ciclo iniziale di ccrt [ 54 ]  . 
 [ 55 ] , nella quale 415 pazienti sono stati trattati dopo la turbt con rt da sola ( 126 pazienti ) o in concomitanza con cddp ( 240 pazienti ) o cddp + 5 - fu ( 49 pazienti ) , con percentuali di risposte complete migliori per rt + cddp - 5 - fu ( 87% ) rispetto al cddp ( 66% ) o rt da sola ( 61% ) ; globalmente , la sg a 5 anni risultata pare a 51% . risultati simili sono stati ottenuti in ulteriori studi con modalit di trattamento combinato per la conservazione della vescica , con eccellenti risposte complete ( ~70% ) , ed una sg a 5 anni ( ~50% ) , con conservazione dellorgano in radiol med ( 2009 ) 114 : 7082 table 1 series of combined modality treatment for bladder preservation series no . 
 [ 70 ] turbt 24 gy at 3 gy + cddp / fu 2 additional cycles of crt ( dose not given ) ( specific cancer ) ng ( overall rate of cystectomy : 25.6% ) danesi et al . 
table 1 summarises the results of these studies [ 5674 ]  . in a recent meta - analysis of fifteen radiation series with different fractionation schedules and total doses , pos et al . [ 75 ] found evidence for an overall doseresponse relationship with an increase in local control by a factor of 1.441.47 for an increment in dose of 10 gy . 
 [ 71 ] recently reported the long - term results of a phase i / ii trial of ct ( mcv ) followed by concomitant cddp + 5 - fu and hyperfractionated irradiation ( three 100cgy fractions / day , for a total dose of 69 gy )  . 
accelerated rt may overcome radioresistance due to tumour - cell repopulation , but a recent randomised trial [ 77 ] reported no improvement of local control or survival rates and increased acute bowel complications . 
there has been only one small phase iii study of curative rt in which survival was inferior in the hypofractionated arm [ 78 ]  . some trials , in an attempt to improve results , explore the use of new chemotherapeutic agents and accelerated or hyperfractionated rt to increase irradiation dose in combination with ct . 
new chemotherapeutic agents , in particular gemcitabine and taxanes , are effective in combination with cddp and rt ( paclitaxel and gemcitabine are good radiosensitisers ) [ 61 , 79 ]  . 
a recent italian study [ 68 ] reported the feasibility of a combined scheme with gemcitabine ( up to 400 mg / m2 ) plus cddp and rt , obtaining 100% cr . 
finally , the rtog 99 - 06 trial introduced concomitant paclitaxel + cddp plus bifractionated rt as induction treatment , followed by adjuvant ct ( gemcitabine and cddp ) in patients with cr ; no specific outcomes are yet available [ 80 ]  . 
in una meta - analisi recente su 15 studi di rt con differenti schemi di frazionamento e dose totale , pos et al . [ 75 ] , hanno evidenziato una relazione globale dose - risposta , con incremento del cl , di un fattore che va da 1 , 44 a 1 , 47 per un aumento di dose di 10 gy . 
una meta - analisi di stuschke e thames [ 76 ] ha indicato che i regimi iper - frazionati mostrano un aumento della sg ( odd ratio di mortalit 0 , 55 ; p = 0 , 002 ) ed una risposta completa ( odd ratio di fallimento di 0 , 44 ; p = 0 , 001 )  . 
 [ 71 ] , recentemente hanno riportato risultati a lungo termine di uno studio di fase ii / ii di ct ( mcv ) seguita da ct a base di cddp + 5 - fu concomitante e rt iper - frazionata ( 1 gy per 3 frazioni al giorno , per una dose totale di 69 gy )  . 
le percentuali a 5 anni di sg , conservazione della vescica , ssm , slm e sopravvivenza senza cistectomia per tutti e 77 i pazienti sono state 58 , 8% , 46 , 6% , 75% , 53 , 5% e 76 , 1% , rispettivamente . 
la rt accelerata pu superare la radioresistenza generata dal ripopolamento delle cellule tumorali , ma in uno studio recente randomizzato [ 77 ] , non riportato alcun incremento delle percentuali del lc o della sg ma sono aumentate le complicanze intestinali acute . 
 alcuni studi , nel tentativo di migliorare i risultati , hanno esplorato luso di nuovi agenti chemioterapici e rt accelerata o iper - frazionata per aumentare la dose di rt in associazione con la ct . 
nuovi agenti chemioterapici , in particolare gemcitabina e taxani , sono efficaci in combinazione con cddp e rt ( paclitaxel e gemcitabina sono buoni radiosensibilizzanti ) [ 61 , 79 ]  . 
un recente studio italiano [ 68 ] , ha riportato la fattibilit di uno schema combinato con gemcitabina ( fino 400 mg / m2 ) pi cddp e rt , ottenendo il 100% di risposta completa . 
infine , lrtog 99 - 06 ha introdotto ct concomitante a base di paclitaxel + cddp pi rt bifrazionata come trattamento di induzione , seguito da ct adiuvante ( gemcitabina e cddp ) nei pazienti con risposta completa ; risultati specifici non sono ancora disponibili [ 80 ]  . conclusions conclusioni radical nephrectomy is the only effective therapy for clinically localised renal cancer . 
la cistectomia di salvataggio usata in caso di risposta parziale dopo terapia di induzione ccrt ( rtog ) o recidiva radiol med ( 2009 ) 114 : 7082 response after induction ccrt treatment ( rtog ) or recurrent disease ( european trials ) : in both cases , surgery can be curative , reporting a 5 - year survival of approximately 50% [ 45 , 50 , 51 , 54 , 55 ]  . 
 trimodality treatment ( turbt , rt , ct ) of invasive bladder cancer may be offered as a valid alternative to radical cystectomy , with no decrease in survival related to the delay in cystectomy , which is currently the gold standard therapy . 
os is comparable with recent surgical data , reporting a 5 - year os of almost 50% , with bladder preservation in three fourths of these patients . di malattia ( studi europei ) : in entrambi i casi la chirurgia pu essere curativa , riportando una sopravvivenza a 5 anni del 50% approssimativamente [ 45 , 50 , 51 , 54 , 55 ]  . 
 il trattamento trimodale ( turbt , rt , ct ) del carcinoma invasivo della vescica pu offrire una valida alternativa alla cistectomia radicale con nessuna diminuzione della sopravvivenza correlata al ritardo della cistectomia , che rimane attualmente la terapia gold standard . 
complete tumour necrosis , confirmed by targeted biopsy , was observed in patients showing no intralesional flow signals and time - intensity curves with low peak of signal intensity and absence of plateau after treatment . 
dopo il trattamento , nei pazienti che presentavano assenza di segnali vascolari intralesionali , curve con basso picco di intensit e assenza di plateau , la biopsia mirata confermava la necrosi completa . 
in base ai risultati ottenuti , le curve con elevato picco di intensit e presenza di plateau , potrebbero rappresentare pattern del tessuto neoplastico intra o perilesionale e fornire quindi importanti informazioni sullefficacia del trattamento . radiol med ( 2009 ) 114 : 3241 keywords power doppler time - intensity curves contrast material focal hepatic lesions hepatocellular carcinoma metastasis parole chiave power doppler curve dintensit - tempo mezzi di contrasto lesioni focali epatiche carcinoma epatocellulare metastasi introduction introduzione in recent years , several nonoperative techniques have been used successfully in the locoregional treatment of hepatocellular carcinoma and metastases , including percutaneous ethanol transcatheter arterial chemoembolisation ( tace ) [ 35 ] , and radiofrequency ablation ( rfa ) [ 69 ]  . injection ( pei ) [ 1 , 2 ] , the follow - up of patients undergoing these treatments is generally based on imaging studies such as magnetic resonance imaging ( mri ) [ 10 , 11 ] or computed tomography ( ct ) [ 12 ] , with only few doubtful cases being studied by biopsy to assess whether complete necrosis of the neoplastic tissue has been achieved . contrast - enhanced colour doppler ultrasound ( cdus ) , thanks to its high sensitivity in detecting weak intratumoural flow signals , has been employed for several years in the differential diagnosis of focal hepatic lesions [ 1315 ]  . recently , it has also been used for the follow - up of intralesional therapy [ 1619 ] , to identify residual areas of intratumoural enhancement that could reflect incomplete necrosis or disease recurrence . 
a study of intralesional and perilesional vascularity with analysis of time - signal intensity curves performed immediately after treatment may help to ascertain whether certain flow signals reflect incomplete treatment . 
the use of contrast - enhanced power doppler us ( pdus ) with timeintensity curves in liver diseases [ 2023 ] , as in focal breast lesions [ 24 ] , may also provide valuable information on the persistence of neoplastic perilesional vascularity and consequently on incomplete necrosis . 
the purpose of this study was to assess the utility of pdus with analysis of time - intensity curves in monitoring focal hepatic lesions ( metastases , hepatocellular carcinoma ) treated with pei or rfa and to correlate the findings with the results of targeted biopsy . materials and methods twenty patients ( twelve men and six women ; age range 55 to 72 years ) with hepatocellular carcinoma and solitary metastases of the liver previously diagnosed by other methods ( ct , mri , biopsy ) underwent intralesional therapy with pei or rfa . 
all patients were studied negli ultimi anni sono state utilizzate diverse tecniche non chirurgiche per il trattamento loco - regionale dellepatocarcinoma e delle metastasi , alcune di queste , molto efficaci come lalcoolizzazione percutanea ( pei ) [ 1 , 2 ] , la chemioembolizzazione ( tace ) [ 35 ] e la radioipertermia ( rfta ) [ 69 ]  . il follow - up dei pazienti sottoposti a questi trattamenti generalmente affidato a metodiche di diagnostica per immagini come la rm [ 10 , 11 ] o la tc [ 12 ] ; solo in alcuni casi dubbi si ricorre alla biopsia per verificare leffettiva necrosi completa del tessuto neoplastico . 
 il color doppler con mdc , grazie allelevata sensibilit nel rilevare i deboli segnali vascolari intralesionali una metodica gi da qualche anno utilizzata per la diagnosi differenziale delle lesioni focali epatiche [ 1315 ] , ed stata gi utilizzata anche per il follow - up dopo la terapia intralesionale [ 1619 ]  . 
spesso subito dopo il trattamento il color doppler con mdc evidenzia una ricca vascolarizzazione perilesionale la cui natura non pu essere determinata se non con la biopsia ; quindi lo studio della vascolarizzazione intra e perilesionale con le curve intensit / tempo , subito dopo il trattamento , potrebbe essere utile poich alcuni segnali vascolari potrebbero essere indice di incompleto trattamento . 
lutilizzo del power doppler con mdc con la determinazione delle curve intensit / tempo nelle malattie del fegato [ 2023 ] o come nel caso delle lesioni focali della mammella [ 24 ] , potrebbe fornire , a nostro giudizio , informazioni preziose anche circa la persistenza di vascolarizzazione neoplastica perilesionale dopo il trattamento e quindi incompleta necrosi della lesione . 
obiettivo di questo lavoro quindi lutilizzo del power doppler con le curve intensit / tempo nel monitoraggio delle lesioni focali epatiche ( metastasi , epatocarcinoma ) sottoposte al trattamento intralesionale con pei e rfta confrontando i risultati con la biopsia mirata . materiali e metodi venti pazienti , dodici maschi e sei femmine , con et compresa tra 55 e 72 anni , affetti da epatocarcinoma e radiol med ( 2009 ) 114 : 3241 by pdus with analysis of time - intensity curves one day before and one day after treatment , followed by targeted biopsy two days later . 
subjects in the control group were imaged by pdus with analysis of time - intensity curves . the us studies were performed on an au cinque idea device ( esaote , genoa , italy ) with a 3.5 mhz convex - array probe . 
in patients the time - intensity curves were derived from a circular region of interest ( roi ) placed over the lesion and large enough to encompass both the intralesional and perilesional vessels . 
il giorno prima e il giorno dopo il trattamento , tutti i pazienti sono stati studiati con esame power doppler con determinazione delle curve intensit / tempo e successivamente , 2 giorni dopo il trattamento , sottoposti a biopsia mirata . 
i pazienti del gruppo di controllo sono stati sottoposti ad esame power doppler con curve intensit / tempo . per lindagine ecografica stato utilizzato un apparecchio au cinque idea ( esaote , genova ) con sonda convex da 3 , 5 mhz . 
nei pazienti patologici la curva intensit / tempo stata ottenuta utilizzando una roi circolare posizionata sulla lesione in modo da comprendere sia la vascolarizzazione intra che perilesionale a tale scopo stato fatto coincidere il diametro della roi con il diametro massimo ottenuto comprendendo la lesione e la vascolarizzazione perilesionale . 
nei pazienti del gruppo di controllo stata utilizzata su tutti i pazienti una roi circolare del diametro di un centimetro posizionata in unarea del parenchima del lobo destro del fegato . 
b the corresponding time - intensity curve shows an elevated peak of signal intensity ( arrowhead ) related to a high concentration of contrast agent and a plateau phase ( arrow ) due to the retention of contrast material within the lesion . 
b la curva intensit / tempo prima del trattamento mostra il raggiungimento di un elevato picco di intensit ( testa di freccia ) , che corrisponde ad unelevata concentrazione di mdc e la presenza di plateau ( freccia ) , dovuti ad una lunga permanenza del mdc nella lesione . 
b the corresponding time - intensity curve shows an elevated peak of intensity ( arrow ) due to high concentration of contrast material and plateaus ( arrow heads ) due to retention of the contrast material in the lesion . 
b la curva intensit / tempo prima del trattamento mostra il raggiungimento di un elevato picco di intensit ( freccia ) , che corrisponde ad unelevata concentrazione di mdc e la presenza di plateau ( teste di freccia ) , dovuti ad una prolungata permanenza del mdc nella lesione . 
b the corresponding time - intensity curve shows an elevated peak of intensity ( arrow ) related to the high concentration of contrast agent and plateaus ( arrowheads ) due to retention of the contrast material within the lesion . 
b la curva intensit / tempo eseguita subito dopo il trattamento mostra il raggiungimento di un elevato picco di intensit ( freccia ) che corrisponde ad unelevata concentrazione di mdc e la presenza di plateau ( teste di freccia ) , dovuto ad una prolungata permanenza del mdc nella lesione . 
b the corresponding time - intensity curve shows an elevated peak of signal intensity ( arrowhead ) related to the high concentration of contrast agent and a plateau phase ( arrow ) due to retention of the contrast in the lesion . 
b la curva intensit / tempo prima del trattamento mostra il raggiungimento di un elevato picco di intensit ( freccia ) , che corrisponde ad unelevata concentrazione di mdc e la presenza di un plateau ( testa di freccia ) , dovuto ad una lunga permanenza del mdc nella lesione . 
d la curva intensit / tempo eseguita subito dopo il trattamento mostra il raggiungimento di un elevato picco di intensit ( testa di freccia ) e la presenza di un plateau ( freccia ) , dovuto ad una prolungata permanenza del mdc nella lesione . in our study , contrast - enhanced pdus performed before treatment revealed intralesional or perilesional flow signals in all patients , so we were able to derive significant time - intensity curves . 
b gruppo di controllo ( paziente sano ) : la curva intensit / tempo eseguita in una porzione del lobo destro del fegato dimostra un basso picco di intensit ( bassa concentrazione di mdc ) ( freccia ) ed un profilo a cupola corrispondente ad un lento accumulo e lento , progressivo rilascio del mdc . performed one day after treatment revealed time - intensity curves without plateaus and with low peaks of signal intensity , confirming complete necrosis of the tumour and success of the treatment . 
this finding was confirmed by the biopsy . instead , in one patient without intralesional flow signals but with persisting perilesional flow signals and time - intensity curve characterised by plateau and high peak of signal intensity , the biopsy revealed residual neoplastic tissue . 
 non si sono verificate reazioni avverse alla somministrazione ev del mdc . discussione le curve intensit / tempo permettono lo studio del comportamento della vascolarizzazione intra e perilesionale dopo la somministrazione ev del mdc e in particolare monitorizzano landamento della concentrazione del mdc nella regione dinteresse in un tempo prestabilito . 
lesistenza di strutture vascolari intra e perilesionali di tipo neoplastico influenza il profilo delle curve in quanto la presenza di shunt artero - venosi e lassenza di sistemi sfinteriali fisiologici , tipiche dei vasi neoformati , si traduce in unintrappolamento del mdc allinterno degli stessi e quindi in una fase di wash - out caratterizzata dai plateau ( fasi stazionamento del mdc ) e presenza di elevati picchi di intensit . nel nostro studio lesame power doppler con mdc ha riscontrato la presenza di segnali vascolari intra o perilesionali in tutti i pazienti prima del trattamento intralesionale ; ci ha permesso lottenimento di curve intensit / tempo significative ; infatti la presenza di enhancement nellarea di interesse una condizione indispensabile e rappresenta quindi un limite della metodica . 
in 15 pazienti , al controllo dopo un giorno dal trattamento , sono state riscontrate curve intensit / tempo con assenza di plateau e basso picco di intensit a sostegno della necrosi completa del tumore e quindi del successo del trattamento ; ci stato confermato dalla biopsia di controllo , mentre , in un paziente che presentava assenza di segnali vascolari intralesionali e persistenza di segnali vascolari perilesionali , in cui la curva intensit / tempo mostrava profilo con plateau ed elevato picco di intensit , la biopsia confermava la persistenza di tessuto neoplastico . 
in questo ultimo caso la curva intensit / tempo ha evidenziato lincompleta necrosi tumorale anche in assenza di segnali vascolari intralesionali dopo il trattamento ; ci fa supporre che la presenza di tessuto neoplastico coinvolgeva la regione perilesionale . 
tale reperto assume notevole importanza , a nostro giudizio , poich il riscontro di una curva come la suddetta dopo il trattamento potrebbe fornire lindicazione per lesecuzione di unaltra seduta terapeutica per ottenere la completa necrosi del tumore . radiol med ( 2009 ) 114 : 3241 an indication for an additional treatment session to achieve complete necrosis of the tumour . 
 in the four patients exhibiting intralesional flow signals after treatment , the time - intensity curves were characterised by plateaus and high peaks of signal intensity , suggesting incomplete necrosis of the tumour , later confirmed by targeted biopsy . 
 the presence of plateaus and high peaks of signal intensity , which characterised all curves before treatment and those of lesions found to be positive at biopsy after treatment , might represent a pattern of neoplastic vascularity . the technique should be further evaluated on a larger number of patients . 
if our findings are confirmed , it could be proposed as an alternative to ct and mri in consideration of its numerous benefits , including limited invasiveness , rapidity , and lack of side - effects of contrast material . nei quattro pazienti che presentavano segnali vascolari intralesionali dopo il trattamento , le corrispondenti curve intensit / tempo mostravano profilo con plateau ed elevato picco di intensit a conferma dellincompleta necrosi del tumore , confermata dalla biopsia mirata . 
passariello1 1 sapienza universit di roma , dipartimento di scienze radiologiche , policlinico umberto i , viale regina elena 324 , 00161 roma , italy 2 sapienza universit di roma , dipartimento di urologia , policlinico umberto i , viale del policlinico 159 , 00161 roma , italy correspondence to : v . 
twenty - eight patients with suspected bladder cancer and ten patients who had undergone transurethral resection of the bladder were studied by ctc and vc in both the supine and prone positions after distending the bladder with air . 
valutare il ruolo della cistografia con tc ( ctc ) e della cistoscopia virtuale ( cv ) utilizzando una tc multidetettore a 64 strati ( tcmd - 64 ) nellidentificazione delle lesioni vescicali e confrontare i risultati ottenuti con la cistoscopia convenzionale . 
ventotto pazienti con sospetta neoplasia vescicale e 10 pazienti in follow - up dopo resezione trans - uretrale della vescica ( turb ) sono stati sottoposti ad esame ctc e cv . 
i valori di sensibilit e specificit delle singole metodiche ctc e cv sono stati costantemente inferiori rispetto a quelli riportati dalla valutazione combinata ( gruppo i : 93 , 10% / 92 , 31% ; gruppo ii : 100% / 100% ; gruppo iii : 100% / 100% )  . 
la cv e la ctc con tcmd - 64 sono tecniche proponibili nellidentificazione delle neoplasie vescicali di dimensioni comprese tra 1 e 5 multeriori studi necessitano di popolazioni pi ampie . 
 radiol med ( 2009 ) 113 : 5269 keywords bladder cancer ct cystography virtual cystoscopy parole chiave neoplasia vescicale cistografia tc cistoscopia virtuale introduction introduzione flexible cystoscopy is the gold standard for diagnosing bladder cancer , not only because of its high sensitivity and specificity in detecting lesions but also the possibility of performing resections or biopsies in real time [ 1 ]  . 
despite the development of new cystoscopes and examination techniques , flexible cystoscopy still remains an invasive investigation that is poorly tolerated by patients and is precluded in subjects with acute bleeding or urethral strictures [ 2 ]  . although noninvasive imaging modalities , such as intravenous urography and transabdominal ultrasound , have been used as first - line investigations in several studies [ 3 , 4 ] , the endoscopic approach , especially when implemented with the new fluorescence techniques that enable detection of flat or very small lesions ( < 5 mm ) [ 5 , 6 ] , remains the mainstay of all international diagnostic protocols . 
the first reports on the use of computed tomography cystography ( ctc ) and virtual cystoscopy ( vc ) for detecting bladder cancers were published in the late 1990s [ 79 ]  . 
with regard to lesion size , it has been shown that single - detector - row , fourdetector - row [ 13 ] and even 16 - detector - row ct performed with thin - slice reconstructions ( 1 mm ) [ 14 ] allow detection of lesions < 5 m incorporation of this modality in new study protocols is a critical decision that is being widely debated among clinicians and diagnostic specialists . 
this aspect does not seem to take into account the potential benefit , in terms of therapeutic timeliness , of early and noninvasive detection of minimal lesions , especially in the 30% of lesions that show rapidly progressive behaviour [ 15 ]  . 
in our opinion , the use of 64 - detector - row ct technology should improve z - axis resolution and increase spatial resolution , thanks to the acquisition of isotropic voxels , such that excellent sensitivity and specificity can be obtained for very small lesions . 
the aim of this study was to evaluate the diagnostic accuracy of ctc with vc in the early detection of bladder lesions 1 mm to 1 cm in size , using flexible cystoscopy as the standard of reference . 
 materials and methods study population between november 2006 and january 2008 , we selected 38 la cistoscopia convenzionale rappresenta attualmente il gold standard diagnostico di neoplasia vescicale non solo per i suoi elevati valori di sensibilit e specificit nellidentificazione di lesioni sospette , ma anche grazie alla possibilit di eseguire in tempo reale sia la resezione che un eventuale prelievo bioptico [ 1 ]  . 
nonostante lo sviluppo di nuovi cistoscopi e metodiche di esecuzione dellindagine , la cistoscopia convenzionale rimane , ancora oggi , un esame invasivo , generalmente poco tollerato dai pazienti e che non pu essere eseguito in tutti i casi come in soggetti che presentano un sanguinamento acuto o una stenosi uretrale [ 2 ]  . 
 le metodiche di imaging diagnostico non invasive , quali lurografia intravenosa , lecografia transaddominale sono state introdotte come primo approccio in diversi studi [ 3 , 4 ] , tuttavia lapproccio endoscopico , in considerazione anche delle nuove tecniche a fluorescenza , con cui stato implementato per lidentificazione delle lesioni piatte o molto piccole ( < 5 mm ) [ 5 , 6 ] , rimane un caposaldo in tutti i protocolli diagnostici internazionali . 
le prime cistografie con tc ( ctc ) e con cistoscopia virtuale ( cv ) per lo studio delle neoplasie vescicali sono state eseguite alla fine degli anni 90 [ 79 ] ; da allora la tecnica stata ampiamente sviluppata e recenti studi [ 1012 ] dimostrano alti potenziali di sensibilit nellidentificazione di neoplasie vescicali . per quanto riguarda le dimensioni delle lesioni vescicali , stato dimostrato che le tc a singolo detettore , a 4 detettori [ 13 ] e perfino a 16 detettori , utilizzando protocolli di studio con ricostruzioni a strato sottile ( 1 mm ) [ 14 ] , permettono lidentificazione di lesioni al di sotto dei 5 mm . linserimento di questa metodica di indagine in nuovi protocolli rimane attualmente una decisione critica dibattuta tra clinici e diagnosti specialisti . 
questo aspetto non sembra quindi rendere conto del vantaggio che lindividuazione precoce , con un metodo non invasivo , di lesioni minime potrebbe rappresentare in termini di tempestivit terapeutica : soprattutto in quel 30% dei casi in cui queste lesioni mostrano un comportamento rapidamente progressivo [ 15 ]  . 
lutilizzo delle apparecchiature tc con tecnologia multistrato a 64 detettori , dovrebbero migliorare la risoluzione lungo lasse z ed aumentare la risoluzione spaziale grazie alla isotropicit dei voxels , cos da offrire unottima sensibilit e specificit diagnostica per lesioni di dimensioni esigue . 
lo scopo di questo studio stato analizzare il grado di accuratezza diagnostica della cistrografia tc con cistoscopia virtuale nellidentificazione precoce di lesioni vescicali di dimensioni comprese tra 1 mm e 1 cm , utilizzando la cistoscopia convenzionale come metodica di riferimento . 
the population was initially divided into 28 patients ( 22 men and six women ) who had not undergone surgery and ten patients ( eight men and two women ) who were being followed up after transurethral resection of the bladder ( turb )  . 
all patients had a medical history suspicious for bladder cancer , including haematuria with negative urine culture , exposure to carcinogenic chemical agents , family history , recurrent or parasitic infections and positive cytology on urinalysis . 
approximately 1 week ( mean 83 ) after the ct scan , each patient was studied with conventional cystoscopy to determine the presence , location , morphology and size of any bladder lesions . 
 patient preparation before ct examination , a three - way 12 - f foley catheter was inserted into the bladder to achieve complete voiding . the bladder was then distended by insufflating 350500 cc of room air with 50 cc syringes , depending on patient tolerance . 
images obtained with these scans were then analysed by a third , independent , radiologist who materiali e metodi popolazione di studio in un periodo compreso fra novembre 2006 e gennaio 2008 , abbiamo selezionato complessivamente 38 pazienti ( 30 di sesso maschile e 8 di sesso femminile ) afferenti presso il centro per le neoplasie dellapparato urinario del dipartimento di urologia del nostro nosocomio . 
la popolazione di studio stata inizialmente suddivisa in 28 pazienti ( 22 uomini e 6 donne ) che non avevano subito interventi chirurgici e 10 ( 8 uomini e 2 donne ) pazienti in fase follow - up dopo resezione trans - uretrale della vescica ( turb )  . 
tutti i pazienti presentavano unanamnesi sospetta per neoplasia vescicale comprendente ematuria con urino - coltura negativa per processi infiammatori aspecifici , fattori di rischio con esposizione ad agenti chimici cancerogeni , familiarit , anamnesi di infezioni ricorrenti o malattia parassitaria della vescica e citologia positiva allesame delle urine . 
a circa una settimana ( media 83 ) dopo lesame tc ciascun paziente stato sottoposto ad esame di cistoscopia convenzionale in cui stata stabilita la presenza , la localizzazione , la morfologia e la dimensione di eventuali lesioni vescicali . 
 preparazione dei pazienti prima dellesecuzione dellesame tc nel lume vescicale stato posizionato un catetere di foley a tre vie ( calibro 12 f ) per il completo svuotamento della vescica ; successivamente stato disteso il lume vescicale insufflando circa 350500 cc di aria tramite siringhe da 50 cc , tenendo conto del grado di tolleranza del paziente . 
dopo lesame e la rimozione del catetere , a tutti i pazienti stata prescritta una terapia antibiotica a scopo profilattico per 3 giorni . apparecchiatura tc e protocolli di scansione tutti gli esami sono stati eseguiti con unapparecchiatura tc spirale multistrato a 64 detettori ( somatom sensation 64 , siemens , germania ) utilizzando i seguenti parametri : radiol med ( 2009 ) 113 : 5269 performed a contrast - enhanced scan ( at 60 s ) directly in the prone position to evaluate the possible extramural extension of any identified mural lesion . 
for each scan , we used multiplanar reconstructions ( mpr ) in the sagittal and coronal planes and volume rendering ( vr ) for endoluminal viewing ( virtual endoscopy )  . each examination was analysed in a double - blind fashion by two radiologists with 10 years experience in virtual endoscopy and who were unaware of patients clinical data . image analysis was performed over three reading sessions held approximately 1 week apart in which the order of the patients was changed each time . 
for each step , the radiologists expressed a diagnostic judgment on any lesion identified based on the following parameters : lesion site ( bladder dome , walls and trigone ) , size and morphology ( sessile where the base was larger than the height , otherwise pedunculated )  . 
bladder distension was evaluated on axial images using a reference score based on maximum anteroposterior diameter ( < 10 cm , poor distension = score 0 ; 1015 cm , satisfactory distension = score 1 ; > 15 cm , optimal distension = score 2 )  . 
bladder lumen and mucosa were studied with a pulmonary window level setting ( 1 , 500 / 700 hu ) ; bladder walls and extraluminal structures were studied with an abdominal - level setting ( 400 / 40 hu )  . 
for virtual cystoscopy , we used the 3d lit fly - through with direct lightshiny rendering and ct fatmusclebone colouring ( window the ct level 1500 / 200 ) supplied with colonoscopy software . 
 statistical analysis the study population was divided into three groups according to the size of bladder lesions identified at flexible cystoscopy : group 1 : lesions with maximum diameter from 1 mm to 5 mm group 2 : lesions between 5.1 mm and 9 mm 50 mas , 120 kv , spessore effettivo della fetta 1 millimetro , collimazione 0 , 6 millimetri , incremento di ricostruzione 0 , 75 millimetri , kernel b30 smooth , velocit di rotazione del tubo 0 , 5 s , risoluzione spaziale ( x ) 0 , 4 mm ( y ) 0 , 4 mm ( z ) 0 , 4 mil campo di vista ( fov ) stato ottimizzato individualmente per la valutazione completa della pelvi , in modo da ottenere contemporaneamente sia una visualizzazione della vescica sia delle strutture extra - vescicali ( media fov 304040 cm )  . 
successivamente sono state effettuate scansioni sia in posizione supina che prona per evitare che eventuali residui fluidi potessero occultare piccole lesioni parietali ; le immagini ottenute da queste scansioni sono state poi analizzate da un terzo radiologo non coinvolto nel presente studio il quale , nel caso in cui identificava lesioni vescicali , eseguiva una scansione supplementare della pelvi direttamente nella posizione prona , dopo somministrazione di mezzo di contrasto iodato non ionico ( a 60 s ) , al fine di evidenziare una possibile estensione extra - parietale della lesione . 
la dose length product ( dlp ) utilizzata stata di 354 mgy / cm , il computed tomography dose index / weight ( ctdiw ) stato di 29 , 06 mgra y e la dose efficace ( de ) stata di 6 , 726 msv . 
in caso di scnsione supplementare della pelvi , il valore dlp stato di 531 mgy / cm , il ctdiw stato di 43 , 59 mgray e il ed stato di 10 , 089 msv . post - processing ed analisi dei dati acquisiti i dati ottenuti con tale protocollo sono stati trasferiti in tempo reale ad una workstation del tipo hp kajak xu 800 double 2 , 8 ghz cpu , 3 , 37 gb di ram , con hard - disk da 140 gb e software dedicato , vitrea ( versione 3.7 , vital images , inc . , plymouth , minnesota , usa ) per lelaborazione e lanalisi dei dati . 
per ogni scansione eseguita sono stati utilizzati algoritmi di ricostruzione multiplanari ( mpr ) sui piani di scansione sagittale e coronale e di volume rendering per la visione endoluminale ( endoscopia virtuale )  . ogni singolo esame stato analizzato in doppio cieco da due radiologi , con 10 anni di esperienza in endoscopia virtuale , i quali non erano a conoscenza dei risultati clinici di preselezione dei pazienti . 
il protocollo di analisi delle immagini si basato su tre sessioni di lettura a distanza di circa una settimana luna dallaltra , nelle quali lordine dei pazienti analizzati stato cambiato ogni volta . 
durante i tre step i radiologi hanno quindi valutato dapprima le immagini assiali 2d nelle scansioni in posizione prona e supina , successivamente le immagini ricostruite con lalgoritmo multiplanare mpr ed infine le ricostruzioni con algoritmo vr ( visione endo - luminale )  . 
per ciascuna fase di analisi i radiologi hanno espresso un giudizio diagnostico sulle eventuali lesioni osservate stimando i seguenti parametri : sede ( cupola , pareti e fondo della vescica ) , dimensioni e morfologia ( sessile ove la base fosse maggiore dellaltezza , peduncolata nel caso contrario )  . 
furthermore , we conducted a detailed analysis of the first group using receiver operating characteristic ( roc ) curves to determine , for each technique taken alone and in combination , the lowest size threshold at which the method showed the best level of diagnostic accuracy . 
histopathology revealed the presence of neoplastic cells in 62 resected specimens , 52 of which were in 28 presurgical patients and ten in post - turb patients ( 48 lesions were transitional - cell carcinomas , ten were clear - cell adenocarcinomas and four were small - cell carcinomas )  . considering the subdivision of lesions into subgroups , 29 positive lesions were identified in 14 patients in group 1 , with lesions < 5 mm [ nine presurgical and five post - turb patients ; size range 1.35 mm ; mean 3.4 mm ; median 3.5 mm ; standard deviation ( sd ) 0.83 ] , 18 positive lesions were identified in 12 patients in group 2 , with lesions between 5.1 and 9 mm ( eight presurgical and four post - turb patients ; size range 5.29 mm ; mean 7.2 mm ; median 7.35 mm ; sd 1.196 ) , and 15 positive lesions were identified in 12 patients in group 3 , with lesions > 9.1 mm ( 11 presurgical patients and one post - turb patient ; size range 9.322 mm ; mean 12.42 mm ; median 11 mm ; sd 3.46 ) ( table 1 )  . 
mean examination time , excluding catheter insertion , was 82 m reconstruction of raw data produced approximately 800 images for each data set ( considering both supine and prone scans ) , with an additional 400 images in the case of contrast - enhanced imaging for staging purposes . 
per la valutazione del lume vescicale e della mucosa stato impostato un livello della finestra ottimale per il parenchima polmonare ( 1500 / 700 hu ) mentre il livello della finestra per laddome ( 400 / 40 hu ) stato utilizzato per la valutazione della parete e delle strutture extra - luminali . 
per la cistoscopia virtuale abbiamo utilizzato il preset fornito nel software dedicato per la colonscopia vituale 3d lit fly - trough con rendering direct light - shiny e colorazione tc fat - muscle - bone ( finestra utilizzata 1500 / 200 )  . 
in particolare sono stati stabiliti tre valori principali di cut - off dimensionale cos distribuiti : gruppo i : lesioni comprese fra 1 mm e 5 mm di diametro massimo ( dm ) ; gruppo ii : lesioni comprese fra 5 , 1 e 9 mm di dm ; gruppo iii : lesioni pi grandi di 9 , 1 mm di dm . lanalisi statistica dei dati raccolti stata realizzata calcolando sensibilit e specificit di ogni singola metodica ( ctc in posizione supina e prona , cv supina e prona , esame completo considerando entrambe ) per ogni sub gruppo di pazienti . 
abbiamo inoltre condotto un analisi accurata del primo gruppo di pazienti utilizzando il metodo delle receiver operating characteristic ( roc ) curve analysis per stabilire , per ciascuna metodica di scansione e per lanalisi combinata delle stesse , quale fosse il limite inferiore dimensionale in cui la metodica mostrava il miglior livello di accuratezza diagnostica . 
come ulteriore controllo abbiamo condotto un analisi incrociata dei risultati ottenuti dai due radiologi nellidentificazione delle lesioni nei tre gruppi , utilizzando il metodo di inter - rater agreement con statistica k per valutare la variabilit interoperatore . 
i seguenti valori di k sono stati utilizzati come cut - off analisi del livello di concordanza statistica : concordanza ; = 0 , 210 , 40 = modesta < 0 , 20 = scarsa = 0 , 410 , 60 = moderata concordanza ; concordanza ; = 0 , 610 , 80 = concordanza buona ; = 0 , 811 , 00 = concordanza completa . 
per tutti i test statistici utilizzati un livello di p < 0 , 05 stato considerato significativo . risultati cistoscopia convenzionale la cistoscopia convenzionale ha visualizzato correttamente un totale di 88 lesioni fra i 38 soggetti esaminati . 
prone vc had a sensitivity of 89.66% , a specificity of 92.31% , a ppv of 96.30% and an npv of 80% , whereas e 10 in pazienti post - turb ( 48 erano carcinomi a cellule transizionali , 10 adenocarcinomi a cellule chiare e 4 carcinomi a piccole cellule )  . 
la suddivisione in sub gruppi ha quindi identificato 29 lesioni positive in 14 pazienti nel i gruppo con neoplasie inferiori a 5 mm ( 9 pazienti preoperatori e 5 post - turb ; dimensioni minime 1 , 3 mm / massime 5 mm ; media 3 , 4 mm ; mediana 3 , 5 mm ; deviazione standard 0 , 83 ) , 18 lesioni positive in 12 pazienti nel ii gruppo con neoplasie comprese tra 5 , 1 e 9 mm ( 8 pazienti preoperatori e 4 post - turb ; dimensioni minime 5 , 2 mm / massime 9 mm ; media 7 , 2 mm ; mediana 7 , 35 mm ; deviazione standard 1 , 196 ) e 15 lesioni positive in 12 pazienti nel iii gruppo con neoplasie superiori a 9 , 1 mm ( 11 pazienti preoperatori e 1 post - turb ; dimensioni minime 9 , 3 mm / massime 22 mm ; media 12 , 42 mm ; mediana 11 mm ; deviazione standard 3 , 46 ) ( tabella 1 )  . 
 cistografia tc e cistoscopia virtuale gli esami tc sono stati generalmente ben tollerati dai pazienti , sebbene in 6 / 38 ( 4 / 10 pazienti dopo turb ) casi si sia verificato un certo disagio dovuto ad un diverso grado radiol med ( 2009 ) 113 : 5269 fig . 
1a - c sagittal multiplanar reproduction ( mpr ) images used for scoring bladder distension : optimal distension coincided with the insufflation of 450350 ml of air ( a ) , whereas satisfactory ( b ) and poor ( c ) distension levels were usually obtained with 350250 ml and < 250 ml . 
1a - c immagini mpr sagittali utilizzate per la valutazione del grado di distensione vescicale : una distensione ottimale coincide con linsufflazione di 350450 ml di aria ( a ) , mentre livelli di distensione soddisfacenti ( b ) e scarsi ( c ) vengono ottenuti di solito con 250350 ml e meno di 250 ml . 
in media il tempo per lesecuzione dellesame , escluso quello relativo al posizionamento del catetere , stato di 82 m la ricostruzione dei dati grezzi ha generato circa 800 immagini per ogni insieme di dati ( considerando entrambe le scansioni prona e supina ) , con un incremento di 400 immagini per l eventuale scansione di staging con mdc . 
il tempo medio della ricostruzione ctc / cv stato di 103 mcon un tempo medio di refertazione di 152 mla distensione della vescica risultata ottimale in 30 casi , soddisfacente in 5 e scarsa in 3 . 
la vc in posizione prona una sensibilit del 89 , 66% , specificit 92 , 31% , ppv 96 , 30% e npv del 80% ; mentre nella posizione supina sensibilit del 82 , 76% , specificit del 92 , 31% , ppv 96% e npv 70 , 59% . 
la valutazione combinata delle immagini ottenute con le diverse metodiche di valutazione nelle diverse posizioni ha riportato valori nettamente superiori con sensibilit del 93 , 10% , specificit 92 , 31% , ppv 96 , 43% e npv del 85 , 71% ( tabella 2 )  . la successiva analisi dei risultati con le curve roc ha evidenziato una tendenza simile alla statistica precedente riportando valori di area sottesa alla curva ( auc ) costantemente maggiori ( maggiore accuratezza diagnostica ) per la valutazione complessiva delle tecniche rispetto ad ogni singola metodica ( ctc prona = 0 , 844 ; ctc supina = 0 , 809 ; vc prona = 0 , 944 ; vc supina = 0 , 901 ; complessiva = 0 , 963 ) ( tabella 3 )  . 
dalle stessa analisi delle curve emerso che la valutazione complessiva abbassa sensibilmente anche il limite inferiore dimensionale per cui si mantiene il livello massimo di accuratezza diagnostica ( ctc prona = 2 , 4 mm ; ctc supina = 2 , 1 mm ; vc prona = 1 , 9 ; vc supina = 2 mm ; complessiva = 1 , 4 mm )  . 
the high - resolution scan protocol and optimal bladder distension allowed clear depiction of the lesion on both the sagittal multiplanar reproduction ( mpr ) ( arrow in a ) and native axial ( arrow in b ) images . 
il protocollo di scansione ad alta risoluzione e lottimale distensione vescicale hanno permesso una chiara visualizzazione della lesione in mpr sia sagittale ( a , freccia ) che assiale nativa ( b , freccia )  . 
nelle immagini vr ( c , testa di freccia ) , la lesione appare come un polipo regolarmente formato con una piccola base di impianto ; lesame istologico dopo resezione tramite cistoscopia convenzionale ( d ) ha dimostrato la presenza di cellule transizionali neoplastiche . 
comparando la scansione ctc supina con lacquisizione in posizione prona , non stata evidenziata una lesione peduncolata di circa 2 mm localizzata a livello del trigono e occultata verosimilmente da residui urinari . 
lanalisi statistica dei risultati riportati dalla valutazione delle immagini ottenute con la sola ctc in posizione prona ha riportato una sensibilit del 88 , 89% , specificit 88 , 89% , ppv 94 , 12% e npv del 80% ; mentre nella posizione supina sensibilit del 94 , 44% , specificit del 88 , 89% , ppv 94 , 44% e npv 88 , 89% . 
la vc in posizione prona una sensibilit del 100% , specificit 88 , 89% , ppv 94 , 74% e npv del 100% ; mentre nella posizione tabella 2 analisi statistica dei risultati ottenuti dalla valutazione singola e combinata di ciascuna metodica nelle differenti posizioni . 
questi risultati debbono comunque essere considerati come indicativi in base al numero di pazienti reclutati nel nostro studio preliminare ctc prona ctc supina vc prona vc supina ct combi confronto tra cistografia e cistoscopia virtuale gruppo 1 risultati gruppo 2 risultati gruppo 3 risultati sensibilit specificit sensibilit specificit sensibilit specificit 75 , 86% 84 , 62% 92 , 00% 61 , 11% 88 , 89% 88 , 89% 94 , 12% 80 , 00% 100% 100% 100% 100% 68 , 97% 76 , 92% 86 , 96% 52 , 63% 94 , 44% 88 , 89% 94 , 44% 88 , 89% 100% 100% 100% 100% 89 , 66% 92 , 31% 96 , 30% 80 , 00% 100% 88 , 89% 94 , 74% 100% 100% 100% 100% 100% 82 , 76% 92 , 31% 96 , 00% 70 , 59% 100% 88 , 89% 94 , 74% 100% 100% 100% 100% 100% 93 , 10% 92 , 31% 96 , 43% 85 , 71% 100% 100% 100% 100% 100% 100% 100% 100% ctc prona ctc supina vc prona vc supina ct combi ctc prona ctc supina vc prona vc supina ct combi ctc , cistografia con tc ; vc , cistografia virtuale ; ppv , valore predittivo positivo ; npv , valore predittivo negativo radiol med ( 2009 ) 113 : 5269 table 3 results of receiver operating characteristic ( roc ) curve analysis in group 1 . 
da notare come i valori di auc ( area under the roc curve ) , rispecchiando i valori di accuracy di ciascun approccio diagnostico ( valutazione singola di ctc e vc e combinata ) , dimostri una costante superiorit dellapproccio combinato . 
la valutazione combinata delle immagini ottenute con le diverse metodiche di valutazione nelle diverse posizioni ha riportato anche in questo gruppo valori nettamente superiori con sensibilit del 100% , specificit 100% , ppv 100% e npv del 100% ( tabella 2 )  . gruppo iii ( lesioni > 9 mm ) nei 12 pazienti nel iii gruppo ( 11 preoperatori e 1 postturb ) le lesioni identificate avevano una dimensione minima di 9 , 3 mm e massima di 22 mm ( media 12 , 42 mm ; mediana 11 mm ; deviazione standard 3 , 46 ) la morfologia delle lesioni identificate stata di tipo peduncolato in 11 fig . 
the area under the curve related to combined computed tomography cystography ( ctc ) and virtual cystoscopy ( vc ) ( ct overall ) is greater compared with those of ctc and vc alone , reflecting the superiority of the combined approach . 
la curva che si riferisce allapproccio combinato ( ct overall ) sottende un area ( auc : area under the roc curve ) maggiore rispetto alle altre curve , riflettendo la superiorit diagnostica dellapproccio combinato . radiol med ( 2009 ) 113 : 5269 fig . 
on sagittal multiplanar reconstruction ( mpr ) ( a ) and native axial ( b ) images , only two out of four lesions are visualised , and the hypertrophic mucosal folds do not allow clear depiction of the remaining polyps . 
in contrast , the volumerendered images ( c ) allow an intuitive discrimination between the long , curved mucosal folds ( arrowheads in c ) and the polyps ( arrows in c )  . 
nelle immagini mpr sagittali ( a ) e assiali ( b ) solo 2 su 4 lesioni totali possono essere visualizzate e le pliche ipertrofiche della mucosa non permettono una chiara individuazione dei rimanenti polipi . 
daltro canto , le immagini in vr ( c ) permettono una intuitiva discriminazione fra le lunghe pliche arrotondate delle pliche mucosa ( c , teste di frecce ) ed i polipi ( c , frecce )  . 
la sensibilit e specificit per lidentificazione delle lesioni stata del 100 % sia per la ctc ( scansione prona e supina ) che per la cv ( scansione prona e supina ) ( tabella 2 )  . 
nella tabella 4 troviamo anche i valori di concordanza parziale nei diversi gruppi ; un livello di concordanza inferiore viene dimostrato soprattutto tra il primo e il secondo gruppo di pazienti , dove le dimensioni delle lesioni hanno probabilmente reso pi diffifig . 
le ricostruzioni in vr ad alta risoluzione spaziale permettono uneccellente definizione delle immagini di cv , le quali sono fortemente confrontabili a quelle dellapproccio endoscopico convenzionale ( d )  . radiol med ( 2009 ) 113 : 5269 radiologist 2 interrater variability statistical analysis of the results of each radiologist revealed a k value of 0.868 ( table 4 ) , indicating complete agreement between the two radiologists . 
lower agreement levels were recorded in groups 1 and 2 in particular , where small lesion size probably hindered correct measurements , leading radiologist 2 to misclassify four group - 1 lesions ( based on measurements made with flexible cystoscopy ) as belonging to group 2 . 
7 , which compares the two radiologists performance in classifying lesions and shows the overall greater accuracy of radiologist 1 in assigning lesions to the groups established with flexible cystoscopy . discussion a number of imaging techniques have been proposed for detecting bladder lesions , but conventional cystoscopy remains the standard of reference in diagnosing bladder carcinoma [ 16 ]  . 
conventional cystoscopy allows not only the detection of lesions < 1 cm but also the opportunity to perform immediate resection ( curative or palliative ) and obtain a tissue sample for histopathological examination . on the other hand , conventional cystoscopy is , after colonoscopy , one of the most invasive and poorly tolerated examinations , often requiring local anaesthesia and sedatives to increase patient compliance . 
the technique is also limited by several intrinsic absolute contraindications that cannot be easily overcome , and it is precluded in patients with active haematuria or urethral strictures [ 2 ]  . 
additionally , even modern fibreoptic cystoscopes are often incapable of visualising correctly the bladder neck and luminal diverticula , especially in the anterior wall sites that are often prone to mucosal dysplasia with malignant degeneration [ 17 ]  . 
il grafico mette confronto visivamente ( nella tabella 4 numericamente ) i risultati ottenuti dal radiologo 1 con quelli del radiologo 2 inserendo nella prima colonna di ciascun gruppo i risultati in accordo e nella seconda quelli in disaccordo . 
 cile un corretta misurazione delle stesse , portando soprattutto per quanto riguarda il radiologo 2 , ad assegnare 4 pazienti del gruppo i ( sulla base della misurazione con cistoscopia convenzionale ) al gruppo ii . 
7 a colonne raggruppate che confronta lattribuzione a ciascun gruppo da parte dei due radiologi nel quale il primo ha dimostrato una accuratezza globale migliore nella misurazione delle lesioni osservate rispetto alla suddivisione in gruppi operata dalla cistoscopia convenzionale . discussione ad oggi sono state proposte diverse metodiche di imaging per lidentificazione delle lesioni vescicali anche se la cistoscopia convenzionale rimane la metodica di riferimento nella diagnosi di carcinoma della vescica [ 16 ]  . 
lapproccio endoscopico convenzionale garantisce oltre allidentificazione di lesioni subcentimetriche , la possibilit di realizzare un trattamento escissionale immediato ( curativo o palliativo ) e di poter ottenere un campione di tessuto per lesame bioptico . 
di contro , la cistoscopia tradizionale rappresenta , dopo la colonscopia , una delle procedure endoscopiche pi invasive e poco tollerate per il paziente al punto che lanestesia locale e leventuale somministrazione di sedativi sono spesso richiesti per aumentare il grado di compliance allesame del paziente . 
la cistoscopia convenzionale presenta inoltre alcune intrinseche intrinseche ed assolute controindicazioni allesecuzione che non possono essere facilmente superate : infatti i pazienti con ematuria in atto o radiol med ( 2009 ) 113 : 5269 involving turb and the size of bladder cancers at the time of initial diagnosis significantly affects treatment decisions . 
in fact , treatment protocols for lesions < 1 cm are essentially based on a conservative approach local chemotherapy ( bacillus of calmette - gurin , doxorubicin , mitomycin , gentamicin ) [ 15 , 19 ] , whereas those for involve neoadjuvant lesions advanced and chemotherapy and radiotherapy followed by surgical resection [ 20 ]  . 
moreover , it should be recalled that lesions < 10 mm carry a statistical risk , albeit low , of concurrent or preexisting invasion of the muscularis propria , which is in turn associated with metastatic spread [ 21 ]  . 
 larger our results suggest that ctc with vc represents a valuable and noninvasive diagnostic method for detecting recurrent or de novo bladder cancers ranging from 2 mm to 9 mm in size . 
furthermore , table 3 interestingly reveals an increase in specificity brought about by vc compared with ctc alone , probably as a result of better delineation of the mucosal surface . these statistical data may appear overly optimistic , and we need to acknowledge a probable bias related to the small number of patients in our series . 
 [ 22 ] identified only 70% of lesions < 1 cm by using a single - detector - row scanner with 3 - mm collimation , and kim et al . 
our analysis of interrater agreement seems in part to reflect the difficulties encountered by song et al . , showing a decreased ability to identify and measure lesions < 5 mhowever , this finding applies to < 30% of patients in group 1 and is , we believe , related to the degree of bladder distension , a crucial factor when evaluating very small lesions . 
in particular , in several cases with irregular appearance of the mucosal surface , bladder - wall evaluation was limited by poor bladder distension : in one of these cases , tumour identification proved impossible , whereas in the other case , it was only possible thanks to the large size of the mass ( 23 mm )  . 
the use of con stenosi uretrale non possono essere sottoposti a questo tipo di indagine [ 2 ] ; inoltre , anche i moderni cistoscopi a fibre ottiche difficilmente riescono a visualizzare correttamente il collo vescicale ed i diverticoli presenti nel lume vescicale , soprattutto della parete anteriore : siti frequentemente esposti a displasia della mucosa con degenerazione neoplastica [ 17 ]  . 
 per ovviare a tali limitazioni , vining ed altri autori hanno introdotto , per primi , nel 1996 la ctc con cv come metodica mini - invasiva per lo studio endoscopico della vescica e per la diagnosi delle lesioni vescicali [ 7 ]  . 
solo un anno pi tardi , hussain e altri autori [ 9 ] hanno proposto la cistoscopia virtuale , in alternativa alla cistoscopia convenzionale , come primo approccio diagnostico in pazienti con ematuria o nel follow - up dopo resezione della neoplasia . oggi , grazie allenorme diffusione delle apparecchiature tc multidettetore , la ctc con cv non rappresenta pi una raffinata evoluzione tecnica , ma sta lentamente cominciando a seguire la strada in passato intrapresa dalla colonscopia virtuale . 
tuttavia , la principale limitazione di questa metodica , rappresentata da unaccuratezza non ottimale nellidentificazione di piccole lesioni , ha continuato , sino ad oggi , a precludere limpiego nella diagnosi precoce delle lesioni vescicali [ 18 ]  . 
infatti , nel caso di lesioni subcentimetriche il protocollo terapeutico si basa essenzialmente su un approccio conservativo con resezione trans - uretrale ( turb ) e con la somministrazione locale di agenti chemioterapici ( bacillo di calmette - guerin , doxorubicina , mitomicina , gentamicina ) [ 15 , 19 ]  . 
al contrario per le neoplasie in stadio avanzato e con dimensioni maggiori i protocolli prevedono dapprima un trattamento neoadiuvante chemioterapico e radioterapico seguito dalla resezione chirurgica [ 20 ]  . 
inoltre bisogna tenere presente che lesioni inferiori a 10 millimetri presentano comunque un rischio statistico seppur basso di invasione concomitante o pre - esistente della muscularis propria che a sua volta si associa a diffusione metastatica della malattia [ 21 ]  . 
 i nostri risultati sembrerebbero dimostrare che la ctc insieme alla cv , rappresentano una metodica diagnostica valida e non invasiva per lidentificazione di lesioni della vescica ricorrenti o di nuova insorgenza con dimensioni comprese fra 29 millimetri . 
in particolare lanalisi dei dati compiuta nel gruppo di lesioni sotto i 5 mm ha dimostrato come un approccio combinato delle tecniche ctc e vc permette non solo di migliorare il grado di sensibilit e specificit statistica nellindividuazione della lesione sospetta ( sensibilit 93 , 10% e specificit 92 , 31% ) , ma soprattutto ridurre sensibilmente il limite dimensionale di identificazione ( 1 , 4 mm con sensibilit di 93 , 1% e specificit de 100% ) , come dimostrato dallanalisi roc ( tabella 3 )  . 
inoltre dalla stessa tabella 3 emerge il dato interessante dellaumento di specificit diagnostica apportato dalla cv rispetto ai valori riportati dalla sola ctc , probabilmente dovuto alla migliore delineazione della superficie total table 4 interrater agreement with kappa statistics . 
questi dati statistici potrebbero apparire oltremodo ottimistici e riconosciamo un probabile bias dovuto allesiguo numero di pazienti reclutato nello studio , tuttavia devono essere intesi come esperienza preliminare in vista di studi su larga scala che dovranno includere necessariamente popolazioni di studio pi ampie ed omogenee rispetto a quella selezionata nel presente studio . inoltre i nostri dati sono sostanzialmente concordi con i risultati di altri studi come quello effettuato da nerumi et al . 
 [ 22 ] il quale ha individuato solo il 70% delle lesioni < 1 cm servendosi di tc a singolo strato con collimazione a 3 mm , mentre kim et al . 
 [ 23 ] ha identificato l82% delle lesioni < 5 mm utilizzando uno scanner mdct a 4 strati ed una collimazione di 1 , 25 mm ; al contrario , song et al . 
 [ 18 ] utilizzando uno scanner mdct a 16 strati ed una collimazione di 3 millimetri , hanno definito inattendibile la metodica ctc insieme alla cv nellidentificazione di lesioni di dimensioni < 5 mm ( solo il 60% delle lesioni )  . 
la nostra analisi della variabilit inter - operatore sembra riflettere in parte le difficolt incontrate in questultimo studio riportando una diminuzione della capacit di individuazione e di misurazione di lesioni sospette inferiori ai 5 mtuttavia questo dato si riferisce a meno del 30% dei pazienti del primo gruppo ed a nostro avviso da correlare al grado di distensione della parete vescicale che assume necessariamente importanza critica in lesioni molto piccole . 
in particolare , in alcuni casi in cui la superficie mucosa si presentava irregolare , il risultato della valutazione della parete vescicale ha risentito dello scarso grado di distensione vescicale : infatti in uno di questi casi con scarsa distensione , lindividuazione del tumore non stata possibile , mentre nellaltro caso , solamente le grandi dimensioni della massa ( 22 mm ) ne hanno permesso lidentificazione . 
si dovrebbe considerare quindi lutilizzo di agenti farmacologici spasmolitici , introdotti con successo negli esami di colografia con tc di routine [ 24 ] in modo da facilitare il raggiungimento di una distensione vescicale adeguata nei pazienti con scarsa compliance e con disordini vescicali . 
altre modalit di distensione , come il riempimento da parte del mezzo di contrasto per via escretoria [ 22 ] o retrograda [ 25 ] , potrebbe rappresentare una valida alternativa ; tuttavia stato provato che linsufflazione di aria sia superiore allopacizzazione positiva per il pi alto gradiente di attenuazione raggiungibile con bassa corrente di tubo ( 100 mas ) , e con ricostruzioni virtuali meno soggette ad artefatti [ 26 ]  . 
una maggiore sensibilit , utilizzando entrambe le tecniche ( 2d e 3d ) , pu essere raggiunta in tutti i pazienti con una distensione ottimale o soddisfacente della vescica ; nel nostro studio , il livello ottimale di distensione ha coinciso con linsufflazione di circa 350450 cc di aria , il livello soddisfacente con 250350 cc e il livello scarso con meno di 250 cc ; ad ogni modo la validit di queste misurazioni soltanto indicativa , poich bisogna prendere atto di alcune considerazioni : unelevata distensione vescicale ( 350450 ml ) riduce lo spessore globale delle pareti vescicali ( nel nostro studio lo spessore medio di parete con distensione stato di 1 , 7 mm ) e pu radiol med ( 2009 ) 113 : 5269 to be superior to positive opacification due to the higher attenuation gradients achievable with low tube current ( 100 mas ) and fewer artefacts on virtual reconstructions [ 26 ]  . 
regardless , the validity of these measurements is only indicative , as several factors need to be considered : a high degree of bladder distension ( 350450 ml ) reduces the overall thickness of the bladder walls ( mean wall thickness of distended bladders was 1.7 mm ) and may enhance the accuracy in identifying focal thickening ( often related to tumour infiltration ) ; on the other hand , a poorly distended bladder ( < 250 ml ) coincides with poor delineation of the mucosal profile and a thickened bladder wall , thus hindering the detection of mural pathology . technical considerations include a short scanning time ( 3.5 s ) , which did not affect image quality with movement artefacts . 
the optimal attenuation gradient achieved between the air - dilated bladder lumen and the surrounding soft tissue allowed both tube current and radiation exposure to be significantly reduced in comparison with other studies [ 13 , 14 , 27 ]  . 
the use of a new - generation 64 - detector - row scanner combined with a high - resolution protocol with low values for slice thickness ( 1 mm ) , collimation ( 0.6 mm ) and reconstruction increment ( 0.4 mm ) enabled accurate , highresolution and fast exploration of the entire region of interest , offering reliable views for lesion detection . 
on the other hand , the high spatial resolution achieved with this thin - slice scanning protocol substantially increases the number of images and the amount of data acquired , requiring a dedicated workstation to efficiently handle data and reconstruct images . 
in contrast , ctc images were evaluated together with mpr reconstructions , as this allowed not only evaluation of axial images but also reliable multidimensional visualisation ( sagittal , coronal and oblique )  . among the various 3d techniques available , we chose vc because it is relatively familiar to radiologists specialising in virtual endoscopy , is widely accepted by clinicians and surgeons owing to its similarity to actual endoscopy and , most importantly , because of the better image quality reported in previous reports [ 2 , 28 ]  . the contribution of the 3d approach was fundamental . not only did it depict all lesions seen on 2d images , it also served as a diagnostic modality for lesions in subjects with suboptimal bladder distension and in two patients with irregular and hypertrophic mucosa . 
in these last two cases , ctc failed to discern between the folds of hypertrophic mucosa and pedunculated lesions , whereas vc allowed correct evaluation of the mucosal folds and a clear distinction between potenziare laccuratezza nellidentificazione dellispessimento focale ( spesso correlato allinfiltrazione tumorale ) ; daltra parte una vescica scarsamente distesa ( < 250 ml ) coincide con una scarsa delineazione del profilo mucoso e con una parete ispessita complicando lindividuazione di processi patologici in questa sede . alcune considerazioni di ordine tecnico riguardano la breve durata della scansione ( 3 , 5 s ) che non ha influenzato la qualit dellimmagine , con artefatti da movimento . 
il gradiente dattenuazione ottimale realizzato fra il lume vescicale dilatato dallaria ed il tessuto molle circostante ha permesso sia una riduzione significativa delle correnti del tubo sia dei valori desposizione alle radiazioni , specie se comparati ad altri studi [ 13 , 14 , 27 ]  . 
lutilizzo di un tomografo dultima generazione multistrato a 64 detettori ( 64mdct ) , in associazione con un protocollo ad alta risoluzione con bassi valori di spessore della fetta ( slice thickness 1 mm ) , di collimazione ( 0 , 6 mm ) ed incremento di ricostruzione ( 0 , 4 mm ) , ha permesso in pochi secondi unesplorazione diagnostica precisa e ad alta risoluzione dellintera regione dinteresse , offrendo una affidabile visione specifica per lidentificazione delle lesioni . 
daltra parte , lalta risoluzione spaziale ottenuta attraverso luso di questo protocollo a strato sottile ha determinato un aumento considerevole del numero di immagini e incrementato drammaticamente il numero di dati acquisiti ; si rende quindi necessario lutilizzo di una workstation dedicata per una gestione efficiente dei dati e per la ricostruzione delle immagini . 
le immagini della ctc , invece , sono state valutate insieme alle ricostruzioni mpr poich ci ha permesso non solo una valutazione delle immagini secondo un piano assiale ma anche una visualizzazione multidimensionale ed esatta ( sagittale , coronale ed obliqua )  . 
fra le diverse tecniche 3d disponibili , abbiamo scelto la cv per la relativa familiarit dutilizzo fra i radiologi che si occupano di endoscopia virtuale , per il largo consenso tra clinici e chirurghi dovuto ad immagini che offrono una visione endoscopica sostanzialmente sovrapponibile a quella in vivo , ma soprattutto , per la migliore qualit delle immagini segnalata in differenti studi [ 2 , 28 ]  . il contributo dellapproccio tridimensionale stato fondamentale poich non solo ha dimostrato tutte le lesioni evidenziate nelle immagini 2d , ma servita come modalit diagnostica in casi di lesioni in soggetti con vescica scarsamente distesa e in due pazienti con mucosa irregolare ed ipertrofica . 
in questi ultimi casi , la ctc non ha propriamente distinto pliche di una mucosa ipertrofica da lesioni peduncolate , mentre la cv ha permesso la corretta valutazione della superficie mucosa plicale e ha chiaramente distinto le lesioni dai tessuti circostanti . 
daltra parte , la cv ha reso possibile per lo studio del collo vescicale , la parete anteriore e , se presenti , i diverticoli vescicali , i quali sono siti scarsamente o per niente accessibili alla cistoscopia convenzionale [ 17 ]  . 
 la ctc e la cv , rispetto alla cistoscopia convenzionale , offrono una minore invasivit consentendo , oltre che la visualizzazione diretta della lesione , la possibilit di misuradiol med ( 2009 ) 113 : 5269 lesions and surrounding tissues . 
moreover , vc allowed detailed inspection of the bladder neck , anterior wall and vesical diverticula , all sites that are recognised as potential blind spots at conventional cystoscopy [ 17 ]  . 
 in comparison with conventional cystoscopy , ctc and vc are less invasive and , in addition to depicting lesions directly , allow precise measurement of lesion size and evaluation of wall thickness . 
the main disadvantages of vc include the inability to perform endoscopic resections or biopsies and acquire in vivo information about the colour and structure of the mucosa , as reported by others [ 2931 ] ; the latter limitation may affect detection rates in flat lesions [ 5 ] , although we were unable to verify this owing to the lack of flat lesions in our series . 
however , the advent of technologically advanced scanners has in part overcome this problem , as radiation doses can be reduced by using the dose - modulation software ( care dose ) supplied with the scanning equipment . 
ctc combined with vc can , however , be used in cases in which conventional cystoscopy is contraindicated or difficult to perform and in the follow - up of patients after surgery . 
in such cases , should vc prove negative , the technique could spare the patient further conventional cystoscopy studies or provide the clinician and endoscopist with basic indications regarding bladder - wall morphology . 
 conclusions ctc combined with vc , performed with 64 - detector - row ct , provides a high level of diagnostic accuracy in detecting both new and recurrent locoregional vesical lesions ranging from 2 mm to 9 mm in size . 
the two approaches ( 2d and 3d ) are complementary , and in some cases , 3d images are absolutely necessary for the diagnosis . evaluation of lesions < 5 mm requires meticulous patient preparation , especially with regard to bladder distension . 
 our study should , however , be considered a preliminary experience owing to the small number of patients examined . further studies with state - of - the - art multidetector equipment need to be conducted on larger and more homogeneous patient populations to achieve higher statistical power . rare esattamente le dimensioni della lesione e di valutare lo spessore della parete vescicale , senza trascurare che lutilizzo delle immagini 2d permette una valutazione dellestensione loco - regionale della malattia . 
ulteriori vantaggi sono rappresentati dalla possibilit di ottenere una visione corretta del lume vescicale in tutti i pazienti , anche in quelli con ostruzione uretrale severa o ematuria . gli svantaggi principali della cistoscopia virtuale sono rappresentati dallincapacit di effettuare resezioni per via endoscopica o biopsie , dallimpossibilit di ottenere informazioni in - vivo in relazione al colore e alla struttura del mucosa , come riportato in altri studi [ 2931 ] : questo limite pu sicuramente influenzare la percentuale dindividuazione di lesioni piatte [ 5 ] anche se nella nostra esperienza non abbiamo potuto constatarlo per lassenza di lesioni di questo tipo nella popolazione studiata . 
infine rimane il problema dellesposizione a radiazioni ionizzanti ; questultimo svantaggio stato parzialmente superato con lutilizzo di moderne apparecchiature , in cui si ha una minor dosaggio di radiazioni , grazie allutilizzo di protocolli con moderne tecniche di care - dose presenti nelle apparecchiature di tecnologia avanzata . 
 attualmente non possiamo offrire una sensibilit del 100% nellidentificazione delle neoplasie della vescica e di conseguenza non possiamo proporre lendoscopia virtuale in sostituzione del metodo endoscopico convenzionale ; tuttavia la metodica ctc , insieme alla cv , possono essere utilizzate a nostro avviso nei casi in cui la cistoscopia convenzionale risulti controindicata o difficile da eseguire e nel follow - up dei pazienti neoplasia post - chirurgici . 
 conclusioni in conclusione , la ctc in combinazione con la cv , con tc spirale multistrato a 64 detettori ( 64 - mdtc ) , rappresenta una metodica di imaging ad elevato livello di accuratezza diagnostica per lidentificazione sia di lesioni di nuova insorgenza che di recidive vescicali locoregionali di dimensioni comprese tra 2 mm e 9 m entrambi gli approcci ( 2d e 3d ) risultano complementari e , in alcuni casi , le immagini 3d sono assolutamente necessarie per la diagnosi . 
benzi 10 , 16132 genova , italy 2department of radiology , university of california san diego ( ucsd ) , san diego , ca , usa 3dipartimento di radiodiagnostica , az . 
it is thus important to recognise common abdominopelvic mr imaging artefacts and know how to choose protocols and modify scan parameters to eliminate or at least minimise themany mr artefacts , on the other hand , provide diagnostically useful information about the underlying tissue , and many powerful mr sequences , such as in - phase or out - of - phase gradient - recalled - echo ( gre ) sequences , may be thought of as imaging artefacts applied creatively . 
to distinguish friend from foe mr artefacts or to convert foes into friends , mr radiologists must recognise and understand the physical basis of such artefacts to take advantage of them for diagnostic purposes . riassunto gli artefatti rappresentano un problema comune a tutte le metodiche di imaging . 
molti artefatti , daltra parte , consentono di ottenere informazioni diagnostiche aggiuntive e alcune sequenze , come ad esempio le gradient - echo ( gre ) in fase e in opposizione di fase , possono essere pensate come artefatti applicati creativamente allimaging clinico . 
per poterli definire amici o nemici , o poterli convertire da svantaggiosi a utili , bisogna conoscerne le basi fisiche in modo da trarre dagli artefatti il potenziale vantaggio diagnostico aggiuntivo . keywords artifacts magnetic resonance body imaging parole chiave artefatti risonanza magnetica body imaging introduction introduzione imaging artefacts are common in magnetic resonance imaging ( mri ) studies of the abdomen and pelvis . 
there are many sources of artefacts in mri [ 14 ] , which can be summarised as follows : gli artefatti sono significativamente presenti negli studi rm di addome e pelvi . 
therefore , it is important to recognise common abdominopelvic mri artefacts and know how to eliminate or at least minimise theon the other hand , many mr artefacts yield diagnostically useful information , and some powerful mr sequences , such as in - phase and opposed - phased ( or out - of - phase ) gradient - recalled - echo ( gre ) imaging , may be thought of as imaging artefacts applied creatively . to distinguish friend from foe mr artefacts or to convert foes into friends , mr radiologists must recognise and understand the physical bases of artefacts to take advantage of them for diagnostic purposes . 
of these many mr artefacts , in this article , we focus on those that can be friends as well as foes in other words , those related to ( 1 ) chemical shift , ( 2 ) magnetic susceptibility , ( 3 ) motion , or ghosts and ( 4 ) flow . 
inoltre alcune sequenze , come ad esempio le gradient - echo ( gre ) in fase ( if ) e in opposizione di fase ( of ) , possono essere pensate come artefatti applicati creativamente allimaging clinico . per poterli definire amici o nemici , o poterli convertire da svantaggiosi a utili , bisogna conoscerne le basi fisiche per trarre dagli artefatti il potenziale vantaggio diagnostico aggiuntivo . 
altre condizioni particolari sono rappresentate dalluso di tecniche di imaging parallelo e dagli effetti di alcuni artefatti su esami di addome e pelvi effettuati con le pi recenti apparecchiature rm a 3 t . chemical shift chemical shift cs artefacts are due to the phenomenon whereby protons from different molecules precess at slightly different frequencies [ 14 ]  . 
in particular , the hydrogen protons in fat and h2o have slightly different precessional frequencies , and the protons in h2o precess faster than do those in fat . this difference in precessional frequency is directly proportional to the main magnetic field strength and has been measured as approximately 3.5 ppm , resulting in a difference of only 73 hz at 0.5 t , about 225 hz at 1.5 t and about 450 hz at 3 t [ 1 , 5 , 6 ]  . 
it is seen only in the frequency - encoding axis and occurs in all sequences spin echo ( se ) or in - phase or opposed - phase gre imaging . 
as the hydrogen protons in h2o precess faster than those in fat , anywhere gli artefatti da chemical shift ( cs ) si basano sul principio che i protoni di molecole differenti precessano a frequenze differenti [ 14 ]  . 
i protoni di idrogeno contenuti nellh2o e nelle molecole di grasso , in particolare , presentano frequenze di precessione lievemente diverse e , in realt , i protoni contenuti nellh2o sono pi veloci rispetto a quelli delle molecole di grasso . 
quello di 1 tipo dovuto a misregistrazione spaziale del segnale , si ha solamente nella direzione della codifica di frequenza , ed comune a tutte le sequenze sia ad echo di spin ( spin - echo , se ) che ad eco di gradiente ( gradient - echo , gre ) , queste ultime tanto con tempi di radiol med ( 2009 ) 114 : 1831 fig . 
this kind of artefact occurs at the interface of water and fat ( arrows ) , where a bright band towards the lower frequencies and a dark band towards the higher frequencies are visible . 
1a , b chemical - shift di 1 tipo . questo tipo di artefatto , alla interfaccia acqua / grasso ( frecce ) , si manifesta come banda di iperintensit alle frequenze pi basse e di ipontensit a quelle pi alte . 
 presente nella sola direzione della codifica di frequenza ed comune a tutte le sequenze , sia ad eco di gradiente ( a ) che ad eco di spin ( b )  . 
the first kind of cs artefact depends on several factors but mainly on magnetic field strength ( b0 ) , frequency range [ bandwidth ( bw ) ] and on pixel size ( matrix )  . 
it occurs in either phaseor frequency - encoding direction and only at specific tes in gre sequences , termed opposed phase or out of phase , when the fat and water spins will be 180 out of phase with consequent signal dropout . 
il cs di 1 tipo dipende da diversi fattori ma in particolare dal campo magnetico esterno ( b0 ) , dal range di frequenze ( bandwidth , bw ) e dalla dimensione del pixel ( matrice )  . 
questo tipo di artefatto , quindi , tanto maggiore quanto maggiore lintensit del campo magnetico ( 3 t > > 1 , 5 t > > 0 , 5 t ) , quanto pi basso il bw selezionato . 
si ha sia nella direzione della codifica di frequenza che di fase , soltanto alle sequenze gre con specifici te definite in opposizione di fase ( of ) ai quali gli spin di acqua e grasso si troveranno in opposizione di fase di 180 , con successiva cancellazione e caduta di segnale . 
the second kind of cs occurs in both the phaseor frequency - encoding direction ( arrows ) only at specific echo times in opposed - phase gradient - recalled echo ( gre ) sequences as a dark band of signal dropout around organs surrounded by fat ( india ink appearance )  . 
questo tipo di artefatto , comune alle sole sequenze gre in opposizione di fase ( of ) , apprezzabile sia nella direzione della codifica di fase che di frequenza ( frecce ) come banda scura intorno agli organi circondati da grasso ( india ink )  . 
potential solutions are , again , to use fat - suppression techniques ( if there is no signal from fat , there can be no cs ) , increase the bw [ with decreasing of signal - to - noise ratio ( snr ) ] and obviously to use a specific te for in - phase imaging . protoni di acqua e grasso sono if a 4 , 4 ms , 8 , 8 ms , e cos via , e sono in of a 2 , 2 ms , 6 , 6 ms , e cos via , e si trovano ciclicamente in fase e in opposizione di fase approssimativamente ogni 2 , 2 ms . 
quindi , ove acqua ( h2o ) e grasso ( tessuti contenenti gruppi - ch , - ch2 e - ch3 come colesterolo e precursori degli acidi grassi ) siano presenti radiol med ( 2009 ) 114 : 1831 fig . 
questo tipo di artefatto , visualizzabile alle sole sequenze gre in of come india ink artifact ( frecce , b ) , consente di diagnosticare con facilit condizioni di steatosi epatica ( si noti la omogenea caduta di segnale del parenchima epatico alle sequenze in of rispetto a quelle if )  . magnetic susceptibility the magnetic susceptibility ( ) of all substances is a measure of how magnetised they become when placed in a magnetic field [ 14 ]  . 
local heterogeneity of the magnetic field causes protons to precess at different frequencies than they normally would , and because of this , the protons are mapped to the incorrect location in the frequency - encoding direction . 
 there are three types of substances , each with different magnetic susceptibility , commonly dealt with in mri : ( 1 ) diamagnetic , ( 2 ) paramagnetic , ( 3 ) ferromagnetic . 
possibili rimedi al cs di 2 tipo sono rappresentati anche in questo caso dallutilizzo di sequenze fat suppression , dallincremento del bw ( con conseguente riduzione del snr ! ) e , ovviamente , dallutilizzo di appropriati te in cui acqua e grasso siano in fase . suscettibilit magnetica la suscettibilit magnetica ( ) di una sostanza la misura di quanto viene magnetizzata questa sostanza una volta posta allinterno di un campo magnetico [ 14 ]  . 
gli artefatti da suscettibilit si hanno qualora tessuti o materiali con differente siano adiacenti tra loro , causando in tal modo disomogeneit del campo magnetico locale . quindi questo fenomeno si ha alle interfacce tra tessuti e aria , tessuti e metalli oppure tessuti e sangue . 
allinterfaccia tra tessuti e aria presente nelle anse intestinali si ha distorsione dellanatomia con vuoto di segnale ( frecce , a e b ) e disomogenea soppressione del segnale del grasso ( freccia curva , b )  . strongly attracted by a magnetic field and have a large positive  . 
they can be seen only in the phase - encoding direction , mainly for a nellimaging a rm si ha a che fare con tre tipi di sostanze , ciascuna con una differente suscettibilit magnetica : diamagnetiche ; paramagnetiche ; ferromagnetiche . 
nonostante le sequenze se e tse siano meno sensibili di quelle gre , leffetto di suscettibilit magnetica comune comunque a tutte le sequenze [ 1 , 11 , 12 ]  . 
la maggior entit dellartefatto alle sequenze gre con te pi alto ( freccia , a ) , rispetto a quella con te pi basso ( b ) , prova la presenza di depositi di ferro ( emosiderinici ) nel parenchima splenico . significant asymmetry in the data space between frequency encoding ( on the order of milliseconds ) and a single phaseencoding step ( in order of seconds )  . 
ghosts are spaced equally , and this spacing depends only on repetition time tica , inoltre , aumenta allaumentare del te utilizzato e questa peculiarit pu rappresentare una fonte di preziose informazioni diagnostiche aggiuntive su particolari materiali o strutture che a volte sono difficili da identificare radiol med ( 2009 ) 114 : 1831 fig . 
for instance , ascites can appear to be peritoneal carcinomatosis , and in mr cholangiopancreatography ( mrcp ) , a flow void in the common bile duct can mimic a stone . 
si propagano solamente nella direzione della codifica di fase , soprattutto per lasimmetrica codifica spaziale dei dati tra la frequenza ( che nellordine di millisecondi ) e la fase ( nellordine invece di secondi )  . 
ad esempio lartefatto da pulsazione in un vaso indica la perviet del vaso stesso anche in assenza del signal void . possibili soluzioni sono rappresentate dal cambio di direzione di fase / frequenza ( lartefatto cambia solo direzione e la risoluzione peggiorata ) , dalluso di bande di presaturazione prossimali ai vasi o applicate sulla parete addominale , dalluso di tecniche di gating cardiaco e / o respiratorio , dalla somministrazione di gadolinio ev ( che accorcia il tempo di rilassamento t1 ) , o dalla sedazione del paziente ( uso pediatrico )  . 
lutilizzo di tecniche single - shot ( ssh ) ( b ) consente di eliminare gli artefatti da movimento respiratorio presenti alla sequenza tse t2 pesata ( a )  . 
the lack of a refocusing pulse in these gre sequences is a useful property that allows this artefact to be avoided . parallel imaging technique parallel imaging is a family of techniques developed shortly after the introduction of phased - array coils in order to reduce acquisition times by taking advantage of the spatial information inherent in multiple radiofrequency coils . 
in particolare nei pazienti non collaboranti consigliabile lutilizzo di sequenze gradient echo 2d con tempo di interpulso ( tipo turbo flash o turbo field echo ) per limaging t1 pree post - contrasto , meno sensibili a questo tipo di artefatti rispetto a quelle 3d . da flusso il flusso dei liquidi ( ascite , liquido amniotico , liquor cefalorachidiano , bile ) causa defasamento dei protoni che , alle sequenze ad eco di spin , si traduce in vuoti di segnale che possono anche mimare delle pseudomasse . 
nei pazienti con ascite , ad esempio , pu apparire come possibile carcinosi peritoneale , e negli studi di colangiopancreatografia a rm il vuoto di flusso nel coledoco pu mimare un calcolo . 
come possibile utilit clinica la perdita del flow void in un vaso , con conseguente iperintensit dello stesso alle immagini t1 pesate pre - contrasto , pu indicare la presenza di un trombo . 
 imaging parallelo limaging parallelo un insieme di tecniche che richiede luso di bobine multicanale ( phased array ) sviluppato relativamente di recente , e che comprende la tecnica smash ( simultaneous acquisition of spatial harmonics ) , asset ( array spatial sensitivity encoding technique ) e sense ( sensitivit encoding ) [ 1318 ]  . 
tale tecnica incrementa la velocit di acquisizione di un fattore ( di accelerazione ) da 1 , 5 a 3 ed oltre sulla maggior parte delle moderne apparecchiature disponibili in commercio [ 13 , 19 ]  . 
 particolarmente utile negli studi cardiaci e di addome - pelvi , dove lapnea frequentemente richiesta al paziente , e la stessa capacit di apnea critica in termini di qualit dellimmagine . 
liquid flow causes proton dephasing and signal loss on spin - echo ( se ) imaging [ such as half - fourier acquisition single - shot turbo spin echo ( haste ) ] ( a ) , which creates pseudomasses ( arrows )  . 
con sequenze ad eco di spin ( come le haste ) ( a ) il defasamento dei protoni allinterno del liquido ascitico crea vuoti di segnale che possono mimare delle pseudomasse ( frecce )  . 
le sequenze ultraveloci ad eco di gradiente , come le fisp ( b ) , non risentono invece di questo artefatto . simultaneous acquisition of spatial harmonics ( smash ) , sensitivity encoding ( sense ) and array spatial sensitivity encoding technique ( asset ) [ 1318 ]  . 
likewise , tse and single - shot fast se sequences can be improved by reducing echo train lengths [ etl , or turbo factor ( tf ) ] and thereby diminishing image blurring . 
if the calibration acquisition does not include a region that is subsequently imaged in the parallel acquisition , this region cannot be reconstructed , and a void will appear in the image . bile incrementare la risoluzione spaziale ; oppure , per le sequenze tse e single - shot fast se , possibile ridurre il blurring dellimmagine riducendo la lunghezza del treno di echi ( etl , o turbo factor [ tf ] )  . 
limaging parallelo altamente flessibile e virtualmente adattabile a qualsiasi sequenza [ 13 ]  . anche limaging parallelo risente di diversi artefatti . molti di essi possono essere eliminati grazie a una attenta cura della tecnica , mentre alcuni rimangono di fatto inevitabili [ 19 ]  . 
tuttavia le pi recenti tecniche di imaging parallelo , msense e grappa ( generalized autocalibrating partially parallel acquisition ) , effettuano una autocalibrazione durante ogni acquisizione per ridurre al minimo questo tipo di artefatti . 
spoiled - gradient - recalled - echo ( gre ) image through the liver with image - based parallel imaging algorithm ( acceleration factor of 2 ) is degraded by incomplete phase unwrapping ( left )  . 
immagine gre assiale del fegato ( a sinistra ) ottenuta con imaging parallelo ( fattore di accelerazione 2 ) che presenta artefatti da ribaltamento lungo lasse delle fasi ; notare sia la presenza di artefatti dovuti allaliasing anteriormente e posteriormente , che quella di una banda di basso rapporto segnale / rumore al centro dellimmagine . 
nellimmagine ripetuta dopo avere adeguatamente incrementato il campo di vista ( a destra ) , la decodifica spaziale lungo lasse delle fasi avviene correttamente e gli artefatti sono quindi eliminati . however , the most recent parallel imaging techniques , msense and generalised autocalibrating partially parallel acquisition ( grappa ) , perform an autocalibration during each acquisition in order to minimise this kind of artefact . artefacts may also appear when the field of view ( fov ) is too small in the parallel acquisition . 
unfortunately , although ultrahigh - field 3.0 - t systems have already been shown to be advantageous for brain and musculoskeletal imaging , only a few published reports have described the use of these systems in the abdomen and pelvis [ 5 , 2224 ]  . 
per diverse ragioni , per , il reale guadagno a 3 , 0 t , se confrontato con un sistema a 1 , 5 t , inferiore al fattore 2 teoricamente aspettato [ 5 , 22 ]  . 
nonostante i sistemi a 3 , 0 t abbiano dimostrato di essere vantaggiosi per limaging neurologico e muscoloscheletrico , sono ancora poche le esperienze riportate in letteratura riguardanti luso e i vantaggi di queste apparecchiature nello studio di addome e pelvi [ 5 , 2224 ]  . 
nellimaging addominale , le sequenze , la dimensione degli organi in esame e alcuni tipi di artefatto , sono significativamente diversi da quelli caratteristici dellimaging muscoloscheletrico o neurologico [ 523 ]  . il chemical shift ( cs ) di primo tipo a 3 , 0 t risulta ampio il doppio rispetto a 1 , 5 t ( tenuti costanti fov , bw , e risoluzione di base ) , ma in genere questo artefatto non rappresenta un problema clinicamente rilevante . 
immagini gre in fase e in opposizione di fase ottenute a 1 , 5 ( in alto ) e 3 t ( in basso ) in paziente portatore di clip metallica vicino allilo epatico . 
si noti anche la dipendenza dal te che in questo caso maggiore nellimmagine in fase a 1 , 5 t e in quella in opposizione di fase a 3 t . organ size and some artefacts differ significantly in abdominal imaging . 
questa maggior differenza in frequenza di risonanza tra acqua e grasso a 3 , 0 t permette una miglior separazione dei loro picchi durante esami di spettroscopia a rm , e consente una migliore e pi rapida tecnica di soppressione del segnale del grasso [ 5 ]  . gli artefatti da suscettibilit magnetica aumentano con il campo magnetico , e a 3 , 0 t presentano un volume circa il doppio rispetto a 1 , 5 t . 
la colografia a rm infatti di sicuro pi critica a 3 , 0 t . importante sottolineare che dispositivi metallici considerati sicuri a intensit di campo magnetico di 1 , 5 t non lo radiol med ( 2009 ) 114 : 1831 fig . 
coronal turbo - spin - echo ( tse ) ( left ) and gradient - recalledecho ( gre ) ( right ) images obtained at 3 t in a patient with a large ascites . 
i cosiddetti effetti dielettrici ( standing wave effect e conductivity artifact ) determinano forti variazioni di segnale in regioni corporee lontane dalla bobina di ricezione , con aree ipointense e buchi neri . 
the so - called standing - wave effect and conductivity artefact result in strong signal variations , especially brightening or dark holes in regions away from the receive coils , and in hypointense areas in the image where the radiofrequency field is partially attenuated [ 5 , 31 ]  . 
 conclusions imaging artefacts are common in mr and can cause image degradation , so it is important to recognise abdominopelvic mri artefacts and know how to eliminate or at least minimise the on the other hand , many mr artefacts provide a diagnostically useful tool and may be thought of as imaging artefacts applied creatively . 
mr radiologists must recognise and understand the physical bases of artefacts in order to be able to modify scan parameters to modulate the detrimental effect of artefacts and take advantage of them for diagnostic purposes . sono necessariamente a 3 , 0 t [ 5 ] , e tutti questi dispositivi necessitano di unattenta valutazione prima di sottoporre il paziente ad esame rm su alto campo [ 2530 ]  . alcuni artefatti , correlati alle pi alte frequenze trasmesse , e non presenti a 1 , 5 t , sono nuovi e peculiari a 3 , 0 t . 
i cosiddetti effetti dielettrici ( standing wave effect e conductivity artifact ) determinano infatti forti variazioni di segnale in regioni corporee particolarmente lontane dalla bobina di ricezione , con aree ipointense laddove il campo di rf risulti parzialmente attenuato [ 5 , 31 ]  . 
infatti per tali effetti degradanti sulle immagini , oltre che per concetti di tipo protezionistico , studi di rm fetale non dovrebbero essere eseguiti a 3 , 0 t [ 5 ]  . conclusioni i comuni artefatti presenti alle indagini di rm possono presentare un effetto degradante sulle immagini , quindi importante saperli riconoscere e possedere gli strumenti per eliminarli o ridurli al minimo . 
signal intensity values were significantly higher for splenic grafts , starting from 40 s after microbubble administration . starting from 90 s after injection , there was no overlapping between enhancement of splenic grafts and peritoneal metastases , and only splenic grafts showed a percent of enhancement higher than 60% of the maximum enhancement . 
sono stati valutati con ecocontrastografia 13 pazienti consecutivi con splenosi peritoneale e 13 con metastasi peritoneali iniettando 2 , 4 ml di sonovue ed osservando lenhancement delle lesioni in tempo reale per 240 s . 
a partire da 90 secondi dopo liniezione non vi era sovrapposizione tra i valori di enhancement degli impianti e delle metastasi , e solo i noduli splenici presentavano un enhancement maggiore del 60% del massimo enhancement . 
 radiol med ( 2009 ) 114 : 4251 considered a valuable alternative to scintigraphy for characterising peritoneal splenic grafts without radiation . keywords spleen us techniques splenosis ultrasound ( us ) contrast media conclusioni . 
lecocontrastografia dopo iniezione di sonovue consente la diagnosi differenziale tra isole di splenosi e metastasi peritoneali . parole chiave milza tecniche ecografiche splenosi ecocontrastografia mezzi di contrasto ecografici introduction introduzione contrary to other microbubble contrast agents , sonovue does not accumulate in the liver or in other organs but is characterised by exclusive spleen - specific uptake [ 1 ]  . 
this characteristic has been demonstrated for the native spleen in which spleen - specific enhancement has been observed for more than 5 min , a time longer than the blood - pool phase [ 1 , 2 ]  . 
spleen - specific uptake has been shown to be useful for characterising ectopic splenic tissue around the spleen [ 2 , 3 ] , in the pancreatic tail [ 4 , 5 ] and within the liver [ 6 ]  . splenosis represents the autotransplantation of splenic tissue after splenic trauma or surgery [ 7 ]  . 
the incidence of splenosis has recently risen following the increasing use of laparoscopic splenectomy [ 8 ] , and splenic autotransplantation in omental pouches is used during splenectomy by some surgeons in an attempt to increase the immune response to infection and prevent overwhelming postsplenectomy sepsis [ 911 ]  . whereas in most cases peritoneal splenosis causes no symptoms and is discovered incidentally during imaging studies performed for other reasons , in a small proportion of cases , it is symptomatic and should be considered in the differential diagnosis of previously splenectomised patients who present with unexplained abdominal masses [ 7 , 12 ] or occult bleeding [ 13 ]  . 
 the purpose of this study was to assess whether the differential diagnosis between peritoneal splenic implants and metastatic deposits can be obtained with contrastenhanced ultrasonography ( ceus ) after sonovue injection , based on the enhancement characteristics during the spleenspecific phase . materials and methods sonovue , contrariamente ad altri mezzi di contrasto ecografici , non si accumula nel fegato e in altri organi , ma presenta un accumulo specifico nel tessuto splenico [ 1 ]  . questo comportamento caratteristico stato dimostrato nella milza nativa nella quale laccumulo specifico delle microbolle consente di rilevarne il segnale per pi di 5 minuti , un tempo pi lungo della fase vascolare [ 1 , 2 ]  . 
 stato dimostrato che laccumulo spleno - specifico utile per la caratterizzazione di tessuto splenico ectopico intorno alla milza [ 2 , 3 ] , nella coda del pancreas [ 4 , 5 ] e nel contesto del parenchima epatico [ 6 ]  . la splenosi lautotrapianto di tessuto splenico che si verifica dopo traumi o chirurgia a carico della milza [ 7 ]  . 
lincidenza di splenosi recentemente aumentata in seguito al ricorso sempre pi frequente alla splenectomia laparoscopica [ 8 ] e per il fatto che alcuni chirurghi , durante lintervento di splenectomia , sono soliti trapiantare frammenti di tessuto splenico allinterno di tasche omentali nel tentativo di migliorare la risposta immunitaria alle infezioni e prevenire linsorgenza di sepsi post - splenectomia [ 911 ]  . nella maggior parte dei casi la splenosi peritoneale asintomatica , individuata accidentalmente durante procedure di imaging eseguite per altre ragioni ; in alcuni casi , tuttavia , pu essere sintomatica . 
opportuno , in particolare , considerare questa possibilit nella diagnosi differenziale dei pazienti precedentemente sottoposti a splenectomia che giungono allosservazione per la caratterizzazione di tumefazioni addominali [ 7 , 12 ] o sanguinamento occulto [ 13 ]  . 
 lo scopo di questo studio valutare se lecocontrastografia dopo iniezione di sonovue consente la diagnosi differenziale tra metastasi peritoneali e impianti peritoneali di tessuto splenico sulla base delle caratteristiche di enhancement in fase spleno - specifica . ceus was performed on 13 consecutive patients who had undergone splenectomy with spontaneous ( n = 2 ) or surgically implanted ( n = 11 ) peritoneal splenic grafts and 13 consecutive patients with peritoneal metastases that developed from different primary sites ( table 1 )  . 
scintigraphy materiali e metodi sono stati studiati con ecocontrastografia 13 pazienti consecutivi con pregressa splenectomia e impianti splenici radiol med ( 2009 ) 114 : 4251 table 1 primary malignancies of patients with peritoneal metastases malignancy no . 
of patients lung cancer pancreatic cancer gastric cancer colonic cancer ovary cancer gallbladder cancer hepatocellular carcinoma carcinoma del polmone carcinoma del pancreas carcinoma gastrico carcinoma del colon carcinoma ovarico carcinoma della colecisti epatocarcinoma tabella 1 neoplasie primitive dei pazienti con metastasi peritoneali neoplasia n di pazienti was performed to confirm the splenic nature of the peritoneal nodules identified in the patients with spontaneous and surgically implanted grafts . 
diagnosis of peritoneal metastases was confirmed with biopsy ( n = 3 ) or postmortem histological confirmation and by documenting disease progression during the follow - up . all examinations were carried out by the same sonologist ( mb ) on an hdi 5000 system ( atl , bothell , wa , usa )  . grey - scale ultrasound ( us ) and color doppler interrogation were performed with a 25 mhz convex probe and a 512 mhz linear probe , and ceus with a 25 mhz convex probe . 
to avoid bias in the retrospective evaluation of nodules , care was taken to avoid visualising ascites , which was present in 8 / 26 patients , within the images . 
digital cine clips were recorded during the entire contrast examinations and stored for offline retrospective analysis . a reader who did not perform the us examinations ( gc ) peritoneali spontanei ( n = 2 ) o chirurgici ( n = 11 ) e 13 pazienti consecutivi con metastasi peritoneali da tumori di diversi organi ( tabella 1 )  . 
la natura splenica dei noduli identificati nei pazienti con impianti splenici peritoneali spontanei o chirurgici stata confermata con la scintigrafia ottenuta eseguendo scansioni delladdome in proiezione anteriore , posteriore e laterale destra 20 minuti dopo liniezione endovenosa di 45 mci di tecnezio - 99m solfuro colloidale . 
la diagnosi di metastasi peritoneali stata confermata alla biopsia ( n = 3 ) o allautopsia e documentando durante il follow - up del paziente la progressione della malattia . tutti gli esami sono stati eseguiti dallo stesso operatore ( m.b. ) utilizzando un ecografo hdi 5000 ( atl , bothell , wa , usa )  . 
per lecografia in scala dei grigi e per lecocolor doppler sono stati utilizzati un trasduttore convex da 25 mhz ed un trasduttore lineare da 512 mhz ; per lecocontrastografia stato utilizzato un trasduttore da 25 mhz . 
lenhancement stato valutato utilizzando una tecnica contrastospecifica non distruttiva ( pulse inversion harmonic imaging , atl , bothell , usa ; indice meccanico 0 , 09 )  . lenhancement stato valutato in tempo reale dal tempo delliniezione del contrasto fino a oltre 4 minuti dopo liniezione . 
 i filmati digitali sono stati processati da un operatore differente da quello che aveva eseguito gli esami ecografici ( g.c. ) e che non era a conoscenza della natura dei noduli . sono state selezionate immagini a intervalli temporali compresi tra il momento delliniezione del contrasto e 4 minuti dopo liniezione ( 0 , 10 , 20 , 30 , 40 , 60 , 90 , 120 , 150 , 180 , 210 , 240 secondi dopo liniezione del contrasto )  . 
lintensit media del segnale stata valutata in tutte le immagini selezionate in unit di intensit video ( viu , range : 0255 ) misurando il livello di grigio medio nellistogramma in una regione dinteresse comprendente lintero nodulo . 
 la forma dei noduli stata valutata misurando sulla stessa immagine ecografica il massimo diametro longitudinale ( l ) e il minimo diametro trasverso ( t ) e calcolando il rapporto l / t [ 14 ]  . 
frames were selected at different time intervals ranging from the time of injection to 4 min after injection ( 0 , 10 , 20 , 30 , 40 , 60 , 90 , 120 , 150 , 180 , 210 , 240 s after microbubble injection )  . 
frames were saved as 8 - bit grey - scale images and stored in tiff format . average signal intensity was evaluated in all selected images in video intensity units ( viu , range : 0255 ) using commercially available software ( adobe photoshop cs2 version 9.0 ) by measuring the average grey level in the histogram in a region of interest encompassing the entire nodule . the shape of each nodule was assessed by measuring the largest longitudinal ( l ) and smallest transverse ( t ) diameters on the same us scan and by calculating the l / t ratio [ 14 ]  . 
to obtain a parameter independent of the variability of lesion enhancement deriving from the type and settings of the equipment used , patient characteristics and lesion depth , the percent rate between the signal intensity and the maximum signal intensity reached by the nodule was calculated using the formula ( sit / simax ) 100 , where sit is enhancement at time t , i.e signal intensity at time t after subtraction of signal intensity at time 0 , which was considered as background intensity , and simax is the maximum enhancement reached by the nodule from the time of injection to 4 min after injection . 
to assess interobserver variability , 45 images were randomly selected and enhancement evaluated by two independent readers ( gc and rz ) , as previously described . statistical analysis was performed with commercially available software ( graphpad prism version 4.00 for windows , graphpad software , san diego , ca , usa )  . differences between the l / t ratio , volume , and enhancement of the splenic nodules and of peritoneal metastases were evaluated using the mannwhitney test . 
probability p values of p < 0.05 were considered statistically significant . results at the time of the ceus examination 3 / 13 patients with peritoneal metastatic disease presented with liver metastases . 
after sonovue injection , splenic grafts were characterised by strong un parametro indipendente dalla variabilit dellenhancement derivante dal tipo e dalla regolazione dellapparecchiatura utilizzata , dalle caratteristiche del paziente , e dalla profondit della lesione stato calcolato il rapporto percentuale tra lintensit di segnale e la massima intensit del segnale raggiunta dal nodulo secondo formula : ( sit / simax ) 100 , dove sit lenhancement al tempo t , cio il valore che si ottiene sottraendo allintensit del segnale al tempo t lintensit del segnale al tempo 0 , considerata come background , e simax il massimo enhancement raggiunto dal nodulo nel tempo tra linizio delliniezione e 4 minuti dopo liniezione . 
 lanalisi statistica stata condotta utilizzando un software commerciale ( graphpad prism version 4.00 per windows , graphpad software , san diego , california , usa )  . il test di mann - whitney stato impiegato per valutare le differenze tra i rapporti l / t , i volumi e lenhancement dei noduli splenici e delle metastasi peritoneali . 
non sono state rilevate differenze statisticamente significative tra il rapporto l / t dei noduli splenici scelti come target ( 1 , 790 , 31 , mediana 1 , 78 , range 1 , 192 , 33 ) e delle metastasi peritoneali ( 1 , 710 , 31 , mediana 1 , 75 , range 1 , 292 , 20 ) : le due classi di noduli presentavano pertanto forma simile . 
non sono state rilevate differenze statisticamente significative tra il volume medio dei noduli splenici ( 32 , 2730 , 27 cm3 , mediana 27 , 88 cm3 , range 5 , 43102 , 96 cm3 ) e delle metastasi ( 29 , 9424 , 78 cm3 , mediana 31 , 10 cm3 , range 2 , 6775 , 73 cm3 )  . 
the average volume of splenic nodules ( 32.2730.27 cm3 , median 27.88 cm3 , range 5.43102.96 cm3 ) and peritoneal metastases ( 29.9424.78 cm3 , median 31.10 cm3 , range 2.6775.73 cm3 ) was not significantly different . 
the rates were significantly higher for splenic grafts starting from 60 s after microbubble injection dellintensit del segnale dei noduli di tessuto splenico e delle metastasi peritoneali nei differenti intervalli di tempo . nella nostra casistica a partire da 40 secondi dopo la somministrazione delle microbolle lintensit del segnale risultata significativamente maggiore per gli impianti splenici ( test di mann - whitney , p < 0 , 001 )  . 
the range of enhancement values is reported in parentheses bp values are by mann - whitney test cno overlapping between the range of enhancement of splenic grafts and of peritoneal metastases 10 s 20 s 30 s 40 s 60 s 90 s 120 sc 150 sc 180 sc 210 sc 240 sc 10 s 20 s 30 s 40 s 60 s 90 s 120 s 150 s 180 s 210 s 240 s 10 s 20 s 30 s 40 s 60 s 90 s 120 s 150 s 180 s 210 s 240 s tabella 2 enhancement allecocontrastografia degli impianti splenici e delle metastasi peritoneali tempo dopo liniezione delle microbolle ( secondi ) impianti splenici ( viu ) a metastasi peritoneali ( viu ) a valori di pb radiol med ( 2009 ) 114 : 4251 aviu , unit di intensit video . 
signal intensity in video intensity units ( viu ) at time t after subtraction of background si in viu at time 0 ] , and simax is the maximum enhancement reached by the nodule from the time of injection to 4 min after injection . 
the range of the percent rates is reported in parentheses bp values are by mann - whitney test tabella 3 rapporto percentuale tra lenhancement degli impianti splenici e delle metastasi peritoneali tempo dopo liniezione delle microbolle ( secondi ) impianti splenicia metastasi peritonealia valori di pb ail rapporto percentuale stato calcolato secondo la formula : ( sit / simax ) 100 , dove sit lenhancement al tempo t ( cio lintensit del segnale in viu al tempo t dopo la sottrazione dellintensit del segnale al tempo 0 , considerata come background ) e simax il massimo enhancement raggiunto dal nodulo dallistante delliniezione fino a 4 minuti dopo liniezione . 
il range e i rapporti percentuali sono riportati tra parentesi bvalori di p ottenuti con il test di mann - whitney radiol med ( 2009 ) 114 : 4251 signal intensity ( viu ) fig . 
in fact , although computed tomography ( ct ) and magnetic resonance imaging ( mri ) may , in splenic implants as in the native spleen , typically show inhomogeneous enhancement in the arterial phase and persistent , homogeneous enhancement during the following vascular phases [ 17 ] , differentiation between splenic implants and metastatic deposits can be problematic in clinical practice if based on radiological imaging alone [ 7 , 12 , 18 ] , when peritoneal nodules are identified accidentally in a patient without other obvious signs of metastatic disease . superparamagnetic iron oxide ( spio ) - enhanced mri allows a confident characterisation of splenic tissue ; this technique , however , is expensive [ 17 ]  . 
whereas ultrasonography allows differentiation between accessory spleens and splenic grafts based on their shape and vascular supply [ 14 ] , morphological and colour doppler appearance of peritoneal splenic nodules and metastases are often similar because both types of nodules are oval with peripheral feeding vessels at colour doppler interrogation . 
a variable amount of ascites is often present in patients with peritoneal metastases ( 7 / 13 patients in our series ) , whereas usually it is problema si presenta in particolare nei pazienti neoplastici quando lascite o altre lesioni secondarie non orientano nella diagnosi differenziale . 
bench la tc e la risonanza magnetica mostrino tipicamente , negli impianti splenici come nella milza nativa , enhancement disomogeneo in fase arteriosa ed enhancement omogeneo e persistente nelle successive fasi vascolari [ 17 ] , la diagnosi differenziale tra impianti splenici e metastasi peritoneali pu essere problematica nella pratica clinica sulla sola base dellimaging radiologico [ 7 , 12 , 18 ] quando i noduli peritoneali sono identificati accidentalmente in un paziente che non presenta altri chiari segni di diffusione metastatica . 
la risonanza magnetica con mezzi di contrasto superparamagnetici composti da nano - particelle di ossido di ferro ( spio ) permette una caratterizzazione accurata del tessuto splenico , questa tecnica , tuttavia , costosa [ 17 ]  . 
lecografia consente di differenziare gli impianti splenici dalle milze accessorie sulla base della morfologia e delle caratteristiche vascolari [ 14 ] , ma gli impianti splenici e le metastasi peritoneali si presentano spesso con morfologia e vascolarizzazione molto simile , cio come noduli di forma ovalare con vasi afferenti periferici . 
i pazienti con metastasi peritoneali si presentano spesso con ascite in quantit variabile ( 7 / 13 pazienti nella nostra casistica ) , mentre in genere , in assenza di complicanze , non si osserva liquido libero in addome nei pazienti con impianti di tessuto splenico . 
 il nostro studio documenta come gli impianti peritoneali di tessuto splenico , analogamente alla milza nativa [ 1 ] , mostrano un accumulo specifico di sonovue che si evidenzia come enhancement persistente per almeno 4 radiol med ( 2009 ) 114 : 4251 not recognised in patients with uncomplicated splenic peritoneal deposits . 
 as shown by our study , peritoneal splenic grafts show spleen - specific uptake after sonovue administration , as occurs for the native spleen [ 1 ] , resulting in contrast enhancement lasting at least 4 min after microbubble administration , a time longer than the blood - pool phase . 
as in our series peritoneal metastases did not present with late enhancement , irrespective of primary neoplasm , this enhancement characteristic should allow differential diagnosis between splenic nodules and metastatic deposits . 
 several limitations of our study should be mentioned . first , the ability of us to detect peritoneal lesions depends on both the skill of the operator and the characteristics of the patient . 
as signal intensity from microbubbles strongly depends on lesion depth and on the performance of the equipment used , our results cannot be generalised because a wider range of signal intensity values is expected using different us machines to image nodules at different depths . 
we believe that evaluating the percent rate between the signal intensity and the maximum signal intensity reached by the nodule could allow characterisation of peritoneal splenic nodules regardless of the equipment and lesion depth . 
bias could have affected the collection process . although the operators who retrospectively processed the digital cine clips and reviewed the images were blinded to the nature of the nodules during the evaluation process , absolute blinding was difficult to attain because the enhancement characteristics were essentially different . finally , the splenic nature of peritoneal nodules was confirmed by using 99mtc sulphur colloid scintigraphy . 
poich nella nostra casistica le metastasi peritoneali non hanno presentato enhancement tardivo , indipendentemente dal tipo di tumore primitivo , questa caratteristica sembra consentire la diagnosi differenziale tra noduli splenici e secondari . 
poich lintensit del segnale dipende fortemente dalla profondit della lesione e dalla performance dellapparecchiatura ecografica utilizzando ecografi diversi per studiare noduli posizionati a profondit differenti ci si aspetta un range pi ampio di valori di intensit di segnale . 
la valutazione del rapporto percentuale tra lintensit del segnale e la massima intensit raggiunta dal nodulo potrebbe pertanto consentire la caratterizzazione dei noduli di tessuto splenico in peritoneo indipendentemente dallapparecchiatura e dalla profondit della lesione . 
una terza limitazione del nostro studio che lenhancement delle lesioni stato valutato misurando il livello di grigio medio in unit video su immagini in scala dei grigi della profondit di 8 bit . 
misurazioni pi accurate possono essere ottenute utilizzando software dedicati progettati elettivamente per quantificare lintensit del segnale in scala lineare , prima della compressione logaritmica necessaria per la presentazione a video . 
bench gli operatori che hanno processato i filmati digitali retrospettivamente e quelli che hanno revisionato le immagini non fossero a conoscenza della natura dei noduli durante il processo di valutazione le caratteristiche di enhancement dei due tipi di noduli sono profondamente differenti ed ottenere unanalisi completamente in cieco difficile . 
questa indagine documenta la captazione del radiocolloide sia da parte del fegato che della milza , ma meno sensibile e specifica rispetto alla scintiradiol med ( 2009 ) 114 : 4251 splenic tissue but is less sensitive and specific compared with heat - damaged red - blood - cell scintigraphy , which allows imaging of the splenic tissue independently from liver [ 20 , 21 ]  . 
these preliminary data , however , need confirmation with a larger series of patients , with a variety of us equipment and contrast - specific modes . in conclusione , nella nostra casistica sonovue ha consentito la diagnosi differenziale tra impianti peritoneali di tessuto splenico e metastasi peritoneali . 
this article provides new evidence that the onset of toh related to pregnancy may occur not only in the third trimester , as described in most articles , but also early after delivery [ 2 ]  . 
to date , postpartum toh had been reported only anecdotally due to the lack of large series [ 2 ]  . most previous reports of pregnancy - related bilateral toh do not specify pain onset was synchronous for both hips or whether there was a side - to - side migration over a period of months . 
 [ 2 ] , who recently reported five patients with synchronous and bilateral pregnancy - related toh . these observations might corroborate the hypothesis that the simultaneous involvement of both hips might occur in some cases of pregnancy - related toh , but not in men , who would be more prone to develop a side - to - side migratory toh . 
lequesne [ 3 ] , in his classic description of toh in a population of ten men , found three cases of bilateral migrating involvement over a period of months , but none of them had a synchronous involvement . when it comes to the images shown in xyda et al.s article , we beg to add some comments . 
first , in the second and third case presented , the images in t1 - weighted sequences would seem to show a subtle subchondral hypointense band paralleling the articular surface . 
in toh , the detection of such subtle linear images may correspond to a epiphyseal fracture , and differentiation from an epiphyseal osteonecrosis may be challenging in some cases [ 4 ]  . 
tale articolo ha il merito di confermare che lesordio dellosteoporosi transitoria dellanca pu osservarsi non solo durante il terzo trimestre di gravidanza , come riportato classicamente in letteratura , ma anche durante il periodo post - parto [ 2 ]  . linsorgenza di unosteoporosi transitoria dellanca era stata documentata in letteratura solo a titolo aneddotico , a causa della mancanza di serie sufficientemente numerose di pazienti [ 2 ]  . 
inoltre , la maggior parte dei precedenti casi riportati di osteoporosi bilaterale dellanca , non specificano se linsorgenza della sintomatologia era sincrona per le due anche o se si trattava , piuttosto , di un quadro di dolore e di impotenza funzionale con migrazione allanca controlaterale realizzatosi nellarco di alcuni mesi . 
linsieme di queste osservazioni potrebbe corroborare lipotesi che il coinvolgimento simultaneo di entrambe le anche si osserva in alcuni casi di osteoporosi transitoria dellanca correlata alla gravidanza , mentre nella popolazione maschile , nelle forme bilaterali , si osserverebbe piuttosto un pattern di tipo migrante con interessamento successivo delle due anche . lequesne [ 3 ] , nella sua classica descrizione dellosteoporosi transitoria dellanca in una popolazione di 10 uomini , descrive 3 casi di coinvolgimento bilaterale migrante , ma nessun caso di coinvolgimento sincrono . dal punto di vista delle immagini fornite nellarticolo , ci permettiamo di aggiungere alcuni commenti . 
in primo luogo , nel caso 2 e 3 , le immagini in ponderazione t1 sembrano mostrare la presenza di una sottile immagine lineare ipointensa a topografia subcondrale , con andamento parallelo alla superficie articolare . 
la differenziazione di tali anomalie epifisarie nei confronti di unosteoneradiol med ( 2009 ) 114 : 168171 the experience of our group [ 4 ] and other authors [ 5 ] , such subchondral lines are frequently seen in toh . 
in our experience , fat suppression should be avoided because these technique obscures subchondral areas of hypointensity in t2 - weighted sequences that , if sufficiently large , are associated with a poor prognosis [ 4 ]  . in conclusion , we congratulate xyda and coworkers on their report of bilateral simultaneous toh in early postpartum , which had been only anecdotally described . 
nella nostra esperienza le sequenze ponderate in t2 senza saturazione rivestono unimportanza prognostica notevole poich la presenza di un territorio subcondrale ipointenso in t2 , di dimensioni sufficientemente estese associata ad una peggior prognosi [ 4 ]  . in conclusione , ci congratuliamo con il xyda et al . per la descrizione di tre casi di osteoporosi transitoria bilaterale dellanca la cui descrizione in letteratura era solo aneddotica . 
cardarelli , via antonio cardarelli 9 , 80138 napoli , italy 3neurological sciences department , second university of naples , piazza miraglia , 80138 naples , italy 4nuclear medicine , pet unit , policlinico s . 
orsola - malpighi , bologna university , italy 5dipartimento di scienze fisiche , universit federico ii , and infn , sezione di napoli , via cinthia , 80126 napoli , italy 6physics department , university of bologna and infn , bologna , italy correspondence to : r . 
the use of animal models in basic and preclinical sciences , for example , offers the possibility of testing diagnostic markers and drugs , which is becoming crucial in the success and timeliness of research and is allowing a more efficient approach in defining study objectives and providing many advantages for both clinical research and the pharmaceutical industry . 
the aim of this article is to review the characteristics of these systems and illustrate their main applications . in preclinical research , importance keywords molecular imaging micro - pet micro - ct optical imaging small - animal imaging riassunto negli ultimi anni si sono rese disponibili nuove apparecchiature per lo studio di piccoli animali . 
lutilizzo di modelli sperimentali nelle scienze di base e pre - cliniche consente di verificare test diagnostici e farmaci , divenendo essenziale per il successo e la tempestivit della ricerca , offrendo un approccio pi efficace nella definizione degli obiettivi da studiare e notevoli vantaggi sia per la ricerca clinica sia per le industrie farmaceutiche . 
lutilizzo di tali tecnologie consente di ottenere informazioni pi predittive riguardo alla distribuzione o allefficacia di una molecola . il topo , in particolare , un modello animale insostituibile per lo studio delle malattie umane . 
 parole chiave imaging molecolare micro - pet microtc imaging ottico imaging per piccoli animali radiol med ( 2009 ) 114 : 152167 introduction introduzione the availability of animal models in which to reproduce human diseases ( neurodegenerative diseases , tumour growth , genetic disorders ) has given rise to new diagnostic and therapeutic approaches . 
experimental models on small animals , and especially on genetically engineered mice , are receiving increasing recognition as a powerful means of analysis in oncological , cardiovascular and neurological research and in testing potential new drugs [ 3 , 4 ]  . imaging methods based on the introduction of very small concentrations of radiotracers have traditionally been used , together with organ dissection and tissue counts [ 5 ]  . 
alternatively , cryosection of the entire body followed by digital autoradiography [ 6 ] can provide anatomical images with very high spatial resolution ( about 50 m ) correlated with a quantitative analysis of the tracer concentration . 
the main limitation of these ex vivo techniques is that each laboratory animal can only be analysed once during the research study , thus rendering the research expensive , time consuming and relatively imprecise given the physiological variation between specimens . 
 the growing number of studies performed in animal models has attracted scientific interest to the point of awarding the inventor of this imaging technology [ 7 ] with the nobel prize for medicine and providing incentives for the development of new imaging systems adapted to the special conditions of an in vivo study . 
a variety of dedicated devices have been developed in this field , including optical imaging , computed tomography ( ct ) , positron emission tomography ( pet ) , single photon emission computed tomography ( spect ) , magnetic resonance imaging ( mri ) and ultrasonography ( us )  . 
today the provision of biomedical imaging techniques for small animals is one of the most important challenges and opportunities of the new millennium , with the possibility of longitudinal research studies repeatedly examining the same animal model before , during and after the experimental procedure . the aim of this paper is to describe the most common applications of these techniques and outline their relative advantages and disadvantages . 
the following techniques will be described in turn : optical imaging ( bioluminescence and fluorescence ) , micro - ct , micro - pet , micro - spect , micro - mri and micro - us . la disponibilit di modelli animali in grado di riprodurre le malattie umane ( malattie neurodegenerative , crescita tumorale , disordini genetici ) ha permesso nuovi approcci diagnostici e terapeutici . 
modelli sperimentali su piccoli animali , ed in particolare topi ingegnerizzati geneticamente , sono sempre pi riconosciuti come un mezzo potente di analisi nel campo della ricerca oncologica , cardiovascolare , neurologica , e nella sperimentazione di potenziali farmaci [ 3 , 4 ]  . 
 metodi di visualizzazione basati sullintroduzione in concentrazioni molto piccole di molecole radio - marcate sono gi tradizionalmente usati , in combinazione con la dissezione dorgano e la conta tissutale [ 5 ]  . 
in alternativa , la criosezione dellintero corpo seguita dallautoradiografia digitale [ 6 ] pu anche fornire immagini anatomiche con una altissima risoluzione spaziale ( circa 50 micron ) correlate ad unanalisi quantitativa della concentrazione di tracciante . 
il punto debole di queste metodiche ex vivo consiste nella possibilit , durante una ricerca , di poter analizzare una sola volta ogni cavia , rendendo cos la ricerca costosa , dispendiosa di tempo , e poco precisa considerata la variazione fisiologica interindividuale . 
 il numero crescente di studi effettuati su modelli animali ha attratto a tal punto linteresse scientifico da far insignire linventore di questa tecnologia [ 7 ] col premio nobel per la medicina e da incentivare lo sviluppo di nuovi sistemi di imaging adatti alle particolari condizioni di uno studio in vivo su di essi . 
oggi , la disponibilit di tecniche di imaging biomediche per piccoli animali rappresenta la pi importante sfida ed opportunit del nuovo millennio per la possibilit di ricerche longitudinali studiando ripetutamente lo stesso modello animale prima , durante , e dopo una procedura sperimentale . obiettivo di questo contributo descrivere le pi comuni applicazioni di queste metodiche , illustrandone limiti e vantaggi . 
saranno illustrate in sequenza limaging ottico ( bioluminescenza e fluorescenza ) , micro - tc , micro - pet , micro - spect , micro - rm e micro - us . strumenti imaging ottico instrumentation optical imaging progress in the field of optics has rekindled interest in i progressi nel campo dellottica hanno riacceso linteresse nellimaging ottico come potente mezzo di applicazione preclinica . 
the first uses a gene that encodes for an enzyme , such as luciferase [ 10 ] , which in the presence of oxygen and adenosine triphosphate ( atp ) generates bioluminescence with an emission peak of around 560 nthe second , in contrast , uses external dyes or fluorescent markers . 
the device for bioluminescence imaging can simply consist of a black box containing a highly sensitive charged coupled device ( ccd ) camera , which provides images of the distribution of light on the surface of the animal . 
fluorescence imaging , instead , requires the addition of an external light source , such as a laser , or a broad - band source , such as a mercury lamp , and multiple filters to obtain a spectrum of the light emitted . 
at these wavelengths there is also greater penetration and limited linear absorption of the animals internal organs . new optical imaging techniques have recently been developed based on infrared fluorescence that can provide not only planar data but also quantitative information and multiplexing studies : time - domain optical imaging [ 12 ] and tomographic optical imaging [ 13 ]  . 
in particular , because bioluminescence does not require stimulation of an external light source , the problem of the excitation beam penetrating the tissue does not exist , as with other techniques , and there is no background autofluorescence to disturb the signal . 
fluorescence imaging , on the other hand , has the advantage of the high signal produced by the fluorophores and the possibility that this is activated by specific biological processes , thus reducing background noise . the disadvantages derive from two fundamental sul mercato sono presenti diversi strumenti con un ampio intervallo di capacit . 
il primo utilizza come rilevatore un gene che codifica per un enzima , come la luciferasi [ 10 ] , che in presenza di ossigeno ed atp , genera la bioluminescenza con un picco in emissione di circa 560 n il secondo , invece , utilizza coloranti esterni o marcatori fluorescenti ; questi ultimi sono molecole che assorbono nella regione del vicino infrarosso ( nir ) oppure una nuova classe emergente , quantum dots [ 11 ]  . 
lo strumento per limaging in bioluminescenza pu essere semplicemente composto da un box nero contenente una camera ccd ad alta sensibilit , ottenendo cos immagini della distribuzione della luce sulla superficie dellanimale . 
limaging in fluorescenza , invece , richiede laggiunta di una sorgente di luce esterna , come un laser , o una sorgente a banda larga , ad esempio , una lampada a mercurio , e di filtri multipli al fine di ottenere uno spettro della luce emessa . 
 necessario utilizzare fluorofori che vengano eccitati nello spettro dellinfrarosso , dal momento che in questo intervallo di frequenze di eccitazione ed emissione si ottengono dei dati pi puliti dato il basso fondo generato . 
a queste frequenze si ha inoltre un maggior potere di penetrazione ed un assorbimento lineare limitato degli organi interni dellanimale . recentemente sono state sviluppate nuove tecnologie di imaging ottico basate sulla fluorescenza nellinfrarosso che consentono di ottenere non solo dati planari , ma anche di avere informazioni quantitative e di effettuare studi multiplexing , time domain optical imaging [ 12 ] ed imaging ottico tomografico [ 13 ]  . 
restano comunque sufficientemente efficaci i sistemi planari di imaging ottico in commercio e a basso costo , per effettuare studi di screening [ 16 ]  . i vantaggi dei metodi di imaging ottico sono luso di radiazioni a bassa energia non ionizzanti , unalta sensibilit con possibilit di rilevare segnali nellordine di micron o nellordine del sub - picomolare , la capacit di acquisizione continua di dati per il monitoraggio in tempo reale e lo sviluppo di accessori economici . 
in particolare , la bioluminescenza non necessita di una stimolazione esterna di luce : non vi quindi il problema della penetrazione del fascio di eccitazione nei tessuti , come nellaltra metodica , e non c un fondo di autofluorescenza a disturbare il segnale . 
limaging in fluorescenza , invece , si avvantaggia dellalto segnale dei fluorofori e dalla possibilit che questo venga attivato da specifici processi biologici , riducendo cos il rumore di fondo . radiol med ( 2009 ) 114 : 152167 properties of light in tissues , which depend on the wavelength and light diffusion and absorption , which influence image resolution and penetration depth of light into tissues , as well as the ability to obtain quantitative data . lastly , optical imaging has no equivalent in clinical practice , which renders correlation and application of the results difficult . 
this technique has been applied in studies monitoring expression of a transgene , progression of an infection , tumour growth and metastasis , transplant , toxicology , viral infection and gene therapy [ 1823 ]  . micro - ct micro - ct is the miniaturisation of ct , a technique widely used in clinical diagnostic imaging . 
small - animal micro - ct ( mice and rats ) is able to supply very high resolution anatomical images ( up to 9 m ) with three - dimensional views [ 24 ]  . 
in daily practice , researchers of some smallanimal - imaging facilities ( saifs ) tend to limit the study to short scans ( around 1 min ) , both to increase proceeds and reduce radiation exposure , which can jeopardise longitudinal studies , although the cost is a lower spatial resolution ( 150200 m )  . micro - ct systems can be found in many imaging facilities and can cover a variety of applications , both with regard to bone and soft tissue . 
in terms of the ct system specifications , particular attention should be paid to the field of view of the detector , which enables those levels of resolution to be achieved . 
a device of this kind is clearly very versatile , with a broad range of applications but with the drawback of delivering an elevated dose to the animal at high resolutions . 
most systems use a cone - beam x - ray source and a solid - state detector that rotates around the animal , allowing imaging of the entire animal in a single scan . 
 the principal drawbacks of first - generation micro - ct systems were poor contrast resolution for soft tissue , even gli svantaggi derivano da due fondamentali propriet della luce nei tessuti che dipendono dalla lunghezza donda , dalla diffusione della luce e dallassorbimento , che condizionano sia la risoluzione dellimmagine che la profondit di penetrazione della luce nei tessuti , oltre che la capacit di ottenere dati quantitativi . infine , limaging ottico non ha un corrispettivo nella pratica clinica , e questo rende difficile la correlazione e lapplicazione dei risultati . 
questa tecnologia stata applicata in studi di monitoraggio dellespressione di un transgene , per la progressione di uninfezione , crescita tumorale e metastasi , trapianto , tossicologia , infezioni virale e terapia genica [ 1823 ]  . micro - tc la micro - tc una miniaturizzazione della nota tc , ampiamente utilizzata nella diagnostica clinica . 
la microtc dedicata a piccoli animali ( topi e ratti ) in grado di fornire un imaging anatomico ad altissima risoluzione ( fino a 9 micron ) e con visualizzazione tridimensionale [ 24 ]  . nella quotidianit , sia per incrementare eventuali ricavi che per ridurre lesposizione a radiazioni ionizzanti che possono pregiudicare studi longitudinali , i ricercatori di alcune saifs tendono a ridurre lindagine a brevi scansioni ( circa 1 minuto ) , a prezzo di una minore risoluzione spaziale ( 150200 micron )  . 
 i sistemi micro - tc sono abbastanza diffusi presso molte facility di imaging e possono coprire diverse applicazioni sia per quanto riguarda studi su ossa che per tessuti molli . relativamente allo studio su reperti ossei , bisogna tenere in considerazione il limite di risoluzione di 15 micron se si vuole effettuare unanalisi a livello delle trabecole ed importante valutare tra le specifiche del sistema tc anche le dimensioni del campo di vista del rivelatore su cui possibile ottenere quelle determinate risoluzioni . 
per quanto riguarda sistemi dedicati a studi in vivo in genere la risoluzione ottimale si aggira intorno ai 50 o 100 micron , con la conseguente riduzione di dosi e la possibilit di effettuare studi longitudinali . 
second - generation micro - ct systems incorporated much of the technology used in clinical practice , including arrays with smaller detector elements and more powerful x - ray tubes , which enable not only faster scans 0.8 s for the entire animal but also the use of clinical contrast agents and the possibility of performing perfusion studies [ 27 ]  . 
in addition , the use of iodinated contrast agents has significantly improved image contrast , enabling visualisation of even small - diameter vessels ( 20 m ) [ 28 ]  . 
in questo caso necessario avere un dispositivo aggiuntivo di controllo respiratorio che minimizza gli artefatti nelle immagini causati dal movimento della cassa toracica dellanimale . la maggior parte dei sistemi utilizza una sorgente conica di raggi x e un detettore solido che ruota intorno la cavia e che permette lacquisizione dellintero animale in una singola scansione . 
gli svantaggi maggiori della prima generazione di micro - tc sono stati una povera risoluzione di contrasto per i tessuti molli , anche dopo luso di mezzi di contrasto , unalta dose di radiazioni , lunghi tempi di scansione , e artefatti nellimmagine , in particolare alle risoluzioni pi alte . 
la generazione successiva di micro - tc ha utilizzato molte delle tecnologie duso clinico , compresi rivelatori sottili e tubi radiogeni pi potenti , che permettono sia di effettuare scansioni pi veloci , 0 , 8 secondi per linterno animale , sia luso di agenti di contrasto clinici con maggiore traslazione degli studi sia la possibilit di effettuare studi di perfusione [ 27 ]  . 
inoltre , luso di mezzi di contrasto iodati , hanno significativamente migliorato il contrasto dellimmagine , visualizzando vasi anche di piccolo diametro ( 20 micron ) [ 28 ]  . 
i sistemi ad alta risoluzione micro - tc sono stati anche utilizzati in maniera soddisfacente nei tumori dei tessuti molli [ 33 ] e nella caratterizzazione fenotipica di malattia in animali transgenici [ 34 ]  . 
 micro - pet la pet , come la spect , consente di ottenere immagini di una funzione o di un processo biochimico del corpo , utilizzando un radiotracciante ad emissione di positroni . 
si supera cos il limite delle tecniche diagnostiche tradizionali , come le indagini radiologiche , tc o rm , che forniscono unimmagine di tipo anatomico sia il limite della variabilit interindividuale , spesso ritrovata negli studi di farmacologia , in quanto ogni animale pu rappresentare il proprio controllo . quasi tutti i sistemi micro - pet utilizzano piccoli la maggior parte bismuto germanato , cristalli , per germanio oxyorthosilicate , lutetium oxyorthosilicate , o cerio - doped lutetium yttrio ortosilicato di 12 mm , accoppiati a fotomoltiplicatori ( pmts ) convenzionali . 
ricostruzione con voxel 93 m . radiol med ( 2009 ) 114 : 152167 micro - pet as with spect , pet produces images of a biochemical function or process of the body with the use of a positronemitting radiotracer . 
the technique overcomes both the limitations of traditional morphological diagnostic techniques , such as ct or mri , and those of intraindividual variability , which is often found in pharmacological studies , in that each animal can serve as its own control . almost all micro - pet systems use small crystals ( 12 mm ) of bismuth germinate , germanium oxyorthosilicate ( gso ) , lutetium oxyorthosilicate or cerium - doped lutetiumyttrium orthosilicate ( lyso ) coupled with conventional photomultipliers ( pmts )  . 
one company has modified the pmt - based design and used solid - state detectors ( photodiodes ) coupled directly to the individual crystals . today , small - animal pet systems have a small gantry with an opening of 1222 cm and a shorter distance between detectors than do clinical pet devices . 
the overall spatial resolution can in general be improved during image reconstruction with the use of physical models to correct for detector blurring , positron - range blurring and other effects [ 37 ]  . the technology recently developed in the field of detectors used in micro - pet has led to significant improvements in terms of resolution uniformity over the field of view . 
the technology used in these systems is based on the presence of a dual ring of scintillation crystals ( gso and lyso ) , which enables greater depth of interaction of photons and therefore reduction in parallax . 
this produces increased and uniform resolution ( 1.4 mm ) throughout the field of view , enabling very effective quantitative analyses [ 38 ]  . another pet modality based on the same concept of coupling two scintillation crystals [ lyso and lutetium yttrium aluminate perovskite ( luyap ) ] is under development . 
this system uses a mobile gantry that is able to accommodate not only small but also medium - sized animals . the use of pet technology is experiencing tremendous expansion , although it is limited by the availability of a cyclotron in the vicinity and a radiopharmacy for production and synthesis of radiopharmaceuticals . 
fluorodeoxyglucose labelled with the isotope 18f is the most commonly used , although new isotopes with a longer half - life are being developed , such as gallium - 68 and copper - 64 for studying hypoxia . lastly , the advent of combined ct - pet systems will further optimise the pet examination by integrating animali si avvantaggiano di un gantry pi piccolo , 1222 cm di apertura , e di una minore distanza tra i rilevatori rispetto alla pet clinica . 
questo si traduce in una pi alta risoluzione spaziale sul volume di almeno 1 , 3 mm e nella capacit di determinare concentrazioni di traccianti nellordine del picomolare in vivo . 
la risoluzione spaziale complessiva pu essere generalmente migliorata durante il processo di ricostruzione dellimmagine attraverso modelli fisici per la correzione degli effetti di blurring del detettore , del range del positrone nellorganismo e di altri effetti [ 37 ]  . 
la tecnologia utilizzata in questi sistemi si basa sulla presenza di un doppio anello di cristalli scintillatori ( gso e lyso ) che consente una maggiore profondit di interazione dei fotoni e quindi una riduzione delleffetto di parallasse . 
in questo modo si possono ottenere elevate risoluzioni ( 1 , 4 mm ) e uniformi lungo il campo di vista , permettendo di fatto unanalisi quantitativa molto efficace [ 38 ]  . unaltra modalit pet in fase di sviluppo ed basato sullo stesso concetto di accoppiamento di 2 cristalli scintillatori ( lyso e luyap ) : questo sistema utilizza un gantry mobile in grado di accogliere non solo piccoli animali , ma anche animali di taglia maggiori . lutilizzo della tecnologia pet in grossa espansione anche se limitata dalla disponibilit di un ciclotrone nelle vicinanze e di una radiofarmacia per la produzione e la sintesi del radiofarmaco . 
attualmente il pi utilizzato il fdg abbinato allisotopo 18f , ma sono in fase di studio nuovi isotopi con emivita pi lunga , quali gallio 68 e rame 64 dedicato allo studio dellipossia . 
 infine , lavvento di sistemi integrati tc - pet consentir un ulteriore ottimizzazione dellindagine pet grazie allintegrazione dellimaging funzionale con quello anatomico della tc . i vantaggi della pet risiedono nella sua specificit , sensibilit , e nella possibilit di quantificare la concentrazione tissutale di un tracciante . 
la sensibilit , pari a 10100 volte la spect , tc e rm , deriva dalluso della coincidenza , dalla conta con collimatori elettronici , e dalla quantificazione conseguenti alla possibilit di correggere la perdita di segnale dovuta allattenuazione dei fotoni [ 39 ]  . 158 radiol med ( 2009 ) 114 : 152167 functional imaging with the morphological information provided by ct . the advantages of pet lie in its specificity , sensitivity and the ability to quantify the tissue concentration of a tracer . 
sensitivity of the technique between 10 and 100 times greater than spect , ct and mr resides in its use of electronic collimators to measure coincidence events and quantification made possible by correcting for signal loss due to photon attenuation [ 39 ]  . 
i pi utilizzati sono 133xe , il 99mtc e lo 123i che decadono emettendo raggi gamma e sono pi facilmente accessibili di quelli usati nella pet [ 46 ]  . 
limaging di piccoli animali , invece , richiede una pi alta risoluzione spaziale per oggetti molto pi piccoli e si ottiene mediante collimatori detti a pinhole , che permettono di raggiungere una risoluzione spaziale molto alta ( < 1 mm ) [ 47 ]  . 
questi sistemi utilizzano un gran numero di radiofarmaci gamma - emittenti , piccole molecole , peptidi e anticorpi , alcuni routinariamente usati nella clinica , altri ancora in fase di sviluppo . fra i limiti della micro - spect si comprendono : una perdita di segnale causata dallattenuazione da parte del collimatore di molti fotoni incidenti ; una elevata attivit del radiofarmaco necessaria per ottenere un segnale utile per la formazione dellimmagine ; la trasmissione di fotoni che decadono attraverso il tessuto . 
con i primi sistemi pinhole spect dedicati ( micro - spect ) , era possibile ottenere una risoluzione spaziale sub - millimetrica , ma il singolo pinhole ne limitava la sensibilit . 
 la prossima disponibilit di nuove tecnologie , in particolare lo sviluppo di nuovi rilevatori quali czt ( cadmio zinc telluride ) , consentir una sensibilit pari alla micropet , conservando una risoluzione sub - millimetrica . 
le fette sono coronali e la freccia rossa indica la assente o ridotta fissazione del radiofarmaco nella sede del danno . rays , are the most widely used and are more readily available than those used in pet [ 46 ]  . 
small - animal systems , in contrast , require much higher spatial resolution for imaging small objects . this is achieved with pinhole collimators capable of producing a spatial resolution < 1 mm [ 47 ]  . 
these systems use a large number of gamma - emitting radiopharmaceuticals , small molecules , peptides and antibodies , some of which are routinely used in clinical practice , and others are under development . the limitations of micro - spect include signal loss caused by collimator - induced attenuation of many incident photons , elevated activity of the radiopharmaceutical required to produce an adequate signal for image formation , and transmission of photons that decay throughout the tissues . 
with these models , the image can be reconstructed by superimposing data , the scan time has been reduced to several minutes and the dose of radiation per animal has been lowered . the upcoming technological developments , in particular , new detectors such as czt ( cadmium zinc telluride ) , will una tecnologia di sicuro interesse e di grosso potenziale applicativo per il futuro . 
la maggior parte dellimaging su piccoli animali con micro - rm effettuato su strumenti con carrello orizzontale , con campi di forza compresi tra i 4 , 7 t e i 9 , 4 t e con lettino di 2040 csono stati anche sperimentati sistemi con campi di forza pi alti come 11 , 7 t e 21 , 1 t [ 51 ]  . 
per esempio , quando si esaminano le differenze tra sostanza grigia e bianca nel cervello , i tempi di rilassamento rispondono in maniera pi stretta rispetto a quelli osservati nei pi bassi campi utilizzati nellimaging umano , e cos le differenze di contrasto grigio - bianco sono peggiori . 
 la risoluzione pari a 10100 micron e il contrasto 160 radiol med ( 2009 ) 114 : 152167 increase sensitivity to the level of micro - pet , all the while maintaining submillimetre resolution . 
the technique can be used to monitor physiological functions [ 49 ] , track metabolic processes and quantify receptor density [ 50 ]  . micro - mri micro - mri is a powerful and versatile imaging modality for studying small animals . 
t1 and t2 relaxation times are dependent on the magnetic field . for example , when the differences between grey and white matter in the brain are examined , relaxation times tend to be much longer and varied than in the lower fields used for human imaging , with a deterioration in greywhite contrast . the resolution of 10100 m and the spatial contrast of micro - mri systems are not the best among preclinical imaging systems . 
however , as with other modalities , hybrid systems are being developed , such as mri / pet , to obtain increasingly complete information from a single device [ 52 ]  . 
micro - mri systems can be used for functional and physiological studies of tissues [ 5355 ]  . micro - us based on the propagation of sound waves for imaging soft tissue , micro - us is in direct competition with the currently available optical systems in terms of reliability and field of application . 
most of these systems consist of clinical instrumentation adapted to preclinical use , and as yet , there are no ultrasound systems dedicated to the in vivo imaging of small animals , with the exception of one system developed around 5 years ago . 
this device uses very high frequencies , 2580 mhz , and produces a resolution below 30 m and a penetration of around 15 min addition , the use of microand nanobubbles with coatings and gas components as a sonographic contrast agent significantly improves sensitivity and enables measurement of flow in vessels 2030 m in diameter . 
tuttavia , come per altre modalit di imaging , si stanno sviluppando sistemi ibridi , come rm / pet , al fine di ottenere informazioni sempre pi complete con un singolo strumento [ 52 ]  . 
i sistemi microrm consentono indagini funzionali e fisiologiche a livello tissutale [ 5355 ]  . micro - us basata sulla propagazione delle onde sonore per limaging dei tessuti molli , la micro - us compete con i sistemi ottici in commercio in termini di affidabilit e campi dapplicazione . la maggior parte di queste apparecchiature consiste in strumenti clinici adattati alluso pre - clinico e non ci sono ancora sistemi ecografici dedicati per limaging in vivo su animale , ad eccezione di uno sviluppato circa 5 anni fa . questo strumento utilizza frequenze molto alte , 2580 mhz , e fornisce una risoluzione inferiore a 30 micron ed una penetrazione di circa 15 m inoltre , con lutilizzo di microe nano - bolle rivestite e riempite di gas come mezzo di contrasto ecografico , possibile migliorare significativamente la sensibilit dellimaging e misurare il flusso in vasi di 2030 micron di diametro . 
anche possibile attaccare ligandi o anticorpi al rivestimento , rendendo il mezzo di contrasto specifico e capace di legarsi selettivamente a citotipi o processi ( ad esempio , angiogenesi o infiammazione ) [ 56 , 57 ]  . lus una tecnica relativamente economica e accessibile , ma limitata alla profondit dei tessuti che possono essere raggiunti e quindi visualizzati . 
come conseguenza , luso degli ultrasuoni nellimaging animale non si diffuso come le altre modalit dimaging , ma si concentrato sullindagine di particolari strutture biologiche accessibili come la vescica [ 58 ] e i vasi sanguigni [ 59 ]  . 
inoltre una specifica potenzialit degli ultrasuoni lo studio dello sviluppo pre - natale [ 60 ] su animali transgenici . stabulazione degli animali un centro saif deve disporre sia di locali destinati ad accogliere le apparecchiature per la diagnostica che di ambienti annessi riservati alla stabulazione degli animali da sottoporre alle indagini . 
as a result , the use of us in smallanimal imaging is not as widespread as other imaging modalities , with efforts being concentrated on the study of accessible biological structures such as the urinary bladder [ 58 ] and the blood vessels [ 59 ]  . 
in addition , one specific potential application is in studying prenatal development of transgenic animals [ 60 ]  . animal housing saifs should have areas both for diagnostic equipment and housing animals to be studied . 
each area should guarantee appropriate air exchange ( every 1520 h ) , temperature regulation ( 222 c ) and control of relative humidity ( 55%10% ) , as well as the alternation of light and dark in the animal housing areas . the animal house should comprise a number of areas separated by filter barriers , including the animal housing area with feed storage and sawdust and other materials ( cages , grids , bottles ) , a procedure room , a cage - washing area , areas for waste disposal and a filter room / changing rooanimals should be housed in an area near to but separate from the procedure room so that animals waiting to undergo experiments are unable to hear the vocalisations of those undergoing procedures or smell blood in the event of surgical procedures . 
the procedure room should be equipped with the following : laminar - flow ventilation hood , housing for the dissecting microscope , airand oxygen - pressure monitoring , anaesthetic vaporiser , work bench , co2 euthanasia chamber , lockable cupboard for the storage of drugs and consumables , telephone and network connection with pc and a sink . lastly , the area for storing special waste and carcasses should be appropriately shielded and house a refrigeration system ( 20 c ) for their storage until their removal . preparation of animals for study all imaging techniques , except for some optical imaging devices , require the animals to be immobilised . 
some researchers inject barbiturates , but most prefer inhalation anaesthetics , such as isoflurane , because these are administered continuously and can be calibrated to ( 55%10% ) ed unalternanza luce / buio nei locali di stabulazione . 
 lo stabulario deve prevede diverse aree accessibili per mezzo di un filtro e comprendenti un locale di stabulazione con deposito mangime e segatura e / o altro materiale ( gabbie , griglie , bottiglie ) , un laboratorio , un locale lavagabbie , uno spazio per il deposito rifiuti , una stanza filtro / spogliatoio . 
gli animali in sosta devono essere allocati in un ambiente vicino , ma separato dal laboratorio per evitare che cavie che attendono di essere sottoposte ad indagini possano ascoltare le eventuali vocalizzazioni di animali sottoposti a manipolazioni o fiutare lodore del sangue in caso di procedure chirurgiche . 
il laboratorio deve disporre di una cappa a flusso laminare con alloggio per microscopio operatorio , flussimetri per aria ed ossigeno , vaporizzatore ed estrattore per gas anestetici , tavolo di lavoro , bocchetta per lerogazione della co2 al fine di poter procedere alleutanasia , armadietto con serratura per il deposito di farmaci e materiale di consumo , impianto telefonico e di rete con postazione pc , lavabo . 
infine , il locale deposito rifiuti speciali e stoccaggio carcasse deve essere opportunamente schermato e poter allocare un frigo 20c per il loro deposito , fino allallontanamento . preparazione degli animali per lindagine tutte le tecniche dimaging , eccetto per alcuni strumenti dimaging ottico , richiedono che i soggetti dello studio siano immobilizzati . 
alcuni ricercatori iniettano barbiturici , ma la maggior parte preferiscono gli anestetici inalatori , come lisofluorano , perch lanestesia inalatoria somministrata in maniera continua e pu essere calibrata sulle specifiche caratteristiche dellanimale e dellesperimento . 
il sistema dellanestesia gassosa , combinato con il sistema di riscaldamento per la prevenzione dellipotermia , stabilizza le funzioni corporee durante i protocolli sperimentali e riduce al minimo leventualit di perdere gli animali durante le diverse procedure . 
use of inhalation anaesthesia , coupled with active warming to prevent hypothermia , stabilises the vital functions during the experiment and reduces the risk of the animal dying during the various procedures . in addition to anaesthesia and the regulation of body temperature , the equipment must ensure the possibility of immobilising the animal during the study session [ 62 ]  . often , many saifs develop their own containers for specific applications , but most systems for small - animal imaging can hold both mice and rats . 
during the acquisition phase , some physiological parameters can and should be monitored , such as heart and respiratory function and internal temperature . facility organisation saifs should provide the six most utilised imaging modalities in scientific research laboratories . 
these modalities are optical imaging , ( bioluminescence and fluorescence ) , micro - pet , micro - ct , micro - spect , micro - mri and micro - us . 
a detailed list of the main saifs throughout the world and the manufacturers of small - animal imaging systems is available at in general , most saifs require the participation of at least a physicist , an engineer , a veterinarian and several technicians with skills in computer operating and programming , data and image acquisition and analysis and animal treatment . 
the use of animal models in basic and preclinical research and the possibility of performing longitudinal studies on the same animal are key factors for the success and timeliness of research . 
in response to this demand , saifs have been set up to perform a number of tasks : to assist researchers in inoltre , alcune tecniche dimaging richiedono una preparazione particolare : gli animali da sottoporre ad us , per esempio , devono essere tricotomizzati . 
nella fase di acquisizione , alcuni parametri fisiologici possono , e dovrebbero , essere monitorati , come la funzione cardiaca e respiratoria e la temperatura interna dellanimale . organizzazione del servizio il servizio di imaging per piccoli animali , saif , dovrebbe fornire un supporto che comprende le sei modalit pi utilizzate nei laboratori per la ricerca scientifica . 
le sei modalit comprendono : limaging ottico ( bioluminescenza e fluorescenza ) , la micro - pet , la micro - tc , la microspect , la micro - rm e la micro - us . 
una lista dettagliata delle principali saif nel mondo e delle ditte produttrici di strumenti per imaging su piccoli animali pu essere visualizzata nel sito generalmente , la maggior parte delle facilities richiedono la collaborazione di almeno un fisico , un ingegnere , un veterinario ed alcuni tecnici con competenza nelle operazioni e nella programmazione di computer , nellacquisizione e analisi dei dati e di immagine e nel trattamento degli animali . 
lutilizzo di modelli animali nelle scienze di base e pre - cliniche , la possibilit di studi longitudinali sullo stesso animale sono un fattore chiave per il successo e la tempestivit della ricerca . 
per soddisfare questa richiesta , sono nate alcuni servizi per limaging di piccoli animali al fine di : assistere i ricercatori nel progetto sperimentale ; sviluppare e fornire strumenti specifici e procedure innovative per limaging ; preparare gli animali per gli studi , inducendo e mantenendo unappropriata anestesia e immobilizzazione durante la sessione dimaging ; marcare in maniera appropriata i tessuti per la correlazione delle caratteristiche macroscopiche , microscopiche e di imaging del tessuto o organo ; processare , analizzare ed interpretare i dati per la pubblicazione . radiol med ( 2009 ) 114 : 152167 experimental projects ; to develop and supply specific instrumentation and innovative procedures for imaging ; to prepare animals for studies by inducing and maintaining appropriate anaesthesia and immobilisation during the imaging session ; to appropriately mark tissues for the correlation of macroscopic , microscopic and imaging characteristics of the tissues or organ ; to process , analyse and interpret data for publication . the facility should be designed to optimise work processes , access to the animals and exposure to radiation and the magnetic field . 
constant upgrading and renewal of the equipment , particularly with the development of new imaging devices and analysis and computational systems , should be a standard feature for improving the technical potential of the facility . 
lastly , a procedure room near the facility and the animal house will make new experimental protocols possible and facilitate analysis before , during or immediately after the surgical procedure [ 65 ]  . in this setting , the six imaging modalities take their place . these modalities are optical imaging , ( bioluminescence and fluorescence ) , micro - pet , micro - ct , micro - spect , micromri and micro - us . 
hybrid technologies , such as ct - pet and ct - spect , and in vivo coregistration of fluorescence imaging with morphological studies performed with ct or mri that compensates for the short path of the fluorescent signal , the limited penetration of the photons in the tissue and the low three - dimensional spatial resolution , provide complementary information [ 66 , 67 ] by correlating functional data with anatomical structures . of particular importance is that the saif should be selfcontained , so that significant and exhaustive results can be achieved with the same facility , the same personnel , the same animal and the same data analysis , thus saving time and money . each in vivo preclinical imaging modality has a specific application , which maximises the potential of each modality [ 68 ] ( table 1 )  . 
for example , us is an excellent instrument for analysing the vascularity of superficial neoplastic masses , micro - pet is extremely sensitive in measuring types of pathological metabolic activity of various processes , micro - mri can provide very high contrast images and optical imaging is the simplest technique for studying gene expression . 
this explains why saifs are becoming equipped with as many imaging modalities as possible , a tendency that is certain to continue increasing . in the setting of the saif , the radiologists and nuclear scientist technological knowledge , familiarity with the similar equipment in clinical practice and ability to correlate the various findings during diagnostic and therapeutic monitoring give him or her a frontline role . 
these skills cannot be substituted by the physicist or other specialist , both of lallocazione del servizio dovrebbe essere predisposta per ottimizzare il lavoro del personale , laccesso agli animali , il percorso e lesposizione alle radiazioni e al campo magnetico . 
un continuo aggiornamento e rinnovo delle apparecchiature , specie con lo sviluppo di nuovi strumenti per limaging e di sistemi di analisi e di calcolo , dovrebbe essere previsto al fine di migliorare le potenzialit tecniche del centro . 
infine , un area chirurgica , vicino alla facility e allo stabulario , permetter nuovi protocolli sperimentali e faciliter lanalisi prima , durante , o immediatamente dopo un intervento chirurgico [ 65 ]  . in questo articolato progetto vanno attentamente inserite le sei modalit dimaging che comprendono : limaging ottico ( bioluminescenza e fluorescenza ) , la micro - pet , la micro - tc , la micro - spect , la micro - rm e la micro - us . 
le tecnologie ibride , come la tc - pet e la tc - spect , e la coregistrazione in vivo dellimaging in fluorescenza con le modalit dimaging anatomico , quali la rm e la tc , che compensa il breve percorso del segnale fluorescente , la limitata penetrazione dei fotoni nel tessuto e la bassa risoluzione spaziale tridimensionale , forniscono informazioni complementari [ 66 , 67 ] correlando dati funzionali con le strutture anatomiche . in questo contesto dunque importante la completezza della saif , cos da ottenere dal medesimo servizio , personale , animale e dalla stessa analisi dei dati , risultati significativi ed esaustivi risparmiando tempo e denaro . 
 ognuna delle metodiche di imaging pre - clinico in vivo trova applicazioni specifiche in cui le potenzialit di ogni singola tecnica sono valorizzate al massimo ( tabella 1 ) [ 68 ]  . 
per esempio lultrasonografia un ottimo strumento per lanalisi della vascolarizzazione di masse neoplastiche superficiali , la micro - pet estremamente sensibile per la misura dellattivit metabolica di vari tipi di processi patologici , la micro - rm consente di ottenere immagini ad elevatissima contrasto , limaging ottico il mezzo pi semplice per lo studio dellespressione genica . 
questo rende ragione del perch le saifs si stiano attrezzando con il maggior numero possibile di metodiche , tendenza destinata sicuramente a crescere nel tempo . in queste saifs , le conoscenze tecnologiche del radiologo e del medico nucleare , la dimestichezza con apparecchiature simili nella pratica clinica e la capacit di correlare i vari reperti nel monitoraggio diagnostico e terapeutico , assegnano al radiologo un ruolo di primo piano . 
 purtroppo , molte apparecchiature sono state acquistate singolarmente ed allocate in dipartimenti estranei alla table 1 main features of commercial small - animal imaging systems technique resolution sensitivity depth of view acquisition time radiol med ( 2009 ) 114 : 152167 ct flat panel spect 10100 m 950 m 150 m < 50 m 12 mm < 1 mm 12 mm ct flat panel spect 10100 m 950 m 150 m < 50 m 12 mm < 1 mm 12 mm - mmol m - cmol m - cmol mmol p - nmol p - nmol p - nmol - mmol m - cmol m - cmol mmol p - nmol p - nmol p - nmol no limit no limit no limit 15 mm no limit no limit < 1 cm nessun limite nessun limite nessun limite 15 mm nessun limite nessun limite < 1 cm 1060 min 560 min 0.5 s3 min 1520 min 10 s20 min 0.5 s20 min 1060 min 560 min secondi 0 , 5 s3 min 1520 min 10 s20 min 0 , 5 s20 min mr , magnetic resonance ; ct , computed tomography ; us , ultrasound ; pet , positron emission tomography ; spect single photon emission computed tomography ; opt , optical imaging tabella 1 principali caratteristiche dei sistemi sul mercato per limaging di piccoli animali strumento risoluzione sensibilit profondit di campo tempo di acquisizione rm , risonanza magnetica ; tc , tomografia computerizzata ; us , ecografia ; pet , tomografia ad emissione di positroni ; spect , tomografia computerizzata a emissione di fotoni singoli ; opt , imaging ottico whom are part of the team but play different roles , optimising and developing the potential of the devices . unfortunately , many devices are acquired separately and allocated to departments outside the sphere of the radiologist . 
in contrast , radiologists and nuclear scientist should act to promote the creation of more complete and functional imaging centres in the mould of the true saif , where their role would come to the fore . 
i radiologi e medici nucleari , invece , devono farsi promotori dellistituzione di centri pi completi e funzionali tendendo alla realizzazione di vere e proprie saifs , dove il loro ruolo esaltato . 
inoltre opportuno che si applichino in queste nuove aree di ricerca , che saranno la linfa del nostra disciplina . infine , i radiologi e medici nucleari pi giovani devono essere allertati sulle potenzialit dimpiego anche in questo genere di strutture . 
questo settore di ricerca richiede la formazione di adeguate figure professionali che abbiamo competenze di tutte le metodiche di imaging ma anche di biologia molecolare , di gestione dei piccoli animali ( ogni specie ha caratteristiche differenti che la rendono pi o meno adatta a ciascun tipo di studio ) , di chimica organica e di fisica . 
 conclusioni conclusions the modalities of optical and nuclear imaging , micro - ct , micro - mri and micro - us are becoming key techniques for in vivo molecular imaging , thanks to their ability to identify molecular events with adequate sensitivity , specificity and temporal and spatial resolution . 
the availability of new le modalit di studio dell imaging ottico e nucleare , micro - tc , micro - rm , e micro - us stanno emergendo come tecniche chiave nellimaging molecolare in vivo grazie alla possibilit di individuare eventi molecolari con sufficiente sensibilit , specificit , risoluzione temporale e spaziale . 
la disponibilit di nuove sonde , hardware e software per limaging e lo sviluppo di differenti modelli transgenici di malattie ha dato un forte impulso a queste tecniche dindaradiol med ( 2009 ) 114 : 152167 imaging probes , hardware and software and the development of different transgenic models of disease have greatly stimulated these imaging modalities , which are complementary and which , despite responding to different needs and applications , require a multimodal approach to ensure coverage of the different applications . 
preclinical imaging will increasingly be used to study different stages of a pathological process and visualise certain tissues , cell types or receptors . the number of universities , pharmaceutical companies and research centres equipped with saifs will continue to grow . gine , che sono complementari tra di loro e che , sebbene rispondono ad esigenze ed applicazioni diverse , necessitano di un approccio multimodale in modo da coprire le diverse esigenze applicative . 
limaging pre - clinico sar sempre pi utilizzato per lo studio di differenti momenti di un processo patologico e per visualizzare un determinato tessuto , citotipo , o recettore di interesse . 
complete tumour necrosis , confirmed by targeted biopsy , was observed in patients showing no intralesional flow signals and time - intensity curves with low peak of signal intensity and absence of plateau after treatment . 
dopo il trattamento , nei pazienti che presentavano assenza di segnali vascolari intralesionali , curve con basso picco di intensit e assenza di plateau , la biopsia mirata confermava la necrosi completa . 
in base ai risultati ottenuti , le curve con elevato picco di intensit e presenza di plateau , potrebbero rappresentare pattern del tessuto neoplastico intra o perilesionale e fornire quindi importanti informazioni sullefficacia del trattamento . radiol med ( 2009 ) 114 : 3241 keywords power doppler time - intensity curves contrast material focal hepatic lesions hepatocellular carcinoma metastasis parole chiave power doppler curve dintensit - tempo mezzi di contrasto lesioni focali epatiche carcinoma epatocellulare metastasi introduction introduzione in recent years , several nonoperative techniques have been used successfully in the locoregional treatment of hepatocellular carcinoma and metastases , including percutaneous ethanol transcatheter arterial chemoembolisation ( tace ) [ 35 ] , and radiofrequency ablation ( rfa ) [ 69 ]  . injection ( pei ) [ 1 , 2 ] , the follow - up of patients undergoing these treatments is generally based on imaging studies such as magnetic resonance imaging ( mri ) [ 10 , 11 ] or computed tomography ( ct ) [ 12 ] , with only few doubtful cases being studied by biopsy to assess whether complete necrosis of the neoplastic tissue has been achieved . contrast - enhanced colour doppler ultrasound ( cdus ) , thanks to its high sensitivity in detecting weak intratumoural flow signals , has been employed for several years in the differential diagnosis of focal hepatic lesions [ 1315 ]  . recently , it has also been used for the follow - up of intralesional therapy [ 1619 ] , to identify residual areas of intratumoural enhancement that could reflect incomplete necrosis or disease recurrence . 
a study of intralesional and perilesional vascularity with analysis of time - signal intensity curves performed immediately after treatment may help to ascertain whether certain flow signals reflect incomplete treatment . 
the use of contrast - enhanced power doppler us ( pdus ) with timeintensity curves in liver diseases [ 2023 ] , as in focal breast lesions [ 24 ] , may also provide valuable information on the persistence of neoplastic perilesional vascularity and consequently on incomplete necrosis . 
the purpose of this study was to assess the utility of pdus with analysis of time - intensity curves in monitoring focal hepatic lesions ( metastases , hepatocellular carcinoma ) treated with pei or rfa and to correlate the findings with the results of targeted biopsy . materials and methods twenty patients ( twelve men and six women ; age range 55 to 72 years ) with hepatocellular carcinoma and solitary metastases of the liver previously diagnosed by other methods ( ct , mri , biopsy ) underwent intralesional therapy with pei or rfa . 
all patients were studied negli ultimi anni sono state utilizzate diverse tecniche non chirurgiche per il trattamento loco - regionale dellepatocarcinoma e delle metastasi , alcune di queste , molto efficaci come lalcoolizzazione percutanea ( pei ) [ 1 , 2 ] , la chemioembolizzazione ( tace ) [ 35 ] e la radioipertermia ( rfta ) [ 69 ]  . il follow - up dei pazienti sottoposti a questi trattamenti generalmente affidato a metodiche di diagnostica per immagini come la rm [ 10 , 11 ] o la tc [ 12 ] ; solo in alcuni casi dubbi si ricorre alla biopsia per verificare leffettiva necrosi completa del tessuto neoplastico . 
 il color doppler con mdc , grazie allelevata sensibilit nel rilevare i deboli segnali vascolari intralesionali una metodica gi da qualche anno utilizzata per la diagnosi differenziale delle lesioni focali epatiche [ 1315 ] , ed stata gi utilizzata anche per il follow - up dopo la terapia intralesionale [ 1619 ]  . 
spesso subito dopo il trattamento il color doppler con mdc evidenzia una ricca vascolarizzazione perilesionale la cui natura non pu essere determinata se non con la biopsia ; quindi lo studio della vascolarizzazione intra e perilesionale con le curve intensit / tempo , subito dopo il trattamento , potrebbe essere utile poich alcuni segnali vascolari potrebbero essere indice di incompleto trattamento . 
lutilizzo del power doppler con mdc con la determinazione delle curve intensit / tempo nelle malattie del fegato [ 2023 ] o come nel caso delle lesioni focali della mammella [ 24 ] , potrebbe fornire , a nostro giudizio , informazioni preziose anche circa la persistenza di vascolarizzazione neoplastica perilesionale dopo il trattamento e quindi incompleta necrosi della lesione . 
obiettivo di questo lavoro quindi lutilizzo del power doppler con le curve intensit / tempo nel monitoraggio delle lesioni focali epatiche ( metastasi , epatocarcinoma ) sottoposte al trattamento intralesionale con pei e rfta confrontando i risultati con la biopsia mirata . materiali e metodi venti pazienti , dodici maschi e sei femmine , con et compresa tra 55 e 72 anni , affetti da epatocarcinoma e radiol med ( 2009 ) 114 : 3241 by pdus with analysis of time - intensity curves one day before and one day after treatment , followed by targeted biopsy two days later . 
subjects in the control group were imaged by pdus with analysis of time - intensity curves . the us studies were performed on an au cinque idea device ( esaote , genoa , italy ) with a 3.5 mhz convex - array probe . 
in patients the time - intensity curves were derived from a circular region of interest ( roi ) placed over the lesion and large enough to encompass both the intralesional and perilesional vessels . 
il giorno prima e il giorno dopo il trattamento , tutti i pazienti sono stati studiati con esame power doppler con determinazione delle curve intensit / tempo e successivamente , 2 giorni dopo il trattamento , sottoposti a biopsia mirata . 
i pazienti del gruppo di controllo sono stati sottoposti ad esame power doppler con curve intensit / tempo . per lindagine ecografica stato utilizzato un apparecchio au cinque idea ( esaote , genova ) con sonda convex da 3 , 5 mhz . 
nei pazienti patologici la curva intensit / tempo stata ottenuta utilizzando una roi circolare posizionata sulla lesione in modo da comprendere sia la vascolarizzazione intra che perilesionale a tale scopo stato fatto coincidere il diametro della roi con il diametro massimo ottenuto comprendendo la lesione e la vascolarizzazione perilesionale . 
nei pazienti del gruppo di controllo stata utilizzata su tutti i pazienti una roi circolare del diametro di un centimetro posizionata in unarea del parenchima del lobo destro del fegato . 
b the corresponding time - intensity curve shows an elevated peak of signal intensity ( arrowhead ) related to a high concentration of contrast agent and a plateau phase ( arrow ) due to the retention of contrast material within the lesion . 
b la curva intensit / tempo prima del trattamento mostra il raggiungimento di un elevato picco di intensit ( testa di freccia ) , che corrisponde ad unelevata concentrazione di mdc e la presenza di plateau ( freccia ) , dovuti ad una lunga permanenza del mdc nella lesione . 
b the corresponding time - intensity curve shows an elevated peak of intensity ( arrow ) due to high concentration of contrast material and plateaus ( arrow heads ) due to retention of the contrast material in the lesion . 
b la curva intensit / tempo prima del trattamento mostra il raggiungimento di un elevato picco di intensit ( freccia ) , che corrisponde ad unelevata concentrazione di mdc e la presenza di plateau ( teste di freccia ) , dovuti ad una prolungata permanenza del mdc nella lesione . 
b the corresponding time - intensity curve shows an elevated peak of intensity ( arrow ) related to the high concentration of contrast agent and plateaus ( arrowheads ) due to retention of the contrast material within the lesion . 
b la curva intensit / tempo eseguita subito dopo il trattamento mostra il raggiungimento di un elevato picco di intensit ( freccia ) che corrisponde ad unelevata concentrazione di mdc e la presenza di plateau ( teste di freccia ) , dovuto ad una prolungata permanenza del mdc nella lesione . 
b the corresponding time - intensity curve shows an elevated peak of signal intensity ( arrowhead ) related to the high concentration of contrast agent and a plateau phase ( arrow ) due to retention of the contrast in the lesion . 
b la curva intensit / tempo prima del trattamento mostra il raggiungimento di un elevato picco di intensit ( freccia ) , che corrisponde ad unelevata concentrazione di mdc e la presenza di un plateau ( testa di freccia ) , dovuto ad una lunga permanenza del mdc nella lesione . 
d la curva intensit / tempo eseguita subito dopo il trattamento mostra il raggiungimento di un elevato picco di intensit ( testa di freccia ) e la presenza di un plateau ( freccia ) , dovuto ad una prolungata permanenza del mdc nella lesione . in our study , contrast - enhanced pdus performed before treatment revealed intralesional or perilesional flow signals in all patients , so we were able to derive significant time - intensity curves . 
b gruppo di controllo ( paziente sano ) : la curva intensit / tempo eseguita in una porzione del lobo destro del fegato dimostra un basso picco di intensit ( bassa concentrazione di mdc ) ( freccia ) ed un profilo a cupola corrispondente ad un lento accumulo e lento , progressivo rilascio del mdc . performed one day after treatment revealed time - intensity curves without plateaus and with low peaks of signal intensity , confirming complete necrosis of the tumour and success of the treatment . 
this finding was confirmed by the biopsy . instead , in one patient without intralesional flow signals but with persisting perilesional flow signals and time - intensity curve characterised by plateau and high peak of signal intensity , the biopsy revealed residual neoplastic tissue . 
 non si sono verificate reazioni avverse alla somministrazione ev del mdc . discussione le curve intensit / tempo permettono lo studio del comportamento della vascolarizzazione intra e perilesionale dopo la somministrazione ev del mdc e in particolare monitorizzano landamento della concentrazione del mdc nella regione dinteresse in un tempo prestabilito . 
lesistenza di strutture vascolari intra e perilesionali di tipo neoplastico influenza il profilo delle curve in quanto la presenza di shunt artero - venosi e lassenza di sistemi sfinteriali fisiologici , tipiche dei vasi neoformati , si traduce in unintrappolamento del mdc allinterno degli stessi e quindi in una fase di wash - out caratterizzata dai plateau ( fasi stazionamento del mdc ) e presenza di elevati picchi di intensit . nel nostro studio lesame power doppler con mdc ha riscontrato la presenza di segnali vascolari intra o perilesionali in tutti i pazienti prima del trattamento intralesionale ; ci ha permesso lottenimento di curve intensit / tempo significative ; infatti la presenza di enhancement nellarea di interesse una condizione indispensabile e rappresenta quindi un limite della metodica . 
in 15 pazienti , al controllo dopo un giorno dal trattamento , sono state riscontrate curve intensit / tempo con assenza di plateau e basso picco di intensit a sostegno della necrosi completa del tumore e quindi del successo del trattamento ; ci stato confermato dalla biopsia di controllo , mentre , in un paziente che presentava assenza di segnali vascolari intralesionali e persistenza di segnali vascolari perilesionali , in cui la curva intensit / tempo mostrava profilo con plateau ed elevato picco di intensit , la biopsia confermava la persistenza di tessuto neoplastico . 
in questo ultimo caso la curva intensit / tempo ha evidenziato lincompleta necrosi tumorale anche in assenza di segnali vascolari intralesionali dopo il trattamento ; ci fa supporre che la presenza di tessuto neoplastico coinvolgeva la regione perilesionale . 
tale reperto assume notevole importanza , a nostro giudizio , poich il riscontro di una curva come la suddetta dopo il trattamento potrebbe fornire lindicazione per lesecuzione di unaltra seduta terapeutica per ottenere la completa necrosi del tumore . radiol med ( 2009 ) 114 : 3241 an indication for an additional treatment session to achieve complete necrosis of the tumour . 
 in the four patients exhibiting intralesional flow signals after treatment , the time - intensity curves were characterised by plateaus and high peaks of signal intensity , suggesting incomplete necrosis of the tumour , later confirmed by targeted biopsy . 
 the presence of plateaus and high peaks of signal intensity , which characterised all curves before treatment and those of lesions found to be positive at biopsy after treatment , might represent a pattern of neoplastic vascularity . the technique should be further evaluated on a larger number of patients . 
if our findings are confirmed , it could be proposed as an alternative to ct and mri in consideration of its numerous benefits , including limited invasiveness , rapidity , and lack of side - effects of contrast material . nei quattro pazienti che presentavano segnali vascolari intralesionali dopo il trattamento , le corrispondenti curve intensit / tempo mostravano profilo con plateau ed elevato picco di intensit a conferma dellincompleta necrosi del tumore , confermata dalla biopsia mirata . 
we initiated a prospective , blinded investigation on 29 patients affected by oncological diseases ( n = 14 ) or lymphoma ( n = 15 ) , who underwent fluorodeoxyglucose ( fdg ) - based pet - ct and whole - body dwibs for restaging purposes . 
reader 1 had a sensitivity of 89.07% , a specificity of 98.5% , and an accuracy of 97.65% , with a positive predictive value ( ppv ) of 85.48% and a negative predictive value ( npv ) of 98.91%. 
abbiamo avviato uno studio prospettico , in cieco , su 29 pazienti oncologici ( 15 con linfoma e 14 con altre neoplasie ) , che si sono sottoposte nel loro normale percorso diagnostico a tac - pet con fluro - desossi - glucosio ( fdg ) e anche ad unindagine con wb - dwibs , per restaging di malattia . 
lagreement tra i due radiologi risultata quasi perfetta ( = 0 , 93 )  . rispettivamente i due radiologi hanno avuto una sensibilit del 89 , 07% e 87 , 39% , specificit del 98 , 5% e del 98 , 39% , accuratezza diagnostica del 97 , 65% e 97 , 8% per i due lettori , ppv del 85 , 48% e 88 , 13% e npv 98 , 91 e 98 , 75% . radiol med ( 2009 ) 114 : 117 conclusions . 
un difetto dello studio sicuramente larruolamento di pazienti con lesioni multiple disseminate , leterogeneit della casistica . keywords diffusion weighted imaging whole - body pet - ct parole chiave diffusion weighted imaging whole body tc - pet introduction introduzione in oncology , tumour therapy and follow - up are strongly based on reliable diagnostic imaging techniques enabling evaluation of the entire body for disease detection and staging . 
whole - body imaging is mainly applied to screening and staging , and the role of magnetic resonance imaging ( mri ) and positron emission tomography computed tomography ( pet - ct ) among whole - body imaging techniques has been well assessed in the literature [ 1 ]  . whole - body mri , intended as a conventional mri technique , has excellent tissue contrast and high spatial resolution and allows detailed morphological evaluation . 
these properties make it particularly suited to the study of bone marrow , very sensitive in detecting and locating skeletal metastasis and superior to ct in depicting bone and parenchymal infiltration [ 2 ] in almost all organs ( though not yet for lung parenchyma )  . since the introduction of pet - ct in 2001 , anatomicmetabolic studies have been found to be extremely sensitive in characterising lymph nodes and detecting metastasis . however , they also tend to be affected by false - positive results mainly related to brown fat , muscle tissue or inflammatory processes given that fluorodeoxyglucose ( fdg ) is not entirely a cancer - specific tracer . 
in addition , bowel muscular activity and urinary tract excretion hinder the interpretation of studies of the gastrointestinal and urinary system [ 3 ]  . the other whole - body technique , 99mtc - phosphonatebased skeletal scintigraphy ( or bone scan ) , is the standard nuclear - medicine method for the initial staging of primary or secondary bone tumours . 
however , it provides limited anatomical detail , has low sensitivity and specificity and only depicts bone metastasis at a relatively late stage when osteoblastic reaction to tumour deposits has already occurred [ 49 ]  . diffusion - weighted imaging ( dwi ) , a validated technique for mr evaluation of the brain , has recently been in oncologia , la terapia ed il controllo della risposta ad essa ( follow - up ) sono strettamente legati alla disponibilit di metodiche di imaging diagnostico , che permettano lanalisi di tutti i distretti corporei potenzialmente interessati dalla patologia e dalla sua diffusione ( whole - body ) , e che siano affidabili nel riconoscere e stadiare la patologia . 
per essere appropriato , lo screening di secondo e terzo livello deve disporre di una metodica caratterizzata da elevata specificit , applicata su una patologia ad elevata prevalenza nella popolazione . 
limaging whole - body ( whole body imaging , wbi ) orientato pertanto principalmente a scopi di screening e staging ; in letteratura [ 1 ] , ben assodato il ruolo dellimaging con risonanza magnetica ( whole body magnetic resonance imaging , wb - mri ) e della tomografia computerizzata abbinata alla tomografia ad emissione di positroni ( tc - pet )  . 
 per wb - mri , si intende lapplicazione total - body della tecnica rm convenzionale ; tale tecnica caratterizzata da eccellente risoluzione di contrasto ed alta risoluzione spaziale , permette una valutazione morfologica dettagliata ; queste caratteristiche rendono la metodica particolarmente idonea allo studio del midollo osseo , molto sensibile nella individuazione e localizzazione delle metastasi scheletriche , superiore alla tc nella definizione dellinfiltrazione ossea e parenchimatosa [ 2 ] , per quasi tutti gli organi ( allo stato attuale , non per il parenchima polmonare )  . dallintroduzione della tc - pet nel 2001 , lo studio anatomo - funzionale risultato particolarmente sensibile per la caratterizzazione dei linfonodi e delle metastasi , sebbene limitato da false positivit . 
queste ultime sono generate prevalentemente dal grasso bruno , da processi infiammatori o da tessuti muscolari metabolicamente attivi , dal momento che il marcatore metabolico il fluorodesossi - glucosio ( fdg ) , marker non specifico per lattivit cellulare neoplastica ; inoltre lattivit muscolare intestinale e lescrezione urinaria del metabolita rendono di difficile interpretazione questi due apparati [ 3 ]  . laltra principale metodica wb disponibile in medicina nucleare la scintigrafia ossea con tecnezio ( bone scan , bs ) , che rappresenta la metodica standard di staging dei radiol med ( 2009 ) 114 : 117 extended to extracranial districts [ 10 , 11 ] , although with several technical difficulties due to hardware limitations and the mobile nature of the organs to be studied . 
this is an echo planar imaging pulse sequence combined with short tau inversion recovery ( epi - stir ) suitable for evaluating extracranial districts and for whole - body imaging , partly owing to the excellent images obtained with postprocessing algorithms . apart from one congress abstract [ 13 ] , no papers have been published on the subject since takaharas report [ 12 ] , so the accuracy of whole - body dwibs in comparison with the pet - ct is as yet unknown . 
 the aim of this study was to assess the overall diagnostic accuracy of whole - body dwibs in comparison with petct , which is considered the standard of reference among whole - body imaging modalities . materials and methods patients from february 2007 to august 2007 , we enrolled 29 consecutive patients undergoing pet - ct for oncological and oncohaematological diseases ( table 1 ) and fulfilling the requisites for the study . inclusion criteria patients were eligible if they met the following criteria : 1 . 
they volunteered for the mr scan , were able to understand and sign an informed consent form informing them that : ( a ) the examination was not necessary but might tumori ossei primitivi o secondari . 
con lo sviluppo di sequenze pi veloci , insieme al potenziamento dei gradienti variabili e allintroduzione della tecnica del tavolo mobile , limaging rm pesato in diffusione , esteso allo studio di tutto il corpo diventato tecnicamente realizzabile . 
 [ 12 ] hanno messo a punto una sequenza per limaging rm pesato in diffusione , denominata diffusion weighted body imaging with background body signal suppression ( dwibs ) , una sequenza echo - planar abbinata alla soppressione del segnale del grasso con tecnica stir , ottimizzata per la valutazione dei distretti extracranici e particolarmente adatta ad un impiego whole - body , grazie anche agli ottimi risultati iconografici ottenuti con opportuni algoritmi di post - processing . 
pertanto , fino ad oggi , laccuratezza diagnostica della tecnica wb - dwibs in confronto con la tc - pet non nota . scopo del presente studio di valutare laccuratezza diagnostica della wb - dwibs in comparazione con il gold standard nellambito delle metodiche di imaging wholebody , la tc - pet . table 1 patient series grouped by type of neoplasm tabella 1 casistica raggruppata per tipologia di neoplasia primary tumour number of patients sedi del tumore primitivo numero di pazienti lymphoma lung breast kidney sarcoma primitive neuroectodermal tumour prostate linfoma polmone mammella intestino rene sarcoma pnet prostata radiol med ( 2009 ) 114 : 117 help to understand or stage their disease in the future , ( b ) the study required no contrast medium injection , ( c ) the study took 20 min , and ( d ) no ionising radiation would be used 2 . 
a q - body coil was used , with the patient positioned feet first on an extended anatomical coverage table , based on rolling - table technology ( mobitrak , philips )  . 
after a scout pulse sequence , we acquired a dwibs pulse sequence and an epi - stir single - shot pulse ( table 2 ) in the axial plane , repeated for up to four stacks to encompass all anatomical districts from the head to at least the distal thigh , similarly to the pet - ct acquisition protocol . 
we adopted two different b - factor values in the diffusion pulse sequence , b = 500 and b = 1000 s / mm2 , but only the latter was used for reading purposes , as suggested by takahara et al . 
the acquisition time for the protocol was 18 min which , summed to the scout sequence time , implied a total room occupation time of 20 min . nuclear medicine pet - ct scans were acquired on a hybrid siemens ( siemens , erlangen , germany ) system consisting of an lutetium oxyorthosilicate ( lso ) pet scanner ( hi - rez ) with pico - 3d electronics and a 16 - row ct device ( somatom sensation 16 )  . 
 image reformatting and analysis after acquisition and the choice of the higher b value , the materiali e metodi pazienti da febbraio 2007 fino ad agosto 2007 abbiamo arruolato 29 pazienti consecutivi , in previsione di eseguire una valutazione tc - pet , per patologie oncologiche , anche del distretto emolinfopoietico ( tabella 1 ) , che soddisfacevano i criteri di inclusione . criteri di inclusione i pazienti sono stati considerati reclutabili in relazione ai seguenti criteri : 1 . 
abbiamo utilizzato la bobina q - body , posizionando il paziente ( in modo che entrasse nel gantry caudocranialmente , feet first ) , su un tavolo porta - paziente a copertura anatomica estesa , basata sulla tecnologia del tavolo mobile ( mobitrak , philips )  . 
dopo una sequenza di centratura , abbiamo acquisito una sequenza dwibs ( stirecho planar , single shot ) ( tabella 2 ) sul piano assiale , ripetuta 4 volte per coprire tutti i distretti anatomici dalla testa alla coscia distale , in modo da replicare la copertura anatomica dellesame tc - pet . 
la sequenza stata pesata con un doppio valore di b ( b = 500 e b = 1000 s / mm2 ) , utilizzando solo il secondo per la refertazione , come suggerito da takahara et al . 
al termine , si provveduto a invertire la scala dei grigi , in modo che su sfondo bianco ( le aree prive di segnale ) si potesse riconoscere la patologia come immagini con diverse intensit di grigio ( le aree con segnale accentuato ) , con tutte le gradazioni intermedie . 
le immagini whole - body mip e mpr cos elaborate sono state poi salvate nel database dello scanner . anche le immagini native assiali sono state separate sulla base del parametro b , e quelle con b = 1000 s / mm2 , dopo aver preparato la finestra di visualizzazione in modo ottimale , sono state salvate nel database delle immagini . 
tutte le immagini , elaborate da un medico specializzando esperto , sono state private delle informazioni anagrafiche e salvate su una memoria di massa mobile ( cd ) , in formato dicom , registrando ogni supporto con il numero progressivo del paziente . 
le sessioni di refertazione sono state condotte , indipendentemente , da due radiologi esperti in rm ( a.s , lettore 1 e a.c. lettore 2 ) , rispettimente con 8 e 18 anni di pratica professionale in rm , reciprocamente inconsapevoli del risultato della refertazione dellaltro , e del risultato della lettura della tc - pet . i due radiologi hanno riempito una scheda di lettura predefinita , costituita da un foglio elettronico ( excel , microsoft , redmond , usa ) , nel quale sono state individuate 40 sedi anatomiche , scheletriche e viscerali , basandosi su unanaloga schematizzazione elaborata da lauenstein et al . radiol med ( 2009 ) 114 : 117 4 - mm - thick multiplanar reformations ( mpr ) in the coronal plane , and ( c ) multiple 4 - mm - thick mpr in the sagittal plane oriented to include the midline as well as the spine and paraspinal regions . 
all reformatted images were then fused by means of mobiview software ( philips ) with the smooth fusion algorithm to obtain whole - body mip and mpr images . the grey scale was subsequently inverted to allow visualisation of abnormalities ( increased signal ) as grey areas of varying intensity against a white background ( without signal )  . all of the whole - body mip and mpr images were saved in the scanners patient - image database . 
the native axial slices were separated on the basis of the b value , and those acquired with b = 1 , 000 s / mm2 were also saved in the image database after optimal window setting . 
all image sets , processed by an experienced trainee radiologist , were rendered anonymous and stored in digital imaging and communications in medicine ( dicom ) format on an optical disc marked with the patients progressive number for subsequent reading . 
images were read independently by two experienced mri radiologists ( reader 1 : as , with 8 years experience ; reader 2 : ac , with 18 years experience ) who were unaware of each others mri report and of the pet - ct results . the two readers noted their findings on a predefined reading grid consisting of a spreadsheet ( excel , microsoft , redmond , usa ) listing 40 skeletal and visceral sites and subsites , modified from the classification proposed by lauenstein et al . 
radiological reading time for each patient was also recorded . the pet - ct study was reported by the nuclear physician on a similar reading grid as that used by the radiologists . 
the nuclear physician first read the source axial pet images separately and then overlaid on axial ct images and next reviewed the coronal and sagittal images reconstructed with the mpr algorith image analysis included assessment of metabolic uptake of the tracer in terms of standardised uptake value ( suv )  . postprocessing and mean reading times were calculated . the cohen constant was calculated to determine the grade of interobserver agreement . 
for each reader , we calculated diagnostic accuracy , sensitivity and specificity of the wholebody dwibs readings together with negative and positive predictive values ( npv and ppv ) and compared the readings with those performed by the nuclear physician on the pet - ct studies . 
 lo specialista medico nucleare ha refertato lesame tcpet , su unanaloga scheda di lettura , valutando le immagini assiali pet , poi sovrapposte alle immagini tc e infine ricostruite nei piani coronale e sagittale tramite algoritmo mpr , utilizzando anche la valutazione dellintensit della captazione metabolica ( suv ) come criterio per definire patologico un reperto . statistica sono stati calcolati il tempo di post - processing e di refertazione . 
per ogni lettore , stata calcolata laccuratezza diagnostica , la sensibilit e la specificit delle letture wb - dwibs , insieme al valore predittivo negativo e positivo ( npv e ppv ) , comparando le letture dei radiologi con quella del medico nucleare eseguita sulle immagini tc - pet . risultati la tecnica wb - dwibs ha comportato un tempo di occupazione della sala rm di circa 20 minuti . 
il tempo di refertazione medio risultato di 20 minuti ( range 1525 minuti ) per il primo lettore e 18 minuti per il secondo ( range 1322 minuti )  . 
il tempo di refertazione ha avuto un trend decrescente per ognuno dei radiologi dal primo paziente fino al ventinovesimo . laccordo tra i due lettori stato quasi perfetto , con un valore di di 0 , 93 . 
i due radiologi , su 29 pazienti ( ognuno analizzato nelle 40 sedi anatomiche ) hanno considerato positive ( affette ) rispettivamente 124 e 128 sedi , delle quali 106 e 104 veri positivi ( vp ) , 18 e 14 falsi positivi ( fp ) , contro il rilievo ct - pet di positivit in 119 sedi . sono state considerate negative dai due radiologi 1196 e 1202 sedi , delle quali rispettivamente 1183 e 1187 veri negativi ( vn ) e 13 e 15 falsi negativi ( fn ) , rispetto alle 1121 sedi considerate negative dalla metodica di riferimento ( tabella 4 )  . 
quattordici dei 29 pazienti hanno avuto un responso esattamente speculare a quello della tc - pet . negli altri 15 pazienti si verificato un mismatch di lettura ( fp e fn ) da 1 a massimo 5 sedi . 
i due radiologi hanno avuto rispettivamente : il primo radiologo una sensibilit del 89 , 07% , specificit del 98 , 5% e accuratezza del 97 , 65% , con vpp e vpn di 85 , 48% e 98 , 91% ; il secondo radiologo una sensibilit del 87 , 39% , specificit del 98 , 39% e accuratezza diagnostica del 97 , 8% , con vpp del 88 , 13% e vpn del 98 , 75% . analizzando le discrepanze tra tc - pet e wb - dwibs , abbiamo rilevato rispettivamente per i due radiologi 18 e 14 falsi positivi , 13 e 15 falsi negativi , distribuiti come evidente nella tabella 5 , dove stata riportata la media tra i due radiol med ( 2009 ) 114 : 117 table 3 categorisation of skeletal and visceral sites used in the radiology report skeletal sites visceral sites skull , orbit and maxillary bones cervical spine thoracic spine lumbar spine sacrum coccyx sternum clavicle scapula humerus forearm ilium - ischium femur lower leg cranio , orbite e massiccio colonna cervicale colonna toracica colonna lombare sacro coccige sterno coste clavicola scapola omero avambraccio ilio - ischio femore arto inferiore neck neck nodes lung mediastinum pleura thoracic nodes breast axillary nodes liver adrenal glands kidneys pancreas spleen gallbladder bladder abdominal nodes retrocrural nodes female pelvis reproductive apparatus male pelvis reproductive apparatus omental / peritoneal pelvic nodes inguinal nodes soft tissues collo linfonodi del collo polmone mediastino pleura linfonodi toracici mammella linfonodi ascellari fegato surreni reni pancreas milza intestino colecisti vescica linfonodi addominali linfonodi retrocrurali pelvi femminile apparato riproduttivo pelvi maschile apparato riproduttivo omento peritoneo linfonodi pelvici linfonodi inguinali tessuti molli tabella 3 elenco delle sedi scheletriche e viscerali adottato per la scheda di lettura sedi scheletriche sedi parenchimali time of approximately 20 mpostprocessing had a mean duration of 15 min ( range 1017 min )  . 
of these sites , 106 and 104 were true positive ( tp ) and 18 and 14 were false positive ( fp ) compared with positive pet - ct findings in 119 sites . 
readers rated 1 , 196 and 1 , 202 sites , respectively , as negative , of which 1 , 183 and 1 , 187 were true negatives ( tn ) and 13 and 15 were false negatives ( fn ) compared with negative pet - ct findings in 1 , 121 sites ( table 4 )  . dallaltra parte , nelle sedi addominali e pelviche , appaiono pi concentrati i reperti di falsa positivit . 
un caso di non completa corrispondenza tra le tc - pet e wb - dwibs , quello di un paziente ( p.l. ) , affetto da sarcoma della coscia , trattato chirurgicamente e poi con chemioterapia . 
i numeri rappresntano la media tra i due lettori distribution of results distribuzione dei risultati false negative falsi negativi skull , orbit and maxillary bones sacrum sternum scapula ilium - ischium lung mediastinum thoracic nodes pancreas retrocrural nodes total thoracic spine scapula femur neck neck nodes breast axillary nodes liver adrenal glands kidneys omental / peritoneal pelvic nodes inguinal nodes soft tissues total cranio , orbite e massiccio sacro sterno scapola ileo - ischio polmone mediastino linfonodi toracici pancreas linfonodi retrocrurali totale colonna dorsale scapola femore collo linfonodi del collo mammella linfonodi ascellari fegato surreni reni omento / peritoneo linfonodi pelvici linfonodi inguinali tessuti molli totale false positive falsi positivi there was a perfect match with the pet - ct findings in 14 / 29 patients ; there was some mismatch , ranging from one site ( fp or fn ) to five sites in the same patient in the remaining 15 / 29 patients . 
 with regard to the sites of disagreement between petct and whole - body dwibs , the two readers provided 18 and 14 false - positive results and 13 and 15 false - negative results , distributed as seen in table 5 , which shows the mean between the two readers . 
table 5 shows that the spine was almost free of fp and fn results , whereas the lung - mediastinum and the skull - maxillary bones recorded more fn results than all the other areas taken as a whole . 
colour multiplanar reformation ( mpr ) fusion of pet and ct data ( a , b )  . single pet and ct axial images before coregistration and fusion ( c , d )  . 
1a - d paziente affetto da sarcoma alla coscia destra , trattato con chirurgia e chemioterapia , attualmente con riscontro di metastasi allilo sinistro e ai polmoni ( frecce ) , oltre a recidiva alla coscia controlaterale ( punta di freccia ) ; a , b fusione a colori mediante algoritmo mpr tra pet e tc ; c , d immagini assiali singole di pet e tc prima della co - registrazione e fusione . 
si notino le calcificazioni lesionali a livello dellilo polmonare ( c ) e del nodulo polmonare ( d )  . thigh sarcoma mismatch between pet - ct findings and dwibs refers to a previous treated with surgery and chemotherapy . 
nella stessa figura , si pu osservare un fp ( a livello renale ) con un mancato riconoscimento della lesione peri - cefalopancreatica , verosimilmente linfonodale ( falso negativo )  . discussione limaging rm a diffusione ( dwi ) , stato inizialmente introdotto nella pratica clinica in ambito neuroradiologico , principalmente per la valutazione dello stroke ischemico . solo recentemente il dwi stato esteso ad applicazioni rm in ambito body [ 11 ] anche per le problematiche tecniche intrinseche al distretto toraco - addominale : movimento , numerosit di interfacce tissutali , contenuto intestinale , contenuto gassoso . 
con il miglioramento tecnologico delle apparecchiature rm , con sequenze sempre pi veloci che hanno permesso lacquisizione delle immagini in respiro libero , la rm body e la diffusione a rm nello stesso ambito , sono divenute tecnicamente fattibili . 
alcuni autori [ 15 ] , inoltre , hanno dimostrato la possibilit di caratterizzare le radiol med ( 2009 ) 114 : 117 b = 500 b = 1000 fig . 
diffusion - weighted imaging with optimised background suppression pulse sequence ( dwibs ) coronal ( a ) , sagittal ( b ) and axial multiplanar reformation ( mpr ) images ( c ) at b = 1 , 000 did not confirm the pet - ct findings ( dotted circles )  . 
dwibs elaborata con mpr in coronale ( a ) , sagittale ( b ) e mpr ( c ) a b = 1000 , senza conferma dei reperti osservati in tac - pet ( circoli punteggiati )  . 
6. the same figure shows an fp result ( kidney ) and a missed , probably nodal , pericephalopancreatic lesion . discussion dwi was originally introduced into clinical practice for neuroimaging purposes and mainly for assessing stroke . lesioni maligne , in ambito body , mediante lo studio della diffusione e la misurazione quantitativa con roi posizionate su mappe adc . 
 [ 12 ] che hanno utilizzato una sequenza echo - planar single - shot accoppiata ad un impulso stir , denominata dwibs : questa sequenza ha permesso una maggiore e pi omogenea soppressione del segnale di fondo e lacquisizione con la tecnica del respiro libero , garantendo cos la possibilit di un tempo di acquisizione pi lungo e per questo con un numero maggiore di radiol med ( 2009 ) 114 : 117 fig . 
3 tac - pet : metastasi ossee diffuse ( frecce ) da primitivo ignoto . only recently has its use been extended to whole - body mr applications [ 11 ] owing to technical problems intrinsic to the thoracoabdominal area , including movement , number of tissue interfaces , bowel content and air content . 
dwi with quantitative measurement of apparent diffusion coefficient ( adc ) has been shown to be able to characterise malignant lesions in whole - body imaging [ 15 ]  . 
more recently , dwi has been improved by the introduction of dwibs , a diffusionweighted pulse sequence paired with a stir sequence [ 11 ]  . this sequence allowed greater homogeneous suppression of the background signal as well as acquisition during free breathing , thus permitting more time for the acquisition , a higher number of signal averages , a better signal - to - noise ratio ( snr ) and acquisition of thinner slices . 
the better fat suppression at the edges of the field of view ( fov ) provided by the stir sequence and the acquisition of thinner slices also allow for better mpr and mip reformamedie del campionamento del segnale , con conseguente incremento del snr e con la possibilit di acquisire sezioni pi sottili . 
in questo modo possibile ottenere migliori immagini mip e mpr , sia per le sezioni pi sottili , sia per la migliore soppressione del grasso ai margini del campo di vista indotta dallimpulso stir . 
in questi ultimi anni , la rm whole body diventata pi accessibile e tecnicamente realizzabile grazie allintroduzione commerciale della tecnologia del tavolo mobile , che insieme anche alla disponibilit del software di fusione di pi pacchetti di acquisizione in ununica immagine , ha permesso lacquisizione e la ricostruzione di immagini whole body . 
questi progressi tecnici sono stati preliminari allintroduzione della tecnica wb - dwibs , nel 2004 . ad oggi , nessuno ha comparato i risultati di questa tecnica con le metodiche di medicina nucleare come la tcpet e la scintigrafia ossea , in pazienti oncologici . 
abbiamo condotto questo studio per comparare la wb - dwibs con la tc - pet , attualmente considerata il gold standard nello staging tnm della patologia neoplastica maligna [ 16 ]  . alcune problematiche relative allimaging wb - dwi , radiol med ( 2009 ) 114 : 117 fig . 
in recent years , whole - body mri has become more accessible and easier to perform , thanks to the advent of rolling or moving table technology that , together with the availability of software allowing the fusion of multiple stacks , has made possible the acquisition and reconstruction of whole - body images . 
all these technical advances paved the way for the introduction of the whole - body dwibs technique in 2004 . to our knowledge , no published study has compared the results of whole - body dw - mri with whole - body nuclear imaging techniques ( pet - ct and bone scan ) in cancer patients . 
this study was thus conducted to compare wholebody dwibs and pet - ct , which is considered the standard of reference in the tumour - node - metastasis ( tnm ) staging system for malignancies [ 16 ]  . limitations of whole - body dwi are the huge amount of data to be postprocessed and interpreted . 
in this study , we had a mean postprocessing time of 15 min , mainly spent for window adjustments before completing image fusion and possono essere sicuramente considerate la notevole quantit di immagini da ri - elaborare nel post - processing e da interpretare . 
nel nostro studio , abbiamo riscontrato un tempo di ri - elaborazione delle immagini piuttosto lungo , circa 15 minuti , prevalentemente spesi nellottimizzazione delle finestre di visualizzazione , prima della fusione dei pacchetti e nella generazione delle ricostruzioni mip e mpr . 
il tempo di lettura medio risultato di 19 minuti , come media tra i due lettori ; le letture sono state molto concordanti e questo garantisce la riproducibilit del risultato , in quanto sembra che le immagini siano facilmente interpretate nello stesso modo da differenti lettori . i nostri risultati dimostrano che la wb - dwibs ha alta specificit ed accuratezza diagnostica ed un elevato valore predittivo negativo ( npv ) , tutti vicino al 98% , quando paragonata alla tc - pet . 
analizzando i falsi positivi e negativi della nostra casistica , si evidenziano tre macroaree critiche , che necessitano ulteriore approfondimento : la regione cranio - mascellare , la regione toracomediastinica e la regione addomino - pelvica . 
in addition , there was high interobserver agreement between the two readers . our results show that whole - body dwibs has high specificity , high accuracy and high npv ( all close to 98% ) when compared with pet - ct . 
analysing the fp and fn results of whole - body dwibs in our series , we observed three critical sites that require further investigation : the skull - maxillary bones , the lung - mediastinum and the abdominopelvic site . 
in particular , the thorax , one of the most investigated districts in cancer screening or staging , requires further investigation by means of dwi with dedicated multichannel coils and a specific pulse protocol . 
specifically , the spine was well assessed , with no fn findings , and only one fp result out of 18 tp results ( table 5 )  . sono responsabili della maggioranza dei fn e fp della nostra casistica . 
in particolare , il torace , che uno dei distretti pi esplorati per screening o staging oncologico , necessita un approfondimento ulteriore , con imaging di diffusione mediante bobine multicanale specifiche ed un protocollo con sequenza ottimizzata per la sua valutazione . invece la tecnica appare estremamente accurata nel riconoscimento di lesioni ossee del rachide e delle ossa lunghe , del cranio e del bacino , e per il rilievo di patologia linfonodale . 
nello specifico , la valutazione della colonna vertebrale appare beneficiare , senza falsi negativi e con un solo falso positivo ( tabella 5 ) su 18 rilievi patologici , delle potenzialit diagnostiche di questa nuova tecnica . 
in a e b si osservano multipli foci di alterazione del segnale a livello latero - cervicale bilaterale , a livello splenico , lomboaortico , pelvico e inguinale sinistro ( freccie nere )  . 
con le punte di freccia si osservano due foci segnalati da entrambi i radiologi a livello renale , dei quali ( a destra , punta di freccia rossa ) uno una localizzazione peri - cefalopancreatica ( errore di sede ) e uno ( a sinistra , punta di freccia verde ) un falso positivo non corrispondente a patologia . 
conferma nelle immagini tc - pet in assiale delle lesioni osservate sia in wb - dwibs che nella ricostruzione tc - pet con mpr in coronale , delle lesioni laterocervicali ( e - g , frecce tratteggiate ) , spleniche ( h - l , frecce intere ) , lombo - aortiche ( m - o , punte di freccia )  . radiol med ( 2009 ) 114 : 117 these preliminary data show that the two major clinical applications could be ( a ) bone lesion detection , and specifically screening or staging of the spine - bony pelvis , and ( b ) lymphoma staging , due to the accurate delineation of nodal disease . 
we will direct our future scientific efforts in these two directions . these data , to be confirmed on a larger number of patients , appear to point to a role for whole - body dwibs in cancer screening , staging , restaging and follow - up , given that the higher the specificity and npv , the better the technique as a screening tool . 
whole - body mri has been shown to be more accurate than pet - ct in evaluating the liver , bone and central nervous system [ 17 , 18 ]  . 
from this point of view , a comparison with pet - ct cannot substitute a comparison based on clinical and pathological follow - up or autopsic confirmation ( the true gold standard ) and , in this sense , our study is limited by a certain degree of bias . 
thus , in presenting our preliminary data , we refer to previous studies in which a direct comparison was made between mri and nuclear medicine . the main limitations of the present study inherent to its pilot nature are a heterogeneous case series , the presence of diffuse disease and the small sample size . 
even though wholebody dwibs is beginning to be used in clinical practice , the standard diagnostic workup of patients enrolled in the study was not modified on the basis of the findings of whole - body dwibs because of the lack of scientific validation . direzioni condurremo i nostri prossimi studi . 
 questi risultati , da confermare su una popolazione di pazienti pi ampia , sembrano indicare un ruolo della wbdwibs nello screening , stadiazione , ristadiazione e followup della malattia tumorale ; infatti , pi alta la specificit e il npv , migliore la tecnica per lo screening . 
osserviamo inoltre che la sensibilit non cos elevata ( < 90% ) come la specificit , sempre in riferimento alla tc - pet . la tc - pet ha comunque le sue limitazioni specifiche , in particolare riguardo ai falsi positivi [ 17 ] , che possono influenzare anche il calcolo dellaccuratezza diagnostica delle tecniche di imaging messe a confronto , e in particolare la sensibilit della wb - dwibs nel nostro studio . 
in molte forme tumorali , la tc - pet pi adatta nella individuazione del tumore ( parametro t ) e nello staging linfonodale ( parametro n ) , ma ci sono alcune forme neoplastiche che hanno scarsa captazione di fdg , come il carcinoma renale o i tumori con frequente diffusione alle ossa , al fegato e al cervello ( come il carcinoma della mammella ) , che sono pi idonei a essere studiati con metodiche whole body alternative : infatti gi stato dimostrato che la rm whole body convenzionale pi accurata della tc - pet nella valutazione del fegato , dellosso e del cervello [ 17 , 18 ]  . 
da questo punto di vista , un confronto con la tc - pet , non pu sostituire un confronto tra una metodica di imaging da validare e il follow - up clinico e patologico o autoptico , che rappresenta il vero gold standard ; pertanto i nostri risultati sono gravati da un certo margine di errore . 
gli autori fanno pertanto riferimento alla letteratura , dove il confronto tra rm e tecniche di medicina nucleare stato pi volte descritto , nel presentare questi dati preliminari . nel nostro studio , anche per le sue caratteristiche di studio pilota , principali bias possono essere considerati la casistica eterogenea , la presenza di patologia neoplastica diffusa e il campione di piccole dimensioni : attualmente stiamo estendendo lo studio ad un campione pi omogeneo , introducendo anche il follow - up clinico - patologico come standard di riferimento . 
caudana1 1diagnostic imaging unit , 2vascular surgery unit , 3orthopaedics and traumatology unit , carlo poma hospital , viale albertoni 1 , 46100 , mantua , italy correspondence to : r . 
if diagnostic uncertainty persists , a bone biopsy is indicated . the inflammatory hyperaemia caused by the ulcer deteriorates the diagnostic quality of 40%50% of mr angiography studies in the infrapopliteal region . 
sedici pazienti diabetici sono stati sottoposti da gennaio 2006 a settembre 2007 a rm del piede per sospetta osteomielite monolaterale ; in 3 / 16 vi erano alterazioni radiografiche da osteo - artropatia neuropatica di charcot . 
la rm ha elevata sensibilit per losteomielite nel piede diabetico , ma pi bassa specificit dovuta soprattutto allosteo - artropatia neuropatica di charcot ; se permane dubbio diagnostico indicata la biopsia ossea . 
liperemia flogistica causata dallulcera deteriora il 40%50% degli studi angio - rm a livello sottopopliteo : in tali casi appropriata larteriografia selettiva per la possibilit di effettuare angioplastica nella stessa seduta . 122 radiol med ( 2009 ) 114 : 121132 keywords diabetic foot osteomyelitis foot mri peripheral vascular disease mr angiography parole chiave piede diabetico osteomielite rm piede vasculopatia periferica angio - rm introduction introduzione diabetic foot complications caused by neuropathy , ischaemia and resulting infection often lead to lower limb amputation , with severe disability and a significant impact on mortality . 
early recognition of osteomyelitis is essential for guiding medical and surgical treatment , which are usually implemented in an inpatient hospital setting [ 1 ]  . ( 89% ) the diagnosis of the clinical diagnosis of the infected foot is problematic in diabetic patients : up to two - thirds of patients may not present with the classic symptoms of deep infection owing to immunodeficiency related to diabetes mellitus . even laboratory examinations are often of little help , as inflammation indices may be absent [ 2 ]  . 
bone probing through skin ulceration is a clinical test with high positive predictive value foot osteomyelitis , although its low sensitivity ( 66% ) means that negative findings do not exclude bone infections [ 3 ]  . 
as a result , diabetic patients with suspected foot osteomyelitis require musculoskeletal imaging , both for the differential diagnosis particularly with neuropathic osteoarthropathy ( charcot foot ) and for an accurate evaluation of the extension of the infection , so as to plan surgery and avoid recurrences . 
moreover , diabetologists recommend that diabetic patients with foot ulcers should always undergo a vascular study to exclude the possibility that healing is prevented by peripheral arterial disease , which often has an insidious clinical onset due to the absence of neuropathic pain [ 4 ]  . our retrospective study presents the modern imaging approach to the infected diabetic foot , emphasizing the important role of magnetic resonance imaging ( mri ) in musculoskeletal and vascular assessment performed with the objective of selecting the most appropriate treatment . le complicanze del piede diabetico causate da neuropatia , ischemia e conseguente infezione portano spesso ad amputazione darto inferiore con grave disabilit e rilevante impatto anche sulla mortalit . 
lulcera del piede rappresenta in genere il primo segnale di allarme nel paziente diabetico e tende a progredire ( 15% dei casi ) verso losteomielite , in prevalenza con meccanismo di diffusione per contiguit . 
il riconoscimento precoce dellinsorgenza di osteomielite essenziale per indirizzare il trattamento sia medico che chirurgico , entrambi in genere attuati in ambiente ospedaliero [ 1 ]  . linquadramento clinico del piede infetto risulta difficile nel paziente diabetico : infatti fino a 2 / 3 dei pazienti possono non presentare i sintomi classici di infezione profonda a causa del deficit immunitario legato al diabete mellito ; anche gli esami di laboratorio spesso non sono di aiuto in quanto gli indici di flogosi possono essere assenti [ 2 ]  . 
il sondaggio osseo con specillo attraverso lulcera cutanea rappresenta un test clinico con elevato valore predittivo positivo ( 89% ) per la diagnosi di osteomielite del piede , ma la sua negativit non esclude linfezione ossea a causa della limitata sensibilit ( 66% ) [ 3 ]  . 
dunque nei pazienti diabetici con sospetta osteomielite del piede le metodiche di imaging muscolo - scheletrico sono necessarie sia per la diagnosi differenziale in particolare con losteo - artropatia neuropatica del piede di charcot sia per laccurato bilancio di estensione dellinfezione , cos da pianificare lintervento chirurgico ed evitare le recidive . 
inoltre , i diabetologi raccomandano di sottoporre sempre a studio vascolare il piede diabetico ulcerato , per escludere che larteriopatia periferica con esordio clinico spesso insidioso per lassenza di dolore dovuta alla neuropatia possa impedire la guarigione dellinfezione [ 4 ]  . lo scopo del nostro studio retrospettivo quello di presentare il moderno approccio imaging al piede diabetico infetto , sottolineando limportanza del ruolo che la risonanza magnetica ( rm ) ha avuto nella nostra esperienza nella valutazione muscolo - scheletrica e vascolare ai fini della scelta terapeutica ottimale . materials and methods materiali e metodi between january 2006 and september 2007 , 16 diabetic patients ( 11 men and five women , mean age 58 years , range 4278 ) with unilateral infected ulcer affecting the forefoot in ten cases , the midfoot in two and the hindfoot in four ( table 1 ) underwent mri of the foot . 
all patients underwent plain radiography of the foot , which was positive for osteomyelitis in 7 / 16 patients owing to the presence of corticoperiosteal thickening and / or of osteolytic foci in the cortical spongiosa close to the skin ulcer . 
tutti i pazienti sono stati sottoposti ad esame radiografico del piede , ritenuto positivo per osteomielite in 7 / 16 pazienti in base alla presenza di ispessimento cortico - periosteale e / o di focolai di osteolisi cortico - spongiosa in adiacenza allulcera cutanea . 
t1 - weighted spin - echo all musculoskeletal mri examinations were conducted with a 1.5 - tesla superconductive unit ( magnetom avanto : siemens ag medical solutions , erlangen , germany ) and an extremity coil . 
the mri protocol , selected on the basis of clinical suspicion and ulcer site , was different for studies of the forefoot compared with those of the midfoot and hindfoot and used a flex surface coil , a smaller field of view ( fov ) and predominantly coronal scans perpendicular to the metatarsal bones as recommended in the literature ( se : tr / te / fa 775 / 10 / 90 ) sequences and short - tau inversion - recovery sequences ( stir : tr / te / ti 5160 - 5760 / 53 / 150 ) were obtained in at least two planes , one of which was always sagittal , for selective fat suppression . 
in a few cases , a double t2and proton - density - weighted turbo - spin - echo sequence ( tse : tr / te / fa 3380 / 11103 / 150 ) was also obtained in the sagittal plane . 
parameters included 3 - mm slice thickness , 0.3 - mm interval for the t1 - weighted se sequences and 0.9 - mm interval for the stir sequences , and 16to 20 - cm fov . 
enhancement was assessed with a t1 - weighted tse sequence with selective ( tse - fs : tr / te / fa 791 / 10 / 150 ) with the same localization parameters as the stir sequences . fat suppression based on the suspicion of peripheral arteriopathy , 12 / 16 patients with infected diabetic foot also underwent mr angiography of the lower limbs with paramagnetic contrast agent ( table 1 ) 3 days to 6 weeks after mri of the foot , using the same scanner . 
this technique , through parallel imaging with dedicated phasedarray coils and high performing gradients , allows acquisithree peripheral arterial regions ( aortoiliac , tion of femoropopliteal , infrapopliteal ) within 42 s only ( excluding table motion )  . 
t1 - weighted 3d fast gradient - recalled - echo ( 3d fast - gre ) sequences were acquired in the coronal plane before and after administration of contrast material to allow subsequent subtraction . 
gadoliniumbenzyloxyproprionic - tetraacetic acid ( gd - bopta ) ( multihance , bracco , milan , italy ) at a dose of 0.15 mmol / kg , equivalent to 0.3 ( 3 / 9 oss . ) erano prevalenti le alterazioni dellosteo - artropatia neuropatica di charcot ( osteorarefazione , fratture , frammentazione ossea , lussazione , reazione osteoblastica riparativa )  . 
un paziente con ulcera del retropiede ed esame radiografico negativo stato sottoposto a biopsia ossea tcguidata del calcagno per interpretare il quadro rm dubbio . la diagnosi finale di osteomielite o la sua esclusione stata ottenuta integrando i rilievi clinico - laboratoristici con quelli imaging in tutti casi ; in 11 / 16 pazienti la diagnosi stata completata con esame batteriologico e / o istologico . tutti gli esami rm muscolo - scheletrici sono stati effettuati con apparecchiatura superconduttiva da 1 , 5 tesla ( magnetom avanto , siemens ag medical solutions , erlangen , germania ) e bobina da estremit . 
il protocollo rm , scelto sulla base del sospetto clinico e della sede dellulcera , stato diverso per lo studio dellavampiede rispetto a quello del medio e retropiede , impiegando bobina flex di superficie , fov pi piccolo e acquisizioni prevalenti sul piano coronale ovvero perpendicolare ai metatarsi come raccomandato in letteratura [ 5 ]  . 
sono state acquisite almeno in due piani , di cui uno sempre sagittale sequenze spin - echo t1 - pesate ( se : tr / te / fa 775 / 10 / 90 ) , sequenze short - inversion - time inversion - recovery per la selettiva soppressione del segnale adiposo ( stir : tr / te / ti 51605760 / 53 / 150 )  . 
in alcuni casi stata acquisita anche la sequenza turbo - spin - echo a doppia pesatura t2 e densit protonica ( tse : tr / te / fa 3380 / 11103 / 150 ) sul piano sagittale . 
sono stati impiegati spessore di fetta 3 mm , intervallo di 0 , 3 mm per le se t1 - pesate e di 0 , 9 mm per le stir , fov compreso tra i 16 cm ed i 20 cin tutti i pazienti lo studio rm stato completato con somministrazione di mezzo di contrasto ( mdc ) paramagnetico per via endovenosa ( dotarem , guerbet , aulnay - sous - bois , francia ) alla dose di 0 , 1 mmol / kg equivalente a 0 , 2 ml / kg ; lenhancement stato valutato con sequenza turbo - spin - echo t1 - pesata a selettiva soppressione del segnale adiposo ( tse - fs : tr / te / fa 791 / 10 / 150 ) con gli stessi parametri di centratura delle sequenze stir . nel sospetto di arteriopatia periferica 12 / 16 pazienti con piede diabetico infetto hanno effettuato anche lo studio angio - rm degli arti inferiori con mdc paramagnetico ( tabella 1 ) , a distanza di tempo variabile ( range : 3 giorni6 settimane ) dallesame rm del piede , utilizzando la stessa apparecchiatura rm . 
langio - rm degli arti inferiori stata effettuata con tecnica bolus - chase a tavolo mobile [ 6 ] : tale tecnica , grazie alla disponibilit dellimaging parallelo con bobine phased - array dedicate e di gradienti pi performanti , consente lacquisizione di 3 distretti arteriosi periferici ( aorto - iliaco , femoro - popliteo , infra - popliteo ) nellarco di soli 42 secondi ( escluso il movimento del tavolo )  . 
le sequenze fast - gradient - recalled - echo tridimensionali ( 3d fast - gre ) t1 - pesate sono state acquisite sul piano coronale prima e dopo somministrazione di mdc per consentire la successiva sottrazione . 
la somministrazione per via endovenosa di gadolinio - bopta ( multihance , bracco , milano , italia ) alla dose di 0 , 15 mmol / kg , equivalente a 0 , 3 ml / kg , radiol med ( 2009 ) 114 : 121132 ml / kg , was administered as an intravenous bolus ( 20 ml on average ) using a two - phase flow injector ( the first half at 1.2 ml / s , the second half at 0.6 ml / s ) , followed by the injection of 20 ml of saline at a flow rate of 1 ml / s . 
acquisition of the 3d gre sequences started when the care bolus technique detected the arrival of the contrast bolus in the aorta . during the postprocessing stage , after the subtraction process , panoramic coronal maximum intensity projection ( mip ) reconstructions were obtained from the aorta to the foot arteries by creating a composite image of the three regions . three radiologists retrospectively reviewed the mri images from the musculoskeletal study of the foot ( ur , ev , rc ) and the vascular study ( ur , at , rc )  . 
the most experienced radiologist ( rc > 15 years ) was considered the reference standard in the event of disagreement among less experienced radiologists ( ur , ev , at < 5 years )  . 
a primary sign of osteomyelitis on mri is evidence of low - signal - intensity areas in the bone marrow on t1 - weighted se images , with higher signal intensity on stir images and enhancement after contrast administration . 
secondary signs are identified close to the altered bone marrow signal and include oedema caused by septic inflammation ( cellulitis or phlegmon ) , soft - tissue abscess , skin ulcer and fistula , with possible interruption of the cortical bone . 
mr angiography of the lower limbs assessed the patency or stenosis - occlusion of the peripheral arteries and the presence of venous contamination artifacts superimposed enhancement of the venous circulation which is one of the main limitations of the technique , as it deteriorates the diagnostic quality of the images . 
with this assessment , the images of each of the three vascular regions were judged as either adequate or inadequate for peripheral revascularization . avvenuta in bolo ( in media 20 ml ) tramite iniettore con velocit di flusso bifasica ( la prima met a 1 , 2 ml / s , la seconda met a 0 , 6 ml / s ) , seguita dalla iniezione di 20 ml di soluzione salina con flusso di 1 ml / s . 
la diagnosi rm di osteomielite stata formulata sulla base della concordanza di segni primari e secondari [ 7 ] : la semeiotica rm della osteomielite considera come segno primario la presenza a livello del tessuto osteo - midollare di aree di ridotta intensit di segnale nelle immagini se t1pesate , con incremento dellintensit di segnale in quelle stir ed enhancement dopo somministrazione di mdc paramagnetico , mentre i segni secondari sono quelli rilevabili in adiacenza allalterazione del segnale osteo - midollare e comprendono ledema dovuto alla flogosi settica ( cellulite o flemmone ) , lascesso dei tessuti molli , lulcera cutanea e la fistola con leventuale interruzione della corticale ossea . nelle indagini angio - rm degli arti inferiori sono state valutate la perviet o la steno - occlusione delle arterie periferiche e la presenza di artefatti da contaminazione venosa ovvero lenhancement sovrapposto del circolo venoso uno dei principali limiti in quanto riduce la qualit diagnostica dellesame . 
i reperti angio - rm evidenziati sulle ricostruzioni mip sono stati analizzati per conferma anche nelle sequenze sottratte con software 3d ; cos valutate , le immagini di ciascuno dei tre distretti vascolari esaminati sono state giudicate sufficienti o insufficienti per la scelta del trattamento di rivascolarizzazione periferica . 
 results risultati in 13 / 16 patients with infected diabetic foot ulcers , the final diagnosis was osteomyelitis , whereas in 3 / 16 patients it was cellulitis ( table 1 )  . 
a the sagittal short - tau inversion - recovery sequence ( stir ) image shows a pathological fracture of the calcaneus and dislocation of the proximal fragment due to retraction of the achilles tendon . 
b magnetic resonance angiography ( mra ) of the right lower limb is hindered by venous contamination in the distal third , whereas the left infrapopliteal arteries are clearly visible up to the plantar arch . 
the mri diagnosis of exclusion of osteomyelitis proved correct in two patients with infected ulcer and cellulitis ( true negatives ) in whom conventional radiology had identified alterations due to neuropathic osteoarthropathy . 
overall , foot mri permitted correct identification or exclusion of osteomyelitis in 15 / 16 patients , with a high accuracy and sensitivity , even in the presence of radiographic evidence of neuropathic osteoarthropathy ( three cases )  . 
mri also proved useful for assessing the extent of osteomyelitis for surgical planning purposes ( four minor and one major amputation ) , for identifying soft - tissue abscesses requiring drainage ( three cases ) as well as other infectious complications including septic arthritis ( four cases ) , pathological fracture ( one case , fig . 1a ) and septic tenosynovitis ( one case )  . non da flogosi settica del tessuto osteo - midollare . 
2a - c reactive bone marrow oedema of the talus and calcaneus due to neuropathy in a diabetic patient with mal perforans and normal radiographic findings [ false - positive magnetic resonance imaging ( mri ) diagnosis of osteomyelitis ]  . 
a the coronal t1 - weighted turbo - spin - echo ( tse ) image with fat suppression after administration of paramagnetic contrast material reveals diffuse changes in signal intensity of bone marrow and soft tissues , as well as moderate contrast enhancement in the talus and in the subtalar portion of the calcaneus , wrongly interpreted as osteomyelitis associated with cellulitis . 
b in the axial short - tau inversion recovery ( stir ) image , the area of increased signal intensity in the calcaneus is identified to perform a bone biopsy . 
2a - c edema osteo - midollare reattivo talo - calcaneare da neuropatia in paziente diabetico con mal perforante e quadro radiografico normale ( errore rm di falsa positivit per la diagnosi di osteomielite )  . 
a nellimmagine coronale tse t1 - pesata con soppressione del segnale adiposo dopo somministrazione di mdc paramagnetico diffuse alterazioni del segnale osteo - midollare e dei tessuti molli in sede talare e nella porzione subtalare del calcagno caratterizzate da discreto enhancement , erroneamente interpretate come osteomielite associata a cellulite . 
c la biopsia ossea sotto guida tc ha escluso linfezione ossea . regions , the diagnostic patients with infected diabetic foot allowed evaluation of the diagnostic quality of the images obtained in the three vascular regions . 
these five patients later underwent selective arteriography of the ulcerated lower limb with an anterograde approach , owing to a suspicion of concomitant distal diabetic arterial disease ( table 1 )  . 
in 4 / 5 patients , arteriosclerotic occlusive lesions of the crural arteries were identified , and percutaneous transluminal angioplasty ( pta ) was performed during the same session . after the mr angiography , 9 / 12 patients underwent three of whom had a surgical vascular surgery , effettuati in 12 pazienti con piede diabetico infetto ha permesso di valutare la qualit diagnostica delle immagini ottenute in ciascuno dei tre distretti vascolari esaminati . 
questi 5 pazienti sono stati successivamente sottoposti ad arteriografia selettiva con approccio anterogrado dellarto inferiore affetto dallulcera , nel sospetto di concomitante arteriopatia diabetica distale ( tabella 1 )  . 
in 4 / 5 pazienti sono state riscontrate lesioni arteriosclerotiche steno - occlusive delle arterie crurali e si proceduto ad angioplastica ( pta ) nella stessa seduta . complessivamente 9 / 12 pazienti sono stati sottoposti dopo lo studio angio - rm a intervento vascolare , di cui 3 mediante by - pass chirurgico femoro - popliteo e 6 con tratta128 radiol med ( 2009 ) 114 : 121132 fig . 
3a - c severe charcot neuropathic osteoarthropathy , infected plantar ulcer and bone infarcts due to peripheral arterial disease [ osteomyelitis excluded by magnetic resonance imaging ( mri ) ]  . 
3a - c grave osteo - artropatia neuropatica di charcot , ulcera infetta plantare e infarti ossei da arteriopatia periferica ( rm negativa per osteomielite )  . a nel radiogramma in incidenza antero - posteriore frammentazione ossea del medio - piede in fase cronica con tipica frattura - dislocazione di lisfranc . 
liperintensit di segnale a livello del primo e secondo dito dovuta ad un artefatto ( insufficiente soppressione del segnale adiposo alla periferia della bobina da estremit )  . femoropopliteal bypass , and six underwent endovascular angioplasty ( three the femoropopliteal and infrapopliteal regions , three in the infrapopliteal region only ) with immediate technical success in 5 / 6 cases . in both mento endovascolare di angioplastica ( 3 a livello sia femoro - popliteo che infra - popliteo , 3 solo in sede infrapoplitea ) con successo tecnico immediato in 5 / 6 casi . discussion conventional radiology is still the first - line modality in the study of the diabetic foot owing to its rapidity , ease of use and inexpensiveness . 
it also allows the diagnosis of clinically overt osteomyelitis , reveals alterations due to charcots neuroarthropathy and is useful for the follow - up . known limits of conventional radiology , especially if performed in the early stage of bone infection , are its low sensitivity ( 60% ) and specificity ( 66% ) [ 8 ]  . 
the former is due to a need for 30%50% bone reabsorption , a process taking up to 23 weeks from the onset of infection , whereas the latter depends on the wide spectrum of diseases entering the differential diagnosis , first of all neuropathic discussione lindagine radiologica convenzionale rimane la metodica di primo livello per lo studio del piede diabetico per rapidit , facilit di esecuzione e bassi costi : oltre a permettere la diagnosi di osteomielite nella fase conclamata rileva le alterazioni da osteo - artropatia neuropatica di charcot ed utile per il follow - up . 
i limiti noti dellindagine radiologica , specie se espletata nelle prime fasi dellinfezione ossea , sono la bassa sensibilit ( 60% ) e specificit ( 66% ) [ 8 ] ; la prima dovuta alla necessit di un riassorbimento osseo pari al 30%50% , processo che impiega fino a 23 settimane dallinizio dellinfezione , mentre la seconda dipende dallampio spettro di patologie coinvolte nella diagnosi differenziale , prima fra tutte losteo - artropatia neuropatica che colpisce fino al 3% dei pazienti diabetici . 
the midfoot and hindfoot bones are the most commonly involved by this disease , which presents with demineralization , fractures , fragmentation , joint dislocation and collapse of the plantar arch , with the classic charcot deformity . 
the use of more sophisticated imaging modalities ( either mri or scintigraphy ) is indicated when radiography demonstrates alterations due to charcot neuroarthropathy or whenever the clinicallaboratory suspicion of osteomyelitis is high , or finally to demonstrate complications . 
bone biopsy , although not routinely performed because of its invasiveness , is considered the gold standard in the diagnosis of osteomyelitis and is still recommended to confirm an uncertain diagnosis and isolate the pathogen [ 9 ]  . mri has come to replace ct , as it is better able to identify bone marrow changes and evaluate septic involvement of the soft tissues , which allows surgery to be planned in such a way as to prevent recurrence of infection after amputation or abscess drainage [ 10 ]  . 
it is thus reserved for patients with contraindications to mri or with doubtful mri findings , or to assess response to antibiotic therapy during the follow - up [ 9 ]  . 
when there is topographic concordance between the primary and secondary mri signs , the mean reported sensitivity and mediopiede e del retropiede sono quelle pi spesso coinvolte da questa patologia che si manifesta con demineralizzazione , fratture , frammentazione , lussazione articolare fino al collasso dellarcata plantare , con la classica deformit del piede di charcot . 
il ricorso a metodiche di imaging pi sofisticate ( rm o scintigrafia ) indicato quando lesame radiologico riscontri alterazioni da osteo - artropatia neuropatica di charcot oppure qualora il sospetto di osteomielite sia elevato dal punto di vista clinico - laboratoristico o , infine , per evidenziare le complicanze dellinfezione . 
la biopsia ossea , bench non praticata di routine a causa dellinvasivit , tuttoggi considerata il gold standard per la diagnosi di osteomielite e viene dunque ancora raccomandata per confermare una diagnosi dubbia e per isolare lagente patogeno [ 9 ]  . la rm oggi ha sostituito la tc per la migliore capacit di identificare le alterazioni osteo - midollari e di valutare il coinvolgimento settico dei tessuti molli , ci che consente di pianificare gli interventi chirurgici al fine di evitare recidive infettive dopo amputazione o drenaggio di ascessi [ 10 ]  . la scintigrafia con granulociti tecneziati presenta accuratezza lievemente inferiore a quella della rm nella diagnosi di osteomielite [ 11 ] e risulta pi indaginosa e costosa ; viene attualmente pertanto riservata ai pazienti che hanno controindicazioni alla rm oppure in quelli con quadro rm dubbio o , infine , nel follow - up per valutare la risposta alla terapia antibiotica [ 9 ]  . 
la rm rappresenta pertanto lindagine preferenziale di secondo livello per la diagnosi di osteomielite : qualora vi sia concordanza topografica tra segni rm primari e secondari i valori medi di sensibilit e 130 radiol med ( 2009 ) 114 : 121132 specificity are 94% ( range 88%99% ) and 83% ( range 78%89% ) , respectively [ 2 , 7 ]  . 
on the other hand , a positive mri study is not always sufficient to establish a diagnosis of osteomyelitis , even when there is topographic concordance between primary and secondary signs . 
administration of paramagnetic contrast material increases specificity because enhancement of bone marrow , as assessed in the t1weighted sequences with selective fat suppression , is regarded as typical of osteomyelitis rather than of neuropathic osteoarthropathy [ 12 ]  . 
nonetheless , some authors have found that neuropathy - related reactive bone marrow oedema often associated with trabecular microfractures and joint effusion may also enhance , thus becoming indistinguishable from osteomyelitis [ 13 ]  . 
 the differential diagnosis between osteomyelitis and neuropathic osteoarthropathy is not a problem when the skin is intact but only when stress - related biomechanical alterations promote skin ulceration and the foot appears swollen , warm and erythematous . 
 in the chronic phase of neuropathic osteoarthropathy , subchondral cysts , osteoporosis and bone fragmentation may appear , with loss of articular relationships and subsequent endosteal and periosteal reparative bone proliferation , which are easily detected even on conventional radiology . in these cases , mri can usually be interpreted correctly if compared with the radiographic pattern [ 1214 ]  . 
the ankle - arm index ( aai ) may overestimate systolic pressure owing to calcified sclerosis of the tunica media hindering compression of the arteries , transcutaneous oximetry may be misleadingly low due to the presence of oedema and , finally , colour doppler ultrasound may not allow adequate evaluation of the infrapopliteal arterial region , the most commonly affected by diabetic arterial disease [ 4 ]  . 
 in genere la rm permette di escludere con affidabilit losteomielite se negativa o se lalterazione del segnale osteo - midollare lontana dallulcera , ma non sempre sufficiente a formulare diagnosi di osteomielite quando positiva , anche se vi concordanza topografica tra i segni rm primari e quelli secondari . 
la somministrazione di mdc paramagnetico risulta utile per aumentare la specificit dellesame dato che lenhancement del tessuto osteo - midollare , valutato nelle sequenze t1 - pesate con selettiva soppressione del segnale adiposo , considerato tipico dellosteomielite pi che delle alterazioni dovute a osteo - artropatia neuropatica [ 12 ]  . 
tuttavia , alcuni autori hanno riscontrato la possibilit che in alcuni casi ledema midollare reattivo da neuropatia spesso associato a micro - fratture trabecolari e versamento articolare possa presentare enhancement , risultando indistinguibile dallosteomielite [ 13 ]  . 
 il problema della diagnosi differenziale tra osteomielite e osteo - artropatia neuropatica non si pone quando la cute integra , ma solo quando le alterazioni bio - meccaniche dovute al carico favoriscono lulcerazione della cute ed il piede appare tumefatto , caldo , eritematoso ; in tale fase acuta la neuro - artropatia pu simulare losteomielite sia dal punto di vista clinico che radiologico . 
pertanto , in tutti i casi con rm positiva , ma discordante dai dati clinici , colturali , laboratoristici e radiologici , necessario ricorrere alla biopsia ossea per conferma , utilizzando la guida fluoroscopica o tc per raggiungere la sede della alterazione del segnale rm , scegliendo un tragitto che attraversi la cute integra , al fine di evitare la possibile contaminazione ossea [ 9 ]  . 
 nellosteo - artropatia neuropatica fase cronica possono comparire cisti subcondrali , osteoporosi e frammentazione ossea con perdita dei rapporti articolari e successiva proliferazione ossea riparativa endo e periosteale , facilmente rilevabili gi allesame radiologico convenzionale . 
 le indagini strumentali vascolari pi comunemente utilizzate per lo screening dellarteriopatia periferica presentano alcuni limiti nel paziente diabetico con osteomielite del piede : lindice caviglia - braccio ( abi ) pu sovrastimare la pressione sistolica per la sclerosi calcifica della tunica media che rende le arterie non comprimibili , lossimetria trans - cutanea pu essere falsamente bassa per la presenza di edema in corso di infezione e , infine , leco - color doppler radiol med ( 2009 ) 114 : 121132 quently , mr angiography plays a crucial role in these highrisk patients , allowing a noninvasive panoramic study of the lower - limb arteries [ 15 ]  . 
the advantages of mr angiography over ct angiography are the low nephrotoxicity of paramagnetic contrast agents , the absence of exposure to ionizing radiation and easier and more accurate postprocessing , especially in patients with diffuse wall calcifications [ 16 ]  . 
 until recently before the implementation of parallel imaging that significantly reduces acquisition times one of the major limits of lower - limb mr angiography was the filling of the venous circulation at the infrapopliteal level , which rendered up to 43% of mr angiography studies nondiagnostic [ 17 ]  . 
there is evidence that cellulitis and ulcers are the only two circumstances capable of significantly accelerating the transit time of contrast material through the peripheral arteries [ 18 ]  . 
in our experience , within the limits shown in the infrapopliteal region , mr angiography proved adequate for planning surgical or endovascular treatment , allowing healing of the ulcer and infection or identification of the level of amputation for critical limb ischaemia . conclusions pu non consentire una adeguata valutazione del distretto arterioso infra - popliteo , il pi frequentemente colpito dallarteriopatia diabetica [ 4 ]  . 
dunque , langio - rm ha assunto un ruolo cruciale in questi pazienti ad alto rischio , consentendo la valutazione panoramica arteriosa degli arti inferiori in maniera non invasiva [ 15 ]  . 
i vantaggi dellangiorm rispetto allangio - tc sono rappresentati dalla bassa nefrotossicit dei mdc paramagnetici , dallassenza di esposizione radiante e dal post - processing pi facile ed accurato , specie nei pazienti con estese calcificazioni parietali [ 16 ]  . 
 fino a pochi anni fa , prima dellapplicazione dellimaging parallelo che permette di ridurre sensibilmente i tempi di acquisizione , uno dei limiti maggiori dello studio angio - rm degli arti inferiori era rappresentato dal riempimento del circolo venoso a livello infra - popliteo : fino al 43% degli esami angio - rm risultavano non diagnostici [ 17 ]  . 
nella nostra esperienza langio - rm , pur con i limiti evidenziati a livello del distretto infrapopliteo , risultata idonea a pianificare la strategia di trattamento chirurgico o endovascolare per permettere la guarigione dellulcera e dellinfezione o comunque a individuare il livello di amputazione nellischemia critica di arto . mri of the diabetic foot is highly sensitive in the study of osteomyelitis and infectious complications . 
bolus - chase mr angiography of the lower limbs is the preferred vascular study in diabetic patients for planning peripheral revascularization . inflammatory hyperaemia of the diabetic foot , however , affects visualization of the infrapopliteal arteries in around 40%50% of limbs with infected ulcers . 
in these cases , in the presence of known patency of the femoropopliteal axis , the use of selective arteriography is justified by the opportunity to perform angioplasty during the same session . conclusioni la rm del piede diabetico ha elevata sensibilit per losteomielite e per le complicanze infettive . 
nei casi dubbi la biopsia ossea con guida imaging resta lunica indagine in grado di discriminare i casi di falsa positivit alla rm dovuti a osteo - artropatia neuropatica di charcot . 
parisi1 1department of radiation oncology , scientific institute hospital casa sollievo della sofferenza , san giovanni rotondo , 71013 foggia , italy 2university of bari , department of radiology , bari , italy 3university of foggia , department of radiology , scientific institute hospital casa sollievo della sofferenza , san giovanni rotondo , 71013 foggia , italy correspondence to : g . 
a systematic review of external radiation therapy studies in urinary cancers was performed . this synthesis of the literature is based on data from metaanalyses , randomised and prospective trials and retrospective studies . 
there are several reports on multimodality treatment in invasive bladder cancer : intravesical surgery and neoadjuvant chemotherapy to radiotherapy or concomitant radiochemotherapy with organ preservation . the conclusions reached for renal cancer are controversial , and data on cancers of the urethra and ureter are few and inconclusive . 
sufficient data now exist in the literature to demonstrate that conservative management with organ preservation is a valuable alternative to radical cystectomy , the traditional gold standard , in invasive bladder cancer , keywords urinary cancers treatment radiotherapy ( rt ) chemotherapy ( ct ) organ preservation overall survival ( os ) riassunto i tumori invasivi del tratto urinario sono relativamente rari ed il loro trattamento pu causare importanti cambiamenti nelle funzioni sessuali , sociali ed urinarie . 
ci sono pochi studi clinici controllati che hanno utilizzato la radioterapia adiuvante o radicalechemioterapia nei tumori del rene , degli ureteri e delluretra ; ci sono diversi studi sul trattamento multimodale del carcinoma invasivo della vescica : chirurgia intravescicale e chemioterapia neoadiuvante alla radioterapia o radio - chemioterapia concomitante con preservazione dellorgano . 
le conclusioni raggiunte sul carcinoma renale sono controverse ; i dati sul carcinoma degli ureteri e delluretra sono pochi e inconclusivi ; invece , vi sono ora in letteratura dati sufficienti che dimostrano come lapproccio con preservazione dellorgano , per il carcinoma vescicale invasivo rappresenta una valida alternativa alla cistectomia radicale , considerata per lungo tempo il gold standard . parole chiave tumore urinario trattamento radioterapia ( rt ) chemioterapia ( ct ) preservazione dellorgano sopravvivenza globale ( sg ) radiol med ( 2009 ) 114 : 7082 introduction surgery is the primary therapeutic option in the curative treatment of clinically localized urinary cancer . 
preoperative irradiation with doses of 2040 gy has been used to facilitate resection , to sterilise well - oxygenated peripheral extensions of the tumour that might be transected during nephrectomy , and to decrease the chance of dissemination at the time of nephrectomy . 
no advantage has been demonstrated in patients receiving preoperative irradiation with doses of 2040 gy with respect to overall survival ( os ) or survival free from distant metastases ( ffdm ) , although improvement in resectability has been noted [ 16 ]  . 
two studies [ 10 , 12 ] have reported excessive bowel and hepatic complications and an irradiation - related mortality rate of 13%19% , explained in part by the fractionation scheme . 
doses of 4550 gy can be given , with acceptable bowel and hepatic complications ; however , a dose of 50 gy is suggested as being the tolerance limit for the upper abdominal gastrointestinal tract [ 13 ]  . 
a recent study [ 15 ] has shown that 5year disease - free survival ( dfs ) is 66% in the radiotherapy introduzione la chirurgia la principale opzione terapeutica nella cura del tumore dellapparato urinario clinicamente localizzato . ad ogno modo , sebbene questappoccio sia associato ad un eccellente controllo locale , non scevro dal rischio di complicanze e , a volte , di una scarsa qualit di vita . 
nel trattamento del cancro invasivo della vescica come alternativa alla cistectomia radicale immediata si pu utilizzare la resezione la resezione transuretrale , la chemioterapia e la radioterapia con cistectomia di salvataggio . 
la maggior parte dei pazienti trattati con terapia trimodale conserva una buona funzione vescicale . rene la nefrectomia radicale resta la sola terapia efficace per il carcinoma renale ( cr ) clinicamente localizzato . 
la rt pre - operatoria con dosi da 20 a 40 gy stata utilizzata per facilitare la resezione , per sterilizzare i margini periferici ben ossigenati del tumore che potrebbero essere sezionati durante la nefrectomia , e per ridurre la possibilit di disseminazione al momento della nefrectomia . 
nessun vantaggio stato dimostrato nei pazienti sottoposti a rt pre - operatoria con dosi da 20 a 40 gy per ci che riguarda la sopravvivenza globale ( sg ) o sopravvivenza libera da metastasi a distanza ( slmd ) , sebbene sia stato notato un miglioramento della resecabilit [ 16 ]  . 
in studi datati , la radioterapia postoperatoria ha dimostrato un vantaggio di circa il 15% sul controllo locale ( cl ) rispetto alla nefrectomia da sola [ 711 ] , ma con risultati insoddisfacenti sulla sopravvivenza . 
due studi [ 10 , 12 ] hanno riportato eccessive complicanze intestinali ed epatiche , ed una percentuale del 13%19% di mortalit legata alla rt , spiegata in parte dallo schema di frazionamento . 
dosi di 4550 gy possono essere erogate con complicanze intestinali ed epatiche accettabili ; comunque suggerita una dose di 50 gy come limite di tolleranza per il tratto gastrointestinale superiore [ 13 ]  . 
 [ 11 ] lirradiazione post nefrectomia con dose mediana di 46 gy ha ridotto la percentuale di recidive locali nei pazienti con tumore in stadio pt3 dal 37% ( nei pazienti non irradiati ) al 10 % ( nei pazienti irradiati ) ( p = 0 , 05 ) ; ma , in un aggiornamento del radiol med ( 2009 ) 114 : 7082 group and 16% in the no - treatment group and has shown reduction of local recurrence , with a significant difference in both univariate and multivariate analyses , although no benefit was noted in patients with earlier - stage disease . another recent study [ 16 ] concluded that adjuvant rt is unlikely to improve treatment outcome . 
however , these series suggest a benefit in patients with a high risk of local recurrence ( incomplete resection t3 / t4 stage , nodal metastases ) , but there is no advantage for os . 
the authors achieved an overall partial / complete response ( cr ) of 90%98% . this new radiation technique is a big challenge , because the side effects are few , and local control is high due to easy dose escalation . 
complete resection is the only known cure for renal cancer , and the value of rt is controversial . results are awaited of multi - institutional prospective randomised trials using modern rt techniques to evaluate the role of rt and its effect on survival , especially in selected patients with a high risk of local or regional failure . ureter and urethra carcinomas of the ureter and urethra are unusual diseases with insufficient clinical experience , and the role of rt in situ not well defined . 
 [ 20 ] reviewed the records of 31 patients with locally advanced transitional cell carcinoma of the renal pelvis and ureter who underwent surgery followed by adjuvant rt with or without concurrent chemotherapy ( ct ) [ methotrexate , cisplatin ( cddp ) and vinblastine ( mcv ) ]  . five - year os and disease - specific survival ( dss ) with adjuvant concurrent chemoradiotherapy ( ccrt ) ( cddp ) improved in patients with resected , locally advanced uppertract urothelial malignancies . 
 the results of 34 women with primary urethral carcinoma were retrospectively reviewed at princess margaret hospital [ 23 ] .these patients were treated with rt with or lavoro , la rt post - operatoria non ha migliorato la sopravvivenza ed ha mostrato tossicit [ 14 ]  . 
un recente studio [ 15 ] ha mostrato che la sopravvivenza libera da malattia ( slm ) a 5 anni del 66% nel gruppo sottoposto a rt e del 16% nel gruppo non sottoposto a rt ed ha mostrato una riduzione della recidiva locale , con una differenza significativa sia allanalisi univariata che multivariata ; ma non stato notato nessun beneficio nei pazienti in stadio iniziale di malattia . 
comunque , tali lavori hanno suggerito un beneficio nei pazienti ad alto rischio di recidiva ( resezione incompleta negli stadi t3 / t4 , metastasi linfonodali ) , ma nessun vantaggio riguardo alla sg . 
 [ 18 ] riguardo lutilizzo della rt stereotassica extracranica nei tumori renali primitivi e metastatici : gli autori hanno riportato il 90%98% di risposte parziali / complete ; questa nuova tecnica di irradiazione una grande sfida perch gli effetti collaterali sono pochi ed il cl alto , dovuto alla facilit di aumentare la dose erogata . 
attendiamo i risultati di studi prospettici randomizzati multi - istituzionali che utilizzano tecniche moderne di rt per valutare il ruolo ed i suoi effetti sulla sopravvivenza , specialmente in pazienti selezionati con alto rischio di recidiva locale o regionale . 
la sg e la ssm a 5 anni con chemio - rt concomitante e adiuvante ( ccrt ) sono migliorate nei pazienti con neoplasie resecate e localmente avanzate del tratto urinario inferiore . 
i pazienti con malattia avanzata trattati con chirurgia da sola hanno avuto una slm pi breve ( 23 , 3 mesi ) rispetto quelli trattati con combinazione di rt - ct ( 45 , 2 mesi )  . grigsby [ 22 ] ha analizzato i dati di 44 donne con carcinoma delluretra . 
brachytherapy reduced the risk of local recurrence , and the beneficial effect was most prominently seen in patients with bulky primary disease . in the conservative management of these tumours , the combination of external beam brachytherapy with or without ct play a significant role , and the multimodality approach appears to be the optimal way to treat these patients , with surgery to be used for biopsy - proven persistent tumours or recurrence [ 24 ]  . radiotherapy bladder radical cystectomy with bilateral pelvic lymph node dissection followed by urinary diversion remains the gold standard for treating muscle - invading bladder cancer and is the single most successful modality , reporting long - term os in the range of 40%60% [ 2528 ]  . 
rt is utilised as single modality treatment , as neoadjuvant or adjuvant to surgery , or combined with ct with or without conservative local surgery . i risultati di 34 pazienti con carcinoma uretrale sono stati analizzati in modo retrospettivo al princess margaret hospital [ 23 ] , trattati con rtbrachiterapia . 
 nella strategia terapeutica conservativa di questi tumori , la combinazione di rt e brachiterapia con o senza ct , riveste un ruolo significativo e , lapproccio multimodale sembra essere il modo ottimale per trattare questi pazienti , riservando la chirurgia per laccertamento istologico della persistenza o recidiva tumorale [ 24 ]  . vescica la cistectomia radicale con linfadenectomia pelvica bilaterale seguita da diversione urinaria rimane il gold standard per il trattamento del carcinoma della vescica invasivo ed la singola modalit pi vantaggiosa che ha riportato una sg a lungo termine del 40%60% [ 2528 ]  . 
such comparisons were very difficult , because patients selected for radical cystectomy had less advanced tumours at diagnosis , were younger and in a better general condition than patients selected for definitive irradiation . 
results reported in the literature are not significant : the 5 - year survival rate for patients treated with rt alone ( dose of 6066 gy at a 1.8to 2 - gy daily fraction ) is 32% ( range 22%50% ) [ 29 , 30 ]  . 
a recent review of treatment outcomes in rt - treated patients showed the following 5 - year survival rates : t1 , 35%71% ; t2 , 10%59% ; t3 , 10%38% ; and t4 , 0%16% [ 31 ]  . 
tali confronti sono molto difficoltosi perch i pazienti selezionati per la cistectomia radicale avevano tumori in stadio meno avanzato alla diagnosi , erano pi giovani ed in migliori condizioni generali rispetto ai pazienti selezionati per la rt radicale . 
in letteratura i risultati non sono significativi : la sopravvivenza a 5 anni per i pazienti trattati con rt da sola ( dosi di 6066 gy , con 1 , 82 gy come frazione giornaliera ) del 32% ( 22%50% ) [ 29 , 30 ]  . 
una review recente riguardo i risultati della terapia nei pazienti sottoposti a rt , ha mostrato la seguente incidenza di sg a 5 anni : t1 , da 35% a 71% ; t2 , da 10% a 59% ; t3 , da 10% a 38% e t4 da 0% a 16% [ 31 ]  . radioterapia pre - operatoria la pi ampia esperienza ( 338 pazienti ) con rt pre - operatoria ha ottenuto una sotto - stadiazione patologica nel 65% dei pazienti ed una risposta patologica completa nel 42% dei casi . 
la sopravvivenza a 5 anni dei pazienti che hanno ricevuto la rt seguita da cistectomia stata 38% vs radiol med ( 2009 ) 114 : 7082 followed by cystectomy was 38% vs 29% for those treated with rt alone ( not statistically significant )  . 
 strong support for the use of preoperative irradiation in patients with t3b bladder cancer was reported by cole et al . [ 34 ] : the 5 - year local control in the preoperative group was 91% compared with 72% for those treated with radical cystectomy alone . 
however , a meta - analysis of five randomised trials showed no statistical difference in treatment outcomes between patients treated with preoperative rt and cystectomy alone [ 35 ]  . 
 preoperative ccrt showed better results than rt alone . in fact , in the nordic cystectomy trial [ 37 ] , the combination of ct ( adriamycin + cddp for two cycles ) plus rt ( 20 gy in four fractions ) followed by cystectomy showed a 5 - year os improvement of 15% only for t3 - t4 disease compared with rt or surgery alone ( p = 0.03 ) , whereas no survival benefit was found for early stages of disease ( t1t2 )  . 
in the canadian randomised study [ 38 ] , concurrent cisplatin improved pelvic disease control with preoperative 29% per quelli trattati con rt da sola ( non statisticamente significativo , nss )  . 
 [ 34 ] : il cl a 5 anni nel gruppo sottoposto a rt preoperatoria stato del 91% rispetto al 72% per quelli trattati con cistectomia radicale e c stato anche un beneficio in termini di sg e slm ( nss )  . 
comunque , una metanalisi di 5 studi randomizzati non ha mostrato differenze statisticamente significative tra i pazienti trattati con rt pre - operatoria e quelli trattati con cistectomia radicale [ 35 ]  . 
una conclusione simile stata raggiunta in uno studio di fase iii su 140 pazienti , che confronta la chirurgia da sola con chirurgia e rt preoperatoria [ 36 ]  . la ccrt pre - operatoria ha mostrato migliori risultati rispetto alla rt da sola . 
infatti , nello studio nordico sulla cistectomia [ 37 ] la combinazione di ct ( adriamicina + cisplatino per 2 cicli ) pi rt ( 20 gy in 4 frazioni ) seguita da cistectomia ha mostrato un incremento della sg a 5 anni del 15% solo per i t3 - t4 rispetto alla rt o chirurgia da sola ( p = 0 , 03 ) , mentre nessun beneficio di sopravvivenza stato trovato negli stadi iniziali di malattia ( t1t2 )  . 
only one randomised study of radical cystectomy alone vs cystectomy and postoperative rt [ 41 ] reported a 5 - year pelvic recurrence of 5% ( 50% in the cystectomy arm ) and an improvement in dfs in patients with schistosomiasis . 
the main disadvantage of postoperative rt is the high rate ( 20%40% ) of serious late gastrointestinal complications [ 39 , 40 , 4244 ] because of the larger volume of small bowel occupying the pelvis after cystectomy . 
patients at high risk of recurrence are probably better treated with ct , which may prevent local and distant relapse and is usually well tolerated . bladder - preservation therapy the relative negative impact on quality of life for urinary diversion and the trend towards organ preservation led many authors to explore the role of conservative management . 
neoadjuvant ct compared with rt or surgery was investigated in an attempt to improve the results of surgery alone or definitive rt maintaining a functioning bladder . in the european organisation for research and treatment of cancer ( eortc ) / medical research council [ 45 ] trial , 976 patients undergoing curative cystectomy or radical rt were randomised to receive three cycles of neoadjuvant ct ( mcv ) in 491 patients or no ct in 485 . 
a recent meta - analysis [ 46 ] confirmed that neoadjuvant platinum - based combination ct reduces mortality risk of 13% , with moderate improvement ( 5% , p = 0.016 ) of 5 - year os , irrespective of the type of local treatment ( surgery or rt alone or rt and cystectomy ) and without differences between subgroups of patients . 
there was no evidence to support the use of single - agent platinum . these studies showed that patients treated with rt ( conservative approach ) have preserved bladder function . over the past 1520 years , several prospective trials have investigated conservative therapy in which all patients were treated with transurethral resection of bladder tumours ( turbt ) with or without ct followed by ccrt . 
alcuni studi hanno riportato una riduzione del rischio di recidive pelviche dal 30% al 50% al 10% al 20% [ 3941 ] , ma alcuni pazienti hanno anche ricevuto rt pre - operatoria . 
solo uno studio randomizzato sulla cistectomia radicale da sola verso cistectomia e rt postoperatoria [ 41 ] , ha riportato recidive pelviche a 5 anni del 5% ( 50% nel braccio della cistectomia ) ed un incremento della slm nei pazienti con schistosomiasi . 
il principale svantaggio della rt post - operatoria lalta percentuale ( 20%40% ) di gravi complicanze gastrointestinali tardive [ 39 , 40 , 4244 ] , poich una gran parte di volume dellintestino tenue occupa la pelvi dopo cistectomia . 
i pazienti ad alto rischio di recidiva sono probabilmente trattati in modo migliore con la ct , che pu prevenire recidive locali e a distanza ed di solito ben tollerata . 
 terapia di preservazione della vescica il relativo impatto negativo sulla qualit della vita per la diversione urinaria e la tendenza verso la preservazione dellorgano , ha permesso a molti autori di esplorare il ruolo di un trattamento conservativo . 
 nello studio delleortc ( european organisation for research and treatment of cancer ) / medical research council [ 45 ] , 976 pazienti sottoposti a cistectomia curativa o rt radicale sono stati randomizzati a ricevere 3 cicli di ct neoadiuvante ( mcv , metotressato , cddp , vinblastina ) in 491 pazienti , oppure nessuna ct in 485 pazienti . 
la differenza assoluta tra i gruppi per quanto riguarda la sg a 3 anni del 5 , 5% ( 55 , 5% per ct vs 50% nessuna ct ) non risultata statisticamente significativa ( p = 0 , 075 ) : la ct neoadiuvante stata associata con una patologica completa pi alta , ma non c stato nessuna chiara evidenza sulla sopravvivenza . 
una recente metanalisi [ 46 ] ha confermato che la combinazione di ct neoadiuvante a base di platino riduce il rischio di mortalit del 13% , con un incremento moderato ( 5% , p = 0 , 016 ) della sg a 5 anni , indipendentemente dal tipo di trattamento locale ( chirurgia o rt da sola o rt e cistectomia ) , e senza differenze tra i sottogruppi di pazienti , non vi stata alcuna evidenza a favore di platino da solo . 
 negli ultimi 1520 anni , diversi studi prospettici hanno analizzato terapie conservative in tutti quei pazienti trattati radiol med ( 2009 ) 114 : 7082 patients with cr , a consolidation rt or ccrt regimen was administered . 
 since 1985 , radiation therapy oncology group ( rtog ) trials have used a trimodality conservative treatment ( turbt , rt , ct ) in patients with t2t4a muscleinvasive bladder cancer who were candidates for cystectomy . 
in three rtog trials [ 4749 ] , patients were treated with neoadjuvant turbt and ccrt ( conventional or altered fractionation ) , whereas in two trials [ 50 , 51 ] , patients were treated with induction by turbt with or without ct and ccrt . 
cystectomy was reserved for incompletely responders or relapse . in the rtog 89 - 03 phase iii study , the combination of neoadjuvant ct ( mcv2 cycles ) with ccrt with cddp failed to show significant benefits in terms of 5 - year local control ( 61% vs 49% ) , os ( 48% vs 49% ) and ffdm ( 33% vs 39% ) compared with ccrt alone , with more toxicity in the mcv arm [ 51 ]  . 
in fact , subsequent studies by the rtog emphasised the impact of adjuvant ct [ mcv or cddp + 5fluorouracil ( fu ) ] following rt ( hypoor hyperfractionated ) [ 49 ] , with good results at 3 years . 
from paris began a prospective trial of preoperative ct using 5 - fu and cddp with concomitant rt ( 3 gy b.i.d. , total dose 24 gy ) , followed by either cystectomy or additional ccrt . 
this treatment strategy resulted in a pathological cr of 77% and may be proposed as conservative treatment in patients with a cr to the initial course of chemoradiation [ 54 ]  . 
globally , os at 5 years was 51% . similar results were obtained in further series of combined modality treatment for bladder preservation with excellent cr ( ~70% ) , and 5 - year os ( ~50% ) , with bladder con resezione transuretrale della vescica ( turbt ) ct seguita da ccrt , e per i pazienti con risposta completa stata somministrata una rt di consolidamento o ccrt ; la cistectomia stata riservata per le risposte incomplete o per le recidive di malattia . 
 dal 1985 , gli studi dellrtog ( radiation therapy oncology group ) hanno utilizzato un trattamento conservativo trimodale ( turbt , rt , ct ) nei pazienti con carcinoma della vescica t2t4a , che erano candidati alla cistectomia . in tre studi dellrtog [ 4749 ] i pazienti sono stati trattati con turbt neoadiuvante e ccrt ( convenzionale o frazionamenti alterati ) ; mentre in 2 studi [ 50 , 51 ] i pazienti sono stati trattati con induzione mediante turbtct e ccrt ; la cistectomia stata riservata per le risposte incomplete o in caso di recidive . 
 nello studio di fase iii dellrtog 89 - 03 , la combinazione di ct neoadiuvante ( mcv2 cicli ) con ccrt mediante cddp non ha mostrato alcun beneficio significativo in termini di cl a 5 anni ( 61% vs 49% ) , sg ( 48% vs 49% ) e slm ( 33% vs 39% ) , rispetto alla ccrt da sola , con una maggiore tossicit nel braccio che ha usato lmcv [ 51 ]  . 
infatti gli studi successivi dellrtog hanno puntualizzato limpatto della ct adiuvante ( mcv o cddp + 5 - fu ) alla rt ( ipoo iper - frazionamenti ) [ 49 ] , con buoni risultati a 3 anni . 
 [ 54 ] di parigi , hanno iniziato uno studio prospettico di ct preoperatoria usando 5 - fu e cddp con rt concomitante ( 3 gy bid , dose totale di 24 gy ) , seguita sia da cistectomia o ccrt addizionale . 
questa strategia di trattamento ha determinato una risposta patologica completa nel 77% dei casi , e pu essere proposta come terapia conservativa nei pazienti con risposta completa al ciclo iniziale di ccrt [ 54 ]  . 
 [ 55 ] , nella quale 415 pazienti sono stati trattati dopo la turbt con rt da sola ( 126 pazienti ) o in concomitanza con cddp ( 240 pazienti ) o cddp + 5 - fu ( 49 pazienti ) , con percentuali di risposte complete migliori per rt + cddp - 5 - fu ( 87% ) rispetto al cddp ( 66% ) o rt da sola ( 61% ) ; globalmente , la sg a 5 anni risultata pare a 51% . risultati simili sono stati ottenuti in ulteriori studi con modalit di trattamento combinato per la conservazione della vescica , con eccellenti risposte complete ( ~70% ) , ed una sg a 5 anni ( ~50% ) , con conservazione dellorgano in radiol med ( 2009 ) 114 : 7082 table 1 series of combined modality treatment for bladder preservation series no . 
 [ 70 ] turbt 24 gy at 3 gy + cddp / fu 2 additional cycles of crt ( dose not given ) ( specific cancer ) ng ( overall rate of cystectomy : 25.6% ) danesi et al . 
table 1 summarises the results of these studies [ 5674 ]  . in a recent meta - analysis of fifteen radiation series with different fractionation schedules and total doses , pos et al . [ 75 ] found evidence for an overall doseresponse relationship with an increase in local control by a factor of 1.441.47 for an increment in dose of 10 gy . 
 [ 71 ] recently reported the long - term results of a phase i / ii trial of ct ( mcv ) followed by concomitant cddp + 5 - fu and hyperfractionated irradiation ( three 100cgy fractions / day , for a total dose of 69 gy )  . 
accelerated rt may overcome radioresistance due to tumour - cell repopulation , but a recent randomised trial [ 77 ] reported no improvement of local control or survival rates and increased acute bowel complications . 
there has been only one small phase iii study of curative rt in which survival was inferior in the hypofractionated arm [ 78 ]  . some trials , in an attempt to improve results , explore the use of new chemotherapeutic agents and accelerated or hyperfractionated rt to increase irradiation dose in combination with ct . 
new chemotherapeutic agents , in particular gemcitabine and taxanes , are effective in combination with cddp and rt ( paclitaxel and gemcitabine are good radiosensitisers ) [ 61 , 79 ]  . 
a recent italian study [ 68 ] reported the feasibility of a combined scheme with gemcitabine ( up to 400 mg / m2 ) plus cddp and rt , obtaining 100% cr . 
finally , the rtog 99 - 06 trial introduced concomitant paclitaxel + cddp plus bifractionated rt as induction treatment , followed by adjuvant ct ( gemcitabine and cddp ) in patients with cr ; no specific outcomes are yet available [ 80 ]  . 
in una meta - analisi recente su 15 studi di rt con differenti schemi di frazionamento e dose totale , pos et al . [ 75 ] , hanno evidenziato una relazione globale dose - risposta , con incremento del cl , di un fattore che va da 1 , 44 a 1 , 47 per un aumento di dose di 10 gy . 
una meta - analisi di stuschke e thames [ 76 ] ha indicato che i regimi iper - frazionati mostrano un aumento della sg ( odd ratio di mortalit 0 , 55 ; p = 0 , 002 ) ed una risposta completa ( odd ratio di fallimento di 0 , 44 ; p = 0 , 001 )  . 
 [ 71 ] , recentemente hanno riportato risultati a lungo termine di uno studio di fase ii / ii di ct ( mcv ) seguita da ct a base di cddp + 5 - fu concomitante e rt iper - frazionata ( 1 gy per 3 frazioni al giorno , per una dose totale di 69 gy )  . 
le percentuali a 5 anni di sg , conservazione della vescica , ssm , slm e sopravvivenza senza cistectomia per tutti e 77 i pazienti sono state 58 , 8% , 46 , 6% , 75% , 53 , 5% e 76 , 1% , rispettivamente . 
la rt accelerata pu superare la radioresistenza generata dal ripopolamento delle cellule tumorali , ma in uno studio recente randomizzato [ 77 ] , non riportato alcun incremento delle percentuali del lc o della sg ma sono aumentate le complicanze intestinali acute . 
 alcuni studi , nel tentativo di migliorare i risultati , hanno esplorato luso di nuovi agenti chemioterapici e rt accelerata o iper - frazionata per aumentare la dose di rt in associazione con la ct . 
nuovi agenti chemioterapici , in particolare gemcitabina e taxani , sono efficaci in combinazione con cddp e rt ( paclitaxel e gemcitabina sono buoni radiosensibilizzanti ) [ 61 , 79 ]  . 
un recente studio italiano [ 68 ] , ha riportato la fattibilit di uno schema combinato con gemcitabina ( fino 400 mg / m2 ) pi cddp e rt , ottenendo il 100% di risposta completa . 
infine , lrtog 99 - 06 ha introdotto ct concomitante a base di paclitaxel + cddp pi rt bifrazionata come trattamento di induzione , seguito da ct adiuvante ( gemcitabina e cddp ) nei pazienti con risposta completa ; risultati specifici non sono ancora disponibili [ 80 ]  . conclusions conclusioni radical nephrectomy is the only effective therapy for clinically localised renal cancer . 
la cistectomia di salvataggio usata in caso di risposta parziale dopo terapia di induzione ccrt ( rtog ) o recidiva radiol med ( 2009 ) 114 : 7082 response after induction ccrt treatment ( rtog ) or recurrent disease ( european trials ) : in both cases , surgery can be curative , reporting a 5 - year survival of approximately 50% [ 45 , 50 , 51 , 54 , 55 ]  . 
 trimodality treatment ( turbt , rt , ct ) of invasive bladder cancer may be offered as a valid alternative to radical cystectomy , with no decrease in survival related to the delay in cystectomy , which is currently the gold standard therapy . 
os is comparable with recent surgical data , reporting a 5 - year os of almost 50% , with bladder preservation in three fourths of these patients . di malattia ( studi europei ) : in entrambi i casi la chirurgia pu essere curativa , riportando una sopravvivenza a 5 anni del 50% approssimativamente [ 45 , 50 , 51 , 54 , 55 ]  . 
 il trattamento trimodale ( turbt , rt , ct ) del carcinoma invasivo della vescica pu offrire una valida alternativa alla cistectomia radicale con nessuna diminuzione della sopravvivenza correlata al ritardo della cistectomia , che rimane attualmente la terapia gold standard . 
san giovanni battista , c.so bramante 88 , 10126 turin , italy 3radiology service , ospedale santa croce e carle , via coppino 26 , 12100 cuneo , italy correspondence to : c . 
le indicazioni alla procedura erano : sanguinamento digestivo ( 52 , 8% ) , ascite refrattaria ( 35 , 3% ) , tutela della perviet portale pre - trapianto epatico ( 3 , 0% ) , trombosi delle vene sovraepatiche ( 2 , 3% )  . 
durante un follow - up di 1500 giorni , lincidenza cumulativa di reintervento sugli stent stata del 25 , 7% ( nitinol ) , 32 , 9% ( wallstent ) e 1 , 8% ( palmaz ) ; la mortalit stata rispettivamente 31 , 1% , 38 , 5% e 56 , 4% . 
tips placement is safe and effective and may act as a bridge to liver transplantation . ultrasonography plays a fundamental role in the preliminary assessment , in portal vein puncture and during the follow - up . 
 parole chiave tips radiologia interventistica insufficienza epatica ipertensione portale keywords tips interventional radiology liver failure portal hypertension introduction introduzione transjugular intrahepatic portosystemic shunt ( tips ) insertion was introduced into clinical practice more than 20 years ago for the treatment of portal hypertension and its complications . 
a number of controlled trials have compared the efficacy of tips versus medical and endoscopic treatment in preventing variceal bleeding and treating refractory ascites , to date the universally accepted indications for tips [ 17 ]  . 
these include portal thrombosis , budd - chiari syndrome , pleural ascites [ 810 ] , hepatopulmonary syndrome ( hps ) [ 11 ] , hepatorenal syndrome ( hrs ) [ 3 , 1217 ] , prophylaxis of intraoperative bleeding in cirrhotic patients with portal hypertension who are candidates for major abdominal surgery [ 8 , 18 , 19 ] and maintenance of portal vein patency prior to orthotopic liver transplantation ( olt ) [ 20 , 21 ]  . we retrospectively reviewed a large series of patients who underwent tips over a 15 - year period at a referral centre and compared the data with the literature with the aim of providing useful radiological and clinical information for multidisciplinary management of cirrhotic patients with complicated portal hypertension . da oltre 20 anni lo shunt intraepatico porto - sistemico ( tips ) stato introdotto nella pratica clinica per il trattamento dellipertensione portale e delle sue complicanze . numerosi trials controllati hanno comparato lefficacia della tips con la terapia medica ed endoscopica nella profilassi del sanguinamento da varici e nel trattamento dellascite refrattaria , che rimangono le principali indicazioni , univocamente condivise [ 17 ] ; le linee guida alla procedura sono state tracciate sulla base di aspetti tecnici angiografici , fattori prognostici clinico - laboratoristici ed incidenza di complicanze . 
indicazioni pi rare ed oggetto di studi non controllati condotti su piccoli gruppi di pazienti , alcune delle quali non uniformemente accettate sono : la trombosi portale , la sindrome di budd - chiari , lascite pleurica [ 810 ] , la sindrome epato - polmonare ( hps ) [ 11 ] , la sindrome epato - renale ( hrs ) [ 3 , 1217 ] , la profilassi del sanguinamento intraoperatorio in pazienti cirrotici con ipertensione portale candidati ad interventi di chirurgia addominale maggiore [ 8 , 18 , 19 ] e la tutela della perviet portale pre - trapianto epatico ( olt ) [ 20 , 21 ]  . scopo di questo lavoro analizzare retrospettivamente , confrontandoli con una revisione della letteratura , i dati di unampia casistica di pazienti sottoposti a tips nellarco di quindici anni presso un centro di riferimento al fine di fornire informazioni tecniche radiologiche e cliniche utili nella gestione multidisciplinare del paziente cirrotico con ipertensione portale complicata . 
 materials and methods between march 1992 and december 2006 , 658 patients ( 432 males and 226 females , age range 485 years , mean age 56.2 years ) with cirrhosis and portal hypertension of varying aetiology ( table 1 ) were referred to our centre for tips placement . 
fourteen patients had previously undergone liver transplantation and were being treated for materiali e metodi dal marzo 1992 al dicembre 2006 sono giunti al nostro istituto per essere sottoposti a tips 658 pazienti , 432 maschi e 226 femmine , det compresa tra 4 e 85 anni , con et media di 56 , 2 anni . 
le indicazioni alla tips sono riassunte in tabella 2 . radiol med ( 2009 ) 114 : 8394 table 1 aetiology of cirrhosis infectious , viral ( hcv , hbv ) alcoholic cryptogenic budd - chiari syndrome biliary cirrhosis number hcv , hepatitis c virus ; hbv , hepatitis b virus tabella 1 eziologia della cirrosi infettiva virale ( hcv , hbv ) alcolica criptogenica sindrome di budd - chiari cirrosi biliare hcv , virus dellepatite c ; hbv , virus dellepatite b refractory ascites ( n = 6 ) , pleural ascites ( n = 4 ) , bleeding ( n = 2 ) and recurrent budd - chiari syndrome ( n = 2 )  . all patients referred electively to our centre had already undergone clinical assessment with echocardiography and portal ultrasonography ( us )  . 
tips procedures were performed in the angiography suite with the following x - ray systems : angio diagnost 3 dvi , multi diagnost 3 and allura xper fd20 ( all philips medical systems , best , the netherlands )  . 
in the case of elective procedures , patients signed the informed consent form and were acute bleeding recurrent bleeding refractory ascites budd - chiari syndrome pleural ascites hepatorenal syndrome hepatopulmonary syndrome pre - olt pre - surgery partial thrombosis of the portal quattordici pazienti erano gi stati sottoposti a trapianto epatico e sono stati trattati per : ascite refrattaria ( 6 casi ) , ascite pleurica ( 4 casi ) , sanguinamento ( 2 casi ) e recidiva di sindrome di budd - chiari ( 2 casi )  . 
per le procedure eseguite in regime di elezione il giorno antecedente lesame il paziente ha firmato un consenso informato ed stato visitato dallanestesista ; sono stati valutati lemocromo e lo screening emocoagulativo completo ed stata intrapresa una profilassi antibiotica ad ampio spettro . 
attraverso un introduttore vascolare posizionato in vena giugulare interna di 10 f di calibro e 35 cm di lunghezza , con lausilio di un catetere angiografico di 4 f e 65 cm di lunghezza ( terumo corporation , tokyo , giappone ) ed una guida idrofila con punta j di 0 , 035 pollici e 180 cm di lunghezza ( terumo corporation , tokyo , giappone ) stata incannulata la vena sovraepatica destra ( 521 pazienti ) o mediana ( 128 pazienti )  . 
all patients underwent complete blood count and coagulation screening tests and received broad - spectrum antibiotics . patients vital signs were monitored continuously throughout the procedure , and an anaesthetistresuscitator was present during all angiographic manoeuvres . 
after placing a 35 - cm - long , 10 - f vascular introducer in the internal jugular vein , the right ( 521 patients ) or middle ( 128 patients ) hepatic vein was cannulated with the aid of a 65cm - long , 4 - f angiographic catheter ( terumo corporation , tokyo , japan ) and a 180 - cm - long , 0.035 - j - tip hydrophilic guidewire ( terumo corporation , tokyo , japan )  . 
us , performed with a 3.5 - mhz convex - array probe and intercostal scans enabled us to withdraw the needle tip in the hepatic vein until a portal branch of adequate size and with favourable anatomical relationships could be identified , and subsequently to guide advancement of the obturator in real time . 
in 520 cases , the portal system was accessed via the right portal vein , in 85 cases via the left portal vein and in 46 cases via the bifurcation . after removing the needle obturator under us and fluoroscopic guidance , the hydrophilic guidewire was advanced to the portal trunk and a 110 - cm - long , 4 - f pigtail catheter was introduced into the guidewire . 
portal ( with the catheter tip in the trunk ) and systemic ( from the side of the vascular introducer sheath with the catheter tip in the hepatic vein ) pressure measurements were then obtained , and subsequently portography was performed after injecting 25 ml of contrast material ( ultravist 370 , shering ag , berlin , germany ) into the splenic vein at a flow rate of 8 ml / s with an automatic injector . 
the parenchymal tract was then dilated until complete distension by using an 8 - mm angioplasty balloon catheter of varying length ( 48 cm ) ( wanda , boston scientific , natick , ma , usa ; smash , boston scientific ; xxl , boston scientific )  . 
subsequently , the palmaz ( mechanically expandable ) , memotherm and luminexx ( nitinol ) , wallstent and wallgraft ( self - expandable ) stents ( table 3 ) were deployed and dilated to approximately 10 mm , except in patients scheduled for olt . 
lecografia , effettuata con sonda convex di 3 , 5 mhz in scansioni intercostali , ha permesso di arretrare in vena sovraepatica la punta dellago sino a visualizzare un ramo portale di calibro adeguato e con rapporti anatomici favorevoli , e successivamente di guidare in tempo reale lavanzamento del mandrino . 
si effettuata la manometria portale ( con apice del catetere posto a livello del tronco ) e sistemica ( dalla via laterale dellintroduttore vascolare con apice in vena sovraepatica ) ; la successiva portografia stata ottenuta infondendo in vena splenica , con lausilio di iniettore automatico , 25 ml di mdc ( ultravist 370 , shering ag , berlin , germania ) , con un flusso di 8 ml / s . 
utilizzando un catetere a palloncino da angioplastica ( wanda , boston scientific corporation , natick , ma , usa ; smash , boston scientific corporation , natick , ma , usa ; xxl boston scientific corporation , natick , ma , usa ) da 8 mm e lunghezza variabile ( 48 cm ) si proceduto quindi alla dilatazione del tramite parenchimale fino alla sua completa distensione . 
sia la puntura portale che la dilatazione del tragitto sono state eseguite con adeguata sedazione ( remifentanyl 0 , 05 - 0 , 1 kg / min e propofol 1 mg / kg / ora )  . 
successivamente si proceduto al rilascio ed alla dilatazione degli stent palmaz ( espandibili meccanicamente ) , memotherm e luminexx ( nitinol stent ) , wallstent e wallgraft ( autoespandibili ) ( tabella 3 )  . 
le trombosi portali sono state trattate con angioplastiche ripetute , utilizzando palloni di diametro variabile tra 8 mm e 14 mm ; tentativi di fibrinolisi con urochinasi ( 200000 ui a bolo seguite da 1 milione di ui nelle successive 24 ore ) sono stati inefficaci . 
 si sono eseguiti infine un controllo flebografico , con i flussi gi descritti , ed una rilevazione delle nuove pressioni portale e sistemica : lopacizzazione esclusiva dei rami portali intraepatici principali e la devascolarizzazione delle varici in corso di portografia insieme ad un gradiente porto - sistemico minore od uguale a 10 mmhg sono stati considerati deponenti per il buon successo emodinamico della procedura . in 45 pazienti ( 6 , 8% ) , di cui 15 sottoposti a tips in regime durgenza , si deciso di eseguire lembolizzazione selettiva di varici gastro - esofagee persistenti con spirali radiol med ( 2009 ) 114 : 8394 repeated angioplasty procedures using 8to 14 - mm balloons . 
attempts at fibrinolysis with urokinase ( 200 , 000 iu as a bolus followed by 1 million iu over 24 h ) were ineffective . finally , postprocedural phlebography was performed with the flow rates described above , and the new portal and systemic pressures were measured : exclusive opacification of the main intrahepatic portal branches and variceal devascularisation at portography along with a portosystemic gradient 10 mmhg were considered evidence of haemodynamic success . in 45 patients ( 6.8% ) , 15 of whom were emergency referrals , we decided to perform selective embolisation of persistent gastro - oesophageal varices with metal coils ( balt , montmorency , france ; cook europe ) measuring 10 mm and 16 mm in diameter . metalliche ( balt , montmorency , francia ; cook , bjaeverskov , danimarca ) di 10 mm e 16 mm di diametro . stato considerato successo tecnico il corretto posizionamento e la regolare perviet dello stent , con rapida opacizzazione dellatrio destro in corso di flebografia . 
in 7 pazienti non si potuto portare a termine la tips per occlusione inveterata delle vene sovraepatiche , per progressione intraepatica della trombosi portale o per rapporti anatomici sfavorevoli tra i rami portali e le vene sovraepatiche . 
in 1 caso di precoce occlusione della tips pluri - recidivata si ottenuta la perviet dello shunt posizionando coassialmente agli stent gi in sede un ulteriore protesi ricoperta ( wallgraft , boston scientific corporation , natick , ma , usa ) di 12 mm di calibro . 
in seven patients , tips placement could not be completed owing to chronic occlusion of the hepatic veins due to intrahepatic progression of portal thrombosis or because of unfavourable anatomical relationships between the portal and hepatic veins . within 48 h of the angiographic procedure , abdominal us was carried out to exclude any possible complications . in one case of recurrent early occlusion of tips , stent patency was achieved by inserting an additional 12 - mmcalibre covered stent coaxially to the previously deployed stents ( wallgraft , boston scientific )  . patients subsequently underwent us every 6 months , or more frequently if clinically indicated . 
in patients treated for hepatic vein thrombosis or hepatopulmonary syndrome , the stent had to be revised within the first year after insertion in 40% and 66.7% of cases , respectively . 
1 si evince inoltre come la mortalit sia stata elevata e precoce ( 56 , 4% , 43 , 9%68 , 7% con ci del 95% ) per i pazienti trattati con stent di palmaz , mentre inferiore , distribuita nel tempo e similare per le tips eseguite con protesi termoespandibili ( 31 , 1% , 24 , 2%37 , 8% con ci del 95% ) ed autoespandibili ( 38 , 5% , 20 , 8%56% con ci del 95% )  . 
 il successo clinico , considerando che 51 pazienti sono andati allolt dopo un periodo medio dalla tips di 6 mesi , stato ottenuto nel 100% dei pazienti con trombosi portale ( regressione dellascite e detensione delle varici ) o candidati alla gastrectomia ( assenza di cospicua emorragia intraoperatoria ) , nel 91% dei sanguinamenti ( stabilit emodinamica ) considerando che 19 / 76 ( 25% ) dei pazienti trattati in urgenza sono deceduti entro 30 giorni dalla tips , nel 60% delle asciti refrattarie ( ascite minima o assente , blanda terapia diuretica ed assenza di paracentesi ) , nel 70% dei pazienti con hrs ( sospensione emodialisi e miglioramento della creatininemia ) , nel 65% delle asciti pleuriche ( incremento dei volumi polmonari e migliore ventilazione ) , nel 47% delle s . 
1 analisi statistica della perviet degli stent e della mortalit dei pazienti . achieved in 100% of patients with portal thrombosis ( ascites regression and variceal devascularisation ) or candidates for gastrectomy ( no substantial intraoperative haemorrhage ) ; 91% of cases of bleeding ( haemodynamic stability ) , considering that 19 / 76 ( 25% ) emergency referrals died within 30 days of tips , 60% of patients with refractory ascites ( minimal or absent ascites , mild diuretic therapy and no paracentesis ) , 70% of patients with hrs ( discontinuation of haemodialysis and improved serum creatinine ) , 65% of cases of pleural ascites ( increased respiratory volumes and improved ventilation ) , 47% of cases of budd - chiari syndrome ( regression of ascites with significant reduction of liver volume ) and 20% of patients with hps ( significant increase in oxygen saturation )  . none of the procedure - related complications proved fatal or required surgical repair . 
si sono verificati : 6 insufficienze cardiache da sovraccarico destro , 6 emobilie , 3 migrazioni accidentali in arteria polmonare di stent palmaz , 2 ematomi intraepatici , 1 emoperitoneo . 
gli ematomi intraparenchimali , verificatisi in pazienti assai scoagulati forse per puntura accidentale di rami arteriosi , non hanno indotto particolare sintomatologia e / o anemizzazione , sono stati diagnosticati dallecografia addominale precoce post - procedura e si sono risolti spontaneamente . 
lemoperitoneo si verificato nelle ore successive alla tips durgenza per stravaso di una varice gastrica persistente non embolizzata in radiol med ( 2009 ) 114 : 8394 haematoma ( n = 2 ) and haemoperitoneum ( n = 1 )  . 
intraparenchymal haematomas , occurring in heavily anticoagulated patients perhaps due to accidental puncture of arterial branches gave rise to no specific symptoms or anaemia , were diagnosed on early postprocedural abdominal us and resolved spontaneously . 
the six cases of haematobilia were caused by accidental puncture of the gall bladder or highorder bile duct branches and were diagnosed on us within 24 h of the procedure ; 3 / 6 patients received blood transfusion . 
in 43 / 651 patients ( 6.6% ) treated for bleeding or refractory ascites , pse resolved following the tips procedure . discussion clinical aspects in our experience , oesophageal , gastric and intestinal bleeding is the most frequent indication for tips placement . 
a number of studies have shown that tips improves the survival rates of patients with massive and / or recurrent haemorrhage against a rebleeding rate at 2 years of 23%40% [ 24 ] , which is mostly promoted by venous systemic pressure exceeding 15 mmhg , stent malfunction and continuing alcohol consumption [ 25 ]  . 
there is no consensus regarding the treatment of varices during tips placement , which can be achieved by several means : sclerosing agents , acrylic glues , coils and the amplatzer vascular plug [ 26 ]  . 
whereas several authors advocate the systematic occlusion of portosystemic collateral vessels [ 27 ] , others report a 65% rate of rebleeding in patients with correctly embolised varices [ 28 ]  . 
our policy of embolising in patients with previous only persistent varices haematemesis after adequate reduction of the portosystemic gradient ( 10 mmhg ) allowed the rate of rebleeding to be maintained < 10% . 
our data confirm the high rate of presenza di sonda emostatica endoscopica ; la complicanza stata sospettata clinicamente , confermata da una tc e trattata con embolizzazione endovascolare selettiva mediante spirali metalliche . 
i 6 casi di emobilia si sono verificati per puntura accidentale della colecisti o di dotti biliari di maggior ordine e sono stati diagnosticati dallecografia a 24 ore dalla procedura ; 3 / 6 pazienti sono stati emotrasfusi . lencefalopatia porto - sistemica ( pse ) comparsa dopo la tips in 77 / 651 pazienti ( 11 , 8% ) ; lincidenza della complicanza stata particolarmente elevata nei casi di ascite pleurica ( 4 / 12 pazienti , 33 , 3% )  . 
non vi unanime consenso circa il trattamento delle varici in corso di tips , attuabile con differenti presidi : sclerosanti , colle acriliche , spirali ed amplatzer vascular plug [ 26 ] ; alcuni autori sostengono lefficacia dellocclusione sistematica dei collaterali porto - sistemici [ 27 ] , mentre altri lavori riportano un 65% di recidiva di sanguinamento in pazienti con varici correttamente embolizzate [ 28 ]  . 
il nostro atteggiamento di embolizzare , in presenza di adeguata riduzione del gradiente porto - sistemico ( 10 mmhg ) , soltanto le varici persistenti di pazienti con pregressi episodi di ematemesi ha consentito di contenere la recidiva di emorragia al di sotto del 10% . 
i nostri dati confermano lelevata mortalit precoce dei pazienti trattati in urgenza , che giungerebbero comunque allexitus in oltre l80% dei casi di sanguinamento non controllato [ 29 ]  . 
 lascite refrattaria , correlata a prognosi sfavorevole oltre che ad incrementato rischio di infezioni ed insufficienza renale [ 30 ] , rappresenta nella nostra casistica la seconda principale indicazione alla tips . 
il risultato clinico della tips , nella nostra ed altrui esperienza [ 31 ] , non influenzato dalla funzionalit epatica basale del paziente e non sono stati dimostrati fattori prognostici positivi certi . 
 riguardo allimpiego della tips nel trattamento radiol med ( 2009 ) 114 : 8394 early deaths among emergency referrals , 80% of whom , however , would have died in any case due to uncontrolled bleeding [ 29 ]  . refractory ascites , correlated to poor outcome and increased risk of infection and kidney failure [ 30 ] , was the second leading indication for tips in our series . 
the clinical outcome of tips , in our experience and that of other authors [ 31 ] , is unrelated to liver function at baseline , and no certain positive prognostic factors have been identified to date . with regard to the use of tips in the treatment of hepatic hydrothorax , our results were similar in terms of clinical outcome [ 32 , 33 ] and pse incidence with those of other nonrandomised , uncontrolled studies [ 34 ]  . 
because the waiting list for olt is relatively short at our hospital ( 6 months on average ) , it was jointly decided to dilate the stent to 8 mm only in order to reduce the risk of liver failure . 
there is evidence that tips may reduce the mortality and morbidity associated with olt [ 21 ]  . no randomised controlled trials exist that establish a protocol for the treatment of budd - chiari syndrome . 
in patients unresponsive to anticoagulation therapy , tips can reduce portal hypertension , improve liver function and control ascites [ 3 ]  . the only established treatment for hps is liver transplantation , which may , however , be risky or contraindicated in cases of severe hypoxaemia [ 11 ]  . 
the therapeutic efficacy of the intrahepatic shunt is still controversial , with some authors suggesting that the extent of arteriovenous pulmonary fistulas before treatment and the portosystemic gradient after tips are the main determinants of outcome . the unsatisfactory clinical impact observed in out series may be explained by the comorbidities affecting some of our patients ( two cases of emphysema , one of a previous specific process ) , whereas we are unable to account for the high incidence of stent revisions . in our patients who received lactulose to regulate bowel function , the pse induced by tips was mostly transient and mild to moderate and rarely required an angiographic reduction procedure . 
pse regression following tips in patients treated for bleeding or intractable ascites may be explained dellidrotorace epatico il nostro studio si allinea per risultato clinico [ 32 , 33 ] ed incidenza di pse con i dati di altri lavori non randomizzati e non controllati [ 34 ] ; in molti casi lo shunt intraepatico rappresenta una soluzione ponte allolt , senza il quale questi pazienti giungono allexitus per lo pi per complicanze correlabili alla progressione dellinsufficienza epatica [ 35 ]  . 
 nei pazienti in lista olt il confezionamento della tips ha garantito la perviet portale ; in considerazione del fatto che presso il nostro centro i pazienti giungono allolt in tempi brevi ( mediamente 6 mesi ) stato deciso collegialmente di dilatare lo stent a soli 8 mm , per ridurre il rischio di insufficienza epatica . 
lefficacia terapeutica dello shunt intraepatico tuttora dibattuta ; alcuni autori suggeriscono come lentit delle fistole artero - venose polmonari prima del trattamento ed il gradiente porto - sistemico post - tips siano i fattori prognostici pi rilevanti . linsoddisfacente impatto clinico emerso nella nostra esperienza potrebbe essere spiegabile con le comorbilit di cui alcuni dei nostri pazienti erano affetti ( 2 casi di enfisema , 1 caso di pregresso processo specifico )  . 
 nei pazienti da noi trattati , con alvo regolato da lattulosio , la pse indotta dalla tips , nella gran parte dei casi temporanea e di grado lieve o moderato , ha reso di rado necessario lintervento angiografico di riduzione . 
la regressione della pse dopo la tips in pazienti trattati per sanguinamento o ascite intrattabile spiegabile rispettivamente con il cessato assorbimento intestinale delle proteine ematiche e con il sensibile decremento della terapia diuretica . 
 i casi di scompenso cardiaco destro da sovraccarico provocati dalla tips ci hanno indotto a sottoporre ad ecocardiogramma tutti i pazienti candidati alla procedura . radiol med ( 2009 ) 114 : 8394 by discontinued intestinal absorption of blood proteins and by a significant reduction of diuretic treatment , respectively . the cases of right - sided cardiac failure following tips induced us to perform echocardiography on all candidates for the procedure . aspetti tecnici technical aspects us enabled correct patient selection , was effective in guiding the portal puncture and helped to avoid major periprocedural complications [ 36 ]  . 
the cases of intraparenchymal haematoma and haematobilia occurred at the end of particularly complex procedures , and their onset was promoted by the blood dyscrasia typical of cirrhosis . pre - tips radiological assessment , in some cases supplemented by a ct study , provides essential information about the nature , extent and age of the portal thrombus and on the haemodynamics of the splenomesenteric system . our experience confirms previously reported findings [ 37 ] regarding the greater technical difficulty of angiographic procedures , sometimes requiring transcaval puncture , and the high incidence of occlusions at 1 year when patients with budd - chiari syndrome are treated with bare stents . 
this finding suggests that us should be performed more frequently every 34 months . the case of haemoperitoneum emphasises the need for accurate phlebography following tips , with injection of contrast material into the proximal splenic vein in order to demonstrate any persistent varices . in our experience , the patency of self - expandable and thermosensitive bare stents was satisfactory but lower than that of modern polytetrafluoroethylene ( ptfe ) - covered stents [ 38 ]  . 
this type of stent - graft , which has inherent technical limitations , was used in the early years of our experience and was characterised by less accurate patient selection , less technical skill of the interventional radiologists and organisational problems in referring patients for olt . conclusions tips placement is a safe and effective first - line procedure to treat complications of portal hypertension that are refractory to medical and endoscopic therapy . 
 la valutazione radiologica pre - tips , talvolta completata da approfondimento tc , fornisce indispensabili informazioni circa la natura , lestensione e let del trombo portale e sullemodinamica del sistema spleno - mesenterico . 
 la nostra esperienza conferma i riscontri della letteratura [ 37 ] circa la maggior difficolt tecnica delle procedure angiografiche , con necessit talvolta di puntura transcavale , e circa lelevata incidenza di occlusione entro 1 anno degli stent nudi nel trattamento di pazienti con sindrome di budd - chiari . 
 il caso di emoperitoneo rimarca la necessit di eseguire un attento studio flebografico post tips con iniezione di mdc in vena splenica prossimale , al fine di dimostrare eventuali varici persistenti . 
la performance dei nitinol stent e degli stent autoespandibili risulta sovrapponibile , a fronte di una mortalit simile . lincidenza cumulativa di reinterventi degli stent palmaz risultata bassa solo perch la mortalit ad essi associata stata molto elevata ; questi tipi di protesi , che presentavano limiti tecnici intrinseci , sono state impiegate nei primi anni del nostro studio caratterizzati da una meno precisa valutazione dei criteri di elezione alla procedura , da una minor abilit tecnica degli radiologi interventisti e dalla difficolt organizzativa di avviare il paziente allolt . 
the literature indicates that modern covered stents ensure better primary patency than do bare stents and that their higher cost is offset by the fewer angiographic revisions required during the follow - up . although there is consensus regarding the effectiveness of tips in treating variceal bleeding , refractory ascites and hydrothorax despite the increased incidence of pse there is still controversy about the use of tips to treat rarer and more complex conditions , such as budd - chiari syndrome , hrs and hps . finally , in some patients , tips can serve as an effective bridge to olt . 
dalla letteratura emerge come i moderni stent ricoperti garantiscano una maggiore perviet primaria rispetto alle protesi nude , a fronte di un costo maggiore , compensato tuttavia dal minor numero di revisioni angiografiche in corso di follow - up . vi unanime consenso circa lefficacia della tips nel trattamento del sanguinamento da varici , dellascite refrattaria e dellidrotorace , a fronte di unincrementata incidenza di encefalopatia porto - sistemica . 
ancora incerti e dibattuti rimangono i risultati del trattamento mediante tips di quadri clinici pi rari e complessi come la sindrome di budd - chiari , la hrs e la hps . in alcuni pazienti infine la tips pu essere considerata unefficace soluzione temporanea in attesa dellolt ; i tempi di attesa di ogni singolo centro trapianti condizionano indicazioni ed aspetti tecnici della procedura . 
fava1 1istituto di radiologia universit di torino sede san luigi , orbassano , italy 2sc radiodiagnostica i e ii ospedale san giovanni battista , torino , italy 3sc radiodiagnostica ospedale s . 
ninety - eight patients with cerebral tumours , 46 of which were primary ( seven grade 0 - i , nine low - grade gliomas , two gliomatosis cerebri , nine lymphomas and 19 high - grade gliomas ) and 52 secondary , underwent conventional magnetic resonance ( mr ) imaging completed with dwi and dynamic contrast susceptibility pwi . 
seventeen of 98 tumours were cystic or necrotic ( 12 / 17 hypointense and 5 / 17 hyperintense on dwi ) ; the adc value of hyperintense cystic areas was 0.970.2310 - 3 mm2 / s . 
novantotto pazienti con tumore cerebrale , 46 con tumore primitivo ( 7 di grado 0 - i , 9 gliomi a basso grado , 2 gliomatosi cerebri , 9 linfomi , 19 gliomi ad alto grado ) e 52 con metastasi sono stati sottoposti ad esame rm convenzionale completato da acquisizione dwi e da studio perfusionale ottenuto durante la somministrazione di un bolo di gadolinio . 
diciassette tumori erano costituiti da una componente centrale cistica che in 5 casi era iperintensa in dwi ( valore medio adc 0 , 970 , 2310 - 3 mm2 / s )  . ladc della parte solida dei tumori aveva valori compresi tra 0 , 64 e 3 , 510 - 3 mm2 / s . 
la pwi fornisce elementi utili nella diagnosi differenziale tra gliomi a basso ed alto grado e tra gliomi ad alto grado e linfomi . parole chiave risonanza magnetica diffusione perfusione tumori cerebrali introduction introduzione the role of conventional magnetic resonance ( mr ) imaging , with or without contrast agent administration , in the diagnosis of brain tumours is widely accepted . 
mr imaging not only has high sensitivity in detecting and locating lesions , it also gives information about phenomena such as mass effect , haemorrhage , oedema or necrosis , thus helping to differentiate brain tumours from other pathological processes and providing indications about the type of tumour [ 1 , 2 ]  . the importance of characterising tumours ( typing and grading ) to plan appropriate treatment and monitor response to treatment early on in the disease has stimulated research in this area , with numerous studies investigating , over the past decade , the application of new techniques such as diffusion - weighted imaging ( dwi ) , perfusion - weighted imaging ( pwi ) and in vivo mr spectroscopy ( mrs ) [ 14 ]  . the term diffusion refers to the random ( brownian ) movement of water molecules in tissues ; the diffusion capacity of water molecules is tissue - specific and may be altered in given pathological conditions . 
dwi enables assessment of this tissue - related parameter , and calculation of the apparent diffusion coefficient ( adc ) provides major diagnostic clues for evaluating intracranial masses [ 2 , 5 ]  . perfusion reflects the delivery of nutrients to tissue and is defined as flow per unit of time . 
the grey matter of a healthy individual is perfused at a rate of 5060 ml / 100 g / min and the flow maintained in a restricted range by cerebral self - regulation mechanisms . 
low - perfusion states may thus lead to cellular ischaemia , whereas high - perfusion states may be related to hypervascular lesions such as certain tumours [ 6 ]  . the most commonly measured perfusion parameters are cerebral blood volume ( cbv ) , which reflects the amount of blood present in a given amount of tissue at a given time ; cerebral blood flow ( cbf ) , which indicates the quantity of blood transiting through a given amount of tissue in a unit of time ; and mean transit time ( mtt )  . 
these parameters are closely linked to one another , with cbf being equal to the il ruolo della risonanza magnetica ( rm ) convenzionale , con e senza mezzo di contrasto ( mdc ) , nella diagnosi dei tumori dellencefalo saldamente consolidato ; la rm non solo possiede unelevata sensibilit nel riconoscimento e nella localizzazione delle lesioni , ma fornisce informazioni su fenomeni quali leffetto massa , lemorragia , ledema o la necrosi dei tessuti che aiutano nella diagnosi differenziale con altri tipi di processi patologici dando anche indicazioni sul tipo di tumore [ 1 , 2 ]  . limportanza della caratterizzazione ( tipo e grading ) dei tumori , al fine della corretta impostazione terapeutica , nonch lutilit di fornire precoci informazioni sulla risposta alla terapia ha stimolato in questi ultimi anni la ricerca e , nellultima decade , sono stati pubblicati numerosi studi riguardanti limpiego delle nuove tecniche quali la diffusione ( dwi ) , la perfusione ( pwi ) e la spettroscopia in vivo ( mrs ) nella caratterizzazione dei tumori cerebrali [ 14 ]  . con il termine diffusione si indica il movimento random ( browniano ) delle molecole di acqua nei tessuti ; la diffusibilit delle molecole dellacqua caratteristica per ogni tessuto e si pu modificare in determinate condizioni patologiche . 
le sequenze pesate in diffusione permettono di valutare questo parametro tessutale ed il calcolo del coefficiente di diffusione apparente ( adc ) fornisce elementi diagnostici importanti nella valutazione delle masse intracraniche [ 2 , 5 ]  . la perfusione rappresenta lapporto nutritivo ematico ai tessuti e viene definita come il flusso per unit di tempo . solitamente viene misurata in ml / 100 g di tessuto / min , o unit di cbf . 
la sostanza grigia delluomo sano viene perfusa ad un ritmo di 5060 ml / 100 g / min e viene mantenuta in un range ristretto dai meccanismi di autoregolazione cerebrale . 
perci stati di bassa perfusione possono sfociare nellischemia cellulare , mentre stati di elevata perfusione possono essere correlati a lesioni ipervascolari , come alcuni tumori [ 6 ]  . i parametri che pi frequentemente vengono misurati sono : il cbv ( cerebral blood volume ) che equivale alla radiol med ( 2009 ) 114 : 645659 ratio between volume and transit time [ 7 ]  . 
the most frequently used parameter in brain tumour imaging is the relative regional cerebral blood volume ( rrcbv ) , which is increased in tumours as a result of neoangiogenesis [ 8 , 9 ]  . this study correlated the adc and rrcbv values obtained by applying echoplanar ( epi ) and gradient - echo ( gre ) sequences in a large series of brain tumours with histological findings to evaluate the usefulness of this technique in characterising brain tumours . materials and methods in this prospective study conducted between january 2003 and august 2006 , patients with brain tumours were studied with conventional mr imaging complemented by dwi and pwi . 
from among these , 98 patients ( 53 men and 45 women ) were selected on the basis of the following exclusion criteria : lack of histological diagnosis , nonneoplastic lesions , previous treatments on the tumours ( surgery , chemotherapy , radiotherapy ) , incorrect examination technique . all lesions were characterised histologically . 
tumours were subdivided into primary and secondary ; the former were grouped by histological type and grade according to the world health organization ( who ) classification , whereas the latter were classified according to the tumour of origin . study protocol mr imaging was performed during a single session in all patients by using a superconducting 1 - tesla scanner ( signa horizon , ge ) with a maximum gradient strength of 23 mt / m and a slew rate of 77 mt / m / s . 
we used 1 m gadobutrol ( gadovist ) injected intravenously with the aid quantit di sangue presente in una data quantit di tessuto in un dato momento ; il cbf ( cerebral blood flow ) che indica la quantit di sangue che attraversa una data quantit di tessuto nellunit di tempo e lmtt ( mean transit time ) o tempo di transito medio . 
il parametro pi utilizzato nello studio dei tumori cerebrali il volume ematico regionale relativo ( rrcbv ) che aumenta nei tumori in rapporto ai fenomeni di neoangiogenesi [ 8 , 9 ]  . in questo lavoro vengono correlati i valori di adc e di rrcbv , ottenuti su unampia serie di tumori cerebrali utilizzando sequenze echo planari ( epi ) gradient echo ( gre ) , con il relativo dato isto - patologico , per valutare lapporto di questa tecnica nella caratterizzazione dei tumori cerebrali . materiali e metodi nel periodo compreso tra gennaio 2003 e agosto 2006 stato effettuato uno studio prospettico che prevedeva di completare lesame rm convenzionale con acquisizioni dwi e pwi nei pazienti con tumore cerebrale . 
tra questi sono stati selezionati 98 pazienti ( 53 maschi e 45 femmine ) utilizzando i seguenti criteri di esclusione : assenza della diagnosi istologica , lesioni non neoplastiche , pregressi trattamenti sul tumore ( chirurgia , chemioterapia , radioterapia ) , non corretta esecuzione dellesame . tutte le lesioni sono state caratterizzate istologicamente ; i tumori sono stati suddivisi in primitivi e secondari ; i primi sono stati raggruppati per istotipo e grado secondo la classificazione della who , mentre i secondi sono stati classificati sulla base del tumore dorigine . protocollo di studio la rm stata eseguita in tutti i pazienti in ununica seduta utilizzando un magnete superconduttivo operante a 1 tesla ( signa horizon , ge ) con potenza massima dei gradienti di 23 mt / m e slew - rate di 77 mt / m / s . 
 stata impiegata la bobina head di ricezione e trasmissione in quadratura . in tutti i pazienti stato eseguito un esame standard con impiego di sequenze se t1 ( tr / te 500 / 12 ms ) , fse pesate in t2 ( tr / te 4000 / 85 ms etl 13 ) e flair ( tr / te 8000 / 90 ms ti 2000 ms )  . 
sono state ottenute immagini sui tre piani dello spazio con sezioni di 45 mm e gap 0 , 51 mm . prima della somministrazione del mdc stata eseguita in tutti i casi una sequenza pesata in diffusione , sul piano 648 radiol med ( 2009 ) 114 : 645659 of a dual - syringe automatic injector ( medrad ) via an 18gauge needle cannula at a flow rate of 5 ml / s . 
in all cases , a few minutes before the dynamic phase , patients were administered 0.05 mmol / kg of gd as a prebolus to attenuate the t1 effects of bloodbrain barrier ( bbb ) disruption . to study the entire area of the lesion , we acquired a set of 12 slices repeated sequentially 40 times , thus obtaining 480 images in 1 min 12 s . 
all axial images were acquired with the same geometry ( fov , slice thickness , gap , angle ) to enable fusion of the dw and pw images with the corresponding flair and / or contrastenhanced t1 - weighted images . 
adc maps inclusive of the adc calculation were generated by positioning 25to 30 - mm2 regions of interest ( roi ) over the tumour areas . the mean adc values of solid and fluid components were obtained separately for each lesion . pw images were processed by applying a formula to calculate , starting from the baseline t2 value of tissues , the drop in signal during the passage of the bolus ( r2 ) and its subsequent return to the baseline level : r2 * = - 1n [ s ( t ) / s ( 0 ) / te ] where s ( t ) corresponds to the signal intensity at time ( t ) , s ( 0 ) represents the mean mr signal before the arrival of the contrast and te to echo time . 
mathematical integration of the area under r2 * curve ( negative enhancement integral ) corresponds to the cbv . to obtain cbv maps , a number of rois 2530 mm2 in diameter were placed over the tumour in such a way as to cover the entire lesion , and the enhancing and nonenhancing areas of tumour were evaluated separately . 
for each lesion , we calculated the mean rcbv of pathological tissue ( rcbvp ) and normal tissue ( rcbvn ) and the ratio between the two means , thus obtaining a value that we called rrcbv [ 10 ]  . 
stato utilizzato il gadobutrolo 1 m ( gadovist ) , iniettato endovena tramite lausilio di un iniettore automatico a doppia siringa ( medrad ) utilizzando unagocannula 18 g con velocit di flusso 5 ml / s . 
la dose somministrata stata per ogni paziente di 0 , 15 mmol / kg , seguita da 20 ml di soluzione fisiologica ; la fase dinamica stata sempre preceduta di pochi minuti dalla somministrazione di un prebolo di 0 , 05 mmol / kg di gadolinio per attenuare gli effetti t1 di alterazione della barriera ematoencefalica ( bee )  . sono state impostate 12 slices in modo da studiare interamente larea lesionale ripetute in maniera sequenziale per 40 volte ottenendo cos 480 immagini in un 112 . 
le prime 4 sequenze ( 48 slices ) , acquisite prima dellinizio della somministrazione del mdc per raggiungere lo stato di steady - state , sono state escluse dalle successive rielaborazioni . 
tutte le immagini assiali sono state acquisite con la stessa geometria : fov , spessore di strato , gap , inclinazione , per permettere la fusione delle immagini acquisite in dwi e pwi con le relative immagini flair e / o t1 post contrasto . 
dopo linfusione del mdc , sono state effettuate scansioni sui tre piani dello spazio ( coronale , sagittale ed assiale ) con sequenze se t1 - pesate . elaborazione delle immagini sono state successivamente rielaborate sia le immagini dwi sia quelle pwi utilizzando , sulla consolle operativa o sulla workstation ( adw 4.0 ) , un software fornito dalla ge ( functool 2 )  . 
per ogni lesione stata ottenuta separatamente la media delladc relativa alle componenti solide e a quelle fluide . le immagini pwi sono state elaborate applicando una formula che , partendo dal valore t2 di base dei tessuti , calcola la caduta di segnale durante il passaggio del bolo ( r2 ) e il successivo ritorno alla stato di partenza . r2 * = - 1n [ s ( t ) / s ( 0 ) / te ] dove s ( t ) corrisponde al segnale al tempo ( t ) ed s ( 0 ) rappresenta la media del segnale rm prima dellarrivo del contrasto e te il tempo di echo . 
the students t test for unpaired data was applied to the mean values obtained ; values of p < 0.0001 were considered to be statistically significant . results of the 98 patients , 46 had primary brain tumours and 52 had one or more secondary lesions ( table 1 )  . 
 of all tumours examined , 17 were morphologically composed of a necroticcystic central component surrounded by a strongly enhancing solid peripheral rim . the necroticcystic component was hypointense on dwi in 12 tumours and hyperintense in five . 
per ogni lesione stata calcolata la media del rcbv del tessuto patologico ( rcbvp ) e del tessuto sano ( rcbvn ) ed stato calcolato il rapporto tra le due medie ottenendo un valore che chiameremo rrcbv [ 10 ]  . 
ai valori delle medie cos ottenute stato applicato il test t di student per dati non appaiati ; sono stati considerati significativi valori di p < 0 , 0001 . risultati dei 98 pazienti 46 erano portatori di un tumore cerebrale primitivo e 52 di una o pi localizzazioni secondarie ( tabella 1 )  . tra tutti i tumori esaminati 17 erano costituiti morfologicamente da una componente centrale necrotico - cistica con spesso cercine periferico solido dotato di intenso contrast enhancement . 
the rrcbv values of the low - grade ( ii ) and high - grade ( iii - iv ) gliomas , as well as the values of highgrade gliomas and lymphomas , were analysed with students t test for unpaired data . 
the results are shown in tables 2 and 3 . twenty - nine of the brain metastases were from lung cancer , whereas the others were distributed as follows : seven from cancers of the uropoietic system ( six renal carcinomas and one bladder carcinoma ) , seven from breast cancer , four from gastrointestinal cancers , one from melanoma and three from lymphoma . 
a scansione assiale t1 pesata dopo somministrazione del mdc : lesione cistica con orletto iperintenso e cospicuo edema digitato della sostanza bianca perilesionale ; b analoga scansione in dwi : la lesione appare iperintensa ; c mappa adc : il valore di adc allinterno della componente cistica della lesione di 0 , 910 - 3 mm2 / s . 10 13 16 19 22 25 28 31 34 37 40 43 46 49 n pazienti metastasi tumori primari fig . 
2 rappresentazione grafica della distribuzione dei valori di adc nei vari tipi di tumori , distinti tra tumori cerebrali primitivi e metastasi . 652 radiol med ( 2009 ) 114 : 645659 fig . 
a contrast - enhanced t1 - weighted image ; b curves of signal decrease in the regions of interest ; c relative regional cerebral blood volume ( rrcbv ) maps . 
5 confronto dellrrcbv tra gliomi grado iii e grado iv . glioma di iv grado glioma di iii grado linfomi cerebrali gliomi ad alto grado 2 3 4 9 10 10 12 13 14 15 16 17 18 19 pazienti fig . 
i valori di rrcbv relativi ai gliomi a basso grado ( ii ) e quelli ad alto grado ( iii - iv ) , cos come i valori dei gliomi ad alto grado e quelli dei linfomi sono stati sottoposti a test statistici utilizzando la t di student per dati non appaiati ed i risultati ottenuti vengono riportati nelle tabelle 2 e 3 . ventinove delle metastasi encefaliche erano da carcinoma polmonare , mentre le altre erano cos ripartite : 7 da tumori dellapparato uropoietico ( 6 carcinomi renali e 1 carcinoma della vescica ) , 7 da carcinoma mammario , 4 da neoplasie dellapparato digerente , 1 da melanoma e 3 da linfoma . 
in the majority of cases , morphological imaging is able to differentiate low - grade from diffusely invasive or frankly malignant lesions , which tend to show significant perilesional oedema and intense contrast enhancement . radiol med ( 2009 ) 114 : 645659 i tumori neuroepiteliali , in particolare i glomi , rappresentano una causa frequente di tumori dellencefalo . 
gli esami radiologici morfologici sono in grado di differenziare nella maggior parte dei casi le lesioni di basso grado da quelle diffusamente infiltranti o chiaramente maligne che presentano generalmente un significativo edema perilesionale ed unelevata impregnazione contrastografica . 
ci nonostante , non tutte le neoplasie gliali ad alto grado presentano necrosi o effetto massa e circa il 40 % non presenta impregnazione contrastografica [ 2 , 11 ]  . il problema si complica se teniamo conto di altri due elementi : in primo luogo dellelevata frequenza delle metastasi cerebrali che si sviluppano in circa il 15%25% di tutti i pazienti con neoplasie maligne [ 12 ] e in secondo luogo dellaumentata incidenza dei linfomi cerebrali primitivi verificatasi nelle ultime due decadi per aumento della popolazione immucompromessa in relazione allincremento del numero di soggetti sottoposti a trapianto dorgano e di quelli con aids [ 13 , 14 ]  . la rm convenzionale dimostra molti limiti nella differenziazione tra i diversi gradi di tumori primari neuroepiteliali , le metastasi e i linfomi . 
nevertheless , not all high - grade glial tumours exhibit necrosis or mass effect , and approximately 40% of them are nonenhancing [ 2 , 11 ]  . the issue becomes more complex if we consider two other factors : first , the high frequency of cerebral metastases occurring in around 15%25% of all patients with malignancies [ 12 ] , and second , the rising incidence over the past two decades of primary cerebral lymphomas as a result of the increasing number of immunocompromised subjects due to organ transplantations and aids [ 13 , 14 ]  . conventional mr imaging has many limitations in distinguishing among the different grades of primary neuroepithelial tumours , metastases and lymphomas . 
for this reason , it is important to have access to a diagnostic imaging technique capable of enabling the rapid and noninvasive diagnosis and tumour monitoring [ 13 , 15 ]  . diffusion imaging by evaluating the mobility of water molecules in tissue , diffusion imaging can provide diagnostic information in several pathological conditions . 
in cerebral masses , the role of dwi and adc mapping in differentiating cystic tumours from abscesses and epidermoid from arachnoid cysts is known and well established [ 1618 ]  . the five cystic tumours with hyperintense fluid component on dwi resembled abscesses in appearance , although the adc values were slightly higher [ 18 , 19 ]  . 
the high mucinous content explains the adc restriction [ 1820 ]  . the adc of the solid component of the tumours showed wide variations without significant differences among the various types of tumour . 
in our series , the mean adc for lymphomas did not exceed 1.410 - 3 mm2 / s , a finding in agreement with previous reports [ 2 , 4 , 16 , 21 ]  . 
in fact , the extent of oedema strongly affects adc values , and it is likely that steroid treatment , frequently initiated at presentation in patients with brain tumours , influences the dwi parameters [ 22 ]  . perfusion imaging brain tumours may stimulate the formation of new vascular networks , and neoangiogenesis is fundamental for tumour growth . 
the increased metabolic demands of the growing tumour and the resulting hypoxia in large tumours stimulate fornire informazioni diagnostiche in svariate condizioni patologiche nel caso di masse cerebrali noto e consolidato il ruolo delle sequenze dwi e delle mappe delladc nel differenziare i tumori cistici dagli ascessi e le cisti epidermoidi dalle cisti aracnoidee [ 1618 ]  . i 5 tumori cistici con componente fluida iperintensa in dwi avevano un aspetto simile a quello degli ascessi , ma con valori di adc lievemente superiori [ 18 , 19 ]  . 
erano tutte metastasi da carcinoma polmonare ; allesame istologico larea cistica di questi tumori era costituita da tessuto mucinoso nel quale galleggiavano cellule neoplastiche , mentre non era evidenziabile contenuto ematico ; lelevata componente mucinosa spiega la restrizione del coefficiente di diffusione apparente [ 1820 ]  . per quanto concerne ladc delle componenti solide dei tumori i dati ottenuti dimostrano unampia variabilit di valori senza significativa differenza tra i vari tipi di neoplasie ; nella maggior parte dei casi tuttavia i valori di adc erano superiori rispetto a quelli del tessuto sano normale . 
dalla osservazione dei dati emerge che tra tutti i tipi tumorali i linfomi sono quelli che mostrano i valori complessivamente inferiori ; in questa casistica la media di adc per i linfomi non supera mai il valore di 1 , 410 - 3 mm2 / s ; questo concorda con i dati della letteratura [ 2 , 4 , 16 , 21 ]  . 
in realt lentit delledema condiziona molto i valori di adc ed probabile che il trattamento con farmaci corticosteroidei , frequentemente adottato gi allesordio della clinica nei pazienti con tumori cerebrali , influisca sui parametri ottenuti in dwi [ 22 ]  . imaging di perfusione i tumori cerebrali possono indurre la formazione di nuovi vasi ; la neoangiogenesi fondamentale per la crescita dei tumori . 
la prima tecnica , detta spin label , ancora in fase di studio e prevede di marcare i globuli rossi durante il passaggio nei vasi epiaortici e successivamente di valutarne il primo passaggio nel tessuto cerebrale [ 23 ]  . 
i mdc esogeni impiegati sono i chelati del gadolinio che non superano normalmente la bee e quindi si comportano come 656 radiol med ( 2009 ) 114 : 645659 the formation of cytokines responsible for angiogenesis [ 1 ]  . this process is closely related to biological aggressiveness and is one of the main parameters considered in the histopathological grading of glial tumours [ 1 ]  . 
the first technique , known as spin labelling , is still under development and involves tagging red blood cells during their passage in the epiaortic vessels and subsequently evaluating their first pass within cerebral tissue [ 23 ]  . 
the contrast agent compartmentalised inside vessels causes , on first pass when concentration is high , a drop in the t2 signal , which is best appreciated in t * gradient - echo sequences that are most heavily affected by magnetic susceptibility effects . 
studies comparing different gd concentrations have demonstrated that at the same dose levels , the use of 1 m gd increases the first - pass magnetic susceptibility effect , leading to curves with higher peaks [ 2426 ]  . one of the problems arising when evaluating concentration curves with dsci is leakage of contrast material into the extravascular space , which occurs in pathological conditions accompanied by bbb abnormalities . 
this problem can be overcome by administering a small dose of contrast material ( 0.05 mmol / kg ) approximately 5 min before initiating perfusion imaging [ 6 , 8 , 27 ]  . because the rcbv values obtained by applying the negative enhancement integral are not absolute , a standard reference is required to enable comparisons among different patients . 
this could be the rcbv obtained at the level of the contralateral white matter , or ipsilateral normal white matter in the case of lesions affecting both cerebral hemispheres . this provides the rrcbv value , which is calculated with the formula rrcbv = rcbvp / rcbvn [ 10 ]  . the results obtained in our series allow us to make a few concluding remarks . 
if we consider glial tumours , there are statistically significant differences ( p < 0.0001 ) between the rrcbv value of lowand high - grade gliomas ( table 2 )  . 
il mdc compartimentalizzato allinterno dei vasi determina , al primo passaggio , quando la concentrazione elevata , un abbattimento del segnale t2 , meglio valutabile utilizzando sequenze gradient - echo t2 * , che maggiormente risentono degli effetti di suscettibilit magnetica ; questa tecnica denominata con lacronimo dsci ( dynamic contrast susceptibility imaging ) [ 6 , 23 ]  . vengono abitualmente utilizzate sequenze epi che permettono di studiare un esteso volume cerebrale ed offrono il vantaggio di unelevata risoluzione temporale . 
in questo studio stato utilizzato gadolinio ( gd ) alla concentrazione 1 m somministrato a 5 ml / s con una dose pari 0 , 15 mmol / kg ; diversi studi condotti comparando concentrazioni diverse di gadolinio , hanno dimostrato che limpiego di gd 1 m , a parit di dose iniettata , aumenta leffetto di suscettibilit magnetica al primo passaggio per cui si ottengono curve con picco pi elevato [ 2426 ]  . uno dei problemi che insorge nella valutazione delle curve di concentrazione con effetto dsci rappresentato dallo stravaso di mdc nello spazio extravascolare che si verifica in quelle condizioni patologiche che si accompagnano ad alterazione della bee . 
un artificio per ovviare a questo problema quello di somministrare una piccola dose di mdc ( 0 , 05 mmol / kg ) circa 5 minuti prima dellacquisizione perfusionale [ 6 , 8 , 27 ]  . i valori di rcbv , ottenuti applicando lintegrale di enhancement negativo , non sono assoluti ; pertanto , per poter effettuare il confronto tra pazienti diversi , necessario utilizzare un riferimento standard che pu essere rappresentato dal valore di rcbv ottenuto a livello della sostanza bianca controlaterale alla lesione o della sostanza bianca sana omolaterale in caso di lesioni che interessano entrambi gli emisferi cerebrali . 
si ottiene in tal modo lrrcbv che si calcola con formula rrcbv = rcbvp / rcbvn [ 10 ]  . la seguente dai risultati ottenuti in questa casistica si possono trarre alcune considerazioni . 
se si considerano i tumori della serie gliale emergono delle differenze statisticamente significative ( p < 0 , 0001 ) del valore di rrcbv tra gliomi a basso grado e gliomi ad alto grado ( tabella 2 )  . 
pertanto se si fa la media del rcbv posizionando roi allinterno di tutto il tumore , ne deriva una riduzione complessiva dei valori di rrcbv nei tumori pi necroradiol med ( 2009 ) 114 : 645659 neoangiogenesis [ 28 ]  . 
all lymphomas were characterised by a low rrcbv value , constantly lower than 2 ( the maximum value observed was 1.53 ) , whereas the majority of high - grade gliomas had high rrcbv values . 
the differential diagnosis between lymphomas and gliomatoses is , however , straightforward at conventional mr imaging and based on morphology and tumour extension [ 30 , 31 ]  . lowest rrcbv values seen with regard to metastases , no statistically significant differences were seen in the perfusion values of the various types of metastases . 
the high rrcbv values seen in meningiomas , however , are in line with previous reports [ 15 , 26 ]  . conclusions diffusionand perfusion - weighted mr imaging in patients with brain tumours provide additional information that tici . 
per questo motivo stato ipotizzato di utilizzare oltre allimaging convenzionale anche le mappe di rrcbv come guida per effettuare le biopsie stereotassiche [ 29 ]  . se si confrontano i gliomi ad alto grado con i linfomi , per i quali la diagnosi differenziale con lrm convenzionale pu talvolta essere problematica , possiamo vedere come in questo studio i valori di perfusione diano risultati statisticamente significativi ( p < 0 , 0001 )  . 
tutti i linfomi erano caratterizzati da un valore di rrcbv basso , sempre inferiore a 2 ( il valore massimo riscontrato stato di 1 , 53 ) , mentre la maggior parte dei gliomi ad alto grado aveva valori di rrcbv elevati . 
la possibilit di discriminare tra tumori gliali ad alto grado e linfomi concorda con quanto gi riportato da altri studi [ 15 , 20 ]  . un discorso a parte va fatto per le gliomatosi cerebri . 
in questo studio sono compresi solo due casi , ed entrambi hanno bassi valori di rrcbv , simili a quelli dei linfomi e dei tumori low - grade ; questo dato correla con la ridotta microvascolarizzazione di questi tumori il cui grading elevato dato dalla elevata estensione . 
la diagnosi differenziale tra linfomi e gliomatosi tuttavia agevole allo studio rm convenzionale sulla base della morfologia e dellestensione del tumore [ 30 , 31 ]  . per quanto concerne le metastasi non sono invece state riscontrate differenze statisticamente significative dei valori di perfusione tra i vari tipi di metastasi . 
i dati ottenuti oscillano su un ampio range ; i valori di rrcbv risultano pi bassi nelle localizzazioni da linfoma ed in alcuni casi di metastasi da carcinoma del polmone o della mammella ; questultimo dato potrebbe essere correlato , almeno in parte , alle terapie a cui tali soggetti erano sottoposti per il tumore dorigine . 
i valori elevati di rrcbv riscontati nei meningiomi sono in linea con quanto riportato in letteratura [ 15 , 26 ]  . conclusioni le tecniche di diffusione e di perfusione con rm se utilizzate nei pazienti con tumore cerebrale forniscono informazioni non ottenibili con lesame convenzionale , possono essere eseguite a completamento dellesame convenzionale utilizzando lo stesso contrasto e con un minimo aumento del tempo di esecuzione . 658 radiol med ( 2009 ) 114 : 645659 cannot be obtained with conventional mr . 
should dwi and pwi prove superior to conventional imaging , they could be used to monitor patients undergoing treatment to obtain an earlier evaluation of response to chemoor radiotherapy and guide the clinicians treatment choices . le immagini dwi hanno rilevante importanza nella diagnosi delle masse cistiche , mentre non sembrano avere un ruolo nella caratterizzazione dei tumori . 
dwi e pwi inoltre si propongono come tecniche promettenti nel management dei tumori cerebrali : il dato relativo alla microvascolarizzazione offerto dalla pwi potrebbe servire come guida al prelievo bioptico , indicando le aree maggiormente aggressive . 
sardanelli servizio di radiologia , dipartimento di scienze medico - chirurgiche , universit degli studi di milano , irccs policlinico san donato , via morandi 30 , 20097 san donato milanese , italy a . 
the mr protocol applied at our institution for both diagnosis and follow - up after surgical or endovascular treatment consists of four steps : morphologic study , cine mr study , flow analysis , and mr angiography ( mra )  . the first three sequences are acquired during breath - hold and with electrocardiographic gating . 
cine true - fast imaging with steady - state precession ( true - fisp ) sequences show not only morphologic features but also blood - flow changes inside the aorta . 
gradient - echo sequences for phase - velocity mapping allow flow analysis . application of bernoullis equation here briefly presented and discussed allows for calculation of the pressure gradient caused by the coarctation . 
mra , acquired with a breath - hold three - dimensional t1weighted gradient - echo sequence and intravenous administration of paramagnetic contrast material , allows for optimal depiction of the aortic lumen , with a panoramic view of the whole aorta , its main branches and possible collateral circulation . 
 keywords aorta aortic coarctation bernoullis equation magnetic resonance ( mr ) imaging mr flow analysis ( phase - velocity mapping ) riassunto la coartazione aortica rappresenta il 5%10% di tutte le patologie congenite del cuore e dei grandi vasi e il 7% dei casi di piccoli cardiopatici in condizioni critiche . 
la risonanza magnetica ( rm ) permette lo studio di questa patologia con una serie di vantaggi rispetto a tecniche alternative quali langiografia convenzionale , lecografia transesofagea e la tomografia computerizzata . 
il nostro protocollo rm , utilizzato sia per la diagnosi , sia per il follow - up dopo trattamento chirurgico o endovascolare , consiste di quattro fasi : studio morfologico , cinetico , flussimetria e angio - rm . 
le sequenze a sangue chiaro true - fast imaging with steady - state precession ( true - fisp ) , oltre a dare informazioni di tipo morfologico , mostrano le alterazioni del flusso ematico allinterno del vaso . 
le sequenze gradient - echo con mappaggio fase - velocit consentono la quantificazione del flusso ; lapplicazione dellequazione di bernoulli ( qui brevemente presentata e discussa ) , consente infatti il calcolo del gradiente pressorio determinato dalla coartazione . 
lo studio angiorm , ottenuto in apnea mediante sequenze gradient - echo tridimensionali t1 - pesate con somministrazione endovenosa di mezzo di contrasto paramagnetico , mostra una visione volumetrica panoramica del lume dellaorta , delle sue principali ramificazioni e , quando presenti , dei circoli collaterali . 
 parole chiave aorta coartazione aortica equazione di bernoulli flussimetria rm risonanza magnetica ( rm ) radiol med ( 2009 ) 114 : 524537 introduction introduzione over recent years , several magnetic resonance ( mr ) techniques have been successfully applied for evaluating congenital heart diseases . 
we summarize here the currently available knowledge about coa , including epidemiology , aetiopathogenesis , topographic classification , and the different mr imaging techniques . negli ultimi anni numerose tecniche di risonanza magnetica ( rm ) sono state applicate con successo alla valutazione delle cardiopatie congenite . 
la coartazione aortica ( coa ) rappresenta un reperto non infrequente nella pratica clinica , pari a circa il 5%10% di tutte le malformazioni congenite del cuore e dei grandi vasi [ 1 ]  . 
sono qui rivisitate le attuali conoscenze sulla malattia relativamente a epidemiologia , eziopatogenesi , classificazione topografica e studio con differenti tecniche rm . definition coarctation is a term derived from the latin coarctatio , meaning narrowing or stricture . 
similar congenital malformations are aortic atresia and the true aortic interruption , mostly located at the arch ( interrupted aortic arch )  . coa , also known as congenital aortic stenosis , was originally noted during autopsy by j.f. 
he collected 200 previously documented cases , but coa was not regularly diagnosed until after 1933 [ 6 ]  . epidemiology and aetiology coa accounts for 5%10% of all congenital heart diseases and represents 7% of critically ill infants with heart disease . it is seven times more common in caucasian than in asian population . 
in atlanta ( usa ) from 1970 to 1983 , the prevalence per 1 , 000 live births was as follows : interruption of the aortic arch , 0.05 ; coarctation , 0.36 ; hypoplasia of the aorta , 0.06 [ 7 ] , giving a total prevalence of 0.47 per 1 , 000 live births . 
in bohemia in 1980 , a prevalence of 6.67 congenital heart malformations per 1 , 000 live births was reported , and 5.77% of these malformations consisted of coa cases [ 9 ] , giving a coa prevalence of 3.84 per 1 , 000 live births . the aetiology of coa is still unclear . 
up to 60% [ 10 ] or 85% [ 1 ] of coa cases are associated with bicuspid aortic valve , supporting the hypothesis of a common cause for the two congenital malformations [ 11 ] or an influence of the bicuspid aortic valve on the development of coa [ 1 ]  . 
pathologic examination of the ascending aorta of patients with definizione il termine coartazione deriva dal latino coarctatio , letteralmente restringimento ovvero , nel caso di una struttura vascolare , riduzione di calibro cui consegue ostruzione al flusso ematico . 
paris [ 4 ] ne forn la prima descrizione accurata nel 1791 mentre j . abbott pubblic una serie autoptica retrospettiva di 200 casi nel 1928 [ 5 ] , ma la malattia non stata diagnosticata con regolarit fino a dopo il 1933 [ 6 ]  . epidemiologia ed eziologia la coa rappresenta il 5%10% di tutte le patologie congenite del cuore e dei grandi vasi e il 7% dei casi di piccoli cardiopatici in condizioni critiche . 
 si osserva una prevalenza maschile con rapporto maschi / femmine tra 1 , 3 e 2 , 1 nella maggior parte delle serie [ 1 ]  . nellarea metropolitana di atlanta ( usa ) stata osservata tra il 1970 e il 1983 una prevalenza su 1000 nati vivi pari a 0 , 05 per linterruzione dellarco , 0 , 36 per la coartazione e 0 , 06 per lipoplasia [ 7 ] , con una prevalenza totale pari al 0 , 47 per 1000 nati vivi . 
una prevalenza pari a 0 , 32 per 1000 nati vivi stata riportata per lislanda dal 1980 al 1994 [ 8 ]  . in boemia nel 1980 stata riportata una prevalenza di 6 , 67 malformazioni congenite cardiache per 1000 nati vivi ; il 5 , 77% di queste era costituito da casi di coa [ 9 ] , per una prevalenza di coa pari a 3 , 84 per 1000 nati vivi . leziologia della coa non ancora stabilita con certezza . 
si associa a bicuspidia valvolare aortica fino al 526 radiol med ( 2009 ) 114 : 524537 bicuspid aortic valve frequently shows loss of smoothmuscle cells and severe degeneration of the medial elastic fibers ( so - called cystic medial necrosis ) [ 11 ]  . 
associated cardiac defects are observed in approximately 50% of patients with coa , mostly being left - sided hypoplastic or obstructive defects and ventricular septal defects [ 1 ]  . 
moreover , the higher coa prevalence in males and a relatively high prevalence in patients with turners syndrome suggest a link between x - chromosome defects and anomalous development of the aorta and valve [ 11 ]  . 
however , this classification is now considered too simplistic because many patients lack symptoms , and coa diagnosis is delayed until adulthood [ 14 ]  . only malformations determining a total obstruction or a severe degree of stenosis ( severe tubular hypoplasia , severe coarctation , vessel atresia or interruption ) or associated with cardiac defects are diagnosed during childhood [ 13 ]  . 
thus , the prevalence in adulthood , including the nonsevere form of coa , should be higher than the incidence in live births . a commonly accepted classification is based on stenosis location : preisthmic , isthmic and postisthmic . 
 a physiopathologic approach is given by the subdivision 60% [ 10 ] o all85% dei casi [ 1 ] , supportando lipotesi di una causa comune alle due malformazioni [ 11 ] o di uninfluenza della bicuspidia valvolare aortica sullo sviluppo della coa [ 1 ]  . 
indagini istologiche dellaorta ascendente di soggetti con bicuspidia valvolare hanno mostrato un ridotto numero di cellule muscolari lisce e severa degenerazione delle fibre elastiche della tonaca media ( cosiddetta necrosi medio - cistica ) [ 11 ]  . 
malformazioni cardiache congenite si associano a coa in circa il 50% dei casi , per lo pi rappresentate da sindromi ipoplasiche o ostruttive delle cavit sinistre o da difetti del setto interventricolare [ 1 ]  . 
dal 3%5% [ 1 ] al 10% [ 12 ] dei pazienti con coa presentano altres aneurismi intracranici , in accordo con lipotesi che la coa sia parte di una patologia arteriosa diffusa , secondaria ad anomalie dello sviluppo della cresta neurale [ 12 ]  . 
inoltre , la maggiore prevalenza di coa nei maschi e nei pazienti con sindrome di turner induce a ipotizzare un legame tra difetti del cromosoma x ed alterazioni dello sviluppo dellaorta e della valvola aortica [ 11 ]  . 
solo le malformazioni che comportano stenosi di grado elevato o ostruzione totale del flusso ematico ( ipoplasia tubulare serrata , coartazione serrata , atresia o interruzione del vaso ) o che si associano a malformazioni cardiache sono diagnosticate in et infantile [ 13 ]  . ne deriva che la prevalenza in et adulta , includendo le forme non severe di coa , dovrebbe essere sensibilmente pi elevata di quella calcolata in base alla diagnosi sui nati vivi . radiol med ( 2009 ) 114 : 524537 fig . 
1a - c angio - rm ( ricostruzioni volumetriche ) di casi di : ipoplasia tubulare dellarco aortico ( freccia in a ) , coartazione istmica ( freccia in b ) e interruzione dellaorta discendente ( freccia in c )  . into preductal , ductal and postductal coa , referring to the location of the botallo ductus arteriosus . 
this kind of coa , observed in approximately 5% of infants with turner syndrome , typically results from a cardiac anomaly during fetal life decreasing blood flow through the left ventricle , leading to hypoplastic development of the aorta . 
in postductal coa , the narrowing is distal to the insertion of the ductus arteriosus . as a consequence , blood flow to the lower body is impaired . it is thought to be the consequence of muscular cells extending from ductus arteriosus into the aorta during fetal life [ 15 ]  . clinical features and treatment the age of clinical presentation is mainly related to the degree of stenosis and to the presence of associated abnormalities . 
nella forma preduttale la coa prossimale allinserzione del dotto ; se severa , il flusso a valle dipende dalla perviet del dotto la cui chiusura , quindi , pu risultare fatale . 
questo tipo di coa , osservato in circa il 5% dei nati affetti da sindrome di turner , tipicamente il risultato di anomalie cardiache fetali con ridotto efflusso dal ventricolo sinistro , cui consegue unipoplasia dellaorta . 
nella forma post - duttale la coa a valle dellinserzione del dotto e ne risulta un ridotto flusso alla porzione inferiore del corpo ; ritenuta essere la conseguenza della migrazione di miocellule dal dotto arterioso allaorta durante la fase fetale [ 15 ]  . 
si dovrebbe sempre considerare lipotesi di coa in soggetti adolescenti o giovani di sesso maschile con 528 radiol med ( 2009 ) 114 : 524537 coa becomes clinically relevant , it can lead to systemic hypertension and secondary left ventricular hypertrophy with heart failure . 
if it is not treated , the patients mean age of death is about 34 years [ 16 ]  . invasive treatment should always be considered in patients with pressure gradients > 30 mmhg on cardiac catheterization [ 17 ]  . 
successful surgical correction of coa by resection of the stenotic tract with end - to - end anastomosis was first described by crafoord and nylin in 1945 [ 18 ]  . 
 perioperative mortality rates are determined largely by age and concomitant congenital heart disease rather than by the choice of operative technique for correcting coa [ 23 ] and are less than 3% for older children and adults [ 24 ]  . several postsurgical complications such as cerebrovascular , cardiac and aortic events may occur . 
the most common complications are cerebrovascular accidents , cerebral haemorrhage , recurrent hypertension , heart failure , endocarditis , endarteritis , coronary artery disease , recoarctation , poststenotic aneurysm , and aortic rupture [ 10 ]  . 
due to successful surgical or endovascular treatments , follow - up of treated adult patients is often required . imaging of coa a posteroanterior chest x - ray examination can show a dilated ascending aorta , low prominence of the first left arch , a notch sign corresponding with the location of coa and small notches on the inferior posterior margin of ribs , mainly from fourth to eighth , due to the osteolytic effect of the collateral hypertrophic intercostal arteries . 
 similarly to aortic dissection [ 26 ] , coa can be diagnosed using transesophageal echocardiography ( tee ) , digital subtraction angiography ( dsa ) , computed tomography ( ct ) , and mr imaging . 
due to the use of ionising radiation and iodinated contrast agents , ct and dsa are not regarded as first - line techniques for diagnosing coa , particularly in children . 
 over recent years , mr imaging has become a well - established diagnostic tool for studying congenital heart diseases and coa [ 27 , 28 ] , offering many advantages over other ipertensione agli arti superiori e ipotensione e polsi deboli agli arti inferiori . 
se non trattata , porta al decesso mediamente a 34 anni di et [ 16 ]  . il trattamento invasivo dovrebbe essere sempre considerato nei pazienti che presentano un gradiente pressorio maggiore di 30 mmhg al cateterismo cardiaco [ 17 ]  . crafoord e nylin [ 16 ] descrissero per primi , nel 1945 , lintervento correttivo che prevede la resezione del tratto stenotico con successiva anastomosi termino - terminale . successivi miglioramenti delle tecniche chirurgiche [ 18 , 20 ] , tra i quali lintroduzione dellaortoplastica con flap dellarteria succlavia , hanno permesso la correzione in caso di varianti anatomiche complesse . 
 i tassi di mortalit peri - operatoria , determinati soprattutto dallet del paziente e da eventuali anomalie associate piuttosto che dalla tecnica utilizzata per la correzione della coa [ 23 ] , sono inferiori al 3% sia nei bambini che negli adulti [ 24 ]  . 
sono riportate complicanze post - chirurgiche quali lesioni cerebrovascolari , emorragie cerebrali , ipertensione recidivante , scompenso cardiaco , endocardite , endoarterite , patologie coronariche , ricoartazione , aneurismi post - stenotici e rottura dellaorta [ 10 ]  . 
il successo del trattamento chirurgico o endovascolare della coa pone oggi lesigenza del follow - up in et adulta dei pazienti trattati . imaging della coa il radiogramma toracico postero - anteriore pu mostrare ectasia dellaorta ascendente , scarsa prominenza del primo arco di sinistra , incisura in corrispondenza della sede di coartazione e piccole incisure sul margine inferiore dellarco posteriore delle coste , per lo pi dalla quarta allottava , dovute alleffetto osteolitico delle arterie intercostali collaterali ipertrofiche . 
 come per la dissezione aortica [ 26 ] , la diagnosi di coa pu essere posta mediante ecocardiografia transesofagea ( ete ) , angiografia digitale con sottrazione dimmagine ( ads ) , tomografia computerizzata ( tc ) e rm . 
the mr examination takes about 30 min ; the postprocessing requires about 30 min . setting up the patient a good electrocardiographic ( ecg ) line resulting from correct placement of four leads over the anterior or posterior left side of the chest is crucial to obtain a good cardiac synchronization ( ecg gating ) [ 35 ]  . 
la tc e lads non sono considerate tecniche di prima scelta per la diagnosi di coa , soprattutto in et pediatrica , a causa dellutilizzo di radiazioni ionizzanti e mezzi di contrasto ( mdc ) iodati . 
essa offre molteplici vantaggi rispetto alle altre tecniche di imaging cardiovascolare tra i quali la possibilit di ottenere immagini native multiplanari senza luso di radiazioni ionizzanti , lelevata risoluzione temporale ( fino allimaging in tempo reale ) e la possibilit di studio flussimetrico [ 2931 ]  . 
 nello studio dei grandi vasi , la rm vede il suo esordio oltre ventanni or sono con semplici sequenze spin - echo ( se ) e gradient - echo ( ge ) con rifocalizzazione degli spin in flusso laminare . 
successivamente sono state introdotte le sequenze turbo - se ( tse ) , ge cine - rm , ge segmentate , half - fourier acquisition single shot turbo spin - echo ( haste ) e true - fast with steady - state precession ( truefisp )  . 
sequenze ge con mappaggio fase - velocit permettono , inoltre , di ottenere dati flussimetrici [ 33 ] mentre sequenze ge tridimensionali con somministrazione endovenosa di mdc paramagnetico permettono di ottenere immagini simil - angiografiche ad ampio campo di vista , sulle quali si possono effettuare misure accurate del lume aortico [ 34 ]  . protocollo rm faremo qui riferimento alla nostra esperienza ( 20032007 ) con apparecchiatura operante a 1 , 5 t ( magnetom sonata maestro class , siemens , erlangen , germania ) presso lirccs policlinico san donato ( san donato milanese , milano , italia ) su pazienti con coa prima del trattamento e nel loro follow - up a breve e lungo termine . 
thus , we prefer the use of breath - hold ecg - gating : dark - blood t2 - weighted haste [ 37 ] , with high il corretto posizionamento di quattro elettrodi sullemilato sinistro del torace , anteriormente o posteriormente , cruciale per una buona sincronizzazione con lelettrocardiogramma ( ecg - gating ) [ 35 ]  . 
the acquisition of an image passing through the ascending and descending aorta ( three parallel oblique lines ) is planned on a scout axial true fast imaging with steady - state precession ( true fisp ) image ( a )  . 
2a , b definizione del piano obliquo anteriore sinistro ( oas ) per lo studio dellaorta toracica . sullimmagine assiale true - fast imaging with steady - state precession ( true - fisp ) di centratura ( a ) rappresentata la pianificazione di una scansione oas passante per laorta ascendente e discendente ( tre linee oblique parallele )  . 
3a , b bright - blood image ( true - fast imaging with steady - state precession ) ( a ) and dark - blood image ( half - fourier acquisition single - shot turbo spin - echo ) ( b ) , along the same left anterior oblique plane passing through an isthmic coarctation ( arrow )  . 
3a , b immagini a sangue chiaro ( true - fast imaging with steady - state precession ) ( a ) e a sangue scuro ( half - fourier acquisition single - shot turbo spin - echo ) ( b ) lungo il piano obliquo anteriore sinistro passante per una coartazione istmica dellaorta ( freccia )  . 
breath - hold , ecg - gated cine true - fisp sequences ( bright blood , hybrid t2 / t1 - weighted ) are acquired for multiple frames along the cardiac cycle . 
la nostra preferenza va a sequenze in apnea e con sincronizzazione ecg : haste a sangue scuro , pesate in t2 [ 37 ] , con elevato radiol med ( 2009 ) 114 : 524537 fig . 
4 immagine cine true - fast imaging with steady - state precession lungo il piano obliquo anteriore sinistro : coartazione istmica dellaorta ( freccia nera ) ; marcato jet anterogrado ( frecce bianche ) da accelerazione di flusso post coartazione . flow study this part of the mr protocol involves the use of ge phasevelocity mapping sequences on planes orthogonal to the longitudinal axis of the vessel ( through - the - plane acquisition )  . 
phase - velocity sequences provide two different set of images : ( 1 ) magnitude images that give anatomical information , and ( 2 ) phase images that give data about flow direction and velocity . 
si utilizzano sequenze true - fisp ( a sangue chiaro , pesatura ibrida t2 / t1 , con sincronizzazione ecg ) acquisite in apnea per molteplici frame lungo le fasi del ciclo cardiaco . 
per studiare il flusso aortico post - coartazione necessario impostare unelevata codifica di velocit ( velocity encoding , venc ) , generalmente tra 350 e 500 ms , onde evitare artefatti ( aliasing ) nel lume aortico . 
lacquisizione fornisce due serie di immagini : una serie con significato anatomico ( immagini di magnitudo o modulo ) e una serie con significato funzionale ( immagini di fase ) , contenente informazioni su direzione e velocit del flusso . 
lacquisizione lungo il piano obliquo anteriore sinistro ( a ) evidenzia minimo restringimento residuo ( freccia ) ; lacquisizione lungo il piano valvolare aortico ( b ) evidenzia la coesistente bicuspidia ( frecce )  . 
since the kinetic energy of the blood is proportional to the square of its velocity , the right side of bernoullis equation represents the difference between the kinetic energy of blood after coa and that before coa . 
siccome lenergia cinetica del sangue proporzionale al quadrato della sua velocit , il membro destro dellequazione di bernoulli rappresenta la differenza tra lenergia cinetica del sangue a valle della coa e quella a monte della coa . 
 al fine di esprimere p in mmhg anzich in pascal ( pa ) , lunit di misura della pressione nel sistema internazionale , occorre introdurre il seguente coefficiente di conversione : 1 pa = 0 , 0075 mmhg infine , ricordando che la densit del sangue d pari a circa 1067 g / l , otteniamo : i.e. 
if v1 and v2 are given in m / s , the coefficient 4 adjusts the measurement units so to allow the pressure gradient p to be expressed as mmhg . 
se le velocit v1 e v2 sono espresse in m / s , il coefradiol med ( 2009 ) 114 : 524537 that the peak velocity before coa ( v1 ) is generally negligible if compared with the peak velocity after coa ( v2 )  . 
first , it works only if the patient is in the supine position , as the nonperfect horizontal position introduces the gravity - force effect ( this limitation is not relevant for mr applications )  . 
in such a context , a p > 20 mmhg is highly suggestive of significant coa [ 41 , 42 ]  . mr angiography ( mra ) contrast - enhanced mra is a reliable technique for evaluating great vessels . 
for a long time , gadolinium chelates have been considered safe contrast agents and have been used in patients allergic to drugs or iodinated contrast agents and in nephropathic or cardiopathic patients [ 43 ]  . 
patients with severe renal failure [ glomerular filtration rate ( gfr ) < 30 ml / min1.73 m2 ] are considered to be at risk [ 44 , 46 ]  . 
in patients with gfr30 ml / min1.73 m2 , a single dose of 0.1 mmol / kg of gadolinium - based contrast agent can be safely administered intravenously [ 46 ]  . 
for diagnosis or follow - up of coa , we used a power injector with an injection rate of 2 ml / s for both the contrast material and the following 20 - ml saline flush . 
we used a three - dimensional breath - hold , not ecg - gated , t1 - weighted ge sequence with a short acquisition time ( nearly 20 s )  . 
in patients with gfr < 30 ml / min1.73 m2 , time - of - flight and phase - contrast sequences can be used , even though image quality is lower and veins are also visualized [ 47 ]  . 
sulla ricostruzione volumetrica posteriore obliqua ( a ) sono riportate le sedi del calcolo dellarea delle sezioni del vaso a monte della coartazione , al livello della coartazione e a valle della coartazione . 
a sinistra sono riportati i valori di tali aree come normal a , minimum e area 01 , rispettivamente ; inoltre riportato come ratio 01 il rapporto percentuale tra larea a monte della coartazione e larea al livello della coartazione ( grado di stenosi )  . 
la linea centrale endoluminale ( frecce in a ) , generata automaticamente , permette la ricostruzione lungo il piano curvo ( b )  . ficiente 4 consente di esprimere il gradiente p in mmhg . unulteriore semplificazione possibile considerando che la velocit di picco a monte della stenosi ( v1 ) in genere trascurabile rispetto alla velocit a valle della stenosi ( v2 ) e , quindi , pu essere ignorata . 
in primo luogo , la formula di bernoulli valida solo se il paziente supino , in quanto la posizione ortostatica introdurrebbe nella formula un termine aggiuntivo che tiene conto della forza di gravit ( tale limitazione non rilevante per usuali applicazioni rm )  . 
i chelati del gadolinio sono stati considerati a lungo mdc privi di effetti collaterali e si riteneva potessero essere impiegati anche in presenza di allergie a farmaci o ai mdc iodati cos come in caso di pazienti nefropatici o cardiopatici [ 43 ]  . 
sono considerati a rischio pazienti con insufficienza renale grave ( gfr , rateo di filtrazione glomerulare < 30 ml / min1 , 73 m2 ) [ 44 , 46 ]  . 
in pazienti con gfr 30 ml / min1 , 73 m2 si pu utilizzare la singola dose endovena ( 0 , 1 mmol / kg ) di un chelato del gadolinio senza rischi per il paziente [ 46 ]  . 
per la diagnosi e il follow - up della coa , noi utilizziamo un flusso di 2 ml / s sia per il mdc , sia per il bolo di 20 ml di soluzione fisiologica , entrambi somministrati con iniettore automatico . 
in caso di gfr < 30 ml / min1 , 73 m2 possibile impiegare sequenze angiografiche senza mdc ( time of flight e phase contrast ) ottenendo per immagini di qualit inferiore comprendenti anche i vasi venosi [ 47 ]  . 
maximum intensity projections allow aortic demonstration in the angiographic - like view . surface shading display and volume - rendering techniques , obtained with standardised reconstruction algorithms , allow reliable measurements . 
in order to correctly measure aorta diameters and assess the degree of stenosis , measurements should be performed both in lao and in planes orthogonal to the longitudinal axis of the vessel . 
the distance of the coa from the left subclavian artery origin is an important variable for surgical or endovascular therapy planning . le rielaborazioni di volume ( volume rendering technique ) e di superficie ( surface shading display ) , con algoritmi di ricostruzione standardizzati , permettono misurazioni accurate . 
la distanza della coa dallorigine dellarteria succlavia sinistra o dai vasi epiaortici un dato importante nel planning della procedura chirurgica o endovascolare . conclusioni conclusions to correctly diagnose coa , establish disease severity and evaluate treatment options , as well as for follow - up during adulthood , a complete mr protocol is required , including morphological , functional , and flow study as well as contrast - enhanced mra and accurate postprocessing . per diagnosticare correttamente i casi di coa , valutare lentit della stenosi e le possibilit terapeutiche , cos come nel follow - up in et adulta , necessario un protocollo rm che comprenda acquisizioni morfologiche , cinetiche , flussimetriche e angiografiche , completato da accurato postprocessing . 
among the various therapies , local radiotherapy is the gold standard for pain palliation from single metastasis , even though the maximum benefit is obtained between 12 and 20 weeks from initiation . 
from november 2003 to may 2008 , ten ablation treatments were performed in ten patients with acute pain from metastatic bone lesions . patient selection and choice of the most appropriate ablation treatment was made based on lesion characteristics . 
three patients were treated with radiofrequency , one with plasma - mediated radiofrequency , two with plasma - mediated radiofrequency and cementoplasty , three with radiofrequency and cementoplasty and one with microwave . 
in both cases , 3 - month follow - up showed a statistically significant reduction of pain no case did local complications occur either during or after treatment . only one patient treated with radiofrequency ( 1 / 9 , 11% ) developed low - grade fever and general malaise during the 6 days following the procedure , compatible with a postradiofrequency syndrome , which was treated with riassunto obiettivo . 
tra le varie terapie , la radioterapia locale il gold - standard nella palliazione del dolore da metastasi singola , anche se il massimo beneficio si ottiene tra le 12 e le 20 settimane dallinizio della terapia . 
la valutazione stata effettuata non solo con imaging , ma anche con una vas score ( visual analoge scale ) per la determinazione del dolore e con la variazione delle dose equivalenti di morfina . 
solamente in un paziente trattato con radiofrequenza ( 1 / 9 , 11% ) abbiamo riscontrato nei 6 giorni successivi al trattamento insorgenza di febbricola e malessere generale compatibile con la sindrome postradiol med ( 2009 ) 114 : 608625 acetaminophen ( paracetamol ) only and resolved on day 7 . 
percutaneous ablation therapies represent a safe and valuable alternative for treating localised pain from single bone metastasis , providing rapid ( 4 - week ) relief of symptoms and a significant reduction in morphine doses . 
le tecniche ablative percutanee rappresentano una sicura e valida alternativa al trattamento del dolore localizzato da metastasi ossee singole , apportando in breve tempo ( 4 settimane ) un miglioramento della sintomatologia e una sensibile riduzione delle dosi di morfina . 
ci concorre a migliorare la qualit della vita in pazienti affetti da malattia metastatica . parole chiave metastasi osso palliazione ablazione introduction introduzione bone metastases are a common problem in cancer patients , affecting up to 30% of patients with epithelial cancers . 
in 70% of cases , the metastases originate from cancers of the prostate or breast , in 20%30% from cancers of the lung or kidney and in 10% from cancers of the rectum , colon , ovary and pancreas [ 1 ]  . the causes of pain in patients with bone metastases are not fully understood but appear to be unrelated to the type of tumour , location , number or size of the metastatic lesions . possible mechanisms of pain include stretching of the periosteum secondary to tumour growth , fractures ( both micro and macro ) , cytokine - mediated osteoclastic bony destruction that results in stimulation of the nerve endings in the endosteum and tumour growth into surrounding nerves and tissues [ 2 ]  . several types of treatment , including chemotherapy , hormonal therapy , surgery , radiotherapy and radiometabolic therapy , are used for palliation purposes . 
however , a number of patients fail to benefit from these conventional treatments , and pain relief , when achieved , does not occur until 412 weeks after treatment [ 3 ]  . 
a variety of alternative strategies have been proposed for treating painful bone metastases , all of which are based on image - guided percutaneous procedures for ablating focal bone lesions [ 1 , 4 , 5 ] , in some cases associated with cementoplasty [ 68 ]  . the aim of this study was to evaluate the technical success , effectiveness and possible complications of percutaneous ablation treatment in patients with painful bone metastases . 
esse nel 70% dei casi insorgono dal tumore della prostata o della mammella , nel 20%30% dal tumore del polmone o del rene , nel 10% dalle neoplasie del retto , colon , ovaio e pancreas [ 1 ]  . le cause di dolore in soggetti affetti da metastasi ossee non sono ancora completamente note , ma non sembrano correlate al tipo di tumore primitivo , alla localizzazione della lesione , al numero e alle dimensioni delle lesioni . 
possibili meccanismi nella genesi del dolore comprendono : stretching del periostio secondario alla crescita del tumore ; fratture ( sia micro - fratture che macro - fratture ) ; distruzione ossea di tipo osteoclastico citochino - mediata che determina una stimolazione delle fibre nervose terminali endostali ; crescita tumorale nelle strutture nervose e nei tessuti adiacenti [ 2 ]  . 
alcuni pazienti non riescono a trarre benefici da questi trattamenti convenzionali e il sollievo dal dolore , quando si verifica , non avviene mai prima di 412 settimane [ 3 ]  . 
sono state proposte varie strategie alternative per il trattamento delle metastasi ossee dolorose , che richiedono metodiche percutanee imaging - guidate finalizzate allablazione delle lesioni focali ossee [ 1 , 4 , 5 ] , in alcuni casi associando la cementoplastica [ 68 ]  . scopo del lavoro stato quello di valutare il successo tecnico , lefficacia e le eventuali complicanze in pazienti affetti da metastasi ossee sintomatiche e sottoposti a terapia ablativa percutanea . 
 610 radiol med ( 2009 ) 114 : 608625 materials and methods materiali e metodi between november 2003 and may 2008 , we performed ten ablation treatments in ten patients ( five men and five women ; mean age 62 years ; range 2882 ) with acute pain from bone metastases of different origin , site ( table 1 ) and size ( mean volume 63.25 cm3 ; range 218.93.3 cm3 ) ( table 2 )  . 
in all of these patients , the pain had proved refractory to conventional approaches . owing to the poor expected response , one patient received no treatment before the ablation procedure ( table 1 )  . 
three patients were treated with radiofrequency ( rf ) alone , one with plasma - mediated rf , two with plasma - mediated rf combined with cementoda novembre 2003 a maggio 2008 sono stati effettuati , nel servizio di radiologia del nostro ospedale 10 trattamenti in 10 pazienti , 5 uomini e 5 donne ( et media 62 anni , range da 28 a 82 anni ) con dolore acuto da lesioni metastatiche ossee di varia origine , sede ( tabella 1 ) e volume ( volume medio 63 , 25 cm3 , range da 218 , 9 a 3 , 3 cm3 ) ( tabella 2 )  . tutte le lesioni trattate erano osteolitiche . 
 quattro pazienti erano stati precedentemente sottoposti a radioterapia e chemioterapia , 1 paziente solo a radioterapia , 4 pazienti solo a chemioterapia : in questi casi si sono dimostrati refrattari alle terapie convenzionali . 
nessun paziente era suscettibile di trattamento chirurgico , per lassenza di rischio di frattura patologica . tre pazienti sono stati trattati solo con radiofrequenza ( rf ) , table 1 personal series patient no . / age / gender primary tumour histology site of metastasis radiotherapy chemotherapy bladder liver liver liver breast lung lung colon - rectum transverse colon uterine cervix carcinoma right 8th rib left 9th rib left ischiopubic ramus right 8th rib infiltrating lobular vertebra l4 carcinoma adenocarcinoma adenocarcinoma adenocarcinoma adenocarcinoma squamous cell carcinoma vertebra l1 left sacroiliac joint left acetabolum left ischiatic spine left iliac wing not responsive not responsive not responsive not responsive not responsive not responsive not responsive not responsive not responsive not responsive not responsive not responsive not responsive n / et / sesso tumore primitivo istologia sede metastasi radioterapia chemioterapia vescica fegato fegato fegato mammella polmone polmone carcinoma carcinoma lobulare vertebra l4 infiltrante adenocarcinoma adenocarcinoma viii arcata costale destra ix arcata costale sinistra branca ischio - pubica sinistra viii arcata costale destra non responsivo non responsivo non responsivo non responsivo non responsivo vertebra l1 articolazione sacroilaca sinistra acetabolo sinistra spina ischiatica sinistra ala iliaca sinistra non responsivo non responsivo non responsivo non responsivo non responsivo non responsivo non responsivo non responsivo colon - retto colon trasverso portio uterina adenocarcinoma adenocarcinoma carcinoma squamocellulare hcc , carcinoma epatocellulare hcc , hepatocellular carcinoma tabella 1 serie personale 1 / 70 / m 2 / 69 / m 3 / 45 / m 4 / 40 / m 5 / 60 / f 6 / 73 / f 7 / 73 / f 8 / 82 / f 9 / 81 / m 10 / 38 / f 1 / 70 / m 2 / 69 / m 3 / 45 / m 4 / 40 / m 5 / 60 / f 6 / 73 / f 7 / 73 / f 8 / 82 / f 9 / 81 / m 10 / 38 / f radiol med ( 2009 ) 114 : 608625 table 2 descriptions of lesions and procedures per patient patient procedure lesion no . 
stato eseguito il calcolo del volume per ciascuna lesione attraverso la formula 4 / 3ab2 , dove a il semiasse maggiore e b il semiasse minore plasty , three with rf combined with cementoplasty and one with microwave ablation . 
 1 con radiofrequenza plasma - mediata , 2 con radiofrequenza plasma - mediata associata a cementoplastica , 3 con radiofrequenza associata a cementoplastica , 1 con microonde . tutti hanno completato il follow - up a 12 settimane . 
 pretreatment assessment before undergoing ablation treatment , patients were evaluated by means of a validated visual analogue scale ( vas ) for pain assessment and by recording any use of analgesics . inclusion criteria were moderate or severe pain rated 4 or more on the 10 - point vas ; patients with lower scores were treated with analgesics . 
pain was localised to the site corresponding to the lesion detected by computed tomography ( ct ) ( aquilon 64 , toshiba , tokyo , japan ) or magnetic resonance valutazione prima del trattamento prima dei trattamenti ablativi i pazienti sono stati valutati usando una codificata visual analogue scale ( vas ) per la determinazione del dolore , ed stato registrato leventuale utilizzo di analgesici . 
criteri di inclusione nello studio sono stati : dolore moderato o severo con valore uguale o superiore a 4 nella scala vas di 10 punti ; i pazienti con punteggio inferiore sono stati trattati con analgesici . 
prima del trattamento , tutti i pazienti inclusi nel nostro studio 612 radiol med ( 2009 ) 114 : 608625 table 3 morphine - equivalent doses for several opioids according to route of administration tabella 3 dosi equivalenti di morfina per diversi oppioidi a seconda della via di somministrazione administration route via di somministrazione parenteral dose equivalents oral dose equivalents parenterale dose equivalenti orale dose equivalenti morphine methadone codeine buprenorphine tramadol fentanyl 10 mg 10 mg 120 mg 0.3 mg 100 mg 25 mcg / hc 30 mga 20 mg 200 mg 0.4 mgb 120 mg morfina metadone codeina buprenorfina tramadolo fentanile 10 mg 10 mg 120 mg 0 , 3 mg 100 mg 25 g / h c 30 mga 20 mg 200 mg 0 , 4 mgb 120 mg achronic use ; bsublingual ; ctransdermal auso cronico ; bsublinguale ; ctransdermica ( mr ) imaging ( 1.5 tesla avanto , siemens , munich , germany )  . 
specifically , nine patients were imaged by contrast - enhanced ct ( iopamiro , bracco , milan , italy ) and one by contrast - enhanced mr imaging ( magnevist , schering , berlin , germany )  . 
exclusion criteria were the presence of more than two symptomatic lesions and safety margins less than 1 cm from the spinal cord , major motor nerve , brain , adamkiewicz artery , bowel and bladder [ 9 ]  . before the ablation treatment , patients were on analgesic therapy ( opioids or nsaids ) , which was translated into morphine - equivalent doses to render the data homogeneous ( table 3 )  . 
analgesic use was monitored before treatment and at 1 , 4 , 8 and 12 weeks thereafter . treatment selection depending on whether the lesion is osteoblastic or osteolytic , the most beneficial ablation method , that is , the one best suited to the individual metastasis , must be selected . both rf and microwave ablation are based on the use of heat , although heat production occurs in two different manners : rf generates heat by delivering electrical current to tissues and causing ion agitation and friction ; electromagnetic microwave generates heat by agitating water molecules in tissue and producing friction [ 10 ]  . 
plasma - mediated rf ( coblation ) consists of using electrical energy to create a field of ionised particles that break the organic bonds within soft tissue ( ablation )  . 
il dolore era localizzato alla sede corrispondente alla lesione evidenziata con indagini eseguite con tomografia computerizzata ( tc ) ( aquilon 64 , toshiba , tokio , giappone ) o risonanza magnetica ( rm ) ( 1 , 5 tesla avanto , siemens , munich , germania ) ; in particolare 9 pazienti sono stati sottoposti a tc con mdc ( iopamino , bracco , milano , italia ) e 1 paziente a rm con mdc paramagnetico ( magnevist , schering , berlin , germania )  . 
criteri di esclusione al trattamento ablativo sono stati : la presenza di pi di due lesioni sintomatiche ; margini di sicurezza inferiori a 1 cm dal midollo spinale , dal nervo motorio maggiore , dallencefalo , dallarteria di adamkiewicz , dallintestino e dalla vescica [ 9 ]  . 
prima del trattamento i pazienti erano in terapia con analgesici ( oppioidi o farmaci anti - infiammatori non steroidei ) , convertiti poi , ai fini di rendere omogenei i dati , in dosi - equivalenti di morfina ( tabella 3 )  . 
tutti i pazienti assumevano dagli 80 ai 100 equivalenti di morfina . lutilizzo di analgesici stato monitorato prima della procedura , alla prima , alla quarta , allottava e alla dodicesima settimana susseguenti la stessa . selezione della tipologia di trattamento in questa fase occorre valutare , a seconda che la lesione sia osteoblastica o osteolitica , quale sia il trattamento ablativo di cui pu giovare il paziente , ovvero il pi adatto a quella specifica metastasi . 
sia lablazione con radiofrequenza che quella con microonde caratterizzata dallimpiego di calore , che avviene con due modalit differenti : la rf genera calore tramite lattrito causato dallagitazione ionica che si ha al passaggio di corrente elettrica , le microonde invece generano calore per le oscillazioni delle molecole dacqua tissutali provocate dallinterazione tra le radiazioni elettromagnetiche e le stesse molecole dacqua [ 10 ]  . 
in particolare la radiofrequenza utilizzabile solo in lesioni osteolitiche o miste , ma non pu essere usata nelle radiol med ( 2009 ) 114 : 608625 there is a need for greater precision and control during the procedure and for treating osteoblastic lesions . 
it may be performed provided that the cortical bone is intact . procedure procedures were carried out with the patient under deep sedation achieved with the cyclic administration of alfentanil ( 0.51 mg ) and midazolam ( 13 mg ) and continuous infusion of propfol ( 50120 mg )  . 
blood pressure was checked every 4 m local anaesthesia ( 1% carbocaine ) was applied to the skin at the access site . different types of imaging were used to guide the procedure : ultrasound ( iu22 , philips , best , the netherlands ) in four cases , ultrasound and fluoroscopy ( allura xper fd , philips ) in one case , ct ( aquilon 64 , toshiba , tokyo , japan ) in three cases , ct fluorography in one case and carm cone - beam ct in one case ( xper ct , allura , philips ) ( table 2 )  . 
1 ago da rf posizionato allinterno di una lesione iliaca . lesioni osteoblastiche , dato che in questo caso non avviene conduzione di calore ; non pu essere impiegata neanche se la lesione si trova in prossimit di organi come il midollo spinale ( esempio metastasi vertebrale ) per il rischio di lesioni da calore [ 11 ] ; lablazione con microonde indicata se la lesione voluminosa e non in prossimit di organi termosensibili . 
la radiofrequenza plasma - mediata ( coblazione ) consiste nellutilizzo dellenergia elettrica atta a creare un campo di particelle ionizzate che spezzano i legami organici allinterno dei tessuti molli ( ablazione )  . 
infine il trattamento combinato con cementoplastica consiste nelliniezione di cemento preceduto da un qualsiasi trattamento ablativo e si pu effettuare a patto che sia integra la corticale ossea . procedura la procedura stata eseguita in sedazione profonda con somministrazione ciclica di alfentanyl ( 0 , 51 mg ) e mydazolam ( 13 mg ) e infusione continua di propofol ( 50120 mg ) ; stata somministrata una ultra short term profilaxis ( 1 g di cefazolina )  . 
 stata effettuata anestesia locale con 1% di carbocaina nella zona cutanea di introduzione dellago . sono state impiegate differenti tipologie di imaging per la guida : in 4 casi stata impiegata guida ecografica ( iu22 , philips , best , olanda ) , in 1 caso guida ecografica e fluoroscopica ( allura xper fd , philips , best , olanda ) , in 3 casi guida tc ( aquilon 64 , toshiba , tokio , giappone ) , in 1 caso guida fluoro - tc e in 1 caso la c - arm cone - beam tc ( xper ct , allura , philips , best , olanda ) ( tabella 2 )  . 
in tutti i casi trattati con radiofrequenza stato utilizzato un ago da 17 gauge con diametro di apertura degli uncini allestremit di 4 cm ; il generatore di rf , durante il passaggio di energia , rileva limpedenza elettrica del tessuto . 
la procedura stata condotta in accordo ai protocolli forniti dalle 614 radiol med ( 2009 ) 114 : 608625 17 - gauge needle with a maximum hook - deployment diameter of 4 cthe rf generator measures electrical impedance of tissue during the passage of energy . 
plasma - mediated rf procedures ( coblation ) were performed with a cavity spinewand kit ( arthrocare corp , sunnyvale , ca , usa )  . the device was directed anteriorly through the tissue to be ablated . 
in the cases undergoing combined treatment with cementoplasty ( fig . 5a , b ) , the rf electrode was introduced through an 11 - gauge simko needle ( simko special vertebroplasty needle , optimed )  . 
after withdrawal of the rf electrode , the simko needle was connected to a system ( cement - king application system , optimed ) for the injection of cement ( osteopal v , biomet , merck )  . 
per la procedura di radiofrequenza plasma - mediata ( coblazione ) , stato usato un kit cavity spinewand ( arthrocare corp , sunnyvale , ca , usa ) ; il dispositivo stato diretto anteriormente attarverso il tessuto da ablare . 
 la fattibilit della procedura combinata con cementoplastica stata sancita dalla possibilit di effettuare un corretto posizionamento dellago allinterno della lesione , mentre il successo tecnico stato inteso come la possibilit sia di effettuare lablazione allinterfaccia tra tessuto molle e osso sia di iniettare cemento nel caso del trattamento combinato . 
2 schema che mostra la modalit di ablazione con movimento rotatorio del dispositivo di radiofrequenza plasma - mediata allinterno della lesione in direzione dei diversi orientamenti orari a ore 3 , 6 , 9 e 12 . radiol med ( 2009 ) 114 : 608625 fig . 
evidente la presenza di cemento nella lesione ablata ( freccia )  . the possibility of achieving correct needle placement inside the lesion , whereas technical success was defined as the ability to achieve ablation at the interface between soft tissue and bone and then to inject cement in the case of combined treatments . 
 risultati il successo tecnico , inteso come la possibilit di effettuare lablazione allinterfaccia tra tessuto molle e osso e di iniettare cemento nel caso del trattamento combinato , stato raggiunto nel 100% dei casi . 
la termoablazione con rf ha raggiunto in tutti i casi dimensioni di necrosi di circa 4 cm , mentre la termoablazione con microonde ha creato unarea di necrosi superiore ai 6 cm in 10 minuti . 
il tempo totale delle procedure ( intese come la sola ablazione o lablazione associata a cementoplastica ) variava da un massimo di 25 minuti a un minimo di 15 minuti . 
solamente in un paziente trattato con radiofrequenza ( 1 / 9 , 11% ) abbiamo riscontrato nei 6 giorni successivi al trattamento insorgenza di febbricola e malessere generale compatibile con la sindrome post - radiofrequenza , risoltisi poi al settimo giorno e trattati solo con acetaminofene ( paracetamolo ) [ 12 ]  . riguardo alla valutazione con scala vas , la risposta di tutti i pazienti nella valutazione del dolore prima della procedura era di 8 punti ( in una scala da 0 a 10 )  . 
attraverso una valutazione a 1 , 4 , 8 e 12 settimane , emerso che il valore medio nella scala vas stato rispettivamente di 4 , 1 la prima settimana e 2 nei radiol med ( 2009 ) 114 : 608625 table 4 results of the follow - up in terms of reduction of pain and morphine - equivalent dose . 
 pain reduction ( vas ) morphine - equivalent dose before 24 h 1 week 4 week 8 week 12 week before 1 week 4 week 8 week 12 week tabella 4 risultati nel follow - up in termini di riduzione del dolore e dose in equivalenti di morfina . 
in due casi ( pazienti n 7 e n 9 ) essa aumentata di 10 equivalenti di morfina tra lottava e la dodicesima settimana dal trattamento ( vedasi testo ) riduzione del dolore ( vas ) dose in equivalenti di morfina prima 24 h sett 1 sett 4 sett 8 sett 12 prima sett 1 sett 4 sett 8 sett 12 complications ( cement leakage )  . 
pain regression was considered statistically significant at a p value of < 0.001. the wilcoxon test for paired data was used to evaluate when this difference was already statistically significant . 
 results technical success , defined as the ability to achieve ablation at the interface between soft tissue and bone and to inject controlli a 4 , 8 e 12 settimane ( tabella 4 )  . 
rf ablation produced an area of necrosis of approximately 4 cm in all cases , whereas microwave ablation produced an area of necrosis > 6 cm in 10 mprocedure times were also shorter with the plasma - mediated rf technique ( 20 min )  . 
total procedure time ( referring to ablation alone or combined ablation and cementoplasty ) ranged regressione lineare logaritmica con p < 0 , 001 statisticamente significativa ; in particolare la differenza tra prima del trattamento e a una settimana dal trattamento significativamente aumentata ( dolore post - procedurale ) , per i motivi precedentemente esposti , mentre vi una significativa diminuzione della dose di morfina tra prima del trattamento e la quarta settimana successiva allo stesso . radiol med ( 2009 ) 114 : 608625 from 25 min to 15 m in no cases did local complications occur either during or after treatment . 
in only one case of rf ablation ( 1 / 9 , 11% ) did the patient develop low - grade fever and general malaise during the 6 days after treatment , compatible with a postradiofrequency syndrome . 
this was treated with acetaminophen only ( paracetamol ) and resolved on day 7 [ 12 ]  . with regard to vas score , all patients had a pain score of 8 ( on a scale from 0 to 10 ) before the procedure . 
after day 1 , the score decreased by 1 point in 9 / 10 patients and by 2 points in the patient treated with microwave ablation . evaluation at 1 , 4 , 8 , and 12 weeks showed that the mean vas score was 4.1 at week 1 , and 2 at 4 , 8 and 12 weeks ( table 4 )  . 
 analgesic dose increased during week 1 ( from 10 to 20 morphine equivalents ) , probably owing to postprocedural pain , but then decreased substantially after 1 month in all patients ( from 50 to 30 morphine equivalents ) ( table 4 )  . 
in two cases ( patients 7 and 9 ) , it increased by ten morphine equivalents between 8 and 12 weeks , most likely because of the appearance of a new bone metastasis at another site from the one previously treated ( table 4 )  . 
in particular , the difference between pretreatment and 1 - week posttreatment increased significantly ( postprocedural pain ) for the reasons described above , whereas there was a significant reduction in the morphineequivalent dose between pretreatment levels and week 4 after treatment . discussion several therapies are used for the palliation of bone metastases , including chemotherapy [ 13 ] , hormonal therapy [ 14 , 15 ] , surgery [ 1618 ] and radiotherapy ( local transcutaneous radiotherapy , hemibody irradiation , radionuclide therapy ) [ 3 , 19 , 20 ]  . 
a number of patients fail to benefit from these conventional treatments , however , and pain relief , when it does occur , is never achieved before 412 weeks [ 3 ]  . 
if , on the one hand , chemotherapy reduces the primary mass and secondary lesions in 20%80% of patients , on the other hand , it leads to drug resistance and recurrence of bone pain . in addition , myelodepression is common due to the toxicity of many drugs used in current treatment protocols . hormonal therapy is only effective in breast and prostate tumours , where it relieves pain from bone metastases in discussione attualmente nel trattamento palliativo delle metastasi ossee vengono utilizzate varie terapie , tra cui la chemioterapia [ 13 ] , la terapia ormonale [ 14 , 15 ] , la chirurgia [ 1618 ] e la radioterapia ( radioterapia transcutanea locale , irradiazione emicorporea , radioisotopoterapia con radionuclidi ) [ 3 , 19 , 20 ]  . 
alcuni pazienti non riescono a trarre benefici da questi trattamenti convenzionali e il sollievo dal dolore , quando si verifica , non avviene mai prima di 412 settimane [ 3 ]  . 
se da un lato la chemioterapia riduce la massa primitiva e le lesioni secondarie nel 20%80% dei pazienti , dallaltro induce farmacoresistenza e ripresa del dolore osseo ; inoltre frequente linsorgenza di mielodepressione data la tossicit di molti farmaci utilizzati negli attuali protocolli terapeutici . 
la terapia ormonale efficace solamente nei tumori della mammella e della prostata , dove tuttavia induce sollievo dal dolore delle metastasi ossee in pi del 70% dei casi [ 14 ]  . 
infine la radioterapia locale gravata nel 60% dei casi da effetti tossici ( nausea , vomito e diarrea ) e la radioisotopoterapia da una parte dimostra efficacia nei soggetti con metastasi ossee multiple , dallaltra non considerata trattamento standard per le metastasi singole . sulla base di queste considerazioni i farmaci analgesici ( oppioidi e fans ) spesso rimangono lunica alternativa nel dolore metastatico e in alcuni casi rimangono lunica alternativa per la palliazione della sintomatologia . 
 noto che gli effetti collaterali , soprattutto nei trattamenti prolungati , sono costipazione , grave limitazione dello stato fisico e mentale , nonch cirrosi e coma epatico . diversi autori hanno studiato varie strategie alternative per il trattamento del dolore da metastasi ossea che comportano lutilizzo di metodiche percutanee imagingguidate con lo scopo di ablare le lesioni focali ossee : etanolo , termoterapia interstiziale laser - indotta , ablazione con radiofrequenza ( rfa ) percutanea e , pi recentemente , crioablazione [ 2 , 4 , 11 ]  . 
la nostra esperienza si riferisce alla risposta di 10 pazienti al trattamento della sintomatologia dolorosa con diverse tecniche di radiologia interventistica che includono tecniche ablative ( termoablazione con rf , termoablazione con radiofrequenza plasma - mediata , termoablazione con microonde ) associate o meno alla 620 radiol med ( 2009 ) 114 : 608625 > 70% of cases [ 14 ]  . 
finally , local radiotherapy produces toxic effects ( nausea , vomiting and diarrhoea ) in 60% of cases , and radionuclide therapy is effective in patients with multiple bone metastases but is not considered standard treatment for a single metastasis . on the basis of these considerations , analgesics ( opioids and nsaids ) are often the only option for metastatic pain , and in some cases for palliation of symptoms . 
the world health organization ( who ) [ 21 ] recommends a three - step approach : ( 1 ) initial treatment with nsaids such as aspirin , ibuprofen , naproxen , ( 2 ) if pain persists , use of mild opioids such as codeine and hydrocodone , ( 3 ) for persisting moderate or severe pain , high doses of opioids such as morphine , hydromorphone , or fentanyl as an intermittent or continuous infusion . 
known side - effects , above all in long - term treatments , are constipation , severe physical and mental impairment , cirrhosis and hepatic coma . several authors have investigated alternative approaches to painful bone metastases , all of which involve using percutaneous image - guided methods to ablate focal bone lesions : ethanol , laser interstitial thermal therapy , percutaneous rf ablation and , more recently , cryoablation [ 2 , 4 , 11 ]  . 
this study reports on the response of ten patients treated with different interventional radiology techniques , namely , ablation techniques ( rf ablation , plasma - mediated rf ablation , microwave ablation ) used alone or in combination with cementoplasty . 
in agreement with the literature [ 11 ] , we treated patients with up to two bone metastases ( lytic or mixed , but not osteoblastic ) causing acute disabling pain . pain was poorly controlled with conventional treatments . ablation techniques are successful in relieving pain only if the needle is placed at the interface between soft tissue and bone . 
the advantage of rf ablation lies in the use of electrodes that have a smaller diameter compared with the microwave antennas , an aspect that should theoretically reduce complications related to electrode deployment . 
microwave treatment has the advantage of shorter procedure times and the ability to ablate larger lesions than those usually treated with rf , thanks to the possibility of placing two antennas simultaneously indeed , in one of our patients , its use was cementoplastica . 
in accordo con le indicazioni della letteratura [ 11 ] , sono stati trattati pazienti che non avessero pi di due metastasi ossee , che queste fossero litiche o miste , ma non blastiche , e che , soprattutto , fossero causa di dolore acuto e invalidante . 
in questi pazienti , il followup non si ferma solo al dato di diagnostica per immagini , ma si concentra sulla efficacia clinica del trattamento , ossia sulla riduzione del dolore . in questo studio sono state trattate tre lesioni solo con radiofrequenza e una solo con microonde . 
gli svantaggi invece rispetto allimpiego di microonde sono dati da tempi lunghi di procedura ( circa 30 minuti ) e dalla necessit di effettuare pi posizionamenti in caso di ampio volume da ablare . 
il trattamento con microonde ha invece il vantaggio di giovare di una procedura pi breve e di poter ablare lesioni di dimensioni maggiori rispetto a quelle usualmente trattate con radiofrequenza , ci per la possibilit di posizionare due antenne contemporaneamente . 
infatti , in una nostra paziente , lampia superficie di contatto softtissue - osso ha giustificato il suo utilizzo : lablazione ha garantito in un tempo inferiore rispetto alla rf un volume di necrosi superiore . 
inoltre , dal punto di vista della qualit dellablazione , studi sperimentali e clinici [ 10 , 22 , 23 ] hanno evidenziato che il trattamento con microonde garantisce il raggiungimento di temperature intra - lesionali pi elevate , una minore dispersione di calore e quindi unefficacia superiore rispetto alla termoablazione con rf nelle lesioni in prossimit dei vasi sanguigni . 
infine con la metodica delle microonde la diminuzione del dolore stata pi sensibile ( 2 punti bpi ) dopo 24 ore dal trattamento rispetto alle altre metodiche utilizzate ( 1 punto bpi ) , per poi allinearsi con i dati ottenuti dalle altre tecniche nelle fasi successive . 
 le metodiche di termoablazione con radiofrequenza e con microonde hanno comunque linconveniente di non poter essere utilizzate nelle metastasi osteoddensanti , dato che il tessuto osseo non favorisce la conduzione di calore ( radiofrequenza ) n lagitazione di molecole dacqua ( microonde )  . nelle metastasi osteoddensanti possibile utilizzare la crioablazione che ha per lo svantaggio di impiegare aghi di grosse dimensioni e di avere tempi lunghi di procedura [ 2 ]  . inoltre sia la radiofrequenza che le microonde possono provocare lesioni termiche ad organi adiacenti . 
per ovviare radiol med ( 2009 ) 114 : 608625 justified by the extensive contact area between soft tissue and bone , and the ablation procedure produced a larger necrosis volume in a shorter time compared with rf . 
in addition , from the viewpoint of the quality of ablation , experimental and clinical studies [ 10 , 22 , 23 ] have shown that microwave treatment provides higher intralesional temperatures and less heat dispersion and consequently better effectiveness compared with rf ablation in lesions located in proximity to blood vessels . 
finally , microwave ablation provided greater pain relief [ 2 points on the brief pain inventory ( bpi ) ] than did the other methods ( 1 point bpi ) 24 h after treatment before becoming aligned with the other methods at subsequent follow - up assessments . 
 rf and microwave ablation methods both suffer the same limitation of not being applicable to osteoblastic lesions , as bone tissue does not allow the conduction of heat ( radiofrequency ) or the agitation of water molecules ( microwaves )  . 
to avoid this complication , several possibilities exist : to create an air ( in microwave ) interface or water ( in rf ablation ) interface to limit the ablation area ; to place thermocouples close to the structures at risk of thermal injury , as these can guide the operator in continuing or discontinuing the procedure by providing feedback on temperature ; to use bipolar rf , which through an electrode thermally shielded by another opposing electrode ensures well - delimited ablations [ 24 ] ; to use plasma - mediated rf ablation [ 25 ]  . a distinctive feature of plasma - mediated rf ablation ( coblation ) is the ability to release , in an extremely precise manner , a minimal amount of heat ( unlike conventional electrosurgery ) , a crucial factor in treating vertebral lesions . the low rf energy ( 100 khz ) is thus used to excite the electrolytes in a conductive medium ( plasma ) , such as saline solution or the aqueous intracellular environment , creating an area of highly ionised particles immediately adjacent to the electrode tip . 
this molecular dissociation process converts the ablated tissue into gas , creating a cavity at the treatment site [ 25 ] , which accounts for its suitability for osteolytic lesions . coblation in this study was used for a metastasis to a rib in a patient who had undergone right liver resection for hepatocarcinoma and had shown , on preprocedural ct , a bowel loop adjacent to the inner aspect of the affected rib . coblation was used to avoid damaging the bowel wall , and indeed , it allowed for extreme precision in delimiting the ablation margins . with regard to our results , in all cases pain , reduction a tale complicanza , si hanno diverse possibilit : creare uninterfaccia o con aria ( nelle microonde ) o con acqua ( nellablazione con rf ) per limitare il territorio di ablazione . 
possibile inoltre posizionare termocoppie a ridosso delle strutture a rischio di danno termico , che misurando la temperatura durante la procedura , sono utili alloperatore nel decidere se interrompere o meno lablazione ; utilizzare la radiofrequenza bipolare , che attraverso un elettrodo schermato termicamente da un secondo elettrodo opposto al primo , garantisce ablazioni ben definite [ 24 ] ; utilizzare la radiofrequenza plasma - mediata [ 25 ]  . nella tecnica di termoablazione con radiofrequenza plasma - mediata ( coblazione ) caratteristica identificativa la capacit di cedere , in maniera molto precisa , una quantit minima di calore ( contrariamente allelettrochirurgia convenzionale ) , fattore importante per esempio nel trattamento di lesioni vertebrali . 
la bassa energia di radiofrequenza ( 100 khz ) viene quindi utilizza per eccitare gli elettroliti in un mezzo conduttivo ( plasma ) , come una soluzione fisiologica o lambiente acquoso intracellulare , in modo da formare unarea di particelle altamente ionizzate nellarea immediatamente adiacente alla punta del dispositivo . 
questo processo di dissociazione molecolare converte il tessuto ablato in gas e in questo modo si viene a creare una cavit nella zona del trattamento [ 25 ] , trovando dunque particolare indicazione nelle lesioni osteolitiche . la coblazione stata utilizzata in questo studio per la lesione costale di un paziente che , in seguito ad epatectomia destra per exeresi di una neoplasia epatica , evidenziava allesame tc , eseguito prima del trattamento , unansa intestinale adiacente alla faccia interna della costa interessata dalla lesione . 
per evitare di danneggiare la parete dellansa , stata impiegata la coblazione che , in effetti , ha dimostrato notevole precisione nella definizione dei limiti dellablazione e ne ha quindi giustificato limpiego . in merito ai risultati , in tutti i casi abbiamo ottenuto un decremento del dolore tale da dimezzare , allottava settimana dal trattamento , la dose di equivalenti di morfina necessari per controllare il dolore . 
qualunque sia la tecnica ablativa utilizzata , il meccanismo di riduzione del dolore non ancora chiaro , ma vi sono alcune ipotesi : distruzione delle fibre sensoriali del periostio e della corticale ; decompressione meccanica del volume tumorale ; distruzione delle cellule tumorali che producono citochine come tnf ( fattore di necrosi tumorale alfa ) e interleuchine che promuovono la trasmissione del dolore ; inibizione dellattivit degli osteoclasti [ 26 , 27 ]  . 
regardless of the ablation technique used , the mechanism by which pain is reduced is not understood , although several hypotheses have been proposed : destruction of the periosteal and cortical sensory nerve fibres , mechanical decompression of the tumour volume , destruction of tumours cells that produce cytokines such as tumour necrosis factor alfa ( tnf - ) and interleukins that promote pain transmission , and inhibition of osteoclast activity [ 26 , 27 ]  . 
 [ 28 ] , in a study of 12 patients , demonstrated that rf is a safe and effective treatment that improves quality of life and reduces analgesic use in patients with bone metastases . 
 [ 29 ] reported major complications in three patients : one second - degree burn at the grounding - pad site , one case of transient bladder and bowel incontinence and one case of fracture of the acetabulum 6 weeks after rf treatment of an acetabular lesion . 
studies of coblation on bone lesions [ 25 ] investigated the use of rf ablation to treat vertebral lesions only ( for which no complications were reported ) , so we are unable to compare the results with our experience , as we used rf treatment in a costal lesion . 
 [ 9 ] presented the first experimental and clinical results of the use of microwave ablation in bone metastases , reporting excellent technical results in terms of volume of necrosis , speed of the procedure and absence of complications . 
 [ 29 ] , there may be a new rise in morphine - dose requirements long after treatment , indicative of the development of pain at other sites of metastases . 
 [ 28 ] , studiando 12 pazienti , hanno dimostrato la sicurezza e lefficacia della rf per la riduzione del dolore , il miglioramento della qualit della vita e la riduzione delluso di analgesici nei pazienti con metastasi che coinvolgevano losso . 
 [ 29 ] che ha considerato 43 pazienti , stato segnalato un incremento della dose di oppiodi al primo giorno dal trattamento e un successivo decremento di pi del 50% . sulla base di unattenta selezione dei pazienti , nella nostra casistica non si sono verificate complicanze probabilmente anche per lesiguo numero di pazienti ( 6 ) trattati con rf . 
 [ 29 ] si sono invece verificate complicanze maggiori in 3 pazienti : uno ha subito unustione di secondo grado nellarea cutanea della messa a terra , un altro ha accusato incontinenza vescicale e intestinale e infine in un paziente si verificata una frattura acetabolare della zona trattata a 6 settimane dal trattamento con rf . 
gli studi effettuati con la coblazione su lesioni ossee [ 25 ] hanno preso in esame solo il trattamento di lesioni vertebrali ( per le quali per altro non sono riportate complicanze ) e quindi non possibile fare un confronto dato che nel nostro studio la metodica stata utilizzata in una lesione costale . 
 [ 9 ] hanno redatto i primi studi sperimentali e clinici sullutilizzo della termoablazione con microonde nelle metastasi ossee , riportando un ottimo risultato tecnico sia in termini di volume di necrosi , sia di rapidit della procedura , sia di assenza complicanze . 
 [ 29 ] , possibile che vi sia , a distanza dalla procedura , un nuovo aumento della dose di morfina , compatibile con linsorgenza di nuove localizzazioni ossee in siti diversi da quello trattato . 
 [ 6 ] , pazienti sottoposti a rfa associata a cementoplastica per voluminose metastasi ossee coinvolgenti anche la regione extra - ossea hanno ottenuto una riduzione della dose di analgesici solo nel 41% dei casi ; le voluminose dimensioni delle lesioni trattate giustificano il divario tra questo risultato e quello ottenuto dallo studio di callstrom et al . 
 [ 28 ] ( trattamento solo con rf )  . nel nostro studio sono state trattate con cementoplastica le lesioni che , in relazione alla sede , necessitavano di una stabilizzazione . 
in all cases , in agreement with the literature , the results achieved were excellent , not only as concerns stabilisation and thus prevention of pathological fractures , but also as concerns pain relief , achieved in 100% of cases . 
 [ 6 ] emphasised that the technique is easy to perform , inexpensive and minimally invasive , even though the effects of combining rf ablation and cementoplasty with radiotherapy are not well known . 
 [ 34 ] believe that the coagulation necrosis induced by rf facilitates even cement distribution within the ablated lesion and that cementoplasty enhances the analgesic effect by stabilising the bone . 
 the results obtained have been excellent , with pain relief being achieved in 100% of cases , both in case reports [ 30 , 31 , 34 ] and in larger series [ 6 , 28 , 32 , 33 ] , as well as in our small patient series . 
it should be emphasised that , whereas studies performed with rf or cementoplasty alone achieved 100% technical success and 80%100% pain relief [ 35 , 36 ] , combined rf and cementoplasty ensured pain relief in all cases but a slightly lower rate of technical success . conclusions there is no agreement as to the best treatment for bone metastases . 
it is clear that the rationale for the use of these treatments is that , when indicated , they ensure considerably faster pain relief compared with conventional treatments , providing a substantial improvement in quality of life in a short time . 
however , further investigations on larger patient populations , as well as randomised controlled studies , are required to validate the effectiveness of these treatments . con cementoplastica dopo rfa e 2 con vertebroplastica dopo radiofrequenza plasma - mediata . 
in tutti i casi , in accordo con i dati della letteratura , sono stati ottenuti ottimi risultati non solo nellambito della stabilizzazione e quindi nella prevenzione della possibile frattura patologica , ma soprattutto in quello della riduzione della sintomatologia dolorosa , che si avuta nel 100% dei casi . 
 [ 34 ] ritengono che la necrosi coagulativa indotta dalla rf faciliti lomogenea distribuzione del cemento allinterno della lesione ablata e che la cementoplastica , per effetto della stabilizzazione ossea , migliori leffetto antalgico . 
 i risultati ottenuti sono stati ottimi , sia nei case report [ 30 , 31 , 34 ] che nelle casistiche [ 6 , 28 , 32 , 33 ] dove la riduzione del dolore si verificata nel 100% dei casi analogamente a quanto emerso nellesperienza personale , peraltro numericamente esigua . 
va sottolineato che , se da un lato negli studi effettuati solo con radiofrequenza o solo con cementoplastica si ottenuto un successo tecnico del 100% a fronte di un risultato in termini di riduzione del dolore compreso tra l80% e il 100% [ 35 , 36 ] , dallaltro la tecnica combinata di radiofrequenza e cementoplastica ha assicurato la riduzione del dolore in tutti i casi trattati , ma un successo tecnico in una percentuale leggermente inferiore . conclusioni attualmente non esistono ancora pareri concordi sulla metodica migliore nel trattamento delle metastasi ossee . 
appare evidente che ci che giustifica il ricorso a queste terapie il fatto che , laddove vi sia lindicazione , esse garantiscono una riduzione della sintomatologia dolorosa notevolmente pi rapida rispetto alle terapie convenzionali apportando , in breve tempo , un sensibile miglioramento della qualit della vita . 
midiri3 1dipartimento di radiologia e cardiologia , azienda ospedaliero - universitaria di parma , parma , italy 2dipartimento di radiologia e cardiologia , erasmus medical center , rotterdam , the netherlands 3dibimel , sezione di scienze radiologiche , universit di palermo , palermo , italy 4dipartimento di ostetricia e ginecologia e scienze radiologiche delluniversit di catania , catania , italy 5dipartimento di radiologia , universit degli studi di trieste , trieste , italy 6dipartimento di radiologia , universit degli studi di verona , verona , italy correspondence to : f . 
cademartiri , dipartimento di radiologia , c / o piastra tecnica piano 0 , azienda ospedaliero - universitaria di parma , via gramsci 14 , parma , italy , tel . : + 39 - 0521 - 703516 / 3756 , fax : + 39 - 0521 - 703630 , e - mail : filippocademartiri@hotmail.com received : 11 february 2008 / accepted : 15 september 2008 / published online : 15 april 2009 springer - verlag 2009 abstract purpose . 
msct - ca was performed in 43 patients ( 31 men , 12 women , mean age 58.87.7 years ) with stable angina after a routine diagnostic workup involving stress ecg and conventional ca . 
lac - tcms stata eseguita in 43 pazienti ( 31 uomini , 12 donne , et media 58 , 87 , 7 anni ) con angina stabile sottoposti ad iter diagnostico comprendente la prova da sforzo con ecg e langiografia coronarica convenzionale . 
lac - tcms ha migliorato la probabilit post - test di una rilevante ca dal 50% fino all86% dopo una prova da sforzo negativa , e 514 radiol med ( 2009 ) 114 : 513523 conclusions . 
lac - tcms uno strumento non invasivo potenzialmente utile nelliter diagnostico dei pazienti con angina stabile in grado di rendere nota od escludere una significativa ca . parole chiave imaging coronaropatia aterosclerotica tomografia computerizzata angiografia coronarica prova da sforzo introduction introduzione stress testing has shown to be of prognostic value in patients with stable angina , as it is useful for selecting patients to be studied with coronary angiography ( ca ) and consequently orienting subsequent revascularisation strategies . 
current guidelines recommend stress testing in the diagnostic workup of patients with stable angina , and exercise electrocardiography ( ecg ) is the first test most patients undergo [ 14 ]  . 
however , the usefulness of msct - ca as an alternative or an adjunct to stress testing in the diagnostic workup of patients with stable angina in clinical practice has not been established . this study aimed to assess the diagnostic value of stress testing and msct - ca , both jointly and independently , in identifying or excluding significant coronary artery disease ( cad ) in a consecutive cohort of patients with stable angina . materials and methods population we evaluated 43 patients ( 31 men , 12 women , mean age 58.87.7 years ) selected according to the following inclusion criteria : stable angina , sinus rhythm and ability to hold breath for at least 12 s ( table 1 )  . 
exclusion criteria were a history of allergic reactions , serum creatinine > 120 mol / l , thyroid disease , ecg abnormalities at rest ( st - segment depression 1 mm at rest , left bundle branch block ) precluding reliable evaluation and inability to perform msct - ca before conventional coronary angiography ( cca ) owing to logistic reasons . 
the study was approved by our institutions ethics committee , and all patients gave their informed consent . la prova da sforzo si dimostrata di valore prognostico nei pazienti con angina stabile poich utile nella selezione dei pazienti da indirizzare verso la coronarografia e conseguentemente in grado di guidare le successive strategie di rivascolarizzazione . 
le attuali linee - guida raccomandano la prova da sforzo nelliter diagnostico dei pazienti con angina stabile ; il test ergometrico con ecg il primo esame effettuato nella maggior parte dei pazienti [ 14 ]  . 
langiografia coronarica mediante tomografia computerizzata multistrato ( ac - tcms ) una tecnica non invasiva che pu valutare con efficacia le stenosi coronariche significative in popolazioni selezionate di pazienti [ 514 ]  . 
tuttavia non ancora noto il valore dellactcms in alternativa o come aggiunta alla prova da sforzo nelliter diagnostico dei pazienti con angina stabile nella routine clinica . con questo studio intendiamo stabilire il valore diagnostico della prova da sforzo e dellac - tcms , insieme ed indipendentemente , in una coorte consecutiva di pazienti con angina stabile , allo scopo di rivelare o escludere una significativa coronaropatia aterosclerotica ( ca )  . materiali e metodi popolazione sono stati valutati 43 pazienti ( 31 uomini , 12 donne , et media 58 , 87 , 7 anni ) nel rispetto dei seguenti criteri dinclusione : angina stabile , ritmo cardiaco sinusale e capacit di mantenere lapnea inspiratoria per almeno 12 secondi ( tabella 1 )  . 
altri criteri di esclusione erano : diatesi allergica , creatinina sierica > 120 mol / l , tireopatie , anomalie ecg a riposo ( sotto - slivellamento 1 mm del tratto st a riposo , blocco di branca sinistro ) precludenti unaffidabile interpretazione , ed impossibilit di eseguire unac - tcms prima dellangiografia coronarica radiol med ( 2009 ) 114 : 513523 table 1 patient characteristics tabella 1 caratteristiche dei pazienti risk factors ( % ) number of patients ( % ) fattori di rischio ( % ) numero pazienti ( % ) hypercholesterolaemia systemic hypertension smoking family history of early onset cad obesity ( bmi 30 ) diabetes mellitus treatment aspirin - blockers ace inhibitors / at - 2 antagonists calcium antagonists nitrates ( systemic ) statins 30 ( 70 ) 29 ( 67 ) 18 ( 42 ) 16 ( 37 ) 11 ( 26 ) 6 ( 14 ) 41 ( 95 ) 36 ( 84 ) 14 ( 33 ) 14 ( 33 ) 13 ( 30 ) 30 ( 70 ) ipercolesterolemia ipertensione sistemica fumo storia familiare di ca precoce obesit ( bmi30 ) diabete mellito trattamento aspirina - bloccanti ace inibitori / at - ii antagonisti calcio - antagonisti nitrati ( per via sistemica ) statine 30 ( 70 ) 29 ( 67 ) 18 ( 42 ) 16 ( 37 ) 11 ( 26 ) 6 ( 14 ) 41 ( 95 ) 36 ( 84 ) 14 ( 33 ) 14 ( 33 ) 13 ( 30 ) 30 ( 70 ) cad , coronary artery disease ; bmi , body mass index ca , coronopatia aterosclerotica ; bmi , body mass index patient preparation patients with sinus rhythm > 70 beats per minute ( bpm ) received 100 mg of metoprolol orally 45 min before the scan unless contraindicated ( severe heart failure or chronic obstructive pulmonary disease )  . scanning parameters , reconstruction and image analysis all patients underwent msct ( sensation 64 , siemens , forchheim , germany ) according to a previously described protocol [ 15 ]  . 
only coronary branches 2 mm in diameter were considered clinically relevant , as smaller ones are not amenable to percutaneous or surgical revascularisation . conventional coronary angiography all ct scans were performed within 4 weeks from cca . 
a single observer blinded to the msct - ca findings determined the diameter of all coronary branches using a quantitative technique ( caas , pie medical , maastricht , the netherlands )  . 
il comitato etico ha approvato il protocollo di studio e tutti i pazienti hanno fornito consenso informato scritto . preparazione dei pazienti i pazienti con un ritmo superiore ai 70 bpm hanno ricevuto per os una dose di 100 mg di metoprololo 45 min prima della scansione , a meno di eventuali controindicazioni ( grave scompenso cardiaco o broncopatia cronica ostruttiva )  . parametri di scansione , ricostruzione e analisi delle immagini tutti i pazienti sono stati sottoposti a tc multistrato ( sensation 64 , siemens , forchheim , germania ) secondo protocollo gi descritto [ 15 ]  . 
tutti i rami coronarici 2 mm sono stati indipendentemente analizzati mediante le convenzionali tecniche di post processing da due osservatori , non a conoscenza dei risultati dellacc ( vedi metodologia e postprocessing gi descritti [ 15 ] )  . 
sono stati valutati solo i rami coronarici di diametro superiore a 2 mm in quanto clinicamente rilevanti , ossia gli unici passibili di eventuale rivascolarizzazione percutanea o chirurgica . angiografia coronarica convenzionale tutte le scansioni tc sono state eseguite entro 4 settimane dallacc . 
un solo osservatore , alloscuro dei risultati 516 exercise stress testing the protocol for the bicycle exercise test involved an initial workload of 25 w followed by a 25 - w increment every 2 mheart rhythm and blood pressure were recorded at rest and at the end of each phase of the test . 
the results of the stress test were classified as positive when the st segment appeared horizontal or had a depression 1 mm for at least 6080 ms after the end of the qrs complex [ 3 ]  . 
cca revealed no significant stenoses in 26% ( 11 / 43 ) , singlevessel disease in 32% ( 14 / 43 ) and multivessel disease in 42% ( 18 / 43 ) of patients . 
in the remaining 37 patients , stress testing to identify significant cad had 79% sensitivity ( 95% ci 6292 ) , 67% specificity ( 95% ci 3490 ) , 88% positive predictive value ( 95% ci 7393 ) and 50% negative predictive value ( 95% ci 2272 )  . only one msct - ca was considered inconclusive as a result of motion artefacts related to high heart rate ( 84 bpm )  . in 42 patients , msct - ca to identify significant cad showed 100% sensitivity ( 95% ci 91100 ) , 90% specificity ( 95% ci 6199 ) , 97% positive predictive value ( 95% ci 8799 ) and 100% negative predictive value ( 95% ci 71100 )  . 
la ca stata ritenuta rilevante quando era presente almeno una stenosi significativa . prova da sforzo il protocollo del ciclo ergometro ha utilizzato un carico iniziale di 25 w , seguito da un incremento di 25 w ogni 2 minuti . 
il ritmo cardiaco e la pressione arteriosa sono stati registrati a riposo e alla fine di ogni fase del test da sforzo . un ecg a 12 derivazioni stato acquisito ogni minuto ; un ecg di monitoraggio del ritmo cardiaco a 3 derivate stato acquisito continuativamente . 
i risultati della prova da sforzo sono stati classificati come positivi quando il tratto st appariva orizzontale o sotto - slivellato 1 mm per almeno 6080 ms dopo la fine del complesso qrs [ 3 ]  . le prove da sforzo negative dei pazienti che non avevano raggiunto gli standard di riferimento stabiliti per et , sesso e peso sono state invece classificate come non conclusive . analisi statistica per valutare il valore diagnostico della prova da sforzo e dellac - tcms allo scopo di individuare o escludere i pazienti con una rilevante ca sono stati calcolati i seguenti parametri : sensibilit , specificit , valore predittivo positivo , valore predittivo negativo , rapporto di verosimiglianza positivo ( sensibilit / [ 1 - specificit ] ) e rapporto di verosimiglianza negativo ( [ 1 - sensibilit ] / specificit ] )  . risultati il 51% ( 22 / 43 ) dei pazienti ha ricevuto b - bloccanti prima della scansione ( frequenza cardiaca media 59 , 38 , 3 bpm )  . lacc ha rivelato lassenza di stenosi significative nel 26% ( 11 / 43 ) , malattia mono - vasale nel 32% ( 14 / 43 ) , e malattia multi - vasale nel 42% ( 18 / 43 ) dei pazienti . 
pertanto , la probabilit pre - test di una ca significativa era del 74% . valore diagnostico del test da sforzo e dellac - tcms sei prove da sforzo sono state classificate come non conclusive . 
patients without significant cad were correctly identified at msct - ca in nine out of 11 cases owing to the presence of one false positive and of one inconclusive scan . diagnostic value of msct - ca in addition to stress testing msct - ca performed after the exercise stress test considerably improved posttest probability . 
in 42 pazienti lac - tcms volta allidentificazione di una ca significativa ha evidenziato : sensibilit 100% ( 95% ic : 91100 ) , specificit 90% ( 95% ic : 6199 ) , valore predittivo positivo 97% ( 95% ic : 8799 ) e valore predittivo negativo 100% ( 95% ic : 71100 )  . 
i pazienti senza ca significativa sono stati correttamente identificati dallac - tcms in 9 degli 11 casi per la presenza di un falso positivo e di una scansione non conclusiva . valore diagnostico dellac - tcms addizionale alla prova da sforzo limpiego dellac - tcms eseguita dopo la prova da sforzo ha prodotto degli apprezzabili miglioramenti nella probabilit post - test . 
limmagine mip mostra una stenosi significativa a livello dellacd prossimale ( testa di freccia ) , confermata dallangiografia coronarica convenzionale ( acc )  . with major cardiovascular risk factors , should undergo stress testing [ 16 , 17 ]  . 
some noninvasive tests have the advantage of providing additional information on ventricular dysfunction , blood pressure and heart rate response to stress , as well as on the degree of ischaemia . 
the choice of the most appropriate noninvasive diagnostic test in patients with known or suspected cad must take into account the patients ability to exercise , the presence of ecg abnormalities at rest and the sensitivity , specificity and costs of the technique . in clinical practice , patients with a positive stress test are subsequently studied with cca to direct patients to coronary angioplasty , surgical bypass or pharmacological treatment . 
conversely , patients with typical chest pain and negative stress test require further noninvasive diagnostic investigations in the context of a periodic clinical follow - up in a day - hospital setting , according to the level of clinical suspicion of cad based on case history , physical examination and ecg at rest . 
in a proportion of cases , when symptoms persist or worsen without other causes accounting for this , cca becomes necessary . recent guidelines / recommendations and publications consider appropriate the use of msct - ca in intermediaterisk patients [ 1922 ]  . 
tutti i pazienti con dolore toracico tipico o atipico , specialmente in presenza dei fattori maggiori di rischio cardiovascolare , dovrebbero essere sottoposti alla prova da sforzo [ 16 , 17 ]  . 
i test non invasivi potrebbero , peraltro , rendere pi accurata la valutazione dei pazienti asintomatici a rischio intermedio [ 18 ]  . tuttavia , lacc spesso necessaria per dirimere la diagnosi quando il test da sforzo non conclusivo . 
alcuni test non invasivi hanno inoltre il vantaggio di fornire informazioni addizionali sulla disfunzione ventricolare , sulla risposta della pressione arteriosa e della frequenza allo stress e sul grado di ischemia . 
la scelta del pi appropriato test diagnostico non invasivo nei pazienti con ca nota o sospetta deve tenere conto della capacit di sostenere uno sforzo fisico , della presenza di anomalie elettrocardiografiche a riposo , della sensibilit , della specificit e dei costi . 
 radiol med ( 2009 ) 114 : 513523 nella routine clinica i pazienti con prova da sforzo positiva sono ulteriormente valutati mediante acc per indirizzare il paziente allangioplastica coronarica , al bypass chirurgico , o al trattamento farmacologico . 
i pazienti con dolore toracico tipico e prova da sforzo negativa , invece , richiedono ulteriori test diagnostici non invasivi nel contesto di un periodico follow - up clinico in regime di day hospital , secondo il livello di sospetto clinico di ca basato sullanamnesi , sullesame obiettivo , e sullecg a riposo . 
in un certo numero di casi , quando la sintomatologia permane o peggiora senza che altre cause possano giustificarla diventa necessaria lesecuzione dellacc . recenti linee guida / raccomandazioni e pubblicazioni riportano come appropriata lapplicazione della ac - tcms in pazienti con rischio intermedio [ 1922 ]  . 
in questa classe di pazienti la distribuzione pressoch dicotomica della malattia rende opportuno un test caratterizzato da alta sensibilit ed ancor pi con elevato valore predittivo negativo . nel nostro studio abbiamo valutato il valore diagnostico della prova da sforzo e dellac - tcms indipendentemente , e in particolare il valore addizionale dellac - tcms dopo prova da sforzo in 43 pazienti consecutivi con angina stabile e una probabilit pre - test medio - alta ( 74% ) di ca significativa . 
i nostri risultati mostrano che la prova da sforzo rivela correttamente la presenza o lassenza di una ca significativa nel 65% ( 28 / 43 ) dei pazienti , mentre lactcms valuta correttamente tutti i pazienti con ca significativa ( 32 )  . 
lac - tcms ha evidenziato valori di sensibilit ( 100% ) , specificit ( 90% ) , valore predittivo positivo ( 97% ) e valore predittivo negativo ( 100% ) in linea con quelli di altri lavori presenti in letteratura [ 7 , 11 , 13 , 14 ]  . 
 [ 10 ] che riporta un valore predittivo negativo moderato ( 75% ) nella valutazione per paziente , tale da suggerire un limitato impatto diagnostico dellac - tcms nelliter diagnostico delle popolazioni ad alto rischio . 
 lac - tcms risulta particolarmente efficace nel migliorare la probabilit post - test di significativa ca dopo prova da sforzo negativa del 50% , un valore che implica unincertezza diagnostica tale da giustificare il proseguimento delliter diagnostico , fino all86% nel caso di ac - tcms positiva e fino allo 0% nel caso di ac - tcms negativa . 
pertanto , le informazioni aggiuntive fornite dallac - tcms hanno la capacit di influenzare il successivo management dei pazienti con angina stabile , ma con test da sforzo negativo ovvero con probabilit pre - test intermedia . nei pazienti con angina stabile e test da sforzo positivo , ovvero ad alta probabilit di significativa ca , lac - tcms risultata capace di migliorare ulteriormente la probabilit post - test fino al 100% nel caso di ac - tcms positiva e fino fig . 
le immagini mip e cmpr indicano la presenza di una stenosi significativa a livello dellarteria circonflessa prossimale , che stata confermata dallangiografia coronarica convenzionale ( acc )  . particular , the additional value of msct - ca after the stress test in 43 consecutive patients with stable angina and a medium - to - high ( 74% ) pretest probability of significant cad . 
our results showed that the stress test correctly identified the presence or absence of significant cad in 65% ( 28 / 43 ) of patients , whereas msct - ca correctly identified all patients with significant cad ( 32 )  . 
msct - ca had sensitivity ( 100% ) , specificity ( 90% ) , positive predictive ( 97% ) and negative predictive ( 100% ) values in line with those reported by previous studies [ 7 , 11 , 13 , 14 ]  . 
conflicts with these data , as they found a moderate negative predictive value ( 75% ) in the evaluation per patient , suggesting a limited diagnostic impact of msct - ca on the diagnostic workup of high - risk patient populations [ 10 ]  . 520 radiol med ( 2009 ) 114 : 513523 msct - ca was particularly effective in improving the posttest probability of significant cad after a negative stress test by 50% , a value that implies a diagnostic uncertainty that calls for further investigation , reaching 86% in the case of positive msct - ca and 0% in the case of negative msct - ca . 
hence , the additional information provided by msct - ca is able to influence the management of patients with stable angina but only in the case of a negative stress test , i.e. 
the current guidelines on stable angina recommend that patients with a high pretest probability should undergo noninvasive investigations followed by diagnostic cca to direct them to the most appropriate treatment . 
however , a targeted approach whereby diagnostic cca is performed in anticipation of an interventional procedure presents several disadvantages compared with a step - wise approach that allows the necessary time to inform the patient about the risks , benefits and therapeutic alternatives while permitting optimal hydration and pretreatment with oral antiaggregants prior performing angioplasty [ 4 ]  . the proposal to introduce msct - ca in the diagnostic algorithm of patients with typical chest pain raises some important questions . 
the cost of performing msct - ca may only be estimated in that it has not yet been assigned a procedure code in the national italian tariff nomenclator identifying it as a specialist service . 
this percentage can be reasonably expected to increase when the population is at high cardiovascular risk and decrease with a decreasing prevalence of the disease ( bayes theorem )  . 
furthermore , when symptoms persist in patients with a negative exercise test , further diagnostic investigations are normally used , such as myocardial perfusional scintigraphy or stress allo 0% nel caso di ac - tcms negativa . 
le attuali lineeguida dellangina stabile suggeriscono che i pazienti con alta probabilit pre - test dovrebbero essere sottoposti ad esami non invasivi e in seguito ad acc diagnostica per indirizzare questi pazienti verso il pi appropriato trattamento . 
un approccio mirato verso lacc diagnostica in previsione di una procedura interventistica presenta tuttavia diversi svantaggi , se confrontato ad un approccio a stadi , che garantisce il tempo necessario ad informare il paziente dei rischi , dei benefici e delle alternative terapeutiche e parimenti consente unidratazione ottimale e il pretrattamento con anti - aggreganti orali prima dellangioplastica [ 4 ]  . 
inoltre , tale sproporzione potrebbe aumentare se come atteso ci sar un necessario adeguamento del rimborso della ac - tcms . tuttavia , come dimostrato dal presente studio , il test ergometrico non ha unelevata predittivit specialmente quando negativo . 
questo determina un ritardo diagnostico in una significativa percentuale di pazienti ad alto rischio . ragionevole attendersi che tale percentuale aumenti quando la popolazione sia ad un rischio cardiovascolare decrescente per la minore prevalenza di malattia ( teorema di bayes )  . 
inoltre , quando tali pazienti con test ergometrico negativo continuano a lamentare sintomi , di regola passano ad ulteriori stadi diagnostici che possono essere costituiti dalla scintigrafia miocardica perfusionale o dallecocardiografia da stress . 
these two techniques , despite a higher diagnostic accuracy than exercise testing , nonetheless have sensitivity and specificity values no higher than 90% . it should be remembered that the correct performance of a stress test remains a basic requirement of clinical medicine . 
furthermore , pharmacological treatment with drugs such as digitalis , nitroderivatives , beta - blockers and some antidepressants needs to be suspended before the test so as not to affect the result . 
in the case of recent myocardial infarction , unstable angina , arrhythmias , severe aortic stenosis , severe heart failure , recent infarction , acute myocarditis / pericarditis and aortic aneurysm , conventional exercise stress testing is contraindicated . thromboembolism / pulmonary a second problem concerns the dose of ionising radiation . 
further studies are needed to better assess the cost - benefit ratio compared with other techniques . inoltre , essere ricordato che la terapia farmacologica con farmaci quali la digitale , i nitroderivati , i betabloccanti ed alcuni antidepressivi dovrebbe essere sospesa prima dellesecuzione del test al fine di non condizionarne il risultato . 
in caso di infarto miocardico recente , angina instabile , aritmie , stenosi aortica severa , scompenso cardiaco severo , trombo - embolia / infarto polmonare recenti , miocardite / pericardite in fase acuta , aneurisma dellaorta , il test da sforzo tradizionale controindicato . una seconda problematica riguarda la dose di radiazioni ionizzanti . 
se vero che la dose riportata pari a 1521 msv superiore a quella di unacc , bisogna sottolineare che questultima comporta un rischio di complicanze maggiori pari al 2% [ 23 ]  . 
se poi effettuiamo il paragone con la scintigrafia miocardica bisogna ricordare che questultima espone il paziente ad una dose di radiazioni ionizzanti analoga [ 24 ]  . studi prospettici pi ampi sono necessari per meglio giudicare il valore diagnostico dellac - tcms addizionale al test da sforzo nei pazienti a bassa probabilit pre - test di ca significativa ( pazienti con angina atipica o dolore toracico non cardiaco ) che teoricamente potrebbero trarre maggior beneficio da una metodica non invasiva . le recenti tecnologie a doppia sorgente hanno introdotto ulteriori miglioramenti dal punto di vista della compliance della metodica ac - tcms nei confronti di frequenze cardiache pi elevate ( ad esempio > 6570 bpm ) e rispetto alle strategie di limitazione della dose di radiazioni ionizzanti . 
questi miglioramenti renderanno progressivamente pi accettabile il compromesso tra rischio necessario della procedura e beneficio dellinformazione diagnostica . conclusioni la prova da sforzo fornisce informazioni aggiuntive che agevolano liter diagnostico dei pazienti con angina stabile , ma appare di limitato valore diagnostico nellindividuazione di una ca significativa , specialmente nella popolazione che ricade nella fascia di rischio intermedio . 
lesecuzione di unac - tcms dopo la prova da sforzo migliora marcatamente la probabilit post - test della presenza di una significativa ca specialmente quando il test ergometrico sia negativo o inconclusivo . 
twenty - six consecutive patients , 14 with liver metastases ( ten from colorectal cancer ; four from carcinoid tumours ) and 12 with biliary cancers ( ten klatskin tumours ; one gallbladder tumour ; one intrahepatic cholangiocarcinoma ) with insufficient predicted future remnant liver ( frl ) underwent right pve to induce hypertrophy of the contralateral hemiliver prior to surgical resection . 
the frl volume increased by 5%25% ( 15% on average ) after right pve in patients with liver metastases and by 9%19% ( 14% on average ) in patients with biliary cancers . 
in all patients , the ratio of frl to functional liver volume was 30% after right pve . no postoperative deaths due to severe liver failure occurred in the 20 patients who underwent extended hepatectomy . 
lo scopo del nostro studio retrospettivo stato di valutare lefficacia dellembolizzazione del ramo portale destro ( rpve ) come trattamento pre - chirurgico per indurre lipertrofia del fegato sinistro in pazienti candidati a resezione epatica . 
ventisei pazienti consecutivi , 14 portatori di metastasi epatiche ( colon - retto , 10 pazienti ; carcinoide , 4 pazienti ) e 12 portatori di neoplasia delle vie biliari ( neoplasia di klatskin , 10 pazienti ; neoplasia della colecisti , 1 paziente ; colangiocarcinoma intraepatico , 1 paziente ) con potenziale riserva funzionale epatica ( frl ) insufficiente sotto stati sottoposti a rpve come trattamento pre - chirurgico . 
il volume del frl ha dimostrato un incremento volumetrico compreso tra il 5%25% ( 15% in media ) dopo rpve nei pazienti con metastasi epatiche e tra il 9%19% ( 14% in media ) in pazienti con neoplasie biliari . 
lembolizzazione portale allarga le indicazioni alla chirurgia resettiva epatica in pazienti con metastasi epatiche e neoplasie delle vie biliari con potenziale riserva funzionale insufficiente . 554 radiol med ( 2009 ) 114 : 553570 keywords right portal vein embolisation hepatic functional reserve ct hepatic volumetry parole chiave embolizzazione del ramo portale destro riserva funzionale epatica tc volumetria epatica introduction introduzione the curative treatment of primary and secondary liver cancers that ensures long - term survival is surgery . 
more than 45% of liver tumours , both primary and secondary , require extended liver resection with disease - free margins . postoperative death after extended hepatectomy , which ranges from 3.2%7% in patients with a normal hepatic functional reserve to 32% in patients with cirrhosis , is most often caused by an insufficient future remnant liver ( frl )  . postoperative liver failure has been shown to be directly related to frl volume , and preoperative procedures to induce liver regeneration have been proposed [ 13 ]  . 
on the basis of experimental studies in 1920 that showed that ligation of a portal branch led to atrophy of the ipsilateral liver parenchyma and hypertrophy of parenchyma with preserved portal flow , makuuchi et al . 
 [ 4 ] in 1990 used portal occlusion as a preoperative treatment to extend the indication for curative liver resection to patients with biliary cancers . embolisation of a portal branch is currently indicated when the volume of the predicted frl is insufficient to ensure an adequate postoperative hepatic functional reserve . 
separate determinations of liver volume and tumour volume makes it possible for hepatic functional reserve to be estimated with computed tomography ( ct ) and magnetic resonance ( mr ) imaging by subtracting tumour volume from total liver volume , i.e. 
the potential functional reserve of frl as a ratio between the frl and the total functional liver mass . the purpose of this retrospective study was to assess the effectiveness of right portal vein embolisation ( pve ) as a preoperative treatment in noncirrhotic patients in whom the predicted functional reserve of the frl , calculated with ct , was insufficient for radical liver resection . 
in pi del 45% dei tumori del fegato , sia primitivi che secondari , necessaria unampia chirurgia resettiva epatica che assicuri margini di resezione liberi da malattia . la principale causa di morte post - chirurgica dopo epatectomie estese , che oscilla tra il 3 , 2% e 7% nei pazienti con normale riserva funzionale epatica e fino al 32% nei pazienti con cirrosi , spesso un insufficiente future remnant liver ( frl )  . 
stato dimostrato che linsufficienza epatica post - chirurgica direttamente correlata al volume del frl e sono state proposte procedure pre - chirurgiche che inducono la rigenerazione epatica [ 13 ]  . 
sulla base di studi sperimentali che nel 1920 avevano dimostrato che la legatura di un ramo portale determinava latrofia del parenchima epatico tributario e lipertrofia del parenchima epatico con flusso portale conservato , makuuchi et al . 
 [ 4 ] gi nel 1990 hanno usato per primi locclusione portale come trattamento pre - chirugico per ampliare lindicazione a chirurgia resettiva epatica curativa in pazienti portatori di neoplasie delle vie biliari . 
lembolizzazione di un ramo portale attualmente indicata quando il volume del potenziale frl inadeguato ad assicurare unadeguata riserva funzionale epatica postchirurgica e quindi il calcolo della volumetria epatica necessario per selezionare i pazienti da sottoporre a trattamento pre - chirurgico . 
il calcolo separato del volume epatico e del volume tumore permette oggi alla tomografia computerizzata ( tc ) ed alla risonanza magnetica ( rm ) di valutare la riserva funzionale epatica sottraendo al volume totale epatico il volume tumore e quindi la potenziale riserva funzionale del frl come rapporto del frl sulla massa epatica totale funzionante . scopo primario di questo studio retrospettivo di valutare lefficacia dellembolizzazione del ramo portale destro ( rpve ) come trattamento pre - chirurgico in pazienti non cirrotici nei quali la potenziale riserva funzionale del frl , calcolata mediante tc , era insufficiente per una chirurgia resettiva epatica oncologicamente radicale . 
 materials and methods materiali e metodi between november 1996 and september 2007 , 26 noncirrhotic patients ( 12 men and 14 women , aged 3581 years ; mean age 58 years ) underwent pve procedures . 
dodici pazienti erano portatori di neoplasie delle vie biliari [ 5 ] : neoplasia di klatskin ( 10 pazienti ) classificati secondo bismuth come tipo ii ( interruzione convergenza biliare principale , 6 pazienti ) , tipo iiia ( interruzione radiol med ( 2009 ) 114 : 553570 confluence , n = 6 ) , type iiia ( obstructed main confluence extending to the right secondary confluence , n = 2 ) , type iv ( obstructed primary and secondary confluence bilaterally , n = 2 ) , gallbladder cancer ( n = 1 ) and intrahepatic cholangiocarcinoma ( n = 1 )  . 
in particular , the lesions were located in the right liver lobe and in segment iv in three patients ( two with colorectal metastases and one with carcinoid metastases ) and in the right liver lobe in segments iv and ii in two patients ( one with colorectal metastases one with carcinoid metastases )  . 
all patients with hepatic metastases from colorectal cancer underwent chemotherapy ( three to six cycles , four cycles on average ) , with partial response ( no less than 30% ) according to the response evaluation criteria in solid tumours ( recist ) criteria [ 6 ] or with stable disease . 
in the patient with bilobar colorectal metastases in segments ii and iv of the left liver , right pve was preceded by excision of the left lobar metastases , a procedure known as two - stage hepatectomy . 
all patients with klatskin tumours underwent percutaneous biliary drainage bilaterally ( n = 6 ) or on the left side only ( n = 4 ) ; a prerequisite for right pve was biliary drainage of the frl with reduction of bilirubin ( < 3 mg / dl )  . 
in particolare le lesioni erano localizzate nel fegato destro e nel iv segmento epatico in 3 pazienti ( 2 da neoplasia colo - rettale e 1 da carcinoide ) ; nel fegato destro , nel iv e nel ii segmento in 2 pazienti ( 1 da neoplasia colo - rettale e 1 da carcinoide )  . tutti i pazienti portatori di metastasi epatiche da neoplasie colo - rettali hanno effettuato chemioterapia ( 36 cicli con una media di 4 cicli ) con una risposta parziale ( non inferiore al 30% ) secondo i criteri recist ( response evaluation criteria in solid tumours ) [ 6 ] o con malattia stabile , che ha preceduto lembolizzazione portale con un intervallo di tempo di almeno 28 giorni . 
nel paziente portatore di metastasi bilobari da neoplasia colo - rettale , localizzate nel fegato sinistro , in corrispondenza del ii oltre che nel iv segmento , la rpve stata preceduta dalla metastesectomia delle metastasi del fegato sinistro , two stage hepatectomy . 
i pazienti con metastasi da carcinoide non sono stati sottoposti a terapia neo - adiuvante : quando possibile , la resezione epatica rappresenta in questi pazienti il miglior trattamento in termini di sopravvivenza a lungo termine [ 7 ]  . tutti i pazienti portatori di neoplasie di klatskin sono stati sottoposti a drenaggio biliare per via transcutanea dei due emisistemi biliari ( 6 pazienti ) o dellemisistema di sinistra ( 4 pazienti ) ; presupposto indispensabile alla rpve stato il drenaggio biliare del frl con riduzione dei valori ematici di bilirubina ( non superiore a 3 mg / dl )  . 
 nel periodo compreso tra il 1996 ed il 2003 gli esami tc sono stati eseguiti in 11 pazienti con tc spirale singolo strato prospeed advantage ( ge medical systems , milwaukee , wis , usa ) come precedentemente descritto [ 8 ]  . nel periodo compreso tra il 2003 ed il 2007 gli esami tc sono stati eseguiti in 15 pazienti mediante tc spirale multistrato lightspeed pro 16 ( ge medical system ) , utilizzando i seguenti parametri : ma automatico ( noise index 14 ) , 120 kvp , collimazione dei detettori 1 , 25 , velocit del tavolo 18 , 75 mm per rotazione , velocit di rotazione del gantry 0 , 6 secondi a rotazione . 
sono stati utilizzati uno spessore di ricostruzione di 2 , 5 mm ed un intervallo di ricostruzione di 2 , 5 mil mezzo di contrasto ( 2 ml / kg di peso corporeo con una concentrazione di 350 mgi / ml ) stato somministrato per via ev attraverso lutilizzo di un iniettore automatico , ad una velocit di 45 ml / s attraverso un ago - cannula di 18 gauge posta in una vena del braccio . 
la scelta dei tempi di ritardo per lacquisizione delle immagini stata ottenuta mediante lutilizzo di un sistema automatico di bolus tracking che prevedeva un singolo sistema di monitoraggio a bassa dose posto a livello di un vaso dinteresse ( aorta addominale ) dopo circa 10 secondi dallinizio delliniezione del mezzo di contrasto ; raggiunto un picco di enhancement di circa 80 uh nella regione dinteresse , circa 10 secondi dopo iniziava lacquisizione della fase arteriosa . 
le fasi portali e tardive venivano calcolate a 70 e 180 secondi dalliniezione del mezzo di contrasto . 556 radiol med ( 2009 ) 114 : 553570 number , location and volume of the liver metastases as well as the resectability criteria based on the evaluation of vascular relationships ensuring adequate perfusion , and venous and biliary drainage of the frl . 
in no patient did the ct scan provide a ratio of hepatic to splenic parenchyma density ratio 1.1 , an indication of moderate - to - severe ( 30% ) steatosis [ 9 , 10 ]  . all patients with klatskin tumours underwent mr cholangiography ( 1.5 - tesla , horizon advantage system , ge medical systems ) to stage the biliary disease . 
volumetric data were obtained from the portal - venous - phase scans , and all examinations were processed by two radiologists on a workstation ( ge medical system 4.1 ) that automatically calculated the volume by multiplying the area by the thickness and also generated 3d reconstructions . the liver parenchyma was isolated from surrounding tissues by using the cursor , and the total liver volume was calculated . 
frl was jointly determined by surgeons and radiologists on the basis of disease extent on imaging ( number , site , vascular relationships of metastases and vascular relationships of biliary cancers ) and according to the surgical plan defined by the surgeons . 
in the volumetric assessment the suprahepatic veins , the teres ligament , the umbilical portion of the left portal branch , the gallbladder and the inferior vena cava were used as anatomical landmarks to delineate the liver segments and separate the liver to be resected from the frl . 
tumour volume was not calculated in patients with biliary cancers , as small size and predominantly biliary involvement made it irrelevant to the evaluation of the ratio of frl volume to total volume of nontumourous liver parenchyma . 
the catheter was advanced to the portal trunk and , after the injection of contrast material , portography was performed to identify the intrahepatic portal branches and any anatomical variants . 
all patients underwent a ct scan 1 week before right pve and , after right pve , within a week before surgery . selection criteria for pve was an frl / total functional liver parenchyma ratio 30% . 
where waiting times exceeded 1 month , this was related to one patient with liver metastases refusing surgery for 2 months , and to our deferring surgery to after having solved the lesame tc prima e dopo rpve ha fornito il numero , la sede , il volume delle metastasi epatiche ed i criteri di resecabilit , basata sulla valutazione dei rapporti vascolari che assicurassero adeguata perfusione , drenaggio venoso e biliare del frl . 
in nessun paziente lesame tc ha rilevato il rapporto della densit parenchima epatico - parenchima splenico 1 , 1 , indice di steatosi moderata - grave ( superiore al 30% ) [ 9 , 10 ]  . tutti i pazienti portatori di neoplasie di klatskin hanno eseguito la colangio - rm per stadiare la malattia sul versante biliare ( stata utilizzata apparecchiatura a 1 , 5 tesla , horizon advantage ge medical systems , milwaukee , wis )  . 
i dati volumetrici sono stati elaborati dalle scansioni eseguite durante la fase portale dellenhancement vascolare e tutti gli esami sono stati elaborati da due radiologi dedicati ad una workstation ( ge medical system 4.1 ) che ci ha fornito il volume mediante il calcolo automatico dellarea x lo spessore , fornendoci anche ricostruzioni 3d . mediante un cursore stato isolato il parenchima epatico dai tessuti circostanti ed stato calcolato il volume epatico totale . 
le vene sovraepatiche , il legamento rotondo , la porzione ombelicale del ramo portale sinistro , la colecisti e la vena cava inferiore sono stati utilizzati come reperi anatomici per delineare i vari segmenti epatici e per separare nella valutazione volumetrica il fegato da resecare dal frl . 
il volume tumore non stato calcolato nei pazienti con neoplasie delle vie biliari per le piccole dimensioni e per il prevalente coinvolgimento biliare , quindi non stato considerato rilevante nella valutazione del rapporto tra volume del frl e volume del parenchima epatico totale funzionante . 
 lembolizzazione del ramo portale destro stata effettuata dopo puntura sotto guida ecografica del ramo portale di sinistra a livello del recesso di rex ; il catetere ha raggiunto quindi il tronco portale e con liniezione del mdc si ottenuto una portografia per individuare i rami portali intra - epatici ed eventuali varianti anatomiche ; successivamente si occluso il ramo portale destro con le sue diramazioni con materiale embolizzante costituito da cianoacrilato e lipiodol . 
tutti i pazienti sono stati sottoposti ad esame tc entro una settimana prima della rpve e dopo rpve entro una settimana prima della chirurgia . il criterio di selezione dei pazienti da sottoporre ad rapporto embolizzazione prechirurgica frl / parenchima epatico totale funzionante inferiore al 30% . 
any volume changes in frl before and after right pve were assessed with the t test , with significance set at p < 0.001. results the injection of cyanoacrylate and lipiodol achieved complete embolisation of the right portal tree in 24 patients . in two patients , preliminary embolisation of the portal branch of segment iv to be resected according to the surgical plan was achieved . 
 the increase a significant change ( p < 0.001 ) in frl volume after right pve was obtained both in patients with liver metastases and in those with biliary cancers . 
in nine patients with hepatic colorectal metastases , in frl volume ( 18571.16 cm3 ) after 2548 days ( 33.8 days on average ) associated with atrophy of the functional liver parenchyma to be resected ( 186.196.62 cm3 ) changed the frl / total functional liver - volume ratio from 18%29% ( 25% on average ) before right pve to 30%51% ( 39% on average ) after right pve . 
after right pve , the volume of the metastases from colorectal cancer in the embolised liver increased by 6%155.5% , whereas in the patient who waited 60 days , the tumour increased in volume by 562.7% ( table 1 )  . 
patients with metastases from carcinoid tumours showed no increase in volume of metastases after right pve in either the embolised or unembolised liver , whereas frl hypertrophy ( 283.7570.1 cm3 ) significantly changed liver parenchyma ratio from 20%27% to 42%52% ( table 1 ) after a period of 3034 days ( 31 days on average )  . 
after a period of 3180 days ( 45.9 days on average ) after right pve , frl hypertrophy ( 187.1671.5 cm3 ) and atrophy of the parenchyma to be resected ( 305.7182.5 cm3 ) changed 30%45% ( table 2 )  . 
le variazioni volumetriche del frl prima e dopo rpve sono state valutate mediante il test t di student ( un valore di p < 0 , 001 stato considerato significativo )  . risultati mediante liniezione di cianoacrilato e lipiodol si ottenuta lembolizzazione completa dellalbero portale di destra in 24 pazienti . 
in 2 pazienti si ottenuta lembolizzazione del ramo portale segmentario del iv segmento da resecare nel programma chirurgico , in uno dei quali la successiva embolizzazione a destra non stata completa ed ha interessato solo il settore anteromediale . 
un paziente stato sottoposto ad una seconda seduta di embolizzazione che ha seguito di 22 giorni la precedente con la quale non si era ottenuta lembolizzazione completa dellalbero portale di destra . 
 la variazione volumetrica dopo rpve del frl stata significativa nei pazienti con metastasi epatiche ( p < 0 , 001 ) e nei pazienti con neoplasie delle vie biliari ( p < 0 , 001 )  . 
in 9 pazienti portatori di metastasi da colon - retto lincremento volumetrico del frl ( 18571 , 16 cm3 ) dopo un periodo compreso tra i 25 ed i 48 giorni ( in media 33 , 8 giorni ) associato allatrofia del parenchima epatico funzionante da resecare ( 186 , 196 , 62 cm3 ) ha modificato il rapporto frl / volume epatico totale funzionante dal 18%29% ( in media 25% ) prima della rpve al 30%51% ( in media 39% )  . 
dopo rpve si riscontrato un incremento volumetrico delle metastasi da colon - retto nel fegato embolizzato compreso tra il 6% e il 155 , 5% , mentre nella paziente che ha atteso 60 giorni lincremento del volume tumore stato del 562 , 7% ( tabella 1 )  . 
nei pazienti portatori di metastasi da carcinoide dopo rpve non si verificato incremento volumetrico delle metastasi sia nel fegato embolizzato che nel fegato non embolizzato , mentre lipertrofia del frl ( 283 , 7570 , 1 cm3 ) ha significativamente cambiato il rapporto frl / parenchima epatico funzionante dal 20%27% al 42%52% ( tabella 1 ) dopo un periodo compreso tra i 30 ed i 34 giorni ( in media 31 giorni )  . 
nei pazienti portatori di neoplasie delle vie biliari lfrl costituiva il 18%28 , 6% del parenchima epatico funzionante , dopo un periodo compreso tra i 31 e gli 80 giorni ( in media 45 , 9 giorni ) dopo rpve lipertrofia del frl ( 187 , 1671 , 5 cm3 ) e latrofia del parenchima da resecare ( 305 , 7182 , 5 cm3 ) ha modificato questo rapporto compreso 30%45% ( tabella 2 )  . 
non progressione di malattia rispetto allesame pre - rpve stata documentata dalla tc nelle neoplasie di klatskin , nella neoplasia della colecisti e 558 radiol med ( 2009 ) 114 : 553570 radiol med ( 2009 ) 114 : 553570 560 radiol med ( 2009 ) 114 : 553570 radiol med ( 2009 ) 114 : 553570 562 radiol med ( 2009 ) 114 : 553570 patient who had undergone metastasectomy during previous surgery . 
eight patients with biliary cancers underwent right hepatectomy extended to segment i ( n = 3 ) and to segments i and iv ( n = 5 )  . preoperative imaging understaged four patients , and radical surgery could not be performed owing to the finding , on exploratory laparotomy , of peritoneal carcinosis ( one patient with metastasis from colon cancer ) , positive lymph nodes at the hepatic hilum ( one patient with type ii klatskin tumour ) , neoplastic lymphangitis along the hepatic pedicle ( one patient with type ii klatskin tumour ) and hepatic artery invasion ( one patient with type iv klatskin tumour )  . 
two patients did not undergo surgical exploration : one with a klatskin tumour because of the onset of ascites after the second embolisation session , and the other with colorectal cancer because of the onset of lung metastases . 
 discussion surgery of biliary cancers is a difficult procedure requiring hepatectomy extended to the caudate lobe , a site of possible recurrence ; resection of the main bile duct with creation of biliodigestive anastomoses ; and possible vascular reconstructions in patients with obstructive jaundice . 
five - year survival in patients with klatskin tumours treated with surgical resection with disease - free margins is between 43% and 46% , but it involves ablation of up to 80% of the liver parenchyma [ 13 ]  . 
in our experience , five patients with biliary cancers who underwent surgical resection after right pve required a right hepatectomy extended to segment iv associated with caudectomy ( necessary for all patients with biliary cancer )  . 
reported that in 132 patients with cholangiocarcinoma who underwent surgical resection after right pve due to insufficient frl volume , the 5 - year survival rate was comparable to that of a control group who underwent surgery without preoperative right pve owing to adequate frl [ 12 ]  . patients with unresected liver metastases from colorectal cancer had a mean survival of 421 months , with a 3 - year survival rate less than 3% . systemic chemotherapy improves quality of life but has limited impact on survival . 
dodici pazienti portatori di metastasi epatiche sono stati sottoposti ad epatectomia destra ( 4 pazienti ) , epatectomia destra allargata a i e iv segmento ( 2 pazienti ) , epatectomia destra allargata al iv ( 6 pazienti ) , uno dei quali con metastesectomia in un precedente tempo chirurgico . 
in tutti i pazienti stata eseguita lecografia intraoperatoria che ha confermato il numero e la sede delle lesioni individuate allesame tc . otto pazienti portatori di neoplasie biliari sono stati sottoposti ad epatectomia destra allargata al i segmento ( 3 pazienti ) e ad epatectomia destra allargata a i e iv segmento ( 5 pazienti )  . quattro pazienti sono stati sottostadiati allimaging preoperatorio e non stato possibile effettuare una chirurgia oncologicamente radicale per la presenza alla laparotomia esplorativa di carcinosi peritoneale ( 1 pazienti portatore di metastasi da neoplasia del colon ) , di linfonodi positivi allilo epatico ( 1 pazienti con tumore di klatskin tipo ii ) , per linfangite neoplastica lungo il peduncolo epatico ( 1 pazienti con tumore di klatskin tipo ii ) , per infiltrazione dellarteria epatica ( 1 pazienti con tumore di klatskin tipo iv )  . 
2 pazienti non sono stati esplorati chirurgicamente , un pazienti portatore di neoplasia di klatskin per la comparsa di ascite dopo la seconda seduta di embolizzazione e laltro pazienti con neoplasia colon - rettale per la comparsa di metastasi polmonari . 
nellimmediato postoperatorio , nella nostra casistica , nessun paziente deceduto per insufficienza epatica grave post - chirurgica ; solo in quattro pazienti si avuta una transitoria lieve insufficienza epatica ( bilirubina > 2 , 9 mg / dl e riduzione del tempo di protrombina < 50% [ 11 ] )  . 
 discussione la chirurgia delle neoplasie delle vie biliari una procedura difficile che richiede estese epatectomie che comprendono sempre il lobo caudato , sede di possibile recidiva , la resezione della via biliare con la ricostruzione di anastomosi biliodigestive , possibili ricostruzioni vascolari , in pazienti con ittero ostruttivo e , quindi , la riduzione dei rischi chirurgici e postchirurgici un sfida per il chirurgo epatobiliare [ 12 ]  . 
la sopravvivenza a 5 anni nei pazienti portatori di neoplasie di klatskin sottoposti ad exeresi chirurgica con margini di resezione libera da malattia compresa tra 43%46% ma prevede lasportazione fino all80% del parenchima epatico [ 13 ]  . 
 [ 12 ] hanno verificato in 132 pazienti con colangiocarcinoma , sottoposti ad exeresi chirurgica dopo rpve per insufficiente frl , che la sopravvivenza a 5 anni sovrapponibile ad un gruppo di controllo sottoposto a chirurgia senza rpve per adeguato frl [ 12 ]  . 
nei pazienti con metastasi epatiche da neoplasie del colon - retto non trattate chirurgicamente , la sopravvivenza media varia dai 4 ai 21 mesi con una sopravvivenza a 3 anni inferiore al 3% . radiol med ( 2009 ) 114 : 553570 fig . 
1a - h computed tomography ( ct ) images obtained before and after right portal vein embolisation ( rpve ) in a 71 - year - old man with gallbladder cancer . 
a - d axial and oblique reformatted ct images obtained before ( a , b ) and after ( c , d ) rpve show the gallbladder cancer invading liver segments iv ( arrows in a and c ) and v ( arrows in b and d ) without hilar invasion . 
e - h twoand three - dimensional maximum intensity projection ( mip ) reformations obtained before ( e , f ) and after rpve ( g , h ) clearly show a proportional twofold atrophy - hypertrophy effect ( decrease in volume of the right liver and increase in volume of the left liver )  . 
a - d le immagini tc assiali e le ricostruzioni oblique ottenute prima ( a , b ) e dopo ( c , d ) rpve documentano la neoplasia delle colecisti che invade iv ( frecce in a e c ) e v segmento epatico ( frecce in b e d ) , senza infiltrazione dellilo . 
e - h le ricostruzioni 2d e 3d mip ottenute prima ( e , f ) e dopo rpve ( g , h ) dimostrano chiaramente un proporzionale duplice effetto atrofia - ipertrofia ( riduzione di volume del fegato destro ed incremento volumetrico del fegato sinistro )  . 
a , b maximum intensity projection ( mip ) reformation in the portal phase ( a ) and axial computed tomography ( ct ) in the delayed phase ( b ) demonstrate an enhancing irregular nodular mass ( arrows in a and b ) adjacent to the right portal vein and invading segment iv with involvement of the right secondary biliary confluence and dilatation of the right intrahepatic bile duct . 
after rpve , left hepatic hypertrophy is evident on 3d mip reconstructions , on anterior view ( c ) and at surgery ( d ) , with evident delimitation caused by the different perfusion of the two hemilivers . 
a , b la ricostruzione mip 2d in fase portale ( a ) e limmagine assiale in fase tardiva ( b ) documentano una formazione nodulare , irregolare e con enhancement ( frecce in a e b ) adiacente al ramo portale di destra che invade il iv segmento con interruzione della convergenza biliare secondaria di destra e dilatazione a monte delle vie biliari intraepatiche dellemisistema di destra . 
lipertrofia del fegato sinistro dopo rpve evidente nelle ricostruzioni mip 3d , visione anteriore ( c ) e allintervento chirurgico ( d ) con evidente demarcazione per la diversa perfusione dei due emifegati . 
the nonresectability of potential candidates for resection may be related to an insufficient frl ( 8% in 746 hepatectomies in elias et al.s experience ) [ 16 ]  . the liver has the ability to regenerate rapidly , a property that provides the rationale for new and innovative surgical techniques for liver resection and organ transplantation . 
one strategy employed by many hepatobiliary surgeons to reduce the risk of complications after extended hepatectomy is pve . occlusion of a portal branch causes the redistribution of the portal flow with a double effect : atrophy of the embolised parenchyma and hypertrophy of the unembolised parenchyma . 
lunico trattamento che migliora la sopravvivenza quello chirurgico : dopo la resezione a 5 anni stimata essere tra il 25% ed il 40% , a 10 anni tra il 22% e il 26% [ 14 , 15 ]  . 
tuttavia lintervento chirurgico possibile solo nel 15%25% dei pazienti con metastasi da colon - retto in relazione allo stadio della malattia , alla localizzazione delle lesioni ripetitive epatiche ed a problemi tecnici ma in pazienti potenzialmente resecabili la non resecabilit pu essere legata ad un insufficiente frl ( 8% in 746 epatectomie nellesperienza di elias ) [ 16 ]  . il fegato possiede la capacit di rigenerare velocemente e su questo principio si basano le pi moderne ed innovative tecniche chirurgiche di resezioni epatiche e di trapianto dorgano . 
locclusione di un ramo portale determina la ridistribuzione del radiol med ( 2009 ) 114 : 553570 to be resected and hypertrophy of the frl is to the advantage of the potential postoperative hepatic functional reserve . immediately after embolisation , the portal flow increases in the unembolised portal territory , as demonstrated by doppler ultrasonography [ 17 ] , leading to parenchymal hypertrophy that correlates with the magnitude of the increase in portal flow [ 8 , 18 , 19 ]  . 
 [ 21 ] , however , showed that not only the amount but also the quality of portal blood affects liver regeneration , and it is indeed the blood flowing back from the pancreatic bed that contains hepatotrophic factors . 
 [ 22 ] reported that the regeneration process of a normal liver after resections occurs in three phases : rapid increase during the first month , decrease during the second month and , finally , extremely slow increase . 
 [ 23 ] recently demonstrated that noncirrhotic livers require a minimum of 3 weeks after right pve owing to hypertrophy of the frl , followed by a plateau lasting between 22 and 56 days . 
noncirrhotic livers have the greatest regenerative capacity ( approximately 1221 cm3 / day 2 weeks after pve , approximately 11 cm3 / day at 4 weeks and 6 cm3 / day at 32 weeks ) [ 2 , 24 , 25 ]  . 
in noncirrhotic patients , a period of 34 weeks is required to assess the efficacy of pve in increasing frl volume . longer waiting periods , related to inflammatory biliary complications in patients with biliary cancers or to the patients refusal , showed that the atrophy of the embolised parenchyma progresses over time , whereas the hypertrophy of the frl is not significantly different . 
 at the beginning of our experience , when surgical planning envisaged a right hepatectomy extended to segment iv , we combined embolisation of the portal branch of segment iv with subsequent right pve . 
 [ 26 ] , we no longer believe this approach to be necessary , in part in consideration of the increased risk of accidental reflux of embolising material into the left portal branch . 
the potential hepatic functional reserve is calculated by using ct determinations of frl volume and total volume of functional liver parenchyma , obtained by subtracting tumour volume from total liver volume , and thus by the frl / total functional liver - volume ratio . 
the method allows for separate determinations of tumour volume and healthy parenchyma , thereby providing an flusso portale con un duplice effetto : latrofia del parenchima epatico embolizzato e lipertrofia del parenchima epatico non embolizzato . 
questo duplice effetto antagonista con atrofia del parenchima epatico da resecare ed ipertrofia del frl a vantaggio della potenziale riserva funzionale epatica post - chirurgica . immediatamente dopo lembolizzazione il flusso portale aumenta , come dimostrato mediante eco - doppler [ 17 ] , nel territorio portale non embolizzato determinando ipertrofia del parenchima epatico che correla con lentit dellincremento del flusso portale [ 8 , 18 , 19 ]  . 
 [ 21 ] hanno dimostrato che non solo la quantit , ma anche la qualit del sangue portale influenza la rigenerazione epatica , infatti il sangue refluo dal letto pancreatico che contiene fattori epatotrofici . 
 [ 22 ] hanno dimostrato che il quadro rigenerativo di un fegato normale dopo resezioni epatiche consiste in tre fasi : un rapido incremento durante il primo mese , un decremento nel secondo e alla fine un lentissimo incremento . 
 [ 23 ] hanno recentemente dimostrato che in fegati non cirrotici un minimo di 3 settimane richiesto dopo rpve per lipertrofia dellfrl seguito da un plateau compreso tra 22 e 56 giorni . 
fegati non cirrotici dimostrano la pi alta capacit rigenerativa ( circa 1221 cm3 / d a 2 settimane dopo lembolizzazione portale , circa 11 cm3 / d a 4 settimane e 6 cm3 / d a 32 settimane ) [ 2 , 24 , 25 ]  . 
fegati cirrotici o pazienti diabetici mostrano il pi basso grado di ricrescita approssimativamente ( 9 cm3 / d a 2 settimane dopo pve ) [ 2 , 25 ]  . 
il periodo di attesa pi lungo , in relazione a complicanze flogistiche biliari nei pazienti portatori di neoplasie delle vie biliari o al rifiuto di una paziente , ha dimostrato che latrofia del parenchima epatico embolizzato progressiva nel tempo mentre non significativamente diversa lipertrofia del frl . 
 allinizio della nostra esperienza , quando il programma chirurgico prevedeva lepatectomia destra allargata al quarto segmento , abbiamo associato lembolizzazione del ramo portale segmentario del iv che precedeva lembolizzazione del ramo portale di destra , ma in accordo con capussotti et al . 
la potenziale riserva funzionale epatica calcolata mediante la volumetria tc del frl e del volume totale del parenchima epatico funzionante , ottenuto sottraendo al volume epatico totale il volume tumore e quindi dal loro rapporto ( frl / volume totale epatico funzionante )  . 
la metodologia da noi usata permette di ottenere la volumetria separata del volume 566 radiol med ( 2009 ) 114 : 553570 accurate estimation of functional parenchyma , particularly in those cases where the liver parenchyma to be resected is extensively involved . 
 [ 27 ] demonstrated that the evaluation of tumour volume in patients with liver metastases significantly affects the evaluation of the hepatic functional reserve . the evaluation of tumour volume before and after right pve also provided us with information on the oncogenic effect of right pve on liver disease . 
in biliary cancers , no local disease progression was detected . our results coincide with previous experiences [ 8 , 28 ] that reported an increase in volume of hypovascular metastases from colorectal cancers after right pve in embolised and unembolised livers and no change in the volume of hypervascular metastases from carcinoid tumours in either tumore e del parenchima sano e quindi fornisce la valutazione reale del parenchima funzionante , in particolare nei casi in cui il parenchima epatico da resecare in gran parte sostituito . 
 [ 27 ] hanno infatti dimostrato che nei pazienti portatori di metastasi epatiche la valutazione del volume tumore incide in maniera significativa nella valutazione della riserva funzionale epatica . la valutazione del volume tumore prima e dopo rpve ci ha inoltre fornito dati sulleffetto oncogenetico della metodica sulla patologia epatica . 
dopo rpve nella patologia secondaria epatica aumentato il volume delle metastasi da colon - retto mentre rimasto immodificato il volume tumore nelle metastasi da carcinoide ; nelle neoplasie delle vie biliari non abbiamo riscontrato progressione locale di malattia . 
i nostri risultati concordano con esperienze precedenti [ 8 , 28 ] che avevano documentato laumento volumetrico delle metastasi , ipovascolari , da colon - retto dopo rpve nel fegato embolizzato e non embolizzato , mentre il volume delle metastasi ipervascolarizzate da carcinoide rimaneva invariato sia nel radiol med ( 2009 ) 114 : 553570 fig . 
a - f axial computed tomography ( ct ) scans obtained in a 54 - year - old man show multiple synchronous liver metastases from colon cancer in the left ( 1 , 2 , 5 ) and right ( 3 , 4 , 6 ) liver , with partial response after six cycles of chemotherapy . 
surgical plan : ablation with free resection margins of the liver lesion located in segment viii ( lesion 3 ) requires right hepatectomy combined with resection of the middle supra hepatic vethe potential frl consists of segments i , ii and iii . 
i - p axial ct scans obtained 29 days after rpve document left hepatic hypertrophy , especially of the caudate lobe , the outcome of previous left metastasectomy ( 1 , 2 , 5 ) and enlarged liver metastases in the right liver lobe ( tumour volume increased from 45 to 115 cm3 ) ( 3 , 4 , 6 )  . 
a - f in un paziente di 54 anni , le immagini assiali dellesame tc documentano multiple metastasi epatiche sincrone da cancro del colon localizzate nel fegato sinistro ( 1 , 2 , 5 ) e nel fegato destro ( 3 , 4 , 6 ) , con risposta parziale dopo 6 cicli di chemioterapia . 
programma chirurgico : lasportazione con margini di resezione liberi della lesione epatica dellviii segmento ( lesione 3 ) necessita di epatectomia destra , associata a resezione della vena sovraepatica media . 
i - p le immagini assiali della tc eseguita 29 giorni dopo lembolizzazione documentano lipertrofia del fegato sinistro , in particolare del lobo caudato , gli esiti della pregressa metastasectomia a sinistra ( 1 , 2 , 5 ) e le metastasi epatiche del fegato destro ( 3 , 4 , 6 ) aumentate di dimensioni ( volume tumore 115 vs 45 cm3 )  . 
 [ 29 ] hypothesised that a cytokine regulating the hepatocyte growth factor ( hgf ) which is altered in patients with colorectal cancer and distant metastases is responsible for the growth of liver metastases in these patients . 
in contrast , preoperative right pve does not affect 5 - year survival rates compared with control groups undergoing resection of liver metastases from colon cancer without right pve [ 16 ]  . new strategies are being proposed for patients with multiple liver metastases from colorectal cancer , which combine the intra - arterial [ 30 , 31 ] or systemic [ 3235 ] infusion of chemotherapeutic agents with selective pve , with the aim of simultaneously obtaining tumour shrinkage and fegato embolizzato che non embolizzato . 
 [ 29 ] hanno ipotizzato che una citochina che regola il fattore di crescita degli epatociti ( hgf ) , alterata nei pazienti con neoplasia colon - rettale e metastasi a distanza , sia responsabile della crescita delle metastasi epatiche in questi pazienti . 
lembolizzazione portale prechirurgica non modifica , invece , la sopravvivenza a 5 anni rispetto a gruppi di controllo sottoposti a chirurgia per metastasi epatiche da neoplasia del colon senza rpve [ 16 ]  . attualmente nuove strategie vengono proposte in pazienti portatori di multiple metastasi epatiche da colon - retto che associano alla selettiva embolizzazione portale infusione intraarteriosa [ 30 , 31 ] o sistemica [ 3235 ] di chemoterapici con lo scopo di ottenere contemporaneamente la 568 radiol med ( 2009 ) 114 : 553570 frl hypertrophy . 
 the second factor that affects the indication for right pve in candidates for liver resection is the possible coexistence of chronic liver disease , as this influences the amount of hepatic functional reserve needed to reduce postoperative morbidity and mortality . 
in patients with klatskin tumours , preventive drainage of the potential frl to resolve the cholestasis ( bilirubin < 2.9 mgdl ) is the essential precondition for pve to obtain an adequate regenerative response of the frl . 
 [ 27 ] , depending on the severity of cirrhosis and on residual liver function assessed with the indocyanine test . the presence of systemic disease , such as diabetes , in which the regenerative capacity of the liver is reduced by the hepatotrophic effect of insulin , and the complexity of surgery are , respectively , the third and fourth factors to be considered when selecting candidates for preoperative right pve . 
the use of lipiodol mixed with cyanoacrylate achieved right pve embolisation without complications and without recanalisation , with the added benefit of radiopacity allowing monitoring of the procedure by fluoroscopy [ 41 , 42 ]  . a potential functional reserve of frl < 30% constitutes an indication for right pve in patients previously exposed to chemotherapy or with previous cholestasis . 
 il secondo fattore che regola lindicazione alla rpve nei candidati a resezione epatiche leventuale concomitanza di epatopatia perch su questo si basa lentit della riserva funzionale epatica necessaria per ridurre la morbilit e mortalit post - chirurgica . 
lasportazione di pi del 75% del parenchima epatico ben sopportata nel paziente giovane ( 40 anni ) , ma la chirurgia resettiva epatica deve essere pi conservativa in relazione allet , alla patologia epatica e sistemica [ 36 ]  . 
 il minimo volume residuo del frl consigliato in pazienti con normale funzionalit epatica tra il 25%30% della massa epatica totale funzionante [ 9 , 19 , 35 , 36 ] anche se sono state effettuate resezioni epatiche con un volume di frl non inferiore al 20% senza complicanze post - chirurgiche [ 22 ]  . 
nei pazienti con neoplasie di klatskin , il preventivo drenaggio del potenziale frl che risolva la colestasi ( livello di bilirubina inferiore a 2 , 9 mgdecilitro ) il presupposto indispensabile allembolizzazione portale perch si ottenga unadeguata risposta rigenerativa del frl . 
la capacit rigenerativa del fegato cirrotico ridotta sia in relazione alla riduzione del flusso portale che alla fibrosi e quindi in questi pazienti il volume minimo del frl necessario pu raggiungere il 50% come dimostrato da kubota ed al . 
 [ 27 ] in relazione alla gravit della cirrosi e quindi alla funzionalit epatica residua valutata mediante il test dellindocianina . la presenza di una malattia sistemica come il diabete che riduce la capacit rigenerativa del fegato in relazione alleffetto epato - trofico dellinsulina e la complessit dellintervento chirurgico sono il terzo ed il quarto fattore da considerare nella selezione dei pazienti da candidare a rpve prechirurgica . 
 in conclusione , il nostro studio retrospettivo ha verificato che lembolizzazione portale pre - chirurgica una procedura sicura ; luso di lipiodol misto a cianoacrilato ha ottenuto lembolizzazione dellalbero portale destro senza complicanze e senza ricanalizzazione con il vantaggio legato alla sua radiopacit di monitorare lembolizzazione mediante fluoroscopia [ 41 , 42 ]  . la potenziale riserva funzionale del frl inferiore al 30% unindicazione alla rpve in pazienti precedentemente sottoposti a terapie chemioterapiche o con precedente colestasi ed allarga le indicazioni alla chirurgia radiol med ( 2009 ) 114 : 553570 extends the indications for liver resection in primary and secondary liver cancers , making radical surgery possible without severe postoperative complications . 
the addition of two - stage hepatectomy appears to be a promising innovation for patients with bilobar liver metastases from colorectal cancer . resettiva epatica in patologie primitive e secondarie epatiche rendendo possibile una chirurgia oncologicamente radicale senza gravi complicanze postchirurgiche . 
although combined ct and fluoroscopic guidance is normally preferred for difficult areas such as the cervical and upper thoracic vertebrae , to ensure operator radiation protection , the technique should also be considered for areas normally treated under fluoroscopic guidance alone . 
however , a larger patient series is needed to correctly evaluate the real contribution of low - dose ct to patient exposure . keywords vertebroplasty dose interventional radiology vertebral compression fracture riassunto obiettivo . 
in base a questo studio si osserva come la dose alloperatore sia in media circa 0 , 8 microsv in corso di vertebroplastica eseguita con guida combinata tc e radioscopica , mentre si attesti attorno a 5 , 8 microsv se eseguita con guida esclusivamente radioscopica ; per quanto riguarda i pazienti la dose di circa 6 msv per la guida combinata e circa 8 msv nel caso di guida esclusivamente radioscopica ; tale differenza non risulta statisticamente significativa . 
se la guida combinata tc e radioscopica risulta preferibile per sedi complesse come il trattamento a livello del rachide cervicale e dorsale alto essa da prendersi in considerazione qualora si privilegi la protezionistica delloperatore anche per quei metameri vertebrali solitamente trattati con esclusiva guida radioscopica , tuttavia serve una pi ampia casistica per poter valutare il reale contributo della tc a basso dosaggio nellesposizione del paziente . parole chiave vertebroplastica dose radiologia interventistica fratture vertebrali da compressione 596 introduction although most operators perform percutaneous vertebroplasty ( pvp ) using fluoroscopic guidance alone , there continues to be controversy as to whether the procedure should be performed under fluoroscopic guidance alone or with a combination of computed tomography ( ct ) and fluoroscopic guidance . 
however , when pvp is performed by experienced operators , fluoroscopy time is reduced to 10 min on average [ 2 ]  . the aim of this study was to evaluate the effective radiation dose to operators and patients during pvp performed under combined ct and fluoroscopic guidance and to compare it with corresponding dose measurements obtained during pvp carried out under fluoroscopic guidance alone . materials and methods dose measurements in previous studies were obtained both by using electronic and thermoluminescent dosimeters ( tld ) [ 1 ] and by recording the dose - area product ( dap ) and the entrance skin dose [ 3 , 4 ]  . 
in our study , operator dose was measured with an electronic dosimeter , whereas patient dose was determined by measuring the dap both with gafchromic films and with an ionisation chamber , from which we derived the effective dose . 
dap is defined as the integral of the absorbed dose to air across the area a of the beam : dap = ( cid : 2 ) adair ( a ) da dap has the useful property of being invariant with distance from the x - ray tube focus . 
this means it can be measured at any plane between the tube and the patient , as long as care is taken to avoid points of measurement with nonnegligible backscattered radiation from the patient and to ensure that the beam is focused on the detector . 
in the case of a uniform beam , as is assumed as a first approximation in diagnostic imaging , the equation becomes : dap dairxa other fundamental concepts are the absorbed dose and , radiol med ( 2009 ) 114 : 595607 introduzione la maggioranza degli operatori esegue la vertebroplastica percutanea ( pvp ) con guida esclusivamente radioscopica tuttavia continuano ad esserci due scuole contrapposte sulla tecnica : esclusiva guida radioscopica , combinata radioscopica e tc guidata , con fautori e detrattori delluna e dellaltra . 
una delle critiche pi frequenti nei confronti di coloro che preferiscono la guida tc che cos facendo la procedura molto pi lenta e che il rischio espositivo del paziente notevolmente superiore . 
in tali lavori si sono evidenziati tempi radioscopici molto variabili fra i 10 ed i 60 minuti ed una elevata esposizione per quanto riguarda loperatore , soprattutto alle estremit [ 1 ]  . tuttavia in mani esperte nei pi recenti lavori il tempo di scopia pu ridursi fino ad una media di 10 minuti [ 2 ]  . scopo del lavoro valutare leffettiva dose radiante a cui sono sottoposti operatore e paziente in corso di pvp con guida combinata radioscopica e tc e confrontarla con misurazioni analoghe eseguite in corso di pvp sotto esclusivo controllo radioscopico . materiali e metodi le misurazioni nei vari studi in letteratura sono state eseguite sia usando dosimetri termoluminescenti ( tld ) ed elettronici [ 1 ] sia registrando la dap ( dose per area ) e la dose in ingresso alla cute [ 3 , 4 ]  . 
per quanto riguarda il nostro studio la dose alloperatore stata misurata mediante dosimetro elettronico , mentre per quanto riguarda la dose al paziente stata misurata la dap sia mediante pellicole gaf - cromic sia con camera a ionizzazione trasmissiva da cui si ricavata la dose efficace ; tale valore stato sommato alla dose efficace in tc nei casi di guida combinata . 
per dap si intende il prodotto dose - area che definito come lintegrale della dose assorbita in aria sullarea a del fascio : dap = ( cid : 2 ) adair ( a ) da essa una grandezza particolarmente utile per la sua invarianza rispetto alla distanza dal fuoco che consente la sua misurazione in qualsiasi piano fra il tubo radiogeno e la posizione del paziente , con lunica avvertenza di evitare punti di misura in cui sia non trascurabile la radiazione retrodiffusa dal paziente e di assicurarsi che il fascio sia compreso nel rilevatore . 
tale grandezza include , oltre a quella di dose , una misura dellarea del fascio e quindi della collimazione impiegata , introducendo informazioni su di un importante fattore di protezione . 
nel caso di fascio uniforme , come in prima approssimazione si ha per la radiol med ( 2009 ) 114 : 595607 directly related to it , the effective dose . 
all the above were during hs [ 6 ] scans , that is , with a pitch of 6 equal to a pitch of 1.5 on single - detector - row machines . 
one limitation to dose reduction was the build of some patients , which obliged us to increase the kilovolts to allow for readable ct scans . the patient was placed prone on the ct table . 
un limite alla riduzione della dose stato lhabitus costituzionale di alcuni pazienti che ci ha costretto ad aumentare i kv per rendere leggibili le scansioni tc . il paziente stato posizionato prono sul lettino della tc . a seguire stato sistemato lateralmente al paziente lintensificatore di brillanza , utile per verificare il posizionamento ed indispensabile per controllare liniezione del cemento allo scopo di identificare la eventuale comparsa di leak extra - somatico vertebrale e / o vascolare e / o epi - durale . 
una volta posizionato il paziente si proceduto ad una prima scansione tc per individuare il / i metameri da trattare e si eseguita una scansione con un repere cutaneo per verificare la via di accesso . 
successivamente si proceduto allanestesia locale con lausilio di un agocannula spinale di 22 g di calibro iniettando lidocaina o , nel caso di vertebre toraciche , una miscela di lidocaina e cortisone ( kenacort ) , cos da ridurre al minimo leventuale disagio relativo allinfiammazione del plesso radicolare in caso di accesso parapeduncolare / intercostotrasversario . 
sono stati quindi inseriti gli aghicannula di calibro compreso fra 10 e 598 radiol med ( 2009 ) 114 : 595607 15 g allinterno del corpo vertebrale con accesso transpeduncolare o intercostotrasversario , aiutandosi nella progressione mediante un piccolo martello ortopedico . 
a questo punto , sotto guida radioscopica , stato infuso il cemento allinterno dei corpi vertebrali . in sala angiografica il paziente stato collocato in decubito prono sul lettino angiografico lasciando le impostazioni automatiche della macchina . 
il posizionamento e la progressione degli aghi da vertebroplastica nel soma , con accesso preferibilmente transpeduncolare , avvenuto sotto controllo radioscopico in antero - posteriore ( ap ) e in latero - laterale ( ll ) in modo da verificare la corretta progressione e il posizionamento dellago attraverso il peduncolo . 
in ap lago non deve uscire dal peduncolo , mentre la ll serve a verificare di quanto progredisce . la strumentazione impiegata nella valutazione di dose stata : camera a ionizzazione trasmissiva a doppio canale diamentor m4 - kdk ; pellicola di tipo gaf - chromic xr tipo t ; dosimetro unfors edd - 30 . 
tale camera radiotrasparente , ha unarea sensibile di 1515 cper la misura del dap il range compreso da 0 , 1 cgycm2 a 106 cgycm2 , la linearit la precisione e la riproducibilit della misura sono comprese entro l1% . 
per la misura del kerma in aria il range compreso fra 0 , 010 mgy e 10 gy ; la linearit e la precisione sono inferiori al 2 , 5% e la riproducibilit entro l1% . 
le caratteristiche principali risposta sono : bassa dipendenza della dallenergia ( < 3% per 80120 kv ) ; bassa dipendenza della risposta dal rateo medio di dose ; elevata risoluzione spaziale ; adeguata equivalenza con lacqua . le pellicole ( di dimensione 1515 cm ) sono state poste sulla finestra di uscita del tubo radiologico , essendo il valore della dap invariante rispetto alla distanza dal fuoco ; ponendo infatti la pellicola sulla finestra di uscita del tubo , piuttosto che sulla cute del paziente , la misura non influenzata dal contributo di radiazione retrodiffusa dal paziente stesso , non di intralcio alla procedura ed inoltre nella guida combinata la pellicola sarebbe stata anche impressionata dal fascio della tc . 
2 vertebroplastica di t12 eseguita con 80 kv e 40 ma , 5 mm di spessore , 30 mm / s lavanzamento del lettino in hs [ 6 ] e rotazione del tubo in 0 , 8 secondi . positioning and monitor the cement injection for possible extravertebral , and / or vascular and / or epidural leakages . 
an initial ct scan was performed to identify the vertebra or vertebrae to be treated , followed by a scan with radiopaque marker on the skin to locate the access route . 
local anaesthesia was achieved by injecting lidocaine or , in the case of thoracic vertebrae , a mixture of lidocaine and cortisone ( kenacort ) with the aid of a 22 - gauge spinal needle to minimise discomfort due to inflammation of the radicular plexus in the case of parapedicular / intercostal transverse approach . 
needle cannulas with 10to 15 - gauge calibre were subsequently inserted into the vertebral body with a transpedicular or intercoastal transverse approach and advanced with the aid of a small orthopaedic hammer . 
at this point , the cement was injected into the vertebral bodies under fluoroscopic guidance . in the angiography room , the patient was placed prone on the angiographic table and the procedure was carried out leaving the machines automatic settings . 
placement and advancement of the vertebroplasty needles , preferably inserted with a transpedicular approach , occurred under anteroposterior ( ap ) and laterolateral ( ll ) fluoroscopic guidance to check correct progression of the needle through the pedicle . 
on ap views , the needle must not be seen to exit from the pedicle , whereas the ll views serve to monitor its progression . the equipment used for dose measurements was as follows : diamentor m4 - kdk dual - channel transmission ion chamber ; type t gafchromic xr films ; unfors edd - 30 dosimeter . 
the chamber radiol med ( 2009 ) 114 : 595607 is radiolucent and has a sensitive area of 1515 cfor dap measurements , the range is between 0.1 cgyxcm2 and 106 cgyxcm2 , and the linearity , precision and reproducibility of measurements are in the range of 1% . 
their main features are low dependence of response on energy ( < 3% for 80120 kv ) , low dependence of response on average dose rate , high spatial resolution and adequate water equivalence . 
 the films ( 1515 cm ) were placed over the output window of the x - ray tube , since the dap value does not vary in relation to distance from the focus . 
in fact , with the film placed over the output window rather than over the patients skin , the measurement is not affected by the backscatter contribution , the film does not interfere with the procedure and , furthermore during procedures using combined ct and fluoroscopic guidance , the film would have been penetrated by the ct beathe irradiated films are read by a scanner at least 24 h following irradiation . from the calibration curve , one can derive the dap and point dose values based on film blackening . 
the values measured with the films and with the transmission ion chamber are comparable . to evaluate effective patient dose when using the image intensifier and the angiographic unit , we utilised the pcxmc15 software programme ( stuk , radiation and nuclear safety authority , helsinki , finland ) , which is based on the monte carlo method ( simulation of the process of energy deposition on a virtual phantom for a large number of incident photons ) and provides a value in terms of deep dose ( hp10 )  . 
the data required are voltage , current , fluoroscopy time , field size , projection , focus - to - skin distance and dap ( obtained from the ion chamber or film measurement )  . to evaluate effective patient dose when using ct , we utilised the ct - expo v1.5 programme , which is also based on the monte carlo method and provides a value of effective fig . 
i dati impiegati sono : tensione di utilizzo , ma , tempo di scopia , dimensione del campo , proiezione , distanza fra il fuoco del tubo radiogeno e la cute del paziente , dap ( ricavato dalla misura della camera a trasmissione o dalla pellicola )  . per la valutazione della dose efficace al paziente nellimpiego della tc si utilizzato il programma ct - expo v . 
i dati impiegati sono : tensione , ma , tempo di rotazione totale , pitch , spessore della slice e numero di slice , numero di serie acquisite e collimazione . 
di fisica sanitaria durante i controlli di qualit annuali sullapparecchiatura . le misurazioni sulla dose alloperatore sono state eseguite mediante un dosimetro unfors edd - 30 indossato dal radiologo interventista inizialmente sotto il camice piombato , poi si deciso che sarebbe stato meglio posizionarlo sopra ed eseguire le dovute correzioni per lo spessore del camice in quanto molto spesso il rateo di dose era inferiore alla minima soglia misurata dallo strumento , che quindi segnava zero ( tabella 1 )  . 
loperatore , essendo sottoposto a sorveglianza fisica per la radioprotezione , munito di dosimetro a film - badge a corpo intero , posizionato sotto il camice piombato , e dosimetro a bracciale a tld , poich tali dosimetri sono indossati dalloperatore non solo durante le procedure di vertebroplastica ma durante tutta lattivit lavorativa e pertanto la misura da essi fornita non impiegabile nella valutazione 600 radiol med ( 2009 ) 114 : 595607 dose in terms of deep dose . 
the data required are voltage , current , total rotation time , pitch , slice thickness and number of slices , number of serial scans and collimation . the ctdi value provided by the equipment is comparable with that measured experimentally with a pencil type ion chamber by the medical physics staff during the annual quality control procedures . measurements of operator dose were carried out with a unfors edd - 30 dosimeter . 
this was initially positioned under the interventional radiologists lead apron and subsequently , because the dose rate was very often lower than the minimum threshold measured by the instrument which showed zero it was decided to place it over the apron and to make the required adjustments for apron thickness ( table 1 )  . 
the dosimeter was placed on the chest to the le owing to continuous physical monitoring for radiation protection purposes , the operator wears a film - badge full - body dosimeter under the lead apron and a tld wrist dosimeter . however , because these dosimeters are worn throughout the working day and not only during vertebroplasty procedures , their measurements could not be used in our evaluation . 
nelle procedure che prevedono solo la guida radioscopica angiografica il paziente in posizione prona sul lettino , il tubo e posto in basso ad una distanza media fra il fuoco del tubo e il ricettore di immagini mediamente 120 cm : il paziente viene quindi investito dal fascio in proiezione antero - posteriore durante il posizionamento dellago e in latero - laterale nelliniezione del cemento ; il tubo e alla destra del paziente mentre loperatore alla sinistra . 
nelle procedure che invece impiegano luso combinato di tc ed intensificatore di brillanza , questultimo posizionato ortogonalmente al lettino tc ( e quindi allasse testa - piedi del paziente ) , il tubo in questo caso alla sinistra del paziente e loperatore a destra . 
the tube is on the patients right and the operator is on the lein procedures performed under combined ct and fluoroscopic guidance , the image intensifier is placed perpendicularly to the ct table ( hence to the headfoot axis of the patient ) , the tube is on the patients left and the operator on the right . 
the distance between the focus and the image intensifier is 100 c during the procedures , the operators wear 0.5 - mm pb - equivalent lead aprons . the unfors edd - 30 dosimeter consists of a 61122 mm sensor placed on a cable connected to a display unit . the sensor has a spherical response that can measure dose independently of the incident angle of radiation . 
 lo strumento ha dimensioni 829821 mm e pesa 200 g , non risulta quindi di minimo intralcio alloperatore . lintervallo di dose accumulata compreso fra 10 nsv e 9999 sv , il minimo valore di soglia del rateo di dose per far partire la misura 0 , 054 msv / h . 
 risultati per ogni paziente abbiamo preso nota delle impostazioni dellapparecchiatura tc come kv , ma , pitch , tempo di rotazione , spessore e numero di slice , collimazione ed il numero delle scansioni eseguite , mentre il contributo dellintensificatore di brillanza ( ib ) stato calcolato considerando i minuti di scopia , i kv , i ma , il fuoco del tubo radiogeno , la distanza fra la cute ed il tubo e calcolando la dose efficace tenendo conto delle misurazioni delle pellicole ( tabella 2 )  . in sala angiografica le misurazioni tenevano conto solo della dose prodotta dallangiografo , in questo caso per tabella 1 dose alloperatore s.c.d.u. 
neuroradiologia , guida radioscopica ds , deviazione standard ; adose alloperatore adattata allo spessore del camice ; t1 , durata dellirraggiamento ( min ) ; t2 , durata delle misurazioni ( min ) ; n.non misurabile 602 results for each patient , we took note of the ct settings , including kv , ma , pitch , rotation time , slice thickness and number , collimation and number of scans , whereas the contribution of the image intensifier was derived from fluoroscopy time , kv , ma , x - ray tube focus , focus - to - skin distance and by calculating the effective dose from the film measurements ( table 2 )  . radiol med ( 2009 ) 114 : 595607 oltre ai valori presi in considerazione per lib le misurazioni sono state calcolate separatamente in base alla proiezione , se ap o ll , e quindi i risultati sono stati sommati ( tabella 3 )  . data lampiezza del campione preso in esame finora abbiamo analizzato i dati applicando il test t di student che ha dimostrato come non ci sia una differenza statisticamente significativa per quanto riguarda la dose al paziente in corso di vertebroplastica quando eseguita sotto table 2 patient dose during vertebroplasty with combined computed tomography ( ct ) and fluoroscopic guidance vertebra / e kv t tot ctdi no . 
bcorrisponde al paziente 2 della tabella 3 trattato per crolli successivi con entrambe le metodiche radiol med ( 2009 ) 114 : 595607 measurements in the angiographic room took into account only the dose produced by the angiographic unit . 
in this case , however , in addition to the values considered for the image intensifier , determinations were made separately for the ap and ll projections , and the results were summed ( table 3 )  . given the size of the sample studied to date , we analysed the data with students t test , which showed that patient dose did not differ significantly between vertebroplasty performed with fluoroscopic guidance alone and with combined ct and fluoroscopic guidance [ t = 0.9726 ; p = 0.3436 with 95% confidence interval ( ci ) ] , whereas operator dose was substantially lower during vertebroplasty performed under combined ct and fluoroscopic guidance ( t = 3.5674 , p = 0.0022 with 95% ci )  . 
this change did not , however , affect the substantial difference in operator dose in favour of combined ct and fluoroscopic guidance ( t = 3.5341 , p = 0.0024 with 95% ci )  . because the ct settings were modified during the procedures , we reanalysed the data with students t test after creating two groups with a cutoff value of 120 kv for ct voltage . 
however , no statistically significant difference emerged between highand low - voltage settings ( t = 2.0956 , p = 0.0656 with 95% ci )  . for each series of values , we then calculated the ratio of dap to effective dose ( and effective dose to dap ) , the mean values and related standard deviations . 
it was particularly interesting to find that the linear coefficient of the straight line was comparable with the mean value of ratio of effective test t di student i dati creando due gruppi e ponendo come cut - off i 120 kv di tensione della tc . 
sono stati quindi posti su grafico i valori di dap ( ascisse ) e di dose efficace ( ordinate ) , interpolando i dati con una correlazione di tipo lineare . 
neuroradiologia , guida radioscopica paziente note dose sv rateo sv / h i valori adattati sono sottolineati ds , deviazione standard ; adose alloperatore adattata allo spessore del camice ; t1 , durata dellirraggiamento ( min ) ; t2 , durata delle misurazioni ( min ) ; n.m. , non misurabile dose to dap . 
 discussione discussion in our series , fluoroscopy time has less variability than that reported in the literature [ 1 ] ( 2.110.53 min for combined guidance and 4.228.4 for fluoroscopic guidance ) , whereas mean fluoroscopy time during procedures with fluoroscopic guidance alone was 10.22 min and thus in line with previous reports [ 2 ]  . 
 our study shows that ct guidance is a valuable alternative for vertebroplasty procedures , as patient dose , generally believed to be too high , can by taking appropriate measures be lowered to similar levels to those imparted in the angiognella casistica presentata il tempo della scopia ha una variabilit molto inferiore rispetto a quanto riportato in letteratura [ 1 ] ( fra 2 , 1 e 10 , 53 minuti per la guida combinata e fra 4 , 2 e 28 , 4 per lesclusiva guida radioscopica ) , mentre il tempo medio di scopia sotto esclusiva guida radioscopica , di 10 , 22 minuti , assolutamente in linea con quanto riportato in letteratura [ 2 ]  . 
 da questo studio si evince come la guida tc sia una valida alternativa per lesecuzione della vertebroplastica in quanto la dose al paziente , ritenuta per lo pi troppo alta , in realt , con gli opportuni accorgimenti , analoga a quella cui il paziente esposto durante una seduta in sala angiografica . 
per quanto riguarda loperatore risultato che la dose , nel caso di procedura eseguita in sala tc , inferiore a quella eseguita in sala angiografica con una differenza statisticamente significativa ( t = 3 , 5674 p = 0 , 0022 con i.c. al 95% ) ; si deve comunque segnalare lesiguit del 606 radiol med ( 2009 ) 114 : 595607 y = 0 , 119x r2 = 0 , 5043 effective dose / dap linear ( effective dose / dap ) y = 0 , 1873x r2 = 0 , 954 relationship dap / effective dose linear ( relationship dap / effective dose ) 10 , 0 15 , 0 20 , 0 25 , 0 30 , 0 35 , 0 dap ( mgyxcm2 ) fig . 
however , it is important to note that the patient sample was small and that in some cases , the operator dose was lower than the minimum threshold detectable by the equipment . 
 further campione ed il fatto che in alcuni casi la dose alloperatore era inferiore al minimo rilevabile dallapparecchiatura . ulteriori studi con un campione di maggiori dimensioni sono pertanto indispensabili per poter individuare leventuale vantaggio della guida tc con bassa tensione ed intensit nel ridurre la dose al paziente . analogamente necessario effettuare un campione pi radiol med ( 2009 ) 114 : 595607 studies on larger patient series are therefore needed to determine the possible benefit of low - voltage , low - current ct guidance in reducing patient doses . likewise , more measurements of operator exposure by placing the dosimeter over the lead apron and correcting the results for apron thickness are required to validate the reduction in dose during pvp under combined ct and fluoroscopic guidance . 
 in view of the quadratic dispersion of the beam , another factor to be considered is the possibility of using devices that allow cement injection without requiring the operator to be in direct contact with the patient or in close proximity to the direct bea numeroso di misurazioni dellirraggiamento alloperatore ponendo il dosimetro sopra il camice ed eseguendo le dovute correzioni per lo spessore del camice stesso al fine di poter confermare o meno la reale riduzione di dose nellesecuzione di pvp sotto guida combinata tc e radioscopica . 
da questo punto di vista anche importante valutare il diverso contributo del tipo di apparecchiature utilizzate ( intensificatori di brillanza , angiografi portatili , applicazione di laser mobili ) , oltre a quello delle possibili modifiche sulle impostazioni delle apparecchiature stesse ; ad esempio : preferire una radioscopia che tendenzialmente usa ma minori di unaltra ( per esempio fluoro1 o fluoro3 negli angiografi philips ) , utilizzare la scopia pulsata al posto della continua durante linfissione degli aghi . 
this study was performed to validate a highresolution whole - body magnetic resonance angiography ( mra ) protocol with parallel imaging and biphasic administration of a single bolus of contrast agent in the preliminary assessment of systemic atherosclerotic burden in patients referred for endovascular procedures . 
forty patients referred for endovascular treatment of atherosclerotic disease of the carotid arteries ( n = 23 ) , peripheral vessels ( n = 14 ) or aorta ( n = 3 ) on the basis of previous clinical and diagnostic examinations underwent high - resolution whole - body mra at 1.5 t with 3d spoiled gradient recalled echo ( gre ) sequences , featuring parallel imaging acquisition technique with 2 acceleration factor . 
ottimizzare un protocollo angio - rm whole - body ( wb ) ad alta risoluzione con tecnica di imaging parallelo e somministrazione bifasica di un singolo bolo di mezzo di contrasto ( mdc ) nello studio della patologia aterosclerotica sistemica ed effettuare una valutazione preliminare del potenziale diagnostico in pazienti candidati a trattamento endovascolare di lesioni arteriose steno - occlusive . 
il mdc stato somministrato con un protocollo di iniezione bifasica a singolo bolo : 10 ml ad una velocit di iniezione di 1 ml / s seguiti da 10 ml alla velocit di 0 , 5 ml / s di gd - bopta . 
la presenza o lassenza di patologia aterosclerotica stata valutata da radiol med ( 2009 ) 114 : 538552 consensus ; segments were classified as having clinically significant disease ( 50% stenosis or an aneurysmal dilatation ) or no significant disease ( < 50% stenosis )  . 
the presence of stenoocclusive disease , determined at all segments , was compared with findings on digital subtraction angiography ( dsa ) , which were interpreted by a third independent reader . 
sensitivity , specificity and concordance of whole - body mra findings with dsa were calculated , and receiver operating characteristic ( roc ) analysis was performed for all vascular territories . 
a total of 1 , 680 arterial segments was evaluated ; 138 ( 8.3% ) were affected by atherosclerotic alterations . carotid lesions were confirmed in 23 patients ( 34 segments ) , involvement of peripheral vessels in 14 ( 57 segments ) and abdominal aneurysms in three . 
subclinical atherosclerotic lesions were evidenced in 25 patients , involving carotid arteries ( 12 segments ) , peripheral vessels ( 21 segments ) and abdominal aorta ( one segment )  . 
the subclinical detection of the total atherosclerotic burden has potential implications for secondary care in this population . due osservatori in consenso ; i segmenti sono stati classificati come patologici ( stenosi 50% o dilatazioni aneurismatiche ) o non patologici ( < 50% )  . 
la capacit di individuare lesioni asintomatiche ha un potenziale nellapproccio di prevenzione secondaria . keywords atherosclerosis mr angiography wholebody imaging systemic vessels parole chiave aterosclerosi angio - rm imaging wholebody vasi sistemici introduction introduzione atherosclerosis is a ubiquitous , multiterritorial arterial disease involving the large vessels and the peripheral circulation whose clinical manifestations lead to adverse social and personal health outcomes in a significant portion of the western population [ 1 , 2 ]  . 
imaging plays a key role in staging the disease and planning appropriate treatment . however , despite the multiterritorial distribution of atherosclerosis , the diagnostic approach relying on conventional modalities such as colour doppler ultrasound ( us ) , computed tomography angiography ( cta ) and conventional angiography has always been limited to investigating symptomatic vascular territories only . 
more recently , magnetic resonance angiography ( mra ) has been successfully proposed for the safe and comprehensive evaluation la malattia aterosclerotica ( ma ) una patologia polidistrettuale con distribuzione ubiquitaria che coinvolge i grossi vasi e la circolazione periferica , con manifestazioni cliniche correlate ad evoluzioni socio - sanitarie sfavorevoli in una quota significativa della popolazione occidentale [ 1 , 2 ]  . 
tuttavia , nonostante la distribuzione polidistrettuale della ma , lapproccio diagnostico , attraverso lutilizzo dei tradizionali metodi quali leco - colordoppler , langio - tc e langiografia convenzionale , stato lungamente confinato ai soli distretti vascolari sintomatici . 
pi di recente langio - rm stata proposta con successo per una sicura e completa valutazione del danno da ma a livello dei 540 radiol med ( 2009 ) 114 : 538552 imaging techniques [ 68 ] and of atherosclerotic burden of all arteries in the body ( wholebody technique ) [ 35 ]  . 
the aim of our study was to validate a high - resolution , whole - body mra study protocol with parallel imaging technique and sequence acquisition during the biphasic administration of a single bolus of a highrelaxivity interstitial contrast agent by performing a preliminary evaluation of its diagnostic accuracy in patients referred for endovascular procedures for atherosclerotic disease . vasi arteriosi di tutto il corpo ( tecnica whole - body ) [ 35 ]  . gran parte di questo successo dovuto soprattutto ai progressi tecnologici nellutilizzo dei sistemi integrati di bobine , delle tecniche di imaging parallelo [ 68 ] ed in parte dei mdc intravascolari [ 911 ]  . 
lobiettivo del nostro studio stato quello di validare un protocollo desame angio - rm whole - body ad alta risoluzione con tecnologia di imaging parallelo e sequenze acquisite durante la somministrazione bifasica di un unico bolo di mdc interstiziale ad alta relassivit , effettuando una valutazione preliminare dellaccuratezza diagnostica in pazienti candidati a trattamento endovascolare per ma . materials and methods population materiali e metodi popolazione between november 2006 and november 2007 , we recruited 40 subjects ( 25 men and 15 women ; mean age 675 years , age range 3284 years ) referred to our hospitals department of vascular surgery for endovascular treatment under angiographic guidance . 
twenty - three patients ( 13 men and 10 women ) had one or more carotid artery lesions , with colour doppler us evidence of a haemodynamically significant and symptomatic stenosis ( 12 drop attacks , 11 minor transient ischaemic attacks )  . 
fourteen patients ( 10 men and 4 women ) had one or more lesions of the peripheral circulation of the lower limbs , so the indication for treatment had been provided by the clinical findings ( ankle - brachial pressure index < 0.90 , refractory claudication or critical ischaemia ) and colour doppler us evidence of haemodynamically significant stenoses . exclusion criteria were general contraindications for mr imaging ( pacemaker or implantable medical devices , metallic fragments , claustrophobia ) , angiography ( congestive heart failure ) and administration of iodineor gadolinium - based contrast agents ( allergy , serum creatinine levels > 1.5 mg / dl )  . 
the study was granted the approval of our institutions ethics committee . dal novembre 2006 al novembre 2007 sono stati reclutati 40 soggetti ( 25 uomini e 15 donne con et media di 675 anni , range det 3284 anni ) ricoverati presso il reparto di chirurgia vascolare del nostro istituto e candidati al trattamento endovascolare sotto guida angiografica per ma . ventitre pazienti ( 13 uomini e 10 donne ) presentavano una o pi lesioni carotidee per cui lindicazione al trattamento era stata posta in seguito alla documentazione con eco - colordoppler di stenosi emodinamicamente significativa e sintomatica ( 12 drop - attack , 11 tia minori )  . 
tre pazienti ( 2 uomini e 1 donna ) presentavano una dilatazione aneurismatica dellaorta addominale per cui lindicazione al trattamento era stata posta in seguito alla diagnosi effettuata con esami tc delladdome eseguiti per altra motivazione ( controllo oncologico )  . 
quattoridici pazienti ( 10 uomini e 4 donne ) presentavano una o pi lesioni a livello del circolo periferico degli arti inferiori per cui lindicazione al trattamento era stata posta sulla base di dati clinici ( indice pressorio sistolico braccio - caviglia inferiore a 0 , 90 , claudicatio non responsiva al trattamento o ischemia critica ) e con esame eco - colordoppler positivo per stenosi emodinamicamente significative . sono stati esclusi dallo studio tutti i pazienti con controindicazioni generali allesame rm ( pace maker o dispositivi medici impiantabili , frammenti metallici , claustrofobia ) , allangiografia ( scompenso cardiaco congestizio ) ed alla somministrazione di mdc iodato o a base di gadolinio ( allergia , livelli di creatinina sierica > 1 , 5 mg / dl )  . 
lo studio ha ricevuto lapprovazione da parte del comitato etico del nostro istituto . all mra studies were conducted on a high - performance 1.5 - t scanner ( magnetom avanto , siemens medical system , erlangen , germany ) with gradient strength of 45 mt / m2 and slew rate of 200 t / m / s . 
the scanning protocol involved the use of the integrated moving - table system and evaluation of four anatomical stations : head / neck , thorax / abdomen , pelvis / thigh , calf / foot . 
the optimal delay between contrast injection and sequence acquisition was calculated by using the bolus - tracking technique , with the start of image acquisition synchronised with the arrival of contrast material in the left cardiac cavities . 
the precontrast images were automatically subtracted from the postcontrast images to increase the contrast between the vasculature and the background . digital subtraction angiography all patients underwent the scheduled angiographic endovascular procedure to treat the previously known vascular lesions . 
all examinations were conducted on a digital subtraction angiography ( dsa ) unit ( integris v5000 , philips medical systems , best , the netherlands )  . in patients undergoing treatment of epiaortic vessels , a transfemoral approach with a 5 - f catheter was used . 
the first injection of contrast agent was given at the aortic arch level , and images were obtained in the two projections ( left antero - oblique and right antero - oblique ) to identify the common carotid , vertebral and subclavian arteries . 
after selective catheterisation of the common carotid arteries , images were obtained in the anteroposterior , lateral and oblique directions ( 45 and + 45 ) , and the intracranial lassetto angio - whole - body ( tecnologia avanto , siemens medical system , erlangen , germania ) con intensit dei gradienti di 45 mt / m2 e tempo di risalita di 200 t / m / s . 
il protocollo di scansione stato impostato utilizzando il sistema integrato di scorrimento continuo del lettino porta - paziente ed ha previsto la valutazione di 4 segmenti anatomici : testa - collo , toraceaddome , pelvi - coscia , gamba - piede . 
le immagini sequenziali pre - contrastografiche sono state ottenute in senso caudo - craniale con scorrimento continuo del lettino porta - paziente , utilizzando sequenze 3d flash ( fast low angle shot ) con integrated parallel acquisition technique ( ipat ) ottimizzate per unelevata risoluzione spaziale e per breve tempo di acquisizione ( tr 3 , 5 ms , te 1 , 18 ms , fa 30 , voxel size 1 , 3 mm0 , 9 mm0 , 9 mm , matrice 256512 , ta 16 s , fattore ipat2 ) secondo sezioni coronali . 
il protocollo stato ripetuto in senso cranio - caudale dopo una singola somministrazione bifasica di mdc tramite iniettore a doppia testata : 0 , 2 mmol / kg di gd - bopta sono stati somministrati in singolo bolo con protocollo di iniezione bifascio ( prima fase di iniezione ad 1 ml / s e seconda fase a 0 , 5 ml / s ) con bolus - chaser di 25 ml di soluzione fisiologica alla velocit di 0 , 5 ml / s . 
il ritardo ottimale tra liniezione del contrasto e lacquisizione stato calcolato con la tecnica del bolus - tracking , con avvio sincronizzato allarrivo del mdc in corrispondenza delle cavit cardiache di sinistra . 
le immagini pre - contrastografiche sono state automaticamente sottratte da quelle contrastografiche , incrementando il contrasto tra il vaso e il background . angiografia con sottrazione digitale in tutti i pazienti stato eseguito il trattamento programmato delle lesioni vascolari note in anamnesi attraverso procedura endovascolare sotto guida angiografica . 
la prima iniezione di contrasto stata condotta a livello dellarco aortico e le 542 radiol med ( 2009 ) 114 : 538552 circulation was visualised in the anteroposterior and lateral projections . 
in all patients , we used a nonionic iodinated contrast agent ( iomeron 300 , bracco , milan , italy ) administered at a dose of 20 ml and flow rate of 20 ml / s for images at the level of the aortic arch and 610 ml at a flow rate of 6 ml / s for selective catheterisation at the level of the common carotids . 
all examinations were performed with a field of view ( fov ) of 33 cm , a matrix of 1.0241.024 , with a spatial resolution of 0.320.32 min patients undergoing treatment of peripheral vessels , we used a transfemoral approach with a 4or 5 - f catheter , depending on the patients characteristics . 
contrast material was administered at a dose of 15 ml and flow rate of 10 ml / s for images at the femoral level and 810 ml at a rate of 6 ml / s for selective catheterisation , for a total of 70 ml ( range 6080 ml ) per patient . examinations were carried out with a fov of 33 cm , a matrix of 1.0241.024 , with a spatial resolution of 0.320.32 mfinally , in patients undergoing treatment of an abdominal aortic aneurysm , we used a transfemoral approach with a 5 - f catheter and administration 30 ml of contrast medium at a flow rate of 25 ml / s , obtaining anteroposterior and laterolateral projections with a fov of 33 cm , and matrix of 1.0241.024 and a spatial resolution of 0.320.32 m postprocessing the mra images were evaluated in real time on an independent workstation ( aquarius , terarecon inc . , san mateo , ca , usa ) dedicated to the 3d reconstruction of vascular data sets . 
to evaluate the anatomy and degree of vascular stenosis and to study the aneurysms , the following reconstruction algorithms were used : maximum intensity projection ( mip ) to gain an overview of the various vascular territories , multiplanar reformation ( mpr ) and curved mpr to measure the degree of stenosis . image assessment and statistical analysis the mra studies were reviewed first , followed by the dsa studies after a 1 - week interval . 
mra results were evaluated in consensus by two radiologists with 8 and 15 years of experience , respectively , in vascular diagnostics ; both observers were unaware of the diagnostic indication for the mra study . 
all dsa examinations were evaluated by an successive immagini , secondo le due proiezioni ( sinistra antero - obliqua e destra antero - obliqua ) , per individuare le carotidi comuni , le arterie vertebrali e le arterie succlavie . dopo una cateterizzazione selettiva delle carotidi comuni sono state ottenute immagini in antero - posteriore , laterale e nelle direzioni oblique ( 45 e + 45 ) e il circolo intracranico stato visualizzato in antero - posteriore e nelle proiezioni laterali . 
in tutti i pazienti stato utilizzato un mdc iodato non ionico ( iomeron 300 , bracco , milano , italia ) al dosaggio di 20 ml con velocit di 20 ml / s per le immagini a livello dellarco aortico e 610 ml sono stati utilizzati , con un flusso di 6 ml / s , per la cateterizzazione selettiva a livello delle carotidi comuni . 
ogni esame stato condotto con un fov di 33 cm , una matrice di 1 , 0241 , 024 , con una risoluzione spaziale di 0 , 320 , 32 m nei pazienti candidati a trattamento di ma dei vasi del circolo periferico stato usato un approccio transfemorale con un catetere da 4 f o 5 f , in base alle caratteristiche del paziente . 
il mdc stato somministrato al dosaggio di 15 ml con velocit di 10 ml / s per le immagini a livello femorale e di 810 ml con velocit di 6 ml / s per la cateterizzazione selettiva , per totale di 70 ml ( range 6080 ml ) di mezzo di contrasto per ciascun paziente . 
per lesame si usato un fov di 33 cm , una matrice di 1 , 0241 , 024 , con una risoluzione spaziale di 0 , 320 , 32 minfine nei pazienti candidati a trattamento per patologia aneurismatica dellaorta addominale stato usato un approccio transfemorale con un catetere da 5 f e somministrazione di mdc al dosaggio di 30 ml con velocit di 25 ml / s , ottenendo proiezioni antero - posteriori e laterolaterali con un fov di 33 cm e matrice di 1 , 0241 , 024 , ad una risoluzione spaziale di 0 , 320 , 32 m post - processing le immagini angio - rm sono state valutate in tempo reale durante la refertazione utilizzando una workstation indipendente ( aquarius , terarecon inc . , san mateo , ca , usa ) dedicata per la ricostruzione volumetrica di datasets di esami vascolari . 
per valutare lanatomia ed il grado di stenosi vascolare ed effettuare lo studio degli aneurismi sono stati impiegati i segeuenti algoritmi di ricostruzione : il mip ( maximum intensity projection ) per la valutazione panoramica dei vari distretti vascolari , lmpr ( multiplanar reformatted projection ) e il curve - mpr per la misurazione del grado di stenosi . radiol med ( 2009 ) 114 : 538552 independent reader unaware of the results of mra at the time of reporting . 
image and diagnostic quality was judged according to a five - point scale : 0 = nondiagnostic ; 1 = suspected diagnosis but insufficient data ; 2 = adequate for diagnosis ; 3 = good - quality images for diagnosis ; 4 = excellent images with definitive diagnosis . segments were considered pathological if they showed a wall dilatation with aneurysmal features or a > 50% reduction in diameter and nonpathological if the stenosis was < 50% . 
in addition , receiver operating characteristic ( roc ) curve analysis was carried out to validate the sensitivity and specificity of mra relative to dsa in all four vascular territories . 
with regard to the segments studied with mra alone , we evaluated the number , severity and prevalence of atherosclerotic lesions . analisi delle immagini e statistica la revisione stata effettuata partendo dallangio - rm e procedendo dopo un intervallo di una settimana con gli esami angiografici . 
i risultati dellangio - rm sono stati valutati in consenso da due radiologi con 8 e 15 anni desperienza nella diagnostica vascolare ; entrambi gli osservatori non erano a conoscienza dellindicazione diagnostica allesame angio - rm . 
la qualit iconografica e diagnostica stata giudicata utilizzando una scala con cinque livelli : 0 = non diagnostico ; 1 = diagnosi sospetta , ma con dati insufficienti a stabilirla ; 2 = sufficiente per la diagnosi ; 3 = immagini di buona qualit per fare diagnosi ; 4 = immagini di qualit eccellente con diagnosi definitiva . 
sono stati considerati patologici tutti quei segmenti che presentavano una dilatazione della parete con caratteristiche aneurismatiche o una riduzione di calibro > 50% e non patologici quelli che avevano stenosi < 50% . 
all mra examinations and angiographic procedures were well - tolerated ; during the days following the examinations , there were no changes in blood chemistry attributable to the administration of contrast material . 
room time for mra studies was 257 min , including patient preparation and positioning ; effective machine time was 135 m room time for the angiographic procedures limited to the diagnostic phase alone without considering stent deployment or angioplasty procedures was 526 min including patient preparation . 
 quantitative assessment of vascular damage for the thorax / abdomen , 3.15 a total of 1 , 680 arterial segments were evaluated , 138 ( 8.3% ) of which were considered pathological ( table 2 )  . the mean quality rating of mra images was 3.48 for the head / neck , 3.56 pelvis / thigh and 2.85 for calf / foot . 
in six patients , image quality was marred by motion artefacts ( n = 4 , carotid ) , incorrect timing ( n = 1 , carotid ) or early venous filling due to the presence of trophic lesions ( n = 2 , tibial )  . 
the total number of images with suboptimal image quality was 49 ( 2% : 27 leg segments , 12 thigh segments , 8 abdominalsplanchnic segments , 2 carotids ) , of which five had a quality rating of 0 , 15 of 1 and 29 of 2 . 
la concordanza statistica tra le due modalit nella valutazione della stenosi stata quantificata utilizzando il test t di student : un valore di p < 0 , 05 stato considerato statisticamente significativo . 
lanalisi statistica stata effettuata con il software statistico spss 13.0 statistical package ( chicago , ill , usa )  . risultati lintervallo medio tra lesecuzione degli studi angio - rm e le procedure angiografiche stato di 42 giorni . 
tutti gli esami angio - rm e le procedure angiografiche sono state ben tollerate ; nei giorni successivi agli esami non si sono evidenziate alterazioni degli esami ematochimici riconducibili alla somministrazione di mdc . 
il room - time per gli esami angiorm stato di circa 257 minuti , inclusa la preparazione ed il posizionamento del paziente ; il tempo - macchina effettivo stato di circa 135 minuti . 
il room - time per le procedure angiografiche , limitatamente allaspetto diagnostico ( non considerando quindi il posizionamento di stent o gli interventi di angioplastica ) , stato di 526 minuti inclusa la preparazione del paziente . 
il tempo impiegato in media per il post - processing degli esami angio - rm stato di 72 minuti . il tempo impiegato in media per la valutazione delle immagini stato di 155 minuti per gli esami angio - rm e di 83 min per quelli angiografici . 
 valutazione quantitativa del danno vascolare sono stati valutati un totale di 1680 segmenti arteriosi , con un totale di segmenti considerati patologici pari a 138 ( 8 , 3% ) ( tabella 2 )  . 
la qualit media delle immagini angiorm stata di 3 , 48 nel distretto testa - collo , 3 , 56 nel distretto torace - addome , 3 , 15 nel distretto pelvi - coscia e 2 , 85 nel distretto gamba - piede . 
in 6 pazienti si verificata una degradazione della qualit delle immgini dovuta ad artefatti da movimento ( n = 4 , carotidi ) , errata valutazione del timing ( n = 1 , carotidi ) , precoce riempimento venoso per presenza di lesioni trofiche ( n = 2 , tibiali ) ; il totale di segmenti in cui la qualit delle immagini risultata sub - ottimale stato di 49 ( 2% , nellordine 27 segmenti di gamba , 12 segmenti di coscia , 8 segmenti addominali - splancnici , 2 carotidi ) , di cui 5 con livello di qualit pari a 0 , 15 con livello di qualit pari a 1 e 29 con livello di qualit pari a 2 . 
in another two cases ( six segments ) , angiography and mra were discordant in their evaluation of lesions affecting the peripheral vasculature ( > 50% stenoses of calf vessels missed at mra )  . 
unanalisi basata su curve roc ( tabella 3 ) stata utilizzata per calcolare sensibilit e specificit dellangio - rm nella valutazione delle lesioni con stenosi superiore al 50% , i valori ottenuti sono stati rispettivamente di 95% e 97% nel distretto testa - collo ( auc = 0 , 948 ) , 100% e 100% nel distretto torace - addome ( auc = 1 ) , 98% e 97% nel distretto pelvi - coscia ( auc = 0 , 973 ) e 84% e 88% nel distretto gamba - piede ( auc = 0 , 856 )  . 
in 2 casi ( 2 segmenti ) vi stata una sottostima di lesioni carotidee da parte dellangio - rm ( stenosi > 85% valutate come 70% ) , uno di questi con qualit delle 546 fig . 
1a - d a 67 - year - old man with high blood pressure and hypercholesterolaemia referred for endovascular procedure on peripheral vessels for leriche - fontaine stage ii intermittent claudication ( fatigue and stress pain of the proximal legs , walking autonomy < 200 m )  . 
whole - body magnetic resonance angiography ( mra ) ( a ) showed a parietal ulcer of the aortic bifurcation ( asterisk ) with severe stenosis of the left common iliac artery ( c )  . 
1a - d uomo di 67 anni , iperteso , dislipidemico , candidato procedure endovascolare a livello del circolo periferico per trattamento di claudicatio intermittens al ii stadio di leriche - fontaine ( dolore sotto sforzo , autonomia inferiore ai 200 metri )  . 
ten of these patients ( six with carotid lesions , two with iliac artery lesions , one with femoral lesions and one with calf - vessel lesions ) were scheduled for additional short - term endovascular treatments to prevent the lesions identified on mra from becoming clinically manifest . 
because the lesions requiring treatment on the basis of mra evidence had not been previously studied in this subgroup of patients , the radiol med ( 2009 ) 114 : 538552 immagini sub - ottimale . 
in altri 2 casi ( 6 segmenti ) vi stata una valutazione non concorde tra angiografia ed angio - rm per quanto riguarda lesioni del distretto periferico ( stenosi > 50% dei vasi di gamba non rilevate allangio - rm )  . 
per quanto riguarda i segmenti studiati unicamente con langio - rm sono state evidenziate lesioni da ma ( stenosi vascolari > 50% o dilatazioni patologiche della parete vascolare ) in 25 pazienti su 40 ( 62 , 5% )  . 
in particolare sono state evidenziate lesioni carotidee misconosciute in 10 casi ( 40% del totale , range di stenosi 60%90% , valore medio 72% , totale segmenti = 12 ) , stenosi significative delle arterie iliache in 7 casi ( 28 % , totale segmenti = 12 ) , stenosi dei vasi femorali in 3 casi ( 12% , totale segmenti = 8 ) , stenosi dei vasi di gamba in 4 casi ( 16% , totale segmenti = 11 ) ed 1 caso di aneurisma dellaorta addominale ( 4% , totale segmenti = 1 ) di dimensioni < 5 cm . in 10 di questi pazienti ( 6 con lesioni delle carotidi , 2 con lesioni delle arterie iliache , 1 con lesioni dei vasi femorali ed 1 con lesioni dei vasi di gamba ) stato programmato unulteriore trattamento endovascolare a breve termine allo scopo di prevenire le manifestazioni cliniche delle lesioni individuate allangio - rm . 
in questo sottogruppo dei pazienti , non essendo state precedentemente studiate le lesioni per cui veniva richiesto il trattamento sulla base dei dati angio - rm , stato effettuato un completamento diagnostico con angio - tc e con ecocolor - doppler ( questultimo nelle carotidi , nei vasi femorali e di gamba ) per fornire unulteriore validazione dei reperti e consentire un planning pre - trattamento pi approfondito . discussione il nostro studio ha permesso di evidenziare come una tecnica angio - rm whole - body allo stato dellarte renda possibile lidentificazione di lesioni misconosciute in pazienti candidati a trattamento endovascolare per ma . 
la recente introduzione delle tecniche di imaging parallelo , supportata dallimpiego di magneti ad alto campo con gradienti altamente performanti e dallavvento delle varie tecniche di bolus - tracking , pu esser considerata un punto di svolta nellevoluzione clinica dellangio - rm : oggi di fatto possibile eseguire un studio whole - body in pochi minuti e con unelevata accuratezza diagnostica . 
tuttavia alcuni distretti vascolari , fra cui gamba e piede dove strutture arteriose di calibro ridotto decorrono in stretta vicinanza di vasi venosi che possono contaminare e degradare lintensit di segnale , presentano ancora delle difficolt nellimaging con angio - rm . 
severe stenosis of the right internal carotid was demonstrated at both digital subtraction angiography ( dsa ) and magnetic resonance angiography ( mra ) ( b , c , arrows )  . 
whole - body mra ( a ) also showed multiple and ulcerated subclinical lesions at the level of the infrarenal aorta and the origin of left common iliac artery ( d , e , arrowheads ) fig . 
oltre la severa riduzione di calibro dellarteria carotide interna destra dimostrata sia allesame angiografico ( b , c , frecce ) che allangiorm , lo studio wb ( a ) ha inoltre evidenziato la presenza di lesioni aterosclerotiche sconosciute ed in gran parte ulcerate a livello dellaorta sottorenale e dellarteria iliaca comune sinistra in sede ostiale ( d , e , punte di freccia )  . 
nella nostra esperienza un fattore di accelerazione ( ipat ) pari a 2 stato sufficiente a ridurre drasticamente il tempo di acquisizione a livello di tutti i segmenti , con maggior beneficio in corrispondenza dei distretti periferici , senza tuttavia limitare la la configurazione utilizzata ha risoluzione spaziale : permesso limpiego di valori di matrice maggiori rispetto a quelli proposti sia negli iniziali studi di angio - rm wholebody [ 4 ] che in lavori pi recenti [ 12 ]  . 
i parametri alla base della fattibilit di questo approccio sono rappresentati pricipalmente dallutilizzo di elementi di bobina con elevat sensibilit e multipli canali indipendenti per la ricezione del segnale associato allelevata performance dei gradienti . inoltre la tecnologia autocalibrante delle bobine evita eventuali interferenze tra lutilizzo dellimaging parallelo e lo spostamento del tavolo durante le varie fasi dellacquisizione . 
rispetto alle sequenze di un protocollo convenzionale , lutilizzo dellipat pu condurre a discreti fenomeni di riduzione del rapporto segnale / rumore , maggiormente evidente in corrispondenza dl circolo periferico ; nella nostra esperienza tuttavia questo inconveniente stato diagnostic workup was completed with cta and colour doppler us ( the latter for the carotids , femoral and calf vessels ) to confirm the findings and permit more in - depth preprocedural planning . discussion our study allowed us to demonstrate that a state - of - the - art whole - body mra technique is able to detect unknown lesions in patients referred for endovascular treatment of atherosclerotic lesions . 
the recent introduction of parallel imaging techniques , supported by the use of high - field magnets with high - performance gradients and the advent of bolus - tracking techniques , may be considered a breakthrough in the clinical evolution of mra . 
some vascular territories , however , including the calf and foot where small arteries run in close proximity to veins that may contaminate and degrade signal intensity , still represent a challenge for mra imaging . 
in these cases , the use of sequences with high spatial resolution , optimal scan timing and high - relaxivity contrast media may greatly improve 548 radiol med ( 2009 ) 114 : 538552 fig . 
whole - body magnetic resonance angiography ( mra ) ( a ) demonstrates severe stenosis of both internal carotid arteries confirmed on digital subtraction angiography ( dsa ) images obtained during the interventional procedure ( b - e , arrows )  . 
in our experience , an acceleration factor ( ipat ) of 2 was sufficient to dramatically reduce acquisition time in all segments with more pronounced benefits in the peripheral vessels without limiting spatial resolution . 
the configuration allowed the use of higher matrix values compared with those proposed by early studies on whole - body mra [ 4 ] and more recent studies [ 12 ]  . 
compared with the sequences of a conventional protocol , the use of ipat may lead to lower signal - to - noise ratio , which is more evident in the peripheral circulation . 
in our experience , this trascurabile dal punto di vista degli effetti sullaccuratezza diagnostica , soprattutto grazie allutilizzo del gd - bopta , un mdc con caratteristiche di relassivit ideali [ 13 ] per gli studi vascolari di primo passaggio ( legame debole con lalbumina plasmatica ) gi impiegato con successo [ 6 ] nei protocolli whole - body . 
nel nostro studio stato utilizzato un approccio con singola iniezione bifasica del mdc , boluschaser e timing calcolato con tecnica di bolus - tracking : nei lavori precedenti [ 47 ] stata generalmente impiegata uniniezione bifasica , pre - miscelazione tra mdc e soluzione fisiologica e timing calcolato con tecnica di test - bolus , oppure stato proposto un approccio con due iniezioni separate di mdc [ 8 ]  . 
 [ 12 ] con risultati sostanzialmente sovrapponibili in termini di qualit ed immagini.unulteriore accuratezza diagnostica delle radiol med ( 2009 ) 114 : 538552 table 3 receiver operating characteristic ( roc ) curve analysis . 
excellent results were also recorded for the head / neck and thigh territories , with sensitivity and specificity levels of 95% and 97% for head / neck and 98% and 97% for thigh . 
results in the remaining segment were slightly worse and less concordant with dsa , with sensitivity and specificity of 84% and 88% , respectively 1specificity 1specificity 1specificity 1specificity problem had little or no effect on diagnostic accuracy , above all thanks to the use of gd - bopta , a contrast agent with ideal relaxivity characteristics [ 13 ] for first - pass vascular studies ( weak affinity for serum albumin ) that has already been successfully used [ 6 ] in whole - body protocols . 
previous studies [ 47 ] generally used either a biphasic injection with premixing of contrast material and saline and timing calculated with the test - bolus technique , or two separate injections of contrast material [ 8 ]  . 
further validation of our contrast administration protocol derives from the evidence that the few cases of suboptimal image quality were related to motion artefacts validazione del protocollo di somministrazione del mdc utilizzato deriva dallevidenza del fatto che i pochi casi in cui ladeguatezza diagnostica delle immagini risultata sub - ottimale sono stati riferibili ad artefatti da movimento o alla presenza di lesioni trofiche dei tessuti molli con conseguente precoce riempimento venoso : in nessun caso abbiamo riscontrato un insufficiente enhancement vascolare per scarso riempimento o unestesa sovrapposizione venosa . 
la rilevanza clinica dei reperti evidenziati nel nostro studio non nuova : gi in precedenza [ 1418 ] stato documentato come la prevalenza di lesioni aterosclerotiche sincrone possa essere accertata virtualmente in qualunque distretto vascolare , indipendentemente dal segmento inizialmente sintomatico . 
la tabella mostra le curve roc calcolate rispettivamente per i distretti : testa - collo , torace - addome , coscia e gamba . lanalisi dimostra come nel distretto toraco - addominale langiorm abbia sensibilit e specificit pari ad 1 , fornendo risultati completamente sovrapponibili alla dsa . 
nel distretto testa - collo e nel distretto coscia il risultato stato leggermente inferiore a quanto ottenuto nel distretto toraco - addominale , ma comunque molto significativo , sensibilit e specificit infatti si sono attestate rispettivamente su valori di 95% e 97% per testa - collo e 98% e 97% per coscia . 
nel rimanente segmento invece langiorm ha mostrato minore concordanza con la dsa con valori di sensibilit e specificit pari 84% ed 88% 1specificit 1specificit 1specificit 1specificit or the presence of trophic lesions with consequent early venous filling . 
in no case was vascular enhancement inadequate because of poor filling or extensive venous overlap . the clinical relevance of our findings is not new ; previous reports [ 1418 ] have already demonstrated that the prevalence of synchronous atherosclerotic lesions may be ascertained in virtually any vascular territory , regardless of the initially symptomatic segment . 
a further important finding concerns treatment planning : in ten patients ( 25% of the study population ) , a subsequent therapeutic intervention was planned in addition to that for which the patient had been referred . these data reflect the true multiterritorial and systemic nature of atherosclerotic disease . 
from the point of view of diagnostic accuracy , the comparison with the reference standard , albeit only available for symptomatic districts interventi terapeutici : in 10 pazienti , ovvero il 25% della popolazione , stato pianificato un ulteriore trattamento successivamente a quello che in un primo tempo ne aveva determinato il ricovero . 
da un punto di vista strettamente pertinente laccuratezza diagnostica della metodica possibile notare come nella nostra popolazione il confronto con la tecnica di riferimento , sia pur limitatamente ai soli territori sintomatici programmati per il trattamento , sia risultato eccellente : in tutti i distretti sono stati raggiunti livelli di sensibilit e specificit superiori al 90% , con la sola eccezione del circolo di gamba che , tuttavia , stato valutato in maniera pienamente soddisfacente , tenendo conto che le cause di degradazione delle immagini sono state quasi esclusivamente riconducibili ad artefatti da movimento o ritorno venoso precoce . 
 oltre ai potenziali , evidenti benefici che una metodica come langio - rm whole - body introduce , devono tuttavia radiol med ( 2009 ) 114 : 538552 scheduled for treatment , showed the technique to have excellent diagnostic accuracy in our study population . 
the levels of sensitivity and specificity achieved were greater than 90% in all districts with the sole exception of the calf , for which assessment was nevertheless fully satisfactory considering that the causes of image degradation were related to motion artefacts or early venous return . beyond the clear potential benefits of a modality such as whole - body mra , a few important remarks are in order . first , the definition of whole - body mra traditionally excludes evaluation of the coronary arteries , a frequent site of synchronous atherosclerotic lesions [ 15 ]  . 
thus , for a comprehensive evaluation of the arterial vasculature with a single examination , the only technologically feasible option is cta , thanks to the high temporal resolution afforded by latest - generation scanners ( with 64 detector rows )  . 
in addition , as concerns treatment of carotid stenoses [ 19 , 20 ] and abdominal aortic aneurysms [ 2124 ] , many conflicting views exist in the literature , and no univocal criteria have been established as to the possible benefits of screening asymptomatic patients . 
first the lack of a reference standard for all segments analysed : as obvious dose concerns preclude conventional angiography of the entire arterial system , and because our protocol did not include any comparison with whole - body cta , we must limit our analysis to the prevalence of atherosclerotic disease in asymptomatic territories without any estimation of the true capability of mra to discriminate between significant and nonsignificant lesions . 
another limitation is related to the fact that our study did not comprise asymptomatic patients : this may have produced an observer bias in that observers were aware that the studies were conducted to confirm a known and / or symptomatic disease in a population with a high pretest likelihood . 
these limitations do not , however , affect the technical aspects that our study emphasises : state - of - the - art equipment , optimised imaging protocols and the currently available contrast media allow for a high - resolution , rapid and relatively safe ( after evaluation of renal function ) study of the entire arterial vasculature , which can help identify a range of asymptomatic abnormalities that may pose a real risk to the patient . 
the technical goal has been achieved , and future research will need to further investigate , on larger patient populations , the possible clinical benefits that mra protocols can provide , even in asymptomatic patients at high risk of cardiovascular events . 
in primo luogo la definizione di whole - body in angio - rm classicamente esclude la valutazione delle arterie coronarie , altra sede in cui si evidenziano frequentemente lesioni da ma sincrone [ 15 ] ; dunque per una integrale valutazione del circolo arterioso nello stesso esame lunica opzione tecnologicamente esplorabile langio - tc , grazie allelevata risoluzione temporale che si ottiene con gli apparecchi di ultima generazione ( con configurazione dei detettori 64 )  . 
inoltre , per quanto concerne il trattamento delle stenosi carotidee [ 19 , 20 ] e degli aneurismi dellaorta addominale [ 2124 ] esistono molte opinioni discordanti in letteratura e non sono stati ancora stabiliti dei criteri univoci per leventuale beneficio apportato da un esame di screening in pazienti asintomatici ; in questi casi potrebbero essere giustificati dei dubbi sulla eventuale selezione dei pazienti . 
 il nostro studio presenta inoltre alcune limitazioni intrinseche , prima fra tutte la mancata disponibilit di un parametro di riferimento su tutti i segmenti analizzati : non essendo possibile effettuare unangiografia convenzionale dellintero sistema arterioso per ovvie motivazioni dosimetriche e non avendo incluso nel protocollo di studio uneventuale comparazione con un protocollo angio - tc whole - body , possiamo limitare la nostra analisi solamente a quella che risulta la prevalenza della ma nei distretti asintomatici , senza alcuna stima della vera capacit dellangio - rm di discriminare tra lesioni significative e non . 
unaltra limitazione deriva dal fatto che non sono stati inclusi nella nostra popolazione dei pazienti asintomatici : pu quindi essersi prodotto negli osservatori un bias dovuto alla conoscienza relativa del fatto che gli esami erano stati condotti per confermare una patologia nota e / o sintomatica , in una popolazione con alta probabilit pre - test . queste limitazioni tuttavia non influenzano quello che laspetto tecnico che il nostro lavoro vorrebbe sottolineare : con apparecchiature allo stato dellarte , protocolli di imaging ottimizzati e mezzi di contrasto attualmente disponibili possibile eseguire uno studio ad elevata risoluzione dei vasi arteriosi dellintero corpo , che allo stesso tempo rapido e relativamente sicuro ( previa valutazione della funzionalit renale ) , evidenziando una gamma di alterazioni asintomatiche che possono costituire un rischio reale per il paziente . 
il limite tecnico praticamente raggiunto ed in futuro dovranno senzaltro essere approfonditi maggiormente e su larga scala gli eventuali benefici clinici che i protocolli angio - rm possono apportare , eventualmente anche in pazienti asintomatici ma ad alto rischio per eventi cardiovascolari . 
bartolozzi1 1radiologia diagnostica e interventistica , dipartimento di oncologia , trapianti e nuove tecnologie in medicina , universit di pisa , via roma 67 , 56100 pisa , italy 2unit operativa di chirurgia iv , azienda ospedaliera universitaria pisana , pisa , italy 3divisione di gastroenterologia , dipartimento di medicina interna , universit di pisa , pisa , italy correspondence to : e . 
in the framework of the 3 - year project of the italian legatumori ( 2003 - 2006 ) , we evaluated the diagnostic accuracy of computed tomography ( ct ) colonography in detecting colorectal lesions in a screening population with positive faecal occult blood test ( fobt )  . 
patients were referred for conventional colonoscopy in the following situations : detection of three or more suspected lesions with maximum diameter 6 mm ; evidence of one or more lesions with maximum diameter > 6 mm ; presence of colonic masses ( maximum diameter > 3 cm )  . 
it generated 93 false positives and 19 false negatives in the identification of diminutive lesions ( 6 mm ) , and 70 false positives and six false negatives in lesions > 6 msensitivity was 83% in smaller lesions and 93% in lesions > 6 mm ; specificity was 45% and 59% , respectively . 
nellambito di un progetto triennale finanziato dalla legatumori ( 20032006 ) stata valutata laccuratezza diagnostica della cv nel rilievo di lesioni colorettali in una popolazione di screening risultata positiva al test per il sof . 
lesame di cc veniva indicato in presenza di : almeno 3 lesioni sospette per polipi con diametro inferiore o uguale a 6 mm , almeno 1 lesione con diametro superiore a 6 mm , o masse coliche ( diametro massimo > 3 cm )  . 
la cv ha consentito la diagnosi di masse coliche in 12 / 135 ( 8% ) pazienti ; ha generato 93 falsi positivi e 19 falsi negativi per polipi di dimensioni minori o uguali a 6 mm , e 70 falsi positivi e 6 falsi negativi per polipi di dimensioni maggiori . 
in una popolazione di screening con sof + , la cv senza fecal tagging e senza ladozione di un cut off dimensionale nella indicazione alla colonscopia tradizionale , ha evidenziato una bassa specificit , a fronte radiol med ( 2009 ) 114 : 586594 very low specificity but high sensitivity in the detection of all colorectal lesions . 
 di una elevata sensibilit nel rilievo di tutte le lesioni . keywords ct colonography colorectal cancer screening parole chiave colonscopia virtuale cancro colorettale screening introduction introduzione screening programmes for the prevention of colorectal cancer ( crc ) envisage the use of faecal occult blood testing ( fobt ) as the first - line investigation , and there is clinical evidence that this test is able to reduce mortality from crc in populations at average risk . 
on four randomised controlled trials and two nonrandomised trials showed that fobt offered to 10 , 000 subjects older than 40 years of age yielded a 16% reduction in mortality from crc [ 1 ]  . 
an italian study , score2 working group , found that in a sample of 28 , 319 asymptomatic subjects ( aged 5564 years ) with average risk of developing crc , adherence to screening by sigmoidoscopy or fobt was similar ( 28% and 30% , respectively ) , whereas the sensitivity of sigmoidoscopy for colorectal lesions was three times higher [ 2 ]  . 
data for the period 20002001 showed an adhesion rate of 41% among 192 , 583 asymptomatic subjects aged 5070 years ; of these , 5.8% were positive and 75.3% agreed to undergo conventional colonoscopy . 
ct colonography ( ctc ) has been proposed as an alternative to conventional colonoscopy in that it has similar sensitivity in detecting significant lesions ( that is , advanced adenomas , with maximum diameter > 1 cm ) but is less invasive and better tolerated . 
a comparison of the three trials shows that the best results , achieved by picki programmi di screening per la prevenzione del carcinoma colorettale ( ccr ) prevedono lutilizzo in prima istanza del test del sangue occulto fecale ( sof ) ed unevidenza clinica che tale test sia in grado di ridurre la mortalit per neoplasia nella popolazione a rischio medio . 
 [ 1 ] , su 4 studi randomizzati e 2 non randomizzati , ha evidenziato che il test del sof offerto a 10000 soggetti di et superiore a 40 anni ha consentito di ridurre la mortalit per carcinoma colorettale del 16% [ 1 ]  . 
uno studio italiano , score2 working group , ha evidenziato che su un campione di 28319 soggetti asintomatici ( et compresa tra 55 e 64 anni ) , con rischio medio di sviluppare un ccr , le percentuali di adesione allo screening mediante sigmoidoscopia o test del sof erano paragonabili , rispettivamente del 28% e 30% ; per contro la sensibilit della sigmoidoscopia nel rilievo di lesioni colorettali era 3 volte superiore [ 2 ]  . 
questi dati sono a favore quindi del test pi invasivo che , a fronte di una simile percentuale di adesione rispetto al sof , presenta una netta superiorit in termini di accuratezza diagnostica . 
la bassa percentuale di adesione confermata da ulteriori programmi di screening , come ad esempio quello effettuato dalla regione toscana ; i dati relativi al periodo 20002001 hanno evidenziato , su 192583 soggetti asintomatici di et compresa tra 50 e 70 anni , una percentuale di adesione del 41% ; di questi il 5 , 8% risultato positivo e il 75 , 3% ha accettato di sottoporsi ad una colonscopia tradizionale di controllo . 
 pertanto gli attuali test di screening del ccr , a fronte di una non invasivit da una parte e elevata accuratezza diagnostica dallaltra , non consentono ancora una significativa adesione al programma di prevenzione da parte della popolazione a rischio . 
la ricerca di un metodo alternativo quindi giustificata allo scopo di identificare un test che ad unaccuratezza diagnostica accettabile , compara588 radiol med ( 2009 ) 114 : 586594 hardt et al . , are accounted for by greater radiologist experience , use of faecal tagging techniques and primary 3d visualisation , which increase the techniques sensitivity [ 57 ]  . 
 because ctc has only recently been introduced into clinical practice , it does not yet feature in crc screening protocols , whereas the use of the double - contrast barium enema is consolidated in cases of incomplete conventional colonoscopy . 
within the framework of a 3 - year project funded by legatumori ( 20032006 ) , we evaluated the diagnostic accuracy of ctc in the detection of colorectal lesions in a screening population with positive fobt . 
 materials and methods patients the study considered 230 asymptomatic subjects ( aged 4580 years ) with positive fobt performed on their physicians advice or in response to the invitation of the tuscany region in the context of a regional crc screening programme [ 3 ]  . 
the patients had been informed about the need for further investigation with conventional colonoscopy but refused to undergo the test and were therefore told about alternative diagnostic tests and referred to the department of diagnostic and interventional radiology to complete screening with ctc . 
 examination protocol and image analysis technique intestinal cleansing was achieved with a low - residue diet during the 2 days before the examination and oral administration of a polyethylene glycol solution ( 3 l ) ( selg 1000 ; promefarm srl , milan , italy ) the day before the examination . 
to reduce bowel peristalsis , promote colonic distension and improve patient tolerance , patients received 20 mg of hyoscine butylbromide ( buscopan , boehringer ingelheim , florence , italy )  . 
subsequently , the colon was insufflated with room air by using a rectal tube and enema bag . the volume of air insufflated , 2 l on average , was adjusted according to patient tolerance . 
the volumetric acquisition was obtained with a multislice scanner ( lightspeedplus ; ge medical systems , usa ) without contrast administration and with low radiation dose with the patient in supine and prone position . 
scanning parameters were as follows : slice thickness 1.25 mm , reconstruction interval 1.25 mm , pitch 6 ( gantry rotation 0.5 s ) , 120 kv , 50 bile a quella della colonscopia convenzionale , unisca un maggior gradimento da parte del paziente . 
la colonscopia virtuale ( cv ) viene proposta come alternativa alla cc per la sua paragonabile sensibilit nel rilievo di lesioni significative ( cio gli adenomi avanzati , con diametro massimo > 1 cm ) , ma meglio tollerata dal paziente perch meno invasiva . 
i pi recenti trials clinici di rockey , cotton e pickhardt , su soggetti asintomatici hanno evidenziato dati discordanti di accuratezza diagnostica della metodica [ 4 ]  . confrontando i 3 studi , emerge che i migliori risultati , relativi allo studio di pichkardt , sono giustificati da una maggior esperienza dei radiologi , dalluso di tecniche di marcatura fecale e dalla tecnica di visualizzazione primaria 3d che consente un incremento della sensibilit [ 57 ]  . 
 data la sua recente introduzione nella pratica clinica , la cv non ancora inserita nei protocolli di screening del ccr ; risulta invece consolidato luso del clisma a doppio contrasto , indicato nei casi di cc incompleta . 
nellambito di un progetto triennale finanziato dalla legatumori ( 20032006 ) stata valutata laccuratezza diagnostica della cv nel rilievo di lesioni colorettali in una popolazione di screening risultata positiva al test per il sof . 
 materiali e metodi pazienti sono stati inclusi nello studio 230 soggetti asintomatici ( di et compresa tra 45 e 80 anni ) , risultati positivi al test del sof , eseguito su consiglio del proprio medico curante o su invito della regione toscana nellambito del protocollo di screening regionale per la prevenzione dei tumori del colon [ 3 ]  . 
i pazienti , informati dal medico curante della necessit di eseguire un approfondimento diagnostico con cc , hanno rifiutato lindagine ; sono stati quindi informati sui possibili test diagnostici alternativi e inviati presso la divisione di radiologia diagnostica e interventistica per completare lo screening con cv . 
 protocollo desame e metodo di analisi delle immagini la pulizia intestinale stata ottenuta mediante dieta priva di fibre nei due giorni antecedenti lesame e , assunzione radiol med ( 2009 ) 114 : 586594 mas ( calculated dose 4.5 mgy per acquisition )  . 
whenever a colonic mass was detected , patients received 120 ml intravenous iodinated contrast material ( visipaque 320 , ge / healthcare ) at a flow rate of 3ml / s . 
all examinations were reviewed with primary 2d analysis , using endoscopic 3d visualisation for equivocal findings ; 2d analysis was carried out with a bone window ( width 1 , 500 , level 150 )  . 
images were reviewed with double reading by two radiologists who , before the start of the study , had interpreted more than 500 ctc examinations , more than 500 of which were with endoscopic control . according to the european society of gastrointestinal and abdominal radiology ( esgar ) consensus statement , the two observers could be considered experts [ 8 ]  . 
 conventional colonoscopy was indicated in the presence of at least three suspected polyps up to 6 mm in diameter , at least one lesion > 6 mm in diameter or colonic masses ( maximum diameter > 3 cm )  . 
the endoscopist was aware of the ctc results , and in all cases , the radiologist or trainee radiologist who had performed ctc attended the conventional colonoscopy examination and indicated the site of the lesion whenever this had not been identified during the first passage of the endoscope . 
 ctc sensitivity and specificity for polyps were calculated using results of conventional colonoscopy as the reference standard both in patients with complete conventional colonoscopy and in those with incomplete examinations , in which case only the examined segments were considered . 
in the case of positive fobt and negative ctc , the patient was offered a 5 - year follow - up visit with repetition of fobt or ctc ( to be chosen by the referring physician and the patient )  . 
ctc examinations were complete in all cases , allowing exploration of the entire colon up to the caecuin 16 / 230 cases , exploration of the sigmoid colon orale di una soluzione acquosa ( 3 l ) di polietilenglicole ( selg 1000 , promefarm srl , milano , italia ) il giorno precedente . 
per ridurre la peristalsi intestinale , favorire la distensione colica e migliorare la tolleranza del paziente sono stati somministrati 20 mg di bromuro di joscina ( buscopan , boehringer ingelheim , firenze , italia ) ; successivamente stato effettuato pneumocolon con aria ambiente , mediante sonda rettale e sacca per clisma . 
lacquisizione volumetrica era ottenuta con appa ( lightspeedplus , ge / medical recchiatura multistrato systems , usa ) , in condizioni basali e bassa dose radiante , nei decubiti supino e prono , utilizzando i seguenti parametri : spessore di strato 1 , 25 mm , intervallo di ricostruzione 1 , 25 mm , pitch 6 ( rotazione del tubo 0 , 5 s ) , 120 kv , 50 mas ( dose calcolata 4 , 5 mgy per acquisizione )  . 
 le immagini relative alla prima acquisizione , condotta in posizione supina , venivano immediatamente analizzate per escludere la presenza di masse coliche ( diametro massimo > 3 cm )  . 
in caso positivo si procedeva alla somministrazione di mezzo di contrasto iodato ev ( visipaque 320 , ge / healthcare ) , per un totale di 120 ml con flusso 3 ml / s . 
gli esami erano valutati con doppia lettura in consenso da due radiologi che , allinizio dello studio , avevano gi letto oltre 500 colonscopie virtuali di cui oltre 150 controllate endoscopicamente . 
 lesame di cc veniva indicato in presenza di : almeno 3 lesioni sospette per polipi con diametro inferiore o uguale a 6 mm , almeno 1 lesione con diametro superiore a 6 mm , o masse coliche ( diametro massimo > 3 cm )  . 
at conventional colonoscopy , the colon could not be explored up to the caecum in 16 / 135 ( 11.9% ) cases owing to patient intolerance ( n = 11 ) , luminal stenosis due to sigmoid diverticulosis ( n = 2 ) and tortuous elongated sigmoid ( n = 2 )  . 
lendoscopista era a conoscenza dei reperti della cv e in tutti i casi il medico radiologo o lo specializzando in radiologia che avevano effettuato lesame di cv erano presenti durante la colonscopia , indicando allendoscopista la sede di lesione nel caso non fosse stata identificata durante il primo passaggio dellendoscopio . 
 la sensibilit e specificit venivano calcolate per polipi , utilizzando i risultati della cc come standard di riferimento , sia nei pazienti in cui la cc era completa , sia in quelli in cui la cc risultava incompleta , limitatamente ai segmenti colici esaminati . 
nei casi di sof positivo e cv negativa veniva prospettato al paziente un follow - up a 5 anni , con ripetizione del sof o dellesame di cv ( a scelta del medico curante e del paziente )  . 
1a - c adenocarcinoma of the ascending colon in a 57 - year - old woman with positive faecal occult blood test ( fobt ) and high familial risk for colorectal cancer . 
in the supine acquisition , the abundant residual fluid in the ascending colon does not allow clear identification of endoluminal lesions . however , a focal thickening of a haustral fold ( arrow ) , partially covered by fluid , can be appreciated in the distal third of the colonic segment ( a )  . 
nel decubito prono ( b ) lo spostamento dei residui fluidi dal colon destro verso i segmenti distali consente il rilievo di una massa ( diametro massimo 3 , 5 cm ) , a sviluppo sessile ( freccia )  . 
a sessile mass ( maximum diameter 4 cm ) located in the distal third of the ascending colon can be appreciated in both the supine ( a ) and prone ( b ) decubitus . 
repositioning of the patient ( from supine to prone ) causes the small amount of fluid evident in the ascending colon to move to the contralateral , more distal , colonic wall ( arrowheads )  . 
nei decubiti supino ( a ) e prono ( b ) apprezzabile una massa ( diametro massimo di 4 cm ) a sviluppo sessile , localizzata nel 1 / 3 medio del colon ascendente . 
si pu notare come nel variare il decubito , la minima quantit di residuo fluido presente nel colon ascendente in supino sia migrata per gravit verso la parete controlaterale e pi distale dello stesso segmento colico ( testa di freccia )  . 
although several semeiotic criteria may be used , including the stato possibile condurre lesplorazione colica fino al cieco per intolleranza del paziente ( n = 11 ) , stenosi del lume in diverticolosi del sigma ( n = 2 ) , angolature in dolicosigma ( n = 2 )  . 
in base al solo decubito supino non possibile identificare la morfologia del polipo che risulta invece chiara nel decubito prono ( b ) , dove sono apprezzabili il peduncolo ( testa di freccia ) e la porzione cefalica ( freccia ) , che presenta diametro massimo di 3 cm . heterogeneous density of residue or the fact that the residue moves as the patient is repositioned , inadequate bowel cleansing will invariably lead to diagnostic doubts . 
 escludendo i polipi inferiori a 6 mm , clinicamente non significativi ( cio con rischio prossimo allo zero nella probabilit di presentare foci di degenerazione neoplastica e quindi non meritevoli di segnalazione alla colonscopia virtuale ) , sono stati rilevati polipi significativi in 57 / 230 ( 19% ) pazienti . 
 discussione il primo dato che emerge dai nostri risultati relativo alla bassa specificit del test per tutti i tipi di polipi considerati . il dato , era a nostro avviso prevedibile e da riferire in primo luogo al mancato utilizzo della marcatura fecale . 
sebbene si possano utilizzare alcuni criteri di semeiotica , come la disomogenea densit dei residui o il fatto che cambiano generalmente posizione al variare del decubito , la scarsa preparazione intestinale genera sempre e comunque un dubbio diagnostico . 
recentemente sia linternational working group on virtual colonoscopy , sia lesgar , hanno riunito i maggiori esperti in materia e raggiunto un consenso , indicando che i polipi con dimensioni inferiori a 6 mm non dovrebbero essere segnalati nel referto onde evitare la generazione di molti falsi positivi [ 7 , 10 ]  . 
in questo studio infatti lelevato numero di falsi positivi da attribuire proprio alla decisione di segnalare le formazioni endoluminali di dimensioni non significative ; situazione che si pu verificare molto spesso in cv senza marcatura fecale e laddove coesiste una scarsa toelette intestiradiol med ( 2009 ) 114 : 586594 fig . 
4a , b false positive result at computed tomographic ( ct ) colonography in a 61 - year - old woman with marked diverticulosis of the sigmoid colon and positive faecal occult blood test ( fobt )  . 
in the supine decubitus ( a ) , a pedunculated lesion with homogeneous density is interpreted as a polyp ( arrows ) ; a coronal multiplanar reconstruction ( b ) further demonstrates the pedunculated morphology of the finding ( arrows ) , which is not , however , confirmed by conventional colonoscopy performed 10 days later . 
nel decubito supino ( a ) si rileva una sospetta lesione parietale a densit omogenea e sviluppo peduncolato ; la ricostruzione multi planare coronale ( b ) evidenzia ulteriormente la morfologia peduncolata del reperto ( frecce ) , che non viene per confermato dalla cc eseguita a distanza di circa 10 giorni . 
 indeed , in this study , the high number of false positive results is to be ascribed primarily to the decision to report endoluminal lesions of nonsignificant size , a situation that may occur very frequently at ctc without faecal tagging and in the presence of poor bowel cleansing . 
even the poor specificity for polyps > 6 mm can be accounted for by the criteria adopted in the study , which had been initially designed in this manner and subsequently not modified to avoid nonuniform interpretation and reporting . 
 [ 11 ] showed that reporting ctc studies with the exclusion of diminutive lesions that is , those < 6 mm would lead to a 77.6% reduction of endoscopic screening procedures and a proportional reduction of related complications . 
this conventional method colonoscopy , which may benefit from a previous ctc , especially in the case of intolerant patients , as the targeted search for a lesion shortens examination times . accuracy of increased the nale . 
anche la scarsa specificit per polipi di dimensioni superiori a 6 mm da attribuire agli stessi criteri dello studio , disegnato inizialmente secondo queste modalit e quindi non modificato per evitare una disomogenea metodologia di interpretazione e refertazione . 
 [ 11 ] , ha evidenziato che la refertazione di esami di cv con esclusione delle lesioni diminutive , cio inferiori a 6 mm , consentirebbe una riduzione del 77 , 6% delle procedure endoscopiche di screening e una proporzionale riduzione delle complicanze ad esse correlate . 
nel presente studio prospettico la cc veniva eseguita in presenza del medico radiologo che aveva interpretato la cv e , che quindi suggeriva di ispezionare il segmento colico in cui aveva indicato la lesione . 
questo metodo consentiva una maggiore accuratezza della cc , che pu pertanto beneficiare di una cv precedente , soprattutto , nei pazienti con scarsa tolleranza allesame , dove la ricerca mirata di una lesione riduce i tempi desame . 594 radiol med ( 2009 ) 114 : 586594 one limitation of this study was the lack of endoscopic control in patients with negative ctc results ( 95 / 230 ; 42% )  . we are therefore unable to provide diagnostic accuracy data on a per - patient basis . 
these limitations may be overcome by larger patient series that are emerging from multicentre trials , among which is the american college of radiology imaging network ( acrin ) 6664 trial ( just completed , with the enrolment of more than 2 , 000 asymptomatic patients ) and the italian society of radiology ( sirm ) - impact study ( completed , with the recruitment of more than 1 , 000 asymptomatic patients ) [ 12 , 13 ]  . 
questi limiti , potranno essere superati da pi ampie casistiche che stanno emergendo da studi multicentrici , di cui lo studio americano acrin 6664 ( appena concluso con il reclutamento di oltre 2000 pazienti asintomatici ) e lo studio italiano sirm - impact ( concluso con il reclutamento di oltre 1000 pazienti asintomatici ) [ 12 , 13 ]  . 
simonetti1 1dipartimento di diagnostica per immagini , imaging molecolare , radiologia interventistica e radioterapia , 2dipartimento di sanit pubblica , cattedra di igiene , policlinico universitario tor vergata , viale oxford 81 , 00133 roma , italy correspondence to : a . 
a total of 467 patients underwent a pet / ct scan using an iodinated contrast mediuwe compared images obtained by the single pet scan , the single ct scan and by the fusion of the two procedures ( pet / ct )  . 
la tecnica pet risultata avere una sensibilit pari al 94 , 05% , una specificit pari al 91 , 60% ed unaccuratezza del 93 , 36% ; la tecnica tc una sensibilit pari al 91 , 07% , una specificit pari al 95 , 42% ed unaccuratezza del 92 , 29% . 
essa risultata particolarmente efficace quando , pur in presenza di un innalzamento progressivo dei marcatori , non era visualizzabile clinicamente o strumentalmente ripresa di malattia , 572 radiol med ( 2009 ) 114 : 571585 specificity , enabling modification of patient treatment , but it is also a unique , high - profile procedure that can produce cost savings . keywords fdg - pet / ct colorectal cancer staging liver metastases recurrence garantendo cos unanticipazione diagnostica . 
la pet / tc non solo offre una performance diagnostica ottimale in termini di sensibilit e specificit , permettendo peraltro di modificare liter terapeutico del paziente , ma riveste anche caratteri di indagine unica di elezione , con conseguenti risparmi economici . parole chiave fdg - pet / tc carcinoma colon - rettale stadiazione metastasi epatiche recidiva introduction introduzione colorectal cancer has an elevated incidence in western countries and is the second most frequent cause of death from cancer in both genders in europe and the united states [ 1 ]  . 
liver metastases are a common occurrence in the history of patients affected by colorectal cancer , with an incidence of 20% at the time of diagnosis and > 50% during the course of the disease [ 3 ]  . 
in 15%30% of cases , the metastases manifest exclusively in the liver at the same time or months or years after diagnosis of the primary tumour of the colon or rectuthe natural history of the disease shows that once they have been identified , liver metastases are destined to increase in size and number . hepatic resection of metastatic lesions is considered the most effective treatment option in terms of increased survival [ 4 , 5 ] , although few patients are able to undergo radical surgery at the time of diagnosis ( owing to lesion site , multifocal involvement and / or insufficient residual liver function ) , and the 5 - year survival rate after surgery is only 20%50% [ 6 ]  . 
to date , the imaging modalities used for this purpose have been ultrasonography , contrast - enhanced computed tomography ( ct ) and magnetic resonance imaging ( mri )  . 
however , given the poor long - term results obtained , these modalities are evidently unable to guarantee an accurate preoperative assessment [ 7 ]  . the impact of positron emission tomography ( pet ) on the quality of pretreatment diagnostic staging has been demonstrated in many tumours , including colorectal carcinoma . 
retrospective studies have shown that pet provides a better preoperative selection of patients with colorectal cancer and secondary lesions , which after resection of liver il cancro del colon - retto presenta unelevata incidenza nel mondo occidentale ed la seconda causa di mortalit per neoplasia in ambedue i sessi sia in europa che negli stati uniti [ 1 ]  . 
in italia , in particolare , i nuovi casi sono ogni anno circa 46000 con sopravvivenza a 5 anni del 58% [ 2 ]  . nella storia dei pazienti affetti da neoplasia del colon - retto , le metastasi epatiche rappresentano unevenienza comune , presentandosi nel 20% dei casi al momento della diagnosi ed in oltre il 50% nel corso della malattia [ 3 ]  . 
nel 15%30% dei casi le metastasi si manifestano esclusivamente al fegato contestualmente o a distanza di mesi o di anni dal momento della diagnosi del tumore primitivo del colon o del retto . 
 la resezione epatica delle lesioni metastatiche lapproccio terapeutico finora ritenuto pi efficace in termini di aumento della sopravvivenza [ 4 , 5 ] , ma solo pochi pazienti sono radicalmente operabili al momento della diagnosi ( in relazione alla localizzazione , al coinvolgimento multifocale e / o in ragione di una insufficiente funzionalit epatica residua ) e la sopravvivenza a 5 anni dopo la chirurgia solo del 20%50% [ 6 ]  . 
finora le tecniche diagnostiche utilizzate a questo scopo sono state lecotomografia , la tomografia computerizzata ( tc ) con mezzo di contrasto ( mdc ) , la risonanza magnetica ( rm ) ma , in considerazione degli scarsi risultati ottenuti a lungo termine , evidentemente esse da sole risultano insufficienti non riuscendo a garantire unaccurata valutazione pre - chirurgica [ 7 ]  . 
 limpatto della pet ( tomografia a emissione di positroni ) sulla qualit dello staging diagnostico pre - terapeutico radiol med ( 2009 ) 114 : 571585 metastases translates into longer survival [ 8 ]  . 
this occurs because the tracer used usually 2 ( 18f - fdg ) , a [ fluorine - 18 ] fluoro - 2 - deoxy - d - glucose glucose analogue labelled with fluorine is avidly taken up by the neoplastic cells , which have a more active glucose metabolism than normal cells [ 9 ]  . 
the metabolic information provided by fdg - pet can therefore be used to obtain a more accurate definition of the areas of malignancy and possible lymph - node involvement , as well as identifying distant metastases , as the examination provides a full - body scan [ 10 ]  . 
furthermore , functional / metabolic alterations can be demonstrated and located earlier than the information provided by other imaging modalities , which are based purely on anatomical dimensional criteria or vascular abnormalities . 
the introduction of new hybrid pet / ct scanners is in this sense a great leap forward [ 1113 ]  . the aim of this study was to compare the diagnostic accuracy of 18f - fdg - pet , ct and pet / ct in the detection of liver metastases during staging and follow - up of patients with colorectal cancer . materials and methods the study involved patients referred to our department between april 2005 and december 2007 with a diagnosis of colorectal cancer and suspected liver metastases . 
the definitive diagnosis was provided by histology and / or clinical / radiological follow - up in all patients , even those who did not undergo biopsy or surgical procedures . pet / ct examination technique all examinations were performed with an integrated pet / ct scanner , discovery st ( ge , general electric medical systems , milwaukee , wi , usa )  . 
before injection of the radiotracer , patients blood glucose levels were evaluated , and 18f - fdg was injected only in conditions of normal glycaemia to avoid reducing the diagnostic potential of pet as a result of the competitive effect created by high blood glucose levels . 
studi retrospettivi hanno dimostrato che la pet permette una migliore selezione pre - operatoria dei pazienti con tumore del colon - retto con localizzazioni secondarie , che si traduce , dopo la resezione di metastasi epatiche , con una pi lunga sopravvivenza [ 8 ]  . 
le informazioni metaboliche che fornisce la fdg - pet permettono dunque una pi accurata definizione delle aree di malignit , delleventuale coinvolgimento linfonodale oltre allidentificazione di lesioni ripetitive a distanza in quanto lindagine consente una scansione di tutto il corpo [ 10 ]  . 
inoltre le alterazioni funzionali / metaboliche possono essere dimostrate e segnalate pi precocemente rispetto a quelle fornite da altre tecniche di imaging che si basano su criteri puramente anatomici dimensionali o su anomalie di vascolarizzazione . le limitate informazioni morfologiche fornite dalla pet , rendono per necessaria lintegrazione con la tc convenzionale . 
lintroduzione di nuovi scanner ibridi pet / tc rappresenta a tal fine un grande passo avanti [ 1113 ]  . lo scopo di questo studio comparare laccuratezza diagnostica della 18f - fdg - pet , della tc e quella della pet / tc nel rilievo di metastasi epatiche in pazienti affetti da neoplasie del colon - retto in fase di stadiazione e nel follow - up . 
 materiali e metodi lo studio ha riguardato i pazienti , giunti presso il nostro dipartimento nel periodo compreso tra aprile 2005 e dicembre 2007 , con diagnosi di neoplasia del colon e del retto con sospetto di metastasi epatiche valutate mediante pet , tc e pet / tc con mdc organo - iodato . 
la diagnosi definitiva stata ottenuta mediante conferma istologica e / o attraverso il follow - up clinico - radiologico di tutti i pazienti , anche di quelli non sottoposti a procedure bioptiche o ad intervento chirurgico . tecnica di esecuzione dellesame pet / tc ms tutti gli esami sono stati eseguiti con uno scanner integrato pet / tc ms discovery st ( ge , general electric , medical systems , milwaukee , wi , usa )  . 
tale sistema combina uno scanner tc modello high speed ultra con 16 file di detettori con un tomografo pet con 10080 cristalli di germinato di bismuto ( bgo ) disposti in 24 anelli . 
during this phase , the patient was kept in a quiet environment to limit muscle activity and thereby reduce radiotracer accumulation in skeletal muscle . the patient was also adequately hydrated with intravenous saline solution ( 250500 ml ) to reduce pooling of the radiotracer in the renal collecting syste at the same time , a single dose of approximately 900 cc of a solution containing contrast material ( gastrografin - 370 mgi / ml , schering ag , berlin , germany ) was administered orally to adequately opacify the bowel loops according to a protocol implemented in the department . the examination was performed with the patient lying supine inside the gantry with arms above the head and began with a scout scan . 
the following scan parameters were used : 140 kv to obtain good quality images at the level of the shoulder and pelvic girdles ; 80 ma ; fov 420500 mm ; ct slice thickness 3.75 mm ( retrospectively reconstructed to 1.25 mm ) to approximate the width of the pet section ; slice interval 3.27 mm , to coincide with the spacing between the pet sections ; scan speed < 1 s per revolution . 
once the ct scan was completed , the pet scan was performed with a 2d technique in the caudocranial direction from the proximal third of the femurs to the head ; five to six bed positions were acquired , with a duration of 4 min per bed . images were reconstructed using a standard iterative algorithm [ ordered subsets expectation maximisation ( osem ) ] , with a mean duration for the pet examination of 2024 mpet acquisition was immediately followed by ct examination performed with the intravenous administration of 100120 ml of nonionic iodinated contrast medium with an injection rate of 23 ml / s ( iomeron - 350 mgi / ml , bracco , milan , italy )  . 
two data sets were acquired : the first included the upper abdomen with a delay of 30 s from the beginning of the contrast injection , and the second extended from the neck to the pubic symphysis with a delay of 6080 s . 
the overall duration of the entire pet / ct acquisition was on average 40 min . image analysis images were processed on an advantage - windows 4.2 workstation ( ge , general electric medical systems ) individually for the pet and ct examinations and with iniettando il 18f - fdg solo in condizioni di normoglicemia per evitare di ridurre il potenziale diagnostico della pet per leffetto competitivo dovuto ad elevati livelli glicemici . nel caso di iperglicemia si invitato il paziente ad incrementare temporaneamente lattivit muscolare , per riportare la glicemia al di sotto di 150 mg / dl . 
durante questa fase il paziente stato tenuto in un ambiente tranquillo per limitare lattivit muscolare onde ridurre , dopo liniezione , laccumulo del radiofarmaco nella muscolatura scheletrica ; si inoltre provveduto ad una adeguata idratazione mediante infusione endovena di soluzione fisiologica ( 250500 ml ) per ridurre il ristagno di tracciante nellemuntorio renale . 
contemporaneamente sono stati somministrati per via orale circa 900 cc di una soluzione contenente mdc ( gastrografin , 370 mgi / ml , schering ag , berlino , germania ) in ununica somministrazione , per unadeguata opacizzazione delle anse intestinali secondo il protocollo praticato nel nostro dipartimento . per lesecuzione dellesame , il paziente viene posizionato allinterno del gantry in decubito supino , con le braccia al di sopra della testa e lindagine inizia con uno scanogramma . viene quindi acquisita una tc in condizioni basali a basso amperaggio ( 80 ma ) necessaria per la correzione dellattenuazione per lo studio pet , utilizzando i seguenti parametri : 140 kv per ottenere immagini di buona qualit a livello del cingolo scapolo - omerale e pelvico ; 80 ma ; fov : 420500 mm ; spessore della sezione tc pari a 3 , 75 mm ( retroricostruibile a 1 , 25 mm ) per approssimare la larghezza della sezione pet ; intervallo tra le sezioni ricostruito pari a 3 , 27 mm , per la coincidenza con la spaziatura tra le sezioni pet ; velocit di scansione inferiore a 1 secondo per rivoluzione . terminata la tc , stato effettuato lesame pet con tecnica 2d in direzione caudo - craniale a partire dal iii prossimale dei femori fino a comprendere il cranio ; sono stati acquisiti 56 lettini con durata di 4 minuti ognuno . 
 le immagini sono state ricostruite utilizzando un algoritmo iterativo standard ( osem , ordered subsets expectation maximization ) , per una durata in media dellindagine pet pari a 2024 minuti circa . 
subito dopo lacquisizione pet viene effettuato lesame tc dopo somministrazione endovena di 100120 ml di mdc organo - iodato non ionico a 23 ml / s ( iomeron - 350 mgi / ml , bracco , milano , italia ) predisponendo due pacchetti susseguenti di scansioni : il primo comprendente laddome superiore con un ritardo di 30 secondi dallinizio delliniezione del mdc , il secondo esteso dal collo fino alla sinfisi pubica con un ritardo di 6080 secondi . 
unulteriore scansione viene effettuata in fase tardiva per una corretta caratterizzazione delle lesioni . infine , dopo avere fatto addurre lungo il tronco gli arti superiori , si acquisiscono scansioni assiali sul cranio . la tecnica di acquisizione stata : 120140 kv , amperaggio automatico ( limite 330350 ma ) , spessore di 3 , 750 mm ( retroricostruibile a 1 , 25 mm ) , modalit di acquisizione radiol med ( 2009 ) 114 : 571585 pet / ct fusion software . 
acquisition data of all ct examinations were processed and retrospectively reconstructed with the following parameters : slice thickness 1.25 mm and interval 3.27 mm to coincide with the spacing between the pet sections . 
attenuation changes of the lesions were then quantified in both baseline conditions and after contrast enhancement . images obtained with ct and pet were independently reviewed by a radiologist and nuclear physician in a doubleblind fashion . 
la durata complessiva dellintera acquisizione pet / tc risultata in media di circa 40 minuti . analisi delle immagini le immagini sono state rielaborate su una workstation advantage - windows 4.2 ( ge , general electric , medical systems ) , singolarmente per lesame pet e tc e mediante ricostruzioni con software di fusione pet / tc . 
i dati di acquisizione di tutti gli esami tc da noi valutati sono stati rielaborati e retroricostruiti con le seguenti indicazioni : spessore delle sezioni pari a 1 , 25 mm ed intervallo tra le sezioni di 3 , 27 mm per la coincidenza con la spaziatura tra le sezioni pet . 
1a - d focal increase of 2 - [ fluorine - 18 ] fluoro - 2 - deoxy - d - glucose ( 18f - fdg ) uptake by two hepatic metastases in the right lobe in a 63 - year - old man with colorectal cancer . 
on coronal maximum intensity projection ( mip ) ( a ) , fdg positron emission tomography ( fdg - pet ) and ( b ) fused pet contrastenhanced computed tomography ( ct ) images show both lesions , although they are located in different planes . 
1a - d iperfissazione del tracciante in corrispondenza di due metastasi epatiche a livello del lobo di destra in un uomo di 63 anni con carcinoma del colon - retto . 
presenza di due lesioni nodulari epatiche localizzate a livello dellviii ( c ) e del v ( d ) segmento . 576 radiol med ( 2009 ) 114 : 571585 fig . 
the axial ( a ) 2 - [ fluorine - 18 ] fluoro - 2 - deoxy - d - glucose positron emission tomography ( 18f - fdg - pet ) image and ( b ) the fusion pet contrast - enhanced computed tomography ( ct ) images show the lesion as a focal increase of fdg uptake that corresponds to a focal hypoattenuating lesion on contrast - enhanced ct in the portal phase ( c )  . 
nellimmagine assiale fdg - pet ( a ) ed in quella di fusione pet / tc ( b ) la lesione riconoscibile come area di aumentato metabolismo in corrispondenza di una formazione ipodensa alla tc ( c ) in fase portale . 
d nella scansione assiale tc basale senza mdc la lesione focale scarsamente riconoscibile . statistical analysis data analysis was performed on the basis of the positive findings in the individual patient . 
for each imaging modality , we evaluated sensitivity , specificity and diagnostic accuracy with 95% confidence intervals ( 95% ci )  . the difference between the individual modalities ( pet and ct ) and the combined technique ( pet / ct ) , with a statistically significant value of p < 0.05 , was estimated with the z lesioni , evidenziate in condizioni basali e nella fase postcontrastografica . 
 le immagini ottenute con la tc e con la pet sono state visionate e valutate indipendentemente , in doppio cieco da uno specialista radiologo e da uno specialista in medicina nucleare . 
abbiamo valutato , per le tecniche in esame , la sensibilit , la specificit e laccuratezza diagnostica con i relativi intervalli di confidenza al 95% ( ic 95% ) ; la differenza tra le singole metodiche ( pet , tc ) e la combinata ( pet / tc ) , con un valore statisticamente significativo di p < 0 , 05 , stata stimata con il test z . 
la diagnosi definitiva stata ottenuta mediante conferma istologica e / o attraverso il follow - up di tutti i pazienti , anche di quelli non sottoposti a procedure bioptiche o ad intervento chirurgico . 
3a - c liver metastases depicted only by 2 - [ fluorine - 18 ] fluoro - 2 - deoxy - dglucose positron emission tomography ( 18f - fdg - pet )  . 
the axial pet scan ( a ) and the fdg - pet / computed tomography ( ct ) fusion image ( b ) show multiple foci of increased fdg uptake in the liver of 59 - year - old woman with colorectal carcinoma . 
la scansione pet assiale ( a ) e limmagine fdg - pet / tc ( b ) con mdc di fusione , documentano aumento focale della captazione di fdg in corrispondenza di multiple metastasi epatiche in una donna di 59 anni con carcinoma del colon - retto . 
the ppv of pet was calculated to be 96.64% ( 95% ci : 94.6998.59 ) , and the npv was 85.71% ( 95% ci : 79.9291.51 ) ( table 2 )  . 
for the combined technique , the ppv was 99.10% ( 95% ci : 98.08100 ) and the npv was 94.81% ( 95% ci : 91.0798.56 ) ( table 4 )  . 
per la tecnica pet / tc combinata il vpp pari al 99 , 10% ( ic 95% : 98 , 08100 ) , il vpn pari al 94 , 81% ( ic 95% : 91 , 0798 , 56 ) ( tabella 4 )  . lindividuazione di una differenza statisticamente significativa per sensibilit , specificit e accuratezza stata ottenuta mediante il test z relativamente alla tecnica combinata ( pet / tc ) vs pet . 
non stata evidenziata alcuna differenza statisticamente significativa di sensibilit , specificit e accuratezza tra le singole tecniche ( pet vs tc ) ( tabella 5 )  . radiol med ( 2009 ) 114 : 571585 fig . 
coronal maximum intensity projection 2 [ fluorine - 18 ] fluoro - 2 - deoxy - d - glucose positron emission tomography ( mip 18f - fdg - pet ) scan ( b ) does not reveal focal uptake of fdg in the liver . 
a la scansione tc , acquisita in fase portale , documenta la presenza di una lesione focale di natura secondaria di 7 mm di diametro localizzata a livello dellviii segmento epatico . 
there was no statistically significant difference in sensitivity , specificity or accuracy between the individual techniques ( pet vs ct ) ( table 5 )  . discussione discussion colorectal cancer is one of the most common cancers in western countries , and liver metastases from the tumour are an enormous challenge for clinical oncology . 
a third of il tumore del colon - retto una delle neoplasie pi frequenti nei paesi occidentali e le metastasi epatiche di tale neoplasia rappresentano un grande problema nelloncologia clinica . 
the high level of clinical transferability of the information provided by pet nello studio delle neoplasie del colon - retto , la pet stata di solito impiegata allo scopo di chiarire la natura di lesioni dubbie gi evidenziate da esami di i livello come la tc oppure quando , soprattutto in corso di follow - up , si osservava un incremento di un marcatore oncologico , in assenza di localizzazioni documentate dai pi convenzionali esami diagnostici [ 14 ]  . 
the z test failed to demonstrate statistically significant differences between the two individual modalities and showed that pet and ct were very much alike in their detection of liver metastases . 
in contrast , combined pet / ct proved superior to pet and ct used individually , with statistically significant differences in all the parameters studied except ct specificity . in our study there was greater concordance among imaging modalities in terms of sensitivity , specificity and diagnostic accuracy than reported in the literature , as most studies to date have used ct without contrast enhancement , which does not enable visualisation of many liver metastases [ 1719 ]  . 
in our study , contrast - enhanced ct improved diagnostic reliability above all for the purposes of correct preoperative staging without negatively influencing the qualitative and quantitative data obtained at pet . although pet and ct are shown by the data to be equivalent in their ability to identify secondary hepatic lesions , the two modalities are evidently complementary . 
in agreement with the literature , the integration of pet with ct makes possible the combination of metabolic data and morphological imaging , thus enabling an accurate definition of the extension of disease at the time of diagnosis [ 20 , 21 ]  . 
the combined technique in fact offers improved localisation of the foci of radiotracer uptake , differentiating the increases in metabolic activity due to physiological or inflammatory processes from those of neoplastic origin [ 22 , 23 ]  . 
in addition , it improves the localisation and characterisation of lesions [ 24 ] , thus reducing the number of lesions of uncertain or indefinite origin . the evaluation of treatment response is also improved by not relying solely on size criteria . 
sebbene tale tracciante non sia specifico della cellula tumorale , la 18f - fdg - pet risulta largamente utilizzata nello studio di molte patologie neoplastiche [ 16 , 17 ] , in quanto considerata migliore dellimaging convenzionale nellidentificazione della patologia tumorale primitiva o secondaria . 
 nel nostro studio abbiamo voluto comparare le informazioni funzionali e morfologiche ottenute rispettivamente mediante la 18f - fdg - pet e la tc , con quelle fornite dalla lesioni secondarie pet / tc , nella valutazione delle epatiche . 
i risultati da noi ottenuti hanno evidenziato valori sovrapponibili di sensibilit , specificit e accuratezza diagnostica tra le due metodiche poste a confronto ed un miglioramento , se considerate le due tecniche associate . 
il test z non ha identificato differenze statisticamente significative tra le due singole tecniche in esame dimostrando una sostanziale equivalenza della pet e della tc nella identificazione delle localizzazioni secondarie epatiche . 
diversamente la tecnica combinata pet / tc risultata superiore alle tecniche diagnostiche pet e tc applicate singolarmente , con valori di significativit statistica in tutti i parametri studiati tranne che per la specificit tc . nel nostro studio , le metodiche mostrano una concordanza con valori di sensibilit , specificit ed accuratezza superiori ai dati della letteratura poich , la maggior parte degli studi finora effettuati utilizzano la tc senza la somministrazione di mdc organo - iodato , che non permette la visualizzazione di molte metastasi epatiche [ 1719 ]  . 
anche se le localizzazioni secondarie epatiche mostrano una evidente ipercaptazione di 18f - fdg alle immagini pet , spesso le lesioni non vengono evidenziate alla tc basale . nel nostro studio lesame tc effettuato con la somministrazione endovenosa di mdc organo - iodato , ha migliorato lattendibilit diagnostica , ai fini soprattutto di un corretto staging pre - operatorio senza inficiare le informazioni qualitative e quantitative ottenute alla pet . sebbene la pet e la tc risultano , dai dati di questo lavoro , equivalenti nella capacit di identificare localizzazioni secondarie epatiche , appare chiaro che le due metodiche sono tra loro complementari . 
in accordo con i dati della letteratura , la possibilit di integrare la pet con la tc consente di combinare linformazione metabolica con il dato anatomico permettendo , al momento della diagnosi , unaccurata definizione della estensione di malattia [ 20 , 21 ]  . 
la tecnica combinata permette infatti una migliore localizzazione dei foci di uptake , differenziando gli aumenti di attivit metabolica di natura fisiologica o flogistica da quelli di natura neoplastica [ 22 , 23 ]  . 
infatti , noto che la riduzione del numero delle radiol med ( 2009 ) 114 : 571585 may not lead to a significant reduction in lesion size , as the neoplastic tissue may be replaced by necrotic tissue or fibrosis , which cannot be differentiated from malignant tissue with conventional diagnostic modalities . 
in addition , treatment planning may be oriented and / or modified by fusion of the metabolic data provided by pet , which is fundamental for identifying secondary lesions located in the peritoneum , mesentery and lymph nodes [ 25 ] where ct shows its major limitations with the morphological data provided by ct which makes possible the examination of fundamental anatomical structures such as the portal vein , the vena cava or the bile ducts [ 26 ]  . accurate preoperative staging is crucial for determining the optimal therapeutic approach in each patient [ 27 ]  . 
the combined technique , which is able to perform precise tumour staging , can orient or rule out the need for further procedures , thus changing the treatment plan [ 18 , 2830 ]  . pet / ct has proven particularly effective even when clinical or imaging techniques fail to demonstrate disease recurrence in the presence of a progressive increase in tumour markers , thus leading to an earlier diagnosis with a positive impact on patient survival . 
the change in the uptake of 18ffdg in fact correlates with serum carcinoembryonic antigen levels , the increase in which is a common finding in these patients [ 25 ]  . the combined modality also minimises the intrinsic limitations of the two techniques responsible for fn findings . 
moreover , the use of pet / ct leads to an overall reduction in costs with respect to the separate use of the two imaging modalities [ 33 ] and improved utilisation of the diagnostic devices [ 34 ]  . 
lastly , by optimising study protocols , the combined pet / ct examination is also able to reduce the overall dose of radiation delivered to the patient [ 33 ] and the overall examination time , thus minimising patient discomfort . cellule neoplastiche pu non comportare una significativa riduzione delle dimensioni della lesione in quanto il tessuto tumorale pu essere sostituito da tessuto necrotico o fibroso , che risulta indistinguibile alla diagnostica convenzionale dal tessuto maligno . 
inoltre la fusione del dato metabolico , fornito dalla pet , fondamentale per evidenziare le localizzazioni secondarie a livello peritoneale , mesenteriale e linfonodale [ 25 ] in cui la tc mostra il suo maggior limite , con il dato morfologico , fornito dalla tc , che permette di esaminare strutture anatomiche fondamentali quali la vena porta , la vena cava o le vie biliari , guida e / o modifica il planning terapeutico [ 26 ]  . 
e la tecnica combinata , in grado di effettuare una precisa stadiazione tumorale , pu guidare o escludere la necessit di ulteriori procedure , cambiando la modalit di trattamento [ 18 , 2830 ]  . 
la pet / tc si dimostrata particolarmente efficace anche quando , in presenza di un innalzamento progressivo dei marcatori tumorali , non era visualizzabile clinicamente o strumentalmente alcuna ripresa di malattia , rendendo possibile unanticipazione diagnostica , con un aumento potenziale dellindice di sopravvivenza . 
il cambiamento nelluptake 18f - fdg correla infatti con i livelli di cea sierico , la cui elevazione sine materia di frequente riscontro in questi pazienti [ 25 ]  . 
infine , ottimizzando i protocolli di studio , con lesame combinato pet / tc si riduce anche la dose complessiva di radiazioni ionizzanti assorbita [ 33 ] , i tempi globali di esecuzione delle indagini , apportando un minore disagio al paziente . conclusions conclusioni pet / ct is able to provide a synergy of functional , anatomical and structural information with considerable optimisation con la pet / tc si ottiene un sinergismo informativo funzionale - anatomico - strutturale con una notevole ottimizzazione 584 radiol med ( 2009 ) 114 : 571585 of the data obtained with the two modalities taken individually . 
the use of pet / ct may be instrumental in reaching an earlier diagnosis , which influences patient management by prompting the performance of procedures of proven effectiveness or the nonperformance of procedures that may prove ineffective or even harmful . 
lastly , the technique has the characteristic of an all - in - one examination if performed with contrast medium , thus leading to significant economic savings . delle informazioni derivanti dalle due modalit pet e tc prese singolarmente . 
la pet / tc pu consentire tra laltro unanticipazione diagnostica che incide sulla gestione del paziente attraverso lesecuzione di interventi di documentata efficacia o la non esecuzione di interventi che potrebbero risultare inefficaci o addirittura dannosi . 
new york , 2009 isbn 978 - 88 - 470 - 1343 - 8 published online : 15 june 2009 springer - verlag 2009 this work presents the entire urogenital system in a methodical and thorough fashion . 
until a few years ago , this technique was considered the reference examination for many renal and urinary tract diseases , whereas today it has largely been replaced by ultrasonography , computed tomography ( ct ) urography and magnetic resonance ( mr ) urography . 
it is clear , however , that the ct and mr studies are able to provide in a single examination information regarding the renal parenchyma and the collecting system , the adjacent organs and structures , and functional and vascular characteristics , all of which cannot be captured in a simple urographic examination . 
 this change has come about not only in intravenous urography , but also in other forms of imaging of the urinary tract , such as antegrade and retrograde pyelography , which today is limited to only a few and principally interventional or intraoperative applications . 
however , the most important change in the imaging of the urogenital system , as with other organs and systems , is the broadening of the diagnostic imaging spectrutoday , the radiologist is involved on a number of fronts , from diagnosis intended as the identification of a lesion , its characterisation and , in the case of oncologic disease , its staging to prognosis and follow - up . added to these are functional evaluations such as can be obtained with doppler ultrasonography , spectroscopy and dynamic studies with contrast media . 
 in this work , olivetti and grazioli manage to provide thorough and updated information on all of these aspects , with a highly topical and modern interpretation of contemporary radiology of the urogenital syste the book is divided into nine sections , beginning with il testo si propone con unimpostazione molto accurata in tutte le sue parti e affronta in modo organico e completo tutto lapparato uro - genitale . 
si tratta di un libro molto attuale perch il progresso che la diagnostica per immagini ha compiuto in questo distretto stato notevole e ha cambiato profondamente il nostro modo di operare e di esaminare la patologia genito - urinaria . lesempio pi evidente di questo cambiamento nella diagnostica per immagini rappresentato dellurografia endovenosa , fino a pochi anni fa considerata lindagine di riferimento per molte patologie renali e della via escretrice , oggi sostituita dallecografia , dallurografia tc o dallurografia rm . 
per evidente che le indagini tc e rm possono dare , in un unico esame , informazioni sul parenchima , sulla via escretrice , su aspetti funzionali , vascolari , nonch sugli organi e le strutture vicine , aspetti che non possono essere colti dalla semplice indagine urografica . questo cambiamento non ha riguardato solo lurografia endovenosa , ma altre forme di visualizzazione della via escretrice , quali le pielografie ascendenti e discendenti , oggi limitate a poche applicazioni , per lo pi interventistiche o intraoperatorie . 
il cambiamento pi importante nel distretto genito - urinario , al pari di quello di altri distretti e altri organi , per lallargamento dellorizzonte della diagnostica per immagini che vede impegnato il radiologo su pi fronti che vanno dalla diagnosi , intesa come riconoscimento di lesione , alla caratterizzazione , alla stadiazione nel caso della patologia oncologica , alla prognosi e al follow - up . a queste si aggiungono oggi valutazioni funzionali come ad esempio quelle ottenibili con il doppler , la spettroscopia , le valutazioni dinamiche con mezzi di contrasto . il testo di olivetti e grazioli riesce a dare una informazione completa e attuale di tutti questi aspetti interpretando radiol med ( 2009 ) 114 : 664665 the anatomy of the urinary tract , and is accompanied by excellent illustrations , some of which can be found in the later chapters . 
the presentation of normal anatomy is followed by radiologic anatomy , which includes urography , angiography , standard ultrasonography , color doppler and contrast - enhanced ultrasonography , ct complete with ct urography and mr with mr angiography and mr urography . 
this is followed by chapters on the normal human anatomy and radiologic anatomy of the male and female reproductive systems , which are organised in the same way as the previous chapters . 
the last section deals with interventional radiology in its various forms and includes chapters on biopsies , the treatment of varicocele , drainage and embolization . my sincere congratulations go to olivetti and grazioli for this fine book which clearly reflects not only their professionalism , but also their passion and enthusiasm for our discipline . in modo molto attuale e moderno la radiologia dellapparato genito - urinario dei nostri giorni . il testo si articola in nove sezioni , iniziando con lanatomia dellapparato urinario , corredata da ottimi disegni che troviamo anche in alcuni capitoli successivi . 
allanatomia classica fa seguito lanatomia radiologica comprendente lurografia , langiografia , lecografia di base , lecografia color doppler e con mezzo di contrasto , la tomografia computerizzata completa di urografia , la risonanza magnetica con langiografia rm e lurografia rm . 
seguono i capitoli di anatomia umana classica e quindi di anatomia radiologica dellapparato genitale maschile e femminile che seguono la stessa impostazione dei capitoli precedenti . le sezioni successive sono dedicate alla clinica e allimaging dei vari organi e apparati . 
guglielmi an issue of the radiologic clinics of north america volume 46 , issue 4 july 2008 elsevier - saunders , 2009 isbn 0033 - 8389 / 08 published online : 15 june 2009 springer - verlag 2009 the ageing of the population worldwide , a result of heavily declining birth rates and the steady increase in the number of elderly individuals , is creating a series of social , economic and health care problems . 
in response to the question : who is the typical geriatric patient ? hazzard some time ago answered : think of the oldest , sickest , most complicated and frail of your patients one normally affected by multiple diseases , often with atypical presentation , and by functional disabilities . 
his or her health problems are chronic , progressive , and only in part reversible . with increasing frequency , health care professionals are confronted with complex clinical scenarios arising in the same patient , which make management difficult and require special competences . 
in the elderly , and especially in geriatric patients , the coexistence of several diseases , the prevalence of involutional and degenerative aspects and a rapidly changing clinical situation together with physical and cognitive problems frequently makes it difficult for the radiologist to provide clinicians with the answers they require . in addition to being typical , all the above disturbances may also be the norm for the elderly . 
however , if we unaware of the specific normality of the elderly , we run the risk of overdiagnosis and overtreatment that is , of putting in place totally inadequate and generally excessive diagnostic and therapeutic measures that may be considered overzealous . 
 it is thus important for the radiologist to be familiar with the most common clinical scenarios in geriatric patients , which often differ from those seen in younger adults , and for the geriatrician to be aware of the potential and limits of linvecchiamento della popolazione mondiale , determinato dalla notevole riduzione del numero delle nascite e dal costante incremento del numero degli anziani , sta progressivamente determinando una serie di problemi sociali , economici e sanitari . 
alla domanda : chi il tipico paziente geriatrico ? qualche tempo fa hazzard rispose : pensa al pi anziano , al pi malato , al pi complicato ed al pi fragile dei tuoi pazienti affetto di solito da multiple malattie , la cui presentazione spesso atipica , e portatore di deficit funzionali . 
 con crescente frequenza gli operatori sanitari al giorno doggi si confrontano con complessi scenari clinici presenti nello stesso paziente , il che comporta una difficile gestione e richiede speciali competenze . 
nellanziano ed in particolare nei pazienti geriatrici , la coesistenza di pi patologie , la prevalenza di aspetti involutivo - degenerativi , una situazione clinica rapidamente mutevole assieme a problemi fisici e cognitivi frequentemente rendono difficile al radiologo fornire ai clinici le risposte che talvolta si aspettano . tutti questi disturbi , oltre ad essere tipici sono nella norma per lanziano ; quindi nellambito della radiologia geriatrica importante sapere distinguere lanziano sano da quello bisognevole di cure , poich quasi tutti si lamentano di qualche disturbo , ma talvolta non conoscendo la normalit specifica dellanziano si corre il rischio di sconfinare nellover diagnosis e nellover treatment , cio di mettere in atto interventi diagnostici e terapeutici del tutto inadeguati , il pi delle volte eccessivi e tali da configurare il cosiddetto accanimento . 
 importante per i radiologi conoscere le situazioni cliniche di pi comune riscontro nel paziente geriatrico , che sono spesso differenti da quelle osservabili negli adulti pi giovani , e per i geriatri essere a conoscenza delle potenzialit e dei limiti della moderna radiologia e delle sue appliradiol med ( 2009 ) 114 : 662663 modern radiology and its applications in geriatric patients . edited by giuseppe guglielmi , this issue of radiologic clinics of north america consists of 11 chapters that , supported by up - to - date references , review all aspects of imaging elderly patients , including the most common cardiovascular , gastrointestinal , neurological , oncohaematological , genitourinary and metabolic disorders . 
questo volume della collana radiologic clinics of north america , coordinato da giuseppe guglielmi , comprende 11 capitoli supportati da una aggiornata bibliografia che focalizzano lattenzione su tutti gli aspetti dellimaging nel paziente anziano , passando in rassegna le pi comuni problematiche cardiovascolari , respiratorie , gastrointestinali , neurologiche , onco - ematologiche , genito - urinarie e metaboliche . 
nieder springer - verlag berlin heidelberg , 2008 isbn 978 - 3 - 540 - 77384 - 9 e - isbn 978 - 3 - 540 - 77385 - 6 in this 650 - pages book written by experts in the field and edited by well - known and respected profs . 
lu and brady the reader will find forty - six chapters divided into 12 sections . the aim of the book is to present to the reader namely radiotherapy experts or radiotherapists in training as well as clinicians an evidence - based approach to the subject . the concept of evidence - based medicine and radiotherapy is explained well in the 16 - page introduction written by the two editors and described throughout the book in the framework of each chapter , according to the type of evidence level and grade of recommendations as found and described in the literature according to tumour type . 
both concepts are delineated well and relevant information for the reader is presented throughout the text . each chapter opens with a summary of important information on introduction and objectives ( clearly indicated in a blue box ) : then different colours are used to highlight the text headings ( blue for diagnosis , staging and prognosis ; red for treatment radiation , surgery , chemotherapy ; green for follow - up and surveillance ) and are summarised in a large algorithm . relevant tables for imaging , laboratory work - ups for diagnosis , tumour typing and tumour tables according to the ajcc and / or who classifications are also listed . an unusually large reference list closes each chapter , some references are as recent as to 2008 . 
 the subject index and two appendixes , one for performance status scales , the other for estimated normal tissue tolerance dose are at the end of the text . throughout the text acronyms are extensively used and this is a source of disappointment since sometimes ( yet not always ! ) their explanation is found later in the text . 
a list of abbreviations would have been useful and is recommended quarantasei capitoli suddivisi in 12 sezioni costituiscono le 650 pagine di questo libro scritto da una serie di esperti nel campo sotto la direzione dei ben noti professori lu e brady . 
 scopo del volume di presentare al lettore soprattutto radioterapisti esperti ed in via di specializzazione e tirocinio , come anche ai clinici un approccio basato sullevidenza riguardo alla materia in studio . il concetto di medicina e radioterapia basate sullevidenza ben spiegato nelle 16 pagine di introduzione a firma dei curatori del volume ed di continuo riscontro nella struttura e nel testo di ciascun capitolo , in funzione del tipo di livello di evidenza e del grado di raccomandazione trovato e descritto nella letteratura e nella pratica clinica per ciascun tipo di tumore . 
entrambi sono ben evidenziati e di importante informazione per il lettore in tutto il testo . ogni capitolo aperto da un riquadro dallo sfondo blu , contenente importanti informazioni circa lintroduzione e gli obiettivi dello stesso . 
poi colori differenti caratterizzano i titoli dei sottocapitoli ( blu per la diagnosi , stadiazione e prognosi ; rosso per la terapia radiante , chirurgica , chemioterapica ; verde per i controlli e la sorveglianza nel tempo ) e sono ripresi nella ampie figure degli algoritmi . vi sono poi tabelle circa le modalit degli studi per immagine , degli esami di laboratorio da eseguire a seconda del tipo di tumore in studio nonch le tabelle con gli elenchi dei sistemi di stadiazione degli stessi secondo le classificazioni della ajcc e / o del who . un elenco di voci bibliografiche sempre piuttosto lungo e talvolta aggiornato fino al 2008 chiude ciascun capitolo . 
il volume termina con lindice analitico e due appendici , una riguardo allo stato di rendimento del paziente , laltro al livello stimato di dose di tolleranza dei tessuti normali . in tutto il testo luso degli acronimi frequente e diffuso ed causa di insoddisfazione dato che la spiegazione degli stessi spesso ( ma talvolta neppure ) rintracciabile a distanza dalla prima citazione . 
pescarini3 1istituto nazionale di riposo e cura dellanziano ( inrca ) sede di roma , via cassia 117 , 00189 roma , italy 2istituto di medicina del lavoro delluniversit cattolica del sacro cuore roma , italy 3istituto oncologico veneto irccs dipartimento di scienze oncologiche e chirurgiche delluniversit degli studi di padova , padova , italy correspondence to : a . 
fileni , via cassia 1167 , 00189 roma , italy , tel . : + 39 - 06 - 30342519 , fax : + 39 - 06 - 30363596 , e - mail : a.fileni@inrca.it received : 12 june 2008 / accepted : 16 september 2008 / published online : 14 may 2009 springer - verlag 2009 abstract purpose . 
the study considered insurance claims filed by radiologists of the italian society of medical radiology ( sirm ) over a 12.5 - year period between 1 january1993 and 30 june 2005 . 
the analysis uncovered a complex problem : although radiologists save many lives through the radiographic diagnosis of breast cancer and consequently contribute to the welfare of society , in practice , they can face real or alleged errors , with serious judicial consequences . 
lo studio riguarda le denunce assicurative dei radiologi iscritti alla sirm dal 01 / 01 / 1993 al 30 / 06 / 2005 per un periodo di 12 , 5 anni . 
nellipotesi che la tendenza a denunciare i radiologi sia costante , si prevede che per il periodo dosservazione giungano ancora 637 nuovi casi , per un totale complessivo di 1725 . per lo studio radiografico della mammella sono attese altre 178 denunce . 
bench il radiologo con la diagnosi mammografica dei tumori della mammella salvi molte vite , contribuendo al bene della societ , nella pratica pu incorrere in errori veri o presunti , con conseguenze giudiziarie sempre pi incombenti . 
 parole chiave mammella denunce mammografia radiologi introduction introduzione from a study of a cohort of radiologists comprising more than half those enrolled in the italian society of medical radiology ( sirm ) , it emerges that failure to diagnose breast cancer is the most frequent cause of medicolegal litigation , which has considerably increased over the past 12 years [ 1 ]  . starting from an analysis of epidemiological data concerning malpractice liability claims against radiologists , we sought to examine error as an adverse event in breast cancer diagnostics with a view to uncovering critical areas and identifying possible strategies to control the malpractice risk connected to the performance of mammography . materials and methods the claims filed by italian radiologists insured through the sirm professional liability insurance scheme were anonymously reviewed . 
the number of insured radiologists increased steadily from 1 , 400 at the beginning of the study period to 4 , 490 in 2005 , for a total of 35 , 081.5 persons / year ( radiologists insured over an entire calendar year )  . 
the incidence and prevalence of claims relating to breast cancer were calculated for the period being examined . to account for the fact that claims for presumed diagnostic errors are lodged after a variable time interval , at times close to the expiry of the prescriptive period for compensation ( 10 years from the event ) , we estimated the number of insurance claims expected to be filed until the end of the prescriptive period by using a method described in a previous paper [ 1 ]  . 
first , starting from the assumption that the tendency to sue physicians is constant over time , we assumed that the trend of claims between 1993 and 1995 , once the effect of late claims was eliminated , expressed the constant trend of claims in the cohort . we then calculated the cumulative percentage of claims filed after 1 , 2 , 3 or more years in the period prior to 1995 . those cumulative percentages were applied as coefficients to the current observations . 
 dallo studio di una coorte di radiologi italiani pari ad oltre la met dei radiologi iscritti alla societ scientifica ( societ italiana di radiologia medica ) risulta evidente che la denuncia per mancata diagnosi di tumore della mammella la causa di contenzioso medico - legale che ha registrato il maggiore incremento negli ultimi dodici anni [ 1 ]  . 
muovendo dallanalisi dei dati epidemiologici relativi alle denunce di responsabilit civile contro i radiologi , ci si proposti di esaminare lerrore quale evento avverso nella diagnostica del tumore della mammella , al fine di riconoscere le problematiche relative e di identificare le possibili strategie per il controllo del rischio di malpractice connesso allesecuzione degli esami mammografici . materiali e metodi sono state analizzate in forma anonima le denunce assicurative sporte dai radiologi italiani che hanno sottoscritto lassicurazione professionale con la sirm . 
gli assicurati , in numero di 1400 allinizio del periodo dosservazione , sono progressivamente aumentati fino a 4490 nel 2005 ; complessivamente quindi le persone / anno ( radiologi assicurati per un intero anno solare ) sono 35081 , 5 . 
si proceduto al calcolo del tasso dincidenza e del tasso di prevalenza nel periodo delle denunce per neoplasia mammaria . per ovviare al fatto che le denunce per i presunti errori diagnostici sono presentate a distanza di tempo variabile dallevento , talvolta anche ai limiti della prescrizione della risarcibilit ( dieci anni ) , si proceduto a stimare il numero delle denunce che perverranno entro i termini di prescrizione con un metodo gi applicato in passato [ 1 ]  . 
in primo luogo , partendo dallipotesi che la propensione a denunciare i medici radiologi sia costante nel tempo , stato assunto che landamento delle denunce nei primi anni dosservazione , tra il 1993 ed il 1995 , una volta esaurito leffetto delle denunce landamento costante delle denunce nella coorte . 
 stata quindi la percentuale cumulativa delle denunce calcolata tardive , esprima 638 results in the study period , 1 , 088 malpractice claims were filed against radiologists ( table 1 ) , 302 of which originated from alleged failure to diagnose cancer . 
of these , 189 ( 62% ) were in regard to breast cancer and mammographic examinations ( table 2 ) , and two to mammography but not breast cancer . comparison with all other cancer sites demonstrated the criticality of mammographic diagnostics ( table 2 )  . 
clearly , in view of the time required for cases to go through three levels of courts in italy , one cannot know whether the event was a consequence of the doctors actions . 
 risultati nel periodo in esame ( 01 / 01 / 199330 / 06 / 2005 ) sono state sporte 1088 denunce contro i radiologi ( tabella 1 ) , di cui 302 originate da presunti errori diagnostici per neoplasie . centottantanove di tali denunce ( 62% ) riguardano la patologia tumorale della mammella e la relativa indagine mammografica ( tabella 2 )  . 
ai fini assicurativi , comunque , questo aspetto irrilevante , in quanto lentit dei premi richiesti al singolo assicurato non commisurata tanto alle somme effettivamente erogate dallente assicuratore , quanto alle richieste di risarcimento della controparte . tenuta ferma lipotesi che la propensione a denunciare i radiologi sia rimasta costante nel periodo dosservazione , stato esaminato separatamente landamento delle denunce degli anni per i quali si concluso il periodo per la prescrizione ( 10 anni )  . 
 stato cos possibile rilevare che solo il 19% delle denunce si riferisce ad eventi occorsi nello stesso anno , mentre nella maggior parte dei casi levento avvenuto in epoche precedenti , fino a 10 anni prima ( tabella 3 )  . landamento storico stato pertanto utilizzato per stimare il numero di denunce che ci si attende pervenga per il periodo di osservazione . 
si ottiene cos la previsione che il numero totale possa raggiungere le 1725 unit , per ulteriori 637 denunce per tutte le cause in aggiunta a quelle finora pervenute ( tabella 4 )  . 
 stata valutata la frequenza di ciascuna categoria delle cause di denuncia , ad uno e pi anni dal sinistro , in modo da stimare a quale gruppo apparterranno le denunce previste in futuro . 
the results indicated that only 19% of claims were related to events that occurred during the same year , whereas the majority of claims were related to events occurring during previous years , up to 10 years earlier ( table 3 )  . 
the total number of claims may reach 1 , 725 , as a further 637 claims for all causes are expected in addition to those already lodged ( table 4 )  . 
the percentage of claims regarding breast cancer filed more than 1 year after the first was 28% of the total ; thus , a further 178 claims relating to breast cancer are expected . 
the mean amount requested for each professional liability claim was 405 , 000 euro , an amount that rose over the last years of the study period . pari al 28% del totale ; si attendono quindi ulteriori 178 denunce per neoplasia mammaria . 
la prevalenza delle denunce gi pervenute , originate da neoplasie mammarie nel periodo in esame , pari al 5 , 4 per mille ( 189 casi per 35081 , 5 persone / anno )  . 
se sar confermata la previsione circa le denunce che saranno ricevute nei prossimi anni , sempre riferite al periodo in esame , si raggiunge un tasso del 10 , 5 per mille . 
limporto medio delle richieste per ciascun procedimento di responsabilit civile stato di 405000 euro ; tale importo in aumento negli ultimi anni dosservazione . discussione i dati esposti corrispondono al rischio malpractice relativo allindagine mammografica e conseguente ad una mancata diagnosi di neoplasia della mammella . 
il notevole incremento di denunce negli anni considerati indica una criticit assoluta che riguarda i radiologi rappresentati nel campione dai numerosi soci della societ scientifica italiana sirm , che scelgono la copertura della polizza assicurativa collegata alliscrizione annuale . lo studio , attendibile quindi per numero e omogeneit , in linea con lindagine di farria [ 2 ] negli usa , tenuto conto di alcune diversit note quali la mentalit , il sistema sanitario e il tasso di litigiosit pi elevato oltreoceano . 
the marked rise in claims over the study period indicates an absolute criticality regarding the radiologists in our sample , that is , members of the sirm who have chosen to subscribe to the professional liability insurance policy attached to the yearly sirm membership . the study , which is robust in terms of sample number and uniformity , is in line with the survey conducted by farria et al . 
if the likelihood of being subject to litigation in italy is equal to one case every 10 years per radiologist , the situation in the usa is even more dramatic : no less than legale in italia di un evento ogni 10 anni per radiologo , negli usa la situazione ancora pi drammatica : ben il 55% dei radiologi occupati in senologia ha ricevuto una denuncia con richiesta dindennizzo negli ultimi 5 anni . inoltre , in un sistema sorretto da strutture private , pi di un terzo delle aziende sanitarie che eseguono mammografie ha chiuso in passivo lultimo esercizio finanziario tanto da sospendere o ridurre il servizio , con un riflesso immediato sul prolungamento delle liste dattesa . 
ancora , unindagine condotta sulla formazione dei radiologi [ 3 ] indica che una delle ragioni principali per non impegnarsi nellattivit mammografica rappresentata dallo stress che si verifica nella visione e interpretazione dei radiogrammi per il timore di denunce . 
additionally , in a system supported by private facilities , more than one third of breast - imaging practices incurred financial losses over the last year to the point that the service was discontinued or reduced , with the immediate effect of longer waiting lists . another survey conducted on radiologist training [ 3 ] indicates that one of the main reasons for not entering the field of breast imaging is the stress related to viewing and interpreting mammograms for the fear of malpractice lawsuits . all this has a negative influence on the practice of radiology , leading to forms of defensive medicine with requests for further investigations or at times with organised aspects such as the publication of databases of the most litigious patients . 
the phenomenon is of great concern in italy owing to the unnecessary costs incurred by the healthcare system as a result of defensive medicine practices [ 46 ]  . the results also show that although the majority of claims related to mammography concerns public facilities , aspetti talora organizzati , quali la pubblicazione di database dei pazienti pi litigiosi . 
il fenomeno di enorme interesse anche nel nostro paese per i costi sostenuti inutilmente dal sistema sanitario a causa della medicina difensiva [ 46 ]  . dai risultati emerge ancora che per quanto riguarda le denunce per la mammografia , bench il maggior numero delle denunce riguardi le strutture pubbliche , vi una litigiosit per esami condotti presso strutture private che appare relativamente elevata in funzione dei minori volumi di attivit : ci potrebbe indicare una maggior propensione al contenzioso da parte delle donne che hanno scelto il professionista e pagato direttamente la prestazione . il riscontro del 69% di casi con decesso indica la gravit della malattia tumorale nei casi che danno origine a denuncia . 
poich la nostra indagine circoscritta alle richieste di assistenza avanzate dai radiologi in risposta alle citazioni , e corrisponde quindi solo allinizio del contenzioso , non possibile sapere se nella realt vi sia 642 radiol med ( 2009 ) 114 : 636644 there is also some degree of litigiousness for examinations performed at private facilities , which appears relatively high in proportion to their small work volumes . 
this could indicate a greater tendency to litigate on the part of women who have chosen their specialist and paid for the service themselves . the finding of 69% of claims in relation to deceased patients reflects the severity of the cancer in the cases producing the claims . 
because our survey is confined to requests for assistance by radiologists who have received summons and thus corresponds only to the early stages of the litigation proceedings , we cannot know whether there indeed was a failure to diagnose the cancer and whether this caused harm to the patient . 
 it should be recalled that apart from possible human error [ 7 ] , the possibility of a false negative ( fn ) result should always be considered in mammography . 
in the self - assessment test of the 2002 sirm national congress [ 10 ] , which may be considered as a test on the mammographic performance of the average italian radiologist , the rate of fn was 31% . 
 [ 11 ] used a test investigating lesion detection and number of recalls , reporting than only 32.7% of radiologists passed at the first attempt . the absolute prevalence of fn cases in mammography also defines the type of error for which the radiologist is sued . 
italian radiologists , like their us counterparts , are sued for not having detected and / or interpreted a mammographic lesion that subsequently turned out to be malignant [ 12 ]  . 
over the past few decades , they are prevalently related to omissive behaviour . in other words , whereas in the past radiologists were sued for having done something wrong , today , they are sued for not having done something appropriate [ 13 ]  . 
 given the considerable possibilities of an increasingly technological medicine , the failure to make use of further investigation methods by those performing and reporting dubious mammographic examinations is the source of claims due to omission . 
on this topic , neither economic nor procedural justifications are normally accepted in court , even when supported by community health interventions that have been validated by an evident reduction in mortality , as occurs in classic screening programmes [ 14 ]  . integrated diagnostics an application that has always resisted in its cultural perspective of maximal clinical effectiveness [ 15 ] with the recent proposal for customised stata una mancata diagnosi e se questa ha determinato un danno alla paziente . 
fanno testo le autorevoli dichiarazioni di kopans [ 8 ] la mammografia non sempre porta a diagnosticare i tumori mammari e di mavroforou [ 9 ] anche ai radiologi pi esperti possono sfuggire lesioni maligne . gli studi in materia sui radiologi italiani mettono evidenza anche lentit delle mancate diagnosi . 
nel test di autovalutazione del congresso nazionale della sirm dellanno 2002 [ 10 ] , che pu essere inteso come verifica della prestazione in mammografia del radiologo medio italiano la quota di fn del 31% . 
 [ 11 ] , con un test tarato sia sul rilevamento delle lesioni che sul numero degli eventuali richiami , segnalano che solo il 32 , 7% dei radiologi in grado di superare la prova al primo approccio . lassoluta prevalenza de casi fn in mammografia definisce in generale anche la tipologia dellerrore per il quale il radiologo chiamato in causa . 
i radiologi italiani , analogamente a quanto avviene negli usa , sono chiamati in giudizio per non aver saputo riconoscere e / o interpretare una lesione mammografica poi rivelatasi maligna [ 12 ]  . 
in sostanza mentre un tempo il radiologo subiva un procedimento per aver fatto qualcosa di sbagliato , oggi lo per non aver fatto qualcosa di appropriato [ 13 ]  . 
 a fronte delle ampie possibilit di una medicina sempre pi tecnologica , il mancato ricorso ad ulteriori metodiche dindagine da parte di chi ha eseguito e refertato lesame mammografico dubbio origina la denuncia di comportamenti omissivi . 
a riguardo , in sede di giudizio , non sono di regola accolte giustificazioni n economiche n procedurali , anche se sostenute da logiche di intervento sanitario di tipo collettivo gi validate da evidente riduzione della mortalit , come avviene nei programmi di screening classico [ 14 ]  . la diagnostica integrata , aspetto applicativo che resiste fin dai primordi nella sua prospettiva culturale di massima efficacia clinica [ 15 ] e con la recente proposta di modelli organizzativi personalizzati [ 16 ] , rappresenta la modalit dapproccio personale pi consona ad evitare censure supportate da omissioni . 
in un recente studio policentrico , lincremento del numero di neoplasie diagnosticate con radiol med ( 2009 ) 114 : 636644 organisation models [ 16 ] constitutes the most suitable personal approach for avoiding litigation caused by omission . 
in a recent multicentre study , the increase in the number of cancers diagnosed through the systematic use of ultrasound combined with mammography in women at high risk was not considered sufficient to compensate for the increased expenditure , the use of highly qualified personnel and the inevitable increase in false positive results [ 17 ]  . the long latency between the diagnostic service and the possible claim indicates a need to seek prospective insurance coverage . 
claims - made policies , for example , while less costly , offer coverage only for claims relating to events that occurred during the policy year and up to a short time thereafter and are therefore unsuitable for protecting against civil liability claims lodged several years after the service was rendered , at times even after retirement . 
another type of policy ( known as loss occurrence ) covers for all claims filed in relation to events that occurred within the policy year and up to the expiry of the 10 - year prescriptive period . it should also be noted that some types of malpractice claims are not covered by the insurance policy . 
 the mean amount of damages , 405 , 000 euro , which appears fairly high , is rising , and this will lead to an increase in insurance premiums unless drastic reforms are made to the healthcare compensation syste to this end , both medical councils and professional medical associations are urging the government to review the judicial and institutional aspects of medical liability in italy . 
in the field of mammography , a recent document of the italian ministry of health [ 18 ] draws attention to all the issues regarding alleged interpretation errors in the reading of mammograms and the finding of interval cancers . 
the document notes that the indications provided for predicting and minimising errors in a context of clinical risk management are inadequate to avert the risk of litigation , in which the review process is usually influenced by the retrospective method of judicial evaluation . limpiego sistematico dellecografia associata alla mammografia in donne a rischio , non stato ritenuto sufficiente a compensare lesborso economico , limpiego di personale altamente qualificato , e linevitabile aumento dei falsi positivi [ 17 ]  . lelevato intervallo di tempo tra la prestazione e leventuale denuncia indica la necessit di provvedere ad una copertura assicurativa valida in termini prospettici . 
le polizze claims made ad esempio , economicamente meno onerose , coprono solo le denunce pervenute nellanno assicurato , o in un breve periodo successivo , e si rivelano quindi inadatte a tutelare dalle denunce di responsabilit civile sporte a distanza di numerosi anni dallesecuzione della prestazione , eventualmente anche dopo la cessazione dellattivit professionale . 
 limporto medio di richiesta dindennizzo di 405000 euro , che gi appare piuttosto elevato , tende a crescere e , con esso , cresceranno i premi richiesti dalla compagnie assicurative , a meno di drastiche riforme del sistema risarcitorio medico - sanitario . 
in campo mammografico un recente documento del ministero della sanit [ 18 ] mette in evidenza tutte le problematiche riguardanti gli errori interpretativi supposti nella lettura della mammografia e il riscontro dei cancri dintervallo . 
si osserva nel documento che le indicazioni a prevedere e a contenere lerrore nellambito della gestione del rischio clinico non appaiono sufficienti a scongiurare il pericolo di un procedimento , nel quale le modalit di revisione risentono di solito di una metodologia di valutazione giudiziaria influenzata dal senno del poi . conclusioni conclusions analysis of professional liability claims filed against italian radiologists in relation to mammography indicates the presence of an extensive and complex problem : on the one hand , the radiologists personal commitment to a field where many lives can be saved and , on the other , the risk of facing the legal consequences of real or alleged errors . 
twenty - six consecutive patients , 14 with liver metastases ( ten from colorectal cancer ; four from carcinoid tumours ) and 12 with biliary cancers ( ten klatskin tumours ; one gallbladder tumour ; one intrahepatic cholangiocarcinoma ) with insufficient predicted future remnant liver ( frl ) underwent right pve to induce hypertrophy of the contralateral hemiliver prior to surgical resection . 
the frl volume increased by 5%25% ( 15% on average ) after right pve in patients with liver metastases and by 9%19% ( 14% on average ) in patients with biliary cancers . 
in all patients , the ratio of frl to functional liver volume was 30% after right pve . no postoperative deaths due to severe liver failure occurred in the 20 patients who underwent extended hepatectomy . 
lo scopo del nostro studio retrospettivo stato di valutare lefficacia dellembolizzazione del ramo portale destro ( rpve ) come trattamento pre - chirurgico per indurre lipertrofia del fegato sinistro in pazienti candidati a resezione epatica . 
ventisei pazienti consecutivi , 14 portatori di metastasi epatiche ( colon - retto , 10 pazienti ; carcinoide , 4 pazienti ) e 12 portatori di neoplasia delle vie biliari ( neoplasia di klatskin , 10 pazienti ; neoplasia della colecisti , 1 paziente ; colangiocarcinoma intraepatico , 1 paziente ) con potenziale riserva funzionale epatica ( frl ) insufficiente sotto stati sottoposti a rpve come trattamento pre - chirurgico . 
il volume del frl ha dimostrato un incremento volumetrico compreso tra il 5%25% ( 15% in media ) dopo rpve nei pazienti con metastasi epatiche e tra il 9%19% ( 14% in media ) in pazienti con neoplasie biliari . 
lembolizzazione portale allarga le indicazioni alla chirurgia resettiva epatica in pazienti con metastasi epatiche e neoplasie delle vie biliari con potenziale riserva funzionale insufficiente . 554 radiol med ( 2009 ) 114 : 553570 keywords right portal vein embolisation hepatic functional reserve ct hepatic volumetry parole chiave embolizzazione del ramo portale destro riserva funzionale epatica tc volumetria epatica introduction introduzione the curative treatment of primary and secondary liver cancers that ensures long - term survival is surgery . 
more than 45% of liver tumours , both primary and secondary , require extended liver resection with disease - free margins . postoperative death after extended hepatectomy , which ranges from 3.2%7% in patients with a normal hepatic functional reserve to 32% in patients with cirrhosis , is most often caused by an insufficient future remnant liver ( frl )  . postoperative liver failure has been shown to be directly related to frl volume , and preoperative procedures to induce liver regeneration have been proposed [ 13 ]  . 
on the basis of experimental studies in 1920 that showed that ligation of a portal branch led to atrophy of the ipsilateral liver parenchyma and hypertrophy of parenchyma with preserved portal flow , makuuchi et al . 
 [ 4 ] in 1990 used portal occlusion as a preoperative treatment to extend the indication for curative liver resection to patients with biliary cancers . embolisation of a portal branch is currently indicated when the volume of the predicted frl is insufficient to ensure an adequate postoperative hepatic functional reserve . 
separate determinations of liver volume and tumour volume makes it possible for hepatic functional reserve to be estimated with computed tomography ( ct ) and magnetic resonance ( mr ) imaging by subtracting tumour volume from total liver volume , i.e. 
the potential functional reserve of frl as a ratio between the frl and the total functional liver mass . the purpose of this retrospective study was to assess the effectiveness of right portal vein embolisation ( pve ) as a preoperative treatment in noncirrhotic patients in whom the predicted functional reserve of the frl , calculated with ct , was insufficient for radical liver resection . 
in pi del 45% dei tumori del fegato , sia primitivi che secondari , necessaria unampia chirurgia resettiva epatica che assicuri margini di resezione liberi da malattia . la principale causa di morte post - chirurgica dopo epatectomie estese , che oscilla tra il 3 , 2% e 7% nei pazienti con normale riserva funzionale epatica e fino al 32% nei pazienti con cirrosi , spesso un insufficiente future remnant liver ( frl )  . 
stato dimostrato che linsufficienza epatica post - chirurgica direttamente correlata al volume del frl e sono state proposte procedure pre - chirurgiche che inducono la rigenerazione epatica [ 13 ]  . 
sulla base di studi sperimentali che nel 1920 avevano dimostrato che la legatura di un ramo portale determinava latrofia del parenchima epatico tributario e lipertrofia del parenchima epatico con flusso portale conservato , makuuchi et al . 
 [ 4 ] gi nel 1990 hanno usato per primi locclusione portale come trattamento pre - chirugico per ampliare lindicazione a chirurgia resettiva epatica curativa in pazienti portatori di neoplasie delle vie biliari . 
lembolizzazione di un ramo portale attualmente indicata quando il volume del potenziale frl inadeguato ad assicurare unadeguata riserva funzionale epatica postchirurgica e quindi il calcolo della volumetria epatica necessario per selezionare i pazienti da sottoporre a trattamento pre - chirurgico . 
il calcolo separato del volume epatico e del volume tumore permette oggi alla tomografia computerizzata ( tc ) ed alla risonanza magnetica ( rm ) di valutare la riserva funzionale epatica sottraendo al volume totale epatico il volume tumore e quindi la potenziale riserva funzionale del frl come rapporto del frl sulla massa epatica totale funzionante . scopo primario di questo studio retrospettivo di valutare lefficacia dellembolizzazione del ramo portale destro ( rpve ) come trattamento pre - chirurgico in pazienti non cirrotici nei quali la potenziale riserva funzionale del frl , calcolata mediante tc , era insufficiente per una chirurgia resettiva epatica oncologicamente radicale . 
 materials and methods materiali e metodi between november 1996 and september 2007 , 26 noncirrhotic patients ( 12 men and 14 women , aged 3581 years ; mean age 58 years ) underwent pve procedures . 
dodici pazienti erano portatori di neoplasie delle vie biliari [ 5 ] : neoplasia di klatskin ( 10 pazienti ) classificati secondo bismuth come tipo ii ( interruzione convergenza biliare principale , 6 pazienti ) , tipo iiia ( interruzione radiol med ( 2009 ) 114 : 553570 confluence , n = 6 ) , type iiia ( obstructed main confluence extending to the right secondary confluence , n = 2 ) , type iv ( obstructed primary and secondary confluence bilaterally , n = 2 ) , gallbladder cancer ( n = 1 ) and intrahepatic cholangiocarcinoma ( n = 1 )  . 
in particular , the lesions were located in the right liver lobe and in segment iv in three patients ( two with colorectal metastases and one with carcinoid metastases ) and in the right liver lobe in segments iv and ii in two patients ( one with colorectal metastases one with carcinoid metastases )  . 
all patients with hepatic metastases from colorectal cancer underwent chemotherapy ( three to six cycles , four cycles on average ) , with partial response ( no less than 30% ) according to the response evaluation criteria in solid tumours ( recist ) criteria [ 6 ] or with stable disease . 
in the patient with bilobar colorectal metastases in segments ii and iv of the left liver , right pve was preceded by excision of the left lobar metastases , a procedure known as two - stage hepatectomy . 
all patients with klatskin tumours underwent percutaneous biliary drainage bilaterally ( n = 6 ) or on the left side only ( n = 4 ) ; a prerequisite for right pve was biliary drainage of the frl with reduction of bilirubin ( < 3 mg / dl )  . 
in particolare le lesioni erano localizzate nel fegato destro e nel iv segmento epatico in 3 pazienti ( 2 da neoplasia colo - rettale e 1 da carcinoide ) ; nel fegato destro , nel iv e nel ii segmento in 2 pazienti ( 1 da neoplasia colo - rettale e 1 da carcinoide )  . tutti i pazienti portatori di metastasi epatiche da neoplasie colo - rettali hanno effettuato chemioterapia ( 36 cicli con una media di 4 cicli ) con una risposta parziale ( non inferiore al 30% ) secondo i criteri recist ( response evaluation criteria in solid tumours ) [ 6 ] o con malattia stabile , che ha preceduto lembolizzazione portale con un intervallo di tempo di almeno 28 giorni . 
nel paziente portatore di metastasi bilobari da neoplasia colo - rettale , localizzate nel fegato sinistro , in corrispondenza del ii oltre che nel iv segmento , la rpve stata preceduta dalla metastesectomia delle metastasi del fegato sinistro , two stage hepatectomy . 
i pazienti con metastasi da carcinoide non sono stati sottoposti a terapia neo - adiuvante : quando possibile , la resezione epatica rappresenta in questi pazienti il miglior trattamento in termini di sopravvivenza a lungo termine [ 7 ]  . tutti i pazienti portatori di neoplasie di klatskin sono stati sottoposti a drenaggio biliare per via transcutanea dei due emisistemi biliari ( 6 pazienti ) o dellemisistema di sinistra ( 4 pazienti ) ; presupposto indispensabile alla rpve stato il drenaggio biliare del frl con riduzione dei valori ematici di bilirubina ( non superiore a 3 mg / dl )  . 
 nel periodo compreso tra il 1996 ed il 2003 gli esami tc sono stati eseguiti in 11 pazienti con tc spirale singolo strato prospeed advantage ( ge medical systems , milwaukee , wis , usa ) come precedentemente descritto [ 8 ]  . nel periodo compreso tra il 2003 ed il 2007 gli esami tc sono stati eseguiti in 15 pazienti mediante tc spirale multistrato lightspeed pro 16 ( ge medical system ) , utilizzando i seguenti parametri : ma automatico ( noise index 14 ) , 120 kvp , collimazione dei detettori 1 , 25 , velocit del tavolo 18 , 75 mm per rotazione , velocit di rotazione del gantry 0 , 6 secondi a rotazione . 
sono stati utilizzati uno spessore di ricostruzione di 2 , 5 mm ed un intervallo di ricostruzione di 2 , 5 mil mezzo di contrasto ( 2 ml / kg di peso corporeo con una concentrazione di 350 mgi / ml ) stato somministrato per via ev attraverso lutilizzo di un iniettore automatico , ad una velocit di 45 ml / s attraverso un ago - cannula di 18 gauge posta in una vena del braccio . 
la scelta dei tempi di ritardo per lacquisizione delle immagini stata ottenuta mediante lutilizzo di un sistema automatico di bolus tracking che prevedeva un singolo sistema di monitoraggio a bassa dose posto a livello di un vaso dinteresse ( aorta addominale ) dopo circa 10 secondi dallinizio delliniezione del mezzo di contrasto ; raggiunto un picco di enhancement di circa 80 uh nella regione dinteresse , circa 10 secondi dopo iniziava lacquisizione della fase arteriosa . 
le fasi portali e tardive venivano calcolate a 70 e 180 secondi dalliniezione del mezzo di contrasto . 556 radiol med ( 2009 ) 114 : 553570 number , location and volume of the liver metastases as well as the resectability criteria based on the evaluation of vascular relationships ensuring adequate perfusion , and venous and biliary drainage of the frl . 
in no patient did the ct scan provide a ratio of hepatic to splenic parenchyma density ratio 1.1 , an indication of moderate - to - severe ( 30% ) steatosis [ 9 , 10 ]  . all patients with klatskin tumours underwent mr cholangiography ( 1.5 - tesla , horizon advantage system , ge medical systems ) to stage the biliary disease . 
volumetric data were obtained from the portal - venous - phase scans , and all examinations were processed by two radiologists on a workstation ( ge medical system 4.1 ) that automatically calculated the volume by multiplying the area by the thickness and also generated 3d reconstructions . the liver parenchyma was isolated from surrounding tissues by using the cursor , and the total liver volume was calculated . 
frl was jointly determined by surgeons and radiologists on the basis of disease extent on imaging ( number , site , vascular relationships of metastases and vascular relationships of biliary cancers ) and according to the surgical plan defined by the surgeons . 
in the volumetric assessment the suprahepatic veins , the teres ligament , the umbilical portion of the left portal branch , the gallbladder and the inferior vena cava were used as anatomical landmarks to delineate the liver segments and separate the liver to be resected from the frl . 
tumour volume was not calculated in patients with biliary cancers , as small size and predominantly biliary involvement made it irrelevant to the evaluation of the ratio of frl volume to total volume of nontumourous liver parenchyma . 
the catheter was advanced to the portal trunk and , after the injection of contrast material , portography was performed to identify the intrahepatic portal branches and any anatomical variants . 
all patients underwent a ct scan 1 week before right pve and , after right pve , within a week before surgery . selection criteria for pve was an frl / total functional liver parenchyma ratio 30% . 
where waiting times exceeded 1 month , this was related to one patient with liver metastases refusing surgery for 2 months , and to our deferring surgery to after having solved the lesame tc prima e dopo rpve ha fornito il numero , la sede , il volume delle metastasi epatiche ed i criteri di resecabilit , basata sulla valutazione dei rapporti vascolari che assicurassero adeguata perfusione , drenaggio venoso e biliare del frl . 
in nessun paziente lesame tc ha rilevato il rapporto della densit parenchima epatico - parenchima splenico 1 , 1 , indice di steatosi moderata - grave ( superiore al 30% ) [ 9 , 10 ]  . tutti i pazienti portatori di neoplasie di klatskin hanno eseguito la colangio - rm per stadiare la malattia sul versante biliare ( stata utilizzata apparecchiatura a 1 , 5 tesla , horizon advantage ge medical systems , milwaukee , wis )  . 
i dati volumetrici sono stati elaborati dalle scansioni eseguite durante la fase portale dellenhancement vascolare e tutti gli esami sono stati elaborati da due radiologi dedicati ad una workstation ( ge medical system 4.1 ) che ci ha fornito il volume mediante il calcolo automatico dellarea x lo spessore , fornendoci anche ricostruzioni 3d . mediante un cursore stato isolato il parenchima epatico dai tessuti circostanti ed stato calcolato il volume epatico totale . 
le vene sovraepatiche , il legamento rotondo , la porzione ombelicale del ramo portale sinistro , la colecisti e la vena cava inferiore sono stati utilizzati come reperi anatomici per delineare i vari segmenti epatici e per separare nella valutazione volumetrica il fegato da resecare dal frl . 
il volume tumore non stato calcolato nei pazienti con neoplasie delle vie biliari per le piccole dimensioni e per il prevalente coinvolgimento biliare , quindi non stato considerato rilevante nella valutazione del rapporto tra volume del frl e volume del parenchima epatico totale funzionante . 
 lembolizzazione del ramo portale destro stata effettuata dopo puntura sotto guida ecografica del ramo portale di sinistra a livello del recesso di rex ; il catetere ha raggiunto quindi il tronco portale e con liniezione del mdc si ottenuto una portografia per individuare i rami portali intra - epatici ed eventuali varianti anatomiche ; successivamente si occluso il ramo portale destro con le sue diramazioni con materiale embolizzante costituito da cianoacrilato e lipiodol . 
tutti i pazienti sono stati sottoposti ad esame tc entro una settimana prima della rpve e dopo rpve entro una settimana prima della chirurgia . il criterio di selezione dei pazienti da sottoporre ad rapporto embolizzazione prechirurgica frl / parenchima epatico totale funzionante inferiore al 30% . 
any volume changes in frl before and after right pve were assessed with the t test , with significance set at p < 0.001. results the injection of cyanoacrylate and lipiodol achieved complete embolisation of the right portal tree in 24 patients . in two patients , preliminary embolisation of the portal branch of segment iv to be resected according to the surgical plan was achieved . 
 the increase a significant change ( p < 0.001 ) in frl volume after right pve was obtained both in patients with liver metastases and in those with biliary cancers . 
in nine patients with hepatic colorectal metastases , in frl volume ( 18571.16 cm3 ) after 2548 days ( 33.8 days on average ) associated with atrophy of the functional liver parenchyma to be resected ( 186.196.62 cm3 ) changed the frl / total functional liver - volume ratio from 18%29% ( 25% on average ) before right pve to 30%51% ( 39% on average ) after right pve . 
after right pve , the volume of the metastases from colorectal cancer in the embolised liver increased by 6%155.5% , whereas in the patient who waited 60 days , the tumour increased in volume by 562.7% ( table 1 )  . 
patients with metastases from carcinoid tumours showed no increase in volume of metastases after right pve in either the embolised or unembolised liver , whereas frl hypertrophy ( 283.7570.1 cm3 ) significantly changed liver parenchyma ratio from 20%27% to 42%52% ( table 1 ) after a period of 3034 days ( 31 days on average )  . 
after a period of 3180 days ( 45.9 days on average ) after right pve , frl hypertrophy ( 187.1671.5 cm3 ) and atrophy of the parenchyma to be resected ( 305.7182.5 cm3 ) changed 30%45% ( table 2 )  . 
le variazioni volumetriche del frl prima e dopo rpve sono state valutate mediante il test t di student ( un valore di p < 0 , 001 stato considerato significativo )  . risultati mediante liniezione di cianoacrilato e lipiodol si ottenuta lembolizzazione completa dellalbero portale di destra in 24 pazienti . 
in 2 pazienti si ottenuta lembolizzazione del ramo portale segmentario del iv segmento da resecare nel programma chirurgico , in uno dei quali la successiva embolizzazione a destra non stata completa ed ha interessato solo il settore anteromediale . 
un paziente stato sottoposto ad una seconda seduta di embolizzazione che ha seguito di 22 giorni la precedente con la quale non si era ottenuta lembolizzazione completa dellalbero portale di destra . 
 la variazione volumetrica dopo rpve del frl stata significativa nei pazienti con metastasi epatiche ( p < 0 , 001 ) e nei pazienti con neoplasie delle vie biliari ( p < 0 , 001 )  . 
in 9 pazienti portatori di metastasi da colon - retto lincremento volumetrico del frl ( 18571 , 16 cm3 ) dopo un periodo compreso tra i 25 ed i 48 giorni ( in media 33 , 8 giorni ) associato allatrofia del parenchima epatico funzionante da resecare ( 186 , 196 , 62 cm3 ) ha modificato il rapporto frl / volume epatico totale funzionante dal 18%29% ( in media 25% ) prima della rpve al 30%51% ( in media 39% )  . 
dopo rpve si riscontrato un incremento volumetrico delle metastasi da colon - retto nel fegato embolizzato compreso tra il 6% e il 155 , 5% , mentre nella paziente che ha atteso 60 giorni lincremento del volume tumore stato del 562 , 7% ( tabella 1 )  . 
nei pazienti portatori di metastasi da carcinoide dopo rpve non si verificato incremento volumetrico delle metastasi sia nel fegato embolizzato che nel fegato non embolizzato , mentre lipertrofia del frl ( 283 , 7570 , 1 cm3 ) ha significativamente cambiato il rapporto frl / parenchima epatico funzionante dal 20%27% al 42%52% ( tabella 1 ) dopo un periodo compreso tra i 30 ed i 34 giorni ( in media 31 giorni )  . 
nei pazienti portatori di neoplasie delle vie biliari lfrl costituiva il 18%28 , 6% del parenchima epatico funzionante , dopo un periodo compreso tra i 31 e gli 80 giorni ( in media 45 , 9 giorni ) dopo rpve lipertrofia del frl ( 187 , 1671 , 5 cm3 ) e latrofia del parenchima da resecare ( 305 , 7182 , 5 cm3 ) ha modificato questo rapporto compreso 30%45% ( tabella 2 )  . 
non progressione di malattia rispetto allesame pre - rpve stata documentata dalla tc nelle neoplasie di klatskin , nella neoplasia della colecisti e 558 radiol med ( 2009 ) 114 : 553570 radiol med ( 2009 ) 114 : 553570 560 radiol med ( 2009 ) 114 : 553570 radiol med ( 2009 ) 114 : 553570 562 radiol med ( 2009 ) 114 : 553570 patient who had undergone metastasectomy during previous surgery . 
eight patients with biliary cancers underwent right hepatectomy extended to segment i ( n = 3 ) and to segments i and iv ( n = 5 )  . preoperative imaging understaged four patients , and radical surgery could not be performed owing to the finding , on exploratory laparotomy , of peritoneal carcinosis ( one patient with metastasis from colon cancer ) , positive lymph nodes at the hepatic hilum ( one patient with type ii klatskin tumour ) , neoplastic lymphangitis along the hepatic pedicle ( one patient with type ii klatskin tumour ) and hepatic artery invasion ( one patient with type iv klatskin tumour )  . 
two patients did not undergo surgical exploration : one with a klatskin tumour because of the onset of ascites after the second embolisation session , and the other with colorectal cancer because of the onset of lung metastases . 
 discussion surgery of biliary cancers is a difficult procedure requiring hepatectomy extended to the caudate lobe , a site of possible recurrence ; resection of the main bile duct with creation of biliodigestive anastomoses ; and possible vascular reconstructions in patients with obstructive jaundice . 
five - year survival in patients with klatskin tumours treated with surgical resection with disease - free margins is between 43% and 46% , but it involves ablation of up to 80% of the liver parenchyma [ 13 ]  . 
in our experience , five patients with biliary cancers who underwent surgical resection after right pve required a right hepatectomy extended to segment iv associated with caudectomy ( necessary for all patients with biliary cancer )  . 
reported that in 132 patients with cholangiocarcinoma who underwent surgical resection after right pve due to insufficient frl volume , the 5 - year survival rate was comparable to that of a control group who underwent surgery without preoperative right pve owing to adequate frl [ 12 ]  . patients with unresected liver metastases from colorectal cancer had a mean survival of 421 months , with a 3 - year survival rate less than 3% . systemic chemotherapy improves quality of life but has limited impact on survival . 
dodici pazienti portatori di metastasi epatiche sono stati sottoposti ad epatectomia destra ( 4 pazienti ) , epatectomia destra allargata a i e iv segmento ( 2 pazienti ) , epatectomia destra allargata al iv ( 6 pazienti ) , uno dei quali con metastesectomia in un precedente tempo chirurgico . 
in tutti i pazienti stata eseguita lecografia intraoperatoria che ha confermato il numero e la sede delle lesioni individuate allesame tc . otto pazienti portatori di neoplasie biliari sono stati sottoposti ad epatectomia destra allargata al i segmento ( 3 pazienti ) e ad epatectomia destra allargata a i e iv segmento ( 5 pazienti )  . quattro pazienti sono stati sottostadiati allimaging preoperatorio e non stato possibile effettuare una chirurgia oncologicamente radicale per la presenza alla laparotomia esplorativa di carcinosi peritoneale ( 1 pazienti portatore di metastasi da neoplasia del colon ) , di linfonodi positivi allilo epatico ( 1 pazienti con tumore di klatskin tipo ii ) , per linfangite neoplastica lungo il peduncolo epatico ( 1 pazienti con tumore di klatskin tipo ii ) , per infiltrazione dellarteria epatica ( 1 pazienti con tumore di klatskin tipo iv )  . 
2 pazienti non sono stati esplorati chirurgicamente , un pazienti portatore di neoplasia di klatskin per la comparsa di ascite dopo la seconda seduta di embolizzazione e laltro pazienti con neoplasia colon - rettale per la comparsa di metastasi polmonari . 
nellimmediato postoperatorio , nella nostra casistica , nessun paziente deceduto per insufficienza epatica grave post - chirurgica ; solo in quattro pazienti si avuta una transitoria lieve insufficienza epatica ( bilirubina > 2 , 9 mg / dl e riduzione del tempo di protrombina < 50% [ 11 ] )  . 
 discussione la chirurgia delle neoplasie delle vie biliari una procedura difficile che richiede estese epatectomie che comprendono sempre il lobo caudato , sede di possibile recidiva , la resezione della via biliare con la ricostruzione di anastomosi biliodigestive , possibili ricostruzioni vascolari , in pazienti con ittero ostruttivo e , quindi , la riduzione dei rischi chirurgici e postchirurgici un sfida per il chirurgo epatobiliare [ 12 ]  . 
la sopravvivenza a 5 anni nei pazienti portatori di neoplasie di klatskin sottoposti ad exeresi chirurgica con margini di resezione libera da malattia compresa tra 43%46% ma prevede lasportazione fino all80% del parenchima epatico [ 13 ]  . 
 [ 12 ] hanno verificato in 132 pazienti con colangiocarcinoma , sottoposti ad exeresi chirurgica dopo rpve per insufficiente frl , che la sopravvivenza a 5 anni sovrapponibile ad un gruppo di controllo sottoposto a chirurgia senza rpve per adeguato frl [ 12 ]  . 
nei pazienti con metastasi epatiche da neoplasie del colon - retto non trattate chirurgicamente , la sopravvivenza media varia dai 4 ai 21 mesi con una sopravvivenza a 3 anni inferiore al 3% . radiol med ( 2009 ) 114 : 553570 fig . 
1a - h computed tomography ( ct ) images obtained before and after right portal vein embolisation ( rpve ) in a 71 - year - old man with gallbladder cancer . 
a - d axial and oblique reformatted ct images obtained before ( a , b ) and after ( c , d ) rpve show the gallbladder cancer invading liver segments iv ( arrows in a and c ) and v ( arrows in b and d ) without hilar invasion . 
e - h twoand three - dimensional maximum intensity projection ( mip ) reformations obtained before ( e , f ) and after rpve ( g , h ) clearly show a proportional twofold atrophy - hypertrophy effect ( decrease in volume of the right liver and increase in volume of the left liver )  . 
a - d le immagini tc assiali e le ricostruzioni oblique ottenute prima ( a , b ) e dopo ( c , d ) rpve documentano la neoplasia delle colecisti che invade iv ( frecce in a e c ) e v segmento epatico ( frecce in b e d ) , senza infiltrazione dellilo . 
e - h le ricostruzioni 2d e 3d mip ottenute prima ( e , f ) e dopo rpve ( g , h ) dimostrano chiaramente un proporzionale duplice effetto atrofia - ipertrofia ( riduzione di volume del fegato destro ed incremento volumetrico del fegato sinistro )  . 
a , b maximum intensity projection ( mip ) reformation in the portal phase ( a ) and axial computed tomography ( ct ) in the delayed phase ( b ) demonstrate an enhancing irregular nodular mass ( arrows in a and b ) adjacent to the right portal vein and invading segment iv with involvement of the right secondary biliary confluence and dilatation of the right intrahepatic bile duct . 
after rpve , left hepatic hypertrophy is evident on 3d mip reconstructions , on anterior view ( c ) and at surgery ( d ) , with evident delimitation caused by the different perfusion of the two hemilivers . 
a , b la ricostruzione mip 2d in fase portale ( a ) e limmagine assiale in fase tardiva ( b ) documentano una formazione nodulare , irregolare e con enhancement ( frecce in a e b ) adiacente al ramo portale di destra che invade il iv segmento con interruzione della convergenza biliare secondaria di destra e dilatazione a monte delle vie biliari intraepatiche dellemisistema di destra . 
lipertrofia del fegato sinistro dopo rpve evidente nelle ricostruzioni mip 3d , visione anteriore ( c ) e allintervento chirurgico ( d ) con evidente demarcazione per la diversa perfusione dei due emifegati . 
the nonresectability of potential candidates for resection may be related to an insufficient frl ( 8% in 746 hepatectomies in elias et al.s experience ) [ 16 ]  . the liver has the ability to regenerate rapidly , a property that provides the rationale for new and innovative surgical techniques for liver resection and organ transplantation . 
one strategy employed by many hepatobiliary surgeons to reduce the risk of complications after extended hepatectomy is pve . occlusion of a portal branch causes the redistribution of the portal flow with a double effect : atrophy of the embolised parenchyma and hypertrophy of the unembolised parenchyma . 
lunico trattamento che migliora la sopravvivenza quello chirurgico : dopo la resezione a 5 anni stimata essere tra il 25% ed il 40% , a 10 anni tra il 22% e il 26% [ 14 , 15 ]  . 
tuttavia lintervento chirurgico possibile solo nel 15%25% dei pazienti con metastasi da colon - retto in relazione allo stadio della malattia , alla localizzazione delle lesioni ripetitive epatiche ed a problemi tecnici ma in pazienti potenzialmente resecabili la non resecabilit pu essere legata ad un insufficiente frl ( 8% in 746 epatectomie nellesperienza di elias ) [ 16 ]  . il fegato possiede la capacit di rigenerare velocemente e su questo principio si basano le pi moderne ed innovative tecniche chirurgiche di resezioni epatiche e di trapianto dorgano . 
locclusione di un ramo portale determina la ridistribuzione del radiol med ( 2009 ) 114 : 553570 to be resected and hypertrophy of the frl is to the advantage of the potential postoperative hepatic functional reserve . immediately after embolisation , the portal flow increases in the unembolised portal territory , as demonstrated by doppler ultrasonography [ 17 ] , leading to parenchymal hypertrophy that correlates with the magnitude of the increase in portal flow [ 8 , 18 , 19 ]  . 
 [ 21 ] , however , showed that not only the amount but also the quality of portal blood affects liver regeneration , and it is indeed the blood flowing back from the pancreatic bed that contains hepatotrophic factors . 
 [ 22 ] reported that the regeneration process of a normal liver after resections occurs in three phases : rapid increase during the first month , decrease during the second month and , finally , extremely slow increase . 
 [ 23 ] recently demonstrated that noncirrhotic livers require a minimum of 3 weeks after right pve owing to hypertrophy of the frl , followed by a plateau lasting between 22 and 56 days . 
noncirrhotic livers have the greatest regenerative capacity ( approximately 1221 cm3 / day 2 weeks after pve , approximately 11 cm3 / day at 4 weeks and 6 cm3 / day at 32 weeks ) [ 2 , 24 , 25 ]  . 
in noncirrhotic patients , a period of 34 weeks is required to assess the efficacy of pve in increasing frl volume . longer waiting periods , related to inflammatory biliary complications in patients with biliary cancers or to the patients refusal , showed that the atrophy of the embolised parenchyma progresses over time , whereas the hypertrophy of the frl is not significantly different . 
 at the beginning of our experience , when surgical planning envisaged a right hepatectomy extended to segment iv , we combined embolisation of the portal branch of segment iv with subsequent right pve . 
 [ 26 ] , we no longer believe this approach to be necessary , in part in consideration of the increased risk of accidental reflux of embolising material into the left portal branch . 
the potential hepatic functional reserve is calculated by using ct determinations of frl volume and total volume of functional liver parenchyma , obtained by subtracting tumour volume from total liver volume , and thus by the frl / total functional liver - volume ratio . 
the method allows for separate determinations of tumour volume and healthy parenchyma , thereby providing an flusso portale con un duplice effetto : latrofia del parenchima epatico embolizzato e lipertrofia del parenchima epatico non embolizzato . 
questo duplice effetto antagonista con atrofia del parenchima epatico da resecare ed ipertrofia del frl a vantaggio della potenziale riserva funzionale epatica post - chirurgica . immediatamente dopo lembolizzazione il flusso portale aumenta , come dimostrato mediante eco - doppler [ 17 ] , nel territorio portale non embolizzato determinando ipertrofia del parenchima epatico che correla con lentit dellincremento del flusso portale [ 8 , 18 , 19 ]  . 
 [ 21 ] hanno dimostrato che non solo la quantit , ma anche la qualit del sangue portale influenza la rigenerazione epatica , infatti il sangue refluo dal letto pancreatico che contiene fattori epatotrofici . 
 [ 22 ] hanno dimostrato che il quadro rigenerativo di un fegato normale dopo resezioni epatiche consiste in tre fasi : un rapido incremento durante il primo mese , un decremento nel secondo e alla fine un lentissimo incremento . 
 [ 23 ] hanno recentemente dimostrato che in fegati non cirrotici un minimo di 3 settimane richiesto dopo rpve per lipertrofia dellfrl seguito da un plateau compreso tra 22 e 56 giorni . 
fegati non cirrotici dimostrano la pi alta capacit rigenerativa ( circa 1221 cm3 / d a 2 settimane dopo lembolizzazione portale , circa 11 cm3 / d a 4 settimane e 6 cm3 / d a 32 settimane ) [ 2 , 24 , 25 ]  . 
fegati cirrotici o pazienti diabetici mostrano il pi basso grado di ricrescita approssimativamente ( 9 cm3 / d a 2 settimane dopo pve ) [ 2 , 25 ]  . 
il periodo di attesa pi lungo , in relazione a complicanze flogistiche biliari nei pazienti portatori di neoplasie delle vie biliari o al rifiuto di una paziente , ha dimostrato che latrofia del parenchima epatico embolizzato progressiva nel tempo mentre non significativamente diversa lipertrofia del frl . 
 allinizio della nostra esperienza , quando il programma chirurgico prevedeva lepatectomia destra allargata al quarto segmento , abbiamo associato lembolizzazione del ramo portale segmentario del iv che precedeva lembolizzazione del ramo portale di destra , ma in accordo con capussotti et al . 
la potenziale riserva funzionale epatica calcolata mediante la volumetria tc del frl e del volume totale del parenchima epatico funzionante , ottenuto sottraendo al volume epatico totale il volume tumore e quindi dal loro rapporto ( frl / volume totale epatico funzionante )  . 
la metodologia da noi usata permette di ottenere la volumetria separata del volume 566 radiol med ( 2009 ) 114 : 553570 accurate estimation of functional parenchyma , particularly in those cases where the liver parenchyma to be resected is extensively involved . 
 [ 27 ] demonstrated that the evaluation of tumour volume in patients with liver metastases significantly affects the evaluation of the hepatic functional reserve . the evaluation of tumour volume before and after right pve also provided us with information on the oncogenic effect of right pve on liver disease . 
in biliary cancers , no local disease progression was detected . our results coincide with previous experiences [ 8 , 28 ] that reported an increase in volume of hypovascular metastases from colorectal cancers after right pve in embolised and unembolised livers and no change in the volume of hypervascular metastases from carcinoid tumours in either tumore e del parenchima sano e quindi fornisce la valutazione reale del parenchima funzionante , in particolare nei casi in cui il parenchima epatico da resecare in gran parte sostituito . 
 [ 27 ] hanno infatti dimostrato che nei pazienti portatori di metastasi epatiche la valutazione del volume tumore incide in maniera significativa nella valutazione della riserva funzionale epatica . la valutazione del volume tumore prima e dopo rpve ci ha inoltre fornito dati sulleffetto oncogenetico della metodica sulla patologia epatica . 
dopo rpve nella patologia secondaria epatica aumentato il volume delle metastasi da colon - retto mentre rimasto immodificato il volume tumore nelle metastasi da carcinoide ; nelle neoplasie delle vie biliari non abbiamo riscontrato progressione locale di malattia . 
i nostri risultati concordano con esperienze precedenti [ 8 , 28 ] che avevano documentato laumento volumetrico delle metastasi , ipovascolari , da colon - retto dopo rpve nel fegato embolizzato e non embolizzato , mentre il volume delle metastasi ipervascolarizzate da carcinoide rimaneva invariato sia nel radiol med ( 2009 ) 114 : 553570 fig . 
a - f axial computed tomography ( ct ) scans obtained in a 54 - year - old man show multiple synchronous liver metastases from colon cancer in the left ( 1 , 2 , 5 ) and right ( 3 , 4 , 6 ) liver , with partial response after six cycles of chemotherapy . 
surgical plan : ablation with free resection margins of the liver lesion located in segment viii ( lesion 3 ) requires right hepatectomy combined with resection of the middle supra hepatic vethe potential frl consists of segments i , ii and iii . 
i - p axial ct scans obtained 29 days after rpve document left hepatic hypertrophy , especially of the caudate lobe , the outcome of previous left metastasectomy ( 1 , 2 , 5 ) and enlarged liver metastases in the right liver lobe ( tumour volume increased from 45 to 115 cm3 ) ( 3 , 4 , 6 )  . 
a - f in un paziente di 54 anni , le immagini assiali dellesame tc documentano multiple metastasi epatiche sincrone da cancro del colon localizzate nel fegato sinistro ( 1 , 2 , 5 ) e nel fegato destro ( 3 , 4 , 6 ) , con risposta parziale dopo 6 cicli di chemioterapia . 
programma chirurgico : lasportazione con margini di resezione liberi della lesione epatica dellviii segmento ( lesione 3 ) necessita di epatectomia destra , associata a resezione della vena sovraepatica media . 
i - p le immagini assiali della tc eseguita 29 giorni dopo lembolizzazione documentano lipertrofia del fegato sinistro , in particolare del lobo caudato , gli esiti della pregressa metastasectomia a sinistra ( 1 , 2 , 5 ) e le metastasi epatiche del fegato destro ( 3 , 4 , 6 ) aumentate di dimensioni ( volume tumore 115 vs 45 cm3 )  . 
 [ 29 ] hypothesised that a cytokine regulating the hepatocyte growth factor ( hgf ) which is altered in patients with colorectal cancer and distant metastases is responsible for the growth of liver metastases in these patients . 
in contrast , preoperative right pve does not affect 5 - year survival rates compared with control groups undergoing resection of liver metastases from colon cancer without right pve [ 16 ]  . new strategies are being proposed for patients with multiple liver metastases from colorectal cancer , which combine the intra - arterial [ 30 , 31 ] or systemic [ 3235 ] infusion of chemotherapeutic agents with selective pve , with the aim of simultaneously obtaining tumour shrinkage and fegato embolizzato che non embolizzato . 
 [ 29 ] hanno ipotizzato che una citochina che regola il fattore di crescita degli epatociti ( hgf ) , alterata nei pazienti con neoplasia colon - rettale e metastasi a distanza , sia responsabile della crescita delle metastasi epatiche in questi pazienti . 
lembolizzazione portale prechirurgica non modifica , invece , la sopravvivenza a 5 anni rispetto a gruppi di controllo sottoposti a chirurgia per metastasi epatiche da neoplasia del colon senza rpve [ 16 ]  . attualmente nuove strategie vengono proposte in pazienti portatori di multiple metastasi epatiche da colon - retto che associano alla selettiva embolizzazione portale infusione intraarteriosa [ 30 , 31 ] o sistemica [ 3235 ] di chemoterapici con lo scopo di ottenere contemporaneamente la 568 radiol med ( 2009 ) 114 : 553570 frl hypertrophy . 
 the second factor that affects the indication for right pve in candidates for liver resection is the possible coexistence of chronic liver disease , as this influences the amount of hepatic functional reserve needed to reduce postoperative morbidity and mortality . 
in patients with klatskin tumours , preventive drainage of the potential frl to resolve the cholestasis ( bilirubin < 2.9 mgdl ) is the essential precondition for pve to obtain an adequate regenerative response of the frl . 
 [ 27 ] , depending on the severity of cirrhosis and on residual liver function assessed with the indocyanine test . the presence of systemic disease , such as diabetes , in which the regenerative capacity of the liver is reduced by the hepatotrophic effect of insulin , and the complexity of surgery are , respectively , the third and fourth factors to be considered when selecting candidates for preoperative right pve . 
the use of lipiodol mixed with cyanoacrylate achieved right pve embolisation without complications and without recanalisation , with the added benefit of radiopacity allowing monitoring of the procedure by fluoroscopy [ 41 , 42 ]  . a potential functional reserve of frl < 30% constitutes an indication for right pve in patients previously exposed to chemotherapy or with previous cholestasis . 
 il secondo fattore che regola lindicazione alla rpve nei candidati a resezione epatiche leventuale concomitanza di epatopatia perch su questo si basa lentit della riserva funzionale epatica necessaria per ridurre la morbilit e mortalit post - chirurgica . 
lasportazione di pi del 75% del parenchima epatico ben sopportata nel paziente giovane ( 40 anni ) , ma la chirurgia resettiva epatica deve essere pi conservativa in relazione allet , alla patologia epatica e sistemica [ 36 ]  . 
 il minimo volume residuo del frl consigliato in pazienti con normale funzionalit epatica tra il 25%30% della massa epatica totale funzionante [ 9 , 19 , 35 , 36 ] anche se sono state effettuate resezioni epatiche con un volume di frl non inferiore al 20% senza complicanze post - chirurgiche [ 22 ]  . 
nei pazienti con neoplasie di klatskin , il preventivo drenaggio del potenziale frl che risolva la colestasi ( livello di bilirubina inferiore a 2 , 9 mgdecilitro ) il presupposto indispensabile allembolizzazione portale perch si ottenga unadeguata risposta rigenerativa del frl . 
la capacit rigenerativa del fegato cirrotico ridotta sia in relazione alla riduzione del flusso portale che alla fibrosi e quindi in questi pazienti il volume minimo del frl necessario pu raggiungere il 50% come dimostrato da kubota ed al . 
 [ 27 ] in relazione alla gravit della cirrosi e quindi alla funzionalit epatica residua valutata mediante il test dellindocianina . la presenza di una malattia sistemica come il diabete che riduce la capacit rigenerativa del fegato in relazione alleffetto epato - trofico dellinsulina e la complessit dellintervento chirurgico sono il terzo ed il quarto fattore da considerare nella selezione dei pazienti da candidare a rpve prechirurgica . 
 in conclusione , il nostro studio retrospettivo ha verificato che lembolizzazione portale pre - chirurgica una procedura sicura ; luso di lipiodol misto a cianoacrilato ha ottenuto lembolizzazione dellalbero portale destro senza complicanze e senza ricanalizzazione con il vantaggio legato alla sua radiopacit di monitorare lembolizzazione mediante fluoroscopia [ 41 , 42 ]  . la potenziale riserva funzionale del frl inferiore al 30% unindicazione alla rpve in pazienti precedentemente sottoposti a terapie chemioterapiche o con precedente colestasi ed allarga le indicazioni alla chirurgia radiol med ( 2009 ) 114 : 553570 extends the indications for liver resection in primary and secondary liver cancers , making radical surgery possible without severe postoperative complications . 
the addition of two - stage hepatectomy appears to be a promising innovation for patients with bilobar liver metastases from colorectal cancer . resettiva epatica in patologie primitive e secondarie epatiche rendendo possibile una chirurgia oncologicamente radicale senza gravi complicanze postchirurgiche . 
sardanelli servizio di radiologia , dipartimento di scienze medico - chirurgiche , universit degli studi di milano , irccs policlinico san donato , via morandi 30 , 20097 san donato milanese , italy a . 
the mr protocol applied at our institution for both diagnosis and follow - up after surgical or endovascular treatment consists of four steps : morphologic study , cine mr study , flow analysis , and mr angiography ( mra )  . the first three sequences are acquired during breath - hold and with electrocardiographic gating . 
cine true - fast imaging with steady - state precession ( true - fisp ) sequences show not only morphologic features but also blood - flow changes inside the aorta . 
gradient - echo sequences for phase - velocity mapping allow flow analysis . application of bernoullis equation here briefly presented and discussed allows for calculation of the pressure gradient caused by the coarctation . 
mra , acquired with a breath - hold three - dimensional t1weighted gradient - echo sequence and intravenous administration of paramagnetic contrast material , allows for optimal depiction of the aortic lumen , with a panoramic view of the whole aorta , its main branches and possible collateral circulation . 
 keywords aorta aortic coarctation bernoullis equation magnetic resonance ( mr ) imaging mr flow analysis ( phase - velocity mapping ) riassunto la coartazione aortica rappresenta il 5%10% di tutte le patologie congenite del cuore e dei grandi vasi e il 7% dei casi di piccoli cardiopatici in condizioni critiche . 
la risonanza magnetica ( rm ) permette lo studio di questa patologia con una serie di vantaggi rispetto a tecniche alternative quali langiografia convenzionale , lecografia transesofagea e la tomografia computerizzata . 
il nostro protocollo rm , utilizzato sia per la diagnosi , sia per il follow - up dopo trattamento chirurgico o endovascolare , consiste di quattro fasi : studio morfologico , cinetico , flussimetria e angio - rm . 
le sequenze a sangue chiaro true - fast imaging with steady - state precession ( true - fisp ) , oltre a dare informazioni di tipo morfologico , mostrano le alterazioni del flusso ematico allinterno del vaso . 
le sequenze gradient - echo con mappaggio fase - velocit consentono la quantificazione del flusso ; lapplicazione dellequazione di bernoulli ( qui brevemente presentata e discussa ) , consente infatti il calcolo del gradiente pressorio determinato dalla coartazione . 
lo studio angiorm , ottenuto in apnea mediante sequenze gradient - echo tridimensionali t1 - pesate con somministrazione endovenosa di mezzo di contrasto paramagnetico , mostra una visione volumetrica panoramica del lume dellaorta , delle sue principali ramificazioni e , quando presenti , dei circoli collaterali . 
 parole chiave aorta coartazione aortica equazione di bernoulli flussimetria rm risonanza magnetica ( rm ) radiol med ( 2009 ) 114 : 524537 introduction introduzione over recent years , several magnetic resonance ( mr ) techniques have been successfully applied for evaluating congenital heart diseases . 
we summarize here the currently available knowledge about coa , including epidemiology , aetiopathogenesis , topographic classification , and the different mr imaging techniques . negli ultimi anni numerose tecniche di risonanza magnetica ( rm ) sono state applicate con successo alla valutazione delle cardiopatie congenite . 
la coartazione aortica ( coa ) rappresenta un reperto non infrequente nella pratica clinica , pari a circa il 5%10% di tutte le malformazioni congenite del cuore e dei grandi vasi [ 1 ]  . 
sono qui rivisitate le attuali conoscenze sulla malattia relativamente a epidemiologia , eziopatogenesi , classificazione topografica e studio con differenti tecniche rm . definition coarctation is a term derived from the latin coarctatio , meaning narrowing or stricture . 
similar congenital malformations are aortic atresia and the true aortic interruption , mostly located at the arch ( interrupted aortic arch )  . coa , also known as congenital aortic stenosis , was originally noted during autopsy by j.f. 
he collected 200 previously documented cases , but coa was not regularly diagnosed until after 1933 [ 6 ]  . epidemiology and aetiology coa accounts for 5%10% of all congenital heart diseases and represents 7% of critically ill infants with heart disease . it is seven times more common in caucasian than in asian population . 
in atlanta ( usa ) from 1970 to 1983 , the prevalence per 1 , 000 live births was as follows : interruption of the aortic arch , 0.05 ; coarctation , 0.36 ; hypoplasia of the aorta , 0.06 [ 7 ] , giving a total prevalence of 0.47 per 1 , 000 live births . 
in bohemia in 1980 , a prevalence of 6.67 congenital heart malformations per 1 , 000 live births was reported , and 5.77% of these malformations consisted of coa cases [ 9 ] , giving a coa prevalence of 3.84 per 1 , 000 live births . the aetiology of coa is still unclear . 
up to 60% [ 10 ] or 85% [ 1 ] of coa cases are associated with bicuspid aortic valve , supporting the hypothesis of a common cause for the two congenital malformations [ 11 ] or an influence of the bicuspid aortic valve on the development of coa [ 1 ]  . 
pathologic examination of the ascending aorta of patients with definizione il termine coartazione deriva dal latino coarctatio , letteralmente restringimento ovvero , nel caso di una struttura vascolare , riduzione di calibro cui consegue ostruzione al flusso ematico . 
paris [ 4 ] ne forn la prima descrizione accurata nel 1791 mentre j . abbott pubblic una serie autoptica retrospettiva di 200 casi nel 1928 [ 5 ] , ma la malattia non stata diagnosticata con regolarit fino a dopo il 1933 [ 6 ]  . epidemiologia ed eziologia la coa rappresenta il 5%10% di tutte le patologie congenite del cuore e dei grandi vasi e il 7% dei casi di piccoli cardiopatici in condizioni critiche . 
 si osserva una prevalenza maschile con rapporto maschi / femmine tra 1 , 3 e 2 , 1 nella maggior parte delle serie [ 1 ]  . nellarea metropolitana di atlanta ( usa ) stata osservata tra il 1970 e il 1983 una prevalenza su 1000 nati vivi pari a 0 , 05 per linterruzione dellarco , 0 , 36 per la coartazione e 0 , 06 per lipoplasia [ 7 ] , con una prevalenza totale pari al 0 , 47 per 1000 nati vivi . 
una prevalenza pari a 0 , 32 per 1000 nati vivi stata riportata per lislanda dal 1980 al 1994 [ 8 ]  . in boemia nel 1980 stata riportata una prevalenza di 6 , 67 malformazioni congenite cardiache per 1000 nati vivi ; il 5 , 77% di queste era costituito da casi di coa [ 9 ] , per una prevalenza di coa pari a 3 , 84 per 1000 nati vivi . leziologia della coa non ancora stabilita con certezza . 
si associa a bicuspidia valvolare aortica fino al 526 radiol med ( 2009 ) 114 : 524537 bicuspid aortic valve frequently shows loss of smoothmuscle cells and severe degeneration of the medial elastic fibers ( so - called cystic medial necrosis ) [ 11 ]  . 
associated cardiac defects are observed in approximately 50% of patients with coa , mostly being left - sided hypoplastic or obstructive defects and ventricular septal defects [ 1 ]  . 
moreover , the higher coa prevalence in males and a relatively high prevalence in patients with turners syndrome suggest a link between x - chromosome defects and anomalous development of the aorta and valve [ 11 ]  . 
however , this classification is now considered too simplistic because many patients lack symptoms , and coa diagnosis is delayed until adulthood [ 14 ]  . only malformations determining a total obstruction or a severe degree of stenosis ( severe tubular hypoplasia , severe coarctation , vessel atresia or interruption ) or associated with cardiac defects are diagnosed during childhood [ 13 ]  . 
thus , the prevalence in adulthood , including the nonsevere form of coa , should be higher than the incidence in live births . a commonly accepted classification is based on stenosis location : preisthmic , isthmic and postisthmic . 
 a physiopathologic approach is given by the subdivision 60% [ 10 ] o all85% dei casi [ 1 ] , supportando lipotesi di una causa comune alle due malformazioni [ 11 ] o di uninfluenza della bicuspidia valvolare aortica sullo sviluppo della coa [ 1 ]  . 
indagini istologiche dellaorta ascendente di soggetti con bicuspidia valvolare hanno mostrato un ridotto numero di cellule muscolari lisce e severa degenerazione delle fibre elastiche della tonaca media ( cosiddetta necrosi medio - cistica ) [ 11 ]  . 
malformazioni cardiache congenite si associano a coa in circa il 50% dei casi , per lo pi rappresentate da sindromi ipoplasiche o ostruttive delle cavit sinistre o da difetti del setto interventricolare [ 1 ]  . 
dal 3%5% [ 1 ] al 10% [ 12 ] dei pazienti con coa presentano altres aneurismi intracranici , in accordo con lipotesi che la coa sia parte di una patologia arteriosa diffusa , secondaria ad anomalie dello sviluppo della cresta neurale [ 12 ]  . 
inoltre , la maggiore prevalenza di coa nei maschi e nei pazienti con sindrome di turner induce a ipotizzare un legame tra difetti del cromosoma x ed alterazioni dello sviluppo dellaorta e della valvola aortica [ 11 ]  . 
solo le malformazioni che comportano stenosi di grado elevato o ostruzione totale del flusso ematico ( ipoplasia tubulare serrata , coartazione serrata , atresia o interruzione del vaso ) o che si associano a malformazioni cardiache sono diagnosticate in et infantile [ 13 ]  . ne deriva che la prevalenza in et adulta , includendo le forme non severe di coa , dovrebbe essere sensibilmente pi elevata di quella calcolata in base alla diagnosi sui nati vivi . radiol med ( 2009 ) 114 : 524537 fig . 
1a - c angio - rm ( ricostruzioni volumetriche ) di casi di : ipoplasia tubulare dellarco aortico ( freccia in a ) , coartazione istmica ( freccia in b ) e interruzione dellaorta discendente ( freccia in c )  . into preductal , ductal and postductal coa , referring to the location of the botallo ductus arteriosus . 
this kind of coa , observed in approximately 5% of infants with turner syndrome , typically results from a cardiac anomaly during fetal life decreasing blood flow through the left ventricle , leading to hypoplastic development of the aorta . 
in postductal coa , the narrowing is distal to the insertion of the ductus arteriosus . as a consequence , blood flow to the lower body is impaired . it is thought to be the consequence of muscular cells extending from ductus arteriosus into the aorta during fetal life [ 15 ]  . clinical features and treatment the age of clinical presentation is mainly related to the degree of stenosis and to the presence of associated abnormalities . 
nella forma preduttale la coa prossimale allinserzione del dotto ; se severa , il flusso a valle dipende dalla perviet del dotto la cui chiusura , quindi , pu risultare fatale . 
questo tipo di coa , osservato in circa il 5% dei nati affetti da sindrome di turner , tipicamente il risultato di anomalie cardiache fetali con ridotto efflusso dal ventricolo sinistro , cui consegue unipoplasia dellaorta . 
nella forma post - duttale la coa a valle dellinserzione del dotto e ne risulta un ridotto flusso alla porzione inferiore del corpo ; ritenuta essere la conseguenza della migrazione di miocellule dal dotto arterioso allaorta durante la fase fetale [ 15 ]  . 
si dovrebbe sempre considerare lipotesi di coa in soggetti adolescenti o giovani di sesso maschile con 528 radiol med ( 2009 ) 114 : 524537 coa becomes clinically relevant , it can lead to systemic hypertension and secondary left ventricular hypertrophy with heart failure . 
if it is not treated , the patients mean age of death is about 34 years [ 16 ]  . invasive treatment should always be considered in patients with pressure gradients > 30 mmhg on cardiac catheterization [ 17 ]  . 
successful surgical correction of coa by resection of the stenotic tract with end - to - end anastomosis was first described by crafoord and nylin in 1945 [ 18 ]  . 
 perioperative mortality rates are determined largely by age and concomitant congenital heart disease rather than by the choice of operative technique for correcting coa [ 23 ] and are less than 3% for older children and adults [ 24 ]  . several postsurgical complications such as cerebrovascular , cardiac and aortic events may occur . 
the most common complications are cerebrovascular accidents , cerebral haemorrhage , recurrent hypertension , heart failure , endocarditis , endarteritis , coronary artery disease , recoarctation , poststenotic aneurysm , and aortic rupture [ 10 ]  . 
due to successful surgical or endovascular treatments , follow - up of treated adult patients is often required . imaging of coa a posteroanterior chest x - ray examination can show a dilated ascending aorta , low prominence of the first left arch , a notch sign corresponding with the location of coa and small notches on the inferior posterior margin of ribs , mainly from fourth to eighth , due to the osteolytic effect of the collateral hypertrophic intercostal arteries . 
 similarly to aortic dissection [ 26 ] , coa can be diagnosed using transesophageal echocardiography ( tee ) , digital subtraction angiography ( dsa ) , computed tomography ( ct ) , and mr imaging . 
due to the use of ionising radiation and iodinated contrast agents , ct and dsa are not regarded as first - line techniques for diagnosing coa , particularly in children . 
 over recent years , mr imaging has become a well - established diagnostic tool for studying congenital heart diseases and coa [ 27 , 28 ] , offering many advantages over other ipertensione agli arti superiori e ipotensione e polsi deboli agli arti inferiori . 
se non trattata , porta al decesso mediamente a 34 anni di et [ 16 ]  . il trattamento invasivo dovrebbe essere sempre considerato nei pazienti che presentano un gradiente pressorio maggiore di 30 mmhg al cateterismo cardiaco [ 17 ]  . crafoord e nylin [ 16 ] descrissero per primi , nel 1945 , lintervento correttivo che prevede la resezione del tratto stenotico con successiva anastomosi termino - terminale . successivi miglioramenti delle tecniche chirurgiche [ 18 , 20 ] , tra i quali lintroduzione dellaortoplastica con flap dellarteria succlavia , hanno permesso la correzione in caso di varianti anatomiche complesse . 
 i tassi di mortalit peri - operatoria , determinati soprattutto dallet del paziente e da eventuali anomalie associate piuttosto che dalla tecnica utilizzata per la correzione della coa [ 23 ] , sono inferiori al 3% sia nei bambini che negli adulti [ 24 ]  . 
sono riportate complicanze post - chirurgiche quali lesioni cerebrovascolari , emorragie cerebrali , ipertensione recidivante , scompenso cardiaco , endocardite , endoarterite , patologie coronariche , ricoartazione , aneurismi post - stenotici e rottura dellaorta [ 10 ]  . 
il successo del trattamento chirurgico o endovascolare della coa pone oggi lesigenza del follow - up in et adulta dei pazienti trattati . imaging della coa il radiogramma toracico postero - anteriore pu mostrare ectasia dellaorta ascendente , scarsa prominenza del primo arco di sinistra , incisura in corrispondenza della sede di coartazione e piccole incisure sul margine inferiore dellarco posteriore delle coste , per lo pi dalla quarta allottava , dovute alleffetto osteolitico delle arterie intercostali collaterali ipertrofiche . 
 come per la dissezione aortica [ 26 ] , la diagnosi di coa pu essere posta mediante ecocardiografia transesofagea ( ete ) , angiografia digitale con sottrazione dimmagine ( ads ) , tomografia computerizzata ( tc ) e rm . 
the mr examination takes about 30 min ; the postprocessing requires about 30 min . setting up the patient a good electrocardiographic ( ecg ) line resulting from correct placement of four leads over the anterior or posterior left side of the chest is crucial to obtain a good cardiac synchronization ( ecg gating ) [ 35 ]  . 
la tc e lads non sono considerate tecniche di prima scelta per la diagnosi di coa , soprattutto in et pediatrica , a causa dellutilizzo di radiazioni ionizzanti e mezzi di contrasto ( mdc ) iodati . 
essa offre molteplici vantaggi rispetto alle altre tecniche di imaging cardiovascolare tra i quali la possibilit di ottenere immagini native multiplanari senza luso di radiazioni ionizzanti , lelevata risoluzione temporale ( fino allimaging in tempo reale ) e la possibilit di studio flussimetrico [ 2931 ]  . 
 nello studio dei grandi vasi , la rm vede il suo esordio oltre ventanni or sono con semplici sequenze spin - echo ( se ) e gradient - echo ( ge ) con rifocalizzazione degli spin in flusso laminare . 
successivamente sono state introdotte le sequenze turbo - se ( tse ) , ge cine - rm , ge segmentate , half - fourier acquisition single shot turbo spin - echo ( haste ) e true - fast with steady - state precession ( truefisp )  . 
sequenze ge con mappaggio fase - velocit permettono , inoltre , di ottenere dati flussimetrici [ 33 ] mentre sequenze ge tridimensionali con somministrazione endovenosa di mdc paramagnetico permettono di ottenere immagini simil - angiografiche ad ampio campo di vista , sulle quali si possono effettuare misure accurate del lume aortico [ 34 ]  . protocollo rm faremo qui riferimento alla nostra esperienza ( 20032007 ) con apparecchiatura operante a 1 , 5 t ( magnetom sonata maestro class , siemens , erlangen , germania ) presso lirccs policlinico san donato ( san donato milanese , milano , italia ) su pazienti con coa prima del trattamento e nel loro follow - up a breve e lungo termine . 
thus , we prefer the use of breath - hold ecg - gating : dark - blood t2 - weighted haste [ 37 ] , with high il corretto posizionamento di quattro elettrodi sullemilato sinistro del torace , anteriormente o posteriormente , cruciale per una buona sincronizzazione con lelettrocardiogramma ( ecg - gating ) [ 35 ]  . 
the acquisition of an image passing through the ascending and descending aorta ( three parallel oblique lines ) is planned on a scout axial true fast imaging with steady - state precession ( true fisp ) image ( a )  . 
2a , b definizione del piano obliquo anteriore sinistro ( oas ) per lo studio dellaorta toracica . sullimmagine assiale true - fast imaging with steady - state precession ( true - fisp ) di centratura ( a ) rappresentata la pianificazione di una scansione oas passante per laorta ascendente e discendente ( tre linee oblique parallele )  . 
3a , b bright - blood image ( true - fast imaging with steady - state precession ) ( a ) and dark - blood image ( half - fourier acquisition single - shot turbo spin - echo ) ( b ) , along the same left anterior oblique plane passing through an isthmic coarctation ( arrow )  . 
3a , b immagini a sangue chiaro ( true - fast imaging with steady - state precession ) ( a ) e a sangue scuro ( half - fourier acquisition single - shot turbo spin - echo ) ( b ) lungo il piano obliquo anteriore sinistro passante per una coartazione istmica dellaorta ( freccia )  . 
breath - hold , ecg - gated cine true - fisp sequences ( bright blood , hybrid t2 / t1 - weighted ) are acquired for multiple frames along the cardiac cycle . 
la nostra preferenza va a sequenze in apnea e con sincronizzazione ecg : haste a sangue scuro , pesate in t2 [ 37 ] , con elevato radiol med ( 2009 ) 114 : 524537 fig . 
4 immagine cine true - fast imaging with steady - state precession lungo il piano obliquo anteriore sinistro : coartazione istmica dellaorta ( freccia nera ) ; marcato jet anterogrado ( frecce bianche ) da accelerazione di flusso post coartazione . flow study this part of the mr protocol involves the use of ge phasevelocity mapping sequences on planes orthogonal to the longitudinal axis of the vessel ( through - the - plane acquisition )  . 
phase - velocity sequences provide two different set of images : ( 1 ) magnitude images that give anatomical information , and ( 2 ) phase images that give data about flow direction and velocity . 
si utilizzano sequenze true - fisp ( a sangue chiaro , pesatura ibrida t2 / t1 , con sincronizzazione ecg ) acquisite in apnea per molteplici frame lungo le fasi del ciclo cardiaco . 
per studiare il flusso aortico post - coartazione necessario impostare unelevata codifica di velocit ( velocity encoding , venc ) , generalmente tra 350 e 500 ms , onde evitare artefatti ( aliasing ) nel lume aortico . 
lacquisizione fornisce due serie di immagini : una serie con significato anatomico ( immagini di magnitudo o modulo ) e una serie con significato funzionale ( immagini di fase ) , contenente informazioni su direzione e velocit del flusso . 
lacquisizione lungo il piano obliquo anteriore sinistro ( a ) evidenzia minimo restringimento residuo ( freccia ) ; lacquisizione lungo il piano valvolare aortico ( b ) evidenzia la coesistente bicuspidia ( frecce )  . 
since the kinetic energy of the blood is proportional to the square of its velocity , the right side of bernoullis equation represents the difference between the kinetic energy of blood after coa and that before coa . 
siccome lenergia cinetica del sangue proporzionale al quadrato della sua velocit , il membro destro dellequazione di bernoulli rappresenta la differenza tra lenergia cinetica del sangue a valle della coa e quella a monte della coa . 
 al fine di esprimere p in mmhg anzich in pascal ( pa ) , lunit di misura della pressione nel sistema internazionale , occorre introdurre il seguente coefficiente di conversione : 1 pa = 0 , 0075 mmhg infine , ricordando che la densit del sangue d pari a circa 1067 g / l , otteniamo : i.e. 
if v1 and v2 are given in m / s , the coefficient 4 adjusts the measurement units so to allow the pressure gradient p to be expressed as mmhg . 
se le velocit v1 e v2 sono espresse in m / s , il coefradiol med ( 2009 ) 114 : 524537 that the peak velocity before coa ( v1 ) is generally negligible if compared with the peak velocity after coa ( v2 )  . 
first , it works only if the patient is in the supine position , as the nonperfect horizontal position introduces the gravity - force effect ( this limitation is not relevant for mr applications )  . 
in such a context , a p > 20 mmhg is highly suggestive of significant coa [ 41 , 42 ]  . mr angiography ( mra ) contrast - enhanced mra is a reliable technique for evaluating great vessels . 
for a long time , gadolinium chelates have been considered safe contrast agents and have been used in patients allergic to drugs or iodinated contrast agents and in nephropathic or cardiopathic patients [ 43 ]  . 
patients with severe renal failure [ glomerular filtration rate ( gfr ) < 30 ml / min1.73 m2 ] are considered to be at risk [ 44 , 46 ]  . 
in patients with gfr30 ml / min1.73 m2 , a single dose of 0.1 mmol / kg of gadolinium - based contrast agent can be safely administered intravenously [ 46 ]  . 
for diagnosis or follow - up of coa , we used a power injector with an injection rate of 2 ml / s for both the contrast material and the following 20 - ml saline flush . 
we used a three - dimensional breath - hold , not ecg - gated , t1 - weighted ge sequence with a short acquisition time ( nearly 20 s )  . 
in patients with gfr < 30 ml / min1.73 m2 , time - of - flight and phase - contrast sequences can be used , even though image quality is lower and veins are also visualized [ 47 ]  . 
sulla ricostruzione volumetrica posteriore obliqua ( a ) sono riportate le sedi del calcolo dellarea delle sezioni del vaso a monte della coartazione , al livello della coartazione e a valle della coartazione . 
a sinistra sono riportati i valori di tali aree come normal a , minimum e area 01 , rispettivamente ; inoltre riportato come ratio 01 il rapporto percentuale tra larea a monte della coartazione e larea al livello della coartazione ( grado di stenosi )  . 
la linea centrale endoluminale ( frecce in a ) , generata automaticamente , permette la ricostruzione lungo il piano curvo ( b )  . ficiente 4 consente di esprimere il gradiente p in mmhg . unulteriore semplificazione possibile considerando che la velocit di picco a monte della stenosi ( v1 ) in genere trascurabile rispetto alla velocit a valle della stenosi ( v2 ) e , quindi , pu essere ignorata . 
in primo luogo , la formula di bernoulli valida solo se il paziente supino , in quanto la posizione ortostatica introdurrebbe nella formula un termine aggiuntivo che tiene conto della forza di gravit ( tale limitazione non rilevante per usuali applicazioni rm )  . 
i chelati del gadolinio sono stati considerati a lungo mdc privi di effetti collaterali e si riteneva potessero essere impiegati anche in presenza di allergie a farmaci o ai mdc iodati cos come in caso di pazienti nefropatici o cardiopatici [ 43 ]  . 
sono considerati a rischio pazienti con insufficienza renale grave ( gfr , rateo di filtrazione glomerulare < 30 ml / min1 , 73 m2 ) [ 44 , 46 ]  . 
in pazienti con gfr 30 ml / min1 , 73 m2 si pu utilizzare la singola dose endovena ( 0 , 1 mmol / kg ) di un chelato del gadolinio senza rischi per il paziente [ 46 ]  . 
per la diagnosi e il follow - up della coa , noi utilizziamo un flusso di 2 ml / s sia per il mdc , sia per il bolo di 20 ml di soluzione fisiologica , entrambi somministrati con iniettore automatico . 
in caso di gfr < 30 ml / min1 , 73 m2 possibile impiegare sequenze angiografiche senza mdc ( time of flight e phase contrast ) ottenendo per immagini di qualit inferiore comprendenti anche i vasi venosi [ 47 ]  . 
maximum intensity projections allow aortic demonstration in the angiographic - like view . surface shading display and volume - rendering techniques , obtained with standardised reconstruction algorithms , allow reliable measurements . 
in order to correctly measure aorta diameters and assess the degree of stenosis , measurements should be performed both in lao and in planes orthogonal to the longitudinal axis of the vessel . 
the distance of the coa from the left subclavian artery origin is an important variable for surgical or endovascular therapy planning . le rielaborazioni di volume ( volume rendering technique ) e di superficie ( surface shading display ) , con algoritmi di ricostruzione standardizzati , permettono misurazioni accurate . 
la distanza della coa dallorigine dellarteria succlavia sinistra o dai vasi epiaortici un dato importante nel planning della procedura chirurgica o endovascolare . conclusioni conclusions to correctly diagnose coa , establish disease severity and evaluate treatment options , as well as for follow - up during adulthood , a complete mr protocol is required , including morphological , functional , and flow study as well as contrast - enhanced mra and accurate postprocessing . per diagnosticare correttamente i casi di coa , valutare lentit della stenosi e le possibilit terapeutiche , cos come nel follow - up in et adulta , necessario un protocollo rm che comprenda acquisizioni morfologiche , cinetiche , flussimetriche e angiografiche , completato da accurato postprocessing . 
fava1 1istituto di radiologia universit di torino sede san luigi , orbassano , italy 2sc radiodiagnostica i e ii ospedale san giovanni battista , torino , italy 3sc radiodiagnostica ospedale s . 
ninety - eight patients with cerebral tumours , 46 of which were primary ( seven grade 0 - i , nine low - grade gliomas , two gliomatosis cerebri , nine lymphomas and 19 high - grade gliomas ) and 52 secondary , underwent conventional magnetic resonance ( mr ) imaging completed with dwi and dynamic contrast susceptibility pwi . 
seventeen of 98 tumours were cystic or necrotic ( 12 / 17 hypointense and 5 / 17 hyperintense on dwi ) ; the adc value of hyperintense cystic areas was 0.970.2310 - 3 mm2 / s . 
novantotto pazienti con tumore cerebrale , 46 con tumore primitivo ( 7 di grado 0 - i , 9 gliomi a basso grado , 2 gliomatosi cerebri , 9 linfomi , 19 gliomi ad alto grado ) e 52 con metastasi sono stati sottoposti ad esame rm convenzionale completato da acquisizione dwi e da studio perfusionale ottenuto durante la somministrazione di un bolo di gadolinio . 
diciassette tumori erano costituiti da una componente centrale cistica che in 5 casi era iperintensa in dwi ( valore medio adc 0 , 970 , 2310 - 3 mm2 / s )  . ladc della parte solida dei tumori aveva valori compresi tra 0 , 64 e 3 , 510 - 3 mm2 / s . 
la pwi fornisce elementi utili nella diagnosi differenziale tra gliomi a basso ed alto grado e tra gliomi ad alto grado e linfomi . parole chiave risonanza magnetica diffusione perfusione tumori cerebrali introduction introduzione the role of conventional magnetic resonance ( mr ) imaging , with or without contrast agent administration , in the diagnosis of brain tumours is widely accepted . 
mr imaging not only has high sensitivity in detecting and locating lesions , it also gives information about phenomena such as mass effect , haemorrhage , oedema or necrosis , thus helping to differentiate brain tumours from other pathological processes and providing indications about the type of tumour [ 1 , 2 ]  . the importance of characterising tumours ( typing and grading ) to plan appropriate treatment and monitor response to treatment early on in the disease has stimulated research in this area , with numerous studies investigating , over the past decade , the application of new techniques such as diffusion - weighted imaging ( dwi ) , perfusion - weighted imaging ( pwi ) and in vivo mr spectroscopy ( mrs ) [ 14 ]  . the term diffusion refers to the random ( brownian ) movement of water molecules in tissues ; the diffusion capacity of water molecules is tissue - specific and may be altered in given pathological conditions . 
dwi enables assessment of this tissue - related parameter , and calculation of the apparent diffusion coefficient ( adc ) provides major diagnostic clues for evaluating intracranial masses [ 2 , 5 ]  . perfusion reflects the delivery of nutrients to tissue and is defined as flow per unit of time . 
the grey matter of a healthy individual is perfused at a rate of 5060 ml / 100 g / min and the flow maintained in a restricted range by cerebral self - regulation mechanisms . 
low - perfusion states may thus lead to cellular ischaemia , whereas high - perfusion states may be related to hypervascular lesions such as certain tumours [ 6 ]  . the most commonly measured perfusion parameters are cerebral blood volume ( cbv ) , which reflects the amount of blood present in a given amount of tissue at a given time ; cerebral blood flow ( cbf ) , which indicates the quantity of blood transiting through a given amount of tissue in a unit of time ; and mean transit time ( mtt )  . 
these parameters are closely linked to one another , with cbf being equal to the il ruolo della risonanza magnetica ( rm ) convenzionale , con e senza mezzo di contrasto ( mdc ) , nella diagnosi dei tumori dellencefalo saldamente consolidato ; la rm non solo possiede unelevata sensibilit nel riconoscimento e nella localizzazione delle lesioni , ma fornisce informazioni su fenomeni quali leffetto massa , lemorragia , ledema o la necrosi dei tessuti che aiutano nella diagnosi differenziale con altri tipi di processi patologici dando anche indicazioni sul tipo di tumore [ 1 , 2 ]  . limportanza della caratterizzazione ( tipo e grading ) dei tumori , al fine della corretta impostazione terapeutica , nonch lutilit di fornire precoci informazioni sulla risposta alla terapia ha stimolato in questi ultimi anni la ricerca e , nellultima decade , sono stati pubblicati numerosi studi riguardanti limpiego delle nuove tecniche quali la diffusione ( dwi ) , la perfusione ( pwi ) e la spettroscopia in vivo ( mrs ) nella caratterizzazione dei tumori cerebrali [ 14 ]  . con il termine diffusione si indica il movimento random ( browniano ) delle molecole di acqua nei tessuti ; la diffusibilit delle molecole dellacqua caratteristica per ogni tessuto e si pu modificare in determinate condizioni patologiche . 
le sequenze pesate in diffusione permettono di valutare questo parametro tessutale ed il calcolo del coefficiente di diffusione apparente ( adc ) fornisce elementi diagnostici importanti nella valutazione delle masse intracraniche [ 2 , 5 ]  . la perfusione rappresenta lapporto nutritivo ematico ai tessuti e viene definita come il flusso per unit di tempo . solitamente viene misurata in ml / 100 g di tessuto / min , o unit di cbf . 
la sostanza grigia delluomo sano viene perfusa ad un ritmo di 5060 ml / 100 g / min e viene mantenuta in un range ristretto dai meccanismi di autoregolazione cerebrale . 
perci stati di bassa perfusione possono sfociare nellischemia cellulare , mentre stati di elevata perfusione possono essere correlati a lesioni ipervascolari , come alcuni tumori [ 6 ]  . i parametri che pi frequentemente vengono misurati sono : il cbv ( cerebral blood volume ) che equivale alla radiol med ( 2009 ) 114 : 645659 ratio between volume and transit time [ 7 ]  . 
the most frequently used parameter in brain tumour imaging is the relative regional cerebral blood volume ( rrcbv ) , which is increased in tumours as a result of neoangiogenesis [ 8 , 9 ]  . this study correlated the adc and rrcbv values obtained by applying echoplanar ( epi ) and gradient - echo ( gre ) sequences in a large series of brain tumours with histological findings to evaluate the usefulness of this technique in characterising brain tumours . materials and methods in this prospective study conducted between january 2003 and august 2006 , patients with brain tumours were studied with conventional mr imaging complemented by dwi and pwi . 
from among these , 98 patients ( 53 men and 45 women ) were selected on the basis of the following exclusion criteria : lack of histological diagnosis , nonneoplastic lesions , previous treatments on the tumours ( surgery , chemotherapy , radiotherapy ) , incorrect examination technique . all lesions were characterised histologically . 
tumours were subdivided into primary and secondary ; the former were grouped by histological type and grade according to the world health organization ( who ) classification , whereas the latter were classified according to the tumour of origin . study protocol mr imaging was performed during a single session in all patients by using a superconducting 1 - tesla scanner ( signa horizon , ge ) with a maximum gradient strength of 23 mt / m and a slew rate of 77 mt / m / s . 
we used 1 m gadobutrol ( gadovist ) injected intravenously with the aid quantit di sangue presente in una data quantit di tessuto in un dato momento ; il cbf ( cerebral blood flow ) che indica la quantit di sangue che attraversa una data quantit di tessuto nellunit di tempo e lmtt ( mean transit time ) o tempo di transito medio . 
il parametro pi utilizzato nello studio dei tumori cerebrali il volume ematico regionale relativo ( rrcbv ) che aumenta nei tumori in rapporto ai fenomeni di neoangiogenesi [ 8 , 9 ]  . in questo lavoro vengono correlati i valori di adc e di rrcbv , ottenuti su unampia serie di tumori cerebrali utilizzando sequenze echo planari ( epi ) gradient echo ( gre ) , con il relativo dato isto - patologico , per valutare lapporto di questa tecnica nella caratterizzazione dei tumori cerebrali . materiali e metodi nel periodo compreso tra gennaio 2003 e agosto 2006 stato effettuato uno studio prospettico che prevedeva di completare lesame rm convenzionale con acquisizioni dwi e pwi nei pazienti con tumore cerebrale . 
tra questi sono stati selezionati 98 pazienti ( 53 maschi e 45 femmine ) utilizzando i seguenti criteri di esclusione : assenza della diagnosi istologica , lesioni non neoplastiche , pregressi trattamenti sul tumore ( chirurgia , chemioterapia , radioterapia ) , non corretta esecuzione dellesame . tutte le lesioni sono state caratterizzate istologicamente ; i tumori sono stati suddivisi in primitivi e secondari ; i primi sono stati raggruppati per istotipo e grado secondo la classificazione della who , mentre i secondi sono stati classificati sulla base del tumore dorigine . protocollo di studio la rm stata eseguita in tutti i pazienti in ununica seduta utilizzando un magnete superconduttivo operante a 1 tesla ( signa horizon , ge ) con potenza massima dei gradienti di 23 mt / m e slew - rate di 77 mt / m / s . 
 stata impiegata la bobina head di ricezione e trasmissione in quadratura . in tutti i pazienti stato eseguito un esame standard con impiego di sequenze se t1 ( tr / te 500 / 12 ms ) , fse pesate in t2 ( tr / te 4000 / 85 ms etl 13 ) e flair ( tr / te 8000 / 90 ms ti 2000 ms )  . 
sono state ottenute immagini sui tre piani dello spazio con sezioni di 45 mm e gap 0 , 51 mm . prima della somministrazione del mdc stata eseguita in tutti i casi una sequenza pesata in diffusione , sul piano 648 radiol med ( 2009 ) 114 : 645659 of a dual - syringe automatic injector ( medrad ) via an 18gauge needle cannula at a flow rate of 5 ml / s . 
in all cases , a few minutes before the dynamic phase , patients were administered 0.05 mmol / kg of gd as a prebolus to attenuate the t1 effects of bloodbrain barrier ( bbb ) disruption . to study the entire area of the lesion , we acquired a set of 12 slices repeated sequentially 40 times , thus obtaining 480 images in 1 min 12 s . 
all axial images were acquired with the same geometry ( fov , slice thickness , gap , angle ) to enable fusion of the dw and pw images with the corresponding flair and / or contrastenhanced t1 - weighted images . 
adc maps inclusive of the adc calculation were generated by positioning 25to 30 - mm2 regions of interest ( roi ) over the tumour areas . the mean adc values of solid and fluid components were obtained separately for each lesion . pw images were processed by applying a formula to calculate , starting from the baseline t2 value of tissues , the drop in signal during the passage of the bolus ( r2 ) and its subsequent return to the baseline level : r2 * = - 1n [ s ( t ) / s ( 0 ) / te ] where s ( t ) corresponds to the signal intensity at time ( t ) , s ( 0 ) represents the mean mr signal before the arrival of the contrast and te to echo time . 
mathematical integration of the area under r2 * curve ( negative enhancement integral ) corresponds to the cbv . to obtain cbv maps , a number of rois 2530 mm2 in diameter were placed over the tumour in such a way as to cover the entire lesion , and the enhancing and nonenhancing areas of tumour were evaluated separately . 
for each lesion , we calculated the mean rcbv of pathological tissue ( rcbvp ) and normal tissue ( rcbvn ) and the ratio between the two means , thus obtaining a value that we called rrcbv [ 10 ]  . 
stato utilizzato il gadobutrolo 1 m ( gadovist ) , iniettato endovena tramite lausilio di un iniettore automatico a doppia siringa ( medrad ) utilizzando unagocannula 18 g con velocit di flusso 5 ml / s . 
la dose somministrata stata per ogni paziente di 0 , 15 mmol / kg , seguita da 20 ml di soluzione fisiologica ; la fase dinamica stata sempre preceduta di pochi minuti dalla somministrazione di un prebolo di 0 , 05 mmol / kg di gadolinio per attenuare gli effetti t1 di alterazione della barriera ematoencefalica ( bee )  . sono state impostate 12 slices in modo da studiare interamente larea lesionale ripetute in maniera sequenziale per 40 volte ottenendo cos 480 immagini in un 112 . 
le prime 4 sequenze ( 48 slices ) , acquisite prima dellinizio della somministrazione del mdc per raggiungere lo stato di steady - state , sono state escluse dalle successive rielaborazioni . 
tutte le immagini assiali sono state acquisite con la stessa geometria : fov , spessore di strato , gap , inclinazione , per permettere la fusione delle immagini acquisite in dwi e pwi con le relative immagini flair e / o t1 post contrasto . 
dopo linfusione del mdc , sono state effettuate scansioni sui tre piani dello spazio ( coronale , sagittale ed assiale ) con sequenze se t1 - pesate . elaborazione delle immagini sono state successivamente rielaborate sia le immagini dwi sia quelle pwi utilizzando , sulla consolle operativa o sulla workstation ( adw 4.0 ) , un software fornito dalla ge ( functool 2 )  . 
per ogni lesione stata ottenuta separatamente la media delladc relativa alle componenti solide e a quelle fluide . le immagini pwi sono state elaborate applicando una formula che , partendo dal valore t2 di base dei tessuti , calcola la caduta di segnale durante il passaggio del bolo ( r2 ) e il successivo ritorno alla stato di partenza . r2 * = - 1n [ s ( t ) / s ( 0 ) / te ] dove s ( t ) corrisponde al segnale al tempo ( t ) ed s ( 0 ) rappresenta la media del segnale rm prima dellarrivo del contrasto e te il tempo di echo . 
the students t test for unpaired data was applied to the mean values obtained ; values of p < 0.0001 were considered to be statistically significant . results of the 98 patients , 46 had primary brain tumours and 52 had one or more secondary lesions ( table 1 )  . 
 of all tumours examined , 17 were morphologically composed of a necroticcystic central component surrounded by a strongly enhancing solid peripheral rim . the necroticcystic component was hypointense on dwi in 12 tumours and hyperintense in five . 
per ogni lesione stata calcolata la media del rcbv del tessuto patologico ( rcbvp ) e del tessuto sano ( rcbvn ) ed stato calcolato il rapporto tra le due medie ottenendo un valore che chiameremo rrcbv [ 10 ]  . 
ai valori delle medie cos ottenute stato applicato il test t di student per dati non appaiati ; sono stati considerati significativi valori di p < 0 , 0001 . risultati dei 98 pazienti 46 erano portatori di un tumore cerebrale primitivo e 52 di una o pi localizzazioni secondarie ( tabella 1 )  . tra tutti i tumori esaminati 17 erano costituiti morfologicamente da una componente centrale necrotico - cistica con spesso cercine periferico solido dotato di intenso contrast enhancement . 
the rrcbv values of the low - grade ( ii ) and high - grade ( iii - iv ) gliomas , as well as the values of highgrade gliomas and lymphomas , were analysed with students t test for unpaired data . 
the results are shown in tables 2 and 3 . twenty - nine of the brain metastases were from lung cancer , whereas the others were distributed as follows : seven from cancers of the uropoietic system ( six renal carcinomas and one bladder carcinoma ) , seven from breast cancer , four from gastrointestinal cancers , one from melanoma and three from lymphoma . 
a scansione assiale t1 pesata dopo somministrazione del mdc : lesione cistica con orletto iperintenso e cospicuo edema digitato della sostanza bianca perilesionale ; b analoga scansione in dwi : la lesione appare iperintensa ; c mappa adc : il valore di adc allinterno della componente cistica della lesione di 0 , 910 - 3 mm2 / s . 10 13 16 19 22 25 28 31 34 37 40 43 46 49 n pazienti metastasi tumori primari fig . 
2 rappresentazione grafica della distribuzione dei valori di adc nei vari tipi di tumori , distinti tra tumori cerebrali primitivi e metastasi . 652 radiol med ( 2009 ) 114 : 645659 fig . 
a contrast - enhanced t1 - weighted image ; b curves of signal decrease in the regions of interest ; c relative regional cerebral blood volume ( rrcbv ) maps . 
5 confronto dellrrcbv tra gliomi grado iii e grado iv . glioma di iv grado glioma di iii grado linfomi cerebrali gliomi ad alto grado 2 3 4 9 10 10 12 13 14 15 16 17 18 19 pazienti fig . 
i valori di rrcbv relativi ai gliomi a basso grado ( ii ) e quelli ad alto grado ( iii - iv ) , cos come i valori dei gliomi ad alto grado e quelli dei linfomi sono stati sottoposti a test statistici utilizzando la t di student per dati non appaiati ed i risultati ottenuti vengono riportati nelle tabelle 2 e 3 . ventinove delle metastasi encefaliche erano da carcinoma polmonare , mentre le altre erano cos ripartite : 7 da tumori dellapparato uropoietico ( 6 carcinomi renali e 1 carcinoma della vescica ) , 7 da carcinoma mammario , 4 da neoplasie dellapparato digerente , 1 da melanoma e 3 da linfoma . 
in the majority of cases , morphological imaging is able to differentiate low - grade from diffusely invasive or frankly malignant lesions , which tend to show significant perilesional oedema and intense contrast enhancement . radiol med ( 2009 ) 114 : 645659 i tumori neuroepiteliali , in particolare i glomi , rappresentano una causa frequente di tumori dellencefalo . 
gli esami radiologici morfologici sono in grado di differenziare nella maggior parte dei casi le lesioni di basso grado da quelle diffusamente infiltranti o chiaramente maligne che presentano generalmente un significativo edema perilesionale ed unelevata impregnazione contrastografica . 
ci nonostante , non tutte le neoplasie gliali ad alto grado presentano necrosi o effetto massa e circa il 40 % non presenta impregnazione contrastografica [ 2 , 11 ]  . il problema si complica se teniamo conto di altri due elementi : in primo luogo dellelevata frequenza delle metastasi cerebrali che si sviluppano in circa il 15%25% di tutti i pazienti con neoplasie maligne [ 12 ] e in secondo luogo dellaumentata incidenza dei linfomi cerebrali primitivi verificatasi nelle ultime due decadi per aumento della popolazione immucompromessa in relazione allincremento del numero di soggetti sottoposti a trapianto dorgano e di quelli con aids [ 13 , 14 ]  . la rm convenzionale dimostra molti limiti nella differenziazione tra i diversi gradi di tumori primari neuroepiteliali , le metastasi e i linfomi . 
nevertheless , not all high - grade glial tumours exhibit necrosis or mass effect , and approximately 40% of them are nonenhancing [ 2 , 11 ]  . the issue becomes more complex if we consider two other factors : first , the high frequency of cerebral metastases occurring in around 15%25% of all patients with malignancies [ 12 ] , and second , the rising incidence over the past two decades of primary cerebral lymphomas as a result of the increasing number of immunocompromised subjects due to organ transplantations and aids [ 13 , 14 ]  . conventional mr imaging has many limitations in distinguishing among the different grades of primary neuroepithelial tumours , metastases and lymphomas . 
for this reason , it is important to have access to a diagnostic imaging technique capable of enabling the rapid and noninvasive diagnosis and tumour monitoring [ 13 , 15 ]  . diffusion imaging by evaluating the mobility of water molecules in tissue , diffusion imaging can provide diagnostic information in several pathological conditions . 
in cerebral masses , the role of dwi and adc mapping in differentiating cystic tumours from abscesses and epidermoid from arachnoid cysts is known and well established [ 1618 ]  . the five cystic tumours with hyperintense fluid component on dwi resembled abscesses in appearance , although the adc values were slightly higher [ 18 , 19 ]  . 
the high mucinous content explains the adc restriction [ 1820 ]  . the adc of the solid component of the tumours showed wide variations without significant differences among the various types of tumour . 
in our series , the mean adc for lymphomas did not exceed 1.410 - 3 mm2 / s , a finding in agreement with previous reports [ 2 , 4 , 16 , 21 ]  . 
in fact , the extent of oedema strongly affects adc values , and it is likely that steroid treatment , frequently initiated at presentation in patients with brain tumours , influences the dwi parameters [ 22 ]  . perfusion imaging brain tumours may stimulate the formation of new vascular networks , and neoangiogenesis is fundamental for tumour growth . 
the increased metabolic demands of the growing tumour and the resulting hypoxia in large tumours stimulate fornire informazioni diagnostiche in svariate condizioni patologiche nel caso di masse cerebrali noto e consolidato il ruolo delle sequenze dwi e delle mappe delladc nel differenziare i tumori cistici dagli ascessi e le cisti epidermoidi dalle cisti aracnoidee [ 1618 ]  . i 5 tumori cistici con componente fluida iperintensa in dwi avevano un aspetto simile a quello degli ascessi , ma con valori di adc lievemente superiori [ 18 , 19 ]  . 
erano tutte metastasi da carcinoma polmonare ; allesame istologico larea cistica di questi tumori era costituita da tessuto mucinoso nel quale galleggiavano cellule neoplastiche , mentre non era evidenziabile contenuto ematico ; lelevata componente mucinosa spiega la restrizione del coefficiente di diffusione apparente [ 1820 ]  . per quanto concerne ladc delle componenti solide dei tumori i dati ottenuti dimostrano unampia variabilit di valori senza significativa differenza tra i vari tipi di neoplasie ; nella maggior parte dei casi tuttavia i valori di adc erano superiori rispetto a quelli del tessuto sano normale . 
dalla osservazione dei dati emerge che tra tutti i tipi tumorali i linfomi sono quelli che mostrano i valori complessivamente inferiori ; in questa casistica la media di adc per i linfomi non supera mai il valore di 1 , 410 - 3 mm2 / s ; questo concorda con i dati della letteratura [ 2 , 4 , 16 , 21 ]  . 
in realt lentit delledema condiziona molto i valori di adc ed probabile che il trattamento con farmaci corticosteroidei , frequentemente adottato gi allesordio della clinica nei pazienti con tumori cerebrali , influisca sui parametri ottenuti in dwi [ 22 ]  . imaging di perfusione i tumori cerebrali possono indurre la formazione di nuovi vasi ; la neoangiogenesi fondamentale per la crescita dei tumori . 
la prima tecnica , detta spin label , ancora in fase di studio e prevede di marcare i globuli rossi durante il passaggio nei vasi epiaortici e successivamente di valutarne il primo passaggio nel tessuto cerebrale [ 23 ]  . 
i mdc esogeni impiegati sono i chelati del gadolinio che non superano normalmente la bee e quindi si comportano come 656 radiol med ( 2009 ) 114 : 645659 the formation of cytokines responsible for angiogenesis [ 1 ]  . this process is closely related to biological aggressiveness and is one of the main parameters considered in the histopathological grading of glial tumours [ 1 ]  . 
the first technique , known as spin labelling , is still under development and involves tagging red blood cells during their passage in the epiaortic vessels and subsequently evaluating their first pass within cerebral tissue [ 23 ]  . 
the contrast agent compartmentalised inside vessels causes , on first pass when concentration is high , a drop in the t2 signal , which is best appreciated in t * gradient - echo sequences that are most heavily affected by magnetic susceptibility effects . 
studies comparing different gd concentrations have demonstrated that at the same dose levels , the use of 1 m gd increases the first - pass magnetic susceptibility effect , leading to curves with higher peaks [ 2426 ]  . one of the problems arising when evaluating concentration curves with dsci is leakage of contrast material into the extravascular space , which occurs in pathological conditions accompanied by bbb abnormalities . 
this problem can be overcome by administering a small dose of contrast material ( 0.05 mmol / kg ) approximately 5 min before initiating perfusion imaging [ 6 , 8 , 27 ]  . because the rcbv values obtained by applying the negative enhancement integral are not absolute , a standard reference is required to enable comparisons among different patients . 
this could be the rcbv obtained at the level of the contralateral white matter , or ipsilateral normal white matter in the case of lesions affecting both cerebral hemispheres . this provides the rrcbv value , which is calculated with the formula rrcbv = rcbvp / rcbvn [ 10 ]  . the results obtained in our series allow us to make a few concluding remarks . 
if we consider glial tumours , there are statistically significant differences ( p < 0.0001 ) between the rrcbv value of lowand high - grade gliomas ( table 2 )  . 
il mdc compartimentalizzato allinterno dei vasi determina , al primo passaggio , quando la concentrazione elevata , un abbattimento del segnale t2 , meglio valutabile utilizzando sequenze gradient - echo t2 * , che maggiormente risentono degli effetti di suscettibilit magnetica ; questa tecnica denominata con lacronimo dsci ( dynamic contrast susceptibility imaging ) [ 6 , 23 ]  . vengono abitualmente utilizzate sequenze epi che permettono di studiare un esteso volume cerebrale ed offrono il vantaggio di unelevata risoluzione temporale . 
in questo studio stato utilizzato gadolinio ( gd ) alla concentrazione 1 m somministrato a 5 ml / s con una dose pari 0 , 15 mmol / kg ; diversi studi condotti comparando concentrazioni diverse di gadolinio , hanno dimostrato che limpiego di gd 1 m , a parit di dose iniettata , aumenta leffetto di suscettibilit magnetica al primo passaggio per cui si ottengono curve con picco pi elevato [ 2426 ]  . uno dei problemi che insorge nella valutazione delle curve di concentrazione con effetto dsci rappresentato dallo stravaso di mdc nello spazio extravascolare che si verifica in quelle condizioni patologiche che si accompagnano ad alterazione della bee . 
un artificio per ovviare a questo problema quello di somministrare una piccola dose di mdc ( 0 , 05 mmol / kg ) circa 5 minuti prima dellacquisizione perfusionale [ 6 , 8 , 27 ]  . i valori di rcbv , ottenuti applicando lintegrale di enhancement negativo , non sono assoluti ; pertanto , per poter effettuare il confronto tra pazienti diversi , necessario utilizzare un riferimento standard che pu essere rappresentato dal valore di rcbv ottenuto a livello della sostanza bianca controlaterale alla lesione o della sostanza bianca sana omolaterale in caso di lesioni che interessano entrambi gli emisferi cerebrali . 
si ottiene in tal modo lrrcbv che si calcola con formula rrcbv = rcbvp / rcbvn [ 10 ]  . la seguente dai risultati ottenuti in questa casistica si possono trarre alcune considerazioni . 
se si considerano i tumori della serie gliale emergono delle differenze statisticamente significative ( p < 0 , 0001 ) del valore di rrcbv tra gliomi a basso grado e gliomi ad alto grado ( tabella 2 )  . 
pertanto se si fa la media del rcbv posizionando roi allinterno di tutto il tumore , ne deriva una riduzione complessiva dei valori di rrcbv nei tumori pi necroradiol med ( 2009 ) 114 : 645659 neoangiogenesis [ 28 ]  . 
all lymphomas were characterised by a low rrcbv value , constantly lower than 2 ( the maximum value observed was 1.53 ) , whereas the majority of high - grade gliomas had high rrcbv values . 
the differential diagnosis between lymphomas and gliomatoses is , however , straightforward at conventional mr imaging and based on morphology and tumour extension [ 30 , 31 ]  . lowest rrcbv values seen with regard to metastases , no statistically significant differences were seen in the perfusion values of the various types of metastases . 
the high rrcbv values seen in meningiomas , however , are in line with previous reports [ 15 , 26 ]  . conclusions diffusionand perfusion - weighted mr imaging in patients with brain tumours provide additional information that tici . 
per questo motivo stato ipotizzato di utilizzare oltre allimaging convenzionale anche le mappe di rrcbv come guida per effettuare le biopsie stereotassiche [ 29 ]  . se si confrontano i gliomi ad alto grado con i linfomi , per i quali la diagnosi differenziale con lrm convenzionale pu talvolta essere problematica , possiamo vedere come in questo studio i valori di perfusione diano risultati statisticamente significativi ( p < 0 , 0001 )  . 
tutti i linfomi erano caratterizzati da un valore di rrcbv basso , sempre inferiore a 2 ( il valore massimo riscontrato stato di 1 , 53 ) , mentre la maggior parte dei gliomi ad alto grado aveva valori di rrcbv elevati . 
la possibilit di discriminare tra tumori gliali ad alto grado e linfomi concorda con quanto gi riportato da altri studi [ 15 , 20 ]  . un discorso a parte va fatto per le gliomatosi cerebri . 
in questo studio sono compresi solo due casi , ed entrambi hanno bassi valori di rrcbv , simili a quelli dei linfomi e dei tumori low - grade ; questo dato correla con la ridotta microvascolarizzazione di questi tumori il cui grading elevato dato dalla elevata estensione . 
la diagnosi differenziale tra linfomi e gliomatosi tuttavia agevole allo studio rm convenzionale sulla base della morfologia e dellestensione del tumore [ 30 , 31 ]  . per quanto concerne le metastasi non sono invece state riscontrate differenze statisticamente significative dei valori di perfusione tra i vari tipi di metastasi . 
i dati ottenuti oscillano su un ampio range ; i valori di rrcbv risultano pi bassi nelle localizzazioni da linfoma ed in alcuni casi di metastasi da carcinoma del polmone o della mammella ; questultimo dato potrebbe essere correlato , almeno in parte , alle terapie a cui tali soggetti erano sottoposti per il tumore dorigine . 
i valori elevati di rrcbv riscontati nei meningiomi sono in linea con quanto riportato in letteratura [ 15 , 26 ]  . conclusioni le tecniche di diffusione e di perfusione con rm se utilizzate nei pazienti con tumore cerebrale forniscono informazioni non ottenibili con lesame convenzionale , possono essere eseguite a completamento dellesame convenzionale utilizzando lo stesso contrasto e con un minimo aumento del tempo di esecuzione . 658 radiol med ( 2009 ) 114 : 645659 cannot be obtained with conventional mr . 
should dwi and pwi prove superior to conventional imaging , they could be used to monitor patients undergoing treatment to obtain an earlier evaluation of response to chemoor radiotherapy and guide the clinicians treatment choices . le immagini dwi hanno rilevante importanza nella diagnosi delle masse cistiche , mentre non sembrano avere un ruolo nella caratterizzazione dei tumori . 
dwi e pwi inoltre si propongono come tecniche promettenti nel management dei tumori cerebrali : il dato relativo alla microvascolarizzazione offerto dalla pwi potrebbe servire come guida al prelievo bioptico , indicando le aree maggiormente aggressive . 
among the various therapies , local radiotherapy is the gold standard for pain palliation from single metastasis , even though the maximum benefit is obtained between 12 and 20 weeks from initiation . 
from november 2003 to may 2008 , ten ablation treatments were performed in ten patients with acute pain from metastatic bone lesions . patient selection and choice of the most appropriate ablation treatment was made based on lesion characteristics . 
three patients were treated with radiofrequency , one with plasma - mediated radiofrequency , two with plasma - mediated radiofrequency and cementoplasty , three with radiofrequency and cementoplasty and one with microwave . 
in both cases , 3 - month follow - up showed a statistically significant reduction of pain no case did local complications occur either during or after treatment . only one patient treated with radiofrequency ( 1 / 9 , 11% ) developed low - grade fever and general malaise during the 6 days following the procedure , compatible with a postradiofrequency syndrome , which was treated with riassunto obiettivo . 
tra le varie terapie , la radioterapia locale il gold - standard nella palliazione del dolore da metastasi singola , anche se il massimo beneficio si ottiene tra le 12 e le 20 settimane dallinizio della terapia . 
la valutazione stata effettuata non solo con imaging , ma anche con una vas score ( visual analoge scale ) per la determinazione del dolore e con la variazione delle dose equivalenti di morfina . 
solamente in un paziente trattato con radiofrequenza ( 1 / 9 , 11% ) abbiamo riscontrato nei 6 giorni successivi al trattamento insorgenza di febbricola e malessere generale compatibile con la sindrome postradiol med ( 2009 ) 114 : 608625 acetaminophen ( paracetamol ) only and resolved on day 7 . 
percutaneous ablation therapies represent a safe and valuable alternative for treating localised pain from single bone metastasis , providing rapid ( 4 - week ) relief of symptoms and a significant reduction in morphine doses . 
le tecniche ablative percutanee rappresentano una sicura e valida alternativa al trattamento del dolore localizzato da metastasi ossee singole , apportando in breve tempo ( 4 settimane ) un miglioramento della sintomatologia e una sensibile riduzione delle dosi di morfina . 
ci concorre a migliorare la qualit della vita in pazienti affetti da malattia metastatica . parole chiave metastasi osso palliazione ablazione introduction introduzione bone metastases are a common problem in cancer patients , affecting up to 30% of patients with epithelial cancers . 
in 70% of cases , the metastases originate from cancers of the prostate or breast , in 20%30% from cancers of the lung or kidney and in 10% from cancers of the rectum , colon , ovary and pancreas [ 1 ]  . the causes of pain in patients with bone metastases are not fully understood but appear to be unrelated to the type of tumour , location , number or size of the metastatic lesions . possible mechanisms of pain include stretching of the periosteum secondary to tumour growth , fractures ( both micro and macro ) , cytokine - mediated osteoclastic bony destruction that results in stimulation of the nerve endings in the endosteum and tumour growth into surrounding nerves and tissues [ 2 ]  . several types of treatment , including chemotherapy , hormonal therapy , surgery , radiotherapy and radiometabolic therapy , are used for palliation purposes . 
however , a number of patients fail to benefit from these conventional treatments , and pain relief , when achieved , does not occur until 412 weeks after treatment [ 3 ]  . 
a variety of alternative strategies have been proposed for treating painful bone metastases , all of which are based on image - guided percutaneous procedures for ablating focal bone lesions [ 1 , 4 , 5 ] , in some cases associated with cementoplasty [ 68 ]  . the aim of this study was to evaluate the technical success , effectiveness and possible complications of percutaneous ablation treatment in patients with painful bone metastases . 
esse nel 70% dei casi insorgono dal tumore della prostata o della mammella , nel 20%30% dal tumore del polmone o del rene , nel 10% dalle neoplasie del retto , colon , ovaio e pancreas [ 1 ]  . le cause di dolore in soggetti affetti da metastasi ossee non sono ancora completamente note , ma non sembrano correlate al tipo di tumore primitivo , alla localizzazione della lesione , al numero e alle dimensioni delle lesioni . 
possibili meccanismi nella genesi del dolore comprendono : stretching del periostio secondario alla crescita del tumore ; fratture ( sia micro - fratture che macro - fratture ) ; distruzione ossea di tipo osteoclastico citochino - mediata che determina una stimolazione delle fibre nervose terminali endostali ; crescita tumorale nelle strutture nervose e nei tessuti adiacenti [ 2 ]  . 
alcuni pazienti non riescono a trarre benefici da questi trattamenti convenzionali e il sollievo dal dolore , quando si verifica , non avviene mai prima di 412 settimane [ 3 ]  . 
sono state proposte varie strategie alternative per il trattamento delle metastasi ossee dolorose , che richiedono metodiche percutanee imaging - guidate finalizzate allablazione delle lesioni focali ossee [ 1 , 4 , 5 ] , in alcuni casi associando la cementoplastica [ 68 ]  . scopo del lavoro stato quello di valutare il successo tecnico , lefficacia e le eventuali complicanze in pazienti affetti da metastasi ossee sintomatiche e sottoposti a terapia ablativa percutanea . 
 610 radiol med ( 2009 ) 114 : 608625 materials and methods materiali e metodi between november 2003 and may 2008 , we performed ten ablation treatments in ten patients ( five men and five women ; mean age 62 years ; range 2882 ) with acute pain from bone metastases of different origin , site ( table 1 ) and size ( mean volume 63.25 cm3 ; range 218.93.3 cm3 ) ( table 2 )  . 
in all of these patients , the pain had proved refractory to conventional approaches . owing to the poor expected response , one patient received no treatment before the ablation procedure ( table 1 )  . 
three patients were treated with radiofrequency ( rf ) alone , one with plasma - mediated rf , two with plasma - mediated rf combined with cementoda novembre 2003 a maggio 2008 sono stati effettuati , nel servizio di radiologia del nostro ospedale 10 trattamenti in 10 pazienti , 5 uomini e 5 donne ( et media 62 anni , range da 28 a 82 anni ) con dolore acuto da lesioni metastatiche ossee di varia origine , sede ( tabella 1 ) e volume ( volume medio 63 , 25 cm3 , range da 218 , 9 a 3 , 3 cm3 ) ( tabella 2 )  . tutte le lesioni trattate erano osteolitiche . 
 quattro pazienti erano stati precedentemente sottoposti a radioterapia e chemioterapia , 1 paziente solo a radioterapia , 4 pazienti solo a chemioterapia : in questi casi si sono dimostrati refrattari alle terapie convenzionali . 
nessun paziente era suscettibile di trattamento chirurgico , per lassenza di rischio di frattura patologica . tre pazienti sono stati trattati solo con radiofrequenza ( rf ) , table 1 personal series patient no . / age / gender primary tumour histology site of metastasis radiotherapy chemotherapy bladder liver liver liver breast lung lung colon - rectum transverse colon uterine cervix carcinoma right 8th rib left 9th rib left ischiopubic ramus right 8th rib infiltrating lobular vertebra l4 carcinoma adenocarcinoma adenocarcinoma adenocarcinoma adenocarcinoma squamous cell carcinoma vertebra l1 left sacroiliac joint left acetabolum left ischiatic spine left iliac wing not responsive not responsive not responsive not responsive not responsive not responsive not responsive not responsive not responsive not responsive not responsive not responsive not responsive n / et / sesso tumore primitivo istologia sede metastasi radioterapia chemioterapia vescica fegato fegato fegato mammella polmone polmone carcinoma carcinoma lobulare vertebra l4 infiltrante adenocarcinoma adenocarcinoma viii arcata costale destra ix arcata costale sinistra branca ischio - pubica sinistra viii arcata costale destra non responsivo non responsivo non responsivo non responsivo non responsivo vertebra l1 articolazione sacroilaca sinistra acetabolo sinistra spina ischiatica sinistra ala iliaca sinistra non responsivo non responsivo non responsivo non responsivo non responsivo non responsivo non responsivo non responsivo colon - retto colon trasverso portio uterina adenocarcinoma adenocarcinoma carcinoma squamocellulare hcc , carcinoma epatocellulare hcc , hepatocellular carcinoma tabella 1 serie personale 1 / 70 / m 2 / 69 / m 3 / 45 / m 4 / 40 / m 5 / 60 / f 6 / 73 / f 7 / 73 / f 8 / 82 / f 9 / 81 / m 10 / 38 / f 1 / 70 / m 2 / 69 / m 3 / 45 / m 4 / 40 / m 5 / 60 / f 6 / 73 / f 7 / 73 / f 8 / 82 / f 9 / 81 / m 10 / 38 / f radiol med ( 2009 ) 114 : 608625 table 2 descriptions of lesions and procedures per patient patient procedure lesion no . 
stato eseguito il calcolo del volume per ciascuna lesione attraverso la formula 4 / 3ab2 , dove a il semiasse maggiore e b il semiasse minore plasty , three with rf combined with cementoplasty and one with microwave ablation . 
 1 con radiofrequenza plasma - mediata , 2 con radiofrequenza plasma - mediata associata a cementoplastica , 3 con radiofrequenza associata a cementoplastica , 1 con microonde . tutti hanno completato il follow - up a 12 settimane . 
 pretreatment assessment before undergoing ablation treatment , patients were evaluated by means of a validated visual analogue scale ( vas ) for pain assessment and by recording any use of analgesics . inclusion criteria were moderate or severe pain rated 4 or more on the 10 - point vas ; patients with lower scores were treated with analgesics . 
pain was localised to the site corresponding to the lesion detected by computed tomography ( ct ) ( aquilon 64 , toshiba , tokyo , japan ) or magnetic resonance valutazione prima del trattamento prima dei trattamenti ablativi i pazienti sono stati valutati usando una codificata visual analogue scale ( vas ) per la determinazione del dolore , ed stato registrato leventuale utilizzo di analgesici . 
criteri di inclusione nello studio sono stati : dolore moderato o severo con valore uguale o superiore a 4 nella scala vas di 10 punti ; i pazienti con punteggio inferiore sono stati trattati con analgesici . 
prima del trattamento , tutti i pazienti inclusi nel nostro studio 612 radiol med ( 2009 ) 114 : 608625 table 3 morphine - equivalent doses for several opioids according to route of administration tabella 3 dosi equivalenti di morfina per diversi oppioidi a seconda della via di somministrazione administration route via di somministrazione parenteral dose equivalents oral dose equivalents parenterale dose equivalenti orale dose equivalenti morphine methadone codeine buprenorphine tramadol fentanyl 10 mg 10 mg 120 mg 0.3 mg 100 mg 25 mcg / hc 30 mga 20 mg 200 mg 0.4 mgb 120 mg morfina metadone codeina buprenorfina tramadolo fentanile 10 mg 10 mg 120 mg 0 , 3 mg 100 mg 25 g / h c 30 mga 20 mg 200 mg 0 , 4 mgb 120 mg achronic use ; bsublingual ; ctransdermal auso cronico ; bsublinguale ; ctransdermica ( mr ) imaging ( 1.5 tesla avanto , siemens , munich , germany )  . 
specifically , nine patients were imaged by contrast - enhanced ct ( iopamiro , bracco , milan , italy ) and one by contrast - enhanced mr imaging ( magnevist , schering , berlin , germany )  . 
exclusion criteria were the presence of more than two symptomatic lesions and safety margins less than 1 cm from the spinal cord , major motor nerve , brain , adamkiewicz artery , bowel and bladder [ 9 ]  . before the ablation treatment , patients were on analgesic therapy ( opioids or nsaids ) , which was translated into morphine - equivalent doses to render the data homogeneous ( table 3 )  . 
analgesic use was monitored before treatment and at 1 , 4 , 8 and 12 weeks thereafter . treatment selection depending on whether the lesion is osteoblastic or osteolytic , the most beneficial ablation method , that is , the one best suited to the individual metastasis , must be selected . both rf and microwave ablation are based on the use of heat , although heat production occurs in two different manners : rf generates heat by delivering electrical current to tissues and causing ion agitation and friction ; electromagnetic microwave generates heat by agitating water molecules in tissue and producing friction [ 10 ]  . 
plasma - mediated rf ( coblation ) consists of using electrical energy to create a field of ionised particles that break the organic bonds within soft tissue ( ablation )  . 
il dolore era localizzato alla sede corrispondente alla lesione evidenziata con indagini eseguite con tomografia computerizzata ( tc ) ( aquilon 64 , toshiba , tokio , giappone ) o risonanza magnetica ( rm ) ( 1 , 5 tesla avanto , siemens , munich , germania ) ; in particolare 9 pazienti sono stati sottoposti a tc con mdc ( iopamino , bracco , milano , italia ) e 1 paziente a rm con mdc paramagnetico ( magnevist , schering , berlin , germania )  . 
criteri di esclusione al trattamento ablativo sono stati : la presenza di pi di due lesioni sintomatiche ; margini di sicurezza inferiori a 1 cm dal midollo spinale , dal nervo motorio maggiore , dallencefalo , dallarteria di adamkiewicz , dallintestino e dalla vescica [ 9 ]  . 
prima del trattamento i pazienti erano in terapia con analgesici ( oppioidi o farmaci anti - infiammatori non steroidei ) , convertiti poi , ai fini di rendere omogenei i dati , in dosi - equivalenti di morfina ( tabella 3 )  . 
tutti i pazienti assumevano dagli 80 ai 100 equivalenti di morfina . lutilizzo di analgesici stato monitorato prima della procedura , alla prima , alla quarta , allottava e alla dodicesima settimana susseguenti la stessa . selezione della tipologia di trattamento in questa fase occorre valutare , a seconda che la lesione sia osteoblastica o osteolitica , quale sia il trattamento ablativo di cui pu giovare il paziente , ovvero il pi adatto a quella specifica metastasi . 
sia lablazione con radiofrequenza che quella con microonde caratterizzata dallimpiego di calore , che avviene con due modalit differenti : la rf genera calore tramite lattrito causato dallagitazione ionica che si ha al passaggio di corrente elettrica , le microonde invece generano calore per le oscillazioni delle molecole dacqua tissutali provocate dallinterazione tra le radiazioni elettromagnetiche e le stesse molecole dacqua [ 10 ]  . 
in particolare la radiofrequenza utilizzabile solo in lesioni osteolitiche o miste , ma non pu essere usata nelle radiol med ( 2009 ) 114 : 608625 there is a need for greater precision and control during the procedure and for treating osteoblastic lesions . 
it may be performed provided that the cortical bone is intact . procedure procedures were carried out with the patient under deep sedation achieved with the cyclic administration of alfentanil ( 0.51 mg ) and midazolam ( 13 mg ) and continuous infusion of propfol ( 50120 mg )  . 
blood pressure was checked every 4 m local anaesthesia ( 1% carbocaine ) was applied to the skin at the access site . different types of imaging were used to guide the procedure : ultrasound ( iu22 , philips , best , the netherlands ) in four cases , ultrasound and fluoroscopy ( allura xper fd , philips ) in one case , ct ( aquilon 64 , toshiba , tokyo , japan ) in three cases , ct fluorography in one case and carm cone - beam ct in one case ( xper ct , allura , philips ) ( table 2 )  . 
1 ago da rf posizionato allinterno di una lesione iliaca . lesioni osteoblastiche , dato che in questo caso non avviene conduzione di calore ; non pu essere impiegata neanche se la lesione si trova in prossimit di organi come il midollo spinale ( esempio metastasi vertebrale ) per il rischio di lesioni da calore [ 11 ] ; lablazione con microonde indicata se la lesione voluminosa e non in prossimit di organi termosensibili . 
la radiofrequenza plasma - mediata ( coblazione ) consiste nellutilizzo dellenergia elettrica atta a creare un campo di particelle ionizzate che spezzano i legami organici allinterno dei tessuti molli ( ablazione )  . 
infine il trattamento combinato con cementoplastica consiste nelliniezione di cemento preceduto da un qualsiasi trattamento ablativo e si pu effettuare a patto che sia integra la corticale ossea . procedura la procedura stata eseguita in sedazione profonda con somministrazione ciclica di alfentanyl ( 0 , 51 mg ) e mydazolam ( 13 mg ) e infusione continua di propofol ( 50120 mg ) ; stata somministrata una ultra short term profilaxis ( 1 g di cefazolina )  . 
 stata effettuata anestesia locale con 1% di carbocaina nella zona cutanea di introduzione dellago . sono state impiegate differenti tipologie di imaging per la guida : in 4 casi stata impiegata guida ecografica ( iu22 , philips , best , olanda ) , in 1 caso guida ecografica e fluoroscopica ( allura xper fd , philips , best , olanda ) , in 3 casi guida tc ( aquilon 64 , toshiba , tokio , giappone ) , in 1 caso guida fluoro - tc e in 1 caso la c - arm cone - beam tc ( xper ct , allura , philips , best , olanda ) ( tabella 2 )  . 
in tutti i casi trattati con radiofrequenza stato utilizzato un ago da 17 gauge con diametro di apertura degli uncini allestremit di 4 cm ; il generatore di rf , durante il passaggio di energia , rileva limpedenza elettrica del tessuto . 
la procedura stata condotta in accordo ai protocolli forniti dalle 614 radiol med ( 2009 ) 114 : 608625 17 - gauge needle with a maximum hook - deployment diameter of 4 cthe rf generator measures electrical impedance of tissue during the passage of energy . 
plasma - mediated rf procedures ( coblation ) were performed with a cavity spinewand kit ( arthrocare corp , sunnyvale , ca , usa )  . the device was directed anteriorly through the tissue to be ablated . 
in the cases undergoing combined treatment with cementoplasty ( fig . 5a , b ) , the rf electrode was introduced through an 11 - gauge simko needle ( simko special vertebroplasty needle , optimed )  . 
after withdrawal of the rf electrode , the simko needle was connected to a system ( cement - king application system , optimed ) for the injection of cement ( osteopal v , biomet , merck )  . 
per la procedura di radiofrequenza plasma - mediata ( coblazione ) , stato usato un kit cavity spinewand ( arthrocare corp , sunnyvale , ca , usa ) ; il dispositivo stato diretto anteriormente attarverso il tessuto da ablare . 
 la fattibilit della procedura combinata con cementoplastica stata sancita dalla possibilit di effettuare un corretto posizionamento dellago allinterno della lesione , mentre il successo tecnico stato inteso come la possibilit sia di effettuare lablazione allinterfaccia tra tessuto molle e osso sia di iniettare cemento nel caso del trattamento combinato . 
2 schema che mostra la modalit di ablazione con movimento rotatorio del dispositivo di radiofrequenza plasma - mediata allinterno della lesione in direzione dei diversi orientamenti orari a ore 3 , 6 , 9 e 12 . radiol med ( 2009 ) 114 : 608625 fig . 
evidente la presenza di cemento nella lesione ablata ( freccia )  . the possibility of achieving correct needle placement inside the lesion , whereas technical success was defined as the ability to achieve ablation at the interface between soft tissue and bone and then to inject cement in the case of combined treatments . 
 risultati il successo tecnico , inteso come la possibilit di effettuare lablazione allinterfaccia tra tessuto molle e osso e di iniettare cemento nel caso del trattamento combinato , stato raggiunto nel 100% dei casi . 
la termoablazione con rf ha raggiunto in tutti i casi dimensioni di necrosi di circa 4 cm , mentre la termoablazione con microonde ha creato unarea di necrosi superiore ai 6 cm in 10 minuti . 
il tempo totale delle procedure ( intese come la sola ablazione o lablazione associata a cementoplastica ) variava da un massimo di 25 minuti a un minimo di 15 minuti . 
solamente in un paziente trattato con radiofrequenza ( 1 / 9 , 11% ) abbiamo riscontrato nei 6 giorni successivi al trattamento insorgenza di febbricola e malessere generale compatibile con la sindrome post - radiofrequenza , risoltisi poi al settimo giorno e trattati solo con acetaminofene ( paracetamolo ) [ 12 ]  . riguardo alla valutazione con scala vas , la risposta di tutti i pazienti nella valutazione del dolore prima della procedura era di 8 punti ( in una scala da 0 a 10 )  . 
attraverso una valutazione a 1 , 4 , 8 e 12 settimane , emerso che il valore medio nella scala vas stato rispettivamente di 4 , 1 la prima settimana e 2 nei radiol med ( 2009 ) 114 : 608625 table 4 results of the follow - up in terms of reduction of pain and morphine - equivalent dose . 
 pain reduction ( vas ) morphine - equivalent dose before 24 h 1 week 4 week 8 week 12 week before 1 week 4 week 8 week 12 week tabella 4 risultati nel follow - up in termini di riduzione del dolore e dose in equivalenti di morfina . 
in due casi ( pazienti n 7 e n 9 ) essa aumentata di 10 equivalenti di morfina tra lottava e la dodicesima settimana dal trattamento ( vedasi testo ) riduzione del dolore ( vas ) dose in equivalenti di morfina prima 24 h sett 1 sett 4 sett 8 sett 12 prima sett 1 sett 4 sett 8 sett 12 complications ( cement leakage )  . 
pain regression was considered statistically significant at a p value of < 0.001. the wilcoxon test for paired data was used to evaluate when this difference was already statistically significant . 
 results technical success , defined as the ability to achieve ablation at the interface between soft tissue and bone and to inject controlli a 4 , 8 e 12 settimane ( tabella 4 )  . 
rf ablation produced an area of necrosis of approximately 4 cm in all cases , whereas microwave ablation produced an area of necrosis > 6 cm in 10 mprocedure times were also shorter with the plasma - mediated rf technique ( 20 min )  . 
total procedure time ( referring to ablation alone or combined ablation and cementoplasty ) ranged regressione lineare logaritmica con p < 0 , 001 statisticamente significativa ; in particolare la differenza tra prima del trattamento e a una settimana dal trattamento significativamente aumentata ( dolore post - procedurale ) , per i motivi precedentemente esposti , mentre vi una significativa diminuzione della dose di morfina tra prima del trattamento e la quarta settimana successiva allo stesso . radiol med ( 2009 ) 114 : 608625 from 25 min to 15 m in no cases did local complications occur either during or after treatment . 
in only one case of rf ablation ( 1 / 9 , 11% ) did the patient develop low - grade fever and general malaise during the 6 days after treatment , compatible with a postradiofrequency syndrome . 
this was treated with acetaminophen only ( paracetamol ) and resolved on day 7 [ 12 ]  . with regard to vas score , all patients had a pain score of 8 ( on a scale from 0 to 10 ) before the procedure . 
after day 1 , the score decreased by 1 point in 9 / 10 patients and by 2 points in the patient treated with microwave ablation . evaluation at 1 , 4 , 8 , and 12 weeks showed that the mean vas score was 4.1 at week 1 , and 2 at 4 , 8 and 12 weeks ( table 4 )  . 
 analgesic dose increased during week 1 ( from 10 to 20 morphine equivalents ) , probably owing to postprocedural pain , but then decreased substantially after 1 month in all patients ( from 50 to 30 morphine equivalents ) ( table 4 )  . 
in two cases ( patients 7 and 9 ) , it increased by ten morphine equivalents between 8 and 12 weeks , most likely because of the appearance of a new bone metastasis at another site from the one previously treated ( table 4 )  . 
in particular , the difference between pretreatment and 1 - week posttreatment increased significantly ( postprocedural pain ) for the reasons described above , whereas there was a significant reduction in the morphineequivalent dose between pretreatment levels and week 4 after treatment . discussion several therapies are used for the palliation of bone metastases , including chemotherapy [ 13 ] , hormonal therapy [ 14 , 15 ] , surgery [ 1618 ] and radiotherapy ( local transcutaneous radiotherapy , hemibody irradiation , radionuclide therapy ) [ 3 , 19 , 20 ]  . 
a number of patients fail to benefit from these conventional treatments , however , and pain relief , when it does occur , is never achieved before 412 weeks [ 3 ]  . 
if , on the one hand , chemotherapy reduces the primary mass and secondary lesions in 20%80% of patients , on the other hand , it leads to drug resistance and recurrence of bone pain . in addition , myelodepression is common due to the toxicity of many drugs used in current treatment protocols . hormonal therapy is only effective in breast and prostate tumours , where it relieves pain from bone metastases in discussione attualmente nel trattamento palliativo delle metastasi ossee vengono utilizzate varie terapie , tra cui la chemioterapia [ 13 ] , la terapia ormonale [ 14 , 15 ] , la chirurgia [ 1618 ] e la radioterapia ( radioterapia transcutanea locale , irradiazione emicorporea , radioisotopoterapia con radionuclidi ) [ 3 , 19 , 20 ]  . 
alcuni pazienti non riescono a trarre benefici da questi trattamenti convenzionali e il sollievo dal dolore , quando si verifica , non avviene mai prima di 412 settimane [ 3 ]  . 
se da un lato la chemioterapia riduce la massa primitiva e le lesioni secondarie nel 20%80% dei pazienti , dallaltro induce farmacoresistenza e ripresa del dolore osseo ; inoltre frequente linsorgenza di mielodepressione data la tossicit di molti farmaci utilizzati negli attuali protocolli terapeutici . 
la terapia ormonale efficace solamente nei tumori della mammella e della prostata , dove tuttavia induce sollievo dal dolore delle metastasi ossee in pi del 70% dei casi [ 14 ]  . 
infine la radioterapia locale gravata nel 60% dei casi da effetti tossici ( nausea , vomito e diarrea ) e la radioisotopoterapia da una parte dimostra efficacia nei soggetti con metastasi ossee multiple , dallaltra non considerata trattamento standard per le metastasi singole . sulla base di queste considerazioni i farmaci analgesici ( oppioidi e fans ) spesso rimangono lunica alternativa nel dolore metastatico e in alcuni casi rimangono lunica alternativa per la palliazione della sintomatologia . 
 noto che gli effetti collaterali , soprattutto nei trattamenti prolungati , sono costipazione , grave limitazione dello stato fisico e mentale , nonch cirrosi e coma epatico . diversi autori hanno studiato varie strategie alternative per il trattamento del dolore da metastasi ossea che comportano lutilizzo di metodiche percutanee imagingguidate con lo scopo di ablare le lesioni focali ossee : etanolo , termoterapia interstiziale laser - indotta , ablazione con radiofrequenza ( rfa ) percutanea e , pi recentemente , crioablazione [ 2 , 4 , 11 ]  . 
la nostra esperienza si riferisce alla risposta di 10 pazienti al trattamento della sintomatologia dolorosa con diverse tecniche di radiologia interventistica che includono tecniche ablative ( termoablazione con rf , termoablazione con radiofrequenza plasma - mediata , termoablazione con microonde ) associate o meno alla 620 radiol med ( 2009 ) 114 : 608625 > 70% of cases [ 14 ]  . 
finally , local radiotherapy produces toxic effects ( nausea , vomiting and diarrhoea ) in 60% of cases , and radionuclide therapy is effective in patients with multiple bone metastases but is not considered standard treatment for a single metastasis . on the basis of these considerations , analgesics ( opioids and nsaids ) are often the only option for metastatic pain , and in some cases for palliation of symptoms . 
the world health organization ( who ) [ 21 ] recommends a three - step approach : ( 1 ) initial treatment with nsaids such as aspirin , ibuprofen , naproxen , ( 2 ) if pain persists , use of mild opioids such as codeine and hydrocodone , ( 3 ) for persisting moderate or severe pain , high doses of opioids such as morphine , hydromorphone , or fentanyl as an intermittent or continuous infusion . 
known side - effects , above all in long - term treatments , are constipation , severe physical and mental impairment , cirrhosis and hepatic coma . several authors have investigated alternative approaches to painful bone metastases , all of which involve using percutaneous image - guided methods to ablate focal bone lesions : ethanol , laser interstitial thermal therapy , percutaneous rf ablation and , more recently , cryoablation [ 2 , 4 , 11 ]  . 
this study reports on the response of ten patients treated with different interventional radiology techniques , namely , ablation techniques ( rf ablation , plasma - mediated rf ablation , microwave ablation ) used alone or in combination with cementoplasty . 
in agreement with the literature [ 11 ] , we treated patients with up to two bone metastases ( lytic or mixed , but not osteoblastic ) causing acute disabling pain . pain was poorly controlled with conventional treatments . ablation techniques are successful in relieving pain only if the needle is placed at the interface between soft tissue and bone . 
the advantage of rf ablation lies in the use of electrodes that have a smaller diameter compared with the microwave antennas , an aspect that should theoretically reduce complications related to electrode deployment . 
microwave treatment has the advantage of shorter procedure times and the ability to ablate larger lesions than those usually treated with rf , thanks to the possibility of placing two antennas simultaneously indeed , in one of our patients , its use was cementoplastica . 
in accordo con le indicazioni della letteratura [ 11 ] , sono stati trattati pazienti che non avessero pi di due metastasi ossee , che queste fossero litiche o miste , ma non blastiche , e che , soprattutto , fossero causa di dolore acuto e invalidante . 
in questi pazienti , il followup non si ferma solo al dato di diagnostica per immagini , ma si concentra sulla efficacia clinica del trattamento , ossia sulla riduzione del dolore . in questo studio sono state trattate tre lesioni solo con radiofrequenza e una solo con microonde . 
gli svantaggi invece rispetto allimpiego di microonde sono dati da tempi lunghi di procedura ( circa 30 minuti ) e dalla necessit di effettuare pi posizionamenti in caso di ampio volume da ablare . 
il trattamento con microonde ha invece il vantaggio di giovare di una procedura pi breve e di poter ablare lesioni di dimensioni maggiori rispetto a quelle usualmente trattate con radiofrequenza , ci per la possibilit di posizionare due antenne contemporaneamente . 
infatti , in una nostra paziente , lampia superficie di contatto softtissue - osso ha giustificato il suo utilizzo : lablazione ha garantito in un tempo inferiore rispetto alla rf un volume di necrosi superiore . 
inoltre , dal punto di vista della qualit dellablazione , studi sperimentali e clinici [ 10 , 22 , 23 ] hanno evidenziato che il trattamento con microonde garantisce il raggiungimento di temperature intra - lesionali pi elevate , una minore dispersione di calore e quindi unefficacia superiore rispetto alla termoablazione con rf nelle lesioni in prossimit dei vasi sanguigni . 
infine con la metodica delle microonde la diminuzione del dolore stata pi sensibile ( 2 punti bpi ) dopo 24 ore dal trattamento rispetto alle altre metodiche utilizzate ( 1 punto bpi ) , per poi allinearsi con i dati ottenuti dalle altre tecniche nelle fasi successive . 
 le metodiche di termoablazione con radiofrequenza e con microonde hanno comunque linconveniente di non poter essere utilizzate nelle metastasi osteoddensanti , dato che il tessuto osseo non favorisce la conduzione di calore ( radiofrequenza ) n lagitazione di molecole dacqua ( microonde )  . nelle metastasi osteoddensanti possibile utilizzare la crioablazione che ha per lo svantaggio di impiegare aghi di grosse dimensioni e di avere tempi lunghi di procedura [ 2 ]  . inoltre sia la radiofrequenza che le microonde possono provocare lesioni termiche ad organi adiacenti . 
per ovviare radiol med ( 2009 ) 114 : 608625 justified by the extensive contact area between soft tissue and bone , and the ablation procedure produced a larger necrosis volume in a shorter time compared with rf . 
in addition , from the viewpoint of the quality of ablation , experimental and clinical studies [ 10 , 22 , 23 ] have shown that microwave treatment provides higher intralesional temperatures and less heat dispersion and consequently better effectiveness compared with rf ablation in lesions located in proximity to blood vessels . 
finally , microwave ablation provided greater pain relief [ 2 points on the brief pain inventory ( bpi ) ] than did the other methods ( 1 point bpi ) 24 h after treatment before becoming aligned with the other methods at subsequent follow - up assessments . 
 rf and microwave ablation methods both suffer the same limitation of not being applicable to osteoblastic lesions , as bone tissue does not allow the conduction of heat ( radiofrequency ) or the agitation of water molecules ( microwaves )  . 
to avoid this complication , several possibilities exist : to create an air ( in microwave ) interface or water ( in rf ablation ) interface to limit the ablation area ; to place thermocouples close to the structures at risk of thermal injury , as these can guide the operator in continuing or discontinuing the procedure by providing feedback on temperature ; to use bipolar rf , which through an electrode thermally shielded by another opposing electrode ensures well - delimited ablations [ 24 ] ; to use plasma - mediated rf ablation [ 25 ]  . a distinctive feature of plasma - mediated rf ablation ( coblation ) is the ability to release , in an extremely precise manner , a minimal amount of heat ( unlike conventional electrosurgery ) , a crucial factor in treating vertebral lesions . the low rf energy ( 100 khz ) is thus used to excite the electrolytes in a conductive medium ( plasma ) , such as saline solution or the aqueous intracellular environment , creating an area of highly ionised particles immediately adjacent to the electrode tip . 
this molecular dissociation process converts the ablated tissue into gas , creating a cavity at the treatment site [ 25 ] , which accounts for its suitability for osteolytic lesions . coblation in this study was used for a metastasis to a rib in a patient who had undergone right liver resection for hepatocarcinoma and had shown , on preprocedural ct , a bowel loop adjacent to the inner aspect of the affected rib . coblation was used to avoid damaging the bowel wall , and indeed , it allowed for extreme precision in delimiting the ablation margins . with regard to our results , in all cases pain , reduction a tale complicanza , si hanno diverse possibilit : creare uninterfaccia o con aria ( nelle microonde ) o con acqua ( nellablazione con rf ) per limitare il territorio di ablazione . 
possibile inoltre posizionare termocoppie a ridosso delle strutture a rischio di danno termico , che misurando la temperatura durante la procedura , sono utili alloperatore nel decidere se interrompere o meno lablazione ; utilizzare la radiofrequenza bipolare , che attraverso un elettrodo schermato termicamente da un secondo elettrodo opposto al primo , garantisce ablazioni ben definite [ 24 ] ; utilizzare la radiofrequenza plasma - mediata [ 25 ]  . nella tecnica di termoablazione con radiofrequenza plasma - mediata ( coblazione ) caratteristica identificativa la capacit di cedere , in maniera molto precisa , una quantit minima di calore ( contrariamente allelettrochirurgia convenzionale ) , fattore importante per esempio nel trattamento di lesioni vertebrali . 
la bassa energia di radiofrequenza ( 100 khz ) viene quindi utilizza per eccitare gli elettroliti in un mezzo conduttivo ( plasma ) , come una soluzione fisiologica o lambiente acquoso intracellulare , in modo da formare unarea di particelle altamente ionizzate nellarea immediatamente adiacente alla punta del dispositivo . 
questo processo di dissociazione molecolare converte il tessuto ablato in gas e in questo modo si viene a creare una cavit nella zona del trattamento [ 25 ] , trovando dunque particolare indicazione nelle lesioni osteolitiche . la coblazione stata utilizzata in questo studio per la lesione costale di un paziente che , in seguito ad epatectomia destra per exeresi di una neoplasia epatica , evidenziava allesame tc , eseguito prima del trattamento , unansa intestinale adiacente alla faccia interna della costa interessata dalla lesione . 
per evitare di danneggiare la parete dellansa , stata impiegata la coblazione che , in effetti , ha dimostrato notevole precisione nella definizione dei limiti dellablazione e ne ha quindi giustificato limpiego . in merito ai risultati , in tutti i casi abbiamo ottenuto un decremento del dolore tale da dimezzare , allottava settimana dal trattamento , la dose di equivalenti di morfina necessari per controllare il dolore . 
qualunque sia la tecnica ablativa utilizzata , il meccanismo di riduzione del dolore non ancora chiaro , ma vi sono alcune ipotesi : distruzione delle fibre sensoriali del periostio e della corticale ; decompressione meccanica del volume tumorale ; distruzione delle cellule tumorali che producono citochine come tnf ( fattore di necrosi tumorale alfa ) e interleuchine che promuovono la trasmissione del dolore ; inibizione dellattivit degli osteoclasti [ 26 , 27 ]  . 
regardless of the ablation technique used , the mechanism by which pain is reduced is not understood , although several hypotheses have been proposed : destruction of the periosteal and cortical sensory nerve fibres , mechanical decompression of the tumour volume , destruction of tumours cells that produce cytokines such as tumour necrosis factor alfa ( tnf - ) and interleukins that promote pain transmission , and inhibition of osteoclast activity [ 26 , 27 ]  . 
 [ 28 ] , in a study of 12 patients , demonstrated that rf is a safe and effective treatment that improves quality of life and reduces analgesic use in patients with bone metastases . 
 [ 29 ] reported major complications in three patients : one second - degree burn at the grounding - pad site , one case of transient bladder and bowel incontinence and one case of fracture of the acetabulum 6 weeks after rf treatment of an acetabular lesion . 
studies of coblation on bone lesions [ 25 ] investigated the use of rf ablation to treat vertebral lesions only ( for which no complications were reported ) , so we are unable to compare the results with our experience , as we used rf treatment in a costal lesion . 
 [ 9 ] presented the first experimental and clinical results of the use of microwave ablation in bone metastases , reporting excellent technical results in terms of volume of necrosis , speed of the procedure and absence of complications . 
 [ 29 ] , there may be a new rise in morphine - dose requirements long after treatment , indicative of the development of pain at other sites of metastases . 
 [ 28 ] , studiando 12 pazienti , hanno dimostrato la sicurezza e lefficacia della rf per la riduzione del dolore , il miglioramento della qualit della vita e la riduzione delluso di analgesici nei pazienti con metastasi che coinvolgevano losso . 
 [ 29 ] che ha considerato 43 pazienti , stato segnalato un incremento della dose di oppiodi al primo giorno dal trattamento e un successivo decremento di pi del 50% . sulla base di unattenta selezione dei pazienti , nella nostra casistica non si sono verificate complicanze probabilmente anche per lesiguo numero di pazienti ( 6 ) trattati con rf . 
 [ 29 ] si sono invece verificate complicanze maggiori in 3 pazienti : uno ha subito unustione di secondo grado nellarea cutanea della messa a terra , un altro ha accusato incontinenza vescicale e intestinale e infine in un paziente si verificata una frattura acetabolare della zona trattata a 6 settimane dal trattamento con rf . 
gli studi effettuati con la coblazione su lesioni ossee [ 25 ] hanno preso in esame solo il trattamento di lesioni vertebrali ( per le quali per altro non sono riportate complicanze ) e quindi non possibile fare un confronto dato che nel nostro studio la metodica stata utilizzata in una lesione costale . 
 [ 9 ] hanno redatto i primi studi sperimentali e clinici sullutilizzo della termoablazione con microonde nelle metastasi ossee , riportando un ottimo risultato tecnico sia in termini di volume di necrosi , sia di rapidit della procedura , sia di assenza complicanze . 
 [ 29 ] , possibile che vi sia , a distanza dalla procedura , un nuovo aumento della dose di morfina , compatibile con linsorgenza di nuove localizzazioni ossee in siti diversi da quello trattato . 
 [ 6 ] , pazienti sottoposti a rfa associata a cementoplastica per voluminose metastasi ossee coinvolgenti anche la regione extra - ossea hanno ottenuto una riduzione della dose di analgesici solo nel 41% dei casi ; le voluminose dimensioni delle lesioni trattate giustificano il divario tra questo risultato e quello ottenuto dallo studio di callstrom et al . 
 [ 28 ] ( trattamento solo con rf )  . nel nostro studio sono state trattate con cementoplastica le lesioni che , in relazione alla sede , necessitavano di una stabilizzazione . 
in all cases , in agreement with the literature , the results achieved were excellent , not only as concerns stabilisation and thus prevention of pathological fractures , but also as concerns pain relief , achieved in 100% of cases . 
 [ 6 ] emphasised that the technique is easy to perform , inexpensive and minimally invasive , even though the effects of combining rf ablation and cementoplasty with radiotherapy are not well known . 
 [ 34 ] believe that the coagulation necrosis induced by rf facilitates even cement distribution within the ablated lesion and that cementoplasty enhances the analgesic effect by stabilising the bone . 
 the results obtained have been excellent , with pain relief being achieved in 100% of cases , both in case reports [ 30 , 31 , 34 ] and in larger series [ 6 , 28 , 32 , 33 ] , as well as in our small patient series . 
it should be emphasised that , whereas studies performed with rf or cementoplasty alone achieved 100% technical success and 80%100% pain relief [ 35 , 36 ] , combined rf and cementoplasty ensured pain relief in all cases but a slightly lower rate of technical success . conclusions there is no agreement as to the best treatment for bone metastases . 
it is clear that the rationale for the use of these treatments is that , when indicated , they ensure considerably faster pain relief compared with conventional treatments , providing a substantial improvement in quality of life in a short time . 
however , further investigations on larger patient populations , as well as randomised controlled studies , are required to validate the effectiveness of these treatments . con cementoplastica dopo rfa e 2 con vertebroplastica dopo radiofrequenza plasma - mediata . 
in tutti i casi , in accordo con i dati della letteratura , sono stati ottenuti ottimi risultati non solo nellambito della stabilizzazione e quindi nella prevenzione della possibile frattura patologica , ma soprattutto in quello della riduzione della sintomatologia dolorosa , che si avuta nel 100% dei casi . 
 [ 34 ] ritengono che la necrosi coagulativa indotta dalla rf faciliti lomogenea distribuzione del cemento allinterno della lesione ablata e che la cementoplastica , per effetto della stabilizzazione ossea , migliori leffetto antalgico . 
 i risultati ottenuti sono stati ottimi , sia nei case report [ 30 , 31 , 34 ] che nelle casistiche [ 6 , 28 , 32 , 33 ] dove la riduzione del dolore si verificata nel 100% dei casi analogamente a quanto emerso nellesperienza personale , peraltro numericamente esigua . 
va sottolineato che , se da un lato negli studi effettuati solo con radiofrequenza o solo con cementoplastica si ottenuto un successo tecnico del 100% a fronte di un risultato in termini di riduzione del dolore compreso tra l80% e il 100% [ 35 , 36 ] , dallaltro la tecnica combinata di radiofrequenza e cementoplastica ha assicurato la riduzione del dolore in tutti i casi trattati , ma un successo tecnico in una percentuale leggermente inferiore . conclusioni attualmente non esistono ancora pareri concordi sulla metodica migliore nel trattamento delle metastasi ossee . 
appare evidente che ci che giustifica il ricorso a queste terapie il fatto che , laddove vi sia lindicazione , esse garantiscono una riduzione della sintomatologia dolorosa notevolmente pi rapida rispetto alle terapie convenzionali apportando , in breve tempo , un sensibile miglioramento della qualit della vita . 
volta 171 , 50133 firenze , italy 2dipartimento di fisica e tecnologie relative , universit di palermo , palermo , italy 3istituto di radiologia , policlinico , palermo , italy 4medicad s.r.l. , palermo , italy correspondence to : s . 
il set contiene 31 mammografie di screening refertate come negative , precedenti la comparsa di carcinomi di intervallo classificati alla revisione come errori di screening o minimal sign , e 89 controlli negativi verificati , scelti a random dalla stessa casistica di screening . 
rispetto ai due sistemi cad a confronto cyclopus risulta pi sensibile ( r2 p = 0 , 14 ; cadx p = 0 , 02 ) , e meno specifico ( r2 p = 0 , 02 ; cadx p = 0 , 64 )  . parole chiave carcinoma mammario diagnosi computer assistita mammografia introduction introduzione false negatives on mammography screening may be due to misperception of radiological abnormalities , and perception could be possibly improved by computer analysis of digitised images identifying mammography sites for review . computer - assisted diagnosis ( cad ) could be particularly useful in screening where the mammograms to be read are many and the abnormalities to be perceived are few , thus easily causing loss of attention or fatigue . 
cad has been the object of several studies demonstrating that it allows a moderate increase in specificity associated with an accettable decrease in specificity [ 17 ]  . different cad systems have been developed , but few have been tested in comparison . 
no doubt , radiologists are mostly interested in the impact of cad on final diagnosis , and studies of cad efficacy are mainly based on comparison of a single reading with or without cad . 
nevertheless the impact of cad depends on its absolute accuracy , that is , the power of marking cancer areas while keeping the rate of marked benign areas as low as possible . 
the aim of this study was to assess the systems diagnostic accuracy by testing it on a training set and to compare its performance with that of two other commercial systems , namely , cadx ( former cadx , now commercialised by icad , inc . , nashua , nh , usa ) and r2 ( r2 technology , inc . , santa clara , ca , usa ) already tested on the same set [ 8 ]  . materials and methods cyclopus was tested on a mammography set developed at i falsi negativi dello screening mammografico possono essere dovuti alla mancata percezione di anormalit radiologiche e la percezione potrebbe essere migliorata con lanalisi computerizzata di immagini mammografiche digitali o digitalizzate , capaci di selezionare aree meritevoli di revisione . 
la diagnosi aiutata dallanalisi computerizzata ( cad ) stata oggetto di numerosi studi che dimostrano come il cad consenta un moderato aumento della sensibilit a fronte di unaccettabile riduzione della specificit [ 17 ]  . 
luso del cad , pertanto , potrebbe essere particolarmente utile nello screening , dove il numero di mammografie da interpretare molto elevato , le anormalit da percepire sono poche e la caduta di attenzione o laffaticamento del lettore pi facile . diversi sistemi cad sono stati sviluppati , ma pochi sono stati testati a confronto . 
indubbiamente quello che interessa al radiologo leffetto di cad sulla diagnosi finale , e per questo gli studi dellefficacia di cad in genere si basano su un confronto tra la lettura singola e quella aiutata da cad . 
peraltro limpatto di cad sulla diagnosi finale dipende dalla sua accuratezza assoluta , dalla capacit cio di segnalare alterazioni mammografiche dovute a cancro e di mantenere al minimo la segnalazione di alterazioni che poi si rivelano non neoplastiche . 
scopo del presente studio stato quello di testare laccuratezza diagnostica di cyclopus applicandolo a una casistica campione , e di confrontare la sua prestazione con due altri sistemi commerciali : cadx ( ora commercializzato da icad , inc . , nashua , nh , usa ) e r2 ( r2 technology , inc . , santa clara , ca , usa ) , gi testati sulla stessa casistica [ 8 ]  . 628 radiol med ( 2009 ) 114 : 626635 the scientific institute for cancer prevention ( ispo ) and used in the past to evaluate the accuracy of other cad systems [ 8 ]  . 
the set consists of 120 one - view ( mediolateral oblique , n = 82 ) or two - view ( mediolateral oblique + craniocaudal , n = 38 ) conventional mammograms and includes previous screening mammograms ( originally reported as negative ) preceding 31 interval cancers ( ic ) and 89 negative controls ( randomly selected from the same screening database and assumed to be negative on the basis of at least one further negative biennial screen )  . 
ic are periodically identified by linkage of florence screening programme archives with the tuscany cancer registry [ 9 ] and are routinely classified by expert radiologists according to the european guidelines [ 10 ] , namely , occult , minimal sign ( ms ) and screening error ( se )  . 
the 31 ic in the set account for all consecutive ic observed in a 5 - year period in the florence screening programme after excluding occult cases : 20 had been classified as ms and 11 as se . all films in the set were digitised at medicad in a blind fashion ( operators were not aware of which cases were ic or controls ) by means of a ccd digitiser operating at 80 m per pixel , 12 - bit greyscale . 
the general architecture of cyclopus includes several steps : pre - processing : the digital image is processed to delimit the mammographic area to be submitted to further analysis segmentation : the cleaned image is segmented to select regions of interest ( roi ) mapping out its contour features extraction : characteristic information extracted from each roi classification : a probability of pathology is assigned to each roi . in cyclopus , in particular , image segmentation is based on the region - growing method in a process of multilevel analysis . 
the region - growing method consists of determining a set of points or initial regions ( seeds ) , which are expanded by incorporating the neighbouring pixels having met a test of similarity with the region . 
for the classification step , artificial neural networks ( ann ) are used , organised on an architecture of neurons and connections . the number of input neurons corresponds to the number of features extracted . 
the ann operation involves a learning materiali e metodi cyclopus stato testato su un set di mammografie sviluppato allistituto scientifico per la prevenzione oncologica ( ispo ) di firenze e in precedenza usato nella valutazione di accuratezza di altri due sistemi cad . 
il set utilizzato per testare il sistema consta di 120 mammografie convenzionali in proiezione singola ( mediolaterale obliqua n = 82 ) o doppia ( mediolaterale obliqua + craniocaudale n = 38 ) , e comprende le mammografie refertate come negative precedenti a 31 casi di carcinoma di intervallo ( ci ) e 89 controlli negativi ( tratti in maniera random dalla stessa casistica di screening , e verificati tali in base ad almeno un controllo successivo biennale negativo )  . 
i 31 casi di ci nel set in studio costituiscono una casistica consecutiva relativa circa 5 anni di attivit del programma di screening di firenze , dopo esclusione dei i casi occulti : 20 erano stati classificati come sm e 11 come es . 
 tutte le mammografie del set sono state digitalizzate da medicad con metodica cieca ( gli operatori non erano al corrente di quali casi fossero ci o controlli ) mediante un digitalizzatore a ccd operante a 80 micron per pixel , 12 bit di scala di grigi . 
il metodo utilizzato da cyclopus comprende varie fasi : pre - processing : inizialmente limmagine viene preprocessata al fine di delimitare la mammella ; segmentazione : la zona delimitata in precedenza viene scomposta in sub - regioni otticamente omogenee e fra queste vengono selezionate quelle interessanti , potenziali sedi di lesioni , region of interest ( roi ) ; estrazione di caratteristiche : ad ogni roi vengono associate alcune grandezze analitiche , che ne descrivono le propriet ; classificazione : ogni roi viene classificata in base al grado di similitudine con patologie tumorali analoghe accertate . in particolare , in cyclopus , la segmentazione delle immagini basata sul metodo di region growing allinterno di un processo di analisi di tipo multi - livello . tale metodo consiste nel determinare un set di punti o regioni iniziali ( seeds ) i quali sono poi espansi incorporando in essi i pixel confinanti che soddisfano ad un test di similarit con la regione . 
per la classificazione delle roi sono utilizzate reti neurali radiol med ( 2009 ) 114 : 626635 phase consisting of iterative training in order to establish the connection parameters between neurons . the system allows a choice among four different working presets ( as to sensitivity / specificity ) , namely , very high sensitivity , high sensitivity , high specificity , and very high specificity . 
on that basis , we assessed the absolute accuracy ( sensitivity , specificity , positive predictive value , number of markings per view among negative controls ) of cyclopus . comparison of performance with the two other commercial systems was performed on the basis of data published in a previous report [ 8 ]  . 
statistical significance of observed differences was calculated with the mcnemar test for paired data and set at p < 0.05. results table 1 shows the results of the case - based diagnostic accuracy of cyclopus . 
data are summarised in table 2 . table 3 shows the diagnostic performance of cyclopus compared with cadx and r2 , as determined in 2004 on the same test set [ 8 ]  . 
il funzionamento di tali reti presuppone una fase di apprendimento , costituita da un addestramento iterativo per la determinazione dei parametri interni di connessione fra i neuroni . il sistema permette di scegliere il punto di lavoro ( in termini di sensibilit / specificit ) tra i quattro possibili : sensibilit molto elevata , sensibilit elevata , specificit elevata , specificit molto elevata , identificati utilizzando un database di propriet della medicad . 
nel presente articolo vengono riportati in dettaglio i risultati ottenuti con il punto sensibilit elevata che risultato quello maggiormente proponibile per luso clinico ; i risultati ottenibili con gli altri punti di lavoro sono riportati in modo riassuntivo . le immagini digitalizzate sono state processate mediante computer , usando applicazioni iterative di cyclopus , al fine di identificare aree di interesse meritevoli di una revisione . i risultati dellanalisi del sistema , in termini di mammografie con marcatura delle roi , sono stati riportati su carta ( calcificazioni e opacit di massa sono contrassegnate con simboli diversi )  . 
 le stampe sono state confrontate con le mammografie originali da uno di noi ( sc ) , per definire quali roi corrispondessero a ci ( veri positivi ) o a aree di benignit ( falsi positivi )  . 
su tale base abbiamo definito laccuratezza diagnostica assoluta di cyclopus ( sensibilit , specificit , valore predittivo positivo , numero di roi per proiezione tra i controlli negativi )  . 
la significativit statistica ( posta a p < 0 , 05 ) delle differenze osservate tra i diversi sistemi a confronto stata calcolata in base al test di mcnemar per dati appaiati . risultati la tabella 1 mostra i risultati di accuratezza diagnostica di cyclopus , basata sui casi , non sulle singole proiezioni . 
la sensibilit stata del 74 , 2% , ed risultata pi elevata per i ci classificati come es ( 90 , 9% ) che per quelli classificati come sm ( 65 , 0% )  . 
i dati sono riassunti nella tabella 2 . la tabella 3 mostra le prestazioni diagnostiche di cyclopus rispetto a r2 e cadx , le prestazioni di questi due 630 radiol med ( 2009 ) 114 : 626635 microcalcifications ( 90 markings for masses , 213 for microcalcifications )  . 
cyclopus had a higher overall sensitivity compared with r2 ( p = 0.14 ) or cadx ( p = 0.02 ) , although the difference with r2 was not significant owing to the limited sample size . 
cyclopus mostra una maggiore sensibilit per le opacit di massa rispetto alle calcificazioni ( 208 roi rispetto a 62 , tra i controlli negativi ) , in maniera pi marcata di cadx ( 192 vs 130 ) , mentre r2 privilegia lidentificazione delle microcalcificazioni ( 90 roi per masse , 213 per calcificazioni )  . 
cyclopus mostra una sensibilit globale superiore a r2 ( p = 0 , 14 ) o cadx ( p = 0 , 02 ) , anche se la differenza con r2 non statisticamente significativa per il limitato campione in esame . 
sample size is no doubt limited compared with screening practice , the prevalence of cancers being substantially higher ( 20% ) compared with the average frequency of ic ( 0.8 [ 10 ] )  . 
such a condition is likely to influence the reader to adopt a lower threshold for suspicion ( aimed at maximising sensitivity ) and is a common bias of studies investigating the impact of cad but is necessary to obtain a set size compatible with a feasible review process . moreover , this study tested the algorithm , which cannot be emotionally influenced by the prevalence of positive cases in the set . 
this applies particularly to controls and to differences in specificity , which nevertheless should not be of major concern , as by definition , cad has a very low specificity that requires the reader to apply a major discount filter to an roi excess . the number of ic in the study set may be apparently low , but this is due to the rarity of ic . 
this study considers all ic reviewed as se or ms observed in a 5 - year period in a screening unit with a workload of 30 , 000 tests per year , among the largest in italy and europe , and thus represents a large realistic sample . 
the proportion of ic reviewed as se or ms is not artificial but real , as this is a consecutive sample , although these values are far from being universal given that attribution to se or ms categories is to some extent subjective [ 12 ]  . 
the choice of excluding ic reviewed as occult from the set was adopted , as the reader is expected to discount the large majority of rois , and this is much more likely to occur with cases that have been reviewed as having no radiological abnormalities . 
cad is unlikely to improve cancer detection rate of ic reviewed as occult . studies of cad impact on conventional reading show an incremental detection rate ranging between 1% and 19 % and a corresponding incremental recall rate ranging between 6% and 35% [ 1222 ]  . 
one particular advantage of cad would be to adopt it as an alternative to double reading , which is recommended by the european guidelines [ 10 ] and causes major radiol med ( 2009 ) 114 : 626635 cyclopus ( 1 , 13 ) , rispetto a r2 ( 0 , 93 ) o cadx ( 0 , 99 )  . la tabella 4 riporta i risultati ottenibili con cyclopus operando con i quattro diversi punti di lavoro . 
la conseguente variazione della sensibilit ( da 45 , 2% a 84 , 9% , p = 0 , 007 ) e delle roi per proiezione ( da 0 , 24 a 1 , 37 , p = 106 ) decisamente importante e significativo . 
le dimensioni del campione in esame sono certamente esigue rispetto alla pratica di screening , con una concentrazione di carcinomi decisamente pi elevata ( 20% ) di quella mediamente riscontrata per i ci ( 0 , 8 [ 10 ] )  . 
tale condizione , che pu certamente influenzare il lettore verso una minore soglia di sospetto ( per aumentare la sensibilit ) , comune alla gran parte degli studi che valutano limpatto di cad sulla lettura umana , per lovvia necessit di mantenere le dimensioni del set entro un margine che renda la valutazione fattibile : peraltro in questo caso ad essere testato il computer , che certamente non influenzato emotivamente dalla frequenza di casi positivi . 
 altres vero che la limitatezza di casi e controlli ha comportato una minore potenza dello studio , che certamente non capace di dimostrare la significativit statistica per differente di modesta entit . 
questo vale soprattutto per la numerosit dei controlli , e quindi per le differenze in specificit , che peraltro non dovrebbero troppo preoccupare dal momento che cad , per definizione , ha una specificit molto modesta , che comunque necessita di un filtro importante da parte del lettore . 
la numerosit dei ci in esame pu sembrare esigua , ma legata alla rarit intrinseca dei ci : in questo caso i ci nel set sono quelli rivalutati come es o sm corrispondenti a circa 5 anni di attivit di una unit di screening di 30000 test annui , tra le pi grandi in italia e in europa , e quindi costituiscono certamente un campione realistico . 
la composizione in es e sm della casistica non artificiale ma reale , in quanto si tratta di un campione di ci consecutivo , anche se non ha un valore universale considerando la soggettivit del revisore nellattribuire queste categorie [ 12 ]  . 
la scelta di escludere dal campione i ci classificati come occulti alla revisione deriva del fatto che , poich nelluso clinico il lettore deve trascurare la gran parte delle roi , molto verosimile che questo avvenga per le regioni che alla revisione sono risultate completamente negative , e che quindi cad non possa aumentare il tasso diagnostico attraverso la identificazione di ci rivalutati come occulti . gli studi sullimpatto di cad in ausilio alla lettura convenzionale mostrano un aumento del tasso diagnostico di carcinoma che varia dal 1% al 19% [ 1222 ] e al radiol med ( 2009 ) 114 : 626635 difficulties due to the lack of trained radiologists , although the existing evidence is not fully encouraging [ 2 ]  . 
such a comparison is not possible on the basis of existing studies of single cad systems because the mammography sets on which algorithms are tested are different in structure and difficulty , thus influencing performance . 
several retrospective studies are biased as cad is tested on screen - detected cancers ( that is , detected without the need for cad ) rather than on ic ( the detection of which is the real goal of cad )  . 
overall , a reliable comparison of different cad systems is still to be done , as very few studies have performed a proper comparison of algorithms on the same training set [ 8 ]  . cad impact on reading diagnostic accuracy is strictly dependent on the absolute accuracy of the systedifferent algorithms could be simply tested on the same training set , comparing them for sensitivity and specificity , independently from the impact of cad marking on the radiologists interpretation . 
a training set made up of negative controls seeded with ic cases , as in this study , is ideal for the purpose . this study evaluates the absolute diagnostic accuracy of cyclopus , a new algorithm , and compares it with two other cad commercial systems , which had been tested on the same training set . 
no doubt , the training set is imperfect , as it is based on screen - film mammograms ( and the future use of cad is likely to be mostly with digital mammography ) and includes a large proportion of singleview mammograms ( single view is suppose to reduce cad performance compared with double view )  . 
however , because the same training set was used to test the three algorithms , its imperfection should equally affect their performance . cyclopus proved to be more sensitive for masses compared with microcalcifications , as was cadx , and this is supposed to be an advantage , at least in theory , as ic are known to be mostly depicted as masses [ 23 ]  . 
setting the high sensitivity working point , cyclopus was substantially more sensitive compared with the two other systems . contempo un aumento del tasso di richiamo che varia dal 6% al 35% [ 1222 ]  . 
un particolare vantaggio offerta dal cad sarebbe quello di impiegarlo in alternativa alla doppia lettura , raccomandata dalle linee guida ue [ 10 ] e fonte di problemi organizzativi per la carenza di radiologi esperti , anche se levidenza finora esistente non del tutto incoraggiante [ 2 ]  . 
certamente la diffusione della mammografia digitale faciliter limpiego del cad , per la possibilit di visionare le marcature cad direttamente sul monitor , decisamente competitiva rispetto alla complessit dellanalisi cad di immagini analogiche . ipotizzando una sempre maggiore diffusione del cad , verosimile che gli acquirenti si interessino , oltre che ai costi , a un confronto tra le performance dei diversi sistemi in commercio . 
il confronto tra diversi algoritmi non pu essere fatto in base ai numerosi studi che impiegano i singoli sistemi cad , perch le casistiche di riferimento , dalla cui composizione e difficolt dipendono le prestazioni del cad , non sono confrontabili . 
molti studi retrospettivi risultano problematici per il fatto che il cad viene testato su carcinomi diagnosticati dallo screening ( per i quali non serviva laiuto di cad ) invece che su carcinomi di intervallo ( che il cad dovrebbe contribuire a ridurre )  . gli studi prospettici di lettura assistita dal cad , impiegando diversi algoritmi , sono pi complessi da realizzare e richiedono tempo per dare risultati validi . 
in sostanza il confronto tra algoritmi sostanzialmente ancora da fare , in quanto sono molto pochi gli studi che confrontano diversi algoritmi sulla stessa casistica campione [ 8 ]  . poich limpatto del cad sullaccuratezza diagnostica deriva in prima istanza dallaccuratezza diagnostica assoluta del sistema , il confronto tra algoritmi potrebbe limitarsi alla analisi di una stessa casistica campione con diversi algoritmi , confrontando la sensibilit e la specificit di questi ultimi a prescindere dallimpatto che le marcature cad possano avere sulla lettura . 
una casistica costruita insemenzando casi normali con carcinomi di intervallo , come in questo studio , costituisce il supporto ideale per tale confronto . il presente studio valuta la performance diagnostica di cyclopus , un nuovo cad commerciale , e la confronta con altri due sistemi commerciali , che in passato erano stati testati sulla stessa casistica . 
indubbiamente la casistica non quella ideale , sia perch analogica ( e luso del cad sar in futuro prevalentemente su sistemi di mammografia digitale ) , sia perch costituita in parte da esami con proiezione singola , che , come noto , pu in qualche modo ridurre la performance del cad . 
il fatto peraltro che la casistica sia la stessa per i tre sistemi suggerisce che i suoi eventuali limiti influenzino in modo paritetico la performance dei sistemi a confronto . cyclopus risulta maggiormente sensibile per la presenza di anomalie caratterizzate da opacit di massa che non da calcificazioni , in analogia a cadx , e questo teoricamente un vantaggio perch noto che la stragrande maggioranza dei ci si associa a opacit di massa e non a calcificazioni 634 radiol med ( 2009 ) 114 : 626635 cyclopus was less specific than the two other systems , as shown by the higher false positive rate and the higher average number of markings per view . 
overall , the balance may look acceptable : the additional cancers marked by cyclopus compared with cadx or r2 were six and ten , and the additional false positive control cases were 12 and two , respectively . 
the aim of our study , however , was to provide a first evaluation of cyclopus , and a comparison with two commercial systems seemed to us appropriate , also considering the difficulty in comparing different cad algorithms ( comparison studies are anecdotal in the literature )  . 
a higher sensitivity ( absolute value ) observed for cyclopus compared with the other two systems is promising , but because of its marginal statistical significance , confirmation by studies with greater power is needed . 
il bilancio pu apparire conveniente : il numero di carcinomi aggiuntivi identificati da cyclopus rispetto a cadx o r2 rispettivamente di 6 e di 10 a fronte di un numero aggiuntivo di controlli falsi positivi rispettivamente di 12 e 2 . 
il tentativo di aumentare la specificit di cyclopus adottando il livello di lavoro specificit elevata consente un notevole miglioramento delle roi per proiezione ( 0 , 48 ) , decisamente migliore di quanto ottenuto con gli altri due sistemi ( r2 = 0 , 93 ; cadx = 0 , 99 ) , ma con una notevole perdita in sensibilit ( 58 , 1% vs 74 , 1% ) , che non riteniamo clinicamente accettabile , anche se la sensibilit risultante paragonabile a quelle degli altri sistemi a confronto ( r2 = 54 , 8% ; cadx = 41 , 9% )  . il campione in esame ha certamente limiti di potenza , ma era in qualche modo obbligato dalla sua disponibilit relativa a uno studio precedente [ 8 ] , effettivamente non disegnato per un puro confronto tra algoritmi , ma per un confronto delleffetto di cad sulla lettura umana . 
scopo dello studio , peraltro , era una prima valutazione del sistema cyclopus , e un confronto con dati gi esistenti per due algoritmi di commercio ci parso opportuno , data la difficolt di poter testare tra loro diversi sistemi ( gli studi tra sistemi sono aneddotici in letteratura )  . 
il confronto tra algoritmi cad dovrebbe avvenire soprattutto in base alla sensibilit , dato che la specificit attesa per definizione molto bassa , e luso clinico di cad prevede che il lettore operi comunque un grossolano sconto sulle roi . 
la maggiore sensibilit ( stima puntuale ) osservata per cyclopus promettente ma con significativit statistica marginale e necessita di conferme con studi di maggiore potenza . auspicabile che altri studi come questo siano condotti in futuro , e che i loro risultati costituiscano una valida base per un giudizio comparativo tra i diversi algoritmi , attualmente pressoch impossibile . conflict of interest statement : gli autori donato cascio , francesco fauci , rosario magro , giuseppe raso e raffaele ienzi sono consulenti tecnico - scientifici e soci della medicad s.r.l. 
twenty - four consecutive patients ( 16 women , eight men ; mean age , 56.5 years ) with a diagnosis of fuo based on routine investigations were retrospectively studied . 
a nal diagnosis was reached in 17 of 24 patients ( vasculitis , n = 5 ; autoimmune disorder , n = 2 ; neoplasm , n = 3 ; infectious disease , n = 6 ; biliary microlithiasis , n = 1 )  . 
if prospective trials on this topic con rm the present ndings , pet / ct should be incorporated in the routine diagnostic work - up of patients with classical fuo . keywords fever of unknown origin pet / ct clinical management diagnosis riassunto obiettivo . 
abbiamo incluso , retrospettivamente , ventiquattro pazienti consecutivi ( 16 femmine e 8 maschi ) di et media 56 , 5 anni , nei quali , dopo gli esami di routine , era stata posta diagnosi di fuo classica . 
in 17 pazienti si raggiunta una diagnosi nale : vasculite ( 5 ) , malattia auto - immune ( 2 ) , neoplasia ( 3 ) , malattia infettiva ( 6 ) , micro - litiasi biliare ( 1 )  . 
se ulteriori studi prospettici su questo argomento confermeranno gli attuali risultati , lesame pet / tc dovrebbe essere incluso nella routine diagnostica di questi pazienti . parole chiave febbre di origine sconosciuta pet / tc gestione clinica diagnosi 810 introduction fever of unknown origin ( fuo ) constitutes a diagnostic challenge owing to both the wide range of possible causes and the frequently inconclusive or misleading ndings of clinical investigations . 
in 1961 , petersdorf and beeson de ned fuo as a fever lasting > 3 weeks with a temperature > 38.3c , measured on several occasions and with no diagnosis after 1 week of inpatient investigation [ 1 ]  . 
nearly 30 years later , in 1992 , petersdorf modi ed the latter criterion into a failure to reach a diagnosis after appropriate examinations in outor inpatients [ 2 ]  . 
in the 1990s , a new four - tiered fuo classi cation was proposed [ 3 ] : ( 1 ) classical fuo ( fuo in nonimmunocompromised patients ) ; ( 2 ) nosocomial fuo ; ( 3 ) fuo in neutropenic patients ; ( 4 ) hiv - associated fuo . causes of classical fuo can be grouped into four major subcategories : infections ; malignancies ; autoimmune noninfectious diseases and miscellaneous [ 4 ]  . 
the healthcare costs related to fuo are very high as a result of the prevalence of the condition and the extent of resources used for its management ( especially in the diagnostic phase )  . 
in addition , 1235% of these patients die of fuo - related problems [ 5 ] , whereas the prognosis is often positive when the cause of the fever is not established after clinical and imaging investigations . 
it is therefore essential to distinguish between patients in whom aggressive treatment is mandatory and patients in whom a wait - and - see approach is more appropriate [ 6 ]  . 
 combined positron emission tomography and computed tomography with [ 18f ] uorodeoxyglucose ( [ 18f ] - fdgpet / ct ) is a noninvasive diagnostic procedure widely used in managing patients with cancer . 
furthermore , glucose and [ 18f ] - fdg are trapped by white blood cells , especially neutrophils and monocytes / macrophages , in response to in ammatory stimuli , as shown by in vitro and in vivo studies [ 7 , 8 ]  . 
several authors have reported the ability of pet to localise periodontal , abdominal or pelvic abscesses , vascular in ammations , tuberculosis and infection of bone , joint , soft tissue and prostheses [ 9 , 10 ]  . 
therefore , [ 18f ] - fdg - pet may represent a highly useful examination not only for identifying the causes of classical fuo radiol med ( 2011 ) 116 : 809820 introduzione la febbre di origine sconosciuta ( fuo ) rappresenta una vera e propria s da in ambito diagnostico sia per lelevato numero dei possibili fattori eziologici coinvolti , sia per i risultati spesso inconcludenti o fuorvianti ottenuti dagli esami clinici eseguiti . 
nel 1961 , petersdorf e beeson de nirono la fuo come febbre di durata maggiore di tre settimane con temperatura superiore ai 38 , 3c misurata in occasioni differenti e senza una diagnosi eziologica dopo una settimana di indagini eseguite in regime di ricovero [ 1 ]  . 
circa 30 anni dopo , nel 1992 , sempre petersdorf ha modi cato lultimo criterio di questa de nizione con : mancata diagnosi dopo appropriati esami in regime di ricovero o ambulatoriale [ 2 ]  . 
negli anni 90 , stata proposta per la fuo una classi cazione su base eziologica con identi cazione di quattro gruppi [ 3 ] : ( 1 ) fuo classica ( fuo in pazienti immunocompetenti ) ; ( 2 ) fuo nosocomiale ; ( 3 ) fuo in pazienti neutropenici ; e ( 4 ) fuo associata ad virus dellimmunodecienza umana ( hiv )  . a loro volta , le cause della fuo classica si possono raggruppare in quattro sottogruppi maggiori : infezioni , neoplasie , malattie autoimmuni e miscellanea [ 4 ]  . 
i costi sanitari legati alla fuo risultano estremamente elevati in considerazione della sua prevalenza e dellentit di risorse impiegate per la sua gestione ( soprattutto nella fase diagnostica )  . 
inoltre , dal 12% al 35% di questi pazienti muoiono per problemi ad essa correlati [ 5 ] , mentre spesso la prognosi risulta positiva se leziologia della fuo non viene identi cata dopo gli esami clinicostrumentali . 
di conseguenza , durante la fase diagnostica risulterebbe estremamente utile poter distinguere i pazienti che necessitano di un trattamento aggressivo da quelli nei quali un approccio wait and see pi appropriato [ 6 ]  . 
 lesame integrato tomogra a ad emissione di positroni / tomogra a computerizzata ( pet / tc ) con ( 18f ) uorodeossiglucosio [ ( 18f ) fdg ] un esame diagnostico non invasivo utilizzato ampiamente nei pazienti oncologici . 
peraltro , glucosio e il ( 18f ) fdg sono utilizzati / intrappolati anche dai leucociti , specialmenradiol med ( 2011 ) 116 : 809820 but also for monitoring treatment response . 
this study was therefore undertaken to compare our results with those reported in the literature , which was extensively analysed and discussed , with a view to de ning the utility of pet / ct in patients with classical fuo . 
fuo was de ned as fever ( > 38c ) for more than 3 weeks without a nal diagnosis after chest x - ray or ct scan , abdominal ultrasonography ( us ) or ct , routine blood chemistry , urinalysis and in - depth examination of any abnormalities detected . 
 pet / ct study pet / ct studies were acquired using the same combined pet / ct scanner ( discovery st , general electric medical system , waukesha , wi , usa )  . 
 the acquisition protocol started with a scout view ( a bidimensional ct projection ) that was used to de ne the body axial extension ( beginning and end position ) over which ct and pet data would be acquired . 
once the scan range was de ned , ct was performed ( voltage , 140 kv ; tube current , 60 mas ) from the proximal femur to the base of the skull . 
pet data on tracer distribute i neutro li e i monociti / macrofagi , quando questi vengono attivati da stimoli ogistici , come stato dimostrato da studi in vitro ed in vivo [ 7 , 8 ]  . 
diversi autori hanno mostrato la capacit dellesame pet di localizzare ascessi periodontali , addominali o pelvici , in ammazioni vascolari , lesioni tubercolari nonch infezioni di protesi , tessuti molli , ossa ed articolazioni [ 9 , 10 ]  . 
queste considerazioni suggeriscono che lesame pet con ( 18f ) fdg possa rappresentare uno strumento estremamente adatto per identi care e chiarire le cause eziologiche della fuo classica ; inoltre lo stesso esame potrebbe essere utilizzato anche per monitorare la risposta al trattamento . 
in questi ultimi anni , il ruolo dellesame pet nei pazienti con fuo classica stato largamente indagato ; tuttavia le conclusioni dei lavori pubblicati non sempre sono concordanti ed dif cile avere un quadro chiaro [ 1114 ]  . 
per questo motivo lo scopo del nostro studio stato prima di tutto di confrontare i nostri dati con quelli riportati in letteratura ( che stata ampiamente analizzata e discussa ) , dopo di che cercare di de nire il ruolo della pet / ct nei pazienti con fuo classica . materiali e metodi selezione dei pazienti in questo studio retrospettivo abbiamo incluso 24 pazienti consecutivi ( 16 femmine e 8 maschi ; et media 56 , 5 anni , deviazione standard 19 , 1 anni ) inviati al nostro centro pet con diagnosi di fuo classica tra marzo 2007 e luglio 2009 ( tabella 1 )  . 
la de nizione di fuo presa in considerazione prevede febbre ( > 38c ) per pi di tre settimane senza una diagnosi nale dopo radiogra a o tc del torace , ecogra a o tc addominale , emocromo , esame delle urine , ed approfondimento delle anomalie riscontrate . 
il follow - up stato realizzato per tutti i pazienti contattando i medici curanti ed stato ottenuto per tutti ; il periodo minimo di follow - up stato di 6 mesi . esame pet / tc lo studio pet stato acquisito con un tomografo pet / tc discovery st ( general electric medical system , waukesha , wisconsin , usa )  . 
i dati emissivi della pet sono stati acquisiti 60 812 table 1 patient characteristics radiol med ( 2011 ) 116 : 809820 sex diagnosis final diagnosis [ 18f ] - fdg - pet / ct pt . 
pet was considered true positive if the nal diagnosis was consistent with the pet ndings and false positive if the pet ndings were not con rmed as the cause of fuo on further investigations . 
a nal diagnosis was reached in 17 cases ( vasculitis , n = 5 ; autoimmune disorder , n = 2 ; neoplasm , n = 3 ; infectious disease , n = 6 ; biliary microlithiasis , n = 1 )  . 
in four cases with [ 18f ] - fdg uptake in lymph node stations and / or the oropharyngeal region , pet scan was interpreted as suggestive of infection and , after further clinical studies , the patients were treated with antimycotics ( n = 1 ) or antibiotics ( n = 3 ) ; all underwent fuo remission after treatment . 
in two patients , an autoimmune disease was diagnosed ; in one patient , the nal diagnosis was haemophagocytosis ( and the fever persisted after 1 year of follow - up )  . 
one patient had biliary microlithiasis that was held responsible for fuo ( in this patient , focal uptake of [ 18f ] - fdg was seen around the biliary prosthesis , but it was considered reactive to the prosthesis itself )  . 
il protocollo di acquisizione inizia con una scout view ( proiezione tc bidimensionale del paziente ) , utilizzata per de nire lestensione assiale del corpo ( posizione di inizio e ne ) sulla quale acquisire i dati tc e pet . 
una volta de nito lintervallo di scansione viene acquisito lesame tc , dalla base cranica alla porzione prossimale del femore ( voltaggio di 140 kv e intensit di corrente di 60 mas )  . 
i dati emissivi della distribuzione del tracciante sono stati acquisiti in modalit 3d ( 89 eld of view [ fov ] di 3 , 5 minuti ciascuno ) , dalla porzione prossimale del femore alla base cranica . 
lesame pet stato considerato : vero positivo se la diagnosi nale era in accordo con lesito pet ; falso positivo se il reperto identi cato come patologico dalla pet non stato confermato come causa della fuo nelle indagini diagnostiche successive . 
2 [ 18f ] uorodeoxyglucose positron emission tomography computed tomography ( [ 18f ] - fdg - pet / ct ) maximum intensity projection ( mip ) of a 31 - year - old women referred for fever of unknown origin ( fuo )  . 
 la febbre scomparsa dopo lintervento chirurgico . presenza di un processo infettivo in atto e , dopo ulteriori approfondimenti clinici , i pazienti sono stati trattati con antimicotici ( un caso ) o antibiotici ( 3 casi )  . 
nei restanti 6 casi ( 6 dei 17 con diagnosi nale ) lesame pet risultato negativo o inutile ai ni della diagnosi : a 2 pazienti stato diagnosticata una malattia auto - immune ; in un caso la diagnosi nale stata di emofagocitosi ed il paziente continua a presentare febbre dopo un anno di terapia ; 1 paziente presentava vasculite degli arti inferiori e bronchiolite obliterante con polmonite ( boop ) ; 1 paziente presentava microlitiasi biliare e questa stata giudicata come la responsabile della fuo ( in questo paziente alla pet si evidenziava un focale accumulo di ( 18f ) fdg intorno alla protesi biliare , ma il reperto stato giudicato come reazione ogistica dovuta alla presenza della stessa )  . 
 di questi pazienti : 3 continuano a presentare febbre dopo due anni di follow - up ( ipotesi diagnostiche : febbre ereditaria , malattia auto - immune ) ; in 3 casi la febbre scomparsa spontaneamente ; 1 paziente morto per cachessia quattro mesi dopo aver eseguito lesame pet . nel nostro studio il valore della pet nellindirizzare la diagnosi stato del 46% ( 11 / 24 )  . 
in due casi lesame pet stato falsamente radiol med ( 2011 ) 116 : 809820 2 years of follow - up ( diagnostic hypotheses of hereditary fever , autoimmune disease ) ; three underwent spontaneous fever remission ; one died of cachexia 4 months after the pet scan . 
in two cases , pet was falsely positive ( one case was the abovementioned patient with biliary microlithiasis and another patient presented a misleading [ 18f ] - fdg uptake in abdominal lymph nodes that was not con rmed by the biopsy )  . 
therefore , of 11 negative pet studies , only one ( 9% ) was followed by a worsening of the clinical condition . discussion in the diagnostic de nition of patients with fuo , the optimal examination is one that has the highest sensitivity to allow identi cation of the aetiology and consequently appropriate treatment planning , enables monitoring of the response to treatment and subsequent follow - up , is also useful in negative cases by identifying patients with a good prognosis for whom the best approach is to wait and see . 
in identifying fuo aetiology , the possible advantages of pet and pet / ct over other diagnostic procedures were largely understood several years ago , and in the last decade , its use in this eld has been extensively evaluated ( table 2 ) [ 1122 ]  . 
pet was helpful in 55% of patients , showing a high positive predictive value ( ppv ) ; furthermore , the authors reported on the usefulness of pet in negative cases also . 
 [ 12 ] , compared the utility of [ 18f ] - fdg - pet and 67ga - citrate scintigraphy to obtain a diagnosis in 58 consecutive cases of fuo . 
pet was used as a second step technique when the cause was not identi ed after medical history and clinical examination , routine laboratory tests , chest radiography and abdominal us . 
 [ 14 ] , in a prospective study of 19 patients with classical fuo recruited from the department of infectious diseases , compared inpositivo ( un primo caso era il paziente sopracitato con la micro - litiasi biliare , un secondo paziente ha presentato un anomalo accumulo in linfonodi addominali reperto che la successiva biopsia non ha confermato come responsabile della febbre )  . 
in de nitiva , di 11 pazienti con esame pet negativo solo 1 ( 9% ) ha mostrato una prognosi negativa . discussione per una gestione clinica ottimale dei pazienti con fuo classica , lesame ideale dovrebbe : avere la massima sensibilit possibile , cos da permettere lidenti cazione delleziologia e di conseguenza la piani cazione del trattamento migliore ; consentire la possibilit di monitorare la risposta al trattamento ed il successivo follow - up ; risultare utile anche in caso di negativit , identi cando quei pazienti con prognosi migliore e dunque nei quali lapproccio migliore quello del wait and see . 
ma i teorici vantaggi dellesame pet e pet / tc rispetto agli altri esami nellidenti care le cause della fuo , sono stati intuiti da diversi autori e nellultima decade il ruolo della pet stato estesamente indagato e discusso ( tabella 2 ) [ 1122 ]  . 
lesame pet era infatti utile a ni diagnostici nel 55% dei casi , con un valore predittivo positivo ( ppv ) elevato ; inoltre , gi in questo lavoro , gli autori sottolineavano lutilit dellesame pet anche nei casi negativi . 
 [ 12 ] hanno confrontato il ruolo della ( 18f ) fdg - pet con quello della scintigra a con 67ga citrato nellidenti cazione delle cause di fuo in un gruppo di 58 pazienti . 
in quello studio , lesame pet stato usato come second step technique , vale a dire in quei casi in cui la causa non era stata identi cata dopo anamnesi , esame obiettivo , esami laboratoristici di routine , radiograa del torace ed ecogra a addominale . 
la pet risultata utile per raggiungere la diagnosi nale nel 69% dei casi , mentre nei pazienti con pet negativa , nessuno studio successivo riuscito ad identi care la causa di fuo . 
 [ 14 ] , nel 2004 , in un studio prospettico hanno confrontato la scintigra a con granulociti marcati e la ( 18f ) fdg - pet , in 19 pazienti con fuo classica ricoverati nel reparto di malattie infettive . 
 [ 15 ] reported a large retrospective study of 74 patients in which pet was performed only if the history , clinical examination , blood chemistry , urinalysis , chest xray and abdominal us failed to provide clear diagnostic indications . 
 [ 15 ] hanno pubblicato un grosso studio retrospettivo su 74 pazienti , nei quali lesame pet era stato effettuato solo dopo che lanamnesi , lesame obiettivo , lemocromo , lesame urine , la radiogra a del torace e lecogra a addominale non avevano portato una chiara indicazione diagnostica . 
lo studio pet risultato pi utile nei pazienti con febbre continua piuttosto che in quelli con febbre intermittente , e nei casi con pcr e ves elevati piuttosto che in quelli con valori normali . 
jaruskova radiol med ( 2011 ) 116 : 809820 in patients with continuous rather than intermittent fever and was found useful in 39% of cases with elevated crp and esr but not in those with normal values . 
 [ 17 ] , in a study in 118 patients with prolonged febrile status ( 76% fuo , 24% suspected postsurgical infections ) , reported that pet or pet / ct was helpful for diagnoses in 36% of the entire population . 
 [ 18 ] evaluated the role of [ 18f ] - fdg - pet / ct in 48 patients , in 46% of which pet identi ed the underlying cause of fuo , with a ppv and a negative predictive value for focal disease of 81% and 100% , respectively . 
a negative [ 18f ] - fdg - pet / ct performed after the initial workup was highly indicative for establishing a wait - and - see strategy and obviated the need for further investigations . 
in this study , the [ 18f ] - fdg - pet / diagnostic contrast - enhanced ct ( cect ) was reported in conference by a radiologist and a nuclear medicine physician . 
 [ 21 ] , performing a similar study in 68 patients , reported a pet / cect diagnostic yield of 56% ( particularly in patients with high levels of crp and esr )  . 
 [ 22 ] , in a study of the role of pet or pet / ct in 44 children with fuo , reported similar results to the studies on adults : 19 scans ( 43% ) were useful for the nal diagnosis . 
patient age did not in uence the accuracy of the technique , whereas crp levels were signi cantly higher in patients with a positive pet scan than in those with a normal scan . 
furthermore , there are some dif culties that limit the use of this examination : rstly , the need for an in vitro labelling process of the white blood cells with its associated risks ( in particular blood contamination ) ; secondly , the special equipment and quali ed staff required ; thirdly , the time required to complete the exam [ 23 ]  . 
on the other hand , the examination is not helpful in the case of malignancies or vasculitis [ 24 , 25 ] , and this limitation is now more important considering the fact that et al . 
 [ 17 ] , in un lavoro su 118 pazienti con prolungato stato febbrile ( 76% fuo e 24% sospette infezioni post - chirurgiche ) hanno riportato che pet o pet / tc erano di aiuto per la diagnosi nel 36% della loro popolazione . 
nel 46% di questi , lesame ha identi cato la causa della fuo ; il ppv e il valore predittivo negativo per lidenti cazione di malattia focale sono risultati rispettivamente del 81% e 100% . 
 [ 20 ] hanno valutato il ruolo dellesame ( 18f ) fdg - pet combinato con tc diagnostica ( cetc ) , refertato in collaborazione tra medico nucleare e radiologo , in 48 pazienti con fuo . 
 [ 21 ] , effettuando un studio molto simile , hanno riportato valori di sensibilit interessanti ma signi cativamente minori , pari al 56% ( in questo studio , la massima sensibilit era nei pazienti con pcr e ves elevati )  . 
non ci sono chiare spiegazioni per giusti care la differenza tra questi due studi , per questo altri lavori sarebbero estremamente utili al ne di stabilire il reale potenziale dellesame pet / tc con tc diagnostica . 
 [ 22 ] studiando il ruolo dellesame pet o pet / tc in 44 bambini con fuo , hanno trovato risultati simili agli studi effettuati negli adulti : 19 esami ( 43% ) sono risultati utili per raggiungere una diagnosi nale . 
let del paziente non in uenzava laccuratezza della tecnica mentre i valori di pcr erano signi cativamente pi elevati nel gruppo dei pazienti con pet positiva rispetto a quelli con pet negativa . come dimostrato da altri autori , lo studio pet non lunico esame di medicina nucleare disponibile per pazienti con fuo . 
peraltro varie dif colt limitano luso di questo esame : primo , la necessit di una marcatura in vitro delle cellule ematiche con tutti i rischi associati ; secondo , sono necessarie una speciale attrezzatura e personale quali cato ; terzo , il tempo necessario per completare lacquisizione dellesame [ 23 ]  . 
 inoltre , in caso di neoplasia o vasculite , questo esame non utile [ 24 , 25 ] e tale limite oggi ancora pi rilevante considerando il fatto che lo spettro delle malattie che causano fuo si sta modi cando con riduzione della prevalenza delle infezioni [ 24 ]  . 
lunico studio che ha confrontato lutilit della ( 18f ) 818 radiol med ( 2011 ) 116 : 809820 the spectrum of diseases causing fuo is changing with a decrease in the proportion of infections [ 24 ]  . 
the only study that compared the utility of [ 18f ] - fdg - pet and white blood cell scans [ 14 ] reported that the latter demonstrated signi cantly greater speci city . 
however , this study had many limitations : the number of patients was very small , the study was performed with pet alone and the ndings were not subsequently con rmed . our study lends support to previous results , con rming and stressing the crucial role of pet / ct in the clinical setting of classical fuo . 
first of all , we can stress the fact that the use of the hybrid scanner adds crucial advantages to pet alone , as the integration of anatomical and morphological images allows improved interpretation of both abnormal [ 18f ] - fdg uptake and suspicious morphological ndings . 
pet / ct was of help in the diagnostic de nition of 11 out of the 24 selected cases ( pet / ct positive in focal in ammations , malignancies and vasculitis ) , but it was also useful in negative cases . 
it should be pointed out that with high probability , a negative scan excludes malignancies , vasculitis and focal infections , and this is helpful both for the referring clinician and for the patient . 
finally , pet / ct is a totalbody , noninvasive , practical and well - tolerated examination , which allows the conclusion of the diagnostic workup in the case of both positive and negative results . 
therefore , during the diagnostic workup , if a ct scan is required , the pet / ct study is equally useful , if not more so . study limitations fdg - pet con quella della scintigra a con globuli bianchi marcati [ 14 ] ha riportato una maggiore speci cit di questultima . 
tuttavia questo studio presentava diversi limiti : il gruppo dei pazienti incluso era estremamente piccolo , lo studio stato effettuato con tomografo pet in assenza di co - registrazione tc , questi dati non sono stati convalidati da altri studi in letteratura . per quello che riguarda la pet / tc , i risultati del nostro studio rafforzano e confermano quelli delle precedenti pubblicazioni , evidenziando il ruolo cruciale di questo esame nella gestione clinica dei pazienti con fuo classica . 
possiamo evidenziare che lesame pet eseguito con tecnica ibrida ( pet / tc ) aggiunge vantaggi cruciali al semplice esame pet grazie al fatto che luso combinato delle immagini anatomiche e di quelle morfologiche consente di migliorare linterpretazione degli accumuli patologici di ( 18f ) fdg e dei reperti anatomici sospetti . 
in questi pazienti , infatti , sia il radiologo che il medico nucleare sono alla ricerca di un indizio e qualsiasi anomalia ( anatomica o funzionale che sia ) potrebbe essere lespressione della causa eziologica . 
dunque , il fatto di poter acquisire simultaneamente e co - registrare entrambi i tipi di immagine porta alla riduzione dei tempi necessari alliter diagnostico e , soprattutto , migliora il risultato nale . 
il valore aggiunto dellesame pet / tc la sua utilit sia nei casi positivi che in quelli negativi ; questa aspetto rinforzato nel nostro studio dal lungo periodo di follow - up . 
lesame pet / tc ha contribuito nellidenti cazione della causa di fuo in 11 dei 24 pazienti ( pet / tc positiva in pazienti con in ammazione focale , neoplasia e vasculite ) ; tuttavia va sottolineato il fatto che lesame risultato utile anche nei casi negativi . 
importante sottolineare che un esame negativo esclude , con alta probabilit , la presenza di neoplasia , vasculite o infezioni focali e questo di aiuto sia per il medico che per il paziente . 
in ne , la pet / tc un esame total body , non invasivo , facile da eseguire e ben tollerato , che consente di giungere alla conclusione delliter diagnostico sia nei casi positivi che in quelli negativi . 
il numero dei pazienti incluso limitato e c una certa eterogeneit tra i radiol med ( 2011 ) 116 : 809820 low , and there was a certain heterogeneity in our population , which was further increased by the fact that the study was undertaken in a diagnostic centre related to different community hospitals . 
questi aspetti potrebbero aver in uenzato la nostra sensibilit complessiva ; tuttavia anche vero che quella presentata nel nostro studio attualmente la situazione pi tipica nella quale si trova ad operare il medico nucleare . 
if larger studies on this topic con rm our ndings , pet / ct , or better , pet / cect , should be included in the routine diagnostic workup of patients with classical fuo . nellultima decade , pi di una dozzina di studi hanno valutato il ruolo dellesame pet e pet / tc nei pazienti con fuo classica . 
 lesame pet / tc risulta utile sia nel caso risulti positivo sia nel caso risulti negativo ; inoltre , nei casi positivi , consente anche di monitorare la risposta al trattamento e di eseguire leventuale follow - up . 
 after the histopathological diagnosis had been obtained , the lesions were retrospectively divided into ve groups : adenocarcinomas ( n = 16 ) , squamous cell carcinomas ( n = 12 ) , chronic pneumonias ( n = 2 ) , malignant mediastinal tumours ( n = 2 ) and typical carcinoids ( n = 2 )  . 
using an adc value of 1.2510 - 3 mm2 / s as a threshold , we were able to differentiate malignant from benign lesions with 91% diagnostic accuracy , 90% sensitivity , 100% speci city , 100% positive predictive value and 57% negative predictive value . 
lobiettivo del nostro lavoro stato valutare retrospettivamente il ruolo delle sequenze pesate in diffusione con soppressione del segnale del grasso e del background nella diagnostica differenziale di lesioni benigne e maligne del distretto toraco - mediastinico , attraverso il calcolo dei valori medi del coef ciente di diffusione apparente ( adc )  . 
trentaquattro pazienti portatori di noduli polmonari / masse mediastiniche , gi sottoposti a tomogra a computerizzata ( tc ) del torace sono stati sottoposti a risonanza magnetica ( rm ) del torace con sequenze pesate in diffusione . 
retrospettivamente , dopo aver ottenuto la diagnosi istopatologica , le lesioni sono state suddivise in cinque gruppi : adenocarcinomi ( n = 16 ) , carcinomi squamocellulari ( n = 12 ) , polmoniti croniche ( n = 2 ) , tumori maligni mediastinici ( n = 2 ) , carcinoidi tipici ( n = 2 )  . 
le sequenze short tau inversion recoveryradiol med ( 2011 ) 116 : 720733 mediastinum provided potentially useful images for the differential diagnosis of benign and malignant lesions . keywords diffusion - weighted imaging ( dwi ) thoracopulmonary mri chest lesions echo - planar imaging ( stir - epi ) applicate al distretto toraco - mediastinico forniscono immagini potenzialmente utili per la diagnosi differenziale di lesioni benigne e maligne . parole chiave imaging pesato in diffusione rm toracopolmonare lesioni toraciche introduction introduzione carr and purcell rst reported that the magnetic resonance ( mr ) signal could be in uenced by the diffusion of water molecules in 1954 . 
the date of this discovery is not surprising , as the magnet they used created such a heterogeneous magnetic eld that it generated a constant eld gradient of around 1 gauss / cm [ 1 ] , resulting in an artefactual signal related to proton motion . 
the rst studies on the application of dw - mri to extracranial sites were disappointing , as image quality was poor , the possibility of adc quanti cation was limited and adc values had low reproducibility . 
in recent years , several investigators have demonstrated the diagnostic potential of dwi in extracranial pathology , and in particular in oncology [ 2 , 3 , 5 , 6 , 810 ]  . 
 in the study of the chest , computed tomography ( ct ) is the modality of choice for evaluating morphology , dimensions , location and structure of pulmonary nodules , as well as the most commonly used noninvasive modality for studying mediastinal masses . 
in recent years , short - tau inversion - recovery echo - planar imaging ( stir - epi ) sequences have been optimised for wb - dw - mri [ 10 , 11 ]  . 
stir - epi sequences allow fat suppression ( with a presaturation inversion pulse ) and suppression of the background body signal , including vessels , muscles and connective tissue ( characterised by high isotropic diffusivity ) by diffusion weighting . 
 these sequences can be applied to the chestmediastinum nel 1954 carr e purcell [ 1 ] notarono per la prima volta che il segnale nella risonanza magnetica ( rm ) poteva essere in uenzato dalla diffusione delle molecole di acqua ; la data di questa scoperta non deve sorprendere in quanto il magnete da loro utilizzato creava un campo magnetico cos disomogeneo da generare un gradiente di campo costante di circa 1 gauss / cm [ 1 ]  . 
nelle sequenze pesate in diffusione il contrasto tissutale dipende infatti dal grado di restrizione del movimento protonico ; tali sequenze permettono inoltre di quanti care il grado di restrizione attraverso il calcolo del coef ciente di diffusione apparente ( adc )  . limaging pesato in diffusione da diversi anni affermato , in campo neuroradiologico ; gli ottimi risultati ottenuti in tale ambito hanno suggerito unestensione dellimpiego di tale tecnica in altri distretti sino alle recentissime applicazioni allintero corpo whole body diffusion magnetic resonance imaging ( wb - dwmri ) [ 211 ]  . 
i primi studi sullapplicazione dellimaging pesato in diffusione ( dwi ) con rm in ambito extracranico hanno avuto risultati poco incoraggianti ; la qualit delle immagini ottenute era scarsa , la possibilit di quanti care ladc molto limitata , i valori calcolati poco riproducibili . 
negli ultimi anni diversi autori hanno documentato le potenzialit diagnostiche del dwi in ambito extracranico , soprattutto nella patologia oncologica [ 2 , 3 , 5 , 6 , 810 ]  . 
 in ambito toracico la tomogra a computerizzata ( tc ) rappresenta la metodica delezione per la valutazione morfologica , dimensionale , topogra ca e strutturale dei noduli polmonari ; la tc inoltre la metodica non invasiva maggiormente utilizzata per lo studio delle masse mediastiniche . 
 materials and methods patients over a period of 30 months ( november 2006 to april 2009 ) , we selected 34 patients ( 25 men and nine women , age range 4773 years , mean age 59 years ) who underwent dw - mri of the chest without administration of contrast material . 
patients were enrolled in the study after undergoing unenhanced and contrast - enhanced ct of the chest , according to the following inclusion criteria : ct evidence of a solitary pulmonary nodule or mediastinal mass requiring characterisation by ct - guided biopsy or mediastinoscopy ; axial dimensions of the pathological nding absence of clear ct features of benignity or malignancy ( in particular , absence of calci cation and necrotic foci , and absence of low - density areas with fat attenuation inside the lesions )  . after mri , 32 patients underwent transthoracic ctguided biopsy and two underwent mediastinoscopy . 
the histopathological diagnosis was considered the standard of reference in all patients . 1 cm , examination technique and image analysis ct examinations were carried out with a light speed 4 system ( ge medical systems , milwaukee , wi , usa )  . 
 scans were acquired during a single breath - hold before and after administration of nonionic iodinated contrast material ( 80100 ml , ow rate 34 ml / s , scan delay 2530 s from the beginning of the injection ) after obtaining the patients informed consent . 
scan parameters were as follows : slice thickness 5 mm ( with retroreconstruction up to 2.5 mm ) ; feed 15 mm / rotation ; rotation speed 0.8 s ; overall acquisition time 1215 s ; 120140 kv ; 180240 ma . 
the mr studies were performed with a 1.5 - t imager ( achieva , philips medical systems , best , the netherlands , release 2.1 ) with a 33 mt / m gradient strength and 160 mt / m / s slew rate by using phased - array sensitivityencoding ( sense ) body coils . 
le sequenze stir - epi sopprimono il segnale del tessuto adiposo ( mediante il pre - impulso di inversione ) e dei tessuti di fondo ( background - suppression ) rappresentati da vasi , muscoli , connettivo ( tessuti caratterizzati da diffusivit isotropica ) mediante la pesatura in diffusione . 
 materiali e metodi pazienti in un periodo di 30 mesi ( da novembre 2006 ad aprile 2009 ) , abbiamo selezionato 34 pazienti ( 25 uomini e 9 donne di et 4773 anni , con et media di 59 anni ) che sono stati sottoposti ad esame diffusion - rm del distretto toracico , senza somministrazione di mezzo di contrasto ( mdc )  . 
i pazienti sono stati arruolati , dopo essere stati sottoposti ad esame tc del torace , con e senza mdc , secondo i seguenti criteri dinclusione : riscontro allesame tc di un nodulo polmonare solitario o di una massa mediastinica da caratterizzare mediante biopsia tc - guidata o mediastinoscopia ; dimensioni assiali del tessuto patologico non inferiori ad 1 cm ; assenza di caratteristiche tomodensitometriche altamente probanti per benignit o malignit ( in particolare : assenza di calci cazioni e di foci necrotici intralesionali , assenza di aree ipodense , con valori densitometrici di tipo adiposo , allinterno delle lesioni )  . dopo lesame rm 32 pazienti sono stati sottoposti a biopsia transtoracica tc - guidata , 2 pazienti a mediastinoscopia . 
adc maps were automatically generated by the software on the basis of the images obtained with a b factor of 1 , 000 s / mm2 and 0 s / mm2 . 
adc values were calculated by a radiologist ( reader 1 , as , with 8 years of experience in mr imaging )  . dwis acquired with b values of 0 , 500 and 1 , 000 s / mm2 were also subjected to qualitative evaluation . 
two radiologists ( reader 2 , as , with 14 years of experience in mri ; reader 3 , ac , with 27 years of experience ) assessed lesion signal intensity , which were displayed by inverting the grey scale to visualise pathological tissue ( increased signal ) as a hypointense area ( grey - black ) in a background devoid of signal . 
the two readers were then asked to independently rate the signal intensity of the pathological areas , according to the following scale : 0 : no signal , equal to signal of the pulmonary parenchyma ; 1 : nearly no signal , slightly higher than that of the pulmonary parenchyma ( lesions just detectable in the dw sequences ) ; 2 : intermediate signal , between 1 and 3 ; 3 : high signal ( areas of de nite hypointensity appearing black on dwi and with similar signal intensity to that of the spinal cord )  . statistical analysis mean adc value was calculated for each lesion by placing an roi on the pathological areas . 
following qualitative evaluation of dwi , the degree of agreement between the two radiologists was assessed by calculating the cohen kappa coefcient . 5 mm ( retroricostruibile no a 2 , 5 mm ) , velocit di avanzamento 15 mm / rotazione , tempo di rotazione di 0 , 8 s , tempo complessivo di acquisizione pari a 1215 s , 120140 kv , 180240 ma . 
gli esami rm sono stati condotti con apparecchiatura da 1 , 5 t ( achieva , philips medical systems , best , olanda , release 2.1 ) , con potenza dei gradienti di 33 mt / m e slew - rate dei gradienti di 160 mt / m / s utilizzando bobine phased - array sense ( sensitivity encoding ) - body per la ricezione del segnale . 
dalle immagini ottenute con pesatura del parametro b di 1000 s / mm2 e 0 s / mm2 sono state generate automaticamente , dal software della rm , le mappe adc . 
il calcolo delladc stato effettuato da un radiologo ( lettore 1 : as , con 8 anni di esperienza in rm )  . le immagini pesate in diffusione acquisite con valori del parametro b di 0 , 500 e 1000 s / mm2 sono state inoltre valutate qualitativamente . 
due radiologi ( lettore 2 : as , con 14 anni di esperienza in rm , lettore 3 : ac con 27 anni di esperienza in rm ) hanno valutato lintensit del segnale delle lesioni , visualizzate invertendo la scala dei grigi in maniera tale da ottenere la visualizzazione del tessuto patologico ( segnale incrementato ) come area ipointensa ( grigio - nera ) su uno sfondo privo di segnale ( rappresentato dal background )  . 
dopo la valutazione delle immagini ai due lettori stato chiesto , indipendentemente , in cieco , di giudicare lintensit di segnale delle aree patologiche , secondo la seguente scala : 0 : intensit di segnale nulla , pari al segnale del parenchima polmonare ; 1 : intensit di segnale quasi nulla , di poco superiore al segnale del parenchima polmonare ( lesioni appena riconoscibili nelle sequenze pesate in diffusione ) ; 2 : intensit di segnale intermedia , tra 1 e 3 ; 3 : elevata intensit di segnale ( aree nettamente ipointense , nere nelle immagini pesate in diffusione , con la stessa intensit di segnale del midollo spinale )  . analisi statistica su ognuna delle lesioni esaminate stato calcolato il 724 results histopathological analysis overall , 30 malignant and four benign lesions were diagnosed . 
there were 16 adenocarcinomas , including undifferentiated ( g4 , n = 1 ) , poorly differentiated ( g3 , n = 7 ) and moderately differentiated ( g2 , n = 8 ) carcinomas ; 12 squamous cell carcinomas , including undifferentiated ( g4 , n = 1 ) , poorly differentiated ( g3 , n = 5 ) , moderately differentiated ( g2 , n = 2 ) , and well - differentiated ( g1 , n = 4 ) carcinomas ; two chronic in ammatory lesions , including one lipoid pneumonia and one eosinophilic pneumonia ; two malignant mediastinal tumours , including one mediastinal teratoma and one thymic carcinoma ; and two typical carcinoids . 
 lesion histopathological diagnosis , location , preoperative staging and grading are reported in table 1 . radiol med ( 2011 ) 116 : 720733 valore medio di adc attraverso il posizionamento di roi sulle aree patologiche . 
stato determinato un valore soglia di adc per discriminare con la massima accuratezza diagnostica le lesioni benigne dalle maligne . lanalisi statistica dei dati stata effettuata utilizzando la versione 13.0 del software spss ( spss , chicago , illinois )  . 
figures 1 and 2 show ct scans , dw images and adc maps ( with rois for adc quanti cation ) of two patients affected by an adenocarcinoma and a squamous cell carcinoma , respectively . 
sono stati diagnosticati 16 adenocarcinomi di cui : carcinomi indifferenziati ( g4 ) n = 1 , carcinomi scarsamente differenziati ( g3 ) n = 7 , moderatamente differenziati ( g2 ) n = 8 ; 12 carcinomi squamocellulari di cui carcinomi indifferenziati ( g4 ) n = 1 , carcinomi scarsamente differenziati ( g3 ) n = 5 , moderatamente differenziati ( g2 ) n = 2 , ben differenziati ( g1 ) n = 4 ; 2 alterazioni ogistiche croniche di cui una polmonite lipoidea , una polmonite eosino la ; 2 tumori maligni mediastinici di cui un teratoma mediastinico , un carcinoma timico ; 2 carcinoidi tipici . 
non sono state riscontrate differenze statisticamente signi cative tra il valore medio di adc calcolato nel gruppo degli adenocarcinomi ( 1 , 36410 - 3 mm2 / s ) ed il valore medio di adc calcolato nel gruppo delle lesioni maligne mediastiniche ( 1 , 05110 - 3 mm2 / s ) ( p > 0 , 05 )  . 
by using a threshold adc value of 1.25103 mm2 / s , we were able to differentiate benign from malignant lesions with the highest diagnostic accuracy ( 91% ) , with 90% sensitivity , 100% speci city , 100% positive predictive value , and 57% negative predictive value ( table 3 )  . qualitative analysis of signal intensity all lesions examined were detectable on dwi , with signal intensity values of 2 to 3 according to the scale adopted . 
no dio di adc calcolato nel gruppo degli adenocarcinomi ( 1 , 36410 - 3 mm2 / s ) si dimostrato signi cativamente inferiore rispetto al valore medio di adc calcolato nel gruppo delle ogosi croniche ( 1 , 85410 - 3 mm2 / s ) ( p < 0 , 05 )  . 
nella figura 4 riportiamo le immagini pesate in diffusione e la relativa mappa adc ( con il posizionamento dellarea roi per la quanti cazione del coef ciente di diffusione apparente ) di un paziente portatore di polmonite lipoidea . adenocarcinomi versus carcinoidi tipici : il valore medio di adc calcolato nel gruppo degli adenocarcinomi ( 1 , 36410 - 3 mm2 / s ) risultato signi cativamente inferiore rispetto al valore medio di adc calcolato nel gruppo dei carcinoidi tipici ( 2 , 20010 - 3 mm2 / s ) ( p < 0 , 05 )  . 
nella figura 5 riportiamo laspetto tc , limaging in diffusione con relativa mappa adc di un paziente portatore di carcinoide tipico . carcinomi squamocellulari versus ogosi croniche : il radiol med ( 2011 ) 116 : 720733 table 2 mean apparent diffusion coef cient ( adc ) values calculated by placing a region of interest ( roi ) over the single lesions examined patient no . 
cohens kappa coef cient for b factor equal to 0 was 0.51 ( poor interrater agreement ) ; for b factor equal to 500 , 0.76 ( indicating substantial agreement ) ; for b factor equal to 1 , 000 , 0.87 ( indicating almost perfect agreement )  . 
previous studies conducted on other body regions have reported statistically signi cant differences in adc values of benign valore medio di adc calcolato nel gruppo delle ogosi croniche ( 1 , 85410 - 3 mm2 / s ) risultato signi cativamente superiore rispetto al valore medio di adc calcolato nel gruppo dei carcinomi squamocellulari ( 1 , 23110 - 3 mm2 / s ) ( p < 0 , 05 )  . 
 carcinomi squamocellulari versus carcinoidi tipici : i valori medi di adc calcolati nel gruppo dei carcinomi squamo cellulari ( 1 , 23110 - 3 mm2 / s ) risultano signi cativamente inferiori rispetto ai valore medi di adc calcolati nel gruppo dei carcinoidi tipici ( 2 , 20010 - 3 mm2 / s ) ( p < 0 , 05 )  . 
 lesioni maligne mediastiniche versus ogosi croniche : il valore medio di adc calcolato nel gruppo delle lesioni maligne mediastiniche ( 1 , 05110 - 3 mm2 / s ) si dimostrato signi cativamente inferiore rispetto al valore medio di adc calcolato nel gruppo delle ogosi croniche ( 1 , 85410 - 3 mm2 / s ) ( p < 0 , 05 )  . 
 lesioni maligne mediastiniche versus carcinoidi tipici : il valore medio di adc calcolato nel gruppo delle lesioni maligne mediastiniche ( 1 , 05110 - 3 mm2 / s ) risulta signicativamente inferiore rispetto al valore medio di adc calcolato nel gruppo dei carcinoidi tipici ( 2 , 20010 - 3 mm2 / s ) ( p < 0 , 05 )  . 
 confronto tra lesioni benigne flogosi croniche versus carcinoidi tipici : non sono state riscontrate differenze statisticamente signi cative tra i valori medi di adc calcolati nel gruppo delle ogosi croniche ( 1 , 85410 - 3 mm2 / s ) e il valore medio di adc calcolato nel gruppo dei carcinoidi tipici ( 2 , 20010 - 3 mm2 / s ; deviazione standard [ ds ] 0 , 338 ) ( p > 0 , 05 )  . 
utilizzando un valore soglia di adc di 1 , 25103 mm2 / s stato possibile differenziare lesioni benigne dalle maligne con la pi alta accuratezza diagnostica ( 91% ) , con valori di sensibilit del 90% , speci cit del 100% , valore predittivo positivo del 100% e valore predittivo negativo del 57% ( tabella 3 )  . 
per un valore di b factor uguale a 0 , la costante di cohen calcolata stata di circa 0 , 51 ( scarso grado di agreement tra i due osservatori )  . 
per valori tabella 2 valori medi di coef ciente di diffusione apparente ( adc ) calcolati mediante il posizionamento delle roi a livello delle singole lesioni esaminate di b factor uguale a 1000 , la costante di cohen risultava pari a 0 , 87 ( grado di agreement tra i due radiologi molto buono )  . 
 radiol med ( 2011 ) 116 : 720733 paziente ( numero ) diagnosi istopatologica ( 103 mm2 / s ) discussione 728 adenocarcinoma adenocarcinoma carcinoma squamocellulare carcinoma squamocellulare polmonite lipoidea adenocarcinoma adenocarcinoma adenocarcinoma carcinoma squamocellulare carcinoma squamocellulare carcinoma squamocellulare carcinoma squamocellulare carcinoma squamocellulare carcinoma squamocellulare adenocarcinoma carcinoma squamocellulare carcinoma timico carcinoma squamocellulare adenocarcinoma adenocarcinoma polonite eosino la adenocarcinoma carcinoma squamocellulare carcinoma squamocellulare adenocarcinoma adenocarcinoma adenocarcinoma teratoma maturo adenocarcinoma carcinoide tipico carcinoide tipico adenocarcinoma adenocarcinoma adenocarcinoma 1 , 360 , 36 1 , 240 , 23 1 , 240 , 81 1 , 530 , 21 1 , 880 , 30 1 , 230 , 02 1 , 450 , 26 1 , 300 , 15 1 , 220 , 18 1 , 230 , 19 1 , 130 , 22 0 , 770 , 12 1 , 270 , 14 1 , 330 , 15 1 , 390 , 17 1 , 280 , 18 1 , 130 , 19 1 , 230 , 24 1 , 40 , 17 1 , 740 , 26 1 , 940 , 34 1 , 280 , 25 1 , 270 , 20 1 , 310 , 15 1 , 450 , 69 1 , 370 , 23 1 , 290 , 26 1 , 040 , 14 1 , 290 , 14 2 , 20 , 33 2 , 10 , 24 1 , 320 , 16 1 , 350 , 18 1 , 280 , 19 and malignant lesions . 
in agreement with previous reports [ 29 ] , our study demonstrated that diffusion is signi cantly restricted in cases of pathological hypercellularity in which extracellular spaces , where proton diffusion is relatively unrestricted , are extremely reduced . 
cell density increases in tumours in comparison with healthy tissue , and the adc consequently decreases , as seen in the patients affected by malignancies with different degrees of cell differentiation . in the thoracopulmonary region , dwi with adc quanti cation proved to be useful in the differential diagnosis between malignant and benign lesions . 
only a limited number of studies have investigated the possible use of dwi in chest imaging [ 1214 ] , and all of them used dw - epi sequences without presaturation pulses . 
le informazioni fornite ri ettono la struttura dei tessuti a livello cellulare ; in particolare lintensit del segnale fortemente in uenzata dalla densit cellulare , dalla presenza di giunzioni strette intercellulari , di membrane cellulari , di bre , di macromolecole , di organuli intracellulari . 
il nostro studio , in accordo con precedenti lavori [ 29 ] , dimostra che la diffusivit signi cativamente ristretta nei casi di patologica ipercellularit tissutale , in cui gli spazi interstiziali extracellulari , ove la diffusivit protonica scarsamente vincolata , risultano estremamente ridotti . 
nei tumori la densit cellulare aumenta rispetto ai tessuti normali , e ladc di conseguenza diminuisce come abbiamo osservato in questo lavoro nei pazienti affetti da neoplasie maligne a vario grado di differenziazione . 
in base alle nostre conoscenze questo studio rappresenta il primo lavoro in cui stato valutato il ruolo di sequenze pesate in diffusione con soppressione del grasso e del background nellimaging del distretto toracico . 
i precedenti studi hanno infatti utilizzato sequenze epi pesate in diffusione , senza pre - impulsi di saturazione . nella nostra esperienza le sequenze stir - epi hanno fornito immagini diagnostiche ed stato sempre possibile quanti care agevolmente il coef ciente di diffusione apparente . 
in tutti i pazienti esaminati , lidenti cazione del tessuto patologico stata semplice e facilitata dallinversione della nestra di visualizzazione . i risultati del nostro lavoro evidenziano una sovrapposizione dei valori di adc di adenocarcinomi e carcinomi squamocellulari , a vario grado di differenziazione ; questi dati differiscono dai risultati precedentemente ottenuti da altri autori [ 12 ]  . 
the lesion results in pathological restriction to tissue diffusivity and appears hyperintense on diffusion - weighted images acquired with a b factor equal to 1 , 000 ( b )  . 
la lesione determina patologica restrizione della diffusivit tissutale e presenta elevato segnale nelle sequenze dwi , acquisite con b factor pari a 1000 ( b ) , nestra di visualizzazione invertita . 
 the lesion results in pathological restriction to tissue diffusivity and is hyperintense in the diffusion - weighted sequences acquired with b factor equal to 1 , 000 ( b )  . 
il posizionamento della roi documenta valori medi di adc relativamente bassi ( 1 , 1710 - 3 mm2 / se ) ( c )  . 730 radiol med ( 2011 ) 116 : 720733 fig . 
3 patient with a mediastinal teratoma : the lesion causes pathological restriction to tissue diffusivity and is hyperintense in the diffusion - weighted sequences acquired with a b factor equal to 1 , 000 ( left panel , inverted display )  . 
la lesione determina patologica restrizione della diffusivit tissutale e risulta iperintensa nelle sequenze dwi , acquisite con b factor pari a 1000 ( immagine a sinistra , nestra di visualizzazione invertita )  . 
4 patient with chronic lipoid pneumonia : the lesion causes mild restriction to tissue diffusivity and has intermediate signal intensity in the diffusionweighted sequences acquired with a b factor equal to 1 , 000 ( left panel )  . 
il posizionamento della roi documenta valori medi di adc relativamente alti ( 1 , 8810 - 3 mm2 / s ) ( immagine a destra )  . quanti cation proved straightforward in all cases . 
the pathological tissue was readily detected in all patients , and detection was facilitated by inversion of the grey - scale display . the results of our study show an overlap of adc values of adenocarcinomas and squamous cell carcinomas with different degrees of cell differentiation . 
 [ 12 ] found signi cantly higher mean adc values among adenocarcinomas compared with squamous sizione dei valori medi di adc dei tumori maligni esaminati ( adenocarcinomi , carcinomi squamocellulari , lesioni maligne mediastiniche )  . 
listotipo ed il grado di differenziazione delle lesioni maligne non vengono determinati in base alla densit cellulare ; tale parametro biologico in uenza invece fortemente il grado di restrizione della diffusivit tissutale e quindi ladc . 
the lesion causes restriction to tissue diffusivity and appears moderately hyperintense in the diffusion - weighted sequences acquired with a b factor equal to 1 , 000 ( b )  . 
this may be explained by the fact that , although the degree of restriction to tissue diffusivity and therefore adc are strongly affected by cellular density , this biological parameter is not used to determined the histological type and grade of malignant lesions , and in histopathological classi cation , tumour grade is mostly established based on other factors , such as come la cheratinizzazione , la strati cazione e le atipie delle cellule , mentre la densit cellulare non un parametro particolarmente considerato , ci pu spiegare il nostro risultato . 
by applying an adc threshold value of 1.25103 mm2 / s , we were able to discriminate malignant from benign lesions with a very high level of diagnostic accuracy ( 91% )  . 
moreover , benign lesions have a reduced mitotic index compared with malignant lesions , and cell lysis and membrane fragmentation are much more limited . our results also demonstrate the poor speci city of the quantitative analysis of signal intensity in differentiating benign from malignant lesions , both of which present high signal intensity in stir - epi sequences ( between 2 and 3 according to the scale adopted )  . 
signal intensity in dw sequences is , in fact , in uenced by the t2 relaxation time of pathological tissues , unlike the adc , which is , instead , a reliable indicator of the degree of restriction to tissue diffusivity . the main limitation to our study is the small number of benign lesions included , so that our ndings require validation by larger series ; in addition , owing to the small size of the overall sample , the groups were compared with a nonparsoglia di adc pari a 1 , 25103 mm2 / s stato possibile discriminare lesioni maligne dalle benigne con la massima accuratezza diagnostica ( 91% )  . 
supponiamo che questo risultato sia legato alla ridotta densit cellulare e al ridotto rapporto nucleo / citoplasma delle lesioni benigne rispetto alle maligne ; inoltre le lesioni benigne hanno un indice mitotico ridotto rispetto alle lesioni maligne ed i fenomeni di lisi cellulare con frammentazione della membrana sono molto pi limitati . i risultati del nostro lavoro evidenziano inoltre la scarsa speci cit dellanalisi qualitativa del segnale nella differenziazione di lesioni benigne e maligne , che presentano indistintamente segnale elevato nelle sequenze stir - epi ( compreso tra 2 e 3 secondo la scala utilizzata )  . 
lintensit di segnale nelle sequenze pesate in diffusione risente infatti del tempo di rilassamento t2 dei tessuti patologici , a differenza del coef ciente di diffusione apparente , che invece un indice fedele del grado di restrizione della diffusivit tissutale . 
 il principale limite del nostro studio la scarsa numerosit campionaria del gruppo delle lesioni benigne , per cui i nostri risultati dovranno essere avvalorati da casistiche pi ampie ; a causa del campione numericamente ridotto i gruppi sono stati confrontati con un test non parametrico per variabili indipendenti ( test u di mann - whitney )  . 
furthermore , the small size of the sample did not allow statistical analysis of malignant lesions subdivided by grade of cell differentiation . zione di pazienti ( lesioni con istotipo , sede e grado di differenziazioni differenti )  . 
la ridotta numerosit del campione non ha inoltre permesso di effettuare unanalisi statistica per sottogruppi delle lesioni maligne a diverso grado di differenziazione . conclusioni conclusions the results of this study conducted on a limited number of patients demonstrate that stir - epi sequences applied to the chestmediastinum provides diagnostic images that are potentially useful for the differential diagnosis between benign and malignant lesions . 
further studies on more numerous and less heterogeneous patient series are required to validate these preliminary results . in conclusione i risultati di questo lavoro , su un ridotto numero di pazienti , dimostrano che le sequenze stirepi applicate al distretto toraco - mediastinico forniscono immagini diagnostiche , potenzialmente utili per la diagnosi differenziale di lesioni benigne e maligne . 
cademartiri1 , 2 1department of radiology and cardiology , c / o piastra tecnica , piano 0 , azienda ospedaliero - universitaria , via gramsci 14 , 43100 parma , italy 2department of radiology and cardiology , erasmus medical center , rotterdam , the netherlands 3department of emergency medicine , azienda ospedaliero - universitaria , parma , italy 4department of cardiology , ospedale san gennaro , asl napoli 1 , naples , italy 5department of cardiology , azienda ospedaliero - universitaria , foggia , italy 6sdn foundation , ircss , naples , italy correspondence to : f . 
a total of 145 consecutive patients ( 75 men ; 6412 years ) with acp were referred from the emergency department for ctca , which was performed with a standard protocol using a 64 - slice scanner . 
patients were strati ed according to the morise clinical score ( low , intermediate , high ) and to the ctca ndings [ absence of coronary artery disease ( cad ) , nonobstructive cad , obstructive cad ]  . 
obstructive cad ( > 50% luminal narrowing ) was detected in 35 ( 24% ) patients ; nonobstructive cad ( 50% luminal narrowing ) in 62 ( 43% ) and absence of cad in 48 ( 33% ) patients . 
sixteen events ( three hard events ) occurred in patients without a history of cad , and four events ( one hard event ) occurred in patients with a history of cad . 
i pazienti sono stati strati cati secondo lo score di morise ( basso , intermedio , alto ) e la ctca ( assenza di coronary artery disease [ cad ] , cad non ostruttiva , cad ostruttiva )  . 
stata rilevata cad ostruttiva ( riduzione del lume > 50% ) in 35 ( 24% ) pazienti ; cad non ostruttiva ( riduzione del lume 50% ) in 62 ( 43% ) pazienti e assenza di cad in 48 ( 33% ) pazienti . 
sedici eventi ( 3 eventi hard ) si sono veri cati in pazienti senza storia di cad e 4 eventi ( 1 evento hard ) si veri cato nei pazienti con storia di cad . 
ctca shows the potential for optimal strati cation of patients with acp . keywords computed tomography coronary angiography prognostic value prognosis acute chest pain con assenza di cad secondo la ctca la frequenza di eventi stata pari allo 0% . 
la ctca ha il potenziale per una ottimale strati cazione del rischio nei pazienti con acp . parole chiave angiogra a coronarica mediante tomogra a computerizzata valore prognostico prognosi dolore toracico acuto introduction introduzione computed tomography coronary angiography ( ctca ) is an emerging technique for noninvasive evaluation of coronary atherosclerosis in patients with suspected and known coronary artery disease ( cad ) [ 16 ]  . 
current - generation ct scanners have a high diagnostic accuracy for detecting and excluding obstructive cad compared with conventional coronary angiography ( cag ) in selected patients [ 16 ]  . 
however , few data are available on the prognostic value of ctca [ 3 , 816 ]  . preliminary reports suggest that ctca has a very high negative predictive value ( npv ) for future cardiovascular events [ 3 , 816 ]  . 
ctca has been proposed for the immediate strati cation of patients presenting at the emergency room ( er ) with acute chest pain ( acp ) [ 1724 ]  . 
of these , 7080% are classi ed as nonanginal pain , 78% as acute coronary syndrome and up to 78% as non - st elevation myocardial infarction ( nstemi ) and / or unstable angina ( ua )  . 
la generazione corrente di apparecchiature di tomogra a computerizzata ( tc ) ha una elevata accuratezza diagnostica nella rilevazione ed esclusione della cad ostruttiva quando confrontata con la coronarogra a convenzionale ( cag ) in popolazioni selezionate di pazienti [ 16 ]  . 
al contrario , pochi dati sono disponibili riguardo il valore prognostico della ctca [ 3 , 816 ]  . studi preliminari mostrano un elevato valore predittivo negativo della ctca per eventi cardiovascolari a distanza [ 3 , 816 ]  . 
la ctca stata proposta per la strati cazione precoce dei pazienti che si presentano nel dipartimento di emergenza ( er ) con dolore toracico acuto ( acp ) [ 1724 ]  . 
lacp rappresenta il 5%10% dei pazienti che si presentano aller ; di questi il 70%80% viene classi cato come dolore toracico non anginoso , il 7%8% viene classi cato come sindrome coronarica acuta , e no al 7%8% viene classi cato come infarti miocardici senza sopra - elevazione del tratto st ( non - st elevation myocardial infarction , nstemi ) e / o angina instabile ( unstable angina , ua )  . 
patients with previous coronary artery bypass surgery ( cabg ) , known previous reaction to iodinated contrast medium and / or renal failure ( creatinine clearance < 60 ml / min ) were excluded . 
the morise risk score was calculated to stratify pretest risk [ 25 , 26 ]  . ctca data acquisition all examinations were performed with a single - source 64 - slice ct system ( somatom sensation 64 cardiac , siemens , forchheim , germany ) [ 4 ]  . 
first , a prospectively triggered coronary calcium ct data set was obtained using standardised ct parameters comprising 201.2 - mm collimation , 330 ms gantry rotation time , 120 kv tube voltage , 150 mas tube current . 
thereafter , ctca was performed after intravenous administration of 100 ml bolus of nonionic contrast material ( iomeron 400mgi / ml , bracco , milan , italy ) at a ow rate of 46 ml / s depending on patient status . 
ctca was performed with 640.6 mm collimation , 330 ms gantry rotation time , 120 kv tube voltage , 900 mas tube current , pitch 0.20 , temporal resolution 165 ms , spatial resolution 0.4 mm3 , reconstruction slice thickness 0.75 mm and reconstruction increment 0.5 m beta - blocker ( atenolol 510 mg ) was administered iv to all patients with a heart rate > 65 beats / min and without contraindications ( asthma or bronchospasm , systolic blood pressure < 100 mmhg )  . 
to obtain optimal image quality , data sets were reconstructed at no less than two points of the cardiac cycle using a retromateriali e metodi pazienti abbiamo arruolato retrospettivamente 145 pazienti consecutivi ( 75 maschi ; 6412 anni ) che si sono presentati al er della nostra istituzione con acp e sono stati inviati a ctca . 
 i pazienti con coronaropatia nota ( per esempio , pregresso infarto miocardico , pregressa rivascolarizzazione mediante angioplastica ) o con cardiopatia nota ( per esempio , scompenso cardiaco ) sono stati inclusi nello studio . 
i pazienti con pregresso bypass aorto - coronarico ( cabg ) , pregressa reazione al mezzo di contrasto iodato e / o insuf cienza renale ( clearance creatinina < 60 ml / min ) sono stati esclusi . 
sono stati considerati i seguenti fattori di rischio : ( 1 ) ipertensione ( de nita come pressione arteriosa [ pa ] 140 / 80 mmhg o necessit di terapia antiipertensiva ) ; ( 2 ) ipercolesterolemia ( colesterolo low density lipoprotein [ ldl ] > 130 mg / dl ) ; ( 3 ) diabete mellito ; ( 4 ) fumo ; ( 5 ) obesit ( body mass index [ bmi ] > 30 ) ; ( 6 ) familiarit per cardiopatia ischemica . 
 per la strati cazione del rischio pre - test stato utilizzato lo score di morise [ 25 , 26 ]  . scansione ctca tutti gli esami ctca sono stati effettuati con un apparecchiatura tc a singola sorgente a 64 strati ( somatom sensation 64 cardiac , siemens , forchheim , germania ) [ 4 ]  . 
 inizialmente , stata effettuata una scansione con triggering elettrocardiogra co ( ecg ) prospettico per la valutazione del calcio coronarico utilizzando parametri tc standard : collimazione 201 , 2 mm , tempo di rotazione del gantry 330 ms , voltaggio del tubo 120 kv , corrente del tubo 150 mas . 
 quindi , la ctca stata effettuata dopo la somministrazione di un bolo endovenoso di 100 ml di mezzo di contrasto non ionico ( iomeron 400 mgi / ml , bracco , milano , italia ) ad un usso di 46 ml / s a seconda delle condizioni del paziente . 
axial data sets were transferred to a remote workstation ( mmwp / leonardo , siemens medical solutions , forchheim , germany ) for postprocessing and subsequent evaluation . ct data analysis coronary artery calcium score the coronary artery calcium score ( cacs ) was assessed with the application of dedicated software ( cascore , siemens medical solutions )  . 
for multivariate analysis , patients were divided into ve groups based on cacs score : group 1 , cacs 010 ; group 2 , 11100 ; group 3 , cacs 101400 ; group 4 , cacs 4011000 ; group 5 , cacs > 1 , 000 . 
patients were nally classi ed as being in one of three groups based on the ctca ndings : ( 1 ) normal coronary arteries , ( 2 ) nonobstructive cad , and ( 3 ) obstructive cad . 
inoltre , in tutti i pazienti senza contro - indicazioni ( stenosi aortica severa , pressione arteriosa sistolica < 100 mmhg ) sono stati somministrati 0 , 3 mg di nitro - glicerina sub - linguale [ 27 ]  . 
per ottenere una qualit di immagine ottimale , i dataset sono stati ricostruiti in almeno due punti del ciclo cardiaco utilizzando un algoritmo ecg retrospettivo ( solitamente una fase tele - diastolica a 350 ms dallonda r ed una fase tele - sistolica a + 275 ms ) , quindi la modulazione prospettica dellamperaggio non stata utilizzata nel nostro studio . 
i dataset assiali sono stati trasferiti ad una workstation ( mmwp / leonardo , siemens medical solutions , forchheim , germania ) per il post - processing . analisi dei dati calcium score coronarico lo score del calcio coronarico ( cacs ) stato valutato con un software dedicato ( cascore , siemens medical solutions , forchheim , germania )  . 
il calcio coronarico stato identi cato automaticamente mediante lapplicazione software come aree di elevata densit lungo il decorso delle arterie coronarie la cui attenuazione risultava 130 unit houns eld ( hu )  . 
per lanalisi multi - variata i pazienti sono stati suddivisi in cinque gruppi a seconda del cacs score : pazienti con cacs 010 ( gruppo 1 ) , pazienti con cacs 11100 ( gruppo 2 ) , pazienti con cacs 101400 ( gruppo 3 ) , pazienti con cacs 4011000 ( gruppo 4 ) , e pazienti con cacs > 1000 ( gruppo 5 )  . 
le placche ostruttive sono state de nite come quelle che determinavano una riduzione del lume > 50% ; le placche non ostruttive sono state de nite tali quando determinavano una riduzione del lume coronarico50% . 
 694 radiol med ( 2011 ) 116 : 690705 in ogni paziente abbiamo valutato il numero di segmenti coronarici con ateromasia , il numero di segmenti con malattia ostruttiva , ed il numero di ogni tipo di placca . 
 in ne , i pazienti sono stati classi cati in 3 gruppi basandosi sulle caratteristiche della ctca : ( 1 ) pazienti con coronarie normali , ( 2 ) pazienti con malattia non ostruttiva , e ( 3 ) pazienti con malattia ostruttiva . 
gli end - point clinici utilizzati erano : ( 1 ) morte cardiaca , ( 2 ) infarto miocardico non fatale , ( 3 ) angina instabile , ( 4 ) rivascolarizzazione miocardica ( pci / cabg )  . 
lanalisi di sopravvivenza stata effettuata per gli eventi totali ( end - point 14 ; major adverse cardiac events [ mace ] e per gli eventi 13 de niti come eventi hard )  . 
le variabili continue , espresse come medie e deviazioni standard ( sd ) , sono state confrontate utilizzando il test t di student a due code e lanalisi della varianza se normalmente distribuite , o mediante il test del chi quadrato ed il kruskal - wallis se non normalmente distribuite . 
le curve degli eventi per lend - point composito ( mace ) e per gli eventi hard ( morte cardiaca , infarto miocardico non fatale ed angina instabile ) sono state confrontate il logrank test . 
gli hazard ratios ( ossia il tasso di eventi / paziente nel tempo ) sono stati calcolati con intervalli di con denza del 95% come una stima del rischio associate con particolari variabili . 
the clinical endpoints were : ( 1 ) cardiac death , ( 2 ) nonfatal myocardial infarction , ( 3 ) ua , and ( 4 ) myocardial revascularisations ( pci ) / cabg ]  . 
 survival analysis was performed for total cardiac events [ endpoints 14 ; major adverse cardiac events ( mace ) ] and for clustered endpoints 13 de ned as hard events . 
 continuous variables , expressed as mean and standard deviation ( sd ) , were compared using the two - tailed t test and analysis of variance ( anova ) if normally distributed or by means of the chi - square and kruskalwallis method if not normally distributed . 
event curves of the composite endpoint ( cardiac death , nonfatal infarction , angina requiring hospitalisation , myocardial revascularisation ; mace ) and hard cardiac events ( cardiac death , nonfatal infarction , ua ) were compared using the log - rank test . 
there were no signi cant differences in terms of mean age ( 63.611.9 years ) , prevalence of men ( n = 75 ; 52% ) , mean bmi ( 26.44.5 ) and heart rate ( 61.19.8 beats / min ) during the ctca scan between the population with and without a history of cad . 
non abbiamo riscontrato differenze signicative in termini di et media ( 63 , 611 , 9 anni ) , prevalenza di maschi ( 75 ; 52% ) , bmi ( 26 , 44 , 5 ) , e frequenza cardiaca ( 61 , 19 , 8 bpm ) durante la scansione ctca , tra la popolazione con e senza storia di malattia coronarica . 
la grande maggioranza dei pazienti si presentava con dolore toracico atipico ( 52 ; 36% ) o altri sintomi come dispnea ( 67 ; 46% ) , mentre solo 26 ( 18% ) pazienti si presentavano con dolore tipico . 
per quanto riguarda la strati cazione del rischio preliminare alla ctca , la maggioranza dei pazienti era a rischio intermedio ( n = 83 ; 57% ) nella popolazione complessiva . 
nel sottogruppo dei pazienti senza storia di cad i pazienti a rischio intermedio erano il 65% ( n = 83 ) ed i pazienti a rischio basso erano il 13% ( n = 16 )  . 
 tutti i pazienti con cad nota sono stati considerati ad alto rischio . table 1 baseline characteristics of overall population , patients without a history of cad and patients with a previous history of cad patients w / o history of cad ( n = 127 ) patients with history of cad ( n = 18 ) clinical characteristics age [ years ; mean ( sd ) ] male gender ( % ) bmi [ kg / m ; mean ( sd ) ] mean heart rate [ bpm ; mean ( sd ) ] follow - up [ months ; mean ( sd ) ] risk factors no . 
in the subgroup without a history of cad , 65% ( n = 83 ) of patients were at intermediate risk , and 13% ( n = 16 ) of patients were at low risk . 
all patients with a history of cad were considered high risk by default . ctca strati cation a total of 127 ( 87.6% ) patients were without a history of cad , and 18 ( 12.4% ) patients had a history of cad . 
signi cant cad ( > 50% luminal narrowing ) was detected in 35 ( 24% ) patients ; nonsigni cant cad ( 50% luminal narrowing ) was detected in 62 ( 43% ) and absence of cad in 48 ( 33% ) patients ( tables 2 and 3 )  . 
the prevalence of obstructive disease , as expected , strati cazione mediante ctca in totale 127 ( 87 , 6% ) pazienti non avevano storia di cad e 18 ( 12 , 4% ) pazienti avevano storia di cad . 
la cad signi cativa ( riduzione del lume > 50% ) stata rilevata in 35 ( 24% ) pazienti ; cad non signi cativa ( riduzione del lume50% ) stata rilevata in 62 ( 43% ) pazienti ed assenza di cad in 48 ( 33% ) pazienti ( tabelle 2 e 3 )  . 
la malattia ostruttiva multi - vasale era presente nell8% ( 12 ) della popolazione totale e prevalentemente distribuita nel sotto - gruppo con storia di cad ( 4 ; 22% )  . follow - up e prognosi abbiamo riscontrato 20 eventi durante un follow - up medio di 203 mesi ; di questi , 4 erano eventi hard e , di questi , 3 morti cardiache ed un infarto miocardico ; gli eventi rimaradiol med ( 2011 ) 116 : 690705 table 2 ctca characteristics of overall population , patients without a history of cad and patients with a previous history of cad patients w / o history of cad ( n = 127 ) patients with history of cad ( n = 18 ) patients absence of cad ( % ) nonobstructive cad ( % ) obstructive cad ( % ) single - vessel disease ( % ) multivessel disease ( % ) obstructive cad in lm / lad ( % ) rca ( % ) lcx ( % ) total agatston cs [ mean ( sd ) ] segments no . 
la maggioranza degli eventi totali erano rivascolarizzazioni ( 16 / 20 ; 80% ) di cui 14 percutanee ( pci ) con stent e 2 bypass aorto - coronarici ( cabg )  . 
sedici eventi ( di cui 3 eventi hard ) si sono veri cati in pazienti senza storia di cad e 4 eventi ( di cui 1 evento hard ) si sono veri cati in pazienti con storia di cad . 
 698 radiol med ( 2011 ) 116 : 690705 table 3 ctca characteristics of overall population , patients without a history of cad and patients with a previous history of cad who experienced a mace during follow - up patients w / o history of cad ( n = 16 ) patients with history of cad ( n = 4 ) patients absence of cad ( % ) nonobstructive cad ( % ) obstructive cad ( % ) single - vessel disease ( % ) multivessel disease ( % ) obstructive cad in lm / lad coronary artery ( % ) rca ( % ) lcx ( % ) agatston score [ mean ( sd ) ] segments no . 
sixteen events ( three hard events ) occurred in patients without a i pazienti sono stati strati cati dalla ctca in coronarie normali ( 48 ; 0 eventi ) , cad non ostruttiva ( 62 ; 6 eventi ) , e cad ostruttiva ( 35 ; 14 eventi ) ; la differenza di prognosi nei tre gruppi risultata sempre signi cativa ( p < 0 , 05 )  . 
un infarto miocardico si veri cato in un paziente con cad ostruttiva . nella popolazione con storia di cad ( n = 127 ) , abbiamo osservato un totale di 16 eventi ( 13 rivascolarizzazioni e 3 eventi hard )  . 
i pazienti sono stati strati cati dalla ctca in coronarie normali ( n = 46 ; 0 eventi ) , cad non ostruttiva ( n = 56 ; 5 eventi ) e cad ostruttiva ( n = 25 ; 11 eventi ) ; la differenza di prognosi nei tre gruppi risultata sempre signi cativa ( p < 0 , 05 )  . 
an acute myocardial infarction occurred in one patient with obstructive cad . in the population without a history of cad ( n = 127 ) , we observed 16 events ( 13 revascularisations and three hard events )  . 
an acute myocardial infarction occurred in a patient with obstructive cad . in the population with a history of cad ( n = 18 ) , we observed four events ( three revascularisations and one hard event )  . 
in multivariate analysis , after adjustment for cardiovascular risk factors , hypercholesterolaemia and obstructive cad were signi cant predictors of events ( p < 0.05 ; table 4 )  . veri cate in pazienti con cad non ostruttiva e 7 in pazienti con cad ostruttiva . 
un infarto miocardico si veri cato in un paziente con cad ostruttiva . nella popolazione con storia di cad ( n = 18 ) , abbiamo osservato un totale di 4 eventi ( 3 rivascolarizzazioni e un evento hard )  . 
i pazienti sono stati strati cati dalla ctca in coronarie normali ( n = 2 ; 0 eventi ) , cad non ostruttiva ( 6 ; 1 eventi ) e cad ostruttiva ( n = 10 ; 3 eventi ) ; la differenza di prognosi nei 3 gruppi non risultata signi cativa a causa del basso numero di pazienti ( p > 0 , 05 )  . 
 nellanalisi multi - variata , dopo correzione per i fattori di rischio cardiovascolari factors , liper - colesterolemia e la cad ostruttiva sono risultati predittore signi cativi di eventi ( p < 0 , 05 ; tabella 4 )  . discussione il nostro studio dimostra che la ctca in grado di strati care in modo af dabile il rischio di eventi cardiovascolari in un follow - up a medio termine in pazienti con acp . 
 la frequenza di eventi cardiaci era signi cativamente pi elevata nei pazienti con cad ostruttiva quando confrontata con i pazienti con cad non ostruttiva , sia per quanto riguarda le rivascolarizzazioni sia per quanto riguarda gli eventi hard . 
the rate of events was signi cantly higher in patients with a known lanalisi multi - variata ha mostrato come gli unici predittori indipendenti di mace erano la presenza di ipercolesterolemia e di cad ostruttiva . 
altri predittori come il calcium score , il diabete mellito , e cos via , non sono emersi probabilmente a causa dei numeri relativamente bassi e del follow - up relativamente breve . 
acute chest pain represented 510% of patients accessing the er ; of those 7080% were classi ed as having nonanginal pain , 78% as having acute coronary syndrome and up to 78% as having nstemi and / or ua [ 21 , 22 ]  . 
the clinical classication between nonanginal chest pain and nstemi / ua is not always easy and sharp , and in such patients , ruling - out strategies may be more effective . 
decision making for patients in this cohort has important consequences at two levels : inappropriate hospital admission resulting in increased costs , and inappropriate discharge resulting in increased risk of cardiac events . 
the requirements for further testing are : simple and fast , reliable and able to detect / exclude signi cant cad and other major cardiothoracic sources of chest paa test with these features may allow us to reduce costs and mistakes / liability and may increase patient satisfaction [ 2124 ]  . it is known from previous studies based on cag that cad severity is an independent prognostic predictor [ 3 , 816 ]  . 
 [ 8 ] con rmed these ndings in a larger patient group ( n = 187 ) without known cad with a longer follow - up ( 20 months )  . 
in that study , the presence of signi cant cad and the extent of cad , de ned by a modi ed duke coronary artery score , were predictors of all - cause mortality [ 14 ]  . 
 [ 12 ] analysed 517 patients with suspected cad who underwent both myocardial perfusion scintigraphy ( mpi ) and ctca and reported an annualised event rate of 4.8% in patients 5%10% dei pazienti che accedono al pronto soccorso ; di questi il 70%80% vengono classi cati come dolore non anginoso , il 7%8% sono classi cati come sindrome coronarica acuta e no al 7%8% vengono classi cati come nstemi e / o ua [ 21 , 22 ]  . 
la distinzione clinica tra dolore toracico non anginoso e nstemi / ua non sempre facile e nitida , e questa una popolazione nella quale le strategie di esclusione ( rule - out ) possono risultare pi ef caci . 
lalgoritmo decisionale in questa coorte ha delle importanti ricadute a due livelli : ricoveri inappropriati che generano aumento dei costi , e dimissioni inappropriate che determinano un aumento del rischio di eventi cardiovascolari . 
i requisiti per ulteriori test sono : semplice e veloce , af dabile , capacit di rilevare e / o escludere cad signi cativa e altre cause cardio - toraciche maggiori di dolore toracico . 
un test con queste propriet potrebbe consentire una riduzione dei costi , degli errori / responsabilit , e potrebbe anche incrementare la soddisfazione dei pazienti [ 2124 ]  . noto da studi precedenti basati sulla cag che la severit della cad un predittore prognostico indipendente [ 3 , 816 ]  . 
 [ 11 ] hanno riportato , in una casistica di 100 pazienti con e senza cad nota , il valore prognostico della ctca ad un anno [ 11 ]  . 
 [ 8 ] hanno confermato queste osservazioni in una popolazione pi ampia ( n = 187 ) senza storia di cad e con un follow - up pi lungo ( 20 mesi )  . 
in questo studio , la presenza di cad ostruttiva e lestensione della cad , de nite mediante uno score coronarico modi cato di duke sono risultati predittore indipendenti per tutte le cause di morte [ 14 ]  . 
 [ 12 ] hanno analizzato 517 pazienti sottoposti a scintigra a miocardica da stress ( mpi ) e ctca con sospetta cad e hanno rilevato frequenza di eventi per anno di 4 , 8% nei pazienti con cad ostruttiva alla ctca e di 1 , 8% nei pazienti con cad moderata o assente . 
 [ 30 ] assessed the prognostic value of 64 - slice ctca in 220 patients with known or suspected cad during a mean follow - up of 144 months [ 30 ]  . 
at 1 year , patients with cad had a 34% event rate , whereas patients with normal coronary arteries had a cardiac event rate of 0% [ 30 ]  . 
in our study , we considered mace and hard events separately , because the rate of cardiac revascularisations during follow - up could be signi cantly in uenced by ctca results . 
short - term ( 30 days ) follow - up in patients with nonsigni cant cad ( as detected by ctca ) was extremely favourable . prognostic studies are important to determine whether patients in whom ctca rules out obstructive cad can be reassured and invasive coronary angiography can be safely avoided . 
in our study , patients without coronary atherosclerosis had an excellent prognosis ( 0% cardiac event rate at 20 months ) , thus con rming the high npv of ctca at follow - up . 
 study limitations the main limitation of our study is that the number of clinical events at follow - up is relatively low ( in particular , for the subgroup with a history of cad ) due to the population characteristics ( population at low to intermediate cardiovascular risk ) and to the relatively short follow - up . 
we included patients with both suspected and known cad , as usually encountered in clinical practice ; however , the proportion of patients with previous acute myocardial infarction or pci was limited ( 12% )  . 
nevertheless , at multivariate analysis , a history of cad was not an independent predictor . high radiation exposure in ctca with retrospective gating is still a matter a concern [ 3137 ]  . 
therefore , ad un anno , i pazienti con presenza di cad avevano una frequenza di eventi del 34% , mentre i pazienti con coronarie normali hanno mostrato 0 eventi [ 30 ]  . 
nel nostro studio abbiamo considerato i mace e gli eventi hard separatamente , poich la frequenza di rivascolarizzazioni coronariche durante il follow - up pu essere signi cativamente in uenzata dai risultati della ctca . 
il follow - up a breve termine ( 30 giorni ) nei pazienti con cad non signi cativa ( alla ctca ) risultato estremamente favorevole . gli studi prognostici sono importanti per determinare se i pazienti nei quali la ctca esclude la cad ostruttiva possono essere rassicurati ed possibile evitare una coronarogra a invasiva . 
nel nostro studio , i pazienti senza aterosclerosi coronarica hanno dimostrato una prognosi eccellente ( frequenza di eventi a 20 mesi pari a 0% ) , confermando lelevato valore predittivo negativo della ctca anche al follow - up . 
 limitazioni dello studio la principale limitazione dello studio consiste nel numero relativamente basso di eventi clinici al follow - up ( in particolare nel gruppo con storia di cad ) , causato dalle caratteristiche della popolazione ( popolazione a rischio bassointermedio ) , e follow - up relativamente breve . 
abbiamo incluso pazienti con cad nota e sospetta nel nostro studio , come accade di osservare nella pratica clinica , tuttavia la proporzione di pazienti con precedente ami o pci era ridotta ( 12% )  . 
in ragione delleterogeneit della popolazione e del numero relativamente basso di eventi , non abbiamo effettuato analisi sullassociazione tra la sede e le caratteristiche delle placche coronariche e la frequenza di eventi . 
tuttavia , allanalisi multi - variata la storia di cad allanalisi multi - variata non risultato un predittore indipendente . la dose di radiazioni elevate della ctca con gating ecg retrospettivo rimane un problema della metodica [ 3137 ]  . 
pertanto il bene cio dellutilizzo della ctca nel 704 radiol med ( 2011 ) 116 : 690705 the bene t of using ctca in the diagnostic workup must be counterbalanced by the possible risks related to radiation exposure . 
recently , a dose - modulation algorithm that reduces tube current by 80% during the systolic phase , and prospective ecg - triggering protocols , have been developed that allow a signi cant reduction of radiation exposure , down to 15 msv , with preserved diagnostic accuracy [ 3137 ]  . 
recentemente , sono stati sviluppati degli algoritmi di modulazione della dose di radiazioni che riducono la corrente del tubo dell80% durante la fase sistolica e dei protocolli di scansione prospettica basati sullecg che consentono una riduzione della dose no a 15 msv con accuratezza diagnostica preservata [ 3137 ]  . 
 conclusions conclusioni this study shows that ctca is able to reliably identify coronary atherosclerotic burden and to stratify cardiovascular risk at follow - up in patients with acp and suspected or known cad . 
bchler published online : 17 june 2011 springer - verlag 2011 dear editor : prognostic value of loss of wall enhancement for bowel infarction school of medicine , ponti cia universidad catlica , lira 44 , santiago , chile correspondence to : p . 
the study stated that loss of bowel wall enhancement was predictive of good outcome in patients with bowel infarction . i believe that a methodological mistake could have led moschetta and colleagues to wrong conclusions . 
a general rule of thumb is to use the fisher test when either the sample size is less than 30 or the expected number of observations in any cell of a 22 contingency table is less than ve [ 2 ]  . i performed the fisher exact test with the data published in table 3 and found a signi cant association with the patient outcome ( dead or alive ) in three out of ten signs evaluated ( wall thickening , intramural pneumatosis and pneumoperitoneum ) , instead of nine reported by the authors . 
the chi - square test showed an approximate p - value of 0.3 ( p = 0.260 ) rather than a p - value of 0.03 , as it is reported in the article . 
1 , east jian she road , zhengzhou , 450052 henan province , pr china correspondence to : x - w han , tel . : + 86 - 0371 - 65165352 , fax : + 86 - 0371 - 66971086 , e - mail : hanxinwei@tom.com. received : 28 april 2010 / accepted : 14 june 2010 / published online : 19 march 2011 springer - verlag 2011 abstract purpose . 
sono stati trattati consecutivamente con sems integrati a y otto pazienti con ostruzioni anastomotiche complesse che coinvolgevano lansa afferente ed efferente dopo gastro - digiuno - anastomosi ( billroth ii )  . 
in base al sistema di punteggio dellostruzione delloutlet gastrico ( gooss ) , il miglioramento in seguito allintervento stato statisticamente signi cativo ( p = 0 , 01 )  . 
durante il followup di 3 , 131 , 81 mesi , tutte le stenosi si sono risolte senza complicanze correlate allo stent e si osservato un miglioramento clinico in tutti gli otto pazienti . 
la media e la mediana dei periodi di sopravvivenza 760 radiol med ( 2011 ) 116 : 759765 proved to be an expedient , simple , safe and minimally invasive procedure for treating complex anastomotic stenoses after gastrojejunostomy ( billroth ii )  . keywords billroth ii stent anastomosis obstruction introduction over the last decade , self - expandable metallic stents ( sems ) have been utilised with increasing frequency in patients with gastric outlet obstruction ( goo ) , providing simple , safe and effective palliation [ 14 ]  . 
the use of an integrated y - sems , which is inserted under uoroscopic or endoscopic monitoring , has not been reported in the literature , with the exception of reports of a y - shaped stent that utilised two sems [ 513 ]  . 
during 2009 , we designed an integrated y - shaped sems ( microtech , nanjing , china ) specialised for patients with multiple anastomotic strictures after gastrojejunostomy ( billroth ii )  . 
 therefore , the purpose of this study was to report our initial experience with an integrated y - sems for patients with complex strictures after billroth ii . materials and methods patients this study was approved by the university committee on human investigation , and informed consent was obtained from each patient . 
from april 2009 to december 2009 , eight patients ( 3876 years , mean age 58.7511.17 years , six men , two women ) who suffered from complex stenoses involving both the afferent and efferent loops were treated with an integrated y - shaped sems ( micro - tech )  . 
il posizionamento di un sems a y integrato si dimostrato semplice , sicuro , minimamente invasivo ed adeguato al trattamento di stenosi anastomotiche complesse dopo gastro - digiuno - anastomosi ( billroth ii )  . parole chiave billroth ii stent anastomosi ostruzione by the interventional radiologist according to the multidetector ct or barium study . stent and delivery system the integrated , self - expandable , y - shaped , metallic stent was woven from threads of 0.24 - mm - diameter highly elastic nitinol ( nickel titanium , naval ordnance laboratory ) wire and consisted of three parts : body , afferent limb and efferent limb , as shown in figure 1 . 
the two limbs were also in a tubular con guration ( 22 mm in diameter , 2080 mm in length , respectively , depending on the patients afferent and efferent size )  . 
indication for use of the inverted y - stent included multiple stenoses or stula near the anastomosis and afferent and the efferent loops . the delivery system was a three - tier - structure y - stent delivery system , and the details of the delivery system have been previously described [ 14 , 15 ]  . 
the longer tubes are long at the proximal end and short at the distal end , whereas the shorter tubes are short at the proximal and long at the distal end . 
the body of the two shorter guiding tubes is also located in the pusher catheter , and the tails extend out of the back of the pusher catheter by about 5 cthe two bronchial stents are bound by two binding threads and xed at the proximal end of the two longer guiding tubes . 
finally , the outer tier is a 27 f ( 9 mm ) - diameter introducer sheath with a radiopaque marker ring at its proximal end and a lateral tube for ventilation at the distal end . 
the patient was placed in a right anterior oblique or supine position with the neck extended , and stenosis was identi ed by an injection of 2050 ml of 30% diluted nonionic contrast medium ( ultravist 300 ; schering , guangzhou , china )  . 
two guide wires ( radiofocus m ; terumo , tokyo , japan ) were inserted into the afferent and efferent loops and were exchanged using catheters ( torcon nb ; cook , bloomington , in , usa ) for two 0.035 - superstiff guidewires ( thsf ; cook , bloomington , in , usa )  . 
c withdrawal of the sheath over the pusher catheter and deployment of the stent while pushing the inverted y - stent to the anastomotic area and the two limbs of the stent into the afferent and efferent loops . 
contrast medium was injected through the catheter into the tracheal stent to assess correct sten placement and expansion . follow - up and evaluation of postoperative outcome a follow - up contrast swallow ( ultravist ) was obtained to evaluate stent patency and migration or expansion 37 days following placement . 
the follow - up protocols were performed at 1 , 3 , 6 and 12 months and annually after by one of the two authors and included a clinical and barium study . 
if follow - up at the outpatient clinic was not practical , patients or their families were contacted by telephone by one of the two authors every 1 or 2 months until the patient died . 
 technical success was de ned as successful stent insertion in the targeted position and the con rmation of patency using a combination of endoscopy and uoroscopy with oral contrast mediuclinical success was de ned as improvement in the obstructive symptoms and oral intake without vomiting 17 days after stent placement . 
the degree of dysphagia was assessed before and after stent placement using an adaptation of the goo scoring system ( gooss ) proposed by adler and baron [ 16 ] , with swallowing ability divided into four categories : 0 no oral intake ; 1 liquids only ; 2 soft solids ; 3 low - residue or full diet . statistical analysis data are presented as mean standard deviation . 
statistical analyses were performed using spss statistical software ( version 10.0 for windows , spss inc . , chicago , il , usa ) ; p < 0.05 was considered statistically signi cant . results technical and initial clinical results stent placement in the anastomotic areas was technically successful and well tolerated in all patients , with no procedure - related complications . 
five patients continued with follow - up health care after the procedures ; the remaining three died of the following causes unrelated to stent insertion : multiple organ failure ( n = 2 ) , diffuse metastasis ( n = 1 )  . 
to date , this is the rst report to describe treatment of multiple anastomotic obstructions after gastrojejunostomy with the use of an integrated y - shaped sems . local recurrence that causes obstruction occurs in approximately 20% of patients after gastrectomy for gastric carcinoma , with or without distant metastasis [ 17 , 18 ]  . 
most of these recurrent tumours are unsuitable for further resective or palliative surgery because of advanced malignancy , poor patient performance status or malnutrition and a high risk of signi cant morbidity and mortality rates [ 19 , 20 ]  . 
when the stenoses exist in the anastomotic area involving the afferent and efferent loops after gastrojejunostomy , a y - stent is often required for 764 radiol med ( 2011 ) 116 : 759765 successful palliation . 
the insertion of a y - stent created by two straight stents , on the one hand , did not t the characteristic of the anastomosis and , on the other hand , could not provide long - term patency of anastomosis due to interference of the stent threads , which resulted in early reobstructions [ 5 , 13 ]  . 
except for our previously designed integrated y - shaped sems and delivery system for treating complex stenoses or stulas in the carinal region , no other report was found in the literature regarding deployment techniques of the integrated y - shaped sems . 
 due to the different structure between the carinal area and the anastomosis after gastrojejunostomy , we devised another integrated yshaped sems specialised for patients with multiple anastomotic strictures after gastrojejunostomy . 
all patients in our study were successfully implanted with the integrated y - shaped sems . compared with the y - stent created by two straight stents , the integrated y - shaped sems has several distinct advantages : it is more suitable for the anatomy of the anastomosis , which provides long - term anastomosis patency and has less in uence on the function of the small bowel ; it is not prone to migration due to the interconnection of the body and the two limbs ; the procedure is relatively simple and rapid due to deployment of one integrated y - shaped sems with one procedure ; the stent can be retrieved easily . this study has the following limitations . 
second , application of the integrated y - shaped sems was not possible in some cases , when in anastomosis , the afferent and efferent loops were completely obstructed and dif cult to recanalise with the guidewire and the catheter . 
fundamentals and technical aspects springer - verlag , new york dordrecht heidelberg london , 2010 isbn 978 - 1 - 4419 - 6520 - 2 ( soft cover ) isbn 978 - 0 - 387 - 73506 - 1 ( hard cover ) e - isbn 978 - 1 - 4419 - 6521 - 9 doi 10.1007 / 978 - 1 - 4419 - 6521 - 9 published online : 8 march 2011 springer - verlag 2011 in this pocket - size , soft cover 241 page book ( hard cover edition published in 2008 ) prof . 
hendrick shares all his experience and knowledge he has gathered over the years as a researcher and teacher in the eld of breast magnetic resonance imaging ( mri )  . the topic is not so simple , yet the way it is addressed ( one - on - one teacher - student approach ) offers a continuous exchange of knowledge between the teacher and pupil driving to the expected mastering of the subject matter . starting from the fundamentals of magnetic resonance breast imaging one is taken through all its different modalities and sequences , learning how to use these and how mri works . 
 the author refers to the mathematically inclined reader in order to help him properly understand some of the presented sometimes at large formulas ; i would also include the physics inclined reader since without this sort of inclination and knowledge one would sometimes get lost . the book will be greatly appreciated by all those who are dealing with breast mri due to its more and more widespread use and its importance in the clinical and diagnostic setting . 
the less than 10 minute procedure time , due to the use of updated , and even dedicated , scanners and proposed protocols , makes breast mri competitive and offers more information ( just to quote the perfusion curves ) than mammography . in questo volumetto in brossura di 241 pagine dalle dimensioni tascabili ( ledizione con copertina rigida stata pubblicata nel 2008 ) , il prof . 
hendrick mette a disposizione del lettore tutta la sua esperienza e conoscenza accumulata nel corso degli anni come ricercatore ed insegnante nel campo della risonanza magnetica ( rm ) della mammella . largomento non dei pi semplici eppure limpostazione della sua esposizione , con un approccio viso a viso insegnante - studente , offre un continuo scambio di conoscenze tra il maestro e lallievo in modo tale da far raggiungere la padronanza voluta dellargomento . 
 iniziando dalle basi dello studio per immagini con risonanza magnetica della mammella , il lettore viene guidato attraverso le sue varie modalit e sequenze , imparando come usarle e , soprattutto , come funziona la rm . 
 lautore fa riferimento a quello che de nisce lettore incline alla matematica per aiutarlo a comprendere in modo adeguato alcune delle formule utilizzate ( talvolta in modo eccessivo ) ; da parte mia aggiungerei anche al lettore incline alla sica poich senza questa inclinazione e conoscenza lo stesso si troverebbe talvolta perso . questo libro sar di sicuro apprezzato da tutti coloro che hanno a che fare con la rm della mammella , visto il sempre pi ampio uso della stessa e la sua importanza nel campo clinico e diagnostico . 
il tempo di indagine inferiore ai 10 minuti , in funzione delluso di attrezzature sempre pi aggiornate , se non propriamente dedicate , nonch i protocolli proposti , rendono la rm della mammella competitiva , offrendo anche maggiori informazioni ( basti pensare alle curve di perfusione ) rispetto alla mammogra a . 824 radiol med ( 2011 ) 116 : 823824 a practical drawback : the way the book is bound makes reading it dif cult ( unless one wants to split open its spine )  . un unico inconveniente : la rilegatura del libro ne rende la lettura dif cile ( salvo il volerne rompere il dorso per facilitarla )  . 
mandale springer , new york heidelberg dordrecht london , 2010 isbn : 978 - 1 - 4419 - 1727 - 0 e - isbn : 978 - 1 - 4419 - 1728 - 7 doi 10.1007 / 978 - 1 - 4419 - 1728 - 7 published online : 8 march 2011 springer - verlag 2011 this book is a review crib and targets the radiologists - intraining preparing for their oral board exam in the usa . 
it encompasses a variety of cases simple as well as dif cult or unusual ones which should and could be of practical use not only to the student but also to the radiologist in the mammographic daily diagnostic work ow . there are 240 pages divided into two parts : the rst and largest reviews 79 mammography and ultrasound cases ; the second 13 magnetic resonance imaging ( mri ) cases . 
an appendix on interventional procedures and high yield facts and a very tight index closes the book . a few pages , at the beginning of each section , deal with mammographic and mri artefacts . each case is presented following a xed framework which presents on one page the patient history , radiology ndings , assessment ( breast imaging reporting and data system [ birads ] whenever possible ) , diagnosis , discussion and references ( no more than ve , usually two , to help the reader widen is the basic knowledge , just in case he / she wants to )  . 
 on the facing page , or sometimes the following page / s , well chosen and carefully reproduced x - ray images , ( coupled with ultrasonography [ us ] , mri and sometimes kinetic curves ) are displayed so making easy to simplify the comparison of clinical data and reported radiological ndings . since the book was supposed to be an aid for the board examination preparation , cases are organized in a random way , as the candidate would nd during his / her oral examination . 
in the same way , case reports and discussions are presented as a report would be written , presenting clinical data , differential diagnoses , possible outcome for the patient etc . 
in this way bi - rads assessment is important and one will nd its categories explained in the appendix . questo volume da considerasi un bigino di revisione rivolto agli specializzandi in radiologia in vista della loro preparazione per gli esami di abilitazione ( board ) negli stati uniti . 
esso comprende una variat di casi , dai semplici ai dif cili ai non comuni , che potrebbero essere di utilit non solo allo studente ma anche al radiologo nello svolgimento del lavoro mammogra co giornaliero . il volume consiste di 240 pagine ed diviso in due parti : la prima e pi consistente rivede 79 casi di mammogra e ed eco - mammogra e ; la seconda 13 casi studiati con risonanza magnetica ( rm )  . 
a chiusura del volume si trova unappendice sulle procedure interventistiche , su dati diagnostici importanti ed un indice analitico coinciso . allinizio di ciascuna parte alcune pagine sono dedicate agli artefatti sia mammogra ci che rm . 
ciascun caso presentato secondo uno schema sso e cio su di una pagina la storia del paziente , i riscontri radiologici , la valutazione ( breast imaging reporting and data system [ bi - rads ] quando possibile ) , la diagnosi , la discussione ed i riferimenti bibliogra ci ( non pi di cinque , di solito due , concepiti come un aiuto ad ampliare le conoscenze di base del lettore , sempre che lui / lei lo voglia )  . sulla pagina a fronte , talvolta sulla / e successiva / e vengono presentate immagini radiologiche ben scelte e riprodotte con cura , corredate da immagini ecogra che , rm e talvolta da curve cinetiche , in modo tale da rendere facile il confronto tra i dati clinici ed i reperti radiologici descritti . 
 dal momento che il volume stato concepito come un aiuto nella preparazione dellesame di abilitazione , i casi sono stati organizzati in modo casuale , tale e quale a quello che il candidato / a troverebbe nel suo esame orale . 
nello stesso modo i reperti e le discussioni sono presentati in maniera analoga a quella in cui un referto dovrebbe essere scritto , descrivendo i dati clinici , le diagnosi differenziali , le possibilit prognostiche per il paziente ecc . 
da questo punto di vista la valutazione bi - rads importante e la descrizione dei vari punti di tale classi cazione riportata nellappendice . in conclusione , questo libro raccomandato agli specia828 radiol med ( 2011 ) 116 : 827828 in conclusion , this book is recommended for trainees in radiology who will approach the dif culties of mammography , not only in view of an examination , but also on practical diagnostic grounds . 
as well it will be useful for grownups and experts and it should be kept on hand in every mammography diagnostic unit . lizzandi in radiologia che dovranno affrontare le dif colt insite nella mammogra a , non solo in funzione di un esame ma , soprattutto , in campo diagnostico pratico . 
simonetti department of diagnostic imaging , molecular imaging , interventional radiology and radiation therapy , policlinico tor vergata , viale oxford 81 , 00133 roma , italy correspondence to : g . 
sixty - four - slice ct coronary angiography provides accurate three - dimensional evaluation of the coronary artery tree with correct visualisation of any coronary anomalies , a relatively common nding that had a prevalence of 5.7% in our study population . 
la tc volumetrica consente unaccurata valutazione tridimensionale del circolo coronarico con visualizzazione delle anomalie coronariche che rappresentano un reperto relativamente comune nella popolazione con prevalenza del 5 , 7% . keywords coronary anomalies ct anatomical variants congenital anomalies parole chiave anomalie coronariche tc coronarica varianti anatomiche anomalie congenite 676 introduction visualisation of the coronary arteries has always constituted a challenge for diagnostic imaging because of their rapid movement during the cardiac cycle and the small size , tortuous course and intrinsic anatomical variability of the cardiac vessels . 
since the early 1990s , increasingly sophisticated technologies have been introduced in coronary diagnostics in an attempt to replace conventional coronary angiography ( ca ) , with the result that cardiac imaging has become a daily problem in routine clinical practice . 
over the past 10 years , multislice ct ( msct ) , especially after the advent of 64 - slice equipment , has rapidly gained credibility for the quality of results and reproducibility in the morphological evaluation of the coronary arteries [ 912 ]  . 
one of the strengths of ctca lies in its ability to provide a three - dimensional visualisation of the epicardial vessels [ 13 ]  . coronary artery anomalies are present at birth , but only a minority of them become manifest during the early years of life . 
nonetheless , some coronary anomalies may manifest with angina pectoris , infarction , syncope , more - or - less severe myocardial arrhythmias or even cardiac arrest and sudden death . 
the indications for the examination were several and included screening for coronary disease in asymptomatic and symptomatic patients , follow - up of patients previously treated with coronary stent or bypass , further investigation following ca or myocardial scintigraphy , electrocardiogram ( ecg ) abnormalities and follow - up of patients after ascending aorta or valve - replacement surgery . 
patients unable to radiol med ( 2011 ) 116 : 675689 introduzione la visualizzazione delle arterie coronarie ha sempre rappresentato una s da per la diagnostica non solo per il loro rapido movimento durante il ciclo cardiaco , ma anche per il calibro ridotto , il decorso tortuoso e lintrinseca variabilit anatomica nella vascolarizzazione cardiaca . 
dallinizio degli anni novanta , diverse tecnologie sempre pi avanzate e alternative alla coronarogra a , si sono affacciate sulla scena della diagnostica delle patologie coronariche , rendendo limaging cardiaco un problema quotidiano nella normale pratica clinica . 
nel corso dellultimo decennio la tomogra a computerizzata multistrato ( tcms ) , soprattutto dopo lintroduzione delle apparecchiature a 64 strati , ha rapidamente guadagnato credibilit per la qualit dei risultati ottenuti e per la riproducibilit nella valutazione morfologica delle arterie coronarie [ 912 ]  . 
uno dei punti di forza dellangio - tc delle coronarie consiste nella capacit di visualizzare in modo tridimensionale lanatomia dei vasi epicardici [ 13 ]  . le anomalie coronariche sono presenti dalla nascita , ma solo una piccola percentuale di esse si rende manifesta nei primi anni di vita . 
tuttavia alcune di esse possono presentarsi con angina pectoris , infarto miocardico , sincope , aritmie pi o meno severe o addirittura arresto cardiaco con gurando il quadro di morte improvvisa . 
in letteratura esistono prevalentemente descrizioni di anomalie coronariche basate sullangiogra a coronarica mentre sono poche quelle basate sulla tcms che presentano peraltro casistiche ridotte o popolazioni di studio gi molto selezionate [ 1417 ]  . 
lo scopo di questo lavoro quello di fornire la prevalenza di varianti e anomalie coronariche in unampia popolazione di pazienti sottoposti ad angiogra a coronarica con tcms a 64 strati . 
 materiali e metodi popolazione in esame da marzo 2005 ad agosto 2009 sono state eseguite 3236 angio - tc diagnostiche delle coronarie su 2133 maschi e 1103 femmine con et compresa tra 1884 anni ( et media 57 , 48 , 3 )  . 
la popolazione in studio eseguiva lesame secondo indicazioni estremamente varie : screening di malattia coronarica in pazienti asintomatici e sintomatici , follow - up in pazienti precedentemente trattati con stent coronarico o bypass , controllo indirizzato dallangiogra a coronarica o dalla scintigra a miocardica , anomalie in pazienti allelettrocardiogramma ( ecg ) , follow - up radiol med ( 2011 ) 116 : 675689 hold their breath for at least 1012 s were also excluded . 
our departments ethics committee gave its approval for the study protocol , and all patients underwent ct imaging after providing their signed informed consent . patient preparation patients with a heart rate > 70 bpm received premedication with oral beta - blockers 45 days before the ct examination . 
patients who continued to have a heart rate between 70 and 80 bpm were given beta - blockers ( 100 mg metoprolol tartrate orally 4550 min prior to the study or 550 mg metoprolol tartrate 1015 min before the study ) under close cardiological monitoring of heart rate and arterial blood pressure , together with benzodiazepines if necessary ( diazepam 12 mg per os 15 min before the examination )  . 
 premedication was only administered to patients who did not have speci c contraindications to the premedication drugs . scan protocol and image reconstruction the msct study was performed using a 64 - slice scanner ( lightspeed 64 ct , ge medical systems , milwaukee , wi , usa )  . 
scan parameters were as follows : axial thickness 2.5 mm ; eight images per rotation ; rotation time 0.35 s ; tube voltage 120 kv ; tube current 300 ma ; dedicated convolution kernel for the calcium score . 
the scan parameters of the postcontrast phase were slice thickness 0.625 mm ; interval 0.625 mm ; rotation time 0.35 s ; tube voltage 120 kv ; tube current 700900 ma for scans with retrospective gating and 400600 ma for those with prospective gating . 
a dose of 100 ml of iodinated contrast material ( 400 mgi / ml , iomeprol , iomeron 400 , bracco , milan , italy ) was administered as a rapid bolus at 5 ml / s , followed by 30 ml of saline solution at the same ow rate . 
contrast material was administered through an automatic injector ( stellant , medrad , pittsburgh , pa , usa ) connected to a 20 - gauge cannula previously placed in an antecubital vein of the right arto optimise opaci cation of the coronary vessels , the smart prep function was used . 
retrospective reconstructions were obtained , as needed , in different phases of the cardiac cycle ( 3090% , 510% increments )  . precedentemente operati allaorta ascendente o di sostituzione valvolare . 
non sono stati inclusi nello studio pazienti con controindicazioni assolute allesecuzione dellesame come : insuf cienza renale , distiroidismi , allergia documentata al mezzo di contrasto organo - iodato , gravidanza . 
il comitato etico del nostro dipartimento ha approvato il protocollo di studio e tutti i pazienti si sono sottoposti allesame dopo aver rmato un consenso informato . preparazione del paziente i pazienti con frequenza cardiaca superiore ai 70 bpm sono stati premedicati con beta - bloccanti per os 45 giorni prima dellesame . 
nei pazienti che continuavano ad avere una frequenza cardiaca compresa tra 7080 bpm si provveduto , sotto videat cardiologico con stretto monitoraggio di frequenza cardiaca e pressione arteriosa , alla somministrazione di beta - bloccanti ( 100 mg di metaprololo tartrato per os 4550 minuti prima dellesame ovvero 550 mg di metoprololo tartrato 1015 minuti prima dellesecuzione dellesame ) e alloccorrenza con benzodiazepine ( diazepam , 12 mg per os 15 minuti prima dellesecuzione dellesame )  . 
sono stati sottoposti a premedicazione solo i soggetti che non presentavano controindicazioni speci che alla somministrazione dei farmaci sopradescritti . protocollo di scansione e ricostruzione delle immagini lo studio tcms stato eseguito con scanner a 64 strati ( lightspeed 64 ct ; ge medical systems , milwaukee , wi , usa )  . 
i parametri utilizzati per la prima scansione sono stati i seguenti : spessore assiale 2 , 5 mm , 8 immagini per rotazione , tempo di rotazione 0 , 35 secondi , voltaggio del tubo radiogeno 120 kv , potenza del tubo radiogeno 300 ma , ltro di convoluzione dedicato per il calcium score . 
 lacquisizione con somministrazione di mezzo di contrasto stata effettuata in 2475 pazienti con gating restrospettivo , mentre nei restanti pazienti che presentavano una frequenza cardiaca stabilmente al di sotto dei 60 battiti al minuto stato utilizzato un gating prospettico per ridurre la dose di radiazioni ionizzanti somministrata . 
i parametri di scansione della fase contrastogra ca sono stati : spessore elicoidale 0 , 625 mm , intervallo 0 , 625 mm , tempo di rotazione 0 , 35 secondi , voltaggio del tubo radiogeno 120 kv , potenza del tubo radiogeno 700900 ma per le acquisizioni con gating retrospettivo , 400600 ma per quelle con gating prospettico . 
sono stati somministrati 100 678 data collection and statistical analysis three observers with 7 years experience with cardiac ct interpreted the examinations and compared their results to reach a diagnostic consensus . 
the 3 , 236 examinations were analysed on a dedicated workstation ( general electric advantage workstation 4.4 , healthcare , waukesha , wi , usa ) using a variety of postprocessing algorithms . 
for each examination , the following reconstructions were obtained : maximum intensity projections ( mip ) , multiplanar reconstructions ( mpr ) , curved mpr , volume rendering ( vr ) and , when needed , advanced vascular analysis ( ava ) and cardiac transparency images to facilitate viewing the coronary anatomy and identify all segments of the coronary tree . 
 the images were assessed for the following parameters : coronary dominance ( right , left , balanced ) ; anatomical variants : presence of intermediate branch , presence and number of diagonal and marginal branches ; coronary anomalies , subdivided anatomically into anomalies of origin and course , intrinsic coronary anomalies and termination anomalies . results are expressed as prevalence in absolute number and percentage of all anomalies identi ed and all ndings reported . results of the 3 , 236 patients , 2 , 851 ( 88.1% ) had right dominance , 275 ( 8.5% ) had left dominance and 110 ( 3.4% ) had balanced , codominant distribution ( table 1 )  . 
marginal branches had a very similar distribution : 51% of patients ( n = 1650 ) had at least one , 41.1% ( n = 1329 ) had two , 6% ( n = 194 ) had more than two and 1.9% ( n = 63 ) had none ( table 1 )  . the number of coronary anomalies amounted to 224 in 186 patients , given that 37 patients had more than one anomaly ( 36 had two ; one had three )  . 
the 89 anomalies of origin and course included : 24 anomalous origins of the right coronary artery ( rca ) , of which eight arose from the ascending aorta , eight radiol med ( 2011 ) 116 : 675689 ml di mezzo di contrasto iodato ( 400 mgi / ml , iomeprol , iomeron 400 , bracco , milano ) in bolo rapido a 5 ml / s , seguiti da un bolo di 30 ml di soluzione siologica alla stessa velocit . 
la somministrazione avvenuta tramite iniettore automatico ( stellant , medrad , pittsburgh , usa ) collegato ad unagocannula da 20 gauge preventivamente posizionata in una vena antecubitale del braccio destro . 
sono state effettuate alloccorrenza ricostruzioni retrospettive in diverse fasi del ciclo cardiaco ( 30%90% , incremento di 5%10% )  . raccolta dati e analisi statistica tre osservatori con unesperienza quotidiana in cardio - tc di 7 anni hanno visualizzato gli esami ottenuti e i singoli risultati sono stati confrontati per ottenere una concordanza diagnostica . 
sono state eseguite per ogni esame ricostruzioni maximun intensity projection ( mip ) , ricostruzioni multiplanari ( mpr ) , mpr - curved , volume rendering ( vr ) e alloccorrenza , advanced vascular analysis ( ava ) e immagini di trasparenza cardiaca , al ne di facilitare la visualizzazione dellanatomia coronarica e identi care la totalit dei segmenti dellalbero coronarico . 
sono stati quindi valutati i seguenti parametri : il bilanciamento della vascolarizzazione ( dominanza coronaria destra , sinistra , bilanciata ) ; le varianti anatomiche : presenza di ramo intermedio , presenza e numero dei rami diagonali e marginali ; le anomalie coronariche , suddividendole sulla base della classi cazione anatomica in : anomalie di origine e decorso , intrinseche della coronaria stessa e di terminazione . i risultati sono stati espressi come prevalenza in numero assoluto , percentuale sul totale delle anomalie riscontrate e sul totale dei reperti descritti . risultati dei 3236 pazienti studiati in 2851 , pari all88 , 1% , la dominanza della vascolarizzazione risultata destra , in 275 sinistra ( 8 , 5% ) ed in 110 bilanciata ( 3 , 4% ) ( tabella 1 )  . 
i dati riguardanti i principali rami marginali sono estremamente simili ai precedenti : il 51% dei soggetti ( n = 1650 ) ne presenta almeno uno , il 41 , 1% ( n = 1329 ) due , il 6% ( n = 194 ) pi di due e nell1 , 9% della popolazione in esame ( n = 63 ) non stato possibile visualizzarne neanche uno ( tabella 1 )  . le anomalie coronariche riscontrate nel nostro studio sono risultate 224 in 186 soggetti , in quanto 37 pazienti erano portatori di pi di unanomalia ( 36 pazienti ne presentavano due , 1 ne presentava tre )  . 
sono state evidenziate : 89 anomalie di origine e decorso ( 2 , 8% ) , 129 anomalie intrinseche della coronaria stessa ( 4% ) e 6 anomalie di terminazione ( 0 , 2% )  . 
i 24 aneurismi , di verosimile origine congenita e non aterosclerotica , erano cos suddivisi sullalbero coronarico : 13 a carico della coronaria destra , 1 sul tronco comune ( tc ) , 5 sulla da , 3 sulla cx e 2 su un ramo marginale . 
 680 radiol med ( 2011 ) 116 : 675689 table 2 prevalence of coronary anomalies expressed in absolute values , as a percentage of all anomalies and of the study population . 
prevalence in number and percentage of principal subtypes observed coronary anomalies number % anomalies % population number % population rca , right coronary artery ; lca , left coronary artery ; lad , left anterior descending artery tabella 2 prevalenza delle anomalie coronariche riscontrate , espresse in : numero assoluto sulla popolazione studiata , percentuale sulla totalit delle anomalie e sulla popolazione in esame . 
the 24 aneurysms , most likely congenital rather than atherosclerotic , were distributed as follows : 13 involved the rca , one the left main stem , ve the lad , three the cx and two a marginal branch . 
nonostante le anomalie coronariche siano spesso soltanto un reperto occasionale , una parte di esse si pu manifestare con una coorte di sintomi estremamente varia : angina pectoris , infarto miocardico , sincope , aritmie e morte improvvisa . 
il quadro di arresto cardiaco con morte improvvisa rappresenta la manifestazione clinica pi drammatica e si stima che il 19% delle morti improvvise in giovani atleti sia correlata con anomalie coronariche [ 18 ]  . radiol med ( 2011 ) 116 : 675689 fig . 
1a - c volume - rendered images of anomalies of origin and course : origin of left main coronary from the ascending aorta ( a , b ) ; origin of both coronary arteries from the ascending aorta ( c ) , the right coronary artery arises more laterally , on the left near the anterior commissure , with angulation and interarterial course ( the pulmonary artery is not visualised )  . 
origine del tronco comune ( tc ) dallaorta ascendente ( a , b ) ; origine di entrambe le coronarie dallaorta ascendente , la coronaria destra ( cdx ) origina in una posizione pi laterale a sinistra , con angolazione e decorso interposto tra aorta e arteria polmonare non visualizzata nellimmagine ( c )  . 
2a - d anomalies of origin and course : maximum intensity projection ( mip ) ( a ) and volume rendering ( vr ) ( b ) images of a circum ex artery arising from the right coronary with a retroaortic course . 
 aorta ( ao ) , arteria polmonare ( ap ) , atrio destro ( adx ) , atrio sinistro ( asn ) , ventricolo destro ( vdx ) , tronco comune ( tc )  . 682 radiol med ( 2011 ) 116 : 675689 fig . 
3a , b anomaly of origin : absence of left main coronary artery : maximum intensity projection ( a ) and volume rendering ( b ) images show a separate origin of the anterior descending artery and circum ex artery from the left sinus of valsalva . 
5a - d anomaly of volume rendering ( a , b ) and multiplanar projection images ( c , d ) demonstrate a thin right coronary artery ( cdx ) arising from the rst segment of a single left anterior descending artery ( da ) ; the left coronary terminates with a 2 - cm ( * ) aneurysm draining directly into the right ventricle . 
5a - d anomalia di immagini vr ( a , b ) e mpr ( c , d ) in un caso di monocoronaria sinistra con origine della coronaria destra ( cdx ) dal primo tratto della discendente anteriore ( da ) che presenta una formazione aneurismatica di 2 cm ( * ) a livello della super cie diaframmatica del cuore stolizzata con il ventricolo destro . 
 although coronary anomalies are often no more than an incidental nding , some may exhibit an extremely varied constellation of symptoms , including angina pectoris , myocardial infarction , syncope , arrhythmias and sudden death . 
cardiac arrest with sudden death is the most dramatic clinical manifestation , and it has been estimated that 19% of sudden deaths among young athletes are related to coronary anomalies [ 18 ]  . 
 criteria for de ning coronary normality / abnormality are as follows : normal , any morphological feature present in > 1% of an unselected population ; normal variant , an alternative , relatively uncommon , morphological feature seen in > 1% of the population ; anomaly , a rare morphological feature seen in < 1% of the population [ 19 ]  . per de nizione si considera : normale ogni condizione presente in pi del 1% di una popolazione non selezionata ; variante normale , unalternativa morfologica relativamente inusuale osservata in pi del 1% della popolazione ; anomalia , quadro morfologico raro osservato in meno del 1% della popolazione [ 19 ]  . la coronarogra a viene attualmente ancora considerata il gold standard per la diagnosi di anomalie coronariche e lincidenza riportata del 1 , 3% [ 15 ]  . 
inoltre la coronarogra a sottostima lincidenza reale dei ponti miocardici , potendo evidenziare solo quelli profondi ed in cui si veri ca una riduzione del lume in sistole [ 20 , 21 ]  . 
altre metodiche quali lecogra a ( sia transtoracica sia transesofagea ) e la risonanza magnetica ( rm ) presentano enormi limiti nella valutazione dellalbero coronarico potendo valutare generalmente solo lorigine o il tratto iniziale . 
any dif culty in catheterising the coronaries or interpreting coronary angiograms should always lead to a suspicion of some anomaly of orig evaluation of the course of the artery is often incorrect , as ca provides only a two - dimensional silhouette of the vessel lumen rather than panoramic , threedimensional images . 
moreover , ca underestimates the true incidence of myocardial bridges , as it is only able to detect deep myocardial bridges in which the lumen is reduced during systole [ 20 , 21 ]  . 
other modalities , such as ultrasonography ( both transthoracic and transoesophageal ) and magnetic resonance ( mr ) imaging are limited in evaluating the coronary tree , as they are generally able to assess only the origin or initial segment of the artery . 
electron - beam tomography has high diagnostic accuracy in the study of the coronary arteries , but its limited availability and high cost have considerably reduced clinical implementation [ 7 , 8 ]  . 
the use of multidetector - row equipment , the possibility of acquiring images synchronised with the electrocardiogram and the availability of several postprocessing protocols ( mip , mpr , curved mpr , vr , ava ) have made it possible for msct to depict the complex and tortuous coronary anatomy , its variants and major diseases [ 16 , 17 ]  . 
coronary dominance was right - sided in 88.1% , left - sided in 8.5% and balanced in 3.4% , in agreement with the main studies reported in the literature ( table 1 ) [ 14 , 15 , 19 ]  . 
moreover , at least two intermediate branches were detected in 0.8% of patients as a result of quadrifurcation of the left main ste our results regarding the number of diagonal and marginal branches of the lca were in part at variance with those reported by cademartiri et al . 
the presence of one or two branches either diagonal or marginal was seen in > 90% of patients in our study , with almost identical prevalence ( 47% ) of one or two branches among the diagonals , in contrast to the greater incidence of two or more diagonal branches reported by cademartiri et al . 
luso infatti di apparecchiature multidetettore , la possibilit di acquisire le immagini con sincronizzazione ecg e la disponibilit di poter usufruire di diversi protocolli di post - processing ( mip , mpr , mpr - curved , vr , ava ) hanno permesso alla tcms di dimostrare agevolmente la complessa e tortuosa anatomia coronarica , le sue varianti e le principali patologie [ 16 , 17 ]  . 
in circa quattro anni e mezzo sono state eseguite 3236 angiotc diagnostiche delle coronarie con tcms a 64 strati , tra queste stata dimostrata una dominanza destra nel 88 , 1% , sinistra nel 8 , 5% e dominanza bilanciata nel 3 , 4% , in accordo con i principali dati riportati in letteratura ( tabella 1 ) [ 14 , 15 , 19 ]  . 
nella nostra casistica la presenza di uno o due rami , sia diagonali che marginali , coinvolge pi del 90% degli individui , con una pressoch totale uguaglianza di prevalenza ( 47% ) tra le due condizioni per quanto riguarda i diagonali , al contrario della maggior incidenza di due o pi rami diagonali riportata dallo studio sopramenzionato . 
tuttavia in tale studio la popolazione era pi selezionata poich 67 soggetti , pari al 12 , 3% della popolazione studiata , eseguivano langio - tc delle coronarie con diagnosi o forte sospetto di anomalia coronarica alla coronarogra a . 
la prevalenza dei ponti miocardici ancora sconosciuta , compresa tra un range dello 0 , 5%4 , 9% negli studi angiogra ci al 15%85% delle autopsie [ 22 ]  . 
 however , their patient population was more selective , as 67 ( 12.3% ) patients underwent ctca due to a diagnosis or strong suspicion of coronary anomaly at conventional ca . 
the actual prevalence of myocardial bridges is probably overestimated by autoptic studies owing to the preparation and selection of the hearts undergoing autopsy , but underestimated by ca , which identi es only indirect signs of bridging such as reduced vessel lumen during systole , a feature present in only a portion of the deeper myocardial bridges . 
along its course within the myocardium , the epicardial vessel may undergo varying degrees of systolic compression , which may manifest as angina , myocardial infarction , left ventricular dysfunction , paroxysmal atrioventricular block and sudden death . 
myocardial bridges are considered to be responsible for infarction in approximately 6% of patients without evidence of atherosclerotic disease ; however , they cannot currently be considered a major additional risk factor for coronary atherosclerosis over and above the classical risk factors . 
an ectopic origin of the coronary arteries from the ascending aorta involving the rca in eight patients , the lca in six and the acute marginal branches in two does not generally give rise to clinical manifestations . 
however , this type of anomaly may cause dif culties in selective catheterisation during ca as a result of the inability to identify the coronary con tcms in pazienti con dolore toracico la prevalenza dei ponti miocardici si distribuisce in un range tra il 3 , 5% e il 30 , 5% . 
gli studi anatomo - patologici verosimilmente sovrastimano la reale prevalenza dei bridge miocardici per motivi di preparazione e di selezione dei cuori sottoposti ad autopsia , mentre la coronarogra a li sottostima , potendo basarsi solo su segni indiretti quale la riduzione del lume vasale in fase sistolica che presente solo in una parte dei ponti miocardici pi profondi . 
il vaso epicardico , nel passaggio allinterno del miocardio che si veri ca in questo tipo di anomalia , pu andare incontro a compressione sistolica di vario grado con possibilit di manifestarsi attraverso angina , infarto del miocardio , disfunzione del ventricolo sinistro , blocco atrio - ventricolare parossistico e morte improvvisa . 
i ponti miocardici sono ritenuti causa di infarto in circa il 6% dei pazienti che non presentano evidenza di malattia aterosclerotica , tuttavia allo stato attuale non possono essere considerati un fattore di rischio rilevante e addizionale per lo sviluppo dellaterosclerosi coronarica rispetto ai fattori di rischio classici della popolazione . 
tra queste sono stati evidenziati 24 casi di origine anomala della coronaria destra , 27 origini anomale della coronaria sinistra , associate in vario modo a 36 anomalie di decorso coronarico . 
le origini ectopiche delle coronarie dallaorta ascendente , riscontrate in 8 soggetti per quanto riguarda la coronaria destra , in 6 per la sinistra ed in 2 per i rami marginali acuti non comportano generalmente disturbi clinici . 
condizioni di questo tipo possono per indurre dif colt nella cateterizzazione selettiva durante coronarogra a , per impossibilit di individuare lostio coronarico nella normale sede bulbare e possono rischiare di essere recise durante interventi sullaorta ascendente o di sostituzione valvolare qualora non si conoscesse il loro decorso anomalo . 
 also associated with a high risk of sudden death , especially in young individuals undergoing intense physical exertion , are some types of ectopic origin of the coronaries from the contralateral sinus of valsalva . 
the ectopic origin of the lca from the right sinus of valsalva is generally associated with a septal course , which is less dangerous than an interarterial course [ 1 ]  . 
in our study , we found 12 cases of ectopic origin of the rca from the left side ( eight from the left sinus of valsalva and four from the lca ) and three cases of ectopic origin of the lca from the right sinus of valsalva . 
speci cally , we found drainage of the rca into the left atrium in one case and into a bronchial vessel in another , two cases of stula between the lad and left ventricle and one of stula between the lad and the pulmonary artery out ow tract , and one case of drainage between the left main stem and the pulmonary artery . coronary stula is an anomaly capable of causing or predisposing to myocardial ischaemia and congestive heart failure in that by draining into the right heart , it may lead to a leftright shunt with volume overload . 
demonstrated that 80% of patients younger than 20 years with coronary stula are asymptomatic on presentation [ 25 ]  . anchessa tuttavia pu essere soggetta a danno iatrogeno in corso di sostituzione valvolare per effetto compressivo . 
nel nostro studio abbiamo riscontrato 12 origini ectopiche della coronaria destra da sinistra ( 8 dal seno di valsalva sinistro e 4 dalla coronaria sinistra ) e 3 origini ectopiche della coronaria sinistra dal seno di valsalva destro . 
 in particolare abbiamo evidenziato : 1 caso di drenaggio della coronaria destra in atrio destro ed 1 caso in un vaso bronchiale ; 2 casi di stola tra da e ventricolo destro ed 1 tra da e tronco di ef usso della polmonare ; 1 caso di drenaggio tra tc e arteria polmonare . 
 la stola coronarica unanomalia in grado di determinare o predisporre ad un evento ischemico miocardico ed a scompenso cardiaco congestizio , in quanto , drenando nelle sezioni cardiache di destra , pu comportare uno shunt sinistro - destro con sovraccarico di volume . 
in primo luogo seppur la nostra popolazione in esame comprendesse individui di diverse razze ed etnie , almeno il 90% dei soggetti studiati era di razza caucasica ( 2133 maschi e 1103 femmine con et compresa tra 1884 anni , et media 57 , 48 , 3 )  . 
moreover , our study focused exclusively on the prevalence of coronary anomalies in the population without discussing their clinical relevance in relation to the literature [ 19 , 28 , 29 ]  . 
the cost - effectiveness of msct in relation to the hanno identi cato nel 52% dei casi valutati unorigine separata dellarteria coronaria discendente e dellarteria circon essa nel seno coronarico sinistro , mentre velican et al . 
 questo studio inoltre valuta esclusivamente la prevalenza nella popolazione delle anomalie coronariche senza valutare la rilevanza clinica delle stesse riferita a quanto noto in letteratura [ 19 , 28 , 29 ]  . 
per quanto riguarda i ponti miocardici sappiamo ad esempio che la rilevanza clinica diversa tra quelli super ciali e quelli profondi e in base alla loro lunghezza [ 20 ]  . 
lesposizione alle radiazioni in corso di un esame di questo tipo generalmente di 1520 msv a discapito dellesame coronarogra co che si pone a valori di esposizione inferiori che dif cilmente superano i 7 msv . 
unindagine di questo tipo pu essere effettuata anche nellinfanzia in pazienti con elevato rischio di malformazioni coronariche con unesposizione estremamente bassa compresa tra 0 , 17 e 0 , 65 msv con modulazione estrema di dose e trigger prospettico come riportato da goo et al . 
 conclusioni i miglioramenti nella risoluzione spaziale e temporale , nelle tecniche di gating ecg - correlate , negli algoritmi e nei software di post - processing hanno reso langio - tc coronarica una metodica accurata e af dabile nella diagnostica vascolare cardiaca con risultati anche superiori rispetto alle tradizionali tecniche diagnostiche invasive e non invasive . 
however , the indication for noninvasive coronary imaging to investigate suspected coronary anomalies or exclude their presence should be carefully considered together with the clinician to avoid exposing young patients to excessive doses of ionising radiation . 
with the availability of extreme dose modulation systems and prospective gating , this problem no longer exists owing to the possibility of obtaining a greater amount of diagnostic information compared with conventional ca , noninvasively , and with an equal or lower dose of ionising radiation . one group of patients who could bene t considerably from a noninvasive imaging modality such as ctca is that of young patients with exertional angina , syncope and ventricular tachyarrhythmias who have a low pretest likelihood of signi cant atherosclerotic disease on the basis of the medical history and clinical and laboratory data . finally , in light of the above considerations , we conclude that msct - ca should become the examination of choice in patients with suspected coronary anomaly . le anomalie coronariche sono un reperto relativamente frequente nel contesto dellimaging non invasivo coronarico . 
tuttavia , lindicazione allesecuzione di unindagine non invasiva coronarogra ca per sospetta anomalia coronarica o per escluderne la presenza deve essere ben ponderata in cooperazione con il clinico , al ne di evitare ad un paziente di giovane et uneccessiva esposizione a radiazioni ionizzanti . 
con gli attuali presidi di modulazione estrema dellesposizione e acquisizioni con trigger prospettico , tale problema non si pone per la possibilit di ottenere maggiori informazioni diagnostiche rispetto alla coronarogra a , in modo non invasivo e con una dose di radiazioni ionizzanti uguale o inferiore . un gruppo particolare di pazienti che potrebbe bene ciare notevolmente di una metodica di imaging non invasiva quale langio - tc coronarica rappresentato da pazienti giovani con angina da sforzo , sincope e tachiaritmie ventricolari e dai quali non ci si aspetta una patologia aterosclerotica signi cativa sulla base dei dati anamnestici e clinico - laboratoristici . alla luce di queste considerazioni possiamo in ne concludere che nel sospetto di anomalia coronarica lesame con tcms dovrebbe essere lindagine di prima scelta . conflict of interest none pediatric surgical diseases . 
 the topic is divided into 8 chapters ( head and neck ; thorax ; abdomen ; genitourinary disorders ; cardiovascular disorders ; musculoskeletal disorders ; emergency and trauma ; oncology ) each of which is headed by ( apart from the emergency and trauma section ) a more or less long introduction . the collection of cases ( 94 from italian hospitals , most from naples , the rest from abroad , even a clavicle fracture ! ) is relevant . each case is presented in one page ( radiological and clinical images ; brief history and salient laboratory data ; questions on how to deal with the problem and in this case further examinations in order to obtain a proper diagnosis ) , the discussion , further images and nal diagnosis are on the following page . this lay - out often requires that the reader must shuf e forward and backward , especially where more than one radiological image is presented . 
shouldnt these chapters have been merged ? is a bone fracture an emergency or a musculoskeletal disorder ? due editori , cinque editori associati , novantasei collaboratori hanno riunito le forze per preparare questo volume che in 530 pagine discute 249 casi clinici . la materia divisa in 8 capitoli ( capo e collo ; torace ; addome ; malattie dellapparato genito - urinario ; malattie cardiovascolari ; malattie muscolo - scheletriche ; urgenze e trauma ; oncologia ) ciascuno preceduto ( eccezion fatta per quello dedicato alle urgenze e trauma ) da una introduzione pi o meno lunga . la raccolta dei casi ( 94 da ospedali italiani , per lo pi da napoli , il resto dallestero , compresa una frattura di clavicola ! ) pertinente . ciascun caso presentato su di una pagina ( immagini radiologiche e cliniche ; breve storia e dati salienti di laboratorio ; quesiti su come comportarsi per risolvere il problema ed in tal caso quali ulteriori indagini richiedere per ottenere una diagnosi corretta ) , mentre la discussione , altre immagini e la diagnosi nale sono sulla pagina successiva . questa impostazione richiede spesso al lettore uno sfogliare avanti ed indietro di pagina , soprattutto nei casi in cui vi sia pi di una immagine radiologica . 
 vi inoltre un ulteriore inconveniente nella presentazione : i casi riguardanti alcune condizioni ( per esempio la stenosi ipertro ca del piloro , linvaginazione o lappendicite ) non sono presentati uno di seguito allaltro in modo tale da fornire al lettore lintero spettro delle loro possibili manifestazioni cliniche e radiologiche . 
questo fatto si ri ette anche nellindice ove la stessa condizione citata pi di una volta ed in modi differenti . motivo di perplessit poi il vedere come le stesse condizioni ( lappendicite per esempio ) siano state trattate nel capitolo dedicato alladdome e poi ancora in quello dedicato allurgenza e trauma . 
non sarebbe stato meglio uni carli ? una frattura unurgenza o una malattia muscoloscheletrica ? 826 radiol med ( 2011 ) 116 : 825826 going through the histories of the presented cases one will often read : the paediatrician , the surgeon or the doctor performed the examination ( a radiological examination )  . 
the paediatrician or the paediatric surgeon request and the radiologist performs the requested examination and makes a diagnosis ! at this point one realizes that only 26 of the presented cases are associated with a radiologists name . most of the images , clinical , surgical , radiological are of good quality , cleverly mixed and organized . 
 furthermore , just to quote some examples , one will nd incorrect image labelling ; images that are described and not to be found ; left for right side re uxing megaureter ; urethral duplication for ureteral duplication ; complete ureteric duplication on the right ( q / a 147 ) when one can only see a grade iii vesico - ureteric re ux etc . i also have some doubts regarding the roviraltas syndrome case ( q / a 92 ) : if one over lls the stomach with contrast medium and there is a tight hypertrophic stenosis as figure 2 shows , it is a rule of thumb that a gastro - oesophageal re ux will follow ( as in figure 1 )  . 
il pediatra o il chirurgo pediatra richiedono ed il radiologo esegue lindagine richiesta fornendo poi la diagnosi ! a questo punto ci si rende conto che solo 26 dei casi presentati sono associati al nome di un radiologo . la maggior parte delle immagini , cliniche , chirurgiche , radiologiche sono di buona qualit , disposte ed organizzate con intelligenza . 
tuttavia , si trovano alcuni brutti esempi di immagini radiologiche : non accuratamente ritagliate ; senza protezione gonadica ; estese a torace e addome in casi di urogra a . inoltre , solo per citare alcuni esempi , si potranno trovare errori nelle didascalie ; immagini che sono descritte e non sono presenti ; sinistro per megauretere re uente destro ; duplicit uretrale per duplicit ureterale ; duplicit ureterale destra completa ( q / a 147 ) , mentre osservabile solo un re usso vescico - ureterale di iii grado ecc . ho poi alcuni dubbi riguardanti il caso di sindrome di roviralta ( q / a 92 ) : se lo stomaco viene riempito in eccesso dal mezzo di contrasto ed presente una stenosi ipertro ca serrata come dimostrato dalla figura 2 , evidente che ne seguir un re usso gastro - esofageo ( come nella figura 1 )  . 
 un reperto conseguente al primo e non vice - versa . la presentazione delle voci bibliogra che discordante : si trovereanno molte e differenti modalit di presentazione , anche nello stesso elenco . 
in group a , the ct scan was performed with conventional bowel preparation ( a full cathartic dose and oral contrast medium to tag any residue in the 3 days preceding the study )  . 
in the second group , ct colonography was performed after a reduced bowel preparation , with the oral contrast medium for residue tagging being administered only on the day of the investigation . 
due gruppi di 40 pazienti asintomatici consecutivi , di et fra i 48 ed i 72 anni , sono stati sottoposti a colon - tc : nel gruppo a lesame tc stato eseguito con preparazione intestinale convenzionale ( catartico a piena dose e mezzo di contrasto orale per la marcatura dei residui nei 3 giorni precedenti lindagine ) ; il secondo gruppo ha eseguito la colon - tc dopo preparazione intestinale ridotta con somministrazione orale di contrasto per la marcatura dei residui solo il giorno dellindagine . 
in pazienti asintomatici , lutilizzo di un software per la sottrazione di liquidi in concomitanza ad 750 radiol med ( 2011 ) 116 : 749758 images in conjunction with reduced bowel preparation does not reduce examination quality or diagnostic performance when compared with the conventional ct colonography technique and is more acceptable to and better tolerated by the patient . una ridotta preparazione intestinale , non comporta una riduzione di qualit e performance diagnostica rispetto alla tecnica colon - tc tradizionale , mentre risulta pi accettabile e meglio tollerato da parte del paziente . parole chiave colon - tc tecnica di esame accettabilit keywords virtual colonoscopy ct colonography imaging technique acceptability introduction introduzione colorectal cancer is the second leading cause of death for cancer in the usa [ 1 ]  . 
the target of screening for colorectal cancer is intermediate - grade adenoma , characterised by a mean size of about 1 cm at diagnosis and the presence of a villous structure equal to at least 25% of the lesion [ 4 ]  . 
computed tomography ( ct ) colonography is able to con rm or exclude the presence of such lesions with levels of accuracy comparable with conventional colonoscopy and is therefore ideal for colorectal cancer screening [ 510 ]  . 
ct colonography performed with conventional bowel preparation , similar to that used for colonoscopy , involves some discomfort for the patient in the days before the examination as well as a risk of side effects , which makes this type of preparation neither suitable nor acceptable for a screening investigation [ 11 , 12 ]  . 
the introduction of oral contrast media for tagging residual stools and uid has led to a signi cant reduction in the level of required intestinal catharsis [ 1115 ] and the development of various combinations regarding the choice of contrast media ( ionic / nonionic ) and timing of contrast administration ( in the days preceding the examination or on the same day ) [ 14 , 19 , 20 , 22 , 23 ]  . 
however , few data are available with regard to the quality , acceptability and the diagnostic performance of ct colonography carried out with conventional preparation compared with simpli ed preparation and administration of oral contrast medium on the day of the ct scan [ 23 ]  . the aim of our study was therefore to compare examination quality , diagnostic performance and acceptability in two groups of patients who were recruited prospectively and studied with ct colonography following simplied bowel preparation and software for electronic residue subtraction , compared with patients receiving conventional bowel preparation . il carcinoma del colon - retto rappresenta la seconda causa di morte per patologie neoplastiche negli stati uniti [ 1 ]  . 
il target dello screening per neoplasia del colon - retto rappresentato dalladenoma di grado medio , caratterizzato da dimensioni medie alla diagnosi di circa 1 cm e dalla presenza di una struttura villosa pari ad almeno il 25% della lesione [ 4 ]  . 
la colon - tomogra a computerizzata ( colon - tc ) capace di confermare o escludere con accuratezza sovrapponibile alla colonscopia tradizionale la presenza di lesioni con tali caratteristiche , e pertanto si candida come indagine ideale per lo screening del carcinoma del colon - retto [ 510 ]  . 
la colon - tc effettuata con preparazione intestinale tradizionale , simile a quella utilizzata per la colonscopia , comporta un disagio per il pazienti nei giorni antecedenti lesame , nonch il rischio di effetti collaterali , rendendo tale preparazione non adatta n accettabile per gli studi di screening [ 11 , 12 ]  . 
recenti lavori hanno pertanto puntato lattenzione sullaumento della accettabilit dellesame da parte del paziente , ottenuto mediante una riduzione della complessit della preparazione intestinale o della restrizione dietetica richiesta [ 1123 ]  . 
lintroduzione dei mezzi di contrasto orali per la marcatura dei residui fecali e uidi ha reso possibile una signi cativa riduzione della catarsi intestinale normalmente richiesta [ 1115 ] e lo svilupparsi di varie combinazioni riguardanti la scelta del mezzo di contrasto da impiegare ( ionico / non ionico ) e le tempistiche di somministrazione degli stessi ( nei giorni precedenti lesame o il giorno stesso ) [ 14 , 19 , 20 , 22 , 23 ]  . 
tuttavia , sono disponibili pochi dati in letteratura riguardo la qualit , laccettabilit e la performance diagnostica della colon - tc effettuata con preparazione intestinale tradizionale standard e mediante preparazione intestinale sempli cata e somministrazione di contrasto orale il giorno stesso dellindagine tc [ 23 ]  . 
 lo scopo del nostro lavoro pertanto quello di confrontare qualit desame , performance diagnostica ed accettabilit dellindagine da parte di due gruppi di pazienti prospetticamente reclutati e sottoposti a colon - tc con proradiol med ( 2011 ) 116 : 749758 materials and methods patients and inclusion and exclusion criteria over a period of 15 months , 150 consecutive patients were considered for inclusion in the study . 
patients with intestinal polyposis ( n = 2 ) , acute abdomen either ongoing or over the previous month ( obstruction , perforation , acute diverticulitis ) ( n = 5 ) and patients with prior colorectal surgery ( n = 63 ) were excluded from the study . 
therefore , 80 patients were prospectively enrolled ( 48 men and 32 women , mean age 59 years , range 4872 years ) who underwent ct colonography for polyp detection . 
the patients underwent ct colonography due to the presence of faecal occult blood ( n = 37 ) , a family history of colorectal cancer ( n = 27 ) or for screening purposes ( n = 16 )  . diet , bowel preparation , residue removal in the 3 days preceding the survey , patients in both groups followed a low - residue diet , avoiding the intake of any bre - containing foods . 
in addition , they were instructed to consume only soup on the day before the ct scan and to fast from any solid food on the day of the scan . 
the rst group ( group a , n = 40 ) performed a conventional cathartic bowel preparation , the goal of which is to make the bowel lumen as free as possible from uid and faecal residue . 
in addition to the dietary restrictions described above , all patients in group a took intestinal cathartics ( isocolan , polyethylene glycol 4 , 000 , giuliani s.p.a , milan , italy ) , one packet of 34.8 g for each of the three main meals of the day , diluted in 500 ml of water in the 2 days preceding the scan ; at each meal , the cathartic was combined with meglumine amidotrizoate ( gastrogra n , bayer schering pharma , milan , italy ; with an iodine concentration of 370 mg / ml ) at a concentration of 10 ml diluted in 100 ml of water . 
in addition , on the day of the scan , all patients in group a received a glycerine enema and a nal dose of gastrogra n ( 25 ml ) during the 3 h preceding the study . the second group ( group b , n = 40 ) followed a simplied or partial bowel preparation with 13.8 g of the oral cathartic polyethylene glycol ( peg macrogol 3 , 350 , movicol , norgine s.r.l , italy ) diluted in 750 ml of water taken with each of the six main meals in the 2 days preceding the study . 
on the day of the examination , an oral contrast medium was administered ( meglumine amidotrizoate , gastrogra n , bayer schering ; iodine concentration of 370 mg / ml ) at a total dose of 50 ml diluted in 1 , 000 ml of water , starting 3 h before the scan . 
patients were also asked to drink the solution in a slow and continuous fashion in tocollo di preparazione intestinale sempli cata ed utilizzo di un software di sottrazione dei liquidi , a confronto con un protocollo di preparazione tradizionale . materiali e metodi pazienti , criteri di inclusione e di esclusione in un arco temporale di 15 mesi , sono stati considerati 150 pazienti consecutivi per linclusione nel nostro studio . 
sono stati esclusi dallo studio i pazienti affetti da sindromi polipose intestinali ( n = 2 ) , qualsiasi forma di addome acuto in atto o nel corso dellultimo mese ( occlusione , perforazione , diverticolite acuta ; n = 5 ) , ed i pazienti con pregressi interventi di chirurgia colo - rettale ( n = 63 )  . 
sono stati pertanto prospetticamente arruolati 80 pazienti ( 48 maschi , 32 femmine ; et media 59 anni , range 4872 anni ) sottoposti a colon - tc per la ricerca di formazioni polipoidi . 
i pazienti arruolati hanno effettuato la colon - tc per la presenza di sangue occulto nelle feci ( n = 37 ) , familiarit per carcinoma del colon - retto ( n = 27 ) , a scopo di screening ( n = 16 )  . 
 dieta , preparazione intestinale , rimozione dei residui nei tre giorni precedenti lindagine , i pazienti di entrambi i gruppi hanno seguito una dieta povera di scorie , evitando lassunzione di qualsiasi prodotto contenente bre . 
in maniera casuale , i pazienti arruolati sono stati suddivisi in due gruppi , in base alla preparazione intestinale effettuata : il primo gruppo ( gruppo a ; n = 40 soggetti ) ha eseguito una preparazione intestinale convenzionale , di tipo catartico , il cui scopo rendere il lume del colon il pi possibile libero da residui uidi e fecali : oltre alle restrizioni dietetiche sopra descritte , tutti i pazienti del gruppo a hanno assunto dei catartici intestinali ( isocolan , polietilenglicole 4000 , giuliani spa , milano , italia ) , 1 bustina da 34 , 8 g per ognuno dei 3 pasti principali della giornata , diluiti in 500 ml dacqua , nei due giorni antecedenti lesame ; ad ogni pasto , il catartico stato associato alla meglumina amidotrizoato ( gastrogra n , bayer schering pharma , milano , italia ; concentrazione di iodio pari a 370 mg / ml ) nella concentrazione di 10 ml , diluito in circa 100 ml dacqua . 
 il giorno dellindagine inoltre tutti i pazienti sono stati sottoposti a clistere di glicerina e somministrazione orale dellultima dose di gastrogra n ( 25 ml ) , in regime ambulatoriale il giorno dellesame , durante le 3 ore antecedenti lindagine . il secondo gruppo ( gruppo b ; n = 40 soggetti ) ha invece eseguito una preparazione intestinale sempli cata o parziale , con assunzione per via orale di un agente catartico 752 radiol med ( 2011 ) 116 : 749758 order to avoid excessive gastric distension and promote a constant intestinal peristalsis , thus ensuring homogeneous distribution of the contrast medium within the intestinal lumen . 
the topogram acquired at the beginning of the study was used to assess the proper progression of the contrast medium and decide on the best timing for the examination . examination protocol all ct colonography studies were performed with a multidetector ct unit ( brilliance 64 , philips , eindhoven , the netherlands ) and two acquisitions with the patient in the prone and supine positions , respectively . 
data were then reconstructed to a slice thickness of 2 mall patients were administered the spasmolytic agent hyoscine butylbromide ( buscopan , boehringer ingelheim , germany ) intramuscularly 10 min before the examination to reduce intestinal peristalsis and improve the quality of the examination . 
with the patient in a left lateral decubitus position , the colon was distended by insuf ating room air manually through a pump connected to a soft rectal tube equipped with a self - retaining balloon . 
 the extent of colonic distension was assessed on a caseby - case basis : colonic insuf ation was interrupted when the patient expressed discomfort , and in all cases , distension was assessed on the scout view before starting the scan and corrected , if necessary , to obtain appropriate distension . image analysis all images were evaluated by two experienced radiologists ( 15 and 16 years of experience in colorectal disease , respectively ) on a dedicated workstation , allowing the simultaneous twoand three - dimensional analysis of the images acquired . 
using bone window settings ( width 3 , 000 hu , centre 300 hu ) , the analysis was performed for each patient taking into account the six colonic segments : caecum , ascending colon , transverse colon , descending colon , sigmoid colon and rectuall studies were also evaluated with and without the use of software for residue subtraction . 
any doubtful ndings were discussed in consensus . examination quality for the two patient groups was evaluated on the basis of a qualitative analysis performed by the two readers , who assessed the following parameters : number of segments free of faecal residue ; residue volume in relation to the total segment volume in segments partially occupied by faecal residue , rated according to three subclasses : ( 1 ) < 10% ; ( 2 ) 1025% ; ( 3 ) > 25% . 
diagnostic performance in the detection of polyps 6 mm in diameter , based on identi cation of true - positive ( tp ) ndings and exclusion of false - positives ( fp ) was evaluated for both ( peg macrogol 3350 , movicol , norgine italia srl , italia ) nella dose di 13 , 8 g in 750 ml di acqua per pasto , nei sei principali pasti delle 2 giornate precedenti lindagine . 
il giorno stesso dellesame stato somministrato un mezzo di contrasto orale ( meglumina amidotrizoato , gastrogra n , bayer schering pharma , milano , italia ; concentrazione di iodio pari a 370 mg / ml ) , in una dose totale di 50 ml , diluito in 1000 ml di acqua , a partire da 3 ore prima dellesame . 
 i pazienti sono stati inoltre invitati ad assumere in maniera lenta e continua la soluzione sopra descritta allo scopo di evitare uneccessiva distensione gastrica e favorire invece una costante peristalsi intestinale che garantisse una distribuzione omogenea del contrasto nel lume intestinale . 
 il topogramma acquisito nelle fasi iniziali dellindagine stato utile a valutare la corretta progressione del liquido di contrasto ed a scegliere il tempismo migliore per lesecuzione dellindagine stessa . protocollo desame tutti gli esami colon - tc sono stati eseguiti con un apparecchio tc multistrato ( brilliance 64 , philips , eindhoven , olanda ) , con 2 acquisizioni rispettivamente con paziente in decubito prono e supino , con i seguenti parametri di scansione : spessore di fetta 1 mm , z = 1 , 120 kvp e 60 mas . 
i dati sono stati poi ricostruiti con uno spessore pari a 2 min tutti i pazienti stato utilizzato un agente spasmolitico ( buscopan , boehringer , ingelheim , germania ) intramuscolo 10 minuti prima dellesame per ridurre la peristalsi intestinale ed aumentare la qualit desame . 
con il paziente in decubito laterale sinistro , il colon stato disteso utilizzando aria ambiente , progressivamente pompata manualmente tramite pompetta collegata ad un tubo rettale morbido dotato di pallone di contenzione . 
lentit della distensione colica stata valutata di caso in caso : linsuf azione colica veniva interrotta nel caso in cui il paziente lamentasse dolore , in tutti i casi era valutata sul topogramma prima dellesecuzione dellindagine ed eventualmente veniva poi corretta no ad ottenere una distensione idonea . 
 analisi delle immagini tutte le immagini sono state valutate da 2 radiologi esperti in patologia colo - rettale ( 15 e 6 anni di esperienza rispettivamente ) , su una workstation dedicata che permetteva lanalisi simultanea bie tridimensionale delle immagini acquisite . 
utilizzando una nestra di visualizzazione per i tessuti ossei ( ampiezza 3000 hu , centro 300 hu ) , lanalisi stata eseguita considerando , per ogni paziente , 6 segmenti colici : cieco , colon ascendente , colon trasverso , colon discendente , colon sigma e retto . 
tutti i rilievi dubbi sono stati discussi in consenso . la qualit desame , per i due gruppi di indagini eseguite con differente protocollo , stata valutata in base ad radiol med ( 2011 ) 116 : 749758 techniques using as a reference - standard breoptic colonoscopy performed only in patients with suspected colorectal polyps on ct colonography . the acceptability of the bowel preparation protocol was assessed with the aid of a self - completed questionnaire and scored on a scale from 1 ( nearly total unacceptability of the protocol ) to 10 ( no discomfort associated with the protocol )  . statistical analysis interobserver variability in determining the presence or absence of polyps and assessing examination quality was evaluated using the statistic . 
a p value < 0.05 was considered statistically signi cant . distribution of faecal residue ( stool and uids ) within the six colonic segments in the two groups of patients is outlined in table 1 . 
in group a , 180 / 240 segments were free una analisi qualitativa espletata dai 2 radiologi coinvolti , i quali hanno valutato i seguenti parametri : numero di segmenti liberi da residui ; nei segmenti parzialmente occupati da residui , volume degli stessi in relazione al volume totale del segmento preso in esame , considerando 3 sottoclassi : ( 1 ) inferiore al 10 % , ( 2 ) dal 10% al 25% , ( 3 ) superiore al 25% . 
la performance diagnostica nellevidenziare polipi con dimensioni uguali o superiori a 6 mm , basata sullidenti cazione di rilievi veri positivi ( vp ) e sullesclusione dei falsi positivi ( fp ) , stata valutata per entrambe le tecniche desame , utilizzando come standard di riferimento la colonscopia ottica , eseguita solo nei pazienti con sospetto di polipi alla colon - tc . 
 laccettabilit della preparazione effettuata stata valutata mediante questionario auto - compilativo da parte dei pazienti coinvolti , considerando una scala di valori di accettabilit compresa fra 1 ( pressoch totale inaccettabilit della preparazione ) e 10 ( nessun fastidio connesso con la preparazione )  . 
 la concordanza inter - osservatore stata considerata scarsa per valori di inferiore o uguale a 0 , 20 , discreta per compresa tra 0 , 21 e 0 , 40 , moderata per tra 0 , 41 e 0 , 60 , table 1 qualitative evaluation of the amount of faecal or uid residue not subtracted or insuf ciently removed by the software in the two study populations undergoing conventional full - dose ( group a ) and reduced - dose ( group b ) bowel preparation residue absent < 10% 10%25% > 25% group a group b group a group b group a group b group a group b tabella 1 stima qualitativa della quantit di residui non sottratti o insuf cientemente rimossi dal software , nelle due popolazioni di studio sottoposte a preparazione intestinale convenzionale ( gruppo a ) e parziale ( gruppo b ) residui assenti < 10% 10%25% > 25% gruppo a gruppo b gruppo a gruppo b gruppo a gruppo b gruppo a gruppo b 180 180 165 165 results caecum ascending transverse descending sigmoid rectum total cieco ascendente trasverso discendente sigma retto totale 754 radiol med ( 2011 ) 116 : 749758 fig . 
1a - d a 56 - year - old woman with a family history of colon cancer undergoing conventional bowel preparation with oral administration of gastrogra n in the 3 days prior to the examination , together with a full dose of the cathartic drug ( group a )  . 
1a - d donna di 56 anni con familiarit per carcinoma del colon sottoposta a preparazione intestinale convenzionale con somministrazione di gastrogra n per os nei tre giorni antecedenti allindagine unitamente allingestione orale dellagente catartico ( gruppo a )  . 
 le immagini di partizione ottenute sui piani assiale e coronale prima ( a , b ) e dopo utilizzo del software di sottrazione ( c , d ) dimostrano una pressoch completa replezione del colon con il mezzo di contrasto orale , correttamente rimosso per azione del software stesso , garantendo una elevata qualit desame . 
in group a , 16 / 17 polyps were identied ( with six fp ) , whereas in group b , 12 / 13 polyps were detected ( with ve fp ) compared with conventional colonoscopy performed on patients with positive or doubtful ndings on ct colonography ( table 2 )  . 
inter - observer agreement was excellent both for identifying polyps ( = 0.94 ) buona per tra 0 , 61e 0 , 80 , ed eccellente per tra 0 , 81 e 1 . 
2a - d a 68 - year - old woman with positive faecal occult blood test undergoing reduced intestinal preparation with oral administration of the contrast material on the day of examination ( group b )  . 
2a - d donna di 68 anni con sangue occulto nelle feci , sottoposta a preparazione intestinale sempli cata con somministrazione di contrasto orale il giorno stesso dellindagine ( gruppo b )  . 
 the self - completed questionnaires revealed greater acceptance patients undergoing the partial bowel preparation protocol compared with patients undergoing conventional preparation ( p = 0.01 ) : the mean score for patients in group a ( conventional preparation ) was 6 ( range 48 ) , whereas that for patients in group b ( partial preparation ) was 9 ( range 610 )  . discussion the technological advances of ct scanners , the relative speed and accessibility of the examination and its good overall accuracy in identifying colorectal polyps make ct colonography a potential candidate for colorectal cancer screening [ 59 ]  . 
conventional bowel preparation , similar to that required for breoptic colonoscopy , ensures a good quality examination but is often associated with marked patient discomfort in the days before the test , leading to poor patient acceptance . 
the presence of faecal residue ( stool or uid ) within the colonic lumen is a major cause of poor examination quality , leading to an increased risk of qualit desame ( p > 0 , 05 )  . 
anche in questo gruppo i residui sono stati mediamente pi abbondanti nel sigma , nel discendente e nel retto . abbiamo riscontrato una differenza non signi cativa per quanto riguarda i settori del colon liberi da residui ( p = 0 , 09 ) , e quelli con maggior quantit di residui ( p = 0 , 124 )  . 
nel gruppo a sono stati identi cati 16 / 17 polipi ( con 6 fp ) , mentre nel gruppo b sono stati identi cati 12 / 13 polipi ( con 5 fp ) a confronto con la colonscopia tradizionale , espletata solo nei pazienti con rilievi positivi o dubbi alla colon - tc ( tabella 2 )  . 
la concordanza inter - osservatore stata eccellente sia per quanto riguarda la identi cazioni di lesioni polipoidi ( = 0 , 94 ) , che per quanto concerne la valutazione della qualit dindagine ( = 0 , 92 )  . 
dai questionari auto - compilativi emersa una maggiore accettabilit da parte dei pazienti sottoposti a preparazione intestinale parziale rispetto a quella convenzionale ( p = 0 , 01 ) : la valutazione media dei pazienti del gruppo a , sottoposti alla preparazione convenzionale , stata di 6 ( range 48 ) , mentre quella del gruppo b , sottoposti a preparazione parziale , stata di 9 ( range 610 )  . 
 756 radiol med ( 2011 ) 116 : 749758 table 2 trueand false - positive ndings in the two subgroups of patients undergoing different intestinal preparations , using as the reference standard breoptic colonoscopy , performed only in the subset of patients presenting positive or doubtful ndings at computed tomography colonography discussione patients true positives , n ( % ) false positives , n group a group b 16 / 17 ( 94.1 ) 12 / 13 ( 92.3 ) tabella 2 rilievi veri positivi e falsi positivi delle due popolazioni di studio , sottoposte a diversa preparazione intestinale , a confronto con i risultati della coloscopia ottica , eseguita nei soli pazienti con rilievi alla colon - tc pazienti veri positivi , n ( % ) falsi positivi , n gruppo a gruppo b 16 / 17 ( 94 , 1 ) 12 / 13 ( 92 , 3 ) error [ both false negatives ( fn ) and fp ] and longer image interpretation times [ 15 , 22 ]  . the introduction of new and less demanding bowel preparation protocols with limited dietary restriction and / or a lower doses of intestinal cathartics in the days preceding the study along with the possibility of administering oral contrast material for faecal tagging on the day of the examination and the development of residue - removal software has made possible a real increase in the acceptability of ct colonography by patients [ 15 , 18 , 23 ]  . 
in agreement with the literature , our results show that there is no signi cant difference in the quality of ct colonography examinations performed after a reducedor full - dose bowel catharsis , thanks to the use of uid - subtraction software . 
although not demonstrated in our study , the success of the simpli ed bowel preparation protocol can be attributed to a greater adherence to the simpli ed preparation on the part of patients , compared with the conventional protocol . 
a further increase in patient acceptance of the various preparation protocols could result from not imposing any dietary restrictions on the study population , as demonstrated by a recent study by liedenbaum et al . 
although those authors concluded that there was no loss of examination quality in the patient group with unrestricted diet compared with the group prescribed a lowbre diet [ 15 ] , we did not introduce this parameter in our analysis because we are convinced that dietary restriction is one of the smaller inconveniences of bowel preparation , with the extent of catharsis being more upsetting in direct proportion to the development of diarrhoea and abdominal pain . nonetheless , despite the absolute quality of investigations obtained following partial preparation being slightly lower grazie alla innovazione tecnologica delle apparecchiature tc , alla relativa rapidit ed accessibilit allindagine , ed alla buona accuratezza globale nellidenti cazione dei polipi colo - rettali , la colon - tc si candida come potenziale indagine per lo screening del carcinoma del colon - retto [ 59 ]  . 
la preparazione intestinale convenzionale , simile a quella richiesta per la colonscopia ottica , garantisce attualmente una buona qualit desame , ma spesso associata ad un marcato disagio da parte dei pazienti nei giorni antecedenti lesame , rendendo laccettabilit dellindagine ancora scarsa . 
la presenza di residui allinterno del lume colico , sia di natura liquida sia di natura fecale , rappresenta unimportante causa di riduzione della qualit desame , condizionando un relativo aumento del rischio di errore sia in negativo ( falsi negativi ) , che in positivo ( falsi positivi ) , e pu portare ad un aumento dei tempi di lettura [ 15 , 22 ]  . 
 lintroduzione di nuovi e meno impegnativi protocolli per la preparazione intestinale , con limitata restrizione dietetica e / o riduzione della dose di catartico intestinale nei giorni antecedenti lindagine , insieme alla possibilit di somministrare il mezzo di contrasto orale per la marcatura fecale il giorno stesso dellesame ed al perfezionamento dei software di rimozione dei residui , ha reso possibile un aumento concreto dellaccettabilit da parte dei pazienti dellindagine colon - tc [ 15 , 18 , 23 ]  . 
in accordo con i dati della letteratura , i nostri risultati dimostrano come non ci sia una differenza signi cativa nella qualit desame colon - tc eseguita previa catarsi intestinale parziale o a piena dose , anche grazie allutilizzo del software di sottrazione liquidi . 
 anche se non dimostrato dal nostro studio , il successo della preparazione intestinale sempli cata da ricercarsi nella maggiore adesione al protocollo di preparazione da parte dei pazienti , rispetto alladesione al protocollo tradizionale . 
nonostante questi autori concludano come non vi sia stata perdita di qualit nel gruppo di pazienti senza restrizioni dietetiche rispetto al gruppo sottoposto a dieta povera di bre [ 15 ] noi non abbiamo introdotto questo parametro nella nostra analisi perch convinti che la restrizione dietetica rappresenti uno dei minori fastidi connessi alla preparazione , essendo fastidio maggiore lentit della catarsi direttamente proporzionale allo sviluppo di diarrea ed addominalgia . 
 in ogni caso , nonostante la qualit assoluta delle indagini acquisite nei pazienti sottoposti a preparazione parziale sia stata lievemente inferiore a quella del gruppo di pazienti sottoposti a preparazione catartica tradizionale , la differenza qualitativa fra le due popolazioni non stata signi catiradiol med ( 2011 ) 116 : 749758 than that obtained after conventional cathartic preparation , the qualitative difference between the two groups was not signi cant . 
speci cally , the sigmoid and descending colon , with the right colon , were the segments with most residue in both groups of patients , with no statistically signi cant difference between groups . 
as expected , and in agreement with the results of other recent reports [ 15 , 22 , 23 ] , our study con rmed greater acceptance of the investigation by the patients in group b when compared with those in group a . although the small sample size renders the statistical validity of our preliminary results rather modest , the overall diagnostic accuracy was comparable between the two groups of patients despite the small qualitative difference encountered between the two protocols being tested . 
an additional advantage of the partial bowel preparation with administration of oral contrast medium on the day of the examination is the possibility of preventing , monitoring and promptly treating any side effects arising as a result of the partial absorption of the oral contrast medium by the intestinal wall [ 24 ]  . this study has its limitations . 
this said , however , the excellent results obtained lay the foundation for re ning the simpli ed intestinal preparation protocol and undertaking further studies involving a larger number of patients . 
 secondly , conventional colonoscopy was performed only in patients with positive ndings at ct colonography , and therefore , the real impact of fn ndings may have been underestimated , giving less reliable estimates of overall accuracy . 
nello speci co , il sigma ed il colon discendente , insieme al colon destro , sono risultati i settori con maggior quantit di residui nei due gruppi di pazienti , senza alcuna differenza statisticamente signi cativa fra i due sottogruppi sottoposti a differente preparazione . 
come atteso , ed in accordo con i risultati di altri lavori recentemente pubblicati [ 15 , 22 , 23 ] , il nostro studio ha confermato un aumento dellaccettabilit dellindagine da parte dei pazienti del gruppo b rispetto al gruppo a . 
 in ne , anche se lesiguit del campione analizzato rende modesta la validit statistica dei risultati preliminari nora ottenuti , laccuratezza diagnostica globale stata sovrapponibile nei due gruppi di pazienti nonostante la modesta differenza qualitativa ottenuta con le due preparazioni testate . 
tra gli altri vantaggi della preparazione intestinale parziale con somministrazione di mezzo di contrasto orale il giorno dellesame vanno annoverati la possibilit di prevenire , monitorare e curare tempestivamente eventuali effetti collaterali che possano insorgere durante lassunzione di contrasto per via orale , in relazione al parziale assorbimento dello stesso dalle pareti intestinali [ 24 ]  . questo studio presenta dei limiti . 
in primo luogo abbiamo arruolato un numero relativamente limitato di pazienti ; tuttavia , gli ottimi risultati ottenuti pongono le basi per il perfezionamento del protocollo di preparazione intestinale sempli cata , e per lesecuzione di ulteriori studi che arruolino un numero pi ampio di pazienti . 
secondo , la colonscopia tradizionale stata eseguita solo nei pazienti con rilievi positivi alla colon - tc e di conseguenza il reale impatto dei rilievi falsi negativi pu essere stato sottostimato , rendendo poco attendibili le stime di accuratezza globale . 
orsi1 1interventional radiology unit of european institute of oncology , via ripamonti 435 , 20141 milan , italy 2clinical center for tumor therapy of 2nd hospital of chongqing university of medical sciences , chongqing 400010 , china 3division of senology of european institute of oncology , via ripamonti 435 , 20141 milan , italy 4school of medicine , university of milan , milan , italy correspondence to : g . 
 from september 2008 to april 2009 , 22 patients with 29 lesions were treated : nine patients with liver and / or softtissue metastases from colorectal carcinoma ( crc ) , six with pancreatic solid lesions , three with liver and / or bone metastases from breast cancer , one with osteosarcoma , one with muscle metastasis from lung cancer , one with iliac metastasis from multiple myeloma and one with abdominal liposarcoma . 
the mean diameter of tumours was 4.2 call patients were evaluated 1 day , 1 month and 3 months after hifu treatment by multidetector computed tomography ( mdct ) , positron - emission tomography ( pet ) - ct and clinical evaluation . 
da settembre 2008 ad aprile 2009 sono stati trattati 22 pazienti con 29 lesioni : 9 pazienti con metastasi epatiche e / o dei tessuti molli da carcinoma del colon retto ( crc ) , sei pazienti con lesioni solide del pancreas , tre con metastasi epatiche e / o ossee da tumore mammario , uno con osteosarcoma , uno con metastasi muscolare da tumore del polmone , uno con lesione iliaca da mieloma multiplo ed uno con liposarcoma addominale . 
il diametro medio era di 4 , 2 ctutti i pazienti sono stati valutati ad 1 giorno , 1 mese e a 3 mesi di distanza dal trattamento hifu con tomogra a computerizzata multidetettore ( mdct ) , tomogra a computerizzata con tomogra a ad emissione di positroni ( pet - ct ) e valutazione clinica . 
pet - ct e / o mdct hanno mostrato risposta completa in 11 / 13 metastasi epatiche ; tutte le lesioni ossee , dei tessuti molli e radiol med ( 2011 ) 116 : 734748 ablated at mri . 
according to our preliminary experience in a small number of patients , we conclude that hifu ablation is a safe and feasible technique for locoregional treatment and is effective in pain control . keywords high - intensity focused ultrasound ( hifu ) solid tumours ablation le lesioni pancreatiche sono state palliate nei sintomi , con risposta completa allesame pet - ct , mdct o risonanza magnetica ( rm ) ; il liposarcoma ha mostrato una ablazione quasi completa allesame rm . 
secondo la nostra esperienza preliminare da un limitato numero di pazienti , lablazione usghifu pu essere considerata una metodica sicura e fattibile in assenza di alternative terapeutiche locoregionali e valida per il controllo del dolore . parole chiave ultrasuoni focalizzati ad elevata intensit ( hifu ) tumori solidi ablazione introduction introduzione high - intensity focused ultrasound ( hifu ) is a highly precise medical procedure that burns and destroys tumour tissue selectively and without harming overlying and adjacent structures within the path of the beathe possibility that fu therapy might be developed as a result of controlling local heating phenomena was introduced by lynn et al . 
the advent of more sophisticated imaging has led to a resurgence of interest in hifu . currently , both b - mode ultrasonography ( us ) and magnetic resonance imaging ( mri ) have been incorporated into hifu devices . 
in the past few years , several clinical hifu projects have been conducted by various research groups , and signi cant results showed that hifu treatment would be safe , effective and feasible in clinical application [ 36 ]  . 
experience from chinese authors with us - imaging - guided hifu ( usghifu ) for treating solid malignant tumours includes primary and metastatic liver cancer , malignant bone tumour , breast cancer , soft - tissue sarcoma , kidney cancer , pancreatic cancer and bone metastatic tumours [ 717 ]  . 
the group from chongqing , china , reported the largest series of hifu clinical applications to date , in which 1 , 038 patients were treated with usghifu in ten centres with both curative and palliative purposes [ 6 ] , showing promising results . 
using either radiological gli ultrasuoni focalizzati ad alta intensit ( hifu ) rappresentano una tecnica altamente precisa che consiste nellutilizzo di ultrasuoni focalizzati per bruciare e distruggere il tessuto tumorale situato in profondit nel corpo , in modo selettivo e senza danneggiare le strutture interposte nel percorso del fascio . 
negli anni passati sono stati condotti diversi progetti clinici hifu da vari gruppi di ricerca e i risultati pi signi cativi hanno dimostrato che il trattamento hifu sarebbe stato sicuro , ef cace e realizzabile nellapplicazione clinica [ 36 ]  . 
lesperienza degli autori cinesi con lhifu che utilizza la guida ecogra ca ( usghifu ) nel trattamento dei tumori solidi maligni include il tumore epatico primitivo e metastatico , il tumore osseo maligno , il cancro della mammella , il sarcoma dei tessuti molli , il tumore renale , il tumore del pancreas e i tumori ossei metastatici [ 717 ]  . 
il gruppo di chongqing no ad oggi ha riportato il maggior numero di applicazioni cliniche per hifu , nelle quali 1038 pazienti sono stati tratti con hifu sotto guida ecogra ca sia con nalit curative che palliative [ 6 ] , mostrando risultati promettenti . 
i risultati hanno mostrato che lablazione 736 radiol med ( 2011 ) 116 : 734748 images , such as mri and contrast - enhanced ultrasound ( ceus ) , 20 of 22 were assessed . 
the results revealed that the adverse event pro le was favourable when compared with open or minimally invasive techniques [ 5 ]  . however , at the present time , there is no report on hifu treatment for solid malignant tumours from a european cancer referral centre for de ning the clinical and technical indications for this technique . 
the aim of our study was to de ne the safety and feasibility of usghifu for treating different type of solid malignant tumours . materials and methods patients this study was de ned as part of the institutional guideline on the minimally invasive management of solid tumours at the european institute of oncology , milan , italy . 
a speci c written informed consent was signed by every patient before selection and treatment . from september 2008 to april 2009 , 22 patients ( eight men and 14 women , age range 1675 years , mean age 61.3 years ) were enrolled . 
all patients were deemed not to be candidates for surgery because of comorbidities ( cardiac and / or renal failure ) and / or local advanced disease , nor suitable radiofrequency ablation ( rfa ) owing to the presence of major blood vessels close to the tumour , tumour location adjacent to viscera ( such as the bowel or gallbladder ) or because of patient refusal to undergo any of those treatments . 
there were ve patients with liver metastases from colorectal cancer ( crc ) , two with chest wall metastasis from crc , one with liver and lung metastases from crc , one with soft - tissue metastasis from crc , one with liver metastasis from breast cancer , one with liver and sternum metastases from breast cancer , one with rib metastasis from breast cancer , one with osteosarcoma , one with leg metastasis from lung cancer , one with iliac metastasis from multiple myeloma , one with abdominal liposarcoma and six with pancreatic lesions . 
with the aim of palliation , we performed hifu on three patients with pancreatic adenocarcinoma and three with pancreatic neuroendocrine tumour ( net ) ; ve of those patients were at the advanced stage with no con ultrasuoni focalizzati con guida rm per lablazione dei bromi uterini fattibile e sicura [ 10 , 11 ]  . 
 attualmente tuttavia , non esiste alcun report sul trattamento hifu dei tumori solidi maligni prodotto da un centro oncologico di riferimento europeo nalizzato alla de nizione delle indicazioni cliniche e tecniche per questa tecnologia . 
 lo scopo del nostro studio stato quello di de nire sicurezza e fattibilit del usghifu nel trattamento di diversi tipi di tumori solidi maligni . materiali e metodi pazienti lo studio stato incluso nelle linee guida istituzionali per il trattamento mininvasivo dei tumori solidi allistituto europeo di oncologia ( milano , italia )  . 
da settembre 2008 ad aprile 2009 stato incluso in questo studio un totale di 22 pazienti ( 8 maschi e 14 femmine , range det 1675 anni , et media 61 , 3 anni )  . 
tutti i pazienti inclusi nello studio non erano n candidabili a chirurgia a causa di comorbilit ( insuf cienza cardiaca e / o renale ) o malattia in stato avanzato , n a radiofrequenza per la vicinanza di grossi vasi alle lesioni da trattare , di visceri cavi come intestino o colecisti o erano stati esclusi per tali trattamenti . tutti i pazienti avevano un punteggio sulla scala di performance di karnofsky di almeno il 70% , in assenza di controindicazioni a sottoporsi ad anestesia generale . 
 tra i 22 pazienti , si segnalano 5 metastasi epatiche da cancro del colon - retto ( crc ) , due metastasi alla parete toracica da crc , un paziente con metastasi epatiche e polmonari da crc , uno con metastasi ai tessuti molli da crc , uno con metastasi epatiche da carcinoma della mammella , uno con metastasi epatiche e sternali da carcinoma mammario , uno con secondarismo costale da carcinoma mammario , uno con osteosarcoma , un paziente con metastasi agli arti inferiori da tumore polmonare , uno con localizzazioni iliache da mieloma multiplo , uno con liposarcoma addominale e sei radiol med ( 2011 ) 116 : 734748 indication for surgical resection , and one had local relapse after surgery . all the lesions scheduled to be treated were detectable at us , and the diagnoses were con rmed with ct / us - guided histology . 
these patients suffered from recurrent episodes of severe nightly hypoglycaemia leading to unconsciousness , episodes that conservative medical therapy with diazoxide ( 300 mg daily ) failed to control . all pancreatic cancer patients had chemotherapy and radiotherapy with advanced local disease at the time of hifu treatment . 
one patient only , admitted with a poor performance status , received supportive care with nonsteroidal anti - in ammatory drugs and narcotic analgesics because she was not a candidate for either chemotherapy for renal failure or surgical resection because of the encasement of main vessels by the tumour . 
 table 1 characteristics of the 22 patients treated with high - intensity focused ultrasound ( hifu ) table 1 caratteristiche dei 22 pazienti trattati con ultrasuoni focalizzati ad elevata intensit ( hifu ) characteristics no . 
di pazienti m / f et ( anni ) valore mediosd dimensione delle lesioni ds , deviazione standard 8 / 14 61.312.5 120 cm 8 / 14 61.312.5 120 cm pazienti con lesioni pancreatiche . 
con nalit palliativa abbiamo eseguito hifu su tre pazienti con adenocarcinoma pancreatico e su tre pazienti con tumore neuro - endocrino ( net ) pancreatico , cinque dei quali in stadio avanzato senza alcuna indicazione alla chirurgia ; un paziente ha mostrato recidiva locale dopo la chirurgia . 
 tutte le lesioni in programma di trattamento erano riconoscibili in ecogra a e la diagnosi stata confermata con esame istologico eseguito sotto guida ecogra ca o della tomogra a computerizzata ( tc )  . 
questi pazienti soffrivamo di episodi ricorrenti di grave ipoglicemia notturna con perdita di coscienza e la terapia medica conservativa con diazoxide ( 300 mg al giorno ) non era in grado di controllare gli episodi notturni di ipoglicemia . 
solo un paziente , incluso nello studio con un basso perfomance status , ha ricevuto terapie di supporto con farmaci antinammatori non steroidei e analgesici narcotici dal momento che non era candidato n alla chemioterapia per insuf cienza renale , n alla resezione chirurgica dal momento che il tumore in ltrava i vasi principali . 
in tutti i pazienti con tumore pancreatico e con metastasi ossee e ai tessuti molli non si era veri cato alcun miglioramento del dolore correlato al cancro nei precedenti trattamenti . 
le caratteristiche dei tumori sono mostrate nella tabelle 1 e 2 . preparazione pre - trattamento esami di routine e preparazioni pre - trattamento sono stati condotti in accordo ai principi della chirurgia . 
prima del trattamento sono stati eseguiti i convenzionali test biochimici epatici , tempo di protrombina , emocromo completo , radiogra a del torace , ecogra a addominale , elettrocardiogramma ( ecg ) ; la funzionalit polmonare stata valutata prima del trattamento . 
stato necessario eseguire una adeguata preparazione intestinale table 2 characteristics of the 22 patients with 29 tumours treated with high - intensity focused ultrasound ( hifu ) site number of tumours patients per i pazienti con tumore pancreatico e per i pazienti con tumori addominali in adiacenza alle anse intestinali . 
ai pazienti stato richiesto il digiuno notturno il giorno prima del trattamento hifu . radiol med ( 2011 ) 116 : 734748 738 liver bone soft tissue pancreas lung total fegato osso tessuti molli pancreas polmone totale table 2 caratteristiche dei 22 pazienti con 29 neoplasie trattati con ultrasuoni focalizzati ad elevata intensit ( hifu ) sede numero delle lesioni pazienti conventional liver biochemical tests , prothrombin time and complete blood cell counts , chest radiography , abdominal us , electrocardiogram ( ecg ) and lung function were evaluated before treatment . 
a mylab70 us imaging device ( esaote , genoa , italy ) coupled with the hifu machine was used as the real - time imaging unit of the systethis 1.08.0 - mhz imaging probe is situated in the centre of the hifu transducer and may allow for realtime us image monitoring during treatment . skin surface overlying the lesion was shaved in every patient to avoid the presence of hairs within the acoustic pathway ; the same skin surface was also degassed by using a vacuum cup aspiration device and degreased with 95 alcohol . 
lenergia terapeutica focalizzata degli ultrasuoni stata prodotta da un trasduttore di 20 cm di diametro con lunghezza focale di 15 cm , prodotti ad una frequenza di 0 , 8 mhz . 
un dispositivo di imaging ad ultrasuoni mylab 70 ( esaote , genova , italia ) , accoppiato con la macchina hifu , stato utilizzato per ottenere immagini in tempo reale . 
questa sonda da 1 , 08 , 0 collocata nel centro del trasduttore di ultrasuoni focalizzati ad alta intensit e permette il monitoraggio del trattamento in tempo reale con limmagine ecogra ca . 
 la super cie corporea che ricopre la lesione stata rasata in ogni paziente , allo scopo di evitare la presenza di peli lungo il percorso acustico ; la stessa super cie stata degassi cata utilizzando un dispositivo di aspirazione a coppa e successivamente sgrassata con alcol a 95 . 
lanestesia generale con intubazione endotracheale e ventilazione meccanica ha svolto anche il ruolo supplementare di permettere la sospensione provvisoria del respiro con il con controllo polmonare dellinspirio , necessario nellablazione di lesioni epatiche poste posteriormente , rispetto alla gabbia toracica , nello spazio intercostale . 
dopo lanestesia , i pazienti sono stati posizionati con cura sopra il letto di trattamento , con particolare attenzione al corretto posizionamento della supercie corporea in corrispondenza della lesione da trattare , a contatto con lacqua degassi cata . 
per tutti i pazienti durante la procedura stato effettuato il monitoraggio respiratorio e della frequenza cardiaca , della pressione sanguigna , della saturazione dellossigeno e dellanidride carbonica . sia per la piani cazione pre - trattamento che per la sonazione stata selezionata la modalit di scansione ad ultrasuoni su di un piano sagittale ; il trattamento stato eseguito con rilascio di energia sia con modalit puntiforme che lineare . 
a ultrasound ( us ) image obtained before high - intensity focused us ( hifu ) treatment shows a small lesion ( bold arrow ) close to the inferior vena cava ( ivc )  . 
b immagine ecogra ca ottenuta immediatamente al termine dellablazione hifu mostra una iperecogenicit a livello dellarea trattata ( freccia )  . anaesthesia to prevent the patient from experiencing pain or discomfort and ensure immobility . 
general anaesthesia with endotracheal intubation and mechanical ventilation also has the supplementary bene t of permitting provisional suspension of breath with controlled pulmonary in ation , as is required for ablating liver lesions behind the rib cage through the intercostal space . 
treatment powers of 60400 w were used for the different tumour types and sites . evaluation of therapeutic ef cacy patients were evaluated by pet - ct , mri , or / and mdct and clinical evaluation . 
in three patients with liver lesions and one with pancreatic cancer , contrast - enhanced ultrasound ( ceus ) was performed at the end of the procedure to assess the vascular feeding after hifu ablation . 
to exclude major complications and evaluate treatment ef cacy , patients underwent mdct within the rst 24 h after hifu treatzate potenze di trattamento da 60 a 400 w in relazione alle diverse tipologie e posizioni dei tumori . valutazione dellef cacia terapeutica i pazienti sono stati valutati con pet - tc , rm e / o tcmd e con la valutazione clinica . 
tutti i pazienti sono stati sottoposti a [ 18f ] uorodesossiglucosio ( fdg ) - pet - tc e / o a tcmd o rm addominale dopo 3 o 4 settimane e a 3 mesi dopo la procedura hifu . 
 interpretazione delle indagini pet - tc e tcmd le [ 18f ] - fdg - pet - tc eseguite in condizioni di base e nei controlli successivi sono state interpretate da un medico specialista in medicina nucleare , alloscuro delle condizioni cliniche e dellesame obiettivo del paziente , degli altri reperti di imaging e dei risultati istopatologici . 
la tcmd stata considerata positiva ogni volta venisse riconosciuta iperdensit irregolare attorno al sito della lesione trattata con hifu e / o 740 radiol med ( 2011 ) 116 : 734748 ment . 
all patients then underwent [ 18f ] uorodeoxyglucose ( fdg ) pet / ct and / or abdominal mdct or mri at 34 weeks and 3 months after the hifu procedure . pet / ct and mdct scan interpretation baseline and follow - up [ 18f ] - fdg - pet / ct scans were read by one experienced nuclear medicine physician blinded to the patients clinical status and physical examination , other imaging ndings and histopathological results . 
detection of hypodensity at hifu ablation site without contrast enhancement on the edges was considered as the sign of lesion treatment , and the mdct scan was therefore interpreted as negative . 
we also calculated sonication time , de ned as exposure time , which was related to tumour size , site and blood supply , which ranged from 1 min 38 s to 166 min ( meansd , 37.442.7 min ) ( table 3 )  . 
the fraction of sonication time over treatment time for liver lesions was 14.3%. grey - scale value changes massive grey - scale changes during treatment , interpreted as tissue necrosis by sonication , were observed in 27 of incremento dimensionale dellarea sottoposta a trattamento . 
 il riconoscimento di ipodensit nel sito di ablazione hifu senza incremento di contrasto nei bordi stato considerato come il segno del trattamento e quindi la tcmd stata interpretata come negativa . 
nei quattro pazienti valutati alla ne della procedura con mezzo di contrasto eco ampli catore ( ceus ) , le caratteristiche di vascolarizzazione della lesione sono risultate in accordo con i reperti tcmd ottenuti il giorno successivo . risultati timing i dettagli riguardo alla durata della procedura sono i seguenti . 
il tempo di sala , che include il tempo di preparazione e il tempo di trattamento , de nito dal momento in cui il paziente giunge nella unit hifu no alluscita del paziente dalla sala hifu , compreso tra 2 ore e 30 minuti e 7 ore e 5 minuti . 
il tempo complessivo di trattamento , de nito dal tempo di inizio della localizzazione no allultima sonicazione , variabile tra 59 minuti e 318 minuti ( mediadeviazione standard [ ds ] , 162 , 783 , 0 min )  . 
abbiamo inoltre calcolato il tempo di sonicazione , inteso come tempo di esposizione , correlato alle dimensioni del tumore , sede del tumore e suo apporto vascolare , variabile da 1 minuto e 38 secondi a 166 minuti ( mediads , 37 , 442 , 7 minuti ) ( tabella 3 )  . 
per 11 pazienti con 13 lesioni epatiche : la media del trattamento complessivo stata di 107 , 2 minuti ( range : 59170 ) ; il tempo medio di sonazione stato di 15 , 3 minuti ( range : 1 , 640 , 7 )  . 
il rapporto tra il tempo di sonazione e il tempo di trattamento stato del 14 , 3% . modi che dei valori nella scala di grigi in 27 delle 29 lesioni ( 93 , 1% ) si veri cata unimportante modi ca dei valori della scala di grigi durante il trattamento , interpretata come necrosi tissutale causata dalla sonazione . 
 radiol med ( 2011 ) 116 : 734748 table 3 data on high - intensity focused ultrasound ( hifu ) procedure performed on 22 patients with solid tumours ( mean valuestandard deviation ) tumour site no . 
grey - scale changes were not observed in two liver metastases . postprocedure evaluation and short - term follow - up mdct or mri performed 1 day after hifu showed complete response in 20 / 22 patients without any injury of the surrounding organs ; as a precautionary measure , all patients were observed in hospital for 2 nights . 
mdct / mri at 24 h after treatment detected no injury of the surrounding organs , and all patients were observed in hospital for 3 days as a precautionary measure . 
amylase levels showed no signi cant elevation over baseline during the 3 days after treatment . the huge retroperitoneal before hifu , all 14 patients with pancreatic tumour and valutazione post - procedura e follow - up a breve tempo la tcmd o la rm eseguite un giorno dopo hifu hanno mostrato completa risposta in venti dei ventidue pazienti senza alcun danno agli organi adiacenti ; tutti i pazienti in via precauzionale sono stati trattenuti in ospedale per due notti . 
la tcmd / rm a 24 ore non ha identi cato alcun danno degli organi adiacenti e tutti i pazienti sono stati trattenuti in via precauzionale in ospedale per 3 giorni dopo il trattamento . prima degli ultrasuoni focalizzati ad alta intensit , tutti i 14 pazienti con tumore pancreatico , lesioni ossee e dei tessuti molli lamentavano dolore cronico per cui era necessaria la somministrazione per via orale di farmaci antidolori ci . 
in tutti i pazienti si osservata riduzione del dolore con remissione completa ( senza necessit di farmaci antidolori ci oppioidi ) entro 2448 ore dalla singola sessione di ultrasuoni focalizzati . 
b lesame tc con mezzo di contrasto eseguito 24 ore dopo trattamento hifu mostra unarea ipodensa priva di contrast enhancement , in corrispondenza della pregressa lesione , in assenza di tessuto patologico residuo . 
b il controllo pet - tc ad un mese mostra la scomparsa di attivit metabolica a livello della lesione ( freccia )  . bone and soft tissues lesions complained of chronic pain that necessitated management with oral analgesic drugs . 
a follow - up postinterventional fasting test was not performed because remission was determined clinically of the basis of the disappearance of the hypoglycaemic episodes and reduction of tumour vascularity at follow - up mdct . stata stabilita dai dati clinici in assenza di ulteriori episodi di ipoglicemia e dalla riduzione nei controlli successivi della vascolarizzazione dei tumori valutata con tcmd . eventi avversi tutti i pazienti sono stati dimessi dopo un intervallo da 1 a 3 giorni di osservazione . 
ledema locale nel sito di esposizione della cute stato lunico effetto collaterale osservato , identi cato in 3 pazienti ( due con metastasi della parete toracica e uno con liposarcoma retro peritoneale )  . 
b controllo pet - tc ad 1 mese con evidente scomparsa dellattivit metabolica in corrispondenza della lesione ( freccia )  . adverse events all patients were discharged after 13 days of observation . 
local oedema at the level of exposed skin surface was the only side effect observed , and it was detected in three patients ( two with chest wall metastases and one with retroperitoneal liposarcoma )  . 
no other postprocedural side effects were observed . one 61 - year - old woman with a liver metastasis located at segment ii showed after 2 months an extrahepatic and subcutaneous sterile in ltration . 
the patient continued to receive chemotherapy , and after 5 weeks , she experienced local pain without high fever , and us and mdct showed a subcutaneous in ltration from liver necrosis . 
no other hifu - related adverse effects were observed after 3 months of follow - up in the responders . discussion minimally invasive therapies , such as radiofrequency locale senza febbre alta e lecogra a e la tcmd hanno mostrato in ltrazione sottocutanea da necrosi epatica . 
nessun altro evento avverso correlato allhifu stato osservato dopo tre mesi di follow - up in questi pazienti che hanno risposto alla terapia . discussione le terapie mininvasive , come la radiofrequenza , la chemioembolizzazione , la crioablazione , lablazione a microonde , la termoterapia interstiziale laser e lhifu , sono state usate per lablazione dei tumori solidi . 
la chirurgia lo standard terapeutico attuale per i pazienti con metastasi epatiche da tumori colorettali , offrendo la possibilit di una radicalit completa con la resezione [ 12 ]  . 
7a , b immagine tc della paziente con metastasi epatica al iv segmento da tumore mammario 1 mese dopo trattamento hifu ( a ) che mostra in ltrato sottocutaneo da necrosi epatica e 3 mesi ( b ) dopo con riassorbimento quasi completo . 
 ablation ( rfa ) , transcatheter arterial chemoembolisation ( tace ) , cryoablation , microwave coagulation , laserinduced interstitial thermotherapy and hifu , have all been used to ablate solid tumours . 
surgery is the current standard of care in patients with colorectal liver metastasis , as resection offers the chance of a complete cure [ 12 ]  . local ablative techniques , such as rfa , cryoablation , microwave coagulation and laser - induced interstitial thermotherapy , also offer potential local tumour control and occasionally achieve long - term disease - free survival [ 1315 ]  . 
a study with a small number of patients has shown that hifu is safe and feasible for treating pancreatic cancer [ 4 ]  . our results show that hifu is safe and could be used to treat solid malignancies . 
local oedema was the most le tecniche di ablazione locale come la radiofrequenza , la crioablazione , lablazione a microonde e la termoterapia interstiziale laser permettono un controllo locale di malattia ed occasionalmente portano a lunghi intervalli di sopravvivenza senza malattia [ 1315 ]  . 
uno studio con un numero limitato di pazienti ha dimostrato che hifu fattibile e privo di complicanze nel trattamento di tumori pancreatici [ 4 ]  . i nostri risultati hanno dimostrato che lablazione con tecnica hifu sicura e pu essere impiegata per il trattamento dei tumori solidi . 
earlier studies reported mild discomfort or local pain was seen in 5480% of treated patients [ 5 , 19 ] , but in our study , no patients reported discomfort or local pain 1 day after hifu treatment . 
a low - grade fever was another common adverse effect in earlier studies [ 5 ] , but we did not observe low - grade fever in any of our patients . 
this event was probably related to the chemotherapy with antiantigenetic drugs that the patient received after hifu . as this was a feasibility study , we enrolled patients who were treated for different purposes and did not focus on the long - term effects of treatment but only evaluated the patients at 1 day and 34 weeks after treatment . 
we treated a total of 13 liver lesions in eight patients ; pet - ct or / and mdct showed that 11 of 13 ( 87.5% ) were completely ablated . 
higher power and more energy are needed for ablating tumours close to the major blood vessels because of the cooling effects from great vessels [ 17 , 20 ]  . our results show that us grey - scale values are reliable to de ne coagulation necrosis . 
studi precedenti al nostro hanno descritto moderato fastidio o dolore locale in 54%80% del pazienti trattati [ 5 , 19 ] , ma nella nostra esperienza non abbiamo avuto pazienti con fastidio o dolore locale il giorno dopo il trattamento . 
tale evento probabilmente da relazionare al trattamento chemioterapico con farmaci antiangiogenetici che la paziente ha ricevuto dopo hifu . nellottica di uno studio di fattibilit abbiamo arruolato pazienti trattati con differenti intenti non concentrandoci sui risultati a lungo termine e rivalutando unicamente i pazienti il giorno dopo e 34 settimane dopo il trattamento . 
abbiamo trattato un totale di 13 lesioni epatiche in otto pazienti e la pet - tc e / o la tcmd hanno dimostrato lablazione completa di 11 / 13 ( 87 , 5% )  . 
sono infatti necessarie unenergia ed una potenza maggiori per i trattamenti di lesioni adiacenti grossi vasi per leffetto di raffreddamento indotto dagli stessi [ 17 , 20 ]  . i nostri risultati dimostrano che i valori di scala di grigio corrispondono alla necrosi coagulativa . 
abbiamo calcolato il tempo di allestimento della sala e del trattamento vero e proprio rilevando che il 51% del tempo di allestimento della sala stato utilizzato per la preparazione del paziente . 
il tempo medio complessivo per lablazione di una lesione con diametro medio di 4 , 2 cm ( range , 1 , 020 , 0 ) variato da 59 a radiol med ( 2011 ) 116 : 734748 treatment time for ablating a mass with a mean diameter of 4.2 cm ( range 1.020.0 ) ranged from 59 min to 318 min ( meansd , 162.783.0 min ) , which is still longer than desirable ( table 3 )  . 
even improving us lesion targeting may result in a shorter treatment time . this study was limited to a small number of patients and short - term follow - up because its aim was to assess the feasibility of this new technique . 
additional studies to compare hifu with other available techniques , such as transcatheter arterial embolization ( tae ) and rfa , are important to allow selection of the most appropriate therapy for individual patients . conclusions the results of this study demonstrate that usghifu can achieve locoregional tumour ablation without signi cant side effects and in fact appears to be feasible and safe for the ablation of solid lesions . 
even though the relative risks and bene ts of hifu ablation must be rigorously measured to better de ne its role in clinical practice in the treatment of solid tumours , we believe it can be used as a cytoreductive measure in an adjuvant setting ( debulking effect ) with the aim of achieving better palliation , especially in locally advanced pancreatic malignancies and bone and soft - tissues lesions . 
however , further studies , such as randomised controlled trials , are needed to select a patient group that would be the best candidate for this noninvasive treatment . 318 minuti ( mediads , 162 , 783 , 0 minuti ) che sempre pi di quanto sia desiderabile ( tabella 3 )  . 
anche il miglioramento nella ricerca della lesione target con gli ultrasuoni pu ridurre ulteriormente i tempi . lo studio attuale limitato ad un numero di pazienti ridotto con un follow - up breve poich nato come valutazione di fattibilit di una nuova tecnica . 
la sopravvivenza ed il tasso di recidiva non sono stati i nostri obiettivi principali ma ulteriori studi in futuro sono necessari per de nire il reale bene cio di questa tecnica . 
paragonandola tuttavia con altre tecniche ablative come lembolizzazione arteriosa e la radiofrequenza ulteriori studi potranno de nire in maniera mirata il trattamento pi adeguato per ogni singolo paziente . conclusioni i risultati di questo studio dimostrano che il trattamento ad ultrasuoni focalizzati eco guidato permette di ottenere un trattamento di tipo loco - regionale senza signi cativi effetti collaterali . 
 anche se il rapporto tra rischi ed i bene ci dellablazione con tecnica hifu devono essere ancora testati per meglio de nire il suo ruolo nella pratica clinica nel trattamento dei tumori solidi , pensiamo che possa essere impiegato con intento citoriduttivo in un contesto adiuvante ( debulking effect ) con lo scopo di una migliore palliazione , in particolare nelle neoplasie pancreatiche localmente avanzate , nelle lesioni ossee e dei tessuti molli . 
lanzara , sezione di radiodiagnostica , seconda universit degli studi di napoli , piazza miraglia 4 , 80100 napoli , italy 3dipartimento di scienze biomorfologiche e funzionali , universit degli studi federico ii di napoli , via pansini 5 , 80134 napoli , italy correspondence to : a . 
our study population comprised 167 ( 91 male , 76 female patients , age range 11 months to 82 years ; mean age 29 years ) out of 1 , 896 patients with throat swab positive for h1n1 and clinical and laboratory ndings indicative of viral in uenza . 
all 167 patients were studied by chest x - ray ( cxr ) , and 20 patients with positive cxr and worsening clinical condition also underwent computed tomography ( ct )  . 
 abnormalities identi ed on cxr , variously combined and distributed , were as follows : 53 ir , 5 n , 13 ggo , 50 cons ; the predominant combination was represented by six ggo with cons . 
 the abnormalities identi ed on ct , variously combined and distributed , were as follows : 14 ir , 2 n , 5 ggo ; the predominant combination was 10 ggo with cons . 
centosessantasette su 1896 casi risultati positivi al tampone faringeo per h1n1 ( 91 m , 76 f , range et 11 mesi82 anni , et media 29 anni ) , con alterazioni clinico - laboratoristiche di in uenza virale , hanno costituito la popolazione del nostro studio . 
tutti i 167 pazienti hanno eseguito radiogra a ( rx ) del torace ; per le pi gravi condizioni cliniche , 20 casi positivi alla rx del torace hanno effettuato tomogra a computerizzata ( tc ) del torace . 
le lesioni polmonari identi cate alla rx , variamente associate e distribuite , sono state : 53 ri , 5 n , 13 ogg , 50 cm ; 6 ogg con cm rappresentavano lassociazione predominante . 
le lesioni polmonari identi cate alla tc , variamente associate e distribuite , sono state : 14 ri , 2 n , 5 ogg ; 10 ogg con cm rappresentavano lassociazione predominante . 
sebbene differentemente identi cati in rx e tc , i segni di sovrainfezione batterica pi frequenti sono stati tree - in - bud , cm con radiol med ( 2011 ) 116 : 706719 bronchogram , and pleural and pericardial effusion . 
la rx e la tc del torace hanno identi cato sede ed estensione delle lesioni polmonari , documentando i segni di sovrainfezione batterica e le complicanze polmonari . keywords in uenza a virus h1n1 subtype computed tomography x - ray pneumonia viral parole chiave virus dellin uenza a sottotipo h1n1 tomogra a computerizzata rx polmonite virale introduction introduzione swine - origin in uenza or swine in uenza ; swine u is an acute respiratory infection caused by the novel in uenza a , subtype h1n1 , which was transmitted to humans from pigs in mexico and the united states in march and april 2009 and rapidly spread worldwide [ 1 , 2 ]  . 
h1n1 virus is a subtype of the in uenza a virus that belongs to the orthomyxoviridae family , of which numerous variants exist that cause u pandemics among animals , such as bird u and swine u . 
genetic analyses suggest that the emergence of the novel h1n1 strain of swine u in humans is the direct consequence of a reassortment of the viral genomes of swine u , bird u and human u in north america and eurasia [ 36 ]  . 
in june 2009 , following the report of the rst human - to - human transmission in two countries in one world health organization ( who ) region and the rapid spread of the virus , the who declared a level - 6 pandemic alert [ 6 , 7 ]  . 
high fever , sore throat , cough , fatigue , joint pain , nausea , vomiting and / or diarrhoea are the principal symptoms of h1n1 in uenza ; more complex cases present with respiratory failure [ 1 , 6 , 8 ]  . 
the speci c tropism of the virus for the respiratory tract has led to the use of imaging to better characterise the location , distribution and type of the primary pulmonary lesions [ 9 ]  . 
chest x - ray ( cxr ) and chest computed tomography ( ct ) have been the investigations of choice for identifying and quantifying , respectively , pulmonary damage in patients affected by in uenza a [ 1012 ]  . 
the aim of this paper is to describe the radiological and clinical ndings in patients hospitalised for a diagnosis of suspected h1n1 swine - origin in uenza . lin uenza virale a di origine suina ( s - oiv , swine - origin in uenza o swine in uenza o swine u ) rappresenta una patologia respiratoria acuta causata dal nuovo sottotipo di virus in uenzale h1n1 , trasmesso alluomo da alcuni allevamenti di maiali del messico e degli stati uniti nei mesi di marzo ed aprile 2009 e diffusosi rapidamente nellintero globo [ 1 , 2 ]  . 
il virus in uenzale h1n1 un sottotipo di in uenzavirus a appartenente alla famiglia delle orthomyxoviridae , di cui sono note numerose varianti che , negli animali , causano forme in uenzali pandemiche , come lin uenza aviaria e la febbre suina . 
analisi genetiche suggeriscono che la comparsa della nuova linea h1n1 dellin uenza suina nelluomo sia diretta conseguenza di un riassortimento dei genomi virali dellin uenza suina , aviaria ed umana del nord america e delleurasia [ 36 ]  . 
 nel mese di giugno 2009 , in seguito alla documentazione pubblicata da parte della organizzazione mondiale della sanit ( oms ) del primo contagio interumano in due stati e alla rapida diffusione del virus , stato dichiarato lo stato di emergenza mondiale di scala 6 ( pandemia in uenzale ) [ 6 , 7 ]  . 
febbre elevata , faringodinia , tosse , astenia , artromialgie , nausea , vomito e / o diarrea hanno costituito i principali sintomi desordio clinico dellin uenza h1n1 ; nei casi pi complessi insorta insuf cienza respiratoria acuta [ 1 , 6 , 8 ]  . 
il peculiare tropismo virale per lapparato respiratorio ha determinato il ricorso alle tecniche di imaging per meglio caratterizzare sede , distribuzione e tipologia delle lesioni elementari polmonari [ 9 ]  . 
 lesame radiogra co del torace ( rx ) e la tomogra a computerizzata ( tc ) del torace hanno rappresentato le indagini 708 materials and methods between may and mid - november 2009 , 3 , 649 patients with suspected swine - origin in uenza a virus infection presented to our hospital . 
the diagnosis of h1n1 viral in uenza was con rmed in 1 , 896 / 3 , 649 ( 51.9% ) cases by reverse transcriptase polymerase chain reaction ( rt - pcr ) for detection of viral rna in pharyngeal swabs . 
 cxr was performed on all 167 patients , 20 of whom ( 11.9% ) were also studied by ct to assess equivocal ndings on cxr or discrepancies between the cxr and clinical ndings . 
cxr was performed using the standard technique ( posteroanterior projection : 10 mas , 70 kv ; lateral projection : 60 mas , 80 kv ; lm - focus distance 180 cm ) with the patient in standing position in 150 cases ( 89.9% ) ; only 17 patients ( 10.1% ) were imaged in a sitting or lying position and with anteroposterior projection due to an inability to maintain a standing position and / or inspiration . 
 ct study was performed with a 64 - detector - row ct scanner immediately after the cxr in 11 / 20 cases and 24 h after cxr in the remaining 9 / 20 cases . 
on clinical request , 18 / 20 patients 12 / 20 large and 6 / 20 poorly cooperative patients underwent unenhanced chest and abdominal ct with the spiral technique : breath - hold volumetric scans , slice thickness 1 mm , kv 120 , ma 180 , pitch 1 . 
only 2 / 18 patients with suspected pulmonary embolism were subsequently imaged after intravenous administration of nonionic iodinated contrast material with the sure start technique at a ow rate of 3 ml / s and dose of 100 ml . 
the resulting ct images were viewed with a lung window ( width 1 , 500 hu , level 700 hu ) and mediastinal window ( width 350 hu , level 40 hu )  . 
the remaining 2 / 20 patients were studied with high - resolution ct of the chest for clinical suspicion of concurrent interstitial pulmonary disease using a standard protocol : collimation 1 mm , interval 10 mm , acquisition time 12 s , high - spatial - frequency reconstruction algorithm , matrix 512 512 , 120 kv , 200 ma , fov encompassing both lungs , window level 600 hu ( range 500 / 900 hu ) , window width 1 , 500 hu ( range 1 , 100 / 2 , 000 hu ) , expiratory scans and patient in prone position when needed . 
two radiologists independently examined and interpreted the cxr and ct images stored in the picture archiving and communications system ( pacs )  . the primary lesions assessed at cxr and chest ct were radiol med ( 2011 ) 116 : 706719 di riferimento ai ni rispettivamente dellindividuazione e quanti cazione del danno polmonare nei pazienti affetti da in uenza virale a [ 1012 ]  . 
scopo del nostro studio de nire il quadro radiologico e clinico dei pazienti ospedalizzati per diagnosi di in uenza virale h1n1 di origine suina . materiali e metodi dal mese di maggio alla met di novembre 2009 , 3649 pazienti con sospetta in uenza virale a di origine suina si sono presentati presso il nostro presidio ospedaliero . 
la diagnosi di in uenza virale h1n1 stata confermata in 1896 / 3649 ( 51 , 9 % ) casi con tecnica reverse transcription - polymerase chain reaction ( rt - pcr ) , mediante ricerca dellrna virale nel materiale prelevato con tampone faringeo . 
di essi , 167 / 261 ( 63 , 9% ) pazienti sono stati sottoposti ad indagini diagnostiche strumentali a causa del persistere e / o dellaggravarsi della sintomatologia respiratoria dopo terapia medica da almeno 3 giorni . 
 hanno eseguito lesame radiogra co del torace 167 pazienti e , di essi , 20 ( 11 , 9% ) sono stati sottoposti ad esame tc , al ne di valutare reperti dubbi o discordanti fra rx del torace e manifestazioni cliniche . 
lesame radiogra co del torace stato effettuato mediante tecnica standard ( proiezione postero - anteriore [ pa ] : 10 mas , 70 kv ; proiezione latero - laterale [ ll ] : 60 mas , 80 kv ; distanza tubo - paziente 180 cm ) ed in posizione ortostatica in 150 casi ( 89 , 9% ) ; solo in 17 casi ( 10 , 1% ) sono stati effettuati radiogrammi in posizione seduta o sdraiata ed in proiezione antero - posteriore ( ap ) per incapacit da parte dei pazienti a mantenere la posizione ortostatica e / o linspirio . 
 lindagine tc stata eseguita mediante apparecchiatura tc multidetettore ( tcmd ) a 64 le di detettori subito dopo la rx del torace in 11 / 20 casi , dopo 24 ore dallesame radiogra co in 9 / 20 casi . 
su richiesta clinica , 18 / 20 pazienti , di cui 12 / 20 di elevata taglia corporea e 6 non collaboranti , sono stati sottoposti ad esame tc torace ed addome senza somministrazione di mezzo di contrasto endovena mediante tecnica spirale : scansioni volumetriche in inspirio , spessore di strato di 1 mm , kv 120 , ma 180 , pitch 1 . 
solo in 2 / 18 pazienti , per sospetto di embolia polmonare , stato successivamente somministrato mezzo di contrasto iodato non ionico endovena mediante tecnica sure start , velocit di usso 3 ml / s , volume di contrasto 100 ml . 
le immagini tc cos acquisite sono state visualizzate mediante nestra radiol med ( 2011 ) 116 : 706719 de ned according to the fleischner society glossary [ 13 ] and recent publications [ 10 , 11 ] describing the main radiological pulmonary manifestations of a / h1n1 in uenza : interstitial reticulation ( ri ; linear opacities of the central and peripheral interstitium appearing as radio - opaque lines on cxr and hyperdensities on ct ) , nodules ( n ; wellor illde ned , rounded opacities / hyperdensities , with maximum diameter of 3 cm . ) , ground - glass opacities ( ggo ; heterogeneous increase in parenchymal opacity with preservation of bronchial and vascular margins ) , consolidation ( cons ; homogenously increased parenchymal attenuation that obscures the margins of the bronchial and vessels walls )  . 
 images were also assessed for signs of bacterial suprainfection : consolidation with air bronchogram ( area of radiolucency at cxr and low attenuation at ct , re ecting the airlled bronchi on a background of opaque or high - attenuation airless lung ) , cavitations ( gaslled spaces , seen as lucencies or low - attenuation areas within a parenchymal consolidation ) , tree - in - bud pattern ( branching centrilobular structures ) , pleural and / or pericardial effusion ( uid in the pleural / pericardial cavity ) , lymphadenopathy ( short - axis diameter > 1 cm for mediastinal nodes and > 3 mm for hilar nodes )  . 
extent of pulmonary damage was de ned univocally at cxr and chest ct : unilateral or bilateral ; symmetrical or asymmetrical ; focal , multifocal or diffuse ; with predominant distribution in the upper , middle or lower lobes . 
all patients had abnormal blood chemistry results : 157 ( 94% ) elevated erythrocyte sedimentation rate ( esr ) , 150 ( 89.8% ) elevated c - reactive protein ( crp ) , 144 ( 86.2% ) elevated lactate dehydrogenase ( ldh ) , 83 ( 49.7% ) polmonare ( ampiezza di nestra 1500 hu , livello di nestra - 700 hu ) e mediastinica ( ampiezza di nestra 350 hu , livello di nestra 40 hu )  . 
i restanti 2 / 20 pazienti sono stati studiati con tomogra a computerizzata ad alta risoluzione ( hrct ) del torace per sospetto clinico di concomitante interstiziopatia polmonare , mediante protocollo standard di acquisizione : collimazione 1 mm , intervallo di acquisizione 10 mm , tempo di scansione 12 s , algoritmo di ricostruzione ad alta frequenza spaziale , matrice 512512 , 120 kv , 200 ma , eld of view ( fov ) contenente ambedue i polmoni , livello di nestra - 600 hu ( range - 500 / 900 hu ) , ampiezza nestra 1500 hu ( range 1100 / 2000 hu ) , scansioni in espirio ed a paziente prono alloccorrenza . 
due radiologi hanno esaminato ed interpretato indipendentemente le immagini radiogra che e tc del torace custodite nellarchivio picture archiving and communications system ( pacs )  . le lesioni elementari individuate alla rx ed alla tc del torace sono state da noi de nite secondo il glossario della fleischner society [ 13 ] e recenti pubblicazioni [ 10 , 11 ] che hanno descritto le principali manifestazioni radiologiche polmonari dellin uenza a / h1n1 : reticolazione dellinterstizio ( ri : opacit a decorso lineare dellinterstizio centrale e periferico che appaiono come strie radiopache sul radiogramma standard e come iperdensit all indagine tc ) , noduli ( n : opacit / iperdensit rotondeggianti , bene o scarsamente de nite , del diametro massimo di 3 cm ) , opacit ground glass ( ogg : aumento disomogeneo della densit parenchimale con conservazione del pro lo dei bronchi e delle strutture vascolari contestuali ) , consolidamenti ( cm : omogeneo incremento dellattenuazione parenchimale polmonare con mancata de nizione delle pareti bronchiali e vascolari contestuali )  . 
 sono stati individuati , inoltre , i segni di sovra - infezione batterica : consolidamento con broncogramma aereo ( area di radiotrasparenza , al radiogramma standard del torace , e di bassa densit , allesame tc , rappresentata dal contenuto aereo dei bronchi nel contesto di unarea di radiopacit / elevata densit del parenchima polmonare , priva daria ) , cavitazioni ( spazi contenenti aria , visibili come aree di trasparenza o bassa attenuazione nellambito di una consolidazione parenchimale ) , albero in ore o tree - in - bud ( strutture rami cate centro - lobulari ) , versamento pleurico e / o pericardico ( lm uido nel cavo pleurico / pericardico ) , linfoadenopatie ( incremento del diametro corto dei linfonodi mediastinici , se esso > 1 cm , dei linfonodi ilari se esso > 3 mm )  . 
l estensione del danno stata de nita univocamente alla rx ed alla tc del torace : unilaterale o bilaterale ; simmetrica o asimmetrica ; focale , multifocale o diffusa ; a predominante distribuzione nei lobi superiori , 710 radiol med ( 2011 ) 116 : 706719 fig . 
a chest x - ray , posteroanterior projection : diffuse and bilateral thickening of the interstitium , particularly visible in the right lower lung eld , where there is an area of ggo . 
a radiogramma del torace in proiezione pa : ispessimento diffuso e bilaterale dellinterstizio polmonare pi evidente nel campo polmonare inferiore di destra in cui si osserva un addensamento parenchimale del tipo vetro smerigliato . 
none of the patients showed isolated pulmonary consolidation , and none of those studied with contrast - enhanced multidetector - row ct ( mdct ) showed direct and / or indirect signs of pulmonary embolis on cxr , ndings suggestive of bacterial suprainfection were seen in various combinations in 14 / 90 patients ( 15.5% ) ( table 1 ) : pleural effusion in 9 / 14 ( 64.2% ) , consolidation with air bronchogram in 4 ( 28.5% ) , lymphadenopathy in 2 ( 14.2% ) , cavitation in 1 ( 7.1% ) , hydropneumothorax in 1 medi o inferiori . 
ricostruzioni multiplanari ( mpr ) e maximum intensity projection ( mip ) sono state eseguite nel post - processing in tutti i pazienti studiati con scansioni tc volumetriche . risultati dei 167 pazienti studiati 161 ( 96 , 4% ) presentavano febbre , 153 ( 91 , 6% ) cefalea , 143 ( 85 , 6% ) tosse , 58 ( 34 , 7% ) faringodinia , 69 ( 41 , 3% ) diarrea , 57 ( 34 , 1% ) vomito . 
113 / 167 ( 67 , 6% ) pazienti presentavano almeno una condizione clinica patologica pregressa : 76 ( 45 , 5% ) erano affetti da asma , 25 ( 14 , 9% ) erano diabetici , 13 ( 7 , 7% ) presentavano broncopneumopatia cronica ostruttiva ( bpco ) , 23 ( 13 , 7% ) immunosoppressione , 65 ( 38 , 9% ) cardiopatie , 17 ( 10 , 1% ) insuf cienza renale cronica , 11 ( 6 , 5% ) disordini congnitivi , 5 ( 2 , 9% ) patologie neuro - muscolari , 3 ( 1 , 7% ) malformazioni scheletriche , 2 ( 1 , 1% ) gravidanza , 1 ( 0 , 5% ) uso di droghe . 
ct con rmed the cxr ndings of bacterial suprainfection in 9 cases ( 9 / 14 , 64.2% ) and identied as false positive ve cases of suspected pleural effusion detected on cxr . 
a chest x - ray performed at the bedside with anteroposterior projection : multiple ggo and small consolidations ( cons ) bilaterally , distributed peripherally and in the middlebasal lung elds . 
a radiogramma del torace , eseguito a letto in proiezione ap : multipli addensamenti con aspetto a ogg e parcellari consolidamenti ( cm ) bilaterali , pi evidenti in sede medio - basale . 
b sezione assiale di mdct del torace : multiple areole di ogg e parcellari cm bilaterali , a prevalente distribuzione mantellare ed in sede lobare inferiore . chogram , which had gone undetected on cxr . 
finally , in 3 / 9 patients in whom ct con rmed the cxr signs of bacterial suprainfection , it also identi ed the presence of pericardial effusion , which had been missed at cxr in all cases owing to very small size . blood and bronchoaspirate culture identi ed staphylococcus aureus in 3 / 9 patients and a mixed bacterial ora in 6 / 9 cases . 
 discussion human in uenza pandemics are caused by in uenza viruses from nonhuman reservoirs : among the in uenza pandemics sede ed estensione ( tabella 2 ) : 14 ( 82 , 3% ) ri , 2 n ( 11 , 7% ) , 5 ( 29 , 4% ) ogg . 
 quattordici / 90 pazienti ( 15 , 5% ) presentavano reperti radiogra ci sospetti per sovrainfezione batterica ( tabella 1 ) , variamente combinati tra di loro : 9 / 14 ( 64 , 2% ) segni di versamento pleurico , 4 ( 28 , 5% ) consolidamenti con broncogramma aereo , 2 ( 14 , 2% ) linfonodi , 1 ( 7 , 1% ) cavitazione , 1 ( 7 , 1% ) idropneumotorace . 
nove / 17 ( 52 , 9% ) casi mostravano segni di sovrainfezione batterica , variamente combinati , allindagine tc ( tabella 2 ) : 9 / 9 ( 100% ) albero in ore , 6 ( 66 , 6% ) consolidamenti con broncogramma aereo , 4 ( 44 , 4% ) versamento pleurico e 3 ( 33 , 3% ) pericardico , 2 ( 22 , 2% ) linfonodi > 1 cm , 1 ( 11 , 1% ) cavitazione , 1 ( 11 , 1% ) idropneumotorace . 
lindagine tc ha confermato in 9 casi i reperti di sovrainfezione batterica identi cati alla rx del torace ( 9 / 14 , 64 , 2% ) ed ha indicato come falsi positivi 5 casi di sospetto versamento pleurico rilevati alla rx del torace . 
inoltre , in due dei 9 casi in cui la tc ha confermato i segni radiogra ci di sovra infezione batterica , essa ha individuato consolidamenti con broncogramma aereo misconosciuti alla rx del torace . 
the infection is due to pigto - human transmission of a viral pathogen produced by the triple genetic reassortment of human , swine and avian viral strains in north america and eurasia ; human - to - human transmission occurs through respiratory droplets or contact with infected surfaces [ 9 , 14 , 16 ]  . 
 according to the who , from the beginning of the pandemic to 15 november 2009 , > 78 , 000 cases of in uenza a h1n1 were noti ed in europe and 190 , 765 in the americas , with a death toll of at least 350 and 4 , 806 , respectively [ 17 ]  . 
from 19 october , when in unet monitoring began in italy [ 18 ] , to 8 november , there were an estimated 1 , 521 , 000 cases of in uenza a / h1n1 in italy sola proiezione ap . 
in ne , in 3 dei 9 pazienti in cui la tc ha confermato i reperti radiogra ci di sovrainfezione batterica , essa ha anche rilevato la presenza di versamento pericardico . 
in tutti i 3 casi esso era stato misconosciuto alla rx del torace a causa della sua modestissima entit . allemocoltura e nel broncoaspirato , in 3 / 9 pazienti stato identi cato il batterio staphylococcus aureus , in 6 / 9 casi una ora batterica mista . 
quindici / 167 pazienti ( 8 , 98% ) hanno necessitato di intubazione oro - tracheale e ventilazione assistita , previo ricovero in unit di terapia intensiva , per il peggioramento delle condizioni clinico - radiologiche . 
 tutti presentavano condizioni patologiche concomitanti rappresentate da ipertensione arteriosa complicata da placenta previa ed atonia uterina post - partum ( 1 / 15 ; 6 , 6% ) , cardiopatia ipertensiva ( 3 / 15 ; 20% ) , en sema ( 3 / 15 ; 20% ) , distro a bollosa ( 1 / 15 ; 6 , 6% ) , malformazioni scheletriche della gabbia toracica ( 2 / 15 ; 13 , 3% ) , obesit ( 4 / 15 ; 26 , 6% ) , diabete ( 2 / 15 ; 13 , 3% ) , uso di droghe ( 1 / 15 ; 6 , 6% )  . 
b sezione assiale di mdct del torace : consolidamenti polmonari bilaterali con associate diffuse alterazioni ground glass sono prevalentemente distribuiti in sede mantellare e posteriore e nel segmento apicale del lobo inferiore . 
sei / 15 ( 40% ) pazienti morirono per linsorgere in 4 casi di acute respiratory distress syndrome ( ards ) , in 2 di multi - organ failure ( mof )  . 
 discussione le pandemie in uenzali umane derivano da virus in uenzali provenienti da reservoir non umani : delle tre pandemie in uenzali del ventesimo secolo , quella del 1918 stata causata da un virus in uenzale di origine aviaria [ 14 ] e le altre due , nel 1957 e nel 1968 , sono state provocate da nuove linee virali risultanti dalla combinazione di virus aviari e umani attraverso un processo di riassortimento [ 15 , 16 ]  . 
 lin uenza virale a rappresenta unepidemia su scala mondiale causata da un nuovo virus in uenzale a / h1n1 che si diffuso nel marzo 2009 dal messico in tutto il mondo . 
linfezione dovuta alla trasmissione , da parte di alcuni allevamenti suini alluomo , di un patogeno virale prodotto dal triplo riassorimento genetico delle tipologie virali umana , suina ed aviaria nel nord america e nei paesi eurasiatici ; il contagio interuomo si realizzerebbe mediante la trasmissione di goccioline di saliva o il contatto delle mani con super ci infette [ 9 , 14 , 16 ]  . 
 secondo loms , dallinizio della pandemia al 15 novembre 2009 , oltre 78000 casi di in uenza a h1n1 sono stati noti cati in europa e 190765 nelle americhe , con un numero di decessi rispettivamente di almeno 350 e 4806 [ 17 ]  . 
dal 19 ottobre , giorno di avvio della sorveglianza in unet in italia [ 18 ] , all8 novembre , sono stimati 1521 mila i casi di in uenza a h1n1 nel nostro paese , con un numero di decessi pari a 53 secondo leuropean centre for disease prevention and control ( ecdc ) [ 19 ]  . 
in italia le regioni dove si registrata la pi ampia diffusione del virus sono state le marche con un incidenza del 2 , 9% seguite da emilia romagna ( 1 , 8% ) , lazio ( 1 , 7% ) , abruzzo ( 1 , 6% ) e campania ( 1 , 6% )  . 
in data 15 novembre 2009 , in cui si registrato il picco di contagio , le vittime correlate alla in uenza a sono salite a 53 di cui 23 in campania , 7 in emilia romagna , 5 in lombardia . 
in un recente lavoro [ 8 ] stato confermato che il virus h1n1 si trasmette caratteristicamente tra bambini e giovani adulti , colpendo nel 45% dei casi giovani di et < 18 anni e solo nel 5% dei casi individui di et > 65 anni . 
sebbene nella nostra personale esperienza la gravit dellimpatto epidemiologico dellin uenza h1n1 sia stato ampiamente ridimensionato rispetto alle aspettative globali , anche la nostra popolazione di studio ha presentato un prevalente interessamento delle fasce di et pi giovanili radiol med ( 2011 ) 116 : 706719 fig . 
the most affected age groups were children and teenagers from birth to 14 years of age ( incidence 3.6% ) , and , to a lesser extent , individuals aged 1564 years ( 0.7% ) and > 65 years ( 0.1 % ) [ 19 ]  . 
a recent paper [ 8 ] con rmed that the h1n1 virus is typically transmitted among children and young adults , affecting individuals < 18 years in 45% of cases and those > 65 years in 5% of cases only . 
la pi alta incidenza nelle fasce di et inferiori ai 18 anni potrebbe essere legata , invece , in particolar modo nei bambini , a meccanismi di immunode cienza e / o di immaturit immunologica [ 21 , 22 ]  . 
 le principali manifestazioni cliniche dellin uenza virale h1n1 descritte in letteratura [ 8 ] sono rappresentate da : febbre ( 95% ) , tosse ( 88% ) , cefalea ( 34% ) , faringodinia ( 31% ) , vomito ( 29% ) , diarrea ( 25% )  . 
in accordo con i dati della letteratura [ 8 ] abbiamo osservato un elevazione degli indici di funzionalit epatica ( 44 , 9% ) , dei leucociti ( 17 , 9% ) e trombocitopenia ( 2 , 9% )  . 
le principali patologie concomitanti sono state bronchite asmatica 716 radiol med ( 2011 ) 116 : 706719 cially as regards children to mechanisms of immunode ciency and / or immunological immaturity [ 21 , 22 ]  . 
 the most important reported [ 8 ] clinical manifestations of h1n1 virus in uenza are : fever ( 95% ) , cough ( 88% ) , headache ( 34% ) , sore throat ( 31% ) , vomiting ( 29% ) and diarrhoea ( 25% )  . 
 to date , few studies addressing chest imaging in patients affected by in uenza a / h1n1 have been published [ 1012 ] , and the presentation of h1n1 virus pneumonia on both cxr and chest ct seems to re ect the general features of viral pneumonia [ 23 ]  . 
one study [ 11 ] reported on the main cxr and chest ct ndings in seven patients affected by in uenza a / h1n1 : bilateral ggo , more frequently associated with focal or multifocal areas of consolidation . 
of the 15 / 66 ( 22.7% ) patients who underwent ct , 9 / 15 ( 60% ) had ggo combined with consolidation , with diffuse or lobar extension in 70% of cases . 
it is likely that the alarmism regarding in uenza infection prompted many patients to seek early medical attention , thus allowing detection of interstitial reticulation , an early nding in viral disease . 
only 5% of patients had allo stato attuale sono stati realizzati pochi studi di imaging del torace nei pazienti affetti da in uenza virale a / h1n1 [ 1012 ]  . 
alcuni autori [ 11 ] hanno descritto le principali alterazioni radiogra che e tc in 7 pazienti affetti da inuenza a / h1n1 : opacit ground - glass bilaterali , pi di frequente associate ad aree di consolidamento a distribuzione multifocale , talora anche focali . 
dei 66 ( 30% ) pazienti che avevano effettuato rx del torace , 28 / 66 ( 42% ) presentavano consolidamenti ( 50% ) , maggiormente distribuiti ai lobi inferiori ; dei 15 / 66 ( 22.7% ) pazienti sottoposti ad esame tc , 9 / 15 ( 60% ) presentavano lassociazione di ground glass e consolidamenti , ad estensione diffusa ed a sede lobare inferiore nel 70% dei pazienti . 
queste ultime presentavano , tuttavia , unestensione prevalentemente monolaterale e focale ( 61 , 5% ) e distribuzione pi evidente in sede medio - basale ( 46 , 1% )  . 
verosimile che lallarmismo per il contagio in uenzale abbia indotto numerosi pazienti a recarsi prontamente allattenzione medica , rendendo precoce losservazione radiogra ca delle ri , reperto iniziale di danno in corso di malattia virale . 
in accordo con i dati della letteratura [ 10 , 11 ] le lesioni elementari evidenziate in tc ( tabella 2 ) sono state ogg associate a cm ( 58 , 8% ) , ambedue ad estensione bilaterale , radiol med ( 2011 ) 116 : 706719 nodules , which showed unilateral and focal extension and basal distribution in 60% of cases . 
the distribution of ggo combined with consolidation was predominant in the lower lobes ( 60% ) and subpleural regions and was associated in 60% of cases with similar peribronchovascular lesions . 
additionally , there were no cases of isolated consolidation without ggo , probably owing to the relatively early observation of the primary pulmonary lesions : in the initial phase of infection , ggos earlier lesions in which bronchial and vessel margins are still discernible manifest alongside consolidations compared with the later phases of disease ( not necessarily evolving to ards ) in which they increase in attenuation and coalesce into consolidations . 
only two cases showed parenchymal nodules , which were focal and unilateral and distributed in the lower lobe ; these nodules had already been identi ed at cxr and were referable to an underlying infectious disease . 
at variance with previous reports [ 10 ] , in the contrast - enhanced examinations requested for suspected pulmonary embolism , we found no thromboembolic phenomena involving the pulmonary arteries or their branches . 
 recent literature [ 2426 ] has extensively described the principal pulmonary abnormalities seen in bacterial infections : consolidations with air bronchogram , tree - in - bud pattern , cavitation , pleural and / or pericardial effusion and lymphadenopathy . 
in agreement with these data [ 2426 ] , although differently identi ed by the two imaging modalities ( tables 1 and 2 ) , the most common signs of bacterial suprainfection in our series were tree - in - bud pattern , consolidation with air bronchogram and pleural and pericardial effusion . 
la distribuzione delle ogg associate ai cm stata predominante ai lobi inferiori ( 60% ) ed in sede subpleurica , associata nel 60% dei casi ad omologhe lesioni in sede peribroncovascolare . 
inoltre non sono stati evidenziati cm isolati in assenza di ogg verosimilmente per la relativa precocit di osservazione delle lesioni elementari polmonari : nella fase iniziale di infezione le ogg , lesioni pi precoci in cui sono ancora distinguibili bronchi e vasi , si manifestano insieme ai cm , rispetto a fasi pi tardive della patologia , non necessariamente evolventi in ards , in cui esse aumentano la loro densitometria e con uiscono interamente in consolidamenti . 
solo in 2 casi sono state identi cate nodulazioni parenchimali , focali e monolaterali , a distribuzione lobare inferiore , peraltro gi identi cate allesame rx del torace , inquadrabili nellambito della patologia infettiva di base . 
contrariamente ad alcuni autori [ 10 ] , negli esami eseguiti con mezzo di contrasto endovena per quesito clinico di embolia polmonare , non abbiamo evidenziato fenomeni di natura tromboembolica a carico delle arterie polmonari e dei suoi rami . 
 la letteratura moderna [ 2426 ] ha ampiamente descritto le principali alterazioni polmonari identi cabili in caso di infezione batterica : consolidazioni con broncogramma aereo , tree - in - bud , cavitazioni , versamento pleurico e / o pericardico , linfoadenomegalie . 
in accordo con i dati della letteratura [ 2426 ] , sebbene differentemente identi cati nelle due metodiche di studio ( tabelle 1 e 2 ) , i segni di sovrainfezione batterica pi frequenti nel nostro gruppo di studio sono stati tree - in - bud , cm con broncogramma aereo , versamento pleurico e pericardico . 
dei 15 / 167 casi ( 8 , 98% ) sottoposti a ventilazione meccanica per peggioramento del quadro clinico - radiologico , tutti presentavano patologie concomitanti , in particolare bpco ( 33 , 3% )  . 
standard cxr and chest ct are the reference investigations in identifying the location and extension of primary pulmonary lesions and documenting the signs of bacterial suprainfection and pulmonary complications of h1n1 in uenza , thus allowing correct diagnostic , prognostic and therapeutic management . 
 radiol med ( 2011 ) 116 : 706719 caratteristiche radiologiche e cliniche di unampia popolazione di pazienti ospedalizzati in un centro di riferimento per le malattie infettive con diagnosi accertata , mediante tampone faringeo , di in uenza virale h1n1 . 
lesame radiogra co standard e la tomogra a computerizzata del torace hanno rappresentato le indagini di riferimento nellindividuazione della sede ed estensione delle lesioni elementari polmonari , documentando i segni di sovrainfezione batterica e le complicanze polmonari dellin uenza h1n1 , ai ni di un corretto inquadramento diagnostico , prognostico e terapeutico . 
mercuri2 1department of interventional angiographic radiology , istituto ortopedico rizzoli , via di barbiano 1 / 10 , 40136 bologna , italy 2department of orthopaedics , istituto ortopedico rizzoli , university of bologna , bologna , italy 3department of radiology , istituto ortopedico rizzoli , bologna , italy 4department of radiology , ospedale sant orsola , bologna , italy 5department of nuclear medicine , maggiore hospital , bologna , italy 6unita operativa di oncologia , ospedale sant orsola , bologna , italy 7istituto toscano tumori , nuovo ospedale san giuseppe , empoli , italy 8poliambulatorio di radiologia zappi , bartalena , imola , italy correspondence to : p . 
 indications for repeat embolisation included pain or imaging evidence of progressive disease : 105 patients had repeat embolisation at the same location at an interval of 13 months ; 260 patients had one embolisation , 78 had two and 29 had three or more . 
in 35 cases , embolisation was not feasible because of poor lesion vascularisation ( 21 patients with bone metastases and two with aneurysmal bone cysts ) , origin of the adamkiewicz artery in the embolisation eld ( four patients with bone metastases and one with aneurysmal bone cyst ) , atheromatosis and arteriosclerosis ( ve patients with bone metastases ) and riassunto obiettivo . 
lembolizzazione stata usata come trattamento primario per pseudo - tumori e tumori benigni , con signi cato adiuvante nel trattamento dei tumori maligni e nelle forme benigne e con signi cato palliativo nel trattamento dei sarcomi dellosso e delle lesioni metastatiche . 
il dolore e levidenza allimaging di una progressione di malattia era lindicazione per la ripetizione dellembolizzazione ; 105 hanno ripetuto lembolizzazione nella stessa sede , ad intervallo di 13 mesi ; 260 pazienti sono stati sottoposti ad una sola embolizzazione , 78 pazienti a due embolizzazioni e 29 pazienti a tre o pi embolizzazioni . 
si ottenuta risposta clinica in 406 procedure ( 97% ) e nessuna risposta in 13 procedure in pazienti con tumori del bacino 794 radiol med ( 2011 ) 116 : 793808 anatomical and technical problems such as small - calibre vessels , many branches and acute vessel angles ( two patients with bone metastases )  . 
in 35 pazienti non stata eseguita alcuna embolizzazione : in 21 pazienti con metastasi e in 2 con cisti aneurismatiche per scarsa vascolarizzazione ; in 4 con metastasi ossea ed in 1 con cisti aneurismatica per la presenza di arteria di adamkiewicz nella vascolarizzazione della lesione ; in 5 pazienti con metastasi ossee per problemi steno - ostruttivi su base ateromasica ; in 2 pazienti con metastasi per problemi tecnici legati al calibro dei vasi . 
lnbca , a nostro avviso , lagente embolizzante pi adatto per locclusione di piccoli vasi in assenza di complicanze maggiori , ma richiede buona esperienza da parte degli operatori . parole chiave embolizzazione arteriosa selettiva nbca tumori ossei introduction introduzione embolisation was rst reported by frieda feldman in 1975 as a useful adjunct for managing selected bone tumours [ 1 ]  . 
ever since , many authors have reported on selective and superselective intra - arterial embolisation for primary bone and soft - tissue tumours and bone metastases from different primary neoplasms [ 217 ]  . 
these reports indicate that embolisation is generally an effective treatment for selected primary tumours and bone metastases and associated with a rapid reduction in pain and tumour volume lasting from 1 to 9 months [ 5 , 7 , 9 , 11 , 18 ]  . embolisation can be palliative or adjunctive , primary or serial . 
reduced bleeding is particularly il primo lavoro sullutilit dellembolizzazione arteriosa come supporto aggiuntivo nel management dei pazienti con tumori ossei , fu prodotto dal dottor frieda feldman nel 1975 [ 1 ]  . 
da allora , molti autori si sono espressi sullapplicazione di questa metodica sia per i tumori primitivi ossei e dei tessuti molli , che per le metastasi ossee da differente neoplasia primitiva [ 217 ]  . 
questi report hanno evidenziato che lembolizzazione in genere uno strumento ef cace per selezionate neoplasie primitive e secondarie , che si associa ad una riduzione del volume tumorale e della sintomatologia dolorosa , con una durata da uno a nove mesi [ 5 , 7 , 9 , 11 , 18 ]  . lembolizzazione pu essere palliativa o adiuvante , primaria o seriale . 
in questo modo si determina una riduzione del volume tumorale , una diminuzione del sanguinamento ed una maggior de nizione del con ne tumore - tessuto circostante , cos da rendere pi agevole la resezione chirurgica . 
il ridotto sanguinamento inoltre radiol med ( 2011 ) 116 : 793808 advantageous for patients with rare blood groups or those prone to transfusion reactions [ 7 , 14 , 19 ]  . 
in this paper , we present our experience with 454 selective transarterial superselective embolisations using n - 2 - butyl cyanoacrylate ( nbca ) for primary , adjuvant or palliative treatment of bone tumours and discuss the clinical and imaging results . materials and methods particolarmente vantaggioso per quei pazienti con gruppi sanguigni rari o per quelli con maggior predisposizione a reazioni avverse trasfusionali [ 7 , 14 , 19 ]  . in questo studio , presenteremo la nostra esperienza di 454 embolizzazioni arteriose selettive e superselettive utilizzando ln - 2 - butil - cianoacrilato ( nbca ) per il trattamento primario , adiuvante o palliativo dei tumori ossei , discutendone i risultati e le implicazioni dal punto di vista clinico e diagnostico . we present 365 patients with primary and metastatic musculoskeletal bone tumours treated with selective transfemoral embolisation using nbca during an 8 - year period from december 2002 to april 2010 . 
primary tumour histology and location varied ( tables 1 and 2 ) ; 105 materiali e metodi durante un periodo di 8 anni , da dicembre 2002 ad aprile 2010 , 365 pazienti , 206 uomini e 159 donne con et media pari a 53 anni ( range da 3 , 3 a 87 anni ) , sono stati sottoposti ad embolizzazione selettiva trans - femorale con nbca per neoplasie primitive o secondarie dellapparato table 1 tumour histology and number of embolisations of the 365 patients in this study primary tumour patients embolisation one embolisation two embolisations three or more embolisations bone metastases aneurysmal bone cyst osteosarcoma giantcell tumour vertebral haemangiomas chordoma haemangioendothelioma of bone osteoblastoma haemangiopericytoma paraganglioma ependymoma malignant schwannoma plasmacytoma metastasi ossee cisti ossee aneurismatiche osteosarcoma tumore a cellule giganti emangioma vertebrale cordoma emangioendotelioma osseo osteoblastoma emangiopericitoma paraganglioma ependimoma schwannoma maligno plasmocitoma 243 243 187 187 tabella 1 istologia tumorale e numero di embolizzazione nei 365 pazienti inclusi in questo studio tumore primitivo pazienti embolizzazione una due tre o pi table 2 site of the 454 embolisations embolisations tabella 2 sito delle 454 embolizzazioni sito embolizzazioni 796 site pelvis and sacrum spine lower limb upper limb thoracic cage pelvi e sacro rachide arto inferiore arto superiore gabbia toracica patients had repeat embolisation at the same location at an interval of 13 months ; 260 patients had one , 78 had two and 29 had three or more . 
the study was approved by the institutional review board / ethics committee of the authors institution . embolisation was indicated in all tumours with favourable vascularity as judged by pre - embolisation angiography . 
patients with bone metastases were the most numerous in the series ; all patients had intense pain , and one patient had a pathological fracture . technique diagnostic digital subtraction angiography ( dsa ) ( contrast medium : iomeprol 300 mg / ml , iomeron , bracco , milan , italy , and iohexol 350 mg / ml , omnipaque , ge healthcare , milan , italy ) was performed before embolisation to identify the feeding vessels . 
angiographic apparatus used were the philips integris v3000 cesar - scp - visub angiographic system ( philips medical systems , eindhoven , the netherlands ) and the siemens angiostar plus / plus or ( siemens ag , medicals engineering , forchheim , germany )  . 
in patients with metastases in the pelvis or lower extremities , the contralateral transfemoral radiol med ( 2011 ) 116 : 793808 muscolo - scheletrico , con lesecuzione , complessivamente , di 454 embolizzazioni . 
listotipo tumorale , cos come la sede , stato variabile ( tabelle 1 e 2 ) : in particolare , 105 pazienti hanno ripetuto la procedura nella stessa sede a distanza di 13 mesi ; 260 pazienti sono stati sottoposti ad una sola embolizzazione , 78 pazienti hanno eseguito 2 embolizzazioni ed in ne 29 pazienti tre o pi embolizzazioni . 
 inoltre , questo studio stato approvato dalla commissione etica del nostro istituto . lembolizzazione stata eseguita in tutti i tumori con una vascolarizzazione giudicata favorevole in base allangiogra a pre - procedura . 
tale procedura stata effettuata come trattamento primario per i tumori ossei benigni , come trattamento adiuvante lintervento chirurgico per tumori ossei sia benigni che maligni , ed in ne a scopo palliativo per i sarcomi ossei e per le metastasi . 
 i pazienti con metastasi ossee sono stati i pi numerosi pazienti arruolati ; in tutti i casi era presente unintensa sintomatologia dolorosa e un paziente presentava una frattura patologica . tecnica al ne di identi care i vasi rifornenti la neoplasia , unangiogra a digitale a sottrazione ( mezzo di contrasto : iomeprol 300 mg / ml [ iomeron , bracco , milano , italia ] e iohexol 350 mg / ml [ omnipaque , ge healthcare , milano , italia ] ) stata eseguita prima della procedura di embolizzazione . 
 lapparecchio angiogra co utilizzato stato il philips integris v3000 cesar - scp - visub angiographic system ( philips medical systems , eindhoven , olanda ) e il siemens angiostar plus / plus o.r. 
in tutti i pazienti adulti , langiogra a e lembolizzazione arteriosa selettiva sono state effettuate sotto anestesia locale , utilizzando la tecnica seldinger attraverso il cateterismo arterioso dellarteria femorale ; in tutti i pazienti pediatrici , langiogra a e lembolizzazione arteriosa selettiva sono state effettuate sotto anestesia generale . 
larteria femorale stata cateterizzata con un introduttore 4 french ( cordis corporation , miami , fl , usa ) o con un introduttore 5 french ( terumo corporation , tokyo , giappone )  . 
a questo punto , nei pazienti con lesioni spinali o pelviche , stata eseguita unaortogra a panoramica con catetere 4 french pigtail ( cordis corporation ) , seguita da una arteriogra a selettiva e superselettiva mediante cateteri cobra , e simmons ( terumo corporation ) e microcateteri di 130 cm , 2 , 7 - 2 , 9 french pre - modellati mc - pp27131 ( coaxial catheters system , terumo corporation )  . 
the femoral artery was catheterised with a 4 - french ( cordis corporation , miami , fl , usa ) or 5 - french ( terumo corporation , tokyo , japan ) introducer . 
from the mixture , 1 ml was aspirated in an insulin ( 1 ml ) syringe ; depending on the pathological vasculature , 0.10.2 ml of the aspirate mixture was injected , sandwiched with 2 + 2 ml of 5% glucosate solution under uoroscopic control . 
the bolus administration in low doses ( 0.10.2 ml ) of the sandwiched embolic agent under uoroscopic control , followed by arteriography and new embolisation if incomplete occlusion or more feeding vessels were observed , provided for the ef cacy and safety of the embolisation procedures . the technical success of embolisation was evaluated by additional angiography after completion of the procedure . 
embolisation was considered technically complete when there was stasis of intravascular contrast material and either complete elimination of the tumours hypervascular staining or 80% elimination of the tumours pathological vasculature compared with the initial diagnostic angiogram [ 7 , 19 ]  . clinical and imaging effects of treatment were evaluated at routine follow - up examinations . 
nei pazienti con lesioni localizzate agli arti , invece , laortogra a non stata eseguita ; in pazienti con lesioni dellarto superiore , lembolizzazione stata eseguita con utilizzo di catetere vertebral ( terumo corporation ) e microcateteri , e in pazienti con lesioni dellarto inferiore con catetere vertebral , cobra e microcateteri . 
in tutti i pazienti , lagente embolizzante impiegato stato lnbca ( glubran 2 , gem , viareggio , italia ) , diluito con lipiodol al 33% ( 1 acone [ 10 ml ] , lipiodol ultra uido , guerber spa , francia ) ed iniettato tra soluzione glucosate al 5% , al ne di prevenire la polimerizzazione . 
di tale miscela , 1 ml stato aspirato in una siringa da insulina ( 1 ml ) e , a seconda della vascolarizzazione patologica , 0 , 10 , 2 ml della miscela aspirata , sono state iniettate , racchiuse in una 2 + 2 ml di soluzione glucosata al 5% , sotto controllo uoroscopico . 
se a causa dellipervascolarizzazione della lesione fosse necessario pi di un acone di nbca ( 1 ml ) , si pu procedere alla preparazione di una nuova miscela con le medesime modalit ; complessivamente , nella nostra casistica , sono stati impiegati in media tra gli 0 , 5 e i 2 ml di nbca con lipiodol per ogni singola embolizzazione . 
il bolo cos composto , somministrato a bassa dose ( 0 , 10 , 2 ml ) sotto controllo uoroscopico , garantisce lef cacia e la sicurezza della procedura . il successo tecnico dellembolizzazione stato valutato da unangiogra a panoramica , eseguita al termine della procedura . 
lembolizzazione stata considerata tecnicamente ben riuscita , laddove era presente stasi intravascolare di mezzo di contrasto , oppure una completa eliminazione dello stesso , o uneliminazione superiore all80% rispetto a quella dellangiogramma iniziale [ 7 , 19 ]  . il risultato dal punto di vista clinico e di imaging stato quindi valutato durante le indagini di routine durante follow - up . 
si de nisce come risposta clinica , una riduzione della sintomatologia dolorosa e dellimpiego di analgesici maggiore o uguale al 50% , come assenza di risposta , una riduzione inferiore al 50% . 
 la risposta dal punto di vista diagnostico stata invece studiata mediante indagini di tomogra a computerizzata 798 radiol med ( 2011 ) 116 : 793808 pain and analgesics use , and no response as < 50% decrease . 
 imaging tumour response was evaluated on computed tomography ( ct ) scans obtained at 3 , 6 and 12 month or at the latest follow - up by hypoattenuating areas within the tumour that resembled necrosis , tumour size and ossi cation . risultati a 3 , 6 , 12 mesi o no allultimo follow - up ed stata de nita dalla valutazione del volume tumorale , dellossi cazione e della comparsa di aree di ipoattenuazione a livello dellarea tumorale , indicanti la comparsa di necrosi . results total of 419 procedures ( 93% ) were technically successful ; selective catheterisation and embolisation of the feeding vessels was achieved in all cases . 
1 pre - embolisation angiography of a 67 - year - old man with a metastatic renal - cell carcinoma of the l3 vertebra shows the adamkiewicz artery originating from the second left lumbar artery ( arrows )  . 
1 langiogra a pre - embolizzazione di un uomo di 67 anni con carcinoma renale metastatico a livello di l3 mostra lorigine dellarteria di adamkiewicz dalla seconda arteria lombare sinistra ( frecce )  . 
 quattrocentodiciannove embolizzazioni ( 93% ) , sono state portate a termine con successo ; la cateterizzazione selettiva e lembolizzazione dei vasi rifornenti la lesione stata raggiunta in tutti i casi . 
inoltre , in tutti i casi , langiogra a post - procedura ha evidenziato , a livello lesionale , una completa interruzione dellapporto vascolare o una riduzione della vascolarizzazione superiore all80% . 
nei pazienti con intensa sintomatologia dolorosa , in particolare nei pazienti con lesioni agli arti dimostratisi essere quelli con una sintomatologia pi intensa prima dellembolizzazione , si raggiunta una risoluzione del dolore pressoch completa . 
 nei pazienti con lesioni spinali , il miglioramento del dolore stato invece di media entit . per quel che riguarda i pazienti con metastasi ossee , si raggiunta una risoluzione della sintomatologia di durata media pari a mesi 8 , 1 ( range da 1 a 12 mesi ) ; la ricomparsa del dolore non ha comunque mai raggiunto i livelli di intensit pre - procedura . 
il massimo diametro tumorale dopo embolizzazione stato in media pari a 5 , 5 cm ( range da 2 a 20 cm ) , contro i 7 , 8 cm ( range da 5 a 30 cm ) prima dellembolizzazione stessa . 
in 65 pazienti ( di cui , 35 con carcinoma renale , figure 1 e 2 , 15 con carcinoma tiroideo , 8 con tumore al seno e 7 con tumore polmonare , figura 3 ) stata documentata la presenza di un variabile grado di ossi cazione : in particolare , si osservata unabbondante ossi cazione in sei pazienti , mentre i restanti hanno evidenziato una iniziale rima periferica di formazione ossea . 
in 12 pazienti radiol med ( 2011 ) 116 : 793808 a clinical response was achieved in 406 procedures ( 97% ) , and no response in 13 procedures in patients with pelvis and sacrum skeletal metastases . 
medium pain relief was observed in patients with spinal lesions . in patients with bone metastases , the mean duration of pain relief was 8.1 ( range 112 ) months ; recurrent pain was never as intense as pre - embolisation pa mean maximal tumour diameter after embolisation was 5.5 cm ( range 220 cm ) compared with 7.8 cm ( range 530 that cm ) before embolisation . 
in 12 patients with bone metastases , surgical treatment was performed after embolisation ; in none of these patients was surgery feasible before embolisation . embolisation was effective in 32 patients with aneurysmal bone cysts ( 94% )  . 
recurrence and / or persistence of the lesion occurred in 14 patients ; nine patients had one recurrence and / or persistence , and ve patients had two recurrences each . 
ten of the 19 recurrences occurred in patients with lesions > 5 c three patients had complications , including skin necrosis ( two patients ) and transient paresis of the sciatic nerve ( one patient )  . 
eighty - one patients ( 22% ) exhibited clinical signs of postembolisation syndrome as diagnosed by fever or chills , nausea and vomiting and increased ischaemic pain at the site of embolcon metastasi ossee , si proceduto allintervento chirurgico dopo lembolizzazione ; in nessuno di essi tale intervento risultava eseguibile prima dellembolizzazione stessa . per quel che riguarda i pazienti con cisti ossee aneurismatiche , lembolizzazione stata ef cace in 32 casi ( 94% )  . 
 la recidiva e / o la persistenza della lesione si veri cata in 14 pazienti , di cui nove hanno presentato una recidiva e / o una persistenza della lesione stessa , e cinque due recidive . 
dieci delle 19 recidive si sono osservate in pazienti con lesioni con diametro maggiore a 5 c tre pazienti hanno presentato complicanze caratterizzate da necrosi cutanea ( due pazienti ) e paresi transitorie del nervo sciatico ( un paziente )  . 
una povert vascolare stata osservata soprattutto in alcuni tumori in ltratanti a derivazione neurogenica , quali schwannomi maligni . nellimmediato periodo post - embolizzazione , si sono registrate 122 complicanze minori e 6 complicanze maggiori . 
ottantuno pazienti ( 22% , di cui 49 con metastasi ossee e 32 con altri tumori ) hanno evidenziato segni clinici della sindrome post - embolizzazione quali febbre , brividi , nausea , vomito e un aumento del dolore , ischemico , a livello del sito dellembolizzazione . 
parestesie transitorie sono state osservate in 41 pazienti ( 11% , di cui 37 con metastasi ossee sacroiliache , 2 con cisti aneurismatiche e 2 con tumori del sacro a cellule giganti ) con lesioni del bacino e del sacro . 
 si segnalano inoltre transitoria paresi del nervo sciatico , veri catasi in un solo paziente ( 0 , 3% ) dopo embolizzazione per cisti ossea aneurismatica della pelvi , e necrosi cutanea e sottocutanea in 5 pazienti ( 1 , 4% ) , di cui due con cisti ossee aneurismatiche della spalla e della pelvi , uno con emangioma dellomero , uno con tumore a cellule giganti del bacino e uno con paraganglioma metastatico a localizzazione pelvica dopo due procedure di embolizzazione . 
2a axial ( left ) and sagittal ( right ) computed tomography of a 32 - year - old man with giant - cell tumour of the sacrub preoperative selective angiography and embolisation of the feeding vessels originating from the internal iliac arteries ( left ) and middle sacral artery ( centre )  . 
3a computed tomography ( left ) and coronal t2 - weighted magnetic resonance imaging ( right ) of the pelvis of a 16 - year - old girl with a large osteosarcoma . 
3a tomogra a computerizzata ( sinistra ) a sezioni coronali t2 - pesate di risonanza magnetica ( destra ) , di un ampio osteosarcoma del bacino , in donna di anni 16 . 
d a due anni dalla diagnosi e dalla chemioterapia , la risonanza magnetica assiale ( sinistra ) e coronale ( destra ) evidenzia una signi cativa riduzione del volume tumorale . radiol med ( 2011 ) 116 : 793808 802 fig . 
transient paraesthesias were observed in 41 patients ( 11% ) with pelvic and sacrum lesions ; there were 37 patients with sacroiliac metastatic bone tumours , two with aneurysmal bone cysts and two with giant - cell tumours of the sacruin all patients , symptoms resolved completely with symptomatic treatment within less than a week . 
skin breakdown and subcutaneous necrosis occurred in ve patients ( 1.4% ) ; there were two patients with aneurysmal bone cysts of the shoulder and pelvis , one with haemangioma of the humerus , one with giant - cell tumour of the pelvis and one with a metastatic paraganglioma of the pelvis after two embolisations . 
the patient with aneurysmal bone cyst complicated by skin necrosis of the shoulder underwent ap reconstruction ; this was considered a major complication . discussione in questo studio , abbiamo esaminato il ruolo dellembolizzazione arteriosa selettiva nei tumori ossei in una grande casistica di pazienti . 
sappiamo , comunque , che un trial prospettico e randomizzato sarebbe pi vantaggioso per de nire lesatto ruolo dellembolizzazione a scopo primario , palliativo o adiuvante per le metastasi ossee , le cisti ossee aneurismatiche e i tumori a cellule giganti , poich noto che in questi pazienti possono essere eseguiti altri approcci terapeutici . 
in 12 pazienti con metastasi ossee , radiol med ( 2011 ) 116 : 793808 discussion in this study , we explored the role of selective transarterial embolisation for bone tumours in a large series of patients . 
we acknowledge that a prospective randomised trial would be more bene cial for de ning the exact value of primary , palliative or adjuvant embolisation for metastatic bone disease , aneurysmal bone cysts and giant - cell tumours , as in these patients , other treatments were also performed . 
preoperative or serial embolisation techniques using different embolic agents can be used as primary or adjuvant treatment to surgery or radiation therapy for patients with metastatic bone disease [ 6 ]  . 
by superselective catheterisation and embolisation of the pathological feeding arteries to the lesion with the most appropriate embolic agent , embolisation can be expected to be successful in up to 90% of cases . 
 patients with renal or thyroid bone metastases have a good chance of undergoing successful treatment with selective vascular occlusion , in contrast to patients with lung metastases to bone [ 7 ]  . 
imaging showed evidence of tumour necrosis in all cases and variable ossialla procedura di embolizzazione seguito il trattamento chirurgico : si precisi che in nessuno di essi la resezione chirurgica era eseguibile prima dellesecuzione dellembolizzazione stessa . le opzioni terapeutiche nei casi di malattia ossea metastatica hanno in genere una nalit palliativa . 
si ricordi inoltre che tutte le lesioni secondarie causano progressivamente una perdita ossea e che molte , se non tutte , si caratterizzano per unipervascolarizzazione , specie in caso di carcinoma primitivo renale e tiroideo [ 5 , 20 ]  . 
le tecniche di embolizzazione pre - operatoria e di embolizzazione seriale , che impiegano differenti agenti embolici , possono essere impiegate nel trattamento primario o adiuvante la chirurgia o la radioterapia proprio per i pazienti con malattia ossea metastatica [ 6 ]  . 
poich tutte le metastasi ossee si caratterizzano per unipervascolarizzazione , tutti i pazienti dovrebbero essere considerati come appropriati canditati per lesecuzione di tale procedura [ 5 , 20 ] , che , tramite il cateterismo superselettivo e lembolizzazione delle arterie tributarie della lesione con un appropriato agente embolico , pu essere espletata con successo in pi del 90% dei casi . 
il periodo di tempo libero dalla sintomatologia dolorosa pu durare da 1 a 9 mesi ; dopo tale intervallo , una ripetizione dellembolizzazione sicura e si pu rendere necessaria [ 21 ]  . 
il successo della procedura dipende , comunque , anche dal tipo e dal comportamento del tumore primitivo ; nei pazienti con metastasi ossee da tumore renale o tiroideo , ci sono buone probabilit che il trattamento attraverso locclusione vascolare selettiva sia portato a termine con successo , a differenza dei pazienti con metastasi da tumore polmonare [ 7 ]  . 
 i nostri dati evidenziano , comunque , che questo effetto palliativo risulta essere solo transitorio ; i sintomi si sono infatti ripresentati a distanza di circa 8 mesi ( range da 1 a 12 mesi )  . 
pu essere oggetto di discussione se la presenza di addizionali terapie possa aver contribuito al successo dellembolizzazione , o se il successo dellembolizzazione combinato ad altri trattamenti possa dipen804 radiol med ( 2011 ) 116 : 793808 cation in 65 patients . 
however , it can be questioned whether the presence of additional therapies may have contributed to the success of embolisation or even that the success of embolisation combined with other therapies may be entirely attributed to these additional therapies . 
as we did not perform the additional treatments in a randomised fashion , we cannot conclude whether they were associated with improved or longer - lasting clinical and imaging results . 
nevertheless , all embolisations were technically successful and were accompanied by an acute improvement in symptoms that cannot be explained by the additional therapies . given the high vascularity and morbidity associated with surgical resection and / or radiation therapy , embolisation has been reported to be a useful primary treatment modality for sacral giant - cell tumours . 
in our series of patients with giant - cell tumour of the sacrum , overall survival to local recurrence was 90% at 60 and 120 months , and local recurrence rate was 10% . 
embolisation has been used for aneurysmal bone cysts in combination with surgery to reduce operative blood loss [ 2830 ] or as primary treatment of sites with dif cult surgical access and in patients at high risk of extensive intraoperative bleeding [ 3139 ]  . 
fourteen patients with recurrent and / or persistent lesions underwent repeat embolisation . long - term series reporting on embolisation for primary treatment of vertebral haemangiomas are lacking [ 4042 ]  . 
poich tali trattamenti aggiuntivi non sono stati studiati in maniera randomizzata , non possiamo concludere se questi possano essere correlati ad una risposta clinica e imaging migliore o pi duratura . 
tuttavia , tutte le embolizzazioni sono state correttamente eseguite dal punto di vista tecnico e si sono associate ad un rapido miglioramento della sintomatologia , che non pu essere spiegato dalle terapie aggiuntive . nei tumori sacrali a cellule giganti , a causa dellalta vascolarizzazione e della morbilit correlata alla resezione chirurgica e / o alla radioterapia , la letteratura riporta che lembolizzazione pu essere unutile opzione terapeutica primaria ; la sopravvivenza no alla recidiva locale a seguito di resezione , terapia radiante ed embolizzazione stata riportata essere tra il 57% e l80% [ 8 , 2326 ]  . 
nei nostri pazienti con tumori sacrali a cellule giganti , la sopravvivenza no alla recidiva locale stata globalmente del 90% a 60 e 120 mesi , ed il tasso di recidive locali stato del 10% . 
nella nostra pratica clinica , il fenolo e lazoto liquido sono impiegati come emostatici nel corso di resezione chirurgica per ridurre il sanguinamento e migliorare il curretage , tranne che nei casi in cui si identi ca una vicinanza con la radice del nervo sacrale [ 27 ]  . 
lembolizzazione stata anche impiegata per il trattamento di cisti ossee aneurismatiche , sia in combinazione con la chirurgia per ridurre le perdita ematiche intra - operatorie [ 2830 ] , che come trattamento primario in siti a dif coltoso accesso chirurgico ed in pazienti ad alto rischio di massivo sanguinamento in corso di intervento [ 3139 ]  . 
quattordici pazienti hanno sviluppato delle recidive e / o evidenziato la persistenza di malattia e sono stati sottoposti a ripetizione del trattamento . mancano invece studi a lungo termine sullapplicazione dellembolizzazione come trattamento primario per gli emangiomi vertebrali [ 4042 ]  . 
in other malignant tumour histologies , embolisation was performed effectively without complications . vascular mapping and the haemodynamic status of the tumour , as well as the tumours anatomical region , must be determined using selective angiography before embolisation . 
arteries feeding the tumour must be catheterised superselectively using microcatheters , and the procedure must be undertaken with the most suitable embolising agent to protect the haemodynamics of normal bone tissues as much as possible . 
when the procedure is followed by surgery , it is recommended that the surgery be performed within 3 days to avoid revascularisation [ 7 , 19 ]  . currently available embolic agents include gelfoam , polyvinyl alcohol ( pva ) particles , liquid ( absolute alcohol ) , coils , tissue adhesives , ethanol , micro brillar collagen and autologous blood clot [ 19 ]  . 
in addition , the use of coils in managing hypervascular bone tumours has been reported to be ineffective because the rich vascularisation of these lesions can open collateral channels within hours [ 17 , 21 , 22 ]  . 
moreover , if particles are used , their size has to be adjusted to the diameter of potential collateral vessels and shunts because these entities are often present in hypervascular malignant bone tumours [ 5 , 10 , 21 , 22 ]  . 
liquid embolic agents offer the advantages of low viscosity for easy injection through small catheters or il mappaggio vascolare e lo stato emodinamico , cos come lanatomia regionale , devono essere determinati attraverso unangiogra a selettiva , prima di procedere allembolizzazione . 
in particolare , devono essere valutati accuratamente le arterie tributarie , le collaterali , i rapporti del tumore con gli adiacenti distretti vascolari e la possibile presenza di stole artero - venose intratumorali . 
 le arterie tributarie della lesione devono essere cateterizzate in maniera superselettiva utilizzando microcateteri e la procedura deve essere eseguita con il pi idoneo agente embolizzante , allo scopo di proteggere il pi possibile lemodinamica del normale tessuto osseo . 
langiogra a post - embolizzazione in ne necessaria per valutare leffetto della procedura : se il vaso tributario occluso , pu essere comunque osservato un vaso adiacente e questo deve essere embolizzato [ 7 , 14 ]  . 
 gli agenti embolici attualmente disponibili includono gelfoam , particelle di alcool polivinile ( pva ) , microsfere , liquidi ( alcool puro e etanolo ) , spirali , adesivi tissutali e micro brille di collagene [ 19 ]  . 
le spirali sono ideali per determinare locclusione di vasi singoli e di grosso calibro ; nel management dei tumori ossei ipervascolarizzati sono inef caci poich determinano una occlusione solo prossimale favorendo le formazione di circoli collaterali nel giro di ore [ 17 , 21 , 22 ]  . 
 tuttavia , la somministrazione di particelle non precisa e il trasporto attraverso i piccoli microcateteri o attraverso distretti vascolari tortuosi pu risultare dif coltoso [ 11 , 14 , 49 , 50 ]  . 
in ne , se si utilizzano le particelle , le loro dimensioni devono essere corrette in base al diametro dei potenziali shunt e vasi collaterali , spesso presenti nei tumori ossei maligni ipervascolari [ 5 , 10 , 21 , 22 ]  . 
gli agenti embolici liquidi offrono invece il vantaggio di una bassa viscosit e quindi di una agevole somministrazione attraverso cateteri piccoli o attraverso cateteri con molte angolazioni per la tortuosit del distretto vascolare . 
lnbca , o colla liquida , 806 radiol med ( 2011 ) 116 : 793808 catheters with many bends through tortuous blood vessels . nbca , or liquid glue , is a liquid embolic agent with distinct advantages as an embolic material . 
 although nbca can pass through bent catheters , thus navigating tortuous blood vessels , it does not permeate all the way to the capillary level , and therefore does not cause tissue death . 
in our practice and in this study , nbca was the preferred embolic agent because we consider it to be the most for controlled embolisation of the pathological tumour vasculature , for permanent occlusion of the target vessels and for complete lesion devascularisation . when embolisation is planned carefully and the vessel is occluded selectively via a securely positioned catheter , complications rarely occur . 
bolus administration of small doses ( 0.10.2 ml ) of sandwiched nbca under uoroscopic control , followed by arteriography , provides for the ef cacy and safety of the procedure . 
postembolisation syndrome , with symptoms such as fever , pain due to ischaemic necrosis of the tumour and malaise , has been reported in 1886% of cases [ 7 , 21 ]  . 
embolisation of adjacent or distant nontargeted vessels can result in a large zone of tissue loss and may be associated with risk of nerve palsy , skin breakdown and subcutaneous or muscle necrosis ; tissue ischaemia may lead to infection . 
care should be taken to the location and vascular supply of at - risk vital structures , such as the femoral region , to avoid embolising supply to the sciatic nerve , the humeral area to avoid circum ex femoral nerve and the lateral cutaneous nerve of the thigh [ 5 , 10 , 16 , 21 , 51 ]  . the occurrence of the adamkiewicz artery that originates between the t5 and l2 vertebra in the embolisation eld should be recognised on the pre - embolisation angiogra in our practice , we encountered the adamkiewicz artery in the embolisation eld during four embolisations for bone metastases and one for aneurysmal bone cyst of the spine . 
in line with the literature , life expectancy is not in uenced by embolisation therapy , and embolisation therapy does not appear to improve the survival of patients with malignant bone tumours [ 3 , 5255 ]  . 
inoltre , sebbene lnbca sia in grado di passare attraverso cateteri curvi utilizzati in distretti vascolari tortuosi , non va incontro a diffusione no al distretto capillare e pertanto non causa morte tissutale . 
 nella nostra pratica clinica e nel presente studio , riteniamo che lnbca sia il miglior agente embolico per il controllo dellembolizzazione nel distretto tumorale , per unocclusione permanente a livello del bersaglio vascolare e per una completa devascolarizzazione della lesione . quando lembolizzazione piani cata con accuratezza e locclusione vascolare selettiva , mediante un sicuro posizionamento del catetere , le complicanze sono rare . 
 la somministrazione di un piccolo bolo di nbca ( 0 , 10 , 2 ml ) sotto controllo uoroscopico , seguito da arteriogra a , effettuato per garantire ef cacia e sicurezza alla procedura . 
lembolizzazione di un distretto vascolare non bersaglio , adiacente o distante , pu determinare una grossa perdita tissutale e pu associarsi al rischio di paralisi , necrosi cutanea , sottocutanea e muscolare ; lischemia tissutale pu inoltre facilitare le infezioni . 
lattenzione dovrebbe quindi essere rivolta alla localizzazione dellapporto vascolare delle strutture vitali a rischio , quali la regione femorale per evitare di embolizzare lapporto ematico al nervo sciatico , al nervo femorale circon esso e al nervo laterale cutaneo della coscia [ 5 , 10 , 16 , 21 , 51 ]  . 
 nella nostra esperienza , abbiamo identi cato tale arteria durante quattro procedure eseguite per metastasi ossee e una procedura eseguita per cisti ossea aneurismatica del rachide ; in questi casi si optato per una sospensione della procedura stessa per evitare il rischio di paraplegia derivante dallocclusione dellarteria di adamkiewicz . in questo studio , a causa delleterogeneit del gruppo dei pazienti e dellistologia tumorale , non abbiamo fatto unanalisi sulla sopravvivenza . 
in linea con la letteratura , laspettativa di vita non in uenzata dallembolizzazione e , daltra parte , lembolizzazione stessa non sembra migliorare la sopravvivenza dei pazienti con tumori ossei maligni [ 3 , 5255 ]  . 
questo dato non deve sorprendere perch , nella maggior parte dei casi , lembolizzazione ha come bersaglio solo una parte della problematica tumoradiol med ( 2011 ) 116 : 793808 most part , embolisation only targets a portion of the tumour burden . 
le terapie locali per il controllo tumorale , quali lembolizzazione , rimangono comunque risorse preziose per preservare la qualit di vita . conclusions conclusioni we recommend embolisation for highly vascular tumours of variable histology as primary or palliative treatment or as an adjunct to surgery to reduce intraoperative blood loss and facilitate resection . 
the low number of complications in this study and the fact that they were minor suggest that strict adherence to the principles of transcatheter embolisation and mini - bolus administration of nbca is important . 
nbca is considered the most appropriate embolic agent for small - vessel occlusion without major causing complications . raccomandiamo lembolizzazione per tumori ipervascolarizzati , di diverso istotipo , come trattamento primario , palliativo o adiuvante la chirurgia , per ridurre il sanguinamento intra - operatorio e facilitare la resezione . 
il basso numero di complicanze , ed il fatto che esse fossero prevalentemente minori , suggerisce che fondamentale una rigorosa adesione ai principi delle tecniche di embolizzazione e della somministrazione del millibolo di nbca . 
bellomi1 , 3 1department of radiology , european institute of oncology , via ripamonti 435 , 20141 milan , italy 2division of epidemiology and biostatistics , european institute of oncology , via ripamonti 435 , 20141 milan , italy 3school of medicine , university of milan , via a . 
 there was no signi cant difference in adc values between responders and nonresponders at the staging mr examination ( p = 0.09 ) and no signi cant correlation between pretreatment adc values and percentage of tumour reduction . 
diciassette pazienti consecutive sono state prospetticamente sottoposte ad esami di risonanza magnetica ( rm ) della pelvi , comprendenti sequenze pesate in diffusione ( dwi ) , prima e dopo terapie non chirurgiche per tumore della cervice uterina . 
le differenze dei valori delle mappe adc tra i due gruppi alla baseline , e nel gruppo di studio prima e dopo terapia sono state valutate con test non parametrici . 
diametro e volume dei tumori cervicali si sono ridotti dopo le terapie in 14 / 17 lesioni ( rispondenti ) , sono invece aumentati in 3 / 17 lesioni ( non rispondenti )  . 
non si sono riscontrate differenze signi cative nei valori di adc alla rm basale tra rispondenti e non rispondenti ( p = 0 , 09 ) , n tra valori adc basali e percentuale di riduzione del tumore . 
i tumori con pi alto incremento percentuale di valori di adc hanno mostrato pi elevata riduzione del volume tumorale , ma questo non risultato signi cativo ( p = 0 , 12 )  . 
i valori di adc del tumore della cervice uterina hanno mostrato signi cativo incremento dopo terapia rispetto ai valori pre - terapia , particolarmente per i tumori rispondenti . parole chiave rm diffusion - weighted imaging cervice tumore risposta introduction introduzione when the radiologist faces uterine cervical cancer treatment options , important but dif cult issues that must be dealt with are evaluation of tumour response to therapy and discrimination between postradiation changes and viable tumour . 
 tumour response to therapy is conventionally addressed by changes in tumour size according to the response evaluation criteria in solid tumours ( recist ) , but changes in size are not always adequate to determine whether or not the tumour is responding to the therapies , and alternative imaging modalities to substitute for the anatomical assessment described in recist are currently under evaluation [ 1 ]  . 
on the other hand , differentiation between residual tumour and radiation changes cannot be made with conventional magnetic resonance ( mr ) images , especially in the rst 3 months after therapy [ 2 , 3 ]  . 
given these dif culties , an early and reliable response marker would have great clinical value for cervical cancer patients to cease ineffective treatment , frequently associated with increased toxicity and development of drug resistance , and to avoid delays in starting alternative , potentially more effective , treatments [ 4 ]  . as changes in tumour size occur as a consequence of biological and molecular changes , functional imaging techniques are under consideration as an adjunctive tool in evaluating many tumour features , including tumour response . 
in oncological imaging , dwi has been linked to lesion aggressiveness and tumour response , with apparent diffusion coefcient ( adc ) measurements being inversely correlated to cellularity and cell membrane integrity [ 7 ]  . 
previous studies evaluated the potential role of dwi in monitoring tumour response to therapy [ 4 , 710 ] and in differentiating normal from cancerous tissue in the uterine cervix [ 11 ]  . 
adc maps usually re ect the complexity of a tumour mass , and during follow - up to therapies , a decreased adc value caused by a decrease of perfusion may be followed by increased adc values caused by necrosis [ 12 ]  . 
therefore , the diffusion characteristics of a tumour would be better evaluated by assessing the adc values of the entire tumour volume rather than on a single most representative slice . quando il radiologo valuta il tumore della cervice uterina dopo terapia , un aspetto importante ma dif cile da valutare rappresentato dalla valutazione della risposta tumorale alla terapia e la distinzione tra le modi cazioni dovute alla radioterapia e il tumore residuo . 
la risposta del tumore alla terapia convenzionalmente indicata da modi cazioni delle dimensioni , in accordo con i criteri response evaluation criteria in solid tumors ( recist ) , ma i cambiamenti dimensionali non sono sempre indicativi di risposta alle terapie , per cui sono in fase di studio modalit alternative di imaging per sostituire i criteri anatomici recist [ 1 ]  . 
 inoltre , la distinzione tra modi cazioni post - radioterapia e tumore residuo , in particolare nei primi 3 mesi dopo terapia , non sempre possibile utilizzando le immagini di risonanza magnetica convenzionale [ 2 , 3 ]  . in considerazione di tali dif colt , individuare un marker di risposta , precoce ed af dabile , avrebbe un enorme valore clinico per le pazienti affette da tumore della cervice , sia per sospendere i trattamenti inef caci , frequentemente associati con un incremento della tossicit e con lo sviluppo di farmaco - resistenza , sia per evitare ritardi nellinizio di trattamenti alternativi e potenzialmente pi ef caci [ 4 ]  . poich i cambiamenti nelle dimensioni del tumore avvengono come conseguenza di modi cazioni biologiche e molecolari , tecniche di imaging funzionale vengono attualmente prese in considerazione come uno strumento aggiuntivo nella valutazione di molti aspetti del tumore , inclusa la risposta tumorale alla terapia . 
nella diagnostica per immagini oncologica , limaging pesato in diffusione , connesso con laggressivit e la risposta del tumore , essendo il valore della mappa del coef ciente di diffusione apparente ( adc ) inversamente proporzionale alla cellularit e allintegrit delle membrane cellulari [ 7 ]  . 
studi precedenti hanno valutato il ruolo potenziale della dwi nel monitoraggio della risposta alle terapie [ 4 , 710 ] , e nella distinzione tra tessuto normale e tumorale nella cervice uterina [ 11 ]  . 
la mappa adc di solito ri ette la complessit della massa tumorale , anche negli esami di follow - up effettuati durante la terapia , nei quali una diminuzione dei valori della mappa adc , causata da una riduzione della perfusione , possono essere seguiti da un aumento nel valore adc dovuto alla 768 radiol med ( 2011 ) 116 : 766780 the purpose of this study was to prospectively assess whether differences of adc values within the entire cervical cancer tumour volume before and after therapy may be considered as a quantitative method to monitor treatment response after nonsurgical therapy . patients and methods patients patients undergoing a rst mr examination for cervical cancer staging and a second examination for end of therapy followup during a 12 - month period between may 2008 and april 2009 were prospectively enrolled . 
written informed consent to undergo the procedure and to the use of anonymised clinical and imaging data for scienti c and / or educational purposes was obtained from all patients . 
for each patient , the following data were recorded on an excel spreadsheet le ( microsoft of ce excel 2003 , richmond , va , usa ) to be further evaluated : mr examination date , age , histological diagnosis obtained from pretherapy biopsy , therapy intent ( neoadjuvant or curative ) and type ( chemotherapy and / or radiotherapy ) , beginning and end therapy dates . 
for the control group , age , adc mean values standard deviations ( sd ) were recorded . mr technique all mr examinations were performed on a 1.5 - t scanner ( avanto , siemens , erlangen , germany )  . 
perci , le caratteristiche di diffusione di un tumore potrebbero essere meglio valutate stimando i valori adc dellintero volume tumorale , piuttosto che unicamente nellimmagine pi rappresentativa del tumore stesso . lo scopo di questo studio stato quello di valutare prospetticamente se le differenze nei valori di adc , considerando lintero volume tumorale della cervice uterina , prima e dopo terapia , possono essere considerate come un metodo quantitativo ef cace nel monitoraggio della risposta alle terapie non chirurgiche . pazienti e metodi pazienti in un periodo di 12 mesi , tra maggio 2008 e aprile 2009 , sono state arruolate pazienti che si sono sottoposte ad una risonanza magnetica basale per la stadiazione del tumore della cervice e ad una seconda valutazione alla ne della terapia . 
tutte le pazienti hanno rmato un consenso informato con il quale acconsentivano alla procedura e allutilizzo dei dati clinici e di imaging , resi anonimi , per scopi scienti ci e / o formativi . 
per ciascuna paziente , i seguenti dati sono stati raccolti in un foglio elettronico in formato excel ( microsoft of ce excel 2003 , richmond , usa ) , per essere successivamente esaminati : data dellesame di risonanza magnetica , et della paziente , diagnosi istologica ottenuta dalla biopsia eseguita prima di iniziare la terapia , data di inizio e ne del trattamento . 
stato riportato lo stadio clinico secondo linternational federation of gynecology and obstetrics ( figo ) alla diagnosi , successivamente modi cato retrospettivamente in accordo alla stadiazione figo modi cata [ 13 ]  . 
in base allo stadio figo le pazienti sono state in seguito anche divise in due sottogruppi comprendenti : tumori in stadio precoce ( tutti gli stadi compresi tra ib1 e iia2 ) , e tumori in stadio avanzato ( tra iib - ivb )  . stato anche memorizzato il diametro assiale massimo ed il volume della lesione cervicale prima e dopo la terapia insieme con il valore medio di adc ( come descritto nel paragrafo sulla misurazione dei valori adc ) ; per il gruppo di controllo sono stati memorizzati et delle pazienti e deviazione standard del valore medio di adc della cervice . tecnica rm tutti gli esami di risonanza magnetica sono stati eseguiti utilizzando una macchina da 1 , 5 t ( avanto , siemens , erlangen , germania )  . 
lesame standard della pelvi ha incluso : sequenze assiali t2 - pesate a respiro libero ( tempo radiol med ( 2011 ) 116 : 766780 mm ; ipat factor = 2 ) ; coronal t2 - weighted sequences ( tr / te 5 , 120 / 174 ms ; 260 - mm fov ; slice thickness 5 mm ; voxel size 1.00.75.0 mm ; ipat factor = 2 ) and para - axial t2 - weighted images , perpendicular to the longitudinal axis of the endocervical canal ( tr / te 5 , 060 / 174 ms ; 220 - mm fov ; slice thickness 3 mm ; voxel size 1.00.93.0 mm ; ipat factor = 2 )  . 
axial dwi sequences were acquired with the following imaging parameters : 40 slices ; voxel 3.02.75.0 mm ; transversal orientation ; fov read 340 mm ; slice thickness 5 mm ; tr / te 6 , 000 / 68 ms ; averages 4 ; ipat factor = 2 ; bandwidth 1 , 446 hz / px ; epi factor 69 ; 5 diffusion weightings ( b - values 0 ; 50 ; 250 ; 500 ; 900 s / mm2 )  . 
the adc value was measured over multiple slices on a four - view screen by uploading the t2 images as morphological images , the b = 0 dwi that most closely re ect the t2 images , thus allowing evaluation of the entire tumour diffusion , and the adc maps . 
following completion of therapy , if there was no residual tumour , the roi was traced on what was considered to be the normal residual cervix , as much as possible in the same area used in the pretherapy mr examination . 
 if there was a remnant of the tumour ( partial response ) or larger tumour ( no response ) , the rois were again traced on each slice to cover the entire tumour volume . 
le sequenze assiali dwi sono state acquisite con i seguenti parametri : 40 immagini ; voxel 3 , 02 , 75 , 0 mm ; orientamento trasversale ; campo di vista ( fov ) 340 mm ; spessore dello strato 5 mm ; tr 6000 ms ; te 68 ms ; averages 4 ; fattore ipat = 2 ; larghezza di banda 1446 hz / px ; fattore echo planar imaging ( epi ) 69 ; 5 pesature in diffusione ( valore - b 0 ; 50 ; 250 ; 500 ; 900 s / mm2 )  . 
le mappe adc sono state automaticamente generate dal software usando tutti i valori di b . se non controindicato , stato somministrato un mezzo di contrasto paramagnetico ( gadobenato dimeglumina [ gd - dtpa ] , 0 , 2 mmol / kg ; velocit di usso = 2 ml / s ) per via endovenosa ed stata acquisita una sequenza gradientecho 3d isotropica , assiale t1 fat - sat post - contrasto ( tr / te 7 , 0 / 2 , 1 ms ; ip angle 12 ; campo di vista di 340 mm ; spessore / intervallo5 / 1 ; dimensioni del voxel 0 , 90 , 90 , 9 mm ; nessun ipat )  . misurazione dei valori adc le caratteristiche di diffusione dellintero volume tumorale cervicale prima e dopo terapia , sono state valutate da un radiologo esperto ( 5 anni di esperienza in radiologia ginecologica ) due volte , per valutare la variabilit intra - osservatore , e da un radiologo non esperto , per valutare la variabilit inter - osservatore . 
il valore adc stato misurato su pi immagini su uno schermo con quattro riquadri , in cui venivano caricate le immagini t2 da usare come immagini morfologiche , le immagini pesate in diffusione con b = 0 , che meglio corrispondono alle immagini t2 e permettono una valutazione della diffusione dellintero volume tumorale e , in ne la mappa adc . 
finally , wilcoxons two - sample test was also performed to evaluate differences in adc values at the staging mr examination between cases and controls , responder and nonresponder tumours and tumours divided according to low and high stage . 
 results patients among 63 women who underwent a pelvic mr imaging for cervical cancer staging during the 1 - year period considered , 15 were excluded because they did not undergo the posttherapy mr examination at our institution , and 31 were excluded because they underwent surgical radical hysterectomy as rst - line therapy . 
therefore , our study cohort comprised 17 women ( median age 50 years ; range 3575 ) with cervical cancer who underwent two routine pelvic mr examinations at our institution , one for staging and one for evaluation after nonsurgical therapy ( chemotherapy and / or radiotherapy )  . 
 the control group comprised 17 women ( median age 46 years ; range 2373 ) who underwent mr imaging for the following indications : intestinal cancer ( n = 10 ) , uterine bromas ( n = 3 ) , retroperitoneal sarcoma ( n = 1 ) , retroperitoneal bromatosis ( n = 1 ) , vaginal cancer ( n = 1 ) , abdominal pain ( n = 1 )  . median interval between beginning of therapy and posttherapy mr evaluation of the study group was 80 ( range 61174 ) days ; median interval between the beginning of therapy and posttherapy mr evaluation was 32 ( range 1684 ) days . 
gadolinium - based contrast medium was administered to all cervical cancer patients : postgadolinium images did not in uence lesion conspicuity , but they helped attain a more precise evaluation of anatomical details in radiol med ( 2011 ) 116 : 766780 termine della terapia , non cera pi tumore residuo , la roi veniva tracciata su ci che era considerato residuo normale della cervice uterina , il pi possibile nella stessa area valutata nella risonanza magnetica pre - terapia . 
se , al termine della terapia , cera tumore residuo ( risposta parziale ) o una lesione tumorale di maggiori dimensioni ( nessuna risposta ) le roi venivano nuovamente tracciate su ciascuna immagine per coprire lintero volume tumorale . 
nel gruppo di controllo , la roi stata tracciata in unarea comprendente tessuto cervicale normale , escludendo lo stroma che normalmente ha un basso segnale sulle immagini t2 - pesate . analisi statistica i valori appaiati di adc prima e dopo terapia sono stati confrontati utilizzando il test non parametrico di wilcoxon . 
in ne , il test di wilcoxon stato anche utilizzato per valutare le differenze nei valori di adc tra i casi e i controlli , tra i tumori rispondenti e non rispondenti alle terapie , e tra i tumori divisi in stadio precoce ed avanzato ; tali valutazioni sono state effettuate solo alla risonanza magnetica di stadiazione . 
le analisi sono state effettuate usando un software standard ( sas versione 8.2 , sas insitute inc . , cary , usa )  . risultati pazienti delle 63 pazienti che sono state sottoposte ad una risonanza magnetica per stadiazione di un tumore della cervice durante il periodo in esame , 15 sono state escluse perch non hanno effettuato la risonanza magnetica post - terapia presso il nostro istituto , e 31 sono state escluse perch sono state sottoposte ad intervento chirurgico di isterectomia radicale come prima linea di trattamento . 
pertanto il nostro studio comprende una popolazione di 17 pazienti ( et media = 50 anni , range 3575 ) con tumore della cervice che hanno effettuato due risonanze magnetiche della pelvi di routine presso il nostro istituto , una per la stadiazione e una per la valutazione della risposta dopo terapie non chirurgiche ( chemioterapia e / o radioterapia )  . 
within the control group , gadolinium was not administered to one patient because of personal history of allergy to gadoliniu median time between the two mr examinations of the study group was 123 ( range 32185 ) days . 
among responders , 9 / 14 had no residual tumour after completion of therapies ( complete response ) and 5 / 14 still had residual tumour ( partial response )  . 
il mezzo di contrasto a base di gadolinio stato somministrato a tutte le pazienti : le immagini post - contrasto non hanno in uenzato la valutazione della lesione , ma hanno permesso una valutazione pi precisa dei dettagli anatomici nelle pazienti candidate ad intervento chirurgico . 
in all patients , the presence or absence of response to therapy was con rmed by clinical examination by the gynaecologist within 1 week after mr examination . interand intraobserver agreement interobserver agreement was assessed by comparing the two sets of measurements made by readers a and b using the bland and altman plot . 
intraobserver agreement was evaluated from the le due indagini di risonanza stato in media di 123 giorni ( range 32185 giorni )  . il diametro assiale massimo delle lesioni tumorali della cervice si ridotto in 14 / 17 lesioni ( rispondenti ) , con un diametro medio prima e dopo la terapia di 43 , 7 mm e 12 , 7 mm , rispettivamente . 
tra le pazienti che hanno mostrato una risposta al trattamento , 9 / 14 non avevano tumore residuo al termine della terapia ( risposta completa ) e 5 / 14 avevano un residuo di malattia ( risposta parziale )  . 
in tutte le pazienti la presenza o assenza di risposta alla terapia stata confermata dallesame clinico ginecologico eseguito entro una settimana dallindagine rm . variabilit intere intra - osservatore adc value measurements the mean number of pixels within the rois , used to average the single adc values over the number of slices , was 2 , 802.8 ( range 6711 , 015 ) for pretherapy mr examinations and 771.4 ( range 874 , 206 ) for posttherapy mr evaluation . 
among responder patients , mean adc values increased on average by 39.4% and tumour la variabilit inter - osservatore stata valutata confrontando i due set di valori ottenuti dai due radiologi a ( esperto ) e b ( non esperto ) usando il metodo di bland e altman . 
il valore medio di adc della cervice uterina nel gruppo di controllo era 1 , 4510 - 3 mm2 / s ( 1 , 241 , 70 ) , valore signi cativamente diverso da quello ottenuto nella popolazione in studio prima della terapia ( p < 0 , 0001 )  . 
4a - c a 58 - year - old woman with international federation of gynecology and obstetrics stage i b2 squamous - cell carcinoma who underwent neoadjuvant chemotherapy : axial t2 - weighted image of the cervix at staging magnetic resonance ( mr ) imaging shows the pretherapy cervical cancer ( a )  . 
diffusion - weighted imaging at b = 0 matched with the t2 - weighted image was used to trace the region if interest ( b ) , which was then applied to the corresponding image of the apparent diffusion coef cient map ( c )  . 
limmagine pesata in dwi a b = 0 , accoppiata allimmagine t2 stata usata per tracciare la roi ( b ) , che stata poi copiata sulla immagine corrispondente nella mappa adc ( c )  . 
 non si evidenziata una differenza statisticamente signicativa dividendo la popolazione in due ulteriori sottogruppi in base alla risposta completa o parziale , con una media dei valori di 1 , 0210 - 3 mm2 / s ( range 0 , 741 , 145 ) e 1 , 0210 - 3 mm2 / s ( range 0 , 971 , 3 ) rispettivamente per le pazienti con risposta completa e parziale ( p = 0 , 29 )  . 
tra le pazienti che hanno mostrato una risposta alla terapia , il valore medio di adc ha mostrato un incremento del 39 , 4% mentre il volume tumorale diminuito dell85 , 8% . 
non c una correlazione statisticamente signi cativa tra i valori di adc pre - terapia e la risposta tumorale post - terapia espressa come percentuale di riduzione del volume tumorale ( coef ciente di spearman 0 , 30 ; p = 0 , 23 )  . 
tumori con un maggiore incremento percentuale dei valori adc hanno mostrato una riduzione percentuale del volume tumorale maggiore , ma questo non stato statisticamente signi caradiol med ( 2011 ) 116 : 766780 fig . 
region of interest traced on the diffusion - weighted image at b = 0 ( b ) was applied to the corresponding apparent diffusion coef cient ( adc ) image ( c )  . 
our data show a signi cant difference in adc values both between normal and cancerous tissue in the cervix and between cervical tumours before and after nonsurgical therapy for all patients ( p = 0.003 ) , with a signi cant increase of adc values for responder tumours ( p = 0.0009 ) and a relative decrease of adc values for nonresponder tumours . 
 [ 11 ] , who demonstrated a signi cant difference in adc values between healthy and cancerous cervix , as well as an increase of adc values in response to therapy in a subgroup of nine cancer patients followed up after therapy , even though they evaluated the adc values only on a single slice rather than on the entire tumour volume . 
in addition to naganawa et al . , other studies showed a signi cant difference in adc values of normal cervix compared with cervical carcinoma [ 16 , 17 ] , but evaluation of the diffusion characteristics of normal and cancerous tissue was made using relatively low ( 600 s / mm2 ) maximum b - values [ 4 , 16 , 17 ] or only one high b - value ( 1 , 000 s / mm2 ) [ 18 , 19 ]  . 
 the information provided by dwi and adc values has been related to cellular density , diffusion of protons within a tissue , tumour structure and intralesional components [ 79 , 20 ]  . 
an inverse correlation between cellular density and adc values has been well described for brain [ 21 ] , prostate [ 22 ] , renal [ 23 ] , breast [ 24 ] and gynaecological [ 16 , 25 ] tumours . 
the choice of b - values in uences the adc measurements , as the adc value is in uenced at low b - values by perfusion from local vasculature [ 7 , 26 , 27 ] and at moderate to high b - values by compartmentalisation in intraand extracellular spaces [ 28 ]  . 
 [ 7 ] , dwi acquisition should be performed with a high maximum b - value ( such as 900 s / mm2 ) and several lower b - values ( 0 ; 50 ; 250 ; 500 s / mm2 ) and , given the heterogeneous cellularity within a tumour , a composite of rois over multiple slices should be delineated if a tumour is being followed over time to better re ect the heterogeneity of the entire + 41 , 8% e - 2 , 5% ( p = 0 , 04 )  . 
non ci sono state differenze statisticamente signi cative nei valori di adc alla rm basale tra i tumori rispondenti e quelli non - rispondenti ( p = 0 , 09 )  . 
non ci sono state differenze statisticamente signi cative nei valori di adc tra i tumori suddivisi in stadio precoce ( n = 9 ) e avanzato ( n = 8 ) ( p = 0 , 96 ) , anche dopo aver escluso i tre tumori non - rispondenti ( p = 0 , 56 )  . discussione le immagini pesate in diffusione sono attualmente utilizzate nella pratica dellimaging oncologico come un possibile strumento per la caratterizzazione tissutale , monitoraggio della risposta alle terapie ed identi cazione di recidive tumorali , senza il bisogno di mezzo di contrasto [ 7 ]  . 
i nostri dati dimostrano una differenza signi cativa dei valori di adc sia tra tessuto normale e tessuto neoplastico nella cervice , sia nel tessuto tumorale prima e dopo terapia non chirurgica per tutte le pazienti ( p = 0 , 003 ) , con un pi signi cativo incremento dei valori di adc per i tumori rispondenti ( p = 0 , 0009 ) , e un relativo decremento dei valori di adc per i tumori non - rispondenti . 
inoltre , i cambiamenti percentuali dei valori di adc prima e dopo terapia erano pi alti nei rispondenti rispetto ai non rispondenti ( p = 0 , 04 )  . 
 [ 11 ] , che hanno dimostrato una differenza signi cativa nei valori di adc tra la cervice sana e quella neoplastica con un incremento dei valori di adc in risposta alla terapia in un sottogruppo di 9 pazienti neoplastiche seguite dopo terapia , sebbene loro avessero valutato il valore di adc solo su una singola immagine invece che sullintero volume tumorale . 
 anche altri studi hanno dimostrato una signi cativa differenza dei valori di adc della cervice normale rispetto alla cervice neoplastica [ 16 , 17 ] , ma la valutazione delle caratteristiche di diffusione del tessuto normale e neoplastico stata fatta utilizzando un valore di b massimo relativamente basso ( 600 s / mm2 ) [ 4 , 16 , 17 ] , oppure utilizzando solo un alto valore di b ( 1000 s / mm2 ) [ 18 , 19 ]  . le informazioni ottenute dalla immagini pesate in diffusione e dai valori della mappa adc sono correlate alla densit cellulare , alla diffusione dei protoni presenti nel tessuto , alla struttura tumorale , ed a componenti intralesionali [ 79 , 20 ]  . 
una correlazione inversa tra la densit cellulare e i valori di adc stata ben descritta per i tumori dellencefalo [ 21 ] , della prostata [ 22 ] , del rene [ 23 ] , della mammella [ 24 ] e per quelli ginecologici [ 16 , 25 ]  . 
la scelta del valore di b in uenza le misurazioni di adc , poich il valore di adc in uenzato : a valori di b bassi dalla perfusione delle strutture vascolari locali [ 7 , 26 , 27 ] , e a valori di radiol med ( 2011 ) 116 : 766780 tumour volume over time [ 7 ]  . 
 [ 4 ] have shown that cellular death and vascular changes caused by therapy could be detected by dwi before a reduction of lesion diameter becomes apparent at morphological imaging . 
according to our results , dwi based on many b - values and adc measurements applied to the entire tumour volume may be used as an adjunctive , but not substitutive , tool to the standard mr imaging examination to assess the response of cervical cancer to nonsurgical therapies . 
nevertheless , the usual morphological images ( t2and t1 - weighted images , with or without administration of contrast medium ) were still necessary to address speci c questions , such as involvement of parametria , endocervix , vaginal fornices and lymph nodes [ 29 ] , as well as to give information about tumour volume and anatomical details of in ltration of adjacent structures in patients for whom surgery or radiotherapy is planned [ 30 ]  . 
thus , the usual management of cervical cancer patients undergoing nonsurgical therapy , based on one mr examination before therapies and one after the end of therapies , was not altered so as not to give additional discomfort to the patients . 
based on our results , further studies , including an earlier mr evaluation , should be performed to assess whether changes in dwi occur earlier than changes in size and , as a consequence , whether the use of dwi may be considered as an early biomarker of tumour response to therapies . 
the possibility of predicting responder from nonresponder uterine cervical tumours , as well as early identi cation of nonresponder tumours , would help in addressing cervical cancer patients to stop ineffective treatments and to avoid delays in starting alternative , potentially more effective , treatments . 
 [ 7 ] le acquisizioni dwi dovrebbero essere eseguite con un elevato valore massimo di b ( come 900 s / mm2 ) e pi valori bassi ( 0 , 50 , 250 , 500 s / mm2 ) e , in considerazione della eterogeneit cellulare presente nel tumore , dovrebbe essere tracciata una serie di roi su multiple sezioni se il tumore viene seguito nel tempo , per meglio ri etterne leterogeneit dellintero volume [ 7 ]  . le sequenze dwi utilizzate in questo studio , come la valutazione delle mappe adc , sono state eseguite in accordo a queste raccomandazioni . 
 [ 4 ] hanno dimostrato che la morte cellulare ed i cambiamenti vascolari indotti dalla terapia possono essere identi cati dalla dwi prima che diventi evidente una riduzione del diametro della lesione alle immagini morfologiche . secondo i nostri risultati , la sequenza dwi basata su pi valori di b e le misurazioni di adc eseguite sullintero volume tumorale possono essere usate in aggiunta , ma non come strumento sostitutivo allo studio rm standard , nel valutare la risposta a terapie non chirurgiche del tumore della cervice uterina . infatti , nella lettura dellesame rm , le sequenze dwi servono ad identi care la presenza e la sede delle lesioni cervicali . 
nonostante ci , le immagini morfologiche standard ( immagini pesate in t1 e t2 , con o senza la somministrazione di mezzo di contrasto ) sono ancora necessarie per rispondere a speci che domande , come il coinvolgimento dei parametri , dellendocervice , dei fornici vaginali e dei linfonodi [ 29 ] , come anche nel dare informazioni sul volume tumorale e sui dettagli anatomici dellin ltrazione di strutture adiacenti , soprattutto in pazienti candidate alla chirurgia e alla radioterapia [ 30 ]  . quando questo studio stato iniziato , non cerano evidenze de nitive sullutilit delle dwi nella valutazione della risposta alla terapia nel tumore della cervice . 
di conseguenza , la gestione normale delle pazienti con questa patologia , candidate a terapia non chirurgica , attualmente basata su uno studio rm eseguito prima delle terapie e uno studio eseguito dopo la ne delle terapie , non stata modicata per non arrecare ulteriori disagi alle pazienti . 
sulla base dei nostri risultati , ulteriori studi , includenti una pi precoce valutazione rm , dovrebbero essere eseguiti per stabilire se le modi cazioni in dwi sono pi precoci rispetto ai cambiamenti di dimensione e , di conseguenza , se lutilizzo delle dwi pu essere considerato come un marcatore precoce della risposta tumorale alle terapie . dzik - jurasz et al . 
 [ 31 ] hanno suggerito che elevati valori di adc prima del trattamento possono rappresentare un indicatore di necrosi che potrebbe essere predittivo di una scarsa 778 radiol med ( 2011 ) 116 : 766780 mcveigh et al . 
 [ 16 ] , we observed no signi cant difference in the median of grouped adc values of responder and nonresponder tumours at the staging mri ( p = 0.09 ) , either in the two readings by the same reader ( intraobserver agreement ) or between readings of the two readers ( interobserver agreement )  . 
median adc values of responders were , in fact , lower ( 1.0210 - 3 mm2 / s ) than those of nonresponders ( 1.1210 - 3 mm2 / s ) , but this difference did not reach signi cance . 
thus , in this study , a cutoff value of adc to distinguish the nonresponder tumours based on the sole dwi information from pretherapy mr examination was not identi ed . 
 the lack of correlation between percent increase in adc values and percent reduction of tumour volumes found in this series may be due to the high number of completeresponder tumours . 
indeed , 9 / 14 tumours showed a 100% reduction , independently of percent increase of adc values , whereas 5 / 14 showed a partial response ( tumour volume reduction lower than 100% ) ; therefore , a statistical analysis in this subgroup of patients would have been underpowered . 
this difference may be due to the different evaluation of the adc maps made over multiple slices to cover the entire volume of the tumour in this study and , in contrast , on the single most representative slice in the study by mcveigh . 
undertaking a more powerful study in a reasonably short time in order to minimise variations in treatment and in mr technique would likely require a multicentric study , which would introduce intersite variability . 
 la possibilit di distinguere i tumori della cervice uterina rispondenti dai non rispondenti , e la conseguente identi cazione precoce dei tumori non rispondenti , potrebbe aiutare ad indirizzare le pazienti con questa patologia ad interrompere trattamenti inef caci e ad evitare ritardi nelliniziare trattamenti alternativi potenzialmente pi ef caci . 
 [ 16 ] , noi non abbiamo osservato nessuna differenza nella mediana dei valori di adc dei tumori rispondenti e di quelli non rispondenti alla rm di stadiazione ( p = 0 , 09 ) , sia nelle due letture dello stesso osservatore ( accordo intra - osservatore ) sia nelle letture dei due osservatori ( accordo inter - osservatore )  . 
la media dei valori di adc dei rispondenti stata infatti pi bassa ( 1 , 0210 - 3 mm2 / s ) rispetto a quella dei non rispondenti ( 1 , 1210 - 3 mm2 / s ) , ma questa differenza non ha raggiunto la signi cativit . 
pertanto , in questo studio non stato identi cato un valore di adc per distinguere i tumori non rispondenti sulla base della sola informazione della dwi dello studio rm preterapia . 
questa mancanza di signi cativit e di speci cit potrebbe essere correlata alla distribuzione dei valori ed al piccolo numero di pazienti non rispondenti ( n = 3 )  . 
 lassenza di correlazione tra lincremento percentuale dei valori di adc e la riduzione percentuale del volume tumorale riscontrato in questo studio , potrebbe essere dovuto allelevato numero di tumori che hanno risposto completamente . 
 infatti , 9 / 14 tumori hanno mostrato una riduzione del 100% , indipendentemente dallincremento percentuale del valore di adc , mentre 5 / 14 hanno mostrato una risposta parziale ( riduzione del volume tumorale < 100% ) ; pertanto unanalisi statistica in questo sottogruppo di pazienti potrebbe essere non adeguata . in contrasto con mcveigh et al . 
 [ 16 ] , i nostri valori di adc non hanno mostrato differenze signi cative tra i tumori raggruppati in stadi precoci ed avanzati ( p = 0 , 96 )  . 
questa discordanza potrebbe essere dovuta alla diversa valutazione delle mappe adc , effettuate su multiple sezioni a coprire lintero volume del tumore in questo studio , e sulla sola sezione pi rappresentativa nello studio di mc veigh et al . 
precedenti studi non hanno dimostrato signi cative differenze nel valore di adc tra i tumori squamocellulari e gli adenocarcinomi [ 4 , 11 , 16 ] , mentre liu et al . 
per il basso numero di adenocarcinomi inclusi in questo gruppo di studio ( 3 / 17 ) , in accordo con la distribuzione di questo tipo istologico , riportata dallistituto nazionale dei tumori degli usa come 25 , 2% contro il 68 , 7% dei tumori squamocellulari [ 32 ] , questa valutazione non stata eseguita e non sono state fatte analisi statistiche . un limite di questo studio il basso numero di pazienti inclusi . 
purtroppo , per intraprendere uno studio pi ampio in tempi ragionevolmente pi brevi , per minimizzare le variazioni nel trattamento e nella tecnica rm , sarebbe stato radiol med ( 2011 ) 116 : 766780 parameters and the same mr machine . 
furthermore , the same method of tracing rois over the slices was applied to all examinations in the three sets of data , acquired twice by the same reader and once by the second reader , thus ensuring optimal intrapatient comparison and consistency of dwi comparison within the cohort . 
however , the aim of this study was demonstrating changes in adc values in cervical cancers before and after therapy and correlating these changes with variations in tumour size rather than assessing whether a negative mr examination matched the absence of residual tumour at surgery . 
 in conclusion , this study showed signi cant differences in adc values of normal and cancerous cervical tissue and of cervical tumours before and after nonsurgical therapies , with a signi cantly higher percent increase of adc values in responders than in nonresponders . 
in the future , dwi may be considered as an additional tool to the standard mr examination of the pelvis in cervical cancer patients to assess response to nonsurgical therapy , with neither exposure to ionising radiation nor additional discomfort for the patient , and no additional need for contrast medium injection . necessario uno studio multicentrico , che avrebbe introdotto una variabilit inter - sede . 
 inoltre , stato applicato a tutti gli esami lo stesso metodo per tracciare le roi con la valutazione di tre serie di dati , valutati due volte dallo stesso lettore e una volta dal secondo lettore , assicurando una ottima comparazione intra - paziente ed anche allinterno della popolazione . 
tuttavia , lo scopo dello studio era dimostrare cambiamenti dei valori di adc nei tumori della cervice prima e dopo terapia , e correlare questi cambiamenti con la variazione dimensionale del tumore , piuttosto che stabilire se una rm negativa corrisponda alla assenza di tumore residuo alla chirurgia . 
 in conclusione , questo studio mostra differenze signicative dei valori di adc tra tessuto cervicale normale e tumorale , e nei tumori della cervice prima e dopo terapie non chirurgiche , con un incremento percentuale del valore di adc signi cativamente pi alto nei rispondenti rispetto ai non rispondenti . 
in futuro , le immagini pesate in diffusione potrebbero essere considerate come strumento aggiuntivo allesame rm standard della pelvi in pazienti con tumore della cervice , per valutare la risposta alle terapie non chirurgiche , senza esposizione a radiazioni ionizzanti , senza disagi aggiuntivi per le pazienti e senza aggiunta di mezzo di contrasto . 
martinoli4 1department of radiology , national institute for cancer research , largo rosanna benzi 10 , 16138 genova , italy 2department of endocrinological & medical sciences ( disem ) and center of excellence for biomedical research , university of genova , genova , italy 3instituto de investigacin biomdica ( iib ) - sant pau , endocrinology / medicine department and centro de investigacin biomdica en red de enfermedades raras ( ciber - er , unidad 747 ) , isciii ; universitat autonoma de barcelona , barcelona , spain 4depatment of radiology dicmi , universit di genova , genova , italy correspondence to : a . 
in the early stage of disease , peripheral nerve enlargement associated with carpal tunnel syndrome or cubital tunnel syndrome and thickening of retinacula , such as a1 pulley in trigger nger , represent the features that may be seen by radiologists and are worthy of an endocrinological evaluation . 
few and nonspeci c symptoms characterise the initial phases of the disease , and therefore , most patients will have generally consulted many specialists ( most frequently musculoskeletal radiologists ) before an adequate endocrinological assessment is performed . 
the rst and most important therapeutic approach to acromegaly is early diagnosis , whereas the therapeutic goals are to eliminate morbidity and reduce mortality to the expected ageand sex - adjusted rates and prevent the development of systemic complications . 
when both radiological and clinical abnormalities are present , an endocrinological workup is useful to diagnose the disease in an early phase . riassunto lobiettivo del lavoro di riassumere le complicanze precoci dellacromegalia . 
nelle fasi precoci della malattia lingrandimento dei nervi periferici associato alla sindrome del tunnel carpale o del tunnel cubitale e lispessimento dei retinacoli come la puleggia a1 nel dito a scatto rappresentano i segni che possono essere notati dal radiologo e che meriterebbero una valutazione specialistica endocrinologica . 
nelle fasi iniziali i sintomi possono essere pochi e aspeci ci , pertanto i pazienti consultano molti specialisti , tra cui il radiologo muscolo - scheletrico , prima di effettuare una visita specialistica endocrinologica . 
 dato che gli obiettivi terapeutici sono la riduzione della morbilit e della mortalit , la diagnosi precoce rimane il miglior approccio terapeutico possibile per prevenire le complicanze sistemiche . parole chiave tunnel carpale tunnel cubitale dito a scatto acromegalia ecogra a complicazioni osteoarticolare 782 radiol med ( 2011 ) 116 : 781792 keywords carpal tunnel cubital tunnel trigger nger acromegaly ultrasound complications musculoskeletal introduction introduzione acromegaly is a rare syndrome that usually develops over many years due to long - term exposure to elevated levels of growth hormone ( gh ) and peripheral insulin - like growth factor 1 ( igf - 1 ) [ 1 ]  . 
however , clinical signs such as somatic dis gurements , voice alterations and extremity enlargement are considered pathognomonic and can suggest the diagnosis during the rst clinical evaluation [ 3 , 4 ] and are as important as symptoms for an early diagnosis . 
nevertheless , the disease still goes undetected because of limited knowledge of the syndrome [ 4 ]  . radiologists are among those specialists who may evaluate a patient affected by acromegaly before the nal diagnosis is achieved , especially for the radiological symptoms . 
in this case , initial recognition of the clinical signs , together with a radiological evaluation of symptoms , could suggest to radiologists the diagnosis of acromegaly and the need to refer the patient for endocrinological evaluation . 
 musculoskeletal manifestations of this syndrome are frequent , and virtually all patients develop symptoms or signs related to arthropathy , but these signs have been poorly studied [ 46 ]  . 
 the aim this review is to highlight the early musculoskeletal complications of acromegaly in order to enable the radiologist to suspect a possible diagnosis in an early phase of the disease and refer the patient for endocrinological assessment . 
 lacromegalia una sindrome rara che si sviluppa lentamente nel corso degli anni in seguito a una prolungata esposizione ad elevati livelli di ormone della crescita ( gh ) e fattore di crescita insulino - simile di tipo i ( igf - 1 ) [ 1 ]  . 
al momento della diagnosi i pazienti generalmente presentano alterazioni somatiche del volto , ingrandimento delle mani e dei piedi , e ipertro a dei tessuti molli [ 2 ]  . 
queste caratteristiche vennero chiamate acromegalia , dal greco acros ( ) , estremit , e megalos ( ) , ingrandite [ 3 ]  . la diagnosi di acromegalia signi cativamente ritardata a causa della natura insidiosa della malattia . 
comunque i segni clinici come le alterazioni somatiche , le alterazioni della voce , lingrandimento di mani e piedi sono considerati patognomonici e possono suggerire la diagnosi durate un primo esame clinico [ 3 , 4 ] e questi segni sono importanti come i sintomi nelleffettuare una diagnosi precoce . 
 le manifestazioni osteoarticolari di questa sindrome sono frequenti e virtualmente tutti i pazienti sviluppano sintomi e segni correlati allartropatia acromegalica , tuttavia queste manifestazioni sono state relativamente poco studiate [ 46 ]  . 
lartropatia acromegalica tipicamente assomiglia allosteoartrosi e colpisce prima che la diagnosi nale sia raggiunta [ 57 ]  . lo scopo di questo articolo di enfatizzare le complicanze muscolo - scheletriche precoci dellacromegalia per rendere il radiologo capace di sospettare la presenza della malattia nelle fasi pi precoci . 
in questo modo verrebbe garantita una pi rapida valutazione endocrinologica . radiol med ( 2011 ) 116 : 781792 radiographic features of acromegaly aspetti radiogra ci dellacromegalia radiographic changes in the peripheral joints are typically widespread . 
 il paziente acromegalico tipicamente presenta : crescita delle ossa acrali con bozze frontali prominenti , aumento della taglia delle mani e dei piedi , slargamento della mandibola e prognatismo , aumento dello spazio tra i denti incisivi [ 8 ]  . 
tra le differenti e non speci che manifestazioni osteoarticolari dellacromegalia , alcune possono suggerire la diagnosi se associate a sintomi e segni clinici tipici ( tabelle 1 e 2 ) [ 10 , 11 ]  . sindrome del tunnel carpale fig . 
note the advanced arthropathy at the level of the rst metatarsophalangeal joint , which is more evident on the left side ( asterisks ) , with hallux valgus , bone erosions and osteophytes . 
a livello della prima articolazione metatarso - falangea si notano i segni di unartropatia avanzata , pi evidente a sinistra ( asterisco ) con alluce valgo , erosioni osse e osteo ti . 
da notare anche laumento di dimensioni dei sesamoidi , tipico dellacromegalia ( teste di freccia )  . i pazienti acromegalici possono soffrire di disturbi sensitivi alle mani come parestesie , bruciore , intorpidimento . 
lassociazione tra acromegalia e sindrome del tunnel carpale stata riconosciuta come una frequente complicanza della malattia con una prevalenza che pu raggiungere il 64% al momento della diagnosi [ 2 , 3 , 8 , 10 ]  . 
alcuni autori hanno riferito che la sindrome del tunnel carpale potrebbe essere causata da un aumento delledema allinterno del nervo mediano 784 radiol med ( 2011 ) 116 : 781792 fig . 
2 sagittal t2 - weighted fat - suppressed magnetic resonance ( mr ) image of the lumbar spine in a 41 - year - old acromegalic patient ( estimated disease duration : 4 years ) showing an increase of intervertebral disc spaces at the level of l3 - l4 and l4 - l5 . 
5 axial t2 - weighted turbo spin - echo magnetic resonance image of a 55 - year - old acromegalic woman demonstrates hypertrophy of the facet joints ( arrow ) , with associated fatty in ltration of the paraspinal muscles ( asterisks )  . 
5 immagine in risonanza magnetica ( rm ) assiale t2 pesata di un paziente di 55 anni con ipertro a delle faccette articolari ( freccia ) e in ltrazione adiposa dei muscoli paraspinali ( asterischi )  . radiol med ( 2011 ) 116 : 781792 table 1 most frequent signs and symptoms present at diagnosis in acromegalic patients sign / symptom percentage hand / foot enlargement facial feature changes hyperhidrosis headache joint pain gonadal dysfunction fatigue macroglossia increased spacing between teeth ingrandimento di mani e piedi cambio della morfologia somatica iperidrosi cefalea dolore articolare disfunzione gonadica astenia macroglossia aumento dello spazio tra i denti tabella 1 sintomi e segni pi frequento nei pazienti con acromegalia al momento della diagnosi segni / sintomi percentuale acromegalic patients typically present with acral bony overgrowth that results in frontal bossing , increased hand and foot size , mandibular enlargement with prognathism and widened space between the lower incisor teeth [ 8 ]  . 
among the different nonspeci c osteoarticular features of acromegaly , some may suggest the diagnosis if related with the clinical signs and symptoms of the disease ( tables 1 and 2 ) [ 10 , 11 ]  . 
association with acromegaly and median neuropathy or carpal tunnel syndrome is recognised as a common condition of this disease , with a prevalence up to 64% of patients at presentation [ 2 , 3 , 8 , 10 ] , and nerve conduction studies reported subclinical abnormalities in the vast majority of patients [ 916 ]  . 
moreover , some authors have reported that the development of a carpal tunnel syndrome is probably due to oedema of the median nerve within the carpal tunnel rather than extrinsic compression from increased volume of the carpal tunnel piuttosto che da una compressione estrinseca allinterno del tunnel [ 13 ]  . 
 stato recentemente dimostrato , utilizzando lecogra a ad alta risoluzione , che nei pazienti con acromegalia , un ingrandimento diffuso dei nervi pu essere responsabile della sindrome del tunnel carpale [ 10 ]  . 
nelle neuropatie da intrappolamento laspetto eco strutturale del nervo diventa uniformemente ipoecogeno con perdita del normale pattern fascicolare a livello della sede di compressione e prossimalmente ad esso [ 10 ]  . 
a livello del tunnel carpale , nei pazienti con acromegalia , larea misurata risultata di 16 , 54 , 4 mm2 , mentre nella popolazione normale larea risulta inferiore ai 10 mm2 . 
 lingrandimento dei nervi periferici nellacromegalia una caratteristica intrinseca della malattia ed correlata alla durata di malattia , al controllo clinico della malattia e ai valori plasmatici di igf - 1 . 
le principali diagnosi differenziali radiologiche includono la lebbra e il diabete mellito in quanto in queste condizioni patologiche pu essere presente un diffuso ingrandimento dei nervi periferici associato alla sindrome del tunnel carpale [ 1720 ]  . 
anche nei pazienti con malattia controllata le dimensioni dei nervi sono pi grandi che nei soggetti normali [ 21 ] , per questo motivo solo una diagnosi precoce e un rapido controllo della malattia pu permettere una normalizzazione dei nervi [ 21 ]  . 
nella nostra casistica il 60% dei pazienti con sindrome del tunnel carpale al 786 radiol med ( 2011 ) 116 : 781792 table 2 radiological ndings of musculoskeletal features of acromegaly radiological ndings site advanced musculoskeletal manifestations decreased bone mineral density vertebral fractures increased sesamoid volume increased heel fat - pad thickness osteoarthritis proliferative enthesopathy baastrups disease bone overgrowth increased thickness of a1 pulley enlargement of distal phalanx diffuse idiopathic skeletal hyperostosis ( dish ) early musculoskeletal manifestations widening of articular spaces increase of intervertebral disc spaces lumbar spine dorsal and lumbar spine foot ( hallux ) plantar foot mcp , mtp , ipj of hand and foot ; spine enthesis especially of the foot lumbar spine acral bones . 
tendon insertions hands fingers thoracic spine mcp , mtp , ipj of hand and foot ; spine spine early musculoskeletal manifestations suggesting an early diagnosis if other clinical features are present trigger nger carpal tunnel syndrome cubital tunnel syndrome diffuse nerve enlargement hands median nerve ulnar nerve ulnar and median nerve mc , metacarpophalangeal ; mtp , metatarsophalangeal ; ipj , interphalangeal joint tabella 2 reperti radiologici delle complicanze muscoloscheletriche dellacromegalia . 
articolazione reperti radiologici sede manifestazioni tardive diminuzione densit ossea fratture vertebrali aumento di volume dei sesamoidi aumento di spessore del grasso della pianta del piede artrosi entesopatia proliferativa malattia di baastrup crescita ossea inserzioni tendinee ispessimento puleggia a1 ingrandimento falangi distali dish manifestazioni precoci ampliamento spazi articolari aumento di spessore dei dischi colonna lombare colonna dorsale e lombare piede ( alluce ) pianta del piede mcp , mtp , ipj di mani e piedi ; colonna entesi del piede colonna lombare ossa acrali mani dita colonna toracia mcp , mtp , ipj mani e piedi ; colonna colonna manifestazioni precoci che suggeriscono ulteriori accertamenti se sono presenti le caratteristiche siche dellacromegalia dito a scatto sindrome del tunnel carpale sindorme del tunnel cubitale diffuso ingrandimento dei nervi mcp , metacarpofalangea ; mtp , metatarsofalange ; ipj , interfalangea mani nervo mediano nervo ulnare nervi mediano e ulnare radiol med ( 2011 ) 116 : 781792 contents [ 13 ]  . 
other studies , on the contrary , have reported that carpal tunnel syndrome in acromegaly is due either to oedematous synovial tissues that compresses the median nerve or to irreversible narrowing of the carpal tunnel [ 12 , 1416 ]  . 
 it has been demonstrated , by using high - resolution ultrasound , that in acromegalic patients , carpal tunnel syndrome may be induced by the diffuse nerve swelling that affects acromegalic limbs , [ 10 ]  . 
in entrapment neuropathies , the nerve echotexture may become uniformly hypoechoic , with loss of the fascicular pattern at the level of the compression and proximal to it [ 10 ]  . 
at the level of the carpal tunnel , the median nerve csa was measured as 16.54.4 mm2 in acromegalic patients , whereas in the normal population , the mean area of the median nerve at the carpal tunnel was < 10 mm2 . 
 main radiological differential diagnoses include leprosy and diabetes , because in these diseases , diffuse enlargement of peripheral nerves associated with carpal tunnel syndrome may be found [ 1720 ]  . 
for this reason , only an early diagnosis and prompt disease control might enable nerve normalisation [ 21 ]  . acromegalic patients often have a history of previous , unsuccessful , surgery for carpal tunnel syndrome . 
a transverse 17 - 5 mhz ultrasound image obtained at the level of the carpal tunnel demonstrates an enlarged median nerve ( cross - sectional area : 14.5 mm2 , normal value < 10 mm2 ) in an asymptomatic patient . 
b the median nerve ( mn ) enlargement persisted ( crosssectional area : 8.2 mm2 , normal value < 5 mm2 ) also at the level of the midforearm between the exor digitorum super cialis and profundus muscles ( ft )  . 
 ecogra a assiale a 17 , 5 mhz a livello del tunnel carpale che dimostra un ingrandimento del nervo mediano ( area 14 , 5 mm2 , normale < 10 mm2 ) in assenza di sintomi in a . 
il nervo mediano ( mn ) risulta ingrandito ( area : 8 , 2 mm2 , normale < 5 mm2 ) anche a livello dellavambraccio tra i tendini essori delle dita ( ft ) in b . 
 un radiologo muscolo scheletrico dovrebbe cercare le tipiche caratteristiche dellacromegalia quando diagnostica un tunnel carpale bilaterale e un diffuso ingrandimento dei nervi , con o senza pregressa chirurgia per sindrome del tunnel carpale . 
se questi indizi coincidono , il paziente dovrebbe essere inviato ad una visita specialistica endocrinologica . sindrome del tunnel cubitale la sindrome del tunnel cubitale , cio lintrappolamento del 788 radiol med ( 2011 ) 116 : 781792 fig . 
transverse 17 - 5 mhz ultrasound image obtained at the level of the carpal tunnel demonstrates a bilaterally enlarged median nerve ( cross - sectional area : 21.5 mm2 and 22 mm2 , normal value < 10 mm2 ) with loss of the normal fascicular echotexture and hypoechoic appearance in a symptomatic patient . 
lecogra a assiale a 17 , 5 mhz a livello del tunnel carpale dimostra un ingrandimento bilaterale del nervo mediano con perdita della normale struttura fascicolare ( area : 21 , 5 mm2 e 22 mm2 , normale < 10 mm2 )  . 
 a musculoskeletal radiologist should look for the typical features of acromegaly when nding a patient with bilateral carpal tunnel and diffuse nerve enlargement , with or without previous surgery for carpal tunnel syndrome : for example , information regarding changes in the size of rings or shoes should be elicited . 
 cubital tunnel syndrome ulnar nerve entrapment at the elbow ( cubital tunnel syndrome ) is the second most common peripheral nerve entrapment syndrome following carpal tunnel syndrome in the normal population . 
moreover it has been found that cubital tunnel syndrome in acromegalic patients has a prevalence of 21% , similar to that for carpal tunnel syndrome , and can be improved with clinical control of the disease . 
inoltre , stato dimostrato che la prevalenza della sindrome del tunnel cubitale nei pazienti affetti da acromegalia raggiunge il 21% , dato simile alla prevalenza del tunnel carpale , e che pu migliorare solo con il controllo clinico della malattia . 
in primo luogo deve differenziare la sindrome del tunnel carpale dalla sindrome del tunnel cubitale , in secondo luogo deve escludere le caratteristiche compatibili con acromegalia nei pazienti con tunnel cubitale . 
in generale , la sindrome del tunnel carpale correlata ad un ingrandimento del nervo mediano prossimale rispetto al tunnel carpale , mentre la sindrome del tunnel cubitale ad un ingrandimento del nervo prossimale rispetto al gomito . 
di fatto la chirurgia pu essere considerata il trattamento di scelta nella sindrome del tunnel carpale , mentre nella sindrome del tunnel cubitale pu essere proposta la terapia conservativa [ 22 ]  . 
a short - axis ultrasound image of the ulnar nerve ( un ) at the cubital tunnel shows a markedly enlarged ulnar nerve in a symptomatic de novo acromegalic patient . 
firstly , carpal tunnel syndrome must be differentiated from cubital tunnel syndrome ; secondly it is necessary to look for typical features of acromegaly in a patient with cubital tunnel syndrome . 
in general , carpal tunnel syndrome is related to an enlarged median nerve proximally to the carpal tunnel , whereas cubital tunnel syndrome is related to an enlarged ulnar nerve proximally to the elbow . 
thus , nerve swelling is one of the predisposing factors to nerve entrapment in both carpal and cubital tunnel syndromes . differentiating between the two entrapment neuropathies is crucial due to the different therapeutic approaches . 
 in fact , in carpal tunnel syndrome , surgery is considered the treatment of choice , whereas in cubital tunnel , conservative therapy including patient education to avoid activities that produce symptoms , such as repetitive elbow exion or direct pressure to the medial epicondyle , splinting and work environment modi cation [ 22 , 23 ]  . 
 tuttavia la patogenesi di questa sindrome sembra simile a quella del tunnel carpale in cui la cronica e abbondante esposizione a gh e igf - 1 determina un diffuso ingrandimento dei nervi . 
nellacromegalia i valori normali dellarea del nervo ulnare sono 9 , 5 mm2 a livello dellavambraccio e 13 , 1 mm2 a livello del braccio , mentre a nella popolazione normale sono 5 , 3 mm2 e 6 , 6 mm2 rispettivamente . 
quindi un consulto specialistico endocrinologico pu essere suggerito in presenza di nervi ingranditi fuori dai canali osteo brosi . 790 radiol med ( 2011 ) 116 : 781792 cubital tunnel syndrome has only recently been recognised as being associated with acromegaly [ 22 ] , and therefore , the literature about this syndrome in relation to acromegaly is poorly represented . 
however , the pathogenesis of this entrapment syndrome seems similar to that of carpal tunnel syndrome , in which the chronic and exaggerated stimulation of gh and igf - 1 determines a diffuse enlargement of the nerves . 
 trigger nger trigger nger , a transient locking of the ngers in exion , is a common condition in which there is thickening and constriction of the proximal part of the exor tendon sheath at the base of the nger or thumb [ 24 ]  . 
this pulley is highly important for pathology : it is not only a stabiliser of the exor tendons during nger exion but it is a target area for trigger nger in different pathological conditions , such as acromegaly and scleroderma [ 24 ]  . 
it is known that the bone and hyaline cartilage are typical targets of gh and igf - 1 excess , and normalisation of gh and igf - 1 levels is only partly effective in reversing advanced arthropathy of acromegaly [ 3 ]  . 
however , the role of gh and igf - 1 on the brous tissue of retinacula , such as the a1 pulley , has only recently been investigated [ 26 ]  . 
measuring the thickdito a scatto il dito a scatto , cio un transitorio blocco del dito in posizione essa , una condizione comune in cui c un ispessimento e una costrizione della parte prossimale dei tendini essori e della loro guaina alla base delle dita o del pollice [ 24 ]  . 
il dito a scatto stato associato anche al diabete mellito , allartrite reumatoide , alla gotta , allinsuf cienza renale con o senza amiloidosi e alla polineuropatia familiare amiloidosica [ 25 ]  . 
la cartilagine ialina e losso sono tipici bersagli di gh e igf - 1 e la normalizzazione di questi valori ormonali non suf ciente a rendere reversibili i segni avanzati dellartropatia acromegalia [ 3 ]  . 
 ecogra a assiale a 12 , 5 mhz a livello della puleggia a1 del terzo dito , ispessita ( frecce )  . radiol med ( 2011 ) 116 : 781792 fig . 
il dito a scatto unaltra frequente e precoce complicanza muscolo - scheletrica dellacromegalia . conclusions conclusioni diffuse peripheral nerve enlargement , carpal tunnel syndrome , cubital tunnel syndrome and trigger nger are the early musculoskeletal complications of acromegaly and represent the features that may be noted by radiologists . 
 lingrandimento diffuso dei nervi periferici , la sindrome del tunnel carpale , la sindrome del tunnel cubitale e il dito a scatto sono le complicanze precoci dellacromegalia che possono essere riconosciute dal radiologo muscolo - scheletrico . 
over the last 4 years , 22 patients ( 14 men and 8 women ; mean age 62.6 years ) affected by 5 acute ( < 14 days ) thrombotic occlusions of the native arteries ( 4 plaque thromboses in the common iliac artery and one on a dissection intimal flap of the external iliac artery ) , 17 subacute and chronic thromboses affecting 4 femoro - popliteal by - pass grafts , 10 stents ( 7 in the common iliac artery and 3 in the superficial femoral artery ) and 3 stents - grafts were studied . 
in the subacute and chronic occlusions , the procedure was completed with percutaneous transluminal angioplasty ( pta ) ( 8 / 22 ) , cutting balloon ( 6 / 22 ) and stenting ( 5 / 22 )  . 
arterial recanalisation with rt is the treatment of choice for acute thrombosis of healthy native arteries ( 47 mm ) ; the treatment of thrombosis complicating calcified plaques or dissection intimal flaps riassunto obiettivo . 
negli ultimi 4 anni sono stati selezionati 22 pazienti ( 14 maschi e 8 femmine ; et media 62 , 6 anni ) portatori di 5 occlusioni trombotiche acute ( < a 14 giorni ) in arteria nativa ( 4 trombosi su placca dellarteria iliaca comune , 1 trombosi su flap di dissezione dellarteria iliaca esterna ) , 17 occlusioni trombotiche subacute e croniche in 4 by - pass femoro - poplitei , 10 stents ( 7 in arteria iliaca comune e 3 in arteria femorale superficiale ) e 3 stents - grafts . 
nelle occlusioni sub - acute e croniche stata associata langioplastica ( pta ) ( 8 / 22 casi ) , cutting ballon ( 6 / 22 ) e stenting ( 5 / 22 )  . 
la ricanalizzazione mediante tr il trattamento di scelta nelle occlusioni trombotiche acute su arteria sana ( 47 mm ) ; il trattamento della trombosi su placca calcifica e su flap di dissezione pu comportare la radiol med ( 2011 ) 116 : 932944 may cause rupture of the arterial wall . 
in subacute and chronic occlusions of by - pass grafts , stents and stent grafts , additional procedures are necessary to achieve complete recanalisation . keywords mechanical thrombectomy rotational thrombectomy thrombotic arterial occlusions rottura della parete arteriosa . 
 parole chiave trombectomia meccanica trombectomia rotazionale occlusioni trombotiche arteriose introduction introduzione acute and subacute thromboembolic occlusions are still a major cause of peripheral ischaemia and limb amputation . conventional treatment includes thrombo - embolectomy with a fogarty thrombectomy catheter and surgical by - pass [ 18 ]  . 
recent years have seen the development of rotational thrombectomy ( rt ) , and many types of devices are available with which to exploit either the vortex principle to fragment the thrombus ( amplatz thrombectomy catheter , angiojet ; arrow - trerotola thrombolysis system ) or the venturi effect to aspirate it ( hydrolyser , thrombektomat ) [ 913 ]  . 
the literature contains reports of experimental studies [ 35 , 14 ] and a few clinical studies [ 6 , 7 , 10 , 11 , 15 ] , all of which have shown promising results . 
la trombectomia rotazionale ( tr ) si sviluppata negli ultimi anni , grazie alla commercializzazione di diversi tipi di device che permettono la frammentazione del trombo sfruttando il vortex principle ( amplatz thrombectomy catheter , angiojet , arrow - trerotola thrombolysis system ) o la sua aspirazione mediante effetto venturi ( hydrolyser , thrombektomat ) [ 913 ]  . 
in letteratura sono attualmente riportati studi sperimentali [ 35 , 14 ] e limitate esperienze cliniche [ 6 , 7 , 10 , 11 , 15 ] che hanno dimostrato promettenti risultati . scopo del nostro lavoro presentare la nostra esperienza nellutilizzo di tale device nel trattamento di occlusioni acute , sub - acute e croniche in vari distretti . 
 materials and methods materiali e metodi between november 2004 and april 2008 , we selected 22 patients ( 14 men and 8 women ; mean age 62.6 years ) affected by 5 acute thrombotic occlusions ( < 14 days ) in native arteries ( 4 thromboses of the common iliac artery , 1 thrombosis of the external iliac artery ) and 17 subacute or chronic occlusions ( > 14 days ) in 4 femoro - popliteal by - pass grafts , 10 stents [ 7 in the common iliac artery and 3 in the superficial femoral artery ( sfa ) ] , 2 femoro - popliteal stentsgrafts and 1 stent - graft in the common iliac artery ( placed to treat aneurysms ) ( table 1 )  . 
the clinical diagnosis was ( us ) and confirmed by colour - doppler ultrasound nel periodo compreso tra novembre 2004 e aprile 2008 sono stati selezionati 22 pazienti ( 14 maschi e 8 femmine ; et media 62 , 6 anni ) portatori di 5 occlusioni trombotiche acute ( < 14 giorni ) in arteria nativa ( 4 trombosi dellarteria iliaca comune , 1 trombosi dellarteria iliaca esterna ) , 17 occlusioni sub - acute o croniche ( > 14 giorni ) in 4 by - pass femoropoplitei , 10 stent ( 7 in arteria iliaca comune e 3 in arteria femorale superficiale ) , 2 stent - graft femoro - poplitei e 1 stentgraft in arteria iliaca comune ( posizionato per aneurisma ) ( tabella 1 )  . 
indications for rt included acute thrombosis in a medium - calibre native artery and subacute or chronic occlusion of by - pass grafts , stents or stent - grafts along the iliac - femoropopliteal arterial axis . 
because the 6 - fr or 8 - fr thrombectomy catheters cannot be used in arteries < 4 mm in calibre , distal trattamento mediante tr nei casi di trombosi acuta su arteria nativa di medio calibro e di occlusione sub - acuta o cronica di bypass , stent o stent - graft lungo lasse arterioso iliacofemoro - politeo . 
il catetere da trombectomia in relazione al calibro ( 6 o 8 fr ) non pu essere utilizzato in arterie di piccolo calibro < a 4 mm , quindi sono state escluse dal trattable 1 rotational thrombectomy with rotarex pt . 
in order to obtain an optimal calibre of the recanalised vessel , in 17 / 22 cases , the procedure was completed by percutaneous transluminal angioplasty ( pta ) , tamento le embolie distali della gamba , con interessamento delle arterie della triforcazione . 
nella nostra esperienza in tutti i casi locclusione stata valicata con filo guida da 0 , 035 idrofilo ( terumo , tokyo , giappone )  . successivamente stato inserito un introduttore vascolare da 8 fr in cross - over , 6 fr o 8 fr in accesso antero - grado ( cordis , johnson & johnson , miami lakes , fl , usa ) mediante catetere da cambio retto da 4 fr ( cordis , johnson & johnson , miami lakes , fl , usa ) con posizionamento , a valle dellocclusione , di una guida da 0 , 018 lunga 260 cm ( terumo , tokyo , giappone )  . 
su tale guida stato fatto scorrere il catetere da tr con un movimento di to - and - fro ( vai e vieni ) lungo locclusione realizzando un lume di ricanalizzazione , mediante spirale metallica rotante ad una velocit di rotazione di 50000 rpm ( rotazioni al minuto )  . 
lazione di tale device ha prodotto la frammentazione del trombo in particelle di circa 100500 m che mediante un vacuum continuo ( > 5 , 8 kpa , 43 mmhg ) sono state aspirate ed eliminate allinterno di una sacca posizionata allestremo prossimale del catetere , con minima perdita di sangue ( < 100 ml / procedura )  . 
a occlusione ( freccia ) ; b trombectomia ; c ricanalizzazione con persistenza delle stenosi alle anastomosi ( frecce ) ; d ricanalizzazione completa delle anastomosi prossimale e distale dopo cutting balloon pta ( frecce )  . 
the recanalisation of occluded stents along the superficial femoral artery ( sfa ) and common iliac artery , in addition to revealing reductions in calibre and stent wall irregularities due to myointimal hyperplasia , showed disease progression in all cases , with stenosis due to an atheromatous plaque both proximal and completata con angioplastica ( pta ) , cutting balloon o stenting . 
la ricanalizzazione degli stents occlusi lungo larteria femorale superficiale e in arteria iliaca comune , oltre ad evidenziare delle riduzioni in calibro con irregolarit delle pareti dello stent da iperplasia miointimale , ha mostrato in tutti i casi una progressione di malattia con stenosi da placca ateromasica a radiol med ( 2011 ) 116 : 932944 fig . 
a langiografia documenta occlusione dello stent - graft ; b trombectomia ( freccia ) ; c ricanalizzazione dello stent - graft con riperfusione della sacca aneurismatica ( frecce ) ; d sotto guida ecografia viene infisso ago ( frecce ) nel contesto della sacca aneurismatica rifornita con iniezione di trombina ; e il controllo documenta ricanalizzazione dello stent - graft e completa esclusione dellaneurisma . 
one of them involved the external iliac artery in a patient in whom a 5 fr vascular introducer had been left in place for several days to monitor intracranial circulation with angiography . 
 sono inoltre state trattate 5 trombosi acute di cui una dellarteria iliaca esterna in una paziente in cui stato lasciato in sede per diversi giorni un introduttore vascolare da 5 fr per controlli angiografici del circolo intra - cranico . rimosso lintroduttore nel sospetto clinico di trombosi acuta 938 radiol med ( 2011 ) 116 : 932944 fig . 
a occlusione dello stent in aic di destra e steno - occlusione dellaic di sinistra in esiti di kissing stenting ; b trombectomia ; c parziale ricanalizzazione , con lume insufficiente ; d kissing balloon pta ; e completa ricanalizzazione degli stents . 
 the follow - up of patients treated with rt for acute thrombosis involved clinical assessment and colour - doppler us at 1 and 3 months and only clinical monitoring thereafter . 
 il follow - up dei pazienti sottoposti a trattamento con tr per trombosi acute stato effettuato inizialmente con valutazione clinica ed eco - color - doppler a 1 e 3 mesi . 
a eco - color doppler ; b dimostrazione angiografica delloccluisone dellaie ; c trombectomia ; d lacerazione dellaie ; e , f trattamento mediante stent - graft ; g tc post - procedura . 
nel caso di occlusioni sub - acute e croniche il follow - up stato eseguito mediante esame clinico ed eco - color doppler a 1 , 3 , 6 e 12 mesi e successivamente annualmente . 
 results in acute occlusions of medium - calibre native arteries ( 4 / 22 cases ) , immediate technical success was achieved with rt and no additional procedures were required . 
in 1 case of acute thrombosis of the external iliac artery , rt produced a vascular lesion that was immediately repaired with a stent grain subacute occlusions of by - pass grafts ( 4 / 17 cases ) , rt was combined with cutting - balloon angioplasty of the anastomotic stenoses . 
in chronic occlusions ( 13 / 17 cases ) of stents and stent - grafts , additional procedures were needed to achieve an optimal lumen size ( 8 / 10 pta ; 2 / 10 cutting balloon ; 3 / 10 stent - in - stent )  . 
in 1 case , rt was not performed because the catheter was unable to cross a thrombotic sfa occlusion in a case of unrecognised fracture of a preexisting stent ; in this case , recanalisation was achieved using cutting - balloon angioplasty and stent - in - stent . risultati nelle occlusioni acute in arteria nativa di medio calibro ( 4 / 22 casi ) stato ottenuto mediante tr un successo tecnico immediato senza procedure aggiuntive . 
in un caso di trombosi acuta in corrispondenzadellarteria iliaca esterna si verificata una lesione vascolare da tr , immediatamente trattata mediante stent - gra nelle occlusioni sub - acute in by - pass ( 4 / 17 casi ) stata la tr stata associata allangioplastica delle stenosi anastomotiche mediante cutting balloon . 
nelle occlusioni croniche ( 13 / 17 casi ) in stents e stent - grafts , per ottenere un lume ottimale , sono state necessarie procedure aggiuntive ( 8 / 10 pta ; 2 / 10 cutting balloon ; 3 / 10 stent in stent )  . 
in the remaining 17 cases of subacute and chronic thrombosis , radiological examinations revealed two stent reocclusions in the sfa at 3 and 12 months , respectively , which were surgically converted into femoro - popliteal by - pass grafts . discussion in recent years , three large prospective randomised studies ( rochester trial , stile trial , and topas trial ) compared intra - arterial fibrinolytic therapy and surgery in the treatment of thrombotic arterial occlusions [ 1618 ]  . 
although comparison of these studies is in part limited by the different protocols and inclusion criteria used ( acute / subacute / chronic ischaemia , thrombotic occlusion / thromboembolic occlusion , proximal / distal occlusions , thromboses of native vessels / bypass grafts , etc . ) , no evidence has emerged indicating the superiority of fibrinolytic therapy over surgery . 
surgical revascularisation is definitely indicated in severe limb ischaemia ( stages iia and iib in the fontaine classification ) , whereas intra - arterial fibrinolysis is a valuable alternative in acute thrombosis ( < 14 days ) with mild to moderate limb ischaemia . 
nei rimanenti 17 casi di trombosi sub - acute e croniche ai controlli radiologici si sono verificate due riocclusioni di stent in afs , rispettivamente a 3 e 12 mesi , convertiti chirurgicamente in by - pass femoropopliteo . 
 discussione negli ultimi anni tre grandi studi prospettici randomizzati ( rochester trial , stile trial e topas trial ) hanno confrontato il trattamento fibrinolitico intrarterioso e la chirurgia nel trattamento delle occlusioni arteriose trombotiche [ 1618 ]  . 
sebbene il confronto tra gli studi citati sia in parte limitato da differenti protocolli e criteri di inclusione nella selezione dei pazienti ( ischemia acuta / subacuta / cronica , occlusione trombotica / occlusione trombo - embolica , occlusioni prossimali / occlusioni distali , trombosi di vasi nativi / trombosi di bypass , ecc . ) , non esiste evidenza di superiorit del trattamento fibrinolitico rispetto a quello chirurgico . 
la rivascolarizzazione chirurgica sicuramente indicata nei casi di severa ischemia dellarto ( classe iia e iib secondo fontaine ) ; al contrario il trattamento fibrinolitico intrarterioso una valida alternativa nei casi di trombosi acuta ( < 14 giorni ) , in presenza di ischemia dellarto di grado lieve - moderato . 
utilizzando questultimo approccio inoltre possibile , mediante langiografia definire con maggiore accuratezza eventuali lesioni stenosanti , possibili responsabili dellevento trombotico acuto , da sottoporre ad un successivo intervento di rivascolarizzazione percutanea o chirurgica [ 1618 ]  . 
 tuttavia la fibrinolisi gravata da un basso ma non trascurabile tasso di complicanze emorragiche ( 1%2% emorragia intracranica ) , che risultano comunque bilanciate come dimostra lo studio rochester da percentuali di mortalit peri - procedurali simili allintervento chirurgico ( 14% fibrinolisi vs 18% chirurgia ) , che al follow - up a 12 mesi tendono a diventare statisticamente significative ( 16% fibrinolisi vs 42% chirurgia ) , poich gravate da un pi alto tasso di complicanze cardio - polmonari ( 16% fibrinolisi vs 49% chirurgia ) [ 16 ]  . 
la tr pu essere considerata una potenziale alternativa alla fibrinolisi loco - regionale ed alla chirurgia per il trattamento delle occlusioni arteriose trombotiche su vasi nativi e su bypass chirurgici . 
in an ischaemic limb , the distal embolisation impairs the microcirculation with persistent or progressive ischaemia , so it is essential that the rt devices cause as little endothelial damage as possible , with rapid thrombus fragmentation and aspiration . 
 compared with fibrinolysis , rt has the advantage of allowing treatment of patients with contraindications to anticoagulation therapy and achieving immediate recanalisation of the occluded segment with prompt resolution of the clinical picture . 
another significant advantage of the new rt catheters ( rotarex ) is the ability to reduce the risk of distal migration of thrombi during the percutaneous procedure , thus significantly lowering the duration of limb ischaemia . on the other hand , rt procedures have the disadvantage of being relatively long and laborious . 
in our experience , the rotarex procedure took 6216 min , its duration being influenced by the extent of thrombosis , device preparation and difficulties in reaching the target site , especially when using a contralateral approach and cross - over technique . 
the rt procedure is complementary to other recanalisation techniques in the case of impassable stenosis or insufficient lumen and in fact cannot be used in arteries < 4 mm in diameter due to catheter calibre ( 6 fr or 8 fr )  . 
the first large series was reported by zeller [ 10 ] , who treated 33 occlusions in native arteries , 58 subacute and chronic utilizzato off - label alcuni di tali device per il trattamento delle occlusioni trombotiche del sistema arterioso periferico . nonostante il differente meccanismo di funzionamento di questi device ( angiojet , amplatz , hydrolyser , oasis ) ed i differenti tassi di perviet primaria ( rispettivamente 61% vs 75% vs 83% vs 11 , 8% ) [ 1922 ] lembolizzazione distale e le lesioni vascolari costituiscono sicuramente le principali complicanze della tr . 
 la tr rispetto alla fibrinolisi ha il vantaggio di poter trattare pazienti con controindicazioni alla terapia anticoagulante e di realizzare unimmediata ricanalizzazione del tratto occluso con pronta risoluzione del quadro clinico . 
altro importante vantaggio dei nuovi cateteri da tr ( rotarex ) la capacit di ridurre il rischio di migrazione distale del trombo durante il trattamento percutaneo , abbassando in modo significativo la durata dellischemia dellarto . 
la durata complessiva della procedura nella nostra esperienza , mediante rotarex di 6216 minuti , appare condizionata dallestensione della trombosi , dai tempi di preparazione del device e dalla difficolt di portare in sede il catetere , specie nel caso di approccio controlaterale in cross - over . 
la procedura di tr complementare ad altre tecniche di ricanalizzazione nei casi di stenosi non valicabili o di lume insufficiente ; infatti non pu essere utilizzata correttamente in arterie con diametro < a 4 mm , in relazione al calibro del catetere ( 6 o 8 fr )  . 
altro importante limite al suo impiego lutilizzo di costosi cateteri monouso da 6 o 8 fr [ circa 2750 euro per catetere rotarex vs 56 , 4 euro per un trattamento fibrinolitico ( 2000000 u / die ) protratto per 72 ore vs 924 , 81002 , 8 euro per terapia fibrinolitica di 72 ore associata a stenting del vaso ] , nonostante la consolle e il motore da tr siano forniti gratuitamente dalla straub . 
 il primo studio clinico che descrive il funzionamento del catetere rotarex quello di schmitt e collaboratori [ 3 ] che hanno dimostrato in fase preliminare , come la tr possa essere tecnica fattibile ed efficace nelle occlusioni trombotiche acute e sub - acute dellarteria femoro - poplitea . 
 [ 6 ] mediante il trattamento di 19 942 radiol med ( 2011 ) 116 : 932944 occlusions in native arteries and 9 by - pass grafts in 98 patients , achieving an immediate technical success rate of 94% , 93% and 78% , respectively , and 18 complications including 1 death ; the other complications were 8 perforations , 7 distal embolisations and 2 amputations . 
the complications reported were 2 reocclusions on day 1 and day 7 , respectively ; 1 haematoma at the access site ; 1 arterio - venous fistula and ten distal embolisations 6 of them significant treated with thromboaspiration . 
 our series was small because we excluded , in agreement with the literature [ 6 , 10 , 12 ] , patients with occlusions on subacute and chronic atheromatous plaques along the iliacfemoral axis and especially along the sfa , as we believe that these occlusions require other recanalisation techniques [ 16 , 17 ]  . 
in these cases , the thrombectomy catheter has a higher risk of perforation , particularly if the plaque is calcified ; in addition , the unrecognised subintimal passage of the angiography wire may cause the thrombectomy catheter to rupture the artery with a risk of arteriovenous fistula and an increased probability inversely proportional to the occluded segment of producing distal embolisation . 
in contrast to previous reports , during the 22 treatments with rt with the rotarex catheter , we had only one case of perforation , which involved a case of acute thrombosis of the external iliac artery in an unrecognised iatrogenic dissection flap caused by an indwelling 5 fr vascular introducer . 
in the case of chronic thrombotic occlusions , an essential precondition for rt is that the guidewire crosses the arterial occlusion , which explains why it is preferable to negotiate long occlusions subintimally and achieve recanalisation with subintimal pta [ 2325 ]  . 
in these cases complete recanalisation was achieved without additional procedures ( pta or stenting ) , which are not , however , unanimously recommended [ 26 ]  . indeed , the technical principle behind angioplasty is to squash the thrombus and spread it along the healthy arterial walls , creating a recanalisation lumen and possible distal migration of the thrombus [ 27 ]  . 
la prima ampia casistica di 98 pazienti quella di zeller [ 10 ] che ha trattato 33 occlusioni in arteria nativa , 58 occlusioni sub - acute e croniche in arterie native e 9 by - pass con un tasso di successo tecnico immediato rispettivamente del 94% , 93% e 78% e 18 complicanze tra cui un decesso ; le altre complicanze riscontrate sono state 8 perforazioni , 7 embolie distali e 2 amputazioni . 
successivamente duc [ 12 ] ha trattato 38 pazienti con trombosi acuta , sub - acuta e cronica dellarteria femorale superficiale con solo 5 procedure aggiuntive , 2 pta e 5 stents , ottenendo un successo tecnico immediato del 100% . 
in questa casistica emergono quali complicanze 2 riocclusioni rispettivamente a 1 e 7 giorni dopo la procedura , 1 ematoma del sito daccesso , 1 fistolaartero venosa ed embolie distali in 10 pazienti di cui 6 rilevanti , trattate con trombo - aspirazione . 
 la nostra casistica esigua in quanto non sono stati selezionati pazienti con occlusioni su placca ateromasica subacuta e cronica lungo lasse iliaco - femorale specie lungo larteria femorale superficiale , come riportato in letteratura , [ 6 , 10 , 12 ] in quanto ormai convinti che questo tipo di occlusioni necessitano di altre tecniche di ricanalizzazione [ 16 , 17 ]  . 
in questi casi il catetere da trombectomia ha un maggior rischio di perforazioni specie su placche calcifiche ; peraltro il passaggio sub - intimale misconosciuto della guida angiografica , pu determinare la fissurazione dellarteria da parte del catetere da trombectomia con il rischio di formazione di fistole artero - venose , nonch la maggiore probabilit , inversamente proporzionale al tratto occluso , di realizzare unembolia distale . 
infatti a differenza dei dati di letteratura durante i 22 trattamenti mediante tr con rotarex nella nostra casistica si verificata una sola perforazione , su trombosi acuta dellarteria iliaca esterna su flap di dissezione iatrogeno misconosciuto , determinato da permanenza in situ di introduttore vascolare da 5 fr . 
nel caso di occlusioni trombotiche croniche , presupposto indispensabile per eseguire la tr che il filo guida valichi locclusione arteriosa ; per tale motivo nei casi di occlusione lunga preferibile valicare locclusione in sede sotto - intimale e realizzare una ricanalizazione mediante pta sub - intimale [ 2325 ]  . 
 i migliori risultati nella nostra serie si sono avuti nel trattamento della tromboembolia su arteria nativa di medio calibro , anche se tale patologia unevenienza abbastanza rara ; in questi casi la ricanalizzazione completa non necessita di procedure aggiuntive ( pta o stenting ) , che peraltro non trovano una indicazione unanime [ 26 ]  . 
infatti il principio tecnico dellangioplastica di schiacciare e spalmare il trombo lungo le pareti arteriose , in questo caso sane , realizzando un lume di ricanalizzazione ed una possibile dislocazione distale del trombo [ 27 ]  . 
this procedure , which is also feasible in patients who have recently undergone surgery or who have contraindications to fibrinolytic therapy , was free of major complications such as vessel perforations and allowed for immediate recanalisation . 
the failure of ultrasound thrombolysis was related to one reocclusion that appeared within only 2 h of the procedure and was subsequently treated with surgery . our series included recanalisation of subacute and chronic occlusions of stents and stent - grafts , an approach that has not been previously reported . 
 conclusions nei casi di ricanalizzazione di by - pass femoro - popliteo , la tr ha immediatamente evidenziato la causa delle stenosi anastomotiche serrate , che viste le caratteristiche morfologiche ( brevi - sub - occludenti da iperplasia miointimale ) abbiamo preferito trattare con cutting balloon ( boston scientific , natick , ma , usa )  . 
tale procedura attuabile anche in pazienti operati di recente o con controindicazioni alla terapia fibrinolitica , risultata priva di complicanze maggiori come la perforazione del vaso , consentendo unimmediata ricanalizzazione vascolare . 
 [ 26 ] hanno trattato 20 pazienti con occlusione acuta di by - pass femoro - popliteo comparando il successo tecnico tra 10 casi di trombectomia mediante catetere rotarex ( tempo medio di 64 , 5 minuti ) e la trombolisi mediante ultrasuoni nei rimanenti 10 casi , con un tempo medio di 904 minuti ; il successo tecnico privo di complicanze peri - procedurali stato del 100% per la tr e del 90% per la lisi ad ultrasuoni . 
 nella nostra casistica abbiamo incluso la ricanalizzazione delle occlusioni sub - acute e croniche di stents e stent - graft ; questo tipo di approccio non viene menzionato in letteratura . 
la ricanalizzazione di stents e stent - graft mediante tr permette unimmediata ricanalizzazione del lume vasale ; in questo caso le maglie stesse dello stent evitano la perforazione della parete arteriosa . 
 technical and clinical success and recanalisation with rt proved to be a valuable alternative to surgery in the treatment of acute thrombotic occlusions in medium - sized native arteries and in sub - acute and chronic occlusions of by - pass grafts , stents and stentgrafts , as the technique is able to restore patency with immediate morbidity rates , thanks to its minimal invasiveness . 
the treatment of subacute and chronic thrombosis in mediumsized native arteries , especially those affected by calcified atheromatous plaques , may lead to rupture of the arterial wall due to the possible subintimal passage of the guidewire . 
because arterial rupture may also occur in acute thromboses secondary to traumatic or iatrogenic conditions due to occluding flaps , rt should not be regarded as the treatment of choice in these cases . rt could become a complementary technique to surgery in the near future . 
on the basis of correct preprocedural planning , the vascular radiologist will be able to choose the most suitable technique according to the location , length and type of thrombosis to be treated , thus minimising complication rates . 
 conclusioni la ricanalizzazione mediante tr si rivelata una valida alternativa terapeutica alla chirurgia nel trattamento delle occlusioni trombotiche acute su arteria nativa di medio calibro e nelle occlusioni sub - acute e croniche di by - pass , stent e stentgraft , in quanto realizza il ripristino della perviet con successo tecnico - clinico immediato e bassi tassi di morbilit legati ad un approccio mininvasivo . 
il trattamento delle trombosi sub - acute e croniche su arteria nativa di medio calibro specie su placca ateromasica calcifica pu comportare la rottura della parete arteriosa per il possibile passaggio sottointimale del filo guida . 
mediante un corretto planning pre - procedurale il radiologo vascolare potr scegliere la tecnica interventistica pi adeguata in relazione alla localizzazione , lunghezza e tipo di trombosi da trattare , riducendo al minimo il tasso di complicanze . 
cardarelli , naples , italy 3department of pathology , maresca hospital , naples , italy 4cytopathology service , section of pathology , department of surgery , national cancer centre , fondazione g . 
although computed tomography ( ct ) is still the gold standard for acute intestinal disorders , over the last few years , magnetic resonance imaging ( mri ) has become a useful alternative tool . 
sebbene attualmente la tomogra a computerizzata ( tc ) rimane la metodica di scelta nelle patologie intestinali acute , negli ultimi anni la risonanza magnetica ( rm ) si proposta come valida alternativa . 
il modello documenta la corretta sequenza degli eventi dellischemia mesenterica arteriosa acuta 830 radiol med ( 2011 ) 116 : 829841 keywords experimental model acute bowel ischaemia micro - mri pathology ( imaa ) e dimostra che la rm pu essere proposta per la sua diagnosi precoce . 
this condition is characterised by sudden occlusion of the superior mesenteric artery ( sma ) and / or inferior mesenteric artery ( ima ) , followed impaired vascular supply to the bowel region [ 1 , 2 ]  . 
this goal , however , is still far from achieved , due to the nonspeci c clinical / laboratory ndings and to the absence of speci c and early radiological ndings typical of ami [ 810 ]  . 
magnetic resonance imaging ( mri ) , on the contrary , is less frequently employed in these patients because of its limited availability , high costs and motion artefacts , which have prevented its use in the diagnostic workup of acute intestinal diseases for several years [ 11 ]  . 
 based on these considerations , a novel animal model of acute intestinal ischaemia was developed with the aim of assessing the effectiveness of 7 - t micro - mri in early diagnosis and to document the correct temporal evolution of these lesions . materials and methods animals the study was conducted on 30 sprague dawley rats weighing 400 g on average , which were randomly assigned to two experimental groups ( 1 and 2 )  . 
questa condizione caratterizzata da unimprovvisa occlusione dellarteria mesenterica superiore ( ams ) e / o inferiore ( ami ) seguita da una compromissione dellapporto vascolare nel distretto intestinale [ 1 , 2 ]  . 
nonostante i progressi raggiunti nelle ultime decadi nellambito della chirurgia e della terapia intensiva , il tasso di mortalit correlato a questa patologia rimane elevato , no a s orare l80% [ 36 ]  . 
questo risultato rimane tuttavia lontano per la presentazione clinica e laboratoristica aspeci ca e per lassenza di reperti radiologici speci ci e precoci di ima [ 8 10 ]  . 
la risonanza magnetica ( rm ) , al contrario , meno utilizzata nella gestione del paziente con patologia addominale acuta , per la sua limitata disponibilit , per i costi elevati e per artefatti da movimento che ne hanno precluso per anni limpiego nelliter diagnostico della patologia intestinale acuta [ 11 ]  . 
 le pi recenti apparecchiature di rm presentano incrementata velocit di acquisizione delle immagini , unaumentata capacit di contrasto per i tessuti molli e maggiore diffusione sul territorio [ 12 ]  . 
sulla base di queste considerazioni stato utilizzato un innovativo modello animale di ischemia intestinale acuta con lo scopo di valutare sia lef cacia della micro - rm a 7 t nella diagnosi precoce sia di documentare la corretta cronologia delle lesioni . materiali e metodi animali lo studio stato condotto su 30 ratti sprague dawley del peso medio di 400 g , assegnati casualmente a due gruppi sperimentali ( gruppo 1 , gruppo 2 )  . 
their body temperatures were tracked using rectal probes and kept at 370.5c. surgical model each rat was studied to analyse lesions secondary to acute arterial mesenteric ischaemia ( arterial ami ) after occlusion of the sma . 
in group 1 ( n = 15 ) , the sma was occluded by laparotomy in a single surgical session and the macroscopic lesions were documented photographically ( nikon coolpix s210 , 8.0 megapixel , iso 2000 ) during the following hours . 
 at time zero ( immediately before sma ligation ) , a rst photograph was taken of the exposed abdominal organs ( jejunum to colon ) to conduct a preliminary morphological evaluation of the bowel loops that would later serve as a control image for the same animal . 
in particular , in three animals , observation was limited to the rst 4 h , and at the end of the test , the intestinal packet was removed for histopathological characterisation of the injury . 
in three other animals , observation lasted 6 h , whereas in the remaining nine , observation was extended to 8 h , followed by resection of the intestinal packet for histological analysis . 
in group 2 ( n = 15 ) , arterial ami was induced over two surgical session approximately 3 days apart , and the lesions were evaluated using 7 - t micro - mr imaging . 
a 3 / 0 silk thread was placed around the origin of the vessel to obtain a loop whose ends were inserted into a silicone tube and later tunnelled subcutaneously from the abdominal cavity to the dorsal cervical region , where the thread ends were secured . 
three days after the rst surgical session , the rats were anaesthetised again with the same protocol , and arterial ami was reproduced by pulling the ends of the thread protruding from the silicone tube . 
the interval of approximately 3 days between the two surgical sessions was suf cient to suppress artefacts due to abdominal free air and manipulation of intestinal loops immediately prior to the operation . 
 7 - t micro - mri each rat in group 2 underwent mri of the abdomen using a 7 - t micro - mri scanner ( biospec 70 / 16us , bruker medical systems , ettlingen , germany )  . 
la temperatura corporea stata monitorata tramite sonda rettale e mantenuta a 370 , 5c . modello chirurgico ciascun ratto stato studiato per analizzare le lesioni conseguenti ad ischemia mesenterica arteriosa acuta ( imaa ) dopo occlusione della ams . 
nei ratti del gruppo 1 ( n = 15 ) lams stata occlusa per via laparotomica in ununica seduta operatoria e nelle successive ore le lesioni macroscopiche sono state documentate con immagini fotogra che ottenute mediante apparecchio nikon coolpix s210 , 8.0 megapixels , iso 2000 . 
al tempo zero ( subito prima della legatura dellams ) , stata acquisita per ciascun ratto una prima immagine fotogra ca degli organi esposti allesterno della cavit addominale ( tratto compreso tra digiuno e colon ) con la nalit di effettuare una valutazione preliminare morfologica delle anse intestinali , da utilizzare poi come immagine di controllo per lo stesso animale . 
in altri 3 animali losservazione stata protratta sino a 6 ore , mentre nei restanti 9 animali losservazione stata estesa no ad 8 ore , con successiva resezione del pacchetto intestinale per lanalisi istologica . 
nel gruppo 2 ( n = 15 ) limaa stata indotta facendo ricorso a due sedute operatorie ad una distanza di tempo di circa 3 giorni luna dallaltra e le lesioni sono state valutate con limaging micro - rm a 7 t . 
un lo di seta 3 / 0 stato posizionato attorno allorigine del vaso in modo da confezionare un cappio le cui estremit sono state inserite in un tubicino di silicone , successivamente tunnelizzato dalla cavit addominale alla regione cervicale dorsale per via sottocutanea . 
dopo 3 giorni dalla prima seduta operatoria , i ratti sono stati nuovamente anestetizzati , seguendo lo stesso protocollo anestesiologico del precedente intervento , e limaa stata riprodotta ponendo in trazione le estremit del lo che fuoriuscivano dal tubicino di sili832 radiol med ( 2011 ) 116 : 829841 characterise any signs of intestinal ischaemia and compare ndings with histological evaluation . 
immediately before inducing infarction , the animals underwent a 7 - t micromri scan for preliminary morphological and functional evaluation of the intestinal loops , which would later serve as a control image for the animal . 
in particular , after a scout view in the three orthogonal planes ( tr 200.0 ms , te 5.0 ms ) , images of the entire abdomen were acquired using an axial t2 - weighted rapid acquisition relation enhancement ( rare ) sequence ( tr 6 , 030.3 ms , te 36 ms 180 ip angle ; 52 slices ; slice thickness 1 mm , interslice distance 1 mm , eld of view 6 cm ; acquisition matrix 256256 )  . 
the same sequence was then used to evaluate the damage in the immediate postischaemic period , 5 min after ligation , and at intervals of 30 min until the end of the observation period . 
 under baseline conditions and 5 min after the sma occlusion , an additional 2d fast low - angle shot time of ight ( flash - tof ) sequence ( tr 12 ms , te 3.5 ms ; 90 ip angle ; 180 slices ; slice thickness 0.4 mm , interslice distance 0.25 mm , eld of view 6 cm ; acquisition matrix 256256 ) was obtained rst to visualise the vascular anatomy then to con rm complete vessel occlusion after a maximum intensity projection ( mip ) reconstruction . 
for group 2 ( n = 15 ) , the scans were repeated up to 4 h after vascular occlusion in three animals , after 6 h in other three and after 8 h in the remaining nine . 
special care was taken in evaluating the mean diameter of the lumen of bowel loops , wall thickness , air uid levels in the affected loops and free uid in the abdomen . at the end of the session , the intestinal packet was removed for histopathological characterisation of the injury and the animal was euthanised as described above ( 0.5 ml tanex administered via the lung )  . histological evaluation pathological characterisation of damage was conducted on all animals in both groups at prede ned time points ( 4 , 6 and 8 h )  . 
for all specimens , the intestinal tract between jejunum and ascending colon was resected before euthanasia and , after being xed in a solution of 10% formalin - acetate for at least 5 days , it was processed for histopathological evaluation . 
conventionally , specimens taken are divided into four portions , each including : ( 1 ) jejunum and the rst 10 cm of the ileum ( rst segment 1s ) ; ( 2 ) central ileal ( second segment 2s ) ; ( 3 ) last 10 cm of ileum ( third segment 3s ) ; ( 4 ) caecum and ascending colon ( fourth segment 4s )  . 
il rispetto di un intervallo di tempo di circa 3 giorni tra le due sedute operatorie risultato suf ciente per abolire gli artefatti dovuti alla presenza di aria libera in addome ed alla manipolazione delle anse nellimmediato periodo postoperatorio . 
 micro - rm a 7 t ciascun ratto appartenente al gruppo2 stato sottoposto a rm delladdome utilizzando un apparecchio micro rm a 7 t ( biospec 70 / 16us , bruker medical systems , ettlingen , germania )  . 
tale metodica veniva utilizzata al ne di confermare lassenza di usso attraverso lams , di identi care e caratterizzare i segni di ischemia intestinale e di comparare questi reperti con lanalisi istologica . 
subito prima di indurre linfarto , gli animali sono stati sottoposti ad una prima scansione micro - rm a 7 t con la nalit di effettuare una valutazione preliminare morfologica e funzionale delle anse intestinali da utilizzare poi come immagine di controllo per lo stesso animale . 
in particolare , dopo una scout view nei tre piani ortogonali ( tempo di ripetizione [ tr ] = 200 , 0 ms , tempo di eco [ te ] = 5 , 0 ms ) , le immagini dellintero addome venivano acquisite mediante una sequenza rapid acquisition with relaxation enhancement ( rare ) t2 in sezione assiale ( tr = 6030 , 3 ms , te = 36 ms ; ip angle 180 ; 52 slices ; slice thickness 1 mm , interslice distance 1 mm , eld of view 6 cm ; acquisition matrix 256256 )  . 
la stessa sequenza veniva poi utilizzata per la valutazione del danno nellimmediato post - ischemico , 5 minuti dopo la legatura , e ad intervalli di 30 minuti no alla ne del periodo di studio . 
in condizioni basali e a 5 minuti dallocclusione dellams una sequenza aggiuntiva fast low angle shot ( flash ) - 2d - time - ofight ( tof ) ( tr = 12 ms , te = 3 , 5 ms ; ip angle 90 ; 180 slices ; slice thickness 0 , 4 mm , interslice distance 0 , 25 mm , eld of view 6 cm ; acquisition matrix 256256 ) veniva effettuata al ne di visualizzare lanatomia vascolare prima e di confermare successivamente locclusione completa del vaso , dopo ricostruzione in maximum intensity projection ( mip )  . 
 anche per questo secondo gruppo sperimentale ( n = 15 ) , le scansioni venivano ripetute sino a 4 ore dalleffettiva occlusione vascolare in 3 animali , 6 ore in altri 3 ratti , ed 8 ore nei restanti 9 animali . 
particolare attenzione veniva posta nella valutazione del diametro medio del lume delle anse intestinali , dello spessore parietale , nellidenti cazione di livelli idro - aerei nelle anse colpite e di versamento libero in addome . al termine della seduta , il pacchetto intestinale veniva rimosso per la caratterizzazione istopatologica del danno e lanimale sottoposto ad eutanasia come precedentemente indicato ( tanex 0 , 5 ml per via intrapolmonare )  . radiol med ( 2011 ) 116 : 829841 from each paraf n block and stained with hematoxilin and eoxin ( h&e )  . 
histopathological evaluation was conducted using a zeiss axioskop 2 light microscope ( zeiss ) equipped with a high - resolution digital microcamera ( c4742 - 95 , hamamatsu photonics , milan , italy )  . 
the ileal loops underwent the same pathological evolution , but the spasm was observed about 1 h after arterial occlusion , with transition to de nite analisi istologica la caratterizzazione anatomopatologica del danno stata effettuata su tutti gli animali del gruppo 1 e del gruppo 2 ai time - point stabiliti ( 4 , 6 e 8 ore )  . 
per tutti i campioni il tratto di intestino compreso tra digiuno e colon ascendente stato resecato prima di procedere alleutanasia e , dopo ssazione per almeno 5 giorni in una soluzione di acetato di formalina al 10% , stato processato per lanalisi istopatologica . 
convenzionalmente , i campioni prelevati sono stati suddivisi in quattro porzioni , ciascuna includendo rispettivamente : ( 1 ) digiuno e i primi 10 cm di ileo ( primo segmento 1s ) ; ( 2 ) segmento centrale di ileo ( secondo segmento 2s ) ; ( 3 ) ultimi 10 cm di ileo ( terzo segmento 3s ) ; ( 4 ) intestino cieco e colon ascendente ( quarto segmento 4s )  . 
per ciascuna di queste regioni di intestino sono state realizzate ed incluse in paraf na delle sezioni trasversali di 3 mm di spessore , a distanza di 10 mm luna dallaltra . 
grazie allausilio di un microtomo , fettine dello spessore di 3 micron sono state effettuate su ciascun blocco di paraf na e successivamente sottoposte a colorazione con ematossilina eosina ( e&e )  . 
spasmo delle anse digiunali a 30 minuti ( freccia in b ) che si protraeva no a 3 ore , per transitare da questo momento in poi in ipotono ( c )  . 
in b aspetto dopo 8 ore di osservazione : mesentere pallido con assottigliamento dei vasi mesenterici ( che esordiva gi a 5 minuti dalla legatura ) ; viraggio cromatico verso il rosso vinaccia della parete intestinale digiuno - ileale . ; ileo ri esso ipotonico diffuso a tutto il tratto di intestino mesenteriale . hypotonia after about 4 h . 
immediately before inducing radiol med ( 2011 ) 116 : 829841 skope 2 light ( zeiss ) , dotato di fotomicrocamera digitale ad alta risoluzione ( c4742 - 95 , hamamatsu photonics , milano , italia )  . 
le anse dellileo hanno presentato la stessa evoluzione patologica , ma lo spasmo stato osservato a circa 1 ora dallocclusione arteriosa con il passaggio a circa 4 ore in ipotono franco . 
subito prima dellinduzione di imaa , 3 giorni dopo lintervento chirurgico in laparotomia , non stata evidenziata la presenza di gas libero nella cavit addominale n segni di irritazione viscerale o mesenterica . 
3a , b maximum intensity projection reconstruction of the vascular tree with identi cation of the superior mesenteric vein ( smv ) and artery ( sma ) a before occlusion ( 2d - fast low - angle shot time of ight )  . 
3a , b ricostruzione mip dellalbero vascolare con identi cazione della vena mesenterica superiore ( smv ) e dellarteria mesenterica superiore ( sma ) prima dellocclusione ( flash 2d - tof ) ( a )  . 
a baseline t2 ; b 2 h after ligation : dilatation of some loops with thinned bowel walls ( region of interest ) ( re ex hypotonic ileus )  . 
in a aspetto basale in t2 ; in b aspetto a 2 ore dalla legatura , dove appare una dilatazione di alcune anse intestinali con assottigliamento della parete delle stesse ( ileo ri esso ipotonico )  . 
alla dilatazione hanno fatto seguito i livelli idro - arei , ben evidenti a 4 ore dalla legatura dellams e uniperintensit della parete delle anse coinvolte ( c )  . 
b increasing free intraperitoneal uid : a small amount of free uid between the loops appeared only 30 min after superior mesenteric vein ( sma ) ligation ( arrows )  . 
il liquido andava crescendo , divenendo meglio apprezzabile dopo 4 ore ( c ) e diffuso in tutto laddome dopo 8 ore ( d )  . arterial ami , 3 days after laparotomic surgery , no free gas within the abdominal cavity nor signs of visceral or mesentery irritation were found . 
 wall damage was more eivdent in the last ileal segment ( 3s ) , where the initial formation of a grunhagen subepithelial space was reported ( chiu 2 )  . the three specimens removed 6 h after sma ligation showed a clear re ex hypotonic ileus , with signi cant dilatation of some loops . 
the mucosa showed discontinuous alterations , with apparently healthy areas alternating with areas undergoing necrobiotic phenomena and regenerative compensatory hyperplasia at the bases of the glandular crypts ( skip lesions )  . 
these ndings were almost identical in the specimens obtained from the two test groups and at different time points . discussion imaging ndings of ami are nonspeci c and heterogeneous , as are aetiology and clinical and laboratory signs [ 14 ]  . 
the diagnostic hypothesis is not considered at all or is considered late for two reasons : first , ami in humans is not always either complete or at the emergence of the vascular axis ; therefore , the pain appears after different time periods in every patient . 
second , every patient presents to the emergency department at different time lines , depending on accessibility of the facility . the radiological approach to diagnosing ami relies on contrast - enhanced ct , which detects morphological changes suggestive of the condition . 
the advantages of mri over ct include higher contrast resolution in soft tissues and no risks from ionising radiation or of nephropathy induced by iodinated infartuali era evidenziata dal viraggio cromatico della sierosa intestinale , variabile dal rosso vinaccia al nero antracite . 
la stessa mostrava alterazioni discontinue , alternando zone di mucosa apparentemente orida con zone in preda a fenomeni necrobiotici ed iperplasia compensatoria rigenerativa della base delle criptae ghiandolari ( skip lesions )  . 
nella regione 3s , in ne , la mucosa appariva francamente sempli cata , oltre a presentare reperti sovrapponibili a quelli gi descritti per la regione 2s ( grado chiu 4 - 5 )  . 
i reperti erano pressoch sovrapponili fra i campioni appartenenti ai due gruppi sperimentali e ai diversi time point . discussione i reperti allimaging di ima sono aspeci ci ed eterogenei , cos come leziologia , i segni clinici e laboratoristici [ 14 ]  . 
il primo perch lima nelluomo non sempre n completa , n allemergenza dellasse vascolare , e per questo la sintomatologia dolorosa si rende manifesta con un intervallo di tempo diverso in ogni paziente . 
 il secondo perch ogni paziente si reca al pronto soccorso dopo che trascorso un intervallo di tempo diverso , condizionato dalla accessibilit della struttura . lapproccio radiologico attuale per la diagnosi di ima basato sul riscontro tc dopo somministrazione di mezzo di contrasto ( mdc ) endovena ( ev ) delle modi cazioni morfologiche suggestive di tale patologia . 
i vantaggi della rm rispetto alla tc includono una maggiore risoluzione di contrasto per i tessuti molli e lassenza di rischio da radiazioni ionizzanti o di nefropatie indotte da mezzi di contrasto iodati . 
lindagine tc mantiene una risoluzione spaziale superiore , ma questa differenza si va riducendo grazie allintroduzione di nuove sequenze per la rm addominale con voxel dellordine di 2 mm per apparecchiature da 1 , 5 t e di 1 mm per apparecchiature da 3 t [ 12 , 15 ]  . 
the ct scan still has a higher spatial resolution , but the difference is becoming less evident with the introduction of new abdominal mri sequences with voxels in the order of 2 mm for 1.5 - t scanners and of 1 mm for 3 - t scanners [ 12 , 15 ]  . 
 our experience is the rst in which 7 - t micro - mri sequences in rats with arterial ami due to sma occlusion were evaluated to establish the typical ndings of the condition and their evolution . 
the micro - mri study method and other diagnostic imaging techniques for small animals are emerging as key techniques in in vivo molecular imaging , given their ability to identify events with suf cient sensitivity , speci city and temporal and spatial resolution . 
in our study , we observed that wall thickening was absent from arterial ami and that the small - bowel loops appeared , on the contrary , extremely thin , resembling sheets of paper , an appearance that has an incidence of 5% in the literature [ 18 ]  . 
its low incidence rate , as well as the reported wide variability of wall thickening in literature reports , are conditioned by the different number of cases with arterial or venous infarctions in their mixed series . 
wall thickening is caused by intramural oedema and / or haemorrhage and / or by superinfection of the ischaemic bowel wall events that are not found in arterial ami [ 25 , 26 ]  . 
 seven - tesla micro - mr imaging never detected oedema of the mesentery , even though neither thinning vascular structures , nor gradual exsanguination of the mesentery could be observed . 
the authors argue that the ct nding described in the literature only applies to infarctions of venous orig the third most frequent nding reported in the literature ( 65% of cases ) is dilatation of bowel loops with air uid levels [ 1924 ]  . 
the la nostra esperienza la prima nella quale sequenze micro - rm a 7 t in ratti con imaa da occlusione dellams sono state analizzate per stabilire quali siano i reperti distintivi della patologia e la loro cronologia . 
la modalit di studio micro - rm , insieme ad altre metodiche di diagnostica per immagini per piccoli animali , stanno emergendo come tecniche chiave nellimaging molecolare in vivo grazie alla possibilit di individuare eventi con suf ciente sensibilit , speci cit , risoluzione temporale e spaziale . 
nel nostro studio abbiamo osservato che lispessimento parietale assente nellimaa e le anse del piccolo intestino si presentano viceversa estremamente assottigliate con aspetto a foglio di carta , che in letteratura descritto con incidenza pari a 5% [ 18 ]  . 
la sua bassa percentuale di incidenza , cos come lampia variabilit riportata per lispessimento parietale , nei contributi presenti in letteratura , sono condizionate dal diverso numero di casi di pazienti con infarto arterioso o venoso nelle loro casistiche miste . 
lispessimento parietale dovuto alledema intramurale e / o allemorragia e / o alla superinfezione della parete intestinale ischemica , che sono assenti in caso di imaa [ 25 , 26 ]  . 
alla micro - rm a 7 t non veniva mai riscontrato edema del mesentere , pur non essendo stato possibile evidenziare lassottigliamento delle strutture vascolari n il progressivo dissanguamento del mesentere . 
 840 radiol med ( 2011 ) 116 : 829841 ileal loops followed the same pattern , but the spasm was observed at around 1 h and hypotonia at 4 h . 
some authors report ascitis in 4988% of cases [ 22 , 23 ] ; others view it as a late nding emerging only after 12 h [ 28 ]  . 
in our experience , using 7 - t micro - mri , we observed a thin amount of uid between the bowel loops after just 30 min , which grew gradually and became more evident after 4 h , extending to the entire abdomen after 8 h . seven - tesla micro - mri without intravenously administered contrast medium revealed hyperintensity of the loops affected by the ischaemic insult after 3 h . 
these observations are in line with reports by other authors [ 29 ] , who believe that unenhanced t2 - weighted mri sequences , conducted ex vivo , are useful in identifying wall hyperintensity of acute intestinal ischaemia , differentiating it from subacute forms . 
our experience con rms the late presentation of this nding , because we observed it in only one rat after 8 h . finally , in arterial ami due to sma occlusion , the earliest ndings are blanching of the mesentery and re ex spastic ileus , which appear at 5 and 15 min , respectively , from onset of the ischaemic insult . 
mri is the most sensitive modality for early diagnosis of arterial ami due to sma occlusion , as it is capable of detecting the ischaemic loop after only 4 h . con ict of interest none lo spasmo una reazione precoce e transitoria allinsulto ischemico delle pliche semilunari che , in anse sottili a foglio di carta , determinano il collasso del lume . 
la micro - rm a 7 t consentiva di documentare lileo ri esso ipotonico , la dilatazione del lume e , dopo 4 ore , i crescenti livelli idro - aerei . 
nella nostra esperienza abbiamo osservato alla micro - rm a 7 t un reperto di sottile falda di liquido tra le anse dopo appena 30 minuti , che andava crescendo divenendo meglio apprezzabile dopo 4 ore e diffuso in tutto laddome dopo 8 ore . 
queste osservazioni sono in accordo con la segnalazione di altri autori [ 29 ] secondo i quali le sequenze rm t2 - pesate senza mdc ev , condotte ex vivo , sono utili per identi care liperintensit parietale dellischemia intestinale acuta distinguendola dalle forme subacute . 
la nostra esperienza conferma la presentazione tardiva di tale reperto essendo stata riscontrata in un solo ratto ad 8 ore . in conclusione , nellimaa da occlusione dellams i reperti pi precoci sono limpallidimento del mesentere e lileo ri esso spastico che esordiscono rispettivamente a 5 e 15 minuti dallinstaurarsi dellinsulto ischemico . 
scaglione3 1dipartimento di diagnostica per immagini , universit degli studi di foggia , azienda ospedaliero - universitaria ospedali riuniti , viale luigi pinto 1 , 71100 foggia , italy 2u.o. 
radiologia muscoloscheletrica , irccs ospedale casa sollievo della sofferenza , viale cappuccini 1 , 71013 san giovanni rotondo ( fg ) , italy 3dipartimento di diagnostica per immagini , presidio ospedaliero pineta grande , via domiziana k30 , 81030 castel volturno ( ce ) , italy 4sezione dipartimentale di medicina legale , universit degli sudi di foggia , ospedale colonnello davanzo , via degli aviatori 1 , 71100 foggia , italy correspondence to : g . 
the aim of this study was to evaluate the impact of conventional radiology on the assessment of causes of death of human beings after a building collapse and to establish whether the radiographic approach is useful and justi able . 
scopo del lavoro stato quello di valutare limpatto della radiologia tradizionale nellaccertamento delle cause del decesso di persone a seguito di un crollo di un edi cio abitativo e stabilire in che termini lapproccio radiogra co stato utile e giusti cabile . 
 nei 3 pazienti del gruppo ii la morte sopraggiunta a causa di as ssia meccanica associata a lesioni cerebrali in tutti i casi , a lesioni addominali in 2 casi e a lesioni cardiache in un caso . 
in questo gruppo di pazienti , i reperti radiogra ci hanno consentito di apprezzare segni di frattura a carico della teca cranica in un caso , aria nelle camere cardiache in un altro e lesione del diaframma in un terzo . 
nei 2 pazienti del gruppo iii sono state evidenziate al tavolo autoptico lesioni incompatibili con la vita a livello cardiaco e addominale in entrambi i casi ed 970 radiol med ( 2011 ) 116 : 969981 victims . 
conventional radiography should be considered inadequate , especially if the potential of the modern software tools available on current computed tomography and magnetic resonance images is considered . keywords plain x - ray radiographs building collapse autopsy ndings noninvasive autopsy forensic pathology postmortem imaging encefalico in uno . 
lapproccio radiogra co tradizionale per laccertamento delle morti di tali pazienti da considerarsi insuf ciente , specie alla luce delle potenzialit dei moderni software collegati alle attuali apparecchiature di tomogra a computerizzata ( tc ) e risonanza magnetica ( rm )  . 
 parole chiave radiologia tradizionale crollo edi cio reperti autoptici autopsia non - invasiva patologia forense post - mortem imaging introduction introduzione collapse of a residential building is an uncommon event resulting from numerous causes ( earthquakes , hurricanes , oods , snow slides , explosions , terrorist attacks , structural failures , design errors , inadequate materials or soil ) [ 1 , 2 ]  . 
buildings such as this , which in part extend below street level and are locally known as the caves , were originally meant for warehousing food and later used as dwellings despite the unsatisfactory hygienic , lighting and ventilation conditions . 
its potential improved markedly as a result of the technological advances of the past 20 years [ ultrasound ( us ) , computed tomography ( ct ) and magnetic resonance ( mr ) technology ] , whereas the objectives remained essentially unchanged : identifying the victim , recording and ascertaining the cause il crollo di un edi cio abitativo un evento raro e riconosce molteplici cause ( terremoti , uragani , allagamenti , slavine , esplosioni , attentati terroristici , insuf cienze strutturali , errori progettuali , inadeguatezza dei materiali o del suolo ) [ 1 , 2 ]  . 
abitazioni come questa , che si sviluppano in parte al di sotto del livello stradale e per questo chiamate cave , erano , in origine , adibite al semplice deposito per la conservazione di alimenti e poi , successivamente , destinate a scopo abitativo nonostante le precarie condizioni igieniche , di esposizione alla luce e alla ventilazione . 
le sue possibilit sono migliorate notevolmente in relazione radiol med ( 2011 ) 116 : 969981 allevoluzione tecnologica che la radiologia ha subito nellultimo ventennio ( tecniche ecogra che , di tomogra a computerizzata e di risonanza magnetica ) , pur restando gli obiettivi principali essenzialmente gli stessi : identi cazione , documentazione e accertamento della causa di morte dei soggetti , ricostruzione dellevento , in formazione e ricerca [ 4 ]  . 
the purpose of this paper was to illustrate the type and frequency of injuries compatible with life , to assess the impact of conventional radiology in ascertaining the cause of death in these victims and to evaluate whether radiography was useful and justi able as a procedure for forensic ascertainment of the causes of death . materials and methods at the time of the catastrophe , the cave was occupied by 14 persons . 
le indagini radiogra che sono state effettuate con apparecchio digitale telecomandato ( opera t90 e , general medical merate spa , seriate , bergamo , italia ) su tutti i distretti corporei e in almeno due proiezioni . 
laddove le lesioni scheletriche erano gi deducibili dallesame obiettivo ( presenza di escoriazioni , ecchimosi o deformit corporee ) e / o nelle proiezioni radiogra che ortogonali si provveduto ad integrare le stesse con ulteriori dettagli , se necessario , per laccertamento della causa di morte . 
radiographs were obtained of all body regions and in at least two projections using remotecontrolled digital equipment ( opera t90 and general medical merate spa , seriate , bg , italy )  . 
when skeletal lesions were noted on physical examination ( presence of excoriations , bruises or physical deformities ) and / or on the radiographic orthogonal views , more detailed information was sought whenever needed for identifying the cause of death . 
the niss is an evolution of the iss and provides a value deriving from the sum of the squares of the three highest scores of the lesions detected , regardless of the anatomical region involved . 
these values were completed classi cati e codi cati utilizzando il punteggio in riferimento allabbreviated injury scale ( ais ) ed il valore del suo derivato new injury severity score ( niss ) [ 68 ]  . 
il niss rappresenta levoluzione delliss e fornisce un numero che deriva della somma dei quadrati dei tre punteggi pi elevati delle lesioni rilevate , indipendentemente dalla regione anatomica di interesse . 
autopsy ndings were compared with radiographic ndings . results to moderate twentyve percent of victims sustained brain , thoracic and abdominal lesions not compatible with life ( niss = 76 ) , whereas 37.5% suffered mild lesions ( niss < 25 )  . 
the chest was the most frequently involved anatomical region ( 32% ) , followed by the head and neck ( 28% ) , the skin ( 22% ) and the abdomen ( 21% )  . 
lesions of the appendicular skeleton were detected in 7% of cases , with a prevalence of femoral - shaft fractures ( 62.5% ) followed by pelvic , humeral and radial lesions . 
il torace stata la regione implicata nel danno ( 32% ) , corporea maggiormente seguita dai distretti testa - collo ( 28% ) , dalla cute ( 22% ) e dalladdome ( 21% )  . 
le lesioni allo scheletro appendicolare sono state rinvenute nel 7% dei casi , con prevalenza delle lesioni della dia si femorale ( 62 , 5% ) seguite dalle lesioni del bacino , dellomero e del radio . 
 i reperti radiogra ci hanno mostrato fratture costali , pneu976 radiol med ( 2011 ) 116 : 969981 classi ed according to the corresponding niss values are reported in table 1 . 
group 3 , which consisted of two cadavers , had the most severe lesions , with an niss score of 76 , a value associated with lesions not compatible with survival , such as myocardial infarction and laceration associated with large multiorgan lacerations of the intraand retroperitoneal organs and massive haemoperitoneuthese lesions were associated with brain lesions , diffuse intra - alveolar haemorrhage and a left hemidiaphragmatic lesion in one case . 
assessing the number , type and severity of lesions observed on the cadavers , revealed by direct necroscopic examination and / or radiography and toxicological analysis , is instrumental for establishing the causes of the event , preventing and reducing risks deriving from such accidents and understanding the death of the victims . 
il gruppo ii , composto da 3 cadaveri , ha mostrato lesioni di grado moderato - grave ( niss compreso tra 26 e 50 ) variamente distribuite nel distretto cranio - facciale , al torace e alladdome . 
in questo gruppo di pazienti la causa di morte stata attribuita ad as ssia meccanica associata a lesioni cerebrali in tutti i casi e a lacerazione dei visceri addominali con emoperitoneo in due casi , di cui uno associato a lesione dellemidiaframma sinistro con erniazione dello stomaco e della essura splenica del colon e laltro associato a lesione del miocardio ventricolare sinistro . 
il gruppo iii , composto da 2 cadaveri , ha presentato il pi alto score di niss ( 76 ) cio un valore relativo a lesioni incompatibili con la sopravvivenza , quali infarto e lacerazioni del miocardio associati a vaste lacerazioni multi - organo dei visceri intrae retro - peritoneali e cospicuo emoperitoneo . 
 discussione i meccanismi , la distribuzione e la tipologia delle lesioni traumatiche a seguito di un crollo di un edi cio abitativo sono molteplici e complessi e vanno principalmente , ma non esclusivamente , posti in relazione alla causa del crollo [ 915 ]  . 
la valutazione del numero , della tipologia e della gravit delle lesioni riscontrate sui cadaveri , evidenziate attraverso lesame necroscopico diretto e / o le indagini radiogra che e tossicologiche , rappresentano un elemento determinante per la comprensione delle cause dellevento , per la prevenzione e la riduzione dei rischi derivanti da tali incidenti ed , in ne , per la comprensione della morte delle vittime . 
infatti noto che a seguito di un crollo di un edi cio abitativo , indipendentemente dalle cause che lo determinano , le caratteristiche tecniche delledi cio e la modalit del danno siano intimamente legati al pattern del danno radiol med ( 2011 ) 116 : 969981 fig . 
b il reperto autoptico mostra la presenza di due ampie lacerazioni sulla super cie esterna del ventricolo cardiaco sinistro ( frecce )  . known that in the event of a building collapse , regardless of its causes , the technical characteristics of the building and the circumstances of the damage are more closely related to the pattern of injury and to the mechanism of the victims deaths rather than to a general increase in the risk of injury [ 810 ]  . 
 nel caso in oggetto , lesplosione di una bombola di gas ha rappresentato levento scatenante , ma non causa esclusiva della tragedia : i rilievi successivi al disastro hanno , infatti , dimostrato notevoli carenze strutturali ed irrego978 radiol med ( 2011 ) 116 : 969981 explosion of a gas cylinder triggered the event , it was not the only cause of the tragedy : in fact , surveys conducted after the catastrophe revealed signi cant structural inadequacies and irregularities in the planning , construction and maintenance of the building [ 4 ]  . 
the frequency and type of the lesions observed were closely related to the factors and cofactors that caused the event and are therefore comparable with events of the same type . 
lesion distribution revealed by autopsy indicated that the chest is the most frequently affected anatomical region and that asphyxia , probably due to dust inhalation following masonry collapse , was the main cause of death among the victims . 
 the paralytic ileus , observed at abdominal autopsy in all cases , should not be considered signi cant and related to the lesions detected at autopsy , as it is more reasonably connected with the death of the victims rather than with the signs of peritoneal irritation secondary to haemoperitoneu conversely , conventional radiology proved to be useful in supporting the evidence of some concurrent causes of death , such as hemidiaphragmatic lesions with visceral herniation , systemic bone disintegration and multiple rib fractures . 
in light of the results obtained from the radiographic assessment of the cause of death of these victims of a building collapse , we can state that radiography is no longer justi ed in that , although it provides complete and exhaustive information about skeletal lesions , it can only reveal the concurrent causes of death . 
 therefore , the use of modern software tools associated with current multislice ct and mr virtopsy techniques would appear more useful , as they provide an important contribution to identifying the victims and establishing causes , force vectors and concurrent causes of death [ 1618 ]  . 
 although it cannot supplant conventional autopsy , and taking into account the panoramic capabilities and shorter examination times of ct , the systematic use of these technologies is helpful in forensic practice , as it prevents or at least markedly reduces the number and duration of conventional autopsies while not altering the state of the cadavers [ 1921 ]  . 
consequently , radiologists with experience in postmortem imaging are needed to guide and support forensic specialists in de nitely establishing causes of death without necessarily having to perform conventional autopsy . 
la frequenza e la tipologia delle lesioni riscontrate risultano intimamente legate a fattori e cofattori che hanno determinato levento stesso ; pertanto , esse sono confrontabili e comparabili con evenienze delle medesima tipologia . 
la distribuzione delle lesioni autoptiche dimostra come il torace sia la regione anatomica maggiormente colpita e come las ssia , legata probabilmente allinalazione delle polveri a seguito del crollo delle opere murarie , sia stata la causa principale della morte delle vittime . 
lileo paralitico , evidenziato allesame diretto delladdome in tutti i casi , non da considerarsi signi cativo e correlabile alle lesioni autoptiche in quanto pi ragionevolmente connesso con lexitus delle vittime piuttosto che ai segni di irritazione peritoneale secondari allemoperitoneo . 
di converso , la radiologia tradizionale si dimostrata utile a supporto dellevidenza di alcune concause di morte quale la lesione dellemidiaframma con radiol med ( 2011 ) 116 : 969981 fig . 
6a skull radiograph : multiple displaced fractures with vertical direction on the front - parietal region of the skull ( arrow ) extending to the skull base and mandible ( arrow )  . 
multiple fratture scomposte a decorso verticale evidenti a livello del tavolato cranico fronto - parietale ( freccia ) con interessamento della base cranica e della mandibola ( freccia )  . 
alla luce dei risultati ottenuti dalla valutazione radiologica tradizionale nellaccertamento delle cause di morte nel decesso di persone coinvolte nel crollo di un edi cio abitativo , possiamo affermare che tale indagine risulta non pi giusti cabile in quanto , mentre per le lesioni scheletriche osteoarticolari fornisce informazioni complete ed esaustive , nellaccertamento dellevento fatale lindagine radiologica tradizionale pu svelare solo le concause di morte delle vittime . 
pertanto auspicabile che si utilizzino i moderni software legati allattuale tecnologia di tomogra a computerizzata ( tc ) multistrato e risonanza magnetica ( rm ) applicata alla virtopsy che offrono attualmente un contributo essenziale nellidenti cazione dei soggetti , nella de nizione degli agenti causali , dei vettori di forza e delle concause di morte delle vittime [ 1618 ]  . 
pur non sostituendosi allautopsia tradizionale e con le opportune differenze in termini di panoramicit e durata di esecuzione delle indagini a favore della tc , lutilizzo sistematico di tali apparecchiature risulta ef cace nella pratica clinica medico - legale perch consente di evitare o ridurre notevolmente il numero ed i tempi di esecuzione delle necroscopie tradizionali , rispettando totalmente lo stato di rilevamento dei corpi delle vittime [ 1921 ]  . 
corrispondentemente , divengono indispensabili radiologi esperti nellimaging post - mortem , al ne di potere indirizzare e sostenere i periti nellaccertamento de nitivo delle cause di decesso , senza dover necessariamente ricorrere al riscontro diretto . 
therefore , conventional radiography in these circumstances must be considered obsolete and inadequate , particularly in light of the potential of modern software tools provided with ct and mr technology . la radiologia tradizionale offre un contributo limitato nellaccertamento delle lesioni letali secondarie al crollo di edi ci abitativi . 
pertanto , lapproccio radiogra co tradizionale per laccertamento delle morti di tali pazienti da considerarsi obsoleto ed insuf ciente , specie alla luce delle potenzialit dei moderni software collegati alle attuali apparecchiature tc e rm . 
cova unit clinico operativa di radiologia , universit degli studi di trieste , ospedale di cattinara , strada di fiume 447 , 34149 trieste , italy correspondence to : f . 
pozzi - mucelli , tel . : + 39 - 040 - 3994372 , fax : + 39 - 040 - 3994500 , e - mail : fabio.pozzimucelli@alice.it received : 26 may 2010 / accepted : 30 august 2010 / published online : 21 april 2011 springer - verlag 2011 abstract purpose . 
different embolisation agents were used : coils ( 17 cases ) , embolisation particles ( 14 cases ) , glue ( one case ) , coated stent ( two cases ) and mechanical occlusion devices ( two cases )  . 
technical success was achieved in 26 / 27 patients ( 96.3% ) ; in one case , embolisation of a polar artery arising from the aneurysmal sac was not possible . 
le indicazioni sono state : macroematuria sintomatica in 9 pazienti ( 33 , 3% ) ( tumorale in 7 casi e iatrogena in 2 ) , tumore renale sintomatico inoperabile in 5 pazienti ( 18 , 5% ) , voluminoso ematoma sottocapsulare o perirenale in 3 casi ( 11 , 1% ) ed aneurisma dellarteria renale principale in 2 casi ( 7 , 4% )  . 
in 8 pazienti ( 29 , 6% ) candidati a posizionamento di endoprotesi aortica addominale ( evar ) si proceduto alla preventiva embolizzazione del ramo renale polare originantesi dalla sacca aneurismatica o dal colletto sottorenale aortico per prevenire la possibile rivascolarizzazione della sacca . 
sono stati utilizzati diversi agenti embolizzanti : spirali ( 17 casi ) , particelle embolizzanti ( 14 casi ) , colla ( 1 caso ) , stent ricoperto ( 2 casi ) , occlusore meccanico ( 2 casi )  . 
one possible new indication is the prophylactic exclusion of the polar artery arising from the neck or the sac of an abdominal aortic aneurysm in patients who are candidates for evar . 
this procedure requires the availability of various materials for performing embolisation and experience in their use . keywords renal arteries kidney angiography embolisation invasiva nel trattamento della macroematuria sintomatica neoplastica / iatrogena e nella palliazione di tumori renali inoperabili . 
questa procedura richiede la disponibilit di diversi materiali per effettuare lembolizzazione ed esperienza nel loro utilizzo . parole chiave arterie renali rene angiogra a embolizzazione introduction introduzione one of the areas in which interventional radiology has demonstrated its ability to offer enormous therapeutic potential with minimal invasiveness regards arterial embolisation procedures . 
although there are many indications for endovascular embolisation or exclusion of the renal arteries and their distal and / or polar branches [ renal artery embolisation ( rae ) ] , these procedures are mainly done for therapeutic or palliative purposes in patients with renal trauma or tumour . 
nonetheless , in recent years , new indications have advanced , including preoperative embolisation of the renal polar arteries in patients who are candidates for endoprosthesis deployment to exclude abdominal aortic aneurysms ( aaa )  . 
different embolisation agents may be used to achieve the desired therapeutic result . the aim of this study was to review our experience with this procedure over the past 10 years to identify the current and emerging indications , the materials used and the results obtained . materials and methods a retrospective review of the interventional radiology procedures performed at our centre between june 1999 and july 2009 revealed 27 patients ( 19 men and eight women ; mean age 74 years ; age range 3793 years ) who had undergone rae . 
tali interventi vengono eseguiti in diversi distretti arteriosi ; a livello addominale tra le varie strutture arteriose che possono essere sottoposte ad embolizzazione vi sono le arterie renali e i suoi rami distali e / o polari . 
le indicazioni allembolizzazione o allesclusione endovascolare delle arterie renali e dei suoi rami distali e / o polari ( ear ) sono molteplici , ma queste procedure vengono effettuate principalmente a scopo terapeutico o palliativo in pazienti con traumi o tumori renali , anche se negli ultimi anni stanno emergendo ulteriori indicazioni quali lembolizzazione preoperatoria delle arterie renali polari nei pazienti candidati al posizionamento di endoprotesi per lesclusione di aneurismi dellaorta addominale . 
diversi sono i materiali embolizzanti che possono venire impiegati per ottenere un risultato terapeutico adeguato . scopo di questo lavoro rivalutare la nostra esperienza di circa 10 anni di attivit in questo tipo di procedure al ne di identi care le indicazioni attuali ed emergenti , i materiali utilizzati ed i risultati ottenuti . materiali e metodi ad una valutazione retrospettiva dellattivit di radiologia interventistica effettuata presso la nostra struttura nel periodo compreso tra giugno 1999 e luglio 2009 , 27 pazienti ( 19 maschi e 8 femmine ; et media : 74 anni ; range : 3793 anni ) sono stati sottoposti ad ear . 
tale procedura stata eseguita con la nalit di prevenire una possibile rivascolarizzazione della sacca aneurismatica dopo il posizionamento dellendoprotesi . la maggior parte delle procedure stata eseguita utilizzando un sistema per angiogra a e radiologia interventistica philips integris allura ( philips medical systems netherlands bv , best , olanda )  . 
 dopo preliminare aortogra a si proceduto a cateterismo selettivo o superselettivo dei rami arteriosi renali interessati e successivo rilascio di agenti embolizzanti quali spirali metalliche a rilascio non controllato ( cook , bloomington , in , usa ; boston scienti c corporation , natick , mass . , usa ; balt , montmorency , francia ) o dispositivi occlusori tipo plug ( amplatzer vascular plug , aga medical , golden valley , usa ) o stent ricoperti ( atrium , atrium medical corporation , hudson , usa ; jostent peripheral graft , jomed , rangendingen , germania ) o particelle embolizzanti non riassorbibili in alcol polivinilico ( pva ) o similare ( contour emboli , boston scienti c corporation , natick , usa ; bead block , terumo medical corp . , somerset , usa ; embosphere , biosphere medical , rockland , usa ) o riassorbibibili ( spon948 radiol med ( 2011 ) 116 : 945959 usa ) or absorbable particles ( spongostan standard ; j&j medical limited , gargrave , skipton , england ; curaspon , curamedical , assendelft , the netherlands ) or liquid embolisation agents , such as glues ( glubran 2 gem srl , viareggio , italy )  . 
in 11 patients ( 40.7% ) , two or three different embolisation agents were used . in all cases , a nal angiogram was obtained to evaluate the effective exclusion of the vascular region subjected to embolisation and the possible embolisation of nontarget segments , with technical success being de ned as the cessation of bleeding or vascular exclusion of the lesion after rae . results the results are reported in table 1 . 
in addition , in the latter case of highow arteriovenous stula , embolisation was initially attempted using multiple coils and , due to the persistence of reduced ow , completed with several coarse fragments of absorbable particles , which completely stopped the ow . 
 in tutti i casi stata eseguita unangiogra a nale di controllo per la valutazione delleffettiva esclusione del territorio vascolare sottoposto ad embolizzazione e di eventuali embolizzazioni di segmenti non bersaglio , de nendo come successo tecnico la cessazione del sanguinamento o lesclusione vascolare della lesione dopo ear . risultati i risultati sono riportati nella tabella 1 . 
in this case , the exclusion of a segmental branch , in addition to the aneurysm , did not produce ischaemic - like symptoms , and a contrast - enhanced colour doppler ultrasound performed 30 days after the procedure showed no areas of renal injury . in the patient in whom selective catheterisation was not possible , the inferior polar artery arose directly from the aneurysmal sac that , at preliminary ct angiography , appeared occupied by a large ulcerated thrombus . 
 in questo caso , lesclusione di un ramo segmentario , oltre allaneurisma , non ha comportato linsorgenza di alcuna sintomatologia di tipo ischemico ed un esame eco - color doppler con mezzo di contrasto , effettuato 30 giorni dopo , non ha evidenziato aree di sofferenza renale . 
 nel paziente in cui non stato possibile cateterizzare selettivamente larteria polare inferiore tale vaso originava direttamente dalla sacca aneurismatica che al preliminare controllo angio - tomogra a computerizzata ( tc ) appariva occupata da una vasta apposizione trombotica ulcerata . 
 solo in un caso ( 3 , 7% ) di paziente affetto da neoplasia renale inoperabile con macroematuria vi stata una recidiva della macroematuria a distanza di 13 mesi ed stato ritrattato con successo . 
polare pre - evar macroematuria in neoplasia renale macroematuria in neoplasia renale fav renale sintomatica neoplasia renale inoperabile sintomatica aneurisma dellarteria renale polare macroematuria in neoplasia renale neoplasia renale inoperabile sintomatica embolizzazione a . 
in another case , during the placement of a coil in a renal polar artery , the slightly oversized coil caused the distal end of the catheter to come out of the vessel . 
in un altro caso durante la fase di posizionamento di una spirale in unarteria polare renale , la spirale lievemente sovradimensionata ha causato la fuoriuscita dellestremit distale del catetere dal vaso . 
a distanza di tempo si osservato un incremento dellemoglobina in 9 / 11 ( 81 , 8% ) dei pazienti con macroematuria o ematoma sottocapsulare , mentre un decremento della creatininemia stato osservato in 9 / 27 ( 33 , 3% ) casi . discussion the indications for embolisation of the renal artery and its intraparenchymal branches are substantially codied and consist of the embolisation of inoperable renal tumours responsible for symptomatic haematuria or signi cant symptoms [ 15 ]  . 
it has in fact been suggested that the embolisation of renal lesions can slow down the le indicazioni allembolizzazione dellarteria renale e dei suoi rami intraparenchimali sono sostanzialmente codi cate e costituite dallembolizzazione di tumori renali inoperabili responsabili di ematurie sintomatiche o di sintomatologie importanti [ 15 ] in quanto stato suggerito che il trattadiscussione radiol med ( 2011 ) 116 : 945959 fig . 
1a - d donna di 69 anni operata 3 giorni prima di nefrectomia parziale al rene destro con anemizzazione e macroematuria , la ricostruzione tridimensionale volume rendering ( a ) mette in evidenza un singolo blush pseudoaneurismatico di mezzo di contrasto di circa 15 mm di diametro nei settori mediosuperiori del rene . 
d post - procedurale , dopo embolizzazione mediante tre spirali metalliche : completa esclusione dei foci pseudoaneurismatici e preservazione dei rami intraparenchimali . progression of metastatic disease [ 6 ]  . 
in agreement with other authors [ 7 ] , we do not perform preoperative embolisation in renal carcinoma patients who are candidates for nephrectomy , as there are no data in the literature to support this practice . 
 occasionally , those lesions can cause substantial bleeding with signi cant haematuria and large subcapsular or perirenal haematomas and produce dramatic , life - threatening mento di embolizzazione delle lesioni renali pu determinare un rallentamento nella progressione della malattia metastatica [ 6 ]  . 
in accordo con quanto sostenuto da altri autori [ 7 ] , non eseguiamo lembolizzazione preoperatoria nei pazienti candidati a nefrectomia per carcinoma renale in quanto non ci sono dati in letteratura che supportino tale pratica . 
on these occasions , the endovascular procedure needs to be extremely rapid [ 12 ] , whereas on other occasions , the symptoms may be absent or vague , with the patient clinically stable such that the endovascular procedure may be done electively [ 7 ]  . the overall results reported in the literature vary broadly according to the characteristics of the various patient popumaturie importanti ed ematomi sottocapsulari o perirenali voluminosi e sono causa di sintomatologie drammatiche che mettono a rischio la vita del paziente e lintervento endovascolare deve essere estremamente tempestivo [ 12 ] , mentre altre volte la sintomatologia pu essere assente o sfumata e il paziente clinicamente stabile , per cui la procedura endovascolare pu essere eseguita in elezione [ 7 ]  . 
c controllo angiogra co dopo embolizzazione mediante particelle riassorbibili e dispositivo occlusore . lations , with the best results in terms of technical success which , when referring to palliation of renal tumours , varies from 90% to 100% , with recurrence and therefore the need for a second embolisation procedure at 218 months from the initial procedure in 510% of cases [ 15 ]  . 
with regard to technical success in cases of acute haemorrhage ( traumatic / iatrogenic or secondary to expansile renal lesions ) , the literature reports values between 85% and 92% , with the need for a second embolisation procedure at 26 days in 1719% of cases [ 815 ]  . the literature [ 1 ] , but also our own experience , suggests that coils are the most frequently used embolisation agent in the treatment of traumatic lesions in that they are technically easier to use , whereas in palliative treatment , the i risultati complessivi riportati in letteratura variano ampiamente a seconda delle caratteristiche della casistica considerata con risultati migliori in termini di successo tecnico che , quando riferito alla palliazione di neoplasie renali , varia dal 90% al 100% con recidive , e quindi necessit di un secondo intervento di embolizzazione , nel 5%10% dei casi a 218 mesi dalla prima procedura [ 15 ]  . 
 per quanto riguarda il successo tecnico in caso di emorragie acute ( traumatiche / iatrogene o secondarie a lesioni espansive renali ) in letteratura sono riportati valori di successo tecnico compresi tra l85% e il 92% con necessit di un secondo intervento di embolizzazione a 26 giorni nel 17%19% dei casi [ 815 ]  . 
4a - c a 65 - year - old man with a history of hypertension , incidental aneurysm of the right renal aneurysa magnetic resonance angiography showing a 2 - cm aneurysm of the main trunk of the right kidney and a polar artery . 
c controllo angiograco dopo posizionamento di stent ricoperto con completa esclusione della lesione aneurismatica . use of embolisation particles is more frequent , as they produce a more distal embolisation [ 2 ]  . 
the literature also suggests greater effectiveness of embolisation treatment when trisacryl microspheres are used rather than pva particles due to their more distal penetration ( for the same diameter ) within the vascular nidus of the lesion [ 1 , 2 ]  . 
it should also be recalled that the literature reports the use of other embolisation agents , the so - called liquids , such as absolute alcohol [ 2 , 16 ] and glues [ 1719 ]  . 
various studies reported on their experience with embolisation at the renal level using an acrylic glue known as n - butyl - 2cyanoacrylate ( nbca ) [ 18 , 19 ]  . 
towards the end of the 1990s , however , it turned out that this use was not of cially authorised for endovascular treatment and was banned [ 20 ]  . 
in later years , however , a new glue derived from the former was introduced , the use of which is permitted emerge che le spirali rappresentano lagente embolizzante pi utilizzato nel trattamento delle lesioni traumatiche in quanto tecnicamente pi facili e maneggevoli da usare , mentre nelle embolizzazioni palliative pi frequente il ricorso alle particelle embolizzanti in quanto consentono unembolizzazione pi distale [ 2 ]  . 
sempre dalla letteratura sembra inoltre emergere una migliore ef cacia del trattamento embolizzante quando impiegate le microsfere trisacriliche piuttosto che le particelle in pva , in virt di una capacit di penetrazione pi distale ( a parit di diametro ) allinterno del nidus vascolare della lesione [ 1 , 2 ]  . 
va ricordato , inoltre , che in letteratura riportata la possibilit di utilizzo di altri agenti embolizzanti , cos detti liquidi , quali lalcol assoluto [ 2 , 16 ] e le colle [ 1719 ]  . 
in passato sono state riportate varie esperienze di embolizzazione a livello renale in cui veniva utilizzata una colla acrilica nota come radiol med ( 2011 ) 116 : 945959 for endovascular treatment . 
the glue is a comonomer consisting of two monomers : nbca and the monomer denominated ms by the manufacturer of the comonomer itself ( gem italy , viareggio , italy )  . 
with respect to nbca , the comonomer has the not insigni cant advantage of polymerising within the vessel in relatively longer times , thus allowing a more controlled embolisation procedure . 
 although this embolisation agent is very effective and enables a lasting and very distal embolisation , its use at the renal level has only been sporadically reported , probably due to the lack of ease in its use and the associated risk of embolising nontarget structures . 
also in our limited experience , we had the feeling of incomplete control of the release of the glue within the artery and , unlike the other patients treated with embolisation particles or mechanical agents , the patient suffered mild pain , which disappeared after a short time . further indications for embolisation of renal arteries are renal artery vascular diseases , such as arteriovenous stulas and acquired or congenital pseudoaneurysms [ 21 , 22 ] and aneurysms . 
encouraging short - term and long - term results have been presented both in case reports and more extensive studies [ 2326 ]  . a less frequently reported indication is the exclusion of an aneurysm of the renal artery . 
in truth , the inclusion of these cases in this series is debatable in that the coated stent guarantees the patency of a vessel , but at the same time , the exclusion of a pathological vascular segment is achieved and the exclusion of splanchnic or renal artery aneurysms with the use of coated stents has been reported as embolisation [ 2729 ]  . 
an alternative to the coated stent is , as mentioned above , the lling of the aneurysmal sac with coils or microcoils , preferably with controlled release , in similar fashion to the treatment of cerebral aneurysms [ 25 ]  . 
nonetheless , this embolisation technique cannot always be used , as in the case of wideneck aneurysms , because the coils may become unstable and protrude into the lumen of the vessel from which the aneurysm arises . 
the rst involves deploying a bare metal stent across the neck of the aneurysm and then lling the sac by passing the microcoils through the stent interstices with a microcatheter [ 30 , 31 ]  . n - butil - 2 - cianoacrilato ( nbca ) [ 18 , 19 ]  . 
si tratta di un comonomero costituito da 2 monomeri : ln - butil - 2 - cianoacrilato e il monomero denominato ms di propriet della ditta produttrice del comonomero stesso ( gemitaly , viareggio , italia ) , che rispetto allnbca ha anche il non trascurabile vantaggio di polimerizzare allinterno del vaso in tempi relativamente pi lunghi , consentendo una manovra di embolizzazione pi controllata . 
sebbene tale agente embolizzante sia molto ef cace e consenta unembolizzazione duratura e molto distale , il suo impiego a livello renale solo sporadicamente riportato e questo verosimilmente a causa del non suo facile impiego e al rischio di causare embolizzazione di strutture non bersaglio . 
anche nella nostra limitata esperienza si avuta la sensazione di un non sicuro controllo del rilascio della colla nellarteria e , a differenza degli altri casi in cui si sono utilizzate particelle embolizzanti o agenti meccanici , il paziente ha comunque avvertito una discreta sintomatologia dolorosa regredita in breve tempo . ulteriori indicazioni allembolizzazione delle arterie renali sono le patologie vascolari dellarteria renale , quali le stole artero - venose , i pseudoaneurismi acquisiti o congeniti [ 21 , 22 ] e gli aneurismi , e risultati incoraggianti a breve e lungo termine sono stati presentati sia in case report che in lavori con casistiche pi consistenti [ 2326 ]  . 
 meno frequente , ma comunque riportata , la procedura di esclusione di un aneurisma dellarteria renale : esistono varie opzioni , quali limpiego delle spirali o microspirali [ 2325 ] o lutilizzo di stent ricoperti [ 26 ]  . 
in realt pu essere discutibile considerare tali casi in questa casistica in quanto lo stent ricoperto garantisce la perviet di un vaso , ma allo stesso tempo si ottiene lesclusione di un segmento vasale patologico ed in letteratura lesclusione di aneurismi splancnici o renali mediante limpiego di stent ricoperti stata riportata come embolizzazione [ 2729 ]  . 
unalternativa allo stent ricoperto , come sopra accennato , il riempimento della sacca aneurismatica con spirali o microspirali , preferibilmente a rilascio controllato , analogamente a quanto viene fatto per gli aneurismi cerebrali [ 25 ]  . 
tuttavia tale tecnica di embolizzazione non sempre pu essere usata , come nel caso di aneurismi a colletto largo , poich le microspirali possono risultare instabili e protrudere nel lume del vaso da cui origina laneurisma . 
due possibili alternative in questi casi , in mancanza di stent ricoperti adeguati o nellimpossibilit di un loro posizionamento , sono il posizionamento di uno 956 radiol med ( 2011 ) 116 : 945959 an alternative method , although data available are still extremely preliminary , sees the use of a new type of multilayer stent composed of several layers of very ne nitinol wires , which preliminary studies show is able to maintain the ow in the vessel within the stent , whereas ow progressively lessens in the aneurysm until its complete thrombosis is achieved [ 32 ]  . a rare indication for rae , seen in our experience and also reported in the literature , is the treatment of haematuria and / or spontaneous haematoma in patients with autosomal - dominant polycystic kidney disease [ 33 ]  . 
a further indication that has appeared in recent years is embolisation of the renal polar branches originating from the aneurysmal sac or infrarenal neck in patients with an aaa who are candidates for evar . 
diffusion of this type of procedure has progressively led to the indication of embolisation of collateral branches , such as the lumbar arteries and the inferior mesenteric artery , to prevent reperfusion of the aneurysmal sac , with results that nonetheless fail to be completely convincing [ 34 , 35 ]  . 
it is instead unclear what attitude should be taken in relation to renal polar branches arising from the aneurysmal sac or the infrarenal neck of the aorta with the aim of preventing possible sac revascularisation . 
a review of the literature shows that some centres , prior to deploying the abdominal aortic endoprosthesis , embolise the renal polar arteries arising from the neck [ 36 , 37 ] , whereas other authors [ 38 , 39 ] who have not performed a preliminary embolisation of the renal polar artery and have evaluated the effect of the coverage by the endoprosthesis of those branches , have not observed consequences such as endoleak due to reperfusion or signi cant damage to the parenchyma and renal function resulting from a segmental infarction of the renal parenchyma . 
in our experience , we have observed that the nonembolisation of a polar branch arising from the aneurysmal sac tends to favour the development of a type 2 endoleak , not so much through the renal polar artery in which ow always remains normally directed , but through a lumbar artery or the inferior mesenteric artery in which the ow changes direction . 
 the procedure is justi ed only in cases of the polar artery arising from the sac , in that polar arteries arising from the neck are adequately closed simply by the deployment of the prosthesis . in our experience the procedure was carried out prior to stent non ricoperto in corrispondenza del colletto dellaneurisma e quindi , dopo essere passati tra le maglie dello stent con un microcatetere , il rilascio in sicurezza delle microspirali no ad ottenere il riempimento della sacca [ 30 , 31 ]  . una nuova alternativa , ma i dati sono ancora estremamente preliminari , data dalla possibilit di impiego di un nuovo tipo di stent , denominato multistrato , costituito da pi strati di li di nitinol molto sottili che , dalle esperienze preliminari , in grado di mantenere il usso nel vaso allinterno dello stent , mentre questo progressivamente si riduce nellaneurisma no alla completa trombosi di questultimo [ 32 ]  . unindicazione pi rara allesecuzione di una ear , osservata anche nella nostra esperienza e riportata anche in letteratura , rappresentata dal trattamento dellematuria e / o dellematoma spontaneo in pazienti con malattia policistica renale autosomica dominante [ 33 ]  . 
unulteriore possibile indicazione , emersa negli ultimi anni , lembolizzazione di rami polari renali originanti dalla sacca aneurismatica o dal colletto sottorenale in pazienti portatori di aneurismi dellaorta addominale candidati a trattamento di esclusione dellaneurisma mediante endoprotesi aortica ( evar )  . 
con la diffusione di tale tipo di intervento si progressivamente posta lindicazione alla procedura di embolizzazione di rami collaterali originantesi dalla sacca aneurismatica , quali le arterie lombari e larteria mesenterica inferiore per prevenire la riperfusione della sacca aneurismatica , con risultati che tuttavia non appaiono ancora del tutto convincenti [ 34 , 35 ]  . 
non invece chiaro latteggiamento da assumere nei confronti dei rami polari renali originantesi dalla sacca aneurismatica o dal colletto sottorenale dellaorta con lo scopo di prevenire possibili rivascolarizzazioni della sacca . 
 dalla revisione della letteratura abbiamo trovato che alcuni centri embolizzano , preventivamente al posizionamento di unendoprotesi aortica addominale , le arterie renali polari originantesi dal colletto [ 36 , 37 ] , mentre altri autori [ 38 , 39 ] , che non hanno eseguito lembolizzazione preliminare dellarteria polare renale e hanno valutato leffetto della copertura da parte dellendoprotesi di tali rami , non hanno osservato conseguenze quali lendoleak da riperfusione o danni signi cativi sul parenchima e la funzione renale quale conseguenza di un infarto segmentario del parenchima renale . 
nella nostra esperienza abbiamo osservato che la mancata embolizzazione di un ramo polare originante dalla sacca aneurismatica costituisce una causa favorente lo sviluppo di un endoleak di tipo 2 , non tanto attraverso larteria polare renale nella quale il usso rimane sempre normo - diretto , ma attraverso unarteria lombare o larteria mesenterica inferiore nelle quali il usso inverte la sua direzione . 
verosimilmente tale procedura giusti cata solo nei casi di arterie polari che originano dalla sacca , in quanto quelle che originano dal colletto vengono chiuse in modo radiol med ( 2011 ) 116 : 945959 fig . 
a langio - tc preprocedurale ( ricostruzione mip ) dimostra la presenza di unarteria polare inferiore accessoria destra in paziente portatore di rene a ferro di cavallo ; b arteriogra a selettiva dellarteria polare inferiore destra . 
c embolizzazione dello stesso vaso mediante 3 spirali metalliche con completa esclusione del usso ematico . the deployment of the endoprosthesis , on the same day or 1 or 2 days before the intervention . 
we preferred to perform the embolisation in the angiography suite rather than in the operating room where the aortic endoprosthesis placement is carried out because the c - arm available to us does not guarantee optimal image quality for precise execution of the procedure . adeguato semplicemente con il posizionamento della protesi . 
si preferito eseguire lembolizzazione nella sala angiogra ca piuttosto che in sala operatoria , dove si esegue il posizionamento dellendoprotesi aortica , in quanto larco a c a nostra disposizione non garantisce la qualit dimmagine ottimale per una precisa esecuzione della manovra . conclusions rae is an effective and minimally invasive procedure with low morbidity rates and short hospital stay . 
it should be considered the treatment of choice in neoplastic / iatrolear rappresenta una terapia ef cace e minimamente invasiva e deve essere considerata il trattamento di scelta conclusioni 958 radiol med ( 2011 ) 116 : 945959 genic symptomatic gross haematuria , in the palliation of inoperable renal tumours , aneurysms and arteriovenous malformations of the main renal artery or its branches and in the prophylactic exclusion of the inferior polar artery in candidates for evar . 
we believe that as tumours require a more distal and less focused embolisation , particles or glues are preferable , whereas coils or microcoils are more suited to vascular lesions ( active bleeding , pseudoaneurysms or stulas ) where a more targeted embolisation is required . 
nonetheless , the use of several agents together may occasionally be necessary . della macroematuria sintomatica neoplastica / iatrogena , nella palliazione di tumori renali inoperabili , di aneurismi e malformazioni arterovenose a carico dellarteria renale principale o dei suoi rami e nellesclusione preventiva dellarteria polare inferiore in pazienti candidati ad evar , con scarsa morbilit e breve degenza intraospedaliera . 
diversi sono gli agenti embolizzanti a disposizione : nella loro scelta sicuramente gioca un ruolo lesperienza delloperatore con il materiale da impiegare : riteniamo che per le lesioni tumorali , dove necessaria unembolizzazione pi distale e meno focalizzata , le particelle o le colle siano da preferirsi , mentre per le lesioni vascolari ( sanguinamenti attivi , pseudoaneurismi o stole ) le spirali o le microspirali , che consentono unembolizzazione pi mirata , siano pi adatte . 
in questo lavoro vengono descritte le caratteristiche in tomogra a computerizzata ( tc ) del sarcoma sinoviale primitivo del polmone ( ppss ) in 3 casi arrivati alla nostra osservazione e sottoposti ad intervento chirurgico , in associazione alla revisione della letteratura relativa a questo raro tumore toracico . 
nei 2 pazienti sottoposti anche a pet - tc , lesame dimostrava patologico accumulo del tracciante solo nella sede della lesione , senza altre localizzazioni toraco - addominali o muscolo - scheletriche . 
il terzo paziente ha avuto la conferma diagnostica di sarcoma sinoviale monofasico radiol med ( 2011 ) 116 : 868875 of monophasic synovial sarcoma in the third patient was con rmed using thoracoscopic biopsy discussion . 
both in the cases described and in those identi ed in the literature review , standard chest x - rays mainly showed a parenchymal mass of pleural origin with either irregular or well - de ned margins . 
ct characteristics are more de nite , with evidence of a mass with regular and sharply de ned margins , occasionally polycyclic , with inhomogeneous density due to the presence of colliquative areas within the tumour . 
sia nei casi descritti che nella revisione della letteratura la radiogra a standard del torace dimostra principalmente una massa parenchimale a margini sia irregolari che netti , a base pleurica . 
sebbene il ppss sia un tumore raro , la presenza di una massa polmonare a densit disomogenea , associata spesso a versamento pleurico ma senza adenopatie , in un paziente asintomatico o paucisintomatico deve indurre a considerare anche questa neoplasia nel range delle diagnosi differenziali dei tumori polmonari , escludendo la presenza di metastasi a partenza dai pi frequenti sarcomi sinoviali a carico dellapparato muscolo - scheletrico . keywords synovial sarcoma lung ct parole chiave sarcoma sinoviale polmone tc introduction introduzione synovial sarcoma is a cancer usually originating in the para - articular soft tissues of the limbs in young adults [ 1 ]  . 
the rise of sophisticated diagnostic techniques in immunohistochemistry , electron microscopy and cytogenetic analysis has led to a more frequent recognition of pssl as a well - de ned histological subtype of sarcoma of the lung [ 4 ]  . 
 we also present a review of the literature regarding this rare thoracic tumour . il sarcoma sinoviale una neoplasia che normalmente origina nei tessuti molli para - articolari delle estremit in giovani adulti [ 1 ]  . 
 [ 3 ] nel 2002 hanno descritto il ppss come una distinta entit anatomica con caratteristiche anatomopatologiche simili a quelle del tumore delle parti molli e la diagnosi deve essere confermata dopo aver escluso altre neoplasie primitive sarcomatose ( per esempio il carcinoma sarcomatoide ) e le metastasi da sarcomi di altri organi . 
 laumento delle tecniche di diagnosi so sticata in immunoistochimica , in microscopia elettronica e nelle analisi citogenetiche , ha portato ad un pi frequente riconoscimento del ppss come un ben de nito sottotipo istologico di sarcoma del polmone [ 4 ]  . 
in questo lavoro vengono descritte le caratteristiche in tomogra a computerizzata ( tc ) del ppss di 3 casi arrivati alla nostra osservazione e sottoposti ad intervento chirurgico , in associazione alla revisione della letteratura relativa a questo raro tumore toracico . 
 materials and methods clinical and radiological ndings of three patients with a diagnosis of pssl were obtained through a review of the le informazioni cliniche e radiologiche di 3 pazienti con materiali e metodi 870 radiol med ( 2011 ) 116 : 868875 clinical records of the department of thoracic surgery and the ct [ and possible positron emission tomography pet - ct ] examinations in the radiology archives , either on the picture archiving and communication system ( pacs ) or on x - ray lall patients were men and aged between 51 and 65 ( mean 58 ) years . 
another patient had undergone thoracoscopic resection of a lesion of the left lower lobe 18 months previously ; the lesion was at rst diagnosed as a brous pleural tumour and then as a monophasic synovial sarcoma . 
the other two patients had symptoms characterised by pain in the hemithorax and shoulder corresponding to the tumour , with fever in one patient only . all patients had undergone both chest x - rays and ct scans , and two were also studied with pet - ct ( siemens biograph - 16 - usa )  . 
the thoracoabdominal ct scans ( all using intravenously administered contrast medium omnipaque 350 , ge healthcare , usa ) were performed with a range of equipment including conventional , single - slice or 16 - slice multidetector ct ( siemens somaton emotion , siemens , germany )  . 
two patients underwent tumour removal , whereas the third was rst operated on ( due to a diagnosis at variance with the nal one ) and then underwent thoracoscopy with a biopsy of the pleural lesions . results in all cases , chest x - ray revealed the presence of lung masses measuring , respectively , 7 , 6 and 4.5 cm ( mean 5.8 cm ) in diameter , two of which were located in the right upper lobe and one in the left lower lobe . 
mediastinal adenopathy , which was diagnosi di ppss sono state ottenute dalla revisione delle cartelle cliniche della chirurgia toracica e degli esami tc e , eventualmente , di tomogra a a emissione di positroni pet - tc , presenti negli archivi radiologici , sia su sistemi picture archiving and communications system ( pacs ) che su pellicola radiogra ca . 
un paziente era gi stato sottoposto a bilobectomia destra 13 anni prima e a resezione di recidiva 10 prima dellattuale neoplasia per blastoma polmonare e un altro paziente era stato sottoposto18 mesi prima a resezione per via toracoscopia di una lesione del lobo inferiore sinistro diagnosticata in prima istanza come tumore broso della pleura e in secondo tempo come sarcoma sinoviale monofasico . 
negli altri 2 pazienti era presente sintomatologia caratterizzata da dolore allemitorace e alla spalla corrispondenti alla neoplasia e a febbre in un solo paziente . tutti avevano eseguito sia la radiogra a del torace che la tc e due anche la pet - tc ( biograph 16 , siemens , usa )  . 
 gli esami tc toraco - addominali ( sempre anche con mezzo di contrasto [ mdc ] endovena [ ev ] ; omnipaque 350 , ge healthcare , usa ) sono stati effettuati con differenti apparecchiature ( tradizionale , singolo strato o multidetettore a 16 strati ; somaton siemens , emotion siemens , germania )  . 
due pazienti sono stati operati con asportazione della neoplasia e 1 stato prima operato ( con diagnosi diversa da quella de nitiva ) e poi sottoposto a toracoscopia con biopsia delle lesioni pleuriche . risultati in tutti i casi la radiogra a del torace era risultata positiva dimostrando la presenza di masse polmonari , rispettivamente di 7 , 6 e 4 , 5 cm ( media 5 , 8 cm ) , localizzate 2 nel lobo superiore destro e una nel lobo inferiore sinistro . 
adenopatie mediastiniche , peraltro non signi cative , erano presenti in un solo paziente e ancora in un unico paziente era presente un nodulo inderadiol med ( 2011 ) 116 : 868875 fig . 
contrast - enhanced ct of the chest ( b ) : bulky , partly colliquated , paramediastinal mass in the upper right lobe without cleavage plane with the posterior tracheal wall and compressing the superior vena cava . 
tc torace con mdc ( b ) : voluminosa massa paramediastinica superiore destra , in parte colliquata , senza piano di clivaggio con la parete postero - laterale della trachea e con compressione sulla vena cava superiore . 
 linfonodi con aspetto reattivo in regione paratracheale inferiore destra . not signi cant , was present in one patient only ; also in one patient , there was an indeterminate nodule 5 mm in diameter close to the main lesion . 
in the two patients who also underwent pet - ct , the examination showed pathological tracer uptake con ned to the lesion site , without other thoracoabdominal or musculoskeletal localisations . ct - guided biopsy , performed in only one patient , proved positive for a mesenchymal tumour . 
in the third patient , who had undergone resection of a pulmonary nodule 18 months previously , the diagnosis of monophasic synovial sarcoma was con rmed by thoracoscopic biopsy ; this patient could not proceed to surgery owing both to lesion extent and to the fact that the lesion was considered a recurrence of pssl arising a short time after initial surgery . 
nei due pazienti sottoposti anche a pet - tc , lesame dimostrava patologico accumulo del tracciante solo nella sede della lesione , senza altre localizzazioni toraco - addominali o muscolo - scheletriche . 
il terzo paziente , quello operato 18 mesi prima per resezione di nodulo polmonare , ha avuto la conferma diagnostica di sarcoma sinoviale monofasico dalle biopsie toracoscopiche , senza poter procedere ad intervento chirurgico sia per lestensione della lesione sia perch la stessa stata considerata come recidiva di ppss a breve distanza dal primo intervento . 
2a , b tc torace con mdc : versamento pleurico sinistro con nodi solidi ( frecce ) e atelettasia da compressione del lobo inferiore per recidiva di sspp asportato 18 mesi prima . 
normally , patients with pssl report chest pain , cough and / or shortness of breath , but in 24% of cases , the tumour is discovered during chest x - rays performed for other reasons , as occurred in one of our patients [ 6 ]  . 
 [ 9 ] reviewed the imaging features of 12 cases of pssl . in our study , standard chest x - rays mostly show a parenchymal mass of pleural origin with margins that are either irregular or sharp . 
ct features are better de ned : the mass always displays sharp margins and a regular , at times polycyclic , outline , with inhomogeneous density due to colliquative areas within the tumour . 
 normalmente i pazienti con ppss riferiscono comparsa di dolore toracico , tosse e / o dispnea , ma nel 24% dei casi la neoplasia viene scoperta casualmente in seguito ad esame radiologico del torace eseguito per altri motivi , come successo in uno dei nostri pazienti [ 6 ]  . 
le caratteristiche tc sono pi de nite : sempre presente una massa a margini netti e contorni regolari , a volte policiclici , con densit disomogenea per presenza di aree di colliquazione allinterno . 
3a - d preoperative contrast - enhanced chest computed tomography ( ct ) ( a , b ) : homogeneously dense mass close to the anterior junction line on the right . 
tc torace eseguita dopo 5 anni dallasportazione della lesione precedente : comparsa nuova massa paramediastinica nella stessa sede ( c ) con adenopatie in parte colliquate sempre in sede precarenale ( d )  . eral pleural effusion is often present , but calci cations and adenopathy are rare . 
 more rarely , pssl can present as a solitary pulmonary nodule , an endobronchial tumour or a mass within the pulmonary arteries . the differential diagnosis must take into account the common primary tumours of the lung as well as metastases arising from tumours elsewhere in the body ( especially musculoskeletal tumours ) , as well as pleural broma , mesothelioma and the rare lung sarcomas ( brosarcoma , sarcomatous carcinoma , leiomyosarcoma or haemangiopericytoma )  . 
 la diagnosi differenziale radiologica deve prendere in considerazione i comuni tumori primitivi del polmone , ma anche le metastasi da neoplasie in altre sedi ( con particolare riguardo a quelle dellapparato muscolo - scheletrico ) , il broma pleurico . 
sicuramente la conoscenza di precedente esposizione ad asbesto indice di insorgenza di mesotelioma e la presenza di adenopatie mediastiniche nettamente pi frequente nel carcinoma broncogeno rispetto al ppss dove , come stato 874 table 1 characteristics at computed tomography size lobe 1 upper right 76 cm 2 lower left 64 cm colliquative colliquative 3 upper right 44.5 cm solid homogeneous tabella 1 caratteristiche tc radiol med ( 2011 ) 116 : 868875 densitometric characteristics other nodules pleural effusion mediastinal adenopathy signs of in ltration prior surgery yes same lobe , 5 mm yes chest wall yes , with solid pleural nodules subcarinal resection of nodule lower left lobe mediastinal median and lower right fat bilobectomy lobo grandezza caratteristiche densitometriche altri noduli versamento pleurico adenopatie mediastiniche segni di in ltrazione pregresso intervento 1 superiore destro 2 inferiore sinistro 76 cm 64 cm 3 superiore destro 44 , 5 cm colliquata colliquata solida omogenea s , stesso lobo , 5 mm si parete toracica s , con noduli pleurici solidi sottocarenale no resezione nodulo lobo inferiore sinistro bilobectomia grasso mediastinico medio - inferiore destra opathy is signi cantly more frequent in bronchogenic carcinoma when compared with pssl where , as already noted , enlarged lymph nodes are rare . in radiological terms , our three patients exhibited the most common form of the cancer , presenting with lung masses and pleural effusion , even though the small lesion excised in one of the patients and diagnosed as a brous tumour was actually a pssl in nodular for perhaps an incorrect diagnosis can also explain the patient with a previous diagnosis of blastoma , the lack of targeted immunohistochemical examinations leading to an evident initial error in lesion interpretation . 
treatment is surgical , sometimes combined with chemotherapy and radiotherapy , which , however , do not provide favourable prognostic outcomes . reported survival is between 6 and 58 months following diagnosis , even though 80% of patients display tumour recurrence in the same hemithorax but without abdominal lesions [ 10 ]  . 
nonehave died , and one is still alive more than 6 years after surgery . given that lung metastases from soft - tissue sarcoma are more common than pssl [ 11 ] , the diagnosis should also be supported by medical history , clinical examination and radiological search for tumours in other sites in the body . 
 i nostri tre pazienti rientrano radiologicamente nella forma pi comune della neoplasia , presentandosi con masse polmonari e con versamento pleurico , anche se la piccola lesione operata in uno dei pazienti e diagnosticata come tumore broso era in realt un ppss in forma nodulare . 
 forse una errata diagnosi si pu ipotizzare anche per il paziente con precedente diagnosi di blastoma : la mancanza di esami immunoistochimici mirati ha portato ad un evidente primo errore interpretativo della lesione . 
 la sopravvivenza riportata in letteratura compresa tra i 6 e i 58 mesi dalla diagnosi , anche se l80% dei pazienti ha mostrano ripresa di malattia nello stesso emitorace di partenza , senza lesioni addominali [ 10 ]  . 
la lunga sopravvivenza , pur in caso di recidiva , documentata anche nei nostri pazienti : nessuno deceduto e uno di loro ancora vivo dopo pi di 6 anni dallintervento . 
 poich la presenza di metastasi polmonari da sarcoma delle parti molli pi comune rispetto al ppss [ 11 ] , la diagnosi deve essere supportata anche dalla storia del paziente , dallesame clinico e dalla ricerca radiologica di neoplasie in altre sedi . 
a questo scopo indicata la pet che mostra un importane aumento dello standard uptake value radiol med ( 2011 ) 116 : 868875 pet - ct is excellent for this purpose , displaying a notable increase in standard uptake value in regions affected by neoplasms . 
a diagnosis of pssl is dif cult to reach with the use of ultrasoundor ct - guided biopsy alone , as the only result obtained is often that of an unspeci ed malignancy of mesenchymal origin , as indeed happened in our patient who underwent this procedure . in conclusion , although pssl is a rare tumour , the presence of a lung mass with inhomogeneous density , often associated with pleural effusion but without adenopathy , in an asymptomatic or poorly symptomatic patient should lead to this tumour being considered in the differential diagnosis after excluding the presence of metastases from the more frequent synovial sarcomas affecting soft tissues . ( suv ) nelle regioni interessate da neoplasia . 
cattaneo labanof , laboratorio di antropologia e odontologia forense , sezione di medicina legale , dmu dipartimento di morfologia umana e scienze biomediche , universit degli studi di milano , via mangiagalli 37 , milano , italy correspondence to : c . 
the increasing applications of forensic radiology and the wide use of conventional radiography and computed tomography ( ct ) in routine clinical practice demonstrate the potential of these technologies as tools for verifying the correspondence between an unidenti ed body and an identity suspect . 
despite the increasing importance of the comparison between radiographic and ct ndings , numerous limitations still need to be overcome , including the fact that few forensic centres have access to sophisticated x - ray technologies and that the reliability of those technologies for detecting speci c morphological traits and bone lesions is a matter of intense debate . 
 in addition , as with other morphological methods for identi cation , comparisons between antemortem and postmortem data require standardisation and statistical analysis , especially in europe where there are very few indications concerning the admission in court of evidence obtained by anthropological and radiological methods . 
in the future , with developments in radiographic technologies and increasing numbers of studies on their application to the forensic setting , radiology will become one of the most useful tools in the eld of personal identi cation . riassunto lidenti cazione personale dei cadaveri senza identit cruciale per motivi etici , giuridici e civilistici , e viene eseguita tramite un confronto fra i dati del pro lo biologico ottenuti dal cadavere e il materiale ante mortem di uno o pi sospetti didentit o persone scomparse . 
le crescenti applicazioni della radiologia forense e lampio uso della radiogra a tradizionale e della tomogra a assiale computerizzata ( tac ) nella comune pratica clinica mostrano la potenziale importanza di tali tecnologie come strumento di veri ca della corrispondenza fra un corpo senza identit ed un sospetto didentit . 
questo studio vuole esporre una revisione della letteratura riguardante lapplicazione della radiologia forense al complesso ambito dellidenti cazione personale ; nonostante la crescente importanza del confronto fra radiogra e ed immagini tac , numerosi limiti aspettano ancora di essere superati ; al momento infatti pochi istituti di medicina legale hanno accesso agli strumenti radiogra ci pi so sticati , e sta sorgendo un ampio dibattito sulla reale af dabilit di tali tecniche nellidenti cazione di speci ci tratti morfologici e lesioni su osso . 
inoltre , cos come altri metodi identi cativi basati sul confronto morfologico , anche il confronto fra dati radiogra ci ante e post mortem richiede una standardizzazione ed un approfondimento statistico , specialmente in europa ove esistono pochissime indicazioni sulla modalit di ammissione di una prova scienti ca ottenuta tramite metodi antropologici e radiologici in un processo . 
in futuro , con lo sviluppo delle tecnologie radiogra che e laumento del numero di studi riguardanti la loro applicazione a contesti forensi , la radiologia diverr uno degli strumenti pi utili nel campo dellidenti cazione personale . radiol med ( 2011 ) 116 : 960968 keywords forensic anthropology forensic radiology biological pro le personal identi cation parole chiave antropologia forense radiologia forense pro lo biologico identi cazione personale introduction introduzione forensic pathologists and anthropologists are often called in to help identify an unknown cadaver . 
the identi cation of an unidenti ed body is crucial both from a juridical point of view in particular for criminal , civil , insurance and administrative reasons and for ethical reasons for the reconciliation of the family with the lost relative . 
 whenever the unknown body is well preserved , bodily features such as height , eye colour , hair colour and marks such as tattoos or scars are usually useful to assist in identity . 
conversely , when the body is badly preserved , mummi ed , skeletonised or even charred , the application of techniques provided by forensic anthropology is mandatory . when the cadaver has a presumed identity and personal identi cation is requested , several disciplines can be applied , such as odontology , genetics and dactyloscopy . 
although plain radiography is widely used by experts [ 28 ] , today , more advanced radiographic techniques such as computed tomography ( ct ) and multidetector ct ( mdct ) are also applied in forensics [ 911 ]  . 
the odontological procedures for personal identi cation are the easiest and fastest to use and can be performed with dental charts , casts and various types of x - ray examinations [ 15 , 16 ]  . 
lidenti cazione di un cadavere senza identit cruciale sia da un punto di vista giuridico , poich coinvolge motivazioni di ordine penale , civile , assicurativo ed amministrativo , sia per esigenze etiche , nalizzate a restituire il corpo del defunto ai propri parenti . 
in caso di cadaveri sconosciuti con un sospetto didentit , tre fattori devono essere attentamente valutati : lo stato di decomposizione del corpo , il materiale ante mortem ( am ) raccolto dal sospetto didentit , e le tecniche disponibili per lidenti cazione , congiuntamente alle indicazioni riguardanti la loro accuratezza . 
 quando un cadavere sconosciuto ben conservato , le caratteristiche corporee come laltezza , il colore degli occhi e dei capelli , contrassegni come cicatrici , tatuaggi , ecc . , sono in genere utili allidenti cazione . 
tuttavia , quando queste ultime non possono essere utilizzate ( ad esempio , la documentazione am non disponibile ) , la radiologia forense diviene lunico strumento per ottenere unidenti cazione positiva [ 1 ]  . 
limmagine radiogra ca ampiamente e comunemente utilizzata in ambito identi cativo [ 28 ] , sebbene attualmente anche le pi avanzate tecniche radiogra che come a tomogra a assiale computerizzata ( tac ) e la multi - detector computed tomography ( mdct ) trovano unutile applicazione in ambito forense [ 911 ]  . 
 nel processo identi cativo i dati radiogra ci am e post mortem ( pm ) sono messi a confronto e la corrispondenza fra le due immagini deve essere ottenuta tramite una meticolosa comparazione dei dettagli [ 1214 ]  . 
questo approccio viene ampiamente utilizzato nel campo dellodontologia forense , ove le caratteristiche anatomiche , patologiche e iatrogene degli elementi dentari contribuiscono a rendere unico il pro lo dentario ; da questo punto di vista , le procedure di identi cazione personale basate su metodi odontologici sono le pi facili e veloci da effettuarsi , e possono essere eseguite tramite schemi dentali , sovrapposizioni dentarie e indagini radiogra che [ 15 , 16 ]  . 
laccuratezza e laf dabilit di tale approccio ampiamente riconosciuto ; nondimeno , tale confronto pu essere eseguito su ogni distretto osseo , anche 962 radiol med ( 2011 ) 116 : 960968 general , it is essential to detect and focus on the distinctive and unchangeable individual features . 
unfortunately , such unique and unchangeable features have not yet been de ned in forensic medicine , nor has any speci c number of concordant traits been established to ascertain an identity [ 18 ]  . 
for example , fischman states that one to four concordant features and no discrepancies are usually considered to claim an identity [ 14 ] ; brogdon [ 17 ] states that a minimum number should not be pursued , as a bunch of common and nonspeci c features is sometimes enough to claim an identity ; kuehn et al . 
in general , the identi cation process is quite easy and straightforward when dealing with bone prosthesis , bone calluses , osteoarthritis and other exceptional traits ; indeed , mann and brogdon state that one feature is enough when it is unique and unmistakable [ 4 , 17 ] , but ascertainment of identity is more dif cult when no particular feature is involved . 
moreover , research carried out on the frequencies of morphological characteristics of the skeleton , such as fractures , pathological conditions and surgical hardware , suggests that some features previously supposed to be suf ciently unique may in fact be quite common [ 20 ]  . 
in fact , the direct comparison of antemortem and postmortem x - rays is widely applied by international experts dealing with personal identi cation [ 2 , 5 , 22 , 23 ]  . 
judges subject forensic science techniques to strict evaluation of scienti c bases , and whenever a method fails to prove reliable , the expert examination may be rejected in court . 
anthropological and radiological methods in fact often deal with morphological assessment of skeletal features , which are based on the personal experience of the single expert witness and the error rate and standard deviation of which are not easily quanti able . 
this means that such methods cannot provide a reliable rate of probability of positive matching , which is requested by the daubert criteria on the admissibility of scienti c evidence in trial [ 24 ]  . 
only in recent years has forensic anthropology found a base for its admission in court , thanks to the kumho sentence , which stated that the daubert criteria are exible and that scienti c evidence se alcuni distretti sembrano pi af dabili di altri ( ad esempio , il torace , il capo , laddome ) [ 17 ]  . 
sfortunatamente , in ambito forense non si ancora raggiunta una de nizione di tali caratteri unici ed immutabili , n un numero speci co di tratti concordanti necessari per stabilire lidentit [ 18 ]  . 
ogni autore suggerisce un diverso numero di caratteristiche necessarie : fischman [ 14 ] sostiene che da uno a quattro tratti in comune e nessuna discrepanza emersa sono comunemente considerati suf cienti per raggiungere unidenti cazione positiva , mentre brogdon [ 17 ] dichiara che un numero minimo di caratteristiche non pu essere stabilito in quanto un insieme di caratteristiche anche solo comuni e non speci che talvolta suf cienti per effettuare unidenti cazione . 
in generale , il processo di identi cazione abbastanza facile e lineare quando riguarda protesi ossee , calli ossei , particolari forme di osteoartrosi ed altri tratti eccezionali ; infatti mann [ 4 ] e brogdon [ 17 ] sostengono che un solo tratto suf ciente se unico e inconfondibile , ma lidenticazione diviene molto pi dif cile quando non coinvolta nessuna caratteristica particolare . 
inoltre , una ricerca effettuata sulla frequenza delle caratteristiche morfologiche dello scheletro , come fratture , condizioni patologiche ed impianti chirurgici , suggerisce che alcuni tratti precedentemente considerati , suf cientemente unici , possono essere piuttosto comuni [ 20 ]  . 
alcuni autori sostengono che la quanti cazione e la standardizzazione delle caratteristiche utili allidenti cazione personale non necessaria e che lopinione del consulente in tali casi valido e suf ciente [ 21 ] ; infatti , la diretta comparazione del materiale radiogra co am e pm ampiamente applicata in ambito internazionale per lidenti cazione personale [ 2 , 5 , 22 , 23 ]  . 
 tuttavia , sfortunatamente questo approccio qualitativo pu mostrare alcuni limiti cruciali nel corso del processo ; infatti i giudici sottopongono le tecniche di indagine forensi ad una stretta valutazione delle basi scienti che e qualora un metodo fallisca nel dimostrarsi af dabile , quella speci ca indagine pu essere rigettata dalla corte ; i metodi antropologici e radiologici infatti spesso riguardano la valutazione morfologica di caratteristiche scheletriche , basata sullesperienza personale del singolo consulente , e il cui margine di errore e deviazione standard non sono facilmente determinabili : questo signi ca che tali metodi non possono fornire una af dabile probabilit riguardante lidenti cazione positiva , che tuttavia richiesta dai criteri stabiliti dalla sentenza daubert riguardati lammissibilit di una prova scienti ca nel processo [ 24 ]  . 
solo negli ultimi anni lantropologia forense ha trovato una base per la sua ammissibilit in aula , grazie alla sentenza kumho , che stabilisce che i criteri della radiol med ( 2011 ) 116 : 960968 may be based on the scienti c experience of the single expert witness [ 25 ]  . 
as a result , there is a strong need to quantify the error rate of the techniques used in forensic anthropology and radiology , and several forensic anthropologists are working to this effect . 
standardisation of the anthropological identi cation methods is particularly needed if one considers that speci c guidelines such as the daubert criteria or kumho sentence are still missing in europe , and that there is therefore no precise indication regarding the admission of scienti c evidence in court . 
 in forensic radiology , research for identi cation is conducted on the skeletal features of several body districts : head ( suture pattern , frontal sinuses ) [ 21 , 26 ] , thorax ( vertebrae , ribs , clavicles ) [ 17 , 19 , 27 ] , abdomen and pelvis ( vertebrae , scoliosis , etc . ) [ 17 , 23 ] , arms and legs [ 2 , 3 ]  . 
this paper presents a review of the classical and more recent applications of forensic radiology in the broad eld of identi cation subdivided into the issues of biological identity and personal identi cation . biological pro le : sex , age , stature at times it is necessary to build a biological pro le when no presumed identity exists . 
ct and mdct are widely explored and applied by several authors to a biological pro le [ 31 ] , also in mass disaster fatalities [ 9 , 11 ]  . 
several authors have demonstrated that information on sex , age and height can be obtained by obtaining ct scans of a dead body [ 9 , 32 ]  . positive or personal identi cation daubert sono essibili e che la prova scienti ca pu essere basata sullesperienza scienti ca del singolo consulente [ 25 ]  . 
per tale ragione , esiste attualmente una forte necessit di quanti care lerrore delle tecniche usate in antropologia e radiologia forense , e diversi autori sono impegnati per trovare una soluzione a tale necessit . 
gli antropologi forensi infatti stanno tentando di dare una quanti cazione ai risultati delle tecniche di identi cazione osteologica , e tale tentativo ancora pi necessario se si pensa che in europa speci che linee guida per lammissibilit della prova scienti ca come i criteri di daubert o la sentenza kumho sono ancora del tutto assenti , e pertanto non esiste ancora alcuna indicazione precisa riguardante tale argomento . 
 in radiologia forense , lapprofondimento dellidenti cazione avviene su criteri scheletrici provenienti da diversi settori corporei : capo ( suture craniche , seni frontali ) [ 21 , 26 ] , torace ( vertebre , coste , clavicole ) [ 17 , 19 . 
questo articolo ha come scopo la presentazione di una revisione della letteratura riguardante le applicazioni classiche e pi recenti della radiologia forense nel campo dellidenti cazione , suddivise nei due ambiti riguardati la de nizione dei caratteri biologici e lidenti cazione personale speci ca . 
la ricostruzione del pro lo biologico include la diagnosi di sesso , stima di et , statura e appartenenza etnica ; lesame radiologico dei distretti scheletrico e dentario sono imprescindibili per determinare let del soggetto se si trova nella fascia di et dellinfanzia e delladolescenza [ 28 ]  . 
la radiologia pu inoltre fornire importanti informazioni riguardanti il sesso ; diverse misure infatti possono essere utilizzate per una valutazione del genere del soggetto [ 29 , 30 ]  . 
la tac e la mdct sono attualmente ampiamente utilizzate ed applicate da diversi autori per la ricostruzione del pro lo biologico [ 31 ] , anche nel caso di disastri di massa [ 9 , 11 ]  . 
diversi autori hanno dimostrato che possibile ottenere informazioni su sesso , et ed altezza usando le scansioni tac su cadaveri [ 9 , 32 ]  . head identi cazione personale positiva capo there is a presumed identity , forensic radiwhen ology is essential for personal identi cation . 
different authors have reported cases of positive identi cation based on x - rays of the head through a pure visual comparison of quando presente un sospetto didentit , la radiologia forense ha unimportanza cruciale per lidenti cazione personale . 
in particolare i seni frontali sono ampiamente utilizzati e studiati , sia nellambito classico che in quello pi innovativo della radiologia : diversi autori 964 radiol med ( 2011 ) 116 : 960968 skeletal features [ 8 ]  . 
other authors focused on the application of advanced techniques to the frontal sinuses , with the aim of de ning an objective and quantitative method of analysis to replace simple visual comparison , with encouraging results [ 26 ]  . 
a similar quantitative approach , applied to cranial suture patterns , was adopted by rogers and allard [ 21 ] , who undertook an extensive statistical study of the number , position , length and direction of the lines , concluding that four consecutive corresponding lines ( for length , position and direction ) are enough to claim a positive identity [ 21 ]  . 
 recent clinical approaches involve the use of ct and magnetic resonance imaging ( mri ) instead of plain radiography , but this tendency makes it even more dif cult to obtain adequate antemortem data . 
solved an identi cation case by comparing the ct images of the bone structures of the skull , and in particular , the details of the frontal and sphenoid sinus , mastoid and ethmoid air cells , torcular and sagittal cranial suture [ 33 ]  . 
 they studied both morphological and metric features , concluding that the morphological feature ( presence or absence of the sinuses , presence , absence or incompleteness of intrasinus septum , etc . ) differed signi cantly in the population . 
altri autori si sono focalizzati sullapplicazione di tecniche pi avanzate ai seni frontali , con lo scopo di raggiungere un obiettivo metodo di analisi quantitativa , che potesse rimpiazzare la semplice comparazione a occhio dei pro li dei seni , con risultati incoraggianti [ 26 ]  . 
un simile approccio quantitativo , applicato al disegno delle suture craniche , fu seguito da rogers e allard [ 21 ] , che intrapresero un ampio studio statistico su numero , posizione , lunghezza e direzione delle linee , concludendo che 4 linee consecutive corrispondenti ( per lunghezza , posizione e direzione ) sono suf cienti per effettuare unidenti cazione positiva . i recenti approcci clinici riguardano luso della tomogra a elettronica a scansione e della risonanza magnetica nucleare ( rmn ) invece delle classiche proiezioni radiogra che , ma questa tendenza rende ancora pi dif cile la ricerca di adeguata documentazione am . 
 [ 33 ] hanno concluso un caso identi cativo tramite comparazione di immagini tac della struttura ossea del cranio , analizzando nel confronto i seni frontali e sferoidale , le celle mastoidee ed etmoidali e la sutura sagittale . 
 [ 10 ] cercarono di sviluppare un metodo facile ed af dabile per lidenti cazione personale sulla base dei seni frontali , analizzando sia gli aspetti morfologici che metrici , e concludendo che le caratteristiche morfologiche ( presenza od assenza dei seni , presenza , assenza od incompletezza del setto , ecc . ) erano signi cativamente differenti nella popolazione . 
 the thorax has proved very useful in several forensic cases because of the remarkable number of features offered by this area and the wide availability of chest x - rays in the population [ 17 ]  . 
 [ 19 ] , in a study using thoracic x - rays , measured the reliability of the visual comparison , established useful features for identi cation process and evaluated possible sources of error . 
the most important source of error proved to be torace il torace un distretto importante per lidenti cazione personale , per la presenza di un elevato numero di caratteristiche morfologiche utilizzabili , nonch per lampia disponibilit di radiogra e toraciche come materiale am nella popolazione generale [ 17 ]  . 
 [ 19 ] condussero uno studio simile su radiogra e del torace , con lo scopo di quanti care laf dabilit del confronto visuale e mettere in evidenza le caratteristiche utili nel processo identi cativo , veri cando anche le possibili fonti di errori . 
 [ 27 ] , and the skeletal features considered to be most useful were the same in both studies . abdomen and pelvis the abdomen and pelvis maintain their anatomical position in the case of an accident , and the complex morphology of vertebrae is useful for identi cation purposes . 
in 1997 , valenzuela reported on a case of identi cation conducted through comparison of lumbar x - rays , emphasising the usefulness of vertebral osteophytes , of the unique shape of transverse and spinous processes , of vertebral - body trabecular patterns , scoliosis and other features [ 23 ]  . 
however , until now , no quantitative study has been conducted on these districts . arms and legs identi cation from a single bone is based on the observation of anomalous development , degenerative disease , tumours , prostheses , trabecular patterns and so on . 
an example of morphological analysis of bone features in the identi cation process is given by dean et al . , who by simulating a comparison between ctitious antemortem and postmortem material using preand postsurgical radiographs of the ankle succeeded in achieving a positive identi cation [ 34 ]  . 
 the features reported to be useful in this research study compare well with those reported in other studies ( bone shape , trabecular bone pattern , shape of radio - opaque and translucent features and degenerative changes ) [ 4 , 36 ]  . 
la pi importante fonte di errore riguardava la qualit dellimmagine radiogra ca : brogdon [ 6 ] consider anche la posizione del cadavere nelle immagini radiogra che pm come unimportante fonte di errore . 
nel 1997 , valenzuela [ 23 ] riport un caso di identi cazione personale effettuata tramite il confronto di radiogra e del distretto lombare , tramite de nizione del pro lo degli osteo ti vertebrali , e la comparazione delle forme uniche dei processi trasversi e spinosi , del disegno trasecolare del corpo vertebrale , delle curve scoliotiche ed altre caratteristiche [ 23 ]  . 
 arti superiori ed inferiori lidenti cazione da un solo osso basata sullosservazione di caratteristiche morfologiche riguardanti un anomalo sviluppo , malattie degenerative , tumori , protesi , disegno trabecolare , ecc . 
 [ 34 ] , che simularono un confronto fra materiale am e pm ttizio utilizzando lastre radiogra che pree post - chirurgiche di anca , con un risultati positivi [ 34 ]  . 
 [ 3 ] pubblicarono un articolo riguardante il confronto visivo di radiogra e della mano ed osservarono che gli operatori con minima esperienza erano meno abili nel processo identi cativo degli operatori pi esperti in tale ambito . 
le caratteristiche registrate come utili allidenti cazione in tale studio coincidono con quanto riportato da altri autori ( forma dellosso , disegno trabecolare osseo , forma delle caratteristiche radiopache e radiotrasparenti e modicazioni degenerative ) [ 4 , 36 ]  . 
laccuratezza del metodo 966 discussion the discovery and development of x - ray technology has given a great contribution to the improvement of medicine and forensic sciences , with x - ray examinations being admitted in court very early after the discovery of x - rays [ 6 ]  . 
the importance of x - ray examination in forensic practice is also supported by developments in the eld of virtopsy and the application of increasingly advanced techniques to forensic cases [ 37 ]  . 
virtopsy involves the use of imaging techniques such as ct and mri to visualise the morphology and lesions of inner viscera and bones and obtain large amounts of information without invasive procedures . 
in addition , the importance of the radiological approach in forensics is demonstrated by its widespread use in cases of child physical abuse , where ascertaining the presence of signs of recent and past trauma is mandatory [ 38 , 39 ]  . the use of radiology in personal identi cation leads to several advantages . 
firstly , x - ray examination is the most common diagnostic test in clinical practice and therefore usually the most frequent antemortem source of information when there is a presumed identity . 
in addition , the same radiograph with the same characteristics of an antemortem x - ray examination can be performed on the unidenti ed corpse , and this means a more reliable comparison between antemortem and postmortem data . 
at the present time , few forensic centres own x - ray and ct equipment , even though odonnell and woodford [ 40 ] suggest that in the next 510 years , most centres will either gain access to ct facilities or install the equipment in their own laboratories . 
the most important limitation , however , consists in the reliability of x - ray imaging , which is widely debated by different authors , especially in the eld of the analysis and description of lesions . 
different authors have in fact remarked that ct and x - ray examinations can detect only a limited percentage of skeletal traits or lesions if compared with the evidence gained from autopsy or after maceration of bones [ 4143 ] , especially in speci c skeletal districts such as the vertebrae [ 44 ]  . 
the issue of precision and accuracy of x - ray technologies is a relevant point of debate and should be analysed in depth to ascertain whether such technologies are reliable in personal identi cation . although reliable , every identi cation procedure based on x - ray technologies similar to other morphological radiol med ( 2011 ) 116 : 960968 presenta una elevata dipendenza dalloperatore , ma lo studio propone una valutazione numerica di tale confronto . 
 discussione la scoperta e lo sviluppo della tecnologia dei raggi x ha dato un grande contributo allo sviluppo della medicina e delle scienze forensi ; infatti molto presto dopo la scoperta dei raggi x gli esami radiogra ci furono ammessi in tribunale [ 6 ]  . 
limportanza dellindagine radiogra ca nella pratica forense provata anche dallo sviluppo crescente registrato nel campo della virtopsy e dallapplicazione di tecnologie sempre pi avanzate ai casi forensi [ 37 ]  . 
la virtopsy nel dettaglio riguarda luso delle tecniche di diagnostica per immagine come la tac e la rmn per la visualizzazione della morfologia e di eventuali lesioni degli organi interni e delle ossa , allo scopo di raggiungere un maggiore numero di informazioni senza utilizzare procedure cruente . 
inoltre , limportanza dellapproccio radiologico provata anche dalla sua diffusione come fondamentale test diagnostico nel caso di abusi sici su bambini , ove laccertamento della presenza di segni di traumatismi recenti e pregressi di fondamentale importanza [ 38 , 39 ]  . luso della radiologia nel campo dellidenti cazione personale comporta numerosi vantaggi ; prima di tutto , gli esami radiogra ci costituiscono il test diagnostico pi comune nella pratica clinica , e pertanto costituiscono la pi frequente forma di informazioni am qualora sia presente un sospetto didentit . 
inoltre , le stesse proiezioni radiogra che del materiale am possono essere ottenute anche sul cadavere da identi care , con una conseguente elevata af dabilit del confronto fra i dati am e pm . 
al momento , gli istituti di medicina legale in possesso di macchine radiogra che o addirittura di strumenti tac sono pochi , anche se odonnell e woodford [ 40 ] suggeriscono che nei prossimi 510 anni la maggior parte degli istituti di medicina legale potr installare tali macchinari nei propri laboratori . 
diversi autori infatti osservano che le tac e le radiogra e classiche possono identi care solo una percentuale limitata di tratti e lesioni scheletriche , se comparate con le stesse percentuali evidenziate durante lautopsia o dopo macerazione delle ossa [ 4143 ] , specialmente in speci ci distretti vertebrali quali le vertebre [ 44 ]  . 
al momento il tema della precisione ed accuratezza dellindagine radiogra ca un punto cruciale di dibattito e dovrebbe essere approfondito per veri care la sua reale af dabilit nel campo dellidenticazione personale . ogni procedura radiogra ca basata su tecnologia radioradiol med ( 2011 ) 116 : 960968 methods for comparing antemortem and postmortem data suffers from a lack of literature data on mean error and the threshold for distinguishing between a correct and an incorrect match . 
in other words , although all skeletons are different , nothing can be said as to how different two skeletons are and how many common radiological features between two bones are needed to con rm a positive identi cation . 
if these limitations are overcome , the easy readability and widespread use of x - rays technology will lead to a heightened role of forensic radiology in the dif cult and crucial issue of personal identi cation . 
 gra ca , cos come per altri metodi morfologici di confronto fra i dati am e pm , sebbene af dabile , soffre della mancanza di dati disponibili in letteratura riguardanti lerrore medio e il livello soglia utile per distinguere unidenti cazione positiva . 
in altre parole , sebbene ogni scheletro sia diverso da qualsiasi altro , nulla pu essere stabilito su quanto due scheletri siano differenti , e quante caratteristiche radiologiche in comune fra due ossa siano suf cienti per confermare unidenti cazione positiva . 
un aiuto in tal senso pu derivare da studi sperimentali condotti su un ampio numero di radiogra e , allo scopo di trovare unindicazione numerica utile a distinguere i confronti con esito positivo ed i confronti con esito negativo . 
 se tali limiti saranno superati , la facile leggibilit e lampia diffusione della tecnologia dei raggi x determiner una crescente importanza della radiologia forense nel dif cile , eppure importante , campo dellidenti cazione personale . 
knauth springer - verlag , berlin heidelberg , 2010 isbn 978 - 3 - 540 - 79879 - 8 e - isbn 978 - 3 - 540 - 79886 - 6 doi 10.1007 / 978 - 3 - 540 - 79886 - 6 published online : 7 march 2011 springer - verlag 2011 this second edition of a well known book is intended to provide the reader with all the most updated information in the rapidly evolving eld of virtual colonoscopy . there are 17 chapters dealing with all the problems and important items the radiologist or more speci cally the colonographer needs when confronting this peculiar and important diagnostic tool . an in depth discussion is devoted to performing , properly evaluating and nally correctly reporting the ndings of a virtual colonoscopy examination . 
in this respect , importance is given to patient preparation ( cathartic drugs are not easily accepted ) , fecal tagging , use of contrast medium , proper computed tomography ( ct ) parameters and dose reduction , plus data interpretation . all of the above are important points , considering that virtual colonoscopy as a screening tool for colonic cancer is strongly advocated by the authors and important international cancer , radiologic and endoscopic societies . in order to avoid pitfalls or devastating consequences for the patient in case of missed lesions all throughout the book the importance of training in virtual colonoscopy image review and evaluation is emphasized . a standard reporting system is advocated to describe the exact location of a suspected or detected lesion , including its appearance , shape , dimension and extension in order to serve as a reference - point for the endoscopist in case of concurrent examination or in view of future check - ups . 2d and 3d images , three transparency views and comquesta seconda edizione di un ben noto volume ha lo scopo precipuo di fornire al lettore le pi aggiornate informazioni in merito al settore in rapida evoluzione della colonscopia virtuale . i diciassette capitoli sono dedicati a tutti i problemi e gli importanti aspetti che il radiologo o meglio il colonradiologo ha bisogno di conoscere quando si confronti con questo importante e peculiare strumento diagnostico . una disamina approfondita dedicata allesecuzione , alla valutazione appropriata ed in ne alla corretta refertazione di quanto riscontrato durante lesecuzione di una colonscopia virtuale . 
sotto questo punto di vista molta importanza deve essere posta nella preparazione del paziente ( le sostanze purganti non sono molto gradite ed accettate ) , alla marcatura delle feci ( fecal tagging ) , alluso del mezzi di contrasto , ai corretti parametri di tomogra a computerizzata ( tc ) per riduzione della dose nonch allinterpretazione dei dati . tutti i punti sopra indicati sono importanti , tenuto anche conto che la colongra a virtuale fortemente indicata quale strumento di screening del cancro del colon sia dagli autori che da importanti societ internazionali di radiologia , endoscopia e per lo studio dei tumori . 
 lungo tutto il volume viene enfatizzata limportanza della preparazione e della pratica necessarie nella revisione e valutazione delle immagini ottenute con la colonscopia virtuale allo scopo di evitare errori o conseguenze devastanti per il paziente nelleventuale mancato riconoscimento di lesioni . viene dunque perorata la creazione di un sistema standard di refertazione per la descrizione dellesatta localizzazione di una lesione sospetta o riconosciuta , completata dalla descrizione del suo aspetto , forma , dimensione ed estensione , tale da servire da punto di riferimento certo per lendoscopista in caso di concomitante indagine endoscopica o in vista di successivi controlli . 988 radiol med ( 2011 ) 116 : 987988 puter aided detection ( cad ) are the bread and butter of virtual colonoscopy . 
in fact virtual colonoscopy reading is time - consuming due to the attention and effort required and one should evaluate no more than six examinations in a session , in order to avoid mistakes and when there are doubts , review by more than one expert radiologist is mandatory . the references are up todate in all chapters ( up to 2008 ) and one 2009 reference is included in chapter 16 . 
 this is an important and impressive book , enriched by an enormous amount of perfectly reproduced images , a must have book for radiologists , endoscopists and surgeons who understand , as stated above , that virtual colonoscopy is a very important diagnostic and screening tool when in the proper hands , or a mouse - trap and source of devastating errors in the hands of others . immagini 2d e 3d , immagini tridimensionali in trasparenza e la diagnosi assistita dal computer ( computer aided detection , cad ) sono il pane quotidiano della colonscopia virtuale . 
in effetti la lettura di un esame di colonscopia virtuale implica una notevole quantit di tempo a causa dellattenzione richiesta e chi referta non dovrebbe superare la soglia di sei esami per seduta , allo scopo di evitare errori : in ogni caso , quando esistano dubbi , si rende necessaria la revisione da parte di pi di un esperto radiologo . le voci bibliogra che sono aggiornate in tutti i capitoli no al 2008 ed una riferita al 2009 inclusa nel capitolo 16 ; tuttavia la loro presentazione discordante : in ordine alfabetico nella maggior parte dei capitoli ; di citazione in altri . 
ciatto published online : 28 july 2011 springer - verlag 2011 b3 core biopsies should be assumed as positive ndings for accuracy purposes le biopsie percutanee b3 dovrebbero essere assunte come reperti positivi nella valutazione di accuratezza corte c brus 1g , 37067 valeggio sul mincio ( vr ) , italy , tel . : + 39 - 348 - 6540748 , e - mail : stefano.ciatto@gmail.com sir , gentile direttore , the paper by luparia et al . 
apart from uncommon pathological gures , such as b3 high prevalence ( 23.6% as compared to 9 - 10% average literature gures ) and a subsequent low positive predictive value ( ppv , 4.2% as compared to 25 - 30% average literature gures ) , or a null ppv for a few b4 cases , i would like to comment on the criteria adopted to assess vab accuracy . 
in particular , assuming b3 ndings as negative is uncommon , as they are currently assumed as positive in the literature [ 2 , 3 ] and usually referred for surgical con rmation [ 3 ]  . 
however , since they still referred to surgery 83% of b3 cases , it seems conrmed that b3 were dealt with as suspicious cases , that means positive for accuracy assessment purposes . 
a parte alcune caratteristiche isto - patologiche non usuali della casistica , come una elevata prevalenza di lesioni b3 ( 23 , 6% rispetto al 9 - 10% mediamente riportato in letteratura ) , un conseguente basso valore predittivo positivo ( ppv ) dei casi b3 ( 4 , 2% rispetto al 25 - 30% mediamente riportato in letteraura ) , e un ppv nullo per un numero limitato di lesioni b4 , mi lascia dubbioso il criterio adottato per la valutazione della accuratezza di vab . 
laver assunto le diagnosi b3 come negative non comune , dal momento che i b3 sono generalmente considerati positivi in letteratura [ 2 , 3 ] e avviati alla conferma chirurgica [ 3 ]  . 
ammetto che sussiste un dibattito sulla possibilit di individuare un sottogruppo di b3 che possano essere solo controllati nel tempo , come certi sottotipi istologici speciali ( lesioni papillari e radial scar ) e / o i b3 senza atipie , e comprendo anche che gli autori , visto il basso vpp dei loro casi b3 , abbiano scelto di discutere collegialmente la necessit di chirurgia in questi casi , ma il fatto che abbiano poi concluso per linvio al chirurgo nell83% dei casi conferma che hanno gestito i casi b3 come sospetti , e positivi per quanto riguarda laccuratezza diagnostica . 
questo soprattutto se gli autori vogliono confrontare i loro risultati con quelli di altri studi ( cosa che riportano in una tabella ad hoc ) che hanno considerato i b3 come positivi . 
ovviamente gli autori possono discutere uno scenario ipotetico nel quale i b3 non vengono mai avviati al chirurgo , consistente con una sensibilit del 94 , 9% e una speci cit del 98 , 3% . 
houssami n , ciatto s , bilous m et al ( 2007 ) borderline breast core needle histology : predictive values for malignancy in lesions of uncertain maligmnat potential ( b3 )  . 
ciatto s , houssami n , ambrogetti d et al ( 2007 ) accuracy and understimation of malignancy of breast core needle biopsy : the florence experience of over 400 consecutive biopsies . 
knauth springer - verlag , berlin heidelberg , 2010 isbn : 978 - 3 - 540 - 78449 - 4 e - isbn : 978 - 3 - 540 - 78450 - 0 doi 10.1007 / 978 - 3 - 540 - 78450 - 0 published online : 7 march 2011 springer - verlag 2011 the aim of this 209 page / 13 chapter book is to introduce all the technical details , opportunities and problems related to the use of digital mammography ( dm ) to the reader . 
 since its introduction about a decade ago , dm has represented a real breakthrough in the diagnostic eld of mammography due to its capabilities in daily work ow , image viewing and storage and ease of communicating these parameters . 
 hints on problems in image processing and in diagnosis when comparing images taken with different digital mammography equipments over time , conventional lm mammography or images taken in different locations with different equipments are presented and discussed . 
 results of clinical trials performed in the usa and europe are taken into account and discussed at length , as well as the impact of visibility and appearance of mammographic signs of malignancy . further improvements in dm , inherent to contrast - enhanced dm , digital breast tomosynthesis and breast computed tomography ( ct ) are discussed in the last two chapters . 
 a long yet incomplete two plus page list of abbreviations intendimento di questo volume , nei suoi 13 capitoli e 209 pagine , di presentare al lettore tutti i dettagli tecnici , le possibilit ed i problemi legati allimpiego della mammogra a digitale ( md )  . dalla sua introduzione , circa una diecina di anni fa , la md ha rappresentato un vero passo in avanti nel campo della diagnostica mammogra ca date le sue potenzialit nella attivit di lavoro giornaliera , le possibilit di valutare ed archiviare le immagini e la facilit di comunicazione di questi parametri . 
 vengono presentati e discussi suggerimenti sui problemi correlati con il trattamento delle immagini e con la diagnosi , quando se ne confrontino serie riprese nel tempo con differenti tipi di attrezzature mammogra che , con pellicole convenzionali oppure immagini ottenute in altre sedi con attrezzature diverse . chi si occupa di md dovrebbe essere preparato a studiare sia la sica che le tecniche proprie della metodica in modo tale da ottenere immagini corrette e che rispettino dei ben precisi criteri di qualit . 
una discussione approfondita dedicata a questi argomenti come anche a problemi pratici come lorganizzazione della sala referti e la sua illuminazione . vengono presi in considerazione e discussi a fondo i risultati di ricerche cliniche svoltesi negli stati uniti ed in europa come pure limpatto rappresentato dalla visibilit ed aspetto dei segni mammogra ci di malignit . 
 negli ultimi due capitoli vengono discussi ulteriori miglioramenti della md insiti nella stessa con aumento di contrasto , la tomosintesi digitale e la mammo - tomogra a computerizzata ( tc )  . 
as advocated in a previous book review it would be advisable to have this list of abbreviations on some loose or hard bound pages to have on hand , to be used ( very often ) in translation of abbreviations . the authors , all well known in their eld of research and daily work , have produced a book which will be of help to all those who are using or will be using dm in the future , to provide the best diagnostic results in the endless ght against breast cancer . una lunga , eppure non completa lista di abbreviazioni ( due pagine e linizio di una terza ) , apre il volume . 
come gi segnalato in una precedente recensione sarebbe consigliabile il poter avere disponibili su pagine separate e mobili , rigide o non , tali elenchi di abbreviazioni da poter utilizzare ( molto spesso ! ) nella traduzione delle stesse . 
 gli autori , tutti ben conosciuti nel loro campo di ricerca e lavoro quotidiano , hanno prodotto un libro che sar di aiuto a tutti coloro che gi usano o useranno in futuro la md , in modo tale da offrire i migliori risultati diagnostici nella lotta senza ne contro il cancro della mammella . 
gourtsoyiannis department of radiology , university hospital of heraklion , medical school of crete , 71110 stavrakia , heraklion , crete , greece department of rheumatology , university hospital of heraklion , medical school of crete , heraklion , crete , greece 3department of preschool education , university of crete , greece correspondence to : e . 
le immagini sono state valutate per la presenza e lestensione di intrappolamento daria , per il coinvolgimento delle vie aeree ( bronchi / bronchioli ) ed il modello hrct dominante . 
la differenza di attenuazione tra le regioni dintrappolamento daria in espirazione e le stesse regioni in inspirazione aveva un range compreso tra 32 e 89 unit di houns eld ( hu )  . 
the nonsigni cant correlation between at extent and inspiratory ndings may suggest at as an additional hrct nding in patients with wg . keywords wegeners granulomatosis bronchiolectasis air trapping risoluzione era patologica in 19 pazienti ( 90 , 4% ) e rappresentata dai seguenti modelli polmonari principali : nodulare , cavitario / non cavitario ( n = 7 , 38 , 9% ) , opacit a vetro smerigliato ( ggo ) ( n = 5 , 26 , 3% ) , masse ( n = 3 , 15 , 8% ) , modello brotico ( n = 3 , 15 , 8% ) e consolidativo con broncogramma aereo ( n = 1 , 5 , 3% )  . 
le regioni dintrappolamento daria nella granulomatosi di wegener , rappresentano un nuovo reperto , indiretto e probabilmente , in rari casi , lunico reperto di coinvolgimento polmonare rilevato in tc ad alta risoluzione . 
la correlazione non statisticamente signi cativa tra lestensione dellintrappolamento daria ed i reperti in fase inspiratoria possono suggerire lintrappolamento daria come un ulteriore reperto nella tc ad alta risoluzione in pazienti con granulomatosi di wegener . 
 parole chiave granulomatosi di wegener bronchiolettasia intrappolamento daria introduction first described by friedrich wegener in the late 1930s , wegeners granulomatosis ( wg ) is a rare systemic autoimmune disorder characterised histologically by a necrotising granulomatous vasculitis that most commonly involves the lungs , nasal pathways , paranasal sinuses and kidneys , but may affect any organ system [ 13 ]  . 
the disease traditionally belongs to the group of diffuse interstitial lung diseases that , according to a recent classi cation , has been renamed diffuse parenchymal lung diseases [ 4 ]  . 
it follows a chronic relapsing course , and the respiratory tract is the most commonly involved syste upper respiratory tract disease is reported in up to 90% and lung involvement in 7090% of patients [ 5 ]  . 
the disease involves the subglottic part of the airways , mainly the trachea and the main bronchi , causing bronchial wall thickening and airway lumen stenosis [ 1 , 2 ]  . 
 the primary aim of this study was to document the inspiratory high - resolution computed tomography ( hrct ) patterns and airway involvement as well as expiratory ndings in patients with known wg . 
disease assessment consisted of documenting the presence and extent of air trapping ( at ) on expiratory hrct scans and correlating it with the inspiratory hrct patterns and with the extent of bronchial and bronchiolar involvement , as studied on deep inspiratory and deep expiratory chest hrct images . materials and methods patients between april 1999 and january 2009 , 23 consecutive patients , 14 women and 9 men with an age range between 18 and 72 ( mean 45 ) years were retrospectively studied . 
twenty - one of these patients were newly diagnosed with wg , whereas two had been diagnosed 7 and 8 years , respectively , before the hrct scans were performed . 
two of these patients were active smokers ( mean 20 pack - years ) and 860 radiol med ( 2011 ) 116 : 858867 were excluded from the study , whereas among the remaining 21 patients , two were former smokers having ceased smoking 7 and 10 years , respectively , before the study began . 
at the time of examination , all 21 patients presented with a positive serum titre for anticytoplasmic antibodies ( canca ) , whereas 12 of them were positive for anti - pr3 antibodies . the clinical diagnosis of wg was established in accordance with the 1990 american college of rheumatology criteria [ 6 ] and the chapel hill criteria [ 7 ]  . 
transbronchial biopsy was performed in four patients , video - assisted thoracoscopy ( vat ) in two , open lung biopsy in one , renal biopsy in seven , nasal mucosa biopsy in four and skin and subcutaneous tissue biopsy in three . 
in each patient , sequential inspiratory scanning sequences were acquired at 10 - mm intervals from the level of the lung apices to below the costophrenic angles , and expiratory hrct scans were acquired with a 20 - mm interval . 
all images were reconstructed using a high - spatial frequency reconstruction algorithm . two experienced chest radiologists independently examined the hrct images in random order and recorded the presence and extent of ndings by visual assessment . 
expiratory scans were studied rst in order to avoid any bias in the interpretation of areas of at documented on expiration from the identi cation of inspiratory ndings , such as nodules and airway involvement . 
 on inspiration , the scans were visualised at two different window settings : one appropriate for lung parenchyma ( width 1 , 400 / level ; 700 hu ) and one ( width 1 , 000 / level ; 600 hu ) for scoring slight attenuation differences in the lung parenchyma . 
 the predominant parenchymal pattern nodules and masses ( cavitated or not ) , ground - glass opacities , consolidation areas and honeycombing as a sign of lung brosis was evaluated on inspiratory hrct scan . 
the presence or absence of bronchial and bronchiolar wall thickening was assessed considering that the wall thickness of conducting bronchi and bronchioles of < 5 mm in calibre should normally measure one sixth to one tenth of their diameter [ 8 ]  . 
hence , in every patient with suspected airway wall thickening , we calculated the ratio between the bronchial wall thickness ( t ) and total diameter of the bronchus ( d ) , which in normal individuals is 0.2 , or 20% , on average [ 9 ]  . 
 to assess possible airway dilation , a comparison was made between the internal diameter of the bronchus and the diameter of the adjacent pulmonary artery branch measured at sea level [ 10 ]  . 
to this end , we calculated the bronchoarterial ratio , de ned as the ratio between the bronchial luminal diameter ( l ) and the diameter ( d ) of its accompanying artery . 
a bronchoarterial ratio > 1 was considered typical of bronchiectasis , as was visualisation of a bronchus within < 2 cm of the costal pleura or abutting the mediastinal pleura . 
according to the nomenclature committee of the fleischner society , at is a pathophysiological term that indicates the retention of excess air in all or part of the lung at any stage of expiration [ 11 ]  . 
to assess the presence or absence of at , small regions of interest ( rois ) 12 cm in diameter including at least 95 pixels each were drawn freehand to measure the attenuation difference in houns eld units between areas of low attenuation on expiration and corresponding areas on inspiration . 
b hrct eseguita in espirazione allo stesso livello presenta delle zone dintrappolamento daria distribuita a carta geogra ca in ambedue le basi polmonari . at extent was classi ed as lobular when composed of hypoattenuation areas corresponding to fewer than three adjacent secondary pulmonary lobules in one or two regions per lung level , as segmental when corresponding to a pulmonary segment or three or more adjacent secondary pulmonary lobules , and as lobar when the hypoattenuation area was more extended than a pulmonary segment . 
in patients showing multiple at patterns , the most extensive one ( lobar rather than segmental ; segmental rather than lobular ) was taken in consideration [ 12 , 13 ]  . subsequently , the extent of at areas was evaluated by visual assessment using a semiquantitative scoring system estimating the percentage of lung that appeared abnormal on each scan [ 14 ]  . 
 [ 16 ] , was used to estimate at on expiratory scans in three different lung elds for each lung to a total of six lung elds for both lungs : upper lungs elds , from the lung apices to just above the level of the carina ; middle lung elds between the level of the carina and the pulmonary veins ; inferior lung elds from the pulmonary vein level to the level of the diaphragat each level and for each lung , a 5 - point scale was used to estimate the percentage of at extent visible to each radiologist : 0 for no at ; 1 for at between 1% and 25% of the cross - sectional area of the affected lung ; 2 for at between 26% and 50% ; 3 for at from 51% to 75% ; 4 for at between 76% and 100% [ 1416 ]  . 
more speci cally , the mann whitney and kruskalwallis tests were used to examine statistically signi cant differences between two mean values or among three or more mean values , respectively . 
the aim of the analyses was to correlate the extent of at on expiration with the predominant inspiratory hrct pattern and its total extent on inspiration as well as with airway involvement . 
 according to the 5 - point scale used for at extent , two patients ( 9.5% ) scored 0 , 16 ( 84.2% ) scored 1 , three ( 14.3% ) scored 2 and none scored 3 or 4 . 
at areas were observed in all six lung elds ( three for each lung ) and were distributed : bilaterally in the lower lung elds in all seven patients ( 100% ) , in the upper lung elds in four ( 51.7% ) and in the middle lung elds in three ( 42.9% ) ; in two , the radiol med ( 2011 ) 116 : 858867 fig . 
in our study group , the attenuation difference between inspiration and expiration ranged from 32 to 89 hu ( mean 6023.5 hu ) , whereas the attenuation measurements in the adjacent normal parenchyma ranged between 710 hu and 885 hu ( mean 795.461.7 hu ) on inspiratory scans and from 640 hu to 775 hu ( mean 69558.9 hu ) on expiratory scans , with a difference between inspiration and expiration that ranged from 100 hu to 250 hu . 
it is important to mention that none of the patients exhibited wall thickening of the trachea or the main bronchi , as commonly mentioned in literature [ 13 , 9 ]  . 
spearmans correlation coef cient between right and left lung at scores was statistically highly signi cant ( r = 0.97 , p < 0.01 ) , indicating that at was diffusely distributed . 
4 inspiratory chest high - resolution computed tomography ( hrct ) scan at the level of the right main bronchus shows thickening of the bronchial wall of the right upper lobar bronchus and its posterior and apical branches . 
4 hrct del torace in inspirio al livello del bronco principale di destra evidenzia ispessimento della parete bronchiale del bronco lobare superiore di destra e dei suoi rami posteriori e apicali . 
b nella nestra per parenchima polmonare la massa circoscritta da opacit a vetro smerigliato ( tc halo sign , che indica emorragia polmonare ) 864 radiol med ( 2011 ) 116 : 858867 fig . 
the disease affects about 3 : 100 , 000 people men and women equally and can occur at any age ( mean 41 years ) and , in comparison with other vasculitides , the lung is the most commonly affected organ , with a very aggressive airway pathology and chronic relapsing course [ 17 , 18 ]  . 
 in contrast to the well - described pulmonary parenchymal involvement in wg , the lower airway disease manifestations are less well recognised by clinicians , and little is known from the literature [ 2123 ]  . 
in the study by travis et al . , open lung biopsy was performed in 67 patients with wg , and a variety of bronchial / bronchiolar lesions were noted , including acute and chronic bronchiolitis ( 51% and 64% , respectively ) , follicular bronchiolitis ( 28% ) and bronchiolitis obliterans ( 31% )  . 
in other studies , small airway abnormalities in wg were characterised as unusual manifestations , including bronchiectasis and peribronchial thickening [ 2528 ]  . interestingly , no patient in our study had tracheal or main bronchial involvement . 
it is worth mentioning that all patients who presented with mild dyspnoea , dry cough , haemoptysis and inspiratory or expiratory wheezing had bronchial and bronchiolar involvement on inspiratory scans . 
based on these results , hrct appears to be capable of demonstrating segmental , subsegmental , bronchial and bronchiolar involvement in patients with wg , and the small airways may also be affected . 
 in normal individuals who are nonsmokers and have no history of exposure to organic or inorganic substances , the attenuation difference increases on expiratory hrct by about 150 hu compared with inspiratory scans [ 15 , 29 ]  . 
it is well known that areas of at show signi cantly less attenuation increase on expiration than in 866 radiol med ( 2011 ) 116 : 858867 normal lung , combined with a lack of volume reduction in these areas [ 30 ]  . 
usually , an attenuation increase of < 80 hu between inspiration and expiration may be indicative of at , so that comparison between inspiratory and expiratory ct scans may be helpful when at is subtle or diffuse [ 30 , 31 ]  . 
the most frequent at distribution pattern was segmental ( n = 4 , 19% ) , which together with the lobar pattern ( n = 1 , 4.7% ) are reported to be pathological and highly suggestive of small - airway disease [ 12 , 13 ]  . 
the probability of these at areas being pathological is higher if we consider that none of these patients was a smoker or suffered from asthma or chronic obstructive pulmonary disease . 
this con rms the diffuse involvement of lung parenchyma due to vasculitis , which is a systemic in ammatory disease , and this explains the geographical distribution of at areas . 
and include : ( a ) parenchymal necrosis , ( b ) vasculitis and ( c ) granulomatous in ammation accompanied by an in ammatory in ltrate composed of a mixture of neutrophils , lymphocytes , plasma cells , histiocytes and eosinophils [ 22 ]  . 
our hypothesis concerning the at nding is that it may be related to endoluminal formation of granulomas as well as to abnormal thickening of the bronchial and bronchiolar walls , causing bronchiectasis and bronchiolectasis and consequently at . 
 this hypothesis clearly needs to be veri ed . finally , the mean values of the total extent of at areas showed no statistically signi cant difference ( p > 0.005 ) between the predominant patterns on inspiratory hrct scans , such as pulmonary nodules , masses , ground - glass opacities , honeycombing and consolidation areas and the extent of airway involvement , including bronchial and bronchiolar wall thickening and the bronchiectasis and bronchiolectasis recorded in these patients . conclusions hrct in wg patients on deep inspiration may present various ndings , such as nodules , ground - glass opacities , consolidation areas and honeycombing . 
especially in the absence of inspiratory ndings , the presence of at may unveil pulmonary involvement in wg , playing an important role in the therapeutic approach to these patients . 
passariello1 1department of radiological sciences , university of rome sapienza , v.le regina elena 324 , 00161 rome , italy 2department of thoracic surgery , university of rome sapienza , rome , italy 3department of nuclear medicine , university of rome sapienza , rome , italy correspondence to : f . 
sono stai determinati i valori di accuratezza , sensibilit , speci cit , valore predittivo positivo ( ppv ) e valore predittivo negativo ( npv ) della tc - ds in confronto alla scintigra a perfusionale : 0 , 88 , 0 , 84 , 0 , 90 , 0 , 93 e 0 , 88 , rispettivamente . 
la tc - ds si presenta accurata e promettente , radiol med ( 2011 ) 116 : 842857 keywords lung cancer perfusion scintigraphy dualsource computed tomography lung perfusion fornendo importanti informazioni di natura funzionale sul polmone . parole chiave tumore del polmone scintigra a perfusionale tc a doppia sorgente radiogena perfusione del polmone introduction introduzione the indication for surgical intervention in patients with from lung cancer is based on both the cancer stage and patients clinical condition . 
when planning the surgical procedure , in particular if this is to be heavily destructive ( pneumonectomy , segmentectomy or lobectomy ) , preliminary pulmonary function tests are needed to obtain a predictive evaluation of the patients haemodynamic and ventilation tolerance [ 1 ]  . 
lung cancer patients candidate for surgical intervention are therefore evaluated in terms of two closely related aspects : morphology and function . computed tomography ( ct ) represents the gold standard in morphological characterisation , localisation and staging of lung cancer , and it is often supported by pulmonary function tests ( pft ) and ventilation / perfusion scintigraphy to measure parenchymal function . 
furthermore , it allows no direct comparison with morphological changes , and it exposes patients to an additional dose of radiation . the new dual - source ct ( dsct ) scanners , equipped with a dual x - ray source , exploit the different attenuation of iodine detectable at different energies and can consequently provide functional information based on the distribution of contrast material during its rst passage through the pulmonary capillary network . 
commercially available software provides both qualitative and quantitative information about iodine distribution and hence about regional perfusion by generating false - colour maps in which areas of the lung parenchyma with different degrees of perfusion appear in different colours ranging from white - yellow ( high degree of perfusion ) to blue - purple ( low degree of perfusion ) [ 57 ]  . 
in addition , integration with the automated study of the distribution of air volumes inside the lung parenchyma and areas of emphysematous change provides information regarding the extent of functional lindicazione allintervento chirurgico nei pazienti affetti da cancro del polmone viene valutata sia in base allo stadio della neoplasia , sia in base alle condizioni cliniche del soggetto . 
nella piani cazione di un intervento chirurgico , in particolare se ampiamente demolitivo , ( pneumonectomia , segmentectomia o lobectomia ) , necessario uno studio funzionale preliminare del parenchima polmonare al ne di effettuare una valutazione predittiva della tolleranza emodinamica e ventilatoria del paziente [ 1 ]  . 
 la tomogra a computerizzata ( tc ) rappresenta il gold standard nel processo di caratterizzazione morfologica , localizzazione e stadiazione del tumore polmonare ; essa viene af ancata dalle prove di funzionalit respiratoria e dalla scintigra a perfusionale polmonare per la valutazione dello stato funzionale del parenchima . 
attualmente , nella maggior parte dei centri di riferimento per la chirurgia del polmone , alle normali prove spirometriche viene infatti associata la valutazione scintigra ca nei pazienti che devono essere sottoposti ad interventi di pneumonectomia o resezioni polmonari ampie . 
il limite principale della tecnica scintigra ca rappresentato , tuttavia , da una limitata risoluzione spaziale e da una conseguente ridotta accuratezza nellindividuazione topogra ca di aree fotopeniche a distribuzione regionale . 
non inoltre possibile effettuare un confronto diretto con le alterazioni morfologiche e sottopone il paziente ad una ulteriore dose di radiazioni . le nuove apparecchiature tc denominate dual - source ( tc - ds ) , dotate di una doppia sorgente radiogena , sono in grado di sfruttare il differente potere di attenuazione dello iodio rilevabile a diverso voltaggio ; questo permette , quindi , di ottenere informazioni funzionali derivate dalla distribuzione del mezzo di contrasto ( mdc ) iodato in corrispondenza della fase di primo passaggio nella rete capillare polmonare . 
 la diffusione commerciale di software dedicati consente di ottenere informazioni sia qualitative che quantitative sulla distribuzione dello iodio e quindi sul grado di perfusione regionale con lelaborazione di mappe di falsi colori , in 844 radiol med ( 2011 ) 116 : 842857 ventilatory impairment [ 810 ]  . the purpose of our study was to determine the diagnostic potential of dsct in assessing pulmonary function in lung cancer patients who are candidates for surgical intervention . 
 data obtained with dsct were then compared with results of perfusion scintigraphy and pft . materials and methods patient selection and study design over a period of 6 months , we recruited 12 patients ( eight men and four women ; mean age 58.37 years ) with biopsyproven lung cancer ( maximum mean transverse diameter of the main lesion 64 cm ) who were candidates for possible surgical intervention . 
after approval from the local ethics committee and patients written consent informed had been obtained , patients were studied by dsct and lung perfusion scintigtable 1 disease stage for patients included in the study patient no . 
 stadio cui le zone di parenchima polmonare con differente grado di perfusione assumono tonalit cromatiche differenti in un range che va dal bianco - giallo ( elevato grado di perfusione ) no al blu - viola ( basso grado di perfusione ) [ 57 ]  . 
lintegrazione con lo studio automatizzato della distribuzione dei volumi aerei allinterno del parenchima polmonare e delle aree con compromissione en sematosa , consente , inoltre , di fornire informazioni per una valutazione del grado di danno funzionale ventilatorio [ 810 ]  . lo scopo del nostro studio stato quello di determinare le potenzialit diagnostiche della tc - ds nella valutazione funzionale del polmone nei pazienti affetti da patologia neoplastica polmonare candidati ad intervento chirurgico ; i dati ottenuti con la tc - ds sono stati confrontati con i risultati della scintigra a perfusionale e con le prove di funzionalit respiratoria . materiali e metodi selezione dei pazienti e progetto di studio durante un periodo di 6 mesi sono stati reclutati 12 pazienti ( 8 uomini , 4 donne ; et media : 58 , 37 ) con una neoplasia polmonare confermata mediante esame bioptico ( diametro trasverso massimo medio della lesione principale : 64 cm )  . 
dopo approvazione del comitato etico locale e sottoscrizione di un consenso informato , i pazienti sono stati sottoposti ad uno studio tc - ds e ad una indagine di scintigra a perfusionale polmonare ad una distanza temporale massima di 5 giorni . 
tutti i pazienti hanno inoltre eseguito le prove di funzionalit respiratoria con spirometria . protocollo tc lesame tc stato eseguito utilizzando una apparecchiatura dual source ( siemens somatom de nition , enlargen , germania )  . 
lacquisizione , condotta secondo un protocollo denominato dual energy , viene effettuata attraverso lutilizzo simultaneo di due tubi radiogeni a differente voltaggio , uno ad 80 kv ( in grado di ricoprire un campo di vista [ fov ] di 26 , 5 cm ) ed uno a 140 kv ( fov di 50 cm ) , con i seguenti parametri tecnici per entrambi : tempo di rotazione 0 , 33 secondi ; collimazione 0 , 6 mm ; spessore di strato 1 , 53 mlamperaggio viene variato utilizzando un protocollo care dose con quality reference impostato a 8 mas per il tubo a 140 kv e 340 mas per il secondo tubo . 
lapplicazione del protocollo care dose permette di ridurre i valori di mas somministrati senza perdita nella qualit dellimmagine : la corrente del tubo viene automaticamente ridotta in quelle posizioni angolari dove il radiol med ( 2011 ) 116 : 842857 raphy over a maximum period of 5 days . 
all patients also underwent pft with spirometry . diametro del paziente , e quindi i valori di attenuazione , sono minori [ 11 ]  . ct protocol ct exams were performed with a dual - source scanner ( siemens somatom de nition , erlangen , germany )  . 
scans were acquired with a dual - energy protocol , which relies on the simultaneous use of two x - ray tubes operated at different voltages : one at 80 kv ( fov 26.5 cm ) and one at 140 kv ( fov , 50 cm )  . 
the technical parameters were as follows : rotation time 0.33 s ; collimation 0.6 mm ; slice thickness 1.53 mmilliamperage was varied by using the care dose protocol with quality reference milliampere set at 8 mas for the 140 - kv tube and 340 mas for the 80 - kv tube . 
application of the care dose protocol reduces milliampere with loss of image quality : tube current is automatically reduced in the angular positions where the diameter of the patient and consequently the attenuation values are smaller [ 11 ]  . a dose of 70 cc of iodinated contrast material was administered at a high ow rate ( 5 cc / s ) , followed by 40 cc of saline solution at the same ow rate . 
synchronisation of ct acquisitions with the passage of contrast agent through the pulmonary circulation was achieved with the bolus - tracking technique ( care bolus ) : a circular region of interest ( roi ) was placed over the pulmonary artery to measure changes in attenuation values from the time of contrast administration ; the scan ( which lasted 56 s approximately ) was started when the contrast agent reached the vessel and the threshold attenuation value of 100 hu was achieved , with an additional 6 - s delay . 
to determine the stage of disease , a second , singleenergy scan was acquired of the chest , abdomen , pelvis and skull during the venous phase after administration of a second bolus of contrast material ( 60 cc at 3 cc / s ) with an additional 40 - s delay . 
 starting from the data set acquired with the dual - energy technique , two groups of images were produced : one with high - resolution reconstruction kernel ( b60 for the study of the lung parenchyma and one with a lower resolution kernel , b30 , for the study of soft tissues such as mediastinum and vascular structures )  . 
all studies were transferred to a workstation and processed using dedicated software . scintigraphy protocol all examinations were obtained with an in nia dual - head sono stati somministrati ad alta velocit di usso ( 5 cc / s ) 70 cc di mdc organo - iodato per ogni paziente , seguiti da un bolo di soluzione siologica di 40 cc alla stessa velocit di usso . 
la sincronizzazione temporale dellacquisizione tc con il passaggio del mdc a livello del circolo polmonare stata effettuata con tecnica di bolus tracking ( care bolus ) : una regione di interesse ( roi ) circolare viene posizionata da un operatore sullarteria polmonare per misurare le variazioni dei valori di hu dal momento della somministrazione del mdc ; la scansione ( della durata di circa 56 secondi ) inizia quando il mdc giunge nel vaso e viene raggiunto un valore soglia di 100 hu , con un ritardo ulteriore di 6 secondi . 
in tal modo lacquisizione viene effettuata in una fase di primo passaggio del mdc nel circolo polmonare : in tale fase vascolare si assiste alla opacizzazione esclusiva del circolo arterioso polmonare . 
ai ni del completamento della stadiazione tumorale , stata effettuata una seconda scansione ( a singola energia ) di torace , addome , pelvi e cranio in fase venosa , dopo somministrazione di un secondo bolo di mdc ( 60 cc a 3 cc / s ) con un ritardo aggiuntivo di 40 secondi . 
 a partire dai dataset acquisiti in doppia energia , sono stati prodotti due gruppi di immagini : uno con ricostruzione ad alta risoluzione per lo studio del parenchima polmonare ( denominato b60 ) ed uno a pi bassa risoluzione per lo studio dei tessuti molli ( mediastino e strutture vascolari , b30 )  . 
gli studi sono stati quindi trasferiti su work station dedicate ed elaborate mediante software ai ni del presente studio . protocollo della scintigra a lesame stato eseguito con apparecchiatura in nia a doppia testa ( ge ) , con collimatore low energy - high resolution dopo somministrazione di un valore dose - attivit di circa 185 mbq ( corrispondenti a circa 5 mc ) di microaggregati di albumina ( maa ) marcati con 99mtc . 
sono state eseguite sei proiezioni standard ( anteriore , posteriore , obliqua destra e sinistra , laterale destra e sinistra ) in un tempo complessivo per paziente variabile fra 10 e 12 minuti . 
 lacquisizione di tutte le proiezioni ha richiesto circa 800 kcounts ( 800.000 colpi ) , utilizzando una energia pari a 140 kev ( con nestra del 20% : range tra 126 e 154 )  . protocollo della spirometria tutti i pazienti hanno eseguito al momento del ricovero una spirometria di base con valutazione dei volumi dinamici : volume espiratorio massimo ( fev1 ) , forced vital capacity 846 radiol med ( 2011 ) 116 : 842857 camera ( ge ) , with low - energy , high - resolution collimator after administration of an activity value of approximately 185 mbq ( equivalent to 5 mc approximately ) of 99mtclabelled macroaggregated albumin ( maa )  . 
acquisition of all projections required approximately 800 kcounts using a 140 kev energy ( with 20% window ; range 126154 )  . ( fvc ) , forced expiratory ow ( fef ) 25%75% . i pazienti che assumevano terapia broncodilatatoria domiciliare hanno eseguito lindagine dopo la somministrazione dei farmaci al ne di effettuare le prove nelle migliori condizioni del paziente e riprodurne le condizioni cliniche di base allingresso in reparto ( dipendenti dalla somministrazione domiciliare dei farmaci broncodilatatori ) [ 12 , 13 ]  . elaborazione degli studi tc con software dedicati spirometry protocol analisi degli studi tc - ds perfusionali all patients underwent initial spirometry with measurement of dynamic volumes [ forced expiratory volume in 1 s ( fev1 ) , forced vital capacity ( fvc ) , forced expiratory ow rate ( fef2575% ) on admission to hospital . 
patients receiving home bronchodilator therapy underwent spirometry after drug administration so they could do the test in the best possible condition and reproduce their condition on admission , which depended on home bronchodilator use [ 12 , 13 ]  . ct postprocessing with dedicated software analysis of dsct perfusion studies evaluation of lung parenchyma perfusion was done on the basis of the dual - energy chest scans reconstructed with the b30 kernel ; data sets were transferred and processed using commercially available software ( siemens dual energy , syngo 2006g , siemens medical solutions )  . 
this scoring system was applied on both the dsct and scintigraphic studies . images were evaluated by two experienced chest radiologists ( ff , 11 years of experience , and cc , 15 years ) in a blinded fashion in order to measure interobserver variability . 
four weeks later , the images were re - evaluated with the same scoring system and the same software by one of the radiologists to measure intraobserver variability . la valutazione perfusionale del parenchima polmonare stata effettuata a partire dagli studi del torace acquisiti in doppia energia e ricostruiti con algoritmo di ricostruzione b30 ; i dataset sono stati trasferiti ed elaborati con un software commercialmente disponibile fornito dalla casa produttrice ( siemens dual energy , syngo 2006g ; siemens medical solutions )  . 
 gli studi sono stati valutati da due esperti radiologi toracici in cieco ( ff , 11 anni di esperienza , cc , 15 anni ) al ne di valutare il livello di variabilit inter - osservatore . 
un radiologo a distanza di 4 settimane ha rivalutato le immagini con lo stesso sistema di scoring , applicando nuovamente lanalisi del software di perfusione al ne di valutare il livello di variabilit intra - osservatore . analisi degli studi tc per la valutazione dei volumi polmonari e del grado di en sema i dataset acquisiti in singola energia ( 140 kv ) e ricostruiti con algoritmo ad alta risoluzione per il parenchima polmonare ( b60 ) sono stati trasferiti su una apposita consolle ed elaborati con un applicativo del programma syngo ( siemens , inspace 4d , lung parenchymal analysis ) , in grado di fornire radiol med ( 2011 ) 116 : 842857 fig . 
1a - c studio perfusionale del parenchima polmonare con tc dual - source : lindagine dimostra multipli difetti di perfusione su un piano sagittale ( a ) , coronale ( b ) e assiale ( c ) , riferibili al quadro bronco - en sematoso del paziente in studio . 
 analysis of ct studies in evaluating lung volumes and extent of emphysema the data sets acquired with the single - energy technique ( 140 kv ) and reconstructed with high - resolution kernel for lung parenchyma ( b60 ) were transferred to a dedicated workstation and processed using a syngo application ( siemens , inspace 4d , lung parenchyma evaluation ) , which automatically calculates values of lung volume and extent of emphysema ; this process does not require a dual - energy study . 
due medici nucleari in consensus hanno valutato le immagini scintigra che identi cando e registrando su una tabella i lobi ipoperfusi secondo il sistema di scoring utilizzato per immagini tc - ds . radiol med ( 2011 ) 116 : 842857 software . 
the scintigrams were evaluated in consensus by nuclear medicine physicians who identi ed and entered on a table the hypoperfused lobes using the scoring system used for the dsct images . analisi statistica statistical analysis statistical analysis was performed with a statistical software package ( spss 13 for macintosh ; spss , chicago , il , usa )  . 
agreement was considered poor for kappa values of 0.210.40 , fair for values of 0.410.60 , good for values of 0.610.80 and excellent for values of 0.811.00. results the mean duration of ct scans was 73 min , including patient preparation and positioning . 
the radiation dose delivered to the patient , calculated as dose - length product ( dlp ) in milligray per centimetre and displayed on the ct console for each patient , had a mean value of 305.429.6 , which is similar to values reported in the literature [ 14 , 15 ]  . 
in particular , as shown in table 2 , of a total of 60 lobes studied , the dsct evaluation reported the absence of perfusion defects in four lobes assessed as hypoperfused at scintigraphy . 
the values of regional lung air content derived from the automatic calculation provided by the ct software and lanalisi statistica stata interamente condotta con un software statistico dedicato ( spss 13 per macintosh ; spss , chicago , ill )  . 
i valori di accuratezza diagnostica , sensibilit , speci cit , valore predittivo positivo ( ppv ) e valore predittivo negativo ( npv ) sono stati calcolati utilizzando come reference standard la scintigra a perfusionale . 
 inoltre , la presenza o assenza di difetti perfusionali rilevati allesame di tc - ds stata confrontata con la presenza o assenza del medesimo reperto alla scintigra a perfusionale tramite il test di mcnemar . 
linter - reader agreement tra i due lettori e lintra - reader agreement tra le due letture di tc - ds , effettuate dal radiologo con maggiore esperienza , sono stati studiati tramite la di cohen . 
lagreement stato giudicato scarso per valori di = 0 , 210 , 40 , moderato per valori di = 0 , 410 , 60 , buono per valori di = 0 , 610 , 80 ed eccellente per valori di = 0 , 811 , 00 . risultati la durata media degli esami tc stata di circa 73 minuti , comprensivi di preparazione e posizionamento del paziente . 
la dose di radiazioni somministrata al paziente , calcolata come dose length product ( dlp ) in mgy per centimetro e riportata dalla tc per ogni paziente , ha presentato un valore medio di 305 , 429 , 6 , sovrapponibile a quelli riportati in letteratura [ 14 , 15 ]  . in tabella 2 sono riportati i dati delle valutazioni qualitative della scintigra a perfusionale e della tc - ds . 
i valori di accuratezza diagnostica , sensibilit , speci cit , ppv e npv della tc - ds nella valutazione qualitativa dei livelli di perfusione sono stati di 0 , 88 , 0 , 84 , 0 , 90 , 0 , 93 e 0 , 88 , rispettivamente . 
in particolare , come mostrato in tabella 2 , su un totale di 60 lobi studiati , la valutazione tc - ds ha riportato lassenza di difetti di perfusione in quattro lobi valutati invece come ipoperfusi alla scintigra a . 
the curves have a reverse trend : the higher the extent of emphysema , the lower the level of fev1 and vice versa . the mcnemar test showed no statistically signi cant difference ( p = 0.09 ) in the identi cation of patients with emphysema . 
 complete agreement was obtained between the rst and second reading by the same radiologist both in the evaluation of perfusion defects and of areas of emphysematous change . discussion spirometrici sono riportati nelle tabelle 3 e 4 . 
il gra co in figura 4 mostra la correlazione tra il grado di en sema e i valori spirometrici ( fev1 ) ; landamento delle curve risulta inverso : maggiore il grado di compromissione en sematica minore il livello di fev1 riportato e viceversa . 
il test di mcnemar non ha evidenziato differenze statisticamente signi cative ( p = 0 , 09 ) nellindividuazione dei pazienti con compromissione en sematosa . linter - reader agreement tra i due lettori nella valutazione della presenza di un difetto di perfusione allesame di tc - ds risultata eccellente ( = 0 , 92 ) ; un risultato analogo stato evidenziato per la valutazione della presenza di compromissione en sematosa ( = 0 , 94 )  . 
una completa concordanza stata ottenuta tra la prima e seconda lettura da parte dello stesso radiologo sia per la valutazione dei difetti di perfusione che per la valutazione delle aree con compromissione en sematosa . ct represents the modality of choice for the morphological evaluation and staging of lung cancer patients . 
these systems provide images of excellent technical and diagnostic quality within a few seconds , allowing imaging of most patients including discussione la tc rappresenta lesame di elezione nella valutazione morfologica e nella stadiazione del paziente con patologia tumorale del polmone . 
tuttavia , alcune informazioni de nite funzionali , rimangono appannaggio di valutazioni clinico - strumentali e della medicina nucleare , in particolare per ci che riguarda la valutazione della perfusione polmonare [ 24 ]  . 
the introduction of dedicated software in clinical practice has allowed us to obtain automatic reconstructions of lung perfusion on which a visual and qualitative analysis of possible hypoperfused areas may be carried out . the results of our study , con rmed by previous reports , millimetriche , in tempi di acquisizione pi rapidi rispetto alle macchine di precedente generazione . 
ci consente di ottenere studi con immagini di eccellente qualit tecnica e diagnostica in un tempo di acquisizione di pochi secondi , quindi effettuabili nella maggior parte dei pazienti , compresi quelli con severa patologia en sematosa o che necessitino di somministrazione di ossigeno permanente [ 16 , 17 ]  . lapplicazione di una doppia sorgente radiogena a differente voltaggio , con due sistemi di acquisizione disposti a 90 rispetto allasse di rotazione del gantry , consente di effettuare una discriminazione sica del tessuto in studio ed in particolare , nella nostra valutazione dello iodio presente durante la fase di primo passaggio del mezzo di 852 radiol med ( 2011 ) 116 : 842857 fig . 
3a - d evaluation of lung perfusion by dual - source computed tomography ( dsct ) and scintigraphy in a patient with lung cancer : both methods show a segmental perfusion defect surrounding the neoplasm ( localised in the basalposterior segment of the right lower lobe ) in a coronal and sagittal plane ( a , c ) in the dsct study and on a posteroanterior projection in the scintigraphic evaluation ( d )  . 
3a - d confronto tra lo studio perfusionale con tc dual - source e scintigra a in un paziente con adenocarcinoma del polmone : entrambe le indagini evidenziano un difetto di perfusione segmentale nella zona periferica della lesione localizzata nel segmento basale - posteriore del lobo inferiore di destra , apprezzabile su un piano coronale e sagittale in tc - ds ( a , c ) ed in proiezione postero - anteriore nello studio scintigra co ( d )  . 
in b si evidenzia un difetto ventilatorio in sede perilesionale . demonstrate that dual - energy ct is an accurate and reproducible technique that can easily be applied in all patients [ 16 , 17 ]  . 
as a result , the contrast agent must be administered at a high ow rate ( > 4.5 cc / s ) to enable detection of a large quantity of iodine inside the pulmonary circulation during the short acquisition time and at a high concentration to obtain more intense and homogeneous enhancement with a small quantity ( 6070 cc approximately per study )  . indeed , a major problem in software - mediated perfusion studies is related to the presence of beam - hardening artefacts at the level of the superior vena cava or of the subclavian and brachiocephalic veins . 
lintroduzione nella pratica clinica di software dedicati consente di ottenere ricostruzioni perfusionali automatizzate del parenchima polmonare su cui effettuare una valutazione visivo / qualitativa di eventuali aree ipoperfuse . i risultati del nostro studio , confermati anche in precedenti valutazioni , hanno dimostrato che lutilizzo della tecnica a doppia energia rappresenta una metodica accurata , riproducibile e di facile applicazione in ogni paziente [ 16 , 17 ] ; tuttavia necessario sottolineare alcuni rilevanti accorgimenti tecnici . 
la perfusione polmonare con tc - ds infatti basata sulla visualizzazione , con ricostruzioni basate su mappe di falsi colori , del passaggio del mdc attraverso la rete capillare polmonare . 
in primo luogo , pertanto necessario che il mdc iniettato sia : somministrato ad alta velocit di usso ( > 4 , 5 cc / s ) , cos da consentire nel breve tempo dacquisizione dellesame il rilevamento di radiol med ( 2011 ) 116 : 842857 table 4 top : mean values derived from the automatic analysis performed by the software in the computed tomography ( ct ) evaluation of emphysema in a topographic approach for the 12 patients . 
the defect una elevata quantit di iodio allinterno del circolo polmonare ; il mdc deve essere ad alta concentrazione , per ottenere un grado di enhancement maggiore ed omogeneo ed in quantit limitata ( circa 6070 cc per studio )  . una delle problematiche pi rilevanti nel processo di 854 radiol med ( 2011 ) 116 : 842857 pft / dsct emphysema fev1 ct - emphysema fig . 
4 il gra co mostra landamento dei valori di fev1 rispetto a quelli riportati automaticamente dal software per il grado di compromissione en sematosa ( valori medi ) per ogni paziente ; landamento delle due curve inverso : laddove il valore di fev1 maggiore , minore il grado di compromissione en sematosa riportato e viceversa ( r = 0 , 657 ; p < 0 , 0001 )  . elaborazione degli studi perfusionali mediato dal software pu derivare , infatti , dalla presenza di artefatti da indurimento del fascio a livello della vena cava superiore o della vena succlavia e brachiocefalica . 
i pazienti affetti da bronco - pneumopatia cronica ostruttiva ( bpco ) , in particolare , possono presentare un tempo di circolo sanguigno signi cativamente rallentato , ed necessario , pertanto , somministrare il mezzo di contrasto in un volume ridotto per evitare che esso sia ancora presente in sede cavale superiore durante il tempo di acquisizione . 
nel nostro studio la tecnica a doppia energia ha erogato al paziente una dose sovrapponibile a quella per un normale studio angio - tc del torace a singola energia ( computed tomography dose index [ ctdi ] : 7 , 14 mgy / cm )  . 
nel nostro studio stato inoltre applicato , per ogni paziente , un protocollo care dose , mantenendo , comunque , un elevato livello di qualit diagnostica . in ne , necessario ricorrere allutilizzo di software di elaborazione dedicati , oggi commercialmente disponibili ed integrabili in consolle af ancate alle apparecchiature tc , per ottenere in maniera rapida elaborazioni delle immagini fig . 
5a , b artefatto da indurimento del fascio nella valutazione perfusionale tc : la presenza del mezzo di contrasto a livello della vena succlavia destra e della vena cava superiore ha reso mal valutabile il parenchima polmonare in sede apicale e paracavale . 
subsequent administration of a saline bolus compacts the contrast bolus and ushes any residual contrast responsible for the artefact . a second factor to be taken into account in performing dual - energy ct imaging concerns the radiation dose delivered to the patient . 
in our study , dual - energy ct delivered a dose almost equivalent to that of normal , single - energy chest ct angiography ( ct dose index 7.14 mgy / cm )  . 
 furthermore , in our study , we used a care dose protocol for all patients while maintaining a high diagnostic quality . lastly , dedicated processing software programmes , which are commercially available and can be integrated into ct workstations , are needed to obtain rapid , reproducible and easy - to - interpret postprocessed images . 
in our study , use of a dsct scanner allowed us to obtain images that were qualitatively very similar to the scintigrams , with the advantage of a higher spatial resolution and topographic de nition of perfusion defects at the level of the pulmonary parenchyma . the selection criteria for choosing candidates for pneumonectomy and lobectomy require an evaluation of respiratory function and consequently of possible defects due to the often associated chronic obstructive pulmonary disease . 
 our preliminary experience reveals a good correlation between data obtained from the automatic evaluation of the extent of emphysema and the functional tests , in line with previous reports [ 1820 ]  . 
this quantitative approach has been investigated in previous studies [ 2123 ] and is particularly useful for : collecting information on the treatment plan by indicating even small changes in parenchymal density undetectable at routine ct ; identifying areas with the most pronounced emphysematous changes that require treatment [ 8 ]  . the combination of morphological and functional both perfusional and ventilatory information afforded by dsct allows the technique to be indicated as the method of choice for managing lung cancer patients scheduled for surgery . 
nel nostro studio , lapplicazione di una apparecchiatura tc dual - source ha consentito di ottenere immagini qualitativamente sovrapponibili a quelle scintigra che , con il vantaggio di una maggiore risoluzione spaziale e di de nizione topogra ca dei difetti di perfusione a livello del parenchima polmonare . la valutazione dei criteri di selezione dei pazienti candidati ad intervento di pneumonectomia e di lobectomia necessita di una valutazione della funzionalit respiratoria e pertanto di eventuali de cit legati alla condizione di bpco spesso associata . 
la nostra esperienza preliminare evidenzia una buona correlazione tra i dati ottenuti dalla valutazione automatizzata del grado di en sema e le prove funzionali , rispecchiando quelle che sono le esperienze note nella letteratura [ 1820 ]  . 
 nel nostro studio abbiamo utilizzato un software in grado di effettuare automaticamente una analisi topograca e quantitativa dei volumi aerei polmonari e del grado di compromissione en sematosa del parenchima . 
in particolare , per la selezione delle aree en sematose , stato scelto come cut - off un range di valori densitometrici compreso tra - 951 e - 1024 uh . 
questo approccio di tipo quantitativo gi stato valutato in studi precedenti [ 2123 ] e si dimostrato particolarmente utile per : ottenere informazioni sullandamento del programma terapeutico evidenziando alterazioni densitometriche parenchimali anche lievi e non valutabili allesame tc di routine ; consentire lindividuazione delle aree con maggior grado di compromissione en sematosa distrettuale su cui praticare un eventuale trattamento [ 8 ]  . la combinazione delle informazioni morfologiche e di quelle funzionali perfusionali e ventilatorie ottenute con la tc dual - source , consente di indicare tale tecnica quale metodica unica nella gestione dei pazienti affetti da patologia neoplastica del polmone candidati alla chirurgia . 
non abbiamo trovato riscontro , inoltre , di nessuno studio precedentemente pubblicato che riportasse una valutazione comparativa tra le informazioni funzionali ventilatorie integrate con i dati di perfusione tc - ds , associata al calcolo automatico del grado di en sema e dei volumi polmonari regionali in pazienti affetti da tumore del polmone . 
 [ 22 ] , in una valutazione simile effettuata su pazienti fumatori affetti da en sema , hanno riportato una correlazione fra il grado di en sema misurato automaticamente ed i difetti di perfusione riscontrati a livello parenchimale ( p = 0 , 0355 ; r = - 0 , 54 ) [ 22 ]  . 
no previous published study has reported a comparative evaluation of ventilatory function data integrated with dsct perfusion data associated with the automatic determination of extent of emphysema and regional lung volumes in lung cancer patients . 
data derived from our preliminary experience are very encouraging and suggest that further studies should be conducted on a larger number of patients . a second limitation is that our study did not include any quantitative assessment of lung perfusion levels in a comparison between scintigraphy and dsct . 
this analysis was not possible owing to the limited fov ( 26.5 cm ) of the low - voltage tube , which caused exclusion from the analysis of a small portion of peripheral pulmonary parenchyma ( in particular at the basallateral level ) in a group of large - build patients ( 8 / 12 )  . 
that way , the overall diagnostic accuracy proved to be adequate , as patients with small perfusion defects at the level of the basal segments ( undetectable because of small fov ) had additional defects in other areas of the same lobe that were depicted by dsct . 
as discussed in previous studies [ 14 , 24 ] , there is as yet no solution to this proble the improvement and evolution of dsct scanners will help to overcome this limitation in the near future . a third limitation is that the scintigraphic studies included a standard acquisition of six projections rather than a single photon emission ct ( spect ) acquisition . 
one example is given by possible areas of atelectasis : in dsct perfusion studies , these appear as nonperfused during the rst passage of the contrast agent owing to peripheral vascular bundles , whereas they appear as regularly perfused on scintigraphy owing to delayed blood lling of the collapsed region . 
hence , for correct interpretation , perfusion studies must always be combined with evaluation of the morphological images . dati derivati dalla nostra valutazione preliminare appaiono molto incoraggianti per un proseguimento dello studio su un numero pi ampio di pazienti selezionati . in secondo luogo nel nostro studio non stata effettuata una valutazione di natura quantitativa dei livelli di perfusione polmonare in un confronto tra scintigra a e tc dualsource . 
questa analisi non stata possibile a causa del limitato campo di vista ( 26 , 5 cm ) del tubo a basso voltaggio , che ha determinato lesclusione dalla elaborazione di una piccola parte della regione periferica del parenchima polmonare ( in particolare a livello della regione basalelaterale dei polmoni ) in un gruppo di pazienti ( 8 / 12 ) di grossa corporatura . 
stato possibile superare questo limite tecnico attraverso una analisi di tipo visivo / qualitativo : in tal modo i livelli complessivi di accuratezza diagnostica sono risultati adeguati in quanto i pazienti che mostravano piccoli difetti perfusionali a livello dei segmenti basali ( non evidenziabili a causa del fov ridotto ) , riportavano difetti addizionali localizzati in altre sedi dello stesso lobo dimostrabili nello studio tc - ds . 
il miglioramento e levoluzione delle apparecchiature tc dual - source consentir , in un prossimo futuro , di superare questo ostacolo . terzo , gli studi scintigra ci effettuati comprendevano una acquisizione standard di 6 proiezioni piuttosto che una acquisizione tomogra a computerizzata a emissione di fotoni singoli ( spect )  . 
 nonostante questa possa essere riferibile alla effettiva perfusione polmonare nei soggetti sani , un ruolo diverso deve esserle af dato nella valutazione della perfusione dei soggetti con patologia distruttiva parenchimale . 
un esempio pu essere dato da una eventuale area di atelectasia : nello studio perfusionale con tc - ds essa si presenta non perfusa durante il primo passaggio del mezzo di contrasto a causa dellaffastellamento dei vasi periferici , mentre apparir regolarmente perfusa allesame scintigra co per via di un riempimento sanguigno pi tardivo della regione collassata . 
per una corretta interpretazione lo studio perfusionale deve , pertanto , essere sempre associato alla valutazione delle immagini morfologiche . nuove apparecchiature dual - source sono in fase di studio e saranno presto disponibili con un incremento dellamradiol med ( 2011 ) 116 : 842857 new dsct scanners are under investigation and will soon be available with increased width of the second integration with high - concentration contrast detector ; agents ( 500 mg / i ) will lead to a signi cant improvement in the diagnostic quality of the technique . in conclusion , dsct is an accurate and promising technique that provides important information on lung function . 
 piezza del secondo detettore ; lintegrazione con mdc ad elevata concentrazione ( 500 mg / i ) consentir un ulteriore miglioramento della qualit diagnostica della metodica . in conclusione , la tecnica tc con doppia energia si presenta accurata e promettente , fornendo importanti informazioni di natura funzionale sul polmone . 
guarise1 1struttura complessa di radiologia , ospedale san bassiano , bassano del grappa , italy 2struttura complessa di chirurgia vascolare , ospedale san bassiano , bassano del grappa , italy correspondence to : g . 
multihole microcatheters allow the thrombolytic agent to be distributed more evenly into the clot and may help to reduce reactive arterial spasm . keywords intra - arterial thrombolysis acute hand ischaemia urokinase microcatheter riassunto obiettivo . 
il microcatetere multiforato un utile supporto che permette una pi omogenea distribuzione dellagente trombolitico allinterno del coagulo e pu ridurre lo spasmo arterioso reattivo . parole chiave trombolisi intra - arteriosa ischemia acuta della mano urochinasi microcatetere 920 introduction acute hand ischaemia is an infrequent occurrence , and the clinical and therapeutic approach is still controversial [ 13 ]  . 
although a few case series of acute ischaemic lesions have been described in the literature , the series are small and do not allow for guidelines regarding the most appropriate therapeutic approach [ 4 , 5 ]  . 
furthermore , no study has as yet compared the results of surgical thrombectomy ( the gold standard in the treatment of thrombotic ischaemia ) [ 5 ] and intra - arterial thrombolysis ( an emerging , minimally invasive method with few local or systemic complications and with good results in other body regions ) [ 13 , 6 ] , nor has the use of multihole microcatheters been described in this particular region of the body . the purpose of this paper is to describe our initial experience in the treatment of acute ischaemic hand lesions with percutaneous intra - arterial thrombolysis with or without multihole microcatheters and compare the results with those available in the literature . materials and methods between march 2005 and may 2009 , three men ( mean age 39 years ; range 2648 years ) presented to the emergency department for acute hand ischaemia following an accidental intra - arterial injection of cocaine ( n = 1 ) or a traumatic event ( n = 2 )  . 
on clinical presentation , all three patients exhibited the typical clinical signs of acute ischaemia : nger pain , pallor , nail cyanosis , coldness , dysfunction , paraesthesia / dysaesthesia . 
preliminary doppler ultrasound ( us ) examination ( iu22 philips , philips healthcare , amsterdam , the netherlands ) con rmed the patency of the radial and ulnar arteries down to the wrist , with no ow signal in the affected digital arteries . all three patients subsequently underwent selective catheterisation of the humeral artery after antegrade access through the humeral artery ( n = 1 ) or retrograde access through the common femoral artery ( n = 2 ) using seldingers technique ( with a 18 - gauge needle cannula ; arterial introducer , radifocus introducer ii , 4 or 5 f , 10 cm , terumo corporation , tokyo , japan ) with placement of a diagnostic catheter ( bern imager ii , 4 or 5 f , 65 or 100 cm , boston scienti c , natick , ma , usa ) into the ipsilateral humeral artery . 
sono stati descritti in letteratura alcuni casi di lesioni ischemiche acute , ma le casistiche riportate sono limitate e non permettono una standardizzazione di linee guida riguardanti lapproccio terapeutico [ 4 , 5 ]  . 
inoltre a tuttoggi non stato compiuto alcuno studio comparativo riguardo i risultati della trombectomia chirurgica ( consolidata come gold standard di trattamento delle ischemie trombotiche ) [ 5 ] con la trombolisi endoarteriosa ( metodica emergente , poco invasiva , con ridotte complicanze sia locali che sistemiche e con buoni risultati gi documentati in altri distretti corporei ) [ 13 , 6 ] , n stato riportato luso di microcatetere multiforato in questo particolare distretto corporeo . scopo di questo lavoro riportare la nostra esperienza iniziale nel trattamento delle lesioni ischemiche acute della mano con trombolisi endoarteriosa percutanea senza o con microcatetere multiforato e confrontare i risultati con quelli della letteratura . materiali e metodi nel periodo compreso fra marzo 2005 e maggio 2009 tre pazienti maschi ( et media 39 anni , intervallo di et 2648 anni ) si sono presentati al pronto soccorso per lesione ischemica acuta della mano secondaria a iniezione accidentale endoarteriosa di cocaina ( 1 caso ) oppure post - traumatica ( 2 casi ) in un intervallo di tempo medio di circa 2 ore dallinsorgenza della sintomatologia acuta . 
alla presentazione clinica tutti e 3 i pazienti manifestavano i tipici segni clinici di ischemia acuta : dolore , pallore , cianosi ungueale , dita fredde , impotenza funzionale , parestesie / disestesie delle dita . 
allesame eco - color doppler preliminare ( iu22 philips , philips healthcare , amsterdam , olanda ) si confermava la perviet delle arterie radiale e ulnare no al polso , con assenza di segnali di usso arterioso a livello delle arterie delle dita interessate . tutti e 3 i pazienti sono stati quindi sottoposti a cateterismo selettivo dellarteria omerale , previo accesso arterioso omerale anterogrado ( 1 caso ) oppure arterioso femorale comune retrogrado ( 2 casi ) con tecnica di seldinger ( agocannula da 18 g ; introduttore arterioso radifocus introducer ii , 4 o 5 f , 10 cm , terumo corporation , tokio , giappone ) e posizionamento di un catetere diagnostico ( bern imager ii , 4 o 5 f , 65 o 100 cm , boston scienti c , natick , ma , usa ) in arteria omerale omolaterale . 
intra - arterial thrombolysis was performed by administering a bolus of urokinase ( crinos , milan , italy , 200 , 000300 , 000 iu ) , combined with a vasodilator ( verapamil , isoptin , knoll , 0.40.8 cc ) in all three cases . 
in the rst case , the procedure was completed with the administration of an intra - arterial bolus of 12 , 500 iu of heparin sodium ( epsoclar , hospira )  . results results are summarised in table 1 . 
in all of them , the arterial occlusion was distal ( ulnar artery , carpal arch ) or ultradistal ( metacarpal arteries , proper digital arteries )  . in all cases , urokinase thrombolysis without ( n = 1 ) or with ( n = 2 ) the microcatheter was completed with the administration of an intra - arterial vasodilator to prevent or treat a reactive spasm , with good angiographic and clinical results . the only local complication ( patient 1 ) , occurring 67 h later , was a haematoma at the site of arterial access resulting from the accidental puncture ( no us guidance ) of an ulnar artery originating from the brachial artery above the elbow joint ( anatomical variant )  . discussion acute hand ischaemia is an infrequent condition , and the few reports in the literature generally regard small case series of posttraumatic ischaemia [ 79 ] or larger series of patients with chronic kidney disease undergoing haemodialysis and with brescia - cimino arteriovenous stulas [ 4 , 10 , 11 ]  . 
nel 1 caso la procedura stata completata con somministrazione di eparina sodica ( epsoclar , hospira ) in bolo endoarterioso ( 12500 ui )  . risultati i risultati sono riassunti in tabella 1 . 
tutti e 3 i pazienti trattati erano maschi , giovani o relativamente giovani , con fattori di rischio traumatici o legati a tossicodipendenza ; nessuno di essi soffriva di insuf cienza renale cronica . 
in tutti e 3 i casi locclusione arteriosa era distale ( arteria ulnare , arcata arteriosa del polso ) o ultra - distale ( arterie metacarpali , arterie digitali proprie )  . il trattamento trombolitico con urochinasi senza ( 1 caso ) o con ( 2 casi ) microcatetere stato completato in tutti e 3 i casi con vasodilatatore intra - arterioso per prevenire / trattare uno spasmo arterioso reattivo , con buoni risultati angiogra ci e clinici . 
si veri cata come unica complicanza locale ( 1 caso ) dopo circa 67 ore un ematoma nel sito di accesso arterioso al braccio per puntura accidentale ( senza guida ecogra ca ) dellarteria ulnare che originava dallarteria brachiale al di sopra dellarticolazione del gomito ( variante anatomica )  . discussione lischemia acuta della mano condizione poco frequente con limitate esperienze riportate in letteratura , riguardanti casistiche generalmente piuttosto esigue ad eziologia posttraumatica [ 79 ] oppure casistiche pi ampie di pazienti in insuf cienza renale cronica in trattamento emodialitico portatori di stola artero - venosa ( a - v ) di brescia - cimino [ 4 , 10 , 11 ]  . 
le cause pi frequenti di ischemia acuta delle estremit distali della mano comprendono stenosi ed occlusioni arteriose distali ( trombotiche o emboliche ) su base aterosclerotica , post - traumatica ( attivit sportive o lavorative con microtraumatismi ripetuti ) [ 79 ] , embolica ( brillazione atriale , valvulopatia cardiaca ed endocardite , 922 radiol med ( 2011 ) 116 : 919931 radiol med ( 2011 ) 116 : 919931 924 radiol med ( 2011 ) 116 : 919931 lation , heart - valve disease and endocarditis , patent ductus arteriosus ) [ 112 ] , accidental intra - arterial introduction of chemical irritants ( drugs ) [ 1317 ] , anatomical abnormalities ( nerve - entrapment syndrome ) causing extrinsic compression [ 7 , 8 ] or arterial steal syndrome . the clinical presentation of acute hand ischaemia includes the typical symptoms and signs of acute ischaemia : nger pain , pallor , nail cyanosis , coldness , sensory and / or motor impairment of the ngers and , in dialysis patients , ischaemic ulcers . 
the differential diagnosis of acute hand ischaemia includes other similar clinical conditions unrelated to impaired arterial supply , such as venous hypertension , neuropathy ( diabetic , uraemic ) or carpal tunnel syndrome , and it is based on an appropriate and detailed history ( including occupational and sports activities ) , accurate physical examination , instrumental investigations such as pulse oximetry , doppler us , computed tomography ( ct ) or magnetic resonance ( mr ) angiography [ 18 ] and , possibly , digital subtraction angiography ( dsa )  . 
 in some reports , percutaneous intra - arterial thrombolysis [ 14 ] is or is not combined with percutaneous transluminal angioplasty ( pta ) [ 10 , 11 ] in cases of acute hand ischaemia ( table 2 ) , with associated vascular stenosis as the risk factor for decreased distal arterial blood ow , which increases the likelihood of thrombotic vessel occlusion . 
the patient populations described in the literature are generally small and heterogeneous , with the inclusion of patients with and without chronic kidney failure and different pharmacological or surgical treatments . 
among reports involving patients without chronic kidney failure treated with intra - arterial brinolysis , patient series ( 612 patients ) comprise various causes of arterial occlusion and , as in the report by widlus et al . 
 however , none of these reports have described the use of microcatheters for superselective catheterisation of the affected arteries . in our admittedly very small patients series , all three patients were relatively young and had no anatomical or familial risk factors for the condition ( there were no cases of chronic kidney failure or arteriovenous stula ) , and no subclinical stenoses requiring dilation ( table 2 )  . 
la diagnosi differenziale di una sindrome ischemica acuta della mano deve essere posta con altre condizioni cliniche simili , ma non associate a de cit dellapporto arterioso , quali ipertensione venosa , neuropatia ( diabetica , uremica ) o sindrome del tunnel carpale , e comprende una corretta e dettagliata anamnesi ( anche lavorativa e sportiva ) , un esame obiettivo accurato e , poi , indagini strumentali quali pulsossimetria , eco - color doppler , angio - tomogra a computerizzata ( tc ) o angiorisonanza magnetica ( rm ) [ 18 ] ed eventuale angiogra a digitale a sottrazione dimmagine ( dsa )  . 
 nel caso di eco - color doppler non diagnostico o dubbio , utile , per decidere la tipologia di trattamento , approfondimento diagnostico con studio angio - tc o angio - rm . 
 in alcuni lavori in letteratura la trombolisi percutanea endoarteriosa [ 14 ] viene associata o meno ad angioplastica transluminale ( pta ) [ 10 , 11 ] per il trattamento di lesioni ischemiche acute della mano ( tabella 2 ) con associata stenosi vascolare , che rappresenta la condizione predisponente ad una riduzione del usso sanguigno arterioso a valle , e , quindi , contribuisce ad aumentare la probabilit di occlusione trombotica del vaso stesso . 
tra quelle riguardanti pazienti senza insuf cienza renale cronica , trattati con terapia brinolitica intra - arteriosa , le serie riportate ( comprendenti da 6 a 12 pazienti ) contemplano cause differenti di occlusione arteriosa e , nel caso del lavoro di widlus et al . 
 [ 12 ] , luso di farmaci brinolitici differenti ( streptochinasi e urochinasi ) ; tuttavia , in nessuna di esse riportato luso di microcatetere per il cateterismo superselettivo delle arterie bersaglio . nella nostra casistica , numericamente molto limitata , tutti e 3 i pazienti erano relativamente giovani e non vi erano fattori anatomici o anamnestici favorenti ( nessuno dei pazienti era affetto da insuf cienza renale cronica , n portatore di stola a - v ) , n stenosi subcliniche meritevoli di eventuale trattamento dilatante associato ( tabella 2 )  . 
le cause identi cabili nella nostra casistica erano la microembolizzazione associata a radiol med ( 2011 ) 116 : 919931 926 radiol med ( 2011 ) 116 : 919931 radiol med ( 2011 ) 116 : 919931 fig . 
b angiography after intraarterial thrombolysis ( urokinase ) , vasodilator ( verapamil ) and heparthe angiogram shows partial restoration of patency of the proper digital arteries to the rst and second digits ( long arrow ) and a reactive spasm of the distal radial artery ( short arrows )  . 
langiogra a mostra parziale recupero della perviet delle arterie digitali proprie del 1 e 2 dito ( freccia lunga ) , e uno spasmo reattivo dellarteria radiale distale ( frecce corte )  . 
c langiogra a mostra lorigine prossimale dellarteria ulnare ( u ) e dellarteria radiale ( r ) dallarteria brachiale ( freccia )  . repeated microtraumas with consequent endothelial damage and superimposed acute thrombosis in the remaining two patients . the peculiarity of the upper limb arteries , which are medium to small in size and course fairly super cially , lies in their sensitivity to mechanical and chemical stimuli to which they respond sharply and immediately with spasms that may be very intense . 
therefore , brinolytic therapy for acute occlusions should involve the combination of the thrombolytic agent with a vasodilator in consideration of the spastic component always associated with such lesions in this anatomic region , in contrast to , for example , the lower limbs , where the reactive spasm is not constant . the type of arterial puncture is another crucial aspect for the outcome of treatment . 
whereas we initially ( patient 1 ) opted for direct antegrade puncture of the humeral artery , which is technically dif cult and not free of complications danno endoteliale da irritazione chimica , nel 1 caso , ed il microtraumatismo ripetuto con conseguente lesione endoteliale e trombosi acuta sovrapposta , nei rimanenti 2 casi . peculiarit delle arterie dellarto superiore ( di medio e piccolo calibro , piuttosto super ciali ) la particolare sensibilit agli stimoli sia meccanici che chimici , ai quali reagiscono vivacemente e subitamente con spasmi reattivi anche molto intensi . 
pertanto il trattamento brinolitico in caso di occlusione acuta , tenendo conto della componente spastica sempre associata in questo tipo di lesioni , dovrebbe contemplare lassociazione del trombolitico con il vasodilatatore , a differenza di altri distretti ( per esempio arti inferiori ) ove , invece , lo spasmo reattivo non sempre costante . il tipo di puntura arteriosa un altro punto cruciale ai ni del risultato del trattamento . 
a preliminary angiography shows occlusion of the palmar arch at the level of the fourth metacarpus ( long arrow ) and occlusion of the ulnar artery at the level of the carpus ( short arrow )  . 
 a langiogra a preliminare mostra occlusione dellarco palmare a livello del 4 metacarpo ( freccia lunga ) e occlusione dellarteria ulnare a livello del carpo ( freccia corta )  . 
d langiogra a nale mostra recupero della perviet dellarco palmare e recupero della perviet dellarteria ulnare distale ectasica ( freccia )  . ( leakage , haematoma , pseudoaneurysm , antegrade dissection , arteriovenous stula ) , in the two following cases , we performed conventional retrograde puncture of the common femoral artery at the groin using seldingers technique and selective catheterisation of the brachial artery . 
this latter technique is easier and better established , although it has the disadvantage of requiring very long catheters ( at least 150 cm ) to arrive as close as possible to the acute thrombotic lesion ( usually very distal )  . 
for this reason , in the last two cases , we used special multihole microcatheters for neuroradiological use ( micromewi , micro therapeutics , risma , dissezione anterograda , stola a - v ) , negli ultimi 2 casi , invece , siamo ricorsi alla puntura tradizionale retrograda dellarteria femorale comune allinguine secondo la tecnica di seldinger e cateterismo selettivo dellarteria brachiale interessata , tecnicamente pi facile e consolidata rispetto alla prima , ma con lo svantaggio di richiedere cateteri molto lunghi ( almeno 150 cm ) per poter arrivare il pi vicino possibile alla lesione trombotica acuta da trattare ( di solito molto distale )  . 
a preliminary angiography shows occlusion of the palmar arch at the level of the fourth and fth ngers ( long white arrow ) , occlusion of the distal ulnar artery ( short white arrow ) and occlusion of the digital arteries to the fourth nger and to the stump of the fth nger ( short black arrows ) b final angiography after intraarterial thrombolysis ( urokinase ) and vasodilator ( verapamil ) using a microcatheter . 
a langiogra a preliminare mostra occlusione dellarco palmare a livello del 4 - 5 dito ( freccia lunga bianca ) , occlusione dellarteria ulnare distale ( freccia corta bianca ) e occlusione delle arterie digitali del 4 dito e del moncone del 5 dito ( frecce corte nere )  . 
these are very thin ( 2.9 f ) and long ( 150180 cm ) , with multiple side holes and two radiopaque markers at the distal end , which can be used coaxially with a standard vertebral or multipurpose diagnostic angiography catheter ( 5 f )  . 
the microcatheters can be placed very distally into small - calibre arteries without causing reactive spasms that would risk invalidating or negatively in uencing the results of the intravascular thrombolysis procedure . 
in addition , they allow better distribution of the brinolytic agent over a larger area of the clot , thus reducing procedure time , patient and operator radiation exposure and brinolytic and vasodilator dose . the most widely used brinolytic drug in this type of procedure is urokinase , usually injected as an intra - arterial bolus at a dose of 100 , 000300 , 000 ui . 
to this we always added a ca2 + antagonist vasodilator ( verapamil , isoptin , knoll , 0.40.8 cc ) in a 1 : 10 dilution in order to prevent or treat the often associated reactive arterial spasms . 
in the two cases treated with a multihole microcatheter , the lower incidence of reactive arterial spasm in response to the passage of guidewires and catheters led to a signi cant reduction in the vasodilator dose . 
 the decision to combine an anticoagulant ( heparin ) as an intra - arterial bolus ( patient 1 ) derived from the hypothesis of a possible synergic effect with the thrombolytic agent , given the underlying chemicalembolic aetiopathogenesis . 
negli ultimi 2 casi con microcatetere multiforato si osservata minore incidenza di spasmo arterioso reattivo al passaggio di li guida e cateteri , conseguentemente una riduzione signi cativa della dose di farmaco vasodilatatore impiegato . 
 la scelta di associare un anticoagulante ( eparina ) in bolo endoarterioso ( 1 caso ) stata suggerita nellipotesi di un possibile effetto sinergico con lagente trombolitico , dato il particolare meccanismo eziopatogenetico chimico - embolico sottostante . 
leventualit di un successivo trattamento trombolitico endoarterioso in infusione lenta continua 930 radiol med ( 2011 ) 116 : 919931 tory , and the patient could be spared the risks of prolonged thrombolysis . in assessing the results , the radiological and clinical pictures need to be distinguished . 
in contrast to the lower limbs , where a good radiological outcome corresponds to a good clinical outcome and vice versa , in the upper limbs , there may be some discrepancy between the ndings , especially in extremely distal occlusions . 
even when the result of postprocedure angiography appeared suboptimal , we refrained from pursuing a better result because we realised that any improvement , even though suboptimal , of the arterial blood supply to the affected region was suf cient to guarantee a partial increase in perfusion with rapid and marked clinical improvements , immediate disappearance of the acute ischaemic symptoms and subsequent functional recovery . 
on the other hand , the appearance of reactive spasms in response to the mere passage of a guidewire or catheter is suf cient to cause occlusion or tight stenosis of a very small artery with the risk of jeopardising the entire procedure , as such spasms often are extremely resistant to vasodilator therapy . in our small case series , there were no systemic complications related to the thrombolytic drug ( such as haemorrhage ) or local complications ( such as dissections , vesselwall rupture , haemorrhage , haematoma )  . 
the only local complication was observed during an antegrade access to the humeral artery ( patient 1 ) and consisted of a haematoma in the access site due to accidental puncture of the ulnar artery , which had a high origin from the humeral artery in the middle third of the arm ( an anatomical abnormality also seen in patient 2 ) [ 20 ] , which required surgical repair . 
this complication could probably be prevented by performing the antegrade puncture under us guidance or , as was done in the two subsequent cases , retrograde catheterisation via the common femoral artery , provided that suf ciently long catheters are available ( 150 cm )  . one limitation of this study is the small number of cases , partially justi ed by the low frequency of the condition . 
anche quando il risultato angiogra co post - procedurale sembrava non ottimale , ci siamo astenuti dal perseguire un risultato migliore perch abbiamo constatato che un miglioramento , seppur non ottimale , della vascolarizzazione arteriosa nel distretto interessato era suf ciente a garantire un parziale aumento dellapporto arterioso con rapido e consistente miglioramento clinico , immediata scomparsa della sintomatologia ischemica acuta e successiva ripresa funzionale . 
come unica complicanza locale abbiamo , tuttavia , riportato , durante laccesso anterogrado allarteria omerale ( 1 caso ) , un ematoma nel sito di accesso arterioso percutaneo in seguito alla puntura accidentale dellarteria ulnare , che presentava unorigine alta dallarteria omerale al tratto medio del braccio ( anomalia presente anche nel caso 2 ) [ 20 ] , che ha richiesto raf a chirurgica . 
tale complicanza si poteva , probabilmente , evitare eseguendo la puntura anterograda sotto guida ecogra ca , oppure , come abbiamo poi fatto nei 2 casi successivi , eseguendo il cateterismo per via retrograda trans - femorale comune , purch siano disponibili cateteri suf cientemente lunghi ( 150 cm )  . tra i limiti di questo lavoro vi in primis lesiguit della casistica , in parte motivata dalla bassa frequenza della patologia considerata . 
it averts or at least reduces the need for surgical thrombectomy or more complex procedures for microvascular reconstruction , which have no doubt proved highly effective in numerous series , especially in posttraumatic or haemodialysis patients [ 5 , 19 , 21 , 22 ]  . the simplest approach with the lowest incidence of complications is retrograde access via the common femoral artery , with particularly long and thin catheters or , better , multihole microcatheters , given the very distal location of the ischaemic lesions . 
these provide the additional bene t of permitting better distribution of the thrombolytic agent and reducing the frequently associated reactive spasms , which can be prevented or treated by combining the brinolytic agent ( urokinase ) with the intra - arterial vasodilator . 
the clinical outcome is generally good , with rapid disappearance of the symptoms of acute ischaemia , even though the immediate radiological results are not always optimal . dellischemia acuta della mano , in quanto migliora il usso arterioso loco - regionale ed il run - off distale , evitando o comunque riducendo la necessit di ricorrere alla trombectomia chirurgica o a procedure pi complesse di ricostruzione microvascolare , che si sono dimostrate certamente valide in numerose casistiche , specie in pazienti post - traumatici o emodializzati [ 5 , 19 , 21 , 22 ]  . lapproccio pi semplice e meno gravato da complicanze quello transfemorale comune per via retrograda , con luso di cateteri o , meglio , di microcateteri multiforati particolarmente lunghi e sottili , in quanto le lesioni ischemiche sono spesso molto distali , con lulteriore vantaggio , da parte di questi ultimi , di consentire una migliore distribuzione dellagente trombolitico e ridurre lo spasmo arterioso reattivo molto spesso associato e prevenibile o trattabile associando al farmaco brinolitico ( urochinasi ) il vasodilatatore intra - arterioso . 
rotondo dipartimento di internistica clinica e sperimentale magrassi - lanzara , sezione di radiodiagnostica e radioterapia , seconda universit degli studi di napoli , p.zza miraglia 2 , 80138 napoli , italy correspondence to : g . 
la sostanziale sovrapposizione tra i parametri mammogra ci e ultrasonogra ci dei broadenomi e dei tumori lloidi e lassenza di caratteristiche patognomoniche non consentono di fare diagnosi differenziale tra i due istotipi . 
la cnb ecoguidata rappresenta un valido strumento nella diagnosi differenziale tra broadenoma e tumore lloide . parole chiave fibroadenoma ecogra a tumore lloide 906 introduction fibroadenoma is the most common benign breast disease in the female population aged 2050 years . 
the aetiopathogenetic factors underlying development of the mass remain unclear , even though the role of oestrogen and progesterone status is undeniable [ 1 ]  . when a broadenoma exceeds 5 cm in diameter , 500 g in weight or occupies more than four fths of the entire mammary gland , it is de ned as being a giant broadenoma . 
giant broadenomas , in particular in the juvenile variant , are perhaps the least common form , with an incidence < 4% of all broadenomas [ 2 , 3 ]  . 
despite the low incidence , however , these lesions are the most common cause of unilateral breast enlargement among teenagers . intralobular stroma also gives rise to phyllodes tumour , a neoplasm with a low incidence ( 0.31.0% of all breast neoplasms ; 23% of broepithelial neoplasms ) , which arises 1020 years later than broadenoma and is generally distinguished into benign or malignant according to its histopathological features . 
the aim of our study was to analyse the mammographic and ultrasonographic ( us ) features of broadenoma and phyllodes tumour and evaluate the diagnostic accuracy of mammography , us and us - guided core needle biopsy ( cnb ) in the differential diagnosis between broadenoma and phyllodes tumour . radiol med ( 2011 ) 116 : 905918 introduzione il broadenoma rappresenta la patologia mammaria benigna di pi comune riscontro nella popolazione femminile di et compresa tra i 20 ed i 50 anni di et ; considerate tutte le patologie della mammella , poi , seconda per incidenza in tutte le fasce det solo alladenocarcinoma . 
 quando un broadenoma supera i 5 centimetri di diametro , i 500 grammi di peso o occupa un volume superiore ai 4 / 5 dellintera ghiandola mammaria , si de nisce gigante . 
il broadenoma gigante , particolarmente nella sua variante giovanile , rappresenta forse la variante di pi rara osservazione , con unincidenza che non supera complessivamente il 4% di tutti i broadenomi [ 2 , 3 ] ; nonostante la bassa incidenza nella popolazione , per , tale lesione costituisce la pi comune causa di aumento volumetrico monolaterale della mammella in et adolescenziale . 
 dallo stroma intralobulare specializzato nasce anche il tumore lloide , neoplasia a bassa incidenza ( 0 , 3%1 , 0% di tutte le neoplasie della mammella , 2%3% delle neoplasie broepiteliali ) , che insorge 1020 anni pi tardi rispetto al broadenoma e che viene generalmente distinto in benigno e maligno in relazione alle caratteristiche anatomopatologiche . 
il tumore lloide presenta un elevato tasso di recidiva locale , nonch una percentuale non trascurabile di degenerazione maligna , motivi per i quali il trattamento di scelta della neoplasia rappresentato dallescissione chirurgica . pertanto , ai ni di un corretto approccio terapeutico , risulta indispensabile la diagnosi differenziale tra le suddette lesioni . 
each patient underwent breast examination , mammography , us , us - guided cnb and excision biopsy , all done by an experienced breast radiologist , which con rmed the presence of 67 broadenomas ( ten giant ) , and 16 phyllodes tumours ( 14 benign ; two malignant )  . 
 images were acquired in the two standard projections ( craniocaudal and mediolateral oblique ) , and spot - compresnel periodo compreso tra il 2001 e il 2008 sono state analizzate 83 lesioni di 83 donne dellet media di 45 , 4 anni ( range 1875 anni , deviazione standard 20 , 3 anni )  . 
ogni paziente stata sottoposta a visita senologica , a mammogra a , a esame ecogra co , a core needle biopsy ecoguidata e a biopsia escissionale che ha confermato la presenza di 67 broadenomi ( 10 dei quali giganti ) e 16 tumori lloidi , ( 14 benigni , 2 maligni )  . 
us was performed using a logiq s6 scanner ( ge healthcare , waukesha , wi , usa ) with a multifrequency matrix - array linear transducer ( 7 - 14 mhz )  . 
sonograms were assessed for the following parameters : diameter ( < 2 , 24 or > 4 cm ) , shape ( oval , rounded , lobular or multilobular ) , contours ( smooth or irregular ) , echogenicity ( hyper - , iso - , hypoechoic ) , echostructure ( homogeneous or heterogeneous ) and posterior acoustic changes ( none ; posterior shadowing ; posterior enhancement ) [ 5 ]  . 
 lesions were considered doubtful ( not broadenomas ) if they had a maximum diameter > 4 cm ( at mammography and us ) , high density ( at mammography ) , multilobulated and / or irregular contours ( at mammography and us ) , rounded shape ( at mammography and us ) and heterogeneous echogenicity with intralesional cystic areas ( at us ) [ 4 , 5 ]  . 
histological examination was performed by an experienced pathologist . statistical analysis we calculated the statistical signi cance of the frequency of the mammographic and us ndings in the differential diagnosis between broadenoma and phyllodes tumour . 
in order to assess the association between the mammographic and us ndings with phyllodes tumour and broadenoma , fishers exact test was performed on 22 tables and the fisherfreemanhalton test on rc tables ( statxact - 7 cytel studio version 7.0.0 21 oct 2005 )  . 
nellanalisi mammogra ca sono stati analizzati i seguenti parametri : forma , dimensioni , contorni , densit , presenza o assenza di calci cazioni o di halo sign [ 4 ]  . 
nellanalisi ecogra ca delle lesioni sono stati analizzati i seguenti parametri : diametro ( < 2 , compreso tra 2 e 4 o > 4 cm ) , forma ( ovale , rotonda , lobulata o plurilobulata ) , margini ( lisci o irregolari ) , ecogenicit ( iper - iso - ipoecogeno ) , ecostruttura ( omogenea o disomogenea ) e alterazioni della trasmissione del fascio ( assenza di alterazioni , attenuazione , accentuazione ) [ 5 ]  . 
sono state considerate dubbie ( non broadenoma ) le lesioni che presentavano : dimensioni massime > 4 cm ( alla mammogra a e allecogra a ) , alta densit ( alla mammogra a ) , pro li lesionali plurilobulati e / o irregolari ( alla mammogra a e allecogra a ) , morfologia rotondeggiante ( alla mammogra a e allecogra a ) ed ecogenicit disomogenea con areole cistiche intralesionali ( allecogra a ) [ 4 , 5 ]  . 
lanalisi istologica stata effettuata da un anatomopatologo dedicato . analisi statistica stata calcolata la signi cativit statistica della frequenza dei reperti mammogra ci ed ecogra ci nella diagnosi differenziale tra broadenoma e tumore lloide . 
per valutare lassociazione dei reperti mammogra ci ed ecogra ci con il tumore lloide e il broadenoma stato effettuato il test esatto di fisher per le tabelle 22 e il test esatto di fisherfreeman - halton nelle tabelle rc , statxact - 7 cytel studio version 7.0.0 ( 21 ottobre , 2005 )  . 
la signi cativit statistica stata calcolata con test di mc nemar . results risultati of the 83 lesions studied , 73 were palpable and ten nonpalpable ; of the 67 broadenomas , 61 were palpable and six nonpalpable ; of the 16 phyllodes tumours , 14 were palpable and two nonpalpable . 
at mammography , 89% of broadenomas appeared as low - density lesions relative to the surrounding parenchyma ( p = 0.0001 ) and only 11% as high - density lesions ; in contrast , 62% of phyllodes delle 83 lesioni analizzate , 73 erano palpabili , 10 non palpabili . 
alla mammogra a , l89% dei broadenomi si presentava come una lesione radiopaca a bassa densit rispetto al parenchima circostante ( p = 0 , 0001 ) e radiol med ( 2011 ) 116 : 905918 908 fig . 
in addition , 94% of broadenomas showed a halo sign ( p = 0.0001 ) , a nding that was seen in only 25% of phyllodes tumours ( table 1 )  . 
inoltre , il 94% dei broadenomi presentava halo sign ( p = 0 , 0001 ) , segno riscontrato soltanto nel 25% dei tumori lloidi ( tabella 1 )  . 
il 56% dei tumori lloidi aveva un diametro traverso massimo compreso tra 2 e 4 cm , il 44% ha un diametro > 4 cm , nessun tumore lloide aveva un diametro traverso massimo < 2 cm ( tabella 2 )  . 
on colour doppler us , no pattern of the spatial distribution of vessels could be identi ed that might help distinguish broadenomas from phyllodes tumours . calculation of the diagnostic accuracy of mammography , us and cnb compared with nal histology in the differential diagnosis between broadenoma and phyllodes tumour yielded the following results : sensitivity 45% for mammography ( range 3456% ) , 50% for us ( range 3961% ) and 81% for cnb ( range 7189% ) ; speci city 50% for mammography ( range 3961% ) , 69% for us ( range 5778% ) and 97% for cnb ( range 9099% ) ; posi49% isoecogeno , il 30% ipoecogeno , il 16% marcatamente ipoecogeno . 
lecostruttura del broadenoma era omogenea nel 44% ( p = 0 , 0001 ) , disomogenea nel 52% , lecostruttura del tumore lloide era omogenea nel 38% dei casi , disomogenea nel 62% e con aree cistiche contestuali nel 44% dei casi . 
 allintegrazione eco - color doppler , non era stato identi cato alcun pattern di distribuzione spaziale dei vasi in grado di differenziare il broadenoma dal tumore lloide . abbiamo , inoltre , calcolato laccuratezza diagnostica della mammogra a , dellecogra a e della core needle biopsy confrontata con listologia de nitiva nella diagnosi differenziale tra broadenoma e tumore lloide . 
a , b ultrasonography performed with multifrequency probe : large hypoechoic mass ( maximum diameter approximately 9 cm ) with homogenous internal echopattern and mass effect on adjacent breast tissue . 
a , b esame ecotomogra co eseguito con sonda multifrequenza : voluminosa lacuna ipoecogena ( diametro trasversale massimo 9 cm circa ) con tappeto di echi endolesionale omogeneo e netto effetto massa sul parenchima mammario adiacente . 
all values were statistically signi cant ( p = 0.001 ) ( table 3 )  . speci cit delle tre indagini era rispettivamente del 50% ( range 39%61% ) , 69% ( range 57%78% ) , 97% ( range 90%99% ) ; il valore predittivo positivo era del 79% per la mammogra a ( range 68%87% ) , dell82% per lecogra a 912 radiol med ( 2011 ) 116 : 905918 table 3 diagnostic accuracy of mammography , ultrasound and core needle biopsy compared with nal histology in the differential diagnosis between broadenoma and phyllodes tumour percent 95% con dence interval p value * lower limit upper limit tabella 3 accuratezza diagnostica della mammogra a , ecogra a e needle biopsy rispetto allistologia de nitiva nella diagnosi differenziale tra broadenoma e tumore lloide ic 95% limite inferiore limite superiore mammography vs nal histology sensitivity speci city positive predictive value ultrasound vs . 
at mammography , broadenomas may appear as low - density or , less commonly , high - density lesions with regular or irregular contours , with or without calci cations and with or without halo sign ( table 1 ) [ 4 , 15 ]  . 
i valori ottenuti sono statisticamente signi cativi ( p = 0 , 001 ) ( tabella 3 )  . discussione il ruolo della diagnostica per immagini nella diagnosi differenziale tra broadenoma e tumore lloide , nonch tra tumore lloide benigno e maligno da sempre controverso [ 424 ]  . 
alla mammogra a il broadenoma si pu presentare come una lesione radiopaca a bassa densit o , pi raramente ad elevata densit , con contorni regolari o irregolari , con o senza calci cazioni contestuali e con o senza halo sign ( tabella 1 ) [ 4 , 15 ] ; la dif colt maggiore radiol med ( 2011 ) 116 : 905918 fig . 
a esame ecogra co di voluminosa massa palpabile tradotta come lacuna ipoecogena ad echo pattern interno disomogeneo , a margini netti ma lobulati che allingrandimento elettronico mostra piccolo incluso similcistico ( freccia nera )  . 
c lesame ecogra co mostrava una lacuna ipoecogena a contorni marcatamente lobulati , ma netti , con tappeto dechi endolesionale omogeneo senza alterazioni del fascio acustico posteriore , priva di chiari elementi di sospetto ( probabile broadenoma )  . 
however , the presence or absence of intralesional calci cations in our study ( table 1 ) or in yilmatz et al.s study [ 4 ] had no statistical signi cance . 
 [ 4 ] affermano che il broadenoma si presenta alla mammogra a come una lesione radiopaca a bassa densit , mentre il tumore lloide come una massa radiopaca a densit maggiore ( p < 0 , 038 ) [ 4 ]  . 
nel nostro studio , nonostante il risultato non sia statisticamente signi cativo , abbiamo riscontrato che il 62% ( 10 / 16 ) dei tumori lloidi si presenta come una massa radiopaca ad elevata densit . 
tuttavia , nel nostro radiol med ( 2011 ) 116 : 905918 a halo sign in 2 / 19 broadenomas and 3 / 12 phyllodes tumours ( p = 0.349 ) [ 4 ]  . in most cases , us can discriminate between broadenomas and cystic or mixed lesions that may have identical clinical and mammographic features . 
however , if we exclude cases of small intralesional cysts , only a limited proportion of masses with similar mammographic appearance ( mostly phyllodes tumours and lymphomas , in particular in immunocompromised patients ) can be clari ed with us [ 5 , 6 , 11 ]  . in our study , broadenomas had a maximum diameter not exceeding 2 cm ( p = 0.0001 ) , consistent with the literature . 
in our study , 83% of broadenomas ( 56 / 67 ) had an oval shape ( p = 0.0001 ) and smooth contours ( p = 0.01 ) , in line with the ndings reported by yilmaz et al . 
the presence of intralesional cystic areas is pathognomonic for phyllodes tumour and improves the limited diagnostic accuracy of us ( 34% sensitivity and 69% speci city in our series , in agreement with the literature ) [ 4 , 5 , 20 , 25 ]  . 
al contrario , il 94% ( 63 / 67 ) dei broadenomi e il 25% ( 4 / 16 ) dei tumori lloidi da noi analizzati presentano halo sign ( p = 0 , 0001 ) ; il dato non in accordo con quello riportato da yilmaz et al . 
 [ 4 ] , che riscontrano questo segno in 2 / 19 broadenomi e in 3 / 12 tumori lloidi ( p = 0 , 349 ) [ 4 ]  . allecogra a possibile , nella maggior parte dei casi , distinguere i broadenomi da lesioni a contenuto cistico o misto che possono assumere aspetti ad essi del tutto sovrapponibili dal punto di vista clinico e mammogra co . 
lecogra a , tuttavia , se si esclude la presenza di piccole formazioni cistiche intralesionali , risolve solo in una limitata percentuale dei casi la diagnosi differenziale con masse con aspetto mammogra co simile ( soprattutto tumori lloidi e linfomi , specie nei pazienti immunocompromessi ) [ 5 , 6 , 11 ]  . 
 nel nostro studio , i broadenomi avevano un diametro massimo non superiore ai 2 cm ( p = 0 , 0001 ) , dato in accordo con la letteratura internazionale . 
 [ 20 ] affermano che i broadenomi si presentano per lo pi come lesioni dal diametro trasverso < 2 cm , i tumori lloidi come lesioni con diametro massimo 2 cm , mentre liberman et al . 
la presenza di aree cistiche contestuali patognomonica per tumore lloide e consente di incrementare la scarsa accuratezza diagnostica dellecogra a ( sensibilit 34% , speci cit 69% nel nostro studio , in accordo con la letteratura ) [ 4 , 5 , 20 , 25 ]  . 
in letteratura descritta la correlazione tra la distribuzione spaziale dei vasi ( capsulari , segmentari ) e la diagnosi anatomopatologica di broadenoma [ 26 ] ; al contrario , non si evidenzia alcuna caratteristica peculiare alleco - color doppler che consenta di porre il sospetto di tumore lloide [ 2729 ]  . 
moreover , from a clinical and imaging point of view , differentiation between giant broadenoma and other conditions associated with breast asymmetry is highly complex : in fact , giant broadenoma is virtually identical to phyllodes tumour in its benign ( high incidence of local recurrence ) and malignant forms ( high recurrence and metastatic rates ) [ 2 ]  . 
in these cases , histopathological examination is mandatory : whereas broadenomas are characterised by low mitotic activity , low stromal cellularity , absence of cellular atypia ( normal diploidy ) and expansile growth with sharp and well - visible contours , phyllodes tumours show an intracanalicular , frond - like growth pattern , moderate stromal hypercellularity at times with necrosis , sclerosis and various types of metaplasia , low mitotic activity and considerable increase in lesion size [ 1 , 8 , 11 ]  . 
contrary to the traditional view that considered phyllodes tumour to be an evolution of broadenoma , the two lesions are now regarded as two distinct disease entities [ 3 ]  . 
therefore , a lesion that shows low density , regular contours and a halo sign on mammography and an oval or lobular shape , smooth contours , hypoechoic echogenicity and a homogeneous echostructure on us may be considered a probable broadenoma . 
conversely , a high - density opacity , with no calci cations or halo sign at mammography , and with rounded or multilobular shape , irregular contours , marked hypoechogenicity ( relative to the surrounding fat ) , with a heterogeneous echostructure and containing cystic areas at us is likely to be a benign lesion ( other than broadenoma )  . despite the presence of features suggestive of the two histological types , the limited diagnostic accuracy of mammography and us ( table 3 ) , the overlap between mammographic and us ndings and the absence of pathognomonic signs do not permit discrimination between broadenoma and phyllodes tumour [ 4 , 5 ]  . 
especially in the case of phyllodes tumours , the results may in fact prove false negative if sampling was performed in an area of hypocellular stroma [ 17 , 18 ]  . 
although only a few studies have investigated the role of cnb in the differential diagnosis between broadenoma and phyllodes tumour [ 19 , 20 ] , they have all reported high ppv and negative predictive values ( npv ) [ 17 , 2123 ]  . 
it is reasonable to believe that immunohistochemical techniques and cell proliferation noma gigante e tumore lloide ( segnale intralesionale nel sequenze t1 e t2 ponderate e cinetica di enhancement post - contrastogra co assolutamente aspeci co ) [ 3 , 11 , 12 , 16 ]  . 
va detto , inoltre , che la diagnosi differenziale sul piano clinico e strumentale del broadenoma nella sua variante gigante nei confronti di altre condizioni associate ad asimmetria mammaria notevolmente complessa : esso infatti frequentemente del tutto indistinguibile dal tumore lloide nelle sue varianti benigna ( alto incide di recidive locali ) e maligna ( elevata capacit di recidiva e metastatica ) [ 2 ]  . 
 in questi casi necessario ricorrere allesame anatomopatologico : mentre il broadenoma si caratterizza per una bassa attivit mitotica cellulare , una bassa cellularit stromale , assenza di atipie cellulari ( corredo nucleare normalmente diploide ) , crescita per espansione con bordi netti e ben visibili , il tumore lloide mostra una crescita intracanalicolare con una estensione frond - like , una ipercellularit stromale modesta , talora con necrosi , sclerosi e vari tipi di metaplasia , bassa attivit mitotica e notevole aumento volumetrico espansivo [ 1 , 8 , 11 ]  . 
quindi , una lesione radiopaca a bassa densit , margini regolari e halo sign alla mammogra a e di forma ovale o lobulata , con margini lisci , ipoecogena e ad ecostruttura omogenea allecogra a , pu essere considerata come un probabile broadenoma . 
 al contrario , unopacit ad elevata densit , senza calci cazioni n halo sign alla mammogra a , che si presenta di forma rotondeggiante o plurilobulata , a margini irregolari , marcatamente ipoecogena ( in relazione al tessuto adiposo circostante ) , ad ecostruttura disomogenea e aree cistiche contestuali allecogra a , pu essere considerata come una probabile lesione benigna ( ma non broadenoma )  . 
 nonostante la presenza di caratteristiche suggestive per i due istotipi , la scarsa accuratezza diagnostica della mammogra a e dellecogra a ( tabella 3 ) , la sovrapposizione tra i parametri mammogra ci ed ecogra ci e lassenza di caratteri patognomonici non consentono di differenziare il broadenoma dal tumore lloide [ 4 , 5 ]  . 
soprattutto in caso di tumore lloide , infatti , il risultato pu essere falsamente negativo se il prelievo viene effettuato in unarea di stroma ipocellulare [ 17 , 18 ]  . 
pochi sono gli studi in letteratura che analizzano il ruolo della cnb nella diagnosi differenziale tra broadenoma e tumore lloide [ 19 , 20 ] , ma tutti riportano un elevato valore predittivo positivo e negativo [ 17 , 2123 ]  . 
 realistico pensare che in futuro tecniche immunoistochimiche e marcatori della proliferazione cellulare , quali p53 e ki 67 , ne renderanno radiol med ( 2011 ) 116 : 905918 markers such as p53 and ki - 67 will facilitate the differential diagnosis and prognostic evaluation in the future [ 17 ]  . in our experience , the diagnostic accuracy of mammography , us and cnb compared with nal histology shows that only cnb is able to differentiate between broadenoma and phyllodes tumour ( 81% sensitivity , 97% speci city and 87% ppv ) , as reported in the literature [ 19 , 20 , 30 , 31 ]  . pi agevole la diagnosi differenziale e la conseguente implicazione prognostica [ 17 ]  . nel nostro contributo i valori di accuratezza diagnostica della mammogra a , dellecogra a e della core needle biopsy , confrontati con listologia de nitiva , dimostrano che soltanto la core needle biopsy in grado di fare diagnosi differenziale tra broadenoma e tumore lloide ( sensibilit 81% , speci cit 97% e valore predittivo positivo 87% ) , cos come dimostrato in letteratura [ 19 , 20 , 30 , 31 ]  . conclusions conclusioni on mammography , broadenomas mostly appear as lowdensity opacities with regular contours and a halo sign ; phyllodes tumours appear as high - density opacities with neither calci cations nor halo sign . 
if a lesion appears on us as an oval or lobular mass with smooth contours , hyperechogenicity relative to the surrounding glandular tissue and homogeneous echostructure , it is likely to be a broadenoma . 
if a lesion appears rounded or multilobular , with irregular contours , marked hypoechogenicity ( relative to the surrounding fat ) , heterogeneous echostructure and cystic areas , it is likely to be a benign lesion ( but not broadenoma )  . 
there is nonetheless a substantial overlap between the mammographic and us parameters of broadenomas and phyllodes tumours , and no pathognomonic signs exist that allow discrimination between the two histological types . 
us - guided cnb represents a valuable tool in the differential diagnosis between broadenoma and phyllodes tumour . in conclusione , alla mammogra a , il broadenoma si presenta prevalentemente come unopacit a bassa densit , a margini regolari e halo sign ; il tumore lloide come unopacit ad elevata densit senza calci cazioni , n halo sign . 
se allesame ecogra co una lesione si presenta di forma ovale o lobulata , margini lisci , ipoecogenicit rispetto al tessuto ghiandolare circostante ed ecostruttura omogenea , pu essere considerata come un probabile broadenoma ; se in presenza di queste caratteristiche , ha un diametro > 5 cm , come un probabile broadenoma gigante ; se di forma rotondeggiante o plurilobulata , a margini irregolari , marcatamente ipoecogena ( in relazione al tessuto adiposo circostante ) , ad ecostruttura disomogenea e aree cistiche contestuali , pu essere considerata come una probabile lesione benigna ( ma non broadenoma )  . 
tuttavia , esiste una sostanziale sovrapposizione tra i parametri mammogra ci e ultrasonogra ci dei broadenomi e dei tumori lloidi e non ci sono caratteristiche patognomoniche che consentano di fare diagnosi differenziale tra i due istotipi . al contrario , la core needle biopsy ecoguidata rappresenta un valido strumento nella diagnosi differenziale tra broadenoma e tumore lloide . 
mr - guided vab was attempted in 29 / 106 lesions ( 27% ) ; in 2 / 29 patients , the procedure could not be performed owing to failure to visualise the lesion . 
lesions with clearly malignant features and borderline lesions ( atypical ductal hyperplasias ) were identi ed in 12 cases ( 44% ) and benign entities in 15 ( 56% )  . 
seven of 12 ( 58% ) malignant lesions were < 10 mamong the 27 successful mr - vab procedures , vab yielded one false - negative diagnosis ( 4% ) and underestimation ( 4% )  . 
sono state avviate al vab , con lutilizzo di un ago da 10 gauge , le pazienti con alterazioni sospette alla rm , non riconoscibili con ecogra a n mammogra a , non palpabili , in cui la tipizzazione non poteva essere ottenuta con metodiche alternative . 
il riscontro di patologia francamente maligna o di lesioni border - line si veri cato in 12 casi ( 44% ) , nei restanti 15 casi ( 56% ) si rilevata patologia benigna . 
sette / 12 ( 58% ) lesioni maligne avevano diametro inferiore o uguale a 10 muno / 27 casi ( 4% ) risultato falso negativo al vab ; 1 / 27 casi ( 4% ) stato sottostimato dal vab . 
complessivamente , il prelievo vab sotto guida rm ha mostrato valori di sensibilit e speci cit rispettivamente del 92% e 100% con valore predittivo positivo del 100% e valore predittivo negativo del 93% . radiol med ( 2011 ) 116 : 876885 keywords breast cancer diagnosis breast mri biopsy vacuum - assisted biopsy ( vab ) conclusioni . 
le dimensioni della lesione inferiori a 1 cm non sono un limite per la metodica . parole chiave carcinoma mammario diagnosi rm della mammella vab biopsia introduction introduzione as its high diagnostic sensitivity , magnetic resonance ( mr ) imaging of the breast provides added value compared with mammography and ultrasonography [ 1 ]  . 
although the clinical impact of breast mr imaging in the workup of breast disease is still controversial [ 26 ] , the growing diffusion of the technique leads to the increasingly frequent nding of incidentally discovered breast lesions without a correlate on conventional breast imaging . 
the wide availability of aspiration cytology with ne ferromagnetic needles and coaxial nonmagnetic needles allows lesions to be characterised noninvasively but is limited by the inadequacy of the samples collected , especially in the case of non - mass - like lesions [ 79 ]  . 
the advent of large calibre needles and mr - compatible materials has made it possible to collect more material with improved precision , at the price , however , of a considerable increase in costs [ 10 ]  . 
 the introduction of a microhistological sampling technique with cutting needles and a vacuum aspiration device vacuum assisted biopsy ( vab ) has marked a new step forward in the direction of more sophisticated sampling technology [ 11 ] , even though the costs have increased further [ 12 ] and no reimbursement by the italian national health system is as yet envisaged . 
 the aim of this study was to evaluate the impact on clinical practice of mr - guided vab as the nal step in the diagnostic workup of breast lesions incidentally detected on mr imaging and to validate the techniques diagnostic accuracy by correlating the microhistological results with the histopathological ndings on the surgical specimen . material and methods in ragione dellelevata sensibilit diagnostica , lindagine di risonanza magnetica ( rm ) della mammella rappresenta una metodica imaging dal valore aggiuntivo rispetto alla mammogra a e allecogra a [ 1 ]  . 
sebbene limpatto clinico dellintroduzione della rm mammaria nelliter diagnostico della patologia mammaria sia ancora controverso [ 26 ] , la crescente diffusione dellindagine rm comporta il sempre pi frequente riscontro di lesioni mammarie incidentalmente identi cate nellindagine rm , prive di corrispettivo allesame senologico tradizionale . 
lampia disponibilit dellesame citologico per aspirazione con ago sottile ferromagnetico e ago coassiale amagnetico rende possibile la diagnosi di natura in maniera incruenta , ma penalizzato da prelievi inadeguati , soprattutto in caso di lesioni non massa [ 79 ]  . 
lavvento di aghi di grosso calibro e di materiali rm compatibili , ha permesso di ottenere materiale pi abbondante e precisione superiore , sebbene con incremento considerevole dei costi [ 10 ]  . 
 lattuale disponibilit di una tecnologia di prelievo microistologico con aghi trincianti e con dispositivo di aspirazione sottovuoto ( vacuum assisted biopsy , vab ) ha rappresentato un nuovo passo avanti verso una tecnologia di prelievo pi so sticata [ 11 ] , sebbene con ulteriore lievitazione dei costi [ 12 ] per la quale in italia non ancora previsto il rimborso da parte del sistema sanitario nazionale ( ssn )  . 
 scopo di questo lavoro valutare lincidenza nella pratica clinica dei prelievi vab sotto guida rm come tappa nale delliter diagnostico nella gestione delle lesioni incidentalmente identi cate allindagine rm e di validarne laccuratezza diagnostica comparando i risultati microistologici con quelli istologici derivanti dal pezzo operatorio . we retrospectively analysed 98 patients ( 97 women and one man ; mean age 49 years ; range 2279 years ) with 106 breast lesions identi ed on mr imaging from september 2007 to march 2009 . 
indications for mr examination included preoperative assessment of patients scheduled for materiali e metodi sono stati retrospettivamente analizzati 98 pazienti ( 97 donne e 1 uomo ; et media 49 anni ; range 2279 anni ) porta878 radiol med ( 2011 ) 116 : 876885 surgery due to malignant disease detected on mammography and ultrasound ( us ) in 53 cases ( 45% ) , evaluation of the surgical scar in 27 ( 27% ) patients and screening of women at high risk of breast cancer in 18 cases ( 18% )  . we included in the analysis cases in which mr imaging identi ed suspicious or highly suspicious lesions , according to classi cation of the american college of radiology breast imaging reporting and data system ( bi - rads ) 4 and 5 , that were nonpalpable and occult on conventional breast imaging and for which we had complete data regarding subsequent diagnostic procedures or follow - up for at least 12 months . 
 the lesions sampled under mr guidance were grouped by mr appearance ( mass - like enhancement , non - masslike enhancement , focus ) [ 1 ] and size ( long axis ) and distributed on the basis of level of suspicion of malignancy ( bi - rads classi cation ) [ 13 ]  . 
patients with a histopathological diagnosis of benign lesion were followed up by mr imaging at 6 months and subsequently referred for standard screening follow - up for at least 12 months . 
the microhistological diagnosis of vab was then correlated with histological diagnosis of the surgical specimen , considered the gold standard . mr - guided biopsy all biopsies were performed after obtaining the patients informed consent and discontinuing any anticoagulation therapy in accordance with the protocol standardised by the current guidelines [ 14 ]  . 
lesion localisation was performed with the patient in the prone position and using an mr - compatible stereotactic system ( siemens , erlangen , germany ) consisting of a support to accommodate the breast and two plates with parallel perforated holes for mediolateral compression of the breast . 
signal reception is guaranteed by a soft , circular surface coil placed between the two bars so as to completely surround the breast . lesion localisation was achieved with t1 - weighted axial sequences before and after administration of contrast material . 
the spatial coordinates were calculated with the breast tori di 106 lesioni identi cate dallesame rm nel periodo compreso tra settembre 2007 e marzo 2009 . lindagine rm stata effettuata per la valutazione preoperatoria in pazienti candidate a intervento per patologia maligna accertata allindagine mammo - ecogra ca in 53 casi ( 45% ) ; per studio di cicatrice in 27 ( 27% ) pazienti sottoposte a precedente intervento ; per sorveglianza di donne ad alto rischio di carcinoma mammario in 18 casi ( 18% )  . sono stati inclusi nellanalisi i casi in cui la rm ha identi cato lesioni sospette o altamente sospette ( breast imaging reporting and data system [ bi - rads ] 4 e 5 ) non palpabili e non visibili allindagine senologica convenzionale , in cui erano disponibili tutte le notizie riguardo al successivo iter diagnostico o al follow - up per almeno 12 mesi . 
 le lesioni sottoposte a prelievo rm - guidato sono state raggruppate per aspetto rm ( enhancement tipo mass , non mass , focus ) [ 1 ] , dimensione ( considerata come maggior asse della lesione ) e distribuite in rapporto al giudizio di sospetto ( secondo la classi cazione bi - rads - american college of radiology ) [ 13 ]  . 
il gold standard stato lesame patologico proveniente dallescissione chirurgica . prelievo sotto guida rm la procedura di prelievo rm guidato stata eseguita con il consenso informato da parte della paziente e dopo sospensione di eventuali terapie anticoagulanti , secondo modalit standardizzate dalle attuali linee guida [ 14 ]  . 
la centratura della lesione avvenuta con paziente in decubito prono e sistema stereotassico rm compatibile ( siemens , erlangen , germania ) costituito da un supporto per il posizionamento della mammella interessata e da due piatti dotati di fessure parallele per la compressione del seno in direzione mediolaterale . 
la ricezione del radiol med ( 2011 ) 116 : 876885 biopsy tool software available on the systeafter establishing the entry hole , a coaxial introducer consisting of a plastic cannula and a titanium stylet was xed to the support syste after removing the stylet , the site was anaesthetised ( 10 cc 2% lidocaine hydrochloride ) and incised with a scalpel , and the biopsy probe was inserted . 
postprocedural mr with an axial t1 - weighted sequence was used to verify clip position and the success of the procedure ( correct targeting ; bleeding )  . results targeted us of the site of the incidental mr lesion identi ed 77 of 106 ( 73% ) lesions identi ed on mr imaging . 
after conventional mr imaging without compression and with a dedicated breast coil , which con rmed nonvisualisation of the lesion presumably re ecting an area of enhancement of functional gland tissue patients were referred for follow - up at 6 months . a total of 27 / 29 ( 93% ) procedures were successfully completed . 
comparing bi - rads assessment with the microhistological diagnosis , in 5 / 12 ( 42% ) malignant lesions , the mr evaluation was highly suspicious ( bi - rads 5 ) and in 7 / 12 ( 58% ) suspicious ( bi - rads 4 )  . 
with regard to the result of pathology segnale garantita da una bobina di super cie , di forma circolare e consistenza morbida , interposta fra le barre in modo da circondare completamente la mammella . per la localizzazione spaziale della lesione sono state acquisite sequenze assiali t1 - dipendenti prima e dopo somministrazione di mezzo di contrasto . 
dopo aver rimosso il mandrino previa anestesia cutanea ( lidocaina cloridrato diluita al 2% , 10 cc ) e incisione con bisturi , stata inserita la sonda per i prelievi . 
la procedura si terminata con il posizionamento di clip in titanio ( micromark ii ; biomarc , ultraclip ) per centratura della sede del prelievo e mediante acquisizione di sequenza t1 assiale stato effettuato il controllo rm post - prelievo del posizionamento della clip e della validit della procedura ( centratura del bersaglio , eventuale comparsa di ematomi )  . risultati nella serie complessiva di 106 lesioni identi cate dallindagine rm , lecogra a mirata nella sede della lesione incidentalmente evidenziata dalla rm ha identi cato 77 su 106 ( 73% )  . 
dopo espletamento dindagine rm convenzionale , senza compressione e con bobina dedicata , che ha confermato la non riconoscibilit della lesione presumibilmente con signi cato di area dimpregnazione ghiandolare funzionale , si optato per il controllo rm dopo 6 mesi . 
il numero dei frustoli prelevati stato variabile 880 radiol med ( 2011 ) 116 : 876885 table 1 distribution of mr pattern , size , level of suspicion in 27 mrguided vab cases study variable variable categories no . 
compared with the previous cancer , the site of the malignant lesion was subcicatricial in one case , ipsilateral but in a different da 9 a 25 cores per ciascuna procedura ( valore medio di 18 frustoli / procedura ) ( tabella 2 )  . 
la comparsa di complicanze si veri cata in una sola paziente , portatrice di una lesione profonda , adesa al pettorale in cui lematoma risultato assai esteso ed ha richiesto terapia antibiotica . 
comparando il giudizio bi - rads con la diagnosi microistologica , in 5 / 12 ( 42% ) delle lesioni maligne stato formulato un giudizio rm di altamente sospetto ( birads 5 ) e in 7 / 12 ( 58% ) giudizio rm di sospetto ( bi - rads 4 )  . 
per quanto riguarda il risultato patologico in rapporto alle dimensioni della lesione , 7 / 12 ( 58% ) lesioni maligne avevano diametro inferiore o uguale a 10 mm e 5 / 12 ( 42% ) lesioni maligne erano estese pi di 10 mm . le 12 pazienti con diagnosi vab di malignit sono state avviate allintervento chirurgico ; 14 / 15 lesioni con diagnosi vab di benignit sono state avviate al follow - up che ne ha confermato la stabilit nel tempo ( periodo minimo di 12 mesi ) ; una paziente con diagnosi vab di benignit stata avviata allintervento chirurgico a causa del persistere del sospetto radiologico . 
rispetto alla neoplasia antecedente , la sede della lesione maligna stata rispettivamente sottocicatriziale in 1 caso , omolaterale alla pregressa neoplasia ma in quadranti diversi in 3 casi e controlaterale in 1 caso . 
of cores per procedure microhistological vab ndings 1 ( 4 ) < 11 21 ( 78 ) 1120 > 20 5 ( 18 ) in ltrating carcinoma 8 ( 30 ) 3 ( 10 ) in situ carcinoma 1 ( 4 ) borderline lesion 15 ( 56 ) benign lesion numero di frustoli < 11 prelevati / procedura 1120 diagnosi microistologica vab lesioni in situ > 20 lesioni in ltranti lesioni borderline lesioni benigne 1 ( 4 ) 21 ( 78 ) 5 ( 18 ) 8 ( 30 ) 3 ( 10 ) 1 ( 4 ) 15 ( 56 ) radiol med ( 2011 ) 116 : 876885 fig . 
axial t1 - weighted contrast - enhanced magnetic resonance ( mr ) image obtained in a screening setting shows a small , irregular , spiculated , enhancing mass spanning 7 mm [ mr diagnostic image ( a ) ]  . 
allesame rm dopo somministrazione di mezzo di contrasto ( mdc ) , espletato per screening , si evidenzia una piccola formazione espansiva di 7 mm , con impregnazione di tipo massa e pro li sfumati ( a immagine durante lesame rm diagnostico )  . 
lesame microistologico ( b5 ) stato confermato dallesame patologico del pezzo operatorio con diagnosi di carcinoma duttale in ltrante ( diametro massimo di in ltrazione 6 mm )  . quadrant in three cases and contralateral in one . 
the mr examination modi ed the surgical operation , and the women underwent bilateral quadrantectomy right upper outer and left lower outer quadrant in a single surgical procedure . correlation between vab results and results of histology of the surgical specimen revealed discordance in four cases : 1 / 27 cases ( 4% ) was false negative at vab ( brocystic disease at vab vs ductal carcinoma in situ at surgery carcinoma duttale in situ [ dcis ] allintervento ; si trattava di lesione di 9 mm nella quale il sospetto radiologico era elevato ) , 1 / 27 casi ( 4% ) stato sottostimato dal vab ( carcinoma duttale in situ al vab vs carcinoma duttale in ltrante allintervento ) ; nei rimanenti 2 casi ( 8% ) risultati dcis al vab lallargamento chirurgico ha rilevato solo proliferazione atipica residua ( trattavasi di 2 lesioni con diametro inferiore al centimetro e con campionamento superiore a 20 frustoli )  . 
complessivamente , il prelievo vab sotto guida rm ha mostrato valori di sensibilit e speci cit rispettivamente del 92% e 100% con valore predittivo positivo del 100% 882 radiol med ( 2011 ) 116 : 876885 fig . 
sagittal t1 - weighted contrast - enhanced magnetic resonance ( mr ) image obtained for preoperative evaluation shows clumped linear and ductal enhancement in the left lower quadrant ( residual disease ) ( f )  . 
 axial t1 - weighted contrast - enhanced mr image of the left breast shows a separate , irregularly shaped , 13 mm , enhancing , non - mass - lesion in the left breast at 12 : 00 oclock position ( g )  . 
allesame mammogra co ( a - e ) , a destra nel versante inferiore del piano sagittale mediano , multiple calci cazioni pleomorfe in area di 2 cle calci cazioni vengono sottoposte a prelievo microistologico sotto guida stereotassica ( b5 )  . 
allesame rm dopo somministrazione di mdc eseguito per valutazione pre - operatoria , nel versante inferiore della mammella destra , sede del prelievo si rileva estesa area di impregnazione a piccoli noduli riferibile a lesione residua ( f )  . 
overall , mr - guided vab had a sensitivity and speci city of 92% and 100% , respectively , with a positive predictive value ( ppv ) of 100% and a negative predictive value ( npv ) of 93% . 
in our series , second - look us resolved the management of a large majority ( 73% ) of mr - visible lesions , a percentage higher than reported in other studies [ 17 , 18 ]  . 
 in our series , in the only case of false - negative result at vab , surgery was dictated by the discrepancy between the benign diagnosis and the high level of radiological suspicion . 
the vab diagnoses of malignant disease proved to be accurate and timely , considering that 58% of the malignant lesions had a diameter < 1 c this nding is in agreement with previous larger series and demonstrates that small lesion size does not constitute a limitation to the procedure [ 16 ]  . 
that atypical proliferative lesions and ductal carcinoma in situ are the most common causes of error is well known and has already been demonstrated for biopsy under stereotactic guidance [ 22 , 23 ]  . 
come evidenziato da precedenti esperienze [ 1720 ] , il second - look ecogra co si conferma essere il primo ragionevole approccio per la identi cazione e tipizzazione delle lesioni incidentalmente evidenziate allindagine rm . 
una possibile spiegazione della discrepanza riguardo al tasso di detection rate del secondlook ecogra co risiede nella diversa diffusione dellimaging ecogra co e nella diversa esperienza degli operatori che lo svolgono . 
in questo studio , il prelievo vab sotto guida rm ha mostrato valori di sensibilit e speci cit rispettivamente del 92% e 100% con valore predittivo positivo del 100% e valore predittivo negativo del 93% . 
 la diagnosi vab di malignit risultata accurata e precoce considerando che il 58% delle lesioni maligne evidenziate avevano diametro inferiore a 1 c tale dato , in accordo con quanto osservato in pi ampie casistiche , dimostra che le dimensioni contenute della lesione non costituiscono un limite della procedura [ 16 ]  . 
in letteratura , come peraltro gi dimostrato per i prelievi sotto guida stereotassica , ben noto che le cause pi frequenti di errore sono rappresentate dalle lesioni proliferative atipiche e dal carcinoma duttale in situ [ 22 , 23 ]  . 
questo risultato conferma lutilit dellesteso campionamento al ne di fornire una 884 radiol med ( 2011 ) 116 : 876885 8% of cases , vab allowed complete removal of the lesion in situ , with only residual foci of a borderline lesion being identi ed at surgery [ 24 ]  . 
this result con rms the usefulness of extended sampling to provide a precise diagnosis and correct possible localisation defects that could affect the adequacy of the sample . in conclusion , mr - guided vab proved to be a welltolerated , simple and reliable method for characterising suspicious nonpalpable breast lesions detected on breast mr imaging and occult on mammography and us . 
this approach allows identi cation of new disease foci , containment of the number of false positive results and planning of de nitive surgery in the case of a microhistological diagnosis of malignancy , all in a single session . 
with regard to costs : economic costs of mr - guided biopsies and the economicsocial costs of biopsies / surgical excisions generated by false - positive mr ndings make it desirable to concentrate all mr - guided procedures in referral centres to which all facilities offering breast mr imaging can direct their patients . 
only by optimising the sampling technique and results can new , possibly less expensive , solutions be proposed . diagnosi precisa e di correggere eventuali vizi di centratura che potrebbero compromettere ladeguatezza del prelievo . in conclusione , il prelievo vab sotto guida rm risultato essere metodica af dabile nella tipizzazione delle lesioni mammarie rm sospette , non palpabili , ecogra camente e mammogra camente occulte , ben tollerato dalle pazienti e di facile procedura . 
il prelievo vab pu pertanto essere proposto in casi selezionati , dopo attenta rivalutazione dellesame senologico convenzionale , per la tipizzazione delle lesioni rm - visibili , rendendo possibile la programmazione del trattamento chirurgico de nitivo in unico tempo . 
tale approccio permette di ottenere lidenti cazione di nuovi foci di malattia , contenimento del numero dei falsi positivi e intervento chirurgico programmato in caso di diagnosi microistologica di malignit . 
in considerazione dei costi , economici per quanto concerne i prelievi rm guidati ed economici - sociali in caso di biopsie / allargamenti chirurgici generati dai reperti rm falsi positivi , si auspica daltra parte laccentramento delle metodiche rm guidate in centri di riferimento a cui possano afferire tutte le strutture ove vengono espletate indagini di rm della mammella . 
between 2004 and 2007 , 182 patients with malignant breast lesions detected on us and / or x - ray mammography and con rmed by cytology / histology underwent preoperative breast contrast - enhanced ( ce ) - mri . 
tra il 2004 ed il 2007 , 182 pazienti con lesioni maligne allecogra a e / o mammogra a e confermate dallesame citologico / istologico sono state sottoposte a rm della mammella con mezzo di contrasto ( mdc ) per stadiazione preoperatoria . 
sensibilit , speci cit , accuratezza , valore predittivo positivo ( vpp ) e valore predittivo negativo ( vpn ) per identi cazione e diagnosi sono risultati del 100% , 88 , 9% , 94 , 6% , 90 , 3% and 100% per la rm e del 64 , 3% , 70 , 4% , 67 , 3% , 69 , 2% e 65 , 5% per il second - look ecogra co . 
il second - look ecogra co un valido metodo radiol med ( 2011 ) 116 : 886904 keywords breast mri second - look ultrasound breast cancer incidental ndings per le lesioni occasionali alla rm della mammella , in particolar modo per i potenziamenti di tipo nodulare . 
 parole chiave rm della mammella second - look ecograco carcinoma della mammella lesioni incidentali introduction introduzione magnetic resonance imaging ( mri ) is well established as the most sensitive diagnostic technique for detecting and staging malignant breast lesions and has recently been recommended by the american cancer society ( acs ) as a breast cancer screening procedure for women with a 2025% or greater lifetime risk of breast cancer , including women with a strong family history of breast or ovarian cancer and women treated for hodgkins disease [ 1 ]  . 
its value as a routine clinical procedure extends beyond the depiction of breast cancer to monitoring the effects of neoadjuvant chemotherapy , presurgical planning and detecting cancer recurrence [ 24 ]  . 
apart from its superiority over whole - breast ultrasound ( us ) and x - ray mammography for evaluating known breast lesions , breast mri has proved particularly valuable for detecting unsuspected multifocal disease and mammographically occult malignant disease [ 57 ] .unfortunately , detecting abnormal enhancing lesions not seen on previous mammography and whole - breast us is potentially problematic for subsequent patient - management decisions in that it is sometimes unclear whether these lesions should be ignored , followed up or referred for biopsy . 
the purpose of this prospective study was to assess the utility of second - look us for evaluating incidental enhancing lesions identi ed on preoperative breast mri performed for staging malignant lesions diagnosed by conventional breast imaging methods ( mammography and / or us ) and con rmed by cytology or core biopsy . materials and methods patients between november 2004 and march 2007 , 182 consecutive patients with suspected breast malignancy on conventional x - ray mammography and / or whole - breast us , and invasive carcinoma subsequently con rmed by cytology or core biopsy , underwent preoperative breast mri for accurate breast cancer staging . 
no patient was undergoing or had undergone chemotherapy or had received any other contrast material in the week prior to stato ampiamente dimostrato come la risonanza magnetica ( rm ) sia la tecnica diagnostica pi sensibile nellidenti cazione e stadiazione delle lesioni mammarie maligne . 
recentemente lamerican cancer society ( acs ) ha raccomandato il suo impiego per lo screening del carcinoma mammario in donne con rischio di carcinoma della mammella pari o superiore al 20%25% , includendo donne con forte familiarit per carcinoma mammario o ovarico e donne sottoposte a terapia per morbo di hodgkin [ 1 ]  . 
nella routine diagnostica il suo ruolo si estende dallindividuazione del carcinoma mammario al monitoraggio della risposta alla chemioterapia neoadiuvante , alla stadiazione preoperatoria ed allidenti cazione di recidiva di malattia [ 24 ]  . 
oltre alla sua superiorit rispetto ad ecogra a e mammogra a nella valutazione delle lesioni mammarie note , la rm si dimostrata particolarmente utile nellindividuazione di patologie multifocali o lesioni maligne non evidenziate mediante le tecniche diagnostiche convenzionali [ 57 ]  . 
sfortunatamente , lidenti cazione di lesioni con impregnazione post - contrastogra ca abnormale e non osservate alla mammogra a e / o ecogra a mammaria , potenzialmente problematica per la scelta della successiva strategia terapeutica da adottare , poich a volte non ben chiaro se tali lesioni debbano essere ignorate , sottoposte a follow - up o a biopsia . 
lobiettivo di questo studio prospettico stato quello di dimostrare lutilit del second - look ecogra co nella valutazione di lesioni con impregnazione post - contrastogra ca evidenziate alla risonanza magnetica preoperatoria della mammella , eseguita per la stadiazione di lesioni mammarie maligne diagnosticate mediante le tecniche diagnostiche convenzionali per la mammella ( mammogra a e / o ecogra a ) e confermate dallesame citologico o dalla biopsia percutanea . materiali e metodi pazienti nel periodo compreso tra novembre 2004 e marzo 2007 , 182 pazienti con sospetto di neoplasia della mammella alla mammogra a e / o ecogra a e successiva diagnosi di carcinoma invasivo allesame citologico o alla biopsia percutanea , sono state sottoposte a rm preoperatoria della mammella per unaccurata stadiazione . 
 likewise , patients with incidental lesions considered de nitely benign on breast mri [ category 2 according to the breast imaging reporting and data system ( bi - rads ) lexicon ] [ 8 ] were not included . 
 mr imaging breast mri was performed on a 1.5 - t imager ( avanto , siemens , erlangen , germany ) using a bilateral breast surface coil with the patient in the prone position . 
transverse threedimensional dynamic t1 - weighted gradient - echo ( gre ) images were acquired before contrast agent administration ( unenhanced images ) and then ve times after contrast agent administration ( contrast - enhanced images )  . 
the imaging parameters for the t1 - weighted gre sequence were identical for all patients , with repetition time 8.1 ms , echo time 4 ms , ip angle 30 ; one signal acquired ; rectangular eld of view < 36 cm and matrix 496 512 . 
contrast agent administration was in all cases performed by power injector ( spectris , medrad , indianola , pa , usa ) via an antecubital venous access at a standard ow rate of 2 ml / s ( bolus ) , followed by a 10 - ml saline ush at the same rate . 
the contrast agent used in all patients was gadobenate dimeglumine ( multihance , bracco imaging spa , milan , italy ) at a nal dose of 0.1 mmol / kg bodyweight , as this agent has previously been shown to have preferential properties for breast imaging [ 912 ]  . 
data acquisition began immediately after contrast agent injection , at the end of the saline ush . second - look ultrasound ultrasound examinations were performed by one of two experienced radiologists using a high - resolution unit ( ssa700a , toshiba , japan ) with a linear array probe centred at rale , le pazienti di et maggiore ai 40 anni anche a mammogra a . 
nessuna di tali pazienti era in corso di chemioterapia neoadiuvante n alcuna di loro aveva ricevuto alcun tipo di mezzo di contrasto nelle due settimane precedenti allo studio con rm . 
le pazienti con lesioni con impregnazione postcontrastogra ca , riscontrate occasionalmente alla rm , sono state sottoposte successivamente a second - look ecogra co mirato allo studio del reperto osservato incidentalmente alla rm . 
allo stesso modo le lesioni occasionali considerate benigne alla rm ( categoria 2 secondo il breast imaging reporting and data system [ bi - rads ] lexicon ) [ 8 ] non sono state incluse in questo studio studio . 
 imaging rm la rm della mammella stata eseguita su un magnete a 1 , 5 t ( avanto , siemens , erlangen , germania ) usando bobine bilaterali dedicate per lo studio della mammella , con la paziente in posizione prona . 
sono state acquisite sequenze assiali 3d dinamiche , t1 pesate ( gradient echo [ gre ] ) prima ( immagini senza impregnazione ) e 5 volte dopo la somministrazione di mezzo di contrasto ( immagini con impregnazione )  . 
in tutti le pazienti per le sequenze t1 ( gre ) pesate sono stati usati gli stessi parametri comprensivi di : un tempo di ripetizione di 8 , 1 ms , tempo di eco di 4 ms , ip angle di 30 , singola acquisizione di segnale , campo di vista rettangolare 36 cm e matrice di 496512 . 
non sono state impiegate sequenze di soppressione del segnale del grasso , per eliminare il segnale proveniente dal grasso stata effettuata una sottrazione delle immagini ( immagini con impregnazione post - contrastogra ca immagini senza impregnazione postcontrastogra ca )  . 
il mezzo di contrasto stato somministrato attraverso un accesso venoso antecubitale , mediante lausilio di un iniettore automatico ( spectris ; medrad , indianola , pa ) al usso standard di 2 ml / s ( bolo ) , seguito dalla somministrazione di 10 ml di soluzione siologica alla stessa velocit . 
come mezzo di contrasto in tutti i pazienti stato impiegato il gadobenate dimeglumine ( multihance , bracco imaging spa , milano , italia ) alla dose di 0 , 1 mmol / kg di peso corporeo , essendo stati gi dimostrati i vantaggi relativi al suo uso nella diagnostica rm della mammella [ 912 ]  . 
the patient was placed in the supine position for lesions detected in the medial parts of the breast and in a contralateral posterior oblique position with the arms raised for lesions detected in lateral parts of the breast . 
 given differences in positioning between prone mri and supine us , targeting for the second - look us exam was in all cases performed by breast quadrant . image interpretation all mr and us images were evaluated prospectively by two radiologists in consensus ( xy , 7 years experience in breast mri and 9 years experience in breast us ; xx , 5 years experience in breast mri and 7 years experience in breast us ) with full knowledge of the patients clinical history and with previous conventional imaging ndings available . 
all images were reviewed electronically using a picture archiving and communication system ( pacs ) ( lifeweb ; ferrania technologies , london , england ) that allowed manual windowing and optimisation of parameters . 
in all cases , source images , subtracted images ( postcontrast minus precontrast unenhanced images ) and maximum intensity projection ( mip ) reconstructions were evaluated , as were signal intensity / time ( si / t ) curves determined at regions of interest ( roi ) placed on enhancing regions within the lesion . 
 type i curves demonstrated persistent enhancement with either a straight si / t line ( ia ) or curve ( ib ) and were more frequently considered indicative of a benign lesion ; type ii curves demonstrated a rapid early increase of si followed by an si plateau and were considered borderline ; type iii curves demonstrated a rapid early increase of si followed by rapid washout of contrast material and were considered indicative of malignancy [ 1315 ]  . 
based on the bi - rads classi cation a spiculated lesion with rim enhancement , nonsmooth borders and rapid washout would typically be considered a bi - rads 5 lesion . 
the size of all detected lesions was recorded for subsequent comparison . lesion biopsy all incidental enhancing lesions detected on breast mri subito dopo linoculazione del mezzo di contrasto , alla ne del usso di soluzione siologica . second - look ecogra co gli esami ecogra ci sono stati eseguiti da uno dei due esperti radiologi , impiegando apparecchi ad alta risoluzione ( ssa700a , toshiba , giappone ) , con sonde lineari con frequenza di 14 mhz . 
per lo studio dei quadranti interni le pazienti sono state sistemate in posizione prona mentre i quadranti esterni sono stati esaminati con la paziente adagiata sul anco controlaterale e con le braccia sollevate . 
 in ogni caso vista la differenza di posizionamento tra risonanza magnetica ed ecogra a , larea studiata attraverso lecogra a mirata sempre coincisa con lintero quadrante della mammella . interpretazione delle immagini tutte le immagini di risonanza magnetica ed ecogra a sono state analizzate da due radiologi in consensus ( xy , sette anni di esperienza nel campo della rm ; xx cinque anni di esperienza nel campo della rm ; xy , nove anni di esperienza in ecogra a mammaria ; xx , sette anni di esperienza in ecogra a mammaria ) con piena conoscenza della storia clinica delle pazienti e con i precedenti esami diagnostici convenzionali a disposizione per confronti . 
tutte le immagini sono state riesaminate elettronicamente attraverso un sistema di archiviazione e trasmissione di immagini ( lifeweb , ferrania technologies , londra , inghilterra ) che ha consentito di modi care manualmente la nestra ed ottimizzare i parametri . 
la valutazione delle immagini di rm avvenuta in base alla morfologia delle lesioni ed alle caratteristiche di potenziamento post - contrastogra co secondo la classi cazione bi - rads [ 8 ]  . 
secondo tale classi cazione le lesioni individuate sono state de nite come lesioni nodulari ( mass - like ) , non nodulari ( non mass - like ) o foci di potenziamento post - contrastogra co . 
in tutti i casi sono stati prese in esame sia le immagini base sia quelle sottratte ( immagini dopo somministrazione di mezzo di contrasto meno le immagini precontrasto ) sia le ricostruzioni di massima intensit di proiezione ( mip ) come anche le curve intensit segnale / tempo ( is / t ) , ottenute attraverso il posizionamento di una regione di interesse ( roi ) in un punto di potenziamento post - contrastogra co allinterno della lesione . 
 le curve di tipo i con un potenziamento di tipo continuo e corrispondenti ad una linea dritta ( ia ) o ad una curva ( ib ) 890 radiol med ( 2011 ) 116 : 886904 that were considered category 3 , 4 or 5 according to the bi - rads lexicon [ 8 ] and that subsequently had a suspicious correlate on second - look us ( bi - rads 45 ) were evaluated histologically immediately after the second - look us examination . 
lesions with a de nite us correlate were evaluated by means of core needle biopsy ( cnb ) under us guidance or by excisional biopsy following us - guided wire localisation . 
incidental enhancing lesions that were considered bi - rads 3 on breast mri that had a correlate lesion on second - look us that was considered benign or probably benign ( bi - rads 23 ) were managed conservatively with initial follow - up breast mri and us at 6 months , followed by repeat follow - up at 12 and 24 months . 
 for suspicious lesions at mri ( bi - rads 45 ) without a us correlate , cnb was performed under mri guidance , whereas lesions considered bi - rads 3 on breast mri were managed conservatively , with initial follow - up breast mri at 6 months followed by repeat follow - up at 12 and 24 months . 
repeat mri was avoided for patients with lesions with similar size and morphology at mri and us that was con rmed as broadenoma on histology after us - guided biopsy . 
 thereafter , the relative abilities of breast mri and secondlook us to accurately characterise incidental enhancing lesions was determined for all histologically proven lesions and all lesions referred for follow - up . 
for this latter assessment , lesions considered bi - rads 4 or 5 on breast mri were considered true positive ( tp ) for malignancy if histology subsequently revealed a malignant lesion but false positive ( fp ) if histology revealed a benign lesion . 
 conversely , lesions classi ed as bi - rads 3 on mri were considered true negative ( tn ) if histology or follow - up subsequently revealed a benign lesion but false negative ( fn ) if histology or follow - up revealed a malignant lesion . 
 likewise , a tp malignant lesion on second - look us was a lesion demonstrating malignant features ( bi - rads 45 ) that was con rmed as malignant on histology ; an fp lesion was one that demonstrated malignant features con rmed as benign at histology . 
finally , a tn lesion on second - look us was one that demonstrated benign features ( bi - rads 23 ) and was con rmed as benign at histology or followup ; an fn lesion was one that demonstrated benign features but was con rmed as malignant at histology . 
sensitivity , sono state associate pi frequentemente a lesioni benigne ; le curve di tipo ii caratterizzate da un rapido e precoce incremento dellintensit del segnale seguito dal raggiungimento di un plateau dellintensit del segnale sono state considerate borderline ; le curve di tipo iii comprendenti curve con rapido e precoce incremento dellintensit di segnale seguito da un rapido washout del mezzo di contrasto sono state invece considerate come indicative di malignit [ 1315 ]  . 
in relazione alla classi cazione bi - rads quindi una lesione con morfologia spiculata , orletto periferico di impregnazione post - contrastogra ca , bordi sfumati e rapido washout , tipicamente dovrebbe essere giudicata come bi - rads 5 . 
di tutte le lesioni sono state documentate le dimensioni in modo da rendere possibile i successivi confronti . biopsia delle lesioni tutte le lesioni riscontrate occasionalmente alla risonanza magnetica , considerate come categorie 3 , 4 o 5 secondo la classi cazione bi - rads [ 8 ] e con caratteristiche di sospetto al second - look ecogra co ( birads 4 - 5 ) , immediatamente dopo il controllo ecogra co mirato sono state sottoposte ad esame istologico . 
le lesioni incidentali , de nite bi - rads 3 alla risonanza magnetica della mammella e con un corrispettivo ecogra co benigno o probabilmente benigno ( bi - rads 2 - 3 ) , sono state indirizzate ad una strategia terapeutica conservativa , comprensiva di un follow - up iniziale con risonanza magnetica ed ecogra a bilaterale delle mammelle a 6 mesi e con la ripetizione di tali esami a 12 e 24 mesi . 
per le lesioni de nite sospette alla risonanza magnetica ( birads 4 - 5 ) , per le quali tuttavia non era stato possibile individuare un corrispettivo ecogra co la biopsia percutanea stata effettuata sotto guida rm , mentre le lesioni classi cate come bi - rads 3 alla risonanza magnetica della mammella sono state trattate in maniera conservativa con uniniziale risonanza magnetica della mammella eseguita a 6 mesi e successivamente a 12 e 24 mesi . 
le pazienti , con reperti benigni aspeci ci alla biopsia percutanea sotto guida ecogra ca , sono state sottoposte nuovamente a rm della mammella dopo un mese , per confermare che fosse stata bioptizzata la lesione corretta . 
in caso di pazienti con lesioni con morfologia e dimensioni simili sia alla rm che allesame ecogra co e confermate broadenomi allesame istologico dopo biopsia percutanea sotto guida ecogra ca , la ripetizione della risonanza magnetica stata evitata . analisi statistica stata valutata lindividuazione delle lesioni al secondradiol med ( 2011 ) 116 : 886904 speci city , accuracy and positive ( ppv ) and negative ( npv ) predictive values of breast mri and second - look us for the characterisation of incidental enhancing lesions was then determined for all incidental lesions detected on mri . 
analysis was performed using a general linear mixed model ( glimmix ; sas version 9.2 ) , adapted to t a model having sensitivity , speci city , accuracy , ppv or npv as the dependent variable ( distributed as a binary and introduced in the model by means of a logit link function ) ; and techniques ( xed effect ) , patients and lesions ( random effects ) as independent variables . 
analyses were also performed to compare detection and characterisation of mass - like and non - mass - like lesions on mri and secondlook us , the relative difference in size of incidental lesions detected on both mri and second - look us and detection of lesions on second - look compared with rst - look us . 
finally , comparison of lesion detection on second - look compared with rst - look us was performed using mcnemars test at a signi cance level of p < 0.05. 
of these 58 patients , 46 ( mean age 50.711.5 years ; range 2175 years ) underwent second - look high - resolution us directed at the site of the incidental nding and had either pathology results or full 24 - month follow - up information available . 
three patients were excluded because second - look us information was not available ( patients changed hospital ) , whereas nine patients were excluded because complete 24 - month follow - up information was missing . 
 quindi attraverso lanalisi di tutte le lesioni sottoposte ad esame istologico , pi tutte quelle sottoposte a follow - up , sia per la rm mammaria che per il second - look ecograco mirato , stata calcolata la capacit di caratterizzare in maniera accurata le lesioni occasionali . 
a tale scopo le lesioni classi cate come bi - rads 4 o 5 alla rm mammaria , sono state considerate come veri positivi ( vp ) per la malignit , se confermate maligne allesame istologico mentre sono state considerate falsi positivi ( fp ) , nel caso in cui lesame istologico abbia mostrato la presenza di una lesione benigna . 
cos al contrario , le lesioni classi cate bi - rads 3 alla rm della mammella , sono state reputate vp , quando lesame istologico o il follow - up abbiano rivelato la loro natura benigna e fp quando abbiano rivelato la loro natura maligna . 
quindi una lesione maligna , vp al second - look ecogra co , stata considerata una lesione con caratteristiche di malignit ( bi - rads 4 o 5 ) , confermata tale allesame istologico , mentre una lesione fp stata considerata una lesione con caratteristiche maligne , ma giudicata benigna allesame istologico . 
in ne stato de nito vn al second - look ecogra co , una lesione con caratteristiche benigne ( bi - rads 2 o 3 ) confermate tali allesame istologico , mentre una lesione fn stata considerata una lesione con caratteristiche di benignit , ma maligna allistologia . 
 per tutte le lesioni riscontrate occasionalmente alla rm della mammella sono stati calcolati sensibilit , speci cit , accuratezza ed i valori predittivo positivo ( vpp ) e negativo ( vpn ) rispettivamente sia della rm mammaria che del second - look ecogra co relativi alla caratterizzazione di tali reperti . 
lanalisi statistica stata condotta mediante limpiego di un modello misto lineare generalizzato ( glimmix , sas version 9.2 ) , adattato ad un modello avente sensibilit , speci cit , accuratezza , vpp o vpn come variabili dipendenti ( distribuite attraverso un sistema binario ed introdotte nel sistema mediante una funzione link logit ) e tecniche ( effetto sso ) e pazienti , lesioni ( effetto casuale ) come variabili indipendenti . 
la sensibilit stata stimata in base al tipo di lesione ( lesione di tipo nodulare o non nodulare ) e confrontata tra i diversi tipi attraverso il test esatto di fisher . 
lanalisi statistica stata compiuta anche per confrontare lindividuazione e caratterizzazione delle lesioni alla rm ed al second - look ecograco , la relativa differenza nelle dimensioni delle lesioni occasionali osservate alla rm ed al second - look ecogra co e per confrontare lindividuazione delle lesioni al secondlook ecogra co rispetto al rst look ecogra co . 
quarantasei di queste 58 pazienti ( et media : 50 , 711 , 5 anni ; intervallo di et 2175 anni ) , con a disposizione i risultati dellesame istologico o del follow - up completo a 24 mesi , sono state sottoposte anche a second - look ecogra co , mirato alla valutazione dellarea , sede del reperto riscontrato occasionalmente . 
tre pazienti sono state escluse per la non disponibilit di informazioni relative al second - look ecogra co ( le pazienti avevano cambiato ospedale ) mentre altre 9 sono state escluse per la mancanza delle informazioni relative al follow - up completo a 24 mesi . 
complessivamente sono state individuate 55 lesioni incidentali con impregnazione postcontrastogra ca , delle quali 24 classi cate come bi - rads 3 , 22 come bi - rads 4 e 9 come bi - rads 5 alla rm . 
queste 55 lesioni sono state identi cate nella stessa mammella in 25 / 46 pazienti ( 26 lesioni ) e nella mammella controlaterale in 21 / 46 pazienti ( 29 lesioni , comprendenti 2 pazienti con patologia multifocale di riscontro occasionale per un totale di 9 lesioni ) ; 39 lesioni erano inoltre di tipo nodulare ( mass - like ) mentre 16 di tipo non nodulare ( non mass - like )  . 
 le dimensioni di tutte lesioni occasionali alla rm della mammella , sono risultate comprese tra 3 e 13 mm ( dimensione media : 72 , 2 mm ) eccetto 5 di dimensioni 1 c il second - look ecogra co stato eseguito su tutte le regioni corrispondenti alle 55 lesioni individuate in maniera occasionale alla rm mammaria . istologia delle lesioni la biopsia percutanea o escissionale stata effettuata su 34 ( 61 , 8% ) delle 55 lesioni incidentali in 31 / 46 pazienti . 
1 diagramma di usso che mostra i criteri di selezione e valutazione dei pazienti . were detected in the ipsilateral breast in 25 / 46 patients ( 26 lesions ) and the contralateral breast in 21 / 46 patients ( 29 lesions , including two patients with incidental multifocal disease , for a total of nine lesions ) and included 39 lesions that were mass - like and 16 that were non - mass - like . 
the size of the incidental lesions detected on breast mri ranged from 313 mm ( mean size 72.2 mm ) , with all but ve lesions 1 cm in diameter . 
second - look us was performed at the sites of all 55 incidental enhancing lesions detected at breast mri . lesion histology core needle or excisional biopsy was performed for 34 ( 61.8% ) of the 55 incidental enhancing lesions in 31 / 46 patients . 
2a - c a 68 - year - old woman with a parenchymal distortion in the upper outer quadrant of the left breast , con rmed to be an invasive lobular carcinoma ( ilc ) at pathology after core biopsy , at mammography and negative ultrasound ( us )  . 
the transverse contrast enhanced t1 - weighted subtracted magnetic resonance image ( mri ) ( tr / te / fa = 8.1ms / 4 ms / 30 ) reveals an enhancing mass ( arrow ) with spiculated margins in the lower outer quadrant of the left breast ( a )  . 
2a - c donna di 68 anni con distorsione parenchimale ( carcinoma lobulare invasivo allesame istologico dopo biopsia percutanea ) nel quadrante superiore esterno ( qse ) della mammella di sinistra alla mammogra a ed ecogra a negativa . 
limmagine di rm ( sequenza t1 pesata , sottratta , dopo somministrazione di mezzo di contrasto ; tr / te / fa = 8 , 1 ms / 4 ms / 30 ) mostra una massa con impregnazione postcontrastogra ca ( freccia ) a margini spiculati nel quadrante inferiore esterno ( qie ) della mammella di sinistra ( a )  . 
la lesione stata sottoposta a resezione dopo posizionamento di repere sotto guida ecogra ca e confermata maligna ( carcinoma lobulare invasivo ) dallesame istologico . performed under us guidance for 31 / 34 lesions in 28 / 31 patients and under mri guidance for the remaining 3 / 34 lesions in 3 / 31 patients . 
however , the positive diagnosis of invasive ductal carcinoma ( idc ) and ductal carcinoma in situ ( dcis ) for the ve biopsied lesions led to both patients undergoing quadrantectomy . 
 mri - guided biopsy was performed in two patients because no lesions were detected on second - look us , whereas in the third patient , mri - guided biopsy was performed for one of two lesions not detected on second - look us despite depiction of two correlate lesions with benign features on second - look us . 
la biopsia stata eseguita sotto guida ecogra ca per 31 / 34 lesioni in 28 / 31 pazienti e sotto guida rm per le rimanenti 3 / 34 lesioni in 3 / 31 pazienti . 
in ogni caso la diagnosi positiva di carcinoma duttale in ltrante ( cdi ) e di carcinoma duttale in situ ( cdis ) per le 5 lesioni sottoposte a biopsia ha fatto s che entrambe le pazienti andassero incontro a quadrantectomia . 
la biopsia rm - guidata stata svolta in 2 pazienti per lassenza di lesioni al second - look ecogra co , mentre nella terza paziente la biopsia sotto guida rm stata eseguita su una delle 2 lesioni non evidenziabile al second - look ecograco , nonostante lindividuazione di 2 lesioni correlate , con caratteristiche benigne , al second - look ecogra co . 
3a - c a 63 - year - old woman with a suspicious lesion in the upper outer quadrant of the left breast [ invasive ductal carcinoma ( idc ) at cytology ] at mammography and ultrasound ( us ) and negative examination in the contralateral breast . 
the transverse contrast - enhanced t1 - weighted gradient - echo subtracted magnetic resonance image ( mri ) ( tr / te / fa = 8.1m / 4 ms / 30 ) reveals an enhancing lesion ( arrow ) in the lower outer quadrant of the right breast ( a )  . 
the lesion has smooth and irregular margins , and the signal intensity / time ( si / t ) curve was considered to be type i [ breast imaging reporting and data system ( bi - rads ) 3 at mri ] ( b )  . 
3a - c donna di 63 anni , recentemente sottoposta a mastoplastica additiva , con lesione sospetta nel qse della mammella di sinistra ( carcinoma duttale invasivo allesame citologico ) alla mammogra a ed ecogra a della mammella controlaterale negativa . 
limmagine di rm ( sequenza gre t1 pesata , sottratta , dopo somministrazione di mezzo di contrasto ; tr / te / fa = 8 , 1 ms / 4 ms / 30 ) rivela una lesione con impregnazione post - contrastogra ca ( freccia ) nel qie della mammella destra ( a )  . 
lesame istologico de nitivo ha rivelato uniperplasia lobulare atipica . hyperplasia , and one ( 3.0% ) each lobular carcinoma in situ ( lcis ) , brocystic dysplasia , atypical lobular hyperplasia , broadenoma , papillomatosis , radial scar , intramammary lymph node ]  . 
three lesions classi ed as bi - rads 4 at mri were subsequently con rmed as benign at histology ( one broadenoma , one intramammary lymph node and one radial scar ) and were considered fp for subsequent analyses . 
tre lesioni classi cate bi - rads 4 alla rm sono state successivamente de nite benigne allesame istologico ( 1 broadenoma , 1 linfonodo intramammario , 1 radial scar ) e quindi considerate fp per le successive valutazioni . 
lesions detected on second - look us included 36 ( 92.3% ) of the 39 su tutte le informazioni radiologiche e cliniche raccolte nei 24 mesi successivi , ha confermato la benignit di tutte e 17 le lesioni . second - look ecogra co il second - look ecogra co ha identi cato 42 delle 55 lesioni aggiuntive in 38 ( 82 , 6% ) delle 46 pazienti . 
considerando che le lesioni evidenti al rst look ecogra co erano apprezzabili anche al second - look ecogra co , lindividuazione delle lesioni al second - look ecogra co , in confronto al primo esame ecogra co , stata estremamente signi cativa tabella 1 confronto tra lindividuazione delle lesioni occasionali , con impregnazione post - contrastogra ca al second - look ecogra co ed alla risonanza magnetica della mammella , per lesioni con conferma allesame istologico lesioni occasionali alla rm della mammella confermate istologicamente reperti al second - look ecogra co paziente no . 
tra le lesioni osservate al secondlook ecogra co vi sono state 36 ( 92 , 3% ) delle 39 lesioni di tipo nodulare evidenziate alla rm , ma solamente 6 ( 37 , 5% ) delle 16 lesioni di tipo non nodulare . 
4a - d a 55 - year - old woman with a cluster of suspicious microcalci cations in the lower outer quadrant of the left breast [ con rmed to be an invasive ductal carcinoma ( idc ) at pathology after core biopsy ] at mammography . 
the transverse contrast - enhanced t1 - weighted gradient - echo ( gre ) subtracted magnetic resonance image ( mri ) ( tr / te / fa = 8.1ms / 4 ms / 30 ) reveals four enhancing nodules with irregular margins in the upper inner quadrant of the right breast ( arrows ) ( a )  . 
despite the si / t curve , the lesions were classi ed as breast imaging reporting and data system ( bi - rads ) 4 on mri because of the morphology . 
4a - d donna di 55 anni con un cluster di microcalci cazioni sospette nel qie della mammella di sinistra ( carcinoma duttale invasivo allesame istologico dopo biopsia percutanea ) alla mammogra a . 
limmagine di rm ( sequenza gre t1 pesata , sottratta , dopo somministrazione di mezzo di contrasto ; tr / te / fa = 8 , 1 ms / 4 ms / 30 ) rivela quattro noduli con impregnazione post - contrastogra ca nel qsi della mammella di destra ( frecce ) ( a )  . 
 le 42 lesioni osservate al second - look ecogra co hanno incluso 21 ( 75 , 0% ) delle 28 ( 24 lesioni sottoposte a biopsia pi 4 lesioni nelle pazienti con patologia multifocale ) lesioni confermate maligne alla rm mammaria e 21 ( 77 , 8% ) delle 27 lesioni che sono state successivamente confermate ( 10 lesioni alla biopsia ) o diagnosticate ( 17 lesioni dopo il follow - up ) come benigne . 
in tutte queste pazienti le lesioni non evidenziabili al second - look ecogra898 radiol med ( 2011 ) 116 : 886904 lesions ( three idc , two dcis ) detected on breast mri and classi ed as bi - rads 4 were present in two patients with incidental multifocal disease who subsequently underwent quadrantectomy . 
at nal diagnosis after follow - up mri , three of these lesions were considered benign , whereas two lesions , which occurred in two premenopausal patients of 32 and 42 years , could not be seen on follow - up mri or us and were considered transient areas of enhancement arising due to the hormonal status of the women at the time of the initial mri examination . 
considering lesions not seen on second - look us as either tn or fn , if the corresponding lesion on mri was tn or tp , respectively , there were 18 tp , eight fp , 19 tn and ten fn lesions on second - look us . 
although evaluation of differences in sensitivity and npv was not possible because of the absence of fn ndings with mri , a signi cant ( p = 0.0021 ) bene t for mri over secondlook us was noted for overall accuracy for depicting lesions . 
differences in the characterisation of mass - like and non - mass - like lesions on second - look us are shown co avevano dimensioni 5 mcomplessivamente il grado di con denza diagnostica del second - look ecogra co per le lesioni presenti sia alla rm che al second - look ecogra co stato pari al 73 , 5% ( 95% intervallo di con denza [ ci ] : 63%86 , 8% ) ma del 92 , 3% ( 95% ci : 79 , 1%98 , 4% ) solo per le lesioni di tipo nodulare . 
tra le 13 lesioni non riscontrabili al second - look vi stato 1 cli di 5 mm classi cato come bi - rads 5 alla rm e 2 casi confermati di cdis di 7 mm precedentemente classi cati come bi - rads 4 ( tabella 1 )  . 
5 ulteriori lesioni ( 3 cdi , 2 cdis ) identi cate alla rm della mammella e classi cate come bi - rads 4 sono state apprezzate nelle due pazienti con patologia multifocale , diagnosticata in via occasionale , che successivamente sono state sottoposte a quadrantectomia . 
second - look us sensitivity ( % ) speci city ( % ) accuracy ( % ) ppv ( % ) npv ( % ) 100 ( 28 / 28 ) 88.9 ( 24 / 27 ) 94.6 ( 52 / 55 ) 90.3 ( 28 / 31 ) 100 ( 24 / 24 ) 64.3 ( 18 / 28 ) 70.4 ( 19 / 27 ) 67.3 ( 37 / 55 ) 69.2 ( 18 / 26 ) 65.5 ( 19 / 29 ) analysis performed using general linear mixed model nc , not calculable ; ppv , positive predictive value ; npv , negative predictive value tabella 2 performance diagnostica del second - look ecogra co confrontata con la rm della mammella per la caratterizzazione di 55 lesioni incidentali alla rm parametri di performance diagnostica second - look ecogra co rm vs . 
the three mass - like lesions among the 13 unseen lesions were 6 , 6 and 7 m the slightly larger size of lesions detected at second - look us compared with mri was con rmed by bland - altman analysis . 
given the increasingly widespread use of breast mri and its acknowledged high sensitivity for detecting lesions [ 1628 ] , a potential dilemma arises when incidental abnormal foci of enhancevp , 3 fp , 24 lesioni vn e nessun fn . 
attribuendo il valore di vn o fn alle lesioni non viste al second - look ecogra co , a seconda che le corrispettive lesioni alla rm fossero vn o vp , rispettivamente , al second - look ecogra co si sono osservate 18 lesioni vp , 8 lesioni fp , 19 lesioni vn e 10 fn . 
sebbene una stima delle differenze nella sensibilit e nel vpn non sia stata possibile a causa del mancato riscontro di reperti fn alla rm , complessivamente stato registrato un vantaggio signi cativo ( p = 0 , 0021 ) nellaccuratezza diagnostica della rm rispetto al second - look ecograco . 
per la caratterizzazione delle lesioni di tipo nodulare rispetto a quelle di tipo non nodulare stata osservata una signi cativa , maggiore performance in termini di sensibilit ( p = 0 , 026 ) ed accuratezza ( p = 0 , 0428 )  . dimensioni delle lesioni la dimensione media delle 42 lesioni evidenziate al secondlook ecogra co stata di 7 , 952 , 56 malla rm le stesse lesioni avevano dimensioni pari a 7 , 382 , 28 mla differenza tra second - look ecogra co e rm ( 0 , 570 , 94 mm ) , nelle dimensioni medie , stata signi cativa ( p = 0 , 0003 )  . 
tra le 13 lesioni non osservate , 3 misuravano 6 , 6 e 7 mil fatto che le dimensioni delle lesioni al second - look ecogra co fossero lievemente maggiori rispetto alla rm stato confermato con il test di bland - altman . discussione il ruolo della rm mammaria ampiamente riconosciuto per numerose situazioni cliniche [ 14 ]  . 
visto il ricorso , sempre pi frequente , alla rm della mammella e vista la sua ben nota , elevata sensibilit , nellidenti cazione delle lesioni [ 1628 ] , il riscontro occasionale di foci di impregnazione post - contrastogra ca , precedentemente non evidenziati agli esami convenzionali di mammogra a ed ecogra a , pu costituire un potenziale dilemma . 
per tale motivo il second - look ecogra co mirato viene considerato sempre di pi un passaggio necessario nella gestione delle pazienti con lesioni individuate incidentalmente alla rm mammaria e non evidenziate mammogra camente ed ecogra camente [ 2633 ]  . 
questo fenomeno legato anche ai recenti risultati che mostrano come le lesioni occasionali alla rm , con un corrispettivo ecogra co , abbiano una maggiore probabilit di essere maligne [ 2629 ] ed in parte al fatto che la biopsia sotto guida ecogra ca sia meno costosa e pi facilmente reperibile rispetto alla biopsia sotto guida rm [ 27 , 3436 ]  . 
in questo senso se una lesione evidenziata alla rm pu essere individuata anche ecogra 900 radiol med ( 2011 ) 116 : 886904 ment are revealed that had previously gone undetected on conventional mammography or us . 
for this reason , second - look - directed us is increasingly considered an integral step in managing patients with mammographically and sonographically occult breast lesions incidentally detected on breast mri [ 2633 ]  . 
in part , this re ects recent ndings that show that incidental enhancing lesions on mri that have a de nite us correlate are more likely to be malignant [ 2629 ] and in part the fact that biopsy under us guidance is less expensive and more readily available than biopsy under mri guidance [ 27 , 3436 ]  . 
in this setting , if a lesion detected at breast mri can be depicted with directed us , it may be amenable to us - guided percutaneous biopsy , which may spare the patient mri - guided biopsy . 
in our study , 55 incidental enhancing lesions , considered bi - rads 3 , 4 or 5 at mri , were detected in 46 ( 25.3% ) of 182 patients referred for preoperative breast mri for histologically con rmed breast cancer depicted on x - ray mammography and / or rst - look wholebreast us . 
 [ 27 ] , who identied a us correlate for 21 ( 23% ) of 93 lesions detected on breast mri , it should be noted that the authors also included patients with lesions classi ed as bi - rads 2 and that those patients comprised the majority of patients who would not have a second - look us correlate . in our study , patients with incidental lesions classi ed as bi - rads 2 were not included . 
 [ 28 ] , who found a de nite correlate on second - look us for 142 ( 82% ) of 173 lesions initially detected only on breast mri ; and those of destounis et al . 
 [ 26 ] , who retrospectively found 31 ( 57% ) of 54 mri - detected and histologically con rmed malignant lesions upon re - evaluation of previous mammograms and sonograms ; and wiratkapun et al . 
 [ 30 ] , who identi ed 46 ( 47% ) of 97 mri - detected lesions on targeted us in 55 women . given that precise topographic evaluation of the site of the incidental lesion is achievable with mri before the second - look us examination , it is perhaps not surprising that directed second - look us should prove effective at depicting even small lesions . 
notably , the mean size of the corresponding lesions on mri was smaller ( 7.382.28 ; p = 0.0003 ) , possibly re ecting the better resolution of camente , possibile pensare di effettuare la biopsia percutanea sotto guida ecogra ca risparmiando alla paziente la biopsia rm - guidata . 
inoltre lesecuzione della biopsia percutanea sotto guida ecogra ca pu accelerare la pianicazione preoperatoria della strategia terapeutica , nel caso in cui siano ottenuti dei risultati maligni [ 2 , 4 ]  . 
nel nostro studio in 46 ( 25 , 3% ) delle 182 pazienti , sottoposte a rm mammaria preoperatoria per la presenza di carcinoma mammario , confermato istologicamente e diagnosticato mammogra camente e / o attraverso lecogra a mammaria bilaterale , stato riscontrato occasionalmente un numero totale di 55 lesioni con potenziamento postcontrastogra co , classi cate come bi - rads 3 , 4 o 5 . 
 [ 27 ] , che hanno identi cato un corrispettivo ecogra co per 21 ( 23% ) delle 93 lesioni evidenziate alla rm , doveroso sottolineare che la trenta et al . 
 [ 27 ] hanno incluso anche pazienti con lesioni de nite bi - rads 2 , che costituiscono la maggior parte delle lesioni prive di un corrispettivo ecogra co al second - look . 
 [ 28 ] che hanno trovato un sicuro corrispettivo ecogra co al second - look per 142 ( 82% ) delle 173 lesioni inizialmente osservate solo alla rm e con quelli di destounis et al . 
 [ 26 ] che retrospettivamente , attraverso la rivalutazione dei precedenti esami mammogra ci ed ecogra ci hanno individuato 31 ( 57% ) delle 54 lesioni evidenti alla rm e confermate maligne allesame istologico e con quelli di wiratkapun et al . 
dal momento che con la rm possibile ottenere una precisa valutazione topogra ca della sede della lesione incidentale prima del second - look ecogra co forse non sorprende lef cacia del second - look ecogra co nellindividuazione anche di lesioni di piccole dimensioni . 
in particolare , la dimensione media delle lesioni corrispondenti alla rm era inferiore ( 7 , 382 , 28 ; p = 0 , 0003 ) , questo forse per la migliore risoluzione della rm . 
 [ 27 ] per le lesioni identi cate sia allesame di rm che ecogra co hanno riportato una dimensione media pari a 9 mm contro gli 8 mm registrati per le lesioni non evidenziabili allesame ecogra co . 
nel nostro studio 24 ( 57 , 1% ) delle 42 lesioni , con un corrispettivo ecogra co , sono state successivamente confermate maligne contro le 7 ( 53 , 4% ) delle 13 , per le quali non era stato possibile apprezzare un corrispettivo ecogra co . 
in our study , 24 ( 57.1% ) of the 42 lesions with a us correlate were subsequently con rmed as malignant compared with seven ( 53.4% ) of 13 lesions that did not have a us correlate . 
although this ratio does not re ect the ndings of previous studies that demonstrated a signi cantly higher likelihood of carcinoma among lesions with a us correlate than among lesions without a us correlate [ 27 , 29 , 33 ] , it can possibly be explained by the fact that the number of lesions without a us correlate was relatively small , and that four of the seven carcinomas that did not have a us correlate were very small ( 5 mm ) and occurred in two patients with incidental enhancing multifocal disease . 
although our ndings suggest that lesion detection on second - look us does not increase the likelihood that a lesion detected on mri is malignant , further work in a larger patient cohort should certainly be performed to clarify these ndings [ 37 ]  . 
discrepancies between us and breast mri among histologically con rmed malignant lesions were noted for two dcis lesions , which were considered to have benign features on second - look us . 
conversely , a broadenoma and an intramammary lymph node , which were misdiagnosed as malignant on breast mri , were both determined to have benign features on second - look us . 
more notable discrepancies between second - look us and breast mri occurred for con rmed benign lesions that were considered to have malignant or , more frequently , doubtful features on second - look us . 
this might have been due to the fact that only women with breast carcinoma were evaluated , which may have negatively in uenced the interpretation of second - look us images . 
a prospective study with randomised mr and second - look us images evaluated separately might have negated a possible bias towards more pessimistic interpretations . possible limitations of our study are : ( i ) evaluation was performed on a lesion - by - lesion basis without focusing i risultati degli studi precedenti , che hanno mostrato una signi cativa maggiore probabilit di malignit tra le lesioni prive di di un corrispettivo ecogra co [ 27 , 29 , 33 ] , in realt pu essere spiegato dal fatto che , il numero delle lesioni non riscontrate al second - look ecogra co stato relativamente basso e che 4 dei 7 carcinomi , che non avevano un corrispettivo ecogra co in realt avevano dimensioni molto piccole ( 5 mm ) e si erano presentati in due pazienti con malattia multifocale incidentale . 
sebbene i risultati del nostro studio suggeriscano che lindividuazione delle lesioni al controllo ecogra co mirato non si associ alla maggiore probabilit di malignit della lesione evidenziata alla rm , per chiarire questo punto sono necessari ulteriori studi condotti su una maggiore coorte di pazienti [ 37 ]  . 
questo per riconducibile al fatto che la nostra valutazione stata condotta solamente su pazienti con carcinoma mammario gi noto , nelle quali il rischio di carcinomi sincroni o omolaterali alla neoplasia nota , maggiore . 
 per quanto riguarda la performance diagnostica , la sensibilit , la speci cit , laccuratezza , il vpp e vpn della risonanza magnetica della mammella con gadobenatedimeglumine nel diagnosticare le lesioni con impregnazione post - contrastogra ca come maligne o benigne , i valori ottenuti sono stati rispettivamente 100% , 88 , 9% , 94 , 5% , 90 , 3% and 100% . 
in confronto per il second - look ecogra co sono stati ottenuti dei valori lievemente inferiori e pari a 90 , 5% , 61 , 9% , 76 , 2% , 70 , 4% ed 86 , 7% . 
tra le lesioni confermate come maligne allesame istologico , si sono riscontrate delle discrepanze tra esame ecogra co e rm mammaria , per due casi di cdis , ai quali al second - look ecogra co erano state attribuite caratteristiche di benignit . 
ulteriori importanti discrepanze tra second - look ecogra co e rm si sono veri cate per lesioni benigne , considerate maligne o comunque con caratteristiche di dubbia interpretazione al second - look ecogra co . 
 possibili limiti del nostro studio sono ( i ) che la valutazione stata condotta lesione per lesione senza mettere a fuoco limpatto esercitato dal rilevamento di lesioni occasionali sulla gestione del paziente , ( ii ) non abbiamo realizzato un sottogruppo per le pazienti con mammella densa , ( iii ) non abbiamo valutato in maniera speci ca pazienti con aumentato rischio di carcinoma mammario o pazienti 902 radiol med ( 2011 ) 116 : 886904 on the impact of the detected incidental enhancing lesions on patient management . 
as regards the latter issues , it is well established that detection of lesions on mammography is less reliable in women with very dense breast parenchyma [ 38 , 39 ] whereas studies have shown that it is not infrequent that benign - appearing lesions on mri may , in fact , be malignant in high - risk women with proven or pro le - like germ - line mutations [ 40 ]  . 
in our study , 24 - month follow - up was considered suf cient to diagnose benign lesions in women with benign - appearing lesions on mri with or without second - look us corroboration . 
nevertheless , future prospective work should be performed to evaluate the role of second - look us speci cally in women with proven or pro le - like germ - line mutations . 
likewise , further work should speci cally address the issue of incidental lesions classi ed as bi - rads 2 on mri . in conclusion , results of our analysis con rm that directed second - look us is a valuable technique for depicting and diagnosing incidental enhancing lesions detected on preoperative mri . 
if a lesion can be depicted with us , it may be amenable to us - guided percutaneous biopsy , which is less expensive and a more straightforward procedure than mri - guided biopsy . 
on the other hand , due to the similar rate of malignancy regardless of whether a de nite correlate is found on secondlook us , the absence of us correlate lesions should not preclude mri - guided biopsy of suspicious ( bi - rads 4 and 5 ) lesions detected on mri . 
per quanto riguarda le ultime questioni ben dimostrato che lidenticazione delle lesioni alla mammogra a , nelle pazienti con mammella densa , meno attendibile [ 38 , 39 ] mentre studi hanno dimostrato come nelle pazienti con elevato rischio di carcinoma mammario , con mutazione provata o pro lo simile a mutazioni della linea germinale , lesioni con caratteristiche di benignit alla rm possano essere invece essere maligne [ 40 ]  . 
nel nostro studio un periodo di follow - up a 24 mesi stato ritenuto suf ciente per considerare benigne lesioni con caratteristiche di benignit alla rm , indipendentemente dalla conferma del second - look ecogra co . 
in ogni caso necessario che nel futuro vengano condotti degli studi per valutare in maniera speci ca il ruolo del second - look ecogra co nella valutazione di donne con mutazione genetica accertata o aventi pro lo simile a mutazioni della linea germinale . 
 in conclusione i risultati del nostro studio confermano che , il second - look ecogra co mirato , costituisce una tecnica attendibile per lidenti cazione e diagnosi di lesioni riscontrate occasionalmente alla risonanza magnetica preoperatoria della mammella . 
marini1 1istituto di scienze radiologiche , policlinico umberto i , universit degli studi sapienza roma , viale regina elena 324 , 00161 roma , italy 2dipartimento di scienze radiologiche , ospedale pediatrico meyer , viale pieraccini 24 , careggi , italy 3chirurgia maxillo - facciale , policlinico umberto i , universit degli studi sapienza roma , viale regina elena 324 , 00161 roma , italy correspondence to : a . 
we enrolled 24 pregnant women ( 27 fetuses ) ( mean gestational age 23.7 weeks ) with a level - two us diagnosis of cleft lip ( cl ) or clp with or without associated central nervous system ( cns ) or facialbone anomalies . 
mri con rmed the diagnosis in 16 / 25 fetuses and added information about the extent of the cleft and the degree of involvement of the anterior and posterior palate in 8 / 25 fetuses . 
in the study of clp fetal , mri is able to de ne the degree of involvement of the posterior palate and the lateral extent of the cleft with higher diagnostic accuracy than us . 
abbiamo prospettivamente arruolato 24 donne in gravidanza ( 27 feti ; et gestazionale media 23 , 7 settimane ) con diagnosi ecogra ca di ii livello di lbs o lps , associata o meno ad anomalie cranio - encefaliche o del massiccio facciale ; le pazienti sono state sottoposte a rm fetale per lo studio del massiccio facciale , oltre che del sistema nervoso centrale ( snc ) e del body fetale . 
la rm ha confermato la diagnosi ecogra ca in 16 / 25 feti ed ha aggiunto informazioni riguardo lestensione della schisi ed il coinvolgimento del palato in 8 / 25 feti ; ha smentito la diagnosi in 1 / 25 feti . 
lo studio rm fetale del massiccio facciale radiol med ( 2011 ) 116 : 11341148 1135 study of the fetal head and biometric development of the facial bones , thus enabling early detection of potential syndromic conditions . keywords fetal mri facial cleft cleft lip and palate prenatal diagnosis nelle lbs - lps de nisce con maggiore accuratezza il grado di coinvolgimento del palato posteriore e la lateralit ; consente , inoltre , una valutazione del distretto cranioencefalico e dello sviluppo biometrico del massiccio facciale al ne di identi care precocemente eventuali condizioni sindromiche . parole chiave rm fetale labioschisi labio - palatoschisi diagnosi prenatale introduction introduzione cleft lip and palate ( clp ) is the most common facial malformation , having an incidence of around 1 / 700 live births [ 1 ]  . 
this malformation may be isolated or associated with genetic syndromes ; in fact , around 11% of patients with cleft lip ( cl ) also have one of more than 170 monogenic syndromes reported in the london dysmorphology database . 
therefore , the presence of an orofacial cleft associated with changes to the main biometric parameters of the fetal face should be considered a warning sign and prompt a fetal chromosome analysis in the search for possible syndromes and multiple anomaly conditions . in this setting , the ultrasound ( us ) examination plays a key role in the early prenatal diagnosis of cl , and especially in the identi cation of clp . 
accurate evaluation of the extension of the cleft underpins a correct diagnosis , parent counselling and treatment planning [ 5 ]  . study of the fetal face has in recent years relied not only on the traditional 2d us examination but also on new 3d and 4d techniques . 
these have made it possible to visualise all of the critical structures of the face ( forehead , eyes , nasal bones and nares , lips , chin ) with single - volume acquisitions , thus markedly increasing the accuracy in the diagnosis of secondary palate anomalies with the possibility of multiplanar reconstructions [ 68 ]  . 
these include surface rendering , which is able to produce images of the facial skin and soft tissues ; and transparency mode , which offers speci c images of the facial bones [ 9 ]  . fetal magnetic resonance imaging ( mri ) is progressively becoming a robust level - three technique in the diagnosis of facial anomalies , thanks to the advent of ultrafast sequences that suffer few motion artefacts and offer excellent spatial resolution . 
the aim of our study was to evaluate the role of fetal mri as a complement to us in diagnosing clp , whether isolated or associated with syndromes and multiple anomaly conditions . la labiopalatoschisi ( lps ) rappresenta lanomalia congenita del massiccio facciale di pi frequente riscontro ; la sua incidenza di circa 1 / 700 nati vivi [ 1 ]  . 
tale malformazione pu essere isolata o rientrare nel contesto di sindromi genetiche ; circa l11% dei pazienti affetti da schisi labiale ( lbs ) , infatti , pu essere inquadrato nel contesto di una delle oltre 170 sindromi monogeniche riportate nel london dysmorphology database . 
di conseguenza , in epoca prenatale , la presenza di schisi facciale associata ad alterazioni dei principali diametri del massiccio deve essere considerata un segnale dallarme ed indurre ad eseguire unanalisi cromosomica fetale e a ricercare eventuali condizioni polimalformative sindromiche e non . in tal senso lesame ecogra co riveste un ruolo chiave per la diagnosi prenatale precoce delle lbs , e soprattutto per lidenti cazione del coinvolgimento palatale ( lps ) ; laccurata de nizione dellestensione del cleft alla base di un corretto inquadramento diagnostico , del counseling con i genitori e della piani cazione terapeutica [ 5 ]  . lo studio del massiccio facciale si avvalso negli ultimi anni , oltre che del tradizionale esame ultrasonograco 2d , delle nuove tecniche 3d e 4d , le quali consentono di visualizzare tutte le strutture critiche della faccia ( fronte , occhi , ossa nasali e narici , labbra , mento ) con singole acquisizioni volumetriche , incrementando notevolmente laccuratezza diagnostica nei difetti del palato secondario con la possibilit di ricostruzioni multiplanari [ 68 ]  . 
inoltre , ad oggi sono disponibili ulteriori tecniche di ricostruzione ecogra che 3d e 4d , quali il surface rendering , che consente di ottenere immagini dei tessuti molli e del piano cutaneo del viso , e il trasparency mode , che permette di ottenere immagini speci che delle strutture ossee facciali [ 9 ]  . la risonanza magnetica ( rm ) fetale , grazie allavvento di sequenze ultraveloci , scarsamente artefattate dal movimento e dotate di ottima risoluzione spaziale , si sta progressivamente affermando , come esame di iii livello , anche 1136 materials and methods between may 2007 and april 2010 , we prospectively enrolled 24 pregnant women ( three twin pregnancies , n = 27 fetuses ) with a level - two us diagnosis of cl or clp with or without associated anomalies of the head , brain or face . 
only 14 / 24 women had undergone amniocentesis , which in all cases de ned a normal karyotype ( 5xy fetuses , 9 xx fetuses ) [ ce1 ]  . imaging technique the fetal mri study was performed with a 1.5 - t system ( siemens somatom avanto ) with 32 - channel phased - array coils used in combination with a spinal coil . 
 lo scopo del nostro studio valutare il ruolo complementare , rispetto allesame ecogra co , della rm fetale nellinquadramento delle labiopalatoschisi isolate e associate a quadri sindromici o polimalformativi . materiali e metodi tra maggio 2007 e aprile 2010 abbiamo prospettivamente arruolato 24 donne in gravidanza ( 3 gravidanze bi - gemellari , 27 feti totali ) con diagnosi ecogra ca di ii livello di lbs o lps , associata o meno ad altre anomalie cranioencefaliche o del massiccio facciale . 
tutte le pazienti sono state sottoposte ad esame di rm fetale per lo studio del massiccio facciale , oltre che del sistema nervoso centrale ( snc ) e del body fetale . 
solamente 14 / 24 pazienti avevano effettuato lamniocentesi de nendo un cariotipo normale in tutti i casi ( 5 feti xy , 9 feti xx )  . tecnica di imaging lesame di rm fetale stato effettuato con ununit a 1 , 5 t ( siemens somatom avanto ) , con bobine di super cie phased - array a 32 canali associate a una bobina spine integrata . 
 tutte le donne sono state sottoposte allesame senza alcun tipo di sedazione , n sulle donne stesse n sul feto , e senza somministrazione di mezzo di contrasto ; preventivamente le pazienti , attraverso la compilazione di un questionario relativo alla gravidanza e allanamnesi personale e familiare , hanno sottoscritto il proprio consenso informato . 
1a - h scanning planes to evaluate the main structures of the splanchnocranium : a - e axial plane ( white arrows : b orbits and eyes , c septum and nasal cavity , d upper jaw with dental roots , e lower jaw with dental roots ) ; f - h sagittal plane ( white arrows : g corpus callosum and palate , h upper lip ) fig . 
1a - h piani di scansione rm per lo studio del massiccio facciale : a - e piano assiale ( frecce bianche : b cavit orbitarie e globi oculari , c fosse nasali e setto nasale , d mascellare superiore con processi alveolari , e mascellare inferiore con processi alveolari ) ; f - h piano sagittale ( frecce bianche : g corpo calloso e palato , h labbro superiore )  . 1138 radiol med ( 2011 ) 116 : 11341148 maxilla and mandible with their tooth buds . 
the sagittal plane is the reference plane for the study of the facial pro le ( particularly useful for evaluating the forehead and its relationship with the nasal structures and the chin ) and the primary and secondary palate , which can best be visualised with a median plane normally used in the brain for studying the corpus callosu this scan is also used to evaluate the tongue and upper airways from the nasal cavities to the pharyngolaryngeal structures . 
the coronal plane is useful for evaluating the entire face and is especially suitable for studying the orbits , nose and nostrils , upper lip and mouth and the ch lastly , the paracoronal plane ( nosemouth scan ) is the reference plane in the evaluation of cl , given its usefulness in the study of the integrity of the upper lip and the normal morphology of the nostrils . 
sul piano assiale abbiamo valutato la posizione e la morfologia delle orbite e dei globi oculari , il setto e le fosse nasali , il mascellare superiore e la mandibola con le relative gemme dentarie . 
 il piano sagittale il piano di riferimento per lo studio del pro lo facciale ( particolarmente utile per la valutazione della fronte , anche in rapporto con le strutture nasali , e del mento ) e del palato anteriore e posteriore attraverso lacquisizione di un piano mediano normalmente utilizzato nellencefalo per lo studio del corpo calloso . 
il piano coronale utile per la valutazione del viso nel suo complesso , in particolare consente lo studio delle orbite , del naso e delle narici , del labbro superiore , della bocca e del mento . 
2a - c scanning planes to evaluate the main structures of the splanchnocranium : a , b coronal plane ( white arrows : b orbits and eyes ) ; c paracoronal plane ( black arrows : nostrils ; white arrow : upper lip ) fig . 
2a - c piani di scansione rm per lo studio del massiccio facciale : a , b piano coronale ( freccia bianca : b cavit orbitarie e globi oculari ) ; c piano paracoronale ( frecce nere : narici ; freccia bianca : labbro superiore )  . radiol med ( 2011 ) 116 : 11341148 1139 with the calculation of the jaw index , de ned as the ratio between apd and bpd ; inferior facial angle ( ifa ) , measured in the midsagittal plane of the head , between a line passing through the frontonasal junction , perpendicular to the forehead , and passing through the anterior border of the upper lip and the chin ; frontomaxillary angle ( fma ) , measured in the midsagittal plane and given by a line passing through the forehead and chin and another passing through the upper border of the palate ; biorbital ( bod ) and intraorbital ( iod ) diameter by tracing a line between the external and internal margins of the two orbits , respectively . results a total of 25 / 27 fetuses had a us diagnosis of cl - clp . 
in 6 / 24 fetuses with cl - clp , the mr study revealed abnormalities in the following parameters with respect to normal development of the fetal face : ifa , fma , iod . 
we considered normal the fetuses with these parameters in the normal range , as de ned by the 2d / 3d us study and reported in the literature [ 12 , 19 , 20 ]  . we found an abnormal ifa in three fetuses with clp riferimento nella valutazione dei cleft labiali , consentendo lo studio dellintegrit del labbro superiore e della normale morfologia delle narici . 
in sede di refertazione , abbiamo inoltre analizzato 7 parametri di riferimento [ 1214 ] per la valutazione biometrica del massiccio facciale : diametro fronto - occipitale osseo ( dfoo ) , misurato su un piano sagittale mediano dellestremo cefalico ; diametro antero - posteriore della mandibola ( dapm ) e diametro biparietale della mandibola ( dbpm ) , misurati su un piano assiale dellestremo cefalico , determinando il jaw index , de nito dal rapporto tra il dapm ed il dbpm ; angolo facciale inferiore ( ifa ) , misurato su un piano sagittale mediano del cranio , tra una linea passante per la giunzione fronto - nasale , perpendicolare alla fronte , e una passante per il bordo anteriore del labbro superiore e il mento ; angolo fronto - mascellare ( fma ) , misurato su un piano sagittale mediano dellestremo cefalico , dato da una linea passante per la fronte e il mento e unaltra passante per il margine superiore del palato ; diametro bisorbitario ( dbo ) ed interorbitario ( dio ) , misurati sul piano coronale , tracciando rispettivamente una linea tra i margini esterni ed interni delle due orbite . risultati venticinque / 27 feti avevano diagnosi ecogra ca di lbs - lps . 
 la rm ha confermato la diagnosi ecogra ca , determinando una concordanza completa tra le due metodiche , in 16 / 25 feti ; ha evidenziato una concordanza parziale in 8 / 25 feti ; ha smentito la diagnosi in 1 / 25 feti . 
labiopalatoschisi completa bilaterale con ampio cleft labiale e del palato anteriore e posteriore ( frecce bianche )  . with associated micrognathia , and an abnormal iod and above normal fma in the fetus with unilateral clp and associated holoprosencephaly , cyclopia and arachnoid cyst of the posterior cranial fossa . 
in addition , the fma was above normal in the fetus with unilateral clp , associated interventricular cardiac defect and borderline ventriculomegaly ; and in the fetus with unilateral cl , involvement of the primary palate , associated lateral shift of the nasal septum and mild bilateral ventriculomegaly . 
mr ndings were con rmed by the postnatal physical examination or autopsy ndings , which were considered the reference standard . discussion the facial skeleton is derived almost entirely from the mesenchyme of the rst three branchial arches , which consist of mesoderm , which develop lateroventrally in the deviazione del setto nasale e ventricolomegalia bilaterale lieve . 
lesame rm ha evidenziato in 6 / 24 feti , positivi per lbs - lps , unalterazione dei seguenti parametri riguardanti il normale sviluppo del massiccio facciale : ifa , fma , dio . 
abbiamo considerato come normali i feti che presentavano tali parametri nellambito dei range di normalit , de niti allesame ecogra co 2d / 3d , della letteratura [ 12 , 19 , 20 ]  . in particolare , abbiamo riscontrato unalterazione dellifa nei tre feti affetti da lps associata a micrognazia , del dio e dellfma , che risultava aumentato , nel feto affetto da lps unilaterale con oloprosencefalia alobare , ciclopia e cisti aracnoidea della fossa cranica posteriore ; inoltre lfma risultava aumentato nel feto affetto da lps unilaterale associata a difetto interventricolare cardiaco e ventricolomegalia bordeline , e nel feto affetto da labioschisi unilaterale con coinvolgimento del palato anteriore associata a laterodeviazione del setto nasale e ventricolomegalia bilaterale lieve . 
5a feto a 27 settimane + 2 giorni affetto da oloprosencefalia alobare con ipotelorismo ( b , riduzione diametro bisorbitario ) , nel quale lo studio rm ha smentito il rilievo ecogra co di labioschisi e dimostrato lintegrit del labbro superiore ( c , d , freccia bianca )  . 
between the 4th and the 10th week of gestation , ve main buds of tissue fuse together : a single median frontonasal process and two paired buds , the maxillary and the mandibular prominences . 
at around the 7th week , the central portion of the nose and the lateral nasal prominences develop into the nostrils , whereas at the 8th week , the structures responsible for the formation of the eyes have developed . 
it is the result of the failure of one or both of the medial nasal prominences to fuse with the maxillary prominences , generally between the 4th and 7th week discussione lo splancnocranio deriva quasi completamente dal mesenchima dei primi tre archi branchiali , costituiti da mesoderma ( blocchi di mesoderma accoppiati e verticali ) , che si sviluppano latero - ventralmente nella regione della testa e del collo , adiacenti allo stomodeo e allorofaringe primitivo . 
tra la 4a e la 10a settimana di gestazione si fondono 5 protuberanze di cui una impari , il processo fronto - nasale , e due pari , le protuberanze mascellari e mandibolari . 
alla 5a settimana si ultima la formazione del processo nasale mediano e laterale , intorno alla 7a settimana la parte centrale del naso e dai processi laterali si sviluppano le narici e all8a settimana si sviluppano le strutture deputate alla formazione degli occhi . 
la schisi di qualsiasi elemento del palato primario , con e senza interessamento del palato secondario , viene de nita come labioschisi o labiopalatoradiol med ( 2011 ) 116 : 11341148 1143 fig . 
cleft secondary palate ( cp ) is the result of a developmental abnormality or failure to fuse of the lateral palatine prominences between the 8th and 12th weeks of gestation [ 1 , 21 ]  . in contrast to cp , which often occurs in isolation , cl alone is a rare event . 
unlike clp , cp ( incidence of 1 / 2000 live births ) shows no remarkable variation among races , whereas the female - to - male ratio is 2 : 1 . 
it has also been noted that males tend to be more frequently affected by the more severe defects and complete forms and are more likely to have bilateral rather than unilateral forms . when analysing factors involved in the aetiology of clp , schisi ed avviene per mancata fusione di uno o pi processi nasali mediani con il processo mascellare generalmente tra la 4a e la 7a settimana di gestazione . 
le schisi del palato secondario avvengono invece per un alterato sviluppo o la mancata fusione dei processi palatini laterali tra l8a e la 12a settimana di gestazione [ 1 , 21 ]  . al contrario delle palatoschisi , che spesso si riscontrano in maniera isolata , le labioschisi isolate sono rare ; infatti circa il 70% di schisi labiali presenta anche schisi palatali . 
rispetto alle labiopalatoschisi , nelle palatoschisi ( incidenza di 1 / 2000 nati vivi ) non vi una contaminazione razziale ed i soggetti nati malformati sono pi femmine che maschi per un rapporto di 2 : 1 . 
per quanto riguarda le lps , quindi , vi una differenza di incidenza delle malformazioni a seconda delle razze ; in particolare lincidenza risulta pi alta nei bambini giapponesi ed ancora maggiore tra gli indigeni americani rispetto alla razza caucasica . 
stato notato anche che tra i maschi sono pi frequenti i difetti pi gravi e le forme complete e 1144 radiol med ( 2011 ) 116 : 11341148 the concept of aetiological heterogeneity is paramount , i.e. 
these factors include : multifactorial inheritance : this is the most important , most common but also the least known type of inheritance pattern underlying many disorders that have a genetic predisposition . inheritance factors : only appropriate genetic counselling is able to establish the in uence exerted by inheritance factors and their role in terms of the risk of recurrence . teratogenic factors : medicinal products and infections are the main factors responsible for congenital defects caused by the environment . 
 lastly , anticonvulsants and alcohol during pregnancy increase the risk of clp in the setting of syndromic conditions ; clefts can be associated with chromosomal defects or syndromes of unknown aetiology : chromosomal defects include down syndrome and trisomy 13 and 18 ; syndromes of unknown aetiology include the pierre robin sequence characterised by micrognathia , glossoptosis and soft - palate cleft . craniomaxillofacial abnormalities can have direct consequences on the survival of the newborn resulting from compromised swallowing and impaired respiratory function . 
at the same time , the fetal brain should be evaluated to identify the presence or otherwise of a syndromic condition . us is the rst - choice examination in the prenatal diagnosis of cl thanks to its low cost and ease of use . 
 additional 3d and 4d us reconstruction techniques , tra queste quelle bilaterali rispetto a quelle monolaterali . nellanalisi dei fattori implicati nelleziologia delle labiopalatoschisi pi che mai da tenere in considerazione il concetto delleterogenicit eziologica , ovvero dellesistenza di molteplici cause per un unico fenomeno . 
i fattori in causa possono essere : lereditariet multifattoriale , che rappresenta il pi importante , il pi comune , ma anche il meno conosciuto tipo di pattern ereditario alla base di molti disordini che riconoscono una predisposzione genetica ; i fattori ereditari : solo una consulenza genetica adeguata in grado di stabilire leffettiva entit dei fattori ereditari e quanto questi incidano in termini di rischio di ricorrenza ; fattori teratogeni : i farmaci e le infezioni sono i principali responsabili di difetti congeniti causati dallambiente . 
 secondo i dati della letteratura le benzodiazepine e gli steroidi possono determinare un modesto aumento del rischio di schisi labiopalatina senza altre anomalie associate ; il diabete materno e lassunzione di anfetamine aumentano il rischio in associazione ad altre anomalie ( ad esempio , cardiopatie congenite ) ; in ne gli antiepilettici e lalcool in gravidanza aumentano il rischio d labiopalatoschisi nellambito di altre sindromi ; in ne le schisi possono anche essere associate a difetti cromosomici , come la sindrome di down , la trisomia 13 e 18 , oppure a sindromi ad eziologia sconosciuta come la sequenza di pierre robin caratterizzata da micrognazia , glossoptosi e schisi del palato molle . 
ai ni della correzione chirurgica risulta necessario un corretto inquadramento della patologia : la monolateralit o la bilateralit , il grado di coinvolgimento del palato duro e le eventuali alterazioni biometriche del massiccio facciale , ottenute mediante la valutazione dei principali parametri . 
risulta inoltre necessaria la contemporanea valutazione dellencefalo per de nire la presenza o meno di una condizione sindromica . lecogra a rappresenta lindagine di prima scelta nella diagnosi prenatale delle labioschisi grazie ai suoi bassi costi e alla facilit di esecuzione . 
tuttavia la sua accuratezza pu essere limitata da condizioni patologiche fetali , quali anidramnios , oligoidramnios , sindromi malformative complesse e condizioni di pertinenza materna , come lobesit , oltre ad essere una metodica operatore - dipendente [ 22 , 23 ]  . 
lesame ecogra co , inoltre , non consente di de nire lesatto coinvolgimento del palato duro in relazione al radiol med ( 2011 ) 116 : 11341148 1145 such as surface rendering and transparency mode , provide improved visualisation of the fetal face and the presence of clefts , particularly in the presence of hard - palate involvement [ 9 ]  . 
 in t2 - weighted sequences , the signal intensity of the amniotic uid swallowed by the fetus provides excellent visualisation of the oropharynx , thus showing involvement of the secondary palate in cases where the volume of the oropharyngeal cavity is reduced by the encumbrance of the tongue . 
 moreover , mri is able to identify the direct communication between the buccal and the nasal cavities [ 21 ]  . in characterising cases of cl - clp , the diagnostic accuracy of mri proved to be higher in eight cases , particularly with regard to de ning the two essential parameters for surgical planning : the laterality and anteroposterior extent of the cleft , which determines the degree of palatal involvement . 
these data are directly correlated with the severity of complications and surgical dif culty in the intraoperative and postoperative period [ 24 ] we also obtained useful information regarding measurement of the fetal face in 6 / 24 fetuses . 
in the cases of associated micoragnathia , multiplanar mri with high anatomical detail played a key role in determining whether intervening immediately in the rst days of life or planning a series of interventions to be performed over time was more appropriate . 
measurements of the fetal face , therefore , may be considered a valid support in the diagnosis of conditions associated with cl - clp , and abnormal measurements should raise the suspicion of a syndromic fetus . results obtained from our population show a diagnostic accuracy of mri equal to 100% in the evaluation of both cl and clp . 
the technique proved able to correctly distinguish unilateral and midline from bilateral defects , as well as involvement of the anterior palate and alveolar processes from involvement of the secondary palate [ 2527 ]  . the main limitation to our study is the small size of the population . 
ulteriori tecniche di ricostruzione ecogra che 3d e 4d , quali il surface rendering ed il trasparency mode , permettono una migliore visualizzazione del viso fetale e di eventuali schisi , soprattutto in presenza di coinvolgimento del piano osseo [ 9 ]  . tuttavia , spesso si rende necessario il ricorso a metodiche di imaging di iii livello : la rm consente la visualizzazione diretta della schisi e la de nizione dellinteressamento del palato primitivo e / o secondario . 
liperintensit di segnale del liquido amniotico ingerito dal feto , nelle sequenze t2 pesate , permette unottima visualizzazione dellorofaringe , de nendo un interessamento del palato secondario nei casi in cui la cavit orofaringea presenta volume ridotto per ingombro della lingua ; inoltre , la rm permette lindividuazione della comunicazione diretta tra la cavit buccale e le cavit nasali [ 21 ]  . nella caratterizzazione dei casi di lbs - lps la rm ha dimostrato una maggiore accuratezza diagnostica in 8 casi , in particolare nella de nizione di due parametri essenziali nella piani cazione chirurgica : la lateralit ed il grado di estensione in senso antero - posteriore della schisi determinando in tal senso il grado di coinvolgimento palatale ; tali dati sono direttamente proporzionali al grado di complicanze e dif colt chirurgiche nellimmediato e nel postoperatorio [ 24 ]  . 
nei casi di associata micrognatia la rm , mediante immagini multiplanari ad alto dettaglio anatomico , permette agli operatori di decidere quando sia preferibile un trattamento immediato nei primi giorni di vita , o quando invece sia pi indicata la piani cazione di una serie di interventi da effettuare nel tempo . 
nei tre feti sindromici , invece , abbiamo riscontrato , unalterazione del dio e dellfma ; la diagnosi prenatale o autoptica hanno confermato la sindromicit di tali feti ; in particolare stato diagnosticato un feto affetto da trisomia 13 , deceduto alla nascita , uno affetto da trisomia 18 ed uno da trisomia 21 ( tabella 2 )  . 
la biometria del massiccio facciale , quindi , pu essere considerata un valido supporto nella diagnosi di patologie associate alla lbs - lps ed una sua alterazione deve porre il sospetto diagnostico di uneventuale condizione sindromica . dai risultati ottenuti dalla nostra casistica emerge come laccuratezza diagnostica dellesame rm risultata essere del 100% sia nella valutazione dei difetti labiali che nella de nizione dellinteressamento palatale , discriminando correttamente i difetti unilaterali e mediani da quelli bilaterali , cos come il coinvolgimento del palato anteriore e dei processi alveolari da quello del palato posteriore o secondario [ 2527 ]  . 
 il limite principale del nostro studio rappresentato dalla casistica ancora limitata ; per quanto concerne la metodica , i limiti sono legati alla dif colt di visualizzazione delle componenti scheletriche e al movimento fetale . 
per lo studio 1146 radiol med ( 2011 ) 116 : 11341148 table 2 magnetic resonance imaging ( mri ) and postnatal diagnosis in syndromic fetuses fetuses , n mri characteristics unilateral clp associated with an interventricular cardiac defect and borderline ventriculomegaly unilateral cl with involvement of the anterior palate associated with a lateral shift of the nasal septum and mild bilateral ventriculomegaly unilateral clp associated with alobar holoprosencephaly , cyclopia and arachnoid cyst of the posterior cranial fossa trisomy 13 ( postmortem diagnosis ) postnatal diagnosis trisomy 18 trisomy 21 cl , cleft lip ; clp , cleft lip and palate tabella 2 rm e diagnosi post - natale in feti sindromici feti , n caratteristiche rm diagnosi post - natale lps unilaterale associata a difetto interventricolare cardiaco e ventricolomegalia bordeline labioschisi unilaterale con coinvolgimento del palato anteriore associata a latero deviazione del setto nasale e ventricolomegalia bilaterale lieve lps unilaterale con oloprosencefalia alobare , ciclopia e cisti aracnoidea della fossa cranica posteriore trisomia 18 trisomia 21 trisomia 13 ( esame autoptico ) rm , risonanza magnetica ; lps , labio - palatoschisi tion . 
the sequences that proved to be diagnostic in terms of facial measurements and identi cation of the malformation were the t2 - weighted haste and the true - fisp sequences . 
both these sequences enabled us to reach the correct diagnosis , although the greater contrast resolution of the t2 - weighted haste sequences better visualised the defects of the palate and soft tissues . 
on the other hand , the true - fisp sequences with their intrinsic characteristics proved particularly useful in the study of particularly mobile fetuses . the images with the highest diagnostic quality , which therefore provided a better and simpler characterisation of the abnormalities found , were acquired on fetuses with advanced gestational age ( after the 27th week of gestation )  . 
 this is largely due to the greater anatomical detail obtained in larger structures and the reduced fetal motion . in conclusion , thanks to an excellent depiction of fetal face anatomy , high - contrast resolution , multiplanar capabilities and large eld of view , mri offers a complete evaluation of abnormalities of the fetal face and bra these advantages therefore enable better treatment planning in the setting of multidisciplinary patient management [ 2830 ]  . del distretto scheletrico , ultimamente , sono state proposte le sequenze epi , le quali per nel nostro lavoro non hanno aggiunto ulteriori informazioni . 
entrambe le sequenze ci hanno permesso di porre diagnosi anche se la maggiore risoluzione di contrasto insita nelle sequenze haste t2 pesate meglio documenta la risoluzione di continuit del palato e delle parti molli . 
daltro canto le sequenze true - fisp sono state particolarmente utili , per le loro caratteristiche intrinseche , nei feti con spiccata motilit . le immagini di maggiore qualit diagnostica , che hanno pertanto permesso una migliore e pi facile caratterizzazione delle alterazioni riscontrate , sono state acquisite su feti con et gestazionale avanzata ( dopo le 27 settimane di gestazione ) ; tale dato da mettere in relazione al miglior dettaglio anatomico derivante da strutture di maggiori dimensioni e alla ridotta mobilit del feto . 
 in conclusione , grazie allottima visualizzazione dellanatomia del massiccio facciale fetale , allelevata risoluzione di contrasto , alla multiplanariet della metodica e allampio campo di vista , la rm consente un completo inquadramento della patologia del massiccio facciale e del distretto encefalico , consentendo una piani cazione del management terapeutico nellottica di una gestione multidisciplinare del paziente [ 2830 ]  . conclusions the mr study of the fetal face is indicated as a level - three study to be performed after level - two us has identi ed an lo studio rm fetale del massiccio facciale trova un ruolo conclusioni radiol med ( 2011 ) 116 : 11341148 1147 abnormality of the fetal lip or palate . 
the aim of this study was to better de ne the laterality and anteroposterior extent of the cleft and thus provide an accurate diagnosis at an early gestational age [ 10 ]  . in addition , mri is able to analyse the entire fetal head , measure the biometric development of the fetal face and identify dif cult - to - characterise associated pathological conditions ( especially involving the cns ) at an early stage , thus prompting suspicion of syndromic conditions . come esame di iii livello dopo un esame ecogra co di ii livello che abbia identi cato il difetto labiale e / o palatale , al ne di meglio de nire il grado di coinvolgimento del palato posteriore ed il grado di lateralit , consentendo una diagnosi accurata gi in et gestazionale precoce [ 10 ]  . inoltre , la rm consente di analizzare in toto il distretto cranio - encefalico , di effettuare una valutazione dello sviluppo biometrico del massiccio facciale e di identi care precocemente eventuali patologie associate di dif cile caratterizzazione ( soprattutto a carico del snc ) , permettendo di sospettare condizioni sindromiche . 
in 19 patients ( 2.6 % ) ultrasound ( us ) showed enlarged lymph nodes in the upper abdomen , and abdominal computed tomography ( ct ) was performed results . 
detecting enlarged lymph nodes in the upper abdomen after liver transplantation is and infrequent occurrence ; however , thorough imaging is required to detect and characterise a wide variety of disorders . 
gli esami istologici hanno rivelato un disturbo linfoproliferativo post - trapianto ( ptld ) in 6 pazienti ( 31 , 5% ) , una neoplasia epatica in 6 pazienti ( 31 , 5% ) , una tubercolosi sistemica disseminata in un caso ( 5 , 2% ) ed una sarcoidosi in un altro caso ( 5 , 2% )  . 
nella maggior parte dei casi le linfoadenomegalie si riveleranno correlate ad un disordine linfoproliferativo post - trapianto o ad una recidiva di malattia . keywords liver hepatic transplantation ct lymph nodes parole chiave fegato trapianto di fegato tc linfonodi 1068 introduction orthotopic liver transplantation ( olt ) is a well - established treatment for a variety of irreversible acute and chronic endstage liver diseases , for which no other satisfactory therapy is available . 
 in cases of inconclusive results , con rmation of anomalies depicted at us or clinical suspicion of a complication despite normal us , helical computed tomography ( ct ) should be performed . 
we review our experience in a single centre on pathologic conditions associated with enlarged lymph nodes after liver transplantation and imaging features that may allow narrowing the differential diagnosis . materials and methods patients between january 1997 and september 2008 , 715 olts were performed at our institution in 585 patients , 488 men and 227 women , between 15 and 73 ( mean 54 ) years of age . 
the main causes for liver transplantation were alcoholic cirrhosis ( n = 149 ) , posthepatitis c cirrhosis ( n = 130 ) , hepatocellular carcinoma ( n = 219 ) , retransplantation ( n = 77 ) , familial amyloidal polyneuropathy ( n = 26 ) , posthepatitis b cirrhosis ( n = 23 ) , primary biliary cirrhosis ( n = 17 ) and others . follow - up after liver transplantation at our institution is basically done with us on the rst and seventh days after the procedure , 1 month after liver transplantation and then every 6 months , and also in the event of clinical symptoms or impaired hepatic biology . 
additionally , an abdominal ct is performed when suspicious ndings are found on us examination . inclusion criterion was the detection of an abnormality on us suggesting lymphadenopathy and re ected in the us report of the hepatic transplantation database . 
in 19 patients ( 2.6% ) , us showed enlarged lymph nodes in the hepatic hilum , celiac region or lesser omentum , in which cases abdominal ct was performed . 
there were 15 men and four women , with ages ranging from 42 to 66 ( mean 55 ) years . patients with a nal diagnosis of posttransplantation lymphoproliferative disorder ( ptld ) , tumoral disease , tuberculosis or sarcoidosis had clinical and / or biological abnormalities ( 76% )  . 
indications for liver transplantation in this group included hepatic cirrhosis of different origin in eight patients ( origin was related to hepatitis c virus in ve patients and to alcohol in three ) , hepatic cirrhosis and hepatocellular carcinoma ( hcc ) in seven patients , cholangiocarcinoma in two patients , liver metastasis of a gastric carcinoid tumour in one patient and familial amyloidal polyneuropathy ( fap ) in one patient . 
in patients in whom hcc was the indication for liver transplantation , the origin of hepatic cirrhosis was related to hepatitis c virus in three , alcohol in three and hepatitis b virus in one . ct technique abdominal ct examinations were performed with two different helical ct units ( nxi , yocogawa , japan , and ct brightspeed , ge healthcare milwaukee , wi , usa ) with 250400 mas and 120 kvp . 
 the incidence of ptld in our series was 0.83% ( 6 / 715 ) , radiol med ( 2011 ) 116 : 10671075 1069 accounting for 31.5% ( 6 / 19 ) of patients with olt and enlarged lymph nodes . 
epstein - barr virus ( ebv ) was positive in three histological specimens ( in two patients with b cell lymphoma and in the patient with plasmacytic hyperplasia )  . 
 axial computed tomography images in arterial ( a ) , portal ( b ) and equilibrium ( c ) phases show a hypovascular , ill - de ned mass encasing hilum structures ( note intrahepatic bile duct dilatation )  . 
le immagini tc assiali in fase arteriosa ( a ) , portale ( b ) e di equilibrio ( c ) mostrano una massa ipovascolarizzata , mal de nita ed in ltrante le strutture ilari ( da notare la dilatazione delle vie biliari intra - epatiche )  . 
il campione istologico risultato positivo per ebv . tumoral disease sarcoidosis four patients with tumoral hepatic disease ( 31.5% ) developed recurrence , which was the indication for liver transplantation ( two hcc , one cholangiocarcinoma and one hepatic metastases of a gastric neuroendocrine tumour )  . 
two patients developed de novo malignancies corresponding to a disseminated squamous - cell carcinoma of the oesophagus and a disseminated mucinous adenocarcinoma of the rectutime from liver transplantation to diagnoses ranged from 3 to 41 months . 
in this group , pathologic lymph nodes were located in more than one region , basically gastrohepatic ligament , celiac region and hepatic hila , but also in the retroperitoneuin all cases except recurrent cholangiocarcinoma , associated hepatic lesions were detected . 
axial computed tomography images in arterial ( a ) and portal ( b ) phases show hypervascular hepatic lesions and hypervascular enlarged lymph nodes located at the porta hepatis and celiac trunk due to massive recurrence of hepatocellular carcinoma . 
le immagini tc assiali in fase arteriosa ( a ) e portale ( b ) mostrano lesioni epatiche ipervascolarizzate associate a linfoadenomegalie ipervascolarizzate localizzate nella porta hepatis e lungo il tronco celiaco dovute ad una massiva recidiva di hcc . 
nine months after the procedure and during re - evaluation for new liver transplantation due to ischaemic cholangitis , a markedly hypodense retroperitoneal enlarged lymph node of 2.5 cm with thin peripheral enhancement and associated bilateral pulmonary pseudonodular lesions were discovered . 
rapid evolution to sepsis caused death 11 months after liver transplantation , and cultures of different specimens at necropsy ( lungs , retroperitoneal lymphadenopathy and spleen ) demonstrated caseating granulomas due to disseminated systemic tuberculosis . 
no signs of hcc progression were demonstrated at necropsy . hyperplasic lymph nodes in a group of ve patients ( 26% ) , cytological study of enlarged lymph nodes located at porta hepatis revealed changes of nonspeci c reactive lymphadenitis without malignant cells . 
mean time of diagnosis ranged from 8 to 12 months in patients with previous medical history of hepatitis c cirrhosis and 36 months in patient undergoing liver transplantation due to familial amyloidal polyneuropathy . 
 in this group , ct images showed isolated , well - de ned , enlarged lymph nodes in porta hepatis ranging from 2 to 3.3 cthey were all homogeneous , but in two cases , they were isodense to the hepatic parenchyma during the portal phase ; in two cases , they were slightly hypodense during the portal phase ; and in one case , the lymph node was hypervascular during the arterial phase . 
3a , b follow - up ultrasound in an asymptomatic 54 year - old woman 2 years after liver transplantation due to hepatitis - c - induced cirrhosis showed enlarged lymph nodes . 
axial ct image in portal phase ( a ) show an ill - de ned hypovascular mass in porta hepatis and celiac trunk without encasement of vascular or biliary structures . 
3a , b follow - up con us di una donna di 54 anni , asintomatica , 2 anni dopo trapianto di fegato per cirrosi epatica c - correlata con comparsa di linfoadenomegalie . 
limmagine tc assiale in fase portale ( a ) mostra una massa ipovascolarizzata , mal de nita , nella porta hepatis ed in adiacenza del tronco celiaco , senza in ltrazione delle strutture vascolari o biliari . 
lo studio del materiale ottenuto con biopsia chirurgica ha evidenziato la presenza di granulomi non - caseosi . lesions were detected , and only in one case were coexisting signs of chronic liver disease detected . 
 ptld is a well - known complication after solid - organ transplantation , with a reported incidence of 210% [ 2 ] , ranging from 23% in adults and > 10% in the paediatric population . 
ebv and immunosuppressive therapy are considered the two major risks factors due to the capability of the virus to stimulate b lymphocyte proliferation without the inhibitory effect of t lymphocytes , which are pharmacologically suppressed [ 3 ]  . 
 these entities are divided into four main histological types according to the latest world health organisation ( who ) reactive plasmacytic hyperplasia classi cation : early and infectious - mononucleosis - like disease , polymorphic ptld , monomorphic ptld and hodgkins and hodgkinlike lymphomas . 
radiologically , ptld can virtually affect any organ , leading to a wide spectrum of ndings , with lymph nodes , liver and gastrointestinal tract being the most frequent sites when considering abdominal disease . 
 [ 6 ] , different patterns of involvement are considered : an in ltrative pattern with heterogeneous ill - de ned low attenuation regions with or without associated hepatomegaly , a tumoral pattern with diffuse hypodense nodular lesions and a porta hepatis mass with frequent encasement of vascular and biliary structures . radiol med ( 2011 ) 116 : 10671075 1073 fig . 
4a ultrasound ( us ) at the level of the porta hepatis in a 39 - year - old patient in whom liver transplantation was performed due to a familial amyloidal polyneuropathy shows isoechogenic lymph node . 
axial ct scan ( b ) at the same level shows well - de ned , hypodense , enlarged lymph nodes close to the main portal veus - guided ne - needle aspiration biopsy showed nonspeci c reactive lymphadenopathy . 
c il follow - up tc 2 anni pi tardi dimostra una stabilit di malattia . in our study , we found no allograft disease , as frequently as reported by pickhardt et al . 
 [ 6 ] the discrepancy may be due to the patterns of allograft disease considered by different authors ; whereas hilar mass is considered as a form of presentation of graft disease by pickhardt et al . 
this histological result probably represents a very initial stage of the disease , considered a nonneoplastic reactive response to ebv infection , which may often disappear spontaneously or with reduced immunosuppression . 
 hepatic tumoral involvement after liver transplantation can be secondary to recurrence of the tumour considered for the indication to the procedure , or less frequently to development of de novo neoplasia ( metastasis of a previously unknown primary tumour )  . 
due to high rates of recurrence , cholangiocarcinoma is no longer considered an indication for liver transplantation by most groups , although some selected cases have been transplanted in our institution . 
 tumoral recurrence of hcc may affect any organ , but most common sites of recurrence in order of frequency are lungs , liver graft and regional lymph nodes , followed by adrenal glands and bone . 
in our series , tumoral recur1074 radiol med ( 2011 ) 116 : 10671075 rence of hcc presented as lung metastasis with multifocal liver involvement and enlarged locoregional lymph nodes . 
 in one patient who was diagnosed 13 months after the procedure , hepatic lesions and lymphadenopathy showed a hyperenhancing pattern , whereas the patient detected 3 months after the procedure presented with hypovascular lesions related to a recurrent poorly differentiated hcc . 
 despite advances in surgical techniques and immunosuppressive treatments leading to a survival increase , patients undergoing organ transplantation are at risk of developing a wide variety of infections , including tuberculosis . 
on the other hand , although mechanisms of transmission may vary among patients , reactivation of a pre - existing latent infection seems to be more frequent than primo - infection or liver - graft transmission radiologically , pulmonary disease is the most frequent pattern seen in transplant recipients , followed by disseminated forms and extrapulmonary disease . 
whereas affected lymph nodes in tuberculosis tend to show a hypodense centre due to necrosis , with peripheral enhancement , pathological lymph nodes in nontreated lymphoma most commonly show homogeneous enhancement after contrast administration [ 12 ]  . 
 some of the proposed causes include in ammatory mediators , in particular , inf [ 12 , 13 ] , a drug widely used as treatment for some neoplasm and for viral infections , especially hepatitis c . 
unfortunately , the patient died approximately 1 year late due to complications of acute hepatitis c recurrence . it is not infrequent to identify enlarged lymph nodes at the hepatic hilum in patients with hepatitis - c - induced cirrhosis , which is the most frequent indication for liver transplantation in europe and the usa . 
these changes are similar to those seen previous to liver transplantation , such as nodularity of liver surface due to regenerating nodules , brous scarring with nonuniform segmental atrophy or hypertrophy of the liver , extrahepatic changes due to portal hypertension and unspeci c lymph nodes at the porta hepatis , more frequently seen in cases of hepatitis c viral infection . 
follow - up usually shows stability or even regression of these nonspeci cally enlarged lymph nodes . in conclusion , detecting enlarged lymph nodes in the upper abdomen after liver transplantation is infrequent , and thorough imaging is required to detect and characterise a wide variety of disorders . 
if liver transplantation is performed due to malignant disease , such as hcc or metastases of neuroendocrine tumours , detection of enlarged lymph nodes associated with liver lesions suggests tumoral recurrence . 
midiri dipartimento di biotecnologie mediche e medicina legale , sezione di diagnostica per immagini , policlinico universitario , via del vespro 127 , 90127 palermo , italy correspondence to : t . 
eighty - four focal breast lesions , 64 benign and 20 malignant ( mean diameter , 15.1 mm ) , detected but not characterised on b - mode us in 72 women , breast imaging reporting and data system ( bi - rads ) us category 3 ( n = 56 ) or category 4 ( n = 28 ) , were studied with us elastography and classi ed in consensus by two radiologists according to a ve - point colour scale . 
a total of 65 / 84 ( 77.4% ) lesions were correctly classi ed as benign or malignant using us elastography , whereas the remaining 19 / 84 ( 22.6% ) were incorrectly assessed . 
the high npv of us elastography may help reduce the use of biopsy in bi - rads 3 lesions , but its low ppv in bi - rads 4 lesions does not allow avoidance of riassunto obiettivo . 
ottantaquattro lesioni focali mammarie , 64 benigne e 20 maligne ( diametro medio : 15 , 1 mm ) , rilevate ma non caratterizzate allecogra a di base in 72 donne punteggio ecogra co breast imaging reporting and data system ( bi - rads ) 3 ( n = 56 ) o bi - rads 4 ( n = 28 ) , sono state sottoposte ad elastosonogra a e classi cate in consenso da due radiologi secondo una scala colorimetrica in cinque punti . 
sessantacinque / 84 ( 77 , 4% ) lesioni sono state correttamente classi cate come benigne o maligne allelastosonogra a , mentre le rimanenti 19 / 84 ( 22 , 6% ) sono state erroneamente valutate . 
lelevato vpn dellelastosonogra a pu essere utile per evitare lestensivo ricorso alla biopsia nelle lesioni mammarie in classe bi - rads 3 , ma il basso 1028 radiol med ( 2011 ) 116 : 10271038 biopsy on the basis of the us elastographic score alone in this group of lesions . vpp in quelle bi - rads 4 non consente di escludere il ricorso alla biopsia sulla scorta del solo punteggio elastosonogra co . keywords breast ultrasound elastography breast neoplasms parole chiave mammella elastosonogra a neoplasie mammarie introduction introduzione the role of grey - scale ultrasound ( us ) has considerably expanded in characterising focal breast lesions . 
 in particular , the development of a categorisation system based on sonographic semiological criteria , such as the breast imaging reporting and data system ( bi - rads ) , has proved useful for describing and classifying both benign and malignant breast lesions [ 2 ]  . 
however , the limited reproducibility of the system in determining the exact likelihood of malignancy of suspicious breast lesions ( category 4 ) , with variability ranging from 3% to 94% , leaves the problem of recourse to more invasive and costly diagnostic procedures , such as biopsy , essentially unchanged [ 3 ]  . 
 furthermore , even though the risk of malignancy in lesions classi ed as bi - rads category 3 should be < 2% , the large number of biopsies performed on benign lesions continues to represent a real problem , especially in the light of the cost - effectiveness of screening programmes [ 2 ]  . the introduction of us elastography into clinical breast imaging in 1997 made it possible to evaluate a mechanical property completely different from echogenicity : that is , tissue elasticity [ 4 ]  . 
tissue elasticity , de ned mathematically by the elastic or young modulus , can be obtained from the pressure required to deform a given tissue or structure [ 5 ]  . 
the recent availability of us units equipped with an elastography module enabling real - time , free - hand imaging has prompted several authors to assess the contribution of us elastography to the characterisation of breast lesions , with encouraging results [ 610 ]  . 
the purpose of this study was to evaluate the usefulness of us elastography in characterising breast lesions classi ed as indeterminate at b - mode materials and methods patients lecogra a in scala di grigi , grazie ai continui progressi tecnologici , che ne hanno notevolmente migliorato sia la risoluzione spaziale sia quella di contrasto , ha notevolmente ampliato il suo ruolo nella caratterizzazione delle lesioni focali mammarie [ 1 ]  . 
in particolare , lelaborazione di un sistema di categorizzazione basato su criteri semeiologici ecogra ci , quale il breast imaging reporting and data system ( bi - rads ) , si dimostrato uno strumento utile ai ni di descrivere e sistematizzare le lesioni mammarie sia benigne che maligne [ 2 ]  . 
tuttavia , la limitata riproducibilit di tale sistema nel de nire lesatta probabilit di malignit di una lesione mammaria sospetta ( categoria 4 ) , con una variabilit che va dal 3% al 94% , lascia sostanzialmente inalterata la problematica relativa al ricorso , in tale ambito , a procedure diagnostiche pi invasive e costose , quali la biopsia [ 3 ]  . 
inoltre , sebbene il rischio di malignit delle lesioni classi cate in categoria bi - rads 3 dovrebbe essere inferiore al 2% , il gran numero di biopsie tuttoggi effettuate su lesioni benigne continua a costituire un reale problema , soprattutto nellottica costo / bene cio dei programmi di screening [ 2 ]  . lintroduzione in ambito clinico - senologico dellelastosonogra a , avvenuta nel 1997 , ha reso possibile valutare una propriet meccanica completamente differente dallecogenicit : lelasticit tissutale [ 4 ]  . 
del tutto recentemente , la disponibilit commerciale di unit ecogra che dotate di modulo elastosonogra co capace di consentire un esame in tempo reale ed a mano libera , ha consentito a diversi autori di cominciare a valutare lapporto dellelastosonogra a nella caratterizzazione delle lesioni mammarie con incoraggianti risultati [ 610 ]  . 
scopo del presente lavoro stato quello di valutare lutilit dellimpiego dellelastosonogra a nella caratterizzazione delle lesioni mammarie indeterminate allecogra a di base . between june 2008 and june 2009 , 72 women ( age range 2284 years ; mean age , 47.512.33 years ) with 84 focal breast lesions ( diameter , 444 mm ; mean , 15.1 mm ) were prospectively evaluated following approval by the ethics ottenuta lapprovazione da parte del comitato etico istituzionale , sono state prospetticamente valutate , nel periodo materiali e metodi pazienti radiol med ( 2011 ) 116 : 10271038 1029 committee . 
thirty - seven were also clinically palpable ( diameter , 844 mm ; mean , 20.9 mm ) , whereas 47 lesions ( diameter 415 mm ; mean , 8 mm ) were nonpalpable and were detected during routine examinations . 
in order to assess the impact of elastography on the diagnostic workup , all lesions with a clear cystic appearance and / or classi ed as bi - rads 2 ( and therefore not requiring biopsy ) were excluded from the analysis . 
the lesions classi ed as bi - rads 5 were also excluded , as they were almost certainly malignant and requiring appropriate treatment . examination technique all investigations were performed using logos hi vision gold us equipment ( esaote / hitachi medical corporation , tokyo , japan ) equipped with a multifrequency linear - array transducer ( 13 - 5 mhz ) and a dedicated us elastography module . 
before the us elastography examination itself , the breast parenchyma was always imaged with grey - scale and colour power doppler us to locate the lesion and select the most appropriate scan plane . 
in particular , the us elastography investigation was conducted in real time using a split - screen display with simultaneous viewing of the b - mode image on the right and the us elastogram superimposed on the same b - mode image on the le the equipment uses a special autocorrelation system ( the extended combined autocorrelation method ) that can be applied in real time to take into account not only changes along the axis of propagation of the us beam but also movements along the lateral axis , thereby reducing artefacts due to lateral displacement of the tissues [ 8 ]  . the signal from the structures being examined and relative to a speci c sampling box placed on the region of interest and extending also to healthy breast tissue is processed in real time and the resulting elastogram is immediately superimposed over the b - mode image and displayed on a colour scale from red ( soft or highly elastic tissue ) to blue ( hard or inelastic tissue )  . 
at the same time , a special indicator light provides real - time feedback on the quality of the examination by scoring it from 1 to 6 and displaying the score alongside the us elastogra scores of 3 or higher indicate a suf cient or good reliability of the elastogra compreso tra giugno 2008 e giugno 2009 , 72 donne di et compresa tra 22 e 84 anni ( et media : 47 , 512 , 33 anni ) con 84 lesioni mammarie focali ( diametro : 444 mm ; media : 15 , 1 mm ) , tutte ecogra camente rilevabili . 
trentasette lesioni erano anche clinicamente palpabili ( diametro : 844 mm ; media : 20 , 9 mm ) , mentre 47 lesioni ( diametro 415 mm ; media : 8 mm ) non erano palpabili e sono state individuate durante controlli di routine . 
tutte le pazienti , quindi , sono state sottoposte a indagine ecogra ca , condotta da due radiologi esperti in ecogra a senologica ( almeno dieci anni ) , i quali hanno classi cato ciascuna lesione , sulla base dei reperti b - mode , in accordo con lo schema bi - rads ecogra co [ 11 ]  . 
al ne di valutare limpatto dellelastosonogra a nel modi care liter diagnostico , sono state escluse dalla presente valutazione tutte le lesioni classi cate con aspetto chiaramente cistico e / o classi cate come bi - rads 2 e , pertanto , non necessitanti di campionamento bioptico . 
anche le lesioni classi cate come bi - rads 5 allindagine ecogra ca di base sono state escluse dal presente studio , in quanto quasi certamente maligne e gi di per s necessitanti di adeguato trattamento . tecnica desame tutte le indagini ecogra che sono state effettuate con apparecchiatura logos hivision gold ( esaote / hitachi medical corporation , tokio , giappone ) dotata di trasduttore lineare multifrequenza ( 135 mhz ) nonch di modulo elastosonogra co dedicato . 
prima dellesame elastosonogra co vero e proprio , stato sempre effettuato uno studio ecogra co del parenchima mammario , sia in scala di grigi che con modulo color e power doppler , al ne di individuare la lesione stessa e selezionare un adeguato piano di scansione . 
in particolare , lindagine elastosonogra ca stata condotta in tempo reale , mediante suddivisione dello schermo ecogra co in due parti e visualizzando contemporaneamente nella parte destra dello schermo la consueta immagine b - mode e , nella parte sinistra , lelastogramma superimposto alla stessa immagine b - mode . 
lapparecchiatura impiegata implementa un particolare sistema di autocorrelazione ( metodo di autocorrelazione estesa combinata ) che pu essere applicato in tempo reale permettendo di tenere conto non solo delle alterazioni lungo lasse di propagazione del fascio ultrasonoro , ma anche dei movimenti lungo lasse laterale , riducendo gli artefatti dovuti , appunto , alla dislocazione laterale dei tessuti [ 8 ]  . 
 1030 radiol med ( 2011 ) 116 : 10271038 in our study , us elastograms were considered acceptable only if they were found to be adequately and continuously superimposed over the b - mode image , with light indicator scores between 4 and 6 . 
two radiologists with at least 10 years experience in breast us and who were not involved in performing the examinations and were unaware of patients clinical history and nal diagnosis , consensually evaluated the images of ine . 
to avoid any recall bias , four image evaluation sessions were scheduled , each 1 week apart . the us elastograms were classi ed using a score ranging from 1 to 5 , according to the scheme proposed by the italian study group ( elasticity score ) based on the classi cation previously proposed by itoh et al . 
 [ 7 ] : score 1 , presence of a three - layered colour pattern of blue , green and red ; score 2 , diffuse green , possibly with a few small inconsistent blue spots ; score 3 , diffuse green with some blue spots ; score 4 , almost completely blue with maybe a few small green spots , especially peripherally ; score 5 , completely blue and with a blue rim surrounding the lesion . 
a score of 1 is considered predominant in liquid lesions ; scores 2 and 3 correspond to lesions that are predominantly elastic and therefore more frequently benign , whereas scores 4 and 5 indicate lesions that are predominantly hard and more frequently malignant [ 7 , 12 ]  . standards of reference the nal diagnosis was obtained through a microhistological biopsy in 67 cases and cytological examination in the remaining 17 . 
all samples were collected under us guidance using 21to 23 - gauge needles for cytological sampling and 14to 18 - gauge needles for microhistological sampling . statistical analysis in order to evaluate the usefulness of us elastography in characterising suspicious breast lesions and therefore to verify its sensitivity , speci city and positive and negative predictive values ( ppv and npv ) , we calculated the true positive ( tp ) , true negative ( tn ) , false positive ( fp ) and false negative ( fn ) results . 
in agreement with the literature , lesions were considered positive ( p ) for malignancy if they had us elastography scores of 4 or 5 and negative ( n ) if they had scores of 1 , 2 or 3 . il segnale proveniente dalle strutture esaminate e relativo ad un apposito box di campionamento posto dalloperatore sulla regione di interesse comprendente anche il tessuto mammario sano viene elaborato in tempo reale e , immediatamente , lelastogramma ottenuto viene sovrapposto allimmagine in b - mode e visualizzato secondo una scala cromatica che va dal rosso ( sof ce ) al blu ( anelastico )  . 
al contempo , un apposito indicatore luminoso indica , in tempo reale , la corretta conduzione dellesame attraverso una scala di valori da 1 a 6 , che viene visualizzata lateralmente allimmagine elastosonogra ca . 
questultima , nel nostro studio , stata considerata accettabile solo nel caso in cui fosse risultata sovrapposta in maniera congrua e continua allimmagine b - mode , con valori assegnato dallapposito indicatore luminoso compresi tra 4 e 6 ; sono state considerate , invece , inesatte le informazioni provenienti da elastogrammi con una sovraimposizione fugace e con valori di scala inferiori a 4 . 
la durata di ogni singola valutazione elastosonogra ca stata , in media , pari a circa 3 minuti ( intervallo : 25 minuti )  . analisi delle immagini tutti gli esami sono stati archiviati come dati grezzi in forma digitale . 
due radiologi esperti in ecogra a senologica ( almeno dieci anni ) , non coinvolti nellesecuzione degli esami e non a conoscenza della storia clinica dei pazienti n della diagnosi nale , hanno valutato in consenso off - line le immagini ottenute . 
per evitare il recall bias sono state previste quattro sessioni di valutazione distanziate da una settimana . le immagini elastogra che sono state classi cate con un punteggio variabile da 1 a 5 , secondo lo schema proposto dal gruppo di studio italiano ( elasticity score ) sulla base della classi cazione precedentemente proposta da itoh et al . 
 [ 7 ] : ( 1 ) presenza di tri - strati cazione cromatica : blu / verde / rossa ; ( 2 ) prevalenza di verde , con eventualmente qualche incostante punto blu ; ( 3 ) prevalenza di verde , con qualche punto blu ; ( 4 ) quasi completamente blu , con eventualmente qualche punto verde , soprattutto perifericamente ; ( 5 ) completamente blu , anche con alone blu perilesionale . 
il punteggio 1 considerato predominante nelle lesioni liquide , i punteggi 2 e 3 corrispondono a lesioni in prevalenza elastiche e , dunque , pi frequentemente benigne , mentre i punteggi 4 e 5 corrispondono a lesioni prevalentemente rigide e pi frequentemente maligne [ 7 , 12 ]  . 
tutti i prelievi sono stati eseguiti sotto guida ecogra a radiol med ( 2011 ) 116 : 10271038 results mediante aghi da 2123 g per lesame citologico e con aghi da 1418 g per lesame microistologico . 1031 the cytohistological examinations of the 84 lesions studied provided a diagnosis of benignity in 64 cases ( 76.2% ) and malignancy in the remaining 20 cases ( 23.8% ) ( table 1 )  . 
 analisi statistica table 1 final diagnosis and ultrasound ( us ) elastography score of the 84 breast lesions examined us elastography score 5 total benign lesions fibroadenoma corpuscular cyst angioma cicatricial brosis adenosis granuloma fat necrosis pseudoangiomatous hyperplasia cold abscess malignant lesions invasive ductal carcinoma ductal carcinoma in situ abscessed ductal carcinoma tubular carcinoma lobular carcinoma mucinous carcinoma sarcoma total 21 2 37 8 tabella 1 diagnosi nale e punteggio elastosonogra co delle 84 lesioni mammarie prese in esame punteggio elastosonogra co 5 totale lesioni benigne fibroadenoma cisti corpuscolata angioma fibrosi cicatriziale adenosi granuloma steatonecrosi iperplasia pseudoangiomatosa ascesso freddo lesioni maligne carcinoma duttale in ltrante carcinoma duttale in situ carcinoma duttale ascessualizzato carcinoma tubulare carcinoma lobulare carcinoma mucinoso sarcoma totale 21 2 37 8 allo scopo di valutare lutilit dellimpiego dellelastosonogra a nella caratterizzazione delle lesioni mammarie sospette e quindi di veri carne la sensibilit , la speci cit ed il valore predittivo positivo ( vpp ) e negativo ( vpn ) abbiamo calcolato : i veri positivi ( vp ) , i veri negativi ( vn ) , i falsi positivi ( fp ) ed i falsi negativi ( fn )  . 
abbiamo considerato , in accordo con i dati della letteratura , positive ( p ) per malignit le lesioni con punteggio elastosonogra co pari a 4 o 5 ; e negative ( n ) le lesioni con un punteggio elastosonogra co pari a 1 , 2 o 3 . risultati lesame cito - istologico delle 84 lesioni oggetto di studio del nostro lavoro ha fornito una diagnosi di benignit in 64 casi ( 76 , 2% ) e di malignit nei rimanenti 20 casi ( 23 , 8% ) ( tabella 1 )  . 
allindagine ecogra ca di base , 56 / 84 lesioni ( 66 , 7% ) sono state classi cate come appartenenti alla classe bi - rads 3 e le rimanenti 28 ( 33 , 3% ) a quella bi - rads 4 . 
complessivamente , 65 / 84 ( 77 , 4% ) lesioni sono state correttamente classi cate come benigne o maligne allelastosonogra a , mentre le rimanenti 19 / 84 ( 22 , 6% ) sono state erroneamente valutate . 
nella serie presa in esame ne derivano , per quanto attiene lelastosonogra a , tassi di sensibilit , speci cit , vpp e vpn pari rispettivamente a 70% , 79 , 6% , 51 , 8% e 89% ( tabella 4 )  . 
parallelamente , considerando come benigne le lesioni in classe ecogra ca bi - rads 3 e maligne quelle in classe bi - rads 4 , la valutazione della sola ecogra a convenzionale ha portato al riscontro di 14 falsi positivi e 6 falsi negativi , con valori di sensibilit , speci cit , vpp e vpn pari rispettivamente a 68 , 4% , 78 , 5% , 48 , 1% e 89 , 5% . 1032 radiol med ( 2011 ) 116 : 10271038 table 2 ultrasound ( us ) elastography scores for the 56 breast imaging reporting and data system 3 lesions us elastography score total cold abscess ( 1 ) corpuscular cysts ( 6 ) granuloma ( 1 ) mucinous carcinoma ( 1 ) fibroadenoma ( 2 ) fibroadenoma ( 19 ) fat necrosis ( 1 ) angioma ( 8 ) fibrosis ( 1 ) corpuscular cysts ( 2 ) fibroadenoma ( 2 ) tubular carcinoma ( 1 ) ductal carcinoma ( 1 ) idc ( 3 ) fat necrosis ( 2 ) fibroadenoma ( 4 ) corpuscular cyst ( 1 ) the number of lesions is shown in brackets idc , invasive ductal carcinoma punteggio elastosonogra co tabella 2 punteggio elastosonogra co delle 56 lesioni mammarie in classe birads 3 totale ascesso freddo ( 1 ) cisti corpuscolata ( 6 ) granuloma ( 1 ) carcinoma mucinoso ( 1 ) fibroadenoma ( 2 ) fibroadenoma ( 19 ) liponecrosi ( 1 ) angioma ( 8 ) fibrosi ( 1 ) cisti corpuscolata ( 2 ) fibroadenoma ( 2 ) carcinoma tubulare ( 1 ) carcinoma duttale ( 1 ) cdi ( 3 ) liponecrosi ( 2 ) fibroadenoma ( 4 ) cisti corpuscolata ( 1 ) tra parentesi indicato il numero di lesioni cdi , carcinoma duttale in ltrante on b - mode us , 56 / 84 lesions ( 66.7% ) were classi ed as bi - rads 3 and the remaining 28 ( 33.3% ) as bi - rads 4 . 
overall , 65 / 84 ( 77.4% ) lesions were correctly classi ed as benign or malignant at us elastography , whereas the remaining 19 / 84 ( 22.6% ) were incorrectly assessed . 
in particolare , sette lesioni benigne ( 2 granulomi su cicatrice , 2 broadenomi , 1 adenosi , 1 iperplasia pseudoangiomatosa , 1 cisti corpuscolata ) classi cate in classe bi - rads 4 sono state correttamente valutate come benigne alla valutazione elastosonogra ca , mentre 3 lesioni maligne ( tutti carcinomi duttali in ltranti ) , classi cate in classe bi - rads 3 , sono state correttamente valutate come maligne allindagine elastosonogra ca . 
di contro , sei lesioni correttamente valutate in classe bi - rads 3 ( 4 broadenomi e 2 aree di steatonecrosi ) sono state erroneamente valutate come maligne alla valutazione elastosonogra ca . 
in ne , tre lesioni maligne ( 1 carcinoma tubulare , 1 carcinoma duttale ascessualizzato e 1 sarcoma ) classi cate come bi - rads 4 sono state invece erroneamente considerate benigne alla valutazione elastosonogra ca . classe ecogra ca bi - rads 3 nella tabella 2 sono riportati i punteggi relativi alla valutazione elastosonogra ca delle 56 lesioni mammarie in classe radiol med ( 2011 ) 116 : 10271038 1033 table 3 ultrasound ( us ) elastography scores for the 28 breast imaging reporting and data system category 4 breast lesions us elastography score total ductal carcinoma ( 1 ) granuloma ( 2 ) sarcoma ( 1 ) fibroadenoma ( 2 ) adenosis ( 1 ) adenosis ( 1 ) tubular carcinoma ( 1 ) pseudoangiomatous hyperplasia ( 1 ) corpuscular cyst ( 1 ) adenosis ( 2 ) fibrosis ( 3 ) corpuscular cyst ( 1 ) idc ( 5 ) ductal carcinomain situ ( 1 ) lobular carcinoma ( 2 ) idc ( 3 ) the number of lesions is shown in brackets idc , invasive ductal carcinoma punteggio elastosonogra co tabella 3 punteggio elastosonogra co delle 28 lesioni mammarie in classe birads 4 carcinoma duttale ( 1 ) granuloma ( 2 ) adenosi ( 1 ) cdi ( 5 ) cdi ( 3 ) sarcoma ( 1 ) fibroadenoma ( 2 ) adenosi ( 1 ) carcinoma tubulare ( 1 ) iperplasia pseudoangiomatosa ( 1 ) cisti corpuscolata ( 1 ) carcinoma duttale in situ ( 1 ) carcinoma lobulare ( 2 ) adenosi ( 2 ) fibrosi ( 3 ) cisti corpuscolata ( 1 ) tra parentesi indicato il numero di lesioni cdi , carcinoma duttale in ltrante totale fig . 
b - mode ultrasound ( us ) ( right ) depicts a heterogeneous hypoechoic lesion with irregular margins and a diameter of 6 mm ( breast imaging reporting and data system us category 4 )  . 
allecogra a di base ( destra ) si apprezza una lesione ipoecogena a margini irregolari del diametro di 6 mm ( classe bi - rads ecogra ca 4 )  . 
a sinistra , la valutazione elastosonogra ca dimostra una marcata rigidit tissutale di tutta la lesione ( punteggio 5 )  . us elastography led to a different judgement from that obtained using b - mode us in 19 / 84 ( 22.6% ) cases , changing the initial b - mode us ndings from benign to malignant in ten cases and from malignant to benign in the remaining nine . 
nessuna di queste ha presentato un punteggio elastosonogra co pari a 5 , tuttavia 10 / 56 ( 17 , 9% ) di queste ultime hanno presentato un punteggio elastosonogra co pari a 4 , deponente quindi per malignit . 
tale giudizio stato confermato dallesame istologico in tre casi di carcinoma duttale in ltrante , ma sconfessato nei rimanenti sette casi ( 4 broadenomi , 2 aree di steatonecrosi , 1 cisti corpuscolata )  . 
 b - mode ultrasound ( us ) ( right ) shows a heterogeneous hypoechoic lesion with a diameter of 6 mm ( breast imaging reporting and data system us category 4 )  . 
 b - mode ultrasound ( us ) ( right ) depicts a homogeneously hypoechoic lesion without posterior acoustic enhancement showing regular margins , 5 mm in diameter ( breast imaging reporting and data system us category 3 )  . 
b - mode ultrasound ( us ) ( right ) depicts an isohypoechoic lesion with ill - de ned margins and a diameter of 15 mm ( breast imaging reporting and data system us category 4 )  . 
allecogra a di base ( destra ) si apprezza una lesione iso - ipoecogena a margini mal de niti del diametro di 15 mm ( classe bi - rads ecogra ca 4 )  . 
in contrast , six lesions correctly assessed as bi - rads 3 ( four broadenomas and two areas of fat necrosis ) were incorrectly assessed as malignant at us elastography . 
finally , three malignancies ( one tubular carcinoma , one abscessed ductal carcinoma and one sarcoma ) classi ed as bi - rads 4 were instead erroneously considered benign at us elastography . classe bi - rads 3 e con punteggio elastosonogra co di benignit ( da 1 a 3 ) , tre carcinomi ( 1 mucinoso , 1 tubulare e 1 duttale in ltrante ) hanno presentato un punteggio elastosonogra co di benignit , pari rispettivamente a 1 per il primo e 3 per i rimanenti due . 
applicando , dunque , il relativo sistema di punteggio alla classe bi - rads 3 lelastosonogra a ha presentato valori di sensibilit , speci cit , vpp e vpn pari rispettivamente a 50% , 86% , 30% e 93 , 5% ( tabella 4 )  . classe ecogra ca bi - rads 4 nella tabella 3 sono riportati i punteggi relativi alla valuradiol med ( 2011 ) 116 : 10271038 1035 table 4 sensitivity , speci city and positive and negative predictive values of ultrasound ( us ) elastography , overall and relative to the breast imaging reporting and data system ( bi - rads ) us classi cation . 
this assessment was con rmed by histology in three cases of invasive ductal carcinoma but refuted in the remaining seven cases ( four broadenomas , two areas of fat necrosis and one case of corpuscular cyst )  . 
 among the remaining 46 bi - rads 3 lesions with benign us elastography scores ( 13 ) , there were three carcinomas : one mucinous ( score 1 ) , one tubular ( score 3 ) and one invasive ductal carcinoma ( score 3 )  . 
therefore , applying the relevant scoring system to bi - rads 3 lesions , us elastography had sensitivity , speci city , ppv and npv of 50% , 86% , 30% and 93.5% , respectively ( table 4 )  . tazione elastosonogra ca delle 28 lesioni mammarie in classe ecogra ca bi - rads 4 . 
in particolare , 17 / 28 ( 60 , 7% ) di queste hanno presentato un punteggio elastosonogra co di malignit ( 4 o 5 ) , confermata in 11 casi allistologia , ma smentita nei rimanenti sei casi ( 3 brosi , 1 adenosi , 1 neoformazione intracistica e 1 iperplasia duttale semplice )  . 
di contro , tra le 11 lesioni in classe bi - rads 4 con punteggio elastosonogra co di benignit ( 13 ) lesame istologico ha confermato la presenza di 1 carcinoma ascessualizzato , 1 sarcoma e 1 carcinoma tubulare , rispettivamente presentanti allindagine elastosonogra ca un punteggio di 1 , 2 e 3 . 
this was con rmed by histology in 11 cases but refuted in the remaining six ( three cases of brosis , one adenosis , one intracystic mass and one simple ductal hyperplasia )  . 
in contrast , among the 11 bi - rads 4 lesions with benign us elastography scores ( 13 ) , histology revealed in accordo con quanto riportato da alcuni autori , nel nostro studio , mirato alla valutazione del ruolo dellelastosonogra a nelle lesione focali mammarie indeterminate allecogra a di base , non abbiamo riscontrato differenze statisticamente signi cative tra le due metodiche in termini di sensibilit , speci cit e valori predittivi , positivo e negativo [ 12 , 13 ]  . 
complessivamente , nella nostra esperienza , lelastosonogra a ha dimostrato una sensibilit del 70% , infe1036 radiol med ( 2011 ) 116 : 10271038 the presence of one abscessed carcinoma , one sarcoma and one tubular carcinoma , with us elastography scores of 1 , 2 and 3 , respectively . 
overall , in our experience , us elastography had a sensitivity of 70% , lower than reported by other authors ( 7996.9% ) and a speci city of 79.6% , within the range reported in the literature ( 7689% ) [ 8 , 12 , 14 , 15 ]  . 
one study only , with a 33.2% prevalence of malignant lesions , reported a sensitivity of 96.9% , but this was obtained using a different us elastography scoring system that is not directly comparable with the one adopted by us [ 15 ]  . 
in the case of indeterminate bi - rads 3 breast lesions , our ndings , in agreement with the literature , support the use of us elastography for its high npv and therefore its ability to con rm the benign nature of the lesion and thus avoid the need for biopsy [ 8 , 16 , 17 ]  . 
in particular , in our experience , us elastography proved particularly useful in corpuscular cysts , which passed from a score of bi - rads 3 ( probably benign ) to a us elastography score of 1 ( highly suggestive of benignity )  . 
in this regard , it should also be noted that riore ai valori riportati da altri autori variabili tra il 79% ed il 96 , 9% e una speci cit del 79 , 6% , compresa nellambito dei valori riportati in letteratura ( 76%89% ) [ 8 , 12 , 14 , 15 ]  . 
 infatti , avendo escluso dalla nostra analisi la classe ecograca bi - rads 5 , per la quale il campionamento bioptico gi indicato , la prevalenza di lesioni maligne risultata , nella nostra casistica , pari al 33 , 8% , dunque nettamente inferiore a quella di altri studi , che presentavano punte anche del 76% [ 6 , 8 , 12 ]  . 
solo in uno studio , con prevalenza di lesioni maligne pari al 33 , 2% , si riscontrata una sensibilit pari al 96 , 9% , ma impiegando un punteggio elastosonogra co diverso e quindi non direttamente comparabile a quello usato nel presente lavoro [ 15 ]  . 
nel caso , dunque , di lesione mammaria indeterminata in classe ecogra ca bi - rads 3 , la valutazione statistica dei nostri dati , in accordo a quanto riportato in letteratura , supporta limpiego dellelastosonogra a per il suo elevato vpn e , quindi , per confermare la natura benigna di una lesione , evitando il ricorso alla biopsia [ 8 , 16 , 17 ]  . 
nella nostra esperienza , in particolare , il contributo della valutazione elastosonogra ca risultato particolarmente utile nel caso delle cisti corpuscolate , passate nella quasi totalit dei casi da un punteggio bi - rads 3 , pur indicativo di probabile benignit , ad uno elastosonogra co 1 , altamente suggestivo di benignit . 
 a tale proposito , appare opportuno ricordare come anche lanalisi color doppler possa fornire utili indicazione al ne di distinguere le cisti non tumorali dai tumori mammari intracistici [ 18 ]  . 
tuttavia , tre carcinomi classi cati come bi - rads 3 ( 1 mucinoso , 1 tubulare e 1 duttale in ltrante ) hanno presentato , nella nostra serie , un punteggio elastosonogra co di benignit , almeno in parte da mettere in relazione alle caratteristiche istologiche , quali lelevato contenuto di mucina , o alle dimensioni superiori a 2 centimetri , situazione questultima che pu condizionare la disoradiol med ( 2011 ) 116 : 10271038 1037 colour doppler can help to distinguish nontumoural cysts from intracystic breast carcinomas [ 18 ]  . 
however , three carcinomas classi ed as bi - rads 3 ( one mucinous , one tubular and one invasive ductal ) had a benign us elastography score , at least in part as a result of histological features such as high mucin content , or a size > 2 cm that can lead to lesion heterogeneity due the presence of necroticcolliquative areas , as demonstrated by giuseppetti et al . 
in this regard , it would seem appropriate to periodically follow - up bi - rads 3 lesions not subjected to biopsy in order to assess their stability over time and thus lower the risk of missing the opportunity of early diagnosis of a possible malignancy [ 19 ]  . 
in contrast , the ppv of us elastography in bi - rads 4 focal breast lesions 64.7% in our experience is not suf ciently high to the need for biopsy on the basis of the us elastography score alone , as suggested by a previous report on nonpalpable breast lesions [ 12 ]  . 
in addition , although all bi - rads 4 lesions with a us elastography score of 5 proved to be ductal carcinomas at histological examination , six of the 14 bi - rads 4 lesions with a us elastography score of 4 turned out to be benign . 
this is con rmed by the fact that an abundant brotic component accounted for a malignant us elastography score in four broadenomas in our series , whereas the majority of broadenomas ( 25 / 29 , 86% ) had a high level of elasticity and consequently a benign us elastography score . there are several limitations to our study . 
first , the reference standard was not histological examination of the surgical specimen in all cases , but the semiological criteria used in cyto - microhistological evaluation , known in the literature and commonly used in clinical practice . 
in addition , as all us elastography examinations were performed by a single operator , it was not possible to assess the interoperator variability of this relatively new us technique . 
further research is needed to evaluate these aspects . in conclusion , by providing additional diagnostic information regarding the elasticity of breast lesions , us elastography completes but does not in any way replace the mogeneit della lesione per la presenza di aree necroticocolliquative , come gi dimostrato da giuseppetti et al . 
 a tale proposito , appare opportuno controllare periodicamente le lesioni classi cate come bi - rads 3 , ancorch non sottoposte a biopsia , al ne valutarne la stabilit nel tempo e , quindi , di ridurre la possibilit di non diagnosticare tempestivamente una eventuale lesione maligna [ 19 ]  . 
di contro , il vpp riportato dallelastosonogra a nellanalisi delle lesioni focali mammarie bi - rads 4 , pari nella nostra esperienza al 64 , 7% , non risulta abbastanza soddisfacente da consentire di escludere il ricorso alla biopsia sulla scorta del solo punteggio elastosonogra co , in accordo con quanto suggerito da un precedente studio condotto su lesioni mammarie non palpabili [ 12 ]  . 
inoltre , sebbene tutte le lesioni in classe bi - rads 4 con punteggio elastosonograco pari a 5 si siano rivelate dei carcinomi duttali allindagine istologica , sei delle 14 lesioni focali in classe bi - rads 4 con punteggio elastosonogra co pari a 4 si sono rivelate in realt benigne . 
a conferma di quanto appena esposto , proprio la presenza di abbondante componente brotica stata alla base del riscontro di un punteggio elastosonogra co di malignit in quattro broadenomi della nostra serie . 
questi ultimi , di contro , hanno presentato nella maggior parte dei casi ( 25 / 29 , 86% ) unelavata elasticit e , di conseguenza , un punteggio elastosonogra co di benignit . 
in primo luogo lo standard di riferimento non stato , in tutti i casi , la valutazione istologica del resecato operatorio , bens i criteri semeiologici alla valutazione cito - microistologia , ancorch noti in letteratura e comunemente impiegati nella pratica clinica . 
inoltre , un solo operatore ha effettuato tutti gli esami elastosonogra ci e , pertanto , non stato possibile valutare la variabilit interoperatore di una metodica di impiego relativamente recente come lelastosonogra a . 
ulteriori studi sono auspicabili per valutare tali aspetti . in conclusione , nella nostra esperienza , lelastosonogra a , fornendo ulteriori informazioni diagnostiche circa lelasticit delle lesioni mammarie , completa ma non sostituisce in alcun modo la valutazione morfologica dellesame ecogra co in b - mode . 
gemelli , largo francesco vito 1 , 00168 roma , italy 2unit operativa di radiodiagnostica , ospedale civile del ceppo , pistoia , italy 3unit di biostatistica , dipartimento di farmacologia clinica ed epidemiologia , consorzio mario negri sud , santa maria imbaro ( ch ) , italy 4unit operativa di ortopedia e traumatologia , ospedale g . 
forty - eight patients ( 40 men , eight women ; mean age , 31 years ) underwent arthroscopic reconstruction of the acl using double - strand semitendinosus and gracilis tendons . 
all patients underwent mr imaging 12 months after surgery and clinical evaluation at 6 and 12 months using the international knee documentation committee ( ikdc ) scoring systemr imaging and clinical features were correlated using the mannwhitney u test for continuous variables and fishers exact test for categorical variables . 
quarantotto pazienti ( 40 uomini e 8 donne , et media : 31 anni ) sono stati sottoposti a ricostruzione artroscopica del lca , utilizzando i tendini semitendinoso e gracile ( dgst )  . 
tutti i pazienti sono stati sottoposti ad esame rm , sono stati valutati 12 mesi dopo lintervento chirurgico e valutati clinicamente a 6 e 12 mesi , in accordo con linternational knee documentation commitee scoring ( ikcd test )  . 
misalignment of the ligament - screw system and the tibial tunnel and the presence of uid in the tibial tunnel appear to be directly correlated with clinical instability . keywords knee mr imaging anterior cruciate ligament tibial tunnel clinicamente instabili vs . 
 72 , 55 , 5 per langolo calcolato sullimmagine coronale del nuovo lca ( p = 0 , 3248 ) ; era presente uido nel tunnel tibiale rispettivamente nel 42 , 9% e nel 9 , 8% ( p = 0 , 2104 )  . 
 parole chiave ginocchio rm legamento crociato anteriore tunnel tibiale introduction introduzione arthroscopic reconstruction of the anterior cruciate ligament ( acl ) is performed by graft xation in tunnels drilled into the tibia and femur . 
 [ 2 ] reported that the direct contact between the graft and the bone inside the tunnel is crucial for integration within 912 weeks . in our study , we refer to the xation system that , following the indications of weil et al . 
the graft is prepared and secured at the proximal extremity using two poly - l - lactic acid screws , whereas a device consisting of a sheath and a conical screw made of 70% poly - l - lactic acid ( plla ) and 30% tricalcium phosphate ( tcp ) is inserted into the tibia [ 37 ]  . 
fixation with bioabsorbable material assists the radiologist in that it avoids interference caused by metal structures that distort magnetic resonance ( mr ) imaging and limit its use in the study of graft healing [ 9 ]  . 
 [ 11 ] observed that central xation of the new ligament is more effective , as it produces a stronger biological stimulus for osteointegration . la ricostruzione artroscopica del legamento crociato anteriore ( lca ) viene attualmente eseguita mediante la ssazione di graft in tunnel costruiti nella tibia e nel femore . 
 il neo - legamento viene preparato e ssato allestremit prossimale con 2 chiodini in acido polilattico , mentre nella tibia si ricorre ad un device costituito da una guaina ed una vite conica composte per il 70% di acido poli - l - lattico ( plla ) e dal 30% di fosfato tricalcico ( tcp ) [ 37 ]  . 
la ssazione con mezzi riassorbili di ausilio al lavoro del radiologo perch si eliminano le interferenze che le strutture metalliche hanno con gli esami strumentali e che modi cano le immagini in risonanza magnetica ( rm ) , limitandone luso nello studio della guarigione dellinnesto [ 9 ]  . 
i mezzi di ssazione in acido polilattico subiscono la degradazione , ma la ssazione primaria permane per il periodo neces1126 radiol med ( 2011 ) 116 : 11241133 mr imaging , with its high speci city ( 85100% ) and sensitivity ( 82100% ) due to the ability to identify direct and indirect signs , is considered the method of choice for characterising acl injuries and is routinely used in clinical practice [ 12 , 13 ]  . 
the site of the tibial tunnel is of crucial importance in acl reconstruction , and in particular , it has been shown that impingement with the anterior condylar roof is one of the leading causes of failure of the procedure [ 1418 ]  . 
the purpose of our study was to evaluate the following with mr imaging : position of the tibial tunnel in relation to blumensaats line in sagittal images and the angle between the ligament - screw system and the tibial surface as measured in coronal images ; presence of uid inside the tunnel and correlation with clinical instability ; alignment between the ligament - screw system and the tibial tunnel ; clinical signs of knee stability in patients treated with arthroscopic acl reconstruction . materials and methods the study was approved by the ethics committee , and all patients gave signed informed consent . 
 from july 2004 to october 2005 , we recruited 48 patients ( 40 men and eight women ; mean age 31 years ; range 1847 years ) who underwent arthroscopic acl reconstruction using the semitendinosus and gracilis tendons as a gra in 16 patients , the orthopaedic surgeon found a traumatic lesion of the internal meniscal brocartilage . graft xation was obtained with a bioabsorbable version of the intra x device , known as bio - intra x ( mitek ) and consisting of 70% poly - l - lactic acid ( plla ) and 30% tricalcium phosphate ( tcp )  . 
assessments were performed by an orthopaedic surgeon with extensive experience in knee disorders and based on the international knee documentation committee ( ikdc ) scoring syste according to the results , the patients were subdivided into three groups ( a , b and c )  . 
the study protocol consisted of the following sequences : proton - density fat - saturated turbo spin - echo ( pd - fs - tse ) with fat suppression in the axial and sagittal sario allosteointegrazione dellimpianto [ 10 ]  . 
 [ 11 ] hanno osservato che la ssazione concentrica del neolegamento pi ef cace poich produce uno stimolo biologico maggiore allosteointegrazione . la rm , presentando unalta speci cit ( 85%100% ) e sensibilit ( 82%100% ) , grazie alla sua capacit di evidenziare segni diretti ed indiretti , considerata metodica delezione per laccertamento delle lesioni del lca e di utilizzo quotidiano nella pratica clinica [ 12 , 13 ]  . 
lalloggiamento del tunnel tibiale risulta di particolare importanza per la ricostruzione del lca , in particolare si rilevato che limpingement con il tetto condilare anteriore tra le pi rilevanti cause di fallimento di questintervento [ 1418 ]  . 
 lobiettivo del nostro studio stato valutare con le immagini rm quanto segue : la posizione del tunnel tibiale rispetto alla linea di blumensaat nelle immagini sagittali e langolo formato dal sistema vite - camicia - ligamento con la super cie tibiale misurato nelle immagini coronali ; la presenza di liquido nel tunnel e la correlazione con lallineamento del sistema vite - camicia - ligamento ed il linstabilit clinica ; tunnel tibiale ; i segni clinici di stabilit del ginocchio in pazienti sottoposti a ricostruzione artroscopica del lca . materiali e metodi per questo studio , stato ottenuto il permesso del comitato etico e tutti i pazienti hanno rmato il consenso informato . 
sono stati reclutati quarantotto pazienti ( 40 uomini e 8 donne , et media 31 anni ; range tra 18 e 47 anni ) , tra luglio 2004 ed ottobre 2005 , e sottoposti ad intervento di ricostruzione artroscopia del lca , utilizzando i tendini semitendinoso e gracile come gra in 16 pazienti il chirurgo ortopedico ha riscontrato lesione traumatica della brocartilagine meniscale interna . la ssazione del graft stata ottenuta con una versione riassorbibile del device , detto intra x , il bio - intra x ( mitek ) , costituito dal 70% di plla e per il 30% da tcp . 
 tutti i pazienti sono stati sottoposti a controllo clinico per valutare il grado di stabilit del ginocchio da chirurgo ortopedico con elevata esperienza in patologia di ginocchio , utilizzando il ikdc test a 6 ( t1 ) e 12 ( t2 ) mesi dopo lintervento . 
we used 3 - mm slice thickness , 0.30.5 interval , 512512 matrix , 14 - cm eld of view ( fov )  . images were analysed by two trainee radiologists , and one consultant radiologist with 18 years of experience in musculoskeletal radiology . 
images were assessed for the position of the tibial tunnel in relation to blumensaats line , the coronal angle between the acl and tibial articular surface , sagittal alignment of the long axis of the ligamentscrew system with the tibial tunnel and the presence of uid inside the tunnel . 
under normal conditions , if a line is drawn tangential to the anterior acl margin , the two lines intersect at the level of the condylar insertion of the acl forming an angle anteriorly [ 19 ]  . 
all those who analysed the mr images were blinded to the results of the clinical assessment . statistical associations between mr parameters and clinical stability were analysed using the mannwhitney u test for continuous variables and fishers exact test for categorical variables . 
il protocollo di studio prevedeva le seguenti sequenze : proton density - turbo spin echo con soppressione del segnale adiposo ( pd - tse ) sui piani assiale e sagittale ( tempo di ripetizione [ tr ] = 4740 , tempo di eco [ te ] = 33 ) , pd - tse sul piano sagittale ( tr = 2000 , te = 32 ) , fat saturated ( fs ) - t1 sul piano coronale ( tr = 1150 , te = 16 )  . 
 stato utilizzato uno spessore di strato di 3 mm , 0 , 30 , 5 di intervallo , matrice di acquisizione 512512 , campo di vista ( fov ) di 14 cm . le immagini sono state studiate da due specializzandi e da uno specialista radiologo con elevata esperienza ( 18 anni ) in radiologia muscolo - scheletrica . 
stata valutata la posizione del tunnel tibiale rispetto alla linea di blumensaat , langolo coronale del lca rispetto alla super cie articolare tibiale , lallineamento dellasse maggiore del sistema vite - camicia - legamento e del tunnel sul piano sagittale , ed in ne la presenza di uido allinterno del tunnel . 
se si traccia una linea tangente al margine anteriore del lca , in condizioni di normalit , le due linee si incrociano normalmente a livello dellinserzione condiloidea del lca , formando un angolo che si apre anteriormente [ 19 ]  . 
1 posizione del tunnel tibiale secondo la classi cazione del metodo di muneta , rispetto alla linea di blumensaat . using sas software ( statistical package release 9.1 sas institute , cary , nc , usa )  . hanno analizzato le immagini non erano a conoscenza della valutazione clinica . radiol med ( 2011 ) 116 : 11241133 1128 results on the basis of the clinical assessment with the ikdc scoring system at 12 months ( table 1 ) , 28 patients were assigned to group a . 
the examiner stands on the side of the injured knee and pulls the tibia anteriorly ; a sensation of giving way or a nonrigid endpoint is a sign of acl injury . 
 mr imaging is the method of choice for the follow - up of acl reconstruction and to assess graft complications [ 14 , sono state analizzate le associazioni statistiche tra i parametri rm e la stabilit clinica del paziente con il mann - whitney u test per le variabili continue ed il fisher exact test per le variabili categoriche . 
tutte le analisi sono state fatte da un esperto in materia , utilizzando il statistical package software ( sas , release 9 , 1 , sas institute , cary , nc , usa )  . risultati sulla base della valutazione clinica con lo schema ikdc a 12 mesi ( tabella 1 ) , ventotto pazienti sono stati inseriti nel gruppo a . 
il test di lachman il pi sensibile e viene eseguito per fare diagnosi clinica di lesione del legamento crociato anteriore con il paziente in posizione supina con il ginocchio a 20 di essione , lesaminatore si pone dal lato del ginocchio leso e tira la tibia in avanti ; una sensazione di cedimento o un punto di blocco non rigido sono indici della lesione del lca . 
2 proton - density fat - saturated turbo spin - echo in the sagittal plane : misalignment between the screw and tibial tunnel ( arrow ) ; oedema is seen inside and on the anterior wall of the tunnel . 
2 scansione pd - tse - fs sul piano sagittale : si noti lanomalo decorso della vite rispetto al tunnel osseo ( freccia ) ; si segnala la presenza di edema nel tunnel e nella parete anteriore . 
3 proton - density fat - saturated turbo spin - echo in the sagittal plane : evident misalignment of the screw , which is enveloped by its expansion sheath ( arrow )  . 
la giusta posizione del tunnel tibiale ha un ruolo importante nelloutcome della ricostruzione , per evitare impingement con il tetto condilare anteriore ed il conseguente fallimento del lca [ 14 , 15 ]  . 
questo valore cut - off in buona correlazione con i nostri risultati 77 , 311 , 3 nel gruppo c e 72 , 55 , 5 nei gruppi a e b . 
 i nostri risultati sulla posizione del tunnel tibiale rispetto alla linea di blumensaat e langolo coronale del graft , comunque , non sono stati statisticamente signi cativi , con una grande deviazione standard rispetto ai valori medi . 
 probabilmente , sarebbe stata necessaria una popolazione pi ampia di pazienti instabili e questo senzaltro il principale limite del nostro studio . abbiamo osservato nel 42 , 9% dei pazienti instabili e nel 9 , 8% dei pazienti stabili una reazione edematosa nella porzione anteriore del tunnel tibiale con in ltrazione di liquido nello spazio tra losso e il graft , probabilmente dovuta ad una perdita della compressione concentrica sul gratale condizione necessita di uno stretto follow - up per fig . 
5 proton - density fat - saturated turbo spin - echo in the axial plane : misalignment between the central portion of the screw and the ligaments displaced to the medial wall of the tunnel ( arrow )  . 
5 scansione pd - tse - fs sul piano assiale : si nota come la porzione centrale della vite sia mal posizionata rispetto ai legamenti disposti tutti sul versante mediale del tunnel ( freccia )  . 
correct placement of the tibial tunnel is crucial for the outcome of the procedure , and in particular for avoiding impingement against the condylar roof anteriorly and subsequent failure of the reconstruction [ 14 , 15 ]  . 
 however , our ndings regarding tibial tunnel position in relation to blumensaats line and the coronal acl angle failed to reach statistical signi cance , showing a high standard deviation of the mean values . 
6a proton - density turbo spin - echo ( pd - tse ) in the sagittal plane : different layers interposed between the bone tunnel and tibial screw are evident . 
6a scansione pd - tse sul piano sagittale : sono ben evidenti le differenti strutture interposte tra il tunnel osseo e la vite tibiale ; procedendo in senso antero - posteriore possiamo documentare la linea ipointensa del tunnel , la stria ipointensa dei legamenti e della camicia , le spire della vite , la parete ed in ne la porzione centrale , cava , della vite ( freccia )  . 
b scansione pd - tse - fs sul piano sagittale : sono ben evidenti le stesse strutture documentate nella scansione in a ; la porzione cava della vite appare iperintensa ( freccia )  . 
 some authors [ 22 , 24 ] report high mr signal intensity in correspondence with the screwbone and ligamentbone interfaces in t2 - weighted images and persistence of uid inside the tibial tunnel , probably due to failed osteointegration , leading to knee instability . 
furthermore , in some cases , it was dif cult to identify the systems expansion valutare se lo slargamento del tunnel pu essere responsabile dellinstabilit del gra alcuni autori [ 22 , 24 ] hanno riscontrato iperintensit del segnale rm in corrispondenza delle interfacce vite - osso e legamento - osso nelle immagini t2 - pesate e persistenza del liquido nel tunnel tibiale , probabilmente dovuti alla mancata osteointegrazione del neo - legamento , causando linstabilit del ginocchio . 
il nostro studio ha mostrato disallineamento del sistema vite - camicia - legamento e tunnel tibiale nel 57 , 1% dei pazienti clinicamente instabili ( gruppo c ) e nel 12 , 2% dei pazienti clinicamente stabili ( gruppi a e b )  . 
in the future , we intend to expand the case series to reach a statistically signi cant correlation between clinical instability and tibial tunnel position in relation to blumensaats line and the coronal angle in unstable patients , and to strengthen our ndings with a 2 - year follow - up of the patients enrolled . 
 osteo - induzione del sistema stesso . in conclusione , il disallineamento del sistema vitecamicia - legamento rispetto al tunnel tibiale e la presenza di liquido nel tunnel sembrano essere direttamente correlati con linstabilit clinica . 
i nostri obiettivi futuri saranno ampliare la casistica per raggiungere una correlazione , statisticamente signi cativa , tra linstabilit clinica con la posizione del tunnel tibiale rispetto alla linea di blumensaat e langolo coronale nei pazienti instabili ed incrementare i risultati con un follow - up a due anni dei pazienti arruolati . 
as it happens in fortunate research elds , three dimensional echocardiography , at rst aiming to add spatial information for better anatomic study , has recently been shown to provide ne physiologic information on myocardial wall motion when coupled with the very new technique of speckle tracking - derived velocity information . 
as such , the medical community is eager to receive solid information on whether the effort and investments have been worthwhile and possibly on how to handle this complex subject . 
the scheme of presentation helps the reader to appreciate the history of the technical evolution , to recognize how limitations in sampling rate have been arranged both for imaging and doppler velocimetry . 
since the learning curve may be strongly in uenced by the capability of physicians to cope with it , the authors devoted a special effort to explain the different modalities and commented on a large number of gures and video - clips . 
the role of 3d - echocardiography emerging in cardiac dyssynchrony is addressed in detail and with caution , especially following the disappointments with the 2d - echo - derived indexes in recent years . 
 the need for an adequate learning curve is correctly stressed , although small studies suggest its usefulness to predict bene t from resynchronization therapy . lecocardiogra a tridimensionale ( ecocardio - 3d ) giunta a maturit per lapplicazione clinica dopo un lungo periodo di progressi tecnici e gli scienziati che hanno iniziato da pionieri questo campo di ricerca possono godere ora del successo dei loro sforzi . 
come si veri ca in campi di ricerca fortunati , lecocardio - 3d che si pre ggeva , alle sue origini , di fornire ulteriori informazioni spaziali per una miglior valutazione anatomica , ha dimostrato ultimamente la capacit di poter fornire ni informazioni siologiche sulla motilit della parete miocardica , se associata con la assai nuova tecnica dellinformazione della velocit derivata dallo speckle tracking . 
appunto perch tale , la comunit medica desiderosa di ricevere informazioni ben precise per riconoscere se gli sforzi e gli investimenti fatti siano stati utili e pro cui e , se possibile , su come utilizzare questa complessa materia . il volume dal titolo three - dimensional echocardiography merita una menzione particolare sia per la capacit che per lottimo e comprensibile modo in cui presenta sinteticamente largomento . la scaletta della presentazione aiuta il lettore ad apprezzare la storia dellevoluzione tecnica ed a riconoscere come le limitazioni delle esempli cazioni siano state ben ordinate sia per le immagini che per la velocimetria doppler . 
dal momento che la curva di apprendimento pu essere in uenzata dalla capacit di adattamento dei medici , gli autori hanno dedicato uno sforzo speciale alla spiegazione delle differenti modalit , dedicando molta attenzione e commenti ad un grande numero di gure e video - clips . 
 viene poi illustrato , sia nel dettaglio che con precauzione , il ruolo emergente dellecocardio - 3d nella valutazione della dissincronia cardiaca , soprattutto a seguito delle disillusioni avute negli ultimi anni relativamente agli indici derivati dallecocardio - 2d . viene evidenziata correttamente la necessit di una adeguata curva di apprendimento della metodica , anche se studi di limitate dimensioni suggeriscono la sua utilit nel predire 1150 radiol med ( 2011 ) 116 : 11491150 the two chapters devoted to the investigation of valve diseases are particularly well written . 
the large number of examples is remarkable and all together constitute a guide to clinical investigation planning , since 3d - echocardiography is far less intuitive than 2d - echo . 
 the fact that 3d - echo may add information in the clinical arena but at the expense of a careful learning curve is well demonstrated with gures and video in the last four chapters on congenital heart diseases , cardiac tumours and sources of embolism and monitoring guiding cardiac interventions and surgery . 
 in summary , this text provides the reader with consistent information in a concise manner that could be useful both to beginners and veterans . bene cio dalla terapia di risincronizzazione . i due capitoli dedicati alla valutazione delle valvulopatie sono particolarmente ben scritti . 
straordinario il gran numero di esempi che nel loro insieme costituiscono una guida nella piani cazione della valutazione clinica , dal momento che lecocardio - 3d assai meno intuitiva dellecocardio - 2d . il fatto che lecocardio - 3d possa aggiungere informazioni nellambito clinico , ma a spese di una appropriata ed accurata curva di apprendimento , ben dimostrato negli ultimi quattro capitoli sulle cardiopatie congenite , i tumori del cuore , le cause dei fenomeni di embolizzazione e nel monitoraggio - guida alle procedure cardiovascolari e agli interventi chirurgici . in conclusione , questo volume fornisce al lettore , in modo succinto , informazioni coerenti , utili sia per il principiante che per lesperto . franco recusani divisione di cardiologia , fondazione irccs policlinico s . 
three hundred and ninety - two patients with liver or / and spleen trauma ( 179 liver and 217 spleen injuries ) , who underwent ceus examinations following contrast - enhanced computed tomography ( ct ) , were enrolled in this retrospective study over a period of > 4 years . 
ceus detected contrast medium extravasation or pooling in 16% ( 63 / 396 ) of liver or spleen lesions in 61 patients , which was con rmed by contrast - enhanced ct . 
of 63 lesions in 61 patients , ceus showed that 74.6% ( 47 / 63 ) ( 21 liver lesions and 26 spleen lesions ) presented contrast medium extravasation or pooling , both in the organ and out the capsule , in 14.3% ( 9 / 63 ) and only outside the capsule in 11.1% ( 7 / 63 )  . 
trecentonovantadue pazienti con trauma epatico e / o splenico ( 179 traumi epatici e 217 splenici ) , sottoposti ad esame ceus seguito da tomogra a computerizzata ( ct ) con mezzo di contrasto , sono stati arruolati in questo studio retrospettivo per un periodo di pi di 4 anni . 
la ceus ha rilevato lo stravaso o laccumulo di mdc nel 16% ( 63 / 396 ) delle lesioni epatiche o spleniche in 61 pazienti , confermato poi dalla tc con mezzo di contrasto . 
le informazioni ottenute dalla chirurgia , sia dal trattamento minimamente invasivo sia da quello conservativo , presi come riferimento standard , e la sensibilit delle due tecniche non si sono dimostrate differenti ( p = 0 , 122 )  . 
 nelle 63 lesioni in 61 pazienti , la ceus ha mostrato che il 74 , 6% ( 47 / 63 ) delle lesioni ( 21 lesioni epatiche e 26 spleniche ) ha presentato stravaso o accumulo di mdc , entrambi allinterno dellorgano o fuori dalla capsula nel 14 , 3% ( 9 / 63 ) e solo fuori dalla capsula nell11 , 1% ( 7 / 63 )  . 
 limaging ceus del sanguinamento attivo da traumi epatici e splenici presenta differenti caratteristiche , e le dimensioni e le forme del sanguinamento attivo dovuto allo stravaso o allaccumulo del mezzo di contrasto sono variabili . 
la ceus pu mostrare il sanguinamento attivo associato a trauma epatico e splenico con diverse caratteristiche di imaging , rendendo quindi possibile diagnosticare il sanguinamento attivo . parole chiave fegato milza trauma sanguinamento attivo mezzo di contrasto ecogra a microbolle introduction active bleeding from hepatic and splenic trauma is a major cause of death and disability . 
digital subtraction angiography ( dsa ) cannot only be used to identify active bleeding but also to arrest bleeding after penetrating trauma to the abdomen [ 5 , 6 ]  . 
additional drawbacks include : ( a ) high expense , ( b ) need for highly trained staff and ( c ) need for patient transfer from the emergency department . 
 with the development of contrast media , contrast - enhanced ultrasound ( ceus ) has improved the sensitivity of conventional us for detecting and characterising focal liver lesions [ 10 ]  . 
performed ceus examinations in 83 traumatic emergencies , and their clinical study showed for the rst time how us can detect contrast medium extravasation , a signi cant indicator of active haemorrhage . 
thus , the purpose of our study was to observe the ceus imaging features of active bleeding from hepatic and splenic trauma . materials and methods the study was approved by the ethics committee of the chinese peoples liberation army general hospital and was designed according to the health insurance portability and accountability act . 
informed consent for both ceus and the use of related data for future research was obtained from each patient before and after the procedure . patients a total of 392 patients with liver and / or spleen trauma ( 179 liver and 217 spleen injuries ) were enrolled in this retrospective study covering a period of > 4 years ( september 2004 to december 2008 )  . 
 ultrasound imaging us contrast agent used in this study was sonovue ( bracco , milan , italy ) , a second - generation contrast medium for diagnostic imaging approved in china in 2003 , which consists of stabilised microbubbles containing an inert gas ( sulfur hexa uoride , 8 l / ml of solution ) and covered by a phospholipid membrane [ 15 ]  . 
sonovue , a dose of 0.025 ml / kg , was administered as a quick bolus through an antecubital ve 1078 ultrasound image interpretation two us specialists , each with 5 years experience in diagnosing abdominal parenchymal organ trauma , performed the examinations . 
for patients not undergoing further diagnostic procedures or surgery , follow - up information was obtained from either the patients chart or the trauma surgeon in charge of the patient . 
differences between group means were determined by analysis of variance ( anova ) ( systat ver 13.0 , spss , inc , chicago , il , usa ) , with the chi - square test for 22 tables , where applicable . 
the 63 liver or spleen lesions with contrast - medium extravasation or pooling indicating active bleeding consisted of 29 liver and 34 spleen lesions . imaging of 63 lesions in 61 patients , ceus identi ed contrast medium extravasation or pooling in 74.6% ( 47 / 63 ) lesions ( 21 liver and 26 spleen ) , both in the lesion and out of the capsule in 14.3% ( 9 / 63 ) and only outside of the capsule in 11.1% ( 7 / 63 )  . 
i siti di emorragia attiva appaiono come spot a banda isolati , iperecogeni ed isoecogeni con dimensioni e forma variabili ( frecce ) b limmagine tc mostra la lesione epatica come unarea di bassa attenuazione ( frecce lunghe ) e dimostra il sanguinamento attivo come area di elevata attenuazione ( frecce corte ) a causa dello stravaso di mezzo di contrasto . brooklet or in a serpentine pattern . 
b within the lesion , there were enhanced perfusion regions , including the remaining normal parenchyma ( ne arrow ) and contrast medium extravasation and pooling ( thick arrow )  . 
a scansione ecogra ca trasversale obliqua della milza che mostra larea lesionata con aspetto anecogeno ed ipoecogeno con difetto di perfusione con margini irregolari nella regione medio - inferiore della milza . 
b allinterno della lesione ci sono regioni che presentano enhancement perfusionale e rappresentano parenchima residuo normale ( freccia sottile ) e stravaso e accumulo di mezzo di contrasto ( frecce spesse in a and b )  . 
the active bleeding disappeared in both patients 20 and 40 min , respectively , after ceus . discussion sonovue , the second - generation contrast medium used in this study , allows boosting of the lesion - to - parenchyma conspicuity , which increases us sensitivity . 
ceus has been largely employed in the evaluation of focal lesions [ 17 , 18 ] but also to improve us accuracy in detecting parenchymal organ injury after blunt abdominal trauma [ 1923 ]  . 
however , active bleeding not only presented as a hyperechoic area but also as a hyperechoic or isoechoic area , in contrast to the ndings of some recent studies [ 11 , 22 ]  . 
in addition , the active bleeding sites presented various shapes depending on the different locations , velocities and quantities . radiol med ( 2011 ) 116 : 10761082 1081 this study showed that the sensitivity of ceus was 72.4%. 
in the diagnosis of active haemorrhage from hepatic or splenic trauma , contrast - enhanced ct has several advantages over ceus , such as screening the entire abdomen and no limitations due to obesity , meteorism or subcutaneous emphysema . 
 in the diagnosis of active bleeding , ceus has several advantages : ( 1 ) it can detect active bleeding from hepatic and splenic trauma with excellent imaging characteristics ; ( 2 ) real - time imaging allows ceus to accurately evaluate active bleeding because active bleeding is characterised by activity . 
 ( 3 ) the examination is rapid , with a room time < 10 mthe use of ceus saves time and is safe . our study has several limitations : ( 1 ) ceus shares with conventional us several limitations , such as patient obesity , meteorism , subcutaneous emphysema etc . 
 ( 2 ) ceus requires rapid and skilful diagnosis because the duration of a single ceus examination is only 68 m ( 3 ) in theory , ceus can show active haemorrhage with any velocity . 
 ( 4 ) the real correlation between bleeding velocity and ceus imaging whether active bleeding from an artery , vein or capillary can be accurately detected or not needs to be investigated by further studies . 
it should not be intended as a substitute for ct but as a selective possibility to boost the role of us in the initial screening of patients with abdominal trauma . in conclusion , our study has shown that active haemorrhage from liver and spleen trauma appeared with various imaging features on ceus , according to which the diagnosis can be established . 
cademartiri1 , 2 1dipartimento di radiologia e del cardio - polmonare , azienda ospedaliero - universitaria di parma , c / o piastra tecnica piano 0 , via gramsci 14 , 43100 parma , italy 2dipartimento di radiologia e cardiologia , erasmus medical center , rotterdam , the netherlands 3dipartimento di radiologia e cardiologia , ospedale san gennaro , napoli , italy 4cpc centro prevenzione cardiovascolare , ospedale san raffaele , milano , italy 5dipartimento di radiologia e cardiologia , azienda asl , carrara , italy 6dipartimento di radiologia , universit di messina , messina , italy 7dipartimento di cardiologia , universit di foggia , foggia , italy 8dipartimento di radiologia , azienda ospedaliero - universitaria san martino , genova , italy correspondence to : f . 
we selected from a population of 1 , 500 patients in a multicentre registry with nstemi - acs who had undergone ctca and cag , ( n = 237 ; 187 men , mean age 6310 years )  . 
scopo del nostro lavoro stato valutare laccuratezza diagnostica dellangiogra a coronarica non invasiva con tomogra a computerizzata ( ctca ) nellindividuazione delle stenosi coronariche signi cative ( riduzione del lume coronarico 50% ) confrontata con la coronarogra a convenzionale ( cag ) nella sindrome coronarica acuta senza elevazione del tratto st ( acs - nstemi ) e nelle sue sottopopolazioni per genere e numero di fattori di rischio ( fdr )  . 
da una popolazione di 1500 pazienti appartenenti ad un registro multicentrico sono stati selezionati i pazienti con acs - nstemi ( 237 , 187 maschi , et media 6310 anni ) sottoposti a ctca e cag . 
ctca is a reliable diagnostic modality with high sensitivity and npv in nstemi - acs patients who are not candidates for early revascularisation , regardless of gender and number of risk factors . keywords ct coronary angiography conventional coronary angiography acute coronary syndrome nstemi risk factors diagnostic accuracy registry popolazione era del 53% . 
nellanalisi per paziente la sensibilit , speci cit , valore predittivo positivo e negativo della ctca sono risultati 100% ( maschi 100% ; femmine 100% ; 0rf 100% ; 1 - 2rf 100% ; > 2rf 100% ) , 95% ( maschi 98% ; femmine 50% ; 0rf nv% ( nv , non valutabile ) ; 12rf 96% ; > 2rf 96% ) , 95% ( maschi 98% ; femmine 91% ; 0rf 91% ; 1 - 2rf 96% ; > 2rf 96% ) , 100% ( maschi 100% ; femmine 100% ; 0rf nv% ; 1 - 2rf 100% ; > 2rf 100% ) , rispettivamente . 
la ctca una metodica diagnostica af dabile sia per lelevata sensibilit che per lelevato valore predittivo negativo nei pazienti con acs - nstemi non candidati ad immediata rivascolarizzazione , indipendentemente dal genere e dal numero di fattori di rischio . parole chiave angiogra a coronarica tc angiogra a coronarica convenzionale sindrome coronarica acuta nstemi fattori di rischio accuratezza diagnostica registro introduction introduzione computed tomography coronary angiography ( ctca ) is an accurate and robust modality for diagnosing and excluding coronary artery disease ( cad ) [ 18 ]  . 
given the limited information and absence of proper guidelines , the creation and sharing of multicentre registries with large patient populations guarantees an adequate response to the emerging questions surrounding the technique [ 2 , 16 ]  . 
 acute coronary syndrome ( acs ) is a clinical situation in which the short times of performing conventional coronary angiography ( cag ) and immediate revascularisation can be crucial [ 2 , 1733 ]  . 
however , acs is characterised by at least two major clinical presentations : acs with elevated st segment ( stemi ) and acs without elevated st segment ( nstemi ) [ 34 , 35 ]  . 
the latter includes unstable angina , which consists of the onset or worsening of typical chest pain , with no increase in markers of myocardial damage or st - segment elevation [ 34 ]  . 
for this category , a reliable diagnostic modality is desirable , and ctca has demonlangiogra a coronarica con tomogra a computerizzata ( ctca ) una metodica accurata e robusta per la diagnosi e lesclusione della malattia coronarica [ 18 ]  . 
la creazione e condivisione di registri multicentrici garantisce la possibilit di rispondere adeguatamente ai quesiti emergenti sulla metodica con ampie popolazioni di pazienti in presenza di scarse informazioni ed in mancanza di vere e proprie linee guida [ 2 , 16 ]  . 
 la sindrome coronarica acuta ( acs ) una situazione clinica nella quale i tempi brevi di esecuzione di una coronarogra a convenzionale e di una rivascolarizzazione immediata possono essere essenziali [ 2 , 1733 ]  . 
tuttavia , la acs costituita da almeno due grandi entit cliniche : la acs con elevazione del tratto st ( stemi ) e la acs senza elevazione del tratto st ( nstemi ) [ 34 , 35 ]  . 
questultima include langina instabile che consiste , in sintesi , nellesordio o nellaggravamento di una sintomatologia dolorosa toracica tipica senza lincremento dei markers di danno miocardico e lelevazione del tratto st [ 34 ]  . 
in questo caso il con ne tra acs e dolore toracico non coro1016 radiol med ( 2011 ) 116 : 10141026 strated signi cant potential in this group of patients [ 2 , 1733 ]  . in this study , we compared the diagnostic accuracy of ctca with cag in a population of patients with nstemiacs who were drawn from a multicentre registry [ 2 ]  . 
we selected patients with nstemi - acs from among the general population of the registry ( n = 237 ; 187 men , 50 women ; mean age 6310 years ; tables 1 and 2 ) [ 34 ]  . 
cdx , coronaria destra ; csx , coronaria sinistra ; tcs , tronco comune sinistro ; acx , arteria coronaria circon essa ; ada , arteria coronaria discendente anteriore . narico pu essere meno de nito ed in questa categoria che una metodica diagnostica af dabile sarebbe auspicabile . 
la ctca ha mostrato di avere un potenziale notevole in questa categoria di pazienti [ 2 , 1733 ]  . in questo lavoro presentiamo laccuratezza diagnostica della ctca a confronto con la coronarogra a convenzionale ( cag ) in una popolazione di pazienti con acsnstemi derivante da un registro multicentrico [ 2 ]  . 
 da maggio 2004 a giugno 2010 , sono stati studiati mediante ctca 1500 pazienti consecutivi ( 882 maschi , 490 femmine , et media 5812 anni ) , candidati allesecuzione di unangiogra a coronarica convenzionale ( cag ) allo scopo di determinare la presenza , la severit e lestensione della malattia coronarica [ 2 ]  . 
dalla popolazione generale del registro sono stati selezionati i pazienti con acs - nstemi ( 237 , 187 maschi , 50 femmine , et media 6310 anni ) ( tabelle 1 e 2 ) [ 34 ]  . 
il comitato etico ha approvato il protocollo di studio e tutti i pazienti hanno fornito un consenso informato . analisi statistica i dati sono presentati come prevalenze , medie e deviazioni standard . 
la performance diagnostica dellangiogra a coronarica mediante ctca nellindividuazione delle lesioni aterosclerotiche coronariche signi cative , utilizzando la cag come tecnica di riferimento di seguito riportata come sensibilit , speci cit , valore predittivo positivo e negativo con intervalli di con denza ( ic ) al 95% calcolati con espansione binomiale . 
tutte le statistiche sono state eseguite per la popolazione totale , per la popolazione maschile e per quella femminile e nelle sotto - popolazioni strati cate per numero di fattori di rischio : 0rf = assenza di fattori di rischio ; 1 - 2rf = presenza di 1 o 2 fattori di rischio ; > 2rf = presenza di pi di 2 fattori di rischio . 
all statistics were calculated for total population , male and female populations and the subpopulations strati ed by number of risk factors ( rf ) ( 0 rf = no risk factors present ; 12 rf = one to two risk factors present ; > 2 rf = more than two risk factors present )  . 
mean heart rate during the examination was 595 bpthe prevalence of obstructive disease was 53% ( 126 / 237 ) , with a marked prevalence among women [ men 45% vs . 
nei sottogruppi suddivisi in base al numero di fattori di rischio si osserva un progressivo aumento dellet media ed un passaggio da una maggiore prevalenza di femmine con 0rf and una netta prevalenza di maschi con 1 - 2rf e > 2rf ( tabella 2 )  . 
subgroups subdivided on the basis of the number of rf showed a progressive increase in age and a shift from a higher prevalence of women with 0 rf and a marked prevalence of men with 12 rf and > 2 rf ( table 2 )  . 
per - patient positive likelihood ratios were optimal in all populations ( lr + between 18 and 27 ) , whereas negative likelihood ratios were excellent ( lr = 0.0 in all cases )  . 
no signi cant differences in diagnostic accuracy between the different subpopulations were observed . discussion acs is a clinical condition in which the short times of performing cag and immediate revascularisation can be crucial [ 2 , 1733 ]  . 
when possible , patients with typical chest pain , positivity for biomarkers of myocardial damage and stemi should be immediately sent to the catheterisation laboratory for prompt revascularisation . ctca has been proposed for early strati cation of patients with acute chest pain [ 16 , 29 , 31 , 33 , 3740 ] , which is present in 5%10% of patients presenting at the casualty ward . 
of these , 70%80% are classi ed as having non - angina - like pain , 7%8% as having acs and up to 7%8% as having nstemi and / or unstable angina . 
ctca may accurately and rapidly rule out signi cant cad in the setting of acute chest pain [ 16 , 31 , 33 , 37 , 38 ]  . acs consists of at least two major clinical entities : acs with stemi and acs with nstemi [ 34 , 35 ]  . 
i quozienti di probabilit positivi per segmento sono risultati ottimali in tutte le popolazioni ( lr + compresi tra 11 e 25 ) , mentre i quozienti di probabilit negativi sono risultati eccellenti ( lrcompresi tra 0 , 0 e 0 , 028 )  . 
i quozienti di probabilit positivi per paziente sono risultati ottimali in tutte le popolazioni ( lr + compresi tra 18 e 27 ) , mentre i quozienti di probabilit negativi sono risultati eccellenti ( lr - = 0 , 0 in tutti i casi )  . 
non sono state osservate differenze signi cative differenze di accuratezza diagnostica tra le sottopopolazioni . discussione la sindrome coronarica acuta ( acs ) una situazione clinica nella quale i tempi di esecuzione di una coronarogra a convenzionale e di una rivascolarizzazione immediata possono essere essenziali [ 2 , 1733 ]  . 
i pazienti con dolore toracico tipico , curva enzimatica di danno miocardico e elevazione del tratto st , quando possibile , dovrebbero essere inviati immediatamente al laboratorio di emodinamica per effettuare una rivascolarizzazione precoce . 
il dolore toracico acuto rappresenta il 5%10% dei pazienti che si presentano al pronto soccorso ; di questi il 70%80% vengono classi cati come dolore non anginoso , il 7%8% vengono classi cati come acs , e no al 7%8% vengono classi cati come nstemi e / o angina instabile . 
 la ctca potrebbe consentire un accurato e veloce ruleout della malattia coronarica signi cativa nel contesto del dolore toracico acuto [ 16 , 31 , 33 , 37 , 38 ]  . la acs costituita da almeno due grandi entit clinica : la acs - stemi e la acs - nstemi [ 34 , 35 ]  . 
questultima include anche lentit clinica denominata angina instabile che consiste , in sintesi , nellesordio di una sintomatologia dolorosa toracica tipica senza lincremento dei markers di danno miocardico e lelevazione del tratto st , oppure nellaggravamento di sintomatologia anginosa in una breve arco temporale [ 34 ]  . 
in questo caso il con ne tra acs e dolore toracico non coronarico pu essere meno de nito ed in questa categoria di pazienti che una metodica diagnostica af dabile sarebbe auspicabile . 
la ctca ha mostrato di avere un potenziale notevole in questa categoria di pazienti [ 2 , 1733 ]  . radiol med ( 2011 ) 116 : 10141026 1021 1022 radiol med ( 2011 ) 116 : 10141026 diagnostic accuracy per segment diagnostic accuracy per patient fig . 
per - patient accuracy shows high sensitivity and negative predictive values in all subgroups , whereas speci city and positive predictive value are lower in the female and 0 rf populations . 
gra co a barre dellaccuratezza diagnostica e dei quozienti di probabilit ( lr ) per segmento e per paziente della angiogra a coronarica con tomogra a computerizzata ( ctca ) nella popolazione totale e nei sottogruppi . 
laccuratezza diagnostica per paziente mostra valori elevati si sensibilit e valore predittivo negativo elevati in tutti i sottogruppi , mentre la speci cit ed il valore predittivo positivo sono inferiori nella popolazione femminile e nella popolazione 0rf . 
for this category of patients , a reliable diagnostic modality is desirable , and ctca has i dati del registro mostrano valori di accuratezza diagnostica ottimali ed in linea con le principali casistiche di validazione in pazienti stabili [ 4144 ]  . 
in particolare la sensibilit ed il valore predittivo per paziente sono risultati del 100% e 100% , rispettivamente , con valori di speci cit e radiol med ( 2011 ) 116 : 10141026 1023 demonstrated signi cant potential in this group of patients [ 2 , 1733 ]  . registry data show optimal accuracy values in line with the major validation series [ 4144 ]  . 
the second is the rule out myocardial infarction using computer assisted tomography ( romicat ) trial [ 29 ] , in which sensitivity and npv of ctca in acs were 100% and 100% , respectively , in 363 patients . 
the authors also suggest that the use of ctca in this category of patients could signi cantly reduce costs [ 25 ]  . evidence in the literature and data from our registry show that in cases of chest pain with suspected coronary origin ( nstemi - acs ) , ctca is able to provide adequate diagnostic accuracy , particularly for ruling out disease . 
 the costbene t ratio must take into account numerous factors , including biological cost ( dose of ionising radiation compared with tests that do not use ionising radiation ) , economic cost ( with respect to less expensive tests ) , geographic distribution of the technique ( compared with more commonly available modalities ) and expertise ( which is still limited in some centres )  . study limitations our series reports data recorded in the period when the most modern dose - reduction systems were not widely available . 
 however , the introduction of new hardware and software able to provide adequate image quality using prospective triggering based on the electrocardiographic signal has brought dose values to < 5 msv [ 912 ]  . 
in questo trial randomizzato controllato la ctca stata confrontata con lo standard of care ( soc , vale a dire stress tomogra a computerizzata a emissione di fotoni singoli [ spect ] )  . 
 [ 24 ] effettuato su 104 pazienti con nstemi gli autori hanno rilevato unottima accuratezza diagnostica della ctca per la rilevazione ed esclusione delle stenosi coronariche critiche ( vale a dire , sensibilit e valore predittivo negativo pari al 100% )  . 
gli autori suggeriscono inoltre che lutilizzo della ctca in questa categoria di pazienti potrebbe portare ad una signi cativa riduzione dei costi [ 25 ]  . le evidenze della letteratura ed i dati del nostro registro evidenziano pertanto che in caso di dolore toracico di sospetta origine coronarica ( acs - nstemi ) la ctca in grado fornire adeguata performance diagnostica soprattutto per lesclusione della malattia . 
il rapporto costo / bene cio deve tenere conto di molti fattori tra cui il costo biologico ( dose di radiazioni rispetto a test che non necessitano di radiazioni ) , il costo economico ( rispetto a test con costi inferiori ) , la disponibilit territoriale della metodica ( rispetto a metodiche diagnostiche molto pi diffuse ) , lexpertise ( ancora limitato ad alcuni centri )  . limiti dello studio la nostra casistica riporta dati provenienti dal periodo nel quale le soluzioni pi moderne per la riduzione della dose non erano largamente disponibili . 
tuttavia , lintroduzione di nuovi hardware e software in grado di fornire adeguata qualit di immagine utilizzando il triggering prospettico basato sul segnale ecg consentono di portare i valori di dose al di sotto dei 5 msv [ 912 ]  . 
una limitazione dello studio deriva dal design del registro che non offre le stesse caratte1024 radiol med ( 2011 ) 116 : 10141026 the study can be found in the registry design , which does not offer the same characteristics of a trial or a prospective study . 
tuttavia , questa anche la forza delle informazioni presentate in quanto pi realistiche rispetto agli studi convenzionali in condizioni ottimali ( per esempio , pazienti ben selezionati , possibilit di valutazione delle immagini estesa nel tempo , ecc )  . conclusioni conclusions data in our registry demonstrate that ctca is a robust and reliable morphological modality for evaluating obstructive cad in patients with acs and without stemi . 
the intrinsic registry design tends to better re ect clinical practice and its characteristic variability and heterogeneity . i dati del nostro registro dimostrano che la ctca una metodica morfologica per la valutazione dellaterosclerosi coronarica ostruttiva nel contesto della sindrome coronarica acuta senza sopra - elevazione del tratto st robusta ed af dabile . 
grassi1 1sezione di radiologia , dipartimento magrassi - lanzara , seconda universit di napoli , naples , italy 2ii clinica neurologica , seconda universit di napoli , naples , italy 3dipartimento di gerontologia , geriatria e malattie del metabolismo , seconda universit di napoli , naples , italy correspondence to : a . 
in 37 patients , contrast agent transport was preserved and safe ; in 19 , we observed penetration of the contrast agent into the laryngeal inlet without aspiration ; in 24 , there was aspiration ( four preswallowing , eight intraswallowing , nine postswallowing , three mixed ) , whereas in one patient no transit was seen . 
penetration without aspiration was resolved by coughing or throat clearing ; aspiration was resolved in 13 patients by assuming the chin - tuck posture and in six by rotating the head ; in ve patients , it was not resolved . 
in caso di transito alterato , penetrazione o aspirazione del mezzo di contrasto ( mdc ) nelle vie aeree , sono state ipotizzate e veri cate durante lesame vfm posture di compenso per la correzione del disturbo deglutitorio . 
in 37 pazienti il transito del mdc era conservato e sicuro , in 19 abbiamo osservato penetrazione del mdc in aditus laringeo senza aspirazione , in 24 aspirazione ( 4 pre - deglutitoria , 8 intra - deglutitoria , 9 post - deglutitoria , 3 mista ) , in 1 non era presente transito . 
la penetrazione senza aspirazione stata risolta con un colpo di tosse o col raclage sostenuto ; laspirazione stata risolta in 13 pazienti con la postura a capo esso , in 6 col capo ruotato , in 5 non stata risolta . 
la vfm , correlando il dato morfologico a quello funzionale , permette di caratterizzare con precisione il disturbo disfagico ed ipotizzare le posture di compenso pi idonee al singolo caso e veri carne lef cacia . 1084 radiol med ( 2011 ) 116 : 10831094 parole chiave video uoromanometria disfagia posture di compenso sclerosi laterale amiotro ca the dysphagic disorder but also to identify the most appropriate compensation posture for each patient and verify its effectiveness . keywords video uoromanometry dysphagia compensatory postures amyotrophic lateral sclerosis introduction introduzione amyotrophic lateral sclerosis ( als ) , or lou gehrigs disease , is a neurodegenerative disease characterised by progressive muscular paralysis re ecting degeneration of motor neurons in the primary motor cortex , brainstem and spinal cord [ 1 ]  . 
in patients with als , dysphagia is caused by degeneration of bulbar motor neurons and leads to an increased risk of aspiration , weight loss , malnutrition and dehydration the latter being also favoured by upper - limb weakness and greater dif culties in meal preparation [ 3 ]  . the rst clinical manifestations of the disease appear when progressive motor neuron loss exceeds the ability of surviving motor neurons to compensate , thus leading to progressive paralysis , with preservation of the cognitive , sensory , sexual and sphincter functions . 
the prognosis is poor for both forms , leading to death by respiratory failure , aspiration pneumonia or malnourishment and dehydration within 23 years for bulbar and 35 years for spinal als [ 4 ]  . early recognition of dysphagia and , in particular , of the different forms of aspiration into the airways , makes it possible to identify and assume compensation postures to improve swallowing and to make appropriate dietary changes to preserve oral feeding and postpone percutaneous endoscopic gastrostomy ( peg ) as long as possible [ 5 , 6 ]  . 
correct diagnostic assessment of dysphagia in patients with als requires a multidisciplinary approach with close collaboration of the neurologist , the speech therapist , the gastroenterologist and the radiologist [ 7 , 8 ]  . evaluation of swallowing by video uoroscopy ( vfs ) represents the gold standard in patients with neurological disorders and enables accurate analysis of the mechanisms involved in the oral and pharyngeal phases of deglutition [ 911 ]  . 
thanks to the simultaneous acquisition of video uoroscopic and manometric data , video uoromanomla sclerosi laterale amiotro ca ( sla ) o malattia di lou gehrig una malattia neurodegenerativa caratterizzata dalla progressiva paralisi muscolare che ri ette la degenerazione dei neuroni motori della corteccia motoria primaria , del tronco encefalico e del midollo spinale [ 1 ]  . 
let media di insorgenza della sla di circa 60 anni con una leggera prevalenza nel sesso maschile ( maschi : femmine1 , 5 : 1 ) [ 2 ]  . 
 la disfagia , nei pazienti con sla , causata dalla degenerazione dei neuroni motori bulbari e determina un aumento del rischio di aspirazione , perdita di peso , malnutrizione e disidratazione , queste ultime favorite anche dalla severa debolezza degli arti superiori e le conseguenti dif colt nella preparazione dei pasti [ 3 ]  . le prime manifestazioni cliniche della malattia compaiono quando la perdita progressiva dei motoneuroni supera la capacit di compenso dei motoneuroni superstiti no ad arrivare ad una progressiva paralisi , con risparmio delle funzioni cognitive , sensoriali , sessuali e s nteriali . 
in entrambe le forme la prognosi infausta conducendo a morte per insuf cienza respiratoria , polmonite ab ingestis o per un grave stato di denutrizione e disidratazione entro 23 anni per i casi di sla bulbare e 35 anni per i casi di sla spinale [ 4 ]  . riconoscere precocemente il disturbo disfagico e , soprattutto , le diverse tipologie di aspirazione nelle vie aeree , consente di ricercare ed adottare posture di compenso della deglutizione e modi care adeguatamente la dieta al ne di mantenere il pi a lungo possibile lalimentazione orale e ritardare il ricorso alla gastrostomia endoscopica percutanea ( peg ) [ 5 , 6 ]  . 
il corretto inquadramento diagnostico della disfagia nei pazienti con sla necessita di un approccio multidisciplinare con stretta sinergia tra neurologo , logopedista , gastroenterologo e radiologo [ 7 , 8 ]  . lo studio della deglutizione mediante video uoroscopia ( vfs ) rappresenta il gold standard nei pazienti affetti da malattie neurologiche e permette unaccurata analisi dei meccanismi della fase orale e faringea della deglutizione [ 911 ]  . 
limpiego della video uromanometria ( vfm ) , grazie allacquisizione contemporanea dei dati video uoradiol med ( 2011 ) 116 : 10831094 1085 etry ( vfm ) allows for a more precise determination of the causes and effects of the pharyngeal dysfunction [ 12 , 13 ] , which cannot be evaluated by using the two techniques separately [ 12 , 14 ] , and it is particularly useful for treatment planning [ 15 , 16 ]  . in the literature , several compensation postures have been described that facilitate food transit and prevent aspiration . 
the purpose of our study was to verify the effectiveness of compensation postures , suggested on the basis of the type of dysphagia identi ed at vfm examination , in ensuring safe oropharyngeal transit . roscopici e manometrici , consente una pi precisa determinazione delle cause e degli effetti della disfunzione faringea [ 12 , 13 ] , non altrimenti valutabili con le singole indagini di cui si compone [ 12 , 14 ] , ed utile soprattutto per la successiva piani cazione terapeutica [ 15 , 16 ]  . in letteratura sono state descritte posture di compenso atte a favorire il transito alimentare e prevenire laspirazione ; scopo del nostro lavoro veri care lef cacia di tali posture di compenso ipotizzate in base alle caratteristiche del disturbo disfagico individuato con vfm , per garantire il transito orofaringeo in sicurezza . materials and methods materiali e metodi eighty - one consecutive patients with als admitted to the department of neurology from january to december 2008 were included in the study . 
all patients underwent speech therapy evaluation according to the most recent guidelines [ 17 , 18 ] , with assessment of clinical history , feeding status , behavioural features , symptoms and problems reported by patients , morphological and functional evaluation and diagnostic summary . 
for this combined study , the dyno compact system ( men s biomedica s.r.l. , bologna , italy ) was used equipped with : ( 1 ) graphics card for to manage ultrasound or radiographic images ; ( 2 ) avius dedicated software package , which enables digital - quality recording ( pal / ntsc , composite video or s - video ) of the vfs study in avi format with 320 240 resolution and 25 hz acquisition frequency ; the delay introduced by the process of image digitalisation is in the order of 200 ms , so for the purposes of analysis , images can be considered synchronised with the manometric recordings . manometry was performed after positioning a manometry probe with ve - channel endoluminal solid - state microtransducers 2 cm apart and with an angle of 120 90 . 
then , a pull - through was performed by retracting the probe until the last transducer was positioned at the level of the upper esophageal sphincter ( ues ) , three inside the pharynx and the rst at the level of passavants ridge . during data acquisition , video images and manometric traces are visualised in real time , with a sliding cursor showing the exact correspondence between theat the end of the examination , video images are analysed either in real time , at increased or reduced speed or frame by frame . 
tutti i pazienti sono stati sottoposti ad un protocollo di valutazione logopedica in sintonia con le pi recenti linee guida [ 17 , 18 ] , corredato cio da anamnesi , stato alimentare , caratteristiche comportamentali , sintomatologia e dif colt riferite dal paziente , valutazione morfofunzionale e sintesi diagnostica . la vfm prevede lesecuzione contemporanea della vfs con la simultanea registrazione di un tracciato manometrico . 
software dedicato , che registra in qualit digitale ( pal / ntsc , video composito o s - video ) la vfs , in lmati avi con risoluzione 320240 e con frequenza di acquisizione di 25 hz ; il ritardo introdotto dal processo di digitalizzazione dellimmagine dellordine dei 200 ms , quindi , ai ni dellanalisi , limmagine si pu considerare sincronizzata con i tracciati pressori . 
 la manometria stata effettuata dopo il posizionamento di un sondino manometrico con microtrasduttori endoluminali allo stato solido a 5 canali , distanziati 2 cm luno dallaltro e con un angolo di 12090 . 
 successivamente stato eseguito un pull - trough ritirando il sondino no a posizionare lultimo trasduttore a livello dello s ntere esofageo superiore ( ses ) , tre in faringe , il primo a livello del cuscinetto del passavant . durante la fase di acquisizione dei dati i lmati e le tracce manometriche sono visualizzate sullo schermo in tempo - reale : un cursore che scorre su di essi mostra lesatta corrispondenza tra il lmato ed i tracciati . 
gli esami sono stati acquisiti con paziente in ortostatismo , salvo i casi in cui la compromissione della funzione statica dei pazienti ha reso necessaria lacquisizione dellesame in posizione assisa . 
lesame 1086 radiol med ( 2011 ) 116 : 10831094 vfm study began with a baseline , unenhanced acquisition to study vocal cord motility and soft palate ; baseline acquisition was followed by a contrast - enhanced scan to evaluate the entire swallowing act , with special attention being paid to aspiration . 
we administered a single bolus of barium ( a pronto bario hd suspension , bracco spa , milan , italy , 250% w / v ) at a dose ranging from 5 to 15 ml optimised for patient status . 
 in the event of altered bolus transit or of bolus penetration or aspiration into the airways , some manoeuvres to correct the swallowing disorder were hypothesised and tested at vfm . 
although indicated to improve bolus transport in the presence of pharyngeal dysfunction [ 19 ] , praxic alterations in als patients of the oral structures involved in bolus retention and swallowing , in particular the tongue [ 20 ] , preclude the application of this manoeuvre . 
it consists of voluntary manual prolongation of laryngeal excursion during swallowing , which is intended to increase the extent of laryngeal elevation and duration of ues opening , thus favouring a safer transit of the food bolus . 
although its use is recommended in cases of ues dysfunction [ 21 ] , the frequent strength de cit associated with atrophy of the distal upper extremities or severe cramps and muscular damage to the upper limbs prevent its use [ 22 ]  . the supraglottic and super - supraglottic swallows are also not applicable to als patients : the former consists of breath - holding during the entire swallowing act and in producing a cough at the end of the swallowing act before starting the next inspiration ; the latter is a variation of the supraglottic swallow in which full closure of the glottis is achieved by contracting the diaphragm muscles [ 23 ]  . 
thus the corrective techniques used in this study to favour adequate and safe bolus transit were the following : chin - down , or chin - tuck , posture , suggested in patients with dif cult bolus management with possible fall below the tongue base or delayed pharyngeal re ex or with pooling of contrast material inside the pharynx [ 24 ] because of the airway protection provided by changes in pharyngeal dimensions [ 25 ] ; head - rotation posture in the presence of failed / reduced vfm iniziava con una valutazione senza mezzo di contrasto ( mdc ) per lo studio della motilit delle corde vocali e la motilit del palato molle ; poi si procedeva con la fase contrastogra ca che valuta tutto latto deglutitorio con particolare attenzione allaspirazione . 
abbiamo somministrato una dose singola di mdc baritato ( una sospensione di prontobario hd , bracco spa , milano ; 250% p / v ) , la cui quantit compresa tra 5 e 15 ml stata ottimizzata in base al paziente . 
 in caso di transito alterato del mdc , di penetrazione o di aspirazione dello stesso nelle vie aeree , sono state ipotizzate e veri cate durante lesame vfm metodiche rimediative per la correzione del disturbo deglutitorio . 
tra le manovre di compenso descritte in letteratura alcune non sono direttamente utilizzabili in caso di sla : la manovra di deglutizione forzata , che consiste nella spinta forzata e prolungata della lingua contro il palato durante latto deglutitorio , indicata per migliorare il trasporto del bolo , in presenza di disfunzioni faringee [ 19 ] , ma non applicabile in questi pazienti perch le alterazioni prassiche delle strutture orali preposte al contenimento e alla deglutizione del bolo , in maggioranza riferibili al distretto linguale [ 20 ] , non consentono di mettere in atto tale manovra . 
anche la manovra di mendelsohn non utilizzabile dai soggetti affetti da sla : consiste nel volontario prolungamento manuale dellelevazione laringea durante la deglutizione che porta ad aumento dellestensione dellelevazione laringea e della durata dellapertura del ses , favorendo un pi sicuro transito del bolo alimentare ; consigliata nei casi di disfunzione dello ses [ 21 ] , ma i frequenti de cit di forza associata ad atro a delle estremit distali superiori , o crampi e danni muscolari severi degli arti superiori ne impediscono lutilizzo [ 22 ]  . non trovano applicazione nei pazienti con sla anche le manovre di deglutizione sopraglottica e supersopraglottica : la prima consiste nel mantenere lapnea durante tutta la deglutizione ed effettuare un colpo di tosse a conclusione dellatto deglutitorio , prima della successiva inspirazione ; la seconda una variante della prima , nella quale si richiede una piena chiusura s nterica della glottide attraverso la contrazione dei muscoli diaframmatici [ 23 ] ; entrambe richiedono un breve training per la loro precisa messa in atto e vengono impiegate nei casi di disfagia secondaria a chirurgia del distretto laringeo . 
in questo studio , pertanto , le ipotesi rimediative impiegate per favorire un transito adeguato del mdc ed in sicurezza sono state : la postura del capo esso , o chin down o chin tuck , suggerita nei casi di dif colt nella gestione del bolo con possibile caduta al basi - lingua o di ritardo nellelicitazione del ri esso faringeo di deglutizione , o di ristagni di mdc in faringe [ 24 ] , per leffetto di protezione fornito alle vie aeree in relazione ai cambiamenti delle dimensioni faringee [ 25 ] ; radiol med ( 2011 ) 116 : 10831094 1087 ues opening , as this produces a fall in ues pressure and delays its closure during the swallowing act [ 26 ] ; hyperextended head posture , which facilitates bolus transit by exploiting gravity ; it is indicated in cases of reduced pharyngeal peristalsis [ 5 ]  . 
 la postura del capo ruotato in presenza di mancata / ridotta apertura del ses , poich produce la caduta pressoria del ses e ne ritarda la chiusura durante latto deglutitorio [ 26 ] ; la postura del capo in iperestensione che facilita il passaggio del bolo sfruttando la forza di gravit ed indicata nei casi di ridotta peristalsi faringea [ 5 ]  . 
 results clinical history and morphological and functional speech therapy assessment risultati of the 81 patients undergoing clinical history speech therapy assessment , 14 ( 17.5% ) reported no swallowing disorders and 67 ( 82% ) reported dysphagia . 
of the six patients with dysphagia for solid food , one showed alterations only during the oral phase of swallowing and ve only during the pharyngeal phase ; the 11 patients with dysphagia for liquids only reported a sensation of liquid bolus going down the wrong way . risultati dellanamnesi e della valutazione morfofunzionale logopedica degli 81 pazienti sottoposti allanamnesi logopedia , 14 ( 17 , 5% ) non riferivano disturbi della deglutizione e 67 ( 82% ) disfagia . 
tra questi ultimi , 6 ( 9% ) presentavano disfagia per solidi , 11 ( 15% ) disfagia per liquidi , e 50 ( 75 , 7% ) riferivano disturbi nella deglutizione di entrambe le consistenze . 
tra i 6 casi di disfagia per solo bolo solido , 1 caso presentava alterazione della sola fase buccale e 5 della sola fase faringea ; gli 11 pazienti che presentavano disfagia per i soli liquidi lamentavano la sensazione del bolo liquido che va di traverso . vfm examination risultati dellesame vfm in 37 of the 81 patients studied , contrast agent transit was preserved and safe . 
in 19 cases ( 23% ) , we observed bolus penetration into the laryngeal lumen only , without aspiration below the glottic level , whereas in 24 ( 34% ) , aspiration was observed . 
of the 39 patients with suspected aspiration at speech therapy assessment , 18 were con rmed at vfm , 13 presented with penetration without aspiration and eight were false positives . 
 vfm showed that in the 24 patients with aspiration , this occurred in the preswallowing phase in four ( 17% ) cases , in the intraswallowing phase in eight ( 30% ) and in the postswallowing phase in nine ( 39% ) ; multiple swallowing disorders were seen in three ( 13% ) , with the concurrent presence of several types of aspiration , which we de ned as mixed aspiration . 
 intraswallowing aspiration was related to pharyngeal hypertonicity in 7 / 8 cases ( 87% ) , accompanied by reduced laryngeal elevation and / or closure in three patients and in four cases by ues dysfunction ; the latter was observed degli 81 pazienti in esame , in 37 il transito del mdc era conservato e sicuro . 
in 19 casi ( 23% ) si osservata la sola penetrazione del mdc nel lume laringeo senza aspirazione dello stesso al di sotto del piano glottico ed in 24 ( 34% ) aspirazione ; tra questi , 6 ( 25% ) non presentavano ri esso tussigeno spontaneo . 
dei 39 soggetti con sospetto di aspirazione alla valutazione logopedica , in 18 stata confermata alla vfm , 13 presentavano penetrazione senza aspirazione e 8 sono risultati dei falsi positivi . alla vfm , nei 24 pazienti con aspirazione , in 4 ( 17% ) si evidenziata aspirazione pre - deglutitoria , in 8 ( 30% ) intradeglutitoria , in 9 ( 39% ) post - deglutitoria ed in 3 ( 13% ) erano evidenti disturbi multipli della deglutizione associati alla presenza contemporanea di diverse tipologie di aspirazione , che per comodit di esposizione abbiamo de nito aspirazione mista . 
laspirazione intra - deglutitoria in 7 casi su 8 ( 87% ) avvenuta in relazione ad ipertono faringeo , accompagnato in 3 soggetti , da ridotta elevazione e / o chiusura laringea e , in 4 casi , da disturbi dello ses ; questi ultimi si sono manifestati in 1 caso con chiusure anticipate e nei restanti 3 con rilasciamenti incompleti . 
study population : january - december 2008 ( 81 patients ) chin tuck compensation postures no compensation head rotation head hyperextension preserved and safe transit penetration without aspiration preswallowing aspiration intraswallowing aspiration postswallowing aspiration mixed aspiration no transit total transito conservato e sicuro penetrazione senza aspirazione aspirazione pre - deglutitoria aspirazione intra - deglutitoria aspirazione post - deglutitoria mista assenza di transito totale 81 tabella 1 correlazione tra disturbo disfagico valutato alla vfm ed ef cacia delle posture di compenso . 
casistica personale : gennaio - dicembre 2008 ( 81 pazienti ) capo esso posture di compenso non compenso capo ruotato capo iperesteso in one case with premature closure and in the remaining three cases with incomplete release . 
in the nine cases of postswallowing aspiration , the bolus was inhaled following contrast - agent pooling inside the pharynx : in two cases , this occurred at the level of the pyriform sinuses , in two at the level of the epiglottic valleculae and in ve at both levels . 
in one patient , the problem was not solved . mixed aspiration was seen in one bedridden patient with hyperextended head and unable to change posture and in two with multiple dysfunctions of the different swallowing phases associated with severe oropharyngeal incoordination . 
nei 9 casi di aspirazione post - deglutitoria il bolo stato inalato in seguito a ristagno di mdc in faringe : in 2 casi si apprezzava a livello dei seni piriformi , in 2 a livello delle vallecole glosso - epiglottiche e in 5 a livello di entrambe le sedi . 
dal punto di vista manometrico in 1 paziente era presente rilasciamento incompleto e in 2 ipotono dello ses , in 2 pazienti era presente ipertono dello ses , negli altri 4 non erano presenti alterazioni . 
 there are 5 endoluminal microtransducers , 2 cm apart from each other , the rst is located at the level of passavants ridge , three inside the pharynx , the last at the level of the upper esophageal sphincter ( ues )  . 
sono presenti 5 microtrasduttori endoluminali , distanziati 2 cm luno dallaltro , il primo posizionato a livello del cuscinetto del passavant , tre in faringe , lultimo a livello dello s ntere esofageo superiore ( ses )  . 
 the second part of the vfm examination , in which the compensatory postures were applied , led to several considerations : in the case of preswallowing aspiration , generally resulting from abnormalities of the oral and propulsive phases , we agree with previous reports that suggest that i risultati dimostrano che i de cit deglutitori in pazienti affetti da sla sono diversi per meccanismo compromesso , in relazione alle caratteristiche della patologia e del decorso . 
la seconda parte dellesame vfm , dove si resa necessaria lapplicazione delle posture di compenso , ha prodotto levidenza di varie considerazioni : nei casi di aspirazione pre - deglutitoria , in genere provocate da alterazioni delle fasi buccali e propulsive , concordiamo con la letteratura per lopportunit di impiegare la postura di compenso del capo esso , che ha radiol med ( 2011 ) 116 : 10831094 1090 fig . 
la base della lingua copre laditus laringeo prevenendo la penetrazione del mdc in laringe . the chin - tuck posture is appropriate , as this resolved the problem in 100% of cases ; in cases of intraswallowing aspiration , the chin - tuck position was effective in patients with reduced laryngeal elevation / closure only , in whom it eliminated aspiration in all cases . 
conversely , head rotation can be more rationally suggested in case of intraswallowing aspiration secondary to cricopharyngeal dysfunction , as it reduces ues tone and delays closure [ 26 ]  . 
in the last case , no satisfactory result was obtained , possibly due to massive intraswallowing aspiration ; postswallowing aspiration occurred in nine patients as a result of penetration into the laryngeal vestibule and pooling inside the epiglottic valleculae and / or the pyriform sinuses . 
five patients bene ted from the chintuck posture [ 24 ] , two of whom had hypotonic ues ; in consentito la risoluzione del disturbo nel 100% dei casi ; nei casi di aspirazione intra - deglutitoria la postura del capo esso si dimostrata ef cace solo in quei soggetti che presentavano ridotta elevazione / chiusura laringea ; ci ha consentito di risolvere laspirazione in tutti i casi che presentavano questo disturbo . 
il suggerimento di rotazione del capo trova , viceversa , unapplicazione pi razionale nei casi di aspirazione intradeglutitoria secondaria a disfunzioni cricofaringee , consigliata per ridurre il tono dello ses e ritardarne la chiusura [ 26 ]  . 
nellultimo caso non si sono ottenuti risultati soddisfacenti presumibilmente per la massiccia aspirazione intradeglutitoria presentata dal paziente ; laspirazione post - deglutitoria nei 9 pazienti si vericata in correlazione a penetrazione nel vestibolo laringeo e stasi nelle vallecole glosso epiglottiche e / o nei seni piriformi . 
il transito avviene senza aspirazione nonostante il passaggio del bolo in faringe non innescchi la peristalsi . contrast , the patient with incomplete release and the two patients with hypertonic ues bene ted from head rotation [ 26 ]  . 
there was no improvement in one case only as a result of massive inhalation of contrast material into the airways ; patients with mixed aspiration were at an advanced stage of disease , with swallowing incoordination and poor cooperation ; in one case only was aspiration reduced with the chin - tuck posture . 
for these patients , compensation postures are known to be of limited bene t [ 27 ] ; in cases of severe swallowing alterations owing to absent lingual pump , the hyperextended head position restored bolus transport [ 5 ] , and vfm validated its safety : although bolus passage failed to trigger the swallowing re ex and pharyngeal peristalsis was uncoordinated with bolus passage , the patient showed no aspiration . postura a capo esso [ 24 ] , tra questi i 2 con ipotono dello ses ; il paziente con rilasciamento incompleto ed i 2 pazienti con ipertono dello ses hanno invece bene ciato del capo ruotato [ 26 ]  . 
non abbiamo ottenuto alcun miglioramento in 1 solo caso in relazione ad inalazione massiva del mdc nelle vie aeree ; i pazienti con aspirazione mista presentavano uno stadio avanzato di patologia con incoordinazione deglutitoria e scarsa collaborazione ; solo in 1 abbiamo ottenuto una riduzione dellaspirazione con capo esso . 
per questi soggetti il bene cio delle metodiche rimediative noto essere molto ridotto [ 27 ] ; nel caso di grave alterazione dellatto deglutitorio per assenza della pompa linguale , la postura del capo in iperestensione ha consentito di ripristinare il transito del mdc [ 5 ] e la vfm ha permesso di veri carne la sicurezza : nonostante il passaggio del bolo non inneschi il ri esso deglutitorio e la peristalsi faringea fosse incoordinata rispetto al passaggio del bolo il soggetto non 1092 radiol med ( 2011 ) 116 : 10831094 separate mention must be made of silent aspiration and penetration without aspiration . 
silent aspiration refers to the passage of ingested material below the vocal cords in the absence of protective cough , and it is estimated to occur in about half of the patients with aspiration [ 28 ]  . 
 in our patient population , it occurred in six cases , that is , in 25% of patients with aspiration three of them with intraand three with postswallowing aspiration with no apparent correlation explaining the absence of protective cough . 
this phenomenon should not be underestimated [ 29 ] owing to the possible associated complications : aspiration pneumonia is three times more frequent in dysphagic than in nondysphagic individuals [ 30 ] and represents the most common cause of death in patients with neurological diseases associated with dysphagia [ 31 ]  . in our series , penetration of contrast material into the laryngeal vestibule occurred in 19 patients ( 23% ) , whereas in the healthy population aged > 50 years , bolus passage into the larynx occurs in 16.8% of swallows [ 32 ]  . 
in patients with als , the need to move the material away from the laryngeal vestibule arises from the consideration that the material may be directly inhaled into the airways at the end of the swallowing act , in particular in the case of deep penetration . 
 conclusions the variety of dysphagic disorders observed in our population of patients with als con rms the need for a complete and individual evaluation allowing identi cation of compensation strategies . 
this type of workup , which involves the collaboration of several different healthcare professionals ( neurologist , speech and language therapist , gastroenterologist , radiologist ) , facilitates the diagnosis and promotes appropriate management according to the needs of the individual patient , taking into consideration that the main feature of als rehabilitation is represented by remediation rather than rehabilitation programmes . in our population of als patients , the alterations observed required only three possible compensation postures : chin tuck , head rotation , and hyperextended head . 
the chin - tuck posture proved useful in the majority of cases , given that it offers a valuable protection mechanism for the airways by opening the valleculae and preventing penetration into the larynx ; above all , with this posture , the tongue base narrows and covers the laryngeal inlet . 
head rotation is indicated in the case of hypertonicity , incomplete release or premature ues closure , as it reduces the resting pressure of the sphincter and presentava aspirazione . due menzioni a parte meritano i fenomeni dellaspirazione silente e della penetrazione senza aspirazione . 
per aspirazione silente si intende il passaggio degli ingesti al di sotto delle corde vocali in assenza di tosse protettiva e si stima intorno alla met dei pazienti con aspirazione [ 28 ] ; nella nostra casistica esso riguarda 6 casi , ossia il 25% dei pazienti che presentavano aspirazione , 3 con aspirazione intrae 3 post - deglutitoria , senza alcuna apparente correlazione che giusti casse lassenza del ri esso tussigeno . 
 questo fenomeno un evento da non sottovalutare [ 29 ] per le possibili complicanze ad esso associate : la polmonite ab ingestis tre volte pi frequente nei soggetti disfagici rispetto ai non disfagici [ 30 ] e rappresenta la pi comune causa di morte nei pazienti con patologie neurologiche associate a disfagia [ 31 ]  . la sola penetrazione del mdc nel vestibolo laringeo nella nostra casistica si veri cata in 19 soggetti ( 23% ) mentre nella popolazione sana con pi di 50 anni , il passaggio del bolo in laringe avviene nel 16 , 8% delle deglutizioni [ 32 ]  . 
 nei pazienti con sla , la necessit di allontanare il materiale dal vestibolo laringeo nasce dalla considerazione che questultimo potr essere aspirato direttamente nelle vie aeree a conclusione dellatto deglutitorio , soprattutto se la penetrazione stata profonda . 
 conclusioni la variet di disturbi disfagici rilevati nella nostra casistica , nei pazienti con sla , ci induce a confermare la necessit di una valutazione individuale e completa , che risponda a requisiti di studio e suggerimenti rimediativi . 
 un percorso cos codi cato , integrato da pi professionalit ( neurologo , logopedista , gastrenterologo , radiologo ) , consente di promuovere una diagnosi e una gestione opportuna in base alle speci cit rilevate , tenendo conto che la principale caratteristica dellassetto riabilitativo nella sla rappresentata dalla metodologia strettamente rimediativa e non riabilitativa . nella sla le alterazioni riscontrate nella nostra casistica hanno condotto a sole 3 le posture di compenso possibili : il capo esso , il capo ruotato e il capo in iperestensione . 
la postura del capo esso si dimostrata utile nella maggior parte dei casi in quanto rappresenta un valido meccanismo di protezione delle vie aeree determinando lampliamento delle vallecole e prevenendo la penetrazione in laringe , ma soprattutto restringe e copre con la base della lingua laditus laringeo . 
la postura del capo ruotato indicata nei casi di ipertonia , rilasciamenti incompleti o chiusure anticipate del ses poich riduce la pressione a riposo dello radiol med ( 2011 ) 116 : 10831094 1093 increases its opening time by moving the cricoid cartilage away from the posterior pharyngeal wall . 
lastly , in the case of penetration without contrast agent aspiration into the laryngeal inlet , we suggest throat clearing every three to four swallowing acts to prevent possible postswallowing inhalation . in conclusion , because vfm enables identi cation and interpretation of alterations underlying swallowing disorders through correlation of morphological and functional data , it can help characterise dysphagic disorders , suggest the most suitable compensation posture for each patient and validate the effectiveness of such postures . s ntere e ne aumenta il tempo di apertura grazie allallontanamento della cartilagine cricoide dalla parete faringea posteriore ; la postura del capo in iperestensione indicata nei casi di assenza della pompa linguale solo se si dimostra che il transito avviene in modo sicuro . 
in ne , nei casi di penetrazione senza aspirazione del mdc in aditus laringeo riteniamo necessario suggerire un raclage sostenuto ogni 34 atti deglutitori , per evitare la possibilit di inalazione post - deglutitoria . 
guglielmi6 , 7 1human nutrition and physiology unit , university of rome tor vergata , via montpellier 1 , 00173 rome , italy 2imaging division , clinical department of radiological and histocytopathological sciences , university of bologna , santorsolamalpighi hospital , via massarenti 9 , 40138 bologna , italy 3department of orthopaedics and traumatology , university hospital policlinico tor vergata , viale oxford 81 , 00133 rome , italy 4endocrinology department , university hospital policlinico tor vergata , viale oxford 81 , 00133 rome , italy 5institute of medical informatics and biometry , university tor vergata , rome , italy 6department of radiology , university of foggia , viale luigi pinto 1 , 71100 foggia , italy 7department of radiology , hospital san giovanni rotondo , 71013 san giovanni rotondo , italy correspondence to : a . 
the knowledge of factors modulating the behaviour of bone mass is crucial for preventing and treating osteoporotic disease ; among these factors , body weight ( bw ) has been shown to be of primary importance in postmenopausal women . 
 our aim was to analyze the relationship between body mass index ( bmi ) , fat and lean mass and bone mineral density ( bmd ) in a large population of women . 
between 2005 and 2008 , weight and height of 6 , 249 italian women ( aged 3080 years ) were measured and bmi was calculated ; furthermore bmd , bone mineral content , fat and lean mass were measured by dual - energy x - ray absorptiometry . 
la conoscenza dei fattori che modulano il comportamento della massa ossea cruciale nella prevenzione e nel trattamento della malattia osteoporotica ; tra questi fattori , il peso corporeo ( bw ) ha dimostrato di essere di primaria importanza nelle donne in postmenopausa . 
il nostro obiettivo stato quello di analizzare la relazione tra lindice di massa corporea ( bmi ) , la massa grassa , la massa magra e la densit minerale ossea ( bmd ) in una vasta popolazione di donne . 
nel periodo compreso tra il 2005 e il 2008 sono stati rilevati peso e altezza di 6249 donne italiane ( et 4080 anni ) , ed stato calcolato il bmi ; inoltre sono stati misurati bmd , contenuto minerale osseo , massa grassa e massa magra attraverso assorbimetria a raggi x a doppia energia . 
an overall examination of our results suggests that both fat and lean body mass can in uence bone mass and that their relative effect on bone could be modulated by their absolute amount and ratio to total bw . 
unanalisi generale dei nostri risultati suggerisce quindi che sia la massa grassa sia la massa magra possono in uenzare la massa ossea e che gli effetti relativi sullosso possono essere modulati dalla loro quantit assoluta e dal rapporto sul peso corporeo complessivo . parole chiave osteoporosi composizione corporea indice di massa corporea densit minerale ossea densitometria ossea a raggi x a doppia energia fotone introduction introduzione osteoporosis is becoming increasingly prevalent with the aging of the world population . 
bmi is inversely related with the risk of osteoporotic hip fracture [ 3 ] , although its relationship with other limb fragility fractures is still debated [ 5 ]  . 
recently suggested that there are no bmi categories that protect against these fragility fractures compared with that of normal bw after adjusting for bone mineral density ( bmd ) and age [ 6 ]  . 
showed that traumatic forces increase with bw , but fracture rates at the hip and central body were less frequent with increasing a causa del progressivo invecchiamento della popolazione mondiale , losteoporosi sta diventando una patologia sempre pi diffusa e rappresenta un grave problema di sanit pubblica associata ad un incremento della mortalit e della morbilit [ 1 ]  . 
 lo studio epidemiologico sulla prevalenza dellosteoporosi in italia ha mostrato che la prevalenza allet di 60 anni al di sopra del 22 , 8% e del 14 , 5% , rispettivamente nelle donne e negli uomini ed responsabile di circa 80.000 nuove fratture ogni anno [ 2 ]  . 
 stata , inoltre , riscontrata una relazione tra le fratture osteoporotiche e lindice di massa corporea ( bmi ) , parametro biometrico espresso come rapporto tra peso ( body weight , bw ) e altezza [ 3 , 4 ]  . 
lindice di massa corporea risultato inversamente proporzionale al rischio di frattura osteoporotica del collo del femore [ 3 ] , anche se ancora oggetto di dibattito leventuale correlazione con altre fratture da fragilit degli arti [ 5 ]  . 
 [ 6 ] hanno recentemente suggerito che , dopo correzione per et e densit minerale ossea ( bone mineral density , bmd ) , non radiol med ( 2011 ) 116 : 11151123 1117 bmi , possibly due to greater soft - tissue padding . 
upper - extremity fractures showed no variation with bmi , and lower - extremity fracture rates were higher only in the overweight population ( bmi 2529.9 kg / m2 ) [ 7 ]  . several changes in body composition may occur with ageing , including reduced fat - free tissue mass and increased fat mass , leading to a higher percentage of body fat at any given bw in older adults . 
however , predictive methods have a relatively large error at an individual level , and thus , if individuals must be classi ed into categories , misclassi cation can occur . 
this study also represents a rst important report on reference standard values for body composition in italian women . materials and methods study participants in a cross - section study , we retrospectively analyzed data of 6 , 249 italian women ranging in age from 30 to 80 years who participated in ongoing studies on body composition and / or energy metabolism at our university from 2005 to mid - 2008 . 
participants were categorised in bmi groups according to world health organisation ( who ) criteria : normal weight , bmi < 24.9 kg / m2 ; overweight , bmi > 25 < 29.9 kg / m2 ; obesity , bmi > 30 kg / m2 . 
in addition , they were classi ed into categories according to body fat percentage ( bf% ) : normal bf% , < 35% in women ; high bf > 35% in women ; very high bf% , > 40% in women . 
 [ 7 ] hanno dimostrato che le forze traumatiche incrementano con laumentare del peso corporeo , ma la percentuale di fratture del rachide e del collo femorale risulta inferiore , probabilmente a causa dellaumento del tessuto adiposo . 
 le fratture alle estremit superiori non hanno evidenziato variazioni rispetto al bmi , mentre le percentuali di fratture alle estremit inferiori sono risultate maggiori solo nei soggetti in sovrappeso ( bmi 2529 , 9 kg / m2 ) [ 7 ]  . linvecchiamento pu comportare molteplici cambiamenti nella composizione corporea ( body composition , bc ) , in particolare pu veri carsi una riduzione della quota di massa magra e un incremento di quella grassa . 
comunque , i metodi predittivi hanno un errore relativamente ampio a livello individuale perci , se i soggetti devono essere inseriti in categorie , pu veri carsi unerrata classi cazione . lo scopo di questo studio stato sia quello di valutare la relazione tra bmi , bc e bmd sia quello di determinare la prevalenza dellosteopenia e dellosteoporosi nella popolazione femminile italiana . 
questo studio rappresenta anche il primo importante contributo scienti co relativo ai valori standard di riferimento per la composizione corporea delle donne italiane . materiali e metodi soggetti in questo studio trasversale abbiamo analizzato retrospettivamente i dati di 6249 donne italiane di et compresa tra 30 e 80 anni . 
the scanner was calibrated daily against the standard calibration block supplied by the manufacturer to control for possible baseline dri dxa measured total bmd and bmc with a precision ( coef cient of variation ) of 0.7%. 
 the diagnosis of osteoporosis was done according to t - score values and diagnosis by who as showed in table 1 . statistical analysis all data analysis was performed using the statistical package for the social sciences windows , version 15.0 ( spss , chicago , il , usa )  . 
i soggetti sono stati inseriti in gruppi di bmi , secondo i criteri dellorganizzazione mondiale della sanit ( who ) : peso normale , bmi < 24 , 9 kg / m2 ; sovrappeso , 25 < bmi < 29 , 9 kg / m2 ; obesit , bmi > 30 kg / m2 . 
inoltre sono stati classi cati in categorie secondo la percentuale di grasso corporeo ( body fat , bf ) nel sesso femminile ( normale bf% , < 35% ; elevata bf% , > 35% ; molto elevata bf% , > 40% )  . 
secondo i criteri who lobesit nei soggetti giovani e di mezza et corrisponde a valori percentuali di bf superiori al 35% nelle donne [ 8 ]  . bmd , contenuto minerale osseo e misurazioni della composizione corporea le misurazioni del bmd , del contenuto minerale osseo ( bmc ) , della massa grassa e di quella magra sono state effettuate in tutte le sedi corporee , utilizzando la tecnica assorbimetrica a raggi x a doppia energia ( dxa , hologic qdr 4500a , hologic inc . , waltham , ma , usa )  . 
la diagnosi di osteoporosi stata effettuata secondo i criteri ed i valori di t - score della who espressi in tabella 1 . analisi statistica lanalisi di tutti i dati stata ottenuta utilizzando il programma statistico social science windows versione 15.0 ( spss , chicago , il , usa )  . 
 le differenze tra le decadi di et per tutte le variabili sono state calcolate usando unanalisi della varianza ( anova ) a una variabile ( one - way ) e la comparazione multipla del test bonferroni . 
vi stato un incremento del bmi in relazione allet , in particolare stato osservato un signi cativo aumento dellobesit ( p < 0 , 001 ) dalla classe 3 di et ( 5059 anni ) alla classe 5 ( 7080 anni )  . 
hence , it clearly appears to be important to determine standard references for body composition values , especially with techniques that will play a prominent role in the next clinical generation . 
the collection and analysis of references for body composition in men and women will be dif cult , as differences in fat mass and lean mass may be marked between countries and populaquanto sia cruciale acquisire dei dati normali di riferimento sulla bmd considerando le varie tecniche utilizzate nella diagnosi della malattia . 
infatti , sono stati raccolti valori standard di riferimento per la dxa , ma anche per la quantitative computed tomography ( qct ) e per la peripheralqct ( pqct ) [ 911 ] ; risaputo quanto questi valori siano essenziali per lapplicazione dei metodi di imaging nella diagnosi delle malattie del metabolismo osseo . 
di conseguenza appare importante determinare gli standard di riferimento per i valori di bc , specialmente per le tecniche che acquisiranno un ruolo prominente nella pratica clinica in un prossimo futuro . 
la raccolta e lanalisi dei dati di riferimento per la bc nei maschi e nelle femmine sar un compito dif cile a causa delle differenze nella massa grassa e muscolare riscontrabili tra paesi e popolazioni differenti tra loro . 
therefore , a national normative database is de nitely necessary on a country basis to provide the speci c phenotype of the population in whole - body dxa analysis of body composition . 
 some authors have performed research on the relationships between bone mass and bw or bmi [ 13 , 14 ] and between bone mass and nonbone mass [ 1517 ]  . 
 the social problem of obesity is becoming better understood , but the endocrine function and the role of adipose tissue is still one of the main subjects of study and debate . 
 hence , body composition analysis may provide an important contribution to understanding how fat is fat and the function and physiopathology of adipose tissue [ 18 , 19 ]  . 
an overall examination of our results therefore suggests that both fat and lean body mass can in uence bone mass and that their relative effect on bone could be modulated by their absolute amount and ratio to total body mass weight . 
an important building block in the reference values of body composition by dxa in italian women has now been laid . per fornire il fenotipo speci co della popolazione in base allanalisi whole - body della bc ottenuta con dxa . al giorno doggi , alcuni autori hanno pubblicato ricerche sulle relazioni tra tessuto osseo e bw o bmi [ 13 , 14 ] , e tra tessuto osseo e parti molli [ 1517 ]  . 
sar una s da prevedere correlazioni signi cative tra i valori di bc e i rischi o i fattori prognostici per un ampio spettro di malattie . il problema sociale dellobesit sta diventando man mano sempre pi conosciuto , ma la funzione endocrina e il ruolo del tessuto adiposo sono ancora oggetto di studio e di dibattito . 
quindi , la bc pu fornire un contributo importante per capire la percentuale di massa grassa , la funzione e la siopatologia del tessuto adiposo [ 18 , 19 ]  . 
una valutazione complessiva dei nostri risultati suggerisce , pertanto , che sia la massa grassa che quella muscolare possono in uenzare il tessuto osseo , e che il loro effetto sul tessuto osseo potrebbe essere modulato dalla loro somma e dalla percentuale rispetto al peso della massa corporea . 
improperly taken x - rays do not help at all in providing clues for a proper diagnosis . this said , a misdiagnosis today could have serious consequences in the short term ; the same child could be referred not long after with more severe lesions , even fatal ones . the book is divided into two sections : section a ( image reproduction , display and interpretation in child abuse ) addresses in one chapter what is de ned as evidence - based radiology in child abuse ; section b ( skeletal injuries in child abuse ) is divided into ve sub - sections addressing axial skeleton ( 3 chapters ) , appendicular skeleton and viscera ( 3 chapters ) , differential diagnoses ( 2 chapters ) , dating fractures ( 1 chapter ) , examples ( 1 chapter ) and ends with the reference list . in section a , evidence - based radiology in child abuse , the reader will nd a great deal of information on : background ; imaging ; skeletal survey ; bone scans and the pitfalls of radionuclide bone scans ; computed tomography ( ct ) scans ; ultrasound ( us ) / magnetic resonance imaging ( mri ) ; post - mortem investigations ; computed / digital vs . 
 il volume ora in discussione , concepito come un atlante di immagini radiologiche raccolte in un ampio arco di tempo , aggiunge ulteriori informazioni ed aiuta nellinterpretazione di tale tipo di immagini . 
le autrici pongono laccento sullimportanza e sulla qualit delle immagini stesse per poter porre una diagnosi adeguata : radiogrammi ripresi in modo improprio non aiutano affatto nelloffrire elementi signi cativi per una diagnosi corretta . detto questo , una diagnosi non corretta oggi , potrebbe avere serie conseguenze nel breve periodo : lo stesso bambino potrebbe infatti essere rivisto dopo non molto tempo con lesioni ancora pi importanti , anche mortali . il volume diviso in due parti : la parte a ( riproduzione delle immagini , presentazione ed interpretazione nellabuso del bambino ) tratta in un solo capitolo quella che de nita radiologia basata sulle prove nellabuso del bambino ; la parte b ( lesioni scheletriche nellabuso ) divisa in cinque sottosezioni che trattano rispettivamente : lo scheletro assiale ( 3 capitoli ) , lo scheletro delle appendici ed i visceri ( 3 capitoli ) , diagnosi differenziali ( 2 capitoli ) , et delle fratture ( 1 capitolo ) , esempi ( 1 capitolo ) e termina con i riferimenti bibliogra ci . nella parte a , radiologia basata sullevidenza nellabuso del bambino , il lettore trover un numero cospicuo di informazioni sul problema di base , il ruolo delle immagini , lo studio dello scheletro , le immagini con scintigra a ossea ed i possibili errori della stessa , la tomogra a computerizzata ( tc ) , lecogra a e la risonanza magnetica ( rm ) , le indagini post - mortem , le immagini della radiologia computerizzata / digitale a fronte di quelle della analogica , le lesioni caratteristiche ( lesioni delle parti molli , fratture meta sarie , dia sarie , del cranio e delle coste , neo - formazione di osso subperiostale , fratture poco comuni e di alta speci cit per abuso ) , la datazione radiologica delle fratture , la diagnosi differenziale e un sommario . 
 1152 radiol med ( 2011 ) 116 : 11511152 in section b , each chapter opens with a few pages of detailed description of fractures affecting the addressed body part ; how and what sort of force is needed to account for the fracture in discussion , its pattern , healing , dating , effects and differential diagnosis . each table exemplifying a fracture presents a key box detailing , according to the type of fracture : age of fracture , degree of force , mechanism , height of fall , general prevalence , prevalence in child abuse and speci city in child abuse , plus a few lines of comment on important clinical or technical rule - of - thumb points . 
this is a pure x - ray lm atlas : where you will encounter the diagnostic dif culties and dilemmas linked to some images , most of all in rib fracture identi cation . 
 going through the chapters , the reader will learn quite a lot , looking at the images , taking into account the very important chapter on the normal variants and pathological conditions as well as the suggestion to repeat the examination at a 14 day interval looking for bone callus to properly assert the presence of a doubtful or suspected fracture improperly x - rayed the rst time . 
 the same can be said about the case examples chapter , a review of some of the cases sent to the authors for their expert opinion : here you can and will realise how poor quality or improper x - ray lm positioning can be the source of dilemma or incorrect diagnoses . 
here the reader will face some questions : what do you think about the images ? how would you report them ? this review can be closed by a few words written by the authors in their preface to the book : which is the lesser error to make a wrongful diagnosis of physical abuse thus removing one infant from loving carers , or to miss the diagnosis and return a baby to an environment in which episodes of abuse may escalate , culminating in that babys death ? both scenarios are distinct possibilities ; the dif culty with making the diagnosis of abuse ( particularly in non - lethal cases ) is that no external gold - standard exists . this book is to be praised and strongly recommended to radiologists ( a copy should be available in all emergency departments ) , orthopaedics , paediatricians and legal medicine physicians . 
 nella parte b , ciascun capitolo si apre con alcune pagine di descrizione dettagliata delle fratture che interessano la regione del corpo di riferimento , come e che tipo di forza sia necessaria per determinare la frattura in discussione , il suo aspetto , la sua guarigione , la sua datazione , le sue conseguenze , per terminare con la diagnosi differenziale . ciascuna presentazione di un esempio di frattura preceduta da una tabella che riporta nei dettagli , a seconda del tipo della frattura , i seguenti dati : et della frattura , entit della forza , meccanismo , altezza della caduta , prevalenza generale della frattura e sua prevalenza e speci cit nellabuso ; dati accompagnati da alcune brevi righe di commento su importanti aspetti clinici o concetti tecnici da non dimenticare . il lettore deve considerare che solo alcune immagini tc e nessuna rm o ecogra a sono presenti o descritte : il volume in questione un puro atlante di immagini radiologiche tradizionali in cui trover le dif colt ed i dilemmi sollevati da alcune immagini , in particolare nellidenti cazione delle fratture costali . nellesaminare i vari capitoli il lettore avr molto da imparare , valutando le immagini , prendendo nella giusta considerazione lassai importante capitolo sulle varianti del normale e su varie condizioni patologiche , nonch tenendo in giusto conto il suggerimento di ripetere le immagini a distanza di 14 giorni alla ricerca della presenza del callo osseo , per sostenere correttamente la presenza di una frattura dubbia o sospetta , riprodotta incorrettamente alla prima indagine . analogo discorso riguarda il capitolo sugli esempi tratti dalla casistisca , una carrellata di alcuni dei casi inviati alle autrici per consulenza specialistica esperta : in queste pagine il lettore pu e si render conto di quanto la scarsa qualit o la non corretta posizione nella ripresa dei radiogrammi possa essere alla base di dilemmi o di diagnosi non corrette . 
in alcuni casi ci si trover di fronte anche ad alcune domande : cosa si pensa delle immagini presentate ? come le si referterebbero ? questa recensione pu essere chiusa con alcune delle parole scritte dalle autrici nella prefazione al volume : quale lerrore di minor importanza : porre una diagnosi errata di abuso sico con la conseguenza di allontanare un infante da amorevoli genitori o sbagliare la diagnosi e riconsegnare un beb ad un ambiente in cui gli episodi di abuso possono aggravarsi per culminare nella morte ? entrambi gli scenari sono possibilit reali : la dif colt nel porre la diagnosi di abuso ( soprattutto nei casi non letali ) sta proprio nel fatto che non esiste una speci ca regola aurea . 
cattaneo1 , 2 1labanof , laboratorio di antropologia e odontologia forense , sezione di medicina legale , universit degli studi di milano , via mangiagalli 37 , milano , italy 2dmu dipartimento di morfologia umana e scienze biomediche , universit degli studi di milano , via mangiagalli 37 , milano , italy 3laboratory of biological and molecular anthropology , institute of experimental biology , faculty of science , masaryk university , 60200 brno , czech republic 4department of radiology , sesto s . 
this study aimed at testing the applicability of the greulich and pyle atlas , the demirjian and the mincer methods on a mixed population to compare skeletal and dental methods of age estimation . 
one hundred and nine orthopantomographs ( opg ) of children aged between 4 and 15.5 years were evaluated by demirjians method ; whenever the highest demirjian score was reached ( 31 cases ) , the mincer method was applied . 
tuttavia , non sempre il gruppo etnico di appartenenza pu essere accertato , per esempio nei casi di cadaveri in avanzato stato di decomposizione : in tali casi la stima dellet deve essere eseguita senza le correzioni fornite dalla letteratura per i diversi gruppi etnici . 
centonove ortopantomogra e di bambini di et compresa fra i 4 ed i 15 , 5 anni sono state valutate con metodo demirjian ; nei casi in cui stato raggiunto il massimo punteggio demirjian , si applicato il metodo mincer . 
la maturazione scheletrica di 54 soggetti di et compresa fra i 7 ed i 19 anni stata quindi determinata con il metodo di greulich e pyle . 1106 radiol med ( 2011 ) 116 : 11051114 results . 
mean variances from the different methods do not signi cantly differ from data reported in the literature and demonstrate that the reliability of demirjian , and greulich and pyle as they stand may be applied satisfactorily to remains or individuals of unknown ethnic orig keywords forensic radiology age estimation greulich and pyle atlas demirjian mincer risultati . 
le differenze medie fra i diversi metodi non differiscono signi cativamente dai dati riportati in letteratura e dimostrano che i metodi demirjian e greulich e pyle possono essere applicati con successo a resti o individui di origine etnica sconosciuta . parole chiave radiologia forense stima dellet atlante di greulich e pyle metodo demirjian metodo mincer introduction introduzione age estimation is one of the most relevant issues for juridical consequences and at the same time a dif cult issue in forensic radiology , both in cases of unknown corpses and in the living ( although with different aims ) from imputability of individuals who do not have legal documents to adoption . 
some reviews of these methods have been made in the last few years , and with time , the rst guidelines have begun to appear in the forensic scenario [ 13 ]  . 
ethnic variability is undoubtedly a relevant issue in age estimation , which is shown also by the increase in the number of population studies , leading in some cases to the development of speci c sections within scienti c although it seems recommendable to apply population - speci c methods , sometimes , ethnicity is not known . 
 in the case of unknown deceased , for example , the speci c ethnic group the subject belongs to can only be hypothesised , and if the corpse is badly preserved , even the main race can be de ned only with dif culty . 
the few studies concerning the epidemiology of unknown deceased individuals state that , on average , one third of all cases concern charred bodies or corpses in advanced decomposition , with clear limits in racial assessment [ 4 ]  . 
but how safely different skeletal and odontological radiological methods can be applied to subjects of unknown ethnicity is not clear . the few guidelines existing in the literature suggest three methods as gold standards for use with the living : the greulich and pyle atlas , the demirjian and the mincer methods . 
 the reference atlas of greulich and pyle is the most widely used method for bone - age determination from hand and wrist la stima dellet uno degli argomenti pi rilevanti in ambito antropologico per le conseguenze giuridiche , e nello stesso tempo una delle applicazioni pi complesse della radiologia forense , sia nellambito del riconoscimento dei cadaveri sconosciuti che nel caso di viventi , seppur con ni differenti . 
diversi metodi di stima dellet , sia scheletrici che dentari , sono stati sviluppati dalla letteratura , e negli ultimi anni sono iniziate a comparire le prime revisioni sui diversi metodi formulati e le prime linee - guida da applicare allo scenario forense [ 13 ]  . 
la variabilit etnica indubbiamente un fattore importante nellambito della stima dellet , come dimostrato anche dal crescente numero di studi di popolazione che hanno portato in alcuni casi allo sviluppo di speci che sezioni nellambito dei giornali scienti ci ; anche se raccomandabile applicare i necessari fattori di correzione derivanti da tali studi , a volte la razza del soggetto di cui necessario eseguire la stima dellet non conosciuta . 
 nel caso di cadaveri sconosciuti ad esempio lo speci co gruppo etnico di appartenenza pu essere solo ipotizzato , e se il cadavere in avanzato stato di decomposizione , persino il gruppo razziale principale pu essere solo de nito con estrema dif colt . 
i pochi studi riguardanti la casistica di cadaveri sconosciuti evidenziano che in media un terzo di tutti i casi riguarda cadaveri carbonizzati o corpi in avanzato stato di decomposizione , con evidenti limiti per la diagnosi di razza [ 4 ]  . 
questo signi ca che la stima dellet in tali casi risulta mancante dellinformazione riguardante lef cienza e laf dabilit degli speci ci metodi scheletrici e dentari in relazione alle variabili etniche . 
tuttavia non ancora chiaro quanto pu essere sicura una stima dellet effettuata tramite metodi scheletrici e dentari basati su immagini radiogra che nel caso di soggetti privi di indicazioni sul gruppo razziale di appartenenza . 
ethnic variability has been considered by the literature : the greulich and pyle method , for example , has shown the widest limits in black female subjects , and some authors suggest that the atlas in these cases should not be applied [ 10 ]  . 
different authors in fact state that other skeletal tests , such as tannerwhitehouse ii , should be the method of choice , thanks to the higher precision shown in different experimental studies [ 11 , 12 ]  . 
however , the literature does not provide a univocal indication , as in some cases , the difference of errors between the two methods does not seem signi cant [ 13 ]  . 
in any case , the greulich and pyle atlas is currently one of the most common methods used for assessing skeletal age not only in forensic , but also in clinical , practice , especially among radiologists since it is easy and fast to use [ 13 , 14 ]  . the demirjian method for assessing dental maturity was rst described in 1973 , applied on a french canadian population , and is widely used and accepted thanks to an easy and user - friendly maturity scoring system , even though the procedure does not consider racial differences . 
a score is given to the dental elements , according to root development , from a to h : a indicates the rst stage of dental development , with the mineralisation of tooth cusps ; h shows that the root growth is complete . 
each stage has an assigned number score ; the sum of scores from the seven permanent teeth ( two incisors , one canine , two premolars and two molars ) gives the age estimation [ 15 ]  . 
different authors have so far applied the method on several populations , and most ascertained that the maturity curves elaborated for the demirjian population did not t [ 1618 ] and that the dental maturity in their respective populations was advanced [ 17 , 1932 ]  . 
 [ 38 ] rst attempted to standardise the demirjian method for a belgian population , although the authors state that the revised version may be not applicable to other geographical contexts . 
other authors pointed out that the demirjian method , although with a general overestimation , shows similar results in different populations , limiting the importance of the racial factor [ 39 , 40 ]  . from demirjian charts for evaluating dental growth , another method was developed based on the study of third molar ( m3 ) development , described by mincer et al . 
their study provides age thresholds for amerletteratura indicano tre metodi come il gold standard per la stima dellet : latlante di greulich e pyle , il metodo demirjian ed il metodo mincer . 
la variabilit etnica stata infatti considerata dalla letteratura : il metodo di greulich e pyle , ad esempio , ha mostrato limiti consistenti quando stato utilizzato per stimare let di femmine negroidi , tanto che diversi autori suggeriscono che latlante in questi casi non dovrebbe essere utilizzato [ 10 ]  . 
 diverse fonti infatti sottolineano che in questi casi altri test come il tanner - whitehouse ii dovrebbero essere la metodiche di scelta , come mostrato dalla maggiore precisione evidenziata dallapplicazione di tale metodo in diversi studi sperimentali [ 11 , 12 ] ; tuttavia attualmente non esiste unindicazione univoca in tal senso , poich in alcuni casi la differenza degli errori mostrata dai due metodi non sembra signi cativa [ 13 ]  . 
in ogni caso , latlante di greulich e pyle attualmente uno dei metodi pi comuni di stima dellet non solo in ambito forense , ma anche nella pratica clinica , specialmente fra i radiologi , poich facile e rapido da usare [ 13 , 14 ]  . il metodo demirjian per la stima dellet dentaria stato descritto per la prima volta nel 1973 , ed stato tarato su una popolazione franco - canadese ; oggi ampiamente accettato ed utilizzato , grazie anche alla facilit di utilizzo del suo metodo a punteggio , sebbene il metodo non prenda in considerazione le differenze etniche . 
un punteggio viene assegnato ad ogni elemento dentario in base al grado di sviluppo della radice , dallo stadio a allh ; a indica il primo stadio di sviluppo dentario , con la mineralizzazione delle cuspidi dentarie , mentre lo stadio h indica che laccrescimento radicolare completo . 
ogni stadio ha un determinato punteggio , variabile per ogni elemento dentario : la somma dei punteggi degli elementi dentari di una emiarcata tranne il terzo molare ( i due incisivi , il canino , i due premolari e i primi due molari ) fornisce la stima dellet [ 15 ]  . 
diversi autori hanno applicato nora il metodo a differenti contesti geogra ci : molti di loro hanno veri cato che le curve di maturit elaborate da demirjian non concordavano con i dati relativi a singoli gruppi etnici [ 1618 ] , e che la maturazione dentaria delle rispettive popolazioni era pi avanzata [ 17 , 1932 ] o , meno frequentemente , veri cavano una sottostima dellet derivante dallapplicazione del metodo [ 3133 ] , sebbene in tali casi i fattori socio - economici potrebbero avere un ruolo non trascurabile . 
in the majority of cases , the method is applicable [ 43 , 44 , 46 , 48 , 50 ] , although some differences in timing of third molar development were observed between populations [ 42 , 49 , 51 , 5254 ] , in part because the m3 is the most variable tooth in the dentition . 
nevertheless , very few studies have been performed to verify their reliability if the race is unknown , as commonly occurs in the case of unknown deceased or the living without documents . 
this study aimed to ascertain the applicability of the three most commonly used methods of age estimation ( greulich and pyle , demirjian , mincer in their original versions ) to a population of unknown ethnic origin in order to verify their reliability . materials and methods one hundred and sixty - seven subjects ( 78 males , 89 females ) of various ethnic origin ( egypt , morocco , senegal , argentina , brazil , colombia , ecuador , peru , albania , bulgaria , moldavia , romania , greece ) aged between 4 and 31 years ( table 1 ) who underwent hand and wrist radiography and an orthopantomography ( opg ) between 1999 and 2008 in the department of radiology of the hospital of sesto s . 
 skeletal maturation of hand and wrist from 54 subjects ( 25 males , 29 females ) aged between 7 and 19 years of age was determined by the greulich and pyle method . 
 [ 38 ] per primi tentarono di standardizzare il metodo demirjian alla popolazione belga , sebbene gli autori stessi osservino che il metodo revisionato potrebbe non essere applicabile ad altri contesti geogra ci [ 38 ]  . 
daltra parte , altri autori sottolineano che il metodo demirjian , sebbene con una generale e costante sovrastima , produce risultati equiparabili in diverse popolazioni , il che limiterebbe limportanza del fattore etnico nellapplicazione di tale metodo [ 39 , 40 ]  . dagli schemi di demirjian per la valutazione dellaccrescimento dentario , un altro metodo stato sviluppato , basato sullo studio del terzo molare ( m3 ) descritto da mincer et al . 
gli stessi stadi ( da a ad h ) creati da demirjian sono stati applicati al terzo molare , allo scopo di ottenere informazioni sullet di soggetti sopra i 16 anni , limite del metodo demirjian . 
gli studi di mincer consentirono di estrapolare i valori di riferimento per la popolazione bianca americana ( dai 14 ai 24 anni ) e per gli afroamericani ( standardizzati solo dallo stadio f allo stadio h )  . 
diversi autori hanno applicato il metodo anche ad alcune popolazioni asiatiche ed europee [ 4251 ] ; nella maggior parte dei casi il metodo applicabile [ 43 , 44 , 46 , 48 , 50 ] , sebbene alcune differenze fra le diverse popolazioni nello sviluppo del terzo molare siano state descritte nelle diverse popolazioni [ 42 , 49 , 51 , 5254 ] , anche a causa delle variabilit di sviluppo di tale elemento dentario . 
 quelli sopra elencati sono comunemente considerati dai diversi autori come i metodi di elezione per la stima dellet e sono attualmente i pi utilizzati in ambito forense ; tuttavia , pochissimi studi sono stati condotti per veri care la loro af dabilit nei casi in cui letnia del soggetto non sia conosciuta , come spesso accade per i cadaveri sconosciuti o per i viventi privi di documenti . 
questo studio mira a veri care lapplicabilit dei tre metodi di stima dellet pi utilizzati in ambito forense ( greulich e pyle , demirjian , mincer nelle loro versioni originali ) , applicati su una popolazione di origine etnica ignota , allo scopo di veri care in tale delicata situazione la loro af dabilit . table 1 details of hand and wrist radiographs and orthopantomographs hand - wrist radiographs , n orthopantomographs , n male female total materiali e metodi tabella 1 dettagli delle radiogra e di mano e polso ed ortopantomogra a ( opt ) selezionati per lo studio maschi femmine totale radiogra e di mano e polso , n 25 opt , n sono stati inclusi nello studio 167 soggetti ( 78 maschi , 89 femmine ) di origine etnica differente ( egitto , marocco , senegal , argentina , brasile , colombia , ecuador , per , albania , bulgaria , moldavia , romania , grecia ) di et compresa fra i 4 ed i 31 anni ( tabella 1 ) che si sottoposero a radiogra a di mano e polso ed ortopantologra a ( opt ) fra il 1999 ed il 2008 presso il dipartimento di radiologia dellospedale di sesto s . 
 results the greulich and pyle method ( table 2 ) showed that age was overall more frequently underestimated ( in 46% of cases ) than overestimated ( in 37% ) ; the agreement of estimated age ( ea ) and chronological age ( ca ) within 1 year was observed in 17% of cases . 
 zione scheletrica di mano e polso stata valutata in 54 soggetti ( 25 maschi , 29 femmine ) di et compresa fra i 7 ed i 19 anni , tramite confronto con atlante di greulich e pyle . 
 le differenze fra et stimata ed et cronologica sono state calcolate per ogni metodo , cos come la differenza media , massima e minima per i maschi e le femmine . 
 risultati lutilizzo dellatlante di greulich e pyle ha mostrato pi frequentemente una sottostima dellet ( 46% ) che una sovrastima ( 37% ) ( tabella 2 ) ; la corrispondenza dellet stimata con let cronologica ( entro lanno ) fu osservata nel 17% dei casi , e fu osservata pi frequentemente nei maschi ( 28% ) che nelle femmine ( 7% )  . 
la differenza media fra et stimata ed et cronologica stata di 0 , 755 anni0 , 522 deviazione standard ( ds ) , nei maschi di 0 , 819 anni0 , 995 ds e nelle femmine di 0 , 7010 , 522 ds ; la differenza massima fra et stimata ed et cronologica fu 3 , 5 anni , la differenza minima di 0 anni . 
 la differenza media fra et stimata ed et cronologica fu di 0 , 824 anni0 , 76 ds , nei maschi 0 , 861 anni0 , 765 ds e nelle femmine 0 , 79 anni0 , 753 ds , la differenza massima fu di 3 , 26 anni e la minima di 0 , 01 anni . 
 tutti i soggetti di et superiore ai 23 anni furono correttamente diagnosticati dal metodo mincer ; la differenza rispetto allet cronologica fu in media di 2 , 95 anni2 , 496 ds . 
la differenza media pi bassa rispetto allet cronologica fu ottenuta dallapplicazione dellatlante di greulich e pyle ( 0 , 755 anni0 , 522 ds ) , seguito dal metodo demirjian con una differenza media di 0 , 824 anni0 , 76 ds . 
il metodo mincer ha mostrato la differenza media pi elevata ( 2 , 95 anni2 , 496 ds )  . negli ultimi anni gli studi sulle metodologie di stima dellet si sono concentrate in particolar modo sulla variabilit etnica . 
in the case of living individuals with no valid documents and with uncertain age and area of provenance , as in cases of unknown deceased or of the living with no identi cation documents , age estimation is important and frequently of radiological relevance , particularly for juveniles and young adults . 
in this scenario , the literature needs to provide not only indications concerning the applicability of single methods to different populations but also to verify whether the results from the same methods are dependent on the ethnic factor . 
this study was performed to verify whether the standards of greulich and pyle , demirjian and mincer methods in their original version are applicable to young adults when their ethnic origin is unknown , in comparison with data from studies performed on populations of known origin . the rst information derives from comparison of the three methods : the greulich and pyle atlas proved to be the most accurate , as con rmed by the literature [ 55 , 56 ] , followed by demirjian and then mincer . 
for example , the literature has shown that the greulich and pyle atlas is less reliable in black females , whereas the error range is more limited in white females and black males [ 8 , 10 ]  . 
the study substantially con rms the indications provided by the literature : the atlas is more prone to yield the best results in female populations , as reported by several authors [ 10 , 57 ]  . 
in 17% of cases , estimated and chronological ages were concordant within 1 year , which shows the fairly good reliability of the atlas as a preliminary method in age assessment , regardless of ethnic origin , in comparison with dental methods . 
this result may be explained by the limited number of x - ray images included in the atlas , whereas the demirjian method , based on the sum of seven different scores , has a wider range of possible results . 
nevertheless , this is a preliminary indication concerning the higher reliability of the greulich and pyle atlas in comparison with dental methods . the demirjian method showed a very similar error range to data provided by the literature in different geographical contexts . 
in tale scenario risulta di notevole importanza ottenere informazioni sullapplicabilit dei singoli metodi su diverse popolazioni , e veri care se i risultati derivanti dagli stessi metodi dipendano dalla variabilit etnica . 
lo studio attuale stato effettuato allo scopo di veri care se i metodi greulich e pyle , demirjian e mincer nella loro versione originale sono applicabili ai giovani adulti nel caso in cui lorigine etnica degli stessi sia sconosciuta . la prima informazione ottenuta deriva dal confronto dei tre metodi : latlante di greulich e pyle risultato il pi af dabile , come peraltro confermato dalla letteratura [ 55 , 56 ] , seguito dal metodo demirjian e mincer . 
per esempio , la letteratura ha mostrato che latlante di greulich e pyle meno af dabile nel caso di femmine negroidi , mentre lerrore medio pi limitato nel caso di femmine caucasoidi e maschi negroidi [ 8 , 10 ]  . 
lo studio attuale ha sostanzialmente confermato le indicazioni fornite dalla letteratura : latlante tende a fornire i risultati migliori nella popolazione femminile , come confermato da diversi autori [ 10 , 57 ]  . 
nel 17% dei casi let stimata corrispondeva allet cronologica con un errore inferiore allanno ; lo studio ha evidenziato una buona af dabilit dellatlante di greulich e pyle come metodo preliminare di stima dellet indipendentemente dal fattore etnico , in paragone ai metodi dentari ; tale risultato potrebbe essere spiegato dal limitato numero di immagini radiogra che incluse nellatlante , mentre il metodo demirjian , basato sulla somma di sette differenti punteggi , ha una pi ampia gamma di possibili risultati : nondimeno , i risultati emersi da questo studio sono ancora preliminari , e richiedono una conferma su una popolazione pi ampia . 
 il metodo demirjian ha mostrato un margine di errore sovrapponibile con le informazioni fornite dalla letteratura sullapplicazione del metodo nei diversi contesti geogra ci , che evidenzia una generale tendenza alla sovrastima . 
lo stesso fenomeno stato osservato infatti nel caso dellapplicazione del metodo su popolazioni saudite [ 23 ] , ungheresi [ 27 ] , nord - est brasiliane [ 26 ] , norvegesi [ 28 ] , nlandesi [ 29 ] , polacche [ 30 ] , cinesi [ 55 ] , olandesi [ 25 ] , iraniane [ 31 ] , australiane [ 19 ] e brasiliane [ 20 ]  . 
i risultati del presente articolo sembrano confermare che il metodo quantitativo sviluppato da demirjian lievemente in uenzato dalla variabilit etnica , come mostrato da diversi autori ( tabella 5 ) [ 39 , 40 ]  . 
unaltra informazione emersa nel corso dello studio riguarda lelevata af dabilit del metodo nella diagnosi dei soggetti di et superiore 1112 radiol med ( 2011 ) 116 : 11051114 table 5 comparison of results of the demirjian method with data provided by the literature on different populations this study farah et al . 
differences shown by different sources seem to con rm the quantitative method developed by demirjian is in uenced slightly by ethnic variation , as stated by different authors [ 39 , 40 ] ( table 5 )  . 
another nding provided by the study concerns the success of the demirjian method in recognising subjects > 16 years , which were all correctly assessed with the highest maturation score . 
the demirjian method can be therefore considered as a good screening method , regardless of ethnic origin , in distinguishing subjects > 16 years , who will then be examined by age estimation methods tested on the late adolescent population , such as the mincer method . 
nevertheless , this procedure should follow an initial age assessment based on skeletal maturation performed by the greulich and pyle method in order to reduce the number of false positive results ( subjects wrongly assessed as being > 16 and 23 years )  . concerning the mincer method , the mean variance was distinctively higher than in the demirjian and greulich and pyle methods . 
 our study therefore con rmed data from the literature ai 16 anni , che sono stati tutti correttamente valutati con il punteggio massimo di maturazione dentaria ; il metodo demirjian pu essere pertanto considerato come un buon metodo di screening indipendentemente dal fattore etnico per distinguere i soggetti di et superiore di 16 anni , che poi saranno ulteriormente esaminati da metodi di stima testati su soggetti tardo - adolescenti e giovani adulti , come il metodo mincer . il metodo di mincer stato in grado di diagnosticare con suf ciente precisione tutti i soggetti di et superiore ai 23 anni . 
nondimeno , tale metodo dovrebbe essere applicato dopo una prima stima dellet basata sul grado di maturazione scheletrica tramite lutilizzo dellatlante di greulich e pyle , allo scopo di ridurre il numero di falsi positivi ( soggetti erroneamente valutati sopra i 16 ed i 23 anni )  . per quanto riguarda il metodo di mincer , la differenza media rispetto allet cronologica fu pi elevata rispetto al metodo demirjian ed allutilizzo dellatlante di greulich e pyle . 
 this information , if con rmed by further studies on wider populations , will assist in de ning a common procedure for age estimation based on the potential of the single methods in order to reduce the estimated age range , even in cases of blind age assessment where individualisation is dif cult to achieve . 
even if it remains true that the smallest error will be achieved when using population - speci c adaptations to methods , blind use of such radiological methods , regardless of the results , may be acceptable , as long as the larger error is taken into consideration with respect to populationspeci c approaches . 
 del soggetto non siano disponibili ; inoltre , i risultati forniti dai singoli metodi evidenziarono unelevata precisione nella diagnosi dei soggetti di et superiore ai 16 anni per il metodo demirjian , e sopra i 23 anni per il metodo mincer ; tale informazione , se confermata da ulteriori studi eseguiti su popolazioni pi ampie , potrebbe fornire un aiuto nella de nizione di una procedura standardizzata per la stima dellet , basata sulla potenzialit di ogni singolo metodo per ridurre lintervallo di stima dellet , anche nei casi in cui la stima dellet sia cieca delle indicazioni sulletnia di appartenenza , ove laderenza del risultato nale al singolo individuo dif cilmente realizzabile . 
anche se rimane scontato che il minor margine di errore viene raggiunto utilizzando applicando i fattori di correzione speci ci per le singole popolazioni etniche , un utilizzo cieco dei metodi radiologici di stima pu essere accettabile . 
avogadro , novara , italy 2universit degli studi di roma campus biomedico , roma , italy 3universit degli studi di roma cattolica , roma , italy 4universit degli studi di sassari , sassari , italy 5universit degli studi di chieti , chieti , italy 6universit degli studi di trieste , trieste , italy 7universit degli studi di catania , catania , italy 8universit degli studi di varese , varese , italy 9universit degli studi di genova , genova , italy 10universit degli studi di foggia , foggia , italy 11universit degli studi di verona , verona , italy 12universit degli studi di milano bicocca , milano , italy 13universit degli studi di modena , modena , italy 14universit degli studi di firenze , firenze italy 15universit degli studi di udine , udine , italy correspondence to : a . 
once a common educational programme and time slot had been identi ed and planned , the programme was delivered via internet - based video conferencing once a week for 2 - h lectures . 
the universities involved were trieste , udine , verona , milano bicocca , novara , varese , genova , sassari , rome campus , rome cattolica , chieti , foggia , catania , modena and firenze . 
scopo del nostro lavoro stato progettare , realizzare e valutare un percorso didattico di e - learning per la formazione degli specializzandi in radiodiagnostica , coinvolgendo pi scuole di specializzazione distribuite su tutto il territorio nazionale , nellottica della condivisione culturale . 
previa identi cazione e piani cazione di un programma didattico temporale condiviso , la modalit tecnica di e - learning in teleconferenza ha previsto un collegamento via internet il mercoled pomeriggio e due ore di lezione , alla lezione seguita una interazione didattica delle singole sedi con il discente . 
le universit che hanno aderito sono state : trieste , udine , verona , milano bicocca , novara , varese , genova , sassari , roma campus , roma cattolica , chieti , foggia , catania , modena , firenze . 
based on our experience , whereas e - learning in radiology has become established and compulsory , there is the need for legislation that on the one hand protects online teaching activity and on the other allows study and continuing medical education ( cme ) credits to be recognised . keywords e - learning video conferencing radiology lezioni , per un totale di 36 ore di lezione effettive . 
relativamente al giudizio sulla qualit del video e , in particolare , sul dettaglio delle immagini radiologiche proposte in diapositiva e / o in lmati il 71% dei discenti ha espresso un giudizio ottimo , il 24% un giudizio buono , il 4 , 5% un giudizio suf ciente non vi stato alcun giudizio insuf ciente . 
da quanto esposto e dallesperienza vissuta se la via delle - learnig in radiologia , a nostro parere , ormai segnata ed obbligatoria , si impone la necessit legislativa di normative che , da una parte , tutelino lattivit didattica in rete e , dallaltra , consentano il riconoscimento di crediti formativi e di educazione continua in medicina ( ecm )  . parole chiave educazione via internet teleconferenza radiologia introduction introduzione the days are long gone when socrates taught with the peripatetic method , whereby each step , or thereabouts , corresponded to a notion that his students were required to appropriate . 
throughout history , teaching has kept pace with the changing times according to patterns , projects and options dictated , for the most part , by technological development [ 1 , 2 ]  . 
with the dawning of the new millennium information , technology ( it ) has become for education and teaching a pillar supporting new teaching strategies , and with it , the term e - learning has been coined [ 1 , 3 ]  . e - learning is short for electronic learning and refers to the possibility of offering teaching and / or education via the internet or world wide web [ 4 , 5 ]  . 
the extent of the role of e - learning soon became evident , such that at the end of 1999 , the european commission launched the eeurope programme , which aimed to accelerate the learning of digital technology in european countries [ 6 ]  . 
in this programme , e - learning is placed at the heart of the european unions cultural policy . as always occurs , the far - reaching spread of new technologies is a slow and gradual process , and the creation of an information technology ( it ) culture took some time [ 7 ]  . 
 technological familiarisation was required , and the results speak for themselves : in 1997 , only 16.7% of italian housesono ormai lontani gli anni in cui socrate educava ed insegnava con il metodo peripatetico , l dove ad ogni passo , o quasi , corrispondeva una nozione che gli allievi , podisti per de nizione , dovevano incamerare . 
nella evoluzione storica anche la didattica stata al passo con i tempi secondo schemi , progetti ed opzioni dettati , soprattutto , dalla evoluzione tecnologica [ 1 , 2 ]  . 
a partire dal nuovo millennio anche leducazione e linsegnamento hanno trovato nellofferta elettronica un pilastro su cui costruire nuove strategie didattiche ed stato coniato il termine e - learning [ 1 , 3 ]  . e - learning lacronimo di electronic learning cio la possibilit di mettere a punto unofferta didattica e / o di educazione tramite internet o world wide web [ 4 , 5 ]  . 
la convinzione che lelectronic learning potesse cambiare le regole del gioco stata immediata , tanto che la commissione europea alla ne dellanno 1999 ha dato vita al programma eeurope , che ha avuto come scopo laccelerazione dellapprendimento della tecnologia digitale nei paesi europei [ 6 ] ; in questo programma viene posto proprio le - learning al centro della politica culturale dellunione . come sempre avviene , il processo di diffusione capillare di nuove tecnologie lento e graduale ed anche la cultura informatica ha necessitato dei suoi tempi [ 7 ] , si parlato di interventi di familiarizzazione tecnologica ed i risultati sono stati tangibili : basti pensare che se nel 1997 solo il 16 , 7% delle famiglie italiane possedeva un personal computer radiol med ( 2011 ) 116 : 989999 holds owned a personal computer , whereas the gure today is said to be around 90% . in 2003 , under the advocacy of the tertiary education and research sectors , the italian society of e - learning ( sie - l ) was founded with the aim of promoting teaching courses at the school , university and postuniversity levels . 
in fact , only 2 short years later , in 2005 , around 80% of italian universities were able to offer either pure or blended e - learning courses [ 1 ]  . in the european setting , italy was quick to realise the prospects of e - learning . 
also in 2003 , a study carried out by assintel ( national association of enterprises operating in the information and communication technology sector ) showed that the e - learning market in italy has grown faster than in other european countries [ 7 ]  . 
 indeed , thanks to the internet , a teacher / trainer may simultaneously reach a greater number of students / trainees in different locations [ 8 , 9 ]  . the advent of teaching over the internet has also enabled the low - cost enhancement of a new concept of cultural sharing . 
this sees the involvement of numerous teachers / trainers representing different working groups who make their contribution and share their own experiences with other colleagues and / or working groups in different locations [ 1012 ]  . 
the aim of our work was to plan , implement and evaluate an e - learning teaching course for the training of radiology residents at numerous specialty schools located throughout italy in the setting of cultural sharing . materials and methods the initial planning phase of the project involved the presentation of the project and aggregation of the participants . 
 an e - mail was sent on 11 may 2009 informing all directors of the italian specialty schools of radiology of the desire to establish an e - learning project for the 2009 / 2010 academic year in the form of single - topic teaching seminars . 
once participation in the project had been acquired , a lesson calendar was drawn up , with seminars attualmente si parla di percentuali vicine al 90% . nel 2003 , sotto la spinta del mondo accademico e della ricerca , stata costituita in italia la societ italiana di e - learning ( sie - l ) , che ha come scopo il favorire percorsi didattici a livello scolastico , universitario e post - universitario ; questo messaggio , di innovazione culturale , stato recepito soprattutto dal mondo accademico tanto che , dopo appena due anni , nel 2005 , circa l80% delle universit italiane erano in grado di offrire percorsi didattici e - learning , puro o blended [ 1 ]  . 
98 del 29 / 04 / 2003 con la legge moratti - stanca si sono costruite le basi per le procedure di accreditamento dei corsi di studio a distanza delle universit statali e non statali e sempre nel 2003 , da uno studio condotto dallassociazione nazionale delle imprese che operano nel settore dellinformatica & communication technology ( assintel ) si evince che il mercato italiano delle - learnig ha presentato il tasso di crescita pi alto rispetto a quello degli altri paesi europei [ 7 ]  . 
internet funge da collante e da veicolo tecnico e culturale ; infatti tramite , e grazie , ad esso possibile per un docente raggiungere , simultaneamente , pi discenti allocati in diverse sedi [ 8 , 9 ]  . lavvento della diffusione didattica , tramite internet , consente , inoltre , di potenziare con costi contenuti , un nuovo concetto di condivisone culturale ; tale concetto vede coinvolti ed impegnati pi docenti , espressione di diversi gruppi di lavoro , che apportano il loro contributo , condividendo la propria esperienza con altri colleghi e / o gruppi di lavoro di diverse sedi [ 1012 ]  . 
scopo del nostro lavoro stato progettare , realizzare e valutare un percorso didattico di e - learning per la formazione degli specializzandi in radiodiagnostica , coinvolgendo pi scuole di specializzazione distribuite su tutto il territorio nazionale , nellottica della condivisione culturale . materiali e metodi la piani cazione del progetto ha previsto una prima fase di presentazione ed aggregazione telematica degli attori . 
 tramite e - mail , in data 11.05.2009 tutti i direttori delle scuole di specializzazione in radiodiagnostica italiane sono stati informati della volont di istituire , per lanno accademico 2009 / 2010 , un progetto di e - learning sotto forma di seminari didattici monotematici . 
acquisite le adesioni al progetto stato piani cato , in base ad esse , un calendario delle lezioni che ha previsto lattivit seminariale a partire dal 21 ottobre 2009 per concludersi a luglio 2010 . 
luigia macarini , luca colosimo , cesare ettorre gian , carlo beomonte zobel , bruno torricelli , pietro cova , maria cova , maria zuiani , chiara mascalchi , mario pozzi - mucelli , roberto sironi , sandro fugazzola , carlo garlaschi , giacomo meloni , gianni carriero , alessandro bonomo lorenzo beomonte zobel bruno storto m . 
per ogni scuola di specializzazione stato , inoltre , richiesto un referente che potesse fungere da tutor di rete per la realizzazione dellintero progetto [ 13 ] , per coinvolgere gli specializzandi stato deciso che il tutor di rete dovesse essere un iscritto alla scuola di specializzazione e radiol med ( 2011 ) 116 : 989999 tutor should be enrolled at the school . 
coordination of the entire network was entrusted to the novara network tutor resident . from the technical point of view , the entire e - learning process was managed by a system administrator with expertise in this sector ( exit srl ) , and the online meeting space microsoft live meeting was chosen for presenting the lessons . 
it requires installation of a software application on all participants computers and provides real - time interaction with participants who may make presentations , launch projects , brainstorm , modify les or collaborate through the use of a virtual whiteboard . 
the programme was chosen for its speed , the excellent audio / video quality and the voice compression characteristics , which guarantee easy listening even in the presence of microinterruptions in the connection . 
in terms of hardware , the minimum requirements for connection and participation in the lessons were the following : personal computer with a 500 - mhz processor or above ( 1 ghz recommended ) operating system windows xp sp2 or sp3 or above ( vista or windows 7 ) at least 256 mb ( 512 mb recommended ) dedicated directx video card compatible with 64 mb of random access memory ( ram ) webcam audio card , microphone , speakers / headphones video projector or other peripheral device for video reproduction , e.g. 
plasma or liquid crystal display ( lcd ) screen , with super video graphics array ( vga ) resolution of at least 800600 ( super vga 1 , 024768 recommended ) fast subscriber line ( adsl ) in order to receive the content , the computers used in the various locations required the following programmes in addition to microsoft live meeting : internet browser ( interned explorer ( ie ) 6 or above , mozilla firefox 3.x , apple safari 3.x ) sun java 1.6.0_11 or above microsoft of ce power point 2002 or above adobe flash player 9 or above . with regard to the server , a hosted service external to the organisation was used , i.e. 
in addition to the part of the server ( hosted ) , the programme was completed with a software client to be installed on all computers participating in the meeting and which could be automatically installed at the rst invitation to participate . 
the client was extremely lightweight , free and constantly updated thanks to the internet connection , e.g. , asymmetric digital la coordinazione tutoriale dellintera rete stata af data allo specializzando tutor di rete di novara . dal punto di vista tecnico tutto il processo di e - learnig stato af dato ad un amministratore di sistema esperto in tale attivit ( exit srl ) e per le lezioni stato valutato e scelto il programma di videoconferenza , microsoft live meeting . 
microsoft live meeting consente di accedere , tramite internet , ad unarea per riunioni in linea ; esso necessita linstallazione di un software su tutti i computer dei partecipanti e consente una reale interazione con i partecipanti che possono effettuare presentazioni , avviare progetti , mettere insieme idee , modi care le , collaborare attraverso una lavagna virtuale . 
 il programma stato selezionato per la sua velocit , lottima resa audio - video e le caratteristiche di compressione della voce che garantiscono uidit nellascolto anche in presenza di microinterruzioni di banda . 
oltre alla parte del server ( hosted ) , il programma si completa con un software client da installare sui computer che partecipano alla riunione , e che pu essere , automaticamente , installato sin dal primo invito di partecipazione . 
una lezione tipo eseguita durante il ciclo seminariale on line ha previsto , in primis , da parte dellamministratore del sistema , la creazione di una conference room alla quale vengono assegnati i partecipanti e generate le relative credenziali di accesso al processo di e - learnig . 
 in data 20 settembre 2009 , presso listituto di radiologia delluniversit del piemonte orientale di novara , si svolta una riunione preliminare , in cui erano presenti i tutor 994 radiol med ( 2011 ) 116 : 989999 windows update facility . 
a typical lesson delivered during the online series of seminars rst involved creation by the system administrator of a virtual conference roo the participants were assigned to the conference room and the relative access credentials for the e - learning process were generated . on 20 september 2009 , a preliminary meeting was held at the universit del piemonte orientale di novara attended by all network tutors of the participating specialty schools . 
for the purposes of assessing the effectiveness of the project , a common multiple - choice questionnaire was devised and approved , to be completed at the end of every lecture at each of the locations . 
the questionnaire sought information regarding number of participants in the classroom , overall assessment of the teaching content , interruptions without reconnection of the internet service , transient interruptions of the internet service and audio and video quality . 
at the end of the series of lessons random suggestions on how to improve the project on the whole were requested from some of the residents . results after identifying and planning a joint teaching programme ( table 1 ) , the technical approach to e - learning in the online meeting space involved an internet connection on wednesday afternoons and a 2 - h lesson , followed by more traditional interaction with the trainee radiologists at the individual locations . 
the following universities participated in the project : trieste , udine , verona , milan bicocca , novara , varese , genoa , sassari , rome campus , rome cattolica , chieti , foggia , catania , modena and florence . 
the university of rome cattolica participated in the project with two locations : rome gemelli university hospital and rome cattolica campobasso campus . overall 18 lessons were held for a total of 36 h of teaching . 
with regard to the overall attendance of the specialty schools to the entire teaching programme , there were absences by the schools in 9% of lessons : three were absent for one lesson , one for seven lessons , one for four lessons and one for six lessons . 
la riunione ha consentito di sviscerare ed affrontare , sia dal punto di vista tecnico che didattico , le varie problematiche ; durante questa , unica , riunione stata quindi costituita la rete organizzativa progettuale . 
 ai ni della valutazione dellef cacia del progetto , stata condivisa ed approvata una scheda comune di valutazione a scelta multipla obbligata , che ogni sede ha compilato alla ne di ogni lezione . 
la scheda ha richiesto delle informazioni relativamente a : numero dei partecipanti in aula , giudizio complessivo sui contenuti didattici , interruzioni senza ripristino della linea internet , interruzione momentanea della linea internet , qualit audio , qualit video . 
alla ne del ciclo didattico sono stati richiesti suggerimenti random ad alcuni discenti , al ne di migliorare il progetto nel suo complesso . risultati previa identi cazione e piani cazione di un programma didattico temporale condiviso ( tabella 1 ) , la modalit tecnica di e - learning in teleconferenza ha previsto un collegamento via internet il mercoled pomeriggio e due ore di lezione , alla lezione seguita una interazione didattica delle singole sedi con il discente . 
le universit che hanno aderito sono state : trieste , udine , verona , milano bicocca , novara , varese , genova , sassari , roma campus , roma cattolica , chieti , foggia , catania , modena , firenze . 
relativamente alla partecipazione complessiva delle scuole di specializzazione allintero programma didattico , nel 9% delle lezioni si sono veri cate assenze da parte delle scuole : tre scuole sono state assenti per una lezione , una scuola per sette lezioni , una scuola per quattro lezioni , una scuola per sei lezioni . 
relativamente al giudizio complessivo dei discenti sui contenuti didattici , l83% ha espresso un giudizio ottimo , il 15% un giudizio buono e lo 0 , 3% un giudizio suf ciente , non vi stato alcun giudizio insuf ciente . relativamente alla interruzione senza ripristino della linea internet , solo in una lezione si veri cata , in una singola sede , una interruzione del collegamento a causa radiol med ( 2011 ) 116 : 989999 pendent of the e - learning process . 
with regard to residents overall opinion of the course content , 83% found the course excellent , 15% good and 0.3% satisfactory ; no trainee expressed an unsatisfactory opinion . an interruption to the internet service without reconnection occurred only on one occasion at one location due to a storm , whereas 1.5% of connections experienced transient interruptions limited to a few minutes and did not signi cantly disrupt learning . 
there were 13 transient interruptions to the internet service for a total of 33 min out of an overall total of 2 , 160 min of internet connection . with regard to residents opinion of video quality , and especially of the detail of the radiological images presented in slides and moving pictures , 71% gave an excellent assessment , 24% good and 4.5% satisfactory ; no trainee expressed an unsatisfactory opinion . 
in this case , the lesson was nonetheless delivered by another location ( novara ) , which at the beginning of the project was nominated as active reserve , ready to intervene and compensate for any technical problems at the location of the scheduled lesson . the overall ( and entirely funded ) costs of the entire series of seminars included the services of a company specialised in webcasting and were equal to 5 , 000 euro . 
the additional cost is dif cult to estimate given the variety of prices and possible con gurations , but it seems reasonable to suggest a minimum amount of 700800 euro . in order to improve the project , 20 random suggestions were requested from the residents who participated in the course . 
 the cases could be presented by the residents at the teaching location for that lesson . provide a nal test on the topic covered . cover a complete topic each year with special emphasis on imaging characteristics ; here emergency medicine was the most proposed topic for a possible future series of seminars . di un temporale , mentre nell1 , 5% dei collegamenti si sono veri cate interruzioni momentanee , contenute nellarco massimo di pochi minuti , che non hanno in ciato la continuit didattica . 
 su un totale di 2160 minuti complessivi di collegamento le interruzioni momentanee della linea internet sono state 13 per un totale di 33 minuti . relativamente al giudizio sulla qualit del video e , in particolare , sul dettaglio delle immagini radiologiche proposte in diapositiva e / o in lmati , il 71% dei discenti ha espresso un giudizio ottimo , il 24% un giudizio buono , il 4 , 5% un giudizio suf ciente , non vi stato alcun giudizio insuf ciente . 
relativamente alla qualit audio , il 72% dei discenti ha espresso un giudizio ottimo , il 25% un giudizio buono , il 3% un giudizio suf ciente , non vi stato alcun giudizio insuf ciente . 
relativamente alla sede di lezione , solo in un caso si veri cato un problema di connessione e caricamento del le con impossibilit a trasmettere la lezione ; in questo caso la lezione stata comunque svolta da unaltra sede didattica ( novara ) che , allinizio del programma di e - learning , era stata prevista come riserva / attiva , pronta ad intervenire per supplire ad eventuali problemi tecnici della sede di lezione programmata in calendario . 
 i costi complessivi , tra laltro nanziati , dellintero ciclo seminariale hanno previsto lassistenza da parte di una ditta specializzata in teletrasmissione , e sono stati pari a 5000 euro . 
tale costo aggiuntivo dif cilmente stimabile , data la variet di prezzi e di possibili con gurazioni , ma ragionevolmente ipotizzabile in un minimo di 700800 euro . al ne di migliorare il progetto sono stati richiesti , in maniera random , 20 suggerimenti da parte dei discenti che hanno partecipato al corso , da questi si evincono i seguenti principali suggerimenti : contenere ad unora il tempo di lezione , al ne di evitare il crollo dellattenzione ; presentare dei casi clinici alla ne di ogni singola lezione , relativi al tema trattato , casi che potrebbero essere presentati dagli specializzandi delle sede didattica di lezione ; predisporre un test nale di apprendimento sullargomento trattato ; trattare ogni anno un argomento completo con particolare attenzione alla semeiotica e , a tal proposito , la maggior esigenza didattica , per il prossimo eventuale ciclo , stata suggerita in tema di patologia dellurgenza . discussione la lezione frontale / ex cattedra , malgrado la sempre maggiore 996 discussion despite the continuing trend towards direct tutorial teaching , traditional lecturing is still an important point of reference [ 14 ]  . 
to keep pace with the times , there is an increasing need to combine a variety of teaching methods to optimise physical space , time and human resources and to maximise cultural gain [ 1 , 9 ]  . 
recently , thanks to the support of new technologies , this need has received an important contribution in the setting of shared culture at low cost [ 15 ]  . 
the sharing of culture makes it possible for opinion leaders of the sector to share professional experience and enrich their own cultural assets through contact with the expertise that is particularly dedicated to the topic . on the other hand , economic dif culties that often arise could seriously jeopardise or at any rate limit direct participation in courses and / or congresses that demand substantial expenses [ 16 ]  . 
this emphasises the concept of safeguarding the need for cultural sharing , enriching and updating with limited costs , by exploiting technological developments and e - learning [ 12 , 1618 ]  . 
in nancial terms , the overall cost of a connection with four centres amounted to 12 , 000 euro , with the setup and management of the point - to - point connection accounting for 8 , 000 euro . 
our preliminary analysis of a satellite conferencing service revealed that , considering booking the satellite and transmitting the signal from the mobile broadcasting station , each 2 - h lesson cost around 3 , 000 euro . recently , the advent of streaming has revolutionised the process of e - learning by making it much more attainable [ 1 , 12 ]  . 
the term streaming indicates the ability to broadcast audio / video data from a single source to one or more reception points , with the signal being conveyed via the internet . 
on - demand streaming indicates that the audio / radiol med ( 2011 ) 116 : 989999 indicazione verso levoluzione tutoriale diretta , rimane , pur sempre , un importante riferimento [ 14 ]  . 
al passo con i tempi , si sente , sempre pi , lesigenza di unire pi forze didattiche al ne di ottimizzare spazi , tempi e risorse umane con il massimo apporto culturale [ 1 , 9 ]  . 
la condivisone culturale consente la possibilit di condividere , con opinion leaders del settore , esperienze professionali , arricchendo il proprio bagaglio culturale grazie al contatto didattico con expertise , che si sono particolarmente dedicati allargomento . daltra parte le dif colt economiche , con cui spesso ci si scontra , potrebbero mandare in crisi , o comunque limitare , la partecipazione diretta a corsi di aggiornamento e / o attivit congressuali che presuppongono costi enormi [ 16 ]  . 
 da qui il concetto di salvaguardare la necessit di condivisione , di arricchimento e di aggiornamento culturale con costi contenuti , sfruttando levoluzione tecnologica , grazie alle - learning [ 12 , 1618 ]  . 
i tentativi di superare le frontiere didattiche , grazie allinsegnamento a distanza , sono stati pi volte descritti dalla letteratura : si passati dai tentativi di collegamento con linea telefonica ai complessi e costosi collegamenti tramite satellite [ 19 , 20 ]  . 
se i collegamenti via satellite sono da sempre stati proibitivi per i costi vivi di esercizio , i collegamenti per via telefonica , nel passato , hanno creato speranze ed illusioni . nellambito delle pregresse esperienze delle scuole di specializzazione in radiodiagnostica in italia , nel 2003 stato tentata unattivit didattica congiunta tra le scuole di specializzazione delluniversit degli studi di novara e di modena . 
a tal ne fu utilizzato un sistema di teletrasmissione per via telefonica tipo polispan fx4 , realizzato con connessione punto - punto ; tale esperienza , malgrado limpegno da parte delle due scuole di specializzazione , non ebbe i risultati attesi , infatti le cadute frequenti della linea telefonica in ciavano pesantemente la qualit didattica ed i costi erano elevati . 
in termini economici basti pensare che una connessione con quattro centri aveva un costo globale di 12000 euro , mentre una connessione punto - punto aveva un costo impianto e gestione pari a 8000 euro . 
la video conferenza tramite satellite ha dei costi poco sostenibili [ 18 , 20 ] sia per la prenotazione del satellite che per la tecnologia di trasmissione ; in una nostra preliminare analisi dei costi , relativa ad un processo di teleconferenza via satellite , abbiamo calcolato che per ogni singola lezione di due ore , tra la prenotazione satellitare e la diffusione con cabina di regia unica su camper mobile , il costo medio si aggira intorno a 3000 euro . 
per streaming si intende la possibilit di veicolare un usso di dati audio / video trasmessi da una sorgente a uno o pi punti di recezione ; il tutto utilizza quale veicolo di diffusione la linea internet . 
lo streaming on demand radiol med ( 2011 ) 116 : 989999 video content is compressed and stored on a server in the form of les , and a user who is connected can request the audio / video content from the server . 
to facilitate distribution of material , the size of video sequences in terms of frames per second needs to be reduced while searching for the appropriate compromise in terms of image and video quality [ 1 , 12 ]  . 
windows media encoder is a highly used encoding application in the windows environment , whereas in linux the most widespread application is mencoder . our pilot e - learning project focused on two priorities ; on the one hand was the need to organise a correct , linear and coherent series of seminars of use to the trainee radiologists and on the other the need for a high - quality product at a reasonable cost . 
in strict teaching terms , the main idea to create a single - theme course on the imaging characteristics of oncological disease proved to be a winning strategy and was particularly appreciated by the residents : 83% found the course excellent , 15% good and 0.3% satisfactory , whereas no participant judged the course to be unsatisfactory . 
this tested single - theme aspect of the course is also fundamental for the purposes of continuing the project , which could develop into a variety of topics over the years . however , in terms of teaching , suggestions from trainee radiologists regarded lesson length . 
in this sense , reducing the length of the lecture to 45 min , to be followed by a session on clinical cases presented by residents at the location of the lecture , could be an alternative solution worth trying out . 
some 7071% of participants judged the overall audiovideo quality to be excellent , which is particularly encouraging considering that small technical modi cations within the individual location could , in the future , have a positive impact at a minimum cost ( optimisation of acoustics in small sound - proofed latest - generation webcams , appropriate video rooms , projectors )  . 
 the entire e - learning process , which also created a minimal sense of cultural belonging , is characterised by presuppone che i contenuti audio / video siano compressi e memorizzati su un server sotto forma di le ed un utente , connesso , possa chiedere al server di ricevere i contenuti audio / video . 
relativamente allattivit didattica in diagnostica per immagini , lencoding costituisce un momento fondamentale ; infatti nelle presentazioni didattiche di diagnostica per immagini la maggiore criticit rappresentata dal peso delle immagini e dai lmati ed necessario disporre di un adeguato programma di encoder . 
per consentire una facile distribuzione del materiale , infatti , necessario ridurre le dimensioni dei lmati , il numero di frame per secondo , trovando un giusto compromesso tra qualit dellimmagine e qualit dei video [ 1 , 12 ]  . 
tutti questi elementi devono essere , necessariamente , ottimizzati , tramite un corretto processo di encoding , in quanto in uenzano , in maniera sensibile , la banda necessaria per poterlo vedere : tra i software dedicati allencoding in ambiente windows , molto diffuso il windows media encoder , mentre in ambito linux il pi diffuso il mencoder . la nostra esperienza pilota di e - learnig ha tenuto conto di due esigenze prioritarie : da una parte la necessit di organizzare una corretta e lineare attivit seminariale organica ed utile ai discenti , dallaltra la necessit di avere un prodotto di elevata qualit a costi contenuti . 
relativamente allaspetto puramente didattico , linput principale di creare un percorso didattico monotematico in tema di semeiotica nella patologia oncologica risultato vincente e particolarmente gradito ai discenti , che nel l83% hanno espresso un giudizio ottimo , nel 15% un giudizio buono e nello 0 , 3% un giudizio suf ciente , senza alcun giudizio insuf ciente . 
 questo aspetto monotematico pilotato , inoltre , fondamentale ai ni del proseguo del progetto che negli anni potrebbe sviluppare diversi argomenti . sempre dal punto di vista didattico , i suggerimenti dei discenti , hanno posto lattenzione sul tempo / lezione ; infatti due ore di lezione sono risultate particolarmente pesanti con calo dellattenzione ; a tal proposito la riduzione del tempo lezione a 45 minuti con unappendice di casi clinici , presentati dai discenti della sede di lezione , potrebbe essere una nuova via didattica suggerita , sicuramente da sperimentare . 
 dal punto di vista tecnico il primo annoso problema delle teleconferenze , cio linterruzione della linea , risultato inin uente ai ni didattici , in particolare le microinterruzioni che si sono veri cate nell1 , 5% dei casi non hanno creato problemi didattici nel complessivo processo di apprendimento . 
il giudizio di qualit complessiva audio - video che , nel 70%71% , stato valutato ottimo , particolarmente incoraggiante , considerato anche il fatto che piccoli accorgimenti tecnici interni alle singole sedi potranno , nel futuro , fare la differenza con un minimo costo ( ottimizzazione dellacustica in sale piccole ed insonorizzate , webcam di ultima generazione , videoproiettori adeguati )  . 998 radiol med ( 2011 ) 116 : 989999 moderate costs . 
in addition , to extend the life of the teaching material , a cd is being prepared that consists of all the lessons , with an associated cost of 2 , 150 euro for 1 , 000 copies . 
this represents a crowning achievement for the entire e - learning process and is particularly important for guaranteeing the continued bene t of this cultural investment . a possible future development of the project is to allow each participant to record the video conferencing lesson , thus creating a le on their own computer containing both the video and audio material . 
in this context , the choice for the moment of not making the lessons available online , despite the possibility of providing password access , is dictated by legislation that fails to protect the concept of copyleft [ 1 , 21 ]  . conclusions the e - learning process has by now become a common feature on the educational scene . 
the innovative nature of e - learning is a useful complement to the traditional educational and training activity ( theoretical and practical ) of each school , which is irreplaceable . 
 the possibility of having the various faculty boards recognise study credits [ creditit formazione universitaria ( cfu ) ] associated with an e - learning project in the setting of specialty - school teaching is a further teaching / institutional goal that could prove to be a valid aid for the teaching body . based on our experience , whereas e - learning in radiology has in our opinion become established and compulsory , there is the need for legislation that on the one hand protects online teaching and on the other allows study and continuing medical education ( cme ) credits to be recognised . lintero processo di e - learnig , che ha anche creato un minimo senso di appartenenza culturale , ha costi contenuti , basti pensare che per ogni lezione avrebbe inciso , su ogni scuola , per un totale di circa 18 euro . 
a latere , al ne di non disperdere il materiale didattico , si sta cercando di creare un cd che possa raccogliere tutte le lezioni , a tal proposito limpatto economico dei costi per costruire il database su cd pari a 2150 euro per 1000 copie ; tale aspetto , a coronamento dellintero processo di e - learnig , particolarmente importante al ne di non disperdere limpegno culturale profuso . in fase di ride nizione del progetto si sta valutando la possibilit , per ogni partecipante , di poter registrare la lezione in videoconferenza , creando un le sul proprio computer che possa contenere sia il materiale video che audio . 
a tal proposito la scelta , al momento , di non mettere in rete le lezioni , malgrado la possibilit di accessi criptati da password , dettata dalla attuale normativa che non tutela il concetto di copyleft [ 1 , 21 ]  . conclusioni il processo di e - learnig fa , ormai , parte integrante della didattica ; esso permette di superare le barriere di diffusione culturale e , con costi particolarmente bassi , permette scambi culturali e condivisioni culturali . 
questa prima esperienza italiana si svolta nello spirito della collaborazione e della sperimentazione , da essa emersa una grande soddisfazione da parte dei docenti e soddisfazione ed entusiasmo da parte dei discenti . 
la didattica in teleconferenza , analogamente a quella dei corsi di aggiornamento post - universitari , migliora complessivamente la qualit della formazione degli specializzandi , che , peraltro , in ciascuna sede deve raggiungere adeguati standard , di cui si deve far carico il direttore con il suo gruppo di docenti . 
cademartiri1 , 2 1dipartimento di radiologia e del cardio - polmonare , azienda ospedaliero - universitaria di parma , c / o piastra tecnica piano 0 , via gramsci 14 , 43100 parma , italy 2dipartimento di radiologia e cardiologia , erasmus medical center , rotterdam , the netherlands 3dipartimento di radiologia e cardiologia , ospedale san gennaro , napoli , italy 4cpc centro prevenzione cardiovascolare , ospedale san raffaele , milano , italy 5dipartimento di radiologia e cardiologia , azienda asl , carrara , italy 6dipartimento di radiologia , universit di messina , messina , italy 7dipartimento di cardiologia , universit di foggia , foggia , italy 8dipartimento di radiologia , azienda ospedaliero - universitaria san martino , genova , italy correspondence to : f . 
a total of 1 , 500 patients ( 928 men , mean age 58.212.5 years ) in sinus rhythm who underwent ctca ( 64 - slice technology ) and cag were enrolled . 
 in the per - patient analysis , sensitivity , speci city , positive predictive value ( ppv ) and negative predictive value ( npv ) of ctca were 99% , 92% , 94% and 99% , respectively . 
obiettivo del nostro lavoro stato valutare laccuratezza diagnostica dellangiogra a coronarica non invasiva con tomogra a computerizzata ( ctca ) nellindividuazione delle stenosi coronariche signi cative ( riduzione del lume coronarico 50% ) confrontata con la coronarogra a convenzionale ( cag ) in base al valore di calcium score ( cacs )  . 
la prevalenza di malattia ostruttiva nella popolazione era del 51% ( 23 , 5% malattia mono - vasale ; 27 , 5% multi - vasale ; con aumento progressivo dal 17 , 9% al 94% nelle diverse classi di cacs )  . 
ctca is a reliable diagnostic modality , with high sensitivity and npv regardless of cacs . keywords ct coronary angiography conventional coronary angiography diagnostic accuracy coronary artery calcium score registry ha mostrato un valore predittivo positivo peggiore ( 76% 77% ) nelle classi di cacs basso ( 110 / 11100 )  . 
la ctca una metodica diagnostica af dabile con elevata sensibilit e valore predittivo negativo indipendentemente dal valore di cacs . parole chiave angiogra a coronarica tc angiogra a coronarica convenzionale accuratezza diagnostica calcio coronarico registro introduction introduzione in the diagnosis and exclusion of coronary artery disease tomography coronary angiography ( cad ) , computed ( ctca ) is an accurate and robust modality that can be performed with low doses of radiation [ 115 ]  . 
 one limitation in evaluating and interpreting coronary artery stenosis with ctca is linked to the presence and quantity of coronary calcium as measured with the coronary artery calcium score ( cacs ) [ 5 , 7 , 1722 ]  . 
however , some studies have shown that a progressively higher cacs tends to reduce the speci city and positive predictive value ( ppv ) rather than cause an overall deterioration in diagnostic performance [ 5 , 7 , 1722 ]  . this report presents a patient population drawn from a multicentre ctca registry and focuses on diagnostic accuracy with conventional coronary angiography ( cag ) as reference . 
 materials and methods registry langiogra a coronarica con tomogra a computerizzata ( ctca ) una indagine accurata e robusta per la diagnosi e lesclusione della malattia coronarica ( cad ) che pu essere effettuata con basse dosi di radiazioni ionizzanti [ 115 ]  . 
 uno dei limiti nella valutazione e nella interpretazione delle stenosi coronariche mediante ctca legato alla presenza ed alla quantit di calcio coronarico ( cacs ) [ 5 , 7 , 1722 ]  . 
parte della letteratura ha cercato di de nire dei valori di cacs al di sopra dei quali non sarebbe opportuno effettuare lo studio angiogra co coronarico mediante tomogra a computerizzata ( tc ) , tuttavia , in alcune casistiche emerge come in realt un cacs progressivamente pi elevato determina una riduzione della speci cit e del valore predittivo positivo pi che un deterioramento globale della performance diagnostica [ 5 , 7 , 1722 ]  . in questo lavoro presentiamo una casistica derivante da un registro multicentrico di ctca focalizzato sullaccuratezza diagnostica della ctca messa a confronto con la coronarogra a convenzionale ( cag ) con particolare attenzione allanalisi della performance diagnostica a seconda del valore di cacs . 
 the participating centres are characterised by : > 300 ctca examinations performed per year ; on - site or directly accessible catheterisation laboratory ; experience of the operators materiali e metodi registro i dati presentati derivano da un registro multicentrico [ 2 ]  . 
i 1002 radiol med ( 2011 ) 116 : 10001013 > 5 years ; ability to maintain a detailed longitudinal prospective database of all the characteristics of patients undergoing ctca ; homogeneous scan protocols and image analysis ( pharmacological preparation of the patient , scan / reconstruction parameters and contrast agent administration ) ; systematic evaluation of the examinations by at least two expert operators in 90% of cases ( double reading )  . study population from may 2004 to june 2010 , 1 , 500 consecutive patients ( 928 men , 572 women ; mean age 58.212.5 years ; median age 59 years ; range 2186 years ; table 1 ) who were candidates for cag were studied with ctca to verify the presence and extension of cad [ 2 ]  . 
with regard to the use of dedicated convolution software , standard lters ( mediumsmooth ) were generally used with cacs < 100 , whereas higher convolution lters ( mediumsharp / sharp ) were used with cacs 100 . 
the ethics committee approved the study , and all patients provided informed consent . statistical analysis data are reported as prevalence , mean and standard deviacentri partecipanti sono caratterizzati da : volume di esami ctca > 300 / anno ; presenza di laboratorio di emodinamica in loco o direttamente afferente ; esperienza degli operatori > 5 anni ; capacit di mantenere un database dettagliato longitudinale prospettico di tutte le caratteristiche dei pazienti che effettuano ctca ; impostazione omogenea dei protocolli di scansione ed analisi delle immagini ( preparazione farmacologica del paziente , parametri scansione / ricostruzione e somministrazione del mezzo di contrasto ) ; valutazione sistematica delle indagini da parte di almeno due operatori esperti nel 90% dei casi ( doppia lettura )  . popolazione studiata da maggio 2004 a giugno 2010 , sono stati studiati mediante ctca 1500 pazienti consecutivi ( 928 di sesso maschile , 572 di sesso femminile , et media 58 , 212 , 5 anni ; mediana 59 anni ; range 2186 anni ) ( tabella 1 ) gi candidati allesecuzione di una cag allo scopo di determinare la presenza e lestensione della malattia coronarica [ 2 ]  . 
il comitato etico ha approvato il protocollo di studio e tutti i pazienti hanno fornito un consenso informato . analisi statistica i dati sono presentati come prevalenze , medie e deviazioni standard . 
la performance diagnostica dellangiogra a coronarica mediante ctca nellindividuazione delle lesioni aterosclerotiche coronariche signi cative , utilizzando la cag come tecnica di riferimento , di seguito riportata come sensibilit , speci cit , valore predittivo positivo ( vpp ) e negativo ( vpn ) con intervalli di con denza ( ic ) al 95% calcolati con espansione binomiale . 
tutte le statistiche sono state eseguite per la popolazione totale e per i sottogruppi con differente cacs ( secondo agatston ) classi cati come segue [ 22 ] : cacs 0 ; cacs 110 ; cacs 11100 ; cacs 101400 ; cacs 401 1000 ; cacs > 1000 . 
the diagnostic performance of ctca in identifying signi cant coronary artery atherosclerotic lesions , with cag used as the reference technique , is reported in terms of sensitivity , speci city , ppv and negative predic1004 radiol med ( 2011 ) 116 : 10001013 tive value ( npv ) , with 95% con dence intervals ( ci ) calculated with binomial expansion . 
all statistics were calculated for the total population and for the classes with different cacs ( agatston score ) , which were classi ed as follows [ 22 ] : 0 ; 110 ; 11100 ; 101400 ; 4011 , 000 ; > 1 , 000 . 
nel gruppo con cacs > 1000 la prevalenza di cad ostruttiva si riduce al 64% come verosimile esito del bias di selezione dei pazienti ( ossia vengono inviati a ctca maggiormente i pazienti nei quali esistono dubbi sulla effettiva presenza di cad ostruttiva )  . 
la speci cit ha mostrato valori meno elevati nei gruppi con cacs > 1000 ( speci cit = 87% ) , ed il valore predittivo positivo ha mostrato valori meno elevati nei gruppi con cacs > 1000 ( vpp = 53% )  . 
la speci cit ha mostrato valori meno elevati nei gruppi con cacs 4011000 ( 77% ) , mentre il valore predittivo positivo ha mostrato valori meno elevati nei gruppi con cacs 0 ( 76% ) e cacs 110 ( 78% )  . 
the literature published to date provides indications that are partly in con ict regarding the use of the total calcium score ( agatston ) for de ning the threshold above which angiographic scans would be inadvisable [ 5 , 7 , 1722 , 2831 ]  . 
on the one hand , to limit the number of false positives , there is an attempt to provide a threshold above which ctca is not recommended [ 5 , 7 , 1722 , 2831 ] , whereas on the other hand , ctca is generally performed to rule out disease following an inconclusive stress test [ 16 , 32 ]  . 
le coronarie sono , infatti , vasi di piccole dimensioni ed il blooming delle calci cazioni ha un impatto maggiore rispetto ad altri vasi di maggiori dimensioni ( per esempio , arterie carotidi interne )  . il ruolo clinico del cacs consiste nella strati cazione del rischio di cad ostruttiva e di eventi coronarici in maniera incrementale , caratteristica non sempre rispettata dai metodi convenzionali come lo score di framigham [ 2427 ]  . 
 la letteratura pubblicata no ad oggi fornisce indicazioni in parte contrastanti sullutilizzo del valore di calcio totale secondo agatston per de nire la soglia oltre la quale non sarebbe opportuno procedere alla scansione angiogra ca [ 5 , 7 , 1722 , 2831 ]  . 
da un lato si vorrebbe fornire una soglia oltre la quale la ctca non andrebbe fatta per limitare il numero di falsi positivi [ 5 , 7 , 1722 , 2831 ] , dallaltro bisogna considerare che la ctca viene generalmente effettuata come test per esclusione di malattia che segue un test provocativo dubbio [ 16 , 32 ]  . 
bisogna considerare , tuttavia , che laumento di cacs si associa allaumento della prevalenza di malattia coronarica ostruttiva e ad un aumento della severit ed estensione della coronaropatia ( vale a dire , una maggiore prevalenza di coronaropatia ostruttiva multi - vasale )  . 
with regard to obstructive disease , a higher prevalence of three - vessel disease can be seen in the extreme cacs classes , which re ects , on the one hand , the selection bias of the patients sent for computed tomography coronary angiography ( cacs < 10 ) and , on the other hand , the natural history of the disease ( cacs > 400 )  . 
focalizzando la valutazione sulla malattia ostruttiva , si osserva una prevalenza di malattia tri - vasale pi elevata nelle classi estreme di cacs che ri ette da un lato il bias di selezione dei pazienti inviati a ctca ( cacs < 10 ) e dallaltro la storia naturale della malattia ( cacs > 400 )  . 
this effect partially attenuates the overestimation of obstructive cad by ctca when shifting from a per - segment to a per - patient analysis . the results of our study based on a large population con rm these observations . 
the prevalence of obstructive cad is undoubtedly higher in patients with a high cacs , but ctca nonetheless is not an appropriate technique for establishing the presence and extent of ischaemia . 
even though the diquesto effetto , in parte , attenua la sovrastima della malattia coronarica ostruttiva da parte della ctca nel passaggio dallanalisi per segmento allanalisi per paziente . i risultati del nostro studio basati su una popolazione ampia confermano queste osservazioni . 
3 per - segment and per - patient diagnostic accuracy of coronary artery computed tomography ( ctca ) in the total population and in the coronary artery calcium score ( cacs ) classes . 
note the low positive predictive value ( ppv ) of the technique in the per - segment evaluation ( upper panel ) , which appears normalised in the per - patient evaluation ( lower panel )  . 
gra co a barre dellaccuratezza diagnostica per segmento e per paziente della angiogra a coronarica con tomogra a computerizzata ( ctca ) nella popolazione totale , e nelle classi di calcium score coronarico ( cacs )  . 
da notare il basso valore predittivo positivo della metodica nella valutazione per segmento ( pannello in alto ) che si normalizza nella valutazione per paziente ( pannello in basso )  . 
 nellanalisi per paziente il valore pi sfavorevole di valore predittivo positivo ( vpp ) si osserva nelle classi a basso cacs ( per esempio , cacs 110 / 11 100 )  . 
on the other hand , in line with the current recommendations , the patient who undergoes ctca will also have been evaluated with at least one other imaging test , and the examination would aim to rule out disease given its npv [ 16 ]  . 
 mance diagnostica della ctca viene in parte deteriorata nelle classi ad elevato cacs , in particolare in termini di speci cit e valore predittivo positivo , i valori rimangono in un range accettabile ( speci cit > 77% e vpp > 76% ) per lapplicabilit clinica della metodica in una popolazione adeguatamente selezionata . i valori quozienti di probabilit ( lr + e lr - ) sono ottimali nei gruppi con cacs basso ed questo un ulteriore punto di forza della metodica che pu essere sfruttato nellambito degli algoritmi diagnostici decisionali clinici . 
daltra parte , seguendo le attuali raccomandazioni , il paziente che arriva a ctca dovrebbe essere stato valutato con almeno un altro test strumentale e lindagine si porrebbe come strumento di esclusione per il suo valore predittivo negativo [ 16 ]  . 
4 likelihood ratios ( lr ) show how weighting of the diagnostic accuracy by disease prevalence produces a reduction in the lr + and lr when going from a per - segment analysis to a per - patient analysis . 
il gra co a barre dei quozienti di probabilit ( lr ) dimostra come la pesatura dellaccuratezza diagnostica per la prevalenza di malattia determini una riduzione del lr + e del lrnel passaggio dalla valutazione per - segmento a quella per - paziente . 
i valori migliori di lr + si ottengono nella popolazione con calcium score coronarico ( cacs ) 0 sia nella valutazione per segmento che in quella per paziente . limitations to the study limiti allo studio our case series reports data recorded in the period when the most modern dose - reduction systems were not available [ 912 ]  . 
tuttavia , questa anche la forza delle informazioni presentate in quanto molto pi realistiche rispetto agli studi convenzionali in condizioni ottimali ( per esempio , pazienti ben selezionati , possibilit di valutazione delle immagini estesa nel tempo , ecc )  . conclusions conclusioni data in our registry demonstrate that ctca is a robust and reliable morphological modality for evaluating obstructive cad in the population , regardless of cacs . 
our data therefore suggest that the introduction of cacs thresholds i dati del nostro registro dimostrano che la ctca una metodica morfologica per la valutazione dellaterosclerosi coronarica ostruttiva robusta ed af dabile nella popolazione indipendentemente dalla quantit di cacs . 
computed tomography coronary angiography ( ctca ) is positive , the probability that a patient effectively has coronary stenosis 50% increases signi cantly , even if the pretest probability is low or very low ( range 020% )  . 
il gra co mostra le probabilit condizionali nella popolazione totale e nelle varie classi di calcium score coronarico ( cacs ) , utilizzando i valori di accuratezza diagnostica e la prevalenza di malattia per paziente . 
 per un test positivo aumenta signicativamente la probabilit che un paziente sia effettivamente portatore di una stenosi coronarica 50% anche con probabilit pre - test bassa o molto bassa ( range 0%20% )  . 
the intrinsic design of the registry tends to better re ect clinical practice and it characteristic variability . non sembra quindi opportuno introdurre soglie di cacs oltre le quali non effettuare la scansione angiogra ca . 
stolzmann p , leschka s , scheffel h et al ( 2008 ) dual - source ct in step - andshoot mode : noninvasive coronary angiography with low radiation dose . 
scheffel h , alkadhi h , leschka s et al ( 2008 ) low - dose ct coronary angiography in the step - and - shoot mode : diagnostic performance . 
achenbach s , marwan m , ropers d et al ( 2010 ) coronary computed tomography angiography with a consistent dose below 1 msv using prospectively electrocardiogramtriggered high - pitch spiral acquisition . 
maffei e , martini c , de crescenzo s et al ( 2010 ) low dose ct of the heart : a quantum leap into a new era of cardiovascular imaging . 
hendel rc , patel mr , kramer cm et al ( 2006 ) accf / acr / scct / scmr / asnc / nasci / scai / sir 2006 appropriateness criteria for cardiac computed tomography and cardiac magnetic resonance imaging : a report of the american college of cardiology foundation quality strategic directions committee appropriateness criteria working group , american college of radiology , society of cardiovascular computed tomography , society for cardiovascular magnetic resonance , american society of nuclear cardiology , north american society for cardiac imaging , society for cardiovascular angiography and interventions , and society of interventional radiology . 
cademartiri f , mollet nr , lemos pa et al ( 2005 ) impact of coronary calcium score on diagnostic accuracy for the detection of signi cant coronary stenosis with multislice computed tomography angiography . 
cornalba1 1dipartimento di radiologia diagnostica ed interventistica , universit degli studi di milano , ospedale san paolo , via di rudin 8 , 20142 milano , italy 2unit di radiologia , irccs policlinico san donato , piazza e . 
malan , san donato milanese , milano , italy 3dipartimento di scienze medico - chirurgiche , universit degli studi di milano , via festa del perdono 7 , milano , italy correspondence to : s . 
forty - nine patients ( 30 men , 19 women ; age range 3082 years ) underwent abdominal contrast - enhanced msct and 3d - us performed with a 3.5 - mhz 3d / 4d convex - array probe . 
analysis of variability per patient between msct and 3d - us showed a bias of 19 ml , a cor of 47 ml and an accuracy of 78% , with an average 3d - us underestimation of 19 ml ( 9% )  . 
three - dimensional us is a valuable technique for monitoring renal volume , whereas msct may be reserved for assessing renal anatomy and relationships with neighbouring organs . keywords ultrasound 3dmsct renal volume riassunto obiettivo . 
quarantanove pazienti ( 30 maschi ; 19 donne ; et 3082 ) , sono stati sottoposti a tcms delladdome con somministrazione di mezzo di contrasto ( mdc ) ed a ecogra a con sonda convex volumetrica 3d / 4d con tecnologia elettromeccanica . 
la volumetria 3d rappresenta una valida metodica nel monitoraggio del volume renale nel tempo , mentre la tcms pu essere riservata ai casi in cui occorra una valutazione panoramica dellanatomia renale e dei rapporti con gli organi circostanti . parole chiave ecotomogra a 3d volumetria renale tcms 1096 introduction determining renal volume is important in numerous clinical situations : during treatment for renal failure , in the followup after renal transplant [ 1 , 2 ] , in the evaluation of renal artery stenoses [ 3 ] , in renovascular hypertension [ 4 ] , in recurrent urinary tract infections [ 5 ] , in the follow - up after unilateral nephrectomy [ 5 ] , in young patients with vesicoureteral re ux [ 6 , 7 ]  . 
given that some treatment decisions are based on serial measurements of renal volume , which provide information about disease progression , stability or regression or about the onset of complications , an accurate and noninvasive method is needed that preferably does not use ionising radiation or contrast material [ 8 ]  . ever since ultrasound ( us ) was described as a useful modality for calculating renal volume [ 4 , 5 , 9 , 10 ] , two techniques have been suggested : measurement of the largest renal diameter [ 10 , 11 ] , and estimation of renal volume based on the ellipsoid formula [ 4 , 5 ]  . 
however , it has been reported that these techniques tend to underestimate renal volume , as measurement of the largest diameter is poorly reproducible and does not correlate with true kidney size [ 4 , 5 ] , whereas the ellipsoid formula fails to take into account the variability of the shape of the kidney [ 5 , 10 ]  . 
in addition , these methods are highly operator dependent , as they involve manually performing the measurements needed to determine renal volume [ 8 ]  . multislice computed tomography ( msct ) [ 12 ] and magnetic resonance imaging ( mri ) , with their ability to analyse volumetric data , are more accurate in determining the volume of abdominal organs [ 8 , 1317 ]  . 
the introduction of 3d - us imaging with dedicated probes allowing direct and automatic breath - hold acquisition of kidney sections reconstructed in the axial , sagittal and coronal planes [ 8 , 1320 ] has opened up new possibilities , in part thanks to limited costs , noninvasiveness , absence of ionising radiation and consequently contraindications . 
 the purpose of our study was to assess the accuracy of 3d - us with a dedicated volumetric probe for determining renal volume in comparison with msct , which is considered the standard of reference . materials and methods population radiol med ( 2011 ) 116 : 10951104 introduzione la determinazione del volume renale assume un ruolo importante in numerose situazioni cliniche : in corso di trattamento dei pazienti con insuf cienza renale , nel follow - up dei pazienti con trapianto renale [ 1 , 2 ] , nella valutazione delle stenosi delle arterie renali [ 3 ] , nellipertensione renovascolare [ 4 ] , nelle infezioni urinarie ricorrenti [ 5 ] , nella valutazione dei pazienti dopo nefrectomia monolaterale [ 5 ] e nei giovani pazienti con re usso vescico - ureterale [ 6 , 7 ]  . 
 dal momento che alcune decisioni terapeutiche possono derivare da misurazioni seriali del volume renale , le quali forniscono informazioni sulla progressione , stabilit o regressione della patologia esistente o sulla insorgenza di eventuali complicanze , si rende utile una metodica che sia accurata , non invasiva e che preferibilmente non utilizzi radiazione ionizzanti o mezzo di contrasto [ 8 ]  . 
 da quando lecogra a stata descritta come metodica utilizzabile per la valutazione del volume renale [ 4 , 5 , 9 , 10 ] sono state proposte due tecniche : la misura del diametro massimo renale [ 10 , 11 ] e la stima del volume renale mediante la formula dellellissoide [ 4 , 5 ]  . 
da quanto riportato in letteratura , con queste differenti tecniche il volume renale appare sottostimato poich la misura del diametro massimo poco riproducibile e non correlata con le reali dimensioni renali [ 4 , 5 ] , mentre la formula ellissoidale non tiene conto della variabilit della forma del rene [ 5 , 10 ]  . 
inoltre , queste tecniche con acquisizione manuale delle varie misure necessarie alla determinazione del volume sono molto operatore dipendenti [ 8 ]  . attualmente la possibilit da parte della tomogra a computerizzata multistrato ( tcms ) [ 12 ] e della risonanza magnetica ( rm ) di analizzare dati volumetrici rende queste tecniche pi accurate nella valutazione dei volumi dei vari organi addominali [ 8 , 1317 ]  . 
la comparsa nella pratica clinica della ecotomogra a tridimensionale ( eco 3d ) con sonda dedicata che permette , durante una singola apnea , lacquisizione diretta ed automatica di sezioni del rene ricostruite nei piani assiali , sagittali e coronali [ 8 , 1320 ] ha aperto una nuova possibilit anche in ragione dei costi contenuti , della non invasivit , dellassenza di radiazioni ionizzanti e , quindi , della mancanza sostanziale di controindicazioni . 
 lo scopo di questo studio stato valutare laccuratezza dellecotomogra a 3d nella misurazione del volume renale utilizzando una sonda volumetrica dedicata e considerando la tcms come standard di riferimento . our study was conducted on 49 consecutive patients ( 30 men and 19 women , age range 3082 years ; mean age 68 years ) who underwent abdominal msct for different clinical indications . 
all patients enrolled in the study were contacted after the ct examination , and informed consent for the 3d - us study was obtained in all cases . acquisition protocol and image analysis all patients had undergone abdominal ct , with scans extending from the upper pole of the kidney to the bladder base ( 120 kvp , automatic mas modulation , 5 - mm slice thickness )  . 
all examinations were performed with a 64 - slice lightspeed vct scanner ( general electric medical system , milwaukee , wi , usa ) following automatic administration of 90110 ml of iomeprol ( iomeron ) at 370 mg / ml iodine concentration . 
 the probe had 192 elements on a single matrix , 40.5 mm radius of curvature , 68 eld of view , 8580 acquisition volume , upper frame rate 35 f / s . 
to understand the cor , consider the following scenario : let us suppose that a patient undergoes ct with renal volume determination and that 6 months later , the determination is repeated with us instead of ct . 
abbiamo deciso di reclutare nello studio solo pazienti con anamnesi negativa per patologia renale allo scopo di testare la metodica su una popolazione priva di alterazioni patologiche che potessero essere considerate un fattore condizionante la misurazione . 
tutti i pazienti arruolati nello studio sono stati contattati al termine della loro indagine tc ed stato acquisito consenso informato allesecuzione dellindagine ecogra ca . protocollo di acquisizione ed analisi di immagine tutti i pazienti avevano effettuato lindagine tc delladdome con scansioni estese dal polo renale superiore al pavimento vescicale ( 120 kvp , modulazione automatica dei mas , spessore di strato 5 mm )  . 
tutte le indagini sono state effettuate con tomografo lightspeed vct 64 strati ( general electric medical system , milwaukee , wi , usa ) e con somministrazione automatica da 90 a 110 ml di iomeron 370 mg / ml . 
tutte le indagini sono state eseguite con un ecotomografo logic q9 ( general electric medical system , milwaukee , wi , usa ) mediante utilizzo di sonda convex volumetrica 3d / 4d con tecnologia elettromeccanica per le acquisizioni volumetriche dotata di banda da 1 , 55 mhz . 
la sonda presenta 192 elementi su singola matrice di elementi , raggio di curvatura 40 , 5 mm , apertura campo di vista 68 , volume di acquisizioni 8580 , frame rate superiore a 35 f / s . 
furthermore , we measured the ratio between this coef cient and the mean renal volume and its complement to 1 ; considering ct as the standard of reference , the value obtained represents the accuracy of 3d - us . 
la variabilit tra le due metodiche di misura stata stimata col metodo bland - altman ed stata riportata in termini di bias e coef ciente di ripetibilit ( cor )  . 
supponiamo che un paziente esegua una tomogra a computerizzata con misura del volume renale e che , dopo un intervallo di sei mesi , ripeta tale misura con ecogra a anzich con tomogra a computerizzata . 
inoltre , abbiamo calcolato il rapporto tra tale coef ciente e il volume renale medio e il suo complemento a 1 : considerando la tc come standard di riferimento , il valore cos ottenuto rappresenta radiol med ( 2011 ) 116 : 10951104 1099 fig . 
analysis of variability per kidney yielded the following results : bias 9 ml , cor 34 ml and accuracy 80% for the right kidney ( n = 48 ) ; 29 ml , 50 ml and 80% , respectively , for the radiol med ( 2011 ) 116 : 10951104 laccuratezza della ecotomogra a 3d . 
inoltre , stata valutata la correlazione bivariata tra lindice di massa corporea ( imc ) , et del paziente , peso e volume medio bilaterale mediante il coef ciente di correlazione di spearman . 
 tutti i calcoli sono stati eseguiti con spss v.17 ( spss inc , chicago , il , usa ) e valori di p < 0 , 05 sono stati considerati come signi cativi . risultati popolazione i dati demogra ci del campione analizzato sono indicati in tabella 1 . 
i dati di 5 reni ( 2 reni di destra e 3 reni di sinistra ) di 5 pazienti diversi sono stati esclusi perch presentano misure estreme , palesemente outlier , distribuite in modo casuale e , pertanto , non costituenti un possibile bias . 
non si osservata alcuna correlazione signi cativa tra limc , et del paziente , peso e volume renale . discussione la stima del volume renale rappresenta un parametro clinico importante nella valutazione della riserva funzionale renale , ed generalmente alla base di alcune decisioni terapeutiche . 
misurazioni seriate del volume renale possono fornire informazioni sulla funzione di ltrazione dellorgano ed assumono particolare importanza nel corso del follow - up per stabilire leventuale stabilit o la progressione della malattia [ 8 ]  . 
 dallanalisi dei dati presenti in letteratura mediante valutazione ecogra ca con la tecnica dellellissoide il volume renale nel maschio compreso tra 110 e 190 ml e nella donna tra 90 e 150 ml [ 21 ]  . 
no signi cant correlation was observed between bmi , age , weight and renal volume . discussion determining renal volume is an important clinical parameter in assessing renal functional reserve and is generally the basis for several treatment decisions . 
serial measurements of renal volume can provide information on renal metodica adeguata per la misurazione del volume renale utilizzando la formula dellellissoide , nonostante sia emerso dal confronto con altre tecniche che tale metodica determina una sottostima del volume compreso tra il 3% ed il 44% [ 9 , 11 , 18 ]  . 
possibili spiegazioni per la scarsa accuratezza della volumetria renale possono essere le seguenti : linadeguata visualizzazione dellorgano dovuta alla presenza di interposto gas intestinale o strutture ossee ( coste ) che limitano la nestra acustica , la non corretta misurazione manuale dei diametri renali , la presenza fig . 
4 gra co di bland - altman per il confronto tra tomogra a computerizzata ed ecotomogra a 3d . 1102 radiol med ( 2011 ) 116 : 10951104 ltration and are especially important during patient followup to assess disease stability or progression [ 8 ]  . the literature regarding sonographic determination of renal volume with the ellipsoid formula indicates renal volumes of 110190 ml in men and 90150 ml in women [ 21 ]  . 
on ct , the mean volume is 193 ml for men and 157 ml for women [ 8 ] , whereas on mri , it ranges from 132 to 276 ml in men and from 87 to 223 ml in women [ 8 ]  . several published studies indicate that us is an adequate technique for determining renal volume using the ellipsoid formula , even though comparison with other modalities has revealed an underestimation of 344% [ 9 , 11 , 18 ]  . 
the reasons for the low us accuracy in renal volume determination may include inadequate visualisation of the organ due to interposed bowel gas or bony structures ( ribs ) limiting the acoustic window , incorrect manual measurements of renal diameters , presence of respiratory artefacts and patient body habitus [ 20 ]  . 
despite this known underestimation , us has been considered adequate for clinical needs given that absolute renal volume is not as important as its variations over time . according to the literature , the 3d technique appears to produce an underestimation of renal volume [ 19 ] , even though this underestimation is lower than that of the ellipsoid formula [ 19 ]  . 
in our study conducted on 49 patients and a total of 98 kidneys , 3d - us revealed higher values compared with msct in 11 cases only ( 11% ) , with a mean difference of 17 ml ; in the remaining 87 cases ( 89% ) , msct yielded higher values ( mean difference 24 ml )  . 
this resulted in 78% accuracy and an average underestimation of 19 ml , which , in relation to the mean values of msct ( 210 ml ) , corresponds to a mean underestimation of 9% , which is consistent with previous ndings [ 9 , 1118 ]  . 
these data are also con rmed by direct comparison between mean values of renal volume measured at msct ( 210 ml ) and those measured at 3d - us ( 192 ml ) : the wilcoxon test yielded a p value < 0.001. the underestimation observed in our study must be understood as a mean value : in clinical practice , this underestimation may be compensated for by adding 9% to the renal volume obtained at 3d - us . 
our study showed cor values of 47 ml , which implies that a difference < 47 ml between the volumes measured with the two techniques ( rst examination and follow - up ) in the individual patient cannot be necessarily interpreted as progression of the clinical condition . the analysis per kidney did not reveal any signi cant difference in accuracy with regard to determining right and left renal volumes : in fact , although the mean underestimation was 9 ml and 29 ml , respectively , in terms of accuracy , we obtained values of 80% in both kidneys . 
tuttavia , nonostante il riconoscimento di questa sottostima , la metodica ecogra ca stata considerata comunque adeguata alle necessit cliniche in quanto non risulta fondamentale il valore assoluto del volume renale ma la sua variazione nel tempo . da quanto noto in letteratura , anche la tecnica 3d appare caratterizzata da una sottostima del valore del volume renale [ 19 ] , ma tale sottostima risulta inferiore a quella che si veri ca con la tecnica dellellisoide [ 19 ]  . 
 ne risultata unaccuratezza del 78% ed una sottostima media di 19 ml che , relativamente al valore medio misurato alla tcms ( 210 ml ) , corrisponde ad una sottostima media del 9% , compatibile con quanto riportato in letteratura [ 9 , 1118 ]  . 
questo dato , inoltre , confermato dal confronto diretto tra la media del volume renale misurato alla tcms ( 210 ml ) e quello misurato alla ecotomogra a 3d ( 192 ml ) : il test di wilcoxon ha fornito un valore di p < 0 , 001 . la sottostima osservata nel nostro studio deve essere intesa come un valore medio : nella pratica clinica , questa sottostima pu essere compensata sommando al volume renale ottenuto alla ecotomogra a 3d il 9% del suo valore . 
dal nostro studio risultato cor = 47 ml , ovvero , nel singolo paziente , differenze tra il volume misurato con le due metodiche ( prima indagine e follow - up ) inferiori a 47 ml non possono essere interpretate necessariamente come progressione dello status clinico del paziente . allanalisi per singolo rene , non emersa una sostanziale differenza di accuratezza nella valutazione del rene destro e del rene sinistro : infatti , sebbene la sottostima media sia risultata di 9 ml e 29 ml , rispettivamente , in termini di accuratezza abbiamo ottenuto un valore pari all80% in entrambi i reni . 
tuttavia , la differenza tra il volume del rene destro misurato alla tcms ( 174 ml ) e quello misurato alla ecotomogra a 3d ( 165 ml ) altamente signi cativa ( p < 0 , 001 ) ; analogamente per il rene sinistro ( 250 ml versus 220 ml , p < 0 , 001 )  . 
lastly , with both techniques , we observed a signi cant difference between mean right and left renal volume ( p < 0.001 , table 2 )  . even assuming that msct [ 22 , 23 ] and mri [ 24 , 25 ] provide an accurate and reproducible estimation of renal volume that is not in uenced by geometric factors [ 8 ] , it should nonetheless be considered that these modalities have some limitations , such as limited availability of equipment , cost of the single examination , long imaging times or use restrictions for mri and use of ionising radiation for ct . 
as a result , us represents a valuable alternative due to the absence of contraindications , its fast examination times and its limited cost per examination , which contribute to making it an ideal , frequently repeatable , examination . 
furthermore , the possibility of using a dedicated probe to obtain the scans needed for volumetric measurement during a single breathhold reduces the incidence of respiratory artefacts [ 8 , 1317 ]  . 
finally , the possibility of obtaining automatic volumetric scans reduces operator dependence , thus improving the potential for reproducibility of the technique . accurata e riproducibile del volume renale non in uenzata da fattori geometrici [ 8 ] , bisogna tuttavia considerare che queste metodiche presentano alcune limitazioni , a partire dalla limitata disponibilit delle apparecchiature ai costi del singolo esame , dalla durata dellesame per la rm alla impossibilit di eseguirla in certe condizioni , dallutilizzo delle radiazioni ionizzanti per la tc . 
tali limitazioni risultano ulteriormente accentuate in quanto lindagine deve essere necessariamente ripetuta pi volte nel tempo nel corso dei vari follow - up per ottenere un monitoraggio della patologia adeguato alle necessit . 
la tecnica ecogra ca conseguentemente rappresenta una valida alternativa per lassenza di controindicazioni , per la velocit di esecuzione e per i costi contenuti per singolo esame , che la rendono ottimale come indagine da ripetere frequentemente nel corso del tempo nello stesso soggetto . 
la possibilit di utilizzare una sonda dedicata che effettui in ununica apnea le scansioni necessarie alla misurazione volumetrica consente inoltre di ridurre gli artefatti respiratori [ 8 , 1317 ]  . 
la possibilit di ottenere la scansione volumetrica in modo automatico permette in ne di ridurre la componente di dipendenza dalloperatore migliorando le potenzialit di riproducibilit della metodica . conclusioni conclusions bearing in mind the almost systematic underestimation 9% , 3d volumetry represents a valuable technique for renal volume determination . 
renal volume determination with msct or mri can be reserved for cases requiring a more precise and accurate assessment and a panoramic overview of the anatomy of the kidney and its relationships with neighbouring organs . tenendo conto della sottostima pressoch sistematica del 9% del volume renale , la volumetria 3d rappresenta una valida metodica nella stima del volume renale . 
fugazzola1 1universit dellinsubria , dipartimento di diagnostica per immagini , ospedale di circolo fondazione macchi , viale borri 57 , 21100 varese , italy 2cattedra di radiologia , dipartimento di scienze per la salute , universit del molise , campobasso , italy correspondence to : g . 
la rfa con effetto citoriduttivo , in pazienti con tumore della mammella rappresenta unef cace metodica di trattamento , in aggiunta alla chemioterapia sistemica e allormonoterapia , delle metastasi epatiche . 
 rfa rappresenta una valida alternativa alla chirurgia nel trattamento loco - regionale delle metastasi da tumore mammario . keywords radiofrequency thermal ablation ( rfa ) breast cancer metastasis parole chiave termoablazione con radiofrequenza ( rfa ) tumore della mammella metastatico 1060 introduction liver metastases have been identi ed at autopsy in as many as 5575% of patients with breast cancer [ 1 ]  . 
 a small group of patients with metastasis con ned to the liver ( 512% ) appear to have an improved prognosis after hepatic resection , and association with hormonal or chemotherapy can be a more effective treatment option [ 36 ]  . in the last 10 years , radiofrequency thermal ablation ( rfa ) has gained wide acceptance for treating patients with nonresectable primary and metastatic liver tumours . 
 rfa has been demonstrated to be an effective cytoreductive strategy for a variety of metastatic liver malignancies , with few side effects and little disruption of patients life [ 711 ]  . 
 the aim of this study was to evaluate the effectiveness of rfa in patients with liver metastasis from breast cancer and its impact on survival . materials and methods between november 2003 and october 2007 , 13 female patients ( age range 3682 years ; median 54.5 years ) underwent rfa at our institution to treat 21 liver metastases from breast cancer . 
inclusion criteria were : ( 1 ) up to three metastases with diameter 7 cm ; ( 2 ) no evidence of extrahepatic disease or stable extrahepatic disease for at least 3 months ; ( 3 ) platelet count > 35 103 / l ; ( 4 ) international normalised ratio ( inr ) < 1.5. 
patient characteristics are listed in tables 1 and 2 . diagnostic workup included contrast - enhanced abdominal computed tomography ( ct ) scan and a biopsy under ultrasound ( us ) guidance . 
all patients were treated with prophylactic antibiotic therapy consisting of cefazolin 1 g iv every 8 h starting 3 days before the procedure and continuing up to 2 days after it . radiol med ( 2011 ) 116 : 10591066 introduzione il 55%75% delle pazienti affette da tumore mammario presenta allesame autoptico metastasi epatiche [ 1 ]  . 
la chemioterapia rimane lopzione terapeutica di scelta per le pazienti con malattia metastatica diffusa , mentre nelle pazienti ( 5%12% ) [ 2 ] con localizzazione metastatica limitata al fegato , si osservata una prognosi migliore dopo resezione epatica in associazione a chemioterapia e ormonoterapia [ 36 ]  . nellultimo decennio , termoablazione mediante radiofrequenza ( rfa ) si dimostrata una terapia diffusamente accettata per il trattamento dei tumori e utilizzata sui tumori epatici metastatici : ha un ef cace potere citoriduttivo con pochi effetti collaterali ed un impatto positivo sulla qualit di vita delle pazienti [ 711 ]  . 
lobiettivo del nostro studio quello di valutare lef cacia della rfa epatica in pazienti affette da metastasi epatiche da carcinoma mammario in chemioterapia o ormonoterapia e il suo impatto sulla sopravvivenza . materiali e metodi tra novembre 2003 e ottobre 2007 , presso il nostro centro , 13 pazienti ( et 3682 anni , mediana 54 , 5 anni ) sono state sottoposte a terapia ablativa tramite radiofrequenza per il trattamento di 21 metastasi epatiche secondarie a tumore mammario . 
i criteri di inclusione nello studio sono stati : limite di 3 metastasi con diametro massimo inferiore o uguale a 7 cm ; nessuna evidenza di malattia extraepatica o malattia extraepatica stabile da almeno 3 mesi ; conta piastrinica > 35103 / l ; international normalized ratio ( inr ) < 1 , 5 . 
prima del trattamento con rfa , 6 / 13 pazienti sono state sottoposte a terapia chemioterapica , 6 / 13 a terapia ormonale e 1 / 13 sia a terapia sia chemioterapica che ormonale . 
le caratteristiche delle pazienti sono elencate nelle tabelle 1 e 2 . le indagini diagnostiche comprendono una tomogra a computerizzata ( tc ) addominale ( aquilion 64 , toshiba ) con mezzo di contrasto e una biopsia sotto guida ecogra ca ( iu22 , philips )  . 
ablations were performed using la procedura ablativa stata condotta in sedazione profonda , in accordo con le linee guida della monitored anaesthesia care : alle pazienti sono stati somministrati propofol ( 50120 mg ) , fentanyl ( 13 mg ) e midazolam ( 13 mg ) ; nella sede dingresso dellago stata effettuata unanestesia locale con lidocaina 1% . 
the choice of electrode size was determined in relation to tumour dimensions , and the safety margin was 5 mlesions with a diameter 3 cm were treated with two or more overlapping ablation treatments , up to a maximum of four for the largest lesion . 
technique effectiveness was evaluated by contrast - enhanced ct at 1 month : complete ablation was considered when no enhancement was observed in the ablated tumour ; irregular or nodular peripheral contrast enhancement was considered as residual tumour [ 12 ]  . 
la dimensione degli elettrodi stata stabilita in relazione alla dimensione della lesione ed al margine di sicurezza di 5 mle lesioni con diametro superiore a 3 cm sono state trattate con 2 o pi trattamenti ablativi sovrapposti , no ad un massimo di 4 per la lesione con diametro maggiore . 
il follow - up stato effettuato mediante tc a 1 , 3 , 6 e 12 mesi . il successo tecnico stato de nito come il corretto posizionamento dellelettrodo durante la procedura . 
lef cacia tecnica stata valutata tramite tc con mezzo di contrasto effettuata ad un mese dallintervento : la mancanza di enhancement nellarea interessata della lesione ablata stata considerata segno di completa ablazione ; enhancement irregolare o periferico stato considerato come tumore residuo [ 12 ]  . 
il metodo di kaplan - meier stato usato per valutare il tasso di insorgenza della prima metaradiol med ( 2011 ) 116 : 10591066 1063 medcalc version 9.4.2.0 ( medcalc software , mariakerke , belgium ) was used for the analyses . results technical success was 100% , and no major or minor complications occurred at the end of the procedure . 
mean follow - up was 12.9 ( range 435 ) months ; at the last follow - up at 12 months , three patients had died for extrahepatic spread of disease , whereas ten were still alive . 
la durata media del follow - up stata di 12 , 9 mesi ( range 435 mesi ) ; allultimo follow - up a 12 mesi , 3 pazienti erano morte per diffusione extraepatica della malattia , mentre 10 erano ancora in vita . 
nella nostra casistica 7 / 21 lesioni in 7 / 13 pazienti sono aumentate dimensionalmente 7 , 18 , 19 e 38 mesi in ordine cronologico ; lintervallo libero da progressione di malattia stato in media di 16 , 6 mesi . 
il 50% della frequenza di comparsa si rileva a 18 mesi , e la curva scende a zero a 38 mesi . 1064 radiol med ( 2011 ) 116 : 10591066 fig . 
most patients are not suitable for surgery because of their poor general condition or the stage of disease . medical therapy for metastatic breast cancer consists of chemotherapy or endocrine therapy because even in the case of isolated metastases in one organ , diffuse tumour - cell dissemination exists . 
in determining technique effectiveness , size of the index tumour and proximity to larger branches of the hepatic and portal veins are also believed to be factors of donne affette da tumore della mammella svilupper metastasi epatiche . 
la resezione chirurgica talvolta associata alla chemioterapia rappresenta lopzione terapeutica di scelta in pazienti selezionate , con singola metastasi epatica e basso rischio chirurgico ; tuttavia solo nel 4%5% delle pazienti si evidenzia una sola metastasi epatica da tumore mammario . 
 la maggior parte delle pazienti non possono essere candidate ad un intervento chirurgico a causa delle loro condizioni generali e dello stato avanzato della malattia . la terapia farmacologia per il tumore della mammella metastatico consiste nella chemioterapia e nelle endocrinoterapia perch anche nel caso di singola lesione metastatica localizzata in un organo , il rischio di disseminazione delle cellule tumorali esiste ; in questo caso la sopravvivenza varia dai 3 ai 6 mesi , e raramente supera i 2 anni [ 17 , 18 ]  . 
attualmente la rfa rappresenta un trattamento alternativo nella gestione delle pazienti ad elevato rischio chirurgico e in quelli con patologia extra epatica stabile , che non sono candidati alla resezione chirurgica . 
la rfa il trattamento complementare di scelta ( in alternativa alla chirurgia ) alla terapia sistemica nelle metastasi epatiche e nei pazienti che hanno sviluppato malattia epatica dopo chemioterapia [ 15 ]  . 
 negli studi esaminati lef cacia tecnica varia dal 79% al radiol med ( 2011 ) 116 : 10591066 1065 greater importance than the site of origin of metastases [ 13 , 14 , 19 , 20 , 2326 ]  . 
 our high rate of technique effectiveness is most probably due to multiple overlapping ablation treatments in lesions with diameter 3 caccording to the literature , the best results in terms of local tumour control have been noted in patients with lesions < 4 cm in diameter . 
 [ 15 ] , rfa is used to reduce tumour bulk in conjunction with systemic therapy and is a relatively simple percutaneous technique demonstrated to be an effective cytoreductive strategy . 
 [ 21 ] recently reported that overall 1 - , 3and 5 - year survival rates after the rst rf ablation were 68% , 43% and 27% , respectively , and that overall 1 - , 3and 5 - year survival rates after diagnosis of the rst liver metastasis were 92% , 60% and 32% , respectively . 
more recently , in a series of 31 patients with isolated breast cancer and liver metastases treated with major liver resections and minor resections with or without rfa , vlastos et al . 
 [ 28 ] demonstrated a median survival of 63 months , overall 2and 5 - year survival rates of 86% and 61% , respectively , and 2and 5 - year diseasefree survival rates of 39% and 31% , respectively . 
nella determinazione dellef cacia tecnica concorrono diversi fattori tra cui le dimensioni del tumore e la vicinanza ai rami di maggior calibro delle vene epatiche e della vena porta [ 13 , 14 , 19 , 20 , 2326 ]  . lelevata ef cacia tecnica da noi ottenuta probabilmente legata ai multipli trattamenti termoablativi nelle pazienti con lesioni di dimensioni superiori ai 4 cm di diametro . 
va comunque sottolineato che i migliori risultati in termine di migliore controllo locale della malattia sono stati evidenziati , in accordo anche ai dati della letteratura , in pazienti con lesioni pi piccole di 4 cm di diametro . 
 [ 13 ] riportano in un follow - up di 444 mesi , un totale di 63% pazienti libere da malattia , tuttavia , in tale studio non sono presenti dati di sopravvivenza totale per cui una discussione e un confronto pi preciso con i nostri dati non realizzabile [ 13 ]  . 
 [ 21 ] hanno presentato una sopravvivenza a 1 , 3 , 5 anni dalla diagnosi di tumore della mammella del 68% , 43% e 27% , mentre una sopravvivenza a 1 , 3 , 5 anni dalla diagnosi di metastasi epatica del 92% , 60% e 32% rispettivamente [ 21 ]  . 
la rfa confrontata con la chirurgia , offre il vantaggio di essere meno costosa e meno invasiva [ 23 , 24 ] ; i nostri dati sono sovrapponibili a quanto ottenuto dal trattamento chirurgico nel lavoro di pocard et al . 
 [ 28 ] in una serie di 31 pazienti con singola lesione epatica da tumore mammario , trattata con resezione chirurgica maggiore o minore , con o senza rfa presenta una sopravvivenza media di 63 mesi e totale a 2 e 5 anni del 86% e 61% rispettivamente . la rfa , ragionevolmente , pu essere utilizzata in alternativa alla chirurgia come terapia locale di prima scelta , in quanto la storia naturale della malattia , ha dimostrato la scarsa ef cacia delle terapie locali ( chirurgia , ablazione del tumore in situ )  . 
luso della rfa non controindica il simultaneo o susseguente espletamento di altri trattamenti complementari . conclusions conclusioni in our experience and according to the literature , rfa appears to be a useful adjunct to systemic chemotherapy and / or hormone therapy in the locoregional treatment of hepatic metastases from breast cancer . 
rfa may also be a less invasive alternative to surgery in the locoregional treatnella nostra esperienza e in accordo con la letteratura , la rfa pu essere associata alla chemioterapia e / o ormonoterapia nel trattamento loco regionale delle metastasi epatiche da tumore della mammella . 
a prospective randomised trial comparing systemic treatment alone and systemic treatment with rfa for selected breast cancer liver metastases is needed to show any advantage of rfa in local and overall disease control when compared with systemic treatment alone . delle metastasi epatiche da umore della mammella . 
risulta quindi necessario uno studio prospettico randomizzato che compari la sola chemioterapia e la chemioterapia in associazione alla rfa atto a dimostrare i vantaggi della rfa nel controllo locale della malattia rispetto al solo trattamento sistemico . 
the aim of this study was to investigate the ef cacy of a dedicated software tool for automated volume measurement of breast lesions in contrast - enhanced ( ce ) magnetic resonance mammography ( mrm )  . 
the volume of all lesions was measured automatically ( avm ) from ce 3d mrm examinations by means of a computer - aided detection ( cad ) system and compared with the size estimates based on maximum diameter measurement ( mdm ) on mrm , ultrasonography ( us ) , mammography and histopathology . 
 this result was similar to mdm ( 3% understimation , not signi cantly different ) but signi cantly better than us and mammographic lesion measurements ( 24% and 33% size underestimation , respectively )  . 
 entrambi i metodi ottengono nella stima della dimensione per lesioni alla mammella valori pi vicini allo standard costituito dalla misura su pezzo operatorio rispetto ad ecogra a e mammogra a . parole chiave risonanza magnetica mammogra ca cad mezzo di contrasto paramagnetico 1040 introduction contrast - enhanced ( ce ) magnetic resonance mammography ( mrm ) is a promising complementary technique to mammography because of its inherent three - dimensionality [ 1 , 2 ]  . 
furthermore , tumourinduced microvessels show structural abnormalities that give rise to leakage of contrast mediuthis is the origin of the characteristic different patterns for malignant and benign lesions [ 15 ]  . image analysis of dynamic series can be manually performed : the radiologist draws regions of interest ( roi ) on areas exhibiting suspicious contrast agent uptake and evaluates the pixel intensity curve as a function of time . 
 however , in recent years , computer - aided detection ( cad ) systems have been developed to help to manage the large number of images produced during the dynamic acquisition [ 6 ]  . 
this is a notable feature , as mr imaging has shown superior potential for quanti cation of tumour size and follow - up after chemotherapy [ 712 ] , in addition to possible improvements in diagnostic accuracy . 
 objective tumour shrinkage has been adopted as a standard end - point for selecting anticancer agents , and size is one of the standard prognostic and therapeutic factors in common use today . 
oltre allinformazione sulla morfologia della lesione , la risonanza magnetica ( rm ) pu evidenziare caratteristiche funzionali dei tessuti , come la perfusione e la cinetica del mezzo di contrasto . 
la cinetica dellimpregnazione del mezzo di contrasto studiata tramite acquisizioni dinamiche : dopo liniezione di un bolo , sono effettuate svariate acquisizioni sul volume delle mammelle e viene considerata la curva temporale pixel per pixel . 
unaumentata densit dei microvasi incrementer il usso sanguigno , che porter ad una evidenziazione del contrasto ; inoltre , i microvasi indotti dal tumore mostrano anomalie strutturali che danno origine a una alterata permeabilit al mezzo di contrasto . 
questa lorigine delle diverse caratteristiche delle lesioni maligne e benigne [ 15 ]  . lanalisi sulle immagini delle serie dinamiche pu essere effettuata manualmente : i medici radiologi sono soliti tracciare regioni di interesse ( roi ) su aree con sospetta impregnazione contrastogra ca e valutare la curva dellintensit del pixel in funzione del tempo . 
tuttavia , negli ultimi anni , sono comparsi sistemi di rilevazione supportata dal computer ( computer aided diagnosis , cad ) , per aiutare i medici radiologi nella gestione dellelevato numero di immagini prodotte durante le acquisizioni dinamiche [ 6 ]  . 
 questi sistemi automatizzano e velocizzano il processo di analisi , in particolare evidenziano le regioni con sospetta impregnazione contrastogra ca e forniscono informazioni addizionali , come ad esempio la dimensione della lesione . 
 questultima una caratteristica rilevante , poich la rm ha dimostrato un potenziale superiore nella quanti cazione della dimensione del tumore e nel follow - up dopo chemioterapia [ 712 ] , oltre a un possibile miglioramento dellaccuratezza diagnostica . 
lesion volume was evaluated according to different methods : automated segmentation on mr imaging , manual volume estimation based on diameter measurement on mr imaging , us and mammography , and volume estimation based on measurement of the diameter on the histopathological specimen . 
of 52 lesions , 44 ( 33 invasive ductal carcinomas , 11 invasive lobular carcinomas ) were malignant , ve were low - grade lesions ( two ductal carcinomas in situ ; three lobular carcinomas in situ ) and three were benign ( one brocistic disease , two sclerosing adenosis )  . 
all remaining 149 patients had lesions classi ed as below category 4 of the breast imaging reporting and data system ( bi - rads ) and hence did not require further workup . 
volume measurements based on mrm , us and mammography were carried out for all 52 histopathologically proven lesions . magnetic resonance imaging examinations were performed on a 1.0 - t scanner ( intera , philips medical systems , pc best , the nederlands )  . 
 the breasts were imaged before and ve times after the intravenous injection ( injection rate 2 cc / s , followed by 15 ml of saline ush ) of 0.2 mmol / kg body weight gadopentate dimeglumine ( magnevist , schering , berlin , germany )  . 
la dimensione della lesione stata valutata secondo diversi metodi : segmentazione automatica in rm , stima manuale del volume basata sulla misura del diametro in rm , ecogra a ( us ) e mammogra a , stima del volume basata sulla misura del diametro sul reperto operatorio . 
delle 52 lesioni , 44 ( 33 carcinomi duttali invasivi e 11 carcinomi lobulari invasivi ) si sono dimostrate maligne , 5 si sono rivelate di basso grado ( 2 carcinomi duttali in situ e 3 carcinomi lobulari in situ ) e 3 benigne ( 1 malattia brocistica e 2 adenosi sclerosanti )  . 
le altre 149 pazienti sono state classi cate al di sotto del grado 4 del breast imaging reporting and data system ( bi - rads ) e perci non hanno necessitato di ulteriore follow - up . 
la misura del volume basata su mrm , ecogra a , mammogra a stata effettuata su tutte le 52 lesioni confermate istologicamente . immagini di risonanza magnetica gli esami sono stati effettuati su unapparecchiatura da 1 , 0 t ( intera , philips , medical systems , pc best , paesi bassi )  . 
le mammelle sono state acquisite prima delliniezione intravenosa e 5 volte dopo la somministrazione di 0 , 2 mmol / kg peso corporeo ( velocit di iniezione 2 cc / s , seguita da 15 ml di lavaggio di soluzione salina ) di gadopentato dimeglumina ( magnevist , schering , berlino , germania )  . 
la scelta del piano coronale , per questo modello di apparecchiatura , stata raccomandata dalla ditta produttrice in fase di impostazione del protocollo di acquisizione , per limitare gli artefatti da movimento . 
 gli esami ecogra ci sono stati effettuati con unapparecchiatura dotata di trasduttore 7 , 55 , 5 mhz ( envisor , philips medical system , pc best , paesi bassi )  . 
i mammogrammi sono stati interpretati secondo il metodo bi - rads e con conoscenza degli esiti clinici . massimo diametro del tumore come stima della dimensione la stima della dimensione della lesione stata valutata misurando il diametro massimo in tutte le modalit di imaging : nelle immagini mammogra che la dimensione della lesione stata misurata direttamente sulla pellicola radiogra ca con un righello . 
 stato poi considerato come dimensione della lesione il valore medio delle misure nelle proiezioni cranio - caudale e medio - laterale obliqua ; negli esami ecogra ci il medico ha direzionato la sonda per visualizzare la lesione sul piano in cui la sua dimensione appariva massima . 
quindi la dimensione stata misurata per mezzo dello strumento di misura delle lunghezze del software dellunit di acquisizione ; le immagini mrm sono state reperite sul sistema picture archiving and communications system ( pacs ) dellospedale ( carestream health , rochester , ny , usa ) e visualizzate su un monitor di refertazione . 
il medico ha quindi misurato la dimensione della lesione sul piano su cui essa appariva massima utilizzando lo strumento per la lunghezza delle distanze nel software del pacs ; le valutazioni con il cad sono state effettuate secondo quanto descritto nella sezione successiva . radiol med ( 2011 ) 116 : 10391049 1043 on mammographic images , lesion size was measured directly on the lm with a ruler . 
the mean value of the measurements in the craniocaudal and oblique mediolateral projections was considered as lesion dimension . on us , the radiologist oriented the transducer to display the lesion in the plane displaying its maximum size . 
 mrm images were retrieved from the hospital picture archiving ( pacs ) and communication system ( carestream health , rochester , ny , usa ) and displayed on a reporting monitor . 
the radiologist measured the lesion size along the plane in which it appeared to be the greatest using the ruler tool of the pacs software . cad evaluations were performed as described in following section . measurements were carried out by three of the authors ( gl , cam and rv ) , with 5 , 28 and 15 years , respectively , of experience in mammographic and us breast imaging , and 5 years of experience in mr breast imaging . volume analysis : automated software programme all mri examinations were processed by a dedicated cad for mrm ( cadstream , merge , hartland , wi , usa )  . 
this software is designed to automate the image processing and analysis functions that are typically performed manually by the mr technologist and radiologist , as previously described [ 15 ]  . 
for breast mr examinations , the software incorporates three mr series into the calculations : an unenhanced series , an early contrast - enhanced series and a delayed contrast - enhanced series . 
in the protocol used in this study , the early contrast - enhanced series was centred over roughly 2 min ( images obtained within 4 min ) and the delayed contrastenhanced series was centred over 47 min ( images obtained within 8 min )  . 
in addition , the software le misure sono state effettuate da 3 degli autori , gl , cam , rv , con rispettivamente 5 , 28 e 15 anni di esperienza in mammogra a ed ecogra a della mammella e tutti e 3 con 5 anni di esperienza nellimaging mr alla mammella . analisi del volume : software automatico tutti gli esami rm sono stati elaborati con un cad dedicato per risonanza magnetica alla mammella , cadstream ( merge , hartland , wi , usa )  . 
il software progettato per elaborare in modo automatico le immagini e le funzioni di analisi che sono tipicamente effettuate manualmente dal medico radiologo o da un tecnico , come precedentemente descritto da levrini et al . 
per esami mrm , il software considera 3 serie dinamiche per il calcolo : una serie pesata in t1 pre - contrasto , una delle prime serie post - contrasto , una serie post - contrasto tardiva . 
nel protocollo utilizzato in questo studio la prima serie post - contrasto utilizzata nel calcolo centrata circa a 2 minuti dalliniezione ( immagini ottenute entro 4 minuti ) e la serie tardiva centrata attorno a 47 minuti ( immagini ottenute entro 8 minuti )  . 
se il valore del pixel cresce oltre una soglia prestabilita , il pixel mostrato colorato sul monitor ( la soglia stata ssata al 100% del valore del pixel nellimmagine pre - contrasto )  . 
inoltre , il programma assegna un colore speci co ai pixel che raggiungono la soglia di impregnazione signicativa ; il colore assegnato al pixel dipende dal cambiamento intercorso tra la prima serie post - contrasto e quella tardiva : se il valore del pixel sulla serie tardiva decresce pi del 10% rispetto alla prima serie post - contrasto , il pixel colorato in rosso , indicando una curva con dismissione rapida ( wash - out ) del contrasto ; se il valore del pixel aumenta pi del 10% , colorato in blu , indicando una curva con impregnazione persistente ; se il valore del pixel non aumenta o diminuisce pi del 10% , colorato in giallo , indicando un andamento a plateau . 1044 radiol med ( 2011 ) 116 : 10391049 assigned a speci c colour to each pixel that reached the threshold for signi cant enhancement . 
the colour assigned depended on the change in pixel values between the initial contrast - enhanced series and the delayed contrast - enhanced series : value on the delayed series decreased by if the pixel > 10% of its initial contrast - enhanced value , that pixel was colour - coded red on the monitor , indicating washout of contrast material . 
 finally , the radiologist may select a speci c area of signi cant enhancement and the software will automatically generate a synopsis of the full volume of that lesion , including the percentage of the lesion that shows washout , plateau and persistent enhancement . 
il risultato nale una mappa colore sovrapposta a ogni strato rm , che indica le regioni di signi cativa impregnazione e fornisce dettagli sulla tipologia dellimpregnazione e sullestensione della stessa . in ne , il radiologo pu selezionare unarea di impregnazione signi cativa e il programma genera automaticamente una sinossi del volume della lesione , che include la percentuale della lesione che mostra wash out , plateau o impregnazione persistente . 
tutte le serie dellesame possono essere visualizzate con o senza mappa colore sovrapposta . analisi statistica lanalisi statistica stata effettuata con il software statistical package for the social sciences ( spss inc . , chicago , il , usa )  . 
i test statistici sono riportati nelle sezioni risultati e discussione . risultati let media delle 43 pazienti con lesioni confermate istologicamente di 50 , 1 anni ( da 33 a 78 anni , range interquartile 43 , 556 anni )  . 
su 52 lesioni , 23 ( 44% ) erano invisibili alla mammogra a , 4 ( 8% ) occulte allecogra a , 1 ( 2% ) stata giudicata non sospetta dallanalisi cad , poich laumento dellintensit del pixel era al di sotto della soglia prestabilita . 
un test di mcnemar stato effettuato per valutare la signi cativit di queste differenze : lanalisi cad signi cativamente pi accurata nella rivelazione di lesioni rispetto alla mammogra a ( p < 0 , 001 ) , mentre la differenza tra cad ed ecogra a non signi cativa ( p = 0 , 37 )  . le misure sul reperto istopatologico sono state considerate come gold standard : il valore mediano stato di 20 mm ( range 370 mm , range interquantile 1033 , 5 mm )  . 
stimata dalla formula : dim x dim h dim h dove a laccuratezza nella misura della dimensione della lesione , dim x la dimensione della lesione misurata nella modalit diagnostica x , dim h la dimensione della lesione valutata sul reperto istopatologico . 
nelle figure 2 e 3 sono riportati esempi di valutaradiol med ( 2011 ) 116 : 10391049 1045 examination can be displayed by the software with or without the colour overlay . table 2 accuracy of the different techniques in evaluating maximum tumour diameter statistical analysis modality accuracy ( mean values ) visible lesions ( n ) statistical analysis was performed with spss software ( spss , chicago , il , usa )  . 
of 52 lesions , 23 ( 44% ) were mammographically occult , four ( 8% ) were sonographically occult and one ( 2% ) was judged as nonsuspicious by cad analysis because pixel intensity enhancement was below the xed threshold . 
accuracy is assessed according to the formula : dim x dim h dim h where a is accuracy in the measurement , dim x is lesion size measured with the diagnostic modality x , dim h is lesion size evaluated on the histopathologic specimen . 
figure 4 shows the cad result that reports lesion size and position . discussion in recent years , mrm has gained increasing importance in diagnosing , staging and surgical planning of breast cancer [ 2 ]  . 
mr examinations offer unique advantages : the study of tumour morphology bene ts from the high spatial resolution and inherent three - dimensional imaging ; differential diagnosis can take advantage of multiparametric information based on t1 and t2 relaxation times . 
moreover , mrm can provide functional information on perfusion and capillary contrast agent leakage : contrast agent washin and washout patterns have shown a high sensitivity in the differmammography ultrasonography magnetic resonance mammography cad analysis 33% 24% 3% 4% cad , computer - aided detection mammogra a ecogra a risonanza magnetica mammogra ca analisi cad 33% 24% 3% 4% cad , computer - aided detection tabella 2 accuratezza delle diverse tecniche nel valutare il diametro massimo del tumore modalit accuratezza ( valori medi ) lesioni visibili ( n ) zione delle dimensioni di una lesione maligna con misura manuale in mrm e con il cad . 
la figura 4 riporta il risultato del cad che indica le dimensioni e la posizione della lesione . discussione negli ultimi anni la risonanza magnetica mammogra ca ha guadagnato unimportanza crescente nella diagnosi dei tumori alla mammella , nella stadiazione e nella piani cazione chirurgica [ 2 ]  . 
gli esami di risonanza magnetica hanno vantaggi peculiari : lo studio della morfologia del tumore trae bene cio dalla elevata risoluzione spaziale e dallintrinseca tridimensionalit dellimaging , la diagnosi differenziale si avvantaggia dellinformazione multiparametrica basata sui tempi di rilassamento t1 e t2 . 
inoltre , mrm pu fornire informazioni funzionali sulla perfusione e la perdita di mezzo di contrasto dai capillari : le curve di accumulo e dismissione del mezzo di contrasto hanno mostrato elevata sensibilit nella diagnosi differenziale tra lesioni benigne e maligne [ 1 ]  . un tipico esame mrm comprende almeno una sequenza pesata in t2 ( con o senza soppressione del grasso ) , unacquisizione dinamica dopo somministrazione di mezzo di contrasto ( inclusa unacquisizione pre - contrasto per sottrarre il grasso e il tessuto stazionario ) ed una sequenza pesata in t1 ad alta risoluzione con soppressione del 1046 radiol med ( 2011 ) 116 : 10391049 fig . 
lacquisizione dinamica fornisce uninformazione funzionale sullangiogenesi del tumore e sulla vascolarizzazione . i sistemi cad possono ottimizzare il usso di lavoro , aiutando il medico nella gestione dellelevata mole di immagini prodotte dalle serie dinamiche . 
sebbene luso di questi sistemi non sia ancora considerato obbligatorio rispetto ad un approccio manuale e il loro valore aggiunto tuttora dibattuto [ 1820 ] , essi si stanno diffondendo in maniera crescente . 
la possibilit di utilizzarli per la determinazione in automatico delle dimensioni della lesione una caratteristica da studiare in dettaglio , in particolare per la valutazione della risposta a trattamenti di chemioterapia neoadiuvante , come indicato da sardanelli et al . 
questa sottostima non statisticamente signi cativa ( test t di student per dati appaiati a due code , p = 0 , 24 ) ed sostanzialmente sovrapponibile a quanto misurato manualmente dal medico direttamente sulle immagini mrm . 
tale valutazione manuale sottostima il diametro massimo in media del 3% e la differenza tra la stima del cad e mrm non signi cativa ( test t di student per dati appaiati a due code , p = 0 , 62 )  . 
la stima nella dimensione della lesione offerta dal cad tanto accurata quanto quella effettuata direttamente su immagini rm , ma lanalisi automatica garantisce una maggiore standardizzazione nella valutazione del volume del tumore e consente un risparmio di tempo . 
la stima del cad della dimensione 33 mm . ential diagnosis of benign and malignant lesions [ 1 ]  . a typical mrm examination comprises at least a t2 - weighted sequence ( with or without fat suppression ) , a dynamic acquisition after contrast agent administration ( including a precontrast acquisition to subtract stationary tissues and fat ) and a high - resolution t1 - weighted sequence with fat suppression to evaluate late enhancement [ 16 , 17 ]  . 
even though the use of cad systems is still not considered mandatory with respect to a manual radiol med ( 2011 ) 116 : 10391049 1047 approach , and its added value is still debated [ 1820 ] , these tools are becoming increasingly wide spread . 
this underestimation was not statistically signi cant ( two - tailed students t test , p = 0.24 ) and it was basically equal to the measurement performed by the radiologist directly on the mrm images . 
in our study , us and mr con rmed their greater accuracy in lesion detection ( even though we did not distinguish among the different lesion types , as our sample was smaller ) , but our results show that mr is suporior to us in the evaluation of tumour size . 
on the contrary , this level of accuracy was accomplished in only 33% of lesions on us examinations and 31% on mammography , which show a general understimation of lesion dimensions . era al di sotto della soglia prestabilita . 
confrontato con le altre modalit lanalisi del cad pi accurata nella valutazione volumetrica . quattro lesioni su 52 sono risultate occulte allesame ecogra co , ma la differenza con le prestazioni del cad non signi cativa . 
tuttavia la valutazione ecogra ca meno accurata nella stima della dimensione del tumore e sottostima questa del 24% in media ( differenza altamente signicativa con il cad , test t di student per dati appaiati a due code , p < 0 , 001 )  . confrontato con gli esami mammogra ci , il sistema cad sia pi accurato nella stima della dimensione del tumore , che pi sensibile nella rivelazione di lesioni : solo 29 lesioni su 52 erano visibili in mammogra a ( la differenza con lanalisi del cad altamente signi cativa , test di mcnemar , p < 0 , 001 ) e per quelle visibili in mammogra a la sottostima nella dimensione della lesione del 33% ( differenza altamente signi cativa con cad e mrm , test t di student per dati appaiati a due code , p < 0 , 001 )  . laccuratezza nella valutazione preoperatoria di tumori alla mammella stata studiata da berg et al . 
 [ 21 ] : gli autori concludono che lecogra a e la risonanza magnetica sono pi accurate della mammogra a nella rivelazione di tumori invasivi , ma rischiano di sovrastimare lestensione della malattia . 
nel nostro studio , lecogra a e la risonanza magnetica confermano di essere pi accurate nella rivelazione delle lesioni ( pur non avendo noi distinto le varie tipologie di tumore essendo il nostro campione pi piccolo ) , ma i nostri risultati mostrano che gli esami di risonanza magnetica sono pi accurati rispetto allecogra a nello stimare le dimensioni della lesione . 
 al contrario questo livello di accuratezza ottenuto solo nel 33% delle lesioni osservate allecogra a e al 31% in mammogra a , che mostrano una generale sottostima delle dimensioni della lesione . poich lobiettivo principale nel trattamento di tumori alla mammella resecabili precocemente sono la conservazione della mammella e la prevenzione di recidive , importante identi care lestensione del tumore prima della chirurgia . 
il volume della massa tumorale pu essere sottostimato , ma questo fatto spesso ben evidenziabile allatto operatorio o al pi tardi determina il riscontro di margini in ltrati da malattia allesame istologico del pezzo . 
 [ 21 ] si attendono lesistenza di una massa tumorale residua approssimativamente nel 19% dei pazienti con carcinoma invasivo se la programmazione dellintervento derivasse unicamente dai riscontri di mammogra a e esame clinico ( ad esempio , allargamento della resezione o mastectomia in caso di margini positivi )  . 
presumibile , ma a sostegno 1048 radiol med ( 2011 ) 116 : 10391049 as breast conservation and prevention of recurrent disease are the main treatment goals in early resectable breast cancer , it is important to reliably determine tumour extent prior to surgery . 
decreasing the theoretical risk of local recurrence comes at the price of an increased risk of unnecessary wider excisions or mastectomy or , in most cases , additional imaging follow - up because of incidental ndings at mr imaging . 
recently , the relation between preoperative mrm and outcome after breast - conserving treatment for early - stage ( t1 or t2 ) and in situ breast carcinoma was determined in a large , nonrandomised cohort study : it was concluded that the use of mrm was not associated with improved outcome after breast - conserving treatment [ 22 ]  . our study has some limitations that need to be addressed . 
it is generally believed that the sensitivity of mrm imaging is lower at 1.0 t than at 1.5 t despite reports that sensitivity at the two eld strengths is equivalent [ 23 ]  . 
 thirdly , the number of patients is relatively small , which makes rm statistical conclusion dif cult ; larger patient samples should be considered in order to strengthen our deductions . 
la riduzione teorica del rischio di recidiva locale ottenuta al costo di un virtuale incremento di resezioni ampie o mastectomie non necessarie o , nella maggioranza dei casi , di indagini di diagnostica per immagini di controllo necessarie per valutare levoluzione di reperti rm incidentali . 
recentemente la relazione tra rm pre - operatoria e lesito del trattamento chirurgico conservativo per il tumore mammario in stadio iniziale ( t1 o t2 ) ed il carcinoma in situ stata descritta in un esteso studio di coorte non randomizzato : le conclusioni sono state che lutilizzo della mrm non associato ad un migliore risultato dopo il trattamento chirurgico conservativo [ 22 ]  . necessario precisare alcune limitazioni di questo studio . 
 opinione diffusa che la sensibilit della mrm inferiore quando viene utilizzata una apparecchiatura a 1 , 0 t rispetto alla abituale da 1 , 5 t , malgrado i dati disponibili [ 23 ] abbiano documentato sovrapponibili sensibilit alle due intensit di campo confrontate . 
terza limitazione costituita dal numero di pazienti relativamente esiguo , fatto che rende dif coltoso ottenere conclusioni statistiche solide ; sarebbe auspicabile valutare un numero di pazienti superiore per rafforzare le nostre deduzioni . 
in ne , tutti gli studi ecograci sono stati effettuati da un radiologo al corrente dei reperti clinici e mammogra ci ; cos le indagini rm sono state valutate alla luce dei reperti mammogra ci , clinici ed ecogra ci . 
 conclusioni in questo studio stato utilizzato un sistema cad per risonanza magnetica mammogra ca , basato sulla dinamica dellimpregnazione contrastogra ca , per stimare le dimensioni di lesioni alla mammella . 
il sistema cad si rivelato accurato e ha fornito misure consistenti con la stima sul reperto istopatologico . un sistema cad pu velocizzare lanalisi di unelevata quantit di immagini che provengono da esami mrm e fornisce informazioni aggiuntive sulla morfologia e funzionalit delle lesioni , come ad esempio la loro dimensione . 
further studies with larger patient samples are required to establish whether cad systems should be used extensively for mrm . tuisce uninformazione rilevante , come ad esempio per valutare la risposta a chemioterapia neoadiuvante . 
biti1 1radiotherapy unit , university of florence , viale morgagni 85 , 50134 florence , italy 2molecular and nutritional epidemiology unit , cancer research and prevention center , scienti c institute of tuscany , florence , italy 3department of medical physics , university of florence , florence , italy 4diagnostic senology unit , university of florence , florence , italy 5department of surgery , university of florence , florence , italy 6department of pathology , university of florence , florence , italy correspondence to : i . 
concerning cardiac assessment , six of the 56 evaluable patients ( 10.7% ) developed an asymptomatic decline of left ventricular ejection fraction > 10% and < 20% of the baseline value . 
per quanto riguarda la tolleranza cardiologica , sei dei 56 pazienti valutabili ( 10 , 7% ) hanno sviluppato un decremento asintomatico della frazione di eiezione ventricolare sinistra compreso tra il 10% e il 20% ( dallinizio del trattamento )  . 
in termini di tossicit non si sono inoltre veri cati effetti collaterali cardiaci severi . parole chiave carcinoma mammario iniziale chemioradioterapia concomitante antracicline tossicit cardiaca radioterapia adiuvante introduction introduzione breast - conserving surgery has been accepted as a validated surgical approach for early breast cancer ( bc )  . 
moreover , a recent meta - analysis of 13 randomised studies involving 22 , 903 patients demonstrated that the addition of taxanes to an ac - based regimen improved disease - free ( dfs ) and overall ( os ) survival in high - risk patients with early bc [ 46 ]  . it seems that the prognosis of bc is associated with the presence or absence of occult distant metastasis but not to the extension of the surgery . 
however , a meta - analysis showed a signi cant risk reduction of death due to bc after breast - conserving surgery and radiotherapy ( rt ) [ 7 ]  . 
the importance of rt timing following surgery to reduce the risk of local recurrence is well known [ 811 ]  . the optimal sequence of ct and rt after breastconserving surgery still remains controversial , although both adjuvant ct and rt can reduce the risk of bc recurrence . 
concurrent administration of ct and rt seems to be an ideal treatment after breast - conserving surgery , but the reported increase in toxicity [ 1012 ] resulted in the infrequent use of this therapeutic approach in clinical practice . we evaluated the feasibility of the concurrent rt and ac - based ct treatment , with particular emphasis on the side of breast disease occurrence . 
we also investigated the impact of treatment on adverse events and , in particular , on cardiac toxicity . la chirurgia conservativa della mammella ( bcs ) comunemente accettata come un valido approccio chirurgico nei tumori in fase iniziale . 
la somministrazione precoce di chemioterapia ( ct ) adiuvante pu ridurre il rischio di recidiva di malattia e la mortalit assoluta , indipendentemente dallet del paziente e dal regime chemioterapico utilizzato [ 1 , 2 ]  . 
i regimi chemioterapici basati sulle antracicline hanno mostrato una superiorit signi cativa quando confrontati con la chemioterapia secondo schema ciclofosfamide , methotrexate e 5uoro uracile ( cmf ) [ 3 ]  . 
inoltre , una recente metanalisi comprendente 13 studi randomizzati che includevano 22.903 pazienti , ha dimostrato che laggiunta dei taxani ad un regime chemioterapico basato sulle antracicline , aumenta la sopravvivenza libera da malattia ( dfs ) e la sopravvivenza globale ( os ) nei pazienti con tumore della mammella in fase iniziale ad alto rischio [ 46 ]  . sembra che la prognosi dei tumori della mammella sia associata alla presenza o meno di metastasi occulte a distanza , ma non allestensione della chirurgia effettuata . 
 daltra parte , una metanalisi ha mostrato una signi cativa riduzione del rischio di morte per tumore della mammella dopo chirurgia conservativa e radioterapia ( rt ) [ 7 ]  . 
 ben nota limportanza della tempestivit della radioterapia dopo chirurgia per ridurre la recidivit locale di malattia [ 811 ]  . la sequenza ottimale con cui eseguire la chemioterapia e la radioterapia dopo la bcs rimane ancora oggi una problematica controversa , sebbene sia noto come la chemioterapia e la radioterapia adiuvanti siano in grado di ridurre il rischio di ricaduta del tumore della mammella . 
la somministrazione concomitante di chemioe radioterapia sembra essere un trattamento ideale dopo la chirurgia conservativa , ma laumento della tossicit riportato in letteratura ha fatto 1052 materials and methods from september 2002 to december 2007 , 60 patients with early bc underwent concurrent adjuvant ac - based ct regimen and rt at our institute . 
according to the protocol , all patients received rt to the entire breast , supraclavicular fossa and axilla with external - beam rt using 6 - mv photon half - beam tangential elds for the breast and lower part of axilla ( mean delivered dose 50 gy ; range 4652 gy ) in 2 - gy daily fractions . 
the supraclavicular fossa and axilla apex were treated with a half - beam direct eld ( 6 - mv photons ) with posterior compensation ( 25 - mv photons )  . 
in all patients , the boost was delivered in 2 - gy daily fractions . ac - based regimens consisted of four cycles of intravenously administered ac ( doxorubicin 60 mg / m2 , cyclophosphamide 600 mg / m2 ) on day 1 , repeated every 21 days , or four cycles of intravenously administered epirubicin ( epi ) 100 mg / m2 on day 1 , repeated every 21 days , followed by four courses of intravenously administered cmf ( cyclophosphamide 600 mg / m2 , methotrexate 40 mg / m2 , and 5uorouracil 600 mg / m2 ) on days 1 and 8 , repeated every 28 days . all patients were required to have normal bone marrow reserve ( granulocytes > 1 , 500 / mm3 , platelets > 100 , 000 / mm3 , haemoglobin > 10 g / dl ) , renal function ( creatinine clearance > 50 ml / min ) , liver function ( bilirubin < 2 mg / dl ) and a left ventricular ejection fraction ( lvef ) > 50% at the baseline evaluation . 
baseline assessments included echocardiography with lvef measurement ; periradiol med ( 2011 ) 116 : 10501058 s che questo approccio terapeutico risulti di non comune utilizzo nella pratica clinica [ 1012 ]  . abbiamo pertanto valutato la fattibilit del trattamento radioterapico concomitante alla chemioterapia contenente antracicline , con particolare enfasi riservata al lato della mammella affetto dalla neoplasia . 
abbiamo inoltre ponderato limpatto del trattamento in termini di effetti collaterali e , in particolare , di tossicit cardiaca . materiali e metodi in un periodo compreso tra settembre 2002 e dicembre 2007 , 60 pazienti con un tumore della mammella in fase iniziale sono state sottoposte presso il nostro istituto a terapia adiuvante concomitante radioe chemioterapica contenente antracicline . 
in questa analisi abbiamo incluso solo le pazienti senza segni radiologici o clinici di recidiva locale o a distanza di malattia , nel momento in cui sono state visitate presso lunit di radioterapia , dopo aver effettuato il trattamento chirurgico conservativo . 
tutte le pazienti sono state trattate con radioterapia a fasci esterni sulla mammella e sulla regione inferiore dellascella utilizzando fotoni da 6 - mv con due campi tangenti ed un frazionamento di 2 gy giornalieri ( dose media 50 gy , range 4652 gy )  . 
la fossa sovraclaveare e lapice dellascella sono stati trattati con un campo diretto half - beam ( fotoni da 6 - mv ) con un compenso posteriore ( fotoni da 25 - mv )  . 
patients main characteristics are summarised in table 1 . acute radiation - related skin toxicity was assessed weekly during rt according to the radiation therapy oncology group scale [ 13 , 14 ]  . 
to record late toxicity , we used the late effects in normal tissues subjective , objective , management and analytic scale ( lent - soma ) [ 15 , 16 ]  . 
 for statistical analysis , we used a logistic model with stepwise selection considering rt interruption as the dependent variable , investigating several parameters ( bc side , age , ct regimen , baseline lvef value ) as independent predictive factors of rt discontinuation . results data regarding acute skin toxicity are shown in table 2 . 
 concomitant ct - rt caused acute skin toxicity g3 in 8.9% of patients ( ve of 56 ) and acute skin toxicity g4 in one normale riserva midollare ( neutro li > 1500 / mm3 , piastrine > 100.000 / mm3 , emoglobina > 10 g / dl ) , una funzione renale ed epatica nella norma ( clearance della creatinina > 50 ml / minuto , bilirubina < 2 mg / dl ) ed una frazione di eiezione ventricolare sinistra ( lvef ) superiore al 50% . 
la valutazione basale consisteva di unecocardiogra a con misurazione del lvef ; indagini ultrasonogra che cardiache sono state eseguite alla ne del trattamento chemioterapico , ogni quattro / sei mesi per i primi due anni dal termine della chemioterapia ed in seguito annualmente . 
le caratteristiche principali dei pazienti sono riassunte nella tabella 1 . la tossicit cutanea acuta da radiazioni stata valutata settimanalmente durante la radioterapia seguendo la scala radiation therapy oncology group ( rtog ) [ 13 , 14 ]  . 
per registrare la tossicit tardiva stata utilizzata la scala late effects in normal tissues subjective , objective , management and analytic ( lent - soma ) [ 15 , 16 ]  . 
rt was stopped in 21.3% of patients ( 12 of 56 ) ; in all cases , the interruption was 7 ( mean 5 ) days and the treatment was subsequently concluded reaching the prescribed total dose . 
in the logistic model with stepwise analysis using rt interruption as dependent variable , none of the evaluated parameters ( age , bc side , baseline lvef value , ct regimen ) emerged as an independent predictive factor of rt discontinuation ( data not shown )  . 
all ct was restarted after the interruption and was subsequently completed . concerning left - side bc patients , four of the 30 ( 13.3% ) developed a decline of lvef > 10% and < 20% of baseline value . 
one year after the end of ct , three of these patients ( including the symptomatic patient ) had unchanged lvef , and one had an increase of 5% of lvef . 
il trattamento concomitante radio - chemioterapico ha causato una tossicit cutanea acuta g3 nell8 , 9% dei pazienti ( 5 su 56 ) e tossicit cutanea acuta g4 in un solo paziente ( 1 , 7% )  . 
la radioterapia stata sospesa nel 21 , 3% dei casi ( 12 su 56 ) ; in tutti i pazienti la sospensione stata 7 giorni ( media 5 giorni ) ed il trattamento stato successivamente concluso , raggiungendo la dose totale prescritta . 
 considerando linterruzione della radioterapia come una variabile dipendente nel modello logistico statistico , nessuno dei parametri esaminati ( et , localizzazione del tumore , lvef basale , chemioterapia effettuata ) si dimostrato un fattore predittivo indipendente di interruzione della radioterapia ( dati non mostrati )  . 
la chemioterapia stata rimandata nel 57 , 1% dei pazienti ( 32 su 56 ) ; in tutti i casi la sospensione stata correlata a tossicit ematologica ed stata 7 giorni . 
tutte le chemioterapie interrotte o rimandate sono state successivamente riprese e poi completate . per quanto riguarda le pazienti con un tumore della mammella localizzato a sinistra , 4 su 30 pazienti ( 13 , 3% ) hanno mostrato un decremento della lvef > 10% e < 20% rispetto ai valori basali . 
ad un anno dal termine della chemioterapia , tre di queste pazienti ( incluso la paziente sintomatica ) hanno mostrato una lvef immutata , mentre una paziente ha mostrato un incremento del 5% della lvef . 
una paziente ha mostrato una riduzione asintomatica della funzionalit cardiaca sinistra , mentre unaltra paziente ha avuto sintomi cardiaci ( brillazione atriale ) , che hanno richiesto una speci ca terapia cardiologica . 
dopo due anni dal termine della chemioterapia questi pazienti hanno mostrato una lvef immutata . prendendo in considerazione le pazienti con tumore della mammella destra , 2 su 26 pazienti ( 7 , 6% ) hanno sviluppato una diminuzione asintomatica della lvef > 10% e < 20% rispetto ai valori basali . 
 dopo due anni dal termine della chemioterapia la paziente mostra una lvef immutata . ad un follow - up medio di 4 , 1 anni ( range 26 anni ) , una paziente ( 1 , 7% ) ha mostrato una recidiva di malattia locoregionale nella fossa sovraclaveare e tre pazienti ( 5 , 4% ) radiol med ( 2011 ) 116 : 10501058 1055 concerning the right - side bc patients , two of the 26 ( 7.6% ) developed an asymptomatic decline of lvef > 10% and < 20% of the baseline value . 
two years after ct , the patient had unchanged lvef . at a median follow up of 4.1 ( range 26 ) years , one patient ( 1.7% ) developed locoregional recurrence in supraclavicular fossa , and three patients ( 5.4% ) developed distant metastases ( two liver and one lung )  . discussion the optimal sequencing of rt and ct in early bc remains controversial [ 17 ]  . 
several authors reported a negative effect of rt on the ability to deliver optimal doses of ct and suggested that this may affect treatment outcome [ 10 , 18 , 19 ]  . 
 [ 26 ] , in a prospective nonrandomised study , reported the acute toxicity of rt alone and rt concurrent with ac ( ac / rt ) and rt concurrent with cmf ( cmf / rt )  . 
this experience was not positive in the concomitant arms : in fact , it was observed that patients treated with ac / rt and cmf / rt had a signi cantly higher incidence of high - grade skin toxicity , oesophagitis , dyspnoea , anorexia , nausea and hospitalisation when compared with those treated exclusively with rt . 
the dose of ct was reduced to < 85% of the planned dose in 11% of patients ; 17% of patients treated with concurrent ct and rt needed admission to hospital . in our experience , all patients received ac - based regimens and rt to the breast and local drains . 
 [ 27 ] ; hanno sviluppato metastasi a distanza ( due localizzazioni epatiche ed una localizzazione polmonare )  . discussione la sequenza ottimale della rt e della ct nei tumori della mammella in fase iniziale continua a rimanere controversa [ 17 ]  . 
diversi autori hanno riportato un effetto negativo della rt sulla possibilit di somministrare dosi ottimali di ct , suggerendo che questo potrebbe in uenzare loutcome del trattamento [ 10 , 18 , 19 ] ; un aumento dei livelli di tossicit potrebbe quindi compromettere la dose ottimale somministrata , sia per quanto riguarda la rt che la ct [ 20 ]  . 
 [ 26 ] , in uno studio prospettico non randomizzato , hanno riportato i dati riguardanti la tossicit acuta della rt da sola o concomitante a doxorubicina - ciclofosfamide ( ac / rt ) o somministrata in regime concomitante con ciclofosfamide , metotrexate e 5uorouracile ( cmf / rt )  . 
questa esperienza non si rilevata positiva nei bracci concomitanti : infatti stato osservato che i pazienti trattati con ac / rt e cmf / rt avevano una incidenza signi cativamente pi elevata di tossicit cutanea di alto grado , esofagite , dispnea , anoressia , nausea e ospedalizzazione , quando confrontati con i pazienti trattati con rt esclusiva . 
la dose della ct era stata ridotta a meno dell85% della dose prestabilita nell11% dei pazienti , il 17% dei pazienti trattati con rt e ct concomitanti hanno richiesto ospedalizzazione . nella nostra esperienza tutti i pazienti hanno ricevuti ct a base di antracicline e rt sulla mammella e sui drenaggi linfatici locali . 
nonostante lampio volume trattato e lutilizzo di antracicline , abbiamo registrato solo il 21 , 4% di interruzioni della rt a causa di tossicit cutanea acuta e inferiore a 7 giorni in tutti i casi ; tossicit cutanea g3 e g4 stata osservata rispettivamente nell8 , 9% e 1 , 7% dei pazienti . 
 ad un follow - up mediano di 76 , 4 mesi ( media 65 , 3 mesi ) , solo una paziente trattata con antracicline ha presentato 1056 radiol med ( 2011 ) 116 : 10501058 the authors compared two adjuvant treatments associating concomitant ct and rt . 
 this preliminary analysis showed the feasibility of this uncommon approach in terms of acute skin toxicity and cardiac safety but does not allow conclusions concerning local bc recurrence and distant failure . we had more ct interruptions compared with ct alone or other ct - rt associations [ 29 , 30 ]  . 
in fact , this group of patients was enrolled in a phase ii study that required rt to the breast and lymph node drain our clinical routine practice , it is not usual to give rt to the lymph node drain independently of the number of positive axillary lymph nodes [ 31 , 32 ]  . a relevant result was represented by the absence of severe cardiac effects . 
these patients were enrolled in a pilot study that investigated the ef cacy and feasibility of sequential and combined doxorubicin / docetaxel / cmf regimens ; their median age was 48 ( range 2965 ) years . 
nelle pazienti trattate con antracicline , la dfs dopo 5 anni era dell80% rispetto al 76 , 4% delle pazienti trattate con cmf ( log - rank test : p = 0 , 136 )  . 
la os dopo 5 anni era rispettivamente dell82 , 5% e dell81 , 1% nelle pazienti trattate con antracicline e cmf ( log - rank test : p = 0 , 428 )  . la sopravvivenza libera da malattia loco - regionale a 5 anni era del 98 , 6% nel gruppo a e del 94% nel gruppo b ( log - rank test : p = 0 , 033 )  . 
le percentuali di anemia di grado 2 e grado 3 erano rispettivamente del 13 , 9% nel gruppo dei pazienti trattati con antracicline e del 6 , 7% nel gruppo cmf ( p = 0 , 009 )  . 
questi risultati non si discostano molto da quelli ottenuti in un nostro precedente studio , in cui avevamo confrontato la rt esclusiva rispetto al trattamento concomitante rt - cmf [ 28 ]  . siamo consapevoli del maggior limite del nostro studio , rappresentato dal breve periodo di follow - up ( 4 , 1 anni )  . 
 questanalisi preliminare dimostra comunque la fattibilit di questo non comune approccio terapeutico , in termini di tossicit cutanea acuta e tossicit cardiaca , ma non permette al momento di formulare nessuna asserzione de nitiva per quanto riguarda il controllo loco - regionale e a distanza della malattia . nella nostra casistica si sono veri cate maggiormente interruzioni della chemioterapia rispetto a regimi chemioterapici esclusivi o ad altre associazioni radio - chemioterapiche [ 29 , 30 ]  . 
infatti i pazienti valutati in questanalisi erano stati arruolati in uno studio di fase ii che prevedeva la rt della mammella e dei drenaggi linfatici locali : nella nostra pratica clinica giornaliera non per comune irradiare i drenaggi linfatici , indipendentemente dal numero di linfonodi ascellari positivi [ 31 , 32 ]  . un importante risultato stato rappresentato dallassenza di importanti effetti collaterali cardiaci . 
per quanto riguarda le pazienti con tumore della mammella localizzato a sinistra , abbiamo osservato un decremento asintomatico del lvef compreso fra il 10%20% rispetto ai valori basali nel 13 , 3% dei casi . 
 queste pazienti sono state incluse in uno studio pilota che valutava lef cacia e la fattibilit di regimi concomitanti e sequenziali con doxorubicina / docetaxel / cmf in pazienti con unet mediana di 48 anni ( range 2965 anni )  . 
 the authors concluded that sequential and combined doxorubicin / docetaxel / cmf regimens plus conventional rt in selected non - high - risk cardiac patients are relatively safe without cardiac toxicity at mid - term follow - up [ 34 ]  . 
 it is possible that this nding is related to the modern rt techniques that offer the opportunity to decrease the risk of cardiac toxicity [ 35 , 36 ] compared with classical techniques [ 37 , 38 ]  . 
probably , partial breast irradiation could be a valid option for selected patients who need concomitant ct - rt , but new randomised clinical trials are necessary . lit cardiaca e sono tornate al loro valore iniziale di lvef durante il follow - up . 
 gli autori hanno quindi concluso che i regimi sequenziali e combinati doxorubicina / docetaxel / cmf con la rt convenzionale in pazienti selezionati non ad alto rischio cardiaco sono relativamente sicuri dopo un follow - up a medio termine [ 34 ]  . 
possibile che questi risultati siano correlati allimplementazione delle moderne tecniche radioterapiche , che offrono lopportunit di diminuire il rischio di tossicit cardiaca [ 35 , 36 ] rispetto alle tecniche classiche [ 37 , 38 ]  . 
probabilmente lirradiazione parziale della mammella potrebbe rappresentare una valida opzione per pazienti selezionati che necessitano di trattamenti concomitanti ct - rt , ma nuovi studi clinici randomizzati sono necessari . conclusions in our experience , concomitant ct - rt did not emerge as a signi cant predictor on rt interruption . 
skin toxicity due to concomitant ac - based regimens and rt was acceptable , and no severe cardiac events were recorded . conclusioni nella nostra esperienza il trattamento concomitante ct - rt non risultato essere un fattore prognostico signi cativo di interruzione della radioterapia . 
la tossicit cutanea dovuta alla somministrazione concomitante di regimi a base di antracicline assieme alla rt risultata essere accettabile e , in particolare , non si sono veri cati eventi cardiaci di grado severo . con ict of interest none radiol med ( 2011 ) 116 : 503 doi 10.1007 / s11547 - 011 - 0646 - 0 erratum the ultrasonographic correlates of carpal tunnel syndrome in patients with normal electrodiagnostic tests correlazioni ecogra che nella sindrome del tunnel carpale nei pazienti con test elettrodiagnostici negativi m . 
kahnouji2 1advanced diagnostic and interventional radiology research center ( adir ) , imaging medical center , imam hospital , tehran university of medical sciences , tehran 14155 - 7146 , iran 2department of neurology , imam hospital , tehran university of medical sciences , p.o. 
bellomi1 , 2 1divisione di radiologia , istituto europeo di oncologia , via ripamonti 435 , 20141 milano , italy 2facolt di medicina e chirurgia , universit degli studi di milano , 20100 milano , italy 3divisione di epidemiologia and biostatistica , istituto europeo di oncologia , via ripamonti 435 , 20141 milano , italy 4divisione di oncologia medica , istituto europeo di oncologia , via ripamonti 435 , 20141 milano , italy 5divisione di anatomia patologica e medicina di laboratorio , istituto europeo di oncologia , via ripamonti 435 , 20141 milano , italy 6dipartimento di medicina del lavoro clinica del lavoro luigi devoto , sezione di statistica medica e biometria ga maccacaro , universit degli studi di milano , milano , italy correspondence to : g . 
twenty - eight patients with solid breast lesions > 10 mm underwent conventional contrastenhanced magnetic resonance imaging ( mri ) and diffusion - weighted mri ( dw - mri )  . 
two observers ( expert and trainee ) segmented the lesion from the surrounding breast tissue on dw images with high b - value ( 1 , 000 s / mm2 )  . 
the mean of the differences was 0.012 10 - 3 mm2 / s , corresponding to an intraobserver variability of 1.1% ( limits of agreement : - 5% / + 8% )  . 
 the mean interobserver difference was 0.022 10 - 3 mm2 / s , corresponding to an interobserver variability of 2% ( limits of agreement : 9% / + 14% )  . 
ventotto pazienti con lesioni mammarie solide > 10 mm sono state sottoposte a risonanza magnetica ( rm ) convenzionale con mezzo di contrasto e a rm pesata in diffusione ( rm - dw )  . 
due osservatori hanno isolato la lesione dal tessuto mammario circostante nelle sequenze con elevata pesatura in diffusione ( b - value = 1000 s / mm2 ) ; tale analisi stata ripetuta da un osservatore dopo 6 mesi . 
stata calcolata una media delle differenze di 0 , 01210 - 3 mm2 / s , corrispondente ad una variabilit intra - osservatore di 1 , 1% ( limiti di accordo - 5% / + 8% )  . 
stata calcolata una media delle differenze di 0 , 02210 - 3 mm2 / s , corrispondente ad una variabilit inter - osservatore di 2% ( limiti di accordo di - 9% / + 14% )  . 
 radiol med ( 2011 ) 116 : 466476 which supports its potential use in routine clinical practice . keywords breast carcinoma diffusion - weighted magnetic resonance imaging intraobserver variability interobserver variability conclusioni . 
in breast cancer , timesignal intensity curves of a region of interest ( roi ) placed over the lesion typically show a high early peak , followed by a plateau or washout phase . 
this feature is due to the presence of highly permeable capillaries , which facilitate the rapid passage of a large amount of contrast material into the interstitial space and its rapid return into the vascular compartment by passive diffusion [ 3 ]  . 
breast mri also assesses the qualitative features of contrast enhancement , which may be homogeneous , heterogeneous or located predominantly at the periphery of the lesions ( rim enhancement )  . 
on the basis of the presence or absence of each of the above features , the breast lesion is given a score , which expresses the likelihood of malignancy according to the categories of the breast imaging reporting and data system ( bi - rads ) for mri [ 4 ]  . although the use of diffusion - weighted ( dw ) mri sequences for studying breast lesions is a relatively recent development , numerous studies have reported on the potential of dw - mri in the workup of breast abnormalities . 
these encouraging results have prompted many radiologists to incorporate dw sequences into conventional mri examinations , given that dw sequences are relatively fast ( generally 56 min ) and do la risonanza magnetica ( rm ) con mezzo di contrasto della mammella entrata a far parte della routine clinica , con performance elevate se eseguita per le corrette indicazioni ( ad esempio , sorveglianza delle donne ad alto rischio , stadiazione locale pre - trattamento chirurgico , valutazione delleffetto della chemioterapia neoadiuvante , studio della cicatrice nella mammella operata per carcinoma , carcinoma occulto della mammella , mammella secernente ) [ 1 , 2 ]  . 
le curve intensit - tempo relative a una regione di interesse ( roi ) posizionata sulla lesione mammaria indagata presentano tipicamente un picco precoce ed elevato , seguito da una fase di plateau o di wash - out nel carcinoma mammario ( cm )  . 
tale caratteristica dovuta alla presenza di capillari molto permeabili nel cm , che favoriscono un veloce ed abbondante passaggio di mezzo di contrasto nellinterstizio e un suo veloce ritorno nel compartimento vascolare per diffusione passiva [ 3 ]  . 
in relazione alla presenza o meno di ciascuna delle caratteristiche semeiotiche descritte attribuito un punteggio alla lesione mammaria , che ne de nisce la probabile malignit secondo le categorie breast imaging reporting and data system ( bi - rads ) rm [ 4 ]  . relativamente recente lutilizzo di sequenze pesate in diffusione ( dw ) per lo studio delle lesioni mammarie . 
one study compared the published experiences of 12 different centres , all of which used a 1.5 - tesla ( t ) mri system but with different parameters for dw sequences [ 11 ]  . 
the aim of our work was therefore to determine the intraand interobserver variability in calculating adc value of breast carcinomas . materials and methods patients the study was approved by the local ethics committee . 
 between may 2008 and july 2009 , a prospective study was conducted in which dw sequences were incorporated into the conventional mri examinations of women with solid breast lesions > 10 mm and a radiological and / or cytological diagnosis of malignancy who were candidates for surgery . 
all patients were enrolled after giving written informed consent . mri technique all mri examinations were performed with a 1.5 - t imager ( avanto , siemens , erlangen , germany )  . 
all patients were imaged lying prone , with both breasts positioned in a dedicated 8 - channel breast coil ( invivo breast array coil , invivo , orlando , fl , usa )  . 
all sequences were acquired with the use of parallel imaging , with the generalized autocalibrating partially parallel acquisitions ( grappa ) algorithm and an acceleration factor of 2 to decrease acquisition time and increase spatial resolution . 
 the dynamic sequence was repeated eight times : one baseline acquisition and seven continuous acquisitions starting after a 10 - s interval , during which gadopentetate dimeglumine ( magnevist , bayer schering , germany ) was administered intravenously with an automatic injector . 
the dose of contrast material was determined based on the patients te stato osservato un valore di adc pi basso nei cm che esprimono recettori per gli estrogeni [ 8 ]  . 
questi incoraggianti risultati hanno spinto molti radiologi ad integrare le sequenze dw allinterno dellesame rm convenzionale , favoriti dal fatto che le sequenze dw hanno una durata relativamente breve ( generalmente , entro i 56 minuti ) e non interferiscono con lesecuzione dellesame rm convenzionale con mezzo di contrasto . 
 uno studio ha confrontato le esperienze pubblicate in letteratura da 12 centri diversi , che hanno utilizzato tutti un apparecchio rm da 1 , 5 t , ma parametri delle sequenze dw non omogenei [ 11 ]  . 
 lo scopo di questo lavoro di determinare la variabilit intra - osservatore e inter - osservatore nel calcolo del valore di adc nei carcinomi mammari . materiali e metodi pazienti lo studio stato approvato dal comitato etico del nostro istituto . 
nel periodo compreso tra maggio 2008 e luglio 2009 stato eseguito uno studio prospettico , che prevedeva di includere sequenze dw allinterno dellesame rm convenzionale effettuato in donne con lesioni solide mammarie superiori ai 10 mm e diagnosi radiologica e / o citologica di malignit , candidate a chirurgia . 
tutte le pazienti sono state arruolate dopo avere ottenuto un consenso informato scritto . tecnica rm gli esami rm sono stati eseguiti con un apparecchio rm da 1 , 5 t ( avanto , siemens , erlangen , germania )  . 
tutte le pazienti sono state esaminate in posizione prona , con entrambe le mammelle adagiate allinterno di una bobina dedicata a 8 canali ( invivo breast array coil , invivo , orlando , florida , usa )  . 
dopo lacquisizione di una survey , sono state acquisite immagini rm nel piano assiale con il seguente ordine : short - tau inversion recovery ( stir ) pesate in t2 ( tempo di ripetizione [ tr ] / tempo di eco [ te ] / tempo di inversione [ ti ] = 3510 / 72 / 170 ms ; spessore , 3 mm ; gap 0 , 3 mm ; matrice 320320 , tempo di acquisizione 3 min 38 s ) ; sequenze dw ( tr / te = 4800 / 71 ms ; spessore , 5 mm ; gap 1 mm ; matrice 15090 , tempo di acquisizione 5 min 04 s ) con 4 b - value ( 0 , 250 , 500 , 1000 s / mm2 ) e soppressione del segnale lipidico mediante tecnica radiol med ( 2011 ) 116 : 466476 weight ( 0.1 mmol / kg ) and administered at a ow rate of 2 ml / s , followed by a standard bolus of saline solution ( 20 ml ) injected at the same rate of 2 ml / s . image analysis included digital subtraction of from the image processing dynamic precontrast sequence the postcontrast sequences ( subtracted images ) ; maximum intensity projection ( mip ) reconstructions in the axial , coronal and sagittal planes ; and adc mapping ( in grey scale , with light shades for high adc values and dark shades for low values ) ; and was done automatically by the mri systems software . 
all examinations were rendered anonymous and analysed by a radiologist experienced in the interpretation of breast mr images ( expert observer ) and a trainee radiologist ( nonexpert observer )  . 
the solid breast lesion was identi ed by using all available images ( stir , subtracted images , mip , dw images and adc maps )  . to calculate adc value ( quantitative analysis ) , dw sequences and adc maps were sent to a pc in digital imaging and communications in medicine ( dicom ) format [ 13 ]  . 
in all cases , the image was manually thresholded to ensure that certain pixels covered the entire area of the lesion ; any pixels outside of the lesion were removed digitally . 
six months after initial evaluation , the expert observer recalculated the adc values after separating the solid lesion from the surrounding breast tissue and selecting the volume of interest , following the same procedure as used for the rst reading . statistical analysis results of quantitative analysis were compared to evaluate intraobserver variability in the expert observer and interobserver variability between the expert and nonexpert observer . 
for each analysis , we calculated the differences between the two measurements with limits of agreespectral selection attenuated inversion recovery ( spair ) ; gradient echo 3d - fast low angle shot ( flash ) ( tr / te 7 , 4 / 4 , 7 ms ; ip angle 25 , matrice 38496 ; spessore 1 , 3 mm , gap 0 , 25 mm , tempo di acquisizione 1 min 02 s )  . 
tutte le sequenze prevedevano lutilizzo dellimaging parallelo , con algoritmo generalized autocalibrating partially parallel acquisitions ( grappa ) e fattore di accelerazione 2 , per diminuire il tempo di acquisizione e incrementare la risoluzione spaziale . 
la sequenza dinamica stata ripetuta per 8 volte : unacquisizione basale e sette acquisizioni continue aventi inizio dopo una pausa di 10 secondi , durante la quale avviene somministrazione endovenosa di gadopentetato dimeglumina ( magnevist , bayer schering , germania ) con un iniettore automatico . 
la dose stata decisa in base al peso della paziente ( 0 , 1 mmol / kg ) e somministrata ad un usso di 2 ml / s , seguita da un bolo standard di soluzione siologica ( 20 ml ) , anchessa ad un usso di 2 ml / s . analisi delle immagini le elaborazioni delle immagini con la sottrazione digitale della sequenza dinamica pre - contrasto dalle sequenze dinamiche post - contrasto ( immagini sottratte ) , le ricostruzioni maximum intensity projection ( mip ) nei piani assiale , coronale e sagittale , e le mappe adc ( in scala di grigi , con colore chiaro per valori elevati e con colore scuro per valori bassi di adc ) sono create in maniera automatica dal software dellapparecchio rm . 
 tutti gli esami sono stati resi anonimi e analizzati da un radiologo esperto nellinterpretazione di esami rm della mammella ( osservatore esperto ) e da un radiologo in formazione ( osservatore inesperto ) ; la lesione solida mammaria stata identi cata utilizzando tutte le immagini disponibili ( stir , sottratte , mip , dw e mappe adc )  . per il calcolo del valore di adc ( analisi quantitativa ) , le sequenze dw e le mappe adc sono state inviate a un pc in formato digital imaging and communications in medicine ( dicom ) [ 13 ]  . 
 stata sempre eseguita una modi ca manuale del threshold dellimmagine , in modo che dei pixel coprissero per intero larea della lesione ; eventuali pixel al di fuori della lesione sono stati rimossi digitalmente . 
i volumi cos ottenuti da entrambi gli osservatori , corrispondenti alle lesioni solide , sono stati salvati ed elaborati con un codice sviluppato nel nostro dipartimento basato su un software disponibile allinterno della functional mri of 470 radiol med ( 2011 ) 116 : 466476 fig . 
1 diffusion - weighted magnetic resonance imaging acquisition produces a series of images with different diffusion weightings according to the b value applied during imaging ( upper row )  . 
signal intensity decay with increasing b values ( lower row , far left ) can be modelled as an exponential equation , and on tting the equation to the data , the adc value is determined for each voxel to produce an adc map ( lower row , centre )  . 
rois were de ned by thresholding the highest b value image ( middle row , far right and centre right ) and then including only the cluster of suprathreshold voxels ( red ) that were part of the lesion ( middle row , centre left ) , as judged by comparison with the postcontrast images . 
to illustrate the stereotypical pattern of hyperintensity on the high b - value image and hypointensity on the adc map , the roi boundary only is overlaid on the corresponding images ( dashed arrows ) , allowing the reader to see the underlying tumour . 
la riduzione dellintensit del segnale allaumento del b - value ( riga inferiore a sinistra ) pu essere modellata come una equazione esponenziale ed integrando i dati allequazione , il valore adc calcolato per ogni voxel per produrre una mappa adc ( riga inferiore , al centro )  . 
le roi sono state disegnate utilizzando un threshold dellimmagine con il b - value maggiore ( riga in mezzo , a destra e centro - destra ) e poi includendo il cluster dei voxel oltre il threshold ( in rosso ) che includevano la lesione . 
per illustrare il pattern tipico di iperintensit sulle immagini con b - value elevato e ipointensit sulle mappe adc , il margine della roi stato sovrapposto sulle rispettive immagini ( freccia tratteggiata ) permettendo allosservatore di identi care il tumore sottostante . 
i valori di adc da ciascun voxel compreso nel volume di interesse sono ottenuti dalle mappe adc e sono stati usati per calcolare la media , la mediana e la deviazione standard per il tumore ( riga inferiore , destra )  . ment , with values expressed in the unit of measurement used , and the ratio between the two measurements with the corresponding limits of agreement to estimate the percentage variation of the two measurements . 
for the latter analysis , logarithmic transformation was applied to adc values , and the analysis was conducted on the differences between logarithms , as suggested by bland and altman [ 16 ]  . 
all patients tolerated the mr examination well , with no cases of adverse reaction to the contrast mediu mean lesion size was 21 ( range 1050 ) mall patients underwent surgery ( 16 mastectomy , 12 quadrantectomy )  . 
 il calcolo del valore di adc delle lesioni mammarie stato ripetuto a una distanza di 6 mesi dalla prima valutazione dallosservatore esperto ; lisolamento della lesione solida dal resto del tessuto mammario e la selezione del volume di interesse sono stati effettuati dallosservatore esperto , seguendo la medesima procedura eseguita per la prima lettura . analisi statistica i risultati dellanalisi quantitativa sono stati paragonati allo scopo di valutare la variabilit intra - osservatore nellosservatore esperto e la variabilit inter - osservatore radiol med ( 2011 ) 116 : 466476 table 1 histology : histological type and tumour node metastasis ( tnm ) staging patient histological typea tra osservatore esperto e non - esperto . 
per entrambe le analisi sono stati calcolati : le differenze tra le due misurazioni con i rispettivi intervalli di accordo , con i valori espressi nellunit di misura utilizzata e il rapporto tra le due misurazioni con i rispettivi intervalli di accordo , per ottenere una stima della variazione percentuale delle due misurazioni effettuate . 
per questultima analisi , i valori di adc sono stati sottoposti a trasformazione logaritmica e lanalisi stata cos condotta sulle differenze tra i logaritmi , come suggerito da bland e altman [ 16 ]  . 
2b. stata calcolata una media delle differenze di 0 , 012103 mm2 / s ( limiti di accordo : - 0 , 059 ; + 0 , 08310 - 3 mm2 / s ) , corrispondente ad una variabilit intra - osservatore pari a 1 , 1% ( limiti di accordo di - 5% / + 8% )  . 
these sequences were optimised to achieve a compromise between image quality requirements for quantitative analysis ( with adequate b value and signal - to - noise ratio ) and the short acquisition time needed for acceptable incorporation into a conventional contrastenhanced mri examination . mean adc value calculated in breast carcinomas in our study ( 1.0710 - 3 mm2 / s ) was comparable with the mean value calculated for seven different centres ( 0.9910 - 3 mm2 / s ) , which used similar mri equipment ( 1.5 t ) and dw sequences ( maximum b value of 1 , 000 or 1 , 074 s / mm2 ) to those used by us [ 11 ]  . 
this similarity in adc value further supports the possibility of comparing the adc value calculated for breast carcinomas in different centres and with different equipment , provided that the same dw sequence parameters are used . 
 queste sono state ottimizzate in modo da trovare un equilibrio tra i requisiti di qualit dellimmagine necessari per unadeguata analisi quantitativa ( con un numero suf ciente di b - value e un adeguato rapporto segnale / rumore ) e i requisiti necessari di brevit , fondamentali per renderne accettabile lintegrazione allinterno di un esame rm convenzionale con mezzo di contrasto . il valore medio di adc che abbiamo calcolato nei cm del nostro studio ( 1 , 0710 - 3 mm2 / s ) paragonabile al valore medio calcolato fra 7 centri diversi ( 0 , 9910 - 3 mm2 / s ) che hanno utilizzato unapparecchiatura rm ( 1 , 5 t ) e una sequenza dw ( b - value massimo di 1000 o 1074 s / mm2 ) simili alle nostre [ 11 ]  . 
questa sovrapponibilit del valore di adc avvalora ulteriormente la possibilit di confrontare il valore di adc calcolato per i cm in centri diversi e con apparecchiature rm diverse , se i parametri delle sequenze dw sono simili . 
tali fonti di variabilit potrebbero rendere elevata la variabilit nel calcolo del valore di adc eseguito da operatori diversi o dallo stesso operatore in momenti diversi , limitando di fatto lutilizzo del valore di adc nella routine clinica . 
 gli autori hanno descritto una buona riproducibilit del parametro misurato ( il valore di adc ) e hanno rappresentato i dati su gra co , senza speci care il dato tuttavia , posnumerico . 
 [ 12 ] , gli autori hanno incluso tutte le lesioni mammarie con dimensioni superiori ai 7 mm , delle quali 22 ( 25% ) erano benigne e 9 carcinoma in situ ( duttale o lobulare )  . 
2a , b both the interrater ( a ) intrarater ( b ) comparisons and showed low mean differences , 0.02210 - 3 mm2 / s and 0.01210 - 3 mm2 / s , respectively . 
2a , b il confronto inter - osservatore ( a ) e intra - osservatore ( b ) ha mostrato piccole differenze nei valori medi ; rispettivamente di 0 , 02210 - 3 mm2 / s e 0 , 01210 - 3 mm2 / s . 
il range dei limiti di accordo del confronto interosservatore quasi il doppio di quello intraosservatore ( - 0 , 10010 - 3 mm2 / s : 0 , 14310 - 3 mm2 / s vs - 0 , 5910 - 3 mm2 / s : 0 , 08310 - 3 mm2 / s )  . random or systematic errors in the use of the image - analysis software could lead to major errors in adc calculation . 
 these sources of variability could lead to high variability in adc determinations done by different operators or by the same operator at different times , thereby limiting the use of adc values in routine clinical practice . 
la natura solida e le dimensioni superiori ai 10 mm delle lesioni mammarie incluse nel nostro studio hanno probabilmente reso pi semplice per gli osservatori la de nizione dei margini della lesione . 
le dimensioni superiori al 10 mm , inoltre , hanno probabilmente limitato gli errori di calcolo dovuti alleffetto di volume parziale che potrebbero veri carsi per il calcolo delladc nelle lesioni mammarie di pochi millimetri , in cui gran parte dei voxel apparentemente relativi alla lesione mammaria sarebbe in effetti condivisa con 474 radiol med ( 2011 ) 116 : 466476 ( interobserver )  . 
 [ 12 ] , all breast lesions > 7 mm where included , of which 22 ( 25% ) were benign and nine were carcinoma in situ ( ductal or lobular )  . 
by contrast , our study considered only solid breast lesions > 10 mm , which were all subsequently found to be in ltrating breast carcinomas ( ductal or lobular )  . 
in addition , the larger size most likely limited calculation errors resulting from possible partial volume effects when determining the adc of lesions measuring a few millimetres only , where many of the voxels apparently belonging to the lesion are in fact shared with the surrounding breast tissue . 
in our study , although all available images ( stir , subtracted images , mip , dw images and adc maps ) were used to identify the lesion , only those with high diffusion weighting ( b value 1 , 000 s / mm2 ) were used to separate the solid lesion from the surrounding breast tissue . 
the differences between the two studies demonstrate the strength of the impact of operator technique in selecting the volume of interest for adc determination in the context of the quantitative analysis of dw - mri of breast carcinomas . 
the breast is less subject to artefacts compared with other body regions , such as the neck and face region ( movement artefacts due to deglutition and carotid pulsatility ) , the upper abdomen ( movement artefacts due to diaphragmatic excursion ) and the pelvis ( movement artefacts due to intestinal peristalsis and magnetic susceptibility due to the interface between bowel gas and surrounding tissues )  . 
in addition , the higher fat content of breast tissue , the signal of which is completely suppressed on dw images , and the use of images with high b value to de ne the roi , which allows il tessuto mammario circostante . 
nel nostro studio , invece , ci siamo serviti di tutte le immagini disponibili ( stir , sottratte , mip , dw e mappe adc ) per lidenti cazione della lesione mammaria , ma abbiamo utilizzato solo le immagini con elevata pesatura in diffusione ( b - value di 1000 s / mm2 ) per isolare la lesione solida dal resto del tessuto mammario . 
le differenze riscontrate tra i due studi mostrano quanto possa essere grande limpatto della tecnica con cui loperatore seleziona il volume di tumore da analizzare ai ni del calcolo del valore di adc , nellambito dellanalisi quantitativa della dw - mr dei cm . 
in ne , un insieme di condizioni che generalmente si veri cano per la rm - dw della mammella pu aver favorito una bassa variabilit nel calcolo del valore di adc nelle lesioni mammarie del nostro studio . 
la mammella meno soggetta ad artefatti rispetto ad altri distretti corporei , come ad esempio il distretto cervicofacciale ( artefatti da movimento per la deglutizione e da pulsatilit carotidea ) , lalto addome ( artefatti da movimento per lescursione del diaframma ) e la pelvi ( artefatti da movimento per la peristalsi intestinale e da suscettibilit magnetica dovuti alle interfacce tra il gas intestinale e i tessuti circostanti )  . 
inoltre , la elevata rappresentazione del tessuto adiposo nella mammella rispetto ad altre regioni anatomiche , il quale del tutto soppresso nelle immagini dw , e lutilizzo nel nostro studio di immagini con elevata pesatura in diffusione per la de nizione delle roi , che consentono un ef cace abbattimento del segnale originato dal tessuto ghiandolare sano , hanno probabilmente favorito lidenti cazione del cm e la delimitazione dei suoi margini con la roi . 
in particolare , la bassa variabilit intra - osservatore mette in evidenza la stabilit nel calcolo del valore di adc nei cm e suggerisce la possibilit da parte dello stesso operadiol med ( 2011 ) 116 : 466476 effective suppression of the signal from the healthy glandular tissue , probably helped our observers to identify the breast carcinoma and delineate its margins with the roi . 
 in particular , the low intraobserver variability re ects the stability of the adc determinations in breast cancers and suggests that the same operator can reliably repeat the measurements at different times , as could be the case in clinical practice for example , in breast cancer patients treated with neoadjuvant chemotherapy . 
the relatively low interobserver variability suggests that , provided image analysis is rigorously standardised , different operators could obtain comparable adc values in breast cancers , as could be the case in routine practice for breast cancer patients whose dw - mr images are analysed by different operators . there are a few limitations to our study . 
 this approach requires collaboration of a specialist in image analysis ( physicist , bioengineer , etc . ) , who is , however , needed for verifying image quality and results in any study involving a quantitative analysis of mr images . 
another limitation could be that a small fraction of the training in radiological mri of the nonexpert observer took place together with the expert observer during routine practice , and this may have contributed to limiting interobserver variability . 
 ratore di ripetere in maniera af dabile tali misure in momenti diversi , come , ad esempio , potrebbe veri carsi nella routine clinica per le pazienti con cm sottoposte a chemioterapia neoadiuvante . 
la variabilit inter - osservatore relativamente bassa suggerisce la possibilit da parte di operatori diversi di ottenere misurazioni sostanzialmente paragonabili del valore di adc sui cm , se lanalisi delle immagini standardizzata in maniera rigorosa , come , ad esempio , potrebbe veri carsi nella routine clinica per le pazienti con cm la cui rm - dw analizzata da radiologi diversi . il nostro studio ha dei limiti . 
questo approccio rende necessario il contributo di uno specialista ( sico , bio - ingegnere , ecc . ) in analisi dimmagini , il quale tuttavia necessario per il controllo della qualit delle immagini e dei risultati negli studi che prevedono lanalisi quantitativa delle immagini rm . 
un altro limite potrebbe essere costituito dal fatto che una piccola parte della formazione radiologica in rm dellosservatore inesperto sia avvenuta con losservatore esperto durante la routine clinica ; questo potrebbe aver contribuito a limitare la variabilit interosservatore . 
 in conclusione , abbiamo dimostrato che il nostro metodo di analisi quantitativa delle immagini dw ha consentito di ottenere basse variabilit intra - osservatore e variabilit inter - osservatore , le quali supportano lutilizzo del valore di adc nella routine clinica per i cm , qualora studi futuri ne validino in maniera de nitiva lutilit clinica . 
torricelli1 1department of radiology , azienda ospedaliera universitaria , policlinico , modena , italy 2department of surgery , azienda ospedaliera universitaria , policlinico , modena , italy 3department of pathology , azienda ospedaliera universitaria , policlinico , modena , italy correspondence to : f . 
thirty patients with histologically proven rectal cancer underwent surface - coil 3t mr imaging with sagittal , paracoronal and para - axial t2 - weighted turbo spin echo ( tse ) sequences . 
in the patients who underwent mr imaging before and after radiotherapy ( group 1 ) , the diagnostic accuracy of 3t mr imaging was 88% for t2 , 94% for t3 and 88% for t4 cancers . 
the high signal - to - noise ratio coupled with a large eld of view enables surface - coil 3t mr imaging to achieve high levels of diagnostic accuracy in the local staging of rectal cancer , and in particular in assessing in ltration of mesorectum and mesorectal fascia . 
sono state eseguite sequenze turbo spin echo ( tse ) t2 sagittali , para - coronali e para - assiali , spessore immagine 3 mm senza gap , eld of view ( fov ) 24 cm , matrice acquisizione 400512 . 
nei pazienti esaminati prima e dopo terapia neoadiuvante ( gruppo 1 ) i valori di accuratezza diagnostica della rm 3 t sono stati 88% per t2 , 94% per t3 , 88% per t4 , valore medio 92% , nei pazienti sottoposti a intervento chirurgico senza terapia neoadiuvante ( gruppo 2 ) laccuratezza diagnostica stata 93% per t2 , 79% per t3 , 86% per t4 , valore medio 86% ; laccuratezza globale stata 90% per t2 , 87% per t3 , 87% per t4 , valore medio 89% . 
 376 radiol med ( 2011 ) 116 : 375388 parole chiave rm 3 t stadiazione loco - regionale carcinoma rettale introduction introduzione colon carcinoma is one of the most common malignancies in industrialised countries , and location in the rectum sigmoid is seen in 60%70% of cases [ 13 ]  . 
once a histological diagnosis of rectal carcinoma has been established , a series of parameters need to be de ned , which are essential for planning treatment and which include depth of wall in ltration , mesorectal in ltration , circumferential resection margin ( crm ) , invasion of surrounding structures and presence of distance metastasis . 
in fact , whereas tumours con ned to the rectal wall can be treated radically by local excision alone , those that in ltrate the mesorectal fat require more extensive surgery , such as total mesorectal excision ( tme ) or abdominoperineal resection [ 39 ]  . 
additionally , stages t3 and t4 cancers [ 1 , 2 ] and those that in ltrate the upper portion of the anal canal are also subjected to neoadjuvant chemoradiotherapy with the aim of downstaging the tumour , a practice that has proved effective in improving outcome and permitting a more conservative surgical treatment [ 2 ]  . accurate preoperative assessment is therefore fundamental for directing management decisions [ 1016 ] , and in this context , imaging plays an increasingly important role . 
 among the imaging modalities used for disease staging , endorectal ultrasound is considered the reference standard for evaluating the depth of wall in ltration ; however , it suffers signi cant diagnostic limitations in evaluating extraparietal disease extent ( mesorectum and adjacent organs ) and regional lymph nodes [ 3 , 17 , 18 ] , as well as being dif cult to perform in severely stenosing tumours . multidetector computed tomography ( mdct ) , despite the signi cant recent improvement in staging effectiveness , still has limitations in contrast resolution that preclude adequate local staging of tumours of the middle and lower rectum , where correct assessment of parietal and perirectal in ltration is problematic [ 1 , 3 ]  . 
better results have been reported for cancers of the upper third of the rectum , where the greater abundance of perirectal fat heightens the natural contrast and therefore makes mdct staging more accurate [ 19 ]  . 
 numerous studies have demonstrated the ability of magnetic resonance ( mr ) imaging with phased - array coil to evaluate in ltration of the rectal layers , extension to the mesorectum and possible involvement of adjacent organs , reporting levels of accuracy that have made it the most accurate imaging technique for staging rectal cancer [ 1 , 3 , 2022 ]  . 
nearly all such studies were , however , conducted with 1.5 - t magnets and very few with systems operating il carcinoma del colon costituisce una delle pi frequenti neoplasie nei paesi industrializzati e la localizzazione al retto - sigma riscontrabile nel 60%70% di tutti i carcinomi del colon [ 13 ]  . 
dal momento in cui viene posta la diagnosi istologica di carcinoma del retto fondamentale de nire una serie di parametri che risultano indispensabili per la programmazione delle strategie terapeutiche e che sono rappresentati dalla profondit di in ltrazione parietale , dallin ltrazione del mesoretto , dal margine di resezione circonferenziale ( crm ) e dallin ltrazione di organi adiacenti oltre che , ovviamente , dalla presenza di metastasi a distanza . 
infatti , mentre i tumori con nati alla parete rettale possono essere trattati in modo radicale con la sola escissione locale , quelli in ltranti il tessuto adiposo mesorettale necessitano di approcci chirurgici pi estesi quali lasportazione totale del mesoretto ( tme ) o la resezione addominoperineale [ 39 ]  . 
inoltre , i tumori agli stadi t3 e t4 [ 1 , 2 ] e quelli che in ltrano la porzione superiore del canale anale , vengono sottoposti anche a chemio - radioterapia neoadiuvante con lo scopo di ridurre lo stadio della neoplasia , prassi che ha dimostrato di poter migliorare la prognosi e rendere possibili trattamenti chirurgici pi conservativi [ 2 ]  . unaccurata valutazione preoperatoria pertanto fondamentale per guidare le decisioni terapeutiche [ 1016 ] ed in tale ambito limaging riveste un ruolo sempre pi determinante . 
tra le metodiche di imaging utilizzabili per la stadiazione , lecogra a endorettale considerata il gold standard nel valutare la profondit di in ltrazione parietale , ma ha signi cativi limiti diagnostici nella valutazione dellestensione extraparietale ( al mesoretto ed organi adiacenti ) e dei linfonodi regionali [ 3 , 17 , 18 ] , oltre ad essere di dif cile esecuzione nelle forme fortemente stenosanti . la tomogra a computerizzata multidetettore ( tcmd ) , pur avendo negli ultimi anni migliorato in modo signile proprie performances stadiative , presenta cativo tuttora limiti di risoluzione di contrasto che non consentono unadeguata stadiazione locale delle neoplasie del retto medio - basso , dove risulta problematica la corretta valutazione dellin ltrazione parietale e perirettale [ 1 , 3 ]  . 
 migliori risultano invece i suoi risultati nelle neoplasie del terzo rettale superiore dove la maggiore presenza di adipe perirettale migliora il contrasto naturale e rende pi adeguate le possibilit stadiative della tcmd [ 19 ]  . 
 numerosi contributi della letteratura hanno dimostrato la capacit della risonanza magnetica ( rm ) con bobina phased array di valutare lin ltrazione tumorale delle radiol med ( 2011 ) 116 : 375388 at higher eld intensities . 
finally , a limited number of investigations conducted with both 1.5 - t and 3 - t magnets have evaluated patients with rectal carcinoma after neoadjuvant therapy [ 27 , 28 ]  . 
 the study population consisted of 30 patients ( 20 men , 10 women ; mean age 63 10 years ) with a diagnosis of rectal carcinoma proved by ultrasound - guided or endoscopic rectal biopsy and who were evaluated between april 2005 and december 2008 . 
twelve tumours were located in the upper third of the rectum , 11 in the middle third and seven in the distal third . mr imaging in all cases , mr imaging was conducted with a 3 - t system ( philips intera 3 - t , philips medical systems , best , the netherlands ) with the use of a pelvic phased - array surface coil . 
approximately 2 h before the mr study , the patients were given a water enema to cleanse the rectum and , at the beginning of the examination , they were administered antispasmodic iv ( buscopan , boheringer ) to reduce artefacts due to bowel peristalsis . 
examinations were performed without the administration of endorectal contrast medium in all but four patients who were affected by cancer of the rectal ampulla that was poorly depicted in the initial sequences and in whom the rectal lumen was distended with warm water delivered through an endorectal tube . 
the following sequences were acquired : axial t2 - weighted turbo spin echo ( tse ) ( time of repetition [ tr ] / time to echo [ te ] 3 , 200 / 100 ms , echo train length 16 ) encompassing the entire pelvis , eld of view 24 cm ; slice thickness 5 mm ; interval 1 mm ; matrix 312400 ; 2 signals acquired ; sensitivity encoding factor 1.5 ; sagittal t2 - weighted tse ( tr / te 3 , 200 / 100 ms , echo train length 18 ) ; eld of view 2224 cm ; slice thickness 3 mm ; no interslice gap ; matrix 400512 ; 2 signals acquired ; tonache rettali , lestensione al tessuto mesorettale e leventuale coinvolgimento degli organi adiacenti , con risultati in termini di accuratezza tali da rendere tale metodica la pi accurata tecnica di imaging per la stadiazione del carcinoma rettale [ 1 , 3 , 2022 ]  . 
la quasi totalit degli studi sovracitati stata condotta con magneti operanti a 1 , 5 t , mentre poco numerosi sono gli studi eseguiti con apparecchiature ad intensit di campo superiore ; in particolare , alcuni studi eseguiti con magnete operante a 3 t [ 2326 ] hanno riportato ottima accuratezza diagnostica , sovente superiore a quella ottenibile con magnete operante a 1 , 5 t . 
pochi studi , in ne , condotti sia con magneti a 1 , 5 t , che a 3 t , hanno valutato pazienti affetti da carcinoma del retto dopo terapia neoadiuvante [ 27 , 28 ]  . 
 lo scopo primario del presente studio valutare laccuratezza della rm a 3 t ( 3t rm ) nella stadiazione del carcinoma del retto ; scopo secondario quello di confrontare laccuratezza ottenibile con 3t rm in pazienti sottoposti a chirurgia dopo terapia neoadiuvante con quella di pazienti sottoposti ad intervento chirurgico senza terapia adiuvante . 
la popolazione dello studio consiste di 30 pazienti ( 20 maschi , 10 femmine ; et media 6310 ) con carcinoma del retto comprovato da biopsia rettale sotto guida ecogra ca o endoscopica , valutati da aprile 2005 a dicembre 2008 . 
dodici tumori erano localizzati nel terzo superiore del retto , 11 nel terzo medio del retto e 7 nel terzo distale . imaging rm la rm stata eseguita in tutti i pazienti impiegando un magnete operante a 3 t ( philips intera 3t , philips medical systems , best , olanda ) , con posizionamento di bobina di super cie phased array pelvica . 
circa 2 ore prima dellesame rm stato eseguito clistere idrico di pulizia dellampolla rettale e , allinizio dellesame , stata somministrato spasmolitico per via endovenosa ( buscopan , boheringer ) per ridurre gli artefatti da peristalsi intestinale . 
tutti gli esami sono stati eseguiti senza limpiego di contrasti endorettali , ad eccezione di 4 pazienti , affetti da carcinoma della porzione ampollare che era risultato mal visualizzabile nelle sequenze iniziali , ed in cui si provveduto a distendere il lume rettale con acqua tiepida , tramite sonda endorettale . 
in the event of very low rectal cancers with possible involvement of the anal canal , we obtained a similar paracoronal sequence parallel to the long axis of the anal canal ; axial t1 - weighted spin echo ( se ) ( tr / te 550 / 10 ms ) before and after administration of paramagnetic contrast medium ( gadolinium dota , dotarem , guerbet , arnoix sous bois , france ) ; eld of view 24 cm ; slice thickness 3 mm ; no interslice gap ; matrix 336512 ; sensitivity encoding factor 1.7 ; 2 signals acquired . 
 overall examination time was 3045 min . image analysis mr images were jointly reviewed prior to surgery by two observers ( fs and pt , radiologists with 5 and 10 years of experience in rectal mr imaging , respectively ) who were aware of the results of endorectal ultrasound and diagnostic biopsy . 
mr staging of local tumour extent was obtained on the basis of all t2 - weighted sequences and , in seven cases , also considering the t1 - weighted sequences with and without intravenous gadolinium administration . 
all tumours showed intermediate signal intensity in the t2 - weighted sequences ( higher relative to muscle and lower relative to submucosa ) in comparison with the rectal wall and mesorectal fat . 
 the observers used the tnm classi cation for tumour staging : stage t1 , if the tumour is con ned to the submucosa ; stage t2 , if the tumour signal intensity extends to the muscular layer without involving the perirectal fat ; stage t3 , if the tumour has intermediate signal and invades the mesorectal fat ; stage t4 , if the tumour extends to surrounding organs ( seminal vesicles , prostate , vaginauterus )  . 
 analisi delle immagini le immagini di rm sono state valutate congiuntamente da 2 osservatori ( fs e pt , medici radiologi , rispettivamente con 5 e 10 anni di esperienza in rm rettale ) prima dellintervento chirurgico . 
 la stadiazione rm dellestensione locale della neoplasia stata ottenuta sulla base dellanalisi di tutte le sequenze t2 pesate e , in 7 casi , anche tramite le sequenze t1 pesate con e senza somministrazione ev di gadolinio . 
le neoplasie hanno dimostrato in tutti casi intensit di segnale intermedia nelle sequenze t2 pesate ( pi alto del muscolo pi basso della sottomucosa ) al confronto con la parete rettale ed il tessuto adiposo mesorettale . 
 gli osservatori hanno utilizzato , a ni stadiativi , la classi cazione tumore - nodulimetastasi ( tnm ) : radiol med ( 2011 ) 116 : 375388 surgery and histopathology all patients underwent surgery consisting of local excision ( n = 11 ) , total mesorectal excision ( n = 16 ) and abdominoperineal resection ( n = 3 )  . 
preoperative neoadjuvant therapy consisted of the application of 45 gy to the pelvis in 1.8 gy fractions ; chemotherapy involved one cycle of 5uorouracil administered by continuous iv infusion at a daily dose of 250 mg / m2 . 
surgery was performed 56 weeks after the end of neoadjuvant therapy . in all 30 patients , histological examination and pathological staging was performed on the surgical specimen through the anatomical study of macrosections and conventional histopathological examination . 
the following two parameters were considered : in ltration of the mesorectal fat , to differentiate stage t2 from t3 ; in ltration of adjacent organs and the mesorectal fascia , which , if present , de ned stage t4 . 
the statistical signi cance of any difference in results between groups 1 and 2 was evaluated using the pearson test . results the mr study was well tolerated by all patients . 
 among patients in group 1 ( table 2 ) , two showed no signs of tumour , eight were staged as t2 , ve as t3 and one as t4 . 
in this group , histological examination established a diagnosis of stage t1 cancer in one case , t2 in stadio t1 , se il tumore non si estende oltre la sottomucosa ; stadio t2 , se il tumore , di segnale maggiore della tonaca muscolare , si estende no alla tonaca muscolare , ma non nel tessuto adiposo perirettale ; stadio t3 , se la neoplasia , di segnale intermedio , in ltra il tessuto adiposo mesorettale ; stadio t4 , se la neoplasia si estende ad organi circostanti : vescichette seminali , prostata , vagina - utero . lidenti cazione di linfonodi a distanza o di metastasi extrapelviche non rientrata tra gli scopi di questo studio . 
 la brosi perirettale stata identi cata come tessuto a margini spiculati ad intensit di segnale bassa nelle sequenze t2 pesate . chirurgia e anatomia patologica tutti i pazienti sono stati sottoposti a intervento chirurgico tramite escissione locale ( 11 ) , escissione totale del mesoretto ( 16 ) e resezione addominoperineale ( 3 )  . 
sedici pazienti sono stati sottoposti a terapia neoadiuvante preoperatoria : radioe chemioterapia ( gruppo 1 ) ed in questo gruppo la rm stata eseguita prima e dopo almeno 1015 giorni il termine della terapia ed entro 10 giorni dallintervento chirurgico . 
la radioterapia preoperatoria neoadiuvante consistita nellapplicazione di 45 gy , applicate alla pelvi in frazioni di 1 , 8 gy ; la chemioterapia stata basata su un ciclo di 5 fu , somministrato per infusione endovenosa continua alla dose giornaliera di 250 mg / m2 . 
lintervento chirurgico stato eseguito 56 settimane dopo il completamento della terapia neoadiuvante . in tutti i 30 pazienti la valutazione istologica e la stadiazione anatomo - patologica stata eseguita sul pezzo operatorio attraverso lo studio anatomico di macrosezioni ed il tradizionale studio istopatologico . 
lanatomo - patologo ha esaminato i campioni senza essere a conoscenza della stadiazione preoperatoria radiologica . analisi dei dati i risultati radiologici sono stati correlati a quelli istopatologici , assunti come standard di riferimento . 
sono stati considerati i 2 parametri seguenti : in ltrazione del tessuto adiposo mesorettale , per differenziare lo stadio t2 dal t3 ; in ltrazione di organi contigui e della fascia mesorettale , che se presenti , hanno de nito lo stadio t4 . 
 la signi cativit statistica della differenza dei risultati tra il gruppo 1 e il gruppo 2 stata valutata statisticamente tramite il test di pearson . table 1 tumour in ltration : comparison between magnetic resonance imaging ( mri ) and histopathological staging ; overall ( group 1 and group 2 ) sensitivity , speci city and accuracy of mr according to the different t stages of the tumour histology no tumour ( n = 2 ) t1 ( n = 1 ) t2 ( n = 12 ) t3 ( n = 11 ) t4 ( n = 4 ) total ( n = 30 ) radiol med ( 2011 ) 116 : 375388 380 mri no tumour stage no tumour mean value mri no tumore stadio no tumore valore medio sensitivity 100% 92% 82% 50% sensibilit 100% 92% 82% 50% speci city 100% 89% 89% 92% speci cit 100% 89% 89% 92% accuracy 100% 90% 87% 87% 89% accuratezza 100% 90% 87% 87% 89% risultati tabella 1 in ltrazione tumorale confronto tra stadiazione tramite rm e quella anatomopatologica ; valori complessivi ( gruppo 1 + gruppo 2 ) di sensibilit , speci cit ed accuratezza della rm in base agli stadi t della neoplasia rettale istologia no tumore ( n = 2 ) t1 ( n = 1 ) t2 ( n = 12 ) t3 ( n = 11 ) t4 ( n = 4 ) totale ( n = 30 ) six , t3 in four and t4 in three ; no residual tumour was detected in two cases . 
six of eight tumours staged as t2 on mr imaging were con rmed by pathology , one case was overstaged ( histological stage t1 ) and one was understaged ( histological stage t4 )  . 
four of ve tumours staged as t3 on mr imaging were con rmed by pathology , one case was overstaged ( histological stage t1 ) and one case was understaged ( histological stage t4 )  . 
mr imaging had a sensitivity , speci city and accuracy of 100% , 80% and 88% , respectively , for stage t2 ; 100% , 92% and 94% for stage t3 ; and 33% , 100% and 88% for stage t4 . 
mean overall accuracy of mr imaging in patients in group 1 was 92% . in the 14 patients in group 2 ( table 3 ) who underwent surgery without prior neoadjuvant therapy , mr imaging lo studio stato ben tollerato da tutti i pazienti . 
 nei pazienti del gruppo 1 ( tabella 2 ) non si sono identi cati segni di localizzazione neoplastica in 2 pazienti , 8 casi sono stati stadiati t2 , 5 casi t3 , 1 caso t4 . 
in questo gruppo lesame istologico ha posto diagnosi di stadio t1 in 1 caso , t2 in 6 casi , t3 in 4 casi , t4 in 3 casi ; in 2 casi non radiol med ( 2011 ) 116 : 375388 table 2 group 1 : comparison between the results of magnetic resonance imaging ( mri ) and histopathology staging after neoadjuvant therapy ; sensitivity , speci city , accuracy and mean value according to the different t stages of the tumour tabella 2 gruppo 1 : confronto tra risultati di rm dopo terapia neoadiuvante con quelli stadiativi dellanatomia patologica ; valori di sensibilit , speci cit , accuratezza e valore medio secondo i vari stadi t della neoplasia rettale patient no . 
 mri postneoadjuvant therapy histological results numero pazienti rm post terapia neoadiuvante risultati istologici stage no tumour t4 mean value fibrosis no tumour sensitivity 100% 100% 100% 33% fibrosis no tumour stadio fibrosi no tumore sensibilit fibrosi no tumore speci city accuracy speci cit accuratezza 100% 80% 92% 100% 100% no neoplasia valore medio 100% 100% 100% 33% 100% 80% 92% 100% 100% staged ve cases as t2 , six as t3 and three as t4 . 
mr imaging had a sensitivity , speci city and accuracy of 83% , 100% and 93% , respectively , for stage t2 ; 71% , 86% and 79% for stage t3 ; and 100% , 85% and 86% for stage t4 . 
sei su 8 tumori stadiati come t2 dalla rm sono stati confermati dallesame anatomopatologico , 1 caso stato sovrastadiato ( stadio istologico : t1 ) ed 1 sottostadiato ( stadio istologico : t4 )  . 
quattro su 5 tumori stadiati come t3 dalla rm sono stati confermati dallesame anatomo - patologico , 1 caso stato sovrastadiato ( stadio istologico : t1 ) ed 1 caso sottostadiato ( stadio istologico : t4 )  . 
la rm ha mostrato sensibilit , speci cit e accuratezza rispettivamente del 100% , 80% , e 88% per lo stadio t2 ; 100% , 92% e 94% per lo stadio t3 ; 33% , 100% , e 88% per lo stadio t4 . 
laccuratezza media complessiva della rm nei pazienti del gruppo 1 stata del 92% . nei 14 pazienti del gruppo 2 ( tabella 3 ) , sottoposti a intervento chirurgico senza terapia neoadiuvante , la rm ha stadiato 5 casi come t2 , 6 come t3 e 3 come t4 . 
quattro su 6 tumori 382 radiol med ( 2011 ) 116 : 375388 table 3 group 2 : comparison between results of magnetic resonance imaging ( mri ) and histopathology staging ; sensitivity , speci city , accuracy and mean value according to the different t stages of the tumour tabella 3 gruppo 2 : confronto tra risultati stadiativi della rm con quelli anatomopatologici ; valori di sensibilit , speci cit , accuratezza e valore medio secondo i vari stadi t della neoplasia rettale patient no . 
 mri staging histological results numero pazienti stadiazione rm risultati istologici stage mean value t3 , 5 mm from fascia t4 , seminal vesicles t4 , seminal vesicles t4 , seminal vesicles sensitivity 83% 71% 100% stadio t3 , 5 mm dalla fascia t4 , vescichette seminali t3 t4 , vescichette seminali t4 t4 , vescichette seminali t3 sensibilit speci cit speci city accuracy accuratezza 100% 86% 85% valore medio 83% 71% 100% 100% 86% 85% the development of high - resolution phased - array coils , have signi cantly improved the performance of mr imaging in the study of pelvic malignancies , making this modality especially suited to the study of rectal tumours where it allows analysis not only of the parietal component of the tumour but also of the surrounding anatomical structures and mesorectal fascia [ 1 ]  . although many authors have reported excellent results with the use of 1.5 - t systems [ 14 , 29 , 30 ] , it became immediately clear that the greater signal - to - noise ratio of 3 - t systems could be exploited to increase spatial resolution and allow acquisition of thin slices , with no interval gap and with high matrices and large elds of view , without impairing image quality [ 1 , 24 ]  . 
however , considering the entire patient cohort , the diagnostic accustadiati alla rm come t3 sono stati confermati dallesame anatomo - patologico , 2 casi sono stati sottostadiati ( stadio istologico t4 )  . 
la rm ha mostrato sensibilit , speci cit , accuratezza rispettivamente del 83% , 100% e 93% per lo stadio t2 ; 71% , 86% e 79% per lo stadio t3 ; 100% , 85% e 86% per lo stadio t4 . 
il valore del test di pearson stato di 0 , 855 , quindi non si dimostrata alcuna signi cativit statistica nella differenza tra i risultati conseguiti nei due gruppi di pazienti . 
 discussione i signi cativi progressi tecnologici delle apparecchiature rm , in particolare i miglioramenti nel campo della tecnologia dei gradienti e lo sviluppo di bobine phased array ad alta risoluzione , hanno signi cativamente migliorato le prestazioni della rm nellambito delle patologie neoplastiche del distretto pelvico , rendendo questa metodica particolarmente indicata per lo studio delle neoplasie rettali dove , oltre alla valutazione della componente tumorale parietale , possibile anche unaccurata analisi delle strutture anatomiche circostanti e della fascia mesorettale [ 1 ]  . sebbene molti autori abbiano riportato ottimi risultati con limpiego di apparecchiature rm operanti a 1 , 5 radiol med ( 2011 ) 116 : 375388 fig . 
1a para - axial t2 - weighted fast spin echo ( fse ) sequence : the image shows diffuse thickening of the rectal wall without precise delineation of the tumour . 
b limmagine assiale t2 pesata ottenuta dopo distensione del lume rettale con acqua mostra una precisa identi cazione del tumore in stadio t2 a livello della parete posterolaterale di destra . t [ 14 , 29 , 30 ] apparso subito evidente come il maggiore rapporto segnale - rumore garantito dalle rm operanti a 3 t avrebbe potuto essere utilmente impiegato per incrementare la risoluzione spaziale e permettere lacquisizione di sezioni sottili , senza intervallo di acquisizione , con matrici elevate ed ampi fov , senza degradazione qualitative delle immagini [ 1 , 24 ] , creando in tal modo le condizioni per unottimale valutazione delle neoplasie pelviche , con particolare riguardo al carcinoma rettale dove lelevata risoluzione spaziale costituisce il presupposto per lottimale valutazione sia dellin ltrazione parietale , che del mesoretto e degli organi endopelvici . in letteratura pochi autori hanno sino ad ora valutato laccuratezza della 3t rm nella stadiazione dei tumore rettale [ 2326 ] , sottolineandone in particolare laccuratezza nella determinazione della resecabilit dello s ntere [ 26 ]  . 
nel nostro studio , nei pazienti gruppo 2 , sottoposto a stadiazione prechirurgica , si avuto una accuratezza diagnostica per lo stadio t2 sovrapponibile ai risultati di kim et al . 
 [ 23 ] , in particolare vi completa sovrapposizione di risultati per lo stadio t2 , mentre per lo stadio t3 i risultati sono molto vicini ( 87% rispetto al 90% ) , mentre nel gruppo di kim et al . 
2 limmagine para - assiale t2 pesata fse mostra un tumore in stadio t3 in ltrante la parete posteriore - destra del retto ed il tessuto adiposo mesorettale ; stata misurata la distanza della neoplasia dalla fascia mesorettale ( freccia )  . racy obtained in our study appears similar to that reported by kim et al . 
a paraaxial t2 - weighted fse image showing , in the mesorectal fat , the presence of low - signal - intensity spiculated tissue ( arrow ) , without signi cant enhancement after intravenous infusion of contrast medium ( b , c before and after contrast administration ) and interpreted as brosis . 
a para - assiale t2 pesata fse mostra la presenza , nel contesto del tessuto adiposo perirettale , di tessuto spiculato ( freccia ) di basso segnale , senza signi cativo incremento del segnale dopo somministrazione e.v. 
 in our opinion , this may depend on the small number of t4 cancers and on the location of the cancers in the two cases of understaging , both of which were anterior supraampullary carcinomas with peritoneal in ltration , locations that are known to be dif cult to evaluate with mr imaging [ 29 ]  . 
in comparison with previous reports , our results are consistent with those obtained with 3 - t systems [ 2326 ] and on average better than those obtained with 1.5 - t equipment [ 1 , 3 , 14 , 15 , 20 , 21 , 31 ]  . 
a nostro parere questo pu dipendere dal basso numero di casi in stadio t4 e dalla particolare localizzazione nei 2 casi sottostadiati , entrambi carcinomi sovrampollari anteriori con in ltrazione neoplastica del peritoneo , sedi riconosciute di dif cile valutazione rm [ 29 ]  . 
inoltre la sovrastadiazione in 2 casi nel gruppo 2 verosimilmente legata alla reazione desmoplastica e alledema reattivo post - attinico , reperti ampiamente descritti nella letteratura corrente [ 24 , 29 ] , e documentati alla valutazione anatomopatologica in entrambi i casi . 
 al confronto con i dati riportati letteratura i nostri risultati sono in linea con quelli ottenuti con apparecchiature a 3 t [ 2326 ] e mediamente superiori ai risultati ottenuti con apparecchiature a 1 , 5 t [ 1 , 3 , 14 , 15 , 20 , 21 , 31 ]  . 
il maggiore limite emerso dalla letteratura scienti ca attuale , negli altri studi con la rm a 1 , 5 t , riguarda la dif colt a differenziare lo stadio t2 dai casi in stadio t3 dove presente solo minima in ltrazione extraviscerale [ 1 , 3 ]  . 
4a para - axial t2 - weighted fast spin echo ( fse ) image of a tumour of the right posterolateral wall of the rectum with invasion of the mesorectal fat and mesorectal fascia . 
however , this staging dif culty with mr imaging has limited clinical impact , as both stage t2 cancer and stage t3 cancer with minimal mesorectal in ltration ( 05 mm ) are treated with the same approach : anterior rectal resection without adjuvant therapy , owing to the good prognosis of both stages [ 32 , 33 ]  . 
in our study , this limitation appeared to have less relevance , as we had only stadiativa della rm ha una ridotta rilevanza clinica , poich sia lo stadio t2 , che quello t3 con minima in ltrazione del mesoretto ( da 0 a 5 mm ) hanno lo stesso approccio terapeutico : resezione anteriore del retto senza terapia adiuvante a seguito della buona prognosi di entrambi questi stadi [ 32 , 33 ]  . 
nel nostro studio questo limite sembrato essere meno rilevante , perch abbiamo avuto solo 2 casi in cui la stadiazione si rilevata errata : un caso nel gruppo 1 dove una neoplasia in stadio t4 per in ltrazione del peritoneo stata sottostadiata a t2 , ed un caso nel gruppo 2 , dove una 386 radiol med ( 2011 ) 116 : 375388 two cases of incorrect mr staging : one in group 1 , where a t4 cancer with peritoneal in ltration was understaged as t2 , and one in group 2 , where a t2 cancer was overstaged as t3 . 
 [ 28 ] found good ppv ( 84% ) and a fair overall diagnostic accuracy ( 79.2% ) in patients with stages t3 and t4 tumours treated with neoadjuvant therapy . 
moreover , in our study , there was no statistically signi cant difference ( p = 0.855 ) between the results achieved in group 1 and group 2 , a nding that appears to con rm the limited impact of the local changes induced by neoadjuvant therapy on the diagnostic accuracy of mr imaging . 
firstly , the number of patients included was small , and their distribution in the two groups was not homogeneous ( there were no patients with stage t1 tumours ) , a fact that might lead to an overestimation of diagnostic accuracy . 
in fact , mr imaging is usually limited in differentiating stage t1 from stage t2 tumours , and the literature demonstrates that endorectal ultrasound is still the modality of choice in these early - stage tumours [ 1 , 5 , 15 , 16 , 23 ]  . 
many reports have demonstrated the importance of describing the relationship between the tumour and the mesorectal fascia , which represents the plane through which tme is usually performed and is an important predictor of survival , the probability of local recurrence and the negativity of excision margins [ 20 , 21 , 29 , 30 , 34 , 35 ]  . 
 dalle nostre ricerche nella letteratura corrente non sono emersi studi che confrontassero laccuratezza della rm tra gruppi di pazienti sottoposti o non a terapia neoadiuvante , mentre solo pochi studi hanno valutato laccuratezza diagnostica della rm a 1 , 5 t dopo terapia neoadiuvante , documentandone una minore ef cacia stadiativa . 
 [ 27 ] hanno mostrato un elevato valore predittivo positivo ( 88%91% ) della rm a 1 , 5 t e buona accuratezza ( 78% ) nella identi cazione dei pazienti sottoposti a terapia neoadiuvante in stadio istologico t0 - t2 , mentre bonomo et al . 
 [ 28 ] in pazienti in stadio t3 e t4 , sottoposti a terapia neoadiuvante hanno riscontrato buoni valori predittivi positivi ( 84% ) e discreta accuratezza complessiva ( 79 , 2% )  . 
nel nostro studio laccuratezza stadiativa della 3t rm dopo terapia neoadiuvante stata complessivamente del 92% con variazioni dal 100% nello stadio t0 all88% nello stadio t2 , risultati che contrastano con quelli di torkzadt et al . 
nel nostro studio , inoltre , non c stata una signi cativa differenza statistica ( p = 0 , 855 ) tra i risultati ottenuti nel gruppo 1 e nel gruppo 2 , dato che conferma la scarsa rilevanza delle alterazioni locali indotte dalla terapia neoadiuvante sulla accuratezza diagnostica della rm , anche se tale riscontro deve essere supportato e confermato da un pi ampio gruppo di pazienti . 
per prima cosa , il numero di pazienti coinvolti nello studio ridotto e la distribuzione dei pazienti nei diversi gruppi non omogenea ( non sono presenti pazienti con tumore in stadio t1 ) , dato che potrebbe portare ad una sovrastima nel calcolo dellaccuratezza . 
di solito infatti in rm dif cile differenziare lo stadio t1 dallo stadio t2 e la letteratura dimostra che lecogra a endorettale ancora la metodica di elezione per questi stadi tumorali iniziali [ 1 , 5 , 15 , 16 , 23 ]  . 
molti lavori hanno dimostrato limportanza di descrivere le relazioni tra neoplasia e fascia mesorettale , rappresentante il piano di resezione principale della tme e importante fattore prognostico di sopravvivenza e di probabilit di recidiva locale e di integrit dei margini di resezione chirurgica dalla neoplasia [ 20 , 21 , 29 , 30 , 34 , 35 ]  . 
knauth springer - verlag , berlin heidelberg , 2010 isbn 978 - 3 - 540 - 97716 - 6 e - isbn 978 - 3 - 540 - 79717 - 3 doi 10.1007 / 978 - 3 - 540 - 79717 - 3 published online : 7 march 2011 springer - verlag 2011 the aim of this 168 page / 13 chapter book is to introduce the latest information and updated ( 2009 ) references in the eld of magnetic resonance angiography ( mra ) to the reader . the content is divided into three main sections : image acquisition techniques and sequences ( 5 chapters ) ; clinical applications ( 6 chapters ) ; special topics : transplants ( 2 chapters ) , plus the subject index . going through the book one realises how important mra and contrast enhanced mra sequences have become , gaining more and more momentum in the clinical diagnostic setting . robust strengths of the book include sequence speci cations ; use of contrast media in different procedures according to body organ and different anatomical districts ; evaluation of images both correct or presenting artefacts . 
the absence of ionizing radiation related to the use of mra is stressed and surely this advantage will increase the demand for mra , as this sort of equipment is made more available , with improved examination speed and reduction in patient time in the gantry . numerous images are presented and are of high quality , perfectly reproduced all throughout the book . 
to properly indicate the addressed problem ? i am also personally proud that this is one of the few books in this important series edited and entirely written by italian crme de la crme radiologists . intendimento di questo volume , nei suoi 13 capitoli e 168 pagine , di fornire al lettore le pi recenti ed aggiornate conoscenze ( voci bibliogra che no al 2009 ) nel campo della angiogra a con risonanza magnetica ( angio - rm )  . 
 il contenuto del volume diviso in tre grandi argomenti : tecniche di acquisizione de lle immagini e loro sequenze ( 5 capitoli ) ; applicazioni cliniche ( 6 capitoli ) ; argomenti speciali : i trapianti ( 2 capitoli ) per terminare con lindice analitico . nello scorrere il volume il lettore si rende conto di quanto siano divenute importanti sia la angio - rm che le sequenze con mezzo di contrasto , che si sono dimostrate di sempre maggior peso nel campo diagnostico clinico . tra i numerosi punti di forza del volume sono da ricordare la descrizione precisa delle sequenze da usare ; luso dei mezzi di contrasto nelle diverse modalit di indagine in funzione dellorgano in studio e dei vari distretti anatomici ; la interpretazione delle immagini sia corrette che in caso di artefatti . 
 viene poi segnalata e messa in evidenza lassenza delle radiazioni conseguente alluso dellangio - rm , cosa che di sicuro incrementer limpiego della stessa man mano che le apparecchiature dedicate saranno pi diffuse , tenuto anche conto dellaumento nella velocit di esecuzione delle indagini e della riduzione del tempo del paziente nellapparecchiatura . vengono presentate numerose immagini , di elevata qualit , perfettamente riprodotte in tutto il volume . 
it is frequently observed on multidetector ct ( mdct ) scans performed during daily clinical practice and may be caused by various pathological conditions , including oedema , inflammation , haemorrhage , neoplastic infiltration or sclerosing mesenteritis . 
otherwise , it may represent an incidental finding on mdct performed for other reasons . this article describes the mdct features of misty mesentery in different diseases in order to provide a rational approach to the differential diagnosis . riassunto il termine mesentere nebuloso indica un incremento patologico della densit del tessuto adiposo mesenterico alla tomografia computerizzata ( tc )  . 
 keywords disease abdomen multidetector ct mesentery parole chiave patologia addome tc multidetettore mesentere introduction introduzione the introduction of multidetector - row technology has greatly enhanced the role of computed tomography ( ct ) in evaluating patients suspected of having abdominal disease . 
furthermore , misty mesentery may represent an incidental finding on multidetector - row ct ( mdct ) performed for other reasons . as a matter of fact , misty mesentery is not an uncommon lavvento della tecnologia multidetettore ha ampliato notevolmente le potenzialit diagnostiche della tomografia computerizzata ( tc ) nella valutazione di pazienti con sospetta patologia addominale ; in tali pazienti il mesentere nebuloso rappresenta frequentemente il segno chiave , espressione della malattia sottostante . 
il mesentere nebuloso pu essere , daltro canto , un riscontro incidentale nel corso di esami tc multidetettore ( tcmd ) 352 radiol med ( 2011 ) 116 : 351365 finding at abdominal mdct , and radiologists must become familiar with this sign in order to understand its diagnostic value . 
we present a review of diseases that can result in misty mesentery , in order to provide a rational approach to the differential diagnosis on the basis of the associated findings . mesentery : normal anatomy mesenteries ( small - bowel mesentery , transverse mesocolon and sigmoid mesentery ) represent the reflection of the peritoneum on the bowel surface and suspend the loops of the small and large intestine from the posterior abdominal wall . they are composed of two layers within which vessels , lymphatics , lymph nodes , nerves and variable amounts of fat and connective tissue are housed . 
un dato di fatto , pertanto , che il mesentere nebuloso un reperto frequente alla tcmd delladdome e che i radiologi devono familiarizzare con tale segno e comprenderne il valore diagnostico . 
con questo lavoro offriamo una revisione delle patologie che possono manifestarsi con il segno del mesentere nebuloso , al fine di fornire un approccio razionale alla diagnosi differenziale , identificando reperti associati e possibili peculiari pattern di presentazione che orientano la diagnosi . mesentere : anatomia normale i mesi ( mesentere , mesocolon trasverso e mesosigma ) sono riflessioni del peritoneo sulla superficie intestinale e sospendono le anse del tenue e del colon ; essi sono formati da due foglietti peritoneali che contengono vasi arteriosi e venosi , linfatici , linfonodi , nervi e una quota variabile di tessuto adiposo e tessuto connettivo . 
 mesentere nebuloso : definizione il termine misty mesentery o mesentere nebuloso , introdotto da mindelzun nel 1996 [ 1 ] , indica un aumento della densit del tessuto adiposo mesenterico alla tc . 
1a , b immagini tc multidetettore assiali che illustrano i normali valori di attenuazione del mesentere ( a ) ed il tipico reperto del mesentere nebuloso , ossia un aumento della densit del tessuto adiposo mesenterico ( b )  . radiol med ( 2011 ) 116 : 351365 mesentere nebuloso : cause e reperti dellimaging il mesentere nebuloso pu essere conseguenza di diverse condizioni , quali ledema , il linfedema , la flogosi , lemorragia , la panniculite o le neoplasie [ 2 ]  . 
ledema mesenterico diffuso si riscontra frequentemente in pazienti con cirrosi epatica : in questi casi il meccanismo eziopatogenetico pi comune delledema lipertensione portale , che causa un aumento della pressione idrostatica allinterno delle vene mesenteriche , con conseguente stravaso di liquidi nel mesentere . 
la caratteristica del mesentere nebuloso nei pazienti cirrotici rappresentata dalla progressione dal solo edema mesenterico , verso una combinazione di edema mesenterico ed omentale , fino alla comparsa contemporanea di edema mesenterico , omentale e retroperitoneale . 
analogamente , la presenza di abbondante ascite senza aspetto nebuloso del mesentere molto rara nei pazienti cirrotici ed in tal caso dovrebbero essere considerate altre ipotesi diagnostiche , quali il carcinoma epatocellulare , locclusione dei rami portali e delle vene sovraepatiche o la peritonite batterica . 
nei pazienti con edema mesenterico sostenuto da patologia sistemica o da cirrosi epatica liperdensit del meso tende ad essere diffusa e si estende dalla superficie sierosa intestinale sino alla radice del mesentere . 
diffuse mesenteric oedema occurs frequently in patients with cirrhosis : in these cases , the most common pathogenic mechanism of oedema is portal hypertension , which causes increased hydrostatic pressure within the mesenteric veins , and consequently , fluid to seep out into the mesentery . 
the ct hallmark of misty mesentery in patients with cirrhosis is represented by a progression from mesenteric oedema alone , through a combination of mesenteric and omental oedema to a combination of mesenteric , omental , and retroperitoneal oedema . 
moreover , as mesenteric oedema in cirrhosis is due to portal hypertension , the presence of severe ascites in a patient with radiol med ( 2011 ) 116 : 351365 fig . 
3a , b arterial contrast - enhanced multidetector - row computed tomography images in a 57 - year - old patient with cirrhosis and a hepatocellular carcinoma nodule treated with transcatheter arterial chemoembolisation ( arrowhead in a )  . 
mild omental oedema ( curved arrow in a ) and ascites ( asterisks in a and b ) are associated with florid , widespread mesenteric oedema ( arrow in b )  . 
3a , b immagini tc multidetettore acquisite in fase arteriosa in un paziente di 57 anni con cirrosi ed un nodulo di epatocarcinoma ( hcc ) trattato con chemioembolizzazione ( testa di freccia in a )  . 
si documenta un modico edema omentale ( freccia curva in a ) con ascite ( asterischi in a e b ) e concomitante diffuso edema mesenterico ( freccia in b )  . 
4a , b portal - venous - enhanced multidetector - row computed tomography images of a 50 - year - old patient with cirrhosis and hepatocellular carcinoma ( arrowhead in a )  . 
ascites and omental soft - tissue nodules ( arrows in a ) cannot be related to oedema from portal hypertension , being associated with normal density of mesenteric fat ( asterisk in b )  . 
lascite e i noduli omentali ( frecce in a ) non sono causati dalledema secondario allipertensione portale , poich la densit del tessuto adiposo mesenterico normale ( asterisco in b ) ; tali reperti possono indicare un coinvolgimento neoplastico del peritoneo , confermato dallanalisi citologica del versamento ascitico . cirrhosis without the ct feature of misty mesentery is so uncommon that additional causes of ascites should be considered ( hepatocellular carcinoma , portal or hepatic venous occlusion , bacterial peritonitis )  . 
in patients with mesenteric oedema due to systemic diseases or cirrhosis , the mesenteric haziness appears to be diffuse and , therefore , extends from the serosal surface of the intestine to the root of the small bowel mesentery at the origin of mesenteric linfedema mesenterico il linfedema mesenterico pu essere primitivo o secondario a varie patologie . 
il reperto caratteristico di tale affezione rappresentato dal diffuso ispessimento radiol med ( 2011 ) 116 : 351365 nodulare della parete delle anse tenuali , conseguente alla dilatazione dei linfatici allinterno dei villi . 
lispessimento parietale dellansa intestinale pu assumere un aspetto stratificato a causa delledema della mucosa o della sottomucosa ; sono generalmente presenti ascite e linfedema mesenterico [ 5 , 6 ]  . 
le linfoadenopatie paraortiche e mesenteriche sono rare e la loro assenza utile per escludere altre patologie , quali ad esempio il linfoma , la malattia di whipple , la malattia celiaca e la tubercolosi , che possono comunque causare diffuso ispessimento parietale del piccolo intestino [ 7 ]  . il linfedema mesenterico secondario dovuto ad ostruzione linfatica conseguente a pericardite costrittiva , anomalie post - attiniche , malattia di crohn , tubercolosi intestinale o linfoma intestinale . flogosi mesenterica la maggior parte delle flogosi acute del tratto gastroenterico , quali colecistite , pancreatite , appendicite , diverticolite , malattie infiammatorie e tubercolosi , coinvolge il meso adiacente , causandone un aspetto nebuloso . 
il mesentere , poich parte integrante del peritoneo , coinvolto nella maggior parte dei pazienti con peritonite tubercolare : i seguenti segni tc sono altamente suggestivi di peritonite tubercolare [ 8 ] : 1 . 
 [ 4 ] , called small intestinal lymphangiectasia and characterised by a congenital abnormality of the lymphatic systethe ct appearance of primary intestinal lymphangiectasia is represented by diffuse nodular thickening of the smallbowel wall , which is the result of the dilated lymphatic channels within the villi . 
small - bowel thickening may assume a typical stratified appearance due to mucosa or submucosa oedema ; ascites and mesenteric lymphoedema are usually present [ 5 , 6 ]  . 
para - aortic and mesenteric lymph nodes are not usually identified , and the absence of such adenopathy was considered to be helpful in excluding other conditions , such as lymphoma , whipples disease , coeliac disease and tuberculosis , which can also produce diffuse small - intestinal - wall thickening [ 7 ]  . 
le frecce indicano anse del tenue distese , con ispessimento stratificato delle pareti . mesenteric inflammation most acute inflammatory diseases of the gastrointestinal tract , such as cholecystitis , pancreatitis , appendicitis , diverticulitis , inflammatory bowel disease and tuberculous peritonitis , involve the adjacent mesentery , causing misty mesentery . 
8 immagine tc multidetettore che documenta il mesentere nebuloso ( asterisco ) in un paziente con pancreatite acuta . rica , caratterizzata da una infiammazione cronica del tessuto adiposo mesenterico , che infiltrato da linfociti e macrofagi ripieni di grasso . 
la causa di tale patologia non chiara ed correlata ad un ampio spettro di affezioni quali vasculite , malattie granulomatose , disordini autoimmuni , radiol med ( 2011 ) 116 : 351365 ischemia , infezioni , traumi , precedenti interventi chirurgici o sindrome paraneoplastica [ 9 , 10 ]  . 
9 immagine tc multidetettore che documenta il mesentere nebuloso localizzato ( asterisco ) in un paziente con appendicite acuta ( freccia )  . mesenteric haemorrhage acute haemorrhage is characterised by high ct attenuation values ( 4060 hu )  . 
12 immagine tc multidetettore di un paziente con tubercolosi addominale che documenta iperdensit del tessuto adiposo mesenterico ( teste di freccia ) , con linfoadenopatie retroperitoneali caratterizzate da necrosi caseosa centrale ( frecce )  . 
the cause of this disease is unclear and has been related to a wide spectrum of conditions such as vasculitis , granulomatous disease , autoimmune disorders , ischaemia , infections , trauma , prior abdominal surgery or paraneoplastic response [ 9 , 10 ]  . 
nella maggior parte dei casi i tumori mesenterici primitivi sono benigni , come i desmoidi , che derivano da una proliferazione benigna di tessuto fibroso e possono manifestarsi in associazione con la sindrome di gardner , nel post - partum o a seguito di interventi chirurgici ; alla tc i desmoidi generalmente si presentano come masse con densit dei tessuti molli , con margini ben demarcati o scarradiol med ( 2011 ) 116 : 351365 fig . 
la tc dimostra sfumata infiltrazione del tessuto adiposo mesenterico o multiple masse solide rotondeggianti che di solito inglobano i vasi mesenterici e producono il caratteristico segno del sandwich , con dislocazione delle anse tenuali . 
15 multidetector - row computed tomography image showing mesenteric panniculitis as a well - circumscribed , inhomogeneous , fatty mass ( arrowheads ) , with higher attenuation values than normal retroperitoneal fat . 
15 immagine tc multidetettore che documenta la presenza di panniculite mesenterica , con una massa a densit adiposa , ben circoscritta , disomogenea ( teste di freccia ) , che mostra densit superiore a quella del normale tessuto adiposo retroperitoneale . 
because it extends along the root of the jejunal mesentery , panniculitis typically has a leftward 360 radiol med ( 2011 ) 116 : 351365 orientation ; there are scattered , well - defined small nodes . the fat - ring sign and a pseudocapsule are present . 
in the retractile form , the fibrosis predominates , and the disease manifests itself as large masses of soft - tissue attenuation that may involve vessels and may contain calcifications . 
when segmental misty mesentery shows the above - described ct features and no underlying disease is detected , mesenteric panniculitis has to be considered . nevertheless , mesenteric panniculitis may be a nonspecific response to an abdominal malignancy [ 11 , 12 ] and , therefore , close clinical follow - up to search for hidden malignancy is warranted in these patients . 
in most cases , primary mesenteric tumours are histologically benign , including desmoid tumours , which result from a benign proliferation of fibrous tissue and may occur in association with gardners syndrome , postpartum or postsurgery . 
direct spread : gastrointestinal carcinoid tumours : desmoplastic reaction from carcinoid tumours of the small bowel is common and occurs as a fixed solid mass with a stellate pattern of linear strands . 
ct features include an ill - defined infiltration of the rounded , mildly mesenteric enhancing masses that usually encase the mesenteric vessels and produce the sandwich sign , a mass loops . 
18 multidetector - row computed tomography image showing an enhancing mass with a coarse calcification in the mesentery ( arrow ) and focal wall thickening of an adjacent bowel loop ( arrowhead )  . 
19 immagine tc multidetettore di un paziente con linfangioma mesenterico . diagnosi differenziale e conclusioni poich il mesentere nebuloso pu essere espressione di patologie benigne e maligne , per la diagnosi differenziale necessario un approccio razionale . 
considerando i reperti precedentemente descritti , si pu dedurre che il coinvolgimento mesenterico , diffuso o focale , a limiti scarsamente definiti e tenuemente iperdenso frequente nelle patologie 362 radiol med ( 2011 ) 116 : 351365 fig . 
21 immagine tc multidetettore di un paziente con mesotelioma peritoneale maligno che documenta abbondante ascite ( asterisco ) e irregolare infiltrazione tumorale lungo i vasi mesenterici ( freccia )  . table 1 misty mesentery and corresponding diagnosis computed tomography features diagnosis diffuse or segmental increase of mesenteric fat attenuation without mass effect mass with increased attenuation showing the fatty - halo sign and a tumour pseudocapsule enhancing mass of soft tissue attenuation with vessel displacement non - neoplastic diseases neoplastic diseases stellate appearance of the mesentery associated with peritoneal nodules tabella 1 caratteristiche tc del mesentere nebuloso e diagnosi corrispondente computed tomography features diagnosis iperdensit diffusa o focale del grasso mesenterico senza effetto massa patologie massa con densit dei tessuti molli , con il segno dellalone adiposo e pseudocapsula massa con densit dei tessuti molli che disloca i vasi patologie non neoplastiche patologie neoplastiche aspetto stellato del mesentere , associato a noduli peritoneali diagnosis . 
according to the aforementioned ct features , diffuse or segmental involvement with ill - defined edges and stranding of mesentery is frequently observed in benign diseases , whereas mass - like , well - defined involvement is observed in neoplastic diseases , with the exception of mesenteric panniculitis ( table 1 )  . 
moreover , the radiologist must keep in mind that the clinical history and associated findings benigne , mentre la presenza di masse a margini ben definiti indica generalmente una patologia neoplastica , con leccezione della panniculite mesenterica nella forma retrattile ( tabella 1 )  . 
il radiologo , inoltre , deve sempre considerare la storia clinica del paziente ed i reperti associati , al fine di ottenere una migliore comprensione delle cause del mesentere nebuloso . 
table 2 provides a rational approach for differential diagnosis : the algorithm aims at reaching the final diagnosis by combining the morphological pattern of mesenteric involvement with specific associated findings . 
this approach could help the radiologist to correctly interpret the sign of misty mesentery , mostly in pathological processes that are characteristically centred in the mesentery adjacent to the bowel wall rather than in the bowel wall itself [ 14 ]  . correct noninvasive diagnosis is important because treatment strategies for these conditions range from monitoring to surgery . 
tale approccio aiuta il radiologo ad interpretare correttamente il segno del mesentere nebuloso , soprattutto nel caso di processi patologici che originano dal mesentere piuttosto che dalle anse intestinali [ 14 ]  . 
gandini istituto di radiologia diagnostica ed interventistica , universit di torino , aso san giovanni battista di torino , sede molinette , via genova 3 , 10126 torino , italy correspondence to : a . 
from january 2004 to december 2008 , we conducted 602 stereotactic , 11 - gauge , vacb procedures on 243 nonpalpable breast lesions categorised as bi - rads r3 , 346 categorised as bi - rads r4 and 13 categorised as bi - rads r5 . 
lesions of uncertain malignant potential ( b3 ) ( 23.6% ) were debated at multidisciplinary meetings , and diagnostic surgical biopsy was recommended for 83.1% of theall malignant lesions ( b4 and b5 ) underwent surgical excision . 
tra gennaio 2004 e dicembre 2008 , sono state eseguite 602 vacb con ago da 11 g , sotto guida stereotassica , su 243 lesioni classi cate come bi - rads r3 , 346 come bi - rads r4 e 13 come bi - rads r5 . 
le lesioni b3 ( 23 , 6% ) sono state discusse in sessioni multidisciplinari ; di queste l83 , 1% stato sottoposto a veri ca chirurgica , come tutte le lesioni risultate b4 e b5 . 
la vacb risultata metodica accurata nella caratterizzazione di lesioni a basso e medio rischio , evitando lintervento chirurgico in oltre la met dei casi con un effettivo risparmio economico . 478 radiol med ( 2011 ) 116 : 477488 keywords breast stereotactic vacuum - assisted core biopsy cost - effectiveness non palpable breast lesions mammotome parole chiave mammella biopsia mammaria stereotassica vacuum - assistita costo - ef cacia lesioni mammarie non palpabili mammotome introduction introduzione percutaneous vacuum - assisted core biopsy ( vacb ) has proven to be an effective and accurate method for histopathological characterisation of nonpalpable breast lesions , showing virtually identical results to diagnostic surgical biopsy , which is considered the reference standard for histological diagnosis of preclinical lesions [ 17 ]  . 
in particular , to quantify the actual cost savings , we evaluated the correlation between abnormalities considered at low ( bi - rads r3 ) and intermediate ( bi - rads r4 ) risk of malignancy and the histopathological diagnosis of benign disease . la biopsia percutanea vacuum assistita ( vacb ) si rivelata una metodica ef cace ed accurata per la caratterizzazione istopatologica di lesioni non palpabili della mammella , con risultati sovrapponibili alla biopsia chirurgica diagnostica , correntemente considerata la procedura gold standard per la diagnosi istologica delle lesioni pre - cliniche [ 17 ]  . 
lesions consisted of microcalci cations as the only suspicious radiological nding in 95.8% of cases ( 577 / 602 ) and microcalci cations associated with other mammographic signs ( structural distortion , asimmetric density , opacities ) in 3% ( 18 / 602 ) ; in 7 / 602 ( 1.2% ) cases only did we obtain biopsies of structural distortions , opacities or thickening for which both core needle biopsy ( cnb ) and ne - needle aspiration cytology ( fnac ) provided inconclusive results ( c1 and b1 )  . 
 mammographic ndings were classi ed according to the breast imaging reporting and data system ( bi - rads ) , 4th edition classi cation of the american college of radiology ( arc ) [ 12 ]  . 
vacb was performed using the mammotome device ( ethicon endo - surgery , cincinnati , oh , usa ) with 11 - gauge needles applied to the prone fischer mammotest stereotactic table ( fischer imaging , denver , co , usa )  . 
biopsy samples were nel periodo compreso tra gennaio 2004 e dicembre 2008 sono state eseguite 602 vacb in pazienti di et compresa tra 35 e 73 anni ( et media : 56 , 3 anni )  . 
le lesioni erano rappresentate nel 95 , 8% dei casi ( 577 / 602 ) da microcalci cazioni come unico sospetto radiogra co , nel 3% ( 18 / 602 ) da microcalci cazioni associate ad altri segni mammogra ci ( distorsione strutturale , addensamenti asimmetrici , opacit ) e in soli 7 / 602 ( 1 , 2% ) casi sono state biopsiate distorsioni , opacit o addensamenti , nelle quali sia la core needle biopsy ( cnb ) che la ne needle aspiration citology ( fnac ) non avevano fornito unadeguata risposta diagnostica ( c1 e b1 )  . 
in 534 / 602 ( 88 , 7% ) lestensione delle lesioni era 10 mm , mentre solo in 68 casi ( 11 , 3% ) le dimensioni erano > 10 mi reperti mammogra ci sono stati classi cati secondo il sistema bi - rads , dellamerican college of radiology [ 12 ]  . 
la vacb stata effettuata utilizzando il dispositivo mammotome ( ethicon endosurgery , cincinnati , oh , usa ) con ago da 11 gauge , applicato al tavolo stereotassico digitale prono fischer mammotest ( fischer imaging , denver , co , usa )  . 
tutti i prelievi radiol med ( 2011 ) 116 : 477488 arranged on absorbent paper according to sampling order , taking care to separate the samples with even numbers from those with odd numbers . 
approximately 2 weeks after the procedure , unilateral two - view mammograms were obtained in all patients to evaluate the correct position of the nonmagnetic metal clip and the presence of any residual microcalci cations . 
all procedures were performed as day cases , and mean procedure time was around 60 mthe results of the microhistological examination were classi ed into ve categories in accordance with the european guidelines [ 13 ] : b1 = normal , b2 = benign , b3 = lesion of uncertain clinical signi cance , b4 = suspicious for malignancy , b5 = positive . 
benign cases ( b2 ) underwent successive follow - up mammograms over the years , all of which have proved negative . cost analysis the second phase of the study involved comparison between the effective and overall costs incurred for the two biopsy methods ( vacb vs diagnostic surgical biopsy ) , taking into account the minimum reimbursement fees ( not including complications and / or comorbidities ) established for the single procedures performed in the departments of radiodiagnostics and general surgery . 
we considered a mean duration of the surgical procedure of around 90 min , with the presence in the operating room of two surgeons , one anaesthetist , one general care nurse , one room nurse and one auxiliary nurse . 
the reimbursement rate for a vacb procedure ( ministerial decree 22 / 7 / 96 : 85.11.3 ) includes the cost of the technology used ( complete vacb kit and operating costs for the stereotactic table ) , consumables ( anaesthesia , gauze swabs , plasters , etc ) and hourly cost of operators based on the salary scale ( one radiologist , one radiographer and one nurse )  . 
sono stati effettuati radiogrammi con ingrandimento dei campioni prelevati , allo scopo di veri care la presenza di microcalci cazioni , e sono stati chinati solo i frustoli contenenti le microcalci cazioni . 
a circa due settimane dallesecuzione della procedura , tutte le pazienti sono state sottoposte a controllo mammograco monolaterale in due proiezioni per valutare il corretto posizionamento della clip metallica amagnetica precedentemente posizionata e leventuale presenza di microcalcicazioni residue . 
lesame microistologico stato classi cato in 5 categorie in accordo con le linee guida europee [ 13 ] : b1 = normale , b2 = benigno , b3 = lesione di incerto signi cato , b4 = sospetto di malignit , b5 = positivo . 
lesame istologico de nitivo stato considerato il gold standard in tutti i casi di lesioni maligne o atipiche sottoposte ad intervento chirurgico ; nei casi in cui allistologia de nitiva non fosse pi presente la lesione maligna , stato rivisto il prelievo da vacb a conferma della diagnosi precedentemente posta . 
i casi benigni ( b2 ) sono stati sottoposti nel corso degli anni a successivi follow - up mammogra ci , risultati in tutti i casi a tuttoggi negativi . valutazione dei costi la seconda fase dello studio ha previsto il confronto tra i costi effettivi e complessivi affrontati per eseguire le due metodiche bioptiche diagnostiche ( vacb vs biopsia chirurgica diagnostica ) , prendendo in considerazione le tariffe di rimborso minime ( non gravate da complicanze e / o comorbilit ) previste per le singole procedure della radiodiagnostica e della chirurgia generale . 
le tariffe di rimborso relative alla biopsia chirurgica sono state derivate dal nomenclatore tariffario regionale delle prestazioni specialistiche ambulatoriali ( drg n262 approvato con deliberazione giunta regione piemonte n50 - 1062 del 10 / 10 / 2005 ) e comprendono il costo della sala operatoria , del ricovero in regime di day - hospital e del reperaggio sotto guida radiologica della lesione da sottoporre a biopsia , il costo dei materiali ( anestesia , materiale per le suture , farmaci vari etc . ) e la retribuzione oraria degli operatori , da stipendio tabellare ( in base alla loro quali ca ed al loro numero )  . 
 stata considerata una durata media della procedura chirurgica di circa 90 minuti , con la presenza in sala operatoria di due chirurghi , un anestesista , un infermiere professionale , uno strumentista e un infermiere generico . 
the correlation between radiological suspicion and microhistological diagnostic category is summarised in table 2 , whereas the correlation between radiological suspicion and nal histology on the surgical specimen is shown in table 3 . vacb proved to be inadequate ( b1 ) in 0.5% of cases because of failure to complete the procedure due to either excessive bleeding ( n = 2 ) or patient anxiety ( n = 1 )  . 
in accordance with the protocol , the patients were scheduled for yearly follow - up if the calci cations were completely removed or 6 - month follow - up if removal were incomplete . 
il 16 , 9% delle b3 non operate stato inviato a follow up annuale ( periodo di osservazione medio 2 , 95 anni / persona intervallo da 1 a 5 anni )  . 
la correlazione tra rischio radiologico e categoria diagnostica istologica riassunta nella tabella 2 , mentre nella tabella 3 riportata la correlazione tra rischio radiologico e diagnosi de nitiva su exeresi chirurgica . 
 la vacb risultata non idonea ( b1 ) nello 0 , 5% dei casi , poich la procedura non stata completata o per eccessivo sanguinamento ( 2 casi ) o per crisi ansiosa della paziente ( 1 caso )  . 
in 2 / 337 casi ( 0 , 6% ) con esito di benignit , in considerazione del rischio radiologico r4 , dellincompleta asportazione delle microcalci cazioni e a seguito della discussione multidisciplinare , stata comunque consigliata veri ca chirurgica . 
delle 150 lesioni proliferative a rischio ( lpr ) , indicate anche come borderline in letteratura [ 1416 ] , 142 sono state classi cate istologicamente come b3 e 8 come b4 . 
tra i 142 casi risultati b3 , 118 ( 83 , 1% ) sono stati sottoposti a veri ca chirurgica : in 112 / 118 casi ( 94 , 9% ) lesame istologico de nitivo ha confermato lesito della vacb di lesioni proliferative non neoplastiche , mentre in 6 casi ( 5 , 1% ) lesame de nitivo stato di carcinoma duttale in situ ( cdis )  . 
tutti questi 6 casi presentavano nei campioni prelevati con la vacb neoplasia lobulare intraepiteliale ( lin ) pura o associata ( 3 casi ) ad altre lesioni proliferative a rischio . 
of the 150 proliferative lesions of uncertain malignant potential , also known as borderline lesions [ 1416 ] , 142 were histologically classi ed as b3 and 8 as b4 . 
among the 142 cases of b3 lesion , 118 ( 83.1% ) were subjected to diagnostic surgical excision : nal histology con rmed the vacb diagnosis of benign proliferative lesion in 112 / 118 cases ( 94.9% ) , whereas in 6 cases ( 5.1% ) , it resulted in a diagnosis of ductal carcinoma in situ ( dcis )  . 
all vacb specimens retrieved in these six cases showed evidence of lobular intraepithelial neoplasm ( lin ) in its pure form or associated with other indeterminate 8 / 602 casi ( 1 , 3% ) di lesioni b4 , tutti appartenenti alla categoria radiologica r4 , stata consigliata la veri ca chirurgica e lesame istopatologico de nitivo stato di lesione benigna . 
in tutti i casi stata confermata la malignit della lesione , tuttavia in 8 ( 10 , 1% ) pazienti la diagnosi su biopsia chirurgica stata di carcinoma invasivo , mentre alla vacb era stata fatta diagnosi di cdis ( tabelle 1 , 4 )  . 
in the other 8 / 602 cases ( 1.3% ) of b4 lesion ( all radiologically classi ed as r4 ) , surgical excision was recommended , and nal histology revealed benign disease . 
the 112 patients with b5 lesions underwent surgical excision , and malignancy was con rmed in all cases ; however , the nal diagnosis in 8 ( 10.1% ) patients was invasive carcinoma , in contrast with vacb that had provided a diagnosis if dcis ( tables 1 , 4 )  . 
given that the regional government reimbursement rate in piedmont is 750.00 euro for each vacb procedure ( ministerial decree 22 / 7 / 96 : 85.11.3 ) , there is substantial parity between the cost of the procedure and the reimbursement . 
la sensibilit della vacb rispetto allesame istologico de nitivo risultata del 94 , 9% ( 112 / 118 ) ( con un intervallo di con denza al 95% compreso tra 94% e 95 , 8% ) , la speci cit del 98 , 3% ( 476 / 484 ) ( con un intervallo di con denza al 95% compreso tra 97 , 8% e 98 , 8% ) , il valore predittivo positivo del 93 , 3% ( 112 / 120 ) , il valore predittivo negativo del 98 , 8% ( 476 / 482 ) , mentre laccuratezza del 97 , 7% ( 588 / 602 )  . 
negli ultimi 5 anni presso il nostro centro sono state complessivamente evitate 335 biopsie chirurgiche su lesioni con rischio radiologico r3 - r4 ; il risparmio complessivo risultato pari a 155440 , 00 euro , corrispondente ad un risparmio annuo di 31088 , 00 euro . discussione le microcalci cazioni rappresentano una buona parte dei segni mammogra ci legati a lesioni del parenchima radiol med ( 2011 ) 116 : 477488 fig . 
1a , b cluster of heterogeneous calci cations , some rounded , with a maximal extension of 6 mm ( bi - rads r3 ) : craniocaudal ( a ) and mediolateral oblique ( b ) magni cation views . 
1a , b gruppo di microcalci cazioni eterogenee , alcune delle quali rotonde , con estensione di 6 mm ( bi - rads r3 ) : ingrandimento mirato nella proiezione cranio - caudale ( a ) e medio - laterale obliqua ( b )  . 
up to 80 morphological descriptors have , in fact , been reported in the literature , with the result that the standardisation of mammographic diagnosis is dif cult [ 12 , 17 , 18 ]  . 
the advent of cnb with manual or semiautomatic devices has substantially improved diagnostic accuracy in microcalci cation clusters , even though a considerable proportion of biopsies still prove to be nondiagnostic or discordant with the radiological classi cation [ 8 , 19 , 20 ]  . although fnac and cnb may represent a good alternative to diagnostic surgical biopsy , neither of them provides conclusive results in all cases . 
con lintroduzione della core needle biopsy mediante dispositivi manuali o semiautomatici , laccuratezza nella diagnosi di natura dei clusters di microcalci cazioni sensibilmente migliorata , pur rimanendo consistente le quota di prelievi non diagnostici o in disaccordo con lipotesi radiologica [ 8 , 19 , 20 ]  . entrambe le metodiche , fnac e cnb , pur rappresentando una buona alternativa alla biopsia chirurgica diagnostica , non sono sempre risolutive . 
 stato dibattuto in letteratura il ricorso ad agobiopsia percutanea come primo approccio a lesioni classi cate bi - rads r3 ( per le quali lacr raccomanda un monitoraggio di almeno 6 mesi )  . 
tuttavia , nel caso delle microcalci cazioni r3 sottoposte a vacb stata osservata in letteratura una malignit variabile tra 0% e 19% , mentre nella nostra casistica stata del 7 , 4% , percentuale bassa ma non trascurabile , che ci pare giusti chi il prelievo ago bioptico [ 1 , 6 , 2123 ]  . 
nei casi classi cati come bi - rads r4 , la malignit riscontrata allesame istologico de nitivo stata del 25 , 1% , similmente ad altre casistiche ( intervallo dal 15 , 3% al 36 , 6% ) radiol med ( 2011 ) 116 : 477488 484 fig . 
2a - c cluster of pleomorphic calci cations , some granular , with a maximal extension of 15 mm ( bi - rads r4 ) , close to the surgical scar in a patient who previously underwent surgical excision for cribriform ductal carcinoma in situ ( dcis ) : craniocaudal ( a ) and mediolateral oblique ( b ) magni cation views . 
stereotactic 11 - gauge vacb result [ magni cation view of specimens with calci cations ( c ) ] : breast parenchyma with intraductal calci cations ( b2 ) [ 13 ]  . 
2a - c gruppo di microcalci cazioni pleomorfe , alcune delle quali granulari , con estensione di 15 mm , localizzate in prossimit della cicatrice chirurgica , in paziente precedentemente sottoposta ad escissione ampia per cdis cribriforme ( bi - rads r4 ) : ingrandimento mirato nella proiezione cranio - caudale ( a ) e medio - laterale obliqua ( b )  . 
 stata eseguita vacb ( radiogramma di alcuni dei frustoli prelevati , comprendenti le microcalci cazioni [ c ] ) con esito microistologico di : frustoli di parenchima mammario con calci cazioni intraduttali ( b2 ) [ 13 ]  . 
however , in the case of r3 microcalci cations sampled with vacb , the literature reports a rate of malignancy ranging from 0% to 19% as against 7.4% in our series , a low but not negligible rate , which we believe justi es a needle biopsy [ 1 , 6 , 2123 ]  . 
in cases classi ed as bi - rads r4 , the rate of malignant disease at nal histology was 25.1% , consistent with previous reports ( range 15.3%36.6% ) [ 6 , 21 , 22 ]  . 
 the bi - rads r5 lesions ( 2.2% ) studied by vacb were characterised by casting microcalci cations not associated with opacities for which fnac and cnb had proven insuf cient ( c1 and b1 )  . 
as reported by previous studies in which casting microcalci cations accounted for 19% of the entire series [ 1 , 6 , 2123 ] , these lesions do not constitute an elective indication for the use of vacb . 
data indicate that microcalci cations classi ed as probably benign were con rmed as such in the majority of cases , whereas the concordance in suspicious cases tended to be lower , [ 6 , 21 , 22 ]  . 
i casi classi cati bi - rads r5 ( 2 , 2% ) sottoposti a vacb seguivano a microcalci cazioni a stampo , non associate ad opacit , per le quali la fnac e la cnb non avevano fornito unadeguata risposta diagnostica ( c1 e b1 ) ; questo tipo di lesioni , similmente a quanto riportato in altri studi , in cui costituivano generalmente dall1% al 9% di tutta la casistica [ 1 , 6 , 2123 ] , non trovano indicazione elettiva allutilizzo della vacb . 
da questi dati si rileva che le microcalci cazioni verosimilmente benigne nella maggior parte dei casi sono state confermate come tali , mentre nei casi sospetti la corrispondenza risultata minore , poich stato comunque trovato un numero considerevole di lesioni benigne . 
il dato morfologico nella categoria bi - rads r4 non trova , infatti , una correlazione con leffettivo risultato istologico : le lesioni maligne corrispondevano prevalentemente a microcalci cazioni con morfologia granulare o nemente pleomorfa , ma anche una buona parte di lesioni benigne presentava tale tipo di morfologia . 
3a , b cluster of pleomorphic calci cations with a maximal extension of 8 mm ( bi - rads r4 ) in a 35 - year - old patient at rst mammography : craniocaudal ( a ) and mediolateral oblique ( b ) magni cation views . 
3a , b gruppo di microcalci cazioni pleomorfe , con estensione di 8 mm ( bi - rads r4 ) riscontrato alla prima mammogra a in paziente di 35 anni con familiarit : ingrandimento mirato nella proiezione cranio - caudale ( a ) e medio - laterale obliqua ( b )  . 
stata eseguita vacb con esito microistologico di cdis di tipo cribriforme micropapillare ( b5a ) [ 13 ] , confermato allesame istologico de nitivo . with many lesions proving to be benign . 
the morphological appearance of bi - rads r4 lesions does not correlate with the histological result : malignant lesions mainly corresponded to granular or ne pleomorphic calci cations , features that were also present in a large number of benign lesions . 
of particular signi cance are the 337 cases in which vacb with the mammotome device demonstrated the benign nature of the microcalci cations , allowing them to be classi ed as b2 . 
avoidance of surgical biopsies thanks to vacb demonstration of the benign nature of the microcalci cations led to considerable economic savings ( 31 , 088.00 euro / year )  . 
even if cost savings are of limited signi cance in themselves , one cannot neglect the savings in terms of time and occupation of the operating rooms and the lower psychological impact of a minimally invasive needle biopsy . 
in our experience , a microhistological diagnosis of borderline lesion was established in 24.9% of cases ( 150 / 602 ) , a higher percentage than reported in other studies ( range 8.1%12% ) [ 1 , 6 ]  . 
 laccuratezza della vacb nella nostra casistica risultata elevata : sensibilit e speci cit sono state rispettivamente del 94 , 9% e 98 , 3% , in linea con i valori riportati in letteratura ( sensibilit 87 , 5%99% , speci cit 86 , 9% 100% ) ( tabella 5 ) [ 2 , 6 , 19 , 24 , 25 ]  . 
le biopsie chirurgiche evitate in seguito a dimostrazione della natura benigna delle microcalci cazioni mediante vacb hanno determinato un discreto risparmio economico ( 31088 , 00 euro / anno )  . 
se il dato economico appare di per s poco signi cativo , non di meno deve essere considerato il risparmio in termini di tempo ed impegno delle sale operatorie e limpatto psicologico sulla paziente , sicuramente inferiore per unagobiopsia mini - invasiva . 
meritevoli di considerazione sono le lesioni classi cate istologicamente come b3 ( lesioni proliferative di incerto signi cato ) : nella nostra esperienza il riscontro microistologico di lpr risultato pari al 24 , 9% dei casi ( 150 / 602 ) , percentuale superiore a quella di altri studi ( valori compresi tra 8 , 1% ed 12% ) [ 1 , 6 ]  . 
 la discussione multidisciplinare che prende in considerazione aspetti clinici , anamnestici , il quadro radiologico e le caratteristiche istologiche di questi casi fondamentale 486 radiol med ( 2011 ) 116 : 477488 table 5 sensitivity and speci city of vacuum - assisted core biopsy ( vacb ) : comparison with the literature study author ( year ) sensitivity ( 95% ci ) speci city ( 95% ci ) no . 
this may have been due to the selection of lesions to be sampled by vacb , as > 95% of them had microcalci cations as the only mammographic sign , in contrast to other studies that included a greater proportion of opacities , distortions and asimmetric densities either alone or associated with microcalci cations [ 46 ]  . 
it should be noted , however , that the sensitivity of mr imaging for in situ lesions , especially if low grade , is not as high as that reported for invasive lesions . the limitations of vacb in our experience were similar to those reported by other studies [ 3134 ] and consist of the possibility of underestimating proliferative lesions of uncertain malignant potential ( b3 ) and malignant lesions in situ . per la corretta gestione della paziente [ 16 , 2628 ]  . 
nella nostra casistica abbiano riscontrato un basso tasso di malignit nelle diagnosi di b3 ( 4% ) rispetto ad altri studi ( 9%15% dei casi ) ; questo dato potrebbe essere dovuto alla selezione delle lesioni da sottoporre a vacb , costituite nella nostra serie in oltre il 95% dei casi da microcalci cazioni come unico segno mammogra co , a differenza di altri studi in cui sono state incluse una maggior quota di lesioni rappresentate da opacit , distorsioni ed addensamenti come uniche lesioni , oppure associate a microcalci cazioni [ 46 ]  . 
nella maggior parte di b3 , abbiamo riscontrato un quadro microistologico di lesioni miste ; la variabile malignit associata e leterogeneit microistologica hanno comportato nella maggior parte dei casi unindicazione alla veri ca chirurgica e solo nei casi con completa escissione delle microcalci cazioni ( radiologicamente confermata ) ( 16% ) al follow - up clinico - radiologico [ 29 ]  . in tal senso , studi preliminari sembrano suggerire che anche limpiego della risonanza magnetica nella valutazione delle lpr diagnosticate microistologicamente possa essere molto utile nellescludere la presenza di patologia maligna associata , con un valore predittivo negativo del 98 , 2% [ 30 ] ( tenendo conto tuttavia che la sensibilit della risonanza per le lesioni in situ , soprattutto se di basso grado , non cos elevata come per le lesioni invasive )  . come emerge da altri studi in letteratura [ 3134 ] , anche nella nostra esperienza i limiti della procedura vacb sono stati costituiti dalla possibilit di sottostima nelle lesioni proliferative a rischio ( b3 ) e nelle lesioni maligne in situ . radiol med ( 2011 ) 116 : 477488 conclusions conclusioni vacb with the mammotome device is an accurate method for characterising microcalci cations at low and intermediate risk of malignancy at radiology , obviating the need for surgery in more than half of the cases with benign results at vacb . 
the effective economic and logistic savings of the operating theatres justi es the use of vacb , even though it is more expensive that conventional core biopsy with semiautomatic devices . la vacb con sistema mammotome risultata una metodica accurata nella caratterizzazione di microcalci cazioni a basso e medio rischio radiologico , evitando lintervento chirurgico in oltre la met dei casi risultati benigni alla vacb . 
di radiologia , dipartimento di scienze mediche , tecnologiche e traslazionali , azienda ospedaliero - universitaria di trieste , strada di fiume 447 , 34149 trieste , italy correspondence to : p . 
image quality was analysed in terms of spatial and contrast resolution on several scans of a phantom performed with automatic dose modulation and different reconstruction kernels and accepted noise level . 
ctu cannot be considered a standard examination : the scan parameters need to be adapted to the image quality required for the speci c clinical problem . keywords multislice computed tomography urography radiation dose reconstruction kernel riassunto obiettivo . 
stata analizzata la qualit di immagine in termini di risoluzione spaziale e di contrasto di diverse scansioni su fantoccio , ripetute con modulazione automatica di dose , variando il ltro di ricostruzione e il livello di rumore accettato ; sono stati valutati i valori di esposizione e confrontato lesito con quanto osservato in 52 pazienti sottoposti ad esame uro - tc . 
la dose di esposizione e la qualit dimmagine variano notevolmente modi cando i parametri di ricostruzione , sebbene non sempre laumento di dose comporti un reale vantaggio in termini di de nizione dimmagine . 
luro - tc non pu essere considerato un esame standardizzato : i parametri di scansione devono essere sempre valutati e modulati in base alla qualit di immagine richiesta dallo speci co problema clinico . parole chiave tomogra a computerizzata multistrato uro - tc dosimetria filtri di ricostruzione introduction introduzione computed tomography urography ( ctu ) is a diagnostic examination optimized for imaging the kidneys , ureters and luro - tomogra a computerizzata ( tc ) un esame diagnostico ottimizzato per limaging dei reni , degli ureteri e della 418 radiol med ( 2011 ) 116 : 417431 bladder . 
the examination involves the use of multidetector ct with thin - slice imaging , intravenous administration of a contrast medium , and imaging in the excretory phase [ 1 ]  . 
 the high resolution provided by the modern multidetector ct ( mdct ) systems allows for the depiction of ndings < 5 mm [ 2 ] and consequently the assessment not only of anatomical variants of the urinary system but also of very early stage in ammatory or neoplastic diseases . 
according to the published data on radiation dose , a ctu protocol involving four acquisition phases ( unenhanced , arterial , parenchymal and excretory ) exposes the patient to a dose of 2535 msv , as against a mean dose for conventional urography of about 5 msv , a dose that varies widely , however , depending on the indication and the operators experience [ 1 , 36 ]  . 
ctu optimisation entails the use of protocols with fewer scans , allowing the dose to be reduced to values of around 10 msv for two - phase protocols ( unenhanced and combined nephrographic and excretory phase ) or even lower values when the study is limited to the combined nephrographic and excretory phase or corticomedullary , nephrographic and excretory phase [ 7 ]  . 
 in 1995 it was calculated that , although accounting for < 5% of all imaging studies , ct examinations contributed 40% of the collective dose to the population from medical x - rays [ 8 ]  . 
therefore , when planning a ct examination , attention should be given as far as possible to reducing radiation dose in accordance with the as low as reasonably acceptable ( alara ) principle established by the international commission on radiological protection ( icrp ) [ 9 ]  . 
to monitor the radiation dose delivered to the patient during a ct examination , two radiation dose indices can be used : the computed tomography dose index ( ctdi ) and the dose - length product ( dlp )  . 
ctdi evaluation can be obtained with a pencil chamber placed inside a standard ct dosimetry phanto weighted ctdi ( ctdiw ) takes into account the different exposure at the periphery and centre of the anatomical region being studied , whereas dlp corresponds to ctdi multiplied by scan length . 
it should be noted that the ctdiw and dlp values are dose estimates that refer to standard - sized dosimetry phantoms rather than to the real size of the patient , so that the dose may be underestimated in thin patients and overestimated in larger patients . 
an estimate of these two values for each examination setup should be displayed on the operators console in accordance with the safety requirements for vescica , che necessita luso di unapparecchiatura tc multidetettore , con acquisizione di immagini a strato sottile , somministrazione endovenosa di mezzo di contrasto ed una scansione ottenuta in fase escretoria [ 1 ]  . 
lelevata risoluzione spaziale offerta dagli attuali sistemi tc multidetettore permette il riconoscimento di reperti di dimensioni inferiori ai 5 mm [ 2 ] e , quindi , lo studio non solo di varianti anatomiche dellapparato urinario , ma anche di condizioni in ammatorie o neoplastiche in fase iniziale . 
luro - tc , in particolare quando eseguita con tecnica multifasica , un esame ad elevata dose di radiazioni ed attualmente questo il vero problema che ne pu limitare lutilizzo . 
in base ai dati di dosimetria desunti dalla letteratura , un protocollo uro - tc che prevede quattro fasi ( diretta , arteriosa , parenchimale ed escretrice ) espone il paziente ad una dose stimabile di 2535 msv , a fronte di una dose media in urogra a tradizionale , pur estremamente variabile in base ai reperti valutati ed allesperienza delloperatore , di circa 5 msv [ 1 , 36 ]  . 
lottimizzazione delluro - tc prevede lutilizzo di protocolli a minor numero di scansioni con diminuzione della dose no a valori di circa 10 msv , per protocolli a due fasi ( diretta e combinata nefro - escretoria ) , o anche inferiori qualora si possa limitare lesame alla sola fase combinata nefro - escretoria o corticomidollare - nefro - escretoria [ 7 ]  . 
 gi nel 1995 si era calcolato che gli esami tc , pur rappresentando meno del 5% di tutti gli esami radiologici , erano responsabili del 40% della dose di esposizione della popolazione alle radiazioni ionizzanti per scopo medico [ 8 ]  . 
negli ultimi decenni la diffusione degli esami tc aumentata esponenzialmente e , di conseguenza , anche il suo impatto sullesposizione , pertanto in fase di piani cazione dellesame necessario focalizzare lattenzione , per quanto possibile , sul risparmio di dose , in linea con il principio di ottimizzazione sancito dallinternational commission on radiological protection ( icrp ) [ 9 ]  . 
per monitorare la dose di radiazioni cui viene sottoposto un paziente durante lesecuzione di un esame tc si possono usare due indicatori di dose il computed tomography dose index ( ctdi ) e il dose lenght product ( dlp )  . 
il ctdi pesato ( ctdiw ) considera la diversa esposizione in periferia e al centro della regione anatomica in corso di studio , mentre il dlp corrisponde al prodotto del ctdi e dellestensione della scansione . 
 da notare come i valori di ctdiw e di dlp siano stime di dose che si riferiscono a fantocci di dimensione ssa , che non tengono in considerazione le reali dimensioni del paziente , con una conseguente sottostima di dose nei pazienti magri ed una sovrastima in quelli con maggiore massa corporea . 
la stima di questi due valori per radiol med ( 2011 ) 116 : 417431 ct systems issued by the international electrotechnical commission ( iec ) [ 10 ]  . in multislice ct , radiation exposure depends on image acquisition and reconstruction parameters . 
on current ct systems the user needs only to select the image quality , which is determined by the reconstruction lter or kernel , slice thickness and accepted noise level . 
once image quality has been selected , the system automatically delivers the optimal tube current for the type of image , calculated on the basis of the density and thickness values obtained with the scout images according to preset maximum threshold values , which cannot be exceeded . 
modulation may be activated along the z axis or in the x - y plane , depending on the how advanced the system is . the aim of this study was to evaluate the possibilities of optimising the ctu examination by modulating reconstruction parameters on the basis of acquisitions performed on a phantom and data from our clinical practice . materials and methods phantom study to evaluate dose and image quality , we imaged a catphan 600 phantom ( phantom laboratory , greenwich , ny , usa ) with repeated scans using a 64 - slice ct system ( aquilion 64 , toshiba medical systems , tokyo , japan )  . 
acquisition parameters were : collimation 640.5 mm , pitch 0.828 , 120 kv , automatic z - axis tube - current modulation based on the scout image using sure exposure software . 
moreover , a scan was acquired with the low - dose protocol suggested by the ciascuna impostazione desame deve essere indicata sulla consolle del tecnico operatore , secondo le speci che per la sicurezza delle apparecchiature tc della international electrotechnical commission ( iec ) [ 10 ]  . in tc multistrato lesposizione a radiazioni del paziente dipende dai parametri di acquisizione e dai parametri di ricostruzione . 
le attuali apparecchiature tc vengono fornite di alcuni software ef caci nella riduzione della dose , come la modulazione automatica dellerogazione della corrente al tubo e i ltri anti - rumore . 
la modulazione automatica della corrente una metodica che consente di adattare la corrente in modo continuo in base alle caratteristiche della regione bersaglio durante la rotazione del tubo [ 11 , 12 ]  . 
nei sistemi tc attuali , sulla consolle deve essere solamente selezionata la qualit di immagine , dipendente dal tipo di ltro o kernel di ricostruzione , dallo spessore di strato e dal livello di rumore che si disposti ad accettare , ed automaticamente viene erogata una corrente al tubo ottimale per quel tipo di immagine , calcolata sulla base delle informazioni di densit e spessore ottenibili dalle immagini scout , secondo valori di soglia massima preimpostati , che comunque non devono essere superati . 
la modulazione pu essere attivata solo lungo lasse z o anche sul piano xy , secondo levoluzione dellapparecchiatura . il nostro studio si propone di valutare le possibilit di ottimizzazione dellesame uro - tc modulando i parametri di ricostruzione mediante acquisizioni su fantoccio e sulla base dei dati registrati nella nostra casistica quotidiana . materiali e metodi studio su fantoccio per la valutazione dosimetrica e di qualit di immagine stato eseguito uno studio su fantoccio tipo catphan 600 ( the phantom laboratory incorporated , greenwich , ny , usa ) sottoposto a scansioni ripetute con apparecchiatura tc a 64 strati ( aquilion 64 , toshiba medical systems , tokyo )  . 
sono stati utilizzati i seguenti parametri di acquisizione : collimazione 640 , 5 mm , pitch 0 , 828 , 120 kv , corrente modulata automaticamente in base allimmagine scout dal software di modulazione automatica ( sure exposure ) sullasse z . 
le immagini assiali sono state ricostruite a spessore di 5 mm , il volume di immagini per le ricostruzioni multiplanari ( mpr ) e volumetriche stato ricostruito a 0 , 5 mm con intervallo di ricostruzione di 0 , 3 mla variazione dei parametri ha interessato unicamente i ltri di ricostruzione e la deviazione standard ( rumore ) accettata . 
sono stati utilizzati rispettivamente il ltro di ricostruzione 11 , corri420 radiol med ( 2011 ) 116 : 417431 manufacturer for studying renal colic , which corresponds to a kernel fc11 with an accepted sd of 15 , to which q01 ( smooth ) noise ltration was applied . 
the ctdiw * is a derived parameter that represents the ctdiw product , the number of detector rows and slice thickness divided by the table feed per rotation . data analysis image volumes were sent to a vitrea version 4.1.2.0 workstation ( vital images inc . , minnetonka , mn , usa ) and evaluated at a slice thickness of 3 mm in the same viewing conditions by two operators with different levels of experience ( 4 and 7 years )  . 
stata inoltre eseguita una scansione con il protocollo low - dose , suggerito dalla ditta costruttrice per lo studio della colica renale , che corrisponde ad un ltro 11 con deviazione standard accettata 15 , a cui applicato il ltro anti - rumore tipo q01 ( smooth )  . 
il ctdiw * un parametro derivato che rappresenta il prodotto del ctdiw , del numero di righe di detettori e dello spessore di strato impostato rapportato al movimento del lettino per rotazione . analisi dei dati i volumi di immagini ottenuti sono stati inviati alla consolle di ricostruzione vitrea versione 4.1.2.0 ( vital images inc . , minnetonka , minnesota , usa ) e valutati ad uno spessore di strato di 3 mm da due operatori con diversa esperienza fig . 
nearly all patients underwent a preliminary low - dose scan at 120 kv with automatic tube - current modulation , extending from the upper pole of the kidney to the base of the bladder . 
for the contrast - enhanced scan , patients received a diuretic injection and were subsequently imaged with the split - bolus technique consisting of a rst injection of 400 mgi kg contrast medium at a rate of 2 ml / s , followed by an interval of 7 min and a second bolus of 200 mgi kg at 2 ml / s . 
assuming as a reasonable approximation that the ctdiw * and dlp values correlate linearly with the bsa , we normalised the values obtained for the bsa and calculated the ctdiw * and dlp derived from these mean values for a theoretical patient with a bsa of 1.8181 , corresponding to the standard patient ( 170 cm tall and 70 kg )  . 
using the conversion factor obtained with the monte carlo method indicated in the european guidelines on quality criteria for computed tomography ( eur 16262 ) [ 13 ] , the effective dose for each scan was calculated in this standard patient . results ctdiw * values recorded on the phantom in the different scans ( table 1 ) demonstrated that , the reconstruction kernel being equal , the tube current selected by the autoradiologica ( 4 e 7 anni ) alle medesime condizioni di visualizzazione . 
in quasi tutti i pazienti stata acquisita una preliminare scansione diretta low - dose , a 120 kv e con modulazione automatica della corrente , estesa dal polo superiore dei reni alla base vescicale . 
 per la scansione contrastogra ca stato utilizzato il metodo split - bolus dopo iniezione di diuretico , con una prima iniezione di mezzo di contrasto di 400 mgi pro kg a 2 ml / s seguita da una pausa di 7 minuti e quindi un secondo bolo di 200 mgi pro kg a 2 ml / s . 
le immagini assiali sono state ricostruite a spessore di 5 mm , il volume di immagini per le ricostruzioni mpr e volumetriche stato ricostruito a 0 , 5 mm con intervallo di ricostruzione di 0 , 3 mm . per ciascun gruppo di pazienti stato calcolato il valore medio di dlp , di ctdiw * e di body surface area ( bsa ) , utilizzando la formula di mosteller [ ( cm * kg ) / 3600 ]  . 
assumendo come approssimazione accettabile che i valori di ctdiw * e di dlp siano correlati in modo lineare con il bsa , i valori ottenuti sono stati normalizzati per il bsa e sono stati calcolati il ctdiw * e il dlp derivabili da questi dati medi per un teorico paziente con bsa pari a 1 , 8181 , corrispondente al paziente tipo di 170 cm e 70 kg . 
2a - d ricostruzione mip ( a , c ) e cmpr lungo il decorso delluretere destro ( b , d ) di due esami uro - tc normali eseguiti con ltro di ricostruzione 11 ( a , b ) e 13 ( c , d )  . matic modulation system increased with decreasing sd . 
 the corresponding ctdiw * values were then compared with one another using as a standard the scan performed with kernel fc13 and sd 12.5 , which represents the scan setting most commonly used for ctu at our institution ( table 2 )  . 
assessment of image quality by the two readers was concordant as regards the detectability of both the highand low - contrast objects ( table 3 )  . in the 52 patients undergoing ctu for whom the recorded dlp and ctdiw * values were displayed on the operator console , the calculated mean dpl and ctdiw * were 306.27 mgy * cm and 10.21 mgy , respectively , for the unenhanced low - dose scan . 
the nephrographicexcretory scan performed with a reconstruction kernel fc11 involved a mean dlp of 313.76 mgy * cm and ctdiw * of il metodo monte carlo tratto da european guidelines of quality criteria for computer tomography ( eur 16262 ) [ 13 ] , sono stati calcolati i valori di dose ef cace in questo paziente tipo derivante per ciascuna scansione . risultati i valori di ctdiw * registrati sul fantoccio nelle diverse scansioni eseguite , riassunti nella tabella 1 , dimostrano , a parit di ltro di ricostruzione , un aumento della corrente al tubo radiogeno selezionata dal modulatore automatico al diminuire della deviazione standard accettata . 
i valori di ctdiw * corrispondenti sono stati poi confrontati fra loro usando come standard la scansione con ltro 13 e deviaradiol med ( 2011 ) 116 : 417431 fig . 
 the mean bsa value of all patients was 1.8654 , whereas that of patients imaged with kernel fc11 was 1.7747 and that of patients imaged with kernel fc13 was 1.8849. 
la valutazione della qualit di immagine stata concorde per i due osservatori sia per la distinzione degli elementi ad alto contrasto sia per la riconoscibilit delle sfere a basso contrasto ( tabella 3 )  . nei 52 pazienti sottoposti ad esame uro - tc di cui possediamo la registrazione dei valori di dlp e di ctdiw * presentati sulla consolle delloperatore stato calcolato per la scansione diretta low - dose un dlp medio pari a 306 , 27 mgy * cm e un valore di ctdiw * pari a 10 , 21 mgy . 
 la scansione nefro - escretoria eseguita con ltro di ricostruzione 11 ha comportato per il paziente in media un dlp di 313 , 76 mgy * cm e un ctdiw * di 10 , 08 mgy , mentre la scansione nefro - escretoria eseguita con ltro di ricostruzione 13 ha comportato in media un dlp di 733 , 61 mgy * cm e un ctdiw * di 22 , 16 mgy . 
il valore di bsa medio di tutti i pazienti corrisponde a 1 , 8654 , quello dei pazienti in cui stato usato il ltro 11 a 1 , 7747 e quello dei pazienti in cui stato usato il ltro 13 a 1 , 8849 . 
 i valori di dlp e di ctdiw * della scansione low - dose nei pazienti in cui stata poi eseguita la scansione nefro - escretoria con ltro 11 corrispondono a 234 , 74 mgy * cm e 8 , 22 mgy rispettivamente . 
in base ai valori di dlp e di ctdiw * medi sono stati calcolati i valori di dlp e di ctdiw * medi normalizzati per il bsa dei singoli gruppi , corrispondenti a 164 , 18 mgy * cm , 176 , 79 mgy * cm e 389 , 20 mgy * cm e 5 , 47 mgy , 5 , 67 mgy e 11 , 75 mgy rispettivamente per le scansioni low - dose , contrastogra che con ltro 11 e con ltro 13 . 
sono stati , quindi , calcolati i valori di dlp e ctdiw * per il paziente tipo , partendo dai valori medi , corrispondenti a 298 , 50 mgy * cm , 321 , 43 mgy * cm e 707 , 61 mgy * cm e 9 , 94 mgy , 10 , 32 mgy e 21 , 37 mgy rispettivamente , come riassunto in tabella 4 ed illustrato nelle figure 4 e 5 . nella nostra casistica si presentato un paziente con perfetta corrispondenza al paziente tipo ( 170 cm e 70 kg )  . 
in un lavoro del 2004 si dimostrato come la maggior parte dei radiologi non sia in grado di fornire una stima della dose di esposizione derivante da un esame tc [ 20 ]  . 
using a published conversion factor [ 13 ] , we estimated the effective dose for each scan in this patient , obtaining a value of 4.8 msv for the low - dose scan , 6.2 msv for the nephrographicexcretory phase with kernel fc11 and a value of 12.7 msv for the nephrographicexcretory scan with kernel fc13 . discussion although the radiology literature has always paid attention to the issue of dosimetry [ 1419 ] , there appears to be little collective awareness of the risks connected to radiation among the medical and radiological community . 
while awaiting the results of a study investigating the real risk of cancer resulting from ct [ 21 ] , it should be recalled that this modality iniziato sul reale rischio di cancro connesso alla tc [ 21 ] , questo va considerato un esame ad alta dose di radiazioni e con potenziale rischio di carcinogenesi , molto pi elevato di quello derivante dalle altre pratiche radiologiche , ed in quanto tale da utilizzare solo quando realmente necessario e nel modo pi appropriato , ricordando di ridurre la dose di esposizione , per quanto possibile . 
in tc multistrato lesposizione a radiazioni del paziente dipende dai parametri di acquisizione e dai parametri di ricostruzione . la maggior parte dei sistemi tc viene preimpostata nello studio delladdome con un voltaggio del tubo radiogeno , per un paziente di media corporatura , pari a 120 kv . 
 [ 19 ] hanno pubblicato uno studio su fantoccio in cui hanno dimostrato una riduzione di dose del 60% impostando il tubo a 80 kv , ma tale protocollo risultato utilizzabile solo quando si studiano elementi ad elevato contrasto , mentre il rumore risulta eccessivo per poter avere una buona sensibilit nellindividuazione delle piccole neoplasie intrarenali o intraureterali . 
la selezione dei valori di corrente erogata al tubo varia molto in letteratura a seconda dei diversi gruppi di studio e dipende fortemente dal numero di strati dellapparecchiatura tc e dallo spessore di strato impostato [ 1 , 3 , radiol med ( 2011 ) 116 : 417431 delivers a high radiation dose with a potential risk of carcinogenesis , which far exceeds that deriving from other radiological procedures . 
as a consequence , ct should be used only when truly necessary and in the most appropriate manner , taking care to minimise the radiation dose as far as possible . in mdct radiation dose depends on acquisition and reconstruction parameters . 
 [ 19 ] published a phantom study in which they demonstrated a 60% dose reduction by setting the tube voltage at 80 kv , but this protocol was found to be utilisable only for studying highcontrast elements , the noise level being too high to achieve good sensitivity for detecting small renal or ureteral neoplasms . 
tube current settings vary widely in the literature and are heavily dependent on the number of detector rows and slice thickness used [ 1 , 3 , 6 ]  . 
spatial resolution in the x - y plane is in uenced by the size of the eld of view and kernel and corresponds to approximately 0.70.8 mm per standardor medium - smooth kernel in the abdomen , but it is also dependent on slice thickness due to presence of partial volume effect [ 8 , 18 ]  . 
clearly , to obtain higher contrast resolution along with improved spatial resolution , a greater amount of current needs to be delivered to reduce quantum noise by increasing the number of incident photons on the detector [ 8 ]  . in addition to modulating the scan parameters , the radiologist can limit the number of scans to be obtained , for example , by using the split - bolus or triple - bolus technique to obtain both angionephrographic and excretory information simultaneously [ 3 , 7 ]  . 
in our practice , ctu is not considered a standard examination , and the number of phases and scan parameters are always adapted to the patients age and to the clinical question being asked . 
 whenever possible , we omit the unenhanced scan and use the split - bolus technique to study both the parenchyma and urinary tract with a single scan ; only if clearly needed do we perform an additional scan , the extension of which is always limited to the equivocal nding . 
il pitch pu essere abbassato no a 0 , 50 , 7 , ottenendo cos maggiori informazioni sullasse z [ 18 ] , anche in questo caso , per , a costo di aumento di dose . 
la risoluzione spaziale sul piano xy in uenzata dalle dimensioni del eld of view ( fov ) e dal kernel e corrisponde a circa 0 , 70 , 8 mm per kernel di tipo standard o mediumsmooth nelladdome , ma dipende anche dallo spessore di strato , per la presenza delleffetto di volume parziale [ 8 , 18 ]  . 
 naturalmente per ottenere una risoluzione di contrasto pi elevata , con miglioramento anche della risoluzione spaziale , necessario erogare maggior corrente al tubo , per ridurre il disturbo del rumore quantico aumentando il numero di fotoni incidenti sul detettore [ 8 ]  . oltre ai parametri tecnici della singola scansione il medico pu agire limitando il numero di scansioni eseguite , ad esempio con lutilizzo della tecnica a doppio o triplo bolo di mezzo di contrasto , per ottenere simultaneamente sia informazioni angio - nefrogra che che escretorie [ 3 , 7 ]  . 
 nella nostra pratica clinica luro - tc non considerato un esame standardizzato , ma viene sempre modulato in base al quesito clinico e allet del paziente sia per quanto riguarda il numero di fasi che i parametri di scansione . 
quando possibile evitiamo di eseguire la scansione diretta e utilizzando la tecnica del doppio bolo otteniamo informazioni sia sul parenchima renale che sulla via escretrice con ununica scansione , riservando solo ai casi realmente necessari lesecuzione di un ulteriore scansione , comunque limitata per estensione allo studio del solo reperto dubbio . 
la scansione senza contrasto impostata sempre a bassa dose , secondo i parametri suggeriti dalla letteratura e dalla ditta costruttrice per lo studio della colica renale , cui corrisponde un valore di dlp nel reale paziente tipo di 170 cm e 70 kg di 284 mgy * cm e di dose ef cace di 4 , 8 msv . 
la scansione nefro - escretoria impostata con parametri differenziati in base al quesito clinico : quando si studiano anomalie anatomiche o patologie benigne caratterizzate da reperti non troppo minuti , viene impostata con ltro di ricostruzione 11 e deviazione standard accettata di 12 , 5 . 
il valore di dlp per il reale paziente tipo di 170 cm e 70 kg di 369 mgy * cm cui corrisponde una dose ef cace di 6 , 2 msv . 
per lo studio della patologia neoplastica e della papilla , il protocollo usato prevede 428 radiol med ( 2011 ) 116 : 417431 eters suggested by the literature and the system manufacturers for studying renal colic . 
the nephrographic - excretory phase is carried out with parameters that are differentiated according to the clinical question : for anatomical abnormalities or benign conditions characterised by ndings that are not excessively small , we use a scan setting with a reconstruction kernel fc11 and an accepted sd of 12.5. 
for studying neoplastic and papillary disease , the protocol involves the use of a kernel fc13 , again with an sd of 12.5 , which allows for less noise and better contrast , thus enabling the detection of small ndings as well . 
as can be noted , the scan done with reconstruction kernel fc13 delivers twice the dose of the scan with kernel fc11 and should thus only be used when absolutely necessary , at least in young patients . 
 when observing data on mean dose in patients undergoing ctu , we noticed that the patients forming the two subgroups were heterogeneous in size : mean bsa of patients studied with kernel fc11 was 1.7587 , whereas that of patients studied with kernel fc13 was 1.8849 , with an overall mean value of 1.8654. 
 to coherently compare the two settings of the contrastenhanced scan , we decided to normalise the dose values to the patients bsa , assuming as a reasonable approximation an undemonstrated linear correlation between bsa variation and dose variation effected by the automatic modulation syste dlp and ctdiw * for the standard patient ( 170 cm and 70 kg ) were derived from the values obtained . in the dosimetric evaluation , it should be noted that the scan range was not uniform in the different techniques : in fact , the unenhanced scan and the contrast - enhanced scan performed with kernel fc11 extended from the upper pole of the kidney to the bladder base , whereas the contrastenhanced scan extended from the hepatic dome to the pubic symphysis in almost all cases . 
 lutilizzo del ltro 13 , sempre con deviazione standard 12 , 5 , che fornisce immagini meno rumorose e , quindi , con miglior de nizione di contrasto , tale da consentire il riconoscimento anche di piccoli reperti . 
ovviamente , per ottenere questa qualit diagnostica la corrente erogata al tubo maggiore e il valore di dlp per il reale paziente tipo di 170 cm e 70 kg di 747 mgy * cm con dose ef cace di 12 , 7 msv . 
come si pu notare la scansione eseguita con ltro 13 comporta per il paziente pi che un raddoppio della dose rispetto alla scansione con ltro 11 , quindi va usata , almeno nei pazienti giovani , solo in caso di reale necessit . 
 osservando i dati di dose media nei pazienti sottoposti ad esame uro - tc abbiamo notato come i pazienti appartenenti ai due sottogruppi non fossero omogenei quanto a dimensioni , infatti il bsa medio dei pazienti studiati con ltro 11 pari a 1 , 7587 e il bsa medio dei pazienti studiati mediante scansione con ltro 13 risulta pari a 1 , 8849 , con media complessiva di 1 , 8654 . 
 per una valutazione di confronto coerente fra le due impostazioni della scansione contrastogra ca si deciso di normalizzare i valori di dose al bsa dei pazienti , assumendo come stima accettabile una correlazione lineare , non dimostrata , fra variazione di bsa e variazione di dose attuata dal modulatore automatico . 
dai valori cos ottenuti stato calcolato il dlp e il ctdiw * per il teorico paziente di 170 cm e 70 kg . nella valutazione dosimetrica da notare come nelle diverse modalit il range di scansione non fosse uniforme , infatti nei pazienti la scansione diretta e la scansione contrastogra ca con ltro 11 sono state limitate dal polo renale superiore alla base vescicale , mentre la scansione contrastogra ca stata , quasi sempre , estesa dalla cupola epatica alla sin si pubica . 
le variazioni di ctdiw * e di dlp sul paziente tipo reale e sulla media dei pazienti appaiono comunque coerenti . dati gli elevati valori degli indicatori di dose nelle indagini in esame , abbiamo valutato la possibilit di cambiare le impostazioni di scansione , per veri care no a che punto la scelta di eseguire esami ad alta dose sia giusti cata dalla qualit diagnostica delle immagini . 
abbiamo veri cato che il rapporto di ctdiw * fra la modalit in ltro 11 e la modalit in ltro 13 risulta rispettivamente 0 , 46 su fantoccio , 0 , 48 sui dati medi normalizzati dei pazienti in esame e 0 , 5 sul paziente tipo reale . 
i valori si presentano , quindi , coerenti fra loro e pertanto in base alle valutazioni sui valori di ctdiw * ottenuti su fantoccio , possiamo ottenere una stima suf cientemente accurata della percentuale di risparmio o aumento di dose connesso alle varie modalit . 
nella scanradiol med ( 2011 ) 116 : 417431 given the high values of the dose indices , we assessed the possibility of changing the scan settings to verify to what extent the decision to perform high - dose examinations is justi ed by the diagnostic quality of the images . 
 we found that the ctdiw * between the technique using kernel fc11 and the technique using kernel fc13 was 0.46 on the phantom , 0.48 on the normalised mean data of the patients and 0.5 on the real standard patient . 
the values are therefore coherent with one another , so that the ctdiw * values obtained on the phantom provide a suf ciently correct estimate of the percentage of dose reduction or increase associated with the different techniques . 
in the low - dose scan ( kernel fc11 and sd 15 ) , application of the smooth denoising lter renders the images visually less noisy and more pleasing but reduces the perception of both highand low - contrast details and is therefore counterproductive , at least in the phantodetectability of highcontrast elements is very similar for all scans performed with a medium denoising lter , whether with reconstruction kernel fc11 ( corresponding to a smooth lter without correction for beam hardening ) or kernel fc13 ( corresponding to a standard medium - smooth lter without beam - hardening correction )  . 
clinical use of the contrastenhanced scan with kernel fc11 when studying ureteral morphology and lithiasis conditions requiring the evaluation of high - contrast structures therefore appears justied and in line with the alara principle , with estimated dose reductions of , respectively , 59% and 54% for techniques using accepted sd of 15 or 12.5 , compared with the standard setting kernel fc13 . all dosimetry studies performed on ctu to date have regarded the opaci ed ureter as a high - contrast structure [ 24 ]  . 
conversely , in view of the dilution of the contrast agent due to the diuretic administered to distend the ureter and distribute the contrast uniformly inside it , our study paid special attention to the evaluation of low - contrast objects . 
depiction of detail does not improve when the accepted noise level is lowered , whereas the dose increases by 10% compared with that associated with the use of the standard kernel fc13 . 
between the scans with an sd of 12.5 and 10 , there is no signi cant improvement in object detectability , even though the estimated increase in dose is around 27% in the less noisy scan . 
to see any improvement in image quality , the sd needs to be further decreased to values of 7.5 , which provide better de nition of edges without , however , improving detectasione a bassa dose ( ltro 11 e deviazione standard 15 ) lapplicazione del ltro antirumore di tipo smooth rende le immagini visivamente meno rumorose e quindi pi gradevoli , ma riduce la percezione sia dei dettagli ad elevato che a basso contrasto , risultando perci controproducente , almeno su fantoccio . 
la riconoscibilit degli elementi ad elevato contrasto sovrapponibile per tutte le scansioni eseguite con un ltro anti - rumore di tipo intermedio , sia con ltro di ricostruzione di tipo 11 , corrispondente ad un ltro smooth senza correzione per lindurimento del fascio radiante , che di tipo 13 , ltro intermedio standard - smooth , senza correzione per lindurimento del fascio radiante . 
luso clinico della scansione contrastogra ca con ltro 11 nello studio della morfologia ureterale e nella malattia litiasica , condizioni in cui si devono valutare strutture ad elevato contrasto , appare pertanto giusti cata e in linea con i principi di ottimizzazione , con un risparmio di dose stimabile , nei confronti dellimpostazione standard a ltro 13 , del 59% e 54% , nelle modalit con deviazione standard accettata rispettivamente di 15 o 12 , 5 . gli studi dosimetrici nora effettuati per la valutazione delluro - tc hanno sempre considerato luretere opacizzato come una struttura ad elevato contrasto [ 24 ]  . 
tenendo conto della diluizione del mezzo di contrasto apportata dalluso del diuretico , utile per ottenere unottimale distensione della via escretrice ed una distribuzione uniforme del contrasto in essa , nel nostro studio abbiamo , invece , posto particolare attenzione alla valutazione del visibilit degli oggetti a basso contrasto . 
la ricostruzione a ltro 11 consente una buona riconoscibilit dei dettagli nellimpostazione con ds 12 , 5 , che non migliora quando viene ridotto il rumore accettato , a fronte di un aumento di dose del 10% rispetto a quello richiesto per luso del ltro 13 standard , pertanto limpostazione di una deviazione standard cos ridotta non appare giusti cata . nelle scansioni eseguite con ltro 13 si osserva un miglioramento nella riconoscibilit degli oggetti a basso contrasto , che sono distinguibili no ad un diametro di 4 mm nelle scansioni con deviazione standard accettata uguale o inferiore a 12 , 5 . 
fra la scansione con deviazione standard di 12 , 5 e di 10 , a fronte di un aumento di dose di esposizione stimabile attorno al 27% nella scansione meno rumorosa , non si osserva un signi cativo miglioramento nella riconoscibilit degli oggetti . 
per osservare un miglioramento nella qualit delle immagini bisogna scendere ulteriormente con la deviazione standard , no a valori di 7 , 5 , ottenendo immagini che si presentano a contorni pi de niti ma che comunque non consentono di riconoscere un numero maggiore di elementi , pur con un aumento di dose che appare davvero considerevole , stimabile attorno al 118% . 
 da questi dati luso dellimpostazione a ltro 11 e ds 15 , con ltro antirumore di tipo intermedio appare un buon compromesso fra dose di esposizione e qualit dellimma430 radiol med ( 2011 ) 116 : 417431 bility ; the increase in dose is indeed very high around 118% . 
 on the basis of these data , using the setting with kernel fc11 with sd 15 and medium denoising lter appears to provide a good trade - off between dose and image quality on the phantom , in contrast with the settings used until now for the unenhanced scan . 
for the contrast - enhanced scan , the decision to use a reconstruction kernel fc11 or fc13 and intermediate accepted noise level ( sd 12.5 ) depending on the clinical question , appears adequate , whereas a further noise reduction provides no diagnostic advantage . conclusions any investigation involving exposure to ionising radiation should be performed after careful evaluation of the clinical indication to ensure that the investigation is justi ed and to select the most appropriate technique in accordance with the alara principle . 
conversely , application of an additional smooth denoising lter for the unenhanced low - dose scan does not appear bene cial , as more information can be gained with the scan performed with the same dose parameters but with the application of a medium lter . optimisation of the ctu study in clinical practice will need to start from the unenhanced scan : an effective dose of 4.8 msv for a scan that provides secondary information compared with the contrast - enhanced scan is not justi ed . 
 a possible immediate solution is to couple the detectors during acquisition , a setup that can also be used for the nephrographicexcretory scan , with a collimation of 321 mm instead of the current 640.5 mthe reconstruction index should never be lower than the spatial resolution in the x - y plane , as the voxels will be shorter in the z axis , and , since noise potentially increases with effective voxel size , the system is forced to deliver higher tube current to maintain the desired signal - to - noise ratio , leading to a 16 - fold increase in dose [ 25 ]  . subsequently , it will be necessary to evaluate image quality obtained with a lower voltage setting , as proposed by coppenrath et al . 
per la scansione contrastogra ca , la scelta nora attuata di un ltro di ricostruzione di tipo 11 o 13 e livello di rumore accettato intermedio ( deviazione standard 12 , 5 ) in base al quesito clinico , appare adeguata , mentre unulteriore riduzione del rumore non comporta un guadagno diagnostico . conclusioni ogni indagine che richiede unesposizione a radiazioni ionizzanti deve essere eseguita dopo attenta valutazione del caso clinico , per accertare la giusti cazione dellesame stesso e valutare la migliore tecnica nel rispetto del principio dellottimizzazione . 
ci tanto pi vero per luro - tc , il cui limite principale la dose di radiazioni relativamente elevata cui il paziente sottoposto , che comporta valori di dose ef cace no a 17 , 5 msv . in base alla valutazione della qualit delle immagini eseguita mediante studio su fantoccio possiamo affermare che le scansioni eseguite con ltro di ricostruzione 11 e 13 , corrispondenti ad un ltro smooth ed intermedio standard - smooth , senza correzione per lindurimento del fascio radiante , con deviazione standard accettata di 12 , 5 , rappresentano un buon compromesso fra dose di esposizione e qualit dimmagine per i differenti quesiti clinici . 
appare , invece , non conveniente lapplicazione di un ulteriore ltro antirumore di tipo smooth per la scansione diretta a bassa dose , potendosi ottenere maggiori informazioni nella scansione eseguita con gli stessi parametri di dose ma con applicazione di un ltro intermedio . lottimizzazione dellesame di uro - tc nella nostra pratica clinica dovr innanzitutto concentrarsi sulla scansione diretta : una dose ef cace pari a 4 , 8 msv per una scansione che fornisce informazioni secondarie rispetto a quella contrastogra ca , non appare giusti cata . 
laccorgimento immediato da adottare potr essere laccoppiamento dei detettori in fase di acquisizione , eventualmente utilizzabile anche nella scansione nefro - escretoria , con una collimazione di 321 mm invece dellattuale 640 , 5 mlindice di ricostruzione non dovrebbe mai essere inferiore alla risoluzione spaziale sul piano xy , perch si ottengono voxel pi corti sullasse z e , dal momento che il rumore aumenta potenzialmente con il volume del voxel effettivo , per mantenere il desiderato rapporto segnale - rumore lapparecchiatura costretta ad erogare una maggiore corrente al tubo , con un aumento di dose al paziente anche di 16 volte [ 25 ]  . in un secondo tempo sar necessario valutare la qualit delle immagini ottenibili impostando una minor tensione al tubo radiogeno , analogamente a quanto proposto da coppenrath et al . 
 [ 19 ] , essendo la scansione diretta necessaria principalmente per studiare elementi ad elevato radiol med ( 2011 ) 116 : 417431 serving mainly to study high - contrast structures such as stones . 
optimisation can then be re ned through more in - depth studies of image quality obtained by modulating the remaining parameters , even with the use of different phantoms having speci c characteristics for ctu . contrasto , quali i calcoli . 
kahnouji2 1advanced diagnostic and interventional radiology research center ( adir ) , imaging medical center , imam hospital , tehran university of medical sciences , tehran 14155 - 7146 , iran 2department of neurology , imam hospital , tehran university of medical sciences , p.o. 
ghasemi esfe , tel : + 98 - 216 - 1192670 , fax : + 98 - 216 - 6910201 , e - mail : areza.ghasemi@gmail.com received : 30 may 2010 / accepted : 9 july 2010 / published online : 4 february 2011 springer - verlag 2011 abstract purpose . 
 nonetheless , in a portion of patients complaining of the typical signs and symptoms of cts , the edt is negative , and yet no paraclinical tool has been acknowledged for con rming the diagnosis . 
 ci nonostante , pur in presenza di sintomi tipici di stc non sempre i ted risultano positivi ed ancora non sono state de nite ed accettate indagini alternative che consentano di confermare la diagnosi . 
larea del nervo mediano calcolata in sezione trasversale a livello del polso ed il rapporto tra questa e larea stimata a livello dellavambraccio sono risultati signi cativamente superiori nel gruppo dei pazienti rispetto ai controlli sani . 
i coef cienti di ipoecogenicit [ odds ratio ( or ) 4 , 317 ; intervallo di con denza ( ic ) del 95% ] e di ipervascolarizzazione [ or 5 , 0004 ; 95% ic 1 , 02 - 21 , 15 ] del nervo mediano sono risultati signi cativamente pi elevati nel gruppo dei pazienti rispetto ai controlli . 
ultrasound imaging is a useful technique in diagnosing cts patients when edt results are not con rmatory and the patient is suspected of having neuropathy . keywords carpal tunnel syndrome electrodiagnostic tests ultrasonography electromyography nel predire la stc mediante sola indagine ecogra ca . 
limaging ecogra co utile nella diagnosi di stc in pazienti con sospetta neuropatia e ted non conclusivi . parole chiave sindrome del tunnel carpale test elettrodiagnostici ecogra a elettromiogra a introduction material and methods carpal tunnel syndrome ( cts ) is the most common form of entrapment neuropathy . 
it is estimated that the prevalence of cts in the adult general population is about 5.8% in women and 0.6% in men , but in a considerable portion of patients the condition remains undetected [ 5 ]  . 
 traditionally , cts is suspected on the basis of its characteristic signs and symptoms , and diagnosis is established by an electrodiagnostic test ( edt ) [ 6 ]  . 
although edt has high speci city in diagnosing cts [ 7 ] , its accuracy is not de nite , and false - negative results have been reported to be about 1020% [ 1 , 811 ]  . 
since that time , ongoing advances in us technology have made it a useful tool with which to obtain better spatial resolution and depict peripheral nerves with higher imaging quality [ 6 ]  . 
nonetheless , as edt does not have de nite accuracy , in a portion of patients complaining of the typical signs and symptoms of cts , edt results are negative , and yet no paraclinical tool has been acknowledged for con rming cts diagnosis in this subgroup of patients . 
considering that us has recently emerged as a practical and noninvasive method with reasonable accuracy in cts diagnosis [ 6 , 1319 ] , it would be interesting to evaluate its value when edt is not helpful , i.e. 
 thirty - four patients with clinical evidence of cts who had normal edt were enrolled in a cross - sectional study that was conducted from january 2008 to july 2009 in the advanced diagnostic and interventional radiology research center , imaging medical center , af liated with tehran university of medical sciences , tehran , iran . 
clinical evidence of cts , evaluated by an experienced neurologist , was de ned as pain , paraesthesia or prickling sensation in at least two of the digits iiii along , with any of the following sign or symptoms : sensory loss in the territory of median nerve , positive tinel or phalen sign , atrophy of the thenar muscles , or weakness of grip . 
patients with a history of surgical treatment for cts , wrist trauma or fracture or other conditions associated with neuropathy ( such as diabetes mellitus , hypothyroidism , chronic renal failure , pregnancy ) or with clinical ndings of any other neuropathy ( such as cervical radiculopathy and polyneuropathy ) were not included . 
electromyography ( emg ) was performed with a 10 - channel medelec synergy t - ep systepatients without evidence of cts on emg according to padua et al.s criteria [ 21 ] were included . 
 healthy individuals ( n = 41 ) were selected from volunteer hospital staff without symptoms and signs of peripheral neuropathy ( such as history of upper - limb pain , paraesthesia , weakness or any abnormality found in a brief neurological examination ) or evidence of any underlying disease associated with peripheral neuropathy . 
emg was performed in all of these volunteers , and those with normal emg ndings were enrolled as the control group . the study was approved by the local ethics review committee at the university . 
1a , b sezione trasversale ecogra ca del tunnel carpale a livello dellosso pisiforme ; caratteristico aspetto punteggiato del nervo mediano in un soggetto sano ( a ) ; marcata ipoecogenicit del nervo in un paziente affetto da sindrome del tunnel carpale ( stc ) ( b )  . in detail to patients and controls , written informed consent was obtained from all participants . 
 ultrasonography an experienced musculoskeletal radiologist blinded to the patient and control groups performed high - resolution us using a 9 - 13 mhz linear - array probe ( sonoline antares , siemens )  . 
peripheral nerves are seen as multiple speckled hypoechoic areas ( groups of fascicles ) surrounded by echogenic perineurium and / or epineuriu nerve oedema increases the hypoechoic signal of the nerve . 
using the ellipsoid area formula ( d1 d2 3.14 / 4 ) , two perpendicular diameters ( maximum anteroposterior and mediolateral diameters ) of the nerve at the level of the pisiform bone were calculated and , presuming the oval shape of the nerve , the area was calculated . 
we performed a double - level measurement of the median nerve at the level of forearm as well as the pisiform bone , as previously proposed by hobson - webb et al . 
the transducer was directly placed on the skin of most patients , but in thin patients with a very super cial nerve , if there was poor image quality to measure the nerve csa , we used a gel pad on the wrists . 
because of the deeper position of the nerve , there was no need to apply gel pads to the forear colour doppler us ( cdus ) was performed with settings suitable for detecting lowow vessels and imaged in power doppler mode . 
pulse repetition frequency was placed at 488 hz , doppler gain was then adjusted to the maximum level , creating no clutter or 492 radiol med ( 2011 ) 116 : 489496 fig . 
2a - c longitudinal view of three median nerves : nerve of a healthy individual without increased intraneural vascularity ( a ) ; intraneural hypervascularity in a patient complaining of clinical signs and symptoms of carpal tunnel syndrome ( cts ) ( b ) ; perineural increased vascularity in a healthy individual ( c )  . 
2a - c sezione longitudinale del nervo mediano in tre differenti casi : nervo di un soggetto sano in assenza di incremento della vascolarizzazione intraneurale ( a ) ; ipervascolarizzazione intraneurale in un paziente con tipici segni e sintomi di sindrome del tunnel carpale ( stc ) ( b ) ; incremento della vascolarizzazione perineurale in un soggetto sano ( c )  . 
if there was perineural vascularity or any prominent vessel near the nerve , we considered it as a normal variant , and it was not considered as intraneural hypervascularity of the median nerve . 
all participants were examined by us within 1 week of emg , and all measurements were performed twice and the average taken as the value . the level of association between continuous variables was measured using pearsons correlation coef cients . 
 the probability of having clinical evidence of cts in an individual with one or a combination of us signs of cts was calculated using the following formula : exp ( logit ) / [ 1 + exp ( logit ) ] , where logit , derived from the logistic regression modelling , is equal to the natural log of the odds of having clinical evidence of cts . 
 comparison of variables between case and control groups was made using chi - square analysis for categorical variables , students sample t test for normally distributed continuous variables , and the mannwhitney u test for continuous variables that deviated from normal distribution . 
the probability of having clinical evidence of cts in a person with one us signs of cts was approximately 35% , approximately 70% in a person with two , and approximately 90% in a person with all three signs . od or the ellipsoid formula . 
csa of the median nerve in the wrist and the wfr were measured either by the tracing method or ellipsoid formula and were signi cantly higher in patients compared with controls . 
in patients but not in controls , d1 / d2 of the median nerve in both forearm and wrist were signi cantly lower in those with hypoechogenicity pattern in the median nerve . 
 with respect to laplaces law , it is believed that the principal sensory bres injured in compressive neuropathies are small and cannot be reliably evaluated with edt [ 27 ]  . 
in fact , edt is more a diagnostic tool to evaluate large myelinated nerve bres rather than small nonmyelinated ones [ 28 , 29 ]  . clinicians usually encounter dilemma when facing patients with the clinical characteristics of cts but with nor494 radiol med ( 2011 ) 116 : 489496 mal edt ndings . 
us is the usually the most commonly recommended alternative to edt for detecting clinically suspicious patients , as it has lower cost and more comfort compared with other imaging techniques , such as computed tomography and magnetic resonance imaging [ 17 , 30 ]  . 
 there are several studies available reporting different us criteria for diagnosing cts in patients with clinical and electrophysiological ndings [ 3 , 6 , 13 , 14 , 16 , 3134 ]  . 
the csa of the median nerve at the level of the pisiform bone is the most commonly discussed criterion for diagnosing cts in suspicious patients [ 6 , 10 , 17 , 18 , 31 , 35 ]  . 
whereas some authors argue that csa of the median nerve has higher sensitivity and speci city in comparison to edt , others do not [ 17 , 35 ]  . 
 in this study , we calculated the csa of the median nerve using either the ellipsoid formula or the tracing method at two levels : wrist and forearwe found that the mean csa of the median nerve at wrist level , calculated with both techniques , was signi cantly higher in patients compared with controls . 
in a study by klauser et al . , the mean csa of the median nerve in 93 wrists of healthy volunteers ( 9.0 mm2 ) was signi cantly smaller than that of 100 cts wrists ( 16.8 mm2 ) [ 22 ]  . 
 normal peripheral nerve is visualised on us as a bundle containing multiple stippled hypoechoic areas surrounded by hyperechoic perineuriuas the nerve becomes oedematous , the hypoechoic signals become intensi ed [ 32 ]  . 
our results may highlight the role of cdus as an emerging tool for cts diagnosis . although , as described above , several studies have evaluated the role of us imaging in cts diagnosis , there is insuf cient evidence as regards the actual implementation of us in clinical practice ( for example , when cts is clinically suspected and edt results have not been clarifying , and the physician has to choose another diagnostic paraclinical tool to con rm the clinical diagnosis )  . 
to the best of our knowledge , the only study that has measured the value of us in detecting cts in patients with negative edt was performed by koyuncuoglu et al . 
they enrolled patients with positive clinical ndings but negative edt results for cts and used csa of the median nerve > 10.5 mm at wrist level as the diagnostic criterion . 
regression models , compared with crude analysis modelling , bene t from the capability of adjustment for the intercorrelation of the determinant factors and can help with readily approximating the probability of the outcome variable when there is more than one positive determinant factor . 
it is notable that the positive us signs were present in a portion of healthy individuals , and it would be important for a physician encountering the same clinical situation to have an approximation of the clinical value for each or a combination of positive us signs . 
among the three determinant factors hypervascularity , hypoechogenicity or high wrist csa of the median nerve we found that the presence of any one of these signs would predict clinical evidence of cts with a probability of approximately 35% . 
in the presence of two phenomena , this probability increases to as high as 70% ; when all the three sings are present , the probability is approximately 90% . our study has some limitations : firstly , us is an operator - dependent imaging tool . 
in this study , to evaluate us imaging in clinically suspected cts without con rmatory edt ndings , we included patients based on the presence of clinical evidence of cts . 
thirdly , to rule out tiny artefactual or noisy pixels produced by lowow settings , one can use spectral doppler recording or rely on visualising the pulsatile ow during systole and diastole within the nerve . 
however , spectral radiol med ( 2011 ) 116 : 489496 doppler is another option for detecting intraneural hypervascularisation . in summary , we showed that us imaging is a useful technique in diagnosing cts patients when edt results are not con rmatory and the patient is suspected of having neuropathy . 
knauth springer - verlag , berlin heidelberg , 2010 isbn 978 - 3 - 540 - 78570 - 0 e - isbn 978 - 3 - 540 - 78576 - 7 doi 10.1007 / 978 - 3 - 540 - 78576 - 7 published online : 7 march 2011 springer - verlag 2011 diffusion - weighted magnetic resonance imaging ( dwmri ) is a well established technique , routinely used in the study and evaluation of intracranial diseases . 
on the contrary , only a few years have passed since it has shown potential in the study of body conditions , gaining increasing momentum in the clinical setting . as stated by the editors in their preface , dw - mri has important advantages over other radiological procedures : short performance times ; no need for contrast medium ; it offers qualitative and quantitative information thus providing unique information on micro - structural and functional alterations in body tissues . the text is presented in 16 chapters , divided into four sections : principles and background ( 3 chapters ) ; non - oncologic application in the body ( 3 chapters ) ; oncological applications in the body ( 8 chapters ) ; future developments ( 2 chapters )  . the reader will note that the bulk of the text deals with dw - mri in oncology , highlighting the pros and cons of the technique in this eld , sometimes dif cult and with convoluted meaning ; yet applications outside this eld of research are also discussed . 
the last two interesting chapters in which multifunctional mri imaging assessment is discussed also look into the future as well as the potential of dw - mri as an imaging biomarker in clinical trials . the gures are impressive ; table lay - out is appropriate and rich in detail on imaging parameters or results of previous data collected from the literature ( references are updated to 2009 )  . in case one is not aware of the how and why of the technique it may be dif cult to properly process the amount of information found throughout the text . 
 la tecnica di risonanza magnetica per diffusione ( dw - mri ) ben riconosciuta ed affermata , di impiego routinario nello studio e valutazione delle cerebropatie ; al contrario , invece , solo pochi anni sono passati da quando ha dimostrato le sue potenzialit nello studio delle affezioni del corpo , guadagnando progressivo impulso nellambito clinico . come ben speci cato dai curatori del volume nella loro prefazione , la dw - mri presenta signi cativi vantaggi rispetto ad altre metodiche radiologiche : breve tempo di esecuzione ; assenza di mezzo di contrasto ; possibilit di offrire informazioni uniche , sia qualitative che quantitative , sulle alterazioni micro - strutturali e funzionali dei tessuti del corpo . il testo organizzato in 16 capitoli , divisi in quattro sezioni : principi e basi ( 3 capitoli ) ; applicazioni non oncologiche nel corpo ( 3 capitoli ) ; applicazioni oncologiche nel corpo ( 8 capitoli ) ; sviluppi futuri ( 2 capitoli )  . il lettore si render conto che il grosso del testo riguarda luso della risonanza magnetica per diffusione in campo oncologico , mettendo in evidenza i pro ed i contro della tecnica in questo campo , talvolta dif cile e con signi cati complicati ; tuttavia sono anche discusse le possibili applicazioni al di fuori di questo campo di ricerca . gli ultimi due interessanti capitoli , in cui vengono discusse le valutazioni dellimaging multifunzionale in risonanza magnetica ( rm ) , si rivolgono al futuro ed anche alla potenzialit della dw - mri utilizzata come biomarcatore per immagine nei trial clinici . le gure sono di grande effetto ; le tavole sono accurate nella loro presentazione e ricche di dettagli sui parametri per ottenere le immagini o sui risultati di dati precedenti raccolti dalla letteratura ( le voci bibliogra che sono aggiornate al 2009 )  . qualora il lettore non sia a conoscenza dei come e dei perch della tecnica , avr dif colt nella valutazione della massa di informazioni contenute nel testo . 
personalmente ho trovato dif cile la digestione dei capitoli riguardanti i principi e le basi della tecnica , lottando anche con gli acronimi usati in abbondanza . 500 radiol med ( 2011 ) 116 : 499500 apart from my personal problems , the book represents a breakthrough in this domain of radiology and will help the reader to better understand and focus on all sorts of problems one might encounter using this technique which is fully open to future development and should become a very important adjuvant in patients care . 
buzzi , via castelvetro 32 , milan , italy 2dipartimento di scienze radiologiche , oncologiche e anatomo - patologiche , sapienza universit di roma , rome , italy 4unit dipartimentale medicina fetale , asl bari , bari , italy correspondence to : f . 
this paper is the result of the work of the fetal magnetic resonance ( fmr ) study group of the italian society of medical radiology ( sirm ) in cooperation with the study group of the italian society of ultrasound in obstetrics and gynaecology ( sieog )  . 
as fmri is undergoing continuous development , and its indications and role are also likely to change over time , the fetal magnetic resonance study group is in agreement with the other scienti c bodies involved in the drafting of this document to propose subsequent modi cations to it when new clinical and scienti c evidence suggest the need . keywords fetal mri brain development fetal ultrasound fetal development riassunto lobiettivo di questo lavoro quello di dare delle risposte , sulla base delle evidenze clinico - scienti che , a quali siano ad oggi le reali indicazioni allo studio con risonanza magnetica fetale ( rmf ) e dove questo debba collocarsi nei protocolli diagnostici prenatali . 
questo lavoro il risultato della attivit del gruppo di studio di risonanza magnetica fetale della societ italiana di radiologia medica ( sirm ) svolto in collaborazione con gruppo di studio della societ italiana di ecogra a ostetrico - ginecologico ( sieog ) e rivisto ed approvato anche dal direttivo della associazione italiana di neuroradiologia ( ainr )  . 
in quanto tecnica in continua evoluzione , anche indicazioni e ruolo della rmf sono destinati verosimilmente a cambiare nel tempo : il gruppo di studio in rmf si propone , in accordo con le altre societ coinvolte , di indicare successive modi che a questo documento quando nuove evidenze clinico - scienti che lo renderanno necessario . parole chiave rm fetale sviluppo cerebrale ecogra a fetale sviluppo fetale introduction introduzione this paper is the nal document drafted by the fetal magnetic resonance ( fmr ) study group of the italian society of medical radiology ( sirm ) in cooperation with the study group of the italian society of ultrasound lavoro rappresenta il documento conclusivo questo elaborato dal gruppo di studio in risonanza magnetica fetale ( rmf ) della societ italiana di radiologia medica ( sirm ) , articolato in collaborazione col gruppo di studio 338 radiol med ( 2011 ) 116 : 337350 in obstetrics and gynaecology ( sieog )  . 
the aim of the document is to provide answers , on the basis of clinical and scienti c evidence , regarding the real indications for fmri today and where it ts in prenatal diagnostic protocols . 
as cooperation between radiologists , neuroradiologists and gynaecologists is the mandatory premise for identifying the correct collocation of fmri in modern fetal imaging , this document was jointly drafted and approved by the executive boards of sirm and sieog and the executive board of the italian association of neuroradiology ( ainr )  . as the technique is undergoing continuous development and the indications and the role of fmri are also likely to change over time , the fmr study group is in agreement with the other scienti c bodies involved in the drafting of this document to revise it when new clinical and scienti c evidence suggests the need . the document is divided into three parts : indications for fmri , study techniques with the various study protocols and safety issues . 
in the rst part , as a premise to the indications for the study , the viewpoint of the three associations involved is emphasised , that is , that fmri is a targeted examination possibly performed following a level ii ultrasound ( us ) assessment . 
instead , it should be considered a study performed with the precise aim of clarifying a diagnostic doubt or query on the basis of the consolidated indications in the literature . 
for the most part , these regard the study of the central nervous system ( cns ) , although with increasing knowledge , other organs and systems can currently be effectively studied with fmri , at all times based on speci c indications . in the section dealing with the study techniques and the various study protocols , the active role of the radiologist is emphasised . 
this role begins with the clinical history of the patient ; includes correctly positioning the patient ; performing the study itself , which must be adapted to each individual and each particular diagnostic query ; and is completed with the reporting phase . 
this latter phase requires not only a qualitative evaluation but also accurate quantitative morphometric measurements , with particular regard to studying the fetal head , which may be compared with morphometric data from the us assessment . the third section presents the most recent evidence regarding the issue of safety . 
to date , however , there are no data available that suggest potential deleterious effects to the fetus or the embryo caused by radiofrequency magnetic elds at the intensity used for clinical purposes . 
lobiettivo del documento quello di dare delle risposte , sulla base delle evidenze clinico - scienti che , a quali siano ad oggi le reali indicazioni allo studio rmf e dove questo debba collocarsi nei protocolli diagnostici prenatali . 
poich la collaborazione fra radiologi , neuroradiologi e ginecologi la premessa necessaria alla identi cazione di una corretta collocazione della rmf nel moderno imaging fetale questo lavoro stato condiviso e approvato oltre che dal direttivo della sirm e della sieog anche dal direttivo della associazione italiana di neuroradiologia ( ainr )  . in quanto tecnica in continua evoluzione , anche indicazioni e ruolo della rmf sono destinati verosimilmente a cambiare nel tempo : il gruppo di studio in rmf si propone , in accordo con le altre societ coinvolte , di indicare successive modi che a questo documento quando nuove evidenze clinico - scienti che lo renderanno necessario . il documento diviso in tre parti : le indicazioni allo studio rmf , le modalit di esecuzione con i diversi protocolli di studio e un capitolo nale sul tema della sicurezza . 
 nella prima parte , come premessa alle indicazioni allo studio , si ribadito il punto di vista delle tre associazioni coinvolte che considera la rmf un esame mirato a seguito di una valutazione ecogra ca possibilmente di 2 livello . 
 non attualmente pensabile , se non in casi selezionati , considerare lo studio del feto come un esame total - body o tanto meno di screening , quanto piuttosto uno studio svolto con il preciso scopo di approfondire un dubbio o un quesito diagnostico sulla base delle indicazioni consolidate dalla letteratura . 
queste sono di gran lunga prevalenti nello studio del sistema nervoso centrale , tuttavia con laumentare delle conoscenze anche altri organi e apparati possono oggi essere utilmente valutati con rmf sempre sulla base di indicazioni speci che . 
 nella parte relativa alla modalit di esecuzione dellesame e ai relativi protocolli di studio , si voluto sottolineare il ruolo attivo del medico radiologo dalla fase di anamnesi a quella del posizionamento della paziente , alla esecuzione stessa dello studio che , a partire dalle sequenze di base , deve essere adattato ad ogni singola paziente e ad ogni particolare quesito , per arrivare alla fase di refertazione che necessita non solo di una valutazione qualitativa , ma anche di una accurata valutazione morfometrica quantitativa , in particolare per il distretto cranio - encefalico , che possa essere di confronto ai dati morfometrici dello studio ecogra co . in ne nellultima parte si riporta la documentazione pi recente in tema di sicurezza . 
non esistono tuttavia ad oggi dati che suggeriscano potenziali effetti dannosi di campi magnetici e radiofrequenze alle intensit utilizzate a scopo clinico sia sul feto che sullemradiol med ( 2011 ) 116 : 337350 basis of this consideration , the american college of radiology ( acr ) recently eliminated all distinctions between the rst and subsequent trimesters . 
sulla base di questa considerazione lamerican college of radiology ( acr ) ha recentemente eliminato ogni differenza fra il primo trimestre ed i successivi , vengono tuttavia mantenute delle misure di prudenza e cautela riassumibili nella valutazione attenta dei costi / bene ci dellesame sul feto e della sua necessit ed inderogabilit . indications for the fmri study when to perform fmri performing the examination is recommended from 19 weeks of gestation ( wg ) onwards with the best technology currently available , obtaining suf cient spatial and contrast resolution to provide diagnostic or at any rate additional information with respect to state - of - the - art us is not considered possible before 19 wg . 
for example , at 19 wg , the hemispheres of the brain have an anteriorposterior diameter of around 50 mtherefore , with a eld of view ( fov ) that on average is between 280 and 340 mm , the effect of the poor spatial resolution becomes extremely critical , and some structures , such as the corpus callosum , may be dif cult to evaluate . 
a titolo esempli cativo , a 19 settimane gli emisferi cerebrali hanno un diametro ant - post di circa 50 mm ; pertanto , con un campo di vista che si colloca mediamente fra i 280 ed i 340 mm leffetto della scarsa risoluzione spaziale diventa assai critico ed alcune strutture , come ad esempio il corpo calloso , rischiano di essere dif cilmente valutabili . 
inoltre va considerato che le tabelle di normalit oggi disponibili in letteratura partono solo dalla 20a sg [ 1 , 2 ] e lesperienza clinica al di sotto di questa et ad oggi molto ridotta . 
non indicato eseguire una rmf per veri care dubbi insorti sulla sola ecogra a di screening effettuata a 1921 settimane di gestazione ( linee guida sieog ) [ 3 ]  . 
la rmf dovrebbe quindi essere considerata una tecnica di iii livello il cui quesito clinico deve essere giusti cato da un approfondimento ecogra co di ii livello eseguito quindi da operatori esperti ( possibilmente in centri di riferimento ) [ 49 ]  . cosa valutare in un esame rmf the fmri study must be targeted , and in general should not be a total - body assessment , except in a minority of speci c cases and only when formulated and agreed upon by the diagnostic team . on the basis of its collocation in the diagnostic pathway , fmri should be targeted at a certain region , which should also be identi ed in the radiological report . 
based on current knowledge , studies on anatomical organs - systemsareas for which there is suf cient agreement in the literature regarding the diagnostic utility of fmri appear to be justied . 
in this setting , two regions can be identi ed : the head and neck , obviously including the brain [ 1013 ] ; and the lo studio rmf deve essere mirato ed in generale non pu assumere carattere di valutazione total - body , tranne che in una minoranza di casi speci ci e preventivamente impostati e concordati con lquipe diagnostica . 
in base alle attuali conoscenze appaiono giusti cati studi su organi - apparati - aree anatomiche per le quali esiste suf ciente accordo in letteratura riguardo lutilit diagnostica della rmf . 
in tale ottica possiamo oggi identi care 2 distretti : quello dellarea testa - collo compreso ovviamente lencefalo [ 1013 ] , e quello toraco340 radiol med ( 2011 ) 116 : 337350 chest and abdomen [ 1417 ]  . 
given the limited evidence in the literature and the dif culty in performing the examination , it is currently not possible to include musculoskeletal structures and in particular the developing limbs and osteocartilaginous components of the vertebral column among the anatomical areas that can be studied with fmri . as fmri examination is by de nition targeted at a certain region , the general rule applying to all radiological examinations clearly holds : if a clear pathological nding belonging to another anatomical region falls within the fov as an incidental nding of the targeted study , it cannot be overlooked . 
this is , of course , provided that the moreor - less evident and unequivocal nature of the incidental nding can be reasonably evaluated on the basis of the quality of the images that were not speci cally targeted and the region of the nding . 
this consideration is also valid for the placenta and the amniotic sac when clear abnormalities not noted on the us scans are identi ed . where to perform a fmri study the fmri study should be performed in centres speci cally equipped for that purpose . the discussion so far suggests that it is dif cult to imagine placing the fmri study on the same level as any other mri examination performed as an outpatient procedure . 
on the one hand , there needs to be direct contact and ongoing cooperation with the referring clinicians / obstetricians , particularly with the team of experts dedicated to studying fetal abnormalities . 
per le scarse evidenze in letteratura e per la dif colt di esecuzione , non sembra oggi possibile includere nei distretti anatomici studiabili anche le strutture muscolo - scheletriche in particolare gli arti e la componente osteo - cartilaginea del rachide in formazione . posto che lesame rmf quindi per de nizione mirato ad un distretto , vale ovviamente la regola generale attuata su qualsiasi esame radiologico : se un chiaro reperto patologico , appartenente ad un altro distretto , ricade nel campo di vista , come evento collaterale dello studio mirato , esso non pu essere trascurato . 
fermo restando che il carattere pi o meno eclatante ed inequivocabile del reperto collaterale possa essere ragionevolmente valutato in base alla qualit delle immagini ( non speci catamente mirate ) ed al distretto sede del reperto . 
collo le patologie di pi frequente riscontro in epoca prenatale sono il linfangioma cistico ed il teratoma [ 40 , 41 ]  . la rmf pu : valutare leventuale estensione allo stretto toracico superiore ; identi care , quando presente , la compressione e / o dislocazione delle vie aeree , ben identi cabili per il loro contenuto uido che le rende iperintense nelle immagini pesate in t2 ; studiare i rapporti della massa con il fascio vascolonervoso del collo . tali informazioni risultano , infatti , dirimenti per decidere la procedura terapeutica pi idonea ( ex utero intrapartum treatment , exit ; resezione della massa )  . 
torace la rmf pu aiutare nella identi cazione di : masse intratoraciche : malattia adenomatoide cistica congenita ( ccam ) , sequestro broncopolmonare , cisti broncogene , sindromi da ostruzione delle vie respiratorie superiori con imaging positivo per segni indiretti ( chaos )  . 
lostruzione in assenza di questi reperti pu non essere direttamente riscontrabile [ 4244 ] ; ernia diaframmatica ( cdh ) [ 45 ] ; 342 radiol med ( 2011 ) 116 : 337350 whatever the intrathoracic disease , fmri provides information not only regarding location , size and morphology of the mass but also regarding the secondary effects it produces , such as developmental abnormalities of the residual and contralateral lung , mediastinal shift , nonimmune hydrops fetalis due to compression of the inferior vena cava and the heart [ 47 , 48 ] and polyhydramnios due to compression of the oesophagus and reduced swallowing of amniotic uid , as well as any associated extrapulmonary conditions . with regard to cdh , fmri should provide the following information : location ( right , left ) ; herniated organs ( bowel ; particularly de ne liver - up and liver - down ) ; lung volume measurements of the residual and contralateral lung and the indices of fetal lung maturity ; signal intensity analysis as a possible expression of lung maturity ( this still requires further validation ) ; possible mediastinal shift ; polyhydramnios and hydrops fetalis ; associated conditions . fmri may be indicated when , after a us study and counselling , the parent ( s ) decides to consider the option of an in - utero procedure . 
abdomen fmri can : identify the position of the visceral organs within the abdomen to verify the cardiacvisceral situs ( situs solitus or situs inversus )  . identify defects of the abdominal wall with herniation of the abdominal organs , lined or otherwise by the peritoneal folds . 
in these cases fmri is able to identify herniated organs , differentiate omphalocele from gastroschisis , measure the abdominal hernia and measure the ratio between the herniated mass and the abdomen ( to date , there is no evidence of greater accuracy than us , and the indication for fmri may be for surgical planning and de ning the manner of birth rather than for diagnostic purposes per se ) [ 4951 ]  . visualise intra - abdominal masses . 
 per quanto concerne la cdh , la rmf deve fornire le seguenti informazioni : localizzazione ( destra , sinistra ) ; organi erniati ( intestino e particolarmente de nire liverup e liver down ) ; volumetria polmonare del polmone residuo e del controlaterale e indici di maturazione polmonare ; analisi dellintensit di segnale possibile espressione della maturazione polmonare ( tale dato necessita ancora di ulteriore validazione ) ; eventuale shift mediastinico ; polidramnios e idrope ; patologie associate . si ritiene che vi sia indicazione allesecuzione della rmf , qualora la coppia , dopo valutazione ecogra ca e counselling , decida di prendere in considerazione la opzione di intervento in utero . 
for example , in differential diagnosis queries for treatment planning purposes , signal hyperintensity in t1 may orient the diagnosis towards a meconium cyst or an enterogenic cyst [ 5255 ]  . evaluate size and signal intensity of parenchymal organs , such as the liver and spleen , which in the event of metabolic or haematologic disorders may be abnormal ( iron overload , haematological diseases with an increase in haematopoiesis )  . when studying the gastrointestinal tract , indications for fmri do not include : identifying the oesophageal atresia . 
the presence of a stomach distended by uid does not rule out the presence of oesophageal atresia given the possibility of tracheooesophageal stulas distal to the obstruction . atresia of the duodenal tract . 
urogenital system the information provided by fmri is important for studying urogenital malformations associated with oligohydramnios or anhydramnios conditions that make the us study technically dif cult [ 5664 ]  . 
 fmri offers a good depiction of : autosomal recessive polycystic kidneys ; multicystic kidneys , renal agenesis , pelvicalyceal dilatations and renal masses ; hydroureteronephrosis ( evaluation of the degree and segments involved ; study of the renal parenchyma possibly with functional index ) ; posterior urethral valves ( puv ) ( evaluation of the degree of hydroureteronephrosis and the renal parenchyma ; renal dysplasia is often associated with puvs ) ; neurogenic bladder with associated conditions ( myelomeningocele ) ; enlarged bladder ( prune - belly syndrome , with study of the abdominal wall , microcolon ) ; renal and adrenal masses ; pathological conditions of the adnexa ; renal pelvic masses . indications for studying the placenta fmri provides a good evaluation of placental insertion ; la visualizzazione delle masse endoaddominali : in tali condizioni la rmf aiuta a determinarne la corretta origine , estensione e volumetria , la tipologia ( cistica o solida ) , lo spessore delle pareti , la presenza di setti o vegetazioni , oltre a caratterizzarne il contenuto distinguendo grazie alla sua multiparametricit il uido sieroso , la componente sebacea , emorragica o proteinacea ( diagnosi differenziale cisti enterogene , linfangioma )  . 
anche se lanalisi della parete , il ne dettaglio dei setti migliore con lecogra a per la sua migliore risoluzione spaziale , importante pu essere lanalisi del segnale sia t2 che t1 . 
per esempio uniperintensit di segnale in t1 pu indirizzare verso lipotesi di cisti da meconio o cisti enterogene in quesiti di diagnosi differenziale ai ni del planning terapeutico [ 5255 ] ; la valutazione delle dimensioni e dellintensit di segnale degli organi parenchimali come il fegato e la milza che nel caso di patologie metaboliche o ematologiche possono risultare alterate ( emocromatosi , malattie ematologiche con incremento dellematopoiesi )  . non costituiscono indicazioni alla rmf : lindividuazione delle atresie esofagee . 
la presenza di uno stomaco disteso da uido non pu escludere la presenza di atresia esofagea per la possibilit di stole tracheo - esofagee a valle dellostruzione ; le atresie del tratto duodenale . 
apparato genitourinario i dati forniti dalla rmf sono importanti per lo studio delle malformazioni del tratto urinario associate ad oligoo anidramnios , condizioni queste che rendono tecnicamente dif cile lo studio ecogra co [ 5664 ]  . sono ben visualizzabili mediante rmf : reni policistici da patologia autosomica recessiva ; reni multicistici , agenesie renali , dilatazioni pelvicaliceali e masse renali ; valutazione delle idroureteronefrosi ( entit , segmenti interessati , studio del parenchima renale con possibile indice funzionale ) ; valvole uretrali posteriori ( vup ; valutazione del grado di idroureteronefrosi , del parenchima renale : spesso nelle vup i reni risultano displasici ) ; vescica neurologica con patologie associate ( mielomeningocele ) ; megavescica ( sindrome di prune belly con studio della parete addominale , microcolon ) ; valutazione masse renali e surrenaliche ; patologia di pertinenza annessiale ; patologia espansiva pelvica . 344 radiol med ( 2011 ) 116 : 337350 extension ; relations with the myometrium ; relations with the internal ori ce of the uterus ; structure , which appears homogeneous between the 20th and 30th wg and becomes relatively inhomogeneous in the third trimester due to the presence of brous bands ; placental infarcts ; and pseudocystic spaces . 
in this sense , it should be noted that as with transvaginal or transanal us , fmri is unable to provide conclusive data , only high suspicion [ 6568 ]  . examination technique and study protocols indicazioni allo studio della placenta la rmf ben valuta linserzione , lestensione , il rapporto con il miometrio , il rapporto con lori zio uterino interno ( oui ) , la struttura , che risulta omogenea tra la 20a e la 30a settimana , diventando relativamente disomogenea nellultimo trimestre per la presenza di strie brotiche , aree infartuali e di lacune pseudocistiche . 
a questo proposito , si sottolinea che anche la rmf , cosi come lecogra a transvaginale ( tv ) o transaddominale ( ta ) , non permette di raggiungere dati conclusivi al riguardo ma solo di elevato sospetto [ 6568 ]  . magnet modalit di esecuzione , protocolli di studio the recommended magnetic eld for obtaining a suf cient signal - to - noise ratio ( snr ) is 1.5 t achieved with traditional superconducting magnets . 
a suf cient snr in terms of vertical geometry can also be obtained with a 1 - t magnet , whereas the use of lower - intensity magnetic elds is not recommended . 
the use of magnetic elds with intensity higher than 1.5 t is currently not considered a routine approach , as this still needs to be validated by speci c research protocols . coils various types of coils can be used in relation to gestational age , size of the amniotic sac and the uterus . 
the highest performing coils are undoubtedly multichannel phasedarray or cardiac surface coils , which enable greater signal in the longitudinal direction , although still limited to around 5060 calternatively , spine coils can be used to study the fetal body , which allow a greater fov for later gestational ages . 
 the following is required before performing an fmri examination : signed informed consent from the expectant mother after consultation with the physician aimed at verifying the absence of the usual contraindications ( pacemaker , metal clips ) , as well as explaining the role of mri in evaluating malformations and informing the patient of speci c contraindications ( safety )  . recording clinical data / history and reviewing the us images that prompted the suspicion of disease . 
the consultation is also aimed at acquiring the active cooperation from the patient in the sequences that require speci c breathing instructions , and the manner in which the fmri examination will be performed is explained ( duration , safety , diagnostic value )  . when possible , the examination is ideally performed magnete bobine il campo magnetico consigliato per ottenere un suf ciente rapporto segnale / rumore quello di 1 , 5 t ottenuto con magneti tradizionali superconduttori . 
un suf ciente rapporto segnale / rumore in ragione della geometria verticale pu essere anche ottenuto con magnete aperto a 1 t , mentre si sconsiglia luso di intensit di campo inferiori . 
 luso di intensit di campo maggiori di 1 , 5 t non oggi considerabile come approccio di routine poich deve essere ancora validato in speci ci protocolli di ricerca . possono essere utilizzate diverse tipologie di bobine anche in relazione allepoca gestazionale , alle dimensioni del sacco gestazionale e dellutero : le pi performanti sono certamente le bobine di super cie multicanale di tipo phasedarray o cardio che permettono di avere maggior segnale per unestensione longitudinale tuttavia limitata intorno ai 5060 cm ; si possono inoltre utilizzare bobine di tipo spine per lo studio del corpo che permettono un maggiore campo di vista per le epoche gestazionali pi tardive . prima di eseguire lesame rmf necessaria : la raccolta del consenso informato dalla gestante da parte del medico dopo un colloquio esplicativo volto allaccertamento dellassenza delle usuali controindicazioni alla rm ( pacemaker cardiaco , clips metalliche ) , del ruolo della rm nella valutazione della patologie malformative , eventuali controindicazioni speci che ( safety ) ; la raccolta dei dati clinico - anamnestici con presa visione della documentazione ecogra ca da cui stato generato il sospetto patologico ; durante tale colloquio viene inoltre stabilito un primo approccio con la paziente volto a conquistarne la collaborazione attiva nelle sequenze con necessit di respirazione e vengono spiegate le modalit di esecuzione della rmf ( durata , safety , valore diagnostico )  . 
alternatively , if a us study is possible immediately prior to the fmri examination to determine the fetal sleepwake phase , it may be bene cial to wait for the sleep phase , as the fetal sleepwake cycles are 30 min in length . the patient is placed in a comfortable position on the table , usually in the supine position , or in cases where this position is not tolerated ( vena cava compression , polyhydramnios , multiple pregnancies ) , then in the left lateral position is preferred . 
in general , no sedatives are used for mother or fetus , and no contrast agent is administered ( see safety issues below )  . the study protocol involves the acquisition of various sequences , some of which are indispensable , whereas others are optionally added according to the clinical query . 
the main fmri sequences are ultrafast t2 - weighted sequences , which enable excellent evaluation of the fetal anatomy thanks to high - contrast resolution and are therefore a good compromise between contrast and spatial resolution . 
the examination technique includes : localiser sequence : fast sequence ( generally a t2 - weighted single - shot fast turbo spin echo ) in the maternal coronal plane to identify fetal position with respect to the mother ( presentation ) and determine the relative position of the fetal head , spine and stomach , and to localise the placenta ( anterior / posterior )  . the standard sequence for an fmri examination is a t2 - weighted fast or turbo spin - echo sequence that in a single tr ( single shot ) acquires a single slice . 
commercially , this sequence is known as half - fourier acquisition single - shot turbo spin echo ( haste ) or single - shot fast spin echo ( ssfse ) or single - shot turbo spin echo ( sstse )  . 
the slice needs to be suf ciently thin ( 34 mm ) , with axial , sagittal and coronal orientation orthogonal to the organ / area of interest to provide detailed evaluation of the fetal anatomy . the optimal compromise between spatial resolution , contrast resolution and snr of these sequences provides excellent visualisation of the fetal anatomy during all stages of pregnancy , and in particular , displays the static uids and structures composed prevalently of uid as hyperintense structures . 
this therefore enables the study of the fetal brain , uid - containing cavities ( nasal and oral cavities , pharynx , trachea , stomach and proximal intestine , urinary tract , gall bladder ) , lungs , placenta and amniotic uid . 
 quando possibile sarebbe preferibile eseguire lesame al mattino dopo un digiuno di almeno 4 ore , poich stato dimostrato che lipoglicemia riduce i movimenti fetali ; alternativamente se possibile eseguire un esame ecograco prima dellesecuzione della rmf , volto a veri care la fase sonno o veglia del feto , si pu aspettare la fase del sonno considerando che il regolare ciclo sonno - veglia fetale prevede unalternanza di tali fasi ogni 30 minuti . la donna viene posta in una posizione confortevole sul lettino , generalmente in decubito supino o , nei casi in cui tale posizione non venga tollerata ( compressione cavale , polidramnios , gravidanze multiple ) , in decubito laterale sinistro e viene fatta riposare alcuni istanti in tale posizione per ridurre il movimento spontaneo fetale . 
 le principali sequenze utilizzate in rmf sono sequenze t2 pesate di tipo veloce - ultraveloce che consentono unottima valutazione dellanatomia fetale grazie allelevata risoluzione di contrasto , rappresentano quindi un buon compromesso tra risoluzione di contrasto e spaziale . 
lo strato deve essere suf cientemente sottile ( 34 mm ) con orientamento multiplanare assiale , sagittale e coronale ortogonale allorgano / distretto di interesse per la valutazione di dettaglio dellanatomia fetale . lottimo compromesso tra la risoluzione spaziale , di contrasto e il rapporto segnale - rumore ( snr ) di queste sequenze oltre alla loro rapidit di esecuzione permettono infatti di visualizzare lanatomia fetale in maniera eccellente durante tutte le fasi della gravidanza ed in particolare di evidenziare i uidi statici e le strutture a prevalente composizione uida come strutture iperintense consentendo 346 radiol med ( 2011 ) 116 : 337350 additional sequences include : gradient echo ( gre ) with steady - state free precession ( ssfp ) for evaluating the cardiac region and great vessels . 
these sequences in fact present intermediate contrast between t1 and t2 , use a very short tr ( < 3 ms ) and are thus not in uenced by motion , and they are able to visualise uid in motion as high - signal - intensity structures ; ssfp cine with both radial and cartesian [ 2d fourier transformation ( ft ) ] k - space sampling . 
these sequences are able to identify the heart and great vessels and can be used as real - time sequences to evaluate certain fetal movements . thick - slab cholangiography ( 4080 mm slice thickness ) heavily weighted in t2 to highlight uidlled structures by freezing an image of all uidlled structures in the entire fetus . t1 - weighted fast spoiled gre single - shot 2d or 3d with and without fat signal saturation , acquired during breathholding . diffusion - weighted ( dw ) echo - planar imaging ( epi ) , with the application of gradients oriented in the three planes in space ( b0 , b200 and b700 s / mm2 ) , for studying the kidneys , lungs , brain and placenta . 
these sequences , which have an acquisition time of around 20 s , provide information on the microscopic motion of the free and bonded water molecules in the biological tissues and enable the study of cerebral , pulmonary and renal maturity . duration of an fmr study depends on a number of factors : number of fetuses , complexity of the malformations present , repositioning of the coils and the amount of fetal motion that may require reacquisition of some sequences in the correct anatomical plane . 
the study usually takes between 20 and 45 min , with a minimum of 15 mit is nonetheless recommended to limit the examination time to avoid maternal heat stress and speci c absorption rate ( sar ) deposition in the fetus . 
using more than one sequence for the study and always including a t1 - weighted sequence in the protocol , even if of limited diagnostic quality , is recommended . reporting the fmri report should include : maternal data ; gestational age as determined from clinical data and us ndings ; type of system , sequences and scan planes used ; clinical indication and region ( s ) targeted by the study ; possibly , measurements of the anatomical structures for each region studied . 
the following measurements are lo studio quindi dellencefalo fetale , delle cavit contenenti uidi ( cavit nasali ed orale , faringe , trachea , stomaco e intestino prossimale , sistema urinario , colecisti ) , dei polmoni , della placenta e del liquido amniotico ( la )  . 
 ulteriori sequenze possono essere : sequenze gradient echo ( gre ) con tecnica steady state free precession ( ssfp ) per la valutazione del distretto cardiaco e dei grossi vasi . 
queste sequenze , infatti , presentano un contrasto intermedio t1 e t2 , utilizzano un tr ultrabreve ( < 3 ms ) non risultando in uenzate dal movimento e permettono di evidenziare come strutture ad elevata intensit di segnale i uidi in movimento ; sequenze cine - rm di tipo ssfp con tecnica di campionamento del k - spazio sia radiale che cartesiana ( 2dft )  . 
 tali sequenze permettono di individuare il cuore e i grossi vasi : possono essere utilizzate come sequenze real time per valutare alcuni movimenti fetali ; sequenze colangiogra che thick slab ( spessore di strato 4080 mm ) altamente pesate in t2 , per mettere in evidenza strutture ripiene di liquido congelando unimmagine delle strutture uide in tutto il feto ; sequenze fast spoiled gre single shot 2d o 3d t1 pesate , con e senza saturazione del segnale del tessuto adiposo , acquisite in apnea respiratoria ; sequenze echo planar imaging ( epi ) pesate in diffusione ( dwi ) , con applicazione di gradienti orientati secondo i 3 piani dello spazio ( b0 , b200 e b700 s / mm2 ) per lo studio dei distretti renale , polmonare , encefalico e della placenta . 
tali sequenze , con tempi di acquisizione di circa 20 secondi , forniscono informazioni sul movimento microscopico delle molecole dacqua , libera e legata , nei tessuti biologici e permettono lo studio della maturazione cerebrale , polmonare e renale . 
 la durata di uno studio di rmf dipende da vari fattori : numero di feti , complessit delle malformazioni eventualmente presenti , riposizionamento della bobina ed entit dei movimenti fetali che possono richiedere la riacquisizione di alcune sequenze sul giusto piano anatomico . 
abitualmente uno studio di rmf richiede dai 20 ai 45 minuti , con un minimo di 15 minuti ; risulta necessario comunque cercare di contenere i tempi dellesame per evitare leccessivo riscaldamento della gestante ed il potenziale trasferimento di energia al feto ( sar )  . 
 consigliabile utilizzare pi di una sequenza per lo studio rmf e includere sempre nel protocollo rmf una sequenza t1 pesata anche se di scarsa qualit diagnostica . refertazione la compilazione del referto prevede : indicazione dei dati materni ; indicazione dellet gestazionale come si evince dai dati anamnestici ed ecogra ci ; tipologia di apparecchiatura utilizzata , sequenze e piani di scansione impiegati ; radiol med ( 2011 ) 116 : 337350 recommended : bone and cerebral biparietal diameter , occipitofrontal diameter , transverse cerebellar diameter , anteroposterior and longitudinal diameter of the vermiform process of the cerebellum , longitudinal diameter of the corpus callosum , atrial diameter of the cerebral ventricles . 
 in addition , an evaluation of the main cerebral ssures is recommended , which is an important parameter for information on brain development and for verifying that the development matches gestational age . 
 in the fetal total - body study , lung - volume measurements are recommended in space - occupying thoracic masses and cdh , whereas kidney measurements are advisable especially in urinary tract disease . 
 is also advised : ( a ) detailed description of the disease in question , with an analysis of the malformation or the expansile lesion being studied ( location , volume , signal analysis , relations with adjacent structures , inability to nd anatomical structures , associated signs ) ; ( b ) conclusions ; where possible these should indicate possible diagnoses in order of probability and follow - up where required . safety limitations the most recent and important documents published on safety available today are the italian national institute for occupational safety and prevention ( ispesl ) document posted on the institutes web site on 6 february 2009 and published in radiologo in april , 2008 , with regard to italy ; and the acr guidance for safe mr practices : 2007 document published in the american journal of roentgenology ( ajr 2007 , 188 : 1 - 27 ) with regard to the united states . 
 02 / 09 / 1991 , which reads : although there is no evidence to suggest the embryo is sensitive to magnetic elds and radiofrequency elds at the strength used in current mr systems for diagnostic purposes ( 1991 ) , caution is warranted in exposing women in the rst trimester of pregnancy , except in cases of real and pressing need assessed by the physician and ordered under her / his direct responsibility . 
the patient shall be informed about the possible risks of the examination beforehand . whereas the need for informed consent has been established , there remains the practical problem of informing the patient of the hypothetical risks of the examination , which to date have neither been con rmed by the literature nor precisely de ned . 
in the chapter titled patient pregnancies , the acr document states : present data have not indicazione clinica e a quale distretto / i rivolto lesame ; per ogni distretto interessato consigliabile la misurazione delle strutture anatomiche . 
si consiglia di misurare il diametro biparietale ( dbp ) osseo e cerebrale , il diametro fronto - occipitale ( dfo ) , il diametro trasverso cerebellare , il diametro antero - posteriore e longitudinale del verme cerebellare , il diametro longitudinale del corpo calloso , il diametro atriale dei ventricoli cerebrali . 
 nello studio del body fetale si raccomanda di misurare la volumetria polmonare nelle patologie toraciche occupanti spazio e nelle cdh ; la biometria renale consigliabile , in particolare in tutte le patologie del tratto urogenitale . 
si consiglia anche di inserire nel referto : ( a ) la descrizione dettagliata della patologia in questione con lanalisi della malformazione o della patologia espansiva in esame ( localizzazione , volumetria , analisi del segnale , rapporti con le strutture contigue , mancato reperimento di strutture anatomiche , segni associati ) ; ( b ) le conclusioni : ove sia possibile indicare le ipotesi diagnostiche in ordine di probabilit ed eventuali controlli . sicurezza limitazioni allesecuzione dellesame i principali e pi recenti documenti sulla sicurezza oggi disponibili sono , per quanto riguarda litalia , il documento dellispesl pubblicato sul sito il 6 / 2 / 09 e ripreso nel numero 4 / 2008 del radiologo e , per quanto riguarda gli stati uniti , la acr guidance for safe mr practices : 2007 pubblicata su ajr 2007 ; 188 : 1 - 27 . 
02.09.1991 : sebbene non esistano evidenze che dimostrino una sensibilit dellembrione ai campi magnetici e ai campi a radiofrequenza di intensit e potenze utilizzate nella attuale strumentazione rm ad uso diagnostico ( 1991 ) , prudente escludere dallesposizione le donne nel primo trimestre di gravidanza , tranne nei casi di effettiva e improrogabile necessit , valutati dal medico , sotto la sua responsabilit . 
stabilita la necessit di un consenso informato , permane il problema pratico di informare su ipotetici rischi che ad oggi non sono n confermati dalla letteratura n de niti con precisione . 
il documento acr riporta invece al capitolo patient pregnancies : 348 radiol med ( 2011 ) 116 : 337350 conclusively documented any deleterious effects of mr imaging exposure on the developing fetus . 
all distinctions between the rst and later trimesters have therefore been eliminated , although caution has been maintained that may be summarised in terms of careful assessment of the costs and bene ts of fetal examination and the substantiated need and pressing nature [ 6976 ]  . present data have not conclusively documented any deleterious effects of mr imaging exposure on the developing fetus . 
 viene quindi abolita ogni differenza fra il primo trimestre ed i successivi , vengono tuttavia mantenute delle misure di prudenza e cautela riassumibili nella valutazione attenta dei costi / bene ci dellesame sul feto e della sua necessit ed inderogabilit [ 6976 ]  . limitations to the use of contrast agents limitazioni alluso del mezzo di contrasto animal studies conducted to date have shown no teratogenic effects of gadolinium - based contrast agents . 
however , there is no evidence of its harmlessness in humans , so its use is not recommended except in cases of absolute necessity , which as a rule are implicitly correlated with maternal health . 
the acr document takes a rather permissive line : mr contrast agents should not be routinely provided to pregnant patients , although thereafter it recalls the concept of riskbene t ratio for the patient . 
when contrast agent use is sporadically found in the literature or reported anecdotally in specialist referral centres , it is generally with reference to cases that nonetheless would have nished with a terminated pregnancy . 
in conclusion , the use of contrast agents should not currently be considered an additional tool for fmri , but rather , solely a special exception in select cases of established clinical need . acknowledgements the authors thank the entire executive board of the italian society of medical radiology , with special thanks to the president , alfredo siani , and francesco sardanelli , for the ongoing support provided to the study group . 
lastly , we thank the sieog study group in fetal imaging , especially vincenzo daddario and dario paladini . gli studi ad oggi compiuti in animali non hanno dimostrato effetti teratogeni dei mdc contenenti gadolinio . 
il documento dellacr di tono pi permissivo : mr contrast agents should not ruotinely provided to pregnant patients , ma nella sostanza richiama di seguito il concetto dei costi / bene ci per la paziente . 
quanto di sporadico e occasionale pu essere rintracciato in letteratura o nellaneddotica dei centri di riferimento di norma riferito a casi che comunque sarebbero andati incontro alla interruzione di gravidanza . 
in conclusione luso dei mezzi di contrasto non pu essere considerato oggi uno strumento aggiuntivo dellimaging rm del feto , ma unicamente una particolare eccezione in casi selezionati dalla necessit clinica . ringraziamenti gli autori desiderano ringraziare tutto il direttivo della societ italiana di radiologia medica ed in particolare il presidente dott . 
nieder springer - verlag , berlin heidelberg , 2010 isbn 978 - 3 - 540 - 92809 - 6 e - isbn 978 - 3 - 540 - 92810 - 2 published online : 7 march 2011 springer - verlag 2011 this book addresses tumour biology and how its role may in uence cancer treatment and related clinical strategies . 
 robust research and clinical results are presented by a team of professionals who , for many years , have been dedicated to the ght against cancer . the topic is fascinating yet very dif cult to follow if one doesnt have sound and up - to - date knowledge of the genetic , biological , and laboratory issues which are the bread and butter of the text and related discussions . my point is that often the reader may get lost among all the acronyms , shortened names , protein names , biological markers , genetic tricks , new terms such as genomic , epigenomics , etc . 
considering the enormous mass of information poured into the text the reader when necessary ( and this happens very often ) will have to struggle with a very concise and poor subject index . 
in another a medley of one lo scopo che si sono pre ssi gli autori di questo volume di studiare la biologia tumorale e come il suo ruolo possa in uenzare le strategie cliniche e terapeutiche nella lotta contro il cancro . 
in questa ottica il gruppo di esperti che da lunghi anni ha dedicato la propria attivit a questo scopo , presenta i risultati di una ricerca di laboratorio e sperimentale continua oltre che dal punto di vista clinico . largomento affascinante , anche se molto dif cile da seguire per un profano ed in assenza di una conoscenza approfondita ed aggiornata dei vari argomenti di genetica , biologia , laboratorio che sono alla base di tutti gli argomenti del testo e delle relative discussioni . dico questo perch spesso il lettore potrebbe avere la sensazione di perdersi tra tutti gli acronimi , termini abbreviati , nomi di proteine e di marcatori biologici , trucchi della genetica , nuovi termini come genomica , epigenomica , ecc . : tenuto poi conto dellenorme massa di informazioni presenti nel testo , in caso di necessit ( e capita spesso ) si scontrer con un indice analitico conciso , povero . se si riescono a superare tutti gli scogli di cui sopra o se si a conoscenza della materia , sar possibile trovare una notevole quantit di informazioni sulla biologia dei tumori e la tumorogenesi , langiogenesi , il ruolo giocato dal sistema immunitario , la proliferazione , la siologia e la ripopolazione cellulare , la diffusione delle metastasi e la capacit delle cellule di sfuggire a tutti i possibili strumenti terapeutici , anche se questi sono sotto forma di armi molecolari . sono ben descritti e discussi lo studio per immagini , la chemioterapia , la terapia molecolare mirata e le possibili strategie radioterapiche , presentandone i pro e contro , alla luce delle ricerche pi avanzate e dei conseguenti risultati clinici . al termine di ogni capitolo presente una bibliogra a ampia ed aggiornata ; la sua impostazione non omogenea : in un capitolo si trover la citazione dei nomi di tutti gli autori ; in un altro un solo nome seguito da et al . ; in un terzo tre nomi e poi et al . ; in un altro ancora un miscuglio 498 radiol med ( 2011 ) 116 : 497498 or more names plus et al.. 
dif cult to understand as well is the fact that each chapters author / s is / are keen to show how much research he / she / they have performed on the relevant matter . 
yet , arent 41 self - citations ( as in chapter 4 ) a bit redundant ? arriving at the end of the book one realizes that much has been achieved in the eld of cancer research and therapy , yet a lot more needs to be done : with every new discovery more problems arise , these need to be addressed , before victory in the eld may be achieved . di uno o pi nomi seguiti da et al.. 
cosa dif cile da capire anche il fatto che in ciascun capitolo lautore / i sia / siano desideroso / i di far conoscere quanto lavoro e ricerca egli / loro abbia / abbiano svolto sullargomento in questione . 
rossi1 1department of clinic sciences , section of radiological sciences , 2department of pathology and laboratory medicine , section of pathology , university of parma , via gramsci 14 , 43100 parma , italy 3department of radiology , santorsola - malpighi hospital , university of bologna , via massarenti 9 , 40138 bologna , italy correspondence to : m . 
de filippo , e - mail : massimo.defilippo@unipr.it received : 22 january 2010 / accepted : 4 april 2010 / published online : 10 february 2011 springer - verlag 2010 abstract purpose . 
lutilizzo di immagini tridimensionali mpr ha permesso in tutti i casi il prelievo di materiale biologico ( 10 di tipo citologico e 4 di tipo istologico ) e in 11 su 14 casi ( 78 , 5% ) il prelievo risultato adeguato per un giudizio diagnostico . 
le immagini mpr ottenute con tcmd hanno permesso di raggiungere lesioni retroperitoneali considerate un tempo difficilmente accessibili o inaccessibili con la guida delle immagini tcmd assiali . limpiego delle immagini mpr , inoltre , non incrementa il tempo di esecuzione del prelievo . 408 radiol med ( 2011 ) 116 : 407416 parole chiave tomografia coomputerizzata biopsia retroperitoneo tumori introduction introduzione radiologically guided percutaneous fine - needle aspiration biopsy ( fnab ) and core biopsy are safe , accurate and minimally invasive procedures allowing cytohistological diagnosis of retroperitoneal lesions for which imaging has failed to provide a definitive diagnosis [ 1 , 2 ]  . 
cytohistological investigation plays an important role in the primary diagnosis of neoplasms but also in staging , selecting tumour - specific therapy and follow - up [ 3 , 4 ]  . 
anatomical and histological complexity makes the retroperitoneum the centre of different primary neoplasms ( many malignant ) arising in the epithelium , soft tissues , nervous tissues and haemolymphopoietic system ; there is also frequent tumour infiltration from adjacent structures ( vertebral column , abdominal wall , chest ) and metastasis [ 57 ]  . 
nowadays , percutaneous fnab biopsy and core biopsy provide minimally invasive options that allow unnecessary surgical resection to be avoided ( as in the case of lymphoma and unresectable masses ) and the planning of alternative strategies , such as neoadjuvant chemotherapy and radiotherapy [ 3 , 9 ]  . 
 the imaging procedures commonly used to guide retroperitoneal biopsy are ultrasound ( us ) and multidetector - row spiral computed tomography ( mdct ) [ 10 , 11 ]  . selecting the more appropriate guidance technique is based on lesion site and size ( lymphadenopathy , free masses , masses or nodules in parenchymatous organs )  . 
these include the lack of ionising radiation , portability , decreased procedure time , real - time visualisation of the needle throughout the procedure and relatively low costs [ 12 , 13 ]  . 
mdct - guided biopsy offers some advantages , especially in small , low - density lesions and lesions located deep in the retroperitoneum that are difficult to reach with us guidance alone [ 3 ]  . 
nevertheless , it is not infrequent for some lesions to be obscured by skeletal segments , abdominal organs , vessels and nervous structures so that mdct - guided biopsy may also prove difficult , if not impossible . 
the introduction of mdct has , however , allowed multiplanar reconstructions ( mpr ) to be obtained that have been shown particularly useful in guiding percutaneous pulmonary biopsies [ 1417 ] the purpose of this paper is to illustrate the utility and advantages of mdct with mpr in guiding fnab and core lagoaspirazione con ago sottile ( fnab ) e lagobiopsia , eseguite con approccio percutaneo , sono procedure scarsamente invasive e di provata sicurezza ed efficacia , spesso impiegate nella definizione cito - istologica di lesioni retroperitoneali nelle quali limaging non stato in grado di fornire una diagnosi di certezza [ 1 , 2 ]  . 
lesame cito - istologico di fondamentale importanza nella diagnosi di natura , ma anche nella fase di stadiazione della neoplasia , nellimpostazione della strategia terapeutica e nel successivo follow - up [ 3 , 4 ]  . il retroperitoneo , per la sua complessit anatomica ed istologica , sede di svariate neoplasie primitive ( molte delle quali maligne ) epiteliali , delle parti molli , neuroectodermiche e del sistema emolinfopoietico ; frequenti sono anche le infiltrazioni tumorali da strutture adiacenti ( ad esempio , colonna vertebrale , pareti addominali , torace ) e le metastasi [ 57 ]  . 
le attuali metodiche agoaspirative e agobioptiche , oltre a ridurre a livelli minimi linvasivit della manovra di prelievo tissutale , consentono di evitare la resezione chirurgica quando non necessaria ( ad esempio , linfomi e masse non resecabili ) e dare cos spazio a strategie terapeutiche alternative tra cui la chemioterapia neoadiuvante e la radioterapia [ 3 , 9 ]  . 
 le tecniche radiologiche di guida allesecuzione dellagobiopsia retroperitoneale sono lecografia ( us ) e la tomografia computerizzata ( tc ) multidettore ( tcmd ) [ 10 , 11 ]  . 
la scelta della guida pi appropriata si basa su fattori strettamente legati alla sede e alle dimensioni della lesione in esame ( ad esempio , linfonodi , masse libere , masse e noduli nel contesto di organi parenchimatosi )  . 
i vantaggi in ambito interventistico dellus , rispetto alla tcmd , sono ben noti ; uno tra tutti la possibilit dellus di guidare in tempo reale la traiettoria dellago [ 12 , 13 ]  . 
la tcmd permette di visualizzare sia lesioni di piccole dimensioni e bassa densit che lesioni situate in zone difficili e profonde nel retroperitoneo , mal visualizzabili con lus [ 3 ]  . 
tuttavia , non di rado , alcune lesioni vengono coperte da segmenti scheletrici , organi addominali , vasi e strutture nervose , e pertanto anche con la guida tcmd , la procedura agobioptica pu risultare difficile e , talvolta , non eseguibile . 
lintroduzione della tcmd ha per consentito di ottenere ricostruzioni di immagini multiplanari ( mpr ) che si sono dimostrate particolarmente vantaggiose nella guida dellagobiopsia percutanea di lesioni polmonari [ 1417 ]  . 
 lo scopo del lavoro stato quello di indagare la possibile utilit dellimpiego delle immagini mpr nelle agobiopsie percutanee tcmd - guidate di lesioni retroperitoneali difficilmente raggiungibili con la sola osservazione delle immagini assiali . 
 radiol med ( 2011 ) 116 : 407416 biopsy of retroperitoneal lesions difficult to reach using axial mdct guidance alone . materiali e metodi pazienti materials and methods patients the study included inpatients and outpatients with us or ct evidence of small retroperitoneal lesions suspicious for malignancy . 
in these lesions , us guidance was of limited use in selecting the optimal path of the biopsy needle owing to the interposition of abdominal organs and vessels between the skin and the target lesion . 
also included in the study were patients in whom axial mdct failed to provide a safe needle trajectory because of the interposition of skeletal structures , vessels and abdominal organs between the skin and the target lesion . patients with small or large retroperitoneal lesions amenable to us or axial mdct guidance , patients with blood coagulation disorders and paediatric patients were excluded . 
 methods a six - slice helical mdct scanner ( somatom emotion 6 , siemens , erlangen , germany ) was used , with the simultaneous acquisition of six slices per rotation and the following technical parameters : 90 mas , 130 kv , 2 - mm collimation , 2.5 - mm slice thickness , 1 - mm reconstruction increment . 
all patients gave written informed consent after receiving verbal and written information regarding the benefits and risks of the procedure . patients were placed on the mdct table in the supine , prone or lateral position depending on the site of the target lesion and its distance from the abdominal wall . 
on the basis of these 3d images , a 2022 gauge chiba needle or an 18gauge microhistological needle ( biomol , hospital service , italy ) was inserted , taking care to select the shortest needle path without the interposition of large vessels , nerves , abdominal organs or bony structures . 
for microhistological biopsies , tissue samples were fixed in formalin . sono stati inclusi tutti i pazienti con lesioni retroperitoneali di piccole dimensioni sospette per malignit allo studio ecografico o mediante tc . 
in tali lesioni la guida ecografica alla biopsia non consentiva unottimale pianificazione della traiettoria dellago , per il sovrapporsi , tra la cute e la lesione bersaglio , di visceri e strutture vascolari . 
sono stati inclusi inoltre tutti i pazienti in cui le immagini tc assiali non consentivano una programmazione sicura della traiettoria dellago per il sovrapporsi , tra la cute e la lesione bersaglio , di strutture scheletriche , visceri e vasi . 
con tali criteri sono stati prospetticamente studiati 14 pazienti det compresa da 26 a 77 anni ( et media 51 , 5 anni ) , dei quali 9 maschi e 5 femmine . 
 tecnica tc stata utilizzata unapparecchiatura a 6 strati tcmd ( somatom emotion 6 , siemens , erlangen , germania ) a scansione elicoidale , con acquisizione simultanea di sei strati per ogni rotazione completa . 
i parametri tecnici utilizzati sono stati i seguenti : 90 mas , 130 kv , collimazione 2 mm , spessore di strato 2 , 5 mm e intervallo di ricostruzione di 1 mlelaborazione delle immagini stata ottenuta con una workstation dedicata ( leonardo , siemens )  . 
ciascun paziente , in regime di ricovero ordinario o di day hospital , ha fornito il proprio consenso firmato previa informazione verbale e scritta sui vantaggi e le possibili complicanze correlate alla procedura . i pazienti sono stati posizionati sul lettino della tcmd in decubito supino , prono , o laterale a seconda della sede della lesione da sottoporre ad agobiopsia e della sua distanza dalla parete addominale . 
sulla base di tali immagini tridimensionali , stato introdotto , un ago chiba point centimetrato di 22 / 20 g ( per prelievo del materiale idoneo allo studio citologico ) o un ago tranciante da 18 g ( biomol , hospital service , italia ) , scegliendo il tragitto pi breve in cui non ci fosse linterposizione , tra la cute e la lesione bersaglio , di grossi vasi , nervi , strutture viscerali o scheletriche , nonch lingresso nello spazio retroperitoneale . con una scansione tcmd successiva stata ottenuta la conferma , che la punta dellago fosse stata correttamente posizionata . 
finally , all patients were followed up until the cytological or histological confirmation of the diagnosis was obtained . biopsie microistologiche , i frustoli di tessuto sono stati fissati in formalina . 
non stata effettuata alcuna restrizione dietetica o idrica aggiuntiva o diversa da quella clinicamente indicata . infine , i pazienti , sono stati seguiti fino a conferma cito / istologica della diagnosi . results risultati anatomical and radiological characteristics of the 14 patients are summarised in table 1 . 
lutilizzo di immagini tridimensionali mpr ha permesso in tutti i casi il prelievo di materiale biologico ( 10 di tipo citologico e 4 di tipo istologico ) e in 11 su 14 casi ( 78 , 5% ) il prelievo risultato adeguato per un giudizio diagnostico . 
i prelievi istologici sono risultati adeguati per la diagnosi in tutti casi , quelli citologici in 7 su 10 casi ( 70% ) ( tabella 1 )  . in 6 casi ( pazienti 3 , 4 , 7 , 1113 ) , le lesioni sottoposte ad agobiopsia erano noduli solidi nel contesto di organi parenchimatosi o strutture muscolari , linfoadenomegalie in 6 casi ( pazienti 1 , 2 , 5 , 6 , 7 , 8 , 9 )  . 
in due casi ( pazienti 10 , 14 ) la biopsia stata eseguita in un surrene lievemente aumentato di volume , in cui era stata documentata una captazione del tracciante radioattivo allesame tc - tomografia ad emissione di positroni ( pet )  . 
in un solo caso si osservata una complicanza rappresentata da una sottile falda di pneumotorace , del tutto asintomatica , a seguito di una biopsia di un linfonodo peripancreatico ( paziente 5 )  . 
 us and mdct are the most commonly used imaging techniques for guiding percutaneous needle biopsy of suspected or known retroperitoneal malignancies [ 10 , 11 ]  . us is considered the technique of choice because it allows real - time visualisation of the needle throughout the procedure , it does not expose the patient to radiobiological risk and it has relatively low costs [ 1214 ]  . 
mdct , with its ability to visualise fine anatomical detail , is preferred for lesions that are difficult to reach with the us beanevertheless , in some cases , the target lesion may prove hard or impossible to reach , even with mdct guidance , because it is obscured by bony structures , vessels and / or abdominal organs . 
in these cases , surgical biopsy or surgical resection is recommended [ 3 ]  . the anatomical obstacles between the skin and the target lesion in our study included abdominal organs in discussione le tecniche radiologiche di guida allesecuzione di agobiopsie percutanee di lesioni retroperitoneali di incerta natura solitamente utilizzate sono lus e la tcmd [ 10 , 11 ]  . 
lus la guida strumentale di prima scelta nelle biopsie percutanee , in quanto permette la guida in tempo reale della traiettoria dellago , non espone il paziente a rischio radiobiologico , ed ha relativi bassi costi [ 1214 ]  . 
tuttavia , in alcuni casi , anche con la guida tcmd , pu essere difficile o non possibile raggiungere il tessuto da sottoporre a biopsia , in quanto mascherato da strutture vascolari , scheletriche e / o viscerali ; in tali situazioni viene suggerita la biopsia escissionale ( open biopsy ) o la resezione chirurgica della lesione [ 3 ]  . radiol med ( 2011 ) 116 : 407416 radiol med ( 2011 ) 116 : 407416 radiol med ( 2011 ) 116 : 407416 fig . 
dimostrazione sul piano assiale nativo della punta dellago ( freccia ) allinterno del surrene sinistro . b , c limmagine mpr assiale obliqua ( b ) e parasagittale sinistra ( c ) rendono possibile la scelta del percorso dellago che eviti la sovrapposizione del rene sinistro e del seno costo - frenico sinistro , suggerendo una doppia inclinazione ( caudo - craniale e medio - laterale )  . 
note how the adrenal lesion is hidden by the proximal end of the left 12th rib ( short arrow ) and in close contact with the abdominal aorta and the upper pole of the left kidney . 
si noti come la lesione surrenalica sia coperta dallestremo prossimale dellultima costa di sinistra ( freccia corta ) e sia a stretto contatto con laorta addominale ed il polo superiore del rene sinistro . 
b limmagine mpr parasagittale rende possibile la scelta del percorso dellago che eviti la penetrazione dellaorta addominale e del rene sinistro , suggerendo una doppia inclinazione ( cranio - caudale e medio laterale )  . 414 radiol med ( 2011 ) 116 : 407416 fig . 
la scansione assiale nativa documenta la punta dellago ( freccia ) allinterno del linfonodo . il linfonodo neoplastico completamente coperto dal processo traverso sinistro della i vertebra lombare ( testa di freccia ) ed a stretto contatto con lilo renale destro . 
a unenhanced axial multidetector computed tomography of the upper abdomen ( patient lying prone ) shows the needle tip ( arrow ) inside the metastatic lymph node ( arrowheads ) , which is hidden by a vertebral transverse process . 
a , b contrast - enhanced axial multidetector computed tomography of the upper abdomen ( patient lying prone ) shows a 10 - mm enhancing node in the left psoas muscle ( arrowheads in a ) , which is more evident in a higher contrast window ( b )  . 
impregnazione nodulare del mdc di 10 mm ( punte di freccia in a ) , nel contesto del muscolo psoas di sinistra , meglio evidente nella finestra ad elevato contrasto ( b ) ; si noti come la lesione sia coperta dal processo traverso della ii vertebra lombare ( freccia in b )  . 
in such cases , double oblique mpr views ( craniocaudal and mediolateral ) enables selection of a needle path that avoids bony structures , vessels and organs . us - guidance of biopsy of the right adrenal gland is particularly straightforward , thanks to the presence of the hepatic acoustic window . 
on the basis of our results , guidance of mdct with mpr enables the selection of alternative routes , thereby increasing the likelihood of reaching sites that would otherwise be difficult to access . riore ai 2 cm , era coperta nelle scansioni assiali tc dallaorta addominale , dal polo renale superiore sinistro e dallarticolazione costo - vertebrale sinistra . 
in questi casi la scelta della migliore traiettoria dellago che potesse evitare strutture scheletriche , vasi o visceri stata ottenuta con una doppia obliquit ( medio - laterale e cranio - caudale ) grazie alla guida delle immagini mpr . 
la guida bioptica tcmd con immagini mpr da tempo entrata nelluso delle unit di radiodiagnostica tuttavia lutilizzo ed i vantaggi sono stati descritti solo per lesioni polmonari [ 1619 ]  . 
sulla base dei nostri risultati , analogamente a quanto gi dimostrato per le lesioni polmonari , la guida tcmd , con lausilio delle ricostruzioni mpr , pu consentire la scelta di piani di accesso alternativi aumentando le possibilit di raggiungere sedi di difficile acceso . 
 416 conclusions conclusioni radiol med ( 2011 ) 116 : 407416 mdct is the imaging examination of choice in the biopsy of retroperitoneal lesions that are difficult to see on us scans . 
in our experience , images did not the use of mpr increase the rate of complications or the procedure time . la tcmd lindagine strumentale di scelta nella guida bioptica delle lesioni retroperitoneali difficilmente documentabili in us . 
rotondo1 1dipartimento di internistica clinica e sperimentale magrassi - lanzara , sezione di radiodiagnostica e radioterapia , seconda universit degli studi di napoli , p.zza miraglia 2 , 80138 napoli , italy 2servizio di radiologia , ospedale san massimo , penne , pescara , italy correspondence to : g . 
the aim of this study was to compare magnetic resonance ( mr ) enteroclysis with mr enterography to verify whether nasoenteric intubation in patients affected by crohns disease can provide supplementary information to that afforded by mr study of the small bowel . 
distension of the small - bowel loops was obtained by administering polyethylene glycol : 15 patients were given the mixture by mouth ( mr enterography ) , whereas the remaining 25 received it via nasoenteric intubation ( mr enteroclysis )  . our study protocol included morphological sequences taken before and after intravenous injection of contrast medium and real - time functional sequences . 
complete distension of the small - bowel loops was obtained in the 25 patients who underwent mr enteroclysis , with the additional advantage of a suitable assessment of those segments involved in the pathological process . 
in un periodo di 12 mesi , 40 pazienti con morbo di crohn ( 28 femmine e 12 maschi ; et media 35 anni ) sono stati sottoposti ad esame rm . 
la distensione delle anse dellintestino tenue stata ottenuta con polietilen glicole ( peg ) : 15 pazienti hanno assunto per os la miscela ( mr - os ) , mentre a 25 pazienti stata iniettata tramite sondino naso - enterico ( mr - e )  . 
nei 25 pazienti sottoposti ad mr - e si ottenuta una completa distensione di tutte le anse dellintestino tenue con unadeguata valutazione dei tratti interessati dal processo patologico , cos non stato nei 15 sottoposti ad mr - os poich le anse del digiuno e del tenue prossimale sono risultate parzialmente collabite . 
la mr - e la tecnica pi efficace nello studio dellintestino tenue nei pazienti affetti da morbo di crohn , fornendo unadeguata valutazione morfologica , ma anche informazioni funzionali . 390 radiol med ( 2011 ) 116 : 389406 keywords crohns disease mr enteroclysis mr enterography bowel distension wall lesions stenosis fistulas parole chiave morbo di crohn enteroclisi rm enterografia rm distensione intestinale lesioni di parete stenosi e fistole introduction introduzione crohns disease is a chronic inflammatory disease of the gastrointestinal tract , which can involve almost any segment from the mouth to the anus . 
the sites most frequently involved are the terminal ileum and the proximal colon [ 1 ]  . although endoscopy with biopsy is considered the reference standard for diagnosing inflammatory bowel diseases such as crohns disease [ 2 , 3 ] , the small bowel is still defined the black box of the gastrointestinal tract due to the difficult access for endoscopic techniques [ 4 ]  . 
various imaging techniques have been proposed for the panoramic study of the small bowel , such as scintigraphy with labelled leukocytes , and computed tomography ( ct ) [ 6 ]  . 
limitations of these techniques include the low contrast resolution of the former and exposure to ionising radiation for both , which in patients with crohns disease given the need for life - long follow - up is biologically significant and limits the repetition of the techniques [ 6 , 7 ]  . magnetic resonance ( mr ) imaging overcomes these limitations . 
the elevated soft - tissue contrast , the lack of ionising radiation and the functional information it can provide make mr imaging an ideal candidate for diagnosis and follow - up of patients with crohns disease [ 8 ]  . however , the study protocol has not yet been standardised , and some controversy remains regarding the value of nasoenteric intubation [ 9 , 10 ]  . the aim of our study was to compare mr enteroclysis ( contrast material administered after the placement of a nasoenteric tube ) with mr enterography ( contrast material administered by mouth ) to evaluate which is more effective in patients with crohns disease . materials and methods in a 12 - month period ( october 2008october 2009 ) , 40 consecutive patients ( 28 women and 12 men , mean age 35 years , range 1860 years ) were referred to our department with a histological diagnosis of crohns disease ( table 1 )  . all 40 patients had undergone conventional enteroclysis ( table 2 ) and underwent mr examination to complete the diagnostic work - up . 
clinically , the patients presented with different levels of disease activity according to the harveybradshaw index ( hbi ) ( table 3 ) and on the basis of laboratory test findings [ white blood count ( wbc ) , haematocrit il morbo di crohn una malattia infiammatoria cronica del tratto gastroenterico , che pu colpire qualsiasi segmento dalla bocca allano . 
sebbene lendoscopia con biopsia sia considerata il gold standard per la diagnosi delle patologie infiammatorie intestinali ( ibd ) , come il morbo di crohn [ 2 , 3 ] , lintestino tenue tuttora definito the black box dellapparato gastroenterico proprio per il difficile accesso alle metodiche endoscopiche [ 4 ]  . 
sono state proposte diverse metodiche di imaging per lo studio panoramico dellintestino tenue , come la scintigrafia con i leucociti marcati e la tomografia computerizzata ( tc ) [ 6 ]  . 
limiti di queste metodiche sono , per la prima , la bassa risoluzione di contrasto e per entrambe , unesposizione a radiazioni ionizzanti , che nei pazienti con morbo di crohn , data la necessit di un follow - up a vita , assumono un significato biologico importante e ne limitano la ripetizione [ 6 , 7 ]  . la risonanza magnetica ( rm ) supera tali limiti . 
lelevata risoluzione di contrasto per i tessuti molli , il non utilizzo di radiazioni ionizzanti e la disponibilit di sequenze funzionali candidano la rm come esame ideale per la diagnosi ed il follow - up dei pazienti con morbo di crohn [ 8 ]  . 
tuttavia il protocollo di studio non ancora standardizzato e rimane soprattutto controverso il valore dellintubazione naso - digiunale [ 9 , 10 ]  . il nostro studio si pone lobiettivo di confrontare lenteroclisi rm con somministrazione di mezzo di contrasto ( mdc ) dopo posizionamento di sondino naso - enterico ( mre ) con lenterografia rm con somministrazione di mdc per os ( mr - os ) , per valutare quale sia pi efficace nei pazienti con il morbo di crohn . materiali e metodi in un periodo di 12 mesi ( ottobre 2008ottobre 2009 ) , 40 pazienti consecutivi ( 28 femmine e 12 maschi ; et media 35 anni ; range 1860 anni ) sono giunti alla nostra attenzione con diagnosi istologica di morbo di crohn ( tabella 1 )  . 
allesame clinico , i pazienti presentavano differente attivit di malattia , secondo lharvey - bradshaw index radiol med ( 2011 ) 116 : 389406 table 1 location and number of lesions detected with endoscopy vs . 
enteroclisi digiuno ileo prossimale ileo terminale ( ultimi 20 cm ) ileo terminale ( ultimi 20 cm ) colon prossimale colon distale - sigma non accessibile non accessibile 1 o > 1 sede pazienti numero di lesioni pazienti numero di lesioni endoscopico enteroclisi 1 or > 1 1 o > 1 table 2 signs and symptoms used to calculate the harvey - bradshaw index ( hbi ) tabella 2 segni e sintomi per calcolare lharvey - bradshaw index ( hbi ) signs and symptoms segni e sintomi general state of health condizioni generali good intermediate poor very bad abdominal pain absent mild moderate serious abdominal mass none not sure sure present and painful good intermediate bad very bad worst absent mild moderate serious dolore addominale massa addominale none not sure sure present and painful bouts of diarrhoea complications ( fistulas , abscesses ) number of stools complications ( fistulas , abscesses ) 1 point for each bout each complication 1 point each stool 1 score each complication 1 score ( ht ) , erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) ] ( table 4 ) [ 11 ]  . 
 two days prior to the examination , all patients took a 2 , 000 cc solution of polyethylene glycol ( peg ) in water ( selg 2000 , milan , italy ) to cleanse the bowel and were invited to follow a semiliquid diet and on the day of the examination to have nothing by mouth . 
distension of the small bowel was obtained with the administra ( hbi ) ( tabella 3 ) e sulla base dei valori di alcuni test di laboratorio ( conta dei globuli bianchi , ematocrito , velocit di eritrosedimentaione [ ves ] e proteina c reattiva ) ( tabella 4 ) [ 11 ]  . 
 due giorni prima dellesame , tutti i pazienti hanno assunto una soluzione acquosa di circa 2000 cc di polietilenglicole ( peg ; selg 2000 , milano , italia ) , allo scopo di ottenere unadeguata pulizia intestinale ed invitati ad una dieta semiliquida , mentre il giorno dellesame al digiuno completo . 
per i pazienti allergici si provveduto alla 392 table 3 disease activity of the sample examined with reference to the harveybradshaw index and laboratory tests patients disease activity tabella 3 attivit di malattia del campione in esame secondo lhbi ed i test di laboratorio pazienti attivit di malattia remission moderate serious remissione moderata severa tion of an 1 , 800 - cc solution of peg in water ( selg 2000 )  . small - bowel distension was achieved in 15 patients with the ingestion of the solution 3040 min prior to entering the scanner ( mr enterography ) , whereas in 25 patients , the solution was injected via the nasoenteric tube after they had entered the scanner ( mr enteroclysis )  . all patients were premedicated with 20 - mg i.v. 
of hyoscine - n - butylbromide to reduce intestinal peristalsis and segmentation of the bowel loops during the examination . this was injected in the patients who underwent mr enterography immediately prior to positioning in the scanner , whereas the patients who underwent mr enteroclysis were injected after the dynamic scan confirmed that the entire small bowel had been enhanced , after around 1 , 000 cc of solution . 
quindici pazienti hanno assunto per os la miscela in un intervallo variabile tra 3040 min prima del posizionamento nello scanner ( mr - os ) , mentre a 25 pazienti la soluzione stata iniettata dopo il loro posizionamento nello scanner , tramite sondino naso - enterico ( mr - e )  . tutti i pazienti sono stati premedicati con 20 mg ev di nbutilbromuro di joscina , per ridurre la peristalsi intestinale e la segmentazione delle anse durante lesecuzione dellesame ; nei pazienti sottoposti a mr - os stato iniettato subito prima del posizionamento nello scanner , mentre ai pazienti sottoposti ad mr - e stato iniettato dopo che , al controllo dinamico , tutto il piccolo intestino risultato opacizzato , dopo circa 1000 cc di soluzione . 
le immagini di mr - e e di mr - os sono state visualizzate ed analizzate in maniera random , indipendentemente , da due differenti radiologi , di cui uno con esperienza di 20 anni sulle patologie intestinali e sulla rm delladdome , laltro con unesperienza di 4 anni . 
i criteri di correttezza dellesecuzione degli esami si sono basati sulla valutazione di 11 parametri : radiol med ( 2011 ) 116 : 389406 table 4 criteria adopted in the 40 patients who underwent magnetic resonance imaging . 
the aim was to verify examination accuracy criteria result score fied at endoscopy and conventional enteroclysis ; fibrofatty proliferation went mr enteroclysis , t2 - weighted dynamic single - shot turbo spin echo ( tse ) functional sequences with fs were acquired in the coronal plane ( matrix 512512 , slice thickness 100 mm ; tr / te 4 , 000 / 299 , flip angle150 ) to monitor distension of the small - bowel loops . the post - contrast acquisitions were processed , and the time - intensity ( t / i ) curves were evaluated . 
mr enteroclysis and mr enterography images were visualised and analysed in a random fashion and independently by two radiologists : one with 20 years of experience in bowel disease and abdominal mr , and the other with 4 years of experience . the accuracy criteria for performing the examinations were based on the evaluation of 11 parameters : 1 . 
degree of distension of the loops ( evaluating both the mean diameter calculated between the two most distended loops and the two least distended loops of the entire length of the small bowel , and distension of all of the small - bowel loops ) ; 2 . 
presence of complications ( fistulas , abscesses ) ; all of these parameters were given a score ( table 4 )  . statistical analysis the accuracy of mr enterography and mr enteroclysis was calculated and compared with endoscopy and conventional enteroclysis . 
of identified lesions stenosis distension wall thickness contrast enhancement time / intensity curve comb sign lymph nodes complications stenosi dilatazione spessore di parete contrast enhancement curva i / t comb sign linfonodi fibro - fatty proliferation complicanze i , intensit ; t , tempo suboptimal optimal 1 or > 1 absent present absent present < 3 mm > 3 mm absent present inconsistent consistent absent present absent present absent present absent present subottimale ottimale 1 o > 1 assente presente assente presente < 3 mm > 3 mm assente presente non significativa 0 significativa assente presente assenti presenti assente presente assenti presenti tabella 4 criteri valutati nei 40 pazienti sottoposti ad esame rm per la valutazione della correttezza della tecnica di esame parametri giudizio score grado di distensione numero delle lesioni identificate results are reported in table 5 for patients who underwent mr enterography and table 6 for patients who underwent mr enteroclysis . 
il grado di distensione delle anse ( valutando sia il diametro medio calcolato fra le due anse pi distese e le due meno distese di tutti i tratti intestinali in esame , sia la distensione di tutte le anse dellintestino tenue ) ; table 5 accuracy criteria in patients who underwent magnetic resonance enterography no . 
la presenza di stenosi ( intesa come la riduzione del calibro dellansa > 80% rispetto al calibro delle anse contigue ) ; radiol med ( 2011 ) 116 : 389406 table 6 accuracy criteria in patients who underwent magnetic resonance enteroclysis no . 
of patients result score criteria distension quality number of identified lesions stenosis distension wall thickness contrast enhancement time - intensity curve comb sign lymph nodes fibrofatty proliferation complications grado di distensione numero delle lesioni identificate stenosi dilatazione spessore di parete contrast enhancement curva i / t comb sign linfonodi fibro - fatty proliferation complicanze i , intensit ; t , tempo suboptimal optimal 1 or > 1 absent present absent present < 3 mm > 3 mm absent present inconsistent consistent absent present absent present absent present absent present subottimale ottimale 1 o > 1 assente presente assente presente < 3 mm > 3 mm assente presente non significativa significativa assente presente assenti presenti assente presente assenti presenti tabella 6 criteri di correttezza nei pazienti sottoposti ad mr - e parametri numero pazienti giudizio score detected by mr enterography and mr enteroclysis . 
la dilatazione delle anse poste a monte rispetto alla stenosi ( incremento del calibro delle anse a monte > 2 / 3 rispetto al calibro delle anse non patologiche ) ; 5 . 
la presenza di complicanze ( fistole , ascessi )  . tabella 7 distensione delle anse intestinali nei pazienti sottoposti ad mros ; valori medi a tutti questi parametri stato assegnato uno score sede distensione digiuno ileo prossimale ileo distale 2 , 5 cm 2 , 2 cm 2 , 15 cm ( tabella 5 )  . analisi statistica stata calcolata laccuratezza diagnostica della mr - os e della mr - e , confrontandola con lendoscopia e con lenteroclisi convenzionale . 
la significativit statistica stata calcolata attraverso il test del 2 con correzione di yates . risultati i risultati ottenuti sono riportati in tabella 6 per i pazienti sottoposti ad mr - os ed in tabella 7 per i pazienti sottoposti ad mr - e . 
 gli esami rm ( mr - os , mr - e ) sono stati confrontati con lendoscopia e con lenteroclisi ; abbiamo costruito le tabelle di contingenza 22 e calcolato laccuratezza diagnostica . 
in particolare , nella tabella 11 sono stati confrontati i risultati dellmr - os con i risultati dellendoscopia e abbiamo ottenuto una sensibilit pari all89% , una specificit pari al 67% , un valore predittivo positivo pari al 94% , un valore predittivo negativo pari al 50% e una prevalenza dell86% . 
nella tabella 12 sono stati confrontati i risultati dellmr - e con i risultati dellendoscopia e abbiamo ottenuto una sensibilit pari al 90% , una specificit pari al 100% , un valore predittivo positivo pari al 100% , un valore predittivo negativo pari al 40% e una prevalenza dell94% . 
nella tabella 13 sono stati confrontati i risultati dellmr - os con i risultati dellenteroclisi e abbiamo ottenuto una sensibilit pari al 89% , una specificit pari al 33% , un valore predittivo positivo pari al 80% , un valore predittivo negativo pari al 50% e una prevalenza dell75% . 
2a - c curva a dispersione , in blu mr - os , in rosso mr - e ; in a rappresentazione della distensione per il digiuno ; in b rappresentazione della distensione per l ileo prossimale ed in c rappresentazione della distensione per l ileo distale . sensitivity , 33% specificity , 80% ppv , 50% npv and 75% prevalence . 
table 13 compares mr enteroclysis with conventional enteroclysis , and the following values were calculated : 91% sensitivity , 100% specificity , 100% ppv , 40% npv and 94% prevalence . 
the chi - squared value was 6.386 with one degree of freedom , and the p value was 0.0115. mr enteroclysis proved to be more effective than mr enterography in evaluating stenosis and its significance and also in evaluating bowel - wall thickness . 
nella tabella 14 sono stati confrontati i risultati dellmr - e con i risultati dellenteroclisi e abbiamo ottenuto una sensibilit pari al 91% , una specificit pari al 100% , un valore predittivo positivo pari al 100% , un valore predittivo negativo pari al 40% e una prevalenza dell94% . 
in addition , mr imaging has the advantage over traditional techniques of visualising the entire thickness of the bowel wall and the perivisceral loose connective tissue [ 16 , 17 ]  . 
bowel distension is the most important technical requirement for all imaging modalities , because a collapsed bowel loop can mask or lalta risoluzione di contrasto per i tessuti molli , lacquisizione multiplanare delle immagini e la possibilit di ottenere informazioni funzionali sono le condizioni che rendono la rm lindagine pi idonea per la valutazione delle malattie infiammatorie dellintestino tenue [ 15 ]  . 
la distensione intestinale il requisito tecnico pi importante da conseguire per tutte le metodiche di imaging , perch unansa collassata pu portare a misconoscere un processo patologico o mimarlo [ 19 ]  . 
bench unansa con pareti marcatamente ispessite si possa considerare patologica anche in assenza di adeguata distensione , nei pazienti con morbo di crohn , per le caratteristiche proprie di questa malattia , devono essere riconosciuti tutti i tratti coinvolti dal processo . 
ne consegue che lottimizzazione della metodica permette lidentificazione del maggior numero di lesioni individuabili con la rm , e pertanto un incremento della sensibilit . alcuni autori [ 20 ] sostengono che sebbene la distensione intestinale sia superiore nei pazienti sottoposti a mr - e rispetto a pazienti sottoposti a mr - os , tuttavia essa non comporta significative differenze gestionali tra i due gruppi . 
although a bowel loop with markedly thickened walls can be considered pathological even in the absence of adequate distension , in patients with crohns disease , due to the particular characteristics of this disease , all of segments involved in the process need to be identified . 
therefore , optimisation of the technique enables identification of the highest number of lesions identifiable with mr , and therefore increases sensitivity . some authors [ 20 ] suggest that although bowel distension is greater in patients undergoing mr enteroclysis than in patients undergoing mr enterography , this produces no significant difference between the two groups . 
in our study , some of the bowel loops , including jejunal and proximal loops , of patients who underwent mr enterography were partially collapsed , and therefore , an evaluation of bowel - wall thickness as an indicator of the presence or absence of disease was not adequate [ 22 , 23 ]  . 
 nel campione in esame la non significativit numerica dei pazienti con localizzazione al digiuno e allileo prossimale , non consente di ottenere dati statisticamente rilevanti , sebbene sia da sottolineare come nel solo paziente in cui era presente un focolaio digiunale e sottoposto ad mr - os la lesione sia stata misconosciuta allesame di rm , cos come sono state misconosciute alla mr - os le due lesioni poste allileo prossimale e note allenteroclisi , mentre stata confermata la lesione ileale prossimale , riscontrata allenteroclisi , nel paziente sottoposto ad mr - e . 
 [ 23 ] , in accordo con la clinica e gli indici di laboratorio , hanno proposto una classificazione della malattia che tenga conto degli aspetti iconografici , identificando quattro sottovarianti , infiammatoria attiva , fistolizzante , fibrostenotica e riparativa - rigenerativa . 
le lesioni ulcerative , aftoidi e penetranti , vengono facilmente identificate con la rm , come delle irregolarit nel profilo della mucosa , rispettivamente come un nidus ad elevata intensit ( cratere ulcerativo ) circondato da un anello ad intensit medio - alta ( edema perilesionale ) , e come una linea ad elevata intensit di segnale che si approfondisce nello spessore parietale , mentre la mucosa sana tra le lesioni pu assumere aspetto pseudopolipoide [ 24 ]  . 
 sia la mr - os che la mr - e nel nostro studio , confrontate con lendoscopia , hanno presentato una sensibilit ed una specificit elevata nellidentificazione delle lesioni di parete poste allileo terminale ( tabelle 11 e 12 ) , con alto valore predittivo positivo e basso valore predittivo negativo , sebbene la mr - e presenti una sensibilit e una specificit superiore rispetto alla mr - os ( p = 0 , 0115 vs 0 , 143 )  . 
diversamente , dai nostri dati , emerge che nella valutazione delle lesioni poste a sede prossimale , e quindi al digiuno ed allileo prossimale , la mr - e abbia una sensibilit , una specificit ed un valore predittivo positivo superiore e un valore predittivo negativo inferiore rispetto alla mr - os ( tabelle 13 e 14 ) , come effetto di uninadeguata distensione delle anse ottenuta con questa ultima tecnica ( p = 0 , 5271 vs 0 , 0115 )  . 
la rm correla bene con la clinica , infatti i pazienti con sintomatologia lieve o moderata ( tabella 3 ) presentano un quadro iconografico e funzionale caratterizzato da stenosi non significative ed assenza di complicanze , diversamente nei pazienti con attivit severa secondo hbi ( tabella 3 ) , i reperti riscontrati , quali ascessi , fistole , occlusione , comb sign hanno presentato una maggiore frequenza rispetto al gruppo moderato ( tabelle 6 e 7 )  . 
a nostro avviso proprio la minor distensione del lume conduce ad una sovrastima del reperto , mentre solo una adeguata valutazione della stenosi indirizza al trattamento pi idoneo , e quindi alla corretta gestione del paziente [ 20 , 21 ]  . 
esame mr - e , sequenza trufi in coronale ; si noti l adeguato grado di distensione digiunale . in our patient population , the nonsignificant number of patients with disease involving the jejunum and the proximal ileum did not allow statistically significant data to be obtained . 
also unrecognised at mr enterography were the two lesions located in the proximal ileum and identified at conventional enteroclysis , whereas the proximal ileal lesion identified at conventional enteroclysis in the patient who underwent mr enteroclysis was confirmed . 
 [ 24 ] , in accordance with clinical and laboratory findings , proposed a classification that takes into account imaging appearance and identifies four subtypes : active inflammatory , fistulizing / perforating , fibrostenotic and reparative or regenerative . 
ulcerating , aphthoid and penetrating lesions are easily identified with mr as irregularities of the mucosa profile , respectively as a nidus of elevated intensity ( ulcerating crater ) surrounded by a ring of mediumhigh intensity ( perilesional oedema ) and as a line of elevated signal intensity that deepens in the wall , whereas the healthy mucosa between the lesions has a pseudopoly402 radiol med ( 2011 ) 116 : 389406 poid appearance [ 25 ]  . 
 some authors have compared mr imaging with the hbi when evaluating disease activity in crohns disease [ 11 ]  . we analysed the same parameters ( table 4 ) and obtained the same results ( tables 5 and 6 )  . 
instead , in patients with severe disease activity according to the hbi ( table 2 ) , findings such as abscesses , fistulas , occlusion and the comb sign are much more frequent than in the moderate group ( tables 5 and 6 )  . 
we suggest that the lesser distension of the lumen leads to an overestimation of the finding , whereas only an adequate evaluation of the stenosis can ideally orient treatment and thus prompt correct patient management [ 20 , 21 ]  . 
once an adequate degree of distension has been achieved , the real degree of stenosis can be quantified by evaluating proximal dilatation and comparing it with the information provided by t2 - weighted and contrastenhanced images ; then , from an analysis of the t / i curves , a fibrous stenosis can be differentiated from an inflammatory stenosis . 
raggiunto un adeguato grado di distensione e valutando la dilatazione a monte possibile quantificare il reale grado di stenosi e confrontando tale dato con le informazioni fornite dalle immagini t2 - w e postcontrasto e quindi dallanalisi delle curve i / t , differenziare una stenosi fibrotica da una infiammatoria . 
nelle immagini t2 - w la sottomucosa di un tratto stenotico tendenzialmente ipointensa , mentre nella stenosi infiammatoria presenta una elevata intensit di segnale , espressione di edema ed infiltrato cellulare . 
le sequenze single shot , attuabili solo in corso di mr - e , radiol med ( 2011 ) 116 : 389406 consentono , quindi una valutazione di ordine dinamicofunzionale , rappresentando un valore aggiunto alla tecnica . unadeguata distensione determina una corretta definizione dello spessore parietale e di conseguenza nella valutazione delle curve di i / t pi semplice lapposizione della regione di interesse ( roi ) , riducendo il rischio di errore nel postprocessing . unulcera transmurale quando supera la tonaca muscolare determina uninfiammazione nel tessuto mesenteriale adiacente , che pu evolvere in un piccolo ascesso peri - intestinale . 
un tramite fistoloso di piccola entit difficilmente identificabile sulle immagini ed apparire come delle irregolarit nodulari o spiculature della superficie sierosa dellansa ; diversamente per fistole di maggiori entit , nelle sequenze t2 - w la penetrazione del mdc nelle stesse che ne consente lidentificazione come delle immagini lineari ad elevata intensit di segnale , perpendicolari alla parete intestinale , mentre nelle sequenze acquisite dopo somministrazione di mdc ev , quando associate a reazioni desmoplastiche del grasso mesenteriale assumono aspetto stellato . 
gli svantaggi della metodica sono legati allintubazione naso - enterica , sia per il costo del sondino sia per lesposizione a radiazioni ionizzanti , necessaria per il controllo del suo posizionamento , sia per il maggior tempo richiesto , sebbene quando condotta da operatore esperto richieda al massimo 5 minuti . 
il nostro studio ha escluso pazienti pediatrici , ove la scarsa collaborazione e compliance dei pazienti giustifica lutilizzo dellmr - os . conclusioni la risonanza magnetica divenuta negli ultimi anni la metodica di imaging che meglio affianca lendoscopia nella prima diagnosi e nel follow - up dei pazienti con morbo di crohn consentendo una valutazione sia di dettaglio che panoramica [ 22 ]  . 
5 paziente donna 30 anni con diagnosi di morbo di crohn ( localizzazione ileo terminale ) , sottoposta ad mr - e : sequenza funzionale single shot idrografica . inflammatory stenosis , signal intensity is increased due to oedema and cellular infiltrate . 
adequate distension makes possible accurate definition of wall thickness and as a result facilitates placing a region of interest ( roi ) when evaluating t / i curves , thus reducing the risk of postprocessing error . a transmural ulcer , when it exceeds the muscular coat , causes inflammation of the adjacent mesentery , which can develop into a small peri - intestinal abscess . 
a small fistula is difficult to identify on the images and appears as a nodular irregularity or spiculation of the serous surface of the loop . with larger fistulas , penetration of the contrast material into the fistula itself makes its identification possible in t2weighted sequences , in which it appears as a linear hyperintensity running perpendicular to the intestinal wall . 
contrast material , when associated with desmoplastic reactions of the mesentery fat , the fistula has a stellate appearance . 404 radiol med ( 2011 ) 116 : 389406 abscesses or inflammatory masses are less common than fistulas , and adequate bowel distension is the most important requirement for identifying enteroenteric fistulas . 
the disadvantages of the technique are related to nasoenteric intubation , not only in terms of tube cost and exposure to ionising radiation needed to monitor tube placement , but also with regard to the longer time required , even though this does not exceed 5 min when performed by an expert operator . 
our study did not include paediatric patients , in whom poor cooperation and compliance justifies the use of mr enterography . conclusions in recent years , mr has become the imaging modality that best supports endoscopy in the initial diagnosis and followup of patients with crohns disease , as it makes possible a detailed and panoramic evaluation [ 22 ]  . 
7 a a 26 - year - old male patient affected by crohns disease located in the terminal ileumagnetic resonance ( mr ) enterography , coronal true fast induction steady - state potential sequence : distinct involvement of the last ileal loop , limited distension of the jejunum folds . 
b enteroclysis : jejunal lesions not identified by mr - os . radiol med ( 2011 ) 116 : 389406 clysis ; the possibility of extraluminal evaluation ; and the absence of ionising radiation justify the growing interest in the technique [ 16 ]  . mr enteroclysis , thanks to the adequate distension it provides of all bowel loops , including the jejunum and the proximal small - bowel loops , is the technique that best enables accurate assessment of crohns disease . 
mr enteroclysis enables identification of a stenotic segment , evaluation of significance or insignificance , definition of characteristics whether inflammatory or fibrous and therefore guidance towards the most appropriate treatment . 
the possibility of obtaining dynamicfunctional information , which can only be achieved with mr enteroclysis , makes it a unique imaging modality . bile a quella fornita dallenteroclisi baritata , la possibilit di una valutazione extraluminale ed il non utilizzo delle radiazioni ionizzanti giustificano il crescente interesse rivolto a tale metodica [ 16 ]  . la mr - e , fornendo unadeguata distensione di tutte le anse del tenue , anche del digiuno e del tenue prossimale , la tecnica che meglio consente una corretta definizione del morbo di crohn . 
con lmr - e possibile individuare un tratto stenotico , ma anche valutarne la significativit o non significativit , definirne le caratteristiche , infiammatoria o fibrotica , e quindi indirizzare il paziente al trattamento pi idoneo . 
6 peoples hospital af liated to shanghai jiaotong university , 600 yishan road , shanghai , 200233 china 2 department of radiology , ruijin hospital af liated to shanghai jiaotong university , school of medicne , 197 ruijin er road , 200025 china 3shanghai jiaotong university , school of medicine , 227 chong qin nan road , shanghai , 200025 china correspondence to : z . 
all ndings were analysed prospectively and correlated with tumour / node / metastasis ( tnm ) staging , dukes staging , histological grading , presence of lymph node metastasis , serosal involvement and mvd . 
a livello del tessuto tumorale e della parete rettale indenne sono stati calcolati i seguenti parametri : costante di transito ( ktrans ) , la percentuale dello spazio di distribuzione extravascolare extracellulare leakage space ( ve ) e la rate constant ( kep )  . la densit microvascolare stata stimata mediante analisi immunoistochimica su sezioni del pezzo operatorio . 
 il valore della costante di transito ( ktrans ) risultato signi cativamente diverso in caso di presenza o meno di coinvolgimento linfonodale di malattia ( p = 0 , 000 ) e nei diversi stadi sia secondo la classi cazione di dukes ( p = 0.04 ) che secondo il ptnm ( p = 0 , 03 )  . 
lo studio dinamico mediante risonanza magnetica 3d rappresenta un valido strumento diagnostico per la valutazione della vascolarizzazione tumorale . parole chiave carcinoma del retto risonanza magnetica studio dinamico post - contrasto angiogenesi prognosi introduction with 655 , 000 deaths worldwide per year , colorectal cancer is the fth most common form of cancer in the united states and the third leading cause of cancer - related death in the western world [ 1 , 2 ]  . 
generally , it is measured by microvessel density ( mvd ) , which depends on the availability of postoperative tissue or adequate biopsy materials and is somewhat invasive [ 6 ]  . 
however , as yet , there is no satisfactory imaging modality to predict prognosis and to assist in the selection of the right patients for extended surgical procedures and trials of adjuvant chemotherapy . 
the microcirculation can be assessed by semiquantitative evaluation of the signal intensity curve or by quanti cation of the change of contrast - agent concentration with kinetic modelling techniques [ 14 ]  . 
semiquantitative evaluation has the advantage of a good intrinsic signal - to - noise ratio ( snr ) , but it does not re ect the rate at which contrast medium passes from the intravascular space to the extravascular extracellular space ( ees ) and then back to the intravascular space over time after bolus injection . 
concentration - time curves ( ctc ) are then mathematically tted using [ 15 ] modelling parameters that include the volume transfer constant ( ktrans ) of the contrast agent , leakage space as a percentage of unit volume of tissue ( ve ) and the rate constant ( kep also called k21 )  . 
as far as we know , the correlation between dce - mr imaging and angiogenesis is controversial , and few studies have investigated the relationship between 3d - dce - mr parameters and angiogenesis [ 6 , 21 , 22 ]  . 
 the primary aim of this prospective study was to evaluate the relationship between 3d - dce - mr parameters and clinicopathological features of rectal cancer and assess their potential as new radiological prognostic predictors . material and methods 368 patients after adequate bowel preparation , 26 patients ( ten men , 16 women ; age range 2875years ; mean 58.310.5 ) with pathologically proved rectal cancer ( from may 2009 to february 2010 ) were prospectively included in this study . 
 magnetic resonance imaging and analysis mr studies were undertaken on a 1.5 - tesla supermagnetic mr scanner ( ge - signa hdx1.5t , ge medical systems , milwaukee , wi , usa ) using a phased - array body coil . 
 after plain scan ( t1 mapping ) , multiphase t1 - weighted dce - mr images were obtained using a spoiled gradientecho sequence ( lava ) in the axial plane . 
the following quantitative kinetic parameters were calculated automatically : volume transfer constant of the contrast agent ( ktrans ) , rate constant ( kep ) and volume as a percentage of the extravascular extracellular leakage space ( ve )  . 
 on the basis of the precontrast images , a region of interest ( roi ) covering the whole lesion and normal rectal wall with exclusion of peripheral fat , artefact and blood vessels was drawn over the tumour by a radiologist with 8 years of experience in gastrointestinal radiology . 
histograms of pixel data were obtained from all acquired tumour slices , and mean values were recorded for further study . microvessel staining and evaluation all patients were evaluated pathologically for the histological diagnosis and mvd counting . 
for surgically resected specimens , tissue blocks were obtained by sectioning each tumour with reference to the plane of imaging of the radiol med ( 2011 ) 116 : 366374 corresponding preoperative mr image as far as possible . 
 tissue blocks were dehydrated in graded alcohols , and microvessel density ( mvd ) was estimated by immunohistochemical staining of surgical specimens with anti - cd34 monoclonal antibody ( dako , denmark ) [ 23 ]  . 
 [ 24 ] : any single brown - staining endothelial cell or small clusters of brown - staining endothelial cells , with or without a lumen , and clearly separated from adjacent microvessels , tumour cells and other connective tissue elements were considered as individual vessels . 
 after screening the areas with intense neovascularised spots in a low power eld ( 40 ) , microvessels in the area with the highest number of discrete microvessels were counted in a 200 eld . 
differences of ktrans , kep and mvd between different histological grading , presence of lymph node metastasis , serosal involvement and dukes staging were compared with the analysis of variance ( anova ) test . 
spearman rank correlation tests were used to determine the strength of the relations between the dce - mr parameters , mvd , ptnm [ tumour / node / metastasis system from the american joint committee on cancer ( ajcc ) ] , dukes staging and histological grade . 
 results following the total mesorectal excision ( tme ) principle , 24 / 26 patients underwent standard rectal cancer resection , including miles and dixons operation , and the remaining two were treated with palliative surgery . 
tumour : transfer constant ( ktrans ) 11.971 / min , leakage space ( ve ) 12.2% and rate constant ( kep ) 98.331 / md detection of endothelial cells by anti - cd34 antibody in poorly differentiated gastric cancer : numerous microvessels are seen ( 200 )  . 
tumore : k trans 11 , 971 / min , leakage space ( ve ) 12 , 2 % e rate constant ( kep ) 98 , 331 / md marcatura delle cellule endoteliali mediante anticorpo anti - cd34 in un carcinoma gastrico scarsamente differenziato : notare la notevole densit microvascolare ( mvd 32 in un campo a 200 )  . 
tumour : transfer constant ( ktrans ) 2.559 / min , leakage space ( ve ) 19.5% and rate constant ( kep ) 13.11 / md detection of endothelial cells by anti - cd34 antibody in moderately differentiated gastric cancer : numerous microvessels are seen ( 200 )  . 
marcatura delle cellule endoteliali mediante anticorpo anti - cd34 in un carcinoma gastrico moderatamente differenziato : notare la notevole densit microvascolare ( mvd 34 in un campo a 200 )  . 
 discussion in previous reports , investigators have suggested that gadolinium - enhanced or dynamic mr imaging may be useful for evaluating ktrans in colorectal [ 26 ] and prostate cancer in selected patients [ 17 , 27 ]  . 
this nding might also be due to the larger tumour size in the late stage , with necrosis in the tumour centre , re ecting the heterogeneity of the tumour in the late stage . 
tumour : transfer constant ( ktrans ) 3.680 / min , leakage space ( ve ) 20.4% and rate constant ( kep ) 18.07 / md detection of endothelial cells by anti - cd34 antibody in well - differentiated gastric cancer : few microvessels are seen ( 200 )  . 
the heterogeneity of tumour predominates the biology in the late stage , and a mixed situation occurs most commonly when neither ow nor permeability surface area product predominates [ 14 ]  . 
another reason could be the case of signet - ring cell carcinoma , a rare form of adenocarcinoma of the large intestine , in which mean mvd was signi cantly lower than mean mvd in adenocarcinoma [ 22 , 28 ]  . 
another case of mucinous cell carcinoma might also have contributed to the decrease of ktrans , as there is excess mucin production and rather lower vessel distribution [ 13 , 22 , 28 ]  . 
 ve re ects the situation in extravascular extracellular space , as the extravascular extracellular space and the vascular space are balanced in size and shape to ensure adequate supply of nutrients and oxygen to the tissue , and this balance could be disturbed in malignant tissues . 
this might be because of tumour angiogenesis , characterised structurally by abnormal blood vessels lacking the usual vessel hierarchy and having a chaotic , heterogeneous distribution , and the extravascular extracellular space could be variable . 
much attention has been paid to the change of ve before and after treatment , and nowadays , ve is regarded as a prognostic parameter for the outcome of cancer treatment , especially chemotherapy or radiotherapy , which was not included in our study [ 29 , 30 ]  . 
 the main impetus of this study was to nd the indicator of prognosis preoperatively and nontraumatically , and the prognosis is determined by tumour in ltration , extension and stage , especially the tnm syste compared with kep , which proved to have a moderate relationship with tnm , ktrans revealed a medium - to - large positive correlation with all of these prognostic indicators . 
 radiol med ( 2011 ) 116 : 366374 microvessel density has also been correlated with colorectal tumour grade and stage in some reports [ 3336 ] , whereas other studies show no relation between microvessel density and patient outcome [ 3739 ]  . 
it might be that tumour vessels are structurally and functionally abnormal , with uneven diameters , increased length and tortuosity , multiple arteriovenous shunts and loss of physiological regulation of blood ow . 
unlike organs such as the uterus and prostate gland , where little change in organ morphology occurs on formalin xation , rectal cancer is more challenging to evaluate because of changes in tumour shape and shrinkage during tissue processing [ 21 ]  . 
in fact , distribution of gd - dtpa is determined not only by mvd but also by vessel permeability and size of the extravascular extracellular space [ 41 , 42 ]  . 
 in this study , the 3d - dce - mr technique enabled greater coverage of tumour volume , with thinner slice thickness ( 4 mm ) and multiple data sets acquired every 8 s rather than 1215 s or 30 s , as in previous reports [ 6 , 19 , 20 ]  . 
 one of the limitations in our study is that the sample size was small , and no estimation of demarcation point of ktrans value could be provided for tnm staging or dukes staging in clinical practice . 
as there are only two patients with signet ring or mucinous cell carcinoma in our study , it is impossible to analyse the changes of ktrans value in the different histological types . 
franco , e - mail : fabiofranco.email@gmail.com received : 17 march 2010 / accepted : 2 june 2010 / published online : 12 january 2011 springer - verlag 2011 abstract purpose . 
during this period , 46 patients were admitted for splenic trauma , of whom 17 were treated surgically , 15 conservatively and 14 with percutaneous embolisation ( 13 men , mean age 44.8 , mean injury severity score 18.5 , six with grade iv and eight with contrast blush )  . 
patients in shock were referred for laparotomy and splenectomy , whereas those who were haemodynamically stable or responsive to uid resuscitation were further evaluated with computed tomography ( ct )  . 
in the presence of imaging evidence of splenic injury ranging from grade i to grade iii ( n = 15 ) a conservative approach was adopted , whereas haemodynamically unstable patients with grade v injury ( n = 17 ) were treated with splenectomy . 
in questo periodo sono stati ammessi 46 pazienti con lesione splenica posttraumatica , di cui 17 sono stati trattati chirurgicamente , 15 con trattamento conservativo e 14 con embolizzazione percutanea ( 13 maschi , et media 44 , 8 , injury severity score [ iss ] 18 , 5 circa , 6 con grado iv e 8 con stravaso di mezzo di contrasto )  . 
i pazienti in stato di shock sono stati immediatamente sottoposti a laparotomia e splenectomia , mentre quelli emodinamicamente stabili o responsivi alla somministrazione di uidi sono stati valutati con tc . 
in base al dato strumentale , in 15 pazienti , con lesioni spleniche di grado i , ii e iii stato considerato il trattamento conservativo , mentre 17 pazienti emodinamicamente instabili , con lesioni di v grado , sono stati sottoposti a splenectomia . 
in 13 casi sono state posizionate spirali rm compatibili con diametro variabile tra 4 e 6 mm ; in un solo paziente sono state iniettate solo radiol med ( 2011 ) 116 : 454465 to reach a quick decision on the most appropriate treatment for patients presenting with blunt abdominal trauma , and splenic artery embolization seems to offer a reliable option in those with high - grade splenic injury or active bleeding . keywords blunt splenic injury , splenic artery embolization nonoperative management spleen splenic salvage particelle riassorbibili . 
in 13 / 14 casi ( 92 , 9% ) non sono state osservate complicanze post - procedurali , mentre in un solo paziente , trattato con particelle riassorbibili , si resa necessaria la splenectomia per recidiva di sanguinamento a 24 ore dallembolizzazione . 
sulla base del nostro algoritmo , possibile una veloce decisione sul trattamento pi opportuno delle lesioni spleniche post - traumatiche e lembolizzazione percutanea sembra offrire unaf dabile opzione in pazienti con lesioni dorgano di grado elevato o con sanguinamento attivo . parole chiave trauma splenico embolizzazione dellarteria splenica management conservativo milza salvataggio di milza introduction introduzione blunt abdominal trauma frequently causes splenic injury , and splenectomy has long been considered the treatment of choice . 
the spleen has important immune functions : it lters the blood , removing particulate antigens , bacteria and damaged red cells ; it produces immunoglobulin , tuftsin , opsonin and properdin and stores white blood cells [ 2 ]  . 
in both children and adults , the asplenic condition is an important risk factor for postsplenectomy infections , such as pneumonia , subphrenic abscess , pancreatitis , meningitis and sepsis , as has emerged from several large series [ 2 , 3 ]  . 
it consists of bed rest associated with close haemodynamic , clinical and imaging evaluation in the attempt to obtain a natural haemostasis noninvasively , reserving the surgical approach for those manifesting a recurrence of bleeding . 
rebleeding itself represents the most frequent cause of failure of this approach [ 511 ]  . splenic artery embolization ( sae ) in blunt abdominal trauma was rst proposed by sclafani et al . 
the aim of this study , which focuses on the periprocedural 30 - day period , was to evaluate the results of our experience with transarterial embolization in patients with blunt splenic injury . i traumi addominali provocano spesso lesioni spleniche e la splenectomia stata considerata per lungo tempo il trattamento di scelta . 
la milza , infatti , ha importanti funzioni , in particolare per limmunit : un ltro per il sangue , rimuove particolari antigeni , batteri e globuli danneggiati , produce immunoglobuline , tuftsina , opsonine , properdina ed un deposito di globuli bianchi [ 2 ]  . 
sia nei bambini che negli adulti , lasplenia un importante fattore di rischio per le infezioni post - splenectomia , come polmoniti , ascessi subfrenici , pancreatiti , meningiti e sepsi , come emerso da diversi studi [ 2 , 3 ]  . 
il management non - operativo ( nom ) si sta affermando , con crescente successo , per preservare le funzioni della milza ed ora considerato unopzione ef ciente nei pazienti emodinamicamente stabili con trauma splenico . 
 esso consiste nel riposo a letto associato a controlli seriati emodinamici , clinici e strumentali , nel tentativo di ottenere unemostasi naturale in modo non invasivo , riservando lapproccio chirurgico solo per coloro che manifestano una recidiva di sanguinamento . 
lobiettivo di questo lavoro , concentrato sul periodo periprocedurale di 30 giorni , quello di valutare i risultati della nostra esperienza di embolizzazione percutanea in pazienti con lesioni spleniche sanguinanti . 
the purpose of the protocol was to accelerate decisions on the proper therapeutic approach on the basis of haemodynamic status , the suspicion of abdominal trauma involving the spleen and the grade of splenic injury . 
according to the algorithm , patients presenting with shock [ blood pressure 90 mmhg ; shock index ( ratio of heart rate to systolic blood pressure ) 1.0 ] were treated with uid resuscitation following the recommendations of advanced trauma life support [ 23 ]  . 
in the presence of splenic damage , treatment was chosen on the basis of injury severity as graded by ct following the american association for the surgery of trauma organ injury scale ( table 1 ) [ 24 ]  . 
scopo del protocollo stato quello di accelerare la scelta di un corretto approccio terapeutico sulla base dello stato emodinamico , il sospetto di trauma addominale con coinvolgimento della milza e il grado di lesione della milza . 
secondo lalgoritmo , i pazienti che presentano shock ( pressione sanguigna 90 mmhg ; indice di shock rapporto tra frequenza cardiaca e pressione sistolica 1 , 0 ) vengono trattati con somministrazione di uidi e secondo le raccomandazioni di advanced trauma life support [ 23 ] , nei casi in cui non si sia ottenuta una risposta pressoria , i pazienti con forte sospetto di lesioni negli organi addominali , sono sottoposti a laparotomia durgenza ed eventuale splenectomia , con la sola valutazione clinica ed ecogra ca . 
in presenza di lesione splenica la terapia stata scelta in base al danno , classi cato alla tc secondo lamerican association for the surgery of trauma splenic injury scale ( tabella 1 ) [ 24 ]  . 
surgery was considered for splenic lesions associated with coexisting abdominal injuries needing laparotomy or for grade v splenic injury or in the case of conservative treatment failure , with bleeding recurrence . mico , riposo a letto , emoglobina sequenziale , ematocrito e monitoraggio ecogra co . 
la chirurgia stata considerata , solo , per lesioni spleniche associate a coesistenti lesioni addominali che necessitavano di laparotomia o per danni splenici di grado v o in caso di fallimento del trattamento conservativo , con recidiva di sanguinamento . 
ct examinations were evaluated by the attending radiologist on a dedicated workstation ( leonardo , siemens ) and integrated by multiplanar and sono state effettuate scansioni con tc spirale a 64 strati ( sensation 64 , siemens , forcheim , germania ) dopo la somministrazione di mezzo di contrasto endovenoso ( 300 mg / ml iomeprolo , iomeron 300 , bracco , milano , italia ) alla dose di 2 , 5 ml / kg peso , per un massimo di 120 ml , a un tasso di 22 , 5 ml / s ; il bolus - tracking stato utilizzato per ottimizzare la scansione per le fasi arteriose e venose . 
gli esami tc sono stati valutati dal medico radiologo su una stazione di lavoro dedicata ( leonardo , siemens , germania ) integrata da rico458 radiol med ( 2011 ) 116 : 454465 volume - rendered reconstructions and assessed for grade of splenic injury . struzioni multiplanari e volume - rendering . 
 transarterial embolization embolizzazione arteriosa percutaneous embolization was performed in 14 patients ( 13 men , mean age 44.8 ) , of whom six had grade iv splenic injuries , and eight ( with grade iiii injuries ) had ct evidence of contrast blush . 
arteriography and subsequent splenic embolization , planned on ct ndings , was achieved in the angiography suite ( integris bn 3000 , philips healthcare , eindhoven , the netherlands ) by an experienced interventional radiologist who was always present or available on call . 
embolisation was achieved through a 45 f cobra or simmons catheter ( glidecath , terumo , tokyo , japan ) or by a coaxial microcatheter ( progreat , terumo , tokyo , japan ) , whereas vessel occlusion was carried out mainly by coil ( 46 mm ) delivery ( mreye , william cook europe , bjaeverskov , denmark ) to exclude the damaged area from blood in ow , as proved by periprocedural angiography . 
in one case only was the artery occluded by injection of gelfoam pledgets ( spongel , yamanouchi , tokyo , japan ) , whereas in another case , occlusion was obtained by the combination of coil implantation and injection of gel - foam pledgets . right in the case of parenchymal injury involving completely the spleen or a large part of the organ , the main splenic artery was occluded ( proximal embolisation )  . 
 in patients treated by embolization , we evaluated the following : immediate technical success , de ned as capacity to reach the damaged vessel or feeding artery until exclusion of direct blood ow to the spleen was proved by angiography ; procedural success , de ned as a permanent haemostasis with no recurrent bleeding needing further interventions ; lembolizzazione percutanea , stata eseguita in 14 pazienti ( 13 maschi , et media 44 , 8 ) , di cui 6 casi con lesioni spleniche di grado iv e in 8 casi ( grading da i a iii ) , la tc ha dimostrato un blush di mdc . 
larteriogra a e la successiva embolizzazione splenica , piani cata in base ai risultati tc , stata realizzata in sala angiogra ca ( integris bn 3000 , philips sanit , eindhoven , olanda ) da un radiologo interventista esperto , sempre reperibile . 
lembolizzazione stata ottenuta attraverso un catetere 45 f cobra o simmons ( glidecath , terumo , tokyo , giappone ) , o da un microcatetere coassiale ( progreat , terumo , tokyo , giappone ) , mentre locclusione di vasi stata effettuata principalmente mediante posizionamento di spirali metalliche rm compatibili con diametro variabile tra 4 e 6 mm ( mreye , william cook europe , bjverskov , danimarca ) , escludendo la zona danneggiata dallaf usso di sangue , come dimostrato da un controllo angiograco periprocedurale . 
in un solo caso , per occludere i vasi sanguinanti , sono state usate particelle riassorbibili ( spongel , yamanouchi , tokyo , giappone ) ed in un altro particelle riassorbibili in combinazione con spirali metalliche . in caso di lesioni parenchimali che coinvolgevano completamente la milza o grande parte dellorgano , stata occlusa larteria splenica principale ( embolizzazione prossimale )  . 
con la punta del catetere allinterno dellarteria splenica , ma distalmente allostio dei maggiori vasi collaterali ( come larteria dorsale del pancreas ) , stata effettuata lembolizzazione no a fermare il usso di sangue attraverso larteria . 
nei pazienti trattati con lembolizzazione , abbiamo valutato : il successo tecnico immediato , de nito come la capacit di ricercare il vaso danneggiato o larteria afferente la lesione , bloccando il usso sanguigno diretto alla milza e comprovarlo al controllo angiogra co periprocedurale ; il successo procedurale , de nito come una emostasi permanente senza risanguinamenti che necessiterebbero ulteriori interventi ; la preservazione della milza , de nito come il mantenimento della vitalit dellorgano no alla dimissione o su 30 giorni di follow - up ; radiol med ( 2011 ) 116 : 454465 table 2 summary of main clinical data and embolization results patient no . 
proximal embolization was chosen in eight cases : six procedures were done with coil implantation , one with injection of gel - foam pledgets and another by a combination of coil implantation and injection of gelfoam pledgets . 
selective embolisation was preferred in six la morbilit - mortalit o complicazioni inerenti alle procedure , de nite come qualsiasi evento avverso rilevante correlato alla procedura ; lintervallo di tempo tra larrivo allospedale e la procedura terapeutica . 
 risultati di 46 pazienti , 17 ( con stato emodinamico instabile ) sono stati trattati chirurgicamente , 15 in modo conservativo ( di cui per 2 stato necessario lintervento chirurgico per risanguinamento ) e 14 sono stati sottoposti ad embolizzazione percutanea . 
lembolizzazione prossimale stata scelta in 8 casi : 6 procedure di posizionamento di spirali , in 1 caso si sono iniettate solo particelle riassorbibili e in un altro caso locclusione stata ottenuta con la combinazione di posizionamento di spirali ed liniezione di particelle riassorbibili ( tabella 2 )  . 
in 13 / 14 cases ( 92.9% ) , no bleeding recurrence was observed ; in one case , the only patient treated with gel - foam particles , urgent splenectomy was required for bleeding recurrence within 24 h of embolization . 
the mean time from hospital arrival to percutaneous embolisation was 171 min ( 120300 min ) ( table 2 )  . ottenuto in tutti i casi , con esclusione completa della lesione dal usso sanguigno ( tabella 2 )  . 
il tempo medio dallarrivo in ospedale allesecuzione dellembolizzazione percutanea stato di 171 minuti ( 120300 min ) ( tabella 2 )  . discussion discussione treatment of blunt splenic injury has evolved in recent decades . 
il management non - operativo ( nom ) stato valutato in diversi studi che indicano globalradiol med ( 2011 ) 116 : 454465 reports , which indicate overall spleen salvage of 57%91% [ 510 , 14 ] , becoming the standard of care in patients presenting with stable haemodynamic values on admission . 
in particular , ct provides rapid and reliable detection of splenic injury and real - time evaluation of its degree , directing the patient to a suitable therapeutic option , namely , nonoperative treatment or percutaneous embolization or surgery . 
in addition , ct is useful for identifying possible associated lesions [ 25 , 26 ]  . with a view to reducing unnecessary surgical interventions , a modi ed algorithm has been adopted to accelerate decisions on proper treatment in patients with blunt abdominal trauma involving the spleen . 
subsequently , on the basis of the ct ndings , patients underwent embolisation in the presence of grade iv splenic injury or , regardless of injury grade , evidence of intraparenchymal contrast blush or posttraumatic pseudoaneurys emergency surgery was reserved for grade v injury or haemodynamically unstable patients . in our experience , the algorithm allows for quick decisions and the opportunity to carry out splenic embolisation in posttraumatic injuries : mean embolisation time from hospital admission to procedure was 171 min ( 120300 min )  . 
embolization of the main splenic artery ( proximal embolisation ) was carried out in the event of diffuse organ damage , and segmentary artery occlusion ( distal embolisation ) was performed on those with localised injury . 
natural repair and splenic vitality is guaranteed through a collateral circulation arising from the greater pancreatic , dorsal pancreatic and short gastric arteries , avoiding direct blood in ow and pressure on the damaged spleen . 
no collateral ow is possible , and the mente un risparmio della milza tra 57%91% [ 510 , 14 ] , diventando , cos , lo standard di cura nei pazienti che presentano valori di emodinamica stabile al momento del ricovero . 
 su queste basi , lintegrazione di embolizzazione percutanea ( sae ) e del nom ha raggiunto un tasso di salvataggio splenico tra 73%96% , come riportato da altre esperienze [ 1321 ]  . 
in particolare , la tc fornisce una detection rapida e af dabile delle lesioni della milza e la valutazione in tempo reale del grado delle lesioni , fornendo al paziente una scelta terapeutica adeguata , vale a dire il nom o sae o la chirurgia . 
 abbiamo , quindi , adottato un algoritmo modi cato con lintento di ridurre inutili interventi chirurgici e decidere pi velocemente il corretto trattamento nei pazienti con traumi addominali che coinvolgono la milza . 
pertanto , sulla base dellesame tc , i pazienti sono stati sottoposti ad embolizzazione in presenza di lesioni spleniche di iv grado o , a prescindere dal grado di lesione , quando cera la presenza di blush vascolare intraparenchimale o uno pseudo - aneurisma post - traumatico . 
 nella nostra esperienza , lalgoritmo ha permesso una rapida decisione sulla possibilit di effettuare lembolizzazione splenica nelle lesioni post - traumatiche : tempo medio dellembolizzazione dal accesso ospedaliero alla procedura stato 171 minuti ( 120300 min )  . 
lembolizzazione dellarteria splenica principale ( embolizzazione prossimale ) stata effettuata in caso di danneggiamento diffuso dellorgano , mentre locclusione delle arterie segmentarie ( embolizzazione distale o selettiva ) in quei pazienti con presenza di una lesione localizzata . 
la riparazione e la vitalit della milza garantita da circoli collaterali derivanti dallarteria grande pancreatica , dalla dorsale del pancreas e dallarterie gastriche brevi , evitando cos lafusso di sangue diretto ed un elevata pressione sanguigna sulla milza danneggiata . 
e controllo angiogra co nale : si osserva vascolarizzazione della milza attraverso vasi collaterali derivanti dallarteria splenica distalmente al sito di occlusione ( freccia )  . treated area is destined to infarction . 
several materials have been proposed for this use : pitressin , autologous clots and , in more recent papers , gel - foam pledgets and metallic coils [ 1222 ]  . 
gel - foam pledgets have a diameter up to 1 , 000 once injected from the catheter , they follow the arterial blood stream and occlude the peripheral vessels , thus stopping the bleeding . 
come contropartita , molti studi segnalano un alto tasso di ri - sanguinamento quando le particelle riassorbibili vengono usate da sole come agente embolizzante [ 17 , 21 ] ; questo si veri cato , nella nostra esperienza , in un solo caso in cui stato embolizzata larteria splenica semplicemente con materiale riassorbibile . 
il paziente , un maschio di 41 anni , ha avuto una lesione splenica di iv grado causata da un incidente dauto che stata trattata con embolizzaradiol med ( 2011 ) 116 : 454465 fig . 
rebleeding may have been due to blood - stream pressure that pushed the gel - foam pledgets located in the main splenic artery distally to the most peripheral vessels , leaving , a large part of the spleen still vascularised . 
questo potrebbe essere dovuto alla pressione sanguigna , che spinge distalmente le particelle riassorbibili , dallarteria principale splenica ai vasi pi periferici , lasciando quindi gran parte della milza ancora vascolarizzata . 
from then on , vessel occlusion was achieved by coil embolization only , which in our opinion , is the technique of choice . an advantage of percutaneous intervention is the possibility of extending the procedure to coexisting lesions [ 17 , 19 ]  . 
among our patients , a 56 - year - old man involved in a car crash was rst embolized for an actively bleeding grade iii splenic lesion and , immediately after , treated for posttraumatic thoracoabdominal aortic dissection by endoprosthesis implantation . 
in our experience , with the exception of one case of post - embolisation re - bleeding , we observed no complications related to the procedure , in agreement with the low morbidity rate described in other reports [ 1215 , 1721 ]  . 
in particular , it was not possible to compare the results of patients who underwent sae with those treated with nom , as these approaches are addressed to substantially different types of splenic injury grade and / or active bleeding . 
additionally , we did not consider the role of contrast - enhanced sonography during admission , monitoring or follow - up , which , according to recent studies [ 27 ] , has proved very effective in identifying and staging abdominal solid - organ injuries . in conclusion , the modi ed algorithm allows rapid direction of patients with blunt abdominal trauma to the most appropriate treatment . 
considering the percutaneous procedure , the mean time from hospital arrival to embolisation was 171 msae can be considered a rapid , effective and safe procedure within the 30 - day period ( our follow - up period after treatment ) and a feasible method of integrating nom . 
more accurate and larger studies are needed to evaluate indications , timing , safety and the role of contrast - enhanced sonography and long - term results of sae . sivamente con spirali che , a nostro avviso , rappresentano il dispositivo di scelta . 
 tra i nostri pazienti , un uomo di 56 anni , coinvolto in un incidente dauto , stato prima embolizzato per una lesione sanguinante della milza di iii grado e , subito dopo , trattato per dissezione post - traumatica dellaorta toraco - addominale mediante impianto endoprotesi . 
nella nostra esperienza , con leccezione di un solo caso di ri - sanguinamento post - embolizzazione , non abbiamo rilevato complicanze connesse alla procedura , in accordo al basso tasso di morbilit descritti in altri studi [ 1215 , 1721 ]  . 
in particolare , non stato possibile confrontare i risultati dei pazienti sottoposti a sae rispetto al nom , dal momento che questi approcci sono rivolti a pazienti sostanzialmente diversi in termini di grado di lesioni spleniche e / o sanguinamento attivo . 
il presente studio si concentrato sui risultati nel periodo peri - procedurale e non abbiamo valutato i pazienti con follow - up a lungo termine per valutare la funzionalit splenica . 
 ulteriore limite del nostro studio di non aver considerato il ruolo dellecogra a con mdc ( ceus ) , che , in base a recenti studi [ 27 ] , si dimostrata molto ef cace nel riconoscimento e nella stadiazione delle lesioni traumatiche degli organi solidi addominali , allammissione , nel monitoraggio e nel follow - up dei pazienti da noi trattati . in conclusione , seguendo il nostro algoritmo modi cato , stato possibile fornire rapidamente il trattamento pi appropriato ai pazienti con traumi addominali . 
lembolizzazione dellarteria splenica pu essere considerata un trattamento rapido , ef cace , sicuro ( secondo i follow - up da noi effettuati nei 30 giorni successivi al trattamento ) ed un metodo pratico per integrare la gestione non operativa . 
selli2 1radiologia diagnostica e interventistica , universit di pisa , pisa , italy 2urologia , universit di pisa , pisa , italy 3scuola superiore santanna , pisa , italy correspondence to : g . 
donatelli , via pesenti 25 , 56124 pisa , italy , tel . : + 39 - 349 - 1581130 , e - mail : graziella_donatelli@hotmail.com received : 23 february 2009 / accepted : 17 september 2009 / published online : 12 february 2011 springer - verlag 2011 abstract purpose . 
the introduction of pharmacological therapy , based on clinical and functional evaluation of the lower urinary tract , is correlated with satisfactory morphofunctional outcomes , reducing moderate - to - severe postvoid residual ( pvr ; p < 0.1 ) and compliance ( p < 0.05 ) at the price of reduced bladder sensation . 
our study con rmed a relationship between detrusor overactivity and hypertonic bladder , bladder diverticula , vesicoureteral re ux , between detrusor underactivity and pvr and between dsd and bladder diverticula . 
questo studio stato condotto con un triplice obiettivo : analizzare i reperti morfo - funzionali vescicos nterici pi frequentemente rilevati con lesame video - urodinamico in pazienti con vescica neurologica secondaria a sclerosi multipla , studiare il ruolo di questo esame nella loro gestione clinica e dimostrare lesistenza di un rapporto di dipendenza tra variabili funzionali e morfologiche . 
lintroduzione della terapia farmacologica basata sulla valutazione clinica e funzionale dellunit vescico - s nterica consente di ottenere risultati morfofunzionali buoni , con il miglioramento del residuo postminzionale ( rpm ) moderato - severo ( p < 0 , 1 ) e della compliance ( p < 0 , 05 ) , a scapito della sensibilit vescicale ( p < 0 , 1 )  . 
la gestione clinica dei pazienti basata anche su valutazioni morfologiche del basso tratto urinario consente di ottenere risultati statisticamente migliori , con la riduzione dei casi di dissinergia detrusore - s ntere esterno ( desd ) e iperattivit detrusoriale con fuga di urina nellesame di controllo . 
i dati ottenuti da questo studio consentono di dimostrare lesistenza di un rapporto di causalit tra iperattivit detrusoriale e ipertono parietale , diverticoli vescicali e re usso vescico - ureterale ( rvu ) , tra ipoattivit detrusoriale e rpm , e tra desd e diverticoli vescicali ; permettono inoltre di avvalorare lutilit dellesame videourodinamico nella valutazione e nel management urologico di questi pazienti . radiol med ( 2011 ) 116 : 432443 keywords videourodynamics neurogenic bladder multiple sclerosis parole chiave video - urodinamica vescica neurologica sclerosi multipla introduction introduzione multiple sclerosis is a demyelinating disease of the central nervous system ( cns ) with multifactorial aetiology , progressive course characterised by periods of remission and relapse and dominated by clinical polymorphism attributed to lesion dissemination in space and time . 
the disease prevalently affects women , with a female - to - male ratio of approximately 3 : 1 , and it appears to be markedly in uenced by environmental factors as well as genetic and immunological factors . 
in fact , different populations have different incidence rates the frequency of the disease rises with increasing distance from the equator , and the relative risk ( rr ) for the individual is linked to the environment in which they spend the rst 15 years of life [ 1 , 4 ]  . 
in 50%90% of patients [ 5 , 6 ] , in different periods of the natural history of the disease but prevalently during the rst 10 years after diagnosis [ 6 , 7 ] , morphofunctional alterations occur to the lower urinary tract . 
 careful management of lower urinary tract dysfunction contributes to improving quality of life in these patients . materials and methods the study was conducted on 75 patients with neurogenic bladder secondary to multiple sclerosis who were observed between july 1998 and may 2008 at the diagnostic and interventional radiology department and the urology department of the santa chiara hospital in pisa . 
the only exclusion criterion applied was the presence of other disease , whether neurogenic or otherwise , that could have been responsible for morphofunctional abnormalities of the lower urinary tract . patients were subdivided into groups on the basis of the number of examinations performed , whether they were undergoing pharmacological treatment at the time of the rst examination or whether treatment was introduced at a later date . 
in this way , ve groups were created : 55 patients la sclerosi multipla una malattia demielinizzante del sistema nervoso centrale ad eziologia multifattoriale , con un decorso progressivo caratterizzato dalla presenza di remissioni e ricadute , dominata da un polimor smo clinico attribuibile al carattere disseminato delle lesioni nello spazio e nel tempo . 
in italia ha una prevalenza di 4070 casi per 100000 individui e unincidenza in costante aumento che nel 1999 ha raggiunto i 4 , 2 casi per 100000 individui [ 3 ]  . 
 infatti popolazioni diverse presentano tassi dincidenza diversi , la frequenza della malattia aumenta con la distanza dallequatore e il rischio relativo assunto dallindividuo relativo allambiente in cui il soggetto trascorre i primi quindici anni di vita [ 1 , 4 ]  . 
nel 50%90% dei pazienti [ 5 , 6 ] , in momenti diversi della storia naturale della malattia ma prevalentemente durante i primi dieci anni dalla diagnosi [ 6 , 7 ] , compaiono alterazioni morfo - funzionali vescico - s nteriche ; esse richiedono uno studio adeguato per poterne de nire le caratteristiche ed intraprendere , se necessario , una terapia appropriata . 
un accurato management delle disfunzioni delle basse vie urinarie contribuisce a migliorare la qualit di vita di tali pazienti . materiali e metodi lo studio stato condotto su 75 pazienti con vescica neurologica secondaria a sclerosi multipla , osservati tra luglio 1998 e maggio 2008 presso lunit operativa ( uo ) di radiologia diagnostica e interventistica e luo di urologia universitaria dellospedale s . 
di essi 47 sono donne , di et compresa fra 39 e 78 anni ( et media 57 anni , = 11 anni ) , e 28 uomini , di et compresa fra 32 e 83 anni ( et media 50 anni , = 11 anni ) ; 21 di essi hanno eseguito due esami video - urodinamici , per un totale di 96 esami valutati . 
lunico criterio di esclusione applicato stato la presenza di altra patologia , neurologica o meno , potenzialmente responsabile di alterazioni morfo - funzionali vescico - s nteriche . i pazienti sono stati suddivisi in pi gruppi in base al numero di esami eseguiti , allassunzione di terapia farmacotable 1 allocation of patients by sex and treatment radiol med ( 2011 ) 116 : 432443 434 men women total patients at rst examination without treatment patients at rst examination with treatment patients who underwent a second examination patients at second examination after introduction of treatment patients second examination after implantation of baclofen pump tabella 1 ripartizione dei pazienti per sesso e percorso terapeutico maschi femmine totale pazienti pazienti al 1 esame senza terapia pazienti al 1 esame con terapia pazienti che hanno eseguito 2 esami pazienti al 2 esame dopo introduzione di terapia pazienti al 2 esame dopo impianto di pompa al baclofene ( 73% ) without treatment at the time of the rst examination ; 20 ( 27% ) undergoing treatment at the time of the rst examination ; 21 ( 28% ) who underwent two examinations in six cases , pharmacological treatment was initiated after the rst examination , and in ve of these ( 83% ) , treatment involved implantation of a baclofen pump ( table 1 )  . videourodynamic study was done in accordance with the international continence society standards [ 8 ]  . 
 flowmetry was judged nonassessable for voided volumes < 50 ml and was not performed in patients with an indwelling catheter or in those who reported an absent bladder sensation at the beginning of the examination . 
videourodynamic study was conducted with a medtronic duet system ; for the cystomanometry and the pressure ow study , 9and 6 - fr double - lumen bladder catheters were used for recording the intravesical pressure , and a 12 - fr rectal balloon catheter was used for measuring abdominal pressure . 
sono stati cos delineati 5 gruppi : 55 pazienti ( 73% ) senza terapia al momento del primo esame ; 20 pazienti ( 27% ) con terapia al primo esame ; 21 pazienti ( 28% ) che hanno eseguito due esami : in 6 casi dopo il primo esame stata introdotta una terapia farmacologica , e in 5 di questi ( 83% ) si trattato dellimpianto di pompa al baclofene ( tabella 1 )  . 
la ussometria risultata non valutabile per volumi minzionali inferiori a 50 ml e non stata eseguita in pazienti portatori di catetere vescicale a permanenza e in coloro che riferivano lassenza di stimolo minzionale allinizio dellesame . 
lesame urodinamico stato eseguito con apparecchio duet medtronic ; per la cistomanometria e lo studio pressioneussso , sono stati utilizzati cateteri vescicali a doppio lume di calibro 9 e 6 fr per la registrazione della pressione endovescicale e cateteri rettali a palloncino del diametro di 12 fr per la misurazione della pressione addominale . 
in particular , one acquisition was obtained at baseline , after urinary owmetry and prior to bladder lling ; two at complete lling with the patient in a lying position , with and without valsalva manoeuvre ; two with the patient in the upright position , with one during the valsalva manoeuvre and one during voiding ; and one after voiding . 
in the case of urine leakage or vesicoureteral re ux ( vur ) , bladder volume and detrusor pressure at the time of the event were recorded with serial morphofunctional acquisitions up to its maximum level of expression . we analysed 18 variables : urgency , frequency , urinary incontinence ( ui ) , recurrent urinary tract infections ( uti ) , urinary retention ( ur ) , bladder sensation , bladder compliance , detrusor overactivity with and without incontinence , detrusor underactivity , acontractile detrusor , vur , hypertonic bladder wall , bladder diverticula , hypertonic bladder neck , hypertonic external urethral sphincter , detrusor sphincter dyssynergy ( dsd ) and postvoid residual ( pvr ) , the latter being subdivided into four subgroups on the basis of volume ( 50150 ml , 151250 ml , 251400 ml , > 400 ml ; pvr < 50 ml was considered nonsigni cant )  . data were analysed with microsoft of ce excel and r 2.5.1. 
i mezzi di contrasto impiegati sono stati visipaque ( iodixanolo , amersham health ; concentrazione di iodio 320 mg / dl ) e ultravist ( iopromide , bayer schering pharma ; concentrazione di iodio 370 mg / dl )  . 
 gli esami sono stati seguiti mediante la registrazione dei tracciati urodinamici e acquisizioni in uoroscopia ottenute periodicamente , in presenza di una sintomatologia riferita dal paziente o alterazioni dei tracciati . 
le immagini acquisite durante gli esami sono quelle ritenute utili dalloperatore a documentare la situazione delle basse vie urinarie ; in particolare sono state fatte una acquisizione in basale , dopo la ussometria e prima del riempimento vescicale ; due a pieno riempimento in clinostatismo , con e senza manovra di valsalva ; due in ortostatismo , di cui una durante manovra di valsalva e una minzionale ; una post - minzionale . 
il riempimento vescicale , le caratteristiche morfologiche delle basse vie urinarie e leventuale fuga di urina iodata durante le contrazioni detrusoriali disinibite o la manovra di valsalva sono state monitorate con la uoroscopia . 
in caso di fuga di urina sono stati registrati il volume di riempimento vescicale e la pressione detrusoriale concomitanti allevento , cos come in presenza di un episodio di re usso vescico - ureterale ( rvu ) , con acquisizione di una documentazione seriale morfo - funzionale no al suo massimo grado di espressione . 
 sono state analizzate 18 variabili : urgenza minzionale ( um ) , frequenza minzionale ( fm ) , incontinenza urinaria ( iu ) , infezioni urinarie ricorrenti ( ivu ) , ritenzione urinaria ( ru ) , sensibilit vescicale , compliance vescicale , iperattivit detrusoriale con e senza fuga di urina ; ipoattivit detrusoriale , acontrattilit detrusoriale , re usso vescicoureterale , ipertono parietale , diverticoli vescicali , ipertono del collo vescicale , ipertono dello s ntere uretrale esterno , dissinergia detrusore - s ntere esterno ( desd ) e residuo post - minzionale ( rpm ) , questultimo suddiviso in quattro sottogruppi in base alla sua entit ( 50150 ml , 151250 ml , 251400 ml , > 400 ml ; lrpm considerato non signi cativo se inferiore a 50 ml )  . i dati sono stati analizzati con microsoft of ce excel 2003 ed r 2.5.1. 
le analisi statistiche sono state condotte utilizzando : il test z per campioni indipendenti nella valutazione delle differenze nelle variabili di interesse tra i pazienti al primo esame video - urodinamico con e senza terapia ( essendo i campioni suf cientemente numerosi , si considerano valide le assunzioni di normalit asintotica degli stimatori ) ; il test di mcnemar per campioni appaiati nel confronto delle variabili di interesse tra campioni appaiati ( in due esami consecutivi , prima e dopo lintroduzione della terapia , prima e dopo limpianto della pompa al baclofene ) ; 436 radiol med ( 2011 ) 116 : 432443 radiol med ( 2011 ) 116 : 432443 438 radiol med ( 2011 ) 116 : 432443 il test chi - quadro nellanalisi della dipendenza tra variabili funzionali e morfologiche . 
 risultati sono state analizzate le frequenze di presentazione delle variabili in studio separatamente nei pazienti che al momento del primo esame video - urodinamico assumevano o meno terapia farmacologica , allo scopo di studiare i reperti pi frequentemente rilevati in questi due gruppi di pazienti e le eventuali differenze tra gli stessi . 
sono invece risultate statisticamente signi cative ( p < 0 , 1 ) le frequenze di presentazione di rpm 251400 ml e normale sensibilit vescicale , pi frequenti nei pazienti non in terapia , e quella delliposensibilit , pi frequente nei pazienti in terapia farmacologica e verosimilmente legata ad essa . 
ancora pi signi cativa la differenza di frequenza della ridotta compliance ( p < 0 , 05 ) , pi rappresentata nei pazienti non in terapia . confrontando i risultati ottenuti nei pazienti studiati prima e dopo lintroduzione della terapia farmacologica ( tabella 2 ) , si evidenzia una signi cativa riduzione della fig . 
statistical signi cance was found ( p < 0.1 ) for pvr 251400 ml and normal bladder sensation , which were more frequent in patients not undergoing treatment ; and reduced bladder sensation , which was more frequent in fig . 
il tracciato cistomanometrico evidenzia repentine uttuazioni della pressione detrusoriale durante la fase di riempimento vescicale , con valori massimi di 78 cmh2o . radiol med ( 2011 ) 116 : 432443 patients undergoing pharmacological treatment and probably linked to it . 
le restanti variabili risultano sostanzialmente stabili in entrambi i casi . confrontando i risultati ottenuti nella valutazione dei pazienti sottoposti a due esami indipendentemente dalle decisioni terapeutiche prese nel frattempo ( tabella 2 ) si osserva una signi cativa riduzione della frequenza di riscontro delliperattivit detrusoriale associata a fuga di urina ( p < 0 , 05 )  . per quanto riguarda lanalisi dei sintomi riferiti dai pazienti al momento dellesame , si nota una frequenza signicativamente minore di urgenza minzionale nei pazienti in terapia al momento del primo esame rispetto a quelli non in terapia , nei pazienti al secondo esame video - urodinamico indipendentemente dal planning terapeutico intrapreso , dopo introduzione di terapia e impianto di pompa al baclofene ( tabella 2 )  . 
allo stesso modo si evidenzia una riduzione dei casi di incontinenza urinaria e infezioni delle vie urinarie dopo introduzione di terapia farmacologica ( tabella 2 )  . come mostrato nella tabella 3 , i dati relativi ai primi esami sostenuti da pazienti non in terapia farmacologica hanno permesso di evidenziare rapporti di dipendenza tra diverse variabili in studio ; in particolare , il rapporto di causa - effetto tra iperattivit detrusoriale e ipertono parietale , diverticoli fig . 
3a , b contrazione detrusoriale disinibita con pressione detrusoriale massima di 44 cmh2o che determina fuga di urina in paziente con diverticoli vescicali . 440 radiol med ( 2011 ) 116 : 432443 vescicali e rvu , tra ipoattivit detrusoriale e rpm , e tra desd e diverticoli vescicali , con vario livello di signi cativit . 
a signi cantly lower frequency of urgency was reported by patients undergoing treatment at the time of the rst examination than by those not undergoing treatment and by patients at the second videourodynamic examination regardless of treatment strategy after the introduction of treatment and implantation of the baclofen pump ( table 2 )  . 
reduction was also observed in patients with ui and uti after the introduction of pharmacological treatment ( table 2 )  . as table 3 shows , data regarding the rst examinations in patients not undergoing pharmacological treatment highlights the dependent relationships between different study variables , particularly the causeeffect relationship between detrusor overactivity and hypertonic bladder wall , between bladder diverticula and vur , between detrusor underactivity and pvr and between dsd and bladder diverticula , with varying levels of signi cance . 
presentation frequency of study variables in patients who at rst videourodynamic examination where not undergoing pharmacological treatment were not in total agreement with data presented in the literature [ 913 ] , which are more heterogeneous . 
 this disagreement could be due to the different degrees of disease severity and different duration of urological symptoms in patients enrolled in the various studies . comparison between patients who at rst videourodynamic evaluation were or were not undergoing pharmacological treatment revealed a more favourable morphofunctional picture in the former group . 
le frequenze di presentazione delle variabili in studio nei pazienti che al momento della prima valutazione video - urodinamica non assumevano terapia farmacologica non sono sempre in accordo con i dati presenti in letteratura [ 913 ] , peraltro non omogenei tra loro ; tale discordanza potrebbe essere imputata al diverso grado di malattia presente nei pazienti reclutati per i vari studi e alla diversa durata dei sintomi urologici negli stessi . 
valutando i risultati ottenuti dal confronto tra pazienti che al momento del primo esame video - urodinamico assumevano o meno terapia farmacologica si evidenzia un quadro morfo - funzionale globalmente migliore per i primi . confrontando i risultati ottenuti nelle analisi con campioni appaiati , in pazienti che al momento del primo esame non assumevano terapia , si evidenzia sempre un miglioramento del quadro morfo - funzionale vescico - s nterico : in particolare la riduzione della frequenza dei casi di desd e di iperattivit detrusoriale associata a fuga di urina al momento del secondo esame . 
sulla base dei risultati riportati in tabella 2 , si pu concludere che il management clinico del paziente con vescica neurologica secondaria a sclerosi multipla integrato dallesame video - urodinamico permette una gestione pi adeguata dellunit vescico - s nterica , consentendo di ottenere miglioramenti morfo - funzionali pi marcati . i pazienti da noi studiati non sono stati strati cati per 442 radiol med ( 2011 ) 116 : 432443 ported in table 2 , we conclude that clinical management combined with videourodynamic examination of the patient with neurogenic bladder secondary to multiple sclerosis improves the management of the lower urinary tract and produces more notable morphofunctional improvements . although the patients we studied were not strati ed according to disease severity or duration of lower urinary tract involvement , the ndings were nonetheless interesting . 
in fact , on the basis of the assumption that the disease can either remain clinically and pathologically stable for a moreor - less lengthy period or worsen but certainly not regress , improvements in one or more functional , morphological or mixed variables can only be the result of the treatment undertaken , whereas stability can also be attributed to remission . 
a more accurate evaluation of this issue requires prospective studies and strati cation of patients according to disease severity and duration of lower urinary tract involvement , with the exclusion of other diseases that could in uence morphology and / or function of the lower urinary tract . 
another limitation of our study was the unavailability of suf cient clinical data ( analytical evaluation of symptoms reported by patients and their correlation with morphofunctional data ) and the lack of adequate diagnostic follow - up of the entire urinary tract . although videourodynamic examination generally includes an emg , which is indicated in patients with neurogenic bladder secondary to multiple sclerosis , this part of the examination was omitted for several reasons : ( a ) the invasiveness of the procedure ( given their greater reliability , needle electrodes tend to be used rather than surface electrodes ) ; ( b ) the need for cooperation of a neurologist both for electrode placement and trace interpretation ( therefore , a larger team is required ) ; ( c ) the nonnegligible frequency of artefacts and risk of electrode displacement during the examination . 
we therefore feel that the experience of operators in the eld of videourodynamics and the correlation of pressure traces with uoroscopic and radiological imaging can compensate for the absence of emg traces . indications for videourodynamic study in patients with neurogenic bladder are still a matter of debate in the absence of guidelines that meet with the consensus of urologists and neurologists [ 14 ]  . 
guidelines of the international consultation on incontinence advise an invasive instrumental approach as a second - line examination after noninvasive clinical and instrumental evaluation and treatment failure [ 2 , 7 , 15 , 16 ] in light of treatment effectiveness in most patients and the low complication rate involving the upper urinary tract ( 0.34% ) [ 7 , 15 , 17 ]  . 
if grado di malattia n per durata dellimpegno delle basse vie urinarie , ma i risultati ottenuti sono ugualmente interessanti ; infatti , partendo dal presupposto che la malattia pu mantenersi clinicamente e anatomo - patologicamente stabile pi o meno a lungo nel tempo o aggravarsi ma certamente non regredire , il miglioramento di una o pi variabili funzionali , morfologiche o miste dovuto unicamente al trattamento instaurato , mentre la loro stabilit pu essere imputabile anche alla fase di remissione della malattia . 
una valutazione pi accurata di questa problematica dovrebbe essere condotta mediante studi prospettici con strati cazione dei pazienti in studio per grado di malattia e durata dellimpegno vescico - s nterico , utilizzando come criterio di esclusione qualunque altra patologia che potrebbe in uenzare la morfologia e / o la funzione delle basse vie urinarie . 
un limite dello studio peraltro rappresentato dalla indisponibilit di suf cienti notizie cliniche ( rilevazione analitica dei sintomi riferiti dai pazienti e della loro correlazione con i dati morfo - funzionali ) e dalla mancanza di un adeguato follow - up diagnostico dellintero apparato escretore urinario . 
 nonostante lesame video - urodinamico includa lesecuzione dellelettromiogra a ed essa sia indicata nei pazienti con vescica neurologica secondaria a sclerosi multipla , questa parte dellesame stata omessa in ragione della sua invasivit ( dovrebbero essere utilizzati elettrodi ad ago anzich a placca perch pi af dabili ) , per la necessit della collaborazione con neurologi sia per il posizionamento degli elettrodi che per linterpretazione dei tracciati ( sarebbe quindi richiesto un team pi numeroso ) , per la frequenza non trascurabile degli artefatti e del rischio di dislocazione degli elettrodi durante lesame . 
riteniamo pertanto che lesperienza degli operatori in campo di video - urodinamica e la correlazione dei tracciati pressori con limaging uoroscopio e radiogra co possano supplire alla mancanza dei tracciati elettromiogra ci . le indicazioni dello studio video - urodinamico nei pazienti con vescica neurologica costituiscono ancora materia di dibattito , in assenza di linee guida che trovino il consenso di urologi e neurologi [ 14 ]  . 
le linee guida dellinternational consultation on incontinence consigliano un approccio strumentale invasivo come indagine di secondo livello dopo una valutazione clinica e strumentale non invasiva e il fallimento della terapia [ 2 , 7 , 15 , 16 ] , in ragione dellef cacia della stessa nella maggior parte dei pazienti e della bassa percentuale di complicanze a carico delle alte vie urinarie ( 0 , 34% ) [ 7 , 15 , 17 ]  . 
eccezioni a questo approccio pratico sono la presenza di un importante rpm o di ivu [ 14 ] , queste ultime spesso indicative di rvu , evidenziabile solo mediante cistogra a minzionale o in corso di iperattivit detrusoriale . 
nel caso in cui si debba ricorrere allo studio radiol med ( 2011 ) 116 : 432443 a more invasive examination is required , the guidelines indicate videourodynamic study as the reference standard [ 6 ]  . invasivo , le linee guida indicano lesame video - urodinamico come il gold standard [ 6 ]  . 
 they support the utility of the morphofunctional evaluation of the lower urinary tract as a second - line study after the noninvasive study of lower urinary tract dysfunction in setting a more complete and adequate clinical management of patients with neurogenic bladder . 
fugazzola 1department of radiology , university of insubria , v.le borri 57 , varese , italy 2department of vascular surgery , university of insubria , v.le borri 57 , varese , italy correspondence to : d . 
fifty - two consecutive patients ( 36 men and 16 women ; mean age 73.6 years ; range 5885 ) with chronic complete sfa occlusion and good distal run - off ( two or three patent vessels ) underwent endovascular recanalisation by direct stenting . 
during the follow - up , 12 reocclusions were observed : eight were treated with mechanical thrombectomy and in - stent angioplasty , and four were converted into femoropopliteal bypasses . 
cinquantadue pazienti consecutivi ( 36 uomini e 16 donne ; et media 76 , 3 anni ; range 5885 ) con occlusione completa dellafs e buon run - off distale ( 2 o 3 vasi pervi ) sono stati sottoposti a ricanalizzazione endovascolare mediante direct stenting . 
il trattamento percutaneo delle occlusioni croniche complete dellafs , nella nostra serie , mostra buoni tassi di perviet primaria e secondaria a breveradiol med ( 2011 ) 116 : 444453 secondary patency rates in the short and medium term , with few periprocedural complications . 
le riocclusioni possono essere trattate con tecnica percutanea , che garantisce un buon tasso di perviet secondaria . keywords super cial femoral artery chronic occlusion percutaneous treatment direct stenting parole chiave arteria femorale super ciale occlusioni croniche trattamento percutaneo direct stenting introduction introduzione treatment of occlusive lesions of the super cial femoral artery ( sfa ) is highly controversial . 
the tasc guidelines generally advise against the use of endovascular angioplasty ( pta ) techniques , sometimes associated with stenting , but they admit that stents may play a limited role in limb salvage in the event of pta failure or complications . 
indications for using stents are therefore restricted to suboptimal results of angioplasty , such as elastic recoil with residual stenosis > 30% , dissection with a ow - limiting ap and failure to maintain initial patency [ 16 ]  . more recently , improvements in techniques and devices have offered new options for percutaneous treatment of sfa occlusions . 
in particular , the use of nitinol stents may provide an effective alternative to surgical revascularisation in patients with severe cardiovascular comorbidities who are not candidates for surgery [ 4 ]  . 
all patients presented with severe claudication ( rutherford stage iii ) , chronic limb ischaemia with pain at rest ( rutherford stage iv ) or ischaemic ulcers ( rutherford stage v )  . 
a fairly good peripheral run - off ( de ned as at least two patent tibioperoneal arteries ) was present il trattamento delle lesioni ostruttive dellarteria femorale super ciale ( afs ) una questione molto controversa . 
le linee guida tasc generalmente scoraggiano luso delle tecniche endovascolari di angioplastica ( pta ) eventualmente associata a stenting , ma ha ammesso che gli stents possono avere un ruolo limitato nel salvataggio darto nei casi di insuccesso o nelle complicanze della pta ; quindi le indicazioni per lutilizzo degli stents sono limitate ai risultati sub - ottimali dellangioplastica come nel caso di ritorno elastico di parete condizionante stenosi residua > 30% , dissezione con ap stenosante o occludente , nonch il mancato mantenimento della perviet iniziale [ 16 ]  . pi recentemente , il miglioramento delle tecniche e dei dispositivi ha fornito nuove opzioni per il trattamento percutaneo delle occlusioni dellafs : in particolare , lutilizzo di stents in nitinol pu rappresentare unef cace alternativa alla rivascolarizzazione chirurgica in pazienti con grave comorbilit cardiovascolare , pertanto non candidati allintervento chirurgico [ 4 ]  . 
1a - f preprocedural angiography : total occlusion of the super cial femoral artery ( sfa ) with recanalisation at the level of the popliteal artery ( a - c )  . 
direct stenting ( d - f ) : posizionamento di stents in nitinol ( edwards lifestent ) con completa ricanalizzazione dellafs . radiol med ( 2011 ) 116 : 444453 fig . 
i - l al termine della procedura si documenta ricanalizzazione completa dellarteria femorale super ciale ( afs ) con perviet delle arterie della triforcazione della gamba . 448 radiol med ( 2011 ) 116 : 444453 in all cases . 
exclusion criteria were acute limb ischaemia , major tissue loss ( rutherford stage vi ) , previous by - pass surgery or sfa stenting , involvement of the iliac arterial axis ( stenoses > 50% or occlusions ) and absolute contraindications to the injection of iodinated contrast material . 
 the contralateral approach was used more frequently to reduce the risk of retroperitoneal haematoma and to avoid compression at the puncture site at the end of the procedure to reduce the risk of acute sfa reocclusion . 
 after a standard transfemoral puncture , a 5f valved introducer sheath ( terumo , tokyo , japan ) was inserted , and the lumen of the contralateral external iliac artery was reached with a 5f hook simmons 1 catheter ( cordis , warren , nj , usa ) and a 0.035hydrophilic guidewire ( terumo , tokyo , japan )  . 
the guidewire was subsequently replaced with a super - stiff guidewire ( amplatz , boston scienti c , natick , ma , usa ) and , after removal of the catheter and introducer sheath , a 45 cm long 6f introducer sheath was inserted ( cordis , warren , nj , usa )  . 
the occlusion was traversed using a stiff , straight or angled hydrophilic guidewire ( glidewire terumo , tokyo , japan ) and with a 4f or 5f straight or vertebral catheter ( cordis , miami , fl , usa )  . in 18 / 52 patients ( 34.6% ) , the occlusion could be easily crossed with the stents , whereas 22 / 52 patients ( 42.3% ) required a catheter with a 2 mm thick and 4 cm long balloon ( cordis , miami , fl , usa )  . 
in the remaining 12 patients ( 23.1% ) , severe atheromatous calci cations did not allow the occlusion to be crossed inside the lumen , so the guidewire and the catheter were advanced through the subintimal space and the lumen was re - entered at the femoro - popliteal junction or above the knee joint at the level of the patent vessel . 
self - expandable nitinol stents 6 or 7 mm in diameter , 10 or 12 cm in length and with a 1 to 2 cm overlap with the next stent were used in 40 cases . 
a total of 152 nitinol stents were employed : 80 luminexx ( bard , nj , usa ) , 48 smart ( cordis , johnson & johnson , miami lakes , fl , iii della classi cazione di rutheford ) , ischemia cronica degli arti con dolore a riposo ( stadio iv della classi cazione di rutheford ) o ulcere ischemiche ( stadio v della classi cazione di rutheford )  . 
i criteri di esclusione sono stati : ischemia acuta degli arti , importanti perdite di tessuto ( stadio vi della classi cazione di rutheford ) , precedente intervento chirurgico di by - pass o stenting dellafs , coinvolgimento dellasse arterioso iliaco ( > 50% stenosi o occlusioni ) ed in ne controindicazioni assolute alliniezione di mezzo di contrasto iodato . 
la ricanalizzazione stato eseguita previa puntura percutanea trans - femorale con approccio retrogrado controlaterale in 44 / 52 casi ( 84 , 6% ) ; nei restanti 8 pazienti ( 15 , 4% ) stato preferito laccesso trans - femorale anterogrado omolaterale . 
abbiamo utilizzato pi di frequente laccesso controlaterale per diminuire il rischio di ematoma retroperitoneale ed evitare , al termine della procedura , la compressione a livello del sito di puntura in modo da ridurre il rischio di riocclusione acuta dellafs . 
dopo puntura trans - femorale comune stato posizionato introduttore valvolato da 5f ( terumo , tokyo , giappone ) e , mediante lutilizzo di catetere tipo hook o simmons1 da 5 f ( cordis , warren , nj , usa ) , stato guadagnato il lume dellarteria iliaca esterna controlaterale con guida idro lica 0 , 035 ( terumo , tokyo , giappone ) ; stata sostituita , quindi , la guida stessa con guida super - stiff ( amplatz , boston scienti c , natick , ma , usa ) e , rimossi il catetere e lintroduttore , con posizionamento di introduttore lungo 45 cm da 6 f ( cordis , warren , nj , usa )  . 
locclusione stata valicata tramite supporto di guida idro lica rigida retta od angolata ( glidewire terumo , tokyo , giappone ) e catetere retto o vertebrale da 4 o 5f ( cordis , miami , fl , usa )  . 
 in 18 / 52 ( 34 , 6% ) pazienti locclusione stata valicata agevolmente dagli stents , in 22 / 52 pazienti ( 42 , 3% ) stato necessario lausilio di un catetere fornito di pallone dilatatore del calibro di 2 mm e della lunghezza di 4 cm ( cordis , miami , fl , usa )  . 
nei restanti 12 casi ( 23 , 1% ) le severe calci cazioni ateromasiche non hanno permesso di valicare locclusione allinterno del lume vasale , quindi stata fatta avanzare guida e catetere in sede subintimale effettuando il rientro al passaggio femoro - popliteo o in sede poplitea sovra - articolare in corrispondenza del vaso pervio . 
 in 40 casi sono stati utilizzati stents autoespandibili in nitinol del calibro di 6 o 7 mm e 10 o 12 cm di lunghezza con overlap di 1 o 2 cm con altro stent . 
la post - dilatazione stata eseguita utilizzando un catetere a palloncino del calibro di 6 mm e lunghezza 10 cm ( cordis , warren , nj , usa )  . 
in the latter eight cases , a genesis balloon - expandable steel stent was used ( cordis , johnson & johnson ) in order to increase precision when delivering it at the femoral bifurcation . all patients received 3 , 000 iu heparin intra - arterially during the procedure and 5 , 000 iu heparin i.v. 
in 250 ml saline solution for 48 h after the procedure , as well as fraxiparine at an anticoagulant dose for 30 days , followed by a double antiaggregant regimen of aspirin ( 100 mg / day ) and clopidogrel ( 75 mg / day ) for 3 months . 
follow - up was performed with clinical evaluation ( classi cation of symptoms and evaluation of peripheral pulses ) and colour doppler ultrasound at 6 , 12 , 18 and 24 months for 36 patients , up to 18 months in four patients and up to 12 months in 12 patients . 
no patient underwent limb amputation in the course of the follow - up . mente sono stati impiegati 152 stents in nitinol : 80 luminexx ( bard , nj , usa ) , 48 smart ( cordis , johnson & johnson , miami lakes , fl , usa ) e 24 edwards lifestent ( edwards lifesciences , irvine , ca , usa )  . 
in questi ultimi ( 8 casi ) stato utilizzato uno stent in acciaio medicale espandibile su pallone genesis ( cordis , johnson & johnson , miami lakes , fl , usa ) per essere molto pi precisi nel rilascio in corrispondenza della biforcazione femorale . durante la procedura , tutti i pazienti hanno ricevuto 3000 ui di eparina per via intra - arteriosa . 
dopo lintervento , tutti i pazienti hanno ricevuto 5000 ui ev di eparina in 250 ml di soluzione siologica per 48 ore , fraxiparina a dose scoagulante per 30 giorni , poi doppio antiaggregante ( aspirina , 100 mg / die e clopidogrel , 75 mg / die per tre mesi )  . 
il followup stato effettuato mediante valutazione clinica ( classi cazione dei sintomi ed esame dei polsi periferici ) ed eco - color doppler a 6 , 12 , 18 e 24 mesi per 36 pazienti , in 4 pazienti no a 18 mesi ed in 12 pazienti no a 12 mesi . 
si sono veri cati 4 casi di embolia distale , trattati mediante trombo - aspirazione con catetere da 6 f lungo 110 cm ( cordis , warren , nj , usa ) in due casi di insuccesso stato lasciato in sede catetere retto da 5 f multiforo ( cordis , johnson & johnson , miami lakes , fl , usa ) per trombolisi loco - regionale intraarteriosa ( uk 500000 ui in 500 ml di soluzione siologica a 60 ml / h ; eparina per via endovenosa 5000 ui in 250 ml di soluzione siologica a 20 ml / h ) per circa 24 ore . nessun paziente stato perso durante il follow - up ( tabella 1 )  . 
la perviet primaria stata del 92 , 3% a 6 mesi ( 4 embolie distali ) e del 76 , 9% a 12 mesi ( 8 occlusioni , trattate con successo con trombectomia meccanica tipo rotarex e angioplastica intra - stent , pertanto , il tasso di perviet secondaria stata del 100% a 6 e 12 mesi )  . 
al follow - up a 24 mesi , il tasso di perviet primaria e secondaria sono risultati non modi cati ( 69 , 2% e 92 , 3% )  . 
several studies have been conducted on the endovascular treatment of sfa occlusions > 10 cm in length ( 16 cm on average ) , and in some cases , only pta was used , achieving a mean primary patency rate of 46% at 12 months [ 79 ]  . 
other studies have reported the results obtained in combination with stenting after angioplasty failure ( secondary stenting ) , with a mean primary patency rate of 55% at 12 months [ 1013 ]  . 
 [ 14 ] found that , in femoral axis stenosis , there is no substantial difference between pta and stenting in terms of long - term patency rate , whereas in the treatment of occlusions especially long ones stenting is superior to angioplasty in term of patency outcomes . the use of stents in the femoro - popliteal region has lowered the rate of early restenoses ( due to elastic recoil , residual stenosis and ow - limiting ap ) , allowing long , complex lesions to be treated even in calci ed arteries [ 4 ]  . 
 despite these encouraging results , however , indications for stenting remain controversial : in fact , the main limitation is myointimal hyperplasia a cause of in - stent restenosis which typically occurs in long stenoses or occlusions or when multiple stents are deployed [ 15 ]  . 
 intimal hyperplasia occurs more frequently after stenting compared with pta alone and is probably due to the constant pressure of the stent on the arterial wall [ 16 ] : ve randomised controlled studies failed to show any bene cial effect of sfa stenting with steel stents compared with pta [ 1721 ]  . 
diversi studi riguardano il trattamento endovascolare delle occlusioni dellafs > 10 cm di lunghezza ( in media 16 cm ) : in alcuni casi stato utilizzata solo la pta , con un tasso di perviet primaria del 46% in media a 12 mesi [ 79 ]  . 
altri studi riportano i risultati ottenuti in associazione allo stenting dopo il fallimento dellangioplastica ( secondary stenting ) con un tasso di perviet primaria del 55% in media a 12 mesi [ 1013 ]  . 
 [ 14 ] ha documentato che , nella patologia stenotica dellasse femorale , non esiste differenza sostanziale tra pta e stent riguardo al tasso di perviet a distanza , mentre nel trattamento delle occlusioni specie lunghe i risultati dello stenting in termini di perviet sono superiori a quelli dellangioplastica [ 14 ]  . limpiego degli stents nel distretto femoro - popliteo ha ridotto la percentuale di restenosi precoci ( dovute al ritorno elastico di parete , a stenosi residua ed a limitazione del usso in caso di ap stenosante ) consentendo il trattamento delle lesioni lunghe e complesse , anche in arterie calci che [ 4 ]  . 
nonostante i risultati incoraggianti , per , le indicazioni alluso degli stents restano incerte : in effetti la principale limitazione rappresentata dalliperplasia miointimale , causa di restenosi intra - stent , che si veri ca in particolare in presenza di stenosi o occlusioni lunghe , o nel caso vengano posizionati pi stents [ 15 ]  . 
liperplasia intimale pi frequente dopo posizionamento di stent rispetto alla sola pta , ed probabile che sia dovuta alla costante pressione dello stent sulla parete arteriosa [ 16 ] : cinque studi randomizzati e controllati non hanno dimostrato un effettivo benecio dello stenting dellafs con stents in acciaio , rispetto alla pta [ 1721 ]  . 
tuttavia , questi studi valutano i risultati radiol med ( 2011 ) 116 : 444453 obtained with the two techniques in patients with short occlusions , in which case excellent results can be obtained with pta alone [ 4 ]  . 
 the recent introduction of nitinol stents appears to have improved the duration of percutaneous treatment in peripheral occlusive arterial diseases of the lower limbs : stent implantation prevents contraction of the elastic bres at the level of the atheromatous plaque , allowing the vessel calibre to remain constant . 
furthermore , thanks to the improvement of stent delivery systems and the smaller calibre of these devices , the most complex lesions , including very long occlusions , can be traversed without predilatation or by using undersized dilatation only if the occlusion cannot be crossed by the stent directly . 
this may reduce the incidence of in - stent restenoses , which are seen on secondary stenting , as well as the risk of embolisation , as reported by mewissen et al . 
in a recent randomised study , the use of nitinol stents proved superior to pta with subsequent stent implantation : at 12 months , restenosis rate was 37% in the rst group ( direct stenting ) and 63% in the second group ( pta ) [ 25 ]  . 
the use of multiple stents in the sfa seems to reduce the number of reocclusions due to the proximal and distal progression of the disease [ 27 , 28 ]  . 
in our experience , it is advisable to use as few stents as possible to cover the length of the occlusion , because their overlapping increases the risk of stent fracture [ 28 ] and consequent thrombotic occlusion . 
in this series , two edwards lifestents ( for each long lesion ) were placed in 12 patients , resulting in regular vascular patency and no complications at 12 months . the main limitation of this study was the small number ottenuti con le due tecniche in pazienti con occlusioni brevi : in questi casi , eccellenti risultati possono essere raggiunti con la sola pta [ 4 ]  . 
 recentemente , tuttavia , lintroduzione di stents in nitinol sembra migliorare la durata del trattamento percutaneo nellarteriopatia obliterante periferica degli arti inferiori : limpianto dello stent previene la contrazione delle bre elastiche in corrispondenza della placca ateromasica per cui il vaso mantiene un calibro costante . 
inoltre , con il miglioramento dei sistemi di rilascio degli stents nonch la riduzione in calibro dei devices , la maggior parte delle lesioni complesse , comprese le occlusioni molto lunghe , pu essere attraversata senza ricorrere alla pre - dilatazione o ricorrere ad una dilatazione sottostimando il vaso solo nei casi in cui non possibile attraversare locclusione direttamente con lo stent . 
la tecnica del direct stenting permette di evitare il trauma della pre - dilatazione ; possibile che questo riduca lincidenza di restenosi intra - stent , osservate con il secondary stenting , riducendo anche la percentuale di rischio embolico , come riportato da mewissen [ 22 , 23 ]  . 
in un recente studio randomizzato , lutilizzo di stents in nitinol ha dato risultati superiori a quelli ottenuti con pta ed impianto successivo di stents : a 12 mesi , i tassi di restenosi erano del 37% nel primo gruppo ( direct stenting ) e del 63% nel secondo gruppo ( pta ) [ 25 ]  . 
 [ 26 ] hanno evidenziato un miglioramento del tasso di perviet primaria a due anni , con limpiego del direct stenting per il trattamento delle occlusioni dellafs confrontato con la pta e secondary stenting ( con tasso di restenosi rispettivamente del 45 , 7% e del 69 , 2% ) , in particolare nei pazienti con ischemia critica degli arti . 
i tassi di perviet primari e secondari sono sostanzialmente sovrapponibili al secondary stenting [ 27 ] con il vantaggio a favore del direct stenting di tempi procedurali pi brevi e di una parziale riduzione delle complicanze emboliche distali peraltro non statisticamente signi cativa ( 7 , 7% vs 9 , 5% )  . 
nella nostra esperienza inoltre meglio utilizzare il minor numero possibile di stents , in relazione alla lunghezza dellocclusione , perch la loro sovrapposizione aumenta il rischio di frattura degli stents [ 28 ] , con conseguente occlusione trombotica degli stessi . 
in questa serie sono stati collocati due edwards lifestents ( per ciascuna lesione lunga ) in 12 pazienti con dimostrazione di regolare perviet vascolare ed assenza di complicanze al follow - up a 12 mesi . la limitazione pi importante relativa a questo studio 452 radiol med ( 2011 ) 116 : 444453 of patients enrolled . 
inoltre , non sono disponibili indagini radiologiche per la valutazione di eventuali fratture di stents , tuttavia , nei pazienti con riocclusioni non si sono evidenziate fratture dei devices impiantati . conclusions conclusioni the optimal treatment of total sfa occlusions is open to debate . 
this said , however , further studies with larger patient populations and longer follow - up periods are needed to validate the method as a primary approach in patients with chronic ischaemia due to total sfa occlusion . il trattamento ottimale delle occlusioni complete dellafs questione di dibattito . 
in questo studio dimostriamo la fattibilit e lef cacia della ricostruzione endoluminale dellafs mediante direct stenting , con un accettabile tasso di complicanze acute e di restenosi a medio - lungo termine . 
policlinico universitario tor vergata , viale oxford 81 , 00133 roma , italy 2dipartimento di medicina nucleare e imaging molecolare , irccs neuromed , via atinense 18 , pozzilli , isernia , italy correspondence to : f . 
the objective of this preliminary study was to examine the effects of combined magnetic resonance / positron emission tomography ( mr - pet ) evaluation in the morphofunctional characterisation of ovarian lesions . 
from june 2008 to september 2010 , we evaluated 24 patients ( mean age 4410 years ; range 2474 ) with ovarian lesions incidentally detected on ultrasonography ( us ) and / or multislice computed tomography ( ct )  . 
the added value of this fusion imaging modality lies in combining the benefits of the morphological evaluation provided by mr imaging and the metabolic assessment provided by pet . keywords mr - pet metabolic evaluation postprocessing ovarian lesions ct - pet fusion imaging riassunto obiettivo . 
scopo di questo studio preliminare stato esaminare gli effetti della valutazione combinata risonanza magnetica ( rm ) / tomografia ad emissione di positroni ( pet ) nella caratterizzazione morfo - funzionale delle lesioni ovariche . 
da giugno 2008 a settembre 2010 sono state valutate 24 pazienti ( et media di 4410 anni ; range 2474 anni ) con riscontro casuale di lesione annessiale mediante esame ecotomografico e / o tramite tomografia computerizzata ( tc ) multistrato . 
 radiol med ( 2011 ) 116 : 12881302 1289 parole chiave rm / pet studio metabolico postprocessing lesioni ovariche tc / pet fusione di immagini introduction introduzione ovarian cancer is the second most common gynaecological malignancy and has the highest mortality rate , with a 5 - year survival rate of 46% [ 1 ]  . 
other risk factors are nulliparity , caucasian race , early menarche , late menopause , gonadal dysgenesis and repeated hormone stimulation in patients undergoing artificial insemination , whereas protective factors include multiparity , the use of combined oral contraceptives and prolonged breastfeeding [ 5 ]  . the international federation of gynecology and obstetrics ( figo ) and the tumour node metastasis ( tnm ) classifications of ovarian cancer are based on intraoperative findings that include both cytological and histological assessment [ 6 ]  . transabdominal or transvaginal colour doppler ultrasound ( us ) is , together with the clinical evaluation , the first - line modality for reaching a diagnosis . 
magnetic resonance ( mr ) imaging , thanks to sequences characterised by high spatial and temporal resolution and dynamic contrastenhanced sequences , is a level - two examination for studying the female pelvis that provides a detailed anatomical depiction of the pelvic organs and permits distinction of the liquid , serous or haemorrhagic content of adnexal masses , thus guiding towards a diagnosis of benignity or malignancy . 
however , given the diverse presentations of ovarian cancers , the definite diagnosis is sometimes only established after exploratory laparotomy [ 8 ]  . computed tomography combined with positron emission tomography ( ct - pet ) with [ 18 ] f - fluorodeoxyglucose ( [ 18 ] f - fdg ) may be a valuable tool in assessing ovarian cancers , as it allows metabolic characterisation of lesions based on the increased glucose metabolism observed in tumour cells [ 9 ]  . 
in this context , even if us remains the method of choice for diagnosing ovarian cancer , mr and ct - pet imaging appear to be the two most promising modalities in this area , although with different features and results . 
ct - pet plays a fundamental role in disease staging , monitoring treatment response and identifying residual disease and recurrence , even though it has low sensitivity for identifying the primary lesion [ 10 ]  . 
this il carcinoma ovarico la seconda pi comune neoplasia maligna ginecologica con il pi elevato tasso di mortalit , presentando una sopravvivenza a 5 anni del 46% [ 1 ] ; ci dovuto anche al fatto che al momento della diagnosi circa il 75 % delle pazienti presenta una patologia in fase avanzata [ 2 ]  . 
fattori di rischio sono rappresentati da nulliparit , razza caucasica , menarca precoce , menopausa tardiva , disgenesia gonadica e ripetute stimolazioni ormonali in pazienti sottoposte a fecondazione assistita , mentre fattori protettivi risultano essere la pluriparit , lutilizzo di anticoncezionali estro - progestinici ed un prolungato periodo di allattamento al seno [ 5 ]  . 
 la classificazione del tumore ovarico da parte della federazione internazionale dei ginecologi e ostetrici ( figo ) e la classificazione tumore - noduli - metastasi ( tnm ) sono basate su reperti intra - operatori che includono sia la valutazione citologica che quella istologica [ 6 ]  . 
la risonanza magnetica ( rm ) , grazie allutilizzo di sequenze ad alta risoluzione spaziale e temporale , alluso di sequenze dinamiche dopo somministrazione di mezzo di contrasto , unindagine di seconda istanza valida nello studio della pelvi femminile , in quanto consente una dettagliata rappresentazione anatomica degli organi dello scavo pelvico e permette di distinguere il contenuto delle masse annessiali determinandone la natura liquida , sierosa o emorragica , indirizzando verso una diagnosi di benignit o di malignit . 
lo sviluppo di tecniche pi recenti ( rm spettroscopia e imaging di diffusione ) pu favorire la caratterizzazione corretta delle lesioni [ 7 ] tuttavia , a causa della eterogeneit di presentazione delle neoplasie ovariche , a volte si perviene alla diagnosi definitiva solo con la laparotomia esplorativa [ 8 ]  . 
 la tomografia computerizzata ( tc ) / tomografia ad emissione di positroni ( pet ) con 18f - fluoro - desossi - glucosio ( 18f - fdg ) pu fornire un valido contributo nella valutazione delle neoplasie ovariche , con la caratterizzazione metabolica delle lesioni in virt dellincremento del metabolismo glucidico nelle cellule tumorali [ 9 ]  . 
considerato quanto sopra detto , se lultrasonografia rimane la metodica di base nella diagnosi delle neoplasie ovariche , la rm e la tc / pet sembrano essere le due pi promettenti metodiche in tale ambito , sebbene con caratteristiche e risultati dif1290 radiol med ( 2011 ) 116 : 12881302 latter role could be played by mr imaging [ 8 ]  . to define malignancy of an adnexal lesion on mr imaging , published studies indicate a series of major criteria ( diameter > 4 cm , solid or predominantly solid lesion , intralesional necrosis , wall thickness > 3 mm , presence of internal vegetation or nodules , septations > 3 mm thick , necrosis ) , and minor criteria ( infiltration of other anatomical structures , presence of ascites , peritoneal involvement and satellite lymphadenopathies ) [ 11 , 12 ]  . the recent development of software that fuses data obtained with mr with the metabolic information provided by pet may have a positive effect on the diagnosis of adnexal lesions , facilitating appropriate surgical and therapeutic planning . 
the purpose of our study was to evaluate the diagnostic accuracy of mr , ct - pet and mr - pet fusion imaging in diagnosing ovarian lesions and the differential diagnosis between benign and malignant lesions . 
 materials and methods from june 2008 to september 2010 , we selected 29 patients ( mean age 4410 years ; range 2474 ) with an adnexal lesion incidentally diagnosed at us and / or multislice ct and no evident clinical symptoms . 
of the 29 selected patients , five were excluded from the study : one with breast cancer and a suspicious serous cystadenoma who underwent neoadjuvant chemotherapy for the breast cancer ; two who could not be studied by ct - pet owing to severe diabetes and two with a diagnosis of benign disease who underwent follow - up imaging . 
the patients finally included in the study underwent blood chemistry analysis with measurement of the specific tumour marker carbohydrate antigen ( ca ) 125 , pelvic mr and ct - pet imaging . 
 magnetic resonance imaging a few minutes before the mr examination , patients received an antispasmodic agent ( buscopan , one vial i.v. , boehringer ingelheim ) to reduce artefacts related to bowel peristalsis . 
 mr imaging was performed using a 3 - t permanent magnet ( achieva , philips medical system , best , the netherlands ) equipped with a slew rate of 150 mt m1 ms and a gradient field strength of 30 mt / m , with multichannel ( 6 channels ) phased - array coil . 
la tc / pet ha un ruolo fondamentale soprattutto nella stadiazione della malattia e nel monitoraggio della risposta alla terapia , ovvero nellindividuare la malattia residua e la presenza di recidive , pur essendo dotata di bassa sensibilit nellindividuazione della lesione primitiva [ 10 ] , ruolo che peraltro potrebbe svolgere la rm [ 8 ]  . 
 in relazione a quanto documentato in letteratura esistono , per definire la malignit di una lesione annessiale in rm , criteri maggiori ( diametro superiore a 4 cm , lesione solida o prevalentemente solida , necrosi intralesionale , spessore della parete maggiore di 3 mm , presenza di vegetazioni o nodularit interne , setti con spessore maggiore di 3 mm , necrosi ) e criteri minori ( infiltrazione di altre strutture anatomiche , presenza di ascite , interessamento peritoneale e linfoadenopatie satelliti ) [ 11 , 12 ]  . il recente sviluppo di un software che consente di fondere i dati ottenuti con la rm e le informazioni metaboliche della pet pu sortire un effetto positivo nella diagnosi delle lesioni annessiali , favorendo un appropriato planning chirurgico - terapeutico . 
scopo del nostro studio stato pertanto quello di valutare laccuratezza diagnostica dellimaging mediante rm , tc / pet e fusione rm / pet nella diagnosi delle lesioni ovariche e nella diagnosi differenziale tra formazioni benigne e maligne . 
 materiali e metodi da giugno 2008 a settembre 2010 sono state selezionate 29 pazienti ( et media : 4410 anni ; range 2474 anni ) con diagnosi di lesione annessiale mediante esame ecotomografico e / o tc multistrato di riscontro casuale , in assenza di sintomatologia clinica evidente . 
delle 29 pazienti selezionate , 5 sono state escluse dal nostro studio : una paziente gi affetta da carcinoma mammario con sospetto cistoadenoma sieroso stata sottoposta a chemioterapia neoadiuvante per la neoplasia della mammella ; due pazienti non sono state sottoposte allesame tc / pet per diabete severo e due pazienti con diagnosi di patologia benigna sono state inviate a follow - up strumentale a distanza . 
le pazienti incluse nello studio hanno eseguito esami ematochimici con dosaggio del marker tumorale specifico carbohydrate antigen 125 ( ca125 ) , esame rm della pelvi ed esame tc / pet . 
the dynamic study was performed with axial 3d t1 - weighted fast - field echo ( ffe ) with fat suppression [ t1 - weighted high - resolution isotropic volume examination ( thrive ) ] , with the possibility of obtaining multiplanar reconstructions ( mpr ) , after intravenous administration of a 0.1 mmol / kg bolus of paramagnetic contrast material [ gadolinium diethylenetriamine pentaacetic acid ( gd - dtpa ) , magnevist , schering , berlin , germany ] at a rate of 2 ml / s . mr images were examined by a radiologist experienced in pelvic mr and then compared with the postoperative histological findings . 
the lesions were considered malignant when they showed at least two of the major criteria described above ; they were considered benign when they showed none or only one of these criteria . 
this system provides 47 images with a 3.75 - mm interval covering a 157 - mm axial field of view ( fov )  . patients received a mean dose of 370 mbq of [ 18 ] f - fdg administered i.v. 
the interval between radiotracer administration and ct - pet imaging was approximately 50 m serum glucose levels were < 150 mg / dl ( mean 98 ; range 77124 ) in all cases . 
for image acquisition , a standard ct - pet protocol was used , from the top to the proximal third of the femur with the patient in the supine position . 
lesame rm stato eseguito con magnete permanente 3 , 0 t ( achieva , philips medical system , best , paesi bassi ) dotato di gradienti rapidi a 150 mt m1 / ms e ripidi di 30 mt / m con bobina phased - array multicanale ( 6 canali )  . 
 stata effettuata una valutazione morfologica della pelvi utilizzando le seguenti sequenze : scout ( gradient echo [ ge ] ) , ref scan per lapplicazione dellimaging parallelo , turbo spin echo ( tse ) - t1 sul piano assiale , tse - t2 sui piani assiale e coronale , tse - t2 - spectral attenuated inversion recovery ( spair ) con soppressione selettiva del segnale del tessuto adiposo sul piano assiale . 
lo studio dinamico stato eseguito con sequenze 3d fast field echo ( ffe ) - t1 con soppressione del grasso ( t1 high - resolution isotropic volume excitation , thrive ) sul piano assiale , con possibilit di ricostruzioni multiplanari , dopo somministrazione endovenosa a bolo di mezzo di contrasto paramagnetico gadobenato dimeglumina ( gd - dtpa ) alla velocit di 2 ml / s e con dosi di 0 , 1 mmol / kg ( magnevist , schering , berlin , germania )  . 
le lesioni sono state considerate maligne quando presentavano almeno due dei criteri maggiori di malignit descritti mentre sono state considerate benigne quando non presentavano alcuno o solo uno di tali criteri . 
lacquisizione delle immagini stata effettuata con tomografo ibrido tc / pet discovery 16st ( ge medical system milwakee , wi , usa ) , che combina uno scanner tc a 16 strati con un tomografo pet che consta di 10080 cristalli di bismuto - germanato ( bgo ) disposti su 24 anelli , con workstation dedicata windows advantage 4.4. 
le dimensioni dei cristalli sono di 6 , 36 , 330 mm3 e sono organizzati in blocchi di 66 cristalli , accoppiati con un singolo tubo fotomoltiplicatore con quattro anodi . 
questo sistema permette di ottenere 47 immagini con un intervallo di 3 , 75 mm , coprendo un campo di vista ( fov ) assiale di 157 m la dose media di 18f - fdg somministrata stata 370 mbq per via endovenosa , a digiuno , successivamente seguita da idratazione parenterale con 500 ml di soluzione fisiologica per ridurre la presenza di radiofarmaco nei reni . 
after pet , total - body ct was ( 120140 ma , automatic milliamperage , performed 3.75 - mm slice thickness with 1.25 - mm reconstruction increment , 0.5 - s rotation time , 512512 matrix ) after opacification of the bowel loops with orally administered contrast material and i.v. 
on the pet images , when sites of abnormal radiotracer uptake were identified , regions of interest ( roi ) were drawn to quantify the increased metabolism by calculating the standardised uptake value ( suvmax )  . 
the comparative analysis of images was therefore achieved by using the software advantage mr - pet fusion ( ge medical systems ) , which is available on the systems workstation ( advantage windows 4.4 , ge medical systems ) and allows simultaneous analysis of images after these have been saved in digital imaging and communication in medicine ( dicom ) format . 
in particular , ct ( obtained by coregistration with pet ) and mr images were first aligned by identifying three or more pairs of corresponding landmarks to align the anatomical images . 
subsequently , once the mr examination was sampled and adequately aligned with ct data , unnecessary ct data were discarded and , using the fusion software , were replaced by pet images . 
lacquisizione tc per lattenuazione , stata effettuata a basso dosaggio , con 80 ma e 120140 kv ad uno spessore di 3 , 75 mla correzione per attenuazione delle immagini pet stata ottenuta adattando le hounsfield units ( hu ) della tc al coefficiente lineare di attenuazione per fotoni di 511 kev . 
alla fine dellacquisizione pet si proceduto allesecuzione di un esame tc total body ( 120140 ma , amperaggio automatico , spessore di strato di 3 , 75 mm ricostruibile fino a 1 , 25 mm , tempo di rotazione di 0 , 5 secondi , matrice 512512 ) previa opacizzazione per os delle anse intestinali con mezzo di contrasto orale e somministrazione endovenosa di contrasto organo - iodato eseguendo una acquisizione in fase precoce delladdomepelvi ed unacquisizione pi tardiva del torace - addomepelvi . 
nelle immagini pet , quando documentati siti di uptake anomalo del radiofarmaco , sono state posizionate delle regions of interest ( roi ) per misurare lincremento del dato metabolico con il calcolo dello standardized uptake value massimo ( suvmax )  . 
tale valore la misura parametrica quantitativa massima della radioattivit misurata nel contesto della lesione in relazione alla dose totale di radiofarmaco somministrata e al peso corporeo , ottenuta con la seguente formula [ 13 ] : [ radioattivit ( kbq ) / volume del tessuto ( ml ) ] / [ dose somministrata ( kbq ) / peso corporeo ( gr ) ] il valore suvmax utilizzato come cut - off per distinguere le formazioni benigne dalle lesioni maligne stato 2 , 5 [ 14 ]  . 
 inoltre , sono state considerate come maligne le neoformazioni pelviche dotate di iperfissazione del radiofarmaco associate a linfonodi loco - regionali anchessi dotati di patologico uptake del 18f - fdg . 
 fusione e valutazione delle immagini rm / pet un tomografo che sia in grado di acquisire in coregistrazione e di fondere automaticamente le scansioni pet ed rm non al momento disponibile , pertanto lanalisi comparativa delle immagini stata possibile mediante softwareadvantage mr - pet fusion ( ge medical systems ) disponibile su consolle di ricostruzione , ( advantage windows 4.4 ; ge medical systems ) in grado di analizzare comparativamente le immagini una volta trasferite in digital imaging and communication in medicine ( dicom )  . 
in particolare si proceduto in prima istanza allallineamento delle immagini tc ( ottenute in coregistrazione con la pet ) radiol med ( 2011 ) 116 : 12881302 1293 final mr - pet diagnosis was obtained in consensus between the two physicians . 
sensitivity , specificity , positive ( ppv ) and negative ( npv ) predictive values were measured for ct - pet , mr and mr - pet fusion imaging using histology as the gold standard . 
suvmax values for malignant lesions were high except in patient numbers 7 , 11 , 12 , 16 and 18 , who were considered false negatives on ct - pet . 
successivamente , una volta campionato e allineato adeguatamente lesame rm con i dati tc , sono stati scartati i dati di tomografia computerizzata non pi necessari e , tramite il software di fusione , sono stati sostituiti dalle immagini pet . 
 nellanalisi delle immagini sono stati impegnati un medico nucleare ed un medico radiologo che hanno prima analizzato separatamente i due esami e poi hanno valutato insieme le due metodiche fuse cercando comparativamente alterazioni morfologiche o aree di alterato enhancement allesame rm cui corrispondessero aree di patologico incremento dellattivit metabolica in pet . 
sono stati calcolati i valori di sensibilit , specificit , valore predittivo positivo ( vpp ) e valore predittivo negativo ( vpn ) dellesame tc / pet , dellesame rm e della fusione delle immagini rm / pet , usando il referto istologico come esame gold standard.i dati del suvmax e del diametro delle lesioni ovariche sono stati confrontati tra i pazienti con lesioni benigne e con lesioni maligne usando il test u di mann - whitney . 
 risultati tutte le pazienti ( n = 24 ) incluse nello studio sono state sottoposte ad intervento chirurgico ( 11 per via laparotomica e 13 per via laparoscopica )  . 
le dimensioni medie delle lesioni maligne sono state di 43 , 629 mm ( range 9140 mm ) , il valore medio di suvmax 42 , 5 ( range 0 , 912 )  . 
 ct - pet detected 14 / 19 malignant lesions ( 74% ) , of which ten were serous cystadenocarcinomas , two were endometrial cystadenocarcinomas and two were mucinous cystadenocarcinomas at histology . 
with regard to the benign masses , ct - pet correctly identified four true negatives ( mean suvmax , 1 ) and one false positive result ( endometriotic cyst ) owing to a high suvmax . 
 mr - pet detected 18 / 19 malignant lesions ( 94% ) , of which 12 were serous cystadenocarcinomas , three were endometrial cystadenocarcinomas and three were mucinous radiol med ( 2011 ) 116 : 12881302 delle lesioni ( p = 0 , 62 ) , mente le lesioni maligne presentavano un suvmax nettamente pi alto rispetto a quello delle formazioni benigne ( p = 0 , 02 )  . tali risultati sono elencati nella tabella 1 per le lesioni maligne , insieme alle dimensioni riscontrate in rm , al valore di captazione semiquantitativo registrato in pet ( suvmax ) e ai risultati dellanalisi integrata rm / pet . 
i valori di suvmax per le lesioni maligne sono risultati elevati ad eccezione delle pazienti 7 , 11 , 12 , 16 e 18 , risultate in tc / pet come falsi negativi . 
per quanto riguarda le formazioni benigne la rm ha presentato 3 veri negativi ( dimensioni medie 28 mm ) e due falsi positivi , in entrambi i casi si trattava di cistoadenoma mucinoso . 
 lo studio tc / pet ha diagnosticato 14 / 19 lesioni maligne ( 74% ) , risultate allesame istologico 10 cistoadenocarcinomi sierosi , 2 cistoadenocarcinomi endometrioidi , 2 cistoadenocarcinoma mucinosi . 
per quanto riguarda le formazioni benigne la tc / pet ha correttamente individuato 4 veri negativi ( suvmax medio 1 ) e un falso positivo ( cisti endometriosica ) , a causa dellelevato suvmax . 
 lo studio rm / pet ha diagnosticato 18 / 19 lesioni maligne ( 94% ) , risultate allesame istologico 12 cistoadenocarcinomi sierosi , 3 cistoadenocarcinomi endometrioidi e 3 cistoadenocarcinomi mucinosi . 
solo un caso ( cistoadenocarcinoma mucinoso ) risultato in rm / pet come falso negativo , perch era negativo alla valutazione pet e presentava dubbie caratteristiche di malignit alla rm . 
 tutte le formazioni risultate allesame istologico come benigne sono state correttamente valutate in rm / pet come veri negativi ( 5 / 5 )  . i valori di sensibilit per rm e tc / pet sono risultati rispettivamente 84% e 74% mentre il valore di sensibilit della rm / pet risultato maggiore ( 94% )  . 
2a - c axial pet image showing pathological uptake of the tracer in the right pelvic region ( a ) , corresponding to an ovarian lesion with mixed fluid and solid component on axial t2 - weighted mr images ( b )  . 
2a - c immagine assiale pet mostra patologica captazione del radiofarmaco in sede pelvica destra ( a ) in corrispondenza di lesione annessiale a componente mista solida e fluida nelle sequenze rm assiali t2 pesate ( b )  . 
listologico ha documentato un cistoadenocarcinoma mucinoso ( paziente 10 in tabella 1 )  . stati rispettivamente del 93% e 44% per la tc / pet , 89% e 50% per la rm e 100% ed 83% per la rm / pet . discussione le pazienti con tumore ovarico vengono spesso sottoposte , in fase di stadiazione , sia ad esame rm della pelvi che ad esame tc / pet . 
considerato questo presupposto interessante valutare se esiste un meccanismo che possa comparare direttamente i rilievi ottenuti con le due metodiche e la sua utilit clinica e diagnostica [ 15 ]  . 
lecografia lesame di prima istanza nella valutazione delle lesioni annessiali , in quanto fornisce informazioni sulla presenza di neoformazioni ovariche , presentando il limite di una non corretta diagnosi differenziale in alcuni casi . 
eventuali alterazioni morfologiche possono inoltre essere valutate anche dopo somministrazione di mezzo di contrasto . per la valutazione morfologica delle lesioni e dellenhancement post - contrastografico fondamentale lesame dei segni diretti e indiretti di neoplasia [ 11 , 12 ]  . 
one case alone ( mucinous cystadenocarcinoma ) proved to be a false negative on mr - pet , as it was negative on pet and showed doubtful signs of malignancy on mr imaging . 
all lesions diagnosed as benign at histology were correctly indicated as true negatives ( 5 / 5 ) on mr - pet . sensitivity values for mr and ct - pet were 84% and 74% , respectively , whereas those of mr - pet were higher ( 94% )  . 
ppv and npv were 93% and 44% for ct - pet , 89% and 50% for mr imaging and 100% and 83% for mr - pet , respectively . discussion during staging , patients with ovarian cancer often undergo both pelvic mr imaging and ct - pet . 
us is the level - one modality for studying adnexal lesions in that it provides information about the presence of ovarian masses , even though with the limitation of an imprecise differential diagnosis in some cases . 
attualmente la rm rappresenta comunque unindagine deccellenza nella diagnosi delle neoplasie ovariche , in quanto ci indirizza ad un adeguato planning terapeutico , risultando utile come metodica problem - solving rispetto al solo esame ecotomografico ed al dosaggio dei markers tumorali [ 16 ]  . 
in addition , any morphological abnormality may also be evaluated after administration of contrast material . in the morphological evaluation of lesions and contrast enhancement , the study of direct and indirect signs of neoplasm is fundamental [ 11 , 12 ]  . 
mr imaging is nonetheless an excellent examination tool in diagnosing ovarian cancer , as it guides towards adequate treatment planning and is a useful problem - solving technique compared with us alone and tumour marker measurement [ 16 ]  . 
 at ct - pet , the increased glucose metabolism of tumour cells is reflected in [ 18 ] f - fdg uptake by neoplastic lesions [ 17 ] where , once in the cytoplasm , it is phosphorylated but , unlike glucose , can no longer represent a substrate for further reactions and remains trapped inside the cell membrane [ 9 ]  . 
for these reasons , and in consideration of its co 18f - fdg allinterno delle lesioni discariocinetiche [ 17 ] ove , una volta trasportato allinterno del citoplasma , viene fosforilato ma , a differenza del glucosio , non pu pi costituire un substrato per ulteriori reazioni e rimane intrappolato allinterno della membrana cellulare [ 9 ]  . 
le applicazioni cliniche dello studio tc / pet nel tumore dellovaio riguardano inoltre non solo la caratterizzazione della lesione primitiva ma anche la stadiazione locoregionale dei linfonodi e la valutazione di metastasi a distanza . 
per queste ragioni e in considerazione dellelevato costo la tc / pet non viene comunemente utilizzata nella diagnosi della lesione primaria , pu tuttavia rappresentare un valido ausilio in pazienti selezionate , in virt del suo elevato potere predittivo positivo . 
la tc / pet risulta inoltre particolarmente utile nella valutazione della recidiva locale e nella diagnosi differenziale tra malattia attiva versus fibrosi ( ad esempio in fase post - attinica ) laddove limaging tradizionale risulta scarsamente dirimente [ 18 ]  . 
questo ruolo appare oggi indiscusso in considerazione della possibilit di sfruttare sia le capacit diagnostiche della tc total body , associata alla somministrazione di mezzi di contrasto orale ed endovenoso , sia il 1298 radiol med ( 2011 ) 116 : 12881302 fig . 
3a - d axial t2 - weighted mr image ( a ) , t2 spectral selection attenuated inversion recovery ( spair ) image ( b ) , t1 - weighted image ( c ) and thrive image after gadolinium administration ( d ) show bulky septated mass in the pelvis . 
3a - d lesame rm con sequenze assiali t2 pesate ( a ) , sequenze assiali t2 spair ( b ) , sequenze assiali t1 pesate ( c ) e sequenze assiali thrive dopo somministrazione di gadolinio ( d ) documenta voluminosa neoformazione in regione annessiale con setti nel contesto . 
listologico ha documentato un cistoadenocarcinoma sieroso ( paziente 3 in tabella 1 )  . high costs , ct - pet is not commonly used for diagnosing the primary lesion ; nonetheless , its high ppv makes it a valuable aid in the study of selected patients . 
furthermore , it has been shown that ct - pet studies can frequently modify the staging obtained with conventional imaging , thus influencing treatment plans and patient prognosis [ 19 ]  . 
 stato inoltre dimostrato che lo studio tc / pet in grado di cambiare frequentemente la stadiazione ottenuta con limaging tradizionale con effetto sullapproccio terapeutico e la prognosi [ 19 ]  . 
in epoca pre - pet il gold standard per stabilire lefficacia di una terapia antiblastica era la valutazione del cambiamento morfologico e dimensionale delle lesioni ; poich tuttavia i cambiamenti metabolici molecolari precedono le variazioni anatomiche , la valutazione quantitativa con suvmax dei diversi valori di captazione del glucosio , prima e dopo trattamento , pu dare una risposta precoce sullefficacia del trattamento iniziato con la possibilit di cambiarlo o correggerlo in itinere [ 18 ]  . 
 altri autori hanno dimostrato una correlazione tra il cambiamento quantitativo della captazione , la risposta clinica e la variazione dei livelli di alcuni markers ( ca125 ) [ 20 ]  . 
 radiol med ( 2011 ) 116 : 12881302 1299 advent of pet imaging , the gold standard for establishing the effectiveness of antineoplastic therapy was the study of morphological and dimensional changes of the lesions ; however , as molecular metabolic changes precede anatomical variations , a quantitative assessment based on suvmax of glucose uptake values before and after treatment may provide an early assessment of treatment effectiveness so that it can be changed or adjusted [ 18 ]  . 
 therefore , in view of the role of mr imaging in diagnosing ovarian lesions , with an accuracy similar to that of postoperative staging [ 21 ] , and the performance of ct - pet in providing a metabolic characterisation of these lesions and evaluating distant metastases , the potential role of combined mr - pet in studying adnexal masses appears clear , even though further studies involving larger patient populations are required . 
with regard to the ppv ( 100% ) and npv ( 83% ) shown by fusion imaging , we can state that a positive result on mr - pet invariably corresponds to a positive histological finding , whereas a negative result cannot rule out a positive histological result , although keeping in mind the limited population in our series . 
 in table 1 , among the histologically proven malignant lesions are two lesions that , due to their small size and predominantly fluid component , were incorrectly considered negative on ct - pet ( patients 7 and 18 ) : in these two cases , the suvmax value was negligible , and the correct diagnosis was obtained with mr imaging , which consequently also affected the mr - pet study . 
pet imaging has limited resolution in diagnosing small neoplasms , having a cutoff of approximately 8 mas a consequence , the difficulty in clinical practice lies in obtaining a metabolic characterisation of lesions < 8 mm that , despite being visible at ct - pet , cannot be studied from a metabolic point of view . 
among benign lesions , ct - pet imaging yielded one false positive result , which turned out to be an endometriconsiderato pertanto il ruolo della rm nella diagnosi delle lesioni ovariche , che presenta accuratezza simile a quella effettiva della stadiazione post - chirurgica [ 21 ] , e in relazione a quanto espresso sulla performance della tc / pet nella caratterizzazione metabolica di tali lesioni e nella valutazione delle metastasi a distanza , appare evidente il ruolo potenziale della valutazione integrata rm / pet nello studio delle neoformazioni annessiali , tuttavia sono necessari ulteriori studi sullargomento su casistiche pi ampie . 
secondo i nostri risultati la rm ha presentato valori di sensibilit superiori a quelli della tc / pet ( 84% rispetto a 74% ) mentre la specificit di questultima metodica si rivelata maggiore ( 80% rispetto a 60% )  . 
si pu affermare che la rm sia pi indicata nella diagnosi delle lesioni maligne e che la tc / pet invece consenta di definire meglio la benignit delle neoformazioni ovariche . 
in relazione al vpp ( 100% ) e al vpn ( 83% ) dimostrati dalla fusione delle due metodiche si pu affermare che lesito positivo della rm / pet sia associato invariabilmente alla positivit del reperto istologico mentre lesito negativo non possa tuttavia escludere la positivit allesame istologico , tenendo presente tuttavia la ridotta popolazione in esame . 
 nella tabella 1 , tra le lesioni maligne allesame istologico , sono presenti due lesioni che , per le loro esigue dimensioni o per la prevalente componente fluida , erano risultate falsamente negative allesame tc / pet ( pazienti 7 e 18 ) : in questi due casi il valore di suvmax documentato era trascurabile e la diagnosi corretta stata ottenuta con la valutazione rm , che di conseguenza ha avuto il suo effetto anche nella valutazione integrata rm / pet . 
il potere di risoluzione della pet nella diagnosi di neoplasie di piccole dimensioni limitato con un cut - off di circa 8 mdi conseguenza la difficolt nella pratica clinica si traduce nel caratterizzare metabolicamente lesioni inferiori a tale diametro che , pur essendo visibili nella tc associata alla pet , non possono essere valutate dal punto di vista metabolico . 
in tal caso ipotizzabile che lelevata captazione del radiofarmaco nel contesto della formazione ( suvmax 3 , 6 ; paziente 1 ; tabella 2 ) potesse esser dovuta allelevato metabolismo espresso dalle cellule infiammatorie [ 22 ]  . in relazione ai falsi negativi della rm tuttavia la caratterizzazione metabolica della tc / pet ha permesso di identificare come maligne due lesioni , identificate come negative radiol med ( 2011 ) 116 : 12881302 1300 fig . 
4a - c axial t2 - weighted mr image ( a ) showing cystic lesion with a wall thickness > 3 mon the axial t2 spair image ( b ) , an internal nodule is evident ( arrow )  . 
4a - c lesame rm con sequenza assiale t2 pesata ( a ) mostra lesione a contenuto fluido con ispessimento della parete maggiore di 3 mm ; nella sequenza t2 assiale spair ( b ) visibile gettone solido ( freccia )  . 
in this case , the strong uptake of contrast material inside the lesion ( suvmax 3.6 ; patient 1 , table 2 ) was probably caused by the accelerated metabolism of inflammatory cells [ 22 ]  . with regard to mr false negative results , the metabolic characterisation of ct - pet allowed detection of two malignant lesions that had strong [ 18 ] f - fdg uptake and that were shown to be two endometrial carcinomas at histology ( patients 8 and 9 ; table 1 )  . 
only combined mr - pet assessment indicated these two cases as true negatives , so it may be stated that the added value of mr - pet was mainly observed in the correct identification of benign masses , with a high specificity ( 100% )  . 
in tc / pet tuttavia risultata falsamente positiva una cisti endometriosica , verosimilmente a causa dellaumentato turn - over cellulare indotto dallinfiammazione : solo la valutazione integrata rm / pet ha permesso di identificare come veri negativi questi ultimi due casi presentati , per cui si pu affermare che il valore aggiunto della rm / pet si osservato in particolare nella identificazione corretta delle formazioni benigne , con specificit elevata ( 100% )  . 
per quanto riguarda i linfonodi il limite della rm rappresentato dal ristretto fov di acquisizione nella valutazione della pelvi , potendo valutare le sole stazioni pelviche e risultando perradiol med ( 2011 ) 116 : 12881302 1301 whole - body ct - pet imaging provided superior longterm staging . 
with regard to lymph nodes , the limitation of mr imaging is its reduced fov , as it can only study the pelvic nodal stations and is thus inferior to ct - pet . 
therefore , ct - pet also plays a key role in studying extrapelvic lymph nodes and evaluating distant metastases , in particular , in lesions > 1 cmr imaging proved to be superior in locoregional staging . 
in consideration of the above , the use of ct - pet is reasonable in all cases with doubtful characterisation on mr and when there is a high suspicion of distant metastases . 
this may also occur on ct - pet , although the time needed for the examination to be reliable is a few weeks only , that is , less than that needed for mr . 
future studies by our department on larger series will ascertain the usefulness of combined pelvic mr fusion imaging and whole - body ct - pet for characterising pelvic masses and in prognostic stratification . 
the major goal is to avoid unnecessary invasive surgical procedures in the case of false positive lesions on mr or ct - pet that turn out to be true negatives on combined mr - pet assessment . 
pertanto la tc / pet ha un ruolo importante anche nello studio delle stazioni linfonodali di drenaggio extra - pelviche e nella valutazione delle localizzazioni a distanza , soprattutto nelle lesioni superiori al centimetro . 
quanto sopra espresso rende ragionevole ricorrere alla tc / pet in tutti quei casi in cui sia dubbia la caratterizzazione delle lesioni in rm e quando il sospetto di metastasi a distanza sia elevato . 
anche in tc / pet la reazione infiammatoria indotta dalla terapia attinica pu creare dei risultati falsamente positivi ; tuttavia il tempo necessario affinch lindagine sia attendibile solo di alcune settimane e comunque inferiore rispetto alla rm . 
futuri studi in programma nel nostro dipartimento su pi ampie casistiche potranno effettivamente chiarire lutilit della valutazione integrata con fusione delle immagini rm della pelvi e tc / pet whole body nella caratterizzazione delle neoformazioni pelviche e nella stratificazione prognostica . 
 in relazione ai nostri risultati la valutazione combinata rm / pet sarebbe auspicabile al fine di implementare i valori di sensibilit e di specificit delle due metodiche in ununica sessione . 
il fine pi importante potrebbe essere quello di evitare interventi chirurgici invasivi non necessari , in riferimento alle lesioni falsamente positive documentate in rm o tc / pet che si sono dimostrate come veri negativi alla valutazione integrata rm / pet . 
cademartiri1 , 2 1department of radiology and cardiology , azienda ospedaliero - universitaria , parma , italy 2department of radiology and cardiology , erasmus medical center , rotterdam , the netherlands 3department of radiology , policlinico g.b. 
giaccone , university of palermo , palermo , italy 5department of radiology , ospedale san gennaro , napoli , italy 6department of radiology , sdn - irccs foundation , napoli , italy correspondence to : f . 
tel. : + 39 - 0521 - 703516 , fax : + 39 - 0521 - 703630 , e - mail : filippocademartiri@gmail.com received : 23 july 2009 / accepted : 24 september 2009 / published online : 4 september 2011 springer - verlag 2011 abstract purpose . 
a total of 722 patients ( 480 men ; 62.710.9 years ) who were referred for further cardiac evaluation underwent cacs and contrast - enhanced ctca to evaluate the presence and severity of cad . 
 patients were stratified according to the morise clinical score ( low , intermediate , high ) , to cacs ( 010 , 11100 , 101400 , 4011 , 000 , > 1 , 000 ) and to ctca ( absence of cad , nonsignificant cad , obstructive cad )  . 
significant cad ( > 50% luminal narrowing ) was detected in 260 ( 36% ) patients ; nonsignificant cad ( < 50% luminal narrowing ) in 250 ( 35% ) and absence of cad in 212 ( 29% )  . 
settecentoventidue pazienti ( 480 maschi ; et media 62 , 710 , 9 anni ) con indicazione ad approfondimento cardiologico sono stati sottoposti a cacs e ct - ca per la valutazione della presenza e severit di cad . 
i pazienti sono stati stratificati secondo lo score di morise ( basso , intermedio , alto ) , secondo il cacs ( 010 , 11100 , 101400 , 4011000 , > 1000 ) e secondo la ct - ca ( assenza di cad , cad non significativa , cad ostruttiva )  . 
cad ostruttiva ( riduzione del lume > 50% ) stata rilevata in 260 ( 36% ) pazienti ; cad non significativa ( riduzione del lume < 50% ) in 250 ( 35% ) e assenza di cad in 212 ( 29% ) pazienti . 
three hard events ( 14% ) occurred in patients with cacs100 and two ( 9.5% ) in patients with intermediate morise score ; one revascularisation was observed in a patient with low morise score . 
cacs 100 and lowintermediate morise score did not exclude the possibility of events at follow - up . keywords computed tomography coronary angiography prognostic value prognosis calcium score morise score nei pazienti con assenza di cad alla ct - ca gli eventi sono stati lo 0% contro l1 , 7% ed il 7 , 3% dei pazienti con cad non ostruttiva e cad ostruttiva , rispettivamente ( p < 0 , 0001 )  . 
due eventi hard ( 9 , 5% , 2 / 21 ) sono stati osservati in pazienti con morise score intermedio ed una rivascolarizzazione in paziente con morise score basso . 
cacs100 ed uno score di morise basso - intermedio non escludono la possibilit di eventi al follow - up . parole chiave angiografia coronarica con tomografia computerizzata valore prognostico prognosi calcium score score di morise introduction introduzione computed tomography coronary angiography ( ctca ) is an emerging technique for the noninvasive evaluation of coronary atherosclerosis in patients with suspected and known coronary artery disease ( cad )  . 
current - generation ct scanners have a high diagnostic accuracy to detect and exclude obstructive cad compared with conventional coronary angiography ( cag ) in selected patients [ 14 ]  . 
however , few data are available on the prognostic value of ctca [ 917 ]  . preliminary reports suggest that ctca has a very high negative predictive value ( npv ) for future cardiovascular events [ 916 ]  . 
the coronary artery calcium score ( cacs ) has also been widely used in north america and asia for stratifying cardiovascular risk in usually asymptomatic patients [ 18 , 19 ]  . very few reports have compared the prognostic value of ctca with other risk stratification strategies [ 1113 , 20 , 21 ]  . 
the aim of this study was therefore to assess the proglangiografia coronarica mediante tomografia computerizzata ( ct - ca ) una metodica diagnostica emergente per la valutazione non invasiva dellaterosclerosi coronarica in pazienti con malattia coronarica ( cad ) sospetta o nota [ 14 ]  . 
lattuale generazione di apparecchiature per la tomografia computerizzata ( tc ) ( 64 strati ) ha una elevata accuratezza diagnostica nella rilevazione della cad ostruttiva se comparata con la coronarografia percutanea ( cag ) in popolazioni selezionate di pazienti [ 14 ]  . 
al contrario , esistono pochi dati sul valore prognostico della ct - ca [ 917 ]  . studi preliminari suggeriscono che la ct - ca abbia un elevato valore predittivo negativo per eventi cardiovascolari [ 916 ]  . 
il calcium score coronarico ( cacs ) stato utilizzato ampiamente in nord america 1190 radiol med ( 2011 ) 116 : 11881202 nostic value of ctca in patients with suspected or known cad and compare it with cacs and a clinical score [ 19 , 22 , 23 ]  . materials and methods patients the study population included 788 consecutive patients ( between april 2006 and december 2007 ) who underwent ctca for suspected or known cad . 
at follow - up , we lost 66 ( 8% ) patients ; therefore , the final study population consisted of 722 patients ( 480 men , 242 women ; mean age 63 11 years )  . 
the following risk factors were considered : ( 1 ) hypertension ( defined as arterial pressure 140 / 80 mmhg or need for antihypertensive therapy ) ; ( 2 ) hypercholesterolemia [ low - density lipoprotein ( ldl ) cholesterol > 130 mg / dl ] ; ( 3 ) diabetes mellitus ; ( 4 ) smoking habit ; ( 5 ) obesity [ body mass index ( bmi ) > 30 ] ; ( 6 ) family history of cardiac disease . 
patients with a history of cad were directly classified with a score > 15 . ctca data acquisition all examinations were performed with a single - source 64 - slice ct system ( somatom sensation 64 cardiac , siemens , forchheim , germany ) [ 24 ]  . 
first , a prospectively ed in asia per la stratificazione del rischio cardiovascolare in pazienti prevalentemente asintomatici [ 18 , 19 ]  . pochi sono gli studi di paragone tra il valore prognostico della ct - ca ed altre strategie di stratificazione del rischio [ 1113 , 20 , 21 ]  . 
lo scopo dello studio quello di valutare il valore prognostico della ct - ca in pazienti con cad sospetta o nota e paragonarlo con il cacs ed uno score clinico [ 19 , 22 , 23 ]  . materiali e metodi pazienti la popolazione studiata consiste di 788 pazienti consecutivi ( periodo aprile 2006 e dicembre 2007 ) sottoposti a ctca per cad sospetta o nota . 
i pazienti sono stati sottoposti a ct - ca a causa di sintomi ( dolore toracico , dispnea ) , test provocativi anormali e / o elevato profilo di rischio cardiovascolare . 
al followup 66 ( 8% ) pazienti sono stati persi , quindi la popolazione finale dello studio consiste di 722 pazienti ( 480 maschi , 242 femmine , et media 6311 anni )  . 
sono stati considerati i seguenti fattori di rischio : ( 1 ) ipertensione ( definita come pressione arteiosa 140 / 80 mmhg o necessit di terapia anti - ipertensiva ) ; ( 2 ) iper - colesterolemia ( colesterolo ldl > 130 mg / dl ) ; ( 3 ) diabete mellito ; ( 4 ) fumo ; ( 5 ) obesit ( indice di massa corporea [ bmi ] > 30 ) ; ( 6 ) familiarit per malattie cardiovascolari . 
 lo score di morise stato calcolato per stratificare il rischio pre - test risk dei pazienti [ 22 , 23 ]  . score di morise lo score di morise [ 23 ] basato sullet , lo stato estrogenico , la storia di angina secondo il metodo di diamond [ 22 ] ed i principali fattori di rischio cardiovascolare ( diabete , dislipidemia , ipertensione , fumo , familiarit )  . 
ad ogni parametro viene assegnato un punteggio ed un punteggio totale pari a 08 rappresenta un rischio basso , un punteggio pari a 915 rappresenta un rischio intermedio , mentre un punteggio > 15 rappresenta un rischio elevato . 
i pazienradiol med ( 2011 ) 116 : 11881202 1191 triggered coronary calcium ct data set was obtained using standard ct parameters comprising 201.2 - mm collimation , 330 ms gantry rotation time , 120 kv tube voltage and 150 mas tube current . 
thereafter , ct coronary angiography was performed after administration of 100 ml nonionic contrast material ( iomeron 400 mgi / ml , bracco , milan , italy ) at a flow rate of 46 ml / s depending on patient status . 
ctca was performed with 640.6 - mm collimation , 330 ms gantry rotation time , 120 kv tube voltage , 900 mas tube current , pitch 0.20 , temporal resolution 165 ms , spatial resolution 0.4mm3 , reconstruction slice thickness 0.75 mm and reconstruction increment 0.5 mintravenously administered beta - blocker ( atenolol 510 mg ) was administered to all patients with a heart rate > 65 bpm and without contraindications ( asthma or bronchospasm , systolic blood pressure < 100 mmhg )  . 
to obtain optimal image quality , data sets were reconstructed at least at two points of the cardiac cycle using a retrospective ecg - gating algorithm ( one diastolic cardiac phase usually at 350 ms from the r waves and one end - systolic phase at + 275 ms ) ; tube current modulation was not used . 
axial data sets were transferred to a remote workstation ( leonardo , siemens medical solutions , forchheim , germany ) for postprocessing and subsequent evaluation . ct data analysis coronary artery calcium score cacs was assessed with dedicated software ( cascore , siemens medical solutions , forchheim , germany )  . 
for multivariate analysis , patients were divided into five groups based on cacs score : group 1 , 010 ; group 2 , 11100 ; group 3 , 101400 ; group 4 , 4011 , 000 ; group 5 , > 1 , 000 . 
 ti con cad nota vengono direttamente classificati con un punteggio > 15 . scansione ct - ca tutte le ct - ca sono state effettuate mediante unapparecchiatura tc a singola sorgente 64 - strati ( somatom sensation 64 cardiac , siemens , forchheim , germania ) [ 24 ]  . 
inizialmente , abbiamo eseguito una scansione con triggering ecg prospettico senza mezzo di contrasto per la quantificazione del calcio coronarico utilizzando parametri standard ( collimazione 201 , 2 mm , tempo di rotazione del gantry 330 ms , 120 kv , corrente del tubo 150 mas )  . 
quindi , abbiamo eseguito la ct - ca dopo la somministrazione endovenosa di 100 ml di mezzo di contrasto non ionico ( iomeron 400 mgi / ml , bracco , milano , italia ) con un flusso di 46 ml / s a seconda delle caratteristiche del paziente . 
la scansione ct - ca stata effettuata con collimazione 64 ( 322 ) 0 , 6 mm , tempo di rotazione del gantry 330 ms , 120 kv , corrente del tubo 900 mas , pitch 0 , 20 , risoluzione temporale 165 ms , e risoluzione spaziale 0 , 4 mm3 , spessore ricostruito degli strati 0 , 75 mm e incremento di ricostruzione 0 , 5 min tutti i pazienti con frequenza cardiaca > 65 bpm ed in assenza di contro - indicazioni ( asma bronchiale o broncospasmo documentati , pressione sistolica < 100 mmhg ) sono stati somministrati beta - bloccanti per via endovenosa ( atenololo 510 mg )  . 
inoltre , in tutti i pazienti senza contro - indicazioni ( stenosi aortica severa , pressione sistolica < 100 mmhg ) sono stati somministrati 0 , 3 mg di nitroglicerina per via sub - linguale [ 25 ]  . 
i dataset sono stati ricostruiti in almeno due punti del ciclo cardiaco utilizzando la tecnica ecg retrospettiva ( una fase diastolica di solito a - 350 msec dallonda r ed una fase sistolica di solito a + 275 msec dallonda r ) al fine di ottenere una migliore qualit di immagine . 
i dataset assiali sono stati trasferiti ad una workstation ( leonardo , siemens medical solutions , forchheim , germania ) per il post - processing e le successive analisi . coronary artery lesion assessment analisi dei dati tc all ctca were evaluated by two experienced observers who were unaware of the patients clinical history . 
patients were finally classified as belonging to one of three groups based on ctca findings : ( 1 ) normal coronary arteries , ( 2 ) nonobstructive cad and ( 3 ) obstructive cad . 
clinical end - points were : ( 1 ) cardiac deathdefined as death caused by acute myocardial infarction , ventricular arrhythmias or refractory heart failure ; ( 2 ) nonfatal myocardial infarction - defined based on criteria of typical chest pain , elevated cardiac enzyme levels and / or typical changes on the ecg ; ( 3 ) unstable angina ; ( 4 ) myocardial revascularisation ( percutaneous coronary intervention / coronary artery bypass grafting )  . survival analysis was performed for total cardiac events [ endpoints 14 ; major adverse cardiac events ( mace ) ] and for clustered endpoints 13 , defined as hard events . 
rca , coronaria destra ; lca , coronaria sinistra ; lm , tronco comune ; cx , coronaria circonflessa ; lad , coronaria discendente anteriore calcio coronarico stato identificato sulle immagini assiali come unarea densa sul tragitto delle arterie coronarie caratterizzata da unattenuazione 130 unit hounsfield ( hu )  . 
per lanalisi multi - variata i pazienti sono stati suddivisi in 5 gruppi a seconda del punteggio cacs : gruppo 1 con cacs 010 , gruppo 2 con cacs 11100 , gruppo 3 con cacs 101400 , gruppo 4 con cacs 4011000 , e gruppo 5 con cacs > 1000 . 
utilizzando i dati precedenti i pazienti sono stati classificati sulla ct - ca in tre gruppi : ( 1 ) pazienti con coronarie normali , ( 2 ) pazienti con cad non ostruttiva , ( 3 ) pazienti con cad ostruttiva . 
gli end - point clinici utilizzati erano : ( 1 ) morte cardiaca , ( 2 ) infarto miocardico non - fatale , ( 3 ) angina instabile , ( 4 ) rivascolarizzazione miocardica ( percutanea / chirurgica , pci / cabg )  . 
lanalisi di sopravvivenza stata effettuata per gli eventi totali ( endpoints 14 ; major adverse cardiac events , mace ) e per gli eventi hard ( endpoints 13 )  . 
 radiol med ( 2011 ) 116 : 11881202 1193 statistical analysis analisi statistica categorical baseline characteristics , expressed as numbers and percentages , were compared using the chi - square test . 
 continuous variables , expressed as mean and standard deviation ( sd ) , were compared using the two - tailed t test and analysis of variance ( anova ) if normally distributed or by means of the chi - square and kruskalwallis method if not normally distributed . 
event curves of the composite endpoint ( cardiac death , nonfatal infarction , angina requiring hospitalisation , myocardial revascularisation ; mace ) and hard events ( cardiac death , nonfatal infarction , unstable angina ) were compared using the log - rank test . 
la variabili continue , espresse come medie e deviazioni standard ( sd ) , sono state confrontate mediante il test t di student a due code e lanalisi della varianza quando caratterizzate da distribuzione normale , e con il test del chi - quadrato e di kruskal - wallis se non normalmente distribuite . 
per la prognosi abbiamo utilizzato un end - point composito caratterizzato da morte cardiaca , infarto miocardico non - fatale , angina instabile , e rivascolarizzazione ( mace )  . 
prevalence of significant cad was 10% ( 43 / 413 ) in the minimal / mild cacs ( 100 ) , 47% ( 48 / 103 ) in the moderate cacs ( 101400 ) and 82% ( 169 / 205 ) in the severe cacs ( > 400 )  . settecento - ventidue pazienti sono stati inclusi nellanalisi finale . 
of risk factors ( meansd ) history of cad , n ( % ) absent present ami revascularisation ami + revascularisation symptoms , n ( % ) asymptomatic typical chest pain atypical chest pain other dyspnoea 62.510.9 480 ( 66 ) 274 6110 bpm 92 ( 12.7 ) 451 ( 62 ) 367 ( 51 ) 256 ( 35 ) 384 ( 53 ) 156 ( 22 ) 2.41.2 511 ( 71 ) 211 ( 29 ) 39 ( 6 ) 68 ( 9 ) 104 ( 14 ) 207 ( 28 ) 143 ( 20 ) 229 ( 32 ) 143 ( 20 ) et ( anni ; mediads ) genere maschile bmi ( kg / m ; mediads ) frequenza cardiaca ( mediads ) fattori di rischio cardiovascolari , n ( % ) 62 , 510 , 9 480 ( 66% ) 274 6110 bpm 92 ( 12 , 7 ) diabete 451 ( 62 ) ipertensione 367 ( 51 ) dislipidemia 256 ( 35 ) fumo 384 ( 53 ) familiarit per cad obesit ( bmi > 30 ) 156 ( 22 ) numero di fattori di rischio ( mediads ) 2 , 41 , 2 storia di cad , n ( % ) assente presente ima rivascolarizzazione ima + rivascolarizzazione simtomi , n ( % ) asintomatici dolore tipico dolore atipico altro dispnea 511 ( 71 ) 211 ( 29 ) 39 ( 6 ) 68 ( 9 ) 104 ( 14 ) 207 ( 28 ) 143 ( 20 ) 229 ( 32 ) 143 ( 20 ) sd , standard deviation ; cad , coronary artery disease ; n , number of patients ; ami , acute myocardial infarction ; bmi , body mass index ds , deviazione standard ; cad , malattia coronarica ; ima , infarto miocardico acuto ; bmi , body mass index ctca stratification a total of 10 , 918 coronary segments were assessed ( 137 ; 1% of nondiagnostic quality )  . 
for the morise score , it is possible to observe that in the two most represented groups ( intermediate and high risk ; 92% of patients ) , ctca can substratify a substantial number of patients without any cad ( 35% in the intermediate risk and 16% in the high risk )  . 
the prevalence of nonobstructive cad remains somewhat stable throughout the three groups , whereas the prevalence of significantly obstructive disease is 3% in the low - risk group , 18% in the intermediate - risk group and 56% in the high - risk group . 
la prevalenza di cad ostruttiva era del 10% ( 43 / 413 ) nei gruppi con cacs100 , del 47% ( 48 / 103 ) nel gruppo con cacs 101400 e del 82% ( 169 / 205 ) nei gruppi con cacs > 400 . stratificazione di malattia secondo la ct - ca sono stati valutati un totale di 10918 segmenti coronarici ( 137 ; 1% di qualit non diagnostica )  . 
riguardo lo score di morise si pu osservare che nei due gruppi pi rappresentati ( rischio intermedio e alto ; corrispondenti al 92% dei pazienti ) , la ct - ca ri - stratifica una consistente quota di pazienti senza alcuna cad ( 35% nel gruppo a rischio intermedio e 16% nel gruppo ad alto rischio )  . 
in particular , the low fraction of patients at low risk using the morise clinical score ( 8% ; a ) compared with cacs ( 40% ; b ) and ctca ( 29% ; c )  . 
in particolare , possiamo osservare la bassa frazione di pazienti a basso rischio del morise score ( 8% ; a ) , a paragone del cacs ( 40% ; b ) e della ct - ca ( 29% ; c )  . 
cad , malattia coronarica ; cacs , calcium score coronarico ; ctca , angiografia coronarica con tomografia computerizzata . 1196 radiol med ( 2011 ) 116 : 11881202 represents 5% in the group with cacs 010 and 85% in the group with cacs > 1 , 000 . 
nonobstructive disease has a prevalence of 22% in the group with cacs 010 and 15% in the group with cacs > 1 , 000 ; however , it represents > 50% of patients in the groups with cacs 11100 and cacs 101400 . 
ami , infarto miocardico acuto ; ua , angina instabile ; ca , arresto cardiaco ; vt , tachicardia ventricolare ; vf , fibrillazione ventricolare ; pci , angioplastica coronarica ; cabg , bypass aortocoronarico . mentre la prevalenza di cad ostruttiva il 3% nel gruppo a basso rischio , del 18% nel gruppo a rischio intermedio e del 56% nel gruppo ad alto rischio . 
la cad non ostruttiva ha una prevalenza del 22% nel gruppo con cacs 010 e del 15% nel gruppo con cacs > 1000 , tuttavia rappresenta > 50% dei pazienti nei gruppi con cacs 11100 e con cacs 101 400 . 
nei pazienti con coro narie normali alla ct - ca la frequenza di mace stata pari 0% versus l1 , 7% nei pazienti con cad non ostruttiva , e il 7 , 3% nei pazienti con cad ostruttiva ( p < 0 , 0001 )  . 
nellanalisi multivariata ( dopo la correzione per i fattori di rischio cardiovascolari ) , il diabete , la cad ostruttiva , ed il cacs > 1000 sono risultati predittori significativi di eventi maggiori ( p < 0 , 05 )  . discussione lo score clinico di morise ha il vantaggio , se confrontato con lo score di rischio di framingham , che richiede pochi parametri , meno dipendente dallet e , cosa pi importante , tiene conto dei sintomi [ 22 , 23 ]  . 
we also observed one revascularisation in the low morise score group ( left panel ) and 26 events ( 24 revascularisations and two hard events ; 9.5% , 2 / 21 ) in the intermediate morise score group ( left and right panels )  . 
abbiamo anche osservato 1 rivascolarizzazione nel gruppo a basso rischio ( pannello di sinistra ) e 26 eventi ( 24 rivascolarizzazioni e 2 eventi hard ; 9 , 5% , 2 / 21 ) nel gruppo a rischio intermedio ( pannelli di sinistra e destra )  . 
we also observed three hard events in the groups with cacs < 100 ( 14% ; 3 / 21 ) : one ami in a patient with cacs 0 ; one ami in a patients with cacs 15 ; one ua in a patient with cacs 56 . 
in particolare , 1 infarto miocardico acuto ( ima ) in un paziente con cacs 0 , 1 ima in un paziente con cacs 15 , ed una angina instabile ( ua ) in un paziente con cacs 56 . 
cardiac event rate pendenti per mace sono la presenza di cad ostruttiva , un cacs > 1000 , ed il diabete . noto da studi precedenti effettuati con cag che la severit della cad un predittore prognostico nei pazienti con malattia ostruttiva . 
in particolare , i pazienti con cad ostruttiva del tronco comune , con cad ostruttiva tri - vasale oppure con cad ostruttiva bi - vasale con un stenosi sulla 1198 radiol med ( 2011 ) 116 : 11881202 survival fig . 
i pazienti con assenza di malattia coronarica ( cad ) alla ct - ca hanno mostrato sopravvivenza priva di eventi al 100% ( sia per gli eventi avversi cardiovascolari maggiori [ mace ] che per gli eventi hard )  . 
multivariate analysis showed that the only independent predictors for mace were the presence of obstructive coronary disease , a cacs > 1 , 000 and diabetes . it is known from previous studies using cag that cad severity is a prognosis predictor in patients with significant cad . 
in particular , patients with significant left main stenosis , three - vessel disease or two - vessel disease with a left anterior descending artery stenosis > 90% are at higher risk of events [ 27 ]  . 
it is known from previous studies using myocardial perfusion scintigraphy or echocardiography stress testing that patients with type - 2 diabetes mellitus have a worse outcome compared with patients without diabetes for any degree of inducible ischaemia [ 28 ]  . 
noto da studi precedenti effettuati con scintigrafia miocardica o ecocardiografia da stress che i pazienti con diabete tipo 2 hanno un outcome peggiore rispetto ai pazienti senza diabete per qualsiasi grado di ischemia inducibile [ 28 ]  . 
i pazienti con infarto miocardico pregresso sono considerati ad alto rischio per la ricorrenza di eventi cardiaci , tuttavia plausibile che il carico aterosclerotico , radiol med ( 2011 ) 116 : 11881202 1199 burden and by a more accelerated progression of the atherosclerotic lesions in diabetics , as observed in an autopsy study [ 29 ]  . 
patients with previous myocardial infarction are considered at high risk for recurrence of a cardiac event ; however , it is plausible that the atherosclerotic burden , which is usually high in these patients , may be the most important prognostic factor . 
in that study , presence , and extent of significant cad defined by a modified duke coronary artery score , were predictors of all - cause mortality [ 10 ]  . 
analysed 517 patients with suspected cad who underwent both myocardial perfusion imaging and ctca and reported an annualised event rate of 4.8% in patients with significant cad at ctca and 1.8% in patients with none or mild cad [ 12 ]  . 
 at 1 year , patients with cad had a 34% event rate , whereas patients with normal coronary arteries had a cardiac event rate of 0% [ 13 ]  . 
in our study , patients without coronary atherosclerosis had an excellent prognosis ( 0% cardiac event rate at 20 months ) , thus confirming the high npv of ctca at follow - up . 
 study limitations the main limitation of our study is that the number of clinical events at follow - up is relatively low due to the population characteristics , which was a population at intermediate cardiovascular risk , and to the relatively limited followup . 
we included patients with both suspected and known cad , as usually encountered in clinical practice ; however , che di solito elevato in questi pazienti , possa essere il pi importante fattore prognostico . 
in questo studio la presenza di cad ostruttiva e lestensione della cad , definite mediante uno score coronarico di duke modificato sono risultati predittori per tutte le cause di mortalit [ 10 ]  . 
 [ 12 ] hanno studiato 517 pazienti con sospetta cad sottoposti a spect e ct - ca ed hanno riportato una frequenza di eventi per anno del 4 , 8% nei pazienti con cad ostruttiva versus l1 , 8% nei pazienti con cad non ostruttiva o senza cad alla ct - ca 1 , 8% . 
ad un anno , i pazienti con cad avevano una frequenza di eventi pari al 34% , mentre i pazienti senza cad avevano una frequenza di eventi pari a allo 0% [ 13 ]  . 
nel nostro studio abbiamo considerato i mace e gli eventi hard anche separatamente , a causa del fatto che la frequenza di rivascolarizzazione durante il follow - up pu essere significativamente condizionata dai risultati della ct - ca . 
 gli studi prognostici sono importanti per determinare se i pazienti , nei quali la ct - ca esclude la presenza di cad ostruttiva , possano essere rassicurati evitando la cag . 
nel nostro studio , i pazienti senza cad ( coronarie normali ) hanno mostrato una prognosi eccellente ( frequenza di eventi a 20 mesi pari allo 0% ) , confermando lelevato valore predittivo negativo della ct - ca anche al follow - up . 
nel nostro studio abbiamo incluso pazienti con cad sospetta o nota , cosa che riflette la pratica clinica , tuttavia la proporzione di pazienti con pregresso infarto miocardico e / o rivascolarizzazione limitata ( 29% )  . 
a causa delle eterogeneit delle popolazione e del numero relativamente basso di eventi , non abbiamo 1200 radiol med ( 2011 ) 116 : 11881202 the proportion of patients with previous acute myocardial infarction or revascularisation was limited ( 29% )  . 
due to the heterogeneous population and the relatively low number of events , we did not perform an association analysis between localisation or characteristics of coronary plaques and event rate . 
nevertheless , at multivariate analysis , cad history was not an independent predictor . high radiation exposure in ctca with retrospective gating is still a matter of concern [ 30 , 31 ]  . 
 recently , a dose modulation algorithm that reduces tube current by 80% during the systolic phase and prospective ecg - triggering protocols were developed that allow significant reduction of radiation exposure up to 34 msv ( or even below ) with preserved diagnostic accuracy [ 3235 ]  . 
 however , so far , the diagnostic accuracy of this new and promising technique is slightly lower compared with coronary angiography and retrospective - gating ct because of motion artefacts and is thus limited to highly selective patient populations . eseguito alcuna analisi per lassociazione tra sede e caratteristiche delle placche aterosclerotiche e la frequenza di eventi . 
tuttavia , nellanalisi multi - variata , la storia di cad non risultato un predittore indipendente . la dose di radiazioni elevata della ct - ca effettuata con tecnica di gating ecg retrospettivo rimane un problema della metodica [ 30 , 31 ]  . 
recentemente sono stati sviluppati protocolli per la modulazione della dose durante la fase sistolica che n consentono una riduzione dell80% e tecniche di triggering prospettico che consentono di effettuare la ct - ca con una dose effettiva di 34 msv ( o meno ) senza condizionare significativamente laccuratezza diagnostica [ 3235 ]  . 
tuttavia , al momento laccuratezza diagnostica ottenuta con queste tecniche leggermente inferiore alla ct - ca con tecnica di gating ecg retrospettivo a causa della limitata capacit di compensazione degli artefatti da movimento e devono essere utilizzate in popolazioni molto selezionate di pazienti . conclusions conclusioni this study shows that ctca reliably identifies coronary atherosclerotic burden and stratifies cardiovascular risk at follow - up in patients with suspected or known cad . 
a morise clinical score with lowintermediate risk and a cacs < 100 or < 10 do not exclude the possibility of future events . il nostro studio dimostra che la ct - ca capace di studiare il carico aterosclerotico coronarico e di stratificare il rischio cardiovascolare al follow - up in pazienti con cad sospetta o nota . 
cademartiri1 , 2 1department of radiology and cardiology , c / o piastra tecnica , piano 0 , azienda ospedaliero - universitaria , via gramsci 14 , 43100 parma , italy 2department of radiology and cardiology , erasmus medical center , rotterdam , the netherlands 3department of radiology and cardiology , ospedale san gennaro , napoli , italy 4department of radiology , universit di messina , messina , italy 5department of radiology , universit di palermo , palermo , italy 6department of radiology , universit di verona , verona , italy 7department of radiology , sdn - irccs foundation , napoli , italy correspondence to : f . 
a total of 183 consecutive asymptomatic individuals ( 92 men ; mean age 5411 years ) with more than one major risk factor ( obesity , hypertension , diabetes , hypercholesterolaemia , family history , smoking ) and an inconclusive or nonfeasible noninvasive stress test result ( stress electrocardiography , stress echocardiography , nuclear stress scintigraphy ) underwent ct - ca in an outpatient setting . 
mean calcium score was 177432 , mean heart rate during the ct - ca scan was 588 bpm and the prevalence ( per - patient ) of obstructive cad was 19% . 
per - patient sensitivity , specificity , positive predictive value and negative predictive value of ct - ca were 100% ( 90100 ) , 98% ( 9699 ) , 97% ( 8599 ) , 100% ( 97100 ) , respectively . 
obiettivo di questo lavoro stato valutare laccuratezza diagnostica dellangiografia coronarica con tomografia computerizzata ( ct - ca ) nellindividuazione delle stenosi coronariche significative ( riduzione del lume coronarico 50% ) confrontata con la coronarografia convenzionale ( cag ) in una popolazione di soggetti asintomatici a rischio intermedio / alto . 
centottantatre soggetti consecutivi asintomatici ( 92 maschi e 91 femmine ; et media 5411 anni ) con pi di un fattore di rischio ( obesit , ipertensione arteriosa , diabete mellito , dislipidemia , familiarit , fumo di sigaretta ) e stress test inconclusivo o non eseguibile ( stress elettrocardiogramma [ ecg ] , stress ecocardiografia , o stress tomografia computerizzata a emissione di fotoni singoli [ spect ] ) sono stati sottoposti a ct - ca . 
il calcium score medio , la frequenza cardiaca media e la prevalenza di malattia per paziente sono risultati 177432 , 588 battiti per minuto ( bpm ) , e 19% , rispettivamente . 
la ct - ca ha mostrato malattia significativa mono - vasale nell9% dei casi , bi - vasale nel 9% dei casi , e tri - vasale in nessun paziente . 
la sensibilit , specificit , valore predittivo positivo della 1162 radiol med ( 2011 ) 116 : 11611173 modality for excluding significant cad in intermediate / high - risk asymptomatic patients with inconclusive or nonfeasible noninvasive stress test . keywords computed tomography coronary angiography conventional coronary angiography asymptomatic primary prevention coronary artery disease ct - ca per paziente sono risultati 100% ( 90%100% ) , 99% ( 96%99% ) , 97% ( 85%99% ) , 100% ( 97%100% ) , rispettivamente . 
la ct - ca un ottimo strumento per lesclusione di malattia coronarica significativa nel asintomatico ad intermedio / alto rischio con stress test in conclusivo o non eseguibile . parole chiave coronarografia con tomografia computerizzata coronarografia convenzionale pazienti asintomatici prevenzione primaria malattia coronarica introduction introduzione computed tomography coronary angiography ( ct - ca ) is a consolidated examination technique for studying the coronary arteries and significant stenoses . 
 the 64 - slice scanner is the current diagnostic standard for ct - ca and is able to explore the entire epicardial coronary tree [ 4 , 5 ]  . 
the main characteristic of ct - ca is its high sensitivity and negative predictive value ( npv ) , enabling exclusion of coronary artery disease ( cad )  . recently , several multicentre trials strengthened data regarding ct - ca and in particular its use in symptomatic patients at intermediate cardiovascular risk [ 69 ]  . 
the population for whom ct - ca is indicated consists of symptomatic patients at intermediate risk after appropriate stratification , including patients with inconclusive or nonfeasible stress test [ stress electrocardiography ( ecg ) , stress echocardiography , stress scintigraphy ] [ 10 ]  . 
 these studies reported good diagnostic performance of ct - ca , even in the presence of low or very low prevalence of significant obstructive disease ( 026% ) [ 1116 ]  . the aim of this study was to evaluate the diagnostic accuracy of ct - ca in asymptomatic patients at intermediate / high cardiovascular risk with inconclusive or nonfeasible stress test compared with conventional coronary angiography ( cag )  . langiografia coronarica con tomografia computerizzata ( ct - ca ) una metodica di indagine consolidata che consente lo studio delle arterie coronarie e delle stenosi significative [ 15 ]  . 
 di recente , alcuni trials multicentrici hanno ulteriormente rafforzato queste informazioni riguardo la ct - ca ed in particolare lutilizzo in pazienti sintomatici a rischio cardiovascolare intermedio [ 69 ]  . 
la popolazione elettiva per la ct - ca consiste dei pazienti sintomatici a rischio intermedio dopo adeguata stratificazione inclusi quelli con test provocativo inconclusivo / non eseguibile ( stress elettrocardiogramma [ ecg ] , ecocardiografia da stress , scintigrafia da stress ) [ 10 ]  . 
in questi studi si evidenzia una buona performance diagnostica della ct - ca anche in presenza di prevalenza di malattia ostruttiva significativa bassa o molto bassa ( 0%26% ) [ 1116 ]  . lo scopo di questo studio valutare laccuratezza diagnostica della ct - ca in pazienti asintomatici a rischio intermedio - alto con test provocativo in conclusivo o non eseguibile confrontata con coronarografia convenzionale ( cag )  . materials and methods patients materiali e metodi over a 36 - month period , 183 consecutive asymptomatic patients [ 92 men , 91 women ; mean agestandard deviation ( sd ) 5411 ] with more than one cardiovascular risk soggetti in un periodo di 36 mesi sono stati arruolati 183 individui radiol med ( 2011 ) 116 : 11611173 1163 factor ( obesity , hypertension , diabetes , dyslipidaemia , family history , smoking ) were enrolled ( table 1 )  . 
i pazienti erano in ritmo sinusale , erano in grado di mantenere una apnea inspiratoria di 12 s e non avevano contro - indicazioni alla somministrazione endovenosa di mezzo di contrasto iodato ( creatinina plasmatica < 120 mg / l , assenza di precedenti reazioni avverse )  . 
lo studio stato approvato da comitato etico ed i pazienti hanno fornito consenso informato . preparazione i pazienti con una frequenza cardiaca allarrivo superiore a 65 battiti per minuto ( bpm ) hanno ricevuto una singola dose endovenosa di atenololo ( 5 mg ) 10 minuti prima dellindagine sotto monitoraggio ecg e pressorio . 
immediatamente prima dellindagine stata somministrata a tutti i pazienti una tavoletta sub - linguale di isosorbide dinitrato ( 0 , 3 mg )  . tutti i pazienti sono stati studiati con ct - ca mediante apparecchiatura a 64 strati ( sensation 64 cardiac , siemens , forchheim , germania )  . 
il calcium score coronarico ( cacs ) stato ottenuto mediante software dedicato ( cascore , siemens , germania ) [ 18 ]  . durante la scansione angiografica stato somministrato per via endovenosa un bolo di 100 ml mezzo di contrasto iodato ( iomeprol , iomeron 400 , bracco , milano , italia ) seguito da 50 ml di soluzione salina attraverso una vena antecubitale del braccio ( velocit 5 ml / s ) e linizio della scansione stata innescata mediante tecnica di bolus tracking [ 19 , 20 ]  . 
 ricostruzione delle immagini le immagini sono state ricostruite con tecnica di gating ecg retrospettivo ( spirale ) con dati provenienti da una singola apnea inspiratoria ed algoritmo di ricostruzione a singolo segmento ( 180 )  . 
i dataset sono stati ricostruiti nelle fasi tele - diastolica ( - 300 / - 450 ms prima della successiva onda r oppure 60% / 70% dellintervallo r - r )  . 
the study was approved by the ethics committee , and all patients provided informed consent . preparation ct protocol patients with a heart rate ( hr ) > 65 bpm received a single intravenous dose of atenolol ( 5 mg ) 10 min prior to the examination under constant ecg and pressure monitoring . 
during the angiography scan , an intravenous bolus of 100 ml of iodinated contrast material ( iomeprol , iomeron 400 , bracco , milan , italy ) followed by 50 ml of saline solution was injected into an antecubital vein at a speed of 5 ml / s , and the scan was triggered automatically with the bolus - tracking technique [ 19 , 20 ]  . image reconstruction images were reconstructed with the ( spiral ) retrospective ecg gating technique , with data being derived from a single breath - hold and with the use of a single - segment reconstruction algorithm ( 180 )  . 
the data sets were reconstructed in the end - diastolic phase ( 300 / 450 ms prior to the subsequent r - wave or 60 / 70% of the r - r interval )  . 
 in the event of inadequate image quality , additional reconstructions were obtained in the end - systolic phase ( + 225 / + 325 ms from the previous r - wave and 25 / 35% of the r - r interval )  . 
le immagini assiali sono state ricostruite con uno spessore individuale di 0 , 75 mm ed un incremento di ricostruzione di 0 , 4 mtutti i dataset sono stati ricostruiti con un filtro di convoluzione intermedio . valutazione immagini tc tutte le scansioni sono state analizzate indipendentemente da due osservatori in consenso e non a conoscenza dei risultati della cag . 
i segmenti sono stati classificati come normali / non significativamente malati ( indenni o con irregolarit del lume o con riduzione del diametro < 50% ) o significativamente malati ( stenosi del lume 50% )  . angiografia coronarica convenzionale la cag stata eseguita entro 4 settimane dalla ct - ca . 
un singolo osservatore , non a conoscenza dei risultati della ct - ca , ha identificato i segmenti coronarici utilizzando la medesima classificazione in 17 segmenti [ 21 ]  . 
i segmenti sono stati classificati come normali ( con pareti e lume regolari ) , non significativamente malati ( con irregolarit del lume o con riduzione del diametro < 50% ) o significativamente malati ( stenosi del lume 50% )  . 
le stenosi coronariche sono state quantificate in due proiezioni ortogonali utilizzando un algoritmo di misurazione validato per langiografia coronarica ( caas , pie medical , maastricht , olanda ) , e classificate come significative se la riduzione del diametro del lume era 50% . 
la performance diagnostica dellangiografia coronarica mediante ct - ca nellindividuazione delle lesioni aterosclerotiche coronariche significative , utilizzando la cag come tecnica di riferimento di seguito riportata come sensibilit , specificit , valore predittivo positivo radiol med ( 2011 ) 116 : 11611173 1165 e negativo con intervalli di confidenza ( ic ) al 95% calcolati con espansione binomiale . 
sono stati calcolati i likelihood ratio positivo e negativo ( lr + e lr - , rispettivamente ) e i valori di probabilit post - test positiva e negativa ( ptp + e ptpcome curve di probabilit condizionale )  . 
 la cag ha dimostrato assenza di malattia significativa in 151 ( 83% ) pazienti e la presenza di almeno una stenosi significativa in 32 ( 19% ) pazienti . individui con stenosi significative e calcio coronarico nella popolazione con almeno una stenosi significativa ( n = 32 ) , un paziente aveva un valore di cacs pari a zero ed il valore medio complessivo era di 697674 ( agatston score )  . 
la dose di radiazioni stimata associate al protocollo stata di calcolata con un software dedicato ( windose , institute of medical physics , erlangen , germania ) ed i valori sono risultati pari a 15 / 21 ( maschi / femmine ) msv . 
la sensibilit , specificit , valore predittivo positive e negative nellanalisi per segmento sono risultati 98% , 97% , 38% , 100% ( lr + 29 ; lr0 , 019 ) , rispettivamente . 
la sensibilit , specificit , valore predittivo positivo e negativo nellanalisi per vaso sono risultati 100% , 95% , 58% , 100% ( lr + 20 ; lr0 , 0 ) , rispettivamente . 
la sensibilit , specificit , valore predittivo positivo e negativo nellanalisi per paziente sono risultati 100% , 99% , 97% , 100% ( lr + 151 ; lr0 , 0 ) , rispettivamente ( tabella 2 )  . 
cdx , coronaria destra ; csx , coronaria sinistra ; tc , tronco comune sinistro ; cx , coronaria circonflessa ; ada , coronaria discendente anteriore . in consensus who were unaware of the cag findings . 
segments were classified as normal / not significantly diseased ( healthy or with lumen irregularities or reduction < 50% ) or significantly diseased ( > 50% lumen reduction )  . conventional coronary angiography cag was performed within 4 weeks of ct - ca . 
the segments were classified as normal ( with regular walls and lumen ) , not significantly diseased ( with lumen irregularities or reduction < 50% ) or significantly diseased ( 50% lumen reduction )  . 
coronary artery stenoses were quantified in two orthogonal views using a measurement algorithm validated for coronary angiography ( caas , pie medical , maastricht , the netherlands ) and classified as significant if lumen reduction was 50% . 1166 statistical analysis data are presented as prevalence , mean and sd . 
the diagnostic performance of ct - ca in identifying significant cad , with cag as the reference standard , is reported in terms of sensitivity , specificity , positive predictive value ( ppv ) and npv , with 95% confidence intervals ( ci ) calculated with binomial expansion . 
the positive ( lr + ) and negative ( lr - ) likelihood ratios were calculated , as were positive and negative posttest probability ( ptp + and ptpas conditional probability curves )  . 
 individuals with significant stenosis and coronary artery calcium in the patient population with at least one significant stenosis ( n = 32 ) , one patient had a cacs value of zero , and the overall mean was 697674 ( agatston score )  . 
estimated radiation dose associated with the protocol was calculated with a dedicated software package ( windose , institute of medical physics , erlangen , germany ) and the values were 15 / 21 ( males / females ) msv . 
a total of 2 , 604 coronary segments were radiol med ( 2011 ) 116 : 11611173 significative , la ct - ca ha correttamente come positivi classificato 53 segmenti . 
i valori di accuratezza diagnostica della ct - ca ( sensibilit 100% e specificit 99% ) in questa popolazione sono risultati al pari o migliori rispetto alle maggiori casistiche riguardanti pazienti sintomatici [ 69 , 2224 ]  . 
 la frequenza di falsi positivi stata dello 0 , 55% ( 1 / 183 ) nellanalisi per paziente , dello 0 , 5% ( 35 / 732 ) nellanalisi per vaso e del 3 , 3% ( 87 / 2604 ) nellanalisi per segmen to . 
tutta via , per qualche motivo questi pazienti erano a rischio intermedio / alto ed avevano effettuato un test provoca tivo oppure non potevano eseguirlo essendo comunque stato indicato dallo specialista . 
il calcio coronarico non sarebbe stato sufficiente ad includere / escludere i pazienti dato che la distribuzione nei pazienti con almeno una stenosi significativa era 13% ( 4 / 32 ) con cacs < 100 , 53% ( 17 / 32 ) con cacs tra 100 e 1000 , e 34% ( 11 / 32 ) con cacs > 1000 ( cacs medio in questo gruppo 668605 , agatston score )  . 
 esistono in letteratura differenti studi che hanno valutato la performance diagnostica della ct - ca in individui radiol med ( 2011 ) 116 : 11611173 1167 1168 radiol med ( 2011 ) 116 : 11611173 cacs in patients with obstructive cad available for evaluation . 
the diagnostic accuracy values of ct - ca ( sensitivity 100% and specificity 99% ) in this population are similar to or better than those in most series regarding symptomatic patients [ 69 , 2224 ]  . the frequency of false positives was 0.55% ( 1 / 183 ) in the per - patient analysis , 0.5% ( 35 / 732 ) in the per - vessel analysis and 3.3% ( 87 / 2 , 604 ) in the per - segment analysis . 
nonetheless , for one reason or another , these patients were at intermediate / high cardiovascular risk and had been referred for a stress test that proved either inconclusive or unfeasible . 
coronary calcium alone would not have been sufficient for including / excluding these patients , given that the distribution in patients with at least one significant stenosis was 13% ( 4 / 32 ) with cacs < 100 , 53% ( 17 / 32 ) with cacs between 100 and 1 , 000 and 34% ( 11 / 32 ) with cacs > 1 , 000 ( mean cacs in this group 668 605 ; agatston score )  . various studies have evaluated the diagnostic performance of ct - ca in intermediate / high cardiovascular risk patients . 
studied the diagnostic accuracy of ct - ca in a cohort of asymptomatic patients at high risk [ 15 ] and reported a disease prevalence of 26% and a sensitivity and specificity of ct - ca of 100% [ 15 ]  . 
tuttavia , una quota non indifferente ( 13% ) di individui aveva un cacs basso ( agatston 1100 ; 10% ) o assente ( agaiston 0 ; 3% )  . 
 in questo studio , senza una validazione con cag , vie ne riportata una prevalenza di malattia ostruttiva del 7% [ 11 ]  . esiste un notevole interesse circa la possibilit di studiare gli individui asintomatici mediante ct - ca . 
la metodica , infatti , consente per la prima volta di valutare non solo la presenza di malattia ostruttiva ma anche il carico di placca coronario in questo gruppo di pazienti [ 26 ]  . 
sensitivity ( sens ) , specificity ( spec ) , positive predictive value ( ppv ) and negative predictive value ( npv ) ( upper panel ) ; positive likelihood ratio ( lr + ; lower left panel ) and negative likelihood ratio ( lr - ; lower right panel ) of the total population that underwent computed tomography coronary angiography ( ct - ca )  . 
il grafico a barre mostra i valori di sensibilit , specificit , valore predittivo positivo e negativo ( pannello superiore ) , e i likelihood ratio ( lr + e lr - ; panelli inferiori ) della popolazione totale sottoposta a ct - ca . 
in that study , done without cag validation , the reported prevalence of obstructive disease was 7% [ 11 ]  . there is significant interest regarding the possibility of using ct - ca to study asymptomatic individuals . 
indeed , for the first time , the modality makes it possible to evaluate not only the presence of obstructive disease but also the coronary plaque burden in this group of patients [ 26 ]  . 
the primary question for which given current knowledge there is no response is : what is the point of evaluating subclinical obstructive atherosclerosis in asymptomatic subjects ? for years , cacs has been used to directly visualise coronary artery atherosclerosis [ 17 ]  . 
la domanda principale , per la quale non esiste allo stato attuale delle conoscenze una risposta , a che cosa serve valutare laterosclerosi sub - clinica ostruttiva o meno nei soggetti asintomatici . 
tuttavia , il rapido e recente sviluppo della tecnologia ct - ca ha portato allattenzione il fatto che la malattia coronaria ostruttiva pu esistere anche in assenza di calcio coronarico [ 25 , 27 ]  . 
 oltre a questo sappiamo dalla letteratura che i test funzionali ( stress ecg , ecocardiografia da stress , stress spect ) non sono in grado di rilevare laterosclerosi sub - clinica e che soffrono di una consistente riduzione dellaccuratezza diagnostica in condizioni di bassa probabilit pretest e / o con individui asintomatici . 
si dimostra come la strategia pi efficace dal punto di vista dei costi sia quella basata sulla ct - ca come primo o secondo esame nella popolazione di 1170 radiol med ( 2011 ) 116 : 11611173 fig . 
ct - ca markedly increases the posttest probability of disease when positive ( light grey line positive test ) and similarly reduces it when negative ( dark grey line negative test ) both in per - patient and per - segment analyses . 
il grafico mostra la relazione tra la probabilit pre - test e post - test di malattia coronarica ostruttiva utilizzando i dati d accuratezza diagnostica ( ct - ca per - patient ; ct - ca per - segment ) e di prevalenza di malattia ( per - patient 19% ; per - vessel 7% ; per - segment 2 , 0% )  . 
la ct - ca aumenta significativamente la probabilit post - test di malattia quando positiva ( linea grigio chiaro ; test positive ) e analogamente la diminuisce quando negativa ( linea grigio scuro ; test negative ) sia nellanalisi per paziente che per segmento . 
shows that the most cost - effective strategy is based on ct - ca as a firstor second - line examination in a patient population with pr - test probability / disease prevalence of 10% [ 22 ]  . 
attualmente la dose di radiazioni media per una ct - ca di circa 12 msv con ampie variazioni legate ai protocolli utilizzati , allesperienza degli operatori , alle apparecchiature [ 28 ]  . 
the mean radiation dose for a ct - ca scan is around 12 msv , with significant variations in relation to the protocols used , operator experience and the system used [ 28 ]  . 
in particular , the implementation of prospective triggering based on the ecg ( instead of spiral retrospective ecg gating ) allows the examination to be performed with a dose of 15 msv depending on the system used [ 2932 ]  . limitations the cad population studied is relatively small , with a relatively low prevalence ( 19% )  . 
radiation dose remains a problem , even though the most recent software and hardware developments suggest the feasibility of ct - ca with doses < 5 msv without significantly compromising accuracy [ 2932 ]  . 
secondly , the ability of ct - ca to accurately evaluate the degree of stenosis is limited , and using a 70% threshold would increase specificity and ppv at the expense of reduced sensitivity and npv . 
la dose di radiazioni rimane un problema della metodica anche se i pi recenti sviluppi tecnologici ( software / hardware ) , suggeriscono la fattibilit della ct - ca con dosi < 5 msv senza significativa compromissione dellaccuratezza diagnostica [ 2932 ]  . 
la discrepanza tra le due metodiche in termini di soglia utilizzata deriva principalmente da due considerazioni : la prima che la ct - ca non una metodica che viene utilizzata per decidere se il paziente deve essere rivascolarizzato a meno ; la seconda che comunque la capacit della ct - ca di valutare accuratamente il grado di stenosi limitata ed utilizzare una soglia del 70% porterebbe ad un aumento della specificit e valore predittivo positivo a prezzo di una riduzione della sensibilit del valore predittivo negativo . 
 inoltre , anche la cag , che costituisce lo standard di riferimento per la quantificazione del grado di stenosi , rimane limitata ed il test provocativo di ischemia associato alla valutazione clinica devono completare comunque la valutazione morfologica . conclusioni conclusions ct - ca has a high diagnostic performance in asymptomatic patients at intermediate / high cardiovascular risk . 
the reservation regarding the use of the technique in this patient population remains the dose of ionising radiation . la ct - ca ha una elevata performance diagnostica nei pazienti asintomatici a rischio intermedio / alto . 
two readers evaluated independently the quality of the dynamic examination ( q : nondiagnostic , diagnostic , optimal ) , condylar motion ( cm : limited , suboptimal , optimal ) , condylar orientation ( co : in - plane , through - plane shift ) , disc visibility and movement ( dv : visible , nonvisible ; dm : normal , reducing , nonreducing dislocation ) and joint effusion ( je : present , absent )  . 
sessantadue pazienti con disfunzione condilo - meniscale ( 124 atm ) sono stati studiati con tecnica rm dinamica dellatm su apparecchio da 1 , 5 t durante movimento fisiologico di apertura e chiusura buccale . 
due lettori hanno valutato indipendentemente la qualit dellesame ( q : non diagnostico , diagnostico , ottimale ) , il movimento condilare ( mc : limitato , subottimale , ottimale ) , lorientamento condilare ( oc : nel piano , fuori dal piano ) , visibilit e movimento del disco ( vd : visibile , non visibile ; m : normale , dislocazione ridotta , dislocazione non ridotta ) e il fluido articolare ( f : presente , assente )  . 
la valutazione qualitativa del movimento condilo - meniscale e della presenza di fluido intraarticolare nellimaging dinamico dellatm solo minimamente dipendente dalla stima dellosservatore , mentre la misurazione del tracciato condilare presenta una variabilit inter - osservatore pari a 30% . parole chiave articolazione temporo - mandibolare variabilit inter - osservatore risonanza magnetica introduction introduzione condyle - disc disorders are the most common abnormalities of the temporomandibular joint ( tmj ) , with an incidence of symptoms of 3572% [ 1 , 2 ]  . 
although disc abnormalities may also be asymptomatic [ 3 ] , patients affected by tmj disorders tend to have more evident signs and symptoms , which may include , in addition to discomfort and articular clicks , joint pain , reduced mandibular movement and limited mouth opening [ 4 , 5 ]  . 
mr study of the tmj is generally performed with a standard protocol [ 14 ] that includes acquisition of static sagittal and coronal images during opening and closing of the mouth . 
 a technical aid to help the patient maintain an openmouth position during mr examination is to use jawopening devices [ 15 , 16 ] that allow acquisition of mr images at maximal mouth opening . 
to verify the condyle disc relationships at the different degrees of mouth opening , the use of an incremental jaw - opening device has been suggested [ 17 ]  . 
moreover , with the use of mechanical jaw le disfunzioni condilo - meniscali sono le pi comuni anomalie dellarticolazione temporo - mandibolare ( atm ) , con incidenza di sintomi stimata del 35%72% [ 1 , 2 ]  . 
 sebbene le alterazioni discali si manifestino anche in soggetti asintomatici [ 3 ] , i pazienti che soffrono di disordini dellatm presentano una sintomatologia pi rilevante , che pu includere oltre a fastidio e rumori articolari ( click ) , anche dolore articolare , ridotta escursione mandibolare e limitata apertura buccale [ 4 , 5 ]  . 
la valutazione radiologica di un sospetto clinico di disfunzione articolare condilo - meniscale si ottiene attraverso limaging di risonanza magnetica ( rm ) [ 6 , 11 , 12 ]  . 
lo studio rm dellatm viene generalmente eseguito con un protocollo standardizzato [ 14 ] che include lacquisizione di immagini sagittali e coronali statiche , in posizione di chiusura ed apertura buccale . 
per verificare i rapporti condilomeniscali a diverse fasi di apertura stato suggerito limradiol med ( 2011 ) 116 : 13031312 1305 openers , it may be difficult to reproduce the optimal mouth opening distance at which the discal dislocation manifests , given that disccondyle incoordination and mouth opening are both prone to wide intersubject variability [ 15 ]  . 
these mr techniques offer additional information on the range of motion , help define the orbit of the disccondyle relationship during repeated movements and allow for evaluation of the dynamic position of the disc and of any disc dislocations [ 15 , 16 ]  . 
to estimate the clinical potential of this technique , it is important to know to what extent the radiologists assessment of dynamic images can affect the variability of the diagnostic result . 
because the reproducibility of the technique has not been investigated , we carried out this study to evaluate interobserver variability in the interpretation of dynamic mr imaging of the tmj . materials and methods patients mr imaging was conducted on 62 symptomatic patients ( 20 men and 42 women ; age range 1266 years ; mean age 33.7 ) selected from a referral centre specialised in the evaluation of disccondyle dysfunction . 
inclusion criteria were a diagnosis of tmj dysfunction based on the presence of articular clicking sound associated with pain , discomfort and / or limited joint motility , with a clinical indication for morphological mr imaging of the tmj . 
con limpiego di distanziatori meccanici pu risultare inoltre indaginoso riprodurre la distanza di apertura buccale ottimale alla quale si manifesta la dislocazione discale , essendo lincoordinazione disco - condilare e la stessa apertura buccale due fenomeni soggetti ad ampia variabilit interindividuale [ 15 ]  . 
queste tecniche rm offrono informazioni aggiuntive sullampiezza del movimento , aiutano a definire lorbita della relazione disco - condilare durante i movimenti ripetuti e consentono una valutazione della posizione dinamica del disco e di sue eventuali dislocazioni [ 15 , 16 ]  . 
al fine di stimare le potenzialit cliniche di questa metodica importante conoscere quanto la valutazione delle immagini dinamiche da parte di un radiologo possa incidere sulla variabilit del risultato diagnostico . 
pertanto , il nostro scopo stato di valutare la variabilit inter - operatore nellinterpretazione dellimaging rm dinamico dellatm . materiali e metodi pazienti sono stati studiati con metodica rm 62 pazienti clinicamente sintomatici ( 20 uomini / 42 donne , range di et 1266 , et media 33 , 7 anni ) selezionati da un centro di riferimento clinico specializzato nella valutazione delle disfunzioni disco - condilari . 
sono stati inclusi nello studio pazienti con diagnosi di disfunzione dellatm basata sulla presenza di rumori articolari associati a dolore , fastidio e / o limitata mobilit articolare , nei quali era stata posta lindicazione clinica ad una valutazione rm morfologica dellatm . 
le 124 atm ( 622 ) includevano 78 casi di incoordinazione ( stadio 1 ) , 22 casi di dislocazione con riduzione ( stadio 2 ) e 24 casi di dislocazione senza riduzione ( stadio 3 ) [ 1921 ]  . risonanza magnetica statica e dinamica dellatm the static images were acquired in the closedand open - mouth positions . 
for the closed - mouth position , lesame delle atm stato effettuato su apparecchi rm da 1 , 5 t ( ge medical system , milwaukee , wi )  . 
i soggetti sono 1306 radiol med ( 2011 ) 116 : 13031312 we acquired ten sagittal images per side ( total 20 ) with a slice thickness of 2 mm , interslice gap 0.3 mm , field of view ( fov ) 12 cm , with fast spin - echo ( fse ) and gradient echo ( gre ) sequences . 
the parameters used for the fse sequence were time to repetition ( tr ) / time to echo ( te ) 4220 / 12 ms and matrix 256224 , whereas those used for the gre sequence were tr / te 600 / 15 ms , flip angle ( fa ) 20 and matrix 256192 . 
 the dynamic study was done with fast imaging steadystate precession ( fisp ) technique in a single sagittal plane localised on the centre of the condyle head identified in the axial plane of the scout image . 
in chiusura buccale sono state acquisite 10 immagini sagittali per ciascun lato ( totale = 20 ) con spessore di immagine di 2 mm , gap tra le immagini di 0 , 3 mm , campo di vista ( fov ) di 12 cm , con tecniche fast spin echo ( fse ) e gradient echo ( gre )  . 
per la sequenza fse sono stati utilizzati i seguenti parametri : tempo di ripetizione ( tr ) / tempo di eco ( te ) 4220 / 12 ms ; matrice 256224 . 
ogni frame ( 130 ) rappresentato nella figura , procedendo da sinistra a destra e dallalto in basso , rappresenta unimmagine acquisita dinamicamente durante il movimento spontaneo di apertura e chiusura buccale ( risoluzione temporale di ogni frame = 0 , 76 s )  . radiol med ( 2011 ) 116 : 13031312 image analysis a preliminary assessment of the quality of the dynamic study was conducted during the examination . 
 using the cine display mode and graphic tools provided with the workstation software , two operators ( fm , lg , with 7 and 2 years of experience , respectively , of mr imaging of the tmj ) independently evaluated quantitative and qualitative parameters . 
the qualitative evaluation took into account diagnostic quality of the examination ( q ) , condylar motion ( cm ) , condylar orientation ( co ) , disc visibility ( dv ) , disc motion ( dm ) and the presence of joint effusion ( je )  . 
cm was evaluated as optimal if the condyle moved beyond the apex of the articular eminence , suboptimal if it aligned with the apex of the articular eminence and limited if it stopped before the articular eminence . 
co was assessed as being in - plane if the condyle remained visible in the scan plane during anterior rotationtranslation and as through - plane shift if the condyle disappeared from the scan plane during movement . 
dm was assessed as normal if no disccondyle alterations were present , reducing if there was a reduction of the anterior dislocation during movement and nonreducing if the anterior dislocation persisted throughout the movement resulting in a discal obstacle . 
this quantitative assessment was made by marking the position of the centre of the condyle in the different frames to trace the curved trajectory covered by the condyle from the mouth - close position to spontaneous maximal opening . 
the condyle pathway in millimetres was measured on each side independently by the two operators . statistical analysis we calculated interobserver variability in the qualitative estimations and quantitative measurements of the condyle path . 
 lo studio dinamico stato effettuato con tecnica fast imaging steady state precession ( fissp ) su singolo piano sagittale , localizzato sul centro della testa del condilo identificata sul piano assiale dello scout . 
nella valutazione qualitativa sono stati considerati : la qualit diagnostica dellesame ( q ) , il movimento condilare ( mc ) , lorientamento condilare ( oc ) , la visibilit del disco ( vd ) , il movimento discale ( md ) , la presenza di fluido intraarticolare ( v )  . 
mc stato valutato secondo le categorie ottimale se il condilo andava oltre lapice delleminenza articolare temporale , subottimale se il condilo si allineava allapice delleminenza articolare , limitato se il condilo si arrestava prima delleminenza articolare . 
oc stato valutato secondo le categorie persistenza nel piano , se durante il movimento rototraslazionale anteriore il condilo era sempre visibile sul piano di scansione , attraversamento nel piano , se il condilo durante il suo movimento dinamico scompariva dal piano di scansione . 
md stato valutato secondo le categorie normale se non erano presenti alterazioni disco - condilari , dislocazione riducibile se si verificava una riduzione della dislocazione anteriore durante il movimento dinamico , dislocazione non riducibile se la dislocazione anteriore persisteva durante tutto il movimento dinamico determinando una condizione di ostacolo meniscale . 
v stato valutato secondo le categorie presente o assente . con gli strumenti grafici del software presenti sulla workstation stato inoltre tracciato il percorso condilare 1308 radiol med ( 2011 ) 116 : 13031312 fig . 
confronto tra un paziente con incoordinazione condilo - meniscale , stadio 1 ( a - e ) ed un paziente con dislocazione discale anteriore non riducibile , stadio 3 ( f - l )  . 
la linea curva che interconnette i punti rappresenta il tracciato condilare misurato in millimetri . percentage differences and the limits of agreement ( la ) , according to the blandaltman method [ 22 , 23 ]  . results the dynamic mr study was completed without difficulty in all patients . 
tale valutazione quantitativa stata effettuata marcando la posizione del centro della testa del condilo nei vari frame in modo da tracciare la distanza curva percorsa dal condilo dalla posizione di chiusura a quella di apertura massimale spontanea . 
il percorso del condilo in mm stato misurato indipendentemente dai due operatori per ogni lato . analisi statistica stata calcolata la variabilit interosservatore nelle stime qualitative e nelle misurazioni del percorso condilare . 
sullasse delle ascisse sono riportate le medie delle misurazioni effettuate dai due operatori ( average ) mentre sulle ordinate riportata la differenza percentualizzata rispetto alla media delle due misurazioni ( difference % )  . 
per misurare la variabilit del percorso condilare stata calcolata la differenza tra le due misurazioni effettuate dai 2 operatori , in percentuale rispetto alla media tra le due misurazioni stesse . 
sono stati quindi calcolati la media tra le differenze percentualizzate e i limiti di concordanza ( lc ) , secondo il metodo di blandaltman [ 22 , 23 ]  . risultati lo studio rm dinamico stato completato senza difficolt in tutti i soggetti . 
la qualit delle immagini , valutata preliminarmente sullapparecchio , risultata sempre soddisfacente da un punto di vista tecnico ( rapporto segnale / rumore , eventuale presenza di artefatti )  . 
il movimento discale stato valutato come normale , dislocazione riducibile e dislocazione non riducibile rispettivamente nel 66 , 1% ( 82 atm ) , 14 , 8% ( 18 atm ) e 19 , 1% ( 24 atm ) ( concordanza buona : = 0 , 64 ; p < 0 , 01 ) ; la presenza di fluido intra - articolare stata giudicata come presente o assente rispettivamente nel 41 , 3% ( 51 atm ) e 58 , 7% ( 73 atm ) ( concordanza buona : = 0 , 67 ; p < 0 , 01 )  . 
lorientamento del condilo stato identificato nel piano o al di fuori del piano di scansione rispettivamente nel 94 , 2% ( 117 atm ) e 5 , 8% ( 7 atm ) ( concordanza moderata : = 0 , 41 ; p < 0 , 01 )  . 
i risultati ottenuti hanno evidenziato una concordanza tra i due operatori elevata nella valutazione della qualit dellesame , del movimento condilare e della visibilit del disco , buona per il movimento discale e per la presenza di fluido intra - articolare , ed una concordanza moderata nella mean of the percentage differences was 1.2% , the lower la was 35% and the upper la was 32.6%. 1310 discussion this study assessed interobserver variability in the evaluation of dynamic mr imaging of the tmj . 
the results obtained by the two operators showed a high level of agreement in evaluating examination quality , condylar motion and disc visibility , a good agreement for disc movement and joint effusion , and moderate agreement in determining condyle orientation . 
in the quantitative evaluation performed on the condylar path , we found a variability of 30% compared with the measurements expressed in millimetres . mr imaging is the reference diagnostic modality for evaluating intracapsular and muscular tmj disorders . 
a serial dynamic acquisition in the same scan plane and with a temporal resolution of 1.25 images per second , during mouth opening and closing may depict in cine mode the movement of condyledisc rotationtranslation . 
 the fisp sequence is particularly suitable for this kind of imaging owing to the high temporal resolution and the good signal - to - noise ration between the bony structures ( hyperintense ) and the discal - ligamentous structures ( hypointense )  . 
moreover , as the movement is voluntary and spontaneous , this type of imaging effectively depicts the condyle - disc relationship and allows the type of discal movement to be defined in a natural and spontaneous manner . the same dynamically acquired images allowed identification of at least two morphological parameters : disc visibility and joint effusion . 
in view of the association existing between increased joint fluid and tmj disorders , a favourable finding was the good level of agreement between the two evaluations of the presence or absence of joint effusion . dynamic evaluation of cine mr acquisitions took into radiol med ( 2011 ) 116 : 13031312 definizione dellorientamento del condilo . 
nella valutazione quantitativa effettuata sul percorso condilare stata misurata una variabilit di 30% rispetto alle misurazioni espresse in millimetri . limaging rm rappresenta la metodica diagnostica di riferimento nella valutazione delle disfunzioni intra - capsulari e muscolari dellatm . 
unacquisizione dinamica , seriata , su uno stesso piano di scansione , con una risoluzione temporale di 1 , 25 immagini per secondo , durante il movimento di apertura e chiusura , pu rappresentare in cine il movimento di rototraslazione condilomeniscale . 
rispetto alle acquisizioni statiche , le immagini visualizzate in modalit cine hanno consentito una valutazione dellintero range di relazione condilo - meniscale , con una qualit diagnostica del 99 , 2% . 
la sequenza fissp , in tal senso , si presta a tale tipo di imaging per lelevata risoluzione temporale ed il buon rapporto segnale - rumore esistente tra le strutture ossee ( iperintense ) e quelle discoligamentose ( ipointense )  . 
il movimento buccale durante lacquisizione dinamica copre tutte le fasi della rototraslazione condilo - meniscale da una condizione di chiusura buccale completa in occlusione fino a quella di unapertura massimale . 
inoltre , essendo il movimento volontario e spontaneo , con questo tipo di tecnica dimaging si rappresenta effettivamente la relazione condilo - meniscale e si definisce il tipo di movimento discale in maniera naturale e spontanea . nelle stesse immagini acquisite in maniera dinamica sono stati identificati almeno due parametri morfologici : la visibilit del disco ed il fluido intra - articolare . 
considerata lassociazione esistente tra laumento del fluido intraarticolare e le disfunzioni dellatm , un dato favorevole stato che nella definizione di presenza o assenza di raccolta fluida articolare la concordanza tra le due valutazioni risultata buona . nella valutazione propriamente dinamica dellacquisizione cine - rm sono stati considerati il movimento del condilo , il movimento del disco e lorientamento condilare . 
however , the range of agreement of 30% between measurements made by the two observers must be related to the extent of condylar excursion , which normally does not exceed 3 c in consideration of the fact that this distance is relatively small , it may be that millimetric differences such as those detected by digital measurements were more relevant compared with those made over greater distances . 
identification of the condylar path using a visual estimate of the position of the condyle centre may have contributed to this variability , which must nonetheless be taken into account before proposing a quantitative method for determining a possible limitation to condyle excursion . there are some potential limitations to this study . 
 in conclusion , qualitative assessment of disc - condyle motion and the presence of joint effusion with dynamic tmj imaging is only minimally operator - dependent , whereas measurement of the condylar path has an interobserver variability of 30% . mento del condilo e del disco risultata rispettivamente ottimale e buona . 
tuttavia il range di concordanza del 30% tra le misurazioni effettuate dai due operatori va tenuto in relazione allentit dellescursione condilare , che normalmente non supera i 3 cconsiderando che questa distanza relativamente contenuta , possibile che differenze millimetriche , quali quelle normalmente presenti nelle misurazioni digitali , siano risultate pi rilevati rispetto a quelle effettuate su distanze maggiori . 
 lidentificazione del tracciato condilare utilizzando una stima visiva della posizione del centro del condilo pu avere inoltre contribuito a tale variabilit , che va considerata se si vuole proporre un metodo quantitativo per stimare uneventuale limitazione dellescursione condilare . in questo studio ci sono delle potenziali limitazioni . 
these additional cancers were located in the same quadrant as the index cancer in 13 women ( 4% ) , in a different quadrant in 12 ( 4% ) and in the contralateral breast in the remaining two ( 1% )  . 
the cancer detection rate in the subgroup of index cancers with lobular histological type was 25% , significantly higher ( p = 0.03 ) than the detection rate of 11% recorded in the subgroup of ductal cancers . 
the cancer detection rate in the subgroup of index cancers > 2 cm was 27% , significantly higher ( p = 0.001 ) than the rate of 8% found in the subgroup of index cancers < 2 c riassunto obiettivo . 
i dati sono stati analizzati con il test esatto di fisher ( p < 0 , 05 ) secondo i seguenti parametri : caratteri istologici della lesione principale ( istotipo tumorale e dimensione ) , densit mammografica ( classificazione breast imaging reporting and data system [ bi - rads ] in categorie da 1 = adiposa a 4 = densa )  . 
le lesioni maligne identificate dalla rm erano nello stesso quadrante rispetto alla lesione principale in 13 pazienti ( 4% ) , interessavano un altro quadrante in 12 pazienti ( 4% ) e la mammella controlaterale nei rimanenti 2 pazienti ( 1% )  . 
una correlazione significativa positiva ( p = 0 , 03 ) emersa tra il tasso di identificazione rm di lesione maligna e istotipo lobulare del carcinoma principale ( lesioni maligne sincrone sono state rilevate nel 25% dei tumori lobulari vs 11% dei tumori duttali )  . 
 una correlazione significativa ( p = 0 , 001 ) stata osservata tra identificazione rm di lesione maligne e dimensione radiol med ( 2011 ) 116 : 12261238 1227 mammographic density was not correlated ( p = 0.48 ) with mr detection of additional cancer , with 14% of additional malignancies being detected in both dense and fatty breasts . 
the use of preoperative mr imaging as an adjunct to conventional breast imaging in women with an infiltrating lobular index cancer and an index cancer > 2 cm is especially beneficial . keywords breast cancer diagnosis breast mri lobular carcinoma dense breast della lesione principale superiore a 2 cm ( lesioni maligne sincrone sono state diagnosticate dalla rm nel 27% dei tumori superiori a 2 cm vs 8% dei tumori inferiori a 2 cm )  . 
il tasso diagnostico superiore quando la neoplasia principale ha istotipo lobulare e dimensioni > 2 c parole chiave tumore mammario diagnosi rm mammaria carcinoma lobulare seno denso introduction introduzione increasing early detection of breast cancer is the primary goal of screening programmes and technological advances . 
conservative surgery necessitates an extremely precise evaluation of disease extent to contain both the number of repeat excisions due to positive surgical margins and local recurrences arising from disease foci outside the radiotherapy field . 
mammography has proved effective as the conventional imaging modality for establishing local disease extent by targeted magnification of the index lesion and of any suspicious abnormalities in the same and contralateral breast [ 3 ]  . 
the use of supplemental screening with us in women with denser breasts quite common in europe and less so in north america is being under evaluation in specifically designed controlled studies [ 5 , 6 ]  . 
 thanks to its high sensitivity [ 7 , 8 ] in detecting invasive breast cancers , magnetic resonance ( mr ) imaging is able to identify neoplasms occult to other imaging modalities [ 9 , 10 ] and has been proposed for local staging of breast cancer [ 11 ] , although this indication is a matter of debate [ 12 , 13 ]  . 
in addition to being a source of anxiety for the patient , false - positive results on mr imaging generate additional costs and delays in definitive surgery and unneclampia diffusione dei programmi di screening e il progresso tecnologico nella diagnostica preventiva per il cancro della mammella hanno comportato il sempre pi frequente riscontro di lesioni mammarie neoplastiche . 
la chirurgia conservativa ha ormai ricevuto definitiva conferma grazie alla dimostrata equivalenza in termini di sopravvivenza della terapia chirurgica conservativa associata a radioterapia e della mastectomia [ 1 , 2 ]  . 
 lespletamento di interventi conservativi imprescindibile da una valutazione estremamente precisa della estensione della malattia onde contenere da una parte il numero di re - escissioni dovute alla presenza di malattia sui margini chirurgici e , dallaltra , le recidive locali che possono insorgere da focolai di malattia esterni al campo della radioterapia . 
lo strumento radiologico tradizionale per il bilancio dellestensione tumorale locale rappresentato dallindagine mammografica con ingrandimenti mirati della lesione principale e delle alterazioni sospette nella stessa mammella e in quella controlaterale [ 3 ]  . 
il carcinoma lobulare e le neoplasie senza microcalcificazioni rappresentano un limite dellindagine mammografica in quanto costituiscono entit che possono essere misconosciute soprattutto in seni con densit elevata [ 4 ]  . 
lassociazione sistematica dellecografia mammaria alla mammografia in donne con seno denso , moderatamente utilizzata in europa e molto meno in usa , attualmente in corso di valutazione in studi controllati appositamente disegnati [ 5 , 6 ]  . 
 in ragione della elevata sensibilit [ 7 , 8 ] nella diagnosi dei tumori mammari invasivi , la risonanza magnetica ( rm ) capace di identificare neoplasie occulte alle altre tecniche di imaging [ 9 , 10 ] e viene proposta per la stadiazione lo1228 radiol med ( 2011 ) 116 : 12261238 essarily extensive surgical procedures . 
to reduce the economic and diagnostic cost , it would be more appropriate to perform preoperative mr imaging only on a subset of patients for whom there is evidence that the examination is beneficial [ 13 ]  . 
 this aim of this study was to verify the existence of parameters allowing selection of patients to be referred to mr evaluation as their risk of additional malignances is significantly higher to justify the use of preoperative mri . 
to this end , we analysed the role of preoperative mr imaging in a series of breast cancers in terms of : ( 1 ) detection rate of synchronous lesions occult to conventional breast imaging ; ( 2 ) distribution of the additional malignant lesions according to size and histological type of index lesion and mammographic density . materials and methods population between january 2006 and november 2009 , we analysed a consecutive series of 291 patients ( age 3387 years ; mean age 54 years ) with a surgical histological diagnosis of invasive breast cancer who had undergone mammography , ultrasound and mr imaging prior to surgery . 
all patients underwent complete breast workup , during which a diagnosis of breast cancer had been established by means of cytological or core biopsy of suspicious abnormalities identified on mammography and ultrasound . 
after undergoing mammography and ultrasound in the same diagnostic centre or , in a minority of cases , in other practices , the patients were referred to our department for preoperative mr staging . 
patients with invasive cancer treated with preoperative adjuvant chemotherapy or hormone therapy , those who had not undergone mammography and ultrasound prior to mr imaging and those with incomplete or unavailable breast cancer documentation at the time of interpreting the mr examination were excluded from the study . histopathology of the surgical specimens revealed invasive ductal carcinoma in 218 ( 75% ) patients , lobular carcinoma in 61 ( 21% ) and other histological types in the remaining patients ( one tubular carcinoma , two mucinous carcinomas , three medullary carcinomas , six papillary carcinomas )  . 
 mr examination and management of additional mr findings mr examinations were performed with the patient in the cale del tumore mammario [ 11 ] , sebbene questa indicazione sia in discussione [ 12 , 13 ]  . 
per ridurre i costi economici e diagnostici , sarebbe ottimale avviare a indagine rm pre - operatoria pazienti selezionate , per le quali vi sia evidenza di beneficio dallespletamento dellesame rm [ 13 ]  . 
 in questo contesto , il presente studio stato condotto allo scopo di verificare lesistenza di parametri che consentano di selezionare le pazienti da avviare a rm , essendo in tali casi significativamente superiore il riscontro di lesioni maligne addizionali . 
pertanto , stato analizzato il ruolo della rm preoperatoria nella serie personale di tumori invasivi della mammella considerando : ( 1 ) il tasso diagnostico di lesioni sincrone occulte alla indagine senologica convenzionale ; ( 2 ) la distribuzione delle lesioni maligne addizionali secondo dimensione e istotipo della lesione principale e secondo la densit mammografica della paziente . materiali e metodi popolazione nel periodo tra gennaio 2006 e novembre 2009 , stata analizzata una serie consecutiva di 291 pazienti ( et 3387 anni ; et media 54 anni ) con diagnosi istologica su pezzo operatorio di carcinoma infiltrante della mammella sottoposte a indagine mammografica , ecografica ed rm prima dellintervento chirurgico . 
tutte le pazienti incluse nello studio hanno effettuato esame senologico completo nel corso del quale stata formulata diagnosi di neoplasia mammaria mediante prelievo per esame citologico o microistologico di alterazioni sospette individuate allesame mammografico ed ecografico . 
dopo aver espletato mammografia ed ecografia nella stessa sede o , in una minor parte dei casi , in altri centri di diagnosi senologica , le pazienti afferivano allindagine rm per la valutazione pre - operatoria dellestensione della malattia . 
sono state escluse dallo studio i casi di neoplasia invasiva avviate a chemioterapia adiuvante o terapia ormonale prima dellintervento chirurgico , quelli che non avevano espletato indagine mammografica ed ecografica prima della rm e quel li in cui la documentazione senologica era incompleta o non disponibile al momento dellinterpretazione dellindagine rm . dallesame patologico del pezzo operatorio , listotipo era duttale infiltrante in 218 ( 75% ) pazienti , lobulare in 61 casi ( 21% ) e altri istotipi nei rimanenti casi ( tubulare in 1 caso , mucinoso in 2 casi , midollare in 3 casi , papillare in 6 casi )  . 
la dimensione media della lesione infiltrante ( deviazione standard ) era di 21 , 511 mm nei casi di istotiradiol med ( 2011 ) 116 : 12261238 1229 prone position in 1.5 - t equipment ( symphony , siemens medical system , erlangen , germany )  . 
the dynamic 3d t1 - weighted gradient echo ( gre ) sequence , performed in the coronal plane until october 2007 ( time to repeat [ tr ] / time to echo [ te ] 12 / 5.65 ms ; flip angle 25 ; matrix 254320 pixels ; field of view [ fov ] 38075 ; thickness 1.24 mm ; acquisition time 60 s ) and in the axial plane thereafter ( tr / te 10 / 4.76 ms ; flip angle 25 ; matrix 38490 pixels ; fov 38591.7 ; thickness , 1.10 mm ; acquisition time 74 s ) was acquired prior to administration of contrast material and repeated six and four times after the intravenous injection of 0.2 ml / kg of gadoterate meglumine ( dotarem ; guerbet s.p.a. ) until december 2008 and after that 0.2 ml / kg of dimeglumine gadobenate ( multihance ; bracco s.p.a. ) at a flow rate of 2.5 ml / s followed by 20 ml of saline solution . 
a region of interest ( roi ) of five to nine pixels was placed on the subtraction images , on the areas of enhancement visualised in the first postcontrast acquisition in the case of coronal sequences and in the second postcontrast acquisition in the case of axial sequences , for timeintensity curve construction . when interpreting the mr images , the readers reviewed patients previous bilateral mammograms and sonograms . 
to establish lesion depth , its distance was measured from the nipple and from the skall radiologists were skilled in breast imaging ( > 3 years of experience ) and breast mr imaging ( > 200 breast mr examinations / year )  . 
areas of enhancement were analysed according to the fischer parameters and in relation to degree of suspicion based on the breast imaging reporting and data system ( bi - rads ) mr imaging classification system of the american college of radiology [ 14 ]  . 
additional lesions detected on mr imaging underwent clinical review , second - look ultrasound and any other appropriate investigations and then monitored for at least 12 months . data analysis results of the clinical examination , imaging investigations , biopsies and surgical procedures were retrospectively reviewed . 
 lesions detected on mr imaging alone were classified as positive for cancer if confirmed by histopathology within 12 months and negative for cancer if they displayed stable features of benignity for at least 12 months . 
malignant lesion detected with mr imaging and interpreted as suspicious at mammography and / or us performed prior to mr imaging po duttale e di 44 , 624 mm nei casi con istotipo lobulare . 
 indagine rm e gestione dei reperti addizionali identificati allindagine rm lindagine rm stata eseguita con paziente in posizione prona e apparecchio da 1 , 5 t ( symphony , siemens medical system , erlagen , germania )  . 
sono state acquisite una sequenza short tau inversion recovery ( stir ) nel piano trasversale e coronale e una sequenza sagittale turbo spin echo ( tse ) t2 - dipendente . 
la sequenza dinamica gradient echo ( gre ) 3d t1 - dipendenti , espletata nel piano coronale fino a ottobre 2007 ( tempo di ripetizione [ tr ] / tempo di eco [ te ] : 12 / 5 , 65 ms ; flip angle 25 ; matrice 254320 pixel ; campo di vista [ fov ] 38075 ; spessore 1 , 24 mm ; tempo di acquisizione 60 secondi ) e successivamente nel piano assiale ( tr / te : 10 / 4 , 76 ms ; flip angle 25 ; matrice 38490 pixel ; fov 38591.7 ; spessore 1 , 10 mm ; tempo di acquisizione 74 secondi ) , stata acquisita prima della somministrazione di mezzo di contrasto ( mdc ) e ripetuta rispettivamente per sei e quattro volte dopo liniezione endovenosa di 0 , 2 ml / kg di gadoterate meglumina ( dotarem , guerbet spa , francia ) fino a dicembre 2008 e successivamente 0 , 2 ml / kg di gadobenato di dimeglumina ( multihance , bracco spa , italia ) di peso corporeo alla velocit di 2 , 5 ml al secondo seguita da 20 ml di fisiologica . 
nelle immagini ottenute per sottrazione , posizionando una regione di interesse ( roi ; 59 pixels ) sulle aree di impregnazione visualizzate nella prima sequenza dopo somministrazione di mdc in caso di acquisizione nel piano coronale e nella seconda sequenza dopo somministrazione di mdc in caso di acquisizione nel piano assiale , sono state costruite curve delle variazioni di segnale intensit / tempo . nella fase di interpretazione delle immagini rm , liconografia relativa alla mammografia bilaterale e alla ecografia immediatamente precedenti stata revisionata . 
per la individuazione della sede della lesione , sono stati presi come punti di riferimento il capezzolo e i piani assiale e sagittale passanti per esso in modo da suddividere la mammella in quadranti . 
tutti i radiologi erano operatori dedicati allimaging senologico ( esperienza in imaging senologico superiore a 3 anni ) esperti di rm mammaria ( espletamento di pi di 200 rm mammarie / anno )  . 
le aree di impregnazione sono state analizzate secondo i parametri di fischer ed in rapporto al giudizio di sospetto secondo la classificazione breast imaging reporting and data system ( bi - rads ) dellamerican college of radiology system [ 14 ]  . 
le lesioni addizionali diagnosticate dallindagine rm sono state sottoposte a rivalutazione dellesame senologico , a second look ecografico e altri 1230 radiol med ( 2011 ) 116 : 12261238 were excluded from the analysis of mr - detected cases , and their detection was ascribed to conventional breast imaging . 
data were analysed with fishers exact test , with significance set a p < 0.05. results synchronous occult malignant lesions detected at mr imaging mr imaging detected 55 malignant lesions synchronous to the index tumour in 38 / 291 patients ( 13% )  . 
in 15 / 55 lesions , the diagnosis of malignancy was established on the basis of ultrasound - guided biopsy , as mr lesions had a sonographic correlate on second - look ultrasound . 
they had a multifocal growth pattern in the same breast quadrant in 13 / 291 ( bifocal in four , trifocal in nine ) , whereas they were located in another quadrant in 12 / 291 ( bifocal in eight , trifocal in four )  . 
histological diagnosis of ipsilateral malignancies was invasive cancer in 17 / 25 cases ( 68% ) and in situ cancer in 8 / 25 ( 32% ) and of contralateral cancer foci was invasive cancer in both cases . 
with regard to lesion size , 5 / 40 ( 12.5% ) malignancies had a diameter 5 mm , 26 / 40 ( 65% ) between 6 and 10 mm , 8 / 40 ( 20% ) between 10 and 20 mm and only 1 / 40 ( 2.5% ) > 20 mm . distribution of malignancies malignant lesions detected with mr imaging were evaluaccertamenti in base alle alterazioni evidenziate , quindi monitorate per un periodo di almeno 12 mesi . analisi dei dati i risultati dellesame clinico , delle metodiche imaging , di biopsie e dellintervento chirurgico sono stati sottoposti a revisione retrospettiva . 
le lesioni identificate dalla sola indagine rm sono state classificate positive per cancro se patologicamente confermate entro 12 mesi e sono state considerate negative per cancro se hanno conservato caratteri di benignit stabili per un periodo di almeno 12 mesi . 
i casi di lesione maligna identificati dalla rm e considerati come portatori di alterazioni di sospetto alla mammografia e / o allecografia espletate prima della rm , sono stati esclusi dalla considerazione dei casi identificati dalla rm e la loro diagnosi stata attribuita allesame senologico convenzionale . 
i dati sono stati analizzati con il test statistico di fisher ( significativit : p < 0 , 05 )  . risultati lesioni maligne occulte sincrone identificate dalla rm lindagine rm ha identificato 55 lesioni maligne sincrone alla neoplasia principale in 38 / 291 pazienti ( 13% )  . 
in 15 / 55 lesioni la diagnosi di malignit stata formulata mediante prelievo sotto guida ecografica trattandosi di lesioni rm con corrispettivo reperto al second look ecografico espletato dopo indagine rm . 
le lesioni maligne sincrone identificate dalla rm erano omolaterali alla lesioradiol med ( 2011 ) 116 : 12261238 1231 table 1 site , histological diagnosis and size of the 40 additional cancers detected with mr imaging study variable category cases , n ( % ) location from the index cancer same quadrant different quadrant contralateral breast histology of mr additional cancer infiltrating carcinoma in situ carcinoma size 5 mm 610 mm > 10 mm 22 ( 55 ) 16 ( 40 ) 2 ( 5 ) 32 ( 80 ) 8 ( 20 ) 5 ( 12.5 ) 26 ( 65 ) 9 ( 22.5 ) tabella 1 sede , diagnosi istologica e dimensioni in 40 lesioni maligne diagnosticate dalla rm , sincrone rispetto al tumore principale ne principale in 25 casi ; avevano crescita multifocale nello stesso quadrante in 13 / 291 ( bifocale in 4 casi , trifocale in 9 casi ) mentre interessavano un altro quadrante in 12 / 291 ( bifocale in 8 casi , trifocale in 4 casi )  . 
la diagnosi istologica delle lesioni maligne omolaterali stata di patologia infiltrante in 17 / 25 casi ( 68% ) e di lesione in situ in 8 / 25 casi ( 32% )  . 
per quanto riguarda il risultato patologico in rapporto alle dimensioni della lesione , 5 / 40 ( 12 , 5% ) lesioni maligne avevano diametro inferiore o uguale a 5 mm , 26 / 40 ( 65% ) avevano diametro compreso tra 6 e 10 mm , 8 / 40 ( 20% ) erano estese tra 10 e 20 mm e solamente 1 / 40 ( 2 , 5% ) lesioni maligne era maggiore di 20 mm . variabile in studio categoria numero di casi ( % ) distribuzione della patologia maligna sede delle lesioni addizionali rispetto al tumore principale stesso quadrante differente quadrante mammella controlaterale 2 ( 5 ) 22 ( 55 ) 16 ( 40 ) diagnosi istotologica lesioni infiltranti lesioni in situ dimensioni 5 mm 610 mm > 10 mm 32 ( 80 ) 8 ( 20 ) 5 ( 12 , 5 ) 26 ( 65 ) 9 ( 22 , 5 ) ated according to histological type of the index cancer and as a mammographic density . 
in particolare , la rm ha evidenziato focolai neoplastici sincroni in 25 / 218 ( 11% ) casi di carcinoma duttale infiltrante versus 15 / 61 ( 25% ) casi di carcinoma lobulare ( p = 0 , 03 )  . 
la dimensione della neoplasia principale si dimostrata correlata con la diagnosi rm di malignit in quanto la rm ha evidenziato tumori sincroni in 14 / 176 ( 8% ) casi in cui il tumore principale aveva dimensioni inferiori a 2 cm versus 26 / 115 ( 27% ) con dimensioni superiori ( p = 0 , 001 )  . 
in 38 donne la biopsia ha confermato la presenza di malignit ( 37% : veri positivi della serie complessiva ) ; nelle rimanenti 64 donne ( 63% falsi positivi della serie complessiva ) la biopsia ha rilevato patologia benigna . 
considerando le lesioni solo rm visibili , prive di corrispettivo al second look ecografico , la verifica istologica ha rilevato patologia maligna in 27 / 52 casi ( 52% : veri positivi rm ) e patologia benigna nei rimanenti 25 / 52 casi ( 48% : falsi positivi rm )  . 
la distribuzione della patologia benigna nella serie complessiva stata la seguente : adenosi ( 24 / 64 : 39% ) e fibroadenomi ( 8 / 64 : 13% ) rappresentano le lesioni pi frequenti , seguite dal mastopatia fibrocistica e da fibro1232 radiol med ( 2011 ) 116 : 12261238 table 2 magnetic resonance imaging ( mri ) detection rate of otherwise occult additional cancers according to histological type and tumour size of the index tumour and mammographic density study variable no . 
in 38 / 102 cases , mr findings were malignant ( 37% true - positive findings for the overall series ) ; in the remaining 64 women ( 63% false - positive findings for the overall series ) biopsy revealed benign disease . 
analyzing lesions visible on mr imaging only ie , without a correlate on second - look ultrasound histological examination revealed malignant disease in 27 / 52 cases ( 52% true - positive mr findings ) and benign disease in the remaining 25 / 52 cases ( 48% false - positive mr findings )  . 
 distribution of benign disease in the overall series was adenosis ( 24 / 64 , 39% ) and fibroadenomas ( 8 / 64 , 13% ) , followed by fibrocystic disease and fibrosis ( 12 / 64 , 19% ) , proliferative sclerotic lesions ( 9 / 64 , 14% ) , papillomas ( 4 / 64 , 6% ) and miscellaneous benign entities ( 6 / 64 , 9% )  . 
 a tale fine stata analizzata la serie consecutiva di 291 carcinomi della mammella alla ricerca di criteri che consentano di radiol med ( 2011 ) 116 : 12261238 1233 fig . 
mammograms show a 3 - cm cluster of pleomorphic calcifications ( arrow ) in the upper inner quadrant of the left breast and in the central area of the right breast . 
preoperative breast mr imaging ( f - i ) ( axial plane , subtracted t1 - weighted , contrast - enhanced images ) shows clumped linear and ductal enhancement adjacent to the biopsy sites , corresponding to residual disease ( f , g )  . 
mr image of the right breast shows additional , irregularly shaped , irregularly marginated , heterogeneously enhancing masses in right lower outer ( h ) and upper inner ( i ) quadrants . 
nel quadrante supero - mediale della mammella sinistra e in regione retroareolare della mammella destra si notano numerose microcalcificazioni pleomorfe estese in unarea di circa 3 cle sedi vengono sottoposte a prelievo microistologico sotto guida stereotassica ( carcinoma duttale infiltrante , in entrambe le sedi )  . 
lindagine rm pre - operatoria ( f - i : piano assiale , immagini di sottrazione con mezzo di contrasto ) documenta una vasta area di impregnazione tipo non massa del parenchima adiacente alla camera bioptica , in entrambe le sedi sottoposte a prelievo compatibile con malattia residua ( f , g )  . 
ulteriori aree di impregnazione , separate da quelle precedenti , si rilevano nei rimanenti quadranti della mammella destra , rispettivamente nel quadrante infero - esterno ( h ) e in quello supero - mediale ( i )  . 
lesame isologico del pezzo operatorio ha confermato la presenza di neoplasia duttale infiltrante , solida e cribriforme , con crescita multicentrica a destra e lesione proliferativa residua a sinistra . 1234 radiol med ( 2011 ) 116 : 12261238 fig . 
preoperative breast mr imaging ( e , f ) ( axial plane , subtracted t1 - weighted contrast - enhanced images ) shows an irregular spiculated heterogeneously enhancing mass ( arrow in e ) corresponding to the index cancer , with an additional irregular spiculated mass in the contralateral breast ( arrow in f )  . 
allindagine mammografica ( a - d ) , nel quadrante supero - esterno della mammella destra , verso il prolungamento ascellare , si nota una formazione espansiva di aumentata densit , con margini spiculati ( frecce in a e c )  . 
lindagine rm preoperatoria ( e , f ) ( piano assiale , immagini di sottrazione dopo somministrazione di mezzo di contrasto ) conferma la lesione proliferativa al prolungamento ascellare di destra ( freccia in e ) e mostra una lesione nel quadrante supero - mediale della mammella sinistra ( freccia in f )  . 
il prelievo sotto guida ecoscopica per esame microistologico depone per lesione maligna sincrona ( carcinoma lobulare infiltrante )  . radiol med ( 2011 ) 116 : 12261238 1235 at 6 months , which confirmed their stability over time ( follow - up range : 1236 months )  . discussion the aim of this study was to verify the benefit of mr imaging in the preoperative assessment of infiltrating breast cancers diagnosed with conventional breast imaging . 
with this in mind , we analysed a consecutive series of 291 breast cancers looking for criteria that could help identify patients who might benefit from breast mr imaging on the grounds of the higher rate of additional cancer foci detected by mr imaging . 
in fact , in addition to being the most reliable tool for determining the index cancer size [ 16 , 17 ] and identifying the associated extensive intraductal component [ 18 , 19 ] , mr imaging detects additional mammographically and sonographically occult cancer foci ipsilateral to the index cancer in 27% of cases [ 20 ] and contralateral foci in 35% of cases [ 21 ]  . in this series , mr imaging detected 55 synchronous malignancies in 38 / 291 ( 13% ) patients . 
to guarantee the effective negativity of conventional breast imaging , it was deemed necessary to distinguish between lesions with a correlate on second - look ultrasound ( n = 15 / 55 ; 27% ) and those effectively visible on mr imaging only that is , the remaining 40 / 55 lesions ( 63% )  . 
 the controversy regarding the contribution of preoperative breast mr imaging revolves around the high number of false - positive findings and the doubtful clinical significance of the additional cancer foci detected with mr imaging in terms of recurrence and prognostic impact . 
in the contralateral breast , the incidence of suspicious mr imaging findings is estimated to be 9.3% , with a positive predictive value of 48% and a false - positive rate of 52% [ 23 ]  . 
 in this series , preoperative mr imaging detected additional findings in 102 / 291 patients ( 35% ) ; histology confirmed the presence of malignancy in 38 / 102 cases ( 37% ) and revealed benign disease in the remaining 64 / 102 cases ( 63% )  . 
the use of second - look ultrasound allowed identification of approximately half ( 50 / 102 cases ) of the findings incidentally detected with mr imaging and consequently reduced the rate of false - positive findings from 63% to 48% . 
 nonetheless , even after second - look ultrasound , the falseidentificare le pazienti che possano giovare dellesecuzione della rm mammaria in quanto il tasso di lesioni maligne sincrone individuate dalla rm maggiore . 
infatti oltre a rappresentare lo strumento pi affidabile per la valutazione dimensionale della lesione principale [ 16 , 17 ] e per la identificazione della estesa componente intraduttale associata [ 18 , 19 ] , lesame rm identifica focolai di malignit occulti alla mammografia ed ecografia omolaterali alla neoplasia principale nel 27% [ 20 ] e controlaterali nel 3%5% dei casi [ 21 ]  . nella presente serie , lindagine rm ha identificato 55 lesioni maligne sincrone alla neoplasia principale in 38 / 291 pazienti pari al 13% . 
a garanzia delleffettiva negativit dellesame senologico convenzionale , si ritenuto opportuno un distinguo tra le lesioni identificate al second - look ecografico e pari a 15 / 55 ( 27% ) e quelle realmente diagnosticate solo dalla rm ovvero le restanti 40 / 55 lesioni ( 63% )  . 
 le questioni tuttora dibattute riguardo al contributo della rm mammaria pre - operatoria vertono attorno allelevato numero di falsi positivi e al dubbio significato clinico dei focolai neoplastici identificati dalla rm , in termini di recidive ed impatto prognostico . 
nella mammella controlaterale , lincidenza di lesioni rm sospette stimata pari al 9 , 3% , con un valore predittivo positivo del 48% e un tasso di falsi positivi del 52% [ 23 ]  . 
 nella serie in esame , lindagine rm pre - operatoria ha evidenziato reperti addizionali complessivamente in 102 / 291 pazienti ( 35% ) ; lesame istologico ha confermato la presenza di malignit in 38 / 102 casi ( 37% ) e di lesione benigna nei restanti 64 / 102 casi ( 63% )  . 
lintroduzione del second look ecografico ha permesso il riconoscimento di circa la met ( 50 / 102 casi ) dei reperti incidentalmente rilevati dalla rm e di conseguenza il contenimento del numero dei falsi positivi dal valore del 63% a quello del 48% . 
anche dopo lapplicazione del second look ecografico , il tasso dei falsi positivi rimane considerevole , ci a dire che circa la met delle lesioni sospette solo rm visibili non sono maligne . 
quindi di essenziale importanza che queste lesioni vengano sottoposte a biopsia prima di qualsiasi decisione terapeutica chirurgica . per quanto riguarda limpatto clinico , nello studio retrospettico di solin et al . 
 [ 24 ] , che analizza il tasso di recidiva , di sopravvivenza e di insorgenza di metastasi a distanza e di neoplasie controlaterali dopo terapia chirurgica conser1236 radiol med ( 2011 ) 116 : 12261238 positive rate resulted high , implying that about half of the suspicious lesions visible on mr imaging alone are not malignant . 
it is therefore mandatory to obtain a biopsy of these lesions before making any decision on surgical treatment . as concerns clinical impact , the retrospective study by solin et al . 
 [ 24 ] , who analysed the rates of recurrence , survival , distant metastasis and contralateral cancers after conservative surgery over an 8 - year follow - up period , found no difference between the group of women who underwent preoperative mr imaging and those who underwent conventional breast imaging alone . 
mr imaging identified synchronous malignancies in both histological types of index cancer , and specifically in 11% of ductal cancers and 25% of lobular cancers , even though there was a statistically significant difference in favour of the lobular type . 
 the data are accounted for by the biology of lobular cancer with its multicentric growth pattern frequently missed at conventional breast imaging and by the high sensitivity of mr imaging in detecting lobular cancer [ 2628 ]  . 
with regard to tumour size , patients with breast cancer who benefit most from mr imaging are those with a cancer > 2 c synchronous lesions were seen in 27% of larger cancers and in only 8% of smaller cancers . 
in fact , increasing the size of the tumor , increases the likelihood of a multifocal growth pattern . considering that the accuracy of mr imaging is unaffected by mammographic density [ 29 ] , we analysed the mr detection rate in dense versus fatty breasts . 
this finding is in disagreement with the theoretical assumptions but is similar to that reported by previous studies [ 20 , 21 ]  . despite the large patient population , our study considers a clinical series of self - referring women . 
furthermore , vativa con follow - up della durata di 8 anni , non sono state evidenziate differenze nel gruppo di donne sottoposte a rm pre - operatoria rispetto a quelle sottoposte al solo esame senologico convenzionale . i risultati preliminari dello studio comparative effectiveness of magnectic resonance imaging in breast cancer ( comice ) provenienti dallo studio randomizzato di 1623 pazienti [ 25 ] , non hanno rilevato differenze nel tasso di reinterventi nelle donne sottoposte a rm pre - operatoria n differenze nel tasso di sopravvivenza libera da malattia fino ad ora , dopo follow - up medio di 3 , 1 anni . 
 in attesa dei risultati definitivi degli studi prospettici riguardo allimpatto clinico della rm , in questo lavoro stata verificata leventuale esistenza di gruppi di pazienti che possano avere maggior beneficio dallindagine rm , considerando come variabili in studio istotipo della lesione principale , dimensioni tumorali e densit radiologica . 
 lindagine rm ha identificato lesioni maligne sincrone in entrambi gli istotipi di lesione primitiva , nell11% delle neoplasie duttali e nel 25% delle neoplasie lobulari , tuttavia con differenza statisticamente significativa a vantaggio dellistotipo lobulare . 
il risultato trova ragione nella biologia del carcinoma lobulare , con pattern di crescita multicentrico , frequentemente misconosciuto allesame senologico convenzionale e nella elevata sensibilit della rm per la diagnosi di carcinoma lobulare [ 2628 ]  . 
in quanto alle dimensioni tumorali , le pazienti con neoplasia mammaria che giovano maggiormente dellesecuzione dellindagine rm sono le pazienti con tumore superiore a 2 clesioni sincrone sono state osservate nel 27% dei tumori con cospicue dimensioni e solo nel 8% dei tumori con dimensioni inferiori . 
infatti , allaumentare delle dimensioni tumorali aumenta la crescita con proliferazione multifocale . considerando che laccuratezza della rm non risente della densit mammografica [ 29 ] stato analizzato il tasso diagnostico rm nei seni densi e nei seni adiposi . 
il dato in disaccordo con i presupposti teorici ma simile a quanto riscontrato in altre esperienze [ 20 , 21 ]  . nonostante la popolazione sia numerosa , la casistica in esame considera una serie clinica con afferenza spontanea . 
nella popolazione in esame , i carcinomi di istotipo duttale rappresentano la grande maggioranza dei casi ( 75% ) e quelli lobulari sono solo circa un quinto della casistica complessiva . 
inoltre , i carcinomi di istotipo lobulare presentano dimensioni medie decisamente superiori a quelle dei carcinomi di istotipo duttale ( 44 , 6 mm versus 21 , 5 mm )  . 
even the literature suggests that lobular carcinomas tend to have a slightly larger mean size compared with ductal carcinomas and that cancers > 5 cm are more likely to be of the lobular type [ 3032 ]  . 
however , we cannot exclude with certainty that the incidence of additional cancer foci was influenced by the larger size of lobular carcinomas , as multifocality is dependent both on the intrinsic growth pattern of lobular cancers and on tumour size . 
in contrast , in our series , mr imaging detected additional malignancies in 25% of cases of lobular carcinoma , and this result is not significantly different from that reported in mann et al . 
if the frequency of additional cancer foci detected in cases of lobular carcinoma had been influenced by the size factor , we would have expected a higher incidence of additional lesions than that reported in the literature . 
overall , this was a retrospective analysis of a consecutive series of infiltrating breast cancers and , as such , it cannot be considered definitive evidence validating the use of preoperative breast mr imaging as a routine investigation . 
 in conclusion , mr imaging detects additional cancer foci to the index cancer that are occult to mammography and ultrasonography in 9% of patients affected by an infiltrating breast cancer . 
the detection rate for malignancies is higher when index cancer shows lobular type histology or size higher than 2 cm , as the mr detection rate for additional cancer foci in these patient groups increased to 25% and 27% , respectively . 
on the other hand , in consideration of the high number of false - positive findings , the detection rate of additional lesions on mr imaging results in a substantial number of further tests and requires correlation with conventional breast imaging as well as confirmation by an imaging - guided pathological examination . 
on the basis of our results , the number of breast mr imaging studies could be contained by selection of patients with a greater likelihood of multifocal and multicentric disease . 
 tuttavia , non si pu escludere con certezza che lincidenza di focolai neoplastici aggiuntivi sia stata influenzata dalle maggiori dimensioni dei lobulari rispetto ai duttali , essendo la multifocalit dipendente dalle caratteristiche intrinseche di crescita tumorale proprie del carcinoma lobulare piuttosto che di altri istotipi tumorali , ma anche dalle dimensioni della neoplasia . 
per contro , nella serie in esame lindagine rm ha diagnosticato lesioni maligne addizionali nel 25% dei casi di carcinoma lobulare e questo risultato diagnostico non si discosta in modo significativo da quello riportato nella revisione di mann et al . 
se la frequenza di lesioni addizionali rilevate nellistotipo lobulare fosse stata influenzata dal fattore dimensionale , nella serie in esame ci si dovrebbe aspettare una frequenza di lesioni addizionali pi elevata rispetto a quella riportata in letteratura . 
nel complesso , si tratta di una analisi retrospettiva condotta in una serie consecutiva di lesioni mammarie infiltranti e come tale non pu costituire un elemento definitivo per validare luso della rm pre - operatoria come esame di routine . 
il tasso diagnostico di lesioni maligne maggiore in presenza di carcinoma lobulare , di neoplasie con dimensioni superiori a 2 cm in quanto in queste categorie di pazienti la identificazione rm di carcinomi sincroni rispettivamente del 25% e del 27% . 
daltra parte in considerazione dellelevata percentuale di falsi positivi , il rilievo di lesioni addizionali allindagine rm genera un cospicuo numero di approfondimenti diagnostici , necessita di correlazione con lindagine senologica mammografica ed ecografica e conferma patologica mediante prelievo sotto guida imaging . 
 conflict of interest none radiol med ( 2011 ) 116 : 1313 doi 10.1007 / s11547 - 011 - 0727 - 0 erratum hypoxic liver perfusion with mitomycin - c for treating multifocal metastases and unresectable primary tumours : a single - centre series of 42 patients infusione intra - arteriosa epatica in ipossia di mitomicina - c per il trattamento di metastasi epatiche multiple e di tumori primitivi inoperabili : esperienza mono - centrica in 42 pazienti a . 
comino , corso re umberto 141 , 10134 torino , italy , tel . : + 39 - 011 - 3181211 , e - mail : mariacom@tiscalinet.it received : 8 october 2010 / accepted : 18 november 2010 / published online : 10 july 2011 springer - verlag 2011 abstract the radiology report has always been considered as the most important step in the clinical radiological act . 
italy aside , this issue has been thoroughly investigated and debated abroad since the 1970s and , in the past as well as more recently , many interesting contributions have been published both on the need for verbal communication , particularly in certain settings , and on the best manner to approach this task . 
the author hopes that in italy , as in other countries , an extensive debate will give rise to a fuller awareness of the issue , thereby providing a range of shared solutions . keywords radiological report patientdoctor communication riassunto il referto radiologico stato considerato da sempre il momento pi significativo dellatto clinico radiologico . 
a differenza che in italia , allestero questo problema stato ampiamente approfondito e dibattuto fin dagli anni 70 e sono stati presentati , in passato come recentemente , numerosi interessanti contributi riguardanti sia la necessit del colloquio verbale specie in determinate circostanze , sia le migliori modalit di esecuzione del colloquio stesso . 
lautore si augura che anche in italia un ampio dibattito sullargomento possa far nascere una migliore presa di coscienza del problema fornendo quindi soluzioni possibili e condivise . parole chiave referto radiologico comunicazione medico - paziente introduction introduzione not so long ago , francesco dalla palma , at the time president of the italian society of medical radiology , stated that the major product of the radiologists profession is the report , which represents the most significant step in the clinical radiological act . 
at the same time , several publications ( mostly by francesco schiavon ) and a large number of conferences extensively discussed the radiology report with the laudable aim of standardising its features and , more importantly , of avoiding omissions or errors . this attention surrounding the report tended , however , to in un passato abbastanza recente , lallora nostro presidente francesco dalla palma affermava che la completa realizzazione della professione del radiologo trova la sua massima espressione nel referto che rappresenta il momento pi significativo dellatto clinico radiologico . 
contemporaneamente , numerosi scritti ( merito soprattutto di francesco schiavon ) e ancor pi numerosi convegni approfondivano in ogni dettaglio il referto con il lodevole scopo di uniformarne le caratteristiche e soprattutto di evitare omissioni o errori . 1154 radiol med ( 2011 ) 116 : 11531160 obscure the indisputable fact that the report does not in any way fulfil the duty of the physician , and therefore of the clinical radiologist , to communicate with the patient , as we are reminded in art . 
taking into account his or her ability to understand .... unfortunately , the tradition of face - to - face communication between the radiologist and the patient was lost in the 1960s as a result of a series of objective factors but also , in my opinion , as a result of new cultural attitudes that i now attempt to delineate . 
the first reason is the expansion of health care , which has dramatically increased the number of radiological procedures ; second , the emergence of the radiology technician , an allied profession that has often deprived us of direct contact with the patient ; third , the development of new technologies ( computed tomography , ultrasound , interventional radiology and magnetic resonance imaging ) , which has further expanded the commitments and responsibilities of the radiologist , taking up more time and making communication with the patient increasingly infrequent and difficult . 
 in addition , the idea has taken hold that it is not essential for the radiologist to communicate with the patient ( now that specialist medicine prioritises communication with the general practitioner or the referring specialist ) , in part because the radiologist may not know the patient and does not know how he or she might react to bad or worrying news . 
the radiologist may also be afraid to say too little or too much , perhaps causing embarrassment to the general practitioner or specialist , and may think that the patient would prefer to have the diagnosis communicated and explained by someone else . finally , todays radiologists are not always adequately trained to communicate with patients or explain a report , being used to maintaining a certain detachment and concentrating on the accuracy and completeness of the diagnosis ; they have long had an almost ancillary status with respect to clinicians to whom the patients refer for interpretation of the report when not of the images themselves . this forced association between the report and its communication has meant that in italy , apart from sporadic mention by a few influential authors [ 14 ] , the question of the desirability or even the need for the radiologist to communicate verbally the results of radiological examinations to patients has never been addressed . 
the first contribution bringing radiologists attention to the increasing lack of information to patients with regard to examination results was the famous and much - quoted editorial by berlin [ 5 ] in 1977 , titled the radiologist : doctors doctor or patients doctor ? at the latest european congress of radiology questa attenzione concentrata sul referto metteva per in ombra il fatto , incontestabile , che il referto non esauriva in alcun modo il dovere del medico , e quindi anche del medico radiologo , di comunicare con il paziente , come ci viene ben ricordato dallart . 
 purtroppo , labitudine al dialogo fra il medico radiologo ed il paziente si perduta negli anni 60 per una serie di fattori oggettivi , ma anche , a mio modesto parere , per nuovi atteggiamenti culturali che cercher ora di ricordare . 
anzitutto , lestendersi dellassistenza sanitaria che ha moltiplicato vertiginosamente gli esami radiologici ; poi , la comparsa della nuova figura professionale del tecnico di radiologia che ci ha privato sovente del contatto diretto con i pazienti ; in seguito , laffermarsi delle nuove tecnologie ( tomografia computerizzata , ecotomografia , radiologia interventistica , risonanza magnetica ) che ha ancora dilatato gli impegni e le responsabilit del medico radiologo impegnandone sempre pi il tempo e rendendo sempre pi difficile e raro il colloquio . oltre a ci , si fatto strada negli anni il concetto che non sia compito essenziale del medico radiologo comunicare con il paziente ( ormai la medicina specialistica considera prioritaria la comunicazione con il medico di base o quantomeno inviante ) , anche perch il medico radiologo non lo conosce o quasi e non sa come potrebbe reagire ad una notizia cattiva o anche solo inquietante , ha inoltre paura di dire troppo o troppo poco mettendo forse in difficolt il curante o un altro specialista e talora pensa che il paziente stesso preferisca ricevere da altri la comunicazione della diagnosi e le spiegazioni ad essa legate . infine , il medico radiologo oggi non sempre adeguatamente preparato a dialogare con il paziente o anche solo a spiegare adeguatamente un referto , abituato a mantenere un certo distacco dal paziente occupandosi di pi dellesattezza e della completezza della diagnosi , mantiene da tempo una condizione quasi ancillare nei confronti del clinico al quale il paziente si rivolge per interpretare il referto quando non le stesse immagini . la forzata identificazione fra referto e comunicazione ha fatto s che in italia , tranne pochi cenni di pochi autorevoli autori [ 14 ] , non fosse mai affrontato il problema dellopportunit o forse della necessit della comunicazione verbale ai pazienti dei risultati delle indagini radiologiche . 
il primo articolo che pose allattenzione dei medici radiologi la sempre crescente disinformazione dei pazienti sui risultati delle loro indagini fu il celebre , citatissimo , editoriale di berlin [ 5 ] del 1977 , dal titolo the radiologist : doctors doctor or patients doctor ? e nel pi recente european congress radiol med ( 2011 ) 116 : 11531160 1155 ( more than 30 years later ! ) the same author again took up and developed what he wrote back in 1977 . of radiology ( dopo pi di trentanni ! ) lo stesso autore ha ripreso e potenziato quanto scritto allora . the numerous papers that followed mainly concerned the two basic aspects of communication : if and why and how . 
according to several authors , the question remains open : the ethical and deontological requirement for communication is not in question ( and improved communication can help avoid malpractice litigation ) , whereas the controversy remains as to whether it should be the radiologist to communicate with the patient and whether this is indeed what the patient wants [ 610 ]  . 
other authors , following surveys conducted both in europe and the united states , show that about 90% of patients want to hear the results of imaging investigations directly from the radiologist in all cases , whereas the remaining 10% want this done only following an explicit request . 
other , especially french , authors have taken for granted the need for verbal communication with the patient to explain the procedures and have moved on to analyse the various possibilities and opportunities of verbal communication [ 1826 ] , dispensing many at times interesting but often obvious suggestions and providing some protocols [ costruire , raliser , ecouter , donner informations , organiser ( credo ) , partnership , excuses ( apology ) , respect , legitimization , empathy , support ( perles ) , setting up , perception , invitation , knowledge , emotions , strategy ( spikes ) ] to improve and standardise communication . what happens in italy ? there are few who deny the right of patients to be informed of the results of imaging procedures , whereas many ( in varying percentages according to the subspecialty ) , for reasons of time and work organisation , overlook this right and consider the report as the only required form of communication . 
to better assess the situation and determine whether and to what extent patients receive verbal communication of the results of procedures in addition to the written report , a brief survey was conducted in the diagnostic imaging unit of a public hospital and a noncontracted private clinic . 
the question put to my colleagues was : was the patient informed verbally of the results of the procedure ? if yes , please specify whether the information was conveyed using a few quick and generic words or via a complete and comprehensive report . 
the results are presented in tables 1 and 2 . as can be seen , the difference between the two situations is substantial as far as conventional radiology is concerned , where the constant and predominant presence of the radiology technician makes any communication with the radiologist almost impossible ; this is much less the case for all the other imaging modalities . 
secondo i diversi autori il problema rimane aperto : lesigenza etica e deontologica della comunicazione non in discussione ( e la maggiore comunicazione pu mettere al riparo dalle denunce per malpractice ) , mentre permane controversa lopportunit che sia proprio il medico radiologo a parlare e che sia proprio questo che i pazienti desiderano [ 610 ]  . 
altri autori , a seguito di indagini svolte in nazioni europee e negli stati uniti , hanno dimostrato che circa il 90% dei pazienti desidera che il medico radiologo comunichi loro sempre lesito degli esami , mentre il restante 10% lo desidera solo a seguito di unespressa richiesta . 
solo il 50% dei medici curanti desidera invece la comunicazione verbale al paziente da parte del medico radiologo , ma la percentuale aumenta considerevolmente ( circa il 70% ) quando si tratta di situazioni gravi o urgenti [ 1117 ]  . 
ancora altri autori , specie francesi , considerata come acquisita la necessit del colloquio con il paziente per la spiegazione degli esami , hanno analizzato le diverse e le migliori possibilit ed opportunit della comunicazione verbale [ 1826 ] dispensando numerosi , talora interessanti ma sovente scontati consigli e fornendo anche alcuni protocolli standard ( construire , raliser , ecouter , donner informations , organiser [ credo ] , partnership , excuses ( apolology ) , respect , legitimization , empathy , support [ perles ] , setting up , perception , invitation , knowledge , emotions , strategy [ spikes ] ) per migliorare e standardizzare il colloquio stesso . 
 cosa avviene in italia ? sono ormai pochi quelli che negano il diritto da parte dei pazienti di essere informati sui risultati delle indagini di diagnostica per immagini , mentre sono moltissimi ( in diversa percentuale a seconda delle sub - specialit ) che per ragioni di tempo e di organizzazione del lavoro non ne tengono alcun conto e considerano il referto la sola forma dovuta di comunicazione . 
al fine di meglio verificare la situazione , stata condotta una breve inchiesta nel servizio di diagnostica per immagini di un ospedale pubblico e di un poliambulatorio privato non convenzionato al fine di verificare se ed in quale misura i pazienti ricevevano , oltre al referto scritto , una comunicazione verbale sui risultati degli esami . 
la domanda formulata al collega era questa : stata data al paziente una comunicazione verbale sullesito dellesame ? in caso positivo si doveva precisare se tale comunicazione era stata fornita con poche rapide e generiche parole ( a ) oppure con una completa ed esauriente relazione ( b )  . 
i risultati sono presentati nelle tabelle 1 e 2 . come si pu osservare , la differenza fra le due situazioni sensibile per quanto riguarda la radiologia tradizionale , dove la costante e prevalente attivit del tecnico radiologo 1156 radiol med ( 2011 ) 116 : 11531160 table 1 san giovanni bosco public hospital , turin , italy table 2 ci . 
 the brief survey showed , however , that in the most serious cases , verbal communication usually does take place . nothing can be said with regard to the quality of the verbal communication , if not to restate that the radiologist is not always sufficiently trained to communicate with patients , given that this tradition has been long lost , and radiology specialty schools offer no opportunity for communication and do not ( yet ? ) teach doctorpatient communication techniques , there being no conviction that this is either useful or necessary . a recent and highly recommended book by de santi and simeon [ 27 ] suggests the characteristics of ideal consultation , lasting not less than 10 min ! here is a summary : 1 . 
welcome : the setting , privacy , the possible assistance of a family member , listening ( patients usually ending what they have to say in 92 s ) ; 3 . 
information : supplied gradually and simply , leaving room for other questions and respecting what the patient does not want to know , to the point of reaching if possible the diagnosis ; rende quasi impossibile il colloquio con il medico ; lo assai meno per tutte le altre metodiche . 
la differenza segnalata fra il colloquio breve e generico e quello pi dettagliato e completo sembra in gran parte legata alla complessit e gravit della patologia riscontrata e in qualche misura anche allimpressione soggettiva del medico radiologo . 
 non possibile alcuna considerazione a proposito della qualit del colloquio , se non ribadendo che il medico radiologo non sempre sufficientemente preparato a parlare con il paziente : se ne da tempo perduta la tradizione , le scuole di specializzazione non ne evidenziano anche solo lopportunit e non insegnano ( ancora ? ) le migliori modalit del colloquio stesso , fa difetto la convinzione che sia davvero utile e necessario . un recente e consigliabilissimo libro italiano di de santi e simeoni [ 27 ] suggerisce le caratteristiche di un colloquio ideale , della durata non inferiore a 10 minuti ! eccone una sintesi : 1 . 
accoglienza : i locali , la privacy , leventuale assistenza di un familiare , lascolto ( di solito il paziente conclude il suo discorso in 92 secondi ) ; 3 . 
support : not only describing the subsequent diagnostic and therapeutic procedures , but also covering the benefits and possible risks , and a general assessment of the prognosis ; 5 . 
conclusion : a friendly goodbye with the offer of further consultations and a telephone number . such an ideal consultation is no doubt difficult , if not impossible , to carry out but , without succumbing to the easy temptation of thinking that it is the duty of clinicians only , certain perhaps necessary considerations need to be made , such as : verbal communication with patients is always appropriate and sometimes indispensable ( in severe cases and emergencies ) ; spazio ad altre domande del paziente e rispettando quanto non vuole sapere fino a giungere se possibile alla diagnosi ; 4 . 
conclusione : un saluto amichevole , la possibilit di un ulteriore colloquio , un numero di telefono . un colloquio ideale come quello proposto certamente difficile e forse impossibile da realizzarsi , ma senza cedere alla comoda tentazione di pensare che sia solo compito dei clinici sono possibili e forse doverose alcune considerazioni : sempre opportuno e talora indispensabile ( casi gravi , urgenze ) il colloquio verbale con i nostri pazienti ; the fact that the referring physician may object to this il fatto che non sempre il curante lo desideri non ci esime activity does not exempt us from this duty ; da questo dovere ; the real problem is mainly to find the time and to acquire il vero problema anzitutto quello di trovare il tempo e and maintain the best possible level of preparation . poi di avere e mantenere la migliore preparazione . preparation la preparazione this is based on two premisses : the first , concerning the radiologists personally , is that the consultation should be considered as an integral part of the examination and not as an added extra to be granted only under certain circumstances ; the other is to ensure that the patient understands that this consultation is his or her right and may prove very useful . 
it is also essential to : humbly read books or magazines that can help to adapt sono necessarie due premesse : la prima , che riguarda personalmente il medico radiologo , quella di considerare il colloquio verbale come parte integrante dellesame e non come un di pi che pu essere concesso solo in determinate circostanze e laltra quella di far comprendere al paziente che tale colloquio un suo diritto e pu essere per lui di grande utilit . 
 time il tempo this is the main problem with one major premiss : it is clear that the radiologist rarely has the time or opportunity to talk to every patient , and this often imposes the difficult choice of whether to say very little to everyone ( or almost everyone ) or something in depth to those who need it most . 
 but which patients have the most need for verbal communication ? apart from the more serious and urgent cases , it is very difficult if not impossible to distinguish those who want to listen and talk from those who cannot wait to get back to their own doctor to talk to him or her , bearing in il principale problema con una sola importante premessa : evidente che ben raramente si ha il tempo e quindi la possibilit di parlare con tutti e sovente si imporr la difficile scelta se dire poco a tutti ( o quasi ) o qualcosa di pi a chi ne ha maggiormente bisogno . 
ma quale paziente ha maggiore bisogno del colloquio verbale ? a parte i casi pi gravi e quelli pi urgenti ben difficile se non impossibile riconoscere chi vorrebbe ascoltare e parlare e chi invece non aspetta che di ritornare dal suo medico per parlare con lui , ben consapevoli che anche il paziente con un radio1158 radiol med ( 2011 ) 116 : 11531160 fig . 
but why not discuss it with the radiologist ? the radiologist is a specialist physician who can guide you as to the most suitable diagnostic pathway for each medical condition . 
dialoga con lui , il confronto davvero importante ( societ italiana di radiologia medica )  . mind that the patient with a normal chest x - ray often needs to know the value and limitations of the examination , when ( or if ) it should be repeated , the risks associated with exposure to so many x - rays , and so forth . 
currently , the choice made in most areas of diagnostic imaging is to speak with those who need it most , although this choice , while often necessary , excludes many ( and all those who have a right to it ) from the possibility of a verbal consultation . 
it should , however , be noted that : gramma toracico normale ha sovente bisogno di conoscere il valore ed i limiti di tale indagine , quando la dovr eventualmente ripetere , se si prendono tanti raggi , ecc . 
 attualmente , la scelta concreta che viene fatta nella gran parte dei servizi di diagnostica per immagini quella di parlare con chi ne ha maggiormente bisogno , ma con tale scelta pur sovente necessaria si toglie a molti che lo desiderano ( e a tutti quegli altri che ne hanno un certo diritto ) la possibilit del colloquio verbale . 
va per ricordato che : radiol med ( 2011 ) 116 : 11531160 1159 the consultation may be deferred to a later date ( a phone number , as recommended in de santi and simeones book on the ideal consultation ) or just slightly delayed if this makes it possible ; it is not strictly necessary that the person reporting on the examination be the same one to engage in the consultation , which may certainly be carried out by a colleague ; it is important that there is a real possibility of a verbal consultation with the radiologist for each patient who wants one . a notice listing the radiologists availability ( as seen in every division of care ) , perhaps accompanied by the posters proposed by the italian society of medical radiology , ought to be displayed at the entrance to each diagnostic imaging department . 
this would be helpful both to remind the radiologist of his or her duty to communicate verbally with patients and to teach patients not to flee or ignore ( as happens not infrequently ) that specialist who can and often wants to communicate numerous things to them . il colloquio verbale pu essere posticipato ( un numero di telefono , come si raccomandava in precedenza nel libro di de santi e simeoni [ 27 ] a proposito del colloquio ideale ) o anche solo differito di poco se questo lo rende possibile ; non strettamente necessario che sia lesecutore del referto ad avere il colloquio verbale con il paziente ma un collega certamente in grado di supplirlo ; importante che vi sia , per ogni paziente che lo desidera , la concreta possibilit del colloquio verbale con il medico radiologo . un cartello con una disponibilit oraria dei medici ( come avviene in ogni divisione di cura ) , magari accompagnato dalle figure proposte dalla sirm auspicabile possa essere affisso allingresso di ogni servizio di diagnostica per immagini . 
sar utile per ricordarci la necessit se non il dovere del colloquio verbale con i pazienti e sar utile a loro che impareranno a non pi sfuggire o ignorare ( come oggi non raramente avviene ) quello specialista che pu e sovente vuole dir loro tante cose . conclusions conclusioni i do not think that these simple musings of an old radiologist ( as i was going to title this short article ) about a problem of the past that has become highly relevant today can provide any easy solutions or resolve uncertainties . 
i do hope , however , that they may help raise awareness of the situation and provide a stimulus for debate in the search for possible and shared solutions . non penso che queste semplici considerazioni di un vecchio radiologo ( cos avevo intenzione di titolare questo breve articolo ) su un problema del passato e oggi pi che mai del presente possano indicare facili soluzioni n risolvere incertezze : spero possano essere unutile presa di coscienza di una realt ed uno stimolo al dibattito ed alla ricerca di soluzioni possibili e condivise . 
 conflict of interest none radiol med ( 2011 ) 116 : 1314 doi 10.1007 / s11547 - 011 - 0726 - 1 erratum adenomyosis : from the sign to the diagnosis . 
senologia clinica e screening mammografico , dipartimento di radiodiagnostica , apss , trento , italy 2dipartimento di radiodiagnostica , apss , trento , italy 3centro di prevenzione senologica ( cps ) , po marzana , ulss 20 , verona , italy 4crr , centro di riferimento regionale per lo screening mammografico , torino , italy correspondence to : s . 
 proportional incidence was determined according to breast cancers expected in the absence of screening , estimated on the basis of patients / year at risk and age - specific incidence . 
 the review of screening mammograms preceding ics was performed by an external ( three radiologists ) and an internal ( five radiologists ) panel and aimed at assessing the proportion of ic reviewed as screening errors . 
ic proportional incidence was 15.90% for the first year ( ec standard < 30% ) and 25.77% for the second year ( ec standard < 50% ) of the interval . 
at external review , 18.4% of cases were reviewed as screening errors ( identified by at least two of three reviewers ) , whereas at internal review ( identified by at least three of five reviewers ) it was 17.4% ( ec standard < 20% )  . 
la revisione dei radiogrammi di screening precedenti i ci stata operata da un panel di radiologi esterno ( 3 radiologi ) e uno interno ( 5 radiologi ) , per la definizione del tasso di ci valutati come errori di screening . 
il tasso proporzionale di ci stato del 15 , 90% nel primo anno dellintervallo ( standard ce < 30% ) e del 25 , 77 % nel secondo anno ( standard ce < 50% )  . 
alla revisione esterna , la frazione di casi rivalutati come errori di screening ( identificati da almeno 2 su 3 revisori ) stata del 18 , 4% : alla revisione interna ( almeno 3 su 5 revisori ) stata del 17 , 4% ( standard ce < 20% )  . 
lapparente equivalenza della revisione esterna e interna suggerisce che la seconda , certamente pi agevole , possa essere impiegata nella routine per la definizione di questo indicatore precoce di efficacia . keywords breast carcinoma diagnosis mammography screening interval cancers parole chiave carcinoma della mammella mammografia diagnosi screening carcinomi di intervallo introduction introduzione mammography screening for early diagnosis of breast cancer has proved effective in reducing breast cancer mortality rates and is recommended by the european community ( ec ) [ 1 ] and implemented in the entire italian territory following specific recommendations of the ministry of health [ 2 ]  . 
as the impact of single screening programmes on mortality rates cannot be determined in the short term , a variety of early indirect indicators of efficacy has been proposed and recommended [ 1 ] for early assessment of screening programme performance [ 3 ]  . 
among these , interval cancer ( ic ) frequency and review to assess screening false negatives are considered as the most reliable indicators of sensitivity and the best indirect indicators of efficacy [ 1 , 3 ]  . 
 the analysis of ic observed in the trento province mammography screening programme was carried out , with a methodology comparable with other italian studies [ 37 ] , based on assessing the proportional incidence of ic and the fraction of ic classified as screening errors at review . 
the results of such analyses are the objects of this report . materials and methods a population - based mammography screening programme was started in trento province in october 2000 , a preliminary 3 - month pilot study being carried out in the district of trento , with further extension to the entire province . 
the screening test was performed at six different sites in trento province , and reading was centralised at the trento screening unit using recommended double reading [ 8 ]  . 
 in 2005 film - screen mammography was abandoned and digital mammography was adopted [ direct ( full - field digital mammography ) at trento centre and indirect ( computer radiography ) at the other screening sites ]  . 
 lo screening mammografico per la diagnosi precoce del carcinoma mammario pratica di provata efficacia , correntemente raccomandata dalla comunit europea ( ce ) [ 1 ] e implementato in tutto il territorio italiano a seguito di raccomandazione del ministero della salute [ 2 ]  . 
vista limpossibilit di definire , sul breve termine , limpatto di un programma di screening sulla mortalit , sono stati suggeriti e raccomandati [ 1 ] una serie di indicatori indiretti di efficacia che consentano una valutazione precoce della performance del singolo programma [ 3 ]  . 
tra questi lanalisi della frequenza dei carcinomi di intervallo ( ci ) e la loro revisione , al fine di definire leventuale errore radiologico , sono considerati i migliori indicatori della sensibilit di un programma di screening e , quindi , indirettamente della sua efficacia [ 1 , 3 ]  . 
in analogia con precedenti esperienze italiane di una simile verifica [ 37 ] , nel programma di screening mammografico della provincia di trento stata effettuata lanalisi dei ci osservati , sia per il calcolo della incidenza proporzionale che per il tasso di errori di screening alla revisione . 
i risultati di tale analisi sono oggetto del presente lavoro . materiali e metodi lo screening mammografico in provincia di trento ha avuto inizio a ottobre del 2000 , con un primo studio pilota condotto per circa tre mesi nel distretto di trento ed in seguito con generalizzazione su tutto il territorio provinciale . 
lesame di screening stato eseguito in sei diverse sedi , dislocate nel territorio provinciale , con centralizzazione delle mammografie presso il presidio di trento dove sono state esaminate da parte di radiologi senologi dedicati secondo la raccomandata procedura della doppia lettura [ 8 ]  . 
dal 2005 dalla mammografia analogica si passati progressivamente alla mammografia digitale , diretta presso il distretto di trento ed indiretta in tutte le altre sedi . gli indicatori di performance del programma , regolarradiol med ( 2011 ) 116 : 12171225 1219 performance indicators for the screening programme are regularly provided , and are available online , to the national screening observatory [ 2 ]  . 
ic were identified by linking screening archives with those of the local cancer registry and the local pathology department and with hospital discharge records ( hdr ) provided by local health authorities . 
all histologically confirmed invasive breast cancers occurring within 2 years of a negative screening episode from 2001 to 2006 ( negative screening test or positive screening test with negative assessment ) were assumed as ic . 
considering that follow - up for ic identification was complete up to june 2008 , ic relative to 2006 screening are underestimated for the second year of the interval , and those relative to 2007 do not contribute to the proportional incidence in the second year of the interval . 
the estimate of expected cancers was obtained by multiplying patient age groups ( 5054 , 5559 , 6064 , 6569 ) by the age - specific breast cancer incidence for trento province , as provided by the local cancer registry and relative to 19962000 . the review of negative screening mammograms preceding ics was performed in a blinded modality [ 9 ] , as recommended by the ministry of health [ 10 ] , by adding negative controls ( at least one subsequent negative screening ) randomly drawn for screening archives and matched to ic with a 3 : 1 ratio . 
external independent review was carried out by a panel of three radiologists with varying screening experience ( fc > 9 years , af > 20 years , sc > 30 years )  . 
internal review was carried out by a panel of five radiologists actively involved in screening reading , with varying screening experience ( db 11 years , sdm 10 years , pt 7 years , cf 3 years , mv 1 year )  . 
the reviewer was requested to mark on a paper scheme of the four mente comunicati e desumibili on line dal sito dellosservatorio nazionale screening [ 2 ] per la survey del 2008 riportano una estensione corretta dell85 , 5% ( biennale 80 , 9% ) , una popolazione bersaglio di 26000 soggetti invitati e 19400 aderenti ( adesione grezza = 74 , 8% , corretta = 79 , 2% )  . 
sono stati considerati ci tutti i carcinomi invasivi di cui sia stata comprovata la diagnosi in data successiva ed entro due anni da una mammografia di screening con esito negativo ( o positivo con successivo approfondimento negativo ) eseguita nellambito del programma di screening da gennaio 2001 a dicembre 2006 . 
poich il follow - up per la identificazione dei ci completo a giugno 2008 , i ci relativi agli esami eseguiti nel 2006 sono sottostimati per il secondo anno dellintervallo , e quelli relativi agli esami eseguiti nel 2007 non contribuiscono alla incidenza relativa per il secondo anno . 
il numero dei ci osservati rispetto a quelli attesi in assenza di screening ha fornito lincidenza proporzionale di ci : i ci attesi sono stati determinati moltiplicando il numero di pazienti / anno per quinquenni di et ( 5054 , 5559 , 6064 , 6569 ) per lincidenza et specifica per carcinoma mammario nella provincia di trento fornita dal registro tumori locale e relativa al periodo 19962000 la revisione dei radiogrammi di screening negativi precedenti i ci stata fatta secondo la modalit della revisione cieca [ 9 ] , in ottemperanza alle raccomandazioni del ministero della salute [ 10 ] , con aggiunta di controlli negativi ( almeno un esame di screening successivo negativo ) tratti a random dal programma di screening e accoppiati in rapporto di tre controlli per ogni caso di ci . 
le immagini ( analogiche , relativamente al periodo in studio ) sono state montate su visori rotanti in modo casuale e il revisione stato chiamato a indicare leventuale anormalit mammografica meritevole di approfondimento su uno schema grafico delle quattro proiezioni mammografiche . 
 in the following evaluation of review results , the exact correspondence of the breast abnormality indicated by the reviewer and the actual site of cancer occurrence was verified for each ic . 
attribution to occult , minimal sign or screening error review categories was based on majority report , that is , if no , one or two to three reviewers ( external review ) or no , one to two or three to five reviewers ( internal review ) had marked the abnormality . results relative to ic proportional incidence and review were compared with ec guidelines [ 1 ] : recommended standards for proportional incidence are < 30% in the first year , < 50% in the second and < 40% on a biennial basis , whereas for ic review , < 20% of cases should be reviewed as screening error . 
observed differences were checked by the chi - square test , with statistical significance being set at p < 0.05. nella successiva valutazione dei risultati della revisione , leffettiva corrispondenza della alterazione mammografica indicata dal revisore con la sede esatta di comparsa della neoplasia stata verificata in tutti i casi di ci . 
lattribuzione alle categorie occulto , segni minimi e errore di screening avvenuta a maggioranza : per la revisione esterna combinando le tre revisioni , rispettivamente nel caso che nessuno , uno solo , o almeno due revisori avessero identificato il ci , e per la revisione interna combinando le cinque revisioni , rispettivamente nel caso che nessuno , 12 o 35 revisori avessero identificato il ci . i risultati in termini di incidenza proporzionale dei ci e della loro revisione sono stati confrontati con la letteratura e con gli standard raccomandati dalla ce [ 1 ] che prevedono per lincidenza proporzionale un massimo del 30% nel primo anno , del 50% nel secondo anno , e del 40% nel biennio rispetto alla incidenza attesa in assenza di screening e , per la revisione , un massimo del 20% dei casi revisionati attribuiti alla categoria errori di screening . 
le differenze osservate sono state valutate mediante il test chi quadrato , ponendo la significativit statistica a un livello di p < 0 , 005 . linkage to the cancer registry , pathology registry and hdr allowed identification of 101 ics during the study period . 
the corresponding sensitivity figures were 84.10% at 1 year , 64.33% at 2 years and 79.61% on a biennial basis . ic review considered a total of 434 cases ( 103 ic , 331 controls )  . 
a slightly higher number of ic compared with those considered to assess proportional incidence is explained by the fact that a few ic cases relative to calendar periods with no follow - up coverage were included in the review set . 
similarly , a slight excess of controls compared with the intended 3 : 1 ratio was retrieved from archives and left in the review set . results of the external review are shown in table 2 . 
overall , ic identified by two to three reviewers and thus classified as screening errors were 18.4% ( 19 / 103 ) , that is , below the maximum acceptable 20% standard recommended by the ec [ 1 ]  . 
lincidenza proporzionale relativa al primo anno dellintervallo risultata quindi del 15 , 90% ( 43 / 270 , 4 ) , quella relativa al secondo anno dellintervallo risultata del 25 , 77% ( 58 / 225 ) , e quella relativa al biennio del 20 , 39% ( 101 / 495 , 3 ) , in tutte le circostanze al di sotto dei valori massimi consentiti dalle raccomandazioni ce . 
il valori di sensibilit equivalenti sono dell84 , 10% a un anno , 64 , 33% a due anni e 79 , 61% nel biennio . la revisione dei ci ha riguardato un totale di 434 casi ( 103 ci e 331 controlli ) : il numero lievemente maggiore di ci rispetto a quelli valutati per il calcolo della incidenza proporzionale risulta dalla inclusione di tutti i ci osservati , anche relativi a periodi con follow - up incompleto . 
anche un lieve eccesso di controlli rispetto alla prevista ratio di 3 : 1 stato acquisito dagli archivi e lasciato nel set di revisione . i risultati della revisione esterna sono riportati nella tabella 2 . 
complessivamente i ci identificati da 2 o 3 lettori e quindi codificati come errori di screening sono stati il 18 , 4% ( 19 / 103 ) , quindi al di sotto del 20% , valore massimo accettabile seconde le raccomandazioni ce [ 1 ]  . 
overall , ic identified by three to five reviewers and thus classified as screening errors were 17.4% ( 18 / 103 ) , that is , below the maximum acceptable 20% standard recommended by the ec [ 1 ] and comparable to what was obtained at external review . 5 indica la probabilit di identificazione di ci di un richiamo ad approfondimento da parte di uno o pi revisori interni . 
complessivamente i ci identificati da 35 lettori e quindi codificati come errori di screening sono stati il 17 , 4% ( 18 / 103 ) , quindi al di sotto del 20% , valore massimo accettabile seconde le raccomandazioni ce [ 1 ] , un tasso non differente da quanto ottenuto con la revisione esterna . discussion this study was based on a sufficiently large series allowing for reliable analysis of ic within the mammography screening programme of trento province . 
the entire province is covered by a single department for breast cancer diagnosis , and symptomatic women referring to any hospital / district in the province and having a previous screening mammography are systematically referred to the central screening unit in trento for diagnostic assessment . 
 this condition definitely made ic identification easier , but the linkage of three archives ( cancer registry , pathology registries , hdr ) would have equally allowed for ic identification , particularly of those with no histological confirmation or those diagnosed / treated outside the province . 
 migration outside the province for treatment is very low , but the identification of cases treated elsewhere is guaranteed through the hdr archive , providing data with only a short delay after discharge . the estimate of cancers expected in the absence of screening appears reliable in this study , as it is based on a local official cancer registry , and the age - specific incidence data used were relative to the period immediately preceding the onset of the screening programme . 
the latter condition allows for a reliable estimate of cancer incidence in the absence of screening , as it excludes major long - term incidence temporal trends and takes into account spontaneous access to mammography available before screening onset . although the ec guidelines indicate that in situ carcinomas should be considered among ic , they cannot be included in the evaluation of the proportional incidence , which is based on the estimate of expected cancers . 
 thus , in situ carcinomas were excluded in order to allow discussione lo studio si basa su una casistica sufficientemente ampia da consentire una valutazione affidabile dei ci nellesperienza di screening della provincia di trento . 
tale centralizzazione garantisce che la donna sintomatica che si presenti in ogni distretto / ospedale diverso da trento , in presenza di una mammografia di screening precedente , venga sistematicamente inviata per gli approfondimenti presso il centro di riferimento . 
peraltro lincrocio dei tre archivi ( registro tumori , anatomia patologica , sdo ) , che garantisce lidentificazione anche di eventuali casi senza identificazione istologica o diagnosticati / trattati al di fuori della provincia , resta il riferimento informativo essenziale ed risultata pi che sufficiente . 
si sottolinea che la migrazione per la terapia verso altre regioni assai modesta in trentino ; anche in quei casi lidentificazione viene per lappunto garantita dalla verifica delle sdo che sono disponibili entro breve tempo dal ricovero . 
 la stima dei ci attesi in assenza di screening , nel nostro caso , appare affidabile sia perch basata su un registro tumori ufficiale , sia perch per lo studio sono stati utilizzati tassi et specifici di incidenza riferibili al periodo immediatamente precedente allo screening , garantendo quindi un quadro affidabile della incidenza attesa in assenza di un programma di screening organizzato : esso infatti esclude variazioni temporali di incidenza sul lungo termine , e tiene conto dellaccesso spontaneo alla mammografia disponibile prima dellavvento del programma di screening . nonostante le linee guida ce indichino che i ci comprendono anche i carcinomi in situ , questi ultimi non possono essere valutati nel calcolo della incidenza proporzionale che si basa sul calcolo dei carcinomi attesi , a loro volta derivati da registri di incidenza , che considerano sono i carcinomi invasivi . 
in situ carcinomas might have been included in the review , but we chose to limit it to invasive lesions : ( a ) for a better comparison with other italian experiences [ 37 ] adopting the same crite rion , and ( b ) as we assume that most in situ carcinomas detected within the interval are usually asymptomatic and identified by spontaneous annual early rescreening . 
 the review of screening mammograms preceding ics was carried out in a blind modality , which has been reported to be the most unbiased condition , better approaching the scenario in which a screening error could potentially occur [ 9 , 10 ]  . 
in fact , the recall rate was higher than in the everyday practice ( external review 5.210.3% ; internal review 5.111.4% ) though less elevated compared with other similar experiences [ 37 ]  . 
at external review , the choice of assuming as screening errors cases with two to three recalls is supported by a high probability of ic being detected and no significant difference between two or three recalls . 
similarly , assuming cases identified by one reviewer only as minimal sign ( a category that does not imply a diagnostic error ) is supported by the low probability of ic being detected in these cases , very similar to that of cases with no recall . 
based on the observed findings , performance of the screening programme was also satisfactory according to the rate of screening errors at review , which was within the limits recommended by the ec [ 1 ] at both external and internal review . overall , the results indicate that the trento province mammography screening programme has a good performance as to sensitivity based on observed ic , and this may predict a good efficacy in reducing breast cancer mortality rates . 
the beneficial effects of screening on mortality rates are visible only some years after screening onset [ 11 ] and bero potuto essere compresi nella revisione , ma abbiamo ritenuto di limitare questultima alle sole forme invasive sia per un migliore confronto con altre esperienze italiane , che hanno adottato lo stesso criterio [ 37 ] , sia anche nella convinzione che i carcinomi in situ diagnosticati nellintervallo , abitualmente asintomatici , siano per la maggior parte relativi alluso di una mammografia intermedia ( annuale ) , di impiego assai varabile nelle diverse realt , e che quindi possano costituire un confondente nella comparazione con altri scenari . 
 la performance del programma di screening in termini di incidenza proporzionale di ci del tutto soddisfacente , decisamente al di sotto dei limiti massimi consentiti dalle raccomandazioni ce . la revisione dei radiogrammi di screening negativi precedenti i ci stata fatta secondo la modalit della revisione cieca , che risulta essere la meno influenzata e la pi vicina alla condizione in cui si pu supporre sia stato commesso un errore diagnostico [ 9 , 10 ]  . 
inevitabilmente i revisori , consci comunque di condurre una revisione a posteriori , hanno probabilmente adottato una soglia di sospetto lievemente inferiore alla norma , come dimostrato dal tasso di richiamo ( revisione esterna = 5 , 2%10 , 3% ; revisione interna = 5 , 1% 11 , 4% ) , che pure risulta abbastanza contenuto in confronto di altre esperienze simili [ 37 ]  . 
 la scelta di attribuire la codifica di eventuale errore di screening non in base ad un unico revisore ma alla maggioranza di un panel di revisori derivata dal desiderio di fornire una valutazione pi bilanciata , dal momento che noto che il giudizio del singolo revisore soffre di una certa soggettivit , come dimostrato anche dalle modeste differenze riscontrate tra le revisioni di questo studio . 
nella revisione esterna , la scelta di codificare come errore di screening i casi nei quali il ci era stato individuato da almeno due lettori confortata dal fatto che la probabilit di identificazione di ci assai elevata e non differisce sia che due o tre revisori abbiano individuato il ci . 
parimenti , laver omologato a segni minimi , categoria che non configura un errore diagnostico , i casi con riconoscimento del ci da parte di un solo revisore confortato dal fatto che la probabilit di identificazione di ci non diverso dai casi negativi per tutti i revisori . 
in base ai risultati dello studio , anche il tasso di ci revisionati e considerati errore di screening risultato essere contenuto entro i limiti consentiti dalle raccomandazioni ce [ 1 ] ad entrambe le revisioni . complessivamente i risultati del presente studio dimostrano che il programma di screening mammografico della provincia di trento ha una buona performance quanto a sensibilit , stimata in base ai ci osservati , e questo consente di prevedere una efficacia altrettanto buona nel ridurre la mortalit per carcinoma della mammella , dato che tende ad essere osservabile con una certa latenza rispetto allinizio radiol med ( 2011 ) 116 : 12171225 1225 will be evaluated in a further study . 
comparison between the external and internal reviews showed similar results , which might suggest the adoption of an internal review , which is easier to implement on a local and regular basis , as a standard quality control procedure . del programma di screening [ 11 ] , ed attualmente in corso di valutazione . 
coronary angiography with multidetector - row computed tomography ( mdct - ca ) allows quantification of coronary artery stenosis with a high level of accuracy ; however , a better estimation of stenosis can be achieved by using appropriate reformatting filters , especially in stents and calcified segments . 
langiografia coronarica ( ac ) con tomografia computerizzata multistrato ( tcms ) permette di quantificare la stenosi delle arterie coronarie con un alto livello di accuratezza ; tuttavia una migliore stima della stenosi pu essere calcolata usando appropriati filtri di ricostruzione , soprattutto nei segmenti con stent o calcificazioni . 
filtri particolari possono aiutare a stimare meglio lesatta percentuale della stenosi . keywords multidetector ct coronary angiography kernel quantitative coronary angiography reformatting filter parole chiave angiografia coronarica - tcms kernel angiografia coronarica quantitativa filtri di ricostruzione introduction introduzione coronary angiography with multidetector - row computed tomography ( mdct - ca ) is a robust and accurate imaging technique for the noninvasive study of coronary arteries [ 1 ]  . 
the correct estimate of coronary artery stenosis is crucial for planning patient treatment from follow - up in cases of nonsignificant stenosis to by - pass surgery in cases of chronic total coronary artery occlusion [ 2 ]  . 
coronary artery evaluation is based on analysis of curved multiplanar reconstructions ( mpr ) , maximum intensity projection ( mip ) reconstructions and volume - rendered ( vr ) images . 
 these techniques , however , are subjective , being based on the quality of the examination and reconstructions obtained and , above all , operator experience [ 3 ]  . 
the objective discrimination of stenosis proves additionally difficult in cases of calcified plaque or coronary artery stents , where the blooming effect alters visualisation of the true lumen of the coronary artery segment . 
 a number of semiautomatic systems for identifying and evaluating stenosis [ quantitative computed tomography angiography ( qcta ) ] have been proposed as postprocessing techniques for objective quantification of stenosis . 
however , in cases in which the stenosis is accompanied by calcified plaque or stents , evaluation with normal convolution filters has been shown to be suboptimal or imprecise [ 5 , 7 ]  . 
 the aim of this study was to evaluate the accuracy of quantitatively estimating coronary artery stenosis using qcta with all available convolution filters . langiografia coronarica ( ac ) con tomografia computerizzata multistrato ( tcms ) rappresenta una valida ed accurata metodica imaging per lo studio non invasivo delle arterie coronarie [ 1 ]  . 
la corretta stima di stenosi coronarica di fondamentale importanza per determinare il destino terapeutico di un paziente dal follow - up in caso di stenosi non significative al by - pass in caso di occlusione totale cronica [ 2 ]  . 
attualmente la valutazione coronarica basata sulla analisi di ricostruzioni multiplanari curvilinee , ricostruzioni in proieizione multiplanare ( mip ) e volume rendering , tuttavia soggettiva , basata sulla qualit dellesame e delle ricostruzioni ottenute e soprattutto sullesperienza delloperatore [ 3 ]  . 
loggettiva discriminazione di stenosi risulta inoltre pi difficoltosa in caso di placche calcifiche o di stent coronarici a causa dellartefatto blooming che altera la percezione del lume vero del segmento arterioso ; per ovviare a tale fenomeno ci si avvale di riformattazioni con filtri di convoluzione specifici consigliati dalle case in modo da ridurre al minimo il blurring originato dal calcio o dalle maglie dello stent [ 46 ]  . 
 esistono sistemi semiautomatici di identificazione e valutazione delle stenosi definiti sistemi di analisi quantitativa computerizzata dei vasi ( qcta ) proposti come tecnica di post - processing per una quantificazione oggettiva del grado di stenosi ; tuttavia in caso di stenosi sostenuta da placche calcifiche o stent , la valutazione con i normali filtri di ricostruzione rimane subottimale o inesatta [ 5 , 7 ]  . 
 scopo del presente lavoro valutare laccuratezza diagnostica nella stima quantitativa di stenosi coronariche utilizzando il qcta con tutti i filtri di convoluzione disponibili . materials and methods study population materiali e metodi popolazione in studio the study population consisted of 17 consecutive patients ( 13 men and four women ; mean age 63.5 years ) who underwent mdct - ca due to suspected coronary artery disease sono stati valutati prospetticamente 17 pazienti consecutivi ( 13 maschi e 4 femmine , et media 63 , 5 anni ) sottoposti radiol med ( 2011 ) 116 : 12031216 1205 ( cad ) between 15 december 2009 and 29 june 2010 . 
all examinations were performed with a 64 - slice system ( brillance 64 , philips , best , the netherlands )  . a ac - tcms per sospetta patologia delle coronarie nel periodo compreso tra il 15 - 12 - 2009 e il 29 - 06 - 2010 . 
tutte le indagini sono state acquisite mediante apparecchiatura tc a 64 strati ( brilliance 64 , philips , best , paesi bassi )  . inclusion and exclusion criteria inclusion criteria were heart rate < 65 bpm and the ability to sustain a breath - hold for 1012 s . 
indications for the examination were an absence of critical cad in patients with atypical chest pain and low - to - medium pretest risk , disagreement between functional tests and patient symptoms and study of stent and bypass graft patency . 
exclusion criteria were refusal to provide informed consent , previous adverse reactions to iodinated contrast material , renal impairment ( creatininaemia > 120 mmol / l ) , pregnancy , respiratory insufficiency , unstable clinical condition or marked cardiac insufficiency , prior angioplasty or cardiac surgery and insufficient diagnostic quality of the data sets . 
the local ethics committee approved the study , and all patients examined provided informed consent . patient preparation patients with heart rate 65 bpm received 515 mg of atenolol intravenously immediately prior to the examination . 
patients were studied in the supine position during an inspiratory breath - hold from the level of the pulmonary arteries up to the proximal abdomen to include the base of the heart . 
in patients with bypass graft , the scan was extended cranially to cover the area from the initial portion of the aortic arch to the base of the heart . scan protocol we intravenously administered 80100 ml of nonionic iodinated contrast material ( iomeprol 400 mgi / ml , iomeron , bracco , milan , italy ) at a rate of 5 ml / s , followed by 40 ml of saline solution at the same speed . 
the contrast material was injected with a dual - head automatic injector ( stellant , medrad , pittsburgh , pa , usa ) into an antecubital vein through a 16 - gauge needle cannula . 
 acquisition and reconstruction parameters are summarised in table 1 [ 9 , 10 ]  . data processing images were retrospectively reconstructed with the ecggated technique [ 11 , 12 ] using data acquired during a single heart beat . 
data sets were reconstructed during the mid - / criteri di inclusione ed esclusione i criteri di inclusione per gli esami ac - tcms comprendevano un ritmo cardiaco < 65 battiti per minuto ( bpm ) e la capacit di trattenere lapnea inspiratoria per 1012 s . 
le indicazioni allesame erano rappresentate da : esclusione di coronaropatia critica in pazienti con dolore toracico atipico in pazienti a basso medio rischio pre - test , discordanza tra prove funzionali e sintomatologia del paziente , controllo perviet stent e by - pass . 
i criteri di esclusione erano rappresentati da : rifiuto al consenso informato per lo studio , precedenti reazioni avverse al mezzo di contrasto iodato , insufficienza renale , ( creatininemia > 120 mmol / l ) , gravidanza , insufficiente funzionalit respiratoria , condizioni cliniche instabili o marcata insufficienza cardiaca , precedente angioplastica o interventi cardiaci e qualit diagnostica insufficiente dei datasets . 
il comitato etico locale ha approvato lo studio e tutti i pazienti esaminati hanno fornito il consenso informato . preparazione del paziente in caso di frequenze cardiache uguali o superiori a 65 bpm , sono stati somministrati 515 mg di atenololo endovena ( ev ) immediatamente prima dellesecuzione dellesame . 
i pazienti sono stati studiati in posizione supina in apnea inspiratoria dal livello delle arterie polmonari fino alla porzione prossimale delladdome in modo tale da comprendere anche la base del cuore . 
in pazienti con bypass il limite della scansione stato innalzato cranialmente alla porzione iniziale dellarco aortico fino alla base del cuore . protocollo di scansione abbiamo somministrato per via endovenosa 80100 ml di mezzo di contrasto non ionico iodato ( iomeprol 400 mgl / ml , iomeron , bracco , milano , italia ) con un flusso di 5 ml / s , seguito da 40 ml di soluzione salina allo stesso flus so . 
il mezzo di contrasto stato introdotto tramite un iniettore automatico a doppia pompa ( stellant , medrad , pittsburgh , pa , usa ) connesso ad una vena antecubitale con unagocannula da 16 g . 
i parametri di acquisizione e di ricostruzione sono riassunti in tabella 1 [ 9 , 10 ]  . 1206 radiol med ( 2011 ) 116 : 12031216 end - diastolic phase , with reconstruction windows from 300 ms to 450 ms from the next r - wave or from 60% to 70% of the r - r interval . 
in cases with insufficient image quality ( three patients ) , additional reconstructions were done during the end - systolic phase ( from 25% to 35% of the r - r interval )  . 
in cases of slightly irregular heart rate , such as bundle - branch block or premature beats , temporal variability in the reconstruction phase was manually compensated with ecg gating . 
the inaccurate detection for rwaves was corrected , and the temporal windows applied to ectopic beats were eliminated , thus compensating the subsequent diastolic interval with one or more windows . 
all diagnostic data sets were then reconstructed from the raw data using the convolution filters available on our systems , with section thickness 0.9 mm , reconstruction increment 0.4 mm and field of view 250 mm . image analysis target lesions were identified for each patient . 
the following criteria were used to select target lesions : ( 1 ) presence of haemodynamically significant stenosis identified at conventional coronary angiography ( cca ) ; ( 2 ) presence of coronary artery stent ; ( 3 ) presence of borderline stenosis . 
the diameter and length of the lesion or stent was defined for each segment . image evaluation visual score data sets used for the score were those corresponding to the standard ( xcc ) in the optimal acquisition window . 
a range of values was obtained in the rotation elaborazione dei dati per la ricostruzione delle immagini stata utilizzata una tecnica retrospettica ecg - gated [ 11 , 12 ]  . 
i pacchetti di dati sono stati ricostruiti durante la fase meso - / tele - diastolica , con finestre di ricostruzione da - 300 ms fino a - 450 ms dalla successiva onda r o 60% fino a 70% dellintervallo r - r . 
in caso di qualit delle immagini non sufficiente ( 3 pazienti ) sono state eseguite ricostruzioni aggiuntive durante la fase tele - sistolica ( 25% fino a 35% dellintervallo r - r )  . 
 stato corretto il rilevamento non accurato delle onde r e sono state eliminate le finestre temporali applicate ai battiti ectopici , compensando il successivo intervallo diastolico con uno o pi finestre . 
lo spessore di sezione acquisito corrispondeva a 0 , 75 mm , con un incremento di ricostruzione di 0 , 4 mtutti i datasets sono stati filtrati con un kernel di ricostruzione medio - morbido . 
tutti i dataset diagnostici sono stati poi ricostruiti a partire dai dati grezzi mediante i filtri di convoluzione disponibili sulla nostra apparecchiatura con spessore di sezione di 0 , 9 mm e incremento di ricostruzione pari a 0 , 4 mm con campo di vista fisso a 250 mm . analisi delle immagini per ogni paziente sono state identificate le lesioni target . 
il criterio di selezione delle lesioni target era il seguente : ( 1 ) presenza di stenosi emodinamicamente significativa riscontrata in angiografia coronarica ; ( 2 ) presenza di stent coronarico ; ( 3 ) presenza di stenosi borderline al limite della significativit . 
per ogni segmento stato definito il calibro e la lunghezza della lesione o dello stent . valutazione delle immagini score visuale il dataset utilizzato per lo score corrispondeva a quello standard ( xcc ) nella finestra di acquisizione ottimale . 
per ogni lesione target stata effettuata una stima di stenosi visuale distinta secondo i criteri internazionali [ 3 , 14 ] : 0%20% : assenza di stenosi o irregolarit parietali ; 20%50% : stenosi non significativa ; 50%70% : stenosi significativa ; 70%100% : stenosi severa occludente o suboccludente . radiol med ( 2011 ) 116 : 12031216 1207 fig . 
1 ricostruzione mpr rettilinea dellarteria discendente anteriore con detezione automatica dei bordi del vaso con qcta ne differenti filtri di convoluzione , utilizzando 15 differenti filtri di ricostruzione da a in alto a sinistra a cd in basso a sinistra ; confronto con angiografia coronarica , in basso a destra . 
 of the main vessel , and these were recorded in absolute terms and later grouped into the stenosis categories described above . qcta conventional coronary angiography cca was performed within 15 days of the mdct - ca examination . 
on the basis of the per - segment analysis , an operator blinded to the mdct - ca findings identified the segments with stenosis between 20% and 70% , including cases of borderline stenosis in mdct - ca and subocclusive stenosis . 
nella rotazione del vaso principale stato ottenuto per ogni singola misurazione un range di valori che sono stati registrati in termini assoluti e successivamente raggruppati nelle categorie di stenosi sopra descritte . angiografia coronarica langiografia coronarica ( ca ) stata eseguita entro 15 1208 radiol med ( 2011 ) 116 : 12031216 table 1 scan parameters of 64 - slice multidetector - row computed tomography coronary angiography ( mdct - ca ) scan parameter detector collimation kilovolt milliampere / s rotation time tube modulation effective time resolution maximal time resolution effective spatial resolution pitch scan time ( interval 120140 mm ) reconstruction effective slice thickness ( mm ) increase reconstruction ( mm ) time window reconstruction cardiac field of view thoracic field of view reformatting filter / kernel contrast medium synchronisation technique region of interest trigger threshold pre - delay preparation time to delay volume flow rate time to contrast administration saline solution venous access 64 slice 0.6250 mm 8001 , 000 330 ms systolic 165 ms 87 ms 0.30.30.4 mm 12 s bolus tracking ascending aorta + 100 hu 10 s 45 s 80100 ml 5 ml / s 15 s 40 ml , 5 ml / s antecubital end diastolic and end systolic 180250 mm entire thorax section medium ( xcc ) quantitative coronary angiography the segments with stenosis were then studied with a software application for the quantitative study of coronary stenosis ( qca ) ( caas , pie medical , maastricht , the netherlands )  . 
the percentage of stenosis was recorded in absolute terms and then grouped in the categories described above . statistical analysis statistical analysis was performed using spss ( version 11.5 , chicago , il , usa 19892002 )  . 
sulla base di una analisi per segmento un operatore in cieco rispetto ai dati rilevati in ac - tcms ha identificato i segmenti con stenosi comprese tra 20% e 70% , comprendendo quindi stenosi borderline in ac - tcms e sub occludenti . 
il valore di stenosi stato poi raggruppato nelle categorie di stenosi sopra descritte . angiografia coronarica quantitativa i segmenti stenotici sono stati poi studiati mediante il software di calcolo quantitativo di stenosi coronarica ( caas , pie medical , maastricht , paesi bassi )  . 
la percentuale di stenosi stata registrata in termini assoluti e poi raggruppata nelle categorie di stenosi sopra descritte . analisi statistica lanalisi statistica stata ottenuta mediante spss ( versione 11.5 , chicago , illinois 19892002 )  . 
gli indici di correlazione tra le radiol med ( 2011 ) 116 : 12031216 1209 tabella 1 parametri di scansione nella angiografia coronarica con tomografia computerizzata ( tc ) 64 strati parametro di scansione detettori collimazione chilovolt milliamperes tempo di rotazione modulazione della tensione del tubo risoluzione temporale effettiva risoluzione temporale massima risoluzione spaziale effettiva pitch tempo di scansione ( intervallo 120140 mm ) ricostruzione spessore di fetta effettivo ( mm ) incremento di ricostruzione ( mm ) finestra temporale campo di vista cardiaco campo di vista torace filtro di convoluzione / kernel mezzo di contrasto tecnica di sincronizzazione roi soglia di attivazione della scansione pre - delay tempo di preparazione al delay volume flusso tempo di somministrazione del mdc soluzione salina accesso venoso tc 64 strati 0 , 6250 mm 8001000 330 ms sistolica 165 ms 87 ms 0 , 30 , 30 , 4 mm 12 s bolus tracking aorta ascendente + 100 hu 10 s 46 s 80100 ml 5 ml / s 15 s 40 ml , 5 ml / s antecubitale tele - diastolica e tele - sistolica 180250 mm lintera sezione del torace medio ( xcc ) roi , regione di interesse ; mdc , mezzo di contrasto the exception of one examination that suffered from motion artefacts at the site of the target lesion , thus precluding both evaluation with qcta and use of the 15 convolution filters available . 
the segments most frequently affected by stenosis and therefore most frequently analysed were the proximal ( 11 cases , 44% ) and intermediate ( four cases , 16% ) tracts of the left anterior descending coronary artery and the proximal ( six cases , 24% ) and intermediate ( four cases , 16% ) tracts of the right coronary artery . 
in particular , comparison between mdct - ca and qca for the 2050% category shows agreement in 83.3% of cases ; for the catdifferenti metodiche sono stati espressi mediante analisi di regressione lineare usando il coefficiente di correlazione di pearson . 
un valore di p inferiore o uguale a 0 , 05 stato considerato statisticamente significativo . risultati tutti gli esami sono risultati di qualit sufficiente per essere analizzati mediante quantificazione visuale ac - tcms , qcta , ac , qca fatta eccezione per un esame soggetto ad artefatti da movimento in corrispondenza della lesione target che non ha quindi permesso lo studio del segmento mediante qcta con i 15 filtri di convoluzione a disposizione . 
i segmenti pi frequentemente soggetti a stenosi e quindi pi frequentemente analizzati sono risultati essere il tratto prossimale ( 11 casi , 44% ) ed intermedio ( 4 casi , 16% ) dellarteria interventricolare anteriore e il tratto prossimale ( 6 casi , 24% ) ed intermedio dellarteria coronaria destra ( 4 casi , 16% )  . 
agreement between mdct - ca and qca was 100% in the per - category analysis of stenosis in both types of plaque risultata pari a 17 , 467 , 43 millimetri e il calibro medio del segmento analizzato stato pari a 2 , 6 mm0 , 91 . le lesioni target sono state distinte a seconda che la stenosi fosse sostenuta da una placca fibrolipidica ( 10 casi ) , mista / calcifica ( 7 casi ) o vi fosse uno stent ( 8 casi )  . 
considerando come standard di riferimento il qca secondo lanalisi per categorie di stenosi vi una concordanza globale del 100% , 0 , 859 , tra ac - tcms e qca . 
per - category comparison between qca and qcta of the various filters in the study of fibrofatty plaques alone showed a low correlation for filter e ( rho = 0.095 ) ; good but not excellent correlation for filters a , b , c , d , cc , ya and xcc ; and excellent correlation for filters cb , cd , xca , xcb and xcd . 
lagreement tra ac - tcms e qca del 100% nellanalisi di stenosi per categoria in entrambi i tipi di placca ( 0 , 700 ; p < 0 , 0001 )  . 
nel confronto per categorie tra qca e qcta dei vari filtri nello studio delle sole placche fibrolipidiche vi una bassa correlazione per il filtro e ( 0 , 095 ) , buona ma non eccellente per i filtri a , b , c , d , cc , ya , xcc , eccellente per i filtri cb , cd , xca , xcb , xcd . 
nel confronto per categorie tra qca e qcta dei vari filtri nello studio delle sole placche miste / calcifiche vi una bassa correlazione per i filtri e , ca , ya , xcc , una correlazione inversa per i filtri a , b , c , cb , cc , cd , xca , xcb , xcd , discreta per il filtro ca , buona ma non eccellente per i filtri d ( 0 , 453 )  . 
objective evaluation of stenosis is , however , fraught with even greater difficulties in cases of calcified plaques or coronary artery stents that cause the blooming artefact and alter the perception of the true lumen of the segment [ 2233 ]  . 
to avoid this phenomenon , the expert operator performs reformatting with specific convolution filters recommended by the system manufacturers in order to minimise blurring caused by the calcium or the stent mesh . 
this system , known as qcta , autonomously traces the outline of the vessel and the segment discussione lac - tcms rappresenta una valida ed accurata metodica di imaging per lo studio non invasivo delle arterie coronarie [ 1621 ]  . 
la corretta stima di stenosi coronarica di fondamentale importanza per determinare il destino terapeutico di un paziente dal follow - up in caso di stenosi non significative al by - pass in caso di occlusione totale cronica . 
la stima di stenosi coronarica ad oggi basata sulla valutazione visuale di ricostruzioni multiplanari curvilinee , ricostruzioni in proiezione di massima intensit ( mip ) e volume rendering che rappresentano le tipiche ricostruzioni di cui il radiologo si serve per linterpretazione dei vasi maggiori , per lidentificazione e la caratterizzazione delle stenosi coronariche . 
la oggettiva valutazione di stenosi risulta inoltre pi difficoltosa in caso di placche calcifiche o di stent coronarici a causa dellartefatto blooming che altera la percezione del lume vero del segmento [ 2233 ]  . 
loperatore esperto per ovviare a tale fenomeno si avvale di riformattazioni con filtri di convoluzione specifici consigliati dalle case in modo da ridurre al minimo il blurring originato dal calcio o dalle maglie dello stent . 
the software , in fact , depends on the initial image ; therefore , if the vessel is poorly enhanced or if the atheromatous plaque being studied is prevalently calcified , the software will provide relatively unreliable information . qcta has been proposed as a postprocessing technique for objective quantification of the degree of stenosis , but often in cases of stenosis characterised by calcified plaques or stents , evaluation with normal reconstruction pacchetto comprehensive cardiac in dotazione alla workstation della tcms brilliance 64 . 
tale software infatti dipendente dalla immagine iniziale e quindi se il vaso sar poco opacizzato o se la placca aterosclerotica in esame sar prevalentemente calcifica il software sar tratto in errore fornendo informazioni poco attendibili . il qcta viene proposto come tecnica di post processing che permetta una quantificazione oggettiva del grado di stenosi ma spesso in caso di stenosi sostenuta da placche calci1214 radiol med ( 2011 ) 116 : 12031216 filters is suboptimal or imprecise . 
the results obtained in the analysis of our patient population show that the visual score obtained with mdct - ca is completely reliable in cases of calcified plaque , fibrofatty plaque and coronary stents . 
the use of qcta is also valid in evaluating fibrofatty plaque and with certain filters ( in our case , ya ) , also for studying stents , but with correlation values far from those required for a broad use of the technique . 
the low correlation between stenosis values identified at qcta and at qca is even more evident in the study of calcified plaque and stents where the reconstruction filters normally recommended are not effective for the mathematical models of qcta that identify vessel edges . 
we did not perform this procedure in order to avoid introducing bias when comparing performances of the various filters associated with qcta , although this technique should always be applied in cases of calcified plaque or stents . 
 whereas vessel edge editing may be considered a source of bias in accurate software validation , it can contribute to limiting errors of overor underestimating the degree of stenosis if performed properly and can even provide data much closer to those produced by qca . 
our study shows that oscillation of absolute values of stenosis quantified by qcta with varying filters is greater in cases of calcified plaques or stents than in cases of fibrofatty plaques . 
only by using the particularly hard filters ya and yb ( without margin correction ) was stent margin evaluation optimal , although the correlation index was still quite far from the value achieved with the visual score . 
although qcta without vessel edge editing did not prove particularly effective in estimating stenosis , it has been reported that optimal quantitative evaluation of the degree of coronary artery stenosis can be achieved with vessel edge editing . 
indeed , an attenuation of in - stent hounsfield unit density of around 37% has been reported with nondedicated filters , thus compromising correct estimation , both visual and automatic , of in - stent restenosis [ 35 , 36 ]  . 
the appropriate filters for stents ( in our case , xcd ) significantly reduce the background noise and increase overall image quality , thus allowing a more objective estimation of stenosis [ 37 ]  . there are a number of limitations to our study . 
lutilizzo del qcta valido daltra parte nella valutazione di placche fibrolipidiche e con particolari filtri duri ( nel nostro caso ya ) anche per lo studio degli stent ma con valori di correlazione lontani da quelli attesi per un largo impiego della tecnica . 
la bassa correlazione tra i valori di stenosi riconosciuti al qcta e al qca risulta ancor pi evidente per lo studio delle placche calcifiche e degli stent dove i filtri di ricostruzione normalmente consigliati non sono efficaci per i modelli matematici di identificazione dei contorni propri del qcta . 
nel nostro studio non abbiamo effettuato tale procedimento in modo da non introdurre un bias nel confronto tra le performance dei vari filtri accoppiati al qcta ma una tecnica che deve essere sempre usata in caso di placca calcifica o stent . 
 lediting dei contorni dei vasi , se da una parte infatti costituisce un bias nella corretta validazione del software , se adeguatamente eseguito contribuisce a limitare lerrore di sovra o sottostima del grado di stenosi e addirittura si avvicina maggiormente ai dati forniti dal qca . 
anche nel nostro lavoro abbiamo rilevato come loscillazione dei valori assoluti di stenosi quantificata dal qcta con i vari filtri sia molto maggiore in caso di placca calcifica o stent rispetto alla placca fibrolipidica . 
solamente con il filtro ya e yb ( senza correzione dei bordi ) particolarmente duro viene ottenuta una valutazione dei bordi dello stent ottimale ma con un indice di correlazione sempre distante da quello espresso dallo score visuale . 
sebbene il qcta senza editing dei contorni del vaso non risulti particolarmente efficace nella stima di stenosi gi stato descritto come mediante editing dei bordi sia possibile una valutazione quantitativa ottimale del grado di stenosi coronarica . 
inoltre lutilizzo di appropriati filtri di convoluzione sempre consigliabile in caso di placche calcifiche e ancor di pi in caso di stent in quanto stato dimostrato come vi sia una attenuazione della densit hounsfield intrastent di circa il 37% con filtri non dedicati , inficiando quindi una corretta stima sia visuale sia automatica della restenosi intrastent [ 35 , 36 ]  . 
i filtri appropriati per stent ( nel nostro caso xcd ) riducono significativamente il rumore di fondo dei dataset incrementando la qualit dellimmagine globale e permettendo cos una stima pi obiettiva della stenosi [ 37 ]  . questo lavoro presenta alcuni limiti . 
 the accuracy of the technique is suboptimal , especially if no vessel edge editing is performed , particularly in cases of calcified plaques or stents . il qcta una metodica promettente per lo studio del grado di stenosi dei segmenti coronarici , e risulta pi efficace se basata su filtri di convoluzione appropriati . 
cademartiri1 , 2 1department of radiology and cardiology , erasmus medical center , rotterdam , the netherlands 2department of radiology and cardiology , azienda ospedaliero - universitaria di parma , parma , italy 3department of radiology , sdn - irccs foundation , napoli , italy 4department of radiology , azienda ospedaliera s . 
cademartiri , department of radiology , azienda ospedaliero - universitaria di parma , via gramsci 14 , 43100 parma , italy , tel . : + 39 - 0521 - 703516 , fax : + 39 - 0521 - 703630 , e - mail : filippocademartiri@gmail.com received : 18 may 2009 / accepted : 08 july 2009 / published online : 4 september 2011 springer - verlag 2011 abstract purpose . 
twenty - one patients with stable and 20 with unstable angina pectoris scheduled for conventional coronary angiography ( cca ) underwent msct - ca using a 64 - slice scanner offering a fast rotation time ( 330 ms ) and higher x - ray tube output ( 900 mas )  . 
to determine the msct coronary plaque burden , we assessed the extent ( number of diseased segments ) , size ( small or large ) , type ( calcific , noncalcific , mixed ) of plaque , its anatomic distribution and angiographic appearance in all available 2 - mm segments . 
ventuno pazienti con angina stabile e 20 pazienti con angina instabile , in attesa di eseguire langiografia coronarica convenzionale ( acc ) , sono stati sottoposti ad angiografia coronarica con tcms a 64 strati caratterizzata da un tempo di rotazione veloce ( 330 ms ) e da una pi elevata emissione del tubo radiogeno ( 900 mas )  . 
langiografia coronarica mediante tcms fornisce importanti informazioni riguardo il carico aterosclerotico coronarico nei pazienti con angina stabile ed instabile . parole chiave malattia coronarica imaging tomografia computerizzata multistrato coronarografia ecografia intra - coronarica introduction introduzione multislice computed tomography ( msct ) is a rapidly developing technique . 
the latest 64 - slice msct scanners have a higher x - ray tube rotation speed and allow scanning with a higher tube current , providing increased temporal resolution and contrast - to - noise ( cnr ) ratio when compared with previous - generation 16 - row scanners . 
preliminary in vivo and ex vivo studies using previous - generation 16 - row scanners showed that msct coronary imaging holds promise for detecting nonobstructive coronary plaques and allows identification of different plaque tissue components [ 38 ]  . 
these observations prompted us to evaluate the msct coronary plaque burden defined as presence , severity , distribution and type of coronary plaques using a 64 - slice msct scanner . 
la tcms a 64 strati di ultima generazione ha una pi elevata velocit di rotazione del tubo radiogeno e consente scansioni con maggiore intensit di corrente del tubo , che si traduce in una maggiore risoluzione spaziale e un miglior rapporto contrasto / rumore rispetto alla precedente generazione di tcms a 16 strati . 
studi preliminari in - vivo ed ex - vivo , condotti con la precedente generazione di tcms a 16 strati , hanno dimostrato che limaging coronarico mediante tcms risulta promettente nella rilevazione di placche coronariche non ostruttive e consente lidentificazione delle diverse componenti tissutali della placca stessa [ 38 ]  . 
queste osservazioni ci hanno spinto a valutare il carico aterosclerotico coronarico mediante tcms , definito come la presenza , la severit , la distribuzione ed il tipo di placche coronariche utilizzando uno scanner tcms a 64 strati . 
abbiamo confrontato questi risultati con quelli ottenuti con langiografia convenzionale e , in un sottogruppo di pazienti , con lecografia intra - coronarica ( icus )  . during a 3 - month period , 21 patients [ 17 men , four women ; mean age ( standard deviation ; sd ) 59.09.2 years ] with stable angina pectoris and 20 patients [ 15 men , 5 women , mean age ( sd ) 60.011.3 years ] with unstable angina pectoris underwent 64 - slice msct coronary angiography materiali e metodi soggetti durante un periodo di tre mesi , 21 pazienti ( 17 maschi , 4 femmine , et mediadeviazione standard [ ds ] = 59 , 09 , 2 ) 1176 radiol med ( 2011 ) 116 : 11741187 ( ca ) prior to diagnostic ca followed , in case of amenable lesions , by percutaneous coronary intervention . 
patients were eligible for inclusion if they were in sinus rhythm , had a heart rate of < 70 bpm ( spontaneously or - blocker induced ) and able to breath - hold for 20 s . 
exclusion criteria were known contrast allergy , significant renal dysfunction ( serum creatinine > 120 mmol / l ) , braunwald class ia , iia , iiia ( unstable angina caused by noncardiac illness ) and previous percutaneous or surgical revascularisation . 
in order of preference , the left anterior descending artery ( lad , n = 8 ) , right coronary artery ( rca , n = 4 ) or circumflex artery ( cx , n = 3 ) were selected . 
 the institutional review board of our centre approved the study , and all patients gave written informed consent . msct - ca all msct examinations were performed using a 64 - slice scanner ( sensation 64 cardiac , siemens , forchheim , germany ) equipped with a faster x - ray tube rotation time ( 330 ms ) and a higher tube output ( up to 900 mas )  . 
other scan parameters were : detector collimation 3220.6 mm , table feed 3.84 mm / rotation , tube voltage 120 kv ; no prospective x - ray tube modulation was applied . 
radiation exposure using this scan protocol was estimated as being between 15 and 21 msv ( male / female ) using dedicated software ( windose , institute of medical physics , erlangen , germany )  . 
patients with a prescan heart rate 65 bpm received a single oral dose of 100 mg metoprolol ( selokeen , astrazeneca pharmaceutics , uk ) 1 h before the scan . 
 a bolus of 100 ml of iodinated contrast material ( iomeron 400 , bracco , milan , italy ) followed by a saline bolus chaser of 40 ml was administered through an arm vein ( flow rate : 45 ml / s )  . 
data sets were reconstructed during different time points of the cardiac phase using a retrospective electrocardiographic ( ecg ) - gated reconstruction algorith this algorithm uses data of a single heart beat obtained in half gantry rotation time , resulting in a temporal resolution of up to 165 ms . 
 con angina pectoris stabile e 20 pazienti ( 15 maschi , 5 femmine , et mediads = 60 , 011 , 3 anni ) con angina pectoris instabile sono stati sottoposti ad angiografia coronarica mediante tcms a 64 strati prima dellesecuzione della coronarografia diagnostica , seguita , in presenza di lesioni suscettibili da trattare , da intervento coronarico percutaneo . 
i pazienti erano eleggibili per linclusione se erano in ritmo sinusale , con frequenza cardiaca < 70 battiti / minuto ( spontanea o indotta mediante somministrazione di beta - bloccanti ) e se erano in grado di trattenere lapnea per 20 secondi . 
 i criteri di esclusione sono stati la presenza di una nota allergia al mezzo di contrasto , una significativa disfunzione renale ( creatinina sierica > 120 mmol / l ) , classe ia , iia , iiia di braunwald ( angina instabile causata da patologia non cardiaca ) e precedente rivascolarizzazione percutanea o chirurgica . 
un sottogruppo di 15 pazienti ( angina stabile : 7 ; angina instabile : 8 ) sono stati sottoposti ad un esame icus di un vaso per cui non era programmato lintervento percutaneo . 
sono state selezionate , in ordine di preferenza , larteria coronaria discendente anteriore sinistra ( lad , n = 8 ) , larteria coronaria destra ( rca , n = 4 ) o larteria circonflessa ( cx , n = 3 )  . 
il comitato etico della nostra istituzione ha approvato lo studio e tutti i pazienti hanno fornito un consenso informato scritto . angiografia coronarica mediante tcms tutti gli esami tcms sono stati eseguiti utilizzando una tcms a 64 strati ( sensation 64 cardiac , siemens , forchheim , germania ) , dotata di una maggiore velocit di rotazione ( 330 ms ) e una pi elevata emissione ( fino a 900 mas ) del tubo radiogeno . 
altri parametri di scansione : collimazione del detettore 3220 , 6 mm , velocit del lettino 3 , 84 mm / rotation , voltaggio del tubo 120 kv , non stata applicata modulazione prospettica della corrente del tubo . 
lesposizione radiogena utilizzando questo protocollo di scansione stata stimata tra i 15 e i 21 msv ( uomo / donna ) usando un software dedicato ( windose , institute of medical physics , erlangen , germania )  . 
ai pazienti che presentavano allarrivo una frequenza cardiaca 65 battiti / minuto stata somministrata per via orale una dose singola di 100 mg di metoprololo ( selokeen , astrazeneca pharmaceutics , uk ) unora prima della scansione . 
 stato iniettato per via endovenosa attraverso una vena del braccio un bolo di 100 ml di mezzo di contrasto iodato ( iomeron 400 , bracco , milano , italia ) seguito da un bolo di 40 ml di soluzione fisiologica ( velocit dinfusione : 45 ml / s )  . 
i datasets ricostruiti allinterno della fase meso - telediastolica generalmente forniscono una qualit dimmagine quasi priva di artefatti da movimento , tuttavia , quando ritenuto necessario , sono state eseguite ricostruzioni in differenradiol med ( 2011 ) 116 : 11741187 1177 the diameters of all coronary segments and branches were measured using a quantitative ca ( qca ) algorithm ( caas , pie medical imaging , maastricht , the netherlands )  . 
 one observer , unaware of the msct results , classified these segments as normal ( no luminal encroachment ) , wall irregularities ( < 20% lumen narrowing ) , moderately diseased ( 20% to < 50% lumen narrowing ) or significantly diseased ( 50% lumen narrowing )  . 
 msct analysis : assessment of the coronary plaque burden in all available 2 - mm coronary segments to evaluate the msct coronary plaque burden , we assessed the extent , type , size and anatomic distribution of plaque along all segments 2 mm , including the main coronary arteries and 2 - mm side branches . 
interand intraobserver variability was calculated for plaque extent , type and size , and differences in stable and unstable patients were compared using the students t test ( p values < 0.05 were considered significant )  . 
the tip of the icus catheter was positioned at least 10 mm distal to the distal landmark , and an automated pullback ( speed 0.5 mm / s ) of the complete roi was performed . 
 tutti i datasets con il minor numero di artefatti da movimento sono stati analizzati su una workstation off - line ( mmwp , siemens , forchheim , germania )  . 
il diametro di tutti i segmenti coronarici e dei rami collaterali stato misurato utilizzando un algoritmo di angiografia coronarografica quantitativa ( qca ; caas , pie medical imaging , maastricht , olanda )  . 
un osservatore , non a conoscenza dei risultati dellesame tcms , ha classificato questi segmenti come normali ( assenza di ristringimento del lume ) , con irregolarit parietale ( restringimento del lume < 20% ) , con malattia moderata ( restringimento del lume 20%50% ) o con malattia significativa : restringimento del lume 50% . analisi tcms : valutazione del carico aterosclerotico coronarico in tutti i segmenti coronarici disponibili 2 mm per determinare il carico aterosclerotico coronarico mediante tcms , abbiamo valutato lestensione , il tipo , la dimensione e la distribuzione anatomica delle placche lungo tutti i segmenti 2 mm , includendo le arterie coronarie principali e i loro rami di calibro 2 mtutti i segmenti coronarici sono stati esaminati per identificare la placca utilizzando immagini multiplanari ricostruite su piani longitudinali e trasversali . 
due osservatori , in cieco rispetto ai risultati delle altre tecniche di imaging , hanno effettuato in modo indipendente tutte le analisi per la valutazione del carico aterosclerotico alla tcms . 
stata selezionata una regione di interesse ( roi ) sullangiogramma , dopo lidenti1178 radiol med ( 2011 ) 116 : 11741187 semiautomated vessel - tracking software package ( circulation , siemens , forchheim , germany ) was used to create a central lumen line throughout the roi . 
plaques with a ct density below the coronary lumen and above the adjacent epicardial fat were classified as noncalcific , whereas plaques with a ct density 130 hu were classified as calcific . 
precision of the overall parameters with respect to plaque detection was expressed with a 95% confidence interval ( ci )  . results fifty - six percent ( 23 / 41 ) of patients was on long - term - blockade . 
demographics of stable and unstable patients are listed in table 1 . evaluation of the msct coronary plaque burden and comparison with conventional angiography a total of 473 coronary segments 2 mm were assessed . 
noncalcific plaques were found in 32% ( 93 / 292 ) , calcific in 25% ( 73 / 292 ) and mixed in 43% ( 126 / 292 ) of diseased segments . 
la punta del catetere icus stata posizionata almeno 10 mm distalmente al repere distale ed stata eseguito un trascinamento retrogrado automatico del catetere ( velocit : 0 , 5 mm / s ) nellintera roi . 
per eseguire tutte le misurazioni icus , stato utilizzato un software precedentemente validato basato su immagini acquisite con gating retrospettivo ( curad , wijk bij duurstede , olanda ) [ 10 ]  . analisi tcms : valutazione delle placche coronariche in una roi selezionata e confronto con licus inizialmente la roi selezionata stata identificata sulle immagini acquisite mediante angiografia coronarica tcms . 
 un software semi - automatico di vessel - tracking ( circulation , siemens , forchheim , germania ) stato utilizzato per creare una linea centrale nel lume allinterno della roi selezionata . 
le placche con densit inferiore al lume coronarico e al tessuto adiposo epicardio adiacente sono state classificate come non calcifiche , mentre le placche con densit 130 unit hounsfield ( uh ) sono state classificate come calcifiche . 
leventuale disaccordo tra i due osservatori stato risolto con il consenso di entrambi in una sessione congiunta . la roi selezionata stata suddivisa in sezioni di 5 mm per confrontare i risultati della tcms e dellicus . 
sono stati considerati significativi valori di p < 0 , 05 bserver agreement regarding plaque extent was 78% and 82% , plaque type was 69% and 74% and plaque size was 70% and 72% , respectively . 
 overall , plaque was found in 71% ( 115 / 161 ) of proximal segments , in 71% ( 79 / 111 ) of mid segments and in 49% ( 98 / 201 ) of distal segments and side branches of the main coronary arteries [ left main ( lm ) , lad , cx , rca ]  . 
al 51% ( 21 / 41 ) stata somministrata una dose ( supplementare ) di beta - bloccanti poich prima della scansione tc presentava una frequenza cardiaca superiore ai 70 battiti / minuto . 
la frequenza cardiaca mediads durante la procedure di scansione in pazienti con angina stabile ed instabile stata rispettivamente di 57 , 02 , 8 e 60 , 711 , 3 . 
i dati demografici dei pazienti con angina stabile ed instabile sono elencati nella tabella 1 . valutazione del carico aterosclerotico coronarico alla tcms e confronto con langiografia convenzionale per la valutazione del carico aterosclerotico coronarico alla tcms stato incluso un totale di 473 segmenti coronarici 2 millimetri . 
placche non calcifiche sono stati riscontrate nel 32% ( 93 / 292 ) dei segmenti malati , placche calcifiche nel 25% ( 73 / 292 ) e placche miste nel 43% ( 126 / 292 )  . 
i valori k per il grado di accordo inter - e intra - osservatore sono stati rispettivamente del 78% e 82% per lestensione delle placche , del 69% e 74% per il tipo di placca e del 70% e 72% per le dimensioni di placca . 
 lestensione , il tipo , le dimensioni , e relativa frequenza dei diversi tipi di placca per vaso coronarico nei pazienti con angina stabile ed instabile sono riportati nella tabella 2 . 
nel complesso , sono state riscontrate placche ateromasiche nel 71% ( 115 / 161 ) dei segmenti prossimali , nel 71% ( 79 / 111 ) dei segmenti medi e nel 49% ( 98 / 201 ) dei segmenti distali e rami collaterali delle arterie coronarie principali ( tronco comune [ lm ] , lad , cx e rca )  . 
inoltre , la presenza di placca stata rilevata nel 33% dei segmenti coronarici classificati come normali allangiografia convenzionale , mentre le irregolarit parietali allangiografia erano associate con il 72% ( 98 / 138 ) dei segmenti che alla tcms presentavano una placca coronarica . 
in general , large plaques were more frequently found in segments with more luminal encroachment on the conventional angiograplaque was detected in nearly all ( 99% , 126 / 128 ) coronary segments performance diagnostiche della tcms vs . 
licus nel rilevare placche significative in una roi selezionata un totale di 92 sezioni ( ognuna di 5 mm ) sono state incluse per il confronto tra tcms e icus . 
cx , arteria coronaria circonflessa ; d1 , primo ramo diagonale ; lad , arteria coronaria discendente anteriore sinistra ; lm , tronco comune ; pda , arteria coronaria discendente posteriore ; prox , prossimale ; rca , arteria coronaria destra . with nonsignificant or significant stenoses . 
as well as in 33% of coronary segments classified as normal with cca ; angiographic wall irregularities were associated with 72% ( 98 / 138 ) of the segments with msct coronary plaque involvement . 
nel complesso , la sensibilit e la specificit dellangiografia coronarica mediante tcms nel rilevare placche coronariche significative sono state rispettivamente dell83% ( 25 / 30 , 95% intervallo di confidenza [ ci ] : 65% 95% ) e 87% ( 54 / 62 , 95% ci : 76%94% )  . 
la sensibilit stata maggiore nei segmenti contenenti una componente calcifica di placca ( placche calcifiche : 91% [ 10 / 11 , 95% ci : 58%97% ] , placche miste : 82% [ 9 / 11 , 95% ci : 48%95% ] ) rispetto ai segmenti con placche costituite da componente esclusivamente non calcifica ( placche non calcifiche : 75% [ 6 / 8 , 95% ci : 34%93% ] )  . 
un esempio che mostra un confronto tra i risultati tcms e icus mostrato in figura 3 . radiol med ( 2011 ) 116 : 11741187 1183 table 4 diagnostic accuracy of multislice computed tomography coronary angiography for detecting coronary plaques compared with intracoronary ultrasound noncalcific calcific mixed overall true positive false positive false negative true negative sensitivity specificity 6 / 8 : 75% ( 3493% ) 10 / 11 : 91% ( 5897% ) 9 / 11 : 82% ( 4895% ) 25 / 30 : 83% ( 6595% ) 54 / 62 : 87% ( 7694% ) tabella 4 accuratezza diagnostica dellangiografia coronarica mediante tcms nellidentificazione delle placche coronariche a confronto con licus non calcifiche calcifiche miste totale veri positivi falsi positivi falsi negativi veri negativi sensibilit specificit 6 / 8 : 75% ( 3493% ) 10 / 11 : 91% ( 5897% ) 9 / 11 : 82% ( 4895% ) 25 / 30 : 83% ( 6595% ) 54 / 62 : 87% ( 7694% ) coronary plaques was 83% ( 25 / 30 , 95% ci 6595% ) and 87% ( 54 / 62 , 95% ci 7694% ) , respectively . 
a higher sensitivity was found in segments containing calcific plaque tissue [ 91% ( 10 / 11 , 95% ci 5897% ) , mixed plaques 82% ( 9 / 11 , 95% ci 4895% ) ] compared with segments with exclusively noncalcific plaque tissue [ 75% ( 6 / 8 , 95% ci 3493% ) ]  . 
a comparison between msct and icus findings is shown in figure 3 . discussion coronary atherosclerosis begins early in life , and it takes several decades for plaques to advance into vulnerable plaques that underlie progression to acute coronary syndromes ( unstable angina pectoris or myocardial infarction ) or progression of coronary stenosis . 
sixtyfour - slice msct - ca has a nearly isotropic submillimetre discussione laterosclerosi coronarica inizia a svilupparsi in et precoce e levoluzione in placche vulnerabili , che sono alla base dello sviluppo di sindromi coronariche acute ( angina pectoris instabile o infarto miocardico ) o la progressione della placca in stenosi coronarica sono processi che richiedono diversi decenni . 
di conseguenza , gli esami icus sono di solito limitati ai tratti prossimali e medi di una sola arteria coronaria , precludendo in tal modo la valutazione del carico aterosclerotico complessivo coronarico . 
questo ha incoraggiato il bisogno di tecniche di imaging non invasive in grado di valutare le placche coronariche , ad esempio la risonanza magnetica nucleare ( rmn ) e la tcms . 
langiografia coronarica mediante tcms a 64 strati ha una risoluzione spaziale sub - millimetrica quasi isotropica che permette la valutazione della parete vasale in tutti i segmenti coronarici 2 mm , fornendo cos una valutazione pi completa , rispetto allicus , dellaterosclerosi coronarica in tutto lalbero coronarico . nel nostro studio abbiamo dimostrato che langiografia coronarica mediante tcms in grado di valutare la presenza , lestensione , la severit , la distribuzione e il tipo di placche coronariche . 
abbiamo riscontrato una elevata sensibilit e specificit ( rispettivamente del 83% e dell87% ) della tcms nel rilevare placche coronariche significative a confronto con licus , dati in linea con gli studi prece1184 radiol med ( 2011 ) 116 : 11741187 fig . 
3 maximum intensity projected computed tomography ( mip - ct ) image showing a nonsignificant obstructive coronary lesion located at the midsection of the right coronary artery ( rca ) , which was also found on the conventional coronary angiogram ( cca )  . 
cross - sectional ct images at the lesion site demonstrate the presence of a large , mixed plaque with both calcific ( a1 ) and noncalcific ( b1 ) plaque tissue , which was confirmed with intracoronary ultrasound ( a2 and b2 )  . 
3 immagine tc di proiezione di massima intensit che mostra una lesione coronarica ostruttiva non significativa localizzata nel tratto medio dellarteria coronaria destra ( rca ) , riscontrata anche allangiografia coronarica convenzionale ( acc )  . 
immagini tc trasversali in corrispondenza della lesione dimostrano la presenza di unestesa placca mista con entrambe le componenti tissutali calcifiche ( riquadro a1 ) e non calcifiche ( riquadro b1 ) , che stata confermata allecografia intra - coronarica ( riquadri a2 e b2 )  . spatial resolution that allows evaluation of the coronary vessel wall in all 2 - mm coronary segments , thereby providing a more comprehensive evaluation compared with icus of coronary atherosclerosis throughout the coronary tree . 
we found a high sensitivity and specificity ( 83% and 87% , respectively ) compared with icus , which is in line with previously published studies [ 6 , 7 ]  . 
this observation may be the result of improved image quality offered by 64 - slice msct scanners due to a higher x - ray tube current , which provides a more favourable cnr . 
 we evaluated the presence of coronary plaques in all available 2 mm coronary segments ( according to the aha classification ) and classified plaques into calcific , noncalcific and mixed [ 48 ]  . 
questo dato pu essere il risultato di una migliore qualit dellimmagine offerta dagli scanners tcms a 64 strati , grazie allapplicazione di una pi elevata corrente del tubo radiogeno che garantisce un miglior rapporto contrasto - rumore . 
in particolare , quando il rumore dellimmagine diventa pi evidente , possono non essere individuate piccole placche non calcifiche . abbiamo valutato la presenza di placche coronariche in tutti i segmenti coronarici disponibili 2mm ( secondo la classificazione dellaha ) e abbiamo classificato le placche come calcifiche , non calcifiche e miste [ 48 ]  . 
stato interessante notare che un terzo delle placche erano non calcifiche e che circa il 40% delle placche era costituito da tessuto non calcifico , mentre il 25% delle placche erano prevalentemente calcifiche . 
preliminary in vivo studies suggest that plaques with predominantly lipid or fibrous tissue could be distinguished on the basis of tissue density differences ( in hounsfield units ) with msct when comparing these tissue - density values ( low , moderate ) with the degree of tissue echogenicity ( hypoor hyperechogenicity ) with icus [ 3 , 5 , 7 , 8 ]  . 
however , classifying noncalcified plaques is limited by high variability in density values within a single plaque , which makes it difficult to reliably distinguish between lipid and fibrous plaque tissue . 
furthermore , distinction between different plaque tissues on the basis of density values is hampered due to partial voluming and interpolation , which may improve when scanners with higher spatial resolution become available [ 11 ]  . plaque extent and distribution was almost similar in stable and unstable patients , but we found significantly more noncalcified plaques in patients with unstable vs . 
 we also demonstrated that coronary plaques were most frequently located in the proximal and mid segments of the large coronary arteries [ lad , left circumflex ( lcx ) , rca ] , as has been demonstrated in earlier pathologic and icus studies . 
 in addition to previous studies , we evaluated the angiographic appearance of coronary plaques detected by msct and found that coronary atherosclerosis was present in one third of angiographically normal coronary arteries , indicating that msct is able to detect earlier stages of coronary artery disease when compared with cca . 
more specifically , plaques without encroachment of the coronary lumen due to compensatory outward remodelling of the vessel wall , thereby alleviating reduction of luminal size ( known as positive remodelling ) remain undetected by cca but have been associated with acute coronary syndromes . 
tuttavia , la classificazione delle placche non calcifiche limitata dallelevata variabilit intraplacca dei valori di densit nel contesto di una singola lesione , che rende difficile distinguere in maniera affidabile tra componente lipidica e fibrosa della placca . 
inoltre , la distinzione tra differenti componenti tissutali della placca sulla base dei valori di densit ostacolata da effetti di volume parziale ed interpolazione , che potranno ridursi con la disponibilit di scanners con maggiore risoluzione spaziale [ 11 ]  . lestensione e la distribuzione delle placche erano pressoch simili nei pazienti con angina stabile ed instabile , tuttavia abbiamo riscontrato una prevalenza significativamente pi elevata di placche non calcifiche nei pazienti con angina pectoris instabile vs . 
 [ 12 ] che ha riscontrato un numero significativamente maggiore di placche non calcifiche nei pazienti con infarto miocardico acuto rispetto ai pazienti con angina stabile [ 1216 ]  . 
tuttavia , da questi risultati non deve derivare linterpretazione che i pazienti con prevalenza di placche calcifiche siano meno vulnerabili , o che una placca calcifica sia stabile . abbiamo inoltre dimostrato che le placche coronariche interessavano pi frequentemente i segmenti medio - prossimali delle principali arterie coronarie ( lad , lcx e rca ) , come stato anche dimostrato in precedenti studi anatomopatologici e mediante icus . in aggiunta rispetto a studi precedenti , abbiamo inoltre valutato laspetto angiografico delle placche coronariche rilevate dalla tcms . 
abbiamo riscontrato che laterosclerosi coronarica era presente in un terzo delle arterie coronarie angiograficamente normali , dimostrando che la tcms in grado di identificare la malattia coronarica in stadi pi precoci rispetto alla coronarografia convenzionale . 
nello specifico , le placche che non impegnano il lume coronarico a causa del rimodellamento compensatorio verso lesterno della parete del vaso , che contrasta la riduzione del diametro luminale ( noto come rimodellamento positivo ) , non vengono rilevate allangiografia convenzionale , tuttavia sono state associate allo sviluppo di sindromi coronariche acute . il nostro studio dimostra che la valutazione del carico aterosclerotico coronarico mediante tcms fornisce importanti informazioni sulla presenza e lestensione dellaterosclerosi coronarica nei pazienti con angina pectoris stabile e instabile . 
ulteriori studi prospettici sono necessari per determinare se la valutazione del carico aterosclerotico coronarico mediante tcms abbia un valore prognostico nellidentificazione di pazienti a rischio di futuri eventi cardiaci . limiti nel nostro studio stata studiata una popolazione di pazienti relativamente ristretta e sono stati inclusi pazienti con ritmo cardiaco lento e regolare , che sono variabili che 1186 limitations we studied a relatively small patient population and included patients with a low and regular heart rhythm , which are variables of high - quality msct imaging . 
it should be noted that the diagnostic performance of msct was compared with the presence of a coronary plaque that had a minimum thickness of 1 mm as determined with icus , excluding smaller plaques from the analysis . 
 although these plaques were not associated with significant coronary lumen obstructions , they should be classified as advanced plaques according to the pathological classification described by stary [ 17 ]  . 
a serious concern is the high radiation exposure during msct - ca , which is estimated as being between 15 and 21 msv ( male / female ) using this scan protocol . 
radiation exposure can be reduced by technical adjustments , such as prospective x - ray tube - current modulation , which reduces radiation exposure by nearly 50% in patients with low heart rates . 
 however , we did not apply this feature because it limits image reconstruction during the early diastolic phase , which can be important for evaluating the rca [ 1 ]  . 
deve essere notato che nel nostro studio la performance diagnostica della tcms stata confrontata con la presenza allesame icus di placche coronariche con spessore minimo di 1 mm , escludendo dallanalisi le placche di dimensioni inferiori . 
sebbene queste placche non erano associate ad una ostruzione significativa del lume coronarico , dovrebbero essere classificate come placche avanzate secondo la classificazione patologica descritta da stary [ 17 ]  . 
questi risultati suggeriscono che la tcms non sia in grado di rilevare gli stadi pi precoci dellaterosclerosi coronarica , che sono associati alla presenza di placche non - calcifiche di piccole di dimensioni . 
un problema rilevante lesposizione ad alte dosi di radiazioni durante langiografia coronarica mediante tcms , stimata tra i 1521 msv ( maschi / femmine ) utilizzando questo protocollo di scansione . 
lesposizione alle radiazioni pu essere ridotta da alcuni adattamenti tecnici come la modulazione prospettica della corrente del tubo radiogeno , che riduce lesposizione alle radiazioni di circa il 50% nei pazienti con bassa frequenza cardiaca . 
tuttavia , non abbiamo applicato questa tecnica perch non consente la ricostruzione delle immagini durante la fase proto - diastolica , che pu essere importante per la valutazione della rca [ 1 ]  . 
 the aim of this pictorial review is to analyse the features of adenomyosis by illustrating the most usual and typical imaging patterns , along with the unusual appearances , seen in a vast array of gynaecological imaging modalities . 
 the different findings of focal and diffuse adenomyosis along with the diagnostic limitations of ultrasound , hysterosalpingography and magnetic resonance imaging are described , as are the pitfalls and differential diagnosis with other pathological conditions that are often misdiagnosed as adenomyosis . 
the role of the different imaging modalities in planning appropriate treatment and their usefulness in monitoring therapy are also discussed . keywords female pelvic pain infertility magnetic resonance imaging transvaginal ultrasound hysterosalpingography riassunto ladenomiosi caratterizzata dallinvasione benigna del miometrio da parte dellendometrio ed spesso sottostimata o diagnosticata con ritardo , nonostante sia invalidante in quanto possibile causa di menorragia , metrorragia , dismenorrea ed infertilit . 
lo scopo del lavoro quello di analizzare gli aspetti diagnostici delladenomiosi mostrando i quadri pi semplici e quelli pi rari e difficili da caratterizzare , raccolti in una ampia galleria che prende in esame tutte le pi note tecniche di imaging ginecologico . 
i limiti e possibilit delle varie tecniche di imaging , ecografia , isterosalpingografia e risonanza magnetica , saranno illustrate ponendo anche particolare attenzione alla diagnostica differenziale con le altre forme morbose pi facilmente equivocabili . 
sar infine discusso il ruolo delle metodiche di imaging nella pianificazione del tipo di trattamento e nel monitoraggio dellefficacia terapeutica . parole chiave dolore pelvico infertilit risonanza magnetica ecografia transvaginale isterosalpingografia 1268 introduction adenomyosis is the benign , non - neoplastic infiltration of the endometrium into the myometrium characterised by ectopic glands and stroma , surrounded by hypertrophic and hyperplastic myometrium [ 1 ]  . 
therefore , the haemorrhagic foci that characterise endometriosis ( patches of ectopic endometrial tissue ) and which are helpful in identifying this disease may not be present in adenomyosis , thus making characterisation difficult [ 2 ]  . 
risk factors for adenomyosis are essentially related to : ( 1 ) reproductive activity , with an increased risk in multiparity , miscarriage and endometriosis ; ( 2 ) lifestyle factors such as smoking ; and ( 3 ) surgical trauma such as caesarean section , induced abortion or curettage [ 4 ]  . 
around 20% of cases of adenomyosis involve women of reproductive age ( < 40 years ) : the remaining 80% of cases involve women between 40 and 50 years , and the most severe symptoms are associated with this age group . 
 the aim of this paper is to illustrate the typical imaging characteristics and the less common and more insidious appearances of adenomyosis , as well as discuss the role of the different imaging modalities and their diagnostic contribution , with particular reference to transvaginal ultrasound ( tvus ) , hysterosalpingography ( hsg ) and magnetic resonance ( mr ) imaging . 
by presenting a series of cases , some of which are illustrated with combined imaging , the fundamental radiological signs useful for the diagnosis are analysed , along with possible diagnostic pitfalls and differential diagnosis . 
traditional and more recent treatment approaches are also described , with reference to the need for a thorough preliminary diagnostic assessment for treatment planning and the utility of imaging in monitoring treatment effectiveness . radiol med ( 2011 ) 116 : 12671287 introduzione viene definita adenomiosi linvasione benigna , non neoplastica , del miometrio da parte dellendometrio caratterizzata da ectopia di ghiandole e stroma , circondati da miometrio ipertrofico e iperplastico [ 1 ]  . 
pertanto , i foci emorragici che caratterizzano lendometriosi ( zolle di tessuto endometriale in sede ectopica ) e che sono di aiuto nellidentificazione di tale patologia , possono non essere presenti nelladenomiosi con conseguenti possibili difficolt di tipizzazione [ 2 ]  . 
 i fattori di rischio delladenomiosi sono essenzialmente legati : ( 1 ) allattivit riproduttiva , con incremento del rischio di adenomiosi nella pluriparit , aborti spontanei ed endometriosi ; ( 2 ) alle abitudini di vita quali il fumo ; ( 3 ) ai traumi chirurgici quali parti cesarei , aborti indotti o raschiamenti [ 4 ]  . 
la presentazione clinica poliforme : in circa 1 / 3 dei casi , ladenomiosi completamente asintomatica ; nei restanti 2 / 3 i sintomi pi frequenti sono la menorragia ( 50% ) , la dismenorrea ( 30% ) e la metrorragia ( 20% ) ; si rilevano anche dispareunia ed infertilit [ 2 , 6 ]  . 
questa patologia interessa nel 20% dei casi donne in et fertile , di et inferiore ai 39 anni , mentre nell80% dei casi sono affette donne tra i 40 e 50 anni , e in questo intervallo di et sono associati i sintomi pi severi . 
 scopo del presente articolo illustrare limaging tipico e i quadri meno usuali e pi insidiosi di adenomiosi di scutendo il ruolo delle differenti metodiche e il loro ap porto diagnostico , con particolare riferimento allecogra fa transvaginale ( etv ) , allisterosalpingografia ( isg ) e alla risonanza magnetica ( rm )  . 
attraverso una serie di casi , illustrati talora con imaging integrato , saran no analizzati gli elementi di semeiotica elementare utili alla diagnosi , i possibili errori diagnostici e la diagno stica differenziale . 
verranno anche descritti i classici e pi recenti presidi terapeutici per la cui pianificazione consigliabile una attenta preliminare valutazione diagnostica , utile anche nel controllo dellefficacia del trattamento . radiol med ( 2011 ) 116 : 12671287 1269 histopathological considerations and introduction to imaging considerazioni istopatologiche e premesse allimaging the endometrium - myometrium junctional zone is usually irregular since the endometrial mucosa is directly joined to the underlying myometrium without interposition of the submucosa [ 7 ]  . 
therefore , in order to speak of adenomyosis and not physiological penetration , the endometrial tissue needs to extend at least 2.5 mm below the junctional zone [ 8 ] or for at least 25% of the myometrial thickness [ 2 ]  . 
 the diagnostic imaging appearances can be best understood by considering that adenomyosis in the uterus can be either diffuse or focal and that due to this variation , it may manifest with more or less insidious characteristics . in the diffuse form of adenomyosis the uterus appears enlarged and globular . 
the focal forms have a different appearance , which can be recognised as follows : the localised change in the myometrium at the level of one of the uterine walls , characterised by the same changes mentioned above , causes local enlargement , which in imaging is defined as pseudowidening ; the adenomyoma is a circumscribed and nodular aggregate of smooth muscle cells , endometrial glands and stroma , which can be identified in the myometrium ; adenomyoma may involve the endometrium by projecting into the cavity in the form of a polyp . 
in around 2% of cases , the appearance is one of an endometrial polyp when in fact the lesion is a polypoid adenomyoma [ 9 ] ; the adenomyotic cyst is the focal form in which there is significant haemorrhagic cavitation caused by repeated bleeding . the broad appearances of the diffuse and various focal forms can be visualised with the various imaging modalities . 
these lesions appear to be confined to the internal third of the cervical wall [ 10 ] and usually manifest in patients with a history of trauma ( curettage or biopsy ) or arise from residual mllerian tissue located in the cervical stroma . 
it is more appropriate to speak of cervical endometriosis rather than adenomyosis because the cervical mucosa is not strictly joined to the fibromuscular portion but is separated from it by the submucosa [ 7 ]  . 
pertanto , perch si possa parlare di adenomiosi , e non di penetrazione cosiddetta fisiologica , necessario che il tessuto endometriale si approfondi per almeno 2 , 5 mm al di sotto del tratto di giunzione [ 8 ] o penetri almeno il 25% dello spessore miometriale [ 2 ]  . 
per meglio comprendere gli aspetti dellimaging diagnostico bene ricordare che ladenomiosi pu interessare lutero in maniera diffusa o focale e che proprio per il suo polimorfismo pu manifestarsi con quadri pi o meno insidiosi . nella forma diffusa di adenomiosi lutero globoso , aumentato in toto in relazione alla presenza di miometrio globalmente ispessito per coesistenza di cisti ghiandolari e ipertrofia delle cellule muscolari lisce a distribuzione non ordinata ; le ghiandole e lo stroma delle zolle adenomiosiche sono spesso di tipo proliferativo [ 2 ]  . 
le forme focali hanno aspetti differenti , che vanno conosciuti : lalterazione del miometrio localizzata a livello di una delle pareti dellutero , caratterizzata dalle medesime alterazioni sopraelencate , provoca un ingrandimento settoriale che allimaging corrisponde a quello che in lingua inglese viene definito pseudowidening ; ladenomioma un circoscritto e nodulare aggregato di cellule muscolari lisce , ghiandole endometriali e stroma , identificabile nel contesto del miometrio ; ladenomioma pu coinvolgere lendometrio protrudendo in cavit sotto forma di polipo e quello che sembra un polipo endometriale in circa il 2% dei casi in realt un adenomioma polipoide [ 9 ] ; la cisti adenomiosica la forma focale in cui c ampia cavitazione emorragica , determinata da sanguinamenti ripetuti . gli aspetti fino ad ora considerati , quello diffuso e le varie differenti forme focali , hanno un corrispettivo nelle varie tecniche di imaging , talora di difficile caratterizzazione , che andremo a valutare di seguito nel dettaglio . 
illustreremo per completezza anche le localizzazioni del collo uterino , anche se in questo caso pi corretto parlare di endometriosi piuttosto che di adenomiosi ; queste appaiono confinate al terzo interno della parete cervicale [ 10 ] e solitamente si manifestano in pazienti con storia di traumi ( curettage o biopsie ) o insorgono su residui mlleriani allocati nello stroma cervicale . 
si parla di endometriosi cervicale e non di adenomiosi perch la mucosa del collo non strettamente congiunta alla porzione fibro - muscolare ma da essa separata mediante la sottomucosa [ 7 ]  . 
 1270 radiol med ( 2011 ) 116 : 12671287 diagnostic imaging and fundamental radiological signs imaging diagnostico e semeiotica elementare not always is diagnostic imaging prompted by the clinical suspicion of adenomyosis . 
nonetheless , mr imaging is particularly useful both in supplementing doubtful tvus cases and in providing a complete evaluation of the disease , thanks to its high contrast resolution , its ability to characterise lesions with blood and fat content and its panoramic views . 
 for example , in cases of pelvic pain that may be a result of adenomyosis or many other causes , tvus is the first - line technique able to raise the diagnostic suspicion of adenomyosis . 
similarly , infertility may prompt imaging workup , and the suspicion of adenomyosis may also be based on hsg , which is traditionally requested in these cases . transvaginal ultrasound the tvus diagnosis of adenomyosis in b - mode is based on changes to the echostructure , which are readily recognised with the intracavitary study and which reflect histological changes . 
the fundamental tvus signs include : increased myometrial echogenicity or linear hyperechoic bands extending deep into the myometrium , which indicates the presence of islets of ectopic endometrial tissue ; hypoechoic areas in the myometrium compatible with hyperplasia of the muscle tissue surrounding the ectopic tissue ; anechoic areas due to glandular dilatation or myometrial cysts ; poor definition of the junctional zone ; enlargement of the uterus with asymmetrical thickening of one of the walls ( pseudowidening )  . finding at least three of these signs is highly suggestive of adenomyosis [ 11 ]  . 
colour or power doppler tvus may reveal hypervascularity in the case of diffuse or focal adenomyosis , but above all it provides important information regarding the course of the non sempre il sospetto clinico di adenomiosi ad indirizzare la paziente allimaging diagnostico . 
tuttavia , la rm risulta particolarmente utile sia a completamento dei casi ecografici dubbi che per linquadramento completo della malattia , in base alle note caratteristiche di elevata risoluzione di contrasto , per la capacit di tipizzazione delle lesioni a contenuto ematico e adiposo e per la panoramicit . 
analogamente , lo stato di infertilit pu motivare un work - up mediante imaging e il sospetto di adenomiosi pu anche essere basato sullisg classicamente richiesta in questi casi . ecografia transvaginale la diagnosi ecografica di adenomiosi in tecnica b - mode si basa sulle alterazioni dellecostruttura , ben apprezzabili con lo studio ecografico endocavitario e che rispecchiano le modificazioni istologiche . 
gli elementi di semeiotica elementare sono : aumentata ecogenicit miometriale o strie lineari iperecogene che si approfondano nel miometrio , espressione della presenza di isole di tessuto endometriale ectopico ; aree ipoecogene nel contesto del miometrio che rispecchiano liperplasia del tessuto muscolare che circonda il tessuto ectopico ; aree anecogene dovute ad ectasia ghiandolare o cisti miometriali ; trio ; scarsa definizione della giunzione endometrio / miomeaumento dimensionale dellutero con ispessimento asimmetrico di una delle pareti ( pseudowidening )  . rilevare almeno 3 di questi segni molto suggestivo per adenomiosi [ 11 ]  . 
i falsi negativi della isg sono legati alla mancanza di comunicazione delle ghiandole con la cavit uterina , e quindi alla loro mancata opacizzazione e conseguente mancato apprezzamento delle spicule ; i falsi positivi sono possibili in presenza di contorni molto irregolari della cavit uterina dovuti a differente patologia , ad esempio esiti flogisti [ 13 ]  . 
la scansione longitudinale mostra i seguenti elementi di semeiotica elementare : scarsa definizione della giunzione endometrio - miometrio ( freccia bianca grande ) ; aumento della ecogenicit miometriale dovuta a piccole aree iperecogene ( frecce bianche piccole ) espressione delle isole di tessuto endometriale ; aree ipoecogene nel contesto del miometrio che rispecchiano liperplasia del tessuto muscolare che circonda il tessuto eterotopico ( freccia vuota piccola ) ; aree anecogene dovute ad ectasia ghiandolare o cisti miometriali ( frecce vuote grandi )  . vessels at the lesion level . 
 if the patches of ectopic endometrium communicate with the cervical lumen , the dilated glandular lumina terminatle con quelle relative alla presenza di un fibroma cervicale ; la diagnostica differenziale isg tra endometriosi e fibroma cervicale , analogamente a quanto riportato per ladenomiosi del corpo e fondo uterino , non agevole e si basa essenzialmente sulla valutazione dei margini lesionali che sono pi sfumati nelladenomioma e pi netti nel fibroma . risonanza magnetica la rm metodica affidabile nella diagnosi di adenomiosi , con sensibilit variabile in letteratura tra 78% e 88% vs . 
la rm particolarmente utile nella tipizzazione delladenomiosi focale poich in grado di dirimere eventuali dubbi ecografici o isterosalpingografici sulla base di precisi elementi di semeiotica elementare e di importanti segni morfologici , utili per la diagnostica differenziale . 
la tipologia di distribuzione dei foci iperintensi nelle sequenze t2 dipendenti nelladenomioma , oltre allaspetto sfumato dei margini , elemento distintivo importante nella diagnostica differenziale con unaltra patologia , rara ma equivocabile , ovvero il tumore adenomatoide . 
oblique view of the uterus shows vessels ( arrows ) surrounding a hypoechoic , well - defined lesion in the wall of the uterine body , corresponding to a leiomyofig . 
la scansione obliqua con studio power doppler ben documenta il decorso dei vasi intra - uterini che abbracciano ( frecce ) una lesione ipoecogena a margini netti localizzata nel contesto del corpo uterino , da riferire a fibroma . ing in a cul - de - sac may be appreciated . 
sagittal transvaginal tvus of the cervix ( a ) demonstrates a iso / hypoechoic , ill - defined mass ( callipers ) with hypoechoic areas embedded , corresponding to ectopic endometrial dilated glands with internal echoic debris ( white arrows ) ; a nabothian cyst is visible near the lesion , appearing as a simple anechoic image ( void arrow )  . 
l ecografia transvaginale , scansione longitudinale ( a ) , documenta in sede cervicale unarea ipoecogena sfumata ( calipers ) nel cui contesto si evidenziano alcune formazioni ipo - / iso - ecogene , riferibile a zolle di endometrio con lumi ghiandolari ectasici e materiale corpuscolato nel loro contesto ( frecce bianche ) ; in sede adiacente una cisti di naboth di aspetto ecografico anecogeno ( freccia vuota )  . 
la scansione rm coronale gradient echo t1 pesata con saturazione del segnale del tessuto adiposo ( b ) , mostra minuti foci iperintensi in regione cervicale ( frecce bianche ) cui si associa formazione iperintensa dellannesso di sinistra , da riferire ad endometrioma ( freccia vuota )  . 
in corrispondenza di tali piccole aree iperintense , nella sequenza assiale fast spin echo t2 pesata ( c ) si osserva una formazione ipointensa a margini sfumati con foci iperintensi nel contesto ( frecce ) , riferibile ad endometrio in posizione ectopica . of faecal matter in the rectum , or to confuse them with signs produced by a cervical fibroma . 
the differential diagnosis with hsg between endometriosis and cervical fibroma , not unlike the situation reported for adenomyosis of the uterine body and fundus , is no simple task and is essentially based on evaluation of lesion margins , which tend to be ill - defined in adenomyoma and better - defined in fibroma . magnetic resonance imaging mr imaging is a reliable modality for diagnosing adenomyosis , with a sensitivity varying in the literature between 78% and 88% vs . 
the classic mr imaging signs [ 12 ] for identifying diffuse adenomyosis include : le ; lanamnesi pu essere di aiuto nella caratterizzazione . la localizzazione cervicale non di facile tipizzazione in rm per la possibile confusione con forme flogistiche ( cervicite glandulo - cistica ) o con la semplice dilatazione cistica delle ghiandole di naboth . 
tuttavia , poich il materiale proteico mostra segnale iperintenso nelle sequenze t1 pesate , analogamente al materiale ematico , un elemento di semeiotica rm utile alla diagnosi lo slargamento dello stroma cervicale nella sede della lesione . 
anteroposterior projection shows small outpouchings ( small white arrows ) perpendicular to internal uterine contour , located in the fundus and right uterine wall , which indicate dilated glandular ducts of the dislocated endometrial tissue . 
la proiezione anteroposteriore documenta , in corrispondenza del fondo e della parete laterale di destra dellutero , la presenza di estroflessioni del contorno terminanti a cul di sacco ( frecce bianche piccole ) , perpendicolari al margine uterino , compatibili con lumi ghiandolari ectasici , che si approfondano nella parete uterina , in rapporto alla presenza di adenomiosi . 
mr imaging is particularly useful in characterising focal adenomyosis because it is able to eliminate doubts raised at tvus or hsg on the basis of precise fundamental imaging signs and important morphological features , which are useful for differential diagfig . 
la proiezione anteroposteriore documenta una estroflessione pseudo - diverticolare del fondo uterino , riferibile ad adenomiosi ( freccia ) con lume ghiandolare particolarmente ectasico della zolla di endometrio indovato nel miometrio , comunicante con la cavit uterina . 
la terapia chirurgica , di tipo radicale o conservativo , molto dipende dal grado di infiltrazione miometriale e la pianificazione del tipo dintervento non pu prescindere dallattenta e preliminare valutazione di eventuale patologia aderenziale pelvica associata , che possibile definire in anticipo . 
 trattamento medico il trattamento medico delladenomiosi ha oggi molteplici opzioni , che spaziano dal trattamento locale con rilascio di farmaci attraverso un dispositivo intra - uterino ( iud ) alle preparazioni di tipo sistemico . 
limpiego di farmaci progestinici rilasciati dallo iud nella prevenzione dei disordini di tipo mestruale , per la riduzione dellentit del sanguinamento nelle donne con mestruazioni particolarmente abbondanti , noto [ 15 ]  . 
tali farmaci , comportando la decidualizzazione dellendometrio [ 16 ] e le conseguenti modificazioni di tipo atrofico che riducono lentit del sanguinamento , riducono anche lentit dei depositi adenomiotici ; ne conseguono laumento della contrattilit uterina , la conseguente riduzione del volume dellutero e il miglioramento del quadro clinico per riduzione della dismenorrea . 
analogamente , stato proposto il rilasciamento attraverso lo iud di ormoni sintetici derivati dal testosterone [ 17 ] la cui azione quella di sopprimere la produzione di gonadotropine inducendo uno stato di pseudo - menopausa . tra i farmaci a somministrazione sistemica , sono stati segnalati gli analoghi dellormone gonadotropo ( agonisti gnrh ) che , avendo una struttura chimica del tutto analoga a questultimo , conducono ad una saturazione dei recettori fig . 
nella sequenza sagittale fse t2 pesata , si segnala utero antiverso con zona di giunzione ispessita ( > 12 mm ) ( freccia bianca piccola ) e fluido a segnale disomogeneo in sede endouterina ( calipers ) ed endovaginale ( freccia bianca grande ) riferibile a materiale ematico in rapporto alla fase mestruale del ciclo . radiol med ( 2011 ) 116 : 12671287 1277 fig . 
sagittal t1 - weighted gradient echo fat - saturated image , high signal intensity foci in the myometrium in the posterior wall of the uterine body ( a , void arrows ) ; sagittal t2weighted fast spin echo image ( b ) ill - defined , low - signal - intensity nodule containing bright foci ( arrows ) corresponding to adenomyoma is visualised in that area ; diffuse widening of the anterior junctional zone is also well visualised . 
la scansione sagittale gre t1 pesata con saturazione del segnale del tessuto adiposo documenta piccoli spots iperintensi nel contesto del miometro della parete posteriore del corpo uterino ( a , frecce vuote ) ; nella scansioni sagittale ( b ) fse t2 pesata , in corrispondenza delle aree iperintense segnalate in a , si osserva nodulo a margini sfumati , di aspetto ellittico e con foci iperintensi diffusamente distribuiti nel suo contesto ( frecce ) relativo ad adenomioma ; si documenta anche lispessimento marcato della zona di giunzione anteriore . 
the distribution of hyperintensive foci in t2 - weighted sequences in adenomyoma , ipofisari del gnrh cui conseguono la riduzione dei livelli del gnrh e uno stato di menopausa indotto . 
se la terapia viene interrotta , tale effetto reversibile e per tale motivo questo tipo di trattamento non pu essere considerato una cura definitiva per ladenomiosi ; di solito , viene protratto per 36 mesi prima dellescissione chirurgica per i vantaggi derivanti dalla riduzione del sanguinamento in corso dintervento e dal minor trauma tissutale [ 16 ]  . 
la soppressione della biosintesi degli estrogeni mediante gli agonisti gnrh e gli inibitori dellaromatase sembra possa significativamente ridurre il rischio di recidive dopo terapia conservativa delladenomiosi [ 15 ]  . 
questo tipo di approccio terapeutico , ovvero di tipo medico , pu essere facilmente supportato da tecniche di monitoraggio diagnostico , rappresentate in particolare dalletv e dalla rm che sono impiegabili per il controllo dimensionale dellutero e dei noduli di adenomiosi in corso di trattamento . chirurgica radicale nelladenomiosi diffusa quando sia in predicato una iste1278 radiol med ( 2011 ) 116 : 12671287 fig . 
la scansione assiali spin echo ( se ) t1 pesata ( a ) documenta una formazione protrudente nel lume endometriale , ad elevata intensit di segnale ( freccia ) come per contenuto ematico ; le scansioni sagittali spoiled gradient echo ( spgr ) t1 pesate con saturazione del segnale del tessuto adiposo dopo somministrazione di mdc , mostrano sia nella fase arteriosa ( b ) , che in quella portale ( c ) e tardiva ( d ) , una ridotta impregnazione della lesione rispetto al miometrio circostante ; la formazione appare inscritta nella zona giunzionale , che aperta a coppa come ben si documenta in fase arteriosa ( b , freccia ) ove essa riconoscibile per il segnale pi marcatamente intenso rispetto al restante miometrio ; il segnale della zona giunzionale si omogeneizza gradatamente col miometrio esterno nelle altre fasi ( d )  . 
tuttavia , a riguardo delle complicanze dellisterectomia transvaginale , in uno studio effettuato su pazienti con leiomiomi e affette da adenomiosi [ 18 ] si riporta un pi elevato rischio di lesioni della vescica nelle pazienti del secondo gruppo . 
la scansione assiale gre t1 pesata con saturazione del segnale del tessuto adiposo ( a ) documenta grossolana formazione ad elevata intensit di segnale , come per presenza di sangue ( freccia ) , dotata di livello endoluminale e localizzata nel contesto della parete miometriale posteriore del fondo , riferibile a cisti adenomiosica ; nella scansione assiale fse t2 pesata ( b ) si conferma la presenza di materiale ematico con sedimento declive ( freccia )  . located and dilated mesothelial tubules . adenomyotic cyst enters the differential diagnosis with the outcomes from recent myomectomy , as blood material may be encountered in the residual uterine cavity and misinterpreted . 
patient history can be helpful in characterising the findings . ectopic endometrial tissue located in the cervix is not easy to characterise with mr imaging given the possible confusion with inflammation ( glandularcystic cervicitis ) or with simple cystic dilatation of the nabothian glands . 
la scansione sagittale fse t2 pesata documenta una lesione ipointensa nel contesto della parete posteriore dellutero , con effetto massa , a margini netti e con foci iperintensi distribuiti perifericamente ( frecce bianche piccole ) , riferibile a tumore adenomatoide ; anteriormente ( freccia vuota ) un fibroma a parziale sviluppo sottosieroso . 1280 radiol med ( 2011 ) 116 : 12671287 fig . 
axial t1 - weighted gradient echo fat - saturated image ( a ) shows a large , hyperintense lesion , indicating haemorrhage ( white arrow ) in the cervix ; an endometrioma can also be seen in the left ovary ( void arrow )  . 
in addition , even aderenze , e una possibile spiegazione nel fatto che ladenomiosi e lendometriosi , nella quale le aderenze pelviche sono pressoch costanti , pur essendo due condizioni patologiche differenti possono coesistere nella stessa paziente . 
 la preliminare valutazione delle aderenze pelviche , quindi , essenziale nella pianificazione del tipo dintervento e la rm particolarmente adatta a tale controllo . chirurgica conservativa la chirurgia conservativa ( escissione / enucleazione chirurgica ) solitamente riservata alladenomiosi focale , ma il tipo di trattamento molto dipende dal tipo di lesione e dal grado di interessamento miometriale della malattia . 
 farquhar e brosens [ 15 ] hanno recentemente riportato i radiol med ( 2011 ) 116 : 12671287 1281 in cases of medical or interventional treatment , a reliable evaluation of the treatment and monitoring its effectiveness are necessary , and imaging can guarantee this . medical treatment medical treatment of adenomyosis has a wide variety of options , which range from local treatment with the release of medications by a intrauterine device ( iud ) to systemically administered treatment . 
these hormones act to suppress the production of gonadotropin , thus inducing a state of pseudomenopause . medications available for systemic administration include gonadotropin - releasing hormone ( gnrh ) agonists , which have a chemical structure analogous to gonadotropin and therefore interact with the gnrh receptors . 
this type of treatment approach ( medical treatment ) can be easily supported by diagnostic monitoring techniques , in particular by tvus and mr , which can be used to monitor the size of the uterus and the adenomyotic nodules being treated . radical surgery in cases of diffuse adenomyosis in which a hysterectomy fig . 
la scansione assiale fast spin echo t2 pesata documenta un grappolo di formazioni iperintense ( freccia ) come per contenuto fluido , riferibili ad estasia cistica delle ghiandole cervicali ( cisti di naboth ) , localizzate medialmente allo stroma . differenti trattamenti chirurgici proposti in letteratura per ladenomiosi focale basati sulla localizzazione delladenomioma , in sede sottomucosa , intramurale , e sub - peritoneale o intra - ligamentosa : ( 1 ) per ladenomioma a sviluppo sottomucoso , ovvero per la forma polipoide a protrusione intra - luminale , e per quella pi superficiale o pseudowidening , vengono proposte rispettivamente lescissione isteroscopica o la resezione endometriale trans cervicale ; ( 2 ) lenucleazione viene suggerita per la localizzazione intramurale , ovvero per ladenomioma che risparmia il contorno uterino ; tuttavia la lesione potrebbe essere vicina alle tube di falloppio o coinvolgere i vasi maggiori e in tal caso viene proposta anche una cito - riduzione preliminare mediante agonisti gnrh ; listerectomia viene proposta se il miometrio comunque interessato in profondit ; ( 3 ) per ladenomioma sottosieroso o sub - peritoneale , localizzato nella parete posteriore dellutero in prossimit di endometriomi ovarici strettamente adesi allutero , viene suggerita lescissione superficiale o anche laparoscopica . intuitivo che la preliminare pianificazione del tipo dintervento mandatoria e che non solo vada accertata preliminarmente leventuale coesistenza di aderenze pelviche ma anche lentit del coinvolgimento miometriale . 1282 radiol med ( 2011 ) 116 : 12671287 is being considered , transvaginal access is preferable to transabdominal access due to the lower morbidity and shorter hospital stay of the former , this of course being dependent on the experience of the surgeon . 
nonetheless , with regard to complications of transvaginal hysterectomy , a study on two groups of patients , one with leiomyomas and the other with adenomyosis [ 18 ] , reported a higher risk of lesions to the urinary bladder in patients from the latter group . 
this may be explained by the fact that despite being two separate pathological conditions , adenomyosis and endometriosis may coexist in the same patient , and pelvic adherences are very frequent in the latter condition . 
a preliminary evaluation for pelvic adherences , therefore , is crucial in planning the type of procedure , and mr imaging is particularly suited to this type of study . conservative surgery conservative surgery ( surgical excision / enucleation ) is usually reserved for focal adenomyosis , but the type of treatment is heavily dependent on the type of lesion and the extent of myometrial involvement . 
farquhar and brosens [ 15 ] recently reported the different surgical procedures proposed in the literature for focal adenomyosis based on adenomyoma location , whether submucosal , intramural , subperitoneal or intraligamentous : ( 1 ) approaches proposed for adenomyoma with submucosal development , i.e. 
the polypoid form with intraluminal protrusion and the more superficial or pseudowidening form , are hysteroscopic or transcervical endometrial excisions , respectively ; ( 2 ) enucleation is suggested for lesions with intramural location , i.e. 
hysterectomy is instead the preferred option if there is deep involvement of the myometrium ; ( 3 ) superficial excision or even laparoscopy are proposed for subserosal adenomyomas located in the posterior wall of the uterus in the vicinity of ovarian endometriomas tightly adhering to the uterus . clearly , preliminary planning intervention type is mandatory , and not only should the coexistence of pelvic adherences be evaluated , but the degree of myometrial involvement also . embolisation embolisation of the uterine artery is a therapeutic approach adopted for treating fibromas . 
adenomyosis and uterine fibromas may embolizzazione lembolizzazione dellarteria uterina noto presidio terapeutico nel trattamento dei fibromi per il controllo della cui efficacia le metodiche di imaging , ed in particolare la rm , sono stati utilmente impiegati [ 19 ]  . 
 tuttavia , nonostante alcuni risultati incoraggianti riportati in letteratura [ 20 , 21 ] la possibilit di fallimento suggerisce ulteriori studi per chiarire il ruolo effettivo di questo tipo di trattamento . ultrasuoni focalizzati labilit delle onde ultrasonore di provocare incremento della temperatura tissutale nota da tempo [ 22 ] ma le onde ultrasonore utilizzate in diagnostica sono distribuite su unampia area e quindi lincremento della temperatura da esse provocata impercettibile . 
se il pattern delle onde ultrasonore viene modificato di modo che esse possano confluire su un unico punto , si pu provocare un incremento localizzato della temperatura in quel punto , con denaturazione delle proteine per temperature che superano i 55c e conseguente morte cellulare irreversibile e necrosi coagulativa [ 23 ]  . 
questa il principio sul quale si basa la terapia ad ultrasuoni focalizzati , per il controllo della quale stato proposto limpiego della rm sia per la sua eccellente risoluzione anatomica che per la sensibilit allimaging termico [ 24 ]  . 
pertanto , nonostante la tecnica possa sembrare una utile alternativa nel trattamento delladenomiosi , essa necessita di ulteriori studi per il chiarimento del suo ruolo effettivo [ 23 ]  . discussione i goals dellimaging in tema di adenomiosi sono essenzialmente : ( 1 ) il corretto inquadramento della malattia ; ( 2 ) la valutazione dellambiente peri - uterino e del grado di interessamento del miometrio , per un corretto approccio terapeutico e ( 3 ) il monitoraggio delle pazienti trattate con terapia conservativa . 
da quanto fino ad ora esposto , emerge che la rm ha un ruolo importante nella diagnosi di adenomiosi , essenziale per linquadramento globale della malattia , a causa dellelevata capacit di caratterizzazione tissutale e per la sua panoramicit . 
however , despite some encouraging results reported in the literature [ 20 , 21 ] , the possibility of failure suggests the need for further studies to clarify the effective role of this treatment . high - intensity focused ultrasound the ability of ultrasound waves to increase tissue temperature has been known for some time [ 22 ] , but the ultrasound waves used in diagnostic imaging are distributed over a wide area and therefore the temperature increase they produce is imperceptible . 
if the pattern of the ultrasound waves is modified such that they are made to converge at a single point , a localised increase in temperature may be produced at that point , with protein denaturation at temperatures > 55c and the consequent irreversible cell death and coagulation necrosis [ 23 ]  . 
this is the principle of high - intensity focused ultrasound , for which mr imaging has been proposed as the guidance technique due to its excellent anatomical resolution and sensitivity to thermal imaging [ 24 ]  . 
therefore , although the technique seems to be a useful alternative , further studies are needed to clarify its effective role [ 23 ]  . discussion the imaging goals in relation to adenomyosis are essentially the following : ( 1 ) correct identification of the disease ; ( 2 ) evaluation of the periuterine environment and extent of myometrial involvement for choosing the most appropriate treatment ; ( 3 ) monitoring patients treated with conservative therapy . as the above demonstrates , mr imaging plays an important role in diagnosing adenomyosis , given its remarkable capacity for tissue characterisation and the panoramic views it offers . 
the accuracy of the technique is , on the whole , greater than that of tvus , with percentages ranging from 85% to 90.5% against the 6886% of tvus [ 12 ]  . 
foci of bleeding may not necessarily be identified in adenomyosis , as only the basal layer of the endometrium , which does not respond to hormonal stimulation , is located ectopically . 
nonetheless , it is always worthwhile to perform the examination in the subacute phase of a possible bleeding , because the hyperintense foci identified a quella della etv , con percentuali pari all85%90 , 5% vs . 
anche se nelladenomiosi possibile non rilevare necessariamente foci di sanguinamento , a causa della posizione ectopica del solo strato basale dellendometrio non rispondente agli stimoli ormonali , sempre bene eseguire lindagine nella fase subacuta di un possibile sanguinamento , perch in tal caso i foci iperintensi nelle sequenze t1 pesate sono in grado di caratterizzare una endometriosi eventualmente associata . 
opportuno , pertanto , osservare con attenzione sempre tutte le sequenze e le varie immagini per evitare errori diagnostici ; lo spasmo non potr essere sempre costantemente rilevato nel corso di una indagine rm , che notoriamente piuttosto lunga . abbiamo anche visto come lapproccio terapeutico necessiti di una corretta pianificazione preliminare . 
la presenza di aderenze pu condizionare lapproccio chirurgico , laparoscopico o classico trans - addominale , e le possibilit della rm nellidentificare le aderenze in maniera indiretta [ 25 ] o mediante cine - rm con immagini dinamiche che documentano la mobilit degli organi , sono note [ 26 ]  . la scelta chirurgica radicale o di tipo conservativo molto dipende anche dal grado di infiltrazione miometriale da parte delladenomiosi e la rm pu accertarlo mediante lattenta valutazione del grado di enhancement lesionale dopo mdc . 
 [ 27 ] ha dimostrato che il grado di interessamento miometriale da parte della neoplasia dellendometrio pi facilmente determinabile nella fase di equilibrio in cui la lesione neoplastica , con segnale pi 1284 radiol med ( 2011 ) 116 : 12671287 in t1 - weighted sequences are able to characterise any associated endometriosis . 
mr imaging identifies adherences indirectly [ 25 ] or with cinemr dynamic images , which demonstrate organ mobility [ 26 ]  . choosing between radical and conservative surgery is also heavily dependent on the extent of myometrial infiltration by the adenomyosis ; mr imaging allows verification of this , with a thorough evaluation of the degree of lesion enhancement after contrast agent administration . 
 [ 27 ] showed that the extent of myometrial involvement by endometrial carcinoma can be easily recognised in the equilibrium phase in which the neoplastic lesion , which has lower signal intensity than the myometrium , is best identified within the myometriu however , if the endometrial carcinoma arises from an adenomyosis , which is also an enhancing lesion and therefore with a less intense signal than the myometrium , it may be useful to exploit junctional zone enhancement in the arterial phase to identify whether the endometrial carcinoma has infiltrated the myometrium [ 5 ]  . 
la scansione assiale t2 pesata mostra a livello della parte posteriore del fondo unarea ipoecogena ( a , frecce ) con foci iperintensi nel contesto , di morfologia ellittica e a margini sfumati ( pseudowidening ) ; la scansione assiale spgr t1 pesata con saturazione del segnale del tessuto adiposo in fase tardiva dellenhacement dopo somministrazione di mdc , conferma la ridotta impregnazione rispetto al circostante miometrio della lesione adenomiosica che si approfond marcatamente nel miometrio ( b , freccia )  . basso rispetto al miometrio , meglio si identifica nel contesto miometriale . 
tuttavia , se la neoplasia endometriale insorge su adenomiosi , anchessa con enhancement e , quindi , con segnale ridotto rispetto al miometrio , pu essere utile sfruttare il potenziamento della jz nella fase arteriosa per identificare leventuale sconfinamento miometriale della lesione neoplastica dellendometrio [ 5 ]  . 
tvus , especially for its ability to evaluate reduction in the size of the uterus and the presence of focal lesions , and mr imaging , for global and complete evaluation of the uterus and its environment , without doubt play a useful role in monitoring this kind of treatment . 
the role that the two imaging modalities , particularly mr imaging , may play in the new interventional techniques still needs to be clearly defined , even though the recent studies appear promising . in conclusion , tvus and above all , mr imaging are particularly useful modalities for studying adenomyosis , both for characterising the disease and monitoring treatment . 
il ruolo delle metodiche di imaging , e in particolare della rm , in relazione alle nuove tecniche terapeutiche di tipo interventistico ancora da definire con certezza , anche se gli studi attuali sembrano promettenti . 
hlpm consists of selective catheterisation of the common hepatic artery , permanent occlusion of the gastroduodenal artery at its origin using metal coils , an inflated balloon catheter placement at the origin of the proper hepatic artery to block blood flow and induce hypoxia for around 10 min , mmc infusion and vascular - bed occlusion through injection of an absorbable haemostatic agent . 
 hlpm consiste nella cateterizzazione selettiva della arteria epatica comune , occlusione permanente della arteria gastro - duodenale alla sua origine mediante spirali metalliche ; posizionamento di un catetere da occlusione con palloncino gonfiato alla origine della arteria epatica propria per bloccare il suo flusso ed indurre ipossia per circa 10 minuti ; infusione di mmc ; occlusione del letto vascolare mediante iniezione di un agente emostatico riassorbibile . 
il ricovero dei pazienti in ospedale durato 1240 radiol med ( 2011 ) 116 : 12391249 a partial response ( pr ) in 29% , stable disease ( sd ) in 45% and progressive disease ( pd ) in 26% of patients . 
it may be considered a palliative treatment option in patients with advanced liver disease in centres with adequately experienced medical teams . keywords hypoxic liver perfusion multifocal liver metastases mitomycin - c in media 10 giorni ( range 715 )  . 
lesame tc condotto dopo 30 giorni dal trattamento ha documentato una risposta parziale ( pr ) nel 29% dei pazienti , una stabilit di malattia ( sd ) nel 45% , ed una progressione di malattia ( pd ) nel 26% . 
essa pu essere considerata una opzione come trattamento palliativo in pazienti con malattia epatica in stato avanzato , nei centri ove sia disponibile team medico dotato di adeguata esperienza . parole chiave infusione intra - arteriosa epatica in ipossia metastasi epatiche multiple mitomicina - c introduction introduzione colorectal cancer ( crc ) is the most frequent cause of liver metastases . 
after systemic chemotherapy , mean survival rates have recently been reported to be approximately 20 months [ 4 , 5 ] , with a 3 - year survival rate of around 10% [ 4 ]  . 
 in addition to crc , liver metastases may complicate the course of many other cancers , such as ocular melanoma and neuroendocrine tumours , and are the main cause of death in the majority of cases . 
only a minority of patients has surgically resectable lesions , most of them being candidates for regional therapies [ 6 ]  . the liver has a double blood supply : the portal vein accounts for 7080% of blood supply , which explains the high rate of metastases from mesenteric regions ; the hepatic artery and its branches allows detection of hypervascular liver metastases . 
infusion therapy consists of administering chemotherapeutic agents directly into the hepatic artery using superselective catheterisation [ hepatic arterial infusion ( hai ) ] ; this may il cancro del colon - retto ( ccr ) la causa pi frequente di metastasi epatiche . 
nel 10%20% dei pazienti con ccr le metastasi epatiche sono sincrone , e rappresentano lunico fattore limitante laspettativa di vita sino a circa il 60% dei pazienti [ 2 ]  . 
dopo chemioterapia sistemica la sopravvivenza media stata recentemente riportata intorno ai 20 mesi [ 4 , 5 ] con una sopravvivenza a 3 anni di circa il 10% [ 4 ]  . 
 oltre al ccr , le metastasi epatiche possono complicare il decorso di molte altre neoplasie , come il melanoma oculare , i tumori neuroendocrini , ed altri ancora , e rappresentano la principale causa di morte nella maggior parte dei casi . 
 il fegato ha un doppio apporto ematico : la vena porta fornisce il 70%80% del sangue e ci spiega lalta frequenza di metastasi che giungono dai territori mesenterici ; larteria epatica ed i suoi rami permettono di evidenziare le metastasi epatiche iper - vascolarizzate . 
la terapia infusionale consiste nella somministrazione di chemioterapici radiol med ( 2011 ) 116 : 12391249 1241 be followed by vascular occlusion [ transarterial chemoembolisation ( tace ) ]  . 
hai , tace and hae have the same palliative and symptomatic indications and contraindications , and in particular , the percentage of hepatic parenchyma involvement ( > 75% ) and the portalvein thrombosis resulting from hepatic artery occlusion by the embolisation agents used in tace . 
 the technique , consisting of comprehensive hepatic arterial infusion of mitomycin - c under hypoxic conditions , followed by embolisation with spongostan [ hypoxic liver perfusion with mmc ( hlpm ) ] was first described by ricci et al . 
 the purpose of our analysis was to evaluate treatment feasibility and toxicity and its impact on os and diseasefree ( dfs ) survival . materials and methods forty - two patients ( 25 men , 17 women ) , mean age 57 ( range 3378 ) years , refractory to other locoregional and / or systemic treatments , were treated with hlpm between june 2001 and may 2009 . 
the patients had multifocal , unresectable liver metastases originating from different primary tumours : crc ( n = 25 ) , neuroendocrine tumour ( n = 2 ) , pancreatic tumour ( n = 3 ) , uveal melanoma ( n = 2 ) , gastric cancer ( n = 2 ) , ovarian cancer ( n = 1 ) and fibrosarcoma ( n = 1 )  . 
in 14 of the 56 procedures , the ecog score was 2 . inclusion criteria were : presence of multifocal liver metastases that were unresectable or nontreatable with local ablative therapies and refractory to at least one direttamente nella arteria epatica mediante un cateterismo super - selettivo ( infusione arteriosa epatica , iae ) ; essa pu essere seguita dalla occlusione vascolare ( chemio - embolizzazione trans - arteriosa , tace )  . 
iae , tace e eae , presentano le stesse indicazioni palliative e sintomatiche , cos come le stesse controindicazioni , con particolare attenzione alla percentuale di coinvolgimento del parenchima epatico ( superiore al 75% ) e alla trombosi della vena porta in relazione alla occlusione della arteria epatica da parte degli agenti embolizzanti usati durante la tace . 
 la tecnica , che consiste nella infusione arteriosa epatica globale con mitomicina - c in condizioni di ipossia , seguita dalla embolizzazione con spongostan ( hypoxic liver perfusion with mmc , hlpm ) stata descritta per la prima volta da ricci et al . 
in questo lavoro , descriviamo la nostra esperienza in 42 pazienti trattati con hlpm . lo scopo della nostra analisi di valutare la fattibilit del trattamento , la tossicit associata al trattamento e limpatto di questo sulla sopravvivenza globale e sulla sopravvivenza libera da malattia . 
 materiali e metodi quarantadue pazienti ( 25 maschi e 17 femmine ) , et media 57 anni ( range 3378 ) , refrattari ad altre terapie locoregionali e / o a trattamenti sistemici , sono stati trattati con hlpm da giugno 2001 a maggio 2009 . 
i pazienti erano affetti da multiple metastasi epatiche non - resecabili , a partenza da tumori primitivi differenti : 25 / 42 erano affetti da ccr , 2 da tumori neuroendocrini , 3 da tumore del pancreas , 2 da melanoma delluvea , 2 da tumore dello stomaco , uno da tumore dellovaio ed uno da fibrosarcoma . 
sei pazienti presentavano tumore epatico primitivo , colangiocarcinoma : uno di questi era stato precedentemente trattato chirurgicamente , mentre gli altri erano stati classificati come inoperabili ( tabella 1 )  . 
 patients with a surgically unresectable primary liver tumour refractory to other locoregional therapies were also considered candidates for treatment . total - body assessment , each patient underwent complete biochemical and anaesthesiological computed tomography ( ct ) and complete analyses of the risks and benefits of hlpm . 
 then , the balloon of the catheter ( occlusion balloon catheter ) was inflated at the origin of the common hepatic artery to block blood flow and infuse mmc diluted in 200 ml of saline solution at 2050 mg . 
finally , vascular occlusion was achieved by injecting the absorbable gelatine sponge ( spongostan , johnson & johnson medical limited , gargrave , skipton , uk ) suspended in 1520 ml of contrast material . 
the overall duration of the procedure varied between 45 and 120 m during the procedure , usually at the embolisation stage , many patients reported abdominal pain and were treated with analgesic therapy managed by the anaesthetist . 
 in ogni paziente stata eseguita una completa valutazione biochimica ed anestesiologica , una tomografia computerizzata ( tc ) total - body , ed una completa pianificazione dei rischi e dei benefici dellhlpm . 
 al termine , stata eseguita locclusione vascolare con la iniezione di gelatina riassorbibile spugnosa ( spongostan , jonson and jonson medical limited , gargrave , skipton , gran bretagna ) solubilizzata con 1520 ml di mezzo di contrasto . 
la risposta biochimica al trattamento stata valutata confrontando i valori del markers ( antigene carcinoembrionario , cea , e antigene carboidratico , ca , 19.9 ) , prima e 30 giorni dopo il trattamento . 
una tc delladdome stata eseguita ad 1 , 3 e 6 mesi dal termine della procedura , e la risposta stata valutata in accordo con i criteri response evaluation criteria in solid tumors ( recist ) [ 11 ]  . 
per valutare lo tossicit epatica e quella sistemica , sono stati confrontati i valori degli enzimi epatici ( ast , alt ) , di - gt , di pseudo - colinesterasi , di bilirubina totale , di lattico - deidrogenasi ( ldh ) e di fosfatasi alcalina prima e dopo la procedura . i valori numerici sono stati espressi come valori medi ed stato indicato il range corrispondente ; le variabili considerate pree post - hlpm sono graficamente rappresentate come box - plots e confrontate usando il wilcoxon ranks test . 
 la sopravvivenza globale ( overall survival , os ) e quella libera da malattia ( progression free - survival , pfs ) sono state valutate usando il metodo di kaplan - meier [ 12 ]  . 
in un paziente con tumore gastrico in stato avanzato ed iniziale lieve insufficienza epatica si registrata una insufficienza epatica ; nellaltro caso il paziente deceduto in seguito ad una emorragia cerebrale non correlabile con la procedura . 
1 limmagine arteriografica mostra il cateterismo selettivo della arteria epatica e la occlusione permanente del tratto prossimale della arteria gastro - duodenale con spirali metalliche . the biochemical response to treatment was assessed by comparing marker values carcinoembryonic antigen ( cea ) and cancer antigen ( ca ) 19.9 before and 30 days after treatment . 
an abdominal ct scan was performed 1 , 3 and 6 months after the procedure , and response was assessed according to the response evaluation criteria in solid tumours ( recist ) criteria [ 11 ]  . 
to assess hepatic and systemic toxicity , we compared the values of liver enzymes [ aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , gamma - glutamyltranspeptidase ( - gt ) , pseudocholinesterase , total bilirubin , lactate dehydrogenase ( ldh ) and alkaline phosphatase ( ap ) before and after the procedure [ 11 ]  . numeric values are expressed as means with the corresponding range ; variables considered before and after hlpm are graphically represented as box plots and compared using the wilcoxon rank test . 
the other died of cerebral haemorrhage unrelated to the procedure . with reference to pain relief , on average , a single dose of a nonsteroid anti - inflammatory drug , four doses of mild opioids and nine doses of major opioids were administered during the hospital stay . 
pre - treatment values of cea [ 62 ng / ml ( sd142.39 ) , range 1507 ng / ml ] compared with those obtained 30 days after treatment [ cea 30 ng / ml ( sd111.6 ) , range 0.60312 ng / ml ] revealed no statistically significant difference . 
four patients were alive at the time of writing this report , one with crc metastases , one with a neuroendocrine tumour and two with liver metastases from uveal melanoma . 
no variable could be correlated to pfs ( table 2 )  . riguardo al trattamento del dolore , una media di una singola dose di farmaci antinfiammatori non steroidei ( fans ) , 4 dosi di oppioidi minori e 9 di oppioidi maggiori sono state somministrate ai nostri pazienti durante il ricovero . 
i valori di cea pre - trattamento ( 62 ng / ml , sd142 , 39 , range 1507 ng / ml ) confrontati con quelli ottenuti 30 giorni posttrattamento ( cea : 30 ng / ml , sd111 , 6 , range 0 , 60312 ng / ml ) non hanno mostrato una differenza statisticamente significativa . 
le variabili statisticamente associate con una migliore os erano : et giovane , intervallo tra la diagnosi del tumore e lhlpm ed intervallo tra la comparsa delle metastasi ed il trattamento in questione . 
nessuna variabile risultata correlabile con la pfs ( tabella 2 )  . discussione i pazienti con ampio coinvolgimento epatico , non responsivi o non candidabili ad altri tipi di trattamento possono radiol med ( 2011 ) 116 : 12391249 1245 fig . 
2a - d le immagini tc documentano un caso di risposta parziale ( pr ) ( a , b ) ed uno di stabilit di malattia ( sd ) ( c , d ) un mese dopo il trattamento . discussion patients with extensive liver involvement unresponsive to or not candidates for other forms of treatment may benefit from intra - arterial infusion of mitomycin c under hypoxic conditions . 
questi dati sono risultati in linea con le nostre aspettative , considerando che la mmc viene somministrata localmente ed in questo modo i rischi di tossicit sistemica sono pi bassi che non con la chemioterapia sistemica . 
la tossicit epatica e sistemica sono state valutate confrontando i valori di transaminasi , - gt , pseudo - colinesterasi , bilirubina totale , ldh e fosfatasi alcalina pree post - trattamento . 
nella nostra esperienza , la procedura ha una mortalit correlata al trattamento del 2% ( 1 / 56 ) : in un paziente affetto da cancro 1246 radiol med ( 2011 ) 116 : 12391249 fig . 
3a - g i diagrammi mostrano : a , b i valori medi di emoglobibna ( hb ) e della conta piastrinica ( plt ) prima e 30 giorni dopo il trattamento ; c - g i livelli di ast , alt , bilirubina , lattico deidrogenasi ( ldh ) e pseudocolinesterasi ( pce ) prima e dopo il trattamento . ment - related mortality rate of 2% ( 1 / 56 ) : one patient with advanced - stage gastric cancer suffered severe acute liver failure ; another died due to causes that were judged unrelated to this treatment . 
in 31 / 56 cases , the ct scan performed at 1 month never showed a complete response , whereas a partial response was obtained in 29% of cases , stable disease in 45% and progressive disease in 26% . 
in 31 / 56 casi , la tc disponibile dopo 1 mese dal termine della procedura , non ha mai mostrato una risposta completa , mentre una risposta parziale si avuta nel 29% dei casi , una stabilit di malattia nel 45% ed una progressione nel 26% . 
4a , b curve di sopravvivenza globale : per tutti i pazienti ( a ) , e solo per i pazienti affetti da carcinoma del colon - retto ( b )  . os ( months ) os ( months ) 4 months ( range 36 ) , and mean os from the diagnosis of liver metastasis was 20 months . 
 1248 radiol med ( 2011 ) 116 : 12391249 comparison with other similar procedures is made harder by the scarcity of data available in the literature concerning the hypoxic procedure and by the difficulty comparing patients with different characteristics . 
in order to obtain statistically comparable results , it may be useful to conduct a randomised trial to compare our data with those of other locoregional treatments ( hai , superselective tace )  . in conclusion , however , intra - arterial liver infusion with mmc can be considered a feasible approach to liver metastases . 
the feasibility of this procedure , even in patients with advanced - stage metastasised liver diseases , makes it an interesting option as a palliative treatment provided that an expert team is available to administer the treatment . tutti i pazienti con ccr erano stati precedentemente trattati con approcci sistemici standard . 
prendendo in considerazione gli studi in cui i pazienti presentavano caratteristiche simili a quelle dei nostri , la os era di 13 , 8 e 23 mesi in quelli trattati rispettivamente con tace e con iae . 
 [ 15 ] hanno riportato una os media di 343 giorni nel trattamento di lesioni epatiche non chirurgiche ; i pazienti sono parte di uno studio multicentrico pertanto difficile delineare esattamente le loro caratteristiche , ma questi erano stati considerati non candidabili per altri trattamenti . 
per avere dei risultati statisticamente confrontabili , pu essere utile , un trial randomizzato per confrontare i nostri dati con quelli ottenuti eseguendo altri trattamenti loco - regionali ( iea , tace super - selettiva )  . tuttavia , in conclusione , linfusione epatica intra - arteriosa di mmc pu essere considerata un approccio possibile alle metastasi epatiche . 
bazzocchi1 1institute of diagnostic radiology , university of udine , p.le santa maria della misericordia 15 , 33100 udine , italy 2dpmsc medical liver transplantation unit , internal medicine , university of udine , udine , italy correspondence to : l . 
this study was performed to assess the role of magnetic resonance cholangiography ( mrc ) in the clinical decision - making process of referring physicians when managing liver - transplanted patients . 
referring physicians were asked to prospectively state , before and after mrc , the leading diagnosis ; the level of confidence ( on a 0100% scale ) ; the most appropriate diagnostic / therapeutic plan . 
in modo prospettico , ai clinici di riferimento stato richiesto di indicare , prima e dopo la crm : la diagnosi presuntiva ; il livello di confidenza ( su scala 0%100% ) ; il piano diagnostico / terapeutico pi appropriato . 
lanalisi dei dati ha valutato : la resa diagnostica ; la proporzione di diagnosi presuntive modificate ; lefficacia terapeutica ( vale a dire , proporzione di piani diagnostico / terapeutici modificati ) ; lefficacia diagnostica presuntiva ( vale a dire , guadagno in confidenza diagnostica )  . 
lanalisi dei dati ha dimostrato : una resa diagnostica del 85 , 7% ; una proporzione di diagnosi presuntive modificate del 19 , 0% ; unefficacia terapeutica del 42 , 8% ; unefficacia diagnostica presuntiva rispettivamente pari a 18 , 8% e 78 , 7% per diagnosi presuntive concordanti e discordanti ( p < 0 , 01 )  . 
la crm radiol med ( 2011 ) 116 : 12501266 1251 keywords liver transplantation magnetic resonance imaging magnetic resonance cholangiography biliary complications clinical decision making ha inoltre determinato una modifica nella gestione dei pazienti in una proporzione significativa di casi , portando ad un beneficio clinico grazie alla sua elevata accuratezza . parole chiave trapianto di fegato risonanza magnetica colangiografia in risonanza magnetica complicanze biliari diagnosi introduction introduzione to date , endoscopic retrograde cholangiography ( erc ) or percutaneous transhepatic cholangiography ( ptc ) remain the standard of reference in evaluating biliary disease [ 1 , 2 ]  . 
in recent years , magnetic resonance cholangiography ( mrc ) has been advocated as an ideal noninvasive alternative to direct cholangiography [ 5 ] , as this highly fluid - sensitive technique allows panoramic and detailed representation of the biliary tree with no need for i.v. 
an increasing amount of evidence suggests that mrc is comparable with direct cholangiography in evaluating a wide spectrum of biliary diseases [ 6 , 7 ] , including biliary complications after orthotopic liver transplantation ( olt ) [ 815 ]  . 
 materials and methods the institutional review board approved this prospecad oggi , la colangiografia retrograda endoscopica ( cre ) e la colangiografia transepatica percutanea ( ctp ) ri mangono gli standard di riferimento nella valutazione della patologia biliare [ 1 , 2 ]  . 
negli ultimi anni , la colangiografia in risonanza magnetica ( crm ) stata proposta come alternativa ideale non invasiva alla colangiografia diretta [ 5 ] , dal momento che questa tecnica , altamente sensibi le ai liquidi , permette una rappresentazione panoramica e dettagliata dellalbero biliare senza bisogno delliniezione endovenosa di mezzo di contrasto . 
una sempre maggiore evidenza scientifica suggerisce che la crm sia comparabile alla colangiografia diretta nella valutazione di un ampio spettro di patologie biliari [ 6 , 7 ] , incluse le complicanze biliari dopo trapianto ortotopico di fegato ( tof ) [ 815 ]  . 
rispetto alla cre o alla ctp , la crm consente una rappresentazione dei dotti biliari sia a monte che a valle dellostruzione , fornendo unaccu rata mappa prima delle procedure interventistiche . 
a nostra conoscenza , nessun precedente studio ha investigato simili aspetti nei pazienti trapiantati di fegato , nei quali le complicanze biliari si verificano frequentemente , ovvero in pi del 30% dei casi [ 8 , 9 ]  . in relazione a ci , lo scopo del nostro studio stato definire il ruolo della crm nella gestione clinica dei pazienti trapiantati di fegato con sospette complicanze biliari . 
in particolare , lanalisi ha avuto lo scopo di investigare se la crm impatti in maniera significativa nel processo di decision - making dei clinici di riferimento e nella determinazione del work - up dei pazienti . 
the study was performed according to the principles of the declaration of helsinki and subsequent amendments . materiali e metodi patient population il comitato etico della struttura ha approvato questo studio di coorte prospettico senza necessit del consenso informato da parte del paziente . 
lo studio stato condotto in accordo ai principi della dichiarazione di helsinki e dei successivi emendamenti . over a 6 - month period ( january 2009june 2009 ) , all livertransplanted patients with suspected biliary complications were referred for mrc by the medical or surgical division of our hospital . 
exclusion criteria included any current contraindication to mr examination . pazienti imaging technique studies were performed on a 1.5 - t scanner ( magnetom avanto , siemens medical system , erlagen , germany ) using the combination of a six - channel abdominal phased - array surface coil and a spine - array coil and with the implementation of the generalized autocalibrating partially parallel acquisitions ( grappa ) algorithm of parallel imaging . 
 mrc was performed with the multislice technique using a heavily t2 - weighted , volumetric , nearly isotropic , respiratory - triggered turbo spin - echo ( tse ) sequence acquired in coronal and in some cases axial planes with the following acquisition parameters : repetition time ( tr ) 2 , 500 ms ; echo time ( te ) , 682 ms ; matrix size , 357384 ; field of view ( fov ) , 380380 mm ; slices 72 ; thickness 1 mm ; thickness in z direction 1.1 mm ; parallel imaging acceleration factor 3 ; nominal acquisition time 2 min 59 s . 
preliminary coronal and axial half - fourier acquisition single - shot turbo spinecho ( haste ) t2 - weighted sequences ( tr 1 , 000 ms ; te 88 ms ; matrix size 192256 ; fov , 178103 mm ; slices 20 ; thickness 5 mm ; slice gap 20% ; acquisition time 10 s ) were performed to allow a panoramic evaluation of the abdominal region . 
both thin slices and maximum intensity projection ( mip ) reconstructions were evaluated on a dedicated workstation . study design and data analysis the study was designed to achieve three main goals . 
with a well - codified approach [ 22 ] , experienced referring hepatologists ( pt , db ) compiled a questionnaire for each patient before in un periodo di 6 mesi ( gennaio 2009luglio 2009 ) , tutti i pazienti trapiantati di fegato con sospette complicanze biliari sono stati indirizzati alla crm dalla clinica medica o chirurgica del nostro ospedale . 
i criteri di esclusione consistevano nella presenza di controindicazioni allesecuzione della risonanza magnetica ( rm )  . tecnica gli studi sono stati realizzati su un magnete da 1 , 5 t ( magnetom avanto , siemens medical system , erlagen , germania ) utilizzando la combinazione di una bobina di superficie a 6 canali di tipo phased - array e di una bobina spine con limplementazione dellalgoritmo di imaging parallelo generalized autocalibrating partially parallel acquisitions ( grappa )  . 
la crm stata eseguita con tecnica multislice , utilizzando una sequenza turbo spinecho ( tse ) volumetrica , quasi isotropica , con trigger respiratorio , fortemente pesata in t2 , acquisita sul piano coronale ed in alcuni casi su quello assiale , con i seguenti parametri di acquisizione : tempo di ripetizione ( tr ) , 2500 ms ; tempo di eco ( te ) , 682 ms ; matrice , 357384 ; campo di vista ( fov ) , 380380 mm ; fette , 72 ; spessore , 1 mm ; spessore nella direzione z , 1 , 1 mm ; fattore di accelerazione di imaging parallelo , 3 ; tempo di acquisizione nominale , 2 min 59 s . 
sono state eseguite sequenze preliminari halffourier acquisition single - shot turbo - spin - echo ( haste ) t2 - pesate coronali ed assiali ( tr , 1000 ms ; te , 88 ms ; matrice , 192256 ; fov , 178103 mm ; fette , 20 ; spessore , 5 mm ; gap , 20% ; tempo di acquisizione , 10 s ) per una valutazione panoramica della regione addominale . 
sono state valutate su workstation dedicata sia le partizioni sottili che le ricostruzioni tridimensionali maximum intensity projection ( mip )  . disegno dello studio ed analisi dei dati lo studio stato mirato ad ottenere tre obiettivi principali . 
after mrc execution , the physicians indicated the post - mrc diagnosis , the level of confidence in it ( on a 0100% scale ) and the diagnostictherapeutic plan taking into account the mrc results . 
criteria to record preand post - mrc diagnoses were based on a previously described [ 14 ] standard classification of biliary complications in livertransplanted patients , as reported in table 1 . 
accordingly , complications were classified into three main categories : ( 1 ) strictures ( intraor extrahepatic ) ; ( 2 ) bile leakage ; ( 3 ) table 1 spectrum of biliary complications in liver - transplanted patients 1 . 
altre complicanze tabella 1 spettro delle complicanze biliari in pazienti trapiantati di fegato abbiamo valutato limpatto della crm nel processo di clinical decision - making dei clinici di riferimento [ 21 ] , ovvero la capacit della crm di modificare la diagnosi pretest ed il management pianificato , indipendentemente dalla sua performance diagnostica . 
dopo lesecuzione della crm , i clinici hanno indicato la diagnosi post - crm , la confidenza in questa ( su scala 0%100% ) ed il piano diagnostico - terapeutico tenendo conto dei risultati della crm . 
i criteri per riportare le diagnosi pre - crm e crm sono stati basati su una classificazione delle complicanze biliari nei pazienti trapiantati di fegato precedentemente descritta [ 14 ] e riportata nella tabella 1 . 
in accordo con tale classificazione , le complicanze sono state divise in tre categorie maggiori : ( 1 ) stenosi ( intrao extraepatiche ) ; ( 2 ) leakage biliare ; ( 3 ) varie , includendo entit patologiche meno frequenti come la cosiddetta vanishing bile duct syndrome ( gruppo di disordini acquisiti ed associati a distruzione progressiva e scomparsa dei dotti biliari intraepatici con colestasi ) [ 23 , 24 ] , la dilatazione non - ostruttiva , la disfunzione ampollare , il kinking , calcoli biliari / sludge / coaguli , le complicanze legate allanastomosi bilioenterica ed il leakage anastomotico . 
alla luce dei risultati della crm , sono state calcolate : ( 1 ) la proporzione delle diagnosi pre - crm che sono state cambiate dai clinici di riferimento dopo limaging ; ( 2 ) la proporzione dei piani diagnostico - terapeutici modificati dopo la crm rispetto alla valutazione pre - crm . 
le diagnosi pree postcrm sono state valutate come concordi quando stato segnalato lo stesso tipo di complicanze biliari , in accordo con la classificazione elencata nella tabella 1 , indipendentemente dalla presenza di altri reperti alla crm . 
 [ 22 ] , lanalisi ha valutato il guadagno in confidenza diagnostica ( la cosiddetta efficacia diagnostica presuntiva ) come segue : quando la diagnosi pree post - crm stata la stessa , il guadagno di confidenza stato calcolato sottraendo il valore assoluto della confidenza pre - crm da quella post - crm . 
in caso di diagnosi pree post - crm discordanti , la confidenza nella diagnosi finale prima della crm stata preliminarmente valutata sulla base dellassunto che la somma delle probabilit della diagnosi scelta e di tutte le diagnosi alternative del 100% . 
se la confidenza diagnostica per la diagnosi formulata prima della crm era eguale o maggiore del 50% , allora la confidenza diagnostica dedotta per la diagnosi rivista stata stimata del 100% meno la probabilit stabilita per la prima scelta diagnostica . 
in the light of the mrc results , the following measures were calculated : ( 1 ) the proportion of pre - mrc diagnoses that were changed by the referring physicians after imaging ; ( 2 ) the proportion of modified diagnostic therapeutic plans post - mrc compared with the pre - mrc evaluation . 
preand post - mrc diagnoses were assessed as concordant when the same type of biliary complication was reported , according to the classification listed in table 1 , regardless of additional mrc findings . 
 [ 22 ] , the analysis evaluated the gain in diagnostic confidence ( so - called diagnostic thinking efficacy ) as follows : if the preand post - mrc diagnosis was the same , the gain in confidence was calculated by subtracting the absolute values of the clinicians post - mrc and pre - mrc confidence . 
in the case of discordant preand post - mrc diagnosis , the confidence in the final diagnosis before mrc was preliminarily assessed based on the assumption that the sum of the probabilities of the chosen diagnosis and all other unstated diagnoses is 100% . 
if the diagnostic confidence for the selected diagnosis before mrc was 50% , then the inferred diagnostic confidence for the revised diagnosis was estimated to be 100% minus the stated probability of the first diagnostic choice . 
on the other hand , if the diagnostic confidence for the selected diagnosis before mrc was < 50% , the inferred diagnostic confidence percentage was estimated to be the same as the diagnostic confidence level given by the physicians . 
all statistical analyses were performed using commercially available software ( spss 16.0.0 for macintosh ; spss , chicago , il , usa )  . finally , mrc performance in the clinical setting was evaluated by calculating sensitivity , specificity , positive ( ppv ) and negative ( npv ) predictive values and overall accuracy after data cross - tabulation . 
direct cholangiographic procedures , such as erc and ptc , hepatic biopsy , and a follow - up period of at least 1 year were considered as standards of reference . results patient population twenty - one liver - transplanted patients ( 18 men , 3 women ; mean age 59 years ; range 4267 years ) underwent mrc in la percentuale di confidenza diagnostica dedotta stata stimata essere pari alla confidenza diagnostica formulata dai clinici . 
tutte le analisi statistiche sono state realizzate utilizzando un software disponibile in commercio ( spss 16.0.0 for macintosh ; spss , chicago , ill )  . infine , stata valutata la performance della crm calcolando la sensibilit , la specificit , i valori predittivi positivo e negativo e laccuratezza globale , dopo una tabulazione incrociata dei dati . 
sono stati considerati come standard di riferimento le procedure di colangiografia diretta , quali cre e ctp , la biopsia epatica ed un periodo di follow - up di almeno un anno . risultati pazienti sono stati considerati 21 pazienti trapiantati di fegato ( 18 uomini , 3 donne ; et media , 59 anni ; range 4267 anni ) che hanno effettuato una crm nel periodo specificato . 
non sono state clinicamente sospettate o riscontrate alla crm altre complicanze . determinazione della resa diagnostica e dellaccuratezza diagnostica sono state considerate positive 18 su 21 crm , con una resa diagnostica pari all85 , 71% ( intervallo di confidenza [ ic ] 95% 70 , 7100 , 0 )  . 
per quanto riguarda i due casi di crm falsi positivi ( pazienti 5 e 13 ) i reperti crm sono stati interpretati come compatibili con stenosi anastomotiche , non confermate alla successiva cre . 
reperti aggiuntivi della crm sono stati un kinking biliare in un paziente con stenosi extraepatica ( paziente 2 ) e due stenosi extraepatiche in pazienti con vanishing bile duct syndrome ( pazienti 4 e 12 )  . 
 nel corso della loro storia clinica , i pazienti privi di reperti biliari alla crm hanno effettuato , per un periodo di follow - up di almeno un anno , multipli esami di diagnostica per immagini tra cui crm ripetute , indagini ecografiche e di radiol med ( 2011 ) 116 : 12501266 1255 the specified period and were therefore considered . 
concerning the two false - positive mrc cases ( patients 5 and 13 ) , mrc findings were interpreted as consistent with anastomotic stricture , not confirmed at subsequent erc . 
additional mrc findings were bile - duct kinking in a patient with extrahepatic stricture ( patient 2 ) and two extrahepatic strictures in patients with vanishing bile - duct syndrome ( patients 4 and 12 )  . 
 patients with no biliary findings at mrc underwent multiple imaging examinations over a follow - up period of at least 1 year , including repeated mrcs , ultrasonography ( us ) and computed tomography ( ct ) scans . 
no biliary complications occurred in any of them . influence of mrc on the clinical decision making process the proportion of presumptive diagnoses modified in the light of mrc was 4 / 21 ( 19% ) patients . 
in three cases , physicians had a clinical suspicion of extrahepatic stricture ( patients 1 , 10 , and 11 ) but subsequently modified their leading diagnosis according to negative mrc . 
in one case ( patient 21 ) , vanishing bile - duct syndrome was clinically suspected , whereas mrc revealed an anastomotic stricture and regular intrahepatic bile ducts , as subsequently confirmed at therapeutic erc . 
 influenza della crm nel processo di clinical decision making la proporzione di diagnosi presuntive modificate alla luce della crm stata di 4 / 21 , corrispondente al 19% dei pazienti . 
in tre casi i clinici avevano sospettato una stenosi extraepatica ( pazienti 1 , 10 e 11 ) , ma hanno successivamente modificato la loro diagnosi in seguito allesito negativo della crm . 
in un caso ( paziente 21 ) a fronte di un sospetto clinico di vanishing bile duct syndrome la crm ha evidenziato una stenosi anastomotica , con dotti biliari intraepatici regolari , come successivamente confermato dalla cre terapeutica . 
dopo il test diagnostico , la confidenza media nella diagnosi crm stata pari al 98 , 7% , con un corrispondente guadagno medio in confidenza diagnostica del 78 , 7% . 
la confidenza media dei clinici nella diagnosi presuntiva nei restanti 17 / 21 casi con uguale diagnosi prima e dopo la crm stata pari a 80% e 98 , 8% rispettivamente , dando luogo cos ad un guadagno medio in confidenza del 18 , 8% ( statisticamente significativo , p = 0 , 0026 )  . 
in particolare la crm ha evitato lesecuzione di 2 cre pianificate in pazienti con esame negativo ( pazienti 10 e 11 ) , ha indotto lesecuzione di 4 cre e 1 ctp in 5 pazienti ( pazienti 2 , 5 , 13 e 14 con diagnosi crm di stenosi extra - epatica e paziente 12 con diagnosi crm di vanishing bile duct syndrome e stenosi extra - epatica ) ed ha modificato lapproccio terapeutico in due casi attraverso la conversione di una ctp in cre e di una cre in ctp ( pazienti 3 e 9 , rispettivamente )  . 
nei rimanenti 12 / 21 pazienti ( 57 , 1% ) ( 4 cre ; 4 ctp ; 4 follow - up ) non c stata modificazione del piano diagnostico - terapeutico prima e dopo la crm . 
 delle procedure colangiografiche invasive previste , 3 ( 2 ctp e 1 cre ) non state effettuate a causa delle condi1256 radiol med ( 2011 ) 116 : 12501266 radiol med ( 2011 ) 116 : 12501266 1257 1258 radiol med ( 2011 ) 116 : 12501266 fig . 
coronal t2 - weighted haste image ( a ) and t2 - weighted thin mip magnetic resonance cholangiography image ( b ) demonstrate a severe bile - duct stenosis at anastomotic site ( arrows )  . 
questi casi sono stati comunque inclusi nellultimo sottogruppo di pazienti descritto sulla base del miglior trattamento possibile cos come programmato dai clinici sulla base dei risultati crm . radiol med ( 2011 ) 116 : 12501266 1259 table 3 confidence rates in concordant and discordant presumptive and magnetic resonance cholangiography ( mrc ) diagnoses , with corresponding gain in confidence patient no . 
 confidence in the presumptive confidence in the mrc diagnosis ( % ) diagnosis ( % ) diagnoses before and after mrc gain in confidence ( % ) a again in confidence was calculated according to carrico et al . 
 [ 22 ] , as explained in the text tabella 3 percentuali di confidenza nelle diagnosi presuntive e colangiografia in risonanza magnetica ( crm ) , concordanti e discordanti , con il rispettivo guadagno in confidenza caso confidenza nella diagnosi presuntiva ( % ) confidenza nella diagnosi crm ( % ) diagnosi prima e dopo la crm guadagno in confidenza ( % ) a 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 discordant concordant concordant concordant concordant concordant concordant concordant concordant discordant discordant concordant concordant concordant concordant concordant concordant concordant concordant concordant discordant discordante concordante concordante concordante concordante concordante concordante concordante concordante discordante discordante concordante concordante concordante concordante concordante concordante concordante concordante concordante discordante 100 100 ail guadagno in confidenza stato calcolato in accordo con quanto descritto da carrico et al . 
 [ 22 ] , come spiegato nel testo 1260 radiol med ( 2011 ) 116 : 12501266 table 4 diagnostic - therapeutic plans before and after mrc , and corresponding standard of reference patient no . 
diagnostic - therapeutic plan before mrc diagnostic - therapeutic plan after mrc standard of reference mrc , magnetic resonance cholangiography ; erc , endoscopic retrograde cholangiography ; ptc , percutaneous transhepatic cholangiography aptc was planned but not performed due to contraindications to interventional procedures . 
la crm eseguita nel follow - up ha confermato i reperti precedenti . cla ctp stata programmata ma non stata eseguita per sanguinamento gastrico acuto e rapido peggioramento delle condizioni del paziente . 
la crm eseguita nel follow - up ha confermato i reperti precedenti . radiol med ( 2011 ) 116 : 12501266 1261 before and after mrc and the final standard of reference for each patient . 
in particular , mrc saved planned ercs in two patients with negative examination ( patients 10 and 11 ) , led to four erc and one ptc in five patients ( patients 2 , 5 , 13 and 14 with an mrc diagnosis of extrahepatic stricture , and patient 12 with an mrc diagnosis of vanishing bile - duct syndrome and extrahepatic stricture ) and modified the therapeutic approach in two cases by converting one ptc to erc and vice versa ( patients 3 and 9 , respectively )  . 
in the remaining 12 / 21 ( 57.1% ) patients ( four erc ; four ptc ; four follow - up ) , there was no modification of the diagnostictherapeutic plan before and after mrc . 
 discussion over the last few years , mrc has gained acceptance as a reliable tool for biliary imaging , providing noninvasive diagnosis and accurate preinterventional road maps for biliary complications after olt [ 14 ]  . 
as mrc is noninvasive , performs better than erc in some settings and reduces unnecessary ercs and ptcs in patients with low or intermediate risk of interventional procedures , it has been advocated as the new diagnostic gold standard for the biliary tract [ 7 ]  . 
thus , even though the role for mrc after olt has been poorly investigated and most studies lack a comparison with direct cholangiography [ 25 , 26 ] , mrc is more than promising in evaluating liver - transplanted patients and provides equivalent diagnostic performance compared with direct cholangiography , at least for strictures [ 13 ]  . nonetheless , the primary goal of this study was to assess the impact of mrc on clinical decisions rather than its diagnostic performance . 
such an impact on patients diagnostic and therapeutic management has been traditionally assessed , for imaging techniques , by the hierarchic model of efficacy proposed by thornbury and fryback [ 27 , 28 ]  . 
 according to the model , we investigated the efficacy of mrc in liver - transplanted patients with a combined analysis of level ii ( diagnostic accuracy ) , level iii ( diagnostic thinking efficacy ) and level iv ( therapeutic efficacy ) using a previously described methodology [ 21 , 22 ]  . 
as far as we know , very few studies evaluated levels of efficacy iii and discussione negli ultimi anni la crm diventata strumento affidabile per limaging delle vie biliari , fornendo , in particolare per quanto riguarda le complicanze biliari dopo tof , la possibilit di ottenere una diagnosi non invasiva ed unadeguata mappa pre - interventistica [ 14 ]  . 
essendo una procedura non invasiva , che in alcuni casi permette di ottenere risultati migliori della cre e che riduce lesecuzione di cre e ctp non necessarie in pazienti a rischio basso o indeterminato per procedure interventistiche , la crm stata proposta come nuovo standard diagnostico di riferimento per le vie biliari [ 7 ]  . 
quindi , anche se il ruolo della crm dopo tof stato poco investigato e nonostante nella maggior parte degli studi manchi un confronto con la colangiografia diretta [ 25 , 26 ] , la crm pi che promettente per la valutazione dei pazienti trapiantati di fegato e fornisce una performance diagnostica equivalente a quella della colangiografia diretta , almeno per quel che riguarda le stenosi [ 13 ]  . lobiettivo primario di questo studio stato valutare limpatto della crm sulle decisioni cliniche , piuttosto che misurare la sua performance diagnostica . 
utilizzando tale modello , abbiamo investigato lefficacia della crm nei pazienti trapiantati con unanalisi congiunta di livello ii ( accuratezza diagnostica ) , livello iii ( efficacia diagnostica presuntiva ) e livello iv ( efficacia terapeutica ) , mediante una metodologia descritta in precedenza [ 21 , 22 ]  . 
a nostra conoscenza , solo pochi studi hanno valutato i livelli iii e iv di efficacia per la crm [ 17 , 20 ] e nessuno nel peculiare contesto dei pazienti trapiantati di fegato . 
di conseguenza , a parte il concetto generale delle potenzialit diagnostiche , in gran parte non noto se la crm incida sulle decisioni cliniche che riguardano i pazienti trapiantati di fegato e sul piano diagnostico - terapeutico adottato qualora questo strumento diagnostico non fosse disponibile . preliminarmente , abbiamo valutato la resa diagnostica della crm , parametro che esprime la proporzione di esami positivi tra i pazienti con una particolare indicazione per lindagine stessa [ 21 ]  . 
come regola generale , si presume che i pazienti per i quali un test stato richiesto abbiano una maggiore probabilit di esame con esito positivo rispetto a quelli per cui non stato richiesto . 
pertanto , poich le complicanze biliari sono relativamente frequenti ( tasso di occorrenza del 5 , 8%30% ) e spesso coesistono con altre complicanze maggiori dopo tof [ 14 ] , i pazienti di questo studio , indirizzati alla crm , avevano elevata probabilit di 1262 radiol med ( 2011 ) 116 : 12501266 iv for mrc [ 17 , 20 ] , and there are none in the peculiar setting of liver - transplanted patients . 
consequently , apart from the general issue of diagnostic capability , it is largely unknown whether mrc affects clinical decisions on livertransplanted patients and modifies the diagnostictherapeutic plan that would have been adopted in the absence of this tool . 
 preliminarily , we investigated the diagnostic yield of mrc , which expresses the proportion of positive examinations among patients with a particular indication for the test [ 21 ]  . 
as a general rule , it is assumed that patients in whom a test is ordered are more likely to have a positive result than those in whom the test is not ordered . 
thus , since biliary complications are relatively frequent ( occurrence rate 5.830% ) and often coexist with other major complications after olt [ 14 ] , patients referred for mrc in this study were likely to show biliary disease . 
in fact , a diagnostic test must have a sufficiently high diagnostic yield to be considered useful in a certain clinical setting , as a majority of negative results would imply that patients do not benefit from the test ( because their management remains unchanged ) [ 21 ]  . 
on the other hand , the high diagnostic yield corresponded to a sensitivity of 100% , ppv of 88.9% , npv of 100% and overall accuracy of 92.3% ( 19 / 21 )  . 
apart from the general aforementioned considerations on mrc diagnostic capability , all patients underwent repeated imaging examinations over a reasonable follow - up time ( at least 1 year ) , showing no development of biliary complications . 
thus , the risk of missing mrc - induced false - negative cases is minimal and in any case lower than that of overtreating with invasive procedures ( such as erc or ptc ) in patients with a low likelihood of having biliary complications . 
on the other hand , such likelihood may be inferred by the confidence values in preand post - mrc clinical diagnosis . overall , the use of mrc led to a change in pretest diagmanifestare patologie biliari . 
infatti , un test diagnostico deve determinare una resa diagnostica sufficientemente alta per essere considerato utile in un dato contesto clinico , dal momento che una maggioranza di risultati negativi implicherebbe che i pazienti non traggono beneficio dal test ( perch la loro gestione non viene modificata ) [ 21 ]  . 
daltra parte , lelevata resa diagnostica stata accompagnata da una sensibilit del 100% , un valore predittivo positivo del 88 , 9% , un valore predittivo negativo del 100% e da unaccuratezza globale del 92 , 3% ( 19 / 21 )  . 
ciononostante , la specificit risultata bassa ( 60% ) , in relazione alla presenza di due casi falsi positivi indotti dalla crm , ovvero di due stenosi biliari lievi non confermate alla successiva cre . 
una possibile spiegazione legata al fatto che la crm tende a sovrastimare il grado e lestensione delle stenosi [ 31 ] e che stenosi lievi non fibrotiche possono essere state non visibili alla cre , perch distese dalliniezione del mezzo di contrasto [ 4 , 32 ]  . 
 discutibile se la biopsia epatica o il follow - up mediante imaging rappresentino un valido standard di riferimento in quei 9 pazienti su 21 nei quali la colangiografia diretta non stata effettuata . 
a parte le considerazioni generali sulle capacit diagnostiche della crm descritte sopra , tutti questi pazienti sono stati sottoposti a ripetuti esami strumentali per un periodo di follow - up ragionevole ( almeno un anno ) , senza che si palesassero complicanze biliari . 
il rischio di perdere casi falsi negativi indotti dalla crm dunque minimo , in ogni caso pi basso di quello di trattare eccessivamente mediante procedure invasive ( come cre o ctp ) pazienti con bassa probabilit di avere complicanze biliari . 
tale probabilit pu essere inoltre dedotta dai valori di confidenza nella diagnosi clinica pree post - crm . complessivamente , il ricorso alla crm ha portato ad un cambiamento della diagnosi pre - test in un basso numero di casi ( 4 / 21 ) , corrispondente al 19% dei pazienti . 
tuttavia , la diagnosi clinica delle complicanze biliari difficile , in quanto i sintomi e le alterazioni biochimiche sono spesso indistinguibili da quelle di diverse altre condizioni , rappresentate in buona parte dal rigetto del trapianto [ 25 , 33 ]  . 
nevertheless , clinical diagnosis of biliary complications is difficult , as symptoms and biochemical features are often indistinguishable from those of several different conditions , mainly represented by graft rejection [ 25 , 33 ]  . 
 consequently , in clinical practice , imaging is required to assess biliary findings requiring interventional or surgical procedures and to provide a preoperative road map [ 4 , 34 ]  . 
 however , our study was performed by expert physicians of a tertiary referral centre , who showed except for few cases a high level of confidence in presumptive diagnosis ( mean rating of 80% )  . 
thus , the crucial issue in the clinical decision - making process was to determine whether mrc further increased physicians confidence in a clinically difficult diagnosis rather than to confirm that clinicians were expert in their field . 
 in the remaining four cases , corresponding to those in which mrc led to a final diagnosis discordant from the pre - mrc diagnosis , clinicians showed confidence ratings ranging from 60100% . 
cases included three suspected extrahepatic bile - duct strictures ( with lithiasis in one case ) assessed as negative mrcs , and one suspected vanishing bile - duct syndrome that was finally assessed as an anastomotic stricture . 
 thus , clinicians appeared to consider mrc as a standard of reference , as the examination increased diagnostic confidence irrespective of concordance with pretest diagnosis [ 7 ]  . analysis of therapeutic efficacy showed that clinicians modified the therapeutic plan that would have been applied in the absence of mrc information in 42.8% ( nine ) cases . 
 first , mrc improved therapeutic planning by modifying the interventional approach in two cases of ptc converted in erc and vice versa , which were effective in determining symptom relief . 
daltra parte , il nostro studio stato effettuato da clinici esperti di un centro di riferimento terziario , che hanno dimostrato ad eccezione di pochi casi elevata confidenza nelle diagnosi presuntive ( valore medio dell80% )  . 
il punto cruciale nel processo clinico decisionale stato quindi determinare se la crm avesse aumentato la confidenza dei clinici nelle diagnosi clinicamente difficili , piuttosto che confermare la loro esperienza nel proprio campo . 
in 17 casi ( 81% ) , le diagnosi pree post - crm sono state similari , portando ad un guadagno in confidenza diagnostica statisticamente significativo e pari al 18 , 8% . 
 nei restanti 4 / 21 casi , corrispondenti a quelli nei quali la crm ha indotto una diagnosi finale discordante rispetto a quella pre - esame , i clinici hanno dimostrato una confidenza variabile dal 60% al 100% . 
i casi comprendevano 3 sospette stenosi extraepatiche ( con associata litiasi in un caso ) valutate come negative alla crm ed una sospetta vanishing bile duct syndrome successivamente inquadrata come stenosi anastomotica . 
i clinici sembrano dunque considerare la crm come uno standard di riferimento , dal momento che lesame ha aumentato la confidenza diagnostica indipendentemente dalla concordanza con la diagnosi pre - test [ 7 ]  . lanalisi di efficacia terapeutica ha dimostrato che i clinici hanno modificato il piano terapeutico che sarebbe stato applicato in assenza delle informazioni fornite dalla crm nel 42 , 8% dei casi ( 9 / 21 )  . 
in primo luogo , la crm ha migliorato il piano terapeutico modificando lapproccio interventistico in 2 casi di conversione da ctp a cre e viceversa , indagini che sono state efficaci nel determinare un miglioramento dei sintomi . 
 [ 35 ] , non abbiamo usato la riduzione del carico di lavoro della cre come outcome per valutare lutilit della crm , dal momento che i pazienti possono trarre beneficio non solo dallevitare procedure invasive ( come successo in due pazienti con esami negativi )  . 
in our opinion , mrc was useful in those in whom imaging follow - up was confirmed , as alternative diagnostic techniques such as us or ct provide less accurate information on the biliary tract , as previously demonstrated [ 3639 ]  . 
in these cases , although mrc cannot be considered useful according to the purposes of the study , it can be argued that indirectly it would avoid invasive monitoring of the treatment . our study has several limitations . 
nevertheless , our results reflect clinical practice because anastomotic and / or nonanastomotic strictures represent the most frequent biliary complications , both early and late , after transplantation [ 14 ]  . 
nevertheless , us has been repeatedly shown to be inaccurate in assessing biliary complications , particularly the site of stricture and biliary dilation upstream , with sensitivity < 50% [ 12 , 36 , 40 ]  . 
 in conclusion , in the clinical setting of liver - transplanted patients , mrc shows a high diagnostic yield due to the prevalence of disease and to the well - established diagnostic accuracy . 
mrc significantly increases diagnostic confidence of referring physicians irrespective of the concordance between the preand posttest diagnosis , although with a limited risk of overtreatment of false - positives cases . 
 moreover , mrc leads to a change in patient management that is effective in at least one third of patients . nostro parere , la crm stata utile nei casi in cui tale indagine ha confermato la scelta pre - test di follow - up mediante imaging , dal momento che le tecniche diagnostiche alternative alla crm ( quali ecografia e tc ) forniscono informazioni sulle vie biliari meno accurate , come precedentemente dimostrato [ 3639 ]  . 
inoltre , nei pazienti nei quali la scelta della colangiografia diretta stata confermata , la crm ha fornito un quadro basale da confrontarsi con le successive rivalutazioni rm post - interventistiche . 
anche se , in questi casi , la crm non pu essere considerata utile secondo gli scopi dello studio , si pu desumere che , indirettamente , essa possa evitare un monitoraggio invasivo post - trattamento . il nostro studio presenta diversi limiti . 
tuttavia i nostri risultati riflettono la pratica clinica , in quanto le stenosi anastomotiche e / o nonanastomotiche sono le complicanze biliari post - trapianto ( precoci e tardive ) pi frequenti [ 14 ]  . 
un limite ancora pi importante legato al fatto che la confidenza pre - crm dei clinici di riferimento possa essere stata falsata dai risultati di precedenti indagini di imaging , ad esempio dallecografia . 
tuttavia , lecografia si pi volte dimostrata poco adeguata nella valutazione delle complicanze biliari , in particolare nella rilevazione della sede della stenosi e della dilatazione a monte , con una sensibilit inferiore al 50% [ 12 , 36 , 40 ]  . 
pertanto , limpatto dellecografia sulla confidenza diagnostica dovrebbe essere ragionevolmente basso e in ogni caso non dovrebbe pregiudicare la capacit della crm di influenzare il processo clinico di decision making . 
 inoltre , dal momento che lecografia una tecnica di primo livello a basso costo e largamente utilizzata nella valutazione dei pazienti dopo tof , sarebbe non etico non farne ricorso . 
complessivamente , ulteriori studi dovrebbero indagare livelli pi elevati di efficacia diagnostica , mediante valutazioni degli effetti sulloutcome dei pazienti o analisi costo - efficacia [ 27 , 28 ]  . in conclusione , nel contesto clinico dei pazienti trapiantati di fegato la crm dimostra unelevata resa diagnostica , grazie alla prevalenza della patologia biliare ed alla ben codificata accuratezza diagnostica . 
la crm incrementa significativamente la confidenza diagnostica dei clinici di riferimento indipendentemente dalla concordanza tra le diagnosi pree post - test , al costo di un limitato rischio di overtreatment per i casi falsi positivi . 
grassi4 1department of radiology , university of foggia , viale luigi pinto 1 , 71100 foggia , italy 2department of radiology , scientific institute hospital css , viale cappuccini 1 , 71013 san giovanni rotondo , foggia , italy 3department of radiology , university of ancona , hospital torrette , via conca 60 , 60100 ancona , italy 4department of rheumatology , universit politecnica delle marche , via dei colli 52 , 60035 jesi , ancona , italy correspondence to : g . 
forty - three consecutive patients with sacroiliitis as the predominant symptom of a rheumatic disorder were retrospectively studied . radiography , computed tomography ( ct ) and magnetic resonance ( mr ) imaging were performed . 
the following imaging findings were evaluated : bone marrow oedema , intra - articular effusion , synovial reaction , joint - space widening , bone sclerosis or hyperostosis , subchondral erosions and , in final stages , joint - space narrowing and ankylosis . 
radiography demonstrated bone sclerosis in 10 patients ( 23% ) , subchondral erosions in 15 ( 34% ) , jointspace widening in 8 ( 18% ) , joint - space narrowing in 17 ( 39% ) and ankylosis in 3 ( 6% )  . 
ct examination showed sclerosis of the sacroiliac joint in 17 patients ( 41% ) , subchondral erosions in 21 ( 53% ) , joint - space widening in 22 ( 53% ) , joint - space narrowing in 18 ( 43% ) and ankylosis in 7 ( 17% )  . 
at mr , we found bone marrow oedema in 25 patients ( 92% ) , intra - articular effusion in 26 ( 96% ) , synovial reaction in 21 ( 77% ) and joint - space widening in 5 ( 18% )  . 
la radiografia convenzionale ha dimostrato la sclerosi ossea in 10 pazienti ( 23% ) , erosioni subcondrali in 15 pazienti ( 34% ) , ampliamento dello spazio articolare in 8 pazienti ( 18% ) e restringimento in 17 pazienti ( 39% ) , anchilosi in 3 pazienti ( 6% )  . 
allesame tc sono stati osservati sclerosi dellarticolazione sacroiliaca in 17 pazienti ( 41% ) , erosioni subcondrali in 21 pazienti ( 53% ) , ampliamento dello spazio articolare in 22 pazienti ( 53% ) , restringimento dello spazio articolare in 18 pazienti ( 43% ) e anchilosi in 7 pazienti ( 17% )  . 
alla rm abbiamo riscontrato edema del midollo osseo in 25 pazienti ( 92% ) , radiol med ( 2011 ) 116 : 292301 problem of difficult diagnostic evaluation due to the complexity of the anatomical region and the variability of radiographic findings . 
the integrated use of conventional radiography , ct and mr imaging is suggested to avoid misdiagnosis . keywords spondyloarthropathies sacroiliac joint sacroiliitis conventional radiography computed tomography magnetic resonance imaging versamento intrarticolare in 26 pazienti ( 96% ) , reazione sinoviale in 21 pazienti ( 77% ) e lampliamento dello spazio articolare in 5 pazienti ( 18% )  . 
lo studio radiologico delle articolazioni sacro - iliache in pazienti affetti da diverse malattie reumatiche rappresenta un problema di difficile valutazione diagnostica sia per la complessit della regione anatomica sia per la variabilit dei risultati radiografici . 
 parole chiave spondilo - artropatie articolazioni sacroiliache sacro - ileite radiologia convenzionale tomografia computerizzata risonanza magnetica introduction introduzione the sacroiliac joints ( sij ) represent an amphidiarthrosis with features between amphiarthrosis and diarthrosis . 
involvement of the sij is a major and characteristic feature of the early spondyloarthropathies ( spa ) , and the most common symptom in adults is inflammatory low back pain due to sacroiliitis [ 2 ]  . included in the spectrum of spa are ankylosing spondylitis ( as ) , psoriatic arthritis ( psa ) , enteropathic spa ( enspa ) , reactive arthritis and undifferentiated spondyloarthropathy ( uspa )  . 
spa is classified according to the european spondyloarthropathy study group criteria ( essg ) [ 5 ] and amor criteria [ 6 ] and as by the modified new york criteria [ 7 ]  . in the case of early as in particular , criteria sets fall short because the classification of as depends on the presence of radiological sacroiliitis , which often appears late in the course of the disease [ 8 , 9 ]  . 
the international assessment in ankylosing spondylitis ( asas ) working group has recognised imaging as an important domain in spa and recommends further development for use in clinical trials [ 11 ]  . magnetic resonance ( mr ) imaging is becoming an important tool for assessing inflammation and structural damage in as . 
the advantages of mr imaging compared with computed tomography ( ct ) and radiography are that it also provides early visualisation of active inflammatory changes , making it possible to diagnose sacroiliitis before le articolazioni sacro - iliache ( asi ) rappresentano un anfidiartrosi con caratteristiche tra lanfiartrosi e la diartrosi . 
il coinvolgimento delle asi una caratteristica importante e tipica delle spondilo - artropatie in fase iniziale , e il sintomo pi comune negli adulti il dolore lombare dovuto alla sacro - ileite [ 2 ]  . sono incluse nello spettro delle spondilo - artropatie la spondilite anchilosante , lartrite psoriasica , le spondilo - artropatie enteropatiche , lartrite reattiva e le spondilo artropatie indifferenziate . 
gli attuali criteri di classificazione delle spondilo - artropatie sono quelli definiti dalleuropean spondyloarthropathy study group ( essg ) [ 5 ] , nonch i criteri amor [ 6 ] e quelli per la classificazione della spondilite anchilosante , i criteri di new york modificati [ 7 ]  . comunque , soprattutto nel caso di una spondilite anchilosante in fase iniziale , i criteri sopra menzionati sono meno presenti , poich la classificazione della spondilite anchilosante dipende dalla presenza di segni radiologici di sacroileite , che appare spesso tardivamente nel corso della malattia [ 8 , 9 ]  . 
linternational assessment in ankylosing spondylitis ( asas ) working group ha riconosciuto limaging come un importante aspetto nella diagnosi radiologica delle spondilo - artropatie e ne raccomanda un ulteriore sviluppo e lutilizzo nelle sperimentazioni cliniche [ 11 ]  . 
la risonanza magnetica ( rm ) sta diventando uno strumento sempre pi importante nella valutazione della flogosi e del 294 radiol med ( 2011 ) 116 : 292301 definite joint destruction is detectable on ct or radiography [ 1215 ]  . the aim of this study was to retrospectively evaluate the role of the different imaging techniques in the study of the sij in patients with spa and other rheumatic conditions and to assess potential pitfalls in the radiological diagnosis . materials and methods patients this retrospective study included 43 consecutive patients ( 19 men and 24 women ; age 2181 years ; mean age 51 years ) who were either inpatients and outpatients of our hospital with clinically confirmed spa and other rheumatic conditions involving the sij and a history of inflammatory back pain ( ibp ) and were evaluated between september 2007 and january 2009 . 
 characteristics , radiological imaging techniques conventional radiography was obtained with a 20 caudal tube tilt and included an anteroposterior projection of the sacru the anatomical sites studied were the entire sacrum and the sij . 
grade 2 , definite early changes including pseudowidening of the joint space and / or focal erosions , sclerosis of the bone usually on both sides of the joint space and frequent blurring of the joint margins ; 2a unilateral changes and 2b bilateral changes . 
grade 3 , severe destructive changes , erosions and pseudowidening of joint space more marked than in grade 2 ; arthritic changes are always bilateral , and there may be small bony bridges . grade 4 , regressive changes with bilateral arthritic changes , narrowing of the joint space with bony bridges in one or both sij and slight sclerosis of the joints . 
grade 5 , terminal changes , with pronounced signs of bony ankylosis in both sij and slight sclerosis of the joints . ct study ( tct 900s toshiba , japan ) was performed under basal conditions only . 
i vantaggi della rm rispetto alla tomografia computerizzata ( tc ) e alla radiografia convenzionale consistono nella capacit della metodica di rilevare precocemente alterazioni infiammatorie attive , rendendo possibile la diagnosi di sacro - ileite prima della diagnosi tardiva caratterizzata da alterazioni articolari rilevabili alla tc o alla radiografia [ 1215 ]  . lobiettivo di questo studio stato quello di valutare retrospettivamente il ruolo delle diverse tecniche di imaging nello studio delle asi , nei pazienti con spondilo - artropatie ed altre patologie reumatiche e per valutare potenziali errori nella diagnosi radiologica . materiali e metodi pazienti in questo studio retrospettivo , sono stati valutati , da settembre 2007 a gennaio 2009 , 43 pazienti consecutivi ( 19 maschi e 24 femmine ) , dai 21 agli 81 anni ( et media 51 anni ) , comprendenti pazienti sia ambulatoriali che in regime di ricovero del nostro ospedale , con spondilo - artropatie ed altre patologie reumatiche coinvolgenti le asi , confermate clinicamente dai colleghi reumatologi e con una storia clinica di dolore lombare irradiato al sacro . 
nella nostra popolazione di studio , 19 pazienti erano affetti da spondilite anchilosante , 11 pazienti da artrite psoriasica , 7 pazienti da spondilo artropatie enteropatiche , 4 pazienti da artrite reattiva e 2 pazienti da spondilo artropatie indifferenziate . tecniche di imaging il radiogramma tradizionale stato ottenuto con uninclinazione caudale del tubo di 20 e comprende la proiezione antero - posteriore del sacro . 
le regioni anatomiche studiate sono state il sacro e le articolazioni sacro - iliache ; le alterazioni morfologiche analizzati sono state : lampliamento dello spazio articolare , la sclerosi ossea o iperostosi , le erosioni subcondrali e , nelle fasi finali , il restringimento degli spazi articolari e lanchilosi . 
 mr images were obtained using a pelvic array coil in the semiaxial and semicoronal planes along the main axis of the sij and with patient lying in the supine decubitus . 
the sequences performed on mr ( signa 1.5 - t unit , ge medical spazio articolare , margini articolari spesso sfumati ; 2a : le alterazioni sopra descritte sono unilaterali ; 2b : le alterazioni descritte sono bilaterali ; grado 3 , severe alterazioni distruttive , erosioni e pseudo - ampliamento dello spazio articolare pi marcato rispetto al grado 2 ; le alterazioni di tipo artritico sono sempre bilaterali ; piccoli ponti ossei possono essere presenti ; grado 4 , alterazioni regressive ; alterazioni di tipo artritico bilaterali , restringimento dello spazio articolare con ponti ossei in una o entrambe le articolazioni , modesta sclerosi sub condrale ; grado 5 , 296 radiol med ( 2011 ) 116 : 292301 systems , milwaukee , wi , usa ) were : semicoronal t1weighted spin echo ( se ) with fat saturation ( tr / te 500 / 20 ; thickness 3 mm ; intersection gap 1 mm ; nex 2 ; matrix 256224 ; fov 18 cm ; time 3 min 52 s ; slices 68 ) , semiaxial t2 - weighted fast spin echo ( fse ) and semicoronal short - tau inversion recovery ( stir ) - fse ( tr / te / etl 3 , 000 / 90 / 8 ; matrix 256256 ; ti 150 ms ; time 3 min24 s ; slices 68 fse and 6 stir - fse ) under basal conditions , and semicoronal and semiaxial t1 - w with fat saturation ( tr / te 500 / 20 ; matrix 256224 ; time 3 min 52 s ; slices 68 ) after intravenous administration of gadolinium diethylenetriamine pentaacetic acid ( gd - dtpa ) ( magnevist , bayer , berlin , germany )  . 
 all imaging studies were independently reviewed by two musculoskeletal radiologists ( gg and ac , the first with more than 20 years of experience in the field and the second with less experience ) who were blinded to the results . 
from a pathological point of view , articular involvement consists of an inflammatory process with proliferation of the serous lining cells , exudative effusion and synovial pannus , which erodes the articular cartilage and penetrates trabecular bone with geode formation . 
in early as and uspa , sacroiliitis table 2 radiological features evaluated with the different imaging techniques and number of patients , expressed as absolute number and percentage , in whom these findings were observed hyperostosis bone sclerosis joint - space widening joint - space narrowing subchondral erosions ankylosis synovial reaction intra - articular effusion bone marrow oedema x - ray 5 ( 11% ) 10 ( 23% ) 8 ( 18% ) 17 ( 39% ) 15 ( 34% ) 3 ( 6% ) 5 ( 18% ) 15 ( 36% ) no 17 ( 41% ) no 22 ( 53% ) 18 ( 43% ) no 22 ( 53% ) no 7 ( 17% ) 21 ( 77% ) 26 ( 96% ) 25 ( 92% ) ct , computed tomography ; mri , magnetic resonance imaging tabella 2 aspetti radiologici valutati con le diverse tecniche di imaging e numero di pazienti , espressi in valore assoluto ed in percentuale , nei quali sono state riscontrate le alterazioni iperostosi sclerosi ossea ampliamento spazio articolare riduzione spazio articolare erosioni subcondrali anchilosi reazione sinoviale versamento intrarticolare edema del midollo osseo 5 ( 11% ) 10 ( 23% ) 8 ( 18% ) 15 ( 36% ) no 17 ( 41% ) no 22 ( 53% ) 5 ( 18% ) 17 ( 39% ) 18 ( 43% ) no 15 ( 34% ) 3 ( 6% ) 22 ( 53% ) no 7 ( 17% ) 21 ( 77% ) 26 ( 96% ) 25 ( 92% ) rx , radiografia ; tc , tomografia computerizzata ; rm , risonanza magnetica alterazioni irreversibili ; segni evidenti di anchilosi ossea in entrambe le articolazioni , lieve sclerosi delle articolazioni . 
le scansioni di serie sono state ottenute con il paziente in posizione supina con una inclinazione craniale del gantry di 2530 per ottenere immagini semicoronali attraverso entrambe le parti cartilaginee e legamentose delle asi e con uno spessore di fetta di 2 mm ; scansioni contigue sono state ottenute a intervalli di 2 millimetri , con tecnica ad alta risoluzione . 
 le immagini rm sono state ottenute utilizzando una bobina pelvica , lungo piani di scansione semiassiali e semicoronali , lungo lasse principale delle articolazioni sacro - iliache e con il paziente in decubito supino . 
alla rm ( signa 1 , 5 t , ge medical systems , milwaukee , wis ) , le sequenze eseguite sono state : semicoronale t1 - pesata con saturazione del grasso ( spin echo [ se ] t1 - w ; tempo di ripetizione [ tr ] / tempo di eco [ te ] 500 / 20 ; spessore 3 mm ; intersection gap 1 mm ; nex 2 ; matrice 256224 , campo di vista [ fov ] 18 cm ; tempo 3 min e 52 s ; 68 slices ) , semiassiale t2 - pesata ( fast spin echo [ fse ] t2 - w ) e semicoronale short - tau inversion recovery ( stir ) radiol med ( 2011 ) 116 : 292301 fse ( tr / te / echo train length [ etl ] 3000 / 90 / 8 ; matrice 256256 ; ti 150 m ; tempo 3 min e 24 s ; 68 slices fse e 6 stir - fse ) in condizioni basali , e semicoronale e semiassiale t1 - pesata con saturazione del grasso ( tr / te500 / 20 ; matrice 256224 ; tempo 3 min e 52 s ; 68 slices ) dopo somministrazione di gadobenato dimeglumina ( gd - dtpa ) ev ( magnevist , bayer , berlino , germania )  . 
 tutte le indagini radiologiche sono state soggette a revisione indipendente da due radiologi muscolo - scheletrici ( gg e ac , il primo con pi di 20 anni di esperienza nel settore , il secondo con meno esperienza ) , che non erano a conoscenza dei risultati . 
lindagine tc ha dimostrato meglio dello studio radiologico tradizionale le lesioni ossee reattive a livello delle articolazioni sacro - iliache , perch non influenzato da problemi proiettivi tipici dellesame radiologico . 
da un punto di vista anatomo - patologico , il coinvolgimento articolare consiste in un processo infiammatorio , con proliferazione delle cellule di rivestimento sieroso , effusione essudativa e nella formazione del panno sinoviale che erode la cartilagine articolare e penetra nell osso trabecolare con la formazione di geodi . 
2 immagine tc semicoronale che dimostra erosioni subcondrali bilaterali e sclerosi , con riduzione dello spazio articolare . is also the most common early clinical finding and the presumed first manifestation of the disease . 
especially in the case of early spa , criteria sets fall short , so a long delay may exist between the start of symptoms and establishing a diagnosis [ 16 , 17 ]  . radiography has a high specificity but a low sensitivity , which can delay the diagnosis for several years . 
5 immagine rm semicoronale t1 - pesata che mostra edema del midollo osseo a livello del sacro . are related to the overlap of the ilium with the sacrum on standard anteroposterior projections , because the s - shaped joint structures course obliquely from a lateral to a medial position . 
4 immagine rm semicoronale t1 - pesata che mostra edema ed iperemia dellosso sub - condrale , principalmente sul versante iliaco delle articolazioni . allanchilosi , inizialmente fibrosa e successivamente ossea [ 13 ]  . 
specialmente nel caso di spondilite anchilosante in fase iniziale , i criteri di classificazione comunemente utilizzati sono meno presenti , cos pu esserci un ritardo tra linizio dei sintomi e la diagnosi [ 16 , 17 ]  . 
i limiti di questa tecnica sono legati alla sovrapposizione dellileo con losso sacro sulla proiezione antero - posteriore standard , perch le strutture articolari , a forma di s , hanno decorso obliquo in senso latero - mediale . nonostante i suoi limiti noti , lo studio radiologico sempre lo strumento iniziale per la valutazione delle articolazioni sacro - iliache [ 18 ]  . 
 la tc e la rm , come dimostrato nel nostro studio , sono superiori al radiogramma per lindividuazione dei segni di base , quali erosioni , sclerosi , distruzione della cartilagine ed edema del midollo osseo e quindi per la diagnosi di sacroileite . 
in particolare , per lindividuazione precoce di alterazioni ossee , come erosioni e anchilosi , la tc si dimostrata superiore alla rm ; daltra parte , la rm permette una valutazione della cartilagine e delledema del midollo osseo , non rilevati alla tc [ 1928 ]  . 
non c un metodo stabilito per la valutazione dei reperti patologici delle articolazioni sacro - iliache alla rm , anche se la necessit di questo evidente come riportato da puhakka et al . radiol med ( 2011 ) 116 : 292301 fig . 
b immagine rm semicoronale con soppressione del grasso t1 - pesata dopo somministrazione di gadolinio che mostra aree di ipervascolarizzazione , segno di infiammazione attiva . imaging ; on the other hand , mr imaging is better for depicting the cartilage and bone marrow oedema , which is not detected by ct [ 1928 ]  . 
 [ 20 ] introduced a method mainly based on signal changes on t1 , t2 , and phase - contrast sequences : type i lesions were characterised by low signal intensity on t1 - weighted images and a high signal intensity on both phase - contrast and t2weighted images . 
 [ 12 ] described a grading system for disease activity based on quantitative evaluation of contrast enhancement at dynamic mr imaging and used the new york criteria to evaluate joint involvement [ 7 ]  . 
 [ 20 ] hanno introdotto un metodo basato principalmente sui cambiamenti del segnale in t1 , t2 e nelle sequenze in contrasto di fase : le lesioni di tipo i sono caratterizzate da una bassa intensit di segnale nelle immagini t1 pesate e da una elevata intensit di segnale sia nelle sequenze in contrasto di fase sia nelle immagini pesate in t2 . 
usando questo sistema di punteggio , si potuto dimostrare che la risonanza magnetica in grado di rilevare le alterazioni patologiche delle articolazioni sacroiliache nei pazienti con scarse o nessuna evidenza clinica di malattia [ 17 ]  . 
nella nostra esperienza , la tc e la rm possono aiutare nella diagnosi precoce e nella diagnosi differenziale da altre condizioni morbose quali 300 radiol med ( 2011 ) 116 : 292301 clinical findings [ 17 ]  . 
in our experience , ct and mr imaging may help in the early diagnosis and differentiation from other conditions such as bone tumours or nerve root pathologies , which can cause sacroiliitis [ 29 ]  . this study has some limitations . 
finally , inflammatory back pain , the most important clinical sign we considered , is not always indicative of sij involvement . despite these limitations , this study confirmed that the diagnosis of sacroiliitis in the course of rheumatic diseases remains a challenge and that integration of the various imaging techniques is needed for correct clinical and radiological classification . tumori ossei o le patologie delle radici nervose che si manifestano clinicamente con una sacro ileite [ 29 ]  . il presente lavoro mostra alcuni limiti . 
tra questi dobbiamo considerare la eterogeneit della popolazione di studio in relazione allet , al sesso , alla durata dei sintomi clinici e alla malattia da cui i pazienti erano affetti . 
pur con queste limitazioni , il presente studio ha confermato che la diagnosi di sacro ileite in corso di patologie reumatiche rappresenta un problema di difficile valutazione diagnostica e che lintegrazione delle diverse tecniche di imaging fondamentale per un corretto inquadramento clinico - radiologico . 
rossi , universit di verona , verona , italy 3radiologia , ospedale misericordia , grosseto , italy 4cardiologia , 5radiologia clinica , ospedale c foncello treviso , treviso , italy correspondence to : a . 
twenty - two consecutive patients with a known diagnosis of hcm were prospectively evaluated , with echocardiography and cardiac mr imaging performed within 2 weeks of each other ( mean interval 7 days , range 214 days )  . 
the following parameters were calculated for both techniques : myocardial mass , wall thickness , end - diastolic volume ( edv ) , end - systolic volume ( esv ) , ejection fraction ( ef ) , systolic anterior motion ( sam ) of the mitral valve and degree of myocardial fibrosis ( based on the ultrasonic reflectivity at echocardiography and degree of late enhancement at cardiac mr imaging )  . 
ventidue pazienti consecutivi con diagnosi nota di cmi sono stati prospetticamente valutati mediante eco - cardiografia e cardio - rm eseguite entro 2 settimane ( scarto medio 7 giorni , range 214 giorni )  . 
per entrambe le tecniche sono stati calcolati massa cardiaca , spessori di parete , volume telediastolico ( vtd ) e telesistolico ( vts ) , frazione di eiezione ( fe ) , spostamento anteriore del lembo mitralico ( sam ) , grado di fibrosi parietale ( basato sul valore di eco - rinfrangenza alleco - cardiografia e sullentit di impregnazione tardiva alla cardio - rm )  . 
la diagnosi di cmi stata confermata in tutti i pazienti con entrambe le metodiche , con concordanza 198 radiol med ( 2011 ) 116 : 197210 terms of the site of disease . 
cardiac mr imaging correlates well with echocardiography in defining the morphological and functional parameters useful for the imaging diagnosis of hcm and therefore , in selected cases ( poor acoustic window , doubtful echocardiography findings ) , it may be a valid alternative to echocardiography . assoluta per quanto riguarda la sede . 
la cardio - rm correla bene con lecocardiografia nella definizione dei parametri morfologici e funzionali utili per la diagnosi strumentale di cmi e pu pertanto rappresentare , in casi selezionati ( scarsa finestra acustica , rilievi eco - cardiografici dubbi ) , una valida alternativa alleco - cardiografia . keywords hypertrophic cardiomyopathy echocardiography cardiac magnetic resonance myocardial fibrosis parole chiave cardiomiopatia ipertrofica ecocardiografia risonanza magnetica cardiaca fibrosi parietale introduction introduzione hypertrophic cardiomyopathy ( hcm ) is an autosomal dominant hereditary disease with variable penetrance and is characterised by left ( lv ) and / or right ( rv ) ventricular hypertrophy in the absence of demonstrable organic causes , both at the cardiac and systemic levels [ 1 , 2 ]  . 
the disease is characterised by possibly fatal cardiac arrhythmias and is an important cause of sudden death in individuals younger than 35 years of age ( 3%6% of cases per year ) [ 3 ]  . 
disease prevalence is around 0.1%0.2% , but these figures are based on screening by physical examination and electrocardiogram , which may show no alterations in patients with hcm [ 4 ]  . echocardiography , which is able to quantify myocardial thickening and evaluate lv motion , is the first - choice imaging technique for diagnosing hcm [ 5 , 6 ]  . 
however , the intrinsic limitations of the technique ( operator dependence and adequate acoustic window ) limit its ability to confirm diagnosis and correctly assess the disease [ 68 ]  . various studies have demonstrated the accuracy of cardiac magnetic resonance ( mr ) imaging in evaluating ventricular disease , thanks to its ability to provide both morphological and functional data , as well as information on myocardial perfusion [ 911 ]  . 
in addition , cardiac mr imaging is useful in diagnosing apical hcm , which , due to the poor acoustic window , is particularly difficult to evaluate with echocardiography [ 7 ]  . 
 the aim of this study , therefore , was to prospectively la cardiomiopatia ipertrofica ( cmi ) una malattia ereditaria autosomica dominante a penetranza variabile che consiste nella presenza di ipertrofia ventricolare sinistra e / o destra in assenza di cause organiche dimostrabili , sia a livello cardiaco che sistemico [ 1 , 2 ]  . 
tale patologia predispone ad aritmie cardiache fatali ed una importante causa di morte improvvisa in et inferiore ai 35 anni ( 3%6% di casi allanno ) [ 3 ]  . la prevalenza della malattia di circa 0 , 1%0 , 2 % , ma questi dati derivano da screening svolti mediante esame obiettivo ed elettrocardiogramma , che possono risultare non alterati in pazienti con cmi [ 4 ]  . leco - cardiografia rappresenta la metodica di prima linea per la diagnosi strumentale di cmi , consentendo di quantificare lispessimento miocardico e valutare la cinetica del ventricolo sinistro ( vsn ) [ 5 , 6 ]  . 
tuttavia , i limiti intrinseci della metodica ( dipendenza dalloperatore e dalla finestra acustica del paziente ) , rendono leco - cardiografia talora insufficiente per una diagnosi di certezza e per un corretto bilancio di malattia [ 68 ]  . 
diversi studi hanno dimostrato laccuratezza della risonanza magnetica cardiaca ( cardio - rm ) nella valutazione delle patologie ventricolari , grazie alla sua capacit di fornire informazioni di tipo morfologico , funzionale , nonch sulla perfusione miocardica [ 911 ]  . 
inoltre la cardio - rm utile nella diagnosi di cmi apicale , forma che per la scarsa finestra acustica spesso particolarmente difficile da valutare con eco - cardiografia [ 7 ]  . 
tuttavia , sono pochi gli studi in letteratura che descrivono il ruolo ed i criteri diagnostici della cardio - rm nella valutazione della cmi [ 10 , 12 , 13 ]  . 
 radiol med ( 2011 ) 116 : 197210 compare morphological and functional findings obtained with echocardiography and cardiac mr imaging to determine which ones are useful in evaluating hcm , with special attention to evaluating associated myocardial fibrosis . 
 pertanto , lobiettivo di questo studio confrontare in maniera prospettica i rilievi morfologici e funzionali ottenuti mediante eco - cardiografia e cardio - rm utili nella valutazione della cmi , con particolare attenzione alla valutazione della fibrosi miocardica associata . 
 materials and methods materiali e metodi patients and inclusion criteria pazienti e criteri di inclusione a review of the cardiology database performed between december 2005 and february 2009 identified 105 patients with hcm who were eligible for inclusion in our study . only patients with a known diagnosis of primary hcm , obtained according to the world health organization criteria ( clinical and m - mode echocardiography demonstrating increased wall thickness in end diastole > 15 mm at the level of the septum or posterior wall ) , in the absence of other demonstrable causes of ventricular hypertrophy [ 14 ] , were included in the study and examined prospectively with echocardiography and cardiac mr imaging within 2 weeks of each other ( mean interval 7 days , range 214 days )  . 
patients affected by possible causes of secondary hcm ( aortic stenosis , amyloidosis or arterial hypertension ; n = 44 ) and those without a comparative study with both techniques ( n = 39 ) were excluded . 
in agreement with the ethics committee , all patients enrolled gave written informed consent before the diagnostic examinations were performed . echocardiography protocol the echocardiography examination was performed with an acuson sequoia 512 device ( mountain view , ca , usa ) with a 3 - v , 2 - c transducer and software for calculating myocardial mass and digitising the epicardial and endocardial profiles in the short axis . 
the lv was then divided into 17 segments according to the guidelines of the american society of echocardiography , and maximum lv wall thickness values were recorded in end diastole and end systole . 
sono stati inclusi solo i pazienti con diagnosi nota di cmi primitiva , ottenuta secondo i criteri della world health organization ( esame clinico ed eco - cardiografico mmode tramite dimostrazione di aumentato spessore parietale in telediastole > 15 mm a livello del setto o della parete posteriore ) , in assenza di altre cause dimostrabili di ipertrofia ventricolare [ 14 ] , studiati prospetticamente mediante ecocardiografia e cardio - rm entro 2 settimane ( scarto medio 7 giorni , range 214 giorni )  . 
sono stati esclusi dallo studio i pazienti affetti da possibili cause di cmi secondaria ( stenosi aortica , amiloidosi o ipertensione arteriosa ; n = 44 ) e quelli che non disponevano di studio comparativo con entrambe le metodiche ( n = 39 )  . 
in accordo con il comitato etico tutti i pazienti arruolati nello studio hanno firmato un consenso informato prima dellesecuzione di entrambi i test diagnostici . protocollo eco - cardiografia per leco - cardiografia stato utilizzato un apparecchio acuson sequoia 512 ( mountain view , ca , usa ) con trasduttore 3v2c dotato di software per il calcolo della massa cardiaca con digitalizzazione dei profili dellepicardio e dellendocardio in asse corto . 
il ventricolo sinistro stato quindi suddiviso in 17 aree , in accordo alle linee guida della societ americana di eco - cardiografia , e sono stati ottenuti i valori di massimo spessore del vsn in telediastole ( td ) ed in telesistole ( ts )  . 
 cardiac mr imaging protocol protocollo cardio - rm all cardiac mr examinations were performed with a 1.5tutte le indagini cardio - rm sono state eseguite con un 200 radiol med ( 2011 ) 116 : 197210 t magnet ( avanto , siemens medical systems , erlangen , germany ) and a phased - array coil . 
images were acquired in the axial and coronal planes with half - fourier acquired single - shot turbo spin - echo ( haste ) sequences and balanced true fast imaging with steady - state precession ( true - fisp ) sequences to obtain cine images along three axes ( short , two - chamber long and four - chamber views ) , with breathhold acquisitions of around 15 cardiac cycles . 
the following parameters were used for the cine sequences : tr / te = 35 ms / 1.8 ms ; flip angle , 60 ; slice thickness , 8 mm ; acquisition time , 1520 s according to heart rate ; receiver bandwidth , 930 hz / pixel ; matrix , 256256 ; fov , 300350 m the images for evaluating delayed enhancement ( de ) were obtained after administration of 0.2 mmol / kg of gadolinium chelates ( dotarem , guerbet , france ) at a flow rate of 2 ml / s . 
images were obtained 10 min after the injection of contrast material in the same planes with t1 - weighted 3d inversion recovery ( ir ) sequences and the following acquisition parameters : tr / te = 7 ms / 3 ms ; flip angle , 15 ; voxel dimensions , 1.41.41.2 mm , 32 lines of the k - space calculated for each r - r interval in end diastole ; fov , 340265 cm ; matrix , 192256 ; 16 cardiac cycles sampled over a single breath - hold . 
to increase diagnostic confidence , the de images were also obtained with phase - sensitive ir ( psir ) true - fisp sequences with the following parameters : tr / te = 2.2 / 11 ; flip angle , 50 ; voxel dimensions , 1.41.41.2 mm ; fov , 340265 cm ; matrix , 192102 ; 16 cardiac cycles sampled during a single breath - hold . mean acquisition time was 30 m postprocessing analysis of the true - fisp sequences to calculate the myocardial mass and ef was performed with a dedicated software package ( argus , siemens , erlangen , germany )  . turbo - flash image analysis echocardiography and cardiac mr studies were evaluated blind by a cardiologist and a radiologist , respectively , who were both expert in cardiac imaging and unaware of the prior clinical and imaging findings . 
on the basis of a series of images acquired during an entire cardiac cycle in the short axis , from the base to apex of the heart , the images magnete da 1 , 5 t ( avanto , siemens medical systems , erlangen , germania ) con bobina phased - array . 
sono state acquisite immagini nei piani assiale e coronale con sequenze half - fourier acquired single - shot turbo spin - echo ( haste ) e successive sequenze bilanciate truefast imaging with steady state precession ( fisp ) per ottenere immagini cine lungo i tre assi ( asse corto , due camere asse lungo e 4 camere ) , con acquisizioni in apnea della durata di circa 15 cicli cardiaci . 
i parametri utilizzati per la sequenza cine sono stati : tempo di ripetizione ( tr ) / tempo di eco ( te ) = 35 ms / 1 , 8 ms ; flip angle : 60 ; spessore di fetta : 8 mm ; tempo di acquisizione : 1520 s a seconda della frequenza cardiaca del paziente ; ampiezza della banda di ricezione : 930 hz / pixel ; matrice 256256 ; field of view ( fov ) : 300350 m la immagini post - contrastografiche per la valutazione del delayed enhancement ( de ) sono state ottenute dopo somministrazione di 0 , 2 mmol / kg di chelati del gadolinio ( dotarem , guerbet francia ) con un flusso di 2 ml / s . 
le immagini sono state ottenute dopo 10 minuti dalla iniezione nei medesimi piani spaziali tramite sequenza 3d turbo - flash inversion recovery ( ir ) t1 - pesata con i seguenti parametri di acquisizione : tr / te = 7 ms / 3 ms ; flip angle : 15 ; dimensioni del voxel : 1 , 41 , 41 , 2 mm , 32 linee del k spazio calcolate per ciascun intervallo r - r in telediastole , fov : 340265 cm ; matrice : 192256 ; 16 cicli cardiaci campionati nellarco di una singola apnea . 
il tempo di inversione ( ti ) stato modificato progressivamente al fine di annullare completamente il segnale del miocardio , con un range di valori compreso tra 220 e 290 ms . 
lanalisi in post - processing delle sequenze true - fisp , al fine di calcolare la massa cardiaca e la frazione di eiezione , stata eseguita tramite software dedicato ( argus , siemens , erlangen , germania )  . analisi delle immagini le indagini eco - cardiografiche e cardio - rm sono state valutate in cieco , rispettivamente da un cardiologo e da un radiologo esperti in imaging cardiaco , entrambi non a conoscenza degli eventuali dati clinici e strumentali precedenti . 
in tutti i casi il profilo dellendocardio stato giudicato riconoscibile : a partire da una serie di immagini radiol med ( 2011 ) 116 : 197210 corresponding to the end diastole and end systole were automatically selected , and the margins of the endocardium and the epicardium were traced on thethe papillary muscles were included in the calculation , whereas trabeculae projecting into the cardiac cavity were excluded , as they were judged to be a possible source of error . 
the software calculates the volume [ area times the slice thickness ( 8 mm ) and the gap between the slices ( 1 mm ) ] and ventricular mass ( myocardial volume times the value of the myocardial density : 1.05 g / ml ) in the phase in question , with evaluation of the curves that define the endocardium and epicardiu edv , esv and ef were calculated with the simpson method ( the volume of an object can be estimated by adding the individual areas in each section and multiplying them by the slice thickness ) [ 14 , 15 ]  . with a parasternal approach and in the m - mode longaxis view and with the cursor perpendicular to the septum , measurements of the septum and posterior wall were manually performed during both diastole and systole . diastolic function was obtained by calculating the peak velocity of the e - wave and a - wave . 
the presence of systolic anterior motion ( sam ) was verified , considered as a displacement of both mitral flaps > 2 mm below the cd line during systole ( overall ventricular systole ) , and the degree of mitral regurgitation was quantified with doppler . 
fibrosis was estimated with a qualitative comparison between the degree of ultrasonic reflectivity in the areas considered to be fibrotic and the parts of the myocardium considered to be healthy [ 14 ]  . 
at cardiac mr imaging , myocardial mass , edv , esv and ef values were calculated during postprocessing on true - fisp sequences with a dedicated software package ( argus , siemens , erlangen , germany ) , considering the curvature of the lv , with the electronic callipers in the short axis for the septum and the long axis for the posterior wall perpendicular to the segment subjectively evaluated most often in end diastole . myocardial thickness was estimated on images acquired in two - chamber short - axis and four - chamber long - axis views in end diastole . 
the degree of myocardial fibrosis was evaluated with the subjective demonstration of intramural areas of late enhancement characterised by signal hyperintensity in a region of hypointense myocardiu no quantitative methods were adopted . 
however , to be acquisite in un intero ciclo cardiaco in asse corto , dalla base allapice del cuore , sono selezionate automaticamente le immagini corrispondenti alla td e alla ts e su queste sono stati tracciati manualmente i bordi dellendocardio e dellepicardio . 
il software calcola il volume ( area per lo spessore della fetta , 8 mm , e del gap tra le fette , 1 mm ) e la massa ventricolare ( volume miocardico moltiplicato per il valore della densit miocardia : 1 , 05 g / ml ) nella fase considerata , valutando larea delle curve che definiscono lendoe lepicardio . 
il volume telediastolico ( vtd ) , il volume telesistolico ( vts ) e la frazione di eiezione ( fe ) sono stati calcolati col metodo di simpson ( il volume di un oggetto pu essere stimato sommando le singole aree in ciascuna sezione e moltiplicandole per lo spessore della fetta ) [ 14 , 15 ]  . con approccio parasternale , nella proiezione asse lungo m - mode e cursore ortogonale al setto , sono state eseguite in modo manuale le misurazioni del setto e della parete posteriore , sia in diastole che in sistole . 
mediante lanalisi doppler stata calcolata la velocit massima e media dellefflusso ventricolare sinistro ed i relativi gradienti ; si ricercato lo spostamento anteriore del lembo mitralico ( sam ) , considerato uno spostamento in sistole dei lembi mitralici > 2 mm al di sotto della linea cd ( sistole ventricolare complessiva ) e quantificato con doppler il grado di rigurgito mitralico . 
alla cardio - rm la massa cardiaca , i valori di vtd , vts e fe sono stati calcolati in post - processing sulle sequenze true - fisp , tramite software dedicato ( argus , siemens , erlangen , germania ) , tenendo conto della curvatura del vsn , con caliper elettronico , rispettivamente in asse corto per il setto ed in asse lungo per la parete posteriore , perpendicolarmente al segmento soggettivamente valutato pi spesso in td . 
il sam stato valutato mediante sequenze a contrasto di fase , insieme con levidenziazione di eventuale insufficienza mitralica associata e in base alla valutazione qualitativa dellescursione valvolare nelle sequenze cine true - fisp . 
la alterazione di segnale 202 radiol med ( 2011 ) 116 : 197210 considered , signal alteration needed to be confirmed in at least two acquisition planes . tuttavia per essere segnalata doveva confermarsi in almeno due piani di acquisizione . statistical analysis analisi statistica the following values were considered normal : myocardial mass between 118 and 238 g ; ef between 56% and 78% ; lv wall thickness at the level of the septum and inferior wall between 8 mm and 10 mcontinuous variables are expressed as mean standard deviation ( sd ) and categorical variables as proportions and percentages . 
statistical correlation was performed using student t test and spearmans correlation coefficient ( r ) for comparison of continuous variables , and fishers exact test was used to compare categorical variables . 
 sono stati considerati normali i seguenti valori : massa cardiaca tra i 118 e i 238 g ; fe tra il 56% ed il 78% ; spessore parietale del vsn a livello del setto e della parete inferiore tra gli 8 mm e 10 mle variabili continue sono state espresse come mediadeviazione standard , le variabili categoriche come proporzioni e percentuali . 
la correlazione statistica stata eseguita usando il test t di student e lindice di correlazione r di spearman per il confronto tra variabili continue ; il test di fisher stato utilizzato per confrontare le variabili categoriche . 
uniforme ispessimento di tutto il miocardio del ventricolo sinistro , particolarmente evidente al setto interventricolare ed alla parete anteriore , evidenziato sia allecocardiografia ( a , b ) che alla cardio - rm ( c , d )  . radiol med ( 2011 ) 116 : 197210 fig . 
2a - d assessment of myocardial mass and ventricular function by means of echocardiography and cardiac magnetic resonance ( mr ) imaging : at echocardiography , in the parasternal short - axis view , at the end - diastolic phase , the epicardial ( a ) and endocardial ( b ) borders are measured , including the papillary muscles , which are often hypertrophic . 
2a - d calcolo della massa e funzione ventricolare in ecocardiografia ( a - c ) e in rm ( d ) : allecocardiografia , in parasternale asse corto in tele - diastole , viene planimetrato il contorno dellepicardio ( a ) e dellendocardio ( b ) , includendo i muscoli papillari ( spesso ipertrofici )  . 
3a - d underestimation of myocardial mass with cardiac mr imaging ; geometric representation of the lv cut by several sections in the short - axis view : the volume could be overestimated or underestimated depending on the position of lv within the section , considering identical thickness ( grey ) and gap ( white spaces ) between sections . 
the most caudal section could be included in assessing the myocardial mass ( a ) or excluded because < 50% of this slice is included in the assessed volume ( b )  . 
3a - d sottostima della massa cardiaca alla cardio - rm : rappresentazione schematica della geometria del vsn tagliato da multiple sezioni in asse corto ( a , b )  . 
pur ammettendo uguali spessori di fetta ( in grigio ) e dei gap tra le sezioni ( spazi bianchi ) , il volume pu essere sia sovra che sottostimato a seconda della posizione del vsn rispetto alla fetta in questione . 
la fetta pi caudale pu venire inclusa ( a ) nel calcolo del volume , mentre potrebbe essere esclusa dal software perch compresa nella zona di interessa per meno del 50% ( b )  . 
si ottenuta una buona correlazione ( r = 0 , 75 ) tra gli spessori del setto calcolati allecocardiografia ( 1236 mm ) ed alla cardio - rm ( 1639 mm ) , mentre non stata verificata correlazione tre le metodiche riguardo lo spessore della parete posteriore ( r = 0 , 04 )  . 
lostruzione dinamica era presente in 9 / 22 pazienti ( 40 , 9% ) analizzati con eco - cardiografia e in 12 / 22 pazienti ( 54 , 5% ) studiati con rm ( p = 0 , 5 )  . 
alla cardio - rm , la concordanza fra la presenza di de con lo spessore massimo in telediastole stata statisticamente non significativa sia per il setto ( p = 0 , 6 ) che per la parete posteriore ( p = 0 , 9 )  . 
il confronto fra i parametri morfologici e funzionali valutati con eco - cardiografia e cardio - rm schematizzato nelle tabelle 1 e 2 . discussion discussione clinical diagnosis of hcm is not simple , as patients tend to be asymptomatic at onset without apparent cause of the la diagnosi clinica di cmi non risulta sempre agevole , trattandosi generalmente di pazienti asintomatici allesordio , radiol med ( 2011 ) 116 : 197210 fig . 
5a - c differenti pattern di fibrosi miocardica alla cardio - rm : distribuzione focale ( a , b ) e diffusa a zolle ( c ) myocardial hypertrophy [ 1 , 2 ]  . 
cardiac mr imaging , which is comparable with echocardiography in terms of diagnostic sensitivity , is more accurate in measuring wall thickness and cardiac motion and can be considered the most reliable technique in demonstrating the presence of disorganisation in muscle structure , inefficient myocardial contraction and myocardial fibrosis [ 7 , 17 , 18 ]  . 
la cardio - rm , paragonabile alleco - cardiografia in termini di sensibilit per la diagnosi , presenta maggior accuratezza nella determinazione degli spessori di parete e della cinetica cardiaca , potendo inoltre essere considerata la metodica pi affidabile nel dimostrare la presenza di disorganizzazione nella struttura muscolare , di uninefficiente contrazione miocardica e di fibrosi parietale [ 7 , 17 , 18 ]  . 
 in accordo con i dati della letteratura [ 5 , 12 , 13 , 16 , 17 , 19 ] , i nostri risultati dimostrano unassoluta concordanza fra eco - cardiografia e cardio - rm nella determinazione della sede e dellestensione di malattia , con differenze statisticamente non significative nella definizione dei valori di vtd ( p = 0 , 31 ) , vts ( p = 0 , 1 ) , fe ( p = 0 , 05 ) , e della presenza di ostruzione dinamica ( p = 0 , 5 )  . 
tale sovrastima relativa da attribuirsi ai differenti metodi di misurazione utilizzati : mentre alleco - cardiografia il calcolo dello spessore automatico , previa acquisizione di immagini mmode in asse lungo , alla rm la misurazione tiene conto della curvatura del vsn , e viene effettuata mediante caliper elettronico , in asse corto per il setto ed in asse lungo per la parete posteriore . 
in our data set , however , the relatively small area at the level of the apical sections produced a minimal amount of error in the quantification of mass and ef , whereas the effect described above is more evident at the base , where the area of the section is greater ( p < 0.001 ) [ 19 ]  . 
the superiority of cardiac mr in diagnosing sam lies in its more objective interpretation and the possibility of obtaining a more precise assessment of its functional repercussions with the acquisition of phasecontrast sequences . 
also important in these cases is the presence of secondary mitral regurgitation , which can be demonstrated as a systolic jet in the left atrium and is quantifiable with phase - contrast sequences [ 8 , 20 ]  . 
the lack of a reference standard , such as a histological examination , prevented absolute verification of the causes of those differences , which we believe are correlated with the radically different methods used for the diagnosis ( increase in ultrasonic reflectivity at echocardiography vs delayed enhancement at mr )  . 
the site of fibrosis in this sense does not seem to correspond with areas supplied by epicardial branches of the coronary arteries , which are thought to be more compromised where the wall is thicker . livello , ed alla base , a causa del movimento verso lapice ( mediamente circa 1 , 3 cm ) che il cuore attua durante la sistole [ 10 ]  . 
nel nostro dataset tuttavia , larea relativamente piccola a livello delle sezioni apicali ha introdotto una minima componente di errore nella quantificazione della massa e della fe , mentre leffetto sopra descritto pi evidente a livello della base , ove larea di sezione maggiore ( p < 0 , 001 ) [ 19 ]  . 
 nella nostra esperienza , anche la delimitazione automatica dei profili cardiaci , del vtd e del vts presenta errori intrinseci al software tali da richiedere un ritocco manuale da parte delloperatore dei contorni endocardici ed epicardici con inevitabile incremento dei tempi necessari per il post - processing . 
 anche se la cardio - rm si rilevata lievemente superiore alla eco - cardiografia nel rilevare il fenomeno del sam , permettendo di individuare anche le forme lievi o in fase iniziale , la differenza di performance non stata significativa ( p = 0 , 1 )  . 
la superiorit della cardio - rm nella diagnosi del sam da ricercare nella maggiore obiettivit interpretativa nonch nella possibilit di poter effettuare una pi precisa valutazione anche delle ripercussioni funzionali del fenomeno con la acquisizione di sequenze a contrasto di fase . 
inoltre importante in questi casi documentare la presenza di una eventuale insufficienza mitralica secondaria , dimostrabile come jet sistolico in atrio sinistro , e quantificabile alla rm mediante sequenze in contrasto di fase [ 8 , 20 ]  . 
in accordo con i dati della letteratura , la cardio - rm si rilevata superiore alla eco - cardiografia nella identificazione della fibrosi parietale [ 9 , 21 ] , e questo aspetto da recenti lavori sembra poter correlarsi alla maggior probabilit di eventi fatali [ 22 ] : tuttavia , la differenza di performance tra le metodiche non stata significativa ( p = 0 , 15 )  . 
la totale concordanza riguardante la sede della fibrosi parietale stata raggiunta in soli 3 / 9 pazienti ( 33% ) , rispettivamente in un caso di localizzazione apicale e due casi di localizzazione diffusa . 
la mancanza di uno standard di riferimento , quale lesame istologico , ha impedito di verificare con certezza le cause di tali differenze che riteniamo comunque correlate ai metodi radicalmente diversi utilizzati per la diagnosi ( aumento della rifrangenza alleco - cardiografia versus delayed enhancement alla rm )  . 
alla cardio - rm , la fibrosi era presente nella maggior parte dei pazienti in quelle sezioni di miocardio ove lipertrofia risultata maggiore : tuttavia , anche il tentativo di cercare una correlazione tra la presenza di impregnazione tardiva con lo spessore massimo in telediastole ( del setto e / o della parete posteriore ) non risultato significativo ( setto p = 0 , 6 ; parete posteriore p = 0 , 9 )  . 
la sede della fibrosi sembrerebbe in questo senso non corrispondere ai territori di perfusione dei rami epicardici delle arterie radiol med ( 2011 ) 116 : 197210 despite the nonsignificant difference in overall performance , in our experience , cardiac mr imaging has the advantage of offering an objective and repeatable evaluation of the morphological parameters ( wall thickness , extent of myocardial fibrosis ) and functional parameters ( edv , esv , sam , ef ) , which is crucial in the follow - up of these patients to facilitate comparison and evaluate disease progression . 
in addition , with the technique of myocardial tagging , mr imaging is better able to demonstrate changes in lv motion resulting from hypertrophy [ 23 ] and better still with a 3 - t system [ 24 ]  . 
 there are , however , some limitations to this study . first , the number of patients enrolled was relatively small insofar as the population can be considered representative , as it included only asymptomatic patients and patients without identifiable causes of cardiac hypertrophy . second , evaluating myocardial fibrosis was based on imaging findings described in the literature , with no reference standard such as histological examination . 
last , clinical and / or radiological follow - up is available for a limited number of patients only , and therefore the findings described cannot be correlated with disease progression and prognostic data . in conclusion , echocardiography and cardiac mr imaging are accurate techniques for diagnosing hcm . given its greater accessibility , echocardiography is the first - line technique , whereas mr being more objective in its measurement , more accurate in identifying sam and more sensitive in recognising myocardial fibrosis is a useful diagnostic integration to be recommended in doubtful cases or in the apical form , in which the poor acoustic window can limit echocardiographic study . 
the correlation between prognosis and the presence of fibrosis in recently published studies [ 22 ] validates the role of mr imaging not only in diagnosing the disease but also , and above all , in the follow - up of these patients , who are often young . coronarie che dovrebbero risultare maggiormente compromessi laddove vi sia maggior spessore di parete . nonostante differenze non significative di performance globale , nella nostra esperienza la cardio - rm presenta il vantaggio di permettere una valutazione obiettiva e ripetibile dei parametri morfologici ( spessore di parete , entit della fibrosi parietale ) , e funzionali ( vtd , vts , sam , fe ) , fatto di fondamentale importanza nel caso di follow - up di questi pazienti per favorire il confronto e la valutazione dellevoluzione del quadro . 
inoltre , mediante la tecnica di tagging miocardico , la rm meglio evidenzia le alterazioni della cinetica del vsn conseguente allipertrofia [ 23 ] , meglio ancora se mediante apparecchiature 3 tesla [ 24 ]  . 
 questo studio tuttavia presenta dei limiti : primo , il numero di pazienti arruolati relativamente basso , per quanto la coorte possa essere ritenuta rappresentativa , comprendendo solo pazienti asintomatici e senza cause evidenziabili di ipertrofia cardiaca . 
secondo , la valutazione della fibrosi parietale si basata su rilievi di imaging descritti in letteratura , mancando uno standard di riferimento di tipo istologico . terzo , uno studio di perfusione miocardica stato eseguito solo in alcuni pazienti , per cui non stato possibile effettuare una correlazione fra fibrosi ed ipoperfusione tissutale . 
infine , un follow - up clinico e / o radiologico disponibile , allo stato attuale , per un numero limitato di pazienti , e pertanto non stato possibile correlare i rilievi descritti con la capacit di evolvere della malattia e con i dati prognostici . in conclusione , eco - cardiografia e cardio - rm rappresentano metodiche accurate per la diagnosi di cmi . 
lecocardiografia , in relazione alla maggiore accessibilit , rappresenta lindagine di prima linea , mentre la rm , grazie alla maggiore obiettivit delle misurazioni , alla maggiore accuratezza nellinquadramento del sam , e alla maggiore sensibilit nel riconoscere la fibrosi parietale rappresenta utile integrazione diagnostica da raccomandarsi in particolare nei casi dubbi o nelle forme apicali laddove la scarsa finestra acustica pu limitare lesame ecografico [ 17 ]  . 
le ricadute prognostiche correlate alla presenza di fibrosi riportate in recenti lavori della letteratura [ 22 ] avvalorano limportanza del ruolo della rm non solo nella diagnosi di malattia ma anche e soprattutto nel follow - up di questi spesso giovani pazienti . conflict of interest none radiol med ( 2011 ) 116 : 336 doi 10.1007 / s11547 - 011 - 0659 - 8 erratum off - label use of intravascular iodinated organic and mr contrast media utilizzo off - label dei mezzi di contrasto organo - iodati e per risonanza magnetica per via iniettiva o . 
david4 1dipartimento di radiologia cardiovascolare , 3dipartimento di cardiochirurgia , 4dipartimento di radiologia demergenza , ospedale san camillo - forlanini , circonvallazione gianicolense 87 , 00152 rome , italy 2dipartimento di scienze radiologiche , universit di roma sapienza , ospedale santandrea , via di grottarossa 1035 , 00189 rome , italy correspondence to : c.n. 
the aim of our work was to compare image quality and radiation dose in a group of patients who underwent cardiac dual - source computed tomography ( dsct ) with prospective electrocardiographic ( ecg ) gating with those of a control group studied with retrospective gating . 
coronary ct with prospective ecg gating , a standard feature on new scanners , allows for a significant reduction in radiation dose without causing any significant decrease in image quality or in the number of segments assessed . 
la dose normalizzata di radiazioni stata di 4 , 910 , 4 msv per il gruppo prospettico , e di 14 , 62 msv4 , 36 per il gruppo retrospettivo ( p < 0 , 01 )  . 
la tc coronarica con tecnica di radiol med ( 2011 ) 116 : 178188 keywords dual - energy computed tomography coronary angiography prospective gating retrospective gating sincronizzazione prospettica , utilizzabile negli scanners di ultima generazione , permette una significativa riduzione della dose di radiazioni somministrata al paziente senza determinare alcuna riduzione rilevante nella qualit delle immagini e nel numero di segmenti valutabili . 
per tale ragione , attualmente consigliabile l utilizzo di tale tecnica nei soggetti che presentano una frequenza cardiaca inferiore ai 70 bpm ed un bmi inferiore ai 30 kg / m2 . parole chiave tomografia computerizzata dual - energy angiografia coronarica gating prospettico gating retrospettivo introduction introduzione multidetector computed tomography ( mdct ) has proved to be an accurate method for evaluating coronary atherosclerosis [ 15 ]  . 
owing to the technological limits of older ct scanners , cardiac ct studies have been carried out until recently with retrospective electrocardiographic ( ecg ) gating , in which the whole heart cycle is acquired to obtain a large number of cardiac phases to assess the coronary arteries [ 8 ]  . 
in this context , different dose - reduction strategies have been developed that rely either on dose - modulation algorithms during the cardiac cycle ( ecg pulsing ) [ 9 ] or on tube current reduction [ 10 ]  . although such strategies have allowed for a significant reduction in radiation exposure , the dose nonetheless remains high , even higher than that delivered by conventional coronary angiography . 
 the most effective dose - reduction technique developed to date is prospective ecg gating , in which only a portion of the cardiac cycle is acquired in a sequential , step - andshoot mode . 
however , these limitations have now been overcome by the latest - generation ct equipment in which different engineering solutions including increased number of x - ray tubes [ 11 ] or detectors [ 12 ] have allowed reliable prospective gating techniques to be developed [ 13 ]  . the purpose of this study was to compare image quality and radiation dose in a group of patients studied with dualsource ct ( dsct ) coronary angiography with prospective ecg gating compared with a control group studied with retrospective gating . la tomografia computerizzata ( tc ) multidetettore ( tcmd ) ha dimostrato di essere una metodica che permette unaccurata valutazione dellaterosclerosi coronarica [ 15 ]  . 
a causa di limitazioni tecnologiche presenti nelle tc di vecchia generazione stata utilizzata sinora una tecnica di sincronizzazione dellelettrocardiogramma ( ecg ) di tipo retrospettivo in cui viene acquisito lintero ciclo cardiaco , in modo da poter disporre di un elevato numero di fasi in cui valutare le coronarie [ 8 ]  . 
per tale motivo sono state sviluppate diverse strategie per la riduzione delle radiazioni basate su algoritmi di modulazione della dose durante il ciclo cardiaco ( ecg pulsing ) [ 9 ] o sulla riduzione dellintensit della corrente del tubo radiogeno [ 10 ]  . 
in tal modo stata ottenuta una significativa riduzione delle radiazioni somministrate al paziente , che per rimangono ancora a livelli elevati , superiori a quelli di unangiografia coronarica tradizionale . attualmente la tecnica pi efficace nella riduzione della dose risulta essere la sincronizzazione prospettica dellecg , in cui viene acquisita solo in una parte del ciclo cardiaco effettuando cos unacquisizione in modalit sequenziale . 
alcuni limitazioni tecniche , quali la velocit di rotazione del gantry , non avevano permesso sinora un ampio utilizzo di questa seconda modalit di acquisizione se non per finalit di ricerca . 
tali limitazioni risultano attualmente superate nelle tc di ultima generazione in cui soluzioni ingegneristiche differenti , come laumento dei tubi radiogeni [ 11 ] o lincremento del numero di detettori [ 12 ] , hanno permesso lo sviluppo di tecniche di sincronizzazione prospettica affidabili [ 13 ]  . 180 materials and methods patient population between january and march 2009 , noninvasive dsct coronary angiography was used to study 60 patients who were randomly assigned to two groups of 30 . 
exclusion criteria were allergy to iodinated contrast material , nephropathy ( creatinine > 120 mmol / l ) , nonsinus rhythm , haemodynamic instability , heart rate > 70 bpm and body mass index ( bmi ) > 30 kg / m2 . 
written informed consent was obtained from all patients enrolled in the study . study protocol examinations were conducted on a dual - source scanner ( somatom definition , siemens medical solutions , forchheim , germany )  . 
the studies were conducted with a biphasic intravenous injection of iodinated contrast material ( iomeron 400 , 400 mgi / ml , bracco , milan , italy ) delivered at high flow rates ( 56 ml / s ) through a dual - head injector . 
group a was studied with the following parameters : detector collimation 320.6 mm ; acquisition slices 640.6 mm with moving focal spot ; voltage 120 kv ; current 506 mas . 
in the prospective acquisition with dsct , the scan angle was extended from 260 to 460 , resulting in 8% of r - r cycle phases to be reconstructed compared with the selected phase . 
in group b patients , scans were performed using the following parameters : detector collimation 320.6 mm ; acquisition slices 640.6 mm acquired with a moving focal spot and adaptive pitch ( 0.20.35 ) ; gantry rotation time 330 ms ; tube voltage 120 kv ; current 350 mas . 
to reduce the dose in group b patients , automatic current modulation ( ecg pulsing ) was used , with maximum tube intensity applied between 30% and 80% of the cardiac cycle . 
this prevented unnecessary exposure during cardiac phases in which the image quality of the coronary arteries is classified as nondiagnostic ( table 1 )  . radiol med ( 2011 ) 116 : 178188 lo scopo di tale lavoro di comparare la qualit dimmagine e la dose di radiazioni di un gruppo di pazienti sottoposto ad angiografia coronarica con tc dual source ( tcds ) con tecnica di sincronizzazione prospettica dellecg rispetto ad un gruppo di controllo studiato con tecnica di sincronizzazione retrospettiva . materiali e metodi popolazione di pazienti nel periodo compreso tra gennaio e marzo 2009 , sono stati studiati mediante angiografia coronarica non invasiva con tcds 60 pazienti randomizzati in due gruppi di 30 pazienti ciascuno . 
i criteri di esclusione sono stati i seguenti : allergia al mezzo di contrasto iodinato , nefropatia ( creatinina > 120 mmol / l ) , ritmo non sinusale , instabilit emodinamica , frequenza cardiaca > 70 battiti per minuto ( bpm ) , body mass index ( bmi ) > 30 kg / m2 . 
tutti i soggetti esaminati hanno fornito il consenso informato scritto . protocollo di studio lesame stato eseguito utilizzando uno scanner dual source ( somatom definition , siemens medical solutions , forchheim , germania )  . 
la scansione stata eseguita con una iniezione intravenosa bifasica con un iniettore a doppia testata di mezzo di contrasto iodato ( iomeron 400 , 400 mgi / ml , bracco , milano , italia ) a flussi elevati ( 56 ml / s ) ad una dose di 70 ml per la scansione retrospettiva e di 8090 ml per la scansione prospettica , il tutto seguito da 50 ml di soluzione salina . 
nel gruppo a sono stati utilizzati i seguenti parametri : collimazione dei detettori 320 , 6 mm ; strati di acquisizione 640 , 6 mm acquisiti mediante macchia focale mobile ; stato applicato un voltaggio di 120 kv ed una intensit di corrente di 506 mas . 
to evaluate calcified segments , reconstructions were obtained with a sharp kernel ( b46f ) [ 15 ]  . image reconstructions were transferred to an external workstation ( leonardo , siemens medical solutions ) equipped with dedicated software for cardiac postprocessing ( syngo circulation ii , siemens medical solutions )  . 
images were evaluated using axial scans , multiplanar reformations ( mpr ) , maximum intensity projections ( mip ) , volume rendering ( vr ) and curved multiplanar reconstructions . two blinded radiologists independently evaluated image quality of the coronary arteries according to the 16 - segment classification of the american heart association [ 16 ]  . 
la scansione stata eseguita nel gruppo b utilizzando i seguenti parametri : collimazione dei detettori 320 , 6 mm ; strati di acquisizione 640 , 6 mm acquisiti mediante macchia focale mobile e pitch adattativo ( 0 , 20 , 35 ) ; tempo di rotazione del gantry 330 ms . 
nel grupppo b , al fine di ridurre la dose somministrata stata utilizzata una modulazione automatica della corrente del tubo ( ecg pulsing ) , con la massima intensit applicata tra il 30% e 80% del ciclo cardiaco , evitando lesposizione del paziente a dosi di radiazioni non necessarie durante le fasi del ciclo cardiaco nelle quali la qualit delle immagini delle arterie coronarie pu risultare non diagnostica ( tabella 1 )  . 
 ricostruzione ed analisi delle immagini per il gruppo a , le immagini sono state ricostruite in fasi del 2% dellintervallo r - r allinterno della finestra di acquisizione prospettica ( 67%83% )  . 
in entrambi i casi le immagini sono state ricostruite utilizzando uno spessore di strato di 0 , 75 mm un incremento di ricostruzione di 0 , 4 mm ed un filtro b26f . 
per la valutazione dei segmenti calcifici sono state effettuate ricostruzioni con l utilizzo di un filtro sharp ( b46f ) [ 15 ]  . le immagini ricostruite sono state trasferite ad una workstation esterna ( leonardo , siemens medical solutions ) equipaggiata con un software dedicato al post - processing cardiaco ( syngo circulation ii , siemens medical solutions )  . per la valutazione delle immagini sono state applicate scansioni assiali , ricostruzioni multiplanari ( mpr ) , proiezioni di massima intensit ( mip ) , immagini in volume rendering ( vr ) e ricostruzioni multiplanari curve . 
due radiologi in cieco hanno valutato indipendentemente la qualit delle immagini delle arterie coronarie basandosi sulla classificazione in 16 segmenti dellamerican heart associations [ 16 ] : larteria coronaria destra include i segmenti 14 , il tronco comune e larteria discendente anteriore comprendono i segmenti 510 e larteria circonflessa i segmenti 1115 . 
a grado 4 , immagine di eccellente qualit diagnostica esente da artefatti ; b grado 3 , immagine di buona qualit diagnostica con artefatti minori ; c grado 2 , immagine di qualit diagnostica sufficiente per escludere patologie coronariche significative ; d grado 1 , immagine non valutabile per la presenza di severi artefatti . evaluation of radiation dose for both groups , we measured the dose - length product ( dlp ) and the scan length in centimetres . 
the conversion factor for the chest [ k = 0.017 msv / ( mgycm ) ] was calculated using human phantoms in a monte carlo simulation [ 17 ]  . since the scan length varied according to the clinical indication , the effective dose was normalised for the length of a standard 12 - cm heart scan [ 18 ]  . 
all statistical analyses were performed using the spss statistical software ( version 14.0 , statistical package for the social sciences spss , chicago , il , usa )  . significative ; grado 3 , immagine con artefatti minori e di buona qualit diagnostica per escludere patologie coronariche e valutare il grado di stenosi ; grado 4 , immagine senza artefatti di eccellente qualit diagnostica . valutazione della dose di radiazioni fattore di conversione per in entrambi i gruppi stato registrato il dose - lenght product ( dlp ) e la lunghezza in cm della scansione . 
dato che la lunghezza della scansione risultata variabile a seconda del tipo di indicazione clinica , la dose effettiva stata normalizzata per la lunghezza di una scansione standard del cuore di 12 cm [ 18 ]  . 
use of the best reconstruction interval for each segment resulted in excellent diagnostic quality ( score 4 ) of images in 40.1% ( 176 / 439 ) of cases in group a and in 42.0% ( 187 / 445 ) of cases in group b . 
minor artefacts ( good image quality , score 3 ) were seen in 54.2% ( 238 / 439 ) and in 55.5% ( 244 / 445 ) and moderate artefacts in 4.3% ( 19 / 439 ) and in 2.5% ( 11 / 445 ) of cases in groups a and b , respectively . 
il test della u di mannwhitney stato utilizzato al fine di rilevare eventuali differenze tra la qualit delle immagini e la dose media di radiazione dei due gruppi in esame . 
tutte le analisi statistiche sono state effettuate utilizzando il software statistico spss ( versione 14.0 , statistical package for the social sciences spss , chicago , illinois , usa )  . risultati langiografia coronarica mediante tcds stata eseguita con successo in tutti i pazienti , senza complicanze . 
non sono state riscontrate differenze significative nella composizione dei gruppi per quanto riguarda il sesso , l et , il bmi , la frequenza cardiaca e la variabilit della frequenza cardiaca ( p > 0 , 05 )  . qualit delle immagini sono stati valutati un totale di 439 segmenti coronarici nel gruppo a e 445 nel gruppo b . 
utilizzando per ogni segmento il miglior intervallo di ricostruzione , stata osservata rispettivamente una eccellente qualit diagnostica dellimmagine ( grado 4 ) rispettivamente nel 40 , 1% ( 176 / 439 ) dei casi del gruppo a e nel 42 , 0% ( 187 / 445 ) nel gruppo b ; si sono riscontrati artefatti minori ( buona qualit immagine , grado 3 ) nel 54 , 2% ( 238 / 439 ) e nel 55 , 5% ( 244 / 445 ) ; artefatti di grado moderato sono stati osservati rispettivamente nel 4 , 3% ( 19 / 439 ) e nel 2 , 5% ( 11 / 445 )  . 
la presenza di artefatti di grado severo che non permetteva la valutazione dei segmenti in esame stata riscontrata nel 1 , 4% ( 6 / 439 ) e nel 0 , 7% ( 3 / 445 ) ( tabella 3 )  . complessivamente , applicando il miglior intervallo di ricostruzione sono risultati diagnostici il 98 , 6% dei segmenti nel gruppo prospettico ed il 99 , 3% in quello retrospettico . 
la dose normalizzata di radiazioni per una scansione di 12 cm stata di 4 , 910 , 4 msv ( range 3 , 765 , 6 msv ) per il gruppo prospettico e di 14 , 62 msv4 , 36 ( 9 , 0818 , 51 msv ) per il gruppo retrospettivo . 
complessivamente , stata ottenuta una riduzione della dose di radiazioni del 62 , 4%9 , 1% ( range 51 , 0%68 , 3% ) ( p < 0.01 ) ( tabella 4 )  . tabella 3 qualit media delle immagini gruppo a ( prospettico ) gruppo b ( retrospettivo ) discussione 40 , 1% 54 , 2% 4 , 3% 1 , 4% 3 , 30 , 6 42 , 0% 55 , 5% 2 , 5% 0 , 7% 3 , 40 , 6 artefacts hindering segment evaluation were present in 1.4% ( 6 / 439 ) and in 0.7% ( 3 / 445 ) ( table 3 ) of patients , respectively . 
tale risultato stato possibile grazie al fatto che nella tecnica prospettica non viene acquisita in realt una sola fase , ma si ha una finestra di 8% rispetto al centro di acquisizione selezionato . 
a coronaria destra ; b arteria discendente anteriore ; c arteria circonflessa . radiation dose mean scan length was 14.12.0 cm ( range 11.218.7 cm ) for the prospective gating group and 13.72.6 cm ( range 11.917.3 cm ) for the retrospective gating group . 
this result was made possible by the fact that with the prospective technique , one acquires not a single phase but a 8% range relative to the selected scan centre . 
this implies the possibility of acquiring additional phases , which although limited in number are fundamental in the event of artefacts . on the other hand , the mean radiation dose was reduced by around 62.4 % , with a dose of 4.9 msv for the prospective technique as against 14.6 msv for the retrospective method . 
con tale dato viene meno il principale argomento a sfavore della tc coronarica che era rappresentato dellelevata dose somministrata . per tale motivo ragionevole aspettarsi in futuro , grazie alla diffusione degli scanners di ultima generazione , unespansione delle indicazioni della tc coronarica ed un notevole aumento del ricorso alla stessa in diagnostica coronarica rispetto alla coronarografia invasiva che attualmente presenta una morbidit ed una mortalit [ 20 ] pi elevate ed una dose di radiazioni comparabile se non superiore . i dati del nostro studio confermano e supportano quelli 186 radiol med ( 2011 ) 116 : 178188 coronary ct , i.e. , its high radiation dose . 
therefore , it can be reasonably expected that the wider diffusion of latestgeneration scanners will expand the indications for coronary ct and considerably increase its use in coronary diagnostics compared with invasive coronary angiography , which has higher morbidity and mortality rates [ 20 ] and similar , if not higher , radiation doses . our study findings confirm and corroborate those of other reports [ 13 , 18 , 2125 ] investigating prospective ecg gating and demonstrate the clinical feasibility of the technique as an effective method to reduce radiation exposure without affecting image quality . 
the prospective gating technique is applied to patients with low heart rates ( < 70 bpm ) and average bmi only to minimise the risk of artefacts that would prove impossible to eliminate given the small number of phases available . 
in our view , the need to administer beta - blockers once again , a need that dual - source technology had considerably reduced is an acceptable price to pay for a significant reduction in the radiation dose delivered , particularly to young patients [ 26 , 27 ]  . 
the technique would thus also allow the study of particular patients , such as heart - transplant patients with a high resting heart rate and reduced response to beta - blockers requiring serial follow - up imaging for possible allograft coronary disease [ 29 ]  . 
 finally , the prospective technique can provide diagnostic - quality images even in patients with arrhythmia , thanks to the sequential nature of the examination that makes it possible to avoid acquiring the segment when an ectopic beat is recognised prior to the scan , or to repeat the scan when the extrasystole occurs during acquisition . 
 conclusions coronary ct and the prospective ecg gating technique featured by the latest - generation scanners allows for a significant reduction in radiation dose , greater than that in conventional angiography , without deterioration of image quality or reduction in the number of assessable segments . this technique can therefore be recommended for patients with a heart rate < 70 bpm and a bmi < 30 kg / m2 . ottenuti da altri gruppi di ricerca [ 13 , 18 , 2125 ] sulla tecnica di sincronizzazione prospettica , e ne dimostrano lapplicabilit nella pratica clinica come metodo efficace nella riduzione della dose di radiazioni somministrata al paziente senza inficiare la qualit dimmagine dello studio . 
infatti , la tecnica di sincronizzazione prospettica viene applicata attualmente in pazienti con frequenze basse ( inferiori ai 70 bpm ) e con un bmi non elevato , al fine di ridurre al minimo la possibilit di artefatti che , data il basso numero di fasi a disposizione , non sarebbe possibile eliminare . 
a nostro giudizio la necessit di betabloccare nuovamente i pazienti , che sembrava essersi notevolmente ridotta con l avvento della tecnologia dual source , ci sembra un prezzo accettabile da pagare a fronte di una significativa riduzione della dose somministrata , in particolare nei soggetti giovani [ 26 , 27 ]  . 
in tal modo sarebbe possibile studiare con tale tecnica anche soggetti particolari come i pazienti sottoposti a trapianto cardiaco in cui presente unelevata frequenza basale ed una scarsa risposta ai farmaci beta - bloccanti e che necessitano di controlli ripetuti nel tempo per leventuale insorgenza della coronaropatia da rigetto [ 29 ]  . 
 infine , da sottolineare come con lutilizzo della tecnica prospettica possibile ottenere esami di qualit diagnostica anche in pazienti aritmici , grazie alla possibilit offerta dalla natura sequenziale dellesame , di evitare lacquisizione del segmento quando viene riconosciuto un battito ectopico prima della scansione o di ripetere la scansione quando lextrasistole avviene durante lacquisizione . 
 conclusioni la tc coronarica con tecnica di sincronizzazione prospettica , utilizzabile negli scanners di ultima generazione , permette una significativa riduzione della dose di radiazioni somministrata al paziente , inferiore anche a quella dellangiografia tradizionale , senza determinare alcuna riduzione rilevante nella qualit delle immagini e nel numero di segmenti valutabili . 
per tale ragione , attualmente consigliabile lutilizzo di tale tecnica nei soggetti che presentano una frequenza cardiaca inferiore ai 70 bpm ed un bmi inferiore ai 30 kg / m2 . conflict of interest none 1 . 
gandini1 1department of radiology , complex operational structure for radiological diagnosis , cardinal guglielmo massaia hospital , via conte verde 125 , 14100 asti , italy 2institute of diagnostic and interventional radiology , university of turin , via genova 3 , 10126 turin , italy 3department of biomedical sciences and human oncology , university of turin , turin , italy correspondence to : c . 
between november 2003 and march 2009 , 98 patients ( 78 women ; 20 men ) with a clinical and imaging suspicion of osteoporotic vertebral compression fractures underwent coaxial biopsy in conjunction with pvp of the thoracic and lumbar vertebrae . 
despite the number of biopsy samples considered insufficient or unsuitable , coaxial biopsy during pvp is useful in verifying the presumed aetiology of vertebral compression fractures , which is often unclear on the basis of clinical and imaging examinations . 
nel periodo compreso fra novembre 2003 e marzo 2009 98 pazienti , 78 femmine e 20 maschi , con sospetto clinico - strumentale di crolli vertebrali porotici sono stati sottoposti a biopsia coassiale in corso di vp di uno o pi metameri dorso - lombari . 
nonostante il discreto numero di prelievi considerati insufficienti / non idonei dall ' anatomopatologo , la biopsia coassiale in corso di vp utile per verificare la sospetta eziologia del crollo vertebrale , che si pu soltanto supporre sulla base degli esami clinici e strumentali . 
 quindi opportuno e consigliabile procedere a biopsia coassiale in tutti i pazienti che vengono sottoposti a vp . radiol med ( 2011 ) 116 : 302309 keywords vertebroplasty coaxial vertebral biopsy osteoporosis vertebral collapses parole chiave vertebroplastica biopsia vertebrale coassiale osteoporosi crolli vertebrali introduction introduzione vertebral fractures are one of the leading causes of hospitalisation both in italy and worldwide [ 1 , 2 ]  . 
most cases of vertebral compression fractures occur in osteoporotic patients . another , albeit less common , cause is malignancy , whether primary ( myelomas , lymphomas and other haematological dyscrasias ) or secondary to nonskeletal primary neoplasms . imaging alone is often unable to distinguish a neoplastic from osteoporotic aetiology [ 3 ]  . 
many patients who develop one or more vertebral compression fractures gain no benefit or pain relief from medication or containment devices ( braces , orthopaedic corsets ) , whereas percutaneous vertebroplasty ( pvp ) has recently been shown to be effective for both pain control and rapid functional recovery [ 47 ]  . 
 pvp is an interventional radiological procedure that involves injection of radio - opaque acrylic cement within the compressed vertebral bodies under fluoroscopic or combined fluoroscopic and computed tomography ( ct ) guidance . 
nonetheless , one should always consider the possibility that the cause is neoplastic , given that this is not a rare occurrence , and misdiagnosis results in delayed treatment and worse prognosis . 
for this reason , it is important to undertake a biopsy of one or more of the vertebral bodies being treated in all patients undergoing pvp for the first time in order to obtain histological confirmation of the clinical and imaging suspicion [ 9 ]  . 
 the aim of this study was to retrospectively analyse our experience to highlight the importance of biopsy sampling during pvp . materials and methods between november 2003 and march 2009 , 98 consecutive patients ( 78 women ; 20 men ) with a mean age of 72.6 ( range 5090 ) years and a total of 182 thoracolumbar vertebral compression fractures of a suspected osteoporotic nature were subjected to pvp and coaxial bone biopsy . 
each patients indications for spinal stabilisation were evaluated according to the guidelines le fratture vertebrali del rachide sono una delle principali cause di ospedalizzazione in italia e nel mondo [ 1 , 2 ]  . nella maggior parte dei casi i crolli vertebrali insorgono in pazienti osteoporotici , laltra principale causa , pur percentualmente inferiore rispetto allosteoporosi , quella neoplastica sia primitiva ( mieloma , linfoma ed altre discrasie ematologiche ) che secondaria a tumori primitivi extraossei . 
molti pazienti che vanno incontro ad uno o pi crolli vertebrali non trovano giovamento o sollievo dalla terapia farmacologica o contentiva ( busti , corsetti ortopedici ) mentre negli ultimi anni la vertebroplastica percutanea ( vp ) si dimostrata efficace sia per il controllo del dolore sia per un rapido recupero funzionale del soggetto [ 47 ]  . la vp una procedura di radiologia interventistica che consiste nelliniezione di cemento acrilico radiopaco , sotto guida radioscopica o combinata radioscopica - tomodensitometrica , allinterno di corpi vertebrali crollati . nella maggior parte dei casi la procedura si avvale del solo ausilio dellanestesia locale e periostale , solo pochi autori preferiscono ricorrere allanestesia generale [ 8 ]  . data la prevalenza dellosteoporosi nella popolazione naturale aspettarsi che alla base dei cedimenti vertebrali vi sia nella maggior parte dei casi una eziologia porotica . tuttavia bisogna sempre considerare la possibilit che la causa del crollo vertebrale sia neoplastica , poich questa un evenienza non rara e le conseguenze di una mancata diagnosi si riflettono in un ritardo terapeutico per il paziente con conseguente peggioramento della prognosi . per tali motivi importante procedere in tutti i pazienti che per la prima volta vengono sottoposti a vp ad un prelievo bioptico su uno o pi corpi vertebrali trattati per ottenere una conferma istologica del sospetto clinico - strumentale [ 9 ]  . lo scopo del lavoro quello di sottolineare limportanza del prelievo bioptico in corso di vp riportando retrospettivamente la nostra esperienza . materiali e metodi nel periodo compreso tra novembre 2003 e marzo 2009 , 98 pazienti consecutivi ( 78 femmine e 20 maschi ) , con una et media di 72 , 6 anni ( range 5090 anni ) ed un totale di 182 304 radiol med ( 2011 ) 116 : 302309 of the society of interventional radiology [ 10 ]  . 
all patients were entered into a database that included their ages , sex , medical history , presumed aetiology of the vertebral compression fracture ( s ) , imaging investigations and the visual analogue scale ( vas ) scores obtained before and after pvp [ 11 ]  . 
in particular , seven patients had a history of breast cancer , four had hepatocarcinoma associated with hepatitis c virus ( hcv ) related cirrhosis , three had a history of lung cancer , three of colorectal cancer , two of prostate cancer , two of stomach cancer , two were being treated for pleural mesothelioma , one had a history of thyroid cancer , one of laryngeal cancer , one of chondrosarcoma of the hand and one of laryngeal cancer . 
 all procedures were carried out by two operators with the patient in the prone position and employing combined fluoroscopic ct guidance using an image intensifier ( oec 9800 plus , ge healthcare , wi , usa ) and a ct scanner ( lightspeed qx / i , ge healthcare , wi , usa )  . 
in each procedure , after two preliminary scout views in the anteroposterior and laterolateral view , a volume was acquired that comprised the metamere immediately above and immediately below the one ( s ) requiring treatment . 
the scan parameters were as follows : helical scanning ; rotation time 0.8 s ; slice thickness 5 mm ; table speed 30 mm / s ; reconstruction interval 5 mm ; fov wide ; 80 kv ; 4060 ma [ 12 ]  . 
as far as fluoroscopy was concerned , we used the systems automatic exposure control . only local subcutaneous or periosteal anaesthesia was used , with 23 ml of 1% lidocaine , or , particularly in vertebral compression fractures of the thoracic spine , with a mixture of 1% lidocaine and triamcinolone . 
the biopsy cannula was 2 cm greater than the vertebroplasty needle , allowing it to penetrate deeply into the vertebral body to collect the bone sample , but this procedure requires careful fluoroscopic monitoring to avoid injury to the vertebral cortex . 
tutti i pazienti sono stati preventivamente informati sugli aspetti procedurali , sui benefici e sui rischi correlati alla vp e alla biopsia vertebrale ed stato chiesto loro il consenso , per iscritto , allesecuzione del trattamento . lindicazione allintervento di stabilizzazione vertebrale stata valutata seguendo le linee guida redatte dalla society of interventional radiology [ 10 ]  . 
tutti i pazienti coinvolti nello studio sono stati inseriti in un database comprendente let , il sesso , lanamnesi patologica remota e prossima , la sospetta eziopatogenesi del / dei cedimento / i vertebrali , le indagini strumentali eseguite ed il visual analogue scale ( vas ) score pree post - vp [ 11 ]  . 
in particolare : 7 pazienti con pregressa neoplasia mammaria , 4 pazienti con epatocarcinoma ( hcc ) in cirrosi positiva al virus dellepatite c ( hcv + ) , 3 pazienti con pregressa neoplasia polmonare , 3 pazienti con pregressa neoplasia del colon - retto , 2 pazienti con pregressa neoplasia prostatica , 2 pazienti con pregressa neoplasia gastrica , 2 pazienti con mesotelioma pleurico in trattamento , 1 paziente con pregressa neoplasia tiroidea , 1 paziente con pregressa neoplasia laringea , 1 paziente con pregresso condrosarcoma della mano ed , infine , 1 paziente con pregressa neoplasia laringea . tutte le procedure sono state condotte con paziente in posizione prona da due operatori con lausilio di una guida combinata radioscopica - tomodensitometrica utilizzando un intensificatore di brillanza ( oec 9800 plus , ge healthcare wis ) ed una tomografia computerizzata ( tc ) ( lightspeed qx / i , ge healthcare wis )  . 
in ogni procedura , dopo aver eseguito due scout preliminari in proiezione anteroposteriore e latero - laterale , si proceduto a confezionare un pacchetto comprendente il metamero immediatamente soprastante e quello immediatamente sottostante a quello / quelli da trattare . 
i parametri di scansione erano : helical scanning , tempo di rotazione 0 , 8 s , spessore di slice 5 mm , velocit del tavolo 30 mm / s , intervallo di ricostruzione 5 mm , campo di vista ( fov ) largo , 80 kv , 4060 ma [ 12 ]  . 
per ci che concerne la scopia si utilizzava lesposizione automatica dellapparecchiatura . stata utilizzata esclusivamente anestesia locale sottocutanea e periostale con 23 ml di lidocaina all1% o , soprattutto nei crolli vertebrali del tratto dorsale del rachide , con una miscela di lidocaina all1% e triamcinolone . 
il prelievo bioptico stato eseguito immediatamente prima dellinfusione del cemento biologico radiol med ( 2011 ) 116 : 302309 utilizzando sempre la tecnica coassiale attraverso lago da vertebroplastica il cui calibro era di 13 g o 10 g . 
essa ha una lunghezza di 2 cm superiore a quella dellago da vertebroplastica , ci permette di penetrare profondamente nel corpo vertebrale per ottenere il frustolo osseo , ma richiede un accurato controllo fluoroscopico intraprocedurale per evitare lesioni della corticale vertebrale . 
il prelievo ottenuto stato fissato in formalina neutra tamponata con tampone fosfato 0 , 05 m e successivamente posto , per 3 ore , in una soluzione di sodio citrato tribasico e acido formico fino a completa demineralizzazione ed infine incluso in paraffina dopo processazione con processatore automatico leica asp200 s . 
per ogni blocchetto contenente il materiale incluso in paraffina sono state allestite delle sezioni dello spessore di 3 micron le quali sono state poi colorate con ematossilina - eosina tramite coloratore automatico leica 5010 . 
for each block containing paraffin - embedded material , 3 - m - thick sections were prepared , which were then stained with haematoxylineosin using an automated leica 5010 stainer . 
immunohistochemical analyses with a ventana benchmark automated immunostainer ( ventana , tucson , az , usa ) employing antibodies against specific tumour antigens were performed in cases requiring further investigation . results in 83 of 98 patients ( 85% ) undergoing pvp , histological examination on the biopsy confirmed the diagnostic suspicion of osteoporotic vertebral compression fracture . 
in 13 cases ( 13% ) , the biopsy nei 98 pazienti sottoposti a vp lesame istologico sul prelievo bioptico ha confermato lipotesi diagnostica in 83 casi ( 85% )  . 
il tempo procedurale aggiuntivo imputabile allesecuzione della biopsia con tecnica coassiale stato in tutti i casi contenuto , compreso fra 3 e 5 minuti , anche in considerazione del fatto che ci si sempre limitati ad 12 prese bioptiche mentre altri autori adottano lesecuzione routinaria di multiple prese bioptiche [ 13 ]  . discussione malgrado vengano effettuate indagini diagnostiche , 306 radiol med ( 2011 ) 116 : 302309 fig . 
in our experience , the majority of patients undergoing pvp had a prebiopsy suspicion of osteoporotic vertebral compression fracture , whereas a smaller proportion had a well - founded suspicion of secondary neoplasm or localisation of a blood dyscrasia . 
nella nostra esperienza la maggior parte dei pazienti che sono andati incontro a vertebroplastica avevano un sospetto prebioptico di crollo vertebrale su base osteoporotica mentre una percentuale inferiore aveva un fondato sospetto prebioptico di tipo neoplastico secondario o di localizzazione di discrasia ematologica . 
in questo lavoro sono stati presi in considerazione solo i pazienti con sospetto pre - bioptico di cedimento vertebrale su base porotica . in letteratura vi sono pochi lavori che descrivono lassociazione tra vp e biopsia con tecnica coassiale [ 9 , 13 , 16 ] , tuttavia il numero di complicanze estremamente basso e comunque non superiore a quello legato alla semplice esecuzione di vp . 
il tempo aggiuntivo per la procedura bioptica assolutamente trascurabile . il razionale della biopsia coassiale quello di confermare la natura del crollo vertebrale , questo dopo aver gi formulato unipotesi sulla base dei dati clinici , laboratoristici e strumentali . 
histological identification of myeloma , lymphoma or spinal metastases in patients linfoma o localizzazioni secondarie al rachide in pazienti con un crollo vertebrale su iniziale sospetta base porotica ha portato diversi autori [ 1416 ] a sostenere la necessit e limportanza di procedere sempre ad un prelievo bioptico in pazienti sottoposti a vp per la prima volta . 
lesame istologico su prelievo bioptico si rilevato utile anche nella nostra esperienza , evidenziando una localizzazione neoplastica al rachide in 2 pazienti ( carcinoma ad origine prostatica e mieloma ) che avevano un sospetto clinico308 radiol med ( 2011 ) 116 : 302309 fig . 
biopsies confirmed diagnoses insufficient or unsuitable material unexpected result 83 ( 85% ) 13 ( 13% ) 2 ( 2% ) tabella 1 risultati istologici dei prelievi bioptici numero biopsie conferme diagnostiche materiale non idoneo o insufficiente risultato non atteso 83 ( 85% ) 13 ( 13% ) 2 ( 2% ) presenting with presumed osteoporotic vertebral compression fracture has led several authors [ 1416 ] to recommend that a biopsy be performed in all patients undergoing pvp for the first time . 
the value of histological examination of the biopsy was confirmed in our experience , as spinal metastases were revealed in two patients ( from prostate cancer and myeloma ) who had a clinical and imaging suspicion of osteoporotic fractures . 
although this percentage of clinically unexpected results is undeniably very small , a correct histological diagnosis can indeed allow a prompt change in the treatment , which is quite different from that for an osteoporotic patient . the number of inadequate or unsuitable samples could be attributed to three factors : firstly , to limit the additional time required for the biopsy , we collected only one or two samples during each pvp procedure , whereas other authors [ 13 ] routinely collect two to six samples . 
certo questa una percentuale esigua di risultati clinicamente inattesi , tuttavia la corretta diagnosi istologica permette di modificare prontamente la terapia , del tutto diversa da quella di un paziente porotico . riguardo al numero di prelievi insufficienti o non idonei vanno considerati tre fattori : in primo luogo nella nostra esperienza ci si limitati ad 12 prelievi bioptici durante vp mentre , come gi detto , alcuni autori [ 13 ] adottano lesecuzione routinaria di un numero maggiore ( 26 ) di prelievi ; il razionale quello di contenere il tempo aggiuntivo legato alla biopsia . 
in secondo luogo nel nostro istituto , per ragioni logistiche e organizzative , non abbiamo la possibilit di avere presente un anatomopatologo al momento della biopsia in grado di valutare estemporaneamente qualit e quantit del prelievo , a differenza di ci che avviene per le biopsie su organi parenchimatosi dove radiol med ( 2011 ) 116 : 302309 centres where biopsies on parenchymatous organs reach adequacy rates of 98%99% , our institute has no on - site pathologist to provide immediate evaluation of the quantity and quality of the sample due to local logistical and organisational reasons . 
the cost related to the histological examination of the biopsy sections is also very small . in conclusion , performing coaxial biopsy in conjunction with pvp should be considered in all patients undergoing pvp for the first time given that it is a safe procedure that does not prolong procedures times excessively , entails only a small additional cost whatever kit is used and , more importantly , is invaluable for characterising vertebral lesions and confirming the clinical and imaging suspicion , thereby helping to establish a correct diagnosis . lidoneit raggiunge percentuali del 98%99% . 
included in the study are patients at the first documented episode of acute pe , with 6 month follow - up . patients with severe pre - existent cardiopulmonary pathology or neoplastic diseases were excluded from the study . 
ct angiography evaluated right ventricular ( rv ) / left ventricular ( lv ) ratio , obstruction index according to qanadli and total clot burden ( ghanima score )  . 
stata riscontrata una significativa associazione tra lo score ghanima e quello di qanadli : i due indici sono risultati equivalenti nel quantificare lostruzione del circolo arterioso polmonare ( p < 0 , 001 )  . 
tra i parametri angio - tc lo score ghanima ha dimostrato di possedere la pi alta accuratezza nellindividuare i pazienti che svilupperanno il radiol med ( 2011 ) 116 : 230245 ( 76% )  . 
this approach allows to obtain , with just one test , both the diagnosis and a rather accurate acute pe risk stratification . keywords pulmonary embolism computed tomography pulmonary artery obstruction index risk stratification cuore polmonare cronico ( 76% )  . 
lo score ghanima pu essere utilizzato nella diagnosi angio - tc in urgenza come marker prognostico per una rapida stratificazione del rischio di cuore polmonare o decesso dei pazienti con ep acuta . 
lutilizzo di questo score consente di ottenere , con un unico esame , sia la diagnosi che una valutazione di gravit abbastanza accurata dellep acuta . parole chiave embolia polmonare angio - tc indice di ostruzione arteria polmonare stratificazione del rischio introduction introduzione pulmonary embolism ( pe ) represents a relatively common cardiovascular emergency that , by occluding the pulmonary arterial bed , may lead to severe acute but potentially reversible right ventricular ( rv ) failure . 
multislice computed tomography ( msct ) pulmonary angiography has recently gained widespread acceptance as a primary imaging technique to confirm the clinical suspicion of acute pe , replacing ventilation - perfusion scintigraphy and conventional pulmonary angiography [ 13 ]  . 
scintigraphy still plays a major role in nuclear medicine facilities by providing round - the - clock emergency imaging , thanks to its high negative predictive value ( npv )  . 
in its new guidelines for diagnosing and treating acute pe , the european society of cardiology recently included ct angiography ( cta ) as the reference standard modality to be used following initial clinical evaluation [ 5 ]  . the development of multidetector scanners has brought a number of technical advantages ( increased spatial and temporal resolution , excellent vascular opacification , reduced image noise , etc . ) and diagnostic improvements ; the possibility of performing a rapid examination in patients with prominent dyspnoea ; minimised motion and breathing artefacts [ 6 ] ; direct visualisation of the thrombus in segmental or subsegmental arteries ; and good interobserver agreement [ 7 ]  . if the examination is extended to include the lower limbs and abdomen , there is the additional possibility of performing ct venography with a single injection of contrast material [ 8 ]  . furthermore , ct provides visualisation of both mediastinal and parenchymal structures , allowing for alternative diagnoses , a particularly important aspect if one considers that the clinical presentation is often nonspecific and the differential diagnosis includes many cardiopulmonary conditions . in recent years , an increasing number of studies has lembolia polmonare ( ep ) rappresenta unemergenza cardiovascolare relativamente comune che , determinando lostruzione del letto arterioso polmonare , pu indurre grave scompenso acuto del ventricolo destro anche se potenzialmente reversibile . 
langio - tomografia computerizzata ( tc ) polmonare multistrato si affermata negli ultimi anni come principale metodica di imaging nel confermare il sospetto clinico di ep acuta , sostituendo la scintigrafia ventilo / perfusionale e langiopneumografia [ 13 ]  . 
la concordanza di risultati tra la scintigrafia e la tc molto elevata in particolare quando le due indagini vengono effettuate entro 2448 ore dallinsorgenza del sospetto clinico [ 4 ]  . 
recentemente , la european society of cardiology ( esc ) nelle nuove linee guida per la diagnosi ed il trattamento della ep acuta , ha inserito langio - tc come indagine gold - standard , dopo liniziale valutazione clinica [ 5 ]  . lo sviluppo delle apparecchiature multidetettore ha portato una serie di vantaggi tecnici ( aumento della risoluzione spaziale e temporale , ottimale opacizzazione vascolare , diminuzione del rumore , ecc . ) e diagnostici : possibilit di eseguire lesame rapidamente anche in pazienti sofferenti ed intensamente dispnoici ; minimizzazione degli artefatti classici della tc generati dal movimento e dagli atti respiratori [ 6 ] ; visualizzazione diretta del trombo , fino ai rami subsegmentari con un buon accordo interobserver [ 7 ]  . 
con lestensione dellesame agli arti inferiori e alladdome vi la possibilit di eseguire anche una venografiatc con ununica iniezione di mezzo di contrasto ( mdc ) [ 8 ] ; inoltre la tc offre la possibilit di visualizzare sia le strutture mediastiniche che parenchimali e consente di formulare anche ipotesi diagnostiche alternative , aspetto 232 radiol med ( 2011 ) 116 : 230245 emphasised that decisions to adopt a more or less aggressive approach to pe should be based on risk stratification . progressive rv failure is the most frequent cause of death related to pe . 
the interventricular septum may protrude into an otherwise normal right ventricle , thus narrowing its lumen and causing incomplete filling , leading to decreased lv output and reduced systemic arterial pressure and coronary artery flow . 
additionally , the increase in rv wall tension produces compression of the right coronary artery and may trigger myocardial ischaemia and rv infarction . the new guidelines of the american college of chest physicians [ 9 ] confirm that treatment during the acute phase should be tailored to the individual patient based on clinical risk . 
lastly , high - risk patients , especially those with evidence of shock or hypotension , should receive thrombolytic therapy . clinical trials have demonstrated that cta can play a major role in evaluating the severity of acute pe , as it helps to assess numerous parameters : in particular , rv / lv ratio [ 1012 ] , interventricular septum displacement [ 11 ] and pulmonary artery [ 11 ] and superior vena cava [ 11 , 13 ] diameter . the presence , position and degree of pulmonary artery occlusion may be quantified by using vascular obstruction indices . 
despite their proven value in quantifying arterial obstruction , their use is limited by their complexity . recently , the qanadli score [ 14 ] , based on evaluation of all arterial branches involved in the obstruction , has been replaced by a score proposed by ghanima et al . 
 [ 15 ] , which is easier to apply in clinical practice . the aim of this study was to analyse the cta findings reflecting pe severity ; correlate them with clinical , laboratory and haemodynamic data ; and determine their value in predicting the development of chronic rv overload . 
lincremento delle resistenze vascolari polmonari determina un aumento della tensione di parete del ventricolo destro con conseguente dilatazione e alterazione funzionale . il setto interventricolare pu protrudere allinterno del ventricolo sinistro , di per s normale , restringendone la cavit e determinando un incompleto riempimento , con riduzione della portata ventricolare sinistra , della pressione arteriosa sistemica e del flusso coronarico . 
laumento della tensione di parete del ventricolo destro provoca inoltre una compressione sullarteria coronaria destra e pu scatenare unischemia cardiaca ed un infarto ventricolare destro . le ultime linee guida dellamerican college of chest physicians ( accp ) [ 9 ] confermano come la terapia in fase acuta dovrebbe essere personalizzata in ogni singolo paziente sulla base del rischio clinico . 
i pazienti a rischio intermedio , in apparenti condizioni di stabilit emodinamica al momento del ricovero , ma con segni di disfunzione ventricolare destra , necessitano di unintensificazione della terapia durante lospedalizzazione . 
infine i pazienti a rischio elevato , soprattutto se con segni di shock o ipotensione , devono essere sottoposti a terapia trombolitica . studi clinici hanno dimostrato come langio - tc possa avere un ruolo importante nella valutazione di gravit dellep acuta attraverso la valutazione di numerosi parametri : in particolare quelli maggiormente utilizzati sono stati il rapporto ventricolo destro ( vd ) / ventricolo sinistro ( vs ) [ 1012 ] , la deviazione del setto interventricolare [ 11 ] , il diametro dell arteria polmonare [ 11 ] , il diametro della vena cava superiore [ 11 , 13 ]  . 
from analysis of the ed registers for ct pulmonary angiography studies performed at the radiology unit of the ed , 79 patients with a diagnosis of acute pe were identified . patients with severe pre - existing cardiopulmonary disease were excluded from the study ( n = 14 ) , as were those with current neoplastic disease ( n = 8 ) , with previous episodes of pe ( n = 4 ) , for whom pe was not the primary diagnosis at presentation ( n = 5 ) and for whom no follow - up examination was carried out ( n = 3 )  . 
therefore , the study included 45 patients 29 women ( 65% ) and 16 men ( 35% ) aged 2692 ( mean 71 ) years at their first documented episode of acute pe and were followed up after 6 months . clinical presentation on admission to the ed was characterised by dyspnoea in 18 patients ( 40% ) , chest pain in ten ( 22% ) , syncope or lipothymia in nine ( 20% ) , asthenia and lower - limb oedema in seven ( 16% ) and shock in one ( 2% )  . in 30 patients ( 67% ) , the presence of deep venous thrombosis was demonstrated by ct venography ( n = 8 ) , lowerlimb compression ultrasound ( n = 25 ) or both ( n = 12 )  . patients clinical records were reviewed for troponin i levels , sonographic findings and length of stay in hospital and in the intensive care unit ( icu )  . 
only one patient underwent thrombolysis with a 10 - mg intravenous bolus of recombinant tissue plasminogen activator ( rtpa ) , followed by 85 mg administered over 2 h . patients were followed up by cta performed 6 months after the acute episode . 
the onset of chronic rv overload ( caused by ventricular free wall thickening and ventricular cavity enlargement ) or death during the hospital stay or within the 6 months prior to the first follow - up examination were considered negative outcomes . ct scanning protocol both diagnostic and follow - up cta was performed with a determinare la loro capacit di predire lo sviluppo di sovraccarico cronico del ventricolo destro . 
si voluto inoltre individuare quale , tra i parametri proposti in letteratura , sia pi affidabile e semplice , per una rapida applicazione alla diagnosi in urgenza . materiali e metodi popolazione di studio il nostro uno studio retrospettivo che ha preso in considerazione i pazienti afferiti consecutivamente al pronto soccorso di un policlinico universitario sede di un dipartimento di emergenza e accettazione ( dea ) di 2 livello , nel periodo compreso tra novembre 2007 e dicembre 2008 . 
consultando i registri degli esami angio - tc per lo studio delle arterie polmonari eseguiti presso la radiologia del pronto soccorso , sono stati individuati 79 pazienti con diagnosi di ep acuta . 
sono stati esclusi dallo studio i pazienti con gravi patologie cardio - polmonari pre - esistenti ( 14 ) , con patologie neoplastiche in atto ( 8 ) , con precedenti episodi di ep ( 4 ) , quelli per cui lep non era la diagnosi principale al momento del ricovero ( 5 ) ed infine quelli nei quali non stato possibile eseguire un esame di controllo ( 3 )  . 
sono stati quindi inclusi nello studio 45 pazienti , di cui 29 donne ( 65% ) e 16 uomini ( 35% ) , di et compresa tra 26 e 92 anni ( et media 71 anni ) al primo episodio documentato di embolia polmonare acuta , con follow - up a 6 mesi . il quadro di presentazione clinica al momento dellingresso in pronto soccorso era caratterizzato da : dispnea in 18 pazienti ( 40% ) , dolore toracico in 10 ( 22% ) , sincope o lipotimia in 9 ( 20% ) , astenia ed edema di arto inferiore in 7 ( 16% ) , shock in 1 ( 2% )  . 
in 30 pazienti ( 67% ) stata dimostrata la presenza di trombosi venosa profonda attraverso venografia - tc ( 8 ) , ultrasonografia compressiva ( cus ) degli arti inferiori ( 25 ) o entrambe le metodiche ( 12 )  . 
nello stesso giorno del ricovero veniva iniziata la terapia anticoagulante orale ( tao ) con warfarin 5 mg con lobiettivo di raggiungere un range terapeutico tale da mantenere il valore dellin234 radiol med ( 2011 ) 116 : 230245 multislice ct ( msct ) scanner ( somatom emotion 6 , siemens , forchheim , germany ) , following the ct protocol for pe [ 16 ]  . 
acquisitions were obtained using the spiral technique before and after intravenous administration of 100 cc nonionic iodinated contrast material ( iomeron 350 , bracco spa , milan , italy ) administered via a vein in the arm at a flow rate of 3 ml / s . the automatic care bolus scan - triggering system was used , with the scan starting 4 s after the contrast medium had reached the common pulmonary artery . 
the study of the lower limbs to identify deep venous thrombosis was done with the sequential technique and image acquisition from the popliteal fossa up to the upper kidney poles , with 10 - mm slice thickness and 180 - s delay . ternational normalized ratio ( inr ) tra 2 e 3 . 
un solo paziente stato sottoposto a trombolisi con attivatore tissutale del plasminogeno ricombinante ( rtpa ) in bolo endovenoso di 10 mg , seguito da infusione di 85 mg in 2 ore . levoluzione a distanza stata valutata attraverso lo studio degli esami angio - tc di controllo effettuati a 6 mesi dallepisodio acuto . 
lo sviluppo di un sovraccarico cronico del ventricolo destro ( determinato dallispessimento della parete libera del ventricolo e dallingrandimento della cavit ventricolare ) o il decesso , durante il periodo di ricovero o entro i 6 mesi fino al primo controllo , sono stati considerati come esiti negativi . protocollo di scansione tc image analysis all cta studies were assessed for the following parameters : rv / lv ratio obtained by calculating the ratio between the diameters of the rv and lv short axes in the axial plane , measured from the endocardial margin of the free wall to the interventricular septum [ 17 ] ; axial diameter of the superior vena cava , 2.5 cm from its entrance into the right atrium [ 11 ] ; maximum diameter of the common trunk of the pulmonary artery in the axial plane [ 11 ] ; vascular obstruction score according to qanadli [ 14 ] ( 0100% obstruction ) defined by the number of obstructed segmental arteries and corrected on the basis of the estimated degree of occlusion of each vessel ( correction factor : 1 = partial obstruction ; 2 = complete obstruction ) ; obstruction score according to ghanima [ 15 ] based on proximal extension of the thrombus relative to the main pulmonary arteries ( clot burden )  . 
the pulmonary artery tree is subdivided into four components , and the score is determined according to the level of the proximal extension of the thrombus in each lung : mediastinal arteries ( 4 points ) , lobar arteries ( 3 points ) , segmental arteries ( 2 points ) and subsegmental ( 1 point )  . 
for a more detailed description of vascular obstruction scores , see appendix 1 . statistical analysis gli esami angio - tc sono stati eseguiti sia in fase diagnostica che durante il controllo evolutivo utilizzando tomografo computerizzato multistrato ( tcmd ) somatom emotion 6 ( siemens , forcheim , germania ) seguendo il protocollo tc embolia polmonare [ 16 ]  . 
le scansioni sono state effettuate senza sincronizzazione elettrocardiografica ( ecg ) , comprendendo il parenchima polmonare dagli apici alle basi con spessore di acquisizione 1 , 25 mm , ricostruzioni a 0 , 8 mm , 120 kv , 110 mas . 
le acquisizioni sono state eseguite con tecnica spirale prima e dopo somministrazione di 100 cc di mezzo di contrasto iodato non ionico ( iomeron 350 , bracco spa , milano , italia ) endovena , al flusso di 3 ml / s introdotto a livello di una vena del braccio . 
il completamento diagnostico a livello degli arti inferiori stato effettuato con tecnica sequenziale mediante acquisizioni dai cavi poplitei fino al margine superiore dei reni , con spessore di strato di 10 mm e delay di 180 s , per identificare eventuali trombosi venose profonde . analisi delle immagini per ogni paziente , dallesame angio - tc sono stati valutati i seguenti parametri : rapporto vd / vs ; ottenuto valutando il rapporto tra i diametri degli assi minori del vd e del vs in sezione assiale misurati dal margine endocardico della parete libera a quello del setto interventricolare [ 17 ] ; diametro assiale della vena cava superiore a 2 , 5 cm dallo sbocco nellatrio destro [ 11 ] ; diametro massimo del tronco comune dellarteria polmonare in sezione assiale [ 11 ] ; the kruskal - wallis test was used to compare the continindice di ostruzione vascolare secondo qanadli [ 14 ] radiol med ( 2011 ) 116 : 230245 uous values of the qanadli scoring system with the groups obtained with the ghanima score . 
fishers exact and chisquare tests were used to assess the significance of the association between outcome and parameters examined . receiver operating characteristic ( roc ) curve was used to identify the cutoff values for the rv / lv ratio and the qanadli score . 
sensitivity , specificity and positive predictive values ( ppv ) and npv were measured using openepi software ( the open source initiative )  . results follow - up cta angiography showed complete embolism resolution in 15 patients ( 33% ) , residual thrombi in some arterial branches in 13 ( 29% ) and chronic rv overload ( pulmonary heart disease ) in 13 ( 29% )  . 
death caused by cardiocirculatory failure occurred in four patients ( 9% )  . altogether , the outcome was positive in 28 patients ( 62% ) and negative in 17 ( 38% )  . 
median obstruction , corresponding to level 1 of the ghanima system , corresponds to 7.5% in the qanadli index ; levels 2 , 3 and 4 correspond to a 20% , 35% and 50% median obstruction , respectively . 
distribution of the study population according to the ghanima score was as ( ostruzione 0%100% ) , definito dal numero di arterie segmentarie ostruite , corretto in base alla stima del grado di occlusione di ogni singolo vaso ( fattore di correzione : 1 ostruzione parziale , 2 ostruzione completa ) ; indice di ostruzione secondo ghanima [ 15 ] , basato sulla prossimalit della trombosi rispetto allarteria polmonare principale ( burden embolico )  . 
lalbero arterioso polmonare suddiviso in 4 componenti : lestensione dellembolo a livello delle arterie mediastiniche conferisce un punteggio di 4 , arterie lobari 3 , arterie segmentali 2 e subsegmentali 1 . 
si individuano pertanto 4 livelli di gravit , in senso crescente dal livello 1 al livello 4 . per una descrizione pi dettagliata degli indici di ostruzione vascolare , vedi appendice 1 . analisi statistiche il test di kruskall - wallis stato utilizzato per confrontare i valori continui dellindice qanadli con i gruppi dellindice ghanima . 
lostruzione mediana corrispondente al grado 1 dello score ghanima pari al 7 , 5% secondo lo score qanadli ; i gradi 2 , 3 e 4 corrispondono rispettivamente ad unostruzione mediana del 20% , 35% e 50% . 
 236 radiol med ( 2011 ) 116 : 230245 risultati lo studio angio - tc di controllo in 15 pazienti ( 33% ) ha documentato una risoluzione completa dellembolia ; in 13 pazienti ( 29% ) erano visibili residui trombotici in alcuni rami arteriosi ; un sovraccarico cronico del ventricolo destro ( cuore polmonare ) stato riscontrato in 13 pazienti ( 29% )  . 
il decesso , per linstaurarsi di uno scompenso cardiocircolatorio , si verificato in 4 pazienti ( 9% )  . complessivamente 28 pazienti ( 62% ) hanno avuto una prognosi positiva e 17 ( 38% ) prognosi negativa . stata riscontrata una significativa associazione tra lo score ghanima e quello di qanadli : i due indici risultano equivalenti nel quantificare lostruzione del circolo arterioso polmonare ( significativit : p < 0 , 001 )  . 
lostruzione mediana corrispondente al grado 1 dello score ghanima pari al 7 , 5% secondo lo score qanadli ; i gradi 2 , 3 e 4 corrispondono rispettivamente ad unostruzione mediana del 20% , 35% , e 50% . 
la popolazione di studio secondo lo score ghanima cos distribuita : 5 pazienti di grado 1 , 5 di grado 2 , 11 di grado 3 e 24 di grado 4 . 
i pazienti a prognosi negativa ( cuore polmonare cronico o decesso ) e quelli a prognosi positiva ( risoluzione completa o risoluzione con residui embolici ) si distribuiscono diversamente nei 4 gradi del nuovo score . 
2 , lo score ghanima permette una stratificazione prognostica dei pazienti gi al momento della diagnosi in urgenza attraverso lo studio delle immagini angio - tc : il grado 4 riunisce l88% dei pazienti a prognosi peggiore ( 15 pazienti ) , in particolare vi appartengono tutti e quattro i casi di decesso . 
considerando come cut - off il grado 4 , in relazione alla prognosi lindice di ghanima ha mostrato sensibilit 88% , specificit 68% , valore predittivo positivo ( vpp ) 62% , valore predittivo negativo ( vpn ) 90% e accuratezza 76% , con il riscontro di 9 falsi positivi e 2 falsi negativi ( tabella 1 )  . analizzando il periodo di ricovero emerso come la degenza media aumenti in relazione al grado di ostruzione vascolare : i pazienti del gruppo 1 hanno avuto una media di 6 giorni di ricovero ( 2 deviazioni standard [ ds ] ) ; quelli del gruppo 2 una media di 8 giorni ( 4 ds ) ; gruppo 3 di 11 giorni ( 8 ds ) ; e gruppo 4 di 13 giorni ( 8 ds )  . 
infatti un solo paziente ( 20% ) al grado 1 ed uno solo ( 20% ) al grado 2 della classificazione di ghanima sono stati ricoverati in reparti di terapia intensiva ed entrambi per una sola giornata ; al grado 3 i ricoveri salgono fig . 
il 18% dei pazienti di grado 3 ( 2 pazienti ) hanno sviluppato un sovraccarico cronico del ventricolo destro ( 12% del totale dei pazienti a prognosi negativa )  . 
2 , the ghanima score allows for a prognostic stratification of patients at the time of diagnosis with emergency cta : level 4 was identified in 88% of patients with the worst outcomes ( n = 15 ) , and in particular in all four cases of death . 
if we consider level 4 as the cutoff value , radiol med ( 2011 ) 116 : 230245 the ghanima score in relation to outcome had 88% sensitivity , 68% specificity , 62% ppv , 90% npv and 76% accuracy , with nine false positive and two false negative results ( table 1 )  . analysis of the length of hospital stay showed that the average hospital stay increases with the degree of vascular obstruction : the average hospital stay was 6 days [ 2 standard deviation ( sd ) ] for level 1 patients , 8 days ( 4 sd ) for level 2 , 11 days ( 8 sd ) for level 3 and 13 days ( 8 sd ) for level 4 . 
the qanadli score cutoff value for poor outcome was set at 40% vascular obstruction , yielding 82% sensitivity , 61% specificity , 56% ppv , 85% npv , 69% accuracy and 11 false positive and three false negative results . 
the cutoff value for the rv / lv ratio was set at 1.4 , yielding 62% sensitivity , 83% specificity , 76% ppv , 71% npv and 73% accuracy , with four false positive and eight false negative results ( table 1 )  . 
cutoff values were consistent with those reported in numerous previous studies [ 10 , 12 , 14 , 15 ]  . troponin i values > 0.01 ng / ml were considered positive . this test had 89% sensitivity , 77% specificity , 74% ppv , 91% npv and 82% accuracy in identifying patients with poor outcome , with six false positive and two false negative results . 
ricordando che valori prossimi a 1 , 0 indicano unelevata accuratezza diagnostica , i nostri risultati hanno portato ad un auroc rispettivamente di 0 , 791 ( 95% indice di confidenza [ ic ] , 0 , 6500 , 932 ) e di 0 , 853 ( 95% ic , 0 , 7460 , 960 )  . 
per la popolazione in studio il cut - off dellindice di qanadli predittivo di esito negativo stato posto al 40% di ostruzione vascolare , con sensibilit 82% , specificit 61% , vpp 56% , vpn 85% , accuratezza 69% e il riscontro di 11 falsi positivi e 3 falsi negativi . 
per il rapporto vd / vs il cut off pari a 1.4 , con sensibilit 62% , specificit 83% , vpp 76% , vpn 71% , accuratezza 73% , con 4 falsi positivi e 8 falsi negativi ( tabella 1 )  . 
i valori cut - off identificati sono risultati in accordo con quelli di numerosi altri studi [ 10 , 12 , 14 , 15 ]  . un valore di troponina i superiore a 0 , 01 ng / ml stato considerato come positivo . 
questo test ha mostrato sensibilit 89% , specificit 77% , vpp 74% , vpn 91% e accuratezza 82% nellindividuare i pazienti a prognosi negativa , con 6 falsi positivi e 2 falsi negativi . 
lecocardiografia ha mostrato sensibilit 79% , specificit 65% , vpp 62% , vpn 81% e accuratezza 71% , con 9 falsi positivi e 4 falsi negativi ( tabella 1 )  . 
i dati relativi al diametro della vena cava superiore e del tronco comune dellarteria polmonare non sono risultati statisticamente significativi ( p > 0 , 05 )  . discussione importante poter quantificare lostruzione vascolare vista con langio - tc , perch la tomografia computerizzata usata quotidianamente come metodo iniziale e al tempo stesso pi accurato per diagnosticare lep acuta . 
il grado di ostruzione permette una stratificazione del rischio : pu prospettare lesito infausto , lo sviluppo di cuore polmonare cronico ed essere di aiuto per le scelte terapeutiche da seguire . 
consentono infatti di differenziare tra unostruzione parziale ed una totale e , nonostante le loro differenze metodologiche , hanno 238 radiol med ( 2011 ) 116 : 230245 radiol med ( 2011 ) 116 : 230245 vd / vs reference line specificity fig . 
auroc rispettivamente di 0 , 791 ( 95% ic , 0 , 6500 , 932 ) e di 0 , 853 ( 95% ic , 0 , 7460 , 960 )  . stratification : it may predict a fatal outcome or onset of chronic pulmonary heart disease and help in selecting the most appropriate treatment . the first scoring systems were those proposed for angiography by miller et al . 
although the scores were also adapted for ct , they were limited by an inability to differentiate partial from complete obstruction of pulmonary artery branches . the scoring systems suggested by qanadli et al . 
in fact , they can facilitate distinguishing partial from complete obstruction and , despite their methodological differences , have shown excellent correlation with each other in several studies [ 21 ] , as well as proving to be reliable for patient stratification . 
 [ 11 ] , patients with severe pe ( with evidence of haemodynamic instability ) had an obstruction index of 54%11% , whereas those with nonsevere pe ( stable and treated with heparin ) had an index of 24%18% . 
 [ 23 ] analysed 33 consecutive cases of massive pe , defined as pe associated with systolic arterial pressure < 90 mmhg or a pressure mostrato uneccellente correlazione reciproca in differenti studi [ 21 ] dimostrandosi affidabili anche nella stratificazione di gravit dei pazienti . 
nello studio di collomb et al . [ 11 ] i pazienti con ep severa ( con segni di instabilit emodinamica ) presentavano un indice di ostruzione di 54%11% , mentre quelli con ep non severa ( stabili e trattati con leparina ) di 24%18% . 
lo studio di qanadli et al . [ 14 ] propone un cut - off del 40% , atto ad individuare pi del 90% dei pazienti con disfunzione acuta del ventricolo destro . 
 [ 10 ] studiarono il rapporto tra la disfunzione del vd e lindice di ostruzione tc nei pazienti con ep acuta emodinamicamente stabili e trovarono che il valore medio di ostruzione era 31 , 8%22 , 9% . 
 [ 23 ] analizzarono 33 casi consecutivi di ep massiva , definita come unep associata a pressione arteriosa sistolica < 90 mmhg o a un calo pressorio 40 mmhg per almeno 15 minuti . 
necessita infatti di unaccurata valutazione delle arterie segmentarie e subsegmentarie , che spesso non possibile effettuare , soprattutto 240 radiol med ( 2011 ) 116 : 230245 drop 40 mmhg for at least 15 min 61% of cases , the embolus affected the main pulmonary artery , and in all cases except one it was bilateral . 
as a result , risk is stratified by using other ct parameters , such as the rv / lv ratio , or by ecg and laboratory tests . an important predictive role is that played by troponins , which are nonetheless limited by the need to wait for the test results , with waiting times that vary according to the medical centre . 
an increase in these biomarkers has been found to correlate with the ecg and cta findings of rv dysfunction , and they have a high npv both in terms of mortality and morbidity [ 24 , 25 ]  . 
ecg , whether transthoracic or transoesophageal , has a high sensitivity in screening patients for ventricular dysfunction , although not all these patients will develop chronic rv overload . furthermore , except in rare cases , this method is unable to provide definite confirmation of the presence of pe and is therefore used as a second - line modality for patient assessment [ 26 ]  . the ghanima score [ 15 ] proved to be easy to calculate and equivalent to the other more complex obstruction indices used to quantify embolisit allows determination of the total clot burden on the basis of the proximal extension of the thrombus relative to the main pulmonary artery and its bilateral presence . 
our study shows that the ghanima score has the best prognostic accuracy compared with other cta parameters , as well an ability to screen patients for chronic pulmonary heart disease that is more accurate than that of ecg . 
lincremento di questi biomarkers stato correlato ai dati ecocardiografici ed angio - tomografici di disfunzione ventricolare destra ed ha alto valore predittivo negativo sia in termini di mortalit che di morbilit [ 24 , 25 ]  . 
inoltre questa metodica non permette , salvo casi eccezionali , di documentare con certezza la presenza di embolia polmonare ed quindi utilizzata come metodica di 2 livello nella valutazione del paziente [ 26 ]  . 
il nostro studio ha dimostrato che lindice di ghanima possiede la migliore accuratezza prognostica rispetto agli altri parametri angio - tc e una capacit superiore anche allecocardiografia di individuare i pazienti che svilupperanno il cuore polmonare cronico . 
lo studio dimostra come lelevazione dei valori della troponina i ( valore cut - off utilizzato > 0 , 08 ng / ml ) ha sensibilit 53 , 8% , specificit 92 , 3% , vpp 70% e vpn 85 , 7% nellevidenziare lostruzione embolica dei rami arteriosi maggiori . le arterie polmonari principali risultano coinvolte nel 70% dei pazienti con elevazione della troponina i e solo nel 14 , 3% dei pazienti con valori sottosoglia . la figura 4 si riferisce allangio - tc di un paziente di 79 anni , con sintomatologia di dispnea ingravescente . 
a , b computed tomography angiography ( cta ) performed as the initial diagnostic investigation : obstruction of middle lobe branch and right basal pyramid ( arrows in a ) ; obstruction of superior lingular segment ( arrow in b )  . 
ghanima score was level 3 ; qanadli obstruction index was 35% ; right ventricular / left ventricular ratio was 1 ; troponin i was 0.01 ng / ml ; echocardiography was normal . 
le immagini a e b si riferiscono allangio - tc eseguita in fase diagnostica iniziale : ostruzione del ramo lobare medio e piramide basale destra ( frecce in a ) ; ostruzione del segmento lingulare superiore ( freccia in b )  . 
fig. 5a , b refers to cta performed on admission and show bilateral obstruction of the central pulmonary arteries ( a ) with dilatation of the right atrium and ventricle ( b )  . 
troponin i level was 0.05 ng / ml , and ecg revealed rv dilatation . figure 5c , d refers to follow - up cta and show the partial resolution of embolism with few persisting segmental and subsegmetal filling defects ( c , arrows )  . 
a , b computed tomography angiography ( cta ) performed as the initial diagnostic investigation : massive pulmonary embolism with bilateral obstruction of the main pulmonary arteries ( a ) ; right atrial and ventricular enlargement ( b )  . 
ghanima score level 3 ; qanadli obstruction index 35% ; right ventricular / left ventricular ratio was 1 ; troponin i was 0.01 ng / ml ; echocardiography was normal . 
score ghanima grado 4 ; indice di ostruzione qanadli 60% ; rapporto vd / vs 2 , 1 ; troponina i 0 , 05 ng / ml ; allecocardiografia dilatazione ventricolo destro . 
le immagini c e d si riferiscono allangio - tc di controllo e mostrano la risoluzione parziale dellembolia con persistenza di alcuni difetti di riempimento segmentari e sub - segmetari ( frecce in c )  . 
il ventricolo destro risulta ancora lievemente dilatato ( d )  . our data suggest that clinical severity and outcome of pe are associated with the proximal extension and bilateral presence of the thrombus . 
pulmonary circulation , which normally has a low resistance , is able to compensate for increases in pressure , even when these are produced by a very marked obstruction of the pulmonary circulation ; however , this is unlikely to occur if both lungs are involved and if the embolism affects , even in part , the major arteries . 
instead , it is much easier and more reliable to determine the most proximal level reached by the thrombus inside the pulmonary artery branches . logistic analysis was not performed owing to the e la bilateralit della trombosi . 
la circolazione polmonare , normalmente a bassa resistenza , riesce a compensare gli aumenti pressori dovuti allostruzione anche di una quota elevata di circolo polmonare , questo per avviene difficilmente se entrambi i polmoni sono colpiti e se lembolia coinvolge , anche parzialmente , le arterie di maggior calibro . 
non risulta pertanto vantaggioso conteggiare ogni singolo embolo per calcolare la percentuale di letto vascolare ostruito , bens molto pi semplice , ma allo stesso tempo affidabile , determinare il livello pi alto raggiunto dal trombo nei rami arteriosi polmonari . le limitazioni dovute alla retrospettivit e al basso numero di casi presi in esame hanno fatto s che lo studio non sia stato completato con unanalisi logistica dei risultati . 
in our opinion , a larger study correlating the ghanima score and troponin determination could provide a diagnostic protocol for rapid stratification of cases of embolism with cardiorespiratory instability . total clot burden could be useful to limit the use of cardiac biomarkers and select patients for ecg , thus streamlining the management of acute pe and resource utilisation . 
radiology reports usually describe the location of the emboli : including an indication of clot burden as well could represent a valuable step in this direction . relazione lo score di ghanima con il dato laboratoristico delle troponine sarebbe possibile , a nostro avviso , identificare un protocollo diagnostico per la stratificazione rapida dei casi di embolia con instabilit cardio - respiratoria . 
il total clot burden potrebbe essere utile per restringere luso dei biomarkers cardiaci e per selezionare i pazienti da inviare allo studio ecocardiografico , permettendo in questo modo di snellire il management dellep acuta e di usufruire in maniera razionale delle risorse disponibili . 
la posizione degli emboli solitamente descritta nei referti radiologici : indicare anche il grado del burden embolico potrebbe essere un importante passo in questa direzione . conclusions conclusioni the extent of embolic burden , determined according to the ghanima score , may be used in cta diagnosis as a prognostic marker for rapid stratification of pulmonary heart disease or death risk in patients with acute pe . 
this approach provides , with the use of a single test , both a diagnosis of pe and a relatively accurate risk stratification . lestensione del burden embolico , calcolata secondo lo score ghanima , pu essere utilizzata nella diagnosi angiotc in urgenza come marker prognostico per una rapida stratificazione del rischio di cuore polmonare o decesso dei pazienti con ep acuta . 
indici di ostruzione vascolare qanadli score qanadli score pulmonary arteries are subdivided into ten segmental arteries in each lung ( three to the upper lobe , two to the middle lobe and lingula , five to the lower lobe )  . 
the presence of an embolus in a segmental artery is scored as 1 point , and more proximal emboli are scored a value equal to the number of segmental branches arising from the affected vessel . 
each score is multiplied by 0 , or 1 , or 2 according to the estimated degree of vascular occlusion ( 0 = no obstruction ; 1 = partial occlusion ; 2 = complete occlusion )  . 
the percentage value is then calculated as : ( nd ) / 40100 [ n = score of the embolus multiplied by the number of dependent segments ( min = 1 ; max = 20 ) ; d = degree of obstruction ( min = 0 , max = 2 ) ]  . le arterie polmonari vengono suddivise in 10 arterie segmentarie per polmone ( 3 per il lobo superiore , 2 per il lobo medio e per la lingula , 5 per il lobo inferiore )  . 
la presenza di un embolo in unarteria segmentaria viene calcolato con 1 punto , la presenza di un embolo pi prossimale viene calcolato con tanti punti quanto sono i vasi segmentari che nascono dal vaso colpito . 
ogni singolo punteggio viene moltiplicato per 0 oppure 1 oppure 2 , in relazione alla stima del grado di occlusione del vaso ( 0 = nessuna ostruzione ; 1 = occlusione parziale ; 2 = occlusione totale )  . 
il rilievo di un embolo subsegmentario viene calcolato come un embolo nella corrispondente arteria segmentaria , con sola parziale occlusione ( valore 11 = 1 )  . lintervallo del qanadli score varia da 0 ( normale ) a 40 ; la misura percentuale viene quindi calcolata : ( nd ) / 40100 ( n = valore embolo per il numero di segmenti dipendenti [ min 1 ; max 20 ] ; d = grado di ostruzione [ min = 0 , max = 2 ] )  . ghanima score ghanima score this score measures the proximal extension of the embolus relative to the main pulmonary artery and its branches . 
the pulmonary artery tree is divided into four components : an embolus at the level of the mediastinal lindice valuta il grado di prossimalit della trombosi polmonare rispetto allarteria polmonare principale e alle sue branche . 
lalbero arterioso polmonare suddiviso in 4 componenti : se lembolo si trova a livello delle arterie 244 radiol med ( 2011 ) 116 : 230245 arteries is scored 4 points , at the lobar arteries 3 points , at the segmental arteries 2 points , and at the subsegmental arteries 1 point . 
the score identifies four levels of severity ranging from 1 to 4 . mediastiniche si applica un punteggio di 4 , arterie lobari 3 , arterie segmentali 2 e subsegmentali 1 . 
in this phase , we exposed a polymethyl - methacrylate ( pmma ) phantom to the two mammography units and recorded the exposure parameters used by thethe phantom was subsequently replaced by a dedicated radcal ionisation chamber on which preliminary dose assessments were conducted . 
scopo del nostro lavoro stato quello di veri care quale tipo di mammografo digitale sia meglio dedicare alle indagini di screening in base al noto principio as low as reasonably achievable ( alara ) della radioprotezione di limitazione della dose . 
si quindi proceduto alla comparazione della qualit delle immagini prodotte avvalendosi di un fantoccio con inserti geometrici impostando , su ciascun mammografo , i parametri di esposizione utilizzati per le valutazioni dosimetriche . 
stato riscontrato un risparmio di dose impartita alle pazienti avvalendosi del sistema con anodo in tungsteno ; il selenia - w stato , per tanto , adibito alle attivit di screening della mammella . 
 parole chiave mammogra a digitale dose radiol med ( 2011 ) 116 : 310318 introduction introduzione the purpose of this study was to compare the performance of two digital mammography units produced by the same company ( hologics lorad selenia model ) , featuring similar structural characteristics but using different materials for their target / lter combinations [ 1 , 2 ]  . 
the second unit was installed in early 2009 , giving us a choice as to which unit to assign to screening activities . when choosing a unit to be allocated to screening mammography , special care should be taken to apply the principle of optimisation [ 1 , 2 ] of radiological procedures . 
the aim is to comply with italian legislative decree 187 / 2000 [ 3 ] , which in article 4 ( optimisation ) and article 9 ( special practices ) focuses in particular on screening programmes , as they involve a large number of potentially healthy people [ 4 ]  . 
as a result , we decided to perform a patient dose and image quality evaluation for the two systems at our disposal . the dose indicators compared were breast entrance surface dose ( esd ) and average glandular dose ( agd )  . 
 the former parameter is used as a reference to optimise the examination for evaluating as envisaged by legislative decree 187 / 00 compliance with reference diagnostic levels ( rdl ) , whereas the latter is recognised internationally as an indicator of radiological risk associated with the examination . 
 scopo del presente lavoro il confronto delle prestazioni offerte da due mammogra digitali prodotti dalla stessa ditta ( hologic lorad , modello selenia ) , con caratteristiche costruttive analoghe , ma diversi materiali per laccoppiata anodo / ltro [ 1 , 2 ] ; il ne ultimo del lavoro decidere quale mammografo sia da preferire per le indagini di screening della popolazione femminile . 
il primo mammografo ( selenia - molibdeno [ mo ] ) , utilizzato nella nostra realt a partire dallanno 2006 , soddisfacendo alle richieste di screening e di mammogra a clinica ; nei primi mesi del 2009 stata installata la seconda apparecchiatura , consentendoci di dedicare un mammografo allattivit di screening . nella scelta dellapparecchiatura da dedicare allo screening mammogra co particolare cura deve essere posta al principio di ottimizzazione [ 1 , 2 ] della pratica radiologica in oggetto ; questo anche al ne di ottemperare al d.lgs. 
4 ( ottimizzazione ) e 9 ( pratiche speciali ) pone particolare attenzione proprio ai programmi di screening dal momento che coinvolgono un gran numero di persone potenzialmente sane [ 4 ]  . 
 gli indicatori di dose confrontati sono la dose in ingresso alla mammella ( esd ) e la dose ghiandolare media ( agd ) ; il primo parametro utilizzato come riferimento per lottimizzazione dellesame ai ni della valutazione prevista dal d.lgs 187 / 00 per il rispetto dei livelli diagnostici di riferimento ( ldr ) , mentre il secondo riconosciuto a livello internazionale come rappresentativo del rischio radiologico associato allesame . 
lo studio sulla qualit dimmagine stato condotto da medici radiologi di comprovata esperienza nella refertazione delle immagini mammogra che ; a tal ne ci si avvalsi delle immagini ottenute dallesposizione di fantocci geometrici e delle immagine cliniche . 
 materials and methods materiali e metodi the mammography units in question are equipped with the same dr detector , made of amorphous selenium [ 5 , 6 ] , 24 30 cm2 in size , but differ in the materials used for targets and additional lters . 
the selenia device , hereafter referred to as selenia - mo , is tted with a molybdenum ( mo ) target and molybdenum and rhodium ( rh ) additional ltration , whereas the selenia - w has a tungsten ( w ) anode and rhodium and silver ( ag ) additional ltration . 
upon receiving the devices , we collaborated with hologics technicians to optimise the response of the digital detector , and a i mammogra oggetto di studio sono dotati dello stesso rivelatore dr , in selenio amorfo [ 5 , 6 ] di dimensioni 2430 cm2 , essi differiscono per il materiale costituente gli anodi ed i ltri aggiuntivi . 
il selenia , indicato nel seguito con seleniamo , corredato di anodo in molibdeno e ltri aggiuntivi di mo e rodio ( rh ) , mentre il selenia - w ha lanodo in tungsteno ( w ) e ltri aggiuntivi di rodio e argento ( ag )  . 
entrambe le apparecchiature sono state sottoposte ad un rigido programma di garanzia di qualit che ha seguito lintero processo diagnostico dallacquisizione delle immagini alla loro visualizzazione su monitor di refertazione e alla stampa su pellicola . 
in fase di accettazione delle apparecchiature si collaborato con i tecnici della ditta hologic per ottimizzare la risposta del rivelatore digitale , si quindi messo a punto un programma di controllo di qualit secondo le indicazioni 312 radiol med ( 2011 ) 116 : 310318 quality assurance programme was devised based on indications of the european guidelines for quality assurance in breast cancer screening [ 7 ]  . 
these guidelines focus mainly on training the radiology technicians involved in the weekly quality checks and period detector calibration [ 8 ]  . the reporting workstation is a 5 - mp double - monitor system calibrated to offer the same response by both monitors . 
after the acquisition chain was optimised and before using the selenia - w clinically , we performed a dosimetric comparison between the two mammography units on a standard breast simulated by a semicircular polymethyl - methacrylate ( pmma ) phantom ( 240 mm in diameter and 45 mm thick )  . 
at this preliminary stage , we exposed the standard phantom in a completely automatic mode , allowing the automatic exposure control of each unit to select the optimal working parameters ( target / lter combination , kv , mas ) , hereunder referred to as the standard exposure parameters . the dose delivered with the standard exposure parameters was measured with a radcal 20 6 - 6 m ionisation chamber placed in the reference point at 60 mm from the chest wall and 45 mm from the breast support table [ 10 ]  . 
after these preliminary evaluations , the seleniaw was introduced into clinical practice and , after nearly 1 month of operation , the doses effectively delivered to patients were assessed . all exposure parameters applied by each mammography unit to a sample of more than 400 women ( around 1 , 600 exposures ) were recorded using the digital imaging and communications in medicine ( dicom ) data sent automatically by the imaging system to the picture archiving and communications system ( pacs )  . 
the dicom header contains the patients name , age , exposure parameters ( kv , mas , projection , target lter combination , compression force ) and thickness of the compressed breast detected by the breast compression device with millimetric accucontenute nelleuropean guidelines for quality assurance in breast cancer screening [ 7 ] ; tale programma pone particolare attenzione alladdestramento dei tecnici sanitari di radiologia medica ( tsrm ) coinvolti nellesecuzione dei controlli settimanali e della periodica calibrazione del detettore [ 8 ]  . la stazione di refertazione costituita da un sistema doppio monitor da 5 mp ; essa stata calibrata in modo da garantire la stessa risposta per i due monitor e periodicamente controllata conformemente alle indicazioni dellamerican association of physicists in medicine ( aapm ) task group ( tg ) 18 [ 9 ]  . 
una volta ottimizzata la catena di acquisizione , prima dellutilizzo clinico del selenia - w , si proceduto ad un confronto dosimetrico tra i due mammogra su mammella standard simulata da fantoccio di polimetilmetacrilato ( pmma ) semicircolare di diametro 240 mm e spessore 45 mm ; in questa fase preliminare si scelto di esporre il fantoccio standard , in modalit totalmente automatica , lasciando al sistema del controllo automatico dellesposizione di ciascun mammografo , la scelta dei parametri ottimali di lavoro ( selezione anodo / ltro , kv , mas ) , nel seguito indicati come parametri di esposizione standard . la dose erogata con i parametri di esposizione standard , stata misurata avvalendosi di una camera a ionizzazione radcal 206 - 6m posta nel punto di riferimento sito a 60 mm dalla parete toracica e 45 mm dal piano di appoggio [ 10 ]  . 
 la qualit dimmagine stata valutata su entrambe le apparecchiature servendosi di un fantoccio contenente inserti geometrici ( leeds , tor max ) ; tale fantoccio , dello spessore di una mammella standard , stato esposto impiegando i parametri standard precedentemente determinati , quindi le immagini prodotte sono state esaminate dai medici radiologi . 
 il fantoccio tor max contiene al suo interno : mire a diversa frequenza spaziale ( da 1 a 20 lp / mm ) per la risoluzione ad alto contrasto nelle due direzioni parallela e ortogonale alla parete toracica , inserti circolari di diametro 5 , 6 mm a contrasto decrescente per la valutazione della sensibilit a basso contrasto , due gruppi di 11 inserti puntiformi di diametro 0 , 5 mm e 0 , 25 mm , per la valutazione dei micro - dettagli ad alto contrasto . 
successivamente a queste valutazioni preliminari , il selenia - w stato introdotto nella pratica clinica e , dopo circa un mese dallinizio del suo impiego , sono state valutate le dosi realmente impartite alle pazienti . stato possibile registrare tutti i parametri di esposizione impiegati su ciascun mammografo per un campione di oltre 400 donne ( circa 1600 esposizioni ) avvalendosi dei dati digital imaging and communications in medicine ( dicom ) inviati automaticamente dal sistema di acquisizione al picture archiving and communications system ( pacs )  . 
the s factor [ 13 , 14 ] corrects for differences between the various x - ray spectra that can be used with the different selected target / lter combinations . 
 results and discussion the exposures of a standard semicircular pmma phantom ( diameter 240 mm , thickness 45 mm ) for a preliminary dose evaluation were performed in a full automatic mode , allowing the unit to select the optimal parameters providing diagnostic images ; every measurement was repeated ten times to test the systems reproducibility . 
these same parameters were used when exposing the geometrical phantom ( tor max ) containing the high and low - contrast resolution inserts ; results of the evaluations conducted by the radiologists on the visible inserts are given in table 1 . the preliminary results of phantom exposures might seem to indicate that the two systems are substantially equivalent in terms of image quality but , at the same della dose in ingresso e della dose ghiandolare media nelle reali condizioni di esposizione . mediante la camera a ionizzazione stato misurato il kerma in aria in ingresso ( esak ) per unit di carico nel punto di riferimento , misurato alle diverse tensioni di utilizzo con tutte le combinazioni anodo / ltro disponibili ( r ) ; successivamente stato possibile stimare la dose in ingresso ( esd ) impartita alle pazienti nelle reali condizioni cliniche , avvalendo della formula : esd = rmas ( d1 / d2 ) 2bsf ( 1 ) dove d1 la distanza fuoco - punto di riferimento , d2 la distanza fuoco - pelle paziente e bsf il fattore di retrodiffusione tabulato in funzione del sev del mammografo [ 10 ]  . per la stima della dose ghiandolare media ( agd ) si utilizzata la formula delleuref [ 10 ] : agd = rmas ( d1 / d2 ) 2gcs ( 2 ) dove g e c sono valori tabulati in funzione dello spessore della mammella e della qualit del fascio ( hvl ) : il fattore g corrisponde ad una mammella costituita per il 50% da tessuto ghiandolare ed derivato dai valori calcolati in [ 11 , 12 ] , mentre il fattore c corregge per ogni variazione dalla composizione tipica del seno ( 50% di tessuto ghiandolare ) in funzione dellet della paziente ( < 50 anni e > 50 anni ) [ 11 , 12 ]  . 
 risultati e discussione le esposizioni su fantoccio standard semicircolare di pmma ( diametro 240 mm e spessore 45 mm ) per una valutazione preliminare delle dosi , sono state eseguite in modalit totalmente automatica lasciando al sistema la selezione dei parametri ottimali per il raggiungimento dellimmagine diagnostica ; ogni misura stata ripetuta 10 volte per testare la riproducibilit del sistema . 
 i valori medi di esposizione standard , registrati per le due apparecchiature , sono schematizzati in tabella 1 : il seleniamo ha sempre selezionato laccoppiamento anodo / ltro mo / mo , una tensione di 28 kv ed un carico medio di 68 , 7 mas ; il selenia - w ha sempre selezionato laccoppiamento anodo / ltro w / rh , una tensione di 28 kv ed un carico medio di 81 , 7 mas . 
questi stessi parametri sono stati impiegati per lesposizione del fantoccio geometrico ( tor max ) contenente gli inserti di risoluzione ad alto e basso contrasto ; i risultati delle valutazioni effettuate dai medici radiologi sugli inserti discriminabili sono schematizzati in tabella 1 . i risultati preliminari delle esposizioni su fantoccio , sembrerebbero indicare una sostanziale equivalenza dei due sistemi in termini di qualit dimmagine e , nel contempo , sembrerebbe sussistere un risparmio in termini di dose mediante limpiego del selenia - w . 
data required for analysis were identi ed within the dicom header , dividing to mammography them according unit , patients age , compressed breast thickness and target / lter combination . 
we found that selenia - w tends to select mainly the w / rh combination , with the w / ag combination being selected only for compression thicknesses of 70 mthe seleniamo , conversely , shows more variability in the combinations it selects , although for compression thicknesses 60 mm , it almost invariably selects the mo / rh . 
i dati necessari per lanalisi , sono stati ricercati allinterno dellheader dicom , suddividendoli in base al mammografo utilizzato , allet della paziente , allo spessore della mammella compressa e dellaccoppiata anodo / ltro utilizzata . 
 i tecnici di radiologia medica , nella pratica clinica , lavorano in modalit totalmente automatica , quindi il sistema a selezionare non solo i parametri di esposizione ottimali ma anche laccoppiata anodo / ltro pi indicata alla densit della mammella . 
abbiamo riscontrato che il selenia - w tende a selezionare prevalentemente laccoppiata w / rh e solo per spessori di compressione maggiori o uguali a 70 mm il sistema imposta laccoppiata w / ag . 
il selenia - mo , invece , presenta una variabilit maggiore nelle accoppiate selezionate , anche se per spessori di compressione superiori od uguali a 60mm , quasi esclusivamente impiegata la mo / rh . 
le valutazioni di dose su entrambi i sistemi sono state eseguite considerando tutte le quattro diverse accoppiate anodo / ltro selezionate nella realt clinica . i risultati riportati in figura 2 evidenziano che il ldr radiol med ( 2011 ) 116 : 310318 fig . 
1a , b distribuzione in classi di frequenza degli spessori di mammella compressa dellintero campione analizzato ( a ) e del campione relativo alla sola proiezione cc ( b )  . ering all four target / lter combinations used in clinical practice . the results given in fig . 
figure 3 shows the agd delivered by the two units , which are , in both cases , within the highest values accepted by the rispettato , con ampio margine , da entrambe le apparecchiature e che la dose impartita dal selenia - w risulta quasi dimezzata rispetto a quella del selenia - mo ; tale differenza risulta statisticamente signi cativa allanalisi di un test t di student condotto con un livello di con denza dell1% ( p = 0 , 00001 )  . lagd per esposizione il parametro dosimetrico correlato al rischio di tumore radioindotto , pertanto riveste fondamentale importanza per la valutazione del rapporto rischio / bene cio nei programma di screening mammogra co . 
in figura 3 si riportano le dosi ghiandolari medie impartite dai due mammogra che risultano , in entrambi i casi , inferiori ai valori massimi accettabili da [ 7 ] , tuttavia il confronto tra le due apparecchiature evidenzia un seppur lieve risparmio di dose ghiandolare media con limpiego del selenia - w . 
2a , b confronto tra selenia - mo e selenia - w in termini di esd impartita per a sola proiezione cc e b tutte le proiezioni . european guidelines for quality assurance in breast cancer screening [ 7 ] , even though the comparison between the two devices indicates a slightly lower ags being delivered by the selenia - w . 
mean agd values estimated for each device were analysed with students t test and a 1% con dence level ( p = 0.00001 ) , con rming the dose reduction obtained with the selenia - w . 
 conclusions the dose evaluations conducted by setting the exposure parameters used for standard breasts revealed given the valori medi di agd stimati per ciascuna apparecchiatura sono stati sottoposti a test t di student all1% ( p = 0 , 00001 ) di con denza che ha confermato la riduzione di dose mediante limpiego del selenia - w . 
in tabella 2 si riporta il riepilogo delle dosi stimate ed impartite dai due sistemi . conclusioni le valutazioni di dose effettuate impostando i parametri di esposizione impiegati per una mammella standard , hanno evidenziato a parit di qualit dimmagine , valutata su fantoccio contenente inserti geometrici , un risparmio di dose in termini di kerma in aria nellutilizzo del selenia - w ; questo dato preliminare ha trovato conferma nella pratica clinica , dove si assiste ad un quasi dimezzamento delle dosi in inradiol med ( 2011 ) 116 : 310318 fig . 
the indisputably lower dose delivered by the selenia - w mammography unit led us to allocate this equipment to the mammography screening examinations performed at our facility . disfano ai limiti di agd suggeriti dalleuropean reference organisation for quality assured breast screening and diagnostic services ( euref ) ; inoltre le immagini prodotte dai due selenia sono qualitativamente sovrapponibili . 
i valori di esd e agd impartiti da ciascun mammografo sono stati confrontati tramite test t di student e le differenze stimate sono risultate statisticamente signi cative con un livello di ducia dell1% . 
triulzi poletto editore srl , vermezzo ( milano ) , 2010 published online : 4 february 2011 springer - verlag 2011 on my desk rests a 647 - page book dedicated to paediatric radiology and neuroradiology . 
more than a third of the endeavour is dedicated to neuroradiology ( the main author and some of his co - authors are well - recognised neuroradiologists ) in its various aspects , from the very early stages of brain development to normal and abnormal ndings ( malformations , cerebralvascular pathology , metabolic and degenerative diseases , in ammation and infectious pathology , skull shape and hydrocephalus , tumours and trauma ) through different age groups . 
further information on spinal problems ( spine and spinal cord development , malformations , acquired lesions , craniocervical junction , anatomical variants and trauma ) is also presented . as magnetic resonance imaging is the ideal diagnostic tool for studying and documenting each of the above , the lions share of images is devoted to it , integrated with ultrasonography ( not extensively represented ) and a few computed tomographic images . 
in this respect , great importance is given to radiation protection for young patients , stressing that radiologists should use the least dangerous available instruments to safely image children . at the end of the neuroradiology section , the reader is introduced to general radiology , where lung , cardiovascular , gastrointestinal , genitourinary , osteoarticular apparatuses and trauma are extensively described . 
as in the neuroradiology section , there is usually an introduction , followed by sulla mia scrivania fa bella mostra di s un volume di 647 pagine dedicato alla radiologia e neuroradiologia pediatrica . 
pi di un terzo dellopera dedicato alla neuroradiologia ( lautore principale ed alcuni dei co - autori sono dei ben noti e rispettati neuroradiologi ) nei suoi vari aspetti , dagli stadi precoci dello sviluppo del cervello , ai reperti normali e patologici ( malformazioni , problemi cerebro - vascolari , malattie metaboliche e degenerative , infezioni e patologia infettiva , morfologia del cranio ed idrocefalia , tumori e trauma ) nelle differenti et . 
inoltre , vengono presentati e discussi dati ed informazioni sui problemi del rachide ( sviluppo del rachide e del midollo , malformazioni , lesioni acquisite , cerniera cranio - cervicale , varianti anatomiche e trauma )  . 
 dal momento che la risonanza magnetica ( rm ) lo strumento ideale per lo studio e la documentazione dei problemi di cui sopra , essa ricopre la parte del leone nelle immagini presentate , integrate da quelle ecogra che ( non in gran numero ) a da saltuarie immagini di tomogra a computerizzata ( tc )  . 
da questo punto di vista viene data grande importanza alla protezione dalle radiazioni , mettendo in evidenza come il radiologo dovrebbe utilizzare le apparecchiature disponibili meno pericolose nello studio per immagini dei bambini . al termine della parte dedicata alla neuroradiologia , il lettore viene messo davanti a quella dedicata alla radiologia generale in cui vengono trattati per esteso gli apparati respiratorio , cardiovascolare , gastrointestinale , genitourinario , osteoarticolare ed il trauma . 
 come gi nella sezione dedicata alla neuroradiologia , anche nelle altre lo schema del testo quello di dare un rapido inquadramento clinico - radiologico , seguito poi da nozioni di embriologia , anatomia e tecniche di immagine e dalla presentazione e discussione della malattia in questioradiol med ( 2011 ) 116 : 334335 embryology , anatomy and imaging techniques , followed by presentation and discussion of the affecting disease , giving attention to the different presentations in newborns ( premature babies , in particular ) and children . 
in this respect , one should note that the latest editions of the cited books are not always reported and also that some iconic texts are omitted ( for instance , see the sections on bone malformation )  . 
 da questo punto di vista il lettore dovrebbe tener conto che non sempre vengono indicate le ultime edizioni dei testi citati e che anche alcuni testi fondamentali sono stati dimenticati ( come per esempio nella sezione sulla malformazioni dello scheletro )  . 
simonetti2 1terapia radiante , universit degli studi di roma tor vergata ( ptv ) , viale oxford 81 , 00133 roma , italy 2dipartimento di diagnostica per immagini , imaging molecolare , radiologia interventistica , roma , italy correspondence to : s . 
lobiettivo del nostro studio di valutare la capacit della risonanza magnetica ( rm ) nellidentificare localizzazione ed estensione del danno ischemico acuto , nel predire la reversibilit del danno e nel distinguere fra aree ischemiche acute e croniche nei pazienti con ischemia miocardica senza elevazione del tratto st ( nstemi )  . 
the use of routine imaging techniques such as echocardiography , which relies on contractility changes and possible myocardial thinning , does not always permit an optimal differential diagnosis , and in many cases , the true condition tends to be underestimated . 
the recent introduction of cardiac magnetic resonance ( mr ) imaging , with sequences for evaluating delayed enhancement , and the use of t2 - weighted blackblood short - tau inversion recovery turbo spin - echo ( t2 bbstir - tse ) acquisitions has made it possible to discriminate between the two pathological conditions by visualising the presence of myocardial oedema , which is usually associated with an ischaemic cardiac event . 
the mechanism underlying the formation of myocardial oedema in acute ischaemia is an injury to the cytoplasmic membrane and changes in transmembrane ion transport , which first trigger intracellular oedema - promoting conditions and then cytotoxic oedema , which involves a marked increase in locoregional extracellular water that can be revealed by t2 stir sequences [ 1 , 2 ]  . 
this condition tends to manifest within the first hours after the ischaemic event ( first 3 h ) and continues to increase over the following 2 weeks , when replacement fibrosis develops to an extent directly proportional to the duration of the ischaemic event [ 15 ]  . 
these pathophysiological mechanisms make it possible to ascertain the true conditions of the ventricular myocardium in the immediate subacute phase once the patients haemodynamic status has been stabilised and a certain and highly reliable prognosis has been established [ 6 ]  . 
in delineating the recovery of ventricular function in a patient who has suffered an acute coronary syndrome ( acs ) , magnetic resonance ( mr ) imaging allows both the identification of nonviable myocardium or myocardial scar from previous , more - or - less recent , episodes of ischaemiainfarction and depiction of any reactive oedema that has formed in the infarcted area . this allows identification of the so - called ischaemic penumbra that could be salvaged by immediate revascularisation [ 79 ]  . 
mr imaging has been shown to identify with a high level of sensitivity and specificity the area of mismatch il poter differenziare una condizione ischemica acuta da un quadro ischemico cronico pre - esistente attualmente il quesito diagnostico che pi frequentemente si propone nella routine clinica , soprattutto nelle condizioni acute o subacute ; ed il poter stabilire con certezza tale differenziazione alla base di una successiva e valida scelta terapeutica . 
ricorrendo alle tecniche strumentali di routine come lecocardiografia , basandosi sulle alterazioni della contrattilit e leventuale assottigliamento miocardico , non sempre si giunge ad una ottimale diagnosi differenziale ed in molte situazioni si tende a sottostimare la reale condizione . 
la recente introduzione della risonanza magnetica ( rm ) cardiaca , con sequenze per la valutazione dellenhancement tardivo e limpiego di scansioni black - blood ( bb ) turbo spin echo ( tse ) t2 ( short tau inversion recovery [ stir ] ) , dai dati presenti in letteratura stato possibile discriminare tra le due entit patologiche sfruttando proprio la possibilit di poter evidenziare la presenza delledema miocardico , che solitamente si associa ad un evento ischemico cardiaco . 
il meccanismo di base dello sviluppo delledema miocardico , negli eventi ischemici acuti , consiste in un danno della membrana citoplasmatica e soprattutto in una alterazione dei trasportatori ionici nei meccanismi di trasporto transmembrana , che dapprima innescano una condizione edemigena intracellulare e successivamente una condizione edemigena citotossica , che comporta un notevole aumento della componente idrica locoregionale extracellulare tale da poter essere rivelata con limpiego di sequenze t2 stir [ 1 , 2 ]  . 
tale condizione tende a manifestarsi nelle prime ore dellevento ischemico ( nelle prime 3 ore ) e successivamente continua ad incrementarsi nelle seguenti 2 settimane , lasciando gradualmente il posto a processi di sostituzione fibrotica del tessuto miocardico con unentit direttamente proporzionale alla durata dellevento ischemico [ 15 ]  . 
proprio sulla base di tali meccanismi fisiopatologici possibile discriminare la reale condizione del miocardio ventricolare nellimmediata fase sub - acuta una volta stabilizzato il quadro emodinamico del soggetto cos da poter delineare una prognosi sicura ed altamente affidabile [ 6 ]  . 
proprio nellottica di poter delineare la reale ripresa della funzione ventricolare di un soggetto colpito da una sindrome coronarica acuta , la rm consente di identificare sia il miocardio non vitale ovvero la scare esitata da pregressi eventi ischemici - infartuali pi o meno recenti e simultaneamente di delineare la condizione edemigena reattiva che si potrebbe innestare sulla stessa pregressa radiol med ( 2011 ) 116 : 163177 between chronic and acute ischaemic events in a noninvasive and highly reliable manner [ 1014 ]  . 
 the 2006 appropriateness criteria for cardiac computed tomography ( ct ) and mr imaging recognised that evaluating the location and extent of myocardial necrosis , including no - reflow regions , was an appropriate indication for cardiac mr viability assessment during the subacute phase of infarction [ 1518 ]  . 
the guidelines clearly also recognised as appropriate viability studies prior to revascularisation procedures , those performed to establish the likelihood of functional recovery , and to quantify viable areas in cases with equivocal results at single - photon - emission computed tomography ( spect ) or at dobutamine stress echocardiography [ 19 ]  . 
in addition to representing the gold standard in evaluating left ventricular wall thickness and kinetics , mr imaging is the only noninvasive technique capable of visualising directly not only the areas of myocardial necrosis through the delayed washout of contrast material but also left ventricular wall oedema through the use of t2 - weighted stir sequences . 
 the aim of this study was to assess the role of mr imaging in identifying the location and extent of acute ischaemic injury , predicting the reversibility or irreversibility of the injury and distinguishing acute from chronic ischaemic regions in patients with myocardial ischaemia with non - st - elevation myocardial infarction ( nstemi ) by using cine - mr imaging , delayed enhancement and t2 bb - stir sequences with fat suppression . 
 materials and methods between november 2006 and december 2007 , cardiac mr was performed on 22 patients ( 14 men and eight women ; mean age 5311 years ) with symptoms and electrocardiographic and cardiac enzyme changes ( troponin ) suggestive of nstemi infarction . 
the diagnosis was confirmed by coronary angiography ( ca ) that , combined with intracoronary ultrasound ( icus ) , allowed identification of the culprit lesion responsible for the acute event . 
on admission to the emergency department , all patients underwent preliminary echocardiography , which demonstrated a reduction in all ventricular function indices and segmental kinetic defects referable to coronary artery disease subsequently identified with ca . 
images were acquired with a 1.5area infartuale ; cos da poter individuare la cosiddetta zona di penombra ischemica , che potrebbe essere salvata dal progredire del fronte donda ischemico da un immediato e repentino trattamento di rivascolarizzazione [ 79 ]  . 
secondo questo punto di vista la rm consente di identificare con elevati valori di sensibilit e specificit larea di mismatch tra levento ischemico cronico ed acuto in modo non invasivo ed altamente affidabile [ 1014 ]  . 
 secondo le linee guida pubblicate nel 2006 sui criteri di appropriatezza nellesecuzione di esami di tomografia computerizzata ( tc ) e rm del cuore stata riconosciuta lappropriatezza degli studi di vitalit con rm nei pazienti nelle fasi post - acute dellinfarto al fine di valutare la localizzazione e lestensione della necrosi miocardica e delle regioni di non - reflow [ 1518 ]  . 
stato ovviamente riconosciuto come appropriato anche lo studio della vitalit prima di interventi di rivascolarizzazione , sia per la stima prognostica del recupero funzionale , sia per la quantificazione delle aree vitali nei casi con risultati dubbi alla tomografia computerizzata a emissione di fotoni singoli ( spect ) o alleco - stress con dobutamina [ 19 ]  . 
 materiali e metodi da novembre 2006 a dicembre 2007 sono stati sottoposti ad esame cardio - rm 22 soggetti ( 14 maschi e 8 femmine ; et media 5311 anni ) con sintomatologia , alterazioni del tracciato elettrocardiografico ( ecg ) e movimento enzimatico ( troponina ) riconducibili ad un quadro di infarto nstemi , confermato allesame coronarografico che integrato con valutazione icus ha consentito di individuare la lesione culprit responsabile dellevento acuto . 
tutti e 22 i pazienti , alla loro accettazione al dipartimento di emergenze , sono stati sottoposti ad un preliminare esame ecocardiografico , che ha documentato una riduzione in toto degli indici di funzionalit ventricolare , con i relativi deficit segmentari di cinetica da correlare con la coronaropatia successivamente individuata dallesame coronarografico . 
da tutti i pazienti stato ottenuto il consenso informato scritto e lo studio stato approvato dal comitato etico interno . 166 radiol med ( 2011 ) 116 : 163177 tesla scanner ( gyroscan intera , philips medical systems , best , the netherlands ) equipped with master gradients ( maximum gradient strength 30 mt ; slew rate 150 mt / m / ms . ) and dedicated five - element phased - array surface coil with sensitivity encoding ( sense ) technique . the examination protocol involved a preliminary cinemr study reduced to two planes biventricular short axis and subsequent four - chamber long axis view with turbo field echo , true fast imaging with steady precession , steady - state free precession ( tfe true - fisp - ssfp ) with retrospective electrocardiographic gating during inspiratory breath - holding , with the following scan parameters : tr 3.8 ms ; te 1.8 ms ; flip angle 70 ; matrix 256256 ; fov 400 ; slab 10 mm ; gap 0 ; sense acceleration factor 2.00 ; cardiac phases / cycle 20 ; retrospective gating ; two slabs per breath - hold ; breath - hold duration 1518 s . subsequently , t2 bb - stir - tse sequences with fat suppression were acquired with prospective electrocardiographic gating , through which we were able to identify and locate the reactive myocardial oedema . 
these scans are respectively obtained along the biventricular short axis ( six slices ) and twoor four - chamber long axis ( three slices ) , depending on the location of the ischaemic area , with the following parameters : tr 1333 ms ; te 100 ms ; epi factor 1 ; turbo factor 33 ; matrix 512512 ; fov 350 ; slab 8 mm ; gap 0.8 ; sense acceleration factor 1.00 ; diastolic phase ; slabs per breath - hold 1 ; breath - hold duration 1518 s . 
then , 1015 min after contrast administration , 3d t1 tfe - ir images were obtained to evaluate delayed enhancement . these sequences were acquired with retrospective electrocardiographic gating in the end - diastolic phase and oriented in the same planes as used for the cine - mr sequences and with the following parameters : tr 4.6 ms ; te 1.4 ms ; flip angle 15 ; matrix 256256 ; fov 400 ; slab 14 mm ; gap 7 mm ; sense acceleration factor 2.00 ; ti 260310 ms ; duration , 16 s . 
the results concerning segmental extent of delayed enhancement or signal hyperintensity in the t2 bb - tse sequences were expressed according to the standard 17 - segment model proposed by the american heart association ( aha ) [ 20 ]  . 
the analysis also included measurement of the indices of global left ventricular function , qualitative and quantitative analysis of end - diastolic wall thickness , systolic wall thickening and wall motility , with the aim of identifying segments with contractile abnormalities . 
 lesame rm stato eseguito circa 12 giorni dopo lo studio coronarografico e leventuale trattamento endovascolare , mentre lintervallo di tempo trascorso fra la comparsa dei sintomi e lesame stato in media di 2 , 7 giorni ( 1 , 4 giorni )  . 
sono stati esclusi dallo studio soggetti con quadro di ischemia miocardica acuta con elevazione del tratto st ( stemi ) , scompenso cardiaco di grado severo , claustrofobia e tutte le controindicazioni assolute allesame rm . 
le immagini sono state acquisite con magnete a 1 , 5 tesla ( gyroscan intera , philips medical systems , best , olanda ) equipaggiato con sistema di gradienti dinamici master ( ampiezza massima 30 mt ; slew rate 150 mt / m / ms ) e bobina di superficie dedicata a 5 elementi con tecnica sense . il protocollo desame risulta composto da un preliminare studio cine - rm ridotto a due soli piani di scansione asse corto bi - ventricolare ed un successivo piano asse lungo 4camere con tecnica turbo field echo ( tfe ) true fast imaging with steady state precession ( fisp ) steady - state free - precession ( ssfp ) con trigger elettrocardiografico retrospettivo in apnea inspiratoria , con i seguenti parametri di scansione : tempo di ripetizione ( tr ) 3 , 8 ms ; tempo di eco ( te ) 1 , 8 ms ; flip angle ( fa ) 70 ; matrice di acquisizione 256256 ; field of view ( fov ) 400 ; spessore di strato 10 mm ; gap 0 ; fattore di accelerazione sense 2.00 ; fasi cardiache / ciclo 20 ; sincronizzazione retrospettiva ; 2 slice ad apnea ; durata apnea 1518 secondi . 
successivamente sono state eseguite scansioni bb t2 pesate a soppressione del grasso ( stir ) con tse con trigger elettrocardiografico prospettico , attraverso le quali stato possibile identificare e localizzare ledema miocardio reattivo . 
tali scansioni vengono eseguite rispettivamente secondo asse corto biventricolare ( 6 slice ) ed asse lungo 2 o 4 camere ( 3 slice ) in relazione alla localizzazione dellarea ischemica , con i seguenti parametri di scansione : tr 1333 ms ; te 100 ms ; fattore epi 1 ; fattore turbo 33 ; matrice di acquisizione 512512 ; fov 350 ; spessore di strato 8 mm ; gap 0 , 8 ; fattore di accelerazione sense 1 , 00 ; fase diastolica ; slice ad apnea 1 ; durata apnea : 1518 secondi . 
successivamente al preliminare studio basale viene somministrato un bolo di mezzo di contrasto paramagnetico pari a 0 , 2 mmoli di gadobenato dimeglumina ( gd - dtpa ) per kg di peso corporeo . 
dopo un intervallo di tempo di 1015 minuti vengono eseguite scansioni inversion recovery ( ir ) 3d tfe t1 pesate per la valutazione del delayed enhancement , ecg - sincronizzate retrospettivamente in fase telediastolica e orientate secondo gli stessi piani delle sequenze cine - rm con i seguenti parametri : tr 4 , 6 ms ; te 1 , 4 ms ; fa 15 ; matrice di acquisizione 256256 ; fov 400 ; spessore di strato 14 mm ; gap 7 mm ; fattore di accelerazione sense 2 , 00 ; tempo di inversione ( ti ) 260310 ms ; durata : 16 secondi . 
 elaborazione dei dati lelaborazione dei dati stata eseguita rispettivamente da radiol med ( 2011 ) 116 : 163177 statistical analysis all the quantitative results [ end - diastolic volume ( edv ) , end - systolic volume ( esv ) and ejection fraction ( ef ) ] were expressed as meanstandard deviation . 
we calculated sensitivity , specificity , diagnostic accuracy , negative predictive value ( npv ) and positive predictive value ( ppv ) of t2 hyperintensity , delayed enhancement and decreased systolic wall thickening , respectively , in identifying areas of acute myocardial infarction and of decreased diastolic wall thickness in identifying myocardial scar . 
results of the latter analysis were expressed as percentages , and sensitivity and specificity values were compared using the mcnemar test . a value of p < 0.05 was considered statistically significant . results mr examination was well tolerated by all patients , and no complications were observed . 
in the 22 patients examined , a total of 374 segments were evaluated , 31 of which ( 8.2% ) showed full - thickness signal hyperintensity in the t2 - weighted sequences . 
twelve patients ( 50% ) showed evidence of myocardial scar from a known previous infarction , which were depicted as areas of delayed enhancement in the viability assessment , for a total of 26 segments ( 6.9% of the total )  . 
contractile abnormalities were identified in 56 segments ( 14.9% ) , of which 28 ( 50% ) were due to acute myocardial ischaemia , 26 ( 46.4% ) to scar from previous infarction and two ( 3.6% ) to left bundle branch block . 
finally , in only 14 ( 53.8% ) of the 26 segments with myocardial scar due to previous infarction was the end - diastolic wall thickness decreased , whereas all healthy segments and those affected by acute myocardial ischaemia showed preserved end - diastolic thickness . 
in all patients , the plaque responsible for the current ischaemic event was identified on the basis of the extent of coronary stenosis or the correspondence between the site of significant stenosis at ca and the electrocardiographic changes ( table 1 )  . 
a comparison between echocardiography performed on admission and subsequent mr imaging demonstrated almost identical results for the function indices , even though the mr study enabled a more analytical and precise assessment of function . 
moreover , mr imaging allowed for a better assessment of ventricular segmental kinetics , especially as regards segments that are difficult to explore with transthoracic un cardiologo ed un radiologo , indipendentemente a doppio cieco . 
stata inoltre condotta la misurazione degli indici di funzionalit globale del ventricolo sinistro e lanalisi qualitativa e quantitativa dello spessore parietale telediastolico , dellispessimento parietale durante la sistole e della motilit parietale , con lo scopo di identificare i segmenti con anomalie contrattili . 
 analisi statistica tutti i risultati quantitativi ( volume telediastolico [ vtd ] , volume telesistolico [ vts ] e frazione di eiezione [ fe ] ) sono stati espressi con valore mediodeviazione standard . 
sono stati calcolati i valori di sensibilit , specificit , accuratezza diagnostica , valore predittivo negativo ( vpn ) e positivo ( vpp ) rispettivamente per liperintensit nelle immagini t2 pesate , il delayed enhancement e la riduzione dellispessimento parietale sistolico nellidentificazione delle aree di infarto miocardico acuto e per il ridotto spessore diastolico nellidentificazione degli esiti di pregresso infarto . 
nei 22 pazienti sottoposti allo studio sono stati valutati un totale di 374 segmenti , di cui 31 ( 8 , 2% ) hanno mostrato iperintensit del segnale a tutto spessore nelle sequenze t2 pesate . 
in un solo paziente ( 4 , 1% ) non stata identificata alcuna area di iperintensit di segnale . in 12 pazienti ( 50% ) sono stati riscontrati esiti di pregresso evento infartuale , noto in anamnesi , visualizzati con aree di iperenhancement tardivo nello studio di vitalit , per un totale di 26 segmenti ( 6 , 9% del totale )  . 
sono state riscontrate anomalie contrattili in 56 segmenti ( 14 , 9% ) , di cui 28 ( 50% ) per lischemia miocardica acuta , 26 ( 46 , 4% ) per esiti di pregressi eventi infartuali e 2 ( 3 , 6% ) per blocco di branca sinistra . 
infine , solo in 14 ( 53 , 8% ) dei 26 segmenti con esiti di pregresso evento infartuale lo spessore parietale telediastolico risultato essere ridotto rispetto alla norma , mentre tutti i segmenti sani o interessati da ischemia miocardica acuta hanno mostrato spessore telediastolico conservato . 
of segments affected segments site of culprit lesion table 1 tabella 1 apical septal middle inferior septal basal inferior apical anterior basal anterior apical lateral basal anterior lateral middle anterior lateral middle inferior lateral apicale settale medio infero - settale basale inferiore apicale anteriore basale anteriore apicale laterale basale antero - laterale medio antero - laterale medio infero - laterale radiol med ( 2011 ) 116 : 163177 proximal third of right coronary middle third of right coronary middle third of right coronary distal third of left anterior descending middle third of left anterior descending middle third of left anterior descending distal third of left circumflex middle third of left circumflex middle third of left circumflex iii prossimale coronaria destra iii medio coronaria destra iii medio coronaria destra iii distale discendente anteriore iii medio discendente anteriore iii medio discendente anteriore iii distale ramo circonflesso arteria coronaria sinistra iii medio ramo circonflesso arteria coronaria sinistra iii medio ramo circonflesso arteria coronaria sinistra numero segmenti segmenti interessati sede placche culprit lesion echocardiography . 
in the detection of myocardial scar due to previous infarction , decreased diastolic wall thickness had a sensitivity and specificity of 53.8% and 100% , respectively , with a ppv of 100% , npv of 96.6% and an overall diagnostic accuracy of 96.7%. 
 discussion acs represents a major diagnostic challenge due to its incidence and high rate of associated mortality and morbidity . more specifically , nstemi infarction is a clinical entity of intermediate severity among the various presentations of acs and one in which the treatment strategy to be adopted is not always clear . 
the use of mr imaging in patients with dellattuale evento ischemico in tutti i pazienti stata identificata sulla base della significativit della stenosi coronarica o della corrispondenza fra la localizzazione della stenosi significativa alla coronarografia e delle alterazioni elettrocardiografiche ( tabella 1 )  . 
lanalisi della funzionalit globale del ventricolo sinistro nei pazienti esaminati ha mostrato un vtd medio di 151 , 232 , 4 ml , un vts medio di 87 , 522 , 4 ml e una fe media di 49 , 367 , 8% . 
un rapido confronto tra il preliminare esame ecocardiografico , eseguito allaccettazione del paziente , ed il successivo studio rm ha documentato una pressocch sovrapposizione dei dati relativi agli indici di funzionalit tra le due metodiche seppur lo studio rm ha permesso una valutazione pi analitica e precisa del quadro funzionale . 
liperintensit nelle immagini t2 pesate , il delayed enhancement e la riduzione dellispessimento parietale sistolico nellidentificazione delle aree di infarto miocardico acuto hanno presentato rispettivamente una sensibilit 96 , 8% , 45 , 8% e 87 , 5% , una dpecificit di 100% , 96 , 9% e 91 , 8% con un vpp di 100% , 68 , 7% e 50% e vpn di 99 , 7% , 92 , 3% e 98 , 7% ed infine unaccuratezza diagnostica complessiva di 99 , 7% , 90 , 3% e 91 , 4% . sono state identificate differenze statisticamente significative fra i valori di sensibilit , valore prognostico positivo e accuratezza diagnostica fra liperintensit del segnale nelle sequenze t2 pesate e le altre due variabili analizzate , radiol med ( 2011 ) 116 : 163177 acs has already been proposed and recognised by the major cardiological and cardioradiological associations as a highly appropriate examination for assessing the extent of necrotic injury due to infarction in the subacute phases of acs and for planning revascularisation procedures , above all in cases in which spect and dobutamine echocardiography have produced equivocal results [ 19 ]  . 
 infarction the results of our study , in agreement with the international literature , showed t2 signal hyperintensity to be an excellent indicator of the presence of acute myocardial ischaemia in patients with nstemi infarction . 
numerous experimental studies have not only documented that myocardial is associated with myocardial oedema , but also clarified that the extent of intramyocardial water accumulation is linearly correlated with signal intensity in t2 - weighted sequences [ 2124 ]  . 
the likely mechanisms leading to increased water content in the myocardial tissue are hyperaemia caused by inflammation resulting from ischaemia , associated with cellular swelling due to changes in cell membranes [ 23 ]  . 
 the correlation of t2 signal hyperintensity with interstitial oedema is also consistent with the observation that the spatial extent of myocardial oedema is usually greater than that of the irreversible myocardial injury ; in fact , the areas showing t2 hyperintensity in the acute phase may subsequently undergo functional recovery , suggesting a mismatch between myocardial oedema and necrosis , which probably indicates myocardium at risk [ 22 , 25 , 26 ]  . numerous clinical studies have demonstrated that delayed enhancement is present in both acute and chronic infarction as a result of the increased volume of distribution of gadolinium chelates due to expansion of the extracellular space in the affected areas : whereas delayed enhancement in acute infarction is related to disruption of cardiac cell membranes with communication between the intraand extracellular space , in chronic infarction , expansion of the extracellular space is due to deposition of collagen matrix in the absence of oedema [ 2729 ]  . 
suggest its persistence for approximately 28 days after coronary occlusion with complete resolution within 36 months , krauss et al . described its persistence over 7 months after an anterior infarction . 
the discrepancy is probably related to the presence or absence of reperfusion in the ischaemic region , which influences the extent and resorption of the interstitial oedema [ 30 , 32 ]  . 
furthermore , the oedema - related t2 cos come fra i valori di specificit solo rispetto allispessimento parietale sistolico e i valori predittivi negativi solo rispetto alliperenhancement tardivo ( p < 0 , 05 )  . 
il ridotto spessore diastolico nellidentificazione degli esiti di pregresso infarto ha invece mostrato una sensibilit e una specificit rispettivamente di 53 , 8% e 100% , con un vpp e un vpn rispettivamente di 100% e 96 , 6% e unaccuratezza diagnostica complessiva di 96 , 7% . 
 discussione la sindrome coronarica acuta ( sca ) rappresenta una sfida di particolare rilievo in ambito cardiologico per lincidenza con cui si manifesta e soprattutto per lelevata percentuale di mortalit e morbidit a cui si accompagna . 
lutilizzo della rm nei pazienti con sca stato gi proposto e riconosciuto dalle pi importanti associazioni di cardiologia e cardioradiologia come esame con elevato grado di appropriatezza nella valutazione dellestensione del danno necrotico infartuale nelle fasi sub - acute della sindrome coronarica acuta , per la programmazione di eventuali interventi di rivascolarizzazione , soprattutto nei casi in cui spect ed eco - stress con dobutamina abbiano conseguito risultati dubbi [ 19 ]  . 
 i dati del nostro studio , in accordo con i dati della letteratura internazionale , hanno mostrato come liperintensit del segnale nelle sequenze t2 pesate sia un ottimo indicatore della presenza di ischemia miocardica acuta nei pazienti con infarto nstemi . 
numerosi studi sperimentali non solo hanno documentato come linfarto miocardico acuto sia associato ad edema miocardico , ma hanno anche chiarito che lentit dellaccumulo intramiocardico di liquidi correli linearmente con lintensit del segnale nelle sequenze t2 pesate [ 2124 ]  . 
verosimilmente i meccanismi che determinano laumento del contenuto idrico del tessuto miocardico sono costituiti dalliperemia indotta dallinfiammazione associata allischemia , in associazione al rigonfiamento cellulare indotto dallalterazione delle membrane cellulari [ 23 ]  . la correlazione delliperintensit del segnale nelle sequenze t2 pesate con ledema interstiziale concorda anche con losservazione che lestensione spaziale delledema miocardico solitamente molto pi ampia dellestensione del danno miocardico irreversibile ; infatti le aree con elevato segnale nelle sequenze t2 pesate nelle fasi acute , possono successivamente andare incontro a recupero funzionale , suggerendo lesistenza di un mismatch fra ledema miocardico e la necrosi che indica probabilmente il miocardio a rischio [ 22 , 25 , 26 ]  . numerosi studi clinici hanno dimostrato come liperenhancement tardivo sia presente nellinfarto miocardico sia acuto che cronico in relazione sostanzialmente allaumento del volume di distribuzione dei chelati del gadolinio 170 radiol med ( 2011 ) 116 : 163177 fig . 
1a - f t2 - weighted black - blood turbo spin echo spectral presaturation inversion recovery images in the biventricular short - axis plane ( a , b )  . 
the most important of these is collagen deposition , a dynamic process that occurs within days of the acute event and continues over the following years : because t2 relaxation time is inversely related to collagen content in muscle , the signal will be significantly lower in mature fibrous tissue [ 32 , 33 ]  . 
the better results can probably be explained in part by the different acquisition paraper lespansione dello spazio extracellulare in tali aree : mentre nellinfarto acuto liper - enhancement tardivo determinato dalla perdita di integrit delle membrane dei miocardiociti edematosi con linstaurarsi di comunicazioni fra lambiente intra ed extra - cellulare , nellinfarto cronico lespansione dello spazio extracellulare derivato dalla deposizione di matrice collagene in assenza di edema [ 2729 ]  . 
in reality , the specificity and ppv were heavily influenced by the low prevalence of disease per segment ( only 32 diseased segments out of a total 374 ) and by patient selection and were both overestimated . 
la discrepanza fra i risultati probabilmente legata alla presenza o meno di riperfusione dellarea ischemica che influenza lentit delledema interstiziale ed il suo riassorbimento [ 30 , 32 ]  . 
inoltre , liperintensit edema - correlata nelle sequenze t2 pesate sensibile ad altri processi che insorgono nella fase di organizzazione dellinfarto , che possono contribuire alla normalizzazione del segnale nel periodo successivo allinfarto , primo fra tutti la deposizione di collagene che un processo dinamico attivo gi pochi giorni dopo levento acuto e persistente negli anni successivi : il tempo di rilassamento t2 inversamente proporzionale alla quota di collagene nel tessuto muscolare , pertanto il segnale risulter significativamente pi basso nel tessuto fibroso maturo [ 32 , 33 ]  . 
the finding of oedema in the t2weighted sequences without delayed enhancement in the viability assessment in some patients with nstemi infarction may be explained by the fact that in the presence of elevated troponin values , the injury does not always cause irreversible segmental or macroscopic subsegmental changes . 
nella nostra casistica con lutilizzo di questa sequenza solamente in un paziente non stato possibile identificare larea di edema miocardico , tuttavia il controllo coronarografico in questo caso ha documentato la presenza di una placca significativa determinante stenosi del lume vasale del 70% a carico del secondo tratto della circonflessa non dominante , con lieve rialzo della sola troponina ed ecg non dirimente per la presenza di alterazioni pre - esistenti del tracciato . 
in realt la specificit ed il vpp sono stati fortemente influenzati dalla bassa prevalenza della patologia per segmento ( solo 32 segmenti colpiti su 374 totali ) e dalla selezione dei pazienti , risultando entrambi sovrastimati . 
di fatto in questi casi , nonostante i risultati di ecg e dosaggio enzimatico siano indicativi di infarto nstemi , i risultati clinici di apparente reversibilit e le caratteristiche miocardiche rilevate dalla rm sarebbero pi affini alle caratteristiche della ua . 
in reality , these values , as with those pertaining to the other myocardial parameters evaluated in our study , were overestimated , particularly as regards specificity and npv , opportuno validare questa ipotesi con controlli rm a distanza di tempo per verificare leffettiva reversibilit del deficit contrattile e la mancata comparsa di delayed enhancement anche nelle fasi croniche . 
se ci fosse dimostrato , si dovrebbe ulteriormente distinguere due categorie di infarti nstemi verosimilmente con due differenti valori prognostici ed indirizzi terapeutici . risultati pi soddisfacenti sono stati ottenuti dallidentificazione del deficit contrattile nelle sequenze cine - rm con sensibilit e specificit rispettivamente di 87 , 5% e 91 , 8% . in realt questi valori , cos come quelli delle altre caratteristiche del miocardio valutate nel nostro studio , risultano sovrastimati , soprattutto riguardo alla specificit ed al valore predittivo negativo , in relazione alla bassa prevalenza segmentaria delle anomalie identificate . 
in questo caso , infatti , il deficit contrattile , identificato nei 26 segmenti con esiti di pregresso infarto del miocardio e nei 2 segmenti settali per la presenza di blocco di branca , radiol med ( 2011 ) 116 : 163177 fig . 
5 a - f immagini su piano asse corto biventricolare ( a , b , f ) e asse lungo quattro camere ( c , d , e ) di sequenza cine - rm balanced - tfe . as a result of the low segmental prevalence of the abnormalities identified . 
in this case , the contractile defect identified in 26 segments with myocardial scar due to previous infarction and in two septal segments due to bundle branch block was a false positive that lead to a specificity of 91.8% in that it compared with 314 segments correctly diagnosed as having normal contractility . 
by contrast , the 50% ppv in identifying segments affected by nstemi infarction is influenced by the low specificity of contractile defects , as both the true positive and false positive segments amounted to 28 . 
 decreased end - diastolic wall thickness , despite having rappresenta un falso positivo che per determina una specificit del 91 , 8% perch contrapposto a 314 segmenti correttamente diagnosticati come normo - contrattili . 
il valore prognostico positivo del 50% nella identificazione dei segmenti colpiti da infarto nstemi risente invece della bassa specificit del deficit contrattile , poich sia i segmenti veri positivi che i falsi positivi sono risultati entrambi 28 . questo significa che il riscontro di un deficit contrattile solo in un paziente su due corrisponde ad unarea ischemica acuta . 
 la riduzione dello spessore parietale telediastolico , sebbene consenta di escludere con elevata accuratezza lischemia acuta , risulta altamente specifico , ma molto poco sensibile nella identificazione dellinfarto cronico con valori rispettivamente di 100% e 53 , 8% , visto che un ridotto radiol med ( 2011 ) 116 : 163177 fig . 
this is explained by the fact that a necrosis involving only a small number of subendocardial myocardial fibres does not necessarily lead to significant myocardial thinning , making this parameter heavily influenced by the intrasegmental extent of the injury . in conclusion , on the basis of data reported in the literature and our preliminary results , we can state that the combination of t2 bb - stir - tse and t1 tfe - ir sequences after administration of contrast material allows precise localisation and differentiation of acute and chronic ischaemic areas . 
although recent reports in the literature have described promising results being achieved with cardiac multidetector computed tomography ( mdct ) in evaluating delayed enhancement [ 35 ] , this option cannot yet be considered satisfactory , especially in view of the radiation dose delivered to patients , which is still definitely too high in relation to the diagnostic information obtained . 
at the same time , it should be noted that to date , mdct is the only noninvasive modality capable of locating the culprit spessore parietale stato riscontrato solo in 14 dei 26 segmenti con esiti infartuali : la spiegazione a questi dati fornita dal fatto che , se la necrosi ha interessato solamente una piccola quota di fibre miocardiche subendocardiche , non necessariamente lo spessore miocardico subisce una significativa riduzione , quindi evidente come questo parametro sia fortemente condizionato dallestensione intrasegmentaria del danno . 
venturi1 1uoc nint neuroimmagini e neurointerventistica , dipartimento di neuroscienze , azienda ospedaliera universitaria senese , policlinico santa maria alle scotte , viale mario bracci , 16 , 53100 siena , italy 2dipartimento di patologia umana ed oncologica , sezione di radiologia universitaria , universit di siena , policlinico santa maria alle scotte , viale mario bracci 16 , 53100 siena , italy 3uoc neurologia malattie neurometaboliche , dipartimento di scienze neurologiche , neurochirurgiche e del comportamento , universit di siena , policlinico santa maria alle scotte , viale mario bracci 16 , 53100 siena , italy 4uoc rianimazione generale , dipartimento di terapia intensiva e anestesia , azienda ospedaliera universitaria senese , policlinico santa maria alle scotte , viale mario bracci 16 , 53100 siena , italy 5dipartimento di neuroscienze , sezione di neurologia , universit di siena , policlinico santa maria alle scotte , viale mario bracci 16 , 53100 siena , italy correspondence to : a . 
the dorsal medulla , red nuclei , cranial nerve nuclei , cerebellum , corpus callosum , frontal and parietal cerebral cortex are less common sites of involvement although they are more frequently affected in nonalcoholic patients . 
paramagnetic contrast material may help to identify lesions not otherwise visible . keywords wernickes encephalopathy computed tomography magnetic resonance riassunto lo scopo di questa rassegna iconogra ca presentare i possibili reperti alla tomogra a computerizzata ( tc ) e alla risonanza magnetica ( rm ) dellencefalopatia di wernicke , una rara grave sindrome neurologica acuta da de cit di tiamina ( vitamina b1 ) , con elevate morbilit e mortalit . 
sedi meno tipiche di interessamento , e pi frequentemente coinvolte nei pazienti non - etilisti , sono la porzione dorsale del bulbo , il nucleo rosso e i nuclei dei nervi cranici , il cervelletto , il corpo calloso e la corteccia fronto - parietale . 
il mezzo di contrasto paramagnetico pu mostrare la presenza di lesioni non altrimenti evidenti . parole chiave encefalopatia di wernicke tomogra a computerizzata risonanza magnetica 320 introduction wernickes encephalopathy is a rare but severe neurological disorder de ned in its various forms by several authors and in particular by karl wernicke in the second half of the nineteenth century [ 15 ]  . 
the classical form is characterised by sudden onset of the clinical triad of eye disorders ( ophthalmoparesis ) , ataxia and cognitive impairment , but the clinical presentation varies widely to the point that it can delay diagnosis and signi cantly worsen the prognosis . 
in the most severe cases , particularly in patients with late diagnosis , there may be progression to persistent severe neurological and psychiatric de ciencies , if not coma and death . 
the study of the brain with computed tomography ( ct ) and especially magnetic resonance imaging ( mri ) plays an essential role in guiding the diagnosis and ruling out other diseases , even though the ndings may be equivocal [ 1034 ]  . 
the purpose of this pictorial essay is to present the ct and mri ndings of wernickes encephalopathy and discuss them in the light of pathogenicity and clinical history . epidemiology wernickes encephalopathy is de nitely more frequent in chronic alcoholics , but it has also been reported to complicate various other conditions , such as uraemia , hyperemesis gravidarum , anorexia nervosa , protracted infectious or febrile conditions , aids , malignancies , dialysis , chronic gastrointestinal disorders , parenteral nutrition or protracted infusional treatment with a glucose solution for instance , following abdominal surgery [ 49 ]  . 
the condition has no racial predilection ; the male sex is the most commonly affected ( m : f ratio = 1.7 : 1 ) , most likely because alcoholism is three to four times more frequent among men than women . 
 aetiopathogenesis the mechanism underlying the development of encephalopradiol med ( 2011 ) 116 : 319333 introduzione lencefalopatia di wernicke una rara ma grave sindrome neurologica de nita nelle sue varie forme cliniche da vari autori , e soprattutto da karl wernicke , nella seconda met del xix secolo [ 15 ]  . 
la malattia consegue a de cit di tiamina ( vitamina b1 ) e colpisce pi frequentemente i soggetti etilisti cronici , anche se pu complicare molte condizioni di malnutrizione in soggetti non - etilisti . 
la forma classica si caratterizza per la comparsa improvvisa della triade clinica costituita da disturbi oculari ( oftalmoparesi ) , atassia e disturbi cognitivi , ma la presentazione clinica estremamente variabile , tale da poterne ritardare la diagnosi e determinarne un notevole peggioramento della prognosi . 
nei casi pi gravi , infatti , ed in particolare nei pazienti con diagnosi tardiva , possibile la progressione verso il coma e la morte o comunque la persistenza di gravi de cit neurologici e psichiatrici . 
il sospetto clinico , la diagnosi precoce e la relativa tempestiva terapia integrativa con tiamina possono consentire , invece , la regressione del danno neurologico e migliorare fortemente la prognosi [ 69 ]  . 
lo studio dellencefalo mediante tomogra a computerizzata ( tc ) e , soprattutto , risonanza magnetica ( rm ) riveste un ruolo fondamentale nellorientamento diagnostico e nellesclusione di altra patologia , seppure i reperti possano non essere univoci [ 1034 ]  . 
lo scopo di questa rassegna iconogra ca presentare i possibili reperti tc e rm dellencefalopatia di wernicke e discuterli alla luce di patogenesi e storia clinica . epidemiologia lencefalopatia di wernicke certamente pi frequente negli etilisti cronici , ma stata descritta come complicanza di molte altre condizioni quali uremia , iperemesi gravidica , anoressia nervosa , condizioni infettive o febbrili protratte , sindrome da immunode cienza umana acquisita ( aids ) , tumori maligni , dialisi , patologie gastrointestinali croniche , nutrizione parenterale o terapia infusionale prolungata con soluzione glucosata come ad esempio nei soggetti sottoposti a chirurgia addominale [ 49 ]  . 
studi autoptici hanno dimostrato lesioni tipiche di encefalopatia di wernicke nello 0 , 8%2 , 8% della popolazione generale ; la frequenza pu arrivare al 12 , 5% nelle popolazioni di etilisti cronici . 
la condizione non ha predilezione razziale ; il sesso maschile pi colpito ( rapporto m : f = 1 , 7 : 1 ) , molto probabilmente poich lalcolismo 34 volte pi frequente negli uomini che nelle donne . 
 radiol med ( 2011 ) 116 : 319333 athy has not been completely elucidated , but it is thought to be dependent on alteration of the metabolism of glial cells and neurons due to the absence of thiamine , a water - soluble vitamin needed to convert glucose into energy . 
in particular , thiamine acts as a cofactor for various enzymes ( transketolase , pyruvate dehydrogenase , ketoglutarate dehydrogenase ) of carbohydrate metabolism and is usually stored in the liver . 
hence , it may be induced by a de cient diet ( typical of populations who mainly eat white rice without the outer husk , i.e. , the part containing most thiamine ) or by alcohol intoxication ( mostly in chronic alcoholics , because alcohol reduces thiamine absorption )  . 
thiamine de ciency causes beriberi ( from the term beri , which in the sinhalese language indicates debilitation ) , a disease causing damage to the nervous system , which then extends to the cardiovascular and gastrointestinal system , even though the most evident symptoms affect one of these systems only . 
 the response to thiamine de ciency tends to be populationspeci c , as asians tend to develop cardiovascular ( or wet ) beriberi , whereas europeans develop neurological ( or dry ) beriberi and wernickes encephalopathy . in the absence of thiamine , the cycles of krebs and of pentose phosphates cannot metabolise glucose , resulting in intracellular accumulation of glutamate , which is subsequently released into the extracellular spaces following homeostatic imbalance and excitotoxic damage [ 23 ]  . 
excess extracellular glutamate may bind to either n - methyl - daspartate ( nmda ) receptors , allowing entry of calcium ( ca2 + ) ions into the cells , with resulting cell injury and death due to necrosis or apoptosis ; or to non - nmda receptors , allowing entry of sodium ( na + ) , with resulting cell injury and death due to cellular oedema ( direct cytotoxic damage )  . 
 the fact that wernickes encephalopathy develops in a few chronic or malnourished alcoholics only has led investigators to hypothesise a genetic predisposition related to variants of the enzymes involved , such as transketolase , but this has not yet been conclusively demonstrated [ 35 ]  . pathology in wernickes encephalopathy the damage occurs more eziopatogenesi il meccanismo attraverso cui si instaura lencefalopatia non stato ancora del tutto chiarito , ma generalmente si ritiene che dipenda dallalterazione del metabolismo energetico delle cellule gliali e dei neuroni indotta dallassenza di tiamina , una vitamina idrosolubile necessaria alla conversione del glucosio in energia . 
in particolare , la tiamina funziona da cofattore per vari enzimi ( transchetolasi , piruvato deidrogenasi , chetoglutarato deidrogenasi ) del metabolismo dei carboidrati e normalmente viene accumulato nel fegato . 
 essa , quindi , pu essere determinata da una dieta carente ( diffusa in popolazioni che si nutrono prevalentemente di riso senza la cuticola , che la parte contiene la maggior parte della tiamina ) , o a unintossicazione da alcool ( per lo pi presente in alcolisti cronici , poich lalcool diminuisce lassorbimento di tiamina ) , ma anche da situazioni particolari , come ipertiroidismo , gravidanza , allattamento o febbre , che portano a una richiesta maggiore di tiamina . 
 la carenza di tiamina provoca beriberi ( dal termine beri della lingua senegalese che indica debilitazione ) , una malattia con danni al sistema nervoso che poi si estendono al sistema cardiovascolare e gastrointestinale , ma i sintomi pi evidenti riguardano uno solo dei sistemi . 
la risposta alla carenza di tiamina tende ad essere popolazione - specica , in quanto gli asiatici tendono a sviluppare soprattutto un beriberi cardiovascolare ( o umido ) , mentre gli europei sviluppano un beriberi neurologico ( o secco ) e lencefalopatia di wernicke . in assenza di tiamina , il ciclo di krebs e dei pentosofosfati non possono metabolizzare il glucosio , con conseguente accumulo intracellulare di glutammato che viene poi rilasciato negli spazi extracellulari per perdita dellomeostasi cellulare e danno citotossico [ 23 ]  . 
leccessivo glutammato extracellulare pu legarsi ai recettori delln - metild - aspartato ( nmda ) , consentendo lingresso di ioni ca2 + nelle cellule che quindi vanno incontro a sofferenza e morte per necrosi o apoptosi , o ai recettori non - nmda , consentendo lingresso intracellulare di na + con conseguente sofferenza e morte da edema cellulare ( danno propriamente citotossico )  . 
involvement of these less typical regions is more frequent in nonalcoholics and is associated in most cases with involvement of the more typical sites [ 32 , 33 ]  . 
1a , b acute wernickes encephalopathy in a nonalcoholic man : serial nonconsecutive iodineenhanced axial computed tomography ( ct ) images obtained at clinical presentation ( a ) , and unenhanced ct images obtained 6 days later ( b )  . 
six days later , they show hypoattenuation in both the mamillary bodies ( arrowhead ) , periaqueductal region ( white arrow ) and both medial thalami ( black arrows ) fig . 
1a , b encefalopatia di wernicke acuta in uomo non - etilista cronico : immagini tc assiali seriali non consecutive dopo iniezione ev di mdc iodato alla presentazione clinica ( a ) e nelle condizioni di base dopo sei giorni ( b )  . 
sei giorni dopo , dimostra ipodensit di entrambi i corpi mammillari ( punta di freccia ) , della regione periacqueduttale ( freccia bianca ) e di entrambi i talami mediali ( frecce nere )  . radiol med ( 2011 ) 116 : 319333 fig . 
2a - e acute wernickes encephalopathy in a nonalcoholic woman : serial nonconsecutive axial ct images ( a ) , and coronal dw ( b ) , adc ( c ) ( b : 1 , 000 s / mm2 ) , axial flair ( d ) and serial nonconsecutive gadolinium - enhanced t1 - weighted ( e ) mri . 
despite negative or doubtful ct images , on flair and gadolinium - enhanced t1 - weighted images , there is slight signal hyperintensity and partial contrast enhancement in both the medial thalami ( black arrows ) , and after i.v. 
2a - e encefalopatia di wernicke acuta in donna non - etilista cronica : immagini tc assiali seriali non consecutive ( a ) e rm coronali dwi ( b ) e adc ( c ) ( b : 1000 s / mm2 ) , assiali flair ( d ) e t1 dipendenti dopo iniezione ev di mdc paramagnetico ( e ) seriali non consecutive . 
a fronte di immagini tc negative o dubbie , alla rm si nota tenue iperintensit di segnale nelle immagini flair e parziale impregnazione dopo mdc dei talami mediali bilateralmente ( frecce nere ) e pi marcata iperintensit , con parziale impregnazione dopo iniezione ev di mdc paramagnetico della corteccia frontale e parietale bilateralmente ( punte di freccia nere )  . 
la regione periacqueduttale ( punta di freccia bianca ) ed i corpi mammillari ( punta di freccia vuota ) presentano solo impregnazione dopo somministrazione di mdc , in assenza di alterazioni dellintensit di segnale . 
the cranial nerve nuclei , on the other hand , would be typically affected in nonalcoholic patients : some authors have even suggested that alcohol protects those regions [ 32 ]  . 
linteressamento dei nuclei dei nervi cranici , invece , sarebbe caratteristico dei pazienti non - etilisti , tanto che alcuni autori ipotizzano un effetto protettivo dellalcol nei confronti di tali regioni [ 32 ]  . 
in et pediatrica , possibile linteressamento della sostanza nera , dei nuclei dei nervi oculomotori , dei nuclei rossi , dei nuclei 324 radiol med ( 2011 ) 116 : 319333 fig . 
high signal intensity is observed in the dorsal medulla ( white arrow ) , facial nerve nuclei ( black arrows ) , periaqueductal grey matter ( white empty arrow ) and mamillary bodies ( black empty arrow ) fig . 
si nota iperintensit di segnale della porzione dorsale del bulbo ( freccia bianca ) delle aree dei nuclei dei nervi facciali ( frecce nere ) , della sostanza grigia periacqueduttale ( freccia vuota bianca ) e dei corpi mammillari ( freccia vuota nera )  . as a result of the high thiamine - dependent metabolism of the nuclear - basal regions in children [ 14 , 21 , 34 ]  . 
 histological ndings include demyelination , alterations of the bloodbrain barrier ( bbb ) with vasogenic oedema and petechial haemorrhage , astrocyte swelling and cellular necrosis in the mamillary bodies , thalamic and hypothalamic nuclei , periaqueductal gray matter , walls of the third and oor of the fourth ventricle and , less frequently , in the caudate nucleus and the frontal and parietal cortex [ 23 , 36 , 37 ]  . 
the progressive loss of neurons in these regions may be responsible for the atrophic alterations found in chronic alcoholics [ 36 ]  . dentati del cervelletto e , soprattutto , dei nuclei della base , ed in particolare del putamen , probabilmente determinato da un elevato metabolismo tiamina - dipendente delle regioni nucleo - basali in et pediatrica [ 14 , 21 , 34 ]  . i rilievi istologici includono demielinizzazione , alterazioni della barriera emato - encefalica con edema vasogenico ed emorragie petecchiali , rigon amento degli astrociti e necrosi cellulare nei corpi mammillari , nei nuclei del talamo e dellipotalamo , sostanza grigia periacqueduttale , pareti del terzo e pavimento del quarto ventricolo e , meno comunemente , nel nucleo caudato e nella corteccia frontale e parietale [ 23 , 36 , 37 ]  . 
la progressiva perdita di neuroni in tali regioni sarebbe responsabile delle alterazioni atro che riscontrate negli etilisti cronici [ 36 ]  . clinical presentation the classic clinical triad of wernickes encephalopathy consists of the abrupt appearance of eye disorders , ataxia and cognitive impairment [ 1 , 2 , 49 ]  . 
the most frequent ocular la triade clinica classica dellencefalopatia di wernicke costituita dalla comparsa improvvisa di disturbi oculari , presentazione clinica radiol med ( 2011 ) 116 : 319333 fig . 
4a - d acute wernickes encephalopathy in a nonalcoholic man : sagittal ( a ) and serial nonconsecutive axial ( b ) t2 - weighted and axial uid attenuated inversion recovery ( c ) and dw ( b : 2 , 500 s / mm2 ) ( d ) images . 
t2 - weighted images show high signal intensity in the dorsal upper medulla , in the area of vestibular nuclei ( short white arrows ) , periaqueductal region ( black arrows ) , both mamillary bodies ( empty arrowheads ) , paramedial nuclei of both thalami ( black arrowheads ) and bilateral frontal and parietal cortex in the central sulci ( long white arrows )  . 
mean adc values of the midbrain and lenticular nuclei , with normal signal intensity , were 0.5310 - 3 mm2 / s and 0.6310 - 3 mm2 / s , respectively . 
4a - d encefalopatia di wernicke acuta in uomo non - etilista cronico : immagini rm sagittale ( a ) e assiali ( b ) t2 - dipendenti seriali non consecutive e assiale flair ( c ) e dwi ( b : 2500 s / mm2 ) ( d )  . 
si nota iperintensit di segnale nelle immagini t2 - dipendenti nella porzione dorsale superiore del bulbo , nella regione dei nuclei vestibolari ( frecce bianche corte ) , della regione periacqueduttale ( frecce nere ) , di entrambi i corpi mammillari ( punta di freccia vuota ) , dei nuclei paramediali di entrambi i talami ( teste di freccia nere ) e della corteccia frontale e parietale in prossimit dei solchi centrali bilateralmente ( frecce bianche lunghe )  . 
i valori medi delladc del mesencefalo e dei nuclei lentiformi di intensit di segnale normali erano di 0 , 5310 - 3 mm2 / s e 0 , 6310 - 3 mm2 / s rispettivamente . 
le lesioni non presentavano impregnazione nelle immagini t1 - dipendenti dopo iniezione ev di mdc paramagnetico ( non mostrate )  . disorders are alterations in eye movements , nystagmus , diplopia and sluggish pupillary re ex ; less frequently there is decreased bilateral visual acuity leading to blindness . 
however , because the classic triad is seen in one third of patients only , it has been suggested that a suspicion of wernickes encephalopathy should be based on two of the following four conditions : malnutrition , oculomotor disorders , cerebellar disorders , altered mental state or even moderate memory defects [ 6 ]  . 
i disturbi oculari pi frequenti sono rappresentati da alterazioni dei movimenti oculari , nistagmo , diplopia , e rallentamento del ri esso pupillare , pi raramente diminuzione bilaterale del visus no alla cecit . 
la triade clinica classica , tuttavia , presente solo in un terzo dei malati , ed stato proposto di sospettare la encefalopatia di wernicke in presenza di due delle seguenti quattro condizioni : malnutrizione , disturbi delloculomozione , disfunzione cerebellare e alterazioni dello stato mentale o difetti mnesici anche moderati [ 6 ]  . 
la sensibilit e la speci cit di tali ultimi criteri , tuttavia , non ancora nota [ 33 ]  . la presentazione clinica , in particolare , estremamente variabile sia in et adulta che in et pediatrica [ 34 ]  . 
5a - d subacute wernickes encephalopathy in a nonalcoholic woman : serial nonconsecutive t2 - weighted ( a , b ) , unenhanced ( b ) and gadolinium - enhanced ( c ) t1 - weighted axial magnetic resonance images . 
t2 - weighted images show swelling and hyperintensity of the mamillary bodies ( empty arrowhead ) and slight signal abnormality of the medial thalami ( short white arrows )  . 
5a - d encefalopatia di wernicke subacuta in donna non - etilista cronica : immagini rm assiali seriali non consecutive t2 - dipendenti ( a , b ) , e t1 prima ( b ) e dopo ( c ) iniezione ev di mdc paramagnetico . 
nelle immagini t2dipendenti si notano tumefazione ed iperintensit di segnale dei corpi mammillari ( punta di freccia vuota ) e pi tenue alterazione di segnale dei talami mediali ( frecce bianche corte )  . 
nelle immagini t1 - dipendenti dopo mdc , si nota dubbia impregnazione dei collicoli inferiori ( frecce nere ) , specie di quello destro . sion , hypothermia , coma , gradual development over several days and without the presence of any element of the classic clinical triad , with bilateral blindness , dysphagia or signs typical of another disease , such as millerfishers syndrome . 
symptoms such as peripheral polyneuropathy ; olfactory discrimination de cits ; bilateral hypacusia ; vestibular paralysis ; cardiovascular signs such as tachycardia , dyspnoea on exertion , postural hypotension ; syncope , hypothermia as an indicator of involvement of the posterior hypothalamus ; but also epileptic seizures may be observed . 
 especially in the later stages of the disease , the appearance of hyperthermia as a sign of involvement of the anterior hypothalamus , pyramidal hemisyndromes ( babinski sign , hyperre exia , muscle hypertonia ) , choreic dyskinesia , and coma are common . 
in these individuals , diagnosis may be achieved before death in only 20% of patients , as the most common presentation is mental confusion without any further symppossibile , ad esempio , lesordio con ipotensione , ipotermia , coma , graduale evoluzione in vari giorni e senza la presenza di nessun elemento della triade clinica classica , con cecit bilaterale , con disfagia oppure con reperti tipici di altra patologia quale , ad esempio , la sindrome di miller - fisher . 
 sono da segnalare sintomi come la polineuropatia periferica , de cit della discriminazione olfattoria , ipoacusia bilaterale , paresi vestibolare , segni cardiovascolari quali tachicardia , dispnea da sforzo , ipotensione posturale , sincope , ipotermia quale indice di coinvolgimento delle regioni posteriori dellipotalamo , ma anche manifestazioni epilettiche . 
soprattutto nelle fasi pi avanzate di malattia , sono comuni la comparsa di ipertermia quale segno di coinvolgimento delle regioni anteriori dellipotalamo , emisindromi piramidali ( segno di babinski , iperre essia , ipertono muscolare ) , discinesie coreiche ed in ne coma . 
in questultima popolazione , infatti , possibile che la diagnosi sia raggiunta prima del decesso solo nel 20% degli affetti , poich lesordio pi frequente radiol med ( 2011 ) 116 : 319333 fig . 
6a - c acute wernickes encephalopathy in a nonalcoholic man : coronal , sagittal and axial uid attenuated inversion recovery ( flair ) ( a ) , axial and sagittal gadolinium - enhanced t1 - weighted ( b ) and late thin - section axial ( c ) magnetic resonance images . 
flair images show high signal intensity not only in typical sites ( periaqueductal area and inferior colliculi , medial thalami and mamillary bodies ) but also in the cerebellar vermis ( full arrowheads ) , dorsal medulla and midbrain ( black arrows ) , fornix ( white arrows ) and frontal - parietal cortex ( empty white arrowheads )  . 
gadolinium - enhanced images show enhancement of mamillary bodies ( black arrows ) and inferior colliculi ( empty black arrowheads ) , which becomes increasingly evident in the late thin - section sequences fig . 
6a - c encefalopatia di wernicke acuta in uomo non - etilista cronico : immagini rm coronale , sagittale e assiale flair ( a ) , assiale e sagittale t1 - dipendenti immediatamente dopo iniezione ev di mdc paramagnetico ( b ) e assiale t1 - dipendente a strato sottile ottenuta successivamente ( in fase tardiva ) ( c )  . 
 nelle immagini flair , oltre che nelle sedi tipiche ( regione periacqueduttale e collicoli inferiori , talami mediali e corpi mammillari ) , si nota iperintensit di segnale nel verme cerebellare ( punte di freccia piene ) , nella porzione dorsale del bulbo e del mesencefalo ( frecce nere ) , nel fornice ( frecce bianche ) e nella corteccia frontale e parietale ( punte di freccia vuote bianche )  . 
dopo somministrazione di mdc si nota impregnazione dei corpi mammillari ( frecce nere ) e dei collicoli inferiori ( punte di freccia vuote nere ) che diviene sempre meglio evidente nelle sequenze acquisite pi tardivamente e con strato sottile . toms [ 6 , 8 , 9 ]  . 
on the other hand , any condition that may mimic wernickes encephalopathy needs to be clinically excluded , especially in individuals at risk presenting with neurological symptoms , such as sleepiness and altered state of consciousness . 
in this situation , the differential diagnosis between pancreatic and wernickes encephalopathy is extremely dif cult , even though thiamine administration can prevent deterioration of the neurological state in individuals with the latter disease . 
daltra parte , clinicamente vanno escluse le condizioni che possono simulare lencefalopatia di wernicke , soprattutto nei soggetti a rischio che si presentino con sintomi neurologici quali la sonnolenza e lalterazione della coscienza . la diagnosi differenziale , quindi , comprende le metastasi ed i tumori cerebrali , le emorragie o lischemia dei tessuti nervosi intracranici , linsuf cienza epatica , la mielinolisi pontina centrale e le infezioni cerebrali . 
in tale condizione , infatti , la diagnosi differenziale tra encefalopatia pancreatica ed encefalopatia di wernicke estremamente dif cile , ma la somministrazione di tiamina pu evitare laggravamento dello stato neurologico nei malati con encefalopatia di wernicke . 
7a - f acute wernickes encephalopathy in an alcoholic man : t2 - weighted sagittal ( a ) , serial nonconsecutive flair ( b , c ) , dw ( d ) and adc ( e ) ( b : 1 , 000 s / mm2 ) axial magnetic resonance images ( mri ) obtained at clinical presentation , and axial flair ( f ) mri obtained 1 week later . 
at clinical presentation , there is high signal intensity on long tr images in the dorsal medulla , in the area of the vestibular nuclei ( full black arrowhead ) , periaqueductal grey matter and inferior colliculi ( white arrow ) and in both medial thalami ( black arrows ) , which show increased diffusion . 
note the atrophy and signal intensity abnormality of the mamillary bodies ( empty arrowheads ) , consistent with a history of chronic alcoholis prompt treatment with thiamine resulted in rapid ( within days ) clinical improvement . 
7a - f encefalopatia di wernicke acuta in uomo etilista cronico : immagini rm sagittale t2 - dipendente ( a ) , assiali seriali non consecutive flair ( b , c ) e dwi ( d ) e adc ( e ) ( b : 1000 s / mm2 ) alla presentazione clinica e assiali flair ( f ) una settimana dopo . 
alla presentazione clinica , si notano alterazione di segnale della porzione dorsale del bulbo nellarea dei nuclei vestibolari ( punta di freccia piena ) , della sostanza grigia peri - acqueduttale e dei collicoli inferiori ( freccia bianca ) e dei talami mediali ( frecce nere ) che presentano incremento della diffusione . 
il controllo rm condotto dopo una settimana dimostra signi cativa regressione dei reperti patologici . wernickes encephalopathy is more complicated , especially in individuals with chronic gastrointestinal diseases , such as crohns , being treated with metronidazole . 
the clinical manifestations of these conditions are very similar , whereas some sites of involvement considered characteristic of metronidazole - induced encephalopathy , such as cerebellar dentate nuclei , cranial nerve nuclei and splenium , may also be affected in wernickes encephalopathy , although they are not typical sites . 
it has been suggested that the neurological damage in these patients is due to both thiamine de ciency and metronidazole neurotoxicity and that the latter may be mediated by mechanisms similar to those responsible for wernickes encephalopathy [ 32 ]  . 
in the most advanced stages of disease , patients may lose their ability to learn new information ( anterograde amnesia ) , lling in the gaps with their past memories without realising their problem , progressing to full - blown psychosis or wernicke korsakoffs syndrome [ 3 , 7 ]  . 
 ziale tra encefalopatia da metronidazolo ed encefalopatia di wernicke , in particolare in soggetti con patologie gastrointestinali croniche , come il morbo di crohn , in trattamento proprio con metronidazolo . 
le manifestazioni cliniche di tali condizioni risultano infatti sostanzialmente sovrapponibili , mentre alcune sedi di lesione considerate caratteristiche della encefalopatia da metronidazolo , quali nuclei dentati del cervelletto , nuclei dei nervi craniali e splenio del corpo calloso , possono essere coinvolte anche nellencefalopatia di wernicke , sebbene non ne rappresentino le sedi tipiche . 
stato ipotizzato che in tali pazienti possa essere presente un danno neurologico conseguente sia alla carenza di tiamina che alla neurotossicit del metronidazolo , nonch la possibilit che questultima possa essere mediata da meccanismi simili a quelli responsabili della encefalopatia di wernicke [ 32 ]  . la prognosi dellencefalopatia di wernicke estremamente variabile , dalla completa remissione della sintomatologia alla irreversibilit delle manifestazioni cliniche [ 59 ]  . 
 nelle fasi pi avanzate i pazienti possono perdere la capacit di apprendere nuove informazioni ( amnesia anterograda ) , riempiendo i vuoti coi ricordi passati , senza rendersi conto del loro problema , sino alla vera e propria psicosi o radiol med ( 2011 ) 116 : 319333 fig . 
8a - e acute wernickes encephalopathy in a nonalcoholic woman : t2 - weighted sagittal and axial ( a ) , serial nonconsecutive coronal uid attenuated inversion recovery ( b ) , gadolinium - enhanced t1 - weighted coronal and axial ( c ) mri at clinical presentation ; and flair coronal ( d ) and gadoliniumenhanced t1 - weighted axial ( e ) mri obtained 10 days later . 
at clinical presentation , high signal intensity areas are visible on long tr images in the inferior colliculi ( black arrows ) and medial thalami ( white arrows ) , with intense contrast enhancement of the inferior colliculi ( empty arrows )  . 
8a - e encefalopatia di wernicke acuta in donna non - etilista cronica : immagini rm sagittale e assiale t2 - dipendenti ( a ) , coronali seriali non consecutive flair ( b ) , coronale e assiale t1 - dipendenti dopo iniezione ev di mdc paramagnetico ( c ) alla presentazione clinica e coronale flair ( d ) e assiale t1 - dipendente dopo iniezione ev di mdc paramagnetico ( e ) dieci giorni dopo . 
alla presentazione clinica , si notano le aree di elevata intensit di segnale nelle immagini a tr lungo nei collicoli inferiori ( frecce nere ) e nei talami mediali ( frecce bianche ) , con intensa impregnazione dopo mdc dei collicoli inferiori ( frecce vuote )  . 
il controllo rm eseguito dieci giorni dopo dimostrava netta regressione dei segni patologici . involvement of less typical sites , especially the cortex , is considered a negative prognostic sign indicating progression and irreversibility of the encephalopathy [ 33 ]  . 
early diagnosis and an adequate supplementation with thiamine ( 100 to 500 mg / day intravenously ) are essential for rapid improvement and to prevent the appearance of irreversible neurological complications and death [ 59 ]  . 
determination of serum thiamine , thiamine monophosphate and erythrocyte transketolase activity can con rm the clinical suspicion , but the tests have limited sensitivity and availability [ 38 ]  . 
neuroradiological investigations are essential for con rming the clinical suspicion and obtaining an early diagnosis [ 8 , 9 ]  . sindrome di wernicke - korsakoff [ 3 , 7 ]  . 
la diagnosi precoce e ladeguato reintegro di tiamina ( da 100 a 500 mg / die per via endovenosa ) sono essenziali per il rapido miglioramento della sintomatologia ed evitano linstaurarsi di complicanze neurologiche irreversibili no al decesso [ 59 ]  . 
il dosaggio della tiamina sierica , della tiamina monofosfato e dellattivit della transchetolasi eritrocitaria possono supportare il dubbio clinico , ma purtroppo sono poco sensibili e scarsamente disponibili [ 38 ]  . 
the absence of neuroradiological ndings does not allow the diagnosis to be excluded , as many cases have been reported of wernickes encephalopathy with negative mri scans [ 12 , 19 ]  . 
 lassenza di reperti neuroradiologici , infatti , non consente di escludere la diagnosi , essendo ampiamente riportati in letteratura casi di encefalopatia di wernicke con esame rm negativo [ 12 , 19 ]  . 
liniezione del mdc paramagnetico , quindi , consente di ridurre la percentuale di falsi negativi dellesame rm e va considerato indicato nel dubbio clinico di encefalopatia di wernicke [ 33 ]  . 
 areas of enhancement in the mamillary bodies and thalami after contrast administration would be more frequent in chronic alcoholics , suggesting a higher sensitivity of these regions to the toxic effect of alcohol [ 32 ]  . 
areas of reduced diffusion are compatible with cytotoxic oedema of astrocytes and neurons due to excitotoxicity , whereas lesions with normal or increased adc values indicate the presence of vasogenic oedema . 
the dwi - adc study is likely to bene t from the availability of images with a thickness < 5 mm and with b values > 1 , 000 s / mm2 , as this has already proved useful in identifying hyperacute and acute lesions of intracranial nerve tissues , both supraand infratentorial [ 39 , 40 ]  . mr spectroscopy the few mr spectroscopy studies performed on wernickes encephalopathy [ 16 , 17 ] have demonstrated an increased lactate peak to be an isolated nding or associated with a reduction in the n - acetylaspartate / creatine ratio . 
le aree di impregnazione dopo mdc di corpi mammillari e talami sarebbero pi frequenti negli etilisti cronici , suggerendo una maggiore sensibilit di tali regioni alleffetto tossico dellalcool [ 32 ] , ma anche tale correlazione non univoca [ 26 ]  . 
 diffusione - rm con misura del coef ciente di diffusione apparente il ruolo dello studio rm in diffusione ( dwi ) con misura del coef ciente di diffusione apparente ( adc ) non ancora de nito . 
proprio per la natura dinamica della siopatologia dellencefalopatia di wernicke , peraltro , possibile la presenza contemporanea di aree di restrizione della diffusione e aree con diffusione normale o aumentata [ 29 ]  . 
le aree di restrizione della diffusione sono compatibili con edema citotossico di astrociti e neuroni da danno eccitotossico , mentre le lesioni con valori di adc normali o aumentati corrispondono alla presenza di edema vasogenico . 
alcuni autori ipotizzano che la presenza di aree di restrizione della diffusione con riduzione dei valori di adc possa rappresentare un fattore prognostico negativo , tuttavia sono necessari ulteriori studi per chiarire se il tipo di alterazioni riscontrate nelle sequenze dwi - adc correlino o meno con la prognosi . 
probabile che lo studio dwi - adc si possa avvantaggiare dalla disponibilit di immagini di spessore inferiore ai 5 mm e con valori di b superiori a 1000 s / mm2 , come gi stato dimostrato utile nellidenti cazione di lesioni iperacute e acute dei tessuti nervosi intracranici , soprae sotto - tentoriali [ 39 , 40 ]  . spettroscopia - rm gli studi di spettroscopia - rm in corso di encefalopatia di wernicke sono pochi [ 16 , 17 ] e generalmente hanno dimostrato lincremento del picco di lattato come reperto isolato o associato a riduzione del rapporto n - acetilaspartato / creatina . 
laumento del picco di lattato , evidente gi in fase acuta , sembra rappresentare unalterazione reversibile , congrua con liperintensit di segnale nelle immagini t2 - pesate ; stato ipotizzato che questo possa dipendere dallaumento del metabolismo anaerobio dei carboidrati conseguente al de cit di tiamina . 
in general , clinical manifestations also regress rapidly after the beginning of the therapy , even though residual de cits may often persist , and clinical progression cannot be excluded if treatment is not timely [ 59 ]  . 
in genere , anche le manifestazioni cliniche regrediscono rapidamente dopo linizio della terapia , ma non rara la persistenza di de cit residui , cos come non pu essere esclusa la progressione clinica in caso di trattamento non suf cientemente tempestivo [ 59 ]  . 
tutto ci conferma la necessit di una diagnosi quanto pi possibile precoce in ogni caso e , comunque , dimostra lopportunit di trattamento preventivo con tiamina nei malati a rischio . conclusions conclusioni wernickes encephalopathy should be suspected not only in chronic alcoholics , but in all clinical conditions involving malabsorption or malnutrition and in patients requiring protracted parenteral nutrition , especially if associated with the infusion of glucose solutions . 
the technique of choice is mri with a dwi - adc study and intravenous injection of gadolinium , bearing in mind that a negative mri does not allow the diagnosis to be excluded . 
mri diagnosis relies on knowledge of typical and atypical sites of involvement and on assessment of clinical and case history ndings . lencefalopatia di wernicke deve essere sospettata non solo negli etilisti cronici , ma in tutte le condizioni cliniche che comportano malassorbimento o malnutrizione e nei malati che necessitano di prolungati periodi di nutrizione parenterale , soprattutto se associata ad infusione di soluzioni glucosate . 
lesame elettivo rappresentato dalla rm comprensivo di studio dwi - adc e studio dopo iniezione ev di mdc paramagnetico , ma la negativit dellesame rm non consente di escludere la diagnosi . 
proportions of cases classified as categories 2 , 3 and 4 were 27.5% ( 90 / 327 ) , 44.3% ( 145 / 327 ) and 7.9% ( 26 / 327 ) , respectively . 
scopo del nostro lavoro stato valutare la resa diagnostica della tomografia computerizzata multidetettore ( tcmd ) del torace nellembolia polmonare acuta ( ep ) e la proporzione di altri reperti clinicamente rilevanti in unampia coorte di pazienti consecutivi , sia ricoverati che provenienti dal pronto soccorso ( ps )  . 
sono stati retrospettivamente considerati 327 referti radiologici di tcmd del torace eseguite per sospetta ep acuta in 327 pazienti ( 158 m , 169 f ; et media , 69 anni , deviazione standard [ ds ] 17 , 33 anni ; 233 ricoverati , 94 provenienti dal ps )  . 
 parole chiave tomografia computerizzata embolia polmonare polmonite introduction introduzione pulmonary embolism ( pe ) is the third most common acute cardiovascular disease and a frequent cause for acute hospital admission , with a reported associated mortality rate exceeding 15% in the first 3 months after diagnosis [ 1 , 2 ]  . 
in clinical practice , the diagnosis of acute pe continues to pose a challenge , because the presenting symptoms and signs are nonspecific and vary widely from one patient to another [ 3 ]  . 
 among diagnostic imaging modalities , multidetector computed tomography ( mdct ) is now considered the technique of choice for depicting the pulmonary vasculature when acute pe is suspected , replacing pulmonary angiography as the reference standard in this clinical scenario [ 5 ]  . 
the widespread availability , noninvasiveness and rapidity of image acquisition are determinant features of mdct , given that an early diagnosis may improve health outcomes and reduce mortality from pe [ 6 , 7 ]  . 
due to its inherent capability to provide detailed visualisation of chest anatomical structures , mdct can also give additional diagnostic the pulmonary parenchyma , mediastinum and thoracic wall , therefore leading to identification of alternative causes for the patients clinical presentation [ 810 ]  . 
 information on given the diagnostic potentialities of chest mdct and its frequent use in clinical practice when acute pe is suspected , the aim of this study was to evaluate over a lengthy period the clinical impact of chest mdct in a large cohort of consecutive patients clinically suspected of having acute pe and referred to our radiology institute . this was done by calculating the diagnostic yield of chest mdct and the proportion of clinically relevant findings other than pe , with particular attention to those requiring intervention . 
a secondary aim was to compare the proportion of chest mdct findings between hospitalised patients ( inpatients ) and patients referred from the emergency department ( outpatients )  . immediate and specific lembolia polmonare ( ep ) considerata la terza pi comune patologia cardiovascolare e rappresenta una causa frequente di accesso ad unit ospedaliera in acuto , con un tasso di mortalit stimato in circa il 15% nei primi tre mesi dalla diagnosi [ 1 , 2 ]  . 
a causa della presentazione con segni e sintomi non specifici , estremamente variabili da paziente a paziente , da un punto di vista clinico la diagnosi rimane un problema aperto [ 3 ]  . 
in soggetti selezionati , la diagnostica di laboratorio si pu avvalere del dosaggio del d - dimero : tale test , dotato di alta sensibilit e di alto valore predittivo negativo , risulta utile specie nellescludere la diagnosi di ep , soprattutto nelle unit di pronto soccorso ( ps ) [ 4 ]  . fra le tecniche di imaging , la tomografia computerizzata multidetettore ( tcmd ) rappresenta attualmente lindagine di prima scelta per la valutazione dellanatomia vascolare polmonare in presenza di sospetto di ep acuta e sostituisce nello scenario clinico odierno langiografia polmonare [ 5 ]  . caratteristiche determinanti della tcmd sono sicuramente la vasta disponibilit , la non invasivit e la rapidit desecuzione , dal momento che una diagnosi precoce fondamentale nel ridurre la mortalit e migliorare loutcome dei pazienti [ 6 , 7 ]  . 
stante la sua intrinseca capacit di fornire informazioni dettagliate anche sul parenchima polmonare e sulle altre strutture toraciche , come il mediastino e la parete , la tcmd pu consentire lindividuazione di eventuali altre cause responsabili della sintomatologia manifestata dal paziente [ 810 ]  . 
 alla luce delle potenzialit espresse dalla tcmd del torace e vista la sua alta frequenza di esecuzione , lo scopo di questo studio stato quello di valutare , in un lungo arco temporale , limpatto clinico della tcmd del torace in una serie consecutiva di pazienti sottoposti allesecuzione della stessa in regime durgenza con sospetto di ep acuta presso il nostro istituto . 
questo stato fatto valutando la resa diagnostica della tcmd e calcolando la proporzione di altri reperti clinicamente rilevanti , ponendo particolare attenzione per quelli che richiedevano un intervento immediato e specifico . 
un obiettivo secondario stato quello di confrontare la proporzione di reperti riscontrati nella sottopopolazione di pazienti ricoverati rispetto a quelli provenienti dallunit di ps . radiol med ( 2011 ) 116 : 219229 materials and methods materiali e metodi the institutional review board approved this retrospective cohort study and waived the patient informed consent requirement . 
the study was conducted in accordance with the principles of the declaration of helsinki . il comitato etico della struttura ha approvato questo studio di coorte retrospettivo senza necessit del consenso informato da parte del paziente . 
lo studio stato condotto in accordo ai principi della dichiarazione di helsinki . study population popolazione di studio through a systematic review of the mdct database of our institute , the examinations of all patients who underwent a chest mdct between january 2003 and june 2009 were considered . 
technical parameters were as follows : tube rotation time 0.5 s ; raw thickness 1 mm ; helical pitch 1.4 ; reconstruction width 1.5 macquisition was made at end - inspiration breath - hold and in the caudalcranial direction to minimise breathing artefacts in the lower lobes , where most emboli are located [ 11 ]  . 
an intravenous injection of 100 ml of 400 mgi / ml iomeprol ( iomeron 400 , bracco diagnostics , italy ) was administered at a flow rate of 4 ml / s , and the bolus - tracking technique ( toshiba sure - start ) was used with a region of interest ( roi ) placed at the level of the main pulmonary artery . 
all images were evaluated using three different window / level settings : one specific for pulmonary angiography ( 700 hu / 100 hu ) , one for pulmonary parenchyma ( 2 , 000 hu / 550 hu ) and one for mediastinum ( 350 hu / 50 hu )  . 
successivamente , sono state selezionate tutte le indagini condotte per sospetto di ep acuta in regime durgenza . stata inoltre valutata la proporzione di pazienti che hanno eseguito lesame provenendo dal pronto soccorso rispetto a quelli ricoverati . tecnica di indagine tutte le indagini sono state acquisite mediante lutilizzo di apparecchiatura tc multidetettore a 4 strati ( aquilion , toshiba medical systems , tokyo , giappone )  . 
successivamente allesecuzione di una scan view comprensiva di tutto il torace , dalla base del collo ai seni costo - frenici , stato posizionato un pacchetto di acquisizione compreso tra la convessit dellarco aortico e le vene polmonari inferiori . 
i parametri tecnici utilizzati sono stati i seguenti : tempo di rotazione 0 , 5 s ; collimazione 1 mm ; pitch 5 , 5 ; intervallo di ricostruzione 1 , 5 mlacquisizione avvenuta ad apnea inspiratoria in direzione caudo - craniale , al fine di minimizzare gli artefatti da respiro ai lobi polmonari inferiori , ove si riscontrano la maggior parte degli emboli [ 11 ]  . 
per ogni esame stato utilizzato un mezzo di contrasto iodato , iomeprolo , in concentrazione pari a 400 mgi / ml ( iomeron 400 , bracco diagnostics , milano , italia ) con un volume di 100 ml e un flusso ottimale di 4 ml / s tramite tecnica bolus tracking ( toshiba sure - start ) , con regione dinteresse posizionata sul tronco comune dellarteria polmonare . 
la documentazione dellesame stata effettuata con tre diverse finestre di visualizzazione : una finestra dedicata per ep ( livello 100 uh , larghezza 700 uh ) , una per parenchima polmonare ( livello - 550 uh , larghezza 2000 uh ) ed una per mediastino ( livello 50 uh , larghezza 350 uh )  . 
 data analysis analisi dei dati two radiologists in consensus retrospectively evaluated all mdct reports , and each examination was classified into one of four different categories using criteria modified from richman et al . 
 [ 8 ] e di seguito citati : categoria 1 : tc positive per embolia polmonare acuta ; categoria 2 : tc negative per embolia polmonare acuta ma positive per altri reperti richiedenti un intervento immediato e specifico ( 2a , sospetta polmonite ; 2b , 222 radiol med ( 2011 ) 116 : 219229 pericardial or pleural effusion ; 2e , lung nodules or masses suspected for lung cancer ; 2f , pneumothorax ; 2g , a new or changed mediastinal mass ; 2h , pneumomediastinum ) ; category 3 : completely negative or positive for findings with potential for significant morbidity requiring specific action on follow - up [ 3a negative ; 3b chronic obstructive pulmonary disease ( copd ) ; 3c stigmata of previous inflammatory / infectious processes ; 3d hepatic , renal or adrenal focal lesions ; 3e hiatal hernia ; 3f small pleural effusion ( 2 cm ) ; 3g atelectasis ; 3h lung metastasis with known cancer or known lung cancer ; 3i lung nodules or masses of uncertain aetiology ; 3j adenopathy ( 1 cm ) ; 3k unhealed rib fractures ; 3l new lung fibrosis ] ; category 4 : indeterminate examination due to degraded images that could not be interpreted ( 4a ) or questionable findings that could not be classified as category 2 or 3 ( including findings labelled as questionable or requiring clinical correlation ) ( 4b )  . if the description of findings in the radiological report was not completely clear , the two radiologists retrospectively reviewed the examination on a dedicated workstation ( vitrea , vital images , minneapolis , mn , usa ) ; any discrepancies were resolved by consensus . 
when findings classified in different categories were described , priority was given to the findings classified as category 1 , followed by category 2 , category 3 and category 4 . 
 statistical analysis the diagnostic yield , defined as the proportion of chest mdct considered positive for acute pe ( corresponding to cases classified as category 1 ) , and the proportions of other findings classified as category 2 and 3 , were calculated for the entire population . 
all statistical analyses were performed using commercially available software ( med - calc , version 9.2.0.1 , mariakerke , belgium )  . results through the systematic review of our mdct database , we identified 8 , 117 mdct examinations performed in the period specified . 
qualora nel referto vi fossero descritti pi reperti appartenenti a diverse categorie , per la classificazione stata attribuita maggior importanza ai reperti ascrivibili alla categoria 1 e successivamente alla categoria 2 e cos di seguito . 
 analisi statistica sono stati calcolati la resa diagnostica , definita come proporzione di casi positivi per riscontro di ep acuta ( casi classificati in categoria 1 ) , e la proporzione degli altri reperti toracici individuati nella popolazione generale . 
si inoltre stimata la proporzione di casi positivi per ep acuta e di casi con altri reperti , rispettivamente nel sottogruppo di pazienti provenienti dal ps e nei pazienti ricoverati . 
absolute values with the corresponding percentages in parenthesis entire population category 1 66 ( 20.18 ) category 2 90 ( 27.52 ) category 3 145 ( 44.34 ) category 4 26 ( 7.95 ) total 327 ( 100 ) tabella 1 ripartizione in categorie degli esami tcmd del torace nellintera popolazione . 
the proportion of examinations negative for pe but positive for other findings requiring specific and immediate intervention ( category 2 ) was 27.52% ( corresponding to 90 / 327 cases )  . 
in this tanti pazienti in regime durgenza con il sospetto clinico di ep acuta ( 158 maschi , 169 femmine ; et media , 69 anni ; deviazione standard ( ds ) , 17 , 33 anni ; range , 2198 anni )  . novantaquattro pazienti ( 28 , 75% ) sono stati sottoposti allindagine provenendo dal ps mentre i rimanenti 233 ( 71 , 25% ) erano pazienti ricoverati . 
la distribuzione dei reperti nelle quattro categorie nellintera popolazione riassunta in tabella 1 . nella popolazione generale , la resa diagnostica della tcmd del torace , proporzione dei reperti classificati in categoria 1 , risultata pari al 20 , 18% ( 66 / 327 casi )  . 
a limmagine tcmd del torace , visualizzata con settaggio angiografico , evidenzia la presenza di un parziale difetto di riempimento circondato da unesile rima di mezzo di contrasto a livello dellarteria per il lobo superiore del polmone destro , reperto diagnostico per embolia polmonare . 
b limmagine tcmd in riformattazione coronale conferma il difetto di riempimento occlusivo e ne dimostra lestensione nei rami segmentari per il lobo superiore . 224 radiol med ( 2011 ) 116 : 219229 fig . 
a chest mdct image displayed with angiographic window / level setting shows no filling defects in the pulmonary artery or its main branches . bilateral consolidation is visible in the posterior region of both lungs . 
b mdct image with parenchymal window / level setting clearly depicts a parenchymal consolidation with air bronchogram in the posterior segment of the right upper lobe , strongly suggestive of pneumonia . 
b limmagine tcmd visualizzata con settaggio per parenchima polmonare dimostra chiaramente la presenza di un consolidamento parenchimale con broncogramma aereo a carico del segmento dorsale del lobo superiore del polmone destro , fortemente suggestivo per polmonite . 
the proportion of chest mdct completely negative or positive for findings with potential for significant morbidity requiring specific action on follow - up ( category 3 ) was 44.34% ( corresponding to 145 / 327 cases )  . 
 in the subgroup of 94 outpatients , the diagnostic yield for the presence of acute pe was 23.40% ( corresponding to the 22 / 94 cases classified as category 1 )  . 
the proportion of examinations negative for pe but positive for other findings requiring specific and immediate intervention ( category 2 ) was 19.15% ( corresponding to 18 / 94 cases )  . 
 in the subgroup of 233 inpatients , the diagnostic yield for the presence of acute pe was 18.88% ( corresponding to the 44 / 233 cases classified as category 1 )  . 
the proportion of examinations negative for pe but positive for other findings requiring specific and immediate intervention ( category 2 ) was 30.90% ( corresponding to 72 / 233 cases )  . 
examinations classified as category 3 were 99 / 233 ( 42.49% ) , and those tivi per reperti richiedenti unazione immediata e specifica ( categoria 2 ) risultata pari a 27 , 52% ( 90 / 327 casi )  . 
in questa categoria , il reperto pi frequente stato quello della sotto - categoria 2a ( sospetta polmonite ) , per un totale di 37 / 327 casi ( 11 , 37% )  . 
la proporzione di tcmd del tutto negative o positive per reperti di potenziale morbilit meritevoli di follow - up ( categoria 3 ) stata del 44 , 34% ( 145 / 327 casi )  . 
 nella sottopopolazione dei 94 pazienti provenienti dal pronto soccorso , la proporzione di casi positivi per ep stata pari al 23 , 40% ( 22 / 94 casi classificati in categoria 1 ) ; quella di casi appartenenti alla categoria 2 pari al 19 , 15% ( 18 / 94 casi ) ; alla categoria 3 pari al 48 , 94% ( 46 / 94 casi ) ed infine pari al 8 , 51% quella dei casi classificati in categoria 4 ( 8 / 94 casi )  . nel sottogruppo dei 233 pazienti ricoverati il 18 , 88% ( 44 / 233 casi ) delle indagini stato classificato in categoria 1 ; il 30 , 90% ( 72 / 233 casi ) in categoria 2 ; il 42 , 49 ( 99 / 233 ) in categoria 3 ed il 7 , 73% ( 18 / 233 casi ) in categoria 4 . 
there were no significant differences in the distribution of mdct findings between inpatients and outpatient . in recent years , ct pulmonary angiography has become a widely accepted first - line imaging technique in evaluating suspected acute pe [ 12 , 13 ]  . 
substantial advances in mdct technology have guaranteed improvement in spatial and temporal resolution and in overall diagnostic performance . data from a variety of studies revealed a sensitivity of i risultati del nostro studio dimostrano che la tcmd del torace ha un grande impatto clinico nei pazienti con sospetta ep acuta , con una resa diagnostica del 20 , 2% e una proporzione aggiuntiva di reperti clinicamente rilevanti con necessit di intervento immediato e specifico del 27 , 5% . non stata dimostrata una differenza statisticamente significativa nella distribuzione dei reperti fra i pazienti provenienti dal pronto soccorso e quelli ricoverati . 
i risultati di diversi studi hanno dimostrato , nella diagnosi di ep acuta , una sensibilit della tcmd del torace compresa fra 83% e 100% e una specificit compresa fra 89% e 98% [ 1417 ]  . 
unelevata accuratezza delle metodiche di imaging di cruciale importanza , in quanto la maggior parte dei casi di decesso per embolia polmonare prevenibili dovuta ad un misconoscimento della diagnosi piuttosto che a un fallimento dellapproccio terapeutico , oramai codificato [ 3 ]  . 
i progressi tecnici e la validazione clinica hanno condotto la fleischner society a stabilire che la 226 radiol med ( 2011 ) 116 : 219229 table 3 division into categories of the chest mdct exams in patients referred from the emergency department ( outpatients ) and in inpatients . 
absolute values with the corresponding percentages in parenthesis category 1 category 2 category 3 category 4 total outpatients inpatients 22 ( 23.40 ) 44 ( 18.88 ) 18 ( 19.15 ) 72 ( 30.90 ) 46 ( 48.94 ) 99 ( 42.49 ) 8 ( 8.51 ) 18 ( 7.73 ) 94 ( 100 ) 233 ( 100 ) tabella 3 ripartizione in categorie degli esami tcmd eseguiti nei pazienti provenienti dal pronto soccorso e nei pazienti ricoverati . 
i valori sono espressi come valori assoluti e fra parentesi riportata la percentuale categoria 1 categoria 2 categoria 3 categoria 4 totale pazienti esterni pazienti interni 22 ( 23 , 40 ) 44 ( 18 , 88 ) 18 ( 19 , 15 ) 72 ( 30 , 90 ) 46 ( 48 , 94 ) 99 ( 42 , 49 ) 8 ( 8 , 51 ) 18 ( 7 , 73 ) 94 ( 100 ) 233 ( 100 ) 83%100% and a specificity of 8998% for chest mdct in diagnosing acute pe [ 1417 ]  . 
a high accuracy of imaging modalities is crucial , as the majority of preventable deaths related to acute pe can be ascribed to a missed diagnosis rather than to a failure of codified therapies [ 3 ]  . 
technical advances and clinical validation of the diagnostic modality led the fleischner society to establish that ct pulmonary angiography has fulfilled the conditions to replace conventional pulmonary angiography as the standard of reference for diagnosing acute pe [ 5 ]  . 
recently , the use of mdct scanners with more than 16 slices has been shown to decrease the incidence of nondiagnostic examinations and to increase the confidence level in the clinical scenario of suspected acute pe [ 10 ]  . 
in agreement with the literature [ 19 ] , we found no statistically significant difference in terms of diagnostic yield between the two subgroups , thus confirming the importance of ct as the first - line diagnostic imaging modality in both inpatients and outpatients . 
when pe is suspected , a further advantage of chest ct over other imaging methods relates to its ability to rule out or detect alternative diagnoses [ 21 ]  . 
 in our study , we classified all chest mdct findings in a tcmd del torace soddisfa tutte le condizioni necessarie per sostituire langiografia polmonare convenzionale come metodica di riferimento nella diagnosi di ep acuta [ 5 ]  . recentemente , inoltre , lutilizzo di apparecchiature tcmd con 16 strati ed oltre ha dimostrato una riduzione delle indagini non diagnostiche ed un aumento della confidenza nella diagnosi nel sospetto clinico di ep acuta [ 10 ]  . la percentuale di casi positivi per ep stata rispettivamente del 23 , 4% in pazienti provenienti dal pronto soccorso e del 18 , 9% nei pazienti ricoverati . 
questo dato in accordo con quanto espresso in precedenti studi , dove le percentuali variano rispettivamente dall8 , 5% al 29 , 8% e dal 19 , 5% al 27 , 4% . 
in accordo con la letteratura [ 19 ] , non stata evidenziata una differenza statisticamente significativa nella distribuzione dei reperti in categoria 1 , a conferma dellimportanza della tcmd del torace quale indagine di prima istanza in regime durgenza sia nei pazienti ricoverati che in quelli provenienti dal pronto soccorso . 
un ulteriore vantaggio della tcmd del torace rispetto ad altre indagini , nel sospetto di ep , quello conseguente alla capacit di consentire eventuali diagnosi alternative [ 21 ]  . 
 stato dimostrato che la tomografia computerizzata ( tc ) aggiunge informazioni diagnostiche che possono suggerire una diagnosi alternativa in ben il 67% di pazienti privi di ep [ 22 ]  . 
 [ 19 ] hanno riscontrato che nei pazienti senza evidenza di ep , i reperti alternativi individuati dalla tcmd del torace variano fra l89 , 8% ed il 76 , 2% del totale dei reperti in una popolazione di pazienti ricoverati e in una di pazienti provenienti dal pronto soccorso , rispettivamente [ 19 ]  . 
lesame ha rivelato la presenza di reperti negativi per ep e compatibili con una diagnosi specifica ed radiol med ( 2011 ) 116 : 219229 categorisation system modified from that proposed by richman et al . 
in that study , a multicentre cohort of outpatients with suspected pe was considered , and chest ct revealed findings compatible with a specific , alternative diagnosis to pe requiring immediate treatment in 7% of cases negative for pe . 
these data are similar to results of a recent study in which acute clinically relevant thoracic non - pe findings were discovered in 25.5% of patients referred from the emergency department for suspected pe [ 10 ]  . the difference between richman et al.s and our series in terms of proportion of outpatients with alternative diagnosis to pe requiring specific and immediate intervention can be partially explained by the fact that , unlike richman et al . , we included in this category four patients with extensive pleural effusion . 
 suspected pneumonia ( category 2a ) was the most frequent alternative diagnosis disclosed ( 14.2% of non - pe findings ) in our study , which in agreement with previous studies that reported frequencies ranging from 6% to 33% of non - pe findings [ 8 , 10 18 , 22 , 23 ]  . 
the presence of such findings confirms that lung cancer is a relatively uncommon but not rare finding on chest mdct studies performed to rule out acute pe [ 9 ]  . 
in agreement with previously reported data [ 24 ] , a per - year analysis of chest mdct requested to rule out acute pe revealed an increasing use of this imaging technique over recent years , without a significant change in the frequency of pe detected . our study has several limitations . 
for this reason , the described distribution of findings is solely dependent on radiological reports . second , all examinations were performed with a four - slice mdct scanner , without evaluating cardiac diseases and with inherent limitations concerning scan time and the alternativa , richiedente intervento immediato , nel 7% dei casi . 
i nostri risultati mostrano che nel 30 , 9% dei pazienti ricoverati e nel 19 , 1% dei pazienti provenienti dal pronto soccorso la tcmd del torace fornisce informazioni riguardanti reperti negativi per embolia ma richiedenti un intervento immediato e specifico ( categoria 2 )  . 
tali dati sono simili a quanto riportato in un recente studio in cui , in un gruppo di pazienti provenienti dal pronto soccorso e sottoposti allesecuzione di tcmd del torace per sospetta ep , emerso un 25 , 5% di reperti toracici acuti alternativi clinicamente rilevanti [ 10 ]  . 
 [ 8 ] e la nostra casistica in termini di pazienti provenienti dal pronto soccorso con reperti alternativi alla ep e meritevoli di intervento specifico ed immediato pu essere in parte attribuita al fatto che , a differenza di richman et al . 
un altro aspetto di cui tenere conto la differenza riscontrata nella proporzione di casi di sospetta polmonite ( il reperto pi frequente fra quelli richiedenti un intervento immediato e specifico in entrambe le casistiche ) , pari al 9 , 6% ( 9 / 94 casi ) nel nostro studio ed al 5 , 3% ( 54 / 1025 casi ) in quello di richman et al . 
 la diagnosi alternativa pi frequente ( 14 , 2% delle tcmd negative per embolia ) stata quella di sospetta polmonite , in accordo con precedenti studi che hanno evidenziato frequenze di polmonite comprese fra il 6% e il 33% di tc negative per embolia [ 8 , 10 , 18 , 22 , 23 ]  . 
nel 2 , 1% dei pazienti ( 7 / 327 ) si sono riscontrate nodularit polmonari o tumefazioni sospette per presenza di processo produttivo primitivo polmonare ( categoria 2e )  . 
la presenza di tali reperti conferma che le neoplasie del polmone sono un reperto alternativo infrequente ma non cos raro in esami tcmd del torace eseguiti per sospetta ep [ 9 ]  . 
in accordo con risultati precedenti [ 24 ] , unanalisi per anno delle richieste di tcmd del torace per sospetta ep ha dimostrato un incremento del ricorso a questa metodica di imaging negli ultimi anni senza significative differenze nella frequenza di ep riscontrate . 
secondo , tutte le indagini sono state eseguite mediante lutilizzo di unapparecchiatura tcmd a 4 strati , senza la possibilit di valutare patologie cardiache e con limiti intrinseci riguardanti la velocit di esecuzione e la presenza di artefatti da movimento respiratorio . 
fourth , only the overall diagnostic yield of acute pe was evaluated , without considering different levels of pretest clinical probability or d - dimer test results , which could have influenced the reported percentage of examinations positive for acute pe . 
the other side of the coin is represented by half cases without an apparent cause justifying the examination in an emergency setting , thus confirming the importance of a specific indication for each single chest mdct performed . 
 acknowledgements authors thank daniela drigo for her valuable contribution to the statistical analysis of this work . riportato in precedenti pu aver influenzato il numero di esami totalmente negativi ( categoria 3 ; 11 , 9% dei casi ) o quello di esami in cui stata rilevata la presenza di reperti non richiedenti un intervento immediato ( categoria 3b - l ; 32 , 1% dei casi ) , con possibile mancata individuazione di condizioni rilevanti ( ad esempio , piccoli emboli polmonari periferici )  . 
nonostante ci , la percentuale di reperti indeterminati ( categoria 4 , 7 , 95% ) simile a quanto lavori ( 6 , 1%10 , 4% ) [ 10 ]  . 
tuttavia , un ridotto volume di acquisizione funzionale a una riduzione della dose [ 25 ] ed a un miglioramento della qualit complessiva delle immagini quando si utilizza una apparecchiatura tcmd a 4 strati . quarto , stata valutata solo la resa diagnostica globale per la presenza di ep , senza considerare diversi livelli di probabilit pre - test da correlare alla clinica o al dosaggio del d - dimero ; ci potrebbe aver influenzato la percentuale di esami positivi per la presenza di ep acuta . in conclusione , in pazienti con sospetta ep acuta la tcmd del torace pu riconoscere sia ep sia reperti che suggeriscano una diagnosi alternativa richiedente un intervento immediato e specifico ( vale a dire , pazienti classificati in categoria 1 e 2 ) in circa il 50% dei casi , senza differenze tra i pazienti ricoverati e quelli provenienti dal pronto soccorso . 
shimizu1 1department of respirology and rheumatology , faculty of medicine , tottori university , 36 - 1 nishimachi , yonago 683 - 8504 , japan 2department of respirology san - in rosai hospital , 1 - 8 - 1 kaikesinden , yonago 683 - 8504 , japan correspondence to : m . 
it has been reported that the prognosis differs between patients who have collagen vascular diseaseassociated interstitial pneumonia ( cvd - ip ) and those with idiopathic ip ( iip )  . 
measuring the llv / tlv ratio by threedimensional ct can help distinguish between cvd - ip and iip at initial diagnosis , especially in patients with cvd - ip who have pulmonary involvement before other organ diseases and symptoms caused by cvd . keywords collagen vascular disease interstitial pneumonia lower - lobe volume total lung volume three - dimensional computed tomography abstract obiettivo . 
non sono state evidenziate differenze significative nel rapporto llv / tlv tra i due gruppi di pazienti . nonostante questo , il rapporto risultava inferiore a 0 , 33 in 9 pazienti su 23 con cvd - ip e in 2 pazienti su 24 affetti da iip , ed stata dimostrata una differenza significativa nella percentuale di pazienti con un rapporto inferiore a 0 , 33 tra i due gruppi ( p = 0 , 001 )  . 
la misurazione del rapporto llv / tlv tramite tc tridimensionale pu aiutare a distinguere , nelle fasi iniziali , la polmonite interstiziale associata a patologia del collagene da quella idiopatica , specialmente nei pazienti con patologie del collagene che hanno una manifestazione polmonare prima dellinizio della sintomatologia e prima del coinvolgimento degli altri organi . parole chiave patologie del collagene vascolare polmonite interstiziale volume del lobo inferiore volume totale del polmone tc tri - dimensionale 212 introduction it has been reported that the prognosis of patients who have collagen vascular disease - associated interstitial pneumonia ( cvd - ip ) is better than that of patients with idiopathic ip ( iip ) [ 15 ]  . 
iip is classified into seven types based on various clinical , radiological and pathological features [ 6 ]  . the majority of patients with iip have idiopathic pulmonary fibrosis ( ipf ) [ 7 ] , whereas the next most common type is nonspecific ip ( nsip ) [ 7 ]  . 
however , most patients with cvd - ip are now classified as having nsip , and only a minority of them shows a uip pattern [ 5 , 811 ]  . 
unlike the case for iip , the prognosis of nsip is not different from that of uip in patients with cvd - ip [ 12 ]  . accordingly , distinguishing cvd - ip from iip is very important for predicting the prognosis , because cvd - ip has a better prognosis than iip , irrespective of the histological pattern of lung disease [ 15 ]  . high - resolution computed tomography ( hrct ) can diagnose ipf with a sensitivity of 4378% and a specificity of 9097% [ 13 ]  . 
furthermore , lung biopsy is performed in < 15% of patients with interstitial lung disease , even though it is an effective method for distinguishing between cvd - ip , nsip and ipf [ 14 ]  . this study was performed to investigate whether differences of ct findings between cvd - ip and iip were helpful for making a differential diagnosis . 
however , there is little available literature about the lower - lobe volume ( llv ) and total lung volume ( tlv ) in either cvd - ip or iip . 
we calculated the llv / tlv ratio 3d computed tomography ( ct ) to investigate the difference of lower - lobe shrinkage between cvd - ip and iip patients at initial diagnosis . materials and methods participants study participants were 23 patients with cvd - ip and 24 with iip . 
for the diagnosis of iip , we used the american thoracic society / european respiratory society consensus classification [ 6 ]  . surgical lung biopsy was performed in seven of 24 patients with iip , including five with uip and two with nsip . 
the diagnosis of iip was made by exclusion of other diffuse parenchymal lung diseases that could be detected with bal and tblb , such as infections , sarcoidosis , hypersensitivity pneumonitis , pneumoconiosis , bronchiolitis obliterans organizing pneumonia , eosinophilic pneumonia and pulmonary histiocytosis x . 
patients were not diagnosed as having iip if they were positive for one or more of the following : ( 1 ) antinuclear antigen ; ( 2 ) rheumatoid factor ; ( 3 ) anti - scleroderma ( scl - 70 ) antibody ; ( 4 ) sjgrens syndrome a or b ; and ( 5 ) jo - 1 antibody . pulmonary dysfunction was defined by a percentage of predicted forced vital capacity ( %fvc ) or percentage of predicted single - breath diffusion capacity for carbon ( %dlco ) < 80% . 
as a result , there were two patients with systemic lupus erythematosus ( sle ) , two with mixed connective tissue disease dermatomyositis / polymyositis ( mctd ) , ( dm / pm ) , one with microscopic polyangiitis ( mpa ) , six with rheumatoid arthritis ( ra ) , one with sjgrens syndrome ( ss ) and six with systemic sclerosis ( ssc )  . 
ten healthy controls who were referred to our hospital because abnormal findings were detected on chest radiographs at other hospitals but who had no significant abnormalities on chest ct were also enrolled . radiol med ( 2011 ) 116 : 211218 pulmonary function tests pulmonary function tests were done three times consecutively with a dry spirometer ( minato , tokyo , japan ) , and the highest values were recorded . 
the following parameters were measured : vital capacity ( vc ) , forced vital capacity ( fvc ) , forced expiratory volume in 1 s ( fev1 ) , residual volume ( rv ) using the helium dilution method , total lung capacity ( tlc ) , single - breath - diffusing capacity for carbon monoxide ( dlco ) and alveolar volume ( va ) using the single - breath method . 
rv was determined in three patients with cvd - ip , 11 with iip and one healthy control . calculation of lung volume chest ct was performed in the supine position with a multislice scanner ( aquilion , toshiba , tokyo , japan )  . 
all images were obtained at maximal inspiration using the following parameters : 120 kv , auto ma , 1 - mm collimation at 5 - mm intervals , window level of 600 hounsfield units ( hu ) and window width of 1 , 600 hu . 
after tlv was measured , the lower lobe was traced by confirming the major fissure on each slice and then the lower - lobe volume ( llv ) was calculated by the software . all procedures were done by two experienced respiratory physicians . data analysis results are shown as the mean standard deviation ( sd )  . associations between tlv and tlc were assessed by linear regression analysis and by creating a bland - altman plot [ 29 ]  . 
spss software ( japanese version 16.0 for windows ; spss japan inc . , tokyo , japan ) was used for statistical analysis and creating receiver - operating - characteristic ( roc ) curves . 
there were significant differences of age , gender , smoking history , %dlco , and partial pressure of arterial carbon dioxide ( paco2 ) between cvd - ip and iip patients . 
however , %fvc and alveoloarterial oxygen difference ( aado2 ) did not differ significantly between the groups . accuracy of calculating tlv by ct volumetry in 15 individuals ( one control , three patients with cvd - ip and 11 patients with iip ) , the tlv calculated by ct volumetry was plotted against the tlc measured by spirometry . 
there was no bias between spirometry and ct volumetry for measurement of lung volume ( p = 0.28 ) , and 93% of values were within the limits of agreement . ct volumetry and roc analysis the mean tlv was 4 , 640225 ml in the ten controls , 3 , 968167 ml in the 24 patients with iip and 3 , 599163 ml in the 23 patients with cvd - ip . 
1a , b relationship between total lung capacity ( tlc ) measured by spirometry and total lung volume ( tlv ) measured by 3d computed tomography ( ct )  . 
closed triangle is a healthy control , closed circles are patients with iip and open circles are patients with collagen vascular disease - associated interstitial pneumonia ( cvdip )  . 
1a , b relazione tra capacit totale polmonare ( tlc ) misurata con spirometria e volume totale del polmone ( tlv ) misurato con tc tridimensionale . correlazione tra tlv e tlc . 
le linee punteggiate orizzontali indicano la media + o 1 , 96 di deviazione standard . radiol med ( 2011 ) 116 : 211218 * * p < 0.001 controls cvd - ip * p < 0.001 cvd - ip fig . 
3 comparison of patients with a lower - lobe volume / total lung volume ( llv / tlv ) ratio < 0.33 in the idiopathic interstitial pneumonia ( iip ) and collagen vascular disease - associated interstitial pneumonia ( cvd - ip ) groups . 
the llv / tlv ratio was < 0.33 in 9 / 23 cvd - ip patients and 2 / 24 iip patients . iip patients with an llv / tlv ratio < 0.33 were dissociated from the other patients . 
3 confronto tra i pazienti con un rapporto llv / tlv < 0 , 33 nel gruppo della polmonite interstiziale idiopatica ( iip ) e nel gruppo della polmonite interstiziale associata a patologie del collagene vascolare ( cvd - ip )  . 
i pazienti con iip che hanno un rapporto < 0 , 33 si presentavano dissociati dagli altri pazienti.si dimostrava una differenza significativa nella frequenza di un rapporto < 0 , 33 tra i due gruppi cdv - ip e iip ( p = 0 , 01 )  . less or more than 0.33 ( table 2 )  . 
2 lower - lobe volume / total lung volume ( llv / tlv ) ratio of controls , idiopathic interstitial pneumonia ( iip ) patients and collagen vascular disease - associated interstitial pneumonia ( cvd - ip ) patients . 
2 volume del lobo inferiore / volume totale ( llv / tlv ) nei controlli , nel gruppo di pazienti con iip e nel gruppo di pazienti con cdv - ip . 
iip contro cvd - ip , non significativo . discussion it has been reported that patients with cvd - ip have a better prognosis than patients with iip [ 15 ] , suggesting that it is important to distinguish cvd - ip from iip . 
in this study , we investigated the difference in frequency of an llv / tlv ratio < 0.33 at initial diagnosis between patients with cvdip and iip using 3d ct . 
we found that there was marked lower - lobe shrinkage in more patients with cvd - ip , suggesting that measuring the llv / tlv ratio by 3d ct may be useful to distinguish between cvd - ip and iip at an early stage without the need for surgical lung biopsy . calculation of lung volume by 3d ct although lung function tests are very useful , it is impossible to assess the volume of each lobe separately . 
when we investigated the validity of calculating the lung volume by 3d ct , a significant correlation was found between tlc measured by spirometry and tlv obtained with 3d ct . 
accordingly , we propose that 3d ct is a useful and reliable method for calculating lung volume . detection of the major fissure was easy on 2d ct and the lower lobe was traced by confirming the major fissure on each ct slice . 
there have also been many reports about measurement of the volume of organs and tumours by tracing [ 26 , 27 ] , indicating that 3d ct can be used to calculate llv . 216 radiol med ( 2011 ) 116 : 211218 fig . 
our results suggest that lower - lobe shrinkage was marked in some patients with cvd - ip at an early stage of disease . an llv / tlv ratio < 0.33 showed a high specificity but low sensitivity for distinguishing cvd - ip from iip at an early stage judging from the results of roc analysis . patients with cvd - ip had a wider range of llv / tlv ratios than patients with iip . 
this difference in the distribution of llv / tlv ratio data explains the high specificity and low sensitivity of a cutoff value of 0.33 for distinguishing cvd - ip from iip . 
therefore , our findings suggest that ip in a patient with an llv / tlv ratio < 0.33 is probably pulmonary involvement of cvd , and a cutoff value llv / tlv ratio < 0.33 is useful to discriminate between early cvd - ip and iip . patients with an llv / tlv ratio < 0.33 nearly all had cvd - ip . 
our findings suggest that patients with ip and an llv / tlv ratio < 0.33 probably have cvd - ip , and thus will have a good prognosis , even if the histological appearance is uip . there are two possible mechanisms by which the lower lobe may shrink so markedly in patients with cvd - ip from an early stage compared with iip . 
one is a difference in the intrapulmonary distribution of ip , and the other is dysfunction of the diaphragthe lower lobes are predominantly involved in ipf , nsip and cvd - ip compared with the other lobes . 
mild dysfunction of the diaphragm might enhance lower - lobe shrinkage , even in patients with early ip , such as our cases 1 , 2 , 6 and 8 ( table 3 )  . 
197 , rui jin er road , shanghai , 200025 , china 2ct laboratory of ge healthcare , beijing economic and technology development area , beijing , china correspondence to : z - l . 
this study compared the performance of prospectively electrocardiographically ( ecg ) - triggered axial computed tomography ( ct ) angiography with retrospective technique in evaluating coronary artery stent restenosis by 64 - slice ct . 
a pulsing cardiac phantom with artificial coronary artery in - stent restenosis was examined by ct angiography with different types of scan modes . the visibility of in - stent restenosis was evaluated with a three - point score . 
artificial lumen narrowing [ ( inner stent diameter - measured lumen diameter ) / inner stent diameter ] , lumen attenuation increase ratio [ ( in - stent attenuationcoronary artery lumen attenuation ) / coronary artery lumen attenuation ] , measurement error of restenosis percent [ ( known restenosis percent - measured restenosis percent ) / known restenosis percent ] and imaging noise were analysed . 
prospective acquisition showed better visibility than retrospective acquisition ( p < 0.05 ) : 61% of in - stent restenoses had good visibility on the prospective acquisition compared with 17% on the retrospective acquisition . 
artificial lumen narrowing ( mean 40% ) , lumen attenuation increase ratio ( mean 33% ) and measurement error of restenosis percent were not different between types of ct acquisitions . 
lacquisizione prospettiva ha dimostrato una migliore identificazione rispetto allacquisizione retrospettiva ( p < 0 , 05 ) : il 61% delle restenosi intrastent ha dimostrato una buona identificazione nelle acquisizioni prospettiche a fronte del 17% ottenuto con le acquisizioni retrospettiche . 
il lume artificiale stenotico ( media del 40% ) , il rapporto di incremento di attenuazione del lume ( media del 33% ) , e lerrore di 190 radiol med ( 2011 ) 116 : 189196 technique , prospective coronary ct angiography offers improved image quality and reduces effective radiation dose in evaluating in - stent restenosis . keywords stent coronary artery cta introduction whereas stent implantation is a successful treatment for coronary artery disease , 9.631.6% of coronary artery stents develop restenosis [ 1 , 2 ]  . 
consequently a noninvasive and available method of stent follow - up becomes indispensable . recently , 64 - slice coronary computed tomography angiography ( cta ) has played an increasing role in assessing coronary arteries and in coronary artery stent imaging [ 35 ]  . however , blooming and streak artefacts from high - attenuation material , such as stent struts that interfere with visualising the stent lumen , are still a common problem for 64slice scanners with improved spatial and temporal resolution . although retrospective electrocardiographically ( ecg ) gated helical ( reh ) scans are mainly used , with advances in technique , prospective ecg - triggered axial ( pea ) scans allow a substantial reduction in radiation dose compared with reh scans ( 15 msv reduced to 1.05 msv ) while having a possible advantage for in - stent restenosis imaging owing to the reduction of artefacts compared with reh scans [ 610 ]  . 
therefore , the purpose of our study was to clarify the influence of scan modes and tube voltage on the in - stent visibility , artificial lumen narrowing and lumen attenuation by using in - stent 50% and 75% restenosis vessel models . 
we also compared in - stent visibility obtained for different heart rates and coronary artery diameters . materials and methods cardiac phantom we used a prototype cardiac phantom ( developed by the ct lab of ge health care , china )  . 
a confronto con la tecnica retrospettiva tradizionale , langio tc conorarica prospettiva offre una migliore qualit delle immagini e riduce la dose di radiazioni effettiva nella valutazione della restenosi intrastent . 
 parole chiave stent arterie coronarie angio - tc coronary artery stent and in - stent restenosis models coronary artery models were made of acrylicsilicon tubes with an inner diameter of 3 or 4 mm and filled with contrast material ( iopamidol 350 , bracco , italy ) diluted to 300 hu . they were closed at both ends and positioned in an orientation parallel to the z - axis of the scanner . 
for prospective scanning , the centre of the temporal window corresponded to 75% of the r - r interval , and the exposure time was set to the minimum ( 233 ms )  . 
in order to compare image quality with different tube voltages under the same radiation dose , 100 kvp and 720 ma , 120 kvp and 500 ma and 140 kvp and 360 ma were used following the equation : ma1 / ma2 = kvp1 2 / kvp2 radiol med ( 2011 ) 116 : 189196 fig . 
2 coronary artery stent and in - stent restenosis models showing configuration of the cypher stent with a strut thickness of 0.14 mthe stent with artificial in - stent stenosis was inserted into a coronary artery model . 
2 modelli di stent coronarici e di restenosi intra - stent mostrano la configurazione di uno stent cypher con spessore delle maglie di 0 , 14 mlo stent con stenosi artificiale era stato inserito in un modello di arteria coronaria . 
il diametro delle arterie coronarie era di 3 o 4 mle arterie erano riempite con mezzo di contrasto diluito a 300 uh e chiuse ad entrambi i lati da un tappo di silicone . image analysis lumen visibility all images were analysed on a separate workstation ( advantage workstation version 4.4 , ge healthcare , waukesha , wi , usa ) by two independent , experienced radiologists blinded to the ct protocols . 
disagreements were resolved by a third observer . 192 radiol med ( 2011 ) 116 : 189196 measurement of artificial lumen narrowing by using electronic callipers , two independent observers measured inner stent diameter and visible lumen diameter with in - stent restenosis , on the line parallel to the x - axis on each image , using the workstation semiautomatic software vessel analysis ( advantage workstation with cardiq software , version 4.4 , ge healthcare , milwaukee , wi , usa )  . 
all measurements were repeated three times and the mean of the measurements by two observers was calculated separately . artificial lumen narrowing was calculated using the following equation [ 9 , 10 ] : artificial lumen narrowing = ( inner stent diameter visible lumen diameter ) / inner stent diameter . lumen attenuation increase ratio to quantify the effects of blooming artefacts on luminal ct attenuation , lumen attenuation increase ratio was calculated according to the following equation : lumen attenuation increase ratio = ( in - stent attenuation coronary artery lumen attenuation ) / coronary artery lumen attenuation [ 9 ]  . attenuation values inside the visible stent lumen were measured using a region of interest ( roi ) , the size of which was drawn as small as possible to avoid stent struts and artefacts . 
two observers plotted out the vessel segment with restenosis , restenosis diameter was measured , the percent was calculated using the following equation : measured restenosis percent = measured restenosis diameter / inner stent diameter . 
measurement errors of the 50% and 75% in - stent restenosis were compared using repeated measurements and analysis of variance ( anova )  . noise was defined as the standard deviation ( sd ) of an roi density measuring the water inside the rubber balloon . measurements were repeated three times to avoid intraobserver variation , and mean value was calculated . 
3 la visibilit della restenosi intrastent stata valutata con un punteggio di qualit dimmagine a tre punti : valore 1 , buono ( chiara visibilit del lume dello stent coronarico con restenosi ) ; valore 2 , moderato ( restenosi visibile ma grado di stenosi non valutabile ) ; valore 3 , basso ( lume dello stent coronarico non valutabile a causa di importanti artefatti )  . consequently , dlp is defined with the assumption that the heart ranges by 14 cm in the z - axis , and the dlp is thus calculated by the following equation : dlp ( mgycm ) = volume computed tomography dose index ( mgy ) 14cm . the effective dose ( e ) was calculated using the following equation : e = kdlp , where k is a conversion coefficient for the anatomical region examined , i.e. 
interobserver reliability for measuring diameter or attenuation was tested by pearsons correlation coefficient . anova for continuous variables and the kruskalwallis test for categorical data were used to compare the effects of the scan type , heart rate , vessel calibre and tube voltage on assessing and measuring stent imaging . 
a two - sided significance level of 0.05 was used in all analyses . results lumen visibility the dose - length product ( dlp ) displayed on the phantom study is not suited for simulating dlp on real patients . kruskal - wallis test proved that there was a significant radiol med ( 2011 ) 116 : 189196 fig . 
one - way anova revealed that this was not different between stent calibres for 75% and 50% restenosis ( p = 0.82 for 75% ; p = 0.71 for 50% ) , heart rates ( p = 0.44 for 75% ; p = 0.93 for 50% ) , scan modes ( p = 0.43 for 75% ; p = 0.59 for 50% ) and tube voltages ( p = 0.27 for 75% ; p = 0.52 for 50% restenosis ) for both 75% and 50% . noise mean noise of water inside the rubber balloon was 24.85 , which was similar to the enhanced aortic root in the clinical routine . 
 radiation dose discussion recent advances in technique of 64 - slice ct , including larger detector coverage , higher temporal and spatial resolution and real - time ecg signal monitoring , make it possible to use pea scans for coronary cta [ 68 ]  . 
for pea scans , x - ray exposure is applied only during the required phase of the cardiac cycle , with complete pausing during the rest of the cardiac cycle . 
accordingly , low - dose pea coronary 64 - slice cta has the important advantage of allowing the follow - up of patients after stent implantation and coronary artery bypass grafts . 
first , as an axial scan mode , the table is stationary during data acquisition of pulmonary artery ( pa ) cta compared with the constant movement of the reh technique during the scan . second , the shorter scan time of pa - cta permit less image degrading due to high and unstable heart rate . due to a combination of partial volume averaging and beam - hardening , the blooming artefacts create a spill - over effect from higher ct attenuation voxels to adjacent lower voxels . 
these two ct artefacts obscured the coronary artery lumen , degraded visibility of in - stent restenosis , produced the artificial lumen narrowing and increased the evaluated thickness of stent struts and in - stent lumen attenuation . 
the visibility of in - stent restenosis with 3.5 - mm stent calibre was significantly better than with the 3 mm , and the artificial lumen narrowing with 3.5 mm was higher than that with 3 m this indicated that coronary artery radiol med ( 2011 ) 116 : 189196 lumen evaluation with bigger stent calibre is more credible than with smaller stent calibre . 
 for pea 64 - slice coronary cta , a low and stable heart rate is an important factor in obtaining high - quality images . the reported ideal heart rate is 56.963 bpm for pea acquisition [ 6 , 16 ]  . 
in our study , there was no difference of restenosis visibility between 50 , 60 and 70 bphowever , there is no accepted heart rate range for pea technique or for in - stent lumen evaluation . 
image quality was degraded by stents with thicker struts ( > 0.14mm ) and stents with smaller diameter ( 3.0 mm ) [ 17 , 18 ] .we tested only one type of stent , the cypher , with a 3and 3.5 - mm inner diameter , 13and 23 - mm length and 0.14 - mm strut thickness . 
second , artificial restenosis consisted of polypropylene with a homogeneous density of approximately 60 hu , whereas in vivo intimal hyperplasia and atheromatous plaque are often heterogeneous . that may limit the extent of our findings in humans . 
gatta2 1unit operativa di senologia , ospedale san paolo , contrada capo scardicchio , bari , italy 2sezione di radiodiagnostica , dipartimento di internistica clinica e sperimentale , seconda universit degli studi di napoli , piazza miraglia 2 , napoli , italy correspondence to : g . 
this study sought to evaluate the accuracy of vacuum - assisted biopsy ( vab ) in the diagnosis of atypical ductal hyperplasia ( adh ) by determining the rate of vab underestimation compared with de nitive histology . 
patients with a diagnosis of adh associated with other lesions ( lobular intraepithelial neoplasia , papilloma ) , atypical lobular hyperplasia , lobular carcinoma in situ and any lesions with a microhistological diagnosis other than adh were excluded . 
mammographic appearance of lesions was as follows : 152 mostly clustered microcalci cations ( 86% ) ; ve opacities with microcalci cations ( 3% ) ; 12 single opacities ( 3% ) ; and eight parenchymal distortions ( 4% ) , of which ve were without and three were with microcalci cations . 
the decision to either perform or not perform surgery was based on combined analysis of the following parameters : patient age ; risk factors in the patients history ; mammographic riassunto obiettivo . 
sono state incluse nel nostro studio 177 pazienti con diagnosi vab di adh pura ; sono state escluse le pazienti con diagnosi di adh associata ad altre lesioni ( neoplasia intraepiteliale lobulare [ lin ] , papilloma ) , iperplasia lobulare atipica ( alh ) , carcinoma lobulare in situ ( lcis ) e tutti le lesioni con diagnosi microistologiche differenti . 
la tipologia mammogra ca di tali lesioni stata la seguente : 152 microcalci cazioni per la maggior parte in cluster ( 86% ) ; 5 opacit con microcalci cazioni ( 3% ) ; 12 opacit singole ( 3% ) ; in ne , 8 distorsioni parenchimali ( 4% ) di cui 5 senza microcalci cazioni e 3 con microcalci cazioni . 
the most relevant parameters affecting the decision to proceed to surgical excision were lesion diameter > 7 mm on mammography , > 2 adh foci , incomplete removal of the calci cations and a family and / or personal history of breast cancer . 
dai dati emersi nel nostro studio i parametri pi rilevanti per la decisione allexeresi della lesione sono stati : diametro mammogra co > 7 mm , estensione delladh > 2 foci , incompleta rimozione delle calci cazioni ed anamnesi familiare e / o personale di neoplasia . 
however , the growing numbers of asymptomatic women self - referring for periodic mammography and the advent of screening programmes have increased the detection rate of subclinical or nonpalpable breast lesions that appear suspicious on diagnostic imaging [ 1 , 2 ]  . 
before the advent of mammography as a screening test , breast carcinoma in situ was diagnosed in only 5% of all cases of breast cancer , and the lesions were usually palpable . 
in parallel with the increased detection rate of subclinical lesions , there has been a higher diagnostic incidence of high - risk proliferative lesions , including atypical ductal hyperplasia ( adh ) [ 3 ]  . 
with the recent world health organisation ( who ) 2003 pathological classi cation , tavassoli introduced the term ductal intraepithelial neoplasia ( din ) to cover the possible changes that lead from epithelial hyperplasia to invasive carcinoma , in agreement with the commonly accepted progression model of breast cancer whereby adh , ductal carcinoma in situ ( dcis ) and in ltrating il carcinoma della mammella la pi frequente malattia tumorale nella popolazione femminile . 
tuttavia , con il costante aumento di donne asintomatiche che spontaneamente e periodicamente si sottopongono alla mammogra a e con lavvento dei programmi di screening organizzato , si determinato un sempre pi frequente riscontro di lesioni mammarie sospette alle metodiche strumentali senza un corrispettivo clinico ( lesioni infracliniche ) , vale a dire lesioni non palpabili [ 1 , 2 ]  . 
prima dellavvento della mammogra a soprattutto come esame di screening , il carcinoma in situ della mammella rappresentava solo il 5% di tutti i tumori mammari e tali lesioni erano comunemente palpabili . 
in contemporanea al maggior riscontro diagnostico di lesioni infracliniche , c stata una sempre maggiore incidenza diagnostica di lesioni proliferative della mammella ad alto - rischio tra cui appunto liperplasia duttale atipica ( adh ) [ 3 ]  . 
considerando che la diagnosi precoce uno degli elementi principali in grado di modicare la prognosi del carcinoma mammario , appare chiaro come tale obiettivo risulti essere una s da sia per il radiologo senologo che per lanatomopatologo . 
tale lesione , considerata un potenziale precursore del cancro invasivo , deve pertanto essere accuratamente e precocemente identi cata al ne di de nirne al meglio il suo signi cato prognostico . 
 con la recente classi cazione anatomo - patologica world 278 radiol med ( 2011 ) 116 : 276291 ductal carcinoma ( idc ) form a continuum of proliferative breast disease [ 4 ]  . 
in the eld of percutaneous biopsy , in 1995 , a particular technique known as vacuumassisted biopsy ( vab ) was proposed that uses a vacuum aspiration system to sample several cores with a single needle pass . the aim of our study was to evaluate the accuracy of vab in adh diagnosis by determining the rate of vab underestimation in comparison with nal histology ( the gold standard ) and to identify any imaging criteria that might help determine appropriate patient management . materials and methods we retrospectively reviewed 1 , 776 percutaneous vab procedures conducted for equivocal / suspicious subclinical breast lesions visible only at mammography on 1 , 765 patients ( age range 3276 years ) between november 1999 and january 2008 all patients had previously undergone mammography in the craniocaudal and oblique projections , supplemented by magni cation views of microcalci cation clusters and / or spot compression views of suspicious ndings . 
in six patients , sampling was bilateral and in ve two biopsies were obtained in different areas of the same breast . the level of suspicion of all lesions had preliminarily been assessed according to the breast imaging reporting and data system ( bi - rads ) classi cation [ 5 , 6 ]  . 
in all cases , percutaneous biopsy was performed with a digital fischer stereotactic prone table unit and a mammotome device ( ethicon endo - surgery , breast care , norstedlt , germany ) , which allows multidirectional ( 360 ) sampling of the lesion with forced aspiration ( 2325 mmhg )  . 
exclusion criteria were : a diagnosis of adh associated with other lesions [ adh with lobular intraepithelial neoplasia ( lin ) , papillomas ] ; atypical lobular hyperplasia ( alh ) ; lobular carcinoma in situ ( lcis ) ; any diagnosis other than adh . 
mammographic pattern heath organization ( who ) 2003 tavassoli [ 4 ] introduce un nuovo termine , quello di neoplasia duttale intraepiteliale ( din ) , per racchiudere in un unico termine quelle possibili lesioni che dalliperplasia epiteliale giungono al carcinoma invasivo secondo un modello evolutivo ormai comunemente accettato per il quale ladh , il carcinoma duttale in situ ( dcis ) ed il carcinoma duttale in ltrante ( cdi ) potrebbero rappresentare un continum di una malattia proliferativa della mammella [ 4 ]  . 
nellambito della biopsia percutanea , nel 1995 stata proposta una particolare tecnica di prelievo istologico percutaneo , chiamata vacuum - assisted ( vab ) , che consente con ununica inserzione dellago il prelievo di pi frustoli tissutali tramite un sistema di aspirazione interna . scopo del nostro lavoro stato dunque quello sia di valutare laccuratezza della vab nella diagnosi di iperplasia duttale atipica , calcolando dunque la percentuale di sottostime diagnostiche della metodica stessa rispetto allesame istologico de nitivo ( gold standard ) , sia di cercare eventuali criteri dimaging utili per il corretto management di tali pazienti . materiali e metodi stata effettuata unanalisi retrospettiva di 1776 biopsie percutanee vacuum assisted per lesioni mammarie dubbie / sospette infracliniche , visibili solo mammogra camente , eseguite nel periodo compreso da novembre 1999 a gennaio 2008 in 1765 pazienti , di et compresa tra 32 e 76 anni . 
 tutte le pazienti avevano precedentemente eseguito una mammogra a nelle due proiezioni cranio - caudale ed obliqua completate da ingrandimenti mirati sui cluster di microcalci cazioni e / o particolari in compressione mirata su altri reperti sospetti . 
in 6 pazienti il prelievo era stato bilaterale ed in altre 5 erano stati eseguiti due prelievi , in zone differenti della stessa mammella . le lesioni erano state preliminarmente valutate , relativamente al grado sospetto , in accordo con la classi cazione breast imaging reporting and data system ( bi - rads ) [ 5 , 6 ]  . 
 in tutti i casi per la microbiopsia percutanea era stato utilizzato un tavolo prono stereotassico digitale fischer associato a sistema mammotome ( ethicon endo - surgery , breast care , norstedlt , germania ) , che permette la campionatura multidirezionale ( 360 ) della lesione con aspirazione forzata ( 23 25 mmhg )  . 
in tutti i casi era stata posizionata sullarea bioptizzata una clip metallica amagnetica per obiettivare lesatta sede del prelievo . il nostro criterio di inclusione stato la diagnosi di adh puro ; i nostri criteri di esclusione sono stati : diagnosi di adh associato ad altre lesioni ( adh con neoplasia intraepiteliale lobulare [ lin ] , papillomi ) ; iperplasia lobulare atipica ( alh ) ; carcinoma lobulare in situ ( lcis ) e qualsiasi altra diagnosi diversa dalladh . 
la tipologia mammogra a di tali lesioni era stata la seguente : 152 microcalci cazioni per la maggior parte in cluster ( 86% ) ; 5 opacit con microcalci cazioni ( 3% ) ; 12 opacit singole ( 3% ) ; 8 distorsioni parenchimali ( 4% ) di cui 5 senza microcalci cazioni e 3 con microcalci cazioni . tutte le biopsie effettuate avevano fornito materiale adeguato per la diagnosi ( 100% )  . 
le pazienti in esame sono state successivamente suddivise in due gruppi : il primo costituito dalle pazienti inviate allintervento chirurgico ; il secondo invece dalle pazienti inviate a follow - up , prima a sei mesi e poi , se negativo , annuale . 
the purpose of this analysis was to identify parameters that might help determine the most appropriate management of patients with a vab diagnosis of adh . similarly , in patients referred for follow - up care , we analysed both the rate of lesions showing progression over time and the imaging , clinical and microhistological features of these lesions to determine the existence of parameters predicting lesion progression . statistical analysis the parameters age , personal and / or family history , bi - rads assessment , lesion diameter , complete or incomplete excision at vab and microhistological extent ( or > 2 adh foci ) were compared in cases underestimated and not underestimated at vab using the two - tailed t and chi - square tests . 
per il gruppo di pazienti sottoposte ad intervento chirurgico stato confrontato , il giudizio microistologico con quello fornito dallistologia de nitiva , considerata come gold standard , al ne di valutare le sottostime diagnostiche con mammotome . 
nei casi sottostimati stata effettuata successivamente unulteriore analisi retrospettiva delle caratteristiche radiologiche e microistologiche ; in particolare stato valutato retrospettivamente come parametro microistologico , fornito pertanto dallanatomopatologo , lestensione delladh allinterno dei dotti e / o lobuli , vale a dire il numero di foci di adh presenti al loro interno in una sezione , suddividendo pertanto le pazienti in due sottogruppi : pazienti con adh2 foci e pazienti con adh > 2 foci ( tabella 2 )  . 
il ne di tale analisi stato di valutare parametri utili per il management dei pazienti con diagnosi vab di adh . con le medesime nalit , anche per il gruppo di pazienti inviate a stretto monitoraggio clinico - strumentale sono state invece valutate sia la percentuale di lesioni evolute nel tempo sia le caratteristiche radiologiche , anamnestiche e microistologiche delle lesioni evolute , al ne dunque di valutare se esistono parametri predittivi di uneventuale evoluzione nel tempo . analisi statistica i parametri et , storia personale e familiare , giudizio birads , diametro delle lesioni , exeresi completa o incompleta alla vab , estensione microistologica ( adh2 o > 2 foci ) sono stati confrontati nelle pazienti sottostimate e non sottostimate alla vab e nelle pazienti stabili ed evolute con t test a due code e 2 . 
stato calcolato il rischio relativo delle pazienti sottoposte a intervento chirurgico e delle pazienti sottoposte a follow - up con lesioni > 15 mi parametri et , storia personale e familiare , giudizio bi - rads , diametro delle lesioni , exeresi completa o incompleta alla vab , estensione microistologica ( adh2 o > 2 foci ) sono stati confrontati nelle pazienti stabili o evolute al follow - up con test t di student a due code e test del 2 . radiol med ( 2011 ) 116 : 276291 fig . 
the patient was referred for surgery owing to the nonexcision of the microcalci cations at vacuumassisted biopsy and microhistological suspicion [ atypical ductal hyperplasia ( adh ) > 2 foci ]  . 
successivamente , dallanalisi retrospettiva delle caratteristiche radiologiche e microistologiche di ogni singola lesione sottostimata emerso che per ladh , i fattori di rischio di malignit signi cativi sono stati ( tabella 3 ) : il giudizio radiologico espresso : nei casi sottostimati il giudizio radiologico bi - rads 4 - 5 stato assegnato nell84% dei casi ( 16 / 19 )  . 
tuttavia anche nel gruppo di radiol med ( 2011 ) 116 : 276291 eter > 15 mm of a total of 46 / 98 patients with mammographic extent 1.5 cm referred for surgery . 
only 6 / 72 follow - up patients ( 8% ) showed a change in mammographic pattern , consisting in all cases of an increase in the number of microcalcications . 
the lesion had progressed to idc in four cases ( 66.7% ) and dcis in two ( 33.3% ) over a mean interval of 56 months ( 2482 months )  . 
a comparative analysis of the radiological and microhistological features of progressed adh lesions ( table 4 ) revealed that parameters predicting malignant disease were , even in this case , the combination of bi - rads category 45 , which was present in 5 / 6 cases ( 83.3% ) ; maximum lesion diameter and > 2 adh foci . 
furthermore , in 5 / 6 cases of lesion progression ( 83.3% ) , excision at vab had been incomplete , a parameter that increases the risk of subsequent progression . in patients referred for surgery , comparison of the main parameters ( age , personal and / or family history , bi - rads assessment , lesion diameter , complete or incomplete excision at vab , microhistological extent or > 2 foci ) between underestimated and nonunderestimated lesions revealed a statistically signi cant difference with regard to patient age ( p = 0.034 ) , lesion extent ( p = 0.007 ) and complete or incomplete lesion excision ( p = 0.010 , table 5 )  . 
in unanalisi comparativa delle caratteristiche radiologiche e microistologiche delle lesioni di adh evolute nel tempo considerato ( tabella 4 ) emerso che i parametri predittivi di malignit sono stati rappresentati , anche in questo caso , dalla combinazione del giudizio radiologico del tipo bi - rads 4 - 5 , presente in 5 / 6 casi ( 83 , 3% ) , diametro massimo della lesione 284 radiol med ( 2011 ) 116 : 276291 lesion extent ( p = 0.001 ) and complete or incomplete lesion excision ( p = 0.006 , table 6 )  . discussion and conclusions dcis is a heterogeneous entity from the point of its histological , molecular and radiological features [ 8 ]  . 
a , c , parenchymal distortion on the internal equator region , with a diameter of 1.5 cm and intralesional lobular microcalci cations ( breast imaging reporting and data system 4 )  . 
a , c distorsione parenchimale a sede equatoriale interna , del diametro di 1 , 5 cm , con microcalci cazioni lobulari intralesionali ( bi - rads 4 )  . 
in particolare lestensione mammogra ca media nei casi evoluti stata di 2 , 1 cnessuna lesione con diametro < 1 cm evoluta nel tempo ( diametro medio delle lesioni 1 , 6 cm ) ed in 5 / 6 casi era presente un adh > 2 ( 83 , 3% )  . 
inoltre , in 5 / 6 lesioni evolute ( 83 , 3% ) lexeresi alla vab era stata incompleta , parametro che pertanto , aumenta il rischio di successiva evoluzione . 
 nelle pazienti sottoposte ad intervento chirurgico , il confronto dei principali parametri ( et , storia personale e familiare , giudizio bi - rads , diametro della lesione , exeresi completa o meno alla vab , estensione microistologica , 2 foci o > 2 foci ) nelle lesioni sottostimate e nelle lesioni non sottostimate ha evidenziato una differenza statisticamente signi cativa relativa allet della paziente ( p = 0 , 034 ) , allestensione della lesione ( p = 0 , 007 ) e allexeresi completa o meno della lesione ( p = 0 , 010 ) ( tabella 5 )  . 
nelle pazienti sottoposte a follow - up il confronto dei principali parametri ( et , storia personale e familiare , giudizio bi - rads , diametro della lesione , exeresi completa o meno alla vab , estensione microistologica , 2 foci o > 2 foci ) nelle lesioni stabili e nelle lesioni evolute ha evidenziato una differenza statisticamente signi cativa relativa alla storia familiare ( p = 0 , 001 ) , allestensione della lesione ( p = 0 , 001 ) e allexeresi completa o meno della lesione ( p = 0 , 006 ) ( tabella 6 )  . discussione e conclusioni il cdis si con gura come unentit eterogenea dal punto di vista delle sue caratteristiche istologiche , molecolari e radiologiche [ 8 ]  . 
accettando oramai un modello di tipo evolutivo del carcinoma mammario , il cdis non compare improvvisamente come tale , ma insorge e si accresce nel contesto di un processo di iperplasia delle cellule epiteliali , accompagnato dalla comparsa di atipie cellulari ( iperplasia atipica , adh )  . 
 utile sottolineare , che sebbene esista un modello progressivo di evoluzione del carcinoma mammario ormai unanimemente accettato , non bisogna dimenticare che non tutti i carcinomi in ltranti seguono questo tipo di storia naturale : alcuni potrebbero avere una fase intraduttale molto breve e quindi divenire invasivi prima che la fase in situ possa venire identi cata , altri potrebbero insorgere ab initio come invasivi [ 9 ]  . 
la precisa distinzione tra adh e cdis dunque importante in quanto ladh considerato come un fattore di rischio per lo sviluppo di carcinoma invasivo , mentre cdis un precursore diretto del cdi e necessita sempre dellexeresi chirurgica . negli ultimi anni lintroduzione del mammotome ( vab ) , vale a dire di sistema di prelievo microistologico multidirezionale con aghi trancianti dotati di un dispositivo aspirante sottovuoto , ha migliorato laf dabilit e la sensibilit delle micro biopsie . 
 however , it is important to recall that , although the progression model of breast cancer has been universally accepted , not all in ltrating carcinomas follow this progression in their natural history : some may have a very short intraductal phase and become invasive before the in - situ phase is identi ed , whereas others may be invasive from the beginning [ 9 ]  . 
precise distinction between adh and dcis is therefore important in that adh is considered a risk factor for the development of invasive carcinoma , whereas dcis is a direct precursor to idc and always requires surgical excision . the recent introduction of the mammotome vab device for multidirectional microhistological biopsy with core needles equipped with a vacuum aspiration system has improved the reliability and sensitivity of core biopsy . 
 in fact , the reported diagnostic accuracy of vab is fully comparable with that achieved by surgical biopsy with marker clips , a method that has been widely used for many years . 
in the case of small lesions , especially those only visible at mammography such as foci of microcalci cations , small opacities or areas of parenchymal distortion accuracy is de nitely higher compared with core needle biopsy thanks to the presence of the aspiration system that allows larger tissue cores ( approximately three times larger ) to be collected , needle calibre being equal [ 10 , 11 ]  . tura , del tutto confrontabile con i dati della biopsia chirurgica su repere , metodica ampiamente utilizzata per lunghi anni . 
nel caso di lesioni di piccole dimensioni , in particolare quelle visibili solo mammogra camente , quali focolai di microcalci cazioni , piccole opacit o aree di distorsione parenchimale , laccuratezza decisamente maggiore rispetto alla core biopsy , grazie alla presenza dei dispositivi di aspirazione che consentono di ottenere , a parit di calibro , frustoli di maggiore volume ( circa 3 volte ) [ 10 , 11 ]  . tuttavia , nonostante laf dabilit diagnostica della metodica , lescissione chirurgica ancora attualmente raccomandata dopo una diagnosi di adh alla vab a causa del rischio di upgrade a cdis o a cancro invasivo allistologia de nitiva ; la percentuale di sottostime riportate in letteratura variano infatti dal 11% al 87% [ 1221 ]  . 
lestrema variabilit delle percentuali di sottostima riportate in letteratura legata da un lato alla diversa metodologia utilizzata ed al diametro dellago utilizzato , dallaltro allesperienza dellanatomopatologo ; percentuali di sottostime pi basse sono state infatti riscontrate sia con lutilizzo dei sistemi vab che con lutilizzo di aghi trancianti di pi grosso calibro ( 11 g o 9 g piuttosto che 14 g ) [ 18 , 2227 ] , accorgimenti che permettono campionamenti di numero e dimensioni ( inteso come peso del frustolo ) maggiori . 
un recente studio di tonegutti e girardi [ 28 ] riporta una sottostima diagnostica di adh della vab rispetto allistologia de nitiva del 19% ( 5 / 27 casi ) , dato perfettamente concordante radiol med ( 2011 ) 116 : 276291 fig . 
istologia de nitiva : iperplasia duttale atipica . however , despite the methods diagnostic reliability , surgical excision is still recommended after a vab diagnosis of adh owing to the risk of an upgrade to dcis or to invasive cancer at nal histology . 
lower underestimation rates have been found both with vab systems and with larger - core needles ( 11or 9 - gauge rather than 14 - gauge ) [ 18 , 2227 ] , which allow collection of more and larger ( intended as weight of the core ) tissue cores . 
a recent study by tonegutti and girardi reported the rate of underestimation of adh by vab compared with nal histology to be 19% ( 5 / 27 ) , which is perfectly in line with the results obtained in our series [ 28 ]  . our study aimed to retrospectively analyse the results obtained with mammotome biopsy to determine not only the possible underestimates but also whether a subset of patients with an adh diagnosis could be identi ed in whom surgical excision could be omitted . 
the current trend when faced with adh is to proceed to surgical excision , although some authors have attempted to establish the conditions that mandate excision and those that warrant follow - up . 
 [ 19 ] , the presence of one of the following parameters should prompt surgical excision : ( a ) personal history of breast cancer , ( b ) lesion diameter > 1 cm , ( c ) presence of adh in the last fragment removed con i risultati da noi ottenuti [ 28 ]  . 
 il nostro lavoro si proposto di analizzare in maniera retrospettiva i risultati ottenute su biopsie con mammotome al ne di individuare , oltre alle sottostime diagnostiche , se esiste un sottogruppo di pazienti con diagnosi di adh che potrebbe non esser sottoposto a escissione chirurgica ; per tale lesione lorientamento attuale infatti quello dellexeresi chirurgica , sebbene alcuni autori abbiano tentato di dettare delle condizioni in cui lexeresi necessaria , riservando il follow - up nei rimanenti casi . 
 [ 29 ] , only patients with complete removal of adh at vab or those > 70 years without additional risk factors were referred for follow - up . in our experience , although personal and / or family history was among the criteria for surgical referral , the parameter was not in uential in the group of underestimates in that only 1 / 19 ( 5% ) underestimated patients had a previous history of breast cancer . 
this parameter was instead in uential in the group of patients referred for follow - up who were found to have progressed to malignancy ( 50% of cases )  . 
furthermore , the parameters predictive of malignancy in the two groups of patients in our study were > 2 adh foci , larger mammographic lesion extent , bi - rads category 45 and incomplete lesion excision at vab . 
 [ 30 ] , in a study of 24 cases of adh at core biopsy , did not identify any case of underestimation among cases with only one or two foci of adh . 
in contrast to ely and sneige , our study identi ed an underestimation rate of 8.7% ( 7 / 80 cases of 2 adh foci ) among lesions with 2 adh foci ; this parameter also had a low incidence among patients referred for follow - up who showed lesion progression ( 1 / 6 , 17% )  . 
the combined analysis of the different radiological and microhistological features of underestimated adh lesions shows that the seven underestimated cases with 2 foci had suspicious radiological features , such as a larger mammographic extent and / or missed excision at vab ( table 3 )  . 
a nding of bi - rads category 45 alone , although very high in the group of underestimates ( 84.2% ) , was not a signi cant parameter in that a high percentage of patients undergoing vab ( 96% ) had been classi ed as bi - rads 45 ( 132 / 144 adh ) ; additionally , the incidence of this parameter was the same ( 85% ) in cases con rmed by nal histology . our study was therefore unable to identify any single radiological criterion predictive of malignancy . 
 [ 20 ] and other authors [ 18 , 21 , 31 , 32 ] reported so carcinoma mammario ; tale dato si invece dimostrato in uente nel gruppo di pazienti che sottoposte a follow - up hanno mostrato una successiva evoluzione della lesione in senso maligno ( 50% dei casi )  . 
nel nostro studio inoltre , i parametri predittivi di malignit nei due gruppi di pazienti sono stati : numero di foci di adh maggiore di 2 , maggiore estensione mammogra ca della lesione , giudizio radiologico bi - rads 4 - 5 e incompleta exeresi della lesione stessa alla vab . 
 [ 30 ] , su un totale di 28 adh non riportano alcun upgrade per lesioni limitate ad 1 o 2 foci usando aghi da 11 e 14 gauge con tecnica vab . 
 [ 20 ] , la sottostima diagnostica per lesioni con adh2 foci stata dell8 , 7% ( 7 / 80 casi di adh2 foci ) ; tale parametro stato comunque basso anche nel gruppo di pazienti che , sottoposte a follow - up , hanno mostrato unevoluzione nel tempo : solo in 1 / 6 di tali lesioni ( 17% ) aveva adh2 foci . 
tuttavia , nellanalisi combinata tra le varie caratteristiche radiologiche e microistologiche delle lesioni sottostimate di adh si pu evincere che i 7 casi di adh2 foci sottostimati possedevano comunque caratteristiche radiologiche sospette , quali una pi estesa lesione mammogra ca e / o mancata exeresi della stessa alla vab ( tabella 3 ) ; analoga osservazione per la singola lesione con adh2 foci evoluta nel tempo ( tabella 4 )  . 
il giudizio radiologico bi - rads 4 - 5 , per quanto sia stato elevato nel gruppo di pazienti sottostimate ( 84 , 2% ) , non un parametro signi cativo da solo , in quanto unalta percentuale delle lesioni sottoposte a vab , pari al 96% , era stata classi cata come bi - rads 4 - 5 ( 132 / 144 adh ) ; inoltre anche nel gruppo di pazienti confermate allistologia de nitiva tale parametro ha avuto la stessa incidenza ( 85% )  . 
 [ 20 ] , cos come altri autori [ 18 , 21 , 31 , 32 ] , hanno riportato nei loro studi unassenza di upgrade delladh per tutte le lesioni completamente escisse alla vab ; nel nostro studio invece , una percentuale seppur bassa , pari al 26% ( 5 / 19 ) , di lesioni completamente escisse hanno dimostrato un successivo upgrade delladh ; tuttavia , in unanalisi combinata emerge che ciascuna di queste 5 lesioni aveva comunque un numero di foci di adh > 2 . 
in our study , by contrast , a low percentage ( 26% , 5 / 19 ) of completely excised lesions showed subsequent upgrade of adh ; however , the combined analysis demonstrated that each of these ve lesions had > 2 adh foci . 
similarly , only 1 / 6 ( 16.7% ) lesions that subsequently showed progression had been removed completely . the combined analysis of all parameters at our disposal shows that the extent of adh is an important indicator for lesions 7 mm and < 15 mm in diameter . 
however , the extent of adh is no longer a relevant parameter with lesions 15 mm in diameter , where surgical excision is prompted by the lesion diameter itself regardless of adh extent . 
this nding is statistically signi cant despite the wide ci ( rr in followup patients 12.17 ; 95% ci 3.1047.73 ) , which re ects only the small size of our study population . 
in con rmation of this , 80% of lesions that had progressed at follow - up also had a diameter 15 mconversely , as none of the underestimated lesions or any adh that progressed had a diameter < 0.7 cm , regardless of the number of adh foci , we considered this parameter to be protective against a possible upgrade . 
in conclusion , although surgical excision is strongly recommended for adh diagnosed at vab with an 11 - gauge needle , it is reasonable to believe that follow - up could constitute a valuable alternative for a subset of patients . in our study , the most relevant parameters leading to a decision to treat the lesion surgically were , in order of importance : ( a ) mammographic lesion diameter > 7 mm ; ( b ) adh extent > 2 ; ( c ) incomplete removal of calci cations ; ( d ) and personal / family history of breast cancer . 
generally , the presence of at least two of these parameters is suf cient to decide on a surgical referral , whereas a high grade of cellular atypia must be considered in itself a parameter determining excision . 
il nostro risultato statisticamente signi cativo nonostante lampio intervallo di con denza ( rr pazienti in follow - up 12 , 17 ic 95% : 3 , 1047 , 73 ) legato esclusivamente alla scarsa numerosit del campione . 
inoltre , il diametro medio di tutte le lesioni di adh stato infatti di 1 , 3 cm versus 1 98 cm per le lesioni di adh sottostimate ; a conferma di ci , anche le lesioni evolute nel follow - up avevano per l80% un diametro 15 mviceversa , poich nessuna lesione sottostimata n nessuna lesione di adh evoluta aveva diametro inferiore a 0 , 7 cm , indipendentemente dal numero di foci di adh , abbiamo ritenuto tale parametro protettivo per un eventuale upgrade . 
in conclusione sebbene lexeresi chirurgica sia attualmente raccomandata per adh diagnosticato alla vab con ago da 11 gauge , si pu ragionevolmente ritenere che il follow - up potrebbe essere valida alternativa per un ristretto gruppo di pazienti . 
 nel nostro studio i parametri pi rilevanti per la decisione allexeresi chirurgica della lesione , sono stati , in ordine dimportanza i seguenti : diametro mammogra co della lesione > 7 mm , estensione delladh > 2 , incompleta rimozione delle calci cazioni ed in ne storia personale / familiare di neoplasia . 
in genere suf ciente che siano presenti almeno due dei suddetti parametri per decidere di inviare la paziente ad exeresi chirurgica , mentre lelevato grado di atipia deve essere da solo considerato parametro decisivo per lexeresi . 
poletti2 1area di radiologia , irstistituto romagnolo studio e cura dei tumori , meldola , forl , italy 2unit operativa di pneumologia interventistica , ospedale pierantoni - morgagni , forl , italy 3istituto di anatomia patologica , universit di verona , verona , italy 4unit operativa di anatomia patologica , ospedale pierantoni - morgagni , forl , italy 5istituto di radiologia , universit campus bio - medico , roma , italy 6unit operativa di radiologia , azienda ospedaliera santa maria di terni , italy correspondence to : s . 
piciucchi , e - mail : s.piciucchi@alice.it received : 13 november 2009 / accepted : 6 may 2010 / published online : 10 february 2011 springer - verlag 2010 abstract purpose . 
transbronchial biopsy was performed in all but four patients ( one case of alveolar haemorrhage and three cases of lipoid pneumonia ) in whom the diagnosis was established with bal . assessment of the hrct images revealed the following patterns : noncardiogenic pulmonary oedema ( n = 13 ) ; organising pneumonia ( op ) ( n = 9 ) ; hypersensitivity riassunto obiettivo . 
tutti i pazienti erano sottoposti a broncoscopia , con biopsia transbronchiale o broncolavaggio ( bal ) in seguito ad una hrct che poneva il sospetto di dr - ild . due radiologi ( un senior ed un junior ) hanno riportato in cieco i dati riguardanti i singoli reperti , la distribuzione e il pattern predominante alla hrct . 
quarantadue pazienti sono stati inclusi nello studio ( 25 maschi ; 17 femmine ; range di et 2084 anni )  . la biopsia transbronchiale stata effettuata in tutti i casi , fatta eccezione per un caso di emorragia alveolare e tre casi di polmonite lipoidea , in cui la diagnosi stata raggiunta con il bal . 
le interpretazioni tc includevano i seguenti pattern : edema polmonare non cardiogeno radiol med ( 2011 ) 116 : 246263 pneumonitis ( hp ) ( n = 2 ) ; alveolar haemorrhage ( ah ) ( n = 2 ) ; nonspecific interstitial pneumonia ( nsip ) ( n = 5 ) ; lipoid pneumonia ( lp ) ( n = 1 ) ; sarcoid - like pattern ( n = 1 )  . cytohistological diagnosis revealed diffuse alveolar damage ( dad ) in 11 patients , op in seven , hp in three , ah in three , chronic interstitial pneumonia ( cip ) in eight , lp in three and pseudosarcoidosis in one . 
in cases in which its specificity was low , hrct was nonetheless useful for biopsy planning and clinicalradiological monitoring after discontinuation of the drug treatment . keywords pulmonary toxicity interstitial lung diseases hrct transbronchial biopsy bronchoscopy ( n = 13 ) ; organizing pneumonia ( op ) ( n = 9 ) ; hypersensitivity pneumonitis ( hp ) ( n = 2 ) ; alveolar haemorrage ( ah ) ( n = 2 ) ; non specific interstitial pneumonia ( nsip ) ( n = 5 ) ; lipoid pneumonia ( lp ) ( n = 1 ) ; sarcoid - like pattern ( n = 1 )  . 
la diagnosi anatomopatologica ha mostrato : danno alveolare diffuso ( dad ) in 11 pazienti , op in 7 , hp in 3 , ah in 3 , polmonite interstiziale cronica ( cip ) in 8 , lp in 3 ; pseudosarcoidosi in 1 . 
dividendo i farmaci in antineoplastici e nonantineoplastici , i pattern pi frequenti sono stati cip ( n = 6 ) ; dad ( n = 2 ) ; op ( n = 2 ) per il gruppo degli antineoplastici . 
nei casi in cui la hrct ha mostrato bassa specificit , stata utile nel planning di una corretta biopsia e nel monitoraggio clnico - radiologico dopo la sospensione del farmaco . parole chiave tossicit polmonare malattia interstiziale del polmone hrct biopsia transbronchiale broncoscopia introduction introduzione drug - related respiratory disease is a major cause of morbidity and mortality , and the relationship between the use of different pharmaceutical drugs and lung disease is increasingly recognised [ 1 ]  . 
many agents have been recognised as being toxic to the pulmonary parenchyma [ 24 ]  . the diagnosis of drug - related interstitial lung disease ( drild ) involves three elements : clinical suspicion , differentiation from other respiratory diseases with similar presentation [ by highresolution computed tomography ( hrct ) and clinical tests ] and a compatible histological pattern . symptoms are often nonspecific and include rapidly developing dyspnoea and a dry , unproductive cough [ 3 , 5 ]  . 
the radiography and ct appearance of the various forms of drild have been variously described in the literature [ 7 , 8 ]  . ct enables precise assessment of the presence , pattern and distribution of parenchymal and airway abnormalities and has higher sensitivity compared with chest radiography , which may be normal in the early stages of dr - ild [ 8 ]  . the radiological manifestations of dr - ild correspond to the underlying histological findings : nonspecific interstitial pneumonia ( nsip ) , usual interstitial pneumonia ( uip ) , hypersensitivity pneumonia ( hp ) , pulmonary oedema , le patologie respiratorie da farmaci rappresentano unimportante causa di morbilit e mortalit . 
la diagnosi di tossicit polmonare da farmaci ( dr - ild ) coinvolge tre elementi : sospetto clinico , la distinzione di patologie respiratorie con manifestazioni simili ( mediante tomografia computerizzata [ tc ] ad alta risoluzione e test clinici ) e infine un pattern istologico . 
la tc offre una precisa valutazione della presenza , pattern e distribuzione delle alterazioni parenchimali e delle vie aeree , con una sensibilit maggiore rispetto alla radiografia , potendo infatti , questultima , essere normale negli stadi iniziali di dr - ild [ 8 ]  . 
la manifestazione radiologica di una tossicit polmonare da farmaci pu avere diversi corrispettivi istologici : polmonite interstiziale non specifica ( nsip ) , usual interstitial pneumonia ( uip ) , polmonite da ipersensibilit 248 radiol med ( 2011 ) 116 : 246263 organising pneumonia ( op ) and alveolar haemorrhage ( ah )  . 
the diagnosis of these patterns has often been obtained by surgical biopsy [ 4 ]  . ( hp ) , edema polmonare polmonite organizzativa ( op ) e emorragia alveolare diffusa ( ah )  . 
spesso la diagnosi di tali pattern stata riservata alla biopsia chirurgica [ 4 ]  . the purpose of this study was to determine the sensitivity and specificity of high - resolution ct ( hrct ) compared with the cytohistological diagnosis provided by bronchoscopy used as the gold standard with the aim of identifying what proportion of patients could be diagnosed with dr - ild by using semi - invasive tests and to what extent ct is specific as well as sensitive in this context . la proposta di questo studio cercare di individuare la specificit e la sensibilit della tc ad alta risoluzione considerando come termine di confronto la diagnosi citoistologica fornita dalla broncoscopia . 
la finalit di questo studio stato infatti cercare di individuare in quale percentuale di pazienti sarebbe effettivamente possibile porre diagnosi di dr - ild con manovre semi - invasive e quanto la tc rappresenta unindagine specifica oltre che sensibile a tal proposito . materials and methods patients patients were included into this prospective study protocol between september 2002 and may 2007 , and the study was approved by the local ethics committee . 
 imaging technique and image analysis non - antineoplastici , causa di tossicit polmonare . sono stati inclusi nello studio i farmaci antineoplastici e hrct ( light speed , ge medical system , milwaukee wi , usa ; aquilon , toshiba ) was performed with volumetric technique , a collimation thickness of 1 or 1.5 mm and scans extending from the lung bases to apices . 
images were reconstructed with a high - spatial - frequency algorithm ( kernel b70 ) with appropriate settings and viewing window ( level 700 hu , width 1 , 0001 , 600 hu )  . 
two chest radiologists ( one senior with > 15 years of experience and one junior with < 2 years of experience ) unaware of the histological patterns analysed all hrct images independently . 
honeycombing. the distribution of findings was recorded as : ( a ) central , tecnica di imaging e analisi delle immagini stata effettuata una tc ( light speed , ge medical systemmilwaukee , aquilon - toshiba ) ad alta risoluzione ( hrct ) con tecnica volumetrica , spessore di collimazione a 1 o 1 , 5 mla scansione si estendeva dalle basi allapice polmonare . 
le immagini erano ricostruite utilizzando un algoritmo con alta frequenza spaziale ( kernel b70 ) con appropriati setting e finestra di visualizzazione ( window level 700 hu , window width 10001600 hu )  . 
due radiologi toracici ( un senior con oltre 15 anni di esperienza ed un junior con esperienza inferiore ai 2 anni ) , non a conoscenza del pattern istologico hanno analizzato indipendentemente tutte le hrct . 
la tc mostra una modesta distorsione dellarchitettura lobulare secondaria nei lobi inferiori ( b ) la tc 3 mesi dopo mostra la riduzione dellattenuazione parenchimale , pur con il persistere del pattern reticolare ( c )  . when predominantly located in the central third of the lung in perihilar location ; ( b ) peripheral , when predominantly seen in the outer third in subpleural location ; ( c ) patchy , when located anywhere in the lung . 
hrct interpretations comprised the following patterns : ( a ) noncardiogenic pulmonary oedema ; ( b ) organising pneumonia ( op ) ; ( c ) nonspecific interstitial pneumonia ( nsip ) ; ( d ) usual interstitial pneumonia ( uip ) ; ( e ) hypersensitivity pneumonitis ( hp ) ; ( f ) lipoid pneumonia ( lp ) ; ( g ) alveolar haemorrhage ( ah )  . 
honeycombing. la distribuzione dei reperti stata registrata come : ( a ) centrale , se i reperti sono localizzati prevalentemente nel terzo centrale del polmone , n sede perilare ; ( b ) periferica , se i reperti sono prevalentemente nel terzo esterno in sede mantellare ; ( c ) patchy , se la distribuzione indifferente a tutto il polmone . 
la diagnosi istologica stata di dad . bronchoscopy broncoscopia bronchoscopy was performed with the patient under deep sedation and with a rigid or flexible bronchoscope . written informed consent was obtained from all patients . bronchoalveolar lavage ( bal ) was performed by instilling 150 ml 0.9% saline solution into the segment to be studied or into the middle lobe if the parenchymal abnormalities were diffuse . 
all samples were reviewed by a pathologist with extensive experience in lung pathology . each histological section was stained with haematoxylin and eoswhere sampling was considered unnecessary , bal alone was carried out . lesame broncoscopico era effettuato in sedazione profonda , con broncoscopio rigido o flessibile . 
il lavaggio bronco - alveolare ( bal ) stato effettuato instillando 150 ml di soluzione salica 0 , 9% , nel segmento da studiare o , in alternativa nel lobo medio , qualora le alterazioni parenchimali fossero diffuse . 
la tc ( a , b ) mostra delle aree di ground glass di tipo patchy e piccole consolidazioni in entrambi i lobi superiori ed ai segmenti apicali dei lobi inferiori . 
una modesta quota di fibrina presente nellalveolo . statistical analysis analisi statistica interobserver agreement of the radiological diagnoses was studied using cohens kappa statistic with a 95% confidence interval ( ci )  . 
in addition , the specificity and sensitivity of hrct was calculated against the cytohistological diagnosis , which was considered the reference standard . linterobserver agreement delle diagnosi radiologiche stato descritto utilizzando lindice kappa di cohen , con un intervallo di confidenza del 95% . 
patients underlying diseases included non - hodgkins lymphoma in seven ( 17% ) , hodgkins lymphoma in one ( 2% ) , psoriatic arthritis in one ( 2% ) , atrial fibrillation in nine ( 21% ) , systemic hypertension in three ( 7% ) , chronic lymphatic leukaemia in two ( 5% ) , depression in two ( 5% ) , colorectal carcinoma in one ( 2% ) , multiple myeloma in one ( 2% ) , constipation in one ( 2% ) , nasal disorders in two ( 5% ) , rheumatoid arthritis in four ( 10% ) , breast cancer in two ( 5% ) , ewings sarcoma in one ( 2% ) , hydrocephalus in one ( 2% ) , bipolar syndrome in one ( 2% ) , hepatitis c virus infection in two ( 5% ) and cannabis - use disorder in one ( 2% )  . 
la biopsia transbronchiale ( tblb ) stata effettuata in tutti i pazienti , eccetto in un caso di emorragia alveolare e tre casi di polmonite lipoidea in cui la diagnosi era ottenuta con il bal . 
le patologie di base comprendevano : linfoma non - hodgkin in 7 pazienti ( 17% ) ; linfoma di hodgkin in 1 ( 2% ) ; artrite psoriasica in 1 ( 2% ) ; fibrillazione atriale in 9 ( 21% ) ; ipertensione sistemica in 3 ( 7% ) ; leucemia linfatica cronica in 2 ( 5% ) ; depressione in 2 ( 5% ) ; carcinoma colon - rettale in 1 ( 2% ) ; mieloma multiplo in 1 ( 2% ) ; costipazione in 1 ( 2% ) , disturbi nasali in 2 ( 5% ) ; artrite reumatoide in 4 ( 10% ) ; carcinoma mammario in 2 ( 5% ) ; sarcoma di ewing in 1 ( 2% ) ; idrocefalo in 1 ( 2% ) ; sindrome bipolare in 1 ( 2% ) ; virus dellepatite c ( hcv ) in 2 ( 5% ) , abuso di cannabinoidi in 1 ( 2% )  . 
la durata dei radiol med ( 2011 ) 116 : 246263 table 1 clinical data patient no . gender underlying diagnosis drug non - hodgkins lymphoma psoriatic arthritis non - hodgkins lymphoma hodgkins lymphoma atrial fibrillation systemic hypertension chronic lymphatic leukaemia major depression non - hodgkins lymphoma colorectal cancer multiple myeloma constipation non - hodgkins lymphoma nasal discomfort rheumatoid arthritis breast cancer non - hodgkins lymphoma ewings sarcoma non - hodgkins lymphoma rheumatoid arthritis hydrocephalus bipolar syndrome atrial fibrillation hepatitis c cannabis abuse atrial fibrillation breast cancer rheumatoid arthritis atrial fibrillation atrial fibrillation atrial fibrillation systemic hypertension chronic lymphatic leukaemia rheumatoid arthritis atrial fibrillation systemic hypertension atrial fibrillation hepatitis c atrial fibrillation major depression nasal disorder atrial fibrillation bleomycin methotrexate macop - b bleomycin , adriamycin , vinblastine amiodarone valsartan fludarabine venlafaxine bleomycin vincristine 5 - fluorouracile oxaliplatin cytosine arabinoside vaseline oil macop - b vaseline oil methotrexate cyclophosphamide adriamycin macop - b , mabthera busulfan , melphalan bleomycin antimonoclonal ab valproic acid lithium amiodarone interferonribavirin cannabinoid amiodarone taxol antimonoclonal ab amiodarone amiodarone amiodarone acebutolol fludarabine leflunomide amiodarone amlodipine amiodarone interferonamiodarone amitriptyline rhinological solution amiodarone macop - b , methotrexate , adriamycin , cyclophosphamide , oncovin , prednisone , and bleomycin four patients died due to the severity of symptoms [ three patients with diffuse alveolar damage ( dad ) and one with a histological diagnosis of chronic interstitial pneumonia ( cip ) ]  . 
interlobular septal thickening was seen in stato documentato un agreement da buono ad eccellente , nella valutazione dei reperti tc e della relativa distribuzione dei reperti ( k = 0 , 72 fino a 1 , 00 )  . 
lispessimento dei setti intertabella 1 caratteristiche cliniche e farmaci somministrati alla presentazione iniziale dei sintomi sesso patologia sottostante farmaco radiol med ( 2011 ) 116 : 246263 linfoma non - hodgkin artrite psoriasica linfoma non - hodgkin linfoma non - hodgkin fibrillazione atriale ipertensione sistemica leucemia lifantica cronica depressione maggiore linfoma non - hodgkin tumore del colon mieloma multiplo costipatione linfoma non - hodgkin rinorrea artrite reumatoide tumore mammario linfoma non - hodgkin sarcoma di ewing linfoma non - hodgkin artrite reumatoide idrocefalo disturbo bipolare fibrillazione atriale abuso di cannabis fibrillazione atriale tumore mammario artrite reumatoide fibrillazione atriale fibrillazione atriale fibrillazione atriale ipertensione sistemica leucemia linfatica cronica artrite reumatoide fibrillazione atriale ipertensione sistemica fibrillazione atriale fibrillazione atriale depressone maggiore rinorrea fibrillazione atriale bleomicina metotrexate macop - b bleomicina , adriamicina , vinblastina amiodarone valsartan fludarabina velfalaxina bleomicina vincristina 5 - fluorouracile oxaliplatino citosina arabinoside olio di vaselina macop - b olio di vaselina metotrexate ciclofosfamide adriamicina maco - b busulfan , alkeran bleomicina anti monoclonal ab acido valproico litio amiodarone interferone ribavirina paraquat amiodarone taxolo anticorpi anti monoclonali amiodarone amiodarone amiodarone acetabulolo anticorpi anti monoclonali leflunomide amiodarone amlodipine amiodarone interferone amiodarone amitriptilina soluzione rinologica amiodarone 25 patients ( 60% ) , centrilobular nodules in three ( 7% ) , peripheral consolidation in nine ( 21% ) , patchy consolidation in 15 ( 35% ) , peripheral ground - glass pattern in six ( 14% ) , central ground - glass pattern in four ( 10% ) , diffuse ground - glass pattern in ten ( 23% ) and ground - glass pattern in ten ( 23% )  . 
there was good interobserver agreement in interpretation of the radiological pattern , in particular with lobulari stato osservato in 25 pazienti ( 60% ) , i noduli centrolobulari in 3 ( 7% ) ; i consolidamenti periferici in 9 ( 21% ) ; i consolidamenti patchy in 15 ( 35% ) ; il ground glass periferico in 6 ( 14% ) ; il ground glass centrale in 4 ( 10% ) ; il ground glass diffuso in 10 ( 23% ) e il ground glass in 10 ( 23% )  . 
noncardiogenic pulmonary oedema was seen in 13 patients ( 30% ) , op in nine ( 21% ) , hp in two ( 5% ) , ah in two ( 5% ) , nsip in five ( 12% ) , lp in one ( 2% ) and pseudosarcoidosis in one ( 2% )  . an indeterminate pattern was identified in seven ( 16% )  . parenchymal findings were confined to the upper lobes in 14% of patients ( n = 6 ) , the lower lobes in 31% ( n = 13 ) and extended to the entire lung in 55% . 
there was no statistical difference between extension of the parenchymal changes and type of drug taken ( antineoplastic or nonantineoplastic )  . cytohistological interpretation pattern radiologico , in particolare per lnsip ( k = 0 , 68 ) ; op ( k = 0 , 67 ) ; dad ( k = 0 , 67 ) ; pattern indeterminato ( k = 0 , 72 ) ; hp ( k = 0 , 78 )  . 
il 14% dei pazienti ( n = 6 ) ha mostrato alterazioni parenchimali ai lobi superiori ; il 31% ( n = 13 ) ai lobi inferiori ed il 55% mostrava un diffuso coinvolgimento del parenchima polmonare . 
non stata registrata alcuna differenza statistica tra la tipologia della estensione delle alterazioni parenchimali e il tipo di farmaco assunto dai pazienti ( antineoplastico o non - antineoplastico )  . histological examination documented dad in 11 patients ( 26% ) , op in seven ( 16% ) , hp in three ( 7% ) , ah in three ( 7% ) , cip in eight ( 19% ) , lp in three ( 7% ) and pseudosarinterpretazione cito - istologica i reperti istologici hanno documentato dad in 11 casi 256 table 2 results of high - resolution computed tomography ( hrct ) and cytohistology for 13 patients treated with antineoplastic drugs patient no . 
in eight patients , histological diagnosis was cip , for which the corresponding radiological pattern was indeterminate pattern ( n = 5 ) ; dad ( n = 2 ) and op ( n = 1 )  . 
dei restanti pattern , nonostante lesiguo numero dei casi per ciascun pattern , si segnala : ah ( sensibilit 0 , 66 ; specificit 1 ) ; hp ( sensibilit e specificit 1 ) ; lp ( sensibilit 0 , 33 ; specificit 0 )  . 
in 8 pazienti , stata posta diagnosi istologica di cip , tale pattern ha presentato come corrispettivo radiologico : pattern indeterminato ( n = 5 ) ; dad ( n = 2 ) ; op ( n = 1 )  . 
lnsip invece ha documentato specificit pari a 1 , contro una sensibilit piuttosto bassa ( 0 , 38 )  . comparison between antineoplastic and nonantineoplastic drugs confronto tra il gruppo dei farmaci antineoplastici e farmaci non - antineoplastici histological patterns documented in the group of antineoplastic drugs were cip ( n = 6 ) and dad ( n = 2 )  . 
distribution of parenchymal findings , histological results and hrct findings are summarised in tables 2 and 3 . il pattern istologico documentato nel gruppo dei farmaci antineoplastici stato : cip ( n = 6 ) e dad ( n = 2 )  . 
la distribuzione delle alterazioni parenchimali , il dato istologico ed i reperti hrct sono sintetizzati nelle tabelle 2 e 3 . table 3 results of high - resolution computed tomography ( hrct ) and cytohistology for 29 patients treated with nonantineoplastic drugs patient no . 
the reason for this includes the use of chest radiography as a first - line modality in evaluating nonspecific dyspnoea , a modality that is often unable to detect interstitial disease [ 9 , 10 ]  . 
whereas idiopathic interstitial pneumonias , as classified by the 2002 consensus of the american thoracic society and european respiratory society [ 10 , 11 ] , tends to show characteristic patterns at hrct , parenchymal changes produced by pharmaceutical drugs are highly variable and often investigated with surgical lung biopsy [ 1215 ]  . in this study , we attempted to compare cytohistological patterns provided by transbronchial biopsy or bal and radiological findings identified with hrct . 
la ragione di questo fenomeno include il ricorso alla radiografia del torace come tecnica di prima scelta nella valutazione di una dispnea non ben specificata che pu spesso non individuare una patologia interstiziale [ 9 , 10 ]  . 
mentre le pneumopatie interstiziali idiopatiche come sono state classificate dal consensus del 2002 [ 10 , 11 ] dallats e dallers , spesso mostrano dei pattern tipici allindagine hrct , i farmaci possono indurre invece delle alterazioni parenchimali altamente variabili , che spesso finiscono alla biopsia chirurgica [ 1215 ]  . in questo studio abbiamo cercato di confrontare il pattern cito - istologico come veniva fornito dalla biopsia trans bronchiale o dal bal ed i reperti radiologici individuati alla tc ad alta risoluzione . 
7 alveolar haemorrhage in a 40 - year - old man affected by non - hodgkins lymphoma and treated with methotrexate , adriamycin , cyclophosphamide , oncovin , prednisone , and bleomyccomputed tomography shows several subpleural consolidations in both lower lobes . 
even though the fibrosing variant of nsip often shows a predominantly reticular pattern [ 24 , 25 ] , radiologists were unable to distinguish between the two subgroups on the basis of hrct findings alone , probably as a result of the wide variability of the nsip pattern . 
in addition , hrct findings of nsip showed significant overlap with those of other patterns , such as dad , hp and ah [ 19 , 25 ]  . radiologica , in particolare per op e dad , con valori che oscillano tra 0 , 8 e 1 . 
anche se la variante fibrosante dellnsip spesso mostra un pattern prevalentemente reticolare [ 24 , 25 ] , i radiologi non hanno potuto distinguerne i due sottogruppi sulla sola base dellaspetto hrct : probabilmente in considerazione della forte variabilit del pattern nsip ; per questo pattern stato registrato il valore pi basso di sensibilit ( 0 , 38 ) , pur mantenendo elevati valori di specificit che raggiungeva lunit . 
il bal mostra le inclusioni di olio nei macrofagi alla colorazione red oil sta it may be that by observing the characteristics of our patient series and emphasising the patchy distribution of the ground - glass pattern and lack of any severe parenchymal disruption , improvement in sensitivity could be obtained . noncardiogenic pulmonary oedema ( figs 3 , 4 ) with its histological counterpart dad was observed in ten patients ( macop - b in one ; amiodarone in four ; busulfan in one ; taxol in one and amlodipine in one )  . 
in our series , imaging findings considered strongly indicative of dad were rapidly progressive ground - glass pattern , normal - sized heart and peripheral thickening of the interlobular septa in the absence of other clinical explanations . 
specificity and sensitivity values obtained for dad were high . hp was observed in three patients ( amitriptyline in one , interferon in one and cytosine arabinoside in one ) ( fig 5 )  . many drugs can cause hp , the most common being methotrexate , bleomycin , penicillamine , cyclophosphamide , nitrofurantoin and carmustine . 
in one patient , we observed a pulmonary pattern that was sarcoid - like , induced by treatment with interferon - alfa ( 2628 ) , and characterised by a diffuse nodular pattern with upper - lobe quali : dad ; hp e ah [ 19 , 25 ]  . 
nel nostro gruppo abbiamo considerato fortemente convincente per dad : un ground glass rapidamente progressivo , in presenza di un cuore di normali dimensioni , con ispessimento periferico dei setti interlobulari , in assenza di altre spiegazioni cliniche . 
molti sono i farmaci che possono indurre una hp , tra questi i pi comuni sono il metotressato , la bleomicina , la penicillamina , la ciclofosfamide , la nitrofurantoina e la carbustina . 
the patient was 78 years old , an unusual age for sarcoidosis , and the clinical findings were a reduction in diffusion lung capacity for carbon monoxide ( dlco ) , coinciding with the administration of ifn -  . 
in seven patients , radiologists were unable to identify any characteristic hrct pattern and defined it as indeterminate pattern ; that is , ground - glass attenuation that did not reflect any specific pattern of interstitial pneumonia . 
in these cases , histological patterns were lp ( n = 1 ) , op ( n = 1 ) and nsip ( n = 5 ) , which further confirms the overlap of the various radiological patterns . despite adequacy of samples obtained with transbronchial lung biopsy , diagnosis proved inconclusive in 14 patients ; eight of them were thus studied with video - assisted thoracoscopy , which yielded four cases of nsip , two of uip and two of hp . 
in una paziente abbiamo osservato un quadro polmonare sarcoid - like , indotto da terapia con interferone - alfa ( 2628 ) , caratterizzato da un pattern nodulare diffuso , con predominanza ai lobi superiori e slargamento dei linfonodi del mediastino . 
inoltre , la paziente aveva 78 anni , unet inusuale per la sarcoidosi , con un quadro clinico caratterizzato da riduzione della diffusione polmonare del monossido di carbonio ( dlco ) , in coincidenza con la somministrazione di ifn . 
sono molti gli studi che hanno documentato la correlazione tra la somministrazione di tale farmaco e la manifestazione polmonare di sarcoidosi , indotta dallattivazione dei macrofagi [ 24 ]  . 
in ben sette pazienti i due radiologi , non potendo meglio caratterizzare il quadro tc , lo hanno definito come pattern indeterminato , cio un ground glass che non configurava alcun quadro specifico di pneumopatia interstiziale . 
il corrispettivo istologico di tali casi era : lp ( n = 1 ) ; op ( n = 1 ) e nsip ( n = 5 ) : questi dati confermano ulteriormente loverlap dei vari pattern radiologici . 
malgrado il buon campionamento della tblb , in 14 pazienti la diagnosi non stata conclusiva e 8 di questi sono stati sottoposti a vats , ottenendone 4 casi di nsip ; 2 uip e 2 hp . 
 262 radiol med ( 2011 ) 116 : 246263 as op , ep or dad , the diagnosis could be fairly straightforward , whereas in cases in which the diagnosis requires larger samples , such as uip , dip and nsip , transbronchial lung biopsy is often insufficient , leading to a need for video - assisted thoracoscopy . 
in particular , for nsip , the difficulties surrounding the microhistological diagnosis have been well described in the literature and will most likely remain one of the main causes of surgical biopsy in dr - ild . 
the first was : what sensitivity and specificity values can be expected of hrct in the diagnosis of dr - ild ? ; the second was : to what extent is transbronchial lung biopsy able to provide a confident diagnosis of dr - ild ? with regard to the first question , we obtained excellent sensitivity , with a loss of specificity for the ground - glass opacity defined as indeterminate pattern ( 16% of cases ) ; nonetheless , in these cases , ct proved to be an excellent aid in planning transbronchial lung biopsy . 
as for the second question , transbronchial lung biopsy proved to be a highly reliable diagnostic tool , especially in cases with a monomorphic histological pattern such as op , dad and hp . 
additionally , no significant complications were observed , and the technique could also be used in patients who were ineligible for any surgical diagnostic approach . there are three main limitations to this study : small series size ; inability to document improvement after drug discontinuation in all patients due to the death of four ( respiratory failure ) ; and it did not add any information about drug interactions . 
in the event of combined administration , demonstration of lung toxicity was empirical and based on suspension of the combination ( e.g. , bleomycin - vincristine ; bleomycin - adriamycin - vinblastine )  . in conclusion , hrct allows the radiologist to make a confident diagnosis , above all in cases with a rapidly progressive picture , such as dad and ah . 
this allows the drug to be promptly discontinued and support treatment to be initiated . la causa della necessit di un approccio chirurgico ( 2932 ) risiede nelle dimensioni del campione istologico ottenuto con la tblb . 
per pattern monomorfi come op , ep o dad la diagnosi potrebbe essere piuttosto agevole , mentre in casi in cui la diagnosi approcciabile spesso su campioni di maggiori dimensioni come uip , dip , nsip , la tblb si pone spesso la necessit di ricorrere a vats . 
in particolare per lnsip , le difficolt correlate alla diagnosi microistologica sono ben descritte in letteratura e probabilmente rimarr tra le cause maggiori di biopsia chirurgica nelle dr - ild . 
 il nostro studio ha cercato di rispondere a due domande . la prima era : che valori di sensibilit e specificit ci si pu aspettare dalla hrct nella diagnosi di dr - ild ; la seconda : quanto la tblb pu essere confidente nella diagnosi di drild . 
per quanto riguarda la prima domanda , abbiamo ottenuto unottima sensibilit , con perdita di specificit di fronte al ground glass definito come pattern indeterminato ( ben il 16% della serie ) , in tali casi la tc ha rappresentato tuttavia un ottimo strumento nel planning della tblb . 
 le limitazioni di questo studio sono state principalmente tre : le dimensioni modeste del campione , limpossibilit di documentare in tutti un miglioramento dopo la sospensione del farmaco , per il decesso di quattro pazienti ( da insufficienza respiratoria ) e il non aver aggiunto alcuna informazione riguardante le interazioni tra farmaci . 
in caso di somministrazione in associazione , la dimostrazione della tossicit stata empirica , basata sulla sospensione della miscela di farmaci ( come ad esempio il caso della associazione bleomicina - vincristina , bleomicina - adriamicina - vinblastina )  . 
ci permette il pronto intervento con sospensione del farmaco e inizio della terapia di supporto . acknowledgements we are very grateful to lawrence r . goodman , department of radiology , medical college of wisconsin , milwaukee and nestor muller , department of radiology , university of british columbia , vancouver , for their revision of the manuscript . ringraziamenti un profondo ringraziamento al lawrence r . 
there were 43 benign lesions ( 13 fibrocystic disease , eight fibroadenoma , seven adenosis , five normal breast tissue , four inflammatory lesions , three intramammary lymph nodes , two scleroelastosis and one fat necrosis ) and 35 malignant lesions ( 30 invasive ductal carcinoma , two invasive lobular carcinoma , one ductal carcinoma in situ , one carcinomatous mastitis and one metastasis from neuroendocrine carcinoma )  . 
with the cutoff value set at 1.52 , dwi sensitivity ( 35 true positive , no false negative ) was 100% and specificity was 86% ( ci 75.7%96.3% ) due to 37 true negatives and six riassunto obiettivo . 
43 lesioni sono risultate benigne ( 13 casi di mastopatia fibrocistica , 8 di fibroadenoma , 7 di adenosi , 5 di tessuto mammario normale , 4 di lesioni flogistiche , 3 di linfonodi intramammari , 2 di scleroelastosi ed 1 di steatonecrosi ) e 35 maligne ( 30 carcinomi duttali infiltranti , 2 carcinomi lobulari infiltranti , 1 carcinoma duttale in situ , 1 mastite carcinomatosa ed 1 metastasi da carcinoma neuroendocrino )  . 
ponendo il cut off di adc pari a 1 , 48 , la dwi ha raggiunto una sensibilit ( 31 veri positivi ; 4 falsi negativi , rappresentati da 3 carcinomi duttali infiltranti e da una mastite carcinomatosa ) dell88 , 6% ( intervallo di confidenza [ ic ] : 78 , 1%99 , 1% ) ed una specificit ( 41 veri negativi ; 2 falsi positivi , dovuti ad un caso di steatonecrosi e ad un fibroadenoma ) del 95 , 3% ( ic : 88 , 9%100% )  . 
dwi is a reliable tool for characterising focal breast lesions . keywords breast cancer magnetic resonance imaging diffusion weighted imaging sensibilit ( 35 veri positivi ; nessun falso negativo ) risultata del 100% e la specificit ( 37 veri negativi ; 6 falsi positivi , per laggiunta ai due falsi positivi riscontrati con cut off di 1 , 48 di 2 fibroadenomi e 2 casi di mastopatia fibrocistica ) dell86% ( ic : 75 , 7%96 , 3% )  . 
con entrambi i cut off laccuratezza globale della dwi ( 72 risultati esatti su 78 casi ) stata del 92 , 3% ( ic : 86 , 4%98 , 2% )  . 
la dwi uno strumento affidabile nella tipizzazione delle lesioni focali mammarie . parole chiave carcinoma mammario risonanza magnetica diffusione rm introduction introduzione specificity ( 68%96% ) with its ability to provide high - resolution morphological imaging combined with information about intralesional blood flow based on spatial and temporal contrastenhancement patterns , conventional dynamic magnetic resonance imaging ( mri ) has widely demonstrated its diagnostic value in breast imaging [ 1 , 2 ]  . 
mri is able to detect tumours missed at mammography or ultrasonography [ 38 ] , stage tumours more accurately ( especially the intraductal component ) and characterise lesions as benign or malignant [ 913 ]  . 
however , there is still room for improvement in breast mri : the need to use a contrast agent prolongs ( albeit not unreasonably ) imaging times , makes the examination expensive and carries a risk of potential toxicity of paramagnetic contrast material . furthermore , although highly sensitive , breast mri has suboptimal in diagnosing neoplasms [ 1418 ]  . 
with the elimination of artefacts related to field nonuniformity [ 21 ] such as magnetic susceptibility and chemical shift [ 22 ] made possible by technological progress and in particular by parallel imaging [ 11 , 19 , 20 ] , diffusion - weighted imaging ( dwi ) has successfully been introduced into routine clinical practice . 
dwi which has higher contrast resolution than mri and does not require contrast administration [ 19 , 23 ] was initially used to diagnose cerebral ischaemia . however , it has also shown to be valuable in characterising expansile lesions in different organs and systems thanks to its ability to reflect structural features ( histological composition and cellularity ) [ 2433 ] that do not affect lesion appearance on conventional mri , which is unable to visualise tumour histology and quantitative cell / stroma ratio [ 19 , 21 ]  . 
although with some minor differences in opinion , numerous studies have reported the potential advantages of dwi compared with mri in evaluating expansile breast lesions [ 14 , 16 , 19 , 31 , 3436 ]  . the aim of this study was to examine the diagnostic grazie alla capacit di desumere dal pattern spaziale e dallandamento temporale della captazione del mezzo di contrasto para - magnetico ( mdc ) dati relativi alla circolazione ematica intra - lesionale e di acquisire parallelamente fini dettagli morfologici in immagini ad elevata risoluzione spaziale , la risonanza magnetica ( rm ) convenzionale dinamica ha ampiamente dimostrato , negli ultimi 15 anni , la sua efficacia diagnostica in ambito senologico [ 1 , 2 ]  . 
la rm infatti in grado di identificare neoplasie non riconoscibili allesame mammo - ecografico [ 38 ] , di valutarne pi accuratamente di questo lestensione ( specie nella componente intraduttale ) e , in molti casi , di definire la natura benigna o maligna delle neoformazioni [ 913 ]  . 
la rm della mammella rimane , tuttavia , unindagine ancora suscettibile di miglioramenti : la necessit di ricorrere al mdc ne prolunga ( seppure in misura non insormontabile ) la durata , la rende costosa e la espone ai rischi potenziali di tossicit impliciti nella somministrazione degli agenti para - magnetici . 
da quando levoluzione tecnologica in seguito soprattutto allintroduzione della tecnica di imaging parallelo [ 11 , 19 , 20 ] stata in grado di rimuovere gli artefatti causati dalla non - uniformit del campo magnetico [ 21 ] quali quelli di suscettibilit e di chemical shift [ 22 ] , la rm con imaging pesato in diffusione ( diffusion - weighted imaging , dwi ) stata introdotta con successo nella pratica clinica . 
inizialmente impiegata nella diagnosi dellischemia cerebrale , la dwi che rispetto alla rm dotata di pi alta risoluzione di contrasto e non presenta gli effetti sfavorevoli connessi alla necessit del ricorso al mdc [ 19 , 23 ] si dimostrata utile nella tipizzazione delle lesioni espansive di diversi organi e apparati , grazie alla sua capacit di riflettere caratteri strutturali delle lesioni stesse ( essenzialmente la composizione istologica e la cellularit ) [ 2433 ] che non ne condizionano 266 radiol med ( 2011 ) 116 : 264275 performance of dwi in a series of patients with focal breast lesions , all studied by mri and cytology and / or histology . materials and methods inclusion criteria between november 2006 and june 2008 , we recruited 241 women with mammographic and sonographic findings of at least one focal breast lesion deserving further investigation with mri . 
four patients were excluded because of contraindications to mri ( two had claustrophobia ; two wore a pacemaker ) ; 17 patients were not eligible , as the long axis of the focal lesion was 6 mm or less on mri . 
in addition , 140 patients had pathognomonic signs of benign disease on mri and dwi so that no pathological examination was required . finally , one patient refused cytological evaluation , and another did not provide consent to participate . 
our series therefore included 78 patients ( mean age 44.1 years ; range : 1877 ) for a total of 78 focal breast lesions with a long axis of 7 mm or more on mri , which were studied by both conventional mri and dwi and for whom an adequate cytological and / or histological examination was available . diagnostic investigations mri all examinations were performed with a 1.5 - t scanner ( signa excite , general electric medical systems , milwaukee , wi , usa ) with axial scans acquired with a four - channel surface coil and the patient in prone position . in our protocol , precontrast conventional mri is carried out with one t2 - weighted fat - suppressed sequence ( tr / te 4000 / 31.6 ms ; acquisition matrix 256256 ; nex = 2 ; field of view 3232 cm ; slice thickness 4 mm with 0.4 interval ; duration 3 h 49 s ) , and one t1 - weighted fast spin - echo sequence ( tr / te 540 / 7.1 ms ; acquisition matrix 320256 ; nex = 2 ; field of view 3434 cm ; slice thickness : 4 mm with 0.4 interval ; duration : 256 )  . 
alcuni lavori apparsi negli ultimi anni in letteratura testimoniano , seppure con qualche marginale discordanza di opinioni , i vantaggi che la dwi potenzialmente in grado di apportare rispetto alla rm nella valutazione delle lesioni espansive della mammella [ 14 , 16 , 19 , 31 , 3436 ]  . scopo di questo lavoro esaminare la performance diagnostica della dwi in una serie di lesioni focali mammarie , tutte sottoposte sia ad indagine rm sia a verifica citologica e / o bioptica . materiali e metodi criteri di inclusione lesione nel periodo compreso fra novembre 2006 e giugno 2008 sono state prese in considerazione , per linclusione in questo studio , 241 pazienti di sesso femminile nelle quali lesame mammo - ecografico aveva messo in evidenza almeno una focale giudicata meritevole di approfondimento diagnostico mediante rm . 
quattro pazienti sono state escluse dallo studio per la loro impossibilit ad espletare lindagine rm ( 2 in quanto claustrofobiche ; 2 portatrici di pace - maker ) ; 17 pazienti sono state giudicate non eleggibili per la valutazione dwi in quanto portatrici di lesioni focali il cui asse maggiore risultato alla rm pari o inferiore a 6 min 140 pazienti , inoltre , sia lindagine rm che quella dwi hanno dato esito a reperti patognomonici di benignit , che non hanno fatto ritenere necessario il ricorso alla tipizzazione anatomo - patologica . infine , una paziente ha rifiutato di sottoporsi allapprofondimento citologico propostole , mentre unaltra non ha espresso il proprio consenso alla propria inclusione nello studio . 
through linear regression analysis of the natural logarithm of the signal intensity versus the diffusion gradient ( b ) used ( 0 and 800 s / mm2 ) inside an operator - defined region of interest ( roi ) , the software quantified the extent of diffusion and produced an apparent diffusion coefficient ( adc ) expressed in millimetres squared per second . reference standard all 78 lesions in the study underwent fine - needle ( 2022 gauge ) aspiration cytology under mammographic or sonographic guidance , depending on which was considered more appropriate by the operator . 
in lesions with inadequate or doubtful cytology and with a high index of suspicion at sonography / mammography and / or mri , the diagnostic evaluation was completed by histological examination of a 14 - gauge cutting - needle core biopsy . 
in the case of cytological or biopsy findings of uncertain malignant potential or suspicious for malignancy ( b3 or higher ) , the lesion was surgically excised , and histological examination was performed on the surgical specimen . statistical analysis for the purposes of this study , lesions were classified as probably malignant or probably benign on the basis of the mri findings . 
statistical significance of the differences in mean adc values between benign and malignant lesions was determined by applying the mann - whitney test . results pathological characterisation was obtained for all 78 lesions : in 49 cases by histology ( in 38 cases on the surgical specimen , in 11 on the biopsy specimen ) and in 29 by cytology . 
among malignant lesions , infiltrating ductal carcinoma not otherwise specified ( nos ) was by far the most frequent histological type , whereas the most frequent benign conditions were fibrocystic mastopathy , fibroadenoma and adenosis ; in five cases , breast tissue showed no pathological abnormality . 
lesame rm convenzionale pre - contrastografico prevede , nel nostro protocollo , una sequenza t2 con tecnica di fat suppression ( tempo di ripetizione [ tr ] / tempo di eco [ te ] : 4000 / 31 , 6 ms ; matrice di acquisizione : 256256 ; nex = 2 ; field of view : 3232 cm ; spessore di fetta : 4 mm con intervallo di 0 , 4 ; durata : 3 min 49 s ) ed una sequenza t1 fast spin - echo ( tr / te : 540 / 7 , 1 ms ; matrice di acquisizione : 320256 ; nex = 2 ; field of view : 3434 cm ; spessore di fetta : 4 mm con intervallo di 0 , 4 ; durata : 2 min 56 s )  . 
lindagine dinamica consiste in una sequenza t1 gradient - echo ( tr / te : 4 , 6 / 2 , 1 ms ; matrice di acquisizione : 256256 ; nex = 1 ; field of view : 3232 cm ; spessore di fetta : 4 mm con intervallo di 0 , 4 ; flip angle : 10 ; durata : 7 min 13 s ) , che prevede una acquisizione basale e 6 acquisizioni successive della durata di circa un minuto ciascuna , dopo la somministrazione endovenosa di mezzo di contrasto a base di acido gadoterico ( dotarem , guerbet , roissy cdg cedex , francia ) alla dose di 0 , 2 ml / kg e con velocit di infusione di 2 , 5 ml / s . dwi le immagini pesate in diffusione sono state acquisite , in scansione assiale , mediante una sequenza echo - planar ( asset ) con tecnica di imaging parallelo ( tr / te : 4000 / 55 , 2 ms ; matrice di acquisizione : 6464 ; nex = 8 ; field of view : 3434 cm ; spessore di fetta : 7 mm con intervallo di 0 , 4 ; durata : 2 min 08 s )  . 
il software , allinterno di una zona di interesse selezionata dalloperatore ( region of interest , roi ) , mediante analisi di regressione lineare del logaritmo naturale dellintensit del segnale versus il gradiente di diffusione ( b ) utilizzato ( 0 e 800 s / mm2 ) , ha quantificato lentit della diffusione ricavando un valore di apparent diffusion coefficient ( adc ) espresso in mm2 / s . 
 reference standard tutte le lesioni incluse nello studio , il cui quadro non sia stato considerato patognomonico di lesione benigna sia allindagine rm che a quella dwi , sono state sottoposte ad esame citologico con ago sottile ( 2022 g ) guidato dalla metodica di imaging ( mammografia o ecografia ) giudicata nel singolo caso pi opportuna dalloperatore . 
nelle lesioni con risultato citologico inadeguato o dubbio in concomitanza di un livello di sospetto elevato allindagine ecomammografica e / o rm liter diagnostico stato proseguito tramite verifica istologica mediante micro - biopsia percutanea con ago tranciante da 14 g . 
in caso di reperto citologico o micro - bioptico indicativo o sospetto di natura maligna ( giudicato in classe b3 o superiore ) la lesione stata asportata chirurgicamente con successiva verifica istologica del pezzo operatorio . 
laffidabilit diagnostica della dwi stata confrontata con il gold standard rappresentato dal reperto citologico e / o istologico utilizzando i due cut off di adc ( 1 , 48 e 1 , 52 10 - 3 mm2 / s ) che secondo lesperienza personale garantiscono adottando le modalit tecniche di esecuzione dellindagine sopra riportate le performance pi efficaci . 
accelerated by membrane permeability between the intraand extracellular compartments and transmembrane transport mechanisms and hindered by risultati di tutte le 78 lesioni comprese in questo studio stata ottenuta la tipizzazione anatomo - patologica : in 49 casi mediante esame istologico ( in 38 casi su analisi istologica del pezzo operatorio , in 11 mediante biopsia percutanea ) , in 29 attraverso indagine citologica . 
although extensive neoangiogenesis implies abundant perfusion [ 6 , 15 , 16 ] , the speed of diffusion is slower in malignancies than in normal tissues and benign lesions [ 14 , 15 ]  . 
this presumably is because of the dominant hindering effect of high cellularity , which reduces the amount of extracellular water and increases barriers to motion [ 14 , 15 , 20 ] , as demonstrated in both spontaneous and experimental tumours [ 34 , 39 ]  . the sensitivity of dwi in detecting breast malignancies varies between being close to that of mri in some reports [ 20 , 23 ] and definitely lower in others [ 16 , 32 ] : in particular , dwi is limited in identifying lesions < 810 mm , for which adc values cannot as yet be determined with certainty [ 15 ]  . 
the risk of not being able to reliably evaluate the speed of water diffusion in small lesions led us to exclude lesions 6 mm , even though we have seen that some 5and 6 - mm masses that are well depicted by mri can also be correctly identified by dwi . in this study , we applied what we have found to be the ( nas ) , mentre le patologie benigne pi rappresentate sono state mastopatia fibrocistica , fibroadenoma ed adenosi ; in 5 casi stato riscontrato tessuto mammario esente da alterazioni anatomo - patologiche . 
la tabella 3 esprime in dettaglio i singoli risultati ottenuti con i due cut off , riportando anche per completezza la performance diagnostica che nella stessa serie di pazienti avrebbe avuto la rm , classificando i reperti esclusivamente come probabilmente benigni o probabilmente maligni . la differenza fra i valori di adc misurati nelle lesioni maligne e in quelle benigne risultata altamente significativa allanalisi statistica ( p < 0 , 001 ) espletata mediante il test di mann - whitney . due impulsi successivi di radiofrequenza di intensit uguale e di direzione opposta , applicati su una sequenza spin echo prima e dopo limpulso rifocalizzante a 180 , destinati ad elidersi a vicenda se interagiscono con bersagli statici , danno luogo a un segnale diverso da zero ( e quantificabile ) nel caso le molecole da essi incontrate siano in movimento [ 28 , 37 ]  . 
su questo presupposto fisico fondato limpiego diagnostico della dwi , che allinterno di una zona di interesse selezionata dalloperatore sullimmagine rm preliminare consente di misurare mediante un apposito parametro numerico ( adc ) la velocit della diffusione , ovvero del movimento casuale dei protoni legati allacqua contenuta nel fluido extra - cellulare [ 14 , 21 , 28 ]  . 
accelerato dalla permeabilit di membrana fra i compartimenti intraed extra - cellulare e dai meccanismi di trasporto attivo trans - membrana ed ostacolato dalla viscosit dei fluidi e dalla quantit di macromolecole e di membrane cellulari presenti [ 38 ] , il movimento di diffusione dipende fondamentalmente dalla perfusione tessutale e dal numero di cellule presenti . nonostante la ricchezza della neoangiogenesi vi comporti una perfusione elevata [ 6 , 15 , 16 ] , nei tumori maligni la velocit della diffusione risulta inferiore a quella osservata nei tessuti normali e nelle lesioni benigne [ 14 , 15 ] , presumibilmente per il prevalere delleffetto di ostacolo esercitato dalla cellularit elevata , che riduce la quantit di acqua extra - cellulare e aumenta le barriere contro il movimento [ 14 , 15 , 20 ] , come stato dimostrato in tumori sia spontanei che sperimentali [ 34 , 39 ]  . 
 la sensibilit della dwi nellidentificare le lesioni maligne della mammella , per quanto prossima a quella radiol med ( 2011 ) 116 : 264275 most appropriate cutoff values for imaging protocols that used and achieved dwi accuracy of 92.3% in differentiating between benign and malignant lesions ( table 3 )  . 
these levels are only slightly different from those achieved with mri , which having to classify the findings as either benign or malignant only produced only one error less than did dwi ( table 3 )  . to our knowledge , all studies on this topic have reported a statistically significant difference between the mean adc values of malignant lesions ( range 0.951.22 , depending on the b value ) and benign lesions ( 1.441.67 ) [ 1416 , 19 , 20 , 23 , 28 , 29 , 32 , 33 ]  . 
in our experience , adc values were moderately higher than those reported in the literature , presumably because our protocol involved lower b values ( 0800 mm / s2 ) than used by most authors ( 01 , 000 ) , a decision dictated by the need to obtain a high signal - to - noise ratio even with a four - channel coil . 
however , we did della rm in alcuni studi [ 20 , 23 ] , secondo altri autori alquanto inferiore [ 16 , 32 ] : in particolare , la dwi dimostra difficolt nel riconoscere lesioni con diametro inferiore a 810 mm , nelle quali non al momento possibile determinare con certezza i valori di adc [ 15 ]  . 
il rischio di una valutazione non del tutto attendibile della velocit di diffusione dellacqua nelle lesioni di dimensioni esigue ha indotto ad escludere da questo studio i reperti con diametro pari o inferiore a 6 mm , sebbene nellesperienza personale alcune formazioni con maggior asse di 5 e 6 mm , ben evidenti allindagine rm , siano state correttamente inquadrate mediante dwi . in questo studio , adottando i valori di cut off pi appropriati secondo lesperienza personale , laccuratezza della dwi nella diagnosi differenziale fra lesioni benigne e maligne risultata pari al 92 , 3% ( tabella 3 ) , ovvero lievemente superiore a quella riportata dai pi ottimistici fra i lavori presenti in letteratura : i dati pubblicati oscillano infatti fra l83 , 6% ed il 91% [ 14 , 15 , 29 , 33 ]  . 
sensibilit e specificit sono ovviamente influenzate dai cut off di adc scelti dai singoli autori , i quali oscillano allinterno di un intervallo piuttosto ampio ( da 1 , 1 a 1 , 610 - 3 mm2 / s ) ; nellesperienza personale si ottengono ferma restando laccuratezza globale del 92 , 3% sensibilit dell88 , 6% e specificit del 95 , 3% ponendo il cut off a 1 , 48 , parametri che diventano rispettivamente 100% e 86% se il valore scelto aumentato a 1 , 52 ( tabella 3 )  . 
1a , b scansioni rm assiali dinamica ( a ) e pesata in diffusione ( b ) di una mammella destra dove si dimostra la presenza di una lesione rotondeggiante a margini spiculati . 
2a , b axial dynamic magnetic resonance image ( a ) and diffusionweighted image ( b ) showing a rounded spiculated lesion in the left breast . both the enhancement pattern and the apparent diffusion coefficient value ( region of interest 1.10 ) are typical of a malignant lesion . 
2a , b scansioni rm assiali dinamica ( a ) e pesata in diffusione ( b ) dove si dimostra la presenza di una lesione rotondeggiante a margini spiculati nella mammella sinistra . 
listologia ha dimostrato un carcinoma duttale nas . not perform this evaluation in our series , as we restricted out analysis to focal lesions and had very few cases of normal breast tissue at histology ( tables 1 and 2 )  . even though relatively few reports exist on the subject , among the possible causes of false negative results in addition to the low cellularity of some histological types such as scirrhous [ 14 ] , lobular [ 29 ] , apocrine [ 21 ] and radiol med ( 2011 ) 116 : 264275 in tutti i lavori di cui siamo a conoscenza risulta statisticamente significativa la differenza fra i valori medi di adc misurati nelle lesioni maligne ( compresi , a seconda dei valori di b adottati , fra 0 , 95 e 1 , 22 ) e in quelle benigne ( 1 , 441 , 67 ) [ 1416 , 19 , 20 , 23 , 28 , 29 , 32 , 33 ]  . 
nellesperienza personale i valori di adc sono modestamente superiori a quanto riportato in letteratura , presumibilmente in conseguenza delladozione , nel nostro protocollo , di valori di b ( 0800 mm / s2 ) pi bassi di quelli utilizzati dalla maggior parte degli autori ( 01000 ) : tale scelta stata motivata dallintenzione di ottenere un rapporto segnale / rumore elevato anche con la bobina a 4 canali impiegata in questo studio . 
la nostra casistica priva di falsi negativi con la scelta del cut off sensibile di 1 , 52 ne annovera 4 adottando il cut off specifico di 1 , 48 . 
in tre evenienze ( tabella 1 ) lerrore stato sostenuto da carcinomi duttali infiltranti nas ( peraltro ampiamente prevalenti fra le lesioni maligne riscontrate in questa casistica ) ; del quarto caso di falsa negativit stata invece fig . 
4a , b axial dynamic mri ( a ) and diffusion - weighted image ( adc ) ( b ) showing a rounded , irregularly margined lesion in the remaining portion of a left breast after quadrantectomy . 
4a , b scansioni rm assiali dinamica ( a ) e pesata in diffusione ( b ) dimostranti la presenza di una lesione rotondeggiante a margini irregolari nella porzione rimanente di una mammella sinistra dopo quadrantectomia . 
laspetto rm suggerisce una recidiva di malattia , mentre il valore di adc ( molto basso : 1 , 32 ) tipico di una lesione maligna ; tuttavia listologia ha dimostrato una zona di steatonecrosi . 
 mucinous [ 15 ] carcinomas is the possible presence of fluid areas capable of raising the adc values , such as areas of haemorrhage and intratumoural necrosis [ 15 , 29 ]  . 
our series , which did not yield any false negative results when the sensitive cutoff value of 1.52 was selected , revealed only four false negatives with the specific cutoff value of 1.48. 
in three cases ( table 1 ) , the error was due to infiltrating ductal carcinomas nos ( the most common malignancy in our series ) ; in the fourth case , the false negative result was related to carcinomatous mastitis , in which it is reasonable to suppose that the lack of a well - defined focal lesion may have misled both the mri and dwi study the latter with regard to the choice of the roi . the high cellularity typical of some benign lesions ( intraductal papilloma and fibrocystic mastopathy ) has been implicated [ 14 , 29 , 31 ] as the possible cause of false positive dwi findings . 
the lowest adc values were recorded in a case of fibrocystic mastopathy ( 1.45 ) , a finding consistent with previous reports [ 14 , 29 ] and , surprisingly , in a case of fat necrosis . 
to our knowledge , the literature offers no explanation for this finding : however , it may be speculated that the naturally hydrophobic complex lipids leaking out of necrotic fat cells form an insurmountable barrier to the motion of water molecules and thus significantly reduce diffusion velocity . in our study , given the small number of patients and the responsabile una forma di mastite carcinomatosa , nella quale lecito supporre che lassenza di una lesione focale ben delimitata abbia verosimilmente ingannato sia lindagine rm che quella dwi , questultima forse nella fase di scelta della regione di interesse . la elevata cellularit di alcune lesioni benigne ( papillomi intra - duttali e mastopatia fibrocistica ) stata invocata in letteratura [ 14 , 29 , 31 ] per giustificare i reperti dwi di falsa positivit . 
nella nostra serie il numero di falsi positivi ( tabella 2 ) , adottando il cut off sensibile di 1 , 52 , pari a 6 ( 3 fibroadenomi , 2 casi di mastopatia fibrocistica ed un caso di steatonecrosi ) ; la cifra si riduce a 2 con il cut off specifico di 1 , 48 . 
i valori pi bassi di adc sono stati misurati in un caso di mastopatia fibrocistica ( 1 , 45 ) , reperto compatibile con i dati della letteratura [ 14 , 29 ] e , piuttosto sorprendentemente , in un caso di steatonecrosi . 
non siamo a conoscenza di spiegazioni pubblicate in merito nella letteratura : possiamo ipotizzare che fuoriusciti dalla lipidi complessi membrana di cellule adipose necrotiche costituiscano , per il loro naturale comportamento idrofobico , un ostacolo difficilmente sormontabile allo spostamento delle molecole di acqua , riducendo pertanto significativamente la velocit di diffusione rilevata . in questo studio , stante la consistenza numerica della casistica e la ripartizione qualitativa delle lesioni maligne ( tabelle 1 e 2 ) , non stato possibile espletare valutazione statistica riguardo alleventuale correlazione tra valori di adc e tipo istologico , correlazione affermata da alcuni 274 radiol med ( 2011 ) 116 : 264275 qualitative distribution of malignant lesions ( tables 1 and 2 ) , we were unable to perform a statistical evaluation of the possible correlation between adc and histology a correlation supported by some authors [ 15 , 35 , 39 ] but refuted by others [ 19 ]  . this study has some limitations . 
because we selected only patients with lesions deserving investigation with mri and , among them , only those requiring pathological characterisation , our series does not contain findings with a low index of suspicion , the presence of which might have reduced the diagnostic performance of dwi . 
the roi in which to determine the adc was selected by the operator on the basis of a subjective judgement on the mri and may not have accurately reflected the behaviour of the entire lesion . in conclusion , we believe that dwi , an inexpensive and relatively rapid technique , is a useful diagnostic complement for characterising focal breast lesions in patients undergoing mri , even though it is as yet unable to avoid the need for pathological characterisation . autori [ 15 , 35 , 39 ] , ma negata da altri [ 19 ]  . 
essendo state selezionate solo pazienti portatrici di lesioni giudicate meritevoli di essere sottoposte a rm e fra queste quelle nelle quali si ritenuto necessario il ricorso alla tipizzazione anatomo - patologica , la casistica non contiene reperti con livello di sospetto basso la cui presenza potrebbe ridurre lefficacia diagnostica della dwi . 
marini4 1radiologia durgenza , ospedale san camillo , c.ne gianicolense 87 , 00152 roma , italy 2dipartimento di otorinolaringoiatria , sapienza universit di roma , italy 3radiologia generale , ospedale san camillo , roma , italy 4dipartimento di scienze radiologiche , sapienza universit di roma , italy correspondence to : a . 
 keywords computed tomography magnetic resonance chronic otitis media preoperative assessment riassunto scopo del presente lavoro puntualizzare limportanza dellimaging integrato con tomografia computerizzata ( tc ) ed risonanza magnetica ( rm ) nella gestione dellotite media cronica , con particolare riguardo alle informazioni chiave necessarie allotoiatra per una corretta pianificazione della terapia chirurgica . parole chiave tomografia computerizzata risonanza magnetica otite media cronica valutazione preoperatoria introduction introduzione due the ever - growing role of computed tomography ( ct ) and magnetic resonance imaging ( mri ) in evaluating the temporal bone , many authors [ 17 ] have studied the middle ear after a surgical procedure , whereas only a few [ 8 , 9 ] , and not recently , have focused on the relevant aspects of these techniques for a more accurate preoperative planning in chronic otitis media ( com )  . 
when treatment regime , either surgical or conservative , a seguito del crescente impiego della tomografia computerizzata ( tc ) e della risonanza magnetica ( rm ) nello studio dellosso temporale , numerosi autori hanno affrontato il tema dellorecchio medio operato [ 17 ] , mentre pochi lavori , e nessuno di recente , sono focalizzati sugli aspetti rilevanti per un corretto planning preoperatorio dellotite media cronica ( omc ) [ 8 , 9 ]  . 
quando il trattamento chirurgico , pur non programmato , radiol med ( 2011 ) 116 : 114124 upon , represents an option to be evaluated after failure of medical treatment . 
a negative ct scan thanks to its high negative predictive value is able to exclude the presence of a significantly sized cholesteatoma in the dry middle ear [ 10 ]  . 
in the chronically inflamed ear , however , different tissue components may be present at the same time , and their characterisation may determine treatment decisions ( whether conservative or surgical )  . 
la tc , grazie ad un elevato potere predittivo negativo in grado di escludere la presenza di un colesteatoma di dimensioni significative nellorecchio medio ben aerato [ 10 ]  . il materiale patologico pi frequentemente riscontrabile con tc in corso di omc il tessuto di granulazione , che non provoca generalmente erosioni n dislocazioni della catena ossiculare [ 11 ]  . 
nellorecchio cronicamente infiammato , tuttavia , possono coesistere varie componenti tissutali , la caratterizzazione delle quali pu condizionare in modo decisivo la scelta di un approccio conservativo o chirurgico : se lorecchio contiene essudato , tessuto di granulazione o fibrosi , il trattamento dipende soprattutto da fattori clinici ( stato uditivo dellorecchio , entit delle secrezioni , ecc . ) , mentre la dimostrazione di un granuloma colesterinico o di un colesteatoma impone generalmente un approccio chirurgico . 
 la diagnosi differenziale tra i tessuti che possono riscontrarsi nellorecchio medio non possibile in tc su rilevazioni densitometriche [ 10 , 12 ] e dovrebbe basarsi sul riconoscimento di precisi segni elementari : ispessimento mucoso , segni di versamento , segni di lesione occupante spazio . 
nellorecchio parzialmente aerato tale semeiotica pu essere impiegata con la massima efficacia , mentre in un orecchio completamente opacato , la discriminazione tra le varie componenti diventa inattendibile [ 13 , 14 ] e richiede limpiego di rm con sequenze in diffusione ed acquisizioni multifasiche dopo somministrazione di mezzo di contrasto ( mdc ) endovena ( ev ) [ 1517 ]  . 116 radiol med ( 2011 ) 116 : 114124 exudate , granulation tissue or fibrosis , treatment depends above all on clinical factors ( auditory state , extent of secretions , etc . ) , whereas demonstration of a cholesterol granuloma or cholesteatoma normally warrants a surgical approach . 
 differential diagnosis among the various tissues that might be detected in the middle ear is not possible by densitometric ct analysis [ 10 , 12 ] and should be based on precise evidence : mucosa thickening ; signs of effusion or a spaceoccupying lesion . 
in a partially aerated ear , such analysis can be effectively carried out , but in an entirely opacified ear , differentiation among the various components is totally unreliable [ 13 , 14 ] and may require the use of mri with diffusion - weighted imaging ( dwi ) and delayed contrastenhanced postgadolinium sequences [ 1517 ]  . ct depiction of the middle - ear mucosal lining always suggests inflammation [ 10 ] , whereas the diagnosis of effusion is based on the presence of gasfluid levels within the partially opacified ear . 
detecting an occupying - space lesion is compatible with both inflammatory ( granulomatous polyps , unusually extensive fibrous scarring foci , cholesterol granulomas , cholesteatomas ) and noninflammatory processes ( such as glomus tympanicum , the most frequent tumour occurring in the middle ear ) and is based identifying , even partially , lesion margins , dislocation / lysis of ossicular elements , surrounding bone walls of the middle ear and protrusion of the tympanic membrane . 
once the presence of a spaceoccupying lesion has been determined , its characterisation may be based on direct ( margins , density , signal , epicentre ) or indirect ( associated changes to the ossicles and middleear walls )  . 
the correlation between the condition of the ossicular chain as depicted by ct and as seen at surgery is controversial [ 2225 ]  . la visibilit del rivestimento mucoso dellorecchio medio sempre segno di flogosi [ 10 ] , mentre la diagnosi di versamento si basa sulla dimostrazione di livelli idroaerei nel contesto dellorecchio parzialmente opacato . la dimostrazione di una lesione occupante spazio compatibile con patologie flogistiche ( polipo granulomatoso , focolai fibrocicatriziali insolitamente ampi , granuloma colesterinico , colesteatoma ) e non flogistiche ( ad esempio , glomo timpanico , il pi frequente tumore dellorecchio medio ) e si basa sul riconoscimento almeno parziale dei margini della lesione , sulla dislocazione / lisi degli elementi ossiculari , sul rimaneggiamento espansivo o litico delle circostanti pareti ossee dellorecchio medio , sullestroflessione della membrana timpanica . 
stabilita la presenza di una lesione occupante spazio , la sua ulteriore caratterizzazione pu essere effettuata su segni diretti ( margini , densit , segnale , epicentro ) ed indiretti ( alterazioni associate degli ossicini e delle pareti dellorecchio medio )  . 
granuloma colesterinico : tessuto epitimpanico iperintenso in t1 e t2 ( non mostrata )  . of particular interest for the surgeon is assessment of the incudostapedial joint and the stapes . 
lapproccio canal - up necessita di una accesso pi ampio , possibilmente attraverso un sistema cellulare ampio , mentre lapproccio canal - down preferibile nelle mastoidi sclerotiche [ 9 ]  . natura dei tessuti la mastoidectomia nelle otiti croniche non colesteatomatose non sembra presentare sicuri vantaggi rispetto alla sola timpanoplastica , associandosi peraltro ad un maggior rischio di complicanze operatorie [ 26 ]  . estensione di malattia nellosso temporale anche quando la diagnosi natura della lesione ( ad esempio , un colesteatoma ) otoscopicamente certa , resta da determinare la reale estensione della patologia : lotoscopia infatti potrebbe non evidenziare che la propaggine pi superficiale di una lesione imprevedibilmente estesa . 
descrivere con propriet la / le sede / i entro i cui limiti confinata la lesione ( cassa timpanica , attico , antro , tuba , labirinto , fossa media ) determinante ai fini della scelta chirurgica . 
the radiologist , however , should be very cautious in providing a diagnostic aetiology in the absence of adequate otoscopic ( lesion colour and pulsatility ) and clinical ( characteristics of otorrhea , tinnitus and type of associated hypoacousia ) findings . preoperative planning temporal - bone cellularity it is important to define the general architecture of the mastoid process by considering a pneumatised mastoid as the opposite of a sclerotic mastoid . 
the approach to a closed and intact canal wall requires a wide access , ideally through a large cellular mastoid , whereas a in smaller canal - wall - down approach is preferable estensione di malattia nellorecchio medio propriamente detto sedi di particolare interesse chirurgico sono il recesso epitimpanico anteriore , il seno timpanico ed il recesso del facciale . 
the modified radical cavity of larger cellular systems holds poorer prospects for healing . equally , the intact canal wall technique in a tiny mastoid cavity is technically difficult because of its poor access [ 9 ]  . fistola labirintica nature of the tissues compared with tympanoplasty alone , mastoidectomy for noncholesteatomatous com does not seem to be advantageous , and procedure - related complications with this technique occur in a higher number of patients [ 26 ]  . 
 disease extension to the temporal bone the diagnostic origin of a lesion ( e.g. , a once cholesteatoma ) is otoscopically confirmed , its real extension should then be determined . 
accurately describing the lesions site ( s ) and boundaries ( tympanic cavity , attic , antrum , eustachian tube , labyrinth , middle fossa ) is vitally important in surgical planning . 
smaller lesions can be treated by targeted and minimally invasive procedures , whereas lesions involving three or more of the abovementioned spaces usually require a radical mastoidectomy [ 2729 ]  . la presenza di una fistola labirintica in caso di colesteatoma varia dal 3 , 6% [ 39 ] al 12 , 9% [ 40 ]  . 
la presenza di pneumolabirinto un segno certo di fistola , ma di raro riscontro . fistole del canale semicircolare laterale ( csl ) o della coclea sono di agevole dimostrazione , perch associate a discontinuit del relativo rivestimento osseo . 
difficile la dimostrazione di fistole della finestra rotonda , che ricoperta da unesile membrana invisibile in tc : il sospetto suggerito dalla sola presenza di tessuto loco - regionale ed avvalorato da aspetti erosivi dellosso circostante ; la dimostrazione di fluido isolato nel seno del timpano ( fluidi endolabirintici scolati nella cassa timpanica ) un segno altamente sospetto . 
anche nel caso di fistola della finestra ovale deve essere valorizzata la semplice presenza di tessuto nellomonima fossa , specialmente in presenza di erosioni ossee ; la compresenza di aspetti erosivi della staffa , la sua frammentazione o dislocazione nella finestra ovale sono particolarmente suggestivi di fistola [ 42 ]  . 
 deiscenza dei tegmina la discontinuit ( deiscenza ) del tetto dellorecchio medio radiol med ( 2011 ) 116 : 114124 disease extension in the middle ear as such the anterior epitympanic recess , the tympanic sinus and the facial recess are anatomical sites of particular surgical interest . 
the presence of a pneumolabyrinth is a definite sign of fistulas , but its detection is unusual ; lateral semicircular canal or cochlear fistulas are instead easily depicted , since their presence is associated with a discontinuity of the related bone layer . 
depicting fistulas of the round window , which is covered by a thin membrane not visualised by ct , is difficult ; a clue is provided by the presence of locoregional tissue , whereas signs of erosion of the surrounding bone corroborate the diagnosis . as an additional sign , demonstration of an isolated collection within the tympanic sinus ( endolabyrinthine fluids draining within the tympanic cavity ) is highly suspicious for fistula formation . 
even in the case of fistulas of the oval window , the mere presence of tissue in the oval fossa should be highlighted , especially if bone erosions are also present . 
fistola del canale semicircolare laterale ( freccia corta ) e superiore ( freccia lunga )  . deve essere segnalata perch si associa al rischio di lesioni infiammatorie durali o cerebrali ( lobo temporale ) nella sovrastante fossa cranica media . 
 complicanze endocraniche tra le possibili complicanze endocraniche dellotite media , meningite , trombosi dei seni durali ed empiema subaracnoideo sono caratteristiche delle otiti medie acute ; lascesso cerebrale ( del lobo temporale o del cervelletto ) invece di solito una complicanze dellotite cronica [ 43 ]  . lascesso facilmente dimostrabile in rm e dovrebbe essere escluso ogni qual volta si apprezzi una deiscenza delle limitanti ossee per la fossa posteriore o per la fossa media ( tegmina ) di un orecchio non precedentemente operato contenente materiale infiammatorio . 
 tegmina dehiscence elementi anatomici caratteristici del paziente any discontinuity ( dehiscence ) of the tegmen tympani should be noted , as it might be associated with the risk of a dural or cerebral ( temporal lobe ) inflammatory lesion in the overlying middle cranial fossa . procidenza della dura madre la dura madre della fossa cranica media generalmente separata dal lume del condotto uditivo estgerno ( cue ) da radiol med ( 2011 ) 116 : 114124 uno strato di cellule aeree dette tegmentali ( cio del tetto del cue ) ; lassenza di tali cellule mette la dura in diretta apposizione con il periostio del cue e limita laccesso alladitus e allepitimpano per il conseguente rischio di lesione iatrogena della dura e del lobo temporale . 
 procidenza e deiscenza del seno laterale il seno laterale separato dalla parete posteriore del cue da una distanza variabile ; considerato procedente quando la sua distanza dalla parete posteriore del cue < 10 mm e costituisce un rischio chirurgico [ 44 ] anomalie di decorso o deiscenze del seno laterale sono riscontrabili in circa il 2%3% dei pazienti [ 45 ]  . il bulbo giugulare si pone in circa il 6% dei casi al di sopra del livello dellannulus timpanico inferiore ( bulbo giugulare alto ) [ 46 ] interferendo potenzialmente con lapproccio chirurgico . 
oltre ad essere altoposto , il bulbo giugulare pu mancare del suo normale rivestimento osseo ( settoipotimpano giugulare ) e trovarsi in diretta apposizione con tessuto patologico nellorecchio medio ( rischio di flebite e trombosi ) oppure a rischio di lesione iatrogena per lesioni ipotimpaniche o interessanti le celle retrofacciali o durante il sollevamento di un flap timpanomeatale . 
 [ 47 , 48 ]  . decorso della carotide interna la deiscenza della carotide rara ( 1 , 4% ) [ 44 ] e mette il paziente a rischio di una grave emorragia arteriosa anche anatomical characteristics of the patient bulging of the dura mater bulbo della giugulare endocranial complications among the various possible endocranial complications of com , meningitis , dural sinus thrombosis and subarachnoid empyema are typical of acute otitis media ; a cerebral abscess ( of the temporal lobe or the cerebellum ) is , instead , usually a complication of chronic otitis [ 43 ]  . 
an abscesseasily depicted by mri - should be excluded whenever posterior or middle cranial fossa are exposed to pathological tissues in the middle ear by dehiscence of the adjacent bone structures . the dura mater of the middle cranial fossa is usually separated by the lumen of the external auditory canal by an aircell layer ( tegmental cells )  . 
the absence of these cells places the dura mater directly in touch with the periosteum of the external auditory canal and limits access to the aditus and the epitympanum due to the related risk of iatrogenic lesions to the dura mater and temporal lobe . 
course anomalies or dehiscences of the lateral sinus are detected in about 2%3% of patients [ 45 ]  . jugular bulb in approximately 6% of cases , the jugular bulb is placed above the level of the lower annulus tympanicus ( high jugular bulb ) [ 46 ] , thus precluding translabyrinthine approaches . 
in addition to this anomaly , if the normal covering bony layer is also missing , the jugular bulb is directly juxtaposed to the pathological tissue of the middle ear , thus being at risk of thrombosis / phlebitis or of an iatrogenic lesion induced by hypotympanic traumas or involving retrofacial cells , or during the raising of a tympanomeatal flap [ 47 , 48 ]  . internal carotid course carotid dehiscence is rare ( 1.4% ) [ 44 ] and may induce a serious arterial haemorrhage , even during minor inpatient fig . 
an aberrant carotid artery is an extremely rare event and is due to the lack of cervical segment development of this vessel [ 45 ] and is frequently associated with the presence of an aberrant stapedial artery . 
una carotide aberrante estremamente rara e dovuta al mancato sviluppo del segmento cervicale della carotide interna [ 45 ] , in associazione alla presenza di una arteria stapediale persistente . un prelievo bioptico o una lesione chirurgica possono in tal caso provocare grave emorragia od ictus cerebrale . conclusions conclusioni imaging techniques provide key elements in managing com : ct is able to reliably exclude the presence of middleear anomalies , whereas characterisation of pathological tissues , which is ideal but not always possible despite the use of both ct and mri , remains mainly dependent on the combined evaluation of clinical , otoscopy and biopsy findings . 
la caratterizzazione dei tessuti patologici altamente desiderabile ma non sempre possibile , nonostante limpiego combinato di tc ed rm , restando spesso legata alla valutazione integrata di dati clinici , otoscopici e bioptici . 
guney1 1department of radiology , 2department of cardiology , 3department of psychiatry , maltepe university school of medicine , feyzullah cad , no : 39 , 34843 , maltepe , istanbul , turkey correspondence to : r . 
2016 , fax : + 90 - 216 - 3830271 , e - mail : rahmicubuk@yahoo.com received : 24 november 2009 / accepted : 2 march 2010 / published online : 17 september 2010 springer - verlag 2010 abstract purpose . 
the participating patients were randomly assigned to one of the two study groups : a control group ( no medication administered for anxiety reduction ) and an anxiolytic medication group , with 30 patients in each group . 
the presence of motion artefacts and image quality for each coronary artery segment were evaluated using a four - point grading systeto estimate hrv , the duration of each heartbeat during mdct data acquisition was measured in each patient . 
i pazienti che hanno partecipato allo studio sono stati casualmente assegnati ad uno dei due gruppi di studio : il gruppo di controllo ( ai quali non veniva somministrato ansiolitico per ridurre lo stato dansia ) ed il gruppo dei pazienti trattati con ansiolitici , questultimo composto da 30 pazienti . 
stata riscontrata una moderata correlazione tra la variabilit della frequenza cardiaca ( hrv ) durante lacquisizione delle immagini mdct e la media della qualit dellimmagine per tutti i segmenti coronarici studiati ( r = 0 , 47 , p < 0 , 01 )  . 
vi era una concordanza tra la variabilit della frequenza cardiaca ( hrv ) ed il livello dansia in tutti i radiol med ( 2011 ) 116 : 4755 that oral premedication to reduce anxiety is also effective in decreasing hrv and improves image quality . 
we also suggest that further studies in larger series are required to validate this finding . keywords multidetector computed tomography coronary angiography heart rate benzodiazepines premedication casi ( r = 0 , 370 , p < 0 , 01 )  . 
la variabilit della frequenza cardiaca ( hrv ) nei pazienti trattati con alprazolam era significativamente pi bassa che nei controlli ( p < 0 , 05 )  . anche la qualit delle immagini nei pazienti trattati con alprazolam era significativamente pi elevata che nei controlli ( p < 0 , 05 )  . 
il riscontro del nostro studio che la premedicazione con ansiolitici per via orale riduce lo stato dansia ed efficace anche nel ridurre la variabilit della frequenza cardiaca ( hrv ) e nel migliorare la qualit delle immagini . 
pertanto , suggeriamo che lutilizzo dellalprazolam in combinazione con gli - litici potrebbe migliorare la qualit delle immagini nei pazienti sottoposti ad angiografia coronarica mediante tc multidetettore ( mdct - ca )  . 
 parole chiave tomografia computerizzata multidetettore angiografia coronarica frequenza cardiaca benzodiazepine premedicazione introduction the main limitation to the use of multidetector row computed tomography ( mdct ) in coronary angiography ( ca ) is temporal resolution [ 13 ]  . 
with the introduction of 64 - detector mdct , the problem of temporal resolution has been substantially resolved , but image quality remains poor in patients with high heart rates ( hr ) [ 35 ]  . 
in another study in which they compared mdct - ca and invasive ca , they reported that a low hrv increases the diagnostic accuracy of 64 - detector mdct - ca [ 7 ]  . 
currently , - blocker administration for premedication is used as the standard procedure to reduce hr in ca performed by mdct with low detector counts , including 64 detectors [ 811 ]  . 
it is reported that blocker administration increases image quality by reducing the hr or , additionally , the hrv [ 6 , 7 ]  . as in many medical procedures , patients can be stressed before undergoing mdct - ca , and the complication rates increase due to impaired patient compliance [ 12 , 13 ]  . 
in addition to the stress originating from mdct imaging , the potentially bad results of the examination can produce high anxiety , especially in patients with coronary artery disease ( cad ) for whom the outcome is of vital importance . we performed a literature review and could find no study evaluating the relationship between the use of anxiolytic premedication before mdct - ca , patients anxiety status and hrv / image quality . 
therefore , the purposes of this study were to investigate the relationship between image quality in 64 - detector mdct and preimaging anxiety status and hrv and to evaluate the efficacy of an orally administered anxiolytic medication on hrv and image quality . materials and methods patient population a total of 91 patients were referred for mdct - ca due to radiol med ( 2011 ) 116 : 4755 table 1 demographic and clinical data of patients with and without alprazolam medication tabella 1 dati demografici e clinici dei pazienti trattati e non trattati con alprazolam characteristics total p value no . 
of patients age ( years ) male - to - female ratio body mass index ( kg / m2 ) average heart rate ( beats / min ) heart rate variability ( beats / min ) a family history hypertension hyperlipidaemia diabetes mellitus smoking no . 
we also excluded 11 patients who had a history of anxiety reduction medication or coronary artery bypass surgery , although they were examined with mdct . at the time of ct , 23 ( 38.3% ) patients were taking receptor antagonists ( n = 18 ) and calcium - channel blockers ( n = 5 )  . 
there were no statistically significant differences in patients ages , body mass index ( bmi ) , gender , average hr , risk factors or chronic consumption of cardiovascular drugs between the two subgroups . 
 anxiety assessment method all patients anxiety levels were assessed with the statetrait anxiety inventory ( stai ) 60 min before the procedure by an investigator ( pg ) [ 19 ]  . 
the stai is a 40 - item self - reported questionnaire ( 20 state anxiety statements and 20 trait anxiety statements )  . responses to each of the 40 questions are given according to a scoring scale from 1 ( not at all ) to 4 ( very much so )  . each anxiety score can range between 20 and 80 . 
the test has been validated in various patient groups and college students [ 19 ]  . the participating patients were randomly assigned to one of the two study groups : a control group ( no medication administered for anxiety reduction ) and an anxiolytic medication group . 
the scan parameters were 640.5 - mm detector collimation , pitch 0.20.45 ( depending on hr ) , rotation time 400 ms , tube voltage 120 kv , current 600900 effective mas . 
electrocardiogram ( ecg ) - dependent tube current modulation was applied in regular hrs < 65 bpm . scan direction was craniocaudal during a single midinspiratory breath - hold . a bolus of 80 ml iodine contrast agent iobitridol ( xenetix 350 , 350mg / ml ; guerbet , france ) was administered intravenously into an antecubital vein at a flow rate of 56 ml / s using a dual - head power injector , followed by 50 - ml saline chaser ( 5 ml / s )  . 
the scanning range covered the entire heart from the level of the tracheal bifurcation to the diaphraga - blocker was given intravenously before the mdct scan if the hr was > 70 beats per min ( 515 ml metoprolol ) , regardless of grouping . 
none of the patients reported any discomfort due to the ct protocol or the use of contrast media or alprazolam medication . mdct image reconstruction and analysis during the scan , ecg was recorded simultaneously . 
images were reconstructed with a section thickness of 0.5 mm , a reconstruction increment of 0.3mm , image matrix 512512 and fov of 180240 m data were transferred to software vitrea 2 workstation ( vital images inc . , plymouth , mn , usa ) , and images were reconstructed with multiple postprocessing methods . mdct images were evaluated and classified by two radiologists ( rc , nt ) experienced in cardiovascular radiology . when the two radiologists had different opinions , the final decision was obtained by consensus . 
segmental evaluation of the coronary arteries was performed according to the american heart associations ( aha ) 15 - segment classification [ 20 ] : right coronary artery ( rca ) 14 ; left main ( lm ) and left anterior descending artery ( lad ) 510 ; left circumflex artery ( lcx ) 1115 . 
the presence of motion artefacts and image quality for each coronary artery segment were evaluated in a semiqualitative fashion using a four - point grading system , where a score of 1 indicates no motion artefacts , radiol med ( 2011 ) 116 : 4755 clear delineation of the segment ; a score of 2 indicates good , with minor artefacts , mild blurring of the segment ; a score of 3 indicates adequate , with moderate artefacts , moderate blurring without structure discontinuity ; and a score of 4 indicates nonevaluable , with doubling or discontinuity in the course of the segment preventing evaluation or vessel structures not differentiable . from the recorded ecg information , the hr in each cardiac cycle was noted by another author ( sy ) , who did not participate in image assessment . 
from this set of measurements , the variability in hr during scanning was calculated as the sd from the average hr [ 6 ]  . statistical analysis the statistical software ncss 2007 & pass 2008 ( ut , usa ) was used for statistical analyses . 
in analysing the study data , students t test was used to evaluate parameters with a normal distribution between groups for quantitative data , besides descriptive statistical methods ( mean , sd , frequency ) ; and the relationships between parameters were tested by pearsons correlation analysis and linear regression analysis . 
chi - square test and fishers exact test were used to analyse categorical data in the two groups . results were evaluated at 95% confidence interval ( ci ) at the significance level p < 0.05. results the average hr was 60.79.6 ( range 4494 beats per min )  . a total of 17 cases ( 28% ) had stable heartbeat , and there was only one to two beats difference during the whole mdct imaging [ 6 ]  . 
heart rate showed mild / moderate variability within a range below and above baseline values in the other cases ( 65% )  . image quality of the coronary arteries a total of 881 coronary artery segments were evaluated . nineteen segments could not be evaluated due to a very low calibre ( < 1.5 mm ) or anatomical variations . 
the best reconstruction interval was used to evaluate image quality . percentages for the coronary segment scores observed were 65% ( 575 / 881 ) for score 1 , 26% ( 226 / 881 ) for score 2 and 8% ( 69 / 881 ) for score 3 . 
1a - d curved multiplanar reformations of 64 - slice multidetector computed tomography ( mdct ) images of right coronary artery ( rca ) in patients with four different semiquantitative four - point image - quality scores . 
1 a - d immagini di ricostruzione mpr - curved , ottenute con tc multidetettore a 64 strati , dellarteria coronaria destra ( rca ) in pazienti con quattro differenti punteggi semiquantitativi di qualit dellimmagine , calcolati in base a quattro punti . 
although there was a weak negative association between hrv and state anxiety scores ( r = - 0.104 , p > 0.05 ) and trait anxiety scores ( r = - 0.315 , p > 0.05 ) in patients who received alprazolam , it was not statistically significant . 
on the other hand , there was a statistically significant positive relationship between hrv and state anxiety scores ( r = 0.636 , p < 0.01 ) and trait anxiety scores ( r = 0.610 , p < 0.01 ) in patients who did not use alprazolam . discussion in bearing with previous studies [ 4 , 6 , 7 ] , a correlation was found between hrv and mean image quality of all coronary segments . 
hrv was lower and image quality higher in patients who received alprazolam when compared with the control group . it has been reported that hrv occurring during mdct imaging influences image quality [ 4 , 6 , 7 ]  . 
in their study using 64 - detectorrow mdct , they reported that hrv is associated with mean image quality of all coronary segments , and in particular , rca is more heavily affected by hrv [ 6 ]  . 
dualsource mdct - ca is reported to be sufficient for diagnosis in high hr and hrv , but image quality deteriorates in the case of both high and variable hrs [ 4 ]  . 
image quality of rca , lad , lm and lcx deteriorated due to hrv in an order from higher to lower . the main limitation to the use of mdct - ca has been temporal resolution and cardiac motion . 
however , there was no statistically significant relationship between trait anxiety scores , which reflect how an individual feels independent of the situation and conditions ( a more general and longstanding condition ) , and hrv . 
it is remarkable that trait anxiety scores and hrv do not change . therefore , we can hypothesise that if we reduce the momentary anxiety in patients prior to mdct imaging , we can decrease the hrv and avoid deterioration in image quality . in our literature search , we could not identify any previous study related to anxiety reduction specifically for mdct - ca . benzodiazepines are the most commonly used anxiolytic drugs in the radiology department and are considered safe , with minimal respiratory and cardiovascular effects [ 12 ]  . alprazolam used in our study is one of the most frequently prescribed benzodiazepines for treating panic disorders [ 24 ]  . benzodiazepines act selectively on central g - aminobutyric acid ( gaba ) - a receptors and enhance response to the gaba inhibitory neurotransmitter . 
benzodiazepines inhibit increased noradrenergic activity in the locus ceruleus as well . according to the american society of anesthesiologists , the definition for minimal sedation ( anxiolysis ) is a drug - induced state during which patients respond normally to verbal commands . 
we think that oral alprazolam used in our study is a feasible anxiolytic premedication agent for use prior to mdct , with its short half - life , rapid effect , inexpensiveness and ease of use when compared with the other benzodiazepines . we achieved anxiolytic effect with oral alprazolam in our study without affecting respiratory and cardiovascular functions . 
nevertheless , it is known that in the presence of other central nervous system depressants , particularly alcohol , they may cause severe respiratory depression , especially in the elderly , children and critically ill patients , as they may result in apnoea [ 27 ]  . 
this kind of effect is out of scope of radiol med ( 2011 ) 116 : 4755 this particular study , as we excluded such patients initially . on the other hand , hrv decreased and image quality increased significantly in patients receiving alprazolam when compared with the control group . 
additionally , whereas hrv increased in parallel with the increase in state anxiety scores in patients not taking alprazolam , there was no such relationship in patients who received alprazolam . we drew the conclusion that alprazolam , used as an anxiolytic , breaks the vicious cycle comprising increased anxiety , increased noradrenergic activity , increased hrv and low image quality . the first limitation in our study was that we could not examine the individual effects of the - blocker and alprazolam on image quality . 
the reasons for this were the relatively small number of patients included in the study , preexisting usage of a - blocker by some patients or our use of a - blocker during mdct to reduce high hr . 
the second limitation was lack of patient demographic data other than age and gender , such as education , marital status and ethnicity , which may be related with stai evaluation . 
however , as our first aim was to lower the hrv and increase image quality rather than evaluate the effect of alprazolam on mood , we neither used combined placebo administration nor collected the postprocedure state anxiety scores of our patients . 
cademartiri1 , 2 1department of radiology and cardiology , azienda ospedaliero - universitaria , parma , italy 2department of radiology and cardiology , erasmus medical center , rotterdam , the netherlands 3healthcare and social agency , regione emilia romagna , bologna , italy 4sdn foundation nuclear diagnostic institute , naples , italy 5department of radiology , university of verona , italy correspondence to : f . 
cademartiri , department of radiology , c / o piastra tecnica piano 0 ct section , azienda ospedalierouniversitaria di parma , via gramsci 14 , 43100 parma , italy , tel . : + 39 - 052 - 1703516 , fax : + 39 - 052 - 1704838 , e - mail : filippocademartiri@hotmail.com received : 31 december 2009 / accepted : 22 february 2010 / published online : 6 october 2010 springer - verlag 2010 abstract purpose . 
the authors investigated the prognostic value of computed tomography coronary angiography ( ctca ) for major adverse cardiac events ( mace ) in patients with suspected or known coronary artery disease ( cad ) , with particular focus on left main ( lm ) disease and obstructive vs . 
a total of 117 mace [ five cardiac deaths , 11 acute myocardial infarctions ( ami ) , five unstable angina , 86 percutaneous coronary interventions , ten coronary artery bypass grafts ] occurred during a mean follow - up of 20 months . 
settecentoventisette pazienti consecutivi ( 485 maschi , et 6211anni ) con cad sospetta ( 514 ; 70 , 1% ) o nota ( 213 ; 29 , 9% ) sono stati sottoposti a ctca . 
sono stati osservati un totale di 117 mace ( 5 morti cardiache , 11 ami , 5 angine instabili , 96 rivascolarizzazioni ) durante un follow - up medio di 20 mesi . 
la valutazione del carico aterosclerotico alla ctca ha un valore prognostico indipendente per i mace . i pazienti con coronarie normali mostrano prognosi eccellente . keywords computed tomography coronary angiography prognosis coronary artery disease left main disease major adverse cardiac events parole chiave angiografia tc delle coronarie prognosi malattia coronarica tronco comune eventi avversi cardiaci maggiori introduction introduzione coronary artery disease ( cad ) is the leading cause of death in the developed world . 
noninvasive imaging is widely used to establish the diagnosis in symptomatic patients with suspected cad and to evaluate cad progression in patients with known cad in order to further stratify the risk of cardiac events beyond clinical data and traditional cardiovascular risk factors . 
noninvasive evaluation is primarily performed using stress nuclear myocardial perfusion imaging and stress echocardiography , given their widely demonstrated diagnostic accuracy and prognostic value tomography coronary angiography [ 13 ]  . 
computed ( ctca ) has demonstrated high diagnostic accuracy for detecting obstructive cad compared with standard coronary angiography in selected populations , and it is increasingly used in the clinical setting as a gatekeeper to coronary angiography [ 47 ]  . 
 the aim of this study was to evaluate the prognostic value of ctca for major adverse cardiac events ( mace ) in patients with suspected or known cad , with particular focus on lm disease and obstructive vs . 
la valutazione di cad mediante test di imaging non invasivo largamente utilizzata nella diagnosi dei pazienti sintomatici con sospetta cad e nella valutazione della progressione di malattia nei pazienti con cad nota , al fine di ottenere una stratificazione del rischio di eventi cardiaci che vada oltre i dati clinici e i tradizionali fattori di rischio cardiovascolare . 
la valutazione viene eseguita , in prima istanza , mediante indagine non invasiva con test - stress nucleare di perfusione miocardica e stress ecocardiografico , per lelevata accuratezza diagnostica e valore prognostico di tali metodiche [ 13 ]  . 
langiografia tramite tomografia computerizzata ( tc ) delle coronarie ( ctca ) ha mostrato unelevata accuratezza diagnostica nella valutazione di cad ostruttiva quando confrontata allangiografia coronarica convenzionale , in una selezionata popolazione di pazienti , ed sempre pi utilizzata nel percorso clinico come apri porte allangiografia coronarica [ 47 ]  . 
 lo scopo di questo studio di valutare il valore prognostico della ctca per eventi avversi cardiovascolari maggiori ( mace ) in pazienti con cad sospetta o nota con particolare attenzione alla malattia del tronco comune ed al confronto tra malattia ostruttiva e non ostruttiva . 
exclusion criteria were renal failure ( creatinine clearance < 60 ml / min ) , known previous reaction to iodinated contrast materiali e metodi disegno dello studio e popolazione di pazienti questo uno studio retrospettivo osservazionale eseguito su 986 pazienti con cad sospetta o nota inviati al nostro istituto per una valutazione con ctca tra gennaio 2005 e aprile 2008 . 
the following risk factors were considered [ 8 ] : hypertension ( defined as arterial pressure 140 / 80 mmhg or need for antihypertensive therapy ) hypercholesterolaemia [ low - density lipoprotein ( ldl ) cholesterol > 130 mg / dl or current treatment with lipidlowering therapy ] diabetes mellitus ( current need for antidiabetic or insulin therapy ) smoking habit obesity [ body mass index ( bmi ) > 30 ] family history of cardiac disease . ( dolore toracico , dispnea sotto sforzo ) , test - stress dubbio o anormale ed elevato profilo di rischio cardiovascolare . invece , i criteri di esclusione sono stati insufficienza renale ( clearance della creatinina < 60 ml / min ) , allergia nota allagente di contrasto iodato e gravidanza . 
the clinical end points were : cardiac death nonfatal acute myocardial infarction ( ami ) unstable angina requiring hospitalisation percutaneous coronary revascularisation ( pci ) coronary artery bypass graft ( cabg )  . 
the end point was the occurrence of one mace at follow - up . ctca all examinations were performed with a single - source 64slice ct system ( sensation 64 cardiac , siemens , germany )  . 
ctca was performed after administration of 100 ml of nonionic contrast material ( iomeprol 400 mgi / ml , iomeron 400 , bracco , milan , italy ) at a flow rate of 45 ml / s depending on patient status . 
all injections were performed by dual - head power injector ( stellant , medrad , usa ) via an antecubital vein and were followed by 50 ml of saline bolus chaser at the same flow rate . 
ctca was performed with ( 322 ) 0.6 - mm collimation , 330 - ms gantry rotation time , i sintomi sono stati classificati come dolore toracico tipico , dolore toracico atipico , dispnea da sforzo . 
le i dati del follow - up sono stati raccolti a seguito di visita ambulatoriale o schede clinico / ospedaliere dei pazienti sono state sottoposte a screening per confermare gli eventi clinici di cui si avuto notizia . 
sono stati considerati i seguenti end - point : morte cardiaca ; infarto miocardico acuto ( ami ) non fatale ; ricovero per angina instabile ; intervento di rivascolarizzazione coronarico percutaneo ( pci ) ; bypass aorto - coronarico ( cabg )  . 
intravenous beta - blocker ( atenolol 510 mg ) was administered to all patients with a heart rate > 65 beats / min and without contraindication ( known asthma or bronchospasm , systolic blood pressure < 100 mmhg )  . 
to obtain optimal image quality , data sets were reconstructed at least at two points of the cardiac cycle using a retrospective ecg gating algorithm ( one diastolic cardiac phase usually at 350 ms from the r waves and one end - systolic phase at + 275 ms )  . 
coronary plaques were defined , as described previously [ 11 ] , as structures of > 1 mm2 within and / or adjacent to the coronary artery lumen , which are clearly distinguishable from the vessel lumen and surrounding pericardial tissue . 
patients were classified as belonging to one of three groups based on ctca findings : ( 1 ) patients with normal coronary arteries , ( 2 ) patients with nonobstructive coronary artery disease and ( 3 ) patients with obstructive coronary artery disease ( presence of at least one plaque > 50% )  . 
la ctca stata eseguita con i seguenti parametri di scansione : collimazione dello strato ( 322 ) 0 , 6 mm , tempo di rotazione del gantry 330 ms , 700900 mas ad una tensione del tubo di 120 kv , pitch 0 , 24 , risoluzione temporale di 165 ms , e risoluzione spaziale isotropica di 0 , 4 mm3 . 
i data - set sono stati ricostruiti con uno spessore di strato di 0 , 75 mm ed un incremento di 0 , 5 m tutti i pazienti con frequenza cardiaca > 65 battiti per minuto ( bpm ) e senza controindicazioni ( asma o broncospasmo noti , pressione sistolica < 100 mmhg ) hanno ricevuto uniniezione intra - venosa di beta - bloccante ( atenololo 510 mg )  . 
inoltre , stata somministrata una dose sublinguale di 0 , 3 mg nitroglicerina a tutti i pazienti senza controindicazioni ( stenosi aortica severa , pressione sistolica < 100 mmhg )  . 
per ottenere una qualit dimmagine ottimale , i data - set sono stati ricostruiti mediante gating elettrocardiografico ( ecg ) retrospettico in due fasi del ciclo cardiaco ( fase telediastolica , 350 ms prima dellonda r successiva , fase tele - sistolica , + 275 ms dopo londa r successiva )  . 
le ricostruzioni in asse corto sono state trasferite su di una workstation dedicata per le successive valutazioni ed il post - processing ( leonardo , siemens medical solutions , forchheim , germania )  . 
le placche coronariche sono state radiol med ( 2011 ) 116 : 1531 statistical analysis categorical baseline characteristics , expressed as numbers and percentages , were compared using the chi - square test . continuous variables , expressed as mean and standard deviation ( sd ) , were compared using the two - tailed t test and analysis of variance if normally distributed or by means of the kruskalwallis method if not normally distributed . 
 results patient population of 986 consecutive patients , 259 ( 26% ) were lost to followup ; therefore , 727 patients ( 485 men ; mean age 6211 years ) were available for the study . 
the majority of patients ( 514 , 70.1% ) were referred for suspected cad , whereas 213 patients ( 29.9% ) had a history of previous mi and / or previous coronary revascularisation by means of cabg or pci . 
patients with no history of cad and nonobstructive disease ( n = 237 ) were subclassified as follows : normal coronary arteries ( subgroup 1 ) , nonobstructive cad ( luminal stenosis < 50% ) in lm ( subgroup 2 ) , nonobstructive cad in other vessels ( subgroup 3 ) and nonobstructive cad in lm and other vessels ( subgroup 4 )  . 
patients with no history of cad and obstructive disease ( n = 103 ) were considered a single cohort . patients with a history of cad and nonobstructive disease ( n = 36 ) were subclassified as follows : normal coronary arteries ( subgroup 1 ) , nonobstructive cad in other vessels ( subgroup 2 ) and nonobstructive cad in lm and other vessels ( subgroup 3 )  . 
patients with a history of cad and obstructive disease ( n = 159 ) were considered a single cohort . definite , come precedentemente descritto [ 11 ] , come strutture > 1 mm2 situate allinterno / o adiacenti al lume del vaso , e chiaramente distinguibili da esso e dal tessuto pericardico circondate . 
le placche ostruttive sono state definite come placche determinanti un restringimento > 50% del lume del vaso ; mentre , le placche non ostruttive sono state definite come placche determinanti un restringimento 50% . 
sono stati messi a confronto i valori e le differenze mediante test t di student per dati appaiati , e mediante analisi della varianza se normalmente distribuiti oppure con il metodo kruskal - wallis . 
la sotto - analisi per la stratificazione pre - test del rischio cardiovascolare stata calcolata con scala morise ( rischio basso , intermedio , e alto )  . risultati popolazione di pazienti nella popolazione di 986 consecutivi pazienti eleggibili per lo studio , 259 ( 26% ) sono stati esclusi al follow - up , radiol med ( 2011 ) 116 : 1531 fig . 
patients with no history of cad and nonobstructive disease : normal coronary arteries ( subgroup 1 ) , nonobstructive cad ( luminal stenosis < 50% ) in lm ( subgroup 2 ) , nonobstructive cad in other vessels ( subgroup 3 ) , nonobstructive cad in lm and other vessels ( subgroup 4 )  . 
patients with history of cad and nonobstructive disease : normal coronary arteries ( subgroup 1 ) , nonobstructive cad in other vessels ( subgroup 2 ) , nonobstructive cad in lm and other vessels ( subgroup 3 )  . 
nei pazienti senza storia di malattia delle coronarie ( cad ) e con cad non ostruttiva : coronarie normali ( sotto - gruppo i ) , cad non - ostruttiva ( stenosi luminare < 50% ) nel tronco comune ( lm ) ( sotto - gruppo ii ) , cad non - ostruttiva negli altri vasi ( sotto - gruppo iii ) e cad non - ostruttiva nel lm e negli altri vasi ( sotto - gruppo iv )  . 
nei pazienti con storia di pregressa cad e cad non ostruttiva : coronarie normali ( sotto - gruppo i ) , cad non - ostruttiva negli altri vasi ( sotto - gruppo ii ) e cad non - ostruttiva nel lm e negli altri vasi ( sotto - gruppo iii )  . 
mace , eventi coronarici avversi maggiori ; 1vd , malattia mono - vasale ; mvd , malattia multi - vasale . the detailed baseline characteristics of the population are given in fig . 
la maggioranza dei pazienti ( 514 pazienti , 70 , 1% ) stata inviata per sospetta cad , mentre 213 pazienti ( 29 , 9% ) hanno avuto un pregresso infarto miocardico e / o un pregresso intervento di rivascolarizzazione coronarica mediante bypass aortocoronarico o angioplastica coronarica percutanea . 
in prima istanza , i pazienti sono stati classificati sulla base della storia di pregressa cad ( assente / presente ) e poi ( non sulla base della ostruttiva / ostruttiva )  . 
i pazienti senza storia di pregressa cad e malattia non ostruttiva sono stati sotto - classificati come segue ( 237 ) : coronarie normali ( sotto - gruppo i ) , cad non - ostruttiva ( stenosi luminare < 50% ) nel lm ( sotto - gruppo ii ) , cad non - ostruttiva negli altri vasi ( sotto - gruppo iii ) e cad non - ostruttiva nel lm e negli altri vasi ( sotto - gruppo iv )  . 
i pazienti senza storia di severit della malattia radiol med ( 2011 ) 116 : 1531 table 1 baseline characteristics of patients with no history of cad and nonobstructive cad subgroup 1 ( n = 204 ) subgroup 2 ( n = 10 ) subgroup 3 ( n = 142 ) subgroup 4 ( n = 55 ) data are presented as mean ( standard deviation ) or number ( percentage )  . 
subgroups : normal coronary arteries ( subgroup 1 ) , nonobstructive cad ( luminal stenosis < 50% ) in lm ( subgroup 2 ) , nonobstructive cad in other vessels ( subgroup 3 ) , nonobstructive cad in lm and other vessels ( subgroup 4 )  . 
 cad , coronary artery disease ; sd , standard deviation ; lm , left main ; bmi , body mass index ; bpm , beats per minute tabella 1 caratteristiche di base dei pazienti senza storia di pregressa malattia delle coronarie ( cad ) e con cad non ostruttiva sotto - gruppo i ( n = 204 ) sotto - gruppo ii ( n = 10 ) sotto - gruppo iii ( n = 142 ) sotto - gruppo iv ( n = 55 ) clinical characteristics age [ years ; mean ( sd ) ] male gender ( % ) bmi [ kg / m2 ; mean ( sd ) ] mean heart rate [ bpm ; mean ( sd ) ] ( sd ) ] follow - up [ months ; mean ( sd ) ] risk factors no . 
ds , deviazione standard ; lm , tronco comune ; bmi , indice di massa corporea ; bpm , battiti per minuto follow - up results the mean follow - up was 204 ( range 527 ) months . 
during the follow - up period , 117 mace ( five cardiac deaths , 11 ami , five unstable angina , 86 pci , ten cabg ) occurred pregressa cad e malattia ostruttiva ( 103 ) sono stati considerati come una unica coorte . 
i pazienti con storia di pregressa cad e malattia non ostruttiva sono stati classificati come segue ( 36 ) : coronarie normali ( sotto - gruppo i ) , cad non - ostruttiva negli altri vasi ( sotto - gruppo ii ) e radiol med ( 2011 ) 116 : 1531 table 2 baseline characteristics of patients with no history of coronary artery disease ( cad ) and obstructive cad data are presented as mean ( sd ) or number ( percentage ) sd , standard deviation ; bmi , body mass index ; bpm , beats per minute tabella 2 caratteristiche di base dei pazienti senza storia di pregressa malattia delle coronarie ( cad ) e con cad ostruttiva . 
nine events occurred in patients with no history of cad and nonobstructive disease ( two cardiac deaths in subgroup 3 and seven pci in subgroups 3 and 4 )  . 
 radiol med ( 2011 ) 116 : 1531 table 3 baseline characteristics of patients with history of coronary artery disease ( cad ) and nonobstructive cad subgroup 1 ( n = 10 ) subgroup 2 ( n = 33 ) subgroup 3 ( n = 11 ) data are presented as mean ( sd ) or number ( percentage )  . 
subgroups : normal coronary arteries ( subgroup 1 ) , nonobstructive cad in other vessels ( subgroup 2 ) , nonobstructive cad in lm and other vessels ( subgroup 3 )  . data are presented as mean ( standard deviation ) or number ( percentage )  . 
 sd , standard deviation ; lm , left main ; bmi , body mass index ; bpm , beats per minute tabella 3 caratteristiche di base dei pazienti con storia di pregressa malattia delle coronarie ( cad ) e cad non ostruttiva sotto - gruppo i ( n = 10 ) sotto - gruppo ii ( n = 33 ) sotto - gruppo iii ( n = 11 ) clinical characteristics age [ years ; mean ( sd ) ] male gender ( % ) bmi [ kg / m2 ; mean ( sd ) ] mean heart rate [ bpm ; mean ( sd ) ] follow - up [ months ; mean ( sd ) ] risk factors no . 
i dati sono riportati come media ( deviazione standard ) o numero ( percentuale )  . ds , deviazione standard ; bmi , indice di massa corporea ; bpm , battiti per minuto radiol med ( 2011 ) 116 : 1531 table 4 baseline characteristics of patients with history of coronary artery disease ( cad ) and obstructive cad patients ( n = 159 ) sd , standard deviation ; bmi , body mass index ; bpm , beats per minute tabella 4 caratteristiche di base dei pazienti con storia di pregressa malattia delle coronarie ( cad ) e cad ostruttiva pazienti ( n = 159 ) clinical characteristics age [ years ; mean ( sd ) ] male gender ( % ) bmi [ kg / m2 ; mean ( sd ) ] mean heart rate [ bpm ; mean ( sd ) ] follow - up [ months ; mean ( sd ) ] risk factors no . 
in patients with a history of cad and obstructive disease , 52 events occurred ( four unstable angina ; four ami ; two cardiac deaths ; 38 pci ; four cabg )  . risultati dellangiografia a tomografia computerizzata delle coronarie la valutazione del carico aterosclerotico stata valutata in un numero totale di 10178 segmenti coronarici . 
tra questi , 158 segmenti ( 1 , 5% ) sono stati esclusi per la presenza di artefatti da movimento cardiaco o di cal cio intra - coronarico o per ridotto lume del vaso . 
durante radiol med ( 2011 ) 116 : 1531 table 5 major adverse cardiac events ( mace ) recorded during the follow - up no history of cad nonobstructive obstructive obstructive history of cad nonobstructive hard events unstable angina death subtotal revascularisations cabg subtotal total eventi maggiori angina instabile morte sub - totale ri - vascolarizzazioni cabg sub - totale totale patients with no history of cad and nonobstructive disease : normal coronary arteries ( subgroup 1 ) , nonobstructive cad ( luminal stenosis < 50% ) in lm ( subgroup 2 ) , nonobstructive cad in other vessels ( subgroup 3 ) , nonobstructive cad in lm and other vessels ( subgroup 4 ) patients with history of cad and nonobstructive disease : normal coronary arteries ( subgroup 1 ) , nonobstructive cad in other vessels ( subgroup 2 ) , nonobstructive cad in lm and other vessels ( subgroup 3 ) cad , coronary artery disease ; ami , acute myocardial infarction ; pci , percutaneous coronary revascularisation ; cabg , coronary artery bypass graft tabella 5 eventi . 
la tabella mostra tutti gli eventi avversi cardiovascolari maggiori ( mace ) raccolti durante il follow - up senza storia di cad non ostruttiva ostruttiva ostruttiva storia di cad non ostruttiva nei pazienti senza storia di pregressa malattia delle coronarie ( cad ) e con cad non ostruttiva : coronarie normali ( sotto - gruppo i ) , cad non - ostruttiva ( stenosi luminare < 50% ) nel tronco comune ( lm ) ( sotto - gruppo ii ) , cad non - ostruttiva negli altri vasi ( sotto - gruppo iii ) e cad non - ostruttiva cad nel lm e negli altri vasi ( sotto - gruppo iv )  . 
among patients with no history of cad and obstructive disease , the prognosis was worse for patients with multivessel disease compared with la ctca la frequenza cardiaca media stata di 6210 bp la ctca ha mostrato assenza di aterosclerosi coronarica in 214 ( 29 , 5% ) pazienti , malattia coronarica non ostruttiva in 251 ( 34 , 5% ) e malattia ostruttiva in uno o pi vasi in 262 ( 36 , 0% )  . 
inoltre , la ctca ha rivelato la presenza di malattia mono - vasale in 116 ( 16 , 0% ) pazienti , e malattia multi - vasale in 146 ( 20 , 1% ) pazienti . 
nei pazienti senza storia di pregressa cad o malattia non ostruttiva sono stati osservati 9 eventi ( 2 morti cardiache nel sotto - gruppo iii e 7 pci nel sotto - gruppo iii e iv )  . 
nei pazienti senza storia di pregressa cad e malattia ostruttiva sono stati osservati 51 eventi ( 1 angina instabile , 5 ami , 1 morte cardiaca , 39 pci , 5 cabg )  . 
nei pazienti con pregressa cad e malattia non ostruttiva sono stati osservati 5 venti ( 2 ami nel sottogruppo ii , 3 pci nel sottogruppo ii e iii )  . 
mentre , nei pazienti con pregressa cad e malattia ostruttiva sono stati osservati 52 eventi ( 4 angine instabili , 4 ami , 2 morti cardiache , 38 pci , 4 cabg )  . 
normal coronary arteries ( subgroup 1 ) , nonobstructive cad ( luminal stenosis < 50% ) in lm ( subgroup 2 ) , nonobstructive cad in other vessels ( subgroup 3 ) , nonobstructive cad in lm and other vessels ( subgroup 4 )  . 
coronarie normali ( sotto - gruppo i ) , cad non - ostruttiva ( stenosi luminare < 50% ) nel tronco comune ( lm ) ( sotto - gruppo ii ) , cad non - ostruttiva negli altri vasi ( sotto - gruppo iii ) e cad non - ostruttiva nel lm e negli altri vasi ( sotto - gruppo iv )  . 
 indipendenti per i mace ( cad ostruttiva hr : 9 , 87 , 95% intervallo di confidenza [ ci ] 3 , 2929 , 57 , p < 0 , 001 ; diabete hr : 3 , 45 95%ci 1 , 438 , 46 , p = 0 , 006 )  . discussione il seguente studio ha dimostrato la capacit della ctca nella stratificazione di eventi di rischio cardiovascolare gi dopo la seconda fase del follow - up . 
bench sia semplice estrapolare dai dati il valore predittivo di et , malattia ostruttiva , storia di pregressa cad , malattia ostruttiva del lm / lad per la definizione della prognosi , il ruolo della malattia del lm in questo senso non fig . 
normal coronary arteries ( subgroup 1 ) , nonobstructive cad in other vessels ( subgroup 2 ) , nonobstructive cad in lm and other vessels ( subgroup 3 )  . 
coronarie normali ( sotto - gruppo i ) , cad non - ostruttiva negli altri vasi ( sotto - gruppo ii ) e cad non - ostruttiva nel lm e negli altri vasi ( sotto - gruppo iii )  . 
the capability to stratify nonobstructive disease may render the identification of lm disease ( both obstructive and nonobstructive ) a more difficult task to perforwhereas it is very easy to extract the role of age from this data , obstructive disease , history of cad , lm / lad obstructive disease and diabetes in determining prognosis , the role of lm disease remains uncertain fact , multivariate analysis showed that the only independent predictors of mace were the presence of obstructive coronary disease and diabetes . 
in particular , patients with significant lm stenosis , three - vessel disease or two - vessel disease with an lad artery stenosis > 90% are at higher risk of events [ 1214 ]  . 
in particolare , i pazienti con stenosi significativa del tronco comune , con malattia trio bi - vasale e coronaria discendente anteriore sinistra con stenosi > 90% sono a pi elevato rischio di eventi [ 1214 ]  . 
in particolare , i nostri dati convalidano il ruolo della ctca come metodica non invasiva per la valutazione dellaterosclerosi coronarica e dimostrano il suo elevato valore prognostico indipendente nella valutazione del carico aterosclerotico . il diabete mellito associato ad un elevato rischio di morbilit e mortalit cardiovascolare ed universalmente riconosciuto come un fattore equivalente alla cad . 
nonostante i pazienti con pregresso infarto miocardico siano considerati ad elevato rischio di ricaduta in eventi cardiaci , probabile che il carico aterosclerotico , radiol med ( 2011 ) 116 : 1531 ings . 
specifically , our study confirms that ctca permits the noninvasive evaluation of coronary atherosclerosis and shows that the atherosclerotic burden detected on ctca is a significant and independent prognostic factor . diabetes mellitus is associated with an elevated risk of cardiovascular morbidity and mortality and is widely recognised as a cad equivalent . 
patients with previous mi are considered at high risk for the recurrence of cardiac events ; however , it is likely that atherosclerotic burden , which is usually high in these patients , may be the most important prognostic factor . 
at 1 year , patients with cad had a 34% event rate , whereas patients with normal coronary arteries had a cardiac event rate of 0% [ 22 ]  . 
 it has been suggested that ctca may be used to rule out the presence of significant coronary disease in patients with suspected cad and intermediate pretest risk [ 47 ]  . 
in fact , a number of studies have demonstrated the very high negative predictive value of ctca ( values ranging from 92% to 99% ) for obstructive cad compared with coronary angiography [ 47 ]  . 
in our study , patients without coronary atherosclerosis had a favourable prognosis , with a 0% cardiac event rate during follow - up , thus confirming the high negative predictive value of ctca . study limitation in our study , the presence of lm or proximal lad disease was not an independent predictor at multivariate analysis . this difference may be explained by the limited number of patients with lm / lad disease and by the different stenosis cutoff used by angiographic scores , which is solitamente elevato in tali pazienti , possa essere il fattore prognostico pi importante . 
in questo studio , la presenza e lestensione di cad significativa , classificati mediante la scala modificata di duke per le coronarie , sono stati considerati predittori di tutte le cause di mortalit [ 19 ]  . 
 [ 23 ] hanno valutato 517 pazienti con sospetta cad sottoposti a mpi e ctca , ed hanno riportato una frequenza di eventi annuali di 4 , 8% nei pazienti con cad significativa alla ctca e 1 , 8% nei pazienti con assenza o non significativa cad [ 23 ]  . 
ad un anno di distanza , i pazienti con cad hanno avuto una frequenza di eventi del 34% , mentre i pazienti con coronarie normali dello 0% [ 22 ]  . 
 la ctca stata proposta come modalit dindagine per escludere la presenza di malattia coronarica significativa nei pazienti con sospetta cad e rischio pre - test intermedio [ 47 ]  . 
infatti , numerosi studi hanno dimostrato che la ctca ha un valore predittivo negativo pi elevato ( range 92%99% ) nella cad ostruttiva rispetto allangiografia coronarica convenzionale [ 47 ]  . 
 gli studi prognostici sono importanti per determinare in quali pazienti la ctca possa escludere con certezza la presenza di cad ostruttiva ed evitare cos un sicuro intervento di angiografia coronarica convenzionale . 
in questo studio , i pazienti senza aterosclerosi coronarica hanno una prognosi favorevole , con una frequenza di eventi cardiaci al follow - up dello 0% , confermando lelevato valore predittivo negative della ctca . 
questa differenza potrebbe esser dovuta al limitato numero di pazienti con malattia del lm / lad e dal differente cut - off utilizzato in angiografia per la valutazione della stenosi , solitamente superiore al cut - off del 50% utilizzato alla ctca . 
ci legato alle caratteristiche di base della popolazione , comprendente una coorte di pazienti ambulatoriali con rischio cardiovascolare principalmente intermedio , e alla durata relativamente breve del follow - up . 
 lelevata esposizione alle radiazioni ionizzanti radiol med ( 2011 ) 116 : 1531 usually higher than the 50% ctca cutoff . the main limitation of our study is that the number of clinical events at follow - up was relatively low . 
recently , dose modulation algorithms that reduce the tube current by 80% during the systolic phase and prospective ecg - gating protocols have been developed that allow a significant reduction of radiation exposure down to 34 msv [ 25 , 26 ] , with preserved diagnostic accuracy [ 27 ]  . 
more recently , with the introduction of newer hardware and software , a potential for ctca with < 1 msv has been shown [ 2830 ]  . erogata durante lesecuzione della ctca eseguita mediante acquisizione gating ecg retrospettivo una questione da risolvere . 
recentemente , sono stati sviluppati degli algoritmi di modulazione della dose in grado di ridurre all80% lerogazione raggi durante la fase sistolica e protocolli gating ecg prospettici che permettono una significativa riduzione di esposizione al di sotto dei 34 msv [ 25 , 26 ] con conservazione dellaccuratezza diagnostica [ 27 ]  . 
pi recentemente , con lintroduzione dei pi innovativi hardware e software ha permesso di ottenere con la ctca una potenziale riduzione della dose < 1 msv [ 2830 ]  . conclusioni conclusions our study shows that ctca is able to reliably identify coronary atherosclerotic burden and to stratify cardiovascular risk at follow - up . 
obstructive and nonobstructive lm disease will probably require larger populations and longer follow - up studies to show their prognostic impact questo studio dimostra labilita della ctca nellidentificazione del carico aterosclerotico coronarico e nella stratificazione del rischio cardiovascolare al follow - up . 
la valutazione della malattia ostruttiva e non ostruttiva del lm necessita di una pi vasta popolazione di pazienti e di una maggior durata del follow - up per dimostrare il loro valore prognostico . 
a retrospective analysis was undertaken of 45 patients ( 26 male , 19 female ; age range 1468 years , mean age 39 years ) with skull - base lesions . diffusion - weighted mr images were acquired with a bfactor of 500 and 1 , 000 s / mm2 using single - shot echoplanar imaging . 
the mean adc value of malignant tumours was ( 1.0020.21 ) 10 - 3 mm2 / s and that of benign tumours was ( 1.630.29 ) 10 - 3 mm2 / s . 
we conclude that diffusion - weighted mr imaging is a promising , noninvasive approach that can be used to characterise skull - base lesions in that it can help differentiate malignant tumours from benign lesions and evaluate the pathological grading of malignant tumours . 
la rm in diffusione rappresenta un approccio promettente e non invasivo che pu essere utilizzato per caratterizzare le lesioni della base cranica e pu essere di aiuto per differenziare i tumori maligni dai benigni e valutare il grading delle lesioni tumorali maligne . keywords diffusion mr imaging skull base tumour parole chiave diffusione risonanza magnetica base cranica tumori 126 introduction tumours of the skull base pose a difficult diagnostic challenge , as the skull base may be involved by a spectrum of intrinsic lesions , systemic diseases and lesions arising in the extracranial head and neck or in the intracranial compartment . 
differentiation of malignant skull - base tumours from benign lesions and definite pathological diagnosis are the cornerstones of proper management of the patient and in prognosis prediction [ 15 ]  . 
besides , bone marrow of the skull base shows different signal intensity on different mr pulse sequences that may be mistaken as a tumour [ 7 , 8 ]  . 
ct - guided biopsy is commonly used but it is an invasive procedure that requires a skilled operator and may give false results with hazards of nerve injury [ 9 ]  . diffusion - weighted echoplanar mr imaging is a technique for evaluating the rate of microscopic water diffusion in tissues . 
eight patients were excluded due to uncertain pathology in five patients and poor image quality due to motion artefacts in three . thus , 45 patients ( 26 male , 19 female ; age range1468 years , mean age 39 years ) were finally included . 
the study was approved by the review board , and informed consent was waived . mr imaging was performed with a 1.5 - tesla mr unit ( symphony , siemens medical systems , erlangen , germany ) using a head and neck circular polarisation surface coil . 
all patients underwent t1 - weighted imaging ( tr / te , 800 / 15 radiol med ( 2011 ) 116 : 125132 ms ) and t2 - weighted fast spin echo ( fse ) imaging ( tr / te 4 , 500 / 80 ms ) with a section thickness of 5 mm , an interslice gap of 12 mm , a field of view ( fov ) of 2025 cm and an acquisition matrix of 256256 . diffusion - weighted mr images were obtained using a multislice single - shot spin - echo echoplanar sequence . imaging parameters were tr / te 10 , 000 / 108 ms ; fov 2025 cm ; acquisition matrix 256128 ; section thickness 5 mm with a 1to 2 - mm interslice gap . 
diffusion - weighted mr images were acquired with a diffusion - weighting b - factor of 500 and 1 , 000 s / mm2 , and adc maps were generated . data acquisition time was 1 mfinally , contrast - enhanced t1 - weighted images ( tr / te 800 / 15 ms ) were obtained after an intravenous bolus injection of 0.1 mmol / kg of body weight of gadopentetate dimeglumine ( magnevist , bayer schering pharma , berlin , germany )  . 
 a quantitative analysis of the adc map was made by a radiologist with 20 years of experience in head - and - neck imaging ( aa ) who was blinded to the pathology and clinical results . 
a region of interest ( roi ) was drawn using an electronic cursor around the margin of the solid portion of the mass , taking care to avoid the cystic parts as far as possible , as these could give falsely elevated adc values . the final diagnosis was made by histopathological examination after surgery ( n = 19 ) or endonasal endoscopy - guided biopsy ( n = 20 )  . 
diagnoses of angiofibroma and glomus tumours ( n = 6 ) were based on the typical mr appearance and angiography in addition to a retrospective review of the pathology results . 
to compare between two groups , students t test was applied . receiver operating characteristic ( roc ) curves were generated to determine the cutoff value , with high accuracy and sensitivity . 
the p value was considered significant if 0.05 at 95% confidence interval ( ci )  . results malignant tumours ( n = 32 ) were squamous - cell carcinoma ( n = 7 ) , chordoma ( n = 7 ) , metastasis ( n = 6 ) , rhabdomyosarcoma ( n = 4 ) , plasmacytoma ( n = 3 ) , mucoepidermoid carcinoma ( n = 2 ) , osteosarcoma ( n = 2 ) and adenoid cystic carciradiol med ( 2011 ) 116 : 125132 fig . 
a axial contrast t1 - weighted image of the skull base shows an enhancing lesion involving the left petrous apex that extends to abut the left cerebellar hemisphere and associated retained secretions in the left mastoid air cells . 
b apparent diffusion coefficient ( adc ) map shows low signal intensity of the mass ( black arrows ) with low adc value ( 0.8710 - 3 mm2 / s ) with high signal intensity and high adc value of the retained secretions in the left mastoid ( white arrow )  . 
a limmagine assiale t1 - dipendente dopo mezzo di contrasto della base cranica evidenzia una lesione con enhancement che coinvolge lapice della rocca petrosa di sinistra e si estende verso lemisfero cerebellare di sinistra , con associate secrezioni ritenute nelle celle mastoidee di sinistra . 
b la mappa del coefficiente di diffusione apparente ( adc ) evidenzia una bassa intensit del segnale ( frecce nere ) con basso valore di adc ( 0.8710 - 3 mm2 / s ) con elevata intensit del segnale e elevato adc delle secrezioni ritenute nella mastoide di sinistra ( freccia bianca )  . 
b la mappa del coefficiente di diffusione apparente ( adc ) evidenzia una bassa intensit del segnale con basso valore di adc ( 0 , 9710 - 3 mm2 / s )  . noma ( n = 1 )  . 
the lowest adc value of malignant tumours was detected in a patient with osteosarcoma ( 0.65 ) 10 - 3mm2 / s . the highest value was seen in a patient with adenoid cystic carcinoma ( 1.64 ) 10 - 3mm2 / s , which was misdiagnosed as a benign tumour . 
the bone marrow of the skull base displays variable patterns of signal intensity depending on the proportion of fat cells and cellular marrow that may be mistaken as a pseudolesion . 
in 2006 [ 17 ] found a statistically significant difference ( p = 0.05 ) in adc values between benign and malignant lesions involving the paranasal sinuses and skull base . 
 [ 18 ] added that the mean adc value of nasal and paranasal sinus malignant lesions ( 1.100.2510 - 3mm2 / s ) is significantly different ( p = 0.001 ) from that of benign lesions ( 1.780.4110 - 3mm2 / s )  . 
moreover , diffusion - weighted mr imaging has been used in diagnosing abscesses of the skull base [ 21 , 22 ] as well as detecting bone marrow infiltration of the lumbar spine . 
tumour infiltration appeared as areas of low signal intensity on the adc map , with a low adc value [ 23 , 24 ]  . in this study , the mean adc value of malignant skullbase tumours was significantly lower ( p = 0.001 ) than that of benign tumours . 
this is explained by the difference in histopathological features of benign and malignant tumours . malignant tumours show structural changes , such as enlarged nuclei , hyperchromatism and angulation of nuclear contour . 
these histological features accompany the reduction of extracellular matrix and the diffusion space of water protons , which lead to an increase in the impediment to the motion of water molecules and a decrease in adc values [ 1017 ]  . radiol med ( 2011 ) 116 : 125132 fig . 
a mean adc tumours values of malignant ( 1.0020.2110 - 3 mm2 / s ) are significantly lower ( p = 0.001 ) than those of benign lesions ( 1.630.2910 - 3 mm2 / s )  . 
b mean adc values ( 1.080.2110 - 3 mm2 / s ) are significantly different ( p = 0.01 ) than those of sarcoma ( 0.840.1310 - 3 mm2 / s )  . 
 [ 26 ] added that squamous - cell carcinoma has a higher adc value than lymphoma because carcinoma may contain small foci of necrosis on histopathological examination , which is not identifiable on mr images . 
in our study , there was a significant difference in the adc value of carcinoma and sarcoma , a finding that may be attributed to the higher cellularity of sarcoma compared with carcinoma [ 1013 ]  . 
 in our study , highly vascular benign tumours such as glomus tumours and angiofibromas showed higher adc values than did the other benign solid tumours owing to excess extracellular spaces with free diffusion within the vascular lesions [ 28 ]  . 
chondromyxoid fibroma exhibited a low adc value that was mistaken for a malignant tumour . this is attributed to the excess fibrous tissue within the tumour , with subsequent restriction of diffusion [ 29 ]  . 
the high adc value of adenoid cystic carcinoma is attributed to its high mucous content and cystic components [ 30 ]  . 130 radiol med ( 2011 ) 116 : 125132 fig . 
a cutoff apparent diffusion coefficient ( adc ) value used for differentiating malignant tumours from benign lesions is 1.310 - 3mm2 / s with area under curve ( auc ) of 0.923. 
a il valore di cutoff utilizzato per differenziare i tumori maligni dai benigni di 1.3x10 - 3mm2 / s con area sotto la curva ( auc ) di 0 , 923 . 
the main trade - off of the echoplanar pulse sequence is that it is very sensitive to radiol med ( 2011 ) 116 : 125132 magnetic susceptibility effects , resulting in geometric distortion artefacts that tend to be more severe with increasing b - values . 
recently , 3 - tesla diffusionweighted mr imaging has been used to evaluate head - andneck lesions , including those in the skull base [ 31 , 32 ]  . 
second , the smallest detected lesion was 1 cm , and further studies are recommended for evaluating smaller skull - base lesions . third , there were few cases for each type of histopathological diagnosis . conclusions diffusion - weighted mr imaging is a promising new , noninvasive imaging approach to characterising skull - base masses , as it can help differentiate malignant tumours from benign lesions . 
mr images were assessed for the following features : skin thickening ( > 4 mm ) , skin oedema , architectural distortion , enhancement pattern ( mass - like / non - mass - like ) , time - signal intensity curve ( continuouspersistent type / wash - out type ) , skin enhancement . 
gli aspetti rm analizzati sono stati : ispessimento della cute ( > 4 mm ) , edema cutaneo , distorsione architetturale , pattern di impregnazione ( tipo massa / tipo non - massa ) , curva dellintensit di segnale nel tempo ( tipo continuativo / persistente / tipo wash - out ) , impregnazione della cute . 
comparando i rilievi rm nel carcinoma infiammatorio rispetto al tumore localmente avanzato sono state osservate significative differenze per quanto riguarda le alterazioni cutanee pi frequenti nel carcinoma infiammatorio e il pattern di impregnazione . 
in particolare , ispessimento cutaneo si rilevato in 16 / 30 ( 53% ) ibc vs 8 / 30 ( 27% ) labc ( p = 0 , 06 ) , edema cutaneo era presente in 26 / 30 ( 87% ) ibc vs 8 / 30 ( 27% ) labc radiol med ( 2011 ) 116 : 7183 suggestive of ibc and should prompt a skin biopsy to confirm or rule out the diagnosis . 
le alterazioni della cute ( ispessimento , edema , impregnazione ) sostenute dallinfiltrazione neoplastica dei linfatici del derma sono segni suggestivi di carcinoma infiammatorio e devono guidare al controllo bioptico della cute per confermare o escluderne la diagnosi . parole chiave tumore mammario imaging tumore mammario localmente avanzato carcinoma infiammatorio della mammella rm introduction introduzione inflammatory breast cancer ( ibc ) and locally advanced breast cancer ( labc ) were once considered inoperable at presentation and / or with poor survival rate if treated with locoregional therapies alone . 
despite being often grouped together , in particular in clinical trials , ibc and labc have different epidemiological features and , more importantly , different clinical and biological presentations [ 1 ]  . 
ibc has a higher incidence of locoregional recurrence and metastasis to soft tissue and bone , has worse outcomes and lower survival rates compared with noninflammatory labc [ 24 ] , and its treatment requires a multidisciplinary radiological , chemotherapeutical and surgical approach . 
ibc and labc are treated in the same manner only in the initial stage , where treatment consists of systemic neoadjuvant therapy to improve surgical options and prolong survival [ 5 ]  . 
subsequently , selected patients with labc , but not those with ibc , can undergo conservative treatment provided it is started early on , at the time of treatment planning [ 1 ]  . 
in routine practice , however , the final diagnosis is only established after surgical biopsy , since the clinical presentation of ibc and labc is similar and preoperative pathology may provide inadequate samples for the correct diagnosis , even when large - core needles are used . identifying ibc and differentiating it from labc remains a challenge in breast imaging . 
increasingly sensitive and accurate diagnostic techniques are needed to identify patients il carcinoma infiammatorio ( inflammatory breast cancer , ibc ) della mammella e il carcinoma mammario localmente avanzato ( locally advanced breast cancer , labc ) sono stati in passato considerati come tumori della mammella inoperabili allesordio e / o con ridotta sopravvivenza se trattati con le sole terapie loco - regionali . 
anche se spesso vengono raggruppati assieme , soprattutto nelle sperimentazioni cliniche , ibc e labc hanno diverse presentazioni epidemiologiche e , fatto pi importante , diversi aspetti clinico - biologici [ 1 ]  . 
il carcinoma infiammatorio ha una pi alta incidenza di recidiva loco - regionale e di metastasi a distanza nei tessuti molli e nelle ossa , risultando in una prognosi peggiore e in un minore tasso di sopravvivenza rispetto al carcinoma mammario localmente avanzato noninfiammatorio [ 24 ] ; il suo trattamento richiede un approccio multidisciplinare radiologico , chemioterapico e chirurgico . 
il trattamento del ibc e del labc similare solo nella fase iniziale e si avvale di una terapia sistemica neoadiuvante allo scopo di aumentare le opzioni chirurgiche e di incrementare la sopravvivenza [ 5 ]  . 
successivamente , in pazienti correttamente selezionate con labc , ma non nelle pazienti portatrici di ibc , pu essere applicata una terapia conservativa che per dovrebbe essere presa in considerazione precocemente , al momento della programmazione del trattamento [ 1 ]  . 
nella pratica quotidiana per la diagnosi definitiva viene raggiunta solo dopo biopsia chirurgica , visto che la clinica pu essere similare in caso di ibc e di labc e che la diagnosi patologica pre - operatoria pu fornire campioni tissutali quantitativamente insufficienti per la corretta diagnosi anche con luso di aghi grossi . 
breast magnetic resonance ( mr ) imaging has become a valuable tool in staging invasive breast cancer [ 7 , 8 ] and monitoring response to chemotherapy [ 911 ]  . although several studies have reported the characteristic signs of ibc [ 1220 ] , few have specifically investigated its mr imaging patterns [ 1215 ] , and only one recent paper has addressed the mr appearance of ibc and the differential diagnosis with labc [ 21 ]  . 
the purpose of this study was to verify the existence of mr imaging patterns specific to ibc and to evaluate their accuracy in the differential diagnosis between ibc and labc . materials and methods patients from january 2004 to january 2009 , 52 patients with a diagnosis of ibc were retrospectively considered candidates for this study . 
inclusion criteria were : ( 1 ) histological diagnosis of ibc ( stage t4d ) characterised by the presence of invasive breast cancer with diffuse tumour embolisation of the subdermal lymph vessels according to the american joint committee on cancer ( ajcc ) guidelines [ 22 ] ; ( 2 ) clinical signs and symptoms of inflammation ( swelling and hardening of the breast , pain , peau dorange ( orange skin ) appearance , increased skin temperature , redness of the skin extending to at least one third of the breast ) ; and ( 3 ) mr imaging performed before surgery and neoadjuvant chemotherapy . 
patients who had undergone mr imaging after chemotherapy ( n = 3 ) or surgery ( n = 2 ) and patients who had not undergone mr imaging or for whom mr images were unavailable ( n = 7 ) were excluded from the study . 
the ibc group consisted of 30 patients ( mean age 49 years , range 3181 ) ; of these , 24 were premenopausal and six postmenopausal , and 24 had had pregnancies with a mean number of two children . 
the labc group included 22 premenopausal and eight postmenopausal women ( mean age 54 years , range 4377 ) ; 22 had had pregnancies , with a mean number of two children . 
the control group was made up of patients with labc with a histological diagnosis of stage iiia and iiib invasive breast cancer according to the ajcc staging system [ 22 ] and who had undergone mr imaging before surgery and chemotherapy . 
in the labc group , there were ten patients with positive lymph nodes ( pn2 ) and tumours ranging in size from 1 to 5 cm ( pt1cpt2 ) ; 14 with tumours > 5 cm ( pt3 ) and six with invasion into the skin ( without infiltration into or involvement of the dermal lymphatics ) or chest wall ( pt4a - c )  . linquadramento del carcinoma infiammatorio e la sua differenziazione dal labc rimane una sfida nella diagnostica per immagini della mammella . 
lindagine di risonanza magnetica ( rm ) della mammella diventata un valido strumento nella stadiazione [ 7 , 8 ] e nel monitoraggio della risposta alla chemioterapia [ 911 ] nei tumori invasivi della mammella . 
 in letteratura , alcuni lavori si sono occupati dei rilievi semeiotici del ibc [ 1220 ] , pochi di questi hanno trattato gli aspetti di imaging rm [ 1215 ] e solo un recente lavoro analizza le manifestazioni rm del ibc e la diagnosi differenziale con il labc [ 21 ]  . 
lo scopo di questo studio quindi verificare lesistenza di rilievi rm specifici di carcinoma infiammatorio e dimostrarne laccuratezza statistica nella diagnosi differenziale tra carcinoma infiammatorio e tumore localmente avanzato della mammella . materiali e metodi pazienti tra gennaio 2004 e gennaio 2009 , 52 pazienti con diagnosi di ibc sono state considerate retrospettivamente come candidate per questo studio . 
i criteri di inclusione sono stati : diagnosi istologica di ibc ( stadio t4d ) caratterizzata dalla presenza di carcinoma mammario invasivo con estesa embolizzazione tumorale dei vasi linfatici subdermici secondo le linee guida dellamerican joint committee on cancer ( ajcc ) [ 22 ] ; il rilievo clinico di sintomi / segni di infiammazione ( aumento volumetrico e indurimento della mammella , dolore , pelle a buccia darancia , aumento della temperatura cutanea , rossore cutaneo di almeno un terzo della mammella ) ; esame rm eseguito prima del trattamento chirurgico e della chemioterapia neoadiuvante . 
sono stati esclusi i casi in cui lindagine rm stata espletata dopo la chemioterapia ( 3 casi ) o dopo il trattamento chirurgico ( 2 casi ) e i casi di ibc in cui lesame rm non stato espletato o non era disponibile ( 7 casi )  . 
il gruppo di ibc in studio quindi costituito da 30 pazienti ( et media 49 anni , range 3181 ) ; 24 donne erano in fase fertile e 6 in menopausa , 24 pazienti hanno avuto gravidanze con un numero medio di 2 figli . 
il gruppo di pazienti controllo con labc costituito da 22 donne in fase fertile e 8 in menopausa ( et media 54 anni , range 4377 ) ; 22 pazienti avevano avuto gravidanze con un numero medio di 2 figli . 
familiarit positiva per tumore della mammella era presente in 15 casi . sono stati inclusi nel gruppo controllo , i casi di labc con radiol med ( 2011 ) 116 : 7183 two experienced breast pathologists ( fb and em ) examined the haematoxylineosin stained surgical specimens to determine tumour histological type , grade and immunohistochemical expression . 
histological type was defined according to the world health organization ( who ) classification [ 23 ] ; grade was established on the basis of the nottingham grading system ( elston - ellis modification of the scarffbloomrichardson system ) [ 24 ]  . 
the presence of prognostic markers such as oestrogen receptor ( er ) , progesterone receptor ( pgr ) , ki - 67 , e - cadherin and human epidermal growth factor receptor ( her ) - 2 / neu protein overexpression was assessed and interpreted as either negative or positive according to the standard international recommendations . 
only the 3 + category was considered positive . image analysis all patients underwent mammography and ultrasonography ( us ) with the exception of two women who did not undergo mammography . 
mammographic findings in the ibc group showed skin thickening in 14 ( 46% ) , asymmetrical density with distorted architecture in ten ( 33% ) , focal masses in ten ( 33% ) and calcifications in eight ( 27% )  . 
in two patients , mammography was attempted but not completed owing to breast paamong ibc patients , us showed the presence of a hypoechoic focal lesion in eight ( 27% ) , an irregular hypoechoic area with posterior shadowing in 14 ( 73% ) , skin thickening in 16 ( 53% ) and lymphatic ectasia in six ( 20% )  . mammographic findings in the labc group were skin thickening in four patients ( 13% ) , asymmetrical density with architectural distortion in six ( 20% ) , presence of a focal mass in 12 ( 40% ) and focal mass associated with calcifications in six ( 20% )  . 
mammography failed to identify any abnormality in one case of extremely dense breast . among labc patients , us showed a hypoechoic focal lesion with ill - defined margins in 12 ( 40% ) , an irregular hypoechoic area with posterior shadowing in 20 ( 67% ) and skin thickening in four ( 13% )  . mr imaging was performed with a 1.5 - t magnet ( symphony ; siemens medical system , erlangen , germany )  . images were acquired with a bilateral breast coil and the patient in the prone position . 
in particolare , vi erano 10 casi di tumore con linfonodi positivi ( pn2 ) e dimensioni comprese tra 1 cm e 5 cm ( pt1c - pt2 ) ; 14 casi di tumore con dimensioni superiori a 5 cm ( pt3 ) e 6 casi di tumore infiltrante la cute ( senza infiltrazione o coinvolgimento dei vasi linfatici dermici ) o la parete toracica ( pt4a - c )  . 
 due anatomo - patologici esperti in patologia mammaria ( fb e em ) hanno esaminato i preparati in ematossilinaeosina allestiti da campioni di pezzi operatori al fine di determinare il tipo istologico , il grado tumorale e lespressione immunoistochimica . 
il tipo istologico tumorale stato definito in accordo con la classificazione world health organization ( who ) [ 23 ] ; il grado stato assegnato sulla base del the nottingham grading system ( the elston - ellis modification of the scarff - bloom - richardson system ) [ 24 ]  . 
la presenza di markers prognostici , quali la sovra - espressione di recettori per gli estrogeni ( er ) , per il progesterone ( pgr ) , del ki - 67 , delle - caderina e della proteina her - 2 / neu stata valutata e interpretata come negativa o positiva sulla base delle raccomandazioni standard internazionali . 
solo la categoria 3 + stata considerata positiva . analisi delle immagini tutte le pazienti sono state sottoposte a indagine mammografica ed ecografica , eccetto due dove non stata eseguita la mammografia . 
i rilievi mammografici nel gruppo di ibc hanno evidenziato ispessimento cutaneo in 14 casi ( 46% ) , densit ghiandolare asimmetrica con distorsione dellarchitettura in 10 casi ( 33% ) , massa focale in 10 ( 33% ) , e calcificazioni in 8 casi ( 27% )  . 
lindagine ecografica nelle pazienti con ibc ha identificato la presenza di una lesione focale ipoecogena in 8 casi ( 27% ) , unarea ipoecogena , irregolare con sbarramento posteriore in 14 casi ( 73% ) , ispessimento cutaneo in 16 casi ( 53% ) e ectasia dei vasi linfatici in 6 casi ( 20% )  . 
nel gruppo dei labc sono stati rilevati i seguenti rilievi mammografici : ispessimento cutaneo in 4 casi ( 13% ) , densit ghiandolare asimmetrica con distorsione dellarchitettura in 6 casi ( 20% ) , presenza di massa focale in 12 casi ( 40% ) e massa focale associata a calcificazioni in 6 casi ( 20% )  . 
the dynamic study was performed with a 3d gradient recalled echo ( gre ) sequence in the coronal plane ( tr / te : 12 / 5.65 ms ; angle 25 ; matrix 254320 pixel ; fov 38075 ; thickness 1.24 mm ; acquisition time 88 s ) acquired before the administration of contrast material and repeated six times after intravenous injection of 0.1 mmol of gadoterate meglumine ( dotarem , guerbet co . ) per kilogram of body weight at a rate of 2.5 ml / s followed by 20 ml of saline solution . baseline sequences were assessed for the following morphological features : skin thickening ( skin thickness > 4 mm ) , skin oedema ( increased signal intensity of the skin on the stir sequences ) , architectural distortion ( subversion of the parenchymal and stromal compartments , appearing as coalescence or retraction ) and axillary lymphadenopathy ( lymph nodes > 1.5 cm , with a rounded shape long - axis / short - axis ratio 0.5 and with loss of hilar fat tissue were considered pathological , whereas those > 1.5 cm with an oval shape and preservation of the hilar fat structure were considered benign and reactive )  . dynamic studies were assessed for areas of increased parenchymal enhancement , time - signal intensity curves and skin enhancement . image postprocessing involved subtraction of precontrast acquisitions from postcontrast images and generation of multiplanar reconstructions ( mpr ) and maximum intensity projections ( mip )  . 
time - signal intensity curves were obtained by placing a region of interest ( roi , 59 pixels in width ) over the areas of enhancement seen on the second postcontrast acquisition . 
the time - signal intensity curve was analysed during the first 2 min after contrast administration ( slow , moderate , fast ) and its course followed over the subsequent minutes , thereby identifying three possible curve patterns : steadily progressive enhancement , type 1 ; rapid wash in followed by plateau , type 2 ; rapid wash in and wash out , type 3 [ 25 ]  . statistical analysis mr findings of ibc and labc were compared with the fishers exact test . 
significance was set at a p < 0.05. results distribution of histological type , grade and immunohistoipoecogena irregolare con sbarramento posteriore in 20 casi ( 67% ) e ispessimento cutaneo in 4 casi ( 13% )  . trasversale lo studio rm stato effettuato con magnete da 1 , 5 t ( symphony , siemens medical system , erlagen , germania )  . le immagini sono state acquisite con paziente in posizione prona e con bobina bilaterale dedicata allo studio della mammella . 
lo studio basale stato condotto con una sequenza short tau inversion recovery ( stir ) nel piano ( tempo di ripetizione [ tr ] / tempo di eco [ te ] : 5960 / 70 ms ; matrice 254320 pixel ; field of view [ fov ] 350100 ; spessore 3 , 00 mm ) , una sequenza stir nel piano coronale ( tr / te : 4840 / 68 ms ; matrice 254320 pixel ; fov 350100 ; spessore 3 , 00 mm ) e una sequenza turbo spin echo ( tse ) nel piano sagittale ( tr / te : 3910 / 95 ms ; matrice 254320 pixel ; fov 320100 ; spessore 3 , 80 mm )  . 
lo studio dinamico stato eseguito con sequenze gradient echo ( gre ) 3d nel piano coronale ( tr / te : 12 / 5 , 65 ms ; angolo 25 ; matrice 254320 pixel ; fov 38075 ; spessore 1 , 24 mm ; tempo di acquisizione 88 secondi ) acquisita prima della somministrazione di mezzo di contrasto e ripetuta per sei volte dopo liniezione endovenosa di 0 , 1 mmol di gadoterate meglumina ( dotarem , guerbet co . ) pro kg di peso corporeo alla velocit di 2 , 5 ml al secondo seguita da 20 ml di soluzione fisiologica . 
 i rilievi morfologici analizzati nelle sequenze basali sono stati : ispessimento cutaneo ( spessore cutaneo > 4 mm ) , edema cutaneo ( aumento dellintensit del segnale della cute nelle sequenze stir ) , distorsione architetturale ( sovvertimento del traliccio parenchimo - stromale con aspetto di convergenza o di retrazione ) , linfoadenopatia ascellare ( sono stati considerati come patologici i linfonodi ascellari con dimensione superiore a 1 , 5 cm , morfologia rotondeggiante , rapporto asse corto - asse lungo 0 , 5 , e con perdita della rappresentazione del tessuto adiposo ilare ; sono stati considerati linfonodi benigni e reattivi quelli con dimensioni > 1 , 5 cm , morfologia ovale e conservazione della struttura adiposa ilare )  . 
i reperti valutati durante lo studio dinamico sono stati i seguenti : presenza di aree di aumentata impregnazione del parenchima ghiandolare , curva intensit / tempo del segnale e impregnazione della cute . le immagini sono state elaborate mediante sottrazione delle acquisizioni pre - contrasto da quelle post - contrasto e con ricostruzioni multiplanari ( mpr ) e in proiezione di massima intensit ( mip )  . 
posizionando una regione dinteresse ( roi , estesa 59 pixels ) sulle aree di impregnazione visualizzate nella seconda acquisizione dopo mezzo di contrasto sono state costruite le curve dellandamento del segnale nel tempo . 
all eight cases of ibc with successivi minuti , configurando cos tre possibili curve intensit - tempo : incremento progressivo e graduale , tipo 1 ; rapido wash in e successivo plateau , tipo 2 ; rapido wash in e wash out , tipo 3 [ 25 ]  . analisi statistica il confronto tra i rilievi rm nel ibc e quelli nel labc stato analizzato mediante il test esatto di fischer . 
the mass - like enhancement pattern was inhomogeneous in 4 / 8 cases ( 50% ) , homogeneous in two ( 25% ) and peripheral ring - like in two ( 25% )  . 
distribution of the non - mass - like rale e del profilo immuno - istochimico stata simile nei casi di carcinoma infiammatorio e nei casi di tumore localmente avanzato ed riportata in dettaglio in tabella 1 . 
b sagittal t2 - weighted turbo spin echo image shows architectural distortion in the upper quadrant . c axial t1 - weighted contrast - enhanced gradient recalled echo subtraction image shows a large , non - mass - like enhancement derived from the coalescence of multiple nodules . 
c sequenza gre - t1 dopo somministrazione di mezzo di contrasto ( mdc ) , immagini di sottrazione , piano assiale : estesa e asimmetrica impregnazione del parenchima ghiandolare sinistro , di tipo non - massa a noduli multipli , confluenti con distribuzione irregolarmente triangolare . 
la cute sovrastante , ispessita , dopo somministrazione di mdc mostra impregnazione disomogenea e settoriale . d ricostruzione mip dopo somministrazione di mdc , piano coronale obliquo : meglio evidente lestensione regionale della impregnazione . enhancement pattern was focal in 4 / 22 cases ( 18% ) , segmental in six ( 27% ) and diffuse in 12 ( 55% )  . 
the masslike enhancement pattern was homogeneous in 8 / 22 cases ( 36% ) , inhomogeneous in 12 ( 55% ) and reticular / dendritic in two ( 9% )  . 
mass - like enhancement pattern was homogeneous in 2 / 18 cases ( 11% ) , inhomogeneous in 12 ( 67% ) and peripheral ring - like in four ( 22% )  . 
il coinvolgimento macroscopico dei linfonodi ascellari allindagine rm stato rilevato in 20 / 30 ( 67% ) casi di ibc e in 26 / 30 ( 86% ) casi di labc ( p = 0 , 12 )  . 
il pattern di impregnazione di tipo massa stato disomogeneo in 4 / 8 ( 50% ) casi , omogeneo in 2 / 8 ( 25% ) casi e con radiol med ( 2011 ) 116 : 7183 fig . 
a sagittal t2 - weighted tse image shows two space - occupying lesions the more cranial one with oval shape and well - defined margins and the caudad one with irregular shape and ill - defined margins . 
a sequenza tse - t2 , piano sagittale : due formazioni espansive , solide , quella craniale con morfologia ovalare e profili netti , laltra caudale con morfologia irregolare e contorni irregolari . 
the non - mass - like enhancement pattern was homogeneous in 4 / 12 cases ( 33% ) , inhomogeneous in six ( 50% ) and reticular / dendritic in two ( 17% )  . 
unifocal growth was seen in 8 / 12 ( 67% ) cases of non - mass - like enhancement . comparison of enhancement patterns between the two segno dellanello periferico in 2 / 8 ( 25% ) casi . 
il pattern di impregnazione di tipo non - massa stato omogeneo in 8 / 22 ( 36% ) casi , non omogeneo in tutti i 12 / 22 casi ( 55% ) e reticolare / dendritico in 2 / 22 ( 9% ) casi . 
il pattern diimpregnazione tipo non - massa stato omogeneo in 4 / 12 ( 50% ) casi e ( 33% ) casi , disomogeneo reticolare / dendritico in 2 / 12 ( 17% ) casi . 
 in 6 / 12 confrontando il pattern di impregnazione nei due gruppi , impregnazione diffusa tipo non - massa stata documentata in 22 / 30 ( 73% ) carcinomi infiammatori vs 12 / 30 ( 40% ) tumori localmente avanzati ( p = 0 , 02 )  . 
la curva intensit / tempo del segnale aveva andamento a wash - out in 22 / 30 ( 73% ) casi di ibc e in 20 / 30 ( 67% ) casi di labc ( p = 0 , 77 )  . nei rimanenti casi landamento della curva intensit / tempo stata a plateau . 
le indagini strumentali classiche ( mammografia ed ecografia ) sottostimano spesso lestensione della malattia e non sono idonee per lo studio della cute , che pu essere valutato solo con lesame clinico analizzando i segni destensione alla cute ( infiltrazione , noduli , ulcerazione ) e ai vasi linfatici intraparenchimali ( edema e / o eritema )  . il carcinoma infiammatorio , la forma pi aggressiva di radiol med ( 2011 ) 116 : 7183 discussion diagnostic imaging plays a crucial role in the study of ibc , where it is used to assess locoregional extension of disease , diagnose distant metastases and monitor the response to treatment [ 911 , 26 ]  . 
conventional imaging techniques ( mammography and us ) often underestimate the extent of disease and are inappropriate for studying the skin , which can only be assessed clinically by detecting signs of skin involvement ( infiltration , nodules , ulcerations ) and intraparenchymal lymphatics ( oedema and / or erythema )  . ibc , the most aggressive form of locally advanced cancer , is a distinct entity , which can be defined clinically on the basis of oedema and erythema extending to the entire mammary gland , often without a palpable mass , and with diffusely increased breast density . 
skin involvement is therefore both a typical finding in ibc even though it is absent in the occult forms and a manifestation of noninflammatory labc , which carries a worse prognosis . in this difficult setting , pathological examination is required to distinguish the underlying disease and help plan the most appropriate treatment strategies . 
in some cases , however , histology fails to identify the pathognomonic sign of ibc namely , neoplastic infiltration into dermal lymphatics owing either to incorrect skin and breast - tissue sampling or inadequate amount of material . 
as a consequence , the patient is often wrongly directed to treatment following labc management protocols and the diagnosis of ibc is only established later , after surgical excision and pathological analysis of the entire breast . our study was therefore designed to verify the existence of mr findings specific to ibc that might be used to differentiate this form from labc . 
in addition , mr imaging revealed the presence of skin oedema in a higher tumore localmente avanzato , rappresenta una malattia a s stante che nella forma tipica clinicamente definibile in base alla presenza di edema ed eritema esteso allintera ghiandola mammaria , spesso senza una massa palpabile ma con un diffuso aumento di consistenza della mammella . 
linteressamento cutaneo rappresenta quindi da una parte il reperto clinico tipico del carcinoma infiammatorio , tuttavia assente nelle forme occulte , e dallaltra la manifestazione del carcinoma non - infiammatorio localmente avanzato con prognosi pi sfavorevole . in questo difficile contesto clinico , lesame anatomopatologico pi che mai opportuno per distinguere la malattia sottostante e quindi lottimale planning terapeutico . 
nel caso di neoplasia localmente avanzata , il prelievo istologico , fornendo le informazioni indispensabili per limpostazione del trattamento , pi utile rispetto al prelievo citologico [ 27 , 28 ]  . 
lesame istologico , tuttavia , non consente in tutti i casi lindividuazione del segno patognomonico di carcinoma infiammatorio linfiltrazione neoplastica dei linfatici cutanei vuoi per il non corretto campionamento della cute oltre che della mammella vuoi per lesigua quantit di materiale . 
di conseguenza accade frequentemente che la paziente venga erroneamente avviata al trattamento secondo i protocolli del carcinoma localmente avanzato e che , solo in un secondo tempo , ovvero alla escissione chirurgica , dopo lesame patologico dellintera mammella venga formulata la diagnosi di carcinoma infiammatorio . il presente studio stato pertanto disegnato con lintento di verificare lesistenza di rilievi rm specifici di carcinoma infiammatorio utili nella diagnosi differenziale tra carcinoma infiammatorio e tumore localmente avanzato della mammella . 
il rilievo pi tipico in corso di carcinoma infiammatorio linteressamento della cute con diffuse alterazioni che sono descritte dagli autori in letteratura in circa il 75% dei casi [ 14 , 21 ]  . 
le alterazioni della cute possono essere associate anche a tumori localmente avanzati , ma in percentuale significativamente minore , pari al 27% ( p < 0 , 0001 )  . 
per tale ragione , le presenza di alterazioni cutanee associate a neoplasia mammaria dovrebbe essere approfondita con esame patologico della cute per confermare o escluderne linteressamento neoplastico , definire il tipo di interessamento cutaneo ( infiammatorio o non infiammatorio ) e quindi radiol med ( 2011 ) 116 : 7183 percentage of ibc cases compared with mammography and us ( 87% vs 53% )  . 
for this reason , the presence of skin changes associated with breast cancer should be investigated with pathological examination to confirm or exclude neoplastic involvement , define the type of involvement ( inflammatory or noninflammatory ) and consequently allow for correct diagnosis and treatment . ibc has been reported to show either mass - like or nonmass - like enhancement , with variable frequencies . 
ibc showed a non - mass - like enhancement pattern in most cases ( 73% ) , whereas labc generally had a mass - like enhancement ( 60% )  . 
with regard to distribution , ibc cases show diffuse breast involvement [ 12 , 13 , 15 , 21 ] , whereas labc appears as single or multifocal lesions with mass - like enhancement . 
this finding was significantly different compared with labc that typically does shows no skin enhancement ( p = 0.02 ) , even though markedly lower than reported in the literature . 
focal skin enhancement in labc cases is rare , although it was detected in 28% of cases in renz et al.s [ 21 ] study and in 7% of cases in our series . previous authors have suggested additional evaluation of the pectoralis major muscle and of coopers ligaments . pectoral enhancement and loss of the fat cleavage plane are reliable signs of neoplastic involvement of the pectoralis muscle [ 21 ]  . 
enhancement of thickened connective septa of coopers ligaments is a useful indicator of infiltration when the need arises to distinguish inflammatory carcinoma from nonneoplastic inflammatory processes of the breast [ 14 ]  . in conclusion , ibc is an aggressive clinical and biological entity , which is separate from labc and shows with distinctive mr imaging features . 
il carcinoma infiammatorio ha presentato impregnazione di tipo non massa nelle maggior parte dei casi ( 73% ) ; il tumore localmente avanzato si manifestato generalmente con impregnazione di tipo massa ( 60% )  . 
per quanto riguarda la distribuzione della crescita neoplastica , i casi di ibc coinvolgono la mammella in modo diffuso [ 12 , 13 , 15 , 21 ] mentre i casi di labc si presentano come lesione focale singola o multipla e con estensione tipo massa . 
nella maggioranza dei casi di ibc e labc , la distribuzione macroscopica della lesione proliferativa e il pattern di enhancement rm sono elementi suggestivi per sospettare il corretto tipo di tumore . 
tale risultato appare statisticamente significativo se confrontato con il labc che di solito non mostra impregnazione della cute ( p = 0 , 02 ) , ma molto inferiore a quanto riportato in letteratura ove descritto con una frequenza pi elevate che in quella presente . 
la presenza di impregnazione delle fibre pettorali e la scomparsa del piano di clivaggio adiposo sono risultati segni affidabili per il coinvolgimento neoplastico del muscolo pettorale [ 21 ]  . 
limpregnazione dei setti connettivali ispessiti dei legamenti di cooper pare essere un indice di infiltrazione utile , qualora ve ne fosse bisogno , nel distinguere il carcinoma infiammatorio dai processi infiammatori non - neoplastici della mammella [ 14 ]  . in conclusione , il carcinoma infiammatorio rappresenta unentit clinica e biologica aggressiva distinta dal carcinoma localmente avanzato risultante in peculiari rilievi allimaging rm . 
la presenza di segni di infiammazione contigui alla neoformazione tumorale elemento suggestivo di embolizzazione neoplastica e nei casi dubbi dovrebbe guidare allesteso esame bioptico della cute . lindagine rm riveste un ruolo importante nella diagnosi radiol med ( 2011 ) 116 : 7183 cases . 
moreover , it is essential in treatment planning , as it supplies information about disease extent . differenziale fornendo criteri indicativi di carcinoma infiammatorio oltre che nel bilancio di estensione della malattia , risultando metodica fondamentale per lottimale planning terapeutico . 
these developmental lesions are often isolated , but the association of two or more anomalies is not infrequent . contrast - enhanced multidetector computed tomography ( mdct ) , thanks to multiplanar and 3d reconstructions , allows for detailed studies of these malformations , achieving better accuracy compared with conventional techniques such as chest x - ray , fluoroscopy , ventilation and perfusion scintigraphy and ultrasonography . 
recently , improved ct scanners and optimised ct protocols have made available to children all the benefits of mdct , thanks to a significant reduction in radiation dose and an improved riskbenefit ratio . 
the aim of our paper was to evaluate mdct in children with bronchopulmonary malformations by reporting our experience ( about 2 , 400 studies in 30 months with a 64 - slice mdct scanner ) and comparing it with the available literature . keywords lung congenital malformations multislice computed tomography riassunto le malformazioni congenite broncopolmonari rappresentano un ampio spettro di patologie che interessano il parenchima polmonare , il sistema vascolare , le vie aeree centrali e lesofago . 
la tomografia computerizzata ( tc ) volumetrica multidettetore ( mdct ) con mezzo di contrasto permette di analizzare dettagliatamente le strutture toraciche coinvolte nelle malformazioni , con ricostruzioni multiplanari e 3d , con unaccuratezza superiore ad altre indagini tradizionalmente utilizzate tra cui il radiogramma toracico , gli studi fluoroscopici , lecografia e la scintigrafia ventilatoria perfusionale . 
recentemente levoluzione tecnologica degli scanner tc e la definizione di protocolli pediatrici dedicati ha permesso una significativa riduzione della dose assorbita , pertanto oggi possibile usufruire delle potenzialit della mdct nello studio del torace del bambino con un rapporto rischio - beneficio ragionevolmente vantaggioso . 
il nostro articolo si propone di valutare i differenti aspetti dellimpiego della tc sul bambino analizzando la letteratura e confrontandola con lesperienza personale , unica in italia : circa 2400 tc del torace in 30 mesi in pazienti compresi tra 1 giorno e 16 anni con un apparecchio a 64 strati . 
 134 radiol med ( 2011 ) 116 : 133151 parole chiave polmone malformazioni congenite tomografia computerizzata multidetettore introduction introduzione the innovation potential of multidetector computed tomography ( mdct ) in chest imaging has been emphasised ever since the first papers on this new modality appeared in the literature [ 1 ]  . 
generally , the limited commercial interest , less funding and greater caution involve delays in transferring to paediatric care any knowledge , drugs and equipment that have become available for adult patients [ 2 ]  . 
in fact , in february 2001 , three studies published in the american journal of roentgenology [ 35 ] , which were immediately echoed by the media , not only alarmed parents worldwide , but also raised awareness of a problem that had been neglected , above all in the usa . 
as a consequence , in november 2001 , the us food and drug administration published a notification on radiation risk to paediatric patients from ct , and in particular from mdct [ 6 ]  . 
in fact , mdct was thought to be a source of higher radiation doses compared with single - detector ct in that it enabled the study of extensive thin - slice areas in multiple phases ( often unnecessary in children ) [ 7 ] and allowed for a greater range of settings in the absence of well - defined paediatric protocols [ 8 ]  . in recent years , the use of new - generation scanners [ 9 ] and the definition of paediatric protocols have led to a significant reduction in radiation dose , especially in chest imaging [ 1012 ]  . 
mdct has therefore changed the study of the airways and thoracic vessels in children , thanks to extensive anatomical coverage with increased contrast enhancement , isotropic acquisitions allowing for multiplanar reconstructions ( mpr ) [ 13 ] , and high spatial and temporal resolution with fewer motion artefacts in studying the heart and lung [ 11 , 1416 ]  . 
although magnetic resonance ( mr ) imaging does not use ionising radiation , it is inaccurate in depicting the lung parenchyma and has long acquisition times , requiring sedation / anaesthesia in the majority of children . 
sedation often does not provide sufficient immobility and may be problematic in patients with airway obstruction , such that general anaesthesia with intubation is often needed . intubation in turn may interfere with depiction of tracheal size and shape . 
as regards contrast enhancement , the use of gadolinium - based agents ( necessary for mr imaging ) is considered a risk factor in children due to the possible onset le possibilit innovatrici della tomografia computerizzata ( tc ) multistrato ( mdct ) nello studio del torace vennero messe in risalto gi nei primi articoli che descrissero la nuova tecnologia [ 1 ]  . 
in generale , scarso interesse commerciale , minori finanziamenti e maggior cautela comportano un ritardo nel trasferire al bambino le conoscenze , i farmaci e le attrezzature divenute disponibili nelladulto [ 2 ]  . 
nel febbraio 2001 , infatti , la pubblicazione sullamerican journal of roentgenology di tre studi sui rischi legati alle radiazioni in et pediatrica [ 35 ] immediatamente ripresi dai mass media provoc non solo timori nei genitori di tutto il mondo , ma anche la presa di coscienza dellesistenza di un problema fino ad allora trascurato specie negli usa . 
in seguito a ci , negli stati uniti la food and drug administration ( fda ) pubblic nel novembre 2001 una notifica sui rischi nel bambino dovuti a radiazione derivante da tc con particolare attenzione verso la mdct [ 6 ]  . 
la mdct sarebbe infatti potuta diventare fonte di maggior irradiazione rispetto alla single detector tc per la possibilit di studiare ampie aree a strato sottile anche con fasi multiple ( spesso inutili nel bambino ) [ 7 ] e per la maggior quantit di settaggi possibili in assenza di protocolli pediatrici definiti [ 8 ]  . nel corso degli ultimi anni limpegno dellindustria con generazioni successive di apparecchiature [ 9 ] e la definizione di protocolli pediatrici hanno portato specie nello studio del torace ad una significativa riduzione della irradiazione [ 1012 ]  . 
la mdct ha cos trasformato lo studio delle vie aeree e dei vasi toracici nel bambino grazie alla sua ampia copertura anatomica con pi consistente contrast enhancement , alla acquisizione isotropica con possibilit di ricostruzione multiplanare [ 13 ] e allalta risoluzione sia temporale ( con minori artefatti da movimento nello studio di cuore e polmone ) che spaziale [ 11 , 1416 ]  . 
la risonanza magnetica ( rm ) non utilizza radiazioni ionizzanti , ma non offre accurate informazioni sul parenchima polmonare e presenta lunghi tempi di acquisizione che richiedono la sedazione / anestesia nella maggior parte dei pazienti pediatrici . 
la sedazione spesso non consente una sufficiente immobilit e rappresenta un problema nei pazienti con ostruzione radiol med ( 2011 ) 116 : 133151 of nephrogenic systemic fibrosis ( nsf ) related to immature renal function [ 17 ]  . 
the choice between these two imaging modalities is therefore based on the individual patients needs , even though the limited radiation dose of modern mdct tends to support the use of this modality . whereas the italian literature contains numerous studies on mdct techniques and applications in the adult population , there are no published studies focusing on its use in children . 
our paper evaluates the use of mdct in children by reviewing the literature and comparing it with our personal experience , unique in italy : approximately 2 , 400 chest ct scans performed over 30 months with a 64 - row scanner in patients 1 day to 16 years of age . 
we discuss the technical and radiation protection aspects and the clinical indications of ct in bronchopulmonary malformations , without inclusion of heart disease as such , which , due to its vastness , would require a separate discussion . mdct : technical considerations the ultimate goal of mdct is to acquire diagnostic images with a radiation dose as low as reasonably achievable ( alara principle ) [ 18 ]  . 
the radiologist must be aware of the clinical question to be able to optimise the examination variables to attain the best possible result . patient preparation on the basis of our experience with 64 - slice mdct , sedation is seldom needed . 
it is usually required in patients 6 months to 4 years of age in whom the approach of specialised personnel together with the help of the parents and patient restraint devices do not ensure sufficient immobility . 
we normally use an automatic injector ( empower cta , e - z - e - m , inc . , lake success , ny , usa ) with the following parameters : 1 ml / s for 24 - gauge catheters ( rarely , with the greatest caution ) , 1.52.0 ml / s for 22 - gauge catheters , 23 ml / s for 20 - gauge catheters , with a pressure limit adjusted to a maximum of 150 psi ( 1 , 034 kpa ) [ 19 ]  . we empirically begin the scan at the end of the injection . ct technique selection of technical parameters is dependent on delle vie aeree , pertanto molto spesso richiesta lanestesia generale con intubazione . 
per quanto concerne limpiego del mezzo di contrasto , ricordiamo come limpiego di prodotti a base di gadolinio ( necessario in rm ) sia considerato fattore di rischio nei pazienti pi piccoli per linsorgenza di fibrosi sistemica nefrogenica ( nephrogenic systemic fibrosis , nsf ) , considerata limmaturit della funzione renale [ 17 ]  . 
perci la scelta tra queste due modalit di imaging si basa sui requisiti del singolo paziente , anche se il contenimento della dose radiante oggi ci fa propendere per la mdct . bench vi sia unampia pubblicistica nella letteratura italiana riguardo le tecniche e le applicazioni della mdct nelladulto , non esiste ad oggi una trattazione focalizzata sullutilizzo in et pediatrica . 
il nostro articolo si propone di valutare limpiego della mdct nel bambino analizzando la letteratura e confrontandola con lesperienza personale , unica in italia : circa 2400 tc del torace eseguite in 30 mesi con un apparecchio a 64 strati in pazienti di et compresa tra 1 giorno e 16 anni . 
approfondiremo gli aspetti tecnici , radioprotezionistici e le indicazioni cliniche , avendo per oggetto le malformazioni bronco - polmonari escludendo la patologia cardiaca strictu sensu che , per la sua vastit , richiederebbe una trattazione a parte . 
 mdct : considerazioni tecniche lesecuzione dellesame deve avere come obiettivo finale lacquisizione di immagini diagnostiche alla pi bassa dose di radiazione ragionevolmente raggiungibile ( principio as low as reasonably achievable , alara ) [ 18 ]  . 
il radiologo deve conoscere il quesito clinico per ottimizzare tutte le variabili dellindagine ai fini del miglior risultato . preparazione del paziente in base alla nostra esperienza con mdct a 64 strati , la sedazione necessaria solo in pochi casi . 
di solito riguarda pazienti di et compresa tra 6 mesi e 4 anni per i quali lapproccio dedicato del personale con laiuto dei genitori e i mezzi di contenzione non permettono di ottenere una sufficiente immobilit . 
in pratica , utilizziamo un mezzo di contrasto non ionico a bassa osmolarit ( 300 mgi / ml ) alla dose di 2 ml / kg fino a 125 ml [ 14 ]  . 136 radiol med ( 2011 ) 116 : 133151 indications . 
high - resolution sequential acquisitions ( stepand - shoot method ; conventional hrct ) , characterised by significant dose reduction , are only used in the follow - up of diffuse lung diseases . 
we use two types of nonelectrocardiographically gated acquisitions focusing on either the vascular anatomy ( high temporal resolution ) or the finer details of the lung parenchyma ( high spatial resolution ) with the following equipment and settings : somaton sensation 64 ( siemens medical solutions , erlangen , germany ) : acquisition 640.6 ( z - flying focal spot ) ; rotation time 0.33 s ; pitch 0.90 ; kvp 80 ( < 15 kg ) 100 ; ref . 
an isotropic data set is thus obtained , which , memorised on the picture archiving and communications system ( pacs ) database , allows for high - quality 2d and 3d reconstructions . postprocessing workstations : syngo ct.3d mm ( siemens medical solutions ) , aquarius ws ( terarecon inc . , san mateo , ca , usa ) , carestream healths 3d virtual desktop ( carestream health , rochester , ny , usa )  . in uncooperative patients , the examination is performed during free breathing . 
anatomical coverage routinely extends from the mediastinal inlet to the diaphragin some cases , we extend the acquisition to include a larger volume ; for example , in suspected extralobar pulmonary sequestration , which may be located in the upper abdomen and / or be supplied by the abdominal aorta , the scan is extended to the plane of the renal arteries [ 20 ]  . in our experience , indications for retrospective ecg gating ( which entails at least a fourfold higher dose , with only a portion of the dose being used for image creation ) are limited , as the origin of the coronary arteries can also be clearly identified in most cases with nongated sequences [ 16 ]  . 
in pratica limitiamo le acquisizioni sequenziali ad alta risoluzione ( step and shoot method ; tc ad alta risoluzione [ hrct ] convenzionale ) , caratterizzate da notevole riduzione della dose , al follow - up delle patologie polmonari diffuse . 
utilizziamo 2 tipi di acquisizione non cardiosincronizzate che privilegiano o lanatomia vascolare ( elevata risoluzione temporale ) o il fine dettaglio del parenchima polmonare ( elevata risoluzione spaziale ) ed i seguenti materiali e settaggi : somaton sensation 64 ( siemens medical solutions , erlangen , germania )  . 
mas : 45 ; 85 . incremento : 1 mricostruzione : 1 mfiltro b30f ; b60f . otteniamo cos un dataset isotropico che , memorizzato nel picture archiving and communication system ( pacs ) , permette ricostruzioni 2d e 3d di alta qualit . workstation di post - elaborazione : syngo ct.3d mm ( siemens medical solutions ) , aquarius ws , ( terarecon inc . , san mateo , usa ) , carestream healths 3d virtual desktop ( carestream health , rochester , usa )  . nei pazienti non collaboranti eseguiamo lesame a respiro libero . 
talora lacquisizione deve essere ampliata , come nel dubbio di sequestro polmonare extralobare che pu essere situata nella cavit addominale superiore e / o avere vascolarizzazione che origina dalla aorta addominale : in tal caso la scansione va estesa al piano delle arterie renali [ 20 ]  . 
volume di computed tomography dose index ( ctdivol ) e dose lenght product ( dlp ) : valori medi e deviazioni standard ( ds ) da noi osservati in 3 differenti fasce det chiaramente individuata nella maggioranza dei casi con sequenze non - gated [ 16 ]  . 
la nostra esperienza invece ancora limitata per quanto concerne le indagini eseguite con cardiosincronizzazione prospettica . il controllo dei fattori di esposizione rappresenta un elemento decisivo per il contenimento della dose . 
passando da 140 a 80 kvp , a corrente di tubo costante , la dose si riduce di un fattore di circa 3 , 4 [ 22 ] con un incremento di contrasto e di rumore ( pochi fotoni prodotti )  . 
nel bambino sottoposto a tc con mezzo di contrasto siegel [ 23 ] ha dimostrato che la ridotta energia del fascio riduce la dose senza incidere in modo significativo sul contrasto dellimmagine . daccordo con herzog [ 24 ] , noi preferiamo utilizzare gli 80 kvp al di sotto dei 15 kg , utilizzando 100 kvp per pesi 15 kg . 
 [ 24 ] ed osservati nel bambino con la nostra stessa apparecchiatura . la dose efficace genericamente riportata in letteratura per la tc del torace in pediatria di 3 msv [ 26 ]  . 
 [ 11 ] , sulla base di unesperienza con apparecchio a 16 strati utilizzato per le acquisizioni angiografiche con collimazione 0 , 75 mm , riferiscono dosi efficaci che si incrementano al crescere del peso da 1 a 2 , 5 msv . 
lo stesso autore attribuisce dosi efficaci variabili da 0 , 21 a 0 , 59 msv per la hrct sequenziale e da 0 , 00487 a 0 , 01769 msv per il radiogramma antero - posteriore ( ap ) del torace . ricordiamo che esistono dei coefficienti di conversione ( et e regione specifici ) da dlp a dose efficace [ 27 ]  . recentemente sono stati pubblicati dati che confermerebbero la affidabilit del metodo [ 28 ]  . 
in tal caso , mentre per il cranio i fattori di conversione sarebbero corretti anche nel bambino , per quanto riguarda il corpo 138 radiol med ( 2011 ) 116 : 133151 we recall that there are ageand region - specific coefficients for conversion from dlp to effective dose [ 27 ]  . 
manufacturers often derive dlp values from adult phantoms with a head diameter of 16 cm and a body diameter of 32 cin this case , whereas for the head , the conversion factors would also apply to children , for the body , the dlp should be multiplied by two before applying the conversion coefficient . postprocessing is an essential part of the investigation . as with adult patients , the common reconstruction techniques are used : 2d mpr and 3d multiplanar volume reconstruction ( mpvr ) , maximum intensity projection ( mip ) , minimum intensity projection ( minip ) , shadedsurface display ( ssd ) , volume rendering technique ( vrt ) and virtual bronchoscopy ( vb )  . 
mpvr ( fast and easy ) is particularly useful in that it combines the spatial resolution of mpr with the visualisation of thicker slices ; in studying the pulmonary parenchyma , it minimises interpretation problems arising from small - size anatomical structures appearing blurred due to the noise of low - dose ct . 
other reconstruction techniques ( some very time consuming ) are also used according to the case to be interpreted [ 11 , 29 ]  . clinical applications according to panicek et al . 
 [ 30 ] , all the most common developmental anomalies of the lung , such as congenital lobar emphysema ( cle ) , bronchogenic cysts , congenital cystic adenomatoid malformation ( ccam ) or congenital pulmonary airway malformation ( cpam ) , pulmonary sequestration , hypogenetic lung syndrome and pulmonary arteriovenous malformation represent a continuum of malformations from cle ( anomalous lung , normal vessels ) to pulmonary arteriovenous malformation ( anomalous vessels , normal lung )  . 
the theory of bronchopulmonary vascular malinosculations suggests a temporal coincidence ( without any causal relationship ) of abnormal connections between respiratory mesenchyma and bronchial epithelium or between rudiments of the foregut , respiratory tract and aortopulmonary vascular system [ 31 ]  . in this light , newman [ 32 ] recently introduced the notion of bronchopulmonary and foregut malformations . 
as a result , there may be associated anomalies , such as pulmonary agenesis / hypoplasia , sequestration , atresia and tracheal bronchus , pulmonary artery sling , alveolar capillary sarebbe opportuno moltiplicare il dlp per un fattore due prima di applicare il coefficiente di conversione . 
 il post - processing una parte fondamentale dellindagine . come per ladulto si utilizzano le comuni tecniche di ricostruzione disponibili sulle consolle : 2 - d : multiplanar reformation o reconstruction ( mpr ) , 3 - d : multiprojective volume reconstruction ( mpvr ) , maximum intensity projection ( mip ) , minimum intensity projection ( minip ) , shaded - surface display ( ssd ) , volume rendering technique ( vrt ) e virtual bronchoscopy ( vb )  . 
la mpvr ( facile e immediata ) particolarmente utile perch unisce la risoluzione spaziale dellmpr con la visualizzazione anatomica di strati pi spessi ; nello studio del parenchima polmonare limita i problemi interpretativi dovuti alle piccole strutture anatomiche confuse nel rumore da bassa dose . 
 [ 30 ] le pi comuni anomalie di sviluppo del polmone come enfisema lobare congenito ( congenital lobar emphysema , cle ) , cisti broncogena , malformazione adenomatoide cistica congenita ( congenital cystic adenomatoid malformation , ccam o congenital pulmonary airway malformation , cpam ) , sequestro polmonare , sindrome del polmone ipogenetico e malformazione arterovenosa polmonare si inseriscono in un continuum malformativo che ha ad una estremit il cle ( polmone anomalo , vasi normali ) e allaltra la malformazione arterovenosa polmonare ( vasi anomali , polmone normale )  . 
la teoria delle bronchopulmonary vascular malinosculations propone la coincidenza temporale ( senza rapporto causaeffetto ) delle anomalie di connessione tra mesenchima respiratorio e epitelio bronchiale o tra abbozzi dellintestino primitivo , dellapparato respiratorio e del sistema vascolare aorto - polmonare [ 31 ]  . 
di conseguenza possono essere associate tra loro anomalie come agenesia / ipoplasia polmonare , sequestro , atresia e bronco tracheale , sling dellarteria polmonare , displasia alveolo capillare , cisti esofagee / neuroenteriche , fistole tracheo - esofagee / diverticoli / stenosi , ecc . 
si deduce che , a fronte di una sospetta anomalia dei vasi toracici e delle vie aeree , limaging deve fornire una valutazione completa di tutte le componenti anatomiche del torace . evidente come la mdct rappresenti la tecnica di imaging elettiva con un ruolo fondamentale nella acquisizione di radiol med ( 2011 ) 116 : 133151 dysplasia , oesophageal / neuroenteric cysts , tracheoesophageal fistula ( tef ) / diverticula / stenoses , etc . 
mdct clearly represents the imaging technique of choice , as it has a primary role in providing knowledge of the natural history , overlapping , terminology , classification and management of these malformations [ 33 ]  . tracheobronchial anomalies always look at the airway is one of the fundamental maxims of paediatric radiology . 
clinical respiratory manifestations often remain unexplained radiologically because the study has only focused on the pulmonary parenchyma [ 34 ]  . tracheomalacia tracheobronchomalacia is a condition deriving from the softening of the tracheobronchial wall or cartilaginous ring , resulting in pathological expiratory collapse . 
 [ 36 ] suggest that the diagnosis be made with mdct with scans acquired during maximum inspiration and expiration . however , this may prove difficult in uncooperative patients , and in these instances , the authors describe a procedure performed under general anaesthesia after intubation , when forced inspiration and expiration are achieved by varying the ventilatory pressure . tracheal stenosis congenital tracheal stenosis is a rare disorder characterised by the presence of single or multiple , complete or nearcomplete , tracheal cartilage rings , resulting in narrowing of the tracheal lumen . 
types of tracheal stenosis include focal stenosis ( 50% of cases ) , generalised stenosis ( 30% of cases ) and segmental carrotor funnel - shaped stenosis ( 20% of cases ) usually located in the lower third of the trachea . given that resistance to laminar airflow increases linearly in proportion to length of stenosis and fourfold relative to calibre decrease , a tight stenosis produces worse symptoms compared with a long and moderate stenosis . 
il problema per si pone di solito nei non collaboranti e , in questo caso , gli stessi autori descrivono una procedura in anestesia generale a paziente intubato con inspirazione ed espirazione forzate ottenute facendo variare la pressione ventilatoria . stenosi tracheale la stenosi tracheale congenita una rara patologia causata dalla presenza di anelli cartilaginei tracheali completi ( o quasi ) , singoli o multipli , causanti restringimento del lume tracheale . 
le tipologie di stenosi tracheale includono la stenosi focale ( 50% dei casi ) , la stenosi generalizzata ( 30% dei casi ) e la stenosi segmentale a forma di carota o ad imbuto ( 20% dei casi ) solitamente localizzata al terzo inferiore tracheale . 
dato che la resistenza al flusso aereo laminare aumenta linearmente con la lunghezza della stenosi e con la quarta potenza della riduzione di calibro , una stenosi serrata provoca sintomi pi importanti di una stenosi lunga e moderata . 
gli anelli tracheali congeniti completi possono presentarsi isolatamente o la loro presenza pu essere associata a sling polmonare ( arteria polmonare sinistra che origina dalla destra e , passando davanti allesofago , circonda il bronco principale di destra e la trachea distale causando compressione )  . 
i tipici reperti alla mdct sono rappresentati dalla riduzione della circonferenza estesa a tutta la trachea , la fusione degli anelli tracheali cartilaginei posteriormente 140 radiol med ( 2011 ) 116 : 133151 fig . 
tracheal bronchus is more common in children affected by other malformations , such as congenital heart defects , tef , tracheal stenosis and down syndrome [ 37 ]  . in the majority of cases , tracheal bronchus consists of a right upper bronchus with anomalous origin or an apical portion with anterior and posterior branches arising from the right mainstem bronchus or , less frequently , a supernumerary bronchus directed towards the right upper lobe territory ( sometimes with a blind ending )  . 
la presenza di bronco tracheale pi comune in bambini con altre malformazioni , come anomalie cardiache congenite , fistola tracheoesofagea , stenosi tracheale e sindrome di down [ 37 ]  . 
nella maggior parte dei casi , il bronco tracheale costituito da un bronco superiore destro che origina in sede anomala , o dal suo ramo apicale con rami anteriori e posteriori che nascono dal bronco principale destro , o raramente un bronco sopranumerario diretto al lobo superiore destro ( talvolta a fondo cieco )  . 
il bronco tracheale spesso un riscontro incidentale in un paziente asintomatico , anche se le dimensioni ridotte della via aerea e il ristagno di secrezioni possono essere causa di stridore espiratorio destro e / o bronchiectasie , atelettasie e ripetute infezioni . 
limmagine segnalata dalla freccia corrisponde al bronco pieno di muco distale al tratto atresico . of the airway and retained secretions may produce right expiratory stridor and / or bronchiectasis , atelectasis and recurrent infections . 
the introduction of mdct has led to a significant increase in the number of cases detected ; indications for the use of mdct include evaluation of heart malformations or study of recurrent bronchopulmonary disease . bronchial atresia congenital bronchial atresia results from atresia or obstruction of a segmental or subsegmental bronchus with normal development of the distal airway . 
although the majority of patients with bronchial atresia are asymptomatic and the diagnosis is made on chest radiographs performed for other reasons , some authors report a history of recurrent pneumonia . 
this should not be mistaken for a nodule or for lung abnormalities such as lobar emphysema or intralobar sequestration with air trapping [ 39 ]  . sono di solito la valutazione di malformazioni cardiache o lo studio di broncopneumopatie ricorrenti . 
spesso lintrappolamento di aria proveniente dallinterstizio e dai pori di kohn provoca liperinsufflazione del segmento polmonare a valle del bronco ostruito . sebbene la maggior parte dei pazienti con atresia bronchiale siano asintomatici e la diagnosi sia ottenuta da un radiogramma del torace eseguito per altri motivi , qualche autore riferisce una storia di polmoniti ricorrenti . 
tale reperto non deve essere confuso con un nodulo o con anomalie polmonari quali enfisema lobare o sequestro intralobare con air trapping [ 39 ]  . cisti broncogene le cisti broncogene sono lesioni congenite che originano dallintestino primitivo ventrale e possono essere localizzate in sede mediastinica ( 60% ) , al iii medio polmonare ( 30% ) o , 142 bronchogenic cysts bronchogenic cysts are congenital lesions that originate from the embryonic foregut and may be located in the mediastinal space ( 60% ) , in the middle third of the lung ( 30% ) or , less frequently , in the lower neck ( 10% )  . 
two - thirds of patients present with cough , stridor , dyspnoea and pneumonia as a result of tracheal and / or bronchial compression . occasionally the mass effect may produce air trapping and / or atelectasis [ 36 ]  . 
a provisional diagnosis of bronchogenic cyst should be considered whenever a well - circumscribed lesion with fluid density is identified in the middle third of the mediastinum adjacent to the bronchial tree . 
only after infection does the cyst show wall enhancement . pulmonary anomalies pulmonary underdevelopment the clinical spectrum of pulmonary underdevelopment includes agenesis of the bronchus , vascular structures and lung ( pulmonary agenesis ) ; the presence of a blind - ending rudimentary bronchus without lung tissue ( pulmonary aplasia ) ; and bronchial hypoplasia with variable reduction in parenchymal lung volume ( pulmonary hypoplasia ) [ 41 ]  . pulmonary agenesis is a rare congenital malformation . mortality rates are high owing to the associated congenital anomalies , which include heart disease , tracheoesophageal atresia , spinal and renal malformations . 
abnormal development of the pulmonary vasculature is frequently associated and includes alveolar capillary dysplasia , pulmonary artery sling , absence of the pulmonary artery , absence of the pulmonary vein and scimitar syndrome [ 42 ]  . 
due terzi dei pazienti sono affetti da tosse , stridore , dispnea , e polmonite in conseguenza della compressione della trachea e / o dei bronchi da parte della cisti . 
 anomalie del polmone iposviluppo polmonare lo spettro dei quadri di iposviluppo polmonare include lagenesia del bronco , delle strutture vascolari e del polmone ( agenesia polmonare ) , la presenza di un abbozzo bronchiale che termina a fondo cieco senza tessuto polmonare ( aplasia polmonare ) e lipoplasia bronchiale con riduzione di volume del parenchima polmonare di grado variabile ( ipoplasia polmonare ) [ 41 ]  . 
frequente la coesistenza di anomalie di sviluppo della vascolarizzazione polmonare tipo displasia capillare alveolare , sling dellarteria polmonare , assenza dellarteria polmonare , assenza della vena polmonare , sindrome della scimitarra [ 42 ]  . 
vena polmonare anomala ( freccia ) che drena nella vena cava inferiore . vaso sistemico arterioso che rifornisce il polmone destro ( freccia aperta )  . ipoplasia dellarco aortico ( asterisco )  . completely ( agenesis ) or partially ( atresia ) absent : in the latter case , the peripheral circulation is normal . 
unilateral agenesis is often associated with cardiac malformations ; if isolated , it may be asymptomatic or manifest over the years as pulmonary hypertension , haemoptysis and recurrent infection [ 43 ]  . 
chest radiographs show a small , harmonious lung ( disharmonious in the case of lobar agenesis / aplasia ) ; the hilum on the affected side is small . mdct demonstrates the absence of the artery , typically on the side contralateral to the aortic arch usually on the right and helps identify associated heart defects and systemic collateral vessels . 
lagenesia unilaterale si associa spesso a malformazioni cardiache ; se isolata pu essere asintomatica o manifestarsi negli anni con ipertensione polmonare , emottisi e infezioni ricorrenti [ 43 ]  . 
la radiografia del torace mostra il cosiddetto piccolo polmone armonico ( disarmonico se agenesia / aplasia lobare ) ; dallo stesso lato lilo piccolo . la mdct evidenzia lassenza dellarteria , tipicamente dal lato opposto dellarco aortico di solito a destra e permette di identificare le anomalie cardiache associate e i vasi sistemici collaterali . 
 sindrome del polmone ipogenetico la sindrome del polmone ipogenetico , anche conosciuta come sindrome venolobare polmonare congenita o sindrome della scimitarra , una forma di connessione venosa 144 radiol med ( 2011 ) 116 : 133151 fig . 
other less common sites of confluence include the superior vena cava , the right atrium , the azygos vein , the portal vein and the hepatic vethanks to mpr , mdct also allows accurate assessment of the anomalous systemic arterial supply . 
images obtained with lung window settings and 3d reconstructions of the central airways can demonstrate the presence of abnormal parenchymal findings , polmonare anomala verso la vena cava inferiore associata ad altre anomalie come polmone destro e arteria polmonare ipoplasici , destrocardia , anomala vascolarizzazione sistemica arteriosa del polmone destro . 
possono essere presenti anche altre malformazioni come difetti del setto interatriale e interventricolare , dotto arterioso di botallo pervio , tetralogia di fallot , anomalie del diaframma , sequestri polmonari e polmoni a ferro di cavallo . i pazienti affetti da sindrome del polmone ipogenetico presentano una gamma di sintomi variabile in relazione allet di presentazione e al grado di shunt . 
le immagini con finestra polmonare e le ricostruzioni 3d delle vie aeree centrali possono dimostrare la presenza di reperti parenchimali polmonari anomali quali consolidazioni , radiol med ( 2011 ) 116 : 133151 such as consolidation , bronchiectasis , abnormal lung lobation and abnormal bronchial branching . bronchiectasie , lobature polmonari anomale e anomalie della diramazione bronchiale . 
 congenital cystic adenomatoid malformation ( ccam ) malformazione adenomatoide cistica congenita ( ccam ) ccam is characterised by a pulmonary mass deriving from the hamartomatous and adenomatoid proliferation of primary bronchioles communicating with the bronchial tree to the detriment of the alveolar component , and it is often associated with airway cartilage defects . 
there is no consensus concerning its classification and nomenclature . recently , the term congenital pulmonary airway malformation ( cpam ) has been favoured , as not all lesions are cystic and only type 3 is adenomatoid [ 33 ]  . 
the natural history of the condition , characterised by regression and disappearance or by persistence , is not completely explained , and there is disagreement as to the best time to operate on these asymptomatic lesions . 
it has been suggested that low - grade cystic pleuropulmonary blastoma , which presents as a cystic mass , could go undetected , as it is virtually indistinguishable from ccam with currently available diagnostic techniques . 
in the case of pleuropulmonary blastoma , the typical mass effect with lobar expansion , the mediastinal shift and the associated parenchymal opacities produce a pattern that is identical to that of ccam type 4 . ccam may be distinguished from sequestration in that it la ccam una massa polmonare che deriva dalla proliferazione amartomatosa e adenomatoide di bronchioli primari comunicanti con lalbero bronchiale a discapito della componente alveolare , ed spesso associata a difetti cartilaginei delle vie aeree . 
recentemente stato preferito il termine malformazione congenita delle vie aeree polmonari ( cpam ) perch non tutte le lesioni sono cistiche e solo il tipo iii adenomatoide [ 33 ]  . 
 [ 45 ] classificarono queste lesioni in tre tipi : tipo i , macrocistica ( > 2 cm ) ; tipo ii , piccole cisti multiple ; tipo iii , forma solida ( cisti microscopiche )  . 
pi recentemente stata proposta una classificazione ampliata ( da tipo 0 a tipo 4 ) per rappresentare le malformazioni delle vie aree di calibro maggiore sino a quelle di calibro minore [ 46 ]  . 
le ccam sono sempre pi frequentemente identificate in epoca prenatale : le dimensioni maggiori si riscontrano durante il ii trimestre per poi ridursi progressivamente sia in senso assoluto che relativo . 
 stato ipotizzato che il blastoma pleuropolmonare cistico di basso grado che si manifesta come una massa cistica possa non essere identificato in quanto praticamente indistinguibile da una ccam con le tecniche diagnostiche attualmente disponibili . 
in caso di blastoma pleuropolmonare il caratteristico effetto massa con espansione lobare , lo shift mediastinico e le opacit parenchimali associate , realizzano 146 radiol med ( 2011 ) 116 : 133151 fig . 
angio tc : a piano assiale e b piano coronale . massa cistica destra con setti interni che determina spostamento controlaterale del mediastino . has no anomalous systemic arterial supply typical of pulmonary sequestration , although ccam and sequestration may coexist and be present as a mixed lesion [ 50 ]  . 
in patients with bacterial superinfection , airfluid levels and parietal contrast enhancement may also be identified [ 32 , 36 ]  . pulmonary sequestration the term sequestration refers to the presence of a portion of lung parenchyma having no connection with the tracheobronchial tree and a systemic vascular supply [ 32 , 36 ]  . 
 la ccam pu essere distinta dal sequestro sulla base dellassenza di un apporto arterioso sistemico anomalo , tipico del sequestro polmonare , sebbene la ccam e il sequestro possano coesistere e presentarsi come lesione mista [ 50 ]  . 
 sequestro polmonare con il termine sequestro si intende la presenza di una porzione di parenchima polmonare priva di connessione con lalbero tracheo - bronchiale e con un apporto vascolare sistemico [ 32 , 36 ]  . 
sono state definite due forme principali : extralobare , rivestita da pleura viscerale , e intralobare , posizionata tra aree di parenchima normale . spesso coesiste un difetto del diaframma , e circa il 15% dei casi dei sequestri extralobari sono addominali . 
presenza di arteria anomala che origina dalla aorta discendente ( freccia )  . sequestrations typically have normal venous connections . nonetheless , any type of arterial or venous vascularisation is possible . un sequestro intralobare solitamente ha connessioni venose normali . 
 the parenchyma of pulmonary sequestration is usually underdeveloped , and it may be associated with cysts covered by columnar or cubic epithelium , or it may be entirely characterised by structures similar to alveolar ducts . mucus can distend the multiple intercommunicating spaces , making the lesion appear radiologically solid or sonographically hyperechoic unless , as in the case of intralobar sequestration , air penetrates through the pores of kohn . 
an increasing number of authors suggest that intralobar sequestration may also derive from recurrent infections that produce anomalous arterial branches from the aorta ( such as the pulmonary ligament arteries ) [ 51 ]  . sequestration is identified in the prenatal period as a hyperechoic mass , usually in the posterior basal hemithorax . colour doppler ultrasonography is useful for demonstrating the presence of anomalous vessels that can be studied from their orig mdct is crucial for the diagnosis and for preoperative planning . 
il muco pu distendere i multipli spazi intercomunicanti , conferendo un aspetto radiologicamente solido alle lesioni o iperecogeno allecografia , a meno che , in caso di sequestro intralobare , laria vi penetri tramite i pori di kohn . 
un sempre maggiore gruppo di autori ipotizza che il sequestro intralobare possa anche derivare da infezioni ricorrenti che producono rami arteriosi anomali dallaorta ( come le arterie del legamento polmonare ) [ 51 ]  . 
 il sequestro identificato in epoca prenatale come una massa iperecogena , solitamente in sede postero - basale . leco - color doppler utile per dimostrare la presenza di vasi anomali che possono essere valutati sino allorigine . 
the typical presentation consists of a limited number of overdistended alveoli ; by contrast , in the recently identified polyalveolar form , there are more alveoli than under normal conditions [ 52 ]  . the most frequent location is the left upper lobe . 
patients usually have respiratory difficulties in the neonatal period , mostly when there is a marked hyperinflation of the affected lobe and a mass effect on the adjacent parenchyma and on the mediastinusurgical lobectomy is indicated above all in cases of progressive hyperinflation , as this condition is potentially fatal . 
once the neonatal lung fluid is reabsorbed from the lymphatic system and replaced by air , the affected lobe becomes hyperlucent , with a mass effect on the adjacent lobes . mdct shows the presence of a hyperinflated lobe with displaced pulmonary vessels . 
when the lesion reaches large dimensions and has a mass effect on the surrounding structures , a contralateral mediastinal dislocation is observed , together with an increase in intercostal spaces and ipsilateral diaphragmatic descent [ 36 , 41 ]  . 
cle may be mistaken for pneumothorax or for simple or acquired cysts : in cle the bronchovascular markings are seen inside the overdistended lobe . lenfisema lobare congenito ( cle ) ora chiamato iperinsufflazione lobare congenita ( congenital lobar hyperinsufflation , clh ) in quanto non c distruzione delle pareti alveolari [ 32 ] descritto come il risultato di una progressiva sovradistensione di un lobo polmonare , secondaria ad ostruzione intrinseca o estrinseca o a deficit di cartilagine bronchiale . la forma classica ha un numero ridotto di alveoli sovradistesi ; invece , nella forma poli - alveolare recentemente descritta esistono pi alveoli del normale [ 52 ]  . 
occasionalmente possono essere interessati o parte di un lobo o pi di un lobo . i pazienti solitamente presentano difficolt respiratorie nel periodo neonatale , specialmente quando c una marcata iperinsufflazione del lobo coinvolto ed effetto massa sul parenchima adiacente e sul mediastino . 
la lobectomia chirurgica indicata soprattutto nei pazienti con progressiva iperinsufflazione , perch pu essere una condizione mortale . la diagnosi di cle dovrebbe essere considerata nei casi in cui unopacit lobare alla rx torace diventa progressivamente ipertrasparente e iperinsufflata dopo la nascita . 
quando la lesione raggiunge ampie dimensioni e ha effetto massa sulle strutture limitrofe si osserva dislocazione mediastinica controlaterale , incremento degli spazi intercostali e abbassamento diaframmatico omolaterale [ 36 , 41 ]  . 
il cle pu essere confuso con il pneumotorace o con cisti semplici o acquisite : nel cle si vede il disegno polmonare allinterno del lobo sovradisteso . oesophageal anomalies anomalie esofagee we previously mentioned the unitary concept involved in defining bronchopulmonary and foregut malformations [ 32 ]  . 
on this subject , we recently reported on two unknown cases of tracheoesophagel fistula ( in two girls 5 and 9 years of age , respectively ) diagnosed at mdct performed for recurrent airway infections . 
in both girls , a marked gaseous distension of the digestive tract was present in the abdomen [ 53 ]  . abbiamo gi accennato alla visione unitaria compresa nella definizione malformazioni bronco - polmonari e dellintestino primitivo [ 32 ]  . 
in tal senso , abbiamo recentemente pubblicato 2 casi misconosciuti di fistola tracheo esofagea ( rispettivamente in 2 bambine di 5 e 9 anni ) diagnosticati con mdct eseguita per infezioni ricorrenti delle vie aeree . 
 vascular anomalies anomalie vascolari we previously mentioned that development of the airways abbiamo gi detto come lo sviluppo delle vie aeree e del radiol med ( 2011 ) 116 : 133151 and circulatory system runs parallel [ 30 , 31 ] and described anomalies such as pulmonary agenesis / hypoplasia , sequestration and scimitar syndrome . 
this section briefly describes the compressions and dislocations of the tracheobronchial tree produced by anomalous anatomical relations , as in the case of the double aortic arch [ 54 ] or dilatation of the heart or pulmonary vessels in a setting of congenital heart defects . these cases can now be studied by mdct and / or mr imaging . 
in our opinion , mdct should be preferred when a study of the pulmonary parenchyma and airways is deemed necessary and in the case of critical patients needing short scanning times without sedation or anaesthesia . 
mr imaging is ideal in conditions requiring serial studies , screening , flow evaluation , biventricular function or intracardiac anatomy [ 16 ]  . sistema vascolare corrano paralleli [ 30 , 31 ] e abbiamo gi descritto anomalie come agenesia / ipoplasia polmonare , sequestro e sindrome della scimitarra . 
facciamo ora un accenno alle compressioni e dislocazioni dellalbero tracheo - bronchiale causate da anomali rapporti anatomici , come nel caso del doppio arco aortico [ 54 ] , o dalla dilatazione del cuore o dei vasi polmonari nellambito di cardiopatie congenite . 
a nostro parere da preferire la mdct quando si ritenga necessaria una valutazione anche del parenchima polmonare e delle vie aeree , e nel caso di paziente critico che necessiti di tempi brevi di scansione che evitino sedazione o anestesia . 
la rm ideale per le condizioni che richiedano studi seriati , screening o valutazione flussi , funzioni biventricolari , anatomia intracardiaca [ 16 ]  . conclusions conclusioni thanks to isotropic resolution and the multiplanar capabilities of mdct , which significantly increase diagnostic confidence even in the most complex cases , the indications for ct in paediatric chest imaging have been significantly extended . 
in the setting of pulmonary malformations , the wide range of associated anomalies requires a multisystem evaluation , an area in which mdct is gaining a key role as a noninvasive approach . 
the maxim : always look at the airway must constantly be borne in mind . la mdct grazie alla risoluzione isotropica e alle conseguenti possibili ricostruzioni multiplanari , che rinforzano significativamente la confidenza diagnostica anche nei casi pi complessi , ha incrementato in modo significativo le indicazioni della tc nellimaging del torace pediatrico . 
intravascular contrast media used in diagnostic imaging are drugs in the complete sense of the term , even though they have unique characteristics which in many ways distinguish them from other pharmacological agents . 
the off - label use of contrast media in diagnostic imaging is a little - investigated field and most commonly , but not exclusively , applies to gadolinium - based contrast media used in mr angiography as well as cardiac and paediatric applications . 
as contrast media are to all effects pharmaceutical agents , their off - label use can be considered admissible within the limitations laid down by the italian law in force ( article 3 of law 94 / 98 ) and its interpretation , i.e. 
the use of contrast media in modern imageriassunto scopo del nostro studio stato analizzare lutilizzo offlabel dei mezzi di contrasto ( mdc ) organo - iodati e per risonanza magnetica ( rm ) utilizzati in diagnostica per immagini e valutare i profili di responsabilit ed i risvolti medico - legali che possono derivare da tale impiego . il termine off - label indica , per lo pi , lutilizzo di farmaci conosciuti , ma per i quali nuove evidenze scientifiche propongono un loro uso con modalit ed in situazioni cliniche non espressamente previste nella scheda tecnica ed al di fuori dei limiti di autorizzazione allimmissione in commercio . 
i mezzi di contrasto endovascolari utilizzati in diagnostica per immagini sono farmaci a tutti gli effetti , seppure con caratteristiche uniche che per molti versi li distinguono dagli altri preparati farmacologici . 
lutilizzo off - label dei mezzi di contrasto in diagnostica per immagini risulta un campo ancora poco esplorato che trova pi spesso , ma non solo , applicazione tra gli agenti di contrasto a base di gadolinio usati in angio - rm , in applicazioni cardiache e pediatriche . 
in cases in which the information contained in the information leaflet is modified and updated in any way whatsoever ( indications , dosage , at others ) , specifically if restrictions are introduced in accordance with the law in force , the pharmaceutical industry must provide formal and timely notification to radiologists . 
laddove vengano modificati ed aggiornati in qualsiasi senso ( indicazione , posologia , ecc . ) , specificatamente in senso restrittivo , gli elementi riportati nel foglio illustrativo , ai sensi delle normative vigenti , lindustria deve darne tempestiva e formale comunicazione ai medici radiologi . 
si propone , infine che nella refertazione venga segnalato non solo il tipo ma anche la dose di mdc utilizzato . parole chiave mezzi di contrasto indicazioni introduction introduzione the prescription and administration of drugs outside their approved indications , route of administration or use , which is defined as off - label , is still an open question in medical and legal settings . 
the term off - label generally indicates the use of known drugs for which new scientific evidence suggests use in a manner and in clinical scenarios not explicitly addressed by the drug data sheet and is outside the indications for which the drug was approved . however , the term off - label also indicates the use of drugs with a different route of administration and dosage from those indicated in the information leaflet . 
in some cases off - label prescriptions provide a valid therapeutic alternative for diseases that do not respond to current treatment . in addition , compassionate drug use is defined as the use of nonapproved pharmaceuticals in a group of patients affected by a life - threatening , chronic or severely disabling disease and who cannot be treated satisfactorily with an approved medication . 
il termine off - label indica , per lo pi , lutilizzo di farmaci conosciuti ma per i quali nuove evidenze scientifiche propongono un loro uso con modalit ed in situazioni cliniche non espressamente previste nella scheda tecnica ed al di fuori dei limiti di autorizzazione allimmissione in commercio . 
648 introduce per la prima volta nellordinamento la possibilit di prescrivere e utilizzare farmaci al di fuori delle indicazioni terapeutiche . scopo di questo lavoro quello di analizzare lutilizzo off - label dei mezzi di contrasto ( mdc ) organo - iodati e per risonanza magnetica ( rm ) utilizzati in diagnostica per immagini e quello di valutare i profili di responsabilit ed i risvolti medico - legali che possono derivare da tale impiego . radiol med ( 2011 ) 116 : 114 characteristics that in many ways distinguish them from other pharmacological agents . regulatory framework definitions as laid down in italian legislative decree no . 
i , article 1 , a drug product or drug is defined as : any substance or compound presented as having curative or prophylactic properties of human diseases ; any substance or compound which can be used in humans or administered to humans with the aim of restoring , correcting or modifying physiological functions , by exerting an immunological or metabolic action , or used to establish a medical diagnosis . 
 ( therefore , the definition fully includes not only the iodinated organic and mr contrast media considered in this paper , but also all other contrast media oral and sonographic and radiotracers . ) intravascular contrast media are pharmaceuticals with special characteristics . 
multidetector computed tomography ( mdct ) ] , almost always with high concentrations and without the aim of producing pharmacological effects . conditions for the use of drugs an approved medication is defined as a reference - listed drug . 
a generic drug is a drug that is bioequivalent to a reference drug with an expired patent and is approved with identical amounts of the same active ingredients , the same formulation / presentation , the same route of administration and the same treatment indications . 
the term generic has proven to be unfortunate in that it is perceived by the general public to mean similar but not the same as the reference drug for the same condition . 
24 april 2006 contains detailed regulations regarding the procedures and the phases of launching and maintaining drugs on the e tanto perch , come preciseremo in seguito , i mezzi di contrasto endovascolari utilizzati in diagnostica per immagini sono farmaci a tutti gli effetti , seppure con caratteristiche uniche che per molti versi li distinguono dagli altri preparati farmacologici . la normativa definizioni ai sensi del dl 24 aprile 2006 , n . 
1 , si definisce prodotto medicinale o medicinale : ogni sostanza o associazione di sostanze presentata come avente propriet curative o profilattiche delle malattie umane ; ogni sostanza o associazione di sostanze che possa essere utilizzata sulluomo o somministrata alluomo allo scopo di ripristinare , correggere o modificare funzioni fisiologiche , esercitando unazione immunologica o metabolica , ovvero di stabilire una diagnosi medica ( quindi tale definizione comprende a pieno diritto non solo i mezzi di contrasto organo - iodati e per rm , oggetto di questo lavoro , ma tutti i mezzi di contrasto digestivi , ecografici ed i radio farmaci )  . i mezzi di contrasto endovascolari sono farmaci con caratteristiche particolari : sono , infatti , farmaci iniettati spesso in larghe dosi ed a velocit estremamente rapida , con limpiego degli iniettori automatici ( ad esempio in tomografia computerizzata multidetettore [ tcmd ] ) , quasi sempre a concentrazioni elevate e non hanno lobiettivo di determinare effetti farmacologici . condizioni di utilizzabilit dei farmaci un medicinale autorizzato viene definito medicinale di riferimento . 
si definisce medicinale generico un medicinale che bioequivalente rispetto ad un medicinale di riferimento , con brevetto scaduto , autorizzato con la stessa composizione quali - quantitativa in principi attivi , la stessa forma farmaceutica , la stessa via di somministrazione e le stesse indicazioni terapeutiche . 
per questa ragione i prodotti generici sono stati ridefiniti medicinali equivalenti ( legge [ l ] 149 del 26 luglio 2005 ) [ 2 ]  . nessun medicinale pu essere immesso in commercio sul territorio nazionale senza aver ottenuto lautorizzazione allimmissione in commercio ( aic ) dallagenzia italiana del farmaco ( aifa ) o unautorizzazione comunitaria a norma del regolamento ( ce ) n . 
219 contiene una dettagliata disciplina relativa alle procedure ed alle fasi di immissione e mantenimento in commercio dei farmaci , disciplinando , non solo le modalit di etichettatura del farmaco e di redaradiol med ( 2011 ) 116 : 114 market , regulating not only the labelling of drugs and the format and contents of the information leaflet , but also modifications regarding drugs that have already received marketing approval and suspension of the marketing of a drug . whereas the reader should refer to the full text of the law in question for a complete listing of all of regulatory points , it seems appropriate , given the aims of this paper , to emphasise that this law provides a detailed regulatory framework regarding the conditions for marketing drugs and the considerations to adopt in terms of safeguarding public health , by laying down specific obligations for the marketing authorisation holder ( which cover drug labelling , the format and content of the information leaflet ) and regulating the manner of disposing of stocks or withdrawing products from the market in the event of approval of modifications with respect to the original prescriptions . 
 zione del foglio illustrativo , quanto anche le ipotesi di modifiche relative ai farmaci di cui sia stato gi autorizzato il commercio e di sospensione della commercializzazione del farmaco . nel rinviare al testo integrale della legge per una esaustiva e puntuale elencazione di tutti i dettami normativi , appare opportuno stante la finalit del presente lavoro evidenziare che con il citato provvedimento normativo viene dettata una dettagliata disciplina in ordine alle condizioni di commercializzazione dei farmaci ed agli accorgimenti da adottare a tutela della salute pubblica , sia prescrivendo specifici obblighi per il titolare dellaic ( tra i quali , letichettatura dei farmaci , le modalit di redazione del foglietto illustrativo ed il contenuto dello stesso ) , sia disciplinando le modalit di smaltimento delle scorte o di ritiro delle confezioni nelle ipotesi di autorizzazione di modifiche rispetto alle prescrizioni originarie . 
 off - label use of drugs off - label use may include a use not present in the product characteristics , or when the clinical indication has been approved but the dosage and route of administration are different from those registered . 
the off - label use of a drug is a risk activity ( with possible consequences for the physician of both a regulatory and civil and / or criminal nature ) that is possible only under certain conditions . 
 the following conditions are , in fact , indispensable : provision of information for the patient and formal consent ; inability of satisfactorily treating the patient with already tested drugs ( and therefore also contrast media ) in rigorous adherence to the specifications of the information leaflet ; an authoritative and documented scientific basis of international standing that supports and endorses the off - label treatment choice [ 5 , 6 ]  . off - label can be classified into : off - evidence : regardless of the indication , there is insufficient information available on safety , activity and efficacy of the drug to establish the riskbenefit ratio ; absolute off - label : the prescription of a drug for a nonapproved indication ; relative off - label : when the indication is approved for the disease in question but the dosage or route of administration or duration are different from those approved [ 7 ]  . uso dei farmaci off - label luso off - label si pu verificare per un impiego non presente nella scheda tecnica , oppure quando lindicazione clinica autorizzata , ma il dosaggio e le modalit di somministrazione sono diverse rispetto a quanto indicato nella registrazione . 
luso off - label di un farmaco unattivit a rischio ( dalla quale possono , peraltro , derivare conseguenze per il medico sia disciplinari , sia civili e / o penali ) possibile solo a determinate condizioni . 
 fin da ora si indica che indispensabile che ricorrano le seguenti condizioni : linformazione al paziente e la formale raccolta del consenso ; limpossibilit di trattare utilmente il paziente attraverso i farmaci ( e quindi anche i mezzi di contrasto ) gi sperimentati , nel rigoroso rispetto di quanto riportato nel foglio illustrativo ; un autorevole documentato fondamento scientifico , di livello internazionale , che sostenga ed avalli la scelta terapeutica off - label [ 5 , 6 ]  . possibile classificare lutilizzo loff - label in : off - evidence : indipendentemente dallindicazione , le informazioni a disposizione sulla sicurezza , attivit ed efficacia del farmaco non sono sufficienti a documentare il rapporto rischio - beneficio ; off - label assoluto : consiste nella prescrizione di un farmaco per un indicazione non prevista ; off - label relativo : quando lindicazione esiste per quella malattia ma , ad esempio , si usa un dosaggio diverso o una diversa via di somministrazione o una durata non prevista [ 7 ]  . regulations the off - label use of drugs has over time been regulated , and although the framework is not systematically strucla pratica di utilizzo dei farmaci off - label ha trovato nel riferimenti normativi radiol med ( 2011 ) 116 : 114 tured , it does have some relatively well - defined components . 
prior to 1998 , the subject was regulated by the general principle of professional responsibility : the physician was free to prescribe any drug to resolve any condition whenever she or he thought it appropriate for the health of the patient . 
23.12.1996 [ 9 ] , introduced into the italian legal system for the first time the possibility of prescribing and using drugs charged to the national health service ( nhs ) outside their indications approved by the regulatory body . 
in particular , article 1 , paragraph 4 of the law states that : ...whenever there is no valid ( therapeutic ) alternative , the following drugs may be fully charged to the national health service : innovator drugs approved for use in other countries but not in italy , drugs undergoing clinical trial but not yet approved and drugs used for a ( therapeutic ) indication different from the approved indication . 
these drugs shall be included in a list which shall be periodically updated by the national drug evaluation board [ commissione unica del farmaco ( cuf ) ] in compliance with the procedures and criteria adopted by the board . the list of drugs consists of medications for which phase ii trials have been conducted and also contains provisions indicating the conditions and manner of use of each individual drug . 
the request to include a drug in the list can be made by the technical scientific committee [ commissione tecnico scientifica ( cts ) ] of aifa or upon request by various associations , scientific bodies , hospital trusts , universities and scientific institutions . 
the request requires that these bodies deliver to the cts detailed documentation demonstrating the seriousness of the disease and the lack of valid treatment options or diagnostic alternatives in the case of contrast media the description of the proposed treatment plan , data indicating the likely cost incurred by the patient for 1 months treatment or for a cycle of treatment , approval of the drug in italy or abroad and all scientific documentation with the relevant clinical data . 
similarly important is the information that needs to be provided by the physician to the patient to obtain written consent , which should state that the patient is aware of the possible risks and benefits of the proposed treatment . 
648 [ 9 ] , viene introdotta per la prima volta nel nostro ordinamento la possibilit di prescrivere e utilizzare , a carico del sistema sanitario nazionale ( ssn ) , farmaci al di fuori delle indicazioni approvate dallautorit regolatoria . 
1 , comma 4 della legge , dispone testualmente che qualora non esista valida alternativa ( terapeutica ) , sono erogabili a totale carico del ssn i medicinali innovativi la cui commercializzazione autorizzata in altri stati ma non sul territorio nazionale , i medicinali non ancora autorizzati ma sottoposti a sperimentazione clinica e i medicinali da impiegare per unindicazione ( terapeutica ) diversa da quella autorizzata , inseriti in apposito elenco predisposto e periodicamente aggiornato dalla commissione unica del farmaco ( cuf ) conformemente alle procedure ed ai criteri adottati dalla stessa . lelenco dei farmaci composto da medicinali che alla loro base hanno studi clinici di fase ii e contiene , anche , provvedimenti e determinazioni attraverso le quali vengono indicate quali sono le condizioni e le modalit duso dei singoli medicamenti . 
 la richiesta di inserimento di un farmaco nella lista , pu avvenire per iniziativa della commissione tecnico scientifica ( cts ) dellaifa o tramite la richiesta da parte di associazioni varie , societ scientifiche , aziende sanitarie , universit e tutte quelle strutture a carattere scientifico . 
per richiedere linserimento necessario che questi enti facciano pervenire alla cts una documentazione articolata , nella quale viene dimostrata la gravit della patologia e lassenza di valide alternative terapeutiche e di alternative diagnostiche in caso di mdc , la descrizione del piano terapeutico proposto , i dati indicativi del costo del trattamento mensile o per ciclo di terapia per paziente , lautorizzazione del medicinale in italia o allestero e tutta la documentazione scientifica con relativi dati clinici . 
di natura altres importante linformazione che deve essere fornita dal medico al paziente per poi acquisire il consenso per iscritto da parte del paziente , dove viene espresso che il paziente a conoscenza degli radiol med ( 2011 ) 116 : 114 april 1998 ( known as the di bella law ) [ 10 ] codified the principle according to which the therapeutic activity of the physician is only fully legitimate when the drug has been previously approved by the regulatory body for the same route of administration , dosage or indication for which it is effectively prescribed to the patient . 
article 3 of this law , in fact , explicitly lays down the conditions according to which drugs may be prescribed under direct responsibility of the physician for indications not contemplated by the drug data sheet : compliance with the approved therapeutic indications article 3 , paragraph 1 : except for the provisions of paragraphs 2 and 3 , when prescribing a speciality medicine or other industrially produced medication , the physician shall comply with the therapeutic indications , the route of administration and dosage outlined in the marketing authorisation issued by the ministry of health . article 3 , paragraph 2 : in individual cases the physician may , under her / his own responsibility and after having informed the patient and having received their consent , use an industrially produced medication for an indication or route of administration or use other than those approved , i.e. 
23 december 1996 , if the physician herself / himself believes , on the basis of documented data , that the patient cannot be satisfactorily treated with drugs and therefore with contrast media for which that therapeutic indication or route of administration or dosage has already been approved and provided that the use is known and in compliance with studies appearing in internationally accredited scientific publications . therefore , this regulation outlines the degree of discretion available to the physician in her / his clinical evaluation provided that international scientific publications are available that confirm that the proposed use is already known and has already been tested . 
in addition to this regulation , it should be noted that the off - label use of drugs is also regulated by the general prescription of the professional code of physicians [ 11 ] , in particular , section iv : diagnostic examinations and therapeutic treatment , article 12 : prescription and therapeutic treatment : ...the prescription of a diagnostic examination and / or treatment involves the professional and ethical responsibility of the physician and must follow an evidence - based diagnosis , or at least a well - founded diagnostic suspicion . 
given this requirement , the physician is free to plan , choose and apply any diagnostic and treatment aid even on an in - patient basis , notwithstanding the freedom of the patient to refuse and accept the responsibility for that refusal... 
94 ( cosiddetta legge di bella ) [ 10 ] ha invece trovato codificazione normativa il principio secondo il quale lattivit curativa del medico reputata pienamente legittima soltanto allorquando il medicinale sia stato preventivamente autorizzato dallautorit regolatoria per le medesime modalit di somministrazione , dosaggi o indicazioni terapeutiche per le quali effettivamente prescritto al paziente . 
larticolo 3 della suddetta legge stabilisce infatti espressamente le condizioni alle quali consentito , sotto diretta responsabilit del medico prescrivere farmaci per indicazioni non contemplate nella scheda tecnica : osservanza delle indicazioni terapeutiche autorizzate : 1 . 
fatto salvo il disposto dei commi 2 e 3 , il medico , nel prescrivere una specialit medicinale o altro medicinale prodotto industrialmente , si attiene alle indicazioni terapeutiche , alle vie e alle modalit di somministrazione previste dallautorizzazione allimmissione in commercio rilasciata dal ministero della sanit . 
in singoli casi il medico pu , sotto la sua diretta responsabilit e previa informazione del paziente e acquisizione del consenso dello stesso , impiegare un medicinale prodotto industrialmente per unindicazione o una via di somministrazione o una modalit di somministrazione o di utilizzazione diversa da quella autorizzata , ovvero riconosciuta agli effetti dellapplicazione dellarticolo 1 , comma 4 , del decreto - legge 21 ottobre 1996 , n . 
536 , convertito dalla legge 23 dicembre 1996 , n . 648 , qualora il medico stesso ritenga , in base a dati documentabili , che il paziente non possa essere utilmente trattato con medicinalie quindi anche mezzi di contrasto per i quali sia gi approvata quella indicazione terapeutica o quella via o modalit di somministrazione e purch tale impiego sia noto e conforme a lavori apparsi su pubblicazioni scientifiche accreditate in campo internazionale . 
 quindi questa norma si premura di delineare i margini di discrezionalit consentiti al medico nella sua valutazione clinica , a condizione che esistano pubblicazioni scientifiche accreditate in campo internazionale , che confermino come limpiego proposto gi noto e sperimentato . 
pur prendendo atto del dato normativo deve tuttavia rilevarsi che lutilizzo off - label dei farmaci regolamentata anche dalle regole e prescrizioni generali dettate dal codice deontologico dei medici [ 11 ] , in particolare al capo iv accertamenti diagnostici e trattamenti terapeutici , allart . 
12 prescrizione e trattamento terapeutico stabilisce che la prescrizione di un accertamento diagnostico e / o di una terapia impegna la responsabilit professionale ed etica del medico e non pu che far seguito a una diagnosi circostanziata o , quantomeno ad un fondato sospetto diagnostico . 
su tale presupposto al medico riconosciuta autonomia nella programmazione , nella scelta e nellapplicazione di ogni radiol med ( 2011 ) 116 : 114 and not approved is allowed provided their efficacy and tolerability have been scientifically documented . 
in these cases , once written consent has been provided by the informed patient , the physician accepts responsibility for the treatment and s / he is held to monitor its effects . 
the physician is obliged to immediately inform the competent bodies of any adverse reactions which may arise during the treatment . off - label use of intravascular gadolinium - based and iodinated organic contrast media and adverse reactions in diagnostic imaging , the off - label use of contrast media [ 12 ] mostly involves gadolinium - based contrast media , especially in mr angiography , cardiac and paediatric applications [ 13 , 14 ] and patients younger than 2 years [ 15 ]  . 
the off - label use must be based on scientific evidence , serve the patients best interest , and requires greater care than would generally be the case in medicalradiological practice [ 16 ]  . 
nephrotoxicity from gadolinium chelates has been documented in both animals and humans , and nephrotoxic effects associated with high doses has been reported following the intra - arterial administration of gadolinium chelates [ 17 ]  . 
given their hypothesised low level of nephrotoxicity ( due to the small osmolar load ) in the past , gadolinium derivatives have been used to substitute iodinated organic contrast media for radiographic and mdct examination in patients at risk of nephrotoxicity from iodinated organic agents [ 15 , 18 , 19 ]  . however , the quality of the images of radiographic examinations obtained with gadolinium - based contrast media is lower than the image quality obtained with iodine - based agents [ 20 ]  . 
in order to deal with the inferior image quality , data regarding the relative toxicity of gadoliniumbased contrast media were extrapolated to the doses used in mr examinations to use them in radiographic procedures [ 21 ]  . 
this fact , together with the suggested role of gadolinium in nephrogenic systemic fibrosis ( nsf ) , prompted the european society of urogenital radiology ( esur ) to contraindicate their use for radiographic procedures [ 22 ]  . 
the use of double or even triple doses of gadolinium - based contrast media ( off - label use ) required in mr angiography procedures may expose patients to greater risk [ 23 , 24 ]  . 
the relationship between gadolinium - based contrast media and nsf has been reported not only in a dose - dependent manner but also in relation to the type of contrast medium presidio diagnostico e terapeutico anche in regime di ricovero , fatta salva la libert del paziente di rifiutare e di assumere la responsabilit del rifiuto stesso . 
obbligo del medico segnalare tempestivamente alle autorit competenti , le reazioni avverse eventualmente comparse durante un trattamento terapeutico . utilizzo off - label dei mezzi di contrasto a base di gadolinio e organoiodati per via iniettiva e reazioni avverse in diagnostica per immagini lutilizzo off - label dei mezzi di contrasto [ 12 ] trova applicazione con maggiore frequenza per quanto riguarda gli agenti di contrasto a base di gadolinio ( gd ) , e segnatamente in angiografia mediante rm , in applicazioni cardiache e pediatriche [ 13 , 14 ] , ed in pazienti di et inferiore ai due anni [ 15 ]  . 
luso off - label si deve basare sulle evidenze scientifiche , utilizzate nel miglior interesse del paziente , e richiede prudenza maggiore di quella che deve essere comunque esercitata nella pratica medico - radiologica in generale [ 16 ]  . 
la nefrotossicit dei chelati di gadolinio stata documentata in uomini ed animali e lincidenza di effetti nefrotossici riportata per lutilizzo di alte dosi , a seguito di somministrazione intra - arteriosa dei chelati di gadolinio [ 17 ]  . 
per la loro ipotizzata bassa nefrotossicit ( dovuta al minimo carico osmolare ) in passato si segnala lutilizzo dei derivati di gadolinio come sostituti dei mdc organoiodati per esami radiografici e tcmd , in pazienti a rischio di nefropatia da contrasto organo - iodati [ 15 , 18 , 19 ]  . 
ma la qualit delle immagini di esami radiografici ottenuta con agenti a base di gadolinio inferiore rispetto alla qualit delle immagini ottenute con mdc a base di iodio [ 20 ]  . 
per far fronte a tale inferiore qualit di immagini , si estrapolarono i dati relativi alla tossicit dei mdc a base di gadolinio alle dosi utilizzate per esami rm , per impiegarli in procedure radiografiche [ 21 ]  . 
essi infatti per avere una migliore capacit di attenuazione del fascio radiante devono essere somministrati a concentrazioni ed a dosi pi elevate che sono , per , anche pi nefrotossiche . 
lutilizzo di dosi doppie o addirittura triple di mdcgd ( uso off - label ) richieste in procedure di angio - rm possono esporre i pazienti ad un maggiore rischio [ 23 , 24 ]  . perci consigliato in linea generale limpiego della dose pi bassa compatibile con le esigenze diagnostiche di gd per evitare anche il rischio di nsf . 
la relazione tra agenti radiol med ( 2011 ) 116 : 114 used on the basis of its physical and chemical properties . this suggests the clear need to avoid inappropriate use , excessive doses , and the need to select the contrast medium with the most appropriate characteristics for the clinical case [ 25 ]  . 
this is in line with the emea press release of 20.11.2009 circulated by aifa [ 26 ]  . apart from the risk of immediate adverse reactions , one of the pathological conditions of greatest concern produced by iodinated organic contrast media is contrastinduced nephropathy ( cin )  . 
the definition of cin states that within 4872 h from the administration of the contrast medium , there is an increase of at least 25% or at least 0.5 mg / dl of creatininaemia with respect to the baseline value [ 27 ]  . 
after having reached a peak in the third / fourth day after injection of contrast medium , creatininaemia falls and returns to baseline values within 1015 days [ 17 , 28 ]  . 
the issue is still wide open , considering also the fact that some authors [ 32 ] have suggested that so - called cin can be induced even without administration of contrast mediu the nephrotoxic potential of various contrast media fundamentally depends on the dose administered . 
it is therefore understandable why the reported incidence of cin varies from values of 1%2% in patients with normal kidney function to 11% in hospitalised patients and up to 20%50% in some at - risk categories ( end - stage kidney failure and diabetes mellitus ) [ 33 , 34 ]  . although the nephrotoxicity of iodinated organic contrast media with normal kidney function is very low , it should be borne in mind that nephrotoxicity is dose dependent ; therefore , the risk of kidney damage is directly proportional to the dose of contrast medium injected [ 35 ]  . with regard to the type of iodinated organic contrast medium , it should be emphasised that data in the literature are not fully in agreement . 
therefore , at present , it is best to assume that all nonionic contrast media , both low osmolarity and iso - osmolar types , can further deteriorate kidney function in patients with impaired kidney function [ 21 ]  . as high doses of iodinated organic contrast media can trigger the onset of cin , caution in the off - label use of such media is advisable . 
even gadolinium - based contrast media can induce nephrotoxicity , especially in high doses in patients with kidney failure , because the pharmacokinetics are very similar to those of iodinated organic contrast media [ 3638 ]  . di contrasto a base di gd e nsf segnalata non solo dose dipendente , ma anche legata al tipo di agente di contrasto impiegato , in base alle caratteristiche fisico - chimiche : pertanto occorre evitare logicamente luso inappropriato , dosi eccessive , selezionando lagente di contrasto con le caratteristiche pi appropriate per il caso clinico [ 25 ]  . 
e tanto anche ai sensi del comunicato stampa emea del 20.11.2009 diffuso dallaifa [ 26 ]  . al di l del rischio di reazioni avverse immediate , una delle condizioni patologiche di maggior interesse indotto dai mdc organo - iodati la nefropatia da mdc . 
la contrast induced nephropahy ( cin ) o nefropatia da mdc organoiodati , definita in genere come un deterioramento acuto della funzione renale susseguente alla somministrazione di mdc in assenza di altre possibili cause di insufficienza renale acuta . 
la definizione di nefropatia da mdc prevede che dopo 4872 ore dalla somministrazione del mdc si verifichi un aumento almeno del 25% o almeno di 0 , 5 mg / dl della creatininemia rispetto al valore basale [ 27 ]  . 
dopo aver raggiunto il picco in terza / quarta giornata dalliniezione del mdc , la creatininemia scende e torna ai valori basali in 1015 giorni [ 17 , 28 ]  . 
la cin rappresenta la terza causa ( 11% ) dei casi di insufficienza renale acuta nei pazienti ospedalizzati , dopo lipoperfusione renale e linsufficienza renale postchirurgica [ 29 ]  . 
comprensibile perci il motivo per cui lincidenza della cin riportata variare da valori di 1%2% in pazienti con normale funzionalit renale all11% nei pazienti ospedalizzati fino al 20%50% in alcune categorie a rischio ( ir allo stato terminale e diabete mellito ) [ 33 , 34 ]  . nonostante la nefrotossicit da mdc organo - iodati in pazienti con normale funzionalit renale sia molto bassa , bene tenere presente che la nefrotossicit dose dipendente , quindi il rischio di danno renale tanto maggiore quanto maggiore la dose di mdc iniettata [ 35 ]  . 
per quanto riguarda il tipo di mdc organo - iodato bene sottolineare che i dati in letteratura non sono univoci : pertanto , al momento attuale bene considerare che tutti i mdc non ionici , sia a bassa osmolarit che iso - osmolari , possono deteriorare ulteriormente la funzionalit renale in pazienti con funzionalit renale gi compromessa [ 21 ]  . 
anche gli agenti di contrasto a base di gd possono indurre nefrotossicit specie ad alte dosi in pazienti in insufficienza renale poich la loro farmacocinetica molto simile a quella dei mdc organo - iodati [ 3638 ]  . radiol med ( 2011 ) 116 : 114 role of the ethics committee for the off - label use of drugs ( and for compassionate use ) ruolo del comitato etico per lutilizzo dei farmaci offlabel ( e per uso compassionevole ) the establishment of an italian national committee for bioethics followed resolution no . 
6 - 00038 , approved on 5 july 1988 by the assembly of the chamber of deputies at the end of a debate on the problems of life that saw the government involved in promoting a dialogue , at both the national and international level , on the state of biomedical and genetic engineering research with a view to ensuring respect for human dignity and freedom [ 39 ]  . 
the committee has been entrusted with the task of guiding the legislative and administrative instruments required for defining the criteria to use in medical and biological practice to protect human rights . 
it also has the task of guaranteeing correct information for public opinion on the problematic aspects and the implications of therapy , diagnostic techniques and progress in the biomedical sciences . 
the opinion expressed by the ethics committee is compulsory and binding for beginning clinical testing , which nonetheless can only begin with approval from the executive directors of the hospital trust . as with all healthcare treatment , informed consent is a fundamental prerequisite for performing diagnostic and therapeutic activity , and even more so for experimental studies . 
therefore , the physician prescribing the drug and in the specific case the radiologist , with indications other than those approved in terms of dosage , frequency of administration , duration or route of administration is directly responsible for the off - label use of the drug , informing the patient and obtaining informed consent as well as for monitoring the patient throughout the treatment . a special aspect regards the off - label use of drugs such as contrast media in emergency conditions . 
in these cases , where there is the so - called state of need , it is the radiologist who takes responsibility for the use of the drugs with dosage and route of administration that , based on both science and conscience , she or he believes is necessary for the individual clinical case . listituzione del comitato nazionale per la bioetica ha fatto seguito alla risoluzione n . 
6 - 00038 , approvata il 5 luglio 1988 dallassemblea della camera dei deputati al termine di un dibattito su i problemi della vita , nella quale il governo veniva impegnato a promuovere un confronto , anche a livello internazionale , sullo stato della ricerca biomedica e dellingegneria genetica nella prospettiva e nel rispetto della libert umana [ 39 ]  . 
ha , inoltre , il compito di garantire una corretta informazione dellopinione pubblica sugli aspetti problematici e sulle implicazioni dei trattamenti terapeutici , delle tecniche diagnostiche e dei progressi delle scienze biomediche . come gi detto , i farmaci disponibili in italia vengono autorizzati allimmissione in commercio dallaifa . 
pertanto il medico prescrittore , e quindi nel caso specifico il medico radiologo ai fini diagnostici , di farmaci con indicazioni diverse da quelle autorizzate , per dosaggio , frequenza di somministrazione , durata o via di somministrazione direttamente responsabile delluso offlabel del farmaco , dellinformazione al paziente e dellacquisizione del consenso informato da parte dello stesso nonch del monitoraggio durante tutto il trattamento . 
un aspetto particolare riguarda leventuale impiego off - label di farmaci , quali i mezzi di contrasto in urgenza indifferibile / emergenza : in questi casi , laddove si configuri il cosiddetto stato di necessit , il medico radiologo che si assume la responsabilit dellimpiego degli stessi farmaci a dosaggio e via di somministrazione che , in scienza e coscienza , riterr utili per il singolo caso clinico . medicolegal aspects : liability of the radiologist in the off - label use of contrast media aspetti medico - legali : la responsabilit del medico radiologo nellimpiego off - label dei mezzi di contrasto the main problem for the radiologist regarding the off - label use of contrast media is undoubtedly related to the liability profiles [ 40 ] resulting from this practice . 
94 / 1998 establishes that when prescribing and therefore using a speciality medicine , the physician shall comply with the therapeutic indications , the route of administration and dosage outlined in the marketing authorisation issued by the ministry of health . 
3 il problema principale per il medico radiologo relativo allutilizzo off - label dei mezzi di contrasto , indubbiamente , legato ai profili di responsabilit [ 40 ] conseguenti alla applicazione di tale pratica . 
come detto , la legge 94 / 1998 stabilisce che il medico nel prescrivere , e quindi nellimpiegare , una specialit medicinale si attiene alle indicazioni terapeutiche , alle vie e alle modalit di somministrazione previste dallautorizzazione allimmissione in commercio rilasciata dal ministero della sanit . 
al comma radiol med ( 2011 ) 116 : 114 of that law establishes that in individual cases , the physician may , under her or his own responsibility and after having informed the patient and having received their consent , use an industrially produced medication for an indication or route of administration or use other than those approved , i.e. recognised in terms of the application of article 1 , paragraph 4 of italian legislative decree no . 
23 december 1996 , if the physician herself / himself believes , on the basis of documented data , that the patient cannot be satisfactorily treated with drugs and therefore with contrast media for which that therapeutic indication or route of administration or dosage has already been approved and provided that the use is known and in compliance with studies appearing in internationally accredited scientific publications . 
 anomalous behaviour , so to speak , by the radiologist must be limited to specific and individually defined hypotheses prompted by the benefit that the patient will presumably enjoy [ 41 ]  . 
648 / 98 that provides for the lawful prescription of nonapproved drugs on the condition that they are already included in the list drawn up by the cuf given the lack of alternative therapies . 
 in addition to creating a regulatory framework for the prescription of drugs outside the approved indications , the law also established the applicable sanctions for the physician who prescribes and / or uses a drug off - label outside the regulations . 
94 / 98 states that the violation by the physician of the dispositions of this article is cause for disciplinary proceedings in accordance with the legislative decree of provisional head of state dd . 
233. in addition to the implicit legal liability of professional activities performed under the supervision of a professional body , the off - label use of drugs entails even more complex forms of civil and criminal liability for the physician who chooses to carry out this practice . 
in fact , in the absence of a clear , organised and systematic regulatory framework , the outlines and limitations to legitimate off - label use of drugs have been traced through the interpretation of the courts [ 41 ]  . 
 the assumption of direct liability for pharmacological treatment and / or diagnostic activity and the preliminary identification of scientific evidence supporting off - label use of a drug are still not enough to satisfy all the obligations a physician must meet for the legitimate use of offlabel drugs . 
3 di tale legge , dispone che in singoli casi il medico pu , sotto la sua diretta responsabilit e previa informazione del paziente e acquisizione del consenso dello stesso , impiegare il medicinale / farmaco prodotto industrialmente per un indicazione o una via di somministrazione o una modalit di somministrazione o di utilizzazione diversa da quella autorizzata , ovvero riconosciuta agli effetti dellapplicazione dellart . 
648 , qualora il medico stesso ritenga , in base a dati documentabili , che il paziente non possa essere utilmente trattato con medicinali per i quali sia gi approvata quella indicazione terapeutica o quella via o modalit di somministrazione e purch tale impiego sia noto e conforme a lavori apparsi su pubblicazioni scientifiche accreditate in campo internazionale . 
 la condotta , per cos dire , anomala , da parte del medico radiologo deve essere circoscritta in relazione ad ipotesi specifiche ed individualmente definite , sulla base dei criteri dettati dal beneficio che ci si potrebbe presumibilmente attendere per il paziente [ 41 ]  . 
1 comma 4 l 648 / 98 in cui prevede la lecita prescrizione di farmaci non autorizzati a condizione che siano gi inclusi in un elenco predisposto dalla commissione unica del farmaco in ragione dellassenza di alternative terapeutiche . 
 la vigente normativa oltre a dettare le regole cui deve soggiacere la prescrizione di medicinali al di fuori delle indicazioni autorizzate , disciplina anche le sanzioni applicabili qualora il medico contravvenga alla specifica disciplina legislativa prescrivendo e / o utilizzando un farmaco off - label al di fuori dei vincoli normativi . 
3 della legge 94 / 98 stabilisce che la violazione da parte del medico delle disposizioni del presente articolo oggetto del procedimento disciplinare ai sensi del decreto legislativo del capo provvisorio dello stato 13 settembre 1946 n 233 . al di l di profili di responsabilit disciplinare connaturali ad attivit professionali espletate sotto il controllo di un organo di categoria , dallutilizzo di medicinali off - label possono derivare ben pi complesse forme di responsabilit civile e penale per il sanitario che ricorre a tale pratica . sotto tale profilo in assenza di un dato normativo di riferimento preciso , organico e sistematico i contorni e i limiti del legittimo utilizzo di medicinali off - label sono stati delineati in via interpretativa dalla giurisprudenza di merito e , soprattutto , di legittimit [ 41 ]  . 
 lassunzione di una responsabilit diretta rispetto alla terapia farmacologica e / o allattivit diagnostica ipotizzata e la preliminare identificazione di un avallo esterno di ordine scientifico non esauriscono tuttavia gli obblighi che il medico deve soddisfare ai fini di un legittimo utilizzo off - label dei farmaci . 
accanto alla complessiva valutazione clinica , infatti , necessario che si proceda ad unappropriata informazione del paziente e allottenimento del suo consenso scritto . presupposto per la validit del consenso per un informaradiol med ( 2011 ) 116 : 114 essence must be honest . 
in particular , in the setting of the off - label use of a drug , the information should cover the characteristics of the proposed treatment , the possibility of choosing a more conventional and consolidated treatment and the side effects and adverse events directly connected with the off - label use of drugs . 
the physician must be the bearer of a simplified truth commensurate with the patients level of comprehension at that particular time and in those circumstances , taking care to describe all important issues , especially in terms of risks and benefits . 
on the other hand , whereas implicit consent may be considered admissible and understood in the doctor - patient relationship during delivery of a routine healthcare service , consent must be explicit when the chosen treatment involves the use of off - label drugs : although the law does not enforce written consent in such circumstances , the provision of such consent by the patient will have clear significance for legal purposes [ 42 ]  . the requirement for potential off - label use is that the radiologists choice , notwithstanding the above - mentioned conditions , is backed up by experimental evidence and / or scientific documents that support the rationality of the choice made , especially with reference to the possible advantages / disadvantages of the known and consolidated use for the indication in question . 
lastly , it should always be borne in mind that off - label prescribing obliges the physician to pay special attention to monitoring side effects or possible inefficacy in relation to the expected results [ 42 ]  . zione da parte del sanitario che deve essere essenzialmente onesta . 
in particolare , nel contesto dellimpiego di un farmaco off - label , linformazione deve riguardare le caratteristiche del trattamento ipotizzata , leventualit di una scelta che confermi un trattamento pi tradizionale e consolidato , ovvero gli effetti collaterali e gli eventi avversi direttamente connessi allimpiego di farmaci off - label . 
il medico deve farsi portatore di una verit semplificata e commisurata al livello di comprensione che il paziente pu avere maturato nello specifico momento storico , ambientale o psicologico , avendo cura di descrivere ogni aspetto significativo , con particolare riguardo al rapporto rischio - beneficio . 
daltra parte , mentre un consenso implicito pu considerarsi ammissibile e sottointeso alla relazione tra medico e paziente qualora la prestazione sanitaria abbia contenuti ordinari , esso dovr essere esplicitato quando il trattamento prescelta attinga ad utilizzo del farmaco non autorizzato , cio off - label : in tal caso , pur non essendovi precetto normativo che imponga la forma scritta , la sottoscrizione del consenso da parte del paziente avr certamente una chiara rilevanza ai fini probatori [ 42 ]  . il presupposto per un utilizzo eventuale in off - label che la scelta del medico radiologo , fatti salvi i presupposti suddetti , sia sostenuta da prove sperimentali e / o documenti scientifici che possono supportare la razionalit delle scelta effettuata soprattutto se riferita ai possibili vantaggi / svantaggi di impiego gi noto e consolidato per lindicazione in oggetto . 
infine , non va mai dimenticato che la prescrizione offlabel impone al sanitario una particolare cura ed attenzione nei confronti di una sorveglianza degli effetti collaterali o di un eventuale inefficacia , rispetto le aspettative attese [ 42 ]  . conclusions conclusioni recent data published in the literature suggest that , in general , the off - label use of drugs is a routine practice in 23% of prescriptions [ 12 ] and that off - label use is widely practised in various specialities to the point of being standard practice in paediatrics and intensive care . 
on the basis of scientific data , under his or her direct responsibility and after having obtained informed consent from the patient , the radiologist may decide to treat the patient with a medication or drug ( contrast medium ) produced for an indication , a dosage or route of administration other than those registered . 
whereas it may appear to be logical that a contrast medium should dai dati pi recenti della letteratura emerge che in generale luso di farmaci off - label una pratica routinaria in circa il 23% delle prescrizioni [ 12 ] e che comunque loff - label ampiamente diffuso in varie specialit rappresentando finanche una pratica standard in et pediatrica ed in terapia intensiva . 
ed a tale proposito si precisa che mancano allo stato dati precisi e sufficienti per definire lentit di questo fenomeno in diagnostica per immagini . nella nostra attivit , lopportunit di usare un farmaco , e quindi di un mezzo di contrasto , off label spetta al medico radiologo , che , sulla base di documentazione scientifica e sotto la sua diretta responsabilit , dopo aver informato il paziente e ottenuto il consenso , pu decidere di trattare il proprio assistito con un medicinale / farmaco ( mezzo di contrasto ) prodotto per una indicazione , un dosaggio ed una modalit di somministrazione diverse da quelle registrate . 
per quanto appare logico che il mezzo di radiol med ( 2011 ) 116 : 114 not be administered in the absence of a clear indication , it is worth recalling that modern image - guided medicine cannot effectively forego the use of contrast media . in light of the concept that the behaviour of different molecules is not identical in terms of microcirculation , capillary permeability and distribution haemodynamics of the various compartments and is further differentiated in terms of the organ or body region being examined , these substantial differences in behaviour suggest the preferential use of one molecule rather than another . 
as contrast media are , to all effects , pharmaceutical agents , their off - label use can be considered admissible within the limitations laid down by the italian law in force ( article 3 of law 94 / 98 ) and its interpretation , i.e. , the following criteria must be present : the lack of a valid diagnostic alternative supported by published data ; written informed consent by the patient ; the presence of scientific publications validated at the international level ; assumption of responsibility by the radiologist . of course it is logical , and also highly desirable , that if the information leaflet of a drug is modified and updated in any sense ( indication , dosage , etc . ) , in accordance with the law in force , the pharmaceutical industry shall promptly and formally inform radiologists . 
in conclusion , while emphasising that modern image - guided medicine cannot forego the use of contrast media , we propose that the radiological report indicate not only the type of contrast medium used but also the dose . contrasto non dovrebbe essere somministrato in assenza di una chiara indicazione , bene tener presente che nella medicina moderna image guided non possibile in concreto rinunciare allimpiego dei mezzi di contrasto . sottolineando il concetto che le diverse molecole hanno un comportamento non sovrapponibile nei confronti del microcircolo , della permeabilit capillare e dellemodinamica di ripartizione dei vari compartimenti , in maniera ulteriormente differenziata secondo lorgano o il distretto in esame , si precisa che queste sostanziali differenze di comportamento suggeriscono limpiego preferenziale di una molecola piuttosto che dellaltra . 
rossi1 1sezione di scienze radiologiche , dipartimento di scienze cliniche , 2sezione di medicina legale , dipartimento di anatomia umana , farmacologia e scienze medico - forensi , universit degli studi di parma , ospedale maggiore di parma , via gramsci 14 , 43100 parma , italy 3istituto di radiologia , policlinico universitario campus di germaneto , viale europa , 88100 catanzaro , italy 4radiologia cardio - toracica , dipartimento cardio - toraco - vascolare , policlinico s . 
some important weaknesses emerged , especially regarding requests for radiological examinations ( 22% lacked the clinical question , 27.6% were inappropriate ) , potentially limiting the effectiveness of the diagnostic process and leading to negative effects on the correct risk management process . 
abbiamo riscontrato una elevata percentuale di richieste inappropriate ( 27 , 6% ) e mancanti del quesito clinico ( 22% ) e una discreta precisione del dato anamnestico ( 80 , 6% ) e della formulazione del quesito diagnostico ( 76 , 8% )  . 
sono emerse importanti criticit , soprattutto nelle richieste di esami radiologici ( 22% di richieste senza quesito clinico ; 27 , 6% di richieste inappropriate ) , in grado di limitare lefficacia del processo diagnostico e comportare ripercussioni negative per la corretta gestione del riskradiol med ( 2011 ) 116 : 152162 keywords radiological request radiological report management . 
si palesa la necessit di una maggiore e migliore collaborazione tra clinico e radiologo . parole chiave richiesta radiologica referto radiologico introduction introduzione clinicalanamnestic evaluation , with an assessment of any previous examinations ; con quesito clinico ; the work of the radiologist involves similar or identical duties of documentation and certification to those of other medical specialist areas , even though diagnostic radiology appears to be characterised by a series of unique operating features that set this discipline apart . 
the radiological medical procedure is a specialist medical service having diagnostic and / or interventional aims [ 1 ] and consisting of a set of closely interdependent elements : a justified request by a referring physician that contains a clinical question ; justification for the proposed examination ( or motivated lack of justification , with possible suggestions for alternative modalities and techniques ) ; information for consent and patients consent ; technical execution of the examination and production of interpretation , reporting , communication and discussion images ; with the clinician ; image archiving . the radiologists diagnostic professionalism has two fundamental and distinct expressions : a physical medium ( the radiological finding ) and a paper - based medium ( the report ) , which must be , for the validity and usefulness of the investigation , exactly complementary to each other and together available for further analysis and possibly reproducible on demand . 
the radiologist is also still the only person responsible for producing a written report of the examination or for interpreting a radiological image with clinical and health purposes [ 2 ]  . 
the radiological report is therefore a scientific statement to be used at a scientific level ; it must be understood as an official , final and written interpretation , which must support each diagnostic imaging study and represents , together with the images produced , its final result . lattivit del radiologo comporta problematiche di documentazione e di certificazione analoghe o identiche a quelle di altri settori medico - specialistici , sebbene la radiologia diagnostica appaia contraddistinta da alcune peculiari ed esclusive necessit operative , che conferiscono a tale disciplina caratteri del tutto propri . 
latto medico radiologico rappresenta una prestazione professionale specialistica medica con finalit diagnostiche e / o interventistiche [ 1 ] , costituita da un insieme di elementi strettamente interdipendenti tra loro : motivata richiesta di prestazione del medico prescrivente inquadramento clinico - anamnestico , con valutazione di eventuali esami precedenti ; giustificazione dellesame proposto ( o non giustificazione motivata , con possibile proposta di tecniche e metodologie sostitutive ) ; informativa per il consenso e relativo consenso ; esecuzione tecnica e produzione del referto iconografico ; interpretazione / refertazione / comunicazione / discussione con il clinico ; archiviazione . la professionalit diagnostica del radiologo si concretizza attraverso due fondamentali e distinte espressioni : un supporto fisico ( il reperto radiologico ) ed un supporto cartaceo ( il referto ) , che devono necessariamente , per la validit e lutilit dellindagine compiuta , essere luno esattamente complementare allaltro ed entrambi disponibili per verifiche ulteriori , nonch eventualmente riproducibili a richiesta . 
se lesecuzione tecnica dellesame pu essere delegata al tecnico sanitario di radiologia medica ( tsrm ) , garante anche della qualit tecnica delliconografia , comunque il medico radiologo lunico responsabile cui competono , sulla base dei principi di giustificazione e ottimizzazione ( rispettivamente indicati dagli articoli 3 e 4 del d.lgs. 187 / 00 ) , la scelta della metodologia e delle tecniche idonee ad ottenere il maggior beneficio clinico con il minore detrimento individuale , nonch la valutazione sulle possibilit di utilizzare tecniche sostitutive non basate su radiazioni ionizzanti . di certo , ancora ed esclusivamente al radiologo compete lonere e la responsabilit della refertazione dellesame in forma scritta , ovvero linterpretazione dellimmagine radiologica con finalit clinico - sanitarie [ 2 ]  . 
il referto radiologico rappresenta dunque una dichiarazione di scienza finalizzata alla sua utilizzazione sul piano scientifico ; essa va intesa come 154 radiol med ( 2011 ) 116 : 152162 the aim of this study was to review a series of radiological requests and their matched radiological reports with a view to analysing their correctness and completeness , with reference to the principles presented in the literature and recommended by the italian national referral guidelines [ 3 ] as a means to achieve a reduction in the number of inappropriately prescribed and performed examinations . materials and methods we retrospectively selected 500 reports from our radiology information systempicture archiving and communications system ( ris - pacs ) and subsequently retrieved the related paper - based clinical request form from our radiological archive . 
a total of 167 computed tomography ( ct ) , 166 ultrasound ( us ) and 167 conventional radiology ( x - ray ) examinations were chosen randomly from among 3 , 100 diagnostic studies performed over 3 months and reported by six radiologists of the university hospital of parma . 
two radiologists , with 8 and 10 years experience , respectively , carried out an analysis of the requests and reports according to the parameters listed below . evaluation criteria following are evaluation criteria for radiology requests and reports , which are summarised in table 1 . table 1 criteria used to evaluate requests and reports requests reports presence / absence of the diagnostic question appropriateness of indication completeness of the information formal aspects presence / absence of answer to the diagnostic question completeness of the response given tabella 1 criteri di valutazione delle richieste e dei referti richieste referti presenza / assenza del quesito diagnostico appropriatezza dellindicazione completezza dellinformazione presenza / assenza risposta al quesito diagnostico completezza della risposta aspetti formali interpretazione ufficiale , finale e scritta , che deve accompagnare tutti gli studi di diagnostica per immagini e che , insieme alliconografia prodotta , ne costituisce il risultato . il nostro lavoro si proposto , mediante una revisione casistica di richieste cliniche e relative refertazioni radiologiche , unanalisi delle problematiche correlate alla correttezza e completezza delle richieste e dei referti radiologici , in riferimento ai principi presenti nella letteratura di merito ed a quanto suggerito dalle linee guida nazionali [ 3 ] , il cui fondamentale obiettivo proprio quello di voler perseguire una riduzione del numero di esami inappropriatamente richiesti ed eseguiti . 
 materiali e metodi abbiamo selezionato retrospettivamente dal nostro sistema informatico ( radiology information system - picture archiving and communications system , ris - pacs ) 500 referti ; successivamente sono state recuperate le rispettive richieste cliniche in cartaceo , presenti nellarchivio radiologico . sono stati scelti con metodica random , su 3100 esami diagnostici eseguiti in 3 mesi e refertati da un totale di 6 medici radiologi dellazienda ospedaliero - universitaria ( aou ) di parma , 167 tomografie computerizzate ( tc ) , 166 ecografie e 167 esami di radiologia tradizionale . 
tra questi , 343 esami erano eseguiti in regime di ricovero ospedaliero , i rimanenti 157 erano esami ambulatoriali ; complessivamente il maggior numero desami ( 80% ) stata condotto in regime di elezione . 
 appropriateness of requests from the critical analysis of the 500 requests , 138 ( 28% ) appeared not to fulfil the appropriateness criteria set out in the national referral guidelines . 
specifically , 78 ( 56.5% ) examinations requested were defined as not indicated due a lack of rationale for the investigation , and the remaining 60 examinations ( 43.5% ) were not routinely indicated , i.e. not supported by incontrovertible arguments . 
with regard to the referrers , whereas the absolute number of inappropriate requests differed slightly among those made by intrahospital specialists ( n = 62 ) and by general practitioners ( n = 76 ) , only 18% ( 62 / 343 ) of intrahospital requests was inappropriate , compared with a significant percentage risultati richieste cliniche assenza o presenza del quesito sul totale delle 500 richieste , in 110 casi ( 22% ) il quesito clinico risultato del tutto assente , pur in presenza di unindicazione della metodica da utilizzare . 
nello specifico 78 ( 56 , 5% ) sono definibili come non indicate per mancanza di una base logica allesecuzione dellindagine stessa mentre le rimanenti 60 ( 43 , 5% ) rivestono caratteri di indagini non indicate di routine , cio non sostenute da motivazioni incontrovertibili . 
 completeness of information regarding the requests rated as appropriate ( 362 / 500 ) , the inclusion of clinicalanamnestic information and data from previous imaging investigations appeared to be parameters of specific relevance for assessment of completeness , and substantial correctness was found with regard to the accuracy of this information . 
previous imaging investigations were mentioned in 98 requests , but the radiological history was only vaguely reported in 61 cases ( 62% )  . another qualitative parameter in judging completeness of the request was identified in the generality or specificity of the question in relation to the clinical diagnostic suspicion . it was found that in 12% of cases , the question other was included in the diagnostic question , maybe an expression of the referrers propensity towards so - called defensive medicine . 
in fact , the question other even assuming a legitimate demand for diagnostic completeness in the answer appears too vague and general and likely to raise expectations that cannot always by met by a radiological response . formal errors although no grammatical errors were identified , 39.6% of requests , especially from intrahospital physicians ( 43.7% ) contained acronyms that , although referring to routine medical terms , were not always easy to understand , in part due to legibility problems . 
in 98 richieste si segnalavano gli esami radiologici effettuati in precedenza dal soggetto , ma in ben 61 casi ( 62% ) tale anamnesi radiologica appariva formulata in maniera imprecisa . 
stato rilevato che , nel 12% dei casi , stato utilizzato linterrogativo altro ? nella formulazione del quesito diagnostico , espressione forse di uneccessiva propensione del medico prescrivente verso la cosiddetta medicina difensiva . 
questo termine infatti , pur presupponendo doverose esigenze di completezza diagnostica nella risposta , appare comunque eccessivamente vago e generico , idoneo a suscitare aspettative non sempre esaudibili da una risposta radiologica . 
 errori formali nonostante non siano emersi errori grammaticali , nel 39 , 6% delle richieste , ed in particolare in quelle provenienti dai medici intraospedalieri ( 43 , 7% dei casi ) , sono state utilizzate sigle e / o acronimi che , seppure legati a termini di uso abituale nella pratica medica , non sempre risultano di cos immediata comprensione anche perch legati alla differente intelligibilit delle varie grafie . 
pu segnalarsi , al riguardo , la mancanza del timbro o della firma del medico nell11% ( 55 / 500 ) delle richieste totali , soprattutto in quelle intraospedaliere . radiol med ( 2011 ) 116 : 152162 form was incompletely filled in 40% of cases , and the physicians stamp or signature was missing in 11% ( 55 / 500 ) of total requests , especially those from intrahospital physicians . referti radiologici risposta al quesito diagnostico radiology reports answer to diagnostic question of 362 requests in which the diagnostic question was appropriately and accurately formulated , 22 were not met with any clear and specific answer to the clinical question in the radiologists report . 
these 362 requests were further divided into appropriate requests ( n = 285 ) and appropriate requests with imprecise question ( n = 77 ) to evaluate whether the report effectively identified the condition for which examination had been requested or whether it identified a different condition from that suspected clinically or even the absence of any pathological findings . 
with regard to reports in response to appropriate requests , 28% ( 80 / 285 ) , most of which concerned conventional radiology studies , ruled out the presence of pathological findings ; a large proportion ( 47.5% , 135 / 285 ) confirmed the suspected diagnosis , and the remaining 24.5% ( 70 / 285 ) identified a different condition from that mentioned in the request . 
of the reports responding to the 77 appropriate requests with imprecise question , 36 ( 46.7% ) described conditions at any rate related to the clinical suspicion , 23 ( 30% ) reported a different diagnosis , and 18 ( 23.3% ) excluded the presence of pathology . 
with regard to this group , it should be noted that in terms of the principle of justification and optimisation , the report ruled out the presence of pathology in about one third of requests lacking a diagnostic question . 
 completeness of report apart from the patients demographic data , which were always present , none of the reports made any mention of the clinical information received , or specified , with the exception of cases of dental arch ct ( dental - scan ) , the equipment used and / or the examination technique ( or software reconstructions )  . 
of 47 reports that mentioned previous radiological examinations , more than half reported them incompletely , with no precise reference to the patients radiological history ( date and type of examination )  . 
finally , the majority of reports lacked diagnostic a fronte di 362 richieste in cui il quesito diagnostico stato formulato con criteri di appropriatezza e precisione , in 22 casi il referto del radiologo non ha fornito nessuna chiara e specifica risposta al quesito clinico . 
queste 362 richieste sono state poi ulteriormente suddivise in prescrizioni appropriate ( 285 ) e in prescrizioni appropriate con quesito impreciso ( 77 ) , e in questi due gruppi si voluto valutare se nelle risposte fosse stata effettivamente riscontrata la patologia per la quale lesame era stato richiesto , ovvero segnalati quadri patologici differenti da quelli sospettati clinicamente o , ancora , lassenza di qualsiasi reperto patologico . per quanto riguarda le prescrizioni appropriate , nel 28% dei casi ( 80 / 285 ) , in gran parte relativi ad accertamenti di radiologia tradizionale , lesame ha escluso la presenza di segni patologici , in una buona parte dei casi ( 47 , 5% , 135 / 285 ) stata confermata lipotesi diagnostica e nel rimanente 24 , 5% ( 70 / 285 ) sono state riscontrate patologie differenti da quelle riportate nella richiesta . 
delle restanti 77 richieste appropriate , in cui il quesito diagnostico era per impreciso , in 36 casi ( 46 , 7% ) sono state fornite risposte contenenti descrizioni di patologie comunque attinenti al quadro clinico , in 23 ( 30% ) stata posta una diagnosi differente da quella sospettata ed in 18 ( 23 , 3% ) la risposta radiologica ha escluso patologie in atto . 
 anche nelle richieste valutate come non appropriate ( 138 / 500 , 27 , 6% ) il radiologo ha comunque compilato un referto ; di questo gruppo da sottolineare , in rapporto al principio di giustificazione e ottimizzazione , come in circa 1 / 3 delle richieste senza quesito la risposta abbia escluso la presenza di qualsiasi patologia . completezza del referto a parte i dati anagrafici del paziente , risultati sempre presenti , in nessun referto stata riportata , nemmeno sinteticamente , linformazione clinica ricevuta , n mai stata precisata , ad eccezione dei casi di tc delle arcate dentarie ( dental - scan ) , lapparecchiatura utilizzata per lindagine e / o la tecnica di esecuzione ( unitamente ad eventuali ricostruzioni con software )  . 
da evidenziare come in 37 referti su 75 ( 49 , 3% ) , in cui , a nostro giudizio , sarebbero stati necessari ulteriori approfondimenti diagnostici , questi non sono stati n consigliati n programmati . 
va infine rimarcata , nella gran parte dei referti , lassenza delle conclusioni diagnostiche , riportate infatti solo nel 9 , 8% dei casi ( 49 / 500 ) , quasi tutti esami tc . 158 radiol med ( 2011 ) 116 : 152162 conclusions , which were present in only 9.8% of cases ( 49 / 500 ) , nearly all of which were ct examinations . errori formali formal errors grammatical errors were detected in 12% ( 60 / 500 ) of reports and in particular in reports of ct examinations , presumably due to the need to provide more in - depth and lengthy answers . 
 discussion there is no doubt that diagnostic imaging is undergoing profound changes , with significant organisational and professional implications for the radiologist , who has had to adapt to these rapid changes brought about by technology and market developments . 
a new concept of the healthcare act is emerging , intended as a complex and articulated set of services , skills and knowledge of several professions as a service provided by a healthcare team not through simultaneous interventions but rather through a series of consequential and concurrent actions performed by different professionals and directed towards the same goal . 
only the clinical radiologist will be able to relate on a par with the clinician and take on that central role as envisaged by the existing laws . it is therefore vital that precise , appropriate and universal communication the professionals take place among involved in requesting , interpreting and reporting [ 4 ]  . 
the radiological medical act represents a professional service that , although characterised by a series of integrated procedures with specific diagnostic / interventional aims , counts the request and the report as its fundamental steps , and there are often difficulties and problems in both interpreting the diagnostic question and in writing up the report . 
the most prevalent cause of these difficulties is often a lack of precise methodological indications , resulting in inconsistencies in the type of requests and high variability in the responses [ 5 ]  . the latter , even though generally adequate to allow for a correct diagnostic classification , sometimes appear at risk of ambiguous and nonunivocal interpretation . 
this leads to a possible negative impact on the patients health and a secondary risk of litigation or malpractice charges against the radiologist [ 6 ] , the incidence of which has recently become almost comparable with that in the united states , gli errori grammaticali si sono rilevati nel 12% ( 60 / 500 ) dei referti ed in particolare tra gli esami tc , presumibilmente in relazione alla necessit di risposte pi articolate e prolisse . 
grazie ai sistemi computerizzati , tutte le risposte sono dattiloscritte e in tutti i referti compare il nome del medico radiologo dattiloscritto , con relativa firma / sigla manoscritta . discussione indubbio che la diagnostica per immagini stia vivendo profondi cambiamenti , con significative ripercussioni organizzative e professionali per lo specialista radiologo che si dovuto adattare ai rapidi mutamenti determinati e condizionati dallevolversi della tecnologia e del mercato . 
 emergente una nuova concezione dellatto sanitario , inteso come un insieme complesso ed articolato di prestazioni , competenze e conoscenze di pi professioni , come una prestazione sanitaria di quipe , non nel senso della contestualit degli interventi , ma nel senso di una serie di atti consequenziali e di azioni concorrenti dirette ad un fine unitario e svolte da diverse figure professionali . 
solo il radiologo clinico potr confrontarsi paritariamente con il clinico , assumendo quel ruolo centrale che le normative vigenti gi gli assegnano . fondamentale dunque una precisa ed adeguata modalit di comunicazione universale tra le varie figure professionali che orbitano attorno alla richiesta , alla stesura e allinterpretazione di tale atto [ 4 ] ; esso rappresenta una prestazione professionale che , pur contraddistinta da una serie di procedure operative integrate tra loro , con specifiche finalit diagnostico / interventistiche , riconosce , quali momenti fondamentali , quello della richiesta e del referto , spesso comportanti problematiche e difficolt sia nellinterpretazione dei dati correlati al quesito diagnostico sia nella compilazione della risposta scritta . 
la causa prevalente di tali difficolt spesso identificabile in una carenza di precise indicazioni metodologiche , con conseguente disomogeneit nella tipologia delle richieste e contestuale notevole variabilit nelle risposte [ 5 ] ; questultime , sebbene generalmente idonee a consentire un corretto inquadramento diagnostico , appaiono talora a rischio di ambigua e non univoca interpretazione dei dati , con possibili ripercussioni negative per la salute del paziente e secondari rischi di contenzioso od accuse per malpractice [ 6 ] , la cui incidenza ha assunto , negli ultimi anni , frequenza quasi paragonabile a quella degli stati uniti , dove il numero di denunce secondo solo a quello delle discipline chirurgiche [ 7 ]  . 
 radiol med ( 2011 ) 116 : 152162 where the number of claims in radiology is second only to that of surgical disciplines [ 7 ]  . richieste cliniche radiology requests the first link between the radiological image and the report is the referring physicians clinical question , which must meet the criteria of appropriateness and relevance . 
it should be noted that 110 / 500 requests in our series ( 22% ) completely lacked a clinical question , thus potentially undermining the very foundation of the radiological procedure , given that the diagnostic question is an essential component of the radiology request as provided for by legislative decree no . 
when a referral is not accompanied by a clinical question or the question is clearly insufficient , the radiologist must contact the requesting physician for details or decide not to conduct the investigation because of the lack of essential information about the patients condition . 
alternatively , he or she must personally obtain the patients history and clinical data to appreciate why the radiological investigation is needed in relation to the patients presumed or suspected condition ; provide justification for the investigation ; and deliver the imaging service . in our study , even in the absence of a clinical question , the radiologist nevertheless wrote up a report , presumably after obtaining the patients history and clinical data , a fact that was not recorded . 
whereas the role of the referring physician is clearly important in proposing the investigation to be undertaken , the radiologist remains , for medicolegal purposes as well , the central reference figure in the diagnostic process the one who has the additional task of informing the patient about the examination and obtaining informed consent . in addition to appropriateness , another essential requirement of the request is accuracy of the anamnestic data , with concise and accurate reconstruction of the patients medical history , which helps the radiologist on the basis of his or her expertise to correctly classify the single case and establish the most appropriate investigation according to justification of the proposed examination . 
when describing the patients clinical history , the il primo collegamento fra limmagine radiologica e la sua refertazione rappresentato dal quesito clinico del medico richiedente , che deve rispondere a criteri di pertinenza e soprattutto al concetto di appropriatezza . 
riguardo tale primo momento , va sottolineato come , nella nostra casistica , in 110 / 500 casi ( 22% ) il quesito clinico era del tutto assente , cos da inficiare potenzialmente la base del procedimento diagnostico - radiologico ; il quesito diagnostico infatti uno dei principi indispensabili della richiesta radiologica previsti dal d.lgs. 
nei casi in cui linvio della persona non sia accompagnato da nessun quesito clinico o questo risulti palesemente insufficiente , al radiologo non resta che contattare in prima persona il medico richiedente per precisazioni a riguardo , o scegliere di non procedere a nessun accertamento in carenza delle indispensabili informazioni sulle condizioni del paziente , qualora queste non siano altrimenti acquisibili ; diversamente dovr effettuare lui stesso un adeguato inquadramento clinico - anamnestico , mirato a comprendere le esigenze di indagine radiografica dello stesso , in rapporto alla presunta patologia di base o al semplice sospetto diagnostico , portando a compimento la prestazione e adoperandosi da professionista a giustificarla . nel nostro studio , anche in assenza di quesiti clinici , lo specialista radiologo ha comunque steso un referto , verosimilmente dopo una fase anamnestica ed un inquadramento clinico che comunque non risultano essere stati documentati . 
pur rilevando infatti limportanza del medico prescrivente nella proposta dellesame da effettuare , lo specialista radiologo , anche ai fini medico legali , la figura essenziale di riferimento nel processo diagnostico , con il compito aggiuntivo dellinformazione al paziente circa la tipologia dellesame e lacquisizione del relativo consenso . oltre al fondamentale requisito dellappropriatezza , riteniamo necessario sottolineare limportanza della precisione del dato anamnestico , con sintetica e puntuale ricostruzione della storia clinica del paziente , utile al radiologo , sulla base delle proprie competenze , per inquadrare correttamente il singolo caso clinico , cos da definire la tipologia dindagine pi idonea ai sensi della giustificazione dellesame proposto . 
against the individual detriment that the exposure might cause , taking into account the efficacy , benefits and risks of available alternative techniques having the same objective but involving less exposure to ionizing radiation . 
this concept also applies to medicolegal exposure ( procedures performed for insurance or legal purposes without a clinical indication ) that does not produce a direct health benefit for the person all doses due to medical exposure ... 
taking into account the diagnostic reference levels ( drl ) in accordance with guidelines indicated in annex ii , and particular care shall be taken to keep the dose delivered during medico - legal exposure as low as reasonably achievable... tabella 3 principi di giustificazione e ottimizzazione , secondo il d.lgs. 
ovvero comportano una minor esposizione alle radiazioni ionizzanti , questo concetto vale anche per lesposizione di persone nellambito di procedure medico - legali ( procedimenti effettuati a fini assicurativi o legali , anche senza indicazione clinica ) che non presentano un beneficio diretto per la salute della persona ... 
this fact could hinder communication of correct and complete clinical information , leading not only to possible difficulty in carrying out del caso , ma anche , e soprattutto , in relazione al principio di giustificazione , gi pi volte menzionato . 
it is the step embodying the radiologists exclusive and specific competence , and it has official medicolegal value [ 9 ]  . the written nature of the radiologists interpretation makes the radiology report similar to a medical certificate , especially in terms of essential requirements [ 10 ]  . 
with regard to the medicolegal aspects , these two documents are quite different in their purposes ( the certificate is a statement of technical facts designed to demonstrate their truth and produces legal certainty ; the report is a scientific statement ) and intended readers ( the patient for the certificate ; the referring physician for the radiology report )  . in our study , we found that radiologists had a good reporting ability . 
they were almost always able to correctly answer the clinical question by correctly framing the condition being investigated , despite the fact that some situations are too complex to allow for a univocal interpretation and that findings may be nonspecific and subject to multiple interpretations that need to be considered in the differential diagnosis and can be better defined with further diagnostic investigations . 
in contrast , we found some lack of accuracy in the clinical information provided , in the description of the examination technique ( including equipment specifications and use of contrast medium ) , in the presence of individual physical factors potentially limiting the accuracy of the survey and in the suggestion for further diagnostic investigations . 
in particular , whereas no report contained any description of the clinicalanamnestic data and only a few described the examination technique , very few cases , indeed , contained the conclusions with suggested diagnoses or recommendations for further investigation , all of which are elements that can help improve communication with the referring physician . il referto rappresenta il momento successivo , indispensabile e complementare allesecuzione del supporto radiologico e pu dirsi il vero e proprio atto medico del radiologo . esso infatti linterpretazione ufficiale , finale e scritta , che deve accompagnare tutti gli studi di diagnostica per immagine e che , insieme alliconografia prodotta , ne costituisce il risultato , configurandosi quale momento di esclusiva e specifica competenza dello specialista radiologo , con valore medico - legale ufficiale [ 9 ]  . il carattere di verbalizzazione scritta del referto radiologico accomuna tale documentazione al certificato medico , specie nei requisiti essenziali di compilazione [ 10 ]  . 
sul piano medico legale invece i due elaborati non possono essere sovrapponibili , sia per differente finalit ( il certificato unattestazione di fatti di natura tecnica destinata a provarne la verit , nonch produttiva di una certezza legale ; il referto una dichiarazione di scienza ) , sia per il diverso destinatario ( il paziente per il certificato , il medico prescrivente per il referto radiologico )  . nel nostro studio abbiamo riscontrato una buona capacit di refertazione da parte degli specialisti , quasi sempre in grado di rispondere correttamente al quesito proposto inquadrando la patologia indagata , anche se , data la complessit di talune problematiche , non sempre possibile fornire interpretazioni e risposte univoche ai quesiti clinici , ma piuttosto evidenziare reperti non sempre specifici , soggetti a pi ipotesi , da porsi fra loro in diagnosi differenziale e meglio definibili con eventuali ulteriori accertamenti . 
in contrapposizione emersa , tuttavia , una certa mancanza di precisione nellinformazione clinica ricevuta , nella descrizione della tecnica di esecuzione dellesame ( comprese le precisazioni circa lapparecchiatura utilizzata e leventuale uso di mdc ) , nella presenza di potenziali fattori fisici individuali limitanti laccuratezza dellesame e nella raccomandazione di ulteriori indagini di approfondimento diagnostico . 
in particolare , se in nessun referto era indicato , neppure sinteticamente , il quadro clinico - anamnestico e solo in alcuni era descritta la metodologia dellesame , in pochissimi casi erano segnalate le conclusioni con eventuali indirizzi diagnostici o suggerimenti al curante per ulteriori indagini , tutti elementi utili per una migliore e pi completa comunicazione con il medico richiedente . conclusions our experience has shown a high percentage of errors in writing requests for radiological examinations ( 50% ) ; 22% of requests lacked the clinical question , and 27.6% most of which were from general practitioners were inappropriate . 
the reports were found to be substantially accurate , nella nostra esperienza emersa unelevata percentuale di errori nella formulazione della richiesta di esami radiologici ( 50% circa ) ; 22% di richieste mancanti del quesito clinico e 27 , 6% di richieste inappropriate , provenienti prevalenteconclusioni 162 radiol med ( 2011 ) 116 : 152162 with only 6% not providing a specific response to the clinical question . in conclusion , the high percentage of inappropriate requests and requests lacking a clinical question identified in this study may potentially undermine the effectiveness of the diagnostic process , with negative consequences on proper risk management [ 11 ]  . 
moreover , audits should be promoted to discuss , with the coordination of the radiologist and the participation of general practitioners and hospital physicians , the pitfalls and advantages of each imaging modality in relation to different clinical questions and to optimise the flow of radiology requests . mente da medici di medicina generale . 
nei referti radiologici si evidenziata una sostanziale correttezza ; solo nel 6% non era presente una risposta specifica al quesito clinico . in conclusione , lelevata percentuale di richieste inappropriate e mancanti di quesito clinico emersa in questo studio pu potenzialmente limitare lefficacia del processo diagnostico e comportare ripercussioni negative in termini di corretta gestione del risk - management [ 11 ]  . 
this study evaluated with ultrasonography ( us ) the presence of epiaortic vessel lesions in hiv - positive individuals receiving highly active antiretroviral therapy ( haart ) and compared them with nave patients and healthy individuals to highlight the differences among the different vascular damage patterns . 
a total of 222 hiv - infected patients receiving haart , 64 hiv - infected patients nave to antiretroviral therapy and 135 hiv - negative control patients underwent us of the carotid vessels . 
scopo dello studio stato quello di valutare , mediante ecografia ( us ) , la presenza di lesioni intimali nei vasi epiaortici di pazienti positivi al virus della immunodeficienza umana ( hiv ) trattati mediante terapia antiretrovirale altamente attiva ( haart ) , confrontando i risultati ottenuti con quelli provenienti dallo studio delle carotidi di pazienti hiv + che non ricevevano haart e di individui sani utilizzati come controllo . 
abbiamo studiato con us le carotidi di 222 pazienti hiv - positivi trattati con haart , di 64 hivpositivi nave per la terapia antiretrovirale e di 135 soggetti hiv - negativi , utilizzati come popolazione di controllo . 
abbiamo rilevato lesioni intimali carotidee in misura significativamente maggiore nei pazienti hivpositivi trattati con haart rispetto a quelli che non ricevano haart ( p < 0 , 0000001 ) , e ai controlli ( p < 0 , 0001 )  . 
in cinque soggetti in terapia haart abbiamo radiol med ( 2011 ) 116 : 6170 that were consistent with arteritis rather than with classic atheroma , but the percentage was too small to suggest any robust hypothesis . 
come gi segnalato da altri autori , abbiamo osservato lesioni carotidee compatibili con arterite piuttosto che con il classico ateroma , ma in una percentuale attualmente troppo esigua ( 0 , 02% ) per supportare valide ipotesi diagnostiche . 
ulteriori studi sono necessari per confermare questultimo dato . parole chiave hiv terapia antiretrovirale ateroscelrosi eco - color doppler placche carotidee introduction introduzione the introduction of new and potent drugs in combination regimens , commonly defined as highly active antiretroviral therapy ( haart ) , has significantly changed the natural history of hiv infection , leading to a significant reduction in morbidity and mortality from hiv / aids and related diseases [ 1 ]  . 
however , concern has been raised about their several adverse metabolic side effects , which include dyslipidaemia , lipodystrophy , insulin resistance and premature atherosclerosis [ 2 , 3 ]  . 
various vascular complications have been described in patients subjected to prolonged treatment with protease inhibitors ( pis ) , an important component of haart , prompting the hypothesis that pis can contribute to atherosclerotic damage of the vascular wall . 
indeed , patients treated with pi - based regimens present significantly higher levels of triglycerides and lower levels high - density lipoprotein ( hdl ) cholesterol [ 49 ]  . 
however , the actual role of these drugs in increasing the risk of atherosclerotic lesions of the carotid vessels in hiv - infected patients is still controversial [ 1013 ]  . 
several factors are related to the onset of vascular lesions in hiv - infected patients , including the direct role of the virus in endothelial damage [ 9 , 1315 ] and the immune reconstitution subsequent to haart [ 16 ]  . 
indeed , whereas some carotid lesions identified are consistent with typical atheromatous plaques , others have a different structure , more similar to inflammatory infiltration of the carotid wall , even though it cannot be excluded that they may subsequently evolve towards the classic features of atheroma . 
 con lintroduzione nellultimo decennio di nuovi efficaci farmaci somministrati in combinazione tra loro ( terapia antiretrovirale altamente attiva , haart ) , la storia naturale dellinfezione da virus dellimmunodeficienza umana ( hiv ) si radicalmente modificata . 
lhaart infatti ha ridotto significativamente morbilit e mortalit da hiv / sindrome da immunodeficienza acquisita ( aids ) e delle malattie correlate [ 1 ] , ma al contempo il suo utilizzo ha provocato la comparsa di molteplici effetti collaterali metabolici come la dislipidemia , la lipodistrofia , la resistenza alla insulina e laterosclerosi prematura [ 2 , 3 ]  . 
questo riscontro ha suggerito lipotesi che gli pis possano contribuire al danno aterosclerotico della parete vascolare , anche se i pazienti trattati con pis presentano livelli pi alti di trigliceridi che non di colesterolo highdensity lipoprotein ( hdl ) [ 49 ]  . 
a tuttoggi rimane tuttavia controverso il reale ruolo di questi farmaci nel determinare linsorgenza di lesioni aterosclerotiche nei pazienti hiv positivi [ 1013 ] la cui genesi potrebbe riconoscere molteplici fattori , come il ruolo diretto del virus sullendotelio [ 9 , 1315 ] o la ricostituzione immunitaria successiva alla haart [ 16 ]  . 
infatti , mentre alcune delle lesioni carotidee riscontrate sono compatibili con placche ateromasiche tipiche , altre presentano una struttura diversa , pi simile allinfiltrazione infiammatoria della parete carotidea , anche se non pu essere esclusa una loro successiva evoluzione verso il classico ateroma . 
 colour - doppler us is a well - established , noninvasive method of assessing the presence and degree of epiaorticvessel pathology and can therefore be used to detect onset leco - color doppler metodica non invasiva , affidabile nella valutazione delle patologie dei vasi epiaortici , e pu essere utilizzata nel paziente hiv positivo per rilevare le radiol med ( 2011 ) 116 : 6170 and progression of carotid lesions in hiv individuals . 
the objective of this study was to use us to evaluate the presence of carotid artery lesions in hiv - positive patients receiving or not receiving haart and to verify whether endothelial damage is more consistent with arteritis or with classic atheromatous plaques . 
 lesioni carotidee iniziali e studiare la loro progressione . obbiettivo di questo studio di valutare mediante ecografia ( us ) la presenza di lesioni intimali nei vasi epiaortici dei pazienti hiv positivi , trattati o non trattati con haart , e verificare se le lesioni endoteliali siano riconducibili a placche aterosclerotiche classiche o a lesioni di tipo infiammatorio , simili a quelle identificabili in corso di arterite . 
 materials and methods between december 2004 and february 2008 , 222 hivinfected patients receiving haart , 64 hiv - infected patients nave to antiretroviral therapy and 135 hiv - negative control patients underwent us of the carotid vessels . the local ethics committee approved the study , and all individuals provided informed consent . 
for each patient , we evaluated risk factors for cardiovascular diseases ( angina , myocardial or cerebral infarction ; transitory ischaemic attack ) , including family history , cigarette smoking , active drug abuse , arterial hypertension , hyperglycaemia , hypercholesterolaemia and hypertriglyceridaemia . 
no patients used lipid - lowering drugs . duplex us grading of the carotid arteries was performed in accordance with the principles established by the atherosclerosis risk in communities ( aric ) study [ 18 ] and the previous consensus meeting regarding quantification of extracranial carotid artery stenosis [ 19 ]  . bilateral carotid artery us was performed by the same specifically trained radiologist ( mc ) , with 15 years experience in colour - doppler imaging , on two scanners equipped with high - frequency broadband linear - array transducers . 
the scanners used were an astro ( esaote biomedica , genoa , italy , 69 mhz ) since 2004 and an iu22 ( philips , eindhoven , the netherlands , 517 mhz ) since 2005 . 
in accordance with the literature data [ 7 , 19 ] , we assessed lesions for echogenicity with respect to the vessel wall and homogeneity , presence of calcifications , parietal and endoluminal surface profiles , presence of ulceration and presence of a cleavage plane . 
a materiali e metodi nel periodo compreso tra dicembre 2004 e febbraio 2008 , presso il nostro istituto , abbiamo valutato prospetticamente con us dei vasi epiaortici 222 pazienti hiv positivi trattati con haart , 64 pazienti hiv positivi nave per la terapia antiretrovirale e 135 soggetti hiv negativi , questi ultimi utilizzati come popolazione di controllo . 
per ogni paziente sono stati valutati i fattori di rischio per patologie cardiovascolari ( angina , infarto del miocardio o cerebrale , attacchi ischemici transitori ) : la storia familiare , il fumo , lassunzione di droghe , lipertensione arteriosa , la iperglicemia , la ipercolesterolemia , la ipetrigliceridemia . 
 lo studio ecografico delle carotidi stato eseguito in conformit con i principi stabiliti dallo studio atherosclerosis risk in communities ( aric ) [ 18 ] e dal precedente consensus meeting sulla quantificazione del grado di stenosi dellarteria carotide extracranica [ 19 ]  . 
lesame eco doppler delle carotidi stato eseguito dallo stesso operatore ( mc ) , specialista radiologo con esperienza di 15 anni nella tecnica eco color doppler , utilizzando due apparecchi dotati di sonde lineari ad alta frequenza , dal 2004 un astro 69 mhz ( esaote biomedica , genova , italia ) , e dal 2005 un u22 517 mhz ( philips , eindhoven , olanda )  . 
lesame morfologico stato eseguito bilateralmente , mediante piani assiali e sagittali con tecnica b - mode , color e power doppler al fine di evidenziare meglio le lesioni e limt . stata eseguita una analisi spettrale pulsed - wave doppler a carico delle arterie carotidi comuni ed interne bilateralmente . 
in base ai dati della letteratura [ 7 , 19 ] abbiamo valutato : lecogenicit delle lesioni in confronto alla parete vascolare e la loro radiol med ( 2011 ) 116 : 6170 fig . 
a p value < 0.05 was considered statistically significant . results omogeneit , soprattutto se fossero presenti calcificazioni ; il profilo della superficie endoluminale e parietale , specialmente la presenza di ulcerazioni e di un piano di clivaggio . abbiamo definito come placca un ispessimento della parete carotidea , focale e > 1 mm : aterosclerotica quando si lispessimento era iso - iperecogeno , senza o con calcificazioni , con superficie parietale ed endoluminale irregolare e con piano di clivaggio ; di tipo infiammatorio quando si lispessimento era iso - ipoecogeno , senza piano di clivaggio , con superficie parietale ed endoluminale liscia e regolare o solo lievemente irregolare . 
la superficie della placca stata definita come irregolare quando abbiamo osservato una variazione di altezza tra 0 , 4 e 2 mm lungo il contorno della lesione . le differenze statistiche fra i pazienti sono state valutate con il test del chi - quadro o con il test di fisher per le variabili qualitative . 
the epidemiological characteristics and cardiovascular risk factors of the three groups of individuals ( hiv - infected patients on haart , hivinfected patients without haart , non - hiv - infected individuals ) are shown in table 2 . 
hiv - positive patients were more frequently cigarette smokers in comparison with hiv - negative patients ( p < 0.0001 ) , but no difference risultati la tabella 1 mostra i dati epidemiologici dei pazienti hiv positivi . 
la tabella 2 documenta i dati epidemiologici e i fattori di rischio cardiovascolari nei tre gruppi in esame ( pazienti hiv positivi in trattamento haart , pazienti hiv positivi non sottoposti a haart , pazienti hiv negativi )  . 
hiv - infected patients on haart had a significantly higher prevalence of hypertriglyceridaemia when compared with either hiv - positive patients nave to antiretroviral therapy or hiv - negative individuals . 
in incidenza di fumatori rispetto ai pazienti hiv negativi ( p < 0 , 0001 ) , mentre non si sono rilevate differenze in tal senso fra i pazienti hiv positivi sottoposti ad haart e quelli nave per la terapia antiretrovirale . 
no alterations in pulsation index , resistance index , minimal speed , peak speed and mean speed were observed in the three groups of patients . discussion the widespread diffusion of haart has deeply modified the characteristics and epidemiology of hiv / aids , leading to a prolonged asymptomatic period after infection , prolonged survival after aids diagnosis and changes in the natural history of hiv - related diseases . 
nei tre gruppi di pazienti non sono stati registrati valori anomali di indice di resistenza , di indice di pulsatilit , di velocit di picco sistolica , diastolica o media . 
 discussione lampia diffusione dellhaart ha profondamente modificato le caratteristiche e lepidemiologia dellhiv / aids , determinando un lungo periodo asintomatico dopo linfezione , un lungo periodo di sopravvivenza dopo la diagnosi di aids e modificando la storia naturale delle patologie associate allhiv . 
3 classica placca ateromasica ulcerata nella carotide comune di un paziente hiv - positivo in trattamento con haart . vascular lesions in hiv - infected patients , including the direct role of the virus and immune reconstitution subsequent to haart , and especially changes in lipid and glycidic metabolism induced by haart and , in particular , by pis [ 6 , 25 ]  . 
pis have been thought to promote atherosclerotic lesion formation independently of dyslipidaemia through cholesterol accumulation in macrophages of the arterial wall , [ 26 ] , although some authors observed that the presence of atherosclerotic plaques in femoral and carotid arteries of hiv - infected patients was more closely related to gender , age , cigarette smoking and plasma cholesterol levels than to pi use [ 27 ]  . 
in our study , there was no difference in age , gender and cholesterol level between hiv - positive and hiv - negative patients . regarding cigarette smoking , hiv - positive patients were significantly more frequently smokers than hiv - negative individuals . 
however , in our hiv - positive population , cigarette smoking did not seem to influence the frequency of carotid lesions , and no difference in cigarette smoking was found between hiv - positive patients on haart and hiv - positive patients nave to antiretroviral therapy . 
haarttreated patients showed a higher prevalence of hypertriglyceridaemia in comparison with either haart - nave or hiv - negative patients , even though hypertriglyceridemia is rarely responsible for premature atherosclerosis [ 28 ]  . 
4 lesione di tipo infiammatorio nella carotide comune di un paziente hiv - positivo in trattamento con haart . coronariche e linfarto del miocardio , sono segnalate con frequenza crescente [ 2124 ] , anche se la dimensione reale del problema ancora poco chiara . molteplici fattori sono stati collegati con linstaurarsi di lesioni vascolari in pazienti hiv positivi : il ruolo diretto del virus e della ricostituzione immunitaria a seguito di haart , ma soprattutto i cambiamenti nel metabolismo lipidico e glucidico indotti dallhaart e dagli pis in particolare [ 6 , 25 ]  . 
stato ipotizzato che gli pis possano causare la formazione di lesioni aterosclerotiche provocando un aumento del colesterolo nei macrofagi della parete arteriosa , indipendentemente dalla dislipidemia [ 26 ] , tuttavia alcuni autori hanno osservato che la presenza di placche aterosclerotiche nelle arterie femorali e carotidee dei pazienti hiv positivi correlata rigorosamente con il sesso , let , il tabagismo ed il livello di colesterolo del plasma piuttosto che alluso di pis [ 27 ]  . 
tuttavia , nella nostra popolazione hiv positiva , il tabagismo non sembra influenzare lincidenza delle lesioni carotidee e nessuna differenza nel tabagismo stata riscontrata fra i pazienti hiv positivi in haart ed i pazienti hiv positivi nave per la terapia antiretrovirale . i pazienti trattati con haart hanno mostrato una pi alta prevalenza di ipertrigliceridemia rispetto sia ai pazienti nave per haart che ai pazienti hiv negativi , anche se la ipertrigliceridemia raramente responsabile della aterosclerosi precoce [ 28 ]  . 
nella nostra casistica nei pazienti hiv positivi trattati con haart sono state riscontrate ecograficamente placche aterosclerotiche nel 32 , 9% dei casi versus il 3 , 2% dei pazienti hiv negativi ( p < 0 , 0001 )  . radiol med ( 2011 ) 116 : 6170 vascular disorders affecting small and medium - sized visceral arteries , and the occurrence of vasculopathy has been reported even in large arteries [ 5 , 13 , 29 ]  . 
in these cases , no evidence of atherosclerotic plaques was reported ; the lesions had histological features characterised by proliferation of vascular channels in the adventitia and the presence of a vasculitis of the vasa vasorum , with or without an intimomedial arteritis . 
however , recent studies failed to find a correlation between the classic mediators of atheromatous lesions , such as c - reactive protein and carotid imt in hiv - positive individuals , reinforcing the hypothesis that there could be an alternative pathway for this damage [ 11 , 30 ]  . 
we observed lesions consistent with inflammatory lesions rather than classic atheroma in five patients on haart . the different us patterns between inflammatory and atheromatous lesions are due to the different pathological substrate . 
furthermore , the atheromatous plaque involves only the endothelium and luminal portion of the intima , which accounts for the presence of the cleavage plane between the lesion and the underlying tissues , and its surface is frequently ulcerated . 
further investigations are warranted to better define the structure and mechanisms of onset of these inflammatory - type lesions and clarify the role of the different antiretroviral drugs in the pathogenesis of coronary and systemic atherosclerosis . 
as suggested by some authors [ 14 , 17 ] , vascular damage may develop in two phases : an initial , inflammatory phase probably triggered by haart directly or through immune reconstitution , and a second phase in which the lesions could be maintained by the classic atheromatous risk factors . 
the characteristics of us make it the ideal tool for the periodic study of the carotid vessels , which should , in our experience , be included in the general follow - up of hiv patients , in particular those with pre - existing cardiovascular risk factors , regardless of their antiretroviral therapy status . 
 i pazienti hiv positivi possono mostrare una vasta gamma di vasculopatie che interessano le arterie viscerali piccole e medie e , recentemente , stato segnalato un caso di vasculopatia anche in una grande arteria [ 5 , 13 , 29 ]  . 
nei casi in cui le lesioni non erano di tipo aterosclerotico , istologicamente erano caratterizzate dalla proliferazione di strutture vascolari nellavventizia e dalla presenza di una vasculite dei vasa vasorum , associata o meno ad unarterite dellintima e della media . 
in letteratura non ci sono ancora riscontri che stabiliscono una correlazione fra i classici mediatori delle lesioni ateromasiche , quali la proteina c reattiva e lispessimento di media ed intima nei pazienti hiv positivi , il che rafforza lipotesi che potrebbero esserci altre cause nella patogenesi di queste lesioni [ 11 , 30 ]  . 
in cinque soggetti della nostra casistica in terapia haart abbiamo documentato la presenza di lesioni carotidee di tipo infiammatorio . il differente substrato anatomo - patologico determina i differenti aspetti ecografici delle placche ateromasiche e di quelle tipo infiammatorio . 
laspetto iperecogeno direttamente proporzionale al contenuto fibroso , la disomogeneit alla presenza di lipidi , fibre e calcio , per questo motivo lateroma , generalmente , iperecogeno e disomogeneo . inoltre , la placca ateromasica coinvolge solo lendotelio e la pozione superficiale dellintima , il che si conferma con la presenza di un piano di clivaggio fra le lesione ed i tessuti sottostanti , e la sua superficie frequentemente ulcerata . ulteriori studi saranno necessari sia per meglio definire la struttura e la patogenesi delle lesioni di tipo infiammatorio , sia per chiarire il ruolo dei differanti farmaci antiretrovirali nella patogenesi della aterosclerosi coronarica e sistemica . ad oggi si ritiene che i farmaci antiretrovirali potrebbero svolgere un ruolo chiave nella patogenesi delle lesioni carotidee che lhaart induce . 
alcuni autori hanno ipotizzato [ 14 , 17 ] che il danno vascolare potrebbe svilupparsi in due fasi : la prima , essenzialmente flogistica , innescata dallhaart , direttamente o tramite la ricostituzione immunitaria indotta , la successiva evoluzione delle lesioni potrebbe essere sostenuta dai fattori di rischio ateromasici classici . 
li contributed equally to this study received : 5 january 2010 / accepted : 15 february 2010 / published online : 6 october 2010 springer - verlag 2010 abstract purpose . 
follow - up inferior vena cavagrams demonstrated complete resolution of the chronic ivc thrombi and full ivc patency , without occurrence of pulmonary embolism at any time during the study . 
questo studio stato condotto per valutare la sicurezza e la fattibilit del trattamento trombolitico con urochinasi dopo pre - dilatazione in pazienti con sindrome di budd - chiari ( bcs ) con trombosi cronica della vena cava inferiore ( ivc )  . 
i risultati immediati ed a lungo termine della tecnica procedurale hanno avuto successo in tutti pazienti . le flebografie di follow - up della vena cava inferiore hanno dimostrato una completa risoluzione della trombosi cronica dell ivc e totale pervieta dell ivc , senza alcun caso di embolia polmonare durante lo studio . 
i nostri risultati preliminari indicano che la trombolisi con infusione continua di urochinasi dopo predilatazione un approccio sicuro e fattibile per il trattamento dei pazienti bcs con trombosi cronica dell ivc . keywords budd - chiari syndrome inferior vena cava thrombus thrombolysis parole chiave sindrome di budd - chiari vena cava inferiore trombosi trombolisi radiol med ( 2011 ) 116 : 5660 introduction primary budd - chiari syndrome ( bcs ) is a rare form of hepatic venous outflow obstruction at the suprahepatic inferior vena cava ( ivc ) , the hepatic veins ( hv ) or both , whereas bcs with ivc thrombosis is even rarer , particularly with fresh ivc thrombosis . 
a number of thrombolytic agents , such as acylated streptokinase - plasminogen complex , urokinase , tissue plasminogen activator and streptokinase have been administered either through peripheral intravenous catheters or through ivc or hepatic catheters for successfully dissolving the fresh clots [ 13 ]  . 
prior to the procedures , during which the patients were monitored continuously for vital signs and bleeding complications , inferior vena cavagraphy of the right groin revealed the location and size of the thrombus and the occluded section of the ivc . 
for predilation , a straight 5 - fr catheter with multiple side holes ( cook , inc . , bloomington , in , usa ) was inserted in the ivc and positioned below the occluded segment . 
to prevent rethrombosis , warfarin was administrated orally 5 mg / day from the second day after the procedure for about 36 months . follow - up evaluation the followup investigations , including final clinical evaluation , colour - doppler sonography or inferior vena cavagraphy , were performed 1 week and 1 , 3 , 6 and 12 months posttreatment and then annually thereafter . 
at each visit , procedural technical , angiographic and ultrasonic outcomes , as well as mortality , morbidity and the final clinical outcome , were evaluated . results primary procedural results predilation and urokinase thrombolysis were technically successful in all patients , without occurrence of any procedure - related complications such as acute pulmonary embolisation . 
 discussion membranous obstruction of the ivc or hv or both is the most common cause of bcs in asia and accounts for up to 6070% of the total number of patients [ 58 ]  . 
due to obstruction of the proximal ivc , the blood flow is slow , turbulent or reverse , which leads , together with the hypercoagulable blood state , to formation of thrombi in these patients . 
mostly , ivc in bcs patients presenting in our hospital is already chronic , since disease onset is often asymptomatic , and thus the onset time is often difficult to indicate . 
 thrombosis current medication or procedures to treat ivc thrombosis include thrombolytic agents [ 1 ] , transjugular intrahepatic portosystemic shunts [ 911 ] , surgical shunts [ 12 ] , balloon angioplasty and metallic stents [ 2 , 3 ] or retrieval stent filtres [ 4 ]  . 
thus , our hypothesis was that local continuous infusion of thrombolytic agents may be beneficial in treating bcs patients with chronic ivc thrombosis after restoring the blood flow in the obstructed ivc by interventional procedures . 
therefore , we attempted a relatively simple thrombolytic approach for our bcs patients by infusing urokinase after predilation of the ivc . acute pulmonary embolisation represents a potential risk after predilation and thrombolysis with urokinase ; however , none of the patients in this study experienced this symptom radiol med ( 2011 ) 116 : 5660 fig . 
1a - i a 55 - year - old man with a 10 - year history of budd - chiari syndrome ( bcs ) , with varices in the thoracicoabdominal region and the legs , as well as hepatomegaly , splenomegaly and pigmentation . 
a colour - doppler sonography exhibits a chronic thrombus in the inferior vena cava ( ivc ) ( arrow ) , with ivc occlusion ( arrowhead ) ; b sagittal - plane multiplanar reconstruction ct image of the ivc showed a low - density ivc thrombus with irregular margins ( arrow ) ; c inferior vena cavagram reveals an irregular chronic thrombus in the left ivc wall ( arrow ) ; d anteroposterior fluoroscopy shows dilation of the occluded ivc by a 16 - mm balloon dilator ( arrow ) after rupture of the membrane ; e inferior vena cavagram displays partial patency of the ivc after predilation . 
1a - i un uomo di 55 anni con una storia di sindrome di budd - chiari da 10 anni ( bcs ) , con varici in regione toracoaddominale e alle gambe , epatomegalia , splenomegalia e pigmentazione . 
a lecografia color doppler ha mostrato trombosi cronica della vena cava inferiore ( ivc ) ( freccia ) , con occlusione dell ivc ; b le immagini di ricostruzione tc multiplanare dell ivc sul piano sagittale , hanno mostrato bassa densit della trombosi dell ivc con margini irregolari ; c la flebografia della vena cava inferiore rivela un irregolare trombosi cronica nella parete sinistra dell ivc ( freccia ) ; d la fluoroscopia antero - posteriore mostra dilatazione dellocclusione dellivc con un dilatatore a palloncino di 16 mm ( freccia ) , dopo la rottura della membrana ; e la flebografia illustra la perviet parziale della vena cava inferiore dopo pre - dilatazione ; f l ecografia color doppler evidenzia una parziale perviet dell ivc con stenosi della membrana allorifizio dellatrio destro e una trombosi cronica dell ivc ( freccia ) ; g la flebografia mostra una perviet parziale dell ivc che priva di trombi a 12 giorni dalla trombolisi ; h la flebografia sottolinea lassenza di trombo in corrispondenza dellivc dopo dilatazione con dilatatore a palloncino di 30mm ; i l ecografia color doppler mostra una perviet da trombi dellivc ad un mese dalla trombolisi . 
the success of our approach may be attributable to the following : ( 1 ) the chronic thrombus was incorporated in the ivc wall and would not easily fall off ; ( 2 ) the fresh thrombus was treated with continuous infusion of urokinase for 30 min at the beginning of thrombolysis ; ( 3 ) predilation of the obstructed ivc with a small dilator ( 1216 mm in diameter ) allowed sufficient blood flow through the occluded ivc to prevent the large thrombus from migrating through the occluded ivc . 
however , this approach is not appropriate for patients with segmental obstruction of the ivc or huge thrombi completely obstructing the ivc . normally , chronic thrombi in occluded ivcs are difficult to dissolve , but dissolving them in dilated ivcs with the help of continuous urokinase infusion has proven successful in our patients . 
the former contains mainly erythrocytes and less leucocytes , platelets and fibr therefore , these thrombi are being formed more loosely and can be dissolved more easily and gradually , for instance , by increasing blood flow in the dilated ivc and by then infusing urokinase for about 730 days . 
also , use of a 30 - mm balloon dilator includes the risk of ivc wall rupture ; thus , the device must be used correctly and with great caution according to the instructions of the manufacturer . in conclusion , our preliminary results indicate that thrombolysis with urokinase after ivc predilation may be a safe and feasible approach for treating bcs patients with chronic ivc the results are thrombosis . 
passariello1 1dipartimento di scienze radiologiche , 2dipartimento di scienze cardiovascolari e respiratorie , sapienza universit di roma , policlinico umberto i , v.le regina elena 324 , 00161 roma , italy 3cardiovascular magnetic resonance unit , royal brompton hospital , imperial college , national heart and lung institute , london , uk correspondence to : m . 
in ischaemic heart disease , t2w - stir represents the technique of choice for detecting oedema in patients with acute myocardial infarction ( mi ) , allowing discrimination of acute and chronic injuries . 
myocardial haemorrhage may also be depicted as a region of signal abnormality characterised by a central hypointense core with a peripheral hyperintense rim , presumably reflecting the presence of intracellular methaemoglobin within the necrotic area . 
in the acute setting , elevated t2 relaxation times in association with regional contractile dysfunction but no signs of delayed enhancement may also signify a reversible ischaemic injury without necrosis . 
in acute myocarditis , the distribution pattern of t2w hyperintensity may be focal in approximately 30% of patients or diffuse in the remaining 70% , and myocardial oedema may be the only marker of disease . 
t2w - stir riassunto in risonanza magnetica cardiaca , le sequenze short tau inversion recovery ( stir ) pesate in t2 rappresentano la tecnica ideale per lidentificazione delledema tissutale grazie allapplicazione di un triplo impulso di presaturazione che consente di combinare la soppressione del segnale dei fluidi in movimento e del tessuto adiposo . scopo di questa pictorial review di illustrare lutilit clinica e lo spettro applicazioni di questa sequenza nella patologia cardiaca ischemica e non ischemica . 
nella cardiopatia ischemica , ledema tissutale rappresenta un marker di danno miocardico acuto e definisce larea di miocardio a rischio che contiene sia tessuto necrotico che segmenti miocardici non contrattili ma vitali . 
lemorragia rappresenta invece la conseguenza della terapia riperfusiva con stravaso di eritrociti nellinterstizio e pu essere identificata in stir t2 come unalterazione caratterizzata da un core centrale ipointenso , che riflette la presenza di metaemoglobina nel contesto dellarea di necrosi . 
ledema tissutale pu essere inoltre osservato in numerose altre cardiopatie non ischemiche , come varie cardiomiopatie primitive e secondarie , pericarditi acute , lipertensione polmonare ed il rigetto di trapianto cardiaco . 
in aggiunta alle tradizionali sequenze radiol med ( 2011 ) 116 : 3246 represents an appealing and versatile technique that can be applied in a wide variety of ischaemic and non - ischaemic conditions , allowing detection of segmental or global increase of myocardial free water content , reflecting an acute myocardial injury . 
 keywords t2w - stir myocardial oedema myocardial infarction myocarditis hypertrophic cardiomyopathy tako - tsubo syndrome pericarditis heart transplantation morfo - funzionali , le sequenze stir t2 costituiscono uninteressante e versatile tecnica di imaging che pu trovare applicazione in una vasta gamma di cardiopatie ischemiche e non , consentendo di identificare un incremento focale o diffuso del contenuto di acqua libera intramiocardica e fornendo molto spesso informazioni di rilevante impatto clinico . parole chiave stir t2 pesate edema miocardico infarto del miocardio miocardite cardiomiopatia ipertrofica sindrome di tako - tsubo pericardite acuta trapianto di cuore introduction introduzione use of t2 - weighted imaging for assessing acutely injured myocardium was initially proposed by higgins et al . 
 [ 1 ] , who described the presence of a linear correlation between t2 - relaxation time and percentage of myocardial free water content in an animal model , allowing depiction of infarcted tissues as areas of increased signal . 
myocardial oedema does not necessarily reflect necrosis and represents a nonspecific marker of acute inflammatory response in numerous other nonischaemic clinical settings , such as tumours , primary cardiomyopathies ( cmp ) , storage disease , cardiac transplant rejection , pulmonary hypertension and acute myocarditis [ 2 , 3 ]  . 
the best imaging approach for oedema - weighted cardiac magnetic resonance imaging ( mri ) is segmented fast spin - echo using a triple inversion recovery preparation module ( t2w - stir ) , which allows signal suppression from flowing blood and from fat [ 2 ]  . 
 [ 1 ] hanno per primi descritto , su modello animale , la presenza di una correlazione lineare tra tempo di rilassamento t2 e quota di acqua libera intramiocardica , identificando la necrosi miocardica acuta come unarea di focale incremento del segnale in t2 dovuta alla presenza di edema tissutale [ 1 ]  . ledema miocardico non necessariamente riflette la presenza di necrosi e rappresenta il segno di una risposta infiammatoria aspecifica del tessuto miocardico e pericardico riscontrabile in numerose cardiopatie di tipo nonischemico , dalle malattia infiammatorie ( miocarditi e pericarditi ) , alla patologia neoplastica , malattie autoimmuni e rigetto dorgano nei trapianti , cardiomiopatie primitive e secondarie ( soprattutto malattie da accumulo ) [ 2 , 3 ]  . 
lo stato dellarte dellimaging pesato in t2 o edema - weighted in rmc si basa sullutilizzo di una tecnica fast spin - echo inversion recovery ( short tau inversion recovery , stir ) , con applicazione di un triplo impulso di presaturazione che consente di combinare la soppressione del segnale dei fluidi in movimento e del tessuto adiposo [ 2 ]  . 
 scopo di questa pictorial review di illustrare lutilit clinica e lo spettro applicazioni di questa sequenza in una variet di condizioni ischemiche e non - ischemiche . design della sequenza la stir t2 una sequenza spin - echo inversion - recovery a sangue nero ( black - blood ) , in cui vengono applicati due impulsi di preparazione ( rispettivamente non - selettivo e selettivo ) a 180 , seguiti da un lungo tempo di inversione ; la soppressione del tessuto adiposo viene successivamente ottenuta con un terzo impulso di presaturazione a tempo di inversione ( ti ) breve e selettivo per il grasso . 
the underlying pathophysiological mechanism leading to accumulation of water within the intraand extracellular spaces of necrotic regions seems to be related to the presence of osmotically active substances and disruption of the adenosine triphosphatase ( atp ) - regulated ionic transport mechanisms across the cell membrane in the acute phase after necrosis [ 5 ]  . 
in the acute phase of mi , regional hyperintensity on t2w - stir is usually larger than true infarct size compared with histology or delayed - enhancement ( de ) imaging measurements [ 6 , 7 ]  . 
 una volta annullata la magnetizzazione del sangue , viene applicato un terzo impulso a 180 , con ti breve per annullare selettivamente il segnale proveniente dal tessuto adiposo ( impulso stir ) , amplificando le differenze di segnale tra tessuti con t1 lungo ( come per esempio il miocardio edematoso ) e tessuti con t1 breve ( come il grasso ) ; il ti di circa 120 ms quando si utilizzano magneti a 1 , 5 t . 
dal punto di vista fisiopatologico , ledema miocardico nellinfarto acuto sia di tipo interstiziale sia intracellulare rispettivamente per accumulo di sostanze osmoticamente attive nellinterstizio che richiamano liquidi dal microcircolo e per la deregolazione delle pompe sodio - potassio atp - mediate con perdita di funzione di membrana dei miociti [ 5 ]  . 
on t2 - weighted short - tau inversion recovery ( a ) , tissue oedema is depicted as a homogeneous transmural area of hyperintense signal located in the inferior and inferoseptal left - ventricular wall ( arrow )  . 
nelle sequenze stir t2 pesate ( a ) , ledema tissutale viene identificato come una zona di omogenea iperintensit del segnale ad estensione transmurale , localizzata a livello della parete inferiore ed infero - settale del ventricolo sinistro ( freccia )  . 
nella stessa sede , le sequenze inversion recovery contrast enhanced ( ir - ce ) mostrano una circoscritta area di potenziamento patologico ad estensione non trans murale ( b )  . 
clearly demonstrated the relationship between t2w abnormality and the area of myocardium at risk using an animal model in which cardiac mri data were compared with histology and the ischaemic region was demarcated by injecting fluorescent microspheres at the time of the induced coronary occlusion [ 8 ]  . 
thus , t2w abnormality in acute mi defines the area at risk at the time of the ischaemic insult , which includes zones of reversibly and irreversibly injured myocardium [ 79 ]  . 
lestensione dellarea edematosa quantificata con stir t2 costantemente maggiore sia rispetto ai rilievi istopatologici che alle misurazioni effettuate con tecnica di delayed enhancement ( de ) in rmc [ 6 , 7 ]  . 
 [ 6 ] hanno ipotizzato come questa differenza tra segmenti iperintensi in stir t2 e segmenti con de potrebbe riflettere la presenza della cosiddetta area perinfartuale contenente segmenti miocardici disfunzionanti radiol med ( 2011 ) 116 : 3246 fig . 
3a , b double acute and chronic mi in a 62 - year - old man in whom a combined approach of t2w - stir ( a ) and ce - ir magnetic resonance image ( b ) allows differentiation an old anterior mi from an area of recent necrosis ( day 5 ) located in the inferoseptal left - ventricular ( lv ) wall and extending to the inferior right ventricular ( rv ) wall . 
chronically infarcted region exhibits lower signal intensity on t2w imaging ( a ) ( arrowheads ) compared with normal adjacent segments , presumably because of the lower free water content of the fibrous scar . 
in questo paziente stato utilizzato un approccio combinato utilizzando sequenze stir t2 pesate ( a ) ed ir - ce ( b ) , per differenziare un pregresso infarto anteriore rispetto ad unarea di necrosi recente ( 5 giorno ) , localizzata in sede infero - settale del ventricolo sinistro e con estensione alla parete inferiore del ventricolo destro . 
nella sede del pregresso infarto , la cicatrice presenta bassa intensit del segnale nelle sequenze t2 pesate ( a ) ( punta di freccia ) , se confrontata con i segmenti contigui , verosimilmente per il minore contenuto di acqua libera nel contesto della cicatrice fibrosa . 
in stir t2 inoltre osservabile lestensione delledema a livello della parete inferiore del ventricolo destro , elemento che ne suggerisce il coinvolgimento , poi confermato dalle immagini in asse corto ir - ce ( b ) ( freccia )  . revascularisation are minimal compared with the extent of reperfusion - related damage [ 1012 ]  . myocardial oedema also represents a promising target of early reperfusion of a thrombotic occlusion preceding the onset of myocardial necrosis in the ischaemic cascade . 
in the acute setting , therefore , elevated t2 relaxation times in association with regional contractile dysfunction but no signs of de might signify a reversible ischaemic injury without tissue necrosis , resulting in an aborted mi , a term first used by gename et al . 
 myocardial haemorrhage is a reperfusion - related injury with severe microvascular damage caused by extravasation of erythrocytes into the interstitium during the reflow period caused by disruption of capillary integrity . 
 [ 14 , 15 ] recently demonstrated with cardiac mri the importance of haemorrhage as an independent and strong predictor of left - ventricular ( lv ) remodelling at short - term follow - up , regardless of the initial infarct size . ma vitali , che possono beneficiare con successo della rivascolarizzazione [ 6 ]  . 
 [ 8 ] hanno ulteriormente chiarito tali osservazioni dimostrando su modello animale la stretta correlazione tra larea di segnale iperintenso in stir t2 e lestensione istopatologica dellarea a rischio utilizzando una tecnica di immunofluorescenza con iniezione intracoronarica di microsfere al momento dellinduzione dellocclusione del vaso [ 8 ]  . 
in tal senso , il concetto fondamentale che le sequenze stir t2 , definiscono ed identificano non tanto lestensione della necrosi quanto larea di tessuto miocardico a rischio interessato dallinsulto ischemico e che comprende sia i segmenti non contrattili ma vitali ( miocardio stordito ) , che quelli danneggiati irreversibilmente ( miocardio necrotico ) [ 79 ]  . 
4a - e haemorrhagic myocardial infarction due to iatrogenic dissection of a first marginal branch of the left circumflex artery during selective coronary angiography . intramyocardial haemorrhage is characterised on t2w - stir ( a , b ) by a central hypointense core with a peripheral hyperintense rim located within the lateral left ventricular wall . regional t2 relaxation time shortening is most likely related to the presence of intracellular methaemoglobin due to haemorrhage and infarct resorption . 
lemorragia intramiocardica caratterizzata nelle sequenze stir t2 ( a , b ) da un core centrale ipointenso , circondato da unarea periferica ad elevate intensit del segnale localizzata nel contesto della parete laterale del ventricolo sinistro ; laccorciamento del tempo di rilassamento t2 verosimilmente legato alla presenza di metaemoglobina intracellulare , secondaria allemorragia ed al rimodellamento infartuale . 
 myocarditis interstitial myocardial oedema is part of the inflammatory response in acute myocarditis , and its presence has been miocardica conseguenza del danno da riperfusione ed espressione dei fenomeni flogistici contestuali alla necrosi con perdita di integrit dellendotelio e distruzione delle arteriole e dei capillari , e conseguente stravaso di eritrociti radiol med ( 2011 ) 116 : 3246 fig . 
in questo paziente di 57 anni , riperfuso tempestivamente su discendente anteriore dopo linsorgenza dei sintomi ( 60 minuti ) , le sequenze stir t2 pesate acquisite in asse corto mostrano imbibizione edematosa della parete antero - settale in sede medioventricolare ( a ) , in assenza di segni di potenziamento nelle immagini ir - ce ( b )  . 
in the diffuse pattern , its presence may be better detected by comparing myocardial signal intensity with that of surrounding normal skeletal muscle in order to obtain an absolute estimate of signal increase ( t2w signal intensity ratio ) [ 3 , 17 ]  . 
 interestingly , it has been shown that in contrast to acute mi , the extent of t2w hyperintensity in myocarditis is very often smaller than de , with lack of correlation between volume of oedema and tissue fibrosis [ 16 ]  . 
furthermore , the t2w abnormality is not always associated with the presence of de , and depending on different stages of disease , oedema or de may be detected separately . 
these observed differences between t2w - stir and de abnormalities in acute mi and myocarditis may be an indirect sign of the different pathophysiological mechanisms underlying processes [ 16 , 17 ]  . 
 [ 13 ] hanno recentemente dimostrato come lemorragia si associ in genere ad una maggiore evidenza dei fenomeni di rimodellamento ventricolare positivo a breve - medio termine , indipendentemente dallestensione iniziale dellinfarto , con prognosi sfavorevole per i pazienti . un ulteriore pattern recentemente descritto e rilevabile in pazienti con ima o sindrome coronarica acuta riperfusi precocemente caratterizzato dalla presenza di edema parietale in stir t2 senza segni di de dopo somministrazione di mezzo di contrasto ( mdc )  . 
tale quadro rappresenta la manifestazione morfologica di un evento ischemico acuto con rivascolarizzazione coronarica precoce prima che si sia manifestato danno tissutale e definisce il cosiddetto aborted myocardial infarction o infarto abortito , termine utilizzato per la prima volta da weaver et al . 
come gi detto per lima , il valore aggiunto fornito dallimaging edema - weighted nelle miocarditi consiste nella possibilit di distinguere i pazienti che hanno radiol med ( 2011 ) 116 : 3246 fig . 
6a - c miocardite focale in un uomo di 21 anni , che si presenta alla nostra osservazione con febbre , doloro toracico acuto e lieve aumento dei livelli di troponina . 
al follow - up a 3 mesi , ledema miocardico completamente risolto ( c )  . primary cardiomyopathies as for ischaemic and inflammatory disease , t2w imaging has been proposed in previous reports in different cardiomyopathies ( cmp ) [ 3 ]  . 
showed that t2w hyperintensity usually matches location and distribution of late - enhancement areas in cmp and hypothesised that this phenomenon could be related to focal oedema accompanying ischaemia and / or inflammation or to different genotypes of the disease or to characteristics of collagen accumulated within the interstitium [ 20 ]  . 
in tako - tsubo cmp , the main hallmark of disease is characterised by a transitory ischaemia with completely reversible regional contractile dysfunction , most frequently involving apical segments with no angiographic signs of coronary artery disease [ 21 ]  . 
the name attributed to this entity relies on the shape of the lv , which appears as the bottle with a round bottom and a narrow neck used for trapping octopus . 
in this entity , t2w - stir could be useful un processo infiammatorio in atto da quelli che presentano forme croniche ; le sequenze t2 pesate offrono inoltre informazioni complementari alla tecnica di delayet enhancement ( de ) per la loro migliore correlazione con i markers biochimici della flogosi e quindi con lo stato di attivit della malattia [ 17 ]  . 
 a differenza dellinfarto acuto , stato inoltre dimostrato come lestensione dellarea di iperintensit del segnale nelle sequenze t2 pesate sia molto spesso inferiore rispetto alla zona di de , e non esista alcuna correlazione tra volume di edema e quota di tessuto fibroso [ 16 ]  . 
le sequenze stir t2 pesate acquisite in 4 camere mostrano una diffusa imbibizione edematosa del miocardio , che coinvolge i segmenti medio - apicali ( t2w ratio 2 , 8 )  . 
previous studies have shown focal infiltrates depicted on t2w images as zones of intramural increased three different from oedema , stages : neamente in pazienti con miocardite acuta come avviene ad esempio nellima ma rappresentano diversi stadi del processo flogistico ( ledema in genere precede la fibrosi ) e non raramente vengono rilevati isolatamente [ 16 , 17 ]  . 
 cardiomiopatie primitive oltre che per la valutazione delle patologie ischemiche ed infiammatorie , le sequenze stir t2 possono essere utilizzate anche in pazienti con cardiomiopatie primitive ( cmp ) [ 3 ]  . nella cmp ipertrofica , ledema potrebbe essere correlato alla presenza di caratteristiche aree di ischemia miocardica radiol med ( 2011 ) 116 : 3246 fig . 
t2w - stir images in the short - axis ( a ) and horizontal long - axis ( b ) planes show severe concentric left - ventricular symmetric hypertrophy of both cardiac chambers , with patchy areas of high signal intensity consistent with the presence of tissue oedema . 
le immagini stir t2 in asse corto ( a ) ed asse lungo orizzontale ( b ) mostrano una severa ipertrofia simmetrica e concentrica del ventricolo sinistro , con alcune aree ad elevata intensit del segnale e a distribuzione irregolare , compatibili con fenomeni di edema . 
le sequenze ir - ce dimostrano un tipico pattern , caratterizzato da multiple aree di potenziamento a distribuzione irregolare , dovuta al disarray delle fibre muscolari . signal intensity reflecting the oedema associated with inflammation [ 3 , 19 ]  . 
 [ 20 ] hanno dimostrato lesistenza di una stretta correlazione in rmc tra i segmenti di edema e di le , ipotizzando come lalterazione del segnale in stir t2 possa essere espressione di deposizione di matrice collagene ad elevato contenuto di acqua nellinterstizio o in alternativa dipendere dalledema che si associa ad eventi ischemici o forme di miocardite focale tipiche di questa cmp [ 20 ]  . 
9a - c tako - tsubo cardiomyopathy in a 49 - year - old woman with atypical chest pain , ischaemic electrocardiographic changes ( st - elevation < 2mm ) and mildly elevated troponin levels . 
end - systolic cine ssfp image on the vertical long axis shows the typical appearance of the apical ballooning phenomenon characterised by apical dyskinesis and basal hyperkinesis ( ejection fraction 41% in the acute phase ) ( a )  . 
9a - c cardiomiopatia di tako - tsubo in una donna di 49 anni , con dolore toracico atipico alterazioni elettrocardiografiche compatibili con patologia ischemica ( sovraslivellamento st < 2 mm ) e lieve incremento dei valori di troponina . 
le sequenze cine - ssfp acquisite in asse lungo in fase tele diastolica mostrano il tipico aspetto dellapical ballooning , caratterizzato da discinesia apicale ed ipercinesia basale ( frazione di eiezione 41% in fase acuta ) ( a )  . le sequenze stir t2 pesate acquisite in asse lungo evidenziano unarea di omogenea iperintensit del segnale coinvolgente i segmenti medio - apicali ( b )  . le immagini di ce - ir acquisite secondo gli stessi piani anatomici non evidenziano segni di potenziamento patologico miocardico . 
durante il follow - up ( immagini non mostrate ) , si riscontrato un buon recupero della funzionalit cardiaca ( fe 64% ) , con completa regressione delledema tissutale . myocardial oedema has been identified in experimental models correlating lv dysfunction with distribution of interstitial water . 
hypothesised that hypertension may affect fluid balance by both increasing filtration and decreasing lymph flow due to impaired lv venous drainage into the coronary sinus caused by increased rightventricle filling pressures [ 25 ]  . 
lipotesi pi probabile per spiegare la presenza di edema interstiziale pu essere legata alla ipersecrezione catecolaminica che si associa al tako - tsubo con edema vasogenico secondario . cardiomiopatie secondarie : sarcoidosi e amiloidosi dal punto di vista fisiopatologico , la sarcoidosi coinvolge il miocardio con pattern di distribuzione focale o diffuso , progredendo attraverso tre stadi , dalledema alla granulomatosi non caseosa fino alla fibrosi . 
inoltre evidente una focale area di potenziamento mesocardico a livello della giunzione interventricolare posteriore ( freccia )  . heart - transplant disease noninvasive detection of acute heart transplant rejection represents a serious clinical challenge , with endomyocardial biopsy remaining the preferred approach for early detection of acute rejection before allograft dysfunction [ 27 ]  . 
myocardial oedema may be an early indicator of acute organ rejection , probably due to an increase in water content caused by oedema and congestion , but it may also be related to the presence of focal haemorrhage and myocyte necrosis [ 27 , descritto aree focali di infiltrazione , visualizzate nelle sequenze t2 pesate come zone di incrementata intensit del segnale ad estensione transmurale , per la presenza di fenomeni edemigeni associati alla flogosi [ 3 , 19 ]  . 
nellamiloidosi cardiaca , ledema stato descritto sia in numerosi lavori di patologia che in studi effettuati con rmc e sembra essere correlato allaccumulo interstiziale di matrice amiloide con richiamo osmotico di fluidi [ 24 ]  . ipertensione polmonare nei pazienti con ipertensione polmonare primitiva o secondaria , ledema miocardico stato identificato attraverso modelli sperimentali che hanno permesso di correlare il grado di disfunzione del ventricolo sinistro ( vs ) con laccumulo di acqua nella matrice interstiziale . 
 [ 25 ] hanno ipotizzato che laumentata pressione nel circolo polmonare pu alterare lequilibrio degli scambi fluidi , incrementando i meccanismi di filtrazione e riducendo il flusso linfatico , a causa dellincompleto ritorno venoso a livello del seno coronarico e delle aumentate pressioni di riempimento del cuore destro [ 25 ]  . 
value of t2w imaging in this field of cardiac mri was emphasised in a previous paper by marie et al . , who found that t2w mri was a sensitive technique for detecting biopsy - proven acute rejections [ 27 ]  . sequenze stir t2 stato sottolineato in uno studio realizzato da marie et al . 
 [ 27 ] , che hanno mostrato con correlazione istologica come questa tecnica abbia elevata sensibilit nellidentificare fenomeni di rigetto acuto . conclusions conclusioni t2 - weighted stir represents an appealing and versatile technique that can be applied in a wide variety of ischaemic and nonischaemic conditions , allowing detection of segmental or global increase of myocardial free water content with high contrast difference between oedele sequenze stir t2 costituiscono uninteressante e versatile tecnica di imaging che pu trovare applicazione in una vasta gamma di patologie ischemiche e non , consentendo di identificare un incremento focale o diffuso del contenuto di acqua libera allinterno del miocardio , con elevata differenza di radiol med ( 2011 ) 116 : 3246 matous myocardium and surrounding tissues . 
raffaele dimiccoli , via ippocrate , 70051 barletta , italy 3department of radiology , scientific institute hospital casa sollievo della sofferenza , viale cappuccini 1 , 71013 san giovanni rotondo , italy correspondence to : s . 
between november 2007 and may 2008 , we performed shoulder mr examinations for shoulder pain or disability on 94 patients ( 46 males , 48 females ; age range 1679 years ; mean age 54.0915.09 years ) for a total of 104 shoulders ( 62 right , 42 left )  . 
rc was more easily detectable in oblique coronal scans where it appeared as a crescent - shaped , regularly marginated structure adjacent to the articular surface of the supraspinatus tendon and medial to the insertion point of this tendon on the greater tuberosity . 
il rc pi agevolmente individuabile nelle scansioni coronali - oblique ove appare come una struttura a forma di semiluna , con margini regolari , adiacente alla superficie articolare del tendine sopraspinato , medialmente al punto dinserzione di questo tendine sulla grande tuberosit . 
in our sample it did not seem to influence shoulder function in patients with cuff lesions . keywords magnetic resonance imaging shoulder rotator cable shoulder pain disability riguardava let tra pazienti con o senza evidenza di rc ( test t di student = 0 , 05 ; p = 0 , 82 )  . 
nel nostro campione di studio esso non sembra influenzare la funzionalit della spalla in pazienti con lesioni di cuffia . parole chiave risonanza magnetica spalla rotator cable dolore di spalla limitazione funzionale introduction introduzione tendon lesions of the rotator cuff are the most frequent cause of shoulder pa the incidence of full - thickness rotator cuff lesions ranges from 18% to 26% . 
this fibrotic structure arises from the coracohumeral ligament , with fibres that are perpendicular to the axis of the supraspinatus tendon , and arches anteriorly and posteriorly to attach onto the humerus [ 35 ]  . 
the latter is a thin sheet of the distal supraspinatus and infraspinatus tendon that is bound proximally by the rc and that corresponds to the [ 6 ] and codman and akerson [ 7 ] , in which cuff lesions occur most frequently . 
burkhart [ 2 ] considered the rc to be a functional system in which there is stress transfer from the rotator cuff to the thick cable and stress shielding of the thin capsular tissue distal to the cable , thus allowing the individual with a cuff lesion to preserve normal shoulder function . 
the aim of our study was to evaluate the rc with magnetic resonance ( mr ) imaging and to investigate its possible role in the biomechanics of the shoulder affected by cuff lesions . zone described by codman critical le lesioni della cuffia dei rotatori sono la causa pi frequente di dolore alla spalla . 
questa struttura fibrotica origina dal legamento coraco - omerale , con fibre perpendicolari allasse maggiore del tendine del sovraspinoso , e si inserisce sullomero con decorso arcuato [ 35 ]  . 
questultimo costituito da un sottile strato delle fibre di inserzione dei tendini sopraspinato e infraspinato , ed prossimamente in continuit con il rc . esso , inoltre , corrisponde alla critical zone descritta da codman [ 6 , 7 ] , in cui si verificherebbero pi frequentemente le lesioni di cuffia . 
burkhart [ 2 ] ha ipotizzato che il rc agisca come un sistema funzionale in cui si ha un trasferimento delle forze tensive dalla cuffia dei rotatori al cable che proteggerebbe il sottile tessuto capsulare situato distalmente al cable consentendo ai soggetti con lesioni di cuffia di conservare la normale funzionalit della spalla . 
secondo gli stessi autori [ 2 , 3 ] il rc tenderebbe ad ispessirsi con lavanzare dellet e ci lo renderebbe pi facilmente identificabile nei soggetti con et superiore ai 65 anni . 
lo scopo del nostro studio stato valutare il rc con la rm e definire il suo possibile ruolo nella biomeccanica della spalla in pazienti con lesione della cuffia dei rotatori . 104 radiol med ( 2011 ) 116 : 102113 fig . 
1 , tendine del sovraspinato ; 2 , rc ; 3 , testa omerale ; 4 , rotator crescent . materials and methods materiali e metodi our study comprised a morphological and a functional phase . abbiamo organizzato il nostro studio in due fasi : una fase morfologica ed una funzionale . morphological phase fase morfologica in this phase , we evaluated the mr appearance of the rc . we studied the morphological features of the structure attempting to establish in which sequences and sections the structure was more easily recognisable . 
moreover , in view of previous reports [ 3 ] of better evidence of the rc in elderly individuals ( > 65 years ) , we investigated whether this result was also confirmed in our sample . in questa fase abbiamo valutato laspetto rm del rc . abbiamo studiato le caratteristiche morfologiche della struttura cercando di individuare le sequenze ed i piani di scansione in cui essa risultava pi facilmente riconoscibile . inoltre , abbiamo valutato se il rc fosse pi evidente in soggetti con et superiore ai 65 anni come riportato in precedenti studi [ 3 ]  . functional phase fase funzionale in this phase , we evaluated whether the rc imaged with mr contributed to preserve shoulder function in patients in questa fase abbiamo cercato di valutare se il rc individuato con la rm sia in grado di preservare la f radiol med ( 2011 ) 116 : 102113 with rotator cuff lesions , as stated by other authors [ 2 , 3 ]  . unzionalit della spalla nei soggetti con lesione della cuffia dei rotatori come descritto da altri autori [ 2 , 3 ]  . patients between november 2007 and may 2008 , we enrolled 94 patients ( 46 males , 48 females ) for a total of 104 shoulders ( 62 right , 42 left )  . 
prior to mr examination , we obtained patient history , in particular , age , sex , reason for the examination and treatment with corticosteroids and / or ultrasoundguided treatment in the period before the examination . patients underwent a clinical examination based on jobes test ( arm abducted to 20 in the plane of the scapula , thumb pointing down ) [ 8 ] , to evaluate the presence of shoulder pain and shoulder disability . 
patients were excluded from the study if they had undergone surgical treatment for rotator cuff injury or were affected by diseases such as bone tumours that could affect shoulder function . mr imaging protocol mr scans were obtained with a 1.5 - t unit ( philips intera achieva , philips medical systems , best , the netherlands ) at centre 1 , a 0.2 - t unit ( esaote e - scan xq , genoa , italy ) at centre 2 and a 1.0 - t unit ( philips gyroscan nt , philips medical systems , best , the netherlands ) at centre 3 . 
patients were placed in a supine decubitus position , with the shoulder and scapula horizontal on the mr table to minimise motion artefacts , and the arm by the side in a neutral position or in mild external rotation . 
 presence or absence of degenerative alterations of the images were assessed for : supraspinatus tendon ; presence or absence of supraspinatus tendon tear , described as partial - thickness or full - thickness tear ; presence or absence of the rc . statistical analysis pazienti tra novembre 2007 e maggio 2008 abbiamo reclutato 94 pazienti ( 46 maschi e 48 femmine ) , per un totale di 104 spalle ( 62 spalle destre e 42 spalle sinistre )  . 
il reclutamento ha coinvolto 3 centri ( centro 1 , centro 2 e centro 3 )  . tutti i pazienti eseguivano lesame di rm della spalla su prescrizione di uno specialista in medicina dello sport o in ortopedia . 
in particolare ci siamo informati su : et , sesso , motivo dellesame , trattamento con cortisonici e / o con ultrasuoni ( us ) effettuato in data anteriore allesame rm . 
lesame clinico prevedeva leffettuazione del test di jobe ( braccio abdotto di 20 , sul piano della scapola , con pollice che punta in basso ) [ 8 ] , per valutare la presenza di : dolore di spalla , limitazione funzionale di spalla . 
abbiamo escluso dal nostro studio : pazienti sottoposti a trattamento chirurgico per lesione della cuffia dei rotatori , pazienti con patologie , quali ad esempio tumori ossei , che potessero alterare la funzionalit della spalla . protocollo rm lesame rm stato eseguito su 104 spalle . 
a p value < 0.05 was considered statistically significant . presenza o assenza di alterazioni degenerative del tendine sovraspinoso ; presenza o assenza di lesioni del tendine sovraspinoso descritte come lesioni parziali o complete ; presenza o assenza di rc . results morphological phase the rc was identified as a hypointense structure with respect to the tendon structures on proton density ( pd ) images . 
in oblique coronal scans , the structure appeared crescent - shaped , with almost regular margins , adjacent to the articular surface of the supraspinatus tendon medial to the tendons insertion point on the greater tuberosity . 
in those shoulders in which rc was observed ( 64 / 104 ) , the structure was identified in the oblique coronal scan in 53 / 64 ( 83% ) cases only , in the axial scan in 1 / 64 cases ( 2% ) only and in analisi statistica lanalisi statistica stata condotta con un software spss , versione 12.0 ( spss , chicago , illinois )  . 
un valore di p < 0 , 05 stato considerato statisticamente significativo . risultati fase morfologica allesame rm , il rc apparso riconoscibile come una struttura ipointensa rispetto alle strutture tendinee nelle immagini a densit protonica ( dp )  . 
nelle scansioni coronali - oblique , la struttura si presentava a forma di semiluna , con margini piuttosto regolari , adiacente alla superficie articolare del tendine sovraspinoso , medialmente al punto dinserzione di questultimo sulla grande tuberosit . 
moreover , in 7 / 64 cases ( 11% ) the structure was recognised in both the oblique coronal scan and the axial scan ; in 2 / 64 cases ( 3% ) , it was observed in both the oblique coronal scan and the sagittal scan . 
 functional phase functional data ( shoulder pain and / or disability on jobes test ) were available for 78 patients for a total of 84 shoulders ( 50 right , 34 left )  . 
a partial - thickness or full - thickness supraspinatus tendon tear was found in 28 / 84 cases ( 33% ) , in which no statistically significant difference was found between the presence or absence of the rc and disability on jobes test ( 2 = 1.17 ; p > 0.05 ) ( table 3 )  . 
similarly , in the remaining group ( 56 / 84 ; 67% ) with no supraspinatus tendon injury , there was no statistically significant difference between the presence or absence of rc and disability on jobes test ( 2 = 2.55 ; p > 0.05 ) , ( table 4 )  . 
nessuna differenza statisticamente significativa relativa allet stata trovata tra pazienti con presenza di rc e con assenza di rc ( test t di student = 0 , 05 ; p = 0 , 82 )  . 
nelle spalle in cui il rc stato individuato ( 64 / 104 ) , la struttura stata riconosciuta in 53 / 64 ( 83% ) casi solo in scansioni coronali - oblique , in 1 / 64 ( 2% ) casi solo in scansioni assiali , in 1 / 64 ( 2% ) casi solo in scansioni sagittali . 
inoltre , in 7 / 64 ( 11% ) casi la struttura stata individuata sia in scansioni coronali - oblique che in scansioni assiali ; in 2 / 64 ( 3% ) casi stata osservata sia in scansioni coronali - oblique che sagittali - oblique . fase funzionale i dati funzionali ( dolore alla spalla e / o limitazione funzionale al test di jobe ) si sono resi disponibili per 78 pazienti per un totale di 84 spalle ( 50 spalle destre e 34 sinistre )  . 
 stata osservata una correlazione statisticamente significativa tra presenza di lesione del sovraspinoso e limitazione funzionale al test di jobe ( beta = 1 , 649 ; p = 0 , 006 )  . 
inoltre , una correlazione statisticamente significativa stata riscontrata tra tendinopatia e dolore di spalla al test di jobe ( beta = 1 , 564 ; p = 0 , 05 )  . 
in questi ultimi non stata osservata alcuna differenza statisticamente significativa tra presenza o assenza di rc e limitazione funzionale al test di jobe ( 2 = 1 , 17 ; p > 0 , 05 ) , ( tabella 3 )  . 
allo stesso modo , nel rimanente gruppo senza evidenza di lesione del tendine alla rm ( 56 / 84 casi ; 67% ) , non stata riscontrata alcuna differenza statisticamente significativa tra radiol med ( 2011 ) 116 : 102113 table 3 functional data for patients with rotator cuff lesion on jobes test jobes test + for d 11 24 jobes test + for d 13 absence of rc presence of rc total rc , rotator cable ; d , disability 2 = 1.17 ; p > 0.05 assenza di rc presenza di rc totale rc , rotator cable ; d , impotenza funzionale 2 = 1 , 17 ; p > 0 , 05 absence of rc presence of rc total rc , rotator cable ; d , disability 2 = 2.55 ; p > 0.05 assenza di rc presenza di rc totale rc , rotator cable ; d , impotenza funzionale 2 = 2 , 55 ; p > 0 , 05 tabella 3 dati funzionali dei pazienti con lesione dei tendini della cuffia dei rotatori al test di jobe jobe test positivo per d jobe test negativo per d totale table 4 functional data for patients with no rotator cuff lesion on jobes test jobes test for d total tabella 4 dati funzionali dei pazienti senza evidenza di lesione dei tendini della cuffia dei rotatori al test di jobe jobe test positivo per d jobe test negativo per d totale jobes test for d 1 4 total fied on mr imaging in > 50% of cases ( 62% )  . 
 in contrast to the findings of burkhart et al . , who reported a mean rc thickness of 4.7 mm [ 2 , 3 ] , the rc reached a mean thickness of 2.8 mm in our sample . 
in our sample , the mean age of individuals with rc evidence at mr imaging was 55.314.9 years versus a mean age of 52.2815.5 years in presenza o assenza del rc e limitazione funzionale al test di jobe ( 2 = 2 , 55 ; p > 0 , 05 ) , ( tabella 4 )  . 
 jobes test is one of the most commonly used clinical tools for investigating shoulder pain and disability . however , its reproducibility is not high , thus leading to subjective evaluation errors . 
thus , jobes test proved to be a reliable tool for clinical examination of the shoulder . moreover , previous investigators [ 1113 ] reported a lack of association between shoulder disability on jobes test and rotator cuff lesions . 
conversely , we found a statistically significant correlation between shoulder disability on jobes test and rotator cuff lesions . shoulder pain is one of the most common symptoms reported by the patient . 
as a consequence , to limit the influence of variable pain on shoulder disability , we investigated individuals who received treatment with corticosteroid agents and / or ultrasound - guided therapy in the period prior to the mr examination . 
moreover , according to his hypothesis [ 2 , 3 ] , the presence of the rc could somehow influence preservation of normal shoulder function in patients with rotator cuff lesions , partly explaining why some individuals with rotator possiamo affermare che il rc una struttura riconoscibile in pi del 50% dei casi ( 62% )  . 
inoltre , attraverso le nostre sequenze di immagine , la sua identificazione risulta pi agevole nelle scansioni coronali - oblique , mentre pi difficoltosa nelle scansioni assiali e sagittali - oblique . 
che hanno riportato uno spessore medio del rc pari a 4 , 7 mm [ 2 , 3 ] , nel nostro campione tale struttura raggiunge uno spessore medio di 2 , 8 mtuttavia i risultati di burkhart et al . 
nel nostro campione , let media dei soggetti con rc alla rm era 55 , 314 , 9 anni contro unet media di 52 , 2815 , 5 anni dei pazienti nel quale il rc non era riconoscibile . 
inoltre , non stata osservata nessuna differenza statisticamente significativa tra questi due gruppi ( p = 0 , 82 )  . il test di jobe uno dei test clinici pi utilizzati per studiare il dolore di spalla e la limitazione funzionale . come noto , esso non ha unelevata riproducibilit essendo operatore - dipendente . 
di conseguenza , per limitare linfluenza della variabile dolore sulla limitazione funzionale di spalla , abbiamo valutato i pazienti che avevano effettuato terapia cortisonica e / o con us in data antecedente lesame di rm . 
nel nostro campione , i 7 / 84 ( 8% ) pazienti che avevano effettuato questo tipo di trattamento non hanno riferito alcun cambiamento significativo nella limitazione funzionale di spalla . 
per di pi , in accordo con la sua ipotesi [ 2 , 3 ] , la presenza del rc potrebbe in qualche modo condizionare la preservazione della normale funzionalit della spalla in pazienti con lesioni di cuffia giustificando in parte perch alcuni soggetti con lesioni della cuffia dei rotatori sono negativi al test di jobe . i nostri dati non mostrano alcuna differenza statisticamente significativa tra presenza e assenza di rc e limitazione funzionale al test di jobe in pazienti con lesioni del tendine sovraspinoso . 
pur considerando che i nostri risultati potrebbero essere stati in parte condizionati dalla bassa numerosit del campione e dal fatto che i macchinari rm a nostra disposizione potrebbero non aver potuto consentire di riconoscere il rc in alcuni casi , i nostri dati sembrano confutare le funzioni biomeccaniche attribuite alla struttura escludendo lipotesi che la presenza del rc potrebbe condizionare la funzionalit della cuffia dei rotatori . 
even considering that our results could be partly influenced by a small study sample and by the fact that mr scanners at our disposal did not allow recognition of the rc in some cases , our data seem to refute the biomechanical functions attributed to the rc and the hypothesis that the presence of this structure could condition rotator cuff function . 
second , our sample had a small number of young individuals ( mean age 54.09 years ) , and we were therefore unable to obtain any reliable estimation of possible evidence of the rc in the young population and of its possible influence on shoulder function . 
third , to make up for the small number of individuals with rotator cuff lesion ( 28 / 84 ) , we analysed our data in terms of presence or absence of lesions , without distinguishing between partialor full - thickness injuries . 
furthermore , mr imaging is known to have a high accuracy in diagnosing full - thickness lesions , reaching a sensitivity of 100% , but its performance in diagnosing partial - thickness tears is lower [ 15 ]  . 
this consideration adds to the fact that in the three centres involved in the study , shoulder mr imaging was performed with three different scanners and study protocols . nonetheless , we noted no substantial differences in shoulder imaging among the three centres . 
an additional limitation was that we had no follow - up data about surgical treatment in patients with rotator cuff injury and rc evidence on mr examination who did not show shoulder disability on jobes test . 
all three individuals had a partial - thickness supraspinatus tendon lesion . this finding , even though not statistically significant , allows us to hypothesise a possible biomechanical role for the rc in the case of partial - thickness tendon lesions in larger samples . 
further studies are needed to determine whether presence of the rc in patients with rotator cuff lesions could play a role in preserving shoulder function and partly influence the surgical management of such patients . 
questo risultato , pur non raggiungendo la significativit statistica , potrebbe far supporre in un campione pi grande un possibile ruolo biomeccanico del rc in caso di lesioni parziali del tendine . il nostro studio ha alcuni limiti . 
per compensare il basso numero di soggetti con lesione della cuffia dei rotatori ( 28 / 84 ) , abbiamo dovuto analizzare i nostri dati in termini di presenza o assenza di lesioni , senza distinguere tra lesioni parziali o complete . 
 inoltre noto che la rm ha unelevata accuratezza nel diagnosticare le lesioni complete ove raggiunge una sensibilit del 100% , ma le sue potenzialit nella diagnosi di lesioni parziali sono inferiori [ 15 ]  . 
questa considerazione si aggiunge al fatto che nei tre centri coinvolti nello studio , gli esami di rm della spalla sono stati effettuati utilizzando macchinari e protocolli di studio differenti . 
ad ogni modo non abbiamo notato differenze sostanziali nellimaging di spalla nei tre diversi centri.inoltre , non abbiamo dati di follow - up relativi ad un eventuale trattamento chirurgico effettuato in quei pazienti con lesioni della cuffia dei rotatori e presenza di rc allesame rm che non mostravano limitazione funzionale al test di jobe . in conclusione , nel nostro campione il rc stato individuato in pi del 60% dei casi . 
lambranzi , 1 , 37142 marzana , verona , italy 2centro di screening mammografico , dipartimento interaziendale di radiologia , azienda ospedaliera , padova , italy 3direzione generale ulss 20 , verona , italy correspondence to : s . 
lapprofondimento diagnostico stato eseguito indipendentemente dallarbitrato e il risultato dellapprofondimento ha costituito lo standard di riferimento ai fini dello studio . ipotizzando che un arbitrato negativo neghi la necessit di approfondimento , limpatto dellarbitrato stato valutato in termini di ridotto tasso di richiamo e di ridotto tasso diagnostico di carcinoma . 
i richiami alla doppia lettura sono stati 528 ( 6 , 8% ) dei quali 230 ( 43 , 5% ) concordanti e 298 ( 56 , 5% ) discordanti . 
complessivamente sono stati diagnosticati 49 carcinomi ( 6 , 39 tra soggetti esaminati , 9 , 2% tra soggetti richiamati ) : 43 carcinomi sono stati diagnosticati in richiami concordanti ( 5 , 6 tra soggetti esaminati , 18 , 6% tra richiami concordanti ) , 6 tra richiami discordanti ( 0 , 7 tra soggetti esaminati , 2 , 0% tra richiami discordanti )  . 
arbitration is a cost - effective procedure that could be employed as a first measure to counterbalance excess recall rate observed in a double - reading scenario . keywords breast cancer screening mammography double reading arbitration avrebbe risparmiato 216 procedure di approfondimento diagnostico ( riduzione del tasso di richiamo : 2 , 8% assoluta , 40 , 9% relativa ) e mancato la diagnosi di un carcinoma ( riduzione del tasso diagnostico : 0 , 13 assoluta , 2 , 0% relativa )  . 
larbitrato una procedura efficace e conveniente dal lato economico , che potrebbe essere impiegata correntemente quale prima misura di correzione ove si osservi un tasso di richiamo eccessivo , specie in scenari che impieghino la doppia lettura . parole chiave carcinoma mammario screening mammografia doppia lettura arbitrato introduction introduzione mammography screening has been demonstrated to be effective in reducing breast cancer mortality [ 1 ] and is currently recommended by the european commission ( ec ) [ 2 ]  . 
full coverage by organised programmes has been achieved or is under way in almost all western countries . in italy , real coverage was estimated to be 62.3% by the end of 2007 according to the last survey of the osservatorio nazionale screening [ 3 ]  . 
double reading , which is also recommended as a standard policy , as it substantially increases sensitivity [ 4 ] , is associated with increased rr [ 5 , 6 ]  . 
arbitration of discordant double readings has been recommended as a measure to reduce excess rr [ 7 , 8 ]  . arbitration of discordant double readings was first evaluated in italy in the florence programme [ 9 ] , and its usefulness in reducing rr was confirmed , together with a very limited and acceptable reduction in cancer detection rate ( dr )  . 
in this study , we evaluated the role of arbitration of discordant double readings in the screening programmes of the cities of verona and padua , veneto region , italy . lo screening mammografico stato dimostrato essere efficace nel ridurre la mortalit per carcinoma mammario [ 1 ] ed correntemente raccomandato dalla comunit europea ( ce ) [ 2 ]  . 
in italia la copertura reale stata stimata essere del 62 , 3% alla fine del 2007 , in base alla ultima analisi diffusa dallosservatorio nazionale screening [ 3 ]  . 
tra questi , il tasso di richiamo ( recall rate , rr ) ad approfondimento diagnostico di casi positivi allo screening non dovrebbe eccedere il 7% al primo screening e il 5% agli screening successivi . 
la doppia lettura , anchessa raccomandata come pratica corrente perch capace di aumentare sostanzialmente la sensibilit [ 4 ] , associata ad un aumento del rr [ 5 , 6 ]  . larbitrato delle doppie letture discordanti stato raccomandato come misura atta a ridurre leccesso di rr causato dalla doppia lettura [ 7 , 8 ]  . 
larbitrato delle doppie letture discordanti stato valutato una prima volta in italia nel programma di firenze [ 9 ] e la sua utilit nel ridurre il rr stata confermata , unitamente ad una riduzione , molto limitata ed accettabile , del tasso diagnostico di carcinoma ( detection rate , dr )  . 
nel presente studio viene valutato il ruolo dellarbitrato delle doppie letture discordanti nei programmi di screening delle citt di verona e padova , regione del veneto . material and methods materiali e metodi verona ( 2008 target - year population = 28 , 760 ) and padua ( 2008 target - year population = 27 , 377 ) mammography screening programmes were implemented in 1999 and i programmi di screening delle citt di verona ( popolazione obiettivo per lanno 2008 , 28760 ) e di padova ( popolazione obiettivo per lanno 2008 , 27377 ) sono in attivit rispettivaradiol med ( 2011 ) 116 : 8491 2001 , respectively . 
overall rr has been reported to be higher than recommended standards at both first and repeat screening [ 10 ] , and a study of the impact of arbitration of discordant double readings by a third reader was designed and performed in 2009 - 2010 . 
recalls to diagnostic assessment by one of two readers only ( henceforth referred to as discordant ) were arbitrated by a third reader ( sc , with > 30 years experience and > 200 , 000 readings in screening mammography )  . 
all cases , irrespective of arbitration result , underwent diagnostic assessment , the result of which was assumed as the reference standard for the study purpose . diagnostic assessment was based on bilateral palpation and ultrasonography in all cases . 
data for each recalled case were patients id , screening date , firstand second - reader report ( negative , recall ) , arbitration report for discordant double readings ( negative , recall ) and final outcome ( cancer , no cancer ) for assessed cases ( concordant or discordant recalls )  . 
 cost analysis of the introduction of arbitration in the screening process was also attempted , although this was not easy , as detailed screening cost estimates in the italian scenario are few and relate to different calendar periods . we chose the 2005 florence scenario as a reference , as data allowing for adjustment by discount rate and conversion to euros were available for this setting only . 
arbitration cost implied adding only a third additional reading : cost per digital mammography reading in the florence scenario was estimated to be 0.25 euros in 2005 by ciatto et al . 
in particolare impiegano entrambi la mammografia digitale diretta con doppia lettura differita : sei radiologi , aventi esperienza di lettura mammografica da 2 a oltre 10 anni , sono attualmente addetti alla lettura delle mammografie di screening . 
il rr complessivo stato riportato essere pi elevato degli standard accettabili raccomandati sia al primo screening che ai successivi [ 10 ] : per questo , uno studio dellimpatto dellarbitrato delle doppie letture discordanti da parte di un terzo lettore stato disegnato e condotto nel periodo 20092010 . 
i richiami richiesti da un solo dei due lettori ( dora in avanti indicati come discordanti ) sono stati arbitrati da un terzo lettore ( sc ) con una esperienza di oltre 30 anni e oltre 200000 mammografie lette . 
tutti i casi , a prescindere dai risultati dellarbitrato , sono comunque stati sottoposti ad accertamento diagnostico , il cui risultato stato assunto come standard di riferimento ai fini dello studio . 
i dati disponibili per ogni caso di richiamo sono stati : identificativo della paziente , data dellesame di screening , diagnosi posta dal primo e dal secondo lettore ( negativo , richiamo ) , risultato dellarbitrato per i casi discordanti ( negativo , richiamo ) , esito finale ( carcinoma , altro ) per i casi sottoposti ad accertamento diagnostico ( concordanti o discordanti alla doppia lettura )  . 
stato in particolare calcolato quale sarebbe stato limpatto dellarbitrato in termini di rr e di dr di carcinoma se i casi discordanti arbitrati come negativi non fossero stati avviati ad approfondimento . stata tentata anche una analisi dei costi conseguenti alla introduzione dellarbitrato nel processo di screening , anche se questo non stato facile , dal momento che stime dettagliate del costo dello screening nello scenario italiano sono state poche e riferite a periodi diversi . 
abbiamo scelto lo scenario del programma di screening di firenze nel 2005 , dal momento che solo i dati di questo studio consentivano lapplicazione del tasso di sconto e la conversione ad euro . 
i costi relativi allarbitrato consistono sostanzialmente nel costo aggiuntivo di una terza lettura : il costo relativo alla lettura con mammografia digitale diretta stato stimato nel 2005 da ciatto et al . 
 [ 12 ] nel 1995 in 83 , 3136 , 3 dollari statunitensi per accertamento : dopo aggiustamento per tasso di sconto 19952005 e tasso di radiol med ( 2011 ) 116 : 8491 $5 , 1807 , 424 in 1995 after adjustment for the 19952005 discount rate and conversion to euros corresponded to 4 , 1956 , 013 erous per screen - detected cancer . 
overall , 49 cancers were detected ( 6.39 among screened , 9.2% among recalled ) : 43 were detected among concordant recalls ( 5.6 among screened , 18.6% among concordant recalls ) and six among discordant recalls ( 0.7 among screened , 2.0% among discordant recalls )  . six were observed among arbitrated cases : five ( 6% ) in positive and 1 ( 4.6 ) in negative arbitrations . 
if discordant recalls arbitrated as negative had not been assessed , returning to periodic screening , 216 assessment procedures would have been spared and one cancer would have been missed . 
the results are summarised in table 1 , where they are compared with those of the only other italian report on the arbitration procedure [ 9 ] , studying a larger series from another population - based screening setting employing different operators . 
the chosen study setting was suitable , as it had a high rr compared with recommended standards : an average rr of 6.8% is high , considering that the large majority of screened women were at repeat screening and that the recommended acceptable highest rr at repeat screening is 5% [ 2 ]  . 
 the choice of arbitrating only discordant double readings proved correct : in fact , discordant double readings conversione dollaro statunitense / euro il costo corrispondente risultato di 67 , 4110 , 4 euro per approfondimento . 
le differenze osservate nello studio sono state valutate mediante test chi - quadrato ( 2 ) , ponendo la significativit statistica a un livello di p < 0 , 05 . 
i richiami totali ad approfondimento alla doppia lettura sono stati 528 ( 6 , 8% ) dei quali 230 ( 43 , 5% ) concordanti e 298 ( 56 , 5% ) discordanti . 
questi ultimi sono stati sottoposti ad arbitrato , risultato rispettivamente negativo in 216 ( 72 , 4% ) e positivo in 82 ( 27 , 6% )  . complessivamente sono stati diagnosticati 49 carcinomi ( 6 , 39 tra soggetti esaminati , 9 , 2% tra soggetti richiamati ) : 43 carcinomi sono stati diagnosticati in richiami concordanti ( 5 , 6 tra soggetti esaminati , 18 , 6% tra richiami concordanti ) , e solo 6 in richiami discordanti ( 0 , 7 tra soggetti esaminati , 2 , 0% tra richiami discordanti )  . 
dei 6 cancri osservati nei casi arbitrati , in 5 ( 6% tra soggetti arbitrati ) larbitrato era stato positivo , e in un solo caso ( 4.6 tra soggetti arbitrati ) era stato negativo . 
se i casi con doppia lettura discordante arbitrati come negativi non fossero stati sottoposti ad approfondimento diagnostico , sarebbero stati risparmiati 216 approfondimenti e non sarebbe stato diagnosticato un carcinoma . 
i risultati sono riassunti nella tabella 1 , dove sono confrontati con quelli ottenuti dallaltro studio italiano in tema di arbitrato [ 9 ] , che aveva valutato una serie assai pi numerosa in un altro scenario di screening coinvolgente altri operatori . 
per quanto riguarda lanalisi dei costi nello scenario di studio , il costo relativo allarbitrato ( 298 terze letture di doppie letture discordanti ) stato pari a 74 euro mentre quello dei 216 approfondimenti risparmiati pari a 14558 , 423346 euro . 
 la scelta di arbitrare solo le doppie letture discordanti si dimostrata corretta : infatti le doppie letture discordanti hanno un valore predittivo positivo molto basso ( 2 , 0% ) rispetto alle concordanti ( 18 , 6% ) e danno il massimo contributo ai richiami inutili ( 60 , 9% )  . 
larbitrato negativo avrebbe consentito una drastica riduzione del rr che sarebbe rientrato negli standard raccomandati : infatti le doppie letture concordanti e gli arbitrati positivi di doppie letture discorradiol med ( 2011 ) 116 : 8491 tion . 
the arbitrated subset setting allows for a biased sensitivity assessment : first , cancers detected only by one of two readers are considered to be difficult , being selected among those with borderline and subtle signs ; second , the aim of arbitration is not to detect ( the lesion has already been detected by one reader ) but to classify another readers finding . 
with all these limitations and using assessment outcome as the reference standard , arbitration had a sensitivity of 83.3% ( five of six cancers in the arbitrated subset )  . 
 that introducing the arbitration procedure is highly convenient from the cost viewpoint is quite evident , although sparing the cost of a substantial proportion of assessment procedures will not ultimately have a major impact on screening costs ( cost per woman screened ) , which are mainly accounted for by organisation and firstscreening level [ 12 ]  . 
the net balance of 23 , 272 eruos in this study setting was much higher than the estimated cost of 4 , 195 - 6 , 013 euros per cancer detected at screening . 
not that we should necessarily consider missing one cancer as a cost to be negotiated ; nevertheless , in a context such as italy , where full real coverage of the eligible population by organised screening is still far from 100% , the money saved by introducing the arbitration procedure could be properly invested in the screening process , allowing for increased coverage . according to our findings , for each cancer missed due to false negative arbitration , the resources saved by arbitration could allow for three to five additional cancers being detected in women who are presently unscreened due to a lack of resources . 
second , assessment of discordant readings , often managed by simple additional views or ultrasonography , is likely to have a lower cost compared with assessment of concordant recalls , which is more likely to prompt costly invasive procedures . 
considering such possible biases in cost estimates , cost analysis was repeated by doubling the cost per mammography reading to 0.50 euros and by reducing the cost per assessment procedure to 50 euros . 
even with these changes , the net balance of introducing the arbitration procedure remains favourable , with an overall saved cost of 10 , 651 euros , which , if invested in screening more women , would allow for two additional cancers detected per each cancer missed by false negative arbitration . danti assommano a 312 casi , pari ad un rr del 4 , 0% . 
da un lato i casi arbitrati vengono comunemente ritenuti difficili , in quanto diagnosticati da uno solo di due lettori e quindi associati a segni radiologici sottili e al limite del normale . dallaltro il fine dellarbitrato non quello di percepire ( la lesione gi stata percepita da uno dei due lettori ) , ma di classificare il giudizio di un altro lettore . 
 che introdurre la pratica dellarbitrato sia conveniente sul piano dei costi ben evidente , anche se risparmiare i costi di un numero sia pure sostanziale di approfondimenti alla fine non avr un impatto ingente sui costi complessivi dello screening ( costo per donna esaminata ) che sono determinati in maggior misura dai costi organizzativi e dal primo livello di screening [ 12 ]  . 
in ogni caso lanalisi dei costi consente un punto di vista particolare per quantizzare la rilevanza delle mancate diagnosi di carcinoma dovute a arbitrato negativo . il bilancio netto di 23272 euro risparmiati per ogni mancata diagnosi di carcinoma osservato in questo studio assai pi elevato del costo per carcinoma diagnosticato allo screening , stimato in 41956013 euro . 
non che questo significhi in assoluto che la mancata diagnosi di un carcinoma debba essere necessariamente negoziata : tuttavia , in un contesto quale quello italiano , dove la copertura totale della popolazione eleggibile da parte di screening organizzato ancora lontana dal 100% , le risorse risparmiate introducendo la pratica dellarbitrato potrebbero essere utilmente reinvestite nel processo di screening , consentendo un aumento della copertura . 
in base alle nostre osservazioni , per ogni carcinoma non diagnosticato con lintroduzione dellarbitrato , il risparmio economico avrebbe consentito la diagnosi di 35 carcinomi aggiuntivi in soggetti attualmente non sottoposti a screening per mancanza di risorse . 
anzitutto questi sono stati stimati in base a quelli determinati in un centro di eccellenza , dove ci si pu attendere che la lettura sia pi efficiente che in uno scenario nazionale medio . 
inoltre lapprofondimento delle doppie letture discordanti , spesso risolto con semplici particolari mammografici o con la sola ecografia , potrebbe avere un costo inferiore a quello dei richiami per doppia lettura concordante , che pi facilmente generano i costi relativi a procedure invasive . 
anche con queste variazioni il bilancio netto relativo alla adozione dellarbitrato resta favorevole , con un costo medio risparmiato di 10651 euro , che , se investito in estensione della copertura da screening , consentirebbe la radiol med ( 2011 ) 116 : 8491 relatively few literature studies address the issue of arbitration of discordant double readings used as a current procedure . 
kopans [ 13 ] reports that a third reading substantially reduces recall rate to diagnostic assessment . in a study comparing computer - aided detection and arbitration of discordant double readings , james and cornford [ 14 ] confirm that arbitration reduces overall recalls and is substantially more specific compared with computer - aided detection while missing a limited number of cancers due to false negativity . 
 [ 7 ] , in which arbitration by a panel of three experts of discordant double readings persisting after consensus reduced discordant recalls by 51% and overall recalls by 14% , with a limited number of false negatives . 
however , this study is based on an extremely low recall rate ( 1% ) , quite common in dutch programmes , and is hardly comparable with other european scenarios . similar comparison of our study findings with the florence experience is particularly interesting . 
although the overall settings were ( organised population - based screening of 50to 69 - year - old women , most at repeat screening , using delayed double reading ) , the operators involved were different ( sc , who did the arbitration in the study reported here , was working in florence in 2005 but had no active role in double reading or arbitration of that series ) and the programmes had a different history : florence was a well - known excellence centre with 35 years experience in population - based screening and 10 years practice of double reading , whereas the verona and padua screening programmes are current service screening entities in which double reading has only recently been introduced . 
nevertheless , a substantial concordance of results observed between the florence experience and our study adds to the reproducibility and generalisability of our study findings to any other italian service screening scenario . 
 in conclusion , arbitration is confirmed to be a costeffective procedure and might be employed as a first measure to counterbalance the excess rr observed in a double - reading scenario . diagnosi di 2 carcinomi aggiuntivi per ogni cancro con mancata diagnosi a causa di arbitrato negativo . relativamente poco stato pubblicato in letteratura in merito alluso corrente dellarbitrato di doppie letture discordanti . 
in un confronto tra diagnosi computer assistita e arbitrato james e cornford [ 14 ] confermano la riduzione del tasso di richiamo operata dallarbitrato , che risulta decisamente pi specifico della computed assisted diagnosis ( cad ) , anche se perde qualche carcinoma per false negativit . 
levidenza pi interessante forse quella riportata da duijm et al . [ 7 ] : larbitrato da parte di un panel di tre esperti delle doppie letture discordanti anche dopo consenso riduce i richiami discordanti del 51% e i richiami complessivi 14% , con un numero limitato di falsi negativi : peraltro tale esperienza , che parte da un tasso di richiamo eccezionalmente basso ( 1% ) come comunemente in uso in olanda , mal confrontabile con le altre esperienze europee . 
anche se lo scenario complessivo simile ( screening organizzato di popolazione di donne residenti dai 50 ai 69 anni di et , per lo pi a screening ripetuto , con doppia lettura differita ) gli operatori coinvolti erano diversi ( sc , che ha eseguito larbitrato nel presente studio , operava nello screening di firenze nel 2005 ma non aveva partecipato n alla doppia lettura n allarbitrato relativamente a quello studio ) e i programmi presentavano differenze . 
firenze era un centro di eccellenza noto , con 35 anni di esperienza di screening di popolazione e 10 anni di pratica corrente di doppia lettura , mentre verona e padova sono , sia pure con buona qualit , realt di screening di servizio dove la doppia lettura differita stata introdotta solo recentemente . 
passariello1 1department of radiological science , university of rome la sapienza policlinico umberto i , viale regina elena 328 , 00161 rome , italy 2igea biophysics laboratory , carpi , modena , italy correspondence to : c.v. 
ad - sos and ubpi were measured at the phalangeal metaphysis using a dbm sonic device . multiple logistic regression analysis was performed to estimate the odds ratio ( or ) for vertebral fractures . 
confronto fra i parametri ultrasonografici quantitativi ( qus ) amplitude dependent speed of sound ( ad - sos ) e ultrasound bone profile index ( ubpi ) a livello delle falangi della mano con la densit minerale ossea ( bmd ) alla colonna lombare e al femore prossimale mediante tecnica dual energy x - ray absorptiometry ( dxa ) nel discriminare le fratture vertebrali . 
fractures resulting from osteoporosis lead to high rates of morbidity and mortality and reduce quality of life and are responsible for a sharp increase in healthcare costs [ 2 , 3 ]  . 
demographic studies also indicate a continuing increase in the proportion of women older than 50 years , and this segment of the population is expected to become predominant in the decades to come . 
in postmenopausal women , it would be both useful and desirable to adopt a preventive approach to the problem , with the aim of slowing disease progression and preventing many fractures [ 5 ]  . 
in order to promote a more rational allocation of resources , a reliable diagnostic method is required that is capable of identifying women at increased risk of fracture . over the past 25 years , many noninvasive methods have been developed that rely on the attenuation of ionising radiation to quantify bone mineral density ( bmd ) at the peripheral , axial and total skeletal level . 
these methods provide a measure of bmd , which is currently considered the best predictor of osteoporotic fractures [ 7 ]  . however , these techniques are still only able to explain 60%80% of the variability in bone strength , and it has been demonstrated that other mechanical aspects of the bone are important in determining fracture risk [ 8 ]  . 
these factors include microarchitectural parameters and geometric and elastic properties of bone tissue , which cannot be assessed using densitometric techniques [ 9 ]  . quantitative ultrasound ( qus ) methods have been developed over the past 10 years to determine bone quality and the state of the skeleton on the basis of various studies that suggest that sonographic parameters were able to provide information not only about bone density but about its structure and elastic properties [ 1012 ]  . 
the qus devices currently available can be applied to different peripheral sites of the skeleton : the calcaneus , the proximal phalanges of the hand and the tibial sha interest in this technique stems from practical , economic and health safety aspects : it is much faster , simpler and more portable than dxa ; it is less expensive , and it does not employ ionising radiation . these features suggest a role for qus as an effective losteoporosi una malattia scheletrica sistemica caratterizzata da ridotta massa ossea e deterioramento della microarchitettura del tessuto , con un conseguente aumento della fragilit ossea e del rischio di frattura [ 1 ]  . 
le fratture conseguenti allosteoporosi portano ad elevata morbilit e mortalit , riducono la qualit della vita degli individui affetti , e sono responsabili di un forte aumento dei costi del sistema sanitario [ 2 , 3 ]  . 
nelle donne in post - menopausa un approccio preventivo al problema , prima dellevento frattura , sarebbe utile e auspicabile , con la finalit di rallentare la progressione della malattia ; in tal modo molte fratture potrebbero essere evitate [ 5 ]  . 
per favorire una pi razionale allocazione delle risorse importante quindi utilizzare una metodica diagnostica affidabile per identificare le donne a maggior rischio di frattura . negli ultimi 25 anni sono state sviluppate molte metodiche non invasive basate sullattenuazione delle radiazioni ionizzanti per quantificare la densit minerale ossea ( bmd ) a livello scheletrico periferico , assiale e totale . 
i metodi maggiormente utilizzati sono : la densitometria a doppio raggio x ( dxa ) e la tomografia quantitativa computerizzata ( qct ) [ 6 ]  . questi metodi forniscono la misura della bmd , che attualemtne considerata il migliore predittore delle fratture osteoporotiche [ 7 ]  . 
comunque , queste tecniche sono in grado di spiegare solo il 60%80% della variabilit della resistenza ossea , inoltre stato evidenziato che altri aspetti meccanici dellosso sono importanti nella determinazione del rischio di frattura [ 8 ]  . 
questi fattori includono i parametri microarchitetturali , le propriet elastiche e geometriche del tessuto osseo , che non sono valutabili mediante le tecniche densitometriche [ 9 ]  . i metodi ultrasonografici quantitative ( qus ) sono stati sviluppati negli ultimi 10 anni per la determinazione della qualit dellosso e dello stato dello scheletro sulla base di varie esperienze che suggerivano che i parametri ultrasonografici potessero fornire informazioni non solo sulla densit ossea , ma anche sulla struttura e sulle propriet elastiche del tessuto [ 1012 ]  . 
linteresse verso questa metodica nasce da aspetti pratici : infatti molto pi veloce , semplice e trasportabile rispetto alla dxa ; da aspetti economici : infatti meno costosa ; da aspetti di sicurezza : non utilizza radiazioni ionizzanti . 
queste caratteristiche suggeriscono per radiol med ( 2011 ) 116 : 92101 screening tool for osteoporosis in postmenopausal women . the purpose of this study was to assess the ability of the ultrasound parameters of amplitude - dependent speed of sound ( ad - sos ) and ultrasound bone profile index ( ubpi ) when compared with those of dxa in identifying women with and without osteoporotic vertebral fractures . la qus una collocazione come efficace strumento di screening per losteoporosi nella popolazione femminile postmenopausale . 
lo scopo di questo studio di valutare labilit dei parametri ultrasonografici amplitude dependent speed of sound ( ad - sos ) e ultrasound bone profile index ( ubpi ) in confronto con i parametri dxa nellidentificare le donne con e senza fratture vertebrali da osteoporosi . materials and methods study population materiali e metodi popolazione a total of 692 postmenopausal italian women aged between 45 and 84 years were enrolled in the study . 
women receiving antiosteoporosis agents , such as calcitonin , oestrogen , raloxifene , bisphosphonates , anabolic steroids , parathyroid hormone , teriparatide , calcium and vitamin d were also excluded , as were all those with a history of severe trauma or previous fractures . 
lindice di massa corporea ( body mass index , bmi ) stato calcolato come rapporto fra peso espresso in kg e il quadrato dellaltezza espresso in metri [ 13 ]  . 
inoltre sono state escluse le donne in trattamento con farmaci utilizzati per la cura dellosteoporosi quali calcitonina , estrogeni , raloxifene , bifosfonati , anabolizzanti , paratormone , teriparatide , calcio e vitamina d . 
lo studio stato approvato dal comitato etico locale . bone densitometry densitometria ossea bone densitometry using a dxa device ( hologic qdr 2000 plus , bedford , ma , usa ) was performed on all individuals . 
a technician with 15 years of experience ( em ) obtained scans of the lumbar spine ( l1l4 ) and proximal femur ( neck and total hip ) in the posteroanterior projection . the individuals for whom it was impossible to assess at least three vertebral bodies were excluded from the analysis . individuals with fractured lumbar vertebrae and vertebrae exhibiting major degenerative changes on radiography were excluded from the analysis to avoid artefacts in the measurement of the bmd . 
results are expressed as bmd ( g / cm2 ) obtained by dividing the mineral content in the region of interest ( roi ) by the area of the region . 
da un tecnico con circa 15 anni di esperienza ( em ) , utilizzando uno strumento dxa ( hologic qdr 2000 plus , bedford , ma , usa )  . 
i risultati sono espressi in termini di bmd ( g / cm2 ) ottenuta dal rapporto fra il contenuto minerale rilevato nella regione di interesse e larea della regione stessa . 
ogni individuo stato classificato sulla base del suo t - score in normale ( t - score > 1 , 0 ) , osteopenico ( t - score compreso tra 1 , 0 e 2 , 5 ) , o osteoporotico ( tscore2 , 5 ) in accordo con i criteri diagnostici della organizzazione mondiale della sanit [ 15 ]  . 
la precisione a lungo termine ( coefficiente di variazione ) stato determinato ripetendo 2 volte sullo stesso soggetto , la scansione entro 5 settimane in 80 donne con et compresa tra 30 e 55 anni . radiol med ( 2011 ) 116 : 92101 phalangeal ultrasonography ultrasound measurements were performed on all individuals at the distal metaphysis of the proximal phalanges of the last four digits of the hand using the dbm sonic bone profiler device ( igea , carpi , italy )  . 
the device consists of two piezo - electric transducers coaxially mounted on a high - precision calliper , the first acting as a transmitter and the second as a receiver . 
the device automatically calculates the speed of signal transmission through the phalanx by measuring the thickness of the digit ( including soft tissues ) and the time required for transmission . 
in this way , the calculated speed is dependent on amplitude and thus termed amplitude - dependent speed of sound ( ad - sos ) [ 17 ]  . we also assessed an additional parameter known as the ultrasound bone profile index ( ubpi ) , which is derived from three sonographic parameters combined to maximise discrimination between individuals with and without fractures , as recently reported [ 14 , 18 ]  . 
to determine reproducibility of the method , the same operator who carried out the study measurements calculated the variation coefficient by performing six repeated measurements on five individuals . assessing vertebral fractures to assess for the presence of vertebral fractures ( either lowenergy or spontaneous ) , lateral radiographs of the thoracolumbar spine were obtained with an x - ray system identical to that employed in thoracic imaging [ 18 ] ; all study participants were subjected to spinal radiography with standardised procedures that we have recently reported [ 19 ]  . radiographs were evaluated by two independent radiologists ( cva and rp ) using the semiquantitative method proposed by genant et al . 
 le misure sono state eseguite sulla mano non dominante , anche se stato osservato in precedenti studi che non ci sono differenze statisticamente significative fra le misure sulle due mani [ 16 ]  . 
lapparecchio costituito da due sonde piezoelettriche montate coassiali su un calibro di precisione , una agisce come emittente e una come ricevente ; le sonde vengono posizionate da una parte e dallaltra rispetto alla falange da misurare sulle superfici latero - mediale della stessa . 
lo strumento calcola automaticamente la velocit di trasmissione del segnale attraverso la falange misurando lo spessore del dito ( inclusi i tessuti molli ) e il tempo di trasmissione del segnale : il rapporto fra le due grandezze fornisce la velocit di trasmissione . 
il tempo di trasmissione definito come il tempo trascorso fra lemissione del segnale da parte della sonda emittente e la ricezione del segnale quando esso raggiunge una ampiezza predeterminata ( 2 mv ) per la prima volta ; in tal modo la velocit calcolata viene detta dipendente dallampiezza e chiamata ad - sos [ 17 ]  . 
 abbiamo inoltre valutato un ulteriore parametro chiamato ubpi che deriva da 3 parametri ultrasonografici combinati per ottimizzare la discriminazione fra soggetti con e senza fratture , come riportato recentemente [ 14 , 18 ]  . 
per determinare la riproducibilit dello strumento , stato calcolato il coefficiente di variazione , ottenuto dallo stesso operatore che ha effettuato le misure dello studio , effettuando 6 misure ripetute su 5 soggetti . valutazione delle fratture vertebrali per valutare la presenza di fratture vertebrali ( a bassa energia o spontanee ) , sono state eseguite delle radiografie del rachide dorso - lombare in proiezione laterale utilizzando lo stesso strumento radiologico per immagini toraciche [ 18 ] ; tutti i partecipanti allo studio sono stati sottoposti a radiografia spinale mediante procedure standardizzate come da noi riportato recentemente [ 19 ]  . 
 quality assurance ulteriori controlli di qualit quotidiani sono stati eseguiti sulle apparecchiature dxa e qus in accordo con le linee guida delle aziende produttrici per verificare la stabilit e calibrazione degli strumenti . 
 radiol med ( 2011 ) 116 : 92101 turers guidelines to verify device stability and calibration . analisi statistica statistical analysis data analysis was performed using spss 15.0 software ( spss inc . , chicago , il , usa )  . 
based on roc analysis , we calculated the area under the curve ( auc ) and its standard errors ( se ) with significance levels using the method of hanley and mcneil [ 21 ]  . 
la regressione logistica multipla stata eseguita per determinare gli odds ratio ( or ) per le fratture vertebrali e i corrispondenti intervalli di confidenza al 95% ( ci )  . 
gli or sono stati aggiustati per le variabili antropometriche risultate significative et , altezza , peso per calcolare il rischio di frattura per diminuzione di una deviazione standard della variabile considerata . 
dallanalisi roc stata calcolata larea sotto la curva ( auc ) e gli errori standard con i relativi livelli di significativit seguendo il metodo di hanley e mcneil [ 21 ]  . 
valori di significativit inferiori a 0 , 05 sono stati considerate statisticamente significativi . risultati i dati raccolti durante le procedure di quality assurance per entrambi gli strumenti non hanno rilevato nessun malfunzionamento o scostamento dai valori di calibrazione durante lintero periodo di durata dello studio . 
la precisione delle misure e lintervallo di confidenza al 95% sono state calcolate per la bmd a livello lombare e femorale rispettivamente : 1 , 0% ( 0 , 9%1.1% ) , 1 , 2% ( 1 , 0%1 , 3% )  . 
although not strong , the correlations were all highly significant ( p < 0.00001 ) , as shown in table 2 . lanalisi della regressione lineare tra le variabili qus e dxa ha evidenziato la migliore correlazione rispettivamente tra il parametro ad - sos e la bmd del rachide lombare ( r = 0 , 31 )  . 
le correlazioni , sebbene non elevate , sono risultate tutte altamente significative ( p < 0 , 00001 ) come riportato in tabella 2 . radiol med ( 2011 ) 116 : 92101 table 1 clinical , anthropometric , dual - energy x - ray absorptiometry ( dxa ) and quantitative ultrasound ( qus ) characteristics in individuals with and without osteoporotic vertebral fractures . 
however , both the dxa and qus variables showed higher levels of significance ( p < 0.0001 ) in discriminating fractures when compared with age and the other anthropometric parameters ( table 3 )  . 
in the multiple logistic model , all dxa and qus measurements contributed significantly to the discrimination of fractures ( p < 0.0001 ) , as can be seen in table 3 . 
comunque sia le misure dxa che qus hanno mostrato un migliore livello di significativit ( p < 0 , 0001 ) nella discriminazione delle fratture rispetto alla variabile et e agli altri parametri antropometrici ( tabella 3 )  . nel modello logistico multiplo tutte le misure dxa e qus hanno mostrato un significativo contributo alla discriminazione delle fratture ( p < 0 , 0001 ) , come si pu osservare nella tabella 3 . 
comunque i valori di ors , che indica laumento del rischio per diminuzione di 1 deviazione standard della misura , sono risultati maggiori per la bmd alla colonna lombare ( or 4 , 01 ) , seguiti dallad - sos ( or 3 , 81 ) e dalla bmd total hip ( or 3 , 77 ) e neck ( or 3 , 62 )  . 
applicando lanalisi roc ai soggetti con e senza fratture vertebrali , la migliore prestazione nella discriminazione stata osservata di nuovo per la bmd lombare ( auc 0 , 840 , 03 ) , la bmd total hip ( auc 0 , 810 , 03 ) , ad - sos ( auc 0 , 790 , 03 ) e per la bmd neck ( auc 0 , 790 , 03 ) ( tabella 3 )  . discussion discussione this paper reports the results of a cross - sectional study conducted on a large cohort of italian women who underwent dxa of the lumbar spine and proximal femur and qus with ad - sos and ubpi of the phalanges to assess the ability of the techniques to discriminate between individuals with and without osteoporotic vertebral fractures . 
the results suggest that bmd measurements at the lumbar spine and proximal femur , as well as the qus parameters adsos and ubpi , are able to distinguish postmenopausal women with vertebral fractures . numerous studies have examined the correlation between dxa and qus parameters measured at various skeletal sites . 
the discriminatory potential in classifying individuals with or without vertebral fractures was tested in cross - sectional studies using a range of qus methods , including qus of the phalanx , and dxa in a sample of older postmenopausal women [ 23 ]  . 
in addition , a multicentre european study assessed the performance of five different ultrasound devices and their association with vertebral fractures compared with dxa in a group of european women [ 24 ]  . analysis of fracture discrimination showed that qus has a similar ability to femoral bmd in identifying vertebral fractures . 
furthermore , after adjusting for age , weight and height , the increased risk arising from a decrease of 1 sd reached the value of 4.20 for ad - sos , practically equivalent to bmd of the lumbar spine . 
la potenzialit discriminatoria nel classificare soggetti con o senza fratture vertebrali stata testata in studi trasversali con vari strumenti qus , inclusa la qus alla falange e la dxa in un campione di donne anziane in postmenopausa [ 23 ]  . 
uno studio multicentrico europeo ha pubblicato inoltre le prestazioni di 5 diversi strumenti a ultrasuoni e la loro associazione con le fratture vertebrali , in confronto con la dxa in un gruppo di donne europee [ 24 ]  . lanalisi di discriminazione delle fratture ha rivelato che la qus possiede una abilit simile alla bmd femorale nellidentificazione delle fratture vertebrali . 
inoltre , aggiustando per et , peso e altezza , laumento di rischio per diminuzione di una deviazione standard raggiunge il valore di 4 , 20 per ad - sos , praticamente equivalente alla bmd del rachide lombare . 
altri studi [ 2325 ] hanno ottenuto valori di or inferiori nella discriminazione delle fratture vertebrali , ci radiol med ( 2011 ) 116 : 92101 100 radiol med ( 2011 ) 116 : 92101 obtained lower or values in discrimination of vertebral fractures , perhaps due to differences in the characteristics of the study populations . 
it should be noted that all these studies provided the same results in terms of comparison with dxa , and they all obtained similar ors , as reported in our study . 
only in the osteoporosis prevention using soy ( opus ) study [ 24 ] was a slight but significant area under the roc curve found for ad - sos when compared with lumbar bmd . 
 [ 26 ] reported very similar or values for ad - sos and ubpi compared with lumbar , distal radius and total - body bmd measurements in discriminating vertebral fractures but lower than those obtained using femoral neck and total hip bmd . the limitations of our study are mainly due to lack of information on important risk factors for osteoporosis unrelated to menopause , age and anthropometric variables . in conclusion , the ad - sos ultrasound parameter demonstrated sensitivity similar to lumbar dxa in discriminating postmenopausal women with osteoporotic vertebral fractures from those without fractures . pu essere dovuto a differenze nelle caratteristiche delle popolazioni in studio : bisogna notare che in tutti questi studi lo stesso risultato stato ottenuto in termini di confronto con la dxa ; infatti gli ors ottenuti in ogni studio fra le due metodiche sono risultati simili fra loro , esattamente come nel nostro studio . 
sartor springer - verlag berlin heidelberg , 2010 isbn 978 - 3 - 540 - 00818 - 7 e - isbn 978 - 3 - 540 - 68889 - 1 doi 10.1007 / 978 - 3 - 540 - 68889 - 1 published online : 8 march 2011 springer - verlag 2011 this is a very special book : it addresses a very dif cult , peculiar topic yet special ; special because the topic is treated with incredible ease , showing the authors profound knowledge of anatomy , clinical situations and ndings , diagnostic procedures and ensuing therapeutic options and end - results . 
 special , also , because the reader in case he is not aware or has poor knowledge of the subject must humbly start from scratch in general , review anatomy , in order to fully appreciate what is poured into the text . there are ten chapters addressing in 311 pages : historical considerations ; anatomy of the cavernous sinus ; classi cation of the cavernous sinus and dural arteriovenous stulas ; etiology , prevalence and natural history of dural cavernous sinus stulas ; neuro - ophthalmology in dural cavernous sinus stulas ; radiological diagnosis ; endovascular treatment ; alternative treatment options ; hemodynamic aspects of dural cavernous sinus stula plus a few summary pages at the end . the bulk of the book ( 176 pages ) includes two chapters on radiological diagnosis and endovascular treatment , where one will nd all possible information on how to properly diagnose and treat these malformations . impressive , among the other chapters , the one on neuroophthalmology : one will nd valuable information regarding signs and symptoms related to the underlying pathology . treatment options and devices to be used for this endeavour are described in depth with a special focus on transvenous embolization rather than the transarterial approach . 
besides high - quality angiographic equipment and suf cient training , profound knowlquesto un libro molto speciale : speciale perch il suo un argomento molto dif cile , eppure speciale ; speciale perch largomento trattato con incredibile facilit a dimostrazione della profonda conoscenza , da parte dellautore , dellanatomia , dei quadri e delle situazioni cliniche , delle procedure diagnostiche , delle conseguenti opzioni terapeutiche e dei loro risultati nali . 
libro speciale anche perch il lettore , in caso non ne sia informato o abbia poca conoscenza dellargomento , deve con umilt affrontarlo partendo dagli elementi di base , rivedendo le sue conoscenza di anatomia , per apprezzare in pieno tutto quanto viene profuso ed insito nel testo . dieci capitoli , per un totale di 311 pagine trattano i seguenti punti : considerazioni storiche ; anatomia del seno cavernoso ; classi cazione delle stole del seno cavernoso e delle stole arterovenose durali ; eziologia , prevalenza e storia naturale delle stole del seno cavernoso durale ; neuro - oftalmologia delle stole del seno cavernoso durale ; diagnosi radiologica ; trattamento endovascolare ; opzioni terapeuriche alternative ; aspetti emodinamici delle stole del seno cavernoso durale per terminare con alcune pagine di sommario nale . la parte del leone del volume ( 176 pagine ) rappresentata dai due capitoli che trattano della diagnosi radiologica e del trattamento endovascolare delle stole del seno cavernoso , capitoli in cui si troveranno tutte le possibili informazioni su come diagnosticare in modo corretto e poi trattare queste malformazioni . di notevole spessore ed importanza tra gli altri capitoli quello sulla neuro - oftalmologia : vi si troveranno assai utili informazioni circa i segni ed i sintomi alla base della patologia in questione . 
oltre ad una attrezzatura angiogra ca di alta qualit e ad una suf ciente preparazio674 radiol med ( 2011 ) 116 : 673674 edge of the vascular anatomy , including its angiographic appearance , remains the essential basis for successful and safe endovascular occlusion of cavernous sinus stulas as well as : good three dimensional understanding of the cavernous sinus are required for optimal use of tools and devices . i would , humbly , also add that when reading the book one needs or must , have a very good memory in order to properly recall the meaning of the enormous number of abbreviations ( two full pages at the beginning ) , used to tag vessels and other paraphernalia . regarding this last point , the author and springer should think about devising and providing some loose or hard bound pages to have on hand , to be used in the translation of abbreviations ( unless one has a pico della mirandolas memory ) instead of making the reader shuf e back and forth from the page with the shortening in question to the abbreviation list . anatomic , radiological , clinical images many in colour are of enviable quality : each caption for most of the compound gures is a tiny booklet in itself due to the precise , in depth speci cations . in summary this is a must have book for all those who deal with this sort of problem : interventional radiologists , neurosurgeons and neurologists , and optional for the general radiologist who wants to keep in touch with the advances in this peculiar eld of radiology . ne , una profonda conoscenza dellanatomia vascolare e del suo aspetto angiogra co , rimangono le basi essenziali per ottenere una occlusione endovascolare riuscita e sicura delle stole del seno cavernoso ed inoltre che : richiesta una buona comprensione dellaspetto tridimensionale del seno cavernoso per un uso ottimale degli strumenti e delle attrezzature . 
 vorrei umilmente aggiungere che leggendo il libro necessario o meglio si deve avere una buona memoria per ricordare in modo corretto il signi cato dellenorme numero di abbreviazioni ( due intere pagine di elenco allinizio del volume ! ) usate per indicare i vasi ed altri elementi accessori . a questultimo proposito sia lautore che la casa editrice dovrebbero pensare di realizzare alcune pagine sfuse o su cartoncino da avere sempre a disposizione per la traduzione / interpretazione delle abbreviazioni ( a meno che non si abbia una memoria da pico della mirandola ) in modo da evitare il continuo andare avanti ed indietro dalla pagina che riporta quelle in questione rispetto alla lista delle stesse . le immagini anatomiche , radiologiche , cliniche , di cui molte a colori , sono di una qualit invidiabile : ogni didascalia , per la maggior parte delle gure composte , un piccolo trattatello a s stante , date le informazioni precise ed accurate che vi si ritrovano . in conclusione , questo un libro da possedere , obbligatorio per tutti coloro che si occupano di questo tipo di problemi : radiologi interventisti , neurochirurghi e neurologi ; facoltativo per il radiologo generale che si vuol tenere in contatto con i progressi in questo campo particolare della radiologia . 
simonetti dipartimento di diagnostica per immagini ed imaging molecolare , radioterapia e radiologia interventistica , fondazione policlinico universitario tor vergata ( ptv ) , viale oxford 81 , 00133 rome , italy correspondence to : e . 
eighteen female patients with a diagnosis of pelvic oor disorder ( physical examination and conventional defecography ) underwent dynamic mr defecography ( mrd ) with a 0.25 - t tilting mr system ( g - scan , esaote )  . 
diciotto pazienti donne con diagnosi di patologia del pavimento pelvico ( esame clinico e defecogra a convenzionale ) hanno eseguito una defecogra a con risonanza magnetica dinamica ( mrd ) , utilizzando un sistema di rm da 0 , 25 t ribaltabile ( g - scan , esaote )  . 
una buona qualit delle immagini stata ottenuta in 15 / 18 pazienti ; 3 pazienti hanno presentato gravi artefatti dovuti al movimento , 3 pazienti presentavano incontinenza che ha impedito gli studi funzionali . 
la mrd dinamica con un sistema di rm aperto a basso campo ribaltabile uno strumento radiol med ( 2011 ) 116 : 620633 keywords mr defecography pelvic oor disorders loweld mr tilt system mr attuabile e promettente per lo studio del pavimento pelvico . 
 parole chiave defecogra a rm patologia del pavimento pelvico rm a basso campo rm a decubito variabile introduction introduzione pelvic oor disorders encompass a spectrum of conditions that are very common in the general population , especially among women and in the older age groups [ 1 , 2 ] , and which greatly affect quality of life [ 3 ]  . 
pelvic oor disorders include a series of anatomical and functional changes affecting the three anatomical compartments of the pelvis : the anterior compartment , which comprises the bladder and the urethra ; the middle compartment , consisting of the uterus , the adnexa and the vaginal fornix ; and the posterior compartment , composed of the rectum and anal canal . 
a survey conducted on a sample of 1 , 200 , 000 italian women aged 5575 years found a prevalence of pelvic oor disorders of approximately 22% [ 4 ]  . 
although conventional defecography has considerably improved the diagnosis of evacuation disorders , the examination often proves inadequate in evaluating abnormalities affecting the other compartments [ 5 , 6 ]  . 
 magnetic resonance defecography ( mrd ) , although still an experimental technique , allows the study of the pelvic oor with the double advantage of sparing the patient exposure to ionising radiation and permitting optimal evaluation of soft tissues and support structures of the pelvic organs . the aim of this study was to investigate the possibility of using an open - con guration , loweld ( 0.25 t ) , tilting mr system normally used for imaging the lumbar spine and knee under weight - bearing conditions in evaluating pelvic oor disorders and to compare results obtained in the same individual but imaged from the supine and orthostatic positions . le disfunzioni del pavimento pelvico rappresentano uno spettro di patologie largamente diffuse nella popolazione , in particolare nel sesso femminile e nelle fasce di et pi avanzate [ 1 , 2 ] , e in uenzano in maniera determinante la qualit di vita dei soggetti che ne soffrono [ 3 ]  . 
tali disturbi comprendono una serie di alterazioni anatomiche e funzionali a carico dei tre compartimenti anatomici della pelvi : il compartimento anteriore , che comprende vescica e uretra ; il compartimento medio , costituito dallutero , dagli annessi e del fornice vaginale ; il compartimento posteriore , formato dal retto e dal canale anale . 
in italia uno studio effettuato su un campione di 1.200.000 donne det compresa tra i 55 e 75 anni ha documentato una prevalenza delle disfunzioni del pavimento pelvico pari a circa il 22% [ 4 ]  . 
la defecograa convenzionale ha migliorato notevolmente la diagnosi dei disturbi della defecazione , ma risulta spesso inadeguata alla valutazione delle anomalie degli altri compartimenti [ 5 , 6 ]  . 
la defeco - risonanza magnetica ( rm ) ( mrd ) seppur rivesta ancora un ruolo sperimentale , consente lo studio del pavimento pelvico e presenta il duplice vantaggio di evitare lesposizione dei pazienti a radiazioni ionizzanti e quello di una ottimale valutazione dei tessuti molli e delle strutture di supporto degli organi pelvici . scopo di questo studio quello di esaminare la possibilit di utilizzare , nella valutazione dei disturbi del pavimento pelvico , un sistema di rm aperto , a basso campo ( 0 , 25 t ) e a decubito variabile , dedicato usualmente alle studio del rachide lombare e del ginocchio sotto carico , confrontando i risultati ottenuti nello stesso soggetto in decubito supino ed in ortostasi . materials and methods patients materiali e metodi pazienti eighteen patients presenting with symptoms due to pelvic oor disorders were enrolled between october 2009 and april 2010 . 
 only two women were nulliparous , and one woman had had a hysterectomy . nel periodo compreso fra ottobre 2009 e aprile 2010 sono stati arruolati nello studio 18 pazienti che riferivano sintomi da disfunzioni del pavimento pelvico . 
tutti i soggetti riferivano sintomi clinici ( tabella 1 ) e da ciascuno di loro stato tabella 1 sintomatologia clinica dei pazienti sottoposti allo studio table 1 clinical symptoms in our patients 622 symptom sense of incomplete evacuation pain during defecation faecal incontinence chronic constipation sintomo senso di incompleta evacuazione dolore durante defecazione incontinenza fecale stipsi cronica imaging techniques mr colpocystodefecography patients , n 10 / 18 9 / 18 3 / 18 12 / 18 pazienti , n 10 / 18 9 / 18 3 / 18 12 / 18 the mr examinations were performed on an open - con guration , tilting - gantry , permanent magnet with a stationary magnetic eld of 0.25 t , a dynamic gradient strength of 20 mt / m and a slew rate of 25 mt / m / s ( g - scan , esaote , italy ) and passive shimming . 
the receiver coil was a lumbar spine dpa surface coil consisting of a rigid base ( width 320 mm , depth 280 mm , height 45 mm ) and a exible belt available in two sizes ( large , 8918.5 cm ; small , 6918.5 cm ) to t the dimensions of the individual to be studied . 
prior to the study , the patients rectum was lled with approximately 200 ml of suspension medium containing 1 ml of gadobutrol paramagnetic contrast medium ( gadovist 1.0 mol / l , bayer schering ag , berlin , germany )  . 
three different suspensions were used : sonographic gel ( n = 2 ) , a psyllium bre compound ( psyllogel ) ( n = 2 ) and mashed potatoes ( prepared by adding water to different commercial products in the examination room ) ( n = 4 )  . 
the bladder was opaci ed with 180 ml of saline solution added to 3 ml of gadobutrol contrast medium ( gadovist 1.0 mol / l , bayer schering ) after having inserted a 16 - french , two - way foley catheter during the examination . 
uno dei soggetti in esame aveva subito unisterectomia . tecniche di imaging colpo - cisto - mrd gli esami rm sono stati eseguiti su magnete permanente aperto a decubito variabile con campo magnetico stazionario di 0 , 25 t gradienti dinamici con potenza di 20 mt / m e slew rate di 25 mt / m / s ( g - scan , esaote , italia ) e shimming passivo . 
il tavolo del magnete provvisto di un meccanismo di ribaltamento da 0 a 90 con passi di 2 che ha consentito lo studio del paziente in decubito supino ed ortostatico . 
 come bobina di ricezione stata utilizzata una bobina di super cie lombar spine dpa , costituita da una base rigida ( lunghezza [ l ] profondit [ p ] altezza [ h ] = 320 mm280 mm45 mm ) e una fascia essibile anteriore di dimensioni variabili ( fascia grande 8918 , 5 cm ; fascia piccola 6918 , 5 cm ) in funzione della taglia del soggetto in esame . 
prima dellesame il retto dei soggetti in esame stato riempito con circa 200 ml di mezzo di sospensione unito ad 1 ml di mezzo di contrasto paramagnetico gadobutrolo ( gadovist 1 , 0 mol / l , bayer schering ag , berlin , germania )  . 
 sono state utilizzate 3 diverse sospensioni : gel ecogra ci ( n = 2 ) , composto di bre ( psyllogel ) ( n = 2 ) , purea di patate ( sono stati utilizati vari prodotti commerciali preparati in sede di esame mediante aggiunta di acqua ) ( n = 4 )  . 
stata opacizzata anche la vescica mediante 180 ml di soluzione siologica unita a 3 ml di mezzo di contrasto paramagnetico gadobutrolo previo posizionamento di catetere foley a doppia via da 16 french , lasciato in sede durante lesame . 
lo studio statico ha previsto lutilizzo sui tre piani ortogonali dello spazio di sequenze 3d hybrid contrast enhanced ( hyce ) , gradient echo ( ge ) bilanciata con le seguenti caratteristiche : tempo di ripetizione ( tr ) 10 ms ; tempo di eco ( te ) 5 ms ; ip angle ( fa ) 90 ; strati 20 ; spessore di strato 2 , 5 mm ; campo di vista ( fov ) 280280 ; matrice 200160 . 
sono state inoltre acquisite immagini statiche sul piano sagittale durante rilassamento , contrazione s nteriale e ponzamento , mediante sequenze ge t1 - pesate con le seguenti caratteristiche : tr 35 ms ; te 10 ms ; fa 90 ; atrati 1 ; spessore di strato 5 , 5 mm ; fov 300300 ; matrice 192128 . 
in ne stato condotto lo studio dinamico durante evacuazioradiol med ( 2011 ) 116 : 620633 static study included acquisition of 3d hybrid contrast - enhanced ( hyce ) gradient echo ( ge ) sequences and three orthogonal spatial planes with the following parameters : repetition time 10 ms ; echo - train length 5 ms ; ip angle 90 ; slices 20 ; slice thickness 2.5 mm ; eld of view 280280 ; matrix , 200160 . 
static images were also acquired in the sagittal plane at rest and during sphincter contraction and straining using t1 - weighted ge sequences with the following parameters : repetition time 35 ms ; echo - train length 10 ms ; ip angle 90 ; slices 1 ; slice thickness 5.5 mm ; eld of view 300300 ; matrix 192128 . 
finally , a dynamic study was performed during rectal evacuation using an axial t1weighted ge sequence with the following parameters : repetition time 30 ms ; echo - train length 6 ms ; ip angle 90 ; slices 1 ; slice thickness 5.5 mm ; eld of view 300300 ; matrix 192128 ; acquisition time 3 s / image . 
overall mr room time for the examinations in the two patient positions , and including re lling of the rectum , was approximately 68 ( range 4293 ) min . image analysis mr images acquired in the sagittal plane were analysed by two radiologists who took the measurements separately . 
to allow measurement standardisation and determine severity of the pelvic oor disorder , we considered the pubococcygeal line ( pcl ) , which delimits the base of the pelvic oor and extends from the inferior border of the pubic symphysis and the last coccygeal joint . 
to evaluate pelvic oor laxity , measurements of the h and m lines were obtained : the h line is measured by tracing a line from the inferior border of the pubic symphysis to the posterior wall of the rectum at the anorectal junction level . 
this line represents the most caudal portion of the levator ani complex ( puborectalis muscle ) and corresponds to the puborectal hiatus , which allows grading of the maximal widening in the anteroposterior direction of the pelvic sling during straining . 
the m line exceeding 2 cm was considered pathological . organ prolapse was de ned as protrusion of any organ ( bladder , urethra , vagina , small bowel ) through the h line . 
anterior rectocele was quanti ed by prolonging upwards to the level of the rectum the tangent to the anterior wall of the anal canal and measuring the disne dellampolla rettale mediante sequenza ge t1 - pesata orientata sul piano sagittale con le seguenti caratteristiche : tr 30 ms ; te 6 ms ; fa 90 ; strati 1 ; spessore di strato 5 , 5 mm ; fov 300300 ; matrice 192128 ; tempo di acquisizione 3 s / immagine . 
il tempo complessivo di occupazione della macchina per la completa acquisizione dellesame nei due decubiti , compreso il secondo riempimento del retto , stato in media di 68 minuti ( range 4293 minuti )  . analisi delle immagini le immagini rm acquisite sul piano sagittale sono state analizzate da due radiologi che hanno effettuato separatamente le rispettive misurazioni . 
per consentire una standardizzazione delle misure e per determinare la gravit delle disfunzioni del pavimento pelvico stata identi cata la linea pubo - coccigea ( lpc ) , delimitante la base del pavimento pelvico , tesa tra il margine inferiore della sin si pubica e lultima articolazione del coccige . 
al ne di valutare la lassit del pavimento pelvico abbiamo misurato la linea h e la linea m : la linea h misurata tracciando una linea dal margine inferiore della sin si pubica alla parete posteriore del retto a livello della giunzione ano - rettale e rappresenta la porzione pi caudale del gruppo dei muscoli elevatori dellano ( muscolo pubo - rettale ) ; inoltre , essa corrisponde allo iato pubo - rettale , che consente la massima apertura in senso antero - posteriore dellanello pelvico durante il ponzamento . 
in accordo con i dati in letteratura abbiamo considerato lampliamento dello iato puborettale patologico quando la linea h supera la lunghezza di 6 cm [ 7 ] ; la linea m si estende perpendicolarmente dal piano della lpc alla ne della linea h . 
abbiamo considerato patologica una lunghezza della linea h superiore ai 2 cm . abbiamo considerato il prolasso come la protrusione di qualsiasi organo ( vescica , uretra , vagina , piccolo intestino ) attraverso lo iato pubo - rettale ( linea h )  . 
la quanti cazione del rettocele anteriore avvenuta prolungando superiormente a livello del retto la tangente alla parete anteriore del canale anale e misurando la distanza tra tale linea e la parete anteriore del bulging nel punto di maggiore ampiezza . 
abbiamo considerato patologico un rettocele di almeno 2 cm [ 7 ]  . qualit delle immagini abbiamo valutato la qualit delle immagini analizzando il rapporto segnale / rumore ed il livello di artefatti mediante 624 radiol med ( 2011 ) 116 : 620633 tance between this line and the anterior wall of the rectal bulge at the point of maximum depth . 
we considered a rectocele of at least 2 cm to be pathological [ 7 ]  . image quality image quality was evaluated by analysing the signal - tonoise ratio ( snr ) and the severity of artefacts on a qualitative scale from 1 to 3 . 
snr was scored 1 if the noise was greater than the signal , 2 if noise and signal were equivalent and 3 if the signal was greater than the noise . 
artefact severity was scored 1 if no artefacts or only minor artefacts were present , 2 if there were artefacts of intermediate severity and 3 in the event of major or severe artefacts . 
these evaluations were performed on both static images obtained with the balanced 3d hyce ( at baseline ) and t1 - weighted gradient echo sequences ( at rest , during contraction and straining ) , and on the single dynamic images . 
riguardo al rapporto segnale / rumore , stato attribuito un punteggio di 1 se il rumore risultava superiore al segnale , 2 in caso di parit e 3 se il segnale era superiore al rumore . 
per quanto riguarda il livello di artefatti , stato assegnato un punteggio di 1 in caso di artefatti minori o assenti , 2 se erano presenti artefatti di grado medio e 3 in caso di artefatti maggiori o gravi . 
tali valutazioni sono state effettuate sia sulle immagini statiche ottenute con le sequenze 3d - hyce bilanciate ( basale ) e ge t1 - pesate ( rilassamento , contrazione e ponzamento ) , sia sulle singole immagini dinamiche . 
ci nonostante , le valutazioni effettuate sul rapporto segnale / rumore e sul livello di artefatti , sono state soddisfacenti per tutte le tipologie di immagini , anche se punteggi inferiori sono stati ottenuti dalle immagini dinamiche ( tabelle 2 e 3 )  . 
 table 3 evaluation of artefacts patient 3d - hyce rest contraction straining cine ( mean ) radiol med ( 2011 ) 116 : 620633 626 mean media hyce , hybrid contrast - enhanced sequence tabella 3 valutazione del livello di artefatti paziente 3d - hyce rilassamento contrazione ponzamento cine ( media ) 1.4 1 , 4 1.5 1 , 5 1.6 1 , 6 1.4 1 , 4 hyce , hybrid contrast - enhanced sequence pelvic oor laxity and anterior rectocele , and compared the results obtained in the orthostatic and supine position . 
the supine position resulted in an underestimation of all disorders : in particular , six bladder prolapses were missed , which were graded as mild in the orthostatic position , whereas two moderate prolapses in the orthostatic position were graded as mild in the supine position . 
sono evidenti bolle daria nelle immagini b e c ma soprattutto in b . moderate prolapses in the orthostatic position , one of which was graded as mild it the supine position ; and three mild prolapses in the orthostatic position , which were all missed in the supine position . 
five cases of severe pelvic oor laxity were graded as severe in the orthostatic position , one of which was underestimated as moderate in the supine position ; one case of moderate laxity in the orthostatic position was graded as mild in the supine position ; and two cases graded as mild laxity in the orthostatic position were missed in the supine position . 
of the 11 anterior rectoceles classied as severe in the orthostatic position , only eight were also graded as severe in the supine position ; three severe and one moderate anterior rectocele were underestimated , whereas one mild rectocele in the orthostatic position was missed in the supine position ( table 4 )  . discussion the true prevalence of pelvic oor disorders is underestimated , as these disorders tend to remain asymptomatic no . 
cinque lassit del pavimento pelvico sono risultate gravi in ortostasi ed 1 stata sottostimata moderata in supino , 1 valutata di grado moderato in ortostasi stata sottostimata lieve in decubito supino e 2 lievi in ortostasi misconosciute in supino . 
degli 11 rettoceli anteriori osservati , classi cati come gravi in decubito ortostatico , solo 8 sono altrettanto severi in supino ; 3 gravi ed 1 moderato sono stati sottostimati , mentre 1 rettocele misconosciuto in decubito supino risultato lieve in ortostasi ( tabella 4 )  . 
 discussione la reale prevalenza delle disfunzioni del pavimento pelvico sottostimata in quanto tali patologie sono spesso asintomatiche per lunghi periodi o paucisintomatiche ( dolore pelvico , senso di peso , sensazione di incompleta evacuazione ) soprattutto in caso di iniziale prolasso degli organi pelvici [ 1 , 2 , 8 ]  . 
lesame sico inoltre spesso insuf ciente e difcoltoso per una completa valutazione anatomica e funzionale della pelvi [ 9 ]  . radiol med ( 2011 ) 116 : 620633 628 fig . 
le immagini ottenute con psyllogel ( b ) e gel ecogra co ( c ) forniscono una ottimale intensit di segnale ma nella fase evacuativa non consentono una suf ciente dilatazione del canale anale lasciandolo liforme ( punte di freccia )  . or poorly symptomatic ( pelvic pain , sense of weight , feeling of incomplete evacuation ) for years , especially in the early stages of pelvic organ prolapse [ 1 , 2 , 8 ]  . 
moreover , physical examination is often dif cult and inadequate for a complete anatomical and functional assessment of the pelvis [ 9 ]  . preoperative planning is based on clinical assessment and data provided by conventional defecography , in some cases supplemented by mrd . 
previous published studies have been conducted with closed - con guration , higheld ( 1.5 - t ) systems and t2 - weighted multiplanar acquisitions , with the patient in the supine position or lateral decubitus with exed knees [ 1012 ]  . 
the development of open - con guration 0.5 - t systems with dedicated supports and suf ciently wide gantry aperture has allowed mrd examinations of the patient in a sitting position [ 13 , 14 ]  . 
all published studies comparing mrd performed with the two patient positions have therefore been conducted using difil planning pre - operatorio si basa sulla visita clinica e sui dati forniti dalla defecogra a tradizionale , coadiuvati talvolta da quelli ottenuti con la defeco - rm . 
i lavori riportati in letteratura sullargomento sono stati eseguiti su magnete chiuso ad alto campo ( 1 , 5 t ) ed acquisizioni t2 pesate su multipli piani dello spazio , con paziente in decubito supino o posto sul anco a ginocchia esse [ 1012 ]  . 
la realizzazione di magneti aperti da 0 , 5 t , dotati di supporti dedicati e apertura del gantry suf cientemente ampia , ha permesso di eseguire lo studio defeco - rm con paziente in posizione seduta [ 13 , 14 ]  . gli studi nora pubblicati in letteratura per la valutazione comparativa degli esami defeco - rm in due differenti decubiti utilizzano pertanto magneti a diversa intensit del campo magnetico stazionario ( 1 , 5 t per il decubito supino e 0 , 5 t per la posizione seduta )  . 
questo , a nostro parere , rappresenta un limite per limpossibilit di compararne i risultati a causa delle differenti performance dei due magneti e radiol med ( 2011 ) 116 : 620633 fig . 
images obtained in the orthostatic position at rest ( a ) , during contraction ( c ) and straining ( e ) and during the same phases in the supine position ( b , d , f )  . 
immagini ottenute in ortostatismo a riposo ( a ) , durante contrazione ( c ) e ponzamento ( e ) ed in posizione supina nelle medesime condizioni ( b , d , f )  . 
il prolasso risulta evidente in fase di ponzamento ma solamente in ortostatismo ( frecce )  . ferent magnetic eld strengths ( 1.5 t for supine decubitus and 0.5 t for the sitting position )  . 
this is a limitation , in our opinion , that precludes comparison of results owing to the different performance of the two magnets and the sequences used [ 13 , 15 ]  . in our study , we used a new open - con guration permanent magnet with a 0.25 - t stationary magnetic eld equipped with a 0 - 90 tilting table , which allowed us to apply the same imaging protocol in the two patient positions . 
nel nostro studio abbiamo utilizzato un magnete permanente aperto di nuova concezione con campo magnetico stazionario di 0 , 25 t , equipaggiato con tavolo ribaltabile da 0 a 90 , che ha consentito di eseguite lo stesso protocollo desame nei due differenti decubiti . 
lesame defeco - rm in ortostasi stato inoltre acquisito con tavolo inclinato a 80 per aumentare la stabilit del soggetto in esame e ridurre gli artefatti da movimento indotti dalla stazione eretta . analogamente allo studio defecogra co convenzionale , sono state acquisite sequenze statiche in fase di riposo , contrazione e ponzamento . 
la possibilit di studiare la fase evatable 4 results obtained in the upright and supine positions condition mild moderate severe total upright supine upright supine upright supine upright supine radiol med ( 2011 ) 116 : 620633 630 bladder pse vaginal prolapse rectal pelvic oor laxity ctocele anterior pse prola prola escicale prolasso aginale prolasso ettale prolasso lassit del pavimento 2 ante rettocele riore tabella 4 valutazione dei reperti ottenuti rispettivamente in ortostasi ed in posizione supina patologia lieve moderato grave totale ortostasi supino ortostasi supino ortostasi supino ortostasi supino patient stability and reduce movement artefacts resulting from the standing position . as in conventional defecography , static sequences were acquired with the patient at rest , during contraction and while straining . 
the possibility of studying the evacuation phase appears essential for increasing diagnostic accuracy of the examination , providing complete clinical assessment and guiding appropriate patient management , given that some pathological ndings are underestimated in the static images acquired during straining . 
the three suspensions we used have different density and texture and , although all three can be used , we found that mashed potatoes achieve greater opening of the anal canal and thus a more accurate assessment of any disorder . 
the suspensions composed of sonographic gel and psyllogel have optimal signal intensity but fail to dilate the anal canal suf ciently in the evacuation phase , leaving it liform , which makes it dif cult , albeit possible , to carry out assessments according to the methods used . 
in our experience , this feature makes it the best medium by which to obtain opacication in our experience . because the mr system we used does not provide steady - state free precession ( balanced ) sequences , which are commonly used for the evacuation phase , we adapted the t1 - weighted turbo eld - echo sequences . 
a reasonable cuativa appare indispensabile per aumentare laccuratezza diagnostica e fornire un completo quadro clinico ed un corretto inquadramento terapeutico del paziente , poich alcuni reperti patologici risultano sottostimati nelle acquisizioni statiche in fase di ponzamento . 
le tre sospensioni utilizzate , hanno una differente densit e consistenza e nonostante risulti possibile utilizzare tutte e tre le sospensioni abbiamo dimostrato come la purea di patate abbia una maggiore capacit di far aprire il canale anale , consentendo una valutazione pi accurata delle eventuali patologie presenti . 
le sospensioni composte dal gel ecogra co e dallo psyllogel danno una ottimale intensit di segnale , ma nella fase evacuativa non dilatano suf cientemente il canale anale lasciandolo liforme , ci rende dif coltoso , seppur possibile , fare le dovute valutazioni secondo i metodi utilizzati . 
 questa sua caratteristica che lo rende nella nostra esperienza il mezzo di opacizzazione migliore . non essendo per il momento disponibili sul magnete utilizzato sequenze di tipo steady state free precession ( balanced ) , attualmente in voga per la fase evacuativa , stato necessario adattare le sequenze turbo eld echo ( tfe ) t1 pesate . 
un compromesso ragionevole fra la risoluzione spaziale , risoluzione temporale e lintensit del segnale ha consentito lottimizzazione della sequenza dinamica utilizzata nella fase evacuatoria con tempi di acquisizione di 3 s / immagine . 
una migliore risoluzione temporale potrebbe proradiol med ( 2011 ) 116 : 620633 compromise between spatial resolution , temporal resolution and signal intensity allowed us to optimise the dynamic sequence used in the evacuation phase with acquisition time of 3 s / image . 
a better temporal resolution could probably be obtained by constructing speci c mrd sequences that do not require calibration between one image and the next and with a suf cient snr to reduce acquisition times , as well as by developing dynamic ( balanced ) steady - state free precession sequences . 
moreover , axial images allowed widening of the puborectal hiatus to be assessed in evaluating pelvic oor laxity . in agreement with previous reports , the comparison of results obtained with the two different patient positions showed that the supine decubitus resulted in an underestimation of the severity of all disorders [ 7 , 11 ] ( table 4 )  . 
in our series , only three out of 18 patients were able to complete the mrd examination in the supine position , whereas all patients succeeded in performing the evacuation phase in the orthostatic position . for quantitative image analysis , we used the h line , m line and organ prolapse with respect to the h line ( hmo ) system in order to de ne , differentiate and grade the severity of organ prolapse and pelvic oor musculature laxity ( table 2 ) [ 1013 , 16 ]  . 
comparison of prolapses and anterior rectocele severity seen in the two imaging positions showed that the supine position resulted in an underestimation in all patients ( table 4 )  . 
the different results of the mrd examination in the two positions are most likely related to the direction of the force of gravity exerted along a vertical axis , bearing completely on the muscularfascial structures of the pelvic oor in the orthostatic position , and along an anteroposterior axis in the supine position . 
in the latter position , the force of gravity is not exerted directly on the pelvic oor , and the underestimation of ndings is related to the fact that the only force bearing on the pelvic oor in the craniocaudal direction is that of bearing down with the abdominal muscles during straining and evacuation . 
our ndings demonstrate that mrd in the orthostatic position , even though not physiological , allows for better evaluation of pelvic oor disorders compared with the patient in the supine position . 
this was due to the con guration of the mr system , which was devised for dynamic osteoarticular studies and is not compatible with a dedicated support due to lack of space ( 35 cm )  . 
a further limitation is related to the sequences used and the relatively babilmente essere facilmente ottenibile grazie alla costruzione di sequenze speci che per lesame defeco - rm esenti dalla calibrazione fra unimmagine e laltra e con rapporto segnale / rumore suf ciente a ridurre i tempi di acquisizione e dalla messa a punto di sequenze dinamiche di tipo steady state free precession ( balanced )  . 
le immagini orientate sul piano assiale hanno inoltre permesso di valutare lo slargamento dello iato pubo - rettale in caso di lassit del pavimento pelvico . nella nostra casistica , in accordo con i dati in letteratura , la comparazione dei risultati nei due differenti decubiti , ha mostrato nel decubito supino una sottostima della gravit per tutte le patologie osservate ( tabella 4 ) [ 7 , 11 ]  . 
un ulteriore limite del decubito supino consiste nella dif colt ad effettuare la fase evacuativa , che risulta invece di fondamentale importanza per un corretto inquadramento clinico - diagnostico del paziente . 
nella nostra casistica infatti solamente 3 soggetti su 8 arruolati sono stati in grado di completare lesame defeco - rm in decubito supino , mentre tutti i soggetti sono riusciti ad effettuare la fase evacuativa in ortostatismo . per lanalisi quantitativa delle immagini ottenute stato utilizzato il sistema hmo ( linea h , linea m e prolasso degli organi pelvici rispetto alla linea h ) , al ne di de nire , differenziare e valutare la gravit del prolasso degli organi pelvici e la lassit della muscolatura del pavimento pelvico stesso ( tabella 2 ) [ 1013 , 16 ]  . 
confrontando la gravit dei prolassi e dei rettoceli anteriori riscontrati nei due decubiti , abbiamo osservato una sottostima del decubito supino in tutti i pazienti ( tabella 4 )  . 
i differenti risultati fra gli esami rm nei due decubiti sono da correlare verosimilmente alla direzione della forza gravitazionale che viene esercitata secondo un asse verticale in ortostasi , gravando interamente sulle strutture muscolo - fasciali del pavimento pelvico , e secondo un asse antero - posteriore in decubito supino . 
in questultimo decubito la forza gravitazionale non viene esercitata direttamente sul pavimento pelvico e la sottostima dei reperti legata al fatto che lunica forza diretta verso il pavimento pelvico in senso cranio - caudale quella del torchio addominale durante ponzamento ed evacuazione . 
i nostri dati dimostrano che lo studio rm in posizione ortostatica , sebbene non siologica , consente una migliore valutazione delle patologie del pavimento pelvico rispetto allesame rm con paziente in decubito supino . 
 il principale limite del nostro studio stato limpossibilit di valutare il paziente in posizione seduta in quanto la conformazione della macchina , concepita per lo studio dinamico osteo - articolare , ha impedito , a causa dellesiguo spazio ( 35 cm ) , di adattare un supporto dedicato . 
the speci cally adapted dynamic sequences do not have optimal temporal resolution , and the presence of a calibration interval between frames could lead to an underestimation of the more transient ndings . 
in addition , the possibility of acquiring only one 5.5 - mm slice during the dynamic phase required not only absolute patients immobility throughout the examination , but also extreme operator precision in setting the geometrical parameters to achieve perfect centring of the axis of the anal canal . 
the use of t1 - weighted sequences in the dynamic study made it necessary to opacify the bladder , vagina and rectum , resulting in increased costs , greater patient discomfort and longer examination time . le sequenze dinamiche adattate allo scopo non possiedono una ottimale risoluzione temporale e la presenza di un intervallo di calibrazione tra un frame ed il successivo potrebbe determinare una sottostima dei reperti pi fugaci . 
la possibilit di acquisire un solo strato di 5 , 5 mm in fase dinamica ha inoltre reso obbligatoria non solo lassoluta immobilit del paziente durante tutto lesame , ma anche una estrema precisione delloperatore nellimpostazione dei parametri geometrici al ne di centrare perfettamente lasse del canale anale . 
lutilizzo di sequenze t1 pesate nello studio dinamico ha reso , inoltre , indispensabile lopacizzazione della vescica , della vagina e del retto , con conseguente aumento dei costi dellesame , del disagio dei pazienti e del tempo complessivo dellesame . conclusions mrd with a permanent 0.25 - t magnet is feasible and provides high diagnostic quality . 
the study in the orthostatic position is more accurate for evaluating pelvic oor disorders compared with the supine position , which may be hampered by dif culties completing the evacuation phase . 
the hmo measurement system in evaluating pelvic oor disorders appears to be the most reliable method ; by evaluating separately two distinct and often coexisting pathological entities ( pelvic oor relaxation and organ prolapse ) , it allows a more accurate clinical assessment and therefore provides information that is vital for selecting the most appropriate treatment strategy . 
 the continuous technological improvements , especially in dedicated dynamic t2 sequences , will improve image diagnostic quality while reducing examination times . conclusioni lo studio defeco - rm risulta fattibile e di elevata qualit diagnostica con un magnete permanente da 0 , 25 t . 
lo studio in posizione ortostatica risulta pi accurato nella valutazione delle patologie del pavimento pelvico rispetto allesame in decubito supino , talvolta in ciato dalla dif colt a completare la fase evacuatoria . 
il sistema di misurazione hmo nella valutazione delle disfunzioni del pavimento pelvico appare , a nostro parere , il metodo pi af dabile perch , valutando separatamente due entit patologiche distinte e spesso coesistenti ( lassit del pavimento e prolasso degli organi ) , consente un pi corretto inquadramento clinico e fornisce quindi informazioni indispensabili per la scelta della migliore strategia terapeutica . 
mdct - ca provides parameters of cardiac function comparable to those found in echocardiography . mdct - ca although used primarily for coronary noninvasive imaging can provide additional information on riassunto obiettivo . 
sono stati analizzati per entrambe le metodiche i seguenti parametri comunemente campionati in ecocardiografia e correlati con quelli ottenuti in actcms : spessore del setto , spessore della parete posteriore , diametro dellaorta ascendente , diametro e volume tele sistolico e tele diastolico , frazione di eiezione , stroke volume , gittata cardiaca e massa cardiaca . 
stata riscontrata una buona correlazione tra le misure dello spessore del setto ( r = 0 , 470 e p = 0 , 001 ) e del diametro dellaorta ascendente ( r = 0 , 777 e p < 0 , 001 ) in ecografia e in tc , mentre una scarsa correlazione tra le misure dello spessore della parete posteriore ( r = 0 , 243 e p = 0 , 104 )  . 
la tcms ha sovrastimato in modo consistente i valori medi dei volumi rispetto allecocardiografia ma i volumi tele diastolico e tele sistolico derivati dalla 2dse e dalla actcms hanno mostrato una correlazione significativa ( rispettivamente r = 0 , 456 e p = 0 , 002 ; r = 0 , 640 e 506 radiol med ( 2011 ) 116 : 505520 ventricular function useful to the diagnostic workup of cardiac patients . keywords mdct coronary angiography ventricular function left ventricle parameters ejection fraction cardiac failure p < 0 , 001 )  . 
come indicatore di funzione sistolica globale del ventricolo sinistro ( vs ) , la frazione di eiezione ( fe ) misurata tramite tcms o ecocardiografia ha mostrato uneccellente correlazione ( r = 0 , 626 e p < 0 , 001 )  . 
la ac - tcms sebbene utilizzata prevalentemente per limaging non invasivo delle arterie coronarie , pu fornire informazioni aggiuntive allo studio delle coronarie utili al work up diagnostico dei pazienti con patologie coronariche e cardiache . 
 parole chiave angiografia coronarica tcms funzione ventricolare parametri ventricolari sinistri frazione di eiezione scompenso cardiaco introduction introduzione morbidity and mortality for cardiac pathologies have an inverse correlation with global left ventricular ( lv ) systolic function [ 1 , 2 ] as preserved ventricular function represents the most important prognostic factor in the short and long ter in routine clinical cardiology practice , critical decision - making such as defibrillator implantation , biventricular pacing and treatment of congestive heart failure is heavily based on ejection fraction ( ef )  . 
for decades , the 2d nature of ultrasound ( us ) imaging has affected the quantification of lv size and function by errors that stemmed mainly from foreshortened views and inaccurate geometric modelling . 
the inadequacy of this methodology has been demonstrated by several investigators who compared 2d echocardiographic ( ecg ) measures against magnetic resonance imaging ( mri ) reference [ 4 ]  . 
 recent multidetector computed tomography ( mdct ) scanners have been introduced in cardiac imaging to provide accurate and reproducible noninvasive visualisation of the coronary tree [ 5 , 6 ]  . 
however , these scanners not only provide a morphological visualisation of the coronaries but also a comprehensive evaluation of the heart during the heart cycle . these scanners are capable of continuous volumetric imaging of the beating heart allowing highly reproducible quantification of ventricular size and function . 
image reconstructions are easily obtained in each phase of the heart cycle from the diastolic to the systolic phase but are usually used only in order to assess the best data set for coronary evaluation , thus utilising only a small part of the large amount of information that is usually available . 
this technique has been increasingly la mortalit e la morbilit per malattie cardiache sono inversamente correlate con la funzione globale del ventricolo sinistro ( vs ) [ 1 , 2 ] dal momento che una funzione ventricolare preservata rappresenta il pi importante fattore prognostico a breve e lungo termine . 
nella pratica clinica cardiologica , la strategia terapeutica in situazioni critiche , come il posizionamento di un defibrillatore , di un pacemaker bi - ventricolare o il trattamento di uninsufficienza cardiaca congestizia principalmente basata sulla frazione deiezione ( fe )  . 
per decenni tale compito stato assolto dalla ecografia , limitata dalla natura bidimensionale dellimaging ecografico a causa di errori principalmente dovuti a poche proiezioni efficaci e allinaccuratezza del modello geometrico . 
linadeguatezza di questa metodica peraltro stata dimostrata da numerosi studi che hanno comparato i parametri ottenuti con lecocardiografia 2d con quelli di riferimento ottenuti con la risonanza magnetica [ 4 ]  . 
la valutazione qualitativa e quantitativa della funzione ventricolare rappresenta la pi comune applicazione dellecocardio nella pratica quotidiana . i pi recenti scanner di tomografia computerizzata multistrato ( tcms ) sono stati introdotti nello studio del cuore al fine di ottenere una visualizzazione accurata dellalbero coronarico , riproducibile , non invasiva [ 5 , 6 ]  . 
questi scanner sono in grado di effettuare un imaging volumetrico continuo del cuore , permettendo radiol med ( 2011 ) 116 : 505520 referred to as a potential tool for combined assessment of coronary anatomy , myocardial perfusion , and lv function [ 7 ] the purpose of this study was to compare global lv systolic function assessment by mdct with ecg in a routine cardiology practice setting . materials and methods patient population between april 2008 and september 2009 , 282 consecutive patients ( 178 men , mean age 64.02 years ) who underwent mdct coronary angiography ( mdct - ca ) for evaluation of coronary artery disease were considered . 
 patients selected for the study were subjected to mdctca according to indications of international guidelines [ 8 ] ( atypical chest pain , equivocal or nondiagnostic exercise test , follow - up of stents or bypass grafts )  . 
 mdct - ca protocol examination all patients included in the protocol were studied with a 64slice mdct scanner ( brilliance 64 , philips , best , the netherlands )  . 
the postcontrast scan was acquired by injecting 80 ml of a high - concentration iodinated contrast agent ( iomeron400 , bracco , milan , italy ) via the antecubital left arm vein at the rate of 5 ml / s followed immediately by a 40 - ml saline bolus . 
the acquisition protocol is summarised in table 2 . raw data obtained were used to retrieve ten different reconstruction datasets every 10% of the r - r interval ranging from systolic to diastolic phases of the cardiac cycle . 
 image analysis images obtained from data sets were analysed in consensus by a radiologist and a cardiologist with six years experience una quantificazione ben riproducibile della dimensione e della funzione ventricolare ; vengono ottenute ricostruzioni di immagini per ogni fase del ciclo cardiaco dalla fase diastolica alla fase sistolica ; normalmente vengono utilizzati solo i migliori dataset per la valutazione delle coronarie , usando quindi una minima parte delle molteplici informazioni generalmente disponibili . 
 la angiografia coronarica ( ac ) - tcms stata considerata sempre di pi , di fatto , come un potenziale strumento per la valutazione combinata della anatomia coronarica , della perfusione miocardica e della funzione del vs [ 7 ]  . 
 lo scopo di questo studio comparare la valutazione della funzione sistolica del vs mediante tcms con lecografia ( eco ) nellambito di una pratica cardiologica routinaria . materiali e metodi popolazione campione nel periodo compreso tra aprile 2008 e settembre 2009 , 282 pazienti ( 178 m , 104 f , et media 64 , 02 anni ) sono stati sottoposti ad ac - tcms per la valutazione di patologie delle arterie coronarie . 
per lo studio finale sono stati quindi considerati 116 pazienti . i pazienti selezionati per lo studio presentavano indicazioni cliniche contemplate nelle guide internazionali [ 8 ] , vale a dire pazienti con dolore toracico atipico , stress test non dirimenti o discordanti , pazienti in follow - up per stent e bypass . 
tutti i pazienti avevano eseguito in precedenza una studio ecocardiografico.il setting clinico della popolazione di pazienti riassunto in tabella 1 . i criteri di esclusione comprendevano insufficienza renale grave ( creatinina 120 mol / l ) , precedenti reazioni allergiche al mezzo di contrasto iodato , gravi malattie polmonari . 
sono stati esclusi quindi i pazienti con aritmie quali fibrillazione atriale cronica con risposta ventricolare elevata e flutter atriale , cos come pazienti con frequenti battiti prematuri atriali e ventricolari ; inoltre pazienti che sono stati sottoposti alla ac - tcms con sincronizzazione prospettica . ac - tcms : tecniche e metodi di indagine tutti i pazienti inclusi nel protocollo sono stati studiati mediante apparecchio con 64 detettori ( brilliance 64 , philips , best , olanda )  . 
images were postprocessed using the philips comprehensive cardiac package included in an off - line external workstation ( ebw64 , brilliance 64 , philips , best , the netherlands )  . 
 stato somministrato un mezzo di contrasto iodato alla concentrazione iodica di 400 mgi / ml ( iomeron 400 , bracco , milano , italia ) , per via endovenosa , attraverso una vena antecubitale del braccio sinistro mediante unagocannula da 1820 gauge somministrato al flusso di 5 ml / s , alla dose standard di 80100 ml seguito da un radiol med ( 2011 ) 116 : 505520 table 2 scan protocol , raw data reconstruction and contrast material administration imaging parameters scan detectors collimation ( mm ) kilovolt mas / slice rotation time ( ms ) scan time ( s ) temporal resolution reconstruction slice thickness ( mm ) reconstruction increment ( mm ) fov ( mm ) convolution filter / kernel contrast material volume ( ml ) flow rate ( ml / s ) iodine concentration [ i ] ( mgi / ml ) bolus chaser venous access 0.625 120 / 80 1201000 912 210 ms 250300 medium ( xcc ) 80 / 100 40 ml@5 ml / s antecubital tabella 2 parametri di scansione utilizzati nella acquisizione ac - tcms scansione detettori collimazione ( mm ) voltaggio ( kv ) amperaggio ( mas ) tempo di rotazione ( ms ) avanzamento ( mm / rot ) tempo per una scansione ( s ) ricostruzione spessore effettivo dello strato ( mm ) indice di ricostruzione ( mm ) campo di vista ( mm ) filtro di convoluzione finestra ( ampiezza / centro ) mezzo di contrasto volume ( ml ) velocit di somministrazione ( ml / s ) tempo di somministrazione ( s ) concentrazione [ i ] ( mgi / ml ) bolus chaser ( soluzione fisiologica ) accesso venoso 0 , 625 120 / 80 1201000 19 , 2 912 250300 medio ( xcc ) 600 / 200 80 / 100 40 ml@5 ml / s antecubitale using simpsons method [ 10 ]  . 
interobserver variability was calculated by means of kappa statistics . end - diastolic volume , end - systolic volume , stroke volume , cardiac mass , ef and cardiac output were automatically available on the biplane view . 
per ogni dataset sono stati tracciati automaticamente i contorni epicardico ed endocardico della cavit ventricolare sinistra in modo da ricavare i volumi del vs attraverso il metodo di simpson [ 10 ]  . 
la variabilit interoperatore stata calcolata tramite statistica k . il volume telediastolico , il volume telesistolico , lo stroke volume , la massa cardiaca , la frazione di eiezione e la portata cardiaca sono risultati cos disponibili per le ricostruzioni biplanari . 
gli operatori hanno misurato manualmente in una sezione sullasse corto , al di sotto del piano mitralico , sia lo spessore del setto interventricolare e della parete posteriore , sia i diametri tele diastolico e tele sistolico del vs . 
stato inoltre calcolato il diametro principale dellaorta ascendente . dalle ricostruzioni 2d e 4d ottenute mediante compattazione di ogni dataset in modalit cine gli operatori sono stati in grado di giudicare la cinetica di parete del ventricolo basandosi sul modello a 17 segmenti dellamerican heart association ( aha ) [ 10 ]  . 
tale modello si basa su una divisione del vs in tre porzioni sullasse corto : basale e media con 6 segmenti , apicale con 4 segmenti ; lapice considerato lultimo segmento . 
2a - d scansioni 2 camere e 4 camere ottenute in ac - tcms in a e b ed ecocardiografia in c e d che dimostrano la parete ventricolare sinistra assottigliata e la camera ventricolare dilatata . ters of septal thickness , posterior lv wall thickness and end - systolic and end - diastolic diameter . 
finally , the mean diameter of the ascending aorta above the valsalva sinuses ( sinotubular junction ) was calculated , as it is a constant parameter reported in each ecg examination . 
 from 2d and 4d cine views obtained by compacting lesame eco e lo studio con ac - tcms sono stati eseguiti radiol med ( 2011 ) 116 : 505520 fig . 
3 epicardial ( arrow ) and endocardial ( arrowhead ) automatic borders of the left ventricular ( lv ) cavity automatically traced to derive lv volume calculations using simpsons method . 
3 bordi automatici lungo la superficie epicardica ( freccia ) ed endocardica ( testa di freccia ) della parete ventricolare sinistra calcolati per valutare i volumi ventricolari con il metodi di simpson . 
 each reconstruction into cine mode , operators were able to judge the wall motion of the ventricle based on the 17segment model of the american heart association ( aha ) [ 10 ] , with six segments each in the basal and mid - lv section , four segments in the apical section and one segment representing the lv apex . 
 nellintervallo di tempo variabile da 1 giorno a un mese . la valutazione ecocardiografica della funzione del vs stata eseguita da un cardiologo esperto , in cieco rispetto ai risultati della ac - tcms . 
the frame at the peak of the r wave was determined as the end - diastolic frame , and the chamber size with the smallest dimension was determined as the end - systolic frame . 
septal thickness and posterior lv wall thickness , end - systolic and end - diastolic diameter and the mean diameter of the ascending aorta were also calculated in a submitral short - axis plane . 
a visual score of 1 if normal , 2 if hypokinetic , 3 if akinetic , 4 if dyskinetic was assigned to each segment . statistics categorical variables were presented as numbers and percentages . 
accuracy of mdct - ca compared with ecg considered as the reference standard was obtained by calculating sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv )  . 
mean heart rate during data acquisition was 62 bpm ranging from 5570 bpthe time required for image analysis of each data set ( including data retrieval ) ranged from 15 to 40 mmanual correction of the epicardial and endocardial borders was necessary to optimise the position of the boundaries in 82% of patients despite the overall good image sistolica . 
la frazione di eiezione stata calcolata dalle immagini convenzionali apicale , a 2 - camere , a 4 - camere , utilizzando il metodo area - lunghezza e un software disponibile in commercio ( ge prosolv analyzer )  . 
sono stati riportati il volume telediastolico , il volume telesistolico , lo stroke volume , la massa cardiaca , la frazione di eiezione e la portata cardiaca ; sono stati misurati in una sezione sullasse corto , al di sotto del piano mitralico , lo spessore del setto interventricolare e della parete posteriore del vs , i diametri tele diastolico e tele sistolico e il diametro principale dellaorta ascendente . 
dai cine 2d registrati , gli operatori hanno giudicato la cinetica del vs , basandosi sullo stesso modello usato in actcms . ad ogni segmento ottenuto stato assegnato un punteggio di 1 se normale , 2 se ipocinetico , 3 se acinetico , 4 se discinetico . analisi statistica le variabili categoriche sono state presentate come numeri e percentuali . 
laccuratezza della ac - tcms nei confronti delleco , lo standard di riferimento , stata ottenuta calcolando la sensibilit , la specificit , il valore predittivo positivo e il valore predittivo negativo . 
la frequenza media durante lacquisizione era di 62 bpm con un range di 55 - 70 bp il tempo necessario per lanalisi delle immagini di ogni dataset ( comprendente caricamento dati ) ha richiesto tra i 15 e i 40 minuti . 
il diametro telediastolico medio risultato di 52 , 82 mm , il diametro telesistolico di 37 , 87 m il volume telediastolico medio risultato di 160 , 61 ml , il volume telesistolico medio di 54.95 ml , la frazione di eiezione media ( lvef ) di 59 , 17% ( range 17%85% )  . 
lo stroke volume , la massa cardiaca e la portata cardiaca medi sono risultati rispettivamente di 81 , 48 ml , 95 , 73 g , radiol med ( 2011 ) 116 : 505520 quality of data sets . 
mean end - diastolic diameter was 52.82 mm and end - systolic diameter 37.87 mmean end - diastolic volume was 160.61 ml , end - systolic volume 54.95 ml and mean lvef 59.17% ( range 17%85% )  . 
mean aortic diameter was 32.94 mm ( range 1949.5 mm ) , mean septum thickness 8.83 mm ( range 2.515.5 mm ) and mean posterior wall 9.67 mm ( range 621 mm )  . 
the coefficient of agreement between operators and software in wall motion analysis was r = 0.7 echocardiography the time required for cine - loop analysis of each patient ranged from 5 to 20 mmean end - diastolic diameter was 52.24 mm and end - systolic diameter 31.57 mmean enddiastolic volume was 99.43 ml , end - systolic volume 43.81 and mean lvef 58.76% ( range 30%75% )  . 
mean aortic diameter was 35.53 mm ( range 2751 mm ) , mean septum thickness 11.03 mm ( range 715 mm ) and mean posterior wall 9.7 mm ( range 715 mm )  . 
the correlation between mdct - ca and ecg in end - diastolic volume , end - systolic volume , stroke volume , ef and cardiac output summarised in table 3 . 
according to blandaltman plots , there was a 74% mean difference between posterior wall on ecg and that on mdct - ca , 2.2% mean difference between interventricular septum , 7.7% between ascending aorta , 5.9% for end - diastolic diameters , 8.7 for end - systolic diameters , 5446 , 68 ml / mil diametro medio dellaorta risultato di 32 , 94 mm ( range 1949 , 5 mm ) , lo spessore medio del setto 8 , 83 mm ( range 2 , 515 , 5 mm ) , della parete posteriore 9 , 67 mm ( range 621 mm )  . 
il diametro telediastolico medio risultato di 52 , 24 mm , il diametro telesistolico di 31 , 57 m il volume telediastolico medio risultato di 99 , 43 ml , il volume telesistolico medio di 43 , 81 , la lvef di 58 , 76% ( range 30%75% )  . 
il diametro aortico medio risultato di 35 , 53 mm ( range 2751 mm ) , lo spessore medio del setto di 11 , 03 mm ( range 715 mm ) , della parete posteriore di 9 , 7 mm ( range 715 mm )  . lo stroke volume , la massa cardiaca e la portata cardiaca medi sono risultati rispettivamente di 78 , 78 ml , 205 , 53 g , 4171 , 79 ml / m la correlazione tra la actcms e lecocardiografia in termini di diametro telediastolico e telesistolico , diametro aortico , spessore del setto e della parete posteriore , volume telediastolico e telesistolico , frazione di eiezione , stroke volume , massa cardiaca e portata cardiaca stata riassunta nella tabella 3 . 
nellanalisi per paziente della cinetica globale del vs la correlazione tra lac - tcms e leco risultata in una concordanza tra le due tecniche nel 73 , 07% dei casi . 
 stata riscontrata una buona correlazione tra le misure dello spessore del setto e del diametro dellaorta ascendente in eco e in tc , mentre una scarsa correlazione tra le misure dello spessore della parete posteriore . 
la tcms ha sovrastimato in modo consistente i valori medi dei volumi rispetto allecocardiografia . in ogni caso , i volumi telediastolico e telesistolico derivati dalla 2dse e dalla tcms hanno mostrato una correlazione significativa . 
come indicatore di funzione sistolica globale del vs , la fe misurata tramite tcms o ecocardiografia ha mostrato uneccellente correlazione . lo stroke volume e la portata cardiaca sono risultati non correlare bene , mentre per lindice di massa cardiaca si 514 radiol med ( 2011 ) 116 : 505520 table 3 comparison between multidetector - row computed tomography coronary angiography ( mdct - ca ) and echocardiography ( ecg ) linear and volumetric parameters in the evaluation of left ventricular function . 
global lv systolic function assessment is performed in daily practice by ecg , although there are other noninvasive imaging techniques such as magnetic resonance imaging ( mri ) , single - photon emission ct ( spect ) and positron emission tomography ( pet ) for evaluation of lv volumes and global systolic function [ 11 ]  . depending on the mdct - ca acquisition technique , assessing cardiac function may be possible [ 9 ]  . 
secondo i grafici di bland - altman le differenze medie tra eco - cardiografia e ac - tcms nella valutazione della parete posteriore , risultata del 7 , 4% , per il setto interventricolare del 2 , 2% , per laorta ascendente del 7 , 7% , per i diametri telediastolici 5 , 9% , telesistolici 8 , 7% , 0% per la gittata cardiaca , 10% per la frazione di eiezione e 2 , 4% per lo stroke volume . 
i risultati sono riassunti in figura 5 . discussione i nostri risultati hanno dimostrato una buona concordanza generale tra i parametri della ac - tcms e quelli rilevati dalleco nella valutazione della funzione sistoradiol med ( 2011 ) 116 : 505520 in our study , only 15 cases were excluded due to prospective triggering , but there was no need for lv analysis . time for lv function analysis with mdct - ca is higher compared with ecg . 
 according to the blandaltman plot , the mean differences between the two techniques are relatively low ; however , not all data fall into sd limits , thus indicating that the results using the two modalities may not be interchangeable . 
 [ 14 ] , although many papers show excellent concordance of mri and ct in assessing lv function , it is very unlikely that ct or mri can be used as first - line investigations . 
however , if the same raw data provided by ct - ca are used to reconstruct a multiphase data set , lv function can be assessed without exposing the patient to any further investigation , and the coronary artery study can be integrated with a functional study that has the same accuracy as mri . 
 our study , compared with previously published reports , investigated several parameters that are routinely recorded on ecg and not on mdct - ca [ 7 ] for example , bansal et al . [ 13 ] compared lv function in evaluating volumes and ef , whereas our study is more comprehensive and considers the diameters of the posterior wall , septal thickness and many other parameters routinely assessed in an ecg study . 
moreover , in our department , aortic root diameter is constantly part of the evaluation of the heart with ecg , and for this reason , this parameter was also retrieved from the data sets . 
la valutazione della funzione del vs viene eseguita nella pratica quotidiana tramite leco , sebbene esistano numerose altre tecniche dimaging non invasive , come la risonanza magnetica ( rm ) , la tomografia computerizzata a emissione di fotoni singoli ( spect ) e la tomografia ad emissione di positroni ( pet ) , per la valutazione dei volumi e della funzione sistolica globale [ 11 ]  . 
a seconda della tecnica di acquisizione actcms , possibile ottenere una valutazione della funzione cardiaca [ 9 ] ; nella sincronizzazione elettrocardiografica ( ecg ) retrospettiva vengono infatti raccolte informazioni durante tutto il ciclo cardiaco che rendono possibile la valutazione funzionale cardiaca , sia da un punto di vista qualitativo che quantitativo . 
dal nostro studio sono stati esclusi 15 casi a causa del triggering prospettico che non ha permesso la valutazione funzionale essendo in questo caso effettuata una acquisizione solo in fase telediastolica . il tempo necessario per lanalisi della funzione ventricolare sinistra con la ac - tcms risultato maggiore rispetto a quello con leco . 
questo fatto dovuto principalmente allacquisizione dei dati e alla necessit dellanalisi dei contorni nella ac - tcms ; va sottolineato per che questa metodica presenta una variabilit irrilevante tra i vari operatori , mentre lecocardiografia rappresenta una metodica fortemente operatore dipendente . 
rispetto a quanto gi riportato da altri autori abbiamo ottenuto simili risultati di buona concordanza tra le due tecniche nella valutazione della frazione di eiezione , diametri , volumi telediastolico e telesistolico , anche se abbiamo riscontrato una sovrastima dei volumi rispetto a quelli misurati con lecocardiografia . 
questa sovrastima , comunque , un fattore noto e riconosciuto e concorda con quanto riportato da altri autori [ 13 , 14 ]  . secondo i grafici di bland - altman le differenze medie tra le due tecniche sono relativamente basse , tuttavia i valori non cadono sempre nei limiti delle deviazioni standard , evidenziando cos come i risultati delle due tecniche possono non essere intercambiabili . 
 the standard of reference in assessing lvef is cardiac eccellente tra rm e tc nella valutazione del vs , non comune che la rm o la tc possano essere utilizzante come metodiche di prima battuta . 
4 risultati della analisi di regressione lineare tra ecocardiografia e ac - tcms nella misurazione dei volumi ventricolari , frazione di eiezione , parametri lineari , gittata cardiaca e massa cardiaca . 
we performed no comparison with this technique , and this can be considered a limitation of the study . however , in our department , mri is performed for cardiac purposes in very selected cases only , whereas ecg is widely used . 
this aspect is not a contraindication to mdct - ca , which also can thus provide important information on lv function in this subgroup of patients . indagini e lo studio delle arterie coronarie pu essere integrato con lo studio funzionale con la stessa accuratezza della rm . il nostro studio si differenzia da quelli precedentemente pubblicati in quanto prende in considerazione pi parametri della funzione cardiaca routinariamente registrati in ecocardiografia [ 7 ]  . 
 [ 12 ] hanno comparato solamente i volumi e la frazione di eiezione , mentre in questo nostro lavoro sono stati valutati parametri lineari quali i diametri , lo spessore della parete posteriore e del setto . 
questo pu essere spiegato dalla differenti tecniche usate in ecocardiografia nel ricavare questi dati , talvolta dipendenti da calcoli automatici ottenuti dalla workstation , talvolta calcolati tramite formule a partire da parametri lineari ; un metodo questo che pu essere responsabile di errori maggiori perch tali formule ricavano volumi e coefficienti a partire da misure lineari ed errori di misura anche minimi possono essere amplificati enormemente [ 15 ]  . lo standard di riferimento nella valutazione della funzione ventricolare sinistra la risonanza magnetica cardiaca [ 1619 ] ; non essendo per stata eseguita in questo lavoro una comparazione tra queste tecniche , sar necessario approfondire questo aspetto nei prossimi studi . 
in ogni caso nel nostro dipartimento la cardio - rm viene eseguita in casi selezionati , mentre lecocardiografia viene utilizzata come indagine di prima battuta per la valutazione della funzione . 
inoltre la rm non pu essere utilizzata in pazienti portatori di pacemaker che spesso hanno uno scompenso cardiaco cronico ; questo aspetto non invece una controindicazione alla actcms . il nostro studio ha preso in considerazione anche lanalisi della cinetica regionale della parete del vs , dimostrando una buona concordanza tra la ac - tcms e lecocardiografia nel valutare il deterioramento della cinetica di parete e nel distinguere le aree acinetiche da quelle ipocinetiche , risultando questa metodica estremamente utile e significativa nella valutazione dellinsufficienza cardiaca cronica [ 20 ]  . 
our study demonstrated an overall good concordance between mdct - ca and ecg in assessing impaired motion and distinguishing stationary areas or an overall impaired function , thus helping assess dilated cardiomyopathy [ 20 ]  . 
on mdct - ca , this rate is higher due to the automated calculation of the borders of the ventricle wall , whereas in ecg , kinetics is judged from a qualitative point of view . 
questo dovuto principalmente al fatto che solo il 3 , 85% di questi segmenti stato giudicato come discinetico in ecocardiografia ; questa percentuale risultata pi alta nella ac - tcms per il calcolo automatico dei bordi della parete del vs , mentre in ecocardiografia la cinetica stata giudicata da un punto di vista qualitativo . 
tuttavia il ruolo definitivo della ac - tcms in questa applicazione funzionale deve essere validata ulteriormente per poter usare questa metodica nella pratica quotidiana . conclusions conclusioni our study demonstrates that mdct - ca provides parameters of cardiac function comparable with those retrieved in ecg . 
thus , mdct - ca can provide additional information to the study of the coronary arteries and is available for diagnostic workup of patients with coronary artery disease and cardiac disease . 
 questo studio dimostra che la ac - tcms ottiene parametri equivalenti rispetto quanto campionato con lecocardiografia nella valutazione della funzione ventricolare quindi la ac - tcms pu essere usata come una valida alternativa per valutare allo stesso tempo la patologia delle arterie coronarie e la funzione cardiaca . radiol med ( 2011 ) 116 : 505520 fig . 
the rst chapter is about the general aspects of fractures in child abuse ( discussion of the differences between paediatric and adult fractures , their differential diagnosis and types of fractures in child abuse )  . 
il primo capitolo tratta degli aspetti generali delle fratture nellabuso ( con discussione delle differenze tra le fratture del bambino e delladulto , la loro diagnosi differenziale ed il tipo di fratture nellabuso )  . 
i successivi otto capitoli trattano : il cranio ; le coste ; le clavicole , le scapole , lo sterno , le vertebre e il bacino ; le ossa lunghe ; il trauma accidentale ; le varianti normali e le malattie congenite ed acquisite ; la radiologia di fronte al sospetto di abuso del bambino ; la datazione della frattura ( alcune pagine di questultimo capitolo 672 radiol med ( 2011 ) 116 : 671672 are also devoted to histological fracture dating of fresh and dried bone tissue from the forensic point of view )  . each chapter follows a rather strict lay - out : introduction , clinical presentation , radiological ndings ; differential diagnosis ; data from the literature ; conclusions and closes with an up - dated literature reference list ( the same reference list is printed twice in the last chapter ! )  . each type of non - accidental trauma can be described more than once according to the relevant literature references , detailing occurrence numbers , percentages etc . image reproduction is quite good and the use of detailed reproduction or photo - enhancement to improve fracture visibility should be praised . 
 there is also a valuable chapter on how to radiograph a child in case of suspected child - abuse related fractures : forget the old fashioned baby - gram and follow the strict rules of the british society of paediatric radiology , implemented by the british society of radiology and those of the american society for pediatric radiology . this is a must - have book , particularly in italy where nonaccidental fractures are not commonly encountered and described ( the very few references from the italian literature are proof of this ) , in order to be prepared , in the unpleasant occurrence of such an event , on how to appraise and report it and to raise awareness on what to do and the related consequences . 
biondetti2 1school of residency , universit degli studi di milano , via di rudin 8 , milano , italy 2foundation irccs , ca granda ospedale maggiore policlinico , via francesco sforza 28 , milano , italy 3dipartimento di scienze dellinformazione ( dsi ) , universit degli studi di milano , via comelico 39 / 41 , milano , italy correspondence to : f . 
this study sought to identify not only hepatodiaphragmatic intestinal interpositions , de ned as chilaiditi , but also other forms of intestinal interposition , which we termed non - chilaiditi . 
moreover , a questionnaire investigating the clinical symptoms reported to be associated with chilaiditi syndrome was given to patients exhibiting any form of intestinal interposition and to a control sample . 
of the 4 , 338 patients examined , 130 ( 3% ) were found to have intestinal interposition , for a total of 143 forms : 90 chilaiditi and 53 non - chilaiditi . 
 in tale studio , sono state evidenziate sia interposizioni di tipo epatodiaframmatico , che sono state de nite chilaiditi come da letteratura , sia altri tipi di interposizione de nite secondo i diversi rapporti anatomici : splenorenale , retrogastrica , epatocavale , retrosplenica e retrorenale , che sono state raggruppate sotto il termine non - chilaiditi . 
su 4338 pazienti sono stati osservati 130 ( 3% ) pazienti con interposizione colica per un totale di 143 manifestazioni , 90 chilaiditi e 53 non - chilaiditi : 30 interposizioni di tipo splenorenale , 12 di tipo retrogastrico , 5 epatocavale , 4 retrosplenico e 2 retrorenale . 
abbiamo inoltre evidenziato che anche le forme non - chilaiditi sono statisticamente pi sintomatiche dei casi controllo . keywords chilaiditi sign chilaiditi syndrome intestinal interposition retrosplenic colon retrogastric colon parole chiave segno di chilaiditi sindrome di chilaiditi interposizione intestinale colon retrosplenico colon retrogastrico introduction introduzione intestinal interposition is a condition in which a section of intestine is temporarily or permanently interposed either between the liver and the diaphragm ( a condition known as chilaiditis sign ) or between the spleen and the diaphragm , the spleen and the left kidney , the stomach and the pancreas , the liver and the inferior vena cava or the kidney and the diaphragm ( these latter conditions being classi ed by us as non - chilaiditi forms )  . 
although colonic interposition is no doubt the most frequent , small - bowel interposition may also be observed ( at the hepatodiaphragmatic level ) , whereas simultaneous interposition of the two segments is rare [ 1 ]  . 
 in 1910 , demetrius chilaiditi [ 2 ] described the radiographic ndings of intermittent hepatodiaphragmatic interposition of the large bowel in three patients , and since then , the term chilaiditis sign has been commonly used to describe this condition [ 3 ]  . 
other forms of colonic and / or small - bowel interposition have only occasionally been investigated [ 4 ] , probably as a result of the fewer symptoms reported and the imaging modalities employed before the age of computed tomography ( ct )  . 
the aim of this paper is therefore to describe the prevalence and clinical impact of non - hepatodiaphragmatic forms of intestinal interposition in an adult population undergoing ct for a range of indications . materials and methods linterposizione intestinale una condizione nella quale un tratto di intestino si trova interposto temporaneamente o permanentemente tra fegato e diaframma ( condizione nota come segno di chilaiditi ) o tra milza e diaframma posteriormente , tra milza e rene sinistro , tra stomaco e pancreas , tra fegato e vena cava inferiore , tra rene e diaframma ( condizioni da noi raggruppate come forme non - chilaiditi )  . 
 linterposizione colica decisamente la pi frequente , ma si osserva talvolta interposizione del piccolo intestino ( in sede epatodiaframmatica ) , mentre linterposizione contemporanea dei due segmenti ancora pi rara [ 1 ]  . nel 1910 demetrius chilaiditi [ 2 ] studi con radiogrammi convenzionali tre pazienti con interposizione epatodiaframmatica intermittente del grosso intestino e da allora il termine segno di chilaiditi stato usato comunemente per descrivere questa condizione [ 3 ]  . 
probabilmente per la minore sintomatologia espressa , nonch per le metodiche utilizzate prima dellera della tomogra a computerizzata ( tc ) , le altre forme di interposizione colica e / o dellintestino tenue non sono state indagate se non saltuariamente [ 4 ]  . 
la sintomatologia clinica eventualmente espressa dai portatori di interposizione epatodiaframmatica , condizione nota come sindrome di chilaiditi [ 5 ] , stata ampiamente trattata , mentre nessun autore si riferisce in tal senso alle forme non - chilaiditi , n vi sono articoli relativi alla loro prevalenza globale nella popolazione generale . 
per tali motivi , in questo articolo abbiamo voluto descrivere la prevalenza delle forme di interposizione intestinale non epatodiaframmatica nonch il loro impatto clinico , in una popolazione adulta sottoposta a tc per differenti patologie . from november 2008 to april 2009 we looked for the presence of intestinal interposition in 4 , 338 adult patients undergoing abdominal , chest ( to mid - kidney level ) or whole - body ct for a range of medical and surgical indications . 
 interposition was de ned as hepatodiaphragmatic when the colon or small bowel were found to lie between the liver and the diaphragm at or above the level of t11 ; splenorenal when the colon was located between the left kidney and spleen at or above the level of the renal hilum ; retrogastric or gastropancreatic when the large bowel lay between the stomach and the pancreas ; hepatocaval when the colon was located between the liver and the inferior vena cava not below the portal plane ; retrosplenic if the colon was interposed between the spleen and the diaphragm at or above the level of the splenic hilum ; and retrorenal when the organ was located between the left kidney and the diaphrag patients who had undergone splenectomy were excluded from the analysis because of the possible anatomical changes brought about by the surgical procedure , as were patients with ascites , given that free uid creates a space between the liver and the diaphragm into which the bowel can easily migrate , as reported in the literature [ 6 ]  . the different conditions were grouped into two classes , separating the better known and studied hepatodiaphragmatic interposition from all the other forms , which we dened as non - chilaiditi . 
 patients with intestinal interposition as well as a control group were administered a questionnaire on the clinical symptoms reported to be associated with chilaiditi syndrome , in particular , abdominal pain , abdominal distension , constipation and atulence . 
linterposizione stata da noi de nita epatodiaframmatica quando il colon o lintestino tenue sono localizzati tra fegato e diaframma non inferiormente a un piano passante per d11 ; splenorenale quando il colon situato tra rene sinistro e milza , non inferiormente a un piano passante per lilo renale ; retrogastrica o gastropancreatica quando lintestino crasso posto tra stomaco e pancreas ; epatocavale quando il colon localizzato tra fegato e vena cava inferiore , non inferiormente al piano portale ; retrosplenica quando il colon si trova interposto tra milza e diaframma posteriormente , non inferiormente a un piano passante per lilo splenico ; retrorenale quando il viscere si trova tra rene sinistro e diaframma . 
 non sono stati inclusi i pazienti sottoposti a splenectomia per le possibili modi cazioni anatomiche dovute alla procedura chirurgica , n i pazienti con ascite poich , come descritto in letteratura [ 6 ] , la presenza di liquido libero crea uno spazio tra fegato e diaframma nel quale lintestino pu facilmente migrare . abbiamo raggruppato queste diverse condizioni in due classi , separando linterposizione epatodiaframmatica , pi conosciuta e studiata , da tutte le altre forme , de nite da noi non - chilaiditi . 
tenendo conto dei differenti rapporti anatomici che in queste si creano , le abbiamo anche considerate distintamente , per valutarne la prevalenza . ai pazienti con interposizione intestinale e ad un campione controllo , stato successivamente sottoposto un questionario relativo ai sintomi clinici che in letteratura risultano legati alla sindrome di chilaiditi , in particolare : dolori addominali , distensione addominale , costipazione e atulenza . 
sono state quindi valutate le manifestazioni cliniche dei pazienti portatori di forme non - chilaiditi , comparandole con la forma di chilaiditi e col gruppo controllo , per veri care se esistessero delle differenze statisticamente signi cative . 610 radiol med ( 2011 ) 116 : 607619 associated with the symptom , assuming the value 1 if the symptom is present and the value 0 where no symptom is present . 
to overcome the small sample size , the yates correction was applied , obtaining a 2 value = 7.3667 , indicating that the difference in symptoms between the three groups was signi cant at a probability level p3% , implying a probability of p97% that the values obtained for the symptoms were not due to chance . results of 4 , 338 patients undergoing ct , intestinal interposition was identi ed in 130 ( 3% ) , of whom 92 were men and 38 women ( age range 3887 years ; mean 66 years )  . 
table 1 shows the intestinal anatomical variants observed in ten patients curiously , all women who presented with more than one interposition , for a total of 23 cases of interposition . overall , the chilaiditi form was observed 90 times . 
in 83 cases , the interposition involved the colon , whereas in the remaining seven cases , it involved the small bowel ( in each case , the ileum )  . 
colon interposto tra fegato e diaframma ( frecce bianche )  . analisi statistica per la valutazione della sintomatologia abbiamo calcolato la media campionaria e la deviazione standard della variabile casuale legata al sintomo , supponendo che essa assuma i valori 1 ( nel caso in cui sia presente un sintomo ) e 0 ( nel caso in cui non ci sia sintomo )  . 
per sopperire alla scarsa numerosit del campione abbiamo applicato la correzione di yates , ottenendo un valore di 2 pari a 7 , 3667 ; ci consente di ritenere che la differenza di sintomatologia fra i tre gruppi sia signi cativa al livello di probabilit p3% , ovvero che ci sia una probabilit p97% che i valori di sintomatologia ottenuti non siano casuali . risultati su 4338 pazienti sottoposti a esami tc abbiamo evidenziato 130 ( 3% ) pazienti portatori di una condizione di interposizione intestinale , 92 maschi e 38 femmine , di et compresa tra i 38 e gli 87 anni , con una media di 66 anni . 
nella tabella 1 sono riportate le varianti anatomiche intestinali dei dieci pazienti , curiosamente solo femmine , che presentavano pi di uninterposizione , per un ammontare di 23 manifestazioni . nel complesso , la forma di chilaiditi stata evidenziata 90 volte , in 83 era interposto lintestino crasso , mentre nei rimanenti 7 casi linterposizione coinvolgeva lintestino tenue ( sempre ileo )  . 
delle 90 forme di chilaiditi , 76 sono state riscontrate in maschi , mentre 14 erano presenti in femmine ; per quanto riguarda le 53 forme non - chilaiditi , 16 erano presenti in maschi e 37 in femmine . da novembre 2008 ad aprile 2009 , 80 pazienti , per un totale di 82 manifestazioni di interposizione intestinale , hanno dovuto sottoporsi a pi esami tc , osservandosi lincostanza di 30 forme , di cui 27 condizioni di chilaiditi e 3 radiol med ( 2011 ) 116 : 607619 fig . 
 stata quindi riscontrata unincostanza della condizione epatodiaframmatica del 42 , 9% e del 15 , 8% per le altre tipologie di interposizione . per quanto riguarda la sintomatologia , nella tabella 3 sono riportati i dati relativi ai disturbi clinici dei tre gruppi ai quali stato sottoposto il questionario ( forme chilaiditi , forme non - chilaiditi , casi controllo ) , con i valori di media e derivazione standard campionari . 
analizzando i valori delle medie campionarie ottenuti per i tre gruppi si deduce che le forme che procurano una maggiore sintomatologia sono le forme di chilaiditi ( media = 0 , 2444 ) , a cui seguono quelle non - chilaiditi ( media = 0 , 1887 ) e in ne quelle dei casi controllo ( media = 0 , 1077 )  . 
paziente con colon retrogastrico ( freccia in a ) , retrosplenico ( fraccia in b ) e splenorenale ( fraccia in c )  . table 1 patients with two or more forms of intestinal interposition patient no . 
 chilaiditi non - chilaiditi splenorenal retrogastric retrosplenic retrorenal radiol med ( 2011 ) 116 : 607619 ileal ileal ileale ileale tabella 1 pazienti portatori di due o pi forme di interposizione intestinale paziente no . 
non - chilaiditi : retrosplenica ( media = 0 , 50 ) e variabilit maggiore rispetto a tutti gli altri gruppi ( ds = 0 , 5774 ) ; 3 . 
non - chilaiditi : retrogastrica e retrorenale ( media = 0 ) , ovvero nessuna frequenza di sintomatologia e nessuna variabilit ( ds = 0 )  . nella tabella 4 abbiamo applicato il test di signi cativit 2 alla matrice delle sintomatologie . 
it should be discussione linterposizione intestinale epatodiaframmatica fu descritta per la prima volta radiologicamente , da antoine bclre nel 1899 , ma fu nel 1910 che demetrius chilaiditi [ 2 ] , radiologo greco nato a vienna , descrisse tre casi di pazienti con temporanea interposizione epatodiaframmatica del colon . 
 prima dellera tc , le condizioni di malposizione dellintestino differenti dalla chilaiditi , da noi de nite come forme non - chilaiditi , sono state oggetto di poche pubblicazioni , nonch descritte di solito singolarmente . nel 1942 poppel e herstone descrivono uninterposizione sinistra ( left - side interposition ) [ 7 ]  . 
nel 1960 poppel descrive una apposizione duodenocolica ( duodenocolic apposition ) [ 8 ] e postula la persistenza di bande peritoneali embrionarie che comporterebbero lo sviluppo di un mesocolon trasverso corto . 
nel 1977 balthazar [ 10 , 11 ] espone una revisione degli aspetti radiologici delle anomalie congenite della posizione anatomica del colon , total ( n = 273 ) asymptomatic , n ( % ) symptomatic , n ( % ) radiol med ( 2011 ) 116 : 607619 table 4 matrix for calculating the signi cance testa chilaiditi ( n = 90 ) non - chilaiditi ( n = 53 ) control group ( n = 130 ) 68 ( 75.6 ) 43 ( 81.1 ) 116 ( 89.2 ) amatrix does not take into account the separation of forms of intestinal interposition into subgroups , as these were too small to allow statistical signi cance to be tested tabella 4 matrice per il calcolo del test di signi cativita totale ( n = 273 ) asintomatici , n ( % ) sintomatici , n ( % ) chilaiditi ( n = 90 ) non - chilaiditi ( n = 53 ) controllo ( n = 130 ) 68 ( 75 , 6 ) 43 ( 81 , 1 ) 116 ( 89 , 2 ) atale matrice non tiene conto della separazione delle forme di interposizione intestinale nei sottogruppi , in quanto queste sono composte da un numero di campioni troppo basso ai ni del calcolo del test di signi cativit 22 ( 24.4 ) 10 ( 18.9 ) 14 ( 10.8 ) 22 ( 24 , 4 ) 10 ( 18 , 9 ) 14 ( 10 , 8 ) ma non parla di colon retrogastrico . 
nel 1993 old eld e wilbur [ 4 ] descrivono il colon retrogastrico alla tc , ma includono sotto questo termine anche la posizione splenorenale e retrosplenica . nel nostro lavoro , partendo dalle forme di interposizione non epatodiaframmatica descritte in letteratura , abbiamo voluto mantenere una divisione maggiore delle varianti anatomiche intestinali , per studiare la prevalenza di ogni singola forma e osservare se ci fossero delle differenze sintomatologiche tra i gruppi . 
 before the age of ct , the literature contained few studies of forms of intestinal malposition other than chilaiditi , and these were usually reports of single cases . in 1942 , poppel and herstone described a case of leftsided interposition [ 7 ]  . 
in 1960 , poppel reported a case of duodenocolic apposition [ 8 ] and postulated the persistence of embryonic peritoneal bands that would lead to the development of a short transverse mesocolon . 
in 1977 , balthazar published a review of the radiological features of congenital anomalies of the anatomical position of the colon but did not mention retrogastric colon [ 10 , 11 ]  . 
in 1993 , old eld and wilbur described retrogastric colon on ct , but included both splenorenal and retrosplenic positions under this term [ 4 ]  . in our study , starting from the forms of non - hepatodifig . 
note colonic air located posteriorly with respect to the portal vein ( white arrow ) , between the liver ( asterisk ) and inferior vena cava ( black arrow ) ( a )  . 
c piano coronale : colon ( freccia nera a sinistra ) , vena cava inferiore ( freccia bianca ) , diaframma ( freccia nera a destra )  . aphragmatic interposition described in the literature , we sought to maintain a greater division of intestinal anatomical variants with the aim of studying the prevalence of each form and ascertaining whether symptoms differed between the groups . 
in particular , among the single non - chilaiditi forms reported in the literature , we separated the splenorenal and retrogastric forms , which were grouped together in the literature [ 4 ] , and the hepatocaval forms , which were previously evaluated alongside retrogastric forms under the grouping of posterior hepatodiaphragmatic interposition [ 13 ]  . 
this said , however , we also considered the cases of non - hepatodiaphragmatic interposition as a single group of non - chilaiditi forms in order to increase the statistical value of data relating to the symptoms . 
 whereas non - hepatodiaphragmatic forms have been correlated with congenital anomalies [ 7 ] , a range of mainly epatocavali , che in letteratura sono state valutate assieme alla forma retrogastrica , con il nome di epatodiaframmatica posteriore [ 13 ]  . 
lidenti cazione delle haustrae o delle pliche conniventi aiuta in questa differenziazione . se le forme non epatodiaframmatiche sono state correlate ad anomalie congenite [ 7 ] , per la condizione di chilaiditi , pi studiata , sono chiamate in causa diverse eziologie , principalmente acquisite . 
choussat e chaussat - clausse [ 14 ] suggeriscono problemi intestinali ( megacolon , motilit anomale del colon , accumulo anomalo di gas ) , diaframmatici ( diaframma alto , degenerazione muscolare , patologie del frenico ) e / o epatici ( fegato piccolo , interventi chirurgici )  . 
 [ 1 ] e murphy [ 15 ] postulano altre cause associate , come la broncopneumopatia cronica ostruttiva , radiol med ( 2011 ) 116 : 607619 acquired aetiologies have been suggested for the more thoroughly investigated chilaiditi forms . 
choussat and choussat - clausse suggested that conditions affecting the bowel ( megacolon , abnormal colonic motility and abnormal gas accumulation ) , the diaphragm ( high diaphragm , muscular degeneration and phrenic diseases ) and / or liver ( small liver , surgery ) were the main causes [ 14 ]  . 
 murphy demonstrated that in obese individuals , fat deposition between the liver and the diaphragm can be a determining factor in the pathogenesis of hepatodiaphragmatic interposition [ 15 ]  . 
other causes include bowel malrotation [ 10 ] dolichocolon , an abnormal length of the mesentery , laxity of hepatic suspensory ligaments [ 16 ] or bowel mal xation [ 14 ]  . 
the literature reports a highly variable prevalence of hepatodiaphragmatic interposition [ 6 ] , from 0.02% to 1% , if one excludes a single study of cirrhotic patients in whom the condition affected 22% , and a distribution dependent on sex ( male : female ratio 4 : 1 ) and age ( being more common in men > 65 years )  . 
 in our series , 2% of patients had hepatodiaphragmatic interposition , a relatively high percentage compared with the literature , and probably justi ed by the fact that most of our patients had some form of liver disease , resulting in abnormal liver morphology . 
nakagawa and toda have shown that the prevalence of the chilaiditi form in a population composed solely of cirrhotic patients is much higher than that found in the general population , reaching 22% in their study [ 6 ]  . 
in our study , in addition , patients with ascites were excluded , as the uid creates a free space between the liver and diaphragm into which the intestines can easily migrate . to our knowledge , no study to date has satisfactorily assessed the prevalence of non - hepatodiaphragmatic forms of interposition . 
this sex distribution is unexpectedly different from the chilaiditi forms , which affected 76 men and 14 women , with a ratio similar to that reported in the literature [ 5 ]  . 
among the non - chilaiditi forms , splenorenal interposition was by far the most frequent , accounting for 56.6% of cases , followed by the retrogastric form with 22.8% , whereas the other non - chilaiditi forms did not exceed 10% . kolju showed that in one third of patients with chilaiditis sign , the condition is temporary , whereas in two thirds , it is permanent and con rmed by subsequent radiographic examinations [ 5 ]  . 
we found a transiency rate of 42.9% for chilaiditi forms , higher than that reported in the literature ( 33% ) , whereas for the non - chilaiditi forms , the transiency rate was 15.8%. 
altre cause possono essere malrotazione [ 10 ] , dolicocolon , anomala lunghezza del mesentere , lassit dei legamenti sospensori del fegato [ 16 ] o mal ssazione [ 14 ]  . 
la letteratura riporta una prevalenza dellinterposizione epatodiaframmatica molto variabile [ 6 ] , dallo 0 , 02% all1% , se si eccettui uno studio effettuato per solo su pazienti cirrotici , in cui si raggiunto il 22% , presentandosi con una distribuzione dipendente dal sesso ( rapporto maschi : femmine 4 : 1 ) e dallet ( pi frequente nei maschi oltre i 65 anni )  . nella nostra casistica il 2% dei pazienti presentava interposizione epatodiaframmatica , percentuale abbastanza alta rispetto alla letteratura , giusti cata verosimilmente dal fatto che la maggior parte dei nostri pazienti sono epatopatici , con conseguente alterazione della morfologia epatica . 
 nakagawa e toda [ 6 ] hanno dimostrato infatti che la prevalenza della forma di chilaiditi in una popolazione di soli cirrotici molto superiore alla popolazione generale , arrivando nel loro studio al 22% . 
in questo lavoro , tra laltro , non sono stati considerati i pazienti con ascite , poich il liquido libero crea uno spazio tra fegato e diaframma nel quale lintestino pu facilmente migrare : per tal motivo abbiamo escluso tali pazienti dalla nostra casistica . dai dati disponibili risulta che ancora nessuno in letteratura abbia valutato soddisfacentemente la prevalenza delle forme di interposizione non epatodiaframmatica . 
questultimo dato si differenzia in modo inaspettato dalle forme di chilaiditi , dove su 90 portatori della condizione epatodiaframmatica , 76 erano maschi e 14 femmine , mostrando un rapporto percentuale come da letteratura [ 5 ]  . 
tra le forme non - chilaiditi quella splenorenale stata di gran lunga la pi frequente , rappresentando il 56 , 6% dei casi , seguita da quella retrogastrica , con il 22 , 8% , mentre le altre forme non - chilaiditi non hanno superato il 10% . kolju [ 5 ] ha dimostrato come in un terzo dei pazienti portatori di chilaiditi tale condizione sia incostante , mentre nei due terzi confermata da controlli radiogra ci a distanza di tempo . 
abbiamo riscontrato unincostanza dei reperti di chilaiditi del 42 , 9% , superiore a quella della letteratura ( 33% ) , mentre per le forme non - chilaiditi lincostanza stata del 15 , 8% . 
questo dato pu essere spiegato dallipotesi , gi presentata in letteratura , di una patogenesi congenita delle forme non epatodiaframmatiche . linterposizione epatodiaframmatica solitamente asintomatica , ma , come descritto in letteratura , pu essere associata ai seguenti sintomi : dolori addominali , disten618 radiol med ( 2011 ) 116 : 607619 pothesis , already presented in the literature , of a congenital pathogenesis of non - hepatodiaphragmatic forms of interposition . hepatodiaphragmatic interposition is usually asymptomatic , but it may be associated with abdominal pain , abdominal distension , atulence , constipation , nausea , vomiting , shortness of breath , substernal pain and anorexia [ 1 , 17 , 18 ]  . 
the symptoms tend to be worse towards the end of the day and when the patient remains sitting or standing for long periods of time , as this facilitates the descent of the liver and the ascent of the distended bowel . 
the symptoms are also more frequent when the interposition involves the small bowel and when it occurs in persons with mental retardation [ 1 , 17 ] , due to aerophagia . 
in our study , the symptoms considered to be discriminant were abdominal pain , abdominal distension , constipation and atulence , given that these are reported to be the most signi cantly correlated with intestinal interposition [ 1 ]  . 
 with regard to the single groups , however , we encountered an unusual pattern of symptom frequency ( the control group showing a higher frequency of symptoms when compared with some of the non - chilaiditi cases ) , but this may be in uenced by the fact that the sample size available for the subgroups of chilaiditi and non - chilaiditi was small and therefore of limited statistical signi cance . it should be noted that the ileal hepatodiaphragmatic forms were found to be more symptomatic than the colonic forms , as reported in the literature [ 1 ]  . 
although the small sample size does not allow for any statistically valid statements regarding these ndings , it would be interesting to assess the presence or absence of symptoms when encountering these forms in other ct studies . 
 conclusions although intestinal interposition is generally infrequent , the lesser known forms ( non - hepatodiaphragmatic ) account sione addominale , atulenza , costipazione , nausea , vomito , respiro corto , dolore sottosternale , anoressia [ 1 , 17 , 18 ]  . 
i sintomi sono peggiori al termine della giornata e quando il paziente sta seduto o rimane in posizione eretta per lungo tempo , poich ci facilita la discesa del fegato e la risalita del viscere intestinale ripieno di gas . 
il trattamento richiesto di solito conservativo , come riposo a letto o decompressione mediante sondino nasogastrico , mentre la chirurgia raramente richiesta , come per esempio nei casi di volvolo intermittente . partendo da tale quadro sintomatologico , abbiamo voluto valutare la prevalenza della sintomatologia nei pazienti con altre forme di interposizione intestinale . 
nel nostro studio abbiamo considerato come sintomi discriminanti il dolore , la distensione addominale , la costipazione e la atulenza , poich , dalla letteratura [ 1 ] , erano quelli pi signi cativamente correlati ai quadri di interposizione intestinale . 
la nostra analisi statistica ha evidenziato come la sintomatologia delle forme di chilaiditi sia superiore sia a quella delle forme non - chilaiditi , sia a quella del gruppo controllo . 
allo stesso modo , le forme non - chilaiditi presentano una sintomatologia statisticamente maggiore rispetto ai casi controllo . per quanto riguarda i singoli gruppi abbiamo invece avuto un insolito ordinamento delle frequenze di sintomi ( i casi controllo presentano una maggiore frequenza di sintomi rispetto ad alcuni casi non - chilaiditi ) , ma ci pu essere in uenzato dal fatto che per i sottogruppi di chilaiditi e non - chilaiditi a volte disponibile un numero di campioni poco elevato e quindi statisticamente poco signi cativo . da rilevare che le forme epatodiaframmatiche ileali sono risultate essere pi sintomatiche delle corrispettive coliche , come da letteratura [ 1 ]  . 
per quanto riguarda le forme nonchilaiditi , quella epatocavale in 5 casi su 5 sempre sintomatica , mentre un comportamento inverso stato riscontrato nelle forme non - chilaiditi retrogastrica e retrorenale . 
tali dati , per la bassa numerosit del campione , non consentono di fare affermazioni statisticamente valide , tuttavia sarebbe interessante , riscontrando tali forme anche in altri studi tc , valutare la presenza o assenza della sintomatologia . conclusioni per quanto le forme di interposizione intestinale siano poco frequenti , da considerare come quelle meno conosciute ( quelle non epatodiaframmatiche ) ne rappresentino comunque una discreta parte , pi della met secondo radiol med ( 2011 ) 116 : 607619 for a fair proportion of cases more than half , according to our study . 
in view of this aspect and of our ndings with respect to symptomatology , we believe it might be useful to indicate as a descriptive nding in the radiology report the presence of anatomical variants of the small and / or large bowel . 
faletti1 1 dipartimento di diagnostica per immagini , uoa di radiologia diagnostica , azienda ospedaliera cto - crf - maria adelaide , torino , italy 2 sezione di scienze radiologiche , di.bi.me.f , universit di palermo , italy 3 dipartimento di traumatologia , ortopedia e medicina del lavoro , azienda ospedaliera cto - crf - maria adelaide , torino , italy 4servizio di anatomia patologica , a.o. 
between january 2003 and may 2010 , 66 suspected neurinomas were evaluated according to their sonographic features ( solid fusiform mass with well - de ned margins located in direct continuity with the nerve that was not always discernible and heterogeneous as a result of the presence of small cystic areas or calci cations )  . 
the lesions were examined using a sonographic contrast medium consisting of sulphur hexa uoride microbubbles and equipment with dedicated contrast - speci c software [ contrast tuned imaging ( cnti ) ]  . 
 of these lesions , ve were excluded from the analysis because the de nitive diagnosis was not available ( in two cases , the follow - up was still in progress , whereas in the remaining three , there was no follow - up )  . 
in 41 / 61 cases ( 67.2% ) , we identi ed an enhancement pattern that we termed reticular owing to the interweaving of blood vessels , of which two subtypes were identi ed depending on whether the interwoven vessels were densely or sparsely packed : loose - knit riassunto obiettivo . 
nel periodo compreso tra gennaio 2003 e maggio 2010 sono state valutate nel nostro dipartimento 66 lesioni sospette per neurinoma in base alle loro caratteristiche ecogra che ( formazioni solide , fusiformi , a margini netti , in diretta continuit con la bra nervosa , non sempre riconoscibile , disomogenee per leventuale presenza di piccole aree similcistiche o calci cazioni ) , ed esaminate con mezzo di contrasto ( mdc ) ecogra co , costituito da microbolle a base di esa uoruro di zolfo e apparecchiatura dotata di software dedicato contrast tuned imaging ( cnti ) contrasto speci co . 
di queste lesioni , 5 sono state escluse dallanalisi in quanto non era disponibile una diagnosi de nitiva ( n = 2 follow - up in corso ; n = 3 assenza di follow - up )  . 
in 41 / 61 ( 67 , 2% ) casi abbiamo identi cato una distribuzione del mezzo di contrasto de nita a reticolo in base allintreccio formato dalle strutture vascolari , in cui si riconoscono due sottotipi : reticolo a maglie larghe in 18 / 41 e reticolo a maglie strette in 23 / 41 , radiol med ( 2011 ) 116 : 634643 reticular in 18 / 41 , and tight - knit reticular in 23 / 41 . 
results showed that all neurinomas studied could be divided into two groups according to the type of enhancement pattern observed : reticular or diffuse heterogeneous . keywords schwannoma ultrasound power doppler imaging contrast media distinti in base allaspetto dellincrocio dei vasi , se pi rado o pi tto . 
i risultati ottenuti in questo studio hanno dimostrato che tutti i neurinomi analizzati possono essere distinti in due gruppi , in base al tipo di pattern vascolare riscontrato : di tipo reticolare o ad impregnazione diffusa disomogenea . parole chiave schwannoma ecogra a power doppler mezzi di contrasto ecogra ci introduction introduzione neurinomas , or schwannomas , are benign tumours that originate from the sheath of peripheral nerves , in relation to which they may align themselves either eccentrically or peripherally [ 1 ]  . 
when stimulated mechanically , they often give rise to pain that feels like an electric shock [ 2 ] , a feature that allows for a well - founded diagnostic hypothesis . 
recent neurinomas are usually fusiform , hypoechoic and homogeneous [ 35 ] , whereas older ones may exhibit cystic areas and , more rarely , calci cations [ 1 , 68 ]  . 
high - resolution us ( hrus ) performed with high - frequency linear - array probes is an excellent modality for visualising and in some cases differentiating peripheral nerve tumours , as in most cases it allows for the easy recognition of direct continuity between the nerve and the two poles of the lesion on longitudinal sections [ 10 ]  . 
power - doppler ( pd ) is an essential complement to baseline us , as the presence of blood vessels within the lesion will in uence any diagnostic suspicion [ 3 ]  . 
however , although power doppler imaging is particularly sensitive to low ows , it is insuf cient to provide a real estimate of lesion vascularity . recent years have seen the increasing use of contrastspeci c us techniques , which optimise recording of the harmonic signal produced by the contrast microbubbles that amplify the signal from the blood vessels [ 11 , 12 ]  . 
 i neurinomi , o schwannomi , sono tumori benigni che prendono origine dalla guaina dei nervi periferici , rispetto ai quali possono disporsi eccentricamente o perifericamente [ 1 ]  . 
sono spesso accompagnati da sintomatologia dolorosa , tipo scossa elettrica [ 2 ] , qualora sollecitati meccanicamente , caratteristica , questa , che ne permette un sospetto diagnostico fondato ; a volte il quadro clinico dubbio o poco signi cativo . 
 la morfologia e lecostruttura dei neurinomi pu variare a seconda del grado di crescita ; quelli di data recente solitamente si presentano fusiformi , ipoecogeni e omogenei [ 35 ] ; quelli di pi vecchia data , invece , possono presentare al loro interno areole similcistiche e , raramente , calci cazioni [ 1 , 68 ]  . 
 lecogra a ad alta risoluzione , ovvero effettuata con sonde lineari ad alta frequenza , costituisce unottima metodica per visualizzare e , talora , differenziare i tumori del sistema nervoso periferico , poich permette agevolmente di dimostrare , nella maggior parte dei casi , la diretta continuazione con la terminazione nervosa ai due poli della lesione , evidenziabile nelle scansioni longitudinali [ 10 ]  . 
 da qualche anno sempre pi frequente limpiego di tecniche ecogra che contrasto speci che , che ottimizzano la registrazione del segnale armonico prodotto dalle microbolle del mezzo di contrasto ( mdc ) ecogra co , le quali consentono un potenziamento del segnale proveniente dai vasi [ 11 , 636 radiol med ( 2011 ) 116 : 634643 unlike diffusive contrast media ( such as those used in magnetic resonance imaging and computed tomography ) that enter the extracellular spaces , us contrast agents remain con ned within the vessels , allowing for clear visualisation of even the smallest vascular structures ( the parenchymal microvasculature )  . 
us contrast agents have a wide range of applications , especially in studying liver and breast focal lesions and in the eld of cardiovascular imaging and haemodynamics [ 1315 ]  . 
few studies have investigated the use of contrast agents in the musculoskeletal system , and no scienti c contributions to date have studied the use of us contrast agents for studying neurinomas [ 16 ]  . 
a differenza dei mezzi di contrasto diffusivi ( utilizzati in risonanza magnetica [ rm ] e tomogra a computerizzata [ tc ] ) , che si dispongono anche negli spazi extracellulari , il mezzo di contrasto ecogra co rimane con nato , per le sue caratteristiche siche , allinterno dei vasi , consentendo cos una precisa visualizzazione anche delle strutture vascolari pi piccole ( microvascolarizzazione parenchimale )  . 
il mdc ecogra co presenta un ampio campo di applicazione soprattutto in ambito epatico e mammario , per lo studio delle lesioni focali , nel distretto cardio - vascolare e nellemodinamica [ 1315 ]  . 
in letteratura limpiego del mdc in ambito muscolo - scheletrico scarsamente rappresentato e non sono stati attualmente riscontrati contributi scienti ci in merito allo studio dei neurinomi con mdc ecogra co [ 16 ]  . 
 scopo del nostro studio valutare se esiste una distribuzione caratteristica dei vasi allinterno dei neurinomi e tentare in tal modo una caratterizzazione degli stessi . materials and methods materiali e metodi between january 2003 and may 2010 , 66 lesions were identi ed as possible neurinomas on the basis of for their us features ( solid , fusiform masses with well - de ned margins , at times in direct continuity with the nerve bre or heterogeneous as a result of the presence of small cystic areas or calci cations )  . 
five of these lesions were excluded from the analysis because a de nitive diagnosis was not available ( n = 2 with follow - up still in progress ; n = 3 without follow - up )  . 
all of these were single lesions except in the case of a woman who had two separate lesions , one in her right thigh ; the other in her left leg . 
these 61 lesions therefore form the basis of our study . the patients ( 35 females , 25 males ) were aged between 15 and 90 ( mean 47 ) years . 
lesion size ranged from 0.9 cm to 7 cm ( table 1 )  . us examinations were performed using an esaote technos mpx unit equipped with la424 14 - 8 mhz , la523 13 - 8 mhz and la532e 8 - 3 mhz linear - array probes and dedicated low - mechanical - index ( 45 kpa ) cnti software , which allows real - time analysis of the signal generated by the contrast mediu all examinations were performed by two operators , both experts in us imaging . 
di queste lesioni , 5 sono state escluse dallanalisi in quanto non era disponibile una diagnosi de nitiva ( n = 2 followup in corso ; n = 3 assenza di follow - up )  . 
le lesioni asportate chirurgicamente e confermate come neurinomi allesame istologico , considerato come gold standard , sono state 61 , tutte uniche tranne nel caso di una donna che presentava due lesioni distinte , una alla coscia destra e una alla gamba sinistra ; dunque unicamente su queste lesioni che si basa il nostro studio . i pazienti ( 35 femmine , 25 maschi ) avevano unet compresa tra 15 e 90 anni ( et media 47 anni ) ; 38 neurinomi erano disposti in sede inframuscolare e 23 sottocutanea ; 24 interessavano larto superiore , 35 larto inferiore , 1 il dorso e 1 la regione cervicale ; le dimensioni variavano da 0 , 9 a 7 cm ( tabella 1 )  . 
 gli esami sono stati condotti con ecografo technos mpx esaote ( biosound esaote , inc , indianapolis , usa ) , dotato di sonde lineari la424 14 - 8 mhz , la523 13 - 8 mhz e la532e 8 - 3 mhz e di software dedicato ( cnti ) a basso indice meccanico ( 45 kpa ) , che consente lanalisi in real time del segnale proveniente dal mezzo di contrasto . 
tutti i pazienti sono stati esaminati in posizione supina o prona , a seconda della sede della lesione , inizialmente con esame ecogra co basale , successivamente con valutazione colorpower doppler . 
at the end of the injection , a timer was started that allowed determination of the time elapsed between the entry of the contrast medium in the circulation and its arrival in the lesion . 
contrast medium time of arrival and distribution pattern was documented on video clips , each lasting about 30 s and which highlighted the most signi cant moments of the study . 
all lesions were studied for at least 3 min ( late phase )  . con 5 cc di soluzione siologica ; ai pazienti non sono stati richiesti digiuno o particolari esami di laboratorio ; in tutti i casi non si sono mai veri cati eventi avversi . 
a ne iniezione viene fatto partire un timer che consente di valutare il tempo che intercorre tra il passaggio in circolo del contrasto e larrivo dello stesso nella lesione esaminata . 
la presenza di spot iperecogeni intraparenchimali dimostra larrivo del mdc nella lesione ; il tempo di arrivo e la modalit di distribuzione sono documentate mediante la registrazione di videoclips , ciascuno della durata di circa 30 secondi , che evidenziano i momenti pi signi cativi dello studio . 
the neurinomas examined at baseline us exhibited a predominantly fusiform appearance ( 40 / 61 ) , with the nerves from which they had arisen being visible at either pole ( 37 / 61 )  . 
sometimes , they appeared as rounded ( 14 / 61 ) or as lobular ( 7 / 61 ) , and it was not possible to visualise the nerve from which they had originated ( 23 / 61 )  . 
often , anechoic areas were observed inside them ( 22 / 61 ) , and , more rarely , ( 3 / 61 ) intralesional calci cation was seen . after us contrast agent administration , neurinomas showed the following characteristics : in 41 / 61 cases , the contrast material distributed from the periphery to the centre , with an interweaving of blood vessels of varying thickness some straight , others tortuous leaving spaces of varying size without visible blood vessels . 
in 20 / 61 lesions , however , the contrast agent did not follow a reticular distribution but diffused within the parenchyma , with densely packed , homogeneously distributed blood vessels , except for the presence of one or more circumscribed areas that exhibited no contrast enhancement . 
this subtype was termed diffuse heterogeneous enhancement ( pattern type 2 ) and was further divided into two subtypes based , in 7 / 21cases , on the presence of a single unenhancing area ( pattern 2a ) , or with more than risultati gli esami sono stati eseguiti da un pool di colleghi esperti della metodica , secondo le indicazioni precedentemente menzionate , ed in seguito revisionati esclusivamente da due di essi , che sono risultati materialmente gli esecutori dellesame nel 88 , 5% dei casi ( 54 / 61 casi )  . 
i neurinomi esaminati , nello studio in basale , hanno presentato prevalentemente un aspetto fusiforme ( 40 / 61 ) ai cui poli si riconosceva la struttura nervosa dalla quale si sviluppavano ( 37 / 61 ) ; talvolta apparivano rotondeggianti ( 14 / 61 ) o bozzuti ( 7 / 61 ) e non si riusciti a visualizzare la struttura nervosa di appartenenza ( 23 / 61 )  . 
spesso , nel loro contesto si sono osservate aree anecogene ( 22 / 61 ) ; pi raramente , in 3 / 61 casi , presentavano calci cazioni intralesionali . dopo somministrazione di mdc ecogra co , i 61 neurinomi esaminati hanno presentato le seguenti caratteristiche : in 41 / 61 casi il mdc si distribuiva dalla periferia al centro , con un intreccio di vasi , costituito da linee pi o meno spesse , a decorso tortuoso o rettilineo , che si intersecano lasciando spazi di differenti dimensioni privi di vasi . 
in 20 / 61 lesioni , invece , il mdc non seguiva una distribuzione di tipo a reticolo , ma si diffondeva allinterno del parenchima con vasi tti , omogeneamente distribuiti , eccezion fatta per la presenza di una o pi aree circoscritte prive di enhancement . 
in tutti i 61 casi losservazione stata protratta no a tre minuti dalliniezione per studiare 640 radiol med ( 2011 ) 116 : 634643 fig.1a , b longitudinal sections of two lesions studied with an 8 - 3 mhz la532 linear probe after intravenous administration of ultrasound contrast material : example of patterns 1a ( a ) loosely - knit reticular and 1b ( b ) tightly - knit reticular . 
1a , b due differenti lesioni studiate mediante scansioni longitudinali dopo somministrazione endovena di mdc ecogra co , con sonda lineare la532 8 - 3 mhz : esempio di pattern 1a ( a ) reticolo a maglie larghe e di pattern 1b ( b ) reticolo a maglie strette . 
i disegni a anco ( a e b ) , chiariscono la distribuzione dei vasi , con il caratteristico intreccio vascolare de nito da noi a reticolo , il cui aspetto , se pi rado o pi tto , permette di contraddistinguere i due sottogruppi . 
we noted that the lesions showing reticular enhancement ( pattern 1 , subtype a or b ) tended to exhibit diffuse and homogeneous distribution of contrast medium during the late venous phases , whereas all neurinomas with diffuse heterogeneous enhancement ( pattern 2 ) maintained the same appearance even 3 min after the injection . discussion according to the international literature , neurinomas , or il comportamento delle lesioni anche nei tempi venosi tardivi . 
 dallanalisi percentuale risulta pertanto che il 67 , 2% dei neurinomi presenta un pattern di tipo 1 , a reticolo , mentre il 32 , 8% presenta un pattern di tipo 2 a impregnazione diffusa disomogenea ( tabella 1 )  . 
abbiamo in ne notato che le lesioni che presentavano un pattern 1 a reticolo , sia di tipo a che di tipo b , nelle fasi venose tardive tendevano ad una diffusa ed omogenea distribuzione del contrasto allinterno della lesione , mentre tutti i neurinomi che appartenevano al gruppo con pattern di tipo 2 impregnazione diffusa disomogenea mantenevano lo stesso aspetto anche a tre minuti dalliniezione . discussione i neurinomi , o schwannomi nella letteratura internazionale , sono tumori benigni a partenza dalla guaina nervosa ; sono riconoscibili come una massa fusiforme disposta eccentricaradiol med ( 2011 ) 116 : 634643 fig . 
2a , b longitudinal sections of two lesions studied with an 8 - 3 mhz la532 linear probe after intravenous administration of ultrasound contrast agent : example of diffuse heterogeneous enhancement , pattern 2a ( a ) showing the presence of a single nonenhancing area and pattern 2b ( b ) due to the presence of several nonenhancing areas . 
2a , b due differenti lesioni studiate mediante scansioni longitudinali dopo somministrazione endovena di mdc ecogra co , con sonda lineare la532 8 - 3 mhz : esempio di pattern 2a ( a ) impregnazione diffusa disomogenea per la presenza di una singola area priva di enhancement e di pattern 2b ( b ) impregnazione diffusa disomogenea per la presenza di pi aree prive di enhancement . 
a anco , i disegni ( a e b ) chiariscono la diffusione del mdc allinterno della lesione attraverso vasi tti e omogeneamente distribuiti ad eccezione delle aree avascolari . schwannomas , are benign tumours originating in the nerve sheath and are recognisable as fusiform masses arranged eccentrically to the nerve and surrounded by a capsule known as the epineuriu the presence of the capsule allows for complete surgical excision of the lesion . 
a location eccentric to the nerve bres is indicative for a diagnosis of neurinoma , but this nding is not very frequent , and the presence of intralesional cystic spaces is typical of cystic neurinomas but very rare in neuro bromas . 
 our research aimed to investigate this aspect , in part in consideration of the fact that few studies have evaluated the vascular appearance of neurinomas and those that have mente al nervo e circondata da una capsula detta epinevrio . 
la localizzazione eccentrica rispetto alle bre nervose suggestiva per la diagnosi di neurinoma , ma questo riscontro non cos frequente ; la presenza di lacune cistiche intralesionali tipica dei neurinomi , molto rara nei neuro bromi . 
nei casi dubbi , per la mancanza della caratteristica morfologia ecogra ca o della tipica sintomatologia , tipo scossa elettrica , la diagnosi risulta meno agevole . attualmente , grazie allimpiego dei moderni ritrovati tecnologici , possibile aggiungere preziose informazioni circa la distribuzione dei vasi allinterno della lesione [ 1720 ] , sfruttando il massimo segnale armonico proveniente dalle microbolle del mezzo di contrasto ecogra co , quando insonate da un fascio ultrasonoro vicino ai 33 , 5 mhz . 
la nostra ricerca si inserisce in questa prospettiva , anche in considerazione del fatto che gli studi in letteratura internazionale , riguardanti la valutazione dellaspetto vascolare dei neurinomi , sono scarsi e riguardano esclusivamente la rm . 642 radiol med ( 2011 ) 116 : 634643 fig . 
3a , b stessa lesione studiata mediante scansioni longitudinali al pd e dopo somministrazione endovena di mdc ecogra co , con sonda lineare la532 8 - 3 mhz : il pd ( a ) visualizza pochi spot vascolari intralesionali , sottostimando la reale vascolarizzazione della lesione , dimostrata dallenhancement della lesione dopo contrasto ( b )  . were conducted with magnetic resonance imaging . 
the added value of us contrast agents is related to the precise evaluation of the distribution of both large and small blood vessels , which allows differentiation into the two main subtypes : reticular , and diffuse heterogeneous enhancement . 
the recognition of either pattern , associated with the morphological characteristics of the lesion , proved useful in directing us towards the diagnosis . conclusions the results obtained in this study show that all neurinomas examined could be divided into two groups according to the type of vascular pattern observed : reticular , or diffuse heterogeneous enhancement . 
il valore aggiunto del mezzo di contrasto ecogra co dato dalla precisa valutazione della distribuzione dei vasi sia di grandi che di piccole dimensioni , in base alla quale sono stati differenziati due principali pattern di distribuzione : a reticolo e a impregnazione diffusa disomogenea . 
il riconoscimento di uno dei due pattern , associato alla caratteristiche morfologiche della lesione , utile ad indirizzarci verso il sospetto diagnostico . conclusioni dai risultati ottenuti in questo studio si evince che tutti i neurinomi analizzati possono essere distinti in due gruppi , in base al tipo di pattern vascolare riscontrato : di tipo reticolare o ad impregnazione diffusa disomogenea . 
dallesperienza acquisita possiamo affermare che un accurato esame ecogra co di base , mirato alla ricerca delle caratteristiche morfo - strutturali dei neurinomi e delleventuale visualizzazione della continuazione delle bre nervose suggestiva per la diagnosi di neurinoma ; se a questo segue poi lo studio con mdc , il riscontro della tipica distribuzione della vascoradiol med ( 2011 ) 116 : 634643 noma . 
if this is followed up by a contrast - enhanced us examination , the nding of a characteristic vascular pattern in the two subtypes identi ed as reticular ( either loose knit or tight knit ) and diffuse heterogeneous enhancement ( due to the presence of one or more nonenhancing areas ) assists in characterising these masses , thus increasing the operators diagnostic con dence . 
this is particularly useful in doubtful cases , especially in lesions that do not exhibit the typical clinical and morphological features of neurinomas and that may mimic more aggressive lesions . larizzazione nei due pattern identi cati come reticolo ( a maglie larghe o a maglie strette ) e impregnazione diffusa disomogenea ( per la presenza di una o pi aree prive di enhancement ) contribuisce a riconoscere come tali queste formazioni , aumentando cos la con denza diagnostica delloperatore . 
bellomi1 , 3 1department of radiology , european institute of oncology , via ripamonti 435 , 20141 milan , italy 2division of epidemiology and biostatistics , european institute of oncology , milan , italy 3school of medicine , university of milan , milan , italy 4division of thoracic surgery , european institute of oncology , milan , italy correspondence to : s . 
 trans ssural course and type of needle were associated with pneumothorax using univariate analysis , whereas trans ssural course was associated with intrapulmonary haemorrhage using both univariate and multivariate analysis . 
sono state valutate caratteristiche relative al paziente , alla lesione , allago e al tragitto intrapolmonare in relazione al risultato patologico e allesperienza degli operatori , alleventuale presenza post - bioptica di pneumotorace e di soffusione parenchimale . 
 il tragitto trans - scissurale dellago spesso associato radiol med ( 2011 ) 116 : 548563 pneumothorax and intrapulmonary haemorrhage , and the type of the needle is related to pneumothorax . con pneumotorace e soffusione , gli aghi citologici con pneumotorace . keywords ct - guided lung biopsy risk factors complications parole chiave biopsie polmonari tc - guidate fattori di rischio complicanze introduction introduzione the growing use of chest computed tomography ( ct ) and the detection of an increasing number of small lung lesions have heightened the interest in percutaneous ct - guided lung biopsy [ 1 ]  . 
when biopsy is used to de ne the diagnostic and therapeutic workup of a lung lesion , the cost bene t ratio must be carefully evaluated for each patient , resulting in a need to take into consideration the most frequent complications of the procedure : pneumothorax and parenchymal haemorrhage [ 2 , 3 ]  . 
although both complications usually resolve spontaneously , the occurrence of pneumothorax during the procedure may lead to an inconclusive result and , in a variable percentage of cases , worsening pneumothorax may require placement of a chest drain whereas parenchymal haemorrhage may require hospitalisation [ 4 ]  . 
knowledge of the possible risk factors for pneumothorax and parenchymal haemorrhage following ct - guided biopsy allows for more accurate communication with patients when obtaining written informed consent , as well as better organisation of the biopsy procedure . 
many variables have been studied as possible risk factors for pneumothorax and parenchymal haemorrhage , with con icting results [ 57 ]  . the purpose of this study was to assess the correlation between pneumothorax and parenchymal haemorrhage following ct - guided lung biopsies and various risk factors related to the patient , the lesion , the needle path , the operators experience and the type of needle employed . luso crescente della tomogra a computerizzata ( tc ) del torace che porta allindividuazione di un numero sempre pi alto di piccole lesioni polmonari , ha portato ad un sempre maggiore interesse per le biopsie percutanee tc - guidate del torace [ 1 ]  . 
quando la biopsia percutanea serve per de nire liter diagnostico - terapeutico di una lesione polmonare , il rapporto costo / bene cio deve essere attentamente valutato per ogni singolo paziente e pertanto le complicanze pi frequenti della procedura , che sono pneumotorace e soffusione parenchimale , vanno prese in considerazione [ 2 , 3 ]  . 
sebbene di solito entrambe le complicanze si risolvano spontaneamente , linsorgenza di pneumotorace durante la procedura pu portare ad un esito non diagnostico e , in una percentuale variabile di casi , lincremento dello pneumotorace potrebbe richiedere il posizionamento di un drenaggio toracico , mentre la soffusione parenchimale potrebbe richiedere lospedalizzazione [ 4 ]  . 
la conoscenza dei possibili fattori di rischio per linsorgenza di pneumotorace e soffusione parenchimale dopo biopsie tc - guidate permette di comunicare ai pazienti informazioni pi precise al momento della rma del consenso informato , e di organizzare al meglio la procedura bioptica . 
molte variabili sono state studiate come possibili fattori di rischio per linsorgenza di pneumotorace e di soffusione parenchimale , con risultati contrastanti [ 57 ]  . lo scopo di questo studio di valutare la correlazione tra linsorgenza di pneumotorace e di soffusione parenchimale dopo biopsie polmonari tc - guidate , con vari fattori di rischio legati ai pazienti , alla lesione , al tragitto della biopsia , allesperienza degli operatori e al tipo di ago . materials and methods patient selection materiali e metodi selezione dei pazienti a retrospective electronic search on our database of all ct scans performed between january 2007 and july 2008 was carried out to select investigations recorded under the institutional code for thoracic interventional procedure . 
the exclusion criteria were previous lung resection ( because of possible postoperative adhesions that may explain the absence of pneumothorax ) , biopsies performed on mediastinal masses , lesions of pleural origin or presence of pleural effusion . una ricerca elettronica retrospettiva dal nostro database di tutti gli esami tc eseguiti tra gennaio 2007 e luglio 2008 stata condotta al ne di selezionare gli esami registrati con il codice istituzionale di procedura interventistica toracica . 
 i criteri di esclusione sono stati : precedenti resezioni polmonari ( per possibili aderenze post - chirurgiche che possono giusti care lassenza di pneumotorace ) ; biopsie eseguite su masse mediastiniche ; lesioni di origine pleurica ; presenza di versamento pleurico . 
 550 ct parameters all examinations were performed on a 16 - slice lightspeed ct system ( ge medical systems , milwaukee , wi , usa ) and were acquired in a craniocaudal direction from the lung apex to the base . 
iodinated contrast agents used in the period under review were iomeron 350 ( bracco , milan , italy ) , xenetix 350 ( guerbet , paris , france ) and visipaque 320 ( bayer schering pharma , germany )  . 
the scans were acquired in the course of a single inspiratory breath - hold with the following standard scan parameters : collimation 160.625 ( 10 mm ) ; thickness 2.5 mm ; eld of view 320360 mm ; 120 kv ; 240380 ma ( automatic tube current modulation ) ; tube rotation speed 0.8 s ; pitch 1.75 ; standard reconstruction lter ; lung window ( centre 450 , width 1 , 300 )  . ct - guided lung biopsy the indication for the procedure was determined by the patients referring physician and agreed to by the radiologist . 
any platelet antiaggregant and anticoagulant treatments were discontinued prior to the procedure and during subsequent recovery in agreement with the indications of the cardiologist or the haematologist . after establishing the ideal needle path from the skin to the lesion , the acquisition of 15 , 2.5 - mm - thick slices was planned to be repeated as many times as required to follow the needle path throughout the procedure . 
the needles employed were chibell ( biopsybell , modena , italy ) for ne - needle sampling and speedybell ( biopsybell , modena , italy ) for histological core biopsies . 
no on - site pathologist was present during the procedure for immediate evaluation of the biopsy sample . the presence of pneumothorax was evaluated using chest radiography during expiration and / or a ct scan after the procedure . 
if , however , it showed signs of increasing , a thoracic surgeon was consulted to assess the need to place a chest drathe onset of parenchymal haemorrhage was diagnosed when ct showed increased lung attenuation with preserved bronradiol med ( 2011 ) 116 : 548563 parametri tc tutti gli esami sono stati eseguiti su una tc 16 - slice lightspeed ( ge medical system , milwaukee wi , usa ) e sono stati acquisiti in direzione cranio - caudale dagli apici alle basi polmonari . 
i mezzi di contrasto iodati in uso nel periodo in esame sono stati : iomeron 350 ( bracco , milano , italia ) , xenetix 350 ( guerbet , paris , fr ) , visipaque 320 ( bayer schering pharma , germania )  . 
le scansioni sono state acquisite nel corso di ununica apnea inspiratoria con i seguenti parametri standardizzati : collimazione 160 , 625 ( 10 mm ) ; spessore 2 , 5 mm ; campo di vista 320360 mm ; 120 kv ; 240380 ma ( modulazione automatica della corrente del tubo ) ; 0 , 8 secondi di rotazione del tubo ; pitch 1 , 75 ; ltro di ricostruzione standard ; nestra di visualizzazione per polmone ( centro 450 , ampiezza 1300 )  . 
la coagulazione dei pazienti stata sempre valutata prima della biopsia attraverso i seguenti esami ematobiochimici : attivit di protrombina ( pa ) , tempo di tromboplastina parziale ( ptt ) e international normalized ratio ( inr ) , con valori normali rispettivamente di : 70%120% , 1726 s , e 0 , 81 , 2 . 
 dopo aver stabilito il tragitto ideale dellago dalla cute alla lesione , stata programmata unacquisizione di 15 fette di 2 , 5 mm di spessore da ripetere tante volte quante necessario per seguire il percorso dellago durante la procedura . 
gli aghi usati sono stati : chibell ( biopsybell , modena , italia ) per prelievi con ago sottile , e speedybell ( biopsybell , modena , italia ) per biopsie istologiche ( corebiopsy )  . 
 nel caso in cui lo pneumotorace fosse stabile o ridotto , il paziente stato dimesso ; nel caso in cui fosse invece incrementato , stato consultato un chirurgo toracico per valutare la possibilit di posizionare un drenaggio toracico . 
 linsorgenza di soffusione parenchimale stata diagnosticata quando la tc ha mostrato , lungo il tragitto dellago , un aumento di densit del polmone , con conservazione dei radiol med ( 2011 ) 116 : 548563 chial and vascular margins along the needle path . 
leventuale ospedalizzazione stata considerata valutando la clinica del paziente . quanti cazione dellen sema emphysema quanti cation data from the chest ct scans obtained at the time of the procedure were transferred from the digital archive to a workstation ( advantage windows , ge medical systems )  . 
 the values for total lung volume , and volume and percentage of emphysema , were obtained with a software package for the semiautomatic calculation of lung density ( volume viewer 2 , ge medical systems ) and entered on an excel spreadsheet ( microsoft of ce excel 2003 , richmond , va , usa )  . 
lung volume was calculated from scans reconstructed at a thickness of 2.5 mm by selecting a threshold between 1023 and 200 houns eld units ( hu ) to exclude both the soft tissue around the lung and the large intrapulmonary vessels . 
the speci c threshold used to discriminate normal from emphysematous lung parenchyma was 950 hu , an experienced radiologist manually selected and excluded measurement of air contained in the colon , trachea and main bronchi if it was included in the reconstructed volume . other factors on the basis of ct images , the following factors were also recorded and taken into account : patient position ( supine , lateral , prone ) ; needle access site ( anterior , lateral , posterior ) ; lesion site ( upper right , middle , lower right , upper left , lower left lobe ) ; lesion type ( solid , partially solid , nonsolid ) and trans ssural needle path ( present or absent )  . 
 elements recorded by the reporting workstation ( advantage windows , ge medical systems ) were : lesion diameter , angle of incidence between needle and pleura ( indicated as perpendicular if equal to 9010 and oblique in all other cases ) and length of the needles intrapulmonary trajectory . 
 needle characteristics noted were : type ( aspiration or core biopsy ) and diameter ( expressed in gauge )  . ct - guided lung biopsies were performed by one of ten radiologists , each with at least 2 years of experience , subdivided into two groups according to experience level : group 1 , with < 150 biopsies ; group 2 , with > 150 biopsies . 
 cytohistological biopsy reports ( provided by the pathologist within 1 week ) were reviewed , along with a short medical history regarding possible previous malignancies and associated therapies . i dati ottenuti dalle scansioni tc del torace acquisite al momento della procedura sono stati trasferiti dallarchivio digitale ad una workstation ( advantage windows , ge medical system , milwaukee , wi , usa )  . 
i valori del volume polmonare totale , del volume dellen sema e della percentuale di en sema sono stati ottenuti utilizzando un software per il calcolo semi - automatico della densit polmonare ( volume viewer 2 , ge medical system , milwaukee , wi , usa ) e registrate su un foglio di lavoro excel ( microsoft of ce excel 2003 , richmond , usa )  . 
il volume polmonare stato calcolato da scansioni ricostruite a spessore di 2 , 5 mm selezionando una soglia compresa tra 1023 e 200 unit houns eld ( hu ) , per escludere sia i tessuti molli attorno al polmone che i grossi vasi intrapolmonari . 
 altri fattori considerati sulla base delle immagini tc , sono stati considerati e registrati anche i seguenti fattori : posizione del paziente ( supina , laterale o prona ) ; posizione di accesso dellago ( anteriore , laterale o posteriore ) ; sede della lesione ( lobo superiore destro , lobo medio , lobo inferiore destro , lobo superiore sinistro , lobo inferiore sinistro ) ; tipo di lesione ( solida , parzialmente solida , non solida ) ; tragitto transscissurale dellago ( presente o assente )  . 
elementi registrati dalla workstation di refertazione ( advantage windows , ge medical system , milwaukee , wi , usa ) sono stati : diametro della lesione ; angolo di incidenza tra ago e pleura ( indicato come perpendicolare se uguale a 9010 e obliquo in tutti gli altri casi ) , lunghezza del tragitto intrapolmonare dellago . 
 le biopsie tc - guidate del polmone sono state eseguite da dieci diversi radiologi con almeno 2 anni di esperienza , suddivisi in base alla esperienza personale in due gruppi : gruppo 1 con esperienza < 150 biopsie , gruppo 2 con esperienza > 150 biopsie . 
il referto cito - istologico della biopsia ( fornito dal patologo entro 1 settimana ) stato valutato insieme ad una breve anamnesi su eventuali precedenti patologie oncologiche e terapie connesse . 
 statistical analysis analisi statistica baseline characteristics of the study population are le caratteristiche basali della popolazione in studio sono 552 radiol med ( 2011 ) 116 : 548563 expressed as meanstandard deviation ( sd ) for continuous variables and as frequencies and percentages for categorical variables . 
the nonparametric wilcoxon test was used to assess whether continuous variables differed signi cantly between patients exhibiting the complications under review and patients without such complications . factors identi ed by univariate analysis as being signi cantly associated with the presence of postbiopsy complications were subsequently included in multivariate logistic regression models to assess their contribution , independent of other variables , to the occurrence of parenchymal haemorrhage and pneumothorax . 
 the risk of having a complication in our sample was estimated as : e ( o + i xi ) l + e ( o + i xi ) where 0 is the intercept calculated in the multivariate logistic regression model and i represent the coef cient of the variable xi . 
the above - described statistical analyses were carried out using sas software , version 8.2 ( sas institute inc . , cary , nc , usa )  . state espresse come media deviazione standard per le variabili continue , come frequenze e percentuali per le variabili categoriche . 
il test non parametrico di wilcoxon stato utilizzato per valutare se le variabili continue erano signi cativamente differenti tra i pazienti con le complicanze in esame rispetto ai pazienti senza complicanze . 
 i fattori identi cati come signi cativamente associati alla presenza di complicanze post - bioptiche nellanalisi univariata sono stati successivamente inclusi in modelli di regressione logistica multivariata , per valutare come contribuivano indipendentemente da altre variabili , allinsorgenza di soffusione parenchimale e di pneumotorace . 
 il rischio di avere una complicanza nel nostro campione e stata stimata come : e ( o + i xi ) l + e ( o + i xi ) dove 0 lintercetta calcolata nella regressione logistica multivariata e i il coef ciente della variabile xi . 
laccuratezza del modello nel predire il rischio di complicanze nel nostro campione stata misurata con la statistica c e rappresentata gra camente da curve roc ( receiver operating characteristics )  . 
le suddescritte analisi statistiche sono state effettuate utilizzando il software sas , versione 8.2 ( sas institute inc . , cary , nc , usa )  . results risultati out of 222 procedures performed , 157 were included in the study . 
none of the values relating to emphysema , assessed in 138 / 157 ( table 1 ) , was found to be signi cant for the occurrence of pneumothorax and effusion . 
patient position , needle site , location of the lobe containing the lesion , type of nodules and angle between the needle and the parietal pleura demonstrated no signi cant association with the onset of parenchymal haemorrhage and pneumothorax . average lesion diameter was 40.1125.58 mm and was nel presente studio sono state incluse 157 / 222 procedure eseguite . 
la posizione dei pazienti , laccesso dellago , la sede del lobo della lesione , il tipo di noduli e langolo di incidenza tra lago e la pleura parietale , non hanno mostrato alcuna associazione signi cativa con linsorgenza di soffusione parenchimale e pneumotorace . 
similarly , pneumothorax rate was lower for larger lesions , ranging from 61% for il diametro medio delle lesioni stato di 40 , 1125 , 58 millimetri , ed risultato associato ( p < 0 , 05 ) con la soffusione parenchimale e con lo pneumotorace sia allanalisi univariata che alla multivariata . 
analogamente , la percentuale di pneumotorace stata inferiore per lesioni di dimensioni maggiori , passando dal 61% per lesioni < 20 mm al 19% per lesioni > 60 mm ( adjusted odds ratio 0 , 16 ( 0 , 050 , 59 ) )  . 
multivariate analysis showed that for every 10 - mm increase in length of the intrapulmonary trajectory increased the risk of pneumothorax and parenchymal haemorrhage by 3% ( adjusted or for both of 1.03 ; 95% ci 1.011.05 ) ] ( table 2 )  . 
lanalisi multivariata ha mostrato che per ogni aumento della lunghezza del tragitto intrapolmonare dellago di 10 mm , aumenta il rischio di soffusione pa556 parenchymal haemorrhage occurring in 9 / 12 ( 75% ) of cases and pneumothorax in 10 / 12 ( 83% )  . 
multivariate analysis showed that a trans ssural path increased the risk of parenchymal haemorrhage and pneumothorax fourfold ( table 2 ) , although this increased risk was statistically signi cant ( p < 0.05 ) for parenchymal haemorrhage only . 
on the other hand , a small but signi cant correlation ( p < 0.04 ) was identi ed between the type of needle used and the type of nodule , as cytology needles were used more frequently than core - biopsy needles for procedures performed on nonsolid nodules . 
on multivariate analysis , only a marginal association was found between the needle type and pneumothorax risk . the risk of a complication developing in our study population was calculated using the coef cients of the complete model with the continuous variables shown in table 2 . 
 for example , a patient with a lesion diameter of 30 mm , an intrapulmonary needle trajectory of 50 mm , the use of a radiol med ( 2011 ) 116 : 548563 fig . 
il tragitto intrapolmonare dellago stato di 77 mm ( a ) e lungo il percorso dellago si veri cata linsorgenza di soffusione parenchimale appena dopo la procedura ( b )  . renchimale e di pneumotorace del 3% ( valori di adjusted odds ratio per entrambi 1 , 03 ( 1 , 011 , 05 ) ) ( tabella 2 )  . 
 il tragitto dellago stato trans - scissurale in 12 casi ( 8% ) ed risultato signi cativamente associato ( p < 0 , 05 ) ad entrambe le complicanze allanalisi univariata : in 9 / 12 ( 75% ) casi si veri cata soffusione parenchimale , in 10 / 12 ( 83% ) pneumotorace . 
il decorso transcissurale ha aumentato di oltre 4 volte il rischio di soffusione parenchimale e pneumotorace ( tabella 2 ) alla analisi multivariata , sebbene tale incremento del rischio sia risultato statisticamente signi cativo ( p < 0 , 05 ) solo per le soffusioni parenchimali . 
2 axial computed tomography image showing the onset of pneumothorax during a lung biopsy performed with a 22 - gauge aspiration needle on a partially solid nodule of the lower right lobe . 
2 immagine tc assiale che mostra linsorgenza di pneumotorace nel corso di una biopsia polmonare eseguita usando un ago per prelievo citologico da 22 g , in un nodulo parzialmente solido del lobo inferiore di destra . 
3 axial computed tomography image showing no pneumothorax during a lung biopsy performed with an 18 - gauge core biopsy needle on a solid nodule of the lower right lobe . 
histological ndings of biopsies showed primary lung cancer in 100 cases , absence of cancer in 22 , metastases in 13 and inadequate material in 22 cases ( 16 / 22 obtained with aspiration biopsy )  . 
4 axial computed tomography image showing a percutaneous lung biopsy of a nonsolid nodule using a 25 - gauge aspiration needle , and onset of subtle pneumothorax during the procedure . 
4 immagine tc assiale che mostra una biopsia polmonare di un nodulo non solido eseguita con un ago citologico da 25 g e linsorgenza di una sottile falda di pneumotorace nel corso della procedura . 
il materiale estratto stato comunque suf ciente al patologo per fare diagnosi di carcinoma polmonare non a piccole cellule . gli aghi per prelievi istologici ( core biopsy ) in 102 / 157 casi ( 65% )  . 
daltra parte , si riscontrata una correlazione leggermente signi cativa ( p < 0 , 04 ) tra il tipo di ago usato e il tipo di nodulo , poich lago per prelievo citologico stato usato pi frequentemente rispetto allago per core biopsy nelle procedure eseguite su noduli non solidi . 
 allanalisi multivariata , si riscontrata unassociazione solo marginale tra il tipo di ago e il rischio di pneumotorace . il rischio di avere una complicanza nei pazienti inclusi nel nostro studio pu essere calcolato usando i coef cienti del modello completo con le variabili continue presentate nella tabella 2 . 
in this study , the rate of pneumothorax fell within this range ( 46% ) , even though a minimal amount air in the pleural cavity was considered evidence of pneumothorax . 
indeed , multivariate analysis shows an increased risk of pneumothorax and parenchymal haemorrhage with decreasing lesion diameter , with a reduction in laccuratezza del modello multivariato nel prevedere il veri carsi di complicanze 0 , 76 e 0 , 79 per le soffusioni parenchimali e per il pneumotorace , rispettivamente . 
le curve roc per il modello completo modi cato e per i modelli che includono solo la dimensione della lesione e il tragitto intrapolmonare dellago , sono presentate nelle figure 5 e 6 . le biopsie polmonari tc - guidate sono state eseguite da dieci radiologi differenti e nessuna correlazione signi cativa stata riscontrata tra lesperienza degli operatori e il veri carsi delle due complicanze in esame . 
i risultati istologici delle biopsie hanno dimostrato un tumore primitivo polmonare in 100 casi ; assenza di patologia oncologica in 22 casi ; metastasi in 13 casi ; materiale inadeguato in 22 casi ( 16 / 22 ottenuti da aspirati citologici )  . 
6 curve roc del modello modi cato completo e dei modelli che includono solo la dimensione della lesione e la biopsia intrapolmonare per linsorgenza di pneumotorace . risk of 4% and 3% , respectively , for each increasing increment of 10 mm in the lesions diameter . 
in the case of pneumothorax , this may be due to the number of pleural needle passes required to obtain correct needle direction , which increases the number of minor pleural injuries ; in the case of parenchymal haemorrhage , it is perhaps a result of the quantity of lung parenchyma traversed by the needle . 
have shown a high rate of complications for smaller lesions , with a particularly signi cant relationship between smaller lesion size and risk of parenchymal haemorrhage [ 3 ]  . 
showed a similar rate of pneumothorax for both small and large pulmonary nodules , with a diameter of 15 mm representing the threshold between the two groups [ 15 ]  . 
il 69 % ( 9 / 13 ) di questi pazienti era stato sottoposto a chemioterapia o ormonoterapia in passato . discussione le percentuali di pneumotorace dopo biopsie polmonari tc - guidate , riportate in letteratura , variano dal 4% [ 8 ] al 62% [ 9 ]  . 
in questo studio , la percentuale di complicanze compresa in questo range ( 46% ) , nonostante anche falde aeree pleuriche di minima entit comparse dopo la procedura siano state considerate come presenza di pneumotorace . 
infatti , lanalisi multivariata mostra un rischio maggiore di pneumotorace e soffusione parenchimale quando si riduce il diametro della 560 radiol med ( 2011 ) 116 : 548563 lesione , con una riduzione del rischio rispettivamente del 4% e del 3% , per ogni incremento di 10 mm del diametro della lesione . 
questo potrebbe essere dovuto : per il pneumotorace al numero di passaggi necessari per ottenere la corretta direzione dallago , che incrementerebbe il numero di piccoli traumi sulla pleura ; per la soffusione emorragica alla quantit di parenchima polmonare attraversata dallago . 
al contrario , yeow et al non hanno riscontrato alcuna associazione tra il diametro della lesione e linsorgenza di pneumotorace , ma hanno comunque mostrato una correlazione tra dimensione della lesione e soffusione allanalisi univariata [ 14 ]  . 
 [ 15 ] hanno mostrato una percentuale di pneumotorace analoga per noduli polmonari piccoli e grandi , usando come limite tra i due gruppi un diametro di 15 mm . la lunghezza del tragitto intrapolmonare risultata associata in modo signi cativo allinsorgenza di soffusione e pneumotorace . 
 [ 14 ] hanno mostrato che un lungo tragitto intrapolmonare dellago il pi importante fattore predittivo di soffusione parenchimale , con la pi alta percentuale di soffusione in particolare per le lesioni distanti pi di 20 mm dalla parete . 
altri studi hanno mostrato che la lunghezza del tragitto intrapolmonare signi cativamente associata con la soffusione [ 16 ] o lo pneumotorace [ 2 , 13 , 17 ]  . 
 [ 18 ] , un tragitto pi lungo dellago potrebbe danneggiare la pleura e il parenchima polmonare con maggior probabilit , giusti cando lalta percentuale di pneumotorace e soffusione intrapolmonare . 
dallanalisi multivariata effettuata sui fattori considerati in questo studio , il rischio di soffusione e pneumotorace aumenta del 3% per ogni aumento di 10 mm della lunghezza del tragitto intrapolmonare dellago . 
 [ 20 ] , rispetto alle nostre ( 12 / 157 ) , si dovrebbero raccogliere pi casi e correlarli al tipo di ago utilizzato come fattori indipendenti . allanalisi univariata abbiamo riscontrato una differenza signi cativa nellinsorgenza di pneumotorace per il fig . 
8 immagine dettagliata e ingrandita della punta di un ago per prelievo istologico ( cortesemente fornita da biopsybell )  . most important predictor of parenchymal haemorrhage , the highest rates of haemorrhage being observed in lesions > 20 mm from the wall [ 14 ]  . 
 other studies have shown that the length of the intrapulmonary trajectory is signi cantly associated with haemorrhage [ 16 ] or pneumothorax [ 2 , 13 , 17 ]  . 
as suggested by kinoshita et al . , a longer needle trajectory may be more likely to damage the pleura and pulmonary parenchyma , explaining the high rate of pneumothorax and intrapulmonary haemorrhage [ 18 ]  . 
multivariate analysis of the factors considered in this study shows that the risk of haemorrhage and pneumothorax increases by 3% with each increase of 10 mm in the length of the intrapulmonary needle trajectory . 
compared with our own experience ( 12 / 157 ) , we need to gather more cases and correlate them with the types of needle used as independent factors . univariate analysis revealed a signi cant difference in the onset of pneumothorax by type of needle used , regardless of its diameter . 
showed a higher rate of pneumothorax occurring with needle aspiration ( 40% ) than with core biopsies ( 24.3% ) [ 21 ]  . in our series , there was a higher rate of parenchymal haemorrhage in lesions that proved to be metastatic , which was unrelated to coagulation disorders in the patient . 
 because of the small number of this subset of patients and the lack of further information on risk factors for bleeding , larger studies are needed to determine whether previous treatments may have damaged the patients entire microvasculature , including pulmonary vessels [ 22 , 23 ]  . strangely , in our series , there was no correlation between emphysema extent and the development of pneumothorax . 
although there is still no unanimous opinion on the threshold in terms of houns eld units to be used to discriminate emphysematous and normal lung parenchyma , we chose a threshold of 950 hu , as it has been shown at both macroscopic and microscopic levels to provide good quanti cation of emphysema extent on thin - slice ct [ 25 , 2730 ]  . 
showed that patients with severe respiratory impairment have a high prevalence of pneumothorax [ 10 ] , but this difference could be related to the use of spirometry values to quantify the emphysema . 
however , in our series , emphysema was calculated for the entire parenchyma and not in particular for the portion of lung parenchyma traversed by the needle . as in previous reports [ 31 , 32 ] , this study found a lower percentage of inadequate samples with core biopsies than with aspiration biopsy . one limitation of this study is that there was no use of coaxial needles , as it was a retrospective study and coaxial needles were not used at our institution during the period tipo di ago usato , indipendentemente dal diametro dellago . 
a causa del piccolo numero di questo sottogruppo di pazienti e dellassenza di maggiori informazioni sui fattori di rischio per sanguinamento , sarebbero necessari studi pi ampi per stabilire se le precedenti terapie possano aver danneggiato la microvascolarizzazione dellintero organismo , ivi inclusi i vasi polmonari [ 22 , 23 ]  . 
sebbene non vi sia ancora un parere unanime sulla soglia in unit houns eld ( uh ) da usare per discriminare il parenchima polmonare en sematoso da quello normale , la soglia di - 950 uh stata scelta in questo studio perch stato dimostrato sia a livello macroscopico che microscopico che quanti ca bene lentit dellen sema in esami tc a strato sottile [ 25 , 2730 ]  . 
 [ 10 ] hanno mostrano che i pazienti con una severa compromissione della funzionalit respiratoria hanno avuto unalta prevalenza di pneumotorace [ 10 ] , ma questa differenza potrebbe essere legata alluso di valori spirometrici per quanti care len sema . 
tuttavia , nella nostra casistica len sema stato calcolato per lintero parenchima , e non in particolare per la parte di parenchima polmonare attraversata dallago . analogamente a studi precedenti [ 31 , 32 ] , in questo studio si ottenuta una percentuale pi bassa di materiale inadeguato per le core biopsies rispetto ai prelievi citologici . un limite di questo studio che non stato incluso alcun utilizzo di aghi co - assiali , poich uno studio retrospettivo e durante il periodo in esame gli aghi coassiali non erano in uso presso la nostra divisione . 
another limitation is that we did not investigate the rationale behind the choice of aspiration or core - biopsy needles for each procedure , and this may have in uenced the occurrence of complications . 
we have , however , shown that in this series , the choice of an aspiration needle was in uences by nodule characteristics rather than by distance of the lesion from the wall , and that an aspiration needle was preferred for nonsolid nodules . 
furthermore , in our series the number of pleural passes was not included among the variables assessed , as a retrospective evaluation of this parameter could perhaps only have been obtained by counting the number of ct series acquired during each procedure . 
however , this number may not re ect the real number of pleural passes , and it was therefore omitted from the collection of data on possible risk factors . permettendoci di eseguire , relativamente alle variabili in esame , unanalisi statistica adeguata . 
un altro limite che non abbiamo indagato il razionale nella scelta degli aghi per prelievo citologico o istologico in ogni singola procedura , e questo potrebbe aver in uenzato linsorgenza di complicanze . 
tuttavia abbiamo dimostrato che in questa casistica la scelta di un ago per prelievo citologico non stata in uenzata dalla distanza della lesione dalla parete , ma dalle caratteristiche del nodulo , ed stato preferito lago per prelievo citologico per noduli non solidi . 
inoltre , nella nostra casistica il numero di passaggi pleurici non stato incluso tra le variabili valutate , poich in una valutazione retrospettiva questo parametro sarebbe stato forse ottenibile solo contando il numero delle serie tc acquisite durante la procedura , ma questo dato potrebbe non ri ettere il numero reale di passaggi pleurici , pertanto stato omesso dalla raccolta dei dati relativa ai possibili fattori di rischio . conclusions statistically signi cant features for the occurrence of pneumothorax and pulmonary haemorrhage following ct - guided lung biopsies were smaller lesions and long intrapulmonary needle trajectory . 
no signi cant association was found between complication onset and the other risk factors investigated , including emphysema , t nodule type , patient position , needle access site , angle between the needle and pleura and needle gauge . conclusioni caratteristiche statisticamente signi cative per linsorgenza di pneumotorace e soffusione polmonare dopo biopsie polmonari tc - guidate sono risultate : piccole dimensioni delle lesioni e lungo tragitto intrapolmonare dellago ; il tragitto transcissurale dellago risultato rilevante per la soffusione intrapolmonare ; il tipo di ago appare invece correlato allinsorgenza di pneumotorace . 
we identi ed 7 / 23 benign lesions ( three myxoid , four nonmyxoid ) and 16 / 23 malignant tumours ( four myxoid , 12 nonmyxoid ) with a mean diameter between 21 mm and 20 cqualitative analysis of dwi showed persistence of high signal intensity for increasing b - values in all malignant tumours . 
abbiamo riscontrato 7 / 23 lesioni benigne ( 3 a matrice mixoide , 4 non mixoide ) e 16 / 23 maligne ( 4 a matrice mixoide , 12 non mixoide ) , con diametro compreso tra 21 mm e 20 clanalisi qualitativa della dwi ha dimostrato persistenza delliperintensit di segnale al crescere dei tre b value in tutte le lesioni maligne . 
 parole chiave rm diffusione dwi tumori dei tessuti molli sarcoma radiol med ( 2011 ) 116 : 644656 introduction introduzione magnetic resonance ( mr ) imaging plays a major role in diagnosis , staging and follow - up of soft - tissue tumours . 
in recent years , dwi has been used to characterise soft - tissue tumours and differentiate benign from malignant lesions [ 7 , 8 ] and to monitor the ef cacy of treatment in patients affected by sarcoma [ 9 , 10 ]  . 
 the aim of this study was to evaluate the role of mr imaging with spin - echo echo - planar diffusion - weighted imaging ( se - epi - dwi ) in characterising primary and secondary tumours of the soft tissues by correlating qualitative and quantitative dwi data with the results of histology . materials and methods between june 2008 and may 2009 , we retrospectively reviewed 23 patients ( 14 men , nine women ; median age 70 years ; range 2587 ) at rst diagnosis of a soft - tissue mass . 
included in the study were also the most common lesions that enter into the differential diagnosis with primary soft - tissue tumours , such as muscular metastasis , lymphomas and schwannomas . 
diagnostic workup prior to mr examination included ultrasound ( us ) in eight patients , us and computed tomography ( ct ) in ve , ct in ve and no examination in four ; one patient had directly undergone biopsy . 
all lesions were subjected to histological assessment following biopsy ( 11 / 23 ) or surgical excision ( 9 / 23 ) ; in 3 / 23 patients , the lesion was surgically excised after biopsy . 
mr imaging was done with a 1.5 - t superconductive magnet ( avanto , siemens medical solutions , forcheim , germany ) , gradient strength 45 mt / m , slew rate 200 t / m / ms and phased - array surface coil . 
lesions were studied at baseline ( precontrast scan ) with the following sequences : axial t1 - se ( tr 610 ms , te 11 ms , fa 150 , matrix 281448 , acceleration factor 2 , thickness 3 mm ) ; axial t1 - se fat la risonanza magnetica ( rm ) riveste un ruolo importante nella diagnosi , nello staging e nel follow - up delle neoplasie dei tessuti molli . 
recentemente la dwi stata utilizzata per caratterizzare i tumori dei tessuti molli , differenziando i tumori benigni dai tumori maligni [ 7 , 8 ] , e controllare i risultati del trattamento in pazienti affetti da sarcoma [ 9 , 10 ]  . 
 lo scopo di questo lavoro valutare il ruolo della rm con sequenze spin echo ( se ) - echo planar imaging ( epi ) diffusion - weighted imaging ( dwi ) nella caratterizzazione dei tumori primitivi e secondari dei tessuti molli , correlando i dati qualitativi e quantitativi offerti dallimaging dwi con i riscontri anatomopatologici ( istologici )  . materiali e metodi sono stati valutati retrospettivamente , nel periodo compreso tra giugno 2008 e maggio 2009 , 23 pazienti ( 14 maschi , 9 femmine , di et compresa tra i 2587 anni , et mediana 70 anni ) con tumefazioni dei tessuti molli al primo riscontro . 
i criteri di esclusione hanno previsto i pazienti gi sottoposti a chemioe radioterapia ed i pazienti con lipoma tipico , prevedendo la sequenza dwi un pre - impulso di radiofrequenza per la soppressione del segnale del grasso . 
 nello studio sono state comprese anche le lesioni pi comuni che entrano in diagnosi differenziale con i tumori primitivi dei tessuti molli come le metastasi muscolari , i linfomi e gli schwannomi . 
nelliter diagnostico precedente allindagine rm , 8 pazienti avevano eseguito una ecogra a , 5 pazienti una ecogra a ed una tomogra a computerizzata ( tc ) , 5 pazienti una tc e 4 pazienti nessun esame ; in un 1 paziente era stata eseguita direttamente la biopsia . 
tutte le lesioni sono state sottoposte ad esame istologico dopo biopsia ( 11 / 23 pazienti ) o ad asportazione chirurgica ( 9 / 23 pazienti ) ; in 3 / 23 pazienti dopo la biopsia si proceduto ad asportazione chirurgica della lesione . 
lindagine rm stata eseguita in tutti i pazienti con apparecchiatura dotata 646 radiol med ( 2011 ) 116 : 644656 saturation ( fat - sat ) ( tr 610 ms , te 11 ms , ti 150 ms , fa 150 , matrix 281448 , acceleration factor 2 , thickness 3 mm ) , coronal t2 - se - fat - sat ( tr 4 , 000 ms , te 265 ms , ti 150 ms , fa 150 , matrix 314448 , acceleration factor 2 , thickness 3 mm ) , single - shot ( ss ) - se - epi - dwi with three different b values ( 50 - 400 - 800s / mm2 ) and parallel imaging with an acceleration factor of 2 [ interactive project for assistive technology ( ipat ) - 2 ]  . 
subsequently , all patients were imaged with t1 - weighted se sequences ( tr 610 ms , te 11 ms , fa 150 , matrix 281448 , acceleration factor 2 , thickness 3 mm ) after the intravenous administration of 0.2 mmol / kg paramagnetic contrast material ( magnevist , bayer schering pharma , berlin , germany )  . 
the ss - se - epi - dwi sequence has the following technical parameters : tr 4 , 100 ms , te 70 ms , ti 150 ms , matrix 160160 , slice thickness 6 mpost - processing was performed on a dedicated workstation ( leonardo , siemens medical solutions , forchheim , germany )  . 
lesions were histologically classi ed into benign and malignant according to the world health organization ( who ) classi cation of soft tissue tumors [ 11 ] and subdivided into myxoid or nonmyxoid depending on the amount of myxoid matrix ( table 1 )  . 
the qualitative analysis of the dwi evaluated the loss of high signal intensity with increasing b values ( from the lowest value of 50s / mm2 to the highest of 800s / mm2 )  . 
persistence of high signal intensity with increasing b values was evaluated semiquantitatively using the following scale : + + + marked persistence of signal ; + + good persistence of signal ; + poor persistence of signal ; absence of signal . quantitative analysis was obtained by mathematically calculating the apparent diffusion coef cient ( adc )  . 
adc maps are automatically generated by the workstation based on the three b values according to the formula adc = ln ( s0 / s1 ) / ( b1 - b0 ) , where s0 and s1 are the signal intensity before and after application of diffusion gradients , and b1 and b0 are the different b values applied . 
values of the considered variables ( b50 , b400 , b800 , adc ) were compared between the group of seven benign lesions and the group of 16 malignant lesions using the nonparametric mannwhitney u test . 
to evaluate di magnete superconduttivo da 1 , 5 t ( avanto , siemens medical solutions , forcheim , germania ) , intensit di risalita dei gradienti di 45 mt / m , slew rate di 200 t / m / ms , bobina di super cie phased - array . 
lo studio delle lesioni stato condotto nelle condizioni di base mediante sequenze t1 - se assiale ( tempo di ripetizione [ tr ] 610 ms , tempo di eco [ te ] 11 ms , ip angle [ fa ] 150 , matrice 281448 , fattore di accelerazione 2 , thickness 3 mm ) , t1 - se - fat - sat assiale ( tr 610 ms , te 11 ms , ti 150 ms , fa 150 , matrice 281x448 , fattore di accelerazione 2 , thickness 3 mm ) , t2 - se con saturazione del grasso ( fat - sat ) coronale ( tr 4000 ms , te 265 ms , tempo di inversione [ ti ] 150 ms , fa 150 , matrice 314448 , fattore di accelerazione 2 , thickness 3 mm ) , single shot ( ss ) - se - epi - dwi con 3 differenti valori di b value ( 50 - 400 - 800 s / mm2 ) ed imaging parallelo con fattore di accelerazione pari a 2 ( ipat - 2 )  . 
successivamente in tutti i pazienti sono state acquisite sequenze se t1 pesate ( tr 610 ms , te 11 ms , fa 150 , matrice 281448 , fattore di accelerazione 2 , thickness 3 mm ) dopo somministrazione ev di 0 , 2 mmol / kg di mezzo di contrasto ( mdc ) paramagnetico ( magnevist , bayer schering pharma , berlino , germania )  . 
la sequenza ss - se - epi - dwi impiegata nello studio prevede i seguenti parametri tecnici : tr 4100 ms , te 70 ms , ti 150 ms , matrice 160160 , slice thichness 6 mil post - processing stato eseguito su consolle dedicata ( leonardo , siemens medical solutions , forcheim , germania )  . 
 lanalisi delle immagini stata effettuata , separatamente , da due radiologi ( eg , mga ) esperti in risonanza magnetica , con particolare riferimento alla patologia muscolo - scheletrica , non sono emerse discordanze tra i risultati . 
le lesioni sono state classi cate istologicamente in benigne e maligne secondo la world health organization ( who ) classi cation of soft tisssue tumors [ 11 ] e suddivise a seconda della loro matrice mixoide e non mixoide ( tabella 1 )  . 
lanalisi qualitativa delle immagini dwi stata effettuata valutando la perdita di iperintensit di segnale nella successione di 3 valori di b value ( dal valore pi basso di 50 s / mm2 , a quello pi alto di 800 s / mm2 )  . 
la persistenza delliperintensit di segnale al crescere dei tre b value stata valutata semi - quantitativamente utilizzando la scala di valori sotto riportata : + + + notevole persistenza di segnale ; + + buona persistenza di segnale ; + scarsa persistenza di segnale ; assenza di segnale . lanalisi quantitativa stata ottenuta mediante calcolo matematico del valore del coef ciente di diffusione apparente ( adc )  . 
i valori delle variabili prese in considerazioni ( b50 , b400 , b800 , adc ) sono state confrontate tra il gruppo delle 7 lesioni benigne e quello delle 16 lesioni maligne con il test non parametrico di mann - whitney . 
 qualitative analysis of dwi demonstrated persistence of high signal intensity with increasing b values ( 50 - 400800 s / mm2 ) in all malignant lesions , although with differences among the various histological types ( table 1 )  . 
le lesioni maligne a matrice mixoide ( 4 pazienti ) hanno compreso i liposarcomi mixoidi g1 e g3 , il mixo brosarcoma ad alto grado , il sarcoma polimorfo con componente mixosarcomatosa . 
low adc values were obtained in lymphomas ( lowest value 0.410.2610 - 3 mm2 / s ) , in muscular metastases from in ltrating ductal breast cancer ( 0.9680.7610 - 3 mm2 / s ) and in the hepatocellular carcinoma ( 1.0100.5010 - 3 mm2 / s ) , all classi ed as nonmyxoid malignant lesions . 
high adc values were recorded in the elasto bromas , with adc between 1.310.4610 - 3mm2 / s and 1.340.510 - 3 mm2 / s , classi ed as nonmyxoid malignant lesions . 
in the single case of softtissue localisation of castlemans disease , we obtained an adc value of 0.780.9010 - 3 mm2 / s , similar to lymphomas , and for this reason , they were included among lesions with benigne a matrice mixoide ( 3 pazienti ) abbiamo inserito lo schwannoma , il neuro broma e langiomixoma . 
 lanalisi qualitativa della dwi ha dimostrato la persistenza delliperintensit di segnale al crescere dei tre b value ( 50 - 400 - 800 s / mm2 ) in tutte le lesioni maligne , anche se in misura differente nei diversi istotipi tumorali ( tabella 1 )  . 
c - e sequenze ssse - epi - dwi acquisite con tre differenti b value ( 50 , 400 , 800 s / mm2 ) : assenza di iperintensit di segnale al crescere del b value . a high grade of biological activity . 
we found a small difference between adc values of the schwannoma ( 1.740.8110 - 3 mm2 / s ) and the myxoid liposarcoma ( 1.560.1710 - 3 mm2 / s ) , but the difference was suf cient to enable distinction between benign and malignant lesions , according to our ndings . 
 statistical analysis conducted with the mannwhitney u test for each parameter considered separately ( b50 , b400 , b800 and adc ) demonstrated a signi cant difference ( p < 0.05 ) between the group of seven benign lesions and that of 16 malignant lesions . 
roc curve analysis allowed us to establish the sensitivity and speci city of each parameter considered ( b50 , b400 , b800 and adc ) in discriminating between benign and malignant lesions . 
 the area under the curve ( auc ) at the cutoff point ( below which are lesions with a low grade of biological activity 0 , 410 , 2610 - 3 mm2 / s ) , nelle metastasi muscolari da carcinoma duttale in ltrante della mammella ( 0 , 9680 , 7610 - 3 mm2 / s ) e nel epatocarcinoma ( hcc ) ( 1 , 0100 , 5010 - 3 mm2 / s ) , classi cati come lesioni maligne non mixoidi . 
elevati valori di adc sono riscontrati negli elasto bromi , con adc compreso tra 1 , 310 , 4610 - 3 mm2 / s e 1 , 340 , 510 - 3 mm2 / s , classi cati come lesioni benigne non mixoidi . 
 nellunico caso di localizzazione nei tessuti molli di malattia di castleman da noi riscontrato abbiamo ottenuto un valore di adc pari a 0 , 780 , 9010 - 3 mm2 / s , simile ai linfomi e per tale motivo incluso nelle lesioni ad alto grado di attivit biologica . 
abbiamo rilevato una ristretta differenza nei valori di adc ottenuti nello schwannoma ( 1 , 740 , 8110 - 3 mm2 / s ) e nel liposarcoma mixoide ( 1 , 560 , 1710 - 3 mm2 / s ) , tuttavia suf ciente , secondo i nostri risultati , a distinguere tra lesioni benigne e maligne . 
according to some authors , lesions with a low grade of biological aggressiveness where the water lanalisi statistica , eseguita con il test di mann - whitney per ogni parametro considerato singolarmente ( b50 , b400 , b800 e adc ) , ha dimostrato una differenza statisticamente signi cativa ( p < 0 , 05 ) tra il gruppo delle 7 lesioni benigne e delle 16 maligne . 
lanalisi statistica mediante curve roc dei dati raccolti ha permesso di stabilire la sensibilit e la speci cit di ciascuno dei parametri considerati ( b50 , b400 , b800 e adc ) nel discriminare tra lesioni benigne e maligne . 
 larea sottesa alla curva al valore di cut - off ( al di sotto del quale si trovano le lesioni a basso grado di attivit biologica e al di sopra del quale quelle ad alto grado di attivit biologica ) rappresenta un parametro fondamentale , in quanto costituisce una misura di accuratezza non dipendente dalla prevalenza . 
qualsiasi variazione del contenuto di protoni di acqua in questi due compartimenti comporta una restrizione della diffusivit , che si traduce in una diversa intensit di segnale nella immagine dwi a diversi b value . 
secondo alcuni autori , allaumentare del b value le lesioni dotate di bassa aggressivit biologica , dove le molecole di h2o sono pi libere di muoversi ( alto coef ciente di diffusione ) perdono intensit di segnale . 
al contrario le lesioni dotate di alta aggressivit biologica , dove le molecole hanno minore libert di movimento ed il coef ciente di diffusione risulta pi basso , persistono iperintense al b value pi alto [ 3 ]  . 
le applicazioni emergenti del dwi in campo oncologico consistono nella caratterizzazione tumorale [ 13 ] ed hanno ottenuto i migliori risultati a livello cerebrale [ 1 , 2 ] ed epatico [ 46 ]  . 
tuttavia in letteratura la maggior parte dei lavori sono incentrati sullef cacia del dwi nel follow - up post - terapia [ 5 , 1430 ] , in particolare nella preradiol med ( 2011 ) 116 : 644656 where molecules have less freedom of movement and the diffusion coef cient is lower , continue to show high signal intensity at higher b values [ 3 ]  . 
the emerging applications of dwi in oncology involve tumour characterization [ 13 ] and have achieved the best results in the brain [ 1 , 2 ] and liver [ 46 ]  . 
however , most published studies have focused on dwi ef cacy for follow - up after treatment [ 5 , 1430 ] , particularly to predict the response to chemotherapy for sarcomas [ 14 , 15 ] and the possibility of discriminating between tumour recurrence and necrosis in musculoskeletal tumours , which are particularly prone to recurrence after surgery [ 1730 ]  . 
 adc , as already widely demonstrated in other body regions , is closely related to the degree of tumour cellularity [ 1 , 2 , 22 , 31 ]  . 
despite there are no unequivocally normal adc values in soft tissues [ 16 ] , the dwi technique appears to have the potential to characterize tissues and de ne their biological activity . 
 [ 8 ] , in a study conducted on 23 patients with histologically proven softtissue tumours , reported signi cantly higher mean adc values in benign ( 1.7110 - 3 mm2 / s ) compared with malignant ( 1.0810 - 3 mm2 / s ) tumours . 
 [ 7 ] , in a small study conducted on 29 patients ( 16 benign lesions and 13 sarcomas ) with two different b values of 0 and 600 s / mm2 , concluded that there is no signi cant difference between the two groups owing to the overlap of adc values in benign lesions ( mean value 1.810 - 3 mm2 / s ) and sarcomas ( mean value 1.710 - 3 mm2 / s )  . 
 [ 17 ] used a sequence called line - scan diffusion - weighted imaging with b values of 5 and 1 , 000 s / mm2 in 44 patients affected by 18 benign and 26 malignant tumours . 
the adc value of myxoid and nonmyxoid tumours ( both benign and malignant ) differs signi cantly , as the structural properties of the extracellular matrix contribute to restricting diffusion . 
the adc of soft - tissue tumours containing myxoid matrix was 1.920.4110 - 3 mm2 / s , whereas that of nonmyxoid tumours was 0.970.3310 - 3 mm2 / s . 
the adc value of benign and malignant soft - tissue tumours was 1.450.5910 - 3 mm2 / s and 1.500.6410 - 3 mm2 / s , respectively , and the difference was not statistically signi cant [ 17 ]  . 
in a larger study of 88 histologically proven lesions studied with se - epi - dwi sequences with b values of 0 and 1 , 000 s / mm2 , nagata et al . 
this is because the adc value is affected by tissue cellularity as well as by the extracellular matrix which , in myxoid tumours , contains large amounts of dizione della risposta dei sarcomi alla chemioterapia , come dimostrano alcuni risultati preliminari [ 14 , 15 ] , e nella possibilit di discriminare tra recidiva e necrosi tumorale nello studio dei tumori del sistema muscoloscheletrico , data la frequenza delle recidive post - chirurgiche [ 1730 ]  . 
nonostante nello studio dwi dei tessuti molli non esistono univoci valori di normalit adc [ 16 ] , questa tecnica sembra avere la potenzialit di tipizzare i tessuti e di de nire la loro attivit biologica . 
 [ 8 ] , in uno studio eseguito su 23 pazienti affetti da tumori dei tessuti molli istologicamente provati , riportano un valore medio di adc signi cativamente maggiore nelle lesioni tumorali benigne ( 1 , 7110 - 3 mm2 / s ) rispetto alle lesioni maligne ( 1 , 0810 - 3 mm2 / s )  . 
 [ 7 ] su una casistica limitata di 29 pazienti ( 16 lesioni benigne e 13 sarcomi ) , usando due diversi b value rispettivamente di 0 e 600 s / mm2 , giungono alla conclusione che non c una differenza signi cativa tra i due gruppi a causa della sovrapposizione dei valori adc nelle lesioni benigne ( valore medio di 1 , 810 - 3 mm2 / s ) e nei sarcomi ( valore medio di 1 , 710 - 3 mm2 / s )  . 
 [ 17 ] hanno utilizzato una sequenza detta line - scan - diffusion - weighted imagingcon due differenti b value di 5 e 1000 s / mm2 in 44 pazienti affetti da 18 tumori benigni e 26 maligni . 
il valore adc tra tumori mixoidi e non mixoidi ( sia benigni che maligni ) differisce in maniera statisticamente signi cativa in quanto le propriet strutturali della matrice extra - cellulare contribuiscono a determinare la restrizione alla diffusione . 
 ladc nei tumori dei tessuti molli contenente matrice mixoide risultava di 1 , 920 , 4110 - 3 mm2 / s , al contrario nei tumori non mixoidi era di 0 , 970 , 3310 - 3 mm2 / s . 
tuttavia , secondo questi autori , il valore adc di tumori benigni e maligni dei tessuti molli rispettivamente di 1 , 450 , 5910 - 3 mm2 / s e di 1 , 500 , 6410 - 3 mm2 / s , con una differenza statisticamente non signi cativa [ 17 ]  . 
 [ 18 ] , su una casistica pi ampia di 88 lesioni istologicamente provate , utilizzando sequenze se - epi - dwi con due b value differenti di 0 e 1000 s / mm2 , affermano che il valore adc discrimina tra lesioni maligne e benigne a matrice non mixoide , mentre non in grado di discriminare tra tumori benigni e maligni a matrice mixoide , poich il valore adc non in uenzato solo dalla cellularit del tessuto , ma anche dalla matrice extra - cellulare , che contiene una grande quantit di acqua nei tumori mixoidi , tale da non consentire la discriminazione tra tumori benigni e maligni . 
ladc riportato nei tumori mixoidi risulta signi cativamente pi elevato rispetto ai non mixoidi , con valori medi rispettivamente di 2 , 080 , 5110 - 3 654 radiol med ( 2011 ) 116 : 644656 water . 
the adc value for myxoid tumours was signi cantly higher than for nonmyxoid tumours , with mean values of 2.080.5110 - 3 mm2 / s and 1.130.4010 - 3 mm2 / s , respectively , in line with the previous study by maeda et al . 
those authors reported no statistically signi cant difference between the mean adc value of benign and malignant myxoid tumours ( 2.100.5010 - 3 mm2 / s and 2.050.5810 - 3 mm2 / s , respectively )  . 
conversely , the adc value of benign nonmyxoid tumours was 1.310.4610 - 3 mm2 / s , which was signi cantly higher ( p < 0.001 ) than that of malignant nonmyxoid tumours ( 0.940.2510 - 3 mm2 / s )  . 
 in our limited experience , the use of three b values ( 50 - 400 - 800 s / mm2 ) enabled us to discriminate the group of seven benign lesions from that of 16 malignant lesions on each parameter considered individually ( b50 , b400 , b800 , adc )  . 
 in our experience , the qualitative analysis of dwi demonstrated an absence of high signal intensity at all b values in benign lesions and persistence of high signal intensity with increasing b value ( 800 s / mm2 ) in all malignant tumours . 
most investigators tend to concentrate exclusively on the adc value for differentiating between benign and malignant tumours [ 7 , 8 , 17 , 18 ] , even though large studies including a variety of tumour types have reported that myxoid lesions show a certain degree of overlap in adc , which nonetheless contributes greatly to the differentiation of malignant and benign nonmyxoid lesions [ 17 , 18 ]  . 
in our opinion , partly in consideration of this overlap , it is useful to combine qualitative and quantitative analyses of dwi , clearly without omitting evaluation of the t1 and t2 morphological sequences . 
the reason for this lies in the high content of mucin and low content of collagenous bres in these lesions , which translates into increased water in the extracellular matrix and high diffusivity . the best correlation between the qualitative / quantitative analysis of dwi and histology was seen in lymphomas and muscular metastases from hepatocellular carcinoma and ductal breast carcinoma , which were characterised as frankly malignant on the basis of the low adc value mm2 / s e 1 , 130 , 4010 - 3 mm2 / s , in linea con il precedente studio di maeda et al . 
non riportata una differenza signi cativa tra il valore medio di adc nei tumori benigni e maligni mixoidi ( rispettivamente 2 , 100 , 5010 - 3 mm2 / s e 2 , 050 , 5810 - 3 mm2 / s )  . 
anche nella nostra casistica il valore di adc pi alto riscontrato nei tumori maligni non mixoidi di 1 , 3410 - 3 mm2 / s , in un sarcoma ad alto grado . 
 nella nostra esperienza , seppur ridotta , utilizzando tre diversi b value ( 50 - 400 - 800 s / mm2 ) , stato possibile discriminare il gruppo delle 7 lesioni benigne dalle 16 maligne su ogni parametro considerato singolarmente ( b50 , b400 , b800 , adc )  . 
 nella nostra esperienza , lanalisi qualitativa della dwi dimostra assenza di ipersegnale in tutti i b value nelle lesioni benigne e persistenza delliperintensit di segnale al crescere del b value ( 800 s / mm2 ) in tutti i tumori maligni . 
 la maggior parte degli autori tendono a concentrare la loro attenzione esclusivamente sul valore di adc nella differenziazione tra tumori benigni e maligni [ 7 , 8 , 17 , 18 ] , ma su casistiche ampie e con diversi tipi di tumori si osservato che le lesioni mixoidi risentono di una certa sovrapposizione tra i valori di adc , che comunque offre un signi cativo contributo nella diagnostica differenziale tra lesioni maligne e benigne a matrice non mixoide [ 17 , 18 ]  . 
a nostro giudizio , anche in relazione a questa sovrapposizione dei valori di adc , utile integrare le analisi qualitativa e quantitativa della dwi , senza prescindere dalle sequenze morfologiche t1 e t2 . 
hence , there is a need for noninvasive methods , such as mr with dwi , which may provide a diagnosis as close as possible to the histological diagnosis , through an evaluation that goes beyond the surgical specimen to include staging . 
 these results form the basis for a promising future in which dwi is able to provide information on the effectiveness of treatment at an earlier stage than with conventional morphological imaging , given that molecular changes precede the macroscopic volumetric changes . 
da qui la necessit di utilizzare metodiche non invasive , quali la rm con dwi , che possano condurre ad una diagnosi il pi vicino possibile a quella istologica , con unanalisi non limitata al solo pezzo anatomico prelevato , ma completa di staging . 
 questi risultati , pongono le basi per un promettente futuro in cui la dwi in grado , pi precocemente dellimaging morfologico convenzionale , di dare informazioni sullef cacia del trattamento , in quanto i cambiamenti molecolari , precedono quelli macroscopici volumetrici . 
 conclusioni conclusions dwi , supplemented with conventional morphological imaging , may offer valuable assistance in the study of softtissue tumours , providing essential information on lesion biological aggressiveness and stage in a single examination . 
in our admittedly limited experience , the use of three different b values enabled correct differentiation between benign and malignant lesions , both myxoid and nonmyxoid , with total agreement with the histological ndings . 
larger and more extensive studies are required to validate this imaging technique and the bene ts deriving from the use of different b values . limaging dwi , corredato al convenzionale studio morfologico , pu fornire un valido aiuto nello studio delle lesioni dei tessuti molli , fornendo in un unico esame preziose informazioni circa laggressivit biologica e la stadiazione della lesione . 
lutilizzo di tre differenti b value ha consentito nella nostra esperienza , bench limitata , una corretta differenziazione tra lesioni benigne e maligne , sia mixoidi che non mixoidi , con una completa concordanza con il risultato istologico . 
 questa sua decisione lo aveva portato da noi per analizzare il funzionamento di un reparto di radiodiagnostica centralizzato di recente istituzione . a ne incontro ci recammo in una tipica trattoria romana . 
nonostante il tempo trascorso , ricordo molto bene quella sosta alla trattoria antico falcone che mentalmente riporto allinizio di un lavoro in corso dopera che , nel tempo , ha prodotto coinvolgenti iniziative formative di gruppo , in ambito toracico , dif cili da dimenticare . parlammo della sua non semplice scelta , di radiologia , e , ovviamente , di radiologia toracica , delle possibili innovazioni della stessa , anche per le prime ricadute dei voli spaziali ( assenza di gravit ) sul circolo polmonare , di radiologia clinica del torace . 
considerazioni che contrastavano con una terminologia radiologica corrente , lontana dalla realt siopatologica , come accentuazione della trama bronco vascolare del polmone , sostituita successivamente da riscontri radiologici strettamente correlati alle nuove cognizioni di siopatologia cardio - polmonare . quel nostro incontro , irrobustito nel tempo dal coinvolgimento attivo di altri quali cati colleghi , port alle giornate pneumo - radiologiche bresciane ( 1975 ) , alla costituzione della sezione di studio di radiologia toracica della sirm ( 1976 ) , allistituzione del premio pigorini ( 1978 ) con il contributo economico della famiglia pigorini , allattivazione dei corsi itineranti regionali di radiologia toracica ( 1979 )  . interventi che , pur nella loro diversit , hanno inciso tutti , nel tempo , sulla formazione professionale del radiologo italiano , annullando o forse anche invertendo , di fatto , un non trascurabile gap culturale di allora . le giornate pneumo - radiologiche bresciane , giunte quasi alla loro ventesima edizione sono , ormai , parte essenziale della radiologia toracica italiana , come possono ben testimoniare radiologi , pneumologi e anatomopatologi bresciani coinvolti in questa iniziativa . in questi lunghi anni di attivit professionale e formativa presso gli spedali civili di brescia , renato bergonzini diventato , di fatto , bresciano di adozione , pur rimanendo profondamente e nostalgicamente emiliano e modenese il ricordo di renato bergonzini mi riporta a uno dei nostri primi incontri , allinizio degli ormai lontani anni settanta , nellistituto di radiologia della facolt di medicina e chirurgia delluniversit cattolica di roma , diretto dal prof . 
 ricordo , per i pi giovani , due dati : il progresso delle conoscenze e delle tecnologie in medicina e in radiologia era , in quegli anni , graduale e lento , a differenza della crescita esponenziale di oggi , e la specializzazione era solo in radiologia . lo specializzando nel suo tirocinio formativo frequentava le tre diverse strutture dellistituto , reparto degenti della radioterapia incluso ; da medico specializzato , poi , aveva possibilit di operare professionalmente , secondo necessit , nei tre diversi ambiti della radiologia . 
 esigenze professionali diverse hanno portato , nel tempo , ad una progressiva diversi cazione delle tre diverse branche della radiologia e alla successiva istituzione di tre diverse scuole di specializzazione , radiodiagnostica ( poi diagnostica per immagini ) , radioterapia e medicina nucleare . renato bergonzini , laureato in medicina e chirurgia e specializzato in radiologia e in oncologia a modena , sua 668 radiol med ( 2011 ) 116 : 667668 nellanimo e nel carattere , sempre pronto a commentare le vicende della rossa ferrari , con tipiche espressioni dialettali modenesi che rispolverava spesso incontrando colleghi della sua citt natale . la sezione di radiologia toracica , concepita nel lontano 1973 , come riporta lo stesso bergonzini su torax del 1997 , ai piedi della montagna incantata di davos da un gruppo di cultori della materia ( barone , bergonzini , corda , marano ) , fu istituita uf cialmente a brescia il 16 ottobre del 1976 , con renato bergonzini primo presidente . 
 il premio luigi pigorini tuttora , per i cultori della materia , il pi importante riconoscimento uf ciale della radiologia toracica italiana ; ha cadenza biennale ed assegnato durante il convegno nazionale della sezione , contemporaneamente alla presentazione della conferenza pigorini , presentata dal vincitore del biennio precedente . con i corsi itineranti , la sezione di radiologia toracica della sirm introdusse uninnovazione metodologica nella formazione professionale del radiologo italiano , coinvolgendolo attivamente nella propria formazione . cesare fava nella sua interessante lettura alle giornate pneumo - radiologiche bresciane del 2005 ricordava che , con lavvio dei corsi itineranti , la sezione di radiologia toracica della sirm completava una manovra a tenaglia nei confronti dei radiologi italiani . 
208 , euro 18 , 00 published online : 22 march 2011 springer - verlag 2011 pasquale marano has occupied , and still occupies , such a prominent place in italian radiological culture that we cannot fail to draw attention to his most recent ( latest , but not least ) book , ritorno al paziente ( back to the patient ) , which was presented at our last national congress . ritorno al paziente is both a wish and a commitment , an anthology that allows us to participate in maranos tribulations as a physician , teacher and faculty dean , in his complex and almost dramatic oscillations between holistic medicine and web 2.0 , past and future , pessimism and hope . 
although there is rare mention of actual facts and exact dates , marano describes the most intimate details and deepest recollections in a story of sentiments and experience that is gripping in its concreteness and almost touching in its empathy . its the diary of a lifetime , as well as a valuable opportunity to gain insight into the evolution of medicine and medical teaching , but with also a keen and interested eye to the possible future . what does the book contain ? a beautiful introduction which is also a synopsis . 
some useful notions of epidemiology ( the current predominance of chronic degenerative diseases ) , the evolution of society and the new doctor - patient relationship , the increasing trust in medicine and simultaneous distrust in doctors , the fragmentation of knowledge and competencies , the dif culties in teaching , the crisis of the ethical - moral values and certainties that once founded western societies... the other chapters offer some beautiful pages on error in medicine and defensive medicine , experimental and clinical research , research and innovation , and the market approach to healthcare , on which subject the author makes an ominous prediction : i fear that by operating in this way the italian healthcare system based on social cohesion and solidarity will follow in the steps taken many years ago by the american healthcare system , with the result of being sold off to insurance companies and private enterprises , among the indifference of the public . pasquale marano ha occupato e occupa un posto di grande importanza nella cultura radiologica italiana , perch possa passare sotto silenzio questa ultima ( latest , but not least ) sua fatica ritorno al paziente che stata messa a nostra disposizione durante il nostro recente congresso nazionale . ritorno al paziente un augurio e un impegno dellautore , uno zibaldone che ci permette di assistere al suo travaglio di medico , di docente e di preside , alle sue oscillazioni complesse e quasi drammatiche fra la medicina olistica ed il web 2.0 , fra il passato e lavvenire , fra il pessimismo e la speranza . 
sono rari i riferimenti a fatti concreti e a date precise , ma marano ssa i particolari pi intimi e le ricordanze pi profonde in una storia di sentimenti e di esperienze appassionante nella sua concretezza e quasi commovente nella sua partecipazione . il diario di una vita , ma anche un prezioso aiuto per capire qualcosa di pi dellevoluzione della medicina , e soprattutto dellinsegnamento della medicina ieri e oggi , con uno sguardo interessato e partecipe anche al possibile futuro . cosa c nel libro ? una bellissima introduzione che ne anche la sintesi . 
qualche utile cenno di epidemiologia ( il predominio attuale delle malattie cronico - degenerative ) , levoluzione della societ e il nuovo rapporto medico - paziente , laccresciuta ducia nella medicina e la contemporanea s ducia nel medico , la frammentazione delle conoscenze e delle competenze , le dif colt dellinsegnamento , la crisi dei valori etico - morali e delle certezze su cui poggiavano le societ occidentali poi , negli altri capitoli che non elenco , alcune bellissime pagine sullerrore in medicina e sulla medicina difensiva , sulla ricerca sperimentale , clinica e sulla cosiddetta ricerca e innovazione , sulla sanit - mercato al cui proposito c una preoccupante previsione : io temo che cos operando si stia portando , nellindifferenza generale , la sanit sociale e solidaristica italiana sulla stessa strada percorsa molti anni fa dalla sanit americana nendo con svenderla ad assicurazioni e imprese private . the second part of the book is devoted to teaching , both la seconda parte del libro dedicata allinsegnamento , 670 radiol med ( 2011 ) 116 : 669670 at university and in other settings . 
marano was perhaps the rst to involve general practitioners and hospital doctors in university teaching ; while in no way repudiating these choices , today he doesnt conceal his bitterness and disappointment for a message that was never understood and only in part implemented . 
he realises that still today the doctors rst duty remains the illness , so that talking to patients , looking after people rather than their illnesses becomes an additional personal characteristic and , as such , one that is not codi ed or compulsory . 
 a book that interested and stimulated me , a book that i found useful and that i think could be useful to many radiologists , above all those who are or have been involved in teaching . 
marano stato forse il primo a coinvolgere medici di base e medici ospedalieri nellinsegnamento universitario ; oggi non rinnega affatto queste scelte anche se non nasconde amarezza e delusione per un messaggio non compreso e solo in parte attuato . 
si rende infatti conto che ancora oggi il compito prioritario del medico resta la malattia per cui il parlare con i pazienti , loccuparsi delle persone e non delle loro malattie diviene un qualcosa di aggiuntivo e personale e , pertanto , non codi cato e non obbligatorio . 
mazziotti , via consolare pompea 1871 , 98165 messina , italy , tel . : + 39 - 090 - 3973213 , fax : + 39 - 090 - 2937427 , e - mail : smazziotti@unime.it accepted : 26 november 2009 / accepted : 15 december 2009 / published online : 19 march 2011 springer - verlag 2011 abstract purpose . 
the entire course of the tympanic canaliculus was identi ed in 80 / 97 petrous pyramids ( 82.4% ) , 57 of which were normal ( 75.4% detection rate ) and 40 pathological ( 90% detection rate )  . 
il canalicolo timpanico stato identi cato in tutto il suo decorso in 80 / 97 rocche petrose ( 82 , 4% ) , 57 normali ( tasso di individuazione del 75 , 4% ) e 40 patologiche ( tasso di individuazione del 90% )  . 
la tc multidetettore rappresenta lunica possibilit di valutare in vivo , in maniera multiplanare e con elevata frequenza ( 82 , 4% ) il canalico timpanico . parole chiave canalicolo timpanico orecchio medio tc multidetettore 658 introduction jacobsons nerve ( jn ) represents the rst efferent branch of the glossopharyngeal nerve above the plane of the jugular foramen [ 1 , 2 ]  . 
in fact , the jn originates from the inferior ganglion of the glossopharyngeal nerve , which lies at the level of the petrosal fossula , located at the level of the anterior border of the petrosal foramen . 
the location of the tc inlet is , however , somewhat variable , as it may lie either at the level of border of the caroticojugular spine or anteriorly or posteriorly to it ; it frequently has a central location , although it may also be found laterally or medially . 
at the level of the tympanic cavity , the tc opens onto the lowermost portion of the hypotympanum , at the base of the promontory , in a position anteroinferior fig . 
1 , forame giugulare ; 2 , setto carotido - giugulare ; 3 , canale carotideo ; freccia lunga , forame dingresso del canalicolo timpanico ; freccia corta , fossula petrosa . 
 radiol med ( 2011 ) 116 : 657666 introduzione il nervo di jacobson ( nj ) rappresenta il primo dei rami ceduti dal nervo glossofaringeo ( ngf ) al di sopra del piano che identi ca il forame giugulare ( fg ) [ 1 , 2 ]  . 
la sede dellorizio di ingresso del ct , tuttavia , alquanto variabile , potendo collocarsi sia a livello del margine della spina carotido - giugulare , che anteriormente o posteriormente ad esso ; pi sovente in sede centrale , ma talvolta anche alquanto laterale o mediale . 
da quanto sopra , appare di tutta evidenza come il decorso complessivo del nj sia suddividibile in un segmento intratimpanico costituito dal plesso omonimo e da un segmento infra - timpanico contenuto interamente nel ct . 
va , in ne , ricordato come il ct dia alloggio oltre che al nj anche alla sottilissima arteria timpanica ( at )  . il presente lavoro , eminentemente di tipo morfologico , nalizzato a valutare le possibilit della tomogra a computerizzata ( tc ) multistrato nella visualizzazione multiplanare del ct , segnatamente alle sue varianti anatomiche ed alle sue caratteristiche morfologiche , sia in condizioni normali che in corso di patologia in ammatoria cronica dellorecchio medio . 
in letteratura esistono solo sporadiche citazioni nelle quali riportata limmagine coronale del ct , senza peraltro alcuna indicazione di ordine tecnico e nelle quali si evidenzia limportanza di distinguere il ct da eventuali rime di frattura [ 35 ]  . 
per il resto , nella letteratura , sia precedente alla introduzione della tc multidetettore , sia in quella pi recente , il ct o non viene citato o ci si riferisce ad esso solo in termini di identi cazione esclusivamente sul piano assiale , nelle immagini acquisite ad alta risoluzione [ 611 ]  . radiol med ( 2011 ) 116 : 657666 to the round window . 
it is clear from this description that the overall course of the jn may be subdivided into an intratympanic segment , represented by the tympanic plexus , and an infratympanic segment , completely enclosed in the tc . 
 this morphological study was aimed at assessing the potential of multidetector computed tomography ( mdct ) for multiplanar imaging of the tc , in particular , as regards its anatomical variations and its morphological features , both in the normal ear and in chronic in ammatory middleear disease . 
to our knowledge , only few published citations exist on coronal imaging of the tc ; they do not , however , provide any technical indications or highlight the importance of differentiating the tc from fracture lines [ 35 ]  . 
 besides these citations , in papers published before or after the introduction of mdct , the tc is either not mentioned at all or only referred to in terms of detection in the axial plane in high - resolution images [ 611 ]  . materials and methods this study consisted of two phases : a preparatory phase and a phase focusing on clinical application . phase 1 to determine beforehand the most effective technique for multiplanar imaging of the tc in living individuals , we conducted a study on three dried skulls in which a very thin copper wire was placed as a landmark in the tc lumen to facilitate its detection . 
 all examinations were performed with a 16 - channel system ( sensation 16 , siemens , erlangen , germany ) with the following technical parameters : collimation 0.75 mm , pitch 0.55 mm , fov 300 mm , reconstruction increment 0.5 mm , high - resolution bone lter . 
on the paracoronal image , a further oblique parasagittal reconmateriali e metodi la metodologia di impostazione del lavoro ha previsto due fasi : un primo approccio di ordine preparatorio , propedeutico alla seconda fase focalizzata allapplicazione clinica . prima fase al ne di determinare preventivamente la tecnica pi ef cace per dimostrare in maniera multiplanare il ct nel vivente , stato realizzato uno studio basato sullimpiego di 3 crani secchi nei quali , al ne di facilitarne la individuazione , il ct stato evidenziato ponendo nel suo lume un repere metallico rappresentato da un sottilissimo lo di rame . tutti gli esami sono stati eseguiti con apparecchiatura con 16 canali di lettura ( sensation 16 , siemens , erlangen , germania ) con i seguenti parametri tecnici : collimazione : 0 , 75 mm , pitch : 0 , 55 mm , campo di vista ( fov ) : 300 mm , indice di ricostruzione : 0 , 5 mm , ltro ad alta risoluzione per losso . 
lacquisizione sul piano assiale stata eseguita senza alcuna angolazione del gantry ( inclinazione del gantry = 0 )  . le procedure di elaborazione post - processing sono state le seguenti : ricostruzione para - assiale obliqua secondo un piano con inclinazione di circa 1520 , orientato secondo la linea basale o canto - meatale . 
quindici pazienti erano affetti da otite cronica non colesteatomatosa , 18 da otite colesteatomatosa di cui 4 portatori degli esiti di pregresso intervento di antro - atticotomia con timpanoplastica ( tre con ogosi della cavit chirurgica ed uno con 660 radiol med ( 2011 ) 116 : 657666 ripresa di malattia colesteatomatosa )  . 
delle 100 rocche petrose studiate , pertanto , ne sono valutate ai ni del presente studio solo 97 ( 57 normali e 40 patologiche ) in quanto le tre , nelle quali si confermata la presenza del tumore glomico timpano - giugulare , sono state escluse , stante le gravi alterazioni ossee presenti nellarea del ct . 
 analisi statistica tutte le analisi statistiche sono state effettuate utilizzando il pacchetto software statistical package for the social sciences ( spss ) 10.0 ( spss , inc . , chicago , il )  . 
 we prospectively recruited 50 patients ( 28 men , 22 women ; age range 2074 years ) who were candidates for ct studies of the temporal bone due to suspected or proven chronic middle ear disease . 
fifteen patients were affected by chronic noncholesteatomatous otitis and 18 by cholesteatomatous otitis , four of whom had previously undergone antroatticotomy followed by tympanoplasty reconstruction ( three with in ammation of the surgical cavity and one with recurrent cholesteatomatous disease )  . 
therefore , of the 100 petrous pyramids examined , only 97 ( 57 normal and 40 pathological ) were included in this study , as the three cases with a con rmation of jugulotympanic glomus tumour were excluded owing to severe bone changes in the tc region . statistical analysis all statistical analyses were performed using the spss 10.0 sono state globalmente valutate 97 rocche petrose . 
per determinare se la differenza nel tasso di individuazione nei due sottogruppi sia statisticamente signi cativa , i dati sono stati analizzati mediante un test non parametrico ( test 2 di pearson )  . 
in tutti i casi lestremit timpanica del ct si collocava alla base del promontorio che , pertanto , ha rappresentato un signi cativo repere anatomico di riferimento . radiol med ( 2011 ) 116 : 657666 fig . 
the p line indicates the oblique paracoronal reconstruction plane centred on the tympanic canal inlet and oriented towards the caroticojugular septub paracoronal reconstruction showing the tympanic canal ( short arrow )  . 
d ricostruzione parasagittale che dimostra il canalicolo timpanico ( freccia corta ) con decorso lievemente obliquo rispetto al piano coronale ( c )  . software package ( spss , inc . , chicago , il , usa )  . 
 pearsons chi - square test was used to determine differences between groups ; cohens kappa test was used to measure interobserver agreement . results a total of 97 petrous pyramids were evaluated . 
in the rst group , the tc detection rate was 42 / 57 ( 73.7% ) , whereas in the second group , pathological pyramids , the detection rate was 36 / 40 ( 90% )  . 
p paracoronal oblique reconstruction plane oriented on the caroticojugular septu b depiction of the tympanic canal ( arrows ) in a paracoronal oblique reconstruction , with approximately 45 obliquity in the horizontal plane , lateromedial and caudocranial inclination . 
 on the basis of the criteria , 61.1% of tc depicted within state dimostrate signi cative differenze di incidenza di ct alterato nei pazienti portatori di otite cronica colesteatomatosa o non colesteatomatosa ( p > 0 , 1 )  . 
 discussione dalla analisi dei risultati ottenuti possibile affermare che la tc multidetettore rappresenta lunica possibilit di valutare in vivo , in maniera multiplanare e con elevata frequenza ( 80 , 4% ) , il ct . fino ad oggi la descrizione del ct era af data alla letteratura anatomica , che basava le proprie valutazioni sullimpiego di tecniche di dissezione . 
la suddetta considerazione spiega , verosimilmente , le differenze di lunghezza del ct riscontrate nel nostro lavoro , che appaiono minori ( media di 7 , 3 mm ) rispetto a quelle riferite dagli studi anatomici ( media 9 , 5 mm )  . 
tali discrepanze , peraltro trascurabili , sono da ricondursi in prima ipotesi , oltre che alla differente metodologia di misura , anche ai diversi reperi anatomici posti alla base delle due modalit di approccio . 
nelle dissezioni anatomiche , infatti , nella misura del ct viene anche inclusa la regione della fossula petrosa . il nostro studio trova , per converso , perfetta coincidenza con le analisi anatomiche nella documentazione dei due gradi di obliquit del ct ( in senso latero - mediale ed antero - posteriore ) con una precisa quanti cazione degli angoli stessi . 
bisogna sottolineare , peraltro , che , nella nostra casistica , in un non trascurabile numero di casi , il ct non presentava alcuna inclinazione antero - posteriore , decorrendo pressoch parallelo ( a 0 ) al piano frontale . 
in fact , the measurement of tc length at anatomical dissection is inclusive of the petrosal fossula . by contrast , our results coincide perfectly with anatomical studies with regard to the nding of the two degrees of tc inclination ( lateromedial and anteroposterior ) , with precise quanti cation of the angles . 
this variation in course has not been described in any anatomical study . another consideration emerging from our study is the dif culty detecting the tc in the presence of extreme pneufig . 
tale problematica trova riscontro anche nella letteratura non radiologica , poich la suddetta condizione rappresenta un ostacolo anche in corso di dissezione anatomica , oltre che durante le manovre chirurgiche nalizzate alla resezione del nj cos che , sovente , in presenza di iperpneumatizzazione infra - timpanica la procedura debba essere abbandonata . 
si rileva nella ricostruzione paracoronale obliqua una modesta quantit di tessuto patologico a livello dellori zio prossimale del canalicolo timpanico ( freccia corta ) , questultimo appare signi cativamente slargato ( freccia lunga )  . 
this problem has also been highlighted in nonradiological studies , as the condition hampers anatomical dissection as well as surgical manoeuvres aimed at jn resection , leading to interruption of the procedure in the presence of infratympanic hyperpneumatisation . our ndings regarding pathological tc are also interesting . 
this is probably due to the very low position of the tc , which facilitates and prolongs the contact of the products of in ammation and in amed tissues with the tympanic opening of the canaliculus . 
the former are caused by a proliferation of cells of the nonchromaf n paraganglial system , which are present in a quantitatively signi cant amount ( almost one fth of the total ) along the intracanalicular course of the jn . 
conversely , tc alterations and jn involvement in the presence of in ammatory changes are in merito alla analisi del ct in condizioni patologiche . il coinvolgimento del ct risulta assai frequente ( 61 , 1% ) in presenza di patologia ogistica , senza alcuna differenza signi cativa tra otite cronica colesteatomatosa e non colesteatomatosa . 
ci verosimilmente in ragione della sua sede estremamente declive che facilita e prolunga il contatto dei prodotti della in ammazione e del tessuto ogistico con lo sbocco timpanico del canalicolo . 
le prime sono secondarie alla proliferazione delle cellule dellapparato non cromaf ne paragangliare , che si localizzano in maniera quantitativamente non trascurabile ( circa un quinto del totale ) lungo il decorso intra - canalicolare del nj . 
 il nj rappresenta uno dei rami terminali del ngf ed pertanto deputato a diverse funzioni nellambito delle atti666 radiol med ( 2011 ) 116 : 657666 less well known and completely lacking from the radiological literature . the jn represents one of the terminal branches of the glossopharyngeal nerve and has therefore several parasympathetic neurovegetative functions [ 2 , 9 ]  . 
in addition , there seems to be a ( not completely clear ) correlation between jn dysfunction and tinnitus , another disorder that was often successfully treated with jn resection . 
anatomical studies , supported by electromyographic ndings , have shown that bres from the jn and the tympanic plexus participate in this function by way of synapses at the level of the pons , from which other efferent bres regulate the tone and function of the elevator and constrictor muscles of the auditory tube . given that our study focused mainly on morphological aspects of the tc , no correlation emerged between the alterations identi ed and the above functional aspects . 
analysis of our results did not take into consideration possible correlations with those clinical aspects ( tinnitus , salivary function alterations , disease duration , etc . ) that could have con rmed or denied the role of jn alterations in the origin and natural history of in ammatory diseases of the middle ear . 
in this perspective , our study was preliminary and preparatory to another study aimed at analysing jn alterations and their correlation with such clinical aspects . vit neuro - vegetative ad impronta parasimpatica [ 2 , 9 ]  . 
studi anatomici , confortati dal riscontro elettromiogra co , hanno dimostrato , infatti , come contingenti di bre del nj e dal plesso timpanico intervengano in tale funzione mediante collegamenti sinaptici a livello del ponte , dal quale altre bre efferenti sono in grado di regolare il tono e la funzionalit dei muscoli elevatore e costrittore della tuba . poich il presente lavoro ha inteso focalizzare principalmente gli aspetti morfologici relativi al ct , da esso non sono emerse correlazioni tra le alterazioni documentate ed i suddetti aspetti funzionali . 
nellanalisi dei risultati , infatti , non sono state prese in considerazione le possibili correlazioni con quegli aspetti clinici ( presenza di tinnito , alterazioni della funzione salivare , durata della malattia , ecc . ) , che avrebbero potuto confermare o smentire il ruolo della alterazione del nj nella con gurazione e nella storia naturale della patologia ogistica dellorecchio medio . 
the learning objectives of this review are to : illustrate radiographic anatomy of the mediastinum with particular attention to mediastinal lines ; describe radiographic signs that allow identi cation of mediastinal abnormalities that are dif cult to detect on conventional chest radiographs ; describe ndings that help localise abnormalities in the anterior , middle or posterior mediastinuthe anterior junction line obliteration , the hilum overlay sign , the preservation of the posterior mediastinal lines and the silhouette sign with the right cardiac border are radiographic signs that allow identi cation and localisation of anterior mediastinal lesions . 
widening of the right paratracheal stripe , distortion of the azygo - oesophageal recess and the convex border of the aortopulmonary window indicate the presence of a middle mediastinal abnormality . 
 gli obiettivi diagnostici della nostra review sono stati : illustrare lanatomia radiogra ca del mediastino con particolare riferimento alle linee mediastiniche ; descrivere i segni che aiutano ad identi care alterazioni mediastiniche di dif cile diagnosi radiogra ca ; descrivere le alterazioni radiogra che che consentono di localizzare la lesione a livello del mediastino anteriore , medio o posteriore . 
la cancellazione della linea di giunzione anteriore , il segno dellilo sovrapposto , la conservazione delle linee mediastiniche posteriori ed il segno della silhouette con il margine destro del cuore sono segni di semeiotica radiogra ca indicativi di patologie del mediastino anteriore . 
 inoltre questo approccio permette di localizzare il radiol med ( 2011 ) 116 : 532547 keywords mediastinal lines mediastinal masses chest radiograph compartimento mediastinico interessato dalla patologia e quindi di porre indicazione al successivo iter diagnostico pi appropriato . 
 preservation , obliteration , thickening or distortion of these lines represent the key to diagnosing and localising mediastinal abnormalities that are dif cult to detect on chest radiographs . the mediastinum is often divided into convenient compartments . 
even if there are no physical boundaries between mediastinal compartments , a simpli ed classi cation can help to more accurately localise an abnormality and may help narrow the differential diagnosis prior to obtaining additional information with chest computed tomography ( ct )  . 
the felson method of division is based on ndings at lateral chest lthe anterior mediastinum is bounded anteriorly by the sternum and posteriorly by a line extending from the thoracic inlet to the diaphragm along the anterior aspect of the trachea and the back of the heart . 
the posterior mediastinum is localised between the line that connects points 1 cm behind the anterior margins of the vertebral bodies and the posterior thoracic wall [ 1 ]  . heitzman divided the mediastinum into the following anatomic regions : thoracic inlet , anterior mediastinum , supra - aortic area ( above the aortic arch ) , infra - aortic area ( below the aortic arch ) , supra - azygos area ( above the azygos arch ) and infra - azygos area ( below the azygos arch ) [ 2 ]  . anterior mediastinum the anterior mediastinum is bounded anteriorly by the sternum , posteriorly by the anterior wall of the trachea and the back of the heart , superiorly by the thoracic inlet and inferiorly by the diaphrag its contents include thymus , lymph nodes , adipose tissue , heart and ascending aorta . 
la conservazione , lobliterazione , lispessimento o la distorsione di queste linee rappresenta la chiave per la diagnosi e la localizzazione di alterazioni mediastiniche di dif cile identi cazioni radiogra ca . il mediastino convenzionalmente suddiviso in alcuni compartimenti . 
sebbene non esistano precise barriere anatomiche che suddividano tali compartimenti , una classi cazione sempli cata pu aiutare nel localizzare in modo pi preciso uneventuale alterazione e nel restringere il ventaglio delle possibilit diagnostiche prima di ottenere ulteriori informazioni con lesame in tomogra a computerizzata ( tc )  . 
il mediastino anteriore delimitato anteriormente dallo sterno e posteriormente da una linea che si estende dalladitus mediastinico al diaframma decorrendo per la parete anteriore della trachea e per il margine posteriore del cuore . 
 il mediastino posteriore compreso tra una linea passante un centimetro dietro i corpi vertebrali e la parete toracica posteriore [ 1 ]  . la classi cazione di heitzman divide il mediastino nelle seguenti regioni : laditus mediastinico , il mediastino anteriore , la regione sovra - aortica ( al di sopra dellarco aortico ) , la regione infra - aortica ( al di sotto dellarco aortico ) , la regione sovra - azygos ( al di sopra dellarco dellazygos ) e la regione infra - azygos ( al di sotto dellarco dellazygos ) [ 2 ]  . mediastino anteriore il mediastino anteriore delimitato anteriormente dallo sterno , posteriormente dalla parete anteriore della trachea e dal pro lo posteriore del cuore , superiormente dalladitus mediastinico ed inferiormente dal diaframma . 
obliteration of the anterior junction line , the hilum overlay sign , preservation of the posterior mediastinal lines and the silhouette sign with the right cardiac border are radiographic signs that allow identi cation and localisation of anterior mediastinal lesions [ 36 ] : anterior junction line : this is seen at frontal chest radiography . 
a small amount of fat is normally found within the line ; however , increased amounts of intervening mediastinal fat and possibly thymus in younger patients may cause the anterior junction line to appear as a stripe . 
anterior mediastinal masses can obliterate the anterior junction line , although it is usually the preservation of more posterior lines at chest radiography that helps to correctly localise an anterior mediastinal mass . hilum overlay sign : this is present when the normal in tale spazio sono contenuti il timo , i linfonodi , il tessuto adiposo , il cuore e laorta ascendente . 
le masse del mediastino anteriore comprendono lesioni timiche ( timoma , carcinoma , iperplasia timica , cisti timiche e timolipoma ) , linfomi , tumori a cellule germinali e cisti pleuro - pericardiche . 
lobliterazione della linea di giunzione anteriore , il segno dellilo sovrapposto , la conservazione delle linee mediastiniche posteriori ed il segno della silhouette con il margine destro del cuore sono segni radiogra ci che permettono di identi care e correttamente localizzare patologie espansive del mediastino anteriore [ 36 ]  . 
 la linea di giunzione anteriore visibile sulla radiogra a del torace in proiezione frontale ; questa linea formata dallapposizione anteriore della pleura viscerale e parietale della porzione antero - mediale dei due polmoni unitamente ad una modesta quota di tessuto adiposo mediastinico interposto . 
una modesta quantit di grasso normalmente presente allinterno della linea ; tuttavia , laumento della quantit di tessuto adiposo e la presenza del timo nei pazienti giovani possono determinare un aspetto a banda della linea di giunzione antefig . 
1a , b drawing on the frontal chest radiograph ( a ) shows the anterior junction line running obliquely from the upper right to the lower left and not extending above the manubriosternal junction . 
1a , b la linea disegnata sulla proiezione frontale del torace ( a ) rappresenta la linea di giunzione anteriore che decorre obliquamente dallalto verso il basso e da destra verso sinistra e non si estende al di sopra dellarticolazione manubrio - sternale . 
la scansione tc ( b ) passante per la porzione superiore del torace mostra il normale aspetto della linea di giunzione anteriore ( freccia ) formata dallaccollarsi della pleura viscerale e parietale dei due polmoni e dal tessuto adiposo interposto . 
posteroanterior chest radiograph ( a ) clearly depicts the hilum ( arrow ) , which indicates that the mass is either anterior or posterior to the hiluin addition , the descending aorta is clearly seen ( arrowhead ) , indicating that the mass is not within the posterior mediastinuchest computed tomography scan ( b ) con rms the presence of an anterior mediastinal mass . 
la visualizzazione dellilo polmonare ( freccia ) nella radiogra a del torace in proiezione postero - anteriore ( a ) indica che la massa localizzata in un piano anteriore o posteriore rispetto allilo . 
an abnormal convex aspect of the cardiac border or the presence of an additional line along the pro le of the heart can also suggest the presence of an anterior mediastinal mass . middle mediastinum the middle mediastinum is bounded anteriorly by the anterior wall of the trachea and the back of the heart , posteriorly by the vertebral bodies , superiorly by the thoracic inlet and inferiorly by the diaphragits contents include the superior vena cava ( svc ) , the inferior vena cava ( ivc ) , brachiocephalic vessels , pulmonary vessels , trachea and main riore . 
la radiogra a del torace ( a ) mostra unopacit a margini netti verso il parenchima polmonare che si proietta a livello della nestra aorto - polmonare ( frecce )  . 
4a , b la proiezione postero - anteriore della radiogra a del torace ( a ) mostra una massa espansiva del mediastino anteriore che determina cancellazione del margine destro del cuore . 
knowledge of the contents of this compartment facilitates development of a differential diagnosis for middle mediastinal masses that includes lymphadenopathy , foregut duplication cysts ( bronchogenic , oesophageal , neurenteric ) , pericardial cyst and neurogenic tumours . 
un anomalo aspetto convesso del margine cardiaco o la presenza di una linea aggiuntiva lungo il pro lo del cuore possono ugualmente essere segni radiogra ci indicativi di una massa localizzata nel mediastino anteriore . 
computed tomography scan ( b ) shows the right wall of the trachea with airsoft tissue interfaces caused by air within the tracheal lumen and right lung ( arrowhead )  . 
la scansione tc ( b ) mostra la parete laterale destra della trachea come uninterfaccia aria - tessuti molli dovuta allaria contenuta allinterno del lume tracheale ed allaria del parenchima polmonare destro adiacente ( testa di freccia )  . 
the right paratracheal stripe should be uniform in width , with a normal width ranging from 1 to 4 mm ; a right paratracheal stripe 5 mm or more wide is considered abnormally enlarged . 
 the lateral aspect of the aortopulmonary window , seen as the aorticpulmonary ( ap ) window re ection , is due to the interface between the left lung and the mediastinu at radiography , the edge of the window extends from the aortic knob to the left pulmonary artery . 
this edge should have a concave or straight border with the adjacent lung , with a straight border being considered normal unless mediastino medio il mediastino medio delimitato anteriormente dalla parete anteriore della trachea e dalla super cie posteriore del cuore , posteriormente dai corpi vertebrali , superiormente dalladitus mediastinico ed inferiormente dal diaframma . 
 esso contiene la vena cava superiore ( vcs ) , la vena cava inferiore ( vci ) , i vasi epiaortici , i vasi polmonari , la trachea , i bronchi principali , lesofago , i linfonodi , il nervo frenico , il nervo vago ed il nervo laringeo ricorrente di sinistra . 
la conoscenza del contenuto di questo compartimento facilita la diagnosi differenziale tra le masse del mediastino medio che includono linfoadenopatie , cisti disembriogenetiche da duplicazione ( broncogene , esofagee , neuroenteriche ) , cisti pericardiche e tumori neurogeni . 
nella radiogra a del torace in proiezione frontale ( a ) la linea paratracheale destra non pi apprezzabile in quanto obliterata da una massa paratracheale destra ( frecce )  . 
la scansione tc ( b ) dimostra la presenza di tumefazioni linfoghiandolari in sede paratracheale destra le quali determinano cancellazione dellinterfaccia aria - tessuti molli tra il parenchima polmonare aerato e la parete laterale destra della trachea . 
anterior to the ap window re ection , the aorticpulmonary re ection extends from the aortic arch to the level of the left main bronchus , where it usually continues the border of the left side of the heart . 
the contents of the ap window include left recurrent laryngeal nerve , left vagus nerve , radiol med ( 2011 ) 116 : 532547 dovrebbe presentare spessore uniforme compreso fra 1 e 4 mm ; una linea paratracheale di spessore superiore o uguale a 5 mm considerata patologicamente slargata . 
la porzione laterale della nestra aorto - polmonare , identi cabile come ri essione della nestra aorto - polmonare , dovuta allinterfaccia tra il parenchima polmonare sinistro ed il mediastino . 
questo margine dovrebbe avere un aspetto concavo o rettilineo rispetto al polmone adiacente ; laspetto rettilineo pu essere considerato normale a meno che esami radiogra ci precedenti non abbiano dimostrato un aspetto concavo . 
anteriormente alla ri essione della nestra ap , la ri essione aorto - polmonare si estende dallarco aortico no a livello del bronco principale sinistro , dove solitamente si continua con il pro lo sinistro del cuore . 
 nella nestra aorto - polmonare sono contenuti il nervo laringeo ricorrente di sinistra , il nervo vago di sinistra , il legamento arterioso , il tessuto adiposo mediastinico , i linfonodi e le arterie bronchiali di sinistra . 
7a - d a 14 - year - old girl with frontal view of the chest radiograph ( a ) shows a slightly convex border of the aortopulmonary window suspicious ( arrow ) for the presence of a mass in the middle mediastinu on ( c ) , computed enlarged lymph nodes are clearly seen in the aortopulmonary window ( arrow ) , causing the abnormality seen on chest radiography . 
la proiezione frontale della radiogra a del torace ( a ) mostra un margine leggermente convesso della nestra aorto - polmonare ( freccia ) sospetto per la presenza di una localizzata massa espansiva nel mediastino medio . 
nella sua porzione inferiore il recesso azygos - esofageo pu essere slargato in caso di patologia esofagea o ernia iatale [ 4 , 13 ]  . mediastino posteriore il mediastino posteriore delimitato anteriormente dallesofago , antero - inferiormente dal diaframma , posteriormente dalla parete toracica posteriore e superiormente dalladitus mediastinico . 
the lateral pro le of the descending aorta maintains a normal airsoft tissue interface with the aerated left lung , thereby preserving the normal radiographic appearance of the paraortic line . 
 la scansione tc assiale dopo somministrazione di contrasto ( c ) evidenzia una massa solida ( freccia ) ben circoscritta localizzata nel mediastino medio in contiguit con larteria polmonare sinistra . 
limmagine in risonanza magnetica ( rm ) t2 - pesata sul piano assiale ( d ) e le immagini t1 - pesate dopo somministrazione di mezzo di contrasto sul piano coronale ( e ) e sagittale ( f ) documentano una massa solida con segnale iperintenso ( frecce ) , risultata essere un tumore neurogeno ( schwannoma )  . della enopathy may cause deviation of the azygo - oesophageal line . 
 posterior mediastinum the posterior mediastinum is bounded anteriorly by the oesophagus , anteroinferiorly by the diaphragm , posteriorly le masse del mediastino posteriore sono prevalentemente causate dai tumori neurogeni , dagli ascessi paraspinali o dalleritropoiesi extramidollare ; queste masse determinano unobliterazione della linea di giunzione posteriore ed ispessimento o deformazione della linea paraortica e / o delle linee paraspinali [ 1417 ]  . 
 la linea di giunzione posteriore una linea mediastinica posteriore visibile al di sopra del livello della vena azygos e dellaorta ; essa determinata dal contatto delle super ci pleuriche viscerale e parietale della porzione radiol med ( 2011 ) 116 : 532547 fig . 
9a , b la radiogra a del torace in proiezione frontale ( a ) mostra un anomalo aspetto convesso della ri essione aorto - polmonare causato da dilatazione dellarteria polmonare sinistra come dimostrato nella scansione tc corrispondente ( b )  . 
10a - d frontal view of the chest radiograph ( a ) shows a normal azygo - oesophageal line ( arrows ) with mild leftwards convexity superiorly and a straight edge inferiorly . 
10a - d la radiogra a del torace in proiezione frontale ( a ) raf gura un aspetto normale della linea azygos - esofagea ( frecce ) che mostra una lieve convessit verso sinistra nella sua porzione superiore ed un andamento rettilineo nella sua porzione inferiore . 
le scansioni tc dopo somministrazione di mezzo di contrasto ( c ) ed rm t1 - pesata sul piano assiale dopo mezzo di contrasto ( d ) documentano la presenza di una formazione espansiva liquida nel mediastino medio ( frecce ) , risultata essere una cisti disembriogenetica . 542 radiol med ( 2011 ) 116 : 532547 fig . 
corresponding computed tomography scans ( c , d ) con rm a middle mediastinal mass ( arrows ) located in the azygooesophageal recess , which proved to be a bronchogenic cyst . 
le scansioni tc corrispondenti ( c , d ) confermano la presenza di una massa espansiva del mediastino medio ( frecce ) localizzata a livello del recesso azygos - esofageo , risultata essere una cisti broncogena . 
il margine destro del cuore ed i corpi vertebrali conservano una normale interfaccia con il parenchima polmonare destro e questo spiega la mancata cancellazione delle linee corrispondenti alla radiogra a del torace . by the posterior chest wall and superiorly by the thoracic inlet . 
posterior mediastinal masses are mostly due to neurogenic tumours , paraspinal abscess or extramedullary haematopoiesis ; these masses determine obliteration of the posterior junction line and thickening or disruption of the paraortic line and / or paraspinal lines [ 1417 ] : posterior junction line : this is a posterior mediastinal line seen above the level of the azygos vein and aorta and is formed by apposition of the visceral and parietal pleura of the posteromedial portion of the lungs posterior to the oesophagus and anterior to the third through fth thoracic vertebrae . 
it appears as a straight or mildly leftwards convex line , typically projecting postero - mediale dei due polmoni , posteriormente allesofago ed anteriormente alle vertebre toraciche dalla terza alla quinta . 
la linea di giunzione posteriore presenta una maggiore estensione craniale rispetto alla linea di giunzione anteriore e , a differenza di questultima , visibile al di sopra delle clavicole e si estende prevalentemente in senso verticale , mentre la linea di giunzione anteriore devia verso sinistra . 
12a - f frontal view of the chest radiograph ( a ) shows normal posterior junction line ( arrows ) as a straight line projecting through the trachea and extending above the clavicles . 
axial nonenhanced computed tomography ( c , d ) and sagittal preand postcontrast t1 - weighted magnetic resonance scans ( e , f ) help con rm the posterior location of the lesion , corresponding to a well - circumscribed solid mass located in the posterosuperior mediastinum between trachea and spine . 
12a - f nella radiogra a del torace in proiezione frontale ( a ) la linea di giunzione posteriore ( frecce ) rappresentata come una linea diritta che si proietta allinterno della trachea e si estende al di sopra delle clavicole . 
le scansioni assiali tc senza contrasto ( c , d ) ed rm t1 - pesate sul piano sagittale prima e dopo somministrazione di contrasto ( e , f ) confermano la localizzazione posteriore della lesione che corrisponde ad una massa solida localizzata nel mediastino posterosuperiore tra la trachea e la colonna vertebrale . 
the posterior junction line demonstrates more cranial extension than the anterior junction line and , unlike its counterpart , is seen above the clavicles and lies almost vertical , whereas the anterior junction line deviates to the lea posterosuperior meditoracica ed il polmone sinistro adiacente normalmente ventilato . 
computed tomography scans in axial ( c ) and sagittal ( d ) planes show a well - circumscribed hypodense lesion ( arrow in c and arrowhead in d ) abutting the posterolateral pro le of the descending aorta . 
le scansioni tc sul piano assiale ( c ) e sagittale ( d ) mostrano una lesione ipodensa ben circoscritta ( freccia in c e testa di freccia in d ) a contatto con il margine posterolaterale dellaorta discendente . 
the left paraspinal line is seen more frequently than the right paraspinal line due to the presence of the descending thoracic aorta on the left , which promotes the tangential contact of the left lung necessary to produce the lungmediastinum interface . 
la linea paraspinale di sinistra determinata dal contatto tangenziale del polmone sinistro e della pleura con il tessuto adiposo del mediastino posteriore , i muscoli paraspinali di sinistra ed i tessuti molli adiacenti . 
la linea paraspinale sinistra pi frequentemente visibile della linea paraspinale destra perch a sinistra la presenza dellaorta toracica discendente agevola il contatto tangenziale del polmone sinistro necessario a determinare linterfaccia polmone - mediastino . 
axial postcontrast computed tomography scans ( c , d ) help con rm a solid mass ( arrows ) located in the middle and posterior mediastinum effacing the left paraspinal and paraortic lines and extending into the spine . 
 le scansioni tc assiali eseguite dopo somministrazione di mezzo di contrasto ( c , d ) mostrano una massa solida ( frecce ) localizzata nel mediastino medio e posteriore , a contatto con le linee paraspinale sinistra e paraortica , la quale mostra estensione intraspinale . 
al contrario , le masse anteriori localizzate al di sopra delle clavicole non hanno uninterfaccia con il parenchima polmonare , e di conseguenza i loro margini non appaiono netti ; queste masse anteriori potrebbero essere totalmente intratoraciche , localizzate tipicamente nel mediastino anteriore , oppure potrebbero rappresentare unestensione intratoracica di masse localizzate nel collo . 
on the lateral chest lm ( b ) , the opacity ( arrow ) projects into the posterior mediastinu computed tomography scan ( c ) shows bilateral soft - tissue paraspinal masses ( arrows ) , representing extramedullary haematopoiesis , in a young thalassaemic patient . 
la proiezione frontale della radiogra a del torace ( a ) mostra una linea aggiuntiva ( freccia ) che decorre medialmente rispetto al margine destro del cuore e che cancella la linea paraspinale destra . 
16a - c frontal chest lm ( a ) shows enlargement of the right superior mediastinuabove the level of the clavicles , the margins of the mass are sharp ( arrow ) due to the interface with adjacent lung , indicating that the mass is located in the posterior mediastinucoronal ( b ) and sagittal ( c ) postcontrast t1 - weighted magnetic resonance scans con rm the presence of a solid inhomogeneous mass located in the posterior mediastinum , with intraforaminal extension . 
al di sopra delle clavicole i margini della lesione appaiono netti ( freccia ) per linterfaccia con il parenchima polmonare adiacente , e ci indica che la massa localizzata nel mediastino posteriore . 
le immagini rm t1 - pesate sui piani coronale ( b ) e sagittale ( c ) acquisite dopo somministrazione di contrasto confermano la presenza di una massa solida a struttura disomogenea localizzata nel mediastino posteriore con estensione intraforaminale . 
tale lesione risultata essere un tumore neurogeno . radiol med ( 2011 ) 116 : 532547 conclusions conclusioni knowledge of the normal radiographic mediastinal anatomy and the mediastinal lines is crucial to identifying subtle mediastinal abnormalities that can be easily missed on conventional radiography . 
moreover this approach allows identi cation on chest radiographs of the involved mediastinal compartment , thereby leading to the most appropriate further diagnostic workup . la conoscenza della normale anatomia radiogra ca del mediastino e delle linee mediastiniche fondamentale per identi care minime alterazioni mediastiniche che potrebbero essere facilmente misconosciute sulla radiogra a convenzionale del torace . 
giglio , cefal , italy 4department of radiology , university of verona , verona , italy 5department of radiology and cardiology , university hospital , parma , italy 6department of radiology and cardiology , erasmus medical center , rotterdam , the netherlands correspondence to : l . 
patients with suspected cad presented the following prognostic outcome ( p < 0.0001 ) : in 41 patients with normal coronary arteries at mdct - ca , the event rate was 0% ; ve of 49 patients with nonobstructive cad had major cardiac events ; two of 35 patients with obstructive cad suffered cardiac death and 19 underwent revascularisation . 
centoventicinque pazienti ( 82 uomini , et media 57 , 410 , 3 anni ) con sospetta mac sottoposti ad ac - tcms sono stati inclusi in un follow - up a 2 anni per eventi cardiaci maggiori . 
i pazienti presentano il seguente outcome prognostico ( p < 0 , 0001 ) : nessun evento in 41 pazienti con coronarie normali alla ac - tcms ; 5 eventi cardiaci maggiori fra i 49 pazienti con mac non ostruttiva ; 2 morti cardiache e 19 rivascolarizzazioni fra i 35 pazienti con mac ostruttiva . 
nella routine clinica la ac - tcms presenta un eccellente valore prognostico a 2 anni in caso di 522 radiol med ( 2011 ) 116 : 521531 with normal coronary arteries . 
il numero di eventi cardiaci maggiori aumenta con la severit della patologia riscontrata . keywords coronary artery disease prognostic value coronary angiography multidetector row computed tomography parole chiave malattia aterosclerotica coronarica valore prognostico angiogra a coronarica tomogra a computerizzata multistrato introduction introduzione noninvasive coronary angiography ( ca ) with multidetector - row computed tomography ( mdct ) is one of the most advanced and technologically inspiring diagnostic imaging applications of the last 10 years . 
other clinical applications of mdct - ca are the follow - up of coronary artery revascularisation procedures with aortocoronary bypass surgery [ 7 ] or stenting of proximal segments [ 8 ] and evaluating the coronary circulation as part of the planning of aortic valve surgery [ 9 ]  . 
in this regard , consensus documents have been drawn up by panels of european and american experts that de ne the appropriateness and reliability of the clinical applications of the technique [ 10 , 11 ]  . 
in terms of routine clinical implementation of the technique , however , evaluating patient outcome should also be considered that is , to what extent can mdct - ca positively in uence prognosis [ 12 ] and reduce the morbidity of major cardiac events [ 13 ]  . 
whereas there are numerous studies in the literature on the diagnostic accuracy of the technique , there are very few that deal with the prognostic impact of mdct - ca [ 1418 ]  . 
the aim of our study was to de ne the prognostic value in routine clinical use over a 2 - year follow - up period of mdct - ca in patients with suspected cad . langiogra a coronarica non invasiva mediante tomogra a computerizzata multistrato ( ac - tcms ) rappresenta una delle applicazioni pi avanzate e tecnologicamente affascinanti dellultimo decennio in ambito di diagnostica per immagini . 
altre applicazioni cliniche della ac - tcms sono il follow - up delle procedure di rivascolarizzazione coronarica , mediante by - pass aorto - coronarici [ 7 ] o stenting dei segmenti prossimali [ 8 ] , e la valutazione del circolo coronarico in previsione di chirurgia della valvola aortica [ 9 ]  . 
a tale proposito , alcuni consensus documents di panel di esperti europei ed americani hanno de nito lappropriatezza e laf dabilit delle applicazioni cliniche della metodica [ 10 , 11 ]  . 
nellottica di una implementazione clinica di routine non deve essere tralasciata , tuttavia , una valutazione delloutcome del paziente ovvero in quale misura lesecuzione di una ac - tcms possa in uire positivamente sulla prognosi [ 12 ] e ridurre la morbilit degli eventi cardiaci maggiori [ 13 ]  . 
 scopo del nostro studio quello di de nire il valore prognostico nella routine clinica della ac - tcms in pazienti con sospetta mac con follow - up a 2 anni . materials and methods population materiali e metodi popolazione one hundred and twentyve patients ( 82 men , mean age 57.410.3 ) with suspected cad who underwent 64 - slice mdct - ca between september 2006 and march 2008 in our department were included in the study . 
patients underwent mdct - ca for the following clinical indications : atypical chest pain ( n = 59 ) , typical angina with inconclusive stress test ( n = 21 ) , high risk of major coronary event ( n = 24 ) and evaluation of coronary artery circulation as a part of planning for aortic valve surgery ( n = 21 )  . 
each patient centoventicinque pazienti ( 82 uomini , et media 57 , 410 , 3 anni ) con sospetta mac sottoposti ad ac - tcms a 64 strati tra settembre 2006 e marzo 2008 nel nostro dipartimento sono stati inclusi nello studio . 
i pazienti sono stati sottoposti ad ac - tcms con le seguenti indicazioni cliniche : dolore toracico atipico ( n = 59 ) , angina tipica con stress - test non conclusivo ( n = 21 ) , alto rischio di eventi coronarici maggiori ( n = 24 ) , valutazione del circolo coronarico in previsione di chirurgia della valvola aortica ( n = 21 )  . 
per ogni paziente stata inizialmente valutata la presenza di fattori di rischio radiol med ( 2011 ) 116 : 521531 was initially evaluated for the presence of cardiovascular risk factors and symptoms ( typical and atypical chest pain , angina - like symptoms )  . 
the following cardiovascular risk factors were considered : hypertension ( de ned as arterial pressure > 140 / 80 or the need for antihypertensive agents ) , hypercholesterolaemia [ low - density lipoprotein ( ldl ) > 130 mg / dl or use of statins ] , diabetes mellitus , tobacco smoking , obesity [ body mass index ( bmi ) > 27 ] and a family history of cardiovascular disease . 
the morise test was calculated for each patient to evaluate the pretest risk [ 19 ] ( table 1 )  . the following exclusion criteria were adopted : refusal to provide informed consent , severely compromised kidneys ( creatininaemia > 120 mol / l ) , known allergy to iodinated cardiovascolare e sintomi ( dolore toracico tipico ed atipico , equivalenti anginosi )  . 
sono stati considerati i seguenti fattori di rischio cardiovascolare : ipertensione ( de nita come pressione arteriosa > 140 / 80 o necessit di terapia antipertensiva ) , ipercolestorelemia ( colesterolo low density lipoprotein [ ldl ] > 130 mg / dl o assunzione di statine ) , diabete mellito , abitudine al fumo , obesit ( body mass index [ bmi ] > 27 ) , anamnesi familiare di malattie cardiovascolari . 
 the ethics committee approved the study protocol , and all patients provided informed consent . tiche , grave compromissione della funzione respiratoria , nota coronary artery disease ( cad ) ( pazienti con pregresso infarto miocardico acuto o pregresse procedure di rivascolarizzazione miocardica mediante stent o by - pass aorto - coronarici )  . 
il comitato etico ha approvato il protocollo di studio e tutti i pazienti hanno fornito consenso informato scritto . parametri di scansione e ricostruzione scan and reconstruction parameters all examinations were done with a 64 - slice ct system ( brilliance 64 , philips medical systems , cleveland , oh , usa )  . 
 cs was measured using a prospectively gated acquisition protocol with the following parameters : fov 140200 mm , kv 120 , mas 100 , slice thickness 3 mmdct - ca scans were performed with the following parameters : slices / collimation 64 / 0.6 mm , rotation time 420 ms , effective temporal resolution ( with 180 algorithm ) 210 ms , 120 kv , 8001040 mas , table feed 11.9 mm / s , effective slice thickness 0.8 mm , reconstruction increment 0.4 mm , fov 140240 mm ( extended cranially only for evaluating ascending aorta aneurysms )  . 
patients with a heart rate > 65 bpm were given a dose of 2040 mg of propranolol by mouth ( inderal , astrazeneca reims , reims , cedex , france ) 1 h prior to the scan to lower the heart rate . 
in the information lea et informing patients on how to prepare for the examination , treatment with 2040 mg propranolol twice daily under physician supervision was recommended in the 3 days prior to the examination to lower and stabilise the heart rate in patients with tachycardia . 
a bolus of 100120 ml of nonionic iodinated contrast agent ( iomeprol , iomeron 400 , bracco , milan , italy ) , dosed according to the scan range , was administered at an injection rate of 5 ml / s using an automatic injector ( stellant , medrad , pittsburgh , pa , usa ) connected to an 18 - gauge needle cannula placed in a right antecubital ve intracoronary enhancement was optimised by using the bolus - tracking technique , which involved placing a region of interest ( roi ) at the level of the ascending aorta in order to synchronise the beginning of the scan with the arrival of the contrast agent in the scan target . 
data were retrospectively reconstructed in the end - diastolic phase ( from 65% to 80% of the rr interval ) and end - systolic phase ( 4045% )  . tutte le indagini sono state eseguite mediante tc a 64 - strati ( brilliance 64 , philips medical systems , cleveland , ohio , usa )  . 
il cs stato eseguito mediante acquisizione cardio - sincronizzata prospettica con i seguenti parametri : eld of view ( fov ) 140200 mm , kv 120 , mas 100 , spessore di strato 3 m le scansioni ac - tcms sono state effettuate con i seguenti parametri : strati / collimazione 64 / 0 , 6 mm , tempo di rotazione 420 ms , effettiva risoluzione temporale ( con algoritmo a 180 ) 210 ms , 120 kv , 8001040 mas , avanzamento del tavolo 11 , 9 mm / s , spessore effettivo di strato 0 , 8 mm , incremento di ricostruzione 0 , 4 mm , fov 140240 mm ( esteso cranialmente solamente per la valutazione di aneurismi dellaorta ascendente )  . 
i pazienti con frequenza cardiaca > 65 battiti per minuto ( bpm ) hanno ricevuto una dose di 2040 mg di propranololo cloridrato per via orale ( inderal , astrazeneca reims , reims , cedex , francia ) unora prima della scansione per ridurre la frequenza cardiaca . 
nella nota informativa preliminare di preparazione allesame era stato consigliato un trattamento con 2040 mg due volte al giorno sotto controllo medico nei tre giorni precedenti lindagine per ridurre e regolarizzare la frequenza cardiaca nei pazienti con tachicardia . 
allintera popolazione stata , altres , somministrata una dose di 2 , 5 mg di diazepam ( tranquirit , aventis pharma , waterford , irlanda ) unora prima della scansione . 
un bolo di 100120 ml di mezzo di contrasto iodato non - ionico ( iomeprol , iomeron 400 , bracco , milano , italia ) , a seconda del range di scansione , stato iniettato con un usso di 5 ml / s mediante iniettore automatico ( stellant , medrad , pittsburgh , usa ) collegato ad unagocannula da 18 gauge , posizionata in una vena antecubitale destra . 
allo scopo di ottimizzare lenhancement intracoronarico la sincronizzazione dellinizio della scansione con il passaggio del bolo di mezzo di contrasto stata eseguita mediante bolus tracking , con una region of interest ( roi ) posizionata al livello dellaorta ascendente . 
i dati sono stati ricostruiti con tecnica retrospettiva in fase telediastolica ( dal 65% all80% dellintervallo r - r ) e telesistolica ( 40%45% )  . image and data analysis analisi delle immagini e dei dati the examinations were evaluated by two radiologists le indagini sono state valutate da due radiologi con esperadiol med ( 2011 ) 116 : 521531 with experience in mdct - ca [ 21 ] who transferred the data sets to a dedicated workstation ( extended brilliance workspace , version 3.0.1.3200 , philips medical systems ) for image postprocessing , with no information on patient history . 
data from the mdct - ca scans were analysed with the following postprocessing techniques : multiplanar reconstructions ( mpr ) , curved mpr ( cmpr ) , maximum intensity projections ( mip ) and volume rendering ( vr )  . 
the atherosclerotic coronary plaques were recognised as structures with a thickness of at least 1 mm2 , either within or adjacent to the coronary artery lumen and clearly distinguishable from the opaci ed lumen and the surrounding epicardial tissue [ 23 ]  . 
obstructive cad was de ned as the presence of plaque causing > 50% lumen reduction ; nonobstructive cad was de ned as the presence of plaque causing 50% lumen reduction . 
patients were therefore classi ed as belonging to the following groups based on the mdct - ca ndings : patients with normal coronary arteries , patients with nonobstructive cad and patients with obstructive cad . follow - up follow - up was carried out with an outpatient visit or by telephone , with additional checks in the archives of the pertinent centre . 
all patients were followed up for 2 years to record the following major cardiac events : cardiac death , myocardial infarction , hospitalisation and myocardial revascularisation procedures . statistical analysis patient characteristics were compared using the chi - squared test . 
statistical analysis was carried out with a dedicated software package ( spss 12.0 , spss , chicago , il , usa )  . rienza in ac - tcms [ 21 ] , che hanno caricato i set di dati in una workstation dedicata ( extended brilliance tm workspace , version 3.0.1.3200 , philips medical systems , cleveland , ohio , usa ) per il post - processing delle immagini , in assenza di notizie anamnestiche . 
tutti i dati delle scansioni ac - tcms sono stati analizzati mediante mezzi di postprocessing quali ricostruzioni multiplanari ( mpr ) , mpr curve ( cmpr ) , proiezioni massima intensit ( mip ) , e volume rendering ( vr )  . 
le placche aterosclerotiche coronariche sono state identi cate come strutture dello spessore di almeno 1 mm2 , entro o adiacenti il lume coronarico , chiaramente distinguibili dal lume opacizzato e dal circostante tessuto epicardico [ 23 ]  . 
la mac ostruttiva stata de nita dalla presenza di placche determinanti > 50% di stenosi del lume ; la mac non ostruttiva stata de nita dalla presenza di placche determinanti 50% di stenosi del lume . 
i pazienti sono stati pertanto classi cati come appartenenti ai seguenti gruppi sulla base dei risultati della ac - tcms : pazienti con coronarie normali , pazienti con mac non ostruttiva , pazienti con mac ostruttiva . follow - up il follow - up stato effettuato mediante visita ambulatoriale o contatto telefonico , con ulteriore veri ca negli archivi dellospedale di riferimento . 
tutti i pazienti sono stati inclusi in un follow - up di 2 anni con attenzione ai seguenti eventi cardiaci maggiori : morte cardiaca , infarto miocardico , ospedalizzazione ed eventuali interventi di rivascolarizzazione miocardica . analisi statistica le caratteristiche dei pazienti sono state confrontate mediante test del chi - quadrato . 
la valutazione statistica stata eseguita con software dedicato ( spss 12.0 , spss , chicago , iiiinois )  . results risultati a total of 1824 segments ( 97.3% ) were evaluated ; 51 sono stati complessivamente valutati 1824 segmenti ( 97 , 3% )  . 
alla ac - tcms evidente una stenosi > 50% ( testa di freccia ) del tratto prossimale della arteria discendente anteriore mediante immagini vr ( a , b ) e mappe coronariche ( c , d )  . 
mdct - ca identi ed 41 patients ( 32.8% ) with normal coronary arteries , 49 ( 39.2% ) with nonobstructive cad and 35 ( 28% ) with obstructive cad . 
single - vessel obstructive cad was found in 19 patients , two - vessel disease in ten and three - vessel disease in six , with a prevalence of disease in the proximal and mid segments of the coronary arteries ( 72% )  . 
la ac - tcms ha evidenziato 41 pazienti ( 32 , 8% ) con coronarie normali , 49 ( 39 , 2% ) con mac non ostruttiva e 35 ( 28% ) con mac ostruttiva . 
stata riscontrata mac ostruttiva monovasale in 19 pazienti , bivasale in 10 pazienti , trivasale in 6 pazienti , con prevalenza nei segmenti prossimali e medi delle arterie coronarie ( 72% )  . 
2 curve di kaplan - meier per tutti gli eventi in pazienti ( n = 125 ) con arterie coronarie normali , con mac non ostruttiva ed ostruttiva ( log rank test , p < 0 , 0001 )  . 
 variabile indipendente messa in evidenza dallanalisi multivariata ( hazard ratio [ hr ] 10 , 1393 , 95% intervallo di con denza [ ic ] 3 , 218931 , 9379 , p < 0 , 0001 )  . 
 other clinical applications of mdct - ca are the followup of coronary artery revascularisation procedures with aortocoronary bypass surgery [ 7 ] or stenting of proximal segments [ 8 ] and evaluating the coronary circulation as part of the planning of aortic valve surgery [ 9 ]  . 
consensus documents have been drawn up by panels of european and american experts that de ne the appropriateness and reliability of the clinical applications [ 10 , 11 ]  . 
 [ 14 ] presented a preliminary demonstration of how mdct - ca can provide independent prognostic that provided by conventional risk factors in predicting cardiac events in patients with suspected or known cad . 
in a study information superior la ac - tcms fornisce risultati eccellenti nella valutazione della patologia aterosclerotica coronarica con elevata sensibilit e notevole valore predittivo negativo , che suggeriscono un impiego della metodica per lesclusione e lidenti cazione della mac [ 15 ]  . 
si rileva , pertanto , un suo sempre maggior utilizzo nella pratica clinica per la valutazione di una sospetta aterosclerosi coronarica con valori di sensibilit e valore predittivo negativo rispettivamente del 97 , 6% e del 93 , 5% [ 6 ]  . 
altre applicazioni cliniche della ac - tcms sono il follow - up delle procedure di rivascolarizzazione coronarica mediante by - pass aorto - coronarico [ 7 ] e stenting dei segmenti prossimali [ 8 ] , nonch la valutazione del circolo coronarico in previsione di chirurgia della valvola aortica [ 9 ]  . 
consensus documents di panel di esperti europei ed americani hanno de nito lappropriatezza diagnostica delle applicazioni cliniche della metodica nel tentativo di delimitarne opportunamente il raggio di azione [ 10 , 11 ]  . 
tuttavia , una implementazione clinica di routine della metodica necessita di una valutazione del suo impatto prognostico a seconda delle speci che possibili indicazioni [ 12 , 13 ]  . 
 [ 14 ] hanno preliminarmente dimostrato come la ac - tcms sia radiol med ( 2011 ) 116 : 521531 done with a 16 - slice scanner and 12 months of follow - up , gilard et al . 
at the same time , electron - beam ct has demonstrated independent incremental value over the conventional risk factors in predicting mortality in patients with suspected cad over a lengthy follow - up period of 78 months [ 16 ]  . 
the presence of obstructive cad takes on a signi cant predictive value with regard to cardiac events in the follow - up . our study ts into this area of research , which still requires thorough investigation . 
we evaluated patients who underwent mdct - ca with precise clinical indications based on the applications laid down in the european and american reference documents [ 10 , 11 ]  . 
the ndings of our 24 months of follow - up are in agreement with previous studies and con rm the excellent prognostic outcome of mdct - ca in patients with healthy coronary arteries at mdct - ca , with independent additional value with respect to conventional risk factors ( table 2 )  . 
in agreement with other recent studies [ 24 , 25 ] , the technique provides an incremental value over cs , as obstructive cad was identi ed in some patients with cs = 0 . 
mdct - ca displays a high prognostic value in cases with the precise clinical indication for the technique and especially in patients at intermediate risk [ 11 , 26 ]  . our study provides no prognostic indications regarding the clinical application of mdct - ca in the follow - up of patients after myocardial revascularisation with stent deployment or aortocoronary bypass surgery [ 27 , 28 ]  . 
 mdct - ca is nonetheless limited in this setting ( stent diameter and material , vessel diameter , massive calci cations , metal clips ) and therefore requires selected clinical indications ( symptomatic patients with chest pain ) [ 10 , 11 ]  . 
prognostic considerations in this setting must take into account these intrinsic limitations , as well as the greater risk of major cardiac events in patients with known cad . the limitations of our study include the medium - term follow - up and the limited number of patients studied considering the heterogeneity of the clinical indications . 
the high prevalence of in grado di fornire informazioni prognostiche indipendenti e superiori rispetto a quelle dei convenzionali fattori di rischio nel predire gli eventi cardiaci in pazienti con sospetta o nota mac attraverso un follow - up a 12 mesi [ 14 ]  . 
 [ 15 ] hanno confermato che una ac - tcms negativa per mac in una popolazione di pazienti con sospetta malattia associata a ridotti tassi di mortalit ( 0% ) ed infarto miocardico ( 0 , 7% ) in un follow - up condotto a 12 mesi . 
 parallelamente la tc a fascio di elettroni ha mostrato un valore addizionale ed indipendente sopra i fattori di rischio convenzionali nel predire le cause di mortalit in pazienti con sospetta mac in un lungo follow - up condotto a 78 mesi [ 16 ]  . 
 [ 17 ] in un follow - up a 144 mesi di pazienti con sospetta o nota mac sottoposti ad ac - tcms a 64 strati hanno riscontrato un tasso di eventi cardiaci del 34% nei pazienti con mac , mentre nessun evento stato rilevato nei pazienti con coronarie normali . 
la presenza di mac ostruttiva assume invece un signi cativo valore predittivo riguardo eventi cardiaci nel follow - up . il nostro studio si inserisce in questo lone di letteratura non ancora estensivamente analizzato . 
 stata presa in considerazione una popolazione di pazienti sottoposta ad ac - tcms con precise indicazioni cliniche sulla base delle applicazioni suggerite dai documenti di riferimento europei ed americani [ 10 , 11 ]  . 
i risultati del nostro followup a 24 mesi sono in linea con gli studi precedenti e confermano leccellente outcome prognostico della ac - tcms in pazienti con coronarie indenni alla ac - tcms , con un valore addizionale indipendente rispetto a quello dei fattori di rischio convenzionali ( tabella 2 )  . 
in accordo con altre recenti esperienze [ 24 , 25 ] , la metodica presenta peraltro un valore incrementale sul cs , dal momento che in alcuni pazienti con cs = 0 stata identi cata mac ostruttiva . 
la ac - tcms trova rilevanti correlati prognostici nella precisa indicazione clinica allindagine ed in particolare nella popolazione a rischio intermedio [ 11 , 26 ]  . la nostra esperienza non fornisce indicazioni prognostiche circa lapplicazione clinica della ac - tcms nel follow - up dei pazienti sottoposti a rivascolarizzazione miocardica mediante posizionamento di stent o confezionamento di by - pass aorto - coronarico [ 27 , 28 ]  . 
la ac - tcms presenta comunque note limitazioni in tale contesto ( diametro e materiale degli stent , calibro dei vasi , massive calci cazioni , clips metalliche ) e , pertanto , richiede selezionate indicazioni cliniche ( pazienti sintomatici con dolore toracico ) [ 10 , 11 ]  . 
considerazioni prognostiche in tale ambito non possono prescindere da tali limitazioni intrinseche e dal rischio superiore di eventi cardiaci maggiori associati a pazienti con nota mac . nel novero delle limitazioni del nostro studio , possono essere inclusi il follow - up effettuato a medio termine ed il 530 radiol med ( 2011 ) 116 : 521531 disease identi ed with mdct - ca ( 67.2% ; nonobstructive cad 39.2% ; obstructive cad 28% ) could be interpreted as a study limitation , although this prevalence may be seen as the result of selecting a clinically justi ed population based on the examination appropriateness criteria . 
the rapid technical progress in multislice technology may further facilitate the use of the technique , especially with the implementation of prospective gating , which markedly reduces the radiation dose delivered [ 2931 ]  . conclusions ridotto numero di pazienti , in considerazione delleterogeneit delle indicazioni cliniche . 
una ulteriore limitazione pu essere costituita dallalta prevalenza di malattia identi cata con ac - tcms ( 67 , 2% ; mac non ostruttiva 39 , 2% , mac ostruttiva 28% ) , sebbene possa essere considerata risultato della selezione di una popolazione clinicamente giusti cata dai criteri di appropriatezza dellindagine . 
il rapido progresso tecnico della tecnologia multistrato potr ulteriormente facilitare lutilizzo clinico della metodica , specialmente con unimplementazione della cardio - sincronizzazione prospettica , che implica una netta riduzione della dose di radiazioni somministrata ai pazienti [ 2931 ]  . the use of mdct - ca in routine clinical practice on the basis of precise indications offers an excellent prognostic value in 2 years of follow - up in cases of normal coronary arteries ; the number of major cardiac events increases with increasing severity of the disease identi ed . 
national and multinational prognostic trials coupled with the adoption of new technological solutions aimed at signi cantly reducing the radiation dose delivered to the patient will further facilitate the clinical use of this technique . conclusioni la ac - tcms eseguita nella routine clinica sulla base di de nite indicazioni presenta un eccellente valore prognostico nel follow - up a 2 anni in caso di coronarie normali ; il numero di eventi cardiaci maggiori aumenta con la severit della patologia riscontrata . 
recall rate and cancer detection rate at rst reading or informed second reading only were assessed in a cohort of 23 , 629 women aged 5069 years screened during 20072008 . 
sono stati valutati il tasso di richiamo alla prima e alla sola seconda lettura informata osservati in una coorte di 23629 donne di et compresa fra 50 e 69 anni , sottoposte a screening durante gli anni 20072008 . 
il tasso di richiamo ad approfondimento stato del 13 , 0% alla prima e del 2 , 7% alla sola seconda lettura ( tasso incrementale di richiamo + 21 , 1% )  . 
il tasso diagnostico di carcinoma stato del 7 , 06 ( 167 casi ) alla prima lettura e dello 0 , 93 ( 22 casi ) alla sola seconda lettura ( tasso incrementale diagnostico di carcinoma + 13 , 1% )  . 
il costo dellaggiunta della seconda lettura stato di + 3 , 65 euro per soggetto sottoposto a screening , o 3.926 , 61 euro per carcinoma aggiuntivo diagnosticato . 576 radiol med ( 2011 ) 116 : 575583 gures are within the range of literature reports on doublereading performance . 
lo studio conferma lutilit della seconda lettura anche in aggiunta alla lettura diretta con approfondimento immediato : il tasso incrementale di richiamo risulta accettabile in considerazione del tasso incrementale diagnostico di carcinoma , con valori entrambi nel range di quanto riportato in letteratura in tema di doppia lettura . 
 parole chiave carcinoma mammario screening mammogra a doppia lettura introduction introduzione double reading has been shown to increase cancer detection rate compared with single reading , at the acceptable cost of a moderate increase in recall rate [ 13 ] , and has been recommended for many years as a standard procedure in mammography screening [ 4 ]  . 
the main features of the programme have been already reported [ 5 ] , as well as a favourable sensitivity estimate , based on the proportional incidence of interval cancers [ 6 ]  . 
double reading has not been adopted for several years on the assumption that immediate assessment , by allowing for an easier , and thus higher , recall rate of minor mammography abnormalities , would equally achieve a good sensitivity level [ 5 ]  . 
 la doppia lettura si dimostrata capace di aumentare il tasso diagnostico di carcinoma sospetto alla lettura singola , al costo di un accettabile aumento del tasso di richiamo [ 13 ] , e viene raccomandata da molti anni come procedura standard per lo screening mammogra co [ 4 ]  . 
le principali caratteristiche del programma sono gi state riportate [ 5 ] , cos come una stima favorevole della sensibilit basata sullincidenza proporzionale di carcinoma di intervallo [ 6 ]  . 
la doppia lettura non stata adottata dal programma per molti anni , in base alla convinzione che la lettura diretta con possibilit di approfondimento immediato , facilitando un maggior tasso di richiamo di anomalie mammogra che , consentisse una buona sensibilit , analoga alla doppia lettura differita [ 5 ]  . 
recentemente le autorit regionali hanno diffuso speci che raccomandazioni per ladozione di protocolli di screening omogenei a livello regionale e la doppia lettura quindi stata adottata a partire da marzo 2007 . 
il presente studio analizza i risultati delladozione di questa seconda lettura differita quando af ancata alla prima lettura diretta con approfondimento immediato . material and methods materiali e metodi during the study period , 23 , 629 women resident in the local health unit and aged 5069 years were invited and examined . 
real - time rst reading with immediate assessment was carried out by one radiologist at a rate of ten cases per hour ( corresponding to the workload of two radiographers ) for a total of 60 cases per 6 - h session . 
 cases with negative rst reading or positive rst reading durante il periodo di studio sono state invitate ed esaminate 23.629 donne residenti nellunit locale socio - sanitaria di et compresa fra 50 e 69 anni . 
la prima lettura diretta con eventuale approfondimento immediato stata condotta da un radiologo in ragione di 10 casi lora ( corrispondente al carico di lavoro di due tecnici di radiologia ) per un totale di 60 casi per sessione di 6 ore . 
le donne con prima lettura negativa o prima lettura radiol med ( 2011 ) 116 : 575583 and negative assessment were informed that second reading would follow and that a nal report would be sent by mail or that they would be recalled for further assessment . 
three radiologists were involved in reading in the study period : two were expert readers ( > 5 years experience in mammography reading ) and one had a lower level of experience ( 1 year )  . 
 all radiologists were involved in rst reading , whereas only the two more expert radiologists were involved in second reading . variables in the study database were patients age , ndings at rst reading and immediate assessment ( if any ) , ndings at second reading and delayed assessment ( if any ) and breast density currently reported according to breast imaging reporting and data system ( bi - rads ) d14 classi cation system [ 7 ]  . 
the nal outcome of immediate or delayed assessment , including excision histology ( if any ) , was available in all cases . the study evaluated incremental recall rate and incremental cancer detection rate at second reading ( cases recalled and cancers detected by the second reader only ) compared with rst reading with immediate assessment . 
assuming an average of 1 min of radiologists working time per case read at delayed second reading [ 8 ] , that is , at least 300 cases read per working session , it was estimated that 23 , 629 second readings could be easily managed within a radiologists yearly workload . 
the cost of incremental assessment [ unit cost of physical breast examination ( pbe ) , ultrasonography ( us ) , ne - needle aspiration cytology ( fnac ) , core needle biopsy ( cnb ) and surgical benign biopsies ] generated by second reading was calculated according to health service of cial tariffs , as previously reported [ 9 ]  . 
 positiva e approfondimento negativo sono state informate che sarebbe seguita una seconda lettura e che il risultato nale sarebbe stato inviato per posta o ci sarebbe stato un richiamo per ulteriore approfondimento . 
tre radiologi sono stati coinvolti nella lettura : due erano lettori esperti ( oltre 5 anni di pratica mammogra ca ) , uno era meno esperto ( 1 anno di pratica mammogra ca )  . 
 tutti i radiologi sono stati coinvolti nella prima lettura , mentre solo i due lettori pi esperti sono stati coinvolti nella seconda lettura . le variabili disponibili nel materiale in studio erano : et della paziente , risultato alla prima lettura e alleventuale approfondimento immediato , risultato alla seconda lettura e alleventuale ulteriore approfondimento , densit del seno ( classi cata secondo la classi cazione breast imaging reporting and data system [ bi - rads ] d1 - 4 [ 7 ] )  . 
il risultato nale delleventuale approfondimento immediato o differito , compresa listologia chirurgica , era disponibile in tutti i casi . lo studio ha valutato il tasso incrementale di richiamo e il tasso incrementale diagnostico di carcinoma generati dalla sola seconda lettura ( casi richiamati e cancri diagnosticati solo grazie alla seconda lettura ) confrontandoli con i risultati della lettura diretta con approfondimento immediato . 
i costi della seconda lettura sono stati equiparati al costo medio annuale di un radiologo ( dirigente di primo livello ) : assumendo un tempo di lettura medio per caso di 1 minuto alla lettura differita [ 8 ] , cio almeno 300 letture per turno giornaliero , si ritenuto che le 23.629 seconde letture potessero facilmente essere erogate in un tempo pari a un anno / radiologo . 
il costo incrementale dellapprofondimento diagnostico ( costo unitario di esame clinico [ ec ] , ecogra a [ us ] , citologia [ ne needle aspiration cytology , fnac ] , core biopsy [ ncb ] , e biopsie chirurgiche benigne ) generato dalla sola seconda lettura stato calcolato in base alle tariffe del servizio sanitario , come riportato in una precedente pubblicazione [ 9 ]  . 
 associata allet ( d3 - 4 per 5059 anni = 25 , 0% , per 6069 anni = 5 , 7% , p < 10 - 6 ) , anche se la frazione complessiva di soggetti con seno denso risultata relativamente bassa ( 17 , 1% )  . 
complessivamente sono stati diagnosticati 189 carcinomi ( 7 , 99 ) , 167 ( 7 , 06 ) alla prima lettura e 22 ( 0 , 93 ) alla sola seconda lettura . 
cost per incremental cancer detected ( n = 22 ) was 3 , 926.61. discussion this study is based on a large consecutive screening series , alla prima stato di + 13 , 1% . 
as previously reported in detail [ 5 ] , real - time reading with immediate assessment is a common procedure in clinical mammography practice but has been rarely adopted in screening , as the recommendation for double reading was issued and diffusely adopted . 
the main reason for this is the dif culty combining real - time rst reading with second reading the latter being necessarily delayed and managing the large workload required by screening , given the limitation in the number of cases screened per session when the immediate assessment approach is adopted . 
other minor negative aspects are represented by : ( a ) the need for a temporary negativity being reported to the woman after rst reading , whereas a de nitive report has to be delayed until second reading , and ( b ) the problematic but unlikely ( about 3% of cases ) event of a woman with a negative rst reading but recalled for a positive second reading : evident discrepancy between readers occasionally caused the rst reader to be blamed for inaccuracy or the second reader to be blamed for excess suspicion when assessment was negative . 
 as expected , the major determinant of recall at rst discussione questo studio si basa su una ampia casistica di screening che consente una valutazione af dabile del contributo della seconda lettura differita aggiunta ad un protocollo di lettura diretta con approfondimento immediato . 
come precedentemente riportato in dettaglio [ 5 ] , la lettura diretta con approfondimento immediato una procedura comune per la mammogra a clinica , ma stata raramente adottata nello screening , da quando la raccomandazione della doppia lettura stata diffusa e recepita . 
la ragione principale sta nella dif colt di combinare la lettura diretta con la seconda lettura , necessariamente differita , e di gestire il massiccio carico di lavoro imposto dallo screening , data la limitazione nel numero di casi esaminati per seduta quando si impieghi la lettura diretta con approfondimento immediato . 
altri aspetti negativi minori sono rappresentati da : ( a ) la necessit di comunicare alla donna una negativit temporanea dopo la prima lettura , mentre la risposta de nitiva rimandata al completamento della seconda lettura ; ( b ) levento problematico , ma raro ( circa il 3% dei casi ) di una donna con una prima lettura negativa , poi richiamata ad approfondimento diagnostico per una seconda lettura positiva . 
la discrepanza tra i due lettori risulta evidente ed ha occasionalmente causato rimostranza per linaccuratezza del primo lettore o , in caso di approfondimento negaradiol med ( 2011 ) 116 : 575583 reading was the presence of a dense breast . 
this is easily explained by the reader being less con dent in issuing a negative report in the presence of multiple superimposed opacities with ill - de ned , masked margins . 
breast density justi ed a higher recall rate in the younger decade , although such a difference was less marked due to the low overall prevalence of dense breasts in both considered age groups . 
such a low overall prevalence of dense breasts is not surprising , as the screening population was mostly postmenopausal , and this explains why most recalls occurred in d1 - 2 breasts . 
the possibility of immediate assessment favoured recall , which was in fact very high ( 13.0% ) , especially considering that most cases in the study series were at repeat screening , which is usually associated with lower recall rates ( reference european commission guidelines standard for repeat screening is < 5% )  . 
incremental recall rate at second reading was not particularly high ( + 2.7% ) , and the increase relative to rst reading ( + 21.1% ) was comparable with what has been previously reported [ 1 ] , although comparisons with the literature may be biased , as the relative increase depends on the high recall rate at rst reading . 
the rationale of facilitating and increasing recall rate to improve sensitivity is challenged by the observation of similar sensitivity estimates with quite different levels of recall rate [ 10 , 11 ] and denied by a study comparing screening practice in the us and uk , where it is quite evident that excess recall rates over recommended standards translate into higher costs but not into higher sensitivity [ 12 ]  . 
other studies investigating the optimal recall rate to improve sensitivity suggest that recall rates > 67% are unlikely to provide cost - effective extra bene ts for cancer detection [ 13 , 14 ]  . in this study , the relative incremental cancer detection rate by second reading ( + 13.1% ) was substantial , at the upper limit of what is commonly reported with conventional double reading [ 13 ]  . 
of course , the performance of rst or second reading as to cancer detection rate depends only on the procedure itself but also on the skill of the radiologists involved . 
it might be argued that in this study , the involvement of one reader with limited experience more likely to have a low sensitivity facilitated a better performance of second reading , but this does not seem to be the case , as a substantial contribution of second reading to cancer detection rate persists even after excluding cases in which the inexperienced reader was involved ( + 9% )  . the study con rms the ef cacy of second reading . 
the incremental recall rate ( + 21.1% ) is acceptable in view of the incremental cancer detection rate ( + 13.1% ) , and both gures are within the range of literature reports on tivo , di eccesso di sospetto per il secondo lettore . come atteso , lelemento che maggiormente in uenza il tasso di richiamo alla prima lettura la presenza di seno denso : questo si spiega facilmente per la minore tranquillit del radiologo nel rilasciare un referto negativo in presenza di opacit multiple sfumate e sovrapposte . 
la densit del seno giusti ca il maggior tasso di richiamo nella decade pi giovane , anche se la differenza per et meno marcata per la frequenza comunque bassa di seni densi nelle due classi di et considerate . 
una bassa frequenza di seni densi non sorprende , trattandosi di una popolazione prevalentemente in menopausa , e spiega perch comunque la maggior parte dei richiami avviene in donne con seno di densit d1 - 2 . 
la possibilit di eseguirlo immediatamente ha favorito il richiamo stesso alla prima lettura , con un tasso molto elevato ( 13 , 0% ) , specie tenendo conto che la maggioranza dei soggetti in studio era ad uno screening ripetuto , in genere associato ad un tasso di richiamo pi basso ( il valore di riferimento delle linee guida della comunit europea per lo screening ripetuto < 5% )  . 
il tasso di richiamo alla sola seconda lettura non stato particolarmente alto ( + 2 , 7% ) , e laumento relativo dei richiami rispetto alla prima lettura ( + 21 , 1% ) paragonabile a quanto riportato in altre esperienze [ 1 ] anche se il confronto con la letteratura discutibile , essendo laumento relativo in uenzato dallelevato tasso di richiamo alla prima lettura . 
il contenimento del tasso di richiamo resta un riferimento per la performance di screening : il razionale di facilitare e aumentare il tasso di richiamo per migliorare la sensibilit contraddetto dallosservazione di sensibilit paragonabili pur con tassi di richiamo molto diversi [ 10 , 11 ] : uno studio ha confrontato pratiche di screening negli usa e nel regno unito , dimostrando chiaramente che un eccesso di richiami oltre gli standard raccomandati si traduce in costi pi elevati , ma non in maggiore sensibilit [ 12 ]  . 
altre esperienze che hanno studiato il tasso di richiamo ottimale per migliorare la sensibilit suggeriscono che tassi di richiamo superiori al 6%7% abbiano poca probabilit di fornire vantaggi aggiuntivi e costo - ef caci quanto a tasso diagnostico di carcinoma [ 13 , 14 ]  . nel presente studio , il tasso incrementale diagnostico di carcinoma relativo alla sola seconda lettura ( + 13 , 1% ) stato piuttosto elevato , ai limiti superiori di quanto stato comunemente riportato in letteratura per la doppia lettura convenzionale [ 13 ]  . 
ovviamente la prestazione della prima o della seconda lettura quanto a tasso diagnostico di carcinoma non dipende solo dalla procedura di per s , ma anche dalla capacit dei radiologi coinvolti . 
si potrebbe osservare che nel presente studio linclusione tra gli addetti alla prima lettura di un radiologo meno esperto , pi esposto al rischio di una bassa sensibilit , potrebbe aver facilitato una buona performance della seconda lettura , ma non sembra che questo sia il caso dal momento che anche escludendo i casi in cui era coinvolto il lettore meno esperto il contributo della seconda lettura resta sostanziale ( + 9% )  . lo studio conferma lef cacia della seconda lettura . 
the positive predictive value at rst reading was 5.4% , largely inferior to recommended standards , and was due to a tendency for higher recall speci c to the readers , further magni ed by the opportunity for immediate assessment . 
the positive predictive value of recall at second reading only was 3.3% , comparable with that reported in other screening experiences with a much higher positive predictive value at rst reading [ 1 ]  . 
a tendency for cancers detected at second reading only to be depicted as isolated microcalci cations or distortions could be explained , as most suspicious presentations , such as irregular mass densities , were easily picked up at rst screening . 
although the sample size does not allow for statistical signi cance , cancers detected at second reading only were less advanced in stage , which adds further value to the bene ts of second reading in terms of incremental cancer detection rate . the direct cost of screening in the programme reported here has not yet been assessed in this study due to the complexity of such an evaluation . 
however , assuming a cost of 50100 per woman screened and of 67 , 000 per cancer detected in accordance to what has been estimated for other italian programmes when single reading was in use [ 15 ] , the incremental costs of 3.65 per woman screened and 3 , 926.61 per incremental cancer detected seem fully acceptable . in conclusion , our study ndings con rm the usefulness of second reading of screening mammograms and the limitations of single reading , even if read in real time with immediate assessment . 
as a consequence of these ndings , in this scenario , the real - time reading plus immediate assessment policy has been abandoned in favour of conventional delayed double reading , as in the rest of italian screening programmes . 
 bile in considerazione del tasso incrementale diagnostico di carcinoma ( + 13 , 1% ) ed entrambi questi valori sono compatibili con quanto riportato in letteratura sulle prestazioni della doppia lettura [ 13 ]  . 
il valore predittivo positivo alla prima lettura stato del 5 , 4% , ampiamente inferiore agli standard raccomandati , ed ascrivibile ad una maggiore tendenza al richiamo dei lettori coinvolti , ulteriormente potenziata dallopportunit dellapprofondimento immediato . 
il valore predittivo positivo alla sola seconda lettura stato del 3 , 3% , confrontabile con quanto riportato in altre esperienze che osservano un valore predittivo positivo pi elevato alla prima lettura [ 1 ] : una caduta sostanziale del valore predittivo positivo alla seconda lettura atteso , considerando la prevalenza pi bassa di carcinomi ancora disponibili per lidenti cazione dopo la prima lettura . 
 la tendenza dei carcinomi identi cati alla sola seconda lettura a apparire in forma di microcalci cazioni e distorsioni si pu spiegare con il fatto che le presentazioni pi sospette , come le densit di massa a margini irregolari , sono tendenzialmente identi cate alla prima lettura . 
anche se il campione in esame non consente di raggiungere la signi cativit statistica , i carcinomi identi cati alla sola seconda lettura risultano in stadio meno avanzato , il che aggiunge ulteriore valore ai bene ci della seconda lettura in termini di diagnosi aggiuntive di carcinoma . il costo diretto dello screening nel presente programma , dal momento che tale valutazione assai complessa , non stato possibile in questo studio e non disponibile un preciso riferimento con cui confrontare i costi della seconda lettura differita nello stesso scenario . 
rispetto a una assunzione approssimativa di 50100 euro per donna esaminata e di 60007000 euro per cancro diagnosticato , in analogia a quanto stato stimato per altri programmi italiani quando la lettura singola differita era ancora in uso [ 15 ] , il costo di 3 , 65 euro per donna esaminata e di 3926 , 61 euro per cancro aggiuntivo diagnosticato appaiono del tutto accettabili . in conclusione , i risultati del presente studio confermano lutilit della seconda lettura delle mammogra e di screening e segnalano i limiti della lettura singola , anche se refertata in diretta con eventuale approfondimento immediato . 
in seguito a questi risultati la pratica della lettura diretta con approfondimento immediato stata abbandonata in favore della doppia lettura differita convenzionale , protocollo comune al resto dei programmi di screening italiani . 
pansini 5 , 80131 napoli , italy 2istituto di bioimmagini e biostrutture ( c.n.r. ) , napoli , italy 3dipartimento di endocrinologia e oncologia molecolare e clinica , universit degli studi di napoli federico ii , napoli , italy 4dipartimento di medicina interna , clinica e sperimentale , seconda universit di napoli ( sun ) , napoli , italy 5u.o.c. 
radiol med 111 ( suppl 1 ) 2006 received : 1 april 2010 / accepted : 25 may 2010 / published online : 1 february 2011 springer - verlag 2011 abstract purpose . 
fourteen consecutive patients ( eight men and six women , aged 2654 years ) with men 1 underwent mdct performed with a 4 ( n = 5 ) or 64 ( n = 9 ) detector - row system and eus done with a radial transducer ( 7.520 mhz ) within 728 days of each other . 
 prior to mdct examination , patients were given 750 cc of water and asked to lie down in the right lateral decubitus for 15 mmultiphase mdct images were acquired both before and after the injection of nonionic iodinated contrast material ( 2 cc / kg ) at an injection rate of 4 ml / s , with technical parameters and scan delay varying in relation to the system used . 
quattordici pazienti consecutivi ( 8 maschi , 6 femmine ; 2654 anni ) affetti da men - 1 sono stati sottoposti sia a tcms , eseguita con 4 ( n = 5 ) o 64 le di detettori ( n = 9 ) , che ad eus eseguita con strumento radiale ( 7 , 520 mhz ) entro 728 giorni . 
gli esami tc sono stati acquisiti con tecnica multifasica previa distensione gastro - duodenale con acqua ed assunzione del decubito laterale destro ( 15 min ) , prima e dopo iniezione a bolo ( 4 ml / s ) di mezzo di contrasto ( mdc ) iodato idro - solubile non ionico ( 2 cc / kg ) con parametri tecnici e ritardi di scansione variabili in funzione del tipo di apparecchiatura . 
alla tcms sono state riscontrate 16 lesioni nodulari ( 518 mm ) a livello del pancreas ( 3 testa , 7 corpo e 6 coda ) e 9 ( 37 mm ) a carico della parete duodenale in 9 pazienti . 
our preliminary data indicate that mdct is complementary to eus in the identi cation of pets in men - 1 patients . 22 sono state identi cate prospetticamente ( 18 iper - , 4 ipo - ecogene o cistiche ) e 3 alla luce del dato endoscopico . 
la tcms rappresenta un utile complemento alleco - endoscopia nella identi cazione dei tepd . keywords multiple endocrine neoplasia ( men ) 1 pancreaticoduodenal endocrine tumours multidetector ct ( mdct ) endoscopic ultrasonography ( eus ) parole chiave multiple endocrine neoplasia ( men ) - 1 tumori endocrini duodeno - pancreatici tomogra a computerizzata multistrato ( tcms ) eco - endoscopia ( eus ) introduction introduzione multiple endocrine neoplasia type 1 ( men 1 ) , also known as wermer syndrome , is a rare autosomal dominant disorder with variable penetrance and clinical manifestations and a prevalence of around 220 cases every 100 , 000 individuals [ 1 ]  . 
the genetic alteration is located on the long arm of chromosome 11 where frameshift and nonsense mutations may be found prevalently at the level of hexons 2 , 7 , 9 and 10 [ 2 ]  . 
 patients affected by men 1 are therefore genetically predisposed to a series of functioning and nonfunctioning tumours arising from the parathyroid glands ( 9095% ) , the pituitary gland ( 2065% ) and the endocrine pancreas and duodenum ( 3080% ) [ 3 ]  . 
in most patients , the rst clinical manifestation is hyperparathyroidism ( hpt ) , which may be the result of hyperplasia of one or all four of the parathyroid glands or parathyroid hormone ( pth ) - secreting adenomas , possibly with ectopic location [ 4 ]  . 
although they are less frequent as a possible cause of clinical onset of the disease , pancreaticoduodenal endocrine tumours ( pets ) are nonetheless the leading cause of mortality , with a mean prevalence that has been increasing over the years and is currently around 6070% [ 5 ]  . 
this is quite likely due to the continuous re nement of imaging examinations and in particular the diffusion of endoscopic ultrasound ( eus ) , which is the technique of choice for the identi cation of these often subcentimetric lesions , especially in asymptomatic patients [ 68 ]  . 
although the treatment of choice for pets in men 1 is still debated , there is a general consensus on the importance of an early diagnosis , as malignancy potential is considered to be closely correlated with tumour size [ 9 ]  . 
where available , eus has proven to have a signi cant impact on the clinical management of these patients [ 10 ]  . the progress achieved with multidetector - row computed tomography ( mdct ) systems and in particular with the use of multiphase protocols has nonetheless markedly increased the diagnostic accuracy of ct in pet diagnosis [ 1114 ] , even la neoplasia endocrina multipla tipo 1 ( men - 1 ) , conosciuta anche come sindrome di wermer , una rara malattia ereditaria a trasmissione autosomica dominante , con penetranza ed espressivit clinica variabile ed una prevalenza di circa 220 casi ogni 100.000 individui [ 1 ]  . 
 lalterazione genetica stata localizzata sul braccio lungo del cromosoma 11 ove possibile riscontrare mutazioni frame - shift e nonsense a livello prevalentemente degli esoni 2 , 7 , 9 e 10 [ 2 ]  . 
i pazienti affetti da men - 1 risultano pertanto geneticamente predisposti ad una serie di lesioni neoplastiche , funzionanti e non funzionanti , a carico delle ghiandole paratiroidi ( 90%95% ) , della adenoipo si ( 20%65% ) , del pancreas endocrino e del duodeno ( 30%80% ) [ 3 ]  . 
nella maggior parte dei pazienti la prima manifestazione clinica rappresentata dalliperparatiroidismo ( ipp ) che pu essere sostenuto sia dalla iperplasia di una o di tutte e quattro le paratiroidi che dalla presenza di adenomi secernenti paratormone ( pth ) , talvolta in sede ectopica [ 4 ]  . 
sebbene meno frequenti come possibile causa di esordio clinico della malattia , i tumori endocrini pancreatico - duodenali ( tepd ) ne rappresentano tuttavia la principale causa di mortalit con una prevalenza media che andata aumentando nel corso degli anni e che attualmente si attesta intorno al 60%70% [ 5 ]  . 
ci verosimilmente in relazione con il continuo af namento delle indagini strumentali ed in particolare con la diffusione delleco - endoscopia che rappresenta la metodica di elezione per la identi cazione di tali lesioni che risultano di dimensioni spesso sub - centimetriche , specie nei pazienti asintomatici [ 68 ]  . 
nonostante il trattamento di scelta per i tepd nella men - 1 sia ancora dibattuto , c un generale consenso nellimportanza della diagnosi precoce di tali neoplasie la cui potenzialit maligna sarebbe strettamente correlata alle dimensioni [ 9 ]  . 
to our knowledge , there are no published works on the role of mdct in the detection of hereditary endocrine tumours , which often are nonfunctioning and as such render their clinical identi cation dif cult [ 8 ]  . the aim of our study was to assess the role of mdct performed with a multiphase protocol in pet screening in men - 1 patients and to compare the ndings with eus . materials and methods population between july 2004 and june 2009 , 14 consecutive patients ( eight men , six women ; mean age 4111 years ) with men 1 and belonging to seven different kindreds were referred to our centre . 
two had zollinger - ellison syndrome with gastrinaemia levels of 750 and 1 , 200 pg / ml ( normal value 080 ) , of whom one had already undergone total parathyroidectomy for primary hyperparathyroidism ( php )  . 
of the remaining 12 patients , three had undergone total parathyroidectomy following php , and eight had clinical and laboratory signs of hyperparathyroidism with pth values between 180 and 213 pg / ml ( normal value < 65 )  . study methods the patients underwent a total of 22 mdct examinations , all performed with a 4 - detector - row ( n = 8 ) or 64 - detectorrow ( n = 14 ) system , with a time interval between 9 and 18 months . 
prior to the mdct examination , patients were given 7501 , 000 cc water and asked to lie down in the right lateral decubitus for 15 min to achieve distension of the duodenum and facilitate the identi cation of lesions in the duodenal wall . 
in particular , mdct ( aquilion , toshiba , japan ) scans were acquired before , during and after the injection of 130150 cc ( 2 cc / kg ) of nonionic iodinated contrast material ( 370 mgi / ml , ultravist , bayer schering pharma , berlin germany ) at an injection rate of 4 ml / s , followed by saline solution ( 80 cc ) with an automatic dual - head injector ( stellant injection system , medrad inc , indianola , ia , usa )  . 
a three - phase protocol was performed with acquisitions in the arterial ( 3040 s ) , pancreatic ( 4555 s ) and portal ( 7080 s ) phases , with scan delay varying in relation to the duration of the injection and the mdct system used [ 19 ]  . 
examinations performed with a 4 - row system used a beam collimation of 3 4 mm dica di notevole impatto sulla gestione clinica di tali pazienti [ 10 ]  . 
 i progressi della tecnologia della tomogra a computerizzata multistrato ( tcms ) ed in particolare limpiego di protocolli multi - fasici hanno tuttavia notevolmente incrementato la accuratezza della tomogra a computerizzata ( tc ) nella diagnosi delle neoplasie del pancreas endocrino [ 1114 ] , anche se alcuni studi hanno sollevato problemi di ordine metodologico [ 14 ]  . 
tali lavori hanno tuttavia riguardato le forme sporadiche ed in particolare gli insulinomi e , al meglio della nostra conoscenza , non ci sono dati in letteratura sul ruolo della tcms nella identi cazione dei tumori endocrini eredo - familiari che risultano spesso non funzionanti ed in quanto tali di dif cile inquadramento clinico [ 8 ]  . scopo del presente studio stato quello di veri care il ruolo della tcms eseguita con tecnica multi - fasica nello screening dei tepd nella men - 1 e di confrontarne i risultati con quelli delleco - endoscopia . materiali e metodi popolazione nel periodo compreso tra luglio 2004 e giugno 2009 sono pervenuti alla nostra osservazione 14 pazienti ( 8 maschi , 6 femmine ; et media 4111 anni ) consecutivi affetti da men - 1 ed appartenenti a 7 diversi ceppi familiari . 
due pazienti erano affetti da sindrome di zollingerellison con valori di gastrinemia di 750 e 1200 pg / ml ( valori normali [ vn ] = 080 ) , di cui uno gi sottoposto a para - tiroidectomia totale per iperparatiroidismo primitivo ( ipp )  . 
dei restanti dodici pazienti , tre erano stati anchessi gi sottoposti a para - tiroidectomia totale per ipp e 8 avevano segni clinici e bio - umorali di iperparatiroidismo con valori di pth compresi tra 180 e 213 pg / ml ( vn < 65 )  . 
 metodiche di studio i pazienti sono stati sottoposti ad un totale di 22 esami tc , tutti eseguiti con tecnologia multistrato con 4 ( n = 8 ) o 64 ( n = 14 ) le di detettori ad intervalli temporali compresi tra i 9 ed i 18 mesi . 
 gli esami tc sono stati tutti eseguiti previa somministrazione orale di acqua ( 7501000 cc ) ed assunzione del decubito laterale destro ( 15 min ) per favorire la distensione dellansa duodenale e facilitare il rilievo di eventuali lesioni a carico della parete duodenale . 
examinations performed with a 64 - row system used a beam collimation of 1 32 mm for the arterial and pancreatic phases , both limited to the pancreatic area ( t11l3 ) and acquired with 25 - cm fov , and 2 16 mm for the baseline and portal phase scans , both with a 35 - cm fov extended to the upper abdomen , and all acquired with 0.75 s gantry rotation speed , 120 kev , 0.87 pitch and automatic dose modulation . 
once the endoscope had been introduced into the gastric lumen , any air or liquid present was aspirated , and the sonographic exploration of the pancreatic body and tail was performed with the transducer in contact with the gastric wall . 
 once the pancreatic study was complete , the duodenal wall was assessed using a frequency of 20 mhz to obtain higher resolution . image analysis mdct images were analysed at a dedicated workstation ( merlino enteprise , rem ) , both prospectively and in light of eus ndings . 
eus criteria for diagnosing endocrine lesions of the pancreas and / or duodenum included identifying hypoechoic nodular areas with homogeneous echotexture , oval or round in shape and with wellde ned margins , located in the pancreatic parenchyma or the second or third layer of the duodenal wall , corresponding to the deep portion of the mucosa and submucosa , respectively . 
 more rarely , endocrine tumours may appear as isoechoic lesions bounded by a hypoechoic rim [ 15 ]  . reference standard in ve patients , ne - needle aspiration was performed on colare , la tcms ( aquilion , toshiba , giappone ) stata eseguita prima , durante e dopo iniezione a bolo ( 4 ml / s ) di 130150 cc ( 2 cc / kg ) di mezzo di contrasto ( mdc ) iodato idro - solubile non - ionico ( 370 mg / i / ml , ultravist , bayer schering pharma , berlin , germany ) seguita da soluzione siologica ( 80 cc ) mediante iniettore automatico a doppia siringa ( stellant injection system , medrad inc . , indianola , usa )  . 
stato eseguito un protocollo trifasico con acquisizioni in fase arteriosa ( 3040 s ) , pancreatica ( 4555 s ) e portale ( 7080 s ) , con ritardi di scansione variabili in funzione della durata delliniezione e del tipo di apparecchiatura [ 19 ]  . 
negli esami eseguiti con tc multistrato ( 4 ) stata impiegata una collimazione del fascio di 34 mm per le fasi arteriosa e pancreatica , entrambe limitate alla loggia pancreatica ( d11 - l3 ) con campo di vista ( fov ) di 25 cm , e 54 mm per la fasi pre - contrastogra ca e portale , entrambe con fov di 35 cm ed estese allintero addome superiore , con correnti al tubo di 350 e 250 mas , rispettivamente , 120 kev , tempo di rotazione di 0 , 5 s , beam pitch 0 , 75 . 
con la tc multistrato con 64 le di detettori la collimazione del fascio impiegata stata di 132 mm per le fasi arteriosa e pancreatica , entrambe limitate alla loggia pancreatica ( d11 - l3 ) ed acquisite con fov di 25 cm e di 216 mm per le fasi pre - contrastogra ca e portale , entrambe estese allintero addome superiore con fov di 35 cm , tutte acquisite con tempo di rotazione di 0 , 75 sec , 120 kev , beam pitch 0 , 87 e modulazione automatica della dose . 
una volta introdotto lo strumento nel lume gastrico si provveduto ad aspirare laria ed il liquido eventualmente presente e si condotta lesplorazione ultrasonogra ca del corpo e della coda del pancreas ponendo il trasduttore a contatto con la parete gastrica . 
per il confronto con i dati delleco - endoscopia sono stati alla ne selezionati 14 esami tc di cui 7 eseguiti con tcms aquilion 4 e 7 con tcms aquilion 64 . 
sono state valutate le sole radiol med ( 2011 ) 116 : 595606 ve lesions ( one duodenal , four pancreatic ) with a 22 - gauge needle , the results of which con rmed their neuroendocrine nature . 
in the remaining nine patients , the eus study was considered the reference standard . results adequate distension of the duodenum was obtained in 11 of the 14 patients examined ( 78% )  . 
in that location , as many as nine nodules were identi ed , of which ve were in the same patient , all hypervascular and between 3 and 7 mnodular formations with similar attenuation but larger in size ( 518 mm ) were also identi ed in the pancreatic parenchyma , i.e. 
i criteri eco - endoscopici per la diagnosi di lesioni endocrine del pancreas e / o del duodeno sono rappresentati dallidenti cazione di aree nodulari ipoecogene ad eco - tessitura omogenea , di morfologia ovalare o rotondeggiante ed a margini netti , situate nel contesto del parenchima pancreatico o a livello del ii o iii strato della parete duodenale , corrispondenti rispettivamente alla porzione profonda della mucosa ed alla sotto - mucosa . 
pi raramente i tumori endocrini possono apparire come lesioni isoecogene delimitate da orletto ipoecogeno [ 15 ]  . gold standard in cinque pazienti altrettante lesioni , duodenali ( 1 ) e pancreatiche ( 4 ) , sono state sottoposte ad ago - aspirazione ( fna ) con ago sottile ( 22 g ) che ne ha confermato la natura neuro - endocrina . 
in particolare , 16 formazioni nodulari ( 518 mm ) sono table 1 multidetector - row computed tomography ( mdct ) in patients with multiple endocrine neoplasia type 1 ( men 1 )  . 
numero , dimensioni e topogra a delle lesioni nodulari identi cate prospetticamente e retrospettivamente in 9 pazienti lesioni nodulari ( n = 25 ; 318 mm ) duodeno ( 37 mm ) testa ( 818 mm ) corpo ( 514 mm ) coda ( 610 mm ) 600 radiol med ( 2011 ) 116 : 595606 fig . 
1a , b four - row multidetector - row computed tomography ( mdct ) and endoscopic ultrasound ( eus ) in multiple endocrine neoplasia type 1 ( men 1 )  . 
alla tc ( a ) , acquisita in fase arteriosa , focale bozzatura del margine ghiandolare anteriore si apprezza a carico del corpo pancreatico ( testa di freccia ) in assenza di alterazioni densitometriche . 
2a , b four - row multidetector - row computed tomography ( mdct ) and endoscopic ultrasound ( eus ) in multiple endocrine neoplasia type 1 ( men 1 )  . 
alla tc ( a ) acquisita in fase arteriosa piccola area nodulare di anomala impregnazione ( testa di freccia ) si apprezza a carico della parete della ii porzione duodenale . 
leco - endoscopia ( b ) documenta a tale livello piccola ( 5 , 2 mm ) formazione nodulare sotto - mucosa ipo - ecogena . detectable in the arterial phase , as the quantitative analysis for the three lesions > 1 cm shows , with absolute attenuation values of 1904 hu and differential values with respect to the surrounding parenchyma of 5912 hu for the arterial phase and 219 hu for the pancreatic phase . 
four ( 16% ) pancreatic lesions had a hypoattenuating ( n = 1 ) or frankly state riscontrate a livello del pancreas , di cui 3 alla testa , 7 al corpo e 6 alla coda , e 9 ( 37 mm ) a carico dellansa duodenale , di cui 1 al bulbo , 4 al ginocchio inferiore , 2 al ginocchio superiore , 1 alla parete mediale della seconda porzione , 1 alla terza porzione duodenale ( tabella 1 )  . 
arterial - phase mdct ( a ) shows a small ( 5 mm ) nodular area of abnormal enhancement on the anterior margin of the pancreatic body , which is still evident but less conspicuous in the pancreatic phase ( b )  . 
in a ben evidente una piccola ( 5 mm ) formazione nodulare ipervascolarizzata a livello del corpo pancreatico che risulta ancora apprezzabile , ma meno evidente , in fase pancreatica ( b )  . 
arterial - phase mdct ( a ) shows a nodular ( 9 mm ) area of abnormal enhancement on the anterior margin of the pancreatic tail ( arrowhead ) , which becomes isoattenuating to the surrounding parenchyma and as such is no longer appreciable in the pancreatic phase ( b )  . 
in a ben evidente formazione nodulare ( 9 mm ) ipervascolarizzata a carico del margine anteriore della coda ghiandolare ( testa di freccia ) che non risulta pi apprezzabile in fase pancreatica in quanto isodensa al circostante parenchima ( b )  . 
la lesione non stata evidenziata dalla eco - endoscopia . cystic ( n = 3 ) appearance and as such were better appreciable in the pancreatic phase ( table 2 )  . the eus study , instead , identi ed 32 nodular pets ( 218 mm ) in 11 patients . 
dei 25 tumori pancreatico - duodenali , 22 sono stati identicati prospetticamente e 3 alla sola luce del dato endoscopico , di cui due isodensi al circostante parenchima ed uno ipodenso . 
in particolare , una formazione nodulare di 12 mm a carico del corpo pancreatico stata identi cata solo retrospettivamente grazie alla focale bozzatura da 602 radiol med ( 2011 ) 116 : 595606 table 2 multidetector - row computed tomography ( mdct ) in patients with multiple endocrine neoplasia type 1 ( men 1 )  . 
number , location and attenuation characteristics of the nodular lesions identi ed prospectively in nine patients nodular lesions ( n = 22 ; 318 mm ) duodenum head body tail tabella 2 tcms nei pazienti con men - 1 . 
numero , dimensioni , sede ed aspetto delle lesioni nodulari identi cate dalla eco - endoscopia in 11 pazienti lesioni nodulari ( n = 32 ; 218 mm ) duodeno ( 212 mm ) testa ( 518 mm ) corpo ( 813 mm ) coda ( 8 mm ) table 3 endoscopic ultrasound ( eus ) in patients with multiple endocrine neoplasia type 1 ( men 1 )  . 
number , size , location and appearance of the nodular lesions identi ed by eus in 11 patients nodular lesions ( n = 32 ; 218 mm ) duodenum ( 212 mm ) head ( 518 mm ) body ( 813 mm ) tail ( 8 mm ) located in the pancreas and 13 in the duodenal wall ; three appeared isoechoic ( 10% ) , of which two were located in the duodenal wall and one in the pancreas ( table 3 )  . 
pets , on the other hand , arise in 6070% of patients with men 1 [ 5 ] , and in 50% of cases , they constitute the clinical onset of the disease [ 16 ]  . 
 le suddette formazioni nodulari sono risultate meglio apprezzabili nella fase arteriosa di studio come supportato dallanalisi quantitativa eseguita per le sole tre formazioni di dimensioni > 1 cm che ha evidenziato valori densitometrici assoluti di 1904 uh e valori differenziali rispetto al circostante parenchima pancreatico di 5912 uh per la fase arteriosa e 219 uh per la fase radiol med ( 2011 ) 116 : 595606 fig . 
5a - c four - row multidetector - row computed tomography ( mdct ) ( a , b ) and endoscopic ultrasound ( eus ) ( c ) in multiple endocrine neoplasia type 1 ( men 1 )  . 
quattro ( 16% ) lesioni pancreatiche risultavano di aspetto ipodenso ( 1 ) o francamente cistico ( 3 ) e come tali meglio apprezzabili nella fase pancreatica ( tabella 2 )  . allesame eco - endoscopico sono state evidenziate invece 32 lesioni nodulari pancreatico - duodenali ( 218 mm ) in 11 pazienti ( 78 , 5% )  . 
in particolare , per i gastrinomi stato dimostrato che lincidenza di metastasi epatiche direttamente correlata alle dimensioni della lesione e risulta signi cativamente pi elevata per lesioni di dimensioni > 2 cm [ 5 ]  . assume pertanto particolare signi cato il monitoraggio di tali lesioni per la cui identi cazione la eco - endoscopia rappresenta sicuramente la metodica pi accurata con una sensibilit del 93%95% [ 10 , 12 ] che ben si correla con i risultati ottenibili con la palpazione bi - manuale del pancreas e risulta solo di poco inferiore a quelli riportati dallecogra a intra - operatoria ( 96 , 4% ) [ 12 ]  . 
 la diffusione della tecnologia multistrato in tc ha tuttavia considerevolmente incrementato la accuratezza della metodica nella diagnosi delle neoplasie endocrine del pancreas grazie alla possibilit di eseguire studi multi604 radiol med ( 2011 ) 116 : 595606 associated with the disease . 
in particular , it has been demonstrated for gastrinomas that the incidence of liver metastases is directly correlated with the lesion size and is signi cantly higher for lesions > 2 cm [ 5 ]  . monitoring these lesions becomes particularly important , and eus is undoubtedly the most accurate technique for their identi cation , with a sensitivity of 9395% [ 10 , 12 ] , which correlates well with the results obtained with bimanual palpation of the pancreas and is only a little lower than intraoperative us ( 96.4% ) [ 12 ]  . 
 the increasing diffusion of mdct systems has nonetheless considerably increased the accuracy of this technique in diagnosing endocrine tumours of the pancreas , thanks to the possibility of performing multiphase studies of the upper abdomen with collimations in the order of 13 mm [ 1114 ]  . 
in our experience , however , mdct proved to be complementary to eus in that it identi ed ve nodular formations of the pancreatic tail that were not detected by eus ( tables 1 , 2 )  . 
this small number does not allow solid conclusions to be drawn regarding the role of mdct , but these observations are supported in the literature , where the lower sensitivity of eus in the exploration of the pancreatic tail when using a radial transducer has been highlighted [ 17 ]  . 
the quantitative analysis performed on the three lesions > 1 cm also displayed higher attenuation differences compared with the surrounding parenchyma in the arterial phase rather than in the portal phase . 
this is in disagreement with the most recent ndings in the literature , according to which pets are said to be better appreciated in the pancreatic phase [ 14 ]  . 
in particular , in our study , the bolus - tracking technique was not used ; rather , a variable scan delay was implemented in relation to the injection rate and the mdct system used , considering a mean transit time of 10 s [ 19 ]  . 
our ndings are also supported by the experience of other authors , who claimed the importance of the arterial phase in the identi cation of endocrine tumours , although sporadic [ 11 , 12 ]  . 
nella nostra esperienza la tcms si tuttavia rivelata complementare alleco - endoscopia avendo identi cato cinque formazioni nodulari a carico della coda pancreatica non rilevate dallesame eco - endoscopico ( tabelle 1 e 2 )  . 
lesiguit di tali numeri non consente di trarre considerazioni conclusive sul ruolo della tcms ma tali osservazioni trovano un riscontro nella letteratura nella quale sottolineata la minore sensibilit della eco - endoscopia nella esplorazione della coda pancreatica quando si utilizza una sonda radiale [ 17 ]  . 
anche lanalisi quantitativa eseguita per le sole tre lesioni di dimensioni sovra - centimetriche ha rivelato valori densitometrici differenziali con il circostante parenchima pancreatico maggiori nella fase arteriosa che in quella portale . 
in particolare , nel presente studio non si fatto ricorso al bolus tracking ma si utilizzato un ritardo di scansione variabile in funzione della durata delliniezione e del tipo di apparecchiatura considerando un tempo medio di transito di 10 s [ 19 ]  . 
nonostante un tale approccio empirico non possa ritenersi rigoroso da un punto di vista metodologico esso trova ampio supporto in letteratura [ 20 ] ed appare giusti cato dalla et media dei nostri pazienti ( 40 , 611 anni )  . 
i nostri dati sono inoltre supportati dallesperienza di altri autori che sostengono limportanza della fase arteriosa nelle identi cazione dei tumori endocrini anche se sporadici [ 11 , 12 ]  . 
 [ 11 ] il 94% ( 18 / 19 ) degli insulinomi identi cati dalla tcms risultato essere ipervascolarizzato e come tale meglio apprezzabile nella fase arteriosa [ 11 ]  . 
per le loro caratteristiche densitometriche tali lesioni sono risulradiol med ( 2011 ) 116 : 595606 al . , 94% ( 18 / 19 ) of insulinomas identi ed by mdct were hypervascular and as such were better appreciated in the arterial phase [ 11 ]  . 
in our study , the combined use of mdct and eus made it possible to identify a total of 37 pets in 11 patients with an incidence of 3.4 lesions per patient and a prevalence of 78.5% , which is in agreement with the most recent data in the literature [ 6 ]  . 
with the exception of the ve lesions subjected to ne - needle aspiration , we do not know which of the lesions identi ed by one or both techniques are in fact endocrine tumours . 
the combined use of the two techniques permitted identi cation of 100% ( 18 / 18 ) of pancreatic insulinomas , all of which were subjected to surgical excision [ 12 ]  . 
this is , however , largely justi ed by the subcentimetric and paracentimetric size of the lesions identi ed , for which the current guidelines recommend only clinical and imaging follow - up [ 9 ]  . 
in fact , no bene t is expected from the surgical treatment of lesions of those dimensions in patients with men 1 , and there is a general consensus to reserve the surgical option only for lesions > 2 cm , for which a higher incidence of liver metastases is reported [ 22 ]  . in conclusion , the results of our study indicate a complementary role of multiphase mdct and eus in the identi cation of hereditary endocrine tumours . tate tutte meglio apprezzabili nella fase pancreatica . 
nel presente studio limpiego combinato della tcms e della eco - endoscopia ha pertanto consentito di identi care un totale di 37 lesioni pancreatico - duodenali in 11 pazienti con una incidenza di circa 3 , 4 lesioni / paziente ed una prevalenza del 78 , 5% che in accordo con i pi recenti dati della letteratura [ 6 ]  . 
 [ 12 ] limpiego combinato delle due metodiche ha consentito di identi care il 100% ( 18 / 18 ) di insulinomi pancreatici , tutti sottoposti ad escissione chirurgica [ 12 ]  . 
nel presente studio non stato possibile confrontarsi con i riscontri anatomo - patologici e ci ne rappresenta il limite principale , in gran parte giusti cato dalle dimensioni sube para - centimetriche delle lesioni identi cate , per le quali le attuali linee guida prevedono il solo follow - up clinico - strumentale [ 9 ]  . 
 non infatti atteso alcun bene cio nel trattamento chirurgico di lesioni di tali dimensioni nei pazienti con men - 1 e c un generale consenso nel riservare lopzione chirurgica solo alle lesioni di dimensioni > 2 cm per le quali sole riportata una maggiore incidenza di metastasi epatiche [ 22 ]  . in conclusione , i risultati del presente studio indicano un ruolo complementare della tcms multifasica e della eco - endoscopia nella identi cazione dei tumori endocrini eredo - familiari . 
del maschio1 1radiology department , vita - salute university , san raffaele scienti c institute , via olgettina 60 , 20132 milan , italy 2nuclear medicine department , vita - salute university , san raffaele scienti c institute , milan , italy 3surgical department , vita - salute university , san raffaele scienti c institute , milan , italy 4pathology department , vita - salute university , san raffaele scienti c institute , milan , italy correspondence to : e . 
allinterno della lesione sono stati iniettati macro - aggregati di albumina umana marcata con tecnezio99 ; nel caso di guida ecogra ca ( 221 / 288 soggetti ) , linoculo ha prodotto iperecogenicit in corrispondenza della lesione ; nel caso di guida mammogra ca ( 67 / 288 soggetti ) , dopo il radiotracciante stato inoculato mezzo di contrasto iodato per il successivo controllo mammogra co . 
la guida radiologica nella roll ha garantito lesito della localizzazione e rimozione di lesioni mammarie singole non palpabili nella maggioranza dei casi . radiol med ( 2011 ) 116 : 564574 keywords breast lesion radioguided localisation mammography ultrasonography parole chiave lesione mammaria localizzazione radioguidata mammogra a ecogra a introduction introduzione a variety of preoperative localisation techniques for nonpalpable breast lesions has been described in the literature : hypodermic needles , wires ( the most widely accepted technique ) , carbon solution , methylene blue dye [ 110 ]  . 
radioguided occult lesion localisation ( roll ) , introduced in 1998 , involves the inoculation of macroaggregates of human serum albumin labelled with radioactive technetium99 ( 99mtc ) directly into the site of a nonpalpable breast lesion , with mammography ( mx ) or ultrasound ( us ) guidance . 
after radiotracer injection , a gamma - detecting probe ( gdp ) was used to locate the lesion during surgery , allowing the surgeon to evaluate its skin projection and decide the best approach with an acceptable cosmetic outcome [ 15 , 16 ]  . 
the same group that described roll for the rst time compared this technique with wire localisation and concluded that roll was rapid , easy and accurate , with reduced excision volume and better lesion centring [ 17 ] , and also safe in terms of radiation exposure [ 18 ]  . following this experience , other groups tested roll alone or in combination with the sentinel node ( sn ) procedure in different clinical settings [ 1922 ]  . 
prospective randomised trials compared roll with wire - guided lumpectomy and concluded that roll was as effective as wire for localising and excising breast lesions , was simple , reduced localisation time and pathologically positive margins and gave better cosmetic results [ 2326 ]  . 
 the aim of this prospective study was to evaluate the methods , technical aspects and impact of patient - tailored preoperative radiological guidance in roll in a large series of women with single , nonpalpable breast lesions . 
 materials and methods study population we evaluated 288 women ( mean age 57.5 years ; range 3288 years ) with single , nonpalpable breast lesions who were sono state descritte in letteratura varie tecniche di localizzazione preoperatoria di lesioni mammarie non palpabili : ago ipodermico , lo guida ( la tecnica pi largamente accettata ) , soluzione di carbone , blu di metilene [ 110 ]  . 
la radioguided occult lesion localization ( roll ) , introdotta nel 1998 , prevede linoculo di macro - aggregati di albumina umana marcata con tecnezio99 radioattivo ( 99mtc ) direttamente nella sede della lesione mammaria non palpabile , con guida mammogra ca o ecogra ca . 
dopo liniezione del radiotracciante , viene utilizzata una sonda per la rilevazione dei raggi gamma ( gdp ) al ne di localizzare la lesione sul tavolo operatorio , permettendo cos al chirurgo di valutarne la proiezione cutanea e di decidere il miglior approccio operatorio , con un buon risultato cosmetico [ 15 , 16 ]  . 
lo stesso gruppo che ha descritto la roll per la prima volta ha poi paragonato questa tecnica con quella del lo guida ed ha concluso che la roll era rapida , facile ed accurata , con ridotto volume di tessuto escisso e migliore centratura della lesione [ 17 ] , ed era inoltre sicura in termini di radioesposizione [ 18 ]  . a seguito di questa esperienza , altri gruppi hanno valutato la roll in differenti situazioni cliniche , anche combinata con la procedura del linfonodo sentinella ( sn ) [ 1922 ]  . 
studi prospettici randomizzati hanno paragonato la roll alla nodulectomia con lo guida ed hanno concluso che la roll era ef cace quanto il lo guida per la localizzazione ed escissione di lesioni mammarie , riduceva il tempo per la localizzazione e la positivit dei margini , e dava migliori risultati cosmetici [ 2326 ]  . 
la maggioranza degli articoli internazionali ha posto lattenzione sulla valutazione della roll dal punto di vista chirurgico , con particolare attenzione alla durata dellintervento , alla scelta dellaccesso , al risultato cosmetico . 
 relativamente poca attenzione stata rivolta al ruolo della guida radiologica con mammogra a ( mx ) e ecogra a ( us ) nella roll . lo scopo di questo studio prospettico stato quello di valutare i metodi , gli aspetti tecnici e limportanza della guida radiologica nella roll , personalizzata per ogni paziente , su ampia casistica di donne con lesione mammaria singola non palpabile . 
when the lesion was detected exclusively or also by us ( 221 / 288 lesions , 76.7% ) , us - guided ne - needle aspiration cytology ( fnac ) or core biopsy ( cb ) were performed . 
 when the lesion was detected by mx only ( 67 / 288 lesions , 23.3% ) and appeared as a group of suspicious microcalcications , it underwent mx - guided stereotactic vacuumassisted biopsy ( vab )  . 
se la lesione era stata evidenziata esclusivamente o anche con us ( 221 / 288 lesioni , 76 , 7% ) , erano state eseguite citologia con ago sottile ( fnac ) o core biopsy ( cb ) sotto guida us . 
se la lesione era stata evidenziata esclusivamente con mx ( 67 / 288 lesioni , 23 , 3% ) e aveva laspetto di un gruppo di microcalci cazioni , esse erano state sottoposte a biopsia vacuum - assisted ( vab ) sotto guida mx . 
i criteri di esclusione dalla roll sono stati evidenza di malattia mammaria multicentrica e stato di gravidanza . radiocomposto e tecniche di medicina nucleare il radiocomposto iniettato consistito in 0 , 10 , 2 cc di macro - aggregati di albumina umana marcati con 3 , 77 , 4 mega - bequerel ( mbq ) di 99mtc . 
questa quantit corrispondeva ad una dose di 100200 micro - curie ( ci ) ed era stata calcolata sulla base della stima del tempo medio intercorrente fra linoculo del radiocomposto e la procedura chirurgica . 
la dimensione dei macro - aggregati variata da 10 a 150 micron ( m ) e , a causa della loro dimensione , la maggioranza delle particelle rimasta allinterno della lesione e non stata diffusibile come le particelle usate per la procedura sn , che ha potuto essere eseguita insieme alla roll senza interferenza [ 21 , 2729 ]  . 
in queste pazienti , sono state iniettate in sede intradermica , nellarea peritumorale , particelle nanocolloidi di albumina umana marcate con 14 , 8 mbq di 99mtc ( dose 400 ci )  . 
la dimensione dei nanocolloidi stata minore di 80 m e ci , come gi menzionato , ha permesso che esse si diffondessero nel sistema linfatico e raggiungessero il sn ascellare . 
dopo liniezione del radiotracciante per la roll , la paziente stata sottoposta a scintigra a biplanare ( valutata da c.c. , con 8 anni di esperienza ) per veri care la corretta localizzazione del 99mtc . 
in these cases , craniocaudal and mediolateral mammographic views were acquired to localise the lesion properly ( senographe essential and senographe dmr + , ge medical systems , cedex , france )  . 
the radiologist decided the best approach to the lesion ( frontal , lateral or medial ) and asked the patient to sit on a chair with arms along the body . 
the position of the 22 g needle , which should be inside the lesion or within 1 cm , was then checked with other orthogonal mammograms ; this was done more than once if the tip of the needle was not in the correct position , which can happen , especially in the case of small clusters of microcalci cations and in large breasts . 
 finally , the nuclear physician inserted on the needle the rst insulin syringe lled with 0.10.2 cc of radioactive tracer , and the radiologist inserted a second syringe with 1 cc of iodinated contrast medium ( iopamiro , iopamidol , bracco , italy )  . 
the cost of the roll procedure , including the biplanar scintigraphy and excluding the surgical intervention , was 250300 euro on average . surgical procedure in the operating room ( or ) , a gdp ( 15 lvr , pol.hi.tech , carsoli , italy ) was held by the surgeon to guide lesion removal . 
the pathological specimen was sent to the radiology department where the presence of the suspicious lesion was checked with us or mx , depending on the radiological guidance technique used previously . 
in questi casi , con la paziente supina e con le braccia estese sopra il capo , il radiologo ha localizzato la lesione ed ha posizionato un ago ipodermico 22 gauge ( g ) direttamente allinterno della lesione o presso di essa ( quando la lesione era troppo dura per iniettare liquido ) sotto guida us real - time eseguita con una sonda lineare da 12 mhz ( technos mp , esaote , genova , italia )  . 
nelle 67 su 288 pazienti ( 23 , 3% ) che erano state sottoposte a biopsia stereotassica mx - guidata preoperatoria , la procedura di localizzazione stata eseguita con guida mx . 
in questi casi , sono stati acquisiti mammogrammi nelle proiezioni cranio - caudale e medio - laterale , per localizzare correttamente la lesione ( senographe essential e senographe dmr + , ge medical systems , cedex , francia )  . 
il radiologo , dopo aver scelto il miglior approccio alla lesione ( frontale , laterale o mediale ) , ha chiesto alla paziente di sedere su una sedia con le braccia lungo il corpo . 
la posizione dellago 22 g , che doveva essere allinterno della lesione o entro 1 cm da essa , stata poi veri cata con altri mammogrammi ortogonali , anche pi di una volta nel caso in cui la punta dellago non fosse nella corretta posizione , e questo si pu veri care in particolare per piccoli gruppi di microcalci cazioni o in mammelle voluminose . 
in ne , il medico nucleare ha inserito sullago la prima siringa da insulina contenente 0 , 10 , 2 cc del tracciante radioattivo , ed il radiologo ha inserito poi una seconda siringa con 1 cc di mezzo di contrasto iodato ( iopamiro , iopamidolo , bracco , italia )  . 
il costo della roll , includendo la scintigra a biplanare ed escludendo lintervento chirurgico , stato in media di circa 250300 euro . procedura chirurgica in sala operatoria ( or ) , il chirurgo ha utilizzato una gdp ( 15 lvr , pol.hi.tech , carsoli , italia ) per guidare la rimozione della lesione . 
il pezzo operatorio stato inviato al dipartimento di radiologia , dove la presenza della lesione sospetta stata veri cata mediante us o mx , a seconda della tecnica di guida radiologica precedentemente adottata . 
il risultato della valutazione radiologica stato comunicato al chirurgo in or , il quale poteva decidere se lescissione era adeguata o doveva essere allargata , a seconda che i margini , orientati con li chirurgici di seta e clips metalliche , fossero radiologicamente liberi ( lesione ad almeno 1 cm da tutti i margini del pezzo operatorio )  . 
 procedura istologica tutti i 288 pezzi operatori sono stati sottoposti a valutazione macroscopica della totalit dei margini , i quali sono stati considerati liberi da malattia se la lesione era almeno ad 1 cm da essi . 
1a - c ultrasound guidance for a 12 - mm nonpalpable lesion ( cytology : c3 ) in the upper outer quadrant of the right breast of a 44 - year - old patient . 
1a - c guida ecogra ca per una lesione non palpabile di 12 mm ( citologia : c3 ) al quadrante supero - esterno della mammella destra di una paziente di 44 anni . 
c valutazione del pezzo operatorio dopo lescissione : la lesione bene visibile ( vedi freccia )  . tutte le procedure sono state ben tollerate e non si sono veri cati eventi avversi importanti . 
in 1 caso la radioattivit del 99mtc scomparsa poich la procedura chirurgica non stata eseguita in tempo ( entro 24 ore ) per problemi tecnici ( sciopero del personale della or )  . 
2a - e mammographic guidance for a cluster of microcalci cations ( histology : ductal carcinoma in situ ) in the upper outer quadrant of the left breast ( para - areolar ) of a 67 - year - old patient . 
2a - e guida mammogra ca per un cluster di microcalci cazioni ( istologia : carcinoma duttale in situ ) nel quadrante supero - esterno , in parareolare , della mammella sinistra di una paziente di 67 anni . 
e radiogramma del pezzo operatorio che mostra che le microcalci cazioni sono contenute nella porzione escissa di mammella ( vedi freccia )  . of the radiological evaluation of the specimen was communicated to the surgeon in the or , who could decide whether the excision was adequate or should be enlarged , depending on the radiological clearance of margins ( lesion 1 cm away from all the specimen margins ) , which were oriented with surgical silk threads and metallic clips . 
in questo sottogruppo , 6 soggetti ( 2 , 6% ) sono stati sottoposti ad una seconda procedura chirurgica poich i margini del tessuto escisso non erano liberi dopo il primo intervento . 
la localizzazione delle lesioni con roll stata corretta in tutti questi 6 casi . in totale , lescissione di sn stata eseguita in 205 diately analysed in all 198 cases in order to decide whether axillary dissection had to be performed in the same surgical session . 570 results all procedures were well tolerated , and no major adverse event occurred . 
in one case , the 99mtc radioactivity declined because the surgical procedure could not be performed in time ( within 24 h ) due to technical problems ( strike of the or personnel )  . 
the surgeons observed no interference between the radioactive signals from the breast lesion and from the sn . discussion breast cancer screening has increased the number of detected nonpalpable breast lesions and , once a suspicious lesion is detected at imaging , a cytological or histological diagnosis is needed . 
our approach to nonpalpable breast lesions is characterised by a preoperative diagnosis obtained either with us - guided fnac or core biopsy or with mx - guided stereotactic vacuum - assisted biopsy . 
it has been demonstrated that even when the breast carcinoma diagnosis is known before surgery , preoperative wire localisation can be inaccurate and lead to reintervention in up to 20% of cases [ 30 , 31 ]  . 
moreover , wire - guided localisation has some well - known drawbacks that affect the entire course of the procedure , such as accidental displacement and need for repositioning , pneumothorax and wire migration into the pleural cavity . 
or availability is fundamental because the radiol med ( 2011 ) 116 : 564574 soggetti su 288 ( 71 , 2% ) , sia nella stessa sessione chirurgica dellescissione della lesione ( 198 casi , 96 , 6% ) , insieme alla roll , che durante una seconda procedura chirurgica ( 7 casi , 3 , 4% )  . 
i chirurghi non hanno osservato alcuna interferenza fra i segnali radioattivi derivanti dalla lesione mammaria e dal sn . discussione i programmi di screening hanno aumentato il numero di lesioni mammarie non palpabili che vengono individuate . 
 stato dimostrato che anche quando la diagnosi di carcinoma mammario nota prima della chirurgia , la localizzazione preoperatoria con lo guida pu essere poco accurata e condurre al re - intervento no al 20% dei casi circa [ 30 , 31 ]  . 
inoltre , la localizzazione con lo guida ha alcuni ben noti problemi che riguardano lintero svolgersi della procedura , come la dislocazione accidentale e la necessit di riposizionamento , lo pneumotorace e la migrazione del lo nella cavit pleurica . 
in or , il lo pu interferire con lincisione chirurgica ed spesso cos sottile che non pu essere seguito nel suo corso dalle mani del chirurgo , o pu essere reciso dal bisturi . 
 in ne , abbiamo gi citato il disagio della paziente , ma si possono anche veri care eventi avversi come reazioni vaso - vagali [ 14 , 15 , 3235 ]  . 
tuttavia , le principali limitazioni di questa tecnica sono la dif coltosa localizzazione delle lesioni in mammelle grandi e dense , la formazione di granulomi se il carbone non viene completamente rimosso durante lintervento chirurgico , la distorsione della lesione e la dif colt della preparazione del pezzo operatorio per la valutazione istologica [ 10 , 36 ]  . 
tuttavia , i reports in merito sono ancora pochi , probabilmente per la limitata radiol med ( 2011 ) 116 : 564574 patient cannot keep the wire in the breast for a long time , as it is uncomfortable and can be accidentally displaced . 
 in the or , the wire can interfere with the surgical incision and is often so thin that its course cannot be followed by the surgeons hands , or it can be cut by the scalpel . 
 the use of vital dyes , such as methylene blue , is anecdotal in the literature and unsatisfactory due to fast dispersion into tissues [ 8 , 10 ]  . 
however , the main limitations of this technique are dif cult lesion localisation in large and dense breasts , granuloma formation if charcoal is not completely removed during surgery , lesion distortion and dif culty in specimen preparation for histological evaluation [ 10 , 36 ]  . 
moreover , seeds can accidentally be lost in the operating room because of their small size [ 13 , 14 ]  . the roll procedure with 99mtc proved to be effective , safe and easy to use . 
in our patients , we used us guidance for the radiotracer injection whenever possible because us allowed us to follow the hypodermic needle in real time throughout the procedure , increasing our con dence in lesion centring . 
 when the lesion was too hard , it was not possible to inject the tracer into the core , so we injected beside it . it is important that the radiologist maintains adequate pressure on the us probe during the injection . 
if not , there is a possibility that the hypodermic needle will come out of the lesion because the breast tissues are no longer compressed , and the tracer is thus injected at a distance from the lesion . 
 this can represent a problem , especially in large breasts , and warrants a new localisation procedure . mx guidance was performed freehand because the stereotactic technique implies very high breast compression , which represents an obstacle during radiotracer injection . 
inoltre , i semini possono accidentalmente essere perduti in or a causa delle loro esigue dimensioni [ 13 , 14 ]  . la procedura di roll con 99mtc si dimostrata ef cace , sicura e facile da utilizzare . 
nelle nostre pazienti stata utilizzata la guida us per liniezione del radiotracciante in tutti i casi in cui stato possibile , poich lus permette di seguire lago ipodermico in tempo reale lungo tutta la procedura , aumentando la con denza riguardo alla centratura della lesione . 
quando la lesione era troppo dura , non stato possibile iniettare il tracciante allinterno della lesione , ed abbiamo iniettato a lato della stessa . importante che il radiologo mantenga adeguata pressione sulla mammella con la sonda us durante liniezione . 
 se ci non accade , esiste la possibilit che lago ipodermico esca dalla lesione perch i tessuti mammari non sono pi compressi ed il tracciante venga iniettato lontano dalla lesione stessa . 
questo pu rappresentare un problema soprattutto nelle mammelle voluminose , e richiede una nuova procedura di localizzazione . la guida mx stata eseguita a mano libera poich la tecnica stereotassica prevede una compressione della mammella assai intensa , che rappresenta un ostacolo durante liniezione del radiotracciante . 
nella nostra esperienza , la procedura a mano libera stata pi facile e sicura . solamente 6 lesioni su 288 ( 2 , 1% ) sono state centrate in modo non corretto . 
lerrore del radiologo stato quello di diminuire la compressione della mammella prima che liniezione del radiotracciante si concludesse , e ci ha causato dislocazione dellago e iniezione del radiotracciante nella sede non corretta . 
in questo caso , il chirurgo non stato in grado di percepire al tatto la lesione e la paziente stata sottoposta ad una ulteriore iniezione del 572 radiol med ( 2011 ) 116 : 564574 the 2 incorrect procedures occurred with mx guidance at the beginning of our experience . 
in this case , the surgeon was not able to feel the lesion , and the patient underwent a further injection of radiotracer a week later , for a second intervention . 
at histology , the specimens turned out to contain diffuse cystic disease , leading us to believe that the radiotracer was dispersed in the acinarlobular syste the remaining case of unsuccessful roll procedure , as mentioned , was due to unavailability of the or because of a personnel strike and subsequent fading of radioactivity at the lesion site . 
roll could be thus considered an almost harmless procedure due to the small diameter of the hypodermic needle ( 22 g ) , so that even when adjustments in needle position were needed , they bore no noticeable consequence . 
moreover , local anaesthesia was unnecessary . it is important to emphasise that when the surgical procedure could not be performed at the scheduled time , radioactivity at the lesion site decayed , with no risks for the patient . 
the surgeons observed no interference between radioactive signals from the breast lesion and from the sn , demonstrating that the two techniques can be used at the same time , with the same gdp , as already stated in the literature [ 21 , 2729 ]  . the main limitations of our study were that we did not perform a side - by - side comparison with conventional wire placement methods ; moreover , the technique requires the presence of a nuclear medicine department in the hospital to ensure correct preparation and handling of the radiotracer . 
 this can be a limitation , especially in small hospitals . in conclusion , radiological guidance in roll guaranteed the reliability , ef cacy and accuracy of the entire procedure of localisation and removal of single nonpalpable breast lesions and ensured the nal outcome in the majority of cases . 
il restante caso di procedura roll senza successo , come gi menzionato , stato dovuto allindisponibilit della or a causa di uno sciopero del personale con conseguente declino della radioattivit nella sede di lesione . 
la roll pu quindi essere considerata una procedura pressoch innocua grazie al piccolo calibro dellago ipodermico ( 22 g ) , cosicch anche quando sono stati necessari aggiustamenti della posizione dellago , essi non hanno causato alcuna signi cativa conseguenza . 
inoltre , non stata necessaria lanestesia locale . importante sottolineare che , quando la procedura chirurgica non stata eseguita in tempo , la radioattivit in sede di lesione declinata e non ci sono stati rischi per la paziente , mentre nel caso di guida con lo , esso avrebbe dovuto essere rimosso , causando ancor pi disagio . 
i chirurghi non hanno osservato alcuna interferenza fra i segnali radioattivi derivanti dalla lesione mammaria e dal sn , dimostrando che le due tecniche possono essere utilizzate contemporaneamente , con la stessa gdp , come gi affermato in letteratura [ 21 , 2729 ]  . i principali limiti dello studio sono stati che non abbiamo eseguito un paragone in parallelo con i metodi convenzionali di posizionamento di lo guida ; inoltre , stato necessario un dipartimento di medicina nucleare allinterno dellospedale per le corrette preparazione e gestione del radiotracciante . 
questo pu essere un limite soprattutto in piccoli ospedali . in conclusione , la guida radiologica nella roll ha garantito laf dabilit , lef cacia e laccuratezza dellintera procedura di localizzazione e rimozione di lesioni mammarie singole non palpabili e ne ha garantito il risultato nale nella maggioranza dei casi . 
simonetti dipartimento di diagnostica per immagini , imaging molecolare , radiologia interventistica e terapia radiante , universit degli studi di roma tor vergata ( ptv ) , viale oxford 81 , 00133 roma , italy correspondence to : c.a. 
il secondo esame ecogra co , eseguito successivamente alla mammogra a , ha confermato 123 / 147 lesioni nodulari solide , 53 / 67 casi di sbarramento del fascio ultrasonoro e 183 / 193 aree di disomogeneit ecostrutturale . 
sono state inoltre individuate 13 ulteriori lesioni nodulari non evidenziate alla preliminare ecogra a ed in due casi abbiamo riscontrato la presenza di substrato nella sede del reperto mammogra co di microcalci cazioni . 
dal nostro studio emerso che lecogra a effettuata senza la possibilit di riscontro mammogra co , ha dato luogo ad erronee interpretazioni diagnostiche intese sia come falsi positivi che come falsi negativi e secondo i nostri dati , qualora eseguita successivamente alla mammogra a , ha consentito di incrementare il valore della detection rate dellindagine ecogra ca da 4 , 16% a 5 , 5% . 
 parole chiave mammella ecogra a mammogra a iter diagnostico radiol med ( 2011 ) 116 : 584594 introduction introduzione in italy , mortality rates from breast cancer among women < 49 years of age have decreased by around 11% over the past 5 years [ 1 ]  . 
conventional imaging modalities , namely , mammography and ultrasound ( us ) , allow early detection of breast disease when performed according to strict methodological criteria and correct temporal sequence [ 2 ]  . 
mammography is the rst - line modality for early diagnosis of breast cancer in women > 3540 years of age , whereas us is an important complementary technique that increases sensitivity and speci city of mammography [ 3 , 4 ] , helps solve possible diagnostic doubts in dense breasts and is the only examination that can be performed in young women or to assess the solid or uid nature of opacities detected on mammography [ 2 , 5 ]  . 
however , the intrinsic limitations of us and its lower sensitivity compared with mammography in detecting suspicious breast lesions , in part as a result of the structural features of the glandular component and the operators experience , prevent this modality from being used as the sole technique for early detection of breast disease [ 69 ]  . sonographic detection of suspicious ndings is often facilitated by previous mammographic detection in relation to site and morphological characteristics . 
 although guidelines have been developed that indicate the correct sequence of diagnostic procedures [ 10 ] , inappropriate requests for us examinations as an alternative or prior to mammography continue to occur in clinical practice . 
consequently , women often appear confused by a lack of clarity regarding the best modality or the correct combination of modalities to be used and are sometimes led to undergo inappropriate investigations and to regard as dangerous and useless examinations that , instead , would effectively contribute to protecting their health . the aim of this study was to evaluate the role of the correct temporal sequence of conventional mammographic and us imaging . 
in particular , we aimed at analysing the increase in sensitivity for detecting suspicious ndings brought about by us performed following mammography . materials and methods from january 2008 to march 2010 , we studied 600 consecutive patients aged from 35 to 75 years referred to our department by their gynaecologist and / or family doctor with an inappropriate request for us to investigate a speci c clinical nding . 
requests were considered inappropriate negli ultimi cinque anni , in italia , la mortalit per carcinoma della mammella nelle donne al di sotto dei 49 anni diminuita di circa l11% [ 1 ]  . 
limaging convenzionale , rappresentato dalla mammogra a e dallecogra a , consente di ottenere una precoce individuazione della patologia mammaria qualora eseguito mediante rigorosi criteri metodologici e corretta successione temporale [ 2 ]  . 
lesame mammograco rappresenta lindagine di prima istanza nella diagnosi precoce del carcinoma della mammella nelle donne di et al di sopra dei 3540 anni , mentre lecogra a costituisce , ad oggi , unimportante tecnica complementare allesame mammogra co in grado di incrementarne la sensibilit e la speci cit [ 3 , 4 ] e dirimerne i dubbi diagnostici nei casi di mammelle a prevalente componente broghiandolare , come unica indagine in donne di giovane et e nella valutazione della natura solida o liquida di radiopacit nodulari evidenziate [ 2 , 5 ]  . 
tuttavia , i limiti intrinseci dellecogra a e la sua inferiore sensibilit rispetto allindagine mammogra ca nellindividuare la presenza di lesioni mammarie sospette , anche in relazione alle caratteristiche strutturali della componente ghiandolare ed allesperienza delloperatore , non consentono di utilizzare tale metodica come unica tecnica per la diagnosi precoce della patologia mammaria [ 69 ]  . 
a tuttoggi , seppur esistano delle linee guida che indicano la corretta successione delle indagini strumentali [ 10 ] , nella pratica clinica continuano a veri carsi richieste non idonee di esami ecogra ci in sostituzione o antecedenti lesame mammograco . 
la donna appare quindi spesso disorientata a seguito della scarsa chiarezza su quale sia la migliore tecnica o sul corretto percorso dintegrazione delle stesse , indotta , talora , a ricorrere ad indagini improprie ed a ritenere pericolosi ed inutili esami che , invece , contribuirebbero ef cacemente alla tutela della sua salute . 
in particolare abbiamo valutato lincremento di sensibilit nellindividuazione di reperti sospetti che lesame ecogra co determina quando effettuato successivamente ad esame mammogra co . 586 radiol med ( 2011 ) 116 : 584594 when no mammographic study had been performed in the previous 12 , 18 , or 24 months , depending on the patients age and structural features of the breast . 
as , according to the current guidelines , us is the rst - line imaging modality for assessing lesions in women < 35 years of age , as well as in breastfeeding and pregnant women , these patients were excluded from our study , as were those with breast implants , so as to obtain the most homogeneous results possible . all patients underwent preliminary us and subsequent mammography in standard projections ( craniocaudal , mediolateral oblique , mediolateral ) , when necessary with targeted compression and / or direct image magni cation . 
all patients with suspicious lesions proceeded to cytology / histology , vacuum - assisted biopsy ( vab ) and / or magnetic resonance ( mr ) imaging to obtain a correct diagnostic assessment , except in the event of ndings that had already been characterised or showed lesion stability over time . 
suspicious us ndings , deemed worthy of further investigation , were solid nodular lesions , posterior acoustic shadowing and areas of heterogeneous echostructure , the latter being de ned as focal areas of alteration of normal breast structure in particular of the regular alternation of supporting structures , fatty tissue and parenchymal component producing a different tissue response to the us beathese ndings could represent direct and / or indirect signs of a tumour . 
for the purposes of our study , we measured the detection rate of preliminary us and that of us performed after mammography . results preliminary us identi ed the presence of 147 hypoisoemateriali e metodi da gennaio 2008 a marzo 2010 abbiamo studiato 600 pazienti consecutive di et compresa tra i 35 ed i 75 anni giunte alla nostra osservazione con richiesta incongrua di esame ecogra co da parte del ginecologo e / o del medico curante al ne di valutare uno speci co reperto clinico . 
la richiesta stata considerata inappropriata in mancanza di un esame mammogra co recente ovvero antecedente ai 12 , 18 o 24 mesi in relazione alla fascia di et della paziente ed alle caratteristiche strutturali della mammella . 
secondo le attuali linee guida , essendo lecogra a lindagine di primo livello per la valutazione di lesioni in donne di et inferiore a 35 anni , in allattamento ed in gravidanza , tali pazienti non sono state incluse nel nostro studio , cos come abbiamo escluso le pazienti con protesi al ne di ottenere risultati il pi omogenei possibile . 
 le pazienti sono state tutte sottoposte , in fase preliminare , ad esame ecogra co e successivamente ad esame mammogra co , condotto mediante proiezioni standard ( cranio - caudale , medio - laterale - obliqua , medio - laterale ) , integrate , qualora necessario , con acquisizioni aggiuntive di dettaglio a compressione mirata e / o con ingrandimenti diretti di immagine . 
in tutti i casi studiati , nella prima indagine ecotomogra ca , non sono stati presi in considerazione i precedenti esami effettuati dalle pazienti e portati in visione , al ne di poter ottenere incondizionatamente un giudizio interpretativo . 
i reperti da noi considerati sospetti allesame ecogra co su cui abbiamo ritenuto opportuno effettuare ulteriori approfondimenti diagnostici , sono stati rilievi ecogra ci di lesioni nodulari solide , sbarramenti del fascio ultrasonoro ed aree di disomogeneit eco - strutturale ; con aree di disomogeradiol med ( 2011 ) 116 : 584594 choic solid nodular lesions ranging in size from 5 to 35 mm , 67 cases of acoustic shadowing ranging in size from 3 to 15 mm and 193 areas of heterogeneous echostructure ranging from 15 to 47 mat mammography , the 147 solid nodular lesions detected with us were depicted as nodular opacities with regular margins and well - de ned borders in 92 / 147 cases and as pseudonodular areas of radiolucency corresponding to fat lobules in 24 / 147 cases ; in 31 / 147 cases of highly dense breasts , no abnormal nding was detected . 
the 67 ndings identi ed as acoustic shadowing on us appeared as stellate radiopacities in 21 / 67 cases and areas of architectural distortion in 32 / 67 cases ; no mammographic correlate was detected in 14 / 67 cases . 
the 193 areas of heterogeneous echostructure proved to be areas of glandular asymmetry in 102 / 193 cases and areas of structural distortion in 81 / 193 ; no mammographic correlate was identi ed in 10 / 193 cases . 
 mammography enabled the detection of 13 additional faint nodular radiopacities in fatty breasts missed at preliminary us and 11 cases of microcalci cations , two of which were associated with an indistinct radiopacity . the second us scan , performed after mammography , con rmed 123 / 147 solid nodular lesions , 92 / 147 corresponding to the nodular opacities depicted by mammography and 31 / 147 missed at mammography in dense breasts . 
areas of heterogeneous echostructure previously identi ed at preliminary us were con rmed in 183 / 193 cases , 102 of which corresponding to areas of glandular asymmetry at mammography and 81 to areas of structural distortion . 
thirteen additional nodular lesions were identi ed , 12 of which were hypoisoechoic and one hyperisoechoic , corresponding to the nodular radiopacities missed at preliminary us owing to their subcentimetre size , hardly visible in a context of fatty breasts . 
in the 113 cases of architectural distortion , neit eco - strutturale intendiamo aree focali di alterazione della normale struttura della mammella , in particolare della regolare alternanza di strutture di sostegno , tessuto adiposo e componente parenchimale , che siano tali da determinare una differente risposta del tessuto al fascio ultrasonoro . 
 ai ni del nostro studio abbiamo quindi valutato il valore della detection rate dellindagine ecogra ca preliminare e di quella successiva alla mammogra a . risultati la prima indagine ecotomogra ca ci ha permesso di individuare la presenza di 147 lesioni nodulari solide ipo - isoecogene delle dimensioni comprese tra 56 mm e 35 mm , 67 sbarramenti del fascio ultrasonoro delle dimensioni comprese tra i 3 ed i 15 mm , e 193 aree di disomogeneit ecostrutturale delle dimensioni comprese tra i 15 mm ed i 47 mallesame mammogra co le 147 lesioni nodulari solide documentate allindagine ecogra ca sono state evidenziate come opacit nodulari a margini regolari e limiti netti in 92 / 147 casi , come aree pseudonodulari di radiotrasparenza riferibili a loggette adipose in 24 / 147 casi ed in ne in 31 / 147 casi non stato individuato alcun reperto in un contesto di mammella ad elevata densit ghiandolare . 
i 67 reperti apprezzabili come sbarramenti del fascio ultrasonoro sono risultati in 21 / 67 casi radiopacit stellariformi , in 32 / 67 casi aree di distorsione architetturale ed in ne in 14 / 67 casi non c stato corrispettivo riscontro mammograco . 
le 193 aree di disomogeneit ecostrutturale si sono rivelate in 102 / 193 casi aree di asimmetria ghiandolare , in 81 / 193 aree di distorsione strutturale ed in 10 / 193 casi non c stato corrispettivo riscontro mammogra co . lindagine mammogra ca ci ha permesso inoltre di individuare ulteriori 13 tenui radiopacit nodulari in mammelle a prevalente componente adiposa , non evidenziate al preliminare esame ecogra co , e 11 casi di microcalci cazioni , delle quali 2 / 11 casi sottesi a sfumate radiopacit . il secondo esame ecogra co , quello eseguito successivamente alla mammogra a , ha confermato 123 / 147 lesioni nodulari solide , di cui 92 / 147 corrispondenti alle opacit nodulari descritte alla mammogra a e 31 / 147 non distinguibili mammogra camente in un contesto di mammella broghiandolare . 
depending on our level of suspicion , 12 / 113 patients were referred for us follow - up at 6 months ( which con rmed lesion stability ) , 12 / 113 for mr imaging ( which excluded neoangiogenic components ) , 9 / 113 for cytology ( grade c2 ) and 11 / 113 for vab . 
seven of the latter 11 cases were brotic ndings , and the remaining four were radial scars ; these ndings were also con rmed by histological analysis of the surgical specimen . the 21 cases of stellate radiopacities underwent histological characterisation with us - guided vab , which revealed in ltrating carcinomas , which were con rmed by surgical histology in all cases . 
of the 13 cases of nodular radiopacities detected at mammography , eight were preexisting nodular formations that had remained virtually mati in 53 / 67 casi , di cui 21 / 67 casi erano rappresentati da radiopacit stellariformi e 32 / 67 casi da aree di distorsione architetturale ; in accordo con la mammogra a , in 14 / 67 casi non c stato corrispettivo riscontro ecogra co . 
le aree di disomogeneit ecostrutturale individuate precedentemente al primo esame ecotomogra co sono state confermate in 183 / 193 casi , di cui 102 / 193 erano riferibili ad aree di asimmetria ghiandolare allindagine mammogra ca e 81 / 193 ad aree di distorsione strutturale . 
sono state inoltre individuate 13 ulteriori lesioni nodulari , di cui 12 / 13 ipo - isoecogene ed 1 / 13 iper - isoecogena , corrispondenti alle radiopacit nodulari descritte , non evidenziate al preliminare esame ecogra co in considerazione delle loro esigue dimensioni subcentimetriche dif cilmente individuabili in un contesto di mammelle a prevalente componente adiposa . 
 nei 113 casi di distorsione architetturale , il confronto con i precedenti esami ci ha permesso di confermare la stabilit dei reperti in 69 / 113 casi , di cui 41 in pazienti gi sottoposte a chirurgia e , in base al nostro grado di sospetto , di inviare 12 / 113 casi al controllo ecogra co a 6 mesi , che ha successivamente confermato la stabilit dei reperti , 12 / 113 casi ad approfondimento diagnostico mediante esame rm , che ne ha escluso la presenza di componenti neoangionetiche , 9 / 113 casi allesame citologico con esito c2 e 11 / 113 casi al prelievo istologico tramite procedura vab . 
di questi ultimi 11 / 113 casi , si sono rivelati reperti di natura brosa 7 / 11 lesioni e radial scar le restanti 4 / 11 lesioni ; tali riscontri sono successivamente stati confermati anche allistologico de nitivo della chirurgia . i 21 casi di radiopacit stellariformi sono stati sottoposti a prelievo istologico mediante procedura vab sotto guida ecogra ca e si sono rivelati tutti carcinomi in ltranti , confermati allistologico chirurgico . 
dei 13 casi di radiopacit nodulari riscontrate alla mammogra a , 8 / 13 erano rappresentate da formazioni nodulari gi presenti e sostanzialmente invariate rispetto ai precedenti controlli , mentre i restanti 5 / 13 casi di primo riscontro sono stati sottoposti a caratterizzazione cito - istologica , il cui esito stato in 2 / 5 casi di benignit , in 2 / 5 casi di carcinoma in situ ed in 1 / 5 casi di carcinoma in ltrante . 
degli 11 casi di microcalci cazioni , in 2 casi si dimostrato un substrato ecogra co in tale sede ed i reperti sono stati quindi sottoposti a caratterizzazione istologica tramite prelievo vab con esito di carcinoma in ltrante ; dei restanti 9 / 11 casi sono state sottoposte a prelievo vab sotto guida mammogra ca 6 / 11 casi , il cui esito stato in 3 / 6 casi di benignit , in 1 / 6 casi di iperplasia duttale atipica ed in 2 / 6 casi di carcinoma in situ , mentre nei restanti 3 / 11 casi le caratteristiche morfologiche delle microcalci cazioni deponevano per la loro benignit . secondo i nostri dati quindi il valore della detection rate dellindagine ecogra ca subisce un incremento qualora questultima sia effettuata successivamente alla mammogra a e guidata da questultima : la detection rate della prelifig . 
1 proiezioni mammogra che medio - laterali - oblique in cui si documenta a destra , in corrispondenza del prolungamento ascellare , una sfumata radiopacit a margini irregolari , che si conferma allesame di dettaglio a compressione mirata . 
la successiva indagine ecotomogra ca ha permesso di evidenziare a tale livello millimetrica area ipoecogena . unchanged since the previous examinations , whereas the remaining ve cases of initial detection underwent cytological and histological characterisation , which revealed a benign lesion in 2 / 5 cases , carcinoma in situ in 2 / 5 and in ltrating carcinoma in 1 / 5 . 
of the 11 cases of microcalci cations , us showed a sonographic correlate at the site in two cases , and the lesions were further characterised with vab , which yielded a diagnosis of in ltrating carcinoma . 
of the remaining nine cases , six underwent mammographically guided vab , which indicated a benign lesion in three cases , atypical ductal hyperplasia in one case and carcinoma in situ in two cases . 
morphology in the remaining three cases indicated a benign nature . according to our results , the detection rate of us increases when the examination follows mammography and is guided by it : the detection rate of preliminary us was 4.16% , whereas that of us performed following mammography rose to 5.5%. 590 radiol med ( 2011 ) 116 : 584594 fig . 
2 proiezioni mammogra che cranio - caudali e medio - laterali in cui si documenta a destra , in corrispondenza del quadrante infero - interno , una centimetrica sfumata radiopacit in un quadro di mammelle a prevalente componente adiposa . 
3 craniocaudal and mediolateral mammograms showing right breast opacity with ill - de ned borders and irregular margins associated with typical irregular calci cations , con rmed by targeted compression . 
3 proiezioni mammogra che cranio - caudali e medio - laterali in cui si documenta a destra , in corrispondenza della porzione di passaggio dei quadranti esterni , una radiopacit a limiti sfumati e margini irregolari , che si conferma allesame di dettaglio a compressione mirata , nel contesto della quale si apprezzano alcune ni calci cazioni . 
la successiva indagine ecotomogra ca ha permesso di evidenziare a tale livello unarea iso - ipoecogena a margini irregolari di circa 1 cm . radiol med ( 2011 ) 116 : 584594 minare indagine ecogra ca risultata del 4 , 16% , mentre la detection rate della seconda ecogra a stata del 5 , 5% . discussione la riduzione della mortalit per carcinoma mammario , cui si assistito negli ultimi anni in italia , dovuta senzaltro allaumento della sensibilit delle donne a sottoporsi spontaneamente a controlli strumentali periodici ed anche alla maggiore diffusione dei programmi di screening , che consentono di diagnosticare questa patologia in fasi sempre pi precoci [ 11 ]  . 
a tale proposito le tecniche ad oggi disponibili per la diagnosi precoce del carcinoma mammario sono rappresentate fondamentalmente dallimaging convenzionale mammogra co ed ecogra co e solo tramite una loro rigorosa e scrupolosa applicazione si giunge ad una diagnosi tempestiva al ne di unef cace prevenzione secondaria [ 10 ]  . 
la mammogra a attualmente lesame di prima istanza in donne di et superiore ai 35 anni , in quanto permette in modo suf cientemente ripetibile e confrontabile , lesplorazione della mammella ; ci fa ritenere lesame mammogra co la tecnica pi importante per lo studio della patologia mammaria [ 2 , 10 , 11 ]  . 
lecogra a rappresenta la pi importante modalit di imaging complementare dopo la mammogra a , in quanto contribuisce insieme allesame clinico ad elevare la sensibilit della diagnosi sino al 90% e la speci cit sino al 98% [ 10 ]  . 
lesame richiede lutilizzo di sonde ad elevata frequenza ( 10 mhz ) di tipo small parts lineare , che assicurano la corretta penetrazione del fascio ultrasonoro anche in mammelle voluminose con una focalizzazione dinamica ed una buona esplorazione del parenchima . 
la speci cit dellindagine ecogra ca inoltre aumentata dallimpiego di tecnologie quali color e power doppler per la valutazione del pattern vascolare peried intralesionale e pi recentemente mediante lausilio della metodica elastosonogra ca . 
 nonostante lecogra a sia diventata strumento diagnostico indispensabile nella valutazione della patologia mammaria , tuttavia necessario puntualizzare che tale metodica non entra in competizione con le altre tecniche diagnostiche , in particolar modo con la mammogra a [ 12 , 13 , 14 ]  . 
lecogra a mammaria deve infatti essere effettuata a complemento della mammogra a , piuttosto che in sostituzione di essa , ed entrambe devono costituire ununit diagnostica nella valutazione della patologia mammaria [ 6 , 12 , 13 , 15 ]  . 
tale ultima considerazione trova ulteriore riscontro nel fatto che negli ultimi 20 anni si assistito ad una crescente incidenza del carcinoma mammario pi signi cativa nelle donne in et fertile , nelle quali lutilizzo della mammogra a richiede necessariamente il completamento ecogra co in relazione alla densit ed alle caratteristiche strutturali della componente ghiandolare [ 1 , 11 , fig . 
4 proiezione mammogra ca cranio - caudale ed esame di dettaglio a compressione mirata della mammella destra in cui si documenta , a livello dei quadranti esterni , una tenue area radiopaca pseudonodulare a limiti sfumati , nel contesto della quale si apprezzano alcune microcalci cazioni . 
 discussion the lower mortality rate of breast cancer recently observed in italy is no doubt a consequence of womens heightened awareness of the need to undergo periodic imaging investigations , as well as the greater availability of screening programmes that allow for increasingly earlier detection of the disease [ 11 ]  . 
the techniques available for an early diagnosis of breast cancer include conventional mammography and us imaging , and only a rigorous and scrupulous application of these modalities can lead to a timely diagnosis and effective secondary prevention [ 10 ]  . 
us represents the main adjunctive imaging modality after mammography , as it helps along with the clinical examination to increase diagnostic sensitivity to 90% and speci city to 98% [ 10 ]  . 
the examination requires the use of high - frequency ( 10 mhz ) small - parts lineararray probes , which ensure correct penetration of the us beam even in large breasts , with dynamic focusing and good parenchymal exploration . 
the speci city of us is further increased by the use of techniques such as colour and power doppler for evaluating periand intralesional vascular patterns and , more recently , by the use of elastosonography . although us has become an essential diagnostic tool 592 radiol med ( 2011 ) 116 : 584594 fig . 
5 proiezioni mammogra che cranio - caudali , medio - laterali ed ingrandimento dimmagine con zoom elettronico in cui si apprezza a sinistra , a livello della porzione di passaggio dei quadranti esterni in corrispondenza della camera posteriore , una tenue radiopacit a limiti sfumati . 
la successiva indagine ecotomogra ca ha documentato , in corrispondenza del reperto mammogra co , la presenza di una formazione nodulare ad ecostruttura disomogenea prevalentemente iperecogena di circa 1 cm . in evaluating breast disease , it must be emphasised that it is not in competition with other diagnostic modalities , notably with mammography [ 12 , 13 , 14 ]  . 
in fact , breast us is to be used as an adjunct rather than a replacement for mammography , with the two modalities forming an integrated diagnostic tool in evaluating breast disease [ 6 , 12 , 13 , 15 ]  . 
this is also supported by the fact that the last 20 years have seen a growing incidence of breast cancers , especially among women of childbearing age in whom mammography necessarily requires supplementation with us in relation to density and structural features of the glandular component [ 1 , 11 , 16 ] , given that mammographic sensitivity is reduced in dense breasts . 
in fact , even though us used as the only imaging technique is able to yield a diagnosis of malignant nodular lesions when these are well de ned , as reported in the literature [ 17 , 18 ] , combined mammography and sonography increases the diagnostic accuracy of the each modality to 95% [ 11 ]  . 
in particular , when us is performed after mammography , it markedly increases in sensitivity and speci city [ 13 ]  . this was also con rmed by our study , which aimed at evaluating the correct temporal sequence of the modalities . 
 in particular , we found that us performed after mammography not only improves the assessment of known mammographic ndings but also in many cases , us ndings are guided by the results of the mammographic study ( lesion site and dimensions )  . 
 [ 19 ] , this observation was found to apply in particular to 16 ] in quanto la sensibilit della mammogra a si riduce se la componente adiposa poco rappresentata . 
sebbene lecogra a utilizzata come unica indagine strumentale sia attualmente in grado di porre diagnosi di lesioni nodulari maligne qualora ben circoscritte , in accordo con alcuni dati della letteratura [ 17 , 18 ] , tuttavia la diagnostica senologica integrata mammo - ecogra ca aumenta no al 95% laccuratezza diagnostica delle singole metodiche [ 11 ] ed in particolare , qualora lecogra a sia eseguita successivamente allesame mammogra co , ne aumenta notevolmente la sensibilit e la speci cit [ 13 ]  . tale ultimo dato stato confermato dal nostro lavoro , che ha avuto lobiettivo di valutare la loro corretta successione temporale ; in particolare abbiamo evidenziato che lecogra a eseguita successivamente allindagine mammogra ca non solo migliora la valutazione di reperti mammogra ci gi noti , ma spesso lo stesso rilievo ecogra co in uenzato dalla conoscenza del reperto mammogra co ( sede e dimensioni della lesione )  . 
 [ 19 ] tale considerazione ha trovato particolare riscontro nella valutazione delle mammelle a prevalente componente broghiandolare [ 7 , 19 ] e nel nostro studio anche nei casi di lesioni focali in mammelle adipose e nella ricerca di un eventuale substrato patologico nei casi di microcalci cazioni . 
 [ 14 ] , lesame ecogra co , qualora necessario , se eseguito successivamente alla mammogra a , aumenta laccuratezza diagnostica della metodica e ne fornisce importante complemento . radiol med ( 2011 ) 116 : 584594 dense breast evaluations [ 7 , 19 ] , as well as in our study to focal lesions in fatty breasts and to the search for a possible pathological substrate in cases of microcalci cations . 
 [ 14 ] , that when needed , us imaging performed after mammography improves its diagnostic accuracy and provides a valuable complement . in our experience , us following mammography enabled the identi cation of 12 cases of small isohypoechoic lesions in prevalently fatty breasts that were missed owing to the limits of us in fatty breasts . 
two cases of hypoechoic areas were identi ed as substrates for microcalci cation clusters only recognisable at mammography , as was one case of a hyperisoechoic nodule that could only be identi ed knowing its anatomical position , which was provided by orthogonal mammographic projections . 
lastly , histology demonstrated that the us detection rate increases when the modality is performed after mammography to further investigate doubtful mammographic ndings rather than as a rst - line imaging test . 
 in addition , our study showed that us performed without mammographic assessment led to diagnostic misinterpretations false positive ( 8% ) or false negative ( 4% ) which may result in additional diagnostic tests that are often unnecessary and burdensome in a context of increasingly scarce healthcare resources . nella nostra esperienza lindagine ecogra ca eseguita successivamente alla mammogra a ha consentito di identi care 12 casi di lesioni di piccole dimensioni iso - / ipoecogene in mammelle a prevalente componente adiposa , misconosciute a causa dei limiti della metodica ecogra ca in mammelle adipose . 
sono stati individuati due casi di aree ipoecogene quali substrati di cluster di microcalci cazioni apprezzabili unicamente allesame mammogra co ed in ne un caso in cui la lesione , rappresentata da nodulo iper - / isoecogeno , poteva essere identi cata unicamente conoscendone la sede anatomica mediante le proiezioni ortogonali mammogra che . 
al contrario , 24 casi di lesioni ipoecogene evidenziate allesame ecogra co si sono rivelate aree di radiotrasparenza alla mammogra a e quindi di nessun signicato patologico da un punto di vista diagnostico . 
in ne , secondo i risultati istologici ottenuti , il valore della detection rate dellindagine ecogra ca subisce un incremento qualora questultima sia effettuata successivamente alla mammogra a , quale approfondimento di reperti mammogra ci dubbi e non come esame di prima istanza : infatti la detection rate della preliminare indagine ecogra ca risultata del 4 , 16% , mentre la detection rate della seconda ecogra a stata del 5 , 5% . 
dal nostro studio emerso inoltre che lindagine ecogra ca effettuata senza la possibilit di riscontro mammogra co , ha dato luogo ad erronee interpretazioni diagnostiche intese sia come falsi positivi , approssimativamente l8% , che come falsi negativi , circa il 4% , che talvolta portano allesecuzione di ulteriori accertamenti diagnostici spesso inutili e gravosi in un contesto sanitario in cui le risorse economiche sono sempre pi carenti . conclusioni conclusions despite an increased awareness of prevention , women are often confused about the most appropriate imaging modality and the best approach for integrating conventional breast imaging techniques . 
they therefore require counselling by a healthcare professional who can direct them towards a correct and timely diagnosis , in consideration of the clinical picture and patient history . in conclusione , sebbene le donne negli ultimi anni siano pi sensibili alla prevenzione , spesso si ritrovano disorientate su quale sia la migliore indagine strumentale da effettuare e quale possa essere il corretto percorso dintegrazione delle stesse . 
the clinical outcome is shown by the use of clinical scores : visual analogue scale ( vas ) , constants score and shoulder pain disability index ( spadi )  . 
boccalini radiology department , school of medicine , universita` di genova , largo rosanna benzi 8 , 16138 genoa , italy common painful condition that occurs in up to 7.5 % of otherwise healthy adults . 
in adhesive capsulitis , different therapeutic ultrasound - guided techniques such as corticosteroid injection , capsular distension ( sodium chlorate solution ; sodium chlorate and corticosteroids ; air ) and viscosupplementation are evaluated . 
the treatment of rotator cuff tendinosis and partial tears with ultrasound - guided injection of concentrated autologous platelets is also described . keywords ultrasound injection ( cid : 2 ) rotator cuff calcication ( cid : 2 ) viscosupplementation ( cid : 2 ) platelet - rich plasma ( cid : 2 ) adhesive capsulitis j . 
martinoli radiology department , dissal , universita` di genova , largo rosanna benzi 8 , 16138 genoa , italy the aim of this review is to describe the techniques of interventional ultrasound for the treatment of shoulder pathology . 
shoulder ultrasonography ( us ) is an accurate method to perform diagnostic studies , because it evaluates both morphology and function by means of dynamic evaluations [ 1 ]  . 
us is also a very radiol med ( 2014 ) 119 : 318326 table 1 constants score combines two subjective variables like pain and pain in relation to daily activities ( value range 035 ) and two objective measurements like strength and range of motion ( value range 065 ) as assessed by a physician item pain none mild moderate severe full work activities of daily living full recreation / sports unaffected sleep hand position up to wrist up to xiphoid up to top of head above head range of motion abduction forward elevation internal rotation external rotation shoulder power pounds to resist total useful tool to guide interventional procedures around the shoulder and sometimes to inject intra - articular contrast media before magnetic resonance ( mr ) arthrography [ 6 , 7 ]  . 
blind injections may fail in a signicant percentage of cases , so the use of a low - cost and safe tool such as us improves the success of the interventional procedures [ 8 ]  . the comparison of us - guided against landmark - guided injection in shoulder pathology shows a statistically signicant difference in pain control [ 9 , 10 ] in favour of usguided methods . mild vagal reactions may be considered the most frequent immediate complications as reported in the literature [ 11 ]  . 
the visual analogue scale ( vas ) , a well - known 10 - cm graduated scale ranging from 0 ( no pain ) to 10 ( unbearable pain ) , is commonly employed in clinical practice [ 11 ]  . 
constants ( score score constant_shoulder_score.html ) combines two subjective variables ( pain and pain in relation to daily activities , value range 035 ) and two objective measurements ( strength and range of movement , value range 065 ) assessed by a physician . 
the shoulder pain and disability index ( spadi ) questionnaire is a self - administered index designed to measure the effects of different pathologic conditions on the functional status of the shoulder [ 12 , 13 ]  . the spadi questionnaire is also used to measure the response to treatment over time . 
the questionnaire consists of 13 items divided into 2 subcategories reecting the disability ( 8 items ) and pain ( 5 items ) associated with the clinical syndrome of painful shoulder . 
all items are rated by using a vas from no pain or no difculty to worst imaginable pain or so difcult it required help . a numeric value was obtained by dividing the scale into 11 segments from 0 to 10 for each itefigure 1 and tables 1 and 2 show different scales to assess shoulder disability . rotator cuff calcication rotator cuff calcication is a common painful condition which occurs in up to 7.5 % of otherwise healthy adults . women are more frequently affected than men , especially during the fth decade of life [ 11 ]  . 
deposition of calcium hydroxyapatite crystal may be due to a relative hypoxia in the so - called critical zone ( about 1 cm from the tendinous insertion of supraspinatus on the greater humeral tuberosity ) [ 14 ]  . it is believed that the decrease of oxygen concentration may be responsible for brocartilaginous metaplasia and cellular necrosis followed by calcium deposit formation . tendon calcications are considered a self - healing condition , but may cause chronic pain and functional disability with a negative impact on everyday life . 
the supraspinatus tendon is most commonly affected , followed by the infraspinatus tendon and the subscapularis tendon [ 1 ]  . the formation of calcication may be considered a disorder that occurs in four stages : stage 2 stage 1 precalcic , with brocartilaginous transformation is usually tendon . 
in this phase 320 table 2 the shoulder pain and disability index ( spadi ) questionnaire is a selfadministered index that consists of 13 items divided into 2 subcategories reecting the disability ( 8 items ) and pain ( 5 items ) associated with the clinical syndrome of painful shoulder all items are rated by using a visual analogue scale from no pain or no difculty to worst imaginable pain or so difcult it required help . 
pushing with the involved arm no pain 0 1 2 3 4 5 6 7 8 9 10 worst pain imaginable disability scale how much difculty did you have : 1 . 
removing something from your back pocket no pain 0 1 2 3 4 5 6 7 8 9 10 worst pain imaginable stage 4 severe pain and restriction of movement could be associated with systemic symptoms . postcalcic phase with self - healing and repair of the rotator cuff . 
this phase lasts several months and may be associated with pain and restricted function [ 1 ]  . three types of calcic deposits have been described on us depending on the amount of calcium in the deposits . type i is hard , rich in calcium and hyperechoic with welldened acoustic shadowing and corresponds to the formative phase ( this kind of calcication is supposed to be present in up to 80 % of cases )  . 
type ii and type iii calcications are dened slurry calcication and correspond to the resorptive phase in which the deposits are almost liquid [ 1 ]  . in the presence of calcications , asymptomatic patients are treated conservatively with no need for preventive interventional procedures . 
the complications of usguided treatment are vasovagal reaction ( during the procedure ) , late painful bursitis and infection ( septic arthritis )  . different techniques are described in literature . 
1 the visual analogue scale ( vas ) is a 10 - cm graduated scale ranging from 0 ( no pain ) to 10 ( unbearable pain ) puncturing it 1015 times and then simultaneously injecting and aspirating saline solution using two 1819 - gauge needles . 
propose a less aggressive technique with a single ne needle ( 22 gauge ) to perform the procedure . this approach consists in puncturing and fragmenting the calcication with intra - calcication injection of saline solution or local anaesthetic and needle beak rotation in a single procedure . 
better results have been obtained in patients who had calcium fragments successfully aspirated from the rotator cuff compared with patients who had no calcium radiol med ( 2014 ) 119 : 318326 fragments aspirated [ 14 ]  . 
 [ 16 ] chose a 20 - gauge needle because the needles with smaller bore tend to become obstructed by the calcic fragments . the rst step of the two - needle technique proposed by serani et al . 
this step is repeated several times ( 1020 , with a total amount of 300400 ml of saline solution injected ) until the ushed uid is completely free of visible calciuone needle is then extracted and the tip of the second needle is inserted into the subacromialsubdeltoid bursa where soluble cortisone is injected . 
this study demonstrated clinical improvement , as shown by the scoring system , at 1 - month follow - up in the treated patients , but not in the controls . 
at 1 - year follow - up , no tendon cuff tear was seen [ 11 ]  . conclusion , percutaneous us - guided therapy improves the clinical outcome and accelerates the healing process . 
however , the use of two needles generates a continuous inand outow of the saline solution , thus allowing control of the saline solution pressure inside the calcication during the injection . 
the ute - beak tip of needle has to be rotated upward . at the end of the procedure , the thin calcic wall is leone or two needles may be used rim , which can cause calcium leakage into the surrounding tissue that correlates with postprocedural bursitis . 
with the one - needle technique , operators were unable to retrieve calcium from all patients [ 6 ] , while with the two - needle technique calcium retrieval was demonstrated in 100 % of the patients [ 11 ]  . 
moreover , the one - needle technique may increase the risk of postprocedural bursitis related to calcication rim disruption . viscosupplementation intra - articular injection of hyaluronic acid ( viscosupplementation ) is a new therapeutic approach for the treatment of shoulder pathology . 
several shoulder pain aetiologies have been evaluated : osteoarthritis ( oa ) , rotator cuff tear and tendinosis , periarthritis , duplays disease , adhesive capsulitis . the prevalence of oa increases with age . 
hand , knee , ankle , spine and hip joints are more frequently involved . more experience has been acquired in oa of the knee . several studies have shown the efcacy of hyaluronic acid in the treatment of oa of the knee , with positive effects on articular function , pain , subjective global assessment and reduction of nsaid consumption . 
the benet is evident within following 3 months 612 months [ 17 ]  . persists hyaluronic acid ( ha ) is a linear polymer formed of the disaccharide units n - acetyl - d - glucosamine and na - d - glucuronate and it is a physiological component of the synovial uid ( of which it represents the major chemical component ) produced by type b synoviocytes and broblasts . 
native ha ( 410 mda ) has viscoelastic properties , due to its high viscosity , which promote joint lubrication that is essential for shock - wave absorption and has protective effects on the articular cartilage and soft tissue surfaces of the joints . 
this would explain why ha has a long - term clinical effect compared to the short half - life of the preparations . nowadays , there are several commercially available ha by - products obtained from rooster combs or with recombinant technology that differ in molecular weight and therefore in rheologic properties [ 18 ]  . molecular preparations ( lmw weight 0.51.5 mda ) demonstrate a good penetration in the extracellular matrix of synovial uid . 
the limit of these 322 radiol med ( 2014 ) 119 : 318326 preparations is the low viscoelasticity compared to the native ha ( in vitro and animal model studies ) [ 17 , 18 ]  . 
for this reason , more recently , attention has been shifted to high molecular weight preparations ( hmw 67 mda ) formed by a cross - linked ha ( hylan f - 20 )  . 
in osteoarthritis , treatment with weekly ha injections for three consecutive weeks improves the clinical scores and achieves pain relief at 6 months follow - up [ 19 ]  . in elderly patients there are several problems in the management of shoulder pathe classic approach is based on nonpharmacological strategies such as the use of orthotics , physiotherapy , exercise and activity modication . 
chronic use of oral nsaids increments the risk of gastrointestinal ( gi ) effects ; the use of selective nsaids reduces the risk of gi effects , but is linked to cardiovascular complications . 
one rare , but severe complication is osteonecrosis . viscosupplementation is indicated in patients where the pain relief strategies have failed , those with intolerance to nsaids and in those with intermediate kellgrenlowrence score ( iiiii )  . 
the contraindications are hypersensitivity to egg proteins and rheumatologic systemic diseases ( rheumatoid arthritis , gout , psoriasis , chondrocalcinosis )  . in particular , an open - label non - randomised trial evaluated the efcacy of us - guided high molecular weight ha injection in elderly patients with massive rotator cuff tear arthropathy and advanced osteoarthritis . 
these were all patients for whom surgery was not feasible because of coexisting medical conditions and because of the poor results expected with any kind of surgical intervention . the injection is performed under us guidance ( this approach is mandatory since blind injection is associated with high failure rate )  . 
then , the physician induces local anaesthesia in the subacromial chloridrate without space 2 % mepivacaine using adrenaline ( maximum 400 mg of local anaesthetic during the entire procedure )  . 
the results showed signicant improvement of the vas and constant score in the rst 4 months of follow - up in the treated patients , but not in the control subjects . 
after 5 months the outcome of treated and nontreated patients was not different [ 20 ]  . in rotator cuff lesions without complete tear , the subacromial injection of sodium hyaluronate is effective . 
in 25 patients , 25 mg / week of sodium hyaluronate was administered into the subacromial bursa for 5 consecutive weeks . the control group was given 2.5 ml of sodium chloride with the same injection protocol . 
the second group did not demonstrate global improvement or signicant differences in the constant and vas scores after the injection [ 21 ]  . the efcacy of ha is also demonstrated in supraspinatus tendinosis . 
the technique differs from subacromial injections in that after determining supraspinatus tendon thickness and its superior border , the needle is introduced as far as the surface of the superior border and then the solution is injected . the patients were divided into two groups : a sodium hyaluronate group and a sodium chloride group . 
note the absence of the needle ( arrows ) and the injection of supraspinatus tendon , hyaluronic acid ( hypoechoic area ) radiol med ( 2014 ) 119 : 318326 group , shoulder disability resolved after three ( 6 out of 28 ) , four ( 13 out of 28 ) and ve ( 6 out of 28 ) injections of sodium hyaluronate . 
in the second group , all patients were unsatised ( no signicant variation of pretreatment vas score at 4 and 12 weeks follow - up ) and none of them reported a sufcient improvement after ve injections of lidocaine and sodium chloride solution [ 22 ]  . the limitations of these open - label studies are the use of nsaids after the procedure , low patient number and short follow - up period . viscosupplementation under us guidance may constitute a valid tool in pain relief and functional recovery in elderly patients with massive rotator cuff tear arthropathy , noncomplete rotator cuff tears , mild osteoarthrosis ( kellgrenlowrence iiiii ) and supraspinatus tendinosis , but more studies , with particular attention to long - term evaluation , are necessary to validate this new therapeutic approach in shoulder pathology . adhesive capsulitis adhesive capsulitis is a condition of varying severity characterised by the gradual development of global limitation of active and passive shoulder motion where radiographic ndings other than osteopenia are absent [ 23 ]  . in 1872 , duplay [ 24 ] described a condition called periarthritis scapulo - humerale , and later codman [ 25 ] described this disorder as frozen shoulder and until now adhesive capsulitis of the shoulder remains a class of cases which are difcult to dene , difcult to treat , and difcult to explain from the point of pathology . 
it has been suggested that frozen shoulder is precipitated by degeneration of the supraspinatus tendon and is associated with chronic inammation of the subacromial bursa and glenohumeral capsule . several conditions are related to adhesive capsulitis such as female sex , trauma and age of more than 40 years , diabetes , prolonged immobilisation , thyroid disease , stroke and myocardial infarction , presence of autoimmune disease , epilepsy , parkinson and dupuytren disease . 
histologic and immunocytochemical investigations have demonstrated active broblastic proliferation accompanied by the transformation of broblasts into myobroblasts [ 26 ]  . this condition is widely reported as a typical middle age disease and is characterised by three phases : a painful phase , lasting between 3 and 8 months , followed by a phase of progressive stiffness or adhesive phase that typically lasts 46 months . 
the nal resolution phase of gradual return of motion usually lasts 524 months [ 27 ]  . the most characteristic mr arthrography ndings in frozen shoulder are the thickening of the coracohumeral ligament and of the capsule at the rotator cuff interval and the complete obliteration of the fat triangle under the coracoid process ( subcoracoid triangle sign ) [ 28 ]  . surgical treatment ( arthroscopic capsulotomy ) is the best choice when conservative treatment fails . injection therapy is often used when ice therapy , oral nsaids and corticosteroids are not enough to improve the clinical outcome . 
corticosteroid injection , ha injection , distension with sodium chlorate , distension with sodium chlorate and steroids and distension with air and physical therapy ( exercise , transcutaneous electrical nerve stimulation , us ) are used for conservative management of adhesive capsulitis . to the biceps interval medial us - guided corticosteroid and lidocaine injection into the tendon shows rotator improvement in primary outcome ( spadi score after 12 weeks ) and in secondary outcome ( increase in active and passive range of motion ) [ 30 ]  . 
capsular distension with lidocaine and ha is effective as well as steroid injection in pain relief and functional improvement [ 31 ]  . comparison of different treatments demonstrates that ha injection is an alternative therapy in adhesive capsulitis . 
isolated intra - articular hyaluronate injection has an equivalent outcome compared to intra - articular corticosteroid injection [ 32 ]  . moreover , us - guided injection associated with shoulder exercises ensures pain relief in the medium - term follow - up with respect to injection alone [ 33 ]  . 
when corticosteroids are contraindicated , hyaluronic acid injection is a proper alternative tool . acromion - clavicular joint osteoarthrosis of the acromionclavicular ( ac ) joint is a common pathology observed in elderly patients and in young adults in some cases . 
the joint is injected with the use of a local anaesthetic have evolved from open repair with transosseous suture to arthroscopic techniques ( single - row , double - row , transosseous - equivalent suture , anchor constructs )  . 
cytokines and growth factors ( including pdgf , tgf - a , tgf - b , interleukin - 1 and broblast growth factor ) play an important role in chemotaxis , matrix synthesis , proliferation and cell differentiation , and a combination of these factors may improve healing of the tendon insertion site . platelet - rich plasma ( prp ) is a fraction of plasma that has been isolated and used to enhance the regeneration of bones and soft tissues . 
blood is taken from the patient ; the prp fraction is separated and transferred to a bottle without air contact to prepare a membrane of autologous brthe nal product , the membrane , obtained by high - speed centrifugation of prp is a thin layer that is very rich in platelets containing approximately 50 times the platelet concentration of initial blood . 
a systematic review demonstrated that prp does not have any effect on overall re - tear rates or shoulder - specic outcomes after arthroscopic rotator cuff repair in patients with full - thickness rotator cuff tears [ 38 ]  . surgical intervention allows debridement of degenerated cuff and partial thickness cuff tears , subacromial bursitis , impinging bone spurs and osteophytes together with rotator cuff repairs . 
repairs of degenerated and torn tissue are often prone to failure due to many intrinsic and extrinsic factors . prp might improve clinical , mechanical and histological outcomes [ 39 ]  . 
thus , the treatment of tendinosis with a usguided injection of concentrated autologous platelets may be a nonsurgical alternative . some studies are trying to validate the role of prp tendon injection . 
in chronic lateral epycondilitis , prp improves pain and function more effectively than corticosteroid injection sustained over a 2 - year follow - up time with no reported complications [ 40 ]  . 
5 needle ( arrows ) in subacromialsubdeltoid bursa ( asterisk )  . the bursa is distended with the use of corticosteroids the level of the ac joint are the typical x - ray ndings in ac osteoarthritis . 
ac injection of steroid and lidocaine solution is indicated in the conservative treatment ( tossy and allman classication type i - ii [ 36 ] ) of painful ac osteoarthrosis . 
clinical improvement evaluated through vas at rest , vas with local pressure ( exerting pressure on the joint space area ) , and constant and murley scores is considerable , especially in the rst week after therapy [ 37 ]  . platelet - rich plasma rotator cuff tears are common injuries that require surgical treatment . 
 [ 42 ] concluded that patients who received prp injection demonstrated modest improvement in functional outcome measures , while the mri appearance of diseased achilles tendons remained largely unchanged following prp injection [ 42 ]  . us guidance can be a valid tool to perform a procedure with a proper selection of injection site , because it allows one to identify the disrupted tendon bres with loss of normal echostructural brillary areas . 
patients were followed up for up to 12 months . results a complete response was obtained in 87 % lesions treated with pla and in 93 % lesions treated with rfa ( p = ns )  . 
angelico epatologia , policlinico universitario tor vergata , viale oxford 81 , 00133 rome , italy conclusions rfa is more effective in the treatment of hcc compared to pla for lesions c21 mhowever , pla should be considered a viable treatment option for incidence of hcc b20 mm , complications . in view of the lower keywords hepatocellular carcinoma ( cid : 2 ) liver cirrhosis ( cid : 2 ) percutaneous laser ablation ( cid : 2 ) radiofrequency thermoablation introduction hepatocellular carcinoma ( hcc ) is the sixth most worldwide malignancy for incidence [ 1 ] and the third leading cause of global cancer mortality [ 2 ] , causing 600 , 000 deaths per year [ 2 , 3 ]  . 
in the western world , more than 90 % of hcc develop in patients with liver cirrhosis [ 4 ]  . surgical resection is the rst - choice treatment , but it is not always feasible due to the location and number of lesions , and the progressive deterioration of liver function in patients with cirrhosis [ 5 , 6 ]  . 
today , the gold standard is liver transplantation ( lt ) , which is , however , difcult to implement for lack of donors [ 7 ]  . over the years , several techniques for locoregional ablation have been developed for the treatment of patients with hcc nodules smaller than 4 cm , who are not candidates for resection or transplantation . 
these techniques include percutaneous ethanol injection ( pei ) , percutaneous injection of acetic acid ( pai ) , microwave thermoablation ( mw ) , cryoablation , high - intensity focused ultrasound ( hifu ) , radiofrequency ablation ( rfa ) and percutaneous laser ablation ( pla ) [ 8 ]  . 
a meta - analysis of prospective randomised trials has shown that rfa of liver radiol med ( 2014 ) 119 : 298308 tumours has greater efciency compared to pei in terms of ease of implementation , safety , applicability and costeffectiveness [ 10 ]  . 
pla can be compared to rfa in the treatment of patients with hcc , in terms of technical reliability and efcacy [ 14 ]  . to our knowledge , few studies have compared and analysed the results obtained with the two techniques [ 15 ]  . the aim of this study was to compare pla and rfa in terms of complete response at 30 days , local recurrencefree survival , complications such as postablation syndrome and postprocedural tnfa production , in cirrhotic patients with unifocal hcc b4 cm . materials and methods this study was approved by the ethics committee of our institution . 
the afetoprotein levels recorded before the procedure were on average 561.67 684 ng / ml ( range 251 , 870 ) ( table 1 )  . all patients were selected during the follow - up for liver cirrhosis , and the choice of percutaneous ablative treatment was made by a multidisciplinary team of hepatologists , surgeons and interventional radiologists [ 16 ]  . 
we selected patients with a single hcc nodule with a maximum diameter of 40 mthe diagnosis of hcc was established in accordance with the guidelines of the european association for the study of the liver ( easl , european association for the study of liver ) [ 17 ] for the diagnosis of hcc in cirrhotic patients . 
exclusion criteria were child class c , poor clotting function ( quick test \50 % , platelets \40 , 000 / ml ) and the presence of multiple lesions . randomisation software was used to allocate each patient to a treatment group . 
to assess any changes in the size of the nodule and / or the appearance of new lesions , within 2 weeks of treatment all patients underwent dynamic multislice ct ( light speed 64 ct , ge medical systems , milwaukee , usa ) with triphasic technique ( 30 , 65 and 180 s ) administration of 125175 ml of 350 mgi / ml iodinated contrast medium , depending on body mass index , injected at a rate of 35 ml / s via automatic injector and followed by 2030 ml of saline solution . 
if necessary , conscious sedation was achieved with fentanyl or midazolam administered intravenously , and drug administration was modulated case by case . local anaesthesia of the insertion point was achieved with the administration of 10 ml of 2 % lidocaine . 
needle introduction was also guided by ceus in the case of iso - echogenic lesions or those poorly detectable with conventional ultrasound . needle size was made in relation to lesion width and desired safety margin ( at least 5 mm )  . 
the needle electrode was introduced under ultrasound guidance ( iu22 ultrasound system philips healthcare , best , the netherlands ) using a 3.5 - mhz convex probe ( c5 - 1 philips healthcare , best , the netherlands )  . 
at the end of the procedure , after automatic cooling of the rf system , the generator was reactivated to ablate the needle tract so as to prevent tumour dissemination . 
an unenhanced ct scan was performed at the end of each treatment . percutaneous laser ablation assessment of treatment effectiveness and follow - up percutaneous laser ablation ( pla ) was done with the echolaser xvg systethis system is composed of a mylab7070xv - l ultrasound scanner ( esaote biomedica , italy ) and an echolaser x4 laser generator ( esaote el.en. , florence , italy ) which uses a neodymium yttriumaluminiumgarnet light source , nd - yag laser , with a continuous wavelength of 1.064 lm 10 ninsertion of the bres within the lesion was performed using the method described by pacella et al . 
the bres were positioned in a square conguration with the tips placed 11.5 cm apart to obtain a correct ablation geometry and complete necrosis with a sufcient safety margin of at least 5 mchiba needles were inserted within the lesion using ultrasound guidance with a 3.5 - mhz convex probe ( ca621 , esaote biomedica , italy ) , through a single point of entry . 
their correct positioning was then evaluated with ct . each stylet was extracted and replaced with a exible , attip , sterile 300 - lm - diameter quartz bre with a bare tip . 
after treatment , the bres were retracted with the laser switched on so as to avoid possible tumour seeding . an unenhanced ct scan was performed at the end of each treatment . radiofrequency thermoablation radiofrequency thermoablation ( rfa ) was performed using an rf 3000 generator ( boston scientic corporation , natick , ma , usa ) , which provides a maximum power of 200 w . 
we used expandable 15to 17 - gauge leveen needles ( boston scientic corporation , natick , ma , usa ) with hook extension ranging from 2 to 5 cm in diameter . 
the image acquisition technique was the same as described for preprocedural evaluation . the pretreatment tumour volumes and the ablation lesion volumes were calculated on a ct console ( advantage workstation 4.3 ge medical systems , milwaukee , usa ) with the use of volume rendering reconstructions . the volumes were selected by means of manual segmentation using the images acquired during the arterial phase , in which the tumour mass was more easily visualised . subsequently , the same phase was used for the evaluation of the parenchyma after thermal ablation . 
the preand postprocedural images were then merged using the same eld of view and six translation and rotation parameters in three reformatting planes , to make them comparable . treatment response was evaluated according to the mrecist criteria [ 18 ]  . 
based on the ct results , tumour ablation and thus response to treatment were dened as complete ( cr , complete response ) when there was disappearance of all signs of lesion and no pathological enhancement was detectable on the edges of the area treated . 
ablation was considered partial or incomplete ( pr , partial response ) in the case of a partial destruction of the lesion , with a reduction of at least 30 % of the volume ; in the case of incomplete ablation , a second treatment was carried out during the following 2 weeks . patients with cr entered the follow - up programme , which included clinical evaluation and a - fetoprotein assay . three - phase dynamic ct at 3 , 6 and 12 months was performed in all patients . 
the medium - term results were assessed according to local progression of disease , meaning the presence of enhancement in proximity to the ablation site and the onset of new lesions at a distance . complications intraand postprocedural complications were evaluated according to the classication of the international society radiol med ( 2014 ) 119 : 298308 of radiology ( sir , society of interventional radiology ) [ 19 ] distinguishing between major complications ( events that lead to substantial morbidity and disability , an increase of care , hospitalisation or a longer hospital stay ) and minor complications . 
the presence of a postablation syndrome , dened as fever , nausea , vomiting and abdominal pain referred to the shoulder , was investigated during the rst 48 h after treatment . evaluation of tnfa production by peripheral blood cells for each treatment , blood samples were obtained 4 h before the beginning of the ablation procedure , and again 24 h and 5 days after the procedure . 
pbmc were cultured at a concentration of 2 9 106 for 18 h in 20 ng / ml of phorbol - 12 - myristate - 13acetate ( pma ) and 500 ng / ml ionomicine ( io ) ( calbiochem - novabiochem intl , la jolla ca )  . 
they were then resuspended at a concentration of 2 9 105 in 20 ll of buffered saline and incubated for 15 min with the monoclonal antibody cy - chrome - conjugated ( cy - chrometm ) anti - cd14 ( pharmingen , san diego , ca )  . 
tnfa production was evaluated cd14 ? tnfa ? cells compared to total cd14 ? cells . five healthy subjects , comparable in terms of age and sex with the two patient groups , were enrolled as controls . calculating percentage statistical analysis the nonparametric mannwhitney test and fischers exact test were used to compare the two treatment groups . 
therefore , a cr was obtained in 87 % of lesions treated with pla and 93 % of those treated with rfa , and a sufcient safety margin was obtained in all of these patients . 
the three patients who failed to achieve a cr , even after the second procedure , were treated with another type of procedure ( chemoembolisation )  . twenty - seven patients entered the follow - up programme of our study . 
in particular , these included pleural effusion ( 1 pla vs 4 rfa ) , perihepatic effusion ( 1 pla vs 3 rfa ) and subcapsular haematoma ( 1 rfa )  . 
4 a 74 - year - old woman affected by chronic hepatitis with hepatitis b virus ( hbv ) - related hepatocellular carcinoma ( hcc ) the level of hepatic segment vi treated with nodule located at percutaneous laser ablation . 
preprocedural computed tomography ( ct ) study : axial ( a ) and volume rendering image ( b ) show the size ( 20 mm ) and location of the nodule ( black arrow )  . 
5 a 69 - year - old woman affected by chronic hepatitis with hepatitis c virus ( hcv ) - related hcc nodule located at the level of hepatic segment vii treated with percutaneous laser ablation . preprocedural ct study : axial ( a ) and volume rendering image ( b ) showing the size ( 24 mm ) and location of the nodule ( black arrow )  . 
ct scan at 30 days : c axial image shows the success of the treatment and volume rendering image shows the size of the area of ablated tumour parenchyma compared to the volume rendering dimensions at preprocedural ct ( d )  . 
ct follow - up at 6 months : e , f axial images show disease recurrence below glissons capsule ( white arrow ) radiol med ( 2014 ) 119 : 298308 fig . 
preprocedural ct study : axial ( a ) and volume rendering image ( b ) show the size ( 23 mm ) and location of the nodule ( white arrow )  . 
 [ 23 ] in a study investigating the variables affecting local progression of disease , which indicates , however , a 3 - cm cutoff under which the best results are obtained . formation of an inadequate safety margin is one of the most signicant risk factors for local disease progression , and many authors agree that this margin should be between 5 and 10 mm [ 24 , 25 ]  . 
as a result , the lower survival rate seen in patients with tumours larger than 2 cm treated with pla may be justied by the absence of an adequate safety margin . this condition may be essentially due to the limited capacity to monitor tissue necrosis formation during treatment . ultrasound and ct are the most widely used low - cost imaging techniques used to monitor ablation procedures . however , the heating of tissue , which occurs during the 306 radiol med ( 2014 ) 119 : 298308 thermal ablation procedures , leads to the formation of gas which , while providing an indication of the tissue destruction , hinders evaluation with ultrasound [ 26 ]  . 
therefore , as it is impossible to use a lowcost imaging technique that is unaffected by this limit , local ablation systems have been developed to try to recreate a volume of tissue necrosis which is easily reproducible , using standard protocols that are independent of the monitoring technique used . 
therefore , a feedback system is necessary to enable tissue changes to be monitored during treatment , and in this sense rfa seems to have a signicant advantage over pla : the continuous recording of tissue impedance during rfa provides , albeit briey , the tumour ablation process . achieving power roll - off , which indicates tissue coagulation , can improve the effectiveness of the locoregional treatment itself [ 29 ]  . 
furthermore , an additional variable that can make it difcult to obtain a precise safety margin might be the relative difculty in placing multiple pla needles in the correct ablation geometry needed to induce an adequate and reproducible necrosis volume [ 23 ]  . 
moreover , the rigidity of the cirrhotic liver [ 30 ] can make it difcult to control the needle trajectory owing to the exibility of the needles , such as the pla needles used in our study , which have a smaller calibre ( 21 gauge ) than the rfa needles ( 1517 gauge )  . information about the main limitations of our study lie in the small size of the population sample ( 30 patients ) and the midterm follow - up ( 12 months )  . 
however , the possibility of carrying out a prospective randomised study , with no evidence of any statistically signicant difference between groups , allowed us to minimise possible bias in the statistical analysis . 
similarly , the fact that we focused on few variables , such as lesion size and type of treatment , made it possible and feasible for us to evaluate results as early as 1 year after treatment . 
in fact , the aim of our study was to evaluate the locoregional effectiveness of the single treatment , because the results of long - term evaluations are often not directly related to the effects of the treatment . 
indeed , especially in hcc , disease - free survival time and overall survival may be related to the aetiology and severity of the liver failure associated with cirrhosis and histological type of tumour . 
in contrast , the end point evaluated in our study , local disease progression - free survival or recurrence - free survival , could be considered a direct expression of the effectiveness of a locoregional ablation procedure [ 31 ]  . with regard to the evaluation of periprocedural complications , both techniques proved to be safe . 
in particular , asymptomatic perihepatic effusion was observed in four patients , pleural effusion in ve patients and subcapsular haematoma in one . these complications occurred more frequently in patients treated with rfa than with pla . 
in particular , in our study we documented the onset of a postablation syndrome in only one of the patients treated with pla , whereas 12 patients treated with rfa developed this syndrome . in this regard , we also examined the possible postprocedural increase of tnfa in the two treatment groups . tnfa is one of the possible mediators of a systemic inammatory response that can occur following treatment with rfa or cryoablation and is therefore one of the factors that inuence the occurrence of postablation syndrome [ 3436 ]  . 
 [ 34 ] , who evaluated the production of serum proinammatory cytokines , including tnfa , in a cohort of 13 patients with liver cancer subjected to pla under mr guidance . in our study , the nding that tnfa production did not increase might account for the lower rate of onset of postablation syndrome in patients undergoing pla . 
this higher concentration of tnfa in subjects treated with rfa may thus in part explain the more frequent occurrence of the postablation syndrome in the rfa compared to the pla group seen in our study . conclusions the ability to induce complete tumour necrosis did not show statistically signicant differences between the two ablation techniques ; however , for lesions with diameter c21 mm rfa yielded better results 1 year after treatment . a small number of minor complications were observed in patients treated with pla . 
morganti received : 20 september 2012 / accepted : 30 january 2013 / published online : 6 december 2013 ( cid : 2 ) italian society of medical radiology 2013 abstract objective this study was done to assess the impact of clinical factors and in particular the use of drugs for concomitant illnesses on late radiation - induced rectal bleeding in patients with prostate cancer . materials and methods patients with histologically proven prostate adenocarcinoma treated with radical radioleast 6 months were therapy and followed up for at selected . 
the correlation between late rectal bleeding and a number of factors was investigated by univariate and multivariate analysis . results a total of 278 patients who underwent radiotherapy at our institution between october 2002 and may 2011 were selected . 
if these results are reconrmed by larger clinical series , calcium channel blockers may be tested as radioprotector agents in clinical trials . keywords radiotherapy ( cid : 2 ) rectal bleeding ( cid : 2 ) radioprotector ( cid : 2 ) hypertension ( cid : 2 ) calcium channel blockers introduction prostate cancer is the most frequently diagnosed cancer in men [ 1 ]  . 
nevertheless , it is likely that the role of modern dose - escalated radiotherapy will further increase even in low and intermediate risk prostate cancer , since recent data suggest it could provide better biochemical disease - free survival than surgery in all risk classes [ 3 ]  . gastrointestinal and bladder complications represent the main limit to radiation dose escalation in prostate cancer . particularly rectal bleeding is the most commonly reported bowel toxicity , with a cumulative incidence of up to 50 % in 5 years in patients undergoing three - dimensional ( 3d ) dose - escalated radiotherapy [ 4 ]  . 344 radiol med ( 2014 ) 119 : 343347 in recent years , several results have demonstrated a clear correlation between rectal dose - volume histograms and the risk of rectal bleeding [ 5 , 6 ]  . 
in order to spare as much volume of rectum as possible while delivering higher radiation dose to the prostate , more advanced and often more expensive techniques , such as intensitymodulated image - guided radiotherapy ( igrt ) have been introduced in clinical practice , leading to a reduction in the risk of rectal bleeding [ 7 ]  . radiotherapy ( imrt ) besides dosimetric factors , also clinical variables such as previous abdominal surgery , presence of haemorrhoids , and use of antihypertensive medications can affect the risk toxicity after radiotherapy [ 8 ]  . 
although of late rectal arterial hypertension or the use of antihypertensive drugs seems to be protective for the development of late effects [ 8 , 9 ] , no clear effect has been shown for any specic antihypertensive drug . the objective of this analysis was to assess the impact of clinical factors and in particular the use of drugs for arterial hypertension on late radiation - induced rectal bleeding . 
the effects of several potentially confounding parameters ( comorbidity , abdominal surgery , radiotherapy dose and technique ) were also analysed . materials and methods study design patients with histologically proven prostate adenocarcinoma treated with either 3d conformal radiotherapy ( 3dcrt ) or imrt for radical intent and followed up for a minimum of 6 months were selected for this retrospective analysis . 
to this end , if no previous prostatectomy had been performed , patients had intra - prostatic gold ducials implanted . comorbidities , previous abdominal / pelvic surgery , use and type of drugs , previous / concomitant locoregional diseases , height and weight , were recorded before radiotherapy . 
rectal toxicity occurring during and within 3 months after the end of radiotherapy , was also recorded according to radiation therapy oncology group ( rtog ) criteria [ 11 ]  . the end of statistical analysis the correlation between late rectal bleeding and a number of factors was investigated by univariate and multivariate analysis . 
the following parameters were considered : body mass index ( [ or b30 ) , pre - treatment morbidities ( arterial hypertension , diabetes mellitus , chronic pulmonary disease ) , hormone therapy , drug prescription during radiotherapy ( use and / or anticoagulants ) , abdominal surgery prior to radiotherapy ( radical prostatectomy or other surgical procedures including rectum - sigma resection , kidney resection , cholecystectomy , appendectomy , prostatic adenomectomy ) , irradiation of pelvic nodes , delivered dose ( equivalent dose in 2 gy per fraction with a / b = 3 , eqd2 [ or b70 gy ) , and technique used ( imrt or 3dcrt )  . antihypertensives and type of standard time - to - event ( survival analysis ) methodology was used to assess the rst reported incidence of toxicity . events were timed from the end of radiotherapy , and the differences between the treatment groups were rst tested using the log - rank test . 
relative risks of late rectal bleeding according to treatment are summarised using hazard ratios from cox ( hr ) with 95 % condence intervals ( ci ) regression models . the difference of the incidence of grade c2 acute rectal toxicity between the treatment groups was assessed using the chi - square test with yates correction . results a total of 278 patients who underwent radiotherapy at our institution between october 2002 and may 2011 were selected for this analysis . 
even the presence of chronic pulmonary disease seemed to favour the development late rectal bleeding , although statistical signicance was not reached . biochemical disease - free survival was not affected by calcium blockers use or radiation dose . discussion in recent years many attempts have been made to protect the rectum against radiation damage by using topical or oral medications [ 1215 ]  . 
both misoprostol rectal suppositories [ 13 ] , and amifostine enemas [ 14 ] seem to exert some protective effect on late proctitis , while oral balsalazide appears to be effective in reducing the symptoms of acute proctitis [ 12 ]  . 
in this analysis , we found that the use of calcium channel blockers for arterial hypertension during and after radiol med ( 2014 ) 119 : 343347 radiotherapy may exert some protective effect against the development of rectal toxicity . 
although many authors have reported that arterial hypertension or antihypertensive medications may be protective for the development of late radiationinduced rectal effects [ 8 , 9 ] , this is the rst time a protective effect was shown for a specic antihypertensive drug . the potential use of calcium antagonists as radioprotectors was suggested by battaini et al . 
furthermore calcium channel blockers have antioxidant properties and have been shown to protect against free radical - mediated injury of cardiovascular cells [ 17 ] suggesting a possible mechanism for radioprotection . until recently , it was considered that the initial damage of the intestinal toxicity of irradiation was the destruction of epithelial stem cells within the crypts of lieberkuhn , causing the destruction and progressive failure of cell renewal , which explains the time delay between rectal mucosa irradiation and the onset of symptoms [ 18 , 19 ]  . more recently it has been suggested that an endothelial injury may occur before crypt stem cell damage in the evolution of the radiation - induced gastrointestinal syndrome [ 20 ]  . 
nifedipine , a calcium channel blocker , has been shown to improve endothelial function in patients with hypertension , at least partly , by enhancing endothelial thus preserving progenitor cell numbers and activity , endothelial integrity [ 21 , 22 ]  . in addition , calcium channel blockers are well tolerated and associated with minimal side effects [ 23 ] , the most common being ushing , headache , hypotension , and pedal oedema . 
adverse effects have been reported in approximately 17 % of patients using nifedipine , in 9 % of patients using verapamil , and in 4 % of those using diltiazem [ 24 ]  . although with the limits of a retrospective analysis , in our experience the use of calcium channel blockers resulted in a protective effect against late rectal bleeding . a recent systematic review of 11 published reports including 4 , 559 patients suggests there is at a minimum no difference , and in many cases superiority , imrt compared with 3dcrt for the radical treatment of localised prostate cancer in terms of acute and late gastrointestinal and genitourinary side effects in the setting of doseescalated ( [ 70 gy / 2 gy fractions ) radiotherapy [ 25 ]  . 
laparoscopy and histopathology anna lia valentini benedetta gui maura micco` maria carla mingote valeria ninivaggi maurizio guido gian franco zannoni eleonora marrucci lorenzo bonomo received : 21 january 2013 / accepted : 20 may 2013 / published online : 3 december 2013 ( cid : 2 ) italian society of medical radiology 2013 abstract objective to verify whether the capability of magnetic resonance imaging ( mri ) in diagnosing deep inltrating colorectal endometriosis ( dice ) is improved using an association of mri ndings . methods and materials the imaging database of our institute of radiology was retrospectively reviewed to identify patients subjected to mri for a suspicion of deep inltrating endometriosis . 
gemelli 8 , 00168 rome , italy the rst section and the second criterion in the second one . mri results were compared with la or histopathology as the gold standard by 2 9 2 tables and statistically analyzed ( k statistics )  . 
likelihood - ratio test was also performed , being independent from the prevalence of the disease . results by consulting case sheets , 33 / 50 females ( ranging age 2439 years , mean age 32.2 years ) who were subjected to mri also underwent la . 
intestinal resection for dice was performed in 11 / 33 patients ; in 22 / 33 supercial intestinal foci , adhesions / nodules in the fat plane were simply removed . 
among the imaging techniques employed in the preoperative evaluation of deep inltrating colorectal endometriosis ( dice ) , transvaginal or transrectal ultrasound as well as computed tomography enteroclysis has been used [ 3 , 4 ]  . 
however , they are limited by the small eld of view and radiation exposure , respectively . magnetic resonance imaging ( mri ) is employed to investigate chronic pelvic pain in women [ 5 ] and is currently used in the assessment of deep endometriosis [ 68 ]  . 
applied lters were : interval technique , mri ; udc ( cost - centre diagnostic unit ) , gynaecology ; uld ( logistic diagnostic unit ) , dysfunctional gynaecological unit ( endometriosis clinic )  . 
the radiologists assessing the mri data were not aware of the patients clinical symptoms or outcome . mri technique the mri protocol applied in our department for the investigation of suspected pelvic endometriosis involves the use of a 1.5 - t superconducting magnet ( vectra , ge medical systems , milwaukee , usa ) with a phased - array coil and no rectal distension . 
to limit intestinal peristalsis and to attenuate uterine contractions , patients receive an intramuscular injection of 1 mg of hyoscine nbutylbromide ( buscopan , schering , berlin , germany ) before the examination and are always studied after the 8th day of the menstrual cycle , to better identify hyperintense bleeding lesions . axial t1 - weighted spin echo images , axial coronal and sagittal t1 - weighted gradient - echo images with fat suppression ( to better evaluate hyperintense bleeding lesions ) , axial , coronal and sagittal t2 - weighted fast spin echo images and axial t2 - weighted fast spin echo images extending to the upper abdomen ( to evaluate hydronephrosis ) are always obtained . 
dynamic axial and delayed phase sagittal images acquired with lava ( liver acquisition with volume acquisition ) sequence ( 3d t1 - weighted gradient echo ) after intravenous injection of gadoliniumbased contrast material are employed in selected cases . 
in particular , lava sequences with fat saturation are usually employed when endometriomas exclude degeneration , or also in the case of adenomyosis to verify the grade of inltration of the myometrial surface . this study was retrospective and images after contrast medium injection ( gadobenate dimeglumine , multihance 0.5 m , bracco , milan , italy ) were not always available . 
for this reason and since no signicant benet of intravenous gadolinium has been reported in the literature for deep pelvic endometriosis [ 8 ] , the mri evaluation was performed only on the t2 - weighted images . 
both types of lesions can be found in the fat plane adjacent to the bowel wall in the case of deep endometriosis , such as fresh nodules in the recto - vaginal septum or brotic lesions at the uterine torus . 
bowel wall thickness was not evaluated in terms of millimetres , as it is a relative rather than absolute evaluation , related to the morphology of the near proximal and distal portion of the same intesthickness of the bowel wall tinal segment . 
the term radial and retracting shape can be considered a 1.5 - t version of the mushroom cap sign [ 2 ] and resembles the fan shape conguration of the bowel wall , as recently described at 1.5 - t imaging for the diagnosis of dice [ 11 ]  . to assess what association of signs improves dice diagnosis , mri evaluation was carried out twice by two radiologists with 10 and 15 years of experience in female pelvic mri , who reviewed the images in consensus in two separate sessions . 
in the rst evaluation ( rst diagnostic criterion ) , any general thickness of the bowel wall combined with nodules or plaque - like lesions in the adjacent fatty plane was considered abnormal , while in the second evaluation ( second diagnostic criterion ) , only the characteristic radial and retracting shape of the thickened bowel wall was considered abnormal when associated with nodules or plaque - like lesions in the adjacent fatty plane . when no nodule or plaque - like lesion was identied in the adjacent fatty plane , the presence of bowel thickness , even without adjacent fat - plane interface , was always considered a normal result . 
la or histopathology specimens in the case of surgical resection were taken as the gold standard . reference standard and statistical analysis la was always performed at the hospital by a single skilled gynaecological surgeon specialised in endometriosis who was assisted by an intestinal surgeon in the case of bowel resection . supercial , or serosal , involvement of the bowel was directly diagnosed by la visualisation and was treated by supercial excision , serosal shaving and adhesion removal . dice was suspected in the event of large or disc - like implants retracting the wall and was treated by segmental bowel resection . all surgical specimens were sent to the hospitals histopathology service and were xed in 10 % formalin for 24 h . 
the diagnosis of endometriosis was made on the basis of the presence of endometrial - type epithelium and endometrial stroma on bowel resection specimens . the mri results in dice diagnosis obtained using the two previously described criteria ( see mri data ) were compared by 2 9 2 tables with the gold standard histopathology in the case of bowel resection for dice . 
comparison of mri results with la was used in patients not subjected to bowel resection because of the absence of disease or presence of supercial foci ( no dice ) and were statistically analysed ( k statistic )  . 
the likelihood ratio test was also applied , being independent of the prevalence of disease . results thirty - three patients ( age range 2439 years ; mean age 32.2 ) met the inclusion criteria . 
first criterion : absence or presence of generally thickened intestinal wall surrounded by fat - plane interface represents a normal result ( no deep inltrating colorectal endometriosis ) ; the simultaneous presence of nodules or hypointense plaque - like lesions in the fat plane and is an abnormal result ( deep generally thickened intestinal wall inltrating colorectal endometriosis )  . 
the better diagnostic performance of the second diagnostic criterion was also conrmed with the likelihood ratio test . to our knowledge , the overall accuracy of t2 - weighted mri is higher in this study ( 97 % ) than in other published reports on the same subject ( 7596 % ) [ 4 , 6 , 7 , 10 , 11 ]  . some reports identied a typical mri morphology of the intestinal wall thickness in the case of dice : the mushroom cap sign was rst reported at 3.0 - t magnet imaging [ 2 ] , but it was not statistically validated because of the small number of patients in that series . 
taking into consideration a combination of signs , or the contemporary presence of nodules or plaque - like lesions in the adjacent fat - tissue plane and the typical bowel thickness morphology for the correct assessment of dice , as this study suggests , can be justied by the course of the disease . 
even if retracting adhesions and muscle layer invasion are responsible for in a typical producing the protrusion which results semicircular morphology of the involved intestinal tract variously known as the mushroom cap sign , the fan shape image or the radial and retracting shapeit should be noted that dice is improbable when no nodules or plaque - like lesions are detectable in the fat - tissue plane closely adjacent to the bowel wall . 
the results of this study conrm the usefulness of this association of signs . including pelvic pain , colorectal endometriosis can cause severe intestinal symptoms , rectal bleeding and problems with defaecation , but needs to be surgically treated by intestinal resection only in the deeply inltrating type , since brosis invading the intestinal wall muscularis does not respond to medical therapy and advanced disease can be complicated by lumen obstruction [ 8 ]  . 
supercial endometriosis lesions located on the serosa surface are not easily evaluated by imaging [ 2 ] , but are negligible since they do not require bowel resection and are usually diagnosed and simply removed during la . 
in the past , the imaging evaluation of colorectal involvement due to endometriosis was especially assessed by dcbe , which seems to correlate with the patients clinical history and clinical ndings and was shown to be capable of detecting bowel wall involvement requiring intestinal surgery [ 9 ]  . 
recently , some reports have demonstrated the ability of mri to detect dice using t2 - weighted images without or with air , 296 radiol med ( 2014 ) 119 : 291297 defaecation problems . 
2 deep inltrating endometriosis of the rectosigmoid juncsuffering from chronic pelvic tion in a 32 - year - old patient pain histopathology . t2 - weighted sagittal mri a shows thickened rectosigmoid junc ( a white tion wall showing radial and retracting shape arrows ) and adhesions ( a void arrow ) which converge on the the uterine torus , as shown hypointense plaque - like lesion at on the t2 - weighted axial fat - saturated image ( b arrow )  . 
histopathology specimen after bowel inltrating type of colorectal endometriosis showing endometrial typical glands ( white arrow ) and stroma ( white star ) within the muscular layer ( void star ) of the large bowel . 
t2 - weighted sagittal mri a shows a thickened colorectal junction wall typically showing a radial and retracting shape ( white arrows ) associated with hypointense plaquelike lesion at the uterine torus ( black arrow )  . 
u uterus , rsj rectosigmoid junction radiol med ( 2014 ) 119 : 291297 water or gel distension of the rectum [ 2 , 7 , 13 , 14 ] , employing contrast - enhanced mri [ 8 ] or mr colonography [ 4 ] or also using 3.0 - t superconductive magnets [ 2 , 13 ]  . 
by contrast , mri is an all - in - one examination able to detect endometriotic foci in different sites , including the colorectal site [ 4 , 6 , 7 , 10 , 11 ] , as well as remaining the examination of choice for the detection of deep pelvic implants [ 4 ]  . 
patient management is improved by using a preoperative assessment of the disease by mri , as decisions to perform intestinal surgery will involve the need for a colorectal surgeon . the retrospective analysis of data could be considered a limitation of this study . 
however , it should be noted that mri and la , as well as the histopathological evaluation , that mri was were always performed at always carried out by the same pool of radiologists using the same study protocol , and that the mr images were reviewed by the same two radiologists with 10 and 15 years of experience in female pelvic mri , who were not aware of the patients specic symptoms and outcome . the hospital , in conclusion , the contemporary presence of nodules or hypointense plaque - like lesions in the fat - tissue plane and thickened intestinal wall characteristically showing a radial and retracting shape improves mri capability in dice diagnosis . 
di mola received : 17 october 2012 / accepted : 17 march 2013 / published online : 12 march 2014 ( cid : 2 ) italian society of medical radiology 2014 abstract purpose a tumour score for venous invasion in patients with pancreatic adenocarcinoma was evaluated by means of computed tomography ( ct ) , in order to improve the assessment of medical treatment and clinical outcome with special attention to borderline resectable disease . materials and methods fifty - six consecutive patients who underwent curative surgical resection for pancreatic cancer were analysed . 
on the basis of ct criteria , tumour involvement of the portal vein ( pv ) and superior mesenteric vein ( smv ) was graded according to an adapted 4 - point scale : score 1 , denite absence of invasion ; score 2 , probable absence of invasion ; score 3 , probable presence of invasion ; score 4 , denite presence of invasion . 
correlations between the venous inltration scores and the patients clinical features were also evaluated . results after radiological evaluation of pv and smv grades of inltration , 21 / 56 ( 37 % ) and 37 / 56 ( 66 % ) patients , respectively , were found to have borderline resectable disease . 
the 4 - point scale achieved a sensitivity of 80 % , a specicity of 96 % and an accuracy of 93 % in the evaluation of the pv , and a sensitivity of 100 % , a specicity of 94 % and an accuracy of 95 % in the evaluation of the smv . 
di mola unit of surgical oncology , g bernabeo hospital , ortona , italy radiol med ( 2014 ) 119 : 334342 keywords pancreatic cancer ( cid : 2 ) vein inltration ( cid : 2 ) prognostic factors ( cid : 2 ) ct scan ( cid : 2 ) borderline resectable lesion abbreviations smv superior mesenteric vein computed tomography portal vein introduction pancreatic carcinoma represents the fourth leading cause of death due to malignant disease [ 1 ]  . 
surgical unresectability may include metastatic disease of the liver and peritoneum , invasion of contiguous structures ( stomach , colon , spleen , left adrenal , kidney or spine ) , invasion of peripancreatic arteries ( common hepatic / proper hepatic artery ; superior mesenteric artery ; celiac axis ) [ 3 ]  . 
otherwise , primary pancreatic cancers with radiological characteristics suggesting limited involvement of the superior mesenteric vein ( smv ) , portal vein ( pv ) , superior mesenteric artery ( sma ) , or celiac axis in the absence of metastatic disease have recently been dened as borderline resectable [ 4 , 5 ]  . 
in addition , considering the involvement of the smv / pv , the impingement and narrowing or segmental venous occlusion should enable a sufcient venous ow above and below the occlusion in order to allow surgical venous reconstruction . with regard to borderline resectable cancers , venous invasion remains a controversial point for pancreaticoduodenectomy , and one of the most difcult steps in preoperative staging is to determine local resectability and to dene a stage - specic therapy [ 6 ]  . 
in addition , in patients with clear venous involvement ( either borderline resectable or locally advanced unresectable ) , the tumour can often be reduced in size and converted to an anatomically resectable lesion with preoperative neoadjuvant therapy , strongly increasing the likelihood of subsequently achieving an r0 resection [ 59 ]  . 
in some cases , tumour ingrowths into the adjacent vessels might be detected only at a late phase of the surgical procedure . spiral computed tomography ( ct ) allows for improved imaging of the pancreas and peripancreatic vessels and is regarded as the most important non - invasive staging technique [ 1315 ]  . 
the accuracy of spiral ct for predicting vascular invasion ranges from 62 to 92 % , suggesting that a lack of complete agreement with regard to vascular inltration exists [ 18 , 19 ]  . in this study we aimed to evaluate a multiparametric score for venous tumour inltration ( portal and superior mesenteric veins ) using multidetector spiral ct , and to assess its inuence on medical treatment and clinical outcome , especially in patients with borderline resectable pancreatic cancer . materials and methods patient population we searched the pathology databases in our electronic medical records system from may 2004 to june 2009 for patients with pathologically proven pancreatic adenocarcinoma . 
in order to have pathologically proven diagnoses associated with intraoperative ndings conrming or not the venous involvement , we included in the analysis 56 consecutive patients who underwent radical surgical resection due to pancreatic cancer ( 23 female , 33 male ; median age 64 years ; range 3582 years )  . tumour localization was pancreatic head ( n = 45 ) , uncinate process ( n = 1 ) , body ( n = 5 ) and in ve patients the tumour involved the head and body or the body and tail as a borderline tumour . 
according to the international classication of the uicc ( union for international cancer control ) there were four , stage ia ; two , stage ib ; 13 , stage iia ; 32 , stage iib ; four , stage iii ; and one , stage iv cancers . pylorus - preserving pancreaticoduodenectomy was performed in 45 patients , left pancreatectomy in 7 and total pancreatectomy in 4 patients . 
nineteen ( 34 % ) patients underwent venous resections due to macroscopic vascular inltration : 10 portal vein ( pv ) and 9 superior mesenteric vein ( smv ) resections . the study was conducted with the approval of the ethics committee of all centres involved in the study and written informed consent was obtained from each patient . 336 ct imaging protocol all examinations were performed with a 16 - row spiral ct system ( siemens somatom sensation 16 , erlangen , germany )  . 
our hydro - ct techniques included intravenous injection of 40 mg n - butyl scopolamine bromide ( buscopan ) for intestinal paralysis , gastric and duodenal wall distension by oral administration of an average of 1.5 l tap water , and right anterior oblique ( rao ) patient positioning [ 16 , 20 ]  . 
then , for arterial and portal phases , we used the care - bolus technique with 120 ml contrast medium ( 5 ml / s , ultravist 370 , bayer )  . 
the arterial ( delay of 8 s after the threshold ) and portal ( delay of 35 s after the threshold ) phases were performed with the following protocol : detector collimation , 0.75 mm ; table speed , 12 mm / rotation ; slice , 3 mm ; 170 mas , 120 kvp . 
in both phases two secondary reconstructions ( 2 and 3 mm ) were performed . image analysis radiol med ( 2014 ) 119 : 334342 table 1 score of venous inltration using computed tomography ( ct ) criteria grade tumour contact with vessel length of tumour contact with vessel circumferential vein involvement stenosis = 0 mm = 0 ( cid : 3 ) no stenosis = 0 ( cid : 3 ) 90 ( cid : 3 ) = 0 ( cid : 3 ) 90 ( cid : 3 ) no stenosis flattened [ 90 ( cid : 3 ) \180 ( cid : 3 ) [ 180 ( cid : 3 ) occlusion thrombus grade grade c \5 mm [ 5 mm grade grade d [ 5 mm grade e or f 1 denitely absence of invasion 2 probably absence of invasion 3 probably presence of invasion 4 denitely presence of invasion type of stenosis : no stenosis attened occlusion thrombus . images were reviewed retrospectively and independently by two abdominal radiologists with 10 years of clinical experience . the criteria considered for venous invasion were : tumour contact with vessel , according to loyer et al . [ 21 ] : grade a : fat plane visible between tumour and vessels grade b : normal pancreatic tissue between tumour and vessels grade c : tumour adjacent to vessel with a convex contour towards vessels grade d : tumour adjacent to vessel with a concave contour towards vessels grade e : circumferential involvement of vessels grade f : vascular occlusion . a 4 - point scale was devised as follows ( table 1 ) : 1 . 
denite absence of invasion : tumour contact = grade ab , length of tumour contact = 0 mm , circumferential involvement of vein = 0 ( cid : 3 ) , no stenosis ; 2 . 
probable absence of invasion : tumour contact = grade c , length of tumour contact \5 mm , circumferential involvement of vein = 0 ( cid : 3 ) 90 ( cid : 3 ) , no stenosis 3 . 
denite presence of invasion : tumour contact grade e or f , circumferential involvement [ 180 ( cid : 3 ) ; narrowing of vessel ; association of tumour convexity d with length [ 5 mm and circumferential [ 90 ( cid : 3 ) \180 ( cid : 3 )  . length of tumour contact with vessel : statistical analysis circumferential involvement of vein : 0 mm \5 mm [ 5 mm . 0 ( cid : 3 ) 0 ( cid : 3 ) 90 ( cid : 3 ) 90 ( cid : 3 ) 180 ( cid : 3 ) [ 180 ( cid : 3 )  . sensitivity , specicity , positive and negative predictive values for vascular involvement were calculated for each reader separately . 
spiral computed tomography ( ct ) ( a , b ) shows a tumour in the pancreatic head with evident fat plane between dilated pancreatic duct and pv ( grade a ) ( arrow ) , without contact between the tumour and vein , which was conrmed intraoperatively ( c )  . 
spiral ct shows a tumour of the pancreatic head ( red arrow ) with a convex contour towards vessel ( grade c ) ( black arrow ) and a grade of contact between 0 ( cid : 3 ) and 90 ( cid : 3 )  . 
spiral ct shows a tumour in the pancreatic head and body with a attened vessel wall ( arrow ) and a length of contact [ 5 miv 52 - year - old man with score 4 . 
spiral ct shows a thrombus ( arrow ) in the conuence between the pv and the splenic vea 75 - year - old man with score 4 ( a )  . 
spiral ct shows a tumour in the head of pancreas with narrowing of pv before the conuence with splenic vein ( b ) radiol med ( 2014 ) 119 : 334342 fig . 
a 66 - year - old man with tumour of the pancreatic head . we evaluated the pv as score 3 ( ab ) ( grade c , length of tumour contact [ 5 mm and attened ) but the surgical results ( c ) demonstrated that the vein was not inltrated but only attached by an inammatory adhesion . 
statistical associations between the patients characteristics ( age , gender , tumour location , grading , tumour staging , classication and status of resection margins ) and each score of venous inltration were assessed using the fisher exact test and the kruskal wallis test for categorical and continuous variables , respectively . 
time - to - event analyses for overall survival ( dened as the time between the diagnosis and the occurrence ) was performed using the kaplanmeier ( km ) method and by log - rank test . 
all analyses were performed using sas release 9.1 ( sas institute , cary , nc , usa )  . results image analysis portal vein there was a high agreement between two readers ; the k value was [ 0.80. evaluation of the pv on the ct images dened 21 / 56 ( 37 % ) as borderline resectable cases . 
we evaluated the pv as score 2 ( ab ) ( tumour convexity grade c , length of tumour contact \5 mm , circumferential involvement of vein = 0 ( cid : 3 ) 90 ( cid : 3 ) , no stenosis ) but surgical exploration demonstrated that the vein was inltrated and a pv en bloc resection was required ( c ) table 2 radiological evaluation of portal vein and superior mesenteric vein inltration using the 4 - point scale 80 % sensitivity , 96 % specicity , 80 % positive predictive value ( ppv ) , 96 % negative predictive value ( npv ) and 93 % overall accuracy ( table 2 )  . true positive true negative false positive false negative accuracy ( % ) sensitivity ( % ) specicity ( % ) ppv ( % ) npv ( % ) pv portal vein , smv superior mesenteric vein , ppv positive predictive value , npv negative predictive value had denite absence of invasion ( score 1 ) , 11 patients had probable absence of invasion ( score 2 ) , 7 patients had probable presence of invasion ( score 3 ) and 3 patients had denite presence of invasion ( score 4 )  . 
in particular , the absence of vascular inltration was then subsequently proved during surgical intervention in 96 % of the cases ( 44 / 46 ) , while portal vein inltration was present during surgical intervention in 80 % of the cases ( 8 / 10 )  . 
using this score for the evaluation of pv inltration and in comparison with the intraoperative assessment , the results were : superior mesenteric vein evaluation of the smv on the ct images classied 37 / 56 ( 66 % ) as borderline resectable cases . in detail , 19 patients had denite absence of invasion ( score 1 ) , 25 patients had probable absence of invasion ( score 2 ) , 6 patients had probable presence of invasion ( score 3 ) and 6 patients had denite presence of invasion ( score 4 )  . 
in detail , the number of patients without evidence of metastasis to distal organs ( m0 ) was higher in patients with low smv scores than in those with high smv scores ( p = 0.028 ) : 56 , 72 , 17 and 17 % of patients with smv scores 1 , 2 , 3 and 4 , respectively . 
score 34 : hr = 0.30 ; 95 % ci 0.051.75 , p = 0.183 and hr = 0.28 ; 95 % ci 0.041.88 , p = 0.192 from univariate and multivariate cox regressions , respectively ; score 2 vs . 
however , this classication is relatively subjective and authors do not differentiate between venous and arterial invasion . we have revised those criteria , including in only one group ( score 1 ) grade a ( fat plane visible between tumour and vessels ) and grade b ( normal pancreatic tissue between tumour and vessels ) because in our opinion there are not many differences between these two grades in terms of surgical outcome since both are clearly resectable . in addition , we have considered also the length of contact between the tumour and vascular wall . 
 [ 25 ] evaluated isolated ct criteria and a combination of two criteria for venous invasion in 65 patients who underwent surgery for pancreatic cancer , considering length of tumour contact , degree of stenosis , circumferential involvement of vein , irregularity of the vessel margin and tumour convexity towards the vessel . 
they concluded that narrowing of the smv and pv seems the most reliable criterion , as well as circumferential involvement of the pv [ 90 ( cid : 3 )  . 
the best combination , however , was tumour concavity with circumferential involvement [ 90 ( cid : 3 ) , with a sensitivity of 60 % and a positive predictive value of 90 % . 
in this study a length of tumour contact [ 5 mm with pv and smv had a sensitivity of 47 and 57 % and a specicity of 80 % , respectively . 
it is worth noting that in order to ensure that the radiological scores were in line with the intraoperative ndings of venous inltration ( associated with the histological report ) we included only patients who underwent radical surgical pancreatic resection . in addition , klauss et al . 
by using this score we have improved the sensitivity , specicity , ppv and npv , with a good correlation with surgical results especially when we considered the group of borderline resectable tumours . 
patients with pv score 34 had shorter survival ( median survival = 10 months ) as compared to those with pv score 1 ( median survival = 13 months )  . log - rank test p value = 0.106 discussion many studies report that multidetector spiral ct has improved pancreatic tumour detection , with an overall accuracy between 89 and 97 % [ 1517 ]  . 
 [ 21 ] have classied tumour contact as forming either a convexity or a concavity against the vessel wall : six different types ( af ) of tumour contact to major vessels ( both veins and arteries ) were described . 
these data suggested that if the tumour is radiologically found in contact with vessels without inltrating the vessel wall , it is 342 radiol med ( 2014 ) 119 : 334342 these data are of interest in addition , when we compared the pv and smv inltration scores with the patients clinical characteristics we found that both scores were associated with metastatic disease and status of resection margins . 
although not surprising , if we consider that sometimes the tumour may be locally resectable but the disease has rapid progression after surgery maybe due to the presence of microscopic metastasis at diagnosis . 
moreover , although it is difcult to imagine that a radiological score might be related to disease - free survival , the log - rank tests suggested that pv inltration scores might correlate with the patients overall survival . 
these data might at least suggest that the present score system is reliable and must be tested in a large number of patients . in conclusion , smv and pv scores can predict resectability , and their correlation with tumour metastatic state and resection margins conrms their reliability in pancreatic cancer . conict of interest tiziana marinelli , antonella filippone , francesca tavano , andrea fontana , fabio pellegrini , jorg koninger , gotz m . 
survival is slightly but steadily increasing , thanks to the early diagnosis of mets and to the availability of new systemic therapies [ 1 ]  . a limit to the usefulness of local treatments of hepatic mets from breast cancer is therefore a tendency towards systemic metastases ( unlike , for example , mets from colorectal carcinoma which tend to affect the liver in an exclusive manner ) [ 2 ]  . therapeutic options include chemotherapy but also metastasectomy and radiofrequency ablation ( rfa ) in selected patients [ 3 ]  . 
until now , hepatic rfa was used primarily for the treatment of hepatocellular carcinoma and mets from colorectal cancer , but a small number of reports also 328 radiol med ( 2014 ) 119 : 327333 concern mets from breast , stomach , kidney and lung carcinoma , and from cholangiocarcinoma and melanoma [ 5 ]  . the aim of this study was to review our personal series of consecutive rfa of hepatic mets from breast cancer , performed on the basis of a multidisciplinary clinical indication , to evaluate the midterm safety , efcacy and clinical utility of this therapy . materials and methods our series is taken from a consecutive series of nearly 1 , 000 rfa procedures performed on mets from breast cancer at the two centres of our academic institution from 1998 to 2011 . includes 45 women ( age range 3381 years ; mean 55 years ) with metastatic disease from breast cancer . 
the study was carried out in accordance with the guidelines for retrospective reviews established by our institutional review board . patients and lesions the indication for treatment was given after multidisciplinary assessment by a medical oncologist , an intervenradiologist and an anaesthesiologist . 
the rfa tional technique was established on the basis of the technical evaluation of the interventional radiologist . the alternative option to surgery was chosen for the following reasons : oncological therapeutic strategy , contraindications to surgery or anaesthesia ( cardiovascular , respiratory , etc . ) , other comorbidities , advanced age , presence of extra - hepatic disease , lesions not surgically resectable , refusal of the patient to undergo surgery . a total of 87 mets were treated in 45 patients ( mean age 56 years ; average 1.9 mets per patient )  . 
six of 45 ( 13 % ) cases were already metastatic at diagnosis of the primary tumour ( synchronous mets ) ; 18 / 45 ( 40 % ) patients also had extra - hepatic mets , already controlled or treated with other therapies . 
the size of the lesions ranged from 10 to 45 mm , with an average of 23 mm . at the time of selection for rfa treatment , breast cancer histology was not considered a selection criterion , nor were previous or current systemic therapies . 
this , because we privileged the potential usefulness of the local treatment in the management of the single patient with a specic stage of disease ; rfa was therefore included as an adjunctive treatment in a constantly multimodal therapy . histology and other treatments were therefore not correlated with the results of rfa . pre - procedural assessments before each treatment , we evaluated age , performance status and cardiorespiratory condition . 
in addition , preprocedural staging with whole - body computed tomography ( ct ) , magnetic resonance ( mr ) imaging , and / or positron emission tomography ( pet ) ct , preferably not earlier than 1 month before the scheduled date for the rfa procedure was always requested . 
each patient underwent coagulation tests ( for possible correction of thrombocytopenia or increased inr / ptt ) , a chest x - ray and electrocardiogram , and an anaesthesiological assessment . 
any anti - coagulation or anti - platelet treatments were adapted or suspended . the indication , risks and potential benets of the procedure were discussed with the patients , who provided written informed consent . procedural aspects rfa was performed in the interventional radiology room , in conditions of local sterility after injection of a local anaesthetic at the site of entry of the needle - electrode ( 10 ml of buffered lidocaine hydrochloride 2 % ) and under anaesthesiological monitoring ( ecg , blood pressure , arterial oxygen saturation ) and analgosedation . all procedures were performed under ultrasound guidance , using esaote technos and mylab scanners ( genoa , italy )  . 
in the case of isoechoic mets , visualisation of the lesion and monitoring of electrode insertion was carried out after intravenous administration of second - generation ultrasound contrast agent ( sonovue , bracco , milan , italy )  . we used expandable needle - electrodes rita starburst ( rita medical systems , inc . , mountain view , ca , usa ) or leveen ( boston scientic , waltham , ma , usa )  . 
it is well known that the ablation protocol is based on temperature for the rita system and tissue impedance for the boston scientic system , so the choice of equipment was linked to their availability rather than to other variables . the average time for each rfa procedure was approximately 20 min , with differences imposed by the size of the radiol med ( 2014 ) 119 : 327333 mets and the different equipment protocols , in accordance with the manufacturers indications . prognostic factors for survival , one of them being maintained local effectiveness ( ca ) : at the end of the procedure , the electrode pathway was ablated during extraction , in order to avoid neoplastic seeding and to achieve haemostasis ; after about 15 min a nal post - procedural ultrasound scan was obtained to exclude immediate complications . patients were kept under observation for one night after the treatment in order to monitor their clinical condition and complete blood count . evaluation of adverse events all adverse events were recorded and categorised according to the society of interventional radiology ( sir ) criteria into minor complications ( low or no clinical relevance ) and major complications ( requiring prolonged hospitalisation and / or interventional radiological or surgical intervention ) , and counted in terms of numbers and percentages . outcome even considering that the clinical goal of rfa was the multimodal management of breast cancer mets rather than achieving local results with radical intent , we considered it useful to assess the outcome at 1 month by means of contrast - enhanced ct to identify any partial ablation ( pa ) with residual tumour tissue . 
where the examination indicated complete ablation ( ca ) , given the high probability of local progression in rfa of liver mets , the ablation site was re - assessed with contrast - enhanced ct 1 year after the treatment in order to verify possible maintained ca and diagnose local recurrences . 
for the assessment of clinical utility , the number of deaths in the studied population was counted . possible local or clinical outcomes of disease were : no evidence of residual / local recurrence ( ca ) , evidence of local residual tumour tissue ( pa ) , evidence of local recurrence ( lr ) , death . on the basis of these parameters , we were able to calculate : the local effectiveness ( ca versus pa ) at initial ct follow - up ( ca versus pa ) and 1 year after the procedure ( ca versus pa ? lr ) , time to progression ( ttp ) , and survival curves in the medium term . also on the basis of our previous experience with rfa of liver mets , we analysed the size of the lesion ( lesion diameter and a threshold diameter of 30 mm ) as potential prognostic factors for local effectiveness at 1 year . given the greater importance of evaluating the clinical impact of the procedure , we analysed several possible single or multiple lesions at the time of treatment , m0 / m1 at the time of diagnosis , absence or presence of extra - hepatic mets , size of the lesions , maintained effectiveness of rfa versus treatment failure ( tf ) at 1 year . statistical analysis all variables were inserted in a microsoft ( microsoft , inc . , redmond , wa ) excel spreadsheet . 
in particular , to identify possible correlations between dimensional variables and local outcome , we applied a multivariate analysis ( the unpaired two - tailed t test to compare averages and the fisher exact test to assess the signicance of threshold diameters ) ; kaplan meier survival curves were stratied based on the variables described above with signicance of the differences being calculated using a multivariate analysis ( cox )  . results complications the periprocedural mortality was nil . 
the rst was a case of protracted pain with evidence of thrombosis of the left portal branch , while the other was a case of sepsis due to serratia temporally related to the procedure . local effectiveness the initial ct follow - up revealed ca in 90 % of cases ( 78 / 87 )  . 
1 mean diameter ( sd ) of metastases ( mets ) with complete ablation ( ca ) maintained 1 year after radiofrequency ablation ( rfa ) ( 22 7.4 mm ) compared with mean diameter of those with treatment failure ( tf ) [ partial ablation ( pa ) or local recurrence ( lr ) ] ( 30 10 mm ) : at the statistical test the difference was considered extremely signicant ( p = 0.0005 , hr 7 , 965 , 95 % ci 3 , 64612 , 284 ) fig . 
more than 50 % of women with breast cancer develop hepatic mets , but only 518 % of patients have metastatic disease conned to the liver [ 7 ]  . 
metastatic breast cancer should therefore be treated by chemotherapy and / or hormone therapy , both standard systemic therapies [ 8 ] , and even so reported survival rarely exceeds 2 years [ 9 ]  . considering the toxicity of chemotherapy , it is increasingly common for patients with limited hepatic disease ( onset or residual after standard therapy ) and controlled extra - hepatic disease , to require a local treatment to be integrated within a broader therapeutic strategy , in a socalled multimodal approach . 
the majority of patients , however , are not eligible for surgery due to the oncologists or the patients decision based on general condition , age , advanced state of the disease or other contraindications . for this reason , in recent years there has developed an increasing interest in ablative therapies , with the aim of managing a given stage of the disease and the hope of improving the prognosis of the patients . in our series , the indications for hepatic rfa were based on what has been said so far . 
similar to previous studies , we therefore rst evaluated the complications and the effectiveness of local rfa , intended as ca demonstrated at contrast - enhanced ct at initial and medium term follow - up . 
with regards to local effectiveness , our results at initial ct follow - up ( 90 % ca ) were broadly in line with those in the few studies reported in the literature , in which ca varies from 79 to 96 % [ 8 , 1014 ]  . 
it was not possible to nd published results comparable with our data ( 74 % ) regarding the local effectiveness ( maintained ca ) at 1 year . in the search for factors that inuence local effectiveness , the literature lists several variables , including the size of the tumour and its proximity to the branches of greater calibre of the hepatic veins and the portal vein [ 15 ]  . 
apart from having limited signicance due to the small size of the sample , interpret : given the nonstatistical signicance of maintained ca as a predictor of survival at 3 years , it seems that survival may depend more on other variables , such as ineffectiveness of systemic therapy and the resulting generalised tumour progression . 
in fact , the vast majority of patients died due to progression of extra - hepatic disease , even though almost all of them underwent systemic therapies until these proved ineffective to stop the disease ( or had to be interrupted due to toxicity )  . these results are difcult thus , considering hepatic rfa as a local therapy that is useful for managing a given stage of metastatic disease , we can conclude with two further considerations . 
as previously mentioned , a large proportion of patients is not eligible for surgery , and a review of the major surgical studies shows that rfa of hepatic mets has a lower mortality and periprocedural complication rate ( table 1 )  . 
on the other hand , due to the poor local effectiveness of rfa in treating mets larger than 3 cm in diameter , surgery remains better for larger lesions . regarding the clinical indications for metastasectomy , a recent literature review attempted to make recommendations in selected patients ( young patients , low surgical risk , long interval between breast cancer surgery and liver metastases , positive hormone receptor status of primary tumour , no extra - hepatic disease , demonstrated disease regression or stability with systemic therapy before resection , normal liver function tests , resection with intent of a complete resection of liver metastases )  . 
owing to the limitations of the few published studies , however , even the impact of surgery on survival is considered to be merely potential [ 3 ]  . the nal consideration is that , even in the light of the lack of correlation between local effectiveness and survival , hepatic rfa should not be used as the only treatment for metastatic breast cancer . 
nonetheless , rfa may be proposed as an alternative to surgery in the context of a multimodal strategy , as it is safe and effective in achieving local control of limited disease , especially when the burden of systemic therapy needs to be decreased , improve the quality of life of patients . in part conclusions rfa of hepatic metastases from breast cancer , intended as a local treatment during multimodal therapeutic management of the disease , has high technical effectiveness in tumours up to 30 mm , but it is not relevant in determining mid - term patient survival . 
the results were analysed statistically using the students t test for independent samples and the k statistic . results among the 97 patients included in the study , a diagnosis of right ventricular infarction was established in 12 , 14 and 24 cases on the basis of the clinical data , the ecg and echocardiography , respectively . 
fedele dipartimento di scienze cardiovascolari e respiratorie , policlinico umberto i , universita` di roma sapienza , viale regina elena 324 , 00161 rome , italy myocardial oedema and late enhancement of the right ventricle in 48 and 32 cases , respectively . 
all patients gave their informed consent before being included in the study . clinical diagnosis of rvmi all patients underwent a complete cardiological assessstandard ment : patient history , physical examination , 12 - lead ecg ( with additional right precordial leads when rvmi was suspected ) and echocardiography . clinically , the contemporary presence of arterial hypotension ( systolic blood pressure \90 mmhg ) , clear lungs on auscultation and jugular venous distension ( kussmaul sign ) [ 14 ] was considered suggestive of rvmi . electrocardiographically , the infarction was dened as anterior when detected on leads v1v4 and inferior on leads ii , iii and avf . 
rvmi was dened as an elevation greater than 1 mm in v1 or v4r within 12 h of symptom onset . the echocardiographic study of the ventricular chambers was performed using standard apical and parasternal projections , with dedicated equipment ( acuson sequoia , siemens , erlangen , germany )  . 
for each mri study , the images were semiquantitatively evaluated using regions of interest as shown below : tissue signal at myocardial oedema ( me ) and late enhancement ( le ) were respectively evaluated on stir and irge sequences in the short axis planes . 
based on the literature data , me was dened by an increase of the pathological myocardial standard deviations ( sd ) as compared to the signal of healthy myocardium ; similarly , assessment of le was based on an increase of the signal of at least 4 sd [ 16 ]  . on each test , the areas of me and le of the left ventricle were manually outlined with dedicated software and quantied in terms of mass and percentage ( injured myocardial mass / myocardial mass ) of the ischaemic area and area at risk ( respectively , ia and aar )  . least the presence of right ventricle le ( rvle ) was identied as an increase of the signal coming from the region of the rv myocardium adjacent to the left ventricle le ( lvle )  . 
as previously reported , a similar criterion was used to evaluate the rv oedema [ 13 ]  . the segmental wall motion assessment was performed on cine - mr using the 17 - segmentation american heart association scheme for the lv , and the isner classication into 12 regions ( 4 basal , 4 mid - ventricular and 4 apical ) for the rv [ 17 ]  . using the semiquantitative evaluation proposed by masci et al . 
 [ 13 ] , each segment was assigned a motion score from 1 to 5 ( 1 = normal , 2 = moderate hypokinesia , 3 = severe hypokinesia , 4 = akinesia , 5 = dyskinesia )  . 
statistical analysis was conducted with dedicated software ( spss 11.0 for windows , spss , chicago , illinois )  . results cardiological assessment nine out of 97 ( 9 % ) patients showed simultaneously the three clinical signs of rv ischaemic involvement . 
thirtyseven patients ( 38 % ) showed ecg signs of inferior infarction , whereas 59 ( 61 % ) showed ecg signs of radiol med ( 2014 ) 119 : 309317 fig . 
t2 - weighted stir ( short - tau inversion recovery ) ( a , c ) and t1weighted irge ( inversion recovery gradient echo ) ( b , d ) images show an extensive area of myocardial oedema at the level of the interventricular septum and the free wall of the right ventricle ( white arrow ) , which corresponds to transmural enhancement in the late enhancement images ( white arrow )  . 
it is associated with a diffuse thickening and enhancement of the pericardial leaets ( white arrowheads ) due to postinfarction inammation ( post - infarction pericarditis epistenocardica ) anterior infarction . 
of the 26 / 97 patients in whom the right leads were acquired , 14 showed an st elevation ( in 13 cases associated with an st elevation in leads dii , diii and / or avf ) in lead v4r . echocardiography revealed an impairment of right ventricular function in 24 cases , and more specically : in 18 cases it showed hypocontractility of at least one segment ( free wall , inferior wall or apex ) , in 17 cases a dilatation of the rv , and in 10 cases a dyskinesia of the interventricular septum ( table 1 )  . magnetic resonance cmr conrmed the presence of a le area within the lv myocardium in 95 / 97 cases . 
in the remaining two patients , both with anterior mi , the mr pattern was characterised by tissue oedema ( hyperintensity in t2 - stir ) in the distribution territory of the culprit lesion in the absence of le ( aborted infarction ) [ 18 ]  . 
2 a 63 - year - old man with acute inferior myocardial infarction due to distal right coronary artery occlusion . t2 - weighted stir ( a ) and t1 - weighted irge ( b ) images acquired in the short axis show a large ischaemic area at the infero - septal and inferior left ventricular segments with typical extension of the area of myocardial oedema ( black arrow ) and late enhancement ( white arrow ) to the inferior wall of the right ventricle fig . 
ad stir images show a wide area of oedema involving the interventricular septum , the left ventricular anterior wall and the right ventricular free wall , both at basal , intermediate and level ( white arrows )  . 
sparing of the free wall ( white arrowhead ) and apical segments ( black arrow ) of the right ventricle conrms the greater the right ventricular myocardium to the ischaemic insult resistance of criteria and the cmr signs is shown in table 1 . 
finally , in le imaging , inversion time to be selected to null the signal of the healthy myocardium may vary between rv and lv , causing artefacts or requiring a double acquisition for each plane with considerably longer duration of the examination [ 22 , 23 ]  . to overcome these difculties , many authors recommend to use high - spatial resolution thin - slice sequences and le sequences with fat suppression , and to consider as pathological only the myocardium which is in continuity with the pathological left ventricular myocardium . in a study by kumar et al . 
 [ 19 ] , among 37 cases of inferior mi subjected to clinical , instrumental and mri investigation , 57 % patients ( 21 / 37 ) showed rvle , while only 19 , 35 and 16 % , respectively , demonstrated signs of rvmi at cardiological examination , on the v4r lead at ecg or at echocardiography . in our population , the rv had an ischaemic involvement in 46 % of cases of inferior mi and in 30 % cases of anterior mi , differing from the values given by jensen et al . [ 21 ] , which were 47 and 65 % , respectively . rv involvement during an anterior mi could be affected by the presence of anatomical variants of the anterior descending artery , with perfusion of the free wall and right para - apical region by particularly long anterior interventricular branches extending beyond the apex , or alternatively from the origin of the moderator branch of the distal anterior descending artery [ 13 ]  . 
this potential anatomical variability may , therefore , account for the discrepancy in the anterior mi values seen in our study population compared to those reported by jensen et al . 
in our population , there was a radiol med ( 2014 ) 119 : 309317 316 radiol med ( 2014 ) 119 : 309317 considerable frequency of rvme in both inferior ( 59 % ) and anterior ( 48 % ) mi , signicantly higher as compared to the frequency of rvle , which was present in 46 and 30 % of patients , respectively . 
therefore , this nding supports the notion of a greater resistance of the rv myocardium to the ischaemic insult as compared to the lv tissue , which for an ischaemia of the same duration showed increased susceptibility to necrotic damage in the at - risk area ( 98 % of patients had lvle )  . as stated by goldstein , the term right ventricle myocardial infarction is to some extent a misnomer because often the acute ischaemic dysfunction of the rv is represented by viable myocardium , in contrast to what happens for the lv [ 4 ]  . multiple reasons contribute to the increased resistance to ischaemia of the rv : the different load conditions due to the lower resistance of the pulmonary circulation , myocardial thickness which results in a lower oxygen demand and a greater oxygen reserve [ 4 ]  . 
furthermore the dual blood supply of the rv free wall , coming from branches of the anterior descending artery and from conal branches of the right coronary , allows for a ready provision in the event of vessel occlusion [ 13 , 26 ]  . 
another limitation is the absence of proper follow - up or clinical monitoring of the study population , which could have provided prognostic information but which we decided not to include in the results because of the limited observation time and because it was the subject of other recently published papers [ 14 , 19 , 24 , 25 ]  . conclusions in our study population , rvle was seen in 25 % of patients with acute mi and , in consideration of the literature data that demonstrates how this nding adversely affects the patients prognosis [ 19 , 24 , 25 ] , it is clear that this is not a negligible element . 
in particular , effective doses were evaluated using the tissue weighting ( wt ) factors reported both in icrp60 ( wt , 60 ) and in icrp103 ( wt , 103 )  . results a decrease in the frequency of pr skull examinations and an increase in the frequency of mammography , ct of the abdomen , chest , and headneck were found during the decade . 
moro 21 , 40127 bologna , italy with the use of wt , 60 until 2007 and wt , 103 from 2008 seem to be consistent and coherent . keywords ct ( cid : 2 ) dose , population ( cid : 2 ) exposure measurements ( cid : 2 ) patient protection ( cid : 2 ) radiography ( cid : 2 ) x - ray introduction the use of ionising radiation in medical radiology is by far the greatest source of radiation exposure of anthropic origin as stated by the united nations scientic committee on the effects of atomic radiation ( unscear ) [ 1 ] and thus the evaluation and quantication of medical exposure is a key subject in radiation protection for many aims : verifying whether there are signicant differences between the dose delivered in different regions or countries ; comparing the contributions from different kinds of x - ray examinations to provide best guidance on where efforts on dose reduction can concentrate ; monitoring the trends in annual per caput doses as associated with changes in radiology practice ; comparing the dose from medical radiology with that due to natural background radiation . many nations have reported population dose estimates through diagnostic imaging procedures [ 16 ] , but no similar study has been reported with regard to italy yet . 
the frequency of examinations ( both inand out - patient ) was based on data provided between 2001 and 2010 directly by all 17 public trusts of the regional health service for each type of procedure , grouped according to national ministry of health nomenclature code , to reduce terminology errors ( table 1 )  . 
to relate those dose quantities to e , conversion coefcients calculated by monte carlo techniques were used : a set of these coefcients was calculated taking into account the wt , 60 and another set was calculated using the wt , 103 . 
to calculate effective dose according to the methods described in the literature [ 1417 ] some data concerning the examination protocols ( kvp , total ltration , etc . ) were also requested : the mean ( weighted for the number of examinations carried out in each hospital ) and the range of these parameters are reported in tables 2 and 3 for pr and ct , respectively . 
in this way the trends of collective effective dose values calculated by three different methods were shown to make a direct comparison . radiol med ( 2014 ) 119 : 348358 results the e percentage contributions of pr and ct procedures to collective dose calculated by different conversion coefcients with regard to the last year of data collection ( 2010 ) are reported in fig . 
the highest relative frequency was found in pr chest examinations ( 35.4 % ) , but they accounted for only 1.4 % of collective effective dose as calculated with both wt , 60 and wt , 103 factors . the total number of examinations per 1 , 000 inhabitants between 2001 and 2010 , subdivided by procedure , are reported in fig . 
in projection radiology , a decrease in the frequency of skull examinations ( - 67 % ) and an increase in the frequency of mammography ( ? 40 % ) were found during the decade . 
as far as the ct procedures are concerned , during the same period ct of abdomen , chest , and headneck increased by 89 , 90 and 57 % , respectively . 
when the same wt factors are used , generally the doses did not change noticeably through the years : the most remarkable decrease and increase in the e / procedure between 2001 and 2010 were found in pr of the skull ( - 22.1 % ) and pelvis ( ? 30.8 % ) , respectively . 
1 contributions to emiliaromagna collective dose ( calculated by different conversion coefcients ) and examinations frequency from projection radiology ( pr ) and computed tomography ( ct ) procedures ( reference year : 2010 ) 352 fig . 
4 : the analogous temporal trends for ct procedures are all e / procedures this shown increased during the decade ( mean percentage increment ? 151 % )  . case , in table 5 the percentage contributions from pr and ct procedures to per caput annual effective dose are reported in detail for each examination : in particular , the ct contributions are calculated with both e103 , rat and e103 , cal . 
the temporal trend of per caput effective dose from 2001 to 2010 ( calculated with both wt , 60 and wt , 103 factors ) for the examinations under consideration is shown in fig . 
5. discussion projection radiology represents the largest contributor to the frequency of examinations in emilia - romagna in 2010 , although ct examinations provide the greatest doses whichever conversion factors for e are used . the trend in the frequency of ct examinations per 1 , 000 inhabitants reported in fig . 
3 temporal trends of effective dose per projection radiology ( pr ) examination between 2001 and 2010 , subdivided for procedure : a abdomen , lumbar spine , pelvis and urography ; b chest , mammography and skull examinations , possibly reecting the recent trend to prefer ct to plain radiography when the patient presents with a cervical trauma [ 2426 ]  . in the years of this study ( 20012010 ) , s evaluated with e60 was always in between the collective effective doses calculated with e103 , rat and e103 , cal : for example , the values of s were 1 , 934.9 man sv2 , 065.4 man sv2 , 196.8 man sv in 2001 and 5 , 855.1 man sv6 , 169.2 man sv6 , 665.5 man sv in 2010 when e103 , cale60e103 , rat were used , respectively . 
the fact that e60 is not denitely higher or lower than e103 reects the fact that the use of wt , 60 or wt , 103 is not a one - way street , and the quantity e / procedure increases or decreases when the old or the new weighting factors are used depending on the anatomic regions irradiated . 
as an example , pr of the skull and pelvis might be compared : in 2010 the doses for pr of the skull calculated with wt , 60 were 36 % lower than those evaluated with wt , 103 , because according to the icrp 103 recommendations the radiosensitive organs in the head are now four ( brain and salivary glands with specic tissue weighting factors ; extrathoracic region and oral mucosa as remainder organs ) , while according to icrp 60 the only structure in the head and neck region explicitly listed as a remainder organ was the braon the other hand , in 2010 the doses 354 fig . 
5 , the line of per caput effective dose calculated with wt , 60 factors lies in between the lines of the same quantity evaluated by different methods ( e103 , rat , e103 , cal ) from wt , 103 coefcients : thus doses calculated with the use of wt , 60 ( until 2007 ) and wt , 103 ( from 2008 ) on the same chart seem to be consistent and coherent . 
in the specic case of the emilia - romagna region reported in this study , this comparison has been shown to be still important and useful to keep the results obtained in the previous surveys : the wt , 60 factors can be used until 2007 , while the wt , 103 coefcients can be used from 2008 . 
thus the emilia - romagna per caput effective dose rose from 0.5 msv in 2001 to 1.5 msv in 2010 : the latter value can be compared with the gure of 3.0 msv quoted in the usa in 2006 [ 29 ] and the corresponding value of 1.9 msv assessed by unscear [ 1 ] as the average dose for healthcare level 1 countries . a major limit of this study lay in the data collection that in the very rst years was not complete : a few trusts did not provide all the parameters requested and many interpolations were needed to have a full picture of frequency of examinations and dose distribution in the region . 
nonetheless , in the last years increasingly complete datasets were returned from the trusts because a culture of patient radiation protection and procedure optimisation became widespread in all hospitals , and the surveys were thus more reliable . 
moreover , radiological information systems ( ris ) are being effectively integrated with the hospital information systems ( his ) and that can provide better and faster the radiological data about departments . 
5 temporal trend of per caput effective dose from 2001 to 2010 calculated with both e60 and e103 conversion factors in pr of the pelvis calculated with wt , 60 were 38 % higher than those evaluated with wt , 103 , due both to the large decrease in the weighting factor for the gonads and to the reduction in the wt for the bladder that compensates for the introduction of the prostate as a remainder organ . the e103 , cal were generally lower than e60 for ct procedures : this might be related to the fact that deak et al . [ 19 ] calculated conversion factors for new ct equipment and not for the ct scanners that were used in the early 1990s like in the european commission report [ 15 ]  . the per caput effective dose due to pr and ct incremented by approximately 170 % from 2001 to 2010 , basically due to increment in both frequency and e per procedure of ct examinations : the contribution of pr to per caput effective dose was 25 % in 2001 and 10 % in 2010 with an increment of 12 % in pr frequency , while ct accounted for 75 and 90 % of per caput effective dose in 2001 and in 2010 , respectively , with an increment of 52 % in the number of total ct procedures . 
therefore , in particular the highest dose - contributor examinations , appropriate strategies with regard to medical exposure justication and optimisation need to be adopted to contain e , and tools for minimising the dose per procedure without affecting the quality of diagnostic information need to be identied , in accordance with the european union and international guidelines [ 8 , 27 , 28 ] : in ct that means , amongst other factors , reducing the number of scan phases , limiting the scan length , using the dose modulation facilities and , last but not least , selecting the appropriate scanning technique . the emilia - romagna annual per caput effective dose from ct was 1.3 msv in 2010 : this value is lower than the corresponding gure of 1.5 msv from ct in the usa [ 29 ] though it is higher than the level of 0.74 msv recently found in canada [ 30 ]  . 
therefore , though the dose level from this specic practice is in good agreement with other countries levels , something in the way those high - dose procedures are carried out can still be done , in accordance radiol med ( 2014 ) 119 : 348358 have been included because italian legislation denes the term radiodiagnostic as pertaining to medical diagnostic radiology and diagnostic nuclear medicine in vivo [ 7 ] , but there are not yet effective dose calculations based on new icrp weighting factors for all the examinations . 
it is still important to study trends in medical exposure of the population but dose estimates preand post - icrp publication 103 must be compared carefully , because changes due to different radiological practices could be confused with changes due to the use of different wts : in this paper indications on how a comparison of per caput and collective effective doses might be carried out have been suggested . 
fileni received : 3 september 2012 / accepted : 30 january 2013 / published online : 3 december 2013 ( cid : 2 ) italian society of medical radiology 2013 abstract purpose since radiologists and radiotherapists can be occupationally exposed to signicant psychosocial risk factors , some may nd themselves in a state of distress . the aim of this study was to investigate the association of work - related stress with the presence of symptoms of anxiety , depression and psychological malaise and to evaluate the risk of psychic disorder in radiologists suffering from work - related stress . methods a total of 654 radiologists responded to our invitation to complete a questionnaire designed to evaluate work - related stress and associated medical conditions : the general health questionnaire and goldbergs anxiety and depression scales . results scores on the anxiety , depression and psychological malaise scales rise with an increase in effort and over - commitment , while control and support exert a protective effect . 
now a major effort must also be made to improve the mental wellbeing of radiologists , both in the interests of the workers themselves , and also in those of their patients and the quality of the treatment they have the right to receive . keywords work - related stress ( cid : 2 ) radiologist ( cid : 2 ) depression ( cid : 2 ) anxiety ( cid : 2 ) distress introduction the work of radiologists and radiotherapists exposes them to considerable occupational risk factors . 
these may be of a structural nature on account of excessive workloads or organisational difculties [ 1 ] , of a compassionate nature in relation to the suffering of patients [ 2 ] , or due to accusations of malpractice or concern about errors committed [ 3 ]  . 
compared with other medical categories , radiologists have the highest levels of work - related stress [ 4 ]  . distress is a mixed and loosely dened term describing a subjective , negative and unpleasant reaction to stress . 
this type of disorder is very widespread ; in europe , it is estimated that the prevalence of mood disorders in the course of working life is 14.0 % , and the prevalence of anxiety 13.6 % , while , in the nal year of work , these become 4.2 and 6 % , respectively [ 6 ]  . distress and its resulting mental disorders are critical factors when work is being performed . 
the percentage of doctors affected by symptoms of depression is very high both in western and eastern countries , although there are relatively few serious cases [ 9 ]  . young physicians are at greater risk [ 10 ]  . 
it has been seen that the pressure of psychosocial factors , such as excessive work demand and low discretionary power , reduce the affective commitment to work and encourage physicians to abandon their occupation [ 11 ]  . there are three reasons why it is important to diagnose srds . 
for example , in italy , it has become compulsory for all employers to evaluate the risk of stress in the work environment and to arrange appropriate measures of prevention should these prove necessary [ 12 ]  . 
at an individual level , the occupational physician can prescribe specic temporary or permanent intervention to facilitate a return to work or to improve the quality of occupational life . theories have been developed to evaluate the negative mental health effects on workers exposed to psychosocial risk factors . 
the most inuential are karaseks demandcontrol - support ( dcs ) model [ 13 ] and the effortreward imbalance ( eri ) model devised by siegrist [ 14 ]  . 
both theories suggest that distress is the result of an imbalance , in the karaseks model between demand and control over ones work , and in the siegrists model between effort and reward . 
the aim of this study was to ascertain whether there is an association between a state of distress in radiologists and the presence of srd : anxiety , depression and psychological malaise . during a national congress of the italian society of medical radiology , radiologists were invited to complete a methods questionnaire that included , amongst other things , a section for evaluating work - related stress and one for estimating its consequences . 
karaseks questionnaire provided scores relating to the demand scale , psycho - physical demands of the job ; the control scale concerning the amount of discretionary power the worker has in performing his work ; and the support scale indicating support from colleagues and superiors . 
job strain can be dened as the proportion of workers who simultaneously psychological workload ( demand values above the median ) and low control ( values below the median )  . 
siegrists questionnaire provided an effort scale ( the psychological work effort or extrinsic stress ) , an over - commitment scale ( high work commitment , or intrinsic stress ) and a reward scale ( rewards received for work performed )  . 
subjects with a score [ 1 were considered to be stressed as they subjectively perceived an imbalance between effort and reward . elevated have the consequences of distress were evaluated using the general health questionnaire ( ghq12 ) and goldbergs anxiety and depression scales , both in their italian versions [ 17 , 18 ]  . 
the level beyond which the risk of suffering from a psychic illness increases signicantly , is set at 2 points since it is the level that provides the best sensitivity and specicity [ 20 ] ; thus from 3 points onwards , the subject could become a case . we should remember that the concept of caseness in social psychiatry has only an epidemiologic value and radiol med ( 2014 ) 119 : 359366 corresponds to a probability , not a psychiatric diagnosis . 
the nal score results from the total number of afrmative answers . caseness , dened as a more than 50 % probability that the diagnosis will be conrmed by a specialist , corresponds to a score of six or more points on the anxiety scale , and three or more points on the depression scale [ 21 ]  . 
in the second and third models , we added the variables indicating work - related stress , by separately adding karaseks ( demand , control , support ) variables and those of siegrist ( effort , reward , over - commitment )  . 
in the nal model , we introduced all the variables relating to stress and the sociodemographic variables simultaneously as predictors of each of the effect variables ( anxiety , depression , psychological malaise )  . 
the standardised correlation coefcient ( b ) indicated the degree of association between the variables . the capacity of each equation to correctly interpret variations in the phenomenon studied was indicated by the adjusted coefcient of determination ( r2 )  . second , we wanted to ascertain what risk radiologists have of suffering from psychic disorders when they are in a state of distress . 
for this purpose , we used binary logistic regression , taking the dependent variable to be the state of anxiety , depression and psychological malaise ( caseness ) as dened above . 
as the independent variable we separately introduced the state of distress evaluated as job strain ( the subject is exposed to high demand and low control ) and the state evaluated as eri ( subjects with eri [ 1 ) , the state of social isolation ( support = below the median ) and that of excessive involvement in work ( over - commitment = above the median )  . 
the value we obtained ( raw or non - adjusted ) was subsequently adjusted by adding to the equation the variables indicating gender , age , length of employment , type of organisation , where subjects were employed , and hierarchical role . 
this enabled us to calculate the odds ratio ( or ) and their 95 % condence intervals ( or 95 % )  . results the characteristics of the study population are illustrated in table 1 . 
table 2 shows the mean values of the variables indicating work - related stress and mental disorders . among the radiologists who completed our questionnaire , there were 286 ( 43.7 % ) probable cases of anxiety ( score of [ 5 on the goldberg a scale ) ; 287 ( 43.9 % ) cases of depression ( score of [ 3 on the d scale ) and 140 ( 21.4 % ) cases of psychological malaise ( ghq [ 2 )  . linear regression analysis ( table 3 ) enabled us to determine to what degree the value of each of the measurements of psychiatric pathology could be predicted using data on sociodemographic factors and the level of work - related stress . 
a signicant association was observed between some of the sociodemographic variables and the scores for anxiety , depression and psychic disorders , but on the whole , sociodemographic factors accounted for a small fraction of the variation in psychic problems ( coefcients of determination r2 ranged from 3 to 7 % )  . 
a positive association was observed between age and depression , but the former was negatively related with acute psychic disorder measured by the ghq12 and this relationship also remained signicant in the more complex model . 
even allowing for rst type error ( false positive ) , we must conclude that the prevalence of psychic disorders in radiologists is not negligible and could exceed values reported in other occupational populations [ 6 ]  . 
moreover , individuals with psychic problems may report working conditions in a less favourable light since they are the type of subjects who complain about everything [ 32 ]  . 
the opposite may also be true ; that is , there are workers who tend to deny the existence of both environmental and health problems , they are the ones who never complain about anything [ 33 ]  . 
the simultaneous presence of these two types of workers lowers the reliability of cross - sectional studies in which participants are asked to make a self - evaluation of both environmental and personal conditions . 
in our the fact particular case , physicians probably increased the objectivity of responses . the interviewees were their that all on the basis of our ndings , we believe that the work of radiologists would benet from primary preventive intervention at a number of levels . 
the dcs model shows that radiologists would benet from programs designed to improve the management of workloads ( especially working hours ) , productive processes and group work , and company policies for enhancing social support . 
the eri model indicates that any reorganisation should include a reduction in extrinsic effort , especially in younger subjects or those who are new to the job , with fairer distribution of workloads , less overtime and a sufcient number of breaks , an increase in rewards ( including immaterial ones ) for work performed . 
it seems to be particularly important to reduce intrinsic stress resulting from over - commitment concentrate on the technical aspects of the job and by teaching subjects to introduce protective factors against stress such as better lifestyles and recreational and sports activities . 
these tasks , which are often disregarded in our country , are an essential part of the general protection that employers , and therefore also public and private health authorities , are legally called upon to implement . 
the mr studies were reviewed according to the mr - bi - rads classication system ; lesions assessed as mr - bi - rads 1 and 2 were considered negative for malignancy , categories mr - bi - rads 3 , 4 and 5 indicated malignant lesions . 
mr sensitivity , specicity , positive predictive value ( ppv ) and npv were calculated . results the nal population consisted of 71 lesions . showed malignancy in six cases histologic analysis ( malignancy rate 8 % )  . 
mr sensitivity was 33 % , specicity 86 % , ppv 18 % and npv 93 % . conclusions because of its relatively low npv , mr imaging is not able to safely exclude malignancy in the a . 
the relatively high malignancy rate found in this study might support svab in the case of bi - rads 3 microcalcications . keywords bi - rads 3 ( cid : 2 ) microcalcications ( cid : 2 ) breast mri ( cid : 2 ) stereotactically guided vacuumassisted - biopsy ( cid : 2 ) dcis introduction the estimated probability of malignancy in breast imaging and reporting data system ( bi - rads ) category 3 mammographic lesions ( including both microcalcications and masses ) is lower than 2 % [ 1 , 2 ] ; therefore , periodic mammography follow - up is recommended in the management of these lesions . 
however , some authors have found disturbingly high rates of malignancy in women with microcalcications assessed as probably benign ( bi - rads 3 ) , ranging from 0 to 19 % [ 37 ]  . 
as a consequence , many bi - rads 3 microcalcications are recommended for stereotactically guided vacuum - assisted biopsy ( svab ) instead of mammographic follow - up , because of referring physician or patient concern that there could be a substantial risk of malignancy [ 4 , 8 ]  . the large majority of patients with bi - rads 3 microcalcications referred for biopsy have benign lesions , in a high number of unnecessary biopsy which result procedures . breast magnetic resonance ( mr ) imaging has very high sensitivity for breast cancer [ 9 ] , and in selected scenarios , a negative breast mr examination is able to safely exclude malignancy [ 10 ]  . 
 [ 11 ] found that mr imaging has sufciently high negative predictive value ( npv ) to rule out malignancy and avoid 394 radiol med ( 2014 ) 119 : 393399 invasive procedures in noncalcied bi - rads 3 mammographic lesions . 
conversely , a denitive conclusion about the role of mr imaging in bi - rads 3 microcalcications could not be drawn because of the small number of microcalcications included in the study [ 11 ]  . 
the purpose of this study was to evaluate whether mr imaging is able to rule out malignancy in the case of bi - rads 3 microcalcications , providing a sufcient npv for early work - up , and therefore to decrease the number of unnecessary svab procedures . materials and methods this study was performed in a university referral hospital for breast diseases . 
all patients provided written informed consent to participate in the study . patient population between september 2006 and june 2011 , we prospectively enrolled consecutive women with screen - detected probably benign ( bi - rads 3 ) microcalcications , who subsequently underwent mr examination of the breast and svab of the microcalcications . 
microcalcications were classied according to bi - rads descriptors for mammographic features including calcication morphology ( punctate , amorphous , pleoregional , morphic , clustered , segmental and linear )  . 
according to the birads lexicon , clusters of punctate / amorphous microcalcications were dened as bi - rads 3 . and distribution ( diffuse , linear ) mr imaging technique breast mr imaging studies were performed on a 1.5 - t scanner ( magnetom avanto , siemens medical system , erlangen , germany ) with a dedicated , bilateral , 7 - channel phased - array coil and the patient in prone position . 
from september 2006 to may 2009 , the dynamic contrast study was obtained using a coronal t1 - weighted three - dimensional ( 3d ) fast low - angle shot ( flash ) pulse sequence with the following parameters : tr / te , 15 / 4.7 ms ( ms ) ; ip angle , 25 ( cid : 3 ) ; matrix , 197 9 448 ; eld of view , 350 9 175 mm ; section thickness , 1.8 mm ; acquisition time , 1 : 28 min . from june 2009 to june 2011 , the dynamic contrast study was obtained using an axial t1 - weighted 3d flash pulse sequence with the following parameters : tr / te , 9 / 4.7 ms ; ip angle , 25 ( cid : 3 ) ; matrix , 512 9 512 ; eld of view , 340 9 340 mm ; section thickness , 2 mm ; acquisition time , 1 : 20 mgadobenate dimeglumine ( gd - boptamultihance , bracco imaging , milan , italy ) was administered intravenously with an automated bolus injection at a dose of 0.1 mmol / kg body weight with a ow rate of 2 ml / s , immediately followed by a 20 - ml saline ush , using an automatic injector ( spectris solaris , medrad )  . 
images were acquired sequentially once before and ve times after injection of contrast agent , beginning 12 s after initiation of the contrast injection and with no interscan delay . postprocessing and construction of dynamic curves were performed by one of the ve radiologists with more than 2 years of experience in breast mr imaging using a syngo multimodality workplace ( leonardo , siemens healthcare , erlangen , germany )  . 
postprocessing included temporal subtraction ( all contrast - enhanced series minus noncontrast - enhanced series ) , and the acquisition of multiplanar reconstructions ( mpr ) and maximum intensity projections ( mip )  . dynamic signal intensitytime curves were constructed for regions of interest ( 3 9 3 pixels ) positioned within the lesion on subjectively determined areas of maximal enhancement . it is our standard of practice to perform targeted sonographic examination ( second - look ) in the case of incidental mr ndings that were occult on mammography or whole - breast initial sonography . mr imaging analysis the study coordinator ( with more than 5 - year experience in breast imaging ) , who was not involved in further mr evaluation , reviewed all imaging available and annotated on a breast map the location of each microcalcication lesion using a clock - face reference and the relative distance radiol med ( 2014 ) 119 : 393399 from the nipple . 
mr evaluation was performed on noncontrast - enhanced and contrast - enhanced series . the mr images were classied as negative / normal if no contrast enhancement was identied in the area of the lesion ( mr - bi - rads 1 ) or if only homogeneous or stippled enhancement representing normal glandular tissue ( mr - bi - rads 2 ) was seen bilaterally . 
lesions that were detected on mr and corresponded to the area of microcalcications were classied as focus , mass enhancement or nonmass enhancement and were assessed as mr - birads 3 , 4 or 5 [ 13 ]  . 
with the patients lying prone on a digital stereotactic table ( mammobed , giotto , ims srl ) , svab was performed using a directional vacuumassisted biopsy ( vab ) device ( mammotome , ethicon endo - surgery , cincinnati , oh , usa ) with an 11 - gauge needle . 
after the procedure , a clip ( mammomark , ethicon endo - surgery , cincinnati , oh , usa ) was placed through the 11 - gauge needle to mark the biopsy site for subsequent surgical excision . if pathologic examination demonstrated a malignancy or a high - risk lesion ( such as atypical ductal hyperplasia ) , surgical excision after placement of a hook - wire under stereotactic guidance was recommended . 
if a benign lesion was pathologically proven on svab , the patient was scheduled for repeat mammography of the ipsilateral breast at 6 months and annual screening mammography was recommended thereafter . data analysis for histologic analysis , we grouped the microcalcications into two main categories , benign and malignant . 
in patients who underwent a surgical procedure after svab ( because of malignant or high - risk lesions demonstrated in biopsy samples ) , the most severe histologic result was considered . in the case of high - risk lesions conrmed at surgical excision , the lesion was considered benign . a malignant lesion was considered to be diagnosed successfully by mr imaging ( true positive ) if contrast uptake in the area of microcalcications was shown ( birads 3 , 4 or 5 )  . 
a false negative nding was dened as the absence of focal contrast uptake in the area of microcalcications ( bi - rads 1 and 2 ) that proved to be malignant at histologic analysis . we calculated the sensitivity , specicity , positive ( ppv ) and npv of mr imaging in diagnosing malignant lesions presenting mammography . 3 microcalcications as bi - rads results patient population seventy - four bi - rads 3 microcalcication lesions were initially considered eligible . 
the mean interval from mr imaging to svab was 9 days ( range 143 ) ; the mean interval from svab to surgical procedure was 20 days ( range 1345 days )  . histologic and follow - up results stereotactically guided vacuum - assisted biopsy demonstrated malignancy in ve cases [ four intermediate - grade ductal carcinoma in situ ( dcis ) and one low - grade dcis ] , that were all conrmed at surgical excision . 
five high - risk lesions were diagnosed at svab ( 3 lobular neoplasia , 1 radial sclerosing lesion and 1 at epithelial atypia ) ; one lobular neoplasia was upgraded to low - grade dcis at surgical excision , while the remaining high - risk lesions were conrmed . 
b , c axial t1 - weighted nonenhanced ( b ) and contrast - enhanced subtracted ( c ) magnetic resonance ( mr ) images ( 9 / 4.7 , 25 ( cid : 3 ) ip angle ) show the absence of pathologic enhancement in the area of microcalcications . 
this examination was assessed as mr - bi - rads 1 ( false negative ) therefore , among the 71 lesions included in the study , nal histologic analysis showed dcis in 6 ( 8 % ) , high - risk lesions in 4 ( 6 % ) , and benign lesions in 61 ( 86 % )  . mr imaging analysis versus reference standard at breast mr analysis , 60 ( 85 % ) lesions were assessed as negative ( 52 mr - bi - rads1 and 8 mr - bi - rads 2 )  . 
of these , 4 ( 7 % ) proved to be malignant at nal histologic examination ( 2 low - grade dcis and 2 intermediate - grade dcis )  . 
of these , 2 ( 18 % ) were malignant . one lesion was assessed as mr - bi - rads 3 and was an intermediate - grade dcis at svab , conrmed at nal pathologic examination . 
2 a 63 - year - old woman with benign disease in the left breast ( stereotactically guided vacuum - assisted - biopsy : brocystic changes ; no calcication progression at 24 - month follow - up )  . 
b , c axial t1weighted nonenhanced ( b ) and contrast - enhanced subtracted ( c ) mr images ( 9 / 4.7 , 25 ( cid : 3 ) ip angle ) show a mass - like lesion with faint enhancement and poorly dened borders ( arrow ) located in the central , prepectoral area of the left breast , corresponding to the location of microcalcications . 
breast mr had a sensitivity of 33 % [ 95 % condence interval ( ci ) 2346 % ] , specicity of 86 % ( 95 % ci 7593 % ) , ppv of 18 % ( 95 % ci 1029 % ) and npv of 93 % ( 95 % ci 8498 % )  . nineteen patients ( 27 % ) underwent second - look sonographic examination because of incidental ndings ( all assessed as mr - bi - rads 3 ) located outside the area of microcalcications and that were occult at mammography and initial whole - breast ultrasound . 
in 18 cases , secondlook examination showed normal or benign ndings ( cysts , intraparenchymal lymph nodes , or broadenomas )  . in these patients , no malignant lesion was seen during follow - up . 
one incidental lesion was occult at second - look ultrasound and was followed up with 6and 12 - month mr examinations that demonstrated no interval changes . 398 discussion our data show that mr imaging does not have sufciently high npv ( 93 % ) to exclude the presence of malignancy in the case of bi - rads 3 microcalcications . 
therefore , mr imaging is not able to guide the work - up ( biopsy or follow - up after 6 months ) of patients with bi - rads 3 lesions . our results are in contrast with those reported by dorrius et al . 
 [ 10 ] , who found a 100 % npv for malignancy in the case bi - rads 3 lesions . however , those studies included only eight cases of birads 3 microcalcications each . several studies evaluated the role of mri in patients with mammographic microcalcications ; however , those studies included also bi - rads 4 [ 14 ] or bi - rads 4 and 5 microcalcications [ 7 ] and global mr performance was calculated , without a by - category analysis . 
these were two low - grade and two intermediategrade dcis , all smaller than 8 mdespite the high sensitivity of mr imaging for invasive cancers and high - grade dcis , an absence of enhancement has been observed in 2060 % of intermediateand low - grade dcis [ 1517 ]  . in addition , it has been demonstrated that mr imaging leads to the detection of incidental benign ndings . 
in our study , in 19 ( 27 % ) of 71 patients , mr imaging detected additional ndings , which resulted in 19 sonographic examinations and 2 follow - up mr examinations in the same patient . 
in none of these cases was malignancy found at additional imaging or at follow - up . in our series , 6 ( 8 % ) of 71 bi - rads category 3 microcalcications were malignant . 
the recommended percentage among bi - rads 3 lesions is 2 % or fewer ; however , these low malignancy rates are based on followup mammograms and not on biopsy series [ 1 , 2 ]  . 
in addition , the stated low malignancy rate in bi - rads 3 lesions is probably related to the broad spectrum of mammographic ndings included in this group , which also includes well - dened masses [ 3 ]  . 
malignancy rates of bi - rads 3 microcalcications in svab series range from 0 to 16 % [ 37 ] , which is not tolerated for lesions managed with short - interval follow - up . radiol med ( 2014 ) 119 : 393399 on the other hand , most of bi - rads 3 microcalcications correspond to intermediateor low - grade dcis at pathologic examination ( 4 and 2 lesions respectively , in our study )  . 
intermediateand low - grade dcis have a benign biologic prole and if left undetected ( and untreated ) , they are likely to remain dormant or to exhibit slow ( decades long ) progression to invasive carcinoma [ 18 ]  . 
therefore , diagnostic delay due to noninvasive management ( imaging follow - up ) would probably not have a substantial effect on prognosis . however , interval changes at follow - up in the number and morphology of probably benign microcalcications may be difcult to assess , in particular if there are sufcient technical differences between the two studies . 
at the same time , the microcalcications seen in low - grade dcis may remain unchanged for many years [ 19 ]  . moreover , a recent study reported a high percentage of noncompliance in women recommended to undergo shortinterval follow - up for probably benign ndings , even in the context of a clinical trial [ 20 ]  . therefore , svab might still be considered an approchoice management bi - rads priate microcalcications . our study has some limitations . 
in addition , because of the difculties and variability in the classication of clusters of punctate / amorphous , an external review of our mammographic cases classied as bi - rads 3 would have been of great benet . in conclusion , we found that , unlike for noncalcied birads lesions , mr imaging is not useful in guiding the management of bi - rads 3 microcalcications . 
at the end of observation , the bowel was excised for histological analysis . results 7t l - mri t2 - w sequences acquired 15 min after sbo , showed early evidence of bowel wall hyperintensity and a small amount of peritoneal free uid . 
at 1 h , a hyperintensity of the loop proximal to the obstruction was found and , after 4 h , free uid between the loops , bowel wall thickening and increased wall hyperintensity were also found . 
after 6 h hypotonic reex ileus ( only gas - lled dilated loops ) was detected , which became paralytic ileus ( dilation with airuid levels ) after 8 h . 
the mri ndings were all conrmed at histological examination . conclusions this study allows denition of the early mri features of sbo ( peritoneal free uid and hyperintensity of the injured bowel ) and their chronological evolution , also conrmed by histological examination . 
the chance to achieve an early detection of bowel injury and to correlate the histological pattern with imaging ndings could contribute to a ner and earlier diagnosis and a more effective treatment . keywords small bowel obstruction ( cid : 2 ) magnetic resonance imaging ( cid : 2 ) experimental study ( cid : 2 ) animal model introduction small bowel obstruction ( sbo ) is a common clinical condition , often presenting with signs and symptoms similar to those found in other acute abdominal disorders . imaging , sbo despite recent advances in abdominal remains complex to diagnose and to treat [ 1 ]  . 
the morbidity and the mortality associated with acute sbo continue to be signicant , accounting for 1216 % of all surgical admissions in patients with acute abdominal conditions [ 1 ]  . imaging is important in the diagnosis and management of patients with sbo because it allows us to determine the presence and severity of obstruction , the transition point , and the cause of obstruction [ 2 ]  . previous investigations have demonstrated that accuracy of abdominal plain lm in determining the presence of obstruction is 4680 % [ 3 ] and that airuid levels are the most specic sign [ 3 ]  . 
unfortunately , air uid levels are not sensitive because they may be absent , especially in the late phase of small bowel volvulus and incarceration , when the loops become completely full of uid [ 4 ]  . 
ultrasonography ( us ) is widespread in several countries but this technique is operator dependent and it has inherent limitations because of the artefacts of gas378 radiol med ( 2014 ) 119 : 377383 containing structures [ 5 ]  . 
however , the ct technique suffers some limitations , the amount of delivered radiation dose and the need to administer iodinated contrast material [ 57 ]  . in particular magnetic resonance imaging ( mri ) , thanks to its excellent soft - tissue contrast and absence of ionising radiation exposure , has several characteristics that would make it an ideal imaging technique for the analysis of the small bowel , resulting in a promising alternative to ct in the evaluation of acute sbo . 
the purpose of this experimental study was to identify the early mri ndings of sbo and to analyse their evolution over time comparing them with histological ndings in an animal model . materials and methods animal preparation all procedures performed on animals were approved by our institutional animal care and use committee . 
ten adult male spraguedawley rats ( 250340 g ; harlan , usa ) , maintained on a 12 / 12 h light / dark cycle and allowed free access to food and water , were randomly divided into two groups of ve rats each ( group 1 and group 2 )  . 
group 1 underwent macroscopic observation , group 2 underwent 7t l - mri examination ; the animals in each group were examined at predetermined time points : basal ( before bowel occlusion ) , 15 min , 1 , 4 , 6 and 8 h after sbo . 
the rats were anaesthetized with ketamine hydrochloride 100 mg / kg im ( cu chemieuetikon gmbh , lahr , germany ) and medetomidine hydrochloride 0.25 mg / kg im ( domitor , pzer , new york , usa ) injections . 
photographs of the exposed bowel were taken after the occlusion at the ve predetermined time points described above . macroscopic analysis was assessed for the following parameters : ( 1 ) spastic reex ileus ( sri ) , consisting in contraction of the semilunar folds ; ( 2 ) hypotonic reex ileus ( hri ) , consisting in bowel wall dilation ( more than 2.5 mm in calibre ) ; ( 3 ) chromatic change of the bowel wall ( which is physiologically pink )  . magnetic resonance imaging group 2 animals underwent mri of the abdomen using a 7t l - mri scanner ( biospec 70 / 16us , bruker medical systems , ettlingen , germany )  . animals turborare sequences ( tr = 4 , 428.7 ms , te = 36 ms ; 180 ( cid : 3 ) ip angle ; 38 slices ; slice thickness , 1 mm ; interslice distance , 1 mm ; eld of view , scan time = 7 min 5 s 156 ms ) were performed just before sbo to act as basal imaging and after sbo at the above - mentioned ve time points . acquisition matrix , 256 9 256 ; 6 cm ; mr images were assessed for the following parameters : ( 1 ) presence of free uid in the abdomen ; ( 2 ) hri , consisting in bowel gas - lled dilation ( more than 2.5 mm in calibre ) ; ( 3 ) gasuid mixed stasis dilated loops ( paralytic ileus , pi ) ; ( 4 ) bowel wall thinning or thickening ( physiological thickness 11.3 ) ; ( 5 ) wall signal intensity on t2weighted sequences . histological analysis in all rats sbo was induced by performing a surgical procedure : after midline laparotomy , the most supercial small bowel loop in the upper right abdomen was isolated for light microscopy , the small bowels in both groups of rats were excised from the duodenum to the cecum , and stored in 10 % buffered formalin acetate for at least 2 days . 
transverse sections 3 lm in thickness were cut and stained with haematoxylineosthe sections were mounted on chrome alum / gelatin - coated slides , dehydrated and coverslipped . slides were imaged with a zeiss axio skope microscope equipped with a high - resolution digital camera ( orcahr c4742 - 95 - 12hr , 10 mp ; hamamatsu photonics k.k. , hamamatsu city , japan )  . results macroscopic evaluation in group 1 imaging the physiological appearance of rat at basal intestine before inducing the obstruction is given in fig . 
2 7 - tesla mri scan of the abdomen : basal image immediately before sbo : the loops appear with normal diameter and normal wall thickening , peritoneal free uid is absent ( a )  . 
at 15 min after bowel ligation , evidence of bowel wall hyperintensity ( arrow ) associated with a small amount of peritoneal free uid ( arrowhead ) ( b )  . 
in ( c ) bowel wall thickening , hyperintense signal in the bowel wall ( arrow ) of the loop proximal to the obstruction and a mild increase of peritoneal free uid ( arrowheads ) 1 h after sbo . 
four hours after sbo , a further increase of free uid among loops and of wall hyperintensity , associated with a mural thickening were detected ( d )  . hri ( arrow ) was appreciated after 6 h ( e )  . 
this study , based on an animal model of sbo , was designed to identify the mri ndings until 8 h of observation and to monitor their evolution in that time comparing them with the histological ndings . 
thus far , the discussion chronological sequence of early obstructive bowel damage has not been described , and the role of mri is still widely debated . the increase of intraluminal there has been a global change in the spectrum of aetiology of acute sbo over the past few years . 
even if some authors divide acute sbo into eight aetiological forms [ 1720 ] ( table 1 ) , just considering the surgical and imaging ndings , sbo can be classied as simple , decompensated and complicated [ 17 , 18 ]  . 
a thin layer of free peritoneal uid between the intestinal loops can be detected , which may increase , lling radiol med ( 2014 ) 119 : 377383 initial fig . 
3 at 15 min after sbo , the histology of the bowel sections showed damage in the proximal segment close to the sbo site , ischaemic damage and initial cleavage of consisting in : supercial epithelial cells . 
in the same region , 1 h after sbo , the histology of the bowel sections showed an increase of blood cell elements within the lamina propria ( b1 ) and in the medium magnication images increased damage of supercial enterocytes was detected ( b2 )  . 
at 6 h after ligation ( 3f ) the lumen was lled with necrotic material , bowel cellular debris and the brovascular axis appeared more congested ( 3d )  . 
at 8 h after bowel ligation , at high magnication , the peak of the villi appeared necrotic and a partial detachment of the villi was clearly visible ( e1 , e2 ) table 1 aetiological mechanisms of acute small bowel obstruction ( sbo ) 1 . 
complex mechanisms the lumen and peritoneal cavity in a more advanced stage . a correlation between the free peritoneal uid and a prompt surgical approach has been previously demonstrated [ 5 , 17 , 19 , 22 ]  . 
this stage requires immediate surgery [ 21 ]  . other authors distinguish two types of sbo , on the basis of vascular involvement : uncomplicated ( without vascular involvement ) and strangulation ( with vascular involvement ) , with a mortality rate varying from 3 to 7 % in uncomplicated cases to around 15 % in cases of strangulation [ 17 , 19 , 23 ]  . levels of severity and medical / surgical approaches [ 17 , 19 ]  . 
active these evolutionary stages exhibit different collaboration between the radiologist , emergency physicians , and general surgeons is necessary to optimise the diagnostic evaluation and management of this common and potentially dangerous problem [ 20 ]  . 
the role of radiologists in sbo is to conrm the obstruction , establish the cause , determine the level , and predict whether strangulation is present [ 24 ]  . at present when sbo is suspected , the diagnostic imaging protocol consists in plain abdominal radiography and ct whereas the utility of mri in acute settings is still a matter of debate . 
the diagnosis of sbo can be made with plain abdominal lms in 5060 % of cases but this technique may be unremarkable in 20 % of cases [ 25 ]  . 
the role of ultrasound ( us ) has been widely applied to evaluate various abdominal diseases and several articles have dealt with the usefulness of us in sbo [ 24 , 26 , 27 ]  . in the last few years , investigations have shown that mri can allow visualisation of the entire bowel , without overlapping bowel loops , and the detection of extraluminal pathological conditions [ 28 ]  . 
moreover , improvements in mri software and hardware have enabled imaging to assume a major role in the evaluation of the small bowel . the ability to provide cross - sectional information combined with the lack of ionising radiation can make mri well suited for small bowel imaging [ 28 ]  . 382 radiol med ( 2014 ) 119 : 377383 previous studies showed that mri using the half - fourier acquisition single - shot turbo spin - echo ( haste ) sequence is accurate for the diagnosis of sbo . 
 [ 29 ] proved mri ability to study the normal appearance of small bowel in haste sequence and to reveal small bowel disease such as crohns and sbo avoiding artefacts related to peristalsis and breathing [ 30 ]  . 
moreover , comparative studies between fast mri and helical ct proved that mri using haste sequences was more accurate than the helical ct ( 96 % with mri vs 71 % for ct ) [ 30 ]  . in this study , we used t2 - weighted mri sequences because , as shown in a previous study [ 31 ] , they allow us to identify , without using contrast media , the hyperintensity of the loop proximal to the obstruction site and peritoneal free uid . in the rats of group 1 , at 15 min after the obstruction , no pathological ndings related to obstructive damage were detected , while , in the rats in group 2 , 7t l - mr imaging without contrast material gave early evidence of bowel wall hyperintensity and mural thickening associated with a small amount of peritoneal free uid . 
the histology of the bowel sections , at this time point , showed initial ischaemic damage and an initial cleavage of the supercial epithelial cells . the submucosal vessels appeared ectasic and congested . in rats of group 1 , 1 h after the obstruction , sri of the loop proximal to the obstruction was observed , which was an early and transient reaction of the semilunar folds to the obstruction insult . 
the histological images showed increase of blood cell elements within the lamina propria and damage of the supercial epithelial cells . at 4 h , in the rats in group 1 , the sri was associated with a chromatic change of the ligated loop . 
within the adipose tissue ecstatic and congested venules were observed and the brovascular axis appeared more congested . in the rats of group 1 some previous ndings were still present at the 8th hour after the obstruction such as dilation and chromatic change . 
in group 2 , the dilation caused the arrest of the progression of the intestinal content , with resulting formation of airuid levels in the lumen ( pi ) , which were evident at the end of observation beyond other reported ndings . 
these results were conrmed at histological examination where a progressive mucosal ( at 15 min after sbo ) and transmural injury ( in the subsequent examination time points ) were found . although ct remains a valid diagnostic tool for the visualisation of signs of sbo [ 32 , 33 ] , mri , which does not require the use of a contrast medium or ionising radiation , can play an important role in the diagnostic and therapeutic management of patients with acute sbo . 
the aim of our paper is to report the frequency of this cerebrovascular disease in a series of patients affected by nf1 , using magnetic resonance angiography ( mra )  . 
in our study , mra proved to be critical , especially for the detection of stenoses in the branches of the circle of willis . conclusion few case series have investigated the incidence of vascular complications of nf1 , and most of them have used mri . 
melis department of pediatrics , university federico ii of naples , naples , italy keywords neurobromatosis type 1 ( cid : 2 ) arterial stenosis ( cid : 2 ) magnetic resonance angiography ( cid : 2 ) magnetic resonance imaging introduction neurobromatosis type 1 ( nf1 ) is a multisystem autosomal dominant disorder that primarily involves the skin and the nervous system , affecting approximately one in 2 , 500 / 3 , 500 individuals [ 1 , 2 ]  . 
it is caused by a mutation of a gene which encodes a protein known as neurobromin , localised on the long arm of chromosome 17 . vascular dysplasia is becoming increasingly recognised as a feature of nf1 with an estimated frequency between 2 and 6 % [ 8 , 9 ] , and the involvement of the circle of willis can be an extremely serious complication [ 4 , 5 ] , especially in the case of development of moya - moya disease . 
the cause of this variability is probably due to the fact that not all the patients with nf1 underwent magnetic resonance imaging and magnetic resonance angiography ( mri / mra ) of the brain [ 7 , 9 ]  . 
3 , all the stenoses of time ( 6 and the intracranial vessels worsened over 12 months follow - up mri / mra scans ) leading to a moya - moya disease clearly evident on the third followfig . 
three - dimensional time - of - ight ( 3d tof ) sequence , maximum intensity projection ( mip ) reconstruction - axial the left middle cerebral artery with view shows occlusion of hypertrophy of the ipsilateral middle meningeal artery up scan . 
1 , 2 , 4 , and 6 , in keeping with the majority of studies on nf1 - related brain vasculopathy , had no neurological signs or symptoms that could be related to the arterial stenoses , while patient no . 
3 had developed a severe mental retardation ( in connection with the worsening of the stenosis of the intracranial vessels )  . on t2 - weighted images all of the six patients showed the typical unidentied bright objects ( ubos ) also known as neurobromatosis bright objects ( nbos ) [ 10 ]  . 
 [ 6 ] classied the nf1 - associated vascular lesions into three types : stenotic , aneurysmal and mixed . all our patients had only stenotic lesions , a nding in agreement with rea et al . 
3d tof coronal mip reconstruction ( left ) shows complete bilateral disappearance of the supraclinoid internal carotid arteries and of the right posterior cerebral artery , with many secondary collaterals , delineating a typical feature of moya - moya syndrome ( puff of smoke ) , more evident on the right side . 
thus , children with nf1 have a far greater risk of having stenotic rather than aneurysmal lesions ( which have been described but are very rare [ 29 ] ) , at least in the cerebral circulation . to our knowledge , there are only ve papers investigating the prevalence of cerebral arteriopathy in populations of patients with a clinical diagnosis of nf1 [ 79 , 27 , 28 ] and few case reports [ 24 , 25 ]  . 
 [ 9 ] , who studied the largest series of children with nf1 , found a prevalence of 6 % ( 17 / 266 children who underwent mri )  . 
cairns and north [ 8 ] reported seven patients with brain arteriopathy out of 144 nf1 children with a clinical indication for a brain mri scan ( 5 % )  . 
even in these two latter studies mra was only performed when vascular abnormalities had already been detected at mri . in our study , we presume that the presence of a wider age range ( 435 years ) does not constitute a bias . 
computed tomography ( ct ) angiography mip reconstruction of the extracranial tract of the right internal carotid artery clearly depicting a severe postbulbar stenosis frequency of vascular dysplasia in younger children [ 8 ] but it also well known that vascular stenosis may become progressive month to years after the diagnosis [ 9 ]  . regarding our higher prevalence of brain vasculopathy , it could be partially due to the fact that mra is not routinely performed in all patients with nf1 . 
mra is not included in the brain mr protocols of patient with nf1 , even in the studies focusing on nf1 - related vasculopathy . all the authors performed mra only when they noted direct or indirect signs of stenosis on mri examination [ 9 ]  . in particular , these were characterised by cortical strokes and / or white matter hyperintensities . 
therefore , mra is critical in detecting arterial narrowing of distal vessels of lesser calibre , which are undetectable by mri only . furthermore , in agreement with other authors , we found an increased prevalence of arteriopathy in patients with optic glioma , detected in four patients ( 66 % ) ; this could , however , result from a selection bias given that the patient 420 radiol med ( 2014 ) 119 : 415421 with optic glioma is often symptomatic and for this reason always undergoes mri examination [ 79 ]  . regarding the clinical status of our group of patients , only the intermittent headache of patient no . 
3 , although probably due to atrophy of the right fronto - sylvian region , could be also related to the fact that the patient had previously undergone radiotherapy for a large optic chiasm glioma . 
it is important to highlight that in all these studies the majority of patients did not have neurological symptoms related to the brain arteriopathy . we performed follow - up mri / mra examinations after 36 months and 1215 months in four patients with vascular dysplasia , nding only in patient no . 
3 that all the stenoses of the intracranial vessels had worsened over time ( 6 and 12 months follow - up mri / mra scans ) leading to a moya - moya disease clearly evident at the third followup scan . a longer follow - up , which is currently underway , is needed to obtain more information about the real progression of brain arteriopathy in patients with nf1 . 
there are no clear indications in the literature about the frequency of the follow - up in patients with nf1 - related vasculopathy because of the low incidence of the condition . 
in addition , we must consider the fact that mra extends the time of a brain mri examination by about 10 % only [ 9 ] , and that the costs of a permanent impairment due to a stroke could be very high . 
indeed the cost of care for a patient with a stroke in only the rst year after diagnosis has been estimated to be $42 , 338 [ 9 , 12 ]  . 
thus , the early identication of a cerebral vasculopathy could reduce the costs of long - term care , especially for children with lifelong disability from stroke . a possible bias in our study is linked to patient no . 
3 because she had previously ( at the age of 5 years ) been treated with radiotherapy for a right optic nerve glioma ( a procedure no longer indicated for treatment of optic gliomas in children [ 13 , 26 ] )  . 
the atrophy could be due either to the radiation therapy or to the right ica stenosis itself or to a coexistence of both the conditions [ 14 , 15 ]  . 
3 was located contralaterally to the irradiation eld , so that it was probably unrelated to the radiotherapy , and should be considered an incidental nding . conclusion we believe that the protocol for patients affected by nf1 with a clinical indication for brain mri should include an mra as well , so as to detect any stenoses of branches of the circle of willis including those of small and moderate degree . 
of these , 92 / 120 nodules ( 76.6 % ; mean diameter 18.4 mm ) showed the typical hcc vascular pattern : 90 / 92 nodules appeared hypointense and 2 / 92 were hyperintense on hepatobiliary phase images . 
an additional 28 / 120 hypointense , nonhypervascular nodules ( 23.3 % ; mean diameter 11 mm ) were detected on hepatobiliary phase images , 15 of which showed hypointensity also on the equilibrium phase images . 
david ospedale santandrea , via di grottarossa 1035 , 00189 rome , italy conclusions gadoxetic acid - enhanced mr imaging is useful for detecting hcc as well as hypovascular nodules with potential progression to hcc . 
it is therefore crucial to identify those patients at high risk of developing hcc by establishing a proper surveillance programme aimed at an early diagnosis . hepatocarcinogenesis should be considered a multistep process arising in a cirrhotic liver ; the tumour develops by evolving from a regenerative to a dysplastic nodule , progressing from an initially well - differentiated hcc ( early to a poorly differentiated hcc and nally to hcc ) undifferentiated neoplasia . 
nodules with a high grade of dysplasia represent pre - neoplastic lesions that evolve into hcc with a reported frequency of 4680 % over 15 years [ 2 ]  . in this scenario , it is important to use diagnostic techniques allowing for the early detection and characterisation 368 radiol med ( 2014 ) 119 : 367376 of focal liver lesions . 
dynamic computed tomography ( ct ) or magnetic resonance ( mr ) imaging , performed with an arterial contrast phase with extracellular contrast agents , are recommended by the american association for the study of liver disease ( aasld ) as the imaging techniques of choice for the detection and characterisation of hcc in cirrhotic patients [ 3 ]  . 
a nding of the typical vascular pattern of hcchypervascularity in the arterial phase , washout in the portal venous and / or equilibrium phasein nodules larger than 1 cm is considered diagnostic , and biopsy is not required . 
however , as a result of the lack of the typical tumour neoangiogenesis , well - differentiated early hccs may appear hypovascular in the arterial phase , and thus easily evade detection on dynamic ct or mr imaging [ 4 ]  . 
great interest is growing around the new mr imaging approaches to the liver and bile ducts made possible by the recent introduction of hepatocytespecic contrast agents , such as the gadoxetic acid disodium ( gd - eob - dtpa )  . 
this is a mixed paramagnetic contrast agent that combined the properties of vascularinterstitial agents and hepatobiliary agents , which are taken up by the hepatocytes and excreted through the biliary systea new imaging phasethe hepato - specic phaseacquired 1520 min after intravenous contrast injection is added to the standard dynamic study , and it seems to improve signicantly the diagnostic performance of mr imaging [ 5 , 6 ]  . the aim of our study was to assess the diagnostic accuracy of gadoxetic acid - enhanced mr imaging in the diagnosis of hcc and in the identication of early - stage lesions , evaluating the evolution and progression of the tumour . materials and methods study population fifty - six patients with hcv - , hbvor alcohol - related liver cirrhosis who underwent gadoxetic acid - enhanced dynamic mr imaging ( gd - eob - dtpa , primovist , bayer healthcare pharmaceuticals ) of the liver between march 2009 and july 2012 were retrospectively reviewed . 
the mr examinations were carried out to characterise indeterminate lesions found on ultrasonography ( us ) or dynamic ct scans performed within the previous 14 weeks , or to investigate elevated a - fetoprotein levels . the criteria for inclusion in the study were the following : ( 1 ) the presence of hypervascular nodules in the arterial phase with washout on the portal and / or equilibrium phase images , or presence of nodules with hypointensity on hepatobiliary phase images , ( 2 ) availability of at least 3 months of dynamic ct and / or gadoxetic acidenhanced mr imaging follow - up . 
the exclusion criteria were lesions with features of cysts , adenomas , focal nodular hyperplasia or haemangiomas ( n = 8 ) ; no evidence of focal liver lesion at mr imaging ( n = 3 ) ; no availability of mr or ct follow - up ( n = 4 )  . 
finally , 41 patients ( 30 men and 11 women ; age range 5287 years ; mean age 70 years ) with a total of 120 nodules were enrolled in the study . the diagnosis of hcc for nodules larger than 1 cm was made according to the aasld criteria of 2010 [ 3 ] , or was based on the histopathological ndings in surgical or biopsy specimens . 
histopathological examination of surgical or biopsy specimens was carried out in seven patients with nodules showing the typical vascular pattern but smaller than 1 cm in size or appearing hyperintense in the hepatobiliary phase . 
the other non - hcc nodules detected during the study underwent gadoxetic acid - enhanced mr imaging follow - up every 3 months for at least 12 months . this retrospective study was approved by the institutional review board of our hospital and informed consent was obtained from all patients . mr imaging protocol mr imaging was performed using a 1.5 - tesla scanner ( sonata siemens , erlangen , germany ) , with a phased - array body surface coil . 
axial t1weighted gradient echo in - phase and out - of - phase and t2weighted turbo spin - echo fat - saturated ( tse - fs ) sequences were obtained as baseline mr images . 
subsequently , volumetric interpolated breath - hold examinations ( vibe ) were acquired before and after intravenous bolus injection of 0.025 mmol / kg of body weight of gd - eob - dtpa , at a rate of 0.81 ml / s , followed by a 10 - cc saline ush at the same injection rate . 
arterial phase images were obtained 2535 s after the contrast medium injection ; portal venous phase and equilibrium phase images were obtained 7080 and 150180 s , the contrast agent injection . 
hepatobiliary phase images were acquired with a delay of 15 and 20 min after gd - eob - dtpa injection , using a vibe sequence ( axial and coronal planes ) and a fat - saturated 2d t1 - weighted high - resolution fast lowangle shot ( flash ) sequence . 
to reduce the examination time , t2 - weighted half - fourier acquisition single - shot ( haste ) fs and t2 - weighted respiratory - triggered tsefs sequences were performed in the interval between the dynamic and hepatobiliary phases . 
the same assessment was performed throughout the follow - up period . a lesion was considered as being an hcc when it showed the following characteristics : ( 1 ) a nodule larger than 10 mm displaying the typical vascular pattern for hcc according to the aasld criteria [ 3 ] ( contrast enhancement on arterial phase , washout on portal and / or equilibrium phase images ) ; ( 2 ) hypointense or hyperintense lesions on hepatobiliary phase images with typical vascular pattern on dynamic mr imaging . a lesion was dened as a dysplastic nodule / early hcc when it showed the following characteristics : ( 1 ) nodule showing hypointensity only on hepatobiliary phase images ; ( 2 ) lesions without hypervascularity on arterial phase images , showing hypointensity on equilibrium ( 150180 s ) and hepatobiliary phase images . statistical analysis statistical analysis was performed using systat software on the group of patients with dysplastic nodules / early hcc . continuous variables were analysed using descriptive statistics . 
given the small sample size , proportions were compared using the nonparametric fishers exact test . a total of 120 nodules were detected in 41 patients ( table 2 )  . 
none of the remaining seven nodules ( 46.6 % ; size range 413 mm ; mean size 8 mm ) showed evolution over the 3to 12 - month follow - up . 
of these six , three were larger than 1 cm at the rst examination , whereas the remaining three were 10 , 10 and 7 mm in size at the rst examination and increased in size ( [ 1 cm ) over the follow - up period . 
at the rst examination , 11 / 28 nodules of the second group were larger than 1 cm in size and 9 / 11 ( 81.8 % ) developed hcc during the follow - up period . 
2 dynamic contrastenhanced mr images show a focal liver lesion ( arrow ) , 20 mm in size , with early arterial enhancement ( a ) and washout in the portal ( b ) and equilibrium phase images ( c )  . the hepatobiliary phase axial ( d ) and coronal ( e ) images show hyperintense nodule ( arrow ) , implying gadoxetic acid uptake . f the histological result shows well - differentiated hcc ( haematoxylineosin stain ) discussion the crucial role of imaging in the evaluation of liver cirrhosis and diagnosis of hcc has already been established as critical improving patient management . 
however , curative treatments at the time of diagnosis , such as liver transplantation , liver resection or loco - regional therapies , are achievable only in a limited number of patients [ 3 , 7 , 8 ]  . mr imaging with its intrinsic high - contrast resolution may be seen as a problem - solving tool , and is now recognised as an accurate method in the study of the liver , with high sensitivity and specicity in the detection and characterisation of focal liver lesions . 
nevertheless , conventional dynamic imaging with extracellular contrast agents may at times provide inconclusive ndings in the characterisation of focal liver lesions . gadoxetic acid disodium ( gd - eob - dtpa ) is a contrast agent characterised by a biphasic nature : approximately 50 % of the administered gd - eob - dtpa is taken up by normal hepatocytes and subsequently excreted into the 372 fig . 
a hypointense nodule ( white arrow ) , 12 mm in size , is seen in the equilibrium phase image ( b )  . c hepatobiliary phase image demonstrates two hypointense nodules ( black arrows ) radiol med ( 2014 ) 119 : 367376 biliary tract , while the remaining 50 % is excreted via the kidney ; therefore , it combines the properties of a conventional extracellular uid contrast agent , allowing accurate dynamic assessment of lesion vascularity , and of a hepatobiliary agent , accumulating in hepatocytes during the information hepatobiliary phase , providing functional about the presence or absence of functioning hepatocytes within the lesion [ 9 ]  . 
the hepatobiliary phase is obtained at 1520 min after intravenous injection of the contrast agent . recent studies [ 1015 ] have shown that the use of gdeob - dtpa does not signicantly increase examination time : gadoxetic acid does not cause signicant effects on t2 - weighted images or dw images , which can therefore be performed immediately after the dynamic study with a total examination time of about 2530 min . in our study , 92 / 120 nodules showed a typical vascular pattern for hcc with enhancement on arterial phase and rapid washout on portal and / or equilibrium phase images . this behaviour is considered pathognomonic for hcc due to a process of arterialisation of the nodule : as its dimension increases , perfusion of the neoplastic lesion is ensured by the development of unpaired arteries originating from the hepatic artery , while the contribution from the portal blood ow is reduced . 
in the hepatobiliary phase , 90 nodules ( 97 % ) appeared hypointense compared to the surrounding liver parenchyma , suggesting the lack of functional hepatocytes , and conrming the diagnosis of hcc . 
hypointensity on hepatobiliary phase images is strongly suggestive of hcc [ 9 , 13 ]  . it should be noted that , in our experience , none of the 92 hypervascular nodules appeared isointense on portal or equilibrium phase images . 
on mr imaging or ct performed using an extracellular contrast agent , small hccs often appear isointense [ 13 , 14 ] causing uncertain differential diagnoses with non - neoplastic arterial - enhancing pseudolesions commonly found in cirrhotic liver , such as arterioportal shunts . 
this behaviour might be caused by the increase in signal intensity of the liver parenchyma already in the portal phase , thanks to the early hepatic uptake of gadoxetic acid , which highlights even faint contrast differences between focal lesions and the liver parenchyma . 
in this group of lesions , the added value of the hepatobiliary phase , compared with dynamic mr imaging , leads to an increase in the diagnostic condence for hcc ; however , it must be emphasised that the failure to nd equivocally isointense nodules during the equilibrium phase is not of secondary importance in that it reduces the incidence of interpretation errors and false negative results . 373 radiol med ( 2014 ) 119 : 367376 fig . 
recent studies [ 16 , 17 ] have demonstrated that gadoxetic acid is transported into the hepatocytes through organic anion transporting polypeptides ( oatps ) and that increased expression of this carrier is responsible for the uptake of contrast agent by well - differentiated hcc . the most interesting nding of our study concerns the group of 28 hypovascular nodules showing hypointensity on hepatobiliary phase images . 
the surveillance programme showed an evolution of 17 / 28 ( 60.7 % ) of these nodules and a process of arterialisation in 14 nodules ( 50 % ) during the 3to 12 - month follow - up . 
in these 14 lesions , the diagnosis of hcc was achieved thanks to evidence of a typical hcc vascular pattern in a nodule larger than 1 cthese ndings are explained by the multistep carcinogenesis process : at the beginning , portal perfusion is still present with no evidence of dominant arterial blood supply ; the portal blood supply then decreases and the development of neoangiogenesis typical of hcc is subsequently evident . 
the hypovascular nodule , without enhancement in the arterial phase and detected due to hypointensity in the portal and equilibrium phase images , can be dened as an early - stage tumour or as early hcc [ 18 , 19 ]  . in our study , the progression to hypervascular hcc was also observed among nodules that appeared hypointense radiol med ( 2014 ) 119 : 367376 fig . 
no focal lesions are depicted in either the arterial ( a ) or equilibrium phase ( b ) images . c hepatobiliary phase image shows 12 mm hypointense nodule ( arrow )  . 
furthermore , our results demonstrated that hypointense nodules on hepatobiliary phase images , greater than 10 mm in size , are at a higher risk of developing hcc than are lesions less than 10 mm in diameter . 
caiazzo mri research center sun - fism , second university of naples , c / o hermitage clinics , via cupa le tozzole , 80138 naples , italy results conrm previous conclusions our reports regarding the increase in vrsd in nonactive phases of ms and support the immunological role of the vrs within the central nervous systethe lack of correlation between vrsd and the degree of gca and their prevailing localisation in atypical sites in ms patients make vrsd a potential marker of inammatory - demyelinating disease . keywords perivascular spaces ( cid : 2 ) virchowrobin spaces ( cid : 2 ) multiple sclerosis ( cid : 2 ) magnetic resonance introduction virchowrobin spaces ( vrs ) are small , pial - lined , interstitial uid - lled channels that house small vessels in their subarachnoid intracerebral course and may be involved in inammatory brain events [ 1 , 2 ]  . 
they are physiological ndings in magnetic resonance ( mr ) studies and , according to some authors [ 57 ] , they are present in 100 % of subjects . vrs are dened as dilated if their short axis is greater than 2 mm [ 5 , 8 ] or , according to some authors , greater than 3 mm [ 6 ]  . 
this is the reason why the number and size of vrs may change in the course of inammatory diseases such as multiple sclerosis ( ms ) [ 1 , 2 , 10 ]  . 
the exclusion criteria were : relapses either ongoing or occurring in the month before the examination , corticosteroid therapy in the month before the examination , presence of other neurological diseases , contraindications to the mr study . 
using this fast and accurate semi - automatic postprocessing application the vrsd of all the patients and controls were identied and evaluated in terms of their morphological and volumetric features and their signal . each individual vrs was identied and manually circumscribed through the placement of a volume of interest ( voi ) with semi - automatic delineation of the contours . 
1 mipav ( medical image processing , analysis and visualisation ) system display screen , with co - registered t1w , t2w and flair sequences and magnied views of the dilated virchowrobin spaces on t2w and t1w images . 
then we calculated the ratio between the three different parts of the brain so as to obtain the brain parenchyma fraction ( bpf ratio ) , an expression of the degree of global cerebral atrophy ( gca )  . statistical analysis the student t test was used to evaluate differences between the ms patients ( msnd and msd ) and the healthy volunteers in terms of vrsd volume , number , and sites , and fig . 
type i is commonly seen at the substantia perforata and anterior commissure along the course of the lenticulostriate arteries : a t2w , b t1w , c flair ( uid attenuated inversion recovery )  . type ii occurs in the white matter of the convexity and semioval centres , along the path of the perforating medullary arteries : d t2w , e t1w , f flair . 
a statistically as regards vrsd distribution , in ms patients the vrsd with typical locations were predominant in the anterior perforated substance ( type i ) compared with the convexity ( type ii ) ; there were no cases of vrsd in the midbrain ( type difference iii ) ( p = 0.0069 ) was found in the frequency of vrsd location at atypical sites : 34 vrsd in ms patients vs one vrsd in controls . 
the fisher test revealed that vrsd location was independent ( p = 0.93 , p = 0.51 ) of the clinical form of disease ( msnd vs msd )  . 
table 2 describes the data in detail . signicant correlation with global cerebral atrophy the volume and number of vrsd , both in patients and controls , are not dependent on the degree of gca , expressed in terms of bpf . 
conversely , with mr imaging , and especially with high - eld mr imaging [ 6 , 19 ] , vrs are routinely observed along the course of intracerebral arteries , arterioles , venules and veins [ 9 , 20 ]  . 
here , the arterioles have an angled trajectory and multiple grouped branches , with a prevalence twice as high [ 10 ] compared to the distal branches of the middle cerebral artery and ten times higher compared to the distal lenticulostriate arteries [ 10 ]  . the immunological rationale for vrs was rst proposed by w . 
his in 1865 , who considered them to be lymphatic spaces of the cns on account of both their structure and cell content ; their relationship with the immune system was further explored in later studies . human brain studies using tracers and histopathological analyses conrm that , in addition to transporting solutes , soluble proteins and leukocytes [ 2 ] , vrs are physiologically [ 6 ] a real route for cerebral lymphatic drainage between the periarterial space and the lymphatic system [ 12 ] , and are implicated in the case of bloodbrain barrier abnormality and inammation [ 18 ]  . 
histopathological studies demonstrate that , in normal conditions , vrs contain , in addition to dendritic cells and perivascular microglia , macrophages and t cells , which give them a key role in immune surveillance [ 2 ]  . the macrophages contained in vrs , besides adjusting some proteins involved in inammatory processes ( mch class ii , interleukins - 1b ) , have contact with the circulating t lymphocytes [ 12 ] from which the immune response starts the periphery of the [ 5 ]  . 
t cell activation begins at radiol med ( 2014 ) 119 : 408414 lymphoid compartment ( extracerebral area ) and then reaches the cns , with the t cells circulating in the vrs [ 2 ]  . immunohistochemical studies compared to post - mortem brain mr imaging studies of patients with demyelinating disease demonstrate the presence in the vrs of cd68positive macrophages in all stages of demyelinating lesions , with perivascular leukocyte inltrates , much more frequent in chronic inactive lesions than in active lesions [ 17 ] and vrs have also been described in experimental ms models [ 2 , 17 ]  . in spite of the growing interest for the role of vrs in immune surveillance , until recently they were underestimated [ 2 ]  . 
ms , a chronic inammatory disease of the cns , is characterised by the presence of immune cells in brain parenchyma , with local inammation , demyelinating lesions and axonal and neuronal damage [ 2 ] and this would in patients with active ms , brain mr explain why , imaging shows dilation of the vrs , which are reserves of inammatory cells [ 6 , 18 ]  . it has been hypothesised that the inammatory activity in ms occurs in the vrs , leading to their increase in size due to inltration by inammatory cells and oedema , resulting in a greater number of vrs being visible in mr imaging studies of the brathis is conrmed by a longitudinal study [ 2 ] which found a signicant increase in both the volume and number of vrs in patients with active ms compared to healthy controls , suggesting the immunological role of vrs in active demyelinating disease , or when the bloodbrain barrier was damaged [ 18 ]  . 
more recent studies indicate nonspecic vrs enlargements as a sign of ms inammatory changes , having observed bihemispheric white matter vrs in 55 % of ms patients and , in 85 % of cases , a vrs number greater than four [ 3 , 4 ]  . in our study , we identied more than four vrsd in each ms patient in 12.5 % of the cases compared with controls ( 0 of 30 subjects ) and more than three vrsd for each ms patient in 17.5 % of the cases compared to controls ( 0 of 30 )  . 
this discrepancy is probably due to the different cutoff size used in our study . unlike previous studies [ 2 ] , in which the number of vrs did not differ between ms patients and healthy subjects in any region of the brain , our study found not only a statistically higher number of vrsd in ms patients compared to controls , but also a signicant increase in the volume and area of the vrsd . we also found a signicantly higher number of vrsd in these ndings may ms patients ; related methodological differences since we only considered vrs with a diameter greater than 2 mm or to differences in the population examined , having assessed only nonacute phase ms patients . the considerable increase in our study in vrsd number , area and volume in patients with nonactive ms compared to controls could result from the possibility of identifying a larger number of perivascular spaces due to their expansion rather than reecting a real increase in their number . another important nding in our study , not previously reported in the literature , is the identication in the group of ms patients compared to healthy controls of a greater number of statistically signicant vrsd located in atypical sites . 
this nding of uncertain signicance could be explained with the need , in this category of patients , of alternative or supplementary cerebral lymphatic drainage pathways , compared with conventional areas . conclusions our mr imaging study of the brain , performed with a 3t system in patients with nonactive ms compared with a group of healthy controls shows that the area , volume and number of dilated vrs are signicantly higher in ms patients than in controls , conrming previous reports of brain mr imaging studies carried out with less powerful equipment and in patients with active ms . the signicance of this nding is validated by the lack of correlation between vrs and bpf , both for ms patients and for control subjects , which rules out any inuence of gca . 
no signicant intragroup difference was observed in the volume and number of vrsd based on degree of disability ( msnd and msd )  . unlike previous investigators [ 13 ] , we did not observe a signicant prevalence in the shape of vrsd ( round or oval ) between patients and controls , the round shape prevailing in both groups . 
the wilcoxon test was used for comparisons . results the least difference between artefact extent and actual valve size ( 0.1 mm ) was obtained with a steady - state free precession ( ssfp ) sequence . 
no signicant differences were found comparing the pg measured with cmr , echocardiography and catheter . conclusions we showed in vitro that the ssfp sequence produced the most accurate valve measurement . 
after ppvi , cmr showed a strong decrease of pg and rf with a signicant improvement of rv function . keywords congenital heart diseases ( cid : 2 ) cardiac magnetic resonance ( cmr ) ( cid : 2 ) percutaneous pulmonary valve implantation ( ppvi ) ( cid : 2 ) right ventricle ( rv ) ( cid : 2 ) pulmonary artery ( pa ) introduction patients with congenital heart defects are usually treated in the rst year of life and they may also undergo multiple surgical interventions over the life course [ 1 ]  . 
malan , 20097 san donato milanese , milan , italy radiol med ( 2014 ) 119 : 400407 implanted to repair numerous congenital heart defects [ 2 ]  . however , the good function of conduits is limited during the life of these patients and multiple re - interventions are often required . 
as a consequence , a surgical procedure may be performed too late for a good rv function recovery [ 7 ]  . in the last decade , bonhoeffer et al . 
 [ 8 ] developed a new approach to treat the rv conduit dysfunction such as stenosis and / or regurgitation : a noninvasive percutaneous pulmonary valve implantation ( ppvi ) using a bovine valve sutured on a platinumiridium alloy stent , with welded joints of gold ( melody , medtronic , inc . , minneapolis , mn , usa )  . 
this assessment may be performed using echocardiography as a rst - line technique . however , cmr has been demonstrated to largely outperform echocardiography in the evaluation of cardiac chambers , in particular the rv , and it is considered as a rst - choice technique in this setting [ 13 ]  . 
moreover , no the pulmonary pressure study has reported data about gradient ( pg ) measured by cmr in these patients . in this study , we rst report the results of a preliminary in vitro mr study of the melody valve aimed at measuring the extent of artefact . 
all 27 patients underwent cmr before ppvi but not all patients performed all the three cmrs after ppvi : 13 / 27 patients were studied after 1 month , 20 / 27 after 3 months , and 21 / 27 after 6 months . cmr protocol we used a 1.5 - t unit with 40 - mt / m gradient power ( magnetom sonata maestro class , siemens , erlangen , germany ) and a four - channel cardio - thoracic phased - array coil . 
before ppvi , the slice was located in the pulmonary conduit , perpendicular to the vessel long axis , immediately distal to the valve ; after ppvi , the slice was located 4 mm distal to the stent artefact . before the implanting valve procedure [ 10 ]  . 
before and 3 months after ppvi conduit , the mean pulmonary pg was calculated with echocardiography by tracing the border of continuous - wave spectral doppler recordings and integrating the area under the curve . 
maximum instantaneous pg was also estimated by echocardiography [ 16 ]  . statistical analysis differences between the two readers of the in vitro study were tested with the wilcoxon test . 
2. catheterisation and echocardiography in vivo study the peak - to - peak rvpa gradient was calculated during the interventional procedure as the difference in systolic pressure between rv outow tract and main pa distal to the conduit all cmr examinations were diagnostic , with metallic artefacts limited to the valve inner space as shown in fig . 
regarding the lv , no signicant differences were found in terms of esvi respectively ; after ppvi , table 2 patients characteristics and spectrum of diseases mean males females total original diagnosis tetralogy of fallot aortic valve disease ( ross procedure ) transposition of great arteries double outlet right ventricle truncus arteriosus pulmonary atresia pulmonary stenosis total rvot conduit type homograft shelhigh matrix hancock contegra monocuspid homograft ionescu - shiley conduit not implanted total rvot right ventricle outow tract , sd standard deviation of the lv and ejection fraction . 
this in vitro / in vivo study showed that : ( 1 ) the artefact from mr sequences is sufciently small not to inuence qualitative and quantitative mr evaluation after ppvi and ( 2 ) there is a signicant reduction in pg and rf and a signicant increase in rv function . single - shot acquisition the low interobserver variability of the artefact size ( cov 3.44.6 % ) showed the reliability of our measurements . 
looking at the artefact size , we showed that ssfp , spin - echo half - fourier ( haste ) , and turbo spin - echo ( tse ) sequences produced the most accurate valve measurement , i.e. 
this means that these mr sequences are suitable for clinical evaluation of the heart with a melody valve , as previously demonstrated for different types of vessel stents in an in vitro model [ 17 ]  . turbo in the in vivo study , we evaluated the pre - treatment and follow - up cmr study of patients treated with a ppvi . 
evolution of right ( rv ) and left ventricle ( lv ) end - diastolic volume index ( edvi ) and end - systolic volume index ( esvi ) up to 6 months after the percutaneous pulmonary valve implantation differences were found for lv . 
however , cmr is commonly considered more reproducible than echocardiography in the evaluation of the right chambers , rv outow tract and pulmonary artery [ 13 , 18 ]  . 
the last limitation is that there is no comparison with a different pulmonary valve stent . in conclusion , the signal void produced in vitro by the metallic structure of melody pulmonary valve is limited to its inner space with a maximal extent to the external space equal to \5 mmoreover , cmr evaluation of patients after 6 months from ppvi shows a drastic decrease of rf and pg and a signicant improvement of all rv parameters . 
pomerri radiologia oncologica , iov , ircss , via gattamelata , 35128 padua , italy radiological documentation ( hrct ) of disease , clinical and radiological follow - up of 1 year and the beginning of any therapy within 1 month from the diagnosis . results among subjects not receiving therapy , the comparison between the two groups showed that the radiological ndings remained stable in subjects with a typical pattern , while they worsened in more than 70 % of cases with atypical appearance . 
re - evaluation of hrct within 1 year revealed no correlation between clinical deterioration and radiological changes . conclusions the ndings of this study suggest that persistence of the inammatory process rather than the radiological pattern at onset is a prognostic factor for recurrence . keywords sarcoidosis ( cid : 2 ) nodular sarcoidosis ( cid : 2 ) hrct introduction sarcoidosis is a multisystem granulomatous syndrome of unknown aetiology . 
it is ubiquitous and has almost equal sex , race and age distribution , with a slight higher prevalence among african american women . it usually affects adults younger than 40 , with a peak in the age group from 20 to 40 years . 
in italy , the prevalence is about 10 per 1 , 00 , 000 population [ 1 ]  . pulmonary involvement with hilar lymphadenopathy is by far the most common manifestation followed by lesions of the eyes and skin ; other organs may also be involved ( liver , spleen , salivary glands , heart , nervous system , radiol med ( 2014 ) 119 : 384392 muscles and bones )  . 
a sudden onset presenting with only erythema nodosum or asymptomatic bilateral hilar lymph node enlargement may predict a self - limiting course , whereas subtle symptoms and the presence of multiple nonpulmonary lesions could lead to progressive brosis of the lungs or of other organs . 
the overall mortality rate is between 1 and 5 % [ 24 ]  . imaging investigations are used to conrm a clinical suspicion , and chest x - ray is always the rst test to be performed . 
high - resolution computed tomography ( hrct ) has 95 % sensitivity and a 99 % specicity , and it enables detection of lung abnormalities which might be missed on a plain lm [ 5 , 6 ]  . 
it has indeed been demonstrated that 15 % of symptomatic patients have normal chest x - ray but pathological ndings on ct scans [ 7 ]  . pulmonary sarcoidosis is typically classied in stages on the basis of the interstitial and hilar - mediastinal abnormalities seen on chest x - ray ( stage 0 : normal ; stage i : bilateral hilar lymph node enlargement with or without paratracheal lymphadenopathy , not associated with visible lung disease ; stage ii : bilateral hilar lymph node enlargement associated with visible lung disease ; stage iii : diffuse lung disease without lymph node enlargement ; stage iv : lung brosis with honeycombing pattern ) [ 1 , 7 ]  . the therapeutic approach is still debated . 
the study compares the incidence of typical and atypical sarcoidosis and the clinical and radiological differences in disease progression between these two groups ; nally , it evaluates whether pharmacological therapy can inuence the evolution of the disease , on the basis of the radiological patterns . materials and methods we retrospectively reviewed the clinical and radiological records of 350 patients with a proven diagnosis of pulmonary sarcoidosis , who were assessed at the institute of immunology and haematology from 1997 to 2010 . inclusion criteria were an hrct study at the time of diagnosis or during an active phase of disease ; diagnosis established by histopathology or immunophenotype analysis on bronchoalveolar lavage ( bal ) ; a 1 - year clinical and radiological follow - up ( the same amount of time for treated and untreated patients ) ; initiation of therapy within 1 month of the diagnosis , in the case of treated patients . patients with a brotic hrct pattern were excluded because of the irreversibility of the ndings . most patients showed a clinical and radiological improvement by the early phase of the follow - up , and only a minority who experienced a relapse or worsening clinical conditions underwent further radiological re - evaluation . therefore , in patients with stable or improved clinical status during the rst year of the follow - up , we assessed the clinical evolution of the disease . 
in patients who developed a clinical relapse and underwent radiological re - evaluation , we assessed the correlation between clinical worsening and the radiological ndings . on the basis of the above inclusion criteria , a total of 56 patients were selected ( 23 males and 33 females ; average age , 49.8 years ; range 2972 years ; all caucasian )  . 
according to the hrct pattern , the patients were classied as being affected by typical or atypical sarcoidosis [ 9 ] ( table 1 )  . table 1 typical and atypical hrct characteristics of pulmonary sarcoidosis in 56 patients typical pattern ( 39 patients ) lymph node enlargement : hilar , bilateral , symmetric and mediastinal ( right paratracheal ) nodules : micronodules ( 14 mm in diameter , well dened , bilateral ) , they can coalesce perilymphatic distribution : peribronchovascular , subpleural , interlobular septa thickening of the interlobular septa mainly distributed in upper lobes and middle lobe ground glass associated with nodules atypical pattern ( 17 patients ) large nodules or masses ( 14 cm in diameter , ill dened , multiple , bilateral ) , with or without air bronchogram galaxy sign lymph node enlargement : as in the typical pattern 386 radiol med ( 2014 ) 119 : 384392 table 2 hrct pattern of typical and atypical sarcoidosis in examined patients typical pattern ( 39 patients ) lymph nodes ( ln ) lymph nodes ? reticulo - nodular ( ln ? rn ) lymph nodes ? reticulo - nodular ? ground glass ( ln ? rn ? gg ) reticulo - nodular ? ground glass ( n ? gg ) atypical pattern ( 17 patients ) lymph nodes ? large nodules / masses ( ln ? nm ) large nodules / masses ( nm ) remission , stability or worsening of the disease was dened on the basis of this comparison . the patients exhibiting typical and atypical sarcoidosis imaging patterns were subdivided into patients who received therapy ( steroids , antimalarial drugs or immunosuppressants ) and those who did not . the clinical course was then analysed in treated and untreated patients , evaluating again improvement / remission , stability or worsening of the symptoms . 
ground - glass opacities superimposed on the reticulonodular pattern . for atypical sarcoidosis , the hrct features considered were large nodules and masses , alone or associated with enlarged lymph nodes ( 14 cm in diameter , large illdened opacities on ct )  . 
small satellite nodules are often visible at the periphery of these masses , leading to the galaxy sign appearance [ 10 ]  . the two main patterns were further divided into subtypes : four subtypes for the typical group and two for the atypical group ( table 2 )  . for the typical pattern , we considered the following abnormalities : lymph nodes ( ln ) lymph nodes ? reticulo - nodular pattern ( ln ? rn ) lymph glass ( ln ? rn ? gg ) reticulo - nodular pattern ? ground glass ( rn ? gg )  . nodes ? reticulo - nodular pattern ? ground for the atypical pattern , the abnormalities considered lymph nodes ? large nodules / masses ( ln ? nm ) large nodules / masses ( nm )  . the course of the radiological picture was assessed by comparing the hrct studies performed at the time of diagnosis with those obtained within 1 year . 
improvement / were : of the 56 patients selected , 39 ( 69.6 % ) had a typical sarcoidosis pattern , whereas 17 ( 30.3 % ) had an atypical pattern . 
among the group with atypical pattern ( n = 17 ) , two patients ( 11.7 % ) presented with respiratory symptoms , nine patients ( 52.9 % ) had extrapulmonary / systemic symptoms , four patients ( 23.5 % ) had pulmonary and extrapulmonary complaints , and two patients ( 11.7 % ) were asymptomatic . the clinical features are listed in table 3 . treatment regimen ( steroids , hydroxychloroquine / chloroquine , methotrexate , singularly or combined ) was administered to 44 ( 78.5 % ) patients : 31 ( 79.4 % ) of them showed a typical radiological pattern , a specic radiol med ( 2014 ) 119 : 384392 fig . 
1 radiological patterns of patients included in the study table 3 characteristics of patients sarcoidosis number of patients mean age ( years ) gender m / f diagnosis made by biopsy / bal pulmonary symptoms pulmonary ? extrapulmonary / systemic symptoms extrapulmonary and / or systemic symptoms asymptomatic typical therapy 11 / 20 27 / 4 atypical therapy 12 / 1 no therapy no therapy table 4 clinical evolution of treated patients resolved symptoms chronic symptoms typical atypical 25 ( 80.6 % ) 10 ( 77 % ) 6 ( 19.4 % ) 3 ( 3 % ) whereas 13 ( 76.4 % ) had an atypical one . 
treatment decisions were based on the reported symptoms and on the involvement of specic organs . spontaneous remission was observed in 10 / 12 ( 83.3 % ) patients who did not receive pharmacological treatment . only two patients affected by typical sarcoidosis ( dry cough ) reported an improvement of the symptoms but not a complete remission . in the typical pattern group , 25 patients ( 80.6 % ) demonstrated complete recovery after therapy , whereas six patients ( 19.4 % ) showed only a mild response . 
2 first high - resolution computed tomography ( hrct ) study . large parenchymal consolidation with air bronchogram in both upper lobes ( a , b ) , with galaxy sign ( arrow ) and small nodules with perilymphatic distribution ( arrowhead )  . 
follow - up hrct after therapy with hydroxychloroquine and steroids , and 1 year of improvement of dyspnea : marked reduction of the consolidations , but progression to brosis in the same location ( c , d ) , with traction bronchiectasis and retraction of ssures table 5 radiological evolution of treated patients remission stability progression typical atypical 16 ( 36.4 % ) 3 ( 6.8 % ) 11 ( 25 % ) 7 ( 15.9 % ) 4 ( 9.1 % ) 3 ( 6.8 % ) prognosis for those with medically treated atypical sarcoidosis ( p = ns )  . 
clinical manifestations subsided in 25 patients with a typical pattern and 21 of them ( 84 % ) further improved or remained stable during the follow - up , whereas 4 worsened . as for the atypical group , of the 10 patients who reported regression of the symptoms , 7 ( 70 % ) had concordant hrct ndings , whereas in 7 patients imaging revealed an aggravation . the hrct pattern was stable or better in all of the nine untreated patients with typical and atypical pattern who reported stationary clinical condition . the course of all the radiological subtypes of typical sarcoidosis was investigated to evaluate whether they could correlate with the prognosis . regression or stability was found in 10 / 11 ( 90.9 % ) patients with a ln ( lymph node involvement ) radiological pattern ( eight of them treated and two untreated )  . 
only one untreated radiological worsening . showed clinical patient extrapulmonary symptoms progressed in two treated patients ( 15.4 % ) with an atypical pattern , whereas the imaging features remained stable ; none of the untreated patient with atypical pattern relapsed or worsened . discussion the radiological abnormalities of sarcoidosis are usually well dened and they include hilar bilateral lymph node enlargement and lung disease , alone or in combination . 
nodules measuring 2 and 5 mm can be seen in both upper lobes ( a , arrows )  . minimum nodular thickening of the septa in the apical segment of the right upper lobe and nodules along the ssures in the upper lobes bilaterally ( b , arrow )  . 
appearance of another nodule in the right upper lobe ( c , arrow ) and increased perilymphatic nodular pattern in the right apex 390 radiol med ( 2014 ) 119 : 384392 typical pattern of sarcoidosis consists of small nodules along the lymphatics in the peribronchovascular interstitial space , sometimes mildly thickened , and in the interlobular septa ( perilymphatic distribution ) [ 11 ]  . 
in this situation , the differential diagnosis is challenging [ 12 ]  . several previous studies have evaluated the prognostic signicance of the radiographic signs of sarcoidosis [ 13 , 14 ]  . the detection of ground - glass opacities , nodules and thickening of the interlobular septa suggests a possible resolution , whereas honeycombing indicates an irreversible course . it is unclear whether the evolution of the typical and atypical patterns is inuenced by sarcoidosis therapy and table 6 radiological evolution of untreated patients remission stability progression typical atypical 3 ( 25 % ) 1 ( 33 % ) 5 ( 41.67 % ) 3 ( 25 % ) stable whether the nding of specic patterns can guide decisions to initiate immunosuppressive treatment . 
5 first hrct study in a patient with dyspnoea and dry cough . typical pattern with perilymphatic nodules in the upper lobes , tending to coalesce in the left parahilar region ( a , b )  . 
marked increase of nodular pattern ( c ) with initial reduction in volume of the right upper lobe ( d ) radiol med ( 2014 ) 119 : 384392 the atypical abnormalities detected in our study were large nodules , masses or areas of pulmonary consolidation : all these features are potentially reversible with drugs . large nodules ( 14 cm in diameter ) were found in 1525 % of patients [ 9 , 10 ]  . in our study , 30 % of patients had an atypical radiological pattern , a nding consistent with the literature data [ 9 ]  . 
this nding seems to be conrmed by the study done by fazzi in 2003 [ 16 ] , in which steroids alone or associated with other drugs ( methotrexate or chloroquine ) were used to treat specic symptoms . 
the presence of clinical features requiring treatment is a greater risk factor for relapse than is the radiological pattern seen at onset . analysis of the efcacy of treatment on radiological progression demonstrates that immunosuppressants are more effective on the typical pattern : 50 % of the typical cases improved compared to 20 % of atypical cases , in which only stability was achieved . another interesting nding , even though partially limited by the small number of cases in our series , is that most of the untreated patients showed a stable radiological course . 
on the other hand , more than 70 % of the untreated patients with atypical appearance experienced a worsening . this result suggests that initiating treatment in patients with an atypical pattern , independently of clinical symptoms or from organ involvement , can arrest the progression of disease . treatment for typical cases should be decided on the basis of clinical parameters because the risk of relapse bears no relation to the radiological pattern . typical patients with lymph node enlargement and those with lung disease ( reticulo - nodular pattern and / or ground glass ) showed no important differences with regard to the progression of disease . 
on the contrary , other recent studies [ 14 ] suggest that ground - glass opacities could predict a chronic course of disease and could precede honeycombing changes . immunosuppressant clinical symptoms in both groups ( typical and atypical ) , leading to complete resolution in 80 % of the cases . therapy improved the our study also indicated that long - term radiological relapses are uncommon , whereas clinical relapses are more frequent ( typical and atypical )  . in conclusion , clinical relapse and worsening are not always related to a radiological progression , but they need to be analysed within the specic clinical context . 
the aim of this study was to investigate the knowledge of radiation exposure doses and risks among interns , resident doctors , and radiographers . materials and methods a questionnaire , consisting of 14 questions in multiple choice format , was distributed to 300 participants ( 100 interns , 100 radiographers , 100 resident doctors ) working in the emergency department . 
the questionnaire was designed to determine the participants knowledge about radiation - related hazards . results none of the radiation doses delivered by the imaging modalities was 100 % correctly estimated . 
this result could imply that we are not aware of the radiation risks , and we are inattentive in informing our patients about the radiation exposure related to the different imaging modalities . keywords radiation awareness ( cid : 2 ) radiological imaging ( cid : 2 ) emergency department ( cid : 2 ) ionising radiation doses introduction in emergency medicine practice , diagnostic imaging techniques and interventional radiological procedures are increasingly used to diagnose a wide range of diseases and injuries , and to provide life - saving treatment . 
according to the international commission on radiological protection ( icrp ) , understanding of the health effects of ionising radiation is central [ 3 ] , but the knowledge of doctors about radiological investigations using ionising radiation is inadequate [ 49 ] , regardless of the eld of expertise . the aim of this study was to investigate the knowledge of radiation exposure doses and risks among interns , resident doctors , and radiographers . materials and methods this prospective observational study was conducted in the emergency department of the ankara university school of medicine . 
the questionnaires , consisting of 14 questions in multiple choice format ( appendix ) , were selfadministered to 100 interns , 100 radiographers , 100 resident doctors ( 10 emergency medicine resident doctors , 10 radiology resident doctors , 40 general surgery resident doctors , and 40 internal medicine resident doctors )  . 
assuming that a single chest radiograph equalled one unit of radiation , the participants were asked to estimate the radiation dose that patients received during the different radiological imaging modalities . 
the participants marking correct intervals were grouped into the correct answer group , the ones who marked higher values into the more than equivalent number of chest x - ray ( overestimated ) group , and the ones who marked lower values were grouped into the less than equivalent number of chest x - ray ( underestimated ) group . 
participants who presented no opinion were grouped into the no idea group . the questionnaire was designed to determine the participants knowledge about the lifetime risks of inducing a fatal cancer , their knowledge of the selection of imaging modalities for pregnant patients , and their knowledge of risks for the foetus [ 11 ]  . 
when the p value obtained from the kruskalwallis anova test was statistically signicant , a multiple comparison test was used to assess which group differed from the others [ 12 ]  . 
a p value less than 0.05 was considered to be signicant . results the answers to the questions related to the radiation doses of various radiological examinations given by the study participants were grouped as correct , more than equivalent number of chest x - rays less than equivalent number of chest x - rays ( underestimated ) , and no idea . 
this was the highest rate for a correct answer , while 3 % stated that us used ionising radiation , and 4.3 % stated they had no idea on the radiation dose of the abdominal us . 
the answers given for the rst question are shown in table 3 . there was no statistically signicant difference between the groups in the rates of correct answers given for abdominal us and abdominal mri . 
when different imaging modalities for the same anatomical region were considered , none of the resident doctors correctly estimated all of the doses for the abdominal examination ( x - ray , us , ct , and mri ) , whereas only one intern correctly estimated the radiation doses in all of these modalities . 
while the correct answer to that question was a pelvic ct , the abdominal ct was the most frequently given answer by all of the participants . discussion it is important that doctors who request diagnostic imaging procedures , and radiographers who perform these requests , are well trained about radiation doses and the risks from radiation exposure . 
this is particularly signicant in the emergency department , because radiological investigations 444 radiol med ( 2014 ) 119 : 440447 using ionising radiation are an accepted part of emergency medicine , and emergency doctors rely heavily on medical imaging procedures in their daily practice . to the best of our knowledge , this is the rst study in turkey that compares resident doctors , interns , and radiographers knowledge about patient exposure to ionising radiation and its risks during radiological imaging examinations in the emergency department . in our study , none of the participants was able to correctly estimate the radiation doses in every single one of the nine different imaging modalities . 
these results were important since they show that the side effects of radiation exposure have potentially been underestimated by clinicians , resulting in an unnecessarily high number of orders . we found that there are still medical practitioners who fail to recognise mri and us as radiation - free modalities . this might be a decit in the knowledge of basic scientic principles . 
these results are consistent with previous studies which reported that the us and mri were associated with radiation by 411 % and 828 % of participants , respectively [ 8 , 9 , 11 , 13 ]  . another result in our study was that the radiographers had a statistically signicant rate of correct estimation of the radiation doses of different imaging studies based on the anatomical regions of the body when compared to other study groups . 
this can be explained by the most common body region for the radiological imaging being the abdomen , and the radiographers knowledge level in particular imaging studies for that body region . the vast majority of patients are unaware of the radiation dose that they are exposed to during radiological investigations and the associated risks [ 6 ]  . 
this is another indication of the radiographers being exposed to radiation at greater levels when compared with other study groups , and a higher level of knowledge about the side effects of radiation exposure and related safety measures to reduce these side effects . the knowledge of the radiology resident doctors is not as good as expected . 
 [ 6 ] mentioned that emergency department physicians did not perceive a possible increased cancer risk associated with a diagnostic ct scan , while nearly half of the radiologists did . 
in our study , we found that emergency resident doctors had a statistically signicantly higher rate of giving correct answers for the lowest and highest radiation sources for a foetus when compared with other groups . 
this may be the result of the more common use of radiological studies in emergency medicine practice , and an increased level of knowledge of the emergency resident doctors in the effects of radiation . our study showed that the doctors , interns , and radiographers knowledge of radiation exposure from radiological investigations and associated risks is poor . 
the question is to ask how knowledge can be improved in all of the emergency departments staff . attendance at training courses can be adequate for radiographers ; however , doctors are likely to benet from a mandatory curriculum on the effects and risks of radiation taught in medical schools . we believe that medical staff working in the emergency department should have this as basic knowledge ; x - rays should be ordered only for real medical indications and should be more cautiously used in paediatric and pregnant patients . 
in addition , the doctors should base their orders on whether the imaging examinations are appropriate to diagnose the patients clinical problems . furthermore , establishing continuous communication between emergency department and radiology staff , developing joint protocols for radiological interventions , and documenting radiation doses of different interventions on the order forms can increase the awareness among clinicians . to increase patients awareness of the effects and risks of radiation , attractive posters can be prepared and presented in the waiting rooms . 
also , presenting basic fact the topic and having an sheets to the patients about radiol med ( 2014 ) 119 : 440447 emergency doctor verbally explain the related risks prior to radiology orders can be helpful . the main limitation of the study is the heterogeneity among resident doctors . 
also , this study cannot be directly compared as the methodology and group of resident doctors / interns / radiographers are different , but it supports the global concern of substandard appreciation of radiation doses given to the patients . 
when the effective dose of a plain chest x - ray is considered 1 unit , how many units would a patient absorb in the following investigations ? 010 1050 50100 100500 [ idea conclusions radiological investigations using ionising radiation are an important part of emergency medicine . 
the main points about protection from the adverse effects of radiation are to educate clinicians and patients on the tolerable levels of radiation exposure , prevent overexposure by increasing awareness , consider irradiation - free modalities like us and mri when these are appropriate alternatives , and remember that all requests for imaging investigations should be expected to do more good than harm . acknowledgments the authors wish to thank atilla elhan for his assistance with the statistical interpretation of the data and nezaket o zgur for contributing to this research . cranial x - ray pelvic x - ray abdominal abdominal x - ray chest ct abdominal abdominal pelvic ct cranial ct 446 radiol med ( 2014 ) 119 : 440447 2 . 
when ordering a diagnostic radiological exam , do you notify your patients about the potential side effects of radiation prior to the examination ? ( ) yes ( ) no 4 . 
images were postprocessed with the software and evaluated independently by three physicians in terms of ramris ( rheumatoid arthritis magnetic resonance imaging score ) , samis ( simplied rheumatoid arthritis magnetic resonance imaging score ) correlated techniques were and ceus grade . 
the most painful joint was consistently found to be the joint with most synovitis . conclusions ce - mr imaging may be used prior treatment and for long - term follow - up . 
ceus might be useful in the short - term follow - up , as it seems to provide an indication of the presence or absence of disease , though not of its severity . 
the software is a very useful tool that can supplement , but not replace , the other techniques . keywords rheumatoid arthritis ( cid : 2 ) mr ( cid : 2 ) ceus ( cid : 2 ) us r . 
raffeiner rheumatology unit , department of medicine , general hospital of bolzano , bolzano , italy introduction rheumatoid arthritis ( ra ) is a chronic inammatory disease characterised by synovial inammation leading to progressive joint involvement with erosions , disabling pain and functional impairment . 
ra almost invariably affects the proximal interphalangeal joints ( pip ) , the metacarpophalangeal joints ( mcp ) and the radiocarpal joints : synovitis in these joints is a marker of generalised joint disease [ 1 ]  . the formation of new vessels is an essential pathogenic feature of progressive synovitis since synovial vascularity correlates with disease activity and aggressiveness [ 24 ]  . 
currently , no quantication system exists that enables the correct assessment of response to therapy and disease progression [ 5 ]  . several studies have demonstrated the usefulness of colour doppler and power doppler ultrasound ( cdus and pdus ) in the evaluation of synovial vascularity , but these radiol med ( 2014 ) 119 : 422431 applications are somewhat limited in detecting the slow ow of very small vessels such as those typically seen in synovial proliferation [ 610 ]  . 
contrast - enhanced ultrasound ( ceus ) has drastically improved the sensitivity of cdus and pdus , enabling the better depiction and quantication of inammation and consequently a more accurate distinction between brous synovial proliferation and active synovitis [ 1113 ] ; however , the technique has yet to become standard in the diagnostic workup of patients with ra . resonance contrast - enhanced magnetic ( ce - mr ) imaging is currently considered the gold standard morphological investigation . 
it is able to depict the synovial pannus , uid distension of the joint , bone erosions and bone oedema , but it is hampered by well - known problems such as high cost , limited availability and considerable discomfort for the patient who has to be imaged in a prone position with arms raised above the head [ 14 , 15 ]  . the aims of this study were to evaluate the reliability of ce - mr imaging and ceus in detecting and quantifying the presence of synovitis ; to compare ceus grade ( scored by three different radiologists ) and clinical severity ( as measured by the das28 score ) in order to determine the level of radiological - rheumatological agreement ; to assess inter - observer agreement for both modalities ( ce - mr and ceus ) ; to compare two mr imaging assessment systems , namely ramris ( rheumatoid arthritis magnetic resonance imaging score ) and samis ( simplied rheumatoid arthritis magnetic resonance imaging score ) [ 1618 ] ; to test the reliability of a specially developed software programme capable of measuring synovial volume . materials and methods we analysed a total of 16 patients affected by ra and receiving treatment with biological agents [ mean age 50 11 years ( 3268 ) ] , 14 of whom were women ( mean age 51 12 years ) and two were men ( mean age 42 6 years )  . average disease duration was 17 12 years ( 137 years )  . the patients met the ra criteria drawn up by the american college of rheumatology ( acr ) in 1987 . 
the same joints were then imaged with pdus to assess synovial vascularity . contrast - enhanced mr imaging the patients underwent ce - mr imaging with a 1.5 - tesla system ( magnetom avanto , siemens , erlangen , germany ) using a body coil ( body matrix coil , siemens , erlangen , germany )  . 
the samis classication is based on the assessment , still in the hand - wrist district , of erosions ( scored 010 ) , bone oedema ( only expressed as the presence or absence , and therefore scored 0 or 1 ) and synovitis ( scored 02 )  . 
the ramris system requires the study of 46 areas ( 23 for each limb ) for the assessment of bone erosions and oedema , and of 14 areas ( 7 per limb ) for the assessment of synovitis . 
conversely , the samis system entails the study of one hand only ( the more painful ) and , in particular , of 15 areas to assess bone erosions and oedema , and 6 areas to assess synovitis [ 16 18 ]  . each patients scores for oedema , erosion and synovitis determined in the various joints considered were calculated based on the data obtained from the two classication systems . the volume of tissue affectedshowing contrast enhancementwas calculated . 
1 magnetic resonance ( mr ) sequences used in the study . coronal isovolumetric t1 3d ( a ) , coronal stir ( b ) , coronal pre ( c ) and postcontrast ( d ) vibe ( gre 3d fs ) , subtraction image between post and precontrast coronal vibe , subsequently reconstructed with maximum intensity projection ( mip ) ( e ) radiol med ( 2014 ) 119 : 422431 and quartiles were calculated for the clinical data , as these were not normally distributed . in order to compare the ceus and the clinical data ( crp , esr , vas , ptga , prga , das28 ( crp ) , das28 ( esr ) , cdai , sdai and haq ) , we applied the wilcoxon rank test after calculating the median values and quartiles of the ceus examination . 
the spearman nonparametric test was used to assess the linear correlation between the two scales ( ramris and samis ) applied to ce - mr reading . statistical signicance was set at 5 % ( p \ 0.05 ) ; correlate the volume of synovitis calculated by the software with the clinical parameters and the sum of the scores assigned to synovitis with the ramris and samis systems ; correlate separately the 28 joints assessed with pdus and the sum of the samis and ramris scores for synovitis , erosions and oedema with the clinical data ( crp , esr , vas , haq , das28 - crp , das28 - esr , cdai , sdai , ptga , prga )  . correlate the sum of erosions with the erosions seen on ultrasound by the rheumatologist at the level of the 28 joints examined . the wilcoxon rank test was used to correlate the synovitis detected with ceus and ce - mr imaging . inter - reader agreement was compared for the ceus examination and for the samis and ramris protocols . we calculated the kendall coefcient of concordance for the ordinal variables ( sum of the samis and ramris scores ) and the kappa coefcient for the nominal variables . given that the ceus scores in this study could take on only one of two values , the kappa coefcient was used to assess the inter - operator agreement of ceus , for which the agreement on each single item is also provided . 
2 enhanced tissue volume after contrast medium administration , based on automatic analysis of mr data in detail , we examined nine radiocarpal joints ( ve rightsided , four left - sided ) , ve metacarpophalangeal ( mcf ) joints ( one fourth mcf and two fth mcf on the left , one second mcf and one fourth mcf on the right ) and two interphalangeal proximal joints ( ifp ) ( third ifp on the left )  . the mechanical index ( mi ) and acoustic pressure ( pa ) were set at 0.1 and 30 kpa , respectively . 
the patients hand was immersed in a basin lled with water , used instead of us gel , at constant temperature ( 28 1 ( cid : 3 ) c ) ; the transducer was placed approximately 1 cm away from the joint and held in place by the bracket of a radiographic stand so as to minimise movement , a method described in previous studies [ 20 , 21 ]  . 
3 graphic representation of contrast - enhanced ultrasound ( ceus ) grade 1 and grade 2 median values compared with c - reactive protein ( crp ) ( a ) and erythrocyte sedimentation rate ( esr ) ( b ) , respectively 75 % ( 12 / 16 patients )  . 
no patient with laboratory ndings of active disease ( das28 [ 2.6 and cdai [ 2.8 ) had a ceus grade 0 . ra is a very widespread and potentially disabling disease , for which different therapeutic approaches are currently available ( from anti - inammatory drugs to biological agents ) [ 2225 ]  . the choice of drug requires adequate disease staging and consequently a sensitive imaging technique . discussion diagnostic imaging has long had a dominant role in rheumatology , but the correct timing and combination of modalities have not yet been standardised [ 26 , 27 ]  . 
previous studies have compared different imaging techniques ( x - ray , us , pdus , ceus , ce - mr ) in an attempt to identify the most appropriate procedure for the diagnosis and follow - up [ 2 , 28 ]  . ce - mr imaging is a reliable , reproducible and non operator - dependent modality . 
ce - mr imaging was superior to us and ceus in the overall assessment of synovitis and erosions , proving to be the most appropriate instrument for evaluating the actual damage produced by radiol med ( 2014 ) 119 : 422431 fig . 
subtraction images therefore make it relatively easy to assess active disease as well as to monitor disease progression at subsequent follow - up examinations . right wrist left 4 mcp right 4 mcp right 3 pip right wrist left 2 mcp left wrist left 5 mcp right wrist right carpus left 5 mcp left 3 pip left wrist right wrist left wrist left wrist 430 radiol med ( 2014 ) 119 : 422431 fig . 
for this reason , calculation of the volume of reactive synovial thickening cannot replace the radiologists assessment but should be used as a diagnostic complement , especially useful for quantitatively assessing the degree of involvement of the single patients joints over time . the nding that the most severely affected joint in each patient corresponded to the one reported as being clinically the most painful suggests that the reliability of this criterion for the choice of which joint should be examined with ceus . according to our results , ceus and ce - mr ( samis and ramris ) did not correlate statistically with each other , just as no correlation was found between the ceus values and the clinical parameters . 
on the other hand , analysis of the hematochemical and clinical data shows that the sample considered was homogeneous with regard to disease activity ( table 2 )  . microscopic examination of synovial biopsies demonstrates that the development of synovial microvasculature is the earliest sign of ra , and previous studies have shown that neoangiogenesis correlates with disease activity [ 3 , 4 ]  . by exploiting the purely intravascular nature of the sonographic contrast medium , the ceus examination is highly sensitive in detecting slow blood ows such as those seen in the synovial microvasculature [ 31 ]  . to conclude , ceus , with its sensitivity of 100 % in detecting active disease , is a highly useful modality for the short - term ( 36 months ) monitoring of treatment response ; the examination should not be considered for radiol med ( 2014 ) 119 : 422431 assessment of disease severity but for evaluating the presence or absence of residual disease activity . 
conversely , ce - mr imaging is a panoramic modality which , by providing comprehensive images of the hand and wrist , allows for a complete assessment of the disease , with detection of not only synovial enhancement but also bone oedema , erosions and other , at times delayed , complications such as subluxations , geodes and tenosynovitis . 
ce - mr imaging is therefore fundamental for the mid long - term ( 12 years ) monitoring of disease , to establish disease severity and extent of damage . the limitation of this study lies in its size . 
surgery is the rst - line approach , and the correct timing of radiation therapy has not yet been dened since early radiation therapy improves relapse - free survival but not overall survival . 
clinical and therapeutic factors were entered into the analysis overall ( os ) and progression - free ( pfs ) survival , and the distribution in two accrual periods identied based on the evolution of imaging procedures and radiotherapy techniques were compared . 
for 6 / 18 long survivors ( ls ) without evidence of disease , neurocognitive evaluation was obtained and the dose to the hippocampus region was retrospectively calculated . results univariate analysis of os showed a statistically signicant advantage for grade 1 and oligodendroglioma histology , better performance status [ karnofsky index ( ki ) ] , age \40 years , radical surgery , no steroid treatment ; a . 
facchetti pathology department , spedali civili hospital , p.le spedali civili 1 , 25123 brescia , italy e - mail : facchettif@gmail.com radiol med ( 2014 ) 119 : 432439 pfs was signicantly related with younger age , better ki and early radiotherapy . 
memory impairment was evident in 4 / 6 of the ls tested ; quality of life was good and executive functions were normal . conclusion radiotherapy remains an essential component in the treatment of low - grade glioma . 
prospective studies are needed to evaluate the relative contributions of the disease itself and of surgery , radiation and chemotherapy to long - term neurocognitive damage . keywords low - grade glioma ( cid : 2 ) radiotherapy ( cid : 2 ) survival ( cid : 2 ) neurocognitive evaluation introduction low - grade gliomas ( lgg ) include tumours arising from neuroepithelial tissue classied as grade i and ii by the who ( 2007 )  . 
they represent 1015 % of gliomas in adults and are almost all grade ii [ 13 ]  . the treatment of lgg and especially the timing of the different modalities are controversial since clinical trials with long - term follow - up are lacking , in contrast with the long survival times of these patients . 
favourable prognostic factors are : age \40 years , tumour size \6 cm , no neurological decits at diagnosis , karnofsky index ( ki ) [ 70 , single hemisphere disease , no enhancement at magnetic resonance ( mr ) imaging and oligodendroglial histology ( 1p / 19q co - deletion )  . 
the european organisation for research and treatment of cancer ( eortc ) 22845 trial demonstrated better progression - free survival ( pfs ) in patients treated with rt early after surgery compared with those treated after disease progression [ 13 ]  . 
no randomised trial has demonstrated a survival advantage for the use of chemotherapy [ 16 ]  . the late neurocognitive toxicity of rt represents another controversial issue , especially in young , potentially table 1 baumanns prognostic classes prognostic class age b40 and ki c70 age b40 and ki \70 age [ 40 , ki c70 and no contrast enhancement age [ 40 , ki c70 and contrast enhancement age [ 40 and ki \70 ki karnofsky index long - surviving patients , as the literature data provides conicting results . 
the aim of this study was to evaluate the evolution of the treatment for in a lgg single institution over the past 25 years , relating the use of rt to survival and toxicity . materials and methods from january 1985 to december 2009 , a total of 95 adult patients with lgg were consecutively treated with rt at the radiation oncology department of the spedali civili , brescia university . all patients were retrospectively classied according to baumanns ve prognostic classes [ 17 ] ( table 1 ) and were divided into two groups : early radiotherapy ( ert ) if radiotherapy was given less than 3 months after the surgery and late radiotherapy ( lrt ) if radiotherapy was given thereafter or at recurrence . 
the cut - off year ( 1996 ) was chosen on the basis of changes in rt and diagnostic work - up ( use of three - dimensional conformal rt and magnetic resonance imaging )  . 
distribution of the diagnostic , clinical and therapeutic features in the two periods was compared with the v2 test . overall survival was the interval between the date of diagnosis and the last follow - up visit or death , while pfs was the interval between the date of diagnosis and the rst evidence of disease progression . 
the remaining six patients were submitted in 2012 table 2 distribution of patients diagnostic , clinical and therapeutic characteristics table 2 continued gender male female 90100 prognostic class steroid therapy antiepileptic therapy histology astrocytoma oligodendroglioma ependymoma other unknown treatment period 19851995 19962009 type of surgery biopsy partial resection total resection no surgery radiotherapy timing post - surgical at recurrence radiotherapy dose b50 gy 5060 gy [ 60 gy radiol med ( 2014 ) 119 : 432439 chemotherapy ki karnofsky index , mri magnetic resonance imaging , who world health organization ( 36 years after rt ) to the following neurocognitive tests : visual analogue scale ( vas ) for pain , sf - 12 health survey questionnaire [ 18 ] , mini mental state examination ( mmse ) [ 19 ] , neuropsychiatric inventory ( npi ) [ 20 ] , rey auditory verbal learning test ( immediately and after 15 min ) [ 21 , 22 ] , trail making test ( tmt ) [ 2325 ] and clock drawing test [ 26 , 27 ]  . 
the sf - 12 is a quality - of - life test ( physical and mental health ) , based on the scores of 12 questions ( 0100 ) , where zero score indicates the lowest level and 100 the highest level of health . 
the mmse , a brief questionnaire used to evaluate cognitive impairment , includes simple questions and problems such as the time and place of the test , repeating lists of words , arithmetic , language , and basic motor skills . 
the clock drawing test represents a screening test for executive dysfunction . for ve of these six patients bilateral hippocampus regions were retrospectively outlined and the dose to these structures calculated . results patients were evenly distributed with regard to sex and age ( median age 41 years )  . 
the type of tumour was astrocytoma in 41 % of patients and other types in 55 % ; histology was unknown in 4 % of cases in whom biopsy radiol med ( 2014 ) 119 : 432439 was judged not possible owing to the site of disease ( table 2 )  . 
no signicant differences were noted in the distribution of these characteristics according to the accrual period . about two - thirds of the patients ( 68 % ) were treated before 1996 . 
most patients were treated on limited volumes : site of disease with margin ( n = 71 ) or site of disease with coning down technique ( n = 16 )  . 
the distribution of patient and disease features ( grading , age and performance status ) in the two groups was not statistically different . overall and pfs after a median follow - up of 12 years ( 2310 months ) , 45 patients ( 49 % ) were alive and 31 % of them ( 14 / 45 ) had no evidence of disease . actuarial os of the entire series was 89 , 60 and 51 % at 1 , 5 and 10 years , respectively . 
in particular , no differences in pfs or os according to recruitment period were observed . multivariate analysis of os conrmed that young age ( p = 0.009 ) , good performance status ( p = 0.001 ) , oligodendroglioma histology ( p = 0.023 ) and the absence of steroids ( p = 0.005 ) were independent factors in predicting better survival . 
good performance status ( p = 0.05 ) and ert ( p = 0.006 ) were independent factors in predicting better pfs ( table 3 )  . neurocognitive assessment and doses to the hippocampus regions the results are shown in table 4 . 
the rey auditory verbal learning test showed pathological values in immediate execution in two patients , pathological memory decit at recall in one patient and values below the standard in other two patients . 
the sf - 12 was administered to all the patients , who reported having a discrete quality of life after rt , with only minor changes in daily living . despite the lack of a randomised controlled trial , the evidence for the benets of extensive surgical resection is discussion growing . 
a prospective randomised clinical trial should be conducted to evaluate the statistical advantages of total gross resection , but even without this evidence it is considered good practice to perform a resection that is as radical as possible without causing functional decits . 
curative surgery , partial or radical , was also the rst treatment in the majority of the patients in our series ( 51 and 27 % , respectively )  . 
the indication for immediate rt can be limited to young patients belonging to baumann prognostic class 1 with mr imaging contrastenhanced disease and subjected to partial resection . to reduce the risk of toxicity , many studies have evaluated a reduction of total and fractional dose and of treatment volumes . 
 [ 29 ] evaluated the adverse long - term effects of radiotherapy and underlined the higher incidence of leukoencephalopathy and cognitive decits in patients treated with whole - brain rt in comparison with patients treated with focal rt . 
also taphoom [ 30 ] and laack [ 31 ] reported that small - volume rt could be linked with a less negative impact on cognition ; accordingly , in our series we commonly prescribed 5054 gy on treatment volumes mostly limited to the disease site . 
the few patients studied with neurocognitive tests are a good example of the radiol med ( 2014 ) 119 : 432439 different factors impacting the risk of toxicity apart from rt ( site of disease , extent of surgery , steroids and antiepileptic drug use )  . the usefulness of chemotherapy for patients progressing after the surgery and rt is relatively well established , with more data available for patients with oligodendroglial 1p / 19q co - deleted tumours [ 16 ]  . 
the results of a phase iii study , comparing radiotherapy with radiotherapy followed by pcv , showed that the addition of chemotherapy provides a benet in pfs but not in os [ 35 ]  . chemotherapy has been tested as post - surgical treatment in high - risk patients but complete responses are generally lacking [ 36 , 37 ]  . 
it is not possible to attribute a prognostic role in terms of survival to this variable . mr imaging is useful for diagnosis , to guide surgery , to plan rt and to monitor response to therapy . 
dynamic susceptibility contrast mri ( dsc - mri ) and measurement of relative cerebral blood volume ( rcbv ) correlates with vascularity and can predict high - grade transformation [ 43 ]  . 
our series reects the increased use of mr imaging in recent years . the late effects of rt might consist of rare episodes of radiation necrosis , multifocal leukoencephalopathy and damage caused by the inclusion in treatment elds of organs such the optic chiasthe leukoencephalopathy may appear months or years after rt and may lead to progressive deterioration in personality , gait and balance , urinary continence , attention , memory and executive function . 
in our series , only three patients had radiologically proven late toxicity , without any symptom : leukoencephalopathy in one patient and cerebral radionecrosis in two others [ 2931 ]  . itself , the cognitive decits may have several sources : the tumour treatments and tumour - related epilepsy , psychological distress . 
 [ 44 ] found neurocognitive decits in 91 % of the patients before the surgery : patients with tumour in the dominant hemisphere have more memory problems and poorer attention , verbal uency and verbal learning and have less chance to normalise following surgery . 
 [ 45 ] reported the analysis of cognitive and quality - oflife outcomes in 65 patients with a 12 - year median followup ; 53 % of patients who received rt had cognitive decits compared to 27 % of nonirradiated patients . 
neurocognitive function is increasingly being incorporated as an outcome measure in clinical trials in patients with lgg . with the limits of the small number of patients , our data conrm prevalent involvement of the memory network and of visual scanning , visual - motor coordination and visualspatial ability , while executive functions appeared to be normal . 
this seems relevant since the patients available for analysis are recently treated patients with a good prognosis . it is important to underline that more studies are needed to correlate cognitive decits with tumour size and site and with the overall treatment burden ( surgery , radiotherapy , chemotherapy )  . 
the italian society of radiation oncology recently published a pattern - of - practice paper on the use of radiotherapy for gliomas [ 48 ] but data on neurocognitive outcome are scarcely available . 
therefore , it may be of interest to organise a multicentric data collection , with information about neurocognitive outcome including also patients subjected to surgery alone . conclusions with the limitations of a retrospective analysis , most of the results obtained are consistent with the literature . 
the already known prognostic factors were conrmed : good performance status , younger age , oligodendroglioma histology and the absence of steroid therapy provide an advantage in terms of os . 
a careful , prospective evaluation of the possible advantages of the use of modern radiation technology for target identication and treatment delivery is therefore needed in order to dene efcacy in sparing the organ at risk ( oar )  . 
sofocleous received : 6 june 2014 / accepted : 11 june 2014 / published online : 1 july 2014 ( cid : 2 ) italian society of medical radiology 2014 abstract surgical resection is the standard treatment for early - stage primary lung cancer and selected cases with pulmonary metastases . 
however , lung resection may not be possible in patients with compromised pulmonary function , concurrent medical conditions , or previous lung resections . image - guided ablation has emerged as a less invasive treatment option for unresectable lung tumors that allows for preservation of pulmonary function and promising efcacy . 
hereby we describe the indications for and outcomes of image - guided ablation performed for the management of pulmonary malignancies . keywords lung ( cid : 2 ) early - stage lung cancer ( cid : 2 ) non - small cell lung cancer ( cid : 2 ) pulmonary metastases ( cid : 2 ) colorectal pulmonary metastases ( cid : 2 ) image - guided ablation ( cid : 2 ) thermal ablation ( cid : 2 ) radiofrequency ablation ( cid : 2 ) microwave ablation ( cid : 2 ) outcomes introduction surgical resection is the standard treatment for stage i nonsmall cell lung cancer ( nsclc ) and confers the best outcomes ; however , only one in four patients is diagnosed in this stage [ 1 ]  . 
sofocleous interventional radiology and image guided therapy , department of radiology , memorial sloan kettering cancer center , room h118 , 1275 york ave , new york , ny 10065 , e - mail : petree@mskcc.org conventional external - beam radiotherapy [ 2 ]  . 
although selected patients may benet from metastasectomy or pneumonectomy for the treatment of metastatic lung disease [ 5 ] , the majority of these patients have diminished pulmonary reserve prohibiting surgery or radiotherapy . unresectable patients with primary or metastatic lung tumors can be successfully treated with non - surgical therapies such as ablation and stereotactic body radiotherapy ( sbrt )  . 
two small retrospective series demonstrated acceptable morbidity and efcacy proles of thermal ablation for the treatment of recurrence after pneumonectomy [ 8 , 9 ] , a clinical scenario that often has no therapeutic option . 
ablation is also suitable for patients who have persistent pulmonary nodules in the setting of 542 radiol med ( 2014 ) 119 : 541548 other lung metastases that are responding to chemo / radiotherapy . finally , ablation can be offered to selected patients with pulmonary metastases that are surgical candidates within the test - of - time concept , as it has been described for liver tumors [ 10 ]  . 
according to this concept patients who could undergo surgery are treated with ablation instead . patients successfully treated by ablation alone and patients who subsequently developed multifocal progression of disease are spared unnecessary surgery with signicant morbidity . several sources of energy have been used for ablation of tissues and tumors . 
more recently , reports on the safety prole and the early clinical experience of irreversible electroporation ( ire ) , a non - thermal modality , have surfaced . pre - procedural planning the decision to treat lung tumors with ablation is generally made by a multidisciplinary team including a thoracic surgeon , an oncologist , a radiation oncologist , a pulmonologist , an anesthesiologist , and an interventional radiologist experienced in oncologic interventions ( interventional oncologist ) , and in particular lung ablation . 
in addition to the medical pre - procedural evaluation , a recent cross - sectional ct and / or pet / ct , preferably within 1 month before the procedure , is necessary in order to assess tumor size , nodal involvement , and proximity to neighboring structures [ 11 ]  . 
patients who are healthy enough to undergo a ctguided needle biopsy of the lung and can tolerate general anesthesia ( ga ) or heavy sedation are generally good candidates for ablation [ 12 ]  . 
ct is also the most frequently used imaging modality for guidance [ 13 ]  . post - ablation imaging ablation produces a rim of ground - glass attenuation ( gga ) around the tumor , seen immediately after the procedure . 
ground - glass attenuation has been regarded as an early indicator of treatment success when it forms a tumor , representing the ablation zone , rim of at least 5 mm all around the ablated lesion , representing the margin of ablation [ 14 ]  . 
subsequent imaging studies every 3 months up to at least 1 year are recommended to detect local or other tumor recurrence [ 15 ]  . pet / ct imaging is a valuable tool follow - up , depending on the [ 18f ] uorodeoxyglucose ( fdg ) characteristics of the index tumor on pre - procedural imaging . a rim of fdg avidity is usually seen surrounding the ablation zone for weeks and even months after ablation . this rim represents hyperemic reactive changes . 
the same pet and ct ndings accompanied by fever , pain , and leukocytosis may indeed indicate the development of abscess as a result of infection of the ablation zone . 
collapse of an ablation cavity may be accompanied by small increase of the suv values in pet without signifying local tumor progression ( ltp )  . subsequent follow - up is critical to show decrease in suv and lesion size . 
when suv increase persists in subsequent follow - ups or when it has a nodular or irregular conguration it may represent ltp . ablation modalities radiofrequency ablation ( rfa ) radiofrequency ablation delivers high - frequency alternating electric current to the target tissue through a needle electrode . 
due to the limited thermal conductivity of the aerated lung [ 16 ] , rfa may result in lower energy deposition into the target tumor , thus preventing adequate ablation with sufcient margin [ 17 ]  . 
another limitation that is inherent to the rfa technique is the heat - sink phenomenon that occurs as a result of adjacent blood ow in nearby vessel or airow in nearby airway that dissipates the thermal energy . 
this perfusion / air mediated cooling is responsible for an ablation zone smaller than it was intended and incomplete treatment or relatively small margins [ 18 ]  . radiol med ( 2014 ) 119 : 541548 fig . 
the light is delivered through 400 - mm long bers terminated by a specially developed diffuser which emits laser light to an effective distance of up to 1215 mm [ 19 ]  . 
the use of thermal mappings allows realtime monitoring under mri . cryoablation the cellular damage caused by cryoablation is the result of complex combination of cellular events during cycles of tissue freezing and thawing . 
crystallization that occurs in the extracellular tissue and intracellular compartment disrupts organellar membranes and causes dehydration and further compromise in cellular function . during the thawing cycle the crystals coalesce into larger sizes , membranes are disrupted , and vessels occlude resulting in cell death [ 20 ]  . cryoablation has several advantages , namely visualization of the ablation zone known as the ice ball . 
this can be visualized on computed tomography ( ct ) and mri and correlates well with the pathologic zone of ablation [ 21 ]  . sequential freezing and thawing cycles result in larger ice ball and extensive ablation zones [ 22 ]  . 
cryoablation preserves the collagenous architecture of the ablated tissue microwave ablation ( mwa ) microwave ablation ( mwa ) utilizes high - frequency waves that cause water molecules to oscillate creating friction , causing heat and tissue destruction by coagulation necrosis similarly to rfa . 
microwave ablation has several theoretical advantages when compared to the technical limitation of rfa : achieves higher temperatures thus limiting the cooling by large vessels or the airway nearby and providing 544 radiol med ( 2014 ) 119 : 541548 that may be particularly benecial when treating lesions adjacent to the tracheobronchial tree and mediastinum [ 23 ]  . ablation in early - stage nsclc the main indications of ablation in early - stage nsclc are high risk , unresectable patients , and those with recurrent tumors after radiation therapy , surgery or chemotherapy [ 2426 ]  . 
tumors \3 cm in largest diameter , located away from the large airways and vessels , surrounded completely by aerated lung are the best candidates for ablation [ 27 ]  . 
another factor associated with local tumor control is the ablation margideally , immediately after ablation , a 10 - mm margin of the gga relative to the ablated tumor should be demonstrated . 
as described before , tumor proximity to large vessels or airways has also been reported as a negative predictive factor for completeness of ablation in lung [ 29 ]  . two recent studies compared ablation to surgery in early - stage nsclc . 
the most recent ( 2011 ) study compared the survival between surgery and rfa in a retrospective review of 22 matched patients and found no difference between the two groups [ 31 ]  . 
the study concluded that there was no signicant difference in outcomes between the three therapeutic modalities . rates after similarly , overall survival image - guided ablation of nsclc are equivalent to those reported after sbrt [ 33 ] , between 40 and 60 % at 3 years for patients with early - stage medically inoperable lung cancer [ 7 , 34 40 ]  . 
along with this consideration , the cost being a fraction of that of sbrt , the ease of use and repeatability and , the safe prole of pulmonary ablation , makes this technique a competitive option for the treatment of inoperable earlystage nsclc . 
ideally , of course , randomized controlled trials should be performed although this is not an easy task . outcomes of thermal ablation in early - stage nsclc are seen in table 1 . ablation in metastatic lung disease ablation is considered in the treatment of selected patients with pulmonary metastases . 
in general ablation is indicated when the number of lesions per hemithorax are \5 , ( preferably \3 ) and the largest lesion diameter is smaller than 3.5 cm surrounded by well - aerated lung [ 24 , 4143 ]  . ablation of metastatic disease should also be contemplated when there is no or limited and controlled extrapulmonary disease . 
more precisely , keywords mrgfus ( cid : 2 ) high - intensity focused ultrasound ( hifu ) ( cid : 2 ) musculoskeletal interventions ( cid : 2 ) benign bone lesions ( cid : 2 ) management of oncologic bone lesions ( cid : 2 ) noninvasive treatment introduction magnetic resonance ( mr ) - guided focused ultrasound surgery ( mrgfus ) is a totally noninvasive ablation technique based on the ability of ultrasound waves to penetrate the tissues and to release energy . 
its limits are , however , due to its indications . currently under investigation is its use for the treatment of breast , pancreas and liver cancer , along with the essential tremor in parkinsons disease . 
a real challenge , considering the social impact , is represented by its use for the treatment of chronic pain in diseases affecting the joints and spine [ 2 ]  . the use of focused ultrasound waves for the treatment of bone lesions has aroused great interest in the scientic community . 
technological advances , however , are needed to reduce treatment times and costs . the management of bone lesions still remains a crucial issue both for the physician and the patient . 
metastases account for the most frequent types of tumoural bone lesions , being a source of pain and functional impairment , due to fractures , medullary compression and hypercalcaemia , all conditions leading to a signicant worsening of life quality . 
the patients take high doses of analgesic drugs such as morphine , and radiol med ( 2014 ) 119 : 470475 therefore , the achievement of pain relief is both a challenge the management of skeletal and the primary aim of metastases [ 5 ]  . 
moreover , since rt is poorly tolerated by the normal tissues , the treatment cannot be repeated in a previously treated area , in the case of recurrence . these problems with rt have urged the researchers to nd alternative procedures . 
several studies have been published on radiofrequency ( rf ) ablation describing palliative treatments of bone metastases , with good results in terms of pain relief [ 79 ]  . 
in these studies , rf ablation is suggested as an alternative to rt to treat painful bone metastases [ 79 ]  . another crucial issue is represented by the management of benign lesions . 
surgical procedure invasiveness and lesion aggressiveness raise the crucial issue of therapeutic management of bone lesions of benign nature , which are nonetheless a source of severe discomfort , functional disability and often invalidating pasince the presence of a benign bone lesion does not necessarily mean lack of aggressiveness , histologically benign lesions may be locally highly aggressive , causing severe discomfort to the patient . 
as a consequence , the therapeutic approach must be as radical as possible to prevent lesion recurrences . depending on the lesion nature and site , surgical treatment includes curettage , excision of a piece of bone and , in the most severe cases , prosthetic implantation . during the last decades , less invasive therapeutic approaches have been developed and performed under radiological guidance . 
the method of choice in the treatment of osteoid osteoma is today represented by thermoablation as an alternative to surgical repair . in more recent times , a new completely noninvasive ablation technique has been developed , mrgfus . 
some authors suggest its use also for the treatment of bone lesions , particularly metastases [ 10 ]  . rationale the focused ultrasound waves are based on two principles , namely denervation of the periosteum , which is quite rich in nervous bres , and ablation of the bone lesion . 
they destroy the tissue by means of heat , by increasing its temperature over 56 ( cid : 3 ) c for at least one second and reaching a coagulative thermal necrosis due to protein denaturation . ultrasound waves are acoustic mechanical waves with higher frequencies than those on average heard by humans ( [ 20 khz )  . 
propagating through matter , they apply pressure to the particles composing it and make them oscillate . the oscillation of the single particles , if observed as a whole , appears as a wave front transmitting ultrasound energy to the matter , through compression and rarefaction . when a transition point is reached , by which the interface between two acoustically and physically different matters is meant , the ultrasound waves are partially reected and partially transmitted . 
either quantity depends on the different tissue impedance : the higher the impedance , the lower the transmission . ultrasonography is based on reection and , particularly , on the possibility that the reected waves be recorded . ultrasound thermoablation is based on the transmission of energy to the tissue . 
this , however , will decrease the beam precision , since the deeper the beam penetrates , the less precisely the area to be treated will be dened and targeted . 
from this description , it becomes clear that great accuracy must be adopted in the choice of the tissue to be targeted by the ultrasound beam and that everything interposing between the skin and the targeted area ( i.e. , bone , air , metallic devices , scars ) must be avoided so as to avoid impairing of the us penetration as well . 
microbubbles between the skin and the transducer must be totally eliminated . 472 radiol med ( 2014 ) 119 : 470475 in principle , these characteristics suggest that mrgfus can be efciently applied to the treatment of bone lesions . timing is of utmost importance in pain management . 
the use of mrgfus provides much shorter times than the other techniques such as rt of bone metastases . mr guidance the high contrast resolution of mr imaging allows for an accurate treatment planning as well as the correct detection of the lesions and the neighbouring sensitive structures to be avoided for the safeguard of their integrity . 
an additional advantage of mr imaging lies in the possibility to use the same technique ( with specic sequences and administration of contrast media ) for imaging and treatment of the lesions . 
moreover , it is possible to monitor in real - time the temperature reached in the target area , thanks to specic sequences ( prf , proton resonance frequency ) that allow detection of mr phase changes , depending on temperature . 
with these sequences , it is possible to subtract the images acquired during the procedure ( as the tissue temperature increases progressively ) from those acquired at time 0 , i.e. 
in this way , it is possible to monitor both the temperature reached in the region of interest and the status of the neighbouring structures . available equipment at present two mr - guided systems are available : the exablate mrgfus system ( insightec ltd , haifa , israel ) , which received us food and drug administration ( fda ) approval for the treatment of uterine broids and bone metastases and ce marks for the treatment of uterine broids and bone metastases . the sonalleve mr - hifu system ( koninklijke philips electronics nv , eindhoven , the netherlands ) , which received ce mark for treatment of uterine broids and bone metastases . 
fda studies for palliative treatment of bone metastases and breast cancer are in phase ii / iii clinical trials . the use of focused ultrasound waves in the treatment of pain related to bone metastases was rst described in 2006 bone metastases by catane et al . 
in 10 out of 13 patients , there was a good pain relief without adverse reactions and with a drastic reduction in medicament ( table 1 )  . these results were conrmed by gianfelice et al . 
in addition , an important and original characteristic of the study was that the authors described the possibility to evaluate bone density demonstrating that the latter showed an increase at the level of the treated lesion after the procedure . 
however , there is still a long way to go to demonstrate that the use of this technique may provide structural effects in addition to the palliation , and further studies on larger series are needed ( table 1 )  . in 2008 , lieberman et al . 
thirty - one patients with 36 bone lesions were submitted to mrgfus , representing both the number of patients and the follow - up duration the driving force of the study . 
twenty - ve out of 31 patients were evaluated at 3 - month follow - up . average visual analogue scale ( vas ) score decreased from 5.9 prior to treatment to 1.8 at 3 - month follow - up . 
in 2013 [ 14 , 15 ] ( table 1 ) reported feasibility and efcacy of mrgfus treatment for pain palliation in patients with both benign ( osteoid osteoma ) and malignant ( bone metastases ) lesions . 
this study was carried out in patients not previously treated with external beam radiation therapy ( ebrt ) in target metastases and demonstrated that mrgfus can be effectively applied as a primary noninvasive technique for pain palliation related to bone metastases . 
of lesions treated decrease in pain scores follow - up bone metastases bone metastases bone metastases bone metastases bone metastases osteoid osteoma 92 % 69 % 100 % 59 days 3 months 3 months up to 3 months 6 months standard pain palliation treatments , including ebrt . 
although this study was performed with a system that was conceptually built for bone pain palliation , they demonstrated a potential role of this noninvasive technique for local tumour control yielded by a reduction in lesion viability following mrgfus procedure and by re - mineralization of spongious bone according to the mda criteria . 
according to the mda criteria , a complete response to treatment was observed in 2 / 18 ( 11.1 % ) patients , a partial response in 4 / 18 ( 22.2 % ) patients , stable disease in 10 / 18 ( 55.6 % ) patients and progressive disease in 2 / 18 ( 11.1 % ) patients [ 14 ]  . other bone lesions the therapeutic management of benign bone lesions such as osteoid osteoma , periosteal chondroma , broangiomas and bromyxomas represents a crucial issue . 
on the one hand , the surgical treatment is quite complex and should be as invasive as possible to avoid recurrence , which is reported to be high in conservative procedures . 
the crucial issue is therefore to reach the right balance between procedural aggressiveness . invasiveness and lesion in recent years , new techniques have been developed with the aim of reducing therapeutic invasiveness . 
interventional radiology nowadays offers a variety of new possibilities such as percutaneous ablation using rf , microwaves , cryoablation . osteoid osteomas are currently treated with satisfactory results by means of interventional radiology procedures , such as percutaneous thermoablation [ 16 ]  . 
almost complete absence of complications is observed using this technique along with a low percentage of minor adverse reactions ( 15 % ) , including reactive synovitis and inammation of soft tissue and nervous roots . the percutaneous treatment of the other benign lesions is scarcely described in literature . 
the lesions described in our study ( periosteal chondroma , broangiomas and myxobromas ) are submitted to surgery only when they cannot be treated with medication and / or present high local aggressiveness . the target in 2013 , napoli et al . 
 [ 15 ] treated six consecutive patients with limited joint function and reduced quality of life due to painful osteoid lesion conrmed at mr and ct . mrgfus was proposed as an alternative treatment among other consolidated modalities , including rf ablation . 
patients with lesions located in the vertebral body were excluded , while patients with lesions in close proximity to joints , tendons , tendon sheaths or neurovascular bundles were individually evaluated for accessibility . 
one of the main problems in the management of these lesions is represented by the presence of a proper acoustic window allowing the us beams to reach the lesion and treat it properly [ 18 ]  . in three cases , mrgfus was employed in combination with surgery . 
this combination , with surgery used both as neo - adjuvant and adjuvant technique , allowed the reduction in surgical aggressiveness . no major complications were observed in keeping with the literature . 
this condition was healed within a few days with administration of anti - inammatory medications , conrming the safety of mrgfus . conclusions mrgfus is a newly developed noninvasive therapeutic technique , mainly employed for the treatment of uterine broids . 
satisfying results have been achieved in the relief of pain caused by metastases [ 14 ]  . the noninvasiveness of the procedure combined with the possibility to check the temperature achieved in the targeted and surrounding areas over time allows drastic reduction in adverse reactions including damage to sensitive organs . there is limited literature about the use of mrgfus in the treatment of benign lesions . 
in one study alone , the authors describe its use in the treatment of osteoid osteomas , with results comparable to ours , particularly in terms of pain relief [ 15 ]  . the treatment of this type of lesions requires specic knowledge and competences both in the clinical as well in the diagnostic elds . 
extremely important , during treatment planning , is the study of the lesion characteristics : growth patterns , matrix , size and location , possible cortical involvement , relationships to the adjacent structures . 
the most sensitive structures in terms of ultrasound - induced damage are the scarcely vascularised tissues , such as ligaments and tendons , followed by nervous roots , which in our experience have never been damaged , though crossed by the beams . 
the lesion must be easily approached by the ultrasound waves , without interposition of scars or bone structures , but it also has to be far from more sensitive structures , such as tendons and ligaments , which absorb well due to the lack of vascularisation . 
as ow helps to lose heat rapidly , vessels are the less sensitive structures . a careful evaluation of the district to be treated has to be carried out , both in terms of anatomy and pathophysiology . in the presence of osteoid osteomas , the targeted area is represented by the periosteal bres and the nidus , source of pain due to inammation . 
as the cortical bone by denition absorbs the ultrasound waves , to achieve the proper temperature increase ( more than 56 ( cid : 3 ) per 1 s ) in the nidus , some technical requirements are to be met . 
in this way , in our experience , a major penetration was obtained as well as a hyperthermal area of 12 mm within the cortical bone . in the rst two cases , high energies were employed . subsequently , we realized that with energies inferior to 1 , 000 j an efcient hyperthermia could be achieved both on the cortex and on the nidus . 
in very young patients , it is extremely important that energies delivered be as low as possible to preserve cartilage growth . radiol med ( 2014 ) 119 : 470475 mrgfus is applied without problems on supercial lesions that do not offer any obstacles to the ultrasound waves . 
these procedures were performed without major complications . on the basis of the safety and efciency in terms of pain relief , it is believed that mrgfus represents a promising technique in the treatment of metastatic and benign bone lesions , though with the limitations due to the peculiarity of the method itself , which requires further studies on larger series . 
naguib abbas darvishi babak bazrafshan emmanuel mbalisike thorsten burkhard stephan zangos received : 19 february 2014 / accepted : 26 march 2014 / published online : 4 june 2014 ( cid : 2 ) italian society of medical radiology 2014 abstract surgery is currently considered the treatment of choice for patients with colorectal cancer liver metastases ( crlm ) when resectable . 
we searched the medical literature to identify studies and reviews relevant to radiofrequency ( rf ) ablation , microwave ( mw ) ablation and laser - induced thermotherapy ( litt ) in terms of local progression , survival indexes and major complications in patients with crlm . 
however , further competitive evaluation should clarify the efcacy and priority of these therapies in patients with colorectal cancer liver metastases . keywords colorectal cancer ( cid : 2 ) liver metastases ( cid : 2 ) radiofrequency ablation ( cid : 2 ) laser - induced thermotherapy ( cid : 2 ) microwave ablation introduction the liver is the second most common site of metastatic spread of epithelial cancers , after lymph nodes . 
systemic chemotherapy with response rates between 36 and 66 % , and a median survival of 1426 months is a good treatment option for unresectable crlm [ 4 ]  . according to variety of tumor locations , liver function and different responses to systemic or loco - regional chemotherapy , ablative therapies have been used in the management of patients with crlm [ 46 ]  . 
the effects of these methods may prolong patients survival and improve quality of life [ 6 ]  . this article was formulated with the aim of reviewing the literature on thermal ablation therapies in the management of patients with crlm . 
searches were conducted via the 452 radiol med ( 2014 ) 119 : 451461 following databases , covering the period from their commencement to 01 / 10 / 2011 : medline , premedline , embase , cochrane library and other databases . 
in this review , the outcome of these techniques and the authors views on the current status of treatments are outlined . thermal ablation therapy thermal ablative techniques have been used to destroy tumor lesions by thermal energy without damaging adjacent vital structures . 
during radiofrequency ( rf ) and microwave ( mw ) ablation and laser - induced thermotherapy , tumor cells can be effectively destroyed by cytotoxic heat from different sources [ 6 , 8 ]  . with all the heating techniques , the aim is to raise the temperature of the tissue between 60 and 100 ( cid : 3 ) c . 
thermal ablation involves placement of a needle ( electrode / antenna / laser ber ) directly into the target tumor , typically under ultrasound ( us ) guidance [ 6 ]  . the ability to generate heat in biological tissue was rst reported in the late nineteenth century . 
rf electrodes such as water - cooled single or cluster electrodes as well as expandable electrodes have been improved , especially in the last three decades with the aim of achieving larger volumes of necrosis specically in tumor lesions [ 510 ]  . microwaves can produce coagulative necrosis with an ultra - high - speed ( 9002 , 450 mhz ) alternating electric eld , causing the rotation of water molecules . 
percutaneous mw ablation was rst used as an adjunct to liver biopsy in 1986 , but since then it has been used for hepatic tumor ablation [ 6 , 8 ]  . lasers have been used successfully in tumor ablation . hyperthermia and necrosis can be produced by photon absorption and heat conduction . 
laser delivery is possible with exible thin bers or a water - cooled laser applicator . a point source from a bare - tip ber will produce a sphere of necrosis , whereas a diffuser ber will produce an elliptical ablation [ 6 , 8 , 11 ]  . radiofrequency ablation electrodes are deployed from the end of the needle into the tissue . 
the size of the ablated area is determined largely by the size of the electrode needle , the temperature of the tissue and the duration of time the energy is applied . 
a sharp boundary separates dead tissue and unaffected surrounding tissue [ 9 , 10 , 14 ]  . three radiofrequency devices are used for tissue ablation . none of the devices is specically for the ablation of hepatic tumors . 
however , just recently the food and drug administration has approved the use of these devices for the treatment of liver tumors that are surgically unresectable [ 9 ]  . radiofrequency ablation has been performed either percutaneously or with laparoscopic surgery . 
the procedure has been used with a cooled - tip or a non - cooled - tip electrode , though the former system is normally preferred in cases of larger lesions as it gives a larger area of ablation due to reduced charring at the tip [ 11 ]  . indications radiofrequency ablation has usually been used for inoperable patients with colorectal cancer liver metastases . in a multidisciplinary concept , rf ablation for isolated colorectal cancer liver metastases may be indicated in patients with the following conditions : ( a ) resectable lesion as an adjunct to resection and ( b ) inoperable lesions which demonstrate complete or partial response after chemotherapy ( c ) recurrent and progressive lesions [ 5 , 915 ]  . contraindications relative contraindications for rf ablation include : tumor located \1 cm from the main biliary duct intrahepatic bile duct dilation anterior exophytic location of the tumor bilioenteric anastomosis absolute contraindications for rf ablation include : radiofrequency ablation has been explained technically in all related studies [ 715 ]  . 
coagulopathy with international normalized ratio radiol med ( 2014 ) 119 : 451461 progression , the development of new hepatic lesions , or the emergence of extra - hepatic disease . 
a recommended follow - up protocol includes ct or mr examinations at 3 , 6 , 9 , and 12 months after the treatment and at 6 - month interval thereafter for the subsequent 3 years [ 19 ]  . figure 2 shows local changes after rf ablation in patient with liver metastases from colorectal cancer . complications radiofrequency ablation ( rfa ) is a relatively low - risk procedure with a low rate of morbidity and mortality [ 22 ]  . few complications are associated with rf ablation [ 2325 ]  . major complications are related to tumor size , number of ablation sessions , electrode type and operator experience . post - ablation syndrome is a common phenomenon after rf ablation of solid abdominal tumors [ 24 ]  . 
most patients should be able to resume normal activity within 710 days . possible complications with an incidence \5 % include shoulder pain , cholecystitis , damage to the bile ducts resulting in biliary obstruction , damage to the bowel , bleeding , hematoma , pneumothorax , pleural effusion , intraperitoneal bleeding or ascites , hemobilia , infection and portal thrombosis , liver abscess , needle tract seeding , collateral damage to proximal vital organs and self - limiting subcutaneous cellulites [ 2325 ]  . in a multicentric study , livraghi et al . 
there were six deaths ( 0.3 % ) , 2.2 % additional major complications including intra - abdominal hemorrhage requiring treatment , tract seeding , liver abscess , intestinal perforation , cardiac arrest , pulmonary embolism , pneumothorax , biloma , and cholecystitis . 
an increased number of rf sessions were related to a higher rate of major complications , whereas the number of complications was not although most institutes have reported treating lesions b5 , there is no contraindication as to the number of lesions [ 9 , 10 , 1416 ]  . imaging techniques radiofrequency ablation is typically used under us - guidance but ctand mr - guided rf ablation are also available in the treatment of crlm patients [ 1018 ]  . 
contrast - enhanced ultrasound can be performed after the end of the procedure for an initial evaluation of treatment effects . ct and mr images obtained 46 weeks after therapy demonstrate successful treatment as a non - enhancing area with or without peripheral enhancing rilater follow - up imaging studies should be aimed at detecting local tumor fig . 
1 transverse t1 - weighted magnetic resonance ( mr ) image ( tr 100 , te 4.76 , st 8 ) in a 53 - year - old female patient with a metastatic lesion from colorectal cancer in segments four and seven of the liver fig . 
some authors consider prfa as the therapeutic standard in the treatment renal neoplasms in non - operable patients due to comorbid conditions and in patients with mild - moderate renal failure , to preserve residual renal functionality . 
the use of prfa has become more and more widespread due to a rise in the incidental detection of renal cell carcinomas with the ever - increasing use of imaging for the study of abdominal diseases . 
lanzara , second university of naples , naples , italy e - mail : antonio.rotondo@unina2.it ablation is an effective therapy with a low level of risk of complications , which provides good results in selected patients over short and medium term periods of time , however up to now few long - term studies have been carried out which can conrm the effectiveness of prfa . keywords renal cell carcinoma ( cid : 2 ) percutaneous radiofrequency thermal ablation introduction in recent years , an increase in renal cell carcinoma ( rcc ) has been observed ( with more than 65.150 new cases expected in the united states in 2014 ) [ 1 ] , due to the everincreasing use of imaging methods . more than half of the new cases are detected incidentally and the number of cases of renal tumors has doubled during the past 50 years [ 25 ]  . for many years , radical nephrectomy surgery was considered the best therapeutic approach for patients with rcc conned to the kidney . there is a low risk of mortality for young patients who undergo surgical resection , however , it is often responsible for serious morbidities [ 6 , 7 ]  . partial resection with the preservation of renal parenchyma has increased for the treatment of small and localized rcc . 
this technique has recently met with success as it has proved to give similar results to total nephrectomy in a 5 - year follow - up reported in an oncological study [ 8 ]  . the preservation of renal functionality is essential in patients with rcc , multiple rcc and / or hereditary renal cancer syndromes and various comorbidities . this requirement has led to the development of minimally invasive ablative techniques , since many tumors 500 radiol med ( 2014 ) 119 : 499511 identied incidentally are low grade , smaller and with rates when detected in asymptomatic longer survival patients compared to other types of cancer [ 9 ]  . for some years , the percutaneous radiofrequency ablation ( rfa ) of renal tumors has been proposed as a therapeutic option for patients with important comorbidities , such as solitary / single functioning kidney , reduced kidney function and transplanted kidney , to prevent the impairment of renal functionality . although radiofrequency needle electrodes can also be used during surgery , most of the benets and experience deriving from this technique were achieved by means of imaging guidance percutaneous procedures [ 10 ]  . this review reports the principles , techniques , indications , effectiveness , complications , the ways of imaging guidance of the needle electrode and the follow - up of the thermal ablation radiofrequency percutaneous treatment of renal neoplasms . physical principles of radiofrequency ablation radiofrequency ablation or radiothermal ablation ( rita , radiofrequency interstitial tissue ablation ) is a tissuedestruction technique which uses thermal energy generated current high - frequency ( 100500 khz )  . alternating electric the current passes from the tip of the needle electrode to the surrounding tissue by means of ionic vibration , which in turn causes the resistive heating of tissues adjacent to the electrode ( joule effect ) ; the needle actively performs at a depth of 12 mm , while the subsequent phenomenon of thermal conduction allows for the heating of much deeper areas . 
when the tissue temperature reaches 60 ( cid : 3 ) c , alterations of the tertiary structure of intracellular proteins occur causing the denaturation of collagen and lipid bilayer damage ; thermal coagulation starts at about 70 ( cid : 3 ) c , while the drying of the tissues occurs at about 95100 ( cid : 3 ) c , causing coagulative necrosis . 
higher temperatures are not recommended as they induce tissue charring , thus causing an increase in impedance and a reduction in the effectiveness of the treatment [ 11 ]  . the amount of thermal damage depends on the diameter of the needle electrode gauge , on the length of the exposed tip and on the duration of the thermal energy administration as well as the average local temperature achieved during the procedure [ 12 ]  . procedural aspects of a radiofrequency generator connected to the main electricity network , a needle electrode ( with a variable gauge ) placed within the lesion to be treated and dispersive electrodes ( ground pads or grounded plates ) closing the circuit [ 13 ]  . needle electrodes are available on the market which release metal hooks that allow for the distribution of thermal energy over a larger area of tissue by means of a special control button located on the handle . 
each application lasts 15 min until the prescribed temperature is reached . the needle electrodes available today are equipped with cooling systems composed of an automated infusion pump connected to a 0 ( cid : 3 ) saline solution which is emitted from each hook to prevent or limit the charring of the surrounding tissue [ 14 ]  . percutaneous treatment technique there are several possible techniques for carrying out radiofrequency treatment : intra - operative , laparoscopic and percutaneous imaging - guided treatment . in this study , we focused our attention on imagingguided percutaneous treatment . 
the advantages and disadvantages of each technique will be discussed in a later section . most of percutaneous treatments are performed in mild sedation , although for certain patients a general anesthetic may be required . according to the imaging technique used , patients are placed in a supine , prone or lateral position , depending on the location of the tumor , the presence of anatomical obstacles ( abdominal viscera , ribs , etc . ) and also on the shortest possible route for the needle electrode . 
the ct and mr guidance usually foresee that the patient is placed in prone position . after capturing a pre - procedural image to locate the tumor , it is necessary to determine the exact point in which to insert the needle electrode before disinfecting the skin and administering a 1 % xylocaine subcutaneous injection . a further image is then obtained to conrm that the needle electrode is in the correct position . 
it is then possible to proceed with the procedure by supplying electricity for 1015 min depending on the technology used [ 15 ]  . if residual vital tumor tissue is detected during a postprocedural imaging check up , the needle electrode may be reused and repositioned during the same session . the procedure requires an instrument which makes the patient part of a closed - circuit electrical network composed as suggested by ogan et al . 
1 left bifocal adenocarcinoma in a 67 - year - old woman with a previous right nephrectomy for rcc , who underwent ct - guidance rf thermal ablation ( double treatment in single session )  . 
14 - mm solid lesion in anterior lip of solitary left kidney ( a , b , arrows ) and 16 - mm solid lesion in interpolar region of the same kidney ( c , d , arrows )  . 24 - month follow - up conrms the complete necrosis of two lesions ( e , f , g , h , arrows ) 502 radiol med ( 2014 ) 119 : 499511 absolute contraindications to percutaneous renal rf ablation include sepsis and severe obesity ; patients with bleeding disorders can only undergo these procedures following coagulopathic correction . 
congestive heart failure should not be considered an absolute contraindication for ablation and the risks of the procedure must be evaluated on an individual basis [ 4 , 21 , 28 , 29 ]  . to obtain good results from prfa , the tumor should not have spread beyond gerotas fascia , into the renal vein or lymph nodes ( stage i or ii in the robson classication system or stage t1 - t3a in the tnm classication system ) [ 19 ]  . 
patients should stop taking aspirin and other antiplatelet agents or anticoagulants a few days before undergoing treatment so that coagulation parameters and renal function can return to normal [ 19 ]  . indicators of outcome the outcome indicators of prfa - treated patients are the size and location of tumor . in a multivariate analysis of 100 prfa of rcc , gervais et al . 
2 47 - year - old male patient with 10 - mm renal adenocarcinoma region of right kidney ( contrastlocated in anterior enhanced ct ) , who underwent ct - guidance rf thermal ablation . prior to rf treatment ( a ) , ct scan in venous phase shows a small interpolar solid lesion with minimum contrast enhancement ( arrow )  . 
30 - day ( b ) and 36 - month ( c ) follow - up contrast - enhanced ct in arterial phase show no evidence of residual neoplastic disease ( arrows ) radiol med ( 2014 ) 119 : 499511 fig . 
3 73 - year - old man with 15 - mm adenocarcinoma originating in the interpolar region of right kidney , with endophytic development , undetectable at unenhanced ct , who underwent ct - guidance rf thermal ablation . 
4 73 - year - old male with a nodular renal adenocarcinoma treated with ct - guidance rf thermal ablation in the presence of an anatomical anomaly ( fusion of two kidneys on the right side )  . contrast - enhanced ct : renal solid rounded 22 - mm lesion , hypervascular and exophytic ( a , arrow )  . 
during the follow - up , contrast - enhanced ct scan at 1 month ( c , arrow ) and contrast - enhanced mr scan at 24 months ( d , arrow ) show no evidence of enhancement of treated lesion in agreement with complete necrosis . 
5 84 - year - old patient with a 38 - mm adenocarcinoma in upper right renal pole , who underwent two sessions of ct - guidance rf thermal ablation . 
one month after treatment , a large necrosis with a small apical - medial eccentric zone was observed by means of contrast enhancement , attributed to residual neoplastic disease ( b , arrow ) , submitted to a second prfa 2 weeks later . 
the placement of the needle electrode during the rst treatment at axial oblique mpr image ( d ) ; the needle electrode placement in apical - medial region ( site of residual neoplastic disease ) during the second prfa session ( e ) ct guidance guarantees a higher level of safety during the procedure compared to the us guidance since ct denes more accurately and comprehensively the relationship between the hooks / electrodes ( from 3 to 9 ) and the anatomical structures that may be damaged by hyperthermia ( bowel loops , large vessels , adrenal gland )  . moreover , ct also enables us to take the shortest posthe needle while avoiding anatomical sible route for obstacles ( ribs , vertebral pedicles , viscera and abdominal vessels ) by carrying out multiplanar reformations ( mpr ) [ 37 , 38 ] ; conversely , ultrasound imaging forces the operator to insert the needle into internal organs that act as an acoustic window ( liver parenchyma for the right kidney , spleen for the left kidney )  . as observed by boss and lewin [ 33 , 3942 ] , mr is well suited for guidance during prfa of renal tumors , due to its intrinsically high soft - tissue contrast and its ability to obtain almost real - time images ( rapid t1 - weighted gradient - echo sequences and t2 - weighted : steady - state free procession sequences )  . 
post - ablation modications in tumor tissue are detectable in both t1and t2 - weighted sequences . the evaluation of small central nodules without repeated injections of contrast medium , as in ct , mr makes it an excellent potential guidance of percutaneous rf ablation of renal malignancies , but the low prevalence of open mr system high - eld - strength and the high cost of non - magnetic materials used in these procedures , currently limit the application of this imaging methods in clinical practice [ 33 , 3942 ]  . role of pre - ablation biopsy some researchers suggest that in most cases biopsy does not signicantly alter diagnosis compared to non - invasive imaging ; furthermore , the percutaneous biopsy of renal neoplasms measuring \3 cm , can be complicated by sampling issues which can cause diagnostic errors [ 30 ]  . with the increased use of percutaneous ablation , the role of pre - procedural biopsy is being reconsidered , as these techniques do not allow for histopathological analysis relative to surgical resection [ 43 ]  . radiol med ( 2014 ) 119 : 499511 fig . 
6 85 - year - old male patient with a 4.5 - cm clear - cell adenocarcinoma originating in right kidney , which presented a peripheral pseudocapsule and a minute zone of central necrosis , who underwent single session of ct - guidance rf thermal ablation . 
contrastenhanced ct oblique axial mpr ( a , c ) and parasagittal mpr ( b , d ) scans in the arterial phase before ( a , b ) and 1 month after ( c , d ) prfa . 
at 1 - month follow - up ct , there is no evidence of enhancement of the treated lesion due to the radicality of treatment ( c , d )  . 
no evidence of enhancement of renal lesion prfatreated ( arrows ) indicates the radicality of the treatment ( b , arrows )  . a small cortical renal cyst coexists ( a e b , head - arrows ) several studies of rcc ablation have in fact included patients diagnosed radiologically with renal cancer who did not undergo a percutaneous biopsy [ 21 , 22 ]  . this might have lead to an overestimation of the positive results of rf as benign tumors such as angiomyolipomas and oncocytomas may have been included in these data [ 43 ]  . 506 radiol med ( 2014 ) 119 : 499511 fig . 
note that the treated lesion is devoid of contrast enhancement with unchanged volume over time , increases in density in unenhanced scans due to an initial calcication of coagulative necrosis ( a , small arrows )  . 
among these , 95 rccs were smaller than 3.7 cm , 21 rcc out of 30 larger tumors ( [ 3.7 cm ) were completely ablated after a second treatment with rf , with 9 ndings of residual tumor at follow - up . only a small number of patients with hereditary forms of renal cancer were included in the various clinical cases mentioned above . in the clinical records studied by hwang et al . 
 [ 45 ] composed of 24 patients with hereditary forms of kidney cancer , 17 patients were successfully treated with rf at average term follow - up ( 1 year )  . 
15 of the 17 patients were suffering from von hippellindau disease , while one had birthogg dube syndrome and another had hereditary rcc . some authors [ 46 ] showed that prfa ( 86 patients ) affects renal function less than partial or radical nephrectomy ( 85 and 71 patients , respectively ) , in whom a greater decrease in gfr was observed at each control . renal function is preserved even in solitary kidney patients as observed by jacobson et al . 
9 66 - year - old woman with a prior right nephrectomy and partial surgical resection of left lower renal pole for adenocarcinoma , who underwent prfa treatment of a residual 20 - mm tumor located in resection margin of solitary left kidney . 
oblique axial ct reconstructions ( mpr ) show the follow - up contrast - enhanced ct , 1 month after positioning of the needle electrode into the lesion ( b )  . 
it is important to note at procedure ( c , arrow ) and at contrast - enhanced mr 3 months after the procedure ( d , arrow ) , avascular tissue indicates complete necrosis . 
36 - month follow - up conrms the radicality of the treatment ; note the no evidence of contrast enhancement ( e , arrow ) hereditary renal neoplasms complications particular attention should be paid when treating patients with familial or hereditary renal malignancies as the ablation procedure must be optimized to take into consideration nephron - sparing , which is an important prerogative as it could lead to the development of metachronous tumors . 
for this purpose , it is necessary to reduce the margin of the treatment to a few millimeters in close vicinity of the tumor . in this type of patients , the impact of thermal ablation on glomerular ltration is minimal compared to surgery according to the urine creatinine levels observed 24 h after the procedure [ 48 ]  . one study analyzed the glomerular ltration rate in patients affected by von hippel - lindau disease who underwent prfa and previous renal surgery , which conrmed a limited decrease in renal functions following the ablation procedure ( 6.4 % increase in serum creatinine and 12.8 % reduction in gfr ) [ 49 ]  . according to observations found in recent literature , the complication rate following prfa of rcc seems to be lower than that observed in partial or radical nephrectomy . prfa is in fact related to lower morbidity and no mortality . 
the incidence of complications arising from partial or total nephrectomy is 1426 % , whereas the complication rate for ct - guided prfa is 011 % [ 3 , 5052 ]  . some authors [ 53 ] divided the complications into two groups , namely major and minor complications , according to the classication system of the society of interventional radiology ( sir ) [ 54 ] table 1 . minor complications ( classes a and b ) include : mild hematomas , neuromuscular complications , pneumothorax , hematuria and bladder globe , tumor seeding of needle electrode track . major complications ( class c ) include ureteral injuries , retroperitoneal hematomas requiring transfusion , bowel 508 radiol med ( 2014 ) 119 : 499511 table 1 complications of radiofrequency thermal ablation of renal tumors minor complications ( a ) no therapy , no consequences evolution ; no consequences major complications ( b ) therapy during hospitalization for observation of clinical ( c ) required therapy , minor hospitalization ( \48 h ) ( d ) required therapy , prolonged hospitalization ( [ 48 h ) permanent consequences ( f ) exitus perforation and perirenal abscess ( treated with percutaneous drainage )  . class a and b complications hematomas are a common complication of prfa with an incidence of 68 % [ 55 ]  . 
 [ 56 ] , caused by local side or paraspinal musculature traumas which occur in 5 % of all thermal ablation procedures . iatrogenic pneumothorax occurs during the prfa treatment of renal tumors located at the upper pole and is more frequent in ultrasound - guided procedures compared to ct - guided treatment . 
a 24 % incidence of this complication was observed in zagorias [ 22 ] clinical cases . another prfa side effect is mild hematuria which is self - limiting within the rst 24 h after the procedure , especially in centralized renal tumors . 
globe bladder rarely occurs [ 57 ]  . the risk of tumor seeding occurring at the entry point of the needle electrode is an extremely rare complication and has only been observed in one case of studies that we examined [ 58 ]  . class c complications the thermal ablation procedure of renal tumors located in close proximity or against the renal pelvis , ureteropelvic junction and proximal ureter can be risky as it can cause damage to the ureteral or collecting system , which can then cause ureteral obstruction and urinoma . these complications were observed by weizer et al . [ 59 ] in 4 % of cases ( one case out of 24 patients ) at a follow - up 1 month after the procedure . hemorrhage or signicant bleeding following prfa occurs in 18 % of the cases with blood transfusion requirements in 12 % , as observed in a multicenter study carried out on 271 patients by zagoria and gervais et al . 
bleeding is caused by mechanical injuries to major vessels and occurred mainly in the past due to the use of larger needle electrodes which were consequently more difcult to insert . 
 [ 21 ] performed the follow - up of the patients with ct or mr imaging with and without intravenous administration of contrast medium at 1 , 3 and 6 months after procedure . 
10 75 - year - old woman with a 35 - mm adenocarcinoma arising from right kidney , who underwent ct - guidance rf thermal ablation . prior to prfa treatment , a strong contrast enhancement of the right renal lesion is observed in which there is a thin rounded calcication and neoplastic tissue in the middle ( a , arrow )  . 
 [ 64 ] have analyzed the dimensional variations of treated renal tumors during follow - up , examining 18 patients at prospectively mr , and observed a 10 % increase in size of the area treated with prfa within the rst 2 weeks and a subsequent 30 % decrease during the following 6 months . hypervascular portion ( c , arrow ) , that could be due to residual disease . 
the mwa has some technical advantages such as the ability to 510 radiol med ( 2014 ) 119 : 499511 treat larger lesions thanks to a wider range of application ; moreover , the diffusion of heat in vital tissue is not limited by the impedance tissue and therefore it is possible to reach higher temperatures within the tumor lesion [ 66 ]  . cryoablation also seems to be an alternative to rfa and has some technical advantages such as the possibility of visualizing the area to be ablated ( well dened and hypodense at ct scans ) during treatment [ 67 ]  . 
over the years , much has been done on prevention and cure of this disease by surgery , chemotherapy and radiotherapy [ 1 ]  . the landscape of medicine is constantly changing , and for the past 40 years , interventional radiologists have been responsible for much of the medical innovation and development of the minimally invasive procedures that are commonplace today . 
thus , interventional radiology ( ir ) can be inserted as the fourth pillar in tumour therapy [ 1 ]  . appropriate treatment of malignancy is dependent on a timely denitive diagnosis and on accurate staging of disimaging techniques have ease . 
biopsies to establish histological diagnosis are increasingly performed using minimally invasive techniques by interventional radiologists . these minimally invasive techniques are applicable to a wide range of biopsy sites and , in most organ systems , have g . 
in an ir technique similar to that used in chemoembolisation , genetic agents may be administered directly into the tumour mass by selective arterial injection , after which the vessel is embolised thus limiting adverse effects and prolonging agent dwell time which is believed to improve genetic transfer rate . 
although there are few studies limited to small patient cohorts , this techniques is a promising one [ 3 ]  . as one can observe ir has improved majorly in recent years and can be inserted as the fourth pillar in cancer therapy alongside surgery , chemotherapy and radiotherapy . in my opinion , the essence of the treatment modality of 450 radiol med ( 2014 ) 119 : 449450 interventional oncology is technical skill , although it may also sometimes depend upon various kinds of devices and drugs . 
modications to the various applications , in particular combining the techniques with highquality imaging and intra - operative approach has enabled real - time treatment monitoring and signicant improvements in safety . 
brunese department of radiology , universita` degli studi del molise , campobasso , italy keywords pancreatic cancer ( cid : 2 ) radiofrequency ablation ( cid : 2 ) microwave ablation ( cid : 2 ) high frequency focused ultrasound ( cid : 2 ) cryoablation ( cid : 2 ) irreversible electroporation ( cid : 2 ) review introduction the term tumor ablation is dened as the direct application of chemical , thermal or electrical energy to a specic focal tumor in an attempt to achieve eradication or cytoreduction inducing cellular necrosis [ 1 ]  . the methods of tumor ablation most commonly used in current practice are divided into two main categories : thermal ablation [ radiofrequency ( rfa ) , microwave ( mwa ) , high - intensity focused ultrasound ( hifu ) and cryoablation ] and chemical ablation . irreversible electroporation ( ire ) is a non - thermal tissue ablation technology that uses very short pulses of highvoltage , low - energy direct current to induce cellular death by creating cellular membrane disruption [ 1 ]  . over the last decade , ablation has become increasingly accepted for the treatment of solid parenchymal lesions , in particular liver tumors [ 2 ]  . 
we identied additional studies through hand searches of bibliographies from primary studies , review articles and key we considered also case reports . we excluded papers that contained data reported previously . the primary endpoint was to investigate safety and complications of these techniques . 
totally , 94 males and 71 females were treated , with a mean age of 65 years old ; 2 articles did not report the sex of 13 patients [ 9 , 16 ]  . 
the tumor size ranging from 13 to 65 mm , with a mean size of 42 mm . tumor was located in the pancreatic headuncinate process in 112 patients and in the bodytail in 55 patients . 
only 4 patients were treated percutaneously with ct guided ; the other 176 underwent laparotomy and open ablation , associated in most cases with palliative surgery : biliary bypass , gastric bypass or double bypass . 
in another case , 10 days after rfa , radioactive 125 i seeds were implanted into the residual lesion area close to the bowel using the same approach [ 4 ]  . 
rfa was performed from 2 to 15 min for open technique and up to 18 min for percutaneous approach , at a temperature variable from 30 to 105 ( cid : 3 ) c . 
the power at which rfa was performed was reported in seven studies and also varied across studies , ranging from 40 to 500 w [ 3 , 5 , 8 , 9 , 11 , 15 , 16 ]  . complications related to rfa included , by incidence : ascites , vomiting , pancreatic stulae , acute pancreatitis , gastrointestinal bleeding , portal vein thrombosis , pleural effusion , pneumonia , duodenal lesion , and pancreatic pseudocystis . 
complications related with surgery included : biliary leak , hemoperitoneum , abdominal uid collection , gastric bypass stula , and gastric ulcer . systemic complications were : septic shock ( 1 / 180 ) , acute renal failure ( 1 / 180 ) , hepatic failure ( 1 / 180 ) , and psychosis ( 1 / 180 )  . 
data regarding long - term pain control and quality - of - life assessment were not available in all the studies selected , and this represented a drawback for the clinical application of rfa . 
registered post - operatively severe lifethreatening complication : a pancreatico - cutaneous stula with bleeding from splenic artery and a necrotizing pancreatitis . discussion rfa of the pancreas was initially applied to animal models . 
 [ 8 ] published the rst clinical study in 2000 . since then , few studies and case reports have been published from various groups of investigators [ 35 , 817 ]  . the revision of the studies revealed some important factors to prevent the occurrence of complications . the temperature at which rfa is performed should not exceed a maximum of 90 ( cid : 3 ) c . 
 [ 17 , 20 ] underlined that temperatures higher than 90 ( cid : 3 ) c carried a double risk of thermal injuries : portal and / or mesenteric vein thrombosis and duodenal ulcer and bleeding . 
whole tumor ablation should be avoided , prevent high temperatures diffusing to healthy surrounding tissues ( the pancreas itself , common bile duct , duodenum )  . a peripheral rim should be left and considered as a safety margin to prevent thermal damage [ 20 ]  . moreover , the distance between the rfa needle and surrounding structures should be [ 5 mm from porto - mesenteric vessels and [ 10 mm from duodenum . furthermore , thermal damage to the duodenum can be prevented by intraluminal duodenal cooling [ 21 ]  . the largest series was reported by cantore et al . 
the authors suggested that the use of a multimodal approach ( rfa , rct and iasc - triple approach strategy ) achieved survival benet in different ways : rct has been shown to be effective in local disease control ; rfa is a local ablative method that destroys tumor by thermal coagulation and protein denaturation ; regional drug delivtumor sites and ery increased drug concentrations at reduced complications due to systemic drug exposure . those patients who received the multimodal treatment had an overall survival of 26 months . 
they nally identied three independent prognostic factors for overall survival : performance status , triple approach strategy , and basal ca 19 - 9 value [ 17 , 20 , 22 ]  . increased evidence indicates that rfa induces the expression of hsp70 which inuences a variety of immunological processes [ 21 ]  . 
the aim of treatment was to obtain a cytoreductive effect of the tumor associated with destruction of celiac ganglia . to our knowledge , there is no other report of palliative surgery associated with rfa of a pancreatic mass and celiac plexus in literature . although the results obtained from rfa studies are very heterogeneous , rfa appears to be a feasible option for the local control of pancreatic tumor . however , safety still remains under debate : the quite high rate of postoperative complications , the thermal damage of the surrounding organs and the inevitable heat - sink effect still limit the application of this method . two retrospective studies involving a total of 25 patients with locally advanced pancreatic cancer treated with mwa were analyzed ( table 2 ) [ 23 , 24 ]  . totally , 16 males and 9 females were treated . 
mwa was performed from 1 to 10 min maintaining a power ranging from 45 to 100 w . one major complication ( 4 % ) related with mwa was registered in one patient that developed one pseudoaneurysm of the gastroduodenal artery , which was treated with an endovascular approach [ 24 ]  . 
over the last three decades , multiple loco - regional approaches such as transarterial chemoembolization or radioembolization were shown to effectively achieve local tumor control , offering signicant survival benets for selected patients with intermediate to advanced - stage disease ( barcelona clinic liver cancer stage b and c )  . 
these therapies provide a dual benet of safely delivering a highly cytotoxic payload directly to the tumor while reducing systemic toxicity . this capability maintained the advantage of iats over conventional systemic chemotherapy . 
the introduction of sorafenib as a systemically applicable drug , the rst of its kind to provide survival benets by means of oral monotherapy , contributed to a paradigm change . 
the idea of combining this novel agent with iats seemed intriguing , and a variety of national and international clinical trials were initiated to explore the potential benets of this exciting new option . 
in this review , we will provide a brief state - of - the - art update on the most frequently used intra - arterial modalities and discuss the molecular mechanism , potential biomarkers as well as the safety prole of sorafenib . 
geschwind ( & ) division of vascular and interventional radiology , russell h . morgan department of radiology and radiological science , the johns hopkins hospital , sheikh zayed tower 7203 , 1800 orleans st , baltimore , md 21287 , usa e - mail : jfg@jhmi.edu keywords liver cancer ( cid : 2 ) hepatocellular carcinoma ( cid : 2 ) tace ( cid : 2 ) deb - tace ( cid : 2 ) radioembolization ( cid : 2 ) intra - arterial therapy ( cid : 2 ) sorafenib introduction hepatocellular carcinoma ( hcc ) is currently a major public health problem worldwide . 
the past 30 years have seen advances in several catheter - based intra - arterial therapies ( iats ) such as transarterial chemoembolization ( tace ) and yttrium ( y ) 90 radioembolization [ 2 ]  . 
the scientic rationale for all iats lies in the fact that in contradistinction to healthy liver tissue which is supplied by the portal vein , most liver tumors draw their blood almost exclusively from the artery . this can then be exploited for selective targeting and treatment . 
indeed , this characteristic allows for tace and radioembolization to deliver a high dose of chemotherapy or radiation directly to the tumor , while preserving normal hepatic parenchyma [ 4 ]  . almost all iats involve the element of embolization as a key step of the procedure , thus preventing the washout of the payload and achieving massive necrosis through radiol med ( 2014 ) 119 : 476482 devascularization [ 5 ]  . 
indeed , the stabilization of the hypoxiainducible factor 1 alpha ( hif - 1 alpha ) causes the expression of the vascular endothelial growth factor ( vegf ) , leading to rebound neovascularization , tumor regrowth , progression and ultimately reduced overall survival [ 6 , 7 ]  . since hcc is known to rely on the formation of new blood vessels for local progression , signicant efforts were made to understand the role of microvascular changes that occur due to the imbalance of proand anti - angiogenic signaling within tumor tissue . 
as a result of these ndings , inhibiting the angiogenic pathway during treatment with tace is a promising goal . since angiogenesis has been rst described as one of the hallmarks of tumor growth , targeting molecular mechanisms of hypoxia and neovascularization has become an organizing principle for drug discovery [ 9 ]  . 
however , no drug with antiangiogenic activity that has entered clinical trials since the 1980s achieved signicant clinical benets in patients with advanced hcc , with one exception : sorafenib [ 10 ]  . 
the successful introduction of sorafenib was preceded by two phase iii trials [ sorafenib hepatocellular carcinoma assessment randomized protocol ( sharp ) and the asia - pacic study ] [ 10 , 11 ]  . 
breaking through the 6 - month median , overall survival ceiling with selected patients surviving for up to a median of 10.7 months , sorafenib quickly received wide acceptance and was adopted throughout multiple disciplines . 
the positive results from the sharp trial lead to the approval of sorafenib by the food and drug administration ( fda ) as well as the european counterpart for the treatment of advanced hcc in 2007 [ 12 ]  . 
given this scientic rationale and the demonstrated impact of this new agent on patient survival , several prospective clinical trials have been designed ( and many are still underway ) to assess the potential of the truly unique combination of iats with sorafenib . in this review , we will provide a brief update on the iats used in this combination and discuss the molecular mechanism , potential biomarkers as well as the safety prole of sorafenib , the knowledge of which has evolved signicantly since the introduction of this drug . 
finally , this review will examine the evidence for clinical outcomes for the combination of different iats with sorafenib . intra - arterial techniques : what are the differences ? conventional tace ( ctace ) the concept of the conventional tace has been introduced over 30 years ago [ 13 ]  . 
during ctace , a mixture of conventional chemotherapeutic agents combined with an oil - based contrast medium ( lipiodol ultrauide ; laboratoire guerbet , france ) is selectively delivered to the tumorfollowed by temporary or permanent feeding artery , embolization . 
in the usa and europe , most reported protocols now include doxorubicin and mitomycin c since the powder form of cisplatin is no longer available . regardless of the drug used , the key component of tace is the mixture of lipiodol as a drug carrier and chemotherapy which results in the formation of an emulsion ( water in oil ) that allows the drug to be delivered to the cancer cells . 
due to the hypervascularized character of most hcc lesions and the absence of kupffer cells , lipiodol can persist within tumor nodules for several weeks after embolizing tumor vasculature up to the capillaries [ 15 , 16 ]  . 
the subsequent administration of embolic material [ such as gelfoam , polyvinyl alcohol ( pa ) particles or trisacryl gelatin ( tg ) microspheres ] serves a dual purpose ; it causes stasis in segmental and sub - segmental arterial branches and prevents washout of the previously deposited emulsion of chemotherapy - lipiodol [ 17 ]  . 
embolization with gelfoam ( a biodegradable gelatin sponge ) has proven safe and effective for the occlusion of larger blood vessels [ 18 ] , but it has been largely replaced by nonbiodegradable tg microspheres ( merit medical , usa ) which can be used to occlude very distal tumor - supplying blood vessels [ 19 ]  . drug - eluting beads - tace ( deb - tace ) the development of polymer - based drug - eluting microspheres ( drug - eluting beads , debs ) as a robust drug delivery system improved on the ability of ctace to deliver higher concentration of cytotoxic agents directly to the tumors while minimizing systemic toxicities caused by chemotherapy [ 20 ]  . 
the pharmacodynamic benets of this technique led to a shift away from ctace toward debtace in the treatment of patients with hcc especially in the usa and europe [ 21 , 22 ]  . 
although two major types of drug - eluting microsphere are available ( dc beads made by biocompatibles / btg in the uk and quadspheres / hepaspheres made by merit medical in the usa ) , clinically neither is approved as a drug delivery system but rather as embolic material . 
these microspheres can be loaded with several ionic agents ; however , doxorubicin ( debdox ) is the most commonly used in the treatment of hcc [ 23 , 24 ]  . 478 radiol med ( 2014 ) 119 : 476482 the bulk of the clinical trials utilized the dc beads that are made of nonbiodegradable materials , such as polyvinyl alcohol ( pva ) and are soft , compressible , spherical particles . 
they range in size from 75 to 900 lthe smaller bead diameters achieve a more distal embolization and more extensive necrosis as compared to larger beads [ 25 ]  . the drug uptake of the dc beads occurs through an ionexchange mechanispharmacokinetic studies have demonstrated that drug elution occurs gradually and only in an ionic environment once the microspheres are delivered to the tumor . 
furthermore , histopathological analysis described the high efciency of debmediated drug delivery and release to the tumor tissue , causing local coagulative necrosis and an inammatory brotic tissue [ 27 ]  . radioembolization radioembolization utilizes the same scientic rationale as the one used for tace . 
a marked difference , however , is the payload in this procedure which consists of radiation in the form of yttrium 90 that is being delivered to the tumor . the infusion of small embolic particles made of or loaded with 90yttrium ( y90 ) , a pure b particle - emitting radionuclide with a half - life of 64.1 h , is a suitable technique to achieve tumoricidal effects while preserving healthy liver tissue and reducing systemic toxicities [ 28 ] that are typically encountered with external beam radiation [ 29 ]  . 
there are two radioembolization devices currently available for clinical use ; the resin - based sir spheres ( sirtex medical ltd . , australia ) and the glass - based theraspheres ( mds nordion , canada ) [ 30 ]  . 
however , only the 2030 lm sized theraspheres are approved for radioembolization of hcc under a human device exemption ( hde ) from the fda . both glass and resin microspheres deliver high cumulative doses to the tumor , which can vary from 100 gy to more than 3 , 000 gy . sorafenib : targets , safety and biomarkers of response sorafenib ( nexavar , bayer health care , berlin , germany ) is an orally applicable multi - kinase inhibitor with activity against raf - 1 , b - raf , vegf2 receptors , pdgf receptors and c - kit receptors [ 10 ]  . 
initially designed as a selective inhibitor of the raf - 1 protein , its broad activity against multiple targets involving several pathways of angiogenesis and tumor apoptosis was soon conrmed , which is also being attributed to a relatively broad toxicity prole [ 31 , 32 ]  . 
although sorafenib was generally well tolerated in the sharp trial as well as in the asia - pacic trial , it caused a range of adverse effects ( aes )  . 
among the grade 3 drug - related aes , diarrhea ( 8 % ) , hand - foot skin reactions ( hfsr 8 % ) , hypertension ( 2 % ) as well as abdominal pain ( 2 % ) were among the most common toxicities [ 10 ]  . 
additional data on the overall safety prole of the drug is currently being collected by the global investigation of therapeutic decisions in hepatocellular carcinoma and of its treatment with sorafenib ( gideon ) , which is an ongoing , prospective phase iv trial . 
this international study investigates the use of sorafenib in advanced - stage , unresectable hcc patients under real - life conditions [ 33 ]  . the recently published second interim analysis conrmed the previously established safety prole of sorafenib across child - pugh and barcelona clinic liver cancer ( bclc ) stages [ 34 ]  . the absence of a reliable biomarker for tumor response in these patients continues to be an unresolved issue in both , the systemic delivery alone as well as combination of sorafenib with iats [ 14 , 35 ]  . 
the results of this large , placebocontrolled study , which was conducted in over 300 patients , appeared to be consistent with the mechanism of angiogenesis - dependent disease progression of hcc . 
while most trials continue to rely on imaging response parameters , such as the response evaluation criteria in solid tumors ( recist ) , modied recist ( mrecist ) as well as the european association for the study of the liver ( easl ) guideline , a recent radiological pathological analysis did not identify one of these markers as a reliable predictor of tumor response to the treatment with sorafenib [ 37 ]  . sequence of administration : what is the rationale ? in order to correctly interpret the plethora of data emerging from the completed and ongoing trials for the combination treatment , three categories of administration regimen have been proposed : sequential , interrupted and continuous [ 38 ]  . the sequential schedule applies a consecutive approach , radiol med ( 2014 ) 119 : 476482 the sequential application might where the systemic therapy is given after the completion of the last session of the intra - arterial treatment . 
the rationale for the sequential schedule lies in the observation that vegf , the main molecular switch to induce neo - angiogenesis in hcc lesions , is upregulated within the rst 36 h after embolization [ 39 ]  . 
furthermore , there is evidence supporting the role of angiogenesis in liver regeneration after parenchymal damage , which is also being inicted by intra - arterial approaches [ 40 ]  . 
although safety seems to be the focus of the interrupted schedule , a clear disadvantage is the sub - therapeutic level of sorafenib within tumor tissue precisely when it is needed the most . the continuous administration of sorafenib during intraarterial therapy is used with the intent to balance the effects toward efcacy , while accepting the risk of increased toxicity . 
this approach may lead to an increased intratumoral level of sorafenib , thus making the tumor more susceptible for the toxic effects of the intra - arterially delivered payload [ 41 ]  . 
thus , it can be assumed that only a prospective , randomized trial will ultimately be able to address all questions arising from the variety of possible combinations . clinical outcome : is there a benet ? intra - arterial therapy and sorafenib one of the rst fda - sanctioned experiences with the combination of deb - tace and sorafenib in the usa was a single - center prospective phase ii trial , conducted in order to evaluate the safety and efcacy of concurrent sorafenib and deb - tace therapy . 
according to the protocol , patients were treated on a 6 - week cycle regimen , in which one cycle consisted of 400 mg sorafenib twice daily , initiated 7 days prior to the rst deb - tace . 
the 35 patients were treated with a total of 128 cycles of therapy . all patients received deb - tace and the mean dose of doxorubicin decreased over time ; cycle one : 75 mg ; two : 60 mg ; three : 49 mg . 
according to easl guidelines for tumor response assessment , 58 % of the patients showed objective tumor response , and the disease control rate was 100 % with no patient showing progression . 
as for the secondary end point of this trial , the published data also demonstrated promising survival outcomes , and follow - up studies will provide a more detailed answer for this important question . the sorafenib or placebo in combination with debtace for intermediate - stage hcc ( space ) trial is the rst international , multicenter attempt to investigate the role of the combination of deb - tace with sorafenib . 
this phase ii randomized , double - blinded , placebo - controlled clinical trial enrolled patients across 85 centers in europe , north america and asia and was recently presented ( not yet published )  . 
here , a total of 307 eligible patients were randomized to receive either sorafenib ( n = 154 ) or placebo ( n = 153 ) in combination with deb - tace . 
the dosage was similar to the trial described above , and patients received a dose of 400 mg sorafenib twice daily or a matching placebo continuously on cycle duration of 4 weeks . 
all patients received deb - tace within the rst week after the rst dose of sorafenib / placebo and subsequently on day 1 of cycle 3 , 7 and 13 , respectively . 
when further stratied , ttp at the 480 radiol med ( 2014 ) 119 : 476482 25th and 75th percentile was 112 days / 88 days and 285 days / 224 days in the sorafenib and placebo groups , respectively . 
interestingly , the hazard ratios were quite low favouring the combination treatment over the placebo arm . however , the overall preliminary results were somewhat disappointing as no statistically signicant benets regarding overall survival and ttp were shown and the authors concluded that the observed differences were not clinically meaningful [ 43 ]  . multiple trials investigating the outcome of ctace in combination with sorafenib are also available . 
in particular , a south korean nonrandomized prospective single - arm phase ii study investigating the combination of transcatheter arterial chemoembolization and sorafenib for patients with unresectable hepatocellular carcinoma ( cotsun ) focused specically on safety , tolerability and ttp . 
in this study , handfoot skin reactions were the most common reason for dose reduction or interruption and 17 out of 50 patients discontinued the combined treatment due to disease progression . 
the nal results regarding overall survival will hopefully provide a nal verdict in this asian population . the interim analysis of the ongoing study in asia of the combination of ctace with sorafenib in patients with hepatocellular carcinoma ( start ) trial provided more insight in a similar subset of patients . 
this prospective phase ii single - arm trial investigates the role of the combination in intermediate - stage hcc and subjects the patients to an interrupted treatment schedule with the ctace sessions being performed on demand . 
with a median ttp of 9 months and an objective tumor response rate of 54 % , the combination of ctace and sorafenib showed a similar trend as compared to deb - tace . 
however , no nal results regarding progression - free and overall survival were presented and can be expected until the end of 2014 [ 45 ]  . multiple further trials are underway combining debtace as well as ctace with sorafenib . 
it might be very well possible that the answer about the combination and treatment regimen of sorafenib and tace will be provided by the only phase iii trial that is already underway in the usa . 
this randomized , double - blinded , placebo - controlled multicenter trial which was proposed by one of cooperative oncology groups ( e1208 ) will compare the outcomes of deb - tace with or without sorafenib in hcc patients with invasion ( nct01004978 )  . 
the results of this trial should be made available at the end of 2014 . or without vascular very little data are available for the combination of radioembolization and sorafenib as most of these trials are at an early stage or have not yet started . 
this truly unique , two - arm study correlated the radiological and pathological tumor response for patients treated with ( n = 7 ) or without ( n = 9 ) sorafenib . 
as a result , the adjunct of sorafenib did not achieve any signicant improvement of radiological or pathological response to radioembolization which in itself is a disappointing outcome for sorafenib [ 37 ]  . other ongoing studies should shed more light as to the potential benet of this combination therapy . summary in summary , the combination of sorafenib and intra - arterial therapy in patients with unresectable hcc is safe and appears to be well tolerated regardless of the sequence of administration . 
only then should the combination of sorafenib and intra - arterial therapy be recommended for patients and included in treatment guidelines . conict of interest julius chapiro has no potential conicts of interest and nothing to disclose . 
filippiadis sean tutton alexis kelekis received : 27 january 2014 / accepted : 1 april 2014 / published online : 4 june 2014 ( cid : 2 ) italian society of medical radiology 2014 abstract benign tumors and metastatic bone lesions can be treated by ablation techniques performed either alone or in combination with other percutaneous techniques . 
ablation techniques include ethanol or acetic acid injection and thermal ablation by means of energy deposition [ including laser , radiofrequency , microwave , cryoablation , radiofrequency ionization and magnetic resonance ( mr ) - guided high - intensity focused ultrasound ( hifu ) ]  . 
depending on the lesions location ablation can be combined with cementation with or without further metallic augmentation ; local tumor control can be enhanced by combining ablation with transarterial bland embolization or chemoembolization . 
tutton division of vascular and interventional radiology , medical college of wisconsin , 9200 west wisconsin av , milwaukee , wi 53226 , usa about safety and effectiveness of percutaneous imagingguided ablation for benign and malignant ( primary and metastatic ) lesions . keywords bone ( cid : 2 ) ablation ( cid : 2 ) thermal ( cid : 2 ) chemical ( cid : 2 ) benign ( cid : 2 ) malignant introduction pain is a common complaint in oncologic patients . 
osseous metastasis is the most common source of pain ( 80 % ) in cancer patients with * 25 % meeting no relief from analgesics [ 1 , 2 ]  . 
benign bone tumors are by far more common than malignant forms [ 4 ]  . ablation techniques aim at generating cytotoxic temperatures ( [ 60 and \ - 20 ( cid : 3 ) c ) ( table 1 )  . 
percutaneous ablation techniques include injection of chemical substances such as ethanol or acetic acid , delivery of energy ( radiofrequency , microwave , laser ) aiming at temperature increase , application of extreme cold ( cryoablation ) and high - intensity focused ultrasound ( usually performed under magnetic resonance ( mr ) guidance which totally lacks any invasive character ) [ 511 ]  . 
mwa offers many benets of other ablation techniques and offers several other advantages : higher intratumoral temperatures , larger tumor ablation volumes , faster ablation times , the ability to use multiple applicators simultaneously , optimal heating of cystic masses and tumors close to the vessels and less procedural pathis review aims to provide the reader with an overview about the state of the art of microwave ablation for renal tumors and to cast a glance on the new development trends of this technique . keywords microwave ( cid : 2 ) renal cell cancer ( cid : 2 ) percutaneous ablation ( cid : 2 ) nephrectomy c . 
brunese department of radiology , university of molise , campobasso , italy introduction for several decades , the most pronounced increase in the incidence of new renal cell carcinoma cases has been for clinically localized , small tumors \2.0 cthis trend for small , low - stage tumors has been proposed to be a reection of earlier diagnosis primarily as a result of the widespread and increasing use of non - invasive abdominal imaging modalities such as ultrasound ( us ) , computerized tomography ( ct ) , and magnetic resonance imaging ( mri )  . this increased utilization of modern imaging techniques has unquestionably altered the clinical landscape of solid renal tumors . 
generally , the majority of all renal tumors will be being detected incidentally and the most of them are small ( \4 cm , clinically stage t1a ) , low grade , with a slow growth rate ( 0.35 cm / year ) , a low metastatic potential [ 1 ]  . 
small renal tumors are often diagnosed in elderly patients , older than 65 years , with medical comorbidities 534 radiol med ( 2014 ) 119 : 533540 whom the risk of surgical complications may pose a greater risk of death than that due to the tumor itself , at least in short term [ 2 , 3 ]  . 
furthermore , the high risk of end - stage renal disease after a radical nephrectomy than with a nephron - sparing surgery has encouraged the development of minimally invasive approach such as ablative modalities for selected patients [ 1 , 4 , 5 ]  . 
the continuous improvements and innovations of thermal ablative modalities , as rening probe design and real - time imaging capabilities , allow to renew the great interest in these techniques , in particular for the treatment of t1 renal malignancies [ 1 ]  . thermal ablation is a less invasive , less morbid treatment option thanks to reduced blood loss , lower incidence of complications during the procedure and a less long convalescence compared with traditional surgical approach . retrospective non - randomized studies have demonstrated good efcacy in oncologic short and intermediate term , however still be inferior than partial nephrectomy ( pn ) [ 6 ]  . finally , ablation has some advantages in terms of cost compared to open partial and radical nephrectomy [ 7 ]  . currently , european guidelines recommend the use of ablative techniques in patients with small renal masses , with comorbidities unt for surgery , and in patients with impaired renal function or with only a functional kidney [ 8 ]  . 
at present , the most widely used thermal ablative techniques are cryoablation ( ca ) , radiofrequency ablation ( rfa ) and microwave ablation ( mwa ) [ 9 ]  . 
cryoablation , using tissue temperatures to between - 20 and - 50 ( cid : 3 ) c , causes ice formation within the extracellular space leading cellular dehydration , cell membrane rupture , and nally tissue ischemia [ 11 ]  . 
differently , rfa utilizes alternating electrical currents to induce thermal injury to a lesion [ 12 ]  . advantages of rfa include its minimally invasiveness , reduced pain , and a shorter hospitalization , among disadvantages we found the lack of long - term clinical data , heat sink effect and charring , leading to decreased ablation zones [ 12 ]  . 
mwa offers many benets of other ablation techniques , in particular , rfa , and offers several other advantages , temperatures , including higher intratumoral larger faster ablation times ( maximum 10 min ) , the ability to use multiple applicators simultaneously , optimal heating of cystic masses and tumors close to the vessels , and less procedural pain [ 14 ]  . thanks to its better convection prole , microwave energy tumor ablation volumes , allows a more uniform cell kill in the ablation zone tending to , thanks to new developments of antennas design , a better roundness [ 14 ]  . 
mwa is a relative new technique and it is starting to emerge in the literature now ; however , only few studies have documented its efcacy and longremains term outcomes and much about mwa still unknown , especially in comparison to other ablative methods [ 9 ]  . the following sections aim to provide the reader with an overview about the state of the art of microwave ablation for renal tumors and to cast a glance on the new development trends . microwave energy : physic principles important microwave radiation lies between infrared and radiowave radiation in the portion of the electromagnetic spectrum between 300 mhz and 30 ghz , and including those most commonly used for microwave ablation procedures : 915 mhz and 2.45 ghz [ 14 , 15 ]  . 
heating of the tissue is based on agitation of water molecules inducing cellular death via coagulation necrosis ; the electrical charge on the water molecule ips back and forth 25 billion times a second , depending on the frequency of the microwave energy [ 14 ]  . 
the most feature is that microwaves propagate through all types of tissues and non - metallic materials including water vapor , dehydrated , charred and desiccated tissues created during the ablative process [ 16 ]  . 
in most tissues , the dielectric properties can be considered isotropic and can change substantially during treatment , but microwave propagation is not hindered by these changes [ 14 ]  . microwaves may also , offering more direct heating than other ablation energies , be more potent in organs with high blood perfusion or near larger vessel thanks to reduced heat sink effect [ 14 ]  . microwave ablation in kidney : experimental studies some preclinical studies have been published to evaluate morphology , size , and histologic features of the ablated areas in animal renal cancer . 
the unique physiology of the kidney has important implications in ablation planning . the kidney is a highly perfused organ with approximately four times the perfusion of the liver [ 17 ]  . 
this difference in radiol med ( 2014 ) 119 : 533540 perfusion signicantly alters the results of the bio - heat equation , which estimates the amount of energy that can be deposited in tissue , by increasing the energy lost [ 18 ]  . convection of heat due to overall perfusion and to perfusion by large central vessels can also contribute to a heat sink effect that may result in under treatment of renal tumors [ 18 ]  . experimental comparison to other thermal ablative techniques microwave and radiofrequency ( rf ) ablation are two techniques that generate heat and induce cellular death by coagulative necrosis in two different ways [ 18 ]  . 
heat in rf ablation is generated by converting electrical current into thermal energy by the creation of a closed - loop circuit [ 19 ]  . application of current results in marked agitation of the ions present in the tissues that immediately surround the electrode with resultant frictional heat and thermal damage to the surrounding tissues [ 12 ]  . 
for adequate destruction of tumor tissue , the entire volume of an index tumor must be treated with temperatures that are above the threshold for cell death , typically 5060 ( cid : 3 ) c [ 19 ]  . 
rfa is a reliable and safe ablation technique that has been used successfully for years in the treatment of small renal cell cancer ( rcc ) [ 1 , 20 ]  . 
however , it has some drawbacks : size and sinus extension of the tumor to be treated can increase the risk of technical failure , difculty to completely ablate irregular - shape tumors , major inuence by heat sink effect by blood circulation , depends on tissue impedance [ 21 ]  . cryoablation has been used in the treatment of skin , breast , liver , brain , and bone tumors during the past four decades [ 1 , 9 , 19 ]  . 
during cryoablation , the freezing and thawing process destroys cell membranes and organelles due to the mechanical stresses associated with phase change and ice formation [ 1 , 9 , 19 ]  . 
subsequently after increasing the intracellular osmotic pressure , an inow of water occurs during thawing resulting in tumor cells bursting [ 9 , 19 ]  . cryoablation has the major advantage of real - time monitoring of the ablation zone by direct visualization of the physical changes caused by freezing , whether using ct , mri or us [ 9 , 19 ]  . there is no denaturing of protein , as in hyperthermic treatment , in the architecture of supporting tissues , particularly urothelial tissue , which is therefore conserve [ 1 , 9 ]  . the disadvantages consist in pain or paresthesia at the probe insertion site and the potential systemic side effects associated with this use [ 1 , 9 ]  . during microwave ablation , tissue heating is induced by extremely fast realigning dipoles generated by an oscillating electric eld [ 18 ]  . 
water molecules are dipoles with unequal electric charge distribution , they attempt to continuously reorient at the same rate in the microwaves oscillating electric eld [ 14 , 18 ]  . 
therefore , temperatures clearly rise due to water molecules friction beyond 60 ( cid : 3 ) c , sufcient to create irreversible cell damage , as demonstrated by sommer et al . 
 [ 22 ] in porcine kidneys . the benets expected from microwave ablation compromise a more rapid and homogeneous ablation , lower sensitivity to local variation in tissue physical properties ( electrical and thermal conductivity ) , less inuenced by heat sink effects of blood circulation as demonstrated by sommer et al . 
studies conducted in rabbits that previously had tumor implantation show that percutaneous microwave ablation can achieve results similar to those of open nephrectomy having thus the potential of being a nephron salvaging treatment for small renal tumors [ 23 ]  . 
however , microwaves have some limitations in particular in relation to the design of the antenna that does not allow a complete control of the lesion during the ablation with an oval shape of coagulation [ 24 , 25 ]  . microwave ablation in kidney : clinical studies in the literature , it is possible to nd clinical studies on mwa for the treatment of rcc in humans since the last decade . 
first in japan , mw technology has been applied to partial nephrectomy to reduce intraoperative bleeding : in this study , a mw tissue coagulator is used not for tumor ablation but is applied peripherally in the healthy parenchyma surrounding the cancer with circumferential punctures producing coagulation of a conical - shaped portion of tissue [ 26 ]  . 
the intent of the study was to test the performance characteristics and safety of a microwave ablation system in the management of renal lesions , so were not placed limitations on the size of the renal lesion to treat [ 27 ]  . 
the results of this phase 1 study 536 radiol med ( 2014 ) 119 : 533540 show that microwave thermal energy can be used to generate reproducible large ablative lesions in 10 - min treatment in solid rcc [ 27 ]  . 
also it was demonstrated , unlike radiofrequency ablation , that microwave ablation can be performed with multiple probes simultaneously , up to three probes , allowing an easily management of large tumors [ 27 ]  . 
histochemical examinations revealed no cell death beyond the ablation area : this is particularly important in renal ablation to preserve healthy parenchyma and , above all , vascular and caliceal structures [ 28 ]  . 
 [ 23 ] published another phase i study with the aim to determine the tolerability of the amica - probe in vivo in patients with solid renal masses and the effects of heating on renal tumors and normal renal parenchyma . 
the results of these studies described have been summarized in table 1 . percutaneous mwa of renal tumors : clinical series after clinical phase i studies , since the last years were published some works about personal experience in vivo for the treatment of small rcc with mwa . 
technical success rate was 100 % , in all cases the antenna was correctly placed in the lesion ; clinical effectiveness was 100 % , no patient showed a recurrence on imaging at follow - up ( table 2 ) [ 30 ]  . 
 [ 31 ] published in 2011 a retrospectively review intermediateterm clinical outcomes after microwave ablation ( mwa ) of renal cell carcinoma ( rcc ) demonstrating , according to carraello et al . 
a very high technical effectiveness ( 98 % ) , with a cancer - specic survival rate of 100 % at 1and 2 - year and of 97.8 % at 3 - year follow - up ( table 2 )  . therefore , the study conrms the safety and efcacy of mwa [ 31 ]  . 
however , open partial nephrectomy ( opn ) and laparoscopic partial nephrectomy ( lpn ) are technically challenging and they may have serious complications , such as excessive blood loss and urinary stula [ 13 ]  . 
in a recent retrospective study was also compared mwa to open radial nephrectomy ( orn ) in the treatment of rcc : although the overall survival after mwa was lower than that after orn ( p = 0.002 ) , rcc - related survival was comparable to orn ( p = 0.78 ) [ 34 ]  . 
estimated 5 - year overall survival rates were 67.3 % after mwa and 97.8 % after orn ; for rcc - related survival , estimated 5 - year rates were 97.1 % after mwa and 97.8 % after orn [ 34 ]  . 
bosniak iii or iv cystic lesions may carry a particular risk for malignancy even if some of these lesions ( particularly bosniak iii lesions ) are proven to be benign after biopsy or surgery [ 35 ]  . 
 [ 35 ] treated seven cystic renal lesions with a total applied energy of 45 w for an ablation time of 10 min and reported a technical success , 538 radiol med ( 2014 ) 119 : 533540 dened as the correct positioning of the antenna into the lesion , of 100 % ; a technical effectiveness , as the absence thermo - ablative residues on cect performed at 1 month after mwa treatment , of 100 % ; and no major complications were recorded . 
this preliminary experience shows a potential role of us / ct - guided percutaneous mwa in treating bosniak category iii or iv cystic renal lesions , as a safe approach to treat selected patients unsuitable for surgery [ 35 ]  . as the most common benign renal mesenchymal neoplasm , renal angiomyolipoma ( aml ) originates from perivascular epithelioid cells and contain a variable proportion of adipose tissue , smooth muscle , and blood vessels [ 37 ]  . 
in 19 lesions treated they found a technical effectiveness of 78.9 % with no aml recurrence observed during the follow - up ( median , 10 months ) [ 37 ]  . complications were recorded in 14.2 % of patients : a stula of the descending colon in one patient was observed , and local infection around the ablation zone was found in another patient [ 37 ]  . 
in conclusion , mwa can provide an effective treatment also for aml with an acceptable percentage of complications [ 37 ] ( table 3 )  . new development trends in mwa to obtain an ablation as complete as possible , it is necessary to improve the ablation technique and also rening imaging guidance . 
imaging guidance improvement currently relies on the use of cone beam ct ( cbct ) which , thanks to a at panel detector and a c - arm gantry , allows to a pathway guidance , to a prediction of ablation area , an immediate imaging control and an evaluation of ablation area , allowing an immediate complication of problem solving . mwa has some technical limitations , which cause disadvantages such as wavelength elongation and unpredictable shape and size of ablation area . 
1 82 - year - old man with left kidney rcc ; a enhanced ct image of renal lesion ; b us image shows the antenna inside the renal lesion ( white arrow ) ; c photo of percutaneous mw antenna placement in left ank ; d follow - up enhanced ct image shows a complete ablation of the left renal lesion with no contrast enhanced of ablated area limitations . 
thermospheretm technology ( covidien ) with its new probes , provides three kinds of spatial energy control thermal , eld and wavelength , holding a predictable spherical ablation zones throughout procedures . 
by employing this system could overcome the uncertain clinical outcomes of other ablative technologies , giving an increased freedom and condence to plan and execute with predictable outcomes in every procedure . 
from this derives the idea that the ablation mw area will be all the more spherical it will be possible to completely ablate the renal lesion . conclusion ablative techniques have been introduced with the aim to control the spreading of a local tumor and to preserve the surrounding parenchymas function , with curative or local control as the primary objectives , when surgery is not feasible [ 79 , 11 , 13 ]  . 
different ablation techniques such as cryoablation , or microwave radiofrequency ablation , ablation have been available for some time , resulting in few complications , most of which are self - limited or readily treated , with great advantages if compared with surgical management [ 1 , 9 ]  . 
this technology can be applied in selected patients who are not candidates for surgery , as an alternative to other ablative techniques [ 11 , 2931 ]  . though mwa achieved comparable results to those obtained with rf ablation or cryoablation , mwa needs a relatively short ablation time and may be more suitable for patients with a variety of comorbidities who cannot tolerate long - time anesthesia [ 31 ]  . 
mwa is a technology that can reliably and reproducibly produce a large ablative lesion of solid renal neoplasms with a uniform tissue necrosis without skip areas [ 27 ]  . 
these recommendations are based on a comprehensive analysis of the current literature , the results of multicenter studies , and expert consensus . keywords thyroid ( cid : 2 ) us ( cid : 2 ) ceus ( cid : 2 ) radiofrequency ( cid : 2 ) ablation ( cid : 2 ) thyroid nodules ( cid : 2 ) thyroid recurrent cancers ( cid : 2 ) intervention radiology introduction thyroid nodules are very common in the adult population with a prevalence of 2076 % [ 1 ]  . 
although most thyroid nodules are benign and do not require treatment , some for associated benign nodules may require treatment symptoms and / or because of cosmetic problems [ 2 , 3 ]  . 
as curative surgery has several drawbacks and the efcacy of thyroid hormone - suppressive therapy has not yet been determined , nonsurgical , minimally invasive treatment modalities , such as ethanol ablation ( ea ) , percutaneous laser ablation , and radiofrequency ( rf ) ablation , have been used to treat thyroid nodules [ 27 ]  . thermal ablation using radiofrequency is a new , minimally invasive modality that may be an alternative to surgery in selected patients with benign thyroid nodules and recurrent thyroid cancers [ 8 ]  . this paper provides information regarding the basic principles , indications , devices , and techniques that have been especially designed to optimize thyroid rf ablation , as well as the clinical results and complications . principles of radiofrequency ablation ( rfa ) rf ablation uses the heat generated from high - frequency alternating electric current oscillating between 200 and 1 , 200 khz [ 2 , 9 ]  . 
although this heat creates immediate damage to tumor tissue , the damage is signicant only in regions very close to ( thus , within a few mm of ) the electrode . 
in addition , ea is a simple and less expensive procedure [ 20 ]  . according to the different clinical problems , it is possible to divide the patients in three group [ 10 , 1517 , 23 , 25 ]  . group 1 symptomatic patients : symptoms as neck pain , dysphasia , foreign body sensation , discomfort , and cough should be directly self - measured by patient using a visual analogue scale from a grade of 0 to a grade of 10 [ 18 , 19 , 35 ]  . group 2 cosmetic problems : a score , based on cosmetic impact of the nodules presence , can be measured by a physician in four grades : grade 1 , no palpable mass ; grade 2 , palpable mass ; grade 3 , a cosmetic problem on swallowing only ; and grade 4 , a readily detected cosmetic problem [ 14 , 16 , 1820 , 35 ]  . group 3 thyrotoxicosis problems : patients with aftn causing problems related to thyrotoxicosis [ 14 , 16 , 17 , 23 , 3640 ]  . recurrent thyroid cancers surgery is the standard treatment for recurrent thyroid cancers as lymph nodes in operation bed , followed by radioactive iodine and thyroid hormone therapy . 
rf ablation , however , can be used in patients with a high - surgical risk and / or in patients who refuse surgery [ 21 , 23 , 26 , 27 ]  . pre - procedural evaluation at least two separate us - guided ne needle aspirations and / or core needle biopsies are necessary to conrm the benign nature of a nodule , and caution should be taken when performing rf ablation of nodules with malignant us features even when there are benign results seen on ne needle aspiration or core needle biopsy [ 12 , 4246 ]  . laboratory tests usually include a complete blood count , a blood coagulation battery , and measurements of thyrotrophin , thyroid autoantibodies , and calcitonin [ 2 ]  . thyroid hormones , if any serum concentrations are abnormal , rf ablation should be performed only after the correction of these abnormal values [ 8 ]  . us examination is important for characterizing and evaluating the surrounding anatomical structures [ 47 ]  . 
in fact with us should be evaluated many characteristics of the nodule : size , shape , margin , proportion of solid / cystic components , echogenicity , calcication , internal , and extracapsular vascularity [ 47 ]  . size could be well dened taking the three orthogonal nodule diameters , including the largest diameter , and the nodule volume could be calculated using the equation : v = pabc / 6 , where v is the volume , a is the maximum diameter , and b and c are the other two perpendicular diameters [ 2 ]  . scintigraphy with 99mtc pertechnetate can be used to differentiate cold nodules from aftns , particularly in with a decreased serum concentration of thyrotrophin [ 2 ]  . prior to the treatment of recurrent thyroid cancers , tumor recurrence should be conrmed by positive us - guided ne 514 radiol med ( 2014 ) 119 : 512520 needle aspiration cytology and measurements of washout thyroglobulin concentration [ 8 ]  . 
a neck ct may be used when appropriate for the evaluation of a recurrent tumor prior to rf ablation [ 8 ]  . patient preparation the patient , during the procedure , should be supine with neck slightly extended [ 48 ]  . to prevent an infection or abscess , the puncture site should be sterilized before rf ablation and prophylactic antibiotics can be used [ 8 ]  . 
local anesthesia could be achieved with an underneath injection of the skin near the cervical - surrounding soft tissue and thyroid capsule with lidocaine or xilocaine [ 16 , 17 , 26 ]  . 
painkillers and sedatives can relieve pain during the ablation but these drugs are not recommended during the ablation because the early detection of complications is impossible in patients under deep sedation , which disturbs communication [ 8 ]  . 
physicians should monitor blood pressure , pulse rate and the voice of each patient by regular conversations during the procedure . if a voice change is suspected during the ablation , the physician should stop the procedure immediately [ 4 ]  . 
an application of an ice bag during the ablation may prevent skin burns at the electrode puncture site . currently , there are above all two types of rf techniques for thyroid nodules [ 4 ]  . the rst technique is called the xed ablation technique , it can be performed using a multitined expandable electrode [ 14 , 16 , 49 ]  . 
in xed ablation , under us guidance , rst lidocaine should be injected into supercial cervical tissue and on the thyroid gland ; then the electrode , always under us guidance , is guardedly inserted along the greatest diameter of the nodule avoiding injury to vital structures [ 14 , 16 , 49 ]  . 
each hook is recommended to be 10 mm away from thyroid capsule , 56 mm from pseudocapsule of the outer edge of nodule , and 15 mm from heat - sensitive cervical structures [ 14 ]  . the correct position of electrode tips and hooks is dened by us exawith xed ablation technique , a spherical ablative zone is usually achieved [ 14 , 16 , 49 ]  . after the ablation , the hooks are retracted and the electrode is slowly withdrawn after the rf energy has been switched off [ 14 , 16 , 49 ]  . the second rf ablative technique is called the moving shot technique , rst described by baek et al . 
a shorter and smaller electrode allows a better control and variation of ablation option in particularly for the treatment of small nodule and / or closed to vital structure closed thyroid nodule . 
in the moving shot technique , the target thyroid nodule is divided into multiple small conceptual ablation units and during the procedure , each conceptual unit is being ablated by the moving ablation electrode tip [ 10 , 22 ]  . 
it is important to preserve from the ablation the danger triangle : the region close to the trachea - esophageal groove to avoid injury to the recurrent laryngeal nerve , trachea , and esophagus . ultrasound monitoring during the ablation is also important to detect hemorrhage [ 3 , 10 , 51 ]  . 
hematomas usually disappear completely within 2 weeks [ 3 , 10 ]  . follow - up patient condition should be monitored after the procedure [ 3 , 10 ]  . 
we recommend that patients be followed up at 12 , 6 and 12 months after rf ablation , as well as every 612 months thereafter , depending on the status of the treated nodules [ 3 , 10 , 18 , 22 ]  . 
additional treatments may be indicated in patients with unresolved clinical symptoms or with a viable growing portion of the nodule detected on imaging [ 3 , 4 , 10 ]  . radiol med ( 2014 ) 119 : 512520 516 clinical results benign nodules rfa therapy has mainly been aimed at decreasing pressure improving the cosmetic results as well as symptoms , resolving thyrotoxic status in hot nodules . 
 [ 11 , 14 , 16 , 1820 , 33 , 34 , 49 , 5254 ]  . three studies were one - armed observational studies utilizing rfa alone [ 14 , 16 , 53 ]  . two studies were one - armed observational studies investigating rfa before and / or after percutaneous ethanol injection ( pei ) [ 19 , 34 ]  . 
one study was a randomized prospective trial comparing the effectiveness of one rfa treatment to two rf ablation treatments [ 20 ]  . the efcacy of rf ablation of cold nodules has been evaluated in terms of reduction of nodule volume , symptoms and cosmetic problems improvement . 
in this study , 20 patients were assigned to the rf ablation group while the other 20 patients to the control group ( conservative treatment ) [ 49 ]  . after 12 months , patients of the rf ablation group had a signicantly decreased mean nodule size ( 13.31.8 ml , p \ 0.001 ) while , in the control group , the mean nodule size was nearly static ( 11.211.8 ml , p [ 0.05 ) [ 49 ]  . 
regrowth of more than 50 % was very uncommon ( 5.6 % ) , it depends on the proportion of cystic component within the ablated nodule [ 54 ]  . 
in cystic nodules with \10 % solid component , rf ablation could achieve a reduction [ 90 % at 6 - month after ablation [ 11 , 53 , 55 ]  . 
1 female45 years , at us and ecd imaging , we found a benign - vascularized nodule in the left lobe with a largest diameter of 41.1 mm and required more sessions and was more expensive [ 11 , 55 ]  . 
in fact the treatment of benign cystic thyroid nodules , the mean volume reduction was 96.9 % in pei while it was 93.3 % in rf ablation , thus demonstrating the superiority of pei to rfa [ 55 ]  . 
authors concluded that pei may be used as the rst - line treatment modality for cystic thyroid nodules , which has comparable therapeutic efcacy to , but is less expensive than , rf ablation [ 55 ]  . 
on the other hand , predominant cystic nodule ( 1050 % solid component ) might be suitable for rf ablation as 6.121 % failure rates in pei were reported for this type of nodules [ 34 , 40 , 56 ]  . rf ablation is generally good for the treatment of the solid component of these refractory nodules [ 19 , 34 ]  . 
in a solid nodule with a predominantly solid component [ 50 % , rf ablation could achieve a 2337 % volume reduction at the 1st month and 5177 % volume reduction at the 6th month [ 22 , 49 , 53 ]  . 
2 female45 years , us image shows during rfa procedure the straight internally cooled electrode contrast - enhanced us ( ceus ) imaging shows a rich vascularization of the benign nodule in the left lobe fig . 
3 female45 years , ( 15 cm length , 17 gauge ) into the benign nodule in the left lobe improved thyroid function reducing the need for antithyroid medication [ 14 , 22 , 49 ]  . 
in a series of 28 patients reported that all patients with pretoxic thyroid nodule and 53 %of patients with toxic thyroid nodule stopped antithyroid medication at 12 - month follow - up after rf ablation . 
therefore , more sessions of rfa are generally needed [ 22 ]  . recurrent thyroid cancers rf ablation can be used for locoregional control of cancer or the treatment of cancer - related symptoms in patients with recurrent thyroid cancer who have a high - surgical risk fig . 
5 female45 years , after a month from rfa , us shows a signicative reduction of the largest diameter ( 33.7 mm ) of the benign nodule of left lobe , compared to fig . 
rf ablation of recurrent thyroid cancers in the neck resulted in a mean volume reduction of 5693 % , with 4258 % of nodules completely disappearing , 64 % of patients experiencing symptom improvement , and serum thyroglobulin concentration decreasing [ 21 , 23 , 26 , 27 ]  . 
however , longterm follow - up data have not been published yet . complications it is necessary to know the broad spectrum of complications of thyroid ra ablation to prevent or minimize complications and sequelae [ 4 , 59 ]  . 
reported complications of thyroid rf ablation include pain , hemorrhage , voice change , skin burn , hypothyroidism , hyperthyroidism , infection and nodule rupture [ 10 , 1416 , 18 , 22 ]  . 
rf ablation does not require general anesthesia , does not induce scarring of the neck , and when performed by 518 radiol med ( 2014 ) 119 : 512520 well - trained physicians , is associated with a low complication rate [ 10 , 1418 ]  . 
during the ablation , most patients could complain of various degrees of pain in the neck and / or radiating to the head , ear , teeth , shoulders , back or chest . 
a voice change is a major complication of rf ablation , caused by the damage of the recurrent laryngeal or vagus nerve ; so , it is necessary to detect vagus nerve before rf ablation [ 60 , 61 ]  . 
to prevent voice change , rf ablation should be performed using the moving shot technique . there is a potential risk of a sympathetic ganglion damage during the extrathyroidal penetration of the electrode if it is not detected during the ablation procedure because it is located posterior to common carotid artery near the thyroid gland [ 8 ]  . 
to avoid this complication , continuous and cautious us - guided tracing of the electrode tip is mandatory during the rf ablation [ 8 ]  . hematomas , resulting from electrode - induced mechanical injury , can occur in the perithyroidal , supcapsular , and intranodular locations [ 59 ]  . 
hematomas can usually be controlled by mild compression of the neck for several minutes , with most hematomas disappearing within 1 or 2 weeks , serious hematomas may compress the airways , close observation is recommended during and after procedure [ 3 ]  . 
hypothyroidism has been reported in patients with nonfunctioning thyroid nodules and those with aftn , thyroid function laboratory analysis are recommended during the following days after rf ablation [ 10 , 15 , 22 , 57 , 62 ]  . 
prophylactic antibiotics and a sterilization of puncture site before rf ablation are recommended to prevent an infection or abscess [ 8 ]  . thyroid nodule rupture after rf ablation may be suspected in patients complaining of sudden neck bulging and pain at the treatment site , us examination usually shows a breakdown of the anterior thyroid capsule and the formation of a new nodule in the anterior neck [ 3 , 59 , 63 ]  . delayed sudden perinodular hemorrhage might be a cause of nodule rupture . 
spontaneous improvement without treatment is possible , but surgery is required when an abscess forms [ 3 , 59 , 63 ]  . skin burns have been reported only at the puncture site , instead the risk of burns at the pad attachment site is relatively low , because rf energy is lower in the thyroid than in the other organ ablation as liver [ 15 , 23 , 59 ]  . 
severe tension , pain , or hypersensitivity to lidocaine could cause nausea and vasovagal reection [ 59 ]  . no life - threatening complications , as injury to trachea and heart attack , have been reported in literature in patients underwent rf ablation of thyroid gland [ 29 , 30 , 33 , 64 , 65 ]  . 
to prevent thermal injury to the esophagus , patients should be asked to swallow cold water during the ablation of a conceptual unit adjacent to the esophagus [ 3 , 29 , 31 , 59 ]  . to prevent life - threatening complications , the operators should strictly trace the electrode tip during the procedure , and should have knowledge of the necks anatomy and experience of an image - guided intervention . conclusions thyroid rf ablation is an effective and safe treatment modality in patients with benign thyroid nodules . 
in this paper , we highlight the technical aspects and clinical applications of cbct imaging and navigation in the most common loco - regional oncological treatments . interventional oncology ( cid : 2 ) cone - beam keywords computed tomography ( cid : 2 ) imaging guidance ( cid : 2 ) percutaneous treatments ( cid : 2 ) embolization ( cid : 2 ) ablation introduction interventional oncology is a growing eld offering new minimally invasive , image - guided treatment options for a variety of primary and metastatic solid tumors . 
imaging is at the core of this approach and selection of the optimal diagnostic and interventional modalities is critical for a safe and effective treatment delivery [ 1 ]  . 
rotondo department of radiology , seconda universita` degli studi , naples , italy 522 radiol med ( 2014 ) 119 : 521532 two - dimensional ( 2d ) angiography is the main imaging technique during embolotherapy . 
ultrasound offers a versatile imaging technology for liver and kidney tumor ablation with real - time imaging . however , tumor and needle or antenna visibility are often suboptimal for deep locations and / or large patients . 
electromagnetic tracking of ablation devices and fusion between real - time ultrasound and preprocedural ct , mri or pet / ct have shown to facilitate targeting of lesions that are sonographically invisible [ 5 ]  . this solution requires dedicated software , hardware and disposables , and does not solve the limitations related to ultrasound for direct thermal ablation monitoring . 
in addition , real time requires ct uoroscopy which is not always available and leads to a signicant increase in radiation exposure to the patient and operator . pet / ct and mri guidance are emerging options for percutaneous tumor ablation , offering real - time imaging , excellent tumor visibility and novel approaches in ablation monitoring [ 7 , 8 ]  . 
however , limited accessibility and increased associated costs have currently limited the use of these modalities and research institutions . to dedicated centers c - arm cone - beam computed tomography ( cbct ) is a new imaging technology that enables acquisition of crosssectional imaging with modern angiographic systems equipped with a at panel detector [ 9 ]  . 
volumetric tomographic images can be combined and co - displayed with conventional 2d angiographic imaging and dedicated software during interventional procedures to plan treatment , navigate / position the catheter or device , monitor the treatment , and assess the nal result or verify margins . 
the motion path includes an acceleration and deceleration phase encompassing a phase of constant speed of 3060 ( cid : 3 ) per second ( s ) and projection acquisition is performed in pulsed mode . 
a complexity for tomographic reconstruction on interventional c - arm systems is the need to accurately measure the true system position , which needs to be taken into account during back - projection . 
for abdominal imaging , typical tube parameters are 510 ms pulses per projection at 120 kv tube voltage including copper ltration with frame rates of 3060 frames per sec . recent detectors cover a planar region of 30 9 40 cm at a high spatial resolution up to 1502 lm2 [ 12 ]  . 
2 schematic representation of a dual - phase cbct acquisition as it is used for tace procedures achieved by adapting the focal spot size , detector readout resolution , and the reconstruction lter . 
satisfactory offset ( off - midline , to include skin entry ) should be taken into account for percutaneous procedures . radiation ceiling - mounted monitors shields should be arranged so that they can be promptly moved and cbct imaging can be performed safely . 
armrest accessories are recommended if available to improve patient comfort during cbct imaging , but should not obstruct rotational movements [ 15 ]  . radiation exposure the x - ray exposure associated to cbct has been the subject of several studies performed on phantoms , animals , patients and the medical team [ 1619 ]  . 
the estimated effective dose to the patient for one cbct scan of the abdomen is approximately 310 msv , generally lower than the comparable abdominal mdct scan ( 1012 msv ) [ 16 , 20 ]  . 
as for all interventional procedures using x - ray exposure , the operators should wear protective devices and leave the examination room when performing 3d scans , and cbct acquisition should be used judiciously . dose therapy planning and navigation software the availability of volumetric datasets showing tumor location , vascular territory and parenchymal environment at the time of the procedure provides an excellent platform to dene a safe and effective route to the lesion and guide device positioning . 
intraarterial therapy planning software ( emboguide , philips healthcare or flight plan for liver , ge healthcare ) is capable to automatically identify the feeding arteries to the targeted lesion ( s )  . 
the basic workow involves the 3d segmentation of the targeted lesion ( s ) on the cbct dataset and the automatic extraction of candidate feeders from a starting point ( normally the micro - catheter tip ) to the targets . 
needle planning software ( xperguide ablation , philips healthcare or innova trackvision , ge healthcare or iguide , siemens healthcare ) provides a simple technique to dene the needle trajectory from the skin entry point to the target . 
the needle paths can be drawn in any direction and multiple paths can be concurrently shown [ 22 ]  . cbct datasets are acquired in a calibrated space where the position of the acquired 3d dataset is known with respect to the x - ray beam generation and the mechanical movements of both c - arm and angiographic table . 
the 3d dataset and the needle or feeding vessel paths follow the rotation and angulation movements of the c - arm , translations of the table , and automatically adjusts to magnication changes . 
the operator can then follow the graphical overlays for device manipulation and / or automatically move the c - arm into pre - dened positions that facilitate catheterization or accurate needle alignment towards the targeted lesion ( s )  . 524 clinical applications cbct and embolotherapy transarterial chemoembolization the use of cbct in the visualization and localization of hepatic lesions at the time of intervention is crucial for effective intra - arterial therapy in seeing and reaching the lesion and in assessing response [ 21 ]  . 
dsa can provide excellent vascular visualization but it has less sensitivity for tumor detection than ct or cbct due to its low softtissue contrast and 2d projection nature [ 15 , 2328 ]  . 
cbct with its 3d nature , soft - tissue contrast , and especially coupled with post - processing software is superior to dsa in lesion detection and tumor feeding vessel identication [ 23 , 3037 ]  . 
dual phase has increased tumor detection versus single - phase cbct alone and is comparable to the gold standard of contrast - enhanced mdct and mri in lesion detection and in predicting therapy response [ 28 , 42 , 43 ]  . with these highlights of cbct in lesion detection , tumor feeding vessel identication , and therapy assessment , the addition of cbct along with dsa can prolong patient survival [ 44 ]  . selective internal radiation therapy selective internal radiation therapy ( sirt ) is a relatively new , catheter - directed treatment modality of both primary [ 45 ] and secondary [ 46 , 47 ] liver tumors . 
in contrast to transarterial chemoembolization ( tace ) , an angiographic work - up is required prior to yttrium - 90 ( y - 90 ) loaded microsphere infusion into the hepatic artery [ 48 ]  . 
angiographic work - up mainly consists in identifying ( and , if indicated , coil - embolizing ) hepatoenteric arteries , originating from the hepatic arteries [ 49 ] ; dening the vascular territory of all targeted hepatic arteries ; and identifying the tumoral lesions within these vascular territories . 
cbct depicts perfused tissue location , which is essential for correct segment classication or pre - treatment portal vein embolization . radiol med ( 2014 ) 119 : 521532 rupture of intratumoral aneurysms . 
cbct has shown potential utility in this procedure to avoid untargeted vessel embolization ( inferior vesical artery or rectal artery with subsequent collateral off - target damage ) and to optimize catheter placement and selective embolization [ 58 ]  . the role of dual - phase cbct with automatic vessel detection software ( emboguide , philips healthcare ) during pae may dene vessels and perfused tissue territory . 
dualphase cbct can be acquired using the following protocol : 24 cc of iodine contrast at 2 cc / s , delay 4 s , considering a single cbct scan duration of 8 s . 
the arterial phase cbct is acquired with a 4 s delay after contrast injection , while the delayed phase cbct is obtained 5 s after the end of the rst cbct scan . 
software - assisted detection of prostatic vessels is feasible and collateral non - target vessels may also be successfully depicted on cbct . cbct and tumor ablation image - guided percutaneous ablation is a minimally invasive , therapeutic option for localized disease . 
note that the lesion depiction on the preand intra - procedural imaging matches with the post - tace imaging , and especially with the hypoenhancement 1 month post - ta renal embolization arterial embolization indications in oncologic kidney therapy are uncommon with the success of partial nephrectomy and percutaneous ablation . 
the main indications are embolization of hypervascular cancers and associated renal vein thrombosis to limit blood loss during renal cell carcinoma ( rcc ) surgery [ 51 ] , or to primarily treat or prevent hemorrhage risk for angiomyolipoma ( aml ) [ 52 ]  . aml is a common benign renal tumor . 
6 ad cbct imaging during y90 work - up in a 60 - year - old patient suffering from chemorefractory colorectal liver metastases clearly shows a small hepatoenteric vessel , feeding the gastric wall : right gastric artery . 
subsequently , proximal coil embolization of the right gastric artery was performed as with embolotherapy , cbct can assist in all steps of thermal ablation ( planning , positioning needles , ablation monitoring , modication of needle plan , post - ablation assessment and verication of completion ) for both thotumors , racic and abdominal tumors . 
for thoracic respiratory gating may minimize motion mis - registration , thus facilitating navigation . indeed , once the patient is positioned on the angiographic table , a pre - procedural cbct is performed to visualize the target lesion and plan the procedure . radiol med ( 2014 ) 119 : 521532 figs . 
ct 1 month after procedure showed complete devascularization of the tumor ( unenhanced ct 9a and enhanced ct 9b ) the operator can segment the lesion and determine the skin entry point as well as the target . 
previous literature concerning cbct - guided lung and bone biopsies demonstrated that unenhanced pre - procedural cbct is sufcient to delineate the lesion and enable segmentation [ 22 , 61 ]  . 
the same could be applied in ablation cases [ 62 ] ; for difcult lung ablations close to vital structures , a contrast enhancement cbct may be needed . most abdominal ablations will also require a contrastenhanced cbct pre - procedurally and post - procedurally . several techniques are possible for contrast injection during cbct scan . in a preliminary study , morimoto et al . 
peripheral intravenous injection is an alternative option which is the method used by the authors during the pre - procedural cbct ( data unpublished ) to provide an optimal visualization of lesion target ( both hepatic and renal tumors ) and adjacent structures , used for ablation planning . moreover , in a preliminary series including 12 hepatic lesions treated with rfa , iwzawa et al . 
 [ 64 ] concluded the efcacy of intravenous contrast - enhanced c - arm ct for assessing ablative areas and safety margins immediately after treatment is nearly equivalent to that of mdct performed 37 day after rfa . contrast - enhanced cbct can be avoided in cases of contrast allergy or renal failure . 
 [ 65 ] , previous diagnostic imaging such as contrast - enhanced ct , pet - ct or mri can be co - registered with unenhanced pre - procedural cbct to plan the procedure and with the post - procedural cbct to assess the completion of the ablation . c - arm cbct intrinsically has also potential disadvantages in terms of contrast resolution , image quality and fov . 
however , the use of co - registration / fusion with diagnostic imaging or enhanced or other phaseenhanced cbct may overcome some of these limitations [ 66 ]  . in addition , the image quality can also be affected by thus , patient setup and support staff motion artifacts ; training are vital to improve image quality [ 67 ]  . radiol med ( 2014 ) 119 : 521532 figs . 
13 ablation planning : mw virtual probe with predicted ablation area ( manufacturer specic ) overlaying to segmented lesion with 5 mm safe margins covered in axial ( 13a ) and progression view visualization ( 13b )  . 
14 intra - procedural cbct : wrong mw probe placement ( 14a , b ) with upper zone of lesion uncovered by the predictable ablation area ( 14b ) ; and subsequent repositioning with all lesion with 5 mm safe margins covered by the predictable ablation area . 
fig. 15 post - procedural cbct , any complication demonstrated 530 conclusion cbct imaging is a new and empowering imaging technology to add to the arsenal of the interventional radiologist / interventional oncologist . 
given the current body of evidence for transarterial chemoembolization and promising future applications such as radioembolization and prostatic arterial embolization , cbct is expected to become a mainstay imaging modality in tumor embolotherapy . 
multimodality navigation , semi - automatic vessel detection , dual - phase cbct , ablation planning , and synergy with ultrasound are early and emerging options , whose added values and potential advantages are in the process of being dened . semi - automatic fusion , certainly , cbct is one of the most interesting and empowering ir / io tools to emerge in recent times . acknowledgments this study supported in part by the intramural research program of the nih and the nih center for interventional oncology ( bjw & naj )  . 
 [ 2 ] found that antero - posterior supine chest radiograph had a sensitivity of only 20.9 % in the detection of pneumothorax if compared with computed tomography ( ct ) , the current gold standard in this setting . thoracic ultrasound ( us ) has proved to be a promising tool [ 3 ] , a bedside technique that can rapidly detect occult pneumothorax , hence avoiding serious potential consequences such as tension pneumothorax , especially in mechanically ventilated patients . 
in patients with major initial , rst - line us examination is generally trauma , radiol med ( 2014 ) 119 : 674680 performed with a fast ( focused assessment with sonography for trauma ) protocol able to depict intraperitoneal collections of free uid that are an indirect sign of solid organ injury and require urgent surgical exploration [ 4 ]  . after the initial fast survey , the us examination may be extended to the thorax to rule out haemothorax and pneumothorax ; when extended to the thorax this examination is known as extended - fast ( e - fast )  . in the trauma setting , the fast examination is usually performed in hypotensive and haemodynamically unstable patients because it helps to determine whether immediate surgery is needed before the patient undergoes a ct evaluation ; in fact , if intra - abdominal bleeding is present , the probability of death increases by about 1 % for every 3 min that elapses before surgical exploration [ 5 ]  . 
in all unstable patients , in addition to the fast acquisition , a right and left longitudinal anterior thoracic view could be quickly obtained to rule out pleural effusion and pneumothorax . 
several authors [ 46 ] have investigated the accuracy of us for the detection of pneumothorax in trauma patients and in those subjected to interventional procedures such as lung biopsy . 
 [ 7 ] who compared the use of antero - posterior chest radiograph with transthoracic us for the diagnosis of pneumothorax , pooled sensitivity and specicity were 88 and 99 % , for us , and 52 and 100 % for chest respectively , radiograph . the aim of this retrospective study was to assess , in our level - 1 trauma centre , the diagnostic accuracy of thoracic us ( e - fast ) in the rapid detection of traumatic pneumothorax in the emergency room in unstable patients with major trauma compared with ct scans and thoracostomy tube placement . the logistic organisation of our level - 1 trauma centre ( emergency room close to the ct room and operating room , staff radiologist present 24 / 24 h and readily available in the emergency room at the arrival of major trauma patients to perform bedside thoracic us ) has allowed us to improve the use of bedside thoracic us in unstable patients . all e - fast examinations were performed by a staff radiologist of our department ( with at least 6 years experience in clinical us and ct ) at the bedside in the emergency room and recorded on videotape . 
because of the need for immobilisation of trauma patients , thoracic us involves scanning only the anterior and lateral wall , not the posterior wall . the presence of pneumothorax is determined on the basis of accepted sonographic criteria ( disappearance of lung sliding and lung pulse , loss of b lines and identication of the lung point )  . 
we dene mild , moderate or massive pneumothorax through the identication ( with lateral scans ) of the lung point on the parasternal line ( mild ) , mid - clavicular line ( moderate ) or anterior / mid / posterior - axillary line ( massive )  . the ct images were interpreted by a staff radiologist without knowledge of the us ndings . 
for every patient , we recorded the time of arrival at the emergency department , the time of e - fast , and the time of multidetector ct ( mdct )  . 
the nal us reports and images were reviewed by all authors in independent reading sessions ( si , vdg , bs , and vm ) and compared with the mdct images for verication ; all images and reports were stored in our ris / pacs system . materials and methods technical equipment study protocol this retrospective case series included 368 consecutive unstable patients admitted to our hospitals emergency department ( a level - 1 trauma centre ) between january 2011 and december 2012 because of a major trauma [ injury severity scale ( iss ) c 15 ]  . 
patients were eligible for inclusion in the study if they had undergone , upon arrival in the emergency department , chest us as part of the e - fast examination before the ct examination , the or ( operating room for damage control ) and before the placement of a thoracostomy tube . 
institutional review board approval was obtained prior to commencement of this study . in our institution e - fast is performed at the bedside , with the patient in the supine position , using a portable us imaging unit ( esaote mylab 75 , italy ) and a 7.5 - mhz linear probe . 
the linear high - frequency probe is suitable for visualisation of the pleural line ; convex probes , working at lower frequencies ( 23.5 mhz ) , are indicated to evaluate the peritoneal cavity , pericardium , and lateral hemithoraces to detect haemothorax . ct is performed with a 16 - channel ct scanning unit ( ct lightspeed 16 , ge medical systems , milwaukee , wis , usa )  . ( visipaque intravenous contrast material 320 mg / ml , ge medical systems , milwaukee , wis , usa ) is always employed for the evaluation of whole - body trauma . 
on the other hand , the visualisation of lung sliding on the anteroinferior chest wall in the supine patient has 100 % negative predictive value in the diagnosis of pneumothorax . another important sign we look for is the presence of b lines , which correspond to thickened interlobular septa in the interstitial syndromes such as pulmonary oedema and lung contusions [ 11 ]  . 
b lines are comet - tail artefacts : they arise from the pleural line , spread up without fading to the edge of the screen and are synchronous with the respiratory movements . 
us detection of the comet - tail artefact allows pneumothorax to be discounted [ 12 ]  . when lung sliding and b lines are absent in the anteroinferior chest area , thus suggesting pneumothorax , the sonologist should check the lateral - inferior chest wall looking for the lung point . 
the lung point conrms the diagnosis of pneumothorax with a specicity of 100 % [ 13 ] and its location provides information about the extent and severity of pneumothorax [ 14 ]  . in some cases , even if lung sliding is absent , sonologist may note the presence of the lung pulse . 
this sign is a vertical movement of the pleural line that is synchronous to the heart beat and it is common in those conditions that cause a consolidated motionless lung ( massive atelectasis and main - stem intubation ) through which the heart movement is transmitted . 
visualisation of lung pulse excludes the diagnosis of pneumothorax because the presence of the air in the pleural space does not allow the cardiac beat to be transmitted [ 8 ]  . 
2. statistical analysis estimates of sensitivity , specicity , positive predictive value , negative predictive value , and overall accuracy were calculated for thoracic us , using mdct as the reference standard for pneumothorax detection in all patients . 
in patients in whom us showed massive pneumothorax and a chest drainage tube was placed before ct , we considered escape or aspiration of intrapleural air at the time of drainage in the emergency room ( as documented in the clinical record ) to be the criterion suggestive of pneumothorax . 
1 thoracic ultrasound : echogenic pleural line ( white arrow ) ; on top , of us ( parasternalline / anterioraxillarline / posterioraxillarline ) scans lines sonographic semeiology in a trauma setting , us evaluation for pneumothorax begins from the antero - inferior chest wall before moving to the lateral chest . 
the pleural line represents either the parietal and visceral layers of the pleura in the normal subject and only the parietal pleura in cases of pneumothorax . the basic sign that indicates normality is the presence of lung sliding , that is , a twinkling movement visible at the pleural line corresponding to the visceral pleura adhering to the parietal pleura . 
when pneumothorax occurs , the air between the two pleural layers causes abolition of the lung line appears motionless . sliding so that according to previous studies [ 810 ] , the abolition of lung sliding does not necessarily conrm pneumothorax because other common conditions occurring in the critically ill the pleural radiol med ( 2014 ) 119 : 674680 fig . 
3 thoracic ultrasound , extended - focused assessment with sonography for trauma ( e - fast ) : mild left pneumothorax in a stabbed pregnant patient ( 24 years old ) ; absent lung sliding and b lines admitted to our emergency department . 
among these , 368 ( 273 men and 95 women ; average age , 25 years ; range , 1668 years ) haemodynamically unstable major trauma patients underwent e - fast upon arrival in the emergency room ( during the primary survey , 010 min after the patients arrival ) for the detection of pneumothorax , haemothorax and haemoperitoneuextended - fast examination , followed by mdct , was performed in all patients . 
these 368 patients , for a total of 736 hemithoraces , represent our study population . the time interval between us and mdct ranged 1075 min , depending on clinical condition on arrival and the need for immediate measures for patient survival . among the 736 lungs in the 368 patients , chest us detected 67 pneumothoraces , while 87 pneumothoraces were either detected by mdct or diagnosed based on the presence of air ush during thoracic decompression in the 678 radiol med ( 2014 ) 119 : 674680 table 1 extended ( to thorax ) values : preliminay results of emergency radiologist parameter thoracic ultrasound sensitivity ( % ) sensitivity during 2011 sensitivity during 2012 specicity ( % ) false positive rate ( % ) false negative rate ( % ) 77 ( 67 / 87 ) 74.5 % ( 38 / 51 ) 80.5 % ( 29 / 36 ) 99.8 ( 668 / 669 ) 0.13 ( 1 / 736 ) 2.7 ( 20 / 736 ) positive predictive value ( % ) 98.5 ( 67 / 68 ) negative predictive value ( % ) 97 ( 668 / 688 ) accuracy ( % ) prevalence ( % ) data pneumothoraces = 87 parentheses 97.2 ( 67 ? 668 / 67 ? 668 ? 1 ? 20 ) 11.8 ( 87 / 736 ) numbers hemithoraces = 736 / emergency room ( 5 / 87 )  . 
us missed 20 / 87 cases of pneumothorax ; 17 of immediately life them were not threatening as they had a thickness less than 5 mm on ct . there was one false positive result in the diagnosis of pneumothorax by us : this was misdiagnosed in the apical region of the left lung in an emphysematous patient . dening the learning curve for thoracic us , all of the false negatives and the single false positive result occurred during the rst ve e - fast examinations performed by each of the staff radiologists ; 13 / 20 false negatives occurred during the rst year ( 2011 ) , and 7 / 20 during the second year ( 2012 ) of our experience . 
therefore , the sensitivity of e - fast for pneumothorax detection was 74.5 % during 2011 but 80.5 % during 2012 ( 668 true negative , negative predictive value 97 % )  . 
in the rst 2 years of experience with chest us in our level - 1 trauma centre ( 2011 - 2012 ) , the overall diagnostic accuracy of this diagnostic tool for the diagnosis of pneumothorax in major trauma in the emergency room was 97.2 % ( table 1 )  . discussion although mdct has been considered the imaging modality of choice in patients with major trauma since the late 1990s , it is often used in conjunction with us for the urgent assessment of patients who have sustained major trauma , particularly in europe . pneumothorax occurs in a large proportion of blunt thoracic traumas [ 1 ] and , even when mild , its detection carries a great clinical relevance because it may quickly progress to cause haemodynamic instability as a consequence of invasive ventilation . 
on the contrary , several studies have demonstrated that lung us has high sensitivity in diagnosing pneumothorax and that the location of the lung point in the supine patient allows evaluation of the extension of pneumothorax . we have investigated , at our level - 1 trauma centre , the use of thoracic us to rapidly detect pneumothorax during the initial resuscitation of unstable patients in the emerthere are limited gency rooin this setting , diagnostic options and even large pneumothoraces could be missed by either clinical examination or antero - posterior chest radiograph ; every us diagnosis was compared with mdct . in fact , in the emergency department of our hospital , critically injured patients are resuscitated by a trauma team guided by a team leader . 
patients suspected of having pneumothorax on physical examination undergo immediate tube or needle thoracostomy without awaiting imaging studies [ 16 ] ; those patients were excluded from the present study . 
patients not requiring immediate invasive intervention undergo a bedside us examination ( e - fast encompassing standard fast and extended thoracic examination ) performed immediately after the physical examination by a staff radiologist ( operational 24 h / day )  . 
although our overall sensitivity was 77 % , if we analyse the 2 years separately , sensitivity was 74 % during 2011 and 80.5 % during 2012 ( a good result for an initial experience with a new method )  . 
in addition , e - fast is performed in only a few minutes , during the primary survey in the emergency room , and this is obviously a important factor to be taken into account compared with other chest us studies , not performed in the same complex conditions . than that of supine chest there was only one false positive result in the diagnosis of pneumothorax by chest us : this was misdiagnosed in the apical region of left lung in an emphysematous patient . subcutaneous in our study , no cases of signicant radiol med ( 2014 ) 119 : 674680 emphysema caused false determination of the presence of pneumothorax . 
in our series , us missed 20 / 87 cases of pneumothorax : 13 / 20 of which were during the rst year ( maybe due to less specic experience ) ; 17 of them were not immediately life - threatening as they had a thickness less than 5 mm on ct ; 3 / 20 moderate pneumothoraces were missed in large habitus patients . 
pneumothorax was bilateral in nine of our cases , but this fact did not affect the sensitivity of the e - fast examination . some limitations to our study should be acknowledged . first , it was a retrospective study , so the us examinations were performed by different operators , with different levels of skill . 
all of the staff radiologists were at their rst experience with thoracic us ( though well - experienced in clinical us ) ; nevertheless the diagnostic performance of e - fast was high . 
a second limitation is that , because this was a retrospective study , we were unable to evaluate the accuracy of us in predicting the extension of pneumothorax in comparison with mdct . 
even if there is no strict correlation between extension on the chest wall and the volume of intrapleural air , the extension of pneumothorax allows semiquantication of the volume , accurately discriminating between large and mild forms . 
during these rst 2 years of experience at our level - 1 trauma centre , we found that examining the chest with us can allow fast recognition ( during the rst 10 min of the golden hour ) of the presence of pneumothorax with very good sensitivity and specicity , either in patients who undergo mdct after us or who need immediate surgical exploration . 
in all cases , the team leader is informed about the presence of pneumothorax and the subsequent need to insert a chest drainage tube if he decides not to insert it immediately in the emergency room . third , with regard to the value of the lung pulse , the sonographic diagnostic algorithm for pneumothorax detection is well codied ; in all reports the lung pulse is the last criterion to be considered because in most cases lung sliding ? b lines ? lung point are sufcient to establish a condent diagnosis . 
in our study , only one patient had left - sided complete absence of lung sliding ? b lines ? lung point but the presence of lung pulse , due to right main - stem intubation with complete left atelectasis . 
we do not have sufcient data to discuss the impact of this sign . conclusions according to our results , bedside thoracic us is a fast and helpful tool with very good accuracy in the workup of unstable major trauma patients , allowing a rapid and highly accurate diagnosis of pneumothorax . 
us does not require sophisticated equipment and it is readily available in the emergency department . in our emergency department , thoracic us is always used during the fast examination in haemodynamically unstable patients , in who time sparing is crucial to allow an immediate treatment . 
we believe that thoracic us , allowing a fast and accurate diagnosis of pneumothorax , is an effective tool during the golden hour , and affects patient survival after a major trauma . 
a protocol limited to the antero - lateral chest wall is sufcient to denitely rule out pneumothorax because , in our series , ct analysis ( with patients in the supine position ) of occult pneumothorax showed that the anterior area was involved in almost all cases . 
the aim of this study was to evaluate correlations between cte ndings with cd activity . materials and methods the cte datasets from 62 retrospectively reviewed for different patients were parameters : bowel wall thickening and hyperenhancement , mesenteric alterations , abdominal free uid and complications related to the disease ( stulas , strictures , abscesses )  . 
activity was assessed using the crohns disease activity index ( cdai ) and some biochemical markers ( creactive protein , erythrocyte sedimentation rate , alpha 2 - globulins , brinogen , platelets , haemoglobin )  . 
in the past , this evaluation was based on clinical indexes , mainly the crohns disease activity index ( cdai ) , integrated by laboratory parameters including c - reactive protein ( crp ) , erythrocyte sedimentation rate ( esr ) , and orosomucoids , and endoscopic ndings [ 2 , 3 ]  . 
indeed , endoscopy can assess only mucosal in a transmural disease like cd , inammation , which , might not be enough [ 4 ]  . recently , a few papers have suggested the role of radiological techniques such as computed tomography enteroclysis or enterography ( cte ) and magnetic resonance enteroclysis or enterography ( mre ) not only in detecting extramural complications but also in the evaluation of disease activity [ 59 ]  . the cte nding of either enteric signs such as mural stratication and hyperenhancement or perienteric signs like mesenteric hypertrophy or engorged vasa recta , the socalled comb sign , has been found to correlate either with radiol med ( 2014 ) 119 : 658666 table 1 demographic , clinical and laboratory features of 62 consecutive crohns disease patients endoscopic or histological inammation or with acutephase proteins , with sometimes discrepant results [ 6 , 7 , 10 12 ]  . 
the importance of recognising the different patterns of disease , especially early in the disease course , may help in tailoring specic therapies , predicting future complicatiming of surgical tions , and possibly offering better interventions [ 13 ]  . 
a good classication of the patient is of primary importance , and sometimes a clinical suspicion is not conrmed on more invasive investigation [ 14 ]  . in this context , cte today appears as a valid technique for the detection of disease complications , and thus a valid alternative for disease staging . 
it seems to be a very powerful technique which can be performed quickly in most radiological departments , even though some concerns have been raised regarding radiation exposure [ 15 , 16 ]  . 
in this respect , correlating cd clinical activity with the cte ndings could help to select patients to be referred for cte , reducing unnecessary radiation exposure [ 9 ]  . however , the relationships between the cte ndings of bowel inammatory activity , and clinical and serum biochemical markers of inammation have not yet been thoroughly investigated . 
the aim of this study was to correlate the cte ndings with cd clinical activity , assessed by cdai , and biochemical activity , assessed by a comprehensive panel of markers including esr , crp , alpha 2 - globulins , brinogen , platelets and haemoglobin . materials and methods patients we retrospectively reviewed all the clinical records of cd patients followed up in our inammatory bowel disease clinic ( a tertiary referral centre for gastroenterological disease ) , between september 2008 and september 2011 . patients were included if they had a denite diagnosis of small bowel cd based on clinical , endoscopic and histological ndings [ 17 ] and had been subjected to cte , ordered as part of routine clinical workup by experienced gastroenterologists for suspicion of strictures or abscesses . exclusion criteria , routinely applied at our department for this type of studies , were age younger than 18 years , renal insufciency ( serum creatinine [ 2 mg / dl ) , pregnancy , intestinal obstruction and documented allergic reaction to iodinated contrast material . 
the study population consisted of 62 patients ( 33 male ; median age 40 years ; range 2066 years ; median duration of disease 52 months ; range 1468 months ) with cd . 
clinical activity was assessed using the cdai . biochemical activity was evaluated with clinical data and biochemical data , obtained within 2 weeks before cte ( esr , crp , complete blood count , serum alpha 2 - globulins and brinogen )  . 
all patients enrolled were 660 radiol med ( 2014 ) 119 : 658666 table 2 computed tomography enterography features of 62 consecutive crohns disease patients cd ( n = 62 ) asked to sign an informed consent form for the processing of their personal data in accordance with the law , and each patient had also read an informative report about tutelage of the personal data , in accordance with the same law . 
statistical analyses were done after removing personal data . cte technique prior to the examination , all patients underwent an intestinal preparation according to the following plan : a diet free of fruit and vegetables for at least 3 days before the examination ; a semiliquid diet and 2 l of water and macrogol 4 , 000 solution ( selg - esse 1000 , promefarm , milan , italy ) the day before the examination . 
to minimise potential artefacts due to peristaltic bowel movement , to obtain homogeneous small bowel distension and to reduce abdominal discomfort , all patients underwent intravenous ( iv ) administration of an anticholinergic compound , 20 mg of n - butyl - joscine bromide ( buscopan , boehringer ingelheim , reggello , florence , italy ) 10 min before the ct scan [ 18 ]  . 
all patients were studied with a 64 - slice mdct scanner ( brilliance 64 , philips medical system , cleveland , ohio , usa ) using the following scan parameters : collimation , 64 9 0.625 mm ; gantry rotation time , 420 ms ; slice thickness , 1.5 mm ; slice increment , 0.7 mm , 140 kv , 250 mas . 
the dose reduction method for all reduction software allowed modulation of the mas parameter during the scan using a maximum of 250 mas with a doselength product mean of 730 mgy * cm with a range of 678923 mgy * cm [ 1921 ]  . 
ct images were analysed in random order and were reviewed by two experienced gastrointestinal radiologists , blinded to all endoscopic and clinical data to ensure objective interpretation of image ndings . 
in each patient we assessed site and number of abnormal bowel segments , bowel wall thickening ( bwt ) , mural hyperenhancement , mural stratication and mesenteric alterations ( table 2 )  . 
mural hyperenhancement was dened as segmental enhancement in all or part ( in the case of mural stratication ) of the small bowel wall , greater than in the adjacent small bowel loops . 
mesenteric alterations included bro - fatty proliferation ( known as creeping fat of the mesentery ) , increased fat density , hypervascularity ( consisting of the presence of the comb sign ) and lymph nodes . 
fibro - fatty proliferation refers to radiol med ( 2014 ) 119 : 658666 loops to noninamed bowel fatty deposition along the mesenteric border of bowel it was dened as a focally segments affected by cd ; increased and inhomogeneous uid attenuation in the mesenteric fat [ ? 20 / ? 60 hounseld units ( hu ) ] , compared with the appearance of subcutaneous fat or perien [ 23 ]  . teric fat adjacent increased fat density refers to uid density in the fat surrounding thickened or abnormally enhancing bowel , resulting from inammatory inltration of the perienteric adipose tissue . 
the comb sign refers to hypervascularity of the involved mesentery because of the presence of dilated and tortuous vasa recta that penetrate the bowel wall perpendicular to the bowel lumen , mimicking the appearance of a comb [ 24 ]  . 
each of these mesenteric effects were graded along a three - point scale as not present ( 0 ) , denitely present to a mild - moderate ( 1 ) or a severe ( 2 ) degree . finally , the presence of abdominal free uid and other complications related to the disease ( stulas , strictures , abscesses ) were assessed . 
all the cte features are summarised on table 2 . statistical analysis all continuous variables were described as median and range , while categorical variables were expressed as frequency and percentage . 
the patients were grouped according to the clinical activity of disease : inactive ( cdai \150 ) ; mild activity ( cdai 150220 ) ; moderate activity ( cdai 220450 )  . 
21 % had nonstricturing nonpenetrating disease , 38 % stricturing , 9 % penetrating , 16 % both stricturing and penetrating disease and ten patients ( 16 % ) had perianal involvement . 
computed tomography enterography shows increased fat density with the comb sign ( arrowhead ) and a bowel stricture ( arrow ) in the axial contrast - enhanced images and in an oblique coronal contrastenhanced reconstruction image fig . 
univariate analysis showed that the presence of lymph nodes ( p = 0.009 ) and comb sign ( p = 0.05 ) were signicantly associated with a high platelet count ( table 4 )  . 
cte provides both anatomical and practical advantages : depiction of the entire bowel wall , multiplanar imaging and no obscuration of small bowel loops due to superimposition , and better evaluation of both luminal and extramural manifestations [ 29 ] ; in addition , it is not an technique [ 30 ]  . 
4 male patient , 52 - year old , with moderate crohns disease activity ( cdai 195 )  . axial and coronal contrastenhanced images of computed tomography enterography show an ileocolic stula ( arrow ) and mural hyperenhancement of the small bowel wall ( arrowhead ) mesenteric adipocytes which are supposed to play a major role in the cd inammatory cascade [ 32 ]  . 
compared to the increased fat density is an bro - fatty proliferation , earlier change and is also more specic than bowel wall thickening , which could give false - positive ndings in the presence of underdistended bowel loops . conicting data have appeared in the medical literature concerning the relationship between radiological signs of inammation ( approached by either cte or mre ) and disease activity in cd . 
 [ 33 ] reported a signicant correlation between mr signs of intestinal inammation ( oedema in the perienteric fat , wall thickening , contrast enhancement ) with a biological activity score which included white blood cells , orosomucoid levels and crp in 20 cd patients . 
 [ 8 ] evaluated both mural signs ( parietal thickness , target sign or alternating rings of low and high density in the bowel wall ) and extraenteric inammation . there was a positive correlation between the target sign and bro - fatty proliferation with the cdai and among wall thickness , the comb sign and perienteric stranding with crp . 
when we explored the relationship between the biomarkers and the cte ndings , we found a correlation of alpha 2 - globulins and platelet count with vascular extraluminal ndings ( comb sign )  . 
alpha 2 - globulins are a fraction of serum proteins containing orosomucoids , in particular alpha 1 - acid glycoprotein , which , though less commonly used in clinical practice , had been shown to have a better correlation with cdai than crp [ 38 ]  . 
axial images of computed tomography enterography show an ileocolic stula ( arrow ) and an abscess ( arrowhead ) perspective , cte has the advantage of being less invasive because it does not require intubation or uncomfortable palpation , it only requires getting on and off the table once , and it is generally faster . 
cte adds useful information to the clinical assessment providing denition of extraluminal involvement characterisation . detection ensuring stricture our study shows that a cte nding of increased fat density has a good correlation with the cdai score . although the cdai has been criticised because of its subjectivity and interobserver variability , it remains the most widely used index for evaluating outcome in clinical trials on crohns disease [ 31 ]  . 
there are some discrepancies between our results and those of previous studies , which could be explained by differences in sample size , patient recruitment and referral and heterogeneity of technical approach . 
our case series has the advantage of being totally homogenous since all patients are referred to the radiology department by a single gastroenterology team dedicated to the care of ibd patients and the technical approach was uniforour study conrms the role of cte as a useful tool not only in the diagnostic workup of suspected cd but also in the evaluation of disease activity , which we believe is an integrated process which involves clinical symptoms and signs , serum acute - phase reactants and endoscopic ndings . to our knowledge , few studies in the literature have correlated cdai , biochemical activity and cte signs with attention focused also on nodal involvement and abdominal free uid . 
in conclusion , cte might help to better address and monitor response to therapy , for example , to dene the so - called window of opportunity for biologics , and this aspect deserves further studies since it has not been evaluated in the present study or in the literature . 
6 computed tomography enterography : axial contrast - enhanced images in a 39 - year - old male with active crohns disease ( cdai 256 ) demonstrating increased fat density and comb sign ( arrowhead ) more advanced and extensive cd , could suggest the presence of prolonged active disease . 
the image characteristics included the number of tumours , tumour location and size , tumour margins , the presence of calcication / necrosis / cavity , the presence of perivascular location , the presence of pleural lesions , tumour homogeneity at contrastenhanced ct , tumour enhancement relative to the adjacent muscle and the presence of extrapulmonary lesions . results multiple nodules / masses with irregular margin were shown in all cases , and reticulonodular opacities and ground - glass opacities were found in one case . 
zhou department of oncology , shanghai medical college , fudan university , shanghai 200032 , china nodules / masses were mostly ( 93 % , 166 / 178 ) located in the subpleural region ( \2 cm from the pleura )  . 
on contrast - enhanced ct , ehe showed a mildly heterogeneous hyperdense appearance . conclusions with predilection for subpleural and perivascular typical pulmonary ehe appears as multiple irregular nodules with punctate calcication and pleural indentation . location , keywords lung ( cid : 2 ) epithelioid hemangioendothelioma ( cid : 2 ) computed tomography introduction pulmonary epithelioid haemangioendothelioma ( ehe ) is a rare endothelium - originating tumour of borderline malignancy [ 1 , 2 ]  . 
ehe was rst described as an intravascular bronchioloalveolar tumour ( ivbat ) by dail and liebow in 1975 ; however , the angiogenic nature of ivbat was revealed by later studies showing that ivbat and epithelioid haemangioendothelioma are different manifestation of the same disease [ 1 , 36 ]  . several studies have reported the ct features of pulmonary ehe [ 25 , 713 ] , but most of them are case reports , so the total reported sample size is small and the ct features of pulmonary ehe , therefore , need further study . 
the aim of this retrospective study was to analyse the clinical and ct features of six cases of pulmonary ehe . radiol med ( 2014 ) 119 : 705713 patients materials and methods ct imaging the ct images of six patients with pathologically proved pulmonary ehe were retrospectively reviewed . 
institutional review board permission was obtained to review patient medical records , and patient informed consent was not required . the clinical features of the six cases of pulmonary ehes are shown in table 1 . 
the serum levels of tumour markers ( a - 1 - fetoprotein , carcinoembryonic antigen , ca19 - 9 , ca125 ) were normal . four patients underwent surgical pulmonary lobectomy and two patients underwent ct - guided transthoracic biopsy using an 18 - gauge needle . 
one patient was lost to follow - up immediately after biopsy ; the remaining ve patients were still alive at the time of writing this paper ( 361 , 197 post - operative days )  . in this study noncontrast ct images were available for three patients and contrast - enhanced ct images for three patients . the ct examinations were performed with an mdct scaner ( somaton sensation 40 or 64 , siemens , germany )  . the ct technical parameters were as follows : tube voltage , 120 kvp ; rotation time , 0.5 s ; pitch , 1.4 ( 60 9 0.6 or 40 9 0.6 ) ; reconstruction slice thickness , 5 mm ( kernel , b70f very sharp and b31f medium smooth ? ) ; tube current was adjusted for each subject by using the care dose 4d technique . 
a bolus contrast material ( 300 mg i / ml of iopamidol at 1.5 ml / kg body weight ) was injected intravenously at a rate of 2 ml / s using an automated injector . nonionic a radiologist evaluated the tumours ct features , including ( 1 ) the number of tumours , ( 2 ) tumour location , ( 3 ) tumour size ( maximum axial diameter ) , ( 4 ) tumour margin , ( 5 ) the presence of calcication , ( 6 ) the presence of necrosis and cavity , ( 7 ) perivascular location , ( 8 ) tumour homogeneity on contrast - enhanced ct and tumour enhancement relative to the adjacent muscle ( hypo - , iso - , or hyper attenuating ) , ( 9 ) the presence of pleural lesions table 1 clinical and computed tomography ( ct ) features of pulmonary epithelioid haemangioendothelioma female chest pain multiple subpleural irregular female none multiple subpleural irregular mild female chest pain multiple subpleural irregular female cough multiple male none multiple subpleural subpleural irregular irregular non - contrast ct heterogeneous no image no image homogeneous no image heterogeneous enhanced ct no image heterogeneous and no image heterogeneous and no image heterogeneous and hyperdense hyperdense hyperdense right frontal bone lesion right thyroid mass multiple bone retroperitoneal mediastinal left inguinal mass lesions mass mass female none multiple subpleural irregular age ( years ) symptom multiple / single location maximum axial diameter ( cm ) margin calcication necrosis cavity perivascular location pleural indentation pleural thickening pleural effusion extrapulmonary lesion radiol med ( 2014 ) 119 : 705713 fig . 
chest computed tomography ( ct ) images show multiple irregular subpleural nodules / masses with the pleural indentation sign ( b , c , e ) , perivascular location ( a , c , e ) , as well as punctate calcication in partial nodules / masses ( d , f ) and pleural thickening ( c , d )  . 
the tumour cells were positive for cd34 , factor - viii - related antigen ( i , j ) ( pleural indentation , pleural thickening , pleural effusion ) and ( 10 ) the presence of extrapulmonary lesions . results the ct ndings of the six ehes are described in table 1 . multiple nodules / masses were shown in all cases and reticulonodular opacities and ground - glass opacities were each found in one case . 
chest ct images show multiple irregular subpleural nodules / masses ( ad ) with pleural indentation ( a , c ) , perivascular location ( a , c ) , as well as punctate calcication in partial nodules / masses ( b , d ) , pleuralthickening ( c , d ) and pleural effusion ( ad )  . 
furthermore , the size of the largest nodules / masses was 5.2 cm in diameter ( b ) and the pulmonary epithelioid haemangioendothelioma showed a mild heterogeneous hyperdense appearance relative to muscle on contrast - enhanced ct ( b ) 709 radiol med ( 2014 ) 119 : 705713 fig . 
chest ct images show multiple irregular subpleural nodules / masses ( ad ) , with pleural indentation sign ( a , c ) , perivascular location ( a , c ) , as well as punctate calcication in partial nodules / masses ( d ) and pleural thickening ( c )  . furthermore , the size of the largest nodules / masses was 3.0 cm in diameter ( a , b ) fig . 
however , in our study the average age was 53 years , in contrast to the ndings of previous reports in which most patients were younger than 40 years [ 1 , 6 , 18 ]  . our results showed that all cases had multiple irregular nodules / masses . 
the average number of nodules / masses in each case was 30 ( range 1170 ) , and most nodules / masses ( 90 % , 160 / 178 ) were \1 cm in diameter . 
these ndings are similar to those reported in past studies which report that the most characteristic feature of ehe on ct is the presence of multiple perivascular nodules with welland ill - dened margins in both lungs and nodules up to 2 cm in size , although most are \1 cm in diameter [ 4 , 5 , 8 , 9 , 11 , 12 , 19 ]  . 
chest ct images show ground - glass opacities ( a ) and multiple irregular subpleural nodules / masses ( af ) with pleural indentation sign ( a , c , e ) , perivascular location ( a , c ) and pleural thickening ( c , e )  . 
the size of the largest nodules / masses was 3.5 cm in diameter bronchioles and lymphatic vessels , especially small arteries , lymphatics and venules of 1 mm or less in diameter within or adjacent to the nodules ; less frequently vessels of several millimetres in diameter may be involved [ 1 , 4 , 9 ]  . to our knowledge , pulmonary hamartoma , leiomyoma and metastasis usually show nodule / mass with smooth margins ; wegener granulomatosis usually appears as multiple nodules / masses with cavity ( size from a few millimetres to over 10 cm ) and the number of lesions are often \10 [ 20 23 ] , which is not similar to that of ehe . in our study , pulmonary ehe showed predominant subpleural location , which was conrmed by gross pathology revealing an increased number of lesions in the immediate subpleural area [ 5 , 24 ]  . 
this feature of ehe was different from pneumoconiosis in which the nodules tend to be most numerous in the upper lobe , from metastatic pulmonary nodules which tend to be distributed in the basal portion of the lung , from wegener granulomatosis which has peribronchovascular , subpleural and angiocentric nodules / masses [ 20 , 23 , 25 , 26 ]  . in our study , the pulmonary ehes showed perivascular reecting the gross pathology nding that location , tumours are usually found in relation to small and mediumsized vessels and bronchi . 
additionally light microscopy shows that tumour vascularity is inapparent , only a rare small capillary can be identied in the tumour itself , and the tumour cells are near vessels and adjacent to alveolar septa , which suggest that ehe has metabolic requirements and its nutrient and oxygen supply is by diffusion [ 1 ]  . 
this ct feature is usually not found in pneumoconiosis , metastatic pulmonary nodule , wegener granulomatosis , pulmonary hamartoma , or benign metastasising leiomyoma [ 2023 , 25 , 26 ]  . in our study , 48 % ( 86 / 178 ) of nodules / masses showed punctate calcication in four of six cases , a feature also reported by other studies [ 7 , 9 , 11 , 13 , 24 ]  . 
nodules of pulmonary ehe show an acellular sclerotic central area and the acellular pale pink centres undergo coagulative necrosis , then dystrophic calcication , chondrication , ossication , and rarely positive congo red staining radiol med ( 2014 ) 119 : 705713 fig . 
chest ct images show multiple irregular subpleural nodules / masses ( af ) with pleural indentation sign ( a , e , f ) , perivascular location ( c , e )  . however , the size of the largest nodules / masses was too small ( 1.1 cm in diameter ) to analyse the enhancement of lesion ( a , b ) reactions can be seen in these necrotic central cores [ 1 ]  . punctate calcication can occur in a metastatic sarcoma , but other ct features including smooth margin , tendency to distribute in the basal portion of the lung may be used for the differential diagnosis [ 26 ]  . in our study , pleural indentation was found in all cases . this corresponds histologically to thickened , brotic connective tissue septations with indrawing of the visceral pleura and reects desmoplastic reaction [ 27 ]  . 
alternatively , the tissue processing shrinkage artefact may be attributed to the fact that the tumour extends into each alveolus from just one focus and a moderate amount of brosis [ 1 ]  . 
usually pneumoconiosis , metastasis , wegener granulomatosis , pulmonary hamartoma and leiomyoma do not show pleural indentation [ 2023 , 25 , 26 ] , unlike pulmonary ehe . in our study , ve cases showed pleural thickening , and two cases showed pleural effusion . 
the ct ndings of pleural ehe are nonspecic and usually demonstrate a pleural effusion with localised or diffuse nodular pleural thickening [ 2 ] , which typically affects older men , induces chest pain and dyspnoea and is easily mistaken for pleural infections or malignant mesothelioma [ 3 , 4 , 28 ]  . 
patients with brinous pleuritis and extrapleural proliferation of tumour cells or spindle tumour cells generally have a worse prognosis [ 18 , 29 ]  . due to the small size and non availability of contrastenhanced ct images , the enhancement of ehe could not be analysed . 
two cases in our study showed that ehe had a mild - heterogeneous hyperdense appearance on contrastenhanced ct , similar to that described in a case report 712 radiol med ( 2014 ) 119 : 705713 revealing a left hilar mass mimicking central lung cancer [ 30 ]  . furthermore , some studies have reported atypical ct manifestations of pulmonary ehe which included diffuse reticulonodular opacities and ground - glass opacities [ 4 , 10 ] ; however , these ct features were each found in one case in our study and are attributed to interstitial inltration presenting focally in interlobular septa or pleura and around bronchopulmonary rays [ 1 ]  . additionally , the extremely long and indolent growth of pulmonary ehe needs to be emphasised . 
some authors reported that pulmonary ehe showed little or no growth on serial radiographs [ 4 , 5 , 31 ] and that even partial spontaneous regression [ 32 ] and life expectancy may be up to 30 years [ 4 , 5 , 31 ]  . some authors have reported the antitumour effects of interferon , chemotherapy using carboplatin and etoposide [ 14 , 33 ]  . 
a monoclonal antibody against vascular endothelial growth factor ( vegf ) ( bevacizumab ) together with chemotherapy was recently used for preventing tumour spread in the early period as these tumours express high levels of vegf [ 34 ]  . 
additionally , the uorodeoxyglucose positron emission tomography ( fdg - pet ) scan can provide useful information concerning the malignant potential of each nodule more than 6 mm in diameter . 
fdg - pet may be useful to select the more clinically malignant nodules , which worsen the prognosis [ 31 , 35 ]  . sometimes ehe involves extrapulmonary organs and may be multifocal ; the liver and bone are the most frequently affected organs [ 6 ]  . 
hepatic ehe typically appears as multifocal conuent peripheral nodules with adjacent capsular retraction and a low - signal halo ; additionally hepatic ehe frequently occurs in older children [ 36 , 37 ]  . bone ehe typically shows a lytic lesion without matrix mineralisation , even though osseous expansile remodelling may be seen [ 19 ]  . the present study was limited by a small sample size because pulmonary ehe is rarely encountered . 
fundamental signs ( extraluminal air , solution of continuity ) and secondary signs ( thickening of the mesentery , free or perilesional uid , wall thickening ) were considered . results ct alterations were found in 31 / 35 ( 88.6 % ) patients : extraluminal air ( 30 / 35 , 85.7 % ) , solution of continuity ( 11 / 35 , 31.4 % ) , intra - abdominal uid ( 27 / 35 , 77.1 % ) , thickening of the mesentery ( 20 / 35 , 57.1 % ) , and wall thickening ( 14 / 35 , 40 % )  . 
in 25 / 35 cases ( 71.4 % ) pneumoperitoneum was detected , associated with secondary signs ( 23 / 25 , 82 % ) , conrmed as free perforations at surgery . 
in 5 / 35 patients ( 14.2 % ) , peri - intestinal air bubbles and secondary signs were evident , while in 1 / 35 cases ( 2.8 % ) only secondary signs were seen , namely covered perforations . 
angelelli dim - interdisciplinary department of medicine , section of diagnostic imaging , aldo moro university of bari medical school , piazza giulio cesare 11 , 70124 bari , italy e - mail : marirosacristallo@alice.it conclusion ct investigation is essential in the recognition of a small - bowel perforation and in the denition of its nature . keywords small bowel ( cid : 2 ) free and covered perforation ( cid : 2 ) introduction jejunalileal perforation represents a pathological condition which can be caused by trauma ( blunt or penetrating trauma , foreign body ingestion , iatrogenic injury ) or by nontraumatic inammatory ( enteritis , crohns disease ) , ischaemic ( mesenteric infarction , volvulus , intussusception , vasculitis ) , and neoplastic conditions [ 1 , 2 ]  . 
it is an uncommon condition , as the small bowel is involved in only 515 % of all closed abdominal traumas and the nontraumatic perforations are even more uncommon with a reported incidence of one case / year / 350 , 000 inhabitants [ 3 , 4 ]  . from a clinical point of view , a jejunalileal perforation is characterised by the appearance of an acute abdomen caused by the peritoneal inammatory reaction secondary to the leakage of intestinal content , although in some cases the clinical features may be nonspecic , for example in patients with covered perforation , those receiving treatment with steroid drugs or immunocompromised patients [ 5 , 6 ]  . in the diagnostic management of these patients , a plain radiography in different positions is usually performed as a rst imaging assessment . 
this tool could be decisive for diagnosis , but it is not very sensitive since it is only rarely able to demonstrate the occurrence of pneumoperitoneum because of the small amount of air in the small bowel as compared to other bowel segments . 
it does not allow identication of a possible covered perforation , which 652 radiol med ( 2014 ) 119 : 651657 arises in the small bowel due to the rapid tendency of the omentum , mesentery and adjacent loops to cover the perforated bowel segment [ 7 , 8 ]  . 
in the event of a clinically suspected bowel perforation , not detected on abdominal x - ray , the contribution of computed tomography ( ct ) is crucial ; it has 92 % sensitivity in detecting perforations in the whole gastrointestinal [ 9 , 10 ]  . 
despite the importance of the issue , only few studies regarding jejunal ileal perforations have been reported in the literature , probably due to the limited incidence of this nding . tract the purpose of this study was to evaluate the ct semeiotics of small - bowel free and covered perforations and to evaluate the potential of ct in recognising their aetiology . materials and methods we retrospectively evaluated 35 patients , 19 females ( 54 % ) and 16 males ( 46 % ) , aged between 21 and 90 years , studied between april 2006 and october 2012 , suffering from surgically proven small - bowel perforations . in 10 cases ( 28.5 % ) , perforation was located at the jejunum while in the remaining 25 ( 71.5 % ) at the ileuthe perforation was due to various causes : loop strangulation ( n = 11 ) , hernia ( n = 3 ) , adhesions ( n = 5 ) , volvulus ( n = 3 ) ; traumatic injuries ( n = 6 ) , including three blunt trauma , two knife lesions and rearm lesion ; foreign bodies ( n = 5 ) , represented by three toothpicks , one phytobezoar , and one shbone ; crohns disease ( n = 4 ) ; metastases ( n = 4 ) ; peritoneal carcinomatosis intestinal tuberculosis ( n = 1 ) ; small - bowel diverticulitis ( n = 1 )  . ( n = 3 ) ; the list of patients was obtained from the computerised registry of the operating room using the keyword jejunal ileal perforation . 
the images were acquired during the arterial phase , with a scan delay of 3040 s from contrast medium injection , and in the venous phase after 6070 s . the ct images were transferred to , and analysed on , a workstation ( hp xw8600 ) equipped with dedicated image reconstruction software ( vitrea fx 2.1 , vital images , minneapolis , minnesota , usa )  . 
the duration of the postprocessing was approximately 15 mthe axial and reconstructed images were assessed in consensus by two experienced radiologists in the eld of gastrointestinal imaging , who were aware of the nal diagnosis at the time of the study . 
the images were evaluated with the usual ct windows for the study of the abdomen and with very large windows ( [ 800 / hu ) similar to those used for the study of the lung parenchyma , to facilitate the identication of extraluminal air . to determine the presence of bowel perforation , the following ct parameters were considered : fundamental signs : extraluminal air , assessing quantity ( air bubbles , free air ) [ 1113 ] and distribution ( supramesocolic , infra - mesocolic compartment , pelvis , retroperitoneum ) [ 14 , 15 ] ; solution of continuity of the wall [ 16 ]  . secondary signs : periluminal mesentery streaking ( increase in the density of the mesentery from - 100 to - 160 hu to - 40 to - 60 hu ) [ 8 , 1720 ] ; free uid or perilesional uid [ 8 ] ; wall thickening . 
they were associated with extraluminal air in 28 / 35 patients ( 80 % ) , with free perforation in 23 ( 82 % ) and with covered perforation in 5 ( 18 % ) ; out of 35 cases , they were isolated in 1 ( 2.8% ) , which surgery conrmed to be a covered perforation by a tip of toothpick . 
in four patients it was possible to obtain only partial information on the nature of the injuries ; these were cases of bowel perforation secondary to metastatic disease . in these patients , a neoplastic disease could be suspected by the presence of circumscribed irregular wall thickening , but a differential diagnosis with a primary lesion was not possible . 
it was represented by pneumoperitoneum in 25 / 35 cases ( 71.4 % ) , always easy to identify , with a maximum thickness of about 1 cm and associated , in 18 / 35 cases ( 51.4 % ) , with peri - intestinal air bubbles . 
with regard to the distribution of the pneumoperitoneum , among the 25 / 35 patients ( 71.4 % ) with pneumoperitoneum , the air small - bowel perforations are uncommon and may represent a complication of a number of inammatory ( crohns disease , small - bowel diverticulitis , tuberculosis ) , neoplastic ( primary or secondary ) , ischaemic ( mesenteric infarction , venous obstruction by loop strangulation , vasculitis ) , and systemic ( paralytic ileus ) diseases or can be caused by trauma ( blunt trauma , knife , rearm , iatrogenic ) or foreign bodies [ 6 , 9 , 2325 ]  . 
regardless of the nature of perforations , they can be divided into covered or free perforations ; this differentiation is crucial in the therapeutic approach . free perforations require immediate surgical treatment , which can be delayed in covered forms [ 8 , 26 ]  . in the diagnostic management of these patients , who typically show clinical signs of an acute abdomen , plain radiography of the abdomen in different positions remains 654 radiol med ( 2014 ) 119 : 651657 fig . 
a transverse ct scan ; b coronal reconstruction ; c sagittal reconstruction . presence of numerous diverticula of the small bowel ( a , b arrows ) ; only extraluminal peri - intestinal air ( arrowheads ) and an increase in the mesenteric density are associated ( b , c empty arrows ) fig . 
the shbone is well evident ( curved arrows ) ; small peri - intestinal air bubbles ( arrowheads ) and an increase in the mesenteric density are associated ( empty arrows ) the rst feasible diagnostic tool . 
in the diagnosis of gastrointestinal perforation , as pointed out in the literature , this investigation has important limitations such as a low sensitivity ( 5070 % ) in the detection of small amounts of extraluminal air , and generally the impossibility of dening the location and nature of the perforation [ 13 , 27 , 28 ]  . ct is today the gold standard in the study of patients with acute abdomen and provides a crucial contribution in radiol med ( 2014 ) 119 : 651657 fig . 
in these cases , appropriate indications for the use of ct are : negative x - ray examination with clinical signs strongly indicative of bowel perforation ; patients difcult to explore with plain radiography due to serious clinical condition ; the need to obtain additional information after a poorly diagnostic x - ray examination [ 22 ]  . potential of ct for suspected intestinal perforation has been widely discussed and an overall sensitivity of 92 % has been reported in the detection of this condition [ 11 ] and values of 86 and 37 % , respectively , in dening the site and nature [ 15 , 32 ]  . in the literature , in our study , ct signs of small - bowel perforation were detected overall in 88.6 % ( 31 / 35 ) of patients , whereas the ct examination was negative in only 11.4 % ( 4 / 35 )  . 
in particular , as shown by the results , ct can show pathological changes in 100 % ( 25 / 25 ) of free perforations and in 60 % ( 6 / 10 ) of the covered forms . with regard to ct semeiotics , extraluminal air was detected in 85.7 % of cases ( 30 / 35 ) , secondary alterations in 82.8 % ( 29 / 35 ) and a solution of continuity of the bowel wall in 31.4 % ( 11 / 35 )  . 
extraluminal air was recognised with greater frequency than reported by some authors , who state that it is possible to recognise a pneumoperitoneum in only 33.3 % of cases of small - bowel perforation [ 33 ]  . 
this discrepancy is probably due to technological developments , which now allow for a slice thickness less than 1 mm , able to highlight even small extraluminal air bubbles , as demonstrated in the literature [ 16 ]  . the analysis of the location of the extraluminal air reveals interesting data , namely the fact that pneumoperitoneum associated or not with peri - intestinal air bubbles is always indicative of free perforation , whereas peri - intestinal air alone is always correlated to covered perforations . therefore , these elements allow the differentiation between free and covered perforations and may contribute to the choice of surgical timing . in the case of a free perforation , the distribution of air within the peritoneal compartments is similar to what has already been reported by other authors . 
 [ 15 ] have pointed out that in the lower perforations ( ileum / colon ) , the air is localised in the supraand inframesocolic compartments , while the predominant presence of air in the supramesocolic compartment orientates towards a gastroduodenal perforation . 
in the examined series , which assessed 25 patients affected by free perforation , the air was localised exclusively in the infra - mesocolic compartment in 16 % of cases ( 4 / 25 ) , in the supraand infra - mesocolic compartments in 64 % ( 16 / 25 ) , in the supraand infra - mesocolic compartments and in the pelvis in 20 % ( 5 / 25 ) of patients . 
in no case was air detected exclusively in the supramesocolic space , in the pelvis or in the retroperitoneutherefore , the recognition of air only in such districts can be considered a clue for the exclusion of small - bowel perforation . the recognition of extraluminal air is not only indicative of bowel perforation , being also observed in physiological conditions , more frequently in women ( sport , sex , catamenial ) , pathological conditions ( extension of a pneumo ( paracentesis , thorax ) or after biopsies , peritoneal dialysis , laparoscopy ) and surgery [ 34 ]  . the differential diagnosis of extraluminal air from gastrointestinal perforation or from other causes is usually readily achieved by considering the patients clinical , medical history and the alterations associated with peritoneal and intestinal walls . iatrogenic procedures as reported in the results , the identication of a solution of continuity of the bowel wall is useful to dene the presence and especially the site of perforation , and this was achieved in 31.4 % of the examined patients ( 11 / 35 ) ; it was 656 radiol med ( 2014 ) 119 : 651657 never the only sign . 
in fact , in 90 % of cases ( 10 / 11 ) it was associated with extraluminal air and secondary signs and in 10 % ( 1 / 11 ) of cases with extraluminal air alone . 
this alteration was never highlighted by other authors [ 33 ] in the case of small - bowel perforation , while it was more frequently observed ( 40 % ) in perforations affecting the remaining gastrointestinal tract [ 15 , 16 , 35 ]  . among the signs seen in the examined series , secondary alterations were recognisable in 82 . 
% of patients ( 29 / 35 ) and were associated with extraluminal air in 51.4 % ( 18 / 35 ) , and extraluminal air and solution of continuity in 28.5 % ( 10 / 35 ) ; only in 2.9 % ( 1 / 35 ) did they represent the only nding . 
the analysis of the results suggests that small foreign bodies cannot be identied , and generally correlate with covered perforations ; all other situations can be fully diagnosed if in the presence of characteristic changes , and sometimes only assumed among different diseases with common ct features . this research conrms the potential of ct in the diagnosis of small - bowel perforations . 
however , it has important is a retrospective analysis performed by radiologists who were aware of the surgical ndings at the time of the study . limitations in that conclusion ct remains the gold standard in the study of small - bowel perforations , as it is able to highlight pathological changes in 100 % of free perforations and in 60 % of covered forms . 
the presence of extraluminal air ( free or periintestinal air ) and solutions of continuity in the bowel wall , generally associated with minor alterations ( intra - abdominal uid , mesenteric thickening , bowel wall thickening ) represents essential ndings for diagnosis . 
the latter ones are involved in development of craniofacial and skull base deformities . materials and methods we retrospectively reviewed 27 children with complex synostosis ( n = 21 ) and anterior synostotic plagiocephaly ( n = 6 )  . 
high - resolution ct images with bone denition algorithm and tridimensional volume rendering reconstructions were assessed . results sutures in 27 children we found different involved in the synostotic process , including both major and minor skull suture synostosis , and synostosis of synchondroses . 
superior orbital rim deformity , nasal root deviation , anterior endocranial axis deviation ( ethmoidal axis ) are found in children with coronal arch synostosis , while reduced size of the posterior fossa and chiari 1 malformation are noted in children with lambdoid arch synostosis . conclusions high - resolution ct allows an accurate identication of both major and minor skull base suture synostosis and it represents the gold standard for the r . 
gemelli 8 , 00168 rome , italy diagnosis of craniostenosis and for planning the proper surgical approach . keywords craniosynostosis ( cid : 2 ) coronal arch ( cid : 2 ) minor sutures introduction the deformity of the skull in the newborn represents a common and signicant diagnostic problem , particularly in distinguishing positional plagiocephaly from deformity caused by craniostenosis [ 1 , 2 ]  . positional plagiocephaly is an alteration of the morphology of the skull in the absence of premature cranial suture synostosis caused by a dynamic distortion of the skull secondary to preand / or postnatal external forces without malformative alterations [ 3 , 4 ]  . 
craniostenosis , by contrast , is a pathological condition of skull deformity caused by premature sutural closure of the vault and / or skull base , occasionally associated with early closure of cranial fontanels [ 5 , 6 ]  . 
sutures are brous bands of tissue that connect the bones of the skull , allowing their growth from the inside to the outside during development , whereas the fontanels are membranes covering parts of the skull where the bones have not yet settled , allowing the skull bones to adapt to brain expansion ( functional mobility during the birth and during the rst year of life ) [ 7 ]  . normally , sutures and fontanels ossify ( that is , they form synostoses ) at different times after the birth . 
specically the metopic suture normally ossies between 9 and 11 months , and the other sutures ( sagittal , coronal and lambdoid ) close completely at approximately 3040 years of age . 
the anterior fontanel ( bregmatic fontanel ) typically ossies within the second year of life , the posterior fontanel ( lambdoid fontanel ) radiol med ( 2014 ) 119 : 694704 fig . 
1 sagittal arch a this is composed of the metopic suture ( white arrow in b1 patent suture ; black arrow in b2 synostotic suture ) , the sagittal suture ( white arrows in c1 patent suture ; black arrows in c2 synostotic suture ) and the ethmoido - frontal ( minor sutures ) ( white arrows in d1 patent sutures ; black arrows in d2 ; synostotic sutures )  . 
2 coronal arch a this begins at the bregma and is composed of coronal sutures ( b white arrow patent suture on the right side ; black arrow synostotic suture on the left side ) , each divided into an anterior branch composed of the fronto - sphenoidal ( minor sutures ) ( c white arrow patent suture on the left side ; black arrow synostotic suture on the right side ) and ethmoido - sphenoidal ( minor sutures ) synchondrosis ( black arrow synostotic suture in d1 ; white arrow patent suture in d2 ) , and a posterior branch composed of the spheno - squamous ( minor sutures ) ( e white arrow patent suture on the right side ; black arrow synostotic suture on the left side ) , the spheno - parietal ( minor sutures ) ( f white arrow patent suture on the left side ; black arrow synostotic suture on the right side ) and spheno - petrosal ( minor sutures ) synchondrosis ( g white arrow patent suture on the right side ; black arrow synostotic suture on the left side ) is no longer palpable after the rst 3 months of life , the sphenoid fontanels ( pteric fontanels ) close after the rst year of life , and the mastoid fontanels ( asteric fontanels ) ossify more often within the rst 18 month of life [ 7 , 8 ]  . in craniostenosis , the welding of the skull sutures , which is sometimes associated with premature closure of the fontanels , occurs in the prenatal period , perinatal epoch , or during early infancy [ 1 , 5 ]  . 
3 lambdoid arch a this starts at lambda and it is composed of the lambdoid sutures ( b white arrow patent suture on the left side ; black arrow : synostotic suture and a copper - beaten appearance of the skull on the right side ) , the spheno - occipital ( minor sutures ) synchondrosis ( c white arrow ) and occipito - mastoid ( minor sutures ) ( d white arrow patent suture on the right side ; black arrow synostotic suture on the left side ) pathological and premature sutural closure , progresses over time ; therefore , timely diagnosis and immediate treatment are required . 
the incidence of craniostenosis is 1 in 2 , 1002 , 500 newborns without any ethnic predilection [ 8 ]  . traditionally craniostenosis has been classied according to the sutures involved , the deformity of the head , the presence or absence of a skull - facial syndrome and the degree of progression of the anomaly [ 5 , 8 , 9 ]  . 
therefore , craniostenosis can be classied as simple ( monosutural ) or complex ( multisutural ) or as a primary defect of ossication or secondary to systemic disorders ( endocrine , haematological , metabolic alterations ) or iatrogenic causes . most cases of craniostenosis ( 84 % ) are found as an isolated defect ( non - syndromic craniostenosis ) , although 15 % of cases are part of a syndromic condition ( syndromic craniostenosis ) [ 5 , 8 , 9 ]  . 
non - syndromic craniostenoses occur more commonly with the premature closure of a single or two major sutures , whereas cases of syndromic craniostenosis more frequently involve the fusion of several sutures and are often associated with facial deformities and brain anomalies [ 10 ]  . 
however , important to note that the severity of the cranial deformity does not reect the severity or the extent of the synostotic process ; indeed , skull symmetry can be preserved when multiple sutures are symmetrically fused [ 11 , 12 ]  . since its introduction , computed tomography ( ct ) has emerged as the technique of choice to conrm the clinical diagnosis , to dene the extent of the synostosis and to exclude any associated anomalies and , consequently , to determine a prompt and accurate treatment . therefore , ct with three - dimensional ( 3d ) reconstructions represents the gold standard in the evaluation of skull sutures but is used , for radioprotective reasons , with a low - dose technique and only when it is deemed clinically necessary . 
several studies have reported the high sensitivity ( 96.4 % ) and specicity ( 100 % ) of 3d ct in the identication of the vault sutures involved by the synostotic process , but no study has ever evaluated the accuracy and utility of spiral ct ( low - dose technique ) in identifying the extension of the synostotic process towards the skull base in children with a clinical suspicion of craniosynostosis [ 5 , 9 ]  . radiol med ( 2014 ) 119 : 694704 fig . 
4 parieto - squamosal arch a this is the junction of the coronal and lambdoid arches and it is responsible for the vertical growth of the skull . parieto - squamous ( b white arrow patent suture on the right side ; black arrow synostotic suture on the left side ) and parieto - mastoid ( minor sutures ) ( c white arrow patent suture on the right side ; black arrow synostotic suture on the left side ) are a part of this arch time along the four the lack of ct data in the involvement of the skull base the synostotic sutures contradicts scientic reports that process progresses over sutural arches of the skull [ 1315 ]  . 
the synchondroses normally ossify ( i.e. , they form synostoses ) before or after birth at different times . the intra - sphenoidal synchondrosis ( is ) in particular , ossies before or spheno - ethmoidal synchondrosis ( se ) ossies by the age of 6 years ; and the spheno - occipital ( so ) between the 17th and 20th year . 
the anterior intra - occipital synchondrosis ( io a ) begins to ossify by year 12 and nishes by year 710 , and the posterior intra - occipital synchondrosis ( io p ) begins to close by year 12 and nishes by year 47 . 
anterior intra - occipital synchondroses ( white arrows in a patent synchondroses ) ; posterior intraoccipital synchondroses ( white arrow in b patent synchondrosis ; black arrow in b synostotic synchondrosis ) ; temporo - occipital synchondroses ( white arrows in c patent synchondroses ) ; anterior intra - sphenoidal synchondroses ( white arrow in d1 patent synchondrosis ; black arrow in d2 : synostotic synchondrosis ) ; spheno - ethmoidal synchondrosis ( white arrow in e1 patent synchondrosis ; black arrow in e2 synostotic synchondrosis ) ; spheno - petrosal synchondroses ( white arrows in f1 patent synchondroses ; black arrows in f2 synostotic synchondroses ) ; spheno - occipital synchondrosis ( white arrow in g1 patent synchondrosis ; black arrow in g2 synostotic synchondrosis ) between the coronal and lambdoid arches . 
in addition , we demonstrate that both the minor sutures and the synchondroses play a remarkable role in determining asymmetries of the cranial fossae and alterations of the facial skeleton [ 18 ]  . materials and methods patients we retrospectively reviewed high - resolution ct images of 27 children with cranio - facial deformities secondary to craniosynostosis . 
twenty - one children ( 14 males , 7 females ) 167 months old ( average , 18 months old ) exhibited complex or multisutural synostoses ( 14 syndromic and 7 non - syndromic children ) , whereas six children ( four males , two females ) between the ages of 7 and 114 months old ( average 37 months old ) exhibited anterior synostotic plagiocephaly ( table 1 )  . radiol med ( 2014 ) 119 : 694704 classication patients diagnosed with table 1 craniosynostosis synostosis syndromic pansynostosis non - syndromic pansynostosis anterior synostotic plagiocephaly m male , f female patients sex age ( months ) the ct scan was performed using a multidetector scanner ( lightspeed pro 64 , ge medical system , milwaukee usa ) without the injection of contrast mediuthe scanner was equipped with automatic tube current modulation techniques . 
image acquisition was performed 100120 kvp , with parameters : following 50280 mas , scan time 1 s , small eld of view ( fov 1820 cm ) , slice thickness 1.25 mm and convolution lter for soft tissue . beginning from the native axial images , the data were processed using multiplanar reconstructions with bone radiol med ( 2014 ) 119 : 694704 table 5 orbital rim deformity in patients with coronal arch synostosis synostosis coronal arch orbital rim deformity coronal suture coronal and fs sutures none elevated ( harlequin deformity ) depressed and recessed syndromic pansynostosis non - syndromic pansynostosis anterior plagiocephaly fs fronto - sphenoidal table 6 chiari 1 malformation in patients with lambdoid arch synostosis synostosis lambdoid arch major sutures minor sutures ( om ) major and minor sutures chiari insertion of the tentorium children exhibited synostosis of the sutures of the coronal arch , ve children exhibited harlequin deformity of the orbit , and one child exhibited superior orbital roof displacement ( table 5 )  . in the group of newborns with complex craniostenosis ( syndromic and non - syndromic ) , in which the synostotic process involves the sutures of the lambdoid arch , 11 out of 15 children exhibited a low insertion of the tentoriuof these 11 children , nine exhibited caudal herniation of the cerebellar tonsils towards the magnum foramen ( chiari 1 ) ( table 6 )  . syndromic pansynostosis nonsyndromic pansynostosis om occipito - mastoid algorithms with a thickness of 0.65 mm and 3d volume rendering reconstructions ( 3d vr )  . using complementary 2d multiplanar images with bony algorithm and 3d vr reconstructions , all of the sutures ( minor and major ) , the synchondroses of the four sutural arches and any synostosis were assessed in each child . moreover , the remaining synchondroses of the skull base and cranial fontanels were also identied . results the results of our study are presented in tables 2 , 3 , 4 , 5 and 6 . 
all of the examined children with craniosynostosis exhibited synostosis of both the major and minor sutures of the four sutural arches of the skull ( table 2 ) as well as synostosis of the synchondroses and the fontanels ( table 3 )  . 
the patients consequently exhibited craniofacial alterations ( cranial deformity , superior orbital rima deformity , nasal root deviation , ethmoidal axis deection ) ( table 4 )  . facial alterations were observed in the majority of children with synostosis of the coronal arch . 
in the group of children with complex craniostenosis ( syndromic and nonsyndromic ) , 18 out of 21 children exhibited synostosis of the sutures of the coronal arch , 10 children exhibited harlequin deformity of the orbit , and only one child exhibited superior orbital roof displacement ( table 5 )  . 
in the group with anterior synostotic plagiocephaly , all discussion the evaluation of cranial deformities in newborns necessitates that clinicians be able to distinguish , above all , between positional plagiocephaly and craniostenosis . 
in particular , 3d ct has revolutionised the diagnostic evaluation , the planning of surgical procedures , the post - surgical evaluation and the follow - up [ 5 , 18 ]  . diagnostic imaging is useful to evaluate the involvement of the sutures in the synostotic process and to assess intracranial alterations that can be associated with craniostenosis . 
however , it should be emphasised that the use of the ct , although it allows the rapid acquisition of images without the use of anaesthesia , exposes newborns to a nonnegligible dose of radiation [ 5 ]  . 
 [ 19 , 20 ] have also demonstrated that this risk dramatically decreases with increasing age , especially during the rst year of life . the diagnostic technique of choice in children with craniostenosis remains a point of controversy and is partially dependent on the skull - facial malformations of the child . 
some authors have suggested the utility of alternative techniques to ct , such as ultrasound and conventional radiography of the skull , in children with cranial deformities associated with a lower clinical suspicion of 702 radiol med ( 2014 ) 119 : 694704 craniosynostosis or in children with the clinical suspicion of simple monosutural craniostenosis [ 19 ]  . 
nevertheless , other authors underline the high sensitivity ( 96.4 % ) and specicity ( 100 % ) of low - dose ct with 3d maximum intensity projection ( mip ) reconstructions for the identication of synostosis of the vault sutures in newborns with craniosynostosis skull - facial deformities ( complex craniostenosis ) [ 18 , 21 ]  . associated with serious in this regard , almost all of the modern mdct scanners are currently equipped with some type of automatic exposure control ( aec ) or automatic tube current modulation ( atcm ) technique [ 22 ]  . 
in our study , in most of the patients , we used the dose modulation technique called automa 3d , whereas more recently in two cases , default asir algorithm was used . 
the authors have also described other 3d reconstruction algorithms , including for craniostenosis , surface projection rendering ( spr ) although this algorithm is not as widely available as 3d mip [ 21 , 23 ]  . to date , however , no study has considered the effectiveness and the importance of low - dose spiral ct in identifying and assessing the vault sutural extension toward the skull base [ 18 , 24 ]  . 
consistent with many previous studies [ 14 , 2529 ] , our data conrmed the high frequency of facial alterations and the hemibase asymmetry of the anterior cranial fossa in all of the newborns in whom the synostotic process involved the sutures of the coronal arch , especially the coronal and the fronto - sphenoidal sutures [ 13 ]  . 
indeed , most of studied children with craniostenosis exhibited synostosis of one or more sutures of the coronal arch ( 24 out of 27 ) and consequently skull shape deformity , median axis deviation of the anterior skull base ( ethmoidal axis ) and orbit deformation ( table 4 )  . the coronal in agreement with current data , our results conrm that the orbital harlequin deformity is present both in children with isolated synostosis of suture ( major suture of coronal ring ) , and in children with associated ipsilateral synostosis of the coronal and frontosphenoidal sutures ( major suture and minor suture of the ba of the coronal ring ) [ 30 ]  . 
nevertheless , unlike anterior synostotic plagiocephalies , newborns with multiple synostosis ( syndromic and nonsyndromic synostosis ) in which coronal ring synostosis was identied , the severity of the facial deformity ( particularly orbital dysmorphism ) and the asymmetry of the anterior skull base was not very evident ; this may be a result of the symmetry of the synostotic process along the sutures of the coronal ring or the time course of the synostotic process . to date , different authors have conrmed the utility of low - dose spiral ct in children with complex or multisutural synostosis in avoiding irradiation in newborns with the clinical diagnosis of monosutural synostosis [ 18 , 19 , 21 ]  . 
however , we believe that low - dose spiral ct can be suitable in children with anterior synostotic plagiocephaly because high - resolution ct has the ability to identify the minor sutures of the coronal arch and , therefore , to radiol med ( 2014 ) 119 : 694704 fig . 
threedimensional computed tomography ( 3d ct ) ( a1 , a2 ) and axial ct image ( a3 , a4 ) show , on the right side , coronal synostosis ( black arrows in a2 , a3 ) and a patent fronto - sphenoidal suture ( arrow heads in a2 , a4 )  . 
note the elevation and relative vertical overgrowth of the right sphenoid , especially in the lateral portion of the greater wing , creating the harlequins eye ( a1 )  . 
3d ct scan ( b1 , b2 ) and axial ct images ( b3 , b4 ) show , on the left side , an absent frontosphenoidal suture ( arrow heads in b2b4 ) and a patent coronal suture ( black arrows in b2 , b3 )  . 
the roof of the left orbit is depressed with obvious retrusion of the lateral part of the supraorbital rim ( b1 )  . coronal and fronto - sphenoidal sutures are patent on the right side ( white arrows in b3 , b4 ) child with left fronto - sphenoidal and coronal sutures synostosis . 
3d ct scan ( c1 , c2 ) and axial ct image ( c3 , c4 ) show , on the left side , both fronto - sphenoidal suture ( arrow head in c2c4 ) and coronal suture synostosis ( black arrow in c2 , c3 )  . 
coronal and fronto - sphenoidal sutures are patent on the right side ( white arrow in c3 , c4 ) evaluate the extension of the synostotic process toward the skull base and the consequent asymmetric deformity of the anterior cranial base . these data can modify the therapeutic choice guiding toward timely and appropriate treatment . 
in our study of 15 children with complex craniostenosis in which the synostotic process involved the sutures of the lambdoid arch , 11 out of 15 children exhibited a low insertion of the tentorium , and 9 out of 15 exhibited caudal herniation of the cerebellar tonsils in the magnum foramen ( chiari 1 malformation ) ( table 6 )  . 
different authors have described the frequent evidence of chiari 1 malformation in children with syndromic multisutural or monosutural craniostenosis , describing the main contribution of the synostosis of the lambdoid suture in this dynamic and complex process [ 31 ]  . other authors have explained the onset of the chiari 1 malformation not only with the reduction in volume / deformity of the posterior cranial fossa secondary to synostosis of the lambdoid suture but also with the altered uid dynamics [ 32 ]  . 
our data conrm the importance of the lambdoid arch as well as the involvement of both the major and minor sutures ( individually or in combination ) in the onset of the chiari 1 malformation . therefore , we are proceeding to volumetrically evaluate , using magnetic resonance ( mr ) imaging , the posterior cranial fossa and the infra - tentorial compartment . 
in the 704 radiol med ( 2014 ) 119 : 694704 same study , we are evaluating some of the morphometric parameters of the posterior cranial fossa ( diameter of the foramen magnum , tentorial angle , and supraoccipital and basioccipital lengths ) in children with craniostenosis and with other syndromic conditions in which chiari 1 malformation is present to better explain the mechanisms of the cerebellar herniation and the variable timing of development . in conclusion , in the study of craniostenosis , low - dose ct with 3d vr reconstructions allows the prompt and precise identication of the extension of the synostotic process towards the skull base , which has important implications for surgical planning . conict of interest r calandrelli , g . 
x - ray ( n = 18 ) , computed tomography ( ct ) ( n = 24 ) and magnetic resoretrospectively nance evaluated . results five tumours were located in the cervical spine , 15 tumours were located in the thoracic spine and 10 tumours in the lumbar spine . 
197 , ruijin 2nd road , shanghai 200025 , china e - mail : rjzhangwb@yahoo.com.cn 682 introduction giant cell tumour ( gct ) is a relatively common skeletal neoplasm , accounting for approximately 45 % of all primary bone tumours and 20 % of benign bone neoplasms in adults [ 1 , 2 ]  . 
they occur infrequently in the spine , mostly in the sacrutheir presence in the mobile spine above the sacrum is even more exceptional , representing only 6.5 % of bone gcts according to the mayo clinic [ 5 ]  . previously published reports on gcts in the mobile spine were predominantly in the orthopaedic and neurosurgical domathere are a few reports of the imaging features of gcts in the mobile spine , which are limited to case reports or a few examples included in general reviews of spinal lesions [ 69 ]  . 
in this article , we discuss and illustrate the radiographic , computed tomography ( ct ) and magnetic resonance ( mr ) features of 30 cases of gct in the mobile spine . 
the purpose is to dene the characteristic imaging features of these unusual lesions , and to discuss our results with respect to the current literature . materials and methods patients we performed a retrospective review of 30 cases of gct occurring in the mobile spine under surgical treatment at 2 institutions between january 1990 and march 2012 . 
all 30 surgical specimens were reviewed by 2 pathologists experienced in musculoskeletal oncology in each institution to reconrm the diagnosis . the patients were evaluated on plain radiograph , ct and / or mr imaging before biopsy or surgery . 
informed consent was not required . imaging procedures anteriorposterior and lateral plain radiographs were performed in 18 patients . ct examination was performed in 24 patients with a 4or 16 - slice multidetector spiral ct scanner ( lightspeed , general electric medical systems , milwaukee , wi , usa ) radiol med ( 2014 ) 119 : 681693 using a routine protocol . 
unenhanced t1weighted spin - echo ( se ) images were obtained with a tr of 500600 ms and a te of 1525 ms , and t2 - weighted fast se or se images were obtained with a tr of 2 , 2004 , 000 ms and a te of 80110 ms . 
contrastenhanced images of sagittal and axial planes were obtained in 15 patients using a t1 - weighted spin - echo sequence with fat suppression . in addition , a plain radiograph or ct of the chest was performed to rule out pulmonary metastasis at presentation . imaging interpretation the imaging studies were evaluated retrospectively and independently by two experienced musculoskeletal radiologists at each institution . 
twenty patients had neurological decits including paralysis ( n = 3 ) , different degree of muscle weakness and numbness ( n = 17 ) , paraesthesia ( n = 6 ) , etc . 
five of the tumours involved the cervical spine , 15 involved the thoracic spine , and 10 the lumbar spine . dened in 13 cases and ill dened in ve cases . 
according to the campanacci grading system [ 10 ] , no case was classied as grade i , 5 cases were classied as grade ii , 13 cases as grade iii . radiographic ndings plain radiograph was performed in 18 cases . 
however , ct scan outlined the cortical alterations in a more accurate way , such as minor cortical erosion , bony ridges , absence of mineralised matrix and margin sclerosis . 
c on computed tomography ( ct ) , peripheral bony ridges ( white arrow ) created by endosteal scalloping of a lobulated tumour gave a radiographic soap bubble appearance which indeed were pseudotrabeculations . 
manipulation and rotation of the 3d images allows the surgeon to better understand the extent of the mass and to plan the operation . mr ndings the mr ndings of 21 cases of spinal gcts are summarised in table 5 . 
according to previous studies [ 9 , 11 ] , areas of low - signal intensity seen on t2 - weighted and gradientecho sequences may result from the presence of hemosiderin which is detected in more than 63 % of gcts . 
a , b plain radiograph showed a lytic and multiloculated lesion with a soap bubble appearance in the vertebral body of t7 , the border of the tumour was partially separated from the surrounding normal bone by an area of sclerosis of thin thickness ( black arrows )  . 
magnetic resonance imaging ( mri ) showed the tumour with homogeneous low signal intensity on t1 - weighted image and markedly high signal intensity on t2 - weighted image , without contrast enhancement , suggesting cystic components which may be consequential to haemorrhages or to secondary aneurysmal bone cyst formation . 
a band of low signal intensity surrounding the tumour on t1and t2 - weighted images ( white arrows ) partially corresponded to both brous capsule or reactive sclerosis seen on radiograph fig . 
a plain radiograph showed a lytic lesion in the t9 vertebral body , which was separated from the surrounding normal bone by a clearly depicted sclerotic border ( black arrow )  . 
peritumoural oedema was uncommon , mainly seen in patients with a history of trauma . radiol med ( 2014 ) 119 : 681693 table 4 computed tomography features of 24 giant cell tumours of the mobile spine case site eccentric pathologic fractures margin sclerosis bony ridges cortical destruction soft - tissue mass tpvb involvement spinal canal involvement right right centre centre left centre left left left left left right right right centre centre centre centre left left left centre left centre ? , local ? , local ? , local ? , short ? , short ? , long ? , short ? , short ? , short ? , short ? , long ? , short ? , long ? , short ? , short ? , short ? , short ? , long ? , long ? , short ? , short ? , short ? , short ? , long plus sign ( ? ) , present ; minus sign ( - ) , absent ; tpvb terminal plate of vertebral body fig . 
the border of the tumour was not well demonstrated due to bowel gas interference . c axial ct scan well depicted a thin area of sclerosis in the posterior margin of the tumour , separating it from the surrounding normal bone ( white arrow )  . 
mri showed the tumour with heterogeneous low signal intensity on t1 - weighted image and markedly high signal intensity on t2 - weighted image , suggesting polycystic components which may be consequential to haemorrhages or to secondary aneurysmal bone cyst formation . 
a band of low signal intensity ( thin black arrows ) was seen in the posterior margin of the tumour , partially corresponding to the sclerotic border seen on ct 688 radiol med ( 2014 ) 119 : 681693 fig . 
c , d ct scan showed a large and ill - dened soft - tissue mass involving the whole vertebra with spinal canal involvement ; contrast - enhanced ct showed intensive and heterogeneous enhancement of the mass ( white arrows )  . 
e , f thin - slice helical acquisitions with 2d and 3d reconstructions were routinely performed in sagittal and coronal planes , which clearly demonstrate the size and the outline of the tumour . 
after the solid intravenous administration of gadolinium , portion was enhanced with a marked and heterogeneous signal pattern discussion gcts of the mobile spine frequently develop 20 and 40 years of age and demonstrate a high female predilection in most series , with ratios ranging from 2.3 : 1 to 2.5 : 1 [ 14 ]  . the peak prevalence of our series is also in the third and fourth decades of life ( 83.3 % , 25 / 30 ) , although no predominance was found in female patients . 
in our series , the thoracic segment was also the most frequent location ( 50 % , 15 / 30 ) , but followed by the ( 33 % , 10 / 30 ) ; only ve cases were lumbar segment detected in the cervical segment ( 17 % , 5 / 30 )  . 
campanacci grade iii tumours [ 10 ] as well as the rate of soft - tissue extension are higher in our series than in patients with typical gct of long bone [ 10 , 17 ]  . 
on the one hand , the diagnosis might be delayed as the initial symptom , spinal pain , is extremely frequent in the orthopaedic practice and can easily be misinterpreted . 
on the other hand , it might be assumed that the gcts of the mobile spine are more aggressive [ 17 ]  . plain radiograph remains the mainstay of the diagnosis of gct . 
for gcts in the epiphyseal - metaphyseal areas of long bones , the location is one of the most important features suggesting the diagnosis [ 12 , 18 ]  . 
as a result , the radiographic presentations are less diagnostic for gcts of the mobile spine . ct scans are more useful in complex - shaped bones such as vertebrae , because the details of the lesion may not be depicted well on radiograph . 
ct improves detection of cortical thinning , pathological fracture , periosteal reaction , radiol med ( 2014 ) 119 : 681693 table 5 magnetic resonance features of 21 giant cell tumours of the mobile spine case site signal on t1weighting signal on t2weighting cystic areas haemorrhage fluid peritumoural oedema tpvb involvement spinal cord compression heterogeneous iso - low with small high area heterogeneous iso - low heterogeneous iso - low heterogeneous heterogeneous iso - low heterogeneous iso - low with small high area with polycystic high heterogeneous with polycystic high heterogeneous iso - low with small high area iso - low with polycystic high with polycystic high heterogeneous iso - low with small high area heterogeneous heterogeneous heterogeneous heterogeneous iso - low iso - low heterogeneous heterogeneous with polycystic high with polycystic high heterogeneous heterogeneous iso - low heterogeneous iso - low heterogeneous markedly high heterogeneous iso - low with small high area heterogeneous with polycystic high heterogeneous heterogeneous heterogeneous iso - low iso - low iso - low heterogeneous iso - low with small high area t10 heterogeneous iso heterogeneous iso t10 heterogeneous iso - low heterogeneous iso - low with small high area t11 heterogeneous iso heterogeneous iso t11 heterogeneous heterogeneous t12 heterogeneous iso - low iso - low with polycystic high heterogeneous iso - low with small high area heterogeneous polycystic marked iso - low high centre low and peripheral iso with small high area centre high and peripheral iso with small high area uid levels softtissue mass large ? , slight ? , slight ? , slight small large small large small large large small ? , slight ? , slight ? , slight ? , moderate ? ? , slight 690 table 5 continued radiol med ( 2014 ) 119 : 681693 case site signal on t1weighting signal on t2weighting cystic areas haemorrhage fluid uid levels softtissue mass peritumoural oedema tpvb involvement spinal cord compression heterogeneous heterogeneous iso - low with polycystic high plus sign ( ? ) , present ; minus sign ( - ) , absent fig . 
c marked and heterogeneous enhancement was seen after intravenous administration of contrast agent ( thin black arrow )  . d sagittal t1 - weighted image , e sagittal t2 - weighted image , f axial t2 - weighted image , g contrast - enhanced sagittal t1 - weighted image . the lesion contained a small high - signal - intensity area on t1weighted images , suggesting relatively recent haemorrhage ( white arrow )  . 
after intravenous administration of gadolinium , the mass was enhanced with a heterogeneous signal pattern degree of osseous expansion and matrix mineralisation compared with radiograph [ 12 , 19 ]  . 
in our series , ct detected minor cortical destruction in two cases ( cases #21 and #27 ) and a small soft - tissue mass in one case ( case #30 ) , which had been missed on radiography . 
on ct , peripheral bony ridges due to endosteal scalloping of a lobulated tumour gave a radiographic soap bubble appearance which were in fact pseudotrabeculations [ 18 , 19 ]  . 
however , the bony ridges in these lesions are usually much longer . mr has a better soft - tissue resolution and multiplanar imaging features , which allows for an accurate evaluation of the soft - tissue extent and the relationship with surrounding tissues . 
a axial and b sagittal t1weighted mr image showed a large and heterogeneous mass presenting a uiduid level with superior / inferior layer signal intensity as high / low pattern ( black arrows ) although artefacted by an internal metal xation structures and blood supply [ 12 , 13 , 15 , 18 ]  . 
the solid components of gct demonstrated low - to - iso signal intensity on t2 - weighted images in the majority of our cases , which is reported to result from hemosiderin deposition or high collagen content [ 12 , 13 , 20 , 21 ]  . 
 [ 11 ] found that in ten cases with low signal intensity on t2 - weighted mr images , neither dense collagenous matrix nor bone formation was prominent in the specimens , whereas large amounts of hemosiderin in the mononuclear stromal cells and multinuclear giant cells were pathologically conrmed . 
the authors believed that the diffusely dispersed extravascular erythrocytes found in their study was a characteristic pathological nding of gct , and was accordingly considered to be an important cause for the frequent and massive hemosiderin deposition in the tumour . 
they concluded that the low - signal - intensity areas on t2weighted mr images were probably the rst radiological evidence of the phagocytic nature of gct and could be a sign of relatively high differentiation or low aggressiveness of the tumour . 
it can also be a key differential feature in excluding other tumours such as metastasis , myeloma , lymphoma , and chordoma that generally demonstrate high signal intensity on t2 - weighted mr images [ 12 , 16 , 22 ]  . gct is the most common lesion associated with secondary abc , accounting for 39 % of the lesions [ 12 , 20 , 2325 ]  . 
mr imaging is also sensitive for detection of cystic components and uiduid levels secondary to abc . contrast - enhanced mr imaging is especially helpful in distinguishing cystic from solid regions of gct , which can be vital for patients treatment for two important reasons : rst , to prevent misdiagnosis of the lesion as a primary abc which contains only cystic components ; and second , to guide biopsy directly to the solid portions of the lesions which harbour diagnostic tissue , as opposed to abc regions which yield nonspecic blood [ 12 ]  . 
 [ 23 ] reported that bone tumours which had uiduid levels with superior / inferior layer signal intensity as high / low on t1 - weighted images were 2.3 - times more likely to be malignant than those with low / high - signal - intensity pattern . 
however , in our series , three lesions ( cases #10 , #14 and #21 ) were partially separated from the surrounding normal bone by a thin sclerotic border on radiograph or ct and a band of low signal intensity on mri , corresponding to both brous capsule and reactive sclerosis [ 11 ]  . 
since bony sclerosis is a host reaction against the tumour and correlates with a less aggressive radiographic appearance , a better prognosis might be predicted when the tumour is contained within this sclerotic margin suggestive of a slow - growing lesion [ 12 , 18 ]  . when encountering a single osteolytic mass in the the differential diagnosis includes abc , mobile spine , brown tumour of hyperparathyroidism , bony metastasis to the spine , haemangioma , chordoma or chondrosarcoma , tuberculosis , etc . 
mr imaging reveals the detailed characteristics of the internal contents with uiduid levels in one or more granular spaces , haemorrhage , and a hypointense circular rim surrounding the entire lesion which correlates with the pathological nding of a brous capsule [ 25 ]  . 
to differentiate a primary abc from a gct with prominent abc components , mr imaging may help by demonstrating a purely cystic appearance in the former lesion , compared with solid components in the latter [ 16 , 22 , 26 ]  . 
brown tumour of hyperparathyroidism is expansile and well marginated and highly likely to show low signal intensity on mr images that can mimic gct , but it must be accompanied by other manifestations of hyperthyroidism and can be excluded by measuring levels of serum calcium and alkaline phosphatase [ 1113 ]  . 
haemangioma is a benign vasoformative neoplasm of endothelial origin , usually with a characteristic presentation as honeycombed or sunburst appearance with spindles radiating towards the periphery [ 28 ]  . 
chordoma or chondrosarcoma are characterised by a high signal on t2weighted images , generally not a feature of gct , so such a nding would favour a diagnosis of chordoma or chondrosarcoma [ 13 ]  . 
the most common site of spinal tuberculosis is the thoracolumbar junction and the most common symptom is back pain , while cough , fever , night sweats and weight loss are less frequent . 
paraspinal , prevertebral or spinal abscess is frequent and the intervertebral discs are often involved [ 30 ]  . although the vast majority of gcts are benign lesions , malignant gcts ( dened as a sarcomatous growth ) account for 510 % of all gcts [ 12 ]  . 
imaging features of gct suggesting local aggressiveness include a large size , rapid growth , illdened boundaries , and invasion of the surrounding soft tissues [ 2 ]  . in conclusion , although rare , gct can occur in the mobile spine . 
radiologia , dipartimento cardio - toraco - vascolare , azienda ospedaliero universitaria , policlinico sant orsolamalpighi , bologna , italy e - mail : giangasparemineo@alice.it subsequently , a clinical - instrumental evaluation was performed , and the response to therapy and histopathological reports were evaluated . results radiological evaluation based on hrct ndings revealed 29 patients as pvod - suspicious and 67 as not pvod - suspicious . 
the ct scan showed 95.5 % sensitivity , 89 % specicity , 72.5 % positive predictive value , and 98.5 % negative predictive value , with a diagnostic accuracy of 90.5 % in identifying patients with pvod . conclusions chest ct can be considered a screening test in the assessment of patients with pah of unknown aetiology , and the radiologist can help the clinician to identify patients with ct ndings that make pvod highly probable . keywords pulmonary veno - occlusive disease ( cid : 2 ) pulmonary hypertension ( cid : 2 ) hrct ( cid : 2 ) differential diagnosis c . 
radiologia , sezione radiologia durgenza , dipartimento di emergenza / urgenza , chirurgia generale e dei trapianti , azienda ospedaliero universitaria , policlinico sant orsola - malpighi , bologna , italy a . 
cardiologia , dipartimento cardio - toraco - vascolare , azienda ospedaliero universitaria , policlinico sant orsola - malpighi , bologna , italy with less than 200 cases described in the literature , pulmonary veno - occlusive disease ( pvod ) is a rare form of pulmonary arterial hypertension ( pah ) [ 1 ] which is associated with a worse prognosis compared to idiopathic pah ( ipah ) [ 2 , 3 ]  . 
although the aetiology has not yet been identied , several risk factors are thought to be involved including infection , chemotherapy toxicity [ 4 ] , radiation , autoimmunity and genetic predisposition [ 58 ]  . signs and symptoms are not specic and mimic those of other disorders ; they include dyspnoea , fatigue , chest pain , haemoptysis , cyanosis , pulmonary crackles on auscultation 668 radiol med ( 2014 ) 119 : 667673 and clubbing on physical examination . 
haemosiderin - laden macrophages in the bronchoalveolar lavage ( bal ) uid , low diffusion of carbon monoxide ( dlco ) in pulmonary function tests as well as low partial pressure of oxygen in arterial blood ( pao2 ) may also be found . the obstructed venous vessel the key histopathological hallmark is a widespread brous intimal proliferation , typically patchy in distribution that predominantly involves the pulmonary venules and small veins , with a gradual reduction of the vascular lumen until complete occlusion [ 9 ] ; the central recanalisation of is frequently observed [ 10 ]  . 
in the lung parenchyma , patchy areas of alveolar capillary dilatation ( socalled loop - like appearance ) are present upstream from the narrowed and occluded veins ; in these areas there is often interstitial brosis , haemorrhage , and intra - alveolar haemosiderin - laden macrophages , probably due to chronic passive congestion . 
with regard to the pulmonary arterial circulation , the muscular pulmonary arteries show medial hypertrophy , while the arterioles become muscularised secondary to post - capillary obstruction of venous drainage [ 11 , 12 ]  . pvod remains a clinical entity difcult to classify ; it shares some features with pre - capillary forms of pah , but there are histopathological , clinical and therapeutic differences . 
table 2 summarises the instrumental ndings suggestive of pvod . in pvod , the x - ray examination only shows kerley b lines and peripheral interstitial thickening in addition to the classic signs of pah ( ectasia of the pulmonary arteries / pulmonary trunk and cardiomegaly with a prevalence of right sections )  . computed tomography ( ct ) , especially the high - resolution study of the lung parenchyma ( hrct ) , is nowadays considered an integral part of the diagnostic workup of pvod [ 16 ] , owing to the detection of smooth thickening of the interlobular septa , ground - glass opacities , increased size of the mediastinal lymph nodes , pleural and pericardial effusions [ 17 , 18 ] ( even though the importance of pleuropericardial effusion is still controversial ) [ 19 ]  . because of the lack of response of ipah to drug therapy which consists in vasodilators ( prostanoids , antagonists for the endothelin receptor , inhibitors of phosphodiesterase type 5 ) and which might even worsen the prognosis due to the onset of severe , at times fatal , pulmonary oedema , the only curative therapy for pvod is represented by cardiopulmonary or lung transplantation [ 20 , 21 ]  . 
therefore , a correct and timely diagnosis becomes crucial to start an adequate medical treatment . the aim of our study was to evaluate the diagnostic accuracy of hrct for povd in a large group of patients with pre - capillary pah of unknown aetiology , and assess the inuence of hrct ndings in determining the diagnosis and subsequent treatment . materials and methods we retrospectively evaluated the ct scans of 131 patients with pre - capillary pah of unknown aetiology performed between 2005 and 2012 , as part of the diagnostic algorithm arterial blood gases pulmonary function tests bronchoalveolar lavage haemosiderin - containing pwp b15 mmhg pao2 \70 mmhg dlco \55 % macrophages ventilation / perfusion lung normal or patchy perfusion defects scan mpap value c25 mmhg at rest , measured by right heart catheterisation , is suggestive of pulmonary hypertension mpap pulmonary arterial pressure , pwp pulmonary wedge pressure , pao2 partial pressure of oxygen , dlco lung diffusion capacity for carbon monoxide pulmonary hypertension of our institute . 
we subsequently evaluated the results of other noninvasive clinical and instrumental investigations , such as pao2 , dlco and the 6 - minute walking test ( 6mwt ) , based on which the therapy was started . 
finally , a post - therapy assessment was performed based on the development of pulmonary oedema secondary to treatment with vasodilators , and on the histological results , if available . statistical analysis the signicance of each hrct nding for the diagnosis of povd was calculated with the fisher exact test . 
sensitivity , specicity , positive predictive value , negative predictive value , diagnostic accuracy of ct and likelihood ratio were calculated using the software openepi ( com , copyright the open source initiative )  . results on the basis of the presence of at least two hrct ndings specic to povd , 29 patients ( 8 men and 21 women ) were considered povd - suspicious ( 30 % , group 1 ) , and 67 not povd - suspicious ( 70 % , group 2 )  . 
all patients had both a dilatation of the pulmonary arteries and right heart chambers , consistent with pah ; in no case was thromboembolism or cardiopulmonary malformations documented . as a result of the clinical - instrumental analysis , among the 29 patients of group 1 , 15 were nally classied as ipah and therefore received treatment with vasodilators ; post - therapy eight patients present complications , while seven patients developed pulmonary oedema leading to a subsequent reclassication of their diagnosis from ipah to povd . 
1 diagnostic efcacy of high - resolution computed tomography ( hrct )  . according to the hrct ndings , patients were subdivided into pulmonary veno - occlusive disease ( pvod ) - suspicious and not pvod - suspicious . 
in none of the six patients with a histological diagnosis of ipah , were septal thickening or enlarged lymph nodes documented , while ground - glass opacities were observed in three cases . discussion and conclusions in the diagnostic classication of patients with evidence of pah of unknown aetiology and pre - capillary pulmonary hypertension at right heart catheterisation , the early identication of those with povd and the differential diagnosis with ipah are crucial . 
moreover , thanks to the high negative predictive value of ct ( 98.5 % ) , the absence of specic hrct signs allows the exclusion of povd . from the comparison between the hrct ndings and the histological examination , we found that all patients with a histological diagnosis of povd showed smooth thickening of the interlobular septa , ground - glass opacities and enlarged lymph nodes . 
pulmonary oedema with bilateral pleural effusion and ground - glass opacities after vasodilatator therapy glass only ; this is further evidence of the importance of the association of the three hrct signs for the diagnosis of povd . our study has some limitations , including the small number of histological examinations and the lack of data from bal , which is not routinely performed . 
in our opinion , bal should be integrated with other radiological and clinical investigations because it is a repeatable and easy examination which allows the identication of haemosiderin - laden macrophages ( frequently observed in povd and rarely in ipah ) [ 2527 ]  . to conclude , chest ct should be considered in the screening of patients with pah of unknown aetiology ; the radiologist could cooperate with the clinician in the recognition of individuals with a suspected povd , whose treatment is different from that of ipah due to the risk of a fatal pulmonary oedema . 
changes in patient body contours were evaluated by comparing the volumes within the external skin outline . results apart from two patients requiring re - planning due to substantial weight loss , our results evidenced no signicant shifts of ptvs and pgs . 
pg volume decreased with a trend in volume reduction for the ipsilateral parotid . no signicant shift of spinal cord at c1 and c6 level was detected , in either the ap or ll direction . 
giovanni battista hospital , torino , italy data demonstrated a trend in external skin contour volume loss . conclusions our preliminary results reect the literature data and indicate that an off - line adaptive rt approach is appropriate in clinical practice . keywords head and neck cancer ( cid : 2 ) intensity - modulated radiotherapy ( cid : 2 ) planning target volumes ( cid : 2 ) organs at risk ( cid : 2 ) computed tomography ( cid : 2 ) adaptive radiotherapy introduction intensity - modulated radiation therapy ( imrt ) is becoming the standard technique for the treatment of head and neck cancer ( h&n ) , because it permits high - dose conformity around complex target volumes while improving sparing of critical structures , leading to an improved therapeutic index . 
through online or off - line interventions at different timescales , adaptive radiation therapy ( art ) is an approach to correct for temporal morphological changes in a patients anatomy during imrt , using computed tomography ( ct ) techniques such as megavoltage ct , ct on rails and cone - beam ct in the treatment room [ 2 , 3 ]  . 
the art process is closely linked to imageguided radiotherapy ( igrt ) , which is commonly interpreted as the use of in - room imaging to perform set - up correction , and aimed to reduce clinical target volume ( ctv ) and planning target volume ( ptv ) margins . the purpose of this prospective study , carried out in an academic department of radiation oncology not equipped with in - room volumetric imaging systems , is to assess the radiol med ( 2014 ) 119 : 714720 volumetric and positional changes of ptvs and selected organs at risk ( oars ) in patients treated with imrt , using consecutive off - board conventional ct scan studies . dosimetric aspects were not investigated in this preliminary study . 
ctv1 was generated by adding a 1 - cm margin to gtv , or by including the postoperative tumour table 1 selected patient , tumour , and therapy characteristics bed in the positive / close margin and dissected lymph node level with extracapsular extension ( ece )  . 
ctv2 was dened as the volume near the tumour at risk of subclinical disease ; in adjuvant settings , ctv2 included the tumour bed plus dissected lymph node levels with positive lymph nodes without ece . 
ctv3 included clinically negative lymph node levels at lower risk . outlined critical structures were optic nerves , optic chiasm , parotid glands , temporomandibular joints , mandible , lens , eye globes , temporal lobes , posterior fossa , inner ear and larynx . 
for the brainstem and spinal cord ( sc ) , an additional 3and 5 - mm margin to create a planning risk volume ( prv ) was added , respectively [ 5 ]  . radiotherapy planning baseline h&n imrt planning involves 7 or 9 coplanar , 6 mv elds using the step - and - shoot technique . 
it is important to underline that our tps supports only rigid body and not deformable image registration , not allowing calculation of cumulative dose distribution on the re - scanned ct sets [ 2 , 6 ]  . 
external contours were delineated in all the new ct scans . positional analysis and volume comparisons based on tps , positional changes of ptvs and sc as well as positional and volumetric changes of pgs were obtained in all patients for each ct set . 
the com coordinates were automatically obtained from tps , asking the system to set the beam isocentre in the geometric centre of the volumes of interest . the positional vectors between the virtual isocentre , dened on the plan - ct and identied by radiopaque markers on each subsequent ct study , and the ptv / pgs com were calculated . the distance d between the virtual isocentre and the com of each volume of interest was simply computed as follows : ( cid : 2 ) x ( cid : 3 ) x1 2 y ( cid : 3 ) y1 2 z ( cid : 3 ) z1 ( cid : 3 ) where ( x , y , z ) are the com coordinates and ( x1 , y1 , z1 ) are the virtual isocentre coordinates . 
1 spinal cord displacements into the spinal canal along the anteriorposterior and latero - lateral axes positive shift sign indicated a sc displacement towards the posterior wall of the vertebral canal and a sc shift towards the left side along the ll axis . as a representation of patient weight loss , the change in the patient contour was evaluated by calculating the volume within the external skin outline [ 7 ]  . statistical analysis linear regression was used to analyse the relative variation in volumes of the parotids and external contour correlated with dose . 
the distribution of shift values during the treatment course of ptvs , pg and sc was simply analysed in terms of mean , standard deviation and range of variability ( 1st and 99th percentile )  . 
the mean value , the p value of the one - sample t test and range of variation of the relative shifts measured for ptvs , pgs and sc are reported in table 2 . 
collected data out of this range are related to signicant weight loss ( [ 5 % ) , which occurred in two patients ( patient #4 and #6 reported in table 1 ) with replacement of the thermoplastic mask and mandatory imrt re - planning ( table 3 )  . 
ptvs and oar contours were imported from the plan - ct to the new image set and re - delineated according both to the new set - up and the anatomical variations . 
to investigate the correlation between ipsilateral pg volume with total dose , the relative volume variation was analysed with linear regression , grouping data between ipsilateral and contralateral pg , both for left and right glands . 
the one - sample t test p values were always [ 0.05 , showing the random data of the shiaccording to these values , the safety 5 - mm margin applied for prv was appropriate . external patient contours the collected data demonstrated a trend towards volume loss of external skin contours , with a mean reduction less than 5 % for all patients . 
in our study population , as weight loss [ 5 % only occurred in 20 % of treated patients , volume reductions of external patient contours did not correlate with nutritional status . 
our data showed a volume loss of both pgs , greater for the ipsilateral pg , of 30 % at an accumulated dose c40 gy , recording no shift for either pg . 
although a signicant variation of the left pg through the course of treatment has been previously reported , this statistically signicant value appeared to be of uncertain clinical meaning , in relation to the small number of patients enrolled and the wide variation in data found for both the left and right pg . the prospective study of bhide et al . 
on 20 h&n cancer patients studied by repeat ct scans at weeks 2 , 3 , 4 , and 5 during induction chemotherapy followed by chemo - imrt , showed a statistically signicant reduction of ctvs volumes at week 2 due to therapeutic effects , resulting in dosimetric changes to the ptvs . 
reported the results of a prospective art program for 23 h&n patients who underwent planned ct at 11 , 22 , and 33 fractions with mandatory re - planning based on comparative dosimetric analyses [ 13 ]  . 
quantify both volumetric and dosimetric changes in selected oars during a course of imrt for 26 oropharyngeal carcinomas using repeat cone - beam ct planned weekly during therapy [ 14 ]  . 
all evaluated oars underwent volumetric modications from baseline and the largest volume reduction ( * 30 % ) was observed for pgs , consistent with our results . the same data about the shrinkage of pg was evidenced in the study by loo et al . 
carried out a study to assess the feasibility of routine art in h&n cancer patients and to determine which patients may be selected for this approach [ 15 ]  . twenty patients undergoing denitive chemo - rt and fig . 
however , older art approaches can be still adopted using implanted ducial markers or repeating the imaging study to guide manual re - planning . as our linear accelerator ( synergy platform ; elekta atlanta , ga , usa ) is not equipped with cone - beam ct , we implemented a protocol of serial ct scans to optimise imrt delivery for h&n cancer patients , reporting the magnitude of volumetric and / or positional changes of ptvs and selected oars . we need to emphasise that our tps is not able to perform cumulative dosimetric plan analysis . 
conducted a study on 14 h&n cancer patients using an integrated ct - accelerator imaging system throughout the rt course to investigate geometric and volumetric changes relative to gtvs and oars [ 1 ]  . 
even though gtv was modied during rt due to tumour shrinkage , the authors concluded that re - planning was not necessary because no signicant dosimetric modications to the target were observed , while the main benet of re - planning is the improved sparing of pgs . 
since target volumes were not redelineated during imrt , our results show that re - planning is only mandatory in patients with a weight loss [ 5 % . 720 radiol med ( 2014 ) 119 : 714720 adjuvant rt were enrolled . 
our data suggest that weight loss is correlated to re - planning benet without difference between patients treated in adjuvant or denitive modality . the literature data show that routine re - planning is probably not necessary in all h&n cancer patients and that signicant weight loss is correlated with positional shifts of ptvs . 
a way to identify weight loss is to evaluate changes in the patients body contour using repeat ct scans [ 16 ]  . probably , the appropriate time for adaptive interventions might be in week 2 after the beginning of irradiation , because most anatomical changes are observed in the second and third week of imrt [ 17 ]  . in clinical practice , online art is the ideal treatment modality but remains a difcult and time - consuming approach [ 17 , 18 ]  . 
on the contrary , off - line art remains more useful , because real - time intervention is not a pressing need as most anatomic variations in h&n cancer patients take place gradually [ 17 ]  . consistent with the literature data , our results suggest that shifts of the ptv and pgs are relatively small . 
bonucci radiotherapy unit ifca , university of florence , florence , italy keywords cyberknife ( cid : 2 ) radiosurgery ( cid : 2 ) brain metastases ( cid : 2 ) brain tumour ( cid : 2 ) stereotactic radiotherapy introduction the development of brain metastases is a dismal event which can occur in up to 20 % of all cancer patients throughout their disease progression [ 1 ]  . 
traditionally , whole - brain radiotherapy [ 2 ] has represented the only treatment modality available in the case of lesions not amenable to surgical resection , patients deemed not operable , or both . 
in the last two decades , stereotactic radiosurgery has emerged as an alternative approach for not operable , single brain metastases or for selected patients with multiple metastatic brain lesions . 
oligometastatic disease refers to a limited metastatic burden , and conventionally authors have dened patients with this entity as any burden from 1 to 5 sites of disease [ 3 ]  . 
it has been level - 1 evidence consistently supports the shown that adoption of different treatment strategies [ surgical resection ( sr ) , stereotactic radiosurgery ( srs ) , whole - brain radiotherapy ( wbrt ) , either alone or combined with one the management of patients with brain another ] metastases [ 4 ]  . the choice of a local treatment alone or in association with whole - brain radiotherapy is usually made in patients with a better prognosis depending on performance status , number of brain metastases , controlled systemic disease and histology [ 5 ]  . 
recently , two large randomised studies have shown similar survival benets and functional independence between patients with 13 brain metastases treated with srs alone and srs plus wbrt [ 6 , 7 ]  . radiosurgery may result in a better local control in patients with radioresistant histology and it can defer the 722 radiol med ( 2014 ) 119 : 721726 different notwithstanding wbrt as salvage treatment . 
the ideal candidate for radiosurgery is the patient with controlled extracranial metastases and an excellent performance status [ 8 , 9 ]  . therapeutic however , approaches attempted , a signicant number of these patients develop a local relapse within the surgical bed or a previously irradiated area . 
for selected cases , when all other treatment modalities are either ineffective or not possible , re - irradiation ( ri ) with cyberknife stereotactic radiosurgery ( csrs ) could be the only viable therapeutic strategy . 
to a lesser extent , the potential of ri by means of csrs has been also explored in the context of well - dened , previously irradiated recurrent high - grade gliomas , with promising results . 
there are few experiences in the literature that report on the efcacy and toxicity of patients who had various courses of radiotherapy within the same radiotherapy eld [ 10 ]  . the literature remains scarce regarding the role of ri for selected patients with recurrent single / oligometastases or primary brain tumours . 
the aim of our study was to retrospectively evaluate the feasibility and clinical benet of csrs in 13 patients treated at florence university for recurrent , pre - irradiated brain lesions . materials and methods from december 2011 to may 2012 , 13 patients underwent ri at the university of florence for a previously irradiated , recurrent brain lesion . 
none of the patients received concurrent chemotherapy , all metastatic patients underwent ri after failure of a chemotherapy regimen and the metronomic chemotherapy was started for these patients at least 15 days from the end of ri . 
in the two patients with high - grade glioma , ri was delivered as salvage treatment due to the haematological toxicity of the systemic therapy . five patients underwent srs as upfront therapy and were re - irradiated with csrs while in six cases wholebrain radiotherapy was followed by srs and at recurrence table 1 patient characteristics characteristics no . 
of patients % c6 m karnofsky performance status time from radiotherapy to cyberknife \6 m irradiation volume \10 cc c10 cc re - irradiation volume \10 cc c10 cc stereotactic radiosurgery dose \20 gy c20 gy no . 
in the two cases of primary brain tumours , 3 - dimensional conformal radiotherapy was delivered to the surgical bed , with a total dose of 60 gy with standard fractionation ( 2 gy / fr ) ; at recurrence , ri was performed with csrs . 
all patients underwent a brain mr scan which showed a local progression at the previously irradiated area . mean tumour volume at csrs measured on planning software was 40.43 cc ( 7374 cc )  . 
the radiation therapy oncology group ( rtog ) central nervous system ( cns ) toxicity criteria [ 11 ] were adopted to evaluate toxicity . the cyberknife system the cyberknife ( accuray incorporated , sunnyvale , ca , usa ) is an image - guided frameless radiosurgery system capable of fractionated treatment . 
a 1 - mm uniform margin from gtv to ptv was applied to take into account the total targeting error ( whole procedure of image guidance and treatment delivery ) as derived by dedicated end - to - end tests performed on an anthropomorphic head phantoplans were optimised using stepwise multicriteria optimisation in the sequential module of the multiplan dedicated treatment planning system ( tps ) ( accuray incorporated , sunnyvale , ca , usa ) , and dose was calculated by a ray tracing algorithdoses were prescribed at the 80 % isodose line . 
together with ptv coverage ( percentage of volume inside the prescription isodose line ) and oar constraints , conformity indexes and doses to healthy brain tissue were also considered during plan optimisation . the conformity index was calculated as the ratio of the volume inside the prescription isodose to the ptv . 
doses to healthy tissue were minimised during the optimisation process by means of automatic shell volumes around the ptv and were quantied using the gradient index ( gi ) dened as the ratio of the volume of half the prescription isodose to the volume of the prescription isodose . treatment planning and plan qa the average value over 13 plans for ptv coverage ( percentage of volume inside the prescription isodose ) was 97.5 % ( max = 100 % ; min = 92.5 % ) ; the lower coverage values were obtained for those plans where the brain stem was tangential to the ptv . 
according to the macdonald criteria , complete response ( cr ) was dened as no visible gross tumour or absence of contrast enhancement , partial response ( pr ) as more than 50 % decrease , no change ( nc ) as \50 % decrease and progressive disease ( pd ) as more than 25 % increase of gtv [ 14 ]  . 
local control and progression - free survival rates were calculated as the time interval between re - irradiation and progression ( kaplan and meier method )  . results in terms of compliance to csrs , the majority of patients did not develop any acute side effects . 
in fact , in 54.6 % of patients steroid medication was not changed and in 30 % it was reduced ( including the replacement of dexamethasone by prednisolone )  . 
in particular , one patient was treated as a rst treatment with srs on 25 cc of tumour volume with 27 gy / 3f at 80 % isodose . the csrs volume was 41.60 cc and the dose delivered was 24 gy / 3f at 80 % . 
the second patient received as a treatment whole - brain radiotherapy ( 30 gy / 10 f ) rst followed by srs on 5 cc tumour volume ( 27 gy / 3f at isodose of 80 % )  . 
the csrs volume was 6.07 cc and the dose delivered was 16 gy to the 80 % isodose in a single fraction . the small number of patients and the short follow - up limit the statistical analysis . 
at the time of analysis we did not report any local progression in the bratwo patients had distant progression with new metastases outside of the braat the time of analysis all patients are alive . discussion recurrent brain lesions present a special treatment challenge for patients already treated with radiation . 
conventional salvage radiation therapy has been tried in selected patients , but whether survival is enhanced without intolerable toxicity is unclear [ 15 , 16 ]  . a rapid treatment response , short treatment time , and reduced doses to normal tissue make radiosurgery a rearecurrent brain lesions ( table 4 )  . sonable option for radiosurgery has shown to be as effective as surgery plus whole - brain radiotherapy ( wbrt ) in patients with a single brain metastasis [ 21 ]  . 
it is thought that prior radiotherapy increases the risk of radiation - induced toxicity from salvage radiosurgery because the cumulative dose to the brain is absolutely higher than that of upfront treatment [ 23 ]  . 
some reports have suggested a short - term benet of salvage radiosurgery for recurrent brain lesions [ 24 , 25 ] but the incidence of long - term radiation toxicity has not been investigated thoroughly and clear guidelines for assessing the risk of radiation toxicity from summated radiation do not exist . 
in order to explore the potential role of re - irradiation with csrs as a possible salvage treatment patients already subjected to srs , our study aimed to analyse the clinical benet and tolerability in this subset of patients . 
even if haemorrhagic transformation ( ht ) of ischaemic brain tissue after rt - pa thrombolysis as detected by magnetic resonance imaging ( mri ) can be asymptomatic , it may impair neustudies have rological recovery [ 3 ]  . 
moreover , mri symptomatic 176 radiol med ( 2014 ) 119 : 175182 suggested that microbleeds ( mb ) may be a potential marker of a haemorrhage - prone state [ 46 ] or of vascular cognitive impairment [ 7 ]  . 
therefore , it is necessary to early and accurately detect mb or ht before and after thrombolysis [ 811 ]  . although mb may be markers of increased risk of secondary haemorrhage after thrombolytic therapy in stroke patients [ 1013 ] , secondary mb or ht from ischaemic infarction is not necessarily associated with poor prognosis . 
the key question is an early and accurate detection of mb or ht , as well as a rapid treatment decision - making according to neuroimaging ndings . these measures are helpful to prevent mb , ht , or parenchymal haemorrhage ( ph ) , and to further decrease the incidence of mb or ht , and to benet neurological recovery . 
only brain imaging can conrm the diagnosis of mb or ht , and differentiate haemorrhage from ischaemia with a high level of accuracy . therefore , we believe that making rapid treatment decisions after thrombolysis according to swi may optimise the patients neurological recovery based on an early detection of mb , ht or ph . 
recently , susceptibilityweighted imaging ( swi ) and t2 - weighted imaging have become sensitivity markers for the accurate detection of mb or ht after ischaemic stroke [ 10 , 12 , 1416 ] , but several studies have shown that swi is superior to t2weighted sequences in detecting mb [ 1719 ]  . 
thus , the purpose of this study was to evaluate the effect of swi for antiplatelet therapy on post - thrombolysis mb . materials and methods study design this prospective clinical study was conducted between january 2009 and december 2011 . 
the institutional review board approved the study , and patient ( or legal representative ) consent was obtained before study enrolment . patients were eligible for thrombolysis if they met the following criteria : ( 1 ) documented clinical signs and symptoms of acute circulation stroke ; ( 2 ) national institutes of health stroke scale ( nihss ) scores [ 20 ] ranging from 4 to 22 at baseline ; ( 3 ) age between 18 and 80 years ; thrombolytic therapy administered within 6 h of symptom onset ; ( 5 ) the absence of intracranial haemorrhage conrmed by computed tomography ( ct ) before and within 24 h of thrombolysis . patients were excluded from thrombolysis if any of the following criteria was present : ( 1 ) intracranial haemorrhage , cerebral stroke of any aetiology , or myocardial infarction in the past 3 months ; ( 2 ) history of alimentary canal or urinary system haemorrhage , or trauma in the last 3 weeks ; ( 3 ) current anticoagulation therapy ; ( 4 ) personal or family history of haemorrhagic tendency or haemorrhagic disease ; ( 5 ) history of circulatory failure or unconrenal , or hepatic trolled hypertension , severe cardiac , inadequacy , severe diabetes , or current pregnancy . addition , patients were also excluded for the following reasons : ( 1 ) lacunar infarctions on mri ; ( 2 ) mri follow - up or clinical scoring was refused ; ( 3 ) mri was incomplete because of technical reasons or the subjects inability to cooperate ; ( 4 ) an apparent diffusion - weighted imaging ( dwi ) lesion exceeding 70 % of the territory of the ipsilateral hemisphere . a total of 204 consecutive patients with acute stroke were screened by mri within 6 h after symptom onset before thrombolysis . 
mri revealed no infarction in 21 patients , lacunar infarction in 19 patients , haemorrhage ( mb = 12 , ht = 2 ) in 14 patients and huge acute ischaemic lesion in four patients . 
the standard dosage for intravenous rt - pa therapy was 0.9 mg / kg body weight , with a maximum dose of 90 mg ; 10 % of the total dosage ( 0.9 mg / kg ) was injected as an intravenous bolus for 1 min , while the remainder was given by continuous intravenous infusion over the next 59 min . after thrombolysis , none of the patients presented sich on ct within 24 h and they were randomly allocated to two groups : group a ( n = 72 ) received antiplatelet therapy 24 h after rt - pa for 10 days , independent of the presence of swi - detected mb or ht . 
the protocol include , dwi , axial fast spinecho ( fse ) with t1and t2 - weighted imaging , threeresonance dimensional time - of - ight magnetic radiol med ( 2014 ) 119 : 175182 angiography ( 3d - tof ) and axial ow - attenuated inversion recovery , and perfusion - weighted imaging ( pwi )  . 
the areas of hyperintensity on dw images were manually traced on each slice , summed and multiplied by the slice thickness and inter - slice gap to obtain the dwi lesion volume [ 21 ]  . denition of different kinds of haemorrhage mb was dened as punctate , homogeneous , rounded , hypointense lesions \0.5 cm in size on swi sequences [ 11 ]  . 
ph was dened as blood clots in the infarcted area with at least slight space - occupying effect . sich was dened as any signs of haemorrhage on followup scans associated with clinical deterioration c4 points on the nihss within 36 h [ 23 ]  . 
the modied rankin scale ( mrs ) [ 24 ] was used together with a semi - structured interview conducted either by telephone or in person to assess clinical outcome at 30 and 90 days after thrombolysis . 
all outcome variables ( favourable outcome , independent outcome , haemorrhage and death ) were assessed independently by physicians unaware of the patients initial stroke severity , time to treatment and imaging modality applied . 
a favourable outcome at day 90 was dened as a mrs score of 1 , and an independent outcome as a mrs score of 2 . statistical analysis after antiplatelet we predicted that favourable outcome at 90 days would be &80 % in group b ( swi - detected haemorrhage patients without thrombolysis ) therapy and &40 % in group a ( swi - detected haemorrhage therapy )  . 
we estimated that patients with antiplatelet 2025 swi - detected haemorrhage patients after thrombolysis in each group would need to be enrolled for the study to have a statistical power of 80 % with a two - sided alpha level of 0.05. 
this power was based on the ability to detect an absolute difference of 40 % in the nal favourable outcome at 90 days . data are presented as mean standard deviation . group comparisons were made using the mannwhitney u test for continuous variables and the fishers exact test for categorical variables . 
all statistical analyses were performed using the spss package , version 13.0 ( spss , chicago , il , usa )  . results demographic data , clinical outcomes and patient ow after thrombolysis of the 146 patients in groups a and b are shown in fig . 
nihss scores at baseline and 6 h after thrombolysis were signicantly worse in group b than in group a , and the hospital stay in group b was shorter than that in group a ( table 1 )  . 
there were no other signicant differences in age , sex , time to thrombolysis , affected hemisphere , nihss score at 24 h , 7 and 14 days 178 fig . 
conrmed diagnosis and differentiation are critical in making acute treatment decisions ; especially , in regard to patient eligibility for thrombolytic and antiplatelet therapy before and after rt - pa thrombolysis [ 25 ]  . this study was designed to evaluate the effect of swi for antiplatelet therapy on post - thrombolysis mb . 
nihss scores at baseline and 6 h after thrombolysis were signicantly worse in group b ( haemorrhage patients not receiving antiplatelet therapy ) than in group a ( haemorrhage patients receiving antiplatelet therapy ) , and hospital stay in group b was shorter than that in group a . 
in addition , the nihss scores at 7 , 14 days and mrs at 30 days after thrombolysis were signicantly lower in the haemorrhage patients in group b than in those in group a ; moreover , hospital stay was signicantly shorter and the rate of favourable outcome at 90 days after thrombolysis was signicantly higher ( table 2 ) in the haemorrhage patients not treated with heparin . a total of 22 ( 78.6 % ) of the 28 haemorrhage patients in group b at 90 days after thrombolysis had a favourable outcome ( mrs b 1 ) ; while 14 ( 63.6 % ) of the 22 haemorrhage patients in group a at 90 days after thrombolysis were moderately disabled ( 2 b mrs b 3 )  . 
although this was an institutional protocol that was not based on solid scientic evidence , the different effect on the patients neurological recovery in swi - detected haemorrhage patients in our two groups with and without antiplatelets conrmed the value of swi - based guidance for antiplatelet therapy after thrombolysis . some studies suggest mb is a potential marker of a haemorrhage - prone state [ 46 ] , and anecdotal case reports have described patients with mb who developed therapyrelated catastrophic post - ischaemic haemorrhage [ 14 ]  . 
in our study , the results demonstrated that mb , ht and ph could progress into each other if no appropriate treatment plan for rt - pa thrombolysis is available . 
the reason for the higher rate of mb is mainly attributed to the application of swi , which may be superior to t2 - weighted imaging in detecting mbs [ 1719 ]  . our study has several limitations : it was a prospective study , but it was not randomised or controlled . 
the mri or ct examinations were performed only in patients with sich or an increase of nihss scores c4 points after rt - pa thrombolysis so as to subsequently stop antiplatelet therapy that sich was detected . 
2 a 69 - year - old female patient from group a with acute ischaemic stroke for 120 min and a baseline nihss ( national institutes of health stroke scale ) score of 12 . 
a , e baseline diffusionweighted imaging ( dwi ) shows a slighter hyperintense area of the right basal ganglia , and susceptibility - weighted imaging ( swi ) shows the haemorrhage - free temporal lobe . 
b , f after 24 h of thrombolysis , dwi shows a mixed hyperintense area of the right basal ganglia , and swi shows the temporal lobe with blurred haemorrhagic transformation ( ht ) at the lateral part of the lenticular nucleus . 
c , g after 7 days of thrombolysis , dwi still shows a mixed hyperintense area of the right basal ganglia , and swi shows the temporal lobe with distinct ht . 
d , h after 14 days of thrombolysis , dwi shows a slighter hyperintense area with some microbleeds , as seen on relatively late for this examination and thus more difcult to identify mb or ht , which further delayed the patients neurological recovery , as occurred in the haemorrhage patients in group a . 
although detection of haemorrhage by mri at 6 and 24 h after rt - pa may be inconvenient , early accurate detection of haemorrhage and prompt treatment decisions according to swi play an important role for the patients neurological recovery . 
another study limitation was that mri scan times were obviously longer than those of ct . in conclusion , our results indicated swi was an effective approach for the guidance of antiplatelet therapy after thrombolysis . 
imaging , along with clinical and laboratory evaluation , is an important tool to reach a correct diagnosis and to provide a precise grading of disease progression , inuencing both clinical management and therapy . 
computed tomography ( ct ) demonstrates with a very high spatial resolution the tiny structural alterations of cortical and spongy bone before they become evident on plain lm radiographs . magnetic resonance imaging ( mri ) is the only modality that provides demonstration of bone marrow oedema , f . 
cimmino dipartimento di medicina interna , clinica reumatologica , universita` degli studi di genova , viale benedetto xv 6 , 16132 genoa , italy which reects vasodilatation and inammatory hyperaemia . 
the primary aim of this review article was to examine the involvement of the spine and sacroiliac joints in spa using a multimodal radiological approach ( radiography , ct , mri ) , providing a practical guide for the differential diagnosis of these conditions . keywords spondyloarthritis ( cid : 2 ) spine ( cid : 2 ) sacroiliac joints ( cid : 2 ) sapho ( cid : 2 ) ankylosing spondylitis introduction ( spa ) seronegative spondyloarthropathies represent a group of chronic articular inammatory diseases which differ from rheumatoid arthritis for the typical absence of rheumatoid factor in serum , and have in common several epidemiological , pathological , clinical and radiological features . 
the positivity for class i human histocompatibility leukocyte antigen ( hla ) b27 is related to a 20 - fold increased risk of developing a spa as compared to the general population [ 1 ]  . 
in 2005 , it was proposed to divide spa into ve subgroups [ 2 ] : ankylosing spondylitis , psoriatic spondyloarthritis , reactive spondyloarthritis ( reiters syndrome ) , ( related to inammatory bowel diseases such as crohns disease and ulcerative colitis ) and undifferentiated spondyloarthropathies . 
regardless of subgroup , the main clinical manifestations of spa include inammatory back pain ( mainly at night , which worsens with rest and improves with movement ) caused by sacroiliitis or inammatory involvement thoracic spine ; peripheral of the lumbar and / or distal arthritis ( often oligoarticular and asymmetric ) ; enthesitis and extra - skeletal manifestations , such as uveitis [ 3 ]  . 
with the development of more extensive and exible criteria by spondyloarthritis enteropathic radiol med ( 2014 ) 119 : 156163 the european spondyloarthropathy study group [ 3 ] , the sapho syndrome ( synovitis , acne , pustulosis , hyperostosis and osteitis ) may also be considered a spa , even though there is no general agreement in this regard . 
sapho syndrome and spa have in common many clinical and laboratory features , primarily seronegativity for rheumatoid factor and inammatory involvement of the axial skeleton , despite only a weak association with hla - b27 antigen having been identied for the sapho syndrome . 
in spa , the most characteristic targets of the autoimmune process are entheses , anatomical structures made up of brous or bro - cartilaginous tissue , which provide anchorage for tendons and ligaments to the bone surface [ 5 , 6 ]  . 
enthesitis is accompanied by the appearance of small erosions in the cortical bone , typically surrounded by subcortical reactive osteosclerosis ( osteitis ) , which can often prevail over bone reabsorption ; in the later stages osteoproliferation leads to the ossication of ligaments , tendons and joint capsules , and , eventually , to ankylosis [ 6 , nonhomologous including ankylosing spondylitis the incidence of ankylosing spondylitis , the archetype of spa , is between 0.1 and 2 % , and the age of onset is less than 30 years in 80 % of patients . 
the classic form ( primary or idiopathic ) must be distinguished from the secondary one , which can appear during the course of psoriasis or enteropathic arthritis [ 9 ]  . 
subchondral osteosclerosis decreases in the following stages , which are characterised by an increase in the fatty content of cancellous bone ( post - inammatory fatty conversion of bone marrow )  . 
the remnants of the joint space are sometimes visible as a thin sclerotic line on plain lm radiographs , which appears hyperdense on ct images [ 12 ]  . in the light of new and promising treatment options , such as anti - tnf ( tumour necrosis factor ) a agents , early diagnosis is of great clinical signicance . 
magnetic resonance imaging ( mri ) is the only imaging modality that may provide an early diagnosis , showing bone marrow oedema , which appears hypointense on t1 - weighted images and hyperintense on uid - sensitive stir ( short - tau inversion recovery ) or t2 - weighted images with fat signal suppression . 
gadolinium - enhanced fat - suppressed t1weighted images can help in the diagnosis by showing contrast enhancement of the subchondral cancellous bone and / or joint space [ 5 , 12 ]  . 
spinal involvement , which may be earlier , contemporary or subsequent to the onset of sacroiliitis , occurs with greater frequency at disco - vertebral junctions and at the level of interapophyseal and costo - vertebral includes enthesitis with bone erosion , post - inammatory fatty conversion of red bone marrow , osteosclerosis and osteoproliferation with ectopic bone formation . 
the enthesitis of the disco - vertebral junction , which occurs at the level of the anterior and posterior corners of vertebral bodies , is associated with erosions , resulting in the typical lesions of romanus . 
osteosclerotic foci develop at the site of corner erosions ( bright corners or shiny corners ) ; they progressively enlarge , causing the squaring of the endplates joints . 158 radiol med ( 2014 ) 119 : 156163 fig . 
a anteroposterior radiograph shows bilateral grade 3 sacroiliitis with diffuse subchondral sclerosis involving the iliac ( antero - inferior ) portion of both sacroiliac joints and widening of the joint space , which is more evident on the left side ( arrows )  . 
c axial ct scan shows bilateral grade 3 sacroiliitis with dense spongiosclerosis along the iliac side of sacroiliac joints ( arrows ) and some left - sided erosions ( small arrows )  . 
in advanced stages the ossication of vertebral ligaments may ( longitudinal , supraand interspinous ) occur , leading to ankylosis [ 5 , 19 ]  . the presence of symmetric syndesmophytes along the whole spine ( cervical spine is the last rachidian region to be leads to the characteristic bamboo spine involved ) appearance . 
in about 8 % of patients with ankylosing spondylitis an extensive inammatory involvement of the intersomatic disc and adjacent vertebral endplates may occur , the so - called andersson spondylodiscitis [ 20 ]  . 
on mri examination spondylodiscitis is characterised by a diffuse hypointense signal on t1 - weighted images involving the intersomatic disc and adjacent vertebral endplates . it appears hyperintense on uid - sensitive sequences and shows signicant enhancement after intravenous administration of gadolinium - based contrast agents [ 7 , 21 ]  . 
3 a , b 37 - year - old man with primary ankylosing spondylitis . a sagittal t2 - weighted and b corresponding t2 - weighted fatsuppressed magnetic resonance ( mr ) images show a focus of high signal intensity at the antero - superior corner of l1 body ( arrow in a and b ) , which is called shiny corner . 
unilateral asymmetric distribution and separation from the lateral aspect of the vertebral bodies are the main radiological features differentiating psoriatic from those of syndesmophytes ( para - syndesmophytes ) ankylosing spondylitis and spondylitis associated with inammatory bowel diseases . 
according to de vlam et al . [ 27 ] , the morphology of syndesmophytes is related to the degree of functionality of the spinal motion segment in which they develop . 
if the interapophyseal joints of this segment maintain a normal mobility , the tensile forces acting on the spine prevent the formation of linear and thin syndesmophytes , and the 160 radiol med ( 2014 ) 119 : 156163 fig . 
5 a , b pathological vertebral fracture in a 58 - year - old man with ankylosing spondylitis of 21 years duration after a minor trauma . a sagittal multiplanar reformation and b corresponding surfacerendered ct image clearly dene the through - and - through fractures ( arrows in a ) involving the anterior longitudinal ligament , intervertebral space , the spinous processes , the interand supraspinous ligaments . 
note the ossication of interspinous and supraspinous ligaments ( thin arrows in a and b ) and the fracture line on the latter ( arrowhead in a and b ) fig . 
a coronal multiplanar reformatted ct image shows subchondral bone sclerosis and multiple erosions involving the iliac aspect of the left sacroiliac joint associated with ( pseudo - ) widening of the joint space ( arrow )  . 
b coronal t2 - weighted fat - suppressed mr image demonstrates a large area of bone marrow oedema on the iliac aspect of the left sacroiliac joint ( asterisk )  . 
these ndings are typical of active sacroiliitis . c anteroposterior radiograph of the lumbar spine and d the corresponding coronal multiplanar reformatted ct image show a left - sided nonmarginal bulky syndesmophyte ( arrow in c and d ) ; its coarse and wavy appearance is different from the short and thin syndesmophyte of ankylosing spondylitis paravertebral ossication has a solid and coarse appearance ( non - marginal bulky syndesmophytes , or para - syndesmophytes ) ; on the contrary , reduced mobility of the interapophyseal joints leads to the development of thin and linear syndesmophytes . 
the authors [ 27 ] observed a direct relation between ankylosis of interapophyseal joints and the development of thin and linear syndesmophytes ( like those of ankylosing spondylitis ) ; on the contrary , they found coarse paravertebral ossication in cases where articular mobility was maintained . the main radiological ndings of psoriatic sacroiliitis , which may also occur in the absence of spondylitis , are : erosions , which are more frequent on the iliac portion of joint space narrowing , and , the joint , osteosclerosis , radiol med ( 2014 ) 119 : 156163 fig . 
7 a , b 52 - year - old man with crohns disease and sacroiliitis . a axial ct enterography image shows thickening and mural stratication with mucosal hyper - enhancement of an ileal loop affected by crohns disease ( arrow in a )  . 
note the total bony ankylosis of the right sacroiliac joint ( arrows ) and partial ankylosis ( thin arrow ) of the left sacroiliac joint rarely , ankylosis [ 28 ]  . 
interapophyseal joints of the cervical spine are frequently involved ( in contrast to what happens in the thoraco - lumbar spine ) , and they may eventually become ankylotic , while anterior atlo - axial subluxation is much more rare [ 29 ]  . reactive spondyloarthritis ( reiters syndrome ) the eponym reiters syndrome indicates a clinical syndrome characterised by conjunctivitis , urethritis or gastroenteritis , followed by the onset of aseptic arthritis after an interval of 14 weeks . 
according to this theory , an infection of the conjunctival , intestinal or urethral - genital mucosa may represent the trigger for a systemic autoimmune reaction which determines an aseptic inammatory process involving the axial skeleton or appendicular joints . the involvement of the appendicular skeleton is typically represented by asymmetrical oligoarthritis , often unilateral , which shows a predilection for the large synovial joints of the lower limbs [ 30 , 31 ]  . 
the involvement of the sacroiliac joints is frequent ( 510 % in the early phase , up to 4060 % in the advanced stages ) and has the same radiological features of psoriatic sacroiliitis [ 30 ]  . 
in fact , a typical early nding is represented by coarse syndesmophytes , which are not distinguishable from those of psoriasis and are located most frequently at the level of the lower three thoracic and the upper three the interapophyseal involvement of lumbar vertebrae . joints is associated with erosions , osteosclerosis and ankylosis , but these ndings are less frequent than those observed in ankylosing spondylitis [ 31 ]  . enteropathic spondyloarthritis ( enteroarthritis ) often transient asymmetrical , enteropathic spondyloarthritis , or enteroarthritis , related to the two major chronic inammatory bowel diseases , ulcerative colitis and crohns disease , of which they are considered extraintestinal manifestations . 
with regard to axial arthritis , the incidence of sacroiliitis varies from 10 to 20 % , while that of spondylitis from 7 to 12 % [ 33 ]  . 
in a recent study [ 34 ] , the review of 221 ct enterography examinations of patients with histologically conrmed crohns disease showed a prevalence of sacroiliitis ( up to 24 % ) higher than that of the other extraintestinal manifestations . 
radiological features of both spondylitis and sacroiliitis are closely similar to those of primitive ankylosing spondylitis [ 35 ]  . sapho syndrome the acronym sapho was rst used by charmot et al . 
according to the more widely accepted pathogenetic hypotheses , sapho syndrome has been considered a reactive osteitis , which in genetically predisposed represents the consequence , 162 radiol med ( 2014 ) 119 : 156163 fig . 
8 ae 24 - year - old man with palmoplantar pustulosis and low back paa sagittal t1 - weighted , b t2 - weighted fat - suppressed , and c contrast - enhanced t1 - weighted fat - suppressed mr images of the lumbar spine show two signal alterations at the level of the anterosuperior corners of l4 and l5 vertebrae ( arrows in a , b and c )  . 
d axial t2 - weighted fat - suppressed mr image passing through l5 body conrms the corner lesion ( arrow ) and e the corresponding axial ct scan clearly demonstrates the corner erosion of the vertebral endplate surrounded by spongiosclerosis ( arrow ) individuals , of a nonspecic and inadequate immune response , triggered by infection with some microorganisms [ 37 ]  . 
sapho is currently considered a rare disease with an incidence of 1 in 10 , 000 individuals , although this gure probably underestimates the real incidence of the disease , because of the wide spectrum of clinical manifestations and the lack of valid diagnostic criteria . 
the syndrome shows a slight preference for females with a mean age at diagnosis of 28.6 years , and a chronic relapsing and remitting course . many clinical features of sapho syndrome , including involvement of the axial skeleton and entheses , seronegativity for rheumatoid factor , and the association with some varieties of psoriasis and enteroarthritis , suggest a link with spa , even if a signicant correlation with histocompatibility antigen hla - b27 has not yet been clearly demonstrated [ 38 ]  . 
according to the criteria dened by kahn et al . [ 39 ] , sapho syndrome should be suspected in patients with pustular skin lesions and musculoskeletal pain caused by an osteoarticular aseptic inammation . 
the bone involvement concerns mainly the sterno - costo - clavicular region ( 7090 % ) , the spine ( 30 % ) and the sacroiliac joints ( 1352 % ) [ 38 ]  . 
franchi department of pathology , university of florence , florence , italy patients underwent wide excision ( 32 out of 38 ) and ve patients had marginal surgery ; only one patient underwent amputation . 
at the time of our analysis 13 patients ( 34.2 % ) had died due to metastatic spread of the disease . in our series , dfs in relation to distant metastases ( dm ) showed a signicant result for lower limb involvement ( p = 0.038 ) and marginal excision ( p = 0.024 ) , both predictors of a worse dfs , histology was statistically signicant although it was not possible to evaluate the risk for specic histology due to the small number of events in the different subtypes . conclusions the results obtained from our study are encouraging with regard to the feasibility and efcacy of preoperative rt in the treatment of soft tissue sarcoma in view of the results obtained in terms of local control , limb sparing and safety . keywords neoadjuvant ( cid : 2 ) chemotherapy ( cid : 2 ) radiation ( cid : 2 ) chemoradiation ( cid : 2 ) soft tissue sarcoma introduction soft tissue sarcomas ( sts ) are rare malignant neoplasms that originate from mesenchymal cells that can differentiate into different types of connective tissues . 
local management of sts usually consists of surgery combined with radiotherapy ( rt )  . for many years local excision of the tumour with postoperative rt was a well - established treatment approach providing good local control and acceptable 196 radiol med ( 2014 ) 119 : 195200 functional outcome [ 2 , 3 ]  . 
in the case of bulky disease or tumours located in critical areas , neoadjuvant rt with or without chemotherapy is employed to improve the chance of limb sparing , local control and functional outcome [ 4 ]  . neoadjuvant rt has the advantage of delivering a lower total dose and involving a smaller target volume than postoperative rt , and this results in less brosis and tissue damage and a theoretically better functional outcome [ 5 ]  . on the other hand , the preservation of function could be impaired due to the higher incidence of wound complications after preoperative rt [ 68 ]  . 
in high - grade sts , the risk of developing distant metastases increases with the increasing tumour size , reaching about 58 % in 15.120 cm lesions [ 9 ]  . 
the aim of our study was to evaluate disease - free survival ( dfs ) , overall survival ( os ) and toxicity of patients who underwent preoperative rt for soft tissue sarcoma . materials and methods neoadjuvant chemo - radiation our study analysed the data of 38 consecutive patients affected by soft tissue sarcoma . 
patients younger than 18 years at diagnosis , histological diagnoses of ewings sarcoma , rhabdomyosarcoma , desmoid tumours , dermatobrosarcoma protuberans , previously irradiated patients or with recurrent disease were not considered eligible for the study . clinical stage was dened according to the enneking surgical staging system for musculoskeletal tumors [ 10 ]  . tumour size , calculated on the major axis of the lesion , was measured on clinical evaluation and conrmed by preoperative imaging . 
before treatment 32 patients underwent a diagnostic magnetic resonance imaging ( mri ) study and 6 of them a computed tomography ( ct ) scan to dene tumour size and extension ; all patients underwent chest ct or radiogram for staging . 
to dene the histological characteristics of the tumour all patients underwent a biopsy , 32 ( 94.7 % ) received an incisional biopsy , 2 an excisional one . preoperative chemotherapy was administered concurrently with rt in the event of high - grade neoplasm , prognostically unfavourable histology , and bulky primary tumour : it consisted of two cycles of epirubicine ( 60 mg / m2 for days 12 ) and ifosfamide ( 1 , 800 mg / m2 for days 15 )  . nrt was performed with 3d conformal technique delivering 2 gy fractions up to total dose of 50 gy . 
at pathological assessment , margin status was assigned using the criteria of enneking [ 10 ]  . histological grade was classied by fcnlcc ( federation nationale des centres de lutte contre le cancer ) fourtiered system ( grades i to iv ) [ 11 ]  . 
occurrence of late toxicity was recorded all along the follow - up period and classied according to the rtog ( radiation therapy oncology group ) toxicity scale . statistical analysis the end of rt was used as the start of observation for the survival analyses . 
stage iii disease predicted a worse prognosis than lower stages , even though this difference did not reach statistical signicance ( p = 0.10 ) at univariate analysis , probably as a result of the small number of events among stage i patients . 
chemotherapy did not reach any signicant value in terms of reducing distant metastases in sarcoma patients . at multivariate analysis , the main independent dm predictors were stage iii and marginal surgery ( table 3 )  . regarding treatment tolerance , acute skin toxicity was mild : only two patients experienced grade 2 toxicity . 
in a recent survey , the choice of a neoadjuvant treatment was shown to be not only inuenced by well - dened treatment volume , increased acute morbidity and decrease in late morbidity but also mostly by specialty and clinical experience or institutional preferences , resulting in lack of clear consensus on eligibility criteria for preoperative treatment [ 15 ]  . 
expected benets from preoperative rt consist of dose reduction with respect to postoperative rt , with a better functional outcome [ 16 ] , narrower treatment elds allowed by lack of surgical contamination , shrinking of tumour boundaries that facilitates resection , and the formation of capsular brosis potentially preventing intraoperative tumour scattering [ 5 , 17 ]  . 
we reported data from 38 consecutive patients who underwent preoperative radiation treatment for soft tissue sarcoma at our institution . local control was achieved in 36 ( 95 % ) of cases , with 2 patients ( 5 % ) showing in - eld relapse . 
in our review , we found that dfs was signicantly inuenced by surgical margins status , histology and tumour site . in sts management , several studies underline the impact of negative surgical margins in predicting the clinical outcome [ 2022 ]  . 
three patients ( all with positive margins ) experienced local failure as rst relapse ( two isolated , one with distant failure ) , and two additional patients ( all with margin \1 mm ) had late local failure after distant metastasis . 
the authors concluded that no local recurrences were observed with surgical margins equal or larger than 1 mm , and it these may be adequate for patients with appears that extremity sts treated with preoperative rt [ 23 ]  . 
sts are heterogeneous diseases , and some histological subtypes can differ in the natural tumour history and prognosis : for example , well - differentiated liposarcomas are considered low - grade tumours owing to the low incidence of distant recurrence , whereas pleomorphic liposarcomas have a poor prognosis [ 28 ]  . 
a strong clinical interest therefore guides the choice of more aggressive treatment approaches depending on the histological type . at the time of our analysis , 13 patients had died due to metastatic spread of the primary disease . 
 [ 29 ] in a retrospective series of 112 cases including surgery alone ( sa ) ( n = 36 ) , ncrt ( n = 39 ) , and neoadjuvant radiotherapy ( nrt ) ( n = 37 ) reported that ncrt did not improve the rate of margin - negative resections over sa or nrt . locoregional relapse - free survival , distant metastasesfree survival , and overall survival ( os ) were not different among the treatment groups . 
patients with relapsed disease ( or 11.6 ; p = 0.01 ) and tumour size greater than 5 cm ( or 9.4 ; p = 0.01 ) were more likely to have a locoregional recurrence on logistic regression analysis . the eortc 62874 trial is the only randomised clinical study that investigated the role of neoadjuvant chemotherapy for the treatment of high - risk soft tissue [ 30 ]  . 
at a median follow - up of 7.3 years no large benet was shown for preoperative chemotherapy , and the chemotherapy group had a slightly higher 5 - year dfs rate and overall survival than the surgery group but the study was not powered for statistical signicance due to slow patient accrual . 
the data for neoadjuvant chemotherapy are still few and controversial , and this is due to the difculty in performing large , randomised trials for a rare disease such as soft tissue sarcoma . 
new drugs such as biological approaches have to be investigated . acute treatment tolerance was generally excellent , even though six patients ( 15.8 % ) had wound complications ( ve cases of wound dehiscence and one of stula ) , with only two requiring minor surgical procedures . 
the multitumours with a limb - salvage modal management of approach could favour impaired wound healing in the postoperative period and the occurrence of long - term complications [ 3133 ]  . 
although preoperative treatment is associated with an increased risk of major wound complications in the order of 35 % [ 31 ] , local anatomy and tumour size are important prognostic factors for acute complications [ 24 , 34 , 35 ]  . the results obtained in our study are encouraging with regard to the feasibility and efcacy of preoperative rt in terms of local control , limbs sparing and safety . conict of interest daniela greto , lorenzo livi , calogero saieva , pierluigi bonomo , icro meattini , mauro loi , lucia di brina , giovanni beltrami , domenico campanacci , guido scoccianti , rodolfo capanna , monica mangoni , fabiola paiar , alessandro franchi , giampaolo biti declare no conict of interest . radiol med ( 2014 ) 119 : 195200 retrospective comparative evaluation of disease outcome . 
al yami a , grifn am , ferguson pc et al ( 2010 ) positive surgical margins in soft tissue sarcoma treated with preoperative radiation : is a postoperative boost necessary ? int j radiat oncol biol phys 77 : 11911197 21 . 
davis am , osullivan b , bell rs et al ( 2002 ) function and health status outcomes in a randomized trial comparing preoperative and postoperative radiotherapy in extremity soft tissue sarcoma . 
curtis kk , ashman jb , beauchamp cp et al ( 2011 ) neoadjuvant chemoradiation compared to neoadjuvant radiation alone and surgery alone for stage ii and iii soft tissue sarcoma of the extremities . 
davidge km , wunder j , tomlinson g et al ( 2010 ) function and health status outcomes following soft tissue reconstruction for limb preservation in extremity soft tissue sarcoma . 
livi l , santoni r , paiar f et al ( 2006 ) late treatment - related complications in 214 patients with extremity soft - tissue sarcoma treated by surgery and postoperative radiation therapy . 
tseng jf , ballo mt , langstein hn et al ( 2006 ) the effect of preoperative radiotherapy and reconstructive surgery on wound complications after resection of extremity soft - tissue sarcomas . ann surg oncol 13 : 12091215 35 . 
the ss and sr were assessed and the results were expressed in terms of sensitivity , specicity , positive predictive value ( ppv ) and negative predictive value ( npv )  . 
simonetti dipartimento di diagnostica per immagini , imaging molecolare , radiologia interventistica e radioterapia , fondazione policlinico universitario tor vergata , viale oxford 81 , 00133 rome , italy e - mail : ire.coco@hotmail.it keywords thyroid nodules ( cid : 2 ) elastography ( cid : 2 ) strain ratio ( cid : 2 ) stiffness score ( cid : 2 ) fnac introduction thyroid nodules are frequently encountered clinically . 
the incidence increases in direct proportion to age , and is greater in women with an f / m ratio of 4 : 1 , in geographic areas with iodine deciency as well as following exposure to ionising radiation [ 1 ]  . although the incidence of thyroid carcinoma is estimated at around 4 / 100 , 000 individuals / year , it should be noted that the majority of thyroid nodules are benign ; in fact , it is known that the percentage of carcinomas in palpable nodules is only 4 % and rises to 8 % if we consider the various surgical series [ 2 ]  . 
therefore , the main objective of the evaluation of thyroid nodules is to exclude malignancy [ 3 ]  . the rigidity of nodular thyroid tissue is considered an important indicator of malignancy . 
elastosonography ( es ) is a newly developed method that provides information on parenchymal elasticity by colour coding the different degrees of mechanical - elastic deformation suffered by the structures being analysed in response to radiofrequency pulses ( rf )  . 
this new ultrasound method therefore aims to provide a real - time noninvasive characterisation of tissue during the same session as conventional ultrasound imaging and without takes advantage of the different mechanical properties of the various tissue components and the loss of the physiological elasticity in many diseases . 
it 150 radiol med ( 2014 ) 119 : 149155 the vascularity of each nodule was assessed by power doppler ( pd ) and classied as : no ow ( pattern i ) , predominantly peri - nodular ow ( pattern ii ) , intra - nodular and peri - nodular ow ( pattern iii )  . elastosonography the es study was carried out together with conventional ultrasound . 
use of the iu22 philips system and probe provided repeatable and nonoperator - dependent measurements since axial compression is applied automatically by the probe and not manually by the operator . deformation of the structures was achieved with mechanical compression obtained by the emission of lowfrequency pulses [ 7 ]  . during the examination , the system calculates the levels of resulting tissue deformation and displays them as a green and grey bar ( feedback bar ) on the side of the image . the bar indicates an appropriate ( green ) or inappropriate ( grey ) tissue deformation in order to obtain a good - quality elastograthe bar displays the tissue deformation in real time , and if the extent of the deformation is excessive the system does not show the corresponding elastography image . 
the data obtained were processed using dedicated q - lab software which employs a suitable algorithm to assess the degree of tissue distortion . in a very short time , the system processes the information coming from the lesion in the form of rf pulses . 
this information is obtained using the normal ultrasound probe and applying a vertical compression to the tissues ; compression can be obtained manually or automatically by the probe , as done in our study . 
this way , the tissues undergo varying degrees of deformation in relation to their elasticity , and return different signals . in recent years , the value of this technique has been demonstrated in the diagnosis of breast nodules , prostate cancer and liver brosis . 
more recently , this method has been proposed for the characterisation of thyroid nodules , and many authors have investigated the usefulness of this method based on the analysis of qualitative colour data [ 5 , 6 ]  . 
this parameter , by exploiting the difference of the elastic coefcients , allows for quantication of the stiffness of the nodule in comparison to the surrounding healthy parenchyma , by studying the deformation ratio ( sr - strain ratio )  . the aim of our study was to determine the diagnostic accuracy of es in the differential diagnosis of thyroid nodules , by means of a qualitative and quantitative evaluation . materials and methods study population the retrospective single - centre study was conducted between december 2010 and march 2012 . 
2 colour score of the thyroid nodules for increasing degree of stiffness ( score 1 for elastic nodules ; score 2 and 3 for intermediate stiffness and score 4 for anelastic nodules ) fig . 
this cut - off value was dened through the analysis of receiver operating characteristic ( roc ) curves in order to maximise the sum of the specicity and sensibility . fine - needle aspiration cytology and histological evaluation all nodules underwent us - guided ne - needle aspiration cytology ( fnac ) , after approval by the local ethics cominformed consent . mittee and obtaining the patients 152 sampling was performed using 21 - gauge needles with a 10 - ml syringe ; the material collected and prepared with the thin - prep method was sent to the medical pathologist together with a statement containing the patients data , the clinical question , the characteristics of the aspirate , and the number of samples taken for each nodule . the results of the cytological study were classied into ve categories , indicated by the abbreviation tir ( siapec consensus review , 2007 ) : tir 1 : nondiagnostic ( inadequate ) ; tir 2 : negative for malignant cells ( benign ) ; tir 3 : indeterminate ( follicular proliferation ) ; tir 4 : suspicious for malignancy ; tir 5 : positive for malignant cells . patients with indeterminate or suspicious for malignancy cytology underwent surgical treatment ; the surgical specimen was subjected to histological analysis to determine the nature of the lesion . statistical analysis continuous variables ( real numbers ) were expressed as mean and standard deviation and discrete variables ( natural numbers ) as number and percentage . 
roc curves were also used to extrapolate the optimum cut - off value for sr . cytological analysis , combined with histological analysis , was used to determine the benignity and malignancy of nodules . 
overall , the cytological and histological analysis revealed 316 benign nodules and 52 malignant nodules . b - mode and power doppler ultrasound the demographic and ultrasonographic characteristics of the study population are shown in table 1 . 
therefore , the differential diagnosis of nodules is crucial to allow the early diagnosis of cancers and ensure appropriate treatment . currently , thyroid ultrasound is the rst - line examination for the identication and follow - up of nodules since it makes it possible to formulate a suspicion of malignancy and select the nodules to be subjected to fnac ( table 4 )  . cytological evaluation is considered the most accurate diagnostic technique [ 9 ]  . 
however , it has two limitations : nondiagnostic results due to inadequate cytology sampling and the presence of follicular proliferation or hartle cells . fnac is also reserved for nodules with a maximum diameter exceeding 10 msmaller nodules are not subjected to fnac unless they show suspicious ultrasonographic features and ow . follicular proliferation or in the presence of hartle cells , cytology does not differentiate adenoma from carcinoma , as the differential diagnosis is based on the presence of capsular or vascular invasion , which can only be evaluated histologically after thyroidectomy . in the case of elastosonography allows for noninvasive , real - time tissue characterisation which provides additional information to b - mode and power doppler ultrasound . 
although sonographic and doppler features are the criteria taken into consideration for fnac performance and allow an accurate identication and follow - up of thyroid nodules , they do not contribute signicantly to their characterisation , which requires cytological evaluation . on this basis , our study compared the information provided by ultrasound and es with the cytological diagnosis achieved by fnac in on a cohort of patients with a sonographic diagnosis of thyroid nodules . the es technique made it possible to evaluate the mechanical - elastic properties of tissues , and in particular the stiffness of the nodules analysed . 
stiffness was analysed in terms of colour map ( ss ) and by comparison with the stiffness of the surrounding healthy parenchyma , by calculating the deformation ratio ( sr )  . 
the results obtained proved to be superior to those of conventional ultrasound . in fact , although the typical ultrasonographic features of malignancymicrocalcications ( p = 0.003 ) and vascularity pattern iii ( p = 0.0016 ) were signicantly present in malignant nodules , with sensitivity of 27 and 63 % and specicity of 96 and 80 % , respectively , degree of malignancy was more strongly correlated with analysis of the es colour scores ( p = 0.0002 ) , reaching 91 % sensitivity and 68 % specicity . the nding of 104 false - positive results , represented by scores c3 , with a ppv of 27 % , indicates an important limitation of this technique and requires caution in drawing conclusions . from another point of view , it should be emphasised that score 1 was found in benign lesions only , with 98 % npv . 
this nding , if conrmed in a larger study , may exclude immediate recourse to fnac , which would be reserved for nodules showing a change in size or morphology over time . with regard to the calculation of the sr , the signicant values reported in recent studies were not reached [ 1012 ] in discriminating benign from malignant forms . 
however , although our case series was limited , it was still possible to the presence of higher values in malignant highlight nodules . a limitation of our study is the failure to assess intraand interobserver variability in both the qualitative and quantitative indexes . in agreement with the research published to date , our study conrmed that the benets of es are many : it is easy to perform , fast , accurate and reproducible ; it provides a qualitative assessment of tissue elasticity in real time ; it provides a semiquantitative evaluation of the sr ; increases the sensitivity , specicity and npv of conventional ultrasound in the diagnosis of thyroid cancer ; it can potentially help to select patients requiring invasive diagnosis ( fnac ) , thus lowering the costs for further investigations . conclusions currently available knowledge indicates that es is a complementary tool in the diagnosis of thyroid cancer , as it radiol med ( 2014 ) 119 : 149155 can complement conventional ultrasound for the identication and follow - up of thyroid nodules . 
we believe that es can be used as an additional imaging method in the differential diagnosis of nodular thyroid disease , since its values has been documented particularly in combination with sonographic features and vascularity . moreover , considering the absence of false - negatives in our study , es could be an alternative in the differential diagnosis in the case of nondiagnostic fnac , in nodules that do not meet the criteria for performing fnac ( e.g. , maximum diameter \10 mm ) , or in the case of technical difculties in fnac execution . compared to es with free - hand compression , the method used in our study has shown certain advantages . 
to evaluate whether these indices may be determinants of success in prosthetic surgery for stress urinary incontinence , we conducted a retrospective study of patients treated with tension - free vaginal tape - obturator ( tvt - o ) surgery and assessed , by measuring the pubourethral distance and angle after tvt - o , whether there was any quantitative difference between the mean values measured in the group of cured patients and uncured patients . materials and methods we selected 51 patients who underwent tvt - o and evaluated the failure rate by means of urogynaecological assessment . 
schettino department of gynaecology , obstetric and reproductive science , second university of studies of naples , largo madonna delle grazie 1 , 80100 naples , italy e - mail : mt.s76@libero.it introduction the mechanisms of urinary continence is ensured by the coexistence of several morphological and functional factors which guarantee regular micturition in its two phases of bladder lling and voiding . 
more specically , these factors are detrusor stability , anatomical and neurovascular integrity of the urethra [ 1 ] , adequate competence and tone of the external urethral sphincter , optimal functional synergy between the pelvic and urogenital diaphragm , and the presence of adequate oestrogen levels . 
stress urinary incontinence ( sui ) is dened by the international continence society ( ics ) as a pathological condition characterised by involuntary urine loss during a rise in intra - abdominal pressure in the absence of detrusor contraction [ 2 ]  . 
the incidence of sui is rising in part as a result of the higher average age of the female population and of the associated risk factors that exasperate the condition , with a signicant impact on daily life due to both incontinence episodes and greater susceptibility to urinary tract infections [ 3 , 4 ]  . in view of the extent of the problem and of the fact that it also affects young women and , to a lesser extent , even young athletes [ 5 ] , in recent decades the treatment of sui has represented one of the most interesting areas of research in the urogynaecological eld . 
evidence that in most cases the defect underlying sui is urethral hypermobility in addition to an intrinsic sphincter deciency induced specialists to look for minimally invasive surgical techniques capable of restoring and preserving a correct urethral axis , allowing adequate dynamic closure of the urethra during exertion . 
for these reasons , the introduction of midurethral slings has revolutionised stress incontinence surgery , becoming the gold standard in the treatment of sui . since the advent over 10 years ago of the rst retropubic midurethral sling , tension - free vaginal tape ( tvt ) , many 190 radiol med ( 2014 ) 119 : 189194 attempts have been made to increase the success rate of these devices by reducing the risk of complications . 
to this end , in 2001 delorme introduced the rst trans - obturator sling ( tvt - o , tension - free vaginal tape obturator ) , which , by involving a trans - obturator approach and avoiding the retropubic space , reduced the risk of vesical , intestinal and vascular lesions , even though the success rates are slightly lower than those of tvt [ 69 ]  . 
success is intended as the restoration of a physiological urethral mobility guaranteeing continence even in the presence of a rise in intra - abdominal pressure . an ability to quantify the degree of urethral mobility by means of imaging modalities would therefore make it possible not only to recognise the exact type of anatomical defect but also to quantify it and rationally stratify the different treatments available , with a view to selecting the most appropriate treatment and optimising the results . urethral mobility is currently evaluated in a general manner using the q - tip test , but the need to standardise this evaluation is becoming increasingly clear , and the international continence society recommends and encourages research aimed at developing diagnostic techniques for the objective assessment of urethral mobility . 
to this end , the introduction of perineal ultrasound in the diagnostic workup of sui is being intensely discussed , as it has shown to be the most sensitive examination for evaluating urethral mobility . 
the technique , in fact , enables measurement of the pubo - urethral distance and angle both under exertion and at rest . the aim of this study was therefore to evaluate whether the degree of urethral mobility can be a determinant of success of a minimally invasive prosthetic procedure . 
a retrospective study of all patients treated with tvt - o for pure sui was conducted to establish whether there was a quantitative difference in the pubo - urethral distance and angle between the group of patients successfully cured with tvt - o and the group of patients with persistent sui despite tvt - o placement . materials and methods the study was approved by our departments ethics committee and informed consent was obtained from all the patients enrolled in the study . we selected 51 patients [ mean age 54.3 ( 3 ) years ] who underwent a tvt - o procedure ( tvt - o ethicon ) between january 2006 and january 2011 to correct urethral hypermobility and restore urinary continence [ 7 ]  . 
we included only patients with a preoperative diagnosis of severe pure sui with no symptoms of urgency , no genital organ prolapse or with mild cystocoele , with a body mass index ( bmi ) of exclusion criteria were age greater 18.5 kg / m2 , parity of 2 or less , no history of pelvic surgery , and no associated chronic respiratory or metabolic disease . than 60 and \48 years , bmi greater than 26 kg / m2 , urge incontinence , hyperactive bladder or detrusor instability , pelvic organ prolapse , previous pelvic surgery , parity [ 2 , previous or current oestrogen therapy , and associated chronic respiratory or metabolic diseases . following tvt - o each patient underwent urogynaecological history and clinical assessment , inclusive of urogynaecological examination , voiding diary , q - tip test , stress test , urodynamic assessment ( cystomanometry , uroowmetry , pressure - ow test ) and perineal pelvic ultrasound . 
the ultrasound examination included a static and dynamic assessment of urethral mobility . the rst goal was to establish the success rate of the tvt - o procedure , dening cure as the total absence of urine leakage on exertion . 
to this end , we divided the patients into two groups : group a , patients cured following tvt - o ; group b , patients with persistent sui after tvt - o . having dened the success rate , we evaluated whether there was any difference in the measurementsboth static and dynamicof the pubo - urethral distance and angle between group a and group b . the pubo - urethral distance represents the line joining the pubis and the midpoint of the urethra . 
reference values for women aged 4860 years are 85.50 ( cid : 3 ) 8.94 ( cid : 3 ) at rest and 105.20 ( cid : 3 ) 10.16 ( cid : 3 ) under stress , as demonstrated by di pietto et al . 
patients were imaged with a half - full bladder ( 250 cc ) so as to avoid excessive lling that might collaboration in performing a valsalva affect their the measurements [ 11 , 12 ]  . 
the manoeuvre and alter examination was conducted with the patient in dorsal lithotomy position and using a 3.5 - mhz convex probe ( volusonr 730 expert , general electric ) , placed longitudinally to the vulvar orice and slightly tilted upwards . 
this position of the probe also allowed for midsagittal scans through the anterior perineum [ 13 , 14 ]  . the pubic bone appears as an oval image surrounded by a regular wrapping ( curved ligament ) , on the left of the screen . the transonic longitudinal structure in the middle of the screen is the urethra , above which are the bladder and , more superior , the uterus . 
the pubic bone is the only xed structure within the eld of observation and is therefore used as a landmark for measuring the pubic - urethral distance and angle [ 15 ]  . 
in the dynamic phase , the increase in intra - abdominal pressure was achieved through coughing and the measurement was obtained on a still image generated at the time of maximum abdominal pressure thanks to the use of the cine - loop technique ( ability to memorise and reproduce images )  . 
it should be noted that the valsalva manoeuvre is not standardised so diagnostic sensitivity is not only operator dependent but also inuenced by the patients ability to cooperate in achieving a valid increase in intra - abdominal pressure . statistical analysis the data are presented as mean and standard deviation . 
this was found to be 10 % , that is , persistent sui after tvt - o was seen in ve patients ( group b ) , whereas the other 46 patients were completely cured ( group a )  . 
7 inclination angle of urethral axis under stress in recurrent ius post tvt - o ( p pubic bone , b bladder , u urethra ) radiol med ( 2014 ) 119 : 189194 discussion in the work - up of patients with pelvic oor dysfunction it is fundamental to obtain an in - depth evaluation of any defects in the individual structures that contribute to the anatomical and functional integrity of the pelvic oor , with a view to dening the most appropriate surgical approach . 
perineal ultrasound , in particular , is the most commonly used technique thanks to ease of use , lack of invasiveness and low cost . ultrasound generally allows for an evaluation of all the anatomical structures making up the pelvic oormuscular components , recesses , and pelvic organs ; more specifically , the perineal approach allows the identication of any qualitative and quantitative abnormalities affecting the structures involved in guaranteeing urinary continence [ 1618 ]  . 
according to the literature , the results of perineal ultrasound demonstrate a close correlation among a diagnosis of sui , bladder neck funnelling and hypermotility of the urethrovesical junction [ 19 , 20 ]  . moreover , this examination also permits a targeted preand postoperative assessment to provide a precise , repeatable and objective index of surgical outcome . 
patients with complex disorders following surgery for stress incontinence or incontinence due to pelvic organ prolapse [ 21 ] should , in fact , undergo ultrasound to study the position of the tape and / or mesh in relation to the urethra and pelvic organs [ 22 , 23 ]  . 
many studies have conrmed the failure of sling insertion techniques , using ultrasound to measure the quantitative reference indices ( pubo - urethral distance and angle ) [ 24 ]  . 
perineal ultrasound therefore represents a useful technique in the diagnosis and follow - up of sui patients who have undergone a sling - placement procedure . starting from these premisses , we carried out perineal ultrasound in all women treated with tvt - o for severe sui , so as to assess whether there was any parameter that might serve as an indicator of surgical failure . 
this suspicion stemmed from the fact that all women had similar preoperative characteristics , had no risk factors associated with treatment failure and had all been operated on by the same surgeon who was highly experienced in the technique . the rst assessment aimed at establishing whether the sling was in the correct position and well suspended , and all patients , both cured and uncured , showed a well - positioned sling . 
we then evaluated the pubo - urethral indices , and found that the mean values of pubo - urethral distance and angle were higher in the group of patients with persistent sui compared to those in cured patients . the nding of a well - positioned sling even in the case of treatment failure and the evidence of greater pubo - urethral angles and distances in uncured women suggests that these patients may have had higher values of both pubo - urethral indices already before sling placement and that therefore the suspension achieved with the trans - obturator technique was inadequate to reduce pubo - urethral angles and distances strongly deviating from the physiological values . this means that very probably , if we had subjected all patients undergoing tvt - o to preoperative perineal ultrasound , we would have realised that the treatment had been successful in the patients with preoperative pubourethral indices closer to what we considered to be the the success of physiological values . 
this suggests that trans - obturator sling placement decreases in proportion to the increase in the mean pubo - urethral distance and angle . confronted with these ndings , we considered whether there had been some evaluation error in selecting tvt - o in cases of failure and whether the classic tvt technique might have ensured complete cure in these patients . this hypothesis is based on the fact that , by producing a greater degree of tension compared to the trans - obturator approach , the retropubic approach may succeed in restoring normal ranges of pubic - urethral indices even when these are considerably increased [ 10 ]  . 
should this be true , it would mean that perineal ultrasound with determination of the pubo - urethral distance and angle should become a necessary diagnostic step for selecting the most appropriate sling - placement technique for sui correction [ 10 , 25 , 26 ] , and one to be included in the diagnostic work - up of patients with pelvic oor dysfunction . for this hypothesis to be conrmed , larger case series are required as are new , prospective studies allowing comparison of preand postoperative pubo - urethral values to dene whether and to what extent these values inuence the likelihood of success of a specic minimally invasive slingplacement procedure for the treatment of sui [ 10 , 24 ]  . conclusions our data show that patients with persistent sui after tvto have a mean pubo - urethral distance and angle greater than those in women with successful treatment . 
rossi sara seitun giulio bovio umberto valente received : 7 august 2012 / accepted : 2 october 2012 / published online : 20 december 2013 ( cid : 2 ) italian society of medical radiology 2013 abstract purpose this study was undertaken to evaluate primary stenting in patients with inferior vena cava torsion after orthotopic liver transplantation performed with modied piggyback technique . materials and methods from november 2003 to october 2010 , six patients developed clinical , laboratory and imaging ndings suggestive of caval stenosis , after a mean period of 21 days from an orthotopic liver transplantation performed with modied piggyback technique . 
all patients were treated with primary stenting followed by in - stent angioplasty in three cases . results in all patients , the stents were successfully positioned at the caval anastomosis and the venous gradient pressure fell from a mean value of 10 to 2 mmhg . 
valente dipartimento di chirurgia addominale e dei trapianti dorgano , irccs azienda ospedaliera ed universitaria san martino , ist - istituto nazionale per la ricerca sul cancro , largo rosanna benzi 10 , 16132 genoa , italy conclusions primary stenting of inferior vena cava stenosis due to torsion of the anastomoses in patients receiving orthotopic liver transplantation with modied piggyback technique is a safe , effective and durable treatment . keywords orthotopic liver transplantation ( cid : 2 ) torsion ( cid : 2 ) inferior vena cava ( cid : 2 ) stent introduction orthotopic liver transplantation has become the treatment of choice for patients suffering from end - stage liver disease . 
the piggyback technique for orthotopic liver transplantation , rst performed by calne and williams [ 1 ] , is an alternative surgical technique that has gained increasing popularity , especially with the advent of paediatric transplants , living donors and other split liver transplantation techniques [ 2 ]  . 
the main advantages of the piggyback technique are the fact that the blood return from the inferior vena cava is maintained during the anhepatic phase , with a signicant increase in haemodynamic stability , reduced cold ischaemia times , less kidney damage and no need for the veno - venous bypass [ 3 , 4 ]  . 
the main complications of the anastomoses between the inferior vena cava and suprahepatic veins may arise in the postoperative period , mainly due to technical factors , torsion or compression of the inferior vena cava , or during follow - up , mainly as a result of brosis or hyperplasia in the anastomotic area [ 5 8 ]  . 
other ndings include ascites , oedema of limbs , hepatomegaly , pleural effusion and altered liver function the lower 184 radiol med ( 2014 ) 119 : 183188 [ 811 ]  . 
arterial and biliary stenoses are the most commonly reported complications [ 5 , 6 ] , which may require a percutaneous and / or endoscopic and / or surgical approach . however , when managing anastomotic complications of the inferior vena cava , most medical reports recommend an exclusively endoscopic approach [ 714 ]  . 
in this study , we present a group of six patients who developed stenosis of the inferior vena cava at the level of suprahepatic veins , due to torsion of the inferior vena cava following orthotopic liver transplantation using the modied piggyback technique . 
we describe our approach to the endovascular treatment by primary stenting using gianturco cook - z vena caval and venous design stent ( cook , bloomington , in , usa ) , with special reference to selection of stent diameter and length , access and the need for primary stenting in view of the poor response to percutaneous angioplasty alone . materials and methods patients and liver transplants from november 2003 to october 2010 , out of a total of 316 orthotopic liver transplantations performed using the modied piggyback technique , 6 patients were referred to our interventional radiology centre with clinical evidence suggesting stenosis of the inferior vena cava with clinical onset between 3 and 94 days ( mean 11 days ) after surgery . these patients , ve of whom had undergone transplantation at our centre and one elsewhere , received endovascular treatment of the inferior vena cava alone . 
the pretransplant liver diseases were : hepatitis c - related cirrhosis ( n = 4 ) , exotoxic cirrhosis ( n = 1 ) , cryptogenic cirrhosis ( n = 1 )  . diagnosis in all six patients , stenosis of the inferior vena cava and of the suprahepatic veins was included in the differential diagnosis on the basis of the clinical presentations consisting of ascites , diffuse abdominal pain , increase in weight , oedema of the lower limbs , pleural effusion , hepato - splenomegaly , and on the basis of liver biochemistry tests such as bilirubinaemia , albuminaemia , coagulation parameters , natraemia and transaminases . 
all patients underwent a colour doppler ultrasound ( us ) examination . the main us ndings such as a reduction in caval diameter in the peri - anastomotic region , monophasicity of the ow velocity wave in the hepatic veins , increase in the ow velocity in the suprahepatic and inferior caval veins , and a reduction in the portal veins , are generally nonspecic , but highly indicative for diagnosing stenosis of the inferior cava [ 15 ]  . 
for the treatment of steno - occlusions of large venous vessels , numerous steel or nitinol stents are available on the market ( e.g. , sinus xl ; optimed , germany , e - xl ; jotec , germany , wallstent ; boston scientic , ma , usa , etc . ) ; however , most of these are closed - cell stents , which may obstruct venous return from the suprahepatic veins and , above all , may cause difculties in passing through the mesh for further procedures . therefore , in all six patients , we selected the gianturco cook - z - vena cava and venous design stent ( cook , bloomington , in , usa )  . 
this is a steel self - expanding stent with 25 - mm z - shaped modules , available with diameters of 20 , 30 and 40 mm and lengths of 25 , 50 and 75 mthe diameter was selected based on the inferior vena cava measurements on preliminary ct angiography and conrmed at pre - stenting cavography . 
ideally , a large literature and the stent manufacturers part of medical themselves recommend oversizing the stent by 1525 % with respect to the maximum diameter of the unaffected vena cava ; however , we preferred a slightly larger oversizing ( by approximately 30 % ) to ensure greater expansile strength of the stent and resolve the stenosis . 
therefore , after obtaining a right transjugular sonographic and uoroscopic access in all six patients , a 9 - fr introducer sheath radiol med ( 2014 ) 119 : 183188 fig . 
1 a axial multidetector computed tomography ( ct ) and b coronal maximum intensity projection ( mip ) reconstruction in a 42 - year - old patient on day 14 after liver transplantation , which demonstrated inferior cava vena steno - torsion at the level of anastomosis ( arrow head ) and dilatation of the hepatic veins ( arrows ) fig . 
the system used to release the z - stent is the radiol med ( 2014 ) 119 : 183188 table 1 patients liver disease , days from transplantation to onset of symptoms , and size and length of the stents and balloon catheters patient no . liver disease days from transplantation to onset of signs and symptoms stent size ( mm ) stent modules ( no . ) pta size / length ( mm ) hcv - related cirrhosis exotoxic cirrosi hcv - related cirrhosis hcv - related cirrhosis hcv - related cirrhosis cryptogenic cirrhosis 40 / 75 30 / 75 40 / 75 30 / 75 30 / 75 30 / 75 no pta no pta 14 / 40 14 / 40 no pta 12 / 40 pta percutaneous transluminal angioplasty , hcv hepatitis c virus introducer set ( cook , bloomington , cook - z stent usa ) , which consists in an introducer with reinforced sheath with dilator and pusher , available in the 10 - , 12and 14 - fr sizes , with a pull - back release systethe 30 - mm stents were released by means of a 12 - fr introducer , while the 40 - mm stents were released by means of a 14 - fr introducer . on two patients , we performed in - stent angioplasty using xxl balloon catheters ( boston scientic , natick , ms , usa ) with diameters of 12 and 14 mm and a length of 40 mm as , despite an optimal radial force , the stent was not sufcient to resolve the steno - torsion satisfactorily by over 50 % . 
3 pressure gradient ( mmhg ) positioning of the stent and iatrogenic vascular lesions . recurrence was dened as an increase in the pressure gradient c3 mmhg , clinical and biochemical signs of liver insufciency , and re - stenosis at the level of the inferior vena cava on colour doppler us . results technical success was achieved in all patients ( 100 % )  . 
an immediate pressure response ( b3 mmhg ) was shown in three patients ( 50 % ) , whereas the remaining three patients ( 50 % ) , showed a persistent high pressure gradient of 7 , 5 and 8 mmhg , respectively . 
no major or recurring complication was found in the other patients . discussion the importance of preserving the graft and thus the management of complications is crucial in this type of patient . as in all surgical procedures requiring anastomosis , orthotopic liver transplantations using the modied piggyback technique are subject to complications , including stenosis [ 5 , 6 ] , which may concern one or more of the four main anastomoses . 
torsion of the inferior vena cava at the level of the anastomosis following orthotopic liver transplantation is an even rarer complication when using the modied piggyback technique , and occurs with a frequency of roughly 12 % [ 58 ]  . 
in general , torsion affects the inferior vena cava at the level of the suprahepatic veins with a tendency to develop below the surgical anastomosis . the colour doppler us ndings are normally nonspecic , variable and unfortunately unable to quantify the haemodynamic signicance of the caval stenosis [ 18 ]  . however colour doppler imaging is highly suggestive for stenosis of the post - transplant suprahepatic veins . 
in these patients , persistence of monophasicity of the wave at the level of the suprahepatic veins is a sensitive , but not specic , sign of inferior caval stenosis at an anastomotic level . persistent phase waves with breathing or three - phase on colour doppler us may exclude the possibility of a consistent stenosis of the suprahepatic veins [ 19 ]  . 
ct angieasily ography , performed on all of our patients , demonstrated stenosis due to torsion of the inferior cava and enabled planning of endovascular treatment thanks to preliminary measurements of the caval diameters , subsequently conrmed by venography . 
venography by image subtraction , with measurement of trans - stenotic pressure gradients , remains the gold standard for the diagnosis of caval anastomotic complications in this type of patient [ 7 ]  . steno - torsion appears as a streaked image on the longitudinal axis , rather than an actual focal stenosis , with varying alterations in density and uniformity of the intraluminal contrast column in the context of the anomaly [ 7 ]  . we considered technical success as a reduction in the pressure gradient of b3 mmhg ; in the literature , however , there is no clear level of pressure gradient above which patients may develop specic symptoms [ 16 , 2022 ]  . 
in our group of patients , the presentation of clinical signs and symptoms developed over an average of 28 days from the transplant ; however , this average was strongly inuenced by a single patient who developed symptoms 94 days after the transplant . 
in our experience , torsion of the inferior vena cava is a typical complication of the early postoperative period , and in this period patients can have a large variety of complicated medical complaints that occur simultaneously , such as ischaemic injury due to reperfusion , rejection , oedema and liver enlargement . 
it is for these very reasons that the treatment should always be patient - specic , considering the clinical scenario , biochemical values , radiological examinations , morphology of the stenosis and above all the trans - stenotic pressure gradients . 
currently , angioplasty and stenting are considered the rst - choice treatments for inferior cava steno - torsion , but most studies agree that angioplasty alone of the inferior vena cava is not sufcient to ensure adequate patency in the long term [ 5 , 9 , 1113 ]  . 
although there is justiable reticence to treating the above - described anastomosis percutaneously by angioplasty , we have found no contribution in the literature regarding the rupture or breakdown of the anastomosis , and therefore we maintain that the risk is decidedly limited . 
in this case , because of the mixed aetiology of both torsion and intimal hyperplasia and because no change in the pressure gradient was detected after angioplasty due to immediate re - stenosis , it was decided to position the stent . 
each patient was treated with three - module stents , for length of 75 mm , because we believe that an a total extended coverage is crucial to avoid recurrence and migration of the torsion to a caudal level [ 7 , 8 , 23 ]  . 
post - stenting angioplasty was carefully considered for each patient on the basis of the response of the stenosis to stenting , and reserved for stenoses with a greater brotic component . we believe that the wait - and - see approach is the most appropriate when choosing to perform angioplasty , and staggering , on the basis of the pressure gradient readings , the dilation procedure over the following days . 
in fact in the three patients who did not undergo angioplasty , the 188 radiol med ( 2014 ) 119 : 183188 stent was sufciently expanded after 3 days and the pressure gradient was below 3 mmhg . 
this approach ensured good stability of the introducer , optimal control on release and avoided the possibility of migration to the right atriuwe found no major procedure - correlated complications , or signs of recurrence on an average follow - up of 49 months . we must stress that the choice of the correct stent characteristicsincluding diameter , radial force , low compressibility and good resistance to pressureis crucial [ 12 , 24 ]  . 
the main limitation of this study was the small number of patients , largely due to the rarity of this complication ; other cases will be needed to conrm the correct choice of technique and materials used . in conclusion , in our case series of steno - torsion of the inferior vena cava following orthotopic liver transplantation using the modied piggy - back technique , the use of large - mesh stents with the transjugular approach was found to be fast and free of complications and ensured a denite and lasting response in symptomatic caval torsion . conict of interest carlo ferro , enzo andorno , andrea guastavino , umberto g . 
imaging of lumbar spine with the patient in a supine , nonweight - bearing position is likely to misrepresent the degree and potential risk of spinal stenosis . materials and methods in the period between september 2008 and may 2011 , we selected 630 symptomatic patients aged 4065 years ( mean age 56 ) who underwent conventional mr in clinostatic position . 
the biomechanical parameters were also considered . changes in the dimension of the neural foramina were compared using the presence of disc and facet degeneration by statistical analysis . results stenosis of the intervertebral foramen was never found in the presence of normal intervertebral discs either in the presence or in the absence of facet disease , in either clinostatic or orthostatic position . 
in all of these cases , disc disease was associated with facet pathology . conclusion our data show that the association between disc pathology and facet osteoarthrosis can cause occult foraminal stenosis . 
gallucci department of radiology , university of laquila , 67100 laquila , italy e - mail : alessandra.splendiani@cc.univaq.it physiological load conditions may improve the clinical diagnosis of radicular pain . keywords dynamic mri ( cid : 2 ) spine pathology ( cid : 2 ) lumbar arthrosis ( cid : 2 ) new technological mr application ( cid : 2 ) weight - bearing mr application introduction foraminal stenosis is a painful and incapacitating condition caused by the pathological narrowing of the intervertebral foramen , with consequent compression of the nerve roots involved . 
the quantity of space available to the nerve along its anatomical path is related to the morphology of the structures delimiting the canal , which are the intervertebral disc , ava ligaments and interapophyseal joints . 
a congenitally narrow canal , possibly associated with increased thickness of the ava ligaments and hypertrophy of the joint facets , asymptomatic in the rst 10 years of life with symptoms often appearing in adult years . 
in fact , the combination of degenerative arthritis with further thickening of the aval ligaments , protrusion of the intervertebral discs , and osteopathy of the joint facets leads to deterioration of the main symptoms , as described in a number of studies [ 1 , 2 ]  . the exact incidence of spinal stenosis in the general population is not well known , although numerous studies have shown that its prevalence increases with age [ 3 , 4 ]  . between 4 and 28 % of asymptomatic patients on computed tomography ( ct ) or magnetic resonance ( mr ) radiol med ( 2014 ) 119 : 164174 fig . 
1 mcgill pain questionnaire examinations show radiological signs of stenosis [ 5 , 6 ]  . however , a large number of diagnostic examinations ( ct and mr ) on symptomatic patients show no signs of the pathology [ 7 ]  . 
therefore , spinal stenosis must represent a dynamic and intermittent process which intervenes when the spinal column is subjected to stress [ 8 ]  . the objective of this study was to assess , in symptomatic patients , the presence of foraminal stenosis not revealed on conventional mr examinations , but detected in the on examinations performed with the patient orthostatic position using dedicated mr equipment able to examine the spinal column in both the orthostatic and particular , we positions . that mr examinations tested clinostatic in weight - bearing hypothesis conditions ( orthostatic position ) are able to detect presence of dynamic stenosis of the intervertebral foramen , as demonstrated by anatomicalpathological studies [ 9 ]  . materials and methods in the period between september 2008 and may 2011 symptomatic ( 33 months ) , we selected a number of 166 radiol med ( 2014 ) 119 : 164174 fig . 
examination in clinostatic position ( 0.25 t tilting permanent magnet g - scan , esaote , genoa , italy ) patients aged between 40 and 65 years ( average age 56 ) who underwent conventional mr in clinostatic position because of reported pain in the lumbosacral region ( total number of patients 630 )  . 
the study excluded patients suffering from pain attributable to the following causes : primary tumours originating from the vertebral canal , metastatic disease from another region , positive clinical intervention on the trauma , surgical history for spinal spine , spondylolysis and spondylolisthesis , arthritic disease affecting more than one metameric level . 
these patients were examined by means of mr examination in the clinostatic and orthostatic positions carried out on a dedicated mr unit ( g - scan , esaote , genoa , italy ) equipped with a tilting system allowing immediate rotation of the patient bed and the magnet . 
these safety straps are also important in the emergency procedure , due to the fact that when changing from the clinostatic to the orthostatic position , some patients may suffer from vaso - vagal or vaso - depressor syncope . 
in the supine position , t1 fast spin echo ( fse ) , t2 relaxation fse , t2 sagittal and 3d scans were acquired ( total duration 17 min ) , whereas in the erect position only the last two types of scan were performed ( duration 11 min )  . 
the three - dimensional ( 3d ) mr images were acquired using a 3d steady - state hybrid contrast - enhanced ( hyce ) sequence with the following parameters : 10 / 5 ( repetition time ms / echo time ms ) , ip angle of 80 ( cid : 3 ) , two sequential scans . 
the other typical acquisition parameters included an acquisition matrix of 180 9 180 for a eld of view of 200 9 200 mm ( from 0 to 512 in both directions to optimise effective resolution ) , a single plate with a thickness of 220 mm , 64 sections and a bandwidth of 185 hz per pixel . 
the a morphological , clinically nonpathological condition of intersomatic discs , joint facets and aval ligaments was evaluated on both levels for a total of 145 ? 145 cases . morphological characterisation of the state of the various structures under examination was performed on the basis of previously described [ 6 ] mr features : radiol med ( 2014 ) 119 : 164174 fig . 
the morphological evaluations were made by two reading panels , each made up of one expert neuroradiologist and two junior neuroradiologists , who examined the images acquired in the orthostatic and clinostatic positions at random and independently from each another . 
the distribution of the patients on the basis of the appearance of intervertebral disc was as follows : 81 / 230 highly degenerated , 34 / 230 degenerated , 35 / 230 normal , 80 / 230 physiologically altered . 
therefore , the discs taken as a reference were at a metameric level below the pathological level in only 25 / 115 cases ( 22 % )  . the interapophyseal joints and aval ligaments were considered pathological in 73 / 230 levels considered . evaluation of all disc - vertebral levels examined ( 230 levels ) showed disc pathology to be associated with a normal appearance of the interapophyseal joints in 54 / 230 levels ; at 61 levels , there was an association between disc abnormality and alteration of the facets ; at 103 levels , the nonpathological disc was associated with a normal appearance of the joint facets ; at 12 levels , there were arthritic changes to the interapophyseal joints in the absence of disc degeneration . the intervertebral foramen was considered , on the basis of the previously stated denitions , to be stenotic at 61 / 230 levels and nonstenotic at 169 / 230 . 
at the level with normal disc and facet appearance , there was no change in the neural foramina from supine to upright position radiol med ( 2014 ) 119 : 164174 fig . 
according to conventional criteria , this difference is considered statistically highly signicant . the spinal parameters were altered in 87 / 115 patients ( 76 % ) , in particular : the lumbar lordosis angle ( with a physiological value of approximately 50 ( cid : 3 ) ) [ 7 ] was altered in 68 patients , of whom 46 had a decreasing angle both in the clinostatic and orthostatic positions ( with an average value of 40 ( cid : 3 ) ) ; 22 patients showed an increased angle ( average value of \60 ( cid : 3 ) ) which was not modied in the orthostatic position ; the lumbosacral angle ( with a physiological value of approximately 120 ( cid : 3 ) 135 ( cid : 3 ) ) [ 7 ] was altered in 47 patients ( 53 % )  . 
in all these patients , we measured an angle greater than 135 ( cid : 3 ) , with no modication in the dynamic study ; both angles were found to be altered in 21 patients . discussion in the industrialised parts of the world , low back pain is extremely common with a prevalence ranging from 60 to 90 % . 
symptoms normally evolve during the 5th or 6th 170 radiol med ( 2014 ) 119 : 164174 decade of life , and are associated with arthritic variations of the vertebral column . 
the societal impact of low back pain in older adults is demonstrated by medicare data from 1991 to 2002 that show a 132 % increase in low back pain patients and 387 % increase in related charges for low back pain . despite the fact that low back pain is common and associated with poor outcomes in vulnerable older adults , little research has focused on low back pain in people over the age of 65 [ 10 ]  . lumbar spinal stenosis is a debilitating chronic pathology , which generally starts with degeneration of intervertebral discs and joint facets , causing a narrowing of the vertebral canal and neural foramen . 
a fundamental contribution to the study of the problem is provided by ct and mri ; in particular , the latter enables a high degree of spatial resolution and contrast , and provides a precise evaluation of intervertebral discs , vertebrae , ligaments , spinal canal and intervertebral foramen . 
despite the unquestionable diagnostic accuracy of these methods for the morphological evaluation of these anatomical structures , when diagnosing foraminal stenosis , the number of false negative results is considerable , as shown in the literature [ 11 ]  . 
by subjecting in vitro cadaveric lumbar segments to movements of axial rotation , extension , bending and lateral curvature , a compression of the spinal nerve was observed , not detected in examinations without the application of weight show that compression results involving the spinal roots is associated with disc and facet pathology with signicant p values , and this conrms that there is an association between the risk factor ( osteoarthritis of facets ) and the disease ( disc pathology )  . 
this correlation agrees with the pathological and biomechanical studies which identify as one of the causes of brosis of the aval ligament the loss of disc elasticity in response to an increase in mechanical stress . 
radial rupture of the annulus brosus or a reduction in disc signal intensity , seen on mri , indicates a serious reduction in elasticity , which leads to brosis and hypertrophy of the aval fig . 
b sagittal t2 - weighted image : 3d hyce reformatted slice on the axial plane . the upright mri study shows an increase in disc protrusion and a reduction of intra - articular uid collection with consequent thickening of the aval ligament which cause a stenosis not seen on supine mri ( white arrow )  . 
in the erect position , the static weight on the spinal column strongly reduces the thickness of the degenerated disc , unlike what occurs on the nonpathological disc , and enlargement and protrusion of the aval ligament can be observed inside the intervertebral foramen . 
the foramen therefore appears to be occupied by the table 2 the p value was calculated with mcnemars test with continuity correction control total case total chi - squared equals 25.470 with one degree of freedothe twotailed p value is less than 0.0001. 
furthermore , in some of the cases , we were able to highlight intersegmental hypermobility with bearing of the facet itself in orthostatic conditions . the advent of dedicated mr has enabled in vivo investigations in orthostatic condition and thus an evaluation of the disc - vertebral structures and of their biomechanical properties . 
the laxity of the ligament also causes shifting of the facet ( black arrow ) structures , and thanks to this that discs in the rear section are subject to greater pressure , which tends to maintain the nuclear gel in a more forward position , thus preventing protrusion [ 17 , 18 ]  . 
the results obtained with dynamic mr under weight - bearing conditions conrm previous studies ( rajnic ) , which show a signicant decrease both in the angle of lumbar lordosis and the sacral angle in patients with disc alterations . 
if the lumbosacral angle is greater than 180 ( cid : 3 ) , this means there is a reduction in lumbar lordosis , with verticalisation of the sacrum and suffering of the front portion , with repercussions on the nuclear content of the disc . 
on the contrary , if the angle is less than 120 ( cid : 3 ) , this means that there is an increase in lumbar lordosis and horizontalisation of the sacrum with an increase in the compression on rear structures . 
in fact , in the erect position , under physiological conditions , the interapophyseal joints support most of the cutting force which acts on the vertebral column [ 19 ] and 16 % of the compression forces [ 20 ]  . 
in the presence of disc pathology ( reduction in disc height , loss of water content , degeneration ) , the facet may enter in close apposition to support 70 % of the compression forces , with overload of the joint . the latter can lead to degenerative arthritis which , as mentioned above , is one of the causes of narrowing of the conjugate foramen [ 20 ]  . radiol med ( 2014 ) 119 : 164174 one of the salient limits to the dynamic investigation of the vertebral column lies in the reproducibility of the cutting planes . 
in fact , movements of the patient and even normal vibrations under the effect of the weight can represent the cause of enormous or small modications to the selected scanning plane . 
for this reason , we used a 3d scan protocol ( 3d hyce both in clinostatic and orthostatic positions ) and precise post - processing reconstruction using the mpr algorithm with an effective thickness of 1 mm , parallel to the vertebral endplate in the coronal and sagittal planes . 
these reformatted images enabled a study of the vertebral foramen and the relationship between the internal structures both in normal and pathological conditions such as the aval ligament ( inside the foramen ) and its relation to the spinal nerve and nerve roots . 
however , in orthostatic conditions may have less the images favourable signal - to - noise ratio compared to those obtained in clinostatic conditions , while maintaining the same diagnostic capacity . 
dynamic imaging techniques may increase our ability to locate the original site of nerve root compression in patients suffering from low back pain or radicular patherefore , the dedicated mr unit used in this study is proposed as an instrument able to provide useful dynamic studies that may help obtain correct diagnoses [ 21 ]  . prompt diagnosis of the site and characteristics of foraminal stenosis may provide useful guidance for correct treatment procedures . 
cbct scans at treatment days 10 , 15 , 20 and 25 were used to transfer the original plan ( cbctplan i , ii , iii , iv , respectively ) using rigid registration between the two . 
the pgs were retrospectively contoured and evaluated with the dosevolume histograthe mean dose , the dose to 50 % of volume , and the percentage of volume receiving 30 and 50 gy were evaluated for each pg . 
the wilcoxon sign ranked test was used to evaluate the effects of dosimetric variations and values \0.05 were taken to be signicant . results from february to june 2011 , ten patients were enrolled and ve imrt plans were evaluated for each patient . 
fiorentino department of radiation oncology , sacro cuore - don calabria hospital , negrar - verona , italy checking the pg volume and dose could be indicated during the third week of treatment . keywords head and neck cancer ( cid : 2 ) imrt ( cid : 2 ) conebeam ct ( cid : 2 ) organ at risk ( cid : 2 ) parotid glands introduction in the treatment approach for patients affected by head and neck ( h&n ) cancer , radiotherapy plays an important role to increase local control and survival . 
the introduction of intensity - modulated radiotherapy ( imrt ) has increased the possibility to deliver higher doses to the target with sparing of the organs at risk ( oar ) compared to conformal radiotherapy [ 1 ]  . volume changes and / or anatomical deformations of both the target and oar are independent of the immobilisation systems used but , due to these changes , there is a risk of administering higher or lower doses compared to the planned dose [ 27 ]  . to reduce the risk of xerostomia , the parotid glands ( pgs ) have been widely studied in terms of volumes and dose changes [ 3 , 4 , 712 ] , and several trials highlighted that imrt re - planning is mandatory for selected patients [ 7 , 10 ]  . the shrinkage of pgs has important implications for the daily dose distribution [ 13 , 14 ]  . 
 [ 10 ] reported similar results : all pg dosimetric values increased during week 3 of treatment . to intercept and correct set - up errors , image - guided radiotherapy ( igrt ) has proved useful [ 1420 ] and in our institution , a trilogy varian medical system cone - beam computed tomography ( cbct ) is available for controlling 202 radiol med ( 2014 ) 119 : 201207 the set - up position of all anatomical regions and for evaluating pg changes [ 3 ]  . moreover , cbct scans have also been used for dose reconstruction , and some authors have reported their value for clinical decisions regarding re - planning [ 21 , 22 ]  . in consideration of the 1530 % volume reduction observed at weeks 34 of treatment [ 3 ] , this study aimed to investigate whether and when re - planning is required to spare the pgs during a course of headneck imrt , by evaluating the dosimetric consequences of pg shrinkage with cbct scans obtained at four time points ( days 10 , 15 , 20 , 25 )  . materials and methods patients patients , with histologically proven headneck cancer , aged more than 18 years and treated with imrt , were analysed . 
patient immobilisation was obtained using a headshoulder thermoplastic mask and type s - fixator ( civco , orange city , iowa , usa )  . treatment plans noncontrast - enhanced ct scans were acquired in the treatment position ( 2.5 mm slice thickness )  . 
all imrt plans were performed with an eclipse 8.6 treatment planning system ( varian medical system , palo alto , ca ) and were based on a seven - eld sliding window technique with a nominal energy of 6 mv . imrt with simultaneous integrated boost dose consisted of 7072 gy to macroscopic disease ( clinical target volume , ctv1 ) ; 6066 gy to microscopic high - risk disease ( ctv2 ) and 5458 gy to microscopic low - risk disease ( ctv3 )  . 
the quantec guidelines were used to evaluate oar [ 23 ]  . every other day during radiotherapy , all patients underwent cbct scans ( slice thickness of 2.5 mm ) to verify the set - up positioning . 
the cbct low - dose head model was used to generate images with full - fan acquisition , with 360 projections being acquired over a gantry rotation of 200 ( cid : 3 )  . 
the position of each patient was adjusted based on the rigid registration between cbct images and planning ct images ( 3d / 3d match )  . 25 ( cbctplan i , cbctplan ii , cbctplan iii and cbctplan iv , respectively )  . 
since this is an observational study , patients continued to be treated according to the original ctplan . statistics plan comparisons were made between the ctplan and the cbctplan i , ii , iii and iv , respectively . 
pg dosimetric variations were analysed for both pgs and , separately , for the ipsilateral and contralateral glands ( ipgs and cpgs , respectively )  . the wilcoxon sign ranked test was used to evaluate the effects of variations and values \0.05 were taken to be signicant . results patients from february to june 2011 , ten patients were analysed ( table 1 )  . 
all but two patients completed imrt : one patient died due to another disease , whereas the other table 1 patients characteristics age / gender subsite stage histology concurrent 41 / f 46 / m 54 / f 60 / f 63 / m 48 / m 77 / m 77 / m 78 / m 75 / m oral cavity oropharynx oral cavity oral cavity hypopharynx oropharynx oropharynx oral cavity larynx larynx t4n0m0 t4n2cm0 t4n2am0 t2n2bm0 t3n0m0 t2n1m0 t3n1m0 t4n1m0 t3n1m0 t4n0m0 the original treatment plan ( ctplan ) was transferred to the corresponding cbct scan at treatment days 10 , 15 , 20 and scc squamous cell carcinoma plan on cbct radiol med ( 2014 ) 119 : 201207 fig . 
1 the increase in average dmean , d50 , v30 and v50 between time 0 ( original plan ) and the subsequent cbct - based plans ( 10152025 fractions )  . 
error bars indicate 95 % condence interval patient missed the last four fractions because of grade 4 skin toxicity . parotid glands forty cbct plans were generated and compared to the corresponding ctplan and , for each patient , ve imrt plans and ten pgs were analysed . 
the average of increase in dmean , d50 , v30 and v50 for both pgs and for ipg and cpg separately are shown in table 2 . the ipg dosimetric variations were higher than those reported for the cpg at each time points , except for the cbctplan i ( table 2 )  . 
2 shows an example of dose change in both pgs . discussion for headneck cancer , imrt has achieved an excellent dose distribution , with a reduction in toxicity [ 16 , 25 ]  . patient immobilisation systems are effective in reducing set - up errors , but due to anatomical and volume variations in the target and oar and the patients weight loss [ 26 ] during treatment , they could increase the risk of missing the target or applying higher doses to the oars . several papers documented that the largest changes occur in the pgs with a volume loss of about 15 % at week 2 of treatment [ 3 , 5 , 11 , 13 ]  . 
 [ 28 ] analysed ten headneck cancer patients and observed that , due to the shrinkage , the delivered dmean increased for both pgs . although several studies have shown an increase in dose over a course of radiation treatment [ 3 , 26 , 28 ] , it is not clear whether and when re - planning should be carried out . thus , the aim of our study was to assess the optimal timing of re - planning to spare the pgs in headneck cancer patients by analysing the dosimetric variations during a course of imrt . in this study , all patients underwent cbct to verify the set - up positioning and , to investigate the correct re - planning time , we selected four time points ( day 10 , 15 , 20 and 25 of treatment ) and the corresponding cbct scans . 
since several studies showed that the imrt dose reconstruction based on cbct was an effective dosimetric basis for clinical decision to replan [ 21 , 22 ] , we transferred the original treatment plan on each cbct to evaluate the pg dose features . we observed that all the dosimetric end - points , except for v50 , increased signicantly if ctplan was compared to cbctplan i and ii . 
 [ 10 ] reported an increase in pg dose at week 3 of treatment in 33 patients with nasopharyngeal carcinoma , who repeated ct scans after 15 5 imrt fractions . 
the authors observed an increase of dmean , d50 and v46 for cpg compared to the original plan ( p = 0.03 , p = 0.04 , p = 0.03 , respectively ) , while the increase in dose for ipg was not signicant . 
the ipg dose was signicantly higher at weeks 4 and 5 ( p = 0.006 , p = 0.01 ) with an average increase compared to the planned dose of 2.7 gy , and the contralateral dmean increased signithe week 4 ( p = 0.03 ) [ 11 ]  . 
2 an example of the change in dose for both parotid glands : a original plan ; b original plan transferred on the cone - beam ct at treatment day 15 only one other study investigated dosimetric monitoring using cbct during parotid - sparing imrt in ten patients with headneck cancer . 
2 and table 3 , we found that the deep part of the pgs could be much more irradiated during an imrt course , there being in fact an increase in the percentage of volume that received 30 and 50 gy . 
in addition , recent papers have shown signicant differences in damage when the cranial or caudal part of pgs was irradiated ; the reduction in ow was much more severe with irradiation of the cranial part of pg [ 30 , 31 ]  . 
our analysis did not evaluate this aspect due to the short follow - up for evaluating xerostomia , but it could be interesting for improving the treatment plan : parotid - sparing imrt is used despite the possibility of parotid recurrences after treatment [ 32 ] , so perhaps cranial - parotid - sparing could be proposed to reduce the damage . another limit of the study was the monitoring of pgs dosimetric changes without using hounseld correction algorithm for cbct . 
 [ 29 ] showed that the differences between dose to the organ structures before and after tissue correction based on the planning ct and cbct were not clinically signicant ( \2 % )  . conclusions the pgs showed major modications in volume , resulting in a signicant dosimetric change during the course of imrt . 
our data showed that cbct is a feasible method to control the dosimetric variations even though the method is still to be validated for dosimetric monitoring and replanning . evaluation of the pgs by monitoring the dose and analysing the different pg regions could be indicated during the imrt course [ 33 ]  . 
the results obtained should be reported , at the end of each working day , on a special dose card , in order to record each workers exposure to the static magnetic eld . moreover , this dose card could be an important tool if long - term effects occur because it provides a complete history of the occupational exposure in an mri site . materials and methods to conduct measurements , three hall - sensor probes were used . 
quadrelli department of biotechnology and life sciences , university of insubria , varese , italy when the strength of the magnetic eld was [ 200 mt the weighted function overestimated the exposure , so that it represents a highly precautionary measure taking into account possible acute and long - term effects . 
in addition , from the data recorded during patient positioning operations by mri staff the db / dt curve was obtained . conclusions the areas obtained from the integral of the weighted static magnetic eld strength over time can be indicative of the global exposure of the occupational staff . these values should be reported on a special dose card that could be considered as an important if long - term effects occur because it provides a complete history of the occupational exposure in an mri site . tool keywords mri staff exposure ( cid : 2 ) mri safety ( cid : 2 ) exposure limits ( cid : 2 ) static magnetic eld introduction the human population is chronically exposed to natural and man - made sources of nonionising radiation . 
recent advances in technologies using static magnetic elds and , particularly , in magnetic resonance imaging ( mri ) , have resulted in increased values of static magnetic elds available for example for applications . 
consequently , diagnostics exposure levels have also increased , and this is especially relevant for mri staff . and medical whilst acute effects are evident , especially for exposure to high - intensity static magnetic elds , [ 1 ] , biological effects derived from chronic exposure to static magnetic elds are less understood . 
the possible interaction mechanisms of static magnetic elds with living tissue and radiol med ( 2014 ) 119 : 208213 resulting detrimental biological effects have been investigated [ 1 ] , highlighting the potential risks of long - term effects due to chronic exposure . 
one related concern is the chronic , repetitive , and lengthy occupational exposure to magnetic elds from mri equipment . currently , international commission on non - ionising radiation protection ( icnirp ) safety regulations [ 2 ] recommend limiting the time - weighted exposure to 200 mt per working day with instantaneous maxima of 5 t for extremities and 2 t for head or trunk . 
the more conservative italian safety regulations1 impose the following limits of exposure to static magnetic elds : ( a ) 200 mt for a maximum of 60 min in one working day ( 8 h ) , and ( b ) 2 t for a maximum of 15 min in one working day . in 2004 , the european union ( eu ) drafted a new directive to be implemented in eu national regulations by 20132 , 3 , 4 on occupational exposure to static magnetic elds , recognising the limitations of present guidelines and the need for new studies to advance knowledge . 
in particular , the new scientic studies on the impact on health of exposure to electromagnetic radiation , made public after the directive was adopted , have been brought to the attention of the european parliament , the council and the commission . 
the results of those scientic studies are currently being examined by the icnirp as part of the ongoing review of its recommendations . the icnirp guidelines on limits of exposure to static magnetic elds recommend that occupational exposure be restricted to a maximum ( acute ) magnetic ux density of 2 t for head / trunk and of 8 t for the limbs . 
the exposure limit of the general public is derived from the former , by applying a reduction factor of 5 , thereby obtaining a maximum acute exposure of 400 mt for any part of the body [ 2 ]  . in the context of static magnetic elds , the evaluation of the dose is complicated because it involves multiple factors related to the characteristics of the eld and its interaction with living matter . 
02 / 08 / 1991 . 2 directive 2004 / 40 / ec of the european parliament and of the council of 29 april 2004 on the minimum health and safety requirements regarding the exposure of workers to the risks arising from physical agents ( electromagnetic elds ) ( 18th individual directive within the meaning of article 16 ( 1 ) of directive 89 / 391 / eec )  . 3 directive 2008 / 46 / ec of the european parliament and of the council of 23 april 2008 amending directive 2004 / 40 / ec on minimum health and safety requirements regarding the exposure of workers to the risks arising from physical agents ( electromagnetic elds ) ( 18th individual directive within the meaning of article 16 ( 1 ) of directive 89 / 391 / eec )  . 4 directive 2012 / 11 / eu . exposure guidelines not only for patients , but also for staff chronically exposed to static magnetic elds . 
from that work , the authors concluded that the adoption of personal dosimeters is essential to both protect the worker and permit the collection of relevant exposure levels and duration to monitor the health of medical and paramedical mri staff . of particular interest in this paper is the category of occasional and permanent mri staff who are chronically exposed to varying levels of magnetic elds during tasks including patient positioning and removal , system verication tests , calibration . 
therefore , the focus in this paper is on the identication of an equivalent exposure level to static magnetic elds , by incorporating information on both magnetic eld strength and exposure time , to estimate the occupational dose for preventative occupational health and safety . exposure is generally dened as the product of time duration and disturbance ( e.g. , the concentration of a toxic chemical ) [ 4 ]  . 
for the denition of exposure to static magnetic elds , the parameter used as disturbance can be interpreted in different ways , for example , in [ 5 ] it is taken as the square of the magnetic ux density normalised to a specic value . 
however , in this paper , it is recognised that to decouple the possible effects from the cause , the simplest approach is to use the magnetic ux density as a measure of the disturbance . the magnetic ux density , although time - independent , typically varies as a function of position . 
by recording the magnetic ux density to which staff are exposed , as a function of time and integrating this information over time , it is possible to monitor occupational exposure levels . the spatial variation of the eld and the relative movement of mri staff within the magnetostatic eld are included in the present formulation by assuming that the magnetic ux density is time - dependent , bt . 
thus , the cumulative exposure , [ 6 ] , eb ( in tesla ( cid : 2 ) hour = t ( cid : 2 ) h ) is dened as the product of the magnetic ux density by the exposure time , as also used in [ 6 ] : eb btdt where the magnetic density given y b2 x b2 , with bx , by , and bz obtained ( cid : 3 ) fw 0 : 8333b 1 ; b ( cid : 3 ) 0 : 2t b [ 0 : 2t measurements are : radiol med ( 2014 ) 119 : 208213 experimentally from the measurements as detailed in the following . in this paper , the authors propose a further precautionary step to protect occupational staff , by introducing a weighting function incorporating the exposure limits imposed by the legislation of interest . 
one interpretation of the above two different exposure limits is that larger magnetic ux density values carry a more signicant risk , i.e. , a heavier weight , by a factor of 2.5 with respect to a unit weight assigned to lower eld levels . 
to include this distinction in the proposed denition of weighted occupational exposure the weighting function is dened , in the rst instance , as for precautionary limits the individual , personal probes should be calibrated using the most conservative exposure limit indicated in the legislation of interest as a maximum exposure limit to dene the weighting function , example , 0.2 t ( cid : 2 ) h in the case of the italian legislation . the above denition of weighted occupational exposure , eq . 
 ( 2 ) , gives the exibility of modifying appropriately the weighting function ( 3 ) in the light of new regulations , and also in the light of possible new quantitative ndings of the biological effects of static magnetic elds . 
the proposed weighted occupational exposure denition enables the integration of the information on magnetic eld levels and exposure times , and the quantication of the magnetic exposure during the working day for healthcare staff . the physical consequence of the implicit time - variation of the magnetic ux density b ( t ) is the induction of currents in body parts of occasional and permanent staff particularly during patient positioning . 
one of the concerns in high - eld mri is related to the induction of in situ electric elds and associated current densities caused by rapid movement of the body within the nonuniform , strong static magnetic eld [ 7 ]  . in such cases , a number of physiological responses have been reported , including headache , nausea , vertigo , phosphene , numbness and tingling , loss of proprioception , and balance [ 8 ]  . 
theoretical models for the evaluation of electric elds and current densities induced in whole body , in male and the methodology followed in the present work involved the following steps : identication of suitable clinical and research mri equipment ; 2 . 
measurement of the magnetic ux density as a function of time over the operating period , using different isotropic personal probes worn by mri staff , following informed consent ; analysis and interpretation of the results in the context of present national and international regulations . the three hall - sensor probes used for the isotropic ( ets ( a ) holaday hi3550 magnetic field monitor lindgren , cedar park , tx , usa ) : range 0.1 mt \ b \ 0.3 t , measuring both instantaneous and time - integrated magnetic eld , with a measurement update period of 3 s . 
the results of the measurements are shown , in real time , on a liquid crystal display . ( b ) metrolab thm1176 magnetometer ( ght photonics srl , italy ) : range b \ 20 t , probe managed by visa labview runtime ( national instruments ) installed on a handheld computer . 
1 results obtained from the measurements using the three probes : thm 1176 ( solid line ) , tsmd ( dotted line ) , hi3550 [ numeric values of the cumulative exposure , eq . 
 ( 2 ) , obtained for the duration of the measurements , expressed in mt ( cid : 2 ) h ] table 1 cumulative exposure derived from the experimental data of fig . 
the same results have been processed and integrated over time for the duration of the measurements , and listed in table 1 . the kruskalwallis test was used to evaluate the consistency of the experimental data presented in fig . 
having assessed the consistency of the probes , for practical reasons the remaining measurements were conducted using only the talete system ( tsmd )  . a typical example of recording obtained during the operation of patient positioning ( duration of approximately 7 min ) in a 0.35 t scanner is presented in fig . 
the cumulative exposure during a single operation of patient positioning was measured to be eb = 0.7 mt ( cid : 2 ) h , well below the limits imposed by the legislation . 
2 a magnetic ux density measured during positioning of the patient ( approximately 7 min ) in a 0.35 t clinical mri scanner , using the tsmd probe , and ( b ) corresponding db / dt curve limits [ 11 ] as they have been quantied to be \17 mt / s over the whole duration of the operation . in contrast , experimental data taken in the same type of operation ( patient positioning , 5 min duration ) , but in a more powerful ( 3t ) mri scanner are presented in fig . 
the resulting ( measured ) cumulative exposure was eb = 26.83 mt ( cid : 2 ) h , corresponding to a weighted occupational exposure ewb = 35.31 mt ( cid : 2 ) h . in addition , the db / dt curve resulting from exposure levels reported in fig . 
as an alternative , a weighted average is proposed here for the denition of occupational exposure to include limits and guidelines specied by regulatory bodies . radiol med ( 2014 ) 119 : 208213 conveniently , the proposed denition permits to account for possible health effects due to chronic exposure to static magnetic elds by simply modifying the weighting function , always complying with the ofcial recommended limits of exposure . the occupational exposure dened here also permits to determine the number of patients that can be positioned and treated by an individual mri worker in a working day , thus providing further precautionary occupational guidelines . 
in fact , while the cumulative exposure of mri staff operating clinical mri equipment ( b \ 2 t ) typically does not exceed the limit value indicated by the italian regulations , 200 mt ( cid : 2 ) h per day , it may indeed exceed such value in the case of advanced / high - denition and research - type mri equipment ( b [ 2 t )  . 
particularly for the italian case , the relevant authority , the italian national institute for occupational safety and prevention ( ispesl ) , now incorporated into the italian workers compensation authority ( inail ) , requires that the methodology for magnetic dosimetry be specied at the institute level . 
therefore , a detailed record of exposure to static magnetic elds for individual mri staff would be essential to provide data for future epidemiological studies on this category of workers for both acute and chronic effects , especially in view of possible changes in ofcial mri protection guidelines . 
by insisting on the use of personal probes for workers operating mri scanners it will be possible to gather a wide and statistically signicant pool of data to assist studies on health hazards due , specically , to chronic exposure to static magnetic elds . 
the recorded data could be stored daily on a dedicated exposure card to provide a complete history of the occupational exposure to mri - originated static magnetic elds , particularly for consultation from occupational health doctors . 
in view of the above considerations , the following data recording sheet is proposed to permit a full record of the occupational exposure to static magnetic elds . acknowledgments the authors wish to thank f . 
liver biopsy , the current gold standard for the diagnosis and staging of brosis and inammation , may be associated with complications such as bleeding , inherent limitations such as sampling error or interobserver variability and is not readily accepted by all patients [ 5 , 6 ]  . including biochemical and haematological noninvasive diagnosis of hepatic brosis has evolved rapidly in recent years [ 13 ]  . 
several noninvasive techtests niques , and a scoring system using a combination of clinical and laboratory tests are used to predict hepatic brosis , but predict their overlapping cannot results 904 radiol med ( 2014 ) 119 : 903909 necroinammatory activity [ 3 , 5 , 7 ]  . 
ultrasonographybased transient elastography , real - time elastography and acoustic radiation force impulse elastography are used for the evaluation of hepatic brosis but they are operator dependent and may be difcult in obese patients with narrow intercostal space [ 8 , 9 ]  . 
limited studies discuss the role of double - contrast magnetic resonance ( mr ) imaging , mr elastography and mr spectroscopy in the assessment of hepatic brosis in adults , but these pulse sequences are not widely available in clinical practice and their results are preliminary [ 10 , 11 ]  . diffusion - weighted mr imaging depends on the microscopic mobility of water protons . 
to our knowledge , no study to date has addressed the role of diffusion mr imaging in quantication of the degree of inammation in children with chronic hepatitis . the aim of this study was to prospectively evaluate the usefulness of the apparent diffusion coefcient ( adc ) value in the diagnosis and grading of hepatic brosis and inammation in children with chronic hepatitis . materials and methods this study was conducted on 53 consecutive untreated children with pathologically proven chronic hepatitis . three patients were excluded from the study due to motion artefacts at diffusion weighted mr imaging . 
informed consent from parents of patients and controls and institutional review board approval were obtained . mr examinations were performed using a 1.5 - t ( magnetom symphony ; siemens , erlangen , germany )  . 
the machine is equipped with a self - shielded gradient set ( 30 mt / m maximum gradient strength and 120 t / m / s slew rate ) for echoplanar imaging . 
the applied parameters were tr of 2 , 800 ms , te of 74 ms , epi factor of 102 , fov of 25 9 25 cm , section thickness of 7 mm , interslice gap of 20 % , acquisition matrix of 192 9 154 and number of excitations of 6 . 
the adc map was automatically calculated from the data set three b - values by commercially available obtained at software ( leonardo , version 2.0 ; siemens ag medical systems , forchheim , germany )  . 
the adc value was calculated according to the following formula : adc_ ( lnsb2 lnsb1 ) / ( b2 b1 ) , where ln is the natural log , and sb1 and sb2 are the signal intensities in the roi placed on sections corresponding to the two different b values ( b1 and b2 ) [ 30 , 31 ]  . 
the mean of these nine values was calculated and represents the nal adc value per subject that was used for statistical analysis . percutaneous liver biopsy was performed by a paediatric hepatologist ( b.t. ) with 7 years experience in liver biopsy . 
the stages of brosis were f0 ( no brosis ) , f1 ( mild expansion of some portal tracts by brosis ) , f2 ( expansion of most portal tracts by brosis ) , f3 ( occasional bridging brosis ) , f4 ( marked bridging brosis ) , f5 ( incomplete cirrhosis ) and f6 ( complete cirrhosis )  . 
the control children were regarded as having necroinammatory activity grade a0 and brosis stage f0 . statistical analysis was done using statistical package for the social sciences ( spss , inc . , chicago , il , usa ) software , version 16 . 
for statistical analysis , the stages of brosis were classied into mild ( f1 , f2 and f3 ) and advanced ( f4 , f5 and f6 ) and the grades of necroinammatory activity were classied into mild ( a1 , a2 ) and advanced ( a3 , a4 )  . receiver operating characteristic ( roc ) curves were done to evaluate the diagnostic capability of the adc value in differentiating hepatic brosis from normal liver , as well as in staging hepatic brosis and grading hepatic necroinammation . 
the area under the curve ( auc ) , sensitivity , specicity , accuracy , positive predictive value ( ppv ) and negative predictive value ( npv ) were calculated . 
diffusion mr imaging has been used for assessment of diffuse diseases in different regions of the body [ 4042 ] as well as in chronic diffuse liver diseases [ 1327 ]  . in the literature , various studies of diffusion mr imaging reported that adc values of hepatic brosis are lower than those of normal livers in adults [ 1421 ] and in children [ 29 ]  . 
in accordance with most of the previous studies [ 1623 ] , the adc values of the study group were also lower than the adc values of the control group . 
the mechanism of restricted diffusion in brosis is deemed to be multifactorial , mostly due to diminished hepatic perfusion and , to a lesser extent , to the presence of increased connective tissue , which contains fewer protons . 
the deposition of brous tissue leads to a reduction in the size or obliteration of small venules , with a resultant increase in portal venous pressure and consequent compensatory increased arterial blood ow [ 1517 ]  . 
the presence of increased connective tissue in the liver in addition to deposition of collagen bres , fatty inltration , hepatitis , cell necrosis / apoptosis and inammatory cell responsible for restricted diffusion with low adc value in patients with hepatic brosis [ 1419 , 29 ]  . inltration is the role of diffusion mr imaging in the study of diffuse liver disease , therefore , is still under debate . 
6 receiver operating characteristic ( roc ) curve of the adc value used for differentiating mild ( a1 , a2 ) from advanced ( a3 , a4 ) grades cutoff adc value was b1.64 9 10 - 3 mm2 / s with an auc of 0.807 , a sensitivity of 87.5 % , a specicity of 61 % and an accuracy of 74 % necroinammation . 
this may be attributed to more accurate calculation of the adc value in this study which was calculated from three b values ( 0 , 400 and 800 s / mm2 ) whereas other studies calculated the adc value only from two b values . 
last , this study was conducted on children who have better adc maps without noise or susceptibility artefacts compared to the adult patients . in this work , the adc value of hepatic parenchyma changed according to the stages of brosis . 
5 scatter plot of the adc value at different grades of hepatic necroinammation 908 radiol med ( 2014 ) 119 : 903909 know the usefulness of adc in distinguishing between the early and intermediate stages . 
also , linear regression analysis showed that hepatic adc values were a good predictor of brosis stage ( r2 = 0.19 , p = 0.0001 ) [ 19 ]  . in this study , there is a negative correlation between the adc values and necroinammatory activity . 
the adc value is signicantly lower in patients with high grades of hepatic inammation compared to those with low grades . the aggressiveness of chronic hepatitis is indicated by the degree of necroinammation , also called the activity ; the more severe the necroinammation , the more aggressive the disease . 
 [ 24 ] found a statistically relationship between the adc values and signicant hepatic activity index scores although this relationship was not as strong as that between adc values and brosis scores . 
in addition , entropy , which is a measure of the variability in the volume histogram analyses of the adc values , showed a greater correlation with the brosis stage and inammatory activity grade than did mean adc , with a sensitivity of 0.91 and a specicity of 0.92 in the detection of inammation [ 27 ]  . 
recent advances in mr software technology such as 3 t scanners with parallel imaging and respiratory triggering will likely continue to improve image quality and also promise the potential application of multiple and higher b values than 1 , 000 mm2 / s [ 43 , 44 ]  . 
also , further studies with application of diffusion tensor mr imaging with multichannel coils will likely improve the results [ 14 ]  . application of advanced postprocessing methods such as entropy [ 27 ] , bi - exponential , non - gaussian ( diffusion kurtosis ) modelling or k - means clustering algorithms [ 4447 ] may provide a better characterisation of diffuse liver disease . there are a few limitations to this study . 
second , we excluded three patients due to susceptibility artefacts , and we expect that future studies with application of parallel imaging will increase the image contrast and decrease susceptibility artefacts . 
if the registration method works correctly , parameters which bring the images into alignment must always be the same . results automatic registration performed by the software did not prove satisfactory , whereas if a specic tool [ volume of interest ( voi ) tool ] allowing the calculation to be limited to the landmark region was used , the registration s . 
stancampiano dipartimento di specialita` medico - chirurgiche , scuola di specializzazione in fisica medica , universita` degli studi di catania , via santa soa 78 , 95123 catania , italy parameters were comparable with those of the manual registration . 
regarding the repeatability of the automatic registration , the software brought the images in the correct alignment performing translations and rotations along the longitudinal axis up to 40 ( cid : 3 ) , while it was not satisfactory for rotations along the transverse axes . conclusion the experimental clinical application automatic registration is reliable if the voi tool that includes visible landmarks in both studies is used . 
however , because the algorithm did not prove sensitive to rotations along the transverse axes , the position of the patient during the examinations plays a crucial role . results showed that keywords radiotherapy treatment planning ( cid : 2 ) computed tomography ( cid : 2 ) magnetic resonance imaging ( cid : 2 ) image registration ( cid : 2 ) phantom ( cid : 2 ) quality assurance introduction radiotherapy relies on images to plan , guide , and assess treatments [ 1 ]  . 
spatially accurate medical images are , indeed , an essential tool in three - dimensional conformal radiation therapy ( 3d - crt ) and intensity - modulated radiation therapy ( imrt ) treatment planning [ 2 ]  . 
computed tomography ( ct ) is the current standard modality for image - based radiation therapy treatment planning because of its ability to convert hounseld units ( hu ) into electron densities [ 3 ]  . a few years after the introduction of ct in routine clinical practice , it became clear that coupling of information provided by ct with that coming from other diagnostic modalities would improve the quality of the denition of tumours [ 4 ]  . 
this is divided into two phases : registration , which achieves the spatial alignment of the two studies , followed by fusion , which consists in the visual representation and combination of data [ 10 ]  . 
the techniques to perform this process are different , but all include the same basic components : the transformation model ( rigid , afne , projective and deformable ) and the metric ( geometry - based and voxel - based )  . another important feature is the level of interaction between the registration algorithm and the user . 
in the semi - automatic case , the interaction can be of two different kinds : the user can initialise the algorithm ( such as segmenting the data ) , or drive the algorithm ( i.e. , by refusing or accepting the suggested registration ) [ 11 , 12 ]  . the timing required in clinical application undoubtedly favours an automated approach to the image registration both as regards the speed of the operation as its standardisation . nevertheless , it requires a careful analysis of the effectiveness of registration , with the purpose of correcting and / or limiting errors that may occur in clinical implementation [ 13 ]  . that each radiotherapy application demands requirements of quality assurance ( qa ) protocols are met . therefore , also image registration applied to planning and execution of treatment requires a verication of results [ 1 ]  . several groups recommend guidelines for qa of registration in radiosurgery and general planning [ 3 , 1418 ]  . recently , the american association of physicists in medicine ( aapm ) formed task group 132 to review techniques for image registration , to identify issues related to their clinical implementation , to determine the best methods to assess accuracy and to outline issues related to acceptance and qa [ 1 ]  . many reports describe methods and evaluations for registration in the head and neck region [ 3 , 17 , 19 , 20 ] ; far fewer describe results for the pelvis [ 6 , 21 , 22 ]  . 
in this case , as the delineation of the target volume is an important source of geometric uncertainty , it is becoming customary to integrate ct and mri studies [ 13 ]  . the main objective of this work was to evaluate the accuracy of the registration of ct and mri images performed by a software dedicated to treatment planning ( focal , elekta )  . 
for this purpose , a phantom [ stylized anatomy with a registration objective ( s.a.r.o. ) ] dedicated to quality control for the registration of images obtained by ct and mri scanners was designed and built . 
it reproduces in a stylised way that the planning target volume ( ptv ) and organs at risk ( oars ) related to the treatment of the prostate . materials and methods the properties of focal software and s.a.r.o. 
phantom and the main steps of the analysis protocol are described below . description of the registration algorithm the registration algorithm of the focal software uses as a metric mutual information ( mi ) , a voxel - based metric which measures the statistical dependence of information between intensities of corresponding voxels in both images . 
it is supposed that mi is maximum when the two studies are geometrically aligned [ 23 ]  . moreover , the registration algorithm uses a model of rigid transformation with six degrees of freedom : three translations and three rotations . 
a rigid transformation can be described by a single matrix equation in homogenous coordinates by : where , xi and yi ( i = 1 , 2 , 3 ) are , respectively , the components of the new and the old coordinate vectors of the voxel , t is an arbitrary translation vector and r is a 3 9 3 rotation matrix dened by : ril r1 i ; j r2 j ; k r3 with r1 r2 r3 sin a1 0 cos a1 ( cid : 3 ) sin a1 cos a1 sin a2 cos a2 ( cid : 3 ) sin a2 cos a2 cos a3 ( cid : 3 ) sin a2 sin a3 cos a3 each matrix r ( i ) rotates the image about the ith axis by an angle ai [ 11 , 12 ]  . 944 radiol med ( 2014 ) 119 : 942950 once chosen the set of reference images , which constitute the primary study and will be kept xed , the algorithm will perform transformations on the other set of images , the secondary study , until the correct alignment is achieved . finally , focal provides that the transformations are carried out through a semi - automatic interaction with the user . 
once the primary and secondary studies are selected , the software proposes a rst registration founding its calculation on the entire content of the images ( intensity of each voxel )  . 
to check the alignment , the software shows the result of the fusion in various modalities ( panels , strips , merging of voxels ) and in each section ( axial , sagittal , coronal )  . 
it also shows the parameters and the transformation matrix as well as the value of mi in the current alignment . to improve the registration quality , it is possible to select a limited region in the three axes [ volume of interest ( voi ) tool ] within which the algorithm calculates the mi and performs its maximisation . 
since mi is a measure of the information common to both images , the selection of a region with well - dened landmarks allows the mi to be increased and the registration to be improved . 
although the selection of voi is user - dependent , even in this case the registration is automatic . finally , the user can customise the registration by directly applying the necessary transformations to the secondary study , assisted by a continuous view of the two studies in fusion on the three sections . description of the phantom the accuracy of the registration algorithm was evaluated by performing a study on a phantom with appropriate inserts of known size and composition . 
it simulates the pelvis region in a stylised way and it is dedicated to the registration of ct and mri images : s.a.r.o. the phantom consists of a cubical tank ( volume of 200 mm3 ) in which the landmarks shown in fig . 
figure 1 shows the axial section of the phantom with the corresponding anatomical landmarks , while table 1 shows the main properties of the inserts . since the two modalities are based on different physical principles , it is necessary to use materials that give signicant signals in both . 
for this reason , inserts are made of pmma , pvc and teon , tissue - equivalent materials in ct acquisition with slightly different densities , but that provide very weak signals in mri acquisition . 
were acquired with a 64 - slice multislice ct600 scanner ( ge medical systems ) using a protocol for radiotherapy treatment of the pelvis . mri images were acquired with a signahdtx scanner ( ge medical systems ) using a protocol suggested by literature regarding the radiotherapy treatment of the prostate [ 14 ]  . 
table 2 shows the main parameters of the ct and mri images . automatic and manual registration the rst part of the study was dedicated to the verication of automatic registration with focal . radiol med ( 2014 ) 119 : 942950 manual registration : the registration is performed manually by means of a visual inspection of the images in fusion . 
the new transformation parameters are marked down to be compared with those of automatic registration . in this way , it is possible to verify if the automatic registration is comparable to the manual registration . repeatability of the registration transformation parameters , a after identifying the best further study was conducted on the repeatability of the transformation performed by the software . for this purpose , a reverse approach was carried out : starting from the ideal alignment , known transformations were performed to the correctly aligned secondary study to verify whether and to what extent the algorithm was able to return to its original position . 
deviations were calculated using the following expression : transformation between where wi , t are the transformation parameters ( translations and rotations along the ith axes ) imposed to the properly aligned secondary study and wi , a are the transformation parameters found by the software . 
a correct transformation corresponds to a situation in which wi , t and wi , a are equal and opposite and , therefore , dwi equal to zero . results the analysis involved testing the as mentioned above , accuracy of the registration algorithm and the repeatability of the transformations the automatic version of ct computed tomography , mri magnetic resonance imaging , fov eld of view dwi wi ; t wi ; a fig . 
phantom during the ct acquisition table 2 main characteristics of the images acquired in the two modalities slice thickness matrix size 5 mm 500 mm 512 9 512 5 mm 400 mm 512 9 512 once the ct series had been selected as the primary study and the mri series as the secondary study , the analysis was divided into three sections . global registration : the software performs an automatic registration basing its calculation on the overall content of images . 
hence , the studies are shown in fusion , and it is possible to assess directly the registration quality . registration with voi tool : through this tool , the region in which the landmarks are visible is selected on the three sections ( axial , coronal , and sagittal ) , as shown in fig . 
the results of the study are shown below . automatic registration the automatic registration performed using the entire contents of the images showed the limits of this procedure . figure 4 shows the fusion of the ct study ( upper panel ) and the mri study ( lower panel ) after this transformation . the overlap of images indeed , the translation returns , belonging to different planes . table 3 shows the transformation parameters and the corresponding matrix . 
the expressions x ( r ? l ) , y ( s ? i ) and z ( a ? p ) indicate , respectively : translation from right ( r ) to left ( l ) , from cranial [ superior ( s ) ] to caudal [ inferior ( i ) ] and from anterior ( a ) to posterior ( p )  . the registration algorithm only ran translations along the three axes . 
as can be seen from this gure , the planes of the two studies coincide , as well as the position of landmarks . the parameters and the transformation matrix obtained through the voi tool are shown in table 4 . as a nal step , translations and rotations were performed manually based on a view of images in fusion . 
the error associated with the manual registration was evaluated by considering the spatial resolution of the images ( 1 mm in the axial plane , 5 mm in the sagittal and coronal planes )  . 
table 5 shows the parameters of the manual transformation and the corresponding matrix . comparing the results obtained with manual registration ( table 5 ) and those of automatic registration obtained by the voi tool ( table 4 ) , it can be noticed that the differences are minimal when compared to the spatial resolution of the images ( calculated from the eld of view size and the sampling matrix )  . 
these differences are included , according to the previously indicated resolution limit , in the error related to the manual registration . to quantitatively evaluate the accuracy of registration , the landmarks were contoured separately in the mri and ct studies . 
6. to further verify the correct operation of the algorithm , a registration of the ct study with itself was performed . the algorithm returned null values for each parameter and the transformation matrix corresponded with the identity matrix . 
for greater rotations , the algorithm was not able to nd the proper registration even with the use of the voi tool . rotations along x : the algorithm failed to nd the correct alignment for small rotations ( 2 ( cid : 3 ) ) even if the voi tool was used . rotations along z : as in rotations along the x axis , the algorithm , running into local maxima , failed to properly register the images with rotations of more than 2 ( cid : 3 )  . complex performing transformations : exclusively translations , the registration algorithm was able to properly align the studies , especially if the voi tool was used . 
table 6 shows some examples of deviations for rotations along y , x , z axis and an arbitrary translation along the three axes . as anticipated , for rotations along the x and z , even with the use of the voi tool , deviations remained signicant . discussion the major advances in radiation therapy have highlighted the need to improve the ability to locate and delineate tumour , healthy tissue and oars accurately . 
the nal image shows that the registration is satisfactory imposed and the for this purpose , the differences between the transfortransformation mation parameters parameters found by the software to properly align the two studies were evaluated . 
although physical spatial resolution along the coronal and sagittal planes is 5 mm , 0.001 mm ( as can be seen in the tables showing the transformation parameters ) , a value that goes far beyond the spatial resolution of the images . 
since , this part of the study was addressed to the ability of the algorithm to nd numerically the same transformation parameters , the same limit was set for all three planes . algorithm computation grid extends to evaluate the automated version of focal , a global registration was rst performed , followed by a registration with voi tool . 
phantom was designed to ensure significant signals in both imaging modalities . first of all , the optimisation procedure of the registration requires , depending on the algorithm , some precautions during image acquisition . 
in addition , since any change in patient position and organ lling represent sources of geometric uncertainties , registration algorithms that use models of rigid transformation must be evaluated carefully to ensure acceptable coherence between the modalities employed . 
respecting these precautions in routine procedures already represents a denite improvement as regards both the quality of the registration and the required timing . during the study , after obtaining images of the phantom according to these precautions , an automatic registration was performed allowing the software to exploit the entire contents of the ct and mri images . 
for this reason , the registration of ct studies with other diagnostic studies is emerging strongly in many centres and it is becoming an integral part of treatment planning . to minimise the possibility of human or software errors , clinical image acquisition and registration processes should be managed where possible in an integrated way to ensure their cogency , sequentiality and reproducibility [ 1 ]  . 
the registration algorithm is , in its automatic version , reliable for rotations along the y - axis up to 40 ( cid : 3 ) only if the voi tool is used . 
the algorithm is indeed able to assure the alignment of the studies if the voi tool is used . on the other hand , automatic registration did not prove satisfactory with regard to rotations along the x and z axis . 
this leads to the criticism of the correct patient position in the two studies as even small differences could affect is therefore desirable to use appropriate outcome . systems for patient immobilization with high precision positioning . 
this objective is certainly favoured by performing the studies on the same day and at a short temporal distance from one another . conclusion this the main objective of this work was to verify the accuracy and the repeatability of the registration of ct and mr images using the automatic version of the focal software . for this purpose , images of the s.a.r.o. 
the results show that , using the manual registration as a reference because it allows one to visually assess the quality of alignment , automatic registration is comparable when the voi tool is in clinical used . applications , the algorithm is reliable and fast if the voi includes that are clearly visible on both modalities . 
the ndings of ceus were compared with those of contrast - enhanced computed tomography scans at level - 1 diagnostic tests . results out of the 22 patients , 21 were diagnosed with blunt pancreatic injury using ceus , including 8 patients with lesions in the neck of pancreas , 9 in the body , 3 in the tail and 1 in the head . 
using ct as a reference standard , the detection rate of ceus in blunt pancreatic trauma was 95.5 % ( 21 / 22 )  . conclusions ceus ndings can be used to provide a reliable diagnosis for blunt pancreatic trauma . 
ceus is thus promising in the assessment of blunt pancreatic trauma , especially in institutions where emergency ceus is used as an initial diagnostic instrument . keywords pancreas ( cid : 2 ) trauma ( cid : 2 ) diagnosis ( cid : 2 ) contrastenhanced ultrasonography ( cid : 2 ) microbubbles introduction trauma [ 1 ]  . 
however , pancreatic trauma is rare , occurring in about 0.2 % of patients with abdominal accompanied with a high mortality rate of 7080 % when injury involves the aorta , the superior mesenteric artery or the vena cava , which is adjacent to the pancreas . 
nie department of radiology , chinese peoples liberation army general hospital , beijing , china e - mail : nieyongkang@gmail.com radiol med ( 2014 ) 119 : 920927 pancreatic injuries result in complications ( severe haemorrhage , pancreatic stula , and abscess ) in 2030 % of cases and mortality in more than 20 % [ 2 ]  . prompt diagnosis of pancreatic injuries has been challenging , particularly in blunt trauma patients . 
diagnosis has relied on amylase levels , computed tomography ( ct ) scans , magnetic resonance imaging ( mri ) and ultrasound ( us ) , with various levels of success . 
this number rises to 84 % following 3 - h elapse between trauma and the time of measurement [ 2 , 7 , 8 ]  . undoubtedly , the best diagnostic tool for pancreatic trauma is ct [ 9 ]  . 
moreover , mr cholangiopancreatography ( mrcp ) can be more useful because it can demonstrate clear delineation of the duct and its integrity . however , ct and mr equipment is large and difcult to use at the bedside for early diagnosis , or at trauma scene for point - ofcare diagnosis . 
however , conventional us cannot sufciently display the location and severity of pancreatic lesions . the introduction of us contrast agents has led to an increase in the diagnostic accuracy of us in many organs , and the technique now has a wide range of applications [ 1115 ]  . 
in 2013 , cokkinos [ 16 ] reported that contrastenhanced us ( ceus ) accurately dened organ injuries , capsular extension and even vascular injury with very good correlation with ct . the portability of ceus makes it a good tool in trauma settings because examinations can be performed quickly at a patients bedside and benets patients with either stable or unstable haemodynamics . 
the purpose of our study was to present the application of ceus in diagnosing adult pancreatic injuries caused by blunt abdominal trauma . materials and methods this study was approved by the ethics committee of the chinese peoples liberation army general hospital and complies with the health insurance portability and ( hipaa )  . regulations , in accordance with our accountability act for ceus institutions examination and use of related data for future research was obtained from each patient ( or the patients relatives if he / she was unconscious )  . informed consent patients from march 2007 to june 2012 at two institutions equipped with modern laboratory , ultrasonographic and radiological equipment , a retrospective review of the charts of 22 consecutive patients with pancreatic injuries due to blunt abdominal trauma assessed by contrast - enhanced ct ( cect ) , was undertaken . the patients demographic data and the mechanism of injury were retrieved from the medical les . 
seven patients had isolated pancreatic injury and 15 patients had complex injury , including 4 with hepatic injury , 6 with splenic injury , 2 with renal injury , 2 with hepatic and splenic injuries , and 1 with renal and splenic injuries . 
the mechanisms of injury included accidental fall , motorcycle crash , motor vehicle crash , and automobile - pedestrian collision . ultrasound imaging the ultrasound contrast agent used in this study was sonovue ( bracco , milan , italy ) , which is a second - generation contrast medium approved in china in 2003 for diagnostic imaging . 
the agent consists of stabilised microbubbles containing inert gas ( sulphur hexauoride , 8 ll / ml of solution ) , and is covered by a phospholipid membrane [ 17 ]  . 
the intravenous injection of this contrast agent can be repeated . conventional us and ceus were performed at patients bedside in the emergency department , using a mobile sequoia 512 machine ( siemens medical solutions , mountain view , ca ) or a portable cx50 system ( philips medical systems , andover , ma )  . 
the scan settings ( including gain , 922 radiol med ( 2014 ) 119 : 920927 scanning depth , and time gain control ) were optimised on each target region independently . 
a lesion involving more than 50 % of the thickness of the pancreas indicated impending disruption [ 18 ] , the diagnosis of pancreatic ductal injury was done indirectly by using ceus . ct imaging following the instruction of the emergency department , all patients were scanned within 1 h of admission by an emergency systematic ct scan after or before ceus examination . 
differences between group means were determined with analysis of variance ( systat ver 13.0 , spss , inc , chicago , il ) with chi - square test , consistency checking t or t test , where applicable . 
using cect as a reference standard , ceus value in the diagnosis of blunt pancreatic trauma was evaluated . kappa values were calculated to assess inter - reader agreement on ceus and us . 
b contrast - enhanced transverse ultrasound image shows the injury site as an anechoic and hypoechoic perfusion defect area with an irregular border in the pancreatic body ( short arrows )  . 
these changes were also shown on the cect images . discussion pancreatic trauma , especially contusion , is a relative enigma , even in modern medical practice with advanced diagnostic technologies . 
however , delayed diagnosis of mild pancreatic injuries could cause major therapeutic challenge to the medical team and potentially disastrous situations for the patients . therefore , improving the sensitivity and specicity of diagnosing pancreatic injuries is essential for effective treatment . ceus has largely improved us accuracy in detecting parenchymal organ injury after blunt abdominal trauma [ 15 , 1921 ]  . 
it can help depict ndings that are not accessible on conventional us , such as tissue hypoperfusion , nonperfusion , hyperaemia and contrast extravasation . in 2006 , valentino et al . 
the portability of ceus makes it a good tool in trauma settings because examinations can be performed quickly at the patients bedside and benets patients with either stable or unstable haemodynamics . 
although a case study has been recently reported , the value of ceus in pancreatic injuries has not yet been evaluated with doubleblind comparisons of ceus and cect . on the ceus images and animations , pancreatic disruption or lacerations appear as anechoic and / or hypoechoic perfusion defect area in arterial and parenchymal phases , ideally with separated structures , which can be missed on conventional us images . 
b contrast - enhanced transverse ultrasound image shows the injury site as an anechoic and hypoechoic perfusion defect area with an irregular border in the pancreatic neck ( arrow )  . 
in this group , 16 patients were considered as having pancreas injuries by us on the basis texture . of pancreas enlargement and heterogeneous however , us did not conrm the lesion size and injury severity because it could not depict the lesion borders clearly . 
while ceus has signicantly improved the ability to visualise the peripancreatic space , the reason for this phenomenon may be the difference in blood supply , which makes it possible for ceus to have a better view of pancreas and peripancreatic microcirculation perfusion . 
of note , ceus visualised pancreatic ductal injury , which was consistent with cect . this study also showed that a ceus examination for pancreatic injuries could be completed in less than 5 min using a single dose of contrast agent with operators skill and experience . 
 ( 2 ) it is portable , therefore it can be performed in the emergency room , ct department and at the bedside in radiol med ( 2014 ) 119 : 920927 fig . 
especially for patients with unstable haemodynamics , ceus is the preferred imaging method . ( 3 ) the examination is timely , with a room time \5 min . ( 4 ) ceus examination can be simultaneously performed while performing other physical examination or resuscitation procedures . some disadvantages of ceus have also emerged in this study . 
 ( 2 ) ceus requires rapid and skilful diagnosis because the duration of a single ceus examination is only 68 min . ( 3 ) timely identication of major pancreatic ductal injury is imperative because delayed diagnosis is largely responsible for the high morbidity and mortality associated with blunt pancreatic trauma . 
either ceus or cect indirectly diagnosed pancreatic ductal injury on the basis of a lesion involving more than 50 % of the thickness of pancreas , and the diagnostic accuracy are both lower . 
endoscopic retrograde cholangiopancreaticography ( ercp ) is the most reliable diagnostic method to accurately dene the continuity of the main pancreatic duct following pancreatic trauma , and it has been strongly suggested as an effective procedure for diagnosis and therapeutic interventions , including stent placement [ 25 ]  . 
however , the invasive nature and associated complications are the major drawbacks of ercp , limiting its use in unstable and uncooperative patients . pancreatic injuries sustained by blunt abdominal trauma are extremely rare . 
it should not be intended as a substitute for ct , but as a selective possibility to boost the role of us in the initial screening of patients with pancreatic trauma . in conclusion , ceus showed the site of pancreatic injury as anechoic and / or hypoechoic perfusion defect region with irregular borders in both the arterial and parenchymal phases , on which basis the diagnosis can be 926 radiol med ( 2014 ) 119 : 920927 fig . 
d conventional us shows that the pancreatic neck injury has become larger , the thin pancreatic parenchyma remains ( arrow ) and a peripancreatic liquid collection has appeared on the 16th day after trauma . 
the purpose of this study was to analyse the radiological and clinicopathological features of sfts in the extracranial head and neck region . materials and methods we retrospectively reviewed the clinical , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and pathological features in 18 patients ( 12 men and 6 women ) , aged 1875 years , with histologically proven sfts in the extracranial head and neck region . fourteen patients underwent ct scanning and nine techniques included underwent mri . 
639 , zhi zao ju road , shanghai 200011 , china e - mail : xiaofengtao2012@126.com enhancing and slow washout pattern on dynamic contrastenhanced mri and dual - phase ct . 
rapidly enhancing and slow washout pattern tics may be additional valuable features . keywords solitary brous tumours ( cid : 2 ) head and neck ( cid : 2 ) magnetic resonance imaging ( cid : 2 ) dynamic contrast enhanced imaging ( cid : 2 ) diffusion - weighted imaging ( cid : 2 ) computed tomography solitary brous tumours computed tomography abbreviations sfts mri magnetic resonance imaging diffusion weighted imaging dynamic contrast enhanced introduction solitary brous tumours ( sfts ) are mesenchymal neoin the pleura [ 1 ]  . frequently occur plasms that most although rare , there has been an increasing incidence of the tumour being found in extrapleural sites such as the abdomen , pelvis , trunk and meninges . 
it can also arise in the extracranial head and neck region such as the orbit , cheek , laryngeal , parapharyngeal space , oral cavity and thyroid gland [ 2 , 3 ]  . radiol med ( 2014 ) 119 : 910919 despite their characteristic , histological and immunohistochemical features , sfts of the extracranial head and neck region remain a diagnostic challenge to both clinicians and radiologists , because they are often poorly recognised and confused with other more common neoplasms . 
here , we conduct a retrospective review of the computed tomography ( ct ) and magnetic resonance ( mr ) imaging features of these 18 cases of sfts and evaluate the clinicopathological features and prognosis . there is only somewhat materials and methods from june 2004 to december 2012 , review of medical records based on the electronic data base of our institution , approved by our institutional review board , revealed 18 patients with pathology - proven sfts occurring in the extracranial head and neck region . 
we retrospectively reviewed the ct and mr ndings obtained from these patients . all ct scans ( 14 patients ) were obtained with a light speed 16 ( ge healthcare , milwaukee , wi , united states ) or brilliance 64 ( philips healthcare , best , the netherlands )  . 
of the 14 patients , 2 underwent plain ct scan only and 12 underwent contrast - enhanced ct scan ( three with single - phase ct with a 45 - s delay and nine with dual - phase ct with 30 and 60 s delay ) [ 4 ]  . 
the scanning parameters were as follows : 5 - mm slice thickness with 11.25 mm reconstructions , 23 cm eld of view ( fov ) , 120140 kv voltage , 200300 ma current , and 256 9 256 matrix . 
a imaging protocols neurovascular array coil was used . contained at least an unenhanced axial t1 - weighted ( tr / te , 600 / 11 ms ) spin - echo sequences , axial t2 - weighted ( tr / te 4 , 700 / 85 ms ) fast spin - echo sequences with fat saturation , and coronal t2 - weighted ( tr / te 3 , 200 / 100 ms ) fast spin - echo sequences ; and contrast - enhanced axial , and coronal t1 - weighted ( tr / te 600 / 11 ms ) sequences . 
an intravenous dose of 0.1 mmol / kg of contrast agent ( gadolinium - dtpa , magnevist ; schering ) had been administered to the patients undergoing conventional contrast - enhanced mri . 
the dwi parameters were as follows : tr / te , 2 , 5003 , 000 / 70 ms for a b factor of 0 and 1 , 000 s / mm2 , thickness / space , 5 / 0.5 mm ; matrix , 128 9 128 ; fov , 24 cm ; and eight acquisitions . the ct and mr images were reviewed by two dedicated head and neck radiologists in consensus . 
we evaluated the imaging ndings with emphasis on the number , size , margin , shape , internal architecture , and pattern and degree of enhancement of the lesion [ 4 ]  . 
as for internal architecture , we compared the attenuation of the lesion on plain ct and the signal intensity of the lesion on t1and t2weighted mr images with that of the neck musculature . we also recorded the presence of any calcications based on plain ct scans . 
as for the degree of enhancement on ct scan , we relied on early - phase contrast - enhanced axial images , viewed with the same window width ( 350 hu ) and level ( 40 hu )  . 
in nine patients examined with dual - phase ct , we also measured the attenuation values in the axial plain , earlyand late - phase ct scans to plot the time - attenuation graph . 
apparent diffusion coefcient ( adc ) values were obtained on dwi , and the time - intensity curves ( tics ) and the maximum slope of increase on dce - mri . clinical data were retrieved from the medical records . sixteen patients underwent complete excision of tumour and two patients underwent partial excision at our institution . 
immunohistochemical included staining for cd34 , vimentin , cd99 , b cell lymphoma protein 2 ( bcl - 2 ) , s - 100 protein , smooth muscle antibody ( sma ) and cytokeratin analysis table 1 summary of clinical ndings in 18 patients case age / sex symptom preoperation diagnosis treatment follow - up ( months ) radiol med ( 2014 ) 119 : 910919 30 / m 27 / m 68 / m 55 / m 23 / f 49 / m 22 / m 59 / f 18 / f 54 / m 31 / m 62 / m 48 / f 46 / m 45 / m 75 / f 24 / f 49 / m proptosis palpable mass facial palsy palpable mass proptosis proptosis sore pain palpable mass sore pain sore pain palpable mass palpable mass palpable mass palpable mass swelling proptosis r ; parotid gland adenolymphoma oropharynx bleeding l ; parapharyngeal space pleomorphic adenoma palpable mass l ; parapharyngeal space schwannoma sfts haemangioma haemangioma haemangioma haemangioma sfts schwannoma location r ; orbit r ; masticator space l ; masticator space r ; orbit r ; orbit r ; maxillary l ; orbit l ; orbit l ; cheek r ; cheek l ; orbit l ; orbit r ; infratemporal fossa r ; cheek malignant neoplasm malignant neoplasm l ; submandibular space haemangioma granulomatous lesions schwannoma / sfts haemangioma / sfts malignant neoplasm schwannoma / haemangioma complete complete complete complete complete complete complete complete complete partial complete partial complete complete complete complete complete complete lost m male , f female , r right , l left , sft solitary brous tumour , complete complete excision , partial partial excision ( ck )  . 
prognosis was assessed by clinic service and telephone interview . operation , 17 patients were in good health with no evidence of recurrent tumour during a follow - up of 2102 months ( mean 36 months )  . results clinical data the clinical features of 18 patients with sfts in the extracranial head and neck region are summarised in table 1 . 
seven of the 18 lesions arose from the orbit , 3 / 18 from the cheek , 2 / 18 from the masticator space , 2 / 18 from the parapharyngeal space , 1 / 18 from the infratemporal fossa , 1 / 18 from the maxillary , 1 / 18 from the submandibular space , and 1 / 18 from the parotid gland . 
sfts were often misdiagnosed as being another type of common tumour , such as cavernous haemangioma ( 7 / 18 ) , schwannoma ( 4 / 18 ) , pleomorphic adenoma adenolymphoma ( 1 / 18 )  . 
after the granulomatous lesions ( 1 / 18 ) , ( 1 / 18 ) imaging ndings the ct and mr imaging features of 18 patients with sfts in the extracranial head and neck region are summarised in table 2 . 
the presence of rounded or linear tissues low - intensity foci was attributable to the collagen content , low cellularity , and associated reduced proton mobility . sfts are vascular tumours that are vigorously enhancing . discussion solitary brous tumours are rare spindle - cell neoplasms originating from mesenchymal tissue . 
although mostly occurring in the pleura , this tumour has also been recently reported in a number of extrapleural sites , including the mediastinum , lung , liver , pancreas , retroperitoneum , kidney , meninges , and extracranial head and neck region such as the orbit , nasal cavity , paranasal sinus , epiglottis , thyroid gland , salivary gland , infratemporal fossa , buccal space and parapharyngeal space [ 2 , 3 ]  . 
in our study , 18 sfts in extracranial head and neck region were located in the orbit , cheek , masticator space , the parapharyngeal space , infratemporal fossa , maxillary , submandibular space and the parotid gland , respectively . clinically , the symptoms and signs of sfts depend on the location of the tumour and the involved organs or tissues , as shown in our series . 
k the inlet shows that the tumour cells and the capillary endothelial cells have immunohistochemically positive cd34 results ( original magnication 9200 )  . l immunohistochemistry of the tumour cells showing positive staining for vimentin ( original magnication 9200 ) space , the initial symptom may not be obvious , and their sizes are usually much bigger . 
patients with sfts occurring in the supercial soft tissues like the parotid gland often present with an asymptomatic palpable mass . sft most commonly present during the fth and sixth decades of life , and there is no signicant sex predilection [ 6 ]  . 
in our study , the age range was also 1875 years , but the incidence was higher in men than in women with a sex ratio of 12 : 6 . 
although local excision is usually curative , sfts have been reported to recur and metastasise . complete surgical excision appears to be the treatment of choice for sfts in the extracranial head and neck region . the prognosis of sfts might be strongly related to initial treatment , and total resection is associated with longer progression - free survival [ 7 ]  . 
in our series , except for one patient who was lost to follow - up , the remaining patients made uneventful recoveries and they are still clinically and radiologically free from tumour recurrence during the follow - up period . microscopically , sfts are well - circumscribed , nonencapsulated tumours that are known to have a characteristic patternless arrangement of alternating hypercellular and hypocellular regions of spindle cells against a collagenous background of variable vascularity . 
in addition to cd34 , sfts are also immunoreactive for mesenchymal markers ( vimentin ) , but they are usually negative for epithelial , vascular , neural and muscle markers [ 8 ]  . 
in our study , sfts were immunoreactive for cd34 , vimentin , partial cd99 and bcl - 2 , but negative for s - 100 protein , ck and sma . 
out of the 18 cases , we were able to identify only 3 cases as having atypical or malignant features . the reported imaging ndings of sfts in the extracranial head and neck region are rather nonspecic [ 9 ]  . 
 [ 11 ] , sfts were mostly isodense on plain ct , homogeneously isointense or mildly hypointense to muscle on the t1 - weighted images , and heterogeneously and mildly hyperintense on the t2 - weighted images . 
 [ 4 ] reported that isointensity to hypointensity on t2weighted images reected brous tissue with high collagen content , and hyperintensity was related to internal haemorrhage , cystic degeneration , or relatively fresh brosis . 916 radiol med ( 2014 ) 119 : 910919 fig . 
a the t1 - weighted image shows a well - dened , irregular mass ( star ) is isointense to muscle with inner hypointense linear septa ( arrow )  . 
we also believe that this variable signal intensity depends mainly on the differences in the main components of the tumour , namely , the amount of cellular components , collagen and broblasts , and also on the presence of degeneration . 
we also observed an interesting nding , that is , the linear septa within the tumours in two cases . those lines corresponded to the hypocellular collagenous stroma intervening in the hypercelluar area . 
even if these hypointense lines were not specic for sfts , we suggest 918 table 3 summary of the histological features of sfts in the present series case benign / malignant cd34 vimentin cd99 bcl - 2 s - 100 benign benign benign benign benign benign benign borderline benign benign malignant malignant benign benign benign benign benign benign radiol med ( 2014 ) 119 : 910919 ? positive ; negative ; partial positivity that they are helpful for including this rare tumour in the differential diagnosis of masses involving the extracranial head and neck region . 
large necrosis or cystic degeneration was present in one case which was proven to be the malignant neoplasm . enhancement of a certain tumour can best be assessed radiologically by dynamic ct or mr imaging . 
in this study , all nine lesions examined with dual - phase ct demonstrated rapid , intense enhancement at early - phase axial scanning , followed by slow washout of contrast material at on late - phase axial images . 
on the dce - mri , the tics also exhibited a rapidly enhancing and slow washout pattern in our cases , which may be an additional clue to establish the correct diagnosis . 
 [ 4 ] reported that the timeattenuation curves of orbital sfts on dual - phase ct demonstrated rapid , intense enhancement at early - phase axial scanning , followed by signicant progressive washout of contrast material at late - phase axial and delayedphase coronal scanning . the dwi ndings of the sfts in the extracranial head and neck region have been rarely reported . 
owing to the limited number of cases , we could not compare adc values between the benign and malignant sfts . on the basis of the aforementioned imaging ndings for sfts of the extracranial head and neck region , this tumour is often misdiagnosed as other common tumours on the initial images , such as cavernous haemangioma , schwannoma , pleomorphic adenoma and adenolymphoma . 
in our cases , cavernous haemangioma and schwannoma should be the main lesions to be included in the differential diagnosis . cavernous haemangiomas may be the most difcult tumours to differentiate from sfts in the extracranial head and neck region on imaging studies . 
their typical feature shows phleboliths in the tumour which are hyperdense on plain ct scan , hypointense on both t1 - weighted and t2 - weighted images and unenhanced on the contrast - enhanced images . 
on the other hand , the other parts show marked hyperintensity on t2weighted images with fat saturation , and delayed pooling of contrast material on dynamic studies [ 15 , 16 ]  . 
they are usually solitary , slowly growing benign lesions and , along with neurobromas , they are the most common nerve sheath tumours . radiol med ( 2014 ) 119 : 910919 extracranial schwannomas have a distinct tendency to originate in the head and neck region . 
unlike sfts , schwannomas have more cystic degeneration , so they are heterogeneously hyperintense on t2 - weighted images and show moderate heterogeneous enhancement on t1 - weighted contrast - enhanced images . 
a solitary , ovoid and well - dened mass with isodensity on plain ct , isointensity on t1 - wi , mild hyperintensity on t2 - wi and strong enhancement after contrast agent injection is suggestive of this diagnosis . 
the prognosis of sfts might be strongly related to initial treatment , and total resection is associated with longer progression - free survival . acknowledgments the authors thank henghua zhou for her help in pathology . 
signal changes and enhancement of vertebral bodies , involvement of paravertebral soft tissues and epidural spaces , nerve root and cord compression and abscess formation were assessed . results of 63 patients with spinal brucellosis , eight had thoracic , 35 had lumbar , ten had cervical vertebral , seven had both thoracic and lumbar , and three had both lumbar and sacral vertebral involvement . 
zhang ( & ) department of orthopaedics , yuncheng central hospital , yuncheng 044000 , shanxi , china e - mail : zhangqin19762@126.com conclusion brucella is still a public health problem in endemic areas . 
mr imaging is a highly sensitive and noninvasive imaging technique which should be rst choice spondylodiscitis . diagnosis imaging early keywords spine ( cid : 2 ) brucella ( cid : 2 ) spondylodiscitis ( cid : 2 ) mri introduction situation became more brucellosis is a systemic infection caused by genus brucella which is a facultative intracellular organism [ 1 ]  . although rarely found in industrialised countries , it is still endemic and considered a public health problem in many developing countries including china [ 2 ]  . 
osteoarticular manifestations , including sacroiliitis , peripheral arthritis , spondylitis , osteomyelitis , and bursitis are the most frequent complications of brucellosis and occur in one - third of the patients [ 5 ]  . 
diagnosing brucellar spondylodiscitis is often difcult ; clinical ndings are usually nonspecic , and radiological features may mimic those of other bacterial , fungal , inammatory , and neoplastic diseases . 
it has been reported that magnetic resonance ( mr ) imaging can differentiate brucellar spondylitis from other spinal infections , along with a good clinical background [ 68 ]  . severe the aim of this retrospective study was to report the clinical features and imaging ndings of patients with brucellar spondylodiscitis . 929 radiol med ( 2014 ) 119 : 928933 fig . 
b the t1 - weighted image reveals hypointense signal intensities corresponding to the same level . c fat - suppressed t2 - weighted image showing hyperintense signal intensities corresponding to the same level . 
d t2weighted axial image showing increased homogeneous signal intensity due to paravertebral abscess materials and methods this retrospective study included 63 cases ( 19 women and 44 men ; mean age 54.7 years ; age range 1874 years ) of brucellar spondylodiscitis , recruited from the archive system of the imaging services among 314 patients with presumed spondylodiscitis by the department of infectious diseases between 2009 and 2012 . 
the diagnosis of brucella infection was considered when seroagglutination tests were positive at a titre of 1 / 160 or higher , and / or brucella spp . was isolated in the blood or sample cultures . 
the diagnosis of spinal brucellosis was made upon ndings consistent with infection in at least one vertebra or intervertebral disc on mr imaging , and positive clinical ndings . each patients medical history , physical examination and laboratory ndings were recorded . 
all of the images were evaluated by two radiologists and a diagnosis was made . results the study involved 19 ( 30 % ) female and 44 ( 70 % ) male patients with spondylodiscitis ( mean age 54.7 , range 1874 )  . 
b the t1 - weighted image reveals hypointense signal intensities corresponding to the same level . c fat - suppressed t2 - weighted image showing hyperintense signal intensities corresponding to the same level . 
thirteen patients had cord compression , six had root compression , four had facet - joint involvement and one had erector spinae muscle involvement at multiple levels . gibbus deformity was not observed in any patient . 
in the acute stage , mr imaging showed low signal intensity on t1 - weighted images and high signal intensity on t2weighted images of the vertebral bodies , endplates and intervertebral disc space . 
infectious spondylitis is an infection by a specic organism of one or more components of the spine , namely , intervertebral discs , paraspinal abnormal soft tissues , and epidural space . 
a the t2 - weighted image shows a focal epidural collection at the posterior aspect of the c6 and c7 bodies . b the t1 - weighted image reveals hypointensity signal intensities at the posterior aspect of the c6 and c7 bodies . 
c a sagittal gadoliniumenhanced , t1 - weighted magnetic resonance imaging scan of the cervical spine shows an epidural uid collection with an enhancing rim , representing an epidural abscess . 
c fat - suppressed t2 - weighted and adjacent image showing hyperintense signal intensities correspond to the same level at the t1 - weighted image erythrocyte sedimentation rate ( esr ) in patients with spondylitis [ 13 ]  . 
the sexes were affected equally . 932 radiol med ( 2014 ) 119 : 928933 according to the time from onset of symptoms in the group with spondylitis , 17 ( 26 % ) brucellosis patients were classied as acute and 31 ( 49 % ) as subacute . 
generally , two vertein six patients , three adjacent brae were involved , but vertebral bodies were involved , these involved vertebrae are mostly contiguous , but noncontiguous spine levels are rare in brucella spondylodiscitis [ 16 ]  . 
cervical involvement , however , is accompanied by more severe manifestations , due to frequent epidural masses with spinal cord compression and neurological disabilities [ 1821 ]  . superior endplate is the rst involved part of the vertebrae because of its rich blood supply and causes bone destruction in the early stage [ 9 , 22 ]  . 
however , epidural masses may be together with spondylodiscitis and sometimes compress nerve roots or spinal cord and mimic disc herniation , which occurred in three of our patients . mr imaging is very useful in differentiating brucellar spondylodiscitis from other spinal pathologies , including tuberculous spondylitis , pyogenic spondylitis , postoperative changes , spinal degenerative diseases , and vertebral metastases [ 9 , 23 ]  . 
in the acute stage , mr imaging depicted low and high signal intensities on t1 and t2 - weighted images , respectively , on vertebral bodies , vertebral end plates and intervertebral discs [ 11 , 22 , 24 , 25 ]  . 
in the subacute and chronic stages , mr imaging revealed hypointense and heterogeneous signal intensities in t1and t2 - weighted images , respectively . contrast - enhanced images reveal contrast enhancement of discs and affected vertebrae in acute , subacute and chronic stages [ 26 ]  . 
paravertebral and / or epidural abscess formation is a rare nding , compared with other infective spondylodiscitis in brucella spondylodiscitis involvement of vertebral mostly unifocal , but multifocal and multilevel involvements may also be seen [ 27 ]  . 
for this reason , the initial clinical evaluation of the patient segments acute [ 5 ]  . with other laboratory ndings like c - reactive protein ( crp ) and esr and follow - up of the patients symptoms and response to therapy are very important in the consideration of spinal involvement . spinal brucellosis may mimic other diseases that affect the spine , such as tuberculous spondylitis , pyogenic osteomyelitis . 
tuberculous ( tb ) spondylitis remains the most important disease in differential diagnosis because of similar mr imaging appearances of both diseases in some cases [ 28 , 29 ]  . 
the size of the paraspinal mass in tb spondylitis is usually larger than paraspinal mass of brucellar spondylitis [ 30 , 31 ]  . imaging modalities and is very useful spondylodiscitis is a rare but important complication of brucellosis which can cause transient or persistent neurological decits resulting in spinal deformities if not appropriately treated . 
the ndings of ceus were compared with those of contrast - enhanced computed tomography scans at level - 1 diagnostic tests . results out of the 22 patients , 21 were diagnosed with blunt pancreatic injury using ceus , including 8 patients with lesions in the neck of pancreas , 9 in the body , 3 in the tail and 1 in the head . 
using ct as a reference standard , the detection rate of ceus in blunt pancreatic trauma was 95.5 % ( 21 / 22 )  . conclusions ceus ndings can be used to provide a reliable diagnosis for blunt pancreatic trauma . 
ceus is thus promising in the assessment of blunt pancreatic trauma , especially in institutions where emergency ceus is used as an initial diagnostic instrument . keywords pancreas ( cid : 2 ) trauma ( cid : 2 ) diagnosis ( cid : 2 ) contrastenhanced ultrasonography ( cid : 2 ) microbubbles introduction trauma [ 1 ]  . 
however , pancreatic trauma is rare , occurring in about 0.2 % of patients with abdominal accompanied with a high mortality rate of 7080 % when injury involves the aorta , the superior mesenteric artery or the vena cava , which is adjacent to the pancreas . 
nie department of radiology , chinese peoples liberation army general hospital , beijing , china e - mail : nieyongkang@gmail.com radiol med ( 2014 ) 119 : 920927 pancreatic injuries result in complications ( severe haemorrhage , pancreatic stula , and abscess ) in 2030 % of cases and mortality in more than 20 % [ 2 ]  . prompt diagnosis of pancreatic injuries has been challenging , particularly in blunt trauma patients . 
diagnosis has relied on amylase levels , computed tomography ( ct ) scans , magnetic resonance imaging ( mri ) and ultrasound ( us ) , with various levels of success . 
this number rises to 84 % following 3 - h elapse between trauma and the time of measurement [ 2 , 7 , 8 ]  . undoubtedly , the best diagnostic tool for pancreatic trauma is ct [ 9 ]  . 
moreover , mr cholangiopancreatography ( mrcp ) can be more useful because it can demonstrate clear delineation of the duct and its integrity . however , ct and mr equipment is large and difcult to use at the bedside for early diagnosis , or at trauma scene for point - ofcare diagnosis . 
however , conventional us cannot sufciently display the location and severity of pancreatic lesions . the introduction of us contrast agents has led to an increase in the diagnostic accuracy of us in many organs , and the technique now has a wide range of applications [ 1115 ]  . 
in 2013 , cokkinos [ 16 ] reported that contrastenhanced us ( ceus ) accurately dened organ injuries , capsular extension and even vascular injury with very good correlation with ct . the portability of ceus makes it a good tool in trauma settings because examinations can be performed quickly at a patients bedside and benets patients with either stable or unstable haemodynamics . 
the purpose of our study was to present the application of ceus in diagnosing adult pancreatic injuries caused by blunt abdominal trauma . materials and methods this study was approved by the ethics committee of the chinese peoples liberation army general hospital and complies with the health insurance portability and ( hipaa )  . regulations , in accordance with our accountability act for ceus institutions examination and use of related data for future research was obtained from each patient ( or the patients relatives if he / she was unconscious )  . informed consent patients from march 2007 to june 2012 at two institutions equipped with modern laboratory , ultrasonographic and radiological equipment , a retrospective review of the charts of 22 consecutive patients with pancreatic injuries due to blunt abdominal trauma assessed by contrast - enhanced ct ( cect ) , was undertaken . the patients demographic data and the mechanism of injury were retrieved from the medical les . 
seven patients had isolated pancreatic injury and 15 patients had complex injury , including 4 with hepatic injury , 6 with splenic injury , 2 with renal injury , 2 with hepatic and splenic injuries , and 1 with renal and splenic injuries . 
the mechanisms of injury included accidental fall , motorcycle crash , motor vehicle crash , and automobile - pedestrian collision . ultrasound imaging the ultrasound contrast agent used in this study was sonovue ( bracco , milan , italy ) , which is a second - generation contrast medium approved in china in 2003 for diagnostic imaging . 
the agent consists of stabilised microbubbles containing inert gas ( sulphur hexauoride , 8 ll / ml of solution ) , and is covered by a phospholipid membrane [ 17 ]  . 
the intravenous injection of this contrast agent can be repeated . conventional us and ceus were performed at patients bedside in the emergency department , using a mobile sequoia 512 machine ( siemens medical solutions , mountain view , ca ) or a portable cx50 system ( philips medical systems , andover , ma )  . 
the scan settings ( including gain , 922 radiol med ( 2014 ) 119 : 920927 scanning depth , and time gain control ) were optimised on each target region independently . 
a lesion involving more than 50 % of the thickness of the pancreas indicated impending disruption [ 18 ] , the diagnosis of pancreatic ductal injury was done indirectly by using ceus . ct imaging following the instruction of the emergency department , all patients were scanned within 1 h of admission by an emergency systematic ct scan after or before ceus examination . 
differences between group means were determined with analysis of variance ( systat ver 13.0 , spss , inc , chicago , il ) with chi - square test , consistency checking t or t test , where applicable . 
using cect as a reference standard , ceus value in the diagnosis of blunt pancreatic trauma was evaluated . kappa values were calculated to assess inter - reader agreement on ceus and us . 
b contrast - enhanced transverse ultrasound image shows the injury site as an anechoic and hypoechoic perfusion defect area with an irregular border in the pancreatic body ( short arrows )  . 
these changes were also shown on the cect images . discussion pancreatic trauma , especially contusion , is a relative enigma , even in modern medical practice with advanced diagnostic technologies . 
however , delayed diagnosis of mild pancreatic injuries could cause major therapeutic challenge to the medical team and potentially disastrous situations for the patients . therefore , improving the sensitivity and specicity of diagnosing pancreatic injuries is essential for effective treatment . ceus has largely improved us accuracy in detecting parenchymal organ injury after blunt abdominal trauma [ 15 , 1921 ]  . 
it can help depict ndings that are not accessible on conventional us , such as tissue hypoperfusion , nonperfusion , hyperaemia and contrast extravasation . in 2006 , valentino et al . 
the portability of ceus makes it a good tool in trauma settings because examinations can be performed quickly at the patients bedside and benets patients with either stable or unstable haemodynamics . 
although a case study has been recently reported , the value of ceus in pancreatic injuries has not yet been evaluated with doubleblind comparisons of ceus and cect . on the ceus images and animations , pancreatic disruption or lacerations appear as anechoic and / or hypoechoic perfusion defect area in arterial and parenchymal phases , ideally with separated structures , which can be missed on conventional us images . 
b contrast - enhanced transverse ultrasound image shows the injury site as an anechoic and hypoechoic perfusion defect area with an irregular border in the pancreatic neck ( arrow )  . 
in this group , 16 patients were considered as having pancreas injuries by us on the basis texture . of pancreas enlargement and heterogeneous however , us did not conrm the lesion size and injury severity because it could not depict the lesion borders clearly . 
while ceus has signicantly improved the ability to visualise the peripancreatic space , the reason for this phenomenon may be the difference in blood supply , which makes it possible for ceus to have a better view of pancreas and peripancreatic microcirculation perfusion . 
of note , ceus visualised pancreatic ductal injury , which was consistent with cect . this study also showed that a ceus examination for pancreatic injuries could be completed in less than 5 min using a single dose of contrast agent with operators skill and experience . 
 ( 2 ) it is portable , therefore it can be performed in the emergency room , ct department and at the bedside in radiol med ( 2014 ) 119 : 920927 fig . 
especially for patients with unstable haemodynamics , ceus is the preferred imaging method . ( 3 ) the examination is timely , with a room time \5 min . ( 4 ) ceus examination can be simultaneously performed while performing other physical examination or resuscitation procedures . some disadvantages of ceus have also emerged in this study . 
 ( 2 ) ceus requires rapid and skilful diagnosis because the duration of a single ceus examination is only 68 min . ( 3 ) timely identication of major pancreatic ductal injury is imperative because delayed diagnosis is largely responsible for the high morbidity and mortality associated with blunt pancreatic trauma . 
either ceus or cect indirectly diagnosed pancreatic ductal injury on the basis of a lesion involving more than 50 % of the thickness of pancreas , and the diagnostic accuracy are both lower . 
endoscopic retrograde cholangiopancreaticography ( ercp ) is the most reliable diagnostic method to accurately dene the continuity of the main pancreatic duct following pancreatic trauma , and it has been strongly suggested as an effective procedure for diagnosis and therapeutic interventions , including stent placement [ 25 ]  . 
however , the invasive nature and associated complications are the major drawbacks of ercp , limiting its use in unstable and uncooperative patients . pancreatic injuries sustained by blunt abdominal trauma are extremely rare . 
it should not be intended as a substitute for ct , but as a selective possibility to boost the role of us in the initial screening of patients with pancreatic trauma . in conclusion , ceus showed the site of pancreatic injury as anechoic and / or hypoechoic perfusion defect region with irregular borders in both the arterial and parenchymal phases , on which basis the diagnosis can be 926 radiol med ( 2014 ) 119 : 920927 fig . 
d conventional us shows that the pancreatic neck injury has become larger , the thin pancreatic parenchyma remains ( arrow ) and a peripancreatic liquid collection has appeared on the 16th day after trauma . 
liver biopsy , the current gold standard for the diagnosis and staging of brosis and inammation , may be associated with complications such as bleeding , inherent limitations such as sampling error or interobserver variability and is not readily accepted by all patients [ 5 , 6 ]  . including biochemical and haematological noninvasive diagnosis of hepatic brosis has evolved rapidly in recent years [ 13 ]  . 
several noninvasive techtests niques , and a scoring system using a combination of clinical and laboratory tests are used to predict hepatic brosis , but predict their overlapping cannot results 904 radiol med ( 2014 ) 119 : 903909 necroinammatory activity [ 3 , 5 , 7 ]  . 
ultrasonographybased transient elastography , real - time elastography and acoustic radiation force impulse elastography are used for the evaluation of hepatic brosis but they are operator dependent and may be difcult in obese patients with narrow intercostal space [ 8 , 9 ]  . 
limited studies discuss the role of double - contrast magnetic resonance ( mr ) imaging , mr elastography and mr spectroscopy in the assessment of hepatic brosis in adults , but these pulse sequences are not widely available in clinical practice and their results are preliminary [ 10 , 11 ]  . diffusion - weighted mr imaging depends on the microscopic mobility of water protons . 
to our knowledge , no study to date has addressed the role of diffusion mr imaging in quantication of the degree of inammation in children with chronic hepatitis . the aim of this study was to prospectively evaluate the usefulness of the apparent diffusion coefcient ( adc ) value in the diagnosis and grading of hepatic brosis and inammation in children with chronic hepatitis . materials and methods this study was conducted on 53 consecutive untreated children with pathologically proven chronic hepatitis . three patients were excluded from the study due to motion artefacts at diffusion weighted mr imaging . 
informed consent from parents of patients and controls and institutional review board approval were obtained . mr examinations were performed using a 1.5 - t ( magnetom symphony ; siemens , erlangen , germany )  . 
the machine is equipped with a self - shielded gradient set ( 30 mt / m maximum gradient strength and 120 t / m / s slew rate ) for echoplanar imaging . 
the applied parameters were tr of 2 , 800 ms , te of 74 ms , epi factor of 102 , fov of 25 9 25 cm , section thickness of 7 mm , interslice gap of 20 % , acquisition matrix of 192 9 154 and number of excitations of 6 . 
the adc map was automatically calculated from the data set three b - values by commercially available obtained at software ( leonardo , version 2.0 ; siemens ag medical systems , forchheim , germany )  . 
the adc value was calculated according to the following formula : adc_ ( lnsb2 lnsb1 ) / ( b2 b1 ) , where ln is the natural log , and sb1 and sb2 are the signal intensities in the roi placed on sections corresponding to the two different b values ( b1 and b2 ) [ 30 , 31 ]  . 
the mean of these nine values was calculated and represents the nal adc value per subject that was used for statistical analysis . percutaneous liver biopsy was performed by a paediatric hepatologist ( b.t. ) with 7 years experience in liver biopsy . 
the stages of brosis were f0 ( no brosis ) , f1 ( mild expansion of some portal tracts by brosis ) , f2 ( expansion of most portal tracts by brosis ) , f3 ( occasional bridging brosis ) , f4 ( marked bridging brosis ) , f5 ( incomplete cirrhosis ) and f6 ( complete cirrhosis )  . 
the control children were regarded as having necroinammatory activity grade a0 and brosis stage f0 . statistical analysis was done using statistical package for the social sciences ( spss , inc . , chicago , il , usa ) software , version 16 . 
for statistical analysis , the stages of brosis were classied into mild ( f1 , f2 and f3 ) and advanced ( f4 , f5 and f6 ) and the grades of necroinammatory activity were classied into mild ( a1 , a2 ) and advanced ( a3 , a4 )  . receiver operating characteristic ( roc ) curves were done to evaluate the diagnostic capability of the adc value in differentiating hepatic brosis from normal liver , as well as in staging hepatic brosis and grading hepatic necroinammation . 
the area under the curve ( auc ) , sensitivity , specicity , accuracy , positive predictive value ( ppv ) and negative predictive value ( npv ) were calculated . 
diffusion mr imaging has been used for assessment of diffuse diseases in different regions of the body [ 4042 ] as well as in chronic diffuse liver diseases [ 1327 ]  . in the literature , various studies of diffusion mr imaging reported that adc values of hepatic brosis are lower than those of normal livers in adults [ 1421 ] and in children [ 29 ]  . 
in accordance with most of the previous studies [ 1623 ] , the adc values of the study group were also lower than the adc values of the control group . 
the mechanism of restricted diffusion in brosis is deemed to be multifactorial , mostly due to diminished hepatic perfusion and , to a lesser extent , to the presence of increased connective tissue , which contains fewer protons . 
the deposition of brous tissue leads to a reduction in the size or obliteration of small venules , with a resultant increase in portal venous pressure and consequent compensatory increased arterial blood ow [ 1517 ]  . 
the presence of increased connective tissue in the liver in addition to deposition of collagen bres , fatty inltration , hepatitis , cell necrosis / apoptosis and inammatory cell responsible for restricted diffusion with low adc value in patients with hepatic brosis [ 1419 , 29 ]  . inltration is the role of diffusion mr imaging in the study of diffuse liver disease , therefore , is still under debate . 
6 receiver operating characteristic ( roc ) curve of the adc value used for differentiating mild ( a1 , a2 ) from advanced ( a3 , a4 ) grades cutoff adc value was b1.64 9 10 - 3 mm2 / s with an auc of 0.807 , a sensitivity of 87.5 % , a specicity of 61 % and an accuracy of 74 % necroinammation . 
this may be attributed to more accurate calculation of the adc value in this study which was calculated from three b values ( 0 , 400 and 800 s / mm2 ) whereas other studies calculated the adc value only from two b values . 
last , this study was conducted on children who have better adc maps without noise or susceptibility artefacts compared to the adult patients . in this work , the adc value of hepatic parenchyma changed according to the stages of brosis . 
5 scatter plot of the adc value at different grades of hepatic necroinammation 908 radiol med ( 2014 ) 119 : 903909 know the usefulness of adc in distinguishing between the early and intermediate stages . 
also , linear regression analysis showed that hepatic adc values were a good predictor of brosis stage ( r2 = 0.19 , p = 0.0001 ) [ 19 ]  . in this study , there is a negative correlation between the adc values and necroinammatory activity . 
the adc value is signicantly lower in patients with high grades of hepatic inammation compared to those with low grades . the aggressiveness of chronic hepatitis is indicated by the degree of necroinammation , also called the activity ; the more severe the necroinammation , the more aggressive the disease . 
 [ 24 ] found a statistically relationship between the adc values and signicant hepatic activity index scores although this relationship was not as strong as that between adc values and brosis scores . 
in addition , entropy , which is a measure of the variability in the volume histogram analyses of the adc values , showed a greater correlation with the brosis stage and inammatory activity grade than did mean adc , with a sensitivity of 0.91 and a specicity of 0.92 in the detection of inammation [ 27 ]  . 
recent advances in mr software technology such as 3 t scanners with parallel imaging and respiratory triggering will likely continue to improve image quality and also promise the potential application of multiple and higher b values than 1 , 000 mm2 / s [ 43 , 44 ]  . 
also , further studies with application of diffusion tensor mr imaging with multichannel coils will likely improve the results [ 14 ]  . application of advanced postprocessing methods such as entropy [ 27 ] , bi - exponential , non - gaussian ( diffusion kurtosis ) modelling or k - means clustering algorithms [ 4447 ] may provide a better characterisation of diffuse liver disease . there are a few limitations to this study . 
second , we excluded three patients due to susceptibility artefacts , and we expect that future studies with application of parallel imaging will increase the image contrast and decrease susceptibility artefacts . 
if the registration method works correctly , parameters which bring the images into alignment must always be the same . results automatic registration performed by the software did not prove satisfactory , whereas if a specic tool [ volume of interest ( voi ) tool ] allowing the calculation to be limited to the landmark region was used , the registration s . 
stancampiano dipartimento di specialita` medico - chirurgiche , scuola di specializzazione in fisica medica , universita` degli studi di catania , via santa soa 78 , 95123 catania , italy parameters were comparable with those of the manual registration . 
regarding the repeatability of the automatic registration , the software brought the images in the correct alignment performing translations and rotations along the longitudinal axis up to 40 ( cid : 3 ) , while it was not satisfactory for rotations along the transverse axes . conclusion the experimental clinical application automatic registration is reliable if the voi tool that includes visible landmarks in both studies is used . 
however , because the algorithm did not prove sensitive to rotations along the transverse axes , the position of the patient during the examinations plays a crucial role . results showed that keywords radiotherapy treatment planning ( cid : 2 ) computed tomography ( cid : 2 ) magnetic resonance imaging ( cid : 2 ) image registration ( cid : 2 ) phantom ( cid : 2 ) quality assurance introduction radiotherapy relies on images to plan , guide , and assess treatments [ 1 ]  . 
spatially accurate medical images are , indeed , an essential tool in three - dimensional conformal radiation therapy ( 3d - crt ) and intensity - modulated radiation therapy ( imrt ) treatment planning [ 2 ]  . 
computed tomography ( ct ) is the current standard modality for image - based radiation therapy treatment planning because of its ability to convert hounseld units ( hu ) into electron densities [ 3 ]  . a few years after the introduction of ct in routine clinical practice , it became clear that coupling of information provided by ct with that coming from other diagnostic modalities would improve the quality of the denition of tumours [ 4 ]  . 
this is divided into two phases : registration , which achieves the spatial alignment of the two studies , followed by fusion , which consists in the visual representation and combination of data [ 10 ]  . 
the techniques to perform this process are different , but all include the same basic components : the transformation model ( rigid , afne , projective and deformable ) and the metric ( geometry - based and voxel - based )  . another important feature is the level of interaction between the registration algorithm and the user . 
in the semi - automatic case , the interaction can be of two different kinds : the user can initialise the algorithm ( such as segmenting the data ) , or drive the algorithm ( i.e. , by refusing or accepting the suggested registration ) [ 11 , 12 ]  . the timing required in clinical application undoubtedly favours an automated approach to the image registration both as regards the speed of the operation as its standardisation . nevertheless , it requires a careful analysis of the effectiveness of registration , with the purpose of correcting and / or limiting errors that may occur in clinical implementation [ 13 ]  . that each radiotherapy application demands requirements of quality assurance ( qa ) protocols are met . therefore , also image registration applied to planning and execution of treatment requires a verication of results [ 1 ]  . several groups recommend guidelines for qa of registration in radiosurgery and general planning [ 3 , 1418 ]  . recently , the american association of physicists in medicine ( aapm ) formed task group 132 to review techniques for image registration , to identify issues related to their clinical implementation , to determine the best methods to assess accuracy and to outline issues related to acceptance and qa [ 1 ]  . many reports describe methods and evaluations for registration in the head and neck region [ 3 , 17 , 19 , 20 ] ; far fewer describe results for the pelvis [ 6 , 21 , 22 ]  . 
in this case , as the delineation of the target volume is an important source of geometric uncertainty , it is becoming customary to integrate ct and mri studies [ 13 ]  . the main objective of this work was to evaluate the accuracy of the registration of ct and mri images performed by a software dedicated to treatment planning ( focal , elekta )  . 
for this purpose , a phantom [ stylized anatomy with a registration objective ( s.a.r.o. ) ] dedicated to quality control for the registration of images obtained by ct and mri scanners was designed and built . 
it reproduces in a stylised way that the planning target volume ( ptv ) and organs at risk ( oars ) related to the treatment of the prostate . materials and methods the properties of focal software and s.a.r.o. 
phantom and the main steps of the analysis protocol are described below . description of the registration algorithm the registration algorithm of the focal software uses as a metric mutual information ( mi ) , a voxel - based metric which measures the statistical dependence of information between intensities of corresponding voxels in both images . 
it is supposed that mi is maximum when the two studies are geometrically aligned [ 23 ]  . moreover , the registration algorithm uses a model of rigid transformation with six degrees of freedom : three translations and three rotations . 
a rigid transformation can be described by a single matrix equation in homogenous coordinates by : where , xi and yi ( i = 1 , 2 , 3 ) are , respectively , the components of the new and the old coordinate vectors of the voxel , t is an arbitrary translation vector and r is a 3 9 3 rotation matrix dened by : ril r1 i ; j r2 j ; k r3 with r1 r2 r3 sin a1 0 cos a1 ( cid : 3 ) sin a1 cos a1 sin a2 cos a2 ( cid : 3 ) sin a2 cos a2 cos a3 ( cid : 3 ) sin a2 sin a3 cos a3 each matrix r ( i ) rotates the image about the ith axis by an angle ai [ 11 , 12 ]  . 944 radiol med ( 2014 ) 119 : 942950 once chosen the set of reference images , which constitute the primary study and will be kept xed , the algorithm will perform transformations on the other set of images , the secondary study , until the correct alignment is achieved . finally , focal provides that the transformations are carried out through a semi - automatic interaction with the user . 
once the primary and secondary studies are selected , the software proposes a rst registration founding its calculation on the entire content of the images ( intensity of each voxel )  . 
to check the alignment , the software shows the result of the fusion in various modalities ( panels , strips , merging of voxels ) and in each section ( axial , sagittal , coronal )  . 
it also shows the parameters and the transformation matrix as well as the value of mi in the current alignment . to improve the registration quality , it is possible to select a limited region in the three axes [ volume of interest ( voi ) tool ] within which the algorithm calculates the mi and performs its maximisation . 
since mi is a measure of the information common to both images , the selection of a region with well - dened landmarks allows the mi to be increased and the registration to be improved . 
although the selection of voi is user - dependent , even in this case the registration is automatic . finally , the user can customise the registration by directly applying the necessary transformations to the secondary study , assisted by a continuous view of the two studies in fusion on the three sections . description of the phantom the accuracy of the registration algorithm was evaluated by performing a study on a phantom with appropriate inserts of known size and composition . 
it simulates the pelvis region in a stylised way and it is dedicated to the registration of ct and mri images : s.a.r.o. the phantom consists of a cubical tank ( volume of 200 mm3 ) in which the landmarks shown in fig . 
figure 1 shows the axial section of the phantom with the corresponding anatomical landmarks , while table 1 shows the main properties of the inserts . since the two modalities are based on different physical principles , it is necessary to use materials that give signicant signals in both . 
for this reason , inserts are made of pmma , pvc and teon , tissue - equivalent materials in ct acquisition with slightly different densities , but that provide very weak signals in mri acquisition . 
were acquired with a 64 - slice multislice ct600 scanner ( ge medical systems ) using a protocol for radiotherapy treatment of the pelvis . mri images were acquired with a signahdtx scanner ( ge medical systems ) using a protocol suggested by literature regarding the radiotherapy treatment of the prostate [ 14 ]  . 
table 2 shows the main parameters of the ct and mri images . automatic and manual registration the rst part of the study was dedicated to the verication of automatic registration with focal . radiol med ( 2014 ) 119 : 942950 manual registration : the registration is performed manually by means of a visual inspection of the images in fusion . 
the new transformation parameters are marked down to be compared with those of automatic registration . in this way , it is possible to verify if the automatic registration is comparable to the manual registration . repeatability of the registration transformation parameters , a after identifying the best further study was conducted on the repeatability of the transformation performed by the software . for this purpose , a reverse approach was carried out : starting from the ideal alignment , known transformations were performed to the correctly aligned secondary study to verify whether and to what extent the algorithm was able to return to its original position . 
deviations were calculated using the following expression : transformation between where wi , t are the transformation parameters ( translations and rotations along the ith axes ) imposed to the properly aligned secondary study and wi , a are the transformation parameters found by the software . 
a correct transformation corresponds to a situation in which wi , t and wi , a are equal and opposite and , therefore , dwi equal to zero . results the analysis involved testing the as mentioned above , accuracy of the registration algorithm and the repeatability of the transformations the automatic version of ct computed tomography , mri magnetic resonance imaging , fov eld of view dwi wi ; t wi ; a fig . 
phantom during the ct acquisition table 2 main characteristics of the images acquired in the two modalities slice thickness matrix size 5 mm 500 mm 512 9 512 5 mm 400 mm 512 9 512 once the ct series had been selected as the primary study and the mri series as the secondary study , the analysis was divided into three sections . global registration : the software performs an automatic registration basing its calculation on the overall content of images . 
hence , the studies are shown in fusion , and it is possible to assess directly the registration quality . registration with voi tool : through this tool , the region in which the landmarks are visible is selected on the three sections ( axial , coronal , and sagittal ) , as shown in fig . 
the results of the study are shown below . automatic registration the automatic registration performed using the entire contents of the images showed the limits of this procedure . figure 4 shows the fusion of the ct study ( upper panel ) and the mri study ( lower panel ) after this transformation . the overlap of images indeed , the translation returns , belonging to different planes . table 3 shows the transformation parameters and the corresponding matrix . 
the expressions x ( r ? l ) , y ( s ? i ) and z ( a ? p ) indicate , respectively : translation from right ( r ) to left ( l ) , from cranial [ superior ( s ) ] to caudal [ inferior ( i ) ] and from anterior ( a ) to posterior ( p )  . the registration algorithm only ran translations along the three axes . 
as can be seen from this gure , the planes of the two studies coincide , as well as the position of landmarks . the parameters and the transformation matrix obtained through the voi tool are shown in table 4 . as a nal step , translations and rotations were performed manually based on a view of images in fusion . 
the error associated with the manual registration was evaluated by considering the spatial resolution of the images ( 1 mm in the axial plane , 5 mm in the sagittal and coronal planes )  . 
table 5 shows the parameters of the manual transformation and the corresponding matrix . comparing the results obtained with manual registration ( table 5 ) and those of automatic registration obtained by the voi tool ( table 4 ) , it can be noticed that the differences are minimal when compared to the spatial resolution of the images ( calculated from the eld of view size and the sampling matrix )  . 
these differences are included , according to the previously indicated resolution limit , in the error related to the manual registration . to quantitatively evaluate the accuracy of registration , the landmarks were contoured separately in the mri and ct studies . 
6. to further verify the correct operation of the algorithm , a registration of the ct study with itself was performed . the algorithm returned null values for each parameter and the transformation matrix corresponded with the identity matrix . 
for greater rotations , the algorithm was not able to nd the proper registration even with the use of the voi tool . rotations along x : the algorithm failed to nd the correct alignment for small rotations ( 2 ( cid : 3 ) ) even if the voi tool was used . rotations along z : as in rotations along the x axis , the algorithm , running into local maxima , failed to properly register the images with rotations of more than 2 ( cid : 3 )  . complex performing transformations : exclusively translations , the registration algorithm was able to properly align the studies , especially if the voi tool was used . 
table 6 shows some examples of deviations for rotations along y , x , z axis and an arbitrary translation along the three axes . as anticipated , for rotations along the x and z , even with the use of the voi tool , deviations remained signicant . discussion the major advances in radiation therapy have highlighted the need to improve the ability to locate and delineate tumour , healthy tissue and oars accurately . 
the nal image shows that the registration is satisfactory imposed and the for this purpose , the differences between the transfortransformation mation parameters parameters found by the software to properly align the two studies were evaluated . 
although physical spatial resolution along the coronal and sagittal planes is 5 mm , 0.001 mm ( as can be seen in the tables showing the transformation parameters ) , a value that goes far beyond the spatial resolution of the images . 
since , this part of the study was addressed to the ability of the algorithm to nd numerically the same transformation parameters , the same limit was set for all three planes . algorithm computation grid extends to evaluate the automated version of focal , a global registration was rst performed , followed by a registration with voi tool . 
phantom was designed to ensure significant signals in both imaging modalities . first of all , the optimisation procedure of the registration requires , depending on the algorithm , some precautions during image acquisition . 
in addition , since any change in patient position and organ lling represent sources of geometric uncertainties , registration algorithms that use models of rigid transformation must be evaluated carefully to ensure acceptable coherence between the modalities employed . 
respecting these precautions in routine procedures already represents a denite improvement as regards both the quality of the registration and the required timing . during the study , after obtaining images of the phantom according to these precautions , an automatic registration was performed allowing the software to exploit the entire contents of the ct and mri images . 
for this reason , the registration of ct studies with other diagnostic studies is emerging strongly in many centres and it is becoming an integral part of treatment planning . to minimise the possibility of human or software errors , clinical image acquisition and registration processes should be managed where possible in an integrated way to ensure their cogency , sequentiality and reproducibility [ 1 ]  . 
the registration algorithm is , in its automatic version , reliable for rotations along the y - axis up to 40 ( cid : 3 ) only if the voi tool is used . 
the algorithm is indeed able to assure the alignment of the studies if the voi tool is used . on the other hand , automatic registration did not prove satisfactory with regard to rotations along the x and z axis . 
this leads to the criticism of the correct patient position in the two studies as even small differences could affect is therefore desirable to use appropriate outcome . systems for patient immobilization with high precision positioning . 
this objective is certainly favoured by performing the studies on the same day and at a short temporal distance from one another . conclusion this the main objective of this work was to verify the accuracy and the repeatability of the registration of ct and mr images using the automatic version of the focal software . for this purpose , images of the s.a.r.o. 
the results show that , using the manual registration as a reference because it allows one to visually assess the quality of alignment , automatic registration is comparable when the voi tool is in clinical used . applications , the algorithm is reliable and fast if the voi includes that are clearly visible on both modalities . 
in females , brain , lumbar spine , and abdominal ct scans , pre - / postcontrast neck ct scans , and post - contrast liver dynamic ct scans were performed . 
breast shielding during neck and liver dynamic ct was the most effective compared with breast or thyroid shielding during other ct scans . conclusions we recommend breast shielding during neck and liver dynamic ct in young female patients to avoid unnecessary radiation exposure . keywords radiation protection ( cid : 2 ) multidetector computed tomography ( cid : 2 ) breast ( cid : 2 ) thyroid introduction increased radiation exposure may be one of the causes of the recent rapid increase in breast cancer in korea [ 1 ] in addition to westernised diet changes , a decreasing birth rate , and an increasing maternal age [ 2 ]  . 
a recent study from the national cancer institute in the united states has projected 29 , 000 excess cancers from the 72 million ct scans performed on americans in 2007 alone [ 6 ]  . 
scans of the abdomen , pelvis , chest , and head were deemed most likely to cause cancer , and 952 radiol med ( 2014 ) 119 : 951957 in female patients , noncontrast - enhanced ct of the brain , lumbar spine , and abdomenpelvis , preand postcontrast neck ct , and preand post - contrast liver dynamic ct were performed . in male patients , noncontrastenhanced brain ct was carried out . 
with this technique , the supercial breast or thyroid skin doses , not the absorbed doses in the deep tissues , were women aged 3554 years were particularly more likely to develop cancer as a result of ct scans than any other age group because of the high frequency of ct use [ 6 ]  . 
twothirds of the excess cancers will occur in women primarily because of the higher frequency of use ( 60 % of scans ) as well as the higher breast and lung cancer risks associated with scans that expose the chest [ 6 ]  . currently , ct scans of the abdomen , lumbar spine , and brain have been performed without shielding of adjacent organs such as the breasts or thyroid glands . 
in general , two - view screening mammography ( \3 mgy standard , equivalent dose for the breast ) , which can cause breast cancer by itself , has not been recommended in women aged less than 35 years because of the radiosensitivity of breast tissue [ 7 ]  . 
during abdominal ct , however , a radiation dose 5 ( single scan ) to 20 times ( post - contrast dynamic scan ) greater than screening mammography is introduced to the abdomen , and a signicant radiation dose is simultaneously exposed to the breast adjacent to the abdomen due to scatter radiation . 
signicant radiation doses can also be introduced to the thyroid and breast during brain ( less than 2 mgy equivalent dose for thyroid ) and neck ct [ 7 ]  . breast tissue is also exposed during lumbar ct . 
therefore , the tissues outside the primary beam should be protected against scatter radiation if the image quality will not be sacriced [ 811 ]  . in the present study , we investigated the efcacy of lead shielding for radiation protection of the breast and thyroid by measurement of the entrance skin dose of radiation exposure during ct of the brain , neck , abdomen , and lumbar spine in young patients . materials and methods institutional review board approval for this prospective study was obtained from our institution , and all patients volunteered to participate and signed the informed consent form . patient selection this study included 150 patients ( 25 patients consecutively in each group ; male : female 25 : 125 , mean age 32.8 years , age range 1545 years ) referred for a ct study of the brain , neck , abdomen , or lumbar spine for objective medical reasons . 
the sensors were also attached to the skin of both thyroids , 2 cm apart from the midline of the lower neck and 45 cm above the suprasternal notch . therefore , two sensors were at the same distance from lumbar spine in each the head , neck , abdomen , or measurement . ct equipment singleor multi - phase , preor post - contrast ct scans were performed using a 16 - row multidetector ct scanner ( mdct ; somatom sensation 64 ; siemens medical solutions , erlangen , germany ) for variable clinical information . 
preand post - contrast ct images were obtained in neck and liver dynamic studies , using intravenous administration of 120150 ml of nonionic iodinated contrast media ( ultravist 300 ; bayer schering pharma ag , berlin , germany ) administered by an automatic injector at 34 ml / s . 
ct parameters used in the variable ct studies are listed in table 1 . body constitution the body mass index ( bmi = body weight / height2 ) was calculated in each patient . 
this unit started dose accumulation , individually for connected sensors , when the dose rate exceeded a trigger level of about 10 lgy / s ( 60 mr / min ) and stopped dose accumulation when the dose rate was below a level of approximately 5 lgy / s ( 30 mr / min )  . 
the sensor is calibrated to entrance skin dose at 90 kvp in gy and the energy dependence is \10 % for the energy range 60110 kvp ( \15 % for the energy range 40140 kvp ) at a tube ltration of 6 mm al . 
regarding thyroid shielding on brain ct , the entrance skin doses in the reduced by 17.9 % ( range shielded thyroids were 1.555.7 % ) in female patients and 20.6 % ( range 1.848.5 % ) in male patients compared with those of unshielded thyroids ( table 2 )  . 
there was no correlation between bmi and the reduced radiation dose in the shielded breast or thyroid . discussion the radiosensitive structures that lie within the primary beam cannot easily be protected , and the organs outside the ct scanning planes are exposed only to scatter radiation . during brain ct examination , internal scatter radiation propagates caudally through the neck , reaching the breasts as well [ 8 ]  . 
a signicant amount of scatter radiation , generated in the head and in the gantry , reaches supercial organs , particularly the thyroid and breasts , from the outside and can be reduced effectively by lead shielding . 
in our study , however , there was no correlation between bmi and the reduced radiation dose in the shielded breasts or thyroid , as reported previously by brnic et al . 
additionally , the shielded and unshielded breasts or thyroids are close , which can cause the possible perturbation effect of the shield in the unshielded portion due to scattering and characteristic x - rays from the shield to the unshielded portion . 
this effect can increase the difference between shielded and unshielded portions with an overestimation of the usefulness of the shield . the radiation dose measured on the surface of the breast or thyroid is obviously higher than the actual glandular dose because of scattered and low - energy x - ray radiation [ 13 , 1921 ]  . 
assuming a weighting factor of 0.05 for the breast and a breast dose of 0.01 gy probability ( 0.01 sv ) , 5.6 9 10 - 2 9 5 9 10 - 2 9 10 - 2 = 2.8 9 10 - 5 . in our study , the mean skin dose on the unshielded breast during preand post - contrast liver dynamic ct was about 0.002 gy ( 2 , 387 lgy )  . 
we believe that causes may be as follows : ( a ) the neck is closer to the breast than the brain , abdomen or lumbar spine , so the breast receives more radiation exposure during neck ct . ( b ) liver dynamic ct consists of four scans : pre - contrast and post - contrast arterial , portal , and equilibrium phase radiation imaging . 
therefore , exposure in liver dynamic ct is higher than other singleor double - phase ct scanning , and breast or thyroid shielding during this ct scan results in more effective elimination of radiation exposure than the unshielded breast or thyroid . the absolute amount of compared to previously published studies , this study differs in several respects . 
 ( a ) this study compared the radiation dose reduction in the multi - organ ct scans ( brain , neck , abdomen , and lumbar spines ) by lead shielding of breasts or thyroids , and investigated which one radiol med ( 2014 ) 119 : 951957 is more effective shielding in the ct scans of different organs . 
 ( b ) we used the unfors psd ( cid : 3 ) unit to measure the entrance skin dose , whereas other studies used a thermoluminescent dosimeter which was relatively more complex to use . 
 ( c ) this study did not use the in - plane shielding such as breast shielding during chest ct or eye lens shielding during brain ct which could evaluate the effect on image quality of using in - plane shielding . our study has several limitations : ( 1 ) neither thermoluminescent dosimeters nor a phantom study to measure the actual glandular dose were included because we only compared the reduction of entrance skin doses in shielded and unshielded organs . 
in the radiation elds of mdct , the direct or scattered radiation from the back or sides of the thorax or neck could not be protected by our shields . in conclusion , breast shielding during preand postcontrast neck ct and liver dynamic ct was most effective in reducing the entrance skin dose compared with breast or thyroid shielding during other ct scanning . 
wholebody dw - mr imaging ndings were compared with results obtained at skeletal scintigraphy and ct bone survey . results the mean examination time for whole - body dwmr imaging was 25.5 mall bone metastases regardless of the size were identied with whole - body dw - mr imaging ; mr imaging depicted more bone metastases than ct . 
skeletal scintigraphy depicted osseous metastases in 13 patients ( with greater sensitivity to the lower limb ) , whereas whole - body dw - mr imaging revealed osseous metastases in 13 patients ( with greater sensitivity to the spine )  . 
schena department of biomedical engineering , campus bio - medico university of rome , rome , italy seen with mr imaging were conrmed at follow - up examinations and some had a change in therapy . 
on a per - patient basis , whole - body dwmr imaging revealed sensitivity and specicity values of 100 % . conclusion whole - body dw - mr imaging was more sensitive in the detection of osseous metastases than were skeletal scintigraphy and ct bone survey . keywords mr ( cid : 2 ) whole - body mr ( cid : 2 ) metastasis ( cid : 2 ) bone lesions ( cid : 2 ) ct ( cid : 2 ) bone scintigraphy introduction diffusion - weighted imaging ( dwi ) is a quantitative magnetic resonance technique that measures the random ( brownian ) motion of water molecules in biological tissues . 
in human tissues , water motion is locally hindered by the presence of cell membranes , vascular structures and brotic tissues , so the degree of restriction to water diffusion is inversely correlated to tissue cellularity and the integrity of the cell membranes . 
widely used in the study of intracranial diseases and cerebrovascular accidents [ 1 ] , dwi is of increasing interest for the detection of primary or metastatic cancers [ 2 , 3 ] , as tumoural tissue consists of densely cellular and highly vascularised structures . radiol med ( 2014 ) 119 : 758766 diagnostic work - up in the evaluation of oncological patients includes some staging procedures that play key therapeutic and prognostic roles . 
since metastases may affect any organ , imaging techniques most commonly involve computed tomography ( ct ) , magnetic resonance ( mr ) , ultrasonography ( us ) and bone scintigraphy ( bs )  . bone scintigraphy is one of the most frequently ordered investigations for suspected bone metastases , particularly during oncological follow - up , and it remains a reference modality for oncologists [ 4 ] , although there are wellknown limitations affecting its specicity and sensitivity [ 5 ]  . 
despite the increasing use of dwi as an adjunct in evaluating tumour patients due to its relatively easy and time - effective application , systematic reports evaluating its technical feasibility , especially in terms of image quality and robustness of apparent diffusion coefcient ( adc ) measurements are rare and only focus on limited parts of the body , with few experiences addressing whole - body dwi . 
moreover , experience with dwi within the concept of whole - body mri including conventional t1w and shorttau inversion recovery ( stir ) sequences in the diagnostic practice is still limited [ 6 ]  . including dwi sequences , the aim of our study was to assess the diagnostic value of whole - body mri , in the detection of skeletal metastases in comparison with wholebody ct and bs to determine the utility of whole - body dwi as a component of the whole - body mr examination for the diagnosis and follow - up in patients affected by bone metastases . 
moreover , we wanted to verify the application and diagnostic value of each technique depending on the different anatomical distributions of skeletal metastases . materials and methods patients from january 2011 to march 2012 , a total of 17 patients ( 11 women and six men ; mean , age 57 years ; age range 4076 years ) with malignant tumours were included in this retrospective study to detect bone metastasis . 
they were examined using whole - body dwmr imaging as well as whole - body ct and bs , with an average interval of 14 days ( range 030 days )  . 
the inclusion criteria were as follows : adults with histologically table 1 patient characteristics patient age ( years ) gender primary tumour breast breast breast breast breast prostate prostate pancreas lung thyroid colon breast lung lymph node proven primary tumour , curative treatment at least 1 year before the examination and no history of another malignancy . the patients characteristics are summarised in table 1 . whole - body dw - mr imaging due to convention derived from various reports in the literature [ 68 ] , the examination was designed as a wholebody examination , although only regions from the head to the middle of the tibia were considered . all mri examinations were performed using a 1.5 - t siemens magnetom avanto system ( siemens healthcare , erlangen , germany ) with the patient in supine and feetrst position with arms in adduction . 
a moving tabletop and tabletop extender were used , generating a longitudinal eld of view ( fov ) of 2 , 000 mm and a transverse fov of 530 mthe larger fov enables head - to - toe scanning of all adult patients without repositioning [ 9 ]  . two sequences were used for the whole - body mri : t1 gradient echo sequences ; single - shot stir spin - echo ( se ) echo - planar imaging sequences for dwi , conducted with two b values of 50 and 500 s / mm2 . 
diffusion weighting was archived by applying pairs of motion - probing gradients before and after the 180 ( cid : 3 ) radiofrequency pulse of the spin - echo t2 - weighted sequence in three orthogonal directions ( x , y and z )  . 
conventional t1 turbo spin - echo ( tse ) and t2 stir were acquired both in the axial plane from the base of the skull to the middle of the tibia and for the whole spine in the sagittal plane , divided into ve stacks . 
the average interpretation time for the whole - body mr images was approximately 9 min . computed tomography all ct exams were performed using a 64 - slice ct scanner ( siemens , somatom sensation )  . 
an intravenous iodine - based contrast medium was administrated and bone lesions were studied during the portal venous phase . the mean acquisition time , including needle insertion for the administration of contrast medium , was 7 min . metastases were classied as osteolitic , mixed and osteoblastic , with regard to their density measured in hounseld units tumour ( hu ) , characteristics . depending primary bone scintigraphy whole - body planar imaging in the anterior and posterior positions was performed 3 h after intravenous injection of 740 mbq of tc99m - hmdp ( schering pharma , brussels , belgium )  . images were acquired using a dual - headed whole - body scanner with a low - energy , high - spatial - resolution collimator . 
the preset scan speed was 12 cm / min and the matrix size 256 9 1 , 024 pixels . image analysis on whole - body mr , a lesion was considered positive for metastatic bone disease when there was a focal , or a diffuse , hypointense bone marrow signal compared with adjacent muscle on t1 - weighted images and intermediate or high signal intensity relative to the surrounding marrow on stir imaging , which could not be explained by a degenerative , inammatory or traumatic cause [ 10 ]  . 
furthermore , criteria for malignancy were periosteal tumour inltration or , for evaluation of the spine , signal changes extending into the pedicles and bulging of the anterior or posterior margin of the vertebral body [ 11 ]  . 
lesions were considered benign if there was a high signal intensity on t1 - weighted images or if they were located close to degenerative changes of the vertebral endplates or near joint surfaces . on dwi , a lesion was considered malignant when there was a focal or diffuse signal intensity increase in conjunction with the b value increment . 
bs was not considered for this classication due to the difculty in obtaining an accurate value of lesion size . on ct , images obtained with the bone window permitted measurement of the hu in healthy and pathological bone tissue . 
in the spine , on bs , a focal lesion was regarded to be malignant when the distribution and pattern of focal tracer accumulation could not be explained by degenerative or posttraumatic changes . a classication system was created for each technique , based on the anatomical distribution of bone metastases . considering the low sensitivity of dw - mr imaging in the evaluation of skeletal metastases in regions such as the skull and ribs and , on the other hand , the high sensitivity of bs in detecting metastases in regions such as skull and thorax bones [ 14 , 15 ] , the division into locations considers three regions for each examination : axial skeleton ( as : cervico - thoracic and lumbo - sacral spine , clavicle , pelvis ) lower limbs ( ll : lower extremities up to the tibial diaphysis ) and chest ( ch : sternum , ribs , scapula , including the proximal humerus and the mandible )  . 
the division by anatomical distribution is intended to ensure maximum uniformity of the data and the best comparison among the radiological techniques considered . the analysis was carried out on each of the 17 patients , for a total of 48 examined areas . radiol med ( 2014 ) 119 : 758766 fig . 
1 a 46 - year - old female patient with breast cancer : whole - body magnetic resonance imaging in the coronal plane shows a vertebral bone lesion in t1 - weighted ( a ) and diffusion - weighted ( b ) sequences ( arrows )  . 
fourteen of 17 patients completed all three of the examinations ; the other three patients , after a diagnosis of bone metastases on ct , showed negative results on mri so these patients were not subjected to bs . 
we compared the results of wholebody dw - mri with those of ct and bs . the total number of lesions identied with the three different procedures was n = 74 for ct , n = 162 for whole - body dw - mri and n = 133 for bs . 
in these two cases , mri detected multiple metastases in the axial region and lower limbs ; the other one ( case #4 ) had negative results on both examinations . radiol med ( 2014 ) 119 : 758766 fig . 
3 a 69 - year - old male patient with prostate cancer : whole - body magnetic resonance imaging in the coronal plane demonstrates a left acetabular lesion in t1 - weighted ( a ) , diffusion - weighted ( b ) and adc maps ( b ) sequences ( white arrows ) , while bone scintigraphy ( d ) was considered negative for bone metastases in the same district for 12 / 14 patients , whole - body mri and bs concordantly demonstrated metastatic lesions , but were disconcordant for lesion number and involvement site in only 1 / 12 ( 8.3 % ) patients ( case #7 )  . 
one patient , conversely , had positive bs and negative whole - body mri ( case #4 ) results ; in this patient , bs detected a total of three metastases , two of them in the axial skeleton ( pelvis ) and the other in the chest ( rib )  . hence , mr identied 22 % more metastatic lesions when compared to bs and 119 % more than ct . 
bone scintigraphy identied 80 % more metastatic lesions when compared to ct . the lesions observed using the different diagnostic procedures are shown in table 2 and are categorised by anatomical site . 
whole - body dw - mri is characterised by an advanced sensitivity in identifying lesions in the lower limbs and axial skeleton , while presenting a limited receptivity in the chest . 
in both whole - body dw - mri and ct , a subdivision based on lesion size was made , underlining how whole - body dwmri had a higher sensitivity in detecting bone metastases independently from size in comparison with ct , and it also showed that in both procedures , the majority of lesions were included in the dimension group beneath 10 mm . patient - by - patient analysis revealed multiple lesions on bs in 11 / 13 ( 84 % ) of the patients with positive mri ndings and multiple lesions on mri in 12 / 13 ( 92 % ) of the patients with positive bs . 
none of the enrolled patients was classied as an equivocal case . particular , regarding the axial district , whole - body dwmri showed a higher number of lesions , followed by bs and then by ct . 
for the chest region , there was a marked higher sensitivity of bs in detecting bone metastases ; otherwise , whole - body dw - mri had a lower sensitivity in detecting bone lesions in the chest . discussion one of the most common sites of distant metastases is the cellular bone marrow ( 5070 % ) , especially in patients with breast and prostate cancer . 
traditionally , bs has been the standard graph 1 anatomical distribution of bone metastases . whole - body dw - mri results are in light grey , bs results are in dark grey and ct results are in black 764 graph 2 detection of bone metastasis considering lesions size . 
whole - body dw - mri versus ct radiol med ( 2014 ) 119 : 758766 imaging investigation used for the detection or exclusion of skeletal metastases , and it is widely used to assess metastatic spread in patients with breast , prostate or lung cancer [ 17 , 18 ] , as bs is more sensitive when sufcient osteoblastic reaction has occurred . 
however , supplemental imaging studies are needed to dene unclear ndings , as bs has limited specicity [ 19 ]  . whole - body mri within a single session has become feasible for the evaluation of the presence of metastases and / or for the evaluation of primary cancers , due to recent advances [ 15 , 20 , 21 ] such as short data - acquisition times , no ionising radiation and no injection of isotopes or contrast mediuin several studies , whole - body mri has shown better results than skeletal scintigraphy [ 12 , 16 , 22 24 ] in the detection of bone metastases . 
 [ 7 ] , high spatial resolution whole - body dwi has become available . the diagnostic added value of whole - body dwi sequences has been established [ 12 ]  . 
in comparison with conventional whole - body mri , its sensitivity , specicity and positive predictive value ( ppv ) in detecting bone metastases are superior to those of both conventional mri and bs . particularly , whole - body dwi excels at bone marrow assessment for diagnosis and for therapy evaluation , where it can potentially address unmet clinical and pharmaceutical needs for reliable measurement of tumour response [ 25 ]  . 
with this modality , no ionising radiation is administered and no injection of any contrast medium is necessary , allowing whole - body dwi to be used in routine clinical practice . moreover , multislice ct has been proposed as an alternative to bs for whole - body screening of the skeletal system due to an equivalence found by groves et al . 
however , a recent study comparing multidetector ct with mri in the detection of osseous metastases of the spine found a greater sensitivity of mri , with equivalent specicities [ 27 ]  . all of these data are in agreement with our study that demonstrates that whole - body dw - mri can be used for assessment of bone metastases in patients with cancer . comparative studies of imaging methods have the major bias that none of them can guarantee 100 % diagnostic accuracy as each may be affected by false - positive and false - negative results . 
validation of a new imaging method , such as whole - body dw - mri , requires a comparison with the most consolidated standard in the clinical management of patients [ 28 ]  . our results also demonstrate that whole - body mri has a higher diagnostic accuracy than ct and a comparable level of agreement to bs in detecting bone metastases , independently from the primary tumour site . 
more importantly , there were two cases in our sample in which bs was completely negative for axial bone metastases , with positive results shown by whole - body dw - mri . 
whole - body dw - mri failed to depict a considerable number of lesions in the ribs but depicted more lesions in the spine , pelvis and lower limbs . thus , in an uncertain diagnosis concerning an axial skeletal metastasis , the availability of dw sequences can help to conrm / deny the suspect lesion . 
it has been suggested that this limitation is due to artefacts related to pulsation and breathing movements in the thorax , which make examination of the ribs , sternum and scapula more difcult . 
motion - related signal intensity decreases may explain why metastatic lesions in the bone marrow of the anterior chest wall are sometimes relatively less conspicuous than lesions found in the spine and paraspinal regions [ 25 ]  . 
in terms of the dimensions of bone metastases , greater the ability of whole - body dw - mri to nd it in comparison to ct . the larger the size of the lesion , in our series , a majority of cases were concerned primary breast and prostate cancers , in which there were too many lesions to count , in some cases hundreds [ 26 ]  . many of these lesions are often very small and not identied on scintigraphy , but can be seen on mri or ct . 
the detection of other extra - small lesions probably makes a difference in patient management , even if it is minimucomparing the results obtained using whole - body mri and ct and classifying them based on lesion dimension , it might be inferred that more lesions are observable using mr regardless of lesion dimension . further , whole - body dw - mri has a more extensive anatomical coverage , which results in an ability to identify also extraskeletal lesions with therapeutic and prognostic signicance . this study has certain limitations . 
also due to ethical reasons , ct examinations , intended as total - body scans , were done not covering the portion between the femoral diaphysis and tibial proximal epiphysis . 
thus , the very high difference in detecting metastases ( in particular in the lower limbs ) between whole - body dw - mri and ct could be a result of this limitation . 
another limitation is related to the heterogeneity of the patients in which metastases are primary tumours , usually depicted when sufcient osteoblastic reaction has occurred , may demonstrate a lower sensitivity in tumours eliciting a particularly poor osteoblastic response [ 30 ]  . in that bs , dwi seems to be a sensitive but rather unspecic modality for the detection of bone metastatic disease . high - quality prospective studies regarding whole - body mri in detecting bone metastases still need to be conducted [ 21 ]  . in conclusion , to the best of the knowledge , no previous studies have compared concurrently ct , whole - body dwmri and bs . 
radiological density was assessed with the birads classication ; a quantra density cut - off value was sought on the 2d images only to discriminate between birads categories 12 and birads 34 . 
use of the automated system requires the adoption of a cut - off value ( set at 21 % ) to effectively discriminate birads 12 and 34 , and could be useful in clinical practice . keywords breast density ( cid : 2 ) digital mammography ( cid : 2 ) computer assessment e . 
bergamasco dipartimento di scienze chirurgiche , azienda ospedaliera citta` della salute e della scienza di torino presidio molinette , universita` di torino , corso bramante 88 , 10126 turin , italy introduction high breast density is an important risk factor for cancer development and reduces the sensitivity of mammographic evaluation [ 14 ]  . over the years numerous methods , both qualitative and quantitative , have been developed for the evaluation of mammographic density . 
in recent years , algorithms for the computerised evaluation of breast density have been proposed . these methods have the advantage of a very high reproducibility compared to traditional visual assessment , and a study of correlation between visual and computerised assessments has recently been published [ 1 ]  . the purpose of our study was to compare the denition of mammographic density between visual assessment with the birads classication and the automatic quantra r2 breast density assessment software ( 1.3 hologic corp , bedford ma , usa )  . 
in particular , the objectives of the study were to evaluate the concordance of the visual assessment of breast density based on conventional ( 2d ) digital mammography and tomosynthesis ( 3d ) ; to determine the correlation between the birads classication of mammographic density and the automatic quantra assessment and choose the best quantra cut - off value for discriminating between birads density categories 12 and 34 ; to estimate correlations between breast density and age , use of hormone therapy and tumour histotypes ; to assess whether the quantra system can be helpful in clinical practice in the case of malignant disease . materials and methods study population the study sample includes 200 cases of women ( mean age 56.04 years ; range 3593 ) who underwent mammography between february 2010 and december 2011 at the diagnostic breast clinic of the radiology university of torino , department of diagnostic imaging and radiotherapy , azienda ospedaliero - universitaria citta` della salute e della scienza di torino . 
of the 200 cases , 100 women ( mean age 60.40 years ; range 3793 ) , who were symptomatic and referred to our clinic for diagnostic mammography or selfreferred for screening mammography in the absence of clinical symptoms , were selected based on the detection of histologically conrmed malignant breast disease ; patients undergoing neoadjuvant chemotherapy , those with breast implants or with a previous mastectomy were excluded . 
the other 100 cases ( mean age 51.79 years ; range 3578 ) were selected from among women who underwent mammography during the same time period and for the same reasons ( symptomatic patients referred for diagnostic mammography or asymptomatic patients self - referred for screening radiological signs of mammograms ) , but negative for malignant breast disease and negative follow - up maintained for at least 1 year ; patients with a previous mastectomy were also excluded in this group . each patients history was collected taking special care to investigate family history of breast disease , previous breast surgery and past and present hormone treatment . image acquisition after giving their informed consent , patients underwent two - dimensional ( 2d ) digital mammography and 3d tomosynthesis with a single compression ( combo mode ) using the same imaging session . 
the mammograms were performed with a lorad selenia dimensions digital mammography system ( hologic corp , bedford ma , usa ) set for tomosynthesis in the two standard craniocaudal ( cc ) and mediolateral oblique ( mlo ) projections . 
for tomosynthesis , the scan was performed along an angle of 15 ( cid : 3 ) ( 7.5 ( cid : 3 ) ) and 15 exposures were acquired ( one exposure for each degree )  . image analysis a radiologist with 23 years of experience in breast imaging and 4 years in tomosynthesis reviewed in a single session the images acquired in 2d and 3d modes and dened visual density according to the american college of radiology ( acr ) birads classication [ 5 ]  . 
only the 2d images were subsequently processed by quantra r2 automatic software ( quantra ( cid : 4 ) 1.3 , hologic corp , bedford ma , usa ) [ 8 ]  . 
this evaluation algorithm is based on a physical model that correlates the attenuation of the x - ray due to the breast tissue and the digital mammography images provided to the radiologist . 
the assessments are based on physical parameters specic to the breast tissue and imaging system , as well as on information relating to individual x - ray exposures including attenuation coefcients for breast tissue [ 9 ] , radiographic spectra of the target material [ 10 ] , kvp , mas and thickness of the tissue reproduced in the images . 
quantra estimates the amount of broglandular and fatty tissue that an x - ray must have crossed to deposit a specic amount of energy on the detector , and it provides a result ( height ) in centimetres of broglandular tissue penetrated corresponding to each pixel of the image . after completing the analysis of the pixels ( excluding the pectoral muscle ) , the system combines the values in the volume of the broglandular tissue , expressed in cubic centimetres . 
the system then calculates the ratio between the volume of the broglandular tissue and the total estimated breast volume in order to determine the percentage of broglandular tissue volume in the breast , for both the left and right side ( combined cc and mlo projections ) radiol med ( 2014 ) 119 : 741749 fig . 
this technique was rst applied to analogue images and then , thanks to semiautomatic procedures , to digital mammograms [ 16 ]  . more recently , a system developed by shepherd et al . 
the image is then analysed to determine breast thickness , percentage of broglandular tissue in each pixel and dense tissue in the mammogram . among fully automated volumetric systems , the standard mammographic form ( smf ) is a technique that divides breast tissue into interesting ( healthy normal tissue and neoplastic tissue ) and uninteresting ( adipose tissue ) and is based on different x - ray attenuation coefcients : the volume resulting from the volume of interesting tissue is regarded as the volume of dense tissue [ 18 ]  . 
to provide a practical criterion and relate the quantra measurements to the birads visual scale , the percentage of quantra breast density which best discriminated dense ( birads 34 ) from nondense breast ( birads 12 ) was sought . 
the rst step was to calculate the two density distributions for the two classes to be discriminated : any technique able to identify the two distributions should only allow for a small overlap . 
therefore , the best quantra density value ( cut - off value ) discriminating between the two birads categories was chosen , evaluating sensitivity , specicity , positive predictive value ( ppv ) , negative predictive value ( npv ) and diagnostic accuracy ( da )  . 
the correlation between breast density and age , both in positive and negative patients , and the correlation between breast density and history of hormone therapy were quantied by pearsons linear correlation coefcient . 
a paired students test was used to analyse , in positive patients and in particular in the subgroup of patients who had or were taking hormone therapy , the difference in quantra density between the pathological ( case ) and healthy side ( control ) , and also subdividing the patients into two age groups ( b50 and [ 50 years )  . 
in all other cases anova analysis of variance was used to 744 table 1 breast density assessed by the visual american college of radiology birads classication : comparison between 2d digital mammography and 3d digital breast tomosynthesis density ( birads ) digital breast tomosynthesis 3d 2d digital mammography d1 fat ( \25 % glandular ) , d2 scattered broglandular ( 2550 % ) , d3 heterogeneously dense ( 5175 % ) , d4 extremely dense ( [ 75 % glandular ) compare continuous variables . 
the average quantra density was 24.89 % ( 95 % ci , 22.8226.96 ) for patients aged \50 years and 17.28 % ( 95 % ci , 16.0118.55 ) for those aged c50 years . 
the anova test on the quantra density values corresponding considering the whole group of patients , 95 out of 200 women reported having taken hormone therapy in a time interval between a few months and 30 years ( oestrogen progestin therapy in 77 cases , hormone replacement therapy in 8 and tamoxifen in 10 )  . 
b linear correlation between quantra density and birads classication . c distribution of the density value measured by the quantra software , considering cases classied as birads 12 and birads 34 . d roc curve in which the value of the area under the curve ( c [ 0.9 ) corresponds to a very good ability of the quantra software to discriminate between birads 12 and birads 34 density analysis of both breasts in relation to breast disease , hormone therapy and age because hormone therapy affects density , we analysed only the positive subgroup of patients with unilateral disease ( n = 94 ) to identify correlations between the presence of malignant disease and density , and evaluated differences in density between healthy breast and diseased breast . 
comparison with paired students t test ( considering the diseased side as a case and the healthy one as a control ) clearly indicated ( p = 0.001 ) that the density of the affected side was statistically signicantly greater than that of the healthy side . 
denitive histological examination revealed the presence of 66 inltrating ductal carcinomas ( idc ) , four idc associated with lobular component ( idc ? ilc ) , one idc with ductal carcinoma in situ component ( dcis ? idc ) , 14 dcis , seven inltrating lobular carcinomas ( ilc ) , one lobular carcinoma in situ ( lcis ) , one inltrating tubular carcinoma ( itc ) , one idc associated with inltrating tubular component ( idc ? itc ) , one papillary carcinoma , and one mucinous carcinoma . 
applying the anova analysis of variance we observed that dcis and idc have equivalent density ( p = 0.44 ) , while a borderline difference ( p = 0.08 ) was noted between them and ilc due to the small number of ilc . all we know today about the meaning and impact of mammographic density on the risk of disease and failure to discussion fig . 
in the left breast mammogram it difcult to assess a clinically palpable lump reported by the patient in the lower quadrants ; at ultrasound examination we found an inhomogeneous hypoechoic area . 
denitive histological examination was invasive ductal carcinoma associated with ductal carcinoma in situ measuring 2 cm . the density values assessed by quantra software were 34 % for the right breast , and 50 % for the left breast ( difference between the right and the left breast was 16 % ) diagnose is based primarily on visual classication . 
while waiting for controlled studies on the value of the automatic density as a risk factor to be repeated , the adoption of a simple system to convert the quantra value into a visual value is currently the only solution if we are to continue using automatic evaluation in current practice . 
the similarity of the results between the two studies also applies to the range of quantra values for the individual birads categories , except for category d4 , in which we observe a discrepancy in the value of the lower range . 
if one applies the same intent to ciatto et als study , the cut - off value that correctly identies all d34 visual cases is \15 % , whereas in our study this corresponds to a cut - off value \9 % . 
 [ 11 ] who used quantra as evaluation software for mammographic density , the pearson correlation coefcient between the age and density parameters was - 0.20 ( p \ 0.0001 ) ; specically , menopausal status showed a signicant correlation with breast density with perior post - menopausal women having a lower density compared to premenopausal women . 
 [ 23 ] in a large meta - analysis including 42 studies used 21 % as a cut - off density value stating that the risk of developing breast cancer in women with dense breasts ( [ 21 % ) was up to 4.6 times higher compared to those with lower density . the literature reports many experiences related to a reduction in the sensitivity of mammography in the detection of breast cancer in dense breasts . 
conducted on women participating in mammography screening from 1988 to 1993 in whom primary invasive breast cancer was diagnosed within 24 months of a screening examination and therefore before the next inspection . 
the odds ratio ( or ) for interval cancers among women with very dense breasts was 6.14 ( 95 % ci , 1.8519.4 ) compared with women with extremely fatty breasts [ 24 ]  . 
specically , a summary or of 3.6 ( 95 % ci , 2.74.8 ) was calculated for the quantitative method , compared to an or of 1.8 ( 95 % ci , 1.52.1 ) for the wolfe method . 
many studies using quantitative methods for the evaluation of breast density reported a higher or , in terms of developing breast cancer , in women with dense breasts compared to studies in which subjective evaluation methods were used . 
 [ 25 , 26 ] conrmed the importance of using precise methods for evaluating mammographic density since they found a 2 % increase in the relative risk of breast cancer for each percentage point increase in breast density . 
in our study , albeit with low test power ( p = 7 % ) , we found that the group of patients aged b50 years and density greater than 21 % had a greater tendency to disease ( probability of 26.9 % ) compared to the group with density \21 % . moreover , comparison between denitive histology and breast density showed that idc and dcis have equivalent density ( p = 0.44 ) , while there was a borderline difference ( p = 0.08 ) between these and ilc due to the small number of ilc which reduced the statistical power ( p = 23 % ) ( probability of false negative , 77 % )  . 
conversely in two other recent studies , the data failed to demonstrate a statistically signicant correlation between density and tumour histology . in particular , in the study by eriksson et al . , which analysed 1 , 747 patients diagnosed with tumour between 1993 and 1995 , the breast density estimated by computerised algorithm was found to be associated with tumour size ( regression coefcient 0.031 , p = 0.017 ) , but not with histopathological classication or receptor status . 
for each of the conditions identied , we dened the overall incidence , the incidence based on the site , gender , average age and age range . results computed tomography examination was positive in 125 out of 143 patients ( 87.4 % ) , 74 men and 51 women , with an average age of 51.1 years , aged between 10 and 90 years . 
we found 79 inammatory / infectious conditions ( 63.2 % of positive cases , 55.2 % of total cases ) , 46 men and 33 women , with an average age of 47 years . 
based on the clinical diagnosis and pathology , they can be divided into subgroups : inammatory / infectious diseases [ 3 , 4 ] , trauma , foreign body ingestion [ 5 ] , tumours [ 6 ] , functional disorders , sensorineural disorders , respiratory diseases , vascular disorders , and spinal and vertebral disorders . with the increasing use of computed tomography ( ct ) to evaluate patients in the emergency department , in our daily activity the number of cases of non - traumatic emergencies of the neck is growing , as demonstrated in a study published in radiology in 2011 [ 7 ]  . 
the aim of this study was to collect information on the incidence and distribution of acute , non - traumatic conditions of the neck seen at the emergency radiology department of our hospital and to review the literature about this topic . radiol med ( 2014 ) 119 : 784789 materials and methods we used our institutions data archives to conduct a descriptive observational study , collecting retrospectively consecutive patients who underwent neck ct in the from 1 december 2008 to 31 emergency department december 2012 . 
during this time , in the emergency radiology department of our hospital , 201 ct examinations of the neck were performed out of a total of 38 , 768 ct examinations ( 0.5 % ) ; considering that we excluded 19 patients whose clinical history was not known and 39 patients ( 19.4 % ) who underwent ct as a result of trauma , we found 143 out of 38 , 768 patients ( 0.37 % of the total emergency ct requests ) , with a non - traumatic condition . among the examined 143 patients , 86 out of 143 were men ( 60.1 % ) and 57 ( 39.9 % ) were women , with an average age of 51.7 years and age range between 10 and 90 years . all the patients underwent multidetector ct scans using a ge lightspeed qxi scanner ( milwaukee , usa ) with section thickness of 1.25 mm and a siemens somatom denition flash scanner ( erlangen , germany ) with a section thickness of 0.6 mm , before and after the intravenous administration of contrast media . patients underwent ct to answer clinical questions that respected the referral guidelines for imaging of european commission directorate - general for the environment in conjunction with the uk royal college of radiologists ( 2000 ) and appropriateness criteria of the american college of radiology . 
we collected demographic and clinical data from the ct requests and medical records . this study was reviewed and approved by the fondazione irccs ca granda maggiore policlinico hospital of milan institutional review board . results the ct examination was negative in 18 out of 143 patients ( 12.6 % ) , 12 men and 6 women , with an average age of 56.1 years and age range between 21 and 88 years . 
computed tomography examination was positive in 125 out of 143 patients ( 87.4 % ) , 74 men out of 125 ( 59.2 % ) and 51 women out of 125 ( 40.8 % ) , with an average age of 51.1 years and age range between 10 and 90 years . 
table 1 shows the incidence of the abscesses based on the site , gender and age . 786 radiol med ( 2014 ) 119 : 784789 table 2 distribution of inammatory alterations based on site , gender and age women average age inammatory alterations submandibular space larynx tonsils parotids submental space masticator and parapharyngeal spaces parapharyngeal space oropharynx and supraglottic larynx paravertebral and parapharyngeal spaces parapharyngeal and laterocervical spaces total 19 out of 79 ( 24.1 % ) 4 out of 19 ( 21.1 % ) 3 out of 19 ( 15.8 % ) 3 out of 19 ( 15.8 % ) 2 out of 19 ( 10.6 % ) 2 out of 19 ( 10.6 % ) 1 out of 19 ( 5.3 % ) 1 out of 19 ( 5.3 % ) 1 out of 19 ( 5.3 % ) 1 out of 19 ( 5.3 % ) 1 out of 19 ( 5.3 % ) inammatory alterations tumours 49.6 ( range 1891 ) 65.2 ( range 2191 ) 66 ( range 5873 ) 29.6 ( range 2436 ) 34.5 ( range 2346 ) 55.5 ( range 3972 ) in this group we considered patients with ct signs of inammation , such as cellulitis , thickening of the fascia , and oedematous swelling of muscles or organs , without documentation of abscesses or phlegmons . 
table 2 shows inammatory alterations based on site , gender and age . phlegmons ct revealed 16 cases : 9 men and 7 women , with an average age of 53.1 years and age range between 23 and 83 years . 
table 3 shows the incidence of phlegmons based on site , gender and age . abscesses together with phlegmons we observed two cases : one man and one woman , with an average age of 56 years and age range between 36 and 76 years . 
the man presented submandibular abscesses together with phlegmons that had spread to the submental and parapharyngeal spaces . table 3 distribution of phlegmons based on site , gender and age ct revealed 26 cases : 19 men and 7 women , with an average age of 68.5 years and age range between 49 and 97 years . 
table 4 shows the incidence of tumours based on site , gender and age . ingestion of foreign bodies ct revealed six cases : three men and three women , with an average age of 64.8 years ( range 3580 years )  . 
all the patients had previously undergone standard x - rays of the airways that had proved non - conclusive . benign swellings computed tomography revealed ve cases : three men and two women , with an average age of 44.8 years and age range between 21 and 79 years . 
1 contrast - enhanced computed tomography ( ct ) in a 37 - yearold patient demonstrates an abscess in the submandibular space ( white arrows ) with strands of the subcutaneous fat . 
2 in a 53 - year - old male patient , unenhanced ct demonstrates marked swelling ( white arrows ) of the larynx , pre - epiglottic space , hypopharynx and retropharyngeal space , with a stula ( black arrow ) between the vertebral body and larynx branchial cyst ( one infected , one not ) , two cases showed a struma , and one case , a lipoma . vascular disorders ct revealed ve cases : two men and three women with an average age of 50.8 years and age range between 33 and 64 years . 
four cases showed a haematoma ( two laterocervical and two para - retro - pharyngeal ) and one case a dissection of the vertebral artery . oedema of the larynx computed tomography revealed four cases : three women and one man , with an average age of 70 years and age range between 49 and 85 years . 
the cause of the oedema was not determined in any of the cases . from 1 december 2008 to 31 december 2012 , requests for ct examinations for non - traumatic conditions of the neck accounted for 0.4 % of all ct scans performed in the emergency radiology department of our hospital . 
although the incidence rate of these requests is low in relation to the total number of ct examinations performed , is not without signicance in the daily activity of our department , with nearly one case every 10 days . 
 [ 16 ] , imaging of infectious / inammatory processes has ve crucial roles : to conrm the clinical hypothesis , to dene the extension , to evaluate the presence of complications , to differentiate a drainable abscess from cellulitis and to follow up the infection progression . sonography allows one to distinguish an abscess from cellulitis , to evaluate the presence of thrombophlebitis in the infrahyoid neck , but gives little information on the anatomical extension . 
it also allows assessment , with bone window setting , of the causative periapical abscess and mandibular cortical dehiscence . patients with tonsillitis and peritonsillar abscess had sore throat , fever , cervical lymphadenopathy , dysphagia and pharyngotonsillar exudate . 
they underwent ct examination when clinical diagnosis was uncertain and when there was a deep neck space extension . unfortunately , the aetiopathogenesis of infectious and inammatory conditions remains unclear in 1767 % of cases [ 14 , 16 , 17 ]  . 
in our study , as many patients were discharged without an aetiological diagnosis , it was not possible to make a correlation between clinical examination , imaging and infectious pathogen ; however , we observed that the majority of the submandibular abscesses ( 7 / 13 cases , 53.8 % ) were due to an odontogenic infection process , with a mainly bacterial aetiology . 
as regards inammatory processes of the pharynx and tonsils , which can deteriorate into tonsillar abscesses and phlegmons in the case of superinfection , the initial aetiology is mainly viral . 
however , all the microbial species responsible for respiratory infections may cause pharyngitis and tonsillitis [ 18 ]  . newly found tumours were the second most frequent pathology after infectious / inammatory diseases . 
these conditions may determine airway obstruction , bleeding , compression of the spinal cord , bilateral palsy of the vocal cords and septic thrombophlebitis , all disorders that require an immediate diagnosis and a rapid intervention of ent specialists , surgeons and radiologists [ 6 ]  . in our study , neoplastic pathology was more frequent in males with an average age of 68.5 years , higher than that of the infectious / inammatory processes . 
3 in a 51 - year - old female patient , contrast - enhanced ct demonstrates a newly diagnosed tumour arising from the right thyroid lobe ( arrows ) , displacing the trachea and vessels and causing airway obstruction fig . 
 [ 11 ] , inammatory and infectious conditions ( abscesses , inammatory alterations and phlegmons ) , were the most commonly observed with 79 cases out of 125 ( 63.2 % ) , mostly in males ( 58.2 % ) and in patients with an average age of 47 years . the three neck spaces most frequently involved in the infectious / inammatory processes were the submandibular space [ 1215 ] , followed by the tonsillar and pharyngeal spaces . among the infectious / inammatory conditions , we found a greater incidence of abscesses ( 53.2 % ) , particularly of the submandibular ( 31 % ) and tonsillar ( 31 % ) spaces . 
patients with submandibular abscess on clinical examination showed a visible swelling in the submandibular region , pain , dysphagia and radiol med ( 2014 ) 119 : 784789 performed ct to evaluate the location and extent of airway obstruction and the degree of intrinsic versus extrinsic compression for therapeutic planning . the third most in a smaller percentage , frequent pathology we examined was ingestion of foreign bodies . usually , the most common foreign bodies are sh bones , chicken bones , small pieces of glass , dentures , coins and needles . 
 [ 5 ] , ct can detect ingested foreign bodies such as slightly calcied objects that are missed by conventional radiographs and it allows the assessment of complications . all the other pathologies of the neck found by various authors had a low incidence . 
our study of emergency ct of non - traumatic conditions of the neck fundamentally revealed infectious / inammatory diseases and newly found neoplasms . imaging may be requested in a patient who presents with an acute condition of the neck and the clinical presentation may vary . 
trovato received : 9 may 2013 / accepted : 22 august 2013 / published online : 5 february 2014 ( cid : 2 ) italian society of medical radiology 2014 abstract interest in transthoracic ultrasound ( us ) procedures increased after the availability of portable us equipment suitable for use at the patients bedside . 
it is possible to detect space - occupying lesions of the pleura , pleural effusion , focal or diffuse pleural thickening and subpleural lesions of the lung , even in emergency settings . transthoracic us is useful as a guidance system for thoracentesis and peripheral lesion biopsy , where it minimises the occurrence of pneumothorax and haemorrhage . 
transthoracic us imaging is strongly inuenced by physical interaction of the ultrasonic beam at the tissue / air interface , which gives rise to reverberations classied as simple ( a - line ) , comet tail and ring down ( b - line ) artifacts . although these artifacts can be suggestive of a disease condition , they are essentially imaging errors present even in normal subjects and in empty - pleura post - pneumonectomy patients . 
in order to clarify some confusion and to report on the state of the art , we present a review of the literature on transthoracic us in diseases of the pleura and peripheral lung regions and our own clinical experience over 3 decades . 
thoracic us is much more than shing for the moon in the well . keywords thoracic ultrasound ( tus ) ( cid : 2 ) contrast - enhanced ultrasound ( ceus ) ( cid : 2 ) lung ( cid : 2 ) pleurisy ( cid : 2 ) copd m . 
feragalli department of clinical sciences and bioimaging , institute of radiology , ss annunziata hospital , chieti , italy e - mail : b.feragalli@unich.it introduction ultrasound ( us ) imaging depends on the different physical interactions between ultrasonic waves and the target tissues : in transthoracic ultrasound the images are strongly inuenced by the features of the chest wall and by air in the lungs . 
their interference gives rise to various artifacts , and more than 95 % of the us beam is reected by the tissue / air interface , so it is not available for further imaging [ 13 ]  . the reverberations generated by this interaction have been classied as simple ( horizontal a - line ) , comet - tail and ring - down ( vertical b - line ) artifacts , although some misunderstanding of these terms is apparent from the literature [ 4 , 5 ]  . 
nevertheless , these artifacts all arise owing to the great difference in acoustic impedance in the pleural spaces and are visible to a certain extent when the lung is lled with air . 
similar artifacts are also visible in the pneumonectomy space , which contains residual air , liquid lms and / or oedema and scar tissue : this fact appears to 730 radiol med ( 2014 ) 119 : 729740 disprove attempts to read such artifacts as specic disease markers [ 6 ] the us examination should always be used in conjunction with standard x - ray or ct scans of the chest and is not in itself able to provide a denitive diagnosis [ 7 ]  . methods and techniques one of the main hindrances to the reproducibility of transthoracic us is the machine settings because available equipment does not provide predetermined settings for this application . 
the frequency will need to be adjusted to suit the application , which means a change from 3 to 8 mhz for the convex probe , as will the probe depth , which should range from 70 to 140 . 
tissue harmonics are preferable , in an effort to reduce the natural artifacts , and the time gain compensation ( tgc ) should not exceed 55 % [ 8 ]  . 
it is also important not to forget that the speed of us in the abdominal organs is on average 1 , 520 m / s , with minimal variation between organs , while in the lungs the speed falls to less than a third ( 450 m / s )  . 
the acoustic impedance of lung tissue is 0.0008 rayl 9 10 - 6 , which is very different from the acoustic impedance of the abdominal ( 1.65 rayl 9 10 - 6 ) and kidney organs like the liver ( 1.62 rayl 9 10 - 6 )  . 
this large difference in acoustic impedance at the interface between the surface tissues and air in the lung ( the so - called pleural line ) generates all the artifacts visible under us ( simple reverberations , ring downs and comet tails )  . 
the us beam should be electronically focused on this pleural line , and similar settings should be pre - set for the high - frequency linear probe ( 812.5 mhz ) [ 9 ]  . 
to the right , this is supplied by the liver , while the spleen provides the window on the lethoracic examination must be completed with laterocervical scans , and scans of the armpit in cases where lymphadenopathy is suspected [ 10 ]  . 
this means that us is useful for viewing pleural and subpleural disease in that portion of the pleura , which corresponds to a very small part of the entire pulmonary tissue [ 11 ]  . 
the diaphragm , however , is highly visible on the right using subcostal scans at the radiol med ( 2014 ) 119 : 729740 level of the posterior face of the liver parenchyma . 
it is seen as a hyperechoic line made up of three separate layers , namely the liver capsule / diaphragm muscle interface ( hyperechoic layer ) , the diaphragm muscle ( hypoechoic layer ) and the mirror effect artifact ( hyperechoic layer )  . at the pleural space , the air in the lungs reects almost all of the us beam ( more than 95 % ) , as there is a great difference in acoustic impedance between the soft tissues and pulmonary air at this point . 
these comprise simple reverberations , also known as horizontal , or a - line artifacts , and comet - tail , ( b - line ) artifacts [ 13 ]  . ring - down and vertical fig . 
the thickness of this line does not correspond anatomically to the parietal or visceral pleura or the pleural space , whose thickness does not exceed 300400 lindeed , a similar line is also visible , albeit xed ( without sliding or gliding sign ) , in the residual cavity of pneumonectomy patients . this is due to a considerable difference in impedance between the soft tissues and the postpneumonectomy cavity , which contains air and uid [ 14 ]  . in addition to these artifacts , others are generated by posterior acoustic shadowing at the dense bones of the thoracic cage ( ribs , sternum and scapula ) [ 15 ] and by the so - called mirror effect . 
mirror effect artifacts are typically seen on right subcostal scans and arise , for example , from hepatic lesions being mirrored on the opposite side of the hyperechoic diaphragm line at the base of the lung [ 16 ]  . in the normal lung , simple reverberations ( horizontal or a - line artifacts ) are seen as multiple hyperechoic signals of decreasing intensity parallel to the pleural line and spaced apart by a xed period that is determined by the time between the emission and reception of the us signal . another type of reverberation , the ring downs ( vertical or b - line artifacts ) , described by avruch and cooperberg [ 17 ] , are very distinctive ( and relatively easy to distinguish from comet tail artifacts )  . 
these can be seen posterior to areas where uid and gases are collected , for example in the bowel loops [ 1719 ] , and they have occasionally been misinterpreted as comet tails [ 20 ]  . 
 [ 4 ] , are broad reverberation echoes that travel distally along the direction of propagation of the us beathey take on a triangular shape with a distal tip and resemble the tail of a comethence the name . 
these comet tails originate at highly echoic interfaces , for example , cholesterol crystals on the anterior wall of the gallbladder , metal clips in the parenchyma and gas bubbles inside the bile duct [ 2123 ]  . 
in pleuropulmonary us , comet tails are also due to the presence of two contiguous tissues with a marked difference in acoustic impedance ( soft tissue / pulmonary air interface ) , and they are found in both the normally aerated lung and in other cases where such interfaces occur , i.e. , distal to pleural lesions ( plaques or pleuritis exudate ) or subpleural lesions of any type [ 23 ]  . the number and intensity of the visible vertical artifacts ( b lines ) depend on the type and frequency of the probe used , as well as the degree of total gain compensation ( tgc )  . 
the erroneous use of a medium - to - low frequency or excessive total gain and the lack of tissue harmonic imaging can generate a large number of such artifacts especially in pleuropulmonary us . 
the number of horizontal artifacts ( a - line ) and / or vertical artifacts ( ringdowns or b - lines ) is determined by the relationship between the curve of the probe used with respect to the curve of the pulmonary surface examined . 
whether tissue harmonics are or are not used can produce discrepancies , or at the very least a lack of homogeneity , between images from the same us scan [ 23 , 24 ]  . 
in - depth knowledge of us physics and artifacts enables an operator appropriately preset the us machine , limiting the poor reproducibility due to the use of unsuitable settings ( frequency , tgc , tissue harmonic and electronic beam focusing ) and / or different probes ( convex , sectoral or linear )  . 
this is fundamental because several pathological conditions of the lung can cause these artifacts to increase in number and intensity . these pictures are usually strikingly different from those of the healthy lung , which features a normal ratio between air in the alveoli , quantity of liquid lm ( uids , exudate ) , vascular circulation and pulmonary parenchyma . 
hence , it is essential that we have good and accurate knowledge of the nature of these artifacts and how they appear on - screen , so that we can avoid false , and potentially detrimental interpretations [ 24 ]  . 
4b , after the image gain has been set correctly , the uid is seen as anechoic ( correct diagnosis )  . pleural and extrapleural disease thoracic us is the method of choice for the assessment of pleural effusion , as the diagnosis is simple and accurate using this technique . 
the effusion will show up as anechoic ( black ) area with distinct margins accompanied by movement deeper into the eld of the pleural line . under this , the aerated , consolidated or atelectasic lung will be visible . 
it is possible to quantify the degree of effusion on us by determining the longitudinal and transversal dimensions of the nonechoic area . greatest radiol med ( 2014 ) 119 : 729740 fig . 
left a : homogeneously hyperechoic pleural effusion with elevated gain level ( diagnostic pitfall )  . right b the image in the same patient with normal gain level in anechoic pleural effusion ( correct diagnosis ) several authors have provided formulae to calculate the volume of the effusion , or the degree of compressive atelectasis and its organisation . 
it allows very small collections of uid to be detected , even \10 ml in the costophrenic angle . pathological processes encroaching on the pleural space ( mesothelioma or metastases ) are easy to identify using us , especially if associated with pleural effusion . 
these are visible as hyperechoic lesions or , in certain cases , as mixed echogenic structures associated with a varying extent with pleural thickening , small discontinuities in the diaphragm and , on some occasions , invasion of the thoracic wall . 
us also enables the identication of pleural adhesions , which are visible as circumscribed hyperechoic lesions , which may on occasion be calcied [ 28 ]  . in the diagnosis of pneumothorax , the negative predictive value of us reaches almost 100 % , as the presence of the sliding sign of the pleural line recognisable at b - mode us in real time and well documented by time - motion ( mmode ) us excludes the possibility of this condition [ 29 , 30 ]  . 
the positive predictive value of us in the diagnosis of pneumothorax , however , is reported to vary between 55 and 90 % , because the absence of the sliding sign associated with a reduction in vertical artifacts is not a denitive clue , being also found in severe pulmonary brosis , brothorax , panlobular emphysema , in some intubated patients and those tted with a thoracotomy drainage tube , in cancer invading the chest wall , in pleurisy sequelae and in other conditions . 
furthermore , it should be taken into account that the sliding sign is scarcely visible in scans of the pulmonary apex of healthy lungs , especially in obese subjects , due to the particular anatomy of the region , namely the presence of three ligaments ( transverse - pleural , costal - pleural and vertebral - pleural ) , which limit the visibility of the sliding sign [ 31 , 32 ]  . certain extrapleural parietal lesions can also be visible in us ; these include cysts , abscesses , haematomas , benign tumours , costal metastases , bacterial and tubercular osteomyelitis and tumours of the soft tissues of the thoracic cage ( myelomas )  . 
5 pleural effusion features a anechoic ; b complex nonloculated ; c homogenous hyperechoic ; d complex loculated units , where chest x - ray performed at the bedside is the most widely used and valuable imaging method in the follow - up of critically ill patients [ 33 ]  . 
6 segmental thickening of hyperechoic pleural line ( 3.9 mm ) subsequent to pleurisy in a patient with a previous pneumonitis enlargement , interruption or dislocation of the pleural line , as well as respiratory immobility . 
this is particularly relevant in intensive care pathological processes of the lung can be examined by means of us when the beam is able to reach the pleura , i.e. , there is no air obstructing the beams passage towards the lesion . 
a layer of air - containing tissue , even as thin as 0.3 cm , is sufcient to block the signal and prevent visualisation of a space - occupying lesion , even if it is very large . 
in all normal peripheral lung diseases , us is only radiol med ( 2014 ) 119 : 729740 the pleural thickening of able to image the lesion in cases in which the surrounding consolidated , atelectatic or effusive parenchyma provides an adequate acoustic window [ 34 ]  . 
however , on occasion , when the subpleural or peripheral pulmonary regions are involved , it is possible to identify subpleural nodules , particularly in the dorsal regions at the apices in silicosis , or plaques at the periphery of the inferior lobes in asbestosis . 
pulmonary atelectases , which are categorised as either central ( caused by some kind of obstruction to the bronchial branches ) , or peripheral ( due to compression of the lung parenchyma by effusion , pneumothorax , trauma , severe brosis or tumours ) can easily be diagnosed using thoracic us . 
in cases of atelectasis , the parenchyma will be visible on us as a fairly homogeneous structure , with an echogenicity equal to or lower than that of the liver . 
on occasion , uid - containing tubular structures with echogenic walls are visible inside these are the pulmonary blood vessels and / or mucus - lled bronchi ( us uid bronchogram )  . 
the sonographic picture of bronchopneumonia densities is characterised by the presence of a hypoechoic / echo - free area , which may be mixed hypo / hyper - echogenic , with ill - dened borders . 
hyperechoic stripes or spotsthe air - lled bronchi ( us air bronchogram ) and / or hypoechoic / echo - free tubular structures trapped blood vessels or oedematous bronchi ( us uid bronchogram ) can be seen as well . 
that being said , us is useful after the diagnosis , in the follow - up of pulmonary densities undergoing treatment , as it enables their healing / regression to be monitored [ 38 ]  . 
also in children , as in adults , after a preliminary and mandatory chest x - ray diagnosis , necessary to avoid misleading understatements or clinical decisions , thoracic us should monitor subpleural pneumonitis consolidation and the response to therapy . 
in this context it is a suitable and repeatable alternative to other laboratory measurements and to further radiation exposure of children . echo - free lesions , which are usually well circumscribed , could also be generated by malformation , bronchogenic or pleural cysts , or cysts caused by parasites , or , very rarely , sterile pulmonary infarcts . 
pulmonary abscesses , which appear as circumscribed , uid lled and corpuscular lesions , are only visible on us when they are peripherally located and in contact with the pleuroparietal surface . 
solid lesions , which typically present slightly irregular margins and varying degrees of hypoechogenicity and uniformity , without a particular typical of neoplastic structure are , generally speaking , masses , although these may also present as either hyperechoic , or hypoechoic / echo - free ( in liquefactive necrosis )  . in lung carcinoma , it is possible to see areas of air and / or uid bronchogram , like those found in inammatory consolidation [ 40 ]  . 
any subpleural lesion identied on us must necessarily be conrmed by radiological techniques like computed tomography ( ct ) , positron emission tomography ( pet ) - ct or magnetic resonance imaging ( mri ) , as there is no denitive diagnostic us pattern . 
in fact , even the use of colour doppler inside the lesion , as well as being impeded by the presence of movement artifacts ( ash artifacts ) is unable to accurately distinguish between neoplastic and non - neoplastic disease [ 41 ]  . 
in some cases , there were unenhanced areas consistent with zones of necrosis and these areas were avoided during ne - needle aspiration biopsy ( fnab ) [ 42 ]  . lung cancers have a different behaviour when challenged by ceus and the ceus pattern is different from that of inammatory conditions , such as pneumonitis . 
differences are signicant both as averages and using the most current appearance of nodule conventional cut - off enhancement , which is \10 s for the so - called early enhancement . 
actually , among all the lung cancers , only small - cell carcinoma has a more distinctive pattern , with very early image enhancement and quite early enhancement disappearance , in comparison both with the other cancer histotypes and pneumonitis , so that this information cannot have a practical use . 
one of them is 736 radiol med ( 2014 ) 119 : 729740 identication of the exact location and the direct guidance of the needle under the eyes of the physician . 
the most reliable biopsy - adapted probes have a central hole through which the biopsy set is introduced and that is followed all the time in its path with an image exactly perpendicular to the target and to the transducer , linear array or convex . skipping necrotic areas is a useful information which is provided by ceus , which enables the choice of the most suitable point ( s )  . 
both provide diagnostically useful information on peripheral lung lesions and increase the diagnostic yield of transthoracic fnab by reducing the risk of inadequate tissue sampling . the densities was greater thoracic us is not suitable for formulating hypotheses based on conjecturally specic clues that are , however , confusing when functional - pathological diagnostic patterns do not exist . 
theories put forward on the basis of signs allegedly visible in pulmonary interstitial disease ( acute pulmonary oedema ) seem to be difcult to demonstrate and their results are not reproducible . 
these theories postulated that in the presence of more than six or seven vertical artifacts described as comet tail ( but in actual fact ringdowns or b - lines ) per intercostal space , this pattern could enable distinction between acute pulmonary oedema from other diffuse lung disease ( for example , acute phase obstructive bronchopneumopathy )  . 
in fact , the idea that a subjective visual count of the number of wandering artifacts can be pathognomonic of any one type of lung disease is , to say the least , scientically weak and open to doubt [ 44 , 45 ]  . 
furthermore , artifacts are in essence imaging errors , dependent on a variety of factors and could not alone be a pathognomonic pattern of a single disease state . in fact , an increase in the number of ring - downs and b - lines per intercostal space is generated every time the us beam is intercepted by an excessive quantity of air and liquid lm or exudate or by the presence of brosis in the pleural space . 
this condition is a feature of many diffuse lung and interstitial diseases of various nature , like pulmonary brosis , acute exacerbation of chronic obstructive pulmonary disease ( copd ) , acute bronchial asthma , uniformly distributed pleural effusion and lymphangitis and cannot , therefore , be considered pathognomonic of a particular disease state ( acute pulmonary oedema )  . 
moreover , this clinical condition has been studied and reported in the literature [ 4446 ] , and no statistical signicance has been attributed to such artifacts in the wet lung of patients affected by acute pulmonary oedema , with respect to those affected by other pleuropulmonary diseases ( acute exacerbations of copd , carcinomatous lymphangitis , acute bronchial asthma , pleural effusions and / or chronic cardiac decompensation )  . 
the need for procedure standardisation is obvious : overall b - line measurements or score , as dened and used by some authors , is only a synthetic personal measure of wandering artifacts [ 47 ]  . 
although paved with good intentions such studies fail to reach their goal , i.e. , lung water measurement , a frail methodological basis and weak data can lead to deceptive conclusions , like shing for the moon in the well . us - guided interventions the earlier experiences with us - guided transthoracic puncture of the pleura or pericardium are reported in haemopericardium when emergency intervention was needed for preventing cardiac tamponade and when the subxyphoid approach is less suitable [ 48 ]  . 
as written above , linear - array or convex probes with a central hole are most suitable for fnab : both allow a biopsy needle or a therapy probe to be inserted through the centre of the transducer . the needle , therefore , enters in the centre of the image , perpendicular to the lesion , facilitating difcult procedures like intercostal biopsies . 
7 large number of ring - downs or b - lines in patients with different conditions : a copd ; b acute pulmonary oedema ; c pulmonary brosis in a patient with systemic sclerosis visible on chest x - ray ( for example , to distinguish between effusion and the opaque base of pulmonary consolidation )  . the international literature reports a percentage of pneumothorax in unassisted thoracenteses as between 7 and 16 % , which falls to 0.5 % when us guidance is used . 
in our 10 - year experience , comprising 2 , 850 consecutive usassisted drainage procedures ( diagnostic and therapeutic ) , it was possible to obtain a diagnosis of neoplastic effusion in 228 patients ( 8 % ) through cytological testing and the identication of neoplastic cells in the drainage uid . 
in this sample , the rate of major complications such as pneumothorax was 0.11 % ( three patients , two of whom suffered minimal pneumothorax and another , a subtotal pneumothoraxboth resolving spontaneously ) [ 49 ]  . ultrasound is also very useful in the emergency department , where it can be used to conrm the clinical suspicion of massive pleural effusion in cases of acute respiratory disease and guide its drainage to reduce the risk of pneumothorax to almost zero . 
8 the tip of the needle is visible within the uid of a left - basal pleural effusion needle beginning at the skin line and allows difcult biopsies to be performed with instant feedback on the precise location of the needle tip . 
furthermore , us enables determination of the type of effusion ( simple , loculated or laminar ) and better characterisation of the opaque areas 738 radiol med ( 2014 ) 119 : 729740 through certied training and the use of accurate up - to - date equipment and settings . 
9 us - guided fnab of a hypoechoic subpleural pulmonary lesion ( right lower - basal ) ; the needle ( 20g ) can be seen inside the lesion during withdrawal of the histological cylinder specimen and cytological material ( squamous carcinoma ) pleural lesions in which us - guided transparietal needle biopsy can be useful are nodular pleural thickening , which appear as micronodular hypoechoic structures , often associated with thickening of the pleural line . 
pleural mesothelioma which appears as a mixed hyperechoic lesion is very often associated with ipsilateral serosanguinous effusion [ 51 ]  . it is rare to nd benign lesions like broma or lipoma . all pulmonary lesions visible on us can be aspirated using 2021g cutting / aspirating needles . 
bronchopneumonic densities or pneumonitis , especially in immunodepressed patients , whose origin is undetermined by the usual battery of tests ( sputum material , other effusions , blood culture , serum and urine tests ) , may need to be identied through us - guided fnab ( 22g ) [ 52 , 53 ]  . in cases of pleural empyema , us - guided drainage is a fundamental procedure , and in lung cancer us - guided needle biopsy allows a diagnostic accuracy of 8596 % , depending on the tumour . 
the complication rate for this procedure was 0.2 % ( one case of spontaneously healed partial pneumothorax and one case of total pneumothorax treated by drainage tube ) [ 49 ]  . despite some confusion , enthusiasm and overstatements in the current medical literature and the advent of more advanced imaging systems , transthoracic us is a valuable diagnostic / treatment tool in pleuropulmonary imaging and interventional medicine . 
all patients were studied with thin - section mr imaging of the eye using surface coils . results high - resolution mr imaging of the eye excluded extrascleral extension of disease in 8 / 12 patients : in 4 / 8 cases it revealed vascular ectasia and in the other 4 / 8 cases the linear hypointensity of the sclera was unbroken . 
seven of these eight patients were followed up by ultrasound , which showed stability of melanoma for at least 2 years , while the last patient underwent enucleation , and the histological examination conrmed the mr diagnosis . 
in 4 / 12 patients , high - resolution mr suggested a diagnosis of extrascleral extension of melanoma , which was conrmed at histological examination after enucleation . conclusion high - resolution mr imaging of the eye with surface coils allowed us to evaluate extrascleral extension therapeutic of uveal melanoma and choose the correct t . 
blasi dipartimento di scienze chirurgiche per le patologie della testa e del collo , istituto di oftalmologia , policlinico agostino gemelli , ucsc , rome , italy approach , avoiding unnecessary enucleation in 7 / 12 patients . keywords malignant melanoma ( c04.557.465.625.650.510 ) ( cid : 2 ) uveal neoplasm ( c04.588.364.978 ) ( cid : 2 ) mri ( e01.370.350.500 ) introduction uveal melanoma arises from melanocytes and accounts for less than 1 % of human cancers ; in spite of its rarity , it is the most common primary intraocular malignancy in the white adult population , where it represents 7988 % of primary intraocular cancers , followed by metastatic carcinoma and retinoblastoma in the paediatric population [ 1 , 2 ]  . 
about 90 % of uveal melanomas arise from the choroid , about 510 % from the ciliary body and 23 % from the iris [ 1 ]  . fewer than 1 % of patients are younger uveal melanoma is uncommon in children and young adults ; than 20 years [ 1 ]  . 
it is more frequently seen in older age groups , with a mean age of 5560 years for both males and females and a progressive age - related incidence with a peak at 70 years ( 24.5 per million in males and 17.8 per million in females ) [ 2 , 3 ]  . 
the highest incidence is reported in northern european populations , especially among scandinavians , with 59 cases per million / year and it is more common in the white race [ 2 ]  . 
choroidal melanomas are more frequent in patients with fair complexion and light coloured eyes ( grey / blue ) than in patients with dark eyes , although the role of the sun in the pathogenesis is still unclear [ 4 , 6 ]  . 776 radiol med ( 2014 ) 119 : 775783 several local factors have an independent effect on tumour shape , tumour size , determining the prognosis : degree of pigmentation , optic nerve invasion , retinal detachment and extrascleral extension [ 1 , 710 ]  . 
extrascleral extension of uveal melanoma is a known negative prognostic factor which reduces patient survival and increases the risk of disease recurrence ; it is found more often in larger choroidal melanomas , although it has also been reported in small ones [ 1 , 11 , 12 ]  . 
these prognostic factors inuence therapy , helping to choose between conservative treatment and enucleation [ 1113 ] ; in particular , according to the collaborative ocular melanoma study group ( coms ) , brachytherapy with plates of 106ru and 125i is currently the most common conservative therapy in patients with medium size melanomas ( thickness of 2.510 mm or maximum diameter \16 mm ) , since there is no difference in survival compared to patients undergoing eye enucleation [ 5 , 6 , 14 ]  . 
at present , enucleation is reserved for larger tumours ( thickness c10 mm or maximum diameter c16 mm ) or tumours with extrascleral extension [ 5 , 14 ]  . because of their posterior anatomical location uveal melanomas are not amenable to biopsy without intraocular surgery , so the diagnosis is usually based on clinical and imaging examination , and especially on transocular ultrasound ( us ) [ 5 , 6 , 9 ]  . transocular us is highly sensitive and inexpensive examination commonly used for the evaluation of intraocular tumours and for detection of scleral inltration . 
it has , however , been shown to have some pitfalls : it cannot always allow for a correct evaluation of extrascleral extension , particularly when choroidal melanomas are close to the optic nerve , because of shading of physiological sheaths ; moreover , it may overestimate tumour inltration when there [ 9 ]  . furthermore there are other signs that can simulate extrascleral extension : vessel congestion , venous ectasia or inammatory phenomena of tenons space and extrinsic muscles [ 6 , 7 , 11 , 15 ]  . 
the insertion of extrinsic muscles , especially the inferior oblique muscle , can be misinterpreted as retrobulbar extension of the tumour [ 13 , 15 , 16 ]  . excavation choroidal magnetic resonance ( mr ) imaging may be useful in doubtful cases and allows better visualisation of tumour invasion of surrounding structures compared to transocular us , a and b scan [ 10 ]  . 
mr imaging with surface coils , given its high spatial resolution , multiplanar capabilities and high intrinsic contrast can be useful in the assessment of intraocular tumour and extrascleral extension , especially in posterior choroidal melanomas [ 5 , 7 , 1724 ]  . 
melanomas show a characteristic signal pattern on mr images , being hyperintense to the vitreous on t1w images , hypointense on t2w images , with enhancement after intravenous contrast medium administration ; this specic signal behaviour helps the differential diagnosis with other ocular tumours . 
because of this correlation between melanin content and signal intensity , melanomas with lower melanin content show a lower signal intensity on t1w images compared to those with high content , which are strongly hyperintense [ 1 , 5 , 8 , 15 , 25 , 26 ]  . the primary aim of this study was to evaluate the role of thin - section mr of the eye with surface coils in patients with a us suspicion of extrascleral spread of uveal melanoma . materials and methods in a prospective study , we enrolled 12 patients with a reliable diagnosis of uveal melanoma ( three men , nine women ; age range 3287 ; median age 64.3 ) , 10 of whom previously treated with brachytherapy . 
the us examination , carried out by a single experienced radiologist at the department of ophthalmology of our university hospital , was performed with the standardised a and b scan method , according to the ossoinig qualitative ( b scan ) and quantitative criteria ( a scan )  . 
in 5 / 12 patients , the transocular us diagnosis was conrmed by histological examination . in 10 previously treated patients , extrascleral extension was suspected on follow - up transocular us examination performed after brachytherapy , while in the remaining two patients it was already suspected at the rst us examination . 
extrascleral growth is generally suspected on transocular us and is based on demonstration of a retrobulbar hypoechoic area , next to the melanoma , even without discontinuity of the scleral wall [ 28 ]  . 
vascular ectasia can simulate extrascleral extension because it appears as a retrobulbar hypoechoic nodule , hardly distinguishable from radiol med ( 2014 ) 119 : 775783 excite 1.5t superconducting system , using a phased - array ( temporomandibular joint ) surface coil formed by two exible receive - only elements , each with a maximum diameter of 11 cm and a central opening of 8 cspecial care was taken to correctly position the surface coils above the eyes , with each exible element perpendicular to the long axis of the respective orbit , in order to avoid an asymmetric signal reduction from the centre to the periphery of the acquisition eld . 
to reduce motion artefacts , the patients head was immobilised with pillows on either side and the patient was instructed to x their gaze on a central spot during image acquisition . 
the axial plane included a stack of sections from the inferior to the superior border of the orbital cavity thereby obtaining a good view of the ocular globe , medial and lateral straight muscles , medial and lateral bone boundaries of the orbital cavity and the optical canal . 
the coronal plane included a stack of sections from the anterior margin of the ocular globe to the posterior margin of the dorsum sellae so as to obtain a good visualisation of the superior , inferior , medial and lateral portion of the ocular globe , all of the straight muscles , and an optimal view of the optic nerve and pituitary gland . 
the study was performed before and after intravenous injection of contrast mediupatients received multihance ( 0.2 mmol / kg ) , followed by 20 ml of saline solution at 1 ml / s ow rate , with a delay of 40 s after the injection of paramagnetic contrast medium , before the start of the 3d - fame sequence . the mr images were read by two neuroradiologists in fig . 
visualisation of vascular structures afferent to the nodule can be an important criterion for the differential diagnosis , but these are not always easily recognisable on transocular us . mr with 3d fast acquisition with multiphase - enhanced fast gradient - echo sequences ( 3d - fame ) after contrast medium injection can easily show the continuity of retrobulbar vascular structures , conrming the benign nature of disease . all 12 patients underwent high - resolution mr to evaluate tumour extension . 
the inclusion criteria were the following : age above 18 years ; no other melanomas in any other part of the body at the time of diagnosis ; extrascleral extension ; transocular us suspicion ( preor post - treatment ) of consensus who evaluated : transocular us maximum diameter of the tumour \16 mm or thickness \10 mm ( larger tumours are always enucleated in accordance with the coms guidelines ) [ 5 , 6 , 14 ] ; no pregnancy . all patients signed a patient consent form , after being accurately instructed on the requirements of mr imaging with surface coils and the need to keep a xed gaze and avoid movements during image acquisition . 
mr imaging of the eyes was performed with the patient supine , with a signa the location of the tumour : anterior vs posterior , taking as reference the equator of the ocular globe . 
in the same patient , the contrastenhanced mr fat - saturated image ( d ) shows an enhancing nodule in the retrobulbar space ( arrowhead ) , unrecognisable on the t1 - weighted unenhanced image ( b ) table 2 patient data : information on uveal melanomas , their evolution and correlation with the histological ndings patient age , location shape max . 
one of these four patients underwent enucleation because of extremely rapid growth of the lesion during transocular us follow - up , even though mr did not show any sign of extrascleral extension . 
these intravenous patients underwent enucleation and in all four cases the mr diagnosis was histopathological conrmed evaluation . in 4 / 12 patients the transocular us suspicion of extrascleral extension was induced by the proximity of the melanoma to the optic nerve ; only in two of these cases was extrascleral extension of disease conrmed by histopathology , and correctly diagnosed by mr . in each case , neoplastic tissue and retrobulbar nodules showed marked enhancement after contrast injection allowing for a clear delineation of the lesion with respect to surrounding structures . 
in 5 / 12 patients , we had to repeat some mr sequences because ocular motion artefacts degraded the images ; however , in all patients the images were denitively diagnostic . clinically stable melanomas are usually monitored by clinical and imaging examination and treatment is required only in the case of proven dimensional growth [ 9 ]  . 
in our study , patients who did not undergo ocular enucleation were re - evaluated every 4 months for at least 24 months by transocular us follow - up performed by the same skilled operator who suspected extrascleral extension . regarding the coms studies , transocular us examination shows the same accuracy as histopathology in the evaluation of uveal melanoma dimensions and growth [ 34 ]  . therefore , the absence of extrascleral extension on mr images , the morphological and dimensional stability of the bulbar melanoma / retrobulbar alteration on transocular us follow - up exclude extrascleral extension [ 28 ]  . discussion in our study , we conrmed the prognostic value of the morphology of uveal melanomas . 
mound - shaped melanomas have a large base implant that is usually observed in advanced stages of disease and 4 / 9 cases were found to be positive for extrascleral extension both with mr and histopathology . 
mushroom - shaped morphology , according to the literature , represents an important sign of tumour progression suggesting interruption of the layer that lies between the retina and uvea ( bruchs membrane ) , but this evidence was not conrmed in the single case observed in our study [ 1 , 5 , 9 ]  . retinal detachment , if associated with uveal melanoma , is a sign of progression of disease because it mainly appears in advanced stages of tumour growth ; however , it can be present in small melanomas for individual and still unknown predisposing factors . 
in our study , the only patient with a mr diagnosis of retinal detachment showed extrascleral extension of disease both on mr imaging and histopathology after enucleation , conrming the evidence from the literature [ 5 , 6 , 22 , 26 , 33 ]  . on the other hand , in our experience the difference of average basal diameters of juxtafoveal compared to nonjuxtafoveal melanomas was not signicant , in contrast to the ndings of other authors , who reported that the diagnosis of juxtafoveal melanoma occurs earlier because of the early onset of more severe visual decits [ 5 ]  . extrascleral extension is the most important factor in the choice of the correct therapeutic management , as its presence increases the risk of metastasis ; therefore , according to the coms guidelines , extrascleral extension is an absolute indication for ocular enucleation [ 5 , 14 ]  . 
in our experience , t1 - weighted fat - saturated images obtained after contrast medium injection were the most useful images for the evaluation of extrascleral extension because of the strong contrast between retrobulbar fat ( signal suppressed ) and enhancing melanoma [ 8 , 25 , 32 , 33 , 35 ]  . unenhanced t1w images allowed a better evaluation of orbital anatomy [ 15 , 33 ]  . 
t2 - weighted images proved to be less useful for the evaluation of extrascleral extension because they are more prone to motion artefacts , and have lower signal - to - noise and contrast - to - noise ratios [ 8 , 15 , 33 ]  . 
we had some difculty evaluating extrascleral extension of melanoma in cases of tumour location close to the bulbar insertion of the extraocular muscles , where generally the and muscle enhancement can simulate an extrascleral extension of disease [ 36 ]  . 
venous ectasia / engorgement can be confused with extrascleral extension of disease as it shows marked enhancement after contrast medium injection ; however , in our experience , high - resolution contrast - enhanced 3d - fame images allowed an immediate distinction of ectasia from neoplastic tissue by depicting small serpiginous vascular structures . sclera becomes thinner the greatest difculties encountered in the use of highresolution mr with surface coils were motion artefacts in noncompliant patients , which in many cases induced us to repeat the mr sequences [ 21 , 23 , 25 , 29 , 37 , 38 ]  . 
nevertheless , high - resolution mr of the eye with surface coils provided a correct diagnosis of deep disease extension in all the patients examined . the limits of our study are essentially two : ( 1 ) the patient sample was too small , even considering the rarity of uveal melanoma and the infrequent use of mr examination in patients with more anterior lesions , in which transocular us is often exhaustive , ( 2 ) the lack of histopathological conrmation of the mr data in 7 / 12 non - enucleated patients , without extrascleral extension on mr and followed up with transocular us for at least 2 years ; however , we assumed that the lack of dimensional growth of the melanoma after a long - term transocular us follow - up can be considered a certain sign of absence of extrascleral extension . in our experience , albeit limited , diagnostic evaluation with high - resolution ocular mr in cases of suspected extrascleral extension of melanoma on transocular us allowed destructive surgery ( bulbar enucleation ) to be avoided in 7 / 12 patients , contributing to improve their quality of life . 
high - resolution mr of the orbit proved to be superior to transocular us especially in cases with vascular ectasia , where the mr demonstration of vascular 782 radiol med ( 2014 ) 119 : 775783 structures led to the correct diagnosis . 
similarly , in lesions adjacent to optic nerve or extraocular muscles , the evaluation of the signal pattern and the greater morphological capability of mr compared to us allowed the transocular suspicion to be solved by demonstrating scleral continuity and no extrascleral nodules / plaques . the results of this preliminary study need to be conrmed by clinical trials involving larger patient samples ; however , they suggest that high - resolution mr with surface coils could assume a primary role in therapeutic planning for patients with a transocular us suspicion of extrascleral extension of disease . 
in our single - centre study , we investigated the efcacy of intravenous ( iv ) administration of rt - pa within 4.5 h of stroke onset , in terms of clinical and radiological outcome , using a 3t magnetic resonance ( mr ) scanner in a cohort of patients similar to that of multicentre clinical trials . materials and methods consecutive patients treated with iv rt - pa were compared with an historical cohort of untreated patients ( controls )  . 
inclusion criteria were : ( 1 ) infarction of the middle cerebral artery territory , ( 2 ) eligibility for iv rt - pa treatment , and ( 3 ) 3t perfusionand diffusion - weighted mr imaging and mr angiography performed within 4.5 h and repeated after 57 days . 
growth of the dwi lesion , saved hypoperfused tissue , and clinical outcome was assessed and compared in treated patients and controls . results forty - three patients treated with rt - pa and 69 controls were eligible for the analysis . 
this practical approach aims to treat a higher number of stroke patients at the earliest possible time , also extending the use of thrombolytic treatment to a higher number of stroke centres . 
single - centre studies have demonstrated the usefulness of magnetic resonance imaging ( mri ) in selecting patients for thrombolytic treatment , as well as its feasibility in monitoring rt - pa safety and efcacy [ 9 , 10 ]  . earlier studies have described the efcacy of iv rt - pa up to 6 h after the onset of symptoms , measured by mri and clinical parameters [ 8 , 11 ]  . 
more recently , the safety and efcacy of an mri - based selection protocol stroke treatment within and beyond 3 h compared with standard ct - based treatment have been underlined [ 12 ]  . in that study , the authors concluded that patient selection is more important for a good outcome . than time - to - treatment however , the usefulness of perfusionand diffusionweighted ( pwidwi ) mri mismatch for predicting response to treatment before iv administration of thrombolytic agents has not yet been demonstrated . 
currently , the international guidelines recommend the use of nonenhanced ct or mri in the selection of patient undergoing iv thrombolysis in the time period b4.5 h from the ischaemic event , to exclude an intracranial haemorrhage . 
perfusion mri and the evaluation of the pwidwi mismatch have a key role not yet clearly dened ; in particular , the guidelines say : mri perfusion and diffusion imaging , including measures of infarct in order core and penumbra , may be considered for the selection of patients for acute reperfusion therapy beyond the time windows for intravenous brinolysis . 
these techniques provide additional information that may improve diagnosis , mechanism , and severity of ischemic stroke and allow more informed clinical decision making [ 13 , 14 ]  . the aim of our single - centre study was to evaluate the safety and efcacy of iv thrombolytic treatment administered up to 4.5 h after onset of symptoms by evaluation of clinical parameters and changes in lesion volume on pwi and dwi using a 3t mri unit . materials and methods study population between july 2008 and june 2012 , 361 patients diagnosed with acute ischaemic stroke presented to our emergency department . 
forty - three ( 12 % ) patients were treated with iv rt - pa within 4.5 h from symptom onset and satised the following criteria : ( 1 ) ischaemic cerebral infarction in the territory of the middle cerebral artery ( mca ) , ( 2 ) availability of pretreatment 3t mri with dwi and pwi sequences and mr angiography ( mra ) , ( 3 ) 3t follow - up mri repeated 57 days after symptom onset , ( 4 ) written informed consent of patients or relatives for the necessary diagnostic procedures and therapies . 
patients treated with iv rt - pa were compared with a historical cohort of 69 untreated patients ( controls )  . among the control group , 43 ( 62 % ) patients were selected among consecutive patients admitted to our stroke unit between january and september 2006 , before iv rt - pa administration had been authorised as the standard treatment for acute ischaemic stroke in our hospital . 
all these patients were admitted between 3 and 4.5 h from the onset . the remaining 26 ( 38 % ) patients were selected among patients admitted to our hospital between 3 and 4.5 h from stroke onset , between october 2006 and june 2008 , before iv rt - pa treatment within 4.5 h from symptom onset had been authorised by our local ethics committee . 
all patients were admitted to our stroke unit and managed according to the current guidelines [ 615 ]  . patient information was prospectively entered into a stroke registry by the stroke teaiv rt - pa treatment was administered according to the international guidelines ( 0.9 mg / kg , max 90 mg dose , 10 % in a bolus and the remainder in a 60 - min infusion ) [ 1 , 7 ]  . 
stroke severity was radiol med ( 2014 ) 119 : 767774 assessed with the national institutes of health stroke scale ( nihss ) at admission , 24 h , discharge , and 3 months after stroke onset . 
for functional outcome , a modied rankin scale ( mrs ) score of 2 or less at 3 months indicated functional independence . image acquisition voxel 320 9 157 ; axial t2 - flair imaging all mr images were obtained with a 3t mr scanner ( achieva , philips medical systems , best , the netherlands ) , with an 8 - channel sense head coil . 
this procedure was performed on images acquired on admission and at the 24 h follow - up mri examination . colour maps of relative cerebral blood volume ( rcbv ) , time and time - to - peak ( ttp ) were obtained by relative cerebral blood ow ( rcbf ) , mean transit ( mtt ) processing the initial perfusion images with a curve t algorithm using the software on the philips viewforum console . 
a follow - up mra was performed after 57 days in patients with evidence of intracranial stenoocclusive disease on the baseline scan for vessel patency . the presence of any haemorrhagic transformation on follow - up mri was recorded . 
symptomatic haemorrhagic transformation was dened according to the national institute of neurological disorders and stroke denition ( i.e. , any intracranial haemorrhage that was not seen on a previous imaging scan and any decline in neurological status )  . statistical analysis descriptive statistics was used for baseline and demographic data in both treated patients and controls . 
haemorrhagic transformation of the ischaemic lesion was observed in 9 out of 43 ( 21 % ) treated patients and in 5 out of 69 ( 7 % ) controls ( p = 0.02 ) ( table 2 )  . 
no stroke recurrences or deaths occurred in the treated group , while the 3 - month follow - up revealed two deaths in the control group . regression analysis performed in untreated patients between mean pwi volume and nal dwi lesion volume ( as measured on the follow - up mri scan ) showed that rcbv and rcbf were the haemodynamic parameters which better correlated with the volume of the nal radiol med ( 2014 ) 119 : 767774 fig . 
hence , signicantly increased rates of haemorrhagic transformation of ischaemic lesions were associated with rt - pa treatment which , however , did not determine a clinical worsening or death in any patient . contrarily to the control group , a signicant clinical improvement was observed in treated patients at each follow - up time point , while functional outcome did not differ signicantly in the two groups after 3 months . although the efcacy of thrombolysis has been evaluated extensively using clinical scores , only a few studies have analysed the effects of thrombolytic treatment through mri evaluation . our results are in accordance with the two largest multicentre studies that used mri to select patients for thrombolysis : the defuse [ 16 ] and the epithet [ 17 ] studies . 
our single - centre study has the advantage of not being subject to the potential bias of multicentric studies . in comparison to defuse [ 16 ] , which was the rst prospective multicentre study to perform mri with diffusion , perfusion , and mra sequences , before and after the administration of a thrombolytic agent in consecutive patients treated 36 h after symptoms onset , our study was performed using a 3t mri systethe second found that multicentre the epithet [ 17 ] , study , 772 radiol med ( 2014 ) 119 : 767774 fig . 
follow - up dwi ( g ) and mra ( h ) after iv thrombolysis show recanalisation of the mca with a nal infarction area that is smaller than the initial dwi lesion alteplase was nonsignicantly associated with lower infarction growth compared to the placebo group ( p = 0.054 ) , but reperfusion was more common with alteplase than with placebo . 
not only does our study conrm these results , but it also demonstrates a signicant reduction in growth of the ischaemic area , as measured by dwi ; these results can be explained by the better quantication of the infarcted area and the ischaemic penumbra using a 3t mri scanner . recently , nagakane et al . 
 [ 18 ] demonstrated that , using coregistration of diffusion and perfusion mri maps , it is possible to evaluate the effects of alteplase vs placebo in a more accurate way : in particular , these authors demonstrated that in patients treated with rt - pa the growth in volume of the infarct , measured in dwi , is signicantly lower than in untreated patients . in our study , we were able to obtain similar results without coregistration . 
a metanalysis elaborated from these studies demonstrated that instead of an increased rate of haemorrhagic transformation , iv rt - pa administration has reduced signicantly the incidence of death / dependence both in patients treated within the rst 3 h and patients treated between 3 and 6 h after stroke onset . our observations conrm previous ndings reported in the literature . 
 [ 11 ] observed that , when compared to untreated patients , stroke patients who received iv rt - pa within 6 h after stroke onset had a higher percentage of vessel recanalisation and a better clinical outcome , as measured by mrs . 
the rate of recanalisation was 66.2 % in treated patients , and 32.7 % in controls , which appears to be similar to our ndings ( 65 and 27.5 % , respectively )  . 
unlike previous evidence [ 2 , 7 , 17 , 21 ] , we could not demonstrate a better functional outcome in patients treated with rt - pa , when compared to controls . more severe strokes ( i.e. , higher baseline nihss scores and larger dwi lesion volumes ) in treated patients versus controls , as well as our relatively small patient population , could explain why in our series similar proportions of treated patients and controls were functionally independent at 3 months ( 62 and 65 % , respectively )  . 
however , not all authors agree upon using volume and ow measures to estimate the mismatch and to predict the volume of nal infarct , suggesting ttp or mtt as preferable haemodynamic parameters [ 2629 ]  . our study shows that iv rt - pa administration can modify the natural evolution of the ischaemic lesion . 
in particular , compared to untreated patients , stroke patients treated with iv rt - pa showed a signicantly smaller enlargement of the initial core lesion , as shown by changes in the volume of the 3t dwi lesion , and a signicant reduction of hypoperfused tissue infarction . although we could not demonstrate a signicantly better functional outcome in treated patients compared with controls , treated patients had a signicant improvement in the nihss at each follow - up time point , and nearly twothirds of them were functionally independent at 3 months . to our knowledge , this is the rst series reporting the efcacy of iv rt - pa administration within 4.5 h from acute ischaemic stroke symptom onset evaluated by combined clinical and mri evaluation using a 3t mri unit . 
boulter zoran rumboldt giuseppe bonaldi mario muto alessandro cianfoni received : 27 august 2012 / accepted : 20 january 2013 / published online : 15 february 2014 ( cid : 2 ) italian society of medical radiology 2014 abstract percutaneous spine procedures can be performed with computed tomography ( ct ) guidance . 
the use of ct guidance is cumbersome for procedures where an oblique needle trajectory is imposed by the spatial orientation of the spine , often requiring complex needle triangulation relative to the true axial scan plane . 
a combination of variable ct gantry tilt , and strategic bolster placement under the patient can be used to obtain optimal imaging planes for guidance along the desired needle trajectory . 
we describe our clinical experience using the modied procedure relative to the conventional technique , and provide representative examples . keywords spine interventions ( cid : 2 ) spine procedures ( cid : 2 ) ct guidance ( cid : 2 ) gantry tilt d . 
cianfoni ( & ) neuroradiology , neurocenter of southern switzerland , ospedale regionale di lugano , via tesserete 46 , 6900 lugano , switzerland e - mail : alessandro.cianfoni@eoc.ch introduction many diagnostic and therapeutic percutaneous spine procedures can be performed under imaging guidance , most commonly with uoroscopy or computed tomography ( ct )  . ct guidance has some advantages over uoroscopy , such as cross - sectional anatomical visualization , precise target localization , and superior depiction of osseous and soft tissue structures [ 1 ]  . 
it is limited , however , by the lack of panoramic view and by more cumbersome procedural aspects , due to the necessity for intermittent scans to verify correct needle placement . 
an additional limitation of ct guidance arises from the fact that the ideal needle trajectory often has a cranio - caudal ( cc ) obliquity different from the true axial scan plane , due to the complex anatomical spatial orientation and curvatures of the spine . with conventional axial ct guidance , the operator must scan a large anatomical region to account for the entire needle course , and then must triangulate the needle trajectory over multiple images to predict the nal tip position following advancement . 
this might lead to cumbersome , lengthy , and at times inaccurate needle placement . as a solution to this commonly encountered problem , we describe a simple procedural modication using two features of modern ct scanners , gantry tilt and laser beams , for performance of ct - guided percutaneous spine procedures . the ct gantry can be tilted in the cc direction parallel to the desired needle trajectory , thereby simulating , at least in part , the en - face or bulls eye views , commonly used with uoroscopic guidance . 
we describe the ct - guided percutaneous access to the l5s1 disc , the sacral wing long - axis , the upper thoracic vertebral body , as well as the cervical anterior vertebral elements and discs , as representative radiol med ( 2014 ) 119 : 750757 procedures to illustrate this technique . 
we also describe our clinical experience using the modied technique in a series of patients relative to the conventional method . technique localizing ct scan : choosing the degree of gantry tilt the patient is positioned prone or supine on the ct cradle , with a metallic grid applied over the skin of the presumed region of needle entry . 
the localizing ct scan is then performed through the region of interest with the chosen gantry tilt , to select the single oblique axial image that shows the entire desired needle path , from the skin entry to the target . 
the size and shape of the bolster can be modied by adding or removing rolled or folded sheets and blankets , depending on the requirements of the individual procedure and body habitus . needle - gantry alignment the ct laser beam projects a planar guidance beam aligned with the tilt of the gantry onto the patient . 
most ct systems project two parallel beams in the same planar orientation , one inside the gantry , corresponding to the scan plane , and another a xed distance away . 
both of these beams can be used interchangeably , however , we prefer to use the beam just outside of the bore to perform the procedures , as it allows greater freedom of movement for the operator . 
the right - to - left needle trajectory is left to the experience and ability of the operator as usual , guided by the localizing ct image of interest . procedural imaging guidance intermittent ct scans are obtained throughout the procedure between needle advancements . 
typically three overlapping low - dose images are acquired at each check , with the central image showing the entire length of the inserted needle , from the skin entry to the tip , with one slice above and one slice below in which no needle is visualized . 
of these , 47 were performed in the lumbosacral , 31 in the thoracic , and 15 in the cervical region . procedures performed included vertebral body access radiol med ( 2014 ) 119 : 750757 fig . 
a bolster under the lower abdomen ( c ) attens the lordotic curvature and reduces the l5s1 disc plane obliquity ; under the chest ( d ) attens the thoracic kyphosis and reduces the upper thoracic pedicles obliquity ; under the hips ( e ) increases the lordosis and brings the long - axis of the sacrum closer to the axial plane . the desired needle path for spinal accesses can be paralleled by gantry tilt ( solid lines on c e ) relative to straight axial orientation ( dashed lines on c e )  . 
f gantry - needle alignment , with laser beam precisely bisecting the needle at the hub and the skin entry point . maintaining this alignment keeps the needle in plane and allows visualization of the entire needle throughout its trajectory on a single ct slice through a pedicular or extra - pedicular approach , access to foramina , discs , epidural space , facet joint spaces , sacro - iliac joint spaces , dural sac , paraand peri - vertebral spaces for the purpose of tissue biopsies , drainages , pain management blocks , radiofrequency denervation , percutaneous disc decompression , and cement augmentation procedures . with the conventional method , an initial axial planning ct scan was performed to cover both the needle access point and desired needle target ( generally at least two vertebral body segments )  . 
the true axial scan plane was used to plan the right - to - left component of needle access . sagittal multiplanar reformatted ( mpr ) images were used to determine the cc distance from the skin access site to the nal needle target . 
following this , adjustments to needle trajectory were made using triangulation in the true axial and mpr sagittal images . gantry tilt after initiating the gantry tilt and gantry - needle alignment technique , 315 procedures were performed over a 2 - year period . 
b straight axial ct images of the l5s1 disc ( dashed lines on a ) , which includes portions of l5 and s1 vertebral bodies on the same ct image . 
disc access can be performed in this plane , with the entire needle visualized on a single ct slice in each case , gantry - needle - target alignment was performed using the technique described in detail previously . results overall , the gantry - needle alignment technique required shorter z - axis ct coverage at each localizing scan . 
operator condence in performing complex accesses , such as cervical antero - lateral , paramaxillary , or high thoracic vertebral bodies , was also improved in the gantry - tilt technique . 
the procedure planning time was roughly the same for both techniques . technical challenges difculties were encountered with both techniques due to patient motion , procedure - related discomfort , or anatomical obstructions to access . 
patient movement interfered with the procedure when it occurred either between the scout view and localizing scan , between localizing scan and skin radiol med ( 2014 ) 119 : 750757 fig . 
b tilting the ct gantry allows the longaxis of the sacrum to be visualized on a single image . c bilateral long - axis sacral access , with the entire needle visualized on a single ct slice marking , or during the procedure . 
the incidence of patient motion was approximately equal with both techniques . among the gantry - tilt procedures , patient motion necessitated a new localizing ct scan with new scout view , gantry tilt , and skin marking in 12 cases . 
in two of these patients , pain occurred during a high thoracic sympathetic block with access to the perivertebral space , which resolved shortly after the procedure in both cases . 
in a third patient pain occurred during an intervertebral disc biopsy , which reportedly persisted for over 1 month post - procedure without motor or sensory decit . follow - up of all patients was performed with a phone call at 24 h and 1 week post - procedure , and / or with a follow - up visit 4 weeks post - procedure , depending on the type of percutaneous spine procedures are often characterised by oblique needle accesses in the cc and right - to - left directions discussion follow - up 755 radiol med ( 2014 ) 119 : 750757 fig . 
note that the pedicles are imaged obliquely , and the plane includes both the disc space , ( arrowheads ) and the vertebral body ( black asterisk ) of the level above . b by placing a wedge beneath the chest and angling the ct gantry , the scan plane now parallels the needle alignment for vertebral body access . c trans - pedicular thoracic vertebral body access , with entire needle course visualized . d extra - pedicular thoracic vertebral body access , with gantry - needle - target alignment to account for the complex spatial anatomy and curvatures of the spine . 
in fact , operators using ct guidance for spine procedures often need to perform complex triangulations to calculate the oblique needle path across multiple axial ct slices , to predict nal needle tip location after a certain depth of needle advancement . 
more recently , application of gantry tilt has been described for ct - guided sacroplasty to better depict the path of the needle and improve the safety of the procedure [ 11 ]  . 
while some operators may commonly use some of the technical additions suggested in this report , to the best of our knowledge a combined gantry tilt and gantry - needle alignment technique has not been formally described for a wide range of ctguided spine procedures . 
d the ct gantry can be tilted to different degrees ( dashed lines ) through a combination of neck exion / extension and bolsters , depending on the intended target ( clivus to c3 )  . e localizing scan obtained during iv contrast injection to depict relevant vascular anatomy . 
tilting the gantry differently , and aligning the needle to the gantry , from the same skin entry point the needle can be directed to c1 ( f ) and c2 ( g )  . 
as an example , an l3 pedicular access to the vertebral body can be modied with a minor degree of gantry tilt if the target is in the inferior versus superior portion of the vertebral body . 
the use of gantry - needle alignment holds valid for each ct - guided spine procedure , whether a gantry tilt is applied or not , as it reproduces , at least in part , the en - face or bulls eye technique used in uoroscopy - guided procedures . we have found that adherence to these recommendations increase the versatility and the precision of ct guidance , ensures greater needle control , while obviating the need for complex , time - consuming , and often unreliable needle triangulations . 
gantry - needle alignment using the ct gantrys laser beam also greatly reduces the number of ct sections required at each ct check ( usually 3 slices are sufcient , compared with an average of 12 slices with the standard technique ) , and consequently has the potential to reduce procedure time and radiation dose . radiol med ( 2014 ) 119 : 750757 conclusions the simple addition of gantry tilt and gantry - needle alignment for ct - guided spine interventions allows precise access to the target while minimizing uncertainty of needle trajectory , with a potential to improve procedure ease and accuracy , reduce procedure time and radiation exposure . conict of interest daniel j . 
there are currently many different therapeutic strategies for these rare tumours and combining several different multi - modality strategies have not proved to have superior long - term clinical results . 
in particular , the role of radiation therapy , treatment - related side effects and the use of modern radiation treatment techniques will be discussed . materials and methods a comprehensive literature search was conducted using pubmed in january 2011 . relevant international articles published from january 1980 to january 2011 were assessed . 
orecchia istituto europeo di oncologia , milan , italy by the authors and selected for inclusion based on relevance to this article . conclusions the addition of radiation therapy ( rt ) to wide surgical excision for rps has improved local control rates when compared with surgery alone . 
emerging therapies that employ charged particles ( such as protons and carbon ions ) are expected to be superior in sparing of normal tissues and efcacy over conventional photon therapy radiation , due to their physical and radiobiological properties . keywords retro - peritoneal sarcoma ( cid : 2 ) imrt ( cid : 2 ) proton therapy ( cid : 2 ) carbon ion therapy introduction soft tissue sarcomas ( sts ) are rare , heterogeneous mesenchymal neoplasms arising from the remnant embryonal mesoderma . 
the common histological variants include liposarcoma , leiomyosarcoma , and malignant brous histiocytoma ( mfh ) , which make up 40 , 30 and 10 % of all rps [ 2 ]  . biological behaviour and histological subtypes the distribution of histologic subtypes in rps differs from that of sts arising at other anatomic sites . 
molecular test results should therefore only be interpreted in the context of the clinical and pathologic features of a sarcoma . improvements in diagnosis with the use of magnetic resonance imaging ( mri ) radiographic imaging is a key component of the evaluation of a patient with a retroperitoneal mass . 
the preferred diagnostic study is a computed tomography ( ct ) scan of the abdomen and pelvis to evaluate the primary site , as well as a chest ct to rule out metastatic disease to the lungs . mri rarely adds important information . 
it can also detect metastases to the liver or peritoneuthe radiographic appearance of the primary tumour on ct can also offer clues as to the histologic subtype and grade , which may guide decisions as to the need for pre - treatment biopsy as well as treatment . 
however , the ability to differentiate benign soft tissue from low or intermediate grade sarcomas is limited , and pet and pet / ct are not routinely recommended for the initial workup of a soft tissue mass . the median survival duration is 74 months and the median 5 - year overall survival ( os ) rate is 3658 % [ 4 ]  . 
clinical symptoms are nonspecic and include vague abdominal discomfort ( from the compression on the gastrointestinal system and neural structures ) , weight loss , early satiety and swelling of the lower extremities . 
a surgical resection is considered oncologically adequate only when the tumour is macroscopically completely excised and negative microscopic margins ( r0 ) have been achieved . this is possible only when diagnoses are made in the early stages . 
this may be explained by the high risk of residual disease ( microscopically and macroscopically ) leading to a high risk for local recurrence ( 3782 % at 5 years )  . 
the role of frontline aggressive surgery and how this may impact on the need for adjunctive radiotherapy is the subject of clinical trials [ 7 ]  . radiotherapy ( rt ) is important for tumour eradication and yet it is difcult to deliver high doses ( [ 60 gy ) to the tumour and keep within the dose tolerance of the adjacent organs ( small bowel , spleen and bone marrow ) [ 9 , 10 ]  . 
charged particles therapy is an emerging therapy for rps and currently only preliminary single - centre experiences have been reported [ 11 ]  . although commonly applied for rps in either the preoperative or postoperative setting , there has been a lack of level - 1 evidence for rt specically in the management of rps . 
table 1 shows the results from published studies involving radiotherapy for the treatment of rps . despite the improvement in diagnostic and therapeutic strategies , patients aficted by rps have a poor prognosis . the results of systemic as well as intraperitoneal chemotherapy are disappointing [ 12 ]  . 
preoperative regional 792 radiol med ( 2014 ) 119 : 790802 radiol med ( 2014 ) 119 : 790802 794 radiol med ( 2014 ) 119 : 790802 hyperthermia with systemic chemotherapy is a therapeutic option for high - risk primary or locally recurrent tumours and is the subject of clinical studies . biological marker - directed therapy tumours ( gists ) more recently , a number of targeted therapies have shown promising results in patients with certain histological types of advanced or metastatic sts ( outside of the retroperitoneal space )  . 
imatinib and sunitinib have shown efcacy in patients with advanced and / or metastatic sts other than gastrointestinal stromal ( alveolar soft part sarcoma , chordoma , pigmented villonodular synovitis / tenosynovial giant cell tumour and solitary brous tumour / haemangiopericytoma )  . 
sirolimus has shown promising results in patients with metastatic perivascular epithelioid cell tumours ( pecomas ) and in patients with recurrent lymphangioleiomyomatosis or angiomyolipomas . bevacizumab in combination with temozolomide was well tolerated and effective in patients with locally advanced , recurrent , and metastatic hemangiopericytoma and malignant solitary brous tumour . this review analyses the available published literature and discusses the role of radiation therapy , the related side effects and how the modern radiation treatment techniques may improve the care of patients with sarcomas of the retroperitoneum . materials and methods a comprehensive literature search was conducted using pubmed in january 2011 . 
the keywords for search purposes were : retroperitoneum , sarcoma , radiotherapy and radiation therapy . the search was limited to articles published in english . only peer - reviewed publications dealing with patient outcome were selected . 
rt planning studies ; short communications ; publications without details on rt technique ; publications on paediatric timing , dose , or tumours ; and publications concerning desmoid tumours , dermatobrosarcoma sarcoma , round - cell sarcoma , and angiosarcoma were excluded , because these histopathology types also are excluded from the american joint committee on cancer ( ajcc ) staging systerhabdomyosarcoma , round - cell sarcoma and primitive neuroectodermal tumour were not included protuberans , kaposi soft because of their well - known radiosensitivity . 
finally , articles were selected for this review according to the following criteria : series with a minimum of ten patients presenting rps with at follow - up and least 24 months of addressing various aspects of preoperative , postopintensity - modulated radiotherapy ( imrt ) , erative , intraoperative radiotherapy ( iort ) , intraoperative electron radiotherapy ( ioert ) , brachytherapy ( bt ) , proton therapy and ion therapy . the outcomes of interest were tumour control and toxicity of radiation therapy . in total , 425 reports related to radiation for rps had been retrieved from the initial pubmed search . 
all articles were read in full by the authors and selected for inclusion based on relevance to this article . results preoperative radiation therapy although the majority of published data are about postoperative rt , preoperative rt has several potential advantages . 
 [ 14 ] enrolled 35 consecutive patients with resectable rps who received preoperative external - beam radiotherapy ( ebrt ) with concomitant doxorubicin in a dose escalation trial of 1850.4 gy , followed by surgery plus iort ( 15 gy )  . 
the chemoradiation regimen was completed in an outpatient fashion in 31 of 35 patients ( 89 % ) , while four patients ( 11 % ) required hospital admission for dehydration and / or anorexia . 
grade 34 gastrointestinal toxicities recorded were : nausea ( no vomiting or diarrhoea ) in three patients ( 20 % ) at the 46.8 gy level , and in two patients ( 18 % ) for 50.4 gy level , while patients at the 41 gy level did not record any toxicity . 
 [ 8 ] analysed 83 patients treated at md anderson cancer centre ( mdacc ) and found no difference in local control regarding rt timing with higher doses ( [ 50 gy ) of ebrt or with the specic use of iort . 
 [ 19 ] combined the data from the two prospective trials conducted at the princess margaret hospital ( pmh ) and mdaac to analyse long - term recurrence and survival rates . 
in both prospective studies , the selection of patients who received iort or bt was based on the surgeons intraoperative assessment . fifty - four patients completed the planned preoperative rt and underwent r0 or r1 resection with curative intent . 
although both bt and ioert offered similar local control , bt was associated with signicantly higher toxicity rates . in 2003 , the american college of surgeons oncology group opened the only multi - institutional phase iii trial ( acosog z9031 ) to determine the effect of preoperative rt on patients with untreated primary rps , but unfortunately it was closed in 2005 due to poor patient accrual . this randomized controlled trial was needed to obtain denitive data about the role of rt . intraoperative radiation therapy ( iort ) iort uses low energy x - rays directed to the tumour bed at the time of surgery , commonly by a mobile linear accelerator , which produces electrons ( ioert )  . 
iort may allow delivery of high rt doses to high - risk areas while limiting small bowel toxicity by direct shielding during iort delivery . to date , a trial performed between 1980 and 1985 at the national cancer institute represents the single published randomized control trial involving rt for patients with rps [ 20 ]  . 
these patients were randomly compared with 15 patients who received intravenous misonidazole ( a radio - sensitizer ) plus ioert ( 20 gy ) , administered using abutting or overlapping elds with high - energy electrons of 1115 megaelectron volts ( mev ) , followed by postoperative ebrt ( 3540 gy )  . 
rt morbidity was more signicant in the iort arm : 60 % of the patients in the ioert arm versus 5 % in the ebrt arm 796 radiol med ( 2014 ) 119 : 790802 developed peripheral neuropathy ; 13 % of patients in iort - ebrt arm versus 50 % in ebrt without iort developed enteritis . 
 [ 21 ] reported on 67 patients who underwent surgery and ioert ( median dose , 15 gy ) ; 45 patients underwent additional postoperative ebrt ( median 45 gy ) and the 5and 10 - year actuarial loco - regional control rates were 40 and 33 % , respectively . these authors emphasised that dose escalation with the combination of ioert and postoperative ebrt decreased the risk of recurrence , after macroscopically complete resection . 
furthermore , the 5 - year local - control rate within the ioert eld was 72 % regardless of the resection status , but increased to 84 % after gross resection and 95 % after complete resection . 
intensity - modulated radiation therapy ( imrt ) utilising a simultaneous integrated boost has been used for further dose escalation to high risk margins [ 6 ]  . postoperative radiation therapy the natural history of rps and the high risk for local recurrences support the use of postoperative local treatments . 
postoperative rt is widely used for its advantages that it does not delay the surgery and it can provide selective treatment in the high - risk group depending on biopsy results or when the complete resection is not possible . 
therefore , it is performed mainly in the cases of high grade , resection margins . resection or incomplete inadequate therefore , rt in addition to surgery is widely accepted in the management of rps . 
thirty - six of 45 patients who had undergone macroscopic total resection and postoperative rt had a delayed time to local recurrence compared to patients treated with surgery alone ( p = 0.06 ) for radiation doses higher than 35 gy . 
when only patients with in - eld difference reached statistical signicance ( p = 0.02 ) , suggesting a theoretical role of further dose escalation and boosting the margins at higher risk of recurrence , to improve local control and survival . recurrences considered , stoeckle et al . 
 [ 25 ] reported on 21 patients ( 19 of 21 irradiated postoperatively ) and found radiation doses [ 55.2 gy yielded 75 % of local control , whereas 62 % was obtained for doses \55 gy . 
the outcomes of postoperative rt for rps and the experiences extrapolated from extremity sarcomas [ 2628 ] support a theoretical optimal tumour killing dose 60 gy or higher , to achieve a signicant improvement of local control . 
the ability to deliver such high doses in the retro - peritoneum is limited by the close proximity of radiosensitive organs such as the small bowel , which cannot tolerate more than 45 gy [ 29 ]  . in a postoperative setting , dose escalation is possible by delivering a boost in a focal area at high risk for local recurrence . 
different delivery techniques have been suggested to overcome the limit related to bowel toxicity , targeting the dose selectively to areas at risk for tumour recurrence . discussion levels of evidence of current protocols as opposed to limb sarcoma , adjuvant rt in rps is not considered a standard , since no randomised trial has addressed the question of surgery combined with adjuvant rt , compared with surgery alone . 
in a multi - institutional study where surgery for rps was classical ( limited to tumour only ) , adjuvant postoperative rt reduced the risk of local relapse by a factor of 3 [ 5 ]  . radiol med ( 2014 ) 119 : 790802 level 2a : based on lower level evidence , there is uniform consensus that the intervention is appropriate for resectable disease surgery iort , biopsy should be performed ( to exclude gist or desmoid tumours ) , surgery ? postoperative radiotherapy chemotherapy . 
if a biopsy was not done , further management will be guided by intraoperative frozen section ( i.e. , in the case of iort ) or nal pathology evaluation . for unresectable or stage iv disease preoperative chemotherapy , radiotherapy or combination may be considered to downsize the tumour and surgery may be considered when the tumour becomes resectable . otherwise , management will be palliative in nature and be guided by patients symptoms and performance status . level 2b : based on lower level evidence , there is consensus that the intervention is appropriate after biopsy , preoperative rt may be considered . 
if the patient had preoperative rt , postoperative boost rt is recommended . highly selected patients ( high risk factors ) with r0 resection may be offered postoperative rt . several factors have contributed to a lack of quality evidence regarding optimal management . 
due to the rarity of this disease and the controversial role of postoperative rt , it would be difcult to conduct a randomised study radiodirectly comparing three - dimensional conformal therapy ( 3dcrt ) , imrt and iort or preand postoperative rt . 
furthermore , it is also unknown how the benet might vary among treatment modalities regarding different disease stages , characterised by tumour size , grade , and lymph node involvement . 
although there have been reports on several cohort studies or case series based on singleinstitution experiences with surgical treatment and rt , it is unclear to what extent these ndings from small numbers of patients can be generalised to most patients with rps . there may be substantial variation among surgical ( margin status ) and radiotherapy techniques ( treatment volumes , rt dose ) in the various series . radiation therapy - related toxicity ( comparison among different approaches ) preoperative rt has been associated with lower morbidity rates compared to postoperative rt . 
 [ 18 ] reported on 37 patients treated at the massachusetts general hospital with preoperative rt . of the 25 patients who received a boost with iort , four patients had severe side effects . 
out of these patients , three ( 12 % ) received iort to 1020 gy and experienced gastrointestinal / genitourinary toxicity versus 8 % of whole series treated with preoperative ebrt ( median dose 45 gy )  . 
patients who received ebrt doses up to 50 gy did not have an increase in the rates of complications . the trials from the princess margaret hospital and the md anderson cancer centre are informative because acute toxicities of preoperative rt were prospectively and separately recorded from other toxicities [ 14 , 31 ]  . 
preoperative ebrt was delivered at a median dose of 45 gy and associated with acute toxicity scores of the radiation therapy oncology group ( rtog ) grade b2 in all patients . 
wound complications were observed in 5 % of patients , whereas acute toxicity of rtog grade c3 was recorded in 39 % of patients after receiving surgery and / or bt . 
moreover , 58 % of patients who underwent bt needed hospitalisation with two deaths and one life - threatening illness owing to upper abdominal bt . consequently , the upper abdominal bt was subsequently excluded from the treatment protocol . regarding radiation tolerance of healthy tissues , krempien et al . 
 [ 21 ] recorded grade 2 or higher late toxicity in 21 % of the patients : 13 % of acute gastrointestinal morbidity , 4.5 % gastrointestinal stulae , 6 % small bowel stenosis and 7 % neuropathies . 
in general , severe gastrointestinal morbidity results in patients treated with iort plus postoperative ebrt using a dose of 4555 gy [ 20 , 798 radiol med ( 2014 ) 119 : 790802 21 , 32 , 33 ] , and drastically decreased for doses \45 gy . such low doses ( \45 gy ) , on the other hand , even if well tolerated by the small bowel , are not sufcient to have a tumoricidal effect [ 20 ]  . 
in the randomised study from the national cancer institute , all patients received the same dose with ioert boosts ( 20 gy ) and the rate of peripheral neuropathy was 60 % . 
conventionally , the radiation beam arrangements that have been employed are antero - posterior and oblique elds using computed tomography ( ct ) treatment planning to conform to the target volume and shield the organs at risk . 
co - registration between pre - surgery and post - surgery ct should be done when possible to facilitate the delineation of the posterior involvement of the tumour in the retroperitoneal space . 
the retroperitoneal surface at higher risk of recurrence is dened as the contact area between the resected preoperative gtv and remaining adjacent organ structures ( ipsilateral psoas muscle , ipsilateral muscles of the posterior abdominal wall , pre - vertebral surface around the great vessels )  . 
1 planning ct scan showing a typical gross tumor volume for a typical retroperitoneal sarcoma ( rps ) radiol med ( 2014 ) 119 : 790802 rt and includes more of the intestinal cavity . 
imrt allows delivering of differential doses to different areas at risk , selectively escalating the dose , and hopefully local control . preoperative imrt has the potential to further improve the therapeutic index by permitting dose escalation to the area of the tumour while minimising the dose to normal tissues at risk for radiation toxicity [ 13 ]  . 
it is possible to reduce overall treatment time using an integrated boost concept with simultaneously increased dose per fraction in parts of the target volume which are at increased risk for incomplete resection during planned surgery . 
other than grade 3 nausea / vomiting , no other severe acute toxicity was noted in 16 patients . however , the small patient numbers and short follow - up period prevented assessment of long - term toxicity to peripheral nerves . 
 [ 13 ] described a novel approach for the denition of the ctv for liposarcoma of the retroperitoneuthe suggested ctv included only the region between the tumour and the posterior abdominal wall . 
eighteen consecutive patients were irradiated preoperatively with the imrt technique : the complication proton beam radiotherapy ( pbrt ) : protons are charged particles that deposit most of their energy at a depth proportional to their energy , resulting in the characteristic dose distribution known as the bragg peak . 
based on qualitative analysis of normal tissue effects in the clinic ( quantec ) benchmarks , the dosimetric advantage of proton therapy may be less gastrointestinal and genitourinary toxicity [ 37 ]  . mgh reported a study of pbrt and / or imrt ( median dose of 50 gy ) with ioert ( single fraction 1020 gy ) in 28 patients with rps [ 38 ]  . 
after a median follow - up of 33 months , only two patients ( 10 % ) with primary disease experienced local recurrence ( lr ) , while three patients ( 37.5 % ) with recurrent disease experienced local recurrence . 
this strategy may minimise radiationrelated morbidity and reduce local recurrence , especially in patients with primary disease . current trials in proton therapy at the moment , there are two active trials in north america to address the issue of proton therapy in rps . 
the university of florida is assessing treatment feasibility of preoperative proton therapy for resectable intermediate or high - grade retroperitoneal sarcoma . high linear energy transfer ( let ) radiotherapy sarcoma has been traditionally considered to be resistant to conventional let radiation . 
the reason may be from the 800 radiol med ( 2014 ) 119 : 790802 mechanism of cell damage : low let radiations generate injuries indirectly through the formation of free radicals and / or directly with ionizations in the patient . 
the formation of radicals depends heavily on oxygenation level . consequently , the sensitivity of tissue to low let radiation depends on the degree of blood supply to the tissues and also on the types of tissues , presence of free radical scavengers and cell cycle phase . the degree of ionisation of low let radiation is sparse in tissue ; the mean distance between two ionisation events is far greater than the diameter of deoxyribonucleic acid ( dna ) double helix . 
the physical advantages are low dose deposition in the entry channel , followed by a steep dose deposition in the target region ( the bragg peak ) , and then a sharp dose fall - off with no exit dose . 
from the biological point of view , heavy ions can produce dense ionisations within the tumour , resulting in multiple clustered dna breaks , which are more difcult to be repaired by the intrinsic cellular repair pathways . 
minor differences between the two are a sharper lateral penumbra and a dose tail beyond the bragg peak for the carbon bealike all charged particles , carbon ions have a variable let that increases with the depth of penetration into tissues . 
carbon ions therefore produce sparse ionisations in the entrance channel and dense ionisations in the bragg peak . in the last part of their track , carbon ions release a higher dose and results in a higher effectiveness in the target volume ( compared to protons )  . 
carbon has an rbe ratio in the bragg peak to ions . rbe in the plateau region higher therefore , carbon ion radiotherapy ( cirt ) has to combine the optimal dose conformality of potential proton therapy with the increased local control of neutron therapy without the increase in unwanted side effects of the latter [ 3941 ]  . 
moreover , cirt possesses various biologic advantages such as high let radiation , decreased oxygen enhancement ratio ( oer ) , diminished capacity for repair of sublethal and potentially lethal damage , and diminished cell cycle - dependent radiosensitivity when compared with those observed with low let radiation . than other from 1993 , cirt has been performed at the national institute of radiological sciences ( nirs ) in chiba , japan . the optimal dose for inoperable , residual or recurrent bone and sts has been determined within dose escalation trials to be at 70.4 gye ( gray equivalent ) in 16 fractions over 4 weeks [ 4244 ]  . 
therefore , cirt could be considered for patients with rps with residual macroscopic disease after surgery due to inltration of surrounding organs or unresectable tumours . existing and planned carbon ion facilities in asia and europe are activating clinical protocols in rps and more time is needed to verify these preliminary experimental data . conclusions a multidisciplinary team with expertise in sts should manage all patients with rps . 
preoperative rt is preferred to postoperative rt especially in patients with high - grade or intermediate - grade rps and for selected cases of lowgrade rps ( e.g. , extremely large or appearing unresectable or borderline resectable )  . 
for patients with either intermediateor high - grade histology or positive margins who have undergone surgical resection without preoperative rt , postoperative rt is a reasonable option . iort with / without preor postoperative ebrt is also a reasonable option , especially for patients with tumour close to critical organs or gross residual disease after surgery . new types and delivery techniques in rt could further improve patient outcomes . 
due to their physical and radiobiological properties , charged particle rt ( such as protons and carbon ions ) are expected to be better in tissues and with higher efcacy than sparing normal radiol med ( 2014 ) 119 : 790802 conventional photon therapy rt . 
however , in the absence of a randomized controlled trial , it is impossible to determine whether any putative improvement in tumour outcome is a consequence of selection bias or other confounding factors . 
in light of the growing awareness of the issue of ionising radiation dose and the possible risk for the individual and the population , there is a need for radiologists , medical physicists and radiographers to s . 
salerno dipartimento di biotecnologie e biotecnologie mediche e forensi , universita` di palermo , aoup policlinico , via del vespro 127 , 90127 palermo , italy e - mail : sergio.salerno@unipa.it play an active role in dose management . 
in this review , the authors try to delineate the problem in a consequential and multifaceted way : radiationpatient interaction , possible mechanisms of damage , main ct dose units , risk and its quantication in the population , with the aim of optimising the acquisition dose without diagnostic drawbacks . 
for an up - to - date use of ct , radiologists must know the dose concerns for the single patient and population , and use the ct apparatus with the best dose care ; substitute ct with other diagnostic techniques when possible , especially in children ; reduce the number / extension of scans and phases , and the dose in single scans and single examinations . keywords ct ( cid : 2 ) radiation exposure ( cid : 2 ) dlp ( cid : 2 ) ctdi ( cid : 2 ) radiation dose ( cid : 2 ) absorbed dose introduction exposure to ionising radiation in computed tomography ( ct ) is a problem that is becoming progressively more important as ct has acquired the role of a rapid , total - body exploratory examination ; is very popular with both patients and clinicians and is considered a defensive tool in the diagnostic setting . 
an increase not only in the absolute number of ct examinations , but also in terms of both length of coverage and number of phases obtained while scanning ( baseline , arterial , sometimes two , venous , late ) has been observed [ 1 , 2 ]  . 
in some places , this is the product of an expedient exchange of the ease and speed of acquisition against appropriateness ; however , there is an abuse of the technique . 
the exposures to be reduced without 804 radiol med ( 2014 ) 119 : 803810 problem is compounded by scanners that seem friendly , but are in fact becoming increasingly complex , favouring the adoption of new practices without giving full regard to the optimal use of dose reduction algorithms . 
it is thus possible that a given instrument , in a single department , as used by various operators for examination of a single body district may yield doses that no longer depend predominantly on the limits of the machine but are operator dependent , with the important implication that some choices of the technicians and radiologists may not be justiable from diagnostic or dose limitation perspectives . the extent of this problem is not entirely known and cannot be adequately addressed without proper knowledge of all the phases that lead to the effective dose calculation [ 3 , 4 ]  . however , there is no doubt that ct investigations are increasing from every point of view : numbers of requests , extension , and scan phases [ 1 , 2 ]  . in light of the growing awareness of the issue of radiation dose and the possible risk for the individual and population there is a need for radiologists , medical physicists and radiographers to take on an active role in dose management . 
the purpose of this review is to delineate the problem in a consequential and multifaceted manner : radiationpatient interaction , possible mechanisms of damage , main ct dose units , risk and its quantication in the population , with the aim of optimising the acquisition dose without diagnostic drawbacks . 
this is extremely important because of the increased awareness of patients , as well as some recent decisions by the american college of radiology and legislative measures by individual american states ( california state dose bill [ 5 ] ) , which change the professional and legal perspective on the practice of ct , establishing a model that might be quickly and uncritically emulated elsewhere . radiation damage table 1 computed tomography dosimetric values acronym symbol unit measure dose in air locally absorbed dose total absorbed dose effective dose ctdiair ctdi100 ctdiw ctdivol mgy cm ctdi computed tomography dose index , dlp dose length product inuenced by the structure ; in fact the transcription zones are repaired faster than the silent zones , using enzymes and co - factors that recognise the damage . 
their lack does not allow a timely repair inuencing the survival and then the probability of inducing mutations and carcinogenesis processes [ 6 ]  . the calculation of dose in ct the rapid evolution of ct technology and the consequent spread of new clinical applications have determined a deep understanding of all information regarding ct dose calculation and awareness of primary denitions of parameters for this estimate , which should be revised following the evolution of technology . 
the ctdi is a basic concept to understand dose measurement in ct and is dened by : ctdi dzdz mgy ionising radiation interacts with the medium in which it propagates yielding its energy . 
in the case of photons ( x - rays ) , low let radiation , it is more probable that the interaction takes place with the water molecules that are present in billions of copies in the cell and represent 80 % of the weight . 
this primary transfer , in living organisms , starts a complex series of chemical reactions that are a prerequisite for cell changes and the possible beginning of pathological alterations . there is solid experimental evidence that dna is the main target of ionising radiation and that the damage is not uniformly distributed along the molecule . 
even repair is where : d ( z ) is the prole of the absorbed dose along the z axis , n is the number of slices acquired in a single axial rotation ; the value of n may be less than or equal to the maximum number of channels available on the system ( for example 64 for a multislice ct detector with 64 rows )  . 
the ctdiair is characteristic for each scanner and depends on tube current intensity and voltage , beam collimation , ltration and the radiol med ( 2014 ) 119 : 803810 complete ct examination and is dened as the product of the ctdivol multiplied by the irradiated scan length l . the dlp expressed in mgy cm is : dlp ctdivol ( cid : 3 ) l the dlp is a comprehensive dose descriptor and allows the assessment of risk by an estimation of the effective dose using the appropriate conversion factors dened by anatomical region . 
these conversion factors have been dened in a document of the european commission [ 7 ] and updated after the release of icrp 103 in 2007 to consider the weighting factors for the different tissues shown in table 2 [ 8 ]  . obviously , this approach does not take into account the individual patient size or the specic examination , but allows a rough estimate of the effective dose for protocol optimisation . 
however , there is a debate in the scientic community about the fact that ctdivol and consequently , dlp are no longer adequate ct dose descriptors for a number of reasons . 
the 100 - mm - long ionisation chamber is not suitable for multislice ct , because it is not long enough to measure the wider beam width or to include the contribution of the tails of the dose prole , resulting in an underestimation of the measured ctdivol . 
finally , the ctdi is not suitable for ct exposures where the patient remains immobile ( or almost immobile ) during acquisition ( cone - beam ct or perfusion studies ) , in which case the value reported on the scanner console is an overestimation of the mean absorbed dose in the scan volume . these criticisms are in fact based on the false assumption that the ctdivol should estimate the dose to the patient . 
in reality , the variety of patient types , scan protocols and clinical applications is such that no single existing phantom is able to accurately estimate the dose in all patients . 
since dose distribution in the phantom is generally not uniform , the measurements are acquired at ve different positions ( in the centre and at the four cardinal points ) , thus introducing the weighted ctdi ( ctdiw )  . ctdiw ctdi100 ; c ctdi100 ; p where ctdi100 , c and ctdi100 , p are measured at the centre and at the periphery of the phantom , respectively , and the index 100 indicates that the ctdi was measured with a 100 - mm - long ionisation chamber . 
the ctdiw , however , does not take into account the pitch used during a spiral acquisition . it was , therefore , necessary to dene the volumetric ctdi , the ctdivol , i.e. 
ctdivol ctdiw pitch it should be noted that the term mas indicated on some manufacturers consoles does not refer to true mas , but to mas per rotation divided by the pitch . 
finally , as we do not acquire a single - slice but a whole volume , we should also consider the length of the scan and introduce a second dose descriptor : the dose length product ( dlp )  . 
furthermore , as the ctdivol is displayed on the console before starting a scan and recorded in the dose report at the end of the examination , many users assume that this is the individual patient dose . 
the ctdivol is no doubt still useful , but it should be clear that it is a standardised phantom measurement of the ct system output , which enables users to optimise and compare radiation output between different protocols or different scanners and not the real dose delivered to the patient . patient dose population dose from ct was a radiation protection concern before the advent of ultrafast multidetector scanners ; in fact , diagnostic reference levels ( drls ) for ct examinations have been dened in european guidelines [ 7 ] and in the italian law d.lgs. 
87 / 00 since 2000 [ 9 ]  . even though drls should not be applied to individual exposures but are reference doses for common examinations , they can help to optimise radiation protection to avoid unnecessarily high doses to the patient . 
for ct , the ctdiw and the dlp were suitable quantities to be used as drls . the italian drls for ct are derived from a european document based on a british study performed in the early 1990s . 
since then , ct has undergone major evolution and while ctdiw was a good metric at the time of single - slice ct scanners , now it has been replaced by the ctdivol commonly displayed by the ct scanner console . in 2006 , shrimpton et al . 
 [ 10 ] published a national survey , a review of patient doses from ct examinations in the uk in 2003 , conducted on the basis of data received from over a quarter of all uk scanners , of which 37 % had multislice capability . 
the mean uk doses for adult patients were in general lower by up to 50 % than previous ones , although doses were slightly higher for multislice relative to single - slice scanners . 
the relative increase in reference dose was larger for scans of the head and the chest ( high resolution )  . these examinations both involve axial scanning with narrow beam collimations , where beam penumbral effects and differences in z axis geometrical efciency between single and multislice scanners were most pronounced [ 10 ]  . a similar investigation , prior to the widespread adoption of multislice ct was conducted in italy in 2004 and published in 2006 [ 11 ]  . 
the survey was carried out for seven adult clinical ct protocols , and showed that ctdiw and dlp were always below the drls set by the european guidelines [ 7 ]  . 
now the rst italian nationwide survey about adult exposures from mdct and including multiphase studies is at last available [ 12 ]  . the subsequent advances in ct technology made it possible to collect multiple images per rotation and to acquire a given volume of data in a much shorter time interval . 
this caused a general increase in the dlp , due to the wide use of multiphase examinations and a larger application in paediatric radiology . in paediatric patients , ct radiation protection became greater concern due to inherently higher radiosensitivity of growing tissues ( such as cartilage , red marrow ) , and childrens longer life expectancy that involves a longer interval in which one can develop a possible neoplasm . analysis of the risks associated with paediatric ct mainly refers to the study by brenner [ 13 ] , which highlighted the increase in the probability of occurrence of tumours when patient age decreased . 
it should be noted that abdominal ct in a child increases the probability of tumour occurrence to more than 20 % . however , the main dosimetric aspects connected with the use of ct in paediatrics are still poorly standardised : 1 . 
the classic ct dose descriptors are based on phantoms with diameter simulating the geometry of an adult and introducing signicant uncertainties in the evaluation of organ doses in children . various tools have been used by different research groups to perform size - dependent dose evaluations . 
 [ 19 ] used the monte carlo n - particle ( mcnp ) radiation transport code to calculate normalised effective dose values for three different scanners and mathematical anthropomorphic phantoms with ages ranging from newborn to adult . 
they demonstrated the high dependence on patient age and size : the effective dose in a newborn was 1.5 times greater than that of an adult for all types of examinations . 
other dosimetric aspects associated with paediatric ct regard optimisation procedures [ 20 ]  . dose reduction systems dose reduction is one of the main problems in ct , and many techniques have been developed to face this problem , such as tube current modulation and voltage reduction . automatic dose modulation is based on the principle that the operator decides on the desired image quality in terms of noise index , reference mas , or reference image , depending on the ct system manufacturer , and the scanner radiol med ( 2014 ) 119 : 803810 table 3 iterative dose reduction system and corresponding acronym , producer and year of commercialisation acronym meaning producer year of commercialisation asir adaptive statistical general 2008 iterative reconstruction electric trade name ( mbir ) general 2009 electric iris image reconstruction in siemens 2009 image space safire sinogram - afrmed siemens 2010 iterative reconstruction idose aidr trade name philips adaptive iterative dose toshiba 2009 2010 reduction automatically selects the right tube currenttime product ( mas )  . 
the system automatically regulates tube current referring to scout images in the z ( longitudinal modulation ) and xy ( transverse modulation ) axes . the limits of these modalities in obtaining additional dose reduction are determined by the inverse correlation between mas value and image noise . 
hence , a new algorithm called iterative reconstruction ( ir ) has been proposed by manufacturersused in the past and abandoned because of the long reconstruction time imposed by older computerswhich is currently capable of additional dose reduction [ 21 ]  . 
 [ 22 ] published an extensive review of the different modalities of dose reduction systems ( table 3 )  . iterative modalities are based on mathematical algorithms , which are not fully accessible because various vendors produce them as black boxes mainly for the commercial impact of these tools . 
however , it is possible to divide these modalities into two types : those working on the control of the images , using a statistical method for noise reduction , and those directly managing the raw data domathe latter allow for a further dose reduction , but imply a complex analysis with longer processing time [ 22 ]  . radiation dose and associated cancer risk although ct accounts for less than 19 % of radiological examinations [ 3 ] , its contribution in radiation dose for the general population is becoming signicantly higher . 
for doses lower than 100 msv ( 100 mgy with wr equal to 1 ) , the risk calculation is based on the linear no - threshold model ( lnt ) that is mainly accepted and adopted by the large majority of international organisations in the eld of radiation protection as icrp , beir , nrpb , unscear , etc . 
103 guideline modied the estimation of cancer risk from low radiation levels . in fact , they increase the risk for the induction of somatic damage in the population up to 6 % per sv received , remaining unchanged at 0.2 % for genetic risk . 
for risk reduction , at low dose levels , in addition to the linear model without threshold ddrf ( dose and dose rate effectiveness factor ) , a factor of 1.5 was used ; conversely , the icrp adopted a factor equal to 2 [ 23 ]  . estimation of cancer risk represents a complex problem and many factors must be considered in cancer induction , not only low - dose x - rays used in ct . 
due to the greater sensitivity of young subjects to radiation damage , the risk factor is 34 times more than in adults [ 27 , 28 ]  . in ct examinations , only body segments and not the entire body ( such as brain , chest and abdomen ) are usually exposed , so the risk calculation should take into account 808 radiol med ( 2014 ) 119 : 803810 tissue - specic weighting factors ( such as the breast in chest exposure , which increases the risk )  . many publications consider it incorrect to calculate a single individual risk , and underline the lack of sound evidence of cancer risk for doses less than 50 msv . 
finally , the risk estimation based on the japanese nuclear bomb survivors compared to the dose commonly used in ct is considered questionable [ 29 ]  . as the icrp guidelines well outline , it is incorrect to extrapolate an individuals cancer risk [ 30 ] , as the guidelines are for the cumulative cancer risk of a population . following brenners publication [ 31 , 32 ] some papers calculated the effective cancer risk with a simple arithmetical calculation ( number of ct examinations multiplied by mean exposure in msv of the population ) resulting in impressive and overestimated rates of cancer related to medical exposures in the general population [ 29 ]  . 
the study determined that the increased cancer incidence rates after ct exposure in these cohorts are mostly due to irradiation [ 35 ]  . incidence rates another additional criticism of exponential cancer risk is related to the evidence of the linear relation of the damage in opposition to the nonlinear relation of the cellular reparative mechanism , with consequent overestimation of the damage [ 36 ]  . 
 [ 38 ] underlined that the accidental chronic exposure to low gamma radiation from cobalt 60 determined protective effects against tumours ; in fact the expected tumour rate in this population is lower than expected . although the scientic community is still debating the effects of low dose levels , some misinterpretations of scientic papers and some questionable ct practices have resulted in internet forums and newspaper articles on ct linked to cancer in single groups of patients . 
the fda has established an initiative to reduce unnecessary radiation exposure from medical imaging ; the california state government has promoted a law imposing a dose report in every ct scan ( dose bill ) [ 5 ] , including cases of overdose and a strict annual control of the dose used for every protocol by the medical physicist . 
moreover , a european community ( ec ) directive aiming to impose the dose report in the radiological report ( council directive 242 , 2012 laying down basic safety standards for protection against dangers arising from exposure to ionising radiation ) is risk denial and risk under evaluation . 
are radiologists and clinicians sufciently prepared on the subject of cancer risk ? dose risk knowledge are all the subjects involved in ct prescription and execution prepared on dose risk ? according to some experiences published in the bmj [ 39 , 40 ] , doctors working in the uk national health service have insufcient knowledge about radiation protection , despite serious continuous education programmes for health professionals and a widely available publication for referring physicians entitled how to make the best use of the diagnostic radiology department , in which the practice of dose reduction is extensively described [ 41 ]  . introduction of radiation protection in medical school programmes is also recommended in european guidelines to all state members ( european commission medical exposure directive ) [ 10 , 42 , 43 ]  . in italy , possible knowledge gaps among referring physicians , radiographers and radiologists have not been investigated despite the fact that since 1995 italy has had detailed legislation regulating the patients radiation protection and operator education and training [ 43 ]  . 
an increased attention to the problem should be adopted in young radiologists , who are more involved in emerging radiation technologies and new applications . in june 2009 , the american college of radiology and the radiological society of north america set up a task force to increase radiation protection knowledge and reduce unnecessary imaging in particular in paediatric patients . radiol med ( 2014 ) 119 : 803810 conclusions dose reduction in ct relies on the correct implementation / optimisation of protocols and everyday practice . we can signicantly reduce unnecessary radiation exposure by being aware of patient risk and using all the available resources in the ct apparatus to the best advantage . 
to synthesise what radiologists are required to do to ensure an up - to - date use of ct , we can state that they should : know the dose concerns for the single patient and population , in the knowledge of possible risks and using the ct apparatus with the best dose utilisation ; involving referring physicians substitute ct with other diagnostic techniques when possible especially in children ; reduce the number / extension of scans , phases , the dose in single scans and examinations , also optimising contrast and reducing costs . glossary and denitions to facilitate reading of this review , this section recalls the unit measures used in radiation protection and dosimetry in alphabetical order . absorbed dose d is the measure of all types of ionising radiation and is dened a d de dm where de represents the mean energy deposited to the mass dm by the ionising radiation . 
the adsorbed dose in si system is measured in joule per kilogram ( j kg - 1 ) and named gray ( gy )  . the absorbed dose is calculated from the energy deposited ( e ) , does not reect the single event of interaction and its value is obtained as a mean on a generic element of mass ( dm )  . the radiation damage for low dose levels is supposed to be correlated with the value of adsorbed dose related to a specic organ or tissue . 
to account for the different modalities of radiation interaction and different radiation sensitivities of tissue two other values need to be introduced : the equivalent dose ( ht ) and the effective dose ( e )  . the unit measure is the same as the adsorbed dose j kg - 1 , but it is called sievert ( sv )  . diagnostic references levels ( drl ) are dened dose levels used in diagnostic and nuclear medicine for typical groups of patients of standard body mass or a standard phantom dened for different systems . 
it may also be interesting to compare doses from different diagnostic the use of similar technologies or procedures and / or technologies or procedures across hospitals or countries to compare technologies for the same medical investigations . the effective dose , however , is not recommended for epidemiological evaluation either for detailed retrospective analysis on exposure and risk of single individuals . the interpretation of effective dose , in patients exposed for medical purposes , remains complex especially when organs or tissue is subjected to partial or heterogeneous exposure . pitch in multislice spiral ct is dened as the ratio of the table increment over the detector collimation . 
one can assume that the detector collimation for each of n detector arrays is the same , excluding cases of either different collimations or combined measurement row . conict of interest stefano colagrande , daniela origgi , giovanna zatelli , andrea giovagnoni , sergio salerno declare no conict of interest . radiol med ( 2014 ) 119 : 803810 23 . 
verdun fr , bochud f , gundinchet f et al ( 2008 ) quality initiatives radiation risk : what you should know to tell your patient . radiographics 28 : 18071816 28 . 
krille l , zeeb h , jahnen a et al ( 2012 ) computed tomographies and cancer risk in children : a literature overview of ct practices , risk estimations and an epidemiologic cohort study proposal . radiat environ biophys 51 : 103111 29 . 
meer ab , basu pa , baker lc , atlas sw ( 2012 ) exposure to ionizing radiation and estimate of secondary cancers in the era of high - speed ct scanning : projections from the medicare population . 
pearce ms , salotti ja , little mp et al ( 2012 ) radiation exposure from ct scans in childhood and subsequent risk of leukaemia and brain tumours : a retrospective cohort study . 
matthews jd , forsythe a , brady z et al ( 2013 ) cancer risk in 680000 people exposed to computed tomography scans in childhood or adolescent : data linkage study of 11 million australians . 
chen wl , luan yc , shieh mc et al ( 2007 ) effects of cobalt - 60 exposure on health of taiwan residents suggest new approach needed in radiation protection . 
royal college of radiologists ( 2007 ) making the best use of clinical radiology services ( mbur ) referral guidelines , 6th edn . royal college of radiologists , london 42 . 
identication of af prior to rfca is paramount to select those patients who are truly amenable to the ablation procedure , which is expensive and not entirely free of risks . keywords atrial brillation ( cid : 2 ) atrial brosis ( cid : 2 ) magnetic resonance ( cid : 2 ) late enhancement introduction atrial brillation ( af ) is the most frequently encountered cardiac arrhythmia in clinical practice , with a prevalence of 0.51 % in the general population and up to 6 % among those older than 65 years [ 13 ]  . 
ablation procedures to electrically disconnect the pv from the left atrium ( la ) have become a common alternative in patients refractory to medical therapy [ 58 ] , and their efcacy is inversely proportional to the extent of atrial brosis produced by the arrhythmia ( electrical , mechanical and anatomical remodtherefore a elling ) [ 912 ]  . 
to integrate the dicom images with the carto3 system , anatomical evaluation of the la - pv complex was carried out on the basis of a three - dimensional ( 3d ) flash angiography sequence acquired during the administration of a 10 ml bolus of gadobenate dimeglumine ( multihance , bracco , milan , italy ; 0.1 mmol / kg ) at a ow rate of 2 ml / s . 
acquisition began 15 min after the injection of the paramagnetic contrast material and the sequences lasted 1520 min on average , depending on respiratory variability and heart rate . on le - mr , brosis was dened as an area of high signal intensity of the atrial wall relative to the normal myocardiuthe la was divided into seven segments ( pv antra , oor , anterior wall and posterior wall - roof )  . 
the presence of brosis was qualitatively evaluated by two radiologists with different levels of experience in cardiac mr imaging ( m.c. , 7 years ; g.c. , 3 years ) who were blinded to any previous ablation procedures . 
the basic anatomical structure was displayed in different colours indicating the voltage levels and correlating with the hearts electrical vitality . the bipolar voltage of the atrial endocardium is normally around 0.39.5 mv ; values \0.5 mv are considered to be low amplitude whereas those lower than 0.05 mv are usually associated with areas of brosis [ 17 ]  . as done in the analysis of the le - mr images , the la was divided into seven segments , each of which was assessed for the presence of brosis . a qualitative analysis was performed by comparing the areas of le on the mr images with the segments radiol med ( 2014 ) 119 : 595600 displaying low voltage values on eam : anatomical agreement was assessed by comparing the segments of brosis and / or healthy tissue detected by the two modalities , with agreement being dened as optimal whenever there was concordance of at least six out of seven segments . sensitivity ( se ) , specicity ( sp ) , positive predictive value ( ppv ) , negative predictive value ( npv ) and diagnostic accuracy were nally calculated . results at the time of the examination , 11 / 37 patients were in af . five of 37 patients ( 14 % ) were excluded because of poor quality of the mr images : in two cases because of failure to suppress the cardiac signal ( incorrect ti choice inuenced , in one case , by the presence of af ) ; in two cases resulting from irregular because of major artefacts breathing and af ; in one case because of inadequate sampling of the thoraco - abdominal interface by the respiratory navigator . 
a , b late - enhancement ( le ) - mr axial images of the left atriuc , d 3d electroanatomical maps integrated with mr angiograms ( carto3 ) in posterior ( c ) and anterior ( d ) views ; the functional information is dened by a colour code that provides an immediate evaluation of atrial electrical activity . 
a , b le - mr axial images of the left atriuc , d 3d electroanatomical maps integrated with mr angiograms ( carto3 ) in posterior ( c ) and anterior ( d ) views . 
the le - mr image ( arrow in a ) does not show pathological late enhancement close to the ostium of the left superior pulmonary vein , whereas the electroanatomical map identied ( arrow in c ) an area of brosis at this level . 
the le - mr image shows a hyperenhanced area ( brosis ) in the anterior wall of the left atrium ( arrow in b ) whereas the electroanatomical map shows normal potential ( purple ) at this location ( arrow in d )  . 
the resulting atrial remodelling promotes the development of af and , with time , makes spontaneous reconversion to sinus rhythm increasingly less likely [ 9 ]  . atrial brosis is therefore both a cause and a consequence of af . 
the effectiveness of the new treatments aiming at electrically disconnecting the pv from the atrium is to the extent of brosis [ 13 ]  . inversely proportional radiol med ( 2014 ) 119 : 595600 detection of brosis is therefore paramount when selecting candidates for af ablation procedures , which are costly and not free of risks [ 14 ]  . in our work , we tried to identify atrial brosis noninvasively , using a le - mr imaging sequence ; in addition , unlike most studies on the subject [ 13 , 14 , 18 ] , we decided not to use post - processing techniques to maximise signal hyperintensity , so as to simplify the diagnostic process of brosis detection . 
in comparison to spragg et al.s work [ 22 ] , which selected patients who had already undergone the ablation procedure , our series consisted of mixed group of patients , 32 % of whom had undergone a previous ablation procedure . 
these two factors may account for the discrepancy in results ; while the se was lower ( 66 vs 84 % ) , the sp was higher ( 87 vs 68 % ) with a resulting npv of 85.5 vs 73 % , which makes the le - mr technique a good candidate for stratifying patients to be treated with ablation . 
in an attempt to eliminate the artefact , at the beginning of the study the navigator was shifted to the more lateral and anterior sectors of the right hemidiaphragm ; however , this approach led to incorrect sampling of right hemidiaphragm excursion , resulting in a longer sequence without eliminating the artefact . in the present study , eam was considered the reference standard . 
several comparative studies have , however , reported a spatial error for eam equal to 510 mm [ 24 , 25 ] ; in addition , by expressing the prevalent voltage of endocardial rather than epicardial myocytes , eam may provide data that are not always correct with reference to the overall thickness of the atrial wall . 
in light of these the le - mr considerations , we could hypothesise that sequence has a greater rate of brosis detection compared to eam . study limitations the poor technical quality of le - mr images ( 14 % of examinations were not assessable ) is dependent on patient compliance , the presence of arrhythmia during the study and the difculty selecting the ti . 
however , these sequences have sufcient signalto - noise ratio only for slice thickness of at least 6 mm , which makes them inappropriate for the study of the atrial wall ( thickness , 13 mm [ 19 ] )  . 
re lucia calculli luca frulloni roberto pozzi mucelli received : 9 august 2012 / accepted : 30 july 2013 / published online : 18 march 2014 ( cid : 2 ) italian society of medical radiology 2014 computed enlargement shows diffuse abstract multidetector - row tomography ( mdct ) and magnetic resonance ( mr ) imaging are currently the most frequently performed imaging modalities for the study of pancreatic disease . 
in cases of suspected autoimmune pancreatitis ( aip ) , a dynamic quadriphasic ( precontrast , contrast - enhanced pancreatic , venous and late phases ) study is recommended in both techniques . 
in the diffuse form of autoimmune pancreatitis ( daip ) , the pancreatic parenchyma appears , during the mdct and mr contrast - enhanced pancreatic phase , diffusely hypodense and hypointense , respectively , compared to the spleen because of lymphoplasmacytic inltration and pancreatic brosis . 
in the focal form of autoimmune pancreatitis ( faip ) , the parenchyma shows segmental enlargement involving the head , the body - tail or the tail , with the same contrast pattern as the diffuse form on both modalities . faip needs to be differentiated from pancreatic adenocarcinoma to avoid unnecessary surgical procedures , since both diseases have similar clinical and imaging presentation . 
finally , in all cases of uncertain diagnosis , mdct and / or mr follow - up short - term treatment ( 23 weeks ) with high - dose steroids can identify a significant reduction in size of the pancreatic parenchyma and , in faip , normalisation of the calibre of the upstream main pancreatic duct . after keywords chronic pancreatitis ( cid : 2 ) autoimmune pancreatitis ( cid : 2 ) acute pancreatitis ( cid : 2 ) computed tomography ( cid : 2 ) magnetic resonance ( cid : 2 ) steroid treatment autoimmune pancreatitis ( aip ) : denition and classication autoimmune pancreatitis ( aip ) is a form of chronic pancreatitis associated with autoimmune processes . 
distinct histological and clinical proles reveal two subtypes of aip , indistinguishable on the basis of imaging alone : type 1 , or lymphoplasmacytic sclerosing pancreatitis ( lpsp ) , radiol med ( 2014 ) 119 : 558571 and type 2 , or idiopathic duct - centric pancreatitis ( idcp ) [ 1 ]  . 
type 1 , lpsp , or aip without granulocyte epithelial lesions , seems to be an igg4 - related multiorgan disease , characterised by elevated serum igg4 levels , multiple extrapancreatic organ involvement and igg4 - rich lymphoplasmacytic inltrate on histology in all the affected organs [ 2 , 3 ]  . 
in type 2 aip , idcp or aip with granulocyte epithelial lesions , there is no extrapancreatic organ involvement or igg4 - rich inltrate on histology , and serum igg4 elevation is unlikely . 
this form appears to be a pancreas - specic disorder and is histologically characterised by the presence of neutrophils with typical granulocyte epithelial lesions . the diagnosis of aip is challenging even at expert centres and many different diagnostic criteria have been developed . 
the comprehensive criteria that must be fullled for the diagnosis include pancreatic imaging of the parenchyma and ductal system , serological and histopathological ndings , other organ involvement and response to steroid treatment [ 1 ]  . the clinical presentation overlaps with other forms of acute and chronic pancreatitis , but without a history of alcohol or tobacco abuse or biliary stone disease . 
aip may vary in its clinical presentation depending on the pancreatic distribution of disease ( focal or diffuse ) and on the specic site involved ( head , body or tail of the pancreas ) [ 421 ]  . 
in diffuse forms or in focal distribution in the pancreatic body - tail , patients may present pancreatic abdominal pain in the epigastric region , irradiating to the back with or without jaundice [ 821 ]  . 
therefore , the clinical presentation of aip may mimic pancreatic adenocarcinoma in the focal forms and pancreatic lymphoma or non - necrotizing acute pancreatitis ( nnap ) in the diffuse forms [ 2226 ]  . in evaluating patients with suspected pancreatic disease , acute or chronic pancreatitis or pancreatic neoplasms , multidetector - row computed tomography ( mdct ) and magnetic resonance ( mr ) are the imaging modalities of choice [ 1517 , 2733 ]  . 
in the specic case of suspected aip , a dynamic quadriphasic study ( precontrast , contrastenhanced arterial , pancreatic , venous and late phases ) is recommended for both techniques . technically , in mdct of the pancreas , contrast medium administration typically involves the use of bolus tracking software with a region of interest ( roi ) placed over the aorta and a predetermined attenuation threshold of 100 hu . pancreatic arterial and portal venous phase images are acquired 2030 and 6070 s after bolus tracking , respectively , and the delayed phase images 180 s after the beginning of the injection . 
the standard reconstruction thickness used during the pancreatic and portal venous phase is 1 mm , with an interval of 0.5 mm , whereas during the late phase it is 21 mm , with a range of 10.5 mpostprocessed coronal and curved mdct multiplanar reconstruction ( mpr ) images are recommended in cases of suspected aip . pancreatic mr and mr cholangiopancreatography ( mrcp ) imaging must be performed on a 1.5 t scanner using a surface phased - array body coil . 
to eliminate overlapping uid - containing organs on t2 - weighted mrcp images , 50150 ml of superparamagnetic iron oxide particles is administered 1020 min before the mr examination . pancreatic mr imaging includes the following sequences : axial t1 - weighted gradient echo , axial t1 - weighted fatsaturated , axial t2 - weighted fat - saturated rapid acquisition with relaxation enhancement ( rare ) , t2 - weighted halffourier rare , coronal true fast imaging in the steady - state precession ( true - fisp ) , axial and coronal , coronal oblique 2d half - fourier rare cholangiopancreatography , axial fat - saturated 3d volumetric gradient echo . 
a quadriphasic dynamic study is performed during injection of 0.1 mmol / kg body weight of gadolinium chelates by means of a power injector at 22.5 ml / s , by acquiring the precontrast - phase , late arterial / pancreatic phase ( 3545 s ) , portal venous phase ( 7580 s ) , and delayed phase ( [ 180 s )  . 
for diffusion - weighted imaging ( dwi ) , a spin - echo echoplanar sequence is performed with a b value of 0 , 50 , 600 s / mm2 . 
the intrapancreatic segment of the common bile duct is slightly dilated ( arrow head ) ; the intrahepatic bile ducts are normal . in the left kidney , a solid cortical lesion is present which later proved to be chronic autoimmune pyelonephritis on biopsy ( black arrows ) ; the right kidney shows a cystic lesion . 
according to some authors , this peripheral rim of delayed contrast enhancement is due to the presence of a chronic inammatory process and brous tissue involving the peripancreatic fat [ 15 , 20 , 21 , 44 ]  . 
the periductal inammatory cell inltration and brous tissue centred around the pancreatic ducts [ 12 , 13 , 4449 ] produce diffuse narrowing of the main pancreatic duct ( mpd )  . 
patients with aip often demonstrate duct the mpd wall on ct imaging ( the enhancement of enhanced duct sign ) [ 42 , 43 ] and this could reect periductal inammatory changes . 
the enhanced duct sign is strongly associated with the abnormal enhancement area of the pancreas in aip and , although relatively uncommon , this nding may be useful for the diagnosis of aip . both the enhanced duct sign and the inability to visualise the mpd lumen within a daip lesion can aid in the diagnosis of aip . 
also peripancreatic stranding , represented by inammatory changes of peripancreatic fat in mild acute non - necrotizing pancreatitis , recorded as a hypodense peripancreatic halo in all phases of ct examination [ 32 , 37 ] is absent in daip [ 50 ]  . daip must be differentiated from nnap and lymphoma since both these pathological conditions show diffuse enlargement of the pancreatic parenchyma [ 19 , 25 , 51 , 52 ]  . in both daip and nnap , pancreatic abdominal pain with epigastric location irradiating to the back , increase of serum amylase and lipase and diffuse pancreatic enlargement are frequently present [ 2831 ]  . 
recently , this vascularisation pattern of daip has been assessed using the rate of relative variation in enhancement from the previous phase or relative enhancement rate across all phases of the mdct study [ 50 ]  . 
a statistically signicant difference in the two groups of patients ( daip and nnap ) regarding the presentation of peripancreatic stranding and retroperitoneal uid has been demonstrated [ 50 ]  . 
in conclusion , the pattern of mdct contrast enhancement of daip and nnap , particularly considering the relative enhancement rate parameters [ 50 ] , provides qualitative and quantitative clues for differentiating the diseases . 
the pancreatic gland is surrounded by a hypodense halo in all mdct phases ( peripancreatic stranding : arrow head ) due to mild inammatory changes of peripancreatic fat . pancreatic lymphoma ( e , f )  . 
secretin is a polypeptide hormone that induces pancreatic bicarbonate - rich uid secretion into the duodenum and increases the tone of the sphincter of oddi with a temporary distension of the pancreatic ducts . 
the pancreatic parenchyma appears hypodense compared to the spleen and unaffected adjacent parenchyma of the body - tail during the contrastenhanced mdct pancreatic phase ( b , d )  . 
the main pancreatic duct is partially invisible because of focal ductal stenosis due to lymphoplasmacytic inltration and areas of brosis in the pancreatic body and head , with upstream main duct dilatation . 
the pancreatic head lesion ( short arrow ) appears more hypodense compared to the spleen and to the body - tail during pancreatic ( b ) , venous ( c ) and late ( d ) phases . 
the mesenteric vein lumen ( arrow head ) near the solid lesion in the pancreatic head has a reduced diameter ( vascular inltration ) mpd stricture resolves after secretin stimulation , this rules out a malignancy and is suggestive of aip ( the so - called duct penetrating sign ) [ 61 ]  . 
on the other hand , dwi and adc values are variable in pancreatic cancer , allowing for this modality to differentiate between massforming focal aip and pancreatic carcinoma . the nal differential diagnosis between focal daip and pancreatic cancer can only be conrmed by a ne - needle aspiration biopsy [ 62 , 69 , 70 ]  . extrapancreatic aip : mdct and mr ndings ct and mr can identify some of the many extrapancreatic manifestations of aip , most commonly biliary , renal and retroperitoneal [ 17 , 71 ]  . biliary involvement , present in up to 80 % of aip patients , is the most common extrapancreatic involvement . both intrahepatic and extrahepatic bile ducts can be involved , showing multifocal stenosis and wall thickening 566 radiol med ( 2014 ) 119 : 558571 fig . 
axial 3d volumetric t1 - weighted fat - suppressed gradient - echo images after intravenous contrast medium administration during pancreatic ( a ) , venous ( b ) and delayed phase ( c )  . 
diffusion - weighted mr steady - state precession ( true - fist ) spin - echo echoplanar dw image with imaging ( dwi ) : axial b value = 600 s / mm2 ( e ) ; axial apparent diffusion coefcient ( adc ) map calculated from baseline images obtained with b value of 0 , 50 , 600 ( f )  . 
the overall pancreatic gland is enlarged with sharp borders ( ad ) and presents decreased enhancement in the arterial pancreatic phase ( a , d ) compared to the spleen , with an increased progressive contrast uptake during the venous ( b ) and the delayed ( c ) phases . 
the affected pancreatic parenchyma appears slightly hyperintense in the dw images ( e ) with lower adc value ( f ) than patients without pancreatitis due to a severe inammatory cellular inltration ( lymphocytes , plasma cells and granulocytes ) resembling primary sclerosing cholangitis . 
this is an important technique to evaluate involvement of the biliary ducts , which is frequently associated because the periductal inltration can extend to the intrapancreatic cbd and even to its suprapancreatic segment . 
axial 3d volumetric t1 - weighted fat - suppressed gradient - echo images before ( a ) and after intravenous contrast medium administration during the pancreatic ( b ) , venous ( c ) and delayed phases ( d , f )  . 
the parenchyma of the pancreatic body - tail shows focal enlargement ( arrows ) with sharp borders and abnormal signal intensity : it appears hypointense in the t1 - weighted images ( a ) compared to the unaffected parenchyma . 
in the dynamic study , the aip - affected parenchyma shows decreased enhancement during the pancreatic phase ( b ) , with a homogeneous and progressive contrast uptake during the venous ( c ) and delayed phases ( d )  . 
the main pancreatic duct is not visible within the faip ( e : arrowhead ) , compressed by the crowded cellularity all around , with an upstream dilation ( e )  . 
in both cases ( a , b , cf ) , diffuse pancreatic enlargement and hypodense pancreatic parenchyma are present during the pancreatic phase . mdct also shows a retroperitoneal soft tissue mass surrounding peripancreatic vessels ( a , b : arrows ) and right side pelvic vessels ( d : arrow )  . 
both in diffuse and focal aip , the pancreatic parenchyma appears isovascular to the spleen in the enhanced radiol med ( 2014 ) 119 : 558571 pancreatic phase and hypovascular in the venous phase , with wash - out in the delayed phase . 
finally , mdct and mr imaging can easily identify normalisation of the calibre of the intraand suprapancreatic common bile duct [ 19 , 20 , 37 ]  . the japan pancreas society has proposed diagnostic criteria for the diagnosis of aip [ 7478 ]  . 
these criteria include : the nding of typical pancreatic imaging results ( enlargement of the pancreatic parenchyma and mpd narrowing ) , positive laboratory tests ( autoantibodies and elevated serum levels of immunoglobulin g4 ) and positive histopathologic al ndings ( lymphoplasmacytic inltrate and pancreatic brosis )  . 
the differential diagnosis of aip is difcult when a patient with typical clinical ndings has other associated autoimmune disorders with only non - specic imaging and / or histopathological ndings . 
in all the cases in which aip is strongly suspected but the diagnosis is uncertain , mdct and mr are useful imaging modalities to suggest the correct diagnosis . repeated mdct and / or mr examinations after short - term treatment ( 23 weeks ) with high - dose steroids can identify a signicant reduction in the size of pancreatic parenchyma , and the normalisation of the calibre of the mpd and bile ducts . 
the response at short - term imaging follow - up after steroid treatment was recently considered a diagnostic criterion of aip [ 1 ]  . conict of interest rgraziani , s . 
we veried whether adding and combining proton mr spectroscopic imaging ( 1h - mrsi ) , diffusion - weighted imaging ( dwi ) and perfusion - weighted imaging ( pwi ) information at 3 tesla facilitate such discrimination . materials and methods twenty - nine patients with histologically veried high - grade gliomas , who had undergone surgical resection and radiotherapy , and had developed new contrast - enhancing lesions close to the treated tumour , underwent mri , 1h - mrsi , dwi and pwi at regular time intervals . 
the metabolite ( cho ) / normal ( n ) chon , n - acetylaspartate ( naa ) / naan , creatine ( cr ) / crn , lactate / lipids ( ll ) / lln , cho / crn , naa / crn , cho / naa , choline ratios naa / cr and cho / cr were derived from 1h - mrsi ; apparent diffusion coefcient ( adc ) from dwi ; and the relative cerebral blood volume ( rcbv ) from pwi . results in serial mri , recurrent gliomas showed a progressive enlargement , and radiation injuries showed regression or no modication . 
tedeschi dipartimento di neuroscienze , seconda universita` di napoli , naples , italy radiol med ( 2014 ) 119 : 616624 keywords radiation injury ( cid : 2 ) recurrent brain tumour ( cid : 2 ) magnetic resonance spectroscopy ( cid : 2 ) diffusion - weighted imaging ( cid : 2 ) perfusion - weighted imaging introduction standard - of - care therapy of high - grade brain gliomas includes surgical resection followed by radiotherapy and chemotherapy , but the tumour most often recurs or progresses after this multiple therapeutic approach [ 1 ]  . 
in a frequency up to 30 % , patients develop a radiation injury that mimics tumour recurrence or progression on conventional magnetic resonance ( mr ) imaging [ 1 , 2 ]  . 
histopathological analysis is currently considered the gold standard for the correct diagnosis [ 2 ] , but it requires the acquisition of tumour samples by open craniotomy or stereotactic biopsy , procedures which place the patients at an increased risk for morbidity and mortality [ 3 ]  . 
thus , a noninvasive technique is highly needed . imaging methods that emphasise metabolic , structural and haemodynamic properties of tissues , namely proton magnetic resonance spectroscopic imaging ( 1h - mrsi ) , diffusionweighted imaging ( dwi ) and perfusion - weighted imaging ( pwi ) respectively , may be helpful for the discrimination of tumour recurrence / progression from radiation effects [ 1 , 4 ] , and could limit the need for brain biopsy . 
considering such limitations , several authors have used a variable combination of 1h - mrs , dwi and / or pwi information in addition to mri features for improving such discrimination [ 612 ] , and some of these studies have been performed at 3 tesla [ 6 , 810 ]  . 
switching to high magnetic eld strengths allows several advantages , such as higher signal - to - noise ratio and better spatial , temporal and spectral resolution [ 13 , 14 ]  . this study aims to further evaluate the usefulness of combining metabolic , diffusion and haemodynamic information in the differentiation between recurrent glioma and radiation injury using a pattern recognition analysis . 
to our knowledge , there is no study in which 3 - t1h - mrsi , dwi and pwi parameters have been combined in a discriminant function - based pattern recognition approach to enhance such differentiation . materials and methods patients and treatments thirty - three patients with pathologically veried highgrade gliomas , who had apparently total surgical resection , radiotherapy and chemotherapy , and had conventional treatment development of new contrast - enhancing lesions in the vicinity of the original treated tumours , were consecutively included in this study . 
the remaining 29 patients ( 18 men and 11 women ; median age , 62.5 years ; range 3874 years ) had a histopathologic diagnosis of glioblastoma multiforme ( who grade iv )  . 
postsurgical radiotherapy ( daily fractions of 2 gy for a total of 60 gy , 5 days a week during 6 weeks ) plus temozolomide at a dose of 75 mg / m2 / day followed by six cycles of adjuvant temozolomide ( 150 mg / m2 / day in the rst cycle and then 200 mg / m2 / day , for 5 days during each 28 - day cycle )  . follow - up mr examinations started between 4 and 8 weeks after completion of radiotherapy and continued at 2to 3 - month intervals during the rst year and at 3to 6 - month intervals during the following years , depending on the clinical course . 
the local ethics committee approved the research protocol and informed consent was obtained from all patients . imaging mr studies were executed with a mr scanner at 3.0 tesla ( general electric medical systems , milwaukee , wis . ) with the standard quadrature head coil . 
mri , dwi , 1h - mrsi and pwi were acquired in that order during the same examination to allow exact comparison of results , and acquisition data were analysed by an automated software package ( functool , ge medical systems ) in a remote workstation . 
all image data were checked for overall image quality and motion artefacts before analysis . the mr imaging protocol included : ( a ) sagittal t1weighted localising images by fast spoiled gradient - echo ( fspgr ) sequence ( slice thickness 5 mm ; gap 1 mm ; repetition time , tr , 225 ms ; echo time , te , 2.5 ms ; eld of view , fov , 24 cm ; ip angle , fa , 75 ( cid : 3 ) ; number of excitations , nex , 2 ; matrix 512 9 256 ; acquisition time 1 min 57 s ) ; ( b ) transverse t2 - weighted fast spin - echo ( fse ) images ( slice thickness 5 mm ; gap 1 mm ; tr 5 , 000 ms ; te 85 ms ; fov 24 cm ; nex 1 ; matrix 448 9 320 ; acquisition time 2 min 39 s ) ; ( c ) transverse uid - attenuated inversion recovery ( flair ) images ( slice thickness 5 mm ; gap 1 mm ; tr 11 , 000 ms ; te 140 ms ; inversion time 2 , 250 ms ; fov 24 cm ; nex 2 ; matrix 288 9 192 ; acquisition time 5 min 53 s ) ; ( d ) gadolinium618 radiol med ( 2014 ) 119 : 616624 enhanced t1 - weighted volume fspgr images ( slice thickness 1.4 mm ; tr 225 ms ; te 3.2 ms ; fov 24 cm ; fa 15 ( cid : 3 ) ; nex 1 ; matrix 320 9 288 ; acquisition time 23 min ) which were acquired nally , after pwi . two - dimensional 1h - mrsi was performed with the proton brain exam - spectroscopic imaging ( probe / si ) protocol ( ge medical systems )  . 
point - resolved spectroscopy ( press ) pulse sequence ( slice thickness 10 mm ; tr 1 , 500 ms ; te 144 ms ; fov 24 cm ; phase 16 ; acquisition time 6 min 53 s ) , preceded by chemical shift selective saturation ( chess ) pulses for water suppression , was used . 
the peak amplitude of each metabolite was obtained by computing the maximum peak values in the range : lactate and / or lipids ( ll ) , 1.581.0 ppm ; n - acetylaspartate ( naa ) , 2.182.01 ppm ; creatine ( cr ) , 3.153.0 ppm ; choline ( cho ) , 3.363.21 ppthe normalized ratios cho / chon , naa / naan , cr / crn , ll / lln , cho / crn and naa / crn ( where n means normal ) were obtained by dividing each metabolite value within the pathological roi by that within an exactly contralateral and symmetrical normal roi , when possible , or within a contralateral representative normal roi . the non - normalized ratios cho / naa , naa / cr and cho / cr were calculated by dividing the metabolite values in the same spectrum [ 19 ]  . diffusion - weighted imaging was performed using single shot , spin - echo type echo - planar imaging resulting in axial images ( slice thickness 5 mm ; gap 1 mm ; tr 11 , 000 ms ; te 66.6 ms ; fov 24 cm ; matrix 164 9 192 ; acquisition time 44 s ) at b = 0 mm2 / s and b = 1 , 000 mm2 / s as the maximum b value applied along the three orthogonal directions . images with b = 0 provided a set of t2 - weighted images , whereas those with b = 1 , 000 mm2 / s provided three sets of true diffusion images in the x , y , and z directions that were geometrically averaged to obtain one diffusion - weighted image . 
immediately before the acquisition , a 0.01 - mmol / kg pre - bolus dose of gadopentetate dimeglumine was administered to minimise contrast leakage and t1 shortening effects from enhancing lesions [ 15 ]  . 
after the rst ten acquisitions , a bolus of gadopentetate dimeglumine ( 0.1 mmol / kg ) was injected with a mechanical injection pump ( 4 ml / s ) through a 20 - gauge intravenous catheter , followed by a 20 - ml saline ush . 
the echo - planar images were transferred to the remote workstation where the software calculated on a voxel by voxel basis : the curves of signal intensity changes over time , the t2 * relaxation rate ( dr2 * ) , the baseline to be subtracted to the dr2 * curve , and nally the area under the tted dr2 * curve . 
the dr2 * was calculated by the equation dr2 ( cid : 3 ) ( cid : 4 ) ln st = s0 = te , where st is the signal intensity at time t , s0 is the pre - contrast signal intensity ( excluding the rst one or two images acquired during the reaching of the steady - state mr signal ) and te is the echo time . 
cbv maps were generated by the numerical integration of dr2 * for the rst - pass bolus through each pixel , and rcbv was calculated by rcbv = cbv [ lesion ] / cbv [ contralateral tissue ]  . 
the enhancing rois were divided into two groups : recurrent glioma , characterised by steady growth or marked enlargement of enhancement and mass effect , despite steroid therapy , on at least two follow - up examinations , and radiation injury , characterised by stable - appearing or regressing regions of enhancement on at least four follow - up examinations . 
measurements derived from the rst examination showing new contrastenhancing lesion ( s ) in the vicinity of the original treated radiol med ( 2014 ) 119 : 616624 tumours were used for the statistical analysis , whereas the following examinations were only used for recurrent glioma or radiation injury attribution . two experienced neuroradiologists identied in consensus the rois , and when in four cases they disagreed , a third neuroradiologist adjudicated . 
however , to reduce errors linked to the presence of distortion in echo - planar images and / or the non - optimal superimposition between images of various types , rois were manually resized and repositioned on the basis of anatomical references . 
the square root of cho / chon and the reciprocal of square root of ll / lln and cho / crn helped to improve the distribution shape . group differences ( recurrent glioma vs . radiation injury rois ) were evaluated by the independent - samples t test , using the levenes test for equality of variances and the bonferroni correction for multiple comparisons . 
with the leave - one - out method , discriminant analysis is performed times , once for each sample in the data set , several including all samples except one . 
the case that is left out is used for testing the classiers elaborated by the analysis . classication accuracy was dened as the ratio between the number of cases correctly classied and the total number of cases in the set . 
all examined parameters showed a large intraand inter - subject variability , with a large overlap between the two groups ( table 1 )  . stepwise discriminant analysis showed that the predictor set including cho / chon , adc and rcbv maximised the discrimination between recurrent glioma and radiation injury rois ( table 2 )  . 
the leave - one - out method , in fact , showed that 79.3 % of the cases were correctly reclassied when considering all 1h - mrsi ratios or just the normalized ratios ( predictor set , cho / crn ) , 86.2 % when considering 1h - mrsi ratios and adc ( predictor set , cho / crn and adc ) , 89.7 % when considering 1h - mrsi ratios and rcbv ( predictor set , cho / crn , cho / cr and rcbv ) , and 96.6 % when considering ratios , adc and rcbv ( predictor set , cho / chon , adc and rcbv ) ( table 2 )  . 
among the single parameters , rcbv showed the best classication accuracy ( 86.2 % ) , followed by adc ( 82.8 % ) and cho / crn ( 79.3 % ) ; naa / crn , ll / lln , naa / cr and cho / cr were not qualied for the analysis ( table 2 )  . discussion in the follow - up of treated high - grade gliomas , the correct classication of newly appearing enhancing lesions on conventional mri is often a challenge . 
both recurrent gliomas and radiation injury , in fact , can show indistinguishable features , such as contrast enhancement , mass effect and vasogenic oedema [ 1 , 2 ]  . 
1h - mrsi , dwi and pwi help to discriminate tumour recurrence from radiation injury [ 1 , 4 ] , but alone have a limited diagnostic accuracy [ 1 , 5 ]  . 
in fact , stepwise discriminant analysis with a leave - one - out method , including 3 - t 1hmrsi ratios , adc and rcbv , allowed us to achieve 96.6 % classication accuracy ( table 2 )  . the metabolic proles of radiation injury and tumour recurrence / progression have been extensively studied , but there is still no consensus about which spectroscopic parameter ( s ) and / or which threshold level ( s ) ensure the better discrimination accuracy [ 6 , 8 , 9 , 1628 ]  . 
nonnormalized ratios have been largely used and several studies have reported that recurrent tumours have higher cho / naa [ 6 , 8 , 1618 , 26 ] and cho / cr [ 6 , 8 , 1618 , 26 ] and lower naa / cr [ 1618 ] than radiation injury . 
taking into account the threshold values that maximised the discriminative power , the reported diagnostic accuracy of cho / naa and / or cho / cr ranged between 81 and 96.2 % [ 6 , 8 , 16 , 18 , 26 ] , and that of naa / cr reached 84 % only in 620 radiol med ( 2014 ) 119 : 616624 fig . 
1 tumour recurrence in a 54 - year - old man who underwent surgery followed by chemoradiation for a left temporal glioblastoma . t2 - weighted ( a ) and contrast - enhanced t1 - weighted ( b ) images , adc ( c ) and rcbv ( d ) maps , localising flair image ( e ) and proton mr spectra ( 14 ) ( f ) from selected rois , acquired 3 months after completion of radiotherapy . 
in our study , cho / naa and cho / cr were found to be higher and naa / cr lower in recurrent glioma than in radiation injury rois , but only cho / naa reached the signicance level ( table 1 )  . 
furthermore , discrimination analysis showed that cho / naa showed a diagnostic accuracy ( 62.1 % ) substantially lower than previously reported , while cho / cr and naa / cr did not enter into any of predictor sets ( table 2 )  . 
our results were in general agreement with those of other studies that reported no signicant differences for cho / naa [ 8 , 9 , 20 ] , cho / cr [ 8 , 9 , 16 , 19 , 24 ] and naa / cr [ 19 , 26 ]  . normalized ratios have been less widely used . nonetheless our results ( table 1 ) are in line with several studies showing that high cho is correlated with tumour recurrence / progression [ 20 , 22 , 23 , 27 ] , whereas low cr [ 21 , 27 ] and low naa [ 21 , 25 , 28 ] are more consistent with radiation injury . 
however , these authors performed the histopathologic analysis in 10 out of 25 patients . studies obtaining histopathologic conrmation in all patients reported good discriminative power for cho / chon [ 20 , 22 , 23 ] and cho / crn [ 19 , 20 ]  . 
cho / crn , in particular , allowed for the correct diagnosis in 16 out of 17 ( 94 % ) radiol med ( 2014 ) 119 : 616624 fig . 
2 radiation injury in a 57 - year - old woman who underwent surgery followed by chemoradiation for a glioblastoma in the left frontal and contrast - enhanced t1 - weighted ( b ) images , adc ( c ) and rcbv ( d ) maps , localising flair image ( e ) and proton mr spectra ( 14 ) ( f ) from selected rois , acquired 6 months after completion of radiotherapy . 
in fact , comparing diffusion features between radiation injury and recurrent tumour groups , some authors [ 7 , 8 , 10 , 29 , 30 ] measured signicantly higher adc values in the rst group , whereas others [ 31 ] found signicantly higher adc values in the second one and others still [ 9 , 32 ] did not nd any signicant differences in mean adc values . 
the high cellularity that generally characterises neoplasms contributes to lower adc in recurrent / progressed gliomas , while necrotic materials concur to higher adc in radiation injury [ 30 , 31 ]  . 
however , inammatory cell invasion , ischaemic cell swelling , gliosis , brosis , hyperviscosity and vascular changes are possible causes of low adc in radiation injury , while necrotic and / or cystic phenomena and vasogenic oedema can cause higher adc in recurrent tumour [ 7 , 31 ]  . 
in the present study , the adc values were higher in radiation injury than in recurrent glioma , but the difference was not signicant after bonferroni correction ( table 1 )  . 
the radiol med ( 2014 ) 119 : 616624 rois mean range mean range table 1 means , standard deviations and ranges of magnetic resonance ( mr ) parameters in the selected regions of interest ( rois ) no . 
in effect , as a consequence of increased metabolic activity and neoangiogenesis , gliomas tend to have higher rcbv values than radiation necrosis that mainly consists of ischaemia - related changes caused by occlusive vasculopathy . 
however , the presence of telangiectasias , aneurysms and vascular elongation in radiation necrosis , which might result in an increase of rcbv , and the radiation - induced micro - bleeding in recurrent tumour , which could cause a decrease of rcbv , might lead to a misclassication of suspicious lesions [ 35 , 36 ]  . 
however , in agreement with several studies [ 6 , 3537 ] , we found signicantly higher rcbv values in recurrent glioma than in radiation injury rois and the diagnostic accuracy we calculated ( 86.2 % ) was similar to that reported in the literature , ranging between 80 and 87.2 % [ 6 , 8 , 35 ]  . the extreme histopathological heterogeneity of gliomas [ 38 ] and radiation injury [ 39 ] , the frequent mixture of both ndings in the same biopsy specimen [ 19 , 20 , 35 ] , the signicant overlap of metabolic , diffusion and perfusion values between the radiation injury and recurrent glioma groups , as reported in most studies , and the differences in the methodological approaches and number of observations are possible explanations for the variability and discrepancy of the above reported results . considering that no single technique is completely reliable , some authors have combined the results acquired radiol med ( 2014 ) 119 : 616624 with the advanced mr techniques to better characterise newly appearing enhancing lesions [ 612 ]  . 
 [ 11 ] , combining information from adc and several 1hmrsi parameters , found that adc increased the odds of correct differentiation between pure tumour and pure necrosis , while the addition of this parameter did not provide further information beyond that of 1hmrsi ratios in distinguishing mixed tumour and necrosis from either pure tumour or pure necrosis . 
 [ 8 ] , combining 3 - t 1hmrsi , dwi and pwi information using a simple , multiparametric scoring system , improved the diagnostic accuracy from 84.6 % for cho / naa and cho / cr , 86.7 % for adc and 86.7 % for rcbv up to 93.3 % [ 8 ]  . 
 [ 6 ] evaluated and compared the diagnostic performances of 1h - mrs , dwi and pwi without combining data in a pattern recognition analysis [ 6 , 9 ]  . 
on the basis of previous studies combining 1h - mrsi [ 40 ] or 1h - mrsi , dwi and pwi [ 41 ] parameters in a discriminant function - based pattern recognition analysis improved the differentiation between tissues , we applied such approach to differentiate radiation injury from tumour recurrence . 
 [ 10 ] who used a similar approach , although they did not add pwi parameters to the analysis . in which we demonstrated that notable limitations to our study are the lack of histological conrmation and the small number of patients . although histopathological diagnosis should be desirable in all patients , it is not always practicable because of increased risk of morbidity and mortality [ 3 ]  . 
patients received ultrasound ( us ) - guided injection of 7 ml of naropin 0.75 % in t7 , t9 and t11 levels , below the costo - vertebral ligament , until we observed an anterior displacement of the parietal pleura . for the subcapsular lesions , a cervical right phrenic nerve block was associated . 
finally , we investigated statistical correlations between pain and lesions ( histological type , site , dimensions ) , and ablation time and technique ( microwave or radiofrequency ablation )  . results technical success was achieved in all patients . despite the correct anaesthetic diffusion during the ablation , c . 
citta` della salute e della scienza , san giovanni battista , turin , italy 10 patients ( 33.3 % ) reported medium / severe pain and intravenous sedation was required . 
we recommend performing a tpvb in the presence of the anaesthetist . keywords paravertebral block ( cid : 2 ) percutaneous thermal ablation ( cid : 2 ) liver tumours introduction coagulative necrosis , induced by the percutaneous ablation of liver tumours , can cause intense pain both during the procedure and in the following hours , particularly in presence of subcapsular nodules [ 1 ]  . conventional analgesia includes , in addition to local anaesthesia with hydrochloride lidocaine , intravenous infusion of remifentanil and midazolam [ 2 ] ; however , a signicant number of patients experience severe pain especially in the following 24 h . 
detailed anatomical knowledge is required [ 7 ] ; furthermore , we believe ultrasound ( us ) guidance can be useful , even though no reports about its effect are present in the literature . the purpose of this paper was to critically report and discuss the results of our preliminary experience with analgesia by means of a unilateral visceral somatic block in patients undergoing percutaneous thermal ablation of hepatic nodules . comorbidities hypertension diabetes arrhythmia obesity hcc nodules mts nodules total materials and methods patients between october 2011 and august 2012 , a total of 30 patients ( 24 males and 6 females aged between 47 and 85 years , mean age 67.5 years ) undergoing percutaneous ablation of 36 primary or secondary liver tumours by radiofrequency ( rfa , 24 nodules ) or microwave ( mwa , 12 nodules ) were given a right paravertebral block prior to the procedure . more precisely , 21 patients had hepatocellular carcinoma ( hcc ) , whereas in 9 cases lesions from metastatic colorectal carcinoma ( mts ) were present . 
all hcc had developed on cirrhotic livers ( three hbv , seven hcv , four alcoholic , one hbv ? hcv and six cryptogenic )  . seventeen patients had a single neoplasia , while four patients had a bifocal cancer . 
lesions were distinguished into parenchymal , more than 10 mm away from the capsule surface and subcapsular ( tables 2 , 3 )  . procedure the day before the procedure all patients gave written informed consent for the anaesthesiological protocol . 
in the us suite , after cannulation of the radial vein , the vital parameters were monitored ( electrocardiogram , pulse oximetry and blood pressure )  . the patient was positioned with the back facing the two operators : a radiologist and an anaesthetist . 
3 the tip of the needle ( arrow ) reaches the paravertebral space ( head arrow ) uid ( csf ) conrmed that no vessels or dural sac had been damaged . 
if the patient reported an nrs score [ 2 , intravenous remifentanil and propofol was administered in the course of the procedure and a therapy with paracetamol ( 1 g every 8 h ) and tramadol ( 1 vial as required for the next 24 h ) was recommended . statistical analysis possible correlations between pain and thermal ablation were evaluated . 
in 10 / 30 cases ( 33.3 % ) , the patient reported burning pain ( nrs [ 2 ) , which required an increase in infusion speed of remifentanil ( from 0.03 up to a maximum of 0.08 c / kg / min ) in combination with administration of midazolam and acetaminophen ( 1 g every 8 h ) and tramadol ( 1 vial as needed every 5 h ) in the following 24 h . 
three patients ( 10 % ) reported a medium pain intensity ( nrs 36 ) , while seven patients ( 23.3 % ) experienced severe pain ( nrs 710 )  . pain irradiation was visceral , sometimes accompanied by nausea ( 5 / 10 ) , epigastric ( 1 / 10 ) and right shoulder pain ( 4 / 10 )  . 
there were no major complications ; subcutaneous haematoma . reported patient small during the study , considering the progressive optimisation of the technique , we obtained a reduction in time fig . 
in patients with subcapsular lesions , an additional right phrenic nerve block was performed ; with the patients head turned to the left , the cervical segment of the phrenic nerve was located next to the anterior scalene muscle under us guidance , and was anaesthetised with 3 cc of 2 % lidocaine . a skin landmark was placed for the insertion of the thermal ablation needle ; at that level , local anaesthesia with 10 ml of carbocaine was performed . 
an expandable ( with curved , deployable tines ranging from 3 to 5 cm ) needle electrode ( med italia , medolla , italy ) was used to perform , for each lesion , two successive cycles between 80 and 190 w , for a total duration of 2045 min , reaching a temperature of 105 ( cid : 3 ) c . as for the microwave technique ( mw ) ( covidien , dublin , ireland and hs amica , aprilia , latina , italy ) , several cycles were performed : power varied between 45 and 80 w for 1020 min , reaching an intratumoral temperature of 180 ( cid : 3 ) c . hypotension , dened as a decrease in systolic blood pressure [ 30 % from baseline , was treated with intravenous uid infusion . 
it may be noted , however , that lesion histology and number are the variables most closely related to the onset of pain . discussion only two experiences are reported in literature on the use of tpvb during hepatic ablation procedures : ning et al . 
in 2011 [ 7 ] , who prospectively evaluated 20 patients with primary and secondary liver lesions treated with radiofrequency , and a case report published by culp et al . 
5 a , b relationship between pain and technique group a : nrs < or = 2 group b : nrs > 2 13 patients 6 patients 7 patients 4 patients nrs , numerical rating scale ; rfa , radiofrequency ablation ; mwa , microwave ablation . 
in particular , a vasovagal reaction can be related to an accidental migration of the anaesthetic into the epidural space : therefore , we believe that the use of us can be very useful for identifying the paravertebral space , guiding for percutaneous puncture and controlling for the distribution of the anaesthetic . 
in fact , in our experience we have seen accidental epidural migration of the drug , which can cause anaesthesia and motor decit in the lower limbs in addition to a vasovagal reaction . for the evaluation of results , we adopted the same rating scale as used by ning ( nrs ) and established a precise cutoff value ( nrs 2 ) to evaluate the effectiveness of analgesia , considering , with this very restrictive criterion , values above this limit as a treatment failure . 
moreover , the comparison of results is complicated by the fact that half of the patients treated in the chinese experience ( 10 / 20 ) was sedated with propofol during the procedure . 
ning also reported using a multiple injection technique , as done in other experiences reported in the literature about vertebral blocks performed for other reasons [ 15 , 16 ] : ning emphasises the importance of this technique in pain control , suggesting that it may also be responsible for the high rate ( 35 % ) of contralateral blocks in his experience , much higher than the previously reported rates of 20 [ 15 ] and 1.1 % [ 16 ]  . in our experience , complete technical success is based on the us evaluation : in all cases , the correct spread of the anaesthetic with pleural detachment was always demonstrated . 
from our point of view , the most likely hypothesis is that the diffuse visceral pain reported by some patients is due to the stimulation of left sympathetic bres and vagal bres ; the liver presents in fact a dual innervation : both orthosympathetic ( from the seventh to tenth thoracic segment of the spinal cord , which reaches the liver through the splanchnic nerves and the celiac plexus ) and parasympathetic [ 17 ]  . 
this could be a limit of the unilateral vertebral block , even if the high rate of contralateral unexpected block reported by ning represents an interesting matter . unlike ning , we tried to correlate the pain sensation to the different characteristics of the patients : analysis of the data showed no statistically signicant relationship between the variables considered ( procedure duration , technique , location , number and histology of the lesions ) and pain onset . 
the most signicant relationship , however , was between lesion number and histology : considering these results , we can hypothesise that patients with multiple metastases are less responsive to the vertebral block and consequently good candidates for alternative forms of analgesia or additional therapies . in view of the absence of factors that can unequivocally be related to the ineffectiveness of the treatment , we hope to develop as soon as possible a block efcacy test to be used before the ablation procedure . 556 radiol med ( 2014 ) 119 : 549557 fig . 
 however , we believe that our study , designed and conducted in collaboration with the anaesthetists of our interventional radiology unit , conrms the results of nings experience , highlighting the versatility , potentiality and advantages of paravertebral block , during algogenic treatments such as percutaneous hepatic ablation . 
this is a pilot study based on respect for the desires of each patient , who were adequately informed of the benets , possible ineffectiveness and complications of the paravertebral block , assuring intravenous analgesia if needed . these results have led us to design a prospective randomised two - arm study ( vertebral block vs intravenous analgesia ) , for which we have already requested the approval of the hospital ethics committee . 
in fact , we believe that the paravertebral block technique could be extended to other interventional radiology procedures , and especially to liver and biliary procedures such as percutaneous biliary drainage , as already reported in the literature [ 18 , 19 ] , portal embolisation , intrahepatic portosystemic shunt . transjugular theoretically , this technique could be managed independently by the radiologist because naropin is a common anaesthetic that does not require the presence of the anaesthetist for its management . 
however , our long and effective collaboration with anaesthetists suggests that a multidisciplinary management is essential , because of the possible need for intravenous analgesia . conclusions the thoracic paravertebral block is a well - tolerated and safe technique for the anaesthesia of patients undergoing percutaneous thermal ablation of malignant liver tumours . 
in particular , we considered the rate of second treatment with interventional technique or conservative or radical surgery , the incidence of postprocedural complications , and the absorbed radiation dose . results immediate technical success , dened as the cessation of active bleeding , was achieved in all cases . 
at the 12 - month follow - up after embolisation , 22 / 49 conservatively treated patients were found to be pregnant and successfully completed their pregnancy . conclusions selective uterine artery embolisation allows for safe and complete control of haemorrhage in patients with obstetric disease , with a very low incidence of complications and preservation of fertility . keywords obstetric haemorrhages ( cid : 2 ) selective embolization ( cid : 2 ) conservative management ( cid : 2 ) postpartum haemorrhage ( cid : 2 ) ectopic pregnancy introduction obstetric haemorrhage has a strong clinical impact because it constitutes a major risk for the health of the woman and the foetus and for preserving the patients fertility [ 1 ]  . the most common causes of haemorrhaging are postpartum haemorrhage ( pph ) [ 2 ] , congenital or acquired arteriovenous malformation ( the latter usually related to uterine curettage or removal of intrauterine devices ) [ 3 ] , instrumental delivery [ 4 ] , or complications in the treatment of ectopic pregnancy [ 5 ]  . 
in obstetrics , pph is the most frequent cause of admissions to intensive care and is associated with a higher mortality and morbidity rate than other types of haemorrhage [ 6 ]  . in recent years , transcatheter or percutaneous interventional radiology techniques have been applied to different pathological conditions , including disorders of the female pelvis and obstetric and gynaecological pathology [ 7 , 8 ]  . 
in table 1 summary of main clinical data and embolisation results embolisation technique sufcient haemostasis effective dose ( e ) uterine salvage complications pregnancies after 12 months 22 yes 4.3 msv none 10 yes radiol med ( 2014 ) 119 : 607615 diagnosis 22 cervical pregnancy fixed angiography 41 pph 38 yes 12 lsv 27 yes none 11 yes mobile angiography oec 32 pva 14 hysterectomies selective 16 pva 5 ibc 1 gelfoam selective 6 ibc 2 gelfoam 1 pva ? coils 3 no ( a second procedure was needed ) pph postpartum haemorrhage , pva polyvinyl alcohol , ibc isobutyl cyanoacrylate particular , transcatheter embolisation was rst used after bilateral ligation of the hypogastric artery [ 9 ] for the control of severe obstetric haemorrhage . 
this procedure is currently considered the rst choice treatment for controlling potentially fatal bleeding , with the aim of avoiding hysterectomy and preserving the fertility of these patients [ 10 ]  . according to some case series [ 11 ] , when used immediately after bleeding onset , embolisation has an efcacy varying between 85 and 95 % . 
of the 1 , 256 patients treated with interventional procedures at our establishment during that period , 104 were treated for a pathology shown to be at high risk of blood loss . 
the transvaginal approach was preferred in patients with abnormal insertion of the placenta and with placenta praevia . in cases of placental anomaly [ 15 ] , either suspected or conrmed on us analysis ( 22 / 63 ) , we proceeded with integration with mr imaging to obtain a fuller characterisation of the placenta and to permit correct planning of the procedure to avoid fatal haemorrhage . mr imaging was carried out with a 1.5 tesla scanner ( eclipse , pickermarconi , philips , eindhoven , netherlands )  . 
t2 - weighted fast spin - echo axial images were also acquired with suppression of fat tissue and t1 - weighted signals . anaesthesiaresuscitaiton protocol the anaesthesiaresuscitation protocol provided for a careful anaesthesiological evaluation with history taking , physical examination and evaluation of comorbidities and standard preoperative tests including haemochrome , coagulation prole [ prothrombin time ( pt ) , partial thromboplastin time / international normalised ratio ( ptt / inr ) , antithrombin iii ( atiii ) ] dimer and brinogen , glycaemia , urea , creatinine , total and fractional bilirubin , transaminase , lactate dehydrogenase ( ldh ) , creatine phosphokinase ( cpk ) , total protein , albumin , electrolytaemia , urinalysis , blood group and blood type screening . 
this phase was completed with the request of at least 4 units of concentrated erythrocytes of cross - matched blood for the operation and provision of a bed in postoperative intensive the interventional care for procedure . intensive monitoring after the intervention was always performed with local anaesthesia , whereas before the caesarean section balanced anaesthesia was introduced using drugs such as remifentanil in continuous infusion ( 0.050.25 mcg / kg / min ) , propofol 45 mg / kg on induction , cisatracurium 0.2 mg / kg and mixtures of anaesthetic gases ( o2 and sevourane )  . intervention protocol arteriography and subsequent superselective uterine artery embolisation were performed both as elective and emergent procedures , in the operating theatre with a mobile angiograph ( oec 9800 plus , general electric company , milan , italy ) , by an expert interventional radiologist on iliac 24 - h duty . 
selective angiography of the internal arteries and superselective angiography of the uterine arteries were always done with femoral access , generally on the right side . embolisation was achieved with a 5 f cobra catheter ( william cook europe , bjverskov , denmark ) with subsequent use of coaxial microcatheters ( progreat , terumo , tokyo , japan )  . 
vessel occlusion was obtained by injecting 700900 l polyvinyl alcohol ( pva ) ( contour se , boston scientic , natick , ma ) ( 48 / 63 cases ) or reabsorbable particles ( spongel , yamanouchi , tokyo , japan ) ( 3 / 63 cases ) to exclude the ow of blood , as shown by periprocedural angiographic imaging . 
in only one case was pva used in combination with metal coils to occlude the blood vessels , whereas in 11 cases , acrylic glue ( isobutyl cyanoacrylate , ibc ) was used ( in ve cases of cervical pregnancy and in six of re - operation for pph after hysterectomy ) ( table 1 )  . where pva was used , the choice of embolising material was dictated by the clear hypervascularity seen at preoperative angiography , which permitted a durable but not permanent treatment . 
in e , f selective catheterisation of the right uterine artery and its embolisation with ibc results superselective embolisation of the uterine arteries was chosen in all cases with complete exclusion of blood ow from the lesion . 
in particular , there were three cases of pph previously treated with pva which required re - operation ; in two of these patients also the cervicalvaginal branches ( pva ) were embolised and in one case of massive haemorrhage also the hypogastric artery ( spongel )  . 
finally , in 14 cases treated for massive pph , it was impossible to perform conservative treatment and hysterectomy was necessary to control the haemorrhage and achieve haemodynamic stability ( table 1 )  . the entire interventional procedure was conducted while limiting as much as possible the dose of ionising 611 radiol med ( 2014 ) 119 : 607615 fig . 
2 case of placenta praevia in a 34 - year - old woman treated with uterine artery embolisation with polyvinyl alcohol ( pva )  . in a , b the longitudinal and coronal t2 - weighted magnetic resonance ( mr ) images show the abnormal placentation without signicant signs of percretisin c , d selective catheterisation of the left uterine artery and its embolisation with pva . 
in e , f selective catheterisation of the right uterine artery and its embolisation with pva radiation absorbed by the patient and particularly by the uterus , with the aim of avoiding iatrogenic damage that could have compromised the fertility and thus the quality of life of these patients . 
after they returned to the obstetrics unit for the following 2448 h . this , at clinical - imaging follow - up at 1 and 6 months , no adverse reactions correlated to the embolisation procedure were observed . 
in g , h , the second treatment performed with catheterisation of the left hypogastric artery ( early and late time point , respectively ) : no signs of haemorrhage were shown . 
in l embolisation with coils radiol med ( 2014 ) 119 : 607615 22 / 49 ( 44.9 % ) patients treated with superselective embolisation of the uterine arteries and preservation of the uterus were found to be pregnant and carried their pregnancy to term ( table 1 )  . 
in the remaining patients , ovulation proceeded regularly from the ovaries after embolisation procedure , as shown by routine ultrasound and from case - history records . discussion in this study , we retrospectively reviewed the angiographic examinations performed from 2009 to 2010 on 63 patients affected by obstetric conditions with a high risk of bleeding and treated with an interventional approach as a rst - line strategy . 
using this approach , immediate technical success was obtained in all cases , with total cessation of the blood ow , as shown by periprocedural angiography , and limitation of the dose of ionising radiation to which the patients were exposed . these results are consistent with those reported in the literature , although therapeutic success in treating the specic causes of obstetric haemorrhaging has often been evaluated on small patient series . 
 [ 5 ] , for example , published the largest study on the conservative treatment of cervical ectopic pregnancy , with only four cases successfully treated using arterial embolisation of branches of the uterine and transverse cervical arteries using gelfoain this study , the 22 cervical ectopic pregnancies were treated with immediate technical success using embolisation with pva , gelfoam or ibc , with no need for a second embolisation for haemostatic control and without encountering any complications at clinicalimaging follow - up at 1 , 6 , and 12 months . recently , delotte et al . 
other authors [ 14 ] , conversely , have compared the efcacy of preventive embolisation treatment with the classic posthaemorrhagic treatment , nding equivalent success rates for the two groups , but with a signicant reduction in the time for embolisation and in the blood loss for the group receiving preventive treatment . 
in our series , an overall success rate of 100 % for the manoeuvre for the prevention of haemorrhage was found in 41 patients treated for pph , with an immediate technical success rate solely for arterial embolisation of 92.68 % ( 38 / 41 )  . 
it is also necessary to consider the different causes of pph [ 17 ] that may also benet , with varying success rates , from superselective embolisation of the uterine arteries . 
in particular , even if the overall success rate for interventional treatment of pph is between 85 and 95 % [ 11 ] , this percentage diminishes in those cases where the pph is due to placental anomalies , with a range that varies from 20 to 100 % [ 18 ]  . at this time , it is not yet clear what determines this wide range of results ; nevertheless , it may be correlated to the time and the intensity of the forces used to remove the placenta , as well as to patients condition at the time of the intervention . 
in this regard , it is interesting to note that in the 14 cases in which hysterectomy was necessary , the pph in 13 of these was due to placenta previa percreta . 
in patients with placental anomalies , where magnetic resonance ( mr ) imaging led to a suspicion of percretism [ 19 , 20 ] , later conrmed at surgery , hysterectomy was done immediately , without waiting for the afterbirth , to limit further possible sources of bleeding . in three cases of pph , positioning of an intrauterine balloon was combined with the embolisation , and in one of these cases the technique of compressive uterine suture , using the method of b - lynch [ 21 ] was used to render the haemostasis even more efcient . 
in one case of uncontrollable pph after spontaneous birth with episiotomy , embolisation of the uterine arteries and the cervicalvaginal branches with pva was performed after application of vaginal tampons and abdominal packing . 
upon removal of these after 48 h , an aorto - iliac angiogram was obtained to verify the loss of further vascular branches . regarding a second embolisation or subsequent surgery for the control of haemostasis , in 4.76 % of cases ( 3 / 63 ) , the embolisation of several vascular branches had to be combined with the primary embolisation treatment , and in 22.22 % ( 14 / 63 ) , surgical treatment with hysterectomy was required . 
these data are consistent with the literature [ 16 , 22 , 23 ] , in which , especially for phh control and resolution of the underlying pathology , hysterectomy must be combined with arterial embolisation . 
the different results of the embolisation treatment in our series may be due to the percentage of placental anomalies included in the pph , which , as analysed previously , respond signicantly less well to interventional treatment , often requiring the integration of a second treatment with hysterectomy . 
retrothe effective dose of spective analysis of radiation delivered to patients treated with the interventional approach has shown slightly higher doses for patients undergoing embolisation for ectopic pregnancy compared to those treated for pph , obtaining in both cases an effective haemostatic control with limited exposure to ionising radiation . 614 radiol med ( 2014 ) 119 : 607615 early and late complications linked to the interventional procedure include fever , vascular perforations , ischaemia of the lower limbs , and necrosis of the uterus or of the walls of the bladder or rectuin our study , at 1and 6 - month follow - up , no complications related to the interventional procedure were observed . 
this information is in keeping with the low complication rate ( about 3 % ) reported in some other studies [ 22 ] , and correlates with the nature of the embolising agent chosen [ 24 ]  . with respect to hysterectomy , considered the standard treatment the control of both pph and obstetric haemorrhages in general , embolisation has proved efcient in guaranteeing and preserving the fertility of the patient treated with superselective embolisation of the uterine arteries and preservation of the uterus [ 2527 ]  . 
some studies [ 10 ] , for example , have reported preservation of the uterus in 95 % of cases of pph treated with embolisation and the return of menstrual ow in all patients , with a pregnancy rate of 20 % in these patients . 
the increased pregnancy rate in our study compared with the current literature [ 10 , 25 , 28 ] can presumably be ascribed to the greater safety of the preventive treatment compared to the standard approach that results in reduced operating times and a lower blood loss [ 14 ] , as well as reduced length of stay in postoperative intensive care ( about 2448 h )  . 
only one case of amenorrhoea due to hypothalamic hypogonadism after embolisation with gelfoam was reported in the literature [ 29 ] , which may be resolved with the aid of hormone stimulation . even though the small series is a limitation of our study , easy access to dedicated instruments in the operating theatre , team work and professional expertise accounted for the high rate of immediate success found in our study . 
in the assessment of long - term outcomes following the interventional approach to pathologies at high risk of bleeding , such as ectopic pregnancy and pph , conicting results continue to be reported with regard the recurrence rate after subsequent pregnancies [ 10 , 28 , 30 ]  . 
this relatively new question is probably the product of the high success rate of the interventional procedure in these pathologies which allows the conservative treatment of patients who would otherwise be subjected to hysterectomy . 
however , the lack , for clinical reasons , of a real control group treated only with hysterectomy and followed in parallel to the embolising treatment , certainly represents the main limitation of this study . 
a longer follow - up period would perhaps have permitted clarication of this aspect and will certainly be the subject of further studies . future prospects for study are thus analysis of this treatment on a greater number of patients and over a longer follow - up period . 
furthermore , given the excellent safety and effectiveness demonstrated by this type of treatment and the low exposure to ionising radiation during the interventional procedure , a pilot study will soon be begun on the possibility of performing the embolisation in the immediate ante - partum period with a view to further reducing the risks of massive haemorrhaging for this type of patient . conclusions the percutaneous method in the prevention / treatment of obstetric haemorrhage is currently an example of the integration of imaging and clinical methods , and it proves extremely useful in reducing the rate , although still high , of mortality linked to postpartum haemorrhage . 
guglielmi department of radiology , university of foggia , viale luigi pinto , 1 , 71100 foggia , italy mechanical properties of achilles tendon in children with clubfoot . keywords real - time elastosonography ( rte ) ( cid : 2 ) clubfoot ( cid : 2 ) achilles tendons introduction congenital clubfoot is a common disorder present at birth that affects the musculoskeletal system with an overall incidence of 1 : 1000 newborns . 
this congenital defect includes four main types of foot alteration : equinus foot , hindfoot varus , forefoot adductus and cavus [ 1 ]  . congenital several theories have been suggested in order to explain clubfoot , idiopathic the pathogenesis of including genetic factors and vascular deciencies . 
abnormal development of leg muscles , genetic defects in growth of the connective tissue , abnormal tendon insertions , and tight deltoid ligaments , have all been described [ 36 ] as possible aetiologies . idiopathic clubfeet usually show resistance to manipulation due to deformity of the affected feet . 
this pathology has been classied into two systems , the dimeglio and the pirani classications , both correlating the severity of this condition with physical ndings [ 7 ]  . 
currently , the ponseti method is the preferred nonsurgical technique used for treatment , and it consists in a deep stretching of the peronei and foot dorsi - exor muscles , in order to normalise foot position and to provide correct posture [ 8 ]  . to the best of our knowledge , there are no studies investigating the properties of mechanical stiffness of achilles tendon in patients affected by unilateral idiopathic 602 radiol med ( 2014 ) 119 : 601606 clubfoot . 
it was observed that unilateral clubfoot might be associated with a lower calf circumference , and this may result in quantitative differences of the muscular masses , when two series of patients with unilateral clubfoot are compared [ 9 ]  . 
moreover , it has been reported by several authors , such as ippolito and ponseti , that a greater presence of brous tissue can be observed in the muscles , in the fascia , in the ligaments and in the tendon sheaths [ 10 ] : this may lead to an increased stiffness of all the involved anatomical structures . recently , real - time elastosonography ( rte ) has been proposed as a technique able to assess the differences in stiffness between diverse tissues using ultrasound ( us )  . 
it has also been suggested for the evaluation of tissue distortion , as well as of muscle and tendon stiffness [ 11 ]  . the aim of our study was to assess the usefulness of rte of the achilles tendon in the clinical evaluation of a cohort of children affected by unilateral clubfoot , not surgically treated . materials and methods study population after the approval from the ethics committee of our institution , 20 young patients ( 8 males and 12 females ) affected by unilateral idiopathic clubfoot were consecutively examined . 
written informed consent was obtained from the parents of all children , in accordance with the declaration of helsinki . the clinical evaluation for all enrolled patients was performed by a single orthopaedic surgeon with 35 years of clinical experience at our institution . 
children who were found to be affected by any systemic condition , any connective tissue disorder , any metabolic and / or endocrine disease , or patients with a history of tendon injury and surgery were excluded from the study group . 
the entire study group was then divided into two subgroups , according to age : the rst group ( group 1 ) included 13 children aged between 0 and 18 months , while the second group ( group 2 ) included 7 children aged from 18 to 60 months . 
b - mode ultrasound was performed along the entire length of the achilles tendon , bilaterally , from the distal enthesis to the proximal myotendinous junction , dening its echotexture and searching for sonographic signs of previous traumatic injury or signs of tendon degeneration . 
the total amount of deformation used to compute the strain of elastogram is represented by the sum of the inherent or physiologic patient - motion and the external compression form the probe . the probe is held in place manually , while the patient is kept at rest . 
a range of colours shows the relative hardness and softness of the roi , conventionally varying from blue to red . first , tendons from the affected clubfoot were evaluated , and then measurements were obtained from the contralateral healthy tendons . 
overall examination time was approximately 14 min for each patient ( 7 min per side )  . the colour - coded images were analysed on a personal computer using the qlab ( developed by philips medical systems ) , which allows the operator to select a 30 - frame images / cine - loop and to position manually two circular rois , with the same area of 4 mm , identied in our study as roi 1 and roi 2 . roi 2 was used as a reference standard and it was positioned on the bone tissue . 
analysis was repeated in quadruplets ( one for each examination ) and nal data were expressed as an average value of the different results . data analysis the students t test and standard deviation ( sd ) were used to assess variation of continuous parameters in order to obtain an average value of the different real - time measurements . 
2 a conventional axial b - mode ultrasound scan and superimposed , b sonoelastogram of the achilles tendon , at the distal third , in clubfoot showing a diffuse hyperechoic pattern , in grey scale , associated with low elasticity of the structure , evaluated by sonoelastography . 
in our study , a sonoelastosonography evaluation was performed to obtain a tensile test which shown a condition of chronic tendinopathy involving the entire achilles tendon . a chronic state of tendinopathy might lead to softening and weakening of collagenic and elastin structures increasing the risk of tendon rupture [ 15 ]  . 
although no signs of achillodynia were present , after rte a decreased elasticity of achilles tendons was observed in clubfoot . tendons while b - mode morphological ultrasound provided structural information , sonoelastography showed a light red colour code along the structure of achilles tendon with reduced values of elasticity comparing to the normal internal control [ 16 ]  . ( healthy ) contralateral therefore us sonoelastography is considered to be a helpful technique for detection and characterisation of achilles tendinopathy for its high sensitivity and specicity in the detection of intratendon alterations [ 17 ]  . 
main rte alterations were observed on the distal portion of the tendon , which was represented by a weaker structure , both at rest and on stress , characterised by decreased values of elasticity and a green colour [ 18 ]  . 
4 a longitudinal b - mode ultrasound scan and superimposed , b sonoelastogram of the achilles tendon , at clubfoot showing a diffuse hyperechoic pattern highlighted in grey scale , associated with low elasticity of the structure , evaluated by sonoelastography . 
the same ratio in group 2 ( patients aged 1860 months patients ) was statistically different , but a lower discrepancy could be observed between the values from the two different groups ( table 1 )  . 
in longitudinal scans , homogenous , hyperechoic , linear continuous shadows with regular margins appeared , in the achilles tendons ; while , in transverse scans , the achilles tendons were elliptical , homogenous and echogenic in appearance . idiopathic clubfoot is one of the most common musculoskeletal congenital disorders with several deformities affecting bone , ligaments and tendons [ 12 , 13 ]  . 
a nonsurgical treatment technique , named the ponseti method , is the currently preferred treatment method and is usually discussion radiol med ( 2014 ) 119 : 601606 to standardise the evaluation of the tendon itself . 
in fact , during the growth of the tendons , these structures undergo morphological changes , such as an increased vascularisation occurring on the distal and proximal thirds and an associated loss of vascularisation in the middle third [ 19 ]  . moreover , de zordo et al . 
 [ 17 ] have observed a distinct softening of symptomatic achilles tendons , occurring on the distal and middle third more frequently than the proximal third of the tendon . a strongly heterogeneous pattern was found , and this result is probably associated with the different mechanical properties of the various tissue components within normal and pathologic tendons [ 18 ]  . 
moreover , we were able to assess both resting and stress conditions of the tendon in real time , using sonoelastography . in our present study , a strong correlation was found between the patients age and rte appearance , without any inuence of the patients sex . one of the main limitations of the study is that intraand inter - observer agreement of rte is still an open question , which has been little investigated in the medical literature . however , all the operators had the same learning curve with regard to rte examination , each with 32 months of experience . 
therefore , we decided to perform the statistical analysis on the mean values of elastic ratios obtained in the different subgroups of patients by three independent operators with the same level of expertise in rte . conclusion rte is a feasible and simple technique , which allows the evaluation of mechanical properties of the achilles tendons in clubfoot , including homogeneous or relatively homogeneous patterns . 
one of the main limitations of this study was a lack of correlation of the us data with the histopathological ndings in achilles tendons from the patients affected by clubfoot . 
another limitation was the overall small cohort of children enrolled , as well as the wide range included in group 2 , ( relatively too small a subgroup for such a broad range )  . 
all examinations were acquired after contrast - agent administration in a craniocaudal direction from the lung apex to base during a single inspiratory breath - hold , with standardised parameters . 
each lesion was scanned with 1315 slices that could be repeated whenever necessary to document the needle track and for lesion centring , by positioning a metallic marker perpendicular to the centring light to indicate the point of needle access . 
berti department of diagnostic sciences , uoc of pathology , san carlo borromeo hospital , via pio ii 3 , 20153 milan , italy results age was found to be a factor inuencing the complications : pneumothorax in young subjects ( 31 % ) and parenchymal haemorrhage in the elderly ( 30 % ) , with cnb but not with fnab . 
we had more complications with the right lung : 50 % of pneumothorax cases in the upper lobe with cnb and 40 % of cases of haemorrhage in the lower lobe with fnab . 
when percutaneous biopsy is used to establish the diagnostic and therapeutic workup of a lung lesion , one must carefully evaluate the costbenet ratio as well as the most frequent complicationspneumothorax and parenchymal haemorrhage [ 5 , 9 , 11 ]  . 
although both complications usually resolve spontaneously , the occurrence of pneumothorax during the procedure may lead to an inconclusive result and , in a variable percentage of cases , worsening pneumothorax may require placement of a chest drain , whereas parenchymal haemorrhage may necessitate hospitalisation [ 12 ]  . the rst aim of this study was to assess the type and incidence of complications arising in ct - guided lung biopsies performed with ne - needle aspiration ( fnab ) or cutting needle ( cnb ) , by analysing the different variables related to the patient ( age ) , the lesion ( dimension , nature , side ) and the procedure ( number of passes , access and needle calibre )  . 
1 lung biopsy with an 18 - g trucut needle of a lesion of the dorsal segment of the right upper lobe , performed with a single pass with posterior access fig . 
2 fine - needle aspiration biopsy ( fnab ) with a 22 - g needle of a 1.2 - cm nodule in the dorsal segment of right lung lower lobe with a single pass and posterior access materials and methods this retrospective study was performed by analysing a series of 121 patients , 15.3 % of them with known oncological disease , who underwent ct - guided lung biopsy using cnb or fnab between 01 - 01 - 2010 and 31 - 12 - 2011 . a total of 122 biopsies were performed in 76 men and 45 574 radiol med ( 2014 ) 119 : 572594 fig . 
any platelet antiaggregant and anticoagulant treatment was suspended a few days prior to the procedure and subsequently resumed by the patient , in accordance with the haematologists indications . all patients had previously undergone a ct or highresolution ct ( hrct ) examination which identied and dened the lesion . 
all the biopsies were carried out as inpatient or day - case procedures . radiol med ( 2014 ) 119 : 572594 all patients received verbal explanations about biopsy procedure and its possible complications , and they subsequently read and signed the written informed consent form , which included consent for using their anonymised clinical data for scientic purposes . the possible complications of a ct - guided lung biopsy are pneumothorax , haemorrhage , haemothorax , haemoptysis , rarely seeding of neoplastic cells along needle path , cardiac effusion , and pulmonary infections ( pneumonia , empyema ) , as well as risks attributable to the administration of the drugs used during the procedure . 
we recorded as pneumothorax any amount of air appearing between the lung and pleura . expiratory chest radiography was performed in all patients 46 h after the procedure to assess the appearance or the growth of a pneumothorax previously detected on postprocedure ct . 
if it became stable or smaller , the patient was discharged , whereas if it increased the surgeons advice was sought for possible placement of a chest drain the event of complications arising in patients fig . 
4 cnb age cnb age . hemoptysis suffusion suffusion + pnx ( 0% ) ( 50% ) 6 ( 30% ) 2 ( 10% ) ( 10% ) young emottisi ( 0% ) ( 50% ) 6 ( 30% ) 2 ( 10% ) ( 10% ) giovane ( 9% ) ( 35% ) ( 17% ) ( 30% ) ( 9% ) elderly ( 9% ) ( 35% ) ( 17% ) ( 30% ) ( 9% ) vecchio hemoptysis suffusion suffusion + pnx 100% 100% young 100% 100% elderly giovane vecchio cnb et . soffusione emorragica soffusione emorragica e pnx emottisi soffusione emorragica soffusione emorragica e pnx 100% 100% 576 radiol med ( 2014 ) 119 : 572594 who underwent the biopsy as a day - case procedure , the need for hospitalisation was assessed in relation to the clinical symptoms and signs . biopsy protocol the examinations were performed on a 16 - slice lightspeed ct system ( ge medical system , milwaukee wi , usa ) and on a 128 - slice somatom denition as ? ct system ( siemens healthcare , erlangen , germany )  . all examinations were acquired in a craniocaudal direction from the lung apex to base , during a single inspiratory breath - hold with the following standardised parameters : thickness 2.5 mm , collimation 10 mm , eld of view 320360 mm , pitch 1.75 , 120 kv ; automatic tube current modulation ( ma ) ; standard reconstruction lter ; lung window . 
patients received iodinated contrast agents : ioversolo 350 ( covidien , italy ) and iopromide 370 ( bayer schering pharma , germany )  . according to lesion location , the ideal needle trajectory from the skin to the lesion was established . 
lesion centring was performed on the axial image of the image stack , in which the lesion presents the greatest extension and needle access is less hampered by the presence of anatomical structures . 
on the chosen image , a metallic marker is placed perpendicular to the centring light , which indicates the point that will be used for needle access . the needles used were chibell ( biopsybell , modena , for fnab italy ) with a calibre of 20 and 22 g , procedures , and biomol ( hp service hospital , latina , italy ) with a calibre of 18 , 20 and 21 g for cnb procedures . 
during the cnb procedures , no pathologist was present in the room , whereas during the fnab procedures , a pathologist was on site to extemporarily evaluate the adequacy of the sample . 
sample adequacy was assessed in real time alternately by two equally experienced pathologists , who subsequently performed also the nal microscope slide . reading of we used test tubes with and without xative , skin disinfectant , bevelled glass slides , cover slips , racks , xatives , haematoxylineosin staining scale , papanicolaou stain , and a diagnostic room set up with a hood and an optical microscope . in relation to the pathological report , the result of each procedure was classied as diagnostic or nondiagnostic . samples were dened nondiagnostic in the event of insufcient , cell - free , or strongly haematic material . diagnostic samples were grouped by histological type . the ct images were assessed for the following parameters : needle access ( anterior , posterior , lateral ) , lesion site ( upper or lower right lobe , middle lobe , upper or lower left lobe ) , lesion size ( large if [ 3.5 cm or small if b3.5 cm ) , patients age [ \65 years ( young ) and c65 years ( elderly ) ] , needle gauge , number of needle passes , and lesion histology ; all variables were correlated to the type and incidence of complications recorded with the two biopsy methods . 
5 fnab with an 18 - g needle of a lesion of the right upper lung lobe with single pass and lateral access ( a ) ; at the end of the procedure , a small parenchymal detachment , and circumscribed haemorrhage along the needle track ( b ) radiol med ( 2014 ) 119 : 572594 statistical analysis results complications the baseline characteristics of the study population were expressed as mean standard deviation for continuous variables , and as percentages for categorical variables . correlation between the variables considered and the presence of ( pneumothorax , parenchymal haemorrhage , haemoptysis ) was calculated using the chisquare test . 
in the graphs on the left , each histogram represents the number of observations classied according to the labels / classes placed under the columns , with an indication , within each label / class , of the type of complication ( the various colours of the histogram )  . 
no case of parenchymal haemorrhage necessitated either hospitalisation ( of the day cases ) or additional treatments ( of the inpatients )  . table 3 fine - needle aspiration biopsy vs . 
6 fnab : age fnab ne - needle aspiration biopsy , cnb core - needle biopsy 578 radiol med ( 2014 ) 119 : 572594 the patients were analysed in relation to age and type of biopsy . 
haemoptysis occurred in 9 % of elderly patients , either immediately or a few hours after the end of the procedure ; no case of haemoptysis was recorded among the young patients . 
7 cnb : lesion size cnb lesion size . hemoptysis suffusion suffusion + pnx hemoptysis suffusion suffusion + pnx 100% ( 6% ) ( 44% ) ( 17% ) ( 28% ) ( 6% ) large ( 6% ) ( 44% ) ( 17% ) ( 28% ) ( 6% ) grande ( 7% ) ( 33% ) ( 27% ) ( 20% ) ( 13% ) small ( 7% ) ( 33% ) ( 27% ) ( 20% ) ( 13% ) piccolo 100% large 100% 100% small 100% grande piccolo cnb dimensione nodulo . emottisi soffusione emorragica soffusione emorragica e pnx emottisi soffusione emorragica soffusione emorragica e pnx 100% radiol med ( 2014 ) 119 : 572594 fig . 
10 fnab : needle approach fnab needle approach . radiol med ( 2014 ) 119 : 572594 suffusion suffusion + pnx 100% 100% 100% suffusion suffusion + pnx ( 50% ) ( 21% ) ( 29% ) ( 0% ) rear ( 50% ) ( 21% ) ( 29% ) ( 0% ) ( 56% ) ( 22% ) ( 11% ) ( 11% ) ( 80% ) ( 20% ) ( 0% ) ( 0% ) anterior lateral ( 56% ) ( 22% ) ( 11% ) ( 11% ) ( 80% ) ( 20% ) ( 0% ) ( 0% ) lateral rear anterior fnab accesso ago . soffusione emorragica soffusione emorragica e pnx soffusione emorragica soffusione emorragica e pnx 100% 100% 100% 100% anteriore laterale posteriore anteriore laterale posteriore in addition , we correlated complications with lesion location , in terms of side and lobe . 
11 cnb : right lung cnb right lung . hemoptysis suffusion suffusion + pnx 100% 100% 100% lower 100% 100% middle emottisi soffusione emorragica soffusione emorragica e pnx 100% 100% upper cnb polmone destro . ( 0% ) ( 20% ) 100% 100% 100% hemoptysis suffusion suffusion + pnx ( 0% ) ( 20% ) ( 50% ) ( 0% ) ( 0% ) ( 0% ) ( 30% ) ( 0% ) lower middle upper emottisi soffusione emorragica soffusione emorragica e pnx ( 14% ) ( 29% ) ( 14% ) ( 29% ) ( 14% ) ( 14% ) ( 29% ) ( 14% ) ( 29% ) ( 14% ) ( 50% ) ( 30% ) ( 0% ) ( 0% ) ( 0% ) ( 0% ) inferiore medio superiore inferiore superiore medio patients undergoing cnb had inammatory disease , or lesion histology since the histological results were widely heterogeneous . the rate of nondiagnostic samples at denitive histological examination , assessed on the basis of the pathology reports , was 18.1 % for cnb and 10.7 % for fnab ( table 6 )  . we then calculated the diagnostic accuracy of the two evaluation revealed that fnab , methods . 
12 fnab : right lung fnab right lung . suffusion suffusion + pnx radiol med ( 2014 ) 119 : 572594 suffusion suffusion + pnx 100% 100% ( 0% ) ( 0% ) ( 17% ) 0 ( 17% ) ( 0% ) middle upper 100% middle fnab polmone destro . soffusione emorragica soffusione emorragica e pnx soffusione emorragica soffusione emorragica e pnx 100% 100% 100% ( 67% ) ( 67% ) 100% lower 100% 100% ( 0% ) ( 0% ) ( 17% ) 0 ( 17% ) ( 0% ) inferiore medio superiore inferiore medio superiore ( 40% ) ( 20% ) ( 40% ) ( 0% ) lower ( 40% ) ( 20% ) ( 40% ) ( 0% ) upper discussion in pulmonary lesions , it is very important to distinguish correctly between benign and malignant lesions , since the pathological diagnosis will determine the treatment to be applied [ 13 ]  . one diagnostic method used in the evaluation of lung cancer is bronchoscopy , but when this is not sufcient for the diagnosis , particularly in peripheral pulmonary lesions or pleural lesions , a trans - thoracic biopsy is performed [ 14 ]  . 
13 cnb : left lung cnb left lung . hemoptysis suffusion suffusion + pnx 100% 100% 100% lower 100% upper cnb polmone sinistro . emottisi soffusione emorragica soffusione emorragica e pnx 100% 100% hemoptysis suffusion suffusion + pnx ( 11% ) ( 56% ) ( 11% ) ( 11% ) ( 11% ) ( 0% ) ( 67% ) ( 17% ) ( 0% ) ( 17% ) lower upper emottisi soffusione emorragica soffusione emorragica e pnx ( 11% ) ( 56% ) ( 11% ) ( 11% ) ( 11% ) ( 0% ) ( 67% ) ( 17% ) ( 0% ) ( 17% ) inferiore superiore inferiore superiore who had inammatory disease . 
this did not allow us to evaluate diagnostic accuracy in relation to the benign or malignant nature of the lesion . the literature contains heterogeneous data on this topic . fnab has a diagnostic accuracy that varies from 64 to 97 % [ 7 ]  . 
this can in part be justied by the severity of the judgement to which the samples were subjected : material lacking malignant cells should not be considered negative for malignancy , unless it is possible to formulate a clear and specic diagnosis of a benign disease [ 19 ]  . in some studies , diagnostic accuracy appears to be higher in the case of larger lesions and of fnab performed in the presence of a pathologist to assess sample adequacy is not [ 4 ]  . 
14 fnab : left lung radiol med ( 2014 ) 119 : 572594 fnab left lung . suffusion ( 56% ) ( 22% ) ( 22% ) lower ( 56% ) ( 22% ) ( 22% ) ( 71% ) ( 14% ) upper ( 71% ) ( 14% ) ( 14% ) ( 14% ) 100% 100% 100% suffusion lower upper fnab polmone sinistro . soffusione emorragica soffusione emorragica 100% 100% 100% inferiore superiore inferiore superiore available for extemporaneous evaluation of the samples . this may lead the operator to use cutting needles even when in the presence of small nodules [ 20 , 21 ]  . charig and phillips [ 20 ] have shown that the diagnostic accuracy of cnb is similar to that of fnab , if performed with a pathologist on site . 
tsukada [ 24 ] found a gradual and progressive decrease of diagnostic accuracy values with decreasing lesion size , from 100 % ( for lesions \5 cm ) to 67 % ( for lesions \1 cm )  . 
we therefore recorded , regardless of the reported rate of pneumothorax after ct - guided lung biopsies ranges from 4 % [ 26 ] to 62 % [ 27 ]  . 
in the present study , where any air ap between the pleura and the lung was considered a pneumothorax , the incidence was 21.3 % and thus falls within the reported range ( table 2 )  . 
17 cnb : number of passes 100% 0%0% 100% hemoptysis suffusion suffusion e pnx 100% 100% emottisi soffusione emorragica soffusione emorragica e pnx 100% 0%0% 100% 100% 100% 100% 100% 590 radiol med ( 2014 ) 119 : 572594 results are consistent with the percentages reported in other studies [ 29 ]  . lesions [ 30 , 31 ] or even a 4 % decrease in risk for each 10 - mm increment in lesion diameter [ 5 ]  . the onset of pneumothorax was found to correlate with variables related to both the procedure and the patient ( table 3 )  . 
18 fnab : number of passes fnab number of passes . suffusion suffusion + pnx suffusion suffusion + pnx 100% 100% 100% ( 55% ) ( 27% ) ( 14% ) ( 5% ) ( 55% ) ( 27% ) ( 14% ) ( 5% ) ( 67% ) ( 33% ) ( 0% ) ( 0% ) ( 67% ) ( 33% ) ( 0% ) ( 0% ) fnab numero passaggi . soffusione emorragica soffusione emorragica e pnx soffusione emorragica soffusione emorragica e pnx 100% 100% 100% radiol med ( 2014 ) 119 : 572594 ( 7% ) ( 29% ) ( 21% ) ( 29% ) ( 14% ) 100% ( 7% ) ( 29% ) ( 21% ) ( 29% ) ( 14% ) 100% cnb histologic type . hemoptysis suffusion suffusion + pnx ( 0% ) ( 0% ) ( 0% ) ( 0% ) ( 0% ) ( 100% ) ( 40% ) ( 0% ) ( 0% ) ( 0% ) ( 20% ) ( 20% ) ( 40% ) ( 40% ) ( 20% ) ( 20% ) ( 0% ) ( 40% ) 2 ( 20% ) notevaluable adenocarcionoma bronchioloalveolar sccl squamos ( 0% ) ( 0% ) inflammation ( 100% ) 100% 100% ( 100% ) 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% adenocarcinoma bronchioloalveolar sccl squamous not evaluable inflammation cnb istologia . emottisi soffusione emorragica soffusione emorragica e pnx ( 0% ) ( 0% ) ( 0% ) ( 0% ) ( 0% ) ( 100% ) ( 40% ) ( 0% ) ( 0% ) ( 0% ) ( 20% ) ( 20% ) ( 40% ) ( 40% ) ( 20% ) ( 20% ) ( 0% ) ( 40% ) 2 ( 20% ) non valutabile k adenocarcinoma k bronchioloalveolare k microcitoma k spinocellulare patologia infiammatoria ( 0% ) ( 0% ) ( 17% ) ( 67% ) 100% ( 17% ) ( 67% ) 100% k adenocarcinoma k bronchioloalveolare k microcitoma k spinocellulare non valutabile patologia infiammatoria fig . 
this can be ascribed to the fact that many of these patients normally took anti - aggregating or anticoagulant therapies and thus suffered from coagulation disorders , a nding in contrast with other authors reports [ 5 ]  . as described in the literature [ 5 ] , posterior access ( table 5 ) carries a greater risk of complications . 
21 diagnostic accuracy of fnab and cnb based on dimension of lesions 594 conclusions fnab seems to be burdened by a lower complication rate than cnb in terms of both absolute values and in relation to variables inherent to the patient , the procedure and the lesion . 
the diagnostic accuracy is higher for fnab and , if this technique is performed in the presence of a pathologist , it is characterised by a lower rate of inadequate samples than cnb . 
a protocol with exposure parameters based on patient body mass index ( bmi ) and with images reconstructed using fbp ( group a ) was compared with a protocol with images acquired using tube current decided by an automatic exposure control system and reconstructed using aidr ( group b )  . 
gravina laboratory of radiobiology , division of radiotherapy , department of biotechnological and applied clinical sciences , university of laquila , via vetoio 1 , 67100 laquila , italy noise was signicantly lower in group b with consequent higher signal - to - noise ( snr ) and contrast - to - noise ( cnr ) ( p \ 0.0001 ) compared with group a . 
subjective quality parameters were also signicantly higher in group b . conclusions comparative analysis by propensity score matching conrms that aidr 3d with automatic exposure control is able to reduce signicantly the mean radiation dose and improve the image quality compared with traditional fbp without exposure modulation . keywords coronary ct angiography ( cid : 2 ) adaptive iterative reconstruction ( cid : 2 ) image noise ( cid : 2 ) radiation dose ( cid : 2 ) image quality introduction coronary artery disease ( cad ) is the leading cause of death in developed countries [ 1 , 2 ] , and cardiac catheterisation remains the standard of reference for the evaluation of the coronary arteries . 
these methods provide signicant reductions in radiation dose and have been reported to result in similar image quality in multicentre studies [ 6 ]  . three recent reports [ 79 ] have indicated that adaptive iterative dose reduction ( aidr 3d ) with built - in automatic exposure control ( sureexposure 3d ) has better performance than traditional ltered back - projection ( fbp ) without automatic dose modulation . 
in order to mitigate methodological biases , the image quality and mean radiation doses of aidr 3d with sureexposure 3d were compared with traditional fbp without automatic dose modulation using a 640 - slice ct scanner and a one - beat prospective acquisition by propensity score matching . 
this powerful statistical approach , attempting to reduce the bias due to confounding variables that could be found in an estimate of the outcome measure obtained from simply comparing outcomes among units belonging to different study groups , simulates a randomisation process by creating an absolute comparability of clinical characteristics among groups . 
thus , the aim of this study was to conrm , using this powerful statistical approach , whether the use of aidr 3d with sureexposure 3d really provides clinical and dosimetric advantages with respect to traditional fbp without dose modulation . materials and methods patients from october 2011 to december 2012 , 272 consecutive patients scheduled for coronary ct were prospectively studied with a 640 - slice ct scanner ( aquilion one , toshiba medical systems )  . 
exclusion criteria were impaired renal function ( serum creatinine [ 1.5 mg / dl ) , previous coronary artery interventions , heart rate [ 65 beats per minute ( bpm ) after beta - blocker premedication and severe calcication of the major coronary arteries . 
images were evaluated for the presence of signicant stenosis , which was dened as a narrowing of the coronary lumen equal to or exceeding 50 % of the cross - sectional area . 
institutional review board approval was obtained and all patients provided informed consent before the procedures . acquisition protocol injector all ct scans were acquired using a 640 - slice ct scanner . one hour before imaging , the patients blood pressure and heart rate were recorded . 
just before the acquisition , 0.3 mg of sublingual nitroglycerin ( isosorbide dinitrate , carvasin 5 mg , wyeth lederle ) was administered to all patients [ 10 ]  . 
sixty millilitres of nonionic contrast medium ( iomeron 400 , bracco diagnostic , milan , italy ) was injected into the antecubital vein at 5 ml / s , followed by 40 ml of saline solution at the same ow rate , using a dual power ( stellant , medrad , indianola , pa , usa )  . 
the timing to start the ct acquisition was determined using bolus tracking ( sure start ) in the descending aorta with a scan start threshold of 300 hounseld units ( hu )  . 
for each patient , the phase with the least artefacts was automatically 644 radiol med ( 2014 ) 119 : 642649 determined by the systea motion map of the sinogram was traced by the system and the phase with the least movement was chosen ( phasexact , toshiba medical systems , tochigi , japan )  . 
the maximum number of reconstructed slices was 640 with 0.5 mm thickness and 0.25 mm interval , obtained by means of the proprietary double - slice technique and cone - beam reconstruction algorithm ( conexact , toshiba medical systems , tochikiken , japan )  . 
issues such as motion and poor gating were not taken into account in the subjective assessment , as they could not be ascribed to the reconstruction algoriththe coronary arteries were subdivided into 15 segments according to the american heart association classication [ 11 ]  . 
the 15 coronary artery segments were categorised into three segmental classes as follows : ( 1 ) proximal [ proximal right coronary artery ( rca ) , left main coronary artery ( lm ) , proximal left circumex artery ( lcx ) , proximal left anterior descending artery ( lad ) ] ; ( 2 ) mid [ mid rca , distal rca , ramus intermedius ( r . intermedius ) , obtuse marginalis ( om ) , rst diagonal branch ( d1 ) , mid lad ] ; and ( 3 ) distal segmental classes [ posterior descending artery ( pda ) , distal lcx , second diagonal branch ( d2 ) , distal lad ] [ 8 ]  . 
1 a , b measurement of signal and noise in the ascending aorta , above the coronary ostia , in two patients with the same body mass index of 26 . 
the signal ( vi ) of the proximal coronary arteries was measured as the mean attenuation value ( hu ) within circular rois drawn in the central portion of both the left main coronary artery and right coronary artery , with care to avoid inclusion of the coronary vessel wall [ 8 ]  . 
ct density of the epicardial fat ( ve ) surrounding the artery was measured by placing a roi immediately next to the artery ( both left main coronary artery and right coronary artery ) [ 10 ]  . 
snr and cnr were calculated as follows : snr = vi / n , cnr = ( vi - ve ) / n ; snr was calculated by dividing the density by the image noise and , as for the cnr , ct density of the epicardial fat ( ve ) was subtracted from signal ( vi ) , which was then divided by the image noise [ 10 ]  . estimation of radiation dose for each patient radiation dose was assessed by multiplying the dlp ( dose length product ) by the conversion coefcient for the chest ( 0.014 msv / mgy cm ) [ 16 ]  . statistical analysis continuous variables were condensed by means , standard deviation ( sd ) , standard error ( se ) or 95 % condence intervals ( ci ) as appropriate . 
a propensity score was arranged using gender , age , weight and bmi as covariates . a 1 : 1 matched analysis was performed where one case was matched with one control . 
twenty - one subjects were excluded due to previous coronary artery interventions ( 15 patients with coronary artery by - pass graft ) and hr [ 64 bpm after beta - blocker premedication ( 6 patients )  . 
in group a , 24 patients ( 24 % ) did not have signicant stenosis , 36 ( 36 % ) had single - vessel disease , 18 ( 18 % ) had two - vessel disease , and 22 ( 22 % ) had threevessel disease . 
among 1 , 600 segments analysed , 257 segments in group a and 242 in group b were considered to be unevaluable because the segment was absent ( 224 and 215 segments , respectively ) , too small ( diameter \1.5 mm ; 30 and 23 segments , respectively ) or completely occluded ( 3 and 4 segments , respectively )  . 
noise was the results of objective image quality analysis are descrihigher ( p = 0.0018 ) in group a while the snr and cnr were signicantly higher in group b ( table 5 )  . 
a previous study reported that coronary ct using a 320 - detector scanner has the potential to signicantly reduce the radiation dose while maintaining high diagnostic accuracy [ 20 ]  . 
moreover , the aec systems now available for multidetector ct scanners are able to adjust the radiation dose according to the patients attenuation while sustaining diagnostic image quality [ 21 ]  . 
sureexposure ( toshiba medical , tokyo , japan ) , the aec system that we used , provides both patient - size and z - axis aec [ 21 ]  . 
the aidr 3d works in both the raw data and the image domafirst , the algorithm works on the raw data performing a noise reduction process of the sinogram based on a projection noise estimation considering a noise model and a scanner model . 
at the end of the iterative process , a weighted blending between the original image and the image obtained by the process is automatically applied by the systeaidr 3d improves the snr , preserving and enhancing the edge of the anatomical structures and the spatial resolution . 
a limit of a general iterative reconstruction is the unnatural look of the images ; aidr 3d gives images a natural look by increasing the snr and the spatial resolution . 
the amount of dose reduction with a xed level of noise is automatically determined by the sureexposure3d with aidr 3d integrated into the acquisition protocols . in this study , we evaluated the effect of aidr 3d and aec on image quality and mean radiation dose in comparison with conventional fbp that employs exposure parameters based on patient bmi , on a 640 slice ct scanner 648 radiol med ( 2014 ) 119 : 642649 [ 8 , 22 ]  . 
its application in ct was limited by the time - consuming repetitive reconstruction process until a few years ago , when advances in computer performance made iterative reconstruction feasible also for ct , which produces much more projection data than emission tomography [ 23 ]  . 
indeed , both subjective and objective image quality analysis showed signicantly higher quality in the aecaidr - 3d group . subjective analysis showed signicant improvement of the image quality in the aidr group images for all the segments evaluated . 
 [ 8 ] found an improvement in image quality using aidr 3d with a built - in aec system but they did not evaluate the effects of these technologies on radiation doses . 
 [ 9 ] also found both a signicant improvement in image quality and a reduction in radiation dose using aidr 3d but the quality assessment was made on a per - patient rather than a per - segment level . 
this is showed because particularly improvement of the image quality in the aidr 3d group , especially in the intermediate and distal segmental class , which are generally the segments with lowest image quality , primarily due to the small calibre of these vessels ( \1.5 mm in diameter )  . 
the aec system , taking into account the aidr3d reconstruction , automatically selects the tube current on the basis of the body prole and the attenuation information derived from the scanogram , to reach the target standard deviation of pixel values for a specied target image quality . 
moreover , aidr 3d improves the snr . important results the main limitation of this study is clearly the estimation of the current reduction generated by the introduction of the aidr 3d with respect to the current chosen from the bmi table . 
the same bmi , indeed , may be present in a patient with a very large abdomen and small chest diameter . in our study we investigated only the dose reduction due to the contribution of aidr 3d . 
the results in dose reduction obtained with aidr 3d suggest that coronary ct imaging could be further improved by modifying the reconstruction kernel [ 24 ] and the contrast injection protocol [ 25 ]  . 
it is worthy of note that aidr 3d is more efcient when the tube voltage is low and , in this study , we use a protocol that employed a tube voltage of 120 kv . 
the next step will be the possible introduction of 80 kv . conclusions the main strength of our study lies in the use of propensity score analysis which helped us to obtain two groups of patients virtually randomised for important clinical characteristics . thus , the comparative analysis by propensity - matched pairs made the results less prone to methodological biases compared with other usual statistical methods and allowed us to conrm that aidr 3d with aec is able to reduce signicantly the mean radiation dose and improve the image quality with respect to traditional fbp without dose modulation . acknowledgments the present work beneted from the input of eng . 
colagrande ( & ) dipartimento di scienze biomediche sperimentali e cliniche , struttura complessa di radiodiagnostica ( sod 2 ) , universita` di firenze , azienda ospedaliero - universitaria careggi , largo brambilla 3 , 50134 florence , italy e - mail : stefano.colagrande@uni.it 626 radiol med ( 2014 ) 119 : 625633 conclusion the pretreatment adc value of a liver metastasis does not seem useful in predicting the cht outcome . 
a trend towards early adc increase , alone or occurring with dimensional decrease , may be a good indicator of a responding lesion . keywords magnetic resonance imaging ( cid : 2 ) diffusionweighted imaging ( cid : 2 ) apparent diffusion coefcient ( cid : 2 ) chemotherapy ( cid : 2 ) liver metastasis least two dimensions , without intralesional necrosis / calcication involving [ 30 % of the volume ; ( b ) a karnofsky performance scale score c60 % ; ( c ) being at least 18 years old ; ( d ) not being pregnant ; ( e ) being scheduled to begin a new cht regimen for metastatic disease , with no contraindications to cht . 
the exclusion criteria were a history of any other malignant disease not included in the entry criteria and contraindications to mr imaging . introduction magnetic resonance diffusion - weighted imaging ( mr - dwi ) allows the quantitative assessment of changes in the diffusion properties of water molecules in vivo by apparent diffusion coefcient ( adc ) calculation . 
mr - dwi acquisition is noninvasive , with no contrast medium administration being required , and possible use in patients with renal dysfunction can be obtained quickly and easily incorporated into routine examination protocols . 
mr - dwi is increasingly being used in extracranial applications , such as in the breast , liver and prostate , as well as in general abdominal imaging , because of its added value in the detection and characterisation of focal lesions , in particular malignant lesions [ 13 ]  . 
mr - dwirelated parameters are appealing as imaging biomarkers because their changes during therapy could potentially be compared with other biomarker changes such as serum tumour markers or with recist and / or who measurements of tumour size . 
in this sense , they could be useful for the early assessment of tumour response to chemotherapy ( cht ) , as already found in previous studies [ 2 , 4 , 5 ]  . given this background , the aim of our multicentric study was to evaluate the potential of mr - dwi in both the prediction and early assessment of response to cht of liver metastases , by itself and along with early dimensional assessment . methods and materials patients the local ethics committee approved the study protocol ( number 470 / 10 , azienda ospedaliera - universitaria careggi , firenze )  . 
the inclusion criteria for patients were : ( a ) the presence of nonconuent liver metastases pancreatic ( including neuroendocrine ) or breast carcinoma diagnosed at the time of primary tumour staging or during the follow - up , histologically conrmed or clinically proved ( by means of serial imaging and serum tumour markers ) , measuring at least 1 cm in diameter and measurable in at from gastrointestinal , imaging technique all patients were scanned by contrast - enhanced ( ce ) - ct and mr - dwi before the beginning of cht ( baseline , time 0 )  . 
the time between these two initial studies did not exceed 1 week . mr - dwi was then repeated 1015 days ( time 1 ) and 2025 days ( time 2 ) after the beginning of the rst cycle of cht . 
the detailed mr protocol is shown in table 1 and included the following sequences : axial breath - hold ge t1w in / out of phase , axial and coronal free - breathing single - shot tse t2w with and without fat suppression , axial free - breathing echo - planar dw multi - b imaging ( b value ranging from 0 to 800 s / mm2 with consecutive steps of 50 s / mm2 )  . 
for each lesion , a dimensional analysis was performed at all times measuring the two maximum perpendicular diameters visible on the axial portal phase contrast - enhanced ct images and on the axial dw images obtained with low b value ( b = 50100 s / mm2 ) where the signal from vessels is erased and lesion conspicuity is greater ; all sums of the diameters were recorded . 
the lesion was again visualised on images obtained at b = 50 s / mm2 ; a region of interest ( roi ) was manually drawn including the entire lesion and taking care to avoid adjacent vessels ; thereafter the same roi was copied and pasted to all remaining b value images acquired . 
an roi of similar size was also drawn in the normal liver parenchyma of the central part of the right lobe ( to avoid cardiac motion artefacts ) where data show higher reproducibility and repeatability [ 6 ] , excluding large blood vessels , biliary vessels and hepatic borders . 
lesion and parenchyma adc values were obtained by tting mono - exponential model to the dwi signal obtained at different b values using the following equation : exp ( cid : 3 ) b ( cid : 4 ) adc assessment of therapeutic response and statistics therapeutic response was determined on a lesion - by - lesion basis by evaluating changes in tumour size at time 3 with respect to baseline , according to recist 1.1 criteria [ 7 ]  . 
a metastatic lesion presenting a 20 % or more increase in the maximum transverse diameter with respect to time 0 was classied as progressive disease ( pd ) ; a lesion showing at least 30 % of reduction in the maximum transverse diameter with respect to time 0 was classied as partial response ( pr )  . 
from a clinical point of view , lesions remaining stable or regressing ( sd and pr , respectively ) were also labelled as responding ( r ) , while those progressing were classied as not responding ( nr )  . 
mean diameter and adc value ( with corresponding standard deviations ) were calculated in pd , pr and sd lesion groups at the different times using mixed linear regression models , with subjects as a random - effect . 
2 box and whisker plots showing distribution of dimensional changes of progressive disease ( pd ) , stable disease ( sd ) and partial response ( pr ) lesions at different times . 
the horizontal line in each box is the median of measured values ; the top and bottom of boxes represent 25th and 75th percentile ; points represent outlier values . although there are several overlaps between groups at different times , some statistically signicant differences were found and are listed in tables 2 and 3 fig . 
the horizontal line in each box is the median of measured values ; the top and bottom of boxes represent the 25th and 75th percentile ; points represent outlier values . 
changes in adc values ( either as absolute values or as lesion / parenchyma ratio ) , early dimensional changes assessed on low - b dw images and a combination of the two were evaluated in pd , pr and sd lesions to determine the performance of these assessments in correctly classifying a lesion . 
table 2 also lists all p values found with analysis of variance ( anova ) using pd group as reference , showing possible differences among the pd , sd and pr groups for each time . 
now , they are even more debated because hospitals around the world are cutting budgets reserved for these treatments and , at the same time , new cht regimens tend to have higher costs . 
early knowledge of whether an expensive new drug will be effective has therefore become crucial . prediction of response based on pre - treatment adc continues to be controversial , as can be seen from the literature . 
tumours with necrotic areas are often surrounded by hypoxic , but viable cells , and were shown to be less sensitive to ionising radiation [ 8 ] , more prone to aggressive behaviour and probably less sensitive to cytotoxic agents [ 9 ]  . 
therefore , it may be hypothesised that such lesions would be presenting higher pre - treatment adc and have a worse treatment outcome . even though some important studies conrm this theory [ 5 , 10 ] , others [ 1113 ] do not show any signicant correlation between pre - treatment adc and response to chemoor radiotherapy . 
in our series , the mean pre - treatment adc value of r ( both sd and pr ) lesions did not turn out to be signicantly different from that of nr ( pd ) lesions ( table 2 ) ; as a consequence , adc failed to predict the cht response of lesions and our result cannot conrm the above hypothesis . early assessment of cht response is the second issue we tried to address in our study : in other words , if dwi would be able to provide early marker ( s ) for treatment efcacy . most published studies [ 3 , 5 , 10 , 1620 ] have shown that adc changes may precede dimensional ones , since early radiol med ( 2014 ) 119 : 625633 diffusion - weighted ( dw ) - mr images fig . 
however , it has also been demonstrated that the timing of imaging is of crucial importance : changes in adc could precede changes in tumour size , but may even disappear after a certain time because of repair mechanisms such as decrease of oedema and organisation of necrosis [ 21 ]  . 
assessment of the cht response of liver metastases in our experience appears feasible after 2025 days since the beginning of cht using both the conventional approach based on evaluation of dimensional changes and adc analysis . 
dimensional assessment is easy , fast and reproducible when performed on dwi acquired with b values of 50100 s / mm2 ; on these images , in fact , changes . 
according to lowenthal et al . [ 22 ] small lesions ( in particular , those with diameter\1 cm ) tend to appear larger on dwi than on mr delayed images after liver - specic contrast administration , and the reason for that remained unclear ; however , this did not affect our results because we compared measurements obtained with the same acquisition sequence . 
as our results show , when assessing lesion diameters 2025 days after the beginning of cht , we should adopt different comparison criteria from those postulated in the recist guidelines . 
because of this and the relatively small sample of lesions analysed , at this point it does not make sense to search for a cutoff increase / decrease in transverse maximum diameters to correctly classify a lesion as r or nr . 
however , the relatively good 632 radiol med ( 2014 ) 119 : 625633 auc value conrms that changes in size at this time directly reect later dimensional changes . when examining adc changes at different times , rst of all we can notice that at time 1 there is an increase in mean values of all groups with respect to the corresponding pretreatment values , so it does not appear possible to discriminate r and nr lesions at this time . 
however , at time 2 and 3 the mean adc values of r lesion groups ( both sd and pr ) become signicantly higher than the corresponding pretreatment values , conrming the hypothesis of reduced cellularity and increased diffusion of water molecule into the extracellular space for r lesions . 
on the other hand , the mean adc of nr lesions , after the above - mentioned initial increase , actually drops to the pre - treatment level , where it remains stable . 
we can therefore conrm that an early ( 2025 days after the beginning of the rst cycle ) evaluation of adc changes appears useful for an early assessment of clinical response . 
nonetheless , moving from a per - group adc analysis to a lesion - by - lesion one ( which means translating these facts into clinical practice ) , things become more difcult and often controversial . 
among the intrinsic factors we should consider the location of the lesion ( in particular , when a lesion is located on or near organs subject to respiratory or other kinds of movement ) , its histotype , the presence of internal necrotic areas and calciuamong the extrinsic factors we should consider those related to cht ( type of drugs used , duration , days past since the beginning , etc ) and others related to the mr - dwi acquisition technique and adc calculation ( in particular , images low signal - to - noise ratio , acquisition artefacts , b value adopted and lesion sampling )  . what the clinician needs is a method capable of overcoming most of these factors and in particular the dependence from primary tumour histotype and type / duration of cht used , to easily manage the scheduling of different patients . besides , both the acquisition process and adc measurement should be kept as quick and reproducible as possible , to be implemented in routine clinical practice . 
based on these considerations , we chose this particular study design , especially with regard to the inclusion of metastatic lesions from a wide range of primaries , the decision to use average time for early evaluations , the choice to acquire multi - b dw images ( to calculate adc by a tting procedure and keep off the dependency from b value ) and the very easy and fast lesion sampling method ( both for adc and for dimensional comparison among times )  . 
all of these variables in our study indeed may have represented the major limitations for us to obtain relevant and not contradictory results , along with the relatively small sample of lesions analysed , in particular nr lesions . 
it should not be completely excluded that inclusion of a larger sample of nr lesions , and thus a comparison between more homogenous lesion groups , would make it possible to extrapolate both analysis method and criteria lesion by lesion allowing early discrimination of clinically r lesions from nr ones . 
the latter point seems to be critical because oncologists usually need to recognise nr patients early on and , if necessary , move them to a different cht regimen , thus avoiding unnecessary side effects and costs . conclusions mr - dwi can provide different tools to perform an early assessment of the response of liver metastases to cht , including the conventional evaluation of dimensional changes and adc analysis . 
however , a trend of early ( 2025 days since the beginning ) adc increase alone or in combination with dimensional decrease may be a good indicator of a responding lesion . 
radiochemotherapy is the treatment of rst choice in non - metastatic disease . intensity - modulated radiation therapy ( imrt ) allows the sparing of parotid glands , improving the toxicity prole . 
the aim of this study was to compare the results obtained with imrt with those obtained with conventional 2d ( 2drt ) and 3d conformal radiation therapy ( 3dcrt ) in terms of tumour control , survival , acute and late toxicity . materials and methods we reviewed the clinical records of 52 patients with histologically proven carcinoma of the nasopharynx ( stage iivb according to the 2002 american joint committee on cancer staging system ) treated with curative intent between january 2003 and august 2011 : 26 patients were treated with 2d or 3d technique ( arm a ) and 26 with imrt technique ( arm b ) with simultaneous integrated boost . 
fifty patients ( 96 % ) received chemotherapy . local control ( lc ) , locoregional control ( lrc ) , diseasefree survival ( dfs ) , overall survival ( os ) , acute and late toxicity were retrospectively evaluated . results after a median follow - up of 37.6 months ( 69 months in arm a and 23 months in arm b ) , 69 % of patients were alive and disease - free , 10 % were alive with disease and 21 % died of disease , with an os of 81 % at 2 years and 79 % at 5 years , a lc rate of 88 % at 2 years and 78 % at 5 years , a lrc rate of 80 % at 2 years and 73 % at 5 years and a dfs of 74 % at 2 years and 65 % at 5 years , with no statistically signicant differences between imrt and 2drt / 3dcrt . 
pecorari dipartimento di scienze chirurgiche , otorinolaringoiatria 1 - u , universita` di torino , turin , italy radiol med ( 2014 ) 119 : 634641 moderately hypofractionated regimens , allows good disease control with better results in terms of late xerostomia , although without statistically signicant differences compared to 2drt and 3dcrt . 
the hypothesis of an impact of imrt on survival has yet to be conrmed . keywords nasopharyngeal cancer ( cid : 2 ) radiotherapy ( cid : 2 ) treatment techniques introduction nasopharyngeal carcinoma ( npc ) is a pathological , epidemiological and clinical entity distinct from other head and neck cancers . 
it is rare in the united states and other western countries , but common in southern china , southeast asia , the middle east and north africa [ 1 , 2 ]  . in these countries , most patients have differentiated nonkeratinizing or undifferentiated carcinoma ( who types 2 or 3 ) , while keratinizing squamous cell carcinoma or who type 1 is rare , but more common in western countries [ 3 , 4 ]  . 
many patients present with locally advanced disease . the standard of care for locally advanced npc is a combination of radiotherapy ( rt ) and chemotherapy [ 2 ]  . the overall survival ( os ) rate at 5 years ranges from 32 to 52 % in large series of patients with locally advanced disease treated with rt alone [ 2 ]  . 
no signicant reduction in the risk of locoregional recurrence and / or distant metastases , and no improvement of os were shown for adjuvant chemotherapy after chemo - radiation . the addition of neoadjuvant chemotherapy to radiation signicantly reduced the risk of locoregional recurrence and distant metastases , but without a signicant improvement in os [ 10 ]  . npc has a relatively good prognosis also in locally advanced cases : the disease is often chemoand radiosensitive and patients are usually younger and less exposed to tobacco and alcohol than the majority of head and neck cancer patients . conformal radiotherapy ( 3dcrt ) was considered the standard technique when intensity - modulated radiation therapy ( imrt ) emerged as a technique able to better conform the dose around target volumes and to better spare organs at risk . 
because of a steep dose gradient and a high conformity index , the risk of target missing with imrt is higher than with conformal technique , as the integral dose . xerostomia is a common and disturbing late effect of conventional rt . 
several authors demonstrated the superiority of imrt over 2drt or 3dcrt in parotid gland sparing and two randomised controlled trials have proved a better salivary toxicity prole [ 11 , 12 ]  . since 2006 , at the radiation oncology department of the university of turin , patients affected by nasopharyngeal cancer were treated with imrt , with or without chemotherapy . 
the aim of the present study was to retrospectively review clinical outcomes ( efcacy and toxicity ) of this subgroup of patients and to compare these results with a previous series of patients treated in the same institution with 2drt and 3dcrt . materials and methods patients all patients affected by histologically proven , stage iivb ( according to the 2002 american joint committee on cancer staging system ) nasopharyngeal carcinoma treated with curative intent between january 2003 and august 2011 at our institution were included , with a minimum follow - up time of 6 months . 
patients with a previous history of cancers other than non - melanomatous skin carcinoma or in situ cervical carcinoma were excluded . fifty - seven patients met the inclusion criteria : 30 patients underwent 2drt or 3drt ( arm a ) and 27 imrt ( arm b )  . five patients ( four from arm a and one from arm b ) were excluded from the analysis because of a history of malignancies . 
patients characteristics are summarised in table 1 . radiation therapy all patients were immobilised with a thermoplastic mask . in the 2drt group , to dene treatment elds , facial cervical radiograms were taken using a conventional x - ray simulator . 
the lower neck was treated with 5054 gy through a single anterior eld with midline shielding . megavoltage photons ( 6 mv ) were used to treat the primary tumour and neck lymph nodes , with high - energy electron boost ( 1020 gy ) for posterior neck lymph nodes . in the 3dcrt group , a planning ct without contrast enhancement was performed . 
radiation elds were chosen according to the extent of the tumour as evaluated by clinical examination , contrast - enhanced ct and / or mri and / or ct - pet . 
the gross tumour volume ( gtv ) included the primary tumour and abnormal lymph nodes . clinical target volume 1 ( ctv 1 ) included the nasopharynx , bilateral upper cervical lymph nodes ( levels ib iiiii , upper part of level v ) and retropharyngeal lymphatics ; ctv2 included low cervical and supraclavicular nodes . 
2d and 3d plans were delivered with a 6 mv linac ( elekta sl - 75 , stockholm , sweden )  . twenty - six patients were treated with imrt . 
twenty of them were treated with a split - eld technique ( with a unique anterior eld for inferior neck and supraclavicular fossa ) and six with a whole - eld technique , to treat the entire volume with a single plan . in the imrt group a planning ct without contrast enhancement was performed . 
ctv1 was generated after a 510 mm expansion of the gtv ; ctv2 included all lymph node levels at risk of subclinical disease ( levels ibiiiiiiv - upper part of v level )  . 
the ptvs in nasopharynx and upper neck were treated using 79 coplanar beams ; a variety of fractionations was used , for example 69.44 gy in 32 fractions , 69.96 gy in 33 fractions or 70 gy in 35 fractions were prescribed to the ptv1 and 51.2 gy in 32 fractions , 54.25 gy in 35 fractions or 59.4 gy in 33 fractions to the node - negative regions ( ptv2 )  . 
imrt plans were delivered with a 6 mv linac ( elekta precise or elekta synergy , stockholm , sweden )  . an optimal sparing of at least one parotid gland was attempted , especially the contralateral one with respect to the tumour or metastatic lymph nodes . 
twelve patients in the arm a and 11 patients in arm b had bilateral neck involvement . chemotherapy all patients but two ( with t1n0m0 and t2bn0m0 disease , respectively ) were treated with chemotherapy ; 33 patients ( 63.5 % ) underwent induction chemotherapy plus concurrent chemotherapy in 31 patients ( table 2 )  . 
for 30 patients a cisplatin - based chemotherapy was scheduled at the dose of 100 mg / m2 every 21 days , and 14 patients were treated with weekly doses ( 30 mg / m2 )  . radiol med ( 2014 ) 119 : 634641 toxicity . 
the follow - up time was counted from the date of completion of radiation therapy . univariate and multivariate analyses were performed for evaluation of the prognostic factors and factors correlated to late toxicity . 
stata statistical package version 11.0 ( statacorp lp , texas , usa ) was used for statistical computations . results after a median follow - up of 37.6 months , 69 months in arm a ( range 7108 ) and 23 months in arm b ( range 874 ) , 36 patients ( 69 % ) were alive and without evidence of disease , 5 ( 10 % ) were alive with disease , 11 ( 21 % ) died from disease . 
twoand ve - year lc rates were 88 and 78 % ; 2and 5 - year lrc rates were 80 and 73 % ; and 2and 5 - year dfs rates were 74 and 65 % , respectively . in arm a , ve patients had persistent disease after rt : a nasopharyngeal mass in one case , lymph node metastases in two cases and disease in both locations in other two cases : three patients with residual nodal disease also developed distant metastases . 
during follow - up , other ve patients relapsed , two locally ( one of whom then developed distant metastases ) , one in the neck , one in both the nasopharynx and neck and another one with bone metastases , respectively . two patients with persistent lymph node metastases underwent salvage surgery , but only one was still alive when the study was concluded . 
one patient underwent photodynamic therapy for a local recurrence , and then a neck dissection was performed for a regional recurrence , without evidence of disease at the last follow - up . table 3 chemotherapy regimens in the two treatment arms timing of chemotherapy drugs neoadjuvant cisplatin ? 5fu patients ( arm a ) patients ( arm b ) concurrent cisplatin ( 100 mg / m2 ) cisplatin ? paclitaxel / docetaxel cisplatin ? 5 - fu ? paclitaxel cisplatin ? epirubicin paclitaxel ? bleomycin cisplatin ( 30 mg / m2 ) cisplatin ? 5 - fu carboplatin paclitaxel adjuvant cisplatin ? 5 - fu in arm a , 14 patients were treated with induction chemotherapy ( median , two cycles ) and 23 patients received concurrent chemotherapy , followed by adjuvant chemotherapy in ve patients . 
tolerance to concurrent chemotherapy was quite good : nine of 14 patients completed the three planned cycles of high - dose cisplatin and four of seven patients completed at least ve cycles of weekly dose cisplatin ve patients , amifostine was used before every rt session . in arm b , 19 patients were treated with induction chemotherapy ( median , two cycles ) followed by concurrent treatment , with good compliance : 13 of 16 patients completed three cycles of high - dose cisplatin and seven of eight patients nished at least ve cycles with weekly dose cisplatthree patients were given additional adjuvant chemotherapy ( table 3 )  . follow - up patients underwent weekly examinations during treatment . the rst follow - up evaluation occurred at 2 months and then every 36 months during the rst 2 years , every 6 months during years 35 and then annually with clinical examination and contrast - enhanced ct or mri , chest radiograms every year and thyroid function tests every year . 
acute and late toxicity was scored according to ctcae v3.0 ( common terminology criteria for adverse events ) and rtog / eortc ( radiation therapy oncology group / european organisation for research and treatment of cancer ) , respectively . statistics the endpoints were os , disease - free survival ( dfs ) , local control ( lc ) , locoregional control ( lrc ) , acute and late fig . 
1 overall survival in the two treatment arms 638 radiol med ( 2014 ) 119 : 634641 on multivariate analysis , age , gender , histology , t stage , n stage , tnm stage , rt technique , biological equivalent dose ( bed ) , high - dose ptv volume , fractionation schedule , overall radiation treatment time ( ott ) , timing of chemotherapy and comorbidities were evaluated and none was correlated with os , but tnm stage iii ( p = 0.021 ) ptv volume c275 ml ( p = 0.014 ) were statistically correlated to better dfs . high - dose there were no treatment - related deaths and no grade 4 acute toxicity . 
parenteral nutrition was necessary in one patient in arm a and in two patients in arm b and enteral feeding tube in two patients in arm a and in three patients in arm b . 
the median treatment interruption was 2 days in arm a and arm b ( range 012 days , the same in the two groups ) , with a median rt overall treatment time ( ott ) of 63.5 days in arm a ( range 4684 ) and 47.5 days in arm b ( range 4264 ) , because a programmed electron boost was administered to 23 patients after treatment with photons in arm a . late toxicity data were unknown for three patients ( two in arm a and one in arm b )  . 
at 5 years of follow - up xerostomia cg2 was 67 and 41 % for arm a and b , respectively ( p = 0.10 ) , chronic dysphagia cg2 was 17 and 12 % ( p = 0.81 ) , hearing loss cg2 was 29 and 35 % ( p = 0.22 ) and dysgeusia cg2 was 4 and 11 % ( p = 0.54 ) , respectively , for arm a and b . possible correlations between late toxicity and variables such as age , gender , stage of disease , high - dose ptv volumes , total radiation dose and fractionation , rt technique , timing of chemotherapy , amifostine use and severe acute toxicity were assessed . 
furthermore imrt was shown to be superior in terms of better target coverage and better sparing of organs at risk [ 11 , 12 ]  . in our study , the median age ( 53 years old ) and the male / female ratio ( 3.7 / 1 ) were the same as in most clinical trials [ 7 , 8 , 1115 ]  . 
in our there was no statistically signicant difference study , between standard and hypofractionated regimen , in terms of os , lc , acute and late toxicity . in our retrospective study , there was no signicant difference in os between 2drt / 3dcrt and imrt techniques , although a marginal survival benet could be expected with imrt because of the better dose distribution and the acceleration of radiation treatment . 
unfortunately , our sample size was too small and follow - up time was quite short in arm b to demonstrate any survival advantage . imrt use did not correlate with a signicant difference in lc , but a weak trend for better lrc was found . 
some data in favour of a better lc with imrt have been reported in the literature [ 20 ]  . in our study , almost all patients were treated with chemotherapy . 
in the treatment of nasopharyngeal cancer , the most recommended neoadjuvant therapy was a combination of two [ 14 , 21 ] or three [ 2224 ] cycles of cisplatin and 5 - fu , but in the literature associations of cisplatin with epirubicin [ 23 , 25 , 26 ] , bleomycin [ 27 ] or taxanes [ 28 , 29 ] are described . 
tpf chemotherapy ( cisplatin - paclitaxel - 5 - fu ) , which correlated with a survival benet in two trials on squamous head and neck tumours [ 30 , 31 ] , is being tested in ongoing international nasopharyngeal cancer trials [ gortec - npc2006 , nct00828386 ; nct01245959 ]  . 
in our study , 82 % of patients underwent induction chemotherapy , almost always two cycles , with a certain variety of schedules and doses ( table 3 )  . 
actually some reports suggested a benet in distant disease control [ 9 , 10 ] , but with potentially higher toxicity and difculty in completing an adequate concomitant phase . in the present study , 92 % of patients were treated with concurrent chemotherapy . 
in our practice , there is a large use of weekly dose cisplatin , due to its low toxicity , but there are some controversies about this schedule [ 3 , 13 , 15 ]  . globally , in our study , it was not possible to show any relationship between chemotherapy timing and patients outcome , but the biases were numerous : the use of several different drugs and schedules , the absence of a control group , the lack of formal restaging after induction , the low number of events and the inadequate number of cycles given after radiation therapy . reports , in historical the 5 - year survival rate was 4050 % with a lc of 6070 % before 1990 [ 32 , 33 ]  . 
in the 90s , improvements in imaging and the use of conformal rt combined with chemotherapy resulted in 5 - year survival rates of 75 % and lc rates of 85 % [ 34 ]  . 
after that , an increasing number of publications reported even better results with the use of imrt , despite the shorter follow - up time : lc up to 90 % and similar survival rates at 3 years [ 1820 , 3538 ]  . 
taking into account the different followup time , the results obtained in our institution compare quite well with those previously reported , particularly with studies using neoadjuvant [ 27 , 29 ] or concurrent and adjuvant chemotherapy [ 58 ]  . the outcome of our patients treated with imrt is similar to that reported in randomised and retrospective trials , suggesting the appropriateness of our practice in terms of radiation doses , target volume denition and chemotherapy . 
patients were staged iiiiv in 75 % of cases ; 70 % of patients were treated with induction plus concurrent chemotherapy and 20 % with concurrent chemotherapy only . no analysed factor was related to os or tumour control , but stage and volumes of ptv treated with high dose were related to dfs . 
in particular , the relationship between higher ptv volumes and a better dfs appears paradoxical . some elements could make this nding a little more sensible : rst , gtvs , ptvs and disease stage are linked in a nonlinear way ; second , ptv delineation is also related to the treatment technique ; third , there was an imbalance between treatment arms : patients treated with imrt also had larger planning treatment volumes and higher prescription doses . the acute toxicity rate in our sample is similar to that of clinical trials and retrospective surveys [ 4 , 11 , 12 , 16 , 18 , 19 ] , even if a underestimation is quite likely because of the retrospective collection of data . two randomised controlled trials [ 11 , 12 ] demonstrated the benet of parotid - sparing imrt in late xerostomia and stimulated salivary ow . 
with regard to late xerostomia , while in the absence of a signicant advantage with imrt , 640 radiol med ( 2014 ) 119 : 634641 our data are similar to those of other studies except for the typically , patients treated with parotid - sparing timing : imrt experience a recovery ; on the contrary late xerostomia after conventional rt is irreversible . 
in our cases , while in arm b , xerostomia had some stabilisation after the toxicity rate little more than 1 year of follow - up , increased with time in arm a , with an advantage that became more evident over time . our dosimetric data compared favourably with published studies : if in our series the mean dose to the spared parotid was about 32 gy , in the randomised study by pow et al . 
the parotid gland sparing imrt technique led to excellent results in terms of late xerostomia , although it was not possible to demonstrate a statistically signicant difference as compared to conventional and conformal rt . 
gemelli 8 , rome , rm , italy pseudoaneurysms and six cases of lower limb ischaemia . five of ten complications occurred in patients presenting calcication of the common femoral arteries , whereas 4 / 10 were in patients with complex groin anatomy . 
it has traditionally been performed 836 radiol med ( 2014 ) 119 : 835841 femoral artery access by surgical ( open , o - evar ) , although recently percutaneous femoral access ( p - evar ) has developed . 
this innovative technique became feasible with the advent of devices for percutaneous closure of large - bore arterial access sheaths , such as prostar xl ( abbott vascular , abbott park , il , usa ) [ 1 ]  . since the development of the totally percutaneous approach , many investigators have discussed the advantages of this technique [ 211 ]  . 
some authors demonstrated a complication rate of 820 % in unselected populations of patients who underwent o - evar [ 4 , 12 , 13 ] , whereas complications of p - evar account for 4.4 % [ 1 ]  . 
in the past , obesity , calcication of the common femoral arteries ( cfa ) , scars in the groin and a large introducer sheath size ( larger than 18 or 20 fr ) were considered to be contraindications to p - evar [ 2 , 3 , 6 , 911 , 14 ] , as they represent the main factors for technical failures . however , more recently , some authors have investigated the possibility to treat with p - evar even patients with obesity , severe calcication of cfa and scarred groin [ 1 , 4 , 8 ]  . the aim of this study was to evaluate the safety and feasibility of p - evar in an unselected population , including patients with calcication of cfa , scarred groin and also obese patients . 
technical success was dened as correct device deployment with closure of the arteriotomy , without the need of conversion to surgery . during each procedure , there were at least two operators , two nurses and one radiologic technologist . 
we performed an accurate dissection from the skin to the femoral arterial wall , by slightly enlarging the incision to allow the advancement of prostar xl deeper into the subcutaneous tissue . 
the guidewire was removed , and the device was advanced until adequate blood marking was achieved through the marker lumen , indicating that the sutures and the needles were inside the arterial lumen . 
1 pre - operative maximum intensity projection ( mip ) image of contrast - enhanced computed tomography ( ct ) scan a shows the presence of an abdominal aortic aneurysm in a 71 - year - old patient with tortuous , ectatic and calcied iliac arteries . 
post - operative mip image of contrast - enhanced ct scan b shows the correct deployment of device with successful exclusion of the abdominal aneurysm and regular patency of the stent grastents were also placed into the renal arteries . 
three - dimensional volume - rendered image ( c ) better depicts the position of aortic and renal stent grafts radiol med ( 2014 ) 119 : 835841 out after inserting a guidewire through the wire lumen . after placing the sutures of the prostar xl closure device , a large vascular sheath compatible with the stent - graft system was introduced into the artery ( sheath size ranged between 18 and 24 fr for the main body access site and between 12 and 18 fr for the contralateral limb access )  . then , the stent - graft system was deployed with a main body access site of 1824 fr and a contralateral limb access site of 1218 fr . 
thus , the sheath was removed , maintaining the guidewire inside the arterial lumen , and the sutures of the closure device were immediately tied using a sliding knot ( rst the white suture and then the green one )  . a knot pusher was used to ensure proper tightening of the knot and positioning of the knot near the vessel wall . patients received a bolus of 5 , 000 iu of heparin during the procedure . all patients were hospitalised for at least 2 days , they received elastic compression of the femoral artery , and they were kept at bed rest for 24 h . 
statistical analysis was performed using spss 12.0 for windows ( spss , chicago , il )  . results among the 200 consecutively enrolled patients , 123 / 200 ( 61.5 % ) were men and 77 / 200 ( 38.5 % ) patients were women . 
in 60 / 200 patients ( 30 % ) a complex groin anatomy was recorded , consisting of patients with a thick pannus in the groin , high femoral bifurcation , small femoral artery calibre or scarred groin from multiple prior catheterisations or surgical procedures . cfa calcications were identied in 144 / 200 patients ( 72 % ) by retrospective review of ct angiographies performed before intervention . 
the patients characteristics are listed in table 1 . the stent grafts used included : 170 zenith cook , three anaconda , six jotec , six lp zenith cook , 14 gore excluder and one ovation . 
we used one prostar xl closure device for each arterial access , except in ve cases which required placement of another device ( primarily because of difculties with nitinol needles and their sutures )  . 
four of ten complications occurred in patients with complex groin anatomy , showing a table 1 characteristics of 200 patients undergoing p - evar using the prostar xl closure device renal impairment : creatinine [ 1.4 mg / dl cfa common femoral artery patient demographics male sex obese renal impairment arterial hypertension 61.5 complex groin anatomy cfa calcications 838 radiol med ( 2014 ) 119 : 835841 fig . 
2 thrombosis of the common femoral artery ( cfa ) thrombosis occurred as an early complication ( few hours ) of p - evar in a 75 - year - old male . 
eight of ten complications ( 80 % of cases ) occurred in the access site of the main body of the prosthesis using a sheath size [ 20 fr , and two in patients with post - surgical scarred grofurther 2 / 10 complications ( 20 % of cases ) regarded the contralateral limb access , using a sheath size of 16 fr . 
no midand long - term complications related to the access sites were recorded during the follow - up period . discussion nowadays , p - evar is more attractive than traditional o - evar because of the wide range of advantages it offers . thanks to the advent of closure devices such as prostar xl , p - evar has become technically feasible , but today the main interest of the interventionist is focused on the patient population for whom it might be performed and on the safety of the technique itself . 
the possible benets of percutaneous closure of the femoral artery are a decreased number of wound complications , a more frequent use of local or regional anaesthesia , shorter operation and hospitalisation times and earlier patient mobilisation [ 10 ]  . 
these advantages of p - evar stand in contrast to the complications of o - evar , such as wound infection , wound dehiscence , lymphocele and haematomas [ 4 , 8 ]  . 
in particular , bleeding , inability to achieve haemostasis and lower limb ischaemia are the main endovascular complications which have been shown to require the conversion to surgery [ 14 ]  . the main end - point of our study was to assess the feasibility and safety of p - evar in a population of patients not selected by any clinical feature ( obesity , previous evar or groin surgery , peripheral arterial disease , type of treatmentelection or emergency ) or anatomical features ( calcications , diameter , groin anatomy and tortuous iliac arteries )  . 
previous studies have shown that severely or circumferentially calcied cfas , large introducer sheath size , scarred groins , obesity or morbid obesity , tortuous iliac arteries and operators inexperience with the procedure increase the likelihood of technical failures or complications [ 4 , 9 , 10 , 17 ]  . 
these risk factors , variously combined , were considered exclusion criteria for using percutaneous closure devices , but more recently a few studies using different percutaneous techniques began to include also patients presenting them [ 3 , 13 , 18 ]  . the technical success obtained in our study ( 100 % ) with an unselected population of patients , accompanied by a complication rate of 2.5 % in all femoral accesses , conrms the feasibility and safety of p - evar in all patients . radiol med ( 2014 ) 119 : 835841 in the literature , the technical success rate with percutaneous closure devices ranges from 66 to 100 % [ 2 , 4 , 8 , 10 , 17 , 19 ]  . 
 [ 1 ] reported that there is no statistically signicant difference in the percutaneous closure success rate between studies that considered all - comers and studies considering the most important exclusion criteria ( i.e. scarred groin and cfa calcications )  . 
in our study , we retrospectively reviewed the ct angiographies performed before the intervention , which revealed the presence of cfa calcications in 144 / 200 ( 72 % ) patients . 
these results show the safety of performing p - evar even on patients with cfa calcications , which are very common in subjects suffering from aortic aneurysms . as regards scarred groins , we experienced two cases of complications in these patients , although it is impossible to determine the concomitant role of larger sheath access size ( [ 20 fr ) used in this subset of patients . 
the majority of complications ( 8 / 10 , 80 % ) reported in our series occurred using sheath size larger than 20 fr , representing 7 % ( 8 / 114 cases ) of all access sites [ 20 fr . 
future advances in stent - graft design will allow for smaller delivery systems , resulting in a possible reduction in the inuence of sheath size on technical failures . however , large sheath size is not the only factor to be considered , as in our experience 2 / 10 complications ( 20 % ) occurred in the contralateral limb access ( 2 / 200 contralateral accesses , 1 % ) , using a sheath size of only 16 fr . 
an accurate dissection from the skin to the femoral arterial wall is fundamental to eliminate all possible brous bands of scar tissue and avoid incorrect device deployment . furthermore , it is very important to tie the sutures of the closure device using an accurate sliding knot , avoiding many loops around the rail end of the suture and allowing sliding of the non - rail end through a loop . 
as reported in other studies [ 2 , 17 , 22 ] , there is a learning curve associated with the use of these closure devices , which may explain the technical errors and the complications that occurred in the initial phase of our experience . 
even if it is not easy to quantify a precise number of interventions to become condent with prostar xl , we hypothesise that 3040 procedures should be performed . in our opinion , a key factor for a successful procedure is the patients groin anatomy . 
indeed , in our series , we observed 4 / 10 ( 40 % ) complications in subjects presenting with a complex groin anatomy , showing a statistically signicant difference between patients with complex groin anatomy and those without overall incidence of complications is very low ( 4 / 200 ; 2 % ) and not signicant in terms of safety of the procedure . 
however , complications in our series , we observed different kinds of complications , including six cases of lower limb ischaemia ( 5 / 6 after a few hours and 1 / 6 after a few days in a patient with peripheral arterial disease ) and four pseudoaneurysms ( 4 / 10 complications , 40 % )  . 
two of ve cases of early lower limb ischaemia occurred in patients with small femoral artery calibre ( complex groin anatomy ) coarse cfa calcications . patients with pseudoaneurysms were treated conservatively ( with usguided compression )  . 
thus , p - evar performed in unselected populations of patients constitutes a feasible and safe procedure that does not require specic selection criteria . furthermore , we recorded this low complication rate among 200 patients , a sizeable population if compared to other published studies . comparing our access - related complication rate with the best o - evar studies published in the literature [ 1 ] , our results demonstrate the attractiveness of p - evar . 
in fact , the o - evar approach for aortic aneurysm repair may be burdened by several complications , such as infection , thrombosis , pseudoaneurysm arterial dissection , arterial lymphoceles and femoral formation , groin haematoma , nerve injury , which occur in up to 14 % of patients and are probably related to the injury that the lymphatic and arterial vessels suffered during the procedure [ 14 , 25 , 26 ]  . some authors have reported a local complication rate between 8 and 20 % in patients who underwent o - evar [ 8 , 2730 ]  . 
the only prospective randomised trial published in the literature comparing the results of o - evar versus p - evar showed a lower complication rate at the access site in the percutaneous group [ 8 ]  . furthermore , o - evar has a much longer and more uncomfortable post - operative stay than p - evar [ 27 , 31 , 32 ] with a longer time to ambulation [ 8 , 27 ]  . 
patients might benet from p - evar time - saving , which may lead to considerable cost reductions for hospitals , in terms of staff resource use and expenses [ 33 ]  . 
costs should be included in a global evaluation of the differences between the two procedures ( o - evar and p - evar ) [ 1 , 31 ] and should include costs of devices and costs of hospitalisation ( procedure time , time to discharge and time to ambulation )  . 
 [ 33 ] found a signicant reduction in total procedure time ( approximately 1 h ) using the prostar xl device compared to surgical cut - down . however , to date , no evidence has been reported on the cost - effectiveness of the prostar xl device compared with surgical cut - down [ 33 ]  . the main limitation of our study is represented by the absence of a control group undergoing o - evar , with subsequent lack of comparison between the two procedures in terms of complications and overall costs . 
however , most published studies about the evar technique were retrospective or prospective non - randomised , with only one randomised controlled trial [ 8 ] ; this should be considered when interpreting results . 
further studies are needed to clearly compare p - evar with o - evar , focusing not only on the cost - effectiveness of prostar xl but also on the operators learning curve . conclusions percutaneous endovascular aneurysm repair in unselected populations is safe and efcient , with a very low risk of access - related complications , comparable to p - evar in selected populations and to the best o - evar series . important complex groin anatomy represents the most factor in the occurrence of complications , though the overall complication rate is very low . the operators experience and their mastery in the use of the prostar xl device are essential for successful outcomes . 
the results obtained in this study need to be conrmed with prospective randomised trials . conict of interest all the authors , sergio petronelli , maria teresa zurlo , silvia giambersio , lucia danieli , mariaelena occhipinti , declare no conict of interest . ethical standard all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1975 , as revised in 2008 . 
technical success , diagnostic accuracy , sensitivity , specicity , positive predictive value ( ppv ) , negative predictive value ( npv ) and complications were evaluated . results of 100 nodules ( mean size 5.19 cm ) , 68 were diagnosed as malignant , 27 as benign , and ve as indeterminate . 
only 33 of 100 patients underwent surgery : the nal pathological diagnosis was concordant with the biopsy diagnosis in 26 cases and discordant in 7 cases ( false negatives )  . 
rotolo center for thoracic surgery , university of insubria , viale borri 57 , 21100 varese , italy conclusions cbct - xperguide navigation is a new , accurate and safe imaging guidance for percutaneous lung biopsies . keywords c - arm cone - beam ct ( cid : 2 ) percutaneous lung biopsy ( cid : 2 ) pulmonary nodule introduction percutaneous transthoracic needle biopsy of lung nodules with image guidance is a safe and accurate diagnostic technique for assessing their benign or malignant nature [ 1 , 2 ]  . 
the use of computed tomography ( ct ) as image guidance does not allow the visualisation of the needle insertion in real time , unless it is carried out using ct uoroscopy ( uoro - ct ) [ 3 ]  . 
cone - beam ct ( cbct ) is a recently developed imaging technique available in c - arm angiography systems equipped with a at - panel detector integrated with a cone beam x - ray . 
the system produces images similar to those of ct providing the operator with the possibility of real - time uoroscopy visualisation and it offers a greater exibility in the orientation of the detector around the patient compared to the closed ct gantry . 
coupled with cbct it is also available a navigation software , vendor specic ( xperguide , philips healthcare , best , the netherlands ) which allows the operator to direct the needle in real time with uoroscopy , once the trajectory has been established by the images acquired in 3d cbct . 
the study was approved by the ethics committee of our hospital and before any procedure patients were adequately informed on the procedure and any possible complications , and all of them signed the informed consent forall patients had undergone a chest ct at our institution or elsewhere which was evaluated by the operator before the procedure to determine the feasibility of the biopsy . 
in particular , the size , location and distance from the pleura were determined for each lesion ( table 1 )  . technical execution all lung biopsies included in the study were performed in the angiography suite equipped with a c - arm cbct system ( philips allura xper fd20 , philips best , the netherlands ) and a dedicated software programme xperguide ( philips healthcare , best , the netherlands )  . 
in particular , with the aid of the xperguide software on the workstation monitor , the radiologist or trainee radiologist established the target point in the context of the lesion ( target point ) and the access point of the needle on the cutaneous plane ( entry point )  . 
1 first cone - beam computed tomography ( cbct ) scan to dene lesion location position of the angiographic c - arm to display the entry point of the needle on the patients skthis phase of the procedure , followed by the angiographic c - arm positioning in the angiography suite was called entry point position . after disinfection of the skin at the area of access and local anaesthesia ( 10 cc of 2 % mepivacaine , angelini , rome , italy ) , via uoroscopy the operator positioned the tip of the needle on the patients skin at the entry point following the cbct image that appeared on a dedicated monitor as a fusion image of the 3d volume previously acquired with cbct and the bi - dimensional plane obtained with realtime uoroscopy . 
3 second sagittal cbct scan to evaluate the correct positioning of the needle if correctly positioned , previously determined and displayed in real time , under uoroscopy on the dedicated monitor . 
in all cases , we used a 20 - gauge trocar ( biopsy bell , medical device , modena , italy ) as a biopsy needle . finally , at the end of the procedure , a third and nal scan was acquired to assess the presence of any complications fig . 
at least 4 h after the biopsy , on advice of the radiologist , a chest x - ray in posteroanterior and maximum expiration projection was performed to highlight possible pneumothorax . data analysis we calculated the rates of technical success , diagnostic accuracy , sensitivity , specicity , positive predictive value ( ppv ) and negative predictive value ( npv )  . 
true positive ( tp ) results were dened as biopsy results of a malignant lesion that agreed with the denitive histological ndings in patients undergoing surgery or with the results of the follow - up ( disease progression or response to radio - chemotherapy established by an oncologist ) in patients not undergoing surgery . 
true negatives ( tn ) were biopsy results of benign disease ( tumour , specic or nonspecic inammatory reaction , brotic tissue ) which resolved or remained unchanged during the follow - up . 
false negatives ( fn ) were patients in which the histological table 2 sir ( society of interventional radiology ) classication of complications table 3 results of cone - beam computed tomography ( cbct ) xperguide percutaneous transthoracic needle pulmonary biopsies radiol med ( 2014 ) 119 : 820827 minor complications ( a ) no therapy , no consequence ( b ) minimal therapy , no consequence major complications ( c ) need therapy , minor hospitalisation ( d ) need major therapy , long hospitalisation ( [ 48 h ) ( e ) permanent sequelae ( f ) death results of the biopsy sample were negative for malignancy and thus were discordant with the examination of the surgical specimen , which instead documented malignancy . complications were classied into major and minor according to the sir classication ( society of interventional radiology ) [ 5 ] ( table 2 )  . 
finally , the technical success rates , diagnostic accuracy , sensitivity , specicity , ppv , npv and complications were analysed by subdividing the data into two subgroups according to lesion size ( b3 and [ 3 cm )  . calculation of radiation dose the dose calculation method with cbct was carried out in collaboration with our hospitals medical physicists . 
the data were acquired through the application of dosimeters on the surface of a phantom ( rando phantom , model ran 10 , churchin associates , smithtown , ny , usa ) and using the pcxmc software ( version 2.0 ) according to the icrp [ 6 ]  . 
depending on the site of the lesion and the patients position ( x - ray tube as close as possible to the lung lesion ) , either a lr or rl projection was chosen . 
a single arc was divided into 43 steps of 5 ( cid : 3 ) each , and the calculation was made by taking into account the patients position ( supine or prone )  . 
forty - four lesions had a diameter b3 cm and 56 a diameter [ 3 cof the 100 lung lesions biopsied using cbct with xperguide software , 68 were diagnosed as malignant and 27 as benign . 
the features and size of the lesions in which the bioptic sample was not diagnostic were : three lesions located in the left upper lobe with the greatest transverse diameter of 9 , 16 and 30 mm ; two lesions located in the right lower lobe with a diameter of 55 and 32 monly 33 / 95 lesions were subjected to surgical resection . 
the 62 cases that were not subjected to surgery underwent clinical and radiological follow - up in collaboration with colleagues from the departments of internal medicine and surgery ( range 126 months ; mean followup period , 14 months )  . 
among the 33 patients who underwent surgery , 24 / 33 patients had malignant disease according to the analysis of the surgical specimen , agreement with the biopsy results ( tp ) , and two patients had benign pathology ndings ( hamartoma and chronic inammation , tn )  . 
these seven cases were judged false negatives ( fn ) , considering the histological examination of the surgical specimen showed the presence of primary or secondary malignant lung lesions ( adenocarcinoma , squamous cell carcinoma , a small cell lung tumour , a sarcomatoid carcinoma , three metastases )  . 
among these patients , the biopsy results revealed the presence of an inoperable malignant lesion in 44 cases ( tp , 26 adenocarcinomas , ve metastases , two carcinoids , four sarcomatoid carcinomas , four squamous cell carcinoma , two small cell lung tumours and one mesothelioma )  . 
there were 18 benign lesions ( seven bronchopneumonia , ve brous tissues , three nonspecic inammation , one tuberculosis and one round atelectasis )  . all 18 patients with benign lesions underwent clinical and radiological follow - up . 
in particular , the radiological follow - up ( range 1226 months , median 15 months ) documented lesion regression in 11 / 18 cases and no change in 7 / 18 cases ; in agreement with the clinical data provided by the clinicians , these cases were all considered true negatives . 
overall diagnostic accuracy was 92.6 % , the sensitivity was 90.9 % , the specicity was 100 % , the ppv was 100 % and the npv was 72 % . 
there were no major complications , while the overall rate of minor complications ( pneumothorax ) was 21 % ( 20 / 95 ) : eight cases of discussion over the last 30 years , numerous studies have demonstrated the diagnostic accuracy and safety of percutaneous transthoracic lung biopsies [ 811 ]  . 
published studies describing the technical aspects of lung biopsies with imaging guidance [ 8 , 9 , 12 ] point out that , despite the different guide used ( uoroscopy , ultrasound , multislice ct , uoro - ct , cbct , cbct with xperguide navigation software ) , the results obtained also depend on the learning curve and , consequently , on the level of condence gained over time by the operator . 
the authors showed that for supercial and voluminous lesions uoroscopic guidance alone may still be usable , having the advantage of exposing the patient to a lower radiation dose ( about 7 times lower than the dose measured in patients undergoing cbct - guided lung biopsy )  . 
other authors performed several studies using ct guidance , demonstrating the safety and diagnostic accuracy for benign and malignant lesions of the chest [ 1 , 11 , 14 ] ; in particular , li et al . 
the authors showed that the diagnostic accuracy for lung biopsies under ct increased signicantly in lesions [ 10 mm and in cases in which the route of the needle from the skin to the lesion was b40 mcurrently , the gold standard technique used to guide percutaneous biopsies of pulmonary nodules is uoro - ct , which is able to follow the progression of the needle from the skin to the target in real time [ 15 ]  . 
compared to conventional ct , uoro - ct allows monitoring of the progress of the needle radiol med ( 2014 ) 119 : 820827 with a smaller number of steps compared to procedures that use the conventional ct guidance [ 12 ]  . 
however , uoroct can lead to higher radiation doses to the patient and to the operator , and it is not available in all radiology services [ 14 , 16 , 17 ]  . 
 [ 15 ] published the result of 99 lung biopsies performed with uoro - ct guidance in 85 patients , reporting accuracy , sensitivity and specicity rates of 96 , 95 and 100 % , respectively , with a complication rate of 26 % . 
these results not only conrmed that the diagnostic accuracy of lung biopsies with uoro - ct guidance was comparable to that of lung biopsies performed under simple ct guidance , but also showed that uoro - ct guidance was indicated especially in small lesions , in which a high complications was diagnostic obtained . 
 [ 22 ] showed that cbct is as reliable as ct an imaging guidance technique in percutaneous interventional procedures because they demonstrated , in a phantom model , that ct and cbct have a similar sensitivity in detecting parenchymal nodules . 
a retrospective analysis showed that two of these four lesions had dimensions of b2 cm and were biopsied in the initial phase of the learning curve and in the other three cases ( lesions c3 cm ) the needle was placed in a peripheral area of the lesion , where necrotic tissue was sampled . excluding the ve nondiagnostic cases , the sensitivity , specicity , accuracy , ppv and npv were 90.9 , 100 , 92.6 , 100 and 72 % , respectively . 
lesion size did not affect the quality of the diagnostic results and safety of the method , as is clear from a comparison of the overall data with those of the two groups of patients divided by lesion size ( b3 and [ 3 cm ) , which showed no signicant differences . 
in the past 2 years , several studies have been published reporting the results obtained in lung biopsies with the guidance of cbct alone [ 13 , 2326 ] and cbct associated with the proprietary xperguide navigation software [ 22 , 2729 ] ( table 5 )  . 
 [ 23 ] reported an initial experience of needle biopsies of 71 pulmonary nodules all b3 cm using cbct guidance alone without navigation : their technical success rate was 100 % , with good sensitivity , specicity and accuracy ( 97 , 100 and 98.4 % , respectively )  . 
on the basis of our results and the data from the literature regarding lung biopsies performed with cbct guidance with or without the aid of the xperguide software , despite the limited amount of currently available data and the lack of homogeneity , a slight reduction in dose could be hypothesised with the use of xperguide guidance . in our opinion the use of xperguide software in cbct guided lung biopsies could reduce the total dose delivered because it can reduce the number of cbct scans needed , by allowing greater precision in the approach to lesion sampling . study has some limitations that need to be acknowledged : the biopsy results of 5 out of 100 nodules were not diagnostic at histological examination ; the followup of patients undergoing surgical verication was not homogeneous ( range 126 months , mean 14 months ) ; nally , the radiation dose was exclusively calculated on the patients and not directly on the operator . 
however , we can 826 radiol med ( 2014 ) 119 : 820827 conclude that in lung biopsies performed using cbct with the aid of the xperguide navigation system the results could be almost similar to those of uoro - ct as concerns the technical success , diagnostic accuracy and complications . 
for cases with the above - mentioned features , we suggest that the higher recurrence rate can be taken into full consideration when choosing appropriate surgical procedures . keywords giant cell tumour ( cid : 2 ) curettage ( cid : 2 ) recurrence ( cid : 2 ) magnetic resonance introduction l . 
197 , ruijin 2nd road , shanghai 200025 , china giant tumour of bone ( gctb ) is a benign bone lesion most often found in the extremities of bones [ 1 , 2 ]  . 
tumours arise in the meta - epiphyseal region of long bones , predominantly in the distal femur and the proximal tibia , but they can occur in the entire skeleton [ 2 ]  . 
histologically , these tumours are classied as a benign neoplastic lesion consisting of three cell types : mononuclear histiocytic cells , multinucleated giant cells that resemble osteoclasts , 862 and neoplastic stromal cells [ 3 ]  . 
en bloc resection has been recommended for aggressive tumours [ 2 ]  . although complete removal of the lesion provides a low recurrence rate , wide resection requires complex reconstruction of the adjacent joints , which increases the rate of surgical complications and disabilities [ 4 , 5 ]  . 
nevertheless , the recurrence rate remains relatively high [ 4 , 6 , 7 ]  . risk factors for local recurrence of gctb after intralesional curettage have been previously studied in depth [ 1 , 8 11 ]  . 
some studies demonstrated that demographic factors ( age , gender ) and disease - related factors ( location , pathological fractures and soft tissue extension ) did not inuence the risk of recurrence [ 1 , 8 ]  . 
however , van der heijden [ 12 ] reported that soft tissue extension strongly increased the risk of local recurrence , whereas age , sex , location and pathological fractures did not . 
the prognostic relevance of soft tissue extension of gctb remains controversial [ 8 ]  . magnetic resonance imaging ( mri ) appears to be more sensitive than computed tomography ( ct ) and radiography in detecting soft tissue extension of gctb for its excellent soft tissue contrast which allows for easier identication of the tumour and its extent . 
in addition , mri can provide visualisation of the number and margins of soft tissue extension and involvement of adjacent tissues , as well as depicting liquefaction and necrosis with the use of intravenous gadolinium injection [ 812 ]  . to our knowledge , all previous studies investigated the soft tissue extension of gctb as one of risk factors for local recurrence . 
in this study , we focused on the soft tissue extension of gctb , with a view to identifying some characteristics that can serve to indicate the recurrence of gctb after intralesional curettage . patients and methods database a total of 48 patients ( 22 women and 26 men ; average age , 30 years ; range 1646 years ) were consecutively enrolled in this study from january 1998 to june 2011 . 
all patients underwent table 1 location of giant cell tumour of bone ( gctb ) with soft tissue extension in 48 patients radiol med ( 2014 ) 119 : 861870 location no . 
of patients distal femur proximal femur femoral shaft distal tibia proximal tibia proximal humerus distal radius sacrum total intralesional curettage with pmma ( polymethylmethacrylate ) , which is the preferred standard treatment for gctb in our hospital , and were all followed up for over 2 years . recurrence occurred in 16 ( 33.33 % ) of the 48 cases . 
the mr examinations were carried out after the patients had been fully informed about the procedure and had given their consent . informed consent was also obtained from all patients for the use of all clinical and imaging data for this study . mr protocols fat - suppressed fast all mr examinations were performed using a 1.5 - t superconducting whole - body imager ( signa , general electric medical system ) with dedicated extremity coils or body phased - array coils . 
all measurements were performed on a postprocessing workstation ( ge healthcare )  . the following sequences were obtained : spin - echo t1weighted ( tr range / te range , 450 - 600 / 15 - 20 ) , fast spinecho t2 - weighted ( tr range / te range , 3500 - 4000 / 80spin - echo t2 - weighted ( tr 120 ) , range / te range 3 , 5004 , 000 / 80120 )  . 
when the volume of soft tissue extension exceeded 50 % of the volume of whole tumour , we dened the soft tissue extension as being large . finding size large small number multiple single margins adjacent tissue involvement with bone envelope integrity without bone envelope integrity signal intensity characteristics t1 : low , homogeneous t1 : intermediate / low , heterogeneous t2 : high , homogeneous t2 : intermediate / high , heterogeneous static enhancement homogeneous enhancement heterogeneous enhancement jaffe grade grade i grade ii grade iii soft tissue extension size of tumour and soft tissue extension the size of gctb and soft tissue extension was measured directly on the contrast - enhanced mr images which best depicted thethe diameters of both the tumour and the soft tissue extension were recorded in three axes : anterioposterior , transverse , and craniocaudal . 
the volume of each tumour and soft tissue extension was calculated using the standard equation for the volume of an ellipsoid : v = ( 4p / 3 ) xyz , in which x , y , number when the number of soft tissue extensions was greater than or equal to two , and the extended soft tissues were isolated in consecutive images , we dened the nding as multiple soft tissue extensions . 
when there was only one soft tissue extension , we dened it as a single soft tissue extension . when the extended soft tissue was expressed as a mass with several lobules , we also dened it as a single soft tissue extension . adjacent tissue involvement ( such as muscle , the absence or presence of adjacent soft tissue involvement ligament , capsula articularis ) , dened as abnormal signal intensity areas and foci ( low on t1 - weighted images and high on t2 - weighted images with fat suppression ) was determined . soft tissue extension margins cortical breach around the soft tissue extension was established when outer cortical integrity was disturbed by the presence of abnormal signal intensity . 
if continuity of the band of low signal intensity on mr images attributed to marginal scleroses was completely broken off , we dened the bone envelope around the extended soft tissue as not having integrity . signal intensities of soft tissue extension relative to muscle on spin - echo t1 - weighted and t2 - weighted images ( higher , equal , or lower ) were determined . static enhancement the pattern of soft tissue extension enhancement on t1weighted gadopentetate dimeglumine - enhanced images ( homogeneous or heterogeneous ) was recorded . jaffe grade gctb was originally described by jaffe et al . 
 [ 13 ] , who classied the tumour into three categories : grade i ( benign ) without appreciable atypia of stromal cells , few mitoses , none abnormal ; grade ii ( intermediate ) with stromal cells showing only slight or more marked atypia , but not enough to justify a diagnosis of malignancy ; and grade iii soft tissue extension was dened as a complete breakthrough of the cortex and additional extension into adjacent soft tissue [ 12 ]  . signal intensity characteristics 864 radiol med ( 2014 ) 119 : 861870 ( malignant ) with obvious features of malignancy . 
on completion of the retrospective review , the mr images were correlated with surgery results . data analysis analysis of the local recurrence rate of gctb with soft tissue extension considered seven aspects including soft tissue extension size , number , margins , adjacent tissue involvement , signal intensity characteristics , static enhancement and jaffe grade . 
when gctb had large soft tissue extension , multiple soft tissue extensions and lacked integrity of the bone envelope around the extended soft tissue , the local recurrence rate after intralesional curettage was higher . 
there was no statistically signicant difference in the groups of adjacent tissue involvement and jaffe grade . giant cell tumour of bone is a benign bone lesion most often found in the extremities of bones [ 1 , 2 ]  . 
tumours arise in the meta - epiphyseal region of long bones , predominantly in the distal femur and the proximal tibia , but they can occur in the entire skeleton [ 2 ]  . 
surgery is the mainstay of for gctb . although en bloc resection provides a low recurrence rate , wide resection requires complex reconstruction of the adjacent joints , which increases the rate of surgical complications and disabilities [ 4 , 5 ]  . 
2 20 - year - old man with gctb with two soft tissue extensions at the sacrua coronal t1 - weighted image shows the soft tissue has low / intermediate signal intensity on t1 - weighted image . 
the image also shows high signal intensity of adjacent tissues around the soft tissue extension . c axial contrast - enhanced t1 - weighted image with fat suppression tissue extension . shows heterogeneous enhancement of the soft d sagittal contrast - enhanced t1 - weighted image with fat suppression shows the two soft tissue extensions . 
some studies demonstrated that demographic factors ( age , gender ) and disease - related factors ( location , pathological fractures and soft tissue extension ) did not inuence the risk of recurrence [ 1 , 8 ]  . 
however , van der heijden [ 12 ] reported that soft tissue extension strongly increased the risk of local recurrence , whereas age , sex , location and pathological fractures did not . 
in this study , we focused on soft tissue extension , to assess the relationship between soft tissue extension of gctb and the recurrence rate after intralesional curettage . there were signicant differences among the groups of size , number , and margins of soft tissue extension . 
the reason might be that for gctb with large or multiple soft tissue extension and lack of bone intralesional curettage proves to be envelope integrity , technically more difcult , and is less likely to completely remove the whole tumour . 
3 35 - year - old woman with gctb with two small soft tissue extensions at the femoral shathe case did not develop recurrence . a , b axial contrast - enhanced t1 - weighted images show two small soft tissue extensions with homogeneous enhancement ( arrow )  . 
on t2 - weighted images , the signal intensities tend to be between those of muscle and fat . most lesions are inhomogeneous due to bleeding , liquefaction and necrosis especially in large soft tissue extension . 
jaffes grading system proved to be difcult to apply in practice and was unable to predict the clinical behaviour and prognosis of gctb [ 16 ]  . 868 radiol med ( 2014 ) 119 : 861870 fig . 
ac axial spin - echo t2 - weighted image with fat suppression , t2 - weighted image , and t1 - weighted image show cortical breach around the soft tissue extension ( arrow )  . 
f photomicrograph of the soft tissue extension shows a number of osteoclast - type giant cells and mononuclear stromal cells ( haematoxylin and eosin 9250 )  . jaffe grade ii intralesional curettage is the least invasive surgical option and usually provides the possibility to save the joint adjacent to the tumour [ 17 ]  . 
intralesional curettage may be combined with the use of local adjuncts , such as pmma void lling , hydrogen peroxide , phenol , and cryotherapy [ 9 , 17 , 18 ] with the intention to further reduce the risk of local recurrence . 
we tissue speculated that extension received intralesional curettage , the recurrence rate would be higher . if all gctb patients with soft tissue extension , multiple soft based on these ndings , we can conclude that gctb with large soft tissue extensions and lacking bone envelope integrity have an increased risk of intralesional curettage . recurrence after local several limitations of this study should be mentioned . first , only a small number of patients were evaluated , which limited the power of the statistical results . 
more patients and multi - institutional studies are necessary for a conrmation of the results with additional statistical power . second , we found that several objective features may appear in one case , such as gctb with large soft tissue extension , mr images of adjacent tissue involvement and a lack of bone envelope integrity . 
we did not analyse the interrelation of objective features because of the small radiol med ( 2014 ) 119 : 861870 table 3 comparison of recurrence rate in each group objective features no . 
 [ adjacent ] represents the adjacent soft tissue involvement around the soft tissue extension * signicant statistical difference ( p \ 0.05 ) the denition and concept of number of cases . 
fourth , we did not sufciently investigate the relationship between histopathological changes and characters on mr images of gctb with soft tissue extension , especially as regards changes of adjacent soft tissue involvement in explaining the mr signal changes . 
fifth , although we found that the local recurrence rate was higher if gctb had large soft tissue extension , multiple soft tissue extensions and a lack of bone envelope integrity , we failed to investigate whether there were any other reasons which might affect the recurrence rate following intralesional curettage . 
this study could provide new clues to identify the characteristics of the soft tissue extension that might best predict recurrence of gctb after intralesional curettage . conclusions in summary , the recurrence rate of gctb with soft tissue extension is higher when the extended soft tissue is large , multiple and lacking bone envelope integrity after intralesional curettage . 
the aim of this study was to compare colonic transit time with radiopaque markers and defaecography in female patients with obstructed defaecation . materials and methods in a prospective observational study , between january 2010 and december 2012 , a total of 30 female patients , mean age 60 years , with symptoms of obstructed defaecation were subjected to colonic transit time and defaecography , and divided into two groups : normal or abnormal colon transit time . 
the results were statistically compared using the chi - square test . results the comparison of data between colonic transit time and defaecography showed the following groups : group 1 ( 6 / 30 = 20 % ) with normal colonic transit time but abnormal defaecography , and group 2 ( 24 / 30 = 80 % ) with abnormal colonic transit time ; the latter was further divided into two subgroups : group 2a ( 4 / 24 = 17 % ) , patients with inertia coli ; group 2b ( 20 / 24 = 83 % ) , m . 
beati dipartimento di scienze chirurgiche , unita` di colo - proctologia , ospedale san carlo borromeo , via pio ii , 3 , 20153 milan , italy patients with impaired defaecation demonstrated at defaecography . 
there was a signicant statistical difference between the radiological ndings in these groups . conclusions this study conrmed the value of both defaecography and colonic transit time in assessing clinically obstructed women . 
the differential diagnosis between colonic slow transit constipation and constipation due to pelvic oor disorders is essential for an adequate strategy of care . keywords colonic transit time ( cid : 2 ) defaecography ( cid : 2 ) constipation ( cid : 2 ) obstructed defaecation introduction normal defaecation requires normal colonic motility ( colonic transit ) , anorectal sensation , expulsion force and a coordinated function of the pelvic oor to evacuate [ 1 ]  . chronic constipation is the second most commonly reported disorder in the western world with an estimated incidence of 20 % [ 2 ] , and it represents a signicant economic burden , often compromising the quality of life [ 3 ]  . 
constipation can be considered not only a disease , but a symptom related to delay in passing of stools . primary constipation can be divided into two forms : constipation due to delayed colonic transit : slow progression of faecal material in the transverse and descending colon . constipation due to pelvic oor dysfunction / alteration , characterised by difculty in expelling the accumulation of stools in the rectum [ 4 ]  . 814 radiol med ( 2014 ) 119 : 813819 table 1 rome iii diagnostic criteria for functional defaecation disorders 1 . 
the aim of this study was to compare colonic transit time with radiopaque markers and defaecography in female patients with obstructed defaecation . materials and methods patients between january 2010 and december 2012 , a total of 30 female patients ( mean age 60 years , range 2986 years ) with symptoms of constipation potentially candidate to surgery were included in this study . 
the surgical procedure may involve a transanal approach like starr , transstarr , or delorme technique in patients with obstructed defaecation or an abdominal approach such as subtotal colectomy with ileorectal anastomosis in patients with severe slow colonic transit time . 
any change in the colonic distribution of markers may indicate one of three patterns of colonic hypomotility : colonic inertia , hindgut dysfunction , or outlet the results of marker studies have obstruction . 
thus , practical consequences in the clinical management of patients with chronic constipation [ 12 ]  . thirty radiopaque markers ( these are inert and nondigestible , radiopaque cylinders 3.5 mm in diameter ) are administered to the patient and the transit is monitored after 24 h and at the fth day by plain abdominal x - ray . the markers are located and counted on the abdominal radiographs with bony landmarks and clear bowel outlines ( spinal processes and imaginary lines from the fth lumbar vertebra to the pelvic outlet serving as landmarks , dened projection zones of the right and left colon and rectosigmoid ) [ 12 ]  . a transit time corresponding to the evacuation of at least 90 % of the markers is assumed to be considered normal ; the retention of more than 20 % of the markers was indicative of slow transit time according to bouchouca et al . 
at least three subtypes of slow colonic transit were identied by colonic transit times , measured on the basis of the distribution of markers visible on abdominal radiographs , although there may have been an overlap between these subtypes : colonic transit is slow , the major site of delay being the ascending colon ( colonic inertia ) ; the delay is predominantly found in the descending colon ( hindgut dysfunction ) ; and marker retention is visible mainly in the rectosigmoid area ( outlet obstruction )  . 
the overlap between patterns of colonic hypomotility may be due to the dysfunction of a colonic segment or to distal faecal impaction , which can cause nonpropagation or backpropagation of the markers [ 14 ]  . the stagnation prevailing in the right colon argues in favour of a signicant alteration of colonic motility ( colonic inertia or functional constipation ) with a prolonged intestinal transit of stools . defaecography defaecography is a morphodynamic examination that simulates the act of defaecation and evaluates some disorders of the rectum and the pelvic oor [ 15 , 16 ]  . 
the entire examination is recorded on videotape and each video clip is analysed using a computer video capture and digitising system combined with an image analysis programme [ 19 , 20 ]  . without any prior bowel preparation , ileal opacication is obtained after oral intake of 300 ml of dilute barium suspension at 60 % ( prontobario 60 % bracco s.p.a. milan , italy ) approximately 3060 min before the examination , opacication of the vagina is obtained with a swab impregnated with iodinated contrast medium to demonstrate the effect of defaecation on the posterior vaginal wall , and rectal ampulla opacication with 180 ml of thick barium sulphate paste of a consistency similar to faeces , injected with a syringe with the patient in the left lateral position . defaecography was divided into pre - evacuation , evacuation and post - evacuation stages and so an anteroposterior radiograph was taken with the patient at rest in the supine position with a focus on the ampulla lled with barium ; after that , ve lateral radiographs were taken : at rest with the patient seated on a comfortable dedicated device in the standard seated position , which is considered more physiological . 
the uoroscopic table is moved into the upright position ; a single latero - lateral radiographic lm is then obtained to document the rectum and pelvic oor conguration at maximum contraction ( on voluntary contraction of the pelvic oor muscles ) and at straining ; a videouorographic recording ( a videotape recorder is also used to allow a good dynamic observation of evacuation ) is obtained at evacuation and a nal image after evacuation . fluoroscopy images were recorded during several such manoeuvres to assess and measure the descent of the pelvic oor and to diagnose any rectocele , enterocele or rectal intussusception . 
perineal descent was determined by measuring the level of the anorectal junction at rest and during straining and was dened as either descent of the anorectal angle ( it was measured at the intersection of the axis of the anal canal and rectal ampulla ) to more than 2 cm below the pubococcygeal line at rest or descent to more than 3 cm below the pubococcygeal line on straining . an enterocele was dened as descent of the small bowel below the pubococcygeal line . 
rectal intussusception was dened as a circumferential descent of the entire thickness of the rectal wall without passing through the anal canal [ 1621 ]  . the examination was considered normal if none of these alterations were present . 
the chi - square test was used for statistical analysis of the colonic transit time an defaecography results . 816 results incomplete the main disorders reported by patients were : reduced frequency of evacuation with multiple unsuccessful attempts ; evacuation efforts ; manual manoeuvres to void ( endorectal or endovaginal ) ; prolonged use of laxatives and enemas for which the patients have complained progressive loss of effectiveness . 
the risk factors for pelvic oor dysfunction include pregnancy , multiparity , advanced age , menopause , obesity and any other factors that result in a chronic rise in intraabdominal pressure . 
4 defaecographic signs in patients with normal ( 6 / 30 ) and abnormal colonic transit time ( 24 / 30 ) , the latter divided into patients ( 4 / 24 ) with inertia coli and patients ( 20 / 24 ) with impaired defaecation gynaecological , and urological evaluations and also the use of panoramic radiological investigations that provide a wide and detailed view of the pelvis [ 22 ]  . in routine practice , the diagnosis of chronic constipation relies primarily on subjective evidence ( rome iii criteria )  . colonic transit study and defaecography play an important role in qualifying and quantifying constipation . 
oral - anal transit time examination is a simple , repeatable , reliable , inexpensive study that uses indigestible objects , with the ability to track delayed transit times in the large intestine [ 23 ]  . 
to assess whether a patients whole gut transit time lies within the normal range , a single type of marker can be used and an abdominal radiograph performed at 12 and 120 h . 
this method provides an overall estimation of the whole gut transit [ 24 ]  . defaecography is a relatively well - tolerated examination , which is fast , highly diagnostic and simple to perform . defaecography can detect structural abnormalities ( rectocele , enterocele , rectal prolapse ) and descending perineum . rectocele and enterocele are common alterations in defaecation disorders . 
particularly , some degree of rectocele formation is present in a large percentage of asymptomatic elderly pluriparous women , suggesting that only rectoceles [ 2 cm in depth should be considered abnormal [ 1 , 25 ]  . 
the wearing out of the rectovaginal septum after vaginal delivery , particularly related to the volume of the babys head , could be a cause of rectoceles [ 2026 ]  . 
large rectoceles do not impede evacuation , but sequestration of stool within a rectocele leads to a sense of incomplete voiding and the need for digital manoeuvres to complete evacuation [ 27 , 28 ]  . 
intussusception associated with rectocele also had a higher incidence in patients with vaginal deliveries [ 29 ]  . obstructed defaecation may be suspected at defaecography : increase or decrease of the anorectal angle during straining and an incomplete opening of the anal canal ( \1 cm diameter ) resulting in delayed and incomplete rectal emptying are considered . 
 [ 32 ] showed that dynamic anorectal endosonography ( dae ) and dynamic magnetic resonance imaging ( mri ) defaecography have a similar accuracy in assessing pelvic oor disorders such as rectocele , perineal descent , and enterocele when conventional defaecography is used as the criterion standard . 
 [ 34 ] have described a very interesting classication of grade of anorectocele observed with three - dimensional dynamic anal ultrasonography ( echodefaecography ) this technique is of particular interest in the assessment of posterior pelvic oor dysfunctions . conventional defaecography is currently the only technique that can be performed with the patient in a physiological defaecation position . 
it not only has the advantage compared with conventional defaecography that it does not involve harmful ionising radiation , but it also can show rectal intussusceptions , which cannot be assessed by dae because of the presence of the probe in the rectum . however , dynamic mri defaecography is a longer , expensive , and not widely available procedure [ 32 ]  . echodefaecography may be used as an alternative method to assess patients with obstructed defaecation because it is minimally invasive , well tolerated , inexpensive , avoids exposure to radiation , and clearly shows all the anatomic structures involved in defaecation [ 34 , 35 ]  . defaecography therefore has still to be considered the rst - line investigation , while echodefaecography and dynamic mri defaecography could add further information in selected patients [ 32 ]  . our study compared defaecography and colonic transit time in women with obstructed defaecation potentially candidate to surgery . 
these patients can benet from pelvic reconstructive surgery , which may greatly improve the abnormal defaecation mechanisthe correction of rectocele and / or rectal prolapse ( starr , trans - starr , delorme technique ) resolves constipation by eliminating terminal obstruction . 
by contrast , 24 of the 30 observed constipated patients ( 80 % ) had abnormal colonic transit time : 17 % of them due to inertia coli , and 83 % due to impaired defaecation . 
this second group shows that a high percentage of obstructed patients with abnormal transit time had defaecographic signs of abnormal defaecation . these data conrm the values of both defaecography and colonic transit time in assessing clinically obstructed women . 
patients with signs of obstructed defaecation may have an abnormal colonic transit time and / or abnormal defaecography . our results showed a subgroup of clinically and defaecographically obstructed patients who probably have a limited benet from surgical procedures to correct abnormal perineal anatomy ( rectocele , enterocele , rectal prolapsed , descending perineum )  . 
descending perineum was the most common defaecographic sign in patients with normal colonic transit time and / or in patients suffering from inertia coli ; these data may be due to continuous straining at defaecation . in patients with obstructed defaecation , defaecography has been considered the rst - line investigation , while colonic transit time has not been routinely performed . 
our study demonstrated that 24 / 30 patients ( 80 % ) had abnormal colonic transit time mainly associated with descending intussusception , prolapse and anterior perineum , rectal rectocele . in 20 % of the obstructed patients , there was no clear correlation between defaecography and colonic transit time . 
in these cases , the obstructed defaecation syndrome is due to pelvic oor dysfunction and should not be correlated with chronic constipation . the differential diagnosis between colonic slow transit constipation and constipation due to pelvic oor dysfunction is essential for an adequate therapeutic strategy . despite the small number of patients , it should be possible to conclude that colonic transit time is useful in female patients with obstructed defaecation and should be associated with defaecography . 
adequacy and false - negative rate were compared specimen with radiological length and complications to assess any statistically relevant association with a multivariate logistic regression model . results a total of 290 / 308 ( 94.1 % ) samples were adequate . 
we encountered 11 / 308 ( 3.5 % ) minor complications and no major complications . conclusion ct - guided biopsy of bone lesions in cancer patients allows for a nal diagnosis in 94 % of cases . 
negative biopsies with positive positron emission tomography or magnetic resonance imaging and specimen shorter than 1 cm should be repeated to avoid a false - negative result . keywords ct - guided bone biopsy ( cid : 2 ) cancer patients ( cid : 2 ) bone metastases introduction bone is a relatively common site of metastases , accounting for more than 50 % of patients with advanced breast cancer [ 1 ]  . 
the suspicion of a bony or soft tissue lesion does not necessarily imply the need for histology [ 2 ] , because the diagnosis of metastases may be made by a combination of symptoms , clinical and radiological signs . 
however , percutaneous bone biopsy usually performed under computed tomography ( ct ) guidance is an accepted procedure and could be safe and consistently ensure the diagnosis of bone metastases [ 3 , 4 ]  . management of metastatic cancer patients with chemotherapy , or immunologic agents is usually based on the characteristics of the primary cancer [ 5 ]  . 
however , given the need for a condent histological diagnosis , the rate of biopsies on metastases is increasing , mainly due to the radiol med ( 2014 ) 119 : 852860 possibility of cancer to change its biological behavior . 
this evidence has been proven for breast cancer and should be carefully considered because of its important impact on treatment choice : any case of systemic progression should be biopsied for possible hormonal and immunological prole modication [ 6 ]  . the purpose of this study was to assess the adequacy and sensitivity of ct - guided bone biopsy in 308 procedures performed in 286 cancer patients with suspected metastases . 
as an advance in knowledge we tried to detect their correlation with radiological features and specimen length to identify any possible element suggesting biopsy repetition in the event of negative result . materials and methods this retrospective evaluation was approved by the institutional review board . 
written informed consent to undergo the procedure , including the consent to the use of anonymised clinical data for scientic and / or educational purposes , was obtained from all patients . the identication of the present study population was performed using the radiology division biopsy database of the european institute of oncology in milan . 
an electronic search of all ct - guided bone biopsies done between january 1997 and december 2012 was retrospectively performed to select patients with a previous history of primary cancer and suspected bone metastases . the assessed baseline characteristics of the patient population were : age at biopsy , sex distribution , and histology of the primary tumor . 
we evaluated biopsy adequacy and sensitivity and their relation with baseline characteristics , with site and morphological ct pattern ( osteolytic , mixed and osteosclerotic ) of bone metastatic lesions , with their mr and pet features and with bone biopsy specimen length . 
post procedural complications were also recorded . biopsy specimens were dened by the pathologist as adequate when they provided enough abnormal tissue to comfortably make a diagnosis and , for breast cancer metastases , to evaluate a reliable receptor status . all patients underwent clinical and radiological followup for at least 1 year after the procedure to assess any recurrence in negative outcomes and to detect any possible late complication . for negative biopsy results , any clinical and imaging evidence of recurrence at the site of biopsy was considered as a false - negative outcome . ct - guided bone biopsy procedure indication to perform the procedure came from the referring oncologist and was approved by the radiologist . patients coagulation was always tested before the bone biopsy by checking prothrombin activity ( pa ) , partial thromboplastin time ( ptt ) , and international normalized ratio ( inr ) ; patients with values out of the following ranges 70120 % , 1726 s , and 0.81.2 , respectively , were excluded . 
a peripheral antecubital vein was preventively accessed before the procedure for the administration of contrast media and / or drugs , if needed . the shortest path from the skin to the lesion was always chosen to avoid neurovascular structures and other solid organs . 
when the axial plane did not include both the optimal entry point of the needle and the target , the gantry was angled to facilitate accurate needle alignment and to avoid traversing other structures . 
all the examinations were performed on a 16 - slice lightspeed ct scanner ( general electric medical system , milwaukee wi , usa )  . scans were acquired during a single breath - hold with the following thickness slice 2.5 mm ; 120 kv ; 240300 ma ; 0.8 s tube rotation ; pitch 1.75. reconstructed parameters : all bone biopsies were performed using local anesthesia ( mepivacaine 200 mg ) that was injected under ct guidance giving special attention to inltrate the periosteum , where the needle was supposed to penetrate the cortical bone layer . we used cutting needles ( speedybell ; biopsybell , modena , it ) when the lesion was soft and the cortical bone was interrupted ; trap - lock 11g needle system ( angiotech ; medical device technologies , gainesville , usa ) , when the lesion showed a normal or thickened bony cortex . 
the samples of osteolytic metastatic lesions were xed in formalin while mixed and osteosclerotic metastatic lesions were decalcied by mielodec ( bio - optica , milan , italy ) according to the manufacturers instructions . 
1 flowchart of selection of patients for the analysis radiol med ( 2014 ) 119 : 852860 286 patients : two biopsies were performed on 22 patients 308 bone biopsies 18 inadequate biopsies for diagnosis 290 adequate biopsies for diagnosis 155 metastases from breast cancer 90 metastases from other or primary 45 normal bone marrow inadequate biopsies 4 inadequate biopsies for receptor assessment 40 with follow - up 5 lost at follow - up antibodies formalin - xed , in the case of breast cancer , er and pgr status , and her2 / neu overexpression were evaluated immunocytochemically . 
a 3 - lm - thick parafnembedded whole tumor sections were incubated following proper heat - induced antigen retrieval with the specic primary mouse monoclonal from dako ( glostrup , denmark ) to er and pgr ( clone1d5 , 1 : 100 dilution and clone 1a6 , 1 : 800 dilution , respectively ) or er / pr pharmdxtm ( dako ) , and a rabbit polyclonal antibody ( 1 / 3 , 200 dilution ; dako ) or herceptesttm ( dako ) to her2 for 30 min at room temperature , and subsequently treated with a high - sensitivity detection kit ( envision plus - hrp , dako ) the manufacturers instructions , using an automatic immunostainer ( autostainer ; dako )  . according statistical analysis fishers exact test was used to evaluate the association between clinical and pathological variables and the two outcomes . 
in the 22 patients with inadequate result , the biopsy was repeated . among adequate diagnoses , 45 / 290 histological ( 15.5 % ) had normal bone marrow as biopsy outcome ; 5 / 45 such patients were lost at 1 - year follow - up , but 40 / 45 were retrospectively evaluated after 1 year , and 10 / 40 ( 25 % ) patients had progression at the site of biopsy identied with ct or mr imaging . 
therefore , 10 / 303 ( 3.3 % ) biopsies were considered false - negative results , and the consequent overall sensitivity of bone biopsy was 96.7 % ( 293 / 303 )  . all biopsies were performed under ct guidance . 
similar , but not statistically signicant ndings ( p = 0.112 ) were seen for pet : among the 10 false - negative biopsies 8 / 10 had a positive pet , 1 / 10 had a negative pet , and 1 / 10 did not undergo a pet scan . finally , the proportion of false - negative biopsies decreased signicantly with increasing age ( p = 0.032 ) and specimen length ( p = 0.002 ) : in the group of specimens longer than 20 mm no false negative was evident . 
odds ratio is estimated using a multivariable fractional polynomial logistic regression models baseline demographic features , site of primary tumor , site of bone lesion , imaging characteristics , specimen length , and their correlation with adequacy are summarized in table 1 . 
for negative biopsies ( n = 40 ) , correlation with the false - negative rate is reported in table 2 . none of the baseline demographic features , site of primary tumor , site of bone lesion , and imaging characteristics showed correlation with biopsy adequacy . 
in contrast , specimen length showed a statistically signicant trend ( p = 0.036 ) : the proportion of unsuccessful biopsies was 13.6 % ( 6 / 44 ) if specimen length was less than 5 mm , percutaneous needle biopsy is a major and valuable tool in the diagnosis of musculoskeletal lesions . 
in a breast cancer patients , ct scan showed a subcortical hyperdense area of the left iliac bone ( a ) and mr showed an hypointense well dened lesion in t1 - weighted sequence ( b )  . ct - guided bone biopsy gave a negative result ( c ) , but after 26 months the patient had mr evidence of progression ( d ) they do not mention sensitivity , specicity and diagnostic accuracy , and the overall rate of diagnostic ct - guided bone biopsy of 69 % is lower than in other series . 
moreover , they use the clinical follow - up mainly to verify the so - called indeterminate biopsy results , while in our study , we followed - up patients with normal bone marrow at bone biopsy , nding a different clinical and / or imaging behavior than expected in 10 / 303 cases , thus ; leading to a sensitivity of 96.7 % ( 293 / 303 )  . 
in a standard clinical setting of suspected bone metastases , an indeterminate result is discussed for further biopsy , while a negative result is usually accepted as absence of bone metastases . 
even if mr - guided biopsies or fusion imaging techniques [ 14 ] may allow for increased accuracy of musculoskeletal biopsies , ct guidance is still considered the reference technique . 
indeed , in this series , we found 19 / 308 samples as performed on lesions undetectable or almost undetectable . among them , 5 / 19 were negative at histology . 
specically , in this subgroup of patients , only 1 / 19 ( 5.3 % ) proved to be inadequate and only 1 / 5 ( 20 % ) was a false negative . 
mrguided biopsies or fusion imaging techniques are available to increase specimen accuracy of such bone lesions [ 14 ] , but those options often are not easily available due to additional costs and because they are more time consuming . 
therefore , there could be some preliminary evidence that ct scan alone can be used for bone biopsy in patients previously studied with pet or mr . in this series , we found a correlation between specimen length and both adequacy and false - negative results . 
5 a case of suspected bone metastasis in the right ischium bone in a breast cancer patient with a hardly detectable softly hyperdense area of the inner bone marrow at ct scan ( a ) , a well - shaped focal lesion hypointense in t1 - weighted sequence ( b ) and hyperintense in stir sequences at mr scan ( c )  . 
a percutaneous ct - guided biopsy ( d ) conrmed the presence of a bone metastasis from breast cancer fragmentation , crush artifact and trabecular distortion . however , adequacy is not mentioned and the maximum specimen length of sample allowed by the in vitro model is 1 cm [ 15 ]  . 
the rst consideration is that one of the two most performing needles for specimen quality was the same as used in our study ( trap - lock 11g needle system ; angiotech ; medical device technologies inc . , gainesville , usa ) for normal or thickened bony cortex lesions . 
furthermore our in vivo analysis was focused also on samples longer than 1 cm revealing a signicant statistical association between specimen length and both the probability of an inadequate result and the probability of a falsenegative result : the longer the specimen , the lower the probability of an inadequate and false - negative result . 
second , the pathologists did not perform a blind evaluation of the specimen being aware of the patients history as in a normal clinical setting and we know this could affect the nal diagnosis . in conclusion , ct - guided bone biopsy in cancer patients with suspected metastases is safe and should be considered as an important tool in the diagnostic workup ; especially , when receptor assessment is needed . 
correlation with surgical and pathological data maria pia bondioni diego gatta vassilios lougaris nicoletta palai marino signorelli silvia michelini giuseppe di gaetano paola tessitore lorella mascaro andrea tironi giovanni boroni roberto maroldi daniele alberti received : 16 october 2013 / accepted : 22 november 2013 / published online : 8 march 2014 ( cid : 2 ) italian society of medical radiology 2014 abstract purpose the aim of this study was to evaluate the diagnostic accuracy of postnatal multidetector computed tomography ( mdct ) compared with prenatal ultrasound ( us ) , surgical ndings , and histology , in 33 patients with congenital cystic lung disease . methods thirty - three patients , 17 males and 16 females , were evaluated by mdct . 
lung lobectomy , segmentectomy , atypical resection , lesion resection were performed in 31 patients and surgical specimens were analysed . results prenatal us and mdct correctly diagnosed 76.9 and 94 % of respectively . 
lougaris pediatrics clinic , department of clinical and experimental sciences , university of brescia , brescia , italy the essential role of imaging , in particular ct , in providing invaluable preoperative information on congenital cystic lung diseases recognised in uterus . keywords multidetector computed tomography ( cid : 2 ) congenital cystic lung disease ( cid : 2 ) congenital lung malformation ( cid : 2 ) prenatal ultrasound introduction the rst bud of the respiratory tract appears around the fourth week of gestation and originates from the ventral anterior primitive intestine ( respiratory diverticulum )  . errors at this stage of development are responsible for congenital lung malformations that can be divided into two groups : ( a ) dysmorphic lung , namely lung agenesis , aplasia and hypoplasia , ( b ) focal malformations or congenital cystic lung diseases ( ccld ) , namely congenital pulmonary airway malformation ( cpam ) , pulmonary sequestralobar emphysema ( cle ) , and tion ( ps ) , congenital n . 
1. according to tukeys notation [ tukey , john ( 1977 ) , exploratory data analysis , addisonwesley ] , reported minimum and maximum values are ltered excluding all the outlier points , i.e. 
these last malformations are the focus of our study [ 1 ]  . cpams make up almost 3040 % of all congenital lung lesions , and are characterised by an excessive overgrowth of the terminal respiratory bronchioles and lung structures with alveolar growth suppression [ 2 ]  . 
genes such as hoxb5 , fgf7 , and platelet - derived growth factor - b ( pdgf - b ) have been implicated in the pathogenesis of cpam [ 35 ]  . 
the rst stocker classication from 1977 [ 6 ] has been modied and now includes lesions from type 0 to type iv based on the origin of the malformation [ 7 ]  . 
ps is the second most common lesion after cpam , characterised by a portion of lung parenchyma that does not connect with the tracheobronchial system and is therefore not ventilated whereas a systemic arterial supply is always present [ 2 ]  . 
based on the age of onset , ps may be distinguished in intralobar and extralobar ; the latter may be associated with other congenital anomalies [ 1 , 8 , 9 ]  . 
finally , bc occurs at a very early age and originates from an abnormal budding tracheobronchial tree , followed by its non - branching process [ 1 , 8 ]  . the rst imaging approach to detect ccld disease is prenatal ultrasound ( pus ) , which is usually performed at 1822 weeks of gestation and allows screening and denition of foetal lung lesions in the majority of cases [ 10 ]  . foetal magnetic resonance imaging is also a valuable tool for prenatal diagnosis of ccld , but for the time being it is only available in few centres . after birth , multidetector computed tomography ( mdct ) plays a key role in the radiological diagnosis of ccld in children , conrming type and number of lesions and their characterisation , and allowing a thorough preoperative evaluation [ 8 ]  . 
mdct offers the highest spatial resolution and enhanced diagnostic quality , together with multiplanar reformation , which is very informative for the surgeon [ 11 ]  . our study aimed to retrospectively evaluate the diagnostic accuracy of mdct in 33 patients with ccld . 
we also compared mdct with pus and with surgical and histological ndings ( and with histology ) to evaluate the diagnostic performance of mdct in dening the right diagnosis and the best surgical planning . materials and methods patient population our institutional review board approved the review of this radiological , surgical , and pathological data for fig . 
samples of the specimens were taken for histology , particularly including cysts , nodules and apparently normal lung when recognised , or random when no gross differences were evident . after sample processing and parafn embedding , slides for microscopy examination were stained with haematoxylin eoshistochemical or immunohistochemical stains were performed when considered appropriate . 
in all cases , dose modulation was used to minimise the radiation dose . mdct was performed after intravenous injection of 2 ml / kg of non - ionic contrast medium ( 320 mg i / ml )  . contrast medium was injected into an antecubital vein with a power injector at a rate of 1.0 ml / s for a 24 - gauge catheter , and 1.52.0 ml / s for a 22 - gauge catheter . 
two patients with intralobar ps had also cpam type i and ii , respectively , for a total of 17 lesions . lesions were detected in the right lung in ten patients ( four cpams were located in the right upper lobe , two in the middle lobe , and four in the right lower lobe ) , and in the left lung in eight patients ( two cpams were located in the upper lobe , one in the lingula , and ve in the lower lobe )  . ps was detected in 14 patients ( 42.4 % ) : ps was intralobar in 4 patients and extralobar in 10 . 
in two patients , the lesions detected 20 / 26 ( 76.9 % ) 31 / 33 ( 94 % ) p = 0.122 lesion was found under the left side of the diaphragm . lesions were located in the left lower thoracic cavity in 50 % of cases ( 7 / 14 ) and in the right lower thoracic cavity in 35.7 % of the cases ( 5 / 14 ) ; in 14.2 % of the cases ( 2 / 14 ) , the lesions were next to the left adrenal gland . in three patients ( 9.1 % ) , the diagnosis was consistent with cle in the left upper and lower lobe and in the right lower lobe , respectively . in one patient ( 3.0 % ) , the diagnosis was consistent with right bc in the right lower lobe . 
the ten patients with extralobar ps underwent lesion resection , the four patients with intralobar ps underwent atypical pulmonary resection including the sequestration in two cases , and lobectomy in two patients . 
patients with cle underwent lobectomy , and the only patient with bc underwent evacuation by puncture and cystic resection . no postoperative complications were observed . 846 radiol med ( 2014 ) 119 : 842851 fig . 
unenhanced mdct scans in axial ( b , c ) and coronal planes ( d ) show , in the left lower lobe , multiple small thin - wall cysts ( arrows )  . 
pus was suggestive of the correct diagnosis in 20 out of 26 lesions ( 76.9 % ) , 11 cpam , eight ps , and one bc , as conrmed by histology . of the two cases of cpam associated with ps , us correctly identied the two different lesions in only one case , while the other case was diagnosed as ps only . 
for cpam detection , pus sensitivity and specicity were respectively 100 and 60 % , whilst they were 66.7 and 100 % respectively for ps . comparison between mdct and histological ndings out of the 33 mdct patients , 31 underwent surgery for a total of 33 lesions . 
postcontrast mdct scans ( b , c ) reveal a hyperdense lesion in the left lower lung ( arrowhead ) with a large artery originating from the aorta ( black arrow )  . 
low power view of intralobar sequestration ( e ) shows pleural brosis and mild inammatory changes in pulmonary parenchyma with mucus in some alveoli ( h&e , 940 ) in the only observed case of bc , both pus and mdct diagnoses were conrmed by histology ; in cle , only two out of three cases in which mdct raised a suspicion were histologically conrmed ( ppv = 66 % ) , the remaining case being a cpam type i . 
in particular , mdct overestimated the extent of the lesions in three patients with cpam . discussion congenital cystic lung diseases include a heterogeneous group of anomalies affecting bronchi , lung parenchyma , arterial supply , and venous drainage . 
the spectrum of these abnormalities includes cpam , ps , cle , and bc [ 1 ]  . the rst diagnostic examination is pus , usually performed at 1822 weeks of gestation , during the rst scheduled morphological evaluation of the foetus [ 14 ]  . 
pus at 21 weeks of gestation ( a , b ) , displays a homogenous hyperechoic lesion ( clippers ) with an arterial vessel ( arrow ) entering the mass . mdct scans after contrast medium administration ( c , d ) show a homogeneously hyperdense mass in the left lower lobe ( asterisk )  . 
a feeding artery arising from the aorta ( white arrowhead ) with a venous drainage through the azygous system ( black arrowheads ) can be seen . mip reconstructions ( e , f ) show a marked vascularity of the lesion ( asterisk ) , and systemic venous drainage ( white arrows )  . 
in our series , lesions increased in size in 30 % of cases , decreased in size in 30 % of cases , and were no longer detectable at the last pus in 40 % of cases . after birth , chest x - ray is no longer the rst imaging approach , as it fails to detect lesions in about 60 % of asymptomatic patients [ 19 ]  . 
our mdct examinations were performed with ageand weight - based protocol in accordance with the literature , achieving a reduced biological aggressiveness in paediatric patients , particularly in newborn babies and little infants , as were many patients in this study [ 2023 ]  . 
furthermore , out of the four patients with no longer detectable lesion at the last pus , mdct showed the presence of cpam in three cases and of ps in one case . the accuracy of pus and mdct diagnosis was then compared with the histological data . 
agreement between pus and histology and mdct and histology was observed in 76.9 % ( 20 / 26 ) and 94 % ( 31 / 33 ) of the cases , respectively . 
overall , the ppv of the survey mdct scans compared with histology was 93.3 % , compared with a reported value of 70 % in the literature [ 19 ]  . 
as most surgeons consider surgical resection of these lesions mandatory , mdct can provide detailed anatomical localisation of lesions , with the possibility of 3d reconstruction and high spatial resolution . 
postprocessing reconstruction can also provide detailed information on vascular afferents , and 3d reconstructions in our study proved to be very helpful to the surgeon during preoperative planning [ 24 , 25 ]  . 
the analysis of our data shows that mdct should be used as rst - step imaging in the postnatal evaluation of patients with ccld , and our work suggests that mdct can be considered the best imaging modality in the postnatal evaluation of patients with ccld detected in uterus , with higher specicity values for cpam lesions and higher sensitivity for ps compared to pus ndings . 
patelli risk management , ospedale san gerardo , via pergolesi , 33 , 20900 monza , italy e - mail : c.patelli@hsgerardo.org nephropathy ( cin ) , when in the angiographic unit , we have used a 20 - item checklist named time - out derived from the cirse sc . 
the main items are : in the time - out phase , active verbal commuidentication of the nication within the team ; correct patient and of the procedure site and side . 
additionally , during the preprocedure ( sign - in ) and postprocedure ( signout ) stages a series of clinical data are collected such as administration of prophylaxis against contrast allergy or contrast - induced needed informed consent , discontinuation of anticoagulation therapy , fasting , correct labelling of biological samples , etc . results preliminary educational sessions were held to sensitise all the staff involved in the time - out project ( physicians , nurses , radiographers ) and ensure satisfactory compliance from the outset in consideration of the increased time and paperwork that checklist implementation would involve . conclusions the use of a checklist in ir , named timeout in our experience was feasible and effective allowing elimination of adverse events in the rst year of use and involvement and awareness of promoting signicant patient safety among the health - care teathe level of implementation , completeness and acceptability of the sc within the team increased after adequate training had been provided . interventional radiology ( cid : 2 ) patient safety ( cid : 2 ) keywords checklist introduction c . 
battisti 3 , 20854 vedano al lambro ( mb ) , italy adverse events are unexpected situations which occur during clinical activities and may result in temporary or permanent , physical , emotive or economic damage to the patient and increased health - care costs to society . radiol med ( 2014 ) 119 : 828834 it has been estimated that 50 % of all in - hospital adverse events are related to surgery and at least 50 % of these could be avoided because they are caused by errors [ 1 , 2 ]  . apart from the surgical risk itself , also the patients stay and transfer to and from the operating theatre , the different specialities and health professionals involved in surgical activity and the complexity of the procedures make the surgical disciplines more prone to error and therefore to adverse events [ 3 , 4 ]  . of all the targeted and effective actions that can be undertaken to reduce adverse events , the use of safety checklists ( scs ) might have a prominent role . 
this checklist led to a signicant reduction in clinical adverse events [ 5 ]  . even similar disciplines to surgery , such as endoscopy or interventional radiology ( ir ) , are potentially at risk of producing adverse events [ 2 ]  . 
ir procedures have many aspects in common with surgical operations ( complexity , fast patient turnover , urgency and emergency procedures , multilevel or diffuse pathologies , teamwork , etc ) and consequently carry a risk for potential error . 
the analysis revealed a lack of clarity regarding the organisation and duties of the various healthcare professionals working in the radiology suite ( denition of roles ) and the procedure for side identication . 
as a corrective measure , together with our hospitals risk manager we decided to plan , develop and implement a radiological sc with the aim of identifying and therefore reducing the probability of avoidable accidents or adverse events , or at least lessening their negative effects . to prepare our sc , we referred to the scientic literature on errors , adverse events and complications in ir , including the available standards and guidelines [ 6 , 8 ]  . 
in november 2011 , a workgroup made up of a radiologist , a nurse and a radiographer , coordinated by our risk manager , drafted a rst sc named time - out that reected our departments logistic reality . 
after completing each phase , the compiler , who is responsible for the check , must sign at the bottom of each block of items . in detail , after the patient is admitted to the ir department , a radiologist checks the blood tests ordered for the procedure and veries that the patient has received the required information regarding the interventional procedure . 
extensive instructions were given on how to ll in the sc correctly , especially in consideration of the time - consuming nature of the task and the need to ensure compliance . 
an initial trial period of 1 month was planned . at the end of the trial period , we addressed any general issues and doubts and assessed the completeness of the sc , making adjustments if necessary . after 3 months , the compilation rates were re - evaluated the team and a questionnaire was administered to all members , investigating satisfaction with the checklist , their experience with the use of the sc and evaluations of patient safety , and eliciting suggestions and ideas for improving the results . 
eight months after the introduction of the sc , another meeting was held to communicate the results of the rst 6 months and the critical points identied , leading to a nal revision of the sc . in december 2012 , data on adverse events related to ir procedures performed during the rst year of implementation of the sc were extracted from the intra - hospital software events . results the results at 1 and 3 months are reported in table 1 as regards the type of procedure performed on different patient groups and in table 2 as regards sc compilation . radiol med ( 2014 ) 119 : 828834 table 2 completeness of time - out safety checklist checklist complete incomplete incomplete hour barcode biological samples ac therapies / prophylaxis procedure signature ward fasting patient name site / side ac anticoagulation 13 january 17 february 100 / 112 62 ( 64.5 % ) 38 ( 35.5 % ) 29 ( 76.3 % ) 23 ( 60.5 % ) 16 ( 42.1 % ) 12 ( 31.5 % ) 4 ( 10.5 % ) 3 ( 7.9 % ) 3 ( 7.9 % ) 3 ( 7.9 % ) 2 ( 5.2 % ) 1 ( 2.6 % ) 13 april 17 may 90 / 101 76 ( 84.4 % ) 14 ( 15.6 % ) 6 ( 42.8 % ) 2 ( 14.2 % ) 2 ( 14.2 % ) 1 ( 7.1 % ) 4 ( 28.5 % ) 1 ( 7.1 % ) during the rst month , scs were compiled for 89.3 % of procedures ( 100 checklists out of 112 procedures )  . 
in particular , in the rst version of the sc , the query time of procedure was omitted in 76.3 % of the cases : considering that this information can be obtained both from the radiological images stored in the pacs and from the nursing assessment form ( used for a long time in the angiographic suite ) , the item was removed . 
other deleted items were the patient requires postprocedure care and insert operative report : in the rst case , because the information is already noted in the nursing assessment form and , in the second , because the operative reported is already contained in the radiological report , the completion and inclusion of which is veried in the sign - out phase . an anonymous survey was distributed to all of the 27 team members ( three physicians , 10 nurses and 14 radiographers ) , 17 of which were collected ( 63 % ) and analysed . 
starting from 2000 in the usa , the institute of medicine supported by the quality of health care in america committee launched a 10 - year project for the development and implementation of strategies to increase the quality and safety of health care by adopting verication procedures ( such as checklists ) and standardising processes to limit reliance on human memory , which is known to be far from infallible [ 9 ]  . a recent systematic review reported that the total average incidence of adverse events in hospitals is 9.2 % ( range 4.612.4 % ) , almost one in ten patients . 
moreover , ir procedures place the team in situations very similar to those experienced in operating theatres ( prolonged stress , operator fatigue , team inexperience ) that can lead to mistakes ( table 3 ) : this explains the need to enhance patient safety measures through greater standardisation of procedures and increased quality of care in ir procedures . in addition to the most commonly recognised errors in medicine , some errors are specic to ir and can potentially occur at any time during the patients stay in the angiographic suite . 
most of them are preventable and are listed in a table of 62 items published by the society of interventional radiology ( sir ) task force on medical error [ 8 ]  . for instance , a patient with bilateral or diffuse vascular disease depicted on ct or mr angiography might undergo different treatments without the appropriate preprocedural communication . 
the orientation of the radiological images must be set according to the patients position on the angiographic bed , which is not constant but related to operative requirements ( head / feet ; right / left ank ; prone / supine ) : if an incorrect setting is not rapidly detected by the team , e.g. , due to large sterile drape masking the anatomical landmarks , this could potentially lead to wrong - side errors . the operator must be aware of most common potential problems occurring before , during and after an interventional procedure , so that the use of a checklist should provide an opportunity for additional reection and evaluation . not many reports exist in the scientic literature on the use of sc in ir . 
since 2003 , at the boston childrens hospital , a time - out checklist has been used in paediatric ir procedures ; this follows the indication of the universal protocol , whose appropriateness is veried monthly by independent professionals [ 12 ]  . 
in 2007 , after an adverse event occurred in ir at the end of a dialysis catheterisation , stecker and mcnamara introduced a short postprocedural checklist so as not to omit administration of intracatheter heparin , afxation of closure caps or placement of dressing on the insertion site [ 13 ]  . an sc can be used in different health - care environments because it includes three important safety goals of the joint commission on accreditation in healthcare organizations ( jcaho ) : ( 1 ) improving accuracy in patient identication ; ( 2 ) improving communication between health - care coworkers ; ( 3 ) eliminating all wrong - site , wrong - patient and wrong - procedure interventions [ 14 ]  . 
the advantage of an sc for ir is that it ensures reduction of human error in terms of omitting key steps in pre - , intraand postoperative care , and therefore minimises potential harm to the patient . two papers recalling the jacho recommendations have described the application of the universal protocol to settings other than the operating theatre , adapting it to ir procedures [ 6 , 12 ]  . 
the universal protocol focuses on prevention of wrong - site / wrong - side procedures , while radiological sc aim to improve the entire ir process , from patient positioning to the use of intraprocedural images for site and side determination . in 2010 , england and wales introduced a specic sc for ir , derived and adapted from the who checklist . 
this list is made up of 28 items divided into three parts : sign - in ( before anaesthesia ) , time - out ( before the beginning of the procedure ) and sign - out ( before any member of the team leaves the angiographic theatre ) and includes a box with all the personal data of the patient . 
each section should be read out loud by one of the team members . in the same year , a radiological sc ( radpass , radiological patient safety system ) was also introduced in holland , at the academic medical centre of amsterdam . developed based on the available literature and expert opinion , the radpass checklist is divided into two parts : part a ( planning and preparation ) and part b ( procedure )  . the latter is divided into a series of checks to be done just before the beginning of the procedure ( b1 ) and items regarding postoperative care immediately after the procedure ( b2 )  . 
this checklist was to be applicable to a variety of interventional procedures , adjustable and modiable to meet the needs of different centres and should be satisfactory to the vast majority of interventional radiologists [ 16 ]  . 
in addition , unlike other scs , it requires the name and signature of the compiler to be inserted at the end of each section . in comparison to the cirse checklist , we decided to produce a more balanced and synthetic checklist to ensure better compliance and completion rates , while guaranteeing high standards of care , compliance with the guidelines and patient safety during all of the phases of ir procedures . out of the 32 items recommended by the cirse checklist , we extracted some essential items that better suited the specic needs of our centre . 
this led to the development of a nal checklist consisting of 20 items ( slightly shorter than the english and the dutch versions , which contain 21 and 27 items , respectively ) , which contributed to eliminating adverse events during the rst year of application , thus conrming its efcacy . the majority of patient safety interventions and some checklists require humans to modify their behaviour . 
we think that the positive effect of an sc is due to several mechanisms : direct prevention of mistakes through checklist performance ; optimisation of processes triggered by sc implementation ; improved team working stimulated by the sc ; improved team communication and attitudes towards safety and quality . the only complaint about checklists regards the amount of time required to perform thehaynes et al . 
these data have been inferred from the respondents comments , such as : explain which information / evaluations are to be done by the nurse , radiographer or physician ; establish , at beginning of each procedure , who does what ; its always the same people who perform the checklist , everybody should perform it . 
these uncertainties about responsibilities were subsequently analysed and claried at a team meeting the content of which was reported in a quality document of the radiological suite ( operative procedure of the angiography suite ) that can be found on our hospitals intranet . in the near future , the integration of checklists in digital patient information systems could help to develop more checklists capable of offering information and links to other sources . 
if the public health mission of ir relied until recently on the dramatic technological development of imaging - guided therapies ( sir , mission statement 2011 , whoweare ) , nowadays we are less centred on technology and more on the patient , in terms of treatment effectiveness , team dynamics , changing health behaviours , constant improvement of the quality of care , standardisation of care and reduction of costs [ 18 ]  . conclusion in conclusion , the use of an sc in ir may constitute a valid tool to maximise patient safety during interventional procedures , by intercepting possible adverse events and reducing or avoiding human errors at key points , such as patient preparation , procedure initiation or postprocedural care . the introduction of an sc ( called time - out in our experience ) into routine ir practice was found to be feasible and helped us to eliminate adverse events during the rst year of implementation , generating strong commitment and greater awareness of patient safety among the health - care teaapplicability , completeness and acceptance of the sc by the team improved after adequate training and after encouraging the different professional categories to communicate , cooperate and share responsibilities , which favoured the realisation of our objectives in terms of safety standards . conict of interest r corso , f . 
the efciency of automatic tube current modulation ( z - dom and d - dom ) , shaping lters ( smartshape and intellibeam ) and asymmetric collimator ( eclipse ) was evaluated using appropriate phantoms . results the scattered radiation contribution from the peripheral regions to the cumulative dose is not negligible . the cnrd is 20 % better compared to a traditional 16 - slice scanner , with a dose reduction of 40 % . 
the application of d - dom and z - dom modulations reduces the currenttime product ( mas ) values by 23 and 18 % , respectively , but they are not yet is not negligible , integrated . 
radiodiagnostica , azienda ospedaliera san gerardo , monza , italy conclusions brilliance ict dose reduction tools are efcient , but should be analysed carefully and used correctly . keywords multislice computed tomography ( cid : 2 ) radiation dosimetry ( cid : 2 ) patient radiation protection ( cid : 2 ) bow - tie lters ( cid : 2 ) over - ranging introduction in the last decade , computed tomography ( ct ) has undergone a remarkable development in particular with the advent of multi - layer technology ( multislice computed tomography , msct ) and , according to italian legislative decree 187 / 2000 , ct examinations are classied as diagnostic procedures involving a high radiation dose to the patient . recent papers emphasise the increase in the number of ct examinations and some of these studies have correlated the dose escalation to the population with the probability of tumour induction . 
in italy , particularly in the emilia romagna region , the contribution to collective effective dose from ct examinations is around 66 % of the total [ 1 ] ; in the us it is 67 % [ 2 ] and in the uk 40 % [ 3 ]  . 
in 1989 the national radiation protection board ( nrpb ) estimated that an abdominal ct scan carried a 1 : 2 , 000 risk of fatal cancer in 1669 - year - old patients [ 4 ]  . an increased risk of cancer induction has been observed in epidemiological studies on atomic bomb survivors , who were exposed to an effective dose of about 10100 msv [ 5 ] : a ct scan may result in an effective dose of the same order and the patient may be subjected to multiple ct examinations during their lifetime . 
in 2007 , approximately 72 million ct scans were performed in the us , grouped for 872 radiol med ( 2014 ) 119 : 871877 effective dose as follows : 78 % low dose ; 20 % moderate exposure ( 320 msv ) ; 2 % high ( [ 20 msv ) and very high ( [ 50 msv ) dose [ 6 , 7 ]  . optimisation of the acquisition technique may substantially reduce the dose per examination and , at the same time , the dose contribution of ct examinations to the population . 
the aim of this study was to provide a dosimetric characterisation of a 256 - slice ct scanner with particular attention to the available dose modulation and reduction tools used in clinical practice . materials and methods the multislice brilliance ict scanner ( philips healthcare , cleveland , oh , usa ) is able to acquire up to 256 slices of 0.625 mm thickness for each x - ray tube rotation , obtaining high quality 3d images . 
at the end of each examination , the volumetric computed tomographic dose index ( ctdivol ) along with the dose - length product ( dlp , dened as the product of ctdivol and scan length ) should be displayed on the control panel and indicated in the dosimetric report [ 9 ]  . 
to dene ctdivol , it is necessary to introduce another physical quantity : the ctdi100 corresponding to the ratio between the dose prole integral and the length of the pencil ionisation chamber ( with 100 mm integration length )  . 
two standard phantoms , consisting of a polymethylmethacrylate ( pmma ) cylinder with a height of 14 cm and a diameter of 16 cm ( head phantom ) or 32 cm ( body phantom ) , were used to measure ctdi100 . 
the ctdi weighted ( ctdiw ) , which expresses the average dose in the scan plane , is calculated from the measurements of ctdi100 at the centre and the periphery of these phantoms [ 10 ]  . 
hence ctdivol is obtained as ctdiw divided by pitch value , because in ct the dose is inversely proportional to the ratio between pitch and collimation . with the introduction of multislice ct scanners , the collimation may also reach values equal to or higher than 80 mm , so the ionisation chamber length ( 100 mm ) is inadequate for ctdiw measurement . 
standard methods are no longer applicable because they neglect the dose contributions of the penumbra and of diffusion from the adjacent tissues placed outside the primary bearecently the american association of physicists in medicine ( aapm ) has suggested to use , as an alternative to the ctdi , the equilibrium dose ( deq ) [ 11 ]  . 
for a xed scan length l along the longitudinal axis , the cumulative dose at the midpoint of scanning lengthdl ( 0 ) includes both the primary dose and the scattered radiation . 
as l increases , dl ( 0 ) increases asymptotically towards an upper limiting value , deq , such that the source of scatter radiation is sufciently remote as to make a negligibly small additional contribution . under the same exposure conditions , dl ( 0 ) was measured for different scan lengths using a 0.6 cm3 ionisation chamber ( 10x5 - 0.6ct , radcal corporation , ca , usa ) and a phantom created by inserting the body phantom between two 32 9 32 9 30 cm3 pmma parallelepipeds . 
to determine the deq , the cumulative dose was measured increasing the scan length from 8 to 45 ca 32 9 32 9 30 cm3 pmma parallelepiped was used as a phantom within which properly calibrated 30 9 1 cm2 gafchromic ( cid : 3 ) xr - qa2 dosimetric lms ( lot number a10071002a , isp technologies inc . , nj , usa ) [ 12 ] were included . 
the lms were digitalised by epson 10000xl atbed scanner 24 h after exposure and dose proles in the longitudinal direction were calculated using picodose xpro software ( tecnologie avanzate , turin , italy )  . contrast - to - noise ratio ( cnr ) the physical quantity for structure and detail identication in msct is the cnr . 
these lters work along with bow - tie lters , named smartshape ( small , medium and large ) , which change the beam intensity as a function of patient size . the bow - tie lters provide more ltration in peripheral regions than in the central one , where patient thickness is greater , to improve dose distribution and standardise image noise . 
furthermore , intellibeam and smartshape lter attenuation proles , expressed as number of counts sampled along a row of the array detectors , were analysed for different acquisition settings using philips proprietary software available on the workstation . values in the xy plane were determined from air - kerma measurements by placing the pencil ionisation chamber at 0 ( cid : 4 ) , 45 ( cid : 4 ) , 90 ( cid : 4 ) , 135 ( cid : 4 ) and 180 ( cid : 4 ) inside the gantry tunnel . 
for each x - ray tube rotation , mas values corresponding to 0 ( cid : 4 ) ( minimum ) and 90 ( cid : 4 ) ( maximum ) were calculated by multiplying the mas average value supplied by the ct scanner by the chamber percentage value equal to 1 for 90 ( cid : 4 ) instead of 0.86 for 0 ( cid : 4 ) and whole phantom or 0.81 for 0 ( cid : 4 ) and half phantom . over - ranging and eclipse asymmetric collimation over - ranging means an increase in scan length due to the additional rotations at the beginning and at the end of a spiral scan required for data interpolation to reconstruct the rst and the last slice of the imaged body region . 
the brilliance ict scanner has an asymmetric collimator , called eclipse , which automatically occurs at the beginning and at the end of the helical acquisition , reducing patient dose without any deterioration in image quality . dose proles of 1 9 30 cm2 gafchromic ( cid : 3 ) xr - qa2 strips irradiated with helical modality at the isocentre were analysed to verify the asymmetric collimator efciency and to estimate over - ranging . 
furthermore , the lms were placed between the cervical and the upper chest region of the anthropomorphic phantom alderson rando surface , to compare the effect of collimation to patient dose reduction . automatic current modulation system dosimetric audit the aim of the automatic current modulation system is to deliver an optimised current ( ma ) value for the type of patient for the anatomical region being studied . 
the brilliance ict scanner ma modulation system is structured as follows : the automatic control system ( acs ) which establishes maximum patient diameter and tissue density on the basis of the scanogram and automatically suggests the optimal x - ray current value to control dose and noise level ; the z - axis dose modulation system ( z - dom ) that modulates ma along the patient longitudinal axis using the attenuation prole ; nally , the dynamic angular dose modulation system ( d - dom ) which modies the ma as a function of patient eccentricity and tissue attenuation for each x - ray tube rotation . 
as there is a direct proportionality between mas and dose , the mas in february , june and september 2011 the dosimetric parameters and the over - ranging of 30 abdominal ct examinations ( 10 examinations / month ) were collected from the ct scanner dosimetric report . 
the dosimetric audit was carried out before installation of the idosetm iterative reconstruction algorithm . the ctdivol and the size - specic dose estimate ( ssde ) were considered to evaluate the patient absorbed dose ; ssde is a dosimetric quantity which takes into consideration corrections based on patient size and is dened as follows : ssde fsize ( cid : 2 ) ctdivol where fsize is the correction factor depending on patient diameter and on ctdivol phantom dimension ( 16 or 32 cm ) [ 14 ]  . 
the patient diameter was calculated by measuring the lateral abdomen dimension on the scanografurthermore , for the upper abdomen single scan , the effective dose was calculated by multiplying the dlp value by 0.015 sv mgy - 1 mm - 1 conversion factor [ 15 ]  . 874 radiol med ( 2014 ) 119 : 871877 figure 2 shows the number of counts sampled along a row of detector array for the ve default acquisition modalities . 
automatic current modulation systems figure 3 shows longitudinal ma prole in the philips calibration phantom as a function of different modulations . the system provides only the average mas for d - dom modulation . 
figure 5 shows the comparison between 64 9 0.625 and 128 9 0.628 mm collimation for mas and dose proles obtained with the alderson rando phantom and gafchromic ( cid : 3 ) xr - qa2 lms . 
the accuracy of mas modulation depends on collimation , especially at the transition of the x - ray beam over adjacent anatomical regions of different anatomical districts ( e.g. , neck - chest )  . 
dosimetric audit figure 6 summarises the ctdivol ( mgy ) , ssde ( mgy ) , effective dose ( msv ) and over - ranging ( mm ) mean values of 10 examinations collected in april , june and september 2011 . discussion an accurate evaluation of ct patient dose reduction tools is necessary for a proper use in clinical practice [ 1618 ]  . the contribution of scatter radiation to the cumulative dose from the phantom / patient peripheral region is not to be underestimated . 
the dl ( 0 ) value arises from 27 mgy ( minimum scanning length ) to 42 mgy ( 45 cm scanning length ) with an increase of 64 % . 
1 cumulative dose dl ( 0 ) at the midpoint of the irradiated length ( a ) and cumulative dose proles in function of irradiated length ( b ) results 1 . 
6 dosimetric audit results : ctdivol ( mgy ) , ssde ( mgy ) , effective dose ( msv ) and overranging ( mm ) mean values the brilliance ict scanner has a better gure of merit than a traditional 16 - slice scanner ( brilliance ct 16p )  . indeed , the new nanopanel3d detector in association with the clearray2d anti - scatter grid allows an improvement in cnrd of approximately 20 % , which means a patient dose reduction of 40 % . the smartshape and intellibeam lters reduce the peripheral patient dose in a variable percentage according to the acquisition modality employed . 
in fact , the dose distribution is not affected by the lter with trauma modality because the full dose is needed to perform a diagnostic examination extending to for body the patients extremities . 
on the contrary , modality and in particular for the cardiac protocol , the lter reduces the dose in the peripheral region , corresponding to a lower diagnostic interest . application of modulation along longitudinal axis ( zdom ) implies a dose reduction less than angle modulation ( d - dom )  . 
the dose to the thyroid increases by about 60 % switching from 64 9 0.625 to 128 9 0.628 mm collimation due to a greater over - ranging and to an inadequate mas modulation . the installation of increasingly complex ct scanners designed for highly specialised performance requires accurate optimisation of acquisition protocols to achieve adequate image quality without involving an unjustied increase in patient dose . 
a total dose of 3651 gy was delivered to the 50 % isodose line covering the planning target volume at 35 gy / fraction , whereas a total dose of 6085 gy was delivered at 57 gy / fraction to the gross target volume . 
the local control rate ( lc ) and overall survival rate ( os ) were calculated using the kaplanmeier method . results patient follow - up ended in march 2013 and the follow - up rate was 100 % . 
xia department of radiation oncology , air force general hospital , beijing 100142 , peoples republic of china e - mail : wangyj9999@163.com individualised treatment and a combination of multiple therapeutic approaches ; this will be a primary research trend in the future . keywords renal cell carcinoma ( cid : 2 ) radiotherapy , stereotactic body radiotherapy ( cid : 2 ) gamma - ray ( cid : 2 ) asynchronous bilateral tumour introduction renal cell carcinoma ( rcc ) is one of the most frequently occurring malignant tumours of the urinary system , and its incidence is rising worldwide [ 1 ]  . 
however , the probability of tumour after rn is developing a contralateral approximately 415 % [ 3 ] and the treatment for this asynchronous bilateral renal cell carcinoma ( brcc ) remains clinically challenging ; there is currently no unied therapeutic scheme . 
however , a considerable number of the patients cannot undergo surgery because of advanced local disease or other medical conditions . renal stereotactic gamma - ray body radiation therapy ( c - sbrt ) is widely known as an independent chinese innovation which is especially effective in the treatment of solid tumours such as lung and pancreatic cancer [ 47 ]  . 
herein , we report our experience regarding c - sbrt in the treatment of nine patients with asynchronous brcc between february 2002 and may 2012 . to our knowledge , there have been no previous published studies that have focused on asynchronous brcc radiol med ( 2014 ) 119 : 878883 treated using c - sbrt . 
the purpose of this retrospective study was a preliminary evaluation of the feasibility , efcacy and toxicity of c - sbrt in the treatment of asynchronous brcc . materials and methods ethics statement this study was approved by the institutional review boards ( irbs ) for the cancer center , air force general hospital , peoples republic of china . 
written informed consent was obtained from all patients in accordance with the irb regulations . general information the clinical data from nine patients with asynchronous brcc who had received c - sbrt between february 2002 and may 2012 were retrospectively evaluated . 
the inclusion criteria were as follows : ( 1 ) patient age 2085 years ; ( 2 ) a history of rn for the primary renal malignancy that was histopathologically or radiologically diagnosed as rcc ; ( 3 ) computed tomography ( ct ) or positron - emission tomography ( pet ) performed to identify any remaining renal lesions that were unresectable or medically unsuitable for resection ; ( 4 ) no loss of renal function ; ( 5 ) no history of radiotherapy involving the abdomen ; ( 6 ) an eastern cooperative oncology group ( ecog ) performance status scale b2 ; and ( 7 ) informed patient consent obtained for radiotherapy . 
patients who suffered from hereditary syndromes or who had a history of another invasive cancer were excluded from the study . the median age of the patients treated with brcc was 70 ( range 5884 ) years ; seven of these patients were male . the median maximum tumour diameter was 4 ( range 2.74.5 ) cthe median disease - free interval was 42 ( range 1302 ) months after surgery . 
tumours were staged according to the international union against cancer tnm classication ( 2006 )  . treatment methods stereotactic gamma - ray body radiation therapy ( ourqgd , shenzhen , china ) was carried out using a body gamma knife ; this consisted of a radiation source , collimator and treatment couch , as previously described [ 4 ]  . the system , which is a co60 stereotactic radiosurgery apparatus , is recognised worldwide as an effective method for the treatment of benign and malignant brain diseases , as well as body tumours [ 510 ]  . patients were immobilised by means of vacuum bags which covered the body from the head to the pelvis in a stereotactic frame ; patients were located in the supine or prone position based on the shorter distance from the lesion to surface . 
quiet breathing ( no special breathing restrictions ) was required throughout the period of slow ct scanning ( 10 s / slice ) with a ct slice thickness and slice interval of 5 mm ( somatom emotion 16 ct scanner : siemens , germany )  . 
then ct images were transferred to the planning system via the internet . the gross tumour volume ( gtv ) was the visible extent of the kidney tumour on the ct images . 
to protect renal function , the planning target volume ( ptv ) was formed from the gtv by enlarging it by 510 mm in accordance with tumour size , patient respiratory movements , positioning system error and the relationship dose . 
normal kidney volume was dened as the total kidney volume minus the gtv . three groups of chambers with collimator aperture diameters of 10 , 30 and 50 mm were used . 
the full width at half - height of the dose - eld range at the target was 10 , 30 and 50 mthe treatment plan used a combination of different collimators in the target area depending on the size of the tumour [ 4 ]  . 
dose - volume planning objectives for the oars were dened as follows : normal kidney , liver , mean dose b18 gy ; small mean dose b15 gy ; fig . 
1 dose distribution in the axial view of a patient who had suffered radical nephrectomy of the right renal malignancy and who underwent stereotactic gamma - ray body radiation therapy for a left renal tumour . 
the 50 % isodose line could cover 100 % of the planning target volume ( ptv ) and the 70 % isodose line could cover 95 % of the gross tumour volume ( gtv ) 880 radiol med ( 2014 ) 119 : 878883 intestine , maximal dose b30 gy ; stomach , maximal dose b35 gy ; and spinal cord , maximal dose \27gy . 
for the small intestine and stomach , the maximal dose was expressed as the d0.5cc. follow - up and curative effect assessment after completion of c - sbrt , all of the surviving patients were regularly examined until death or the last clinical follow - up . 
follow - up included medical history , physical examination , blood work and a ct scan on the 1st , 3rd , 6th and 12th month in the rst year after treatment and every 612 months ; thereafter . 
the information regarding recurrence , toxicity and survival were recorded via telephone , mail and local hospital imaging reports . clinical effects were classied on the basis of single diameter measurement and solid tumour response evaluation criteria [ response evaluation criteria in solid tumours [ 11 ] as complete remission ( cr ) , partial ( recist ) ] remission ( pr ) , stable disease ( sd ) and progressive disease ( pd ) according to the ct images . 
reaction to irradiation was classied as early or late side effects according to the radiation therapy oncology group and the european organization for research and treatment of cancer toxicity criteria . statistical analysis statistical analysis was performed using the spss 13.0 software packages ( spss inc , chicago , usa )  . 
the follow - up period was dened as the time from the date of c - sbrt to the date of either death or the last clinical follow - up in months . 
comparison of the renal function index of the nine patients before and after c - sbrt was analysed using the paired t test ; a p value \0.05 was considered statistically signicant . results technical and dosimetric ndings the planning objectives were reached in all of the c - sbrt plans and all of the patients completed radiotherapy . 
2 dose - volume histograms ( dvh ) for stereotactic gamma - ray body radiation therapy illustrating the delivery of a high radiation dose to the gross tumour while sparing adjacent normal structures . 
left kidney mean dose , 13.6 gy ; spinal cord maximum dose , 12.5 gy ; stomach maximum dose , 10.0 gy ; small intestine maximum dose , 9.8 gy ; liver mean dose , 0.1 gy radiol med ( 2014 ) 119 : 878883 table 1 tumour features and treatment outcomes case sex age ( years ) pathology of the primary renal tumour stage clinical effects follow - up ( months ) survival m 71 m 67 m 70 m 77 m 73 m 84 m 58 c clear cell carcinoma , g granular cell carcinoma , p papillary renal cell carcinoma , pr partial remission , sd stable disease , pd progressive disease , pm pulmonary metastasis , mm multiple metastasis , c cardiac diseases , s survival these patients was gradually phased out of the use of analgesics within 3 months after c - sbrt , and the pain experienced by the other two patients was in cr after treatment with oral drugs at low maintenance doses . 
seven patients had died by the end of the follow - up period ; death was caused by multiple metastases in three patients , respiratory failure in three patients as a result of pulmonary metastasis and fig . 
neither temporary nor permanent dialysis was required . discussion the incidence of brcc has been reported to vary from 1.811.0 % [ 12 ] , and it can be subdivided into synchronous and asynchronous tumour types according to the occurrence over time [ 13 ]  . 
the clinical symptoms of brcc are similar to those of unilateral rcc in showing a typical trilogy including lumbago , gross haematuria and a palpable mass , or paraneoplastic syndrome such as fever , hypertension and others . 
however , the applications of nss or minimally invasive surgical techniques are currently limited in patients with large tumours , cardiopulmonary comorbidities , and adhesions between the lesion and the surrounding intestine , or in patients who are unwilling to undergo surgery . 
in these patients , ongoing maintenance haemodialysis after rn results in a poor quality of life . renal cell carcinoma has traditionally been labelled as radioresistant when treated with conventional external beam radiotherapy . 
with constant improvements and the wide application of modern imaging technologies and radiotherapy , clinical outcomes have been further improved . our retrospective study demonstrated that c - sbrt is a promising treatment for local disease control with only minor toxicities in the management of asynchronous brcc . the prominent characteristics of c - sbrt can be summarised as higher precision , higher target dose , higher target conformity and delivery in a lower number of fractions . 
in the present study , a total dose of 3651 gy was delivered to the 50 % isodose line covering 100 % of the ptv at 35 gy per fraction , with 6085 gy to the 70 % isodose line covering 95 % of the gtv . 
the biological equivalent dose ( bed ; a / b = 3 ) regarding the gtv was 160227 gy , which represents an obvious increase as compared with conventional radiotherapy . 
the toxicity of c - sbrt in the current study was very tolerable . the side effects of therapy were mainly myelosuppression and digestive tract reaction which was relieved after symptomatic treatment . 
the gfr had not signicantly declined relative to previously measured values ( p [ 0.05 ) , which indicated that c - sbrt was safe and could effectively preserve renal function . untreated brcc carries a poor prognosis with no survival beyond 1 year . 
the 5 - year survival rate in our study was 35.6 % , which was similar to a 5 - year survival rate of 38 % reported in the literature [ 15 ]  . 
the survival of one of our patients treated using c - sbrt combined with interleukin - 2 ( il - 2 ) was 122 months ; another patient who underwent c - sbrt for pulmonary metastasis as well as combined il - 2 and sorafenib after radiotherapy survived for 65 months , underlining the necessity of comprehensive treatment . the pathologies of brcc are not always the same [ 16 ] and gene mutation may lead to inconsistency [ 17 ]  . 
 [ 18 ] suggested that the characteristics of contralateral metastatic disease were : ( 1 ) multiplicity of tumours on the contralateral side ; ( 2 ) a similar histological pattern ; ( 3 ) absence of encapsulation ; and ( 4 ) occurrence at a short interval after the primary tumours . 
analysis of the clinical information in this series indicated that : ( 1 ) the interval between the tumours appearances on the contralateral side was relatively long ( 42 months ) ; and ( 2 ) the tumours on the contralateral side were relatively large ( 4 cm ) and they were mostly solitary . 
it was also uncertain as to whether the different toxicities were related to the doses received by the oars because of a lack of grade 3 or 4 toxicity in our cohort ; thus , a prospective randomised controlled study is required to further conrm the value of c - sbrt in these patients . ours is the rst study that has focused on asynchronous brcc treated using c - sbrt . 
the results revealed that csbrt could be a feasible treatment for asynchronous radiol med ( 2014 ) 119 : 878883 brcc , especially for the patients who are unable to undergo surgery . 
imaging evaluation of fai , mainly based on plain lm and magnetic resonance evaluation , must be performed according to precise guidelines and is fundamental for reaching a nal diagnosis . 
sconenza dipartimento di scienze biomediche per la salute , universita` degli studi di milano , via morandi 30 , 20097 san donato , milanese ( mi ) , italy imaging techniques used in the diagnosis , and the main radiological signs that may be encountered . keywords hip ( cid : 2 ) femoro - acetabular impingement ( cid : 2 ) pelvis radiography ( cid : 2 ) magnetic resonance arthrography introduction first described by the swiss orthopaedic surgeon reinhold ganz et al . 
 [ 1 ] , femoro - acetabular impingement ( fai ) is a pathological condition that is frequently seen in young active subjects , often in conjunction with top - level sport activities , in which bony components of the hip joint do not match correctly . 
this pathological condition can be caused by an anomalous junction between the femoral head and neck , by an anomalous acetabular shape or orientation , or by a combination of both factors ( the latter event being the most common , occurring in up to 86 % of cases ) [ 2 ]  . femoro - acetabular impingement is currently considered one of the main causes of early hip osteoarthritis , especially in young active subjects [ 2 ]  . despite the extensive literature on this topic , the diagnosis of fai is still challenging and is based on a combination of hip pain , reduced range of movement , positive conict tests , and specic radiological ndings . 
in this setting , normal movements performed when an abnormal anatomical situation is present lead to abnormal contact between the articular surfaces , causing important degenerative abnormalities [ 3 ]  . 
finally , cam - type fai may be also associated with an acetabular undercoverage , which represents residual dysplasia and may have different the load is grades of in this condition , severity . radiol med ( 2014 ) 119 : 103112 fig . 
1 femoro - acetabular impingement : a normal hip ; b cam type ; c pincer type concentrated over a limited area of cartilage , thus favouring early damage and instability , in turn leading to the typical morphostructural alterations [ 9 ]  . symptoms femoro - acetabular impingement generally has a subtle onset . 
sometimes , the hip may snap or partially block due to labral tears [ 10 ]  . radiol med ( 2014 ) 119 : 103112 clinical tests clinical examination of a patient with suspected fai includes three tests [ 11 ]  . ( a ) anterior conict test ( exion , adduction , intra - rotation = fadir ) is considered the most sensitive and specic test . 
with the patient lying supine on the table , the physician exes 90 ( cid : 3 ) the patients knee and rotates it internally , then adding an adduction movement . 
with the patient lying on the table , the physician moves the patients foot of the affected side on the contralateral knee , then applying a mediallateral pressure on the knee of the affected side . 
when positive , the test elicits posterior - lateral gluteal pain . imaging x - ray evaluation patients with a clinical suspicion of fai generally undergo x - ray evaluation rst . 
the anterior acetabular margin is lateral to the posterior margsolid line bottom of the acetabulum ; dotted line ilioischiatic line ; dashed line anterior acetabular margin ; dasheddotted line posterior acetabular margin this condition , both on the femur and on the acetabuluat the beginning of the disease , subtle fai ndings can be seen , while characteristic signs of hip osteoarthritis are not already visible [ 2 ]  . anteriorposterior ( preferably performed with patient standing ) and axial 45 ( cid : 3 ) femoral exion ( also known as 45 ( cid : 3 ) dunn view ) projections should be always performed . 
radiographic evaluation must be always performed on both hips [ 2 , 12 ]  . ( a ) anteriorposterior projection : the patient stands with the lower limbs in 15 ( cid : 3 ) of internal rotation . 
the anterior bump can be detected only in the anteriorposterior projection when located on the superior - lateral headneck junction ; only in the axial projection when located anterior to the headneck junction ; or in both projections when it has a larger extension . 
such abnormality can be quantied by calculating the angle between the major axis of the femoral neck and the base of the femoral headneck junction abnormality [ 1 ]  . 
what was once dened as a herniation pit is now considered as bro - osteitis at the site of impact with the acetabulum in cam - type fai [ 15 ]  . 
for example , an anterior bony bump can be frequently seen in elderly patients affected by plain hip osteoarthritis . the diagnostic performance of radiographic evaluation in fai demonstrated variable results depending on the anteriorposterior the projection considered . 
the bottom of the acetabulum ( arrowheads ) is medial to the ilio - ischiatic line ( dotted line ) when the femoral head is projected medially to the ilioischiatic line , a protrusion acetabuli can be diagnosed . the relationship between the margin of the anterior and posterior acetabular proles on the anteriorposterior projection allows for assessing the physiological acetabular anteversion or retroversion . 
moreover , a lateral pelvic projection is useful to assess the anteriorposterior tilt ( normal value approximately 60 ( cid : 3 ) ) [ 14 ]  . radiol med ( 2014 ) 119 : 103112 fig . 
a the antero - posterior view shows a cross of the anterior ( dashed line ) and the posterior ( dashed dotted line ) acetabular margins . the typical 8 - like pattern can be seen . 
6 wibergs angle can be calculated by tracing a line perpendicular to the transverse pelvic axis and passing over the centre of the femoral head and another line that joins the superior - lateral acetabular roof and the centre of the femoral head . 
for the axial hip projection , the same data were 7074 % and 63 % , respectively , while the dunn projection reached values of 9169 % and 88 % , respectively [ 17 , 18 ]  . 
a recent paper on acetabular retroversion reported that the evaluation of such parameter is burdened by strong interobserver variability , which progressively decreases with increasing observers experience [ 19 ]  . ultrasonography the importance of ultrasonography in the diagnosis of fai is limited . 
7 acetabular intersecting a line that connects the acetabular drops ( arrows ) and another line that courses along the superior - lateral margin of the acetabulum and the femoral head . 
values over 10 ( cid : 3 ) indicate potential instability evaluation of the neonatal hip [ 20 ] , in adults ultrasonography is limited to the detection of intra - articular effusion , evaluation of inammatory , degenerative or post - traumatic conditions , or to the guidance of diagnostic or therapeutic intra - articular injections [ 21 ]  . because the hip is a deep joint , ultrasonography is commonly performed using a low - frequency convex probe . some authors reported that ultrasonography was able to detect most acetabular degenerative tears [ 22 ]  . 
 [ 23 ] , who suggested three qualitative criteria to evaluate the anterior and anteriorsuperior proles of the femoral headneck junction , and to quantify the a angle in cam - type fai . the features of ultrasonography ( low cost , wide availability and lack of ionising radiation ) would make it the ideal imaging modality for preliminary cam - type fai screening . 
however , the results [ 23 ] do not support such a hypothesis , particularly because the evaluation of the a angle alone may not be sufcient for the diagnosis of fai [ 23 ]  . 
finally , as it is unable to evaluate intra - articular 108 radiol med ( 2014 ) 119 : 103112 multiplanar reconstructions oriented along the neck axis , with submillimetre slice thickness . 
computed tomography allows for an accurate evaluation of calcic metaplasia of the labrum , scarcely detectable using magnetic resonance imaging ( mri )  . conversely , mri allows for a more precise evaluation of subcortical bone damage , represented with low signal on t1 - weighted sequences and high signal on t2 - weighted sequences . 
however , the simple mri evaluation allows for a limited analysis of the labrum and of the cartilagelabrum complex , which are of paramount importance when a surgical approach is anticipated [ 24 , 25 ]  . 
it is advisable to perform intra - articular injection under imaging guidance , as it has been demonstrated that hip injection without guidance is burdened by a 40 % rate of failure [ 27 ]  . 
the contrast agent must be injected very slowly , explaining to the patient that the procedure may be variably painful and may be associated with lameness lasting from few minutes to 7 days . within the magnet , the patient is positioned in the supine position , and the feet should be bound together with thus allowing for adhesive tape in 15 ( cid : 3 ) intra - rotation , muscle arthrography examination starts with axial and coronal t1and t2weighted fat - saturated and non - fat - saturated sequences , including the iliac wings and the lesser femoral trochanter . then , t1 - weighted or proton density sequences should be performed , with axial and coronal oblique orientation centred on the femoral neck and with double oblique sagittal orientation centred on the labruthe examination can be completed with radial sequences oriented along the major axis of the femoral neck : these sequences can be included in the standard package provided with the mr system , or isotropic volumetric t1 - weighted sequences can be reformatted in radial planes . 
these sequences have the greater advantage of sections that are always perpendicular to the labrum , with a low occurrence of artefacts , thus allowing a 360 ( cid : 3 ) evaluation of femoral headneck junction [ 28 ]  . 
magnetic resonance in the diagnostic setting of fai , several aspects should be considered . ( a ) evaluation of skeletal morphology : presence of pistol grip - like headneck complex deformity and bumplike irregularity of the femoral headneck junction fig . 
9 computed tomography axial scan ( slice thickness = 3 mm )  . presence of a typically round - shaped os acetabuli ( arrow ) and calcic metaplasia of the anterior labrum ( arrowhead ) abnormalities , ultrasonography has a limited value in the assessment of patients with painful hip . 
10 proton density - weighted fat - saturated coronal oblique scan ( te = 15 ms ; tr = 3 , 310 ms ; slice thickness = 3 mm )  . 
b proton density - weighted fat - saturated coronal oblique scan ( te = 15 ms ; tr = 3 , 310 ms ; slice thickness = 3 mm )  . 
this parameter can be also evaluated on routine noncontrast sequences . ( b ) evaluation of chondral erosion and damage of the femoral head and acetabular surface : it is important to describe the articular rim ( width and symmetry ) [ 29 ] and calculate the extent of chondral damage ( as a percentage or in centimetres )  . 
these parameters cannot be adequately evaluated using standard mri , except for advanced chondropathy associated with subchondral oedema ( hyperintense on t2 - weighted sequences ) , which is extremely important for surgical planning . 
regarding the site , chondral damage occurs more frequently on the anterior superior portion of the acetabulum in cam - type fai , while an posterior inferior localisation is more frequent in pincer - type 110 radiol med ( 2014 ) 119 : 103112 therapeutic options in the literature , there is little agreement on the use of drugs and physiotherapy in patients with fai . 
however , it is a widely held opinion that conservative treatment , being unable to modify the morphological aspects of this condition , cannot represent a real solution [ 33 ]  . patients with hip pain lasting over 6 months , unresponsive to conservative treatment , in whom radiographic signs of fai are present , should undergo surgical treatment [ 34 ]  . three surgical techniques are available for treating the morphological abnormalities underlying fai and the associated lesions : ( a ) open surgery : rst described by ganz et al . 
some orthopaedic surgeons also perform cartilage repair using mitmcroperforations or chondroplasty [ 36 ]  . ( b ) arthroscopy : this is a less invasive technique , but more difcult to perforwithin the articular space , it is possible to treat cartilage tears and reduce the acetabular margin , detaching and re - inserting the labrum ; outside the joint space , it is possible to reduce the femoral bump , checking the disappearance of conict by using dynamic manoeuvres [ 37 ]  . ( c ) mini - open technique : this is a mixed technique , in which femoral neck osteoplasty is performed in open luxation through a small anterior surgery without incision , while intra - articular abnormalities are treated using arthroscopy [ 38 ]  . at similar rates of post - surgical shortand long - term success , arthroscopy has a lower complication rate [ 39 ]  . 
in open surgery , possible complications are trochanter pseudoarthrosis and trochanteric bursitis , heterotopic calcications , and capsular brosis , while in mini - open surgery lateral femorocutaneous nerve lesion is the most frequent complication ( 1745 % ) , followed by transitory neuroapraxis of the pudendal and femoral nerve [ 39 ]  . in patients in whom fai led to advanced osteoarthritis , conservative surgery is contraindicated and hip prosthesis represents the only solution . the role of the general radiologist in consideration of the above , it is clear that fai diagnosis is extremely complex and involves a series of different parameters that cannot be summarised in a single radiological examination . 
13 proton density - weighted fat - saturated coronal oblique scan ( te = 15 ms ; tr = 3 , 310 ms ; slice thickness = 3 mm )  . 
similarly to what happens for the shoulder , conventional mri is able to detect large abnormalities of the labrum , especially when associated with joint effusion that may mimic the arthrographic effect . 
previous studies have shown that conventional mri has 30 % sensitivity and 36 % accuracy in the detection of labral tears , in comparison to 90 and 91 % , respectively , of mra . radiol med ( 2014 ) 119 : 103112 a few years , the scientic community is now facing a possible problem of overdiagnosis . 
indeed , abnormalities that are found in fai are present in a number of asymptomatic subjects and can lead to an incorrect diagnosis if not closely correlated to clinical symptoms [ 2 ]  . in this setting , the general radiologist has a crucial role . on the one hand , some subtle diagnostic signs are not clearly visible on routine radiological examinations . 
this is particularly important , as it is thought that early conservative treatment is able to slow the progression of osteoarthritis , although long - term studies on this topic are still lacking [ 2 , 7 ]  . information to request conclusions fai is a pathological condition with multiple clinical and diagnostic aspects that affect the hip joint . 
orsola - malpighi , bologna , italy results saber - sheath trachea was found in 18 / 71 ( 25.4 % ) patients , with a greater prevalence in patients with lower tiffenau index ( p = 0.02 ) , gold stages iiiiv and visual severity score 3 ( severe ) on chest ct . 
moreover , 52 / 71 ( 73.2 % ) had a chest computed tomography ( ct ) scan available for review ( 16 - slice somatom sensation cardiac 16 , siemens , forchheim , germany )  . images were assessed for the presence of saber - sheath trachea ; radiographic / ct pattern ( negative , emphysematous , bronchitic , mixed ) ; in patients with ct : visual severity score ( 1 = mild , 2 = moderate , 3 = severe ) , visual emphysema score , tracheo - bronchial diverticula , bronchiectases , signs of inammation of the small airways , ossication of cartilage rings , tracheomalacia on expiratory scans , if acquired . 
the nonparametric wilcoxons test for independent samples and the v2 test or fishers exact test were used to compare the continuous and categorical variables with the presence or absence , respectively , of saber - sheath trachea . linear regression was used to verify the relationship between the tracheal index at ct and radiography . 
similarly , the prevalence of saber - sheath trachea was higher among patients with severe - to - very severe copd ( gold stages iiiiv ) as compared to those with mild - to - moderate copd ( gold stages iii ) , with a trend towards statistical signicance ( p = 0.05 , table 3 )  . 
by contrast , no signicant difference was found between patients with and without saber - sheath trachea and the parameters age , sex and radiological pattern of disease . finally , the serial evaluation of patients with more than one available chest radiogram ( 32 / 71 , 45.1 % ) demonstrated that the tracheal index tended to remain constant over time without signicant dimensional changes . at ct , considered the gold standard , 18 / 52 ( 34.6 % ) patients showed saber - sheath trachea . 
he explained that the nding was a result of aging and did not put it in relation to obstructive airway disease , even though the majority of the cadavers examined had emphysema . 
it was not until the 60s that this sign was also reported on standard chest radiography and correlated with copd [ 7 ] , and thus added to the wide range of airway abnormalities that may be encountered in patients with copd [ 813 ]  . nowadays saber - sheath trachea is known to be a specic sign ( specicity , 92.9 % ) of copd , although its reported sensitivity is low ( 39.1 % ) [ 14 ]  . 
among the possible causes of airway remodelling with tracheal narrowing we should recall : stenoses ( post - infectious or iatrogenic ) , neoplasms ( benign , malignant and metastases ) , other conditions ( amyloidosis , sarcoidosis , behcet syndrome , wegener granulomatosis , tracheorelapsing polychrondritis , ulcerative bronchitis associated with crohns disease , etc . ) [ 1 , 15 , 16 ]  . only a few , older , studies have attempted to correlate the presence of saber - sheath trachea with functional parameters : is still a matter of debate whether its development is to be ascribed to parenchyma hyperinsufation [ 3 , 14 , 1719 ]  . 
5 saber - sheath trachea ( st ) is not linked to the radiological pattern of copd : a , b coronal ct reconstruction and axial image in a patient with copd with severe emphysema phenotype ( emphysema visual score , 63 % ) and st ( tracheal index 0.51 ) , c , d coronal ct reconstruction and axial image in a patient with copd with mild bronchitic phenotype and st ( tracheal index 0.56 ) radiol med ( 2014 ) 119 : 9096 fig . 
6 axial inspiratory ( a ) and expiratory ( b ) ct scans in a patient with saber - sheath trachea : tracheomalacia is not documented the results of our study show that saber - sheath trachea is not a constant sign in patients with copd ( 25.4 % prevalence ) , but it is correlated with functional severity , being more frequent in patients with low tiffenau index and with gold stages iiiiv . 
moreover , it is independent of the patients age and sex , in contrast to previous reports , which found a clear predominance among men [ 3 ]  . saber - sheath trachea is therefore a prognostic element that can be detected with no more than standard chest radiography . 
thus , these results require conrmation by a larger study with prospective design . in conclusion , saber - sheath trachea seems to constitute one of the many radiological subphenotypes that copd patients may present with , it is independent from the other radiological signs of disease and it represents an excellent marker of functional severity . 
anxiety may affect ctca image quality in women . comfort , overall greater keywords coronary artery disease ( cid : 2 ) anxiety ( cid : 2 ) conventional coronary angiography ( cid : 2 ) ct coronary angiography introduction computed angiography with tomography coronary ( ctca ) has emerged as one of the most technologically fascinating imaging tools over the last years [ 13 ]  . 
ctca has achieved high sensitivity and negative predictive value in the evaluation of coronary artery disease with applications extending to various clinical settings [ 49 ] and recently reinforced by the excellent prognostic outcome [ 1012 ]  . however , the point of view of the patient and the social acceptance of a method should also be tested for a comprehensive assessment of a modern medical technology the success of a method is undeniably [ 13 ]  . 
in fact , radiol med ( 2014 ) 119 : 128134 associated with the intrinsic acceptance of the method by the patients , who are increasingly pursuing noninvasive and outpatient diagnostic examinations [ 14 ]  . 
exclusion criteria were : refusal to provide informed consent , severe renal impairment ( creatininaemia [ 120 mmol / l ) , known allergy to iodinated contrast media , possible pregnancy , presence of hyperkinetic arrhythmias , severe impairment of respiratory function . 
patients underwent ctca with the following clinical indications : suspected coronary artery disease ( n = 299 ) , atypical chest pain ( n = 152 , 34 % ) , typical angina with inconclusive stress test ( n = 47 , 11 % ) , high cardiovascular risk prole ( n = 52 , 12 % ) , or candidates for aortic valve replacement surgery ( n = 48 , 11 % ) ; follow - up of myocardial revascularisation procedures performed at least 2 years before ctca ( n = 143 ) , including the assessment of the patency of proximal stents ( n = 75 , 17 % ) and bypass grafts ( n = 68 , 15 % )  . 
a history of cca was present in 181 patients . all patients underwent the endler multidimensional assessment of anxiety ( endler multimodality anxiety scales , emas ) before and after ctca , and a multiple choice questionnaire with a ve - point likert scale for the evaluation of acceptance after ctca . 
ct scans were performed with the following parameters : slices / collimation 64 / 0.6 mm , rotation time 420 ms , effective temporal resolution ( with 180 ( cid : 3 ) algorithm ) 210 ms , 120 kv , 8001040 mas , table feed 11.9 mm / s , effective slice thickness 0.8 mm , reconstruction increment 0.4 mm , eld of view ( fov ) 140240 mm ( extended cranially only for evaluating ascending aorta aneurysms and by - pass grafts )  . 
patients with a heart rate [ 65 beats per minute ( bpm ) were given a dose of 2040 mg of propranolol by mouth ( inderal , astrazeneca reims , reims cedex , france ) 1 h prior to the scan to lower the heart rate . 
in the leaet - informing patients on how to prepare for the examination , treatment with 2040 mg propranolol twice daily under physician supervision was recommended in the 3 days prior to the examination to lower and stabilise the heart rate in patients with tachycardia . 
a bolus of 100120 ml of nonionic iodinated contrast agent ( iomeprol , iomeron 400 , bracco , milan , italy ) , according to the scan range , was administered at an injection rate of 5 ml / s using an automatic injector ( stellant , medrad , pittsburgh , pa , usa ) connected to an 18 - gauge needle cannula placed in a right antecubital vein . intracoronary enhancement was optimised by using the bolus - tracking technique , with a region of interest ( roi ) positioned at the level of the ascending aorta in order to synchronise the start of the scan with the arrival of the contrast agent . 
the emas test , while maintaining the traditional constructs of the distinction between state and trait anxiety , represents a multidimensional scale of anxiety that can accurately assess individual responses and anxiety reactions in different situations . 
the emas test is composed of three parts : the emas - state ( emas - s ) , which evaluates state anxiety in relation to autonomic emotional and cognitive components ; the emas - trait ( emas - t ) , which assesses anxiety proneness in four different general contexts [ social evaluation ( se ) , physical danger ( pd ) , ambiguous situations ( am ) , and daily routine ( dr ) ] ; the emas - perception of the situation ( emas - p ) , fundamental control in a research context , which represents a measure of the respondents subjective perception of the type of situation and degree of threat evoked by that situation at the time of testing [ 25 ]  . 
in particular , the following aspects were evaluated : preparation and information before the imaging examination , degree of preceding concern , comfort , helplessness during the examination , pain experienced , degree of overall satisfaction . 
the same questionnaire was formulated also with reference to cca and given to the 181 patients who reported having a history of cca imaging . statistical analysis in all patients before the results of the emas - s test ( emas - si ) and after the performance of ctca ( emassii ) were compared by using students t test ( p \ 0.01 ) , further dividing subjects into subgroups based on gender and presence of cca history . 
the process of the health technology assessment ( hta ) investigates not only the technical reliability and diagnostic performance of a new method , but also aims to assess its prognostic , economic , and social impact , with the primary goal of improving health and wellbeing of the population , by integrating both medical and nonmedical multidisciplinary knowledge and strategies [ 13 ]  . 
therefore , hta needs to combine clinical and scientic evidence with social , cultural and ethical aspects , largely involving the patients , who are the protagonists and beneciaries of a health service [ 27 ]  . 
it is now recognised that full acceptance of a medical procedure or a diagnostic tool by the patient provides more satisfaction , better clinical results , and radiol med ( 2014 ) 119 : 128134 fig . 
in this respect , the following missions are essential for the spread of a modern imaging technique : adequate information and preparation of the patient in a recovered doctorpatient the adoption of noninvasive investigations relationship ; reducing associated anxiety and pain ; less time spent in health facilities , with lower impact on the patients psyche and work activities [ 28 ]  . several studies exist in the radiological literature that investigate the patients acceptance of new noninvasive it was demonstrated that imaging methods [ 1517 ]  . noninvasive methods such as ct virtual colonoscopy [ 15 ] , mr cholangiopancreatography [ 16 ] , and mr angiography of the carotid arteries [ 17 ] are more widely accepted by patients than the traditional invasive approaches [ 1823 ]  . computed tomography coronary angiography provides excellent results in the evaluation of atherosclerotic coronary artery disease due to high sensitivity and negative predictive value , which have enabled its clinical implementation [ 19 ]  . 
the effective 132 radiol med ( 2014 ) 119 : 128134 strategies to reduce the dose of radiation introduced by the latest generation of scanners have helped to further improve the clinical applicability of ctca [ 30 ]  . in our study , the focus was instead oriented on examining the psychological impact of ctca on the patients anxiety and the acceptance of the technique . 
we used the emas test , a tool considered the gold standard for the assessment of anxiety in a clinical / hospital and research context , due to the excellent psychometric properties and the several validation studies of validity and reliability , as well as the presence of italian standardised norms [ 31 , 32 ]  . the advantages of the emas test include : the ease and speed of administration , which is pivotal in a clinical / hospital setting , where the patient is already unavoidably concerned about his own health and the medical procedure ; a very accurate assessment of anxiety in relation to different contexts ; the exibility of using three measurement scales ; the ability to apply the control of perceived anxiety of the situation ( emas - p ) to state anxiety scale ( emas - s ) [ 25 ]  . anxiety was more intense prior to ctca than after , and higher in patients with a previous history of cca . 
ctca had an excellent prole of acceptance by the patient , and better than cca with respect to parameters such as preparation and information , comfort , degree of concern , helplessness , pain experienced , and overall satisfaction [ 14 , 19 ]  . there are a number of limitations to our study . 
the heart rate parameter was inuenced by the pharmacological preparation ( beta - blockers , benzodiazepines ) ; however , diazepam was given before the administration of the emas test in order to equalise the heart rate - state anxiety readings . 
another limitation is that the surveys carried out with specic reference to cca were exclusively performed in 181 patients on the basis of their previous medical history ; the comparison was made only in this subgroup and not on a population undergoing both methods because of the compulsory containment of biological costs of ionising radiation [ 30 ]  . finally , it seems appropriate to emphasise that recovery of the traditional doctorpatient relationship in radiology should be built on the balance between diagnostic performance and patient acceptance , avoiding the consolidation of distorted alliances based on the use of defensive medicine , corporate interests , and patient self - referrals inuenced by the mass - media [ 3638 ]  . conclusions from the point of view of a thorough and multidisciplinary evaluation of diagnostic imaging , ctca , as seen by patients , proved to have an excellent acceptance prole . higher levels of anxiety should be taken into account in women undergoing the examination , because they may partially affect image quality . 
chai ( & ) department of radiology , ruijin hospital , shanghai jiao tong university , school of medicine , ruijin er road 197 , shanghai 200025 , china e - mail : anchorzjrj@yahoo.cn small , prominently solid tumours showing enhancement patterns typical of spts may suggest a diagnosis of spt . keywords solid pseudopapillary tumour ( cid : 2 ) pancreas ( cid : 2 ) computed tomography ( cid : 2 ) male introduction solid pseudopapillary tumour ( spt ) of the pancreas was rst reported in 1933 [ 1 ] , described very well by frantz [ 2 ] , and then dened by the world health organization in 1996 as solid pseudopapillary tumours of the pancreas . 
spt is a relatively indolent tumour ; the prognosis for spt is excellent , with 5 - year survival rates reaching 95100 % [ 3 ]  . solid pseudopapillary tumour is an exceptionally rare neoplasm in males . 
the radiological characteristics of spts have yet to be fully claried in male patients ; thus , spts in men present a diagnostic problein this paper , the imaging features and clinical characteristics of spt in males are described and compared with those of females . 
the ndings obtained will help radiologists recognise distinct characteristics and provide more accurate diagnoses . materials and methods patient selection the institutional review board approved the present retrospective study , and the requirement for informed consent was waived . 
given the unavailability of computed tomography ( ct ) studies for seven patients , only the clinical data and ct studies of 102 patients were included in the present review . 
patients were divided into two groups on the basis of gender : 16 were males ( mean age , 38.5 years ; range 1567 years ) and 86 were females ( mean age 28.7 years ; range 1163 years )  . available records of clinical manifestations , as well as surgical and pathological reports , were reviewed for each patient . 
after each operation , patients were assessed clinically and then followed up by ultrasonography and multidetector ct ( mdct ) for a period of 2162 months ( mean , 97 months )  . ct technique all 102 patients underwent mdct of the abdomen ; different ct machines were used . 
twelve patients underwent 4 - slice , 40 underwent 16 - slice , and the rest underwent 64 - slice mdct scan ( lightspeed 4 , 16 , 64 ; ge medical systems , milwaukee , wi , usa )  . 
after fasting for at least 4 h , all patients were administered 500800 ml of water 30 min prior to the study , and an additional 250300 ml of water was provided immediately prior to the study to achieve adequate distension of the stomach and duodenum . nonionic iodinated contrast material ( omnipaque 300 mg i / ml , ge healthcare ) was administered to all of the patients by a power injector ( ulrich medical , germany ) at a rate of 2.53.5 ml / s through an 18 - gauge intravenous catheter placed in an antecubital vethe dose of contrast material delivered was 1.52 ml / kg body weight . 
sixty patients underwent dual - phase ct during the unenhanced , pancreatic , and hepatic venous phases , while 42 patients underwent triple - phase ct during the unenhanced , arterial , pancreatic , and hepatic venous phases . 
the items included in the imaging analysis were the following : location ( head , neck , body , or tail ) , shape ( round , oval , or lobulated ) , encapsulation ( present or absent ) , and calcication ( present or absent )  . 
the tumours were also evaluated and classied into one of four types according to the existence and proportion of solid and cystic components : purely solid , solid with minor cystic component ( \10 % of tumour with cystic component ) , mixed solid and cystic , and purely cystic . 
the internal ct attenuation characteristics of the tumours were compared with those of surrounding pancreatic tissues and described as follows : hypo - , iso - , or hyperattenuation on enhanced pancreatic phase images ; enhancement pattern ( peripheral or complete ) ; dilatation of the pancreatic duct ; or atrophy of the pancreatic parenchyma . 
images were also evaluated for bile duct dilatation and spread to regional vasculature , lymph nodes , adjacent organs , and distant abdominal sites . statistical analysis quantitative variables were expressed as mean standard deviation . 
no signicant differences were observed between the imaging features of spts in male and female patients with respect to the shape , encapsulation , attenuation , enhancement grade , enhancement pattern , and calcication of tumours . 
radical surgery was performed on 10 male patients , including distal pancreatectomy without splenic preservation on two , whipples operation on three , and concurrent resection of other organs on ve . 
portal vein involvement was seen in four male patients , who subsequently underwent venous resection and reconstruction . thirteen male cases were histopathologically diagnosed as benign spts , whereas the rest were found to be malignant . no signicant differences were found between male and female groups with respect to surgical procedures or malignant spts . 
1 pathologically conrmed benign solid pseudopapillary neoplasms ( spt ) in a 67 - year - old man . a unenhanced computed tomography ( ct ) scan showing a 4.3 cm oval neoplasm in the head of the pancreas with solid lesions . 
b , c pancreatic and hepatic parenchyma phases , respectively : dynamic contrastenhanced ct reveals an illdened mass in the head of the pancreas with initially weak enhancement and slightly progressive ll - in pattern . 
the tumour invaded the adjacent duodenud coronal reconstruction image showing a blurred interface between the tumour and the adjacent duodenumthe tumour had apparently inltrated the duodenum unenhanced and enhanced images . 
c computed axial tomography scan of a pancreas obtained during the hepatic parenchyma phase revealing an almost isodense tumour ( compared with surrounding pancreatic parenchyma )  . d coronal arterial phase ct image shows a hypodense lesion with clear borders fig . 
4 pathologically conrmed benign spt in a 63 - year - old man . a unenhanced ct scan showing a 3.8 cm oval neoplasm in the head of the pancreas with solid lesions with minor cystic components . calcication is seen in the periphery and centre of the lesion . b , c pancreatic and hepatic parenchyma phases , respectively ; dynamic contrast - enhanced ct reveals solid components with progressive ll - in enhanced pattern , however , minor cystic remained unenhanced . 
d coronal pancreatic phase ct image shows minor cystic components in the head of the pancreas characteristics of the pancreatic parenchyma , pancreatic showed tumours with hyperattenuation phase images ( n = 1 ) , isoattenuation ( n = 2 ) , and hypoattenuation ( n = 13 ) in the male patients . 
no statistical difference was observed between male and female patients in terms of calcication . no marked differences were observed in the prevalence of parenchymal atrophy or pancreatic ductal dilatation between the two groups . 
in the male group , parenchymal atrophy ( 2 / 16 ; 12.5 % ) and pancreatic duct dilatation ( 1 / 16 ; 6.2 % ) were present . was achieved in only three patients because of mixed components . discussion solid pseudopapillary tumours currently constitutes about 23 % of all primary pancreatic tumours [ 6 ]  . 
spt is a very rare pancreatic tumour that mainly aficts young females , and only 8.313.5 % of all cases discovered involve males [ 3 , 4 , 911 ]  . 
in 2009 , a total of 1 , 014 cases were reported in a large review of english publications ; only 137 of these were males [ 4 ]  . 
to the best of the authors knowledge , the present study is the largest single - centre study to have analysed the imaging features and clinical characteristics of spts in males . 
furthermore , no prior report comparing the imaging features of spts in males and females has yet been published . the mean age of male patients at the time of diagnosis was signicantly higher than that of female patients [ 46 ]  . our ndings generally agree with this statistic . 
in our retrospective study , the male patients tended to have solid masses ( eight cases had purely solid lesions ) ; this nding agrees with other reports [ 9 , 12 ]  . in the present study , calcication was observed in all parts the tumours , including their periphery , centre , and capsule . 
in our series , none of the patients had a denitive preoperative diagnosis and a correct diagnosis radiol med ( 2014 ) 119 : 8389 a classic ct image of an spt of a young woman showed a large , well - encapsulated mass with internal haemorrhage , solid and cystic components ( often ) brous pseudocapsule and peripheral contrast enhancement , which supports the diagnosis of spt [ 12 , 13 , 15 , 16 ]  . 
imaging ndings for other factors , including shape , encapsulation , attenuation , enhancement grade , and enhancement pattern of the spt , in men are very similar to those in women [ 11 , 12 , 15 , 17 ]  . 
awareness of the radiological and clinical features of spts in males is extremely important in establishing an accurate diagnosis before operation and further operative plans . surgery is the main therapeutic modality for lowgrade malignant spts . 
a good prognosis is expected after surgical resection of primary tumours ; even patients who exhibit distant metastasis have good prognoses as long as the metastatic lesions are resected completely [ 18 , 19 ]  . 
its incidence has been for years considered very low , but the considerable increase in patients receiving anticoagulation therapy for various diseases [ 59 ] ( atrial brillation , valvular heart disease , prophylaxis of deep vein thrombosis / pulmonary thrombo - embolism in addition to the development of increasingly effective anticoagulant , antiplatelet and antithrombotic agents ) has made this complication highly relevant [ 1012 ] , with the annual incidence of major bleeding in patients on oral anticoagulant therapy currently estimated to be 0.23 % [ 13 , 14 ]  . seh treatment consists in suspending anticoagulation therapy with correction of coagulation and restoration of blood volume through uid replacement and blood transfusions [ 3 , 4 ]  . in the case of haemorrhage refractory to transfusions and of arterial bleeding , transcatheter embolisation represents the most valuable therapeutic option because it is able to stop the bleeding with less intraprocedural risk as compared to open surgery [ 3 , 15 ]  . few studies have evaluated the efcacy of endovascular treatment of seh and most of them are limited by small patients series [ 3 , 1621 ]  . 
the objective of this study was to analyse our experience with transcatheter embolisation 122 radiol med ( 2014 ) 119 : 121127 in the treatment of seh to evaluate its effectiveness in terms of immediate and late clinical success , and to compare our data with those reported in the literature . table 1 risk factors risk factors comorbidities materials and methods thirty patients , who underwent endovascular treatment for seh between january 2005 and december 2010 were retrospectively included in the study . 
the study protocol consisted of axial scans at the site of suspected haemorrhage and scans before and after the administration of contrast medium , with post - processing ( table 2 )  . 
on baseline ct , bleeding site was identied as a hyperdense mass attributable to the presence of fresh blood with attenuation values ranging from 60 to 80 hu ; after contrast administration , active bleeding was displayed as a focal area of high density in the arterial phase . in 2 / 30 cases ( 6.6 % ) , the ct study was not performed because of haemodynamic instability ( systolic blood pressure \90 mmhg ) , and the patients underwent digital subtraction angiography ( dsa )  . 
dsa was performed in all patients in the angiography room ( integris v5000 philips , eindhoven , the netherlands ) , with approach through the common femoral artery ( 5f introducer terumo corp . , tokyo , japan ) and local anaesthesia ( 10 ml of lidocaine 2 % )  . 
in cases , where the ct study clearly indicated the bleeding vessel , the treatment involved selective catheterisation of the vessels by means of angiographic catheters ( cobra and vertebral , terumo corp . , tokyo , japan ) ; where the ct scan was not conclusive , angiography with a pigtail catheter ( terumo corp , tokyo , japan ) followed by selective catheterisation of the injured vessel was performed . in the presence of bleeding from small vessels superselective catheterisation was achieved using 0.025 a progreat microcatheter ( terumo corp . , tokyo , japan ) and its 0.021 micro - guidewire which allowed embolisation no . 
of patients anticoagulant therapy ( heparin ) deep vein thrombosis , coronary artery disease , cardiac valve replacement anticoagulant atrial brillation , cardiac valve therapy ( warfarin ) replacement anticoagulant therapy ( fondaparinux ) deep vein thrombosis , coronary artery disease , unstable angina with microcoils ( boston scientic , natick , ma , usa ) and microparticles ( contour 150250 , 250355 , 355500 lm , boston scientic , natick , ma , usa )  . 
when the active bleeding vessels could not be reached , we proceeded to embolise the main afferent vessel with coils of larger calibre ( balt extrusion , montmorency , france )  . the treatment algorithm was as follows : in the case of a bleeding vessel calibre of\1.5 mm ( calculated on the basis of the calibre of the 5f catheter used in the diagnostic phase ) we employed microparticle embolisation , whereas in the case of a calibre larger than 1.5 mm we always used coils , with preliminary use of microparticle injection before coil release in the presence of multiple haemorrhagic foci . 
preliminary contrast - enhanced ct scan shows the presence of a large haematoma in the anterolateral abdominal wall with active bleeding from the supercial circumex artery ( a ) , successfully treated by transcatheter embolisation with particles and coils ( b , c ) inammatory diseases ; especially , of the kidneys or adrenal glands [ 26 ]  . 
in recent years , the incidence of this condition has greatly increased because of anticoagulant therapy and related risks [ 10 ] : in our experience , we have observed a progressive increase in the incidence of seh , with doubling of the cases observed from four cases in 2005 to eight cases in 2010 . endovascular arterial embolisation is nowadays an established therapeutic option in the management of many haemorrhagic conditions [ 2832 ]  . 
there are numerous requests for embolisation of spontaneously bleeding vessels in patients on oral anticoagulants / heparin : we therefore considered it important to present our experience in terms of clinical and therapeutic management and results . not much is known about seh pathophysiology : supposed risk factors are atherosclerosis of small vessels , minor trauma , occult vascular disease of the kidneys and adrenal glands and immune - mediated microangiopathy induced by heparin [ 33 ]  . identifying the site of bleeding and the blood vessels responsible [ 20 ]  . 
in agreement with the literature [ 34 ] , in the case of negative ct , dsa has to be considered the rst option for a correct diagnostic and therapeutic interpretation . the rst - line treatment of seh is conservative : suspension , when possible , of anticoagulant therapy , identication and correction of coagulation disorders , uid infusion and blood transfusion [ 3 , 4 ]  . endovascular treatment with transcatheter embolisation has become the rst choice in seh management because surgery is limited by the difculty identifying and treating bleeding vessels the context of a massive haematoma and the risk that surgical manoeuvres may increase bleeding or re - start a tamponaded bleed [ 30 ]  . the indication for angiography in emergency / urgent settings is established in the presence of persistent anaemia despite adequate transfusion therapy that can be identied in [ 4 u of packed red blood cells in 24 h or [ 6 u of packed red blood cells in 48 h [ 28 ] and on the basis of the considerable experience gained with the treatment of posttraumatic haemorrhage [ 3739 ]  . the diagnosis of seh is essentially based on clinical and laboratory ndings ; ct , however , plays a key role in the diagnostic and therapeutic effectiveness of dsa in the treatment of seh has been documented by recent radiol med ( 2014 ) 119 : 121127 fig . 
dsa is indicated in the case of negative ct examination if required by the patients clinical condition ; in haemodynamically unstable patients , dsa should be performed as the rst - choice examination as the time factor is essential in this setting [ 40 ]  . treatment modalities depend on the operators condence with different materials : in our experience we favoured the use of coils and microparticles as we believe these materials to be more easily handled . 
other reports , however , have widely described the use of absorbable gel ( gelfoam , pharmacia and upjohn , kalamazoo , michigan ) with optimal results [ 40 ]  . 
in cases of difcult micromultiple bleeding vessels catheterisation , proximal embolisation with larger calibre coils is performed . in our experience , transcatheter embolisation achieved complete ( 100 % ) technical success . 
the therapeutic efcacy of dsa was also demonstrated by the statistically signicant reduction of transfusion requirements : is also consistent with the literature [ 27 ] and demonstrates that the technical success of the treatment ( cessation of active contrast leakage at nal angiography ) is associated with clinical success . this result concordance of all published studies on the effectiveness of arterial embolisation in the management of seh allows us to consider this approach the rst - choice treatment option . despite a technical success of 100 % , we observed a postoperative mortality of 10 % ( 3 / 30 patients )  . 
one of 126 radiol med ( 2014 ) 119 : 121127 these patients had arrived with substantially compromised cardiovascular condition ( haemodynamic shock ) and died at 24 h due to multiorgan failure , despite correct embolisation of the bleeding vessels . 
the other two patients died at 48 h due to massive bleeding from new , previously unaffected sites as the patients failed to reach the angiography suite in time for further treatment . 
 [ 33 ] , that spontaneous haemorrhage can be related to a widespread small - vessel disease in coagulated patients which , associated with microtrauma , can produce massive haemorrhagic manifestations . mortality at 6 and 12 months was 14.8 % ( 4 / 27 patients ) and 26 % ( 6 / 23 patients ) , respectively , and not related to haemorrhage or embolic treatment in all cases . this result is consistent with the literature data , documenting a high rate of mortality post - embolisation : pathi et al . 
 [ 20 ] reported that 3 / 4 patients with spontaneous haemorrhage successfully treated endovascularly , died within 30 days of treatment for reasons not related to embolisation or its complications ; rimola et al . [ 17 ] observed that , compared with a complete technical success of embolisation in 12 patients with seh , three patients died within 8 days and four patients within 30 days of treatment , again for reasons not related to in a recently published study , haemorrhage . 
 [ 40 ] , in a total of 25 patients with seh successfully treated with embolisation , observed one postoperative death due to massive bleeding from other sites not previously treated and a 48 % mortality rate at 12 months due to co - morbid conditions . 
these results indicate that even though the treatment is associated with a high technical success rate and a low postoperative mortality , seh occurs in patients with heavily compromised general condition and is , therefore , associated with a poor prognosis [ 40 ]  . in conclusion , on the basis of our experience and the literature data , endovascular treatment of seh represents the treatment of choice , as it can provide complete therapeutic success . 
all prostheses were imaged with both a clinical 1 tesla ( t ) ( signa horizon , general electrics ) and 1.5 t ( achieva , philips ) mri system , using spin echo ( se ) and gradient echo ( gre ) sequences : sagittal t1 se , coronal t2 fast se ( fse ) , axial t1 se , axial t2 fse , sagittal t2 gre , axial t2 * gre , coronal t1 gre , axial t1 gre . 
magnetic resonance imaging ( mri ) may be useful , but it is limited by magnetic susceptibility artefacts that hinder the evaluation of periprosthetic structures , local complications of arthroplasty such as mechanical loosening , prosthetic or periprosthetic fracture , dislocation , infections , heterotopic bone formation and foreign body granulomatosis . 
in computed tomography ( ct ) , beam - hardening artefacts degrade image quality causing errors in image reconstruction [ 3 ]  . mri has a role in the detection of complications owing to its ability to characterise soft tissues and potential bone 114 radiol med ( 2014 ) 119 : 113120 fig . 
1 a prosthesis in cobaltchromemolybdenum ( head and stem )  . a1 axial t2 gre ( gradient echo ) ; a2 coronal t2 fse ( fast spin echo ) ; a3 coronal t1 gre . 
in mri of periprosthetic tissues , some studies have focused on sequence optimisation and on the use of techniques and strategies to reduce ferromagnetic artefacts [ 8 , 11 , 1319 ]  . 
with identical imaging parameters for the different materials , cobalt chrome produced the largest artefacts both in se and gre sequences , followed by titanium and cobaltchrome molybdenum ; ceramic produced the smallest artefacts in all sequences . 
the arrows show the direction of the frequency encoding gradient [ 9 ] but also due to misregistration artefact ; in fact , the clover - leaf pattern became more pronounced with the cobaltchrome prosthesis . around the femoral stem , the signal void followed the longitudinal axis of the prosthesis , with a variable degree of artefact in the direction of the frequency - encoding gradient , as expected [ 10 , 11 ]  . 
substances can be classied as diamagnetic , paramagnetic , superparamagnetic , or ferromagnetic [ 10 ] and the resulting artefacts are different depending on these characteristics . the major mri artefacts associated with implants include geometric distortion of signal intensity in the frequency - encoding direction ( also known as misregistration ) , section thickness variation due to altered excitation proles in two - dimensional imaging , increased signal intensity loss due to diffusion effects from the paramagnetic implant and increased dephasing at ge imaging [ 11 , 12 ]  . misregistration in the frequency - encoding direction creates characteristic signal intensity variations , depending on the implants shape and orientation . 
there is no misregistration in the phase - encoding direction because phase encoding performed with variable amplitudes is immune to misregistration . variation in section thickness is a result of altered excitation proles in a distorted primary magnetic eld . 
in the presence of a mildly distorted primary magnetic eld , an excited section takes on a warped appearance , as it follows isomagnetic lines within the imaging volume . diffusion - related signal - intensity loss increases in the proximity of an implant due to randomly moving water molecules which , in the presence of very large magnetic eld gradients , undergo increased spin dephasing that is not completely recoverable with a refocusing pulse . 
therefore , near an orthopaedic implant , a long echo time , such as that in t2 - weighted se imaging , causes increased signal intensity loss from random processes such as diffusion . there is additional unfortunately , in ge imaging of implants there is no refocusing pulse so that intravoxel dephasing ( t2 * ) which creates marked signal voids . although decreasing the voxel size or shortening the echo time can improve gradient - echo imaging to some degree , the extent of intravoxel dephasing with this sequence usually precludes its use in the evaluation of tissue around the implants . in mri of periprosthetic tissues , some studies have focused on sequence optimisation and on the use of strategies to reduce ferromagnetic artefacts [ 8 , 11 , 1322 ]  . 
in addition , new materials have been introduced . in this study , a comparison of magnetic susceptibility artefacts depending on the physical composition of prostheses was performed , and the utility of some techniques to minimise artefacts produced by orthopaedic hip prostheses has been conrmed . with regard to the physical composition of prostheses , it is important to bear in mind that cobalt is ferromagnetic , radiol med ( 2014 ) 119 : 113120 whereas chrome and molybdenum are not , having molar susceptibilities of 167 9 10 - 6 and 72 9 10 - 6 cm3 / mol - 1 , respectively [ 23 ]  . 
this was followed in decreasing order by titanium which showed a slightly greater artefact size than cobaltchromemolybdenum , both in se and in gre sequences , even though titanium is not ferromagnetic ; by contrast , ceramic had the smallest artefact size . with regard to the inuence of the type of sequence adopted , cobaltchrome was again the material showing the greatest discrepancy in signal loss between gre and se sequences ; thus , cobaltchrome is the most affected by the intravoxel additional dephasing ( t2 * ) characteristic of gre sequences . imaging of periprosthetic structures should avoid gre acquisitions , because of the severity of associated magnetic susceptibility artefacts . titanium instead showed a greater susceptibility to increasing magnetic eld , with greater increases in artefact size at increasing magnetic eld strength . 
in general , all materials showed susceptibility to increases in eld strength ; thus , it could be useful to decrease the eld strength , as this would proportionally reduce both the geometric distortion artefact with the same frequency - encoding gradient strengths , and signal - to - noise resolution . the head of these prostheses has a spherical geometry . the signal void around the head has a clover leaf - like pattern , because of the variations in section thickness around a spherical source , related to the complex section thickness variations at this level , as reported by white et al . [ 1 , 5 ] , but also due to the misregistration artefact ; in fact , the clover leaf pattern becomes more pronounced with cobaltchrome prostheses . 
around the femoral stem , the signal void followed the longitudinal axis of the prostheses , with a variable degree of artefact in the direction of the frequency - encoding gradient , as expected [ 6 , 7 ]  . 
therefore , at clinical mri of a joint prosthesis , it is very important to choose the frequency encoding direction away from the intra - articular structures of interest , and to position the patient with the long axis of the prosthetic implant parallel to the primary magnetic eld [ 14 , 19 ] to disclose them as possible . this study presents some limitations . 
the aim of this retrospective study was to assess the impact of pet / ct on the planning of appropriate treatment for known recurrent disease in operated dtc patients . materials and methods the study concerned 44 consecutive dtc patients ( 36 papillary , 8 follicular ) , who underwent total thyroidectomy and thyroid remnant ablation with 131i and pet / ct . 
all patients had proven or strongly suspected recurrent disease judging from neck ultrasound ( us ) and ne - needle aspiration cytology , and detectable basal tg levels . results pet / ct ndings were positive in 25 / 44 patients ( 56.81 % ) and negative in 19 . 
a positive pet / ct result predicted resectable tumour recurrences in 19 / 25 patients , but also detected additional tumour sites that prompted changes to the treatment plan in 6 / 25 patients ( 24 % )  . 
local recurrences in the thyroid bed or cervical lymph nodes do occur , however , in 1435 % of patients with dtc [ 24 ]  . the clinical follow - up of treated dtc patients is usually based on measuring serum tg levels and various imaging modalities [ 5 , 6 ]  . 
measuring serum tg has its weaknesses ; however , tg autoantibodies cause false low serum tg measurements ; serum tg measurements obtained during thyroid hormone suppression of tsh may fail to identify patients with small residual tumours ; and patients may have aggressive poorly differentiated tumours , despite low tg levels [ 79 ]  . 
procedures for locating recurrences or metastases in the routine followup of dtc patients primarily involve neck ultrasound ( us ) and 131i whole - body scan ( wbs ) [ 5 , 10 ]  . 
neck us has radiol med ( 2014 ) 119 : 97102 proved highly sensitive and specic in assessing the thyroid bed and locoregional lymph nodes [ 1012 ] and is considered the rst - line technique for identifying suspected cervical recurrences during follow - up [ 11 ] ; us - guided neneedle aspiration cytology ( fnac ) can also be performed to increase its sensitivity [ 13 ]  . 
on the other hand , us cannot easily detect recurrences in the central compartment of the neck or in cases of metastatic lesions outside the neck , making it necessary to perform a total body assessment of the extent of the disease by other diagnostic means . wbs has been considered one of the most sensitive diagnostic tools in the follow - up of dtc [ 14 ] , but the ndings are negative in 1030 % of patients with detectable serum tg levels because the tumour cells may lose the ability to trap radioiodine , or the tumours volume may be too small to be detectable at scintigraphy [ 3 , 1517 ]  . 
several studies have shown that 18f - uorodeoxyglucose ( fdg ) - positron emission tomography / computed tomography ( pet / ct ) can identify locally recurrent disease or distant metastases in iodine - negative and tg - positive dtc patients [ 1829 ] , but the diagnostic accuracy of pet / ct vis - a` - vis tg levels is still a matter of debate . 
some authors found that pet / ct performed better in patients with high tg levels [ 20 , 23 , 25 ] , others found it helpful even in cases with low tg levels [ 20 , 22 ] , and some investigators reported nding no correlation between the results of pet / ct and serum tg levels [ 28 ]  . 
there is also no denite cut off at which an optimal accuracy of pet / ct can be assumed [ 24 , 27 ]  . although several studies have focused on the diagnostic accuracy of pet / ct in operated dtc patients with high serum tg levels , but negative wbs ndings , there is still a paucity of information available on other possible indications for pet / ct . 
the aim of this retrospective study was to ascertain whether pet / ct can contribute to the planning of therapy for known local recurrent disease in operated dtc patients , irrespective of their serum tg levels . materials and methods patients this study was conducted in full accordance with the guidelines issued by the local institutional review board . the need for direct informed consent was waived . 
the study was a retrospective analysis of the medical records of patients with dtc who underwent total thyroidectomy and had at least one cycle of rai therapy for thyroid remnant ablation between january 2008 and december 2009 . 
overall , 44 / 104 dtc patients ( 42.30 % ) met these inclusion criteria and gender female male histology papillary follicular stage i stage ii stage iii stage iv surgery 30 ( 68.18 % ) 14 ( 31.81 % ) 57 ( 2881 ) 36 ( 81.81 % ) 8 ( 18.18 % ) 10 ( 22.72 % ) 3 ( 6.81 % ) 19 ( 43.18 % ) 12 ( 27.27 % ) 40 ( 90.90 % ) 4 ( 9.10 % ) table 1 patient characteristics characteristics total ( n = 44 ) median age , range ( years ) initial disease staging ( according to 6th edition ajcc ) a total thyroidectomy total thyroidectomy plus lymphadenectomyb 7 ( 15.90 % ) 37 ( 84.09 % ) rai ablative activity ( gbq ) a american joint committee on cancer ( 2002 ) b unilateral cervical lymph node dissection formed our study sample , which included 14 men and 30 women ( median age 57 years )  . 
the characteristics of the study population are reported in table 1 . tg measurement blood samples for serum tg ( and anti - tg antibody ) evaluation were collected during thyroxine suppressive therapy . 
serum tg levels were considered pathological if measurable ( value [ 1 ng / ml )  . pet / ct technique all pet / ct studies were performed at our institution on a biography 16 scanner ( siemens medical solutions , il , us )  . 
together with the pet system , the ct scanner is used both to correct radiol med ( 2014 ) 119 : 97102 the attenuation of the pet data and to locate fdg uptake on the pet images . 
patients fasted for at least 6 h prior to imaging and were asked to drink mineral water freely within the fdg uptake time to stimulate diuresis and obtain an adequate distension of the intestinal walls . 
the gaussian lter was applied to the reconstructed image in the axial and transaxial directions . the data were reconstructed over a 128 9 128 matrix with a pixel size of 5.25 mm and a slice thickness of 5 mthe processed images were displayed in the coronal , transverse , and sagittal planes . the pet / ct images were interpreted by two experienced physicians . 
a standardized uptake value ( suv ) was measured for all positive lesions and presented as the maximal suv ( suvmax )  . wbs technique diagnostic wbs was performed after thyroxine had been discontinued for at least 4 weeks and a low - iodine diet followed for at least 6 weeks . 
in brief , wbs was performed 72 h after the oral administration of 185 mbq of radioiodine , using a dual - head gamma camera ( ecam ; siemens medical solution , il , us ) equipped with a high - energy collimator . anterior and posterior wbss were obtained , plus spot images of the neck and chest ( acquisition with matrix 256 9 256 , zoom 1 )  . 
finally , progressive disease was considered in the presence of increased serum tg levels and / or evidence of disease progression on imaging . a patient - based analysis was performed . 
cohens k statistics was used to measure agreement between procedures . results in all 44 patients , the use of pet / ct was based on the nding or strong suspicion of recurrent disease at a single neck site after us and fnac . 
on a patient - based analysis , pet / ct conrmed recurrent disease at a single neck site in 25 / 44 patients ( 56.81 % ) who underwent this test for the purposes of planning curative re - surgery ; the method also detected additional tumour sites that prompted changes to the treatment plan in 6 / 25 patients ( 24 % )  . 
among these 19 patients with negative pet / ct ndings , 11 were only followed - up , 5 had further rai therapy , and 3 underwent repeat surgery with radical intent . at the time of our analysis , overall 19 of the 44 patients ( 43.18 % ) were disease free , 9 ( 20.45 % ) had a stable disease , 11 ( 25 % ) had a progression of disease and 5 ( 11.36 % ) were lost . 
neck us is considered the most sensitive diagnostic tool for local recurrences in dtc patients [ 32 ] , but the chances of curative surgery in cases with localised disease justify the search for residual cancer at sites inaccessible to us - guided fnac , or unexpected metastases outside the neck . to date , there have been few studies involving pet or pet / ct in the work - up of dtc patients with recurrences detected by various other imaging techniques . 
although some authors observed no correlation between fdg uptake and thyroid hormone levels [ 22 , 28 , 34 ] , other studies showed that the chances of positive ndings on pet / ct increase with high or rising tg levels [ 20 , 21 , 24 , 35 ]  . 
another explanation for the number of patients found negative at pet / ct in our series lies in the fact that patients were not selected in the light of an increase in their tg levels between two successive measurements . 
our results also indicate , however , that pet / ct ndings could be positive in patients with serum tg levels below 5 ng / ml as well , which is consistent with the fact that there is still no precise tg cut off for selecting dtc patients that warrant pet / ct examination . we found that pet / ct had a clinical impact even when its outcome was negative : the absence of fdg uptake correlated with a less aggressive disease and prompted a follow - up more conservative treatment approach ( i.e. , alone in most cases )  . there was a lack of agreement between pet / ct and wbs ndings in this study ( k = 0.1 ) , conrming that the sensitivity of these two techniques in thyroid cancer depends on the cells metabolic activity [ 24 , 36 ] , and suggesting that one of the two methods cannot be used in lieu of the other . it would be most interesting to know whether using pet / ct in patients whose us - guided fnac demonstrate or raise a strong suspicion of recurrent disease at a single neck site would inuence their clinical course and ultimate survival , but this question remains to be answered . limitations of the present study are the short follow - up period ( since dtc is known to grow slowly ) , and the small number of subjects evaluated . 
for this reason , our ndings should be validated on a larger series of patients . to conclude , pet / ct can add to the information provided by neck us and fnac when it comes to diagnosing recurrent or metastatic dtc , thus helping to improve the clinical management of patients after thyroidectomy and rai therapy . 
stavropoulos soa anagnostopoulou received : 19 april 2012 / accepted : 28 may 2012 / published online : 3 december 2013 ( cid : 2 ) italian society of medical radiology 2013 abstract purpose this paper presents a technique of ultrasoundguided localisation and block of the musculocutaneous nerve through the anterior wall of the axilla . materials and methods twenty patients ( 7 males and 13 females ; mean age , 35 years ) had axillary nerve block for upper extremity trauma . 
with the arm adducted , ultrasound probe was positioned on the anterior axillary wall ; the axillary artery , coracobrachialis and pectoralis major muscles and lateral cord of brachial plexus were visualised in cross section . 
with continuous imaging of the axillary artery in cross section , the ultrasound probe was slowly moved towards the biceps muscle until the musculocutaneous nerve appeared crossing the coracobrachialocalisation lis muscle . 
the quality of sensory and motor nerve block , as well as of ultrasound imaging were evaluated . results ultrasound - guided block of the musculocutaneous nerve was excellent and complete in 18 of the 20 z . 
no patient experienced pain or tourniquet discomfort during surgery , or any other nerve block - related complication . conclusion the anterior axillary ultrasound view provides for complete nerve block and imaging of the entire course of the musculocutaneous nerve and its relations with adjacent structures with excellent quality . keywords musculocutaneous nerve ( cid : 2 ) ultrasound imaging ( cid : 2 ) axillary block ( cid : 2 ) anterior axilla wall introduction the axillary block is the most distal block performed on the brachial plexus . 
however , because the musculocutaneous nerve leaves the brachial plexus sheath proximal to the site of injection , axillary block often results in inconsistent coverage for tourniquet pain , as well as anaesthesia of the volar aspect of the skin below the elbow that extends to the thenar eminence [ 17 ]  . the musculocutaneous nerve arises from the lateral cord of the brachial plexus , opposite the lower border of the pectoralis major muscle , its bres being derived from c5 , c6 and c7 nerve roots . 
the median , ulnar and radial nerves all travel with the axillary artery within the axillary sheath [ 14 ] , whereas the musculocutaneous nerve has already left this sheath , crossing the coracobrachialis muscle [ 5 ]  . therefore , the musculocutaneous nerve must be blocked separately during an axillary nerve block . 
however , 136 radiol med ( 2014 ) 119 : 135141 variations and irregularities of the musculocutaneous nerve , such as double , short or absent nerve , adherence or fusion with the median nerve and variable course through or beneath the coracobrachialis and biceps brachii muscle may complicate localisation and blockade of the musculocutaneous nerve in the axilla [ 1 , 2 , 6 , 7 ]  . ultrasound - guided nerve block has greatly facilitated nerve localisation and blockade in regional anaesthesia [ 8 10 ] , especially for nerves such as the musculocutaneous nerve that do not have predictable relationship to easily identiable vascular structures [ 1014 ]  . 
using a standard axillary ultrasound - guided approach to the musculocutaneous nerve , the patient is placed in the supine position with the arm abducted and externally rotated ; the ultrasound probe is positioned at the base of the axilla , at the junction of the pectoralis major and biceps muscle [ 1012 ]  . the musculocutaneous nerve is found at the juncture between the biceps and coracobrachiales muscle [ 1012 ]  . however , anatomical and ultrasound studies have described variations in the exact anatomical location of the musculocutaneous nerve in 022 % of cases [ 7 , 1526 ] ; in these cases , the nerve was outside the coracobrachialis muscle , near the axillary artery , or more frequently near or fused with the median nerve in a common trunk [ 17 , 18 ]  . 
in addition , using a standard axillary ultrasound - guided approach to the musculocutaneous nerve the position of the nerve outside the coracobrachialis muscle and its course from the lateral cord of the brachial plexus to the biceps brachii muscle could not be detailed [ 26 ]  . 
moreover , the injection needle of the local anaesthetic is brought into the ultrasound beam in the axial section , and could be easily missed as it appears as a small dot in the ultrasound images . 
to address these problems , we performed this study to present a technique for axillary nerve block of the musculocutaneous nerve using an anterior axillary ultrasoundguided approach . materials and methods twenty patients had an axillary nerve block for surgical treatment of upper extremity trauma including distal radius fractures ( 14 patients ) and scaphoid fractures ( 6 patients ) from february 2009 to april 2010 . 
all axillary nerve blocks were performed using the same technique as described below , by the same anaesthesiologist ( t.s. ) with an 8 - year experience in ultrasound - guided regional anaesthesia . 
motor and sensory block was graded as normal motion / sensation or incomplete block , decreased block and complete block . inability to move the ipsilateral biceps brachii muscle against gravity was considered the motor block endpoint . complete absence of pinprick sensation to the lateral aspect of the forearm ( lateral antebrachial cutaneous nerve ) was considered the sensory block endpoint . 
the quality of ultrasound imaging of the entire course of the musculocutaneous nerve in the axilla , the axillary artery and vein , and the coracobrachialis , pectoralis major and biceps brachii muscles was graded as excellent , difcult or unfeasible . 
in both these patients , the nerve block was repeated successfully with the same technique . the quality of ultrasound imaging of the musculocutaneous nerve , the axillary artery and vein , the coracobrachialis , biceps brachii and pectoralis major muscles was excellent in all patients , without any difference between the two ultrasound scanners and probes with different resolution used in this series . 
the musculocutaneous nerve was clearly seen along its entire course from the lateral cord of the brachial plexus , crossing coracobrachialis muscle , to the junction with the biceps brachii muscle . 
fusion of the musculocutaneous with the median nerve was observed in one patient , while in another patient the nerve was not found and was probably also fused with the median nerve . 
in addition , the next day no residual patient anaesthesia , or any other nerve block related complication . dysaesthesia , paraesthesia , reported discussion traditionally , anatomical landmarks have been used to identify needle insertion sites for nerve blockade [ 5 ]  . however , the degree to which these landmarks are appreciated depends on multiple variables , such as body habitus , distortion of anatomy from prior surgical operations , ability to position the patient and vascular disease . 
therefore , alternative techniques for nerve localisation , such as ultrasound - guided techniques for regional anaesthesia in both the upper and lower extremities have evolved [ 12 , 14 , 27 , 28 ]  . 
inset pulse wave doppler used for the identication of the axillary vessels ( v axillary vein , a axillary artery , m - mcm musculocutaneous - median nerve , p posterior cord , l lateral cord ) that it affords the anaesthesiologist the real - time ability to visualise the target nerve of interest , direct the needle and witness local anaesthetic spread [ 27 ]  . 
the musculocutaneous nerve does not have predictable relationship to adjacent structures in the axilla [ 1014 ] ; localisation of the nerve is atypical in up to 22 % of axillary blocks [ 7 ]  . 
our results showed that this technique provides for excellent localisation of the musculoskeletal nerve and its entire course from the lateral cord of the brachial plexus to the biceps muscle , and complete motor and sensory nerve block . at the axilla , the lateral , medial and posterior cords of the brachial plexus are divided into their terminal nerve branches . 
the lateral cord divides into the musculocutaneous nerve and radiol med ( 2014 ) 119 : 135141 the lateral portion of the median nerve , the medial cord divides into the ulnar nerve and the medial portion of the median nerve , and the posterior cord divides into the radial nerve and axillary nerve . 
in most arms , the nerve pierces the coracobrachialis muscle and then passes between the biceps brachii and brachialis muscle , supplying each of these muscles [ 7 , 29 , 30 ]  . however , in 830 % of arms , the musculocutaneous nerve may accompany or fuse with the median nerve , as occurred in 2 of the 20 patients in this series , without entering the coracobrachialis muscle [ 13 ]  . 
it may adhere for some distance to the median nerve and then pass outwards , beneath the biceps brachii , instead of through the coracobrachialis muscle , or it may pass under the coracobrachialis or through the biceps brachii muscle [ 1 , 2 , 6 ]  . 
these variations may complicate localisation and blockade of the musculocutaneous nerve in the axilla [ 7 ]  . currently , multiple techniques are used to localise the musculocutaneous nerve for local anaesthetic nerve block . these techniques include direct injection into the coracobrachialis muscle , injection along the humerus , paraesthesia seeking , generation of a biceps contraction by use of nerve stimulation , ultrasound , perivascular and transarterial techniques [ 5 , 31 ]  . 
with the paraesthesia - seeking and nerve - stimulating approaches , all four nerves ( median , ulnar , radial and musculocutaneous ) can be individually identied and anaesthetised in the axilla ; both these methods seem to be equally successful . however , in procedures using the nerve - stimulation technique , actual stimulation of the musculocutaneous nerve leads to a more successful outcome than a simple injection into the coracobrachialis muscle . 
moreover , the aforementioned techniques rely on anatomical assumptions of normal neural anatomy and on palpation of internal anatomy , such as the coracobrachialis muscle , biceps muscle and axillary artery to guide needle placement [ 12 , 32 ]  . 
the degree to which these landmarks are appreciated depends on multiple variables , such as body habitus , prior surgical distortion of anatomy , ability to position the patient and vascular disease [ 12 ]  . ultrasound guidance is an attractive option for neural structures , such as the musculocutaneous nerve , that do not have predictable relationships to easily identiable vascular structures [ 12 ]  . 
needle insertion can be done with the in - plane approach ( the needle itself is imaged in its long - axis ; complete needle visualisation ) , or the out - of - plane approach ( the needle is imaged in its short - axis ; limited view , the operator cannot be assured that the needle tip is being imaged in contrast to part of the shaft )  . 
the main downside to the in - plane technique is that the ultrasound beam is very thin and it can be frustrating and difcult to continuously maintain needle imaging [ 32 ]  . the frequency of the ultrasound system is directly related to the resolution of the system [ 7 ]  . 
however , higher frequency systems do not penetrate deeply into the body , thus preventing effective imaging of deep structures because of ultrasound attenuation ; higher frequency ultrasound attenuates at more supercial depths than lower frequency ultrasound [ 7 ]  . since the initial report of a high - resolution ultrasound technique to visualise and block the musculocutaneous nerve [ 12 ] , no large clinical study on musculocutaneous nerve localisation in the axillary area has been available . 
in that study [ 12 ] , the authors identied the musculocutaneous nerve by placing the transducer over the base of the axillary pyramid where the musculocutaneous nerve seems to exit the axillary cavity by piercing the coracobrachialis muscle . an ultrasound study in healthy volunteers described median , ulnar and radial nerve localisations around the axillary artery , but their limited ultrasound resolution rate precluded any musculocutaneous nerve localisation [ 26 ]  . other authors described the ultrasound appearance of the musculocutaneous nerve in the axilla to suggest potential areas to target neural block [ 13 ]  . 
however , placing the ultrasound probe at the base of the axilla , as per the standard ultrasoundguidance techniques , often makes it difcult to accurately visualise the origin of the musculocutaneous nerve from 140 radiol med ( 2014 ) 119 : 135141 the lateral cord of the brachial plexus , and its relationship with the coracobrachialis muscle [ 12 , 26 , 38 ]  . 
by sliding the ultrasound probe at the base of the axilla and trying to follow the musculocutaneous nerve to the apex , the axillary cavity further narrows ; hence , it is almost unfeasible to depict the entire anatomical course of the musculocutaneous nerve from the lateral cord of the brachial plexus to the biceps brachii muscle without having to angulate the ultrasound probe considerably . 
placing the probe at the base of the axilla , also makes it difcult to visualise the lateral cord of the plexus , the exit of the musculocutaneous nerve from the lateral cord , the coracobrachialis muscle and the axillary artery because these structures are located deep in the axillary fossa [ 7 , 12 , 29 , 30 , 38 ]  . 
last , during injection of the local anaesthetic , if the needle is brought into the ultrasound beam in the axial section , the needle shaft appears as a small dot which can be easily missed . 
in the present study and our practice , positioning the ultrasound probe on the anterior wall of the axilla provides for crosssection visualisation of the lateral cord of the brachial plexus , the musculocutaneous nerve , the axillary artery and vein and the coracobrachialis muscle . 
although the probe is slowly brought towards the biceps brachii muscle , the entire course of the musculocutaneous nerve from the brachial plexus cords , crossing the coracobrachialis muscle to its terminal branches , its relationship with the anatomical structures of the axilla , and any anatomical variations can be clearly recognised . 
finally , the injection needle is advanced in the longitudinal plane of the ultrasound beam ( in - plane technique ) ; therefore , the operator can see the entire needle and make adjustments as needed . we see three limitations in this study : rst , this study did not include a control group of patients , and has not been compared with anatomical dissection . 
second , the resolution of our ultrasound device may have not allowed us to explore the thin connections between musculocutaneous and median nerves , which could be observed in up to 46 % of arms in cadavers [ 23 , 25 ]  . 
furthermore , by increasing the resolution of the system ( higher mhz ) , the depth of penetration would have been reduced [ 23 , 25 ]  . third , we used two different ultrasound scanners and probes with different resolution . 
however , with the probes used in the present series , no difference was observed regarding imaging quality during the procedure ; both ultrasound probes are new generation ones and of the same frequency bandwidth . in conclusion , the technique presented uses an anterior ultrasound axillary view of the musculocutaneous nerve . the advantages of this approach are ( 1 ) the musculocutaneous nerve can be followed throughout its course in the axilla , from the cords to the terminal branches , thus identifying possible variations and abnormalities , ( 2 ) it allows cross - section visualisation of the lateral cord of the plexus , the musculocutaneous nerve , the axillary artery and vein , and the coracobrachialis muscle and ( 3 ) the injection needle is advanced in the longitudinal plane of the ultrasound beam , so the operator can see the entire needle and make adjustments as needed . 
in this study , our aim was to investigate the relationship between work stress and metabolic syndrome in a population of radiologists . materials and methods radiologists and radiotherapists taking part in scientic conferences were invited to compile a questionnaire to evaluate work stress and the main parameters for diagnosing metabolic syndrome ( obesity , hypertension , elevated cholesterol level , elevated triglycerides , and hyperglycemia )  . results most of the doctors taking part in the survey ( n = 383 , 58.6 % ) were found to have at least one pathological component ; 47 subjects ( 7.1 % ) had metabolic syndrome . 
all the variables indicating work stress , whether derived from karaseks demand / control model or from the effort / reward model devised by siegrist , were signicant predictors of metabolic syndrome components . 
in fact , it is thought that the latter together with life styles accounts for approximately one - third of the effect of stress on the risk of cardiovascular disease [ 4 ]  . radiologists can be exposed to considerable environmental pressure [ 5 , 6 ] that may lead to a prolonged state of occupational stress [ 7 ] and a reduction in job satisfaction [ 8 ]  . 
our cross - sectional study is a rst attempt to address this issue . materials and methods since italian radiologists and radiotherapists are increasingly being accused of alleged malpractice [ 1018 ] , we decided to use a questionnaire to investigate the possible health effects resulting from such accusations [ 19 ]  . 
a total of 654 completed responses were returned . the questionnaire examined the perception of occupational stress through the simultaneous use of the two commonest stress models : the karasek demandcontrol support ( dcs ) model [ 20 ] and the effortreward imbalance ( eri ) model devised by siegrist [ 21 ]  . according to the dcs model , a work environment where there is a high level of psychological demand ( frenetic pace , contradictory , and excessive demands ) and a low level of job control , exposes the worker to job stress . 
high strain jobs characterized by elevated demand and low control , lead to a greater risk of negative mental and physical health effects as compared to the other three groups . 
in active jobs , characterized by high demand and high decisional power , enhanced autonomy enables workers to avoid becoming the victims of stress . similarly , in the other two categories , workers with low strain and high discretionary power , or passive workers ( with low demand and low control ) are not exposed to occupational strathe four groups are established by dividing the scores of the demand and control scales at the median . 
the dcs model was the rst to demonstrate the association between stress and pathologies ; much of the literature is based on this model [ 23 27 ]  . according to the eri model , stress originates from an imbalance between the effort required to perform work and the material or immaterial rewards obtained thereby . 
in this model , imbalance is calculated once more as a weighted ratio between effort and reward and is a continuous variable that can be divided at the median to obtain categories of workers who are more or less exposed to stress . 
although the karasek model provides a perfect picture of the repetitive tasks of the rst industrial revolution when work was conditioned by machinery , the siegrist model seems better suited to interpret the occupational conditions of the second industrial revolution where work has taken on a more immaterial aspect and is more invasive . 
in fact , in recent years , there has been a considerable increase in the number of studies based on this model . their ndings have shown that the combination of high effort and low reward constitutes a risk factor in numerous illnesses [ 2933 ]  . 
however , the two models offer a complementary rather than alternative solution , so a combination of the two questionnaires can provide better information on the relationship between work and health [ 34 , 35 ]  . both questionnaires are available in different versions . despite their different composition , these provide homogeneous results [ 3638 ]  . 
the questionnaire included a third , overcommitment , scale ( alpha = 0.91 ) made up of six questions ( e.g. , people who know me well say that i am too committed to my work )  . 
the responses were scored on a 5 - point scale where a value of 1 corresponded to : strongly disagree , does not apply ; a value of 2 to : agree but i am not distressed and a value of 5 to : strongly agree , i am greatly distressed . metabolic factors were investigated using the following questions : level ? ( threshold : 40 mg / dl males , 50 mg / dl females )  . 
what is is your hdl - cholesterol what 144 radiol med ( 2014 ) 119 : 142148 table 1 characteristics of the study population your blood pressure ? ( threshold : 130 / 85 mmhg )  . 
what is your fasting glycemia level ? ( threshold : 100 mg / dl )  . what is your waist circumference ? ( threshold : 102 cm males , \88 females )  . 
table 1 illustrates the characteristics of the study population . a total of 274 subjects ( 41.9 % ) reported abnormal hdl cholesterol levels , while 74 subjects ( 11.3 % ) reported an increase in triglycerides . 
forty - six subjects ( 7.1 % ) who manifested three or more simultaneous pathological abnormalities were diagnosed as having metabolic syndrome ( table 2 )  . by analyzing the variables correlated to work stress ( table 3 ) , we were able to build up a prole of this category of workers . 
on average , radiologists claimed that they had a high workload ( the mean score for the demand variable was close to maximum values for the scale ) , but a good level of control over their work . 
on the other hand , those who worked at an excessively fast pace and were unable to adequately control their work ow were exposed to risk . the mean score for the job strain variable was higher than one , indicating excessive psychological strain compared to that could be exercised . 
the tendency towards overcommitment was quite widespread and this aggravated the situation . linear regression analysis indicated that all the occupational stress variables were signicantly associated with a prevalence of metabolic syndrome components . 
the increase in demand , effort , overcommitment , job strain or effort / reward imbalance , and the reduction in support or reward were associated with more frequent signs of pathology . 
data adjustment for age , gender , and lifestyles did not alter these associations ( table 4 )  . logistic regression provided proof that radiologists with higher stress levels , measured by both models , were at greater risk of manifesting a complete metabolic syndrome . even after correction for confounding factors ( sex , age , and the relationship was still statistically highly lifestyles ) , signicant . 
the mean psychological effort needed to perform tasks was not held to be excessively elevated ( we must consider that we are dealing with highly qualied and professional physicians ) , but the immaterial rewards they receive from their work were not always satisfactory . 
this meant that a signicant number of radiologists worked in a state of effortreward imbalance , as they obtained less than they our study demonstrated that radiologists with higher stress levels run a fourfold to sixfold greater risk of being affected by metabolic syndrome than radiologists with lower levels of stress . 
to our knowledge , this was the rst study to take this category of workers into consideration to examine the association between stress and metabolic diseases . our study ndings are in agreement with the literature . stressful life events have been found to induce the onset of metabolic syndrome in the general population [ 40 ]  . 
in workers who develop a post - traumatic disorder after 146 radiol med ( 2014 ) 119 : 142148 exposure to traumatic events , there was a signicant increase in metabolic syndrome [ 41 ]  . 
other occupational risk factors , such as loss of sleep due to shift work [ 42 , 43 ] and depression , which is also often associated with stress , are linked to metabolic syndrome [ 44 ]  . 
workers affected by burnout show a signicant increase in the prevalence of metabolic syndrome components , such as hypercholesterolemia , high blood pressure , and obesity [ 47 ]  . the relationship between stress and obesity is rather complex and there are conicting ndings in the literature . a study conducted in finland with the two stress models we used in this study showed that the lowest control and demand values and the highest levels of job strain and effort / reward imbalance are associated with an increase in body mass index ( bmi ) in males [ 48 ]  . 
these discrepancies can be explained by the fact that researchers used a cross - sectional study design or , in longitudinal studies , failed to take into consideration the physical condition of the workers at the beginning of the experiment . 
findings from the whitehall ii study indicated that thin subjects who were exposed to high job strain and low control tend to get thinner , while those who are initially overweight tend to put on more weight when they are under stress [ 50 ]  . 
a recent analysis summarizing the ndings of 13 studies conducted in europe , conrmed that there is a u - shaped association between stress and body weight , whereby thin subjects lose weight , while obese individuals get fatter [ 51 ]  . clearly the relationship between stress and metabolic syndrome is so complex that it would be difcult for a single cross - sectional study to draw any denite conclusions . 
our single study is not sufcient to exclude the possibility of inverse causality , i.e. , that it is the onset of metabolic syndrome or its components that are responsible for an increase in work stress , even though knowledge of the literature would seem to exclude such a hypothesis . the second important in our study regards the voluntary participation of the radiologists . because the cases were not randomized , the prevalence of the disorders reported cannot be extended to all italian radiologists . 
a study conducted in the marche region of italy found a metabolic syndrome prevalence of 11.5 % in the 3642 age group and of 22.5 % in subjects between the age of 43 and 60 years [ 54 ]  . 
this demonstrated that sample group at least , the health level of italian doctors was higher than that of the general populationa fact that had already been ascertained in previous epidemiological studies [ 55 ]  . 
it also indicated that the subjects taking part in our study were not prompted to do so because they were suffering from one of the conditions under investigation . this was important since we know that being affected by any morbid condition induces individuals to be more precise when recalling all the possible causal factors for that illness . 
our statistics were , however , also adjusted for gender . since there was no collection of analytical or objective data in our study , the third limitation concerns the fact that it was not possible to verify the accuracy of the responses given . 
in epidemiological studies where self - assessment is made of both the risk factors and their consequences , what is known as common method variance may occur , i.e. , individuals with negative affectivity tend to give a negative answer to all the questions , thus , inuencing the strength of the association between risk factors and outcome . 
however , it has been shown that this is not an automatic process and that when it occurs it has little effect [ 56 ] and tends to diminish the strength of the association rather than boost it [ 57 ]  . 
in our case , the fact that the radiol med ( 2014 ) 119 : 142148 individuals questioned were all doctors makes us condent with regard to the accuracy of the clinical parameters reported . our study demonstrated that radiologists are an occupational category that is exposed to considerable number of critical situations and that they often suffer from stress even though they carry out their work in a highly protected and controlled environment . 
the prevalence of blood pressure , obesity , dyslipidemia , and diabetes was higher in subjects with a more elevated level of job strathis nding should encourage researchers to carry out more prospective studies . 
depending on their position relative to the heart , they are classied into ( 1 ) an anterior group situated between radiol med ( 2014 ) 119 : 2026 fig . 
3 axial computed tomography ( ct ) scan showing the position of the extrapleural lymph nodes ( white ) in adipose paraspinal tissue near the costovertebral joints they receive afferent vessels from the parietal pleura , diaphragm , liver and abdominal wall [ 6 , 7 ]  . 
extrapleural lymph nodes are located in a triangular area at the costovertebral joint , near the head of the rib , in the adipose paraspinal tissue [ 7 ]  . 
the retrocrural space communicates with the posterior mediastinum above and with the retroperitoneal space below , without clear anatomical limitations , representing a potential conduit for the spread of pathological processes between thorax and abdomen [ 8 ]  . 
lymphatics of the posterior mediastinum , the posterior portion of the diaphragm , the spine and the posterior parietal pleura drain into the extrapleural lymph nodes ; those of the anterior parietal pleura drain into the internal mammary chain for the upper and middle part of the thorax and into the cardiophrenic lymph nodes for the lower part [ 7 ]  . 
several neoplastic and nonneoplastic diseases can spread easily from thoracic organs to the abdomen along these lymphatic chains [ 912 ]  . thoracic lymph nodes can be evaluated with various methods , such as computed tomography ( ct ) , magnetic resonance imaging ( mri ) and uorodeoxyglucose - positron emission tomography ( fdg - pet )  . 
lymph node abnormalities are depicted by ct and mri as an increase in nodal size and / or number , and patterns of thoracic lymph node involvement can provide important clues in the diagnosis of many pulmonary and extrapulmonary diseases . 
2 axial computed tomography ( ct ) scan showing the pericardium ( arrow ) as a relatively hyperdense line that separates the epicardial adipose tissue ( ? ) from the cardiophrenic space ( asterisk ) the pericardium posteriorly and the xiphoid process of the sternum and costochondral articulation of the seventh rib anteriorly ; ( 2 ) a middle or lateral group located between the pericardium medially and lung hilum laterally in the region where the phrenic nerves run ; and ( 3 ) a posterior group located near the oesophagus , posteromedial to the inferior vena cava , which goes down to the aortic hiatus [ 5 ]  . 
generally , lymph nodes with a short - axis diameter [ 1 cm are considered pathological [ 13 , 14 ]  . there are little data on the normal size of cardiophrenic and extrapleural lymph nodes , for which no denite cutoff value has yet been established . materials and methods patient population we included in our study 750 consecutive patients ( 430 men , 320 women ; mean age , 59 6 years ) referred to our institution between january and september 2009 for multidetector - row ct ( mdct ) of the chest for different clinical reasons . 
according to their primary disease , these patients were divided into four groups : 340 nonneoplastic patients , 270 patients with extrathoracic neoplasms , 120 patients with intrathoracic neoplasms and 20 patients with pleural metastasis . 
we also included 91 patients ( 61 men , 30 women ; mean age , 67 9 years ) with histologically proven malignant pleural mesothelioma ( mpm ) who had undergone mdct of the chest at our institute between january 2001 and september 2009 . scan protocol and image acquisition ct studies were performed on a four - channel mdct ( somatom volume zoom , siemens medical solutions , forchheim , germany ) using standard parameters mdct of the chest : 120 kv ; effective 400 ma ; gantry rotation time , 500 ms ; table speed , 6 mm / rotation . 
images were reconstructed with a slice width of 1 mm and reconstruction interval of 1 mm . multiplanar reconstructions were obtained in selected cases , especially in mpm patients , for staging purposes . a dose of 90100 ml of nonionic contrast medium ( iomeron 300 mg iodine / ml , bracco , milan , italy ) was injected via an antecubital vein at 23 ml / s . 
a standard scan delay of 25 s was selected in all patients ; in patients with mpm , we performed another acquisition with a 45to 50 - s scan delay to obtain better enhancement of pleural surfaces and disease . imaging evaluation and statistical analysis axial ct images were independently evaluated by two chest radiologists who assessed the presence , number ( single or multiple ) and short - axis diameter ( b5 mm ; 610 mm ; [ 10 mm ) of the extrapleural and cardiophrenic nodes . 
images were analysed using a mediastinal window ( width , 400 hu ; level , 40 hu )  . for statistical purposes , the prevalence of extrapleural and cardiophrenic nodes of each size category ( b5 mm ; 610 mm ; [ 10 mm ) was calculated in patients with mpm and in each patient group ( nonneoplastic , extrathoracic neoplasm , intrathoracic neoplasm , and pleural metastasis )  . comparison of the prevalence between patients with mpm and the remaining four groups was done using the chi square test , with signicance set at p \ 0.05. 
interobserver agreement was analysed using kappa statistics . results the 750 patients in the study comprised 340 nonneoplastic , 270 with extrathoracic neoplasm , 120 with intrathoracic neoplasm and 20 with pleural metastasis . cardiophrenic lymph nodes results regarding cardiophrenic lymph nodes are summarised in table 1 . 
cardiophrenic nodes , independent of their size , were found in 66 ( 8.8 % ) of 750 patients assessed ; in particular , they were b5 mm in 47 patients , 610 mm in 15 and [ 10 mm in the remaining four . considering each category of patients , cardiophrenic nodes were present in 26 of 340 ( 7.6 % ) nonneoplastic patients ( b5 mm in 19 , 610 mm in ve , [ 10 mm in two ) , in 22 of 270 ( 8.1 % ) patients with extrathoracic neoplasm ( b5 mm in 15 , 610 mm in six , [ 10 mm in one ) , in 16 of 120 ( 13.3 % ) patients with intrathoracic neoplasm ( b5 mm in 12 , 610 mm in three , [ 10 mm in one ) and in 13 of 20 ( 65 % ) patients with pleural metastasis ( b5 mm in two , 610 mm in six , [ 10 mm in ve )  . 
extrapleural nodes , independent of their size , were present in only eight ( 1.3 % ) of 750 patients assessed ; in particular , they were b5 mm in four patients , 610 mm in two and [ 10 mm in the remaining two . considering each category of patients , extrapleural lymph nodes were present in three of 340 ( 0.9 % ) nonneoplastic patients ( b5 mm in one , 610 mm in one , [ 10 mm in one ) , in two of 270 ( 0.7 % ) patients with extrathoracic neoplasm ( both b5 mm ) , in two of 120 ( 1.6 % ) patients fig . 
4 axial computed tomography ( ct ) scan showing three lymph nodes \5 mm in the right cardiophrenic angle ( arrows ) in a 52 - yearold woman without known disease fig . 
5 axial computed tomography ( ct ) scans showing the presence of nodes [ 5 mm in the right cardiophrenic space in a patient with right pleural effusion related to malignant pleural mesothelioma ( mpm ) radiol med ( 2014 ) 119 : 2026 table 2 results regarding extrapleural nodes nonneoplastic pts . 
6 axial and coronal computed tomography ( ct ) images show typical extrapleural nodes located in the paraspinal extrapleural fat tissue adjacent to the rib heads in a patient with circumferential pleural involvement due to malignant pleural mesothelioma ( mpm ) with intrathoracic neoplasm ( b5 mm in one , 610 mm in one ) and in one of 20 ( 5 % ) patients with pleural metastasis ( 610 mm )  . 
despite the well - known limits , node size is the main criterion used to evaluate the possible involvement of these nodes [ 13 ] : hilar and mediastinal nodes are considered involved if the short - axis diameter is c10 mm [ 15 , 16 ]  . 
whereas this dimensional criterion is well accepted for hilar and mediastinal nodes , to our knowledge , no cutoff value has been established to date for cardiophrenic and extrapleural nodes . on the basis of the study by dorfman et al . , cardiophrenic nodes with a short - axis diameter [ 8 mm should be considered pathological [ 17 ] , but other studies indicate a different cutoff of 5 mm [ 18 ]  . 
no standard dimensional criteria are available for extrapleural and internal mammary lymph nodes , which are always considered pathological when present , even when the short - axis diameter is \5 mm [ 9 ]  . several papers about nodal involvement in mpm have recently been published . 
they concluded that hilar node involvement , in contrast with the tnm staging system of mpm , is secondary to parenchymal inltration and not due to direct spread from the pleura , whereas extrapleural and cardiophrenic nodes are primarily involved in the presence of mpm . 
 [ 20 ] conducted a retrospective study on 92 patients with a histological diagnosis of mpm and found a prevalence of cardiophrenic radiol med ( 2014 ) 119 : 2026 nodes of 46 % ( 42 / 92 )  . 
this study did not , however , evaluate extrapleural nodes . diagnosis of malignant pleural diseases , whether primary or secondary . our department conducted a study on 91 patients with histologically proven mpm to verify the frequency of early ct signs of this neoplasthe most frequent ct signs in patients with mpm were diffuse or focal thickening of the mediastinal pleura ( 96 % ) , thickening of the costal pleura ( 87 % ) , unilateral pleural effusion ( 85 % ) , volume loss of the pathological hemithorax ( 80 % ) and presence of extrapleural ( 68 % ) and cardiophrenic ( 67 % ) lymphadenopathy . 
we concluded that the presence of extrapleural and cardiophrenic nodes , associated with other signs of mpm and especially involvement of the mediastinal pleura and volume loss of the affected hemithorax , could have an important diagnostic role in the early diagnosis of this neoplasm . the few published reports concerning involvement of cardiophrenic and extrapleural nodes led us to investigate their prevalence in patients with different neoplastic and nonneoplastic diseases and to dene a dimensional cutoff value for these nodes . 
the overall prevalence of these nodes was 1.3 % , with a minimum value of 0.7 % in patients with extrathoracic neoplasm and a maximum value of 5 % in patients with pleural metastasis . by contrast , the prevalence of these nodes was signicantly higher in patients with mpm ; in particular , 62 of 91 patients with mpm ( 68 % ) showed involvement of extrapleural nodes , 18 ( 20 % ) with a diameter b5 mm , 25 ( 27 % ) , with a diameter 610 mm and 19 ( 21 % ) with a diameter [ 10 mthis means that extrapleural nodes should be considered pathological independently of their size , even when \5 mm ; considering that they are extremely rare in all categories of patients except those with mpm , these nodes could have an important diagnostic value in mpm patients when associated with other ndings of this disease , such as unilateral pleural effusion , hemithorax volume loss and mediastinal pleura thickening . regarding the prevalence of cardiophrenic nodes b5 mm , we found no signicant differences between patients with mpm and the other four groups , including patients without neoplaswhen we consider nodes [ 5 mm , the prevalence of these nodes in mpm patients was signicantly higher than in any other group , except for patients with pleural metastasis . 
this means that cardiophrenic nodes , unlike extrapleural nodes , should be considered pathological only when they are [ 5 mm in size . because they are enlarged both in mpm and in pleural metastasis , these nodes could have an important role in the conclusion extrapleural nodes of b5 mm and cardiophrenic nodes of [ 5 mm have signicant diagnostic value in both primary and secondary malignant pleural disease , whereas they are extremely rare in other diseases , whether neoplastic or not . conict of interest b feragalli , c . 
two different techniques were used according to lesion size ( cine mode ; sequential mode )  . after 24 h , each study was repeated to assess repeatability . lesion blood volume ( bv ) , blood ow ( bf ) , mean transit time ( mtt ) and peak enhancement intensity ( pei ) were automatically calculated by two chest radiologists in two different reading sessions . 
inhibition of tumour - related angiogenesis is now an attractive target for anticancer therapy . lung cancer ( nsclc ) , antiangiogenic therapies are yielding promising results in terms of response rate , overall survival ( os ) and progression - free survival ( pfs ) when combined with chemotherapy [ 2 , 3 ]  . 
to evaluate the effects of antiangiogenic drugs , it is advisable to estimate tumour vascularity . in patients with non - small - cell an emerging non - invasive imaging modality that allows in vivo assessment of tumour vascularization is computed tomography perfusion ( ctp ) with a multidetector - row scanner [ 4 , 5 ]  . 
a correlation between ctp parameters and histological markers of tumour neovascularisation , such as microvessel density or vascular endothelial growth factor ( vegf ) , has been demonstrated in lung cancer [ 68 ]  . 
ctp allows the processing of dynamic image data and generates radiol med ( 2014 ) 119 : 412 functional perfusion parameters based on changes in tissue attenuation over time during the injection of a contrast medium bolus . 
during the rst - pass phase ( within the rst 4050 s from contrast medium injection ) , contrast material is predominantly intravascular , and tumour contrast enhancement essentially reects blood ow ( bf ) and blood volume ( bv ) , without interference of blood recirculation on perfusion quantication . 
delayed enhancement ( after 60 s from the start of contrast medium injection ) is determined by the passage of contrast material into and out of the extravascular space and is used to calculate tumour vascular permeability [ 5 ]  . evidence exists that ctp allows monitoring the therapeutic effect of antiangiogenic treatment in lung cancer [ 4 , 9 , 10 ]  . 
 [ 9 ] demonstrated that perfusion parameters calculated with 64 - detector - row ct were higher in patients with lung adenocarcinoma that responded to chemotherapy associated with antiangiogenic drugs than in non - responding patients . good reproducibility and repeatability of any quantitative measurement repeated on the same patient is an essential prerequisite in clinical practise , as in estimating tumour treatment response . 
in the last few years , there has been considerable improvement in the ctp technique , moving from single - tumour - level studies to whole - tumour perfusion studies , providing more reliable results in terms of reproducibility [ 1113 ]  . 
in particular , to the best of our knowledge , no previous studies have been published on the repeatability of wholetumour rst - pass perfusion with 64 - detector - row ct in lung cancer . 
therefore , the purpose of our study was to assess repeatability and intraand interobserver variability of whole - tumour rst - pass perfusion technique in nsclc carried out with a 64 - detector - row ct scanner and commercial software for image postprocessing . materials and methods patient population this study received the approval of the local medical ethics committee and was performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki . 
the cine - mode technique consists of 80 acquisitions encompassing the lesion site and was carried out with the following parameters : detector conguration 64 9 0.625 mm ( total coverage , 40 mm ) ; reconstruction slice thickness , 5 mm ( eight images per rotation ) ; tube voltage , 80 kvp ; xed tube current , 120 ma ; tube rotation time , 0.5 s ; total scan duration , 40 s . the total number of ct images was 640 ( eight images per rotation 9 80 acquisitions )  . 
after 24 h , each patient underwent a second ctp acquisition using the same technical and contrast material injection parameters to assess repeatability of both perfusion techniques . technical adequacy of dynamic ct fullled the following criteria : ( a ) absence of contrast medium extravasation at the site of injection , ( b ) appropriate enhancement of vascular structures ( enhancement c150 hu ) , ( c ) no marked reaction to contrast medium that interfered with image acquisition , ( d ) adequate z - axis coverage of the whole lesion and ( e ) absence of signicant patient respiratory misregistration that hampered the automatic calculation of perfusion parameters . image postprocessing and analysis all 24 ctp scans , 12 of which were obtained at time 0 ( rst scan , t0 ) and 12 at time 1 ( second scan , t1 ) were transferred to a commercial workstation ( aw4.4 , ge medical systems ) for quantitative assessment of perfusion parameters using a commercial ctp software ( ct - perfusion 4 , ge medical systems )  . 
firstly , a circular region of interest ( roi ) was manually placed at the centre of the thoracic aorta included in the tumour scan volume with the aim of obtaining a timeattenuation curve ( arterial input ) expressed in hounseld units per second . 
the reading session was repeated by the same two radiologists with the same modality 1 month following ( second reading session ) the rst evaluation ( rst reading session ) to assess intraobserver variability . statistical analysis mean and standard deviation ( sd ) were used for descriptive statistics . 
differences between measurements yielded at the rst ( t0 ) and at the second ( t1 ) scans were plotted against the average of the differences using data collected from the rst reading session . 
computed tomography ( ct ) native images at mediastinal window and colourimetric images of blood volume ( bv ) , blood ow ( bf ) and mean transit time ( mtt ) are shown at a similar position along the patient z - axis on rst ( t0 ) and second ( t1 ) scans . 
computed tomography ( ct ) native images at mediastinal window and colourimetric images of blood volume ( bv ) , blood ow ( bf ) and mean transit time ( mtt ) are shown at a similar position along the patient z - axis on t0 and t1 scans . 
for this purpose , cronbachs a coefcient was calculated [ 17 ]  . interobserver variability was assessed by evaluating intraclass correlation coefcient ( icc ) with 95 % condence intervals ( 95 % ci ) by comparing measurements obtained by the two observers at each ctp scan ( t0 ; t1 )  . 
ten of 12 tumours were located in the upper lobes and two in the lower lobes ; nine tumours were peripheral and three were centrally located . all dynamic ct scans were considered technically adequate , without signicant patient respiratory misregistration , and included the entire tumour . 
all ctp scans were successfully analysed by the software . mean and sd of the four ctp measurements obtained by the two observers at t0 and t1 on both reading sessions are shown in table 1 . 
2 and 3 , showing two lesions located at the left lung base , a site that could potentially be affected by respiratory and cardiac motion artefacts . radiol med ( 2014 ) 119 : 412 analysis of intraobserver variability showed a high repeatability of measurements for each of the four parameters , as demonstrated by cronbachs a coefcient reported in table 2 . 
tumour vasculature shows temporal heterogeneity [ 19 ] , which cannot be exceeded in practise ; and spatial heterogeneous distribution , which seems to be compensated by increasing the tumour volume coverage assessed with the ctp technique [ 12 , 13 , 20 ]  . 
technique repeatability and intraand interobserver variability assessment of perfusion measurements is clearly relevant for accurate quantitative estimation of lung cancer perfusion , especially when considering treatment effect . until now , published studies on ctp technique repeatability have been performed using a 16 - detector - row ct scanner and different techniques [ 12 , 13 , 21 ]  . 
 [ 12 , 13 ] , a non - commercial software was used to assess whole - tumour perfusion parameters , and only bv and permeability were evaluated due to the low temporal resolution of the scanner , which did not allow whole - tumour bf measurement . 
 [ 21 ] , a rst - phase cine - mode scan for assessing rst - pass perfusion parameters , which covered only 20 mm of volume , was followed by a delayed phase , with a total duration scan time of 125 s . 
in that study , the absolute values and reproducibility of perfusion parameters were markedly inuenced by motion artefacts and long acquisition time , for permeability [ 21 ]  . indeed , the main advantage of rst - pass perfusion over permeability assessment in the delayed phase is the short scan time , which limits motion artefacts [ 22 ]  . in particular the use of 64 - detector - row ct evaluating lung cancer to improve misregistration perfusion has the potential fig . 
6 blandaltman agreement plot of mean transit time ( mtt )  . the plot shows the difference in mtt between the rst ( t0 ) and second ( t1 ) scans against the mean of mtt values . 
 [ 23 ] assessed the feasibility of rst - pass whole - tumour perfusion with 64 - detector - row ct but only tested intraobserver variability , not technique repeatability . 
the technique used in that study consisted of 12 repeated helical ct scans with a total acquisition time of * 55 s , a temporal resolution of 4.6 s and a maximum covered volume of up to 5 cm . in our study , we tested the repeatability and reproducthe rst - pass perfusion performed with a ibility of 64 - detector - row ct scanner that enabled coverage of tumours with a diameter of up to 8 cm with higher temporal resolution ( 0.5 s for cine mode ; 2.7 s for shuttle ) and shorter scan duration time ( \50 s ) than did the abovementioned study . 
we observed good overall repeatability of the whole lung tumour rst - pass perfusion technique with low coefcients of variation and narrow limits of agreement for all parameters , in particular for bv and mtt . 
moreover , we noted better results in comparison with those reported in the three previous studies performed using a 16 - row ct scanner [ 12 , 13 , 21 ] , which enrolled a similar overall number of patients ( respectively 10 , 10 and 11 patients ) to our study ( 12 patients )  . 
improved perfusion parameter repeatability measured using 64 - detector - row ct , in our experience , is mainly due to the higher temporal resolution of the scanner and perfusion protocols used , which allowed technically adequate ct scans without relevant patient respiratory misregistration . 
in addition , limits of agreement for the cine - mode technique measurements in our study were narrower than those for the sequential - mode technique , which may be explained by the better performance in terms of temporal resolution of the cine - mode technique . 
our results in terms of good repeatability could be also due to the patient respiratory training performed before starting the examination and to the prevalent location of tumours in the upper lobes ( 10 / 12 , 83 % ) , which are less affected by respiratory motion artefacts . in the clinical context of assessing lung cancer response to treatment , the intrinsic variability of perfusion measurements should be small enough to allow the identication of changes in tumour vascularization induced by the treatment . 
in a study that used the ctp technique to determine the effects of an antiangiogenic drug ( combretastatin a4 phosphate ; ca4p ) on tumour vascularisation in patients with advanced nsclc , a reduction in bv of 29.4 % was demonstrated after 72 h from ca4p administration [ 10 ]  . 
the intrinsic variability we observed for bv in the ctp technique was 26 % , a value small enough to assess bv changes induced by the therapeutic agent , according to data mentioned above . 
this result suggests that the ctp technique may be considered quite repeatable for detecting perfusion parameter changes and that it could have a clinical application in assessing therapy response . as regards intraobserver agreement , the high consisresults demonstrated in our study for both tency of observers and all parameters may be explained by the adequate period of training in the ctp technique received by the two radiologists . 
this result was expected , considering the variability due to manual drawing of rois along the tumour margins , which is closely operator dependent , but did not affect the interobserver reliability . 
similar results were reported for colorectal cancer perfusion technique [ 24 ]  . in summary , all our results , in terms of technique repeatability and agreement among observers , are indicative of the possible clinical use of the ctp techniques tested , with the advantage of employing commercial software for postprocessing . 
first is the small number of patients enrolled , even though other published studies [ 12 , 13 , 21 ] on the same topic also included few patients ( 10 , 10 and 11 , respectively )  . 
to assess repeatability of the ctp technique , two examinations were performed at t0 and t1 after 24 h , with a considerable overall effective radiation dose to each patient , in particular for the cine - mode technique ( 25 msv )  . 
secondly , patient clinical outcome on the basis of perfusion parameters was not analysed , as this was beyond our aiin fact , this study represented only a laboratory test of technique repeatability . 
largescale trials are required to investigate the usefulness of the rst - pass perfusion ct technique in assessing prognosis and survival of lung cancer patients . in conclusion , rst - pass perfusion with 64 - detector - row ct proved to be feasible to quantitatively assess wholetumour perfusion in lung lesions measuring up to 8 cm , with good technique repeatability and high intraand interobserver agreement . 
pneumologia , ospedale morgagni - pierantoni , via c.forlanini 34 , forl` , italy their diagnosis is a challenge for clinicians , pathologists , and radiologists , and any additional knowledge about the ct ndings may help the diagnosis in the case of patients affected by ph of unknown origin . keywords pulmonary hypertension ( cid : 2 ) tumour embolism ( cid : 2 ) pulmonary artery sarcomas introduction pulmonary hypertension ( ph ) is dened as an increase in mean pulmonary arterial pressure ( pap ) c25 mmhg at rest as assessed by right heart catheterisation , in the presence of normal left atrial pressure . 
chest radiography , echocardiography , and electrocardiography are run as rst level investigations but in many cases the diagnostic process requires further investigations , including a computed tomography ( ct ) study . 
ph can be associated with several clinical conditions , which have been classied into six groups according to specic characteristics ( dana point 2008 ) [ 1 ] : 1 . 
pulmonary hypertension with multifactorial mechanisms . the relevant literature and presenting our personal series of 26 cases . in order to adopt recently published guidelines by the european society of cardiology and the european respiratory society emphasise the importance of a correct diagnosis in patients the appropriate affected by ph [ 1 ] , therapeutic approach . 
whereas thromboendarterectomy is a potentially curative option for cteph , non - embolic ph can be treated by specic drugs and supportive therapy or , in the case of inadequate clinical response , by balloon atrial septostomy and / or heartlung transplantation . 
it is also important to recognise patients affected by pvod or pch , because the administration of vasodilators in such patients can lead to a fatal pulmonary oedema [ 1 , 2 ]  . histology may be required to conrm the diagnosis , but lung biopsies are high - risk procedures and , therefore , not recommended [ 2 , 3 ] ; hence , the need to nd a less invasive approach for a correct aetiopathogenetic diagnosis of patients . under these circumstances , high - resolution computed tomography ( hrct ) of the chest , especially in the angiographic phase after injection of contrast material ( ct pulmonary angiography , ctpa ) , plays a central role because it allows both evaluation of indirect signs of ph and identication of any underlying disease that might lead to secondary ph . ct features common to all forms of ph [ 4 , 5 ] are : dilatation of the pulmonary artery trunk , which may exceed that of the ascending aorta ( ratio greater than 1 : 1 )  . 
the presence of a diameter greater than or equal to 29 mm has a positive predictive value ( ppv ) [ 95 % and a specicity [ 90 % in the diagnosis of ph in the absence of diffuse pulmonary brosis . 
its aetiology is not wellknown , and among the suggested hypotheses are immunemediated causes ; viral aetiology ; an excessive immune response to chemotherapy or radiation or bone marrow transplants in cancer patients [ 6 ] ; smoke and exposure to anorectic agents derived from fenuramine [ 2 ]  . 
both the clinical and haemodynamic features of pvod are non - specic but radiology is of great help because the signs of pulmonary hypertension and of interstitial oedema are evident , even when the anatomical obstruction is post - capillary ( pap is high and pulmonary wedge pressure is normal or low )  . pvod is histologically characterised by intimal brosis , which causes narrowing and occlusion of the pulmonary veins of any size , with a preference for venules with a diffuse or patchy distribution [ 7 ]  . 
however , the absence of the typical radiological ndings does not exclude a diagnosis of pvod and cardiac catheterisation remains necessary , with precise documentation of the transcapillary wedge pressure [ 9 ]  . recent reports have highlighted the usefulness of pulmonary function tests in the diagnostic workup of pvod , as they typically show a decrease in diffusing capacity of the lung for carbon monoxide ( dlco ) , hypoxaemia at rest in arterial blood gases analysis and signicant desaturation at the 6 - minute walk test ( 6mwt )  . bronchoalveolar lavage ( bal ) might show macrophages containing haemosiderin , which is a manifestation of occult intra - alveolar haemorrhage [ 2 , 3 ]  . ct and clinical - laboratory ndings allow for a sufciently reliable diagnosis of pvod , thus averting the need radiol med ( 2014 ) 119 : 4153 fig . 
1 our ct ndings are in agreement with literature ; indeed all 6 patients had smooth septal thickening ( a , b ) , groundglass opacities with preferential centrilobular distribution ( a , b ) and mediastinal adenopathy ( c ) ; 3 patients had also pericardial and pleural effusion for lung biopsy and consenting the planning of a treatment schedule and the addition of patients to transplant waiting lists [ 2 , 9 ]  . capillary haemangiomatosis pulmonary capillary haemangiomatosis ( pch ) , just one personal case , is an extremely rare form of post - capillary ph , identied for the rst time in 1978 by wagenvoort . pch affects mainly young adults ( 2040 years ) and it shows a median survival of 3 years [ 6 , 9 ]  . 
a high frequency of haemoptysis and pleural effusion ( haemorrhagic in many cases ) are clinically reported ; pulmonary function tests show the reduction of dlco and hypoxaemia [ 2 ]  . 
nodular opacities probably reect focal angiomatous proliferation within bronchovascular structures along the alveolar septa and within the vessel walls , while the ground - glass background can be ascribed to the small vessel growth [ 10 ]  . septal thickening , pleural effusion ( clinically in 50 % of cases ) , and mediastinal node enlargement are rarely present , as in pvod . 
with disease progression , common signs of pulmonary arterial hypertension , such as dilation of the main pulmonary artery and right heart chambers , start to arise [ 11 ]  . pulmonary hypertension due to non - thrombotic pulmonary embolism non - thromboembolic pulmonary embolism is a rare cause of precapillary ph . 
it is dened as the embolisation of the pulmonary circulation by different cell types ( adipocytes , amniotic , cancer , etc . ) , bacteria , fungi , foreign material or gas [ 1 ]  . 
in contrast embolism , the effects of non - thrombotic emboli are not only purely mechanical but also related to the nature of the embolic agent . chest x - ray may be in the norm or shows signs of ph such as enlargement of central pulmonary arteries and cardiomegaly . 
2 hrct : diffuse centrilobular ground glass opacities with some nodules superimposable ( a c ) emphasized by min - ip reconstruction ( d ) ; no mediastinal adenopathies or pleural effusion ( d ) radiol med ( 2014 ) 119 : 4153 artery ( fleischner sign ) ; peripheral area of oligaemia with minimal change in lung volume ( westermark sign ) or diaphragm elevation . 
the presence of single or multiple pulmonary infarcts , most frequent in the basal regions , may be noticed as well : it is classically described as hamptons hump and consists of a homogeneous wedge - shaped consolidation in the lung periphery with a base contiguous to a visceral pleural surface and a rounded convex apex directed towards the hilum [ 12 , 14 ]  . frequent ct features common to cteph [ 4 , 6 , 15 ] include : complete or partial obstruction of the vessel lumen . characteristic mosaic pattern of perfusion in the pulmonary parenchyma , due to the blood ow redistribution and the presence of hypoperfused areas with low attenuation high hyperperfused attenuation . areas enlargement of both bronchial and non - bronchial systemic arteries ( phrenic , mammary , intercostal ) with a diameter [ 2 mm , which helps to keep the blood ow in the presence of pulmonary arterial vascular obstruction by participating in gas exchange . 
blood ow in these arteries , which in normal conditions represents 1 % of the cardiac output , may reach almost 30 % of the systemic blood ow [ 16 ]  . lymphadenopathy , pleural effusion and pleura - based triangle opacities with thickening of parenchymal adjoining visceral pleura or parenchymal bands , representing scars of pulmonary infarcts . it is important to emphasise that chronic thrombi , after the injection of the contrast agent , often show signicant enhancement , depending on the thrombus organisation [ 17 ]  . tumour embolism a case of ph caused by tumour emboli in the pulmonary circulation was rst reported in 1937 . 
many types of malignant tumour can give rise to this picture , the most frequent of which is gastric cancer , followed by renal cell carcinoma , breast , prostate , hepatocellular carcinoma , and choriocarcinoma [ 12 , 18 , 19 ]  . tumour thrombi are frequently formed by malignant cells , platelets , and brin , and are highly resistant to recanalisation and lead to a progressive and irreversible obstruction ; in many cases , they are associated with vascular tissue reaction with intimal proliferation and brosis ( thrombotic microangiopathy )  . 
when 6080 % of pulmonary arterial bed is involved , cor pulmonale occurs which leads the patient to death [ 18 , 20 ]  . ct in the angiographic phase , as in the single case we observed , shows the presence of intraluminal lling defects or multifocal dilatations and strictures in affected segmental and subsegmental arteries associated with hypopshow carcinomatous erfused areas ; ct can also radiol med ( 2014 ) 119 : 4153 fig . 
common ndings in these cases consist of : a or b kerley lines ( expression of thickening septa ) ; thickening with unior bilateral reticular pattern with or without adenopathy and , in some cases , thickening and / or pleural or pericardial effusion . 
hrct enables the detection of the disease in more than 50 % of patients with compatible symptoms and negative x - ray : it shows nodular thickening of the septa , subpleural and peribronchovascular interstitium , in the absence of distortion of the brotic lung parenchyma . 
this nding may be focal or bilateral and diffuse [ 22 ]  . a tree - in - bud - like pattern has recently been described as an expression of tumour intravascular pulmonary embolisin these cases , two different pathogenetic mechanisms are involved : the lling of centrilobular arterioles by tumour cells and the so - called carcinomatous endarteritis , which is a rare form pathologically characterised by marked intimal hyperplasia brocells initiated by the presence of microemboli tumour [ 19 , 23 ]  . hydatid disease hydatid disease is a parasitic disease , particularly widespread in the mediterranean , most frequently caused by larvae or cysts of echinococcus granulosus . 
the parasite can reach any part of the body , but the liver ( 75 % ) and lung ( 15 % ) are the most frequent sites of infection [ 24 , 25 ]  . pulmonary embolism is a rare complication of heart or liver echinococcosis resulting from the rupture of a hydatid cyst in the right heart or , more rarely , because of haematogenous dissemination after the rupture of a cyst in the hepatic veins and inferior vena cava . 
in many cases , a chronic course with acute recurrences can be observed [ 19 ]  . ct in the angiographic phase , as in the single case we observed , shows occlusion of the pulmonary arterial branches caused by cystic lesions that appear as multiple bilateral nodules of variable diameter and well - dened margins with characteristic hypodense homogeneous postcontrast enhancement . 
embolism of numerous segmental and subsegmental arteries ending in hydatid cysts , some of which partially calcied ( b , c ) ; two partially calcied hydatid cysts are present in the mediastinum : one between aortic root and right atrium , the other prior to right atrium ( d ) radiol med ( 2014 ) 119 : 4153 magnetic resonance ( mr ) imaging can be useful in the diagnostic workup of these patients both for the characterisation of cardiac hydatid cysts and for the evaluation of lung nodules : homogeneous hyperintense signal with hypointense peripheral rim , which represents the pericystic capsule , on t2 weighted images and hypointense on t1 weighted images [ 26 ]  . 
knowledge of the radiological characteristics is important because urgent surgical treatment ( endoarterectomy or lung transplantation ) is required , where possible , to avoid the occurrence of complications or death . talcosis there are several forms of lung talcosis ( talcosilicosis , talcoasbestosis and talcosis alone ) caused by inhalation of talc , a mineral widely used in factories ( ceramic , cosmetic , paper , plastic and paint ) [ 27 ]  . 
pulmonary talcosis affects drug abusers who inject drugs made out of crushed tablets containing talc as a binding agent , and the disease progresses with the chronic intravenous injection of such substances , as in the single case we observed . 
clinically , most patients are asymptomatic or show non - specic symptoms such as worsening dyspnoea , cough , and chest pain , but there are also fulminant forms ; the clinical ndings are dose - related but may progress even after cessation of drug abuse . 
when injected intravenously , talc is trapped in the pulmonary arterioles because of its insolubility , resulting in thrombosis and inammation ; as a consequence , brosis and architectural distortion of the lung may arise , especially in the upper lobes [ 12 , 20 ]  . chest x - ray may show varying ndings , from small nodules 13 mm in diameter in the early stages to extensive areas of increased opacity , predominantly located in the perihilar region with sparing of the apical regions and costophrenic sinuses which may enter the differential diagnosis with the progressive massive brosis affecting patients with silicosis , coal workers pneumoconiosis and stage iv sarcoidosis . 
elevation of the hila , hyperlucency of the lung bases and signs of ph are also reported [ 27 ]  . in the early stages , ct also dedepicts perilymphatic micronodules with both centrilobular and lymphatic distribution and ground - glass areas that can evolve into areas radiol med ( 2014 ) 119 : 4153 fig . 
5 ( a , b , c , d ) : widespread and severe interstitial lung disease ( with involvement of approximately 75 % of lung parenchyma ) characterized by ground - glass opacities and thickening of interlobular septa with sparing of some secondary lobules . 
hilar or mediastinal nodal enlargements are rare [ 19 , 27 , 28 ]  . bronchoalveolar typically shows the lavage ( bal ) presence of lymphocytosis with a prevalence of cd8 lymphocytes and macrophages with intracellular microcrystals of talc which have a typical birefringent appearance under polarisation microscopy [ 20 , 27 ]  . pulmonary artery sarcomas pulmonary artery sarcoma ( pas ) is a rare malignant intraluminal mesenchymal tumour rst described during autopsy by mandelstamm in 1923 . 
pas tends to grow quickly and has an unfavourable prognosis , with an average survival of 12 months in surgical patients ; death is usually due to heart failure caused by obstruction of the pulmonary arteries [ 29 , 30 ]  . clinical presentation is not specic and the most common symptoms are : progressive dyspnoea ( 67 % ) , chest pain ( 54 % ) , cough ( 42 % ) , and haemoptysis ( 22 % ) ; weight loss , anaemia , and fever are not common symptoms and tend to suggest malignant disease rather than pulmonary embolism [ 3032 ]  . 
the correct diagnosis is often delayed because pas generally mimics acute or chronic pulmonary embolism , lung cancer or mediastinal masses , with a consequent delay in surgical treatment and worse prognosis [ 30 , 31 ]  . many pas involve , and probably arise from , the pulmonary artery trunk near the pulmonary valve and often involve the main pulmonary artery [ 29 , 31 ]  . the chest x - ray may be normal or , more frequently , show hilar masses , lung consolidation or oligaemia , heart and pulmonary artery enlargement or evidence of metastasic disease , but such ndings are generally considered non - specic [ 31 ]  . ct can help to differentiate pas from pulmonary embolism by showing a low - attenuation lling defect which occupies the entire luminal diameter of the main pulmonary trunk and / or main branches of the pulmonary artery with extraluminal tumour extension in the mediastinugenerally , this defect shows inhomogeneous contrast enhancement because of the presence of internal areas of necrosis or thrombi covering the tumour ; this heterogeneous appearance can be due to the presence of haemorrhagic foci , cysts or intralesional calcic spots [ 29 , 30 , 33 ]  . 
cases without any signicant contrast enhancement have also been observed . the presence of extraluminal tumour extension or mediastinal , pulmonary or distant metastases allows the late denitive differential diagnosis , but these fig . 
6 ctpa : large lling defect in the pulmonary artery trunk and its branches ( main , lobular and segmental ) ( a , b , c )  . the tissue has contrast enhancement and there is fdg uptake in the pet - ct study ( d ) radiol med ( 2014 ) 119 : 4153 manifestations [ 33 ]  . 
besides , pet plays a central role in the diagnostic process of pas associated with thrombosis as it may be used to isolate pas from thrombosis by showing site , size , and the extension of the tumour , which presents intense contrast enhancement as opposed to the thrombotic area , which is not fdg - avid because thrombi contain few macrophages and inammatory cells [ 30 , 34 ]  . neurobromatosis neurobromatosis type 1 ( nf1 ) , known also as von recklinghausens disease , is a genetic autosomal dominant disorder characterised by nf1 tumour suppressor gene mutation on chromosome 17 . 
7 widespread alteration of lung parenchyma , which has cystic appearance in the upper lobes ( b ) and reticular appearance , with areas of ground - glass , in the lower lobes ( c , d ) ; extension bigger than 50% of the lung parenchyma ; no ndings typical ct of ph . diagnosis of ph at heart catheterization ( papm 35 mmhg , pwp 10 mmhg ) pulmonary brosis has also been reported [ 38 ]  . 
the disease has an estimated incidence of 1035 cases / 100 , 000 people in the usa , with a mortality of 15 % and an average age at diagnosis of 40 years [ 39 ]  . the typical lesion is a non - caseating sarcoidosis granuloma consisting of a central core of t lymphocytes surrounded by epithelioid and giant multinucleated cells ; broblasts and brosis that evolve along a centripetal direction to ll the granuloma during the course of the disease are situated in the external area . frequently , the diagnosis is made with chest x - ray , because of the presence of hilar and mediastinal lymph node enlargement , with or without lung parenchymal involvement . 
the affected lung has a typical nodular interstitial pattern characterised by several solid nodules varying from 1 to 5 mm , associated with linear and irregular opacities [ 22 ]  . subpleural distribution , the hrct features can vary and mimic other diffuse inltrative diseases ; characteristic ndings of active sarcoidosis include several small well - dened nodules showing perilymphatic along the interlobular septa and bronchovascular bundles , often predominant in the upper lobes . 
the earliest nding of brosis is the posterior displacement of the upper lobar bronchi associated with reticular septal thickening due to the evolution of the brotic lesions which can develop from peribronchial brosis into perihilar masses , which distort the bronchi and vessels and cause traction bronchiectasis [ 22 ]  . 
the nding of areas of honeycombing is rare [ 14 , 40 ]  . once considered a rare manifestation of sarcoidosis , ph associated with sarcoidosis ( saph ) , of which we have observed six cases , is now acknowledged as an important complication that signicantly worsens morbidity and mortality . in the recently published guidelines by the european society of cardiology and the european respiratory society , ph associated with lung diseases or hypoxaemia is classied in group iii , while saph is included in group v ( miscellaneous ) because its pathogenesis is complex and multifactorial [ 1 ]  . 
the presence of ph despite the absence of hypoxaemia or a diffuse parenchymal damage can be caused by : pvod , a recognised complication of sarcoidosis characterised by an occlusive venopathy with intimal brosis and haemosiderosis [ 39 ]  . intrinsic sarcoid vasculopathy , granulomatous involvement of the pulmonary vessel wall occurring in 70100 % of patients with saph ; in fact , because of the perilymphatic distribution of sarcoidosis , granulomas are located close to arterial or venous pulmonary septa vessels , and they may penetrate the intima , media or adventitia of the arteries causing occlusion , or they may affect the veins , haemodynamically mimicking pvod [ 45 , 46 ]  . other causes include : extrinsic compression of pulmonary vessels by mediastinal or hilar lymphadenopathy or mediastinal brosis [ 40 ] ; myocardial dysfunction for direct myocardial involvement by sarcoidosis [ 42 ] ; portopulmonary hypertension resulting from hepatic sarcoid inltration with consequent cirrhosis and portopulmonary hypertension [ 39 , 40 ] ; obstructive sleep apnea syndrome ( osas ) , which is common in patients with sarcoidosis and can contribute to a high pulmonary pressure [ 39 ]  . is important to identify the occurrence of ph in patients with sarcoidosis as early as possible because ph signicantly worsens the prognosis , with a median 5 - year survival rate of less than 60 % [ 46 ]  . ph should be suspected in all patients with sarcoidosis with dyspnoea , hypoxaemia or clinical ndings compatible with right heart failure ( such as peripheral oedema or syncope ) , especially if these symptoms are out of proportion to the extent of pulmonary parenchymal damage . some parameters and clinical laboratory parameters predictive of ph have been studied including : increased need for supplemental oxygen , dlco \30 % in patients with stage iv sarcoidosis on chest x - ray or dlco \50 % in radiographic evidence of pulmonary patients without radiol med ( 2014 ) 119 : 4153 fig . 
 ( b , c , d ) : hrct permits clearer imaging of diffuse and irregular cystic pattern ( involving more than 50% of lung parenchyma ) that simmetrically involves the middle and upper lung zones and sparing the costophrenic sulci brosis [ 47 ] , or oxygen saturation at the 6mwt \90 % and dlco \60 % [ 48 ]  . debate about the usefulness of a specic therapy for ph is still open , but it has been shown that vasodilators should be used with caution in patients with a pvod - like pattern because they may precipitate pulmonary oedema [ 42 , 46 ]  . hrct is therefore fundamental as it can raise a suspicion of pvod in the presence of its typical features ( smooth septal thickening , ground - glass opacities and mediastinal nodal enlargement ) , which are frequently present in patients with non - brotic sarcoidosis and ph [ 45 ]  . pulmonary langerhans cell histiocytosis pulmonary langerhans cell histiocytosis ( plch ) or histiocystosis x or eosinophilic granuloma is a rare smokerelated disease predominantly affecting young people ( 2040 years ) [ 49 ]  . plch belongs to the group of langerhans cell histiocytosis ( lch ) , an idiopathic disorder that affects the specialised immune langerhans cells , which can accumulate in tissues as a result of local reaction to inammatory or neoplastic stimuli [ 50 ]  . 
its course is unpredictable because it can improve or even reverse with smoking cessation or corticosteroid therapy , but in a minority of cases it may progress to pulmonary brosis that can lead to death or lung transplantation [ 51 , 52 ]  . 
the cessation of smoking does not always lead to the resolution of the pathology [ 49 , 50 ]  . from a histological point of view , the characteristic lesion of plch is the bronchiole - centric inammation supported by aggregates of langerhans cells , inammatory cells and brosis [ 52 ] , similar to a sheath extending proximally and distally along the damaged bronchioles . chest radiography shows nodules with irregular margins of variable size ( 110 mm ) , usually with bilateral and symmetrical distribution predominantly in the upper and middle lobe , with sparing of the costophrenic sinuses [ 50 , 51 ]  . hrct , as we observed in our ve cases , is much more sensitive and specic than chest x - ray in the assessment of the extent and activity of the disease , and it leads to a correct diagnosis in 90 % of patients . 
in particular , the initial stages of the disease are characterised by a nodular pattern that reects the presence of bronchiolocentric granuloma : nodules usually sized less than 5 mm with typical peribronchiolar and centrilobular distribution , in variable number depending on the degree of disease activity . 
during disease progression , the nodules become cavitated and the thickening of bronchiolar walls and the formation of cystic spaces with walls of variable thickness from perceptibility to some millimetres become clear . 
cysts are the most typical features of this disease ; they may be of variable shapes ( round , lobular or bizarre ) , usually smaller than 10 mm , but they may coalesce or break into the pleural space causing pneumothorax [ 22 ]  . 
numerous studies have shown that , in the advanced stages of plch , the decrease in exercise capacity does not appear to be related to ventilatory limitation , but rather to a severe and widespread vascular pulmonary disease . 
in fact , as opposed to patients with chronic obstructive pulmonary disease ( copd ) or other interstitial lung disease ( ild ) , patients with plch have ph out of proportion to their indices of ventilator or gas - exchange limitation . 
this theory is supported by the fact that , while the ph secondary to chronic obstructive or other interstitial brotic diseases rarely exceeds 35 mmhg , in plch the pulmonary artery pressure is 60 mmhg on average , that is , similar to that observed in idiopathic ph [ 50 ]  . histopathological observations conrm that pulmonary vascular involvement may be a central process in advanced plch . 
the causes of such vascular remodelling is still unknown [ 50 , 52 ]  . conclusion ph is a frequent clinical diagnosis often associated with high patient morbidity and mortality . 
diseases leading to ph cause a wide spectrum of ct ndings , and the recognition and further knowledge of the ct features of rare forms of ph are important because , when combined with the clinical and laboratory data , they can help radiologists to provide a sufciently reliable diagnosis without lung an appropriate biopsy . 
mcloud received : 27 october 2013 / accepted : 4 november 2013 / published online : 22 november 2013 ( cid : 2 ) italian society of medical radiology 2013 lung cancer remains the leading cause of death from malignancy with approximately 1.3 million deaths occurring world wide per year [ 1 ]  . 
the overall 5 - year survival still remains very low at only 14 % [ 1 ]  . lung cancer , however , which is detected early in its course , demonstrates much better survival data . 
survival in non - small - cell lung cancer stage i is greater than 70 % and small peripheral less than 1 cm lung cancers have survival rates of greater than 80 % [ 2 ]  . 
diagnosis of lung cancer early in preclinical stages has a higher cure rate and , therefore , high - risk asymptomatic individuals are likely to benet from screening . the introduction of helical and subsequently multidetector ct in the 1990s led to a number of feasibility studies in which ct was used as a screening tool for the detection of early asymptomatic lung cancer . 
mcloud ( & ) thoracic radiology , massachusetts general hospital , harvard medical school , 55 fruit street , boston , ma 02114 , usa e - mail : tmcloud@partners.org least in regard to diagnostic algorithms , three times that of standard positive rate was at radiographs . these studies provided evidence that peripheral nodules greater investigation than 810 mm in size required further because of the high likelihood of lung cancer . 
smaller nodules ( \8 mm diameter ) could be managed with ct follow - up to determine interval growth indicating the likelihood of malignancy . these single arm feasibility and observational studies the effectiveness of provided compelling evidence of helical and mdct as a screening tool for early detection of lung cancer . 
only randomized controlled trials can determine effectiveness of screening where the end point must be a decrease in mortality in the screened group as compared to a non - screened control . subsequently , several prospective randomized controlled trials were undertaken both in the united states and europe . 
these included the national lung cancer screening trial ( nlst ) in the united states [ 10 ] and the nelson trial and others in europe [ 1115 ]  . 
it was a randomized national study and the population group included individuals between the ages of 55 and 74 with history of c30 - pack years of current or prior smoking . the enrollees underwent three annual screens . 
the study was designed to determine lung cancer - specic mortality and had a 90 % power to detect a 20 % reduction in lung cancer in the screened group . 
a positive screen consisted of the detection of a nodule or nodules greater than 4 mm in diameter or other abnormalities suspicious of lung cancer . follow - up was recommended based on nodule size and larger nodules required diagnostic workup . 
there were few major complications from diagnostic workups conducted on cases suspicious for lung cancer . however , there were a number of important disadvantages of screening reported by this study . 
39 % of subjects in the ct arm had a positive scan over the course of the 3 - year screening period and 95 % of the positive screens were false positives with no evidence of conrmed lung cancer . 
however , the technique delivered a mean whole body effective dose of 1.4 msv in the nlst trial which represents about one - fth of the dose of a diagnostic chest ct scan [ 16 ]  . the potential benets of ct screening for high - risk individuals outweigh the small risk of cancer deaths related to cumulative radiation exposure [ 17 ]  . there are methods to improve the positive predictive value and decrease the large number of false positives encountered with ct screening for lung cancer . 
the nelson trial in europe is a two - step process which has been able to decrease the number of false positive ndings based on nodule size [ 11 ]  . 
another method of improving the positive predictive value is to better dene risk proling . although the greatest risk for lung cancer is smoking , the risk is less than 20 % for the development of lung cancer . however , patients with copd may have up to a sixfold increased risk for malignancy . 
it is also possible that medical biomarkers may be used to further identify highrisk populations . many american professional societies have recommended lung cancer screening for individuals meeting the criteria of the nlst trial with some slight modications . these include the american college of chest physicians , the american cancer society , the american association of thoracic surgery , and the national comprehensive cancer network [ 17 , 18 ]  . 
this will ultimately mean that in the us , there will be insurance coverage for lung cancer screening probably within the course of a year . the cost - effectiveness data from the nlst trial have not yet been published . 
estimates of the cost - effectiveness of screening ct have relied on decision analysis modeling with estimates ranging from $50 , 000 up to $2 , 000 , 000 per quality - adjusted life year gained [ 19 ]  . is extremely important for institutions to develop programs for lung cancer screening . 
in europe , there has been much discussion of the possibility of training physician extenders ( such as technologists or radiographers ) , interpretation of to provide the initial screening examinations . 
evidence - based infrastructure must be in place to secure that patients have access to uniform quality care and a similar life saving benet from ct examinations that was demonstrated in the national lung cancer screening trial . 
on the basis of the clinicalradiological evolution , we analysed complete ablation ( ca ) versus partial ablation ( pa ) at the rst computed tomography ( ct ) scan and during the follow - up ( mean 23 months ) , time to progression ( ttp ) and survival . 
a threshold of 30 mm ( p = 0.0030 ) and the histological type ( nsclc 75 % / mts 94 % ; p = 0.0305 ) were also predictive of ca . 
filippini dipartimento di scienze chirurgiche , universita` di torino , corso bramante 88 , 10126 turin , italy conclusion rfa for thoracic malignancies is accurate for lesions up to 30 mm , especially if metastatic ; survival is more closely related to staging factors than to the local effectiveness of rfa . keywords radiofrequency ( cid : 2 ) thermal ablation ( cid : 2 ) tumour ( cid : 2 ) lung ( cid : 2 ) thorax introduction primary tumours of the lung are the leading cause of cancer mortality worldwide , amounting to 1.2 million deaths each year [ 1 ]  . 
improving survival in nonsmall - cell lung cancer ( nsclc ) requires early diagnosis and effective treatment . lobectomy with hilar and mediastinal lymph node sampling is still the gold standard treatment for nsclc in stages i and ii . 
unfortunately , many patients are not good candidates for lobar resection [ 2 ]  . pulmonary metastases ( mts ) are present in 2530 % of patients suffering from different types of cancer ; for some patients with oligometastatic pulmonary disease , metastasectomy seems to be associated with improved survival [ 3 ]  . radiotherapy ( rt ) has been offered as an alternative to surgery for both nsclc and mts ; however , the 5 - year survival after traditional rt remains low ( 1520 % ) , with a high rate of local recurrence ( lr ) [ 4 ]  . the last few decades have seen an increase in the detection of smaller neoplasms ( in part as a result of low - dose ct screening for nsclc [ 5 ] ) and , at the same time , the development of new local therapeutic possibilities , e.g. 
initially applied to other solid tumours , it has been also used in the chest to increase the possibility of local control of the tumour in inoperable patients [ 7 ]  . radiol med ( 2014 ) 119 : 3340 this study discusses the safety , efcacy and clinical utility of thermal ablation of nsclcs and pulmonary mts ( mainly from colorectal cancercrc ) by presenting our results in terms of survival and prognostic factors and comparing them with the literature data . materials and methods of a total of 1 , 591 patients who underwent rfa at our institution since the late 1990s , 94 ( 5.9 % ) were treated for thoracic cancers . 
to assess the signicance of rfa in this type of cancers , we conducted a retrospective study on a series treated between 2002 and 2011 , consisting of 81 consecutive patients who underwent rfa for 100 thoracic lesions . the study was carried out in accordance with the guidelines on retrospective analyses established by our hospitals institutional review board . patients and lesions each patient was referred for treatment on the basis of clinical indications , after a multidisciplinary assessment by an interventional radiologist , a thoracic surgeon , a medical oncologist and an anaesthesiologist . 
the rfa technique was established based on the interventional radiologists technical evaluation . the lesions treated with rfa were not candidates for surgery due to the following reasons : reduced pulmonary function ; compromised general and / or cardiovascular condition ; advanced age of the patient ; location of the lesions in both lungs ; presence of extrapulmonary disease , but of limited extent ; refusal of the patient to undergo surgery . depending on the histological type of neoplasm , our patients can be divided into two groups : a group suffering from primary lung cancer ( nsclc ) and a second group with pulmonary mts , mainly from crc . at the time of rfa , 61 of the 81 patients were male ( 75 % ) with a mean age of 61.6 years ( median 66 ) and 20 were females ( 25 % ) with a mean age of 68.7 years the average age was 61.7 years ( median 70.5 ) ; overall ( range 1788 years ; median 66 )  . fifty - ve of the eighty - one patients ( 67.9 % ) had received other treatments prior to thoracic rfa ( including six liver rfa ) ; nine patients suffering from mts from crc underwent liver rfa synchronous to the pulmonary rfa . 
thirty - four of the eighty - one patients ( 42 % ) were also affected by extrapulmonary mts . one hundred lesions were treated : 30 nsclc ( 30 % ) and 70 mts [ 51 / 70 from crc ( 72.8 % ) , 6 / 70 from sarcomas ( 8.5 % ) , four from hepatocellular carcinoma , two from gastric carcinoma and the remaining seven from cholangiocarcinoma , laryngeal , breast , ovary , parotid , prostate and kidney cancer , respectively ]  . 
specically 79 lesions ( 79 % ) a diameter \3 cm , and 40 lesions ( 40 % ) had a diameter \2 cm . the lung lesions were solitary in 48 cases ( 48 % ) and multiple in 52 cases ( 52 % )  . preprocedural assessment before treatment , general and cardiorespiratory condition ( performance status ) was evaluated . 
in addition , preprocedural ct or positron - emission tomography ( pet ) ct staging was requested , preferably not more than 1 month before the procedure . pathological diagnoses were available for all of the nsclcs ; the mts were mostly recognised as such on the basis of their clinical evolution and imaging characteristics ; in a few cases only ( e.g. those with atypical imaging appearance ) biopsies were performed . for each patient we requested clotting tests ( in case they needed correction of thrombocytopenia or inr or ptt elongation ) and electrocardiograsubsequently , an anaesthesiological consultation was held . 
finally , any antitherapy was modied or coagulation or antiplatelet suspended . the indications , potential benets and risks of the procedure were discussed with the patients , who always signed a consent form . procedure the rfa was performed percutaneously in all 81 patients . the needle electrodes used for thoracic rfa were always of the expandable type . 
in particular , the systems used were rita ( rita medical systems , usa ) ( 15 / 100 cases ) , leveen ( boston scientic corporation , usa ) ( 75 / 100 ) and meditalia ( meditalia biomedica , italy ) ( 10 / 100 ) , with different opening diameters . 
the rita system is regulated by the temperature reached in the tissues ( based on the application time ) , the leveen system is impedance based , and the meditalia system can be based on either impedance or temperature . 
this , in addition to preventing neoplastic seeding , allows drainage of any pneumothorax during withdrawal . four hours after the procedure , a chest x - ray was obtained in all cases . 
in any case , patients were kept under observation for one night after treatment to monitor their clinical condition and blood count , and follow the possible evolution of pneumothorax . all adverse events were recorded , categorised according to the sir criteria into minor events ( clinically relevant ) and major complications ( requiring interventional radiological or surgical intervention ) [ 7 ] , and counted in terms of numbers and percentages . follow - up the result of the rfa was evaluated by performing contrast - enhanced ct 1 month after the procedure , to highlight the possible presence of residual viable tissue . 
in the case of complete ablation ( ca ) at the rst ct scan , patients were placed on a protocol of follow - up with 4 monthly ct studies ( but for patients with mts , follow - up intervals were sometimes changed on the basis of the evolution of metastatic disease and / or in relation to systemic therapy ) ; doubtful cases were examined by petct to establish possible lr . the possible local or general evolutions of pathology were considered as : no evidence of residual / local recurrence of disease ( ca ) ; evidence of residual local disease ( partial ablation , pa ) ; evidence of local recurrence of disease ( lr ) ; death . in particular , the ct appearance of ca was assessed by applying the ve possible patterns indicated in a recent classication by palussiere et al . 
 [ 8 ] ( atelectasis , cavitation , disappearance , nodule and brosis )  . based on these parameters , it was possible to calculate : local effectiveness ( ca versus pa ) at the rst ct scan ; follow - up ( range 1115 months ; local effectiveness at mean 23 months ) ( ca versus pa ? lr ) , time to progression ( ttp ) , and mid - term survival . 
in this regard , since there was a problem of patients being affected by two different neoplasms ( nsclc and mts ) , we were unable to carry out a study of cancer - specic survival ( to be further divided , in the case of mts , according to the different primary tumours ) , because the numbers would be so small fig . 
at the follow - up ct scan , appearance of a uid collection , with a small air - uid level : the patient required prolonged hospitalisation and antibiotic therapy fig . 
at ct scan 1 month later , favourable appearance of the ablated area ( mixed nodular - atelectatic evolution ) , and regression of the pneumatocele discussion among all the rfa applications , the indication for treatment of thoracic tumours is still marginal , as attested by our experience ( only 6 % of all patients undergoing rfa at our institution , despite it receiving referrals from important regional thoracic surgery and oncology centres ) and the fact that the sir reporting standard for these procedures was only published in 2009 , while more general ones were issued in 2003 . 
furthermore , with regard to nsclc , no evidence - based approach is available to date which claries the role of various treatments in general or the specic indications for rfa [ 7 ]  . rfa is therefore suggested in patients with primary lung cancers or mts ( especially from crc ) who are considered inoperable due to medical conditions or to the unfavourable position of the tumour [ 9 , 10 ]  . 
at the rst follow - up ct scan , complete ablation is diagnosed on the basis of the absence of contrast enhancement ; a subsequent ct scan shows evidence of local progression of the tumour on the anteriormedial side of the lesion fig . 
5 bulky metastasis from humeral osteosarcoma at the apex of the left the follow - up ct scan , after multiple needle placements , an enhancing ring of viable tissue can still be seen , due to partial ablation ( pa ) lung : at regards the treatment of mts , the choice was the result of a multidisciplinary assessment and it was included in a multimodal therapeutic strategy . during the rfa sessions , all patients were treated using conscious sedation rather than general anaesthesia . 
this implies some advantages , such as the partial respiratory collaboration of the patient and the possibility of using the usual radiological roonot being able to place the patient in a prone position constitutes the main disadvantage , which can be overcome by having patients lie in the lateral position . 
in the literature , comparisons of general anaesthesia and conscious sedation during thoracic rfa did not show signicant differences in terms of feasibility , technical success and complication rate [ 11 ]  . rfa of thoracic tumours is an excellent option to prehealthy function , without sacricing lung serve fig . 
the absence of extrapulmonary metastases was a favourable predictive factor for survival at 3 years ( p = 0.0261 ) parenchyma , and it is a relatively safe procedure , with low mortality ( about 0.4 % ) [ 12 ]  . 
these data are similar to those reported by de baere [ 17 ] , who described a higher rate of local control for mts from crc compared with nsclc , renal cell carcinoma or hepatocellular carcinoma ( p = 0.023 ) ; however , multivariate analysis showed that the relative risk of local progression of a particular type of cancer was comparable to that of other histological types . 
 [ 15 ] showed a better local control in the treatment of mts from crc compared to nsclc ( and also to mts from other types of cancer ) , but without reaching a statistically signicant correlation . in our study , overall survival at 1 , 2 and 3 years after treatment was 84 , 65 and 51 % , respectively . the rst stratication curves , based on the different tumour histological types , showed no statistically signicant differences between nsclc and mts , but this nding could be affected by the relatively short average follow - up and by the limited sample size , especially for nsclc . 
the local efcacy of radiofrequency ablation was not signicant for survival at 3 years , but the observation of the two survival curves suggests an advantage for patients with maintained ca at shorter intervals our results are in line with those of the best series reported in the literature , in particular as regards the rate of major complications ( 7 % ) ; in fact , the development of a limited pneumothorax , which affects a large percentage of patients , often resolves without any medical intervention or with a simple drainage and could therefore be counted among the minor complications [ 15 ]  . in the literature , while a consensus regarding the denition of ca has been reached , there are different denitions for pa . 
when there is radiographic evidence that ca has been reached at the rst follow - up after rfa , ca shall be deemed to be maintained until the new development of viable tumour during the follow - up , in which case it is commonly called lr , even though the term local tumour progression should be preferred , since the lr is essentially a lesion not completely ablated [ 16 ]  . in our study , we used univariate analysis to evaluate possible variables that can inuence the effectiveness of local rfa . 
 [ 17 ] indicated , as favourable factors for local effectiveness , a tumour size threshold of 2 cm and the use of expandable needle electrodes ; conversely , proximity with radiol med ( 2014 ) 119 : 3340 7 . 
rose sc , dupuy de , gervais da , for the technology assessment committee of the society of interventional radiology et al ( 2009 ) research reporting standards for percutaneous thermal ablation of lung neoplasms . 
hoffmann rt , jakobs tf , lubienski a et al ( 2006 ) percutaneous radiofrequency ablation of pulmonary tumors : is there a difference between treatment under general anaesthesia and under conscious sedation ? eur j radiol 59 : 168174 12 . 
de bae`re t , palussie`re j , auperin a et al ( 2006 ) mid - term local efcacy and survival after radiofrequency ablation of lung tumors with minimum follow - up of 1 year : prospective evaluation . radiology 240 : 587589 13 . 
hiraki t , sakurai j , tsuda t et al ( 2006 ) risk factors for local progression after percutaneous radiofrequency ablation of lung tumors : evaluation based on a preliminary review of 342 tumors . cancer 107 : 28732888 16 . 
goldberg sn , grassi cj , cardella jf , for the society of interventional radiology technology assessment committee and the international working group on image - guided tumor ablation et al ( 2009 ) image - guided tumor ablation : standardization of terminology and reporting criteria . 
fernando hc , de hoyos a , landreneau rj et al ( 2005 ) radiofrequency ablation for the treatment of non - small - cell lung cancer in marginal surgical candidates . 
lencioni r , crocetti l , cioni r et al ( 2008 ) response to radiofrequency ablation of pulmonary tumors : a prospective , intentionto - treat , multicentric clinical trial ( the rapture study )  . 
remembering that diameter is one of the cornerstones of tumour staging , at least for nsclcs , the better survival rate of patients with smaller lesions is believed to be partly related to the natural history of the disease . 
to verify the real impact of the rfa on survival , we therefore stratied the patients by local effectiveness of rfa : the difference between the survival curves at 3 years of ca versus treatment failures ( pa and lr ) was not statistically signicant , consistent with what was reported until the last decade [ 23 ] ; the observation of a trend in favour of effective treatment in the rst 2 years , however , lets us hope that the clinical utility of these treatments can be demonstrated later , preferably by randomised trials . conclusions the growing experience with rfa of thoracic tumours , including our rst 100 cases , demonstrates the safety and efcacy of local treatment for small tumours ; the predictive factors for a longer survival are mainly related to staging ( tumour diameter and presence of extrapulmonary disease ) , as for the other cancer therapies . 
the accuracy , sensitivity , specicity , positive predictive value ( ppv ) , and negative predictive value ( npv ) of measures used to detect ps were determined by patient - based and aberrant systemic artery - based evaluations . 
the capability of cta to provide a working view and the accuracy of measurements in choosing a coil were also assessed . results digital subtraction angiography and / or surgery revealed ps in 38 patients , and 7 patients had no ps . 
the patient - based evaluation yielded an accuracy of 97.8 % , sensitivity of 97.4 % , specicity of 100 % , ppv of 100 % and npv of 87.5 % , in the detection of ps . 
li department of interventional radiology , the first afliated hospital , zhengzhou university , no.1 , east jian she road , zhengzhou 450052 , henan , republic of china e - mail : rjzjrk@126.com conclusions 64 - row cta appears to be effective in terms of supporting accurate diagnosis and pre - treatment planning in ps . 
cta is not only able to provide clear visualisation of aberrant systemic arteries but also provides detailed images of abnormal lung parenchyma and the airways . keywords pulmonary sequestration ( cid : 2 ) angiography ( cid : 2 ) dsa ( cid : 2 ) endovascular introduction pulmonary sequestration ( ps ) is a rare congenital pulmonary disorder dened by an area of dysplastic and nonfunctioning pulmonary tissue which has an anomalous systemic blood supply [ 1 ]  . 
in recent years , less invasive imaging techniques , particularly helical multislice computed tomographic angiography ( cta ) , have proved to be equally effective and safer alternatives to dsa [ 313 ]  . 
cta can provide the precise visualisation of the site , size and number of aberrant systemic arteries and their relationship to the aorta , which are fundamental factors that must be assessed before choosing between endovascular and neurosurgical treatments . previous reports in the literature have assessed the sensitivity and specicity of cta compared with dsa [ 3 13 ]  . 
however , these investigations have not analysed the clinical implications of a protocol that replaces dsa with cta as the only diagnostic and pre - treatment planning tool for patients with ps . 
therefore , the purpose of the current study was to evaluate the clinical implications and clinical results of a prospective protocol using 64 - row cta for the diagnosis and endovascular treatment of ps . materials and methods study design the institutional review board approved the study protocol , and patients or qualifying family members provided informed consent before participation . 
the site of origin , distribution , and course of ps were evaluated on the basis of mpr , mip , and vr by adjusting the value of translucency or slab thickness . 
conventional 2d - dsa was performed on a monoplanar unit ( axiom artis vb22n , siemens ) with a 1 , 024 9 1 , 024 matrix and a 17 9 20 cm eld of view . the access routes for angiography were the umbilical artery ( neonates ) and the femoral artery . 
this was classied as insufcient information provided , endovascular treatment probably possible or probably not possible , or endovascular treatment denitely possible , or denitely not possible . the treatment which was actually carried out was recorded in each case . 
in the case of surgery , direct conrmation was obtained by means of visualisation of the aneurysin the case of endovascular treatment , indirect conrmation was partially provided using the following two additional parameters , which were evaluated using mpr : 1 . 
the possibility of obtaining a view on cta of the aberrant systemic artery in ps in relation to the aorta that could be reproduced on the c arthis was classied as either possible or impossible . 
this parameter was classied as the correct or incorrect choice of coil . statistical analyses the categorical demographic and basic characteristic variables , expressed as numbers and percentages , were compared using the chi squared test . 
the diagnostic performance parameters of cta for the diagnosis of pulmonary sequestration compared with those of dsa [ that is , accuracy , sensitivity , specicity , positive predictive values ( ppv ) , and negative predictive values ( npv ) ] were expressed as percentages ( 95 % ci )  . 
aberrant systemic arteries originating from the thoracic artery were evident in 38 patients , and no aberrant systemic artery was exhibited in the remaining 7 patients . dsa or surgery results according to the reference standard , ps was detected in 38 patients and 7 patients had no demonstrable ps . 
dsa revealed 46 aberrant systemic arteries in 38 of the 44 patients : in 2 patients , 3 arteries were detected ; 4 patients each had 2 arteries detected ; and a single artery was detected in 32 patients . diagnostic performance of cta the diagnostic accuracy , sensitivity , specicity , ppv , and npv of cta in the patient - based and aberrant systemic artery - based evaluation on cta are detailed in table 2 . single - patient sample images acquired with cta and dsa are shown in fig . 
cta also revealed 45 aberrant systemic arteries in 38 of the 45 patients : in 1 patient , 3 arteries were detected ; 5 patients each had 2 arteries detected ; and a single artery was detected in 32 patients . 
a axial multiplanar reconstruction ( mpr ) image demonstrates a homogenous solid mass in the posterobasal segment of the right lower lobe with an aberrant systemic artery ( arrow )  . 
b coronal maximum intensity projection ( mip ) exhibits an aberrant systemic artery ( arrow ) arising from the hepatic artery supplying the mass in the right lower lobe . c pre - embolisation aortography shows the aberrant artery ( arrow ) from the hepatic artery . d post - embolisation angiography conrms complete occlusion of the aberrant artery with coils ( arrow ) aberrant systemic arteries detected in one patient was falsenegative on cta . potential for endovascular treatment in 38 patients with ps , mpr images provided sufcient information on the site , number and size of aberrant systemic arteries in 37 ( 97 % ) of 38 patients , and insufcient information in one patient with multiple aberrant systemic arteries . 
in fact , endovascular treatment was performed in 36 patients with ps , and 2 patients opted for surgery due to other complications . radiol med ( 2014 ) 119 : 2732 the surgery carried out in these patients demonstrated that cta ndings about the aberrant systemic arteries had been correct . view for performing endovascular treatment ( working view ) in 36 patients with ps where endovascular treatment was possible , mpr analysis allowed us to easily nd the working view that clearly demonstrated the location of the aberrant systemic artery and could be used to perform endovascular treatment . 
this working view was then reproduced on the c arthe resultant cta series was always similar to the dsa view and satisfactory for performing endovascular treatment . size of the aberrant systemic artery in the 36 patients with pss suitable for coil embolisation , measurements the size of the aberrant systemic artery were performed on mpr . 
the coil selected was appropriate in 35 ( 97 % ) of 36 treated patients with ps . discussion this prospective study was based on our hypothesis that cta could replace dsa as a reliable diagnostic and pretreatment planning tool for patients with ps . 
in this study , the high accuracy ( [ 95 % ) and sensitivity ( [ 95 % ) of cta for detecting ps was equivalent to the gold standard , dsa , indicating that cta cannot only replace the dsa for the diagnosis of ps but can also differentiate ps from other similar conditions . 
when compared with actual treatment decisions made using dsa or surgery , cta provided an effective diagnostic performance , delineating aberrant systemic arteries and enabling appropriate coil selection prior to embolisation in 37 of 38 patients with ps . 
this suggests the potential of cta as an effective tool which could be used alone in treatment planning for most patients with ps . pulmonary sequestration is a rare congenital malformation that represents 0.156.4 % of all pulmonary malformations [ 14 , 15 ]  . 
traditionally , the diagnosis of ps requires arteriography to identify abnormal systemic vessels feeding the abnormal portion of the lung and to provide valuable information for preoperative planning [ 2 , 16 ]  . 
recently , the quality and accuracy of cta , particularly helical multi - slice cta , has been seen to be approaching that of catheter dsa , which has been the gold standard diagnostic tool malformations . in patients with pulmonary the advantages of cta are that , compared with dsa , it is noninvasive , has a much lower risk of complication or morbidity , can be performed much more quickly , requires is not fewer resources ( stafng , equipment , and cost ) , painful to the patient , does not require sedation ( in most patients ) , and is more suitable for critically ill or unstable patients . 
conversely , while dsa can clearly identify aberrant feeding arteries in ps , it is invasive , associated with radiation , time - consuming , relatively expensive , and requires hospitalisation . although there have been many reports in the literature that assess the validity of cta as a diagnostic technique for the detection of ps , few have evaluated the clinical implications of a protocol that uses cta instead of dsa as a diagnostic and pre - treatment planning tool . 
if cta is to serve as a non - invasive replacement for dsa in pre - treatment planning , it must provide precise visualisation of the site and number of aberrant systemic arteries and their relationships to the aorta . 
in addition , it is essential to know the shape and the size of the feeding artery before performing endovascular treatment using coil embolisation . our study showed that therapeutic decisions could be based on information provided by cta alone in most cases because 64 - section cta is capable of three - dimensional reconstructions in unlimited projections . 
we found that certain reconstruction algorithms , such as vr reconstructed images and mprs , may provide useful anatomical information about the ps that closely approximates intraoperative ndings . in the investigation of a suspected case of ps , ct , and cta have two principal objectives : to rule out other pathologies and to conrm the presence of an anomalous arterial supply [ 4 , 9 , 16 ]  . 
cta is able not only to identify the systemic artery supply , but also yields the maximum information about the lung parenchyma including the airsuch as bronchiectasis , atelectasis ways . 
these are not amenable to diagnosis by colour doppler sonography , magnetic resonance imaging or dsa , whereas they can be detected with high accuracy by ct [ 17 ]  . 
in other words , cta is the sole diagnostic method which can delineate the origin and course of anomalous systemic arteries and venous drainage accurately as well as evaluating abnormal lung parenchyma and the airways . recently , endovascular coil embolisation has provided a less invasive alternative to surgery [ 1823 ] , especially for neonates or children . 
while cta facilitates a simple and reproducible angiographic protocol , dsa has been part of our angiographic protocol in undetermined cases . for endovascular treatment , one must use a twodimensional view that clearly depicts the aberrant systemic artery in relation to adjacent vessels and could be used to perform treatment in optimal conditions . 
precise measurements of the size of the aberrant systemic artery that were obtained by using mprs on cta imaging helped us in this important and difcult choice , especially in determining the coil diameter . 
last , the dose of the contrast material may be high for paediatric patients ; therefore , a more extensive evaluation of patient dose should be performed taking age into consideration . in conclusion , 64 - row cta appears to be effective in terms of supporting accurate diagnosis and pre - treatment planning in ps . 
we aimed to investigate the impact of a breathing - control system on irradiated volumes and dosimetric parameters in three - dimensional conformal radiotherapy ( 3d - crt ) and stereotactic radiotherapy ( srt ) treatments . materials and methods twelve patients were scheduled for radical radiotherapy : ve for srt and seven for 3dcrt . 
signicant differences were found both in srt and in 3d - crt , peripheral and apical lesions . conclusion in our experience , abc has the potential to reduce lung toxicity in the treatment of lung cancer ; it can allow the prescribed dose to be alternatively , increased while maintaining the same risk of lung toxicity . keywords active breathing control ( abc ) ( cid : 2 ) internal target volume ( itv ) ( cid : 2 ) lung dose ( mld ) ( cid : 2 ) radiotherapy ( cid : 2 ) target volumes introduction treating lung cancer with radiotherapy ( rt ) or radiochemotherapy ( rct ) is hindered by the tolerance of normal tissues in the thorax [ 1 ]  . 
local control is known to be correlated with improved overall survival , both in nsclc and in sclc . in conventional 3d - conformal rt ( 3d - crt ) , treatment eld sizes are usually large enough to take into account tumour motion . 
the reduction of organ motion and the correlated increased accuracy in patient positioning is under investigation in stereotactic radiotherapy ( srt ) [ 9 ]  . radiol med ( 2014 ) 119 : 1319 table 1 clinicalpathological features of the 12 lung cancer patients several methods exist to coordinate respiratory motion , including active breathing control or coordination ( abc ) [ 10 ] , real - time tumour - tracking systems [ 11 ] , respiratory gating systems , voluntary deep inspiration breath - hold ( dibh ) [ 12 , 13 ] and abdominal compression . 
the aim of this study was to evaluate the impact of abc on treatment volumes and to correlate the variations in predicting lung toxicity . materials and methods from march to october 2011 , 12 patients ( nine men and three women ) , 5 scheduled for srt and 7 for 3d - crt for lung lesions , were selected for our study . 
the active breathing coordinator device ( abc , elekta oncology systems ltd . , crawley , west sussex , uk ) consists of a mouthpiece connected to a ow meter system to measure the volume of air that moves through the transducer as a function of time , thus monitoring patient respiration . 
no breath - hold was performed , but after recording normal respiration , the patient was asked to breathe in a window as narrow as possible . this window was established in a rst training phase . 
for each patient affected by lung lesions , together with the freebreathing ct scan , four additional sets of ct slices were acquired : two in the maximum and minimum phases of a free - breathing cycle and two in the maximum and minimum phases of a breathing cycle controlled by the abc system . the tumour was outlined by only one operator . 
the freebreathing internal target volume ( itv ) [ 14 ] was created from enlargement of lesion visualised in the free - breathing ct scan with fusion images of the tumour in the maximum and minimum phases of a free - breathing cycle . 
the controlled - breathing itv was created from the sum of the tumour volume in free - breathing ct scan and maximum and minimum phases of a controlled - breathing cycle . 
in the case of 3d - crt treatment , gross tumour volume ( gtv ) for primary lesion ( gtvt ) and a gtv for metastatic lymph nodes ( gtvn ) were contoured . 
the planning target volume ( ptv ) was obtained by an isotropic automatic expansion of 1 cm to ctvt ( ptvt ) and 0.7 cm to ctvn ( ptvn )  . 
for srt , gtv was contoured on the lung window , and ptv was obtained by an isotropic automatic expansion of 1 cspinal cord , normal affected lung ( dened as normal tissue of affected lung minus ptv ) , normal unaffected lung ( dened as lung volume opposite the target lesion ) , oesophagus and heart were outlined as organs at risk . 
in particular , abc allowed for a signicant reduction in volumes in both srt and 3d - crt treatments . results all 12 selected patients were able to tolerate the abc device during the study procedures . target volumes ( itv and ptv ) figure 1 shows itv for both free and controlled breathing in all patients . 
signicant differences in v20 were found in 3dlesions crt plans ( p = 0.034 ) and in apex lesions ( p = 0.012 ) ( table 4 )  . in peripheral ( p = 0.026 ) , target figure 3 shows mld values for both free and controlled breathing in all patients . 
many discussion studies show that radiation dose and volume to lungs are associated with the risk of radiation pneumonitis , such as mld [ 17 ] , normal tissue complication probability ( ntcp ) value and relative volume of lung receiving more than a threshold dose ( v dose ) [ 18 ]  . 
in the royal marsden experience [ 20 ] , the majority of patients suitable for radical radiotherapy tolerated abc ; compliance was conrmed in our study , where all patients were able to understand the instructions and follow the procedures for the abc device . 
our data show a 1734 % reduction in v20 in both srt and 3d - crt treatments , whereas the advantage is smaller but still statistically signicant in mld reduction ( 6 and 9 % reduction srt and 3d - crt , respectively )  . 
previous studies show that lower - lobe tumours tend to move more than upper - lobe tumours during free breathing [ 21 ] : these data are apparently in contrast with our results . 
however , we believe that the greater variability in inter - breath - hold itv and the smaller number ( four vs . eight ) of lower - lobe tumours in our series can be a bias in the interpretation of our results . 
v20 is the metric or surrogate for the risk of lung toxicity [ 22 ] , and the reduction in tumour motion is a key concept that is correlated with this dosimetric parameter in the dosevolume histogram . reduced v20 can be considered a primary endpoint for obtaining a safe dose escalation in treating lung cancer . alternatively , abc may allow dose escalation while maintaining the same risk of lung toxicity [ 23 ]  . radiol med ( 2014 ) 119 : 1319 radiol med ( 2014 ) 119 : 1319 luca cardillo , g . 
lesions with a diameter b3 cm were treated with a single antenna , lesions with a diameter [ 3 cm were treated by positioning two or more antennae , simultaneously . 
all patients underwent computed tomography ( ct ) follow - up with and without contrast administration at 1 , 3 and 6 months and then yearly in combination with complete blood and metabolic tests . results technical success was 100 % . 
partial necrosis was obtained in 30.8 % ( 8 / 26 lesions ) ; in one case ( 3.8 % ) a progression of the disease was recorded and in another case ( 3.8 % ) a stability was observed . conclusions our preliminary experience may be considered in accordance with literature dates , in terms of efcacy and safety . keywords microwave ablation ( cid : 2 ) lung tumours ( cid : 2 ) technical results introduction surgery remains the optimal treatment for early - stage lung cancer , with a reported 5 - year survival rate of approximately 70 % , but the majority of patients present with advanced disease are not suitable for surgery [ 1 ]  . the lungs are also a common site of metastatic spread , and to date , the standard treatment for isolated pulmonary metastasis has been surgical resection with an overall 5 - year survival after resection ranged 2040 % [ 2 ]  . while the treatment of choice has historically been resection , newer therapies that do not require pulmonary resection are arising such as ablation techniques to enable local control of unresectable tumours . 
over the years , various ablation techniques for the treatment of lung tumours have been developed , including ethanol ablation , laser ablation , cryoablation , radiofrequency ( rf ) ablation and microwave ( mw ) ablation . radiol med ( 2014 ) 119 : 7582 mw ablation offers all the benets of rf ablation as well as some substantial advantages , such as a larger volume of cell necrosis in a shorter procedure time , a larger zone of active heating , less susceptibility to heat - sink effect and less intraprocedural pamw ablation uses dielectric hysteresis to produce heat [ 3 , 4 ]  . 
heating of the tissue is based on agitation of water molecules inducing cellular death via coagulation necrosis ; the electrical charge on the water molecule ips back and forth 25 billion times per second , depending on the frequency of the microwave energy [ 5 , 6 ]  . 
this mechanism of heating differs from rf ablation , which creates heat via resistive heating when electrical current passes through the ionic tissue medium , resulting in heating of tissues only immediately adjacent to the electrode [ 3 ]  . comparing ablation zones obtained with a 14 - gauge microwave antenna vs those obtained with a 17 - gauge radiofrequency electrode in an in vivo normal porcine lung model , that one generated with the use of microwaves was 25 % larger in mean diameter and signicantly more circular , and developed faster than those achieved with radiofrequency energy [ 6 , 7 ]  . the aim of this study was to demonstrate the technical success , safety and efcacy of mw ablation in patients affected by unresectable lung cancer . materials and methods this retrospective study was approved by the ethics committee of our centre . 
twenty - four patients ( 15 men , 9 women ) with a mean age of 71.7 years ( range 4683 ) who underwent percutaneous mw ablation of 26 intraparenchymal pulmonary masses ( mean 1.08 lesions per patient ) in 36 ablation sessions between 26 november 2007 and 12 august 2011 were enroled in the study . a central tumour was dened as being entirely located within the inner two - thirds of the lung . 
a tumour was dened as peripheral if a portion of it resided in the outer one - third of the lung . all selected patients had neoplastic disease that was judged to be inoperable on the basis of tumour stage , comorbidities , advanced age and / or refusal to undergo surgery . 
the coagulation prole was in the normal range for all patients . patients undergoing anticoagulant and / or antiaggregant therapy interrupted treatment at least 7 days before the procedure , with the introduction of fractionated heparin when necessary . 
written informed consent was obtained from all patients . baseline imaging pre - treatment imaging consisted of a 64 multidetector - row computed tomography ( mdct ) chest scan extending to the abdomen ( aquilion 64 , toshiba , japan ) with and without contrast medium administration . 
the contrast - enhanced scans were acquired after injection of 100 ml of iodinated contrast agent ( visipaque 320 , ge healthcare , usa ) at an injection rate of 3 ml / s , followed by injection of 40 ml saline at a rate of 2 ml / s . 
one week prior to the ablation treatment , all patients underwent percutaneous pulmonary biopsy under computed tomography ( ct ) guidance , with histological conrmation of malignancy for all lesions . pre - treatment procedure the patient is in supine or prone position according to the site of the lesion . 
each patient is kept in a state of moderate sedation through intravenous administration of a combination of midazolam ( ipnovel 15 ; roche , milan , italy ) ( 0.070.08 mg / kg ) , propofol ( diprivan , astrazeneca spa . , caponago , italy ) ( 0.52.0 mg / kg / h ) and fentanyl ( fentanest ; pharmacia & upjohn , milan , italy ) ( 12 lg / kg )  . heart rate , electrocardiographic trace , oxygen saturation , respiratory frequency and blood pressure are continuously monitored throughout the procedure . 
at the end of the procedure , a ct scan or a cbct was performed to evaluate the presence of pneumothorax ; if present , the patients underwent chest radiography 2 h later to monitor the pneumothorax . follow - up yearly in combination with complete blood and metabolic tests . the following imaging characteristics were evaluated : total volume of the ablated area , presence of cavitation , absent enhancement of the lesion ( \15 hu ) , pleural effusion and adenopathy ( short axis [ 1 cm ) , according to the response evaluation criteria in solid tumours ( recist ) [ 3 , 4 , 8 ]  . all complications were recorded and classied in accordance with the common terminology criteria for adverse events version 4.0 ( ctcae ) [ 9 ]  . outcomes : technical success , safety and efcacy of the technique all patients underwent ct follow - up with and without contrast administration at 1 , 3 and 6 months and technical success was dened as the correct positioning of the antennae within the lesion . 
the efcacy of the technique was evaluated on the basis of imaging characteristics , using the recist criteria [ 8 , 11 ]  . efcacy statistical analysis overall survival ( os ) was estimated using the kaplan meier method [ 12 ]  . 
a wilcoxon matched - pairs test was used to compare the size of the tumours before and after ablation using the medcalc 12.0 statistical package ( medcalc software , broekstraat 52 , 9030 mariakerke , belgium )  . 
a difference with p value \0.05 was considered statistically signicant . results tumours were measured in the three dimensions ( transverse , anteriorposterior and longitudinal ) obtaining a mean index of the maximum diameter of 30.96 mm ( range 8100 mm )  . 
histological analysis showed that the lesions were : 14 non - small cell lung carcinoma ( nsclc ) ( 6 squamous cell pulmonary carcinomas , 7 adenocarcinomas and 1 neuroendocrine carcinoma ) , 11 metastasis ( from primary tumours located elsewhere ) and 1 microcytoma . safety no major complications were recorded in the intraand peri - procedural period . 
there were no cases of symptomatic grade - 2 pneumothorax , nor were there any cases of skin burns induced by microwave - related thermal damage [ 13 , 14 ]  . 
additional ct scans were performed at intervals of 1 ( all patients ) , 3 ( 23 / 24 patients ) , 6 months ( 6 / 24 patients ) and yearly ( 11 / 24 patients )  . after an initial increase of the maximum diameters , there was a persistent reduction in diameter of the ablated areas at subsequent examinations , consistent with consolidation of the pulmonary parenchyma . 
no statistically signicant correlation ( p \ 0.1 ) was found between tumour size ( \ or c3 cm ) , location ( central or peripheral ) , and type ( primary tumour or metastasis ) and os . 
c ct scan reveals cavitation ( star ) of the lesion after treatment mw ablation is a relatively new technique that can be applied to different types of tumour and offers all the benets of rf ablation as well as some substantial advantages . 
 [ 14 ] showed that , with a microwave ablation system tuned for the lung , large circular ablation zones in porcine lungs in vivo could be obtained with a single antenna . 
coagulation zones created with bronchial occlusion and multiple antennas placed and activated simultaneously were signicantly larger than those seen without bronchial occlusion and with a single antenna , respectively [ 7 , 14 ]  . 
the electromagnetic waves associated with mw are not limited by lower thermal conductivity of lung , or by the increased impedance of charred tissues [ 7 , 16 ]  . 
 [ 13 ] percutaneously treated 82 lung masses ( primary lung tumours and metastatic disease ) in 50 patients , using a 915 - mhz generator ( evident / covidien , boulder , co , usa ) and obtained a 67 % local control rate ( based on serial follow - up imaging with ct and positron emission tomography ) at 1 year , with a mean time to rst they observed an actuarial recurrence of 16.2 months ; survival of 65 % at 1 year , 55 % at 2 years , and 45 % at 3 years from ablation . 
cancer - specic mortality yielded a 1 - year survival of 83 % , a 2 - year survival of 73 % , and a 3 - year survival of 61 % ; these values were not signicantly affected by an index size of larger than 3 cm or 3 cm or smaller or presence of residual disease . 
pneumothorax was observed in 39 % of cases but 66 % of them were not drained . thermal ablation incites an inammatory / cytotoxic response in adjacent normal lung tissue that appears as ground - glass opacity or frank consolidation that initially increases the apparent size of the tumour . 
in fact , the size of the ground - glass opacity surrounding the ablated lesion on immediate post - ablation imaging has been shown to be predictive of an effective ablation . 
cavitation has been reported in up to 30 % of patients following mw and rf ablation and is associated with effective ablation and further complicates interpretation of post - ablation imaging [ 10 , 11 ]  . 
given the apparent increase in lesion volume following ablation , the authors follow - up protocol includes a post - ablation ct scan within 1 month of the procedure ( to serve as a new baseline ) and then contrast - enhanced ct at 3 - month intervals to determine treatment response . 
tumour morphology , volume , and contrast enhancement can be followed over time , and the authors consider tumours that are stable or decreasing in size and do not demonstrate more than 10 hu of contrast enhancement to be completely ablated [ 19 , 20 ]  . radiol med ( 2014 ) 119 : 7582 fig . 
f ct scan performed after the 2nd session of microwave ablation reveals complete necrosis in the recent and largest study to date on the mid - term outcomes of lung metastasis treated with mw ablation ( 130 lesions in 90 patients ) , vogl et al . 
 [ 21 ] showed successful tumour ablation was signicantly more frequent for lesions with a maximal axial diameter of 3 cm or smaller than for larger lesions and for peripheral lesions than for centrally located lesions . 
our results showed an os after mw ablation of 75 % after 12 months and 55 % after 24 months ; the overall 30 - day mortality rate was 0 % . 
3 overall survival ( os ) after microwave ablation was 75 % after 12 months and 55 % after 24 months radiol med ( 2014 ) 119 : 7582 fig . 
4 multiple logistic regression analysis ( p \ 0.02 ) showed a trend towards improvement of os in patients with metastasis ( a ) , with a diameter \3 cm ( b ) , and located peripherally ( c ) ( m metastasis , pr primary tumour , c central lesion , p peripheral lesion ) ( primary tumours and metastasis ) , our results may be considered to be in agreement with those of vogl [ 21 ]  . moreover , vogl et al . [ 21 ] observed a volumetric increase in size of the ablated metastases in all lesions at the 24 - hour follow - up study , attributed to the inammatory response and to the oedema created by the heat energy applied during the ablation process itself . 
the authors suggest the 3 - month follow - up study theoretically shows the true tumour volume after complete subsidence of the ablation - associated inammation response . in our experience as well there was a volumetric increase of the lesions after ablation . that positron emission tomography ( pet ) - ct has also been employed after ablation , and it can be advantageous as it is not limited to an anatomical evaluation . 
fluorodeoxy - glucose ( fdg ) uptake should decrease in tumours as early as 2 months following ablation , and nodules with a percentage reduction in fdg uptake \60 , or an absolute suv of 3.0 or greater at 2 months should be considered incompletely ablated and possibly requiring further treatment [ 20 , 22 ]  . 
given the above , we could hypothesise an important role for pet - ct in following up pulmonary ablations during the rst 3 months , to be followed by ct after the rst 3 months . a median 28 % ( range 4.561.1 % ) was reported for rf ablation in a recent metaanalysis with approximately 11 % ( range 3.338.9 % ) of these requiring chest tube placement [ 23 ]  . 
however , differential diagnosis is not always straightforward and the diagnosis of granulomatous disease , considering separately the clinical , radiological and pathological aspects , is at times incomplete or uncertain and requires multiin this paper , we propose a disciplinary assessment . combined hrct - pathological approach to assess both the topographical and morphological features of the lesions . based on topography , we can distinguish between granulomatous lesions distributed along the lymphatic vessels , with random distribution or centred on the airways . 
barozzi servizio di radiologia , ospedale bellaria maggiore , ausl di bologna , bologna , italy distribution of granulomas are those with haematogenous spread , the most typical of which is miliary tuberculosis ( tb )  . 
the anatomical approach is completed by the assessment of the morphological aspects of the lesions and associated signs , reecting both the possible mechanisms of spread and the different types of pathological and / or reparative tissue related to the disease . keywords granulomatous lung disease ( cid : 2 ) sarcoidosis ( cid : 2 ) tuberculosis ( cid : 2 ) hypersensitivity pneumonitis ( cid : 2 ) hrct introduction granulomatous lung diseases form a heterogeneous group of clinical entities which share the presence of granulomas presenting in the form of nodular clusters of macrophages , usually with epithelioid appearance , possibly associated with giant cells as a manifestation of t immunity ( cellmediated )  . 
they represent a frequent nding for pathologists and may be seen in the lung in a number of conditions , more commonly diffuse [ 1 ]  . there are several granulomatous lung diseases which have been classied according to different criteria . 
the most common classication is based on aetiology , with the diseases being divided into infectious and noninfectious ( table 1 ) ; this criterion is crucial for the implications on frequent and management and treatment . 
granulomas may be distributed along the lymphatics , randomly or along the small airways [ 3 , 4 ]  . the topographical approach is completed with the evaluation of lesions and a description of associated lesions , which are sometimes predominant and valuable to the diagnosis of these diseases . the morphological aspects of parasitic infections sarcoidosis dirolaria intravenous or inhalational talcosis vasculitis ( wegeners granulomatosis , churgstrauss syndrome ) lymphatic distribution the clinical diagnostic approach to this wide spectrum of diseases always includes imaging and in particular computed tomography ( ct )  . 
high - resolution ct ( hrct ) is the most commonly used investigation because granulomatous lung diseases are often diffused diseases and hrct provides a precise in vivo depiction , with accurate demonstration of their distribution and morphological features . 
less commonly , adenopathy affects internal mammary , paraverteberal perilymphatic distribution with a predilection for the upper lung zones , together with typically located adenopathies , is highly suggestive of sarcoidosis and helps to differentiate sarcoidosis from other granulomatous or nongranulomatous conditions with lymphatic distribution such as carcinomatous lymphangitis and lymphoproliferative disorders . 
the radiological differential diagnosis with berylliosis and silicosis may be impossible . berylliosis chronic berylliosis is caused by prolonged inhalation of dusts or fumes containing beryllium - based compounds or products . 
the pathological and hrct patterns are similar to those of sarcoidosis ; the differential diagnosis requires a history of exposure to beryllium or beryllium compounds [ 3 , 17 ]  . lymphocytic interstitial pneumonia ( lip ) granulomas are commonly present in lymphocytic interstitial pneumonia , even though this entity cannot be formally considered a granulomatous disease , as it is classied fig . 
the ct image with mediastinal window shows symmetrically enlarged bilateral hilar and subcarinal lymph nodes with amorphous calcications inside ( b ) radiol med ( 2014 ) 119 : 5463 fig . 
in over 50 % of the cases granulomas are scattered , small and located along the lymphatics or in the peribronchiolar interstitium [ 18 ]  . on hrct , the subpleural nodules are dense , with sharp margins and often associated with thin - walled cysts . 
they are the result of air trapping due to peribronchiolar lymphoid inltration [ 19 ]  . random distribution in the random distribution pattern , granulomas appear uniformly distributed within the secondary lobule and lung parenchyma . 
similarly to lymphatic nodules , random nodules show high density and sharp margins ( like opaque beads ) as they are interstitially located ; they are often small ( miliary ) and may be cavitated , as a result of necrosis suggestive of an infectious process . the prototype of random granulomatous lung disease is including miliary tb . 
even haematogenous metastases can mimic haematogenous granulomatous diseases . miliary infections ( tb and fungal infections ) infections , in particular those due to mycobacteria and fungi ( including pneumocystis jiroveci ) , are still considered the most common cause of lung granulomas so that the detection of granulomas should suggest a thorough search for microorganisms . 
the attempt to isolate the micro - organisms starts with the rst examination of the slide with haematoxylin eosin and proceeds with special stains and in particular ziehlneelsen ( for tb ) and grocott ( for fungi ) stains . 
4 miliary infection ( tb )  . the hrct sagittal view shows innumerable tiny nodules quite uniformly scattered throughout both lungs with a random distribution , only some of them touching the pleura ( a )  . 
5 airway - centered ( centrilobular ) soft nodules appear both pathologically ( a ) and radiologically ( b ) separated from the pleural surfaces and ssures by several millimetres ( pavid of pleura ) disease with nodular pattern is extrinsic allergic alveolitis , also called hypersensitivity pneumonitis . 
the branching opacities ( tree ) reect the presence of dilated and thickened bronchioles , whereas the nodular opacities ( buds ) represent clusters of partially or completely lled alveoli ( centrilobular nodules )  . 
these changes may have an acute or chronic infectious nature ( often in mycobacteriosis , especially when atypical ) or noninfectious nature ( allergic bronchopulmonary aspergillosis , aspiration syndrome , bronchiolitis associated with chronic intestinal infections , bronchocentric granulomatosis , hot tub lung and inhalation talcosis ) ( table 1 )  . 
the tree - in - bud sign , however , may also be present in other nongranulomatous conditions ( diffuse panbronchiolitis , follicular bronchiolitis , some cases of organising bronchiolitis , neoplasms and the so - called vascular tree in bud in thrombotic microangiopathy due to haematogenous micrometastasis ) [ 23 ]  . extrinsic allergic alveolitis ( hypersensitivity pneumonitis ) extrinsic allergic alveolitis ( also known as hypersensitivity pneumonitis ) in its subacute form is pathologically characterised by the presence of interstitial pneumonia , foci of bronchiolitis and small , poorly formed interstitial granulomas centred along the small airways [ 24 ]  . 
the branching opacities ( the tree ) reect the presence of dilated thickened bronchioles , whereas the nodular opacities ( the buds ) are due to clusters of lled alveoli ( centrilobular nodules ) fig . 
the ct image depicts ill - dened lowdensity centrilobular nodules ( snowake - like ) ; the nodules are diffusely and uniformly distributed through both lungs ( a )  . 
in approximately 80 % of the patients , there is also a diffuse ground - glass opacity secondary to extensive diffuse interstitial pneumonia . the possible combination of ground - glass opacity , reduced pulmonary attenuation and areas of normal parenchyma may give rise to an unusual appearance dened as head - cheese pattern , which is not pathognomonic but only suggestive of the disease in patients with a history of allergy [ 25 , 26 ]  . hot tub lung hot tub lung is caused by exposure to water contaminated by nontuberculous mycobacteria ( mycobacterium avium complex , mac )  . 
pathologically , this form presents several similarities with hypersensitivity pneumonitis ( interstitial inltrate , bronchiolar - centred but also random granulomas which are rarely necrotising ) [ 27 ]  . 
consistent with the pathological features , the hrct signs are similar to those seen in extrinsic allergic alveolitis and characterised by low - density centrilobular nodules , ground - glass opacities and possible lobular air trapping [ 28 ]  . mycobacterial infection the histological features of mycobacteriosis are several necrotising or non - necrotising granulomas distributed along the airways ( bronchi and bronchioles )  . 
it is often impossible to distinguish tuberculous from nontuberculous mycobacteria on the basis of morphology alone ; the differential diagnosis therefore relies on special stains , cultures or molecular biology techniques [ 21 , 29 ]  . radiologically , airway involvement manifests through tree - in - bud appearance resulting from granulomatous cellular bronchiolitis and bronchial wall thickening with or without bronchiectases , dense nodules and bronchocentric consolidations , at times cavitated due to necrosis . 
the nonclassic variant of atypical mycobacterioses is predominantly in the middle lobe and lingula in the form of bronchiectases , bronchiolectases and centrilobular nodules associated with mosaic hypoperfusion due to oblitrative bronchiolitis . 
in the classic form , the ct coronal view shows tree - in - bud pattern and sporadic airway - centred nodules in the upper lobes ( a )  . in another female patient with nonclassic form ( lady windermere syndrome ) , bronchiectases are prevalent in the middle and lingular lobes ; mosaic hypoperfusion also coexists in the lower lobes ( b ) fig . 
there may also be consolidations , at times transient [ 32 ]  . chronic aspiration syndrome aspiration often concerns particles , such as food remnants ( especially vegetables ) , pill fragments , excipients , drugs or lipids . 
alcoholism is probably the most exogenous important predisposing factor in adults , even though other factors ( for example , structural abnormalities of the pharynx or oesophagus , neuromuscular disorders and deglutition abnormalities ) may also play a role in chronic aspiration . 
in the chronic form , ct may show a typical pattern consisting of fatty density in the context of parenchymal consolidations / nodules / masses , and often symmetrically which are bronchocentric associated with inammatory radiol med ( 2014 ) 119 : 5463 fig . 
bilateral gravitational areas with bronchial thickening and minimal tree - in - bud pattern ( a )  . ct scan with mediastinal window demonstrates oesophageal dilatation with an air - uid level ( b ) arranged in the posterior - basal segments . 
the presence of fatty attenuation values is noted in 7080 % of patients ; in the others , the radiological ndings are nonspecic [ 36 , 37 ]  . granulomatous vasculitides ( churgstrauss , wegener ) the granulomatous forms of vasculitis are characterised by an inammatory process affecting the vessel wall as a main feature ; rarely they manifest pathologically as a granulomatous interstitial pneumonia . churgstrauss syndrome ( css ) churgstrauss syndrome ( css ) , also known as allergic granulomatous angiitis , is a rare multisystem disease characterised by chronic rhinosinusitis , asthma and prominent eosinophilia in peripheral blood . 
the granulomas are well formed , necrotising , rich in eosinophils , and readily identied both in the vessel walls and around the bronchioles [ 38 , 39 ]  . radiologically , the most common manifestations are bilateral consolidations and ground - glass opacities which may be migratory and peripheral without predilection for site , a pathological expression of inammatory eosinophil or granulomatous involvement . 
in some cases , there may be associated mediastinal lymphoadenopathy due to reactive hyperplasia and eosinophil inltration [ 39 , 40 ]  . wegeners granulomatosis ( wg ) wegeners granulomatosis ( wg ) is a systemic necrotising vasculitis , which manifests with the classic triad of involvement of the upper and lower airways and kidney . the most common pathological nding is a combination of vasculitis and necrotising granulomatous inammation in a background of intense lymphoplasmacytic inltrate . 
the necrosis consists of large geographic areas and typically appears basophilic ( blue ) at low power [ 4 , 38 ]  . radiologically , the necrosis may appear in form of cavitation in the context of unior bilateral nodules or masses , which are multiple in about 85 % of the cases . these manifestations are often the initial signs of pulmonary disease . 
the alveolar haemorrhage may be diffuse in about 10 % of patients as a result of necrotising capillaritis , and present on ct as diffuse areas of parenchymal consolidation , ground - glass opacities and scattered low - density nodules ( 2550 % of cases )  . 
involvement of the airways is a late complication and may be present in 1525 % of patients . tracheal stenosis most frequently involves the subglottic portion ; it may be smooth or irregular [ 42 ]  . conclusions the heterogeneity of pulmonary granulomatous diseases may present to the clinician with patterns that are difcult to diagnose . 
correct diagnosis and treatment often relies on an interdisciplinary approach including clinical , imaging and pathological aspects . a combined pathological - hrct approach can help to understand and diagnose these diseases , similar to what has radiol med ( 2014 ) 119 : 5463 been demonstrated by other authors with regard to interstitial lung diseases . 
radiodiagnostica , universita` degli studi di bari , bari , italy 18fdg - pet / ct is useful in evaluating the conclusions extent of sarcoidosis and recognising lesions at different sites , including lymph nodes , lungs , liver , spleen and bone . it also improves the interpretation of the morphological lesions seen on mdct and depicts a larger number of lesions . 
therefore , 18fdg - pet / ct could be used to complement other more traditional techniques for the restaging and follow - up in patients with sarcoidosis . keywords sarcoidosis ( cid : 2 ) 18fdg ( cid : 2 ) pet / ct ( cid : 2 ) mdct ( cid : 2 ) follow - up introduction first described by hutchinson in 1877 [ 1 , 2 ] , sarcoidosis is a chronic , systemic , idiopathic disease ; the pathological hallmark of which is non - caseating granuloma characterised by macrophages surrounded by a rim of mononuclear cells . 
although sarcoidosis may involve any organ or system , it most commonly affects the chest structures and the upper respiratory tract , such that the lungs and mediastinal lymph nodes are almost invariably affected , even when the patient is asymptomatic [ 1 , 3 ]  . 
extrathoracic manifestations , usually associated with thoracic involvement , are seen in 2550 % of cases [ 1 ] and are represented by cervical , axillary and inguinal lymphadenopathies in one - third of patients with sarcoidosis . sarcoidosis has a chronic - progressive course in 30 % of cases , whereas 4090 % of patients , depending on the series , show spontaneous regression . 
in 15 % of cases , cardiorespiratory complications may lead to death , whereas a subtle and non - specic onset , associated with different possible localisations , often underlies the failure in obtaining an early diagnosis [ 17 ]  . radiol med ( 2014 ) 119 : 6474 in the early stages of disease , the diagnosis requires biopsy , whereas diagnostic imaging and in particular chest radiography ( cxr ) and computed tomography ( ct ) can provide highly suggestive , though not pathognomonic , ndings and represent a crucial tool for staging the disease , planning treatment and predicting the prognosis [ 8 , 9 ]  . numerous retrospective studies since 1990s have dened the role of cxr , ct , high - resolution ct ( hrct ) and magnetic resonance ( mr ) imaging in chest sarcoidosis and in the assessment of extrathoracic lesions [ 10 , 11 ]  . 
in recent years , the potential of diagnostic imaging in the study of this disease has expanded with the 18uorodeoxyglucose positron - emission introduction of tomography / ct ( 18fdg - pet / ct )  . 
some authors have proposed 18fdg - pet / ct for the differential diagnosis of solid lesions of the chest and for evaluating inammation and response to corticosteroids in patients with sarcoidosis [ 4 , 14 ]  . the published studies about thoracic and systemic sarcoidosis are retrospective and conducted on small series and heterogeneous groups of patients without precise inclusion criteria [ 13 , 1522 ] , and the usefulness of wholebody 18fdg - pet / ct in the diagnosis and follow - up of patients with thoracic and systemic sarcoidosis has not yet been completely dened . 
multidetector ct ( mdct ) is currently the standard of reference for the diagnosis and follow - up of this disease , but no comparative study with 18fdg - pet / ct for the restaging and follow - up of sarcoidosis has been conducted to date . the aim of this study was to evaluate , through a retrospective study of a rigorously selected and homogeneous clinical series , the role of 18fdg - pet / ct in restaging and follow - up of patients with histologically proven sarcoidosis . 
the results obtained with 18fdg - pet / ct were compared with those of mdct in the management of patients with thoracic and systemic sarcoidosis . materials and methods we carried out a retrospective evaluation of 39 patients with histologically conrmed sarcoidosis referred to us for clinical and radiological assessment for suspected relapse or disease follow - up over a period of 49 months ( from september 2006 to march 2010 )  . 
from the initial group of 39 patients , 21 patients were selected who met the following inclusion criteria : histologically proven diagnosis of sarcoidosis ; stage ii and iii sarcoidosis ; 18fdg - pet / ct performed for restaging and follow - up of sarcoidosis ; time interval between mdct and 18fdg - pet / ct not exceeding 40 days . the group of 21 patients included 16 women and ve men aged 2680 years ( mean 57.28 years ) who had , at the time of 18fdg - pet / ct , histological evidence of non - caseating granuloma ( sarcoid ) on a biopsy obtained from at least one lesion site ( table 1 )  . 
all cases were assessed for clinical course [ symptoms , serum angiotensin - converting enzyme ( ace ) , kveim reaction ] and morphological imaging ndings ( cxr , ct and mri ) useful for the follow - up of sarcoidosis . 
at the time of the 18fdg - pet / ct study , all patients had given their consent for their demographic , clinical and diagnostic data to be used in clinical research . 
all patients had suspended all forms of therapy at least 30 days before both mdct and 18fdgpet / ct . ct technique ct studies were performed with a 16 - slice mdct scanner ( tsx - 101 , aquilion 16 , toshiba medical system , tokyo , japan ) , using the following acquisition parameters : slice thickness 1 mm , pitch 1.75 , table feed 0.6 mm , gantry rotation time 0.5 s , kv / mas 120 / 250 . 
all patients were administered 1.5 cc / kg contrast medium through an antecubital vein at a ow rate of 3.55 cc / s , with postcontrast acquisitions starting 60 s after injection . the beginning of 18fdg - pet / ct technique pet - ct images were acquired with a combined pet - ct scanner ( ge , discovery lsa ) incorporating a pet scanner ( advance nxi ) and a 16 - slice ct system ( light speed plus )  . 
prior to the administration of uorine - 18 - fdg , all patients , who had fasted for at least 8 h , underwent capillary glucose sampling ( \160 mg / ml ) and , to avoid artefacts due to muscle activity , they were instructed not to do any physical activity prior to the scan . image acquisition was started approximately 50 min after the intravenous injection of 37 mbq / 10 kg of 18fdg . during this time all patients were instructed to drink at least 500 ml of water and to void their bladders as needed . table 1 characteristics of patients considered patient sex age symptoms other imaging studies biopsy site radiol med ( 2014 ) 119 : 6474 history tumour treatment for sarcoidosis ( cortisone ) ( years ) respiratory respiratory , evening fever , night sweats and weight loss anorexia , vomiting , slight evening fever anorexia , vomiting magnetic resonance imaging of brain : multiple lesions in both cerebral hemispheres and hypophyseal pedicle ultrasound of neck and abdomen : bilateral latero - cervical and retroperitoneal lymphadenopathies , advanced chronic liver disease , splenomegaly normal 67 ga - scintigraphy scan 18fdg pet / ct with pathological uptake at liver hilum , celiacmesenteric , para - aortic and inguinal regions bilaterally , hilar - mediastinal , right latero - cervical , left retroclavicular , right diaphragmatic pillar and right subphrenic peritoneal and pelvic regions mediastinal lymph node latero - cervical lymph node bone latero - cervical lymph node latero - cervical lymph node latero - cervical lymph node mediastinal lymph node liver latero - cervical lymph node mediastinal lymph node lung node lung lung mediastinal lymph node lung inguinal lymph node mediastinal lymph node lung mediastinal lymph node lung respiratory whole - body 67 ga scintigraphy : axillary lymph tracer uptake in the lungs and hilarperihilar and mediastinal region respiratory respiratory radiol med ( 2014 ) 119 : 6474 no muscle relaxants were administered . 
the low - dose ct ( ldct ) acquisition parameters : 340 ma ( auto ) , 120 kv ; slice thickness , 3.75 mm ; gantry rotation time , 0.8 ms ; eld of view ( fov ) collimation , 50 cct images were reconstructed with ltered backprojection . 
pet acquisition was obtained in the caudo - cranial direction , via three - dimensional interactive technique reconstruction . acquisition with before injecting the fdg , patients to take their medical histories , view previous diagnostic examinations and to provide information about the examination procedure ( informed consent )  . the physicians met image analysis all images obtained with the two modalities ( mdct and 18fdg - pet / ct ) were evaluated independently by two mixed groups of radiologists and nuclear medicine specialists with similar levels of experience ; the readers in the two groups were not aware of the patients clinical history , but only of the diagnostic suspicion . 
in case of doubt , the 18fdg - pet / ct images were reviewed by consensus reading . mdct images were assessed for the presence , distribution and predominance of radiological evidence of sarcoidosis in all body regions [ 6 ]  . 18fdg - pet / ct images were visualised in the three orthogonal planes , as pet images , ct images and fusion images . 
pet / ct images , used for the detection of lesion site and type and for tracer uptake ( focal vs diffuse ) , were evaluated both separately and as fusion images . moreover , as a quantitative index of 18fdg uptake , we determined the body - weighted standard uptake value ( suvbw ) , expressed in g / ml , using commercial software that calculates it as the ratio between 18fdg ( mbq / ml ) in a region of interest ( drawn on the attenuation - corrected images ) and the administered activity ( mbq ) per body weight ( kg )  . data analysis for all patients without signs of relapse or progression of disease , the longest available follow - up , always greater than 12 months , was taken as the reference standard . 
patients were considered false positive if , despite being positive on imaging , they did not show signs of relapse during the follow - up . patients were considered true negative if they had negative imaging results conrmed by an absence of relapse on follow - up . 
false - negative patients had clinical evidence of disease relapse or progression at follow - up , but negative results on imaging . statistical analysis sensitivity , specicity and accuracy of both pet / ct and mdct were evaluated in relation to the follow - up , taken as the standard of reference . 
the results of the comparison between mdct and 18fdg - pet / ct with follow - up are given in table 2 , which provides the sensitivity , specicity and accuracy both for the entire group of 21 patients and for the subgroup of 16 patients who underwent whole - body mdct . 
the disease most commonly occurs between the ages of 20 and 50 years , and the incidence is slightly higher in women than in men [ 6 ]  . the clinical presentation is highly variable and nonspecic , and instrumental diagnosis , consisting of standard blood chemistry tests ( including ace ) , cxr and chest ct does not permit a denitive diagnosis . radiol med ( 2014 ) 119 : 6474 fig . 
1 whole - body 18fdgpet / ct ( 18uorodeoxyglucose positron - emission tomography / computed tomography ) maximum intensity projection ( mip ) ( a ) and pet / ct coronal fusion image ( b ) , multidetector ct ( mdct ) ( c ) and pet / ct fusion image of the neck ( c , d ) , chest ( e , f ) and abdomen ( g , h ) trans - axial sections of a patient with systemic sarcoidosis diagnosed by biopsy of the latero - cervical lymph nodes . 
both techniques revealed multiple lesions of the supraclavicular and thoracic lymph nodes , lungs , abdominal lymph nodes , liver and spleen ct is the most commonly used technique , and to date the standard of reference ; in routine clinical practice the examination is limited to the chest without whole - body imaging . 
when positive , the results of diagnostic imaging are fundamental for choosing the biopsy site and evaluating disease extent . in the past , lung and whole - body 67 ga scintigraphy has been used to identify thoracic sarcoidosis , including cardiac localisations , and lesions in other body regions [ 21 ]  . 
metabolic imaging has also been proposed for distinguishing active lesions from the nal stage of irreversible brosis . 18fdg - pet / ct has recently been proposed for differentiating solid lesions of the chest seen on ct into malignant and benign based on the 18fdg metabolism [ 24 ]  . 
the possibility of identifying active sites of sarcoidosis is based on the capability of 18fdg to accumulate within the sarcoid granuloma characterised by the presence of a rich inltrate of lymphocytes and macrophages [ 25 , 26 ]  . 
all selected patients had stage ii and table 3 comparison of pet / ct and mdct by lesion site iii sarcoidosis , in which active lesions are present but there is no end - stage irreversible brosis . unlike previous reported series , the population included in our study was a selected and homogeneous group of patients , all undergoing follow - up and with a histological diagnosis of sarcoidosis . 
our study evaluated the possibility of detecting sites of sarcoid lesions with 18fdg - pet / ct and compared the data with mdct , which is normally performed as the examination of reference in the standard management of these patients . 
in particular , in patients with sarcoidosis mdct can reveal enlarged lymph nodes and pulmonary inltrates with predominantly perihilar distribution , even though in cases of negative mdct ndings for thoracic or mediastinal lesions there was histopathological evidence of disease on transbronchial biopsy samples [ 11 ]  . 
it should also be noted that , despite its panoramic capabilities , mdct is very often used for regional imaging of the chest , which explains the smaller number of extrathoracic localisations detected . 18fdg - pet / ct is a whole - body examination , which always allows the exploration of the entire body including the brain , with the possibility of detecting all sites of disease and affected organs [ 27 ]  . 
its use has been proposed not only for staging but also for identifying occult sites of disease to be sampled for histological examination [ 28 ]  . ours was a retrospective study that considered the standard clinical use of two imaging modalitiesthe more commonly used mdct with 18fdg - pet / ct performed as a complementary examinationto evaluate their current role in the restaging and follow - up of sarcoidosis . 
the two modalities revealed lymph nodes in the superior mediastinuwhole - body pet / ct also showed 18fdg uptake in the supraclavicular , axillary and abdominal lymph nodes and in the spleen . 
pet / ct revealed a greater extent of disease whole - body imaging techniques are difcult to organise in the context of diagnostic imaging with ionising radiation . while allowing a rigorous comparison by site of disease , they do not reect real clinical practice . in our study , one patient with negative whole - body mdct was found to be positive on 18fdg - pet / ct and showed disease relapse during the follow - up ( true positive )  . 
nine patients with positive mdct ndings showed a greater number of lesion sites on 18fdg - pet / ct ; eight of these nine patients had disease relapse at follow - up ( true positive )  . 
mdct depicts the morphological changes of active or acute inammation and end - stage brosis , whereas 18fdg - pet / ct describes only the inammation of the active stage , and the patients included in our study were in stage ii or iii . frequent the mdct follow - up studies in patients with pulmonary sarcoidosis demonstrated that nodular opacities are the most ( 80100 % ) and potentially reversible lesions , whereas cystic airspaces and architectural distortion are usually irreversible regardless of treatment [ 30 32 ]  . 
findings of ground - glass and linear opacities are considered both reversible and irreversible [ 30 , 31 , 33 , 34 ]  . 18fdg - pet / ct showed 17 thoracic localisations compared to the 23 detected by mdct , with moderate concordance for neck lymph nodes and fair concordance for mediastinal , hilar and pulmonary nodes , whereas there was no concordance for bony lesions of the cervico - thoracic region ( table 3 )  . parenchymal pulmonary lesions were detected with a fair level of concordance between the two techniques ( one out of six patients was false positive at 18fdg - pet / ct , two out of eight patients were false positive at mdct )  . in sarcoidosis , asymptomatic involvement of the liver and spleen occurs in respectively 4080 and 3877 % of patients , but organ - related symptoms are rare ( 26 % ) [ 6 ]  . in our study , there was no concordance between the two modalities for liver localisations , and poor concordance for splenic lesions . 
the literature reports that ct scans of the abdomen in addition to the chest are used to assess intraabdominal extent of disease and response to therapy . abdominal lymphadenopathy is the most frequent nding in 76 % of cases , the most common sites being the portal vein ( 86 % ) , the para - aortic region ( 77 % ) and the coeliac axis ( 59 % )  . 
although the morphological and dimensional criteria ( greater / less than 1 cm ) for dening positive nodal status at ct have often been questioned , our study found the level of concordance with 18fdg - pet / ct to be moderate for cervical nodes , fair for mediastinal nodes , and poor for lymph nodes at other sites . in our series , 18fdg - pet / ct revealed 26 abdominal localisations compared to the nine detected by mdct , with poor concordance between the two examinations ( table 3 )  . radiol med ( 2014 ) 119 : 6474 fig . 
both modalities depicted localisations of disease in the pulmonary hilar lymph nodes , liver and splenic hilum radiol med ( 2014 ) 119 : 6474 however , it should be noted that this observation is limited by the fact that only 16 out of 21 patients had a ct scan extending to the abdomen and pelvis . the literature reports that 18fdg - pet / ct can be useful in some patients with bone , neurological and cardiac involvement [ 35 ]  . 
bone lesions were detected at 18fdgpet / ct alone in patients who were found to be true positive at follow - up . brauns study [ 5 ] as well reported that 18fdg - pet / ct proved to be particularly useful in describing site of extrathoracic involvement . 
in another cohort of 20 patients with biopsy - proven sarcoidosis , 18fdg - pet / ct had a sensitivity of 87 % and was useful for the evaluation of response to therapy in patients treated with corticosteroids [ 5 ]  . moreover , sarcoid lesions may coexist with sites of malignancy in 413 % of cancer patients , and it important to bear in mind that the presence of sarcoid lesions showing 18fdg uptake may cause problems in interpreting 18fdg - pet / ct studies obtained for cancer staging and restaging [ 36 ]  . 18fdg uptake by sarcoid lesions is not specic and may mimic other pathological processes , including lymphomas and diffuse metastatic disease [ 37 ]  . 
the use of the suvbw is still being debated ; in that no specic guidelines exist that provide a cut - off value for differentiating benign from malignant lesions [ 38 ]  . 
in the three patients in our study who had a diagnosis of both sarcoidosis and malignant disease ( endometrial carcinoma , biliary carcinoma ) , histological examination of the surgical specimen conrmed that the lesions affecting the cervical , mediastinal and abdominal lymph nodes and the spleen were indeed due to sarcoidosis . non - invasive overall evaluation of disease activity by means of whole - body 18fdg - pet / ct may prove to be increasingly important in helping to choose the best site for the biopsy [ 5 , 17 , 19 , 29 ]  . in 18fdg - pet / ct , irradiation depends on the dose administered and not on the body section being imaged , while in mdct the scans are performed on the body segments suspected of harbouring disease , so as to limit the dose of ionising radiation [ 39 ]  . 
in 18fdg - pet / ct , however , the dose delivered by ldct performed for attenuation correction and suv determination must be added to the radiation dose resulting from the radio - pharmaceutical . the possibility of using 18fdg - pet / ct combined with is an contrast - enhanced ct for diagnostic purposes example of excellent synergy in the eld of diagnostic imaging , which requires a pet scanner combined with shows , pet / ct 8 / 16 - slice ct . 
as our experience combined with mdct is not yet a clinical reality in the follow - up of patients with sarcoidosis , but recent studies have demonstrated the advantages of multimodal imaging for the diagnosis of severe neoplastic diseases , which are difcult to dene [ 28 , 40 , 41 ]  . moreover , techniques are being developed to lower the dose delivered by ct while obtaining all the possible diagnostic information in a single examination [ 42 ]  . therefore , further clinical studies are needed to establish the most effective diagnostic strategy in this eld . the role of 18fdg - pet / ct in the follow - up of patients with sarcoidosis is currently still being dened , but the literature supports its value as an important aid for clinicians [ 37 ]  . 
of particular interest is its use to avoid repeated mdct studies of the abdomen and pelvis in patients with thoracic sarcoidosis , considering that it detects active and reversible lesions in all body regions [ 43 ]  . however , thanks to the growing availability of pet / ct scanners and recognition of its usefulness in depicting occult disease and identifying sites amenable to biopsy , and 18fdg - pet / ct demonstrating appears to be very promising for the functional evaluation of sarcoidosis . response therapy , some limitations to our study need to be acknowledged . the case series included only 21 patients ; however , they were rigorously selected based on the histological diagnosis and active stage of disease , and no larger series with such selection criteria have been published in the literature . histological conrmation was obtained on a single site and not on all localisations identied with the two diagnostic modalities , but this was in part due to the fact that it is not requested for either initiating treatment . the diagnosis or conclusions whole - body 18fdg - pet / ct depicts a larger number of disease localisations compared with mdct , in part as a result of the fact that the latter is often limited to the single body sections only . 
combined pet / ct facilitates the localisation of hypermetabolic areas and anatomical improves the interpretation of the morphological lesions depicted by mdct , allowing an accurate assessment of disease extent . 
therefore , even though not necessary for the diagnosis , 18fdg - pet / ct could be used in the future to complement other , more traditional modalities for assessing the course of stage ii and iii sarcoidosis and for guiding patient management . 
integration of 18fdg - pet / ct and mdct into a single examination , even though more complex to organise , deserves greater attention in clinic research and further studies are required to evaluate its impact on the management of patients with sarcoidosis . radiol med ( 2014 ) 119 : 6474 conict of interest g rubini , s cappabianca , c . 
zuiani institute of diagnostic radiology , university of udine , p.le santa maria della misericordia 15 , 33100 udine , italy e - mail : clauser.p@hotmail.it provide valuable information for the preoperative evaluation of lesions . keywords breast cancer ( cid : 2 ) breast ultrasound ( cid : 2 ) breast mri 3 - dimensional ultrasonography ( cid : 2 ) introduction ultrasound ( 2d - us ) in breast imaging proved to be an accurate , available , noninvasive and inexpensive one which is frequently used in the study of breast disease . 
it can be combined with mammography especially in women with dense breasts , in the differential diagnosis of palpable lesions or in presence of equivocal mammographic ndings [ 1 , 2 ]  . 
however , it is an operator - dependent examination and its quality is strongly related to the doctors expertise and knowledge , with limited possibilities for standardisation [ 1 ]  . when a space - occupying lesion is identied on ultrasound , its volume can be estimated using the three orthogonal diameters [ antero - posterior ( ap ) , latero - lateral ( ll ) and cranio - caudal ( cc ) ] and applying the formula for calculating the volume of a sphere ; however , this may be only an approximation when the lesion has irregular morphology . in the last few years , 3d ultrasound ( 3d - us ) has been increasingly used in clinical practice , also in breast imaging [ 3 , 4 ]  . 
images can be archived and then visualised and reviewed at a later date . the possibility of saving a series of images of the lesion , rather than a single image , makes the examination easier to radiol med ( 2014 ) 119 : 240248 review and less operator - dependent . 
currently , the bulkiness of 3d probes with square or circular arrays makes their use difcult in everyday clinical practice : 2d - us is still irreplaceable for the identication of suspicious lesions . magnetic resonance imaging ( mri ) of the breast is a very good technique for the detection and staging of disease prior to surgery in women with breast cancer [ 6 ]  . 
in these nine women , the biopsy was performed because of suspicious ndings on mri . images obtained with a 3d probe were collected for 26 lesions ( 72.0 % ) before biopsy and for 10 lesions ( 28.0 % ) when the patient came for positioning of the needle - wire , about 1 month after biopsy . 
histology revealed 28 invasive ductal carcinomas ( idc ) ( 9 grade i , 15 grade ii and 4 grade iii , one of which with a widespread intraductal component ) , one high - grade ductal carcinoma in situ ( dcis ) , two intermediate grade dcis with a lobular component , two grade - ii invasive lobular carcinomas ( ilc ) , two mucinous carcinomas ( one grade i and the other grade ii ) , and one papillary carcinoma . 
these ndings were conrmed by analysis of the surgical specimen ( 23 quadrantectomies and 13 mastectomies )  . imaging evaluation ultrasound all patients underwent 2d - us , performed by a resident following specic training in breast imaging and by a radiologist experienced in breast imaging , with the aim of locating and characterising lesions , and measuring the three diameters ( antero - posterior , latero - lateral and cranio - caudal )  . 
then , before the biopsy or while positioning the wire , the resident acquired the volumetric images on the same ultrasound unit using a 616 mhz 3d4d rsp squarearray probe , which automatically scans the area of interest . 
this probe is able to obtain 3d images on an area with a maximum diameter of 4.0 cm ; lesions larger than 4.0 cm cannot be correctly assessed . breast magnetic resonance imaging magnetic resonance imaging was performed on a 1.5 - t system ( magnetom avanto , siemens medical system , erlangen , germany ) using a dedicated bilateral multichannel coil . 
patients were studied in prone position . axial t1 - weighted images were obtained with a 3d flash sequence with the following parameters : tr / te , 9 / 4.7 ms ; ip angle , 25 ( cid : 3 ) ; matrix , 512 9 512 ; eld of view , 340 9 340 mm ; slice thickness , 2 mm ; acquisition time , 80 s . 
of averages , 1 ; oversampling , 7 ; acquisition time , 239 s . volume measurement with 3d - us volumes were calculated at the end of the session by the same resident who performed the 3d4d acquisition . specic software was used ( vocal , virtual organ computer - aided analysis , 4d view ; ge healthcare , kretztechnik , zipf , austria )  . 
of the three planes available for image display [ axial ( a ) , sagittal , ( b ) , and coronal ( c ) ] , we selected plane a to trace the lesion margins . 
the same trainee radiologist who measured the volumes with 3d - us traced the lesion borders in each axial plane , using the rst acquisition of subtracted post - contrast images . volume evaluation based on the three diameters on the images acquired with breast mri , the cranio - caudal , latero - lateral and anteroposterior diameters of each lesion were measured , and the mean diameter was calculated . 
the same test was then used to identify a signicant difference in volumes evaluated with imaging and at histopathologic examination . concordance was evaluated between the volumes calculated by : 3d - us and 2d - us ; 3d - us and mri and between those three imaging modalities and histology using the interclass correlation coefcient ( icc )  . 
the gure is visualised in three planes ; the axial and longitudinal planes can also be obtained with a 2d probe , while the third plane is a reconstruction in the coronal plane . after the operator traces the borders of the lesion , dedicated software ( vocal , virtual organ computer - aided diagnosis ) calculates the volume and represents it with a surface rendering modality ( b ) radiol med ( 2014 ) 119 : 240248 results the maximum diameters of the lesions considered are described in table 1 , in terms of range , mean and standard deviation . 
concordance between imaging and histology was also good , with 0.79 for 3d - us , 0.82 for 2d - us and 0.79 for mri , respectively . lesions with a volume \1 cm3 were slightly overestimated by 3d - us ( 16 / 20 cases ) , while 12 of 16 lesions with a volume c1 cm3 were underestimated . 
image processing performed by a single operator to calculate volumes took 24 min , depending on dimensions and morphological features of the area . discussion 2d - us is an easy and useful approach , which is irreplaceable in recognising and characterising breast lesions . once identied , the lesion can be studied with a 3d probe and the area of interest can be acquired and stored with a volumetric acquisition that includes the lesion and a small portion of surrounding tissues . 
this can be achieved with a liner - array probe that scans the area of interest , with or without systems to localise ( tracked freehand systems or untracked freehand systems ) , obtaining 2d - images from which volumetric images are reconstructed [ 4 ]  . as an alternative , one can use a probe with a square or circular 2d - matrix ( two - dimensional arrays ) that is kept still while it automatically scans the area of interest to give 3d real - time information [ 4 ]  . 
the images acquired can be presented with surface rendering ( showing the acquired volume with 3d appearance ) , multiplanar reformatting ( showing the three perpendicular planes , with the possibility to move within the slices acquired ) or volume rendering [ 4 ]  . 
several studies comparing the sensitivity , specicity , predictive values and accuracy of 3d - us with those 2d imaging in the differential diagnosis of breast masses showed that 3dus does not improve diagnostic accuracy [ 14 ]  . 
cho [ 14 ] found a high level of concordance between different operators in identifying a lesion as benign or malignant using 3d probes , and concluded that this technique could improve diagnostic condence and reproducibility . 
d volume is calculated on breast mr with dedicated software ( vitrea 2 - vital images , plymouth , mn , usa ) , after drawing the area of the lesions in all the slices . 
histology of the lesion after surgery conrmed the dimensions identied by 3d - us and breast mr imaging than one study underlined the usefulness of the coronal plane ( c ) , which is perpendicular to the two planes usually visualised on 2d ultrasound . 
the mathematical estimation of volume has also lower intraand inter - operator reproducibility . many authors have shown that reproducibility in volume measurement is better when using a volumetric probe , with an inter - operator concordance of almost 100 % , example , in the study of thyroid or focal liver lesions [ 17 , 19 ]  . 
the use of ultrasound with 2d probes has also been proposed for measuring renal volume in healthy subjects and in patients with impaired renal function : the accuracy , radiol med ( 2014 ) 119 : 240248 fig . 
6 graph showing the results of blandaltman analysis applied in the comparison of the volumes measured with 3d ultrasound and histology reproducibility and correlation with other techniques such as computed tomography were interesting [ 15 , 16 ]  . the limits of measuring the three diameters are particularly evident for masses or organs with irregular margins and morphology [ 17 , 18 ] , which is a typical aspect of malignant breast lesions [ 11 ]  . acquiring 3d images requires only a few seconds ( 36 s ) , and overall the examination is extended by no more than 1 min , necessary to activate the probe and identify the lesion . 
this operation entails a short learning curve : the operator has to become familiar with a slightly different image representation from that of the 2d probe . the volume measurement can be performed at the end of the session after the acquisition , by a different operator and by more than one operator , as can the morphological evaluation of the lesion and surrounding tissues . 
volume measurement requires a maximum of 4 min , especially in lesions with a greater diameter and undened margins . the vocal software used in our study has already been reported to have good accuracy in the volumetric measurement of irregular lesions , especially when margins are traced manually and not automatically [ 5 ]  . 
its reliability improves with the number of planes used to describe the the same time margins of the area of interest , but at increases the time needed for the analysis . 
 [ 20 ] as well as our own experience , we decided to use a 30 ( cid : 3 ) angle , which appears to be a good compromise , as it is simple , with no signicant differences with respect to measurements obtained at 6 ( cid : 3 ) , 9 ( cid : 3 ) or 15 ( cid : 3 ) , but at the same time less time - consuming . 246 radiol med ( 2014 ) 119 : 240248 it has already been underlined that mri can be superior in describing lesion characteristics and extension : for this reason , we decided to compare also the volumes obtained with this technique [ 7 , 8 , 21 ] , even though our standard of reference has been histology of the specimen . in the literature , we have found only one article [ 22 ] comparing the volumes of benign and malignant breast lesions measured with 2d - us , 3d - us and mammography , and the authors reported a good correlation . we are not aware of studies that evaluated the correspondence of 2d - us , 3d - us , mri and histology for malignant breast lesions . 
c , d in this case the volume calculated with the three methods was surgery ( 2dcomparable to that obtained by histology after us = 1.2 cm3 ; breast mr = 0.87 cm3 ; histology = 0.9 cm3 ) 3d - us = 1.05 cm3 ; radiol med ( 2014 ) 119 : 240248 histology comparable to that of mri , which is considered the standard of reference among imaging modalities for a correct evaluation of lesion extension . 
 [ 7 ] , 2d - ultrasound underestimated tumour diameter during the follow - up of patients treated with neoadjuvant chemotherapy , while mri showed a better performance with a slight tendency to overestimation . however , the particular situation and hence the modications related to chemotherapy could probably explain the limited ability of us in identifying post - therapy changes , especially compared to mri , which is able to visualise contrast - enhancement only where there is vascularisation . the 95 % limits of agreement calculated with blandaltman analysis between imaging and histology overlapped for all modalities , proving that 3d - us is at least comparable to other imaging techniques in evaluating the volume of malignant breast lesions . in the literature , other possible applications of 3d - us can be found . 
for example , its use in interventional procedures : 3d images allow a better evaluation of needle position , thus improving the accuracy of tissue sampling and reducing the number of cores necessary [ 24 , 25 ]  . in addition , the possibility of using cad ( computeraided diagnosis ) on volumetric images acquired with 3dus has already been evaluated , with the aim of improving sensitivity and specicity in the identication of lesions , especially those classied as bi - rads 3 [ 26 ]  . conclusions on the basis of our initial experience , 3d ultrasound appears to be a reliable and easy - to - use method which is at least as accurate as 2d ultrasound and mri in measuring volumes of malignant breast lesions . 
 [ 3 ] reported that in men the mean age at diagnosis was signicantly higher , the size was signicantly smaller and fnh showed more often atypical than those in women . 
furthermore , was not studies have demonstrated that detection of the typical imaging features of fnh is often heavily dependent on lesion size , being less frequent in lesions smaller than therefore , undertook this study in an 3 cm [ 57 ]  . 
we , attempt to clarify whether radiologists should expect any gender differences in the imaging ndings of fnh detected in men or women . radiol med ( 2014 ) 119 : 222230 materials and methods patient population and imaging techniques institutional review board approval was obtained and full informed consent was waived for this retrospective study . our study complied with the terms of the declaration of helsinki [ 8 ]  . we searched our hospitals medical records ( radiology , pathology , surgical pathology and discharge summary ) to identify patients with fnh treated during a 6 - year period ( from january 2006 to december 2011 )  . 
patients were eligible for enrolment on the basis of the following inclusion criteria : ( 1 ) a conclusive diagnosis of fnh ( see reference standard ) ; ( 2 ) they had undergone at least one of the following imaging studies : ( a ) baseline and contrastenhanced ultrasound ( ceus ) scan ; ( b ) multiphase multidetector computed tomography ( mdct ) scan ; ( c ) magnetic resonance imaging ( mri ) with hepatocellular - specic contrast agent . 
patients were excluded from the analysis if the imaging protocol was suboptimal or if the images could not be retrieved from our imaging archives . computed tomography ct studies were performed with a 64 - slice mdct scanner ( brilliance 64 , philips medical systems , eindhoven , the netherlands )  . 
to determine the scanning delay for the hepatic arterial phase , the time - topeak aortic enhancement was assessed using an automatic bolus - tracking technique with automated scan - triggering software ( bolus pro ultra , philips medical systems , eindhoven , the netherlands )  . 
a triphasic dynamic contrast - enhanced study was obtained after the administration of an iv bolus of 0.1 mmol / kg of gadobenate dimeglumine ( multihance , bracco , italy ) into an antecubital vein at a ow rate of 2 ml / s through a 20 - gauge intravenous catheter by means of a power injector ( mr spectris ; medrad , pittsburgh , pa , usa ) and ushed by 20 ml of sterile saline solution . 
images were acquired using an automated bolus - detection technique ( smartprep technique , ge healthcare ) during the arterial ( 14 s after bolus injection ) , hepatic venous and delayed phase ( 60 and 180 s after bolus injection , respectively )  . the dynamic study was followed by a hepatocellular - specic phase obtained 2 h after the injection of contrast material , with the same scanning parameters . contrast - enhanced ultrasound two experienced radiologists ( more than 5 years of ceus of the liver ) , who were aware of the patients clinical histories , performed us scanning using either an hdi 5000 ( atl , bothell , wash , usa ) or an iu22 unit ( philips ultrasound , bothell , wash , usa ) , both of them equipped with c5 - 2 / c5 - 1 convex - array probes and pulse inversion imaging software . 
the us contrast agent used in the present study was sonovue ( bracco , milan , italy ) , which was injected intravenously as a 2.4 ml bolus followed by 10 ml of normal sterile saline ush using a 20or 22 - gauge peripheral intravenous cannula . 
in patients with multiple lesions , a 2.4 ml further bolus of sonovue was administered for each lesion , with an interval time at least of 15 min to allow for clearance of the previously injected contrast . digital cineloops were recorded during both baseline and postcontrast us in the arterial , hepatic venous and extended hepatic venous or late phase ( 540 s , 5590 s and injection , up to 200300 s from the beginning of 224 radiol med ( 2014 ) 119 : 222230 respectively )  . 
all images and cineloops were digitally stored as raw data in a pc - based workstation connected to the us units via a standard ethernet link and sent to our pacs ( impax , agfa - gevaert , milan , italy )  . image analysis two abdominal radiologists ( more than 10 years of experience ) randomly reviewed all imaging studies by consensus . 
neither of the readers was involved in the scanning and both were blinded to the nal diagnosis , as well as to the identity , clinical histories and other imaging ndings of the patients . 
five consecutive interpretation sessions , with a seven - day interval to prevent recall bias , were held to complete the review process . on each imaging modality , the two readers were asked to report on the size and segment location of each lesion according to the couinaud classication system and to visually assess echogenicity / attenuation / signal intensity ( the latter in all mri sequences ) in comparison with adjacent liver parenchyma on both the unenhanced and the contrast - enhanced images obtained during the arterial , hepatic venous and delayed ( or extended hepatic venous for ceus ) phases . 
for us and ceus studies , the colour doppler images and spectral waveforms were also evaluated for each lesion , as were the following parameters [ 911 ] : central scar : a central or eccentric hypoor hyperechoic area at baseline us and / or unhenancing at ceus in the arterial and hepatic venous and extended hepatic venous phase , also showing distinctly different attenuation / intensity on unenhanced scans or at different phases of enhancement . feeding vessel : an arterial vessel , appreciable at baseline cd / pd and / or at ceus in the arterial phase , branching from the hepatic arterial tree and directed towards the lesion and penetrating it ; spoke - wheel sign : a radial arterial vascularity within the lesion appreciable at baseline colour and power doppler and / or centrifugal enhancement of the lesion with a central vessel branching from the centre towards the periphery at ceus in the arterial phase . presence and type ( arterial or venous ) of any intralesional ow , other than the above signs , at baseline colour and power doppler examination . reference standard the nal diagnosis was established by core biopsy performed with an 18 - g needle ( n = 1 ) or by demonstrating that fnh was isoor hyperintense to the surrounding liver in the hepatobiliary phase of mri ( n = 81 )  . 
for the remaining 29 lesions , a combination of size stability during a follow - up period of at least 1 year and imaging features consistent with fnh was used . the ceus diagnostic criteria for fnh were based on arterial phase centrifugal lling and stellate vascularity , followed by sustained contrast enhancement on the hepatic venous phase and extended hepatic venous or late phase [ 10 ]  . 
the ct diagnostic criteria were : mild hypoattenuation or isoattenuation on precontrast scan ; rapid , homogeneous and strong enhancement in the arterial phase except for the central scar ; near isoattenuation to liver parenchyma in the hepatic venous and delayed phases . 
to assess the statistical signicance of the difference between two genders with respect to us , colour doppler , ceus , mdct and mri patterns and with respect to the presence and appearance of the central scar , the z test for proportions or the fischer exact test were used , as appropriate . 
statistical signicance was considered to be present at a p value of \0.05. results overall , 195 complete imaging studies pertaining to 111 fnhs ( size range 0.59.2 cm , mean 3 1.8 cm ) in 91 patients were retrieved ( table 1 )  . 
b during the arterial phase a markedly hypervascular lesion ( arrow ) is evident in segment viii showing a tiny hypoattenuating central area corresponding to the central scar ( arrowhead )  . 
seventy - three ( 65.8 % ) lesions were located in the right liver and the remaining 38 ( 33.2 % ) in the left liver . computed tomography eleven out of the 17 ( 64.7 % ) fnhs studied by means of mdct were slightly hypoattenuating , whereas 4 / 17 226 radiol med ( 2014 ) 119 : 222230 table 2 multidetector ct pattern of 17 focal nodular hyperplasia ( fnh ) lesions no . 
in the delayed phase , these four lesions were either isoattenuating ( n = 3 ) or hyperattenuating ( n = 1 )  . a central scar was depicted on mdct images as an unenhancing hypoattenuating central area in 7 / 15 ( 46.7 % ) fnhs occuring in women and in one of the two ( 50 % ) fnhs in men . 
a unenhanced mr shows a 3.5 - cm isointense ( arrow ) lesion in segment vi which shows a small central scar that is hypointense with respect to the surrounding liver parenchyma on t1 - weighted imaging ( arrowhead )  . 
further multicentric studies sampling a larger population may be warranted to elucidate this issue . in our series , no statistically signicant differences between men and women were found on unenhanced images from us , mdct and mri . after contrast agent injection , our ndings conrm the previously reported strong arterial enhancement on dynamic contrast - enhanced images followed by sustained enhancement , without any statistically signicant difference among imaging modalities [ 5 , 7 , 10 , 13 ]  . 
interestingly , in that study the two men with fnh without central stellate area on mr images had small lesions ( 25 and 30 mm in diameter , respectively ) , with no central brous area on gross examination of the surgical specimen . several studies have demonstrated that the detection of typical imaging features of fnh is often strongly , heavily dependent on lesion size , being less frequent in lesions smaller than 3 cm [ 57 ]  . 
consequently , considering our series , it might be hypothesised that the difference found in that study may be , at least in part , more closely related to the different lesion size reported in the two different groups of male and female patients than to real pathological differences . 
did not use gadolinium chelate with hepatocellular - specic properties , so the hepatobiliary phase could not be exploited and , eventually , the seven male patients with fnhs lacking at least one typical nding on mri underwent surgery . 
it is noteworthy that all the fnhs studied in our serieswhether in men or womenshowed uptake of hepatocellular - specic contrast agent , demonstrating the hepatocellular nature of lesions and , in the proper clinical setting , their substantial benignity . 
hence , our data also support the hypothesis made by other researchers that exploiting the properties of the newer hepatocellular - specic mr contrast agents enables us to depict the morphological and functional characteristics of fnh noninvasively and may aid in the differential diagnosis of these lesions , thus reducing the need for biopsy and even surgery [ 1315 ]  . this retrospective study has some important limitations . first , we had a selection bias . 
consequently , those with fnh lesions lacking the characteristic imaging ndings may have been missed . nevertheless , if atypical appearance of fnh is a feature mainly observed in men we should have observed a dramatic reduction of typical fnhs found in men in comparison with women , but this was not the case . 
however , all lesions were well characterised at ceus , multiphase contrast - enhanced ct and / or mri on the basis of the typical enhancement patterns , which we considered to be established diagnostic criteria , as done in other studies [ 14 ]  . 
in one patient selective renal arteriography was negative probably because the bleeding observed at ct angiography was self - limited . twenty - one embolisation procedures were performed in 20 patients ; one patient required a second embolisation 3 h after the rst one . 
no statistically signicant differences in egfr or renal function stage appeared after rae . conclusions percutaneous treatment can be proposed as a injuries , rst - line treatment resulting in a safe and effective procedure . in iatrogenic renal arterial keywords transarterial embolization ( cid : 2 ) iatrogenic renal vascular injuries ( cid : 2 ) emergency embolization introduction the kidney is the most common injured genitourinary and abdominal organ . 
advances in imaging and treatment strategies over the past 20 years 262 radiol med ( 2014 ) 119 : 261268 have decreased the need for surgical increased renal preservation . intervention and iatrogenic renal artery injuries are usually reported after renal artery angioplasty or stenting , and have an incidence of 1.6 % [ 2 ]  . 
in addition , as nephron - sparing surgery ( nss ) has recently become the procedure of choice to treat localised renal tumours in selected patients , complications are more frequent , affecting mainly the renal vascular and collecting system [ 4 , 5 ]  . 
furthermore , although relatively uncommon , iatrogenic renovascular injuries have increased due to the increasing number of interventional procedures . possible radiological ndings in iatrogenic vascular lesions are arteriovenous stulae , pseudoaneurysms , arterial dissection , or contrast extravasation . 
traditional therapy for acute iatrogenic rupture of the main renal artery has been renal artery ligation followed by bypass grafting or nephrectomy , but nowadays , approaches conservative or minimally invasive increasingly favoured over surgery , including selective transarterial embolisation ( tae ) and the placement of stents / stent grafts . tae is a well - established nonsurgical endovascular treatment for iatrogenic renal arterial injuries , with many advantages , such as rapid recovery , short hospital stay and early resumption of physical activities [ 1 ]  . 
the aim of our study was to review our experience and long - term followup in the treatment of iatrogenic renal vascular injuries using transcatheter embolisation . materials and methods patients the medical les and imaging data of all patients who renal arterial embolisation ( rae ) between underwent january 2006 and july 2012 in two centres with established interventional retrospectively radiology service were reviewed . 
indications for diagnostic angiography and endovascular treatment included : signs and symptoms suggestive of vascular injury , ( such as haemodynamic instability necessitating blood transfusion , non - resolving haematuria in a haemodynamically stable patient , and non - remitting ank pain ) , suspicious laboratory ndings ( decrease in haematocrit or haemoglobin levels ) ; uncontrolled intraoperative blood loss ; imaging evidence of vascular injury ( pseudoaneurysm formation , active extravasation of contrast medium , creation of arteriovenous stula , etc . ) ( table 1 )  . 
haemodynamically stable patient means stabilisation of blood pressure ( bp ) and haematocrit level , further decrease of haemoglobin value by no more than 1.5 g / dl , and haemodynamic stabilisation without the need for blood transfusion . 
moreover , rae was implemented only when renal bleeding was not controlled by conservative measures , such as nephrostomy tube clamping , adequate hydration , and correction of coagulation disorders , or in any case of severe bleeding leading to haemodynamic instability . written informed consent was obtained from all patients except those deemed non - competent who were in hypovolaemic shock . baseline imaging computed tomography ( ct ) angiography ( aquilion 64 , toshiba , japan ) ( 16 - siemens , erlangen , germany ) was performed with a baseline non - contrast acquisition and an arterial contrast phase following the administration of 120 ml of 320 mgi / ml iodinated contrast medium ( visipaque 320 , ge healthcare , usa ) at an injection rate of 4 ml / s , immediately followed by 40 ml of normal saline solution at the same injection rate , using a double head automatic injector ( stellant d , medrad , usa )  . 
optimal arterial enhancement was achieved using the bolus - tracking technique , with a region of interest ( roi ) placed on the abdominal aorta ( at level ) with an enhancement threshold 100 hu higher than the baseline arterial density . 
when clinical conditions permitted , a delayed enhancement phase was acquired 5 min after contrast medium administration . on ct angiography , haemorrhage was dened as an active extravasation of contrast media , pseudoaneurysm as a round or ovoid cavity communicating with a ruptured vessel wall , arteriovenous stula as an early or simultaneous opacication of one or more intraparenchymal arteries and veins during the arterial phase , and arteriocalyceal stula as the presence of contrast medium in a calyceal group during the arterial phase . the suprarenal procedure all endovascular procedures were performed by an interventional radiologist . 
1 selective left renal arteriography shows a psedoaneurysm ( psa ) in a patient with a percutaneous nephrostomy ( a ) ; the angiogram performed at the end of the embolisation procedure ( carried out with microcoils ) revealed complete exclusion of the psa the procedure was performed in the angiography suite ( bv 300 allura xper fd20 ; philips and axiom artis siemens ) under local anaesthesia and continuous cardiovascular and respiratory monitoring . 
before treatment each patient received an intravenous dose of 5 % mannitol , 3 ml / kg / min of dopamine plus 600 mg of intravenous n - acetylcysteine as a protection against the ischaemia - related production of free radicals and contrast medium - induced renal damage . 
shortterm antibiotic prophylaxis with a rst - generation cephalosporin ( cefazolin 2 gr b.i.d. ) was initiated at the beginning of each endovascular procedure . in all patients , from a common femoral approach , a unilateral renal angiography was initially performed , using a 5f pig - tail catheter , followed by selective renal digital subtraction angiography of the affected side using a selective cobra - type or sim - 1 catheter . 
the interval between completion of the intervention and rst imaging follow - up was in the range of 3 days2 months . a minimum 6 - month follow - up was available for all of our patients who underwent rae . 
values of egfr ( estimated glomerular ltration rate ) were assessed and classied according to the national kidney foundation scale [ 7 ]  . in each patient , the egfr before injury was compared with the egfr after rae . 
the last egfr measurement reported in the patients medical records within the following 6 months after rae was selected as a reference , to avoid interference with other variables such as hypotension and contrast agent - induced nephrotoxicity after renal bleeding and embolisation . 
patients without a follow - up egfr measurement within 6 months after rae were excluded from the analysis of renal function because a decrease in egfr observed beyond that period could be associated with other factors . 
individual changes in egfr and renal function stage before renal artery injury and after rae were evaluated by the student t test . outcomes the technical success , clinical success and complications were independently evaluated . 
clinical success after rae was dened as the absence of clinical or imaging evidence of further bleeding as determined by the resolution of haematuria , retroperitoneal bleeding , and hypotension after rae , in 24 h and within 1 week after the fig . 
2 selective right renal arteriography shows early opacication of draining veins ( white arrow ) during the arterial phase due to postnephrostomy iatrogenic arteriovenous stula ( a ) ; an 8 mm amplatzer plug inside the delivery catheter ( b ) ; performed at arteriovenous stula ( c ) the angiogram the end of the procedure revealed closure of the radiol med ( 2014 ) 119 : 261268 in the absence of endovascular procedure . follow - up imaging , bleeding cessation was dened as stabilisation of blood pressure ( bp ) and haematocrit level , further decrease of haemoglobin value by no more than 1.5 g / dl , and haemodynamic stabilisation without the need for blood transfusion . 
all complications were recoraccording interventional radiology classication [ 8 ]  . the society classied results renal injuries complicating biopsy ( n = 4 ) , percutaneous eswl ( n = 4 ) , nephron - sparing surgery ( n = 4 ) , guidewire - induced arterial perforation during coronary angiography or renal stenting ( n = 3 ) , percutaneous nephrostomy renal endopyelotomy / pyeloplasty ( n = 2 ) and ( n = 3 ) , surgical nephrectomy ( n = 1 ) , were all causes of iatrogenic vascular lesions necessitating endovascular treatment . 
the procedures involved 20 native kidneys and one renal allograall patients presented with haemoglobin / haematocrit drop , which was associated with persistent macroscopic haematuria in 16 ( 76.2 % ) and ank pain in 11 patients ( 524 % )  . 
in the case of severe renal failure ( n = 3 ) , colour doppler visualisation of a pseudoaneurysm in the site of previous renal biopsy in association with laboratory data and clinical signs , were considered sufcient to justify a digital subtraction angiography ( dsa ) examination . diagnostic renal angiography revealed nine actively bleeding vessels , six pseudoaneurysms , four arteriovenous stulas and one arteriocalyceal stula . 
in all cases the injuries were intraparenchymal . twenty - one embolisation procedures were performed in 20 patients ; one patient required a second embolisation 3 h after the rst rae for persistent pain and evidence of persistent bleeding at ct scan . the technical success rate was 100 % , as shown by the complete exclusion of bleeding demonstrated with angiography performed at the end of the procedure . 
one patient who was referred late to the interventional radiology unit required massive after percutaneous biopsy , renal transfusions , died 48 h later in the intensive care unit due to disseminated intravascular coagulation . 
during this period , no recurrence of bleeding or complications related to rae were observed . overall , endovascular treatment was well tolerated , even though minor complications such as vasovagal reactions or temporary spasm of a catheterised artery were not registered . the long - term effects of rae on renal function could not be reliably assessed in all of our patients ; because in four cases the follow - up egfr assessment was done after more than 1 year following rae and this measurement could be affected by other causes . 
in available patients , no statistically signicant differences in egfr or renal function stage appeared after rae . discussion as the number of interventional procedures in modern clinical practice increases , so does the number of iatrogenic renovascular injuries , which have become the most common ( [ 50 % ) cause of renal vascular lesions [ 4 , 5 ]  . 
the present study was performed to evaluate the immediate radiological and clinical success and the long - term followup of transcatheter embolisation of iatrogenic renal vasis worth noting that during the early cular injuries . 
it postoperative period after partial nephrectomy , major haemorrhage is the most common complication , with a reported incidence between 0 and 5.26 % [ 3 , 9 , 10 ]  . in a retrospective series of 21 iatrogenic renovascular lesions resulting from surgical ( nephron - sparing surgery ) , minimally invasive surgical or percutaneous procedures we found that rae is a safe and effective treatment for either the prevention or treatment of renal haemorrhage . 
in a patient presenting with gross haematuria or acute ank pain 266 radiol med ( 2014 ) 119 : 261268 after invasive and non - invasive renal procedures , examinations such as contrast - enhanced ct , colour doppler sonography , or magnetic resonance angiography should be performed . 
the advantage of ct is that it enables imaging of the entire urinary tract [ 10 ]  . most vascular injuries are minor with spontaneous healing in the majority of patients , so conservative management is preferred . 
in the event of massive bleeding , renal haemorrhage persisting for more than 72 h , or progressively deteriorating renal function , surgical or percutaneous treatment of vascular lesions has been recommended [ 11 , 12 ]  . the alternative treatment method to percutaneous embolisation is emergent nephrectomy or clamping of the renal artery which results in kidney loss [ 1 , 5 ]  . 
previous studies have also shown that embolisation of iatrogenic renal arterial injuries is a safe , tissuepreserving treatment method for renovascular injuries , associated with high technical and clinical success rates , using various agents for embolisation [ 1316 ]  . 
 [ 16 ] reported a similar series ( 15 patients with the same iatrogenic lesions and causes ) treated with superselective embolisation using coils or n - butyl cyanoacrylate ( nbca ) iodised oil mixture or pva particles or nbca iodised oil mixture when the lesion could not be superselectively catheterised . 
as in our series , embolisation was performed as superselectively as possible ; when possible , we preferred coils or plug to limit as much as possible non - target embolisation and we usually use pva particles and ncba iodised oil mixture only in superselective embolisation , even though reported experiences [ 1316 ] have not described non - target embolisation . 
furthermore , in agreement with our results , percutaneous embolotherapy had a high success rate and a low complication rate ( i.e. , major parenchymal infarction ) [ 17 ] in the management of iatrogenic haemorrhage in cases of vascular injuries of renal allografts . considering that the choice of embolisation material is important for efcient treatment of vascular injuries , the material should be chosen according to the site , size and the ow pattern of the vessels to be occluded , material availability , and the experience of the interventionalist . percutaneous angiography with selective coil embolisation is the initial treatment of choice in renal arterial haemorrhage [ 18 ]  . 
superselective embolisation as distally as possible is mandatory , at least at the level of the interlobar arteries , to keep parenchymal loss to a minimuthis can usually be achieved using microcatheters and microcoils as embolic agents [ 13 ]  . 
the main disadvantage of coil embolisation is that usually more than one coil is required for adequate occlusion , which increases the cost and time of the procedure [ 5 ]  . 
one arteriovenous stula ( avf ) in our series was embolised using a single amplatzer vascular plug iv of 8 mm in diameter ( aga medical corporation , usa ) , delivered through a 5f catheter successfully placed immediately proximal to the site of interest . 
in this case , the avf occlusion was immediate and safe and the total cost may be comparable to the combined cost of several coils . control of the polyvinyl alcohol particles ( pva ) during injection is difcult , and inadvertent embolisation may occur [ 5 ]  . 
even when more than one feeding artery needs to be occluded ( e.g. , in the case of multiple bleeding sites or pseudoaneurysms or multiple arteries contributing to the bleeding site ) , only minor renal parenchymal loss post embolisation is still possible . 
additional renal arterial trauma , inadvertent or nonselective embolisation of the main renal artery or occlusion of non - target sites may lead to postembolisation functional syndrome , systemic hypertension , and renal impairment as the most frequent possible complications . these complications have not been noted in recent reports using superselective embolisation [ 5 , 14 , 2123 ]  . 
we did not encounter any of these complications in our series . no long - term renal function impairment was found in our patients , which is also indicative of the limited renal parenchymal loss after embolisation . 
as reported in the literature [ 14 ] , patients who present with iatrogenic renal vascular trauma often manifest other comorbidities , such as diabetes or pre - existing hypertension and renal disease , which are known to be associated with worsening renal function . 
finally , we have to consider that the initial vascular trauma even though deemed renal sparing , may nonetheless radiol med ( 2014 ) 119 : 261268 be responsible for some loss of renal tissue . 
in the literature , a transient elevation of the serum creatinine level during the days following the procedure is described , but this may reect the larger amount of contrast material used in some more complex procedures [ 10 ]  . potential limitations and difculties associated with this technique are impaired renal function , vascular anatomical difculties , or stenosis of the main renal artery [ 5 , 14 ]  . bleeding from a segmental artery is rare ; however , when this more proximal artery is occluded , keeping major parenchymal loss to a minimum may be a probleif renal function before embolisation was not impaired , more extensive parenchymal loss is still a preferred solution over total nephrectomy [ 5 , 14 ]  . limitations of the present study are the small number of patients and the lack of comparison with a cohort of patients treated surgically . 
because of the rarity of this type of lesions , it is unlikely that a prospective randomised trial with a high number of patients will be conducted to compare the percutaneous and open approaches . in conclusion , perirenal haemorrhage , intrarenal bleeding with pseudoaneurysm formation or the occurrence of an arteriocalyceal stula are all serious iatrogenic renovascular complications requiring prompt diagnosis and treatment . 
giannitrapani department of clinical medicine and emerging pathologies , university of palermo , via del vespro 127 , 90147 palermo , italy resonance imaging , during the unenhanced phase , hepatic arterial phase and venous phase were recorded . 
no statistically signicant difference in venous enhancement was found among the mrecist groups . conclusions mrecist showed a more favourable response compared to recist 1.1 in patients with unresectable hcc receiving sorafenib . keywords liver ( cid : 2 ) ct ( cid : 2 ) mr ( cid : 2 ) hcc ( cid : 2 ) sorafenib introduction hepatocellular carcinoma ( hcc ) is a major health problem worldwide and the main cause of death among cirrhotic patients [ 1 ]  . 
the introduction of this new therapy has led to search for new radiological endpoints / biomarkers able to measure and predict tumour response to treatment [ 3 , 511 ]  . response evaluation criteria in solid tumours ( recist ) are typically used to assess response to cytotoxic therapy . 216 radiol med ( 2014 ) 119 : 215221 however , these criteria may not adequately reect tumour response to sorafenib , because changes in tumour vascularity and necrosis may occur without major changes in tumour size [ 6 , 8 , 1115 ]  . 
therefore , modied recist ( mrecist ) have been developed which differ from recist in that the target lesion measured is not the whole lesion but only the viable tumour , dened as the contrast - enhanced portion of the tumour on hepatic arterial phase images [ 9 , 12 , 16 , 17 ]  . we hypothesised that the anti - angiogenic effect of sorafenib could be measured on contrast - enhanced computed tomography ( ct ) and magnetic resonance ( mr ) imaging as changes in tumour enhancement . 
therefore , the purpose of our study was to compare tumour response according to recist and mrecist and to describe changes in hcc enhancement before and after sorafenib treatment . patients and methods study population review board approval was obtained , with a waiver for informed patient consent . through a computerised search of our gastroenterology department database from december 2007 to january identied 40 patients who 2010 , a study coordinator received anti - angiogenic treatment with sorafenib ( nexavar , bayer healthcare , germany ) in a standard dose of 800 mg daily for unresectable hcc . 
these patients were deemed inoperable or untransplantable either because tumour burden exceeded the united network of organ sharing ( unos ) or university of california sans francisco ( ucsf ) criteria or because of comorbidities . 
patients with previous locoregional treatment ( n = 15 ) and patients without available baseline or follow - up contrast - enhanced ct / mr imaging studies ( n = 4 ) were excluded . 
there were 12 men ( mean age 68 years ; range 5879 years ) and ve women ( mean age 73 years ; range 6778 years ) with an overall mean age of 69 years ( range 5879 years )  . 
the underlying cause of cirrhosis was hepatitis c in 11 ( 64 % ) patients , hepatitis b in three ( 18 % ) in three ( 18 % ) patients and ethanol patients . 
seven ( 41 % ) patients had one measurable lesion and 10 ( 59 % ) patients had multiple measurable lesions . the study coordinator noted the following clinical data for each patient : alphafetoprotein level , performance status , child - pugh class , barcelona clinic liver cancer ( bclc ) stage , treatment starting date and duration , previous vascular surgery and association with other vascular hepatic diseases . imaging studies baseline examinations were performed at a median of 30 days ( range 2836 days ) before the start of treatment . thirteen ( 76 % ) patients had one follow - up imaging study available , two ( 12 % ) patients had two follow - up imaging studies available , and two ( 12 % ) patients had three followup imaging studies available . 
the baseline examination was performed with either ct ( n = 10 ) or mr imaging ( n = 7 )  . computed tomography ct imaging was performed using a 64 - section scanner ( philips brilliance , royal philips electronics , eindhoven , the netherlands )  . 
the following ct parameters were used : detector conguration , 64 9 0.625 mm ; peak , 120 kvp ; scan time , 4 s ; mas , 250300 ; section thickness , 3 mm ; no section overlap . 
the contrast medium was administered with a mechanical power injector ( invision ct , medrad ) at a rate of 35 ml / s through an 18to 20 - gauge intravenous catheter inserted into an arm vein , followed by a ush of 25 ml of saline administered at the same injection rate . automatic bolus - tracking techniques with automated scantriggering software ( bolus pro ultra , philips medical systems ) were used . 
hepatic arterial phase and venous phase scanning were started automatically 18 and 58 s , respectively , after the trigger threshold ( 150 hu ) was reached at the level of the suprarenal abdominal aorta . 
the delayed phase was started 180 s after the start of contrast material injection . mr imaging mr imaging was performed using a 1.5 - t scanner ( signa excite hdxt , general electric , healthcare , milwaukee , wi , usa )  . 
contrast administration consisted of 0.1 mmol / kg body weight of gadobenate dimeglumine ( multihance ; bracco imaging , milan , italy ) in six patients or 0.025 mmol / kg of gadoxetate disodium ( primovist , bayer , berlin , germany ) in one patient , injected at 12 ml / s through a 20 - gauge intravenous catheter with a power injector ( spectris ; medrad , pittsburgh , pa , usa ) , followed by 20 - ml saline ush at the same rate . 
if a patient had multiple follow - up ct or mr imaging studies , the latest was considered for analysis . diagnosis of hcc was made using a combination of imaging and laboratory criteria ( i.e. , size [ 1 cm ; enhancement during the hepatic arterial phase and washout during hepatic venous and / or delayed phase ; interval growth ; mild hyperintensity on t2 - weighted imaging ; signal loss on out - ofphase imaging due to the presence of lipids ; tumour invasion of the portal vein ; elevated alphafetoprotein ) [ 1 ]  . 
when multiple hcc lesions were noted at baseline ct or mr examinations , a maximum of two target lesions were selected for further analysis ; a target lesion was selected if the following conditions were satised : ( a ) the lesion could be accurately measured in at least one dimension ; ( b ) it was suitable for repeat measurement ; ( c ) it showed intratumoural arterial enhancement on contrast - enhanced ct or mr imaging . 
the roi values of lesion attenuation measured on the ct images or the signal intensity values measured on the mr images during the unenhanced , hepatic arterial and venous phase were recorded . 
enhancement values during the hepatic arterial phase and venous phase were calculated for each lesion as : hepatic arterial phase enhancement = [ ( attenuation / signal intensity during hepatic arterial intensity during unenhanced phase attenuation / signal unenhanced phase ) / attenuation / signal phase ] 9 100 ; venous phase enhancement = [ ( attenuation / signal intensity during venous phase attenuation / signal intensity during unenhanced phase ) / attenuation / signal intensity during unenhanced phase ] 9 100 . 
no patient had been treated with transjugular intrahepatic portosystemic shunt or surgical spleno - renal shunt . recist vs mrecist response was slightly different depending on whether recist or mrecist was used . 
according to recist , two of 17 ( 12 % ) patients showed partial response , ten of 17 ( 59 % ) patients showed stable disease and ve of 17 ( 29 % ) showed progression disease . 
according to mrecist , three of 17 ( 18 % ) patients showed partial response , ten of 17 ( 59 % ) patients showed stable disease and four of 17 ( 23 % ) patients showed progression disease . 
similarly , patients with partial response had a greater decrease in venous phase enhancement than did those with stable disease or progressive disease , but the differences were not statistically signicant . 
four patiens had tumor invasion of the portal vein and one patient had bland portal vein thrombosis . discussion in this study , we compared recist and mrecist in assessing response in 17 patients with unresectable hccs treated with sorafenib . 
since mrecist measures only the viable ( i.e. , the enhancing ) portion of the tumour , as opposed to recist 1.1 , which measures the whole tumour regardless of the enhancing components , the results of our study are not surprising , because sorafenib acts as a cytostatic drug causing a reduction in tumour vascularity rather than in tumour size . 
those authors compared recist and mrecist in 70 patients with unresectable hccs treated with sorafenib and found a good agreement between the two methods ( pearsons coefcient r = 0.818 ) , whereas ten of 70 patients ( 18 % ) 219 radiol med ( 2014 ) 119 : 215221 fig . 
1 transverse unenhanced ( a , d ) and contrast - enhanced fatsuppressed three - dimensional gradient - echo images acquired in the hepatic arterial phase ( b , e ) and venous phase ( c , f ) in a 61 - year - old man with hepatitis c virus ( hcv ) - related cirrhosis and multiple hepatocellular carcinomas . 
a target lesion in liver segment iv is shown before ( ac ) and 180 days after anti - angiogenic treatment ( de )  . thick lines indicate mrecist evaluation ( b , e )  . 
this case represents an example of discordance between mrecist and recist criteria and demonstrates that arterial enhancement variation after sorafenib is a useful marker of therapy response who had stable disease according to recist had partial response according to mrecist [ 9 ]  . 
in our study , no patient showed complete response according to either recist and mrecist , possibly as a result of the relatively small size of our study population . the second purpose of our study was to describe changes in hcc enhancement before and after sorafenib treatment . 
in our series , patients with partial response had a statistically signicant decrease in hepatic arterial phase enhancement at followup in comparison to patients with stable disease and progressive disease . 
our results are , however , preliminary and should be analysed with caution because the low number of subjects included in this study prevents any denitive conclusion . we also compared venous phase enhancement among the mrecist groups . 
 [ 16 , 17 ] reported that changes in tumour vascularity were the most specic indicators of treatment response in patients with gastrointestinal stromal tumours ( gist ) on imatinib and proposed new criteria based on evaluation of either tumour size or tumour attenuation after contrast enhancement . 
these and our results suggest that a different method for measuring hcc response after cytostatic therapy , taking into account the changes in enhancement and not only the size of the viable tumour portion , should be incorporated into clinical practice . 
however , this procedure is more time consuming in comparison to recist and mrecist , because three rois within each target lesion need to be recorded on nonenhanced and postcontrast images and compared at baseline and follow - up . 
furthermore , roi placement can be more prone to bias than a single tumour dimension measurement . some authors have reported the potential of perfusion ct and mr imaging examinations as an emerging tool for response to anti - angiogenic the evaluation of tumour fig . 
however , long delivered by ct , breathing and cardiac motion , postprocessing time required to obtain perfusion parameters [ 19 ] and measurement variability are all limitations to the dissemination of this technique . 
one additional means for monitoring the effects of targeted therapy agents on hcc with mr imaging is diffusion - weighted imaging , whose potential , however , has still to be further investigated [ 8 ]  . there are several limitations to our study . 
moreover , contrast media were administered at different rates for ct ( 35 ml / s ) and mr ( 12 ml / s ) imaging , and different mr contrast agents with different doses of gadolinium were also administered . 
fifth , the clinical importance of these ndings is limited owing to the small study population , and further prospective studies would require a more systematic design and a precise timing to evaluate response to treatment using mrecist criteria on the basis of ct or mr changes in tumour density or intensity , respectively . in conclusion , our preliminary results suggest that , in comparison to recist 1.1 , mrecist may show a more favourable treatment response in patients with unresectable hccs treated with sorafenib . 
ulivieri francesco sardanelli received : 28 july 2012 / accepted : 20 february 2013 / published online : 3 december 2013 ( cid : 2 ) italian society of medical radiology 2013 abstract purpose our aim was to estimate the in vivo reproducibility of bone mineral density ( bmd ) at dual - energy x - ray absorptiometry ( dxa ) and to compare fast array , array , and high - denition scan modes . materials and methods a total of 378 patients ( 38 males and 340 females ; mean age 63 9 years ) underwent dxa using a qdr - discovery a densitometer ( hologic )  . 
considering the three scan modes on lumbar spine and right femur , six independent groups of 30 patients were examined twice ( for a total of 180 patients )  . 
the remaining 198 patients underwent three scans of the lumbar spine ( n = 92 ) or of the right femur ( n = 106 ) , one for each scan mode . 
bandirali facolta` di medicina e chirurgia , scuola di specializzazione in radiodiagnostica , universita` degli studi di milano , via festa del perdono 7 , 20122 milan , italy l . 
as a consequence , although the absolute reduction in time and radiation dose is relatively low , when bmd measurement is the aim of dxa , fast array can be generally preferred . keywords dual - energy x - ray absorptiometry ( cid : 2 ) bone mineral density ( cid : 2 ) scan modes ( cid : 2 ) reproducibility osteoporosis is the most common metabolic bone disorder , characterised by low bone mass and microarchitectural deterioration , which leads to diminished biomechanical competence of the skeleton , with a subsequent increase in bone fragility and susceptibility to low - trauma or atraumatic fracture . 
such fractures can occur in any site but are most common in the spine , wrist and hip , and all regions of the skeleton with a high percentage of trabecular bone . osteoporosis is asymptomatic and not clinically manifested until a patient suffers a fracture [ 1 ]  . 258 radiol med ( 2014 ) 119 : 257260 dual - energy x - ray absorptiometry ( dxa ) is recognised as the reference method to measure bone mineral density ( bmd ) accurately and reproducibly , capable of detecting bone mineral loss at an early stage [ 2 ]  . 
moreover , the world health organization ( who ) has established dxa as the best densitometric technique for estimating bmd in postmenopausal women and based the denitions of osteopenia and osteoporosis on dxa results [ 3 ]  . hologic qdr - discovery a , like other densitometers , allows for various scan modalities , i.e. 
however , to the best of our knowledge , a comparison among the three scan modes has never been performed on patients with known or suspected osteoporosis undergoing dxa in clinical practice . since small differences in magnitude of bmd may change the diagnosis and since patient monitoring is based on the capability of detecting such small differences , there is a rationale for estimating the reproducibility of these three scan modes and assessing possible differences among them . our aim was to estimate the in vivo reproducibility of bmd measurements obtained using dxa and to compare the three scan modes of the hologic qdr - discovery a densitometer . each of the three scan modes , without repositioning . 
finally , the percent |dbmd| was obtained . all examinations were performed using a qdr - discovery a densitometer ( hologic , rome , italy ) and processed by a technician with 3 years experience with dxa . scan times were recorded and radiation doses were estimated using the weighting factors issued by the icrp60 [ 6 ]  . results the mean bmd of right femur and lumbar spine obtained with the three scan modes on the six independent subgroups is shown in table 1 . 
however , all |dbmd| were lower than the best sdd , while percent |dbmd| was lower than all lscs except for the lsc of the femur obtained with the fast array scan mode . materials and methods discussion this study was approved by the local ethics committee . patient consent was obtained for using the results of dxa scans for scientic purposes . 
a total of 378 patients ( 38 males and 340 females ; mean age 63 9 years ) underwent a dxa examination for known or suspected osteoporosis . considering the combination of the three scan modes and two anatomical sites ( lumbar spine and right femur ) , six independent subgroups of 30 patients were considered , for a total of 180 patients who repeated the scans twice without repositioning . 
these data were used to estimate intra - scan mode bmd reproducibility . reproducibility was expressed in terms of least signicant change ( lsc ) and smallest detectable difference ( sdd )  . 
in particular , lsc was estimated using the method outlined in the ofcial paper issued by the international society of clinical densitometry [ 4 ] , while sdd was estimated using the blandaltman method [ 5 ]  . 
for sdd calculation , we did the following : with reference to the difference of the two compared datasets , we calculated the coefcient of repeatability as 1.96 multiplied by the standard deviation . the remaining 198 patients underwent one scan of the lumbar spine ( n = 92 ) or of the right femur ( n = 106 ) for the who has established dxa as the best densitometric technique for measuring bmd in postmenopausal women , and has consequently based the denitions of osteopenia and osteoporosis on dxa results [ 7 ]  . 
there is a large body of evidence on osteoporosis that led to the publication of the international society for clinical densitometry 2007 adult and pediatric ofcial positions [ 2 ]  . 
a summary of the advantages of dxa over other densitometric techniques may be found in the review by blake and fogelman [ 8 ]  . most dxa densitometers provide the operator with different scan modes , which differ from each other in terms of spatial resolution , scan time , and patient radiation exposure . 
the densitometer installed at our institution is produced by one of the manufacturers ( hologic ) ofcially accepted by the frax model for fracture risk assessment . regarding the choice of the appropriate scan mode , each manufacturer / model has its own policy . 
for example , our densitometer is set up to always propose the fast array mode and the operator is allowed to change it based on a subjective evaluation of the patients anthropometric characteristics . 
however , as it is well known , it is not feasible to obtain the true patients bmd value in clinical practice . considering that in clinical practice very small bmd differences may result in a different who diagnosis , our study was aimed to assess whether a difference does exist among the three scan modes of our densitometer . besides accuracy there is reproducibility , which is important for the patient follow - up . 
in clinical practice , the most common situation is the comparison of two bmd measurements of a given individual obtained at different times using the same instrument . when a second measurement is performed on a patient , the clinician needs to identify a true change in bmd and , in this regard , only changes greater than the lsc can be ascribed to treatment effects or worsening of the disease . smaller changes may be due to the measurement error . 
in this scenario , the precision of the measurement is more important than accuracy . in the present study , we obtained a very high intra - scan mode reproducibility for all of the three scan modes . 
5.3 % for the values recommended by the iscd , lumbar spine and 5.0 % for the total hip [ 2 ]  . apart from the methodology proposed by the iscd , we also calculated the sdd using the blandaltman method . 
moreover , it has been demonstrated that , to express variability on a percentage basis using the coefcient of variation leads to underestimated variability in patients with low bmd and to overestimated variability in patients with high bmd [ 10 ]  . 260 radiol med ( 2014 ) 119 : 257260 the comparison among the three scan modes highlighted a substantial homogeneity of data . 
moreover , this result holds for both the spine and the femur . the above considerations on precision and bmd comparisons should be integrated with comments on scan time and radiation dose . 
regarding the scan time , we should note that , once the scan mode is chosen , the moving speed of the x - ray tube / detector system is constant in time , i.e. 
even though both scan time and radiation exposure are very low for each scan mode when compared with other x - ray or magnetic resonance examination , the fast array mode has the lowest radiation dose and is the fastest one . from this study , we may conclude that , in clinical practice , the fast array scan mode may be preferred over the other two scan modes . 
however , this general conclusion may be reviewed for particular patients ( such as obese patients or those with a marked arthritis ) , in which the clinicians may decide to use a more reliable scan mode like hd . 
cova received : 24 april 2012 / accepted : 23 january 2013 / published online : 12 december 2013 ( cid : 2 ) italian society of medical radiology 2013 abstract purpose this study was undertaken to compare the different acquisition protocols available in a last - generation multislice computed tomography scanner used for cardiovascular studies , with particular attention to dosimetric aspects . materials and methods our study compared prospective and retrospective electrocardiographic - gating techniques for cardiac imaging . 
for each patient , we performed in vivo dose measurements , using gafchromic lwe compared the effective dose values estimated from the experimental measurements and the dose data reported on the ct console . 
image quality was also assessed . results prospective acquisition allows for major dose savings compared to retrospective acquisition ( mean effective dose , 4.5 msv with prospective acquisition versus 27.5 msv with retrospective acquisition )  . 
fisica sanitaria , ospedale maggiore , azienda ospedalierouniversitaria ospedali riuniti di trieste , strada di fiume 447 , trieste , italy keywords multislice computed tomography ( cid : 2 ) ct coronary angiography ( cid : 2 ) radiation dose ( cid : 2 ) gafcromich lm introduction coronary angiography with multislice computed tomogrpahy ( msct - ca ) is one of the most important innovations in medical diagnostics of the past 10 years [ 1 ]  . since its introduction into clinical practice in 1998 , diagnostic imaging of the heart and coronary arteries has constituted one the most attractive applications of msct [ 2 , 3 ]  . 
despite the major epidemiologic and health economics ischemic heart disease [ 4 ] , intrinsic limitations signicantly hindered the noninvasive study of the coronary tree for several decades , with cardiac and coronary imaging being almost exclusively left to selective coronary angiography , a procedure burdened by non - negligible rates of mortality and morbidity [ 57 ]  . implications of the high spatial and temporal resolution achieved especially by 16 - , 32 - , 40and 64 - detector - row ct scanners and the possibility of obtaining adequate cardiac synchronization have allowed at least some of the limitations to be overcome , leading to a revolution in the management of patients with coronary heart disease [ 8 , 9 ]  . 
the numerous clinical studies published in recent years have widely demonstrated that msct has achieved high levels of diagnostic accuracy , both in the assessment of coronary stenosis and for the follow - up of the patency of aortocoronary bypass grafts [ 1017 ]  . the main pitfalls of msct are related to limitations in resolution . 
as a result , as emphasized by achenbach in the key to obtaining an increasingly accurate 2004 , 250 radiol med ( 2014 ) 119 : 249256 table 1 characteristics of the patients and clinical indications table 2 scanning and reconstruction parameters ; contrast medium clinical indications thoracic aorta evaluation known or suggested coronaropathy evaluation of left atrium and pulmonary veins stable or unstable angina patients with surgical indications asymptomatic patients with multiple risk factors previous bypass planned ptca previous stent placement ptca percutaneous transluminal coronary angioplasty 10 ( 22 ) 8 ( 18 ) 7 ( 16 ) 6 ( 14 ) 6 ( 14 ) 4 ( 9 ) 1 ( 2 ) 1 ( 2 ) 1 ( 2 ) diagnostic performance is to increase the techniques spatial and temporal resolution [ 18 ]  . the rapid technological advances of msct have indeed increased the diagnostic accuracy of noninvasive coronary imaging , improving image quality in terms of both spatial and temporal resolution . the increasingly widespread use of msct - ca has highlighted the need for controlling the radiation dose delivered to the patient in the course of these imaging procedures . 
ever since the early years of msct - ca , the development of the modality has been inuenced by the issue of radiation dose [ 19 , 20 ] , with dose being one of its major problems . 
in the absence of contraindications , sublingual nitrate was administered ( 0.61.2 mg of nitroglycerin , natispray , teofarma srl , salimbene , italy ) a few minutes before the scan . materials and methods scan protocols between november 2010 and may 2011 , we enrolled a total of 41 patients ( 29 men and 12 women , mean age 63 years , age range 3489 years ) who underwent msctca with a 256 - slice ct scanner ( brillance ict , philips healthcare , eindhoven ; the netherlands )  . for some patients ( n = 16 ) , when requested by the referring physician , we obtained a pre - contrast scan for the quantication of coronary calciuthe amount of calcium at the level of the coronary artery walls was assessed using the agatston protocol for calcium scoring . radiol med ( 2014 ) 119 : 249256 during the post - contrast phase , a high - concentration iodinated contrast agent ( iomeprol 400 mgi / ml , iomeron 400 , bracco , italy ) was administered through an 18 - gauge needle cannula into an antecubital vein of the right arm ; the contrast agent was injected at a ow rate of 46 ml / s , for a total volume ( tv ) calculated with the following formula : tv flow ( cid : 3 ) scan delay scan time 10 cc differences in ow rate were dependent on the availability of a viable peripheral access . 
administration of the contrast agent was followed by a 40 ml bolus of saline injected at the same rate , using a dual - head stellant automatic injector ( medrad , indianola , usa )  . the scan was synchronized with the contrast bolus by using a proprietary bolus tracking protocol , which involves drawing a region of interest ( roi ) over the ascending aorta and setting a threshold enhancement value of 100 hu above baseline . 
examinations were acquired with prospective triggering ( with axial scans generally centered at 78 % of the rr interval ) or retrospective gating ( based on a helical scan with retrospective reconstruction of images obtained during the same cardiac phase ) , depending on the clinical needs and the patients heart rate after the betablocker . 
the scan parameters are summarized in table 2 . dose evaluations in addition to recording the ct dose index ( ctdi ) and dose - length product ( dlp ) at the console , in vivo dose measurements were obtained for each patient using special self - developing lm , commercially known as gafchromic xr - qa , which becomes progressively darker during exposure . the lm , cut into 1 - cm - wide strips and protected by a plastic sheet to avoid contact with the skin , was placed and left on the patients throughout the examination , at the level of the sternum , along the right and left middle axillary line and in the interscapular space . 
the strips placed on the chest had a length of * 24 cm , and the others were of 10 cm . the lm is calibrated to provide the dose as a function of the degree of lm darkening obtained by acquiring an image with a scanner . 
to calculate the desper , the dlp value was calculated as the product of the experimental dose indicator ( dsin ) multiplied by the scan range . results of the 41 patients initially enrolled in the study , ve were excluded owing to technical problems related both to the lm reading system and to failures of the software generating the ct dose report . 
the population nally eligible for the study therefore comprised 36 patients ( 24 men and 12 women )  . on the basis of each patients diagnostic needs and physique , the examinations were carried out using ve different acquisition protocols : as shown in table 3 , 42 % ( 15 / 36 ) of patients were imaged with the prospective pro ( prospective alone or prospective with calcium tocol score ) , 50 % ( 18 / 36 ) with the retrospective protocol ( also divided into retrospective alone or retrospective with calcium score ) , mainly reserved for patients with elevated heart rate ( [ 75 bpm ) or frequent extrasystoles or when imaging of the entire chest was required ( indications for heart surgery or follow - up of internal mammary artery bypass grafts )  . 
a small minority of patients ( 3 / 36 ) underwent two imaging procedures because of extrasystoles and movement artefacts causing non - diagnostic results of the prospective protocol ; in this situation , the retrospective protocol allows safer management of extrasystoles , especially in view of the need for a second contrast agent injection , which was always administered after verifying that the total dose of contrast medium was compatible with the patients renal function . 
in these cases , we decided to perform a second scan with retrospective gatingmore likely to provide diagnostic results ( through postprocessing ) partly on account of the inability to administer a 252 radiol med ( 2014 ) 119 : 249256 fig . 
b complex pattern of the dosimetric curve of an axial scan with acquisition of four slabs , the last of which is only partially included in the graph as it extends beyond the gafchromic l ( in this patient , the entire thorax was acquired as the clinical question required investigation of the coronary arteries and thoracic aorta ) the high temporal resolution of the 256 - slice ct system used in our study allows for cardiac ct investigations to be carried out with the prospective technique also in patients with high heart rate , who are difcult to assess with older - generation systems . 
the dose savings are evident , even though it should be pointed out that the mean effective dose estimated for the group of patients investigated with the retrospective protocol was particularly high due to the extent of the mean scan range ( mean 30.6 cm for studies without calcium score and 19.1 cm for those with calcium score )  . 
in several cases , in fact , to investigate specic problems , we selected larger areas extending beyond the cardiac region alone , since in routine clinical practice it is often necessary to assess also the thoracic aorta or the course of internal mammary artery bypass grafts , in addition to the coronary arteries . 
if the effective dose is renormalized to a typical scan range for a cardiac study ( around 16 cm ) , the estimated mean desper value is 15.1 msv , which is identical to the doses reported in the literature ( 1418 msv ) [ 3133 ]  . 
pharmacological management with betablocking agents is therefore mandatory [ 36 , 37 ]  . one of the main arguments raised against msct - ca by the cardiological community is that the patient exposure is greater than that involved in conventional coronary angiography ( cca ) ; this issue would therefore be overcome by the possibility of using the prospective technique , characterized by a low radiation dose . 
it should , however , be borne in mind that this is a merely diagnostic examination , which requires a careful selection of patients who should have a high pre - test probability of no disease [ 32 , 34 ]  . the discriminating factors in selecting the acquisition protocol are regularity of the ecg trace and the heart rate , which should not exceed 75 bpm with our ct system . control of the heart rate is therefore a fundamental aspect of the msct - ca protocol [ 35 ] , without which the advantages offered by better - performing equipment cannot be fully exploited and the results will fall short of the if we subdivide the examinations by tube voltage applied , we can appreciate important dose savings with those performed at low voltage ( 80100 kv ) compared to high voltage ( 120 kv )  . the mean value of the percentage variations between desper and dephilips , lower than 10 % , indicates that the estimates of effective dose obtained with the in vivo dose measurements and the ct system dose reports , respectively , have excellent agreement , considering that degree of uncertainty of gafchromic lm measurements is in the order of 1012 % . 
the ct dose report provided by the systems software can therefore be considered reliable . the analysis of the skin dose proles obtained by scanning the 24 - cm - long lm strip placed on the sternum proved instrumental to understanding the methodological principles underlying the two techniques . radiol med ( 2014 ) 119 : 249256 fig . 
this seems to depend on the temporal duration of the tracker scan , in turn related to the single patients circulation time , with higher doses in patients with impaired pump function . this observation allowed us to optimize the imaging protocol by lengthening the interval between tracker scans ( from 1 scan / 0.5 s to 1 scan / s ) and allowing the patient dose to be reduced . the scanner in use at our institute , having a temporal resolution of 135 ms , allows for safe prospective imaging of patients with heart rates up to 75 bpm , with substantial dose savings . 
use of a beta - blocker thus appears necessary not only to improve the quality of the investigation , but also to extend low - dose prospective imaging to a larger number of patients . 
the entrance surface air kerma values for the hospitals of this survey exceeded the korean reference level of 100 lgy . conclusions the different reference levels might be appropriate for the same examination conducted on children of different ages . 
jeon department of radiology , seoul national university hospital , seoul 110 - 744 , korea e - mail : radiologistray@nate.com pediatric patients are different from typical adult patients and the difference is very important in radiological diagnostic imaging . 
in general , pediatric radiological examinations use as short exposure times and as high ma as possible to minimize image blurring that may result from patient motion because pediatric patients are uncooperative . 
pediatric patients vary in size and a small pediatric patient is smaller than the size of the automatic exposure control ( aec ) chambers , so that for pediatric examinations manual technique is selected . 
selecting the imaging modality , using the appropriate positioning and technical parameters , proper geometry , immobilizing the patient , and using gonadal shielding should be considered in pediatric 232 radiol med ( 2014 ) 119 : 231239 tool projection imaging . 
the concept of drl was subsequently adopted by the international commission on radiological protection in icrp publications 60 and 73 [ 3 , 4 ] and provides guidance on appropriate dose levels for typical examinations for groups of standard - sized patients or standard phantoms [ 5 ]  . 
in korea , a recent survey of pediatric patient doses in chest radiological examinations has been carried out on 135 institutions nationwide by the national institute of food and drug safety evaluation ( nifds ) and the drl which is derived from median values of dose distributions is 100 lgy for chest examinations of the average dimensions of 5 - year - old children [ 7 ]  . however , the entrance surface air kerma ( esak ) values in a signicant number of pediatric patients were larger than the korean reference levels . 
in general , higher detective quantum efciency ( dqe ) , which is a measure of the overall efciency of the detector , offers the potential to reduce patient dose or enable better quality images at the same dose [ 1 , 8 ]  . the main purpose of this study was to investigate the technical parameters for dose reduction in chest radiological examinations by evaluating effective detective quantum efciency ( edqe ) including the scatter radiation from the object , the blur caused by the focal spot , geometric magnication and detector characteristics . 
the secondary objective was to measure the esak values for the standard pediatric chest phantom based on data collected from major hospitals of korea and to compare them with the esak values for pediatric chest radiological examinations from data published in the nrpb - report 318 . methods and materials equipment a typical digital radiographic imaging system was used as a platform for dose optimization in pediatric chest examinations . 
this phantom was designed by the food and drug administration ( fda ) for use in the nationwide evaluation of x - ray trends ( next ) program [ 9 , 10 ]  . 
the bsf for diagnostic x - ray beam qualities are reported in the literature [ 11 ] and extrapolation and interpolation of the bsf data , when necessary , were used . average technical parameters and esak values for each hospital are presented in table 1 . effective dose the patient dose is described by the esak , as illustrated in a previous publication , because of the simplicity of its measurement [ 6 , 7 ]  . 
this may be reasonable in some cases , but it is not sufcient for comparing patient doses of different x - ray examination techniques because the esak is not directly related to radiation risk . 
the effective dose quanties the stochastic radiation risk to a patient undergoing any diagnostic x - ray examination and takes into account the dose and the relative radiosensitivity of all irradiated organs [ 2 , 12 ]  . 
pcxmc is a monte carlo program for calculating the doses in 25 organs and the effective doses in pediatric and adult patients of various ages , heights and weights in medical x - ray examinations [ 13 ]  . 
for tube voltages ranging from 40 to 90 kv in 10 kv increments at the fdd of 100 , 110 , 120 , 150 , 180 cm , the entrance air kerma was measured and converted into an effective dose by using each conversion factor which was obtained as the effective dose per mgy . 
in this study , we examined the effects of the radiation eld size , fdd and choice of projection by comparing the effective dose simulated by pcxmc . effective detective quantum efciency ( edqe ) linear system analysis metrics such as detective quantum efciency ( dqe ) had limited usefulness in characterizing the quality of the image obtained with realistic clinical conditions . 
 [ 14 , 15 ] developed the concept of edqe including the scatter radiation from the object , the blur caused by the focal spot , geometric magnication and detector characteristics and the edqe was determined using the following equation : edqef 0 emtff 021 ( cid : 3 ) sf2 ennpsf 0 ( cid : 2 ) tf ( cid : 2 ) k ( cid : 2 ) q where emtf is an effective modulation transfer function ; ennps is an effective normalized noise power spectra ; sf is a scatter fraction ; tf is a transmission factor through the phantom ; k is an incident air kerma corrected to the detector plane ( mr ) ; q is a number of photons per mm2 / mr ; f0 = magnication - corrected spatial frequency . 
the edge test device was placed on the front face of the phantom and was tilted at about 23 ( cid : 3 ) with respect to the detector pixel array to generate a composite edge spread function ( esf ) , which had an than the pixel pitch . 
2 a side - view of the setup system for measurement of scatter fraction , b an image of a beam - stop array device composed of a 14 9 16 array of 224 6 - mm - thick and 3 - mm - diameter lead cylinders smoothed and differentiated to obtain the line spread function ( lsf ) , as described in a previous publication [ 16 , 17 ]  . 
the rois used in the centers of the beam - stop shadows were 10 - pixel - wide squares , and similar rois adjacent to the beam stop were used . we investigated the effects of the radiation eld size to the area of interest , fdd and choice of projection by using results pcxmc . 
the radiation eld size with xed exposure parameters of 50 kv and 2 mas at a fdd of 100 cm was collimated to approximately 17 9 20 , 19 9 22 and 21 9 24 cm2 . 
the effective dose of 156.70 lsv , was calculated for 50 kv and l mgy of entrance air kerma without backscatter fraction in pcxmc dose calculation , was taken to be the standard dose for various fdds . 
figure 3 shows a comparison of the organ doses resulting from chest anteroposterior ( ap ) and posteroanterior ( pa ) projections for pediatric chest phantoall doses correspond to an entrance air kerma of 1 gy without the backscatter fraction . 
technical parameters of 50 kv with a focus - to - detector distance ( fdd ) of 100 cm and ltration of 2.7 mmal are used for 1 - year - old patient phantom various tube voltages . 
6a , the conventional mtf for the system evaluated in this study was also evaluated using standard beam quality , rqa 5 described by an international electrotechnical commission ( iec ) standard [ 20 ]  . 
4 scatter fraction a for the standard pediatric chest phantom with and without an anti - scatter grid as a function of tube voltage and b for the phantom without an anti - scatter grid as a function of fdd . the solid line is a least square t to a second - order polynomial fig . 
this result indicates that the edqe reects the performance of the whole system , while conventional dqe is reective of detector performance alone . the effective dose quanties the radiation risk to a patient undergoing any diagnostic x - ray examination and includes the ability to compare patient doses associated with various in this study , we types of radiographic examination . investigated the effective dose for various technical parameters in pediatric chest examinations using pcxmc dose calculation version 1.5.1. 
although tight collimation can be difcult because a substantial fraction of pediatric patients are uncooperative , radiation eld size should be reduced in order to reduce unnecessary patient dose and scatter radiation . 
the conventional mtf estimate in the horizontal direction obtained with an edge test device at 71 kvp and 21 mmal ltration ( iec - specied rqa 5 beam quality )  . 
b measured emtf with an antiscatter grid as a function of tube voltage discussion pediatric chest phantom , thus reducing the blur caused by focal spot magnication . figure 8 illustrates the edqe results without and with an anti - scatter grid , as well as the effect of variations in tube voltage . 
on the other hand , when an anti - scatter gird was used the largest peak edqe was similar at 70 kvp and the edqe was smallest at 40 kvp . 
the edqe increased with increasing fdd because greater emtf with the lower radiol med ( 2014 ) 119 : 231239 the effect of pediatric chest ap and pa projections by comparing the effective and organ doses . 
these results indicate that organs in the radiation eld are shielded by the rest of the body . thus , the effective and organ doses can be reduced signicantly with proper choice of projection . average technical parameters and esak values from seven major hospitals in korea for the chest radiological examination of a 15 - month - old infant are presented in table 1 . 
we investigated the effect of various technical parameters for dose reduction in pediatric chest radiological examinations by evaluating edqe including the scatter radiation from the object , the blur caused by the focal spot , geometric magnication and detector characteristics . 
comparing the results of the conventional mtf and emtf , the emtfs were notably lower , and these emtf results reect the actual resolutions that might affect chest images obtained using this system in a clinical setting . 
9. the conventional dqe reects the signal - to - noise ratio ( snr ) performance of the image detector alone , while the edqe reects the performance of the overall imaging systethis suggests that the edqe , as a comprehensive metric of image quality , has the possibility of being used for optimization of various technical parameters . 
in this study , the edqe was largest at 60 kvp without and with an anti - scatter grid when comparing the edqe at different is similar when compared to tube voltage . 
the other edqes were evaluated at 50 kvp and a gaussian t was performed 238 radiol med ( 2014 ) 119 : 231239 primary radiation by the grid in order to compensate for a decrease in the snr ratio . 
for small pediatric patients , the amount of scattered radiation is lower , and the amount of grid attenuation increased unnecessary radiation dose . when the grid is not used the edqe increased with increasing fdd because of the greater emtf with the lower focal spot blurring . 
however , most of the major hospitals in korea employed a short fdd of 100 cm with an anti - scatter grid for the chest radiological examination of a 15 - month - old infant , as shown in table 1 . 
the radiation safety management series for pediatric chest radiographic imaging has been released by nifds in korea and drl which are derived from median values of dose distributions is 100 lgy for chest examinations of the average dimensions of 5 - year - old children [ 7 ]  . 
this is because the chest x - ray examinations for 5 - year - olds are usually performed at a longer fdd which usually varies between 150 and 180 cm , while the chest x - ray examinations for the standard pediatric chest phantom representing a 15 - month - old , 24 - pound infant are used at a shorter fdd of 100 cmoreover , the chest ap projection for a 15 - month - old infant is used because the patient cannot be turned . 
it is necessary to rene the technical parameters to perform pediatric chest examinations in korea . conclusion the majority of esak measured in pediatric chest examinations for a 15 - month - old infant are found to be higher than the korean reference level . 
the purpose of this paper was to investigate the effect of these technical parameters by evaluating the edqe , which was designed to reect the actual snr performance of the entire x - ray imaging systeas a result , some of the reasons for the high esak were found to be use of the grid , short fdd and high mas . 
if an anti - scatter grid is required , for older or more obese children , the composition and grid ratio of the device should be considered to keep doses as low as possible . 
dalba unit of innovation , development , strategic planning and management control , university hospital tor vergata , rome , italy conclusions nonmedical costs heavily affect patients nances as well as having an indirect impact on national health expenditure . 
our results show that petct performed with standard dose cect in a single session provides benets in terms of both medical and nonmedical costs . keywords computed tomography ( cid : 2 ) positronemission tomography ( cid : 2 ) costbenet analysis ( cid : 2 ) petct ( cid : 2 ) image fusion introduction in modern oncology , combined imaging based on positronemission tomography and computed tomography ( petct ) plays a central role in the diagnosis , staging and post - treatment follow - up of patients with neoplastic disease [ 14 ]  . two different ct protocols for combined petct are currently used in diagnostic imaging departments . 
the second uses standard contrast - enhanced ct ( cect ) protocols with intravenous or oral administration of contrast medium in order to obtain high - quality diagnostic images and thus make the examination fully diagnostic . several studies have in fact demonstrated the increase in diagnostic value resulting from combined petct . 
recent studies have shown that petct is the best imaging modality for tumour staging , superior both to pet and to pet and ct performed separately [ 6 ]  . by combining diagnostic morphostructural and vascularity information with metabolic data , petct increases 284 radiol med ( 2014 ) 119 : 283289 diagnostic accuracy in the staging and restaging of lung cancer , gastrointestinal tract cancer , breast cancer , kidney and genitourinary tract cancer , melanoma , head and neck cancer , and lymphoma [ 79 ]  . 
according to the authors , the combined petct examination changed the clinical management in 23.1 % of patients , increasing the sensitivity and specicity of the diagnosis in doubtful cases of recurrent cervical carcinoma at pet alone . 
therefore , in addition to enabling a better clinicalradiological management of patients , petct also has a signicant impact on disease - free survival [ 12 ]  . at our institute , close collaboration between radiologists and nuclear medicine specialists and the availability of the appropriate technology enables us to perform petct with cect , when not contraindicated . this petct acquisition technique is used to improve the diagnostic value of the examination and to implement the concept of one - stop - shop recently introduced in modern medicine . 
the possibility of performing tnm staging with a single examination provides a full diagnosis immediately and reduces the time required for the patient to undergo separate diagnostic examinations [ 13 ]  . 
moreover , in a period of general economic difculty , the evaluation of economic resources and their use is important for effective and efcient healthcare management . different types of costs exist that can be evaluated in the healthcare setting : ( 1 ) direct costs , related to prevention , diagnosis and treatment ; ( 2 ) indirect costs , also known as nonmedical costs , which patients and / or family members have to face to receive care ( cost of transport , cost of lost productivity )  . 
loss of productivity should be recorded for both the patient undergoing the test and those who accompany him . their consequently , the choice of partial or total inclusion of the different cost categories in economic analyses depends on the perspective from which the analysis is conducted . the national health system considers only the direct medical costs , while the service users ( the patients ) bear both the direct and indirect costs . 
the literature shows , however , that even studies with a social perspective often fail to include estimates of loss of productivity , even though this represents on average about half of the total cost of a disease . 
these data were obtained from our hospitals accounts department . as regards personnel costs , we started from the contractual annual salary which we divided by the number of working hours and then minutes to arrive at the cost per minute . 
to calculate the duration of the examination and the use of medical personnel , we used the information provided by the italian society of medical radiology ( sirm ) in its document on the qualitativequantitative appropriateness criteria in diagnostic imaging [ 14 ]  . nonmedical costs of combined petct and cect our survey was conducted on a random sample of 100 patients referred to our institute between october and november 2010 . 
the questions elicited current working status at the time of the questionnaire and the type of work done , the distance from home to the institute , and the means of transport used ( public or private )  . 
moreover , to evaluate the costs of informal care , they were asked to indicate whether someone accompanied them to the institute and the type of work done by the person . 
the question on the type of work done was aimed at dening a mean value for each patients working day . in the assessment of transport costs , where the patient used public transport , this cost was approximated to the cost of the fare indicated in the questionnaire . 
where the patient used their own transport , we referred to the italian automobile club tables to identify the cost per kilometre ( km ) for vehicles with the corresponding cylinder size . 
we therefore calculated the cost of transport by multiplying the cost per km by the distance travelled . statistical analysis a general descriptive statistical analysis was performed by considering gender , age , working status of the patient , presence of person accompanying them and their occupation . 
the results of the statistical evaluation , expressed as mean standard deviation and percentage , were obtained using a microsoft ofce excel 2000 spreadsheet . results medical costs of examinations to calculate the costs of the different types of diagnostic examination , we noted that the performance of ct alone involves the use of the ct room for at least 20 min addition , the medical staff is employed for 30 min , the technical staff for 30 min , the nursing staff for 20 min and the auxiliary staff for 15 min ( table 1 )  . 
performance of a petct examination involves the use of a medical doctor for 90 min , a radiology technician for 40 min , a nurse for 25 min and auxiliary staff for 15 min ( table 1 )  . 
all questionnaires were correctly lled in and the data were subsequently recorded correctly in 100 % of cases . our analysis showed that , at the time of the survey , 35 / 100 patients were currently employed , 51 / 100 were retired , 3 / 100 were unemployed , 4 / 100 were students and 7 / 100 were economically inactive ( table 3 )  . 
to reach our centre , each patient travelled an average of 67.4 km for a total travel time of 186 mproductivity loss in monetary terms was estimated on the whole working day , in consideration of the time needed to travel to and from our centre and the overall duration ( about 3 h ) of the examination . 
the cost of a working day was calculated based on data from the european and italian statistical ofces , eurostat - istat , which indicate a median yearly salary of 22 , 701 euro for the industrial sector , 24 , 843 euro for the tradetransport catering sectors , 27 , 134 euro for the insurance , research and service sectors and 23 , 406 euro for the private sector . for an 8 - h working day lost productivity amounted to 102.16 euro ( range 94.58113.05 ) ( table 4 )  . the 35 currently employed patients , 23 were accompanied by employed individuals and two by pensioners . 
in the evaluation of informal care , among the 80 carers we identied 57 currently employed persons , one student and 23 pensioners ( table 3 )  . the productivity loss for these currently employed patients and carers was calculated by estimating the loss of daily income sustained to attend hospital for the examination or to accompany the patient . 
our evaluation shows that 13.5 % of this expenditure is fully borne by the patients and their families . discussion the aims of oncological imaging are to detect and localise lesions , including their relation with adjacent anatomical radiol med ( 2014 ) 119 : 283289 structures and vessels , characterise the lesions , stage the disease and evaluate response to treatment . 
iodinated contrast mediumoreover , the use of pet with specic tracers for the measurement of tumour cell metabolism allows one to dene the grade of malignancy , the presence and distribution of distant metastases , and the effect of chemoand / or radiotherapy , as well as to differentiate between tumour recurrence and necrosis after radiation therapy . 
with this examination it is possible to obtain a clinical diagnosis and more accurate staging , limiting the number of invasive diagnostic procedures . the main limitations of pet are represented by a poor anatomical depiction , which makes the detection of lesions difcult , especially small size ones , and physiological tracer uptake by normal organs and tissues such as the brain , muscle , salivary glands , thyroid , myocardium , gastrointestinal tract and urinary tract , which makes it difcult to interpret images when lesions are situated in such organs . moreover , there may be no tracer uptake by tumours with low metabolism ( neuroendocrine tumours , bronchioloalveolar carcinoma ) and increased uptake in nonmalignant diseases , such as benign or inammatory lesions . it is recognised that fdg - pet and ct are complementary modalities , and that their combined use is essential in oncological clinical practice . 
the introduction of combined petct has made it possible to reduce the noise during attenuation correction making the procedure faster with a 2030 % reduction in acquisition times and improving image quality . 
moreover , fusion of the highimages of ct with the pet resolution morphological images helps to localise the anatomical site of increased tracer uptake on the pet images ( corresponding to a tumour ) , while ct can provide additional information to complete the clinical picture , such as tumour size and the presence of small lesions not identiable on pet . 
at the same time , the ct data can be used to correct the limitations of pet , as in the calculation of the suv ( standardised uptake value ) or correction of the partial volume effect . few authors have investigated the value and efcacy of combined petct performed with simultaneous diagnostic ct . 
diagnostic multiphase cect as part of the combined petct protocol increased petct sensitivity by detecting lesions that were negative on petct in 17 % of patients and exactly identifying the location of 39 % of tumours in suspected sites , especially in patients inuences clinical management with gastrointestinal tract cancers . 
the same authors [ 16 ] , analysing the results obtained in patients with non - smallcell lung cancer , found that diagnostic ct as part of a pet / ct protocol in a substantial number of patients , especially as a result of better planning of surgical interventions and radiotherapy . 
 [ 17 ] also consider petct with diagnostic ct as the examination of choice in patients who require re - evaluation for recurrent cancer of the colon and rectum . additionally , in the assessment of response to chemotherapy in patients with hodgkins lymphoma , orlacchio et al . 
in fact , according to the authors , the use of contrast agents can discriminate between lymph nodes and physiological uptake of 18f - fdg in the urinary and intestinal tract or uptake in the vascular systestill according to the same authors , assessment with cect is also indispensable in the staging of lymphomatous diseases with extranodal involvement , as in this case pet with nonenhanced ct is not always able to detect focal sub - centimetre lesions due to disease persistence . it is therefore clear that pet and ct support each other and that their combined use leads to signicant benets . 
in diagnostic imaging centres with appropriate technology and competence , petct should be performed with a diagnostic ct protocol both because of the undoubted diagnostic value and to reduce the dose of ionising radiation . 
in fact performing the examinations separately could lead to a diagnostic delay which in turn could delay treatment decisions , with unquantiable clinical and economic disadvantages . according to our experience , there was no evidence of any increase in waiting lists when performing petct with cect compared to petct without cect . 
the examination time is in fact the same for the different techniques ( about 90 min ) and the possibility to perform diagnostic ct at the same time as pet allows a reduction of the daily ct lists . 
the nal result is a possible reduction in the 288 radiol med ( 2014 ) 119 : 283289 time to complete diagnosis and savings in terms of both medical and nonmedical costs ( working days )  . the aim of our study was to evaluate the costs of performing petct with diagnostic ct , both in terms of medical and nonmedical costs . 
as a result , these studies tend to be incomplete and imprecise because they only evaluate the direct medical costs without considering the specic situation of the single patient [ 21 ]  . however , even if the costs borne by the patient are invisible to the national health system , in an economically difcult period it is necessary to perform a detailed analysis of costs deriving from the provision of a health service , of whatever kind it may be [ 22 ]  . regarding medical costs , our evaluation shows clearly that the costs of combined petct are lower than the sum of the costs of the two examinations performed separately . our study shows the importance of nonmedical costs and informal care in economic evaluations in health care . 
in fact , our results show that these costs amount to 13.5 % of the costs deriving from the examination . to our knowledge , there are no other studies that correlate the nonmedical costs of petct and ct examinations . 
consequently , hypofractionation would result in a reduction of nonmedical costs and , when the patient is accompanied , also of informal care costs . with this assessment we can deduce that reducing the number of hospital visits by performing the examination in a single session is useful for lowering the total costs , both medical and nonmedical . performance of petct with cect , instead of separate petct and cect , results in a signicant decrease in nonmedical and informal care costs in addition to reducing the direct medical costs . 
our experience also emphasises the need for dedicated resources for carrying out cost analyses and for a close interaction between clinical and economic researchers , with periodic meetings to update the cost assessments of the various examinations . with respect to costs , the cost of petct with cect is lower than the sum of the costs of the two examinations performed separately . 
moreover , petct with cect could also allow for lower nonmedical costs compared to the sum of cect and petct performed on separate occasions ( cost of transport and loss of productivity for patients )  . these evaluations can be extended to other diagnostic methods . 
also in this case , it would be appropriate for mri to be of diagnostic value rather than just useful for pet attenuation correction and the anatomical localisation of pet ndings . conclusions the petct examination performed with cect should be offered in centres that have the appropriate technology , on account of both its high sensitivity and specicity and the possibility of reducing patient radiation exposure . moreover , the results of our study indicate that nonmedical costs are considerable , so that petct with cect performed during the same session rather than at different times is absolutely efcient and provides signicant advantages in terms of costbenet for the patient , because it halves the costs related to transport and loss of productivity . 
to evaluate the effectiveness of this process , t cells loaded with 111in - radiolabelled aunps were injected directly into the right lung of nude mice for in vivo imaging by micro - spect / ct . 
t cells loaded with 64cu - radiolabelled aunps were then injected into the tail vein of nude mice and imaged by micro - pet / ct . results high uptake signals were observed in the right lung following the direct injection of t cells containing 111in - labelled aunps . 
cui college of science , university of shanghai for science and technology , shanghai , china the dynamic biodistribution of t cells containing 64cu - labelled aunps when compared with 64cu - labelled aunps alone . conclusions this study demonstrated the feasibility of the in vivo imaging of t cells loaded with radiolabelled aunps . keywords t cells ( cid : 2 ) gold nanoparticles ( cid : 2 ) in vivo imaging introduction in the human immune system , lymphocytes with cytolytic functions include natural killer ( nk ) cells and cytolytic t cells . 
the basic principle of adoptive immunotherapy involves the separation of lymphocytes from either the peripheral blood of cancer patients or surgically resected tumour tissue . these lymphocytes are then cultured in vitro in the presence of interleukin - 2 ( il - 2 ) , which stimulates lymphocyte [ 1 ]  . proliferation and enhances anti - tumour adoptive transfer of these lymphocytes into cancer patients results in tumour recession and prevents metastasis , thus achieving the therapeutic purpose . 
therefore , tracking the reinfusion of such lymphocytes in vivo is critical . functions t cells play a central role in cell - mediated immunity and can be genetically modied to target specic receptors expressed on tumour cells . 
1 the aunp labelling process radiol med ( 2014 ) 119 : 269276 materials and methods laboratory animals eleven male balb / c - nu / nu nude mice ( aged 6 weeks , 1822 g ) were acquired from the national cancer institute and housed in micro - isolator cages . 
the synthesis and analysis methods of these two aunp types were similar and , therefore , the method used the purposes of for dota alone is described for illustration . bifunctional opss - peg - nhs stable rod - like gold aunps were prepared using the cetyl trimethyl ammonium bromide ( ctab ) method described by sau et al . 
p - nh2 - bn - dota ( s - 2 - ( 4 - aminobenzyl ) - ) was conjugated ( opyrglycol - n - hydroxysuccinimide idyldisulde - polyethylene ester )  . 
p - nh2 - bn - dota and opss - peg - nhs were mixed at a 1 : 1 molar ratio and the mixture was incubated overnight at room temperature . 
the resulting opss - pegdota was then added to the aunps solution in 10 mm phosphate buffer ( ph 7 ) at a 10 , 000 : 1 molar ratio followed by overnight incubation at room temperature . 
solutions were then incubated at 37 ( cid : 3 ) c for 90 min followed by the addition of the peg - sh blocking agent at an approximate 300 , 000 : 1 molar ratio and incubated at room temperature for 1 h . reaction mixtures were then centrifuged to remove unconjugated 64cucl2 and peg . 
labelling efciencies were calculated by the following formula : ( cid : 2 ) activity in pellet = activity in supernatant activity in pellet ( cid : 3 ) ( cid : 3 ) 100 : radiolabelling stability was also investigated by incubating 64cu - nps in pbs and serum at 37 ( cid : 3 ) c for up to 5 h . 
percentages of 64cu remaining on the nps were calculated by the following formula : ( cid : 2 ) activity in pellet = activity in supernatant activity in pellet ( cid : 3 ) ( cid : 3 ) 100 : the control 64cu - dota sample was prepared by mixing 64cucl2 ( 2.0 mci ) with 200 ll 0.20 mm dota solution ( ph 6.5 ) and incubating at 37 ( cid : 3 ) c for 90 mthe 64cu - dota - peg was prepared by mixing 64cucl2 ( 2.0 mci ) with 200 ll 0.20 mm dota - peg solution ( ph 6.5 ) and incubating at 37 ( cid : 3 ) c for 90 mthe formation of 64cu complexes was veried by radio thinlayer chromatography using a mobile phase that consisted of 50 : 50 meoh / 10 % ammonium acetate on silica plates . and incubated on ice for 510 melectroporation was conducted using a btx cm830 device with the following settings : 1 kv / cm , 4 ms duration , single square pulse . subsequently , the electroporation cup was incubated on ice for a further 510 min before the cells were transferred to 1 - ml culture medium containing 10 % calf serum and incubated at 37 ( cid : 3 ) c in an atmosphere containing 5 % co2 for 2 h . 
preliminary spect and ct images were acquired in axial , sagittal and coronal planes and fusion images were obtained on the workstation . analysis of the biodistribution of t cells containing aunps by micro - pet / ct the other ten mice were randomly divided into two groups . in group 1 , t cells ( 0.5 9 106 cells ) transformed with 64cu - aunps were administered intravenously ( iv ) into ve mice and in vivo micro - pet / ct images were acquired at three time points : immediately after injection , 2 and 18 h after injection . 
all mice were anaesthetised , and pet and ct fusion images were acquired as described above . results transferring aunps into t cells via electroporation all cell handling was aseptically performed in a laminar ow hood . 
t cells were separated from the peripheral blood of normal individuals and the trypsinized cell suspension was transferred to 1.5 ml ep tubes containing dmem medium supplemented with 10 % calf serum and centrifuged at 1 , 000 rpm for 5 min at room temperature . 
the cell suspension and synthesised aunps were mixed at a concentration of 2 9 107 / ml in a 4 - mm electroporation cup the maximum absorption wavelength of the aunps was identied as * 740 nm by uvvis spectrophotometry . zetasizer measurements showed that the particle size of the primary aunps was * 25 nm in diameter with a zeta potential of approximately - 50 mv . 
t cells were injected into mice via the tail vein and micro - pet / ct images were acquired . the biodistribution of t cells containing 64cu - labelled aunps was tracked in group 1 mice . 
this technique involves the adoptive transfer of specic immunity in the form of sensitised lymphocytes or the products of these cells into patients lacking some aspects of cellular immune function , thus providing anti - tumour immunity . 
many groups [ 5 , 6 ] are now committed to generate a variety of t cell chimeric antigen receptors ( car ) that render t cells capable of targeting tumours in an antigen - specic manner . 
the ability to observe such lymphocytes in vivo is critical for the real - time monitoring of their biodistribution , viability and the occurrence of rejection and allows the more accurate assessment of adoptive immunotherapy efcacy [ 7 ]  . surface - modied nanoparticles possess controllable biological characteristics and versatility [ 810 ]  . 
these include the amplication of imaging signals that emanate from the encapsulated imaging agent , molecular recognition of specic antigen and receptor interactions of target cells by the coupling of antibodies or ligands , transport of one or more treatment factors , and the avoidance of biobarriers , such as by pegylation to increase their circulation time for the avoidance of macrophage uptake . 
therefore , surface - modied nanoparticles concurrently perform a variety of important functions in disease treatment and diagnosis [ 11 ]  . aunps exhibit many favourable features [ 1214 ] such as controllable shape and size , mild surface chemistry and radiol med ( 2014 ) 119 : 269276 fig . 
3 micro - pet / ct images showing the dynamic biodistribution of t cells carrying 64cu - labelled aunps good biocompatibility , coupled with a unique plasma surface absorption , light scattering and other physical properties . 
to date , the majority are\50 nm in diameter , with uniform size and good colloidal stability . these aunps are spherical , shell - like , rod - like or cage - like and the surface is easily modied with targeting proteins , specic biological markers and aptamers . 
furthermore , gold is easily identied and characterised by commonly used elemental analysis methods , thus enabling the improved validation of imaging data . in this study , a conjugation procedure was developed to label nps with 64cu ( t1 / 2 = 12.7 h ) , using bifunctional opss - peg - nhs and the bifunctional chelating agent , dota - nh2 . 
5 kinetic analysis of imaging agent uptake in the lung and liver tissues of group 1 and group 2 mice showing marked differences in the biodistribution of t cells containing nanoparticles compared with nanoparticles alone radiol med ( 2014 ) 119 : 269276 liver rapid clearance the ability to transfer nanoparticles and a wide range of other materials into t cells via electroporation can be exploited to adapt t cells as biological delivery vehicles capable of targeting specic tissues , such as tumour tissue , and to facilitate the in vivo imaging of these tissues [ 18 ]  . furthermore , aunps can be used to target the delivery of therapeutic genes and chemotherapeutics for tumour therapy [ 19 , 20 ]  . 
this improvement in local retention can also enhance the efcacy of the therapeutic effect [ 21 ]  . electroporation involves the subjection of cells to a controlled electric eld , such as a transient high - intensity electric eld , which results in reversible pore formation in the cell membrane . 
in general , the electroporation transfection efciency and cell viability are affected by factors that include pulse amplitude and duration , temperature , buffer composition , cell status and volume [ 23 , 24 ] , with pulse amplitude being the key parameter . 
in contrast , if the pulse amplitude is too high , the viability of transfected cells is greatly affected , which in turn decreases the transfection efciency . in the initial phase of our experiment , 111in - labelled aunps were transferred into t cells via electroporation , and directly injected into the right lung of mouse 1 . 
after the injection of transfected t cells into the tail vein of group 1 mice , the acquisition of micropet / ct images at three time points clearly showed the dynamic biodistribution of t cells loaded with 64culabelled aunps in the body of normal nude mice . 
control images of 64cu - aunp alone from group 2 mice showed a marked difference in the biodistribution of radiolabelled nanoparticles alone when compared with t cells loaded with 64cu - labelled aunps . these preliminary studies indicated that the in vivo imaging of t cells loaded with radiolabelled aunps was feasible . 
they represent a basis for the further development of imaging protocols that can assess the delivery of multifunctional aunps by genetically - modied t cells into tumour - bearing nude mice [ 25 ]  . 
the clinical and technical characteristics of the rst ten proton beam radiotherapy treatments are reported . materials and methods ten patients , six males and four females ( age range 2773 years , median 55.5 ) , were treated with proton beam radiotherapy . 
after one to two surgical procedures , seven patients received a histological diagnosis of chordoma ( of the skull base in three cases , the cervical spine in one case and the sacrum in three cases ) r . 
prescribed doses were 74 gye for chordoma and 70 gye for chondrosarcoma at 2 gye / fraction delivered 5 days per week . results treatment was well tolerated without toxicityrelated interruptions . 
after 612 months of follow - up , no patient developed tumour progression . conclusions the analysis of the rst ten patients treated with proton therapy at cnao showed that this treatment was feasible and safe . 
currently , patient accrual into these as well as other approved protocols is continuing , and a longer follow - up period is needed to assess tumour control and late toxicity . keywords proton therapy ( cid : 2 ) skull base ( cid : 2 ) spine ( cid : 2 ) chordoma ( cid : 2 ) chondrosarcoma ( cid : 2 ) hadrontherapy introduction the italian national centre for oncological hadrontherapy ( centro nazionale di adroterapia oncologica , cnao ) was established in 2001 by the italian ministry of health , and a project was launched to realise a centre designed for the use of charged particles for cancer treatment . 
the facility is equipped with a synchrotron to accelerate protons and ions and with three treatment rooms , two with xed horizontal and one with both horizontal and vertical beam lines . 
after relevant commissioning tests , clinical activity started with protons in september 2011 and with carbon ions in november 2012 . 278 radiol med ( 2014 ) 119 : 277282 we report the results of the rst ten patients affected by skull base and spine chordoma and chondrosarcoma who have completed proton beam radiotherapy , with an emphasis on the clinical and technical characteristics of the treatment . inclusion into the approved protocols for proton therapy . each patient received in - depth information regarding the potentially expected outcome and side effects and gave their informed consent . materials and methods patient population to start clinical activity , prospective trials for proton beam radiotherapy were designed for established indications as per existing literature and included skull base and spine chordoma and chondrosarcoma and intracranial meningioma . 
they were conducted in agreement with the criteria of the helsinki declaration . we report on the rst ten patients , with a minimum of 6 months follow - up , treated in phase ii trials for skull base and spine chordoma and chondrosarcoma of grade iii . 
the main patients characteristics are shown in table 1 . clinical symptoms and signs were dependent on the site of the disease and included headache , cranial nerve decits , visual and auditory and hypothalamuspituitary dysfunclesions , and mobility tion in patients with intracranial problems and bowel or bladder incontinence for patients with spine tumour locations . five out of ten patients were referred after the rst surgical operation , 3 / 10 were referred after having been reoperated either for gross residual disease or for tumour relapse , and 2 / 10 , deemed unresectable at the time of diagnosis , were referred for proton therapy after undergoing biopsy for diagnostic purposes . 
the ct scan was performed with a slice and interslice thickness of 2 mm . magnetic resonance imaging ( mri ) with a 3.0 t unit ( magnetom verio , siemens , germany ) was also performed in the same position as the simulation ct , using the same immobilisation device . 
the sequences obtained were contrast and noncontrast t1 , t2 images along with fat suppression , flair ( uid - attenuated inversion recovery ) , stir ( short tau inversion recovery ) and adc ( apparent diffusion coefcient ) sequences . 
both ct and mri datasets were transferred via the dicom protocol to the treatment planning system ( tps ) ( syngo vb10 , siemens , germany ) for image fusion prior to segmentation . 
the structures delineated were gross tumour volume ( gtv ) , clinical target volume ( ctv ) , both low dose and high dose , planning target volume ( ptv ) both low and high dose , and organs at risk ( oars )  . 
1 setup verication in a patient with skull base chordoma before treatment delivery radiol med ( 2014 ) 119 : 277282 tumour location and included brainstem , temporal lobes , pituitary , optic chiasm , optic nerves , eyes , cochlea , spinal cord , parotid glands , pharyngeal sphincters , larynx for the skull base region , and rectal wall , sacral nerves , spinal cord , kidneys and small bowel for the spine location ; the skin was delineated as an oar in all cases . 
ctv to ptv expansion was 2 mm for skull base and 4 mm for spine tumours , gures that were obtained after accurate in - house estimation of the patient setup . 
the high - dose ctv values for skull base and spine tumour locations are reported in table 2 . doses were measured in gray equivalent ( gye ) considering a relative biological effectiveness value of 1.1 compared to that of photons . 
in the second phase , the prescribed dose was 20 gye for chordoma and 16 gye for chondrosarcoma at 2 gye per fraction , and sbo was used in two cases and impt in seven cases . 
the maximal acute toxicities recorded are listed in table 3 and consisted of grade 2 oropharyngeal mucositis and nausea and vomiting for skull base tumours , and grade 2 dermatitis for sacral tumours . 
2 relative dose distribution on axial , sagittal and coronal computed tomography ( ct ) slices depicting planning target volume and healthy structures in a case of skull base chordoma 280 radiol med ( 2014 ) 119 : 277282 table 3 maximal acute toxicities during treatment none grade 1 grade 2 grade 3 skull base / cervical spine ( n = 7 ) oropharyngeal mucositis dermatitis alopecia headache nausea vomiting dysphagia dermatitis proctitis dysuria sacrum ( n = 3 ) fully recovered within a few weeks after completion . treatment after a minimum and maximum follow - up interval of 6 and 12 months , all but one patient ( 9 / 10 ) showed sd at mri studies . 
as for subtypes are chondrosarcoma , the usual histological conventional and mesenchymal , which are further classied as grades iiii based on the differentiation [ 14 ]  . grades iii ( low - grade ) are quite indolent and tend to recur locally , while grade iii is more aggressive with a high metastatic potential [ 2 , 3 ]  . 
complete surgical resection offers the best chance of control , but is rarely achievable due to the proximity of nearby critical structures which risk irreversible damage in the case of aggressive resection . consequently , in most cases gross or microscopic residual disease is the norm , which translates into high recurrence rates . 
in our series , only partial resection could be achieved in skull base cases , even though repeat resection was attempted in two out of seven cases . combining surgery with radiation has improved the local control rates in literature studies . 
these tumours are considered to be relatively radioresistant and a minimum useful photon dose is considered to be 6065 gy , or even higher , especially in series where charged particle irradiation was used [ 2 , 3 , 57 ]  . 
in part , these high local recurrence rates in older literature series could be attributed to limitations of imaging for target identication , but even contemporary series reported poor control rates with conventional radiation at dose levels of 5064 gy [ 11 ]  . 
 [ 12 ] , in a study of patient outcome after stereotactic fractionated photon radiotherapy used to treat skull base chordoma and low - grade chondrosarcoma with doses of 66.6 gy ( for chordoma ) and 64.9 gy ( for chondrosarcoma ) , reported 5 - year local control rates of 50 and 100 % , respectively . analysis of predictors for local failure after proton beam radiotherapy by terahara et al . 
this can be achieved by combining adequate surgical resection with protons which can offer a better dose distribution compared to photons . when performing surgery on sacral tumours , care has to be taken to preserve the sacral nerves and cauda equina . chordomas are slow - growing tumours which can reach considerable dimensions in the pelvis before the symptoms appear and the diagnosis can be obtained . 
additionally , in these cases , proton therapy offered a chance to preserve bladder and bowel function while controlling tumour progression . in terms of dose distribution , protons have an edge over photons due to the inherent radiation physics , although radiobiologically they do not differ greatly from photons , having a radiobiological effective value of 1.1 as compared to 1 of photons . 
the characterising feature is the energy deposition in the patient tissues located in the bragg peak that can be opportunely spread out , thereby sparing most of the traversed and the deeper located tissues . 
this high spatial selectivity enables better sparing of the healthy tissues [ 16 , 17 ] and can result in delivery of 1020 % higher doses to the tumour as compared to conventional radiotherapy [ 12 , 18 ]  . 
it has also been reported that protons deliver 1.53 times lower integral dose outside the target volume as compared to x - rays [ 19 ]  . protons can be delivered as either a passive scattered beam or an active spot - scanning technique . 
this way , the high - dose region is limited to the target alone and , unlike the passive scattering systems , there is no extra dose deposition proximal to the target itself [ 20 ]  . 
in the given set of patients , a combination of both sbo and impt techniques was used in most cases . consequent to the superior dose conformation , sparing of normal tissues was possible to a higher extent as compared to conventional radiotherapy , which translated into a low incidence of acute side effects reported in this set of patients , with maximal toxicities being bgrade 2 overall . 
it should be emphasised that , apart from dermatitis , more common in cases of sacral lesions approaching the skin surface , a major proportion of patients had no toxicity at all during the course of treatment . after a minimum of 6 months follow - up , 9 / 10 patients had sd and 1 / 10 was labelled as having pr as per the protocol . 
after radiotherapy , no dramatic shrinkage of the tumour is expected to be seen on imaging studies . accordingly , the general consensus is to dene tumour control as a state of no clinical or radiological signs of tumour progression . late toxicity was assessed in all patients , although the follow - up was limited to 612 months ; two patients were scored as having grade 2 toxicity as per ctcae v 4.0. 
one patient with sacral chordoma had episodic rectal bleeding persisting for 34 weeks that resolved spontaneously , and the other patient , who had skull base chordoma , had an expected hypothalamicpituitary failure and is currently on hormone replacement therapy . 
early toxicity and preliminary ndings of late side effects are in the range of the other literature reports [ 2 , 7 , 14 ]  . conclusion the analysis of the rst ten patients treated with spotscanning proton therapy at cnao showed that this treatment was feasible and could be delivered safely with mild toxicity . 
currently , patient accrual into these as well as other approved protocols is continuing and a longer followup is needed to assess denitive local tumour control rate and late toxicity . the technical and scientic committee , acknowledgments the authors would like to acknowledge the president , the advisory board , the general secretary , the ofce of communications , the department of safety prevention and environment service , department of legal affairs and human resources , the department of radioprotection , the ofce of quality , the accelerator department , the department of infrastructure , the department of administration and finance , the radiation technologist unit and the nursing staff for their contribution towards making it possible to initiate clinical activity at cnao . conict of interest roberto orecchia , v . 
the type of claim was examined in each of the 26 cases , with as many as 15 being related to radiotherapy injury to tissues surrounding the neoplasm , thus confirming this to be the most frequent cause for insurance claims . 
now that the historical causes of error that led to incorrect radiotherapy treatment have been eliminated through strict adherence to protocols , risk management in radiotherapy identifies side effects as the main source of risk for patients and physicians . 
data analysis confirms the need to implement risk management procedures in radiotherapy in which insurance claims ( 15 / 26 cases ) are motivated by side effects of treatment . key words risk management radiotherapy side effects insurance claims riassunto obiettivo . 
si utilizzata unanalisi delle denunce assicurative , nellarco di un periodo congruo di 10 anni , sulla base dei radioterapisti , 256 nel 2004 , assicurati con la societ scientifica . 
stata analizzata la tipologia delle 26 denunce e di queste ben 15 riguardavano danni da radioterapia a tessuti circostanti la neoplasia confermando come tale fattore , analizzato nelle cause di risk management , sia quello pi frequente di denuncia . 
eliminate , con ladesione puntuale ai protocolli , le cause storiche di errore che portavano ad un trattamento radioterapico errato , attualmente il risk management in radioterapia individua negli effetti collaterali la fonte di maggiore rischio per il paziente ed il medico . 
lanalisi dei dati conferma la necessit di attuare norme di risk management in radioterapia nella quale il contenzioso , 15 casi su 26 , riferibile agli esiti collaterali del trattamento radioterapico . parole chiave risk management radioterapia effetti collaterali denunce assicurative f . 
fileni : risk management in radiotherapy introduction introduzione all human activity has associated risk factors , and the awareness of this has led to undertaking measures for protection and prevention . 
risk factors are particularly numerous in medicine and depend on the nature of diseases treated , increasing average patient age , the high number of patients treated each year , the number of professionals involved in treatment and complexity of each individual therapeutic procedure . for a number of years now , awareness has been increasing of the need to assess the various risk factors of hospitalisation , particularly with regard to patient care and treatment , both to implement prevention measures and to reduce the number of litigations and , as a consequence , the amount of insurance premiums , particularly for hospitals that implement forms of risk prevention [ 1 ]  . 
in other words , risk management involves all of the coordinated activities undertaken by a healthcare facility to define work procedures and exclude foreseeable and avoidable risks [ 2 ]  . 
this type of definition and interpretation , which applies to high - risk activities ( above all in aeronautics ) , should also be applied in healthcare , the specific field of action of which involves the patient , treatment of disease and risks the patient may run when treated in a healthcare facility . applying criteria of risk management to medical practice allows identification of all activities carrying an intrinsic risk both those due to the organisation of the healthcare facility and to the actions of healthcare workers themselves and identification of tools necessary for assessing , managing and eliminating those risks . 
these actions are aimed at improving overall healthcare practice since risk management in healthcare means attaining quality and safety in the patients interest [ 3 , 4 ]  . a further aspect of risk management is aimed at specific assessment of medical activity to minimise professional risks related to the various specialties , with the same goal of recognising and preventing thethe concept of safe practice should be seen in the context of clinical governance , considered as healthcare workers monitoring and self - assessment of their practices , which is also aimed at safeguarding the healthcare workers themselves , both physicians and nonphysicians alike , who work within the systeso as to be completely effective , clinical governance must therefore implement risk management programmes within each department or division . 
assessment should therefore include the interaction between risk and clinical governance that is distinctive of the various specialties , thus making all healthcare professionals more responsible for reducing risk . this objective appears to be more ambitious and a greater safeguard for patients than the objective that has been set for several years in the united states , where risk management has the economic - financial aim of reducing economic loss ( incurred by hospitals and physicians ) due to errors and injury to patients , and of reducing insurance premiums as a consequence . ogni attivit umana presenta potenzialmente dei fattori di rischio e la coscienza di ci ha spinto ad intraprendere azioni di tutela e prevenzione . 
in campo medico tali fattori sono particolarmente numerosi : ci dipende dalla natura stessa delle malattie , dallinnalzamento dellet media , dallelevato numero di pazienti trattati ogni anno , dalla pluralit degli operatori coinvolti e dalla complessit ormai raggiunta da ogni singola attivit terapeutica . da diversi anni ci si resi conto della necessit di valutare i diversi fattori di rischio nella ospedalizzazione , specie nella attivit assistenziale e medica , sia per effettuare unopera di prevenzione e sia al fine di ridurre il fenomeno del contenzioso medico - legale e , conseguentemente , anche limporto dei premi assicurativi , specificatamente per gli ospedali che attuavano forme di prevenzione del rischio [ 1 ]  . con il termine risk management si intende quindi linsieme delle attivit con le quali si conosce e gestisce il rischio e grazie alle quali si attuano le procedure per prevenirlo ; rappresenta cio linsieme delle azioni coordinate intraprese in una azienda sanitaria al fine di guidare unorganizzazione lavorativa ad escludere i rischi prevedibili ed evitabili [ 2 ]  . questo tipo di definizione ed interpretazione , valida nelle comuni imprese ed attivit ad alto rischio ( massima in quella aeronautica ) , deve essere applicata anche nellambito sanitario che ha il suo campo specifico di azione verso il paziente , verso il trattamento della malattia , e verso i rischi nei quali il paziente pu incorrere quando trattato in ambito clinico - sanitario . applicare anche nellattivit medica i criteri del risk management consente di evidenziarne tutti gli atti intrinsecamente rischiosi , da parte sia della organizzazione della struttura sia degli atti propri degli operatori , ricercando gli strumenti che ne consentano la valutazione con il fine ultimo di governarli ed eliminarli . 
con tali azioni si persegue il miglioramento dellattivit aziendale nel suo complesso , perch la gestione del rischio clinico in sanit si configura come raggiungimento della qualit e della sicurezza a tutela dei pazienti [ 3 , 4 ]  . un ulteriore aspetto del risk management rivolto anche alla specifica valutazione dellattivit medica per ridurre al minimo i rischi professionali legati alle varie specialit sempre al fine di conoscerli e prevenirli . 
inoltre il concetto di attivit sicura si deve inserire in quello che oggi il governo clinico , inteso anche come controllo ed autovalutazione della propria attivit da parte degli operatori della sanit , e ci a tutela anche degli operatori , medici e non , che in tale sistema operano . il governo clinico , per essere completamente efficace , deve tradurre operativamente allinterno di ogni unit operativa anche il programma di risk management . 
sono quindi da valutare anche gli aspetti di interazione tra rischio e governo clinico ( clinical governance ) che sono peculiari delle varie specialit aumentando la responsabilizzazione di tutti i professionisti e operatori sanitari finalizzata alla diminuzione del rischio . tale obiettivo appare pi ambizioso e tutelante rispetto a quello applicato gi da diversi anni negli usa , nel quale il risk management in radiotherapy radiotherapy has distinctive risk features owing to the invasiveness of radiating techniques used and to the seriousness of neoplastic disease [ 57 ]  . radiotherapy applications in nonneoplastic disease will not be considered here , both because these are numerically more limited and , above all , because the radiotherapists safety margin is much broader since these diseases do not require high radiation doses to control the tumour . 
it is therefore essential to achieve a therapeutic compromise based on appropriateness of radiation treatment required for recovery , personalised for each individual patient affected by a specific type of cancer , and with minimal , even if inevitable , damage to surrounding healthy tissues [ 8 ]  . risk management for the specialist oncological radiotherapist applies to the first assessment of the patient , monitoring the type and course of disease , choice of treatment plan , sharing and acceptance by the patient of both radiotherapy and possible side effects , and during treatment cycles close observation of premonitory symptoms of side effects , all of which is decided on the basis of disease stage and general condition of the patient [ 9 ]  . the considerable changes in the attitude of patients in recent years with regard to their disease , and in particular in the case of cancer , should be borne in mind . 
thanks to the increasing diffusion of news by various media , there is today a much more widespread knowledge of medical topics . just a few decades ago , patients tended to be passively resigned to having cancer and undergoing the necessary treatment whereas today , they are much more informed , or even fully aware , of their disease . 
they are , nonetheless , less well informed regarding discomfort , risks and side effects that treatment may entail , be it surgery , radiotherapy or chemotherapy . increasing survival times and the possibility of complete recovery have , on the one hand , clearly led to increased trust in the positive outcome of treatment while , on the other , they have led to an increasing demand to reduce the toxic effects of treatment and to nonacceptance of their inevitability . 
for decades now , radiotherapy has been performed taking for granted that the diagnosis of cancer , supported by correct clinical , imaging and histological assessment , is certafor this reason , diagnosis of a tumour must be based on definite histological findings . 
this has ( or should have ) led to elimination of the first three types of risk in radiotherapy that were present in the early days of the discipline : ( 1 ) treatment of a healthy patient ; ( 2 ) treatment of an unhealthy patient not suffering from cancer ; ( 3 ) incorrect treatment . given that the conditions have been satisfied for eliminating the risk of inappropriate indication of treatment , the risk inherent in the method of performing treatment , which is still potentially possible , needs to be examined . 
fileni : risk management in radiotherapy risk management ha una finalit economico - finanziaria per ridurre le perdite economiche ( degli ospedali e dei medici ) dovute a errori e danni al paziente nelle cure e , conseguentemente , per ridurre gli importi dei premi assicurativi . il risk management in radioterapia la radioterapia presenta peculiari e specifici aspetti di rischio per linvasivit delle metodiche radianti utilizzate e per la gravit delle patologie neoplastiche trattate [ 57 ]  . non verranno qui considerate le applicazioni della radioterapia in patologie non neoplastiche , sia perch numericamente pi limitate sia soprattutto perch il margine di sicurezza del radioterapista pi ampio non essendo condizionato dalla necessit di erogare una dose elevata per ottenere il controllo della neoplasia . 
in ambito oncologico invece ogni trattamento richiede la ricerca di un equilibrio ottimale determinato dal rapporto tra effetto terapeutico delle radiazioni sulle cellule neoplastiche ed il minor danno ai tessuti sani circostanti la neoplasia . caratteristica fondamentale quindi il raggiungimento di un compromesso terapeutico basato sulladeguatezza del trattamento radiante , al fine della guarigione , personalizzato sul singolo paziente affetto dalla specifica tipologia di cancro , con la minima , anche se inevitabile , compromissione dei tessuti circostanti sani [ 8 ]  . il risk management dello specialista radioterapista oncologo legato alle varie fasi che vanno dal primo approccio con il paziente , al controllo del tipo e decorso della malattia , alla scelta dei percorsi e piani terapeutici , alla condivisione ed accettazione da parte del paziente sia della radioterapia che dei possibili effetti secondari o collaterali , e , durante i cicli di terapia , anche losservazione attenta dei sintomi premonitori degli effetti collaterali ed il tutto sempre deciso in base dello stadio di malattia e alle condizioni generali del paziente [ 9 ]  . bisogna considerare che negli anni latteggiamento dei pazienti nei confronti della malattia , ed in particolare nel caso di tumore , notevolmente mutato . 
solo pochi decenni fa i pazienti manifestavano una rassegnazione passiva nei confronti del cancro e delle necessarie terapie ; oggi invece sono sempre pi informati , se non perfettamente consapevoli , della loro malattia . 
sono tuttavia sicuramente meno informati e consapevoli dei disagi , dei rischi e degli effetti collaterali che qualsiasi trattamento ( chirurgico , radio o chemioterapico ) comporta . evidente che il sempre maggiore allungamento dei tempi di sopravvivenza , se non la guarigione stessa , da un lato , porta ad una maggiore fiducia di risultati positivi da parte dei trattamenti instaurati , dallaltro , rende sempre pi alta la richiesta di ridurre gli effetti tossici del trattamento stesso e la non accettazione della loro inevitabilit . 
ormai da decenni la radioterapia viene attuata dando come fattore acquisito che la diagnosi di cancro , supportata da una corretta valutazione clinico - strumentale ed istologica , sia di certezza e per tale motivo i criteri di diagnosi di neoplasia def . 
fileni : risk management in radiotherapy without the patient having given consent or being correctly informed of the risks and possible ( serious ) side effects ; ( 2 ) correct radiotherapy treatment but not accompanied by a satisfactory therapeutic response in terms of tumour control and symptom reduction or remission ; ( 3 ) incorrect radiotherapy treatment . an example of the first type of possible litigation , which regards informed consent , could be a case of radical radiotherapy of the cervix , which , given the high doses of radiation required for it to be completely effective , can produce more or less serious side effects with possible damage to the small bowel and colon . 
once a methodological error has been ruled out , possible subjective biological factors or unrecognised variables can be considered , which negatively affected the therapeutic response [ 11 ]  . such cases of limited therapeutic response are most commonly attributable to tumour aggressiveness . 
there is , however , the inevitable risk that the patient may compare their own with other situations and be critical of the treatment received ( an attitude supported at times by the physicians tendency to criticise , often in the absence of the necessary information for making a fair judgment )  . the third case incorrect treatment falls into the class of malpractice due to inexperience , carelessness or negligence . 
to prove this , there are methods to analyse a posteriori , independent of the therapeutic result , the correctness of the radiotherapy technique performed and the potential risk factors [ 12 ]  . 
this review is fundamental in that an analysis of real or presumed errors is the basis for implementing risk management . criteria for assessing treatment correctness are the following : ( 1 ) correspondence of the treatment schedule for administration of radiation dose and technical performance by the radiographer ; ( 2 ) identification of the most restricted field of radiation possible taking into consideration the assistance provided by modern diagnostic imaging devices in preparing vono basarsi su dati istologici definitivi . 
ci ha ( o dovrebbe avere ) determinato la scomparsa delle prime tre tipologie di rischio nella attivit radioterapia presenti alla nascita e allo sviluppo della disciplina : ( 1 ) trattare un paziente non malato ; ( 2 ) trattare un paziente malato , ma non di cancro ; ( 3 ) trattare in modo errato . poste le condizioni per eliminare il rischio di inappropriatezza dellindicazione al trattamento , resta da analizzare il rischio inerente alle modalit di esecuzione che ancora potenzialmente possibile . 
questo lattuale risk management radioterapico . la invasivit e tossicit di trattamento pu infatti dare origine a tre tipologie di rischio e di conseguente contenzioso : ( 1 ) trattamento radioterapico corretto , ma senza che il paziente sia consenziente e correttamente informato dei rischi e dei possibili effetti collaterali , anche gravi ; ( 2 ) trattamento radioterapico corretto , ma non accompagnato da una soddisfacente risposta terapeutica in termini di controllo della neoplasia e di riduzione o remissione dei sintomi ; ( 3 ) trattamento radioterapico non corretto . per esemplificare la prima tipologia di possibile contenzioso , che attiene alla sfera del consenso informato , si pu considerare la radioterapia radicale della cervice uterina che , alle dosi radianti necessarie per la sua completa efficacia , produce effetti collaterali pi o meno gravi con possibili danni allintestino tenue ed al colon . 
necessario quindi spiegare sempre al paziente le conseguenze collaterali della terapia radiante , allo scopo di informarlo e discuterne le possibili alternative terapeutiche , e al fine di avere un consenso informato che dovr prudentemente essere documentato con firma di assenso al fine di evitare una fonte pretestuosa di contenzioso [ 10 ]  . sicuramente le condizioni di ansiet del paziente neoplastico possono allungare i tempi di approccio terapeutico , ed anche le alternative proposte possono provocare latenze , pi lunghe anche del dovuto , prima di iniziare il trattamento radioterapico , ma la mancata comunicazione della prevedibilit e non evitabilit dei possibili danni e delle alternative per limitarli , uno dei motivi pi frequenti fra le possibili cause di contenzioso medico - legale . 
in addition , the definition of patient management protocols for tumour type eliminates , or minimises , the possibility of excessive or insufficient dose , in part associated with variations caused by incorrect positioning . 
specific competence of the radiotherapist for the organ or system being treated has become currently accepted among criteria for treatment quality . in this regard , it is unfortunately realistic to state that the radiotherapy culture , particularly linked to specific technological development , hinders absolute understanding of all diseases and the relative optimal treatment plans by a single operator , and as a result , subspecialties have been developed for individual tumours or homogenous groups of tumours . 
if correctly monitored and performed , all the specific points covered enable risk management in radiotherapy . risk assessment in italy through an analysis of insurance claims cases of unsafe behaviour or real or presumed malpractice in radiotherapy are infrequent and little known . 
this is due to the fact that radiotherapists have not overcome the fear that the study of unsafe behaviour is not aimed at identifying causes underlying the behaviour , unsafe action or injury but , rather , at finding the guilty party . a real appreciation of the phenomenon in italy is therefore not possible owing to the absence of national or regional figures regarding such cases . 
nonetheless , we can indirectly gain insight into the phenomenon and type of services most at risk of litigation through analysis of insurance claims made to the insurance company that , according to an agreement , insures a high number of radiotherapists . materials and methods the analysis of litigation through the use of insurance claims has already been performed in radiodiagnostics on a high number of radiologists , accounting for about 50% of italian radiologists , with highly indicative results [ 13 ]  . 
for radiotherapy , data we analysed refer to a 10 - year period , from 1995 to 2004 , with the number of radiotherapists insured increasing from 123 in 1995 to 256 in 2004 ( table 1 )  . results the number of insurance claims made over the 10 years was f . 
per provare ci esistono modalit per analizzare a posteriori , indipendentemente dal risultato terapeutico , la correttezza tecnica di un trattamento radioterapeutico ed i fattori di potenziale rischio [ 12 ]  . 
questa rivalutazione fondamentale , in quanto dallanalisi dei veri o presunti errori che si pu attuare una vera opera di risk management . i criteri per valutare la correttezza del trattamento sono : ( 1 ) la corrispondenza del piano terapeutico elaborato per la somministrazione della dose radiante e la sua esecuzione tecnica da parte del tsrm ; ( 2 ) lindividuazione del campo di irradiazione pi ridotto possibile in considerazione dellaiuto , per lelaborazione del piano di trattamento e di centratura , dato oggigiorno dallutilizzo delle pi moderne apparecchiature di diagnostica per immagine ; ( 3 ) la sicurezza nel controllo di qualit delle apparecchiature e lesattezza della dose erogata , di pertinenza del fisico sanitario . fondamentale definire protocolli organizzativi che indichino le modalit con cui procedere alla verifica costante delle apparecchiature identificando anche il responsabile di ogni processo . 
inoltre , la definizione di protocolli di gestione del paziente per tipologia di neoplasia elimina , o riduce al minimo tollerabile , le possibilit di sovradosaggio o sottodosaggio radiante legato anche a variazioni per errato posizionamento . la competenza specifica per organo o apparato da parte del radioterapista ormai opinione acquisita tra i criteri di qualit dei trattamenti . 
a questo proposito purtroppo realistico affermare che la cultura radioterapica , soprattutto legata allo sviluppo tecnologico specifico , impedisca una conoscenza assoluta di tutte le patologie e dei relativi trattamenti ottimali da parte di un singolo operatore , per cui si sono andate sviluppando nel tempo sottospecializzazioni con indirizzo specifico per singole neoplasie o gruppi omogenei delle stesse . tutti gli specifici punti trattati , se correttamente controllati ed eseguiti , consentono la gestione del risk management in radioterapia . valutazione del rischio in italia tramite analisi del contenzioso assicurativo i casi di comportamento rischioso , o di vera o presunta malpractice , in radioterapia sono occasionali e poco conosciuti . non esiste una raccolta nazionale di dati relativi ad errori o danni in radioterapia . ci dovuto al fatto che non stato superato dagli operatori il timore che lo studio dei comportamenti a rischio non sia finalizzato alla individuazione delle cause che hanno f . 
fileni : risk management in radiotherapy table 1 insured radiotherapists table 2 number and cause of insurance claims in radiotherapy insured , n cause year 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 anno 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 tabella 1 radioterapisti assicurati assicurati , n 26 , of which two were made by the same patient for different reasons ( table 2 )  . in five cases , the cause of the claim was unspecified , while in the remaining 21 , motivations were explicit : four cases related to patient death ( three postradiotherapy : one 2 years after treatment and one during the stay in the radiotherapy department ) , and one case regarded a lesion treated that proved not to be a tumour ; in addition to the unmotivated radiotherapy injury , a claim was also made for lack of informed consent to the treatment itself ( dual claim )  . 
the remaining 15 cases related to radiotherapy outcomes of varying nature ( table 3 )  . discussion and conclusions data analysed confirm what was stated above regarding risk management in radiotherapy , that is , risks of radiotherapy treatment ( and therefore risk of litigation ) pertain to the outcome or complications of treatment . 
in addition , the claim made for incorrect treatment of a nontumorous lesion , if shown to be true , demonstrates that there is still the possibility of radiotherapy treatment of a lesion that turns out not to be a tumour ( or caused by incorrect histological diagnosis that led to the unjustified treatment )  . clearly , the insurance claim is a precautionary , but obligatory , act of reporting to the insurance company the beginning of legal action in which the healthcare worker is involved . 
as such , the insurance claim is not an index of guilt or malpractice which can only be confirmed at the various nontumorous lesion death during or after radiotherapy postradiotherapy injury or outcomes not specified failure to obtain consent total cause lesione risultata non tumorale decessi durante o dopo radioterapia danni o esiti post - radioterapia non specificate mancato consenso totale tabella 2 numero e specifica delle denunce assicurative in radioterapia determinato il comportamento , lazione rischiosa o il danno , ma bens a quella di ricercare un colpevole . non vi quindi possibilit di una raccolta di tale casistica in ambito regionale o nazionale per avere la reale dimensione del fenomeno in italia . possiamo , in maniera indiretta , analizzare le denunce assicurative pervenute alla societ assicurativa che , tramite convenzione , assicura un numero elevato di radioterapisti , per avere la percezione del fenomeno e la tipologia delle prestazioni a maggior rischio di denuncia . materiali e metodi lanalisi del contenzioso , utilizzando le denunce assicurative , gi stata effettuata in radiodiagnostica su un elevato numero di radiologi che rappresenta circa il 50% dei radiologi italiani con dati estremamente indicativi [ 13 ]  . 
per la radioterapia , i dati in nostro possesso si riferiscono ad un periodo di 10 anni , dal 1995 al 2004 , con una adesione del numero di radioterapisti che passata dai 123 del 1995 ai 256 del 2004 ( tabella 1 )  . risultati il numero di denunce assicurative pervenute nel decennio di 26 , delle quali due dello stesso paziente per motivi diversi ( tabella 2 )  . 
delle 26 denunce , in 5 non erano specificate le cause mentre nelle restanti 21 , in cui erano specificate le motivazioni , 4 riguardavano decessi di pazienti ( 3 post - radioterapia , dei quali 1 a due anni dal trattamento , ed 1 durante la degenza in radioterapia ) ; unaltra si riferiva a lesione trattata , ma poi risultata non tumorale , e , oltre al danno da radioterapia non motivata , si denunciava anche il mancato consenso informato al trattamento stesso ( denuncia table 3 reasons for insurance claims due to radiotherapy damage doppia )  . 
fileni : risk management in radiotherapy reasons incorrect dose sores or burns bone marrow damage female genital sphere ( vaginal stenosis , sterility ) pulmonary fibrosis retinal damage osteomyelitis total tabella 3 motivazioni delle denunce per danni radioterapici motivazione errato dosaggio piaghe e ustioni danni midollari sfera genitale fem ( stenosi vag . , sterilit ) fibrosi polmonare danni retinici osteomielite totale stages of the hearing right up to the final sentence but does , however , allow one to assess the scale of the phenomenon . only at the end of legal proceedings , if concluded with a conviction , is there the legal certainty of medical malpractice in the radiotherapy procedure due to either inexperience , carelessness or negligence . the slow pace of the italian legal system and the safeguarding of privacy do not allow us to know the outcome of the insurance claims studied . 
we can , however , state that the number of claims is statistically low ( 1.4% of radiotherapists ) compared with the number of insured physicians and the number of claims , but this does confirm the need highlighted by risk management to carefully follow pretreatment radiotherapy protocols to contain the phenomenon . 
with the elimination of historical causes of error that led to incorrect radiotherapy treatment , thanks to careful observance of protocols , risk management in radiotherapy currently identifies the main source of risk for the patient and the healthcare worker to be side effects induced by treatment . 
in addition , the claim made for radiotherapy treatment of a lesion that proves not to be a tumour , if shown to be true , demonstrates that this is still possible , despite being caused by incorrect behaviour of another specialist and probably with no malpractice on the part of the radiotherapist . discussione e conclusioni i dati rilevati confermano quanto osservato in precedenza nel risk management radioterapico ; cio che i rischi dei trattamenti radioterapici ( e quindi anche delle denunce ) riguardano gli esiti del trattamento e / o le complicanze dello stesso . inoltre la denuncia per trattamento di lesione risultata non tumorale , se provata , insegna che ancora esiste la possibilit di trattamento radioterapico in lesione non tumorale ( o causata da diagnosi istologica errata che ha portato al trattamento risultato non giustificato )  . sicuramente la denuncia assicurativa un atto cautelativo , ma obbligatorio , di segnalazione alla compagnia assicurativa dellapertura di un contenzioso nel quale il sanitario risulta coinvolto . come tale la denuncia assicurativa non un indice di colpa o di prestazione errata che dovr eventualmente essere confermata nei vari gradi di giudizio fino a sentenza definitiva ma consente comunque di valutare le dimensioni del fenomeno . solo al termine dei vari gradi di giudizio , se conclusi con sentenza di condanna , si avr la certezza giuridica della colpa del medico nella prestazione radioterapica , per imperizia , negligenza o imprudenza . la lentezza del sistema giudiziario italiano e la tutela della privacy non ci consentono di conoscere lesito di tali denunce assicurative . possiamo affermare che il numero delle denunce statisticamente basso ( 1 , 4% dei radioterapisti ) correlato al numero dei medici assicurati e al numero delle denunce , ma conferma la necessit , evidenziata dallo studio del risk management , di seguire puntualmente i protocolli pre - trattamento radioterapico al fine di contenere il fenomeno . 
lagalla sezione di diagnostica per immagini , dipartimento di biotecnologie mediche e medicina legale , policlinico universitario , via del vespro 127 , i - 90127 palermo , italy , correspondence to : t.v. 
thirteen patients with histologically proven gist ( size : 430 cm ; mean : 9 cm ) underwent 40 - slice mdct after the ingestion of 1 , 000 ml of water . 
seven out of 13 gists were located in the jejunumileum , 3 / 13 in the stomach , 2 / 13 in the rectum and in one case , the origin remained unknown . 
thirteen out of 13 exhibited inhomogeneous enhancement due to areas of necrosis and cystic degeneration . direct invasion to adjacent organs ( n = 3 ) , ascites ( n = 3 ) , and liver ( n = 1 ) and peritoneal ( n = 1 ) metastases were also detected . 
tredici pazienti affetti da tgis ( dimensioni : 430 cm ; media : 9 cm ) istologicamente confermati sono stati sottoposti a tcmd con una unit 40 - strati , previa assunzione per os di 1000 ml di acqua , prima e dopo somministrazione ev di 120 ml di mdc iodato . 
le immagini sono state esaminate in consenso da due esperti radiologi valutando , per ciascuna lesione , sede , dimensioni , margini , densit , tipologia di crescita e aspetto contrastografico , nonch leventuale presenza di ascite , linfadenopatie , invasione loco - regionale e / o a distanza . 
in prevalenza , i tgis presentavano margini netti e regolari ( 10 / 13 ) , sviluppo extraluminale ( 11 / 13 ) ed un aspetto contrastografico disomogeneo ( 13 / 13 )  . 
la tcmd consente un ottimale bilancio preoperatorio dei tgis , fornendo elementi utili per la loro caratterizzazione . parole chiave tumore gastrointestinale stromale tratto gastrointestinale tc tumori introduction introduzione gastrointestinal stromal tumours ( gists ) are relatively uncommon mesenchymal neoplasms ( 0.1%3% of all gastrointestinal neoplasms ) that originate in the muscularis propria [ 1 ]  . 
in particular , they account for 1%3% of gastric tumours , 20% of small - bowel tumours and 0.2%1% of coli tumori stromali del tratto gastro - intestinale ( tgis ) sono neoplasie mesenchimali nel complesso poco frequenti 0 , 1%3% di tutte le neoplasie gastrointestinali che traggono origine dalla tonaca muscolare propria [ 1 ]  . 
among these , mitotic index , size and site of origin are especially important , with prognosis being worse for tumours with intestinal rather than gastric origin [ 3 ]  . 
although small - bowel enema may depict the endoluminal component of the tumour , computed tomography ( ct ) is able to accurately demonstrate both wall involvement and extraparietal growth , including possible invasion of adjacent structures or the presence of metastatic lesions [ 47 ]  . 
recent studies have shown that the introduction of multidetector ct ( mdct ) can be useful both in characterising gist through depiction of the exophytic component and in evaluating extent of disease through detection of possible distant metastasis or vascular invasion [ 7 , 8 ]  . 
the aim of this study was to describe the ct features of gists studied by mdct ( 40 - slice ) and evaluate the contribution of the technique to preoperative assessment . materials and methods patients and lesions after obtaining approval from our institutions ethics committee , we retrospectively reviewed the ct studies of 13 patients , eight men and five women , aged between 25 and 75 ( mean : 54.6 ) years with a diagnosis of gastrointestinal stromal tumour confirmed by surgical excision and / or biopsy and immunohistochemistry . 
twelve patients had a nonspecific clinical presentation with diffuse abdominal pain ( n = 7 ) , sense of weight in the abdomen ( n = 4 ) , palpable mass ( n = 2 ) , anaemia ( n = 5 ) , haematemesis ( n = 1 ) , melaena ( n = 1 ) and retrosternal pyrosis ( n = 1 )  . 
before the ct study , all patients gave their written informed consent . examination technique ct images were available on our institutes pacs ( impax , agfa - gevaert , milan , italy ) and had been obtained with a multidetector ( 40 - slice ) philips brilliance scanner ( royal philips electronics , andover ma , usa ) using the following study technique : after distending the gastrointestinal tract with 1 , 000 ml of oral water to ensure correct visualization of the vascular tree on the three - dimensional ( 3d ) reconstructions , we made volumetric acquisitions before and after intravenous administration of 120 ml of water - soluble iodinated nonionic contrast material ( iomeron 400 , bracco spa , milan , italy ) infused at a rate of 34 ml / s via a 18 - gauge needle cannula in an antecubital vein of the right arm ; scan delay was about 70 s . 
technical parameters were as follows : 120 kev ; 380 mas ; collimation 400.625 ; slice thickness 0.9 mm ; increment 0.7 mm ; pitch 1.2 ; gantry rotation time 420 ms ; matrix 512512 ; reconstruction algorithm 180 . 
sebbene gi il clisma del tenue possa evidenziare la componente endoluminale del tumore , la tomografia computerizzata ( tc ) in grado di dimostrare compiutamente sia il coinvolgimento di parete che laccrescimento extra - parietale , incluse leventuale invasione delle strutture anatomiche contigue o la presenza di localizzazioni metastatiche [ 47 ]  . 
recenti studi hanno indicato come lintroduzione delle unit tc multidetettore ( tcmd ) possa risultare utile ai fini sia della caratterizzazione , mediante il riconoscimento della componente esofitica , sia di un corretto bilancio destensione , valutando leventuale presenza di metastasi a distanza o di invasione vascolare [ 7 , 8 ]  . 
scopo di questo lavoro stato quello di descrivere gli aspetti dei tgis studiati con tc multidetettore ( 40 - strati ) e di valutare il contributo di questultima nel bilancio preoperatorio . materiali e metodi pazienti e lesioni ottenuta lapprovazione da parte del comitato etico istituzionale , sono stati valutati retrospettivamente gli esami tc di 13 pazienti , 8 uomini e 5 donne , con et compresa tra 25 e 75 anni , ( et media 54 , 6 anni ) , giunti alla nostra osservazione presso la sezione di scienze radiologiche delluniversit degli studi di palermo e affetti da neoplasie stromali gastrointestinali , confermate mediante escissione chirurgica e / o biopsia e analisi immunoistochimica . 
dodici pazienti presentavano un quadro clinico aspecifico , caratterizzato da dolore addominale diffuso ( n = 7 ) , senso di peso in sede addominale ( n = 4 ) , massa palpabile ( n = 2 ) , anemia ( n = 5 ) , ematemesi ( n = 1 ) , melena ( n = 1 ) e , infine , pirosi retrosternale ( n = 1 )  . 
prima dellesecuzione dello studio tc tutti i pazienti hanno fornito il loro consenso informato sotto forma scritta . tecnica desame la documentazione tc era disponibile sul sistema pacs in dotazione alla nostra istituzione ( impax , agfa - gevaert , milano , italia ) ed stata ottenuta con apparecchiatura multidetettore ( 40 - strati ) philips brilliance ( royal philips electronics andover , mass . , usa ) mediante la seguente tecnica di studio : previa distensione del tratto gastroenterico con 1000 ml di acqua per os al fine di non interferire con la corretta visualizzazione dellalbero vascolare con le tecniche di ricostruzione tridimensionale , sono state effettuate acquisizioni volumetriche prima e dopo somministrazione endovena ed work station ( royal philips electronics ) to visualise axial , multiplanar reconstruction ( mpr ) , maximum intensity projection ( mip ) and 3d volume rendering ( vr ) images . image analysis images were evaluated in consensus by two expert radiologists ( with over 15 years experience in abdominal radiology ) blinded to the histological diagnoses . 
 clinical , laboratory and histological investigations were saved separately and used for statistical evaluation using the chi - square and fischer exact test , with statistical significance established at p < 0.05. reference standard definitive diagnosis was obtained by surgical excision in 11 cases and biopsy and subsequent histological typing ( cd117 positivity ) in the remaining two cases [ 1 ]  . results results are summarised in table 1 . 
in order of frequency , 7 / 13 gists ( 53.8% ) were located in the jejunum - ileum , 3 / 13 ( 23% ) in the stomach and 2 / 13 ( 15.4% ) in the rectum ; in the remaining case , mdct was unable to identify the site of origtumour size ranged from 4 cm to 30 cm ( mean : 9 cm )  . most gists ( 11 / 13 ) were of medium size ( > 5 cm and < 10 cm ) ; one was larger than 10 cm , and one was smaller than 5 cin 76.9% of cases ( 10 / 13 ) , the gist had well - defined , sharp margins whereas in the remaining 23.1% ( 3 / 13 ) , the margins were ill defined and irregular . 
sono stati utilizzati i seguenti parametri tecnici : 120 kev ; 380 mas ; collimazione 400 , 625 ; spessore di strato 0 , 9 mm ; incremento 0 , 7 mm ; pitch 1 , 2 ; tempo di rotazione 420 ms ; matrice 512512 ; algoritmo di ricostruzione 180 . 
in 3 / 13 ( 23% ) cases , the tumour appeared to be in close contact with adjacent organs , as there was no clear cleavage plane , and in particular with the bladder dome , small - bowel loops , sigmoid colon and uterus . 
the absence of vascular involvement was confirmed at surgery in 11 cases whereas in the remaining two cases both nonresectable , prevalently giuno - ileale , 3 / 13 ( 23% ) in sede gastrica ed 2 / 13 ( 15 , 4% ) in corrispondenza del retto , mentre nel rimanente caso non stato possibile , allo studio tcmd , identificare con esattezza la sede dorigine . 
la maggioranza dei tgis osservati ( 11 / 13 ) presentava dimensioni intermedie ( > 5 cm e < 10 cm ) ; 1 tgis superava i 10 cm , mentre il rimanente era inferiore a 5 cnel 76 , 9% dei casi ( 10 / 13 ) i tgis presentavano margini netti e definiti , mentre , nel rimanente 23 , 1% ( 3 / 13 ) , erano sfumati ed irregolari . 
in condizioni di base tutte le lesioni presentavano densit disomogenea e , in extraluminal tumours analysis was limited to the images obtained in the portal phase only . liver metastases ( n = 9 ) with a diameter of 0.520 mm and hypervascular appearance in the arterial phase were detected in one case whereas 3 / 13 ( 23% ) patients had minimal ascites in perihepatic locations ( two cases ) and the pouch of douglas ( one case )  . 
no statistically significant correlations were identified between onset of gist and clinical , laboratory or histological parameters . discussion gists are rare gastrointestinal mesenchymal tumours made up of spindle , epithelioid or , more rarely , pleomorphic cells that present a tyrosine - kinase receptor for growth factor cd117 or c - kit , which is useful for histological characterisation [ 9 ]  . 
some authors have reported an increased risk of gist in patients with type i neurofibromatosis , in whom the neoplasm tends to be multifocal , whereas no correlation has been demonstrated with race , gender or occupation [ 4 ]  . 
this observation , at least as concerns the last two variables , was confirmed by our experience . reported sites of origin are , in order of frequency , stomach ( 60% ) , small bowel ( 30% ) , colon - rectum ( 5% ) and oesophagus ( < 5% )  . 
in our series , however , the majority of lesions ( 7 / 13 ) originated in the small - bowel loops , few in the stomach or rectum , and none in the colon or oesophagus . 
this discrepancy is probably connected to the relative rarity of the tumour and consequently to the small number of patients that can be investigated in a single - centre study such as ours . moreover , the higher incidence of small - bowel gists in our series may also related to the considerable size ( 610 cm ) of the majority of tumours observed . in most cases , gists are asymptomatic . 
however , the presence of clinical symptoms , even if variable and nonspecific , is often correlated with the finding of large tumours , which carry a poor prognosis and tend to recur after surgical excision [ 3 , 8 ]  . 
the tumours in our series had a mean diameter of 9 cm , and 12 / 13 patients had clinically appreciable symptoms and signs whereas in the remaining patient , the tumour was discovered incidentally during abdominal ultrasound . 
the clinical picture was totally nonspecific and characterised by abdominal pain ( 7 / 13 cases ) ; this was associated with a sense of weight in four cases and correlated with an exophytic growth pattern observed in six patients , two of whom had a palpable abdominal mass . 
in 3 / 13 ( 23% ) casi la massa tumorale assumeva rapporti di stretta contiguit , senza la presenza di sicuri piani di clivaggio , con gli organi adiacenti costituiti , in particolare , dalla cupola vescicale , alcune anse del piccolo intestino , sigma e utero . 
in accordo con i dati di letteratura , secondo i quali i tgis si manifestano pi frequentemente in soggetti di et media compresa tra i 50 e i 60 anni , nella nostra casistica let media risultata pari a 56 anni [ 2 ]  . 
alcuni autori riportano un rischio maggiore di sviluppare un tgis nei pazienti affetti da neurofibromatosi di tipo 1 nei quali , peraltro , tale neoplasia si manifesterebbe in forma multifocale , mentre , di contro , non stata finora dimostrata alcuna correlazione con la razza , il sesso o lattivit lavorativa [ 4 ]  . 
1a - c extraluminal gastric gastrointestinal stromal tumour ( gist ) in a 68 - year - old man with abdominal paa transverse unenhanced computed tomography ( ct ) scan shows a well - defined and homogeneous mass arising from the stomach , with extraluminal growth ( arrows )  . 
a unenhanced axial multidetector computed tomography ( mdct ) scan shows a slightly inhomogeneous intraluminal mass with well - defined borders arising from the inner wall of the greater curvature and extending to the lesser curvature ( arrow )  . 
a scansione assiale precontrastografica dimostrante una massa a sviluppo endofitico , a margini netti e sfumatamente disomogenea , che origina dalla parete della grande curvatura gastrica estendendosi allinterno del lume sino alla piccola curvatura ( freccia )  . 
pi raramente , i tgis possono coinvolgere direttamente lomento , il mesentere o il retroperitoneo [ 4 , 9 , 10 ]  . a differenza di quanto appena riportato , nella nostra serie la maggioranza delle lesioni ( 7 / 13 ) aveva come sede dinsorgenza le anse del piccolo intestino , mentre le sedi gastrica e rettale hanno presentato unincidenza minore . 
tale discrepanza , verosimilmente , da mettere in relazione alla relativa rarit della lesione e , di conseguenza , al limitato numero di pazienti che possibile esaminare in uno studio fig . 
3a - c extraluminal invasive gastrointestinal stromal tumours ( gist ) of unknown origin in a 60 - year - old man with a palpable abdominal mass and suffering from diffuse abdominal paa axial unenhanced multidetector computed tomography ( mdct ) reveals a bulky , irregular and inhomogeneous tumour ( arrows )  . 
b contrast - enhanced mdct shows the mass to be mainly hypodense in comparison with adjacent muscles and in close contact with the left kidney which appears dislocated and some bowel loops . 
b dopo somministrazione di mdc iodato , la massa si presenta ipodensa rispetto ai piani muscolari adiacenti e contrae stretti rapporti di contiguit con il rene sinistro che appare dislocato e alcune anse del piccolo e del grosso intestino . 
inoltre , la maggior frequenza di tgis a sede nel piccolo intestino riscontrata nella nostra casistica pu anche essere posta in relazione alle considerevoli dimensioni ( tra 6 e 10 cm ) della maggior parte dei tumori osservati . nella maggioranza dei casi , i tgis non si associano ad alcun sintomo . 
tuttavia , la presenza di una sintomatologia clinica , ancorch variabile ed aspecifica , risulta spesso correlata al riscontro di eteroplasie voluminose , le quali non godono di una prognosi favorevole e tendono a recidivare dopo escissione chirurgica [ 3 , 8 ]  . 
nella nostra serie , caratterizzata da lesioni con un diametro medio pari a 9 cm , un apprezzabile quadro clinico risultato presente in 12 / 13 pazienti , mentre nel rimanente caso il riscontro della massa tumorale stato del tutto occasionale , essendo avvenuto durante lesecuzione di un esame ecografico addominale . 
il quadro clinico , peraltro del tutto aspecifico , risultato caratterizzato in primo luogo ( 7 / 13 casi ) dal dolore addominale , questultimo associato in quattro casi a senso di peso e correlato ad una modalit di crescita di tipo esofitico , riscontrata in sei di tali pazienti , due dei quali presentavano una massa addominale palpabile . tuttavia , in ulteriori quattro casi a crescita esofitica non era presente alcuna sintomatologia dolorosa . 
tale osservazione potrebbe essere messa in relazione al diametro medio pi piccolo ( 6 , 7 cm ) di questultimo gruppo di lesioni rispetto al precedente ( 11 , 8 cm )  . 
lascite , descritta nella nostra serie in tre casi , un reperto occasionale , suggerendo che , in genere , questi tumori non sono responsabili di una significativa reazione flogistica [ 1 ]  . 
le metastasi sono unevenienza meno infrequente ( fino al 50% dei casi allesordio ) ma , qualora presenti , esse possono presentarsi con aspetto prevalentemente ipervascolare e si localizzano pi frequentemente in sede epatica e peritoneale , come nei casi da noi riportati , mentre raro il loro riscontro in sede retroperitoneale , pleurica , polmonare , ossea e sottocutanea [ 1 , 2 , 6 , 9 , 11 ]  . a tale proposito , mentre alcuni autori ritengono sufficiente lacquisizione delle scansioni tc nella sola fase portale , altri , invece , preferiscono anche lacquisizione di una fase arteriosa epatica perch convinti che possa portare ad un miglioramento nella detezione di quella quota parte di metastasi epatiche che appaiono riccamente vascolarizzate , come nel caso da noi descritto [ 6 ]  . 
in nessun paziente stata da noi evidenziata la presenza di coinvolgimento linfonodale , reperto peraltro a tal punto incostante da non giustificare lesecuzione routinaria di una linfoadenectomia in tali pazienti [ 12 ]  . tuttavia , il coinvolgimento linfonodale , loco - regionale o a distanza , rientra tra i criteri classificativi impiegati per definire la prognosi di tali tumori , tra i quali ricordiamo lindice mitotico , la sede , le dimensioni , i margini , la gi descritta modalit di crescita o il comportamento dopo somministrazione ev di mdc , come ad esempio la dimostrazione di fenomeni degenerativi intralesionali [ 3 , 13 , 14 ]  . 
4a - c peritoneal metastasis from rectal gastrointestinal stromal tumour ( gist ) in a 33 - year - old woman with a palpable abdominal mass and suffering from diffuse abdominal paa contrast - enhanced axial multidetector computed tomography ( mdct ) reveals a huge , well - defined and inhomogeneous mass ( arrows )  . 
a tali fini la tc , grazie alla sua panoramicit e alla possibilit di consentire un pi accurato studio della parete viscerale , rispetto ad esempio al clisma del tenue , si rivelata un mezzo particolarmente utile per poter indirizzare il paziente ad un adeguato trattamento , sia esso chirurgico o chemioterapico [ 15 ]  . limpossibilit , infatti , di definire chiaramente i margini della lesione o la dimostrazione tc dellinvasione degli organi contigui o di metastasi a distanza consentono di indirizzare il trattamento , anzich alla chirurgia , verso una terapia farmacologica con glivec [ 2 , 46 ]  . 
in particolare , la nostra esperienza suggerisce che la qualit delle ricostrumatemesis , melaena and retrosternal pyrosis in two cases . ascites , seen in three cases in our series , is an uncommon finding , suggesting that these tumours do not generally produce a significant inflammatory reaction [ 1 ]  . 
metastases are less uncommon ( up to 50% of cases at presentation ) but , when present , appear prevalently hypervascular and often involve the liver and peritoneum , as seen in our series . 
on this subject , whereas some authors consider portal - phase scans sufficient , others prefer to acquire images in the hepatic arterial phase as well , as they believe this improves detection of richly vascular hepatic lesions , as in the case described in our study [ 6 ]  . 
none of the patients in our series had nodal involvement , a finding that is so inconstant that routine lymphadenectomy is not justified in these patients [ 12 ]  . 
nevertheless , locoregional or distant nodal involvement is one of the criteria for defining the prognosis of these tumours ; others include mitotic index , site , size , margins , growth pattern or behaviour after i.v. 
administration of contrast material , as well as demonstration of internal degenerative changes [ 3 , 13 , 14 ]  . about 30% of tumours smaller than 6 cm cause symptoms and signs and show malignant behaviour [ 8 ]  . 
it follows that early diagnosis and accurate staging are extremely important for planning appropriate treatment and improving prognosis . in this context , the panoramic capabilities of ct and its ability to visualise the visceral wall more accurately than , for example , small - bowel enema , have made the technique particularly useful in selecting the most appropriate treatment , whether that be surgery or chemotherapy [ 15 ]  . 
inability to clearly define lesion margins or the ct demonstration of invasion of adjacent organs or distant metastasis will orient the decision towards pharmacological therapy with glivec rather than surgery [ 2 , 46 ]  . 
our experience suggests that the high - quality mpr , mip and 3d vr images obtained with the recent isotropic mdct scanners enable evaluation of the complete abdominal vascular tree especially the more distal branches with mip while providing concise views that are particularly appreciated by clinicians and surgeons for preoperative planning [ 2 ]  . most tumours observed in our study were well - circumscribed masses , which were found at histology to be confined to the serosa . 
however , even when these tumours do display malignant behaviour , as seen in our experience , they tend to preserve relatively well - defined , lobulated margins or give rise to fingerlike extensions rather than infiltrate the surrounding organs in an irregular fashion . 
however , the patients clinical history and some ct signs may provide useful information , thanks to the ready depiction of exophytic or intraand extraparietal dumbbell growth patterns [ 17 ]  . 
carcinomas , for example , tend to give rise to luminal stenosis or substenosis ; they have irregular margins , strongly inhomogeneous enhancement and marked tendency towards contiguous and distant nodal and visceral invasion . 
lymphomas , like carcinomas , produce concentric stenosis of the lumen or form bulky masses , which become ulcerated , extend to the adjacent mesentery , and often involve locoregional and distant lymph nodes . 
as regards differentiation with other mesenchymal tumours such as leiomyomas , leiomyoblastomas , leiomyosarcomas , schwannomas and neurofibromas useful diagnostic data will generally be provided by immunohistochemistry and electronic microscopy [ 7 ]  . 
however , even in this case , tumour site may provide some indication , given that leiomyomas commonly develop in the oesophagus and remain confined within the wall [ 10 ]  . 
negative attenuation values on nonenhanced ct scans may indicate the presence of fat , suggesting a diagnosis of gastric lipoma [ 18 ]  . there are some limitations to our study . 
tali tumori , tuttavia , anche quando presentino caratteri di invasivit , cos come riscontrato nella nostra esperienza , pi che infiltrare gli organi circostanti in modo irregolare , tendono a mantenere dei margini abbastanza ben definiti , lobulati , o a produrre delle espansioni digitiformi . 
queste caratteristiche , tra laltro , ne rendono pi semplice lescissione chirurgica [ 12 ]  . nella nostra serie , la maggioranza delle lesioni osservate ha mostrato un aspetto contrastografico disomogeneo , attribuibile alla presenza di aree di necrosi , di emorragia o di degenerazione cistica . per quanto attiene la diagnosi differenziale con le altre neoplasie che originano nellapparato gastro - intestinale , essa non infrequentemente risulta difficoltosa e , spesso , di pertinenza dellanatomo - patologo . 
tuttavia , la storia clinica del paziente e alcune caratteristiche semeiologiche dellindagine tc possono fornire utili indicazioni a tale fine , grazie allagevole riconoscimento della modalit di crescita esofitica o intraed extra - parietale a clessidra [ 17 ]  . 
i carcinomi , ad esempio , tendono a determinare pi frequentemente la stenosi o la sub - stenosi del lume , hanno margini irregolari , captazione fortemente disomogenea del mdc , una spiccata tendenza allinvasivit linfonodale e viscerale , contigua e a distanza . i linfomi , cos come i carcinomi , determinano pi frequentemente una stenosi concentrica del lume del viscere o la formazione di masse di dimensioni notevoli che tendono ad ulcerarsi , oltre che ad estendersi al mesentere contiguo , comportando , inoltre , un frequente coinvolgimento linfonodale loco - regionale e a distanza . i carcinoidi si localizzano solitamente in corrispondenza dellileo terminale determinando anche una reazione desmoplasica locale . 
per quanto pi propriamente riguarda , invece , la diagnosi differenziale con le altre neoplasie di origine mesenchimale quali leiomiomi , leiomioblastomi , leiomiosarcomi , schwannomi e neurofibromi sono sostanzialmente limmunoistochimica e la microscopia elettronica a fornire utili elementi diagnostici [ 7 ]  . tuttavia , anche in questo caso la sede del tumore pu fornire qualche indizio , infatti i leiomiomi sono neoplasie pi frequenti a livello esofageo , dove rimangono spesso confinati nello spessore della parete [ 10 ]  . 
valori densitometrici intralesionali negativi in condizioni di base possono testimoniare la presenza di grasso , suggerendo la diagnosi di lipomi gastrici [ 18 ]  . il nostro studio presenta dei limiti e , in particolare , la sua natura retrospettiva ha comportato limpossibilit di analizzare , tranne che in un caso , le immagini in fase arteriosa . 
as a result , vascular invasion was assessed on the basis of the portal - venous phase alone and the absence of a fat plane separating the lesion from the vessel ; in addition , identification of hypervascular liver metastases was limited by the possibility that these appear isodense to the surrounding liver parenchyma in the portal phase and therefore go undetected [ 9 ]  . in conclusion , mdct provides an important contribution to the staging of gists before treatment , as it is able to identify small , typically hypervascular , liver metastases that , together with peritoneal metastases , are crucial for correct staging of the disease . 
zompatori , sezione di diagnostica per immagini , padiglione barbieri , ospedale maggiore di parma , via gramsci 14 , i - 43100 parma , tel . : + 39 - 0521 - 703432 , e - mail : maurizio.zompatori@unipr.it received : 20 january 2006 / accepted : 20 february 2006 / published online : 29 june 2006 abstract up to now , neither chest radiograph nor high - resolution computed tomography ( hrct ) has much to offer to the diagnosis of patients with chronic bronchitis ( cb )  . 
a lot of hrct findings can be observed , but they cannot be regarded as specific for pure cb . their evaluation is often subjective , and measurements are poorly reproducible . 
a better comprehension of the large airways abnormalities is important because it is likely that the same pathophysiologic process that causes small - airway obstruction also takes place in larger airways , where it has less functional effect . key words chronic bronchitis chronic obstructive pulmonary disease smoking emphysema riassunto fino ad ora n la radiografia del torace , n la tomografia computerizzata ad alta risoluzione ( hrtc ) hanno contribuito molto alla diagnosi di bronchite cronica ( bc )  . 
lispessimento delle pareti bronchiali dovrebbe essere considerato un reperto non sensibile oltre che non specifico ; la bc non dovrebbe essere equiparata solamente ad un ispessimento delle pareti bronchiali visto che lo spessore pu variare attraverso i diversi stadi di sviluppo della malattia . usando la tc volumetrica a strato sottile , si possono notare frequentemente delle piccole fossette lungo la superficie interna dei bronchi centrali nei pazienti con bc . 
importante una miglior comprensione delle alterazioni a carico delle grandi vie aeree , poich il processo pato - fisiologico sembra essere lo stesso di quello che causa lostruzione a livello delle piccole vie , anche se con conseguenze ridotte dal punto di vista funzionale . parole chiave bronchite cronica affezione ostruttiva polmonare cronica fumo enfisema chronic bronchitis ( cb ) is a clinical diagnosis characterized by chronic productive cough on most days during at least three consecutive months for not less than two consecutive years , after having excluded other causes of chronic cough , such as bronchiectasis or tuberculosis [ 1 ]  . 
the mucuscontaining exudate produced by inflammatory response la diagnosi di bronchite cronica ( bc ) clinica e si basa sulla presenza di tosse cronica produttiva per pi di tre mesi e per almeno due anni consecutivi , dopo aver escluso altre cause di tosse cronica come le bronchiectasie e la tubercolosi [ 1 ]  . 
cb may precede or accompany pulmonary emphysema . pure cb is not necessarily associated with functional impairment , but reduced expiratory flow is often present , usually increased by the coexistent emphysema and bronchiolitis . 
obstruction , which is usually concentrated in small airways , has both reversible components ( mucous plugging , smooth muscle hypertrophy ) and irreversible ones ( fibrosis and stenosis ) [ 2 ]  . 
the main pathologic changes are always described in the mucus - secreting elements : hypertrophy and hyperplasia of the bronchial glands , which develop enlarged ducts , and goblet - cell hyperplasia . 
the enlargement of mucous glands can be measured histologically using different indexes , such as the time - honoured reid index , which is the ratio of gland thickness to bronchial wall thickness measured from the epithelial basement membrane to the perichondrium [ 4 ]  . 
the reid index correlates rather well with the severity of mucous hypersecretion [ 5 ] , but there is overlap between normal subjects and patients with cb [ 6 ]  . 
the absolute increase in gland thickness is small , and the mean reid index in the unselected adult population is 0.35 mm ( range : 0.260.44 ) and in cb is 0.51 mm ( range : 0.430.59 ) [ 7 ]  . 
 [ 11 ] , using chest x - ray , were able to demonstrate that the wall thickness of the bronchi visualized end - on in the parahilar regions is of limited value in distinguishing patients with cb from normal subjects . 
the increased lung markings give rise to the appearance of so - called dirty lung , though we currently know that it can also be related to other smoking - related diseases , such as respiratory bronchiolitis with interstitial lung disease [ 12 , 13 ]  . 
la bc non necessariamente associata ad una modificazione funzionale , sebbene una alterazione dei test di funzionalit respiratoria di tipo ostruttivo sia di frequente riscontro , incrementata soprattutto dalla concomitante presenza di enfisema e bronchiolite . 
lostruzione , che principalmente causata dalla patologia delle piccole vie aeree , presenta aspetti sia di tipo reversibile ( tappi mucosi , ipertrofia della muscolatura liscia delle pareti bronchiali ) che di tipo irreversibile ( fibrosi e stenosi ) [ 2 ]  . 
specifiche correlazioni patologiche con la sindrome clinica propria della bc sono difficili da reperire . sebbene alcuni pazienti con bc possano non dimostrare alcuna anormalit istologica , la maggior parte di essi presenta modificazioni a carico delle vie aeree di medio - grande calibro ; in alcuni casi di bc il processo infiammatorio si estende fino a coinvolgere anche i bronchioli pi piccoli [ 3 ]  . le principali alterazioni patologiche si osservano a carico degli elementi muco - secernenti : ipertrofia e iperplasia delle ghiandole bronchiali con dilatazione dei dotti e iperplasia delle cellule caliciformi . lingrandimento delle ghiandole mucose pu essere misurato su preparati istologici ricorrendo a diversi indici , il pi noto dei quali lindice di reid che corrisponde al rapporto tra lo spessore della ghiandola bronchiale e lo spessore della parete bronchiale misurato dalla membrana basale al pericondrio [ 4 ]  . 
lindice di reid si correla significativamente con la gravit dellipersecrezione mucoide [ 5 ] , ma esiste una certa sovrapposizione tra i valori riscontrati in individui asintomatici ed in pazienti affetti da bc [ 6 ]  . 
laumento assoluto dello spessore ghiandolare di minima entit e lindice di reid nella popolazione generale in et adulta pari in media a 0 , 35 mm ( range : 0 , 260 , 44 ) mentre , nei bronchitici cronici , in media pari a 0 , 51 mm ( range : 0 , 430 , 59 ) [ 7 ]  . altri reperti patologici includono : infiltrato di cellule infiammatorie croniche a livello della mucosa bronchiale , metaplasia squamosa epiteliale , ipertrofia muscolare liscia delle pareti bronchiali e bronchiolite cronica . 
la presenza di atrofia cartilaginea , di esiti cicatriziali e di tratti stenotici bronchiali alternati ad altri dilatati , pu essere inoltre riscontrata negli stadi pi avanzati della malattia . fino ad ora n la radiografia del torace , n la tomografia computerizzata ad alta risoluzione ( hrtc ) hanno contribuito molto alla diagnosi di bc . 
furthermore , an objective evaluation of bronchial wall thickness is difficult to obtain ; airways measurements are influenced by the angle between the bronchial long axis and the plane of section and by other variables , such as lung volume and window settings . 
dedicated softwares may provide more objective measurements , but they are not widely available and might still overestimate wall thickness , underestimating the luminal area as well as the inner diameter [ 16 , 17 ]  . 
although patients with emphysema had significantly lower mean lung attenuation at 90% of vital capacity than normal subjects or patients with cb , attenuation was the same for normal subjects and patients with cb [ 18 ]  . variable emphysema extents can be seen ( centrilobular and / or paraseptal ) , and emphysema may be the predominant feature in patients with symptoms of cb . 
as would be expected , evidence of air trapping may be identified on expiratory images in patients with cb , mostly due to mast - cell and neutrophil infiltration of the smooth muscle layer in smokers [ 19 ]  . 
in order to better characterise hrct features of cb , further efforts are warranted . firstly , the finding of bronchial wall thickening may even be considered nonsensitive as well as nonspecific ; cb should not only be equated with narrowing of the airway since thickness of the bronchial wall may vary among different stages of development [ 20 , 21 ] : chronic catarrhal bronchitis , chronic catarrhal - fibrotic bronchitis , chronic granulating bronchitis , chronic purulent bronchitis , and chronic fibrotic bronchitis . 
although longitudinal studies are lacking , the bronchial wall might also grow thinner during disease progression , as subjects with more severe emphysema have less airway thickening than those with less severe emphysema [ 16 ]  . although bronchography is almost completely outdated and bronchographic findings are mainly of historic interest , radiologists of the first half of the twentieth century using that technique were able to illustrate a complex and refined set of delle pareti bronchiali ( viste come opacit ad anello o a binario ) e laccentuazione del disegno parenchimale polmonare dovuta alla sovrapposizione delle pareti bronchiali ispessite e alla malattia delle piccole vie aeree [ 8 , 10 ]  . 
 [ 11 ] , utilizzando il radiogramma del torace , furono in grado di dimostrare che la valutazione dello spessore parietale dei bronchi visti di infilata in sede para - ilare scarsamente utile per discriminare tra pazienti normali e pazienti con bc . laccentuazione del disegno parenchimale polmonare nota anche con il termine di polmone sporco , che attualmente sappiamo essere presente anche in pazienti affetti da altre malattie connesse al fumo , come la bronchiolite respiratoria associata a patologia polmonare interstiziale [ 12 , 13 ]  . 
altri segni radiografici associati comprendono : lieve iperinsufflazione polmonare con oligoemia ( usualmente dovuta allenfisema associato ) , trachea a fodero di sciabola , dilatazione del cuore e delle arterie polmonari centrali per ipertensione arteriosa polmonare . non molto si sa dei reperti hrtc riscontrabili nella bc . poich le alterazioni sono localizzate a livello delle vie aeree , le ricerche sono state focalizzate soprattutto sullispessimento delle pareti bronchiali , sebbene questo sia un reperto non specifico [ 14 ]  . 
 [ 14 ] hanno rilevato un ispessimento delle pareti bronchiali in un terzo dei pazienti fumatori , anche se questa alterazione non era comunque specifica , vista la sua presenza nel 18% dei non fumatori . 
inoltre , una misura oggettiva dello spessore delle pareti bronchiali difficile da ottenere ; le misurazioni dipendono dallangolo tra il maggior asse bronchiale ed il piano di scansione assiale e da altre variabili come il volume polmonare e i valori di finestra elettronica . 
alcuni software dedicati potrebbero offrire qualche vantaggio , ma non sono ampiamente diffusi e possono portare a sovrastime dello spessore parietale , sottostimando larea endoluminale ed il diametro interno [ 16 , 17 ]  . misure densitometriche eseguite su esami tc , spirometricamente controllati al 90% e al 10% della capacit vitale , sono state impiegate per distinguere i pazienti con enfisema da quelli con bc ; sebbene i pazienti con enfisema avessero al 90% della capacit vitale valori di attenuazione media polmonare significativamente pi bassi dei pazienti normali o con bc , non vi era differenza tra quelli dei pazienti normali e quelli dei soggetti con bc [ 18 ]  . si possono notare gradi di estensione di enfisema ( centrolobulare e / o parasettale ) variabili , e lenfisema pu essere lalterazione dominante nei pazienti che presentano sintomi di bc . 
come facilmente ci si potrebbe aspettare , la presenza di aree di intrappolamento aereo pu essere evidenziata nelle scansioni espiratorie , soprattutto a causa della infiltrazione infiammatoria di mastociti e neutrofili della muscolatura liscia dei piccoli bronchi dei fumatori [ 19 ] ; questo processo potrebbe progressivamente condurre al rimodellamento permanente delle piccole vie aeree . 
1 la broncografia mostra piccole estroflessioni lungo il bronco lobare medio ( freccia nera ) e superiore di destra ; si osserva inoltre la perdita del nomale affusolamento bronchiale e la dilatazione e lincompleto riempimento delle vie aeree periferiche con terminazioni brusche . 
2 piccola depressione ( freccia nera ) lungo la parete del bronco lingulare in un paziente affetto da bronchite cronica . findings related to cb , which included [ 22 , 23 ] : loss of bronchial tapering , intraluminal secretions ( mucous plugs ) , dilatation or incomplete filling of the peripheral airways with abrupt terminations , excessive collapse during expiration ( or cough ) due to bronchomalacia , accordion - like pattern of the bronchial inner surface due to filling of dilated mucus gland ducts , and pseudodiverticular projections that were analogous to the visualization of paraurethral ducts in cases of urethritis . the latter abnormalities appeared to start as submicroscopic depressions and dilatations of the bronchial gland ducts on the mucosal surface [ 24 ]  . 
3 la ricostruzione coronale a strato sottile mette in evidenza multiple fossette che appaiono come diverticoli lungo il bronco principale di destra . prevalenza della tracheo - broncomalacia non nota ( probabilmente non meno del 10% dei soggetti con bpco )  . 
altri reperti hrtc comprendono : secrezioni endoluminali tracheo - bronchiali , noduli centrolobulari , opacit ad albero in fiore ( tree in bud ) , aree di opacit a vetro smerigliato ( ground - glass ) o zone consolidative dovute alla sovrainfezioni . in sostanza , diversi reperti hrtc possono essere riscontrati , ma essi non possono essere considerati specifici per la diagnosi di bc ; la loro interpretazione spesso soggettiva e le misurazioni sono scarsamente riproducibili . 
innanzitutto , lispessimento delle pareti bronchiali dovrebbe essere considerato un reperto non sensibile oltre che non specifico ; la bc non dovrebbe essere equiparata solamente ad un ispessimento delle pareti bronchiali , visto che lo spessore pu variare attraverso i diversi stadi di sviluppo della malattia [ 20 , 21 ] : bronchite cronica catarrale , bronchite cronica catarrale - fibrotica , bronchite cronica granulomatosa , bronchite cronica purulenta e bronchite cronica fibrotica . 
sebbene manchino studi longitudinali , le pareti bronchiali potrebbero anche assottigliarsi con il progredire della malattia , dato che stato osservato che i soggetti con enfisema pi esteso presentano pareti bronchiali meno spesse di quelli con enfisema meno severo [ 16 ]  . sebbene la broncografia sia ormai quasi completamente dimenticata e i reperti broncografici abbiano un interesse storico , i radiologi operanti nella prima met del xx secolo furono in grado di sviluppare un corpus di segni broncografici collegati alla bc rilevante per raffinatezza e complessit [ 22 , 23 ] : perdita di normale affusolamento bronchiale verso m . 
these signs were observed mostly in the large bronchi of patients with cb although some authors recorded such findings in normal adults as well as in patients affected by tuberculosis and bronchiectasis [ 25 , 26 ]  . 
a better comprehension of large airways abnormalities is important because it is likely that the same pathophysiologic process that causes small - airways obstruction also takes place in larger airways , where it has less functional effect [ 27 ]  . 
although the presence of bronchial pits is rarely challenged , the use of thin - collimations , multiplanar reconstructions and different reconstruction algorithms , such as minimum - intensity projection ( minip ) , are recommended to improve detection of such bronchial abnormalities . 
multiple dilatazioni e depressioni si fondevano fino a creare un diverticolo che erniava attraverso i fasci della muscolatura liscia ; la rottura di questultima determinava quindi la formazione di ampi diverticoli . 
the purpose of this study was to evaluate the efficacy of postoperative radiotherapy in reducing the incidence of prostate carcinoma ( pca ) recurrences after radical prostatectomy ( rp ) , define the importance of the time interval between surgery and radiotherapy for prognosis and the toxicity of the treatment in comparison with radiotherapy or surgery alone . 
the treatment was considered adjuvant if was conducted less than 6 months after rp , if there was no macroscopic residual disease and if there was no progressive increase in serum prostate - specific antigen ( psa ) and salvage if performed more than 6 months after rp , for the presence of macroscopic recurrence or with rising psa . 
the best results are obtained with early postoperative treatment ( adjuvant ) ; adjuvant radiotherapy of high - risk forms yields better results if performed with psa less than or equal to 1 ng / ml . key words prostate carcinoma radical prostatectomy postoperative radiotherapy riassunto obiettivo . 
il trattamento stato considerato adiuvante se condotto meno di 6 mesi dopo pr , se non era presente residuo macroscopico di malattia e in assenza di incremento progressivo dellantigene prostatico specifico ( psa ) serico ; di salvataggio se effettuato pi di 6 mesi dopo pr , per la presenza di malattia macroscopica recidiva o con psa in crescita . 
la sopravvivenza libera da ricadute ( rfs ) a 5 anni e 10 anni risultata rispettivamente pari a 538% e 3214 , 2% per lintera casistica , a 769% e 382 , 7% nel gruppo di pazienti trattati con radioterapia adiuvante e a 3610% e 2810% nei pazienti sottoposti a radioterapia di salvataggio ( p < 0 , 01 )  . 
inoltre , la rfs a 5 anni risultata migliore nel gruppo di pazienti trattati con radioterapia adiuvante e psa inferiore o eguale a 1 ng / ml ( p < 0 , 05 )  . 
i risultati migliori si ottengono con un trattamento precoce dopo chirurgia ( adiuvante ) ; inoltre , la radioterapia adiuvante delle forme ad alto rischio ha migliori risultati se effettuata con psa1 ng / ml . parole chiave carcinoma prostatico prostatectomia radicale radioterapia post - operatoria b . 
however , although radical surgery has proved to be just as effective as radiotherapy alone for intracapsular forms , around 15%40% of patients with stage t1 - t2 prostate cancer develop a recurrence [ 1 , 2 ]  . 
moreover , in about one third of patients with recurrence , the disease progresses to distant metastasis [ 1 , 3 ]  . the risk of local recurrence after prostatectomy alone has been estimated at 15%45% at 5 years in patients with locally advanced prostate carcinoma ( pca ) ( pt3 ) and at 20%60% and 40%65% in the presence of positive margins and involvement of the seminal vesicles . 
it is therefore possible to identify prognostic factors that correlate with a high risk of local recurrence , including preoperative serum prostate - specific antigen ( psa ) , margin positivity , gleason score and clinical and pathological stage . the combination of all of these factors increases the risk of recurrence by around 50% [ 58 ]  . numerous studies have highlighted the efficacy of postoperative radiotherapy in reducing the incidence of local recurrence in high - risk cases [ 911 ]  . 
the timing of radiotherapy is , however , still controversial : is adjuvant or salvage radiotherapy more effective ? adjuvant treatment is generally performed within 6 months of surgery in the presence of positive margins and / or extracapsular spread ; salvage radiotherapy refers to radiotherapy initiated when a macroscopic recurrence appears or with rising psa , generally later than 6 months after surgery . 
 this paper reports the results of a retrospective review carried out on a single - institution case series of 97 patients treated with surgery for pca in order to evaluate the efficacy of postoperative radiotherapy in reducing the incidence of locoregional recurrence , define the best timing for radiotherapy and assess late toxicity of postoperative treatment compared with that seen after radiotherapy alone and after surgery alone . 
it was considered salvage if performed more than 6 months after rp , for the presence of macroscopic disease or progressively rising psa values . patient age was 6070 years in 80% of cases ; 78% of patients had a karnofsky performance status ( ki ) equal to or greater than 80 . 
tuttavia , sebbene la chirurgia radicale si sia dimostrata un trattamento altrettanto efficace della radioterapia esclusiva per le forme intracapsulari , circa il 15%40% dei pazienti portatori di carcinoma prostatico in stadio t1 - t2 sviluppano una recidiva [ 1 , 2 ]  . 
inoltre , circa un terzo dei pazienti con recidiva progrediscono con la comparsa di metastasi a distanza [ 1 , stato stimato che il rischio di ricaduta locale dopo sola prostatectomia pari al 15%45% a 5 anni in pazienti portatori di carcinoma prostatico localmente avanzato ( pt3 ) e rispettivamente al 20%60% ed al 40%65% in presenza di margini positivi e in caso di interessamento delle vescicole seminali . 
possibile , quindi , identificare dei fattori prognostici che si correlano ad un elevato rischio di recidiva locale , tra cui il valore del psa pre - chirurgia , la positivit dei margini , lo score di gleason , lo stadio clinico e patologico . 
lassociazione di tutti questi fattori aumenta il rischio di recidiva di circa il 50% [ 58 ]  . numerosi studi hanno evidenziato lefficacia della radioterapia post - operatoria nel ridurre lincidenza delle recidive locali nei casi ad alto rischio [ 911 ]  . 
risulta , per , ancora controverso il timing radioterapico : pi efficace la radioterapia adiuvante o quella di salvataggio ? il trattamento adiuvante viene in genere effettuato entro sei mesi dalla chirurgia , in presenza di margini positivi e / o di extracapsularit ; mentre considerata di salvataggio la radioterapia iniziata alla comparsa di recidiva macroscopica o con psa in crescita progressiva , in genere oltre i sei mesi dallintervento chirurgico . in questo lavoro vengono riportati i risultati ottenuti dallanalisi retrospettiva di una casistica monoistituzionale di 97 pazienti trattati con chirurgia per carcinoma prostatico , al fine di valutare lefficacia della radioterapia post - operatoria nel ridurre lincidenza delle recidive loco - regionali , definire il timing ottimale della radioterapia e valutare la tossicit tardiva del trattamento post - operatorio , nel confronto con quella che si osserva dopo trattamento radioterapico esclusivo o dopo sola chirurgia . materiali e metodi dal 1980 al 2003 sono stati trattati consecutivamente 97 pazienti portatori di carcinoma prostatico in esiti di prostatectomia radicale ( pr )  . 
il trattamento radioterapico ( rtt ) stato considerato adiuvante se condotto meno di 6 mesi dopo pr e in assenza di malattia macroscopica residua o recidiva locale e di innalzamento progressivo dei valori del psa . 
in 13 patients , this information was not available ( during the first years of the period considered , psa testing was not available at our institution )  . as a result , data analysis for this parameter was done excluding these patients . 
there were no significant differences between the two treatment groups as regards age , ki or gleason score . of the 41 patients treated with adjuvant radiotherapy , only 22% had disease - free surgical margins ; extracapsular spread was present in 80% of cases . 
treatment volume was limited to the surgical bed in 76% of cases , with a total target dose less than 70 gy in 54% of cases . of the 56 patients treated with salvage radiotherapy , 82% had disease - free surgical margins and only 46% had extracapsular spread . 
treatment volume was limited to the surgical bed in 82% of cases , with a total target dose less than 70 gy in 12% of cases only . the main clinical and treatment differences between the two groups undergoing adjuvant and salvage radiotherapy are summarised in table 1 . 
after radiotherapy , patients were followed up by means of psa monitoring and physical examination every 6 months . further investigations ( bone scintigraphy , chest x - ray and prostate ultrasound ) were requested for patients showing rising psa level . 
the appearance of clinically documented disease , local or distant , was considered as the event of interest . comparison between the two groups was carried out with the log - rank test ( cox multivariate analysis being impossible due to the small number of events in the subgroups )  . 
late toxicity of rtt was documented and described according to the radiation therapy oncology group ( rtog ) scale . pi di 6 mesi dopo pr , per la presenza di malattia macroscopica o per innalzamento progressivo del valore del psa . nellintera casistica , let era compresa tra 60 e 70 anni nell80% dei casi ; il 78% dei pazienti presentava un performance status secondo karnosfky ( ik ) maggiore o uguale a 80 . 
in 13 pazienti tale dato non era disponibile ( nei pazienti reclutati nei primi anni del periodo considerato , quando il dosaggio del psa non era disponibile presso la nostra istituzione )  . 
non sono state documentate differenze significative tra i due gruppi terapeutici per ci che concerne et , ik , gleason score . dei 41 pazienti trattati con radioterapia adiuvante , solo il 22% presentava margini chirurgici liberi da malattia ; unestensione extracapsulare era presente nell80% dei casi . la maggior parte dei pazienti ( 68% ) aveva un psa pre - radioterapia inferiore o eguale a 1 ng / ml . 
le principali differenze nella distribuzione dei pazienti nei due gruppi trattati con radioterapia adiuvante e di salvataggio , a seconda delle pi importanti caratteristiche patologiche e terapeutiche sono riassunte nella tabella 1 . 
la grande maggioranza dei casi ( circa l80% ) stata sottoposta anche a trattamento ormonale , senza differenze significative tra i pazienti trattati con radioterapia adiuvante e quelli trattati con radioterapia di salvataggio . 
this finding is more significant if we consider that the total dose was lower in the group treated with adjuvant radiotherapy ( table 1 )  . no significant differences in rfs were observed in relation to gleason score , surgical margin positivity or extracapsular spread either in the series as a whole or in the two subgroups . a preradiotherapy psa level greater than 1 ng / ml was found to correlate significantly with a worse rfs only in the adjuvant radiotherapy group [ 50% vs . 
patients treated with salvage rtt therefore showed table 1 clinical and treatment characteristics of the case series studied analisi statistica lend point principale per lanalisi dei risultati stato la sopravvivenza libera da ricaduta ( rfs ) , calcolata con metodo attuariale di kaplan - meier . 
il confronto tra sottogruppi stato effettuato con il log - rank test ( non essendo possibile impiegare lanalisi multivariata secondo il modello di cox per lesiguit numerica degli eventi nei vari sottogruppi )  . 
la tossicit tardiva del trattamento radioterapico stata documentata e descritta in accordo con la scala del radiation therapy oncology group ( rtog )  . risultati sopravvivenza libera da ricaduta la sopravvivenza attuariale non corretta per altre cause di morte ( os ) a 5 e 10 anni dalla fine della radioterapia e per lintera casistica stata del 75%8% e 26%2% , quella corretta per altre cause di morte ( dss ) dell83%7% e 38%28% , e quella libera da ricadute ( rfs ) del 53%8% e 32%14 , 2% . 
la rfs stata correlata a vari fattori prognostici , tra cui il tipo di radioterapia ( adiuvante o di salvataggio ) , il gleason score , lestensione extracapsulare , la positivit dei margini chirurgici ed il valore del psa pre - radioterapia . 
questo dato assume magadjuvant rtt , % ( n = 41 ) salvage rtt , % ( n = 56 ) rtt , radiotherapy treatment ; psa , prostate - specific antigen ; rt , radiotherapy apsa data available for only 84 / 97 patients ( see text ) tabella 1 caratteristiche cliniche e del trattamento radioterapico della casistica in esame rtt adiuvante , % ( n = 41 ) rtt di salvataggio , % ( n = 56 ) free surgical margins extracapsular spread psa pre - rt 1 ng / mla total dose < 70 gy rtt limited to surgical bed associated hormone therapy margini chirurgici liberi estensione extracapsulare psa pre rt 1 ng / mla dose totale < 70 gy rtt solo letto operatorio ormonoterapia associata rtt , trattamento di radioterapia ; psa , antigene prostatico specifico ; rt , radioterapia ail dato sul psa disponibile solo in 84 / 97 pazienti ( vedi testo ) b . 
only two cases ( 2% ) presented gastrointestinal toxicity ( grade less than or equal to 2 )  . discussion this study evaluated the results of postoperative radiotherapy for prostate cancer and in particular compared adjuvant and salvage radiotherapy . 
non si sono invece osservate differenze significative nella rfs a seconda del gleason score , della positivit dei margini chirurgici , della presenza di malattia extracapsulare , sia nella intera casistica che nei due sottogruppi terapeutici . 
69% , p ( log - rank ) = 0 , 05 ]  . sede e incidenza delle ricadute nellintera casistica si sono verificate 27 ricadute ( 28% ) , cos distribuite : 11 recidive locali , 12 metastasi a distanza , 2 ricadute loco - regionali isolate e 2 associate a metastasi a distanza . 
si osservata una tossicit urinaria in 6 / 97 casi ( 6% ) , in un solo caso ( cistite emorragica ) di grado > 2 secondo la scala rtog . 
solo 2 casi ( 2% ) hanno presentato tossicit gastrointestinale , di grado2 . discussione in questo studio , abbiamo valutato i risultati della radioterapia post - operatoria per il cancro prostatico ed in particolare abbiamo confrontato la radioterapia adiuvante con quella di salvataggio . 
i pazienti sottoposti a radioterapia adiuvante hanno manifestato una miglior rfs rispetto al gruppo di pazienti trattato con radioterapia di salvataggio , con una differenza statisticamente significativa ( p < 0 , 01 )  . 
infatti , nella casistica in esame ( 97 casi ) , abbiamo osservato 27 recidive ( 28% ) : 7 / 41 ( 17% ) nel gruppo della radioterapia adiuvante e 20 / 56 ( 36% ) in quello della radioterapia di salvataggio , nonostante che la dose totale sia stata meno elevata nei pazienti trattati con rtt adiuvante . 
sebbene lanalisi univariata sia stata estesa a tutti gli altri diversi fattori prognostici , anche separatamente nei due diversi sottogruppi terapeutici , la rfs risultata significativamente migliore solo nel gruppo in cui il psa pre - radioterapia era 1 ng / ml , e ci solo nel sottogruppo trattato con radioterapia adiuvante . 
limportanza prognostica del psa pre - rtt stata anche recentemente confermata dalla letteratura [ 12 ] e rappresenta un altro elemento a favore di un precoce inizio della radioterapia post - operatoria nei sottogruppi a rischio . 
our data are therefore in agreement with previous studies that testify to the efficacy of postoperative rtt . these include retrospective studies comparing the results of surgery alone with those of surgery combined with radiotherapy [ 13 ] and , above all , the randomised study of european organisation for research and treatment of cancer ( eortc ) 22911 , which confirms the improvement in biochemical and clinical disease - free survival [ 14 ] with the use of adjuvant postoperative rtt in pt3 cases . 
ours was a retrospective study and therefore suffers some limitations : the groups were not homogeneous , treatment changed over the years and surgical indications have evolved . in our series , radiotherapy was generally well tolerated . late toxicity was acceptable , with only one case of g4 urinary toxicity ( haemorrhagic cystitis )  . there were no major chronic gastrointestinal sequelae . comparison of the late sequelae reported for surgery alone with those observed after postoperative rtt ( in our series and others ) identified no major differences [ 15 ]  . 
likewise , in terms of late sequelae , radiotherapy alone for prostate cancer has similar toxicity to postoperative rtt , as shown by the results of a recent italian multicentre study [ 16 ]  . hormone treatment , with different drugs and duration , was given to the majority of patients receiving either adjuvant or salvage radiotherapy : this precludes any reliable assessment of the therapeutic role of androgen block in our series . 
si tratta sia di studi retrospettivi , in cui si paragonano i risultati della sola chirurgia con quelli in cui si associa al trattamento chirurgico quello radiante [ 13 ] e soprattutto dello studio randomizzato delleortc 22911 , in cui si conferma il miglioramento della sopravvivenza libera da malattia sia biochimica che clinica [ 14 ] con luso della rtt adiuvante postoperatoria nelle forme pt3 . 
il nostro uno studio retrospettivo con tutte le limitazioni correlate : la non omogeneit del gruppo , le modifiche dellapproccio terapeutico in un lungo periodo di tempo e levoluzione delle indicazioni chirurgiche . 
confrontando le sequele tardive della sola chirurgia , documentate in letteratura , con quelle osservate dopo rtt post - operatoria ( in questa come in altre casistiche ) , non si sono osservate differenze sostanziali [ 15 ]  . allo stesso modo , in termini di sequele tardive , il trattamento radioterapico esclusivo del cancro prostatico gravato da una tossicit simile a quella della rtt post - operatoria , come si pu ad esempio osservare considerando i risultati di un recente studio multicentrico italiano [ 16 ]  . 
il trattamento radioterapico adiuvante post - operatorio , tuttavia , trova indicazione solo nelle forme clinicamente intracapsulari che , allesame istologico del pezzo operatorio , risultino extracapsulari o con positivit marginale . 
thirty - two out of 36 patients met the following inclusion criteria : transabdominal and endocavitary ( us ) examination and mr imaging , followed by laparoscopy performed within 2 weeks . 
sensitivity and specificity of the two imaging techniques in the recognition of the different locations were 58% and 25% , respectively , for us and 56% and 50% , respectively , for mr imaging . 
us examination is the primary evaluation in cases of suspected disease and for the rectovaginal septumr examination is recommended for correct classification in doubtful cases and in cases of suspected extrapelvic lesions and adhesions . key words endometriosis magnetic resonance ultrasound riassunto obiettivo . 
sono state studiate consecutivamente 36 pazienti con sospetto o diagnosi clinica di endometriosi . trentadue / 36 pazienti rispondevano ai seguenti criteri di inclusione e sono state comprese in questo studio : esecuzione di indagine eg con approccio sovra - pubico e trans - cavitario , di indagine rm e intervento laparoscopico entro le 2 settimane successive . 
lindagine rm preferibile per la tipizzazione della lesione nei casi dubbi , per lo studio delle localizzazioni extra - pelviche e nel caso di aderenze . parole chiave endometriosi risonanza magnetica ecografia g . 
distinction on the basis of lesion morphology rather than lesion depth justifies the importance that imaging techniques , and in particular magnetic resonance ( mr ) , are assuming in defining different lesion components [ 1 ]  . 
for the same reason , although laparoscopy remains the gold standard in the diagnosis of endometriosis , several recent studies evaluated the role of noninvasive imaging techniques in the attempt to overcome the intrinsic limits of the laparoscopic approach ( evaluation of posterior pelvis ; superficial implants on intestinal loops or the bladder ; localisations in the ureter or rectovaginal septum ; extrapelvic localisations , presence of adhesions ) [ 18 ]  . 
the purpose of this study was to define the real contribution of imaging , in particular ultrasound ( us ) and mr , in diagnosis and local staging of endometriosis by comparing results with those of laparoscopy . materials and methods population between july 2003 and may 2005 ( overall duration 22 months ) , we consecutively evaluated 36 women with suspected or clinically diagnosed endometriosis . 
all patients underwent transabdominal and endocavitary us and mr ( both performed in the preovulatory phase within the 12th day of the menstrual cycle ) and laparoscopy within 15 days from mr without interposition of menstrual cycle . 
patients who did not give informed consent or did not undergo laparoscopy within 2 weeks of the imaging studies were excluded . patients ages ranged from 24 to 51 ( mean 33 ) years ; 18 / 36 patients had previously undergone appendicectomy whereas 17 / 36 had been operated on for endometriosis . 
only 4 / 36 patients had had a pregnancy whereas 4 / 36 were sterile with normal partner spermiogra rectosigmoidoscopy , performed in 21 / 36 patients before surgery , was negative for endometriosis in all cases ; in 7 / 36 patients , it showed hyperaemia of the mucosa due to inflammation , with hyperplastic polyps in two cases . ultrasound us examination was carried out with a transabdominal and lendometriosi una malattia comune nelle giovani donne e si associa a dolore pelvico cronico ed infertilit . 
tale distinzione importante , dal momento che lendometriosi peritoneale e quella ovarica rappresentano lesioni prevalentemente emorragiche , mentre le localizzazioni alla pelvi posteriore , allintestino ed alla vescica rappresentano prevalentemente lesioni di tipo adenomiosico . 
la distinzione delle lesioni sulla base dellaspetto morfologico piuttosto che in relazione alla profondit dellimpianto rende ragione dellimportanza che le metodiche di imaging , in particolare la risonanza magnetica ( rm ) , stanno assumendo nel determinare le componenti lesionali [ 1 ]  . 
 anche per questa ragione , sebbene la laparoscopia rimanga il gold standard nella diagnosi di endometriosi , recentemente numerosi studi hanno valutato il ruolo delle metodiche di imaging non invasivo , nel tentativo di ovviare alle limitazioni intrinseche dellapproccio laparoscopico ( valutazione della pelvi posteriore ; impianti superficiali su anse intestinali o vescicali ; localizzazioni ureterali o nel setto retto - vaginale ; localizzazioni extra pelviche , presenza di adesioni ) [ 18 ]  . scopo di questo studio definire leffettivo contributo dellimaging , in particolare dellecografia ( eg ) e della risonanza magnetica ( rm ) , nella diagnosi e nella valutazione di estensione dellendometriosi , attraverso il confronto dei dati ottenuti mediante tali metodiche con i risultati dellintervento chirurgico laparoscopico cui sono state sottoposte tutte le pazienti considerate . materiali e metodi popolazione in studio nel periodo compreso fra luglio 2003 e maggio 2005 ( durata complessiva 22 mesi ) sono state studiate in maniera consecutiva 36 pazienti giunte alla osservazione con sospetto o diagnosi clinica di endometriosi . 
le pazienti incluse in questo studio sono state sottoposte sia ad indagine eg con approccio sovrapubico e trans - cavitario che ad indagine mr ( espletate nel periodo pre - ovulatorio entro il 12 giorno del ciclo mestruale ) ed a intervento laparoscopico entro 15 giorni dalla esecuzione dello studio mr e senza interposizione di ciclo mestruale . 
i criteri di esclusione sono stati : pazienti che non hanno espresso il consenso informato e pazienti in cui non stato possibile realizzare latto laparoscopico entro 2 settimane dallo studio imaging . le pazienti esaminate avevano et compresa tra 2451 anni ( et media di 33 anni ) ; di queste , 18 / 36 erano state in precedenza sottoposte ad appendicectomia e 17 / 36 ad intervento chirurgico per endometriosi . 
transabdominal us was performed with a realtime unit ( sequoia , acuson , siemens , erlangen , germany ) using convex or sectoral 3to 5 - mhz probes , depending on the patients build . 
in some cases , to better evaluate possible involvement of rectal walls , vaginal fornix and rectovaginal septum , as well as the presence of adhesions , the rectum was distended using a water - filled balloon . 
in some cases , the colour doppler technique , available on both transabdominal and endocavitary probe , was performed to differentiate between endometriotic implants and normal anatomical structures ( vessels )  . magnetic resonance mr imaging was performed with a 1.5 - t superconductive unit ( symphony , siemens , erlangen , germany )  . 
t2 fast spin echo sequences ( tr / te : 6 , 400 / 96 ; etl : 16 ) were acquired with a 256256 matrix in the sagittal , axial and sometimes coronal plane . 
t1 gre breath - hold sequences ( tr / te : 100 / 2.66 ; fa : 70 ) were performed with and without fat saturation in the three spatial planes . 
bolus was reserved for selected patients only when it was necessary to demonstrate the possible neoplastic nature of a solid component detected within the mass ( three cases )  . data analysis in all patients studied with us and mr , we evaluated lesion presence , number , location , dimensions and morphology . results were compared with findings of laparoscopy performed within 2 weeks of the mr examination and without intervening menstrual period . 
four patients were excluded owing to refusal ( n = 1 ) , obesity ( n = 2 ) and excessive time lag between imaging and surgery ( n = 1 )  . 
la rettosigmoidoscopia effettuata in 21 / 36 pazienti prima dellintervento era risultata in tutti i casi negativa per endometriosi ; in 7 / 36 casi era stata rilevata la presenza di iperemia della mucosa per fenomeni flogistici , associata al riscontro di polipi iperplastici in 2 casi . ecografia lindagine ecografica stata realizzata mediante approccio trans - addominale ed endo - cavitario , in particolare endo - rettale e / o endovaginale . 
per lesame ecografico trans - addominale stato utilizzato apparecchio real - time con 512 canali digitali ( sequoia , acuson , siemens , erlangen , germania ) , impiegando sonde convex o settoriali , da 35 mhz , a seconda della costituzione fisica della paziente . 
la preparazione ha richiesto distensione della vescica con il duplice fine di spostare verso lalto le anse intestinali , riducendo lo sbarramento del fascio ad opera del meteorismo , e ridurre la normale antiversione dellutero . a quello trans - addominale stato sempre affiancato lesame ecografico endo - cavitario per via trans - rettale o trans - vaginale , per la visualizzazione della parete rettale e dei suoi rapporti con il setto retto - vaginale , dei parametri e dei legamenti utero - sacrali . 
in alcuni casi , per meglio valutare leventuale coinvolgimento delle pareti rettali , del fornice vaginale o del setto retto - vaginale , nonch la presenza di aderenze , si ottenuta distensione del retto con palloncino pieno dacqua . lo studio stato realizzato sempre dallo stesso operatore particolarmente esperto . 
la tecnica color doppler , disponibile sia sulla sonda trans - addominale che endo - cavitaria , stata utilizzata in alcuni casi al fine di conseguire la diagnosi differenziale fra le localizzazioni endometriosiche e le strutture anatomiche normali ( vasi )  . risonanza magnetica lindagine rm stata realizzata utilizzando un magnete superconduttivo a 1.5 t ( symphony , siemens , erlangen , germania )  . 
la preparazione della paziente ha richiesto clistere di pulizia la mattina dellesame , al fine di limitare la presenza di residui fecali in corrispondenza del retto e del sigma . al momento dellesame stato somministrato farmaco ipotonizzante ( buscopam , 1 fl ev ) al fine di ridurre gli artefatti legati alla peristalsi intestinale . 
lindagine stata condotta impiegando bobine di superficie di tipo phased - array . lesame nel complesso stato realizzato utilizzando spessore di strato di 4 mm ed intervallo di 0 , 10 , 2 mle sequenze t2 fast spin echo ( tr / te : 6400 / 96 ; etl : 16 ) sono state realizzate con matrice 256256 nei piani sagittale , assiale e g . 
in particular , adhesion was considered as a lack of fatty cleavage plane between adjacent organs , with stretching of their walls and / or distortion of an intestinal loop [ 4 ]  . cul - de - sac obliteration was defined as the presence of signs of adhesion , such as stretching of the rectum towards the uterus with or without a fibrous plaque , and elevation of the posterior vaginal fornix [ 4 ]  . 
comparative data analysis provided sensitivity , specificity and predictive values of imaging techniques relative to different disease locations identified at laparoscopy . results ultrasound us examination showed the presence of endometriotic lesions in all 32 patients examined . 
these patients had a total of 101 lesions , with the following anatomical distribution : 31 in the rectovaginal septum , 37 in the ovary , 14 in the rectum , four in the vagina , three in the posterior vaginal fornix , five in the posterior cul - de - sac , two in the salpinx , two in the left uterosacral ligament , one on the bladder wall and one case of cul - de - sac obliteration . 
with regard to number , there were 84 / 101 single lesions and 17 / 101 multiple lesions ; single lesions were most frequent in the ovary ( n = 37 ) and rectovaginal septum ( n = 31 ) whereas multiple lesions were prevalently located in the rectovaginal septum ( n = 10 )  . 
 small nodules ( 43 / 101 ) , with diameter between 3 mm and 18 mm ( mean 6.6 mm ) , were prevalently located in the rectovaginal septum ( 28 / 43 )  . 
laminar lesions ( 13 / 101 ) , which had a greater axis of 1040 mm ( mean diameter , 25 mm ) , were seen in the rectovaginal septum ( two cases ) , rectum wall ( eight cases ) , posterior cul - de - sac ( two cases ) and vaginal fornix ( one case ) and were all hypoechoic . 
of the cystic lesions , 38 / 39 had internal echogenic components whereas 1 / 39 , located in the rectovaginal septum and with a diameter of 3 mm , was thick walled and with low - level internal echoes . 
le sequenze t1 gre breath - hold ( tr / te : 100 / 2 , 66 ; fa : 70 ) sono state espletate senza e con soppressione del grasso nei tre piani spaziali . 
in particolare , stato utilizzato solo in presenza , nellambito della massa , di componente solida al fine di dimostrarne la eventuale natura neoplastica ( 3 casi )  . analisi dei dati in tutte le pazienti esaminate con eg e rm stata valutata la presenza , il numero , la sede , le dimensioni e la morfologia delle lesioni eventualmente presenti . 
pertanto delle 36 pazienti esaminate , ne sono state selezionate e sono oggetto di questo studio 32 pazienti , sottoposte ad indagine eg con approccio sovrapubico e trans - cavitario e a rm , ed infine ad intervento chirurgico . quattro pazienti sono state escluse dallo studio di cui una per rifiuto da parte della paziente , 2 per obesit e una per il ritardo intercorso fra limaging e latto laparoscopico . 
ogni lesione identificata con indagine eg e rm stata classificata in base alla sede anatomica , al numero ( uniche o multiple ) e ad una delle seguenti caratteristiche morfologiche come : piccolo nodulo ( diametro inferiore a 15 mm ) , grande nodulo ( diametro superiore a 15 mm ) , lesione laminare , cisti , neoformazione complessa , adesione e obliterazione del cavo di douglas , sulla base degli aspetti recentemente riportati in letteratura [ 3 , 4 ]  . 
in particolare , abbiamo inteso come adesione la mancata visualizzazione del piano adiposo di clivaggio tra organi adiacenti con stiramento della parete degli stessi e / o distorsione di unansa intestinale [ 4 ]  . inoltre , abbiamo definito obliterazione del cavo di douglas la situazione in cui sono stati visualizzati segni di adesione come stiramento del retto verso lutero con o senza placca fibrotica e innalzamento del fornice vaginale posteriore [ 4 ]  . tale reperto che configura coinvolgimento del cavo di douglas , senza tuttavia lesione occupante spazio , non consente approccio laparoscopico anteriore . 
lanalisi comparativa dei dati ha fornito i valori di sensibilit , specificit e di predittivit delle metodiche di imaging utilizzate , relativamente alle singole localizzazioni di malattia riscontrate alla laparoscopia . risultati ecografia lindagine ecografica ha documentato la presenza di lesioni endometriosiche in tutte le 32 pazienti esaminate . 
finally , cul - de - sac obliteration was visualised as hypoechoic in only one case ( 1 / 101 )  . magnetic resonance mr examination showed the presence of endometriotic lesions in all 32 patients considered . 
overall , it demonstrated 92 lesions with the following anatomical distribution : 23 in the rectovaginal septum , 41 in the ovary ( 16 on the right and 25 on the left ) , nine in the rectum , three in the vagina , two in the posterior cul - de - sac , three in the posterior vaginal fornix , two in the large ligaments , one in the vesicouterine fold , one in the terminal ileum , two in the uterus , one in the bladder wall , two cases of cul - de - sac obliteration and two cases of adhesions . 
the majority of single lesions were seen in the ovary ( n = 18 ) , followed by the rectovaginal septum ( n = 13 ) and rectum ( n = 8 )  . 
all lesions located in the vagina , posterior cul - de - sac , posterior vaginal fornix , large ligaments , vesicouterine fold , ileum , uterus and bladder wall as well as the two cases of cul - de - sac obliteration and adhesions appeared as single . 
the 92 lesions were classified on the basis of their morphology . small nodules ( 40 / 92 ) , with diameter between 3 mm and 15 mm ( mean 8 mm ) , were prevalently seen in the rectovaginal septum ( 20 / 40 observations )  . 
of these , 36 / 40 had homogeneous appearance in t1and t2 - weighted sequences . in particular , 36 lesions were hyperintense in t1 - weighted sequences , 20 / 36 hypointense in t2 - weighted sequences , 14 / 36 hyperintense and 2 / 36 isointense . 
the 4 / 40 small nodules exhibiting inhomogeneous appearance were all hyperintense in t1 - weighted sequences ( one had an isointense marginal halo ) whereas in t2 - weighted sequences , they had a peripheral halo that was hypointense ( two cases ) and hyperintense ( two cases )  . 
 large nodules ( 3 / 92 ) , with diameter between 15 mm and 30 mm ( mean 23 mm ) , were seen in the vaginal fornix ( one case ) , rectovaginal septum ( one case ) and bladder ( one case )  . these lesions were isohypointense in t1 - weighted sequences , with small foci of signal hyperintensity . 
 laminar lesions ( 8 / 92 ) were encountered in the rectovaginal septum , large ligament and posterior cul - de - sac ; the last one was described as haematic paint . 
all these lesions had hyperintense signal in t1 - weighted sequences ; in t2 - weighted sequences , they were poorly detectable because of their isointensity in comparison with adjacent tissues , with the exception of one case that was hyperintense . 
 cystic lesions ( 32 / 92 ) , which had diameter between 4 mm and 80 mm ( mean 29 mm ) , were almost exclusively seen in the ovary ( 31 / 32 observations )  . 
per quanto concerne il numero delle 101 lesioni riscontrate , 84 sono state descritte come singole , con sede di maggiore riscontro in corrispondenza dellovaio ( n = 37 ) , seguita dal setto rettovaginale ( n = 31 )  . 
analizzando le 101 lesioni e suddividendole in rapporto al loro aspetto eg , emerge quanto segue . i piccoli noduli ( 43 / 101 ) , con diametro compreso tra 3 mm e 18 mm ( diametro medio 6 , 6 mm ) , sono risultati localizzati prevalentemente in corrispondenza del setto retto - vaginale ( 28 / 43 )  . 
questa , localizzata in corrispondenza della vescica , possedeva diametro di 30 mm ed aspetto ipoecogeno . le lesioni laminari ( 13 / 101 ) , che presentavano maggior asse compreso tra 10 mm e 40 mm ( diametro medio di 25 mm ) , sono state descritte in corrispondenza del setto retto - vaginale ( 2 casi ) , della parete del retto ( 8 casi ) , del cavo di douglas ( 2 casi ) e del fornice vaginale ( 1 caso ) e mostravano sempre aspetto ipoecogeno . le lesioni cistiche ( 39 / 101 ) , con diametro compreso tra 3 mm e 80 mm ( diametro medio 24 mm ) sono state localizzate prevalentemente a livello dellovaio ( 34 / 39 di cui 14 a destra e 20 a sinistra )  . 
di tutte le lesioni , 38 / 39 possedevano contenuto corpuscolato , mentre 1 / 39 , localizzata in corrispondenza del setto retto - vaginale , con diametro di 3 mm , stata descritta con parete spessa a contenuto finemente ecogeno . 
le neoformazioni complesse ( 4 / 101 ) , con diametro compreso tra 55 e 70 mm ( diametro medio di 64 mm ) erano localizzate in corrispondenza dellovaio ( 3 / 4 ) e del cavo del douglas ( 1 / 4 )  . 
tali lesioni presentavano sempre contenuto corpuscolato ed aspetto ecografico complesso , per la contemporanea presenza di zone ipoed iper - ecogene , per il riscontro di pareti particolarmente spesse o di gettoni solidi allinterno della formazione cistica . 
infine , lobliterazione del cavo di douglas stata visualizzata con aspetto ipoecogeno in un unico caso ( 1 / 101 )  . risonanza magnetica lindagine rm ha documentato la presenza di lesioni endometriosiche in tutte le 32 pazienti . 
nelle pazienti esaminate , sono state complessivamente riscontrate 92 lesioni con la seguente sede anatomica : 23 al setto retto - vaginale , 41 in corrispondenza dellovaio di cui 16 a destra e 25 a sinistra , 9 al retto , 3 alla vagina , 2 al cavo del douglas , 3 al fornice vaginale posteriore , 2 in corrispondenza dei legamenti larghi , 1 alla plica vescico - uterina , 1 allileo terminale , 2 allutero , 1 alla parete vescicale , obliterazione del cavo di douglas in due casi e aderenze in altri 2 casi . 
the latter two lesions showed a peripheral halo of signal hyperintensity , and in t2 weighted sequences , they had a hyperintense signal with a hypointense marginal halo with varying conspicuity . 
 adhesions ( 2 / 92 ) were observed between the rectum , an intestinal loop and the uterus in one case and between the uterus and sigmoid colon in the other case . 
 posterior cul - de - sac obliteration ( 2 / 92 ) was visualised as isointense , with foci of hyperintensity in t1 - weighted sequences and homogeneous hypointensity in t2 - weighted sequences . data analysis after being analysed separately , imaging findings in the 32 patients were compared with the results of laparoscopy in order to define the number of correct diagnoses ( true positives and true negatives ) , underestimated lesions ( false negatives ) and overestimated lesions ( false positives ) in common for the two techniques and separately for each one . among the 101 lesions detected by us and the 92 lesions detected by mr , the total number of lesions in the same anatomical site diagnosed with both us and mr and confirmed by surgery was 67 ( table 1 )  . 
of these 67 true positive findings , 33 were located in the ovaries , 20 in the rectovaginal septum , eight on the rectal wall , two in the vagina , one in the vaginal fornix , one in the posterior cul - de - sac , one in the bladder and one was a cul - de - sac obliteration . 
both us and mr misdiagnosed 38 lesions , most of which involved the large ( ten cases ) and uterosacral ( eight cases ) ligaments ( false negatives for us and mr )  . 
tutte le lesioni con sede anatomica alla vagina , al cavo del douglas , al fornice vaginale posteriore , in corrispondenza dei legamenti larghi , alla plica vescico - uterina , allileo , allutero , alla parete vescicale , lobliterazione del cavo di douglas e le aderenze sono state rilevate come singole . 
trentaquattro lesioni sono state rilevate come multiple e sono state localizzate in corrispondenza del setto retto - vaginale ( 10 casi ) e dellovaio ( 23 casi ) e del retto ( 1 caso )  . 
le 92 lesioni sono state suddivise ed analizzate in rapporto al loro aspetto . i piccoli noduli ( 40 / 92 ) , con diametro compreso tra 3 mm e 15 mm ( diametro medio 8 mm ) , sono stati individuati prevalentemente in corrispondenza del setto retto - vaginale ( 20 / 40 osservazioni )  . 
in particolare 36 lesioni erano iperintense nelle sequenze t1 pesate , 20 / 36 mostravano ipointensit di segnale nelle sequenze t2 pesate , 14 / 36 iperintensit e 2 / 36 isointensit . 
i 4 / 40 piccoli noduli che invece presentavano disomogeneit del segnale apparivano tutti iperintensi nelle sequenze t1 pesate ( 1 di essi mostrava alone marginale di isointensit di segnale ) , mentre nelle sequenze t2 pesate erano caratterizzati da alone periferico , rispettivamente ipointenso ( 2 casi ) ed iperintenso ( 2 casi )  . i grandi noduli ( 3 / 92 ) , che presentavano diametro compreso tra 15 mm e 30 mm ( diametro medio 23 mm ) , sono stati riscontrati in corrispondenza del fornice vaginale ( 1 caso ) , del setto retto - vaginale ( 1 caso ) e della vescica ( 1 caso )  . 
nelle sequenze pesate in t2 solo 1 / 3 dei grandi noduli presentava iperintesit di segnale con alone marginale ipointenso . le lesioni laminari ( 8 / 92 ) , sono state descritte in corrispondenza del setto retto - vaginale , del legamento largo e del cavo del douglas ; questultima era descritta come verniciatura ematica . 
tali lesioni erano tutte caratterizzate da iperintensit di segnale in t1 ; nella sequenza pesata in t2 erano mal riconoscibili in quanto iso - intense rispetto ai tessuti adiacenti ad eccezione di 1 caso , in cui la lesione appariva iperintensa . le lesioni cistiche ( 32 / 92 ) , che presentavano diametro compreso tra 4 mm e 80 mm ( diametro medio 29 mm ) , sono state individuate quasi esclusivamente a carico dellovaio ( 31 / 32 osservazioni )  . 
di queste 25 / 32 presentavano aspetto omogeneo nelle sequenze t1 e t2 pesate , caratterizzato sempre da iperintensit di segnale in t1 , mentre in t2 solo 9 / 25 apparivano iperintense , mostrando le restanti 16 lesioni ipointensit di segnale . 
le stesse lesioni presentavano iperintensit di segnale nelle sequenze pesate in t2 con coesistenza di alone marginale ipointenso pi o meno rappresentato , fino ad apparire come spessa banda . le neoformazioni complesse ( 5 / 92 ) , con diametro compreso tra 20 mm ed 80 mm ( diametro medio 44 mm ) , erano g . 
in contrast , 12 lesions were detected by us but not confirmed by mr or surgery ( table 1 ) localizzate in corrispondenza dellovaio ( 4 casi ) e del douglas ( 1 caso )  . 
nelle sequenze pesate in t2 in tutte le osservazioni sono stati rilevati noduli parietali , mentre il contenuto ha presentato aspetto variabile . le aderenze ( 2 / 92 ) sono state osservate fra retto , ansa table 1 differences in ultrasound ( us ) and magnetic resonance ( mr ) findings by location site of lesions identified at laparoscopy concordant diagnoses us + mr discordant diagnoses tp , true positive ; fn , false negative ; tn , true negative ; fp , false positive tabella 1 differenze dei rilievi alle diverse metodiche , suddivise per sede sede delle lesioni identificate alla lps rectovaginal septum ovary large ligaments uterosacral ligaments rectum vagina and vaginal fornix uterus douglas cul - de - sac vesicouterine fold salpinx lymph node bladder pararectal fossa peritoneum ileum sigmoid colon adhesions cul - de - sac obliteration total setto retto - vaginale ovaio legamenti larghi legamenti utero - sacrali retto vagina e fornice vaginale utero douglas plica vescico - uterina tuba linfonodo vescica fossa para - rettale peritoneo ileo sigma aderenze obliterazione douglas totale diagnosi coincidenti us + rm diagnosi discordanti vp , vero positivo ; fn , falso negativo ; vn , vero negativo ; fp , falso positivo g . 
us had greater accuracy in evaluating the rectovaginal septum , thanks to the endorectal approach that made it possible to identify lesions affecting the rectal wall and vagina . ileale e utero in un caso e fra utero e sigma nellaltro caso . le caratteristiche di segnale in entrambi i casi sono state iperintensit con banda periferica nelle sequenze pesate in t1 e ipodensit di segnale nelle sequenze pesate in t2 . lobliterazione del cavo di douglas ( 2 / 92 ) stata visualizzata con segnale isointenso con focolai di iperintensit nelle sequenze pesate in t1 e con omogenea ipointensit nelle sequenze pesate in t2 . ( cid : 2 ) ( cid : 2 ) fig . 
in this case , a peritoneal lesion was interpreted as bowel content ( white arrow in b ) at mr imaging , but us correctly identified the lesion ( a )  . 
in questa paziente non stata riconosciuta lesione peritoneale ( freccia bianca in b ) , identificata invece dalla indagine ecografica ( a )  . demonstration of lesions of large ligaments and uterosacral ligaments proved difficult , if not impossible , for both techniques ( table 2 )  . 
in particular , although us was more sensitive than mr in demonstrating lesions of the rectovaginal septum , its specificity in this site was lower ( table 2 )  . discussion endometriosis refers to the presence of functioning endometrial tissue in a heterotopic site , more frequently in the ovaries , peritoneum , posterior cul - de - sac , uterosacral ligaments and serous surface of the rectosigmoid flexure or uterus . 
di questi 67 rilievi veri positivi , 33 sono stati identificati a carico delle ovaie , 20 a carico del setto rettovaginale , 8 a livello della parete rettale , 2 in corrispondenza della vagina , 1 a livello del fornice vaginale , 1 in corrispondenza del cavo del douglas , 1 sulla parete della vescica , ed 1 come obliterazione del cavo di douglas . 
entrambe le metodiche non hanno identificato ben 38 lesioni , localizzate in prevalenza nei legamenti larghi ( 10 casi ) ed in quelli uterosacrali ( 8 casi ) ( falsi negativi eg e rm )  . 
leg si dimostrata pi attendibile nello studio del setto retto - vaginale in virt dellapproccio trans - rettale , che peraltro ha favorito anche lidentificazione di lesioni a carico della parete rettale e della vagina . 
lindagine eg ( a ) evidenza una lesione ipoecogena a livello della parete anteriore della ampolla rettale , ma non riconosce lobliterazione del cavo di douglas , identificata dalla rm ( sequenza gre t1 fs ) ( b ) e confermata dalla laparoscopia . 
nel cavo di douglas apprezzabile nodulo ipointenso , stiramento delle anse intestinali e obliterazione dello spazio retto - uterino ( b )  . paroscopic classification of the american fertility society ( afs ) ( 1985 ) based on evaluation of the presence , size and depth of peritoneal or ovarian implants , density of adhesions and degree of cul - de - sac obliteration [ 7 , 12 ]  . 
nevertheless , inspection by physical examination is frequently inadequate for assessing extension of the disease to the posterior pelvis or the presence of adhesions , so that imaging plays a major role in diagnosing endometriosis in the posterior pelvis , bowel and bladder [ 1 , 13 ]  . 
the radiological diagnosis of endometriosis is largely based on the demonstration of endometriotic cysts or endometrioma , consisting of blood degradation products accumulated after repeated bleeding . endocavitary us , particularly with a transvaginal approach , proved to be very useful in diagnosing and differentiating endometriomas from other ovarian masses , with sensitivity of 83%88% and specificity of 89%90% [ 1416 ]  . accuracy of transvaginal us is better in endometriomas larger than 2 cm and in patients with a low risk of recurrent hemorrhagic cysts [ 1 ]  . 
endorectal us had sensitivity of 97% and specificity of 96% in the diagnosis of endometriosis of the rectovaginal septum and of 80% and 97% , respectively , in the assessment of infiltration of the uterosacral ligaments [ 17 ]  . 
a recent study found similar accuracy levels for transvaginal us and endorectal us in the evaluation of rectal endometriosis and proposed using transvaginal us as the firstline examination and endorectal us to investigate suspected intestinal involvement or before surgery [ 18 ]  . 
mr is cheaper than laparoscopy , can be repeated over time [ 19 ] , is not operator delizzazioni a carico dei legamenti larghi e / o utero - sacrali ( tabella 2 )  . la specificit delle due metodiche appare significativamente diversa , nettamente a favore della rm ( 25% eg vs . 50% rm , tabella 2 )  . tali insoddisfacenti risultati , anche per quanto riguarda la rm , trovano giustificazione da un lato nella quota relativamente elevata di falsi positivi e dallaltra nellesiguo numero di veri negativi . 
in particolare leg , dimostratasi pi sensibile della rm nella dimostrazione di localizzazioni nel setto retto - vaginale , risultata meno specifica proprio in questa sede ( tabella 2 )  . discussione con il termine endometriosi si definisce la presenza di tessuto endometriale funzionante in sede eterotopica , pi comunemente a carico delle ovaie , peritoneo , cavo di douglas , legamenti utero - sacrali , superficie sierosa del retto - sigma o dellutero . 
possono essere pur raramente interessati anche altri organi pelvici o extra - pelvici [ 911 ]  . lattuale stadiazione della malattia comunemente accettata quella laparoscopica proposta dallamerican fertility society ( 1985 ) basata su presenza , dimensioni , profondit di penetrazione degli impianti peritoneali e ovarici , sulla estensione delle aderenze , e sul grado di obliterazione del cavo di douglas [ 7 , 12 ]  . tuttavia , lispezione con esame obiettivo si dimostra frequentemente inadeguata per valutare lestensione della malattia alla pelvi posteriore , come anche la presenza di adesioni , perci limaging acquista un ruolo importante per la diagnosi nella pelvi posteriore , nellintestino e nella vescica [ 1 , 13 ]  . 
the left ovarian fossa shows a hyperintense lesion in the t1weighted sequence ( a ) , which is isointense and surrounded by a hypointense peripheral ring in the t2 - weighted sequence ( b )  . 
lindagine rm , meno costosa della laparoscopia , pu essere ripetuta pi volte nel tempo [ 19 ] , non operatore - dipendente , meno invasiva della ecografia con approccio endo - cavitario e non risente dello stato di riempimento vescicale . 
alcuni autori sostengono che il ruolo della rm nella diagnosi di endometriosi dipenda dalla sede degli impianti , riconoscendole ruolo utile nella identificazione di quelli localizzati in corrispondenza dei legamenti utero - sacrali e del setto rettovaginale [ 21 ]  . confrontando la stadiazione della malattia basata sul punteggio afs con la stadiazione proposta con limpiego dello studio rm emerge una concordanza del 89% [ 22 ]  . 
la metodica rm valuta in modo accurato i primi due parametri della stadiazione laparoscopica , ma il limite maggiore dellindagine rm risiede nella valutazione ed estensione delle aderenze [ 2224 ]  . non ci risulta che sino ad oggi siano stati effettuati studi di confronto della metodica rm con quella ecografica , in particolare quella effettuata mediante approccio endocavitario trans - rettale , allo scopo di valutare leffettivo apporto delle due metodiche nel riconoscimento e nella valutazione di estensione della malattia . in questo lavoro si cercato di verificare i risultati di ciascuna metodica nel definire la presenza , la sede e le dimensioni delle singole lesioni , mettendole a confronto fra loro e con la laparoscopia . 
ma tale lesione non confermata dalla indagine rm , sequenza t2 tse nel piano sagittale ( b ) n dallatto laparoscopico . pendent , is less invasive than us with endocavitary approach and is not influenced by degree of filling of the urinary bladder . 
some authors believe the role of mr in the diagnosis of endometriosis depends on the site of the implants and recognise its usefulness in detecting lesions located in the uterosacral ligaments and rectovaginal septum [ 21 ]  . 
mr is accurate in evaluating the first two parameters of the laparoscopic classification but is limited in the assessment of adhesions [ 2224 ]  . to our knowledge , no studies have compared mr and us , and in particular , endocavitary us with the transrectal approach , to evaluate the real contribution of the two techniques to identifying and staging the disease . 
the present study evaluated the results of each technique in assessing presence , location and dimension of each lesion and compared the techniques with each other and with laparoscopy . to this aim , we selected only patients with endometriosis , which limits the statistical value of the data . 
another limitation is that confirmation of the radiological diagnosis was obtained by laparoscopy , which is not a foolproof method . with these limitations , sensitivity and specificity of the two techniques in the diagnosis of disease were of 100% . 
in fact , all patients with a diagnosis of endometriosis were true positive at surgery , but if we consider the detection of single lesions , the results of the two techniques are more disappointing . 
overall , us demonstrated sensitivity of 58% ( table 2 ) , specificity of 25% ( table 2 ) , positive and negative predictive value of 82% and 9% and accuracy of 53% . 
mr demonstrated sensitivity of 56% ( table 2 ) , specificity of 50% ( table 2 ) , positive and negative predictive value of 90% and 16% , respectively , and accuracy of 55% . 
the two techniques therefore have excellent sensitivity for identification of endometriosis , but their performance in assessing disease extent is unsatisfactory owing to the possible misinterpretation of many la validit statistica dei risultati . 
considerando per il riconoscimento delle singole lesioni i risultati delle due metodiche sono meno brillanti . delle 143 lesioni confermate chirurgicamente infatti , ne sono state rilevate solo 83 con eg e 80 con rm . 
pertanto alla eg la sensibilit stata del 58% ( tabella 2 ) , la specificit del 25% ( tabella 2 ) , i valori predittivi positivi e negativi rispettivamente del 82% e del 9% , laccuratezza del 53% . 
alla rm la sensibilit stata del 56% ( tabella 2 ) , la specificit del 50% ( tabella 2 ) , i valori predittivi positivi e negativi rispettivamente del 90% e del 16% , laccuratezza del 55% . si deve pertanto ritenere che le due metodiche abbiano ottima sensibilit nella identificazione della endometriosi , mentre il loro bilancio di estensione della malattia assai meno accurato in quanto molte localizzazioni , identificate laparoscopicamente , possono essere misconosciute . 
endorectal us can in part overcome the field - of - view limitations of the transvaginal approach , as progress of the probe into the pelvis is not hindered by the vaginal fornices . 
at the same time there was a high incidence of false positive results for us in the diagnosis of very small lesions , especially those located in the rectovaginal septu because this site contains no anatomical structures that could resemble small endometriotic foci , it is more likely that they should go undetected during laparoscopy . 
in the rare cases that a single small lesion is detected , it is treated medically rather than surgically ; instead , where such lesions are associated with larger ones , detectable with the gynaecological examination , surgery is required , above all in the presence of pain . false positive us results might also be caused by the misdiagnosis of endometrioma in the presence of a haemorrhagic ovarian cyst , a tuboovarian abscess or an ovarian neoplasia [ 27 ]  . 
the false negativity of mr is accounted for by the very small dimensions of some endometriotic implants ; instead , its false positivity results from vessels and bowel content , both of which cause signal hyperintensity in t1weighted sequences , especially in those with fat suppression . conclusions to date , diagnostic accuracy of endorectal us and mr in the study of endometriosis is not significantly different . 
in contrast , in the case of a diagnostic doubt as to the real nature of the lesion , mr and , in particular , t1 - weighted sequences with fat suppression will prole lesioni pelviche , localizzate nel campo di vista del fascio ultrasonoro , e dalla maggiore esplorabilit e discriminazione tissutale consentita dalla indagine rm . 
lindagine ecografica , con approccio endo - cavitario trans - rettale , pu parzialmente ovviare alle limitazioni di campo di vista intrinseche alla metodica trans - vaginale in rapporto al mancato ostacolo , rappresentato dai fornici vaginali , alla risalita della sonda nello scavo pelvico . 
nel contempo si registrata elevata incidenza di false positivit degli us proprio nella diagnosi di lesioni di assai piccole dimensioni , localizzate soprattutto nello spessore del setto retto - vaginale . 
poich non esistono in tale sede strutture anatomiche che possano presentare aspetto simile ai piccoli focolai endometriosici descritti , parrebbe pi verosimile il loro mancato riconoscimento in corso di laparoscopia . in ogni caso , lesioni di cos piccole dimensioni presentano scarso valore clinico . 
infatti , nei rari casi di localizzazione unica , queste lesioni non sono suscettibili di trattamento chirurgico , ma solo farmacologico ; quando sono associate a lesioni di pi grosse dimensioni , valutabili peraltro con la sola visita ginecologica , comunque si impone il trattamento chirurgico , soprattutto in presenza di dolore . la falsa positivit ecografica pu inoltre derivare dalla errata diagnosi di endometrioma in presenza di cisti emorragica dellovaio , ascesso tubo ovarico o tumore ovarico [ 27 ]  . 
gli errori di falsa negativit della rm trovano il pi delle volte giustificazione nelle assai esigue dimensioni di alcune localizzazioni endometriosiche . per la falsa positivit rm giocano invece ruolo fondamentale i vasi ed il contenuto intestinale responsabili entrambi di iperintensit di segnale nelle sequenze t1 pesate , soprattutto in quelle con soppressione del grasso . conclusioni allo stato attuale leg , per via trans - rettale , e la rm non dimostrano significative differenze di accuratezza diagnostica nello studio della endometriosi . 
in particolare nel caso di documentata lesione pelvica , unica o multipla , con chiare caratteristiche di endometriosi , il ricorso alla rm appare superfluo dovendosi procedere direttamente con la laparoscopia . 
nel caso invece vi siano dubbi diagnostici circa la effettiva natura della lesione identificata , preferibile il ricorso alla rm che consente pi accurata analisi strutturale grazie soprattutto alle sequenze pesate in t1 con soppressione del grasso . 
di radiologia , universit degli studi , ospedale di cattinara , strada di fiume 447 , i - 34149 trieste , italy , tel . : + 39 - 040 - 910947 , fax : + 39 - 040 - 910921 , e - mail : dallap@units.it received : 12 march 2006 / accepted : 25 march 2006 / published online : 29 june 2006 abstract todays radiology is experiencing two major trends , one negative and one positive . 
the first is the so - called turf war , in other words , the progressive invasion of the imaging domain by other specialists such as cardiologists , urologists , gastroenterologists , gynaecologists etc . 
in this process , they are aided by new technologies such as picture archiving and communication systems ( pacs ) and computed - aided diagnosis cad and by radiology technologists who collaborate with them , replacing radiologists . 
the positive aspect is the outstanding technological evolution : the advent of molecular imaging , optical imaging , nanotechnologies , teleradiology and percutaneous gene therapy . while dramatically expanding the diagnostic possibilities down to the subcellular level , these techniques demand new forms of training in radiology and interdisciplinary cooperation . tomorrows radiologist will need to acquire appropriate clinical knowledge , restore contact with the patient to take on a prominent role in the diagnostic process , learn the basic sciences , foster a multidisciplinary approach and finally be able to use the internet for learning and continuing education . 
il primo rappresentato dalla cosiddetta guerra di campo o turf war ovverosia dalla progressiva invasione del campo dellimaging da parte di altri specialisti quali il cardiologo , lurologo , il gastroenterologo , il ginecologo etc . , che si vanno impossessando di varie tecniche , in primis lecografia e via via della tac e della rm . 
in questo sono favoriti dalle nuove tecnologie quali il pacs e le cad e dalla collaborazione con tecnici di radiologia che si sostituiscono , loro fianco , ai radiologi . laspetto positivo rappresentato dalla strepitosa evoluzione tecnologica : lavvento dellimaging molecolare , dellimaging a luce ottica , delle nanotecnologie , della teleradiologia , della terapia genica percutanea le quali se allargano enormemente le possibilit diagnostiche sino a livello subcellulare richiedono peraltro al futuro radiologo una nuova formazione e una collaborazione interdisciplinare . 
egli dovr acquisire una appropriata cultura clinica , ritrovare il contatto con il paziente per acquisire un ruolo preminente nelliter diagnostico , apprendere le scienze di base , favorire lapproccio operativo multidisciplinare e infine saper navigare in internet per favorire lapprendimento e laggiornamento . 
il radiologo del futuro sopravvivr se sapr reinventarsi . key words future radiology turf war imaging new technologies continuing education parole chiave radiologia , futuro guerra di campo nuove tecnologie dimmagini educazione continua introduction introduzione before considering possible future scenarios for radiologists in the new century , i find it useful to describe the current situation , outline recent developments in radiology and predict future developments : with radiology constantly evolving , how will radiologists survive ? my presentation therefore deals with : radiologists today : turf wars the radiology revolution radiologists tomorrow : what future ? trattare il tema di quale potr essere il futuro del radiologo in questo nuovo secolo implica iniziare con la fotografia della situazione attuale dei radiologi , delineare i recenti sviluppi della radiologia e prevedere i successivi per poi entrare nei vivo del tema : in una radiologia in continua evoluzione come sopravvivr il radiologo ? tratterr quindi in successione : il radiologo oggi : la guerra di campo la rivoluzione della radiologia il radiologo domani : quale futuro ? l . 
dalla palma : tomorrows radiologist : what future ? radiologists today : turf wars il radiologo oggi : guerra di campo in a recent article , levin and rao [ 1 ] wrote : we in radiology generally believe that for a number of reasons , we are the ones who should perform imaging on patients . 
we are familiar with all imaging modalities and are thus able to consult with ordering physicians and steer their patients to other studies that may be more appropriate for the clinical questions at hand.... 
and last but not least , we are trained in radiation and magnetic resonance imaging safety . despite the inherent truth and logic of this argument , many of our physician colleagues in other specialties do not agree with it . 
and this interest lies at the heart of the so - called turf battles or wars . the most blatant example is that of cardiologists , who have virtually taken over both coronary angiography and percutaneous coronary interventions ( angioplasty and stenting )  . 
radiologists have progressively abandoned cardioradiology . less striking but equally pressing is the turf war concerning percutaneous angioplasty and peripheral artery stenting . interesting data in this respect have been reported by levin et al . 
the authors evaluated the number of procedures carried out from 1997 to 2002 subdivided by specialists performing the while interventional vascular procedures done by radiologists increased by 29% , those done by cardiologists increased by 181% , those done by vascular surgeons by 398% and those done by other specialists by 195% . 
a clear sign of this is the recent creation of the in un loro recente articolo levin e rao [ 1 ] scrivono : noi in radiologia generalmente crediamo per un insieme di ragioni di essere quelli che dovrebbero acquisire le immagini dei pazienti . 
noi abbiamo familiarit con tutte le tecniche di imaging e siamo quindi nelle condizioni di consultare il medico richiedente e dirottare i loro pazienti ad altri tipi di indagini pi adatte a risolvere i quesiti clinici . 
nasce da questo interesse quella che in inglese viene definita turf battle or war ovverossia battaglia o guerra di campo . lesempio pi clamoroso lo abbiamo con i cardiologi che di fatto si sono impossessati pressoch totalmente sia della coronarografia che degli interventi percutanei coronarici ( angioplastica e stent )  . 
vi stato un progressivo allontanamento dei radiologi dalla cardioradiologia . meno importante , per incalzante , loccupazione di campo per quanto riguarda langioplastica percutanea e la collocazione di stents nelle arterie periferiche . 
mentre il numero di procedure intervenzionali vascolari dei radiologi aumentato del 29% , quello dei cardiologi del 181% , dei chirurghi vascolari del 398% e di altri specialisti del 195% . 
valutando il numero complessivo per specialit nei due anni di osservazione si nota come il numero di procedure eseguite da radiologi si sia abbassato dal 63 , 3% al 49% , quelle dei cardiologi si alzato dal 25 , 2% al 36 , 3% e di pari passo per gli altri specialisti . lavanzamento nel campo cardiovascolare sta profilandosi preoccupante per le tecniche non invasive quali la tc e la rm . 
ne un forte segnale la recente costituzione della society of cardiovascular computed tomography ( scct ) forsociety of cardiovascular computed tomography ( scct ) , composed of cardiologists , radiologists and vascular surgeons but with a predominance of cardiologist . 
the same is happening in mri with the creation of the society for cardiovascular magnetic resonance whose 2005 congress saw an increase in both scientific communications and posters compared with the previous 2 years . 
in europe , the european society of urologic imaging has been set up , with the more - or - less unwitting connivance of some radiologists ; society members so far include experts in ultrasonography ( us ) , ct and mri . a very lively battle is being fought over us , above all in certain fields ; the battle over cardiology has been lost whereas the situation is compromised in other fields such as obstetrics . 
european radiologists have controlled general us ( abdomen , pelvis , soft tissue , breast , etc . ) since the mid - 1970s , but the invasion by nonradiologists is becoming more aggressive on the grounds that sonography refines the clinical assessment . 
on the basis of the data collected , they divided the hospitals into four categories : in group 1 , radiologists performed 96%100% of examinations with an average of 2 , 800 exams per machine / year . 
in group 2 , radiologists performed 60%95% of exams with an average of 2 , 0003 , 000 exams per machine / year ; nonradiologists performed 300400 exams per machine / year . 
in group 3 , radiologists controlled 20%50% of the work with 2 , 5003 , 200 exams per machine / year ; nonradiologists performed an average of 300600 exams per machine / year . 
in group 4 , the authors list the hospitals where the situation is hopeless : at the university hospital of frankfurt , radiologists performed 1 , 500 exams per year on five sonography machines whereas it was assumed that nonradiologists , with 38 sonography machines , performed 35 , 00040 , 000 exams / year . 
according to the authors , this example is not the only one in germany and reflects the tendency to lose control over us , as is also happening in switzerland and austria . data gathered for the usa are also interesting . 
 [ 6 ] used the medicare database to collect data on the number of us examinations done by radiologists , cardiologists and other physicians from 1993 to 2001 per 1 , 000 medicare beneficiaries . 
analogo orientamento gi in atto per la rm con la costituzione della society for cardiovascular magnetic resonance che nel suo congresso del 2005 ha visto un aumento rispetto ai due anni precedenti sia delle comunicazioni scientifiche che dei posters . 
negli stati uniti da tempo pure attiva la north american society of cardiac imaging ( nasci )  . lo stesso fenomeno sta profilandosi in campo urologico . a livello europeo si infatti costituita , con la connivenza pi o meno ingenua di alcuni colleghi radiologi , la european society of urologic imaging che per ora si limita ad annoverare esperti di ecografia e cultori di tc ed rm . molto vivace invece lo scontro per lecografia soprattutto in alcuni campi ; mentre in cardiologia la battaglia perduta , in altri ancora , quali lostetricia , compromessa . con gli urologi la battaglia molto accesa mentre meno accesa con i gastroenterologi . i radiologi in europa hanno preso posizione nellecografia generale ( addome , pelvi , parti molli , mammella etc . ) sin dalla met degli anni 70 ma attualmente nel mondo non radiologico loccupazione di campo sta avanzando alla luce del concetto che la sonda ecografica consente di perfezionare lesame semeiologico clinico . 
su questa base , sulla miglior conoscenza del quadro clinico i medici specialisti autoprescrivono e conducono lesame ecografico : particolarmente aggressivi sono i medici del dipartimento di emergenza ed i gastroenterologi . 
nella categoria 3 i radiologi controllano il 20%50% dellattivit con un numero di esami per apparecchio / anno di 25003200 ; i non radiologi eseguono mediamente tra i 300600 esami apparecchio / anno . 
 [ 6 ] hanno raccolto il numero di esami ecografici eseguiti da radiologi , cardiologi e altri medici negli anni 1993 e 2001 attraverso il servizio medicare calcolando il numero su 1000 utilizzatori del servizio . 
i radiologi sono passati da 132 , 9 a 166 , 3 per mille con un aumento del 25% ; i cardiologi da 190 , 3 e 356 , 1 con un aumento dell85% ( nella grandissima maggioranza si trattava di ecocardiografie ) ; altri medici sono passati da 116 , 9 a 167 con un aumento del 43% . 
dalla palma : tomorrows radiologist : what future ? an 85% increase ( the vast majority were echocardiograms ) ; other physicians increased from 116.9 to 167 , a 43% increase . 
in other words , the number of examinations for nonradiologists ( excluding cardiologists ) grew twice as rapidly as it did for radiologists , a phenomenon driven by self - referrals , which lead to overutilisation and higher costs . 
the situation in italy is even bleaker given that the siumb ( italian society for ultrasonography in medicine and biology ) has also involved general practitioners , and it was facilitated in this by industry , which introduced portable us machines . a turf battle may also be looming for ct and mri at the level of both large hospitals and private practices . 
another example is mobile outsourcing where mri and ct machines are installed on vehicles that travel from hospital to hospital to make up for staff and / or technology shortages . 
here , the competition is not based on examination quality or staff professionalism , both of which have to be above standard in public facilities : the superspecialisation seen in public facilities surely fosters a better clinical - radiological interpretation . 
a typical example of teleradiology reporting centres can be seen in bangalore , india , which does plenty of work for the united states . let us now consider the situation in nonvascular interventional radiology , a domain that has been thoroughly investigated by schnyder et al . 
 in the meantime , radiologists devised new percutaneous drainage techniques , stenting procedures ( mostly biliary ) and balloon dilatation procedures and subsequently new abdommero di esami tra i non radiologi ( esclusi i cardiologi ) cresciuto due volte rispetto a quello dei radiologi , fenomeno sul quale incide lautoprescrizione che porta ad una superutilizzazione e a maggiori costi . 
in italia il fenomeno ancora pi grave dal momento che la siumb ha coinvolto come operatori i medici di medicina generale e lindustria li ha facilitati con la produzione di apparecchi portatili . la battaglia di campo pu profilarsi anche per la tc e rm sia a livello di grandi ospedali sia nellattivit privata . 
tipico lesempio dellimprenditore finanziario che attiva strutture tecnologicamente dotate e gestite da radiologi non sempre allaltezza ; tali strutture si mettono in concorrenza con la struttura pubblica , magari molto qualificata ma non sempre in grado di soddisfare tempestivamente il paziente esterno . 
caratteristico lesempio delloutsourcing mobile basato sullinstallazione di macchine rm e tc che si spostano di ospedale in ospedale per sopperire carenze di organico e / o di struttura . unaltra forma di outsourcing rappresentata dalla possibile creazione di centri di refertazione teleradiologici gestiti da radiologi cui far afferire immagini radiologiche digitali acquisite da colleghi di altre discipline . 
in questo caso la competizione non pu che basarsi sulla qualit dellesame e sulla professionalit dello staff che nella struttura pubblica devono essere di livello superiori : la superspecialit presente in questa struttura favorisce certamente una migliore interpretazione clinico - radiologica . 
un tipico esempio di tali centri lo riscontriamo in india - bangalore che ha unintensa attivit con gli stati uniti . passo ora a descrivere la situazione nel campo della radiologia interventistica non vascolare , aspetto ben analizzato da schnyder et al . 
la radiologia interventistica esplosa attorno agli anni 80 quando venne introdotta laspirazione percutanea di alcuni visceri addominali , di masse e raccolte per esami citologici e batteriologici e successivamente estesa al torace e incrementata con le biopsie con aghi di maggior calibro per esami istologici , attivit facilitate dalla guida con gli us e con la tc . 
pochi anni dopo inizi la battaglia , favorita appunto dallimpiego dellecografia , con i gastroenterologi per le procedure epatiche e biliari , con gli urologi per le nefrostomie e le litotrissie percutanee : questi specialisti stanno progressivamente avanzando . 
nel frattempo i radiologi mettevano a punto nuove tecniche di drenaggio percutaneo , procedure di stenting , prevalentemente biliare , e di dilatazione mediante palloncino ; successivamente a livello addominale altre tecniche apparivano , grazie ai radiologi , quali la tip e il posizionamento di filtri cavali . 
the current situation should be basically unchanged , favoured as we are by the advances in imaging technology such as ct fluoroscopy and open mri , which ensure precise guidance in the insertion of needles , catheters and probes . i end this section by illustrating yet another invasion that is occurring in england where , due to the small number of radiologists and increased workloads , the door has been opened for radiographers to report on chest and skeletal radiographs and carry out and report on contrast - enhanced exams such as barium enemas and urograms . 
the decision has been justified by the need for radiologists to devote their time to more complex and specialised examinations and by the opportunity given to radiographers to increase their motivation and to develop professionally . 
a recent metaanalysis of a homogeneous selection of 12 articles [ 8 ] demonstrated that the reporting accuracy of skeletal radiographs in emergency department patients did not differ between welltrained radiographers and radiologists with varying levels of experience . 
the degree of radiographer training only affected specificity , meaning that the distinction between normal and pathological requires a good level of training . another recent study found that the accuracy of radiographers in reporting on urographic examinations reached 92% compared with an expert uroradiologist [ 9 ]  . 
the trend is undoubtedly alarming as regards what could happen in italy where the law has given the head radiographer management responsibilities and set up degree courses for radiographers . it should also be noted that radiographers are being employed by other specialists , in particular , cardiologists and orthopaedic surgeons [ 11 ] , which broadens the gap between these clinical domains and radiology and turns the turf battle into a real war . the radiology revolution this is the aspect that will most influence the future of radiologists . 
and the magnitude of these advances also emerged in various sections of this excellent congress of our society , which has done admirably well in handling the developments of radiology from congress to congress . 
dalla palma : tomorrows radiologist : what future ? dovrebbe essere molto diversa al giorno doggi favoriti come siamo dallavanzamento della tecnologia di imaging quale la fluorotc e la rm a campo aperto che facilitano la guida precisa nellintroduzione di aghi , di cateteri e di sonde . chiudo questo capitolo segnalando unaltra invasione che in atto soprattutto in inghilterra dove per lo scarso numero di radiologi e laumento dei carichi di lavoro sono state aperte le porte ai tecnici radiologi nella refertazione di esami radiografici del torace e dello scheletro e nellesecuzione e refertazione di esami contrastografici quali il clisma e lurografia . 
questa decisione viene giustificata dalla necessit che hanno i radiologi di dedicarsi ad indagini pi complesse e pi specialistiche e dallopportunit che viene offerta ai tecnici di sentirsi pi motivati e di migliorare la loro professionalit . 
una recente valutazione metaanalitica su 12 articoli selezionati in modo omogeneo [ 8 ] ha documentato che laccuratezza nella refertazione di esami radiografici dello scheletro in pazienti provenienti dal pronto soccorso e dal dipartimento di emergenza non apparsa diversa tra tecnici radiologi ben addestrati e radiologi di varia anzianit . 
il grado di addestramento dei tecnici ha influenzato solamente la specificit per cui la distinzione tra normale e patologico richiede loro un buon training . un altro recente articolo ha riportato che laccuratezza del tecnico nel refertare esami urografici ha raggiunto il 92% messo a confronto con un esperto uroradiologo [ 9 ]  . tale iniziativa in atto anche in alcune universit degli stati uniti [ 10 ]  . 
non c dubbio che il fenomeno allarmante per quanto potrebbe succedere nel nostro paese dove la legge ha responsabilizzato il capotecnico nella gestione del reparto e ha attivato i corsi di laurea per tecnici . va inoltre tenuto presente che i tecnici radiologi vengono arruolati da altri specialisti , in particolare cardiologi ed ortopedici [ 11 ] aggravando il distacco delle rispettive attivit dalla radiologia e trasformando la battaglia in vera e propria guerra di campo . la rivoluzione della radiologia questo il capitolo che condizioner maggiormente il radiologo del futuro . 
in quale direzione stiamo andando ?  . per quanto concerne la radiologia del futuro trovo interessante riportare quanto ha scritto arenson [ 13 ] , chairman del dipartimento di radiologia delluniversit di san francisco , in un articolo dal titolo the practice of medicine and radiology in 2020 dove lautore descrive il futuribile che sintetizzo . 
dalla palma : tomorrows radiologist : what future ? as regards the future of radiology , the predictions made by arenson [ 13 ] , chairman of the department of radiology of the university of san francisco in an article titled the practice of medicine and radiology in 2020 are worth summarising . 
firstly , telemedicine will undergo incredible development with the use of videoconferencing , permitting a variety of interactions , including two - way audio and video communications between patients at home and the medical facilities where they have been examined . 
there will be considerable ageing of the population , with a mean age around 100 years for women and 90 years for men : some will survive until the age of 120 years , so there will be a preponderance of geriatric diseases and a corresponding decrease in paediatric diseases . plain film radiography will be supplanted by ct and mri and digital radiography as the first - line approach in emergency settings and the bedside modality in hospitals and ambulances . 
in interventional radiology , endovascular mri will guide not only catheters but also remote - controlled electronic devices that serve to launch a variety of devices that , like microscopic missiles , travel through vessels to reach organs or tissues adjacent to the vessels . 
picture archiving and communication systems ( pacs ) systems will be so efficient and widespread as to allow the transmission of images , connected to the patients smart card , to any part of the world . 
in this scenario , it is the radiologists who have expertise in electronics and informatics who will control the imaging of the future . as a postscript to my article , i briefly describe what is happening in the united states [ 14 ]  . 
recently , the national institutes of health ( nih ) provided funding for three broad lines of research , one titled new pathways to discovery , within which a number of bioinformatics and computational biology projects were selected . 
one of these is based at the brigham and womens hospital of boston , which operates through the national alliance for medical imaging computinterazioni ivi compreso la comunicazione audio e video a due vie che metter in contatto i pazienti da casa con la struttura medica dove stato esaminato . 
vi sar un invecchiamento notevole della popolazione con unet media attorno ai 100 anni per le donne e 90 anni per gli uomini : alcuni sopravviveranno sino ai 120 anni , per cui vi sar una preponderanza di patologia geriatrica , con una concomitante diminuzione della patologia pediatrica . lesame radiografico ceder il passo allesame con tc ed rm e allesame diretto digitale come primo esame nella patologia durgenza , e quale esame al letto del paziente e nelle ambulanze . 
a livello interventistico , la rm endovascolare guider non solo cateteri ma anche dispositivi elettronici controllabili a distanza e predisposti al lancio di congegni vari che come micromissili viaggiano nei vasi sino a raggiungere organi o comunque strutture tissutali adiacenti ai vasi . 
in questa evoluzione il radiologo che avr una formazione professionale elettronica ed informatica potr tenere sotto controllo limaging del futuro . in appendice a quanto ho scritto nel mio articolo ritengo opportuno aggiornarvi su quanto sta attualmente succedendo negli stati uniti [ 14 ]  . 
il brigham and womens hospital di boston opera attraverso il centro national alliance for medical imaging computing che costituito da un team multi - istituzionale che comprende computer scienziati , ingegneri software e ricercatori medici che si focalizzano sullo sviluppo di metodi computazionali per analizzare e visualizzare i dati di immagini mediche . 
per avanzare la frontiera della conoscenza di questa patologia mentale verranno utilizzati nuovi metodi computazionali , un robusto software integrato con imaging di risonanza magnetica strutturale , funzionale e di diffusione , elettroencefalografia quantitativa e pet . ing , a multiinstitutional team of computer scientists , software engineers and medical investigators who develop computational tools for analysis and visualisation of medical image data . 
focusing on schizophrenia , the research project aims at increasing knowledge about the disease by using new computational methods , a robust software integrated with structural , functional and diffusion mri , quantitative electroencephalography and positron emission tomography ( pet )  . thanks to research by image scientists , radiologists will find themselves handling the diagnosis and treatment of a large part of tomorrows medicine . tomorrows radiologist : what future ? we need to make a distinction between the near future and the distant future in relation to which radiologists will have to change many of their attitudes and rethink their professional training to accommodate the dramatic revolution and evolution of radiology . to face the near future , we need to reconsider our strategy in fighting the various turf wars described above . one of the main reasons why radiologists are losing many turf battles is no doubt their inadequate if not inexistent clinical culture : a high level of technical training is not sufficient for dealing with clinicians and their clinical queries . medical practice is becoming increasingly interdisciplinary due to the vastness of knowledge involved . 
good clinical training will enable radiologists to interact on a par with clinicians , even in view of the fact that clinicians are moving further and further away from the patient , being fascinated by the disease in contrast with the spirit of medicine passed down by hippocrates . 
we have all found ourselves reporting without any clinical data because the patient was referred to the radiology department before being examined . clinicalisation is therefore one of the main steps . 
clearly , this approach entails privileging organ pathology , which should be learned after having acquired a basic grounding . each staff radiologist working in a complex facility will have to be superspecialised in the imaging of a specific organ system ( nervous , musculoskeletal , chest , abdomen ) to be able to take part in an interdisciplinary discussion as a key player rather than merely an image reader unable to tackle a clinical differential diagnosis . 
clinicians will feel the need to seek the opinion of radiologists with good clinical knowledge ; otherwise , pacs and teleradiology will prevail , with images arriving directly in the clinical departments and bypassing the radiologist . 
dalla palma : tomorrows radiologist : what future ? il radiologo quindi , grazie alle ricerche dello scienziato dellimmagine , si trover a gestire diagnosi e terapia di una parte cospicua della medicina del futuro . il radiologo domani : quale futuro ? distinguo anzitutto un futuro immediato e un futuro in proiezione in rapporto ai quali i radiologi devono anzitutto modificare molti dei loro atteggiamenti e in secondo luogo devono reinventarsi una nuova formazione professionale che affronti la eccezionale rivoluzione ed evoluzione della radiologia . il futuro immediato va affrontato riorganizzando la strategia con la quale combattere le varie battaglie di campo che ho ora trattato . 
uno dei fattori principali che trovano i radiologi in ritirata in molte battaglie di campo certamente nella maggior parte dei casi la modesta per non dire assente cultura clinica : una elevata preparazione tecnica infatti non sufficiente per affrontare il collega clinico con i suoi quesiti . 
nellattivit medica che sta diventando sempre pi interdisciplinare per la vastit delle conoscenze che necessario saper dominare , una buona conoscenza della clinica consente un dialogo allo stesso livello con il clinico anche perch questi si sta sempre pi allontanando dal malato affascinato invece dalla malattia in contrasto con lo spirito della medicina tramandato da ippocrate . 
nellorganico di una struttura complessa quindi opportuno che ognuno dei radiologi sia super specializzato nella radiologia di un apparato ( nervoso , muscolo - scheletrico , torace , addome ) in modo da poter partecipare a un dibattito interdisciplinare come una figura di primo piano e non come un semplice lettore di immagini incapace di gestire una diagnostica differenziale clinica . 
un buon esempio di quanto valida sia la preparazione clinica la troviamo nel neuroradiologo la cui conoscenza della neurologia anatomo - clinica di ottimo livello , il che garantisce un ottimo dialogo con il neurologo e con il neurochirurgo . 
un radiologo con buona cultura clinica far sentire la necessit di essere interpellato dal collega ; in caso contrario prender il sopravvento la tecnologia pacs e teleradiologica con le immagini che arrivano direttamente nei reparti clinici bypassando il radiologo : se ci inevitabile per la casistica semplice , non cos avverr per la casistica complessa che non pu prescindere dal dibattito collegiale . 
la clinicizzazione del radiologo sar completa quanto pi egli riprender il contatto con il paziente ; questo si verifica con lecografia quando il radiologo conducendo lesame dialoga con il paziente e si trasforma , nel momento in cui gestisce la sonda , in un vero e proprio semeiologo clinico . 
this is the case in us where the radiologist talks to the patient while performing the examination and becomes , as he or she handles the probe , a true clinical semiologist . 
but for all the other modalities , despite the physicians respect for the radiologist who guides the diagnosis and treatment and documents treatment efficacy through follow - up examinations , patients do not perceive the importance of the role because of the minimal contact they have with the radiologist . 
as a result , the patient is convinced that it is the referring doctor who reads the examination , even more so today as pacs allows him or her to view the image . 
margulis and sostman [ 15 ] , however , state that radiologists need to restore the doctor - patient relationship , take the history and give patients information on the examination results so that patients may perceive they have been dealing with a doctor and not with a technician . 
i have had this experience in my private practice where this approach meant that the patients often wanted to see me before the clinician , who paradoxically devotes increasingly less time to the patient . 
this anecdotal data is confirmed by margulis and sostman [ 15 ] , who mention private ct screening centres in the united states where the radiologist - patient relationship is becoming more and more popular . 
this should not be merely the bureaucratic signing of a form but the signing of a consent based on correct information that justifies performance of the examination despite the risks associated with it and with a view to solving the clinical problem troubling the patient . 
contact with outpatients can be further improved by using internet technology : for example , by putting the report online as soon as it is ready , possibly together with the most significant images . already in place in the united states , this system was found to increase patient satisfaction and foster acknowledgement of the radiologist as the professional actually responsible for the examination and result [ 16 ]  . clinicalisation of radiology will encourage medical graduates to choose radiology as their speciality given that diagnostic radiology is often discarded precisely because it is considered exclusively technical , increasingly complex and without contact with patients , the latter being the most meaningful aspect of medicine . 
exclusively technical radiologists are destined to be progressively replaced by graduate radiographers and / or physicists with a sound medical knowledge . grado il ruolo del radiologo sia rispettato dal medico perch la sua interpretazione guida la diagnosi e la successiva terapia e documenta lefficacia di questultima con il follow - up , ci malgrado il paziente non percepisce questo ruolo proprio per il minimo contatto che ha con il radiologo , affermano margulis e sostman [ 15 ] che documentano questa affermazione sulla base di un questionario cui hanno risposto 42 dipartimenti universitari e 24 centri privati . 
egli invece , sostengono margulis e sostman , deve ritrovare il contatto con il paziente , raccogliere lanamnesi , dargli le prime informazioni sullesito dellesame in modo che abbia la percezione che ha avuto a che fare con un medico e non con un tecnico . 
io ho avuto questa esperienza nella mia attivit privata dove con questa impostazione mi sono trovato molto spesso ad essere richiesto dal paziente prima del clinico che paradossalmente dedica sempre meno tempo al paziente . 
questo dato aneddotico peraltro confermato da margulis e sostman che citano i centri tc di screening privati negli stati uniti dove il contatto paziente - radiologo la regola con un ritorno di popolarit . 
tale rapporto peraltro la regola nei centri senologici . il rapporto con il paziente una delle responsabilit etiche del radiologo , rapporto che nasce al momento del consenso informato : questo non deve essere una semplice firma burocratica su un modulo bens la firma ad un consenso basato su una informazione corretta che giustifica lesame pur con i rischi ad esso connessi , nella logica di risolvere il problema clinico che angoscia il paziente . 
il contatto con il paziente esterno ancora perfezionabile ricorrendo alluso della tecnologia internet e mettendo quindi in linea il referto non appena questo pronto ed eventualmente le immagini pi significative dellesame : tale sistema gi operativo negli stati uniti dove si notato una maggior soddisfazione del paziente che riconosce , fra laltro , nel radiologo , il vero responsabile dellesame e del risultato [ 16 ]  . la clinicizzazione del radiologo favorir la scelta della nostra specialit da parte del medico neolaureato che nel suo orientamento molto spesso scarta la radiologia diagnostica proprio perch considerata esclusivamente tecnica e sempre pi complessa e senza rapporto con il paziente che rappresenta il momento pi pregnante in medicina . 
il radiologo esclusivamente tecnico destinato a essere progressivamente soppiantato dal tecnico radiologo laureato e / o dal fisico con buone conoscenze mediche : questi sono favoriti dallimporsi della tecnologia pacs e della teleradiologia nonch dei computers con software dedicati al riconoscimento automatico dimmagine ( cad )  . 
ovvio che il clinico operante nei grossi complessi ospedalieri in mancanza di un radiologo con cultura clinica sar portato a gestirsi linquadramento diagnostico su immagini acquisite da altre figure tecniche . this is facilitated by pacs technology and teleradiology and by computers with software for automated image detection ( cad )  . 
clearly , the lack of radiologists with clinical knowledge will lead clinicians in large hospitals to make their diagnoses on images acquired by other technical professionals . clinicalised radiology also entails a need to recover the role of interventional radiologists who will need both hospital beds and a follow - up clinic for their work . patients undergoing percutaneous interventional procedures require adequate preparation and aftercare , as well as careful follow - up to check the success of the radiologists intervention . 
even in this case , patients will perceive that the radiologist has been crucial to the solution of their problem and can be relied on for the follow - up ; in other words , the radiologist becomes their treating physician as well . 
it should be noted that radiologists actually have the edge in the turf wars , as they have access to an array of imaging modalities to guide the procedure , and hospital management will consider the concentration of interventional procedures in a single facility devoted full - time to this activity as the economically soundest solution . 
this means that an interventional procedure facility that works part time is underutilised and therefore has higher costs . another limitation of nonradiologist specialists doing interventional procedures is the fact that they generally do not carry out all types of interventional procedures but only those pertaining to their speciality . 
in other words , when modernising the facility , they will have to devote the same attention and effort to locating resources and improving organisation as they do to upgrading diagnostic technologies . in order to survive , the radiologist of the near future will have to face up to the challenge of the dramatic technological revolution of the past few years . 
the advent of clinical spectroscopy , functional techniques in mri and us , quantitative and volumetric analyses with various digital techniques , optical imaging [ 20 ] , molecular imaging [ 21 ] , nanotechnologies [ 22 ] , the internet revolution [ 23 ] , percutaneous gene therapy [ 24 ] and the need for strategic planning [ 25 ] , all this calls for a parallel revolution in professional education and training . 
this is the task of the speciality schools that , to ensure the survival of radiologists , need to foster a diagnostic culture that goes beyond macroscopic anatomy and l . 
dalla palma : tomorrows radiologist : what future ? nel quadro della clinicizzazione si inserisce anche la necessit di recuperare il ruolo del radiologo intervenzionale che per la sua attivit necessita di alcuni letti di degenza e di un ambulatorio per il follow - up : il paziente che deve essere sottoposto ad una procedura intervenzionale percutanea deve essere adeguatamente preparato prima dellintervento e adeguatamente assistito dopo la procedura e tenuto sotto controllo nel follow - up per stabilire lefficacia o meno di quanto il radiologo ha fatto . 
anche in questo caso il paziente avr del radiologo la percezione che stato determinante nella soluzione del suo problema e che pu affidarsi a lui anche per il follow - up successivo : in altre parole diviene anche il suo medico curante . 
ovvio che il radiologo o lo staff radiologico deve essere competente per tutte le procedure intervenzionali vascolari , urologiche , gastroenterologiche , etc . , avere il suo laboratorio cateteri e assicurare un costante controllo di qualit . 
va tenuto presente che nella battaglia di campo il radiologo ha il vantaggio di disporre di varie tecniche di imaging utili a guidare la procedura intervenzionale e pu godere del favore degli amministratori dellospedale che in unattivit concentrata in ununica struttura dedicata full - time vedranno la soluzione pi convincente da un punto di vista economico . 
lattivit infatti condotta da chirurghi vascolari o addominali unattivit part - time perch il maggior tempo dedicato alla sala operatoria e / o al reparto : ne deriva che la struttura per lattivit intervenzionale che lavora part - time sottoutilizzata e comporta quindi un maggior costo di gestione . 
un altro limite degli specialisti non radiologi che eseguono attivit intervenzionale rappresentato dal fatto che in generale questo specialista non esercita tutta lattivit intervenzionale ma si limita a quella specifica del proprio settore di competenza . 
in altre parole nellaggiornamento della struttura il responsabile dovr dedicare alla radiologia interventistica la stessa attenzione e lo stesso impegno nella ricerca delle risorse e nel miglioramento dellorganizzazione che vengono dedicati allaggiornamento della tecnologia diagnostica . il radiologo del futuro oramai alle porte per sopravvivere deve saper affrontare e gestire la strepitosa rivoluzione tecnologica di questi ultimi anni . 
lavvento della spettroscopia clinica , delle tecniche funzionali in risonanza magnetica e in ecografia , delle analisi quantitative e volumetriche con le varie tecniche digitali , dellimaging a luce ottica [ 20 ] , dellimaging molecolare [ 21 ] , delle nanotecnologie [ 22 ] , della rivoluzione elettronica con luso di internet [ 23 ] , lavvento della terapia genica percutanea [ 24 ] , lesigenza di una cultura gestionale strategica [ 25 ] , tutto ci richiede una parallela rivoluzione nella formazione professionale . 
ci compito delle scuole di specializzazione che devono realizzare , per la sopravvivenza del radiologo , una cultura diagnostica che non deve limitarsi allanatomia e patologia macroscopica ma deve allargarsi alle strutture microscopiche e submicroscopiche : limaging molecolare ne un esempio . 
dalla palma : tomorrows radiologist : what future ? pathology to incorporate all microscopic and submicroscopic structures : molecular imaging is one example . this form of diagnostics based on microscopic or submicroscopic images opens the way for diagnosing diseases in the early , asymptomatic stage at the cellular level . 
we therefore need to strengthen the teaching of the basic sciences chemistry , biochemistry , physics , informatics and genetics by relying on experts who cannot limit themselves to teaching theory but will have to help the radiologist apply these new techniques in clinical practice . 
it has been reported that 90% of pet machines sold in europe are pet / ct machines [ 26 ]  . with regard to this technology , there is the problem of reporting , which in most cases is done by the nuclear medicine specialist and the radiologist for their respective competences . 
if most of this activity is currently represented by oncological diagnostics ( detection , staging and follow - up of lesions ) , the most convincing research on molecular imaging has been carried out with pet , as well as with very - high - field mri ( 47 tesla ) for experimental studies . how will the radiologist survive this revolution ? surely by acquiring a new culture but above all by interacting closely with the nuclear physician [ 27 ]  . 
in some countries , such as italy , these new technologies reopen the need to bring together diagnostic imaging and nuclear medicine in the same department , as is already the case in most north american and british hospitals . 
the mere number of modalities available for morphofunctional diagnostic imaging makes the need to combine a basic specialist culture with superspecialist organ - specific knowledge even more pressing . radiology residents need to be involved not only in learning but also in research on the grounds that todays research is tomorrows clinical practice [ 28 ]  . 
until now , research has taken its cue from the advent and evolution of digital technologies and has focused on the creation of a new semiology and on the evaluation of diagnostic accuracy . 
as marano states [ 29 ] , this is research funded by the market rather than by the university and scientific institutions on projects and protocols endorsed by them . there is virtually a complete lack of hypothesis - driven basic research . 
the reasons for this are several , the most important being a lack of specific training in specialty schools and a lack of funds , which have been mostly diverted towards biological , scientific , and clinical centres . 
if we want competitive radiologists capable of driving the development of diagnostic imaging , a field that clinicians find so appealing as to want to invade it , we need to reverse the situation and train ranostro ruolo nella diagnosi precoce mediante tecniche quali ecografia , tac ed rm . 
necessario quindi potenziare linsegnamento delle scienze di base chimica , chimica biologica , fisica , informatica e genetica coinvolgendo gli esperti di queste discipline che non devono limitarsi ad un insegnamento teorico ma devono affiancare il nuovo radiologo nella messa a punto clinica delle nuove tecniche . 
linsegnamento di queste discipline fondamentale ad esempio per una corretta gestione della tomografia ad emissione di positroni ( pet ) oggi sempre pi abbinata alla tomografia computerizzata ( tc )  . si ritiene che in europa il 90% delle macchine pet vendute sia rappresentato da macchine pet / tc [ 26 ]  . 
se al momento la maggior parte di questa attivit rappresentata dalla diagnostica oncologica ( riconoscimento di lesione , stadiazione e follow - up ) , alla pet fanno capo le ricerche pi convincenti di imaging molecolare , cui si affiancano , a livello sperimentale , quelle con rm ad altissimo campo , 47 tesla . 
queste nuove tecnologie riaprono in alcuni paesi come il nostro la necessit di vedere nello stesso dipartimento la radiodiagnostica e la medicina nucleare , organizzazione gi operante nella maggioranza degli ospedali nord - americani ed inglesi . 
il numero delle tecnologie disponibili per la diagnostica dimmagine morfo - funzionale rende ancor pi attuale lesigenza di una cultura specialistica di base affiancata da una cultura superspecialistica dorgano . fondamentale che lo specializzando venga coinvolto oltre che nellinsegnamento anche nella ricerca ricordando che la ricerca oggi pratica clinica domani [ 28 ]  . 
sinora la ricerca ha preso spunto dalla comparsa ed evoluzione delle tecnologie digitali e si basata sulla messa a punto di una nuova semeiotica e sulla valutazione dellaccuratezza nella diagnosi di patologia . 
si tratta nella gran parte dei casi di ricerca sponsorizzata dallindustria e da questa ispirata , e quindi di ricerca finalizzata ad ottenere ricadute socio - economiche a breve e medio termine . 
si tratta quindi , come dice marano [ 29 ] , di una ricerca finanziata dal marketing e non dalluniversit e istituzioni scientifiche su progetti e protocolli da queste avvallati . 
certamente in radiologia abbiamo sofferto questo aspetto per diverse ragioni , anzitutto per la mancanza di training specifico nelle scuole di specializzazione e in secondo luogo per la mancanza di finanziamenti , diretti prevalentemente alle strutture biologiche , scientifiche e cliniche . 
se vogliamo avere radiologi competitivi allaltezza di guidare lo sviluppo della diagnostica per immagini , che sempre pi affascina i nostri colleghi clinici al punto da invadere il campo dellimaging , necessario rovesciare la situazione formando un gruppo di radiologi preparati alla ricerca . 
the research training model devised by arenson , dunnick and hillman in the united states [ 30 ] and discussed and approved by the society of chairmen of academic radiology departments is interesting . 
in this case , residents follow programme 2 of the previous class . after the presentation , the society of chairmen of academic radiology departments drew the following conclusions : 1 . 
nellambito di un programma nazionale di educazione alla ricerca sono state identificate 3 categorie di dipartimento e precisamente : categoria 1 : comprende dipartimenti di radiologia con forte storia di ricerca competitiva e dotati di finanziamenti per supportare lavviamento e la formazione di ricercatori . il programma in questo caso prevede almeno un anno e preferibilmente due anni di ricerca che comprende : 1 . 
insegnamento a preparare richieste di finanziamento per progetti di ricerca . categoria 2 : comprende dipartimenti che hanno annualmente finanziamenti per coprire i costi dello sviluppo , di ricerca e di training di ricerca . 
almeno 3 mesi di esperienza nella ricerca seguiti dalla preparazione di un manoscritto da pubblicare che pu esser pronto 3 mesi dopo aver completato il tirocinio di ricerca ; 2 . 
un ciclo di lezioni , di presentazioni di ricerca ed esercizi di pensiero critico che sottolinea l ' importanza della ricerca nella pratica clinica . categoria 3 : comprende dipartimenti con modeste risorse umane , di ricerca e finanziarie per educare adeguatamente ricercatori competitivi . 
ogni dipartimento viene richiesto di assegnarsi la categoria di appartenenza e si invitano quelli di categoria inferiore ad adoperarsi per un avanzamento . gli autori cos concludono : il futuro della radiologia l . 
dalla palma : tomorrows radiologist : what future ? the authors conclude : the future of radiology depends on the training of a new class of radiologists who are clinicians and scientists . continuing education is extremely important . 
web - based learning is very appealing : education is widely available , inexpensive ( or free ) , always accessible , regularly updated and a huge amount of educational materials are available [ 23 ]  . 
constant technological developments and critical review of knowledge force radiologists to use web sites for their continuing education , but not all web sites undergo quality control and peer review . 
accuracy of the presentation is especially important . the authors also present four broad categories of web sites for radiology : ( 1 ) metalink sites provide a large number of links on a single portal ; ( 2 ) sites with teaching files , similar to our eurorad ; ( 3 ) subspecialty sites , such as those devoted to skeletal radiology , nuclear medicine etc . ; ( 4 ) technique - based sites , such as those devoted to multislice ct . 
the internet provides radiologists with an infinite learning resource that allows them to keep up - to - date with recent developments . in conclusion , to survive the tendency of other specialists to invade and occupy the radiology turf , the radiologist of the very near future will have to live up to the challenge by : 1 . 
becoming familiar with the basic sciences , physics , chemistry , biochemistry , genetics , informatics and nandipende dalla formazione di una nuova categoria di radiologi clinici e scienziati . estremamente importante l ' aggiornamento professionale : oggi una larga parte dei congressi , convegni e corsi sono accreditati e quindi sono la garanzia di un aggiornamento serio . 
l ' apprendimento basato sulla ricerca web molto attraente : l ' aggiornamento disponibile su larga scala , poco costoso ( o gratuito ) , accessibile in ogni momento , regolarmente aggiornato ed disponibile una vasta quantit di materiale educativo [ 23 ]  . 
la costante evoluzione tecnologica e revisione critica del sapere costringe il radiologo ad utilizzare per l ' aggiornamento i siti web che peraltro non hanno tutti un controllo di qualit e quindi un comitato di revisori . 
il radiologo insomma trova in internet una fonte di apprendimento inesauribile che gli consente un aggiornamento costante . in conclusione il radiologo del futuro che gi iniziato se vuole sopravvivere alla tendenza di altri specialisti di invadere ed occupare il campo della diagnostica per immagini deve affrontare nuove realt quali : 1 . 
dalla palma : tomorrows radiologist : what future ? otechnologies to be able to take on board new imaging techniques , such as molecular imaging , and new forms of percutaneous treatment , such as gene therapy 4 . 
fostering a multidisciplinary approach in his or her practice , made increasingly complex by technological advances : interaction with the nuclear physician is one of the first approaches determined by pet technology and pet / ct 5 . 
acquisire la dimestichezza di navigare in internet per favorire l ' apprendimento e l ' aggiornamento su siti web dedicati e disponibili in ogni momento . l ' apprendimento costante in una disciplina in continua evoluzione imperativo . 
salvatore dipartimento di scienze biomorfologiche e funzionali ( dsbmf ) , universit degli studi di napoli federico ii ( unina ) , istituto di biostrutture e bioimmagini ( ibb ) , consiglio nazionale delle ricerche ( cnr ) , napoli , italy correspondence to : s . 
maurea , via raffaele de cesare 7 , i - 80132 napoli , italy , tel . : + 39 - 081 - 7463560 / 2356 , fax : + 39 - 081 - 5457081 / 2296117 , e - mail : simone.maurea.@unina.it received : 26 september 2005 / accepted : 8 march 2006 / published online : 29 june 2006 abstract purpose . 
histology ( n = 19 ) , biopsy ( n = 3 ) or clinical - imaging follow - up ( n = 19 ) demonstrated 29 adenomas , five pheochromocytomas , three cysts and four carcinomas . 
axial and coronal imaging planes were used , with a slice thickness of 35 mmr images were qualitatively assessed for signal intensity of the adrenal mass relative to the liver on t1 , t2 , cs and t1 - gd scans ; diagnostic criteria for adenomas were isointensity or hypointensity on both t1 and t2 scans , out - of - phase cs signal loss and mild transient enhancement after gd . 
analysis of t1t2 signal intensity showed diagnostic accuracy , sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) of 80% , 72% , 100% , 100% and 60% , respectively . 
sono stati arruolati 36 pazienti ( 9 m , 27 f , et media = 51 , 314 , 4 anni ) con tumori surrenalici a sede unilaterale ( n = 31 ) o bilaterale ( n = 5 ) identificati incidentalmente nel corso dellesecuzione di altri esami dimaging ( ecografia , tc ) eseguiti per altre indicazioni cliniche . 
i risultati del nostro studio suggeriscono che la sequenza cs migliora significativamente laccuratezza diagnostica della rm nella identificazione degli adenomi surrenalici in confronto alle sequenze convenzionali t1 e t2 s . 
in particular , adenomas showed signal hypointensity relative to liver parenchyma on both t1 and t2 scans whereas pheochromocytoma , cysts and adrenal metastasis displayed signal hypointensity on t1 scans and hyperintensity on t2 scans . 
quantitative analysis of signal intensity of adrenal lesions compared with that of the liver , fat , muscle and image background was subsequently shown to improve characterisation of adrenal masses compared with qualitative analysis of signal variations [ 15 ]  . 
 the use of gadolinium ( gd ) , an intravenous paramagnetic contrast agent with extracellular distribution , made it possible to assess the enhancement pattern of tumor masses on the basis of signal variations induced by the contrast agent [ 1 , 7 ]  . compared with metastatic lesions , adrenal adenomas showed early arterial - phase enhancement followed by rapid washout [ 1 ]  . 
recently , a new mr imaging sequence known as chemical shift ( cs ) has been proposed , which is based on the different lipid content of adrenal lesions of a different histological nature [ 1 , 2 , 8 , 9 ]  . we report our experience with patients with adrenal masses to assess diagnostic accuracy of the cs sequence in differential diagnosis between adenoma and non adenoma lesions compared with conventional precontrast t1 and t2 scans and t1 scans after the administration of paramagnetic contrast material . materials and methods population a total of 36 patients ( 9 men , 27 women , mean age 51.314.4 years ) with adrenal masses were prospectively studied by mr of the upper abdomen . 
patients with adrenal masses documented by imaging studies were included ; in particular , the masses had been incidentally discovered during ultrasound ( us ) and / or ct performed for other clinical indications ( table 1 )  . 
a total of 41 materiali e metodi popolazione la risonanza magnetica ( rm ) stata proposta come valida alternativa alla tomografia computerizzata ( tc ) come tecnica di diagnostica per immagini per la caratterizzazione delle formazioni espansive a sede surrenalica [ 13 ]  . 
inizialmente , lesecuzione dellesame stata proposta mediante limpiego di sequenze t1 e t2 pesate che consentivano di valutare le variazioni del segnale rm come indice di tipizzazione tessutale [ 1 ] ; in particolare , gli adenomi hanno mostrato una ipointensit di segnale rispetto al parenchima epatico sia in t1 che t2 ; al contrario , il feocromocitoma , la cisti e le metastasi surrenaliche hanno mostrato unipointensit di segnale in t1 associata ad una iperintensit di segnale in t2 . 
successivamente , lanalisi quantitativa dellintensit del segnale rm delle lesioni surrenaliche in rapporto a quella del tessuto epatico , adiposo , muscolare e del fondo dellimmagine stata suggerita come pi valido approccio per la caratterizzazione delle masse surrenaliche rispetto allanalisi qualitativa delle modifiche del segnale [ 15 ] ; tuttavia anche questo tipo di analisi ha mostrato dei risultati non completamente soddisfacenti in quanto sovrapponibili tra lesioni di diversa natura [ 6 ]  . 
lutilizzo del gadolinio , mezzo di contrasto paramagnetico a distribuzione extra - cellulare somministrato per via endovenosa , ha consentito di valutare la dinamica del grado di impregnazione delle formazioni espansive in base alle modifiche del segnale indotte dal mezzo di contrasto [ 1 , 7 ] ; a tale proposito , gli adenomi surrenalici hanno mostrato una precoce impregnazione di gadolinio in fase arteriosa associata ad una rapida dismissione del mezzo di contrasto paramagnetico rispetto alle lesioni metastatiche [ 1 ]  . 
negli ultimi anni , stata proposta una nuova sequenza di acquisizione delle immagini rm , la sequenza chemical - shift ( cs ) , basata sulle differenze nel contenuto lipidico di lesioni surrenaliche di diversa natura istologica [ 1 , 2 , 8 , 9 ]  . 
riportiamo in questo studio la nostra esperienza in pazienti con formazioni espansive a sede surrenalica allo scopo di valutare laccuratezza diagnostica della sequenza cs per la diagnosi differenziale tra lesioni adenomatose e non - adenomatose in confronto alle sequenze convenzionali t1 e t2 pesate eseguite in fase pre - contrastografica e alla sequenza t1 dopo mezzo di contrasto paramagnetico . sono stati studiati prospetticamente con rm delladdome sus . 
in the remaining 19 patients with lesions smaller than 3 cm , clinical - imaging follow - up yielded a diagnosis of adenoma ( n = 17 ) or pheochromocytoma ( n = 2 )  . periore 36 pazienti con formazioni espansive a sede surrenalica ( 9 m , 27 f , et media = 51 , 314 , 4 anni ) , di cui 31 con lesioni a sede monolaterale e 5 con lesioni a sede bilaterale ; il criterio di inclusione dei pazienti nel gruppo di studio consisteva nellevidenza di lesioni espansive a sede surrenalica , documentate dal risultato di esami di diagnostica per immagini ; in particolare , i tumori surrenalici sono stati identificati incidentalmente nel corso di ecografia e / o tc eseguiti per altre indicazioni cliniche ( tabella 1 )  . 
sono state valutate in totale 41 lesioni ( diametro medio 3 , 02 , 2 cm ) e sono stati utilizzati come standard di riferimento per la diagnosi di natura i risultati istologici ( n = 19 ) , bioptici ( n = 3 ) o del follow - up clinico - strumentale ( n = 19 ) ; il follow - up clinicostrumentale comprendeva i dosaggi ormonali specifici della funzione ghiandolare ( corticale e / o midollare ) surrenalica , s . 
all examinations were carried out with 3to 5mm - thick axial and coronal scans ; more specifically , slice thickness was 5 mm for axial scans and 3 mm for coronal scans . 
the studies were integrated with cs t1 - dual - ffe sequences with a tr of 236 ms and double te of 4.6 ms in phase and 2.3 ms out of phase in the axial plane . 
axial t1 - ffe - bh il monitoraggio dimensionale delle lesioni mediante ecografia e / o tc ed esame scintigrafico della zona corticale e / o della zona midollare dei surreni ; in particolare , la mediana della durata del follow - up stata uguale a 5 mesi ( intervallo compreso tra 3 e 8 mesi )  . 
lesame istologico ha mostrato la presenza di adenoma ( n = 10 ) , feocromocitoma ( n = 3 ) , carcinoma ( n = 3 ) e cisti ( n = 3 ) ; lesame bioptico ha dimostrato la presenza di 2 adenomi e 1 carcinoma ; nei rimanenti 19 pazienti , con lesioni di dimensioni inferiori ai 3 cm , il followup clinico - strumentale ha consentito di formulare la diagnosi di adenoma ( n = 17 ) o di feocromocitoma ( n = 2 )  . risonanza magnetica gli esami rm sono stati effettuati utilizzando un apparecs . 
administration of extracellular paramagnetic contrast agent ( gadolinium chelates , 0.1 mmol / kg body weight ) using a dynamic technique with preset scan times and image acquisition in the arterial ( 30 s ) , portal ( 60 s ) and late equilibrium phases ( 180300 s )  . image and data analysis mr images were qualitatively analysed by two radiologists who assessed both the preand postcontrast signal intensity of adrenal lesions relative to that of the liver parenchyma ; for three cases in which there was no agreement , a third radiologist was consulted . 
imaging signs considered diagnostic of adenoma were : t1 and t2 isoor hypointensity , cs signal loss ( definite hypointensity ) on out - of - phase images relative to in - phase images and early enhancement during the postcontrast arterial phase with rapid washout during the following phases [ 13 ]  . results of the qualitative analysis of t1t2 , cs and postcontrast t1 mr images were compared with histological , biopsy and follow - up data defining the nature of the adrenal lesions ( pheochromocytoma , adenoma , cyst , myelolipoma and malignancies )  . 
in particular , we compared the results of t1t2 images , cs in - phase and outof - phase images and postcontrast t1 images . we calculated accuracy , sensitivity and specificity values , as well as positive ( ppv ) and negative predictive values ( npv ) in the three series of images ( t1t2 , in - phase and out - of - phase cs and postcontrast t1 )  . statistical analysis to assess statistical significance of the differences observed in accuracy , sensitivity , specificity , ppv and npv among the three series of mr images ( t1t2 , cs and postcontrast t1 ) , we used the mcnemar test ; a value of p < 0.05 was taken to be statistically significant . results results of qualitative analysis of t1t2 signal intensity performed for each lesion are shown in table 2 . 
gli esami sono stati integrati con sequenza cs t1 - dual - ffe con tr di 236 ms e doppio te di 4 , 6 ms in - fase e 2 , 3 ms in - opposizione di fase secondo il piano assiale . la sequenza t1 - ffe - bh , in piani di scansione assiali stata ripetuta dopo somministrazione endovenosa di mezzo di contrasto ( mdc ) paramagnetico a distribuzione extra - cellulare ( chelati di gadolinio , 0 , 1 mmol / kg di peso corporeo ) con tecnica dinamica a tempi pre - fissati ed acquisizione di immagini in fase arteriosa ( 30 s ) , in fase portale ( 60 s ) e in fase tardiva allequilibrio ( 180300 s )  . analisi delle immagini e dei dati le immagini rm sono state analizzate qualitativamente da due medici radiologi valutando le caratteristiche di intensit del segnale delle lesioni surrenaliche , sia in fase preche post - contrastografica , in rapporto allintensit del segnale del parenchima epatico ; in tre casi in cui si verificata discordanza di interpretazione delle immagini , stato consultato un terzo medico radiologo . 
lintensit del segnale rm delle lesioni surrenaliche in t1 e t2 stata classificata come isointensa , ipointensa , lievemente o nettamente iperintensa rispetto a quella del tessuto epatico . gli studi rm acquisiti con sequenza cs sono stati valutati qualitativamente in base alla variazione di intensit del segnale delle lesioni surrenaliche tra le immagini in - fase e quelle in - opposizione di fase ; stata considerata come significativa una variazione dellintensit del segnale rm in termini di netta ipointensit ( perdita di segnale ) in - opposizione di fase rispetto alle immagini in - fase . 
sono state , inoltre , valutate le modifiche dellintensit di segnale delle lesioni surrenaliche nelle diverse fasi ( arteriosa , portale e tardiva allequilibrio ) post - contrastografiche ; il grado di impregnazione di mdc stato classificato come assente , lieve o netto . sono stati considerati criteri semeiologici diagnostici per adenoma lisoo lipo - intensit sia in t1 che in t2 , la perdita di segnale ( netta ipointensit ) in - opposizione di fase rispetto alle immagini in - fase in sequenza cs e la precoce impregnazione di mdc in fase arteriosa post - contrastografica con rapida dismissione di mdc nelle successive fasi di studio [ 13 ]  . 
one of the aims of medical research in this field is to investigate the possibility of characterising adrenal lesions by using imaging methods so as to avoid the need for biopsies [ 12 ]  . mr is one of the most accurate methods for morphofunctional evaluation of adrenal masses [ 1 ] , and one of its advananalisi statistica allo scopo di valutare la significativit statistica delle differenze osservate nei valori di accuratezza , sensibilit e specificit diagnostica , e dei valori predittivo positivo ( vpp ) e negativo ( vpn ) tra le tre serie di immagini rm ( t1 - t2 , cs e t1 dopo mdc ) stato impiegato il test di mc nemar ; un valore della p < 0 , 05 stato considerato statisticamente significativo . risultati i risultati dellanalisi qualitativa delle intensit di segnale s . 
in addition , qualitative and quantitative analyses of the mr signal , the use of paramagnetic contrast material and cs imaging constitute specific methods that allow characterisation of adrenal masses [ 1 , 48 ]  . 
the cs sequence relies on the different resonance frequencies of the hydrogen protons in tissues with a prevalent water or lipid component as a result of the electron shell around the nucleus . 
by creating a local magnetic field , this electron shell forms a shield that depends on the density and spatial distribution of electrons and interferes with the main magnetic field [ 9 ]  . 
by modifying the te , water and fat protons may be in phase ( longer te ) or out of phase ( shorter te ) , leading to differences in signal intensity that allow identification of prevalently fatty adrenal adenomas out of phase , with significant signal reduction with respect to in - phase images . in our experience , use of the cs sequence or postcontrast t1 sequence succeeded in reducing the rate of false negatives in the identification of adrenal adenomas compared with conventional t1and t2 - weighted sequences . 
in dettaglio , la differente incidenza di risultati falsamente negativi nel confronto tra le immagini rm t1 - t2 , in cs e t1 dopo mdc ha determinato la significativit statistica in termini di accuratezza , sensibilit e vpn tra le tre serie di immagini rm . discussione in pazienti con formazioni espansive a sede surrenalica , laspetto clinico fondamentale rappresentato dal riconos . 
la lesione si presenta isointensa al parenchima epatico sia nelle scansioni assiali ffe t1 pesate ( a ) che in quelle tse t2 pesate ( b ) : risultato t1 - t2 vero positivo . zation of adrenal adenomas and obviates the need for the postcontrast t1 sequence . clinical applications of the cs sequence are based on the different lipid content of benign and malignant adrenal lesions [ 1117 ] ; in particular , adrenal adenomas have a higher lipid concentration than metastases . 
the variability of fat content of adenomas may lead to heterogeneous signal alterations on out - of - phase images relative to in - phase images [ 13 ]  . 
in addition , the possible presence of a lipid component in malignant adrenal lesions may result in reduced out - ofphase signal similar to that seen in adenomas and therefore lead to false positive results at cs imaging [ 14 ]  . 
we had three cases of false positive adenomas at cs imaging ; in these patients , lower fat content might have accounted for the different pattern out of phase [ 13 ]  . 
nonetheless , the low false negative rate of cs imaging led to significantly better accuracy and sensitivity values compared with conventional t1t2 sequence such that the cs sequence increased the diagnostic potential of the mr examination . 
accuracy , sensitivity and specificity values in this study were 94% , 89% and 99% , respectively , resulting in high predictive values for the cs sequence in the characterization of adrenal adenomas . a further possibility offered by the cs sequence is the scimento della natura benigna o maligna di tali lesioni allo scopo di definire la terapia adeguata [ 10 ]  . 
numerose tecniche di diagnostica per immagini possono essere utilizzate nella valutazione morfo - funzionale di tali pazienti , quali la tc , la rm e le metodiche radionuclidiche [ 1 , 6 , 11 ]  . 
uno degli obiettivi della ricerca medica in tale settore rappresentato dalla possibilit o meno di ottenere informazioni per la caratterizzazione tessutale delle lesioni surrenaliche utilizzando queste metodiche strumentali allo scopo di evitare lesecuzione di un esame bioptico [ 12 ]  . 
la rm rappresenta una delle metodiche pi accurate per la valutazione morfofunzionale delle lesioni espansive a sede surrenalica [ 1 ] , in particolare , le scansioni multiplanari rappresentano uno dei vantaggi di questa tecnica rispetto alle altre metodiche ; inoltre lanalisi qualitativa e quantitativa del segnale rm , limpiego del mdc paramagnetico e le sequenze cs costituiscono aspetti tecnici specifici per la caratterizzazione tissutale delle masse surrenaliche [ 1 , 48 ]  . 
la sequenza chemicalshift si basa , in particolare , sulla differente frequenza di risonanza dei protoni di idrogeno nei tessuti a prevalente componente fluida o adiposa in dipendenza della nube elettronica che circonda il nucleo ; tale nube elettronica , creando un campo magnetico locale , determina una schermatura , dipendente dalla densit e dalla distribuzione spaziale degli elettroni , che interferisce con lazione del campo magnetico principale [ 9 ]  . 
i protoni di idrogeno dei tessuti di tipo fluido sono meno schermati verso il campo magnetico esterno rispetto a quelli dei tessuti a componente adiposa ; ci determina che i protoni dei tessuti di tipo fluido hanno una maggiore frequenza di risonanza e , dunque , un segnale pi elevato . 
modificando il tempo di eco ( te ) della sequenza possibile che i protoni dei tessuti di tipo fluido e quelli di un tessuto a prevalente contenuto adiposo siano in - fase ( te pi lungo ) o in - opposizione di fase ( te pi breve ) realizzando delle differenze di intensit tali da poter identificare in - opposizione di fase gli adenomi surrenalici a prevalente componente adiposa e , dunque , con significativa riduzione di segnale rispetto alle immagini in - fase . 
nella casistica riportata ed in base alla nostra esperienza limpiego della sequenza cs o della sequenza t1 dopo mdc ha consentito di ridurre lincidenza dei risultati rm falsamente negativi , rispetto alle immagini convenzionali t1 e t2 pesate , nella corretta identificazione delle lesioni adenomatose delle ghiandole surrenaliche . 
la significativit statistica delle differenze in accuratezza , sensibilit e vpn della sequenza cs e t1 dopo mdc rispetto alle sequenze convenzionali t1 - t2 suggerisce certamente un ruolo clinico diagnostico dellimaging rm in cs per la caratterizzazione degli adenomi surrenalici senza la necessit di ricorrere allimpiego della sequenza t1 post - contrastografica . 
le applicazioni cliniche della sequenza cs si basano sulle differenze del contenuto lipidico tra lesioni surrenaliche benigne e maligne [ 1117 ] ; in particolare , stata osservata una maggiore concentrazione di lipidi negli adenomi surrenalici rispetto alle metastasi . la sequenza cs pu , tuttavia , presentare alcune limitazioni ; infatti , il contenuto lipidico variabile degli adenomi pu determinare una eterogeneit delle modifiche del segnale rm in - opposizione di fase rispetto alle immagini infase [ 13 ]  . 
the index is calculated on the basis of the difference between signal intensity in phase and out of phase divided by signal intensity in phase [ 2 , 16 ]  . 
in more detail , adenomas were shown to have an index of 0.36 , corresponding to 27% fat , whereas metastatic lesions or pheochromocytomas had negative signal - intensity indexes , with values corresponding to 0% fat [ 16 ]  . 
this method for quantitative assessment of signal intensity was found to be more accurate than calculating the differences in signal intensity between adrenal lesions and that of other locoregional tissues , such as liver , spleen and muscle [ 17 ]  . 
i nostri risultati hanno mostrato tre casi falsamente negativi per lesione adenomatose in sequenza cs ; in tali pazienti , la minore concentrazione lipidica potrebbe giustificare il diverso comportamento in - opposizione di fase [ 13 ]  . 
tuttavia la bassa incidenza di risultati falsi negativi in sequenza cs ha determinato valori di accuratezza e sensibilit significativamente superiori rispetto alla sequenza convenzionale t1 - t2 , tali da realizzare un incremento delle potenzialit diagnostiche dellesame rm utilizzando tale sequenza . 
i risultati del nostro studio concordano con quelli di uno studio recentemente pubblicato riguardo un significativo gruppo di pazienti con masse surrenaliche valutate qualitativamente con la sequenza cs [ 15 ] ; in tale esperienza , gli autori hanno studiato 182 pas . 
t1 - fast field echo ( ffe ) axial dynamic scans with fat suppression before gadolinium ( gd ) administration ( a ) , and in arterial ( b ) and delayed ( c ) phases after gd administration . 
scansioni assiali in sequenza dinamica ffe t1 pesata con soppressione del segnale del tessuto adiposo in fase pre - contrastografica ( a ) , in fase arteriosa post - contrastografica dopo gadolinio ( b ) e in fase tardiva post - contrastografica dopo gadolinio ( c )  . 
la lesione mostra precoce impregnazione contrastografica in fase arteriosa ( b ) con associata rapida dismissione contrastografica in fase tardiva ( c ) : risultato rm t1 - gadolinio vero positivo . zienti riportando valori di accuratezza , sensibilit e specificit diagnostica della sequenza cs analoghi a quelli da noi osservati , rispettivamente 94% , 89% e 99% che comportavano elevati valori di predittivit diagnostica della sequenza cs per la caratterizzazione degli adenomi surrenalici . una ulteriore possibilit metodologica della sequenza rm in cs rappresentata dalla valutazione quantitativa delle variazioni dellintensit di segnale in - opposizione di fase rispetto alle immagini in - fase mediante il calcolo dellindice di intensit di segnale di una lesione surrenalica adenomatosa o non - adenomatosa ; tale indice viene calcolato in base alla differenza tra lintensit di segnale in - fase e quella inopposizione di fase diviso lintensit di segnale in - fase [ 2 , 16 ] ; stato dimostrato che tale indice espressione della percentuale in contenuto adiposo della lesione , tale che lindice di intensit di segnale pari a 1 corrisponde ad una quota adiposa di circa il 90% ; in particolare , gli adenomi hanno mostrato un indice pari a 0 , 36 che corrispondeva ad una frazione adiposa di circa il 27% , mentre le lesioni metastatiche o i feocromocitomi mostravano indici di intensit di segnale negativi con valori corrispondenti di percentuale adiposa pari a zero [ 16 ]  . 
questo metodo di valutazione quantitativa dellintensit di segnale risultato essere pi accurato rispetto al calcolo dei rapporti tra lintensit di segnale della lesione surrenalica e quella di altri tessuti loco - regionali , quali il tessuto epatico , splenico e muscolare [ 17 ]  . 
in base ai recenti dati riportati in letteratura [ 18 ] , la rm con sequenza cs ha mostrato un suo ruolo specifico nei casi in cui la tc non consente di caratterizzare con certezza una les . 
da ronch , istituto di radiologia apugd , via colugna 50 , i - 33100 udine , italy , e - mail : tdaronch@sirm.org received : 9 june 2005 / accepted : 17 november 2005 / published online : 29 june 2006 abstract purpose . 
between august 2000 and september 2004 , we conducted a retrospective study of the abdominal ct of 15 patients with histological diagnosis of gist that is , the immunopositivity for c - kit ( cd117 ) on a surgical specimen of the primary disease . 
we used a helical ct single - slice ( toshiba , asteion ; rotation time 0.75 s ) in ten cases and helical ct multislice ( toshiba , aquilion ; 4 slices / rotation ; rotation time 0.5 s ) in five cases . 
size , partial or total extraluminal tumour growth , homogeneous or inhomogeneous lesion enhancement and the eventual presence of calcifications were assumed to be criteria for characterisation of a gist . 
we demonstrated one substantial concordance between radiological and pathological valuation of site , morphology , tumour size and absence of intralesional calcifications of gist . nine of ten gist smaller than 5 cm , the two 510 cm and the three larger than 10 cm presented extraluminal growth . enhancement was inhomogeneous in five of ten lesions smaller than 5 cm and in all cases larger than 5 cat pathological analysis , in all cases of inhomogeneous enhancement , solid , hemorrhagic , necrotic and cystic areas were found . 
of seven tumours of intermediate malignancy , six were smaller than 5 cm and only one larger than 10 cm ; the two 510 cm were of high malignancy and all tumours superior of 10 cm were at intermediate or high malignancy . 
si sono prese in considerazione , mediante studio retrospettivo , le tc addominali di 15 pazienti con diagnosi istologica di gist sul pezzo operatorio in malattia primitiva , in un periodo compreso tra agosto 2000 e settembre 2004 . 
le apparecchiature tc usate sono state una spirale a singolo strato ( tcss ) ( toshiba , asteion ; tempo di rotazione 0 , 75 s ) in 10 casi ed una multistrato ( tcms ) ( toshiba , aquilion ; 4 strati / rotazione ; tempo di rotazione 0 , 5 s ) in 5 . 
in tutti i casi stato utilizzato un mezzo di contrasto ( mdc ) organo - iodato non ionico ad una concentrazione di iodio variabile da 350 a 400 mgi / ml per via endovenosa . 
la sede , la morfologia e le dimensioni ( classificate in tre gruppi : inferiori a 5 cm , comprese tra 5 e 10 cm e superiori a 10 cm ) sono stati considerati parametri per lidentificazione della neoplasia ; le dimensioni , lo sviluppo parzialmente o francamente estrinseco , lenhancement omogeneo o disomogeneo della lesione e leventuale presenza di calcificazioni al suo interno sono stati assunti come criteri di caratterizzazione di un gist ; la presenza di metastasi epatiche , peritoneali e / o linfonodali stata considerata parametro di malignit intermedia o elevata . 
si dimostrata una sostanziale concordanza tra valutazione radiologica ed anatomo - patologica per quanto riguarda la sede , la morfologia , le dimensioni dei gist e lassenza di calcificazioni intralesionali . 
tumour size established with ct was not sufficient to determine the degree of malignancy . metastases at the time of diagnosis were indicative of intermediate or high malignancy , but the absence of metastases did not allow classification of gist in the group of low and very low risk of malignancy . key words gist helical ct stomach bowel liver tutti i casi di enhancement disomogeneo , allanalisi del pezzo operatorio si sono trovate aree sia solide , che necrotiche , emorragiche e / o cistiche . 
delle 7 neoplasie a rischio intermedio di malignit 6 erano inferiori a 5 cm e solo 1 superiore a 10 cm , 2 su 2 di dimensioni tra 5 e 10 cm rientravano nel gruppo a rischio elevato e tutte quelle superiori a 10 cm rientravano nel gruppo a rischio intermedio o in quello a rischio elevato . 
la diagnosi di certezza dei gist richiede la dimostrazione dellimmunopositivit al cd117 , ma la diagnostica per immagini ed , in particolare , la tc spirale svolge un ruolo di primaria importanza . 
da questo studio emerso che la tc si rivelata attendibile nellidentificare la neoplasia , che tutti i tumori di dimensioni superiori a 5 cm presentavano uno sviluppo estrinseco , un enhancement disomogeneo , lassenza di calcificazioni e di interessamento linfonodale e che anche quelli inferiori a 5 cm tendono a presentare uno sviluppo estrinseco ed una densit disomogenea . 
after more than 50 years of controversial classification , the term gist is currently reserved for gists that are immunopositive for c - kit ( cd117 ) , a receptor for the tyrosine - kinase growth factor [ 3 , 5 , 69 , 11 ]  . 
with the exception of the presence of metastasis at diagnosis , an invariably negative prognostic factor , tumour size and mitotic index alone seem to be predictive of the benign or malignant behaviour of gist [ 3 , 8 ] , and these parameters are commonly used by pathologists for prognostic evaluation as proposed by fletcher et al . 
 [ 3 ] ( table 1 )  . gist tends to affect individuals aged over 50 years , without any gender predilection [ 7 , 9 , 12 ]  . 
although they may affect any level of the gastrointestinal tract , the most frequent site is the stomach ( 60%70% ) followed by the small bowel ( 20%30% ) , the anusrectum ( 7% ) , the colon ( 5% ) and the oesophagus ( 2% )  . 
unlike the case with many other neoplasms , the lymph nodes are not the main route of spread [ 1 , 7 , 11 , 1315 , 17 , 18 ]  . the most frequent clinical presentations are haematemesis , melaena and signs of anaemia ; bowel obstruction ; abi tumori stromali gastrointestinali ( gist ) sono le pi comuni neoplasie mesenchimali del tratto gastroenterico . 
attualmente il termine gist , dopo oltre cinquantanni di classificazioni alquanto dibattute , indica esclusivamente i tumori stromali gastrointestinali che presentano limmunopositivit al c - kit ( cd117 ) , un recettore per il fattore di crescita tirosino - kinasi [ 3 , 5 , 69 , 11 ]  . 
eccetto il fattore prognostico invariabilmente sfavorevole della presenza di metastasi al momento della diagnosi , solo le dimensioni della neoplasia ed il tasso di mitosi sembrano essere criteri predittivi di un comportamento benigno o maligno dei gist [ 3 , 8 ]  . 
the use of fast ct scanners allowing thin - slice acquisitions and high - quality multiplanar reconstructions associated with optimisation of iv contrast material enables detailed evaluation of the gastrointestinal tract as well as staging of a tumour in this area . 
in gist in particular , despite the fact that data are few and often imprecise as a result of recent introduction of immunohistochemical classification , there seem to be highly characteristics signs for these neoplasms [ 2 , 4 , 7 , 10 , 14 , 15 , 2224 ]  . as regards treatment , surgical resection when feasible is the procedure of choice given that the response rate to conventional radioand chemotherapy is negligible [ 2 , 11 , 13 ]  . however , sti - 571 [ imatinib ( gleevec ) , novartis basel , switzerland ] has recently been introduced , a drug that acts specifically on the biomolecular change at the origin of the disease by targeting tumour cells exclusively [ 1 , 5 , 7 , 12 , 13 , 2528 ]  . this drug , which appears to make the disease stable in at least 80% of cases [ 1 ] , is used in cases of inoperable gist , whether metastatic or not , and in patients with metastatic disease who have undergone surgery . 
meno frequenti sono lostruzione intestinale , il reperto palpatorio di massa addominale e il riscontro incidentale in corso di indagini eseguite per altri motivi [ 4 , 5 , 7 , 9 , 15 ]  . la diagnosi definitiva di gist istologica , per , la diagnostica per immagini ed , in particolare , la tc spirale di fondamentale importanza . 
infatti , luso di apparecchiature tc con elevata velocit di acquisizione che consentono scansioni di spessore sottile e ricostruzioni multiplanari di notevole qualit , associato allottimizzazione delliniezione endovenosa di mdc , permette una valutazione dettagliata del tratto gastrointestinale oltre che un preciso bilancio destensione di una patologia di tale distretto . 
in particolare , per quanto riguarda i gist , nonostante dati non numerosi e non sempre precisi , in relazione allintroduzione piuttosto recente del criterio immunoistochimico di classificazione , sembrano esserci reperti semeiologici altamente caratteristici di queste neoplasie [ 2 , 4 , 7 , 10 , 14 , 15 , 2224 ]  . in ambito terapeutico , la resezione chirurgica , quando praticabile , la procedura di prima scelta , anche perch il tasso di risposta alla radio e chemioterapia convenzionali insignificante [ 2 , 11 , 13 ]  . 
recentemente , per , stato introdotto il sti - 571 ( imatinib , gleevec , novartis , basel , switzerland ) un farmaco che agisce specificatamente sullalterazione biomolecolare allorigine della neoplasia aggredendo esclusivamente le cellule neoplastiche [ 1 , 5 , 7 , 12 , 13 , 2528 ]  . 
tale farmaco , che sembra stabilizzare la malattia in almeno l80% dei casi [ 1 ] , usato nei pazienti con gist inoperabili sia metastatici che non ed in quelli sottoposti ad intervento chirurgico , ma metastatici . 
essendo la definizione istogenetica dei gist di recente introduzione [ 3 ] , in letteratura non ci sono ancora numerosi studi sulle possibilit offerte dalla tc spirale nella valutazione di queste neoplasie . lo scopo di questo lavoro , pertanto , stato valutare laccuratezza diagnostica della tc spirale nella identificazione , caratterizzazione e nel bilancio destensione dei gist mediante la correlazione con alcuni aspetti anatomo - patologici degli stessi . materiali e metodi si sono prese in considerazione , mediante studio retrospettivo , le tc addominali di 15 pazienti , di cui 8 maschi e 7 femmine di et compresa tra 23 anni e 84 anni ( et media 64 anni ) , con diagnosi istologica di gist sul pezzo operatorio in malattia primitiva , in un periodo compreso tra agosto 2000 e settembre 2004 . 
criteri di esclusione sono stati la diagnosi di gist su prelievo bioptico della neoplasia e / o su lesioni recidivanti ; inoltre , un altro parametro di esclusione stata lesecuzione della tc in altre t . 
the aim of this study was to assess the diagnostic accuracy of spiral ct in detection , characterisation and staging of gist by correlating ct findings with pathology reports . materials and methods we retrospectively reviewed abdominal ct scans of 15 patients ( eight men and seven women , age range 2384 years , mean age 64 years ) with histological diagnosis of primary gist obtained on a surgical specimen . 
all of the patients underwent surgical treatment ; two also underwent medical treatment with sti - 571 but at 8 and 10 months after surgery following the appearance of liver metastasis . since this retrospective study considered ct examinations carried out over a period of 4 years , ct imaging techniques used were different . 
a single - slice spiral ct ( ssct ) scanner ( toshiba , asteion ; rotation time 0.75 s ) was used in ten cases and a multislice ct ( msct ) scanner ( toshiba , aquilion ; 4 slices / rotation ; rotation time 0.5 s ) in five cases . all cases underwent a complete abdominal study ( from the costophrenic angles to the inguinal regions )  . 
ssct studies were performed using a protocol that involved a slice thickness of 7 mm , a table feed of 10 mm ( pitch 1.4 ) and a reconstruction interval of 7 mm with 120 kv and 190 mas . 
the msct study protocol involved a slice thickness of 3 mm with a reconstruction interval of 5 mm in three cases and a slice thickness of 5 mm and reconstruction interval of 7 mm in the other two , with 120 kv and 125200 mas . all studies were carried out after iv administration of nonionic iodinated contrast material with an iodine concentration of 350400 mgi / ml : five patients received 140 ml at a rate of 3.5 ml / s ( for a study including the arterial phase ) or a rate of 2 ml / s ( portal phase only ) whereas in the other cases , such technical data were not available . 
all patients had a portal - phase study ( 70 - s scan delay ) ; eight had a non - contrast - enhanced study ; three had an arterial - phase study ( 30 - s scan delay ) and ten a late study in the equilibrium phase ( 3 min after iv injection of contrast material )  . 
ten patients received oral , water - soluble radiopaque contrast material ( gastrografin ) diluted in water , four received only water ( 500 ml ) whereas one received no contrast material as he was polytraumatised and intubated . 
 two radiologists independently assessed the scans for : site ( stomach , duodenum , small bowel , colon or peritoneum ) , size ( classed into three groups : less than 5 cm , 510 cm , and larger than 10 cm ) , morphology ( rounded or oval , multilobulated or not multilobulated ) , intrinsic or extrinsic growth relative to the organ of origin , homogeneous or inhomogeneous enhancement , possible presence of calcification , presence of liver , peritoneal and / or nodal metastasis and presence of intraabdominal fluid collection . 
tutti i suddetti pazienti sono stati sottoposti a trattamento chirurgico , 2 a terapia medica con sti - 571 , ma a distanza di 8 e 10 mesi dallintervento chirurgico in seguito alla comparsa di lesioni secondarie a livello epatico . essendo questo uno studio retrospettivo su esami eseguiti nellarco di quattro anni , le tecniche dindagine mediante tc utilizzate sono state pi duna . 
le apparecchiature tc usate sono state una spirale a singolo strato ( tcss ) ( toshiba , asteion ; tempo di rotazione 0 , 75 s ) in 10 casi ed una multistrato ( tcms ) ( toshiba , aquilion ; 4 strati / rotazione ; tempo di rotazione 0 , 5 s ) in 5 . 
gli studi condotti con lapparecchiatura tcss sono stati effettuati mediante un protocollo che prevedeva un row - thickness di 7 mm , un avanzamento del lettino di 10 mm ( pitch di 1 , 4 ) ed un intervallo di ricostruzione di 7 mm con 120 kv e 190 mas . 
in quelli condotti con tcms il protocollo prevedeva un row - thickness di 3 mm con un intervallo di ricostruzione di 5 mm in 3 casi e rispettivamente row - thickness di 5 mm e intervallo di ricostruzione di 7 mm in altri 2 , con 120 kv e con valori di mas compresi tra un minimo di 125 ed un massimo di 200 . tutte le indagini sono state eseguite con mezzo di contrasto ( mdc ) per via endovenosa : il mdc utilizzato stato un organo - iodato non ionico ad una concentrazione di iodio variabile da 350 a 400 mgi / ml : in 5 casi la quantit iniettata stata di 140 ml alla velocit di 3 , 5 ml / s ( se effettuato anche uno studio in fase arteriosa ) o alla velocit di 2 ml / s ( per la sola fase portale ) , mentre negli altri casi non stato possibile risalire ai suddetti dati tecnici . 
in tutti i casi stato eseguito lo studio in fase portale ( scan - delay di 70 s ) ; in 8 casi stato eseguito anche lo studio diretto ( senza mdc ) ; in 3 casi anche lo studio in fase arteriosa ( scan - delay di 30 s ) ed in 10 lo studio tardivo nella fase dequilibrio ( a 3 min dalliniezione endovenosa di mdc )  . 
a 10 pazienti stato somministrato per os mdc radiopaco idrosolubile ( gastrografin ) diluito con acqua , a 4 solamente acqua ( 500 ml ) , mentre a 1 paziente nessun mdc , poich politraumatizzato intubato . due radiologi indipendentemente hanno valutato : la sede ( stomaco , duodeno , tenue , colon o peritoneo ) , le dimensioni ( classificate in tre gruppi : inferiori a 5 cm , comprese tra 5 e 10 cm e superiori a 10 cm ) , la morfologia ( rotondeggiante o ovalare , polilobata o non polilobata ) , lo sviluppo intrinseco o estrinseco rispetto allorgano di origine , lenhancement omogeneo o disomogeneo della neoplasia , leventuale presenza al suo interno di calcificazioni , il riscontro di lesioni metastatiche a livello epatico , peritoneale e / o linfonodale e la presenza di versamento endoaddominale . 
la sede , la morfologia e le dimensioni sono stati considerati parametri per lidentificazione della neoplasia ; le dimensioni , lo sviluppo parzialmente o francamente estrinseco , limpregnazione di mdc omogenea o disomogenea della lesione e leventuale presenza di calcificazioni al suo interno sono stati assunti come criteri di caratterizzazione di un gist ; la presenza di metastasi epatiche , peritoneali e / o linfonodali e / o di versamento libero endoaddominale stata considerata parametro di malignit intermedia o elevata . i risultati ottenuti sono stati successivamente correlati phology and size were considered parameters for identification of the neoplasm ; size , partially or frankly extrinsic growth , homogeneous or inhomogeneous enhancement and the possible presence of intralesional calcifications were considered criteria for gist characterisation ; the presence of liver , peritoneal and / or nodal metastasis and / or the presence of free intraabdominal fluid were considered parameters of intermediate or high malignancy . the results obtained were subsequently correlated with some pathological findings obtained on the surgical specimen , in particular , with gross pathology findings such as lesion site , morphology , and size before fixation and with its various tissue components ( solid , haemorrhagic , cystic , ulcerative or calcific )  . 
comparison was based on correspondence between radiological and histopathological assessment of site , morphology and size and some criteria reported in the literature whereby gists are noncalcific lesions that exhibit homogeneous density and a predominantly intrinsic growth pattern if smaller than 5 cm , and inhomogeneous density and a prevalently extrinsic growth pattern in those larger than 5 cin particular , we evaluated whether inhomogeneous density , which was more evident after iv injection of contrast material and characterised by areas of high attenuation in the nonenhanced study and low attenuation in the contrast - enhanced study and areas of fluid or fluid - fluid low attenuation in all phases of the study , correlated with the presence of haemorrhagic , cystic and / or ulcerative areas , respectively . furthermore , we evaluated whether size and the presence of metastasis and / or intraabdominal fluid could be considered indicative of an intermediateor high - grade of malignancy . all malignancy grades are included in the classification proposed by fletcher et al . 
 [ 3 ] ( table 1 ) that , in addition to the macroscopic parameter of size in centimetres , also uses a microscopic parameter such as the number of mitoses per 50 high - power fields ( hpf ) and is currently the only prognostic tool available . 
no multivariate statistical analysis was carried out because patient samples were relatively small . results in the 15 cases considered , at gross pathology , the disease was found to affect the stomach in nine ( 60% ) and the small bowel in six ( 40% ) , of which one was in the duodenum . there were no cases of primary location of gist in other sites of the gastrointestinal systein the four cases of larger lesions ( three larger than 10 cm and one measuring 9 cm ) , at abdominal ct , the site of origin was hypothesised to be the stomach , but the hypothesis was considered uncertain three of these cases , the hypothesis corresponded to the pathology report whereas for the 9 - cm neoplasm , the site of origin was the ileu in the remaining 11 cases ( lesions smaller than 9 cm ) , the site was defined with a good degree of certainty and confirmed by the gross pathology report . as regards lesion morphology and size ten gists smaller than 5 cm , two 510 cm , and three larger than 10 cm ( table 2 ) ct findings essentially agreed with those of gross pathology . 
il confronto si basato sulla corrispondenza tra valutazione radiologica ed anatomo - patologica di sede , morfologia e dimensioni e su alcuni criteri riportati in letteratura secondo cui i gist sarebbero lesioni prive di calcificazioni , a densit omogenea e sviluppo prevalentemente allinterno dellorgano di origine se inferiori a 5 cm , a densit disomogenea e sviluppo preferenzialmente estrinseco se superiori a 5 cin particolare , si valutato se la disomogenea densit , pi evidente dopo iniezione endovenosa di mdc , caratterizzata da aree iperdense in fase diretta ed ipodense dopo mdc ed aree ipodense di tipo liquido o sovraliquido in tutte le fasi di studio , si correlava alla presenza di aree rispettivamente emorragiche e cistiche e / o ulcerative . 
inoltre , si valutato se le dimensioni e la presenza di metastasi e / o di versamento endoaddominale , potessero essere criteri indicativi di un grado di malignit intermedio o elevato . 
nel 2002 [ 3 ] ( tabella 1 ) che , oltre ad un parametro macroscopico come le dimensioni espresse in cm , utilizza anche un parametro microscopico quale il numero di mitosi per 50 campi ad alta risoluzione ( hpf ) e che attualmente lunico strumento in uso per una valutazione prognostica . 
non si effettuata unanalisi statistica multivariata dei risultati , poich il campione dei pazienti stato ritenuto di dimensioni relativamente piccole . risultati nei 15 casi presi in considerazione , allesame anatomo - patologico la localizzazione di malattia risultata lo stomaco in 9 casi ( 60% ) ed il piccolo intestino in 6 ( 40% ) di cui il duodeno in 1 caso . 
nei 4 casi di lesioni di maggiori dimensioni ( di cui 3 superiori a 10 cm e 1 di 9 cm ) alla rivalutazione della tc addominale , la sede dorigine stata ipotizzata essere lo stomaco , ma posta come dubbia . 
nei rimanenti 11 casi ( di dimensioni inferiori a 9 cm ) , invece , la sede stata definita con un certo grado si sicurezza ed effettivamente stata confermata dallanalisi anatomo - patologica . per quanto concerne la morfologia delle lesioni e le loro dimensioni , 10 gist inferiori a 5 cm , 2 gist con dimensioni comprese tra 5 e 10 cm , 3 con dimensioni superiori a 10 cm ( tabella 2 ) , i risultati sono sostanzialmente concordi con quelli dellanatomia patologica . 
in tc , inoltre , nessuna neoplasia presentava calcificazioni intralesionali ed anche questo rilievo stato confermato dallesame istopatologico . per quanto riguarda lo sviluppo della lesione , in 9 casi sui 10 inferiori a 5 cm esso risultato alla tc parzialmente t . 
in all cases of homogeneous density , histopathology revealed the almost exclusive presence of solid areas whereas in all those with inhomogeneous contrast enhancement ( solid , haematic and / or fluid attenuation ) , it revealed solid , haemorrhagic , necrotic and / or cystic areas . the correlation between malignancy size and grade according to the predictive criteria proposed by fletcher et al . [ 3 ] revealed that of the seven tumours with intermediate malignancy grade , six were smaller than 5 cm and only one larger than 10 cm , that two out of two measuring 510 cm were high malignancy grade and that all those larger than 10 cm were intermediate or high malignancy grade ( table 3 )  . ct demonstrated liver metastasis in two cases , peritoneal and / or nodal metastasis in none , and intraabdominal fluid in one . 
in tutti i casi di densit omogenea lanalisi patologica ha rivelato la presenza quasi esclusivamente di aree solide , mentre in tutti quelli con impregnazione di mdc disomogenea per la presenza di aree a densit di tipo solido , ematico e / o liquido sono state riscontrate aree sia solide , che emorragiche , necrotiche e / o cistiche . la correlazione tra dimensioni e grado di malignit , secondo i criteri predittivi proposti da fletcher et al . 
 [ 3 ] , ha messo in evidenza che delle 7 neoplasie a rischio intermedio 6 erano inferiori a 5 cm e solo 1 superiore a 10 cm , che 2 su 2 di dimensioni tra 5 e 10 cm rientravano nel gruppo a rischio elevato e che tutte quelle superiori a 10 cm rientravano , invece , nel gruppo a rischio intermedio o in quello a rischio elevato di malignit ( tabella 3 )  . in 2 casi alla tc si sono individuate metastasi epatiche , in nessun caso peritoneali e / o linfonodali , in 1 versamento endoaddominale . 
negli altri casi di malignit intermedia o elevata non erano presenti metastasi al momento della diagnosi . discussion a final diagnosis of gist requires demonstration of cd117 positivity , but diagnostic imaging and spiral ct in particular la diagnosi di certezza dei gist richiede la dimostrazione dellimmunopositivit al cd117 , ma la diagnostica per immagini ed , in particolare , la tc spirale , svolge comunque un discussione table 2 results of correlation between size , development , enhancement and calcifications size , cm total , n development extrinsic , n intrinsic , n enhancement inhomogeneous , n homogeneous , n no calcifications , n tabella 2 risultati della correlazione tra dimensioni , sviluppo , enhancement e presenza di calcificazioni dimensioni , cm totale , n svilupppo enhancement assenza di calcificazioni , n estrinseco , n intrinseco , n disomogeneo , n omogeneo , n > 5 - < 10 > 5 e < 10 t . 
abdominal computed tomography ( ct ) , axial images : without iv contrast agent , arterial and portal phases , respectively ; a roundish formation , lobate , with partial extraluminal growth . density is inhomogeneous , particularly after contrast agent and particularly in the portal phase . 
abdominal computed tomography ( ct ) , axial images : arterial , portal and late phases , respectively ; oval formation ( arrow ) of inhomogeneous density with extraluminal growth . 
e aspetto macroscopico del gist allanalisi del pezzo operatorio ; presenza di necrosi centrale ( freccia ) ed aree ulcerate ; sviluppo allesterno della parete gastrica ( freccia )  . is nonetheless instrumental in the diagnostic workup [ 10 , 15 , 22 , 29 ]  . 
however , one study [ 20 ] has shown that , thanks to its high - contrast resolution , mri is reliable in depicting the various components of larger gists ( 728 cm ) , and thanks to its multiplanar capabilities , it is effective in assessing the relationship between the lesion and adjacent organs . as regards endoscopy , although in a very small proportion of patients diagnosis is made through biopsy , in the majority of patients , histology is negative owing to the tendent . 
a , b tc delladdome , immagini assiali , fase portale : formazione che mostra enhancement disomogeneo per la presenza di aree a densit di tipo solido ed altre francamente ipodense . 
c , d ricostruzioni sul piano coronale e sagittale che consentono una buona valutazione dellestensione della lesione e dei rapporti con le strutture adiacenti . ruolo fondamentale nelliter diagnostico [ 10 , 15 , 22 , 29 ]  . esami di vario livello sono lendoscopia , lecografia delladdome , lo studio radiologico del tubo digerente con mezzo di contrasto ( mdc ) e la tc addominale . 
comunque , da uno studio riportato in letteratura [ 20 ] , emerge che la rm , grazie allelevata risoluzione di contrasto , attendibile nellevidenziare le varie componenti dei gist di maggiori dimensioni ( da 7 a 28 cm ) e , grazie alla multiplanariet , nel valutare i rapporti tra la lesione neoplastica e gli organi adiacenti . per quanto riguarda lendoscopia , mentre esiste una piccolissima quota di pazienti in cui la diagnosi viene fatta mediante prelievi bioptici , nella maggioranza dei casi listologia negativa a causa della tendenza di queste neoplasie ad avere uno sviluppo estrinseco rispetto alla parete . 
i limiti dellecografia , invece , quali la costituzione fisica del paziente , il meteorismo intestinale , loperatore - dipendenza e la scarsa panoramicit spesso non consentono la caratterizzazione e soprattutto un corretto bilancio destensione di una massa addominale . 
lo studio radiologico del tubo digerente a singolo o doppio contrasto in grado di mettere in evidenza alterazioni parietali e della plicatura e lesioni aggettanti nel lume riferibili ad una neoplasia , consentendo anche di stabilire se essa intrao extra - murale , per , non permette una valutazione extra - luminale della patologia [ 15 ]  . lintroduzione della tc spirale ed , in particolare , di quella multistrato , invece , grazie allelevata velocit desecuzione dellesame , allutilizzo di scansioni con spessore molto sottile con possibilit di sovrapposizione e di ottenere ricostruzioni multiplanari di elevata qualit , ha offerto nuot . 
the limitations of ultrasound instead , such as patient build , intestinal gas , operator dependency and limited field of view , often prevent characterisation and , above all , correct staging of abdominal masses . 
singleor double - contrast barium studies are able to detect changes in the wall and folds and identify exophytic lesions referable to a tumour , even allowing establishment of whether the lesion is intraor extramural ; however , they do not enable an extraluminal evaluation of the disease [ 15 ]  . 
 introduction of spiral ct and in particular msct thanks to the short examination times , the use of very thin slices with the possibility of overlapping slices and obtaining highquality multiplanar reconstructions has provided new opportunities in gastrointestinal imaging , allowing detailed evaluation of the stomach , small and large bowel and relations with adjacent structures . 
spiral abdominal ct has long been used as a routine study in patients with gastrointestinal ve opportunit nello studio per immagini dellapparato gastrointestinale consentendo valutazioni dettagliate di stomaco , piccolo e grande intestino e dei rapporti con le strutture adiacenti . 
la tc spirale delladdome , da tempo , unindagine usata di routine in pazienti con tumori delle vie digestive per il bilancio destensione della malattia , nonch nel follow - up del paziente operato . 
pi recentemente , limpiego della tc , grazie alle innovazioni tecnologiche , stato esteso anche alla identificazione e caratterizzazione di neoplasie dellapparato digerente , in particolari situazioni rappresentate da masse di origine ignota o a partenza da aree del tratto digerente non raggiungibili con lendoscopia . 
inoltre , proprio per quanto riguarda i gist , sembra che queste neoplasie presentino in tc aspetti semeiologici , se non patognomonici , perlomeno altamente caratteristici [ 4 , 7 , 10 , 14 , 15 , 19 , 2124 ]  . 
per consentire risultati validi in tale ambito , va condotta unadeguata tecnica tc che deve prevedere una buona distensione gastrica ed intestinale e unottimizzazione size , cm > 5 < 10 total > 5 e < 10 totale very low medium high molto basso basso intermedio elevato tumours , both for disease staging and follow - up after surgery . 
thanks to the more recent technological innovations , the use of ct has been extended to the identification and characterisation of digestive tract neoplasms in specific situations , such as masses with unknown origin or originating in areas of the digestive tract not amenable to endoscopic techniques . 
in addition , as regards gists in particular , these tumours seem to exhibit highly characteristic , if not pathognomic , ct patterns [ 4 , 7 , 10 , 14 , 15 , 19 , 2124 ]  . 
 for results to be reliable in this field , a correct ct technique is needed that involves good gastric and intestinal distension and optimisation of iv injection of contrast material . adequate distension of the stomach and bowel loops , preferably with low - attenuation agents and possibly water , is essential , as a collapsed wall may mimic a nonexistent alteration or mask existing disease [ 10 , 21 , 22 ]  . 
 with regard to the study phases after iv contrast injection , some authors [ 21 , 22 ] agree on carrying out an arterial - phase study of the abdominal sectors comprising the stomach and liver only on the basis of precise requests for a vascular study and a portal - phase study of the entire abdomen ( from the costophrenic angles to the pubic symphysis ) ; the latter phase is considered indispensable for the diagnosis of gastrointestinal disease and at times sufficient [ 21 , 22 ]  . 
even in our study , arterial - phase scans were not acquired in all cases whereas portal - phase scans were . as concerns ct evaluation of gist , it should be noted that because the immunohistochemical criterion for tumour identification was only recently introduced , the studies published to date have included a relatively small number of patients and not all have paid special attention to tumour size . this information , which can be obtained both at ct and at gross pathology , is the most important prognostic factor currently used , together with the histopathological mitotic count [ 4 ]  . 
 as regards site and growth pattern , since gists originate in the muscularis propria , they commonly have exophytic growth from the wall and therefore appear at ct as a mass prevalently located outside the affected organ . predominantly intraluminal forms are rare and mostly related to small size and wall thickening , which makes this sign uncharacteristic for these tumours [ 4 , 7 , 10 , 14 , 21 ]  . 
in particular , stomach gists , which tend to affect the gastric body and fundus , exhibit extragastric extension in a wide majority of cases ( 86% in the series by levy et al . ) [ 7 ] , and those affecting the small bowel may have primary presentation in extraserous sites ( 22% in the same series )  . 
 [ 4 ] showed that , regardless of site , gists smaller than 5 cm tend to have predominantly intraluminal growth , those measuring 510 cm combined intraand extraluminal growth and those greater than 10 cm prevalently extraluminal growth with a tendency to invade adjacent organs [ 4 ]  . in contrast , in our series , even tumours smaller than 5 cm exhibited prevalently extrinsic growth ( only one case out of ten was contained in the affected organ ) , as did the larger t . 
infatti , unadeguata distensione dello stomaco e delle anse intestinali , preferenzialmente con agenti a bassa attenuazione ed , in particolare lacqua , di fondamentale importanza , poich un lume collassato pu mimare unalterazione inesistente o mascherare una patologia realmente presente [ 10 , 21 , 22 ]  . 
dal punto di vista delle fasi di studio dopo iniezione ev di mdc alcuni autori [ 21 , 22 ] concordano nellesecuzione di una fase di tipo arterioso dei settori delladdome comprendenti stomaco e fegato solo sulla base di precise richieste di uno studio vascolare , e di una fase portale di tutto laddome ( dai seni costofrenici alla sinfisi pubica ) : questultima ritenuta indispensabile per una diagnosi di patologia gastrointestinale , ma talvolta sufficiente [ 21 , 22 ]  . 
per quanto riguarda la valutazione dei gist mediante tc va sottolineato che , essendo recente il criterio immunoistochimico di identificazione di queste neoplasie , gli studi finora pubblicati hanno compreso un numero relativamente basso di pazienti e che non tutti hanno posto particolare attenzione alle dimensioni del tumore : questo elemento , per , ricavabile sia quale reperto semeiologico in tc sia dalla valutazione macroscopica del pezzo operatorio il pi importante fattore prognostico attualmente usato assieme allelemento istopatologico della conta mitotica [ 4 ]  . 
per quanto riguarda la sede ed il tipo di crescita , poich i gist prendono origine nella muscolare propria , molto comunemente hanno uno sviluppo esofitico rispetto alla parete e , quindi , in tc si presentano come massa localizzata prevalentemente allesterno dellorgano interessato . 
le forme principalmente intra - luminali sono rare , per lo pi legate alle piccole dimensioni e lispessimento parietale , dunque , non un reperto semeiologico tipico di questi tumori [ 4 , 7 , 10 , 14 , 21 ]  . 
 [ 4 ] emerso che , indipendentemente dalla sede , i gist inferiori a 5 cm tendono a presentare una crescita preferenzialmente intraluminale , quelli tra 5 e 10 cm una combinata intraed extra - luminale e quelli maggiori di 10 cm una prevalentemente extraluminale con tendenza allinfiltrazione degli organi adiacenti [ 4 ]  . 
nella nostra casistica , invece , anche i tumori inferiori a 5 cm mostrano uno sviluppo prevalentemente estrinseco ( solo in 1 caso su 10 era allinterno dellorgano colpito ) , come quelli di maggiori dimensioni ( 2 su 2 di quelli tra 5 e 10 cm e 3 su 3 di quelli superiori a 10 cm )  . 
 ( 3% ) [ 4 , 7 ] : i risultati del nostro studio sono concordi con tale rilievo , poich in nessuna delle neoplasie in esame si evidenziata la presenza di calcificazioni al suo interno . 
homogeneous enhancement is instead present in a minority of cases only ( 8% ) and is typical of small lesions [ 4 , 7 , 14 , 2124 , 30 ]  . 
in our study , only 50% of tumours smaller than 5 cm ( five cases out of ten ) had homogeneous density whereas the remaining 50% had markedly or slightly inhomogeneous density ; all cases of gist larger than 5 cm exhibited frankly inhomogeneous density , as reported in the literature . 
in all cases of homogeneous enhancement , however , analysis of the surgical specimen demonstrated the presence of solid areas only whereas in all those of inhomogeneous enhancement , it found solid , necrotic , haemorrhagic and / or cystic areas . 
ct differential diagnosis of gastric gist with adenocarcinoma and lymphoma relies on the fact that they have exophytic growth and are almost never associated with perigastric adenopathy [ 7 ]  . 
gist metastasis to the mesentery and / or omentum enter the differential diagnosis with peritoneal carcinomatosis , lymphomatosis and disseminated peritoneal leiomyomatosis [ 4 , 7 , 11 , 16 ]  . 
liver metastasis , typically hypovascular , appear at ct as hypodense areas , at times with a hyperdense rim ( similar to secondary lesions from other neoplasms ) [ 1 , 4 , 7 , 14 ]  . 
the ability of ct to identify recurrences is essential in the decision to initiate treatment with gleevec , as the drug is reserved for patients with inoperable or metastatic gist . conclusions final diagnosis of gist requires demonstration of positivity for cd117 , but spiral ct plays a fundamental role because gists exhibit signs that are strongly characteristic , if not pathognomic , and ct enables correct grading of the disease , which is crucial in deciding upon resection and / or medical dei gist dopo somministrazione endovenosa di mdc , simile indipendentemente dal sito di origine , va data enfasi al fatto che la gran parte dei tumori di maggiori dimensioni ( 92% secondo levy et al . ) tendono ad avere una presa di contrasto periferica e delle zone ipodense centrali espressione dei reperti patologici macroscopici rispettivamente di tumore vitale e di aree di emorragia , necrosi o degenerazione cistica ( per cui si parla di massa complessa al taglio )  . 
unassunzione omogenea di contrasto , invece , presente solo in una minoranza di casi ( 8% ) ed tipica di lesioni piccole [ 4 , 7 , 14 , 2124 , 30 ]  . 
nel nostro studio solo nel 50% dei casi di tumori inferiori a 5 cm ( 5 casi su 10 ) la densit omogenea , mentre nel restante 50% marcatamente o perlomeno lievemente disomogenea ; in tutti i casi di gist superiori a 5 cm essa , invece , francamente disomogenea come riportato in letteratura . 
in tutti i casi , comunque , dimpregnazione omogenea di mdc lanalisi del pezzo operatorio ha rivelato la presenza solamente di aree solide , mentre in tutti quelli dimpregnazione disomogenea sono state riscontrate aree sia solide , che necrotiche , emorragiche e / o cistiche . 
nella quasi totalit dei casi non presente un interessamento metastatico dei linfonodi loco - regionali e / o a distanza [ 4 , 14 , 15 , 30 ] : elemento confermato dal nostro studio nel quale in nessun caso si sono rilevati linfonodi aumentati di volume . 
la diagnosi differenziale dei gist in tc , in ambito gastrico , con ladenocarcinoma ed il linfoma si basa sul fatto che mostrano una crescita esofitica e non sono quasi mai associati ad adenopatie perigastriche [ 7 ]  . 
per quanto riguarda il piccolo intestino anche il linfoma pu presentarsi come massa disomogenea che si estende al mesentere adiacente : ci che permette lipotesi diagnostica di un gist , per , anche in questo caso , lassenza di interessamento linfonodale [ 7 , 30 ]  . 
le metastasi da gist a livello di mesentere e / o omento vanno in diagnosi differenziale con la carcinomatosi peritoneale , la linfomatosi e la leiomiomatosi peritoneale disseminata [ 4 , 7 , 11 , 16 ]  . 
le metastasi epatiche , tipicamente ipovascolarizzate , presentano un aspetto alla tc di aree ipodense talvolta con cercine iperdenso ( caratteristiche comuni a lesioni secondarie ad altri tipi di neoplasie ) [ 1 , 4 , 7 , 14 ]  . tale aspetto era presente anche nei nostri due casi di secondariet epatiche . 
la capacit della tc di individuare lesioni ripetitive fondamentale al fine della scelta del trattamento medico con gleevec in quanto farmaco riservato oltre che ai pazienti con gist inoperabili anche a quelli metastatici . conclusioni la diagnosi di certezza dei gist richiede la dimostrazione dellimmunopositivit al cd117 , ma la tc spirale svolge un ruolo di primaria importanza poich i gist mostrano aspetti semeiologici se non patognomonici perlomeno fortemente caratteristici e poich permette un corretto bilancio destensione , elemento fondamentale per la scelta di resecabilit e / o trattamento medico . 
tumour size , as established by ct , even though not corresponding to the true size of the tumour , is not a sufficient criterion to predict the grade of malignancy . 
while the presence of metastases at the time of diagnosis may be indicative of intermediateor high - grade malignancy , their absence , like the absence of intraabdominal fluid , does not allow one to consider the gist to be low or very low malignancy grade . 
mentre la presenza di metastasi al momento della diagnosi pu essere indicativa di un grado di malignit intermedio o elevato , la loro assenza , come la non evidenziazione di un versamento endoaddominale , non consente di collocare i gist nel gruppo a basso o molto basso rischio . 
at contrastenhanced ct , they were inhomogeneous in 21 cases ( 91.3% ) , with geographic pattern ( 61.9% of cases ) , serpiginous linear areas of enhancement ( intralesional vessels ) ( 23.8% ) , rounded ( 52.4% ) or linear ( 33.3% ) areas of low attenuation ( necrosis )  . 
il sospetto di lesione pleurica comporta una distinzione in base alle sue dimensioni : in presenza di una piccola neoformazione con margini netti , contorni lisci , angoli di raccordo retti / ottusi e densit omogenea , facile ipotizzare la diagnosi di tfsp ; qualora la neoplasia sia pi voluminosa , la presenza di margini netti e contorni lobulati , angoli di raccordo acuti dolci ed enhancement disomogeneo nelle scansioni con contrasto possono permettere di operare una distinzione da una massa di pertinenza polmonare . key words solitary fibrous tumors of the pleura ct pleural neoplasm parole chiave tumore fibroso solitario della pleura tc neoplasie pleuriche introduction introduzione solitary fibrous tumour of the pleura ( sftp ) is a rare neoplasm that accounts for fewer than 5% of all pleural tumours [ 1 ]  . 
over 50% of patients are asymptomatic at presentation , and the lesion is usually discovered inil tumore fibroso solitario della pleura ( tfsp ) una neoplasia rara che rappresenta meno del 5% di tutti i tumori pleurici [ 1 ]  . 
oltre il 50% dei pazienti asintomatico al momento della diagnosi e la lesione riscontrata in cidentally on chest radiographs performed for other reasons [ 1 , 2 , 5 , 7 , 8 ]  . 
patients may , however , also present with nonspecific symptoms ( cough , chest pain , dyspnoea and fatigue ) [ 1 , 2 , 8 ] or a neoplastic syndrome ( digital clubbing , hypertrophic osteoarthropathy and refractory hypoglycaemia ) [ 9 , 10 ]  . 
sftp generally appears as a solid , sharply marginated , well - circumscribed solitary lesion located at the periphery of the chest or close to a fissure ; it can become very large and occupy even the entire hemithorax [ 1 , 7 , 11 , 12 ]  . 
the tumour has benign behaviour in 80% of cases , and prognosis is generally excellent after surgical resection provided that this is radical [ 2 , 4 , 7 , 8 , 12 , 15 ]  . 
despite the use of transthoracic needle biopsy [ fine - needle aspiration ( fna ) ] [ 16 , 17 , 18 ] and fibreoptic bronchoscopy [ 9 , 12 ] , as well as all the imaging modalities , diagnosis is rarely straightforward . 
in some cases , a definite diagnosis may be established preoperatively with a cutting - needle biopsy , but in most , diagnosis is provided by postoperative histology and immunohistochemistry on the surgical specimen [ 3 , 4 ]  . the primary aim of this study was to review computed tomography ( ct ) features of sftp in order to : ( 1 ) identify a specific ct pattern that could allow a diagnosis of sftp ; ( 2 ) search for radiological signs capable of suggesting lesions of pleural origin or , at least , identifying the benign nature of the lesion . materials and methods between november 1989 and january 2005 , 29 consecutive patients with a histological and immunohistochemical diagnosis of sftp underwent surgical resection of the lesion at the thoracic surgery department of our hospital . 
as such lesions grow within the lung parenchyma without projecting into the pleural cavity , they cannot be assimilated to the classic forms and therefore were not considered suitable for the purposes of this study . 
the study was therefore conducted on 26 patients . we collected clinical data of possible radiological relevance , such as mode of lesion discovery ( incidental / symptomatic ) , presence or absence of respiratory symptoms , transthoracic needle biopsy ( fna ) under fluoroscopic or ct guidance and its results , and preoperative clinical diagnosis . data from surgical reports concerning sessile or pedunculated tumour base , gross pathology features , site of origin ( visceral , parietal , mediastinal or diaphragmatic pleura ) , presence of inflammatory adhesions and / or infiltration of adjacent tissues or organs . 
dal punto di vista radiologico , lelemento pressoch patognomonico delle forme peduncolate il cambiamento di forma e posizione durante le fasi della dinamica respiratoria [ 9 , 13 , 14 ]  . il comportamento di tale neoplasia benigno nell80% dei casi e la prognosi generalmente ottima dopo intervento chirurgico di exeresi , purch radicale [ 2 , 4 , 7 , 8 , 12 , 15 ]  . nonostante lutilizzo dellagobiopsia transtoracica ( fna ) [ 1618 ] e della fibrobroncoscopia [ 9 , 12 ] , nonch di tutte le metodiche di imaging , la diagnosi risulta spesso difficoltosa . 
talvolta una diagnosi preoperatoria di certezza pu essere ottenuta con la biopsia ad ago tranciante , ma nella maggior parte dei casi post - chirurgica , frutto dellesame istologico e dellanalisi immuno - istochimica sul pezzo operatorio [ 3 , 4 ]  . lobiettivo primario di questo studio stato la valutazione dei quadri tc allo scopo di ( 1 ) identificare un quadro tomodensitometrico specifico che permetta di porre diagnosi di tfsp ; ( 2 ) ricercare i segni radiologici indicativi di una lesione di origine pleurica o , quanto meno , individuare la natura benigna della lesione in esame . materiali e metodi nel periodo compreso tra novembre 1989 e gennaio 2005 , 29 pazienti consecutivi con diagnosi definitiva , istologica e immunoistochimica , di tfsp sono stati sottoposti ad intervento chirurgico di resezione presso la s.c.d.u. 
dal momento che tali lesioni crescono indovate nel contesto del parenchima polmonare anzich estrinsecarsi nel cavo pleurico , non possono presentare per definizione un aspetto assimilabile alle forme classiche e pertanto non si adattano allo scopo di questo studio . 
pertanto 26 pazienti costituiscono loggetto dellindagine . sono stati desunti i dati anamnestici di possibile interesse radiologico , quali la modalit del riscontro ( occasionale / sintomatico ) , la presenza o meno di sintomi respiratori , lesecuzione di agobiopsia trans - toracica ( fna ) sotto controllo fluoroscopico o tc , la diagnosi clinica pre - operatoria . 
administration of contrast medium and 14 at baseline ( 11 studies were performed before and after contrast - medium administration )  . statistical analysis the data collected ( clinical , surgical , histopathological and radiological ) were entered into an excel spreadsheet for comparative evaluation . 
di mezzo di contrasto e 14 in condizioni basali ( 11 studi sono stati eseguiti prima e dopo somministrazione del contrasto )  . analisi statistica le informazioni ottenute ( cliniche , chirurgiche , anatomopatologiche e radiologiche ) sono state inserite in un foglio di lavoro elettronico excel per le debite valutazioni comparative . 
 stato considerato significativo un valore di p0 , 05 . risultati dati clinici i 26 pazienti oggetto di questo studio , operati nel periodo tra novembre 1989 e gennaio 2005 , presentano unet media di 64 , 9 anni ( mediana 68 anni ; range 4081 anni )  . 
diciotto pazienti ( 69 , 2% ) erano totalmente asintomatici al momento della diagnosi e il tfsp stato riscontrato occasionalmente nella radiografia del torace eseguita per altre ragioni ( routine preoperatoria o esame eseguito in occasione di traumi )  . 
in tutti i pazienti la neoplasia pleurica era evidente nei radiogrammi standard del torace , localizzata a destra in 14 casi ( 53 , 8% ) , a sinistra nei restanti 12 ( 46 , 2% )  . la fibrobroncoscopia stata eseguita in 18 pazienti ( 69 , 2% ) : una compressione estrinseca delle strutture bronchiali , sempre confermata alla tc , era presente in 9 casi ( 50% )  . 
una diagnosi istologica pre - operatoria corretta stata raggiunta in 10 casi ( 38 , 4% ) ; nei restanti 10 casi , il prelievo risultato non diagnostico o suggestivo per mesotelioma maligno . allesplorazione chirurgica , il tumore risultato originare dalla pleura viscerale in 20 pazienti ( 83 , 3% ) : 18 lesioni ( 90% ) sono peduncolate , 2 ( 10% ) sessili . 
presentano pi frequentemente una stretta base dimpianto i tfsp a partenza dalla pleura viscerale , mentre quelli originanti dalla pleura parietale sono pi spesso sessili : tale correlazione risultata significativa dal punto di vista statistico ( p = 0 , 018 )  . 
aderenze di natura flogistica tra il tfsp e le strutture adiacenti sono state riscontrate intraoperatoriamente in 15 ( 57 , 7% ) pazienti ( pleura mediastinica 2 , pleura parietale 8 , pleura viscerale di parenchima polmonare contiguo 12 ) ; in 11 ( 42 , 3% ) la lesione apparsa , invece , ben delimitata e facilmente enucleabile . dati tomodensitometrici la valutazione morfologica - densitometrica dei tfsp stata eseguita sulle 26 tc pre - operatorie disponibili . 
as regards the second parameter , two angles were evaluated for each tumour , and the results are shown in figures 2 and 3 . although 19 / 26 ( 73% ) lesions were found to be pediculated at surgery , there was no ct evidence of the presence of a pedicle . 
an ipsilateral pleural effusion was seen in 5 patients ( 19.2% ) whereas no ct scan demonstrated enlarged hilarmediastinal lymph nodes . analysis of the available unenhanced ct scans ( n = 14 ) alsono pi voluminose ( media 16 , 1 cm ; range 14 , 520 )  . 
i margini delle neoplasie sono risultati a carattere netto in tutti i casi : in 12 ( 46 , 1% ) con contorni lobulati , in 14 ( 53 , 9% ) lisci . 
per quanto riguarda , invece , il secondo parametro , per ogni tumore sono stati valutati due angoli di raccordo , con i risultati riportati nelle figure 2 e 3 . 
in 17 tc ( 65 , 4% ) sono stati rilevati segni di compressione sulle strutture circostanti ; in particolare , leffetto massa si reso evidente sul parenchima polmonare in tutti i casi , associato in due casi a dislocazione mediastinica . 
un versamento pleurico omolaterale associato stato riscontrato in 5 pazienti ( 19 , 2% ) , mentre in nessuna tc sono stati obiettivati linfonodi ilo - mediastinici ingranditi . lanalisi delle scansioni basali disponibili ( 14 tc ) ha permesso di valutare le caratteristiche tomodensitometriche dei tfsp . 
dodici tumori ( 85 , 7% ) dimostrano unattenuazione simile a quella dei muscoli della parete toracica , 2 ( 14 , 3% ) mostrano unipodensit relativa , mentre nessun tfsp risultato iperdenso rispetto ai muscoli . 
all lesions larger than 7 cm showed heterogeneous enhancement after administration of contrast mediuenhancement patterns of sftp after administration of contrast material are summarised in table 3 . certain features of sftp were correlated to the size of the tumour , and the results are shown in table 4 . 
lanalisi delle risultanze derivate dalla clinica , dalle procedure bioptiche e dai referti radiologici originali ha permesso di porre diagnosi di lesione pleurica in 17 pazienti ( n = 26 ; 65 , 4% ) e di identificare con certezza il tfsp in 12 di essi ( n = 17 ; 70 , 6% )  . 
its rarity is confirmed by our experience , as the 26 cases considered were encountered over a period of 16 years , despite the fact that our department of thoracic surgery is a regional referral centre . 
sftp are discovered incidentally on routine chest radiographs performed for other reasons [ 2 , 7 , 8 , 9 ] , and they tend to have an indolent course . 
when possible , definitive diagnosis is made preoperatively on the basis of fna cytology [ 17 , 18 ] and confirmed by the absence of infiltration of the bronchial tree as ascertained by fibreoptic bronchoscopy [ 9 , 12 ]  . 
 on chest radiographs and chest ct scans , sftp appear as well - delineated solitary lesions located in the chest periphery or within a fissure [ 1 , 2 , 9 , 12 , 21 ]  . 
they may become very large and at times occupy an entire hemithorax , giving rise to extensive opacities and causing respiratory problems [ 1 ]  . typically , they present as lesions , with their maximum diameter abutting the chest wall , with which they form obtuse , right or acute angles with a smooth tapering margin [ 22 , 23 ]  . a pathognomic finding in pedunculated lesions is the mobility of the tumour with changes in patient position [ 3 , 9 , 11 , 13 , 14 ]  . 
the usefulness of this finding is , however , limited by the size of the mass : the larger the sftp , the more firmly it is attached to adjacent structures by adherences ( 84.6% of tumours > 10 cm in our series ) , which makes the mass less mobile [ 9 , 20 , 24 ]  . 
in addition , another limitation inherent to the retrospective design of our study is that this sign was never elicited by changing patient position during the ct scans . finally , the right , obtuse or acute angles with smooth tapering contours allowed us to confirm that the origin of the lesion is pleural rather than parenchymal ( 73% of our cases ) [ 1 , 3 , 17 , 25 ]  . 
dal punto di vista clinico , le caratteristiche dei nostri tfsp sono del tutto analoghe a quelle di altri studi [ 4 ]  . noto che spesso i tfsp sono scoperti incidentalmente in radiografie del torace eseguite di routine [ 2 , 79 ] e che il decorso di tali neoplasie in genere indolente . 
la diagnosi di certezza posta , quando possibile , preoperatoriamente grazie allesame citologico sul materiale prelevato in corso di fna [ 17 , 18 ] , corroborata dallassenza di segni di infiltrazione dellalbero bronchiale alla fibrobroncoscopia [ 9 , 12 ]  . 
quando , invece , lfna non sia eseguita o risulti non diagnostica , lanalisi anatomopatologica , istologica ed immunoistochimica sul pezzo operatorio , a confermare la natura della lesione [ 17 ]  . 
al radiogramma ed alla tc del torace , i tfsp appaiono generalmente come lesioni solitarie , a margini netti , situate alla periferia del torace o in relazione ad una scissura [ 1 , 2 , 9 , 12 , 21 ]  . 
talvolta possono raggiungere dimensioni notevoli fino ad occupare un intero emitorace , producendo grossolane opacit e causando disturbi respiratori al paziente [ 1 ]  . caratteristicamente si presentano come lesioni con il diametro massimo a contatto con la parete toracica , con la quale formano angoli di raccordo ottusi , retti o acuti dolci [ 22 , 23 ]  . 
tale aspetto , per , trova un limite alla sua utilizzazione nelle dimensioni della massa : infatti , pi il tfsp voluminoso pi ancorato alle strutture adiacenti per mezzo di tenaci aderenze ( lo l84 , 6% delle neoplasie di dimensioni > 10 cm nella nostra casistica ) e quindi meno risulta essere mobile [ 9 , 20 , 24 ]  . 
a questo si aggiunge un ulteriore limite intrinseco al nostro studio : essendo un lavoro retrospettivo , nessuno ha mai ricercato questo segno con lacquisizione di scansioni tc al variare del decubito dei pazienti . gli angoli di raccordo retti , ottusi o , infine , acuti dolci ci permettono di confermare che tale lesione di origine pleurica e non parenchimale ( 73% dei nostri casi ) [ 1 , 3 , 17 , l . 
at least one acute angle with smooth tapering margins , first described by dedrick et al . [ 1 ] in five out of six lesions examined , was present in 57% of lesions in our series . 
the different incidence of this parameter is , in our opinion , largely due to the small size of the series examined by dedrick et al . evaluation of relationships between the lesion and adjacent structures is another important element . 
tale caratteristica presente in quasi tutte le lesioni di piccole dimensioni ed in una buona percentuale di masse di dimensioni superiori , tenendo conto del fatto che langolo acuto dolce ha , nei tumori pi voluminosi ( diametro massimo medio 13 , 5 cm ) , significato analogo a quello degli angoli retti / ottusi nei pi piccoli ( diametro medio 7 , 25 cm )  . 
inoltre , nel 65 , 4% dei casi stato possibile osservare i segni delleffetto massa sulle strutture circostanti , in particolare sul parenchima polmonare e sul mediastino [ 1 , 26 ]  . 
tale caratteristica , assente in caso di lesioni parenchimali , risente delle dimensioni del tfsp : risulta , infatti , maggiormente evidente con lincremento dimensionale ( 100% delle lesioni di dimensioni > 10 cm )  . la valutazione delle tc a nostra disposizione individua nelle caratteristiche dellenhancement un ulteriore aspetto distintivo dei tfsp . 
il 91 , 3% delle lesioni disomogenea dopo somministrazione del mezzo di contrasto e tale disomogeneit si configura nel 76 , 2% dei casi nei pattern tipici [ 3 , 11 , 21 , 24 , 25 ]  . 
7 woman with dyspnoea and a bulky solitary fibrous tumours of the pleura ( sftp ) occupying almost the entire right hemithorax with lobulated contours , smooth tapering margins and heterogeneous enhancement . 
due to their rich vascularity , the lesions show high attenuation relative to muscle on contrast - enhanced ct scans [ 4 ] , and their enhancement pattern is often heterogeneous with a geographic pattern , with areas of necrosis and depiction of serpiginous vessels [ 3 , 11 , 21 , 24 , 25 ]  . in very large tumours , the discrepancy between the scarcity of clinical findings and the dramatic radiological picture is in itself indicative of a slow - growing lesion ( lung tumours rarely grow so large without causing symptoms )  . absence of lymph node involvement [ 21 ] and preservation of cleavage planes with adjacent structures , despite the fact that these masses may occupy an entire hemithorax , provide confirmation of the benign nature of the lesion . 
when lesion arises from the mediastinal pleura , a suggestive finding is displacement of the thymic space , which does not occur in the presence of thymomas and lymphomas . ct therefore proves to be a very reliable imaging modality , especially when integrated with clinical and biopsy findings . se alle dimensioni , al fine di porre il sospetto diagnostico . 
la densit omogenea , simile a quella della muscolatura adiacente ; ci aiuta nella diagnosi differenziale con lesioni adipose o con raccolte liquide saccate [ 4 , 11 , 27 ]  . 
in virt della ricca vascolarizzazione delle lesioni , la loro densit superiore a quella dei muscoli dopo somministrazione di mezzo di contrasto [ 4 ] e lenhancement pi spesso disomogeneo a carta geografica , con aree di necrosi e immagini di vasi dallaspetto serpiginoso [ 3 , 11 , 21 , 24 , 25 ]  . di fronte a tumori molto voluminosi , inoltre , la discrepanza fra la povert della presentazione clinica e limportanza del quadro radiologico sono gi orientative di una lesione a lenta crescita ( i tumori polmonari raramente raggiungono tali dimensioni in assenza di un corredo sintomatologico )  . 
lassenza di un coinvolgimento linfonodale [ 21 ] e la conservazione di piani di clivaggio con le strutture adiacenti , nonostante le masse possano giungere ad occupare un intero emitorace , sono la conferma della benignit della lesione . 
di chirurgia , universit degli studi dellinsubria , viale borri 57 , i - 21100 varese , italy 3unit operativa di nefrologia , azienda ospedaliera ospedale di circolo e fondazione macchi , varese , italy correspondence to : g . 
over the past 2 years , we have observed 80 patients with stenotic haemodialysis accesses ; 24 of these ( mean age 66.4 years , range 5081 ) with 26 stenoses of 24 accesses ( 21 cimino - brescia fistulas and 3 dialysis loops ) were selected for cutting balloon angioplasty . 
in 11 cases , the cutting balloon device was used after failure to dilate the access with a high - pressure balloon whereas in 15 cases ( 10 focal stenoses and 5 restenoses ) , it was used as a first choice . 
follow - up examinations ( range 324 months , mean 18.2 months ) demonstrated patency of the vascular access and its good functioning during dialysis in 23 / 24 cases ( 95% )  . 
cutting balloon angioplasty is safe and effective in the treatment of haemodialysis access stenosis , especially in cases of severe stenosis , with low restenosis rate both in the short and medium ter key words cutting balloon arteriovenous fistula pta riassunto obiettivo . 
negli ultimi 2 anni sono giunti alla nostra osservazione 80 pazienti con stenosi dellaccesso per emodialisi ; 24 di essi ( et media 66 , 4 anni , range 5081 ) portatori di 26 stenosi di 24 accessi ( 21 fistole di cimino - brescia e 3 loops dialitici ) sono stati selezionati per un trattamento di angioplastica mediante cutting balloon . 
in 11 casi , il cutting balloon stato utilizzato dopo il fallimento della dilatazione con un pallone ad alta pressione , mentre in 15 casi ( 10 stenosi focali e 5 restenosi ) stato utilizzato di prima intenzione . 
langioplastica mediante cutting balloon sicura ed efficace nel trattamento delle stenosi degli accessi vascolari per dialisi , particolarmente nei casi di stenosi severe con bassi tassi di restenosi anche nel medio periodo . parole chiave cutting balloon fistola artero - venosa angioplastica percutanea introduction introduzione g . 
interventional radiology has taken on an increasingly important role in the prevention and treatment of complications affecting vascular haemodialysis accesses ; in particular , percutaneous angioplasty ( pta ) has become the treatment of choice in stenosis of access grafts [ 1 , 2 ]  . 
however , these stenoses , which are caused by intimal hyperplasia and proliferation of fibrous scar tissue , often resist dilatation with conventional angioplasty balloons [ 35 ] and high - pressure balloons [ 6 ]  . 
more recently , new applications in extracardiac vascular surgery have been proposed [ 1013 ] , in particular , in the treatment of stenoses of dialysis access sites [ 3 , 6 , 14 , 15 ]  . 
 materials and methods over the past 2 years , 80 patients were referred to our department for suspected stenosis of vascular haemodialysis accesses on the basis of the following clinical criteria : decreased thrill , increased pulsatility , oedema of the hand or forearm , low arterial pressure during dialysis . 
for this retrospective study , we collected data concerning 24 patients ( mean age 66.4 years , range 5081 ) presenting with 26 stenoses ( double stenosis in two patients ) in 24 haemodialysis accesses ( 21 cimino - brescia fistulae and 3 dialysis loops ) , which were treated by cutting balloon pta as a first choice or after failed conventional balloon angioplasty . 
twenty - three patients had a radiocephalic access and one had a femoral access . all patients had a failing fistula with a flow capacity < 300 ml / min as measured by the dialysis equipment and resulting difficulty in completing the dialysis procedure . before treatment , all patients underwent evaluation by colour doppler ultrasound ( cdus ) ( philips atl 5000 )  . stenosis was considered to be greater than 50% when peak systolic velocity was higher than 4 m / s and if the maximum velocity ratio was greater than 3 : 1 . 
subsequently , all patients underwent preprocedural angiography , with catheterisation of the humeral artery in 23 / 24 patients and of the common femoral artery in one patient ( using a 0.035 terumo hydrophilic guidewire and 4f terumo introducer sheath )  . 
la radiologia interventistica ha assunto negli ultimi anni un ruolo sempre pi importante nella prevenzione e nel trattamento della complicanze degli accessi vascolari per emodialisi ; in particolare langioplastica percutanea ( pta ) diventata il trattamento di scelta in caso di stenosi dei grafts con protesi [ 1 , 2 ]  . 
nellultimo decennio sono stati pubblicati numerosi lavori riguardanti questo device ; la maggior parte delle casistiche riguarda lutilizzo dello stesso in ambito cardiologico [ 8 ] ed endo - urologico [ 9 ]  . 
pi recentemente sono state proposte diverse applicazioni del cutting balloon in ambito vascolare extra - cardiaco [ 1013 ] e in particolare nel trattamento delle stenosi degli accessi dialitici [ 3 , 6 , 14 , 15 ]  . materiali e metodi durante gli ultimi 2 anni sono stati inviati alla nostra osservazione 80 pazienti per sospetta stenosi dellaccesso vascolare per emodialisi sulla base dei seguenti criteri clinici : diminuito thrill , aumentata pulsabilit , edema della mano e dellavambraccio , bassa pressione arteriosa durante la dialisi ; in 10 pazienti non erano presenti stenosi morfologicamente superiori al 30% e pertanto non meritevoli di trattamento percutaneo ; 46 pazienti sono stati trattati con angioplastica con pallone convenzionale ; in questo studio retrospettivo sono stati raccolti i dati relativi a 24 pazienti ( et media 66 , 4 anni , range 5081 ) portatori di 26 stenosi ( duplice stenosi in 2 pazienti ) in 24 accessi per emodialisi ( 21 fistole di cimino - brescia e 3 loops dialitici ) che sono stati trattati mediante cutting balloon pta di prima intenzione o dopo fallimento di angioplastica con pallone convenzionale . ventitr pazienti erano portatori di accesso radio - cefalico e 1 / 24 di accesso femorale . tutti i pazienti presentavano una failing fistula con portata della pompa misurata dallapparecchiatura di dialisi < 300 ml / min e conseguente difficolt allespletamento della procedura dialitica . 
la valutazione pre - trattamento stata effettuata in tutti i casi , con eco - color doppler ( philips atl 5000 ) : si considerato una stenosi superiore al 50% quando il picco di velocit sistolica era superiore a 4 m / s e se il rapporto di massima velocit era superiore a 3 : 1 ; successivamente tutti i pazienti sono stati sottoposti a studio angiografico preliminare alla procedura , espletato mediante cateterismo dellarteria omerale in 23 / 24 pazienti e dellarg . 
lo studio preliminare stato condotto tramite cateterismo arterioso in quanto nella nostra esperienza liniezione di mdc da tale versante consente di visualizzare in maniera ottimale laccesso per emodialisi lungo tutto il suo decorso e non crea intralcio al successivo cateterismo venoso per lespletamento di una eventuale procedura di angioplastica . 
il grado delle stenosi trattate era compreso tra 30% e 90% . previo consenso informato da parte del paziente , abbiamo proceduto allincannulamento del versante venoso con superamento della stenosi che stato conseguito con lutilizzo di guida idrofilica 0 , 035 ( terumo ) e catetere idrofilo ( 4f glidecath / terumo )  . 
successivamente stato posizionato un introduttore 6 / 7f ( terumo ) e si provveduto alla sostituzione della guida 0 , 035 con una guida 0 , 018 ( sv wire / cordis ) mediante catetere retto 5 f ( cordis )  . 
nei pazienti con duplice stenosi abbiamo impiegato lo stesso device per il trattamento di entrambe . la dilatazione stata eseguita mediante siringa dotata di manometro ( medflator ii / medex medical ) con insufflazione fino a 20 atm , mantenuta per 1 minuto , nel caso di un pallone ad alta pressione , e con insufflazione graduale del pallone ( 1 atm / 5 s ) fino ad una pressione di 6 atm ( tranne in 3 casi in cui si raggiunta una pressione di 78 atm determinante rottura del pallone ) con conseguente sgonfiaggio una volta raggiunta la pressione desiderata quando stato impiegato il cutting balloon . 
il follow - up stato pertanto espletato mediante valutazione clinica e con eco - color doppler a 1 , 3 , 6 , 12 , 18 e 24 mesi . risultati il successo tecnico immediato stato documentato in tutte le fig . 
1 cutting balloon : pallone da angioplastica non convenzionale con 3 o 4 aterotomi ( lame microchirurgiche ) montate longitudinalmente sulla superficie esterna ( cortesia boston scientific )  . inary study was carried out using arterial catheterisation because we have found that arterial injection of contrast material allows for optimal visualisation of the entire course of the haemodialysis access and does not interfere with possible subsequent venous catheterisation carried out for angioplasty procedures . 
the degree of stenosis ranged from 30% to 90% . after obtaining the patients informed consent , we proceeded to cannulate the vein crossing the stenosis using a 0.035 terumo hydrophilic guidewire and a 4f hydrophilic catheter ( terumo glidecath )  . 
a 6 / 7f terumo introducer sheath was subsequently inserted , and the 0.035 guidewire was exchanged for a 0.018 guidewire ( sv wire , cordis ) through a straight 5f catheter ( cordis )  . 
in patients with two stenoses , these were both treated using the same device . dilatation was carried out using a syringe equipped with a manometer ( medflator ii , medex medical )  . if high - pressure balloons were used , these were inflated up to a pressure of 20 atm , maintained for 1 min , whereas if the cutting balloon was used , it was inflated gradually ( 1 atm / 5 s ) up to 6 atm ( except for three cases in which a pressure of 78 atm was reached , causing balloon rupture ) g . 
when this was the case , we first negotiated the stenosis with a stiff guidewire ( amplatz super stiff , boston scientific ) and then advanced the balloon through the introducer sheath , which was withdrawn before inflation . there were no minor complications or early thromboses . in 3 cases , the balloon ruptured owing to inflation to 78 atin all 3 cases , the device could be removed in one piece without difficulty or complications . 
 clinical success was achieved in all patients ( 24 / 24 ) , with restored function of the access ( 24 / 24 ) used for dialysis within 2 days of the procedure . 
during follow - up ( mean 18.2 months , range 324 ) there was one case of restenosis which was not haemodynamically significant ( < 20% ) and did not impair access function . 
 discussion a functioning vascular access for haemodialysis is of crucial importance for efficient dialysis procedures and requires accurate monitoring , as it may be affected by a series of problems with patency rates at 1 year of 41%75% [ 16 ]  . 
 fibrotic changes resulting from repeated punctures [ 15 ] and haemodynamic conditions arising above all in the anastomoses and efferent vein through intimal hyperplasia induced by turbulent flow and high pressure applied on the efferent vein underlie the formation of stenoses . this reduction in calibre entails decreased access capacity with resulting impaired efficiency of the access during dialysis , which requires a flow capacity of at least 300 ml / min [ 19 ]  . 
monitoring access flow and prompt treatment of haemodynamically significant stenoses ( > 50% ) are required to increase the life span of an access site and decrease its rate of thrombosis [ 20 , 21 ]  . endovascular treatments have taken on a major role in the management and salvage of vascular dialysis accesses and now constitute the treatment of choice [ 20 ] due to the lower relazione alla rigidit del device e al ridotto calibro della guida portante : in questo caso stato dapprima avanzato lintroduttore oltre la stenosi con lausilio di una guida rigida ( amplatz super stiff , boston scientific ) ; successivamente il pallone stato avanzato allinterno dellintroduttore che stato poi ritirato prima dellinsufflazione . 
in 3 casi si verificata la rottura del pallone quando lo stesso stato insufflato a 78 atm ; in tutti i 3 casi il device stato rimosso integro , senza difficolt e senza complicanze . 
in tutti i pazienti ( 24 / 24 ) si ottenuto anche un successo clinico con ripristino della funzionalit dellaccesso ( 24 / 24 ) che stata utilizzata per la procedura dialitica entro 2 giorni dal trattamento . 
in 23 / 24 pazienti laccesso si pertanto mantenuto pervio e funzionante ed stata utilizzato durante le procedura dialitica con una perviet primaria del 95% . discussione la disponibilit di un accesso vascolare per emodialisi ben funzionante un elemento di grande importanza per lespletamento della procedura dialitica e richiede una gestione molto accurata in quanto gravata da una serie di problemi con tassi di perviet a 1 anno del 41%75% [ 16 ]  . 
la presenza di una stenosi il fattore responsabile del malfunzionamento e della trombosi dello stesso in oltre il 90% dei casi [ 1 , 2 , 17 , 18 ]  . 
le alterazioni fibrotiche dovute ai traumatismi legati alle punture ripetute [ 15 ] e le condizioni emodinamiche che si creano , soprattutto in corrispondenza delle anastomosi e del versante efferente dellaccesso , rappresentano , attraverso liperplasia miointimale indotta dal flusso turbolento e dalle elevate pressioni cui viene esposta la parete della vena efferente , il substrato eziopatogenetico della stenosi . 
tale riduzione di calibro comporta una diminuzione della portata dellaccesso con conseguente compromissione della efficienza dello stesso durante la dialisi che richiede un accesso con portata di almeno 300 ml / min [ 19 ]  . 
il monitoraggio del flusso dellaccesso e il trattamento tempestivo delle stenosi emodinamicamente significative ( > 50% ) sono necessari al fine di aumentare la vita dellaccesso e di diminuirne la percentuale di trombosi [ 20 , 21 ]  . 
i trattamenti endovascolari hanno assunto un ruolo fondamentale nella gestione e nel salvataggio degli accessi vascolari dialitici e rappresentano oggi lapproccio terapeutico di prima scelta [ 20 ] , in quanto gravato da minori tassi di complicanze rispetto alla chirurgia [ 5 ]  . 
la pta la procedura endovascolare di scelta [ 1 , 2 ] per il trattamento delle stenosi degli accessi vascolari con tassi di successo tecnico compresi tra 85% e 94% [ 2224 ]  . 
pta is the endovascular treatment of choice [ 1 , 2 ] for vascular access stenosis , with rates of technical success ranging from 85% to 94% [ 2224 ]  . 
pta nonetheless suffers some limitations , especially as regards anastomotic stenoses [ 3 ] and the high incidence of restenosis with a 2 - year patency rate of 20%30% [ 2224 ]  . the high incidence of post - pta recoil , caused by the pathogenesis of this type of stenosis [ 3 , 4 ] , has stimulated the search for new devices and alterative endovascular procedures [ 19 ]  . 
as regards pta , proposals include the use of high - pressure balloons [ 5 , 6 ] and prolonged inflation with devices that allow simultaneous perfusion of the fistula [ 3 ]  . 
in addition , the presence of the stent increases the incidence of thrombosis limiti legati alle stenosi resistenti , soprattutto in prossimit dellanastomosi [ 3 ] e dallelevata incidenza di restenosi con tasso di perviet a 2 anni del 20%30% [ 2224 ]  . 
lelevata frequenza di recoil post - pta , determinata dalla particolare patogenesi di questo tipo di stenosi [ 3 , 4 ] , ha favorito la ricerca di nuovi devices e di procedure endovascolari alternative [ 19 ]  . 
per quanto riguarda la pta stato proposto lutilizzo di palloni ad alta pressione [ 5 , 6 ] e linsufflazione prolungata con dispositivi che consentono la contemporanea perfusione della fistola [ 3 ]  . 
lintegrazione della pta con lo stenting non sembra migliorare i risultati dellangioplastica [ 25 ] ; inoltre la presenza dello stent aumenta lincidenza di trombosi e riduce larea disponibile per la puntura [ 26 ]  . 
il cutting balloon , device che unisce i principi della microchirurgia e dellangioplastica percutanea , stato proposto per la prima volta nel 1991 da barath per pta coronarica al fine di cong . 
 the cutting balloon , a device that combines the principles of microsurgery with those of percutaneous angioplasty , was first proposed by barath in 1991 to control intimal hyperplasia and decrease restenosis rate in coronary pta [ 7 ]  . 
it consists of a noncompliant balloon equipped with three or four microblades ( atherotomes ) mounted longitudinally on the outer surface of the balloon . balloon expansion exposes the microblades , which cut into the intima in a controlled manner , allowing vessel dilatation without the irregular intimal tears caused by conventional balloons [ 27 ]  . the regular incisions involve minimal exposure of the media with reduced procoagulation and local inflammatory responses and lesser extent of repair process [ 7 , 28 ]  . 
it appears that after cutting balloon dilatation , there is less expression of neutrophil adhesion molecules and the expression of cell growth and proliferation factors is not circumferential but limited to the intimal incisions . 
these factors are apparently responsible for the lower rates of restenosis [ 28 ]  . the cutting action of the microblades creates an area of least resistance in the fibrous strand at the level of the stenosis , enabling balloon inflation . 
the cutting balloon may therefore also enhance the efficacy of conventional pta by allowing greater balloon expansion [ 3 ]  . beginning in 1991 , many papers have addressed the use of the device in coronary arteries [ 8 ]  . 
more recently , other studies have demonstrated its good results in other districts , above all when the stenosis is caused by intimal hyperplasia and fibrosis [ 1013 ]  . applicability of the device to the venous system and in particular to the treatment of stenotic fistulae was demonstrated by vorwerk et al . 
 [ 3 ] published the results achieved in 15 cimino - brescia fistulae and four haemodialytic shunts showing a primary patency of 100% postprocedure of 94% at 6 months and of 64% at 69 months . 
in 2005 , singer - jordan published the interesting results of a prospective study of 29 patients with arteriovenous fistulae for a total of 42 stenoses in which the immediate technical success was 100% , primary patency was 76% at 6 months and secondary patency was 93% [ 29 ]  . a previous paper of ours reported our experience with cutting balloon pta in 21 patients followed up for mean period of 11.1 months [ 30 ]  . 
regular monitoring of these patients allowed us to verify the efficacy of the treatment , even in the medium term ( 18.2 months ) , confirming a primary patency of 95% . at long - term follow - up , we recorded only one case of haemodynamically significant restenosis in a patient in whom vessel diameter had been originally underestimated . the stenosis was treated after 6 months with a repeat cutting balloon pta procedure , which had immediate technical success . 
in all patients , the cutting balloon expanded rapidly to its full diameter without the need for prolonged dilatations , trollare liperplasia intimale e diminuire il tasso di restenosi [ 7 ]  . 
quando questo viene insufflato si realizza lesposizione delle microlame che provocano una incisione controllata dellintima in modo da permettere la dilatazione del vaso evitando le lacerazioni irregolari dellintima causate dai palloni convenzionali [ 27 ]  . 
le incisioni regolari comportano una minima esposizione della media con ridotta risposta procoagulante e infiammatoria locale e minore entit dei processi riparativi [ 7 , 28 ] ; sembra infatti che dopo dilatazione con cutting balloon si verifichi una minore espressione di molecole di adesione per i neutrofili ; inoltre lespressione di fattori di crescita e la proliferazione cellulare non sono circonferenziali , ma limitati alle incisioni dellintima . 
a partire dal 1991 sono stati pubblicati numerosi lavori riguardanti lutilizzo del device a livello coronarico [ 8 ]  . pi recentemente altri studi ne hanno dimostrato i buoni risultati in stenosi resistenti in altri distretti , soprattutto laddove la riduzione di calibro sostenuta da iperplasia intimale e fibrosi [ 1013 ]  . 
lapplicabilit del device al distretto venoso ed in particolare al trattamento delle stenosi delle fistole stata dimostrata da vorwerk nel 1995 [ 15 ] ; successivamente lo stesso vorwerk nel 1996 [ 3 ] ha pubblicato i risultati riguardanti 15 fistole di cimino - brescia e 4 shunts emodialitici con una perviet primaria del 100% alla fine della procedura , del 94% a distanza di 6 mesi e del 64% a 69 mesi ; bittl e feldman [ 6 ] hanno trattato 3 stenosi di graft con successo tecnico immediato del 100% e tasso di restenosi del 16 , 6% durante un follow - up medio di 6 , 7 mesi ; ryan et al . 
nel 2005 singer - jordan ha pubblicato degli interessanti risultati di uno studio prospettico di 29 pazienti con fistola artero - venosa per un totale di 42 stenosi riportando un successo tecnico immediato del 100% , una perviet primaria del 76% a 6 mesi e una perviet secondaria del 93% [ 29 ]  . 
in un precedente lavoro abbiamo gi riportato la nostra esperienza riguardante la pta con cutting balloon in 21 pazienti seguiti in media per 11 , 1 mesi [ 30 ]  . 
durante il follow - up prolungato abbiamo riportato solo un caso di ristenosi emodinamicamente significativa in una paziente in cui avevamo inizialmente sottostimato il diametro del vaso ; la stenosi stata trattata a 6 mesi di distanza con una nuova cutting balloon pta con successo tecnico immediato . 
in tutti i pazienti , il cutting balloon si espanso rapidamente al proprio diametro senza necessit di dilatazione prolungate , tranne nei 3 casi in cui si dovuto sovradilatare fino a 78 atm ( pressione di rottura del pallone ) in quanto a 6 atm la divulsione della stenosi non era iconograficamente soddisfacente : tale comportamento verosimilmente da mettere in relazione al fatto che lincisione g . 
 conclusions on the basis of our experience and previous reports , we can state that cutting balloon angioplasty is a safe and effective procedure for the treatment of haemodialysis - access stenosis with low rates of restenosis in the short and medium term in that it provides excellent results even in focal , tight stenoses that are resistant to dilatation with high - pressure balloons . nonetheless , randomised controlled studies on larger patient populations are required to confirm our encouraging results on the efficacy of the device in prolonging access patency . 
in addition , the economic impact of the use of cutting balloons should be assessed given that , despite having a higher unit cost compared with conventional pta balloons , these devices require fewer maintenance procedures . dellintima riduce il barotrauma sulla parete vasale , richiedendo pressioni di insufflazione minori . 
questo aspetto particolarmente vantaggioso se consideriamo che nella nostra serie i pazienti trattati con palloni convenzionali ad alta pressione avvertono dolore che richiede sedazione o supporto anestesiologico . conclusioni in base a quanto emerge dallesperienza personale e dai dati riportati in letteratura , possibile affermare che langioplastica mediante cutting balloon una procedura sicura ed efficace nel trattamento delle stenosi degli accessi vascolari per emodialisi , in quanto consente di ottenere ottimi risultati anche nelle stenosi focali serrate , resistenti alla dilatazione con palloni ad alta pressione , con bassi tassi di restenosi a breve e medio termine . 
sono comunque necessari ulteriori studi , randomizzati e controllati , numericamente pi consistenti al fine di confermare gli incoraggianti risultati della nostra serie riguardante lefficacia del device nel prolungare la perviet degli accessi . 
solivetti , via citt della pieve 19 , i - 00191 roma , italy , tel . : + 39 - 06 - 52666447 , fax : + 39 - 06 - 52663937 , e - mail : solivetti@tiscalinet.it received : 15 september 2005 / accepted : 17 february 2006 / published online : 29 june 2006 abstract purpose . 
following a review of the relevant literature , we present the results of a retrospective study based on 2 , 000 malignant melanoma patients with complete case records . of these , we selected 600 patients who had a thick melanoma ( > 1 mm ) at presentation but were clinically free of in - transit or satellite melanoma metastases during follow - up . 
a total of 95 lesions were identified . average lesion diameter was 0.7 mm , and only four were larger than 1 call suspected lesions were confirmed by surgery , follow - up or us - guided fine - needle aspiration ( fna ) with 22gauge needles using a freehand technique and exploiting the capillarity principle . 
in this series , there were apparently no false positive or false negative us results although inclusion criteria precluded correct evaluation of possible false negatives . minimum lesion diameter allowing sonographic detection appears to be around 0.4 mus features of in - transit metastases have been well documented . 
dopo revisione della letteratura di pertinenza , gli autori presentano i risultati di uno studio retrospettivo basato su un archivio di dati clinico - radiologici completi relativi a 2000 pazienti . 
nellambito di tale ampia casistica sono stati presi in considerazione 600 casi , riguardanti pazienti che avevano , al momento della valutazione iniziale , una lesione melanomatosa di maggior spessore ( > 1 mm ) , ma riscontrati clinicamente negativi per metastasi in transito o satelliti di melanoma nel follow - up clinico . 
nellambito di tale popolazione di pazienti clinicamente negativi , in 63 casi , il sospetto di metastasi in transito o satellite stato posto ecograficamente , e sono state individuate 95 lesioni , con dimensioni medie di 0 , 7 mm e solo in quattro casi di dimensioni > 1 ctutte le lesioni sospette sono state confermate chirurgicamente , con follow - up o mediante esame cito - aspirativo , eseguendo il prelievo citologico sotto guida ecografica , con aghi da 22 g , sfruttando il principio di aspirazione per capillarit . 
sotto il profilo ecografico le dimensioni minime accettabili per poter identificare una lesione metastatica sembrano essere intorno a 0 , 4 mle caratteristiche ecografiche delle metastasi da melanoma sono ben documentate in letteratura . 
solitamente si tratta di lesioni solide ipoecogene , rispetto al tessuto adiposo sottocutaneo circostante , con margini abbastanza netti e definiti , con una buona trasmissione degli ultrasuoni ( us )  . 
queste caratteristiche ecografiche in ecografia dermatologica sono piuttosto aspecifiche per tutti i tipi di metastasi , ma , tra le patologie benigne , soltanto site of a malignant melanoma should be more widely used and associated with us - guided fna biopsy . key words ultrasound in - transit metastasis cutaneous malignant melanoma f.m. 
gli autori desiderano promuovere ed incoraggiare un pi ampio impiego dellecografia nel follow - up dei tessuti molli circostnati la sede iniziale dei melanomi , associata al prelievo citologico sotto guida ecografica . parole chiave ultrasuoni metastasi in transito melanoma maligno cutaneo introduction introduzione cutaneous malignant melanoma ( mm ) is a relatively common malignancy . 
its incidence is rapidly increasing , with rates doubling every 1020 years [ 1 , 2 ] , and it is the third most frequent cancer to give rise to cutaneous metastases , with an incidence of 24% [ 3 ]  . melanoma recurrences in soft tissue are known as satellite metastases when located within 2 cm from the primary lesion and in - transit metastases when located in the cutaneous or subcutaneous tissues more than 2 cm from the primary tumour . 
in particular , the use of probes with frequencies between 7.5 and 20 mhz has enabled a more accurate differential diagnosis between mm recurrences / metastases and skin metastases from other malignancies and even between mm metastases and other conditions , such as fibromas , lipomas and aberrant scars [ 5 ]  . the aim of this study was to assess the potential of us in detection of in - transit or satellite metastases on the basis of a large case series . il melanoma maligno cutaneo ( mm ) una patologia aggressiva relativamente comune , con frequenza in incremento ; stato osservato che il suo tasso dincidenza raddoppia ogni 1020 anni [ 1 , 2 ] ; esso rappresenta inoltre la terza neoplasia che , in ordine di frequenza , determina metastasi a livello cutaneo , con unincidenza del 24% [ 3 ]  . le recidive di melanoma nei tessuti molli sono chiamate satelliti se sono nel raggio di 2 cm dalla lesione primitiva , mentre quelle cutanee e sottocutanee lontane pi di 2 cm sono definite come metastasi in transito . 
esse si sviluppano lungo la via linfatica tra la lesione primitiva e la stazione linfonodale di drenaggio [ 4 ]  . la diagnosi precoce di queste recidive stata incrementata dalluso dellecografia ( us ) , con sonde ad alta frequenza . 
in particolare , luso di sonde di frequenza compresa tra 7 , 5 e 20 mhz ha reso possibile una pi accurata diagnosi differenziale tra recidive - metastasi da mm e metastasi cutanee da altre patologie maligne od anche tra metastasi da mm ed altre ipotesi diagnostiche , quali fibromi , lipomi e cicatrici aberranti [ 5 ]  . lo scopo del lavoro di verificare le attuali possibilit dellecografia nel riconoscimento di metastasi in transito e satelliti sulla base dei dati di una casistica molto numerosa . materials and methods a total of 600 patients were selected on the basis of the following inclusion criteria : ( 1 ) patients affected by mm with a thickness greater than 1 mm ; ( 2 ) patients who underwent wide surgical excision and assessment of sentinel node , and removal of the draining lymph node station in the case of positive sentinel node . 
during follow - up , all study patients underwent clinical assessment every 6 months , and if the findings were negative , a yearly us examination of the surgical scar and surrounding tissue within a radius of 10 cm from the tumour materiali e metodi nel nostro studio abbiamo selezionato 600 pazienti , sulla base dei seguenti criteri di inclusione : ( 1 ) pazienti affetti da mm di spessore superiore ad 1 mm ; ( 2 ) pazienti trattati chirurgicamente con escissione ampia della lesione , valutazione del linfonodo sentinella ed , in caso di positivit dello stesso , svuotamento della stazione linfonodale di drenaggio . 
i 600 pazienti erano 336 di sesso femminile e 264 di sesso maschile , con et media di 54 anni ; di essi il 60% ha raggiunto i previsti 5 anni di follow - up . 
 all us examinations were carried out with an esaote au5 unit ( genoa , italy ) using a 20 - mhz annular array probe and 13 and 7.5 mhz linear probes ; standard chest radiographs and total - body computed tomography ct scans were also performed , as per protocol . 
colour - doppler us was performed using settings allowing assessment of slowflow conditions ( therefore using the most sensitive low - cut filter and the units highest sensitivity setting ; flow velocities that can be detected are in the order of a few centimetres per second ) and taking multiple samplings in different areas of the lesion ; the sampling volume for pulsed doppler us was chosen in relation to the expected vessel size , below 1 mm . fine - needle aspiration ( fna ) was carried out after obtaining written informed consent and using a freehand technique under sonographic guidance ; samples were taken with 23gauge needles exploiting the capillarity principle thus without applying vacuum and making two passes for each nodule . 
 results out of 600 patients who were clinically free of local recurrence , us examination raised the suspicion of in - transit metastasis ( 39 cases ) or satellite lesion ( 24 cases ) in 63 patients . 
in these patients , the primary lesion had a mean thickness of 2 mm and was located on the limbs in 41 cases and on the trunk in 22 cases . 
the time interval between detection of the new lesion and surgical excision of the primary tumour was about 2 years , with a range of 6 months to 5 years . 
of the 32 patients who underwent fna , cytology provided a definitive diagnosis of metastasottoposti ad esame clinico ogni 6 mesi e , se obbiettivamente negativi , ad ecografia annuale dei tessuti molli dellarea cicatriziale e della zona ad essa circostante , in un raggio di circa 10 cm , intorno alla sede originaria della neoplasia . parte rilevante dei pazienti positivi sono stati trattati senza ulteriori indagini diagnostiche per immagini locoregionali . tutti gli esami ecografici sono stati eseguiti con ecografo esaote au5 ( genova , italia ) , impiegando sonda anular array da 20 mhz e sonda lineare da 13 e 7 , 5 mhz ; sono stati inoltre effettuati radiogrammi toracici standard ed esami tc total body , come da protocollo . lesame color doppler stato eseguito con impostazione dei parametri tale da consentire la valutazione dei flussi pi bassi ( quindi impiegando il filtro taglia - basso pi sensibile e con la sensibilit in ricezione pi alta consentita dallapparecchiatura in uso ; le velocit di flusso rilevabili sono nellordine di pochi cm / s ) ; effettuando inoltre plurimi campionamenti in aree diverse della lesione ; per ci che concerne il doppler pulsato , il volume campione stato scelto in relazione alle dimensioni attese dei vasi , al di sotto del millimetro . per la fna si sempre ottenuto consenso informato preliminare scritto e si impiegata una tecnica a mano libera , sotto assistenza ecografica , con aghi sottili da 23 g , utilizzando il principio della capillarit e senza quindi applicare il vuoto ed effettuando normalmente due successivi prelievi per ogni nodulo . risultati tra questi 600 pazienti , in 63 casi - clinicamente negativi per ricorrenza locale stata posta diagnosi ecografia di sospetta metastasi in transito ( 39 casi ) o lesione satellite ( 24 casi )  . 
in questi 63 pazienti , la lesione primitiva aveva uno spessore medio di 2 mm , era localizzata in 41 casi agli arti ed in 22 casi al tronco ; lintervallo di tempo tra la documentazione della nuova patologia ed il trattamento chirurgico della lesione primitiva era di circa 2 anni , con un minimo di 6 mesi ed un massimo di 5 anni . in questi 63 pazienti stato trovato un totale di 95 lesioni , con un massimo di 4 noduli per paziente . 
le lesioni campionate con fna avevano un diametro medio di 7 mm , con un minimo di 5 mm ed un massimo di 13 mnon vi sono stati casi falsi positivi . discussione la ricerca e la diagnosi di localizzazioni secondarie , in pazienti affetti da mm , rappresentano ovviamente un punto cardine nel percorso terapeutico . 
gi da tempo gli ultrasuoni sono stati utilizzati in questo specifico settore ; in particolare , il primo a proporre il ricorso allecografia per la ricerca di metastasi , satelliti od in transito , stato fornage [ 6 ]  . 
il suo studio , basato su tre casi , descriveva tali lesioni come solide , ipoecogene e con contorni netti ; altri autori [ 7 ] , in seguito , hanno descritto ulteriori caratteristiche ecografiche di tali metastasi quali , ad esempio , la presenza di segnali vascolari allinterno del nodulo , evidenziabili con il color doppler e , assai meglio , con il power doppler , oppure la presenza di piccole aree fluide interne , dovute a fenomeni di necrosi . nella nostra esperienza abbiamo potuto verificare inoltre come la maggior parte di queste lesioni si localizzi in un raggio di 10 cm dalla lesione primitiva e , in riferimento alla nostra casistica , costituita da pazienti con melanoma di spessore superiore ad 1 mm , tale metodologia dindagine ci ha consentito di identificare lesioni metastatiche nel 10% dei casi nel corso del follow - up . abbiamo potuto verificare come le caratteristiche di queste lesioni coincidano con quanto descritto in letteratura ; in particolare : la presenza di aree fluide interne sembra legata a fenomeni di necrosi e quindi pi frequente nelle patologie di maggiori dimensioni , dove il flusso ematico insufficiente ; tali ripetizioni possono inoltre mostrare contorni netti ma leggermente irregolari ; esse possono infine mostrare buona trasmissione del fascio ultrasonoro e rinforzo di parete posteriore , con frequentemente segnali vascolari interni al power doppler . per le modeste dimensioni delle lesioni e per i limiti tecnici delle apparecchiature impiegate , non possibile esprimere un definitivo giudizio circa il vero pattern vascolare di questo tipo di patologia.una corretta stima dei falsi negatifig . 
2 la sonda lineare da 13 mhz documenta la presenza di una formazione nodulare , ipoecogena , a margini lievemente sfumati . sis in 25 and a probable diagnosis of metastasis in seven . 
lesions sampled with fna had a mean diameter of 7 mm , with a range of 513 mthere were no cases of false positive results . discussion in patients with mm , detection and diagnosis of metastases are clearly crucial steps in the therapeutic process , and us has long been used for this purpose . 
fornage was the first to propose us for the search for satellite or in - transit metastases [ 6 ]  . his study , based on three cases , described the lesions as being solid , hypoechoic and well circumscribed . 
3 la sonda lineare da 13 mhz documenta la presenza di una formazione nodulare , modestamente disomogenea , per la presenza di qualche millimetrica area fluida al suo interno . doppler us and especially with power - doppler us or the presence of tiny internal fluid areas due to necrosis . 
in our experience we also found that most of these lesions are located within 10 cm from the primary lesion and , as regards patients with melanoma with a thickness > 1 mm , that us enabled detection of metastasis in 10% of cases during follow - up . sonographic features of the metastatic lesions identified in our study were in agreement with the literature . 
more specifically : the presence of internal fluid areas appears to be related to necrosis and is therefore more common in larger lesions , where blood flow is insufficient metastatic lesions may also exhibit well - defined but slightly irregular contours . metastatic lesions may also show good sound - wave transmission and posterior shadowing , frequently with internal vascular signals at power - doppler imaging . given the small size of the lesions and the technical limitations of the us equipment used , we are unable to express a definitive judgement on the vascular pattern of the disease . 
likewise , correct estimation of sonographic false negatives , despite long - term follow - up , cannot be provided owing to the retrospective nature of the study , the yearly intervals for us followup and the relatively high rate of patients lost to follow - up ; every year , about 8% of our patients fail to attend routine follow - up appointments though they appeared to be in good health at the previous examination . 
it should be recalled that by including patients with mm with a thickness greater than 1 mm and excluding those with a clinical diagnosis of recurrent disease , this retrospective study does not allow assessment of the true incidence of satellite and in - transit metastases . 
for detection of metastatic lesions , it is important to use multiple us probes , from 7.5 up to 20 mhz , or an appropriate multifrequency probe , if possible , because depending on its site and size a lesion may be well visualised at a given frequency but totally invisible at another . 
as regards differential diagnoses in this phase , an abnormal scar shows blurred contours and is often avascular whereas a granuloma often incorporates the foreign body and tends to be hypovascular or avascular at colour imaging . 
on the basis of our experience , given that we detected no lesions smaller than 4 mm , it seems reasonable to consider 4 mm as the threshold value for the identification of such lesions . as previously stated , the characteristics of our study population do not allow us to exclude the existence of false negative results . 
even though some of the lesions identified at the second , third , fourth or fifth us follow - up examination might have been present earlier but were too small to detect , the doubling time of these tumours makes this hypothesis unlikely . 
in our series , 52 patients out of 63 were negative at the first follow - up and became positive during the follow - up period . fornage [ 6 ] was the first to propose us as a modality to guide fna . 
fanelli , tel : + 39 - 06 - 4455602 , fax : + 39 - 06 - 490243 , e - mail : fabrizio.fanelli@uniroma1.it received : 7 july 2005 / accepted : 27 january 2006 / published online : 29 june 2006 abstract transjugular intrahepatic portosystemic shunt ( tips ) is a nonoperative therapeutic option for the management of portal hypertension , variceal bleeding , recurrent ascites , budd - chiari syndrome . 
in view of the many issues surrounding the use of tips , in 1994 the us national digestive diseases advisory board convened a scientific conference to review the current data available and to establish the indications and controindications for this procedure . 
however there are still unsolved problems especially short primary patency of the shunt due to intimal hyperplasia , which causes a reduction of the shunt lumen thus favoring a return of the portal hypertension with recurrent variceal bleeding . 
in our experience more then 100 patients were treated with the viatorr stent - graft . after a follow - up ranging from 1 to 50 months we reported a 1 - year primary patency rate of 83.8%. 
in case of hepatic hencefalopathy refractory to the conventional medical therapy , tips reduction should be performed . key words tips portal hypertension covered stents hepatic encephalopathy viatorr riassunto lo shunt transgiugulare intraepatico portosistemico ( tips ) una procedura terapeutica , non chirurgica , per il trattamento dellipertensione portale , del sanguinamento da varici esofagee , dellascite refrattaria e della sindrome di budd - chiari . dallintroduzione della tips nella pratica clinica , sono state avanzate diverse teorie sullutilizzo di tale metodica . 
per tale motivo , nel 1994 la national digestive diseases advisory board americana si riunita in una conferenza per stabilire , sulla base dei dati disponibili in letteratura , le linee guida con indicazioni e controindicazioni alla procedura . 
tuttavia esistono ancora dei problemi irrisolti correlati principalmente con la ridotta perviet primaria conseguente ad iperplasia neo - intimale con progressiva riduzione del lume dello shunt che favorisce la ricomparsa dei sintomi . 
nella nostra esperienza pi di 100 pazienti sono stati trattati con il viatorr e dopo un follow - up compreso tra 1 e 50 mesi la perviet primaria stata del 83.8%. 
in caso di encefalopatia epatica refrattaria alla terapia medica convenzionale , necessario effettuare una riduzione dello shunt . parole chiave tips ipertensione portale stent ricoperti encefalopatia epatica viatorr introduction introduzione the transjugular intrahepatic portosystemic shunt ( tips ) is a nonoperative therapeutic option for the management of portal hypertension , variceal bleeding , recurrent ascites and budd - chiari syndrome . 
the portal venous system is partially decompressed by the creation of a parenchymal tract between the portal ( pv ) and hepatic ( hv ) veins followed by reinforcement of the tract with a metallic stent . 
from 1969 , when this procedure was used for the first time in dogs lo shunt transgiugulare intraepatico portosistemico ( tips ) una procedura terapeutica , non chirurgica , per il trattamento dellipertensione portale , del sanguinamento da varici esofagee , dellascite refrattaria e della sindrome di budd - chiari . il sistema venoso portale parzialmente decompresso mediante la creazione di un tramite intraparenchimale tra la vena porta e le vene sovraepatiche , rinforzato da uno stent metallico . 
in the 1980s , metallic stents were developed and used by palmaz and rosch , and in 1983 , colapinto performed for the first time this procedure in a human [ 2 ]  . 
however , the initial enthusiasm was subsequently tempered by several complications represented by hepatic encephalopathy ( he ) and shunt stenosis . patient selection in view of the many issues surrounding the use of tips , in 1994 , the us national digestive diseases advisory board convened a scientific conference to review the available data and establish indications and contraindications for this procedure [ 3 , 4 ]  . 
after tips , immediate cessation of bleeding is reported in 73%96% of cases [ 6 ] , with a recurrent variceal bleeding rate of 15%20% at 1 year [ 5 ]  . tips is particularly helpful when bleeding occurs from inaccessible intestinal or gastric varices or is the result of severe portal hypertensive gastropathy [ 3 ]  . 
tips has been found to be valuable in managing patients who have developed rebleeding despite receiving adequate medical or endoscopic therapy [ 3 , 8 ]  . many trials were published comparing tips with endoscopic treatment for the prevention of recurrent oesophageal variceal bleeding . 
a metaanalysis of these trials reported that tips is associated with a 28% reduction in recurrent bleeding compared with endoscopic therapy [ 5 ]  . la prima volta in un cane da rosch , molti studi sono stati effettuati per migliorare la tecnica e la perviet dello shunt . nel 1980 stato impiantato da palmaz e rosch uno stent metallico e , nel 1983 , colapinto effettu la procedura su un uomo per la prima volta [ 2 ]  . finalmente negli ultimi tre anni sono stati introdotti stent ricoperti per prolungare la perviet dello shunt evitando malfunzioni dovute alliperplasia intimale . 
tuttavia lentusiasmo iniziale si gradualmente stemperato a causa delle gravi complicanze rappresentate dallencefalopatia epatica e dalla stenosi dello shunt . selezione dei pazienti dallintroduzione della tips nella pratica clinica , sono state avanzate diverse teorie sullutilizzo di tale metodica . 
dopo tips la scomparsa immediata del sanguinamento riportata nel 73%96% dei casi [ 6 ] , con una percentuale di ricorrenza del sanguinamento ad un anno del 15%20% [ 5 ]  . la tips particolarmente efficace quando il sanguinamento origina da varici intestinali inaccessibili o da varici gastriche o quando il risultato di una severa gastropatia conseguente ad ipertensione portale [ 3 ]  . 
sia la scleroterapia che la terapia farmacologica con - bloccanti sono ben tollerate ed efficaci nel prevenire il sanguinamento ricorrente da varici ma i pazienti che hanno sviluppato un nuovo sanguinamento dopo terapia medica o endoscopica sono da ritenersi candidati alla tips [ 3 , 8 ]  . 
refractory ascites is defined as recurrent tense ascites that cannot be improved by routine medical care , including sodium restriction and diuretics , or that requires frequent paracenteses [ 6 ]  . 
ascites can represent , also , a technical problem because it pushes the liver up and makes the angle between the hepatic and the pvs more acute and more difficult for the needle catheter to traverse . 
the effect of tips in these patients is associated with a marked improvement of sodium and water handling of renal perfusion with a general clinical improvement [ 8 ]  . 
tips has been used successfully in the treatment of hepatic hydrothorax ; this is secondary to refractory ascites due to migration of ascitic fluid into the thoracic cavity along a pressure gradient . 
budd - chiari syndrome ( bcs ) results from obstruction of the hepatic venous outflow tract due to occlusion of hvs and / or inferior vena cava ( ivc )  . tips decreases portal congestion by creating a new hepatic venous outflow tract and improving hepatic blood flow with an increase of arterial blood supply to the liver . technically , tips placement in bcs is frequently challenging due to difficulties in identifying an hv as take - off point for the shunt . 
si definisce ascite refrattaria una ascite tesa che non pu essere migliorata attraverso i comuni accorgimenti medici compresi la restrizione sodica ed i diuretici , o una ascite che richiede frequenti paracentesi [ 6 ]  . 
lascite pu anche rappresentare un problema tecnico durante lesecuzione della tips perch spinge il fegato in alto e crea quindi un angolo pi acuto tra la vena porta e la vena epatica , che rende pi difficoltoso il passaggio dellago . 
per questo , nei pazienti con severa ascite consigliabile effettuare una paracentesi evacuativa , subito prima della procedura , per facilitare il successo tecnico . leffetto della tips in questi pazienti associato ad un marcato miglioramento della ritenzione di sodio e acqua , della perfusione renale , con un miglioramento clinico generale [ 8 ]  . 
la tips in grado di ridurre il gradiente pressorio porto - sistemico del 46%63% , con una risposta al trattamento che varia dal 50% al 92% [ 6 ]  . 
on the other hand , encephalopathy precipitated by acute , uncontrolled haemorrhage may be improved when tips is used to treat active variceal haemorrhage refractory to conventional therapy [ 3 , 6 ]  . 
 tips procedure tips is a multistep procedure that includes four key points : jugular - vein puncture , hepatic - vein incannulation , portalvein puncture and stent selection and position . 
the procedures were performed with the patient under deep sedation using a laryngeal mask with a mixture of o2 , n2o , and isoflurane ( 1%2% ) [ 12 ]  . jugular vein puncture the right internal jugular vein ( jv ) is the preferred access because the path to the liver is straight , with minimal angulation controindicazioni assolute 1 . 
il posizionamento della tips determina dei cambiamenti fisiologici anche sui pazienti in buone condizioni cliniche . la creazione con la tips di un canale intraepatico a bassa resistenza causa un flusso preferenziale attraverso lo stent diretto dalle varici e dai sinusoidi epatici attraverso lo shunt . 
in casi selezionati , viene eseguita una tc spirale per ottenere una migliore valutazione dellanatomia del fegato e delle patologie associate . procedura la tips una procedura multi - fasica che include quattro fasi : la puntura della vena giugulare , lincannulazione della vena epatica , la puntura della vena porta , la selezione dello stent e il suo posizionamento . 
needle guidance with real - time sonography is a very simple method that can be used in difficult cases and can also reduce the risk of haematoma or adverse carotid artery puncture . 
an 18 - gauge needle is directed towards the ipsilateral nipple and approximately to the medial third of the clavicle , performing a single - wall puncture with gentle aspiration . 
a 12 - f introducer sheath ( 35to 40 - cm long , daig , minnetonka , mn , usa ) is advanced into the right atrium for pressure measurement and then into the ivc . 
the hv is then catheterised directly using the curved metallic cannula of the tips set ( angiodynamics , queensberry , ny , usa ) and the glidewire ( terumo )  . 
a venogram is then performed in order to visualise the morphology of the hepatic vegenerally , we prefer to create the shunt from the right hv to the right portal branch . 
however , a short , straight path is preferred because of less trauma to the liver parenchyma . portal - vein puncture this is the most difficult and critical step of the tips procedure and requires special attention to avoid the risk of intraperitoneal haemorrhage secondary to liver capsule perforation . 
the puncture is generally performed through the wall of the hv 13 cm from its origonce the needle is into the hv , it is turned anteriorly and advanced into the liver parenchyma for 45 cit is then slowly retracted while performing a gentle aspiration with a 10 - cc syringe . 
many methods have been proposed to locate the pv : arterial portography , ct , us , and wedge hepatic portal venography with contrast media or with co2 [ 14 , 15 ]  . 
in our experience , a blind puncture was performed in 101 / 105 patients while in four cases ( 3.8% ) , us guidance was necessary to reach the pv . 
we prefer to create the shunt into the right pv 23 cm from its orig however , selection of the left portal branch is sometimes necessary in patients with advance cirrhosis because atrophy of the right hepatic lobe is f . 
il giorno prima della procedura deve essere intrapreso un trattamento antibiotico ev ( ceftriaxone , rocefin , hoffmann - la roche , kaiseraugust , ch ) e continuato per 48 ore . puntura della vena giugulare la principale via di accesso rappresentato dalla vena giugulare interna destra in modo che lago possa effettuare un percorso pi rettilineo possibile durante la puntura della vena porta . 
la vena giugulare interna destra localizzata 0 , 5 cm lateralmente allarteria carotide comune . la puntura viene praticata di solito con tecnica alla cieca , pungendo allapice dellangolo formato dai due capi del muscolo sternoscleidomastoideo . 
quando la giugulare interna destra non accessibile per agenesia , occlusione o legatura chirurgica , devono essere prese in considerazione altre vie di accesso quali la vena giugulare esterna destra , la vena giugulare sinistra o la vena succlavia . una volta punta la vena giugulare , una guida angolata idrofilica 0 , 035 ( terumo co , tokyo , giappone ) viene avanzata allinterno della giugulare fino alla vena cava inferiore . a questo livello la guida deve essere manipolata con cautela onde evitare la comparsa di extrasistoli . 
un introduttore da 12 f ( 3540 cm di lunghezza , daig co , minnesota , usa ) viene avanzato fino allatrio destro per effettuare la misurazione della pressione venosa centrale . 
la vena epatica viene cateterizzata direttamente usando la canula metallica curva del set tips ( angiodynamics inc , queensberry , ny , usa ) e la guida idrofilica ( terumo co , tokyo , giappone )  . 
tuttavia sempre preferibile praticare un tramite corto e diretto cos da ridurre il trauma del parenchima epatico . puntura della vena porta rappresenta la fase pi delicata di tutta la procedura e richiede unattenzione particolare per evitare il rischio di emorragia intraperitoneale secondaria ad uneventuale perforazione della capsula epatica . 
1 puntura della vena porta : dopo lincannulazione della branca principale della vena porta si esegue una flebografia per valutare il sito di puntura e la morfologia della vena porta . 
una volta avanzato lago allinterno della vena epatica , questo viene ruotato anteriormente e avanzato attraverso il parenchima epatico per 45 cm . lago viene quindi lentamente retratto praticando una leggera aspirazione con una siringa da 10 ml . 
sono stati proposti molti metodi per localizzare la vena porta : portografia arteriosa , tc , ecd , venografia portale epatica con mezzo di contrasto o co2 [ 14 , 15 ]  . nella nostra esperienza una puntura alla cieca stata praticata in 101 / 105 pazienti mentre in 4 casi ( 3 , 8% ) stato necessario utilizzare una guida ecografica per individuare la vena porta . 
per evitare complicanze , noi preferiamo creare lo shunt nel ramo destro della vena porta , a 23 cm dalla sua origine . la selezione del ramo sinistro della porta necessaria talvolta nei pazienti con cirrosi avanzata perch latrofia del lobo epatico di destra associato ad una posizione pi mediale del ramo destro che non pu essere raggiunto attraverso la vena epatica di destra . 
dato che il catetere percorre generalmente il tratto pi corto dello shunt , consigliabile selezionare uno stent circa 1 cm pi lungo rispetto alla misura ottenuta con il catetere centimetrato . successivamente , una guida super - stiff ( amplatz , boston scientific co , natick , ma , usa ) inserita nella vena splenica o mesenterica ed il tratto intraepatico viene dilatato con un catetere a palloncino a basso profilo ( wanda , boston scientific co , natick , ma , usa ) delle dimensioni corrispondenti al diametro dello stent . scelta dello stent il diametro dello stent deve essere stabilito in base a vari fattori : gradiente portosistemico , et del paziente , condizioni cliniche , grado di encefalopatia , funzionalit cardiaca . 
 afterwards , a super - stiff guidewire ( amplatz , boston scientific , natick , ma , usa ) is inserted into the splenic or mesenteric vein , and the intrahepatic tract is dilated using a low - profile balloon ( wanda , boston scientific ) of the size corresponding to the stent diameter or , in sicker patients , only to 8 mm . stent - graft selection the diameter of the stent must be tailored to the patients portosystemic gradient , age , clinical condition , preexisting encephalopathy , cardiac function and indication . 
following our experience , in older patients with reduced liver and cardiac functions , a small - calibre stent graft ( 8 mm ) can be used to decrease the incidence of encephalopathy . stent - graft design the viatorr stent graft consists of a self - expanding nitinol fig . 
3 misurazione del tratto intra - epatico : per ottenere la corretta lunghezza del tratto intra - epatico dello shunt viene avanzato in vena porta un catetere centimetrato pig - tail . 
la superficie esterna del rivestimento composta da una pellicola in eptfe modificato , a ridotta permeabilit per bile e mucina , che inibisce la crescita iperplastica di tessuto e permette un rinforzo radiale della superficie interna . 
la superficie interna a contatto con il sangue composta da e - ptfe con propriet meccaniche e microstruttura simile a quella degli stent vascolari prodotti con gore - tex . il viatorr ha un design costituito da una parte rivestita in e - ptfe per il tratto intraepatico e da una porzione scoperta , lunga 2 cm , per la regione portale ; disponibile in commercio in differenti diametri ( 8 , 10 , 12 mm ) e in differenti lunghezze ( 6 , 7 , 8 cm )  . 
in pi un altro piccolo marker in oro incorporato nellestremit prossimale della porzione ricoperta per migliorare limmagine fluoroscopica durante il posizionamento del viatorr . rilascio dello stent il viatorr ( wl gore e associati , flagstaff , az , usa ) rappresenta a tuttoggi lunico stent ricoperto disponibile sul mercato specificatamente progettato per la tips . 
un introduttore da 12 f deve essere avanzato nel sistema portale per almeno 3 c una volta avanzato lo stent al suo interno , fino allestremo distale , in porta , lintroduttore viene retratto delicatamente per permettere il corretto rilascio della porzione non rivestita allinterno della vena porta . va sottolineato che non possibile riposizionare il segmento scoperto della protesi in caso di rilascio non corretto . 
una volta confermato il corretto posizionamento della porzione non ricoperta allinterno della vena porta , lintroduttore viene retratto fino allestremo prossimale della protesi , in corrispondenza della giunzione tra vena epatica e vena cava inferiore , e , tenendo fermo il sistema , la porzione ricoperta viene rilasciata tirando lapposito filo . la protesi viene poi dilatata con il pallone per angioplastica utilizzato precedentemente per la dilatazione del tramite intraepetico . successivamente , viene effettuata una venografia per valutare il corretto funzionamento dello shunt . 
4a - d deployment of the viatorr stent graonce the introducer is advanced into the portal vein ( pv ) for at least 3 cm , the uncovered portion of the stent graft should be deployed by retrieving the introducer ( a )  . 
the device is gently retracted until resistance is felt , thus indicating that the proximal portion of the bare stent has reached the junction of the pv with the intrahepatic tract ( b )  . 
4a - d rilascio dello stent viatorr : una volta che lintroduttore stato avanzato nella vena porta , per almeno 3 cm , la parte non ricoperta dello stent pu essere rilasciata retraendo lintroduttore ( a )  . 
tutto il dispositivo viene retratto delicatamente fino a che non si avverte una resistenza , questa indica che la porzione prossimale del tratto non ricoperto dello stent ha raggiunto la giunzione tra vena porta e tratto intraepatico ( b )  . 
the outer surface of the tube is composed of a modified e - ptfe film that minimises transmural permeation of bile and mucin to inhibit hyperplastic tissue ingrowth and provide radial reinforcement to the inner surface . 
the inner blood - contacting surface is made of e - ptfe and has a microstructure and mechanical properties similar to vascular stent grafts manufactured with gore - tex . the viatorr presents a characteristic structural design composed of an uncovered portion 2 - cm in length for the portal region and an e - ptfe - covered portion for the intrahepatic tract , available in different diameters ( 8 , 10 , 12 mm ) and different lengths ( 48 mm )  . 
dopo la procedura , nei pazienti con parametri della coagulazione normali , per ridurre lincidenza di occlusione dello shunt viene prescritta una terapia anticoagulante con 12500 ui di eparina in 500 ml di soluzione fisiologica per le prime 24 ore e successivamente i pazienti vengono sottoposti a terapia antiagregions . 
the stent graft is mounted on a very low profile 10 fr device where the covered portion is secured with a constraining e - ptfe sleeve while the self - expandable uncovered part is secured with an introducer sleeve . stent - graft deployment deployment of the viatorr can be divided in two parts : 1 . 
the 12 - f introducer sheath should be advanced into the portal system for at least 3 cm and then gently retracted to permit release of the uncovered portion within the pv . 
all patients underwent postimplantation therapy with 12 , 500 ui of heparin diluted in 500 ml of saline for the first 24 h , and thereafter 0.5 mg of low - molecular - weight heparin are administered twice a day for at least 1 week . technical and periprocedural complications the most frequent complications that can occur during tips procedure are represented by [ 11 ] : neck haematoma for inadvertent carotid artery puncture ( 1% ) f . 
i fattori associati ad una prognosi negativa dopo il posizionamento della tips per sanguinamento di varici comprendono liponatremia , lencefalopatia , la cirrosi child c e una procedura urgente di tips per ascite refrattaria , comprendente littero e linsufficienza renale [ 17 ]  . 
le maggiori complicanze che possono accorrere pi frequentemente dopo la tips sono rappresentate dallencefalopatia epatica e dellinsufficienza dello shunt [ 17 ]  . encefalopatia epatica lencefalopatia epatica si manifesta nel 15%30% [ 18 , 19 ] dei pazienti dopo tips effettuata utilizzando stent non ricoperti , mentre con lutilizzo degli stent ricoperti tale incidenza arriva al 47% [ 20 ]  . 
sono stati identificati diversi fattori predittivi , inclusa una storia precedente di encefalopatia epatica , una patologia epatica non alcolica , let maggiore di 60 anni , il diametro dello shunt . 
unanalisi comparativa retrospettiva dei pazienti trattati con stent non ricoperti e dei pazienti trattati con viatorr , ha evidenziato una maggiore perviet a lungo termine dello stent ricoperto con ridotta iperplasia intimale , associata per ad una maggiore incidenza di encefalopatia ( 47 , 7% )  . insufficienza dello stent pneumothorax following too low a puncture site ( < 1% ) hepatic capsula laceration during pv puncture linsufficienza dello shunt pu essere secondaria a stenosi o ad occlusione dello stent dovuta ad iperplasia intimale con f . 
5 lo stent viatorr costituito da due porzioni differenti separate da un anello doro : una parte non ricoperta lunga 2 cm , per la porzione portale dello shunt e una parte ricoperta in e - ptfe per il tratto intraepatico . hepatic - artery puncture secondary to a too anterior direction of the needle ( 1% ) bile - duct puncture ( 10% ) extrahepatic puncture of the pv ( 2% ) thirty - day mortality rates after tips placement range from 3% to 44% , and 1 - year mortality rates range from 10% to 58% [ 17 ]  . 
factors associated with a poor prognosis after tips placement for variceal bleeding include hyponatremia , encephalopathy , childs class c cirrhosis , and urgent tips placement for refractory ascites , including jaundice and renal insufficiency [ 17 ]  . 
the two major complications that can occur more frequently after tips procedure are represented by he and shunt insufficiency [ 17 ]  . hepatic encephalopathy hepatic encephalopathy ( he ) is present in the 15%30% [ 18 , 19 ] of patients after tips performed with conventional bare stents ; using a covered stent , a higher incidence is generally reported ( 47% ) [ 20 ]  . 
in case of severe encephalopathy refractory to conventional medical therapy , a shunt reduction should be performed using a covered stent ( jostent , abbot vascular , rangendingen , germany ) released inside the viatorr with an hourglass shape . 
6 la riduzione della tips : in caso di encefalopatia , refrattaria alla comune terapia medica , necessario ricorrere alla riduzione dello shunt mediante lutilizzo di uno stent ricoperto ( jostent , abbot vascular , rangendingen , germany ) , opportunamente conformato a clessidra , da rilasciare allinterno del viatorr . 
dopo il rilascio dello stent si osserva un aumento immediato del psg con miglioramento delle condizioni cliniche . riduzione del lume dello shunt , che favorisce il ripristino dellipertensione portale ed il sanguinamento da varici . come riportato in letteratura , gli stent non ricoperti presentano una perviet primaria del 40%50% ad un anno e del 15%30% a due anni [ 6 , 21 , 22 ]  . 
tale problema pu richiedere ripetute revisioni della tips , che prolungano certamente la perviet dello shunt , ma incrementano notevolmente il costo della procedura , sottoponendo inoltre il paziente a maggiori complicanze . 
per superare questa importante complicanza , che aveva fatto ridurre il numero delle procedure per anno , molti autori hanno sperimentato lutilizzo di stent ricoperti fatti in casa , prima negli animali e poi nelluomo . 
molti materiali sono stati utilizzati per ricoprire lo stent , come il politetrafluoroetilene ( ptfe ) , il polietilene tereftalato ( pte , commercialemente conosciuto come dacron ) , e poliuretanolo ( pu )  . 
i risultati dei tentativi con dacron e pu non sono stati molto soddisfacenti per lalta incidenza di occlusione precoce , ma gli esperimenti eseguiti con il ptfe hanno evidenziato una alta percentuale di perviet primaria a 12 mesi [ 23 , 24 ]  . valutazione post - procedurale del paziente il successo della tips correlato alla perviet dello shunt . shunt insufficiency shunt insufficiency is secondary to stent stenosis or occlusion . 
this is due to the almost constant presence of progressive increase of pseudointimal hyperplasia , which causes reduction of the shunt lumen , thus favouring return of portal hypertension with recurrent variceal bleeding . 
as reported in the literature , using bare stents , primary patency rates are almost constant at approximately 40%50% at 1 year and 15%30% at 2 years [ 6 , 21 , 22 ]  . the problem can be partially resolved with repeated tips revisions , which certainly prolong stent patency , but they are very expensive and require extended patient compliance . 
various materials have been used to cover the stent , such as ptfe , polyethylene terephthalate ( pte , commercially known as dacron ) , and polyurethanol ( pu )  . 
results of attempts with dacron and pu were not very good because of the high incidence of early occlusions , but attempts made using ptfe were very promising , with a high primary patency rate at 12 months [ 23 , 24 ] postprocedure patient evaluation tips success is correlate with shunt patency . 
shunt function can be assessed by cdus , angiography with pressure measurement or spiral ct angiography ( cta ) [ 11 ]  . cdus is a noninvasive , inexpensive procedure that can be easily performed in intervals of 36 months , with sensitivity of 53%100% and specificity of 62%98% [ 6 , 22 ]  . 
however , this procedure is time consuming , difficult to perform and highly operator dependent [ 22 ]  . angiography with measurement of portal - venous pressure gradient represent the gold standard in the diagnosis of shunt failure . 
however , ionising radiation and relatively high doses of intravascular contrast media can represent a limitation of cta . our experience from january 2000 , 105 consecutive patients with a mean f . 
la funzionalit dello shunt pu essere valutata con eco - color doppler ( ecd ) , angiografia con misurazione della pressione o angio - tc spirale [ 11 ]  . 
lecd una metodica non invasiva , non costosa , che pu essere facilmente praticata con intervalli di 3 e 6 mesi , con una sensibilit del 53%100% e specificit del 62%98% [ 6 , 22 ]  . 
in un recente studio pubblicato da chopra [ 25 ] stata riportata una sensibilit di questa metodica del 97% , una specificit dell89% e unaccuratezza del 94% . tuttavia lutilizzo delle radiazioni ionizzanti e la relativa alta dose di mezzo di contrasto endovenoso possono rappresentare delle limitazione allutilizzo della tc durante il follow - up . la nostra esperienza da gennaio 2000 , 105 pazienti con unet media di 56 , 513 , 8 anni ( range 1675 anni ) sono stati trattati con uno stent viatorr . 
le indicazioni a tale trattamento erano sanguinamento ( n = 56 ) , asciti refrattarie ( n = 44 ) e sindrome budd - chiari ( n = 5 )  . limpianto dello stent viatorr ha avuto successo in tutti i pazienti , con una immediata rifuzione della psg da 23 , 36 , 5 mmhg ( range : 1342 mmhg ) a 7 , 133 , 1 mmhg ( range : 216 mmhg )  . in 4 pazienti ( 3 , 8% ) , un sanguinamento intra - addominale a seguito della puntura della vena porta stato immediatamente arrestato tramite rilascio dello stent . 
in un paziente trattato per sanguinamento delle varici , lo stent viatorr stato impiantato mediante puntura della vena porta sinistra . una settimana dopo limpianto si registrata una ostruzione del dotto biliare sinistro con aumento del livello di billirubina , per compressione causata dallo stent . i sintomi si sono risolti completamente dopo impianto di una endoprotesi biliare ; il paziente tuttavia deceduto dopo 19 mesi . 
three - dimensional ( 3d ) images obtained with maximum intensity projection ( mip ) and vr ( volume - rendering ) algorithms performed at 12 months follow - up showed complete patency of the stent graft , with no signs of intimal hyperplasia . 
 in four patients ( 3.8% ) , extrahepatic pv puncture with intraabdominal extravasation of contrast media was treated with the rapid deployment of the viatorr stent graft , with immediate arrest of intraabdominal bleeding . 
the patient was treated with implantation of a plastic biliary endoprosthesis , and symptoms resolved completely until the patients death 19 months later . after a mean follow - up of 23.89.3 ( range 150 ) months , 72 / 105 ( 68.6% ) patients were in good condition with a patent shunt ; 22 / 105 ( 20.9% ) had died from multiorgan failure while 11 / 105 ( 10.5% ) underwent orthotopic liver transplantation olt without procedural difficulties . 
twelve reinterventions were performed for implantation of too short a viatorr not completely covering the intraparenchymal tract ( n = 7 ) , pv stenosis ( n = 1 ) , hv stenosis ( n = 2 ) and increase of psg value with no evidence of shunt stenosis ( n = 2 )  . 
 the 1 - year primary patency rate was 83.8% , with a secpatency sufficienza sistemica , mentre 11 / 105 pazienti ( 10 , 5% ) sono stati sottoposti a trapianto di fegato senza complicazioni procedurali . 
nove dei 105 patients ( 8 , 6% ) sono stati sottoposti a reimpianto dello shunt a causa di malfunzionamento rilevato tramite ecd , endoscopia o evidenza clinica ( presenza di varici o asciti )  . 
in 7 pazienti stata necessaria una revisione , in un paziente sono state necessarie 2 revisioni e 3 revisioni in un ultimo paziente . dodici reinterventi sono stati effettuati a causa di impianti di stent viatorr troppo corti e non in grado di coprire completamente il tratto intraparenchimale ( n = 7 ) , stenosi della vena porta ( n = 1 ) , stenosi della vena epatica ( n = 2 ) e incremento del psg senza evidenza di stenosi dello shunt ( n = 2 )  . 
se , tuttavia , calcoliamo separatamente la percentuale di perviet per quei pazienti con uno stent della lunghezza adeguata otteniamo una perviet primaria ad 1 anno del 90 , 8% , mentre per il gruppo di pazienti con uno stent troppo corto la percentuale di perviet primaria era 79 , 5% . 
 encefalopatia nella nostra esperienza , durante un periodo di follow - up medio di 23 , 89 , 3 mesi , 49 / 105 pazienti ( 46 , 6% ) trattati con uno stent ptfe ( viatorr ) hanno sviluppato encefalopatia epatica . 
in 13 pazienti , lencefalopatia stata considerata di grado moderato ( grado iii ) e in 12 di grado severo ( grado iiiiv )  . dodici pazienti con encefalopatia sono stati ricoverati . ondary patency rate of 98.1%. 
if , however , we calculate separately the patency rate for those patients with a stent graft of the proper length , we obtain a 1 - year primary patency rate of 90.8% while in the group with too short a stent graft , the primary patency rate was only 79.5%. 
 encephalopathy in our experience , during a mean follow - up of 23.89.3 months , 49 / 105 patients ( 46.6 % ) treated with an e - ptfecovered stent graft ( viatorr ) developed he . 
however , statistical analysis showed no significant correlation between the incidence of encephalopathy , psg values and stent diameter ( ttest , p = 0.27 ; chi square test , p = 0.88 ; with identical values for logistic regression analysis )  . in 37 of 49 patients , encephalopathy was successfully treated medically with lactulose and dietary restriction . 
shunt reduction was required in the remaining 12 cases ( 24.4% ) of severe encephalopathy refractory to medical treatment after a mean period of 6.3 months ( range 6 days to 37 months )  . 
an immediate increase of psg value was observed in all cases , and up to the time of writing of this article , 8 / 12 patients ( 66.6% ) were alive and in good health while three had died . only in one case ( 8.3% ) was redilatation of the reduced shunt performed for rebleeding . orthotopic liver transplantation in our series , 11 patients ( 10.5% ) underwent olt without procedural difficulties . 
an initial belief that longer stents would cause difficulty during surgery was later discarded because the covered portion of the stent graft was not attached to the liver parenchyma and easily displaced for clamping . 
histopathologic analyses of explanted viatorrs showed patent stent grafts lined internally with a thin layer of fibrno signs of endothelialisation or intimal hyperplasia were observed in the covered portion of the viatorrs , and the stents were easily removed from the liver parenchyma . 
una riduzione dello shunt stata necessaria nei rimanenti 12 casi ( 24 , 4% ) che presentavano encefalopatia di grado severo refrattaria a terapia medica dopo 6 , 3 mesi ( range : 6 giorni37 mesi )  . 
in tutti i casi stato osservato un immediato incremento del valore del psg e , al momento in cui questo articolo stato redatto , 8 / 12 pazienti ( 66 , 6% ) erano vivi e in buone condizioni di salute , mentre 3 erano deceduti . 
solo in un caso ( 8 , 3% ) stata necessaria una ri - dilatazione dello shunt a causa di un nuovo episodio di sanguinamento . trapianto di fegato nella nostra casistica , 11 pazienti ( 10 , 5% ) sono stati sottoposti a trapianto di fegato senza complicazioni procedurali . contrariamente a quanto si era creduto inizialmente , gli stent lunghi non sono stati causa di complicazioni chirurgiche , perch la porzione ricoperta dello stent non era adesa al parenchima epatico e pertanto stata facilmente rimossa . lanalisi istopatologica ha mostrato la perviet degli stent viatorr espiantati che erano rivestiti internamente da un sottile strato di fibrina . non sono stati osservati segni di endotelializzazione o iperplasia intimale nella porzione ricoperta degli stent viatorr e gli stent sono stati facilmente rimossi dal parenchima epatico . 
la porzione di stent non ricoperta era invece strettamente adesa alla parete della vena porta e solo in un caso ( 9 , 1% ) risultata troppo lunga e , attraversando la linea di sezione della vena porta , ha complicato la procedura . mortalit a breve termine durante il periodo di follow - up , la mortalit a 30 giorni risultata del 3 , 8% ( 4 / 105 pazienti ) : un paziente deceduto per insufficienza cardiovascolare , uno per sindrome epato - renale e gli altri per insufficienza sistemica . mortalit a lungo termine escludendo gli 11 pazienti sottoposti a trapianto , abbiamo registrato una percentuale di sopravvivenza del 76 , 6% ( 38 / 94 ) e una mortalit del 23 , 4% ( 22 / 94 pazienti ) ad un periodo medio di follow - up di 23 , 82 mesi . 
 excluding the 11 patients who underwent transplantation , we had a survival rate of 76.6% ( 38 / 94 ) and a total mortality of 23.4% ( 22 / 94 patients ) , with a mean of 23.82 months of follow - up . la tips pu essere considerata una modalit particolarmente interessante per il trattamento dellipertensione portale , del sanguinamento da varici , dellascite refrattaria e conclusioni f . 
however , there are still unsolved problems , especially short primary patency of the shunt due to intimal hyperplasia , which causes reduction of the shunt lumen , thus favouring a return of portal hypertension with recurrent variceal bleeding . 
with the use of new ptfe - covered stents , we hope to prolong primary shunt patency and that tips will regain its lost popularity in the treatment of portal hypertension . 
further studies are necessary to compare these two imaging modalities . della sindrome di budd - chiari . sono stati praticati molti studi per valutare lefficacia e il rapporto costo - benificio di questa tecnica in relazione alle altre modalit terapeutiche . 
comunque rimangono ancora alcuni problemi irrisolti , soprattutto per quanto riguarda la breve perviet primaria dello shunt conseguente allo sviluppo delliperplasia intimale , che causa una riduzione del lume dello shunt , favorendo il ripristino dellipertensione portale . con lutilizzo dei nuovi stent ricoperti in ptfe noi auspichiamo di poter prolungare la perviet primaria dello shunt cosicch la tips possa incrementare la sua popolarit per il trattamento dellipertensione portale . lecd rimane la modalit di scelta per il follow - up dei pazienti con tips , mentre langio - tc spirale ritenuta vantaggiosa per la sua elevata sensibilit e specificit . 
an accessory finding devoid of pathological meaning in most cases , it can , however , be a possible cause of voiding disorders in some instances . key words urethra children cowpers ducts and glands riassunto obiettivo . 
due diaframmatiche e due bulbari o accessorie , le ghiandole di cowper sono piccole formazioni lobulate , di tipo tubulo - alveolare composto , disposte ai lati delluretra , tra i foglietti del diaframma urogenitale le prime ; nel corpo spongioso , lungo luretra bulbare g . 
le diaframmatiche ( di cui la destra prevalente sulla sinistra , spesso inesistente ) sono simili ad un pisello , con dimensione di circa 0 , 5 cm , maggiore rispetto a quello delle bulbari , pi piccole . 
i dotti escretori delle ghiandole diaframmatiche hanno lunghezza di circa 3 cm e diametro medio di 0 , 7 mm , attraversano la faccia inferiore del trigono urogenitale e sboccano sulla faccia inferiore delluretra bulbare , mentre quelli delle ghiandole bulbari o accessorie , sottili e brevi , sboccano direttamente nelluretra o si fondono con il dotto escretore principale del lato corrispondente . 
pu essere invece pi frequente in caso di stenosi uretrali o di problemi intrinseci ai dotti stessi che assumono laspetto di dilatazione cistica venendo cos descritti con il termine di siringocele [ 5 , 6 , 1316 ] anche se lo stesso termine non universalmente accettato e considerato causa di confusione [ lebowitz rl ( 2005 ) , cofig . 
diaphragmatic gland ducts are 3 - cm long , with an average diameter of 0.7 mthey run through the inferior side of the urogenital trigonum and open onto the inferior side of the bulbar urethra . 
5a , b dotto di cowper semplice a monte del collare di cobb : a durante la fase minzionale , b alluretrografia . as syringocele [ 5 , 6 , 1316 ] , a definition considered misleading by some authors [ lebowitz rl ( 2005 ) , personal communication ]  . 
lopacizzazione del dotto spesso rapida , transitoria ( per cui importante la ripresa delle immagini con spot - film a cadenza rapida ) e richiede una certa esperienza nella sua dimostrazione ed interpretazione . 
 il dotto di cowper appare come una sottile struttura allungata , a margini netti , parallela al margine inferiore delluretra membranosa / bulbare , che si dirige in senso cefalico risalendo fino al diaframma urogenitale . 
lack of opacification of the corresponding gland can be a source of diagnostic doubt [ 2 , 6 , 20 ]  . the real frequency of cowpers glands and ducts lesions is at present unknown since most of them are asymptomatic , with a reported rate of about 1.5% in paediatric vcu [ 25 ]  . autopsic studies have shown a 2.3% rate of occurrence in males in all age groups [ 26 , 28 ]  . 
a possible association with urethral valves has been reported [ 11 , 13 , 14 ]  . the term syringocele ( from the greek syrinx , tubular formation , and cele , swelling ) was first used by maizels in 1983 [ 13 ] to define the congenital or acquired dilatation of cowpers ducts . 
la reale incidenza delle lesioni dei dotti e delle ghiandole di cowper sconosciuta dal momento che la maggior parte delle stesse asintomatica con unincidenza variabile tra l1 , 5% in corso di esami cisto - uretrografici in et pediatrica [ 25 ] ed il 2 , 3% dei soggetti maschi in tutti i gruppi di et in casi autoptici [ 2628 ]  . 
in tali situazioni si potr avere non solo lopacizzazione del dotto , di calibro superiore a quanto osservabile in condizioni di normalit in funzione della componente ostruttiva , ma anche delle ghiandole . 
il termine siringocele ( da syrinx = formazione tubulare e cele = dilatazione ) stato coniato nel 1983 da maizels per definire la dilatazione , congenita od acquisita , del dotto di cowper [ 13 ]  . 
 [ 14 ] ne stata segnalata una frequenza dello 0 , 065% ( 14 casi in 21.384 pazienti adulti e bambini ) e dello 0 , 18% nella presente ( anche se basata su di una popolazione assai pi ridotta ) , mentre riportion of the duct imperforated or cystic syringocele : the duct orifice is closed causing dilatation of the distal duct , which appears as a filling defect or a mass that compresses the floor of the bulbar urethra , sometimes resulting in urethral stenosis and dilatation of its proximal portion perforated syringocele : the dilated distal duct looks like a diverticulum of the bulbar urethra into which the duct drains through a large opening ; opacification of cowpers glands is possible ruptured syringocele : the bulbar portion of the duct is dilated , like a cyst that has lost connection with its proximal portion ; radiologic images show thin filling defects in the radiolucent bulbar urethra due to remnants of the dilated duct transperineal ultrasound ( us ) in the sagittal and longitudinal scans show an anechoic double - channel tubular structure , the nearest to the probe representing the dilated cowpers duct , the more distal the urethra [ 21 , 22 ]  . 
the most important advantage of mri for patients consists of it being free of ionising radiation ; it is noninvasive and allows multiplanar imaging and precise evaluation of the relationship between the cyst and adjacent tissues and the ventral urethra [ 23 , 24 ]  . 
 [ 14 ] reported the finding in 0.065% of an adult and paediatric population ( 14 cases in 21 , 384 patients )  . this rare occurrence is in accordance with the 0.18% recorded in our population , even if smaller than the previous reports , while according to dewan , it can be found in 1.5% of children [ 25 ]  . 
a possible explanation of the higher percentage of cowpers syringocele in children than in adults may be the greater number of radiological procedures performed to exclude congenital malformations of the urinary tract or vesicoureteric reflux when recurrent urinary tract infections are present . 
considering these facts , it is not easy or even possible to establish clear - cut percentage differences amongst different types of syringocele according to different age groups . results during the past 24 years , 3 , 096 children ( 1 , 676 males , 1 , 420 females , age : newborn to 18 years ) were studied by vcu in our department of paediatric radiology . 
in six of them ( 0.35% ) , aged 6 months to 10 years and undergoing vcu for urinary tract infection , opacification of cowpers ducts appeared as a thin , clear - cut , radiopaque image running parallel to the inferior membranous / bulbar urethral edge . 
la possibile spiegazione del perch il riscontro del siringocele pi elevato nei bambini che nelladulto pu essere determinata dalla frequenza delle indagini eseguite per lesclusione di malformazioni congenite dellapparato urinario o di reflussi vescico - ureterali in caso di infezioni ricorrenti delle vie urinarie nei bambini , mentre nelladulto motivi dello studio sono cause traumatiche o post - infettive [ 2 , 7 ] e perci meno frequenti . 
in vista di ci non facile o possibile stabilire differenze percentuali tra le varie forme in funzione dellet . risultati in 24 anni di attivit della nostra sezione di radiologia pediatrica , sono stati sottoposti a cisto - uretrografia minzionale 3096 bambini ( 1676 maschi , 1420 femmine ; et neonatale18 anni ) per un totale di 4321 indagini di cui 2340 nei maschi e 1981 nelle femmine ed stata osservata in 9 soggetti ( 0 , 53% ) lopacizzazione del dotto di cowper . 
in 6 di questi ( 0 , 35% ) , di et variabile tra i 6 mesi ed i 10 anni , sottoposti ad indagine per infezione delle vie urinarie , si osservata opacizzazione del dotto sotto forma di sottile immagine radiopaca a margini netti , parallela al margine inferiore delluretra membranosa / bulbare . 
la visualizzazione , in proiezione obliqua o laterale , di una immagine canalicolare grossolanamente parallela alluretra , e labituale assenza di opacizzazione del corpo ghiandolare , possono rendere difficile lattribuzione della stessa al dotto della ghiandola di cowper . 
le principali diagnosi differenziali in caso di dubbio sono rappresentate da tragitti fistolosi uretrali , dai margini irregolari e con tendenza espansiva ; da spandimenti e stravasi di mezzo di contrasto ; da false immagini [ 6 ] , da duplicazioni e sovrapposizioni di immagini [ 2 , 14 ] nonch da diverticoli delluretra bulbare , di forma ovalare , dai contorni sottili e netti , ma g . 
one of the two other patients had been treated in the neonatal period due to the presence of posterior urethral valves : at the age of 4 years , he presented haematuria and urinary tract infection ; the second one was admitted at the age of 14 years , being affected by distal urethral stenosis . 
in utero si manifesta sotto forma di ostruzione transitoria o meno della vescica le cui pareti possono essere ispessite con associata idronefrosi quando lostruzione uretrale provocata da un dotto ostruito [ 28 ] ; nel neonato sono frequenti forme francamente ostruttive , con alterazioni del mitto , gocciolamento di urina , an incidental finding during urologic radiological procedures in children and adults , and such finding should not lead to incorrect diagnoses . 
a typical finding in the rare cases of duct opacification due to contrast reflux secondary to stenosis of the distal urethra is an image of the excretory duct and its corresponding glandular structure [ 6 ] , which is easily differentiated from the prostatic canal or from an abnormal course . 
syringocele can cause hydronephrosis in utero [ 28 ] ; in the newborn , there may be frank obstruction often followed by renal insufficiency , as is the case in the presence of posterior urethral valves [ 11 , 15 ]  . 
perforated and ruptured syringocele very often traumatic or postinfectious may rarely lead to dysuria due to local irritative phenomena , haematuria probably due to traumatic local lesions during micturition , posturinary incontinence and urinary tract infections due to bacterial colonisation of mucus and urine stagnating in the syringocele [ 15 16 , 19 , 28 ]  . 
treatment comprises symptomatic medical treatment and , in the case of obstructive syringoceles , endoscopic unroofing as first - choice option or open surgery [ 11 , 15 , 19 ]  . 
9 diverticolo delluretra bulbare in paziente sottoposto a ripetuti interventi di correzione di ipospadia di alto grado . globo vescicale , dilatazioni del tratto urinario superiore e , spesso , insufficienza renale : nel complesso , un quadro assimilabile a quello osservabile in presenza di valvole uretrali [ 11 , 15 ]  . 
nelle altre forme di siringocele , quasi sempre di natura traumatica o post - infiammatoria , sono possibili disuria , attribuibile a fenomeni irritativi locali , ematuria , presumibilmente secondaria a lesione traumatica della fragile parete del siringocele durante la minzione , infezioni urinarie , conseguenti a colonizzazione batterica di muco ed urina ristagnanti nel contesto del siringocele stesso [ 15 ] , piuria ed incontinenza urinaria post - minzionale [ 16 , 19 , 28 ]  . 
sonographic features of 256 histologically confirmed ( 148 benign , 108 malignant ) breast lesions were retrospectively and independently reviewed by three radiologists unaware of mammographic findings and pathology results . 
analysis of the sonographic features predictive of malignant disease , taken as a whole , showed that only irregular margins and marked hypoechogenicity maintain their predictive value independent of lesion size . 
i segni ecografici relativi a 256 lesioni mammarie con verifica istologica ( 148 benigne , 108 maligne ) , sono stati analizzati retrospettivamente e indipendentemente da tre radiologi , che non erano a conoscenza n della mammografia n dei risultati istologici relativi alla lesione in esame . 
i criteri normalmente utilizzati in ecografia a questo scopo presentato una accuratezza significativamente ridotta nella caratterizzazione delle lesioni di piccole dimensioni . parole chiave mammella ecografia segni ecografici carcinoma mammario c . 
with the recent advances in technology and standardisation of sonographic findings , breast us has improved significantly and is now used to characterise solid masses through analysis of their features [ 1 ]  . the remarkable technical improvements of us instrumentation compared with the machines used in the early studies on breast sonography have significantly increased sensitivity and specificity of this diagnostic tool [ 26 ]  . 
the current importance of breast us in characterising breast lesions has also been the result of standardisation of examination technique and evaluation of sonographic findings that may be useful in differentiating benign from malignant lesions . 
the authors compared sonographic findings of 750 solid breast lesions classified by the radiologist as benign , malignant or indeterminate with histological findings , and they analysed the frequency of each finding and its positive and negative predictive values . 
the reproducibility of their findings in routine clinical practice has since been evaluated by several authors [ 810 ] who considered the different us equipment , experience of sonographers and interobserver variability in the classification of findings to show how all these elements affect the final result . 
the purpose of this study was to retrospectively evaluate the accuracy of breast us alone in characterising solid lesions and identify sonographic features that may be predictive of benign or malignant disease . 
we also evaluated interobserver variability in classifying the lesions and the influence of lesion size on the above aspects . materials and methods selection of cases sonographic images of 256 consecutive solid breast lesions , which underwent us - guided core biopsy , were retrospectively examined . 
the reasons for biopsy were : breast imaging reporting and data systems ( birads ) 4 and 5 lesions , birads 2 and 3 lesions in patients with a previous history of breast carcinoma , strong family history of breast carcinoma or highly anxious patients . 
negli anni settanta ed ottanta questa tecnica era utilizzata esclusivamente allo scopo di differenziare le opacit a margini ben definiti , identificate alla mammografia , distinguendo le lesioni cistiche dalle lesioni solide . 
nei recenti anni passati , grazie agli avanzamenti nella tecnologia e alla standardizzazione dei segni ecografici , lecografia mammaria migliorata significativamente , essendo attualmente utilizzata anche nella caratterizzazione delle lesioni grazie allanalisi degli aspetti ecografici [ 1 ]  . i notevoli avanzamenti tecnici ottenuti negli anni recenti per quanto concerne le apparecchiature ecografiche , in confronto con gli ecografi utilizzati nei primi studi eseguiti mediante ecografia mammaria , ha permesso di ottenere un incremento significativo di sensibilit e specificit di questa metodica [ 26 ]  . 
nondimeno , il ruolo fondamentale recentemente ottenuto dallecografia mammaria nella caratterizzazione delle lesioni mammarie , stato ottenuto anche grazie alla standardizzazione dei metodi di esame e alla valutazione dei segni ecografici , che risultano utili nel differenziale le lesioni benigne dalle lesioni maligne . 
gli autori hanno comparato i segni ecografici relativi a 750 lesioni solide mammarie , che erano state classificate dai radiologi come maligne , benigne o indeterminate , correlandoli con listologia e analizzando la frequenza di ogni segno ecografico valutandone il valore predittivo positivo e negativo . 
 [ 7 ] , utilizzando il loro sistema di classificazione , hanno ottenuto valori di sensibilit , specificit , accuratezza , valore predittivo positivo e negativo rispettivamente 98 , 4% , 67 , 8% , 72 , 9% , 38% e 99 , 5% . 
nella routine clinica , considerando differenti apparecchiature , lesperienza degli ecografisti e la variabilit interosservatore nella classificazione dei segni ecografici , dimostrando come tutti questi elementi influenzino il risultato finale . per quanto a noi noto , linfluenza della dimensione delle lesioni nella valutazione di questi segni ecografici utili nella differenziazione tra lesioni benigne e maligne , non stata ancora valutata . 
lobiettivo di questo lavoro stato quello di valutare laccuratezza dellecografia mammaria nella caratterizzazione delle lesioni solide mammarie identificando i segni ecografici potenzialmente utili nella diagnosi di lesione benigna o maligna . 
surgical biopsy was performed in 2% of benign lesions because of patient anxiety or recommendation by the pathologist ; in these cases , the diagnosis of benign lesion was always confirmed . 
all other benign lesions were unchanged at follow - up after at least 24 months . evaluation of sonographic images three radiologists experienced in breast imaging ( 4 , 5 and 8 years of practice , respectively ) retrospectively and independently evaluated sonographic images of the 256 selected cases . 
the radiologists assessed each lesion for shape ( round , ellipsoid , irregular ) , margins ( regular , ill - defined , microlobulated , angular , spiculated , with fewer or more than three well - defined macrolobulations , branch pattern ) , texture ( homogeneous , heterogeneous ) , echogenicity ( mild or marked hypoechogenicity , hyperechogenicity , isoechogenicity , anechogenicity ) , posterior acoustic transmission ( increased , regular , reduced ) and presence or absence of lateral acoustic shadows , pseudocapsule and microcalcification . 
each radiologist expressed a degree of suspicion for each lesion : benign , probably benign , probably malignant and malignant . statistical analysis and study design association between lesion morphological features and histological diagnosis was evaluated using the chi - square test after combining some categories , if necessary . 
finally , we assessed the accuracy of breast us in characterising solid lesions by comparing the degree of radiological suspicion expressed by each reader and the final pathology report . results histological analysis demonstrated that 148 / 256 ( 58% ) lesions were benign and 108 / 256 ( 42% ) were malignant . 
benign lesions were classified as follows : 101 fibroadenomas ( 68% ) , 15 fibrocystic disease ( 10% ) and 32 different benign lesions ( 22% ) such as focal fibrosis , typical ductal hyperplasia , radial scar , sclerosing adenosis , granuloma , lipoma , hamartoma , benign phyllodes tumour , papilloma and adenografiche di 256 consecutive lesioni solide mammarie , sottoposte ad agobiopsia sotto guida ecografica . 
la biopsia chirurgica stata eseguita nel 2% delle lesioni benigne in seguito ad ansiet delle pazienti e a raccomandazione del patologo ; anche in questi casi la diagnosi preliminare dellagobiopsia stata confermata . 
tutte le altre lesioni benigne risultavano invariate al follow - up di ameno 24 mesi . valutazione delle immagini ecografiche tre radiologi , esperti in senologia ( rispettivamente con quattro , cinque e otto anni di esperienza ) , hanno valutato indipendentemente e retrospettivamente , le immagini ecografiche relative ai 256 casi selezionati . 
ogni radiologo ha descritto per ogni lesione la forma ( rotonda , ovalare , irregolare ) , i margini ( regolari , indefiniti , microlobulati , angolati , spiculati , con meno di tre o pi di tre macrolobulazioni , ramificati ) , la tessitura ( omogenea , disomogenea ) , lecogenicit ( ipoecogenicit moderata o marcata , iperecogenicit , isoecogenicit , anecogenicit ) , la trasmissione del segnale acustico posteriore ( aumentata , normale , ridotta ) , e la presenza o assenza delle ombre acustiche laterali , della pseudocapsula e delle microcalcificazioni . 
ogni radiologo ha poi espresso il grado di sospetto relativo ad ogni lesione , classificandole come benigne , probabilmente benigne , probabilmente maligne o maligne . analisi statistica e disegno dello studio lassociazione tra gli aspetti morfologici delle lesioni e la diagnosi istologica stata valutata mediante il test ( cid : 2 ) 2 , dopo aver combinato alcune categorie se ritenuto necessario . 
al fine di analizzare linfluenza della dimensione della lesione sui singoli segni , utili a predire la diagnosi istologica di benignit o malignit , per ogni singolo segno stata identificata la soglia al di sopra della quale esso risulti ancora in grado di esprimec . 
malignant lesions were classified as follows : 85 invasive ductal carcinomas ( 79% ) , eight invasive lobular carcinomas ( 7% ) , two in situ ductal carcinomas ( 2% ) and 13 different neoplastic lesions ( 12% ) such as invasive papillary carcinoma , invasive medullary carcinoma , invasive ductal - lobular carcinoma and mixed in situ invasive carcinoma . 
 the logistic model applied to the features as a whole yielded an odds ratio ( or ) = 2.34 [ 95% confidence interval ( ci ) ( 1.025.40 ) ] for marked hypoechogenicity and an or = 112.29 [ 95% ci ( 37.6334.71 ) ] for irregular margins . impact of lesion size the predictive value of each feature relative to lesion size is shown in table 3 . 
infine , abbiamo calcolato laccuratezza dellecografia mammaria nel caratterizzare le lesioni solide , comparando il sospetto radiologico di ogni lettore con la diagnosi istologica definitiva . risultati lanalisi istologica ha dimostrato che 148 / 256 ( 58% ) lesioni erano benigne , mentre 108 / 256 ( 42% ) lesioni erano maligne . 
le lesioni benigne sono state classificate come di seguito : 101 fibroadenomi ( 68% ) , 15 malattia fibro - cistica ( 10% ) , 32 differenti lesioni benigne ( 22% ) come fibrosi focale , iperplasia duttale tipica , radial scar , adenosi sclerosante , granuloma , lipoma , amartoma , tumore fillode benigno , papilloma , adenoma . 
le lesioni maligne sono state classificate come di seguito : 85 carcinoma duttale infiltrante ( 79% ) , 8 carcinoma lobulare infiltrante ( 7% ) , 2 carcinoma duttale in situ ( 2% ) , e 13 differenti lesioni neoplastiche maligne ( 12% ) quali carcinoma papillare infiltrante , carcinoma midollare infiltrante , carcinoma duttulo - lobulare infiltrante , carcinoma duttale misto invasivo e intraduttale . 
let delle pazienti risulta compresa tra i 18 e gli 88 anni ( et media 53 , 2 anni , mediana 51 anni )  . la dimensione massima delle lesioni risultava compresa tra i 3 ed i 100 mm ( mediana 13 , 79 mm , mediana 12 mm , ds 9 , 09 mm ) ; in particolare , la dimensione delle lesioni benigne era compresa tra i 4 e i 45 mm ( media 13 , 11 mm , mediana 12 mm , ds 6 , 88 mm ) , mentre la dimensione delle lesioni maligne era compresa tra i 3 e i 100 mm ( media 14 , 71 mm , mediana 13 mm , ds 11 , 42 mm )  . la distribuzione dei diversi aspetti ecografici tra lesioni benigne e maligne riportata nella tabella 1 ; peraltro , per lanalisi dei dati stato necessario combinare in categorie alcuni segni ecografici . 
in particolare i segni relativi ai margini sono stati raggruppati come di seguito : margini regolari , o con macrolobulazioni sono stati raggruppati nei margini regolari ; margini indefiniti , microlobulati , angolati , spiculati , ramificati sono stati raggruppati nei margini irregolari . analizzando i dati riportati in tabella 1 relativi alle lesioni benigne , si evince come i segni ecografici pi frequenti per ogni categoria siano la forma ovoidale ( 74 , 3% ) , i margini regolari ( 54 , 7% ) , la moderata ipoecogenicit ( 46 , 6% ) , la presenza di una pseudocapsula ( 67 , 6% ) , una normale trasmissione acustica posteriore ( 62 , 8% ) , e la presenza delle ombre acustiche laterali ( 55 , 4% )  . 
sullaltro versante , per le lesioni maligne , i segni ecografici pi frequenti per ogni categoria sono risultati essere la forma ( 45 , 4% ) , i margini microlobulati ( 43 , 5% ) , la marcata ipoecogenicit ( 75 , 9% ) , e la trasmissione acustica posteriore ridotta ( 53 , 7% )  . 
sia le lesioni benigne che maligne hanno presentato pi frequentemente una tessitura omogenea . il valore di ogni singolo segno , considerato singolarmente , nella predizione della diagnosi istologica di benignit o malignit stato riportato in tabella 2 . 
un assorbimento acustico posteriore , una tessitura disomogenea e la presenza di microcalcificazioni . il modello logistico applicato alla globalit dei segni ecografici ha dimostrato una odds ratio ( or ) = 2 , 34 ( 95% ci ipoecogenicit e una [ 1 , 025 , 40 ] ) per or = 112 , 29 ( 95% ci [ 37 , 67334 , 71 ] ) per i margini irregolari . la marcata influenza delle dimensioni sulla lesione il valore di ogni singolo segno nella predittivit della diagnosi istologica di benignit o malignit , in relazione alle dimensioni delle lesioni , riportato in tabella 3 . 
la presenza di una pseudocapsula risulta associata pi frequentemente con diagnosi di benignit solo per lesioni uguali o maggiori di 7 mconsiderando la trasmissione acustica posteriore normale come riferimento , un assorbimento acustico posteriore in grado di predire la diagnosi di malignit solo per lesioni maggiori o uguali ai 7 mm , mentre un rinforzo acustico posteriore diventa segno di benignit solo per lesioni solide maggiori di 30 mla presenza delle ombre acustiche laterali diventa segno di benignit per lesioni di almeno 6 mm di diametro , mentre una tessitura disomogenea risulta segno predittivo di malignit per lesioni con massimo diametro di almeno 7 mlanalisi relativa alla presenza di microcalcificazioni deve essere interpretata con cautela in relazione al ridotto numero di casi , dal momento che tale reperto risulta difficilmente identificato ecograficamente . 
the lesions measure 3 mm ( a ) and 4 mm ( b ) in diameter , and both present mild hypoechogenicity , ill - defined margins and regular posterior acoustic transmission . 
1a , b vengono messe a confronto le immagini di due lesioni mammarie , riscontrate in due differenti pazienti , del diametro rispettivamente di 3 ( a ) e 4 ( b ) mm , con caratteristiche semeiologiche simili . 
dopo agobiopsia percutanea , la lesione in aa risultata essere un carcinoma duttale infiltrante di grado 1 , mentre la lesione in bb una mastopatia fibrosocistica . with benign diagnoses for lesions equal to or greater than 7 mif we take regular posterior acoustic transmission as a reference , reduced transmission is able to predict malignancy for lesions equal to or larger than 7 mm whereas increased transmission becomes a sign of benign disease for lesions at least 30 mm in diameter . 
the presence of lateral acoustic shadows indicates benign disease in lesions at least 6 mm in size whereas heterogeneous texture indicates malignancy for lesions at least 7 mm in size . 
sensitivity , specificity and positive predictive , negative predictive and accuracy values for each reader and on the lesions as a whole and by size are shown in table 6 . 
these data became particularly interesting when we considered the readers ability to correctly classify the lesions into the four categories ( benign , probably benign , probably malignant and malignant )  . 
the results , shown in tables 7 and 8 , indicate a significantly larger number of lesions classified in the intermediate categories ( probably benign and probably malignant ) in concordanza interosservatore e accuratezza la concordanza interosservatore per quanto concerne il sospetto ecografico attribuito a ogni lesione , quando calcolata indipendentemente dalle dimensioni delle lesioni , risultata essere sempre buona o eccellente , secondo i criteri classificativi di landis e koch [ 11 ] , risultando compresa tra 0 , 71 e 0 , 83 , come dimostrato in tabella 4 . 
considerando invece due gruppi di lesioni con diametro massimo inferiore o uguale ai 7 mm e diametro massimo maggiore di 7 mm , la concordanza interosservatore ha dimostrato risultati sostanzialmente simili come dimostrato in tabella 5 . 
i valori di sensibilit , specificit , valore predittivo positivo e negativo e accuratezza per ogni singolo lettore considerati nella globalit o relativamente alle dimensioni sono riportati in tabella 6 . 
questi dati sono risultati interessanti quando abbiamo preso in considerazione la capacit del lettore in classificare correttamente le lesioni nelle quattro categorie ( benigne , probabilmente benigne , probabilmente maligne e maligne )  . 
 finally , the results of logistic analysis , interobserver agreement and accuracy of breast us show that a maximum diameter of 7 mm is the best threshold beyond which the sonographic features are able to express their predictive value . 
although breast us has not been demonstrated to be an alternative to mammography in breast cancer screening , it can be helpful for detecting a significant number of lesions missed at mammography in patients with dense breasts [ 12 , 13 ]  . differentiation between benign and malignant lesions , on the contrary , has never been considered an indication for breast gnificativamente pi numerose nel gruppo di lesioni con diametro inferiore o uguale ai 7 mm rispetto alle lesioni con diametro maggiore di 7 mal contrario , le lesioni con diametro massimo maggiore di 7 mm sono state classificate pi frequentemente nelle categorie benigne o maligne , con una riduzione della percentuale di lesioni classificate come indeterminate . infine , prendendo in considerazione i risultati dellanalisi logistica , la concordanza interosservatore e laccuratezza dellecografia mammaria , si evinto come la soglia dei 7 mm per il massimo diametro delle lesioni sia quella pi idonea , al di sopra della quale gli aspetti ecografici possano esprimere il loro valore predittivo . discussione lecografia mammaria , nel caso in cui venga eseguita con tecnica ottimizzata , rappresenta la tecnica ancillare pi utile in approfondimento alla mammografia . 
percutaneous biopsy of all solid lesions was therefore recommended in the past [ 1416 ] , with breast us being used to distinguish between solid and cystic lesions detected by mammography . in the 1990s , several studies appeared that demonstrated the ability of us to characterise breast lesions . 
the application of digital compound technology [ sono - computed tomography ( ct ) ] has improved contrast resolution and eliminated several artefacts typical of conventional sonography [ 4 , 5 ]  . sono - ct , for example , allows better definition of margins , pseudocapsule , galactophorous ducts and connective fibres [ 5 ]  . 
it also improves visualisation of low - contrast lesions and of the internal architecture of solid and cystic lesions [ 4 , 5 ]  . microcalcifications are better detected thanks to elimination of the clutter artefact [ 4 ]  . 
 the role of breast us in lesion characterisation has also alla mammografia nello screening del tumore della mammella , pu essere utile nellidentificazione di un numero significativo di lesioni non visualizzabili alla mammografia , in particolare nelle pazienti con seni densi mammograficamente [ 12 , 13 ]  . 
la differenziazione tra lesioni mammarie benigne e maligne , al contrario , per lungo tempo non stata considerata una corretta indicazione per lecografia mammaria , in relazione allampia sovrapposizione tra i vari aspetti e segni ecografici . 
pertanto , in passato , era raccomandata la biopsia percutanea di tutte le lesioni solide mammarie [ 1416 ] , mentre lecografia veniva utilizzata solo al fine di distinguere tra le lesioni identificate mammograficamente , quelle solide da quelle cistiche . 
questo risultato stato ottenuto soprattutto grazie agli avanzamenti tecnologici , allutilizzo di sonde ad ampio spettro , di algoritmi compound , e alla standardizzazione della metodologia ecografica e degli aspetti semeiologici . 
lapplicazione dellalgoritmo compound digitale ( sono - ct ) ha permesso di ottenere un incremento della risoluzione spaziale , eliminando diversi artefatti tipici della ecografia convenzionale [ 4 , 5 ]  . 
essa consente per esempio di ottenere una miglior definizione dei margini delle lesioni , della pseudocapsula , dei dotti galattofori e delle fibre connettivali [ 5 ]  . inoltre risultano meglio visualizzabili le lesioni a basso contrasto , nonch larchitettura interna delle lesioni sia solide che cistiche [ 4 , 5 ]  . 
infine le microcalcificazioni , quando evidenziabili , risultano meglio visibili grazie alleliminazione dellartefatto clutter [ 4 ]  . il ruolo dellecografia mammaria nella caratterizzazione delle lesioni quindi cresciuto nel corso del tempo anche grazie alla chiara identificazione di alcuni segni semeiologici , come riportato da stavros et al . 
successivamente , correlando la classificazione ecografica con i risultati istologici , hanno calcolato dei valori di sensibilit , specificit , valore predittivo positivo e negativo , e accuratezza rispettivamente 98 , 4% , 67 , 8% , 38% , 99 , 5% e 72 , 9% . 
i margini spiculati , angolati o microlobulati , la presenza di microcalcificazioni , la marcata ipoecogenicit , lassorbimento acustico posteriore , la crescita verticale , lestensione intraduttale e laspetto ramificato sono stati considerati segni predittivi di malignit . 
lassenza dei segni predittivi di malignit , la marcata iperecogenicit , la forma ovalare , la presenza di macrolobulazioni ben circoscritte e la presenza di pseudocapsula , sono stati considerati segni predittivi di benignit . 
spiculated , angular or microlobulated margins , microcalcifications , marked hypoechogenicity , shadowing , vertical growth , intraductal extension and branch pattern were considered predictive of malignancy ; the absence of malignant findings , marked hyperechogenicity , ellipsoid shape , presence of wellcircumscribed macrolobulations and presence of a pseudocapsule were considered predictive features for benign lesions ; indeterminate features were lesion size , isoechogenicity , mild hypoechogenicity , regular or increased posterior acoustic transmission and texture ( homogeneous or heterogeneous )  . 
 [ 7 ] was followed by several other papers analysing the same [ 9 ] or largely similar [ 8 , 19 , diverse pubblicazioni su simili argomenti sono seguite al lavoro di stavros et al . 
 [ 7 ] , che hanno preso in considerazione gli stessi segni ecografici [ 9 ] o comunque in gran parte simili [ 8 , 19 , 20 ] ottenendo analoghi risultati [ 8 , 9 , 19 , 20 ]  . 
inoltre , gli autori hanno cercato di quantizzare la capacit di questi segni ecografici nel caratterizzare le lesioni , analizzando la frequenza di presentazione di ogni singolo segno e valutando la variabilit interosservatore relativa a ciascuno di essi . 
 [ 8 ] hanno dimostrato come il segno pi predittivo di benignit , presente frequentemente e affetto da bassa variabilit interosservatore , siano i margini regolari o macrolobulari , nonch la morfologia rotondeggiante o ovalare . 
 [ 9 ] hanno messo in evidenza come la morfologia delle lesioni sia il segno con la maggior concordanza interosservatore , mentre al contrario la presenza della pseudocapsula presenti la pi bassa concordanza interosservatore , rimanendo comunque bassa o modesta anche la concordanza per quanto concerne i margini , lecogenicit e la trasmissione c . 
 [ 8 ] demonstrated that the most predictive findings for benign lesions , with high frequency and low interobserver variability , were regular or macrolobulated margins and round or ellipsoid shape . 
 [ 9 ] identified shape as the finding with the highest interobserver agreement whereas the presence of a pseudocapsule had the lowest interobserver agreement ; interobserver agreement for margins , echogenicity and posterior acoustic transmission was always low or moderate . 
sonographic findings that were found to be most frequent and most strongly predictive of malignant lesions ( or risk factors ) were irregular shape , microlobulated margins , marked hypoechogenicity and reduced posterior acoustic emission . 
the most frequent and most predictive findings for benign lesions ( or protective factors ) were ellipsoid shape , regular margins , presence of a pseudocapsule , regular posterior acoustic transmission and lateral acoustic shadows . 
the only differences between our results and those reported in the literature concern mild hypoechogenicity and regular or increased posterior acoustic transmission , which we classify as protective factors , and heterogeneous texture , which we classify as a risk factor ; these features were not considacustica posteriore . per quanto a noi noto , fino ad oggi , non stata presa in considerazione linfluenza delle dimensioni delle lesioni , sulla frequenza o sul valore predittivo dei diversi segni ecografici presi in esame . 
i segni ecografici che sono risultati essere pi predittivi di malignit ( o cosidetti fattori di rischio ) e pi frequenti , sono la morfologia irregolare , i margini microlobulati , la marcata ipoecogenicit , e lassorbimento acustico ; mentre i segni ecografici che sono risultati essere pi predittivi di benignit ( o fattori protettivi ) nonch pi frequenti , sono la morfologia ovalare , i margini regolari , la presenza di una pseudocapsula , la trasmissione acustica posteriore e la presenza di ombre acustiche laterali . 
lunica differenza tra i nostri risultati e quelli riportati in letteratura , concerne la moderata ipoecogenicit e la trasmissione acustica posteriore regolare o aumentata , che per noi risultano essere fattori protettivi e la tessitura disomogenea , che noi classifichiamo con fattore di rischio ; questi segni in realt non sono stati considerati utili per una caratterizzazione dagli autori precedenti [ 79 ]  . nondimeno , dai nostri dati emerge come correlando questi segni con le dimensioni delle lesioni , il loro valore predittivo diminuisce progressivamente , proporzionalmente alla riduzione del massimo diametro delle lesioni . 
siamo stati quindi in grado di identificare dei valori soglia relativi alle dimensioni , per ogni singolo segno , al di sotto dei quali , esso non risulta pi predittivo . 
other findings , such as the presence of pseudocapsule and lateral acoustic shadows , were instead found to be protective factors only for lesions equal to or larger than 7 mm and 6 mm , respectively , whereas findings such as round or irregular shape , decreased posterior acoustic transmission and heterogeneous texture were risk factors only for lesions equal to or larger than 7 mm , and marked hypoechogenicity for lesions equal to or larger than 11 mm . these results indicate that the distribution of sonographic findings among small lesions , whether benign or malignant , is more homogeneous , making their characterisation more difficult . 
there is a significant difference in the frequency of each finding in lesions larger or smaller than 7 mm , whether benign or malignant , and this was particularly true for shape , echogenicity and texture . 
in detail , the percentage of lesions classified in the more indeterminate , central categories ( probably benign and probably malignant ) is significantly higher for lesions equal to or smaller than 7 mm compared with those larger than 7 mm . consequently , the percentage of lesions correctly classified as benign or malignant was significantly higher for lesions larger than 7 mm compared with smaller ones ( 63% and 73% compared with 44.8% and 48% , respectively )  . 
in conclusion , while confirming the potential of breast us in characterising solid breast lesions , thanks to the definition of features predictive of benign or malignant lesions , our results revealed that the method suffers limitations in characterising small lesions . 
therefore , even in the absence of suspicious findings , solid lesions smaller than 7 mm should be monitored with short - term follow - up or be subjected to percutaneous biopsy ( fine - needle aspiration or core needle biopsy )  . acustiche laterali sono risultati essere predittivi di benignit solo per lesioni con diametro massimo maggiore o uguale rispettivamente a 7 e 6 mm , mentre la morfologia tondeggiante o irregolare , lassorbimento acustico posteriore , la tessitura disomogenea sono risultati essere predittivi di malignit solo per lesioni con diametro massimo maggiore o uguale a 7 mm , mentre la marcata ipoecogenicit solo per lesioni con diametro massimo maggiore o uguale a 11 m questi risultati mettono in evidenza come la distribuzione dei segni ecografici nelle lesioni di piccole dimensioni , sia pi omogenea , sia nelle lesioni benigne che in quelle maligne , rendendone pi difficoltosa la caratterizzazione . 
infatti abbiamo dimostrato come vi sia una significativa differenza di frequenza dei diversi segni ecografici sia nelle lesioni benigne che nelle lesioni maligne , per diametri maggiori o inferiori / uguali ai 7 mm , in particolare per quanto concerne la morfologia , lecogenicit e la tessitura . 
in particolare , la percentuale delle lesioni classificate nelle due categorie centrali pi indeterminate ( probabilmente benigne e probabilmente maligne ) significativamente maggiore per lesioni inferiori o uguali a 7 mm rispetto alle lesioni maggiori di 7 mconseguentemente , la percentuale di lesioni classificate correttamente nelle due categorie benigna e maligna risultata significativamente maggiore nelle lesioni con diametro maggiore ai 7 mm rispetto alle lesioni pi piccole ( 63% e 73% rispetto a 44 , 8% e 48% )  . 
la sua brillante intelligenza e il suo interesse per la medicina e le scienze matematiche gli hanno consentito di conseguire con il massimo dei voti , presso luniversit cattolica di roma , una laurea in medicina e chirurgia e tre specializzazioni in radiologia diagnostica , radioterapia ed oncologia . il suo cruccio fu quello di non potersi laureare in scienze matematiche , ma gi gli argomenti della sua tesi di laurea e di specializzazione in radiologia ( langiografia digitale : sviluppo di una classica metodica e nuove prospettive offerte dalluso del computer ; considerazioni dosimetriche in angiografia digitale ) dimostrano la sua propensione ad affrontare e comprendere immediatamente argomenti che avessero a che fare con numeri . io ho avuto lonore di collaborare , durante la sua formazione sia alluniversit che alla scuola di specializzazione , con un professionista geniale e nello stesso tempo semplice come tutti i grandi . 
ferrero di cambiano 17 , i - 10024 moncalieri ( to ) , italy , tel . : + 39 - 011 - 6615506 , e - mail : ago.depascale@libero.it received : 10 march 2006 / accepted : 7 april 2006 / published online : 11 august 2006 abstract purpose . 
computed tomography ( ct ) is of great value in the evaluation of the mediastinum , which is frequently involved by the disease , but carries a high radiation load . 
x - ray proved to be superior in hilar adenopathies only . us provides qualitative information ( hypoechoic or hyperechoic tissues ) in addition to quantitative data ( maximum diameter of each lymph node ) it shares with ct . 
compared with x - ray , us allows for a better evaluation of the anterior mediastinal regions , showing small , central adenopathies that do not alter the mediastinal lines on x - ray . it is , however , of very limited value in the evaluation of posterior compartments because of their deep location . 
us may have a specific role in monitoring patients with mediastinal adenopathies , providing early indications on possible response to treatment and allowing the frequency of ct follow - up scans to be reduced . 
il mediastino , frequentemente coinvolto dalla patologia , studiabile in modo ottimale con la tc , indagine che utilizza dosi importanti di radiazioni ionizzanti : alcuni autori hanno pertanto proposto di avvalersi anche dellecotomografia per valutare la risposta al trattamento gi dalle prime settimane , in virt dei peculiari vantaggi dellesame , caratterizzato da buona compliance e assenza di rischi per il paziente , bassi costi , agevole ripetibilit , immagini dinamiche che permettono valutazioni multiplanari , criteri qualitativi oltre che quantitativi . 
nel corso del 2005 , in 12 pazienti stato monitorato , in momenti successivi , il coinvolgimento mediastinico mediante radiografia standard del torace in due proiezioni , ecotomografia mediastinica e tc spirale con mdc ( gold standard )  . 
rispetto alla radiografia , lo studio ultrasonografico consente di valutare meglio le regioni del mediastino anteriore , evidenziando adenopatie piccole e centrali , che non modificano le linee mediastiniche sul radiogramma ; le regioni posteriori sono invece scarsamente valutabili a causa della loro maggiore a . 
lecotomografia pu rivestire un ruolo specifico durante il trattamento di pazienti con adenopatie mediastiniche , fornendo indicazioni precoci sul trend di risposta e consentendo di ridurre e distanziare nel tempo i controlli tc , e diventare cos complementare alla stessa tc , pur non potendo sostituirla ( la tc deve infatti continuare ad essere considerata la tecnica gold standard )  . parole chiave linfoma ecotomografia mediastino introduction introduzione lymphomas refer to a wide range of multicentric , solid malignancies of the lymphatic system , which at the thoracic level occur almost exclusively in the mediastinum [ 1 , 2 ]  . 
the mediastinum has been traditionally studied by using different imaging techniques , each of which has a different indication in diagnosis , treatment and follow - up [ 3 ]  . 
at present , the most commonly used diagnostic modalities are standard chest x - ray a firstline examination characterised by a large number of false negatives that does not provide a detailed description of mediastinal masses and computed tomography ( ct ) [ 35 ]  . 
although permitting accurate morphostructural and topographic evaluation in this complex anatomical region , ct involves high exposure to ionising radiation and requires the use of iodinated contrast material , with possible adverse reactions [ 3 , 4 , 6 ]  . 
nuclear imaging provides other very useful diagnostic tools , in particular positron - emission tomography ( pet ) - ct , which combines functional information and precise topographic and morphostructural localisation of the lesion [ 4 , 7 , 8 ]  . 
however , similarly to magnetic resonance imaging ( mri ) ( in which diagnostic detail is nearly identical to ct without the disadvantages ) , this technique is not widely used because of its limited availability [ 4 , 6 , 9 ]  . it has been suggested that these modalities should be integrated with ultrasonography ( us ) , a technique characterised by good compliance , absence of patient risks , low cost , easy reproducibility , multiplanar images and qualitative and quantitative lesion assessment [ 1021 ]  . 
though recognising that the anatomical characteristics of the mediastinum impair penetration of the us beam into all mediastinal districts i linfomi , ampia gamma di neoplasie maligne solide multicentriche del sistema linfatico , a livello toracico si sviluppano quasi esclusivamente nel contesto del mediastino [ 1 , 2 ]  . per la corretta gestione clinica di questi pazienti , spesso giovani , che necessitano di trattamenti articolati , emerge la fondamentale importanza della diagnostica radiologica , il cui impatto non trascurabile si associa agli importanti effetti tossici che gravano le terapie . 
lo studio del mediastino si avvale tradizionalmente di diverse tecniche di imaging , che trovano differente indicazione nel decorso della malattia : in corso di diagnosi , durante il trattamento , nel follow - up successivo [ 3 ]  . 
attualmente le metodiche di diagnostica per immagini pi utilizzate sono la radiografia standard , esame di prima istanza , ma caratterizzato da un elevato numerosi di falsi negativi e che non permette una descrizione dettagliata delle masse mediastiniche , e la tc [ 35 ]  . 
questultima consente unaccurata valutazione morfostrutturale e topografica in un distretto anatomico complesso , ma gravata da unimportante esposizione a radiazioni ionizzanti , oltre a prevedere lutilizzo di mezzo di contrasto organoiodato , non scevro dal rischio di reazioni avverse [ 3 , 4 , 6 ]  . 
di grande ausilio diagnostico sono sicuramente le indagini medico - nucleari , e tra queste in particolare offre notevoli potenzialit di impiego la pet - tc , che associa informazioni di carattere funzionale ad altre di precisa collocazione topografica e morfostrutturali della patologia [ 4 , 7 , 8 ]  . 
tuttavia tale ultima indagine , cos come la rm ( metodica in grado di fornire dettagli diagnostici quasi sovrapponibili alla tc in assenza degli inconvenienti sopramenzionati ) non diffusamente impiegata per la minore disponibilit sul territorio [ 4 , 6 , 9 ]  . alcuni autori hanno suggerito di affiancare alle precedenti metodiche lecotomografia , indagine caratterizzata da a . 
pur riconoscendo le importanti implicazioni negative dovute alle caratteristiche anatomiche del mediastino , che non consente la progressione del fascio ultrasonoro in tutti i distretti e quindi una valutazione diagnostica completa , lo scopo del nostro lavoro rilevare il possibile ruolo di tale metodica come esame integrativo nello studio della risposta al trattamento in corso di terapia di pazienti con localizzazione mediastinica di linfoma . materiali e metodi presso il servizio di ecotomografia della scdu di radiologia dellospedale s . 
luigi di orbassano ( to ) , nel corso del 2005 , sono stati esaminati 12 pazienti , 4 donne e 8 uomini , di et compresa tra 17 e 68 anni , con localizzazione mediastiniche di linfoma ( 4 linfomi di hodgkin , lh , e 8 linfomi nonhodgkin , lnh ) , diagnosi sempre formulata in base allesame istologico seguito da stadiazione mediante tc . 
ogni paziente stato valutato in quattro momenti successivi ( tabella 1 ) , per un totale di 44 esami ecotomografici : prima dellinizio della chemioterapia ; alla fine del primo ciclo di trattamento , quando dovrebbero essere evidenziabili i primi effetti terapeutici ; al termine del quarto ciclo di chemioterapia ( 8 pazienti ) o subito prima dellautotrapianto ( 4 pazienti ) , a seconda dello schema di trattamento impostato ; al termine dellintero percorso terapeutico . 
 solo 10 pazienti sono giunti a completare il follow - up , in and consequently hinder a thorough diagnostic evaluation , we carried out this study to assess the possible role of us as a complementary modality to evaluate response to treatment in patients with mediastinal lymphomas . 
 materials and methods in 2005 , we studied 12 patients ( four women , eight men ; age range : 1768 years ) with mediastinal lymphoma [ four hodgkins lymphomas ( hl ) and eight non - hodgkins lymphomas ( nhl ) ] at the department of ultrasonography of the s.c.d.u. 
each patient was studied on four different occasions ( table 1 ) , for a total of 44 us examinations : before initiating chemotherapy ; after the first treatment cycle ( when the first therapeutic effects should be visible ) ; after the fourth chemotherapy cycle ( eight patients ) or just before autologous transplantation ( four patients ) , depending on the treatment regimen ; and on completion of treatment . 
in the majority of cases , 3040 days elapsed between the first and second follow - up examination , 35 months between the second and third and 12 months between the third and fourth . 
for two patients whose treatment regimen involved longer intervals between cycles , the followup period was longer ( approximately 2 months between the first and second follow - up , 67 months between the second and third and approximately 3 months between the third and fourth )  . 
the radiologist systematically explored the mediastinal regions in search of disease locations and accurately determined size , morphology and structure of each adenopathy , which was then classified based on location . 
us examinations were performed on a sonoline elegra siemens device using 3.5 - mhz convex , 7.5to 10 - mhz linear and 6.5 - mhz sector transducers following the approaches outlined by wernecke et al . 
in the suprasternal approach , the patient lies supine with a cushion under the shoulders to enable full extension of the head ; the transducer is placed in the jugular fossa , and scans are made in the coronal , sagittal and semisagittal planes , providing easy exploration of the aortopulmonary window and supraaortic and paratracheal regions . 
in the parasternal approach , the patient lies in the right and left lateral position ; the transducer is placed in the intercostal spaces , and scans are made in the quanto 2 sono deceduti durante il periodo di osservazione . nella maggior parte dei casi , tra il primo ed il secondo controllo sono intercorsi 3040 giorni , 35 mesi tra il secondo ed il terzo , 12 mesi tra il terzo e lultimo . 
per 2 pazienti , invece , per i quali era stato scelto uno schema di terapia con cicli pi distanti nel tempo , il periodo di follow - up stato pi lungo ( circa 2 mesi tra il primo ed il secondo controllo , 67 mesi tra il secondo ed il terzo e circa 3 mesi tra il terzo e lultimo )  . 
ad ogni controllo , in pochi giorni i pazienti sono stati tutti sottoposti a radiografia standard del torace in proiezione pa e ll , a tc spirale in corso di somministrazione di m.d.c. 
organoiodato ( pitch 1 , 5 , spessore di strato 5 , 5 mm , ritardo di acquisizione delle immagini 3035 s , ricostruzioni ogni 5 scansioni ) e ad ecotomografia mediastinica . gli esami ecotomografici sono stati eseguiti tutti dallo stesso radiologo , non a conoscenza dellesito delle altre due indagini , che ha proceduto ad una sistematica esplorazione delle regioni mediastiniche , alla ricerca di localizzazioni della malattia di base , ed ad unaccurata misurazione dimensionale e valutazione morfostrutturale di ogni adenopatia riscontrata , classificata poi in base alla sede . 
il materiale iconografico rx e tc raccolto per ogni paziente stato poi attentamente rivalutato da due radiologi operanti presso i servizi di radiologia convenzionale e di tc : questi dati sono stati quindi confrontati con quelli preventivamente riscontrati in corso di ecotomografia . 
sono state infine calcolate con metodo statistico le correlazioni diagnostiche di radiologia convenzionale ed ecotomografia rispetto alla tc quale indagine di riferimento ( gold - standard )  . lindagine ecotomografica stata eseguita con attrezzatura sonoline elegra siemens , utilizzando le sonde convex 3 , 5 mhz , lineare 7 , 510 mhz , settoriale 6 , 5 mhz , seguendo gli approcci descritti da wernecke et al . 
per lapproccio sovrasternale , il paziente in posizione supina con un cuscino sotto le spalle per favorire liperestensione del capo ; ponendo la sonda a livello della fossa giugulare , con scansioni lungo i piani coronali , sagittali e semisagittali , sono agevolmente esplorabili la finestra aorto - polmonare e le regioni sovraaortica e paratracheale . 
per lesplorazione parasternale , invece , il paziente deve assumere il decubito laterale , destro e sinistro ; ponendo la sonda a livello degli spazi intercostali , attraverso la nuova finestra acustica che si crea al di sotto dello sterno , con scansioni lungo il piano sagittale e altri pi o meno prossimi al trasversale , si valutano le regioni prevascolare e subcarinale ed il mediastino posteriore . 
prevascular and subcarinal regions are evaluated together with the posterior mediastinum through the acoustic window just beneath the sternumediastinal exploration is easier if the breath is held during forced expiration , as the width of the window changes with respiration . 
us yielded 38 / 41 true positives ( tp ) , 3 / 3 true negatives ( tn ) and three false negatives ( fn ) ; standard radiography yielded 27 / 41 tp , 3 / 3 linfoadenopatie in 41 / 44 casi ( tabella 2 )  . 
con gli ultrasuoni rilevabile una maggior frequenza di esami veri positivi ( vp ) e veri negativi ( vn ) nelle regioni sovraaortica ( vp 28 / 29 e vn 15 / 15 ) , prevascolare ( vp 30 / 31 e vn 13 / 13 ) , paratracheale ( vp 26 / 30 e vn 14 / 14 ) e nella finestra aortopolmonare ( vp 12 / 15 e vn 29 / 29 ) , rispetto allesame radiografico che presenta un numero maggiore di falsi negativi ( fn ) nelle regioni sovraaortica ( 13 / 29 ) , prevascolare ( 19 / 31 ) , nella finestra aortopolmonare ( 15 / 15 ) e nella regione subcarinale ( 19 / 19 )  . 
per quanto riguarda lo studio ultrasonografico delle localizzazioni pi profonde , si osservato 1 / 19 vp per la regione subcarinale e 41 / 41 vn per il mediastino posteriore ; lesame radiografico ha evidenziato 19 / 19 e 3 / 3 fn rispettivamente . i maggiori valori di sensibilit e accuratezza diagnostica ( tabella 5 ) sono stati osservati per lecotomografia a carico delle regioni sovraaortica ( 97% e 98% rispettivamente ) , prevascolare ( 97% e 98% ) , paratracheale ( 87% e 91% ) e della finestra aortopolmonare ( 80% e 93% ) , superiori a quelli calcolati per lesame radiografico . le adenopatie mediastiniche evidenziate sono risultate ipoecogene in 92 / 97 ( 95% ) casi . 
negli altri 5 casi ( 5% ) , invece , lecotomografia mostrava , a livello del mediastino anteriore ( in 2 casi a carico della regione paratracheale ed in 3 della regione prevascolare ) , formazioni solide ad ecogenicit aumentata rispetto ai controlli precedenti e con scarsa delimitazione di vere e proprie lesioni adenopatiche . 
subcarinale mediastino posteriore totale regions supraaortic region , % prevascular region , % paratracheal region , % aortopulmonary window , % 80 subcarinal window , % posterior mediastinum , % total , % regioni r . 
thus , sensitivity and diagnostic accuracy ( da ) of us were both 93% compared with 66% and 68% , respectively , for standard x - ray ( table 3 )  . 
results shown in table 2 were subsequently classified according to due pazienti presentavano un habitus costituzionale sfavorevole allesplorazione ecotomografica : il primo per una conformazione dello stretto toracico superiore priva di adeguate finestre acustiche ultrasonografiche , il secondo per a . 
in tutti gli esami eseguiti stata utilizzata la funzione thi ( tissue harmonic imaging ) , che ha favorito la corretta valutazione delle regioni anatomiche pi superficiali ; lattenuazione del fascio ultrasonoro ha penalizzato lievemente lesplorazione di distretti pi profondi . 
il colordoppler stato di ausilio solo in alcuni casi , per gli inevitabili artefatti legati alla pulsazione vigorosa dei grossi vasi arteriosi . discussione la metodica ecotomografica tradizionalmente considerata non idoneo allo studio del distretto toraco - mediastinico , territorio senza dubbio ostico per lesplorazione con gli ultrasuoni a causa delle numerose strutture anatomiche che ne limitano la progressione . 
tuttavia , grazie allimportante progresso tecnologico , con la commercializzazione di attrezzature pi sofisticate dotate di sonde sempre pi adatte alla valutazione di compartimenti anatomici con finestre acustiche limitate , e al crescente interesse manifestato dallambiente radiologico , negli ultimi anni si assistito ad un ampliamento nellutilizzo della metodica , che sta progressivamente acquisendo un ruolo importante nello studio di patologie del cavo pleurico , del parenchima polmonare , della parete toracica , del diaframma [ 10 , 21 ]  . la letteratura pi comunemente reperibile riporta studi che confrontano lecotomografia unicamente con la radiografia tradizionale e la tc . 
il nostro studio , condotto su una popolazione di pazienti affetti da lh e lnh con localizzazione mediastinica , ha evidenziato il positivo ruolo dellecotomografia come indagine di monitoraggio , e si affianca ad altri eseguiti in passato su pazienti affetti da differenti tipi di tumori mediastinici ; come nel lavoro di wernecke et al . 
 ( 1990 ) [ 12 ] , sono state stimate sensibilit e specificit dellecotomografia e della radiografia standard del torace in ogni regione in cui stato suddiviso il mediastino , assumendo la tc quale esame di riferimento ( gold standard )  . 
dai risultati da noi ottenuti , sensibilit e specificit dellecotomografia ( 93% e 100% ) sono nettamente superiori rispetto alla radiografia ( 66% e 100% ) come mostrato in tabella 3 . 
confluenza delle vene anonime nella vena cava superiore : importanza del color - doppler nel riconoscimento dei vasi mediastinici ( c )  . anatomical region ( table 4 )  . 
by contrast , x - ray had more fn results ( fn ) in the supraaortic ( 13 / 29 ) and prevascular ( 19 / 31 ) regions , in the aortopulmonary window ( 15 / 15 ) and in the subcarinal rea . 
scansione sovrasternale : arco aortico e tessuto iperecogeno ( di verosimile natura fibrosa ) a carico della finestra aortopolmonare ( c )  . gion ( 19 / 19 )  . 
us of deeper mediastinal masses produced 1 / 19 tp in the subcarinal region and 41 / 41 tn in the posterior mediastinum ; x - ray revealed 19 / 19 and 3 / 3 fn , respectively . the highest sensitivity and da values ( table 5 ) were obtained by us in the supraaortic ( 97% and 98% , respectively ) , prevascular ( 97% and 98% ) and paratracheal ( 87% and 91% ) regions and aortopulmonary window ( 80% and 93% )  . 
4a , b adenopatie in sede sovraaortica : scansione sovrasternale ( a ) e tc con mdc ( b )  . sono infatti solo 3 i falsi negativi , con una sensibilit del 80% , presentando una performance diagnostica di poco inferiore , per tale distretto , rispetto alla tc ( ad 93% )  . 
lindagine ecotomografica non offre invece adeguata attendibilit diagnostica nella valutazione di distretti mediastinici pi profondi , quali la regione subcarinale ed il mediastino posteriore ( valori di sensibilit pari a 5% e 0% )  . 
tali risultati , pur di scarsa rilevanza diagnostica , sono comunque superiori a quelli riportati con lo studio radiografico standard ( sensibilit pressoch nulla )  . in accordo con gli studi di wernecke et al . 
 ( 1995 , 1997 ) [ 15 , 23 ] sullaccuratezza e lattuabilit di unindagine ecotomografica del mediastino finalizzata a rilevare la presenza di linfoadenopatie , i dati statistici dello studio da noi condotto mettono in evidenza , in confronto con la tc , una buona sensibilit , specificit e accuratezza diagnostica per lesioni localizzate a livello sovraaortico ( 97% , 100% , 98% ) , prevascolare ( 97% , 100% , 98% ) e paratracheale ( 87% , 100% , 91% ) , con differenze di sensibilit nei confronti dellesame radiografico tradizionale del 10 - 58% a favore dellecotomografia . 
in queste sedi lecotomografia appare dunque pressoch sovrapponibile alla tc . anterior mediastinum ( paratracheal region in two cases and prevascular region in three cases ) demonstrated solid masses with increased echogenicity compared with the previous examinations and poor resemblance to adenopathic lesions . 
 two patients had a body habit that was unfavourable for us : the first due to inadequate acoustic windows in the thoracic outlet and the second due to severe spondyloarthrosis with dorsal kyphosis preventing the head hyperextension needed for suprajugular examination . 
tissue harmonic imaging was performed in all cases , allowing for accurate evaluation of the more superficial anatomical regions ; attenuation of the us beam slightly hindered exploration of the deeper districts . 
si infatti osservata una netta superiorit dellaccuratezza diagnostica dellecotomografia mediastinica rispetto alla radiografia standard del torace : lo studio ultrasonografico del mediastino consente infatti di valutare meglio le diverse regioni e di evidenziare linfoadenopatie pi piccole e centrali , che non determinano alcuna modificazione delle linee mediastiniche sul radiogramma . 
tale lieve discrepanza pu essere legata alla diversa tipologia di studio , con valutazione , nei lavori segnalati , di tumori mediastinici anche non linfomatosi , e quindi caratterizzati da struttura con gradiente di ecogenicit maggiore rispetto ai tessuti circostanti . di notevole interesse , inoltre , il risultato ottenuto per lo studio della finestra aortopolmonare ( sensibilit e specificit pari rispettivamente a 80% e 100% ) , lievemente sopra la media rispetto ai lavori presenti in letteratura . 
i migliori risultati da noi ottenuti in termini di sensibilit sono probabilmente ascrivibili alla migliore qualit delle sonde oggi disponibili e allutilizzo della funzione thi , che consentono una migliore valutazione di questo distretto anatomico , tipifig . 
this modality has acquired an important role in the evaluation of diseases of the pleural cavity , pulmonary parenchyma , chest wall and diaphragm [ 10 , 21 ]  . previous studies have compared us with x - ray and ct only , so the value and potential of us have been analysed in relation to the results obtained with these two imaging modalities . 
our study , conducted on patients with mediastinal hl and nhl , revealed the positive role of us for surveillance and adds to other previous studies on different types of mediastinal malignancies . 
our results indicate that sensitivity and specificity ( 93% and 100% ) of us were higher than those of x - ray ( 66% and 100% ) , as shown in table 3 . 
moreover , us showed remarkable sensitivity in evaluation of the aortopulmonary window , yielding only three false negatives and achieving 80% sensitivity and a da only slightly lower than ct ( da 93% )  . 
us does not have sufficient da in the evaluation of deeper mediastinal regions , such as the subcarinal region and the posterior mediastinum ( sensitivity of 5% and 0% )  . 
on accuracy and feasibility of us in detection of mediastinal lymph nodes [ 14 , 15 , 22 , 23 ] , our statistical data revealed good sensitivity , specificity and da compared with ct for lesions in the supraaortic ( 97% , 100% , 98% ) , prevascular ( 97% , 100% , 98% ) and paratracheal ( 87% , 100% , 91% ) regions , respectively . 
 our data substantially agree with those reported in the studies by wernecke et al . , which evaluated all mediastinal tumours and not only lymphomas [ 12 , 24 , 25 ]  . 
the da of mediastinal us was clearly superior to that of standard chest x - ray : mediastinal us allows better evaluation of the different regions and detection of smaller and more central lymphadenopathies that do not affect the mediastinal lines on radiograms . 
this slight discrepancy may be related to the different study decamente poco esplorabile per la profondit e per la scarsa accessibilit , ma che gode dellottima finestra acustica costituita dallarco aortico che la delimita superiormente . 
per le stesse ragioni di maggiore profondit e scarsa accessibilit , la sensibilit della metodica di molto inferiore se si valutano la regione subcarinale , il mediastino posteriore e le regioni paravertebrali ( sensibilit 0% - 5% )  . 
per quanto riguarda il mediastino posteriore e le regioni paravertebrali , lelevata distanza dalla sonda , con importante assorbimento del fascio ultrasonoro , e le caratteristiche delle finestre acustiche disponibili , di dimensioni ridotte e forma sfavorevole , rendono queste regioni scarsamente accessibili con gli ultrasuoni . 
anche il valore predittivo negativo dellecotomografia , riscontrato pari al 50% , pu essere motivato dalla presenza di regioni pi difficilmente esplorabili con gli ultrasuoni , ed in particolare dal maggior numero di falsi negativi riscontrato a carico di regioni quali la subcarinale . lunica situazione in cui abbiamo constatato una superiorit dallesame radiografico sullecotomografia riguardava linteressamento degli ili polmonari : le stazioni ilari , per le loro peculiari caratteristiche anatomiche di sede e rapporti , sono infatti ben valutabili sul radiogramma , ma non esplorabili con gli ultrasuoni . 
alcuni autori sostengono che la scarsa accessibilit di alcune regioni mediastiniche agli ultrasuoni non debba essere considerata limitante per lutilizzo dellecotomografia , perch il successo del trattamento analogo a livello di tutte le localizzazioni di malattia [ 14 , 18 ]  . 
 ( 1991 ) hanno osservato che tutti i linfonodi non evidenziati con lecotomografia mostravano risposta alla terapia identica a quella delle adenopatie accessibili agli ultrasuoni . un aspetto di notevole importanza legato alla peculiare capacit dellecotomografia di studiare con notevole dettaglio le strutture superficiali . 
in alcuni casi lindagine ultrasonografica stata agevolmente estesa alla valutazione di adenopatie a livello sovraclaveare , latero - cervicale ed ascellare , presenti rispettivamente in 17 , 4 e 2 esami . 
sono tutte possibili localizzazioni della malattia , per la loro sede non facilmente rilevate con altre indagini , che risultano invece di facile esplorazione con gli ultrasuoni , diventando cos reperti aggiuntivi nella valutazione della risposta alla terapia . bench lunico criterio di differenziazione tra linfonodi normali e patologici in tc sia di tipo esclusivamente dimensionale , sono stati osservati casi di linfonodi ingranditi non coinvolti in processi neoplastici n flogistici classificati come patologici dallesame tc e non evidenziati in ecotomografia , e linfonodi invece patologici di dimensioni 1 cm non considerati come tali dalla tc . 
inoltre , il confronto tra le dimensioni di adenopatie mediastiniche misurate mediante tc con quelle ricavate da esame ecotomografico non sempre ugualmente attendibile : molti linfonodi , infatti , sono disposti in senso longitudinale nel mediastino [ 23 ]  . 
our better sensitivity values are probably the result of the improved quality of current transducers and the use of the thi function , which enable better evaluation of this deep and poorly accessible anatomical region in which the aortic arch provides , however , an excellent acoustic window . 
a significant distance from the probe , with intense absorption of the us beam , together with the small and suboptimal acoustic windows , make the posterior mediastinum and paravertebral regions barely amenable to sonography . 
the negative predictive value ( npv ) of sonography ( 50% ) and , in particular , the high number of false negatives at the subcarinal level may be due to the presence of regions that are difficult to explore by us . 
 one site in which chest x - ray proved superior to us was the pulmonary hiludue to their peculiar anatomic characteristics , hilar stations are clearly visualised on x - ray but not on us . 
some authors maintain that the poor accessibility of some mediastinal regions should not limit the use of us since treatment is successful for all disease locations [ 14 , 18 ]  . 
 [ 18 ] noted that all lymph nodes missed by us had identical response to treatment to those that were visible . a very important aspect is the ability of us to visualise superficial structures . 
in some cases , assessment was extended to adenopathies in the supraclavicular , laterocervical and axillary regions , present in 17 , four and two of our patients , respectively . 
 though size is the only ct criterion for discriminating between normal and pathological nodes , enlarged lymph nodes uninvolved by neoplastic or inflammatory processes were classified as pathological on ct but were not visualised on us ; likewise , pathological lymph nodes 1 cm were not classified as such by ct . 
moreover , comparison of the size of mediastinal adenopathies measured on ct and us is not always reliable : many lymph nodes are arranged longitudinally in the mediastinum [ 23 ]  . 
tale discrepanza pu essere oggi superata grazie alle efficaci ricostruzioni multiplanari ottenibili con la tc multislice ( msct ) , metodica per altro meno diffusamente disponibile rispetto alla tc con una o due spirali . la misurazione ultrasonografica , condotta sistematicamente ed in modo dettagliato durante gli esami eseguiti per il nostro studio , correla meglio con le reali dimensioni del conglomerato adenopatico . 
chiaro che la metodica ultrasonografica non pu assolutamente competere , per quanto riguarda laccuratezza diagnostica , con la tc , che infatti nel nostro lavoro viene considerata indagine di riferimento : per sicuramente utile come esame complementare nello studio delle masse linfoadenopatiche in corso di terapia , anche solo per il semplice controllo dimensionale , parametro sempre importante nella valutazione della risposta al trattamento . lecotomografia stata inoltre in grado di aggiungere un criterio di valutazione qualitativo ( lecogenicit ) a quello quantitativo tipico della tc ( il diametro massimo ) [ 13 , 16 ] , che sembra essere un indicatore pi affidabile dellattivit tumorale rispetto alla sola valutazione dimensionale . 
nel nostro lavoro la metodica ultrasonografica risultata in alcuni casi pi precisa e dettagliata rispetto alla tc nella valutazione strutturale di tessuti a livello del mediastino anteriore . nel corso della chemioterapia , si proceduto infatti alla valutazione ultrasonografica della struttura delle linfoadenopatie , che risultata ipoecogena in 92 / 97 ( 95% ) casi . 
negli altri 5 casi ( 5% ) , nei quali la tc rilevava la presenza di formazioni patologiche riferite verosimilmente a linfoadenopatie a livello del mediastino anteriore ( in 2 casi a carico della regione paratracheale ed in 3 della regione prevascolare ) , lecotomografia mostrava formazioni solide ad ecogenicit aumentata rispetto ai controlli precedenti e con scarsa delimitazione di vere e proprie lesioni adenopatiche . tale reperto contrastava con quello tc , che segnalava formazioni di verosimile natura adenopatica con caratteristiche densitometriche non specifiche . 
molti studi confermano infatti la possibilit di differenziare , con lecotomografia , il tessuto adenopatico attivo da residui fibrosi conseguenti alla terapia , distinzione non consentita dalla tc [ 14 , 18 ]  . linfonodi infiltrati da cellule neoplastiche , infatti , appaiono ipoecogeni rispetto al tessuto adiposo ed al connettivo circostanti . 
se rispondenti alla terapia , assumono di nuovo , progressivamente , la loro naturale ecogenicit , fino al punto da non essere pi distinguibili se si ottiene la remissione completa della patologia . 
 us has added a qualitative criterion for evaluation ( echogenicity ) to the quantitative criterion of ct ( maximum diameter ) [ 13 , 16 ] , and echogenicity appears to be a more reliable indicator of tumour activity compared with size . 
in some cases , us proved more precise and detailed than ct in the structural evaluation of tissues within the anterior mediastinu during chemotherapy , us appearance of adenopathic masses was hypoechoic in 92 / 97 cases ( 95% )  . 
in the remaining five cases ( 5% ) in which ct showed the presence of pathological masses in the anterior mediastinum ( in the paratracheal and prevascular regions in two and three cases , respectively ) , us detected solid masses with increased echogenicity compared with the previous follow - up exams and poor resemblance to adenopathic lesions . 
others , such as schulte - altedorneburg et al . , believe that the use of us contrast material does not significantly add to the information obtained with conventional us ( or colour power doppler ) despite the good depiction of intranodal vascularity [ 27 ]  . 
on the basis of our preliminary observations , we believe that the use of sonographic contrast material in mediastinal lymphadenopathies may improve da , above all for evaluating the activity of residual tissue . our study indicates that mediastinal us , a technique requiring regular practice and specific training , offers a number of advantages : it is inexpensive , widely available and reproducible an ideal condition for frequent use during follow - up and is generally characterised by good compliance given that there are no side effects due to ionising radiation or iodinated contrast material . 
us has good da for evaluation of some mediastinal regions only , with deeper lesions being more difficult to distinguish due to the high rate of absorption of the us beam . the uncertain acoustic window prevents exploration of the posterior mediastinal regions . 
 ( 2004 ) , sulla base delle caratteristiche di perfusione osservate , sostengono una maggiore accuratezza diagnostica dellindagine condotta con la somministrazione del mdc [ 26 ]  . si contrappongono coloro che , come schulte - altedorneburg et al . 
 ( 2003 ) , ritengono che lutilizzo del mdc non comporti un incremento significativo delle informazioni fornite dallecotomografia condotta tradizionalmente , eventualmente con lausilio del color - power - doppler , nonostante la buona visualizzazione della vascolarizzazione intranodale [ 27 ]  . nostra opinione , in base ad osservazioni preliminari da noi condotte , che lutilizzo del mdc su linfoadenopatie mediastiniche possa migliorare lattendibilit diagnostica della metodica ecotomografica , soprattutto nella valutazione dellattivit di tessuti residui , evidenziando uno specifico pattern di enhancement . dalla nostra sperimentazione abbiamo inoltre evidenziato come lecotomografia mediastinica , esame che richiede una pratica assidua e una preparazione specifica delloperatore , presenti numerosi vantaggi di carattere generale : un esame poco costoso , facilmente disponibile e ripetibile , condizione ideali per un frequente utilizzo in corso di followup , ed generalmente caratterizzato da una buona compliance , trattandosi di unindagine priva di effetti collaterali in quanto non ricorre allutilizzo di radiazioni ionizzanti n di mdc organoiodato . abbiamo tuttavia osservato alcuni limiti , strettamente legati alla metodica . 
lesame presenta buona attendibilit diagnostica per lo studio solo di alcune regioni mediastiniche , mentre lesioni situate in distretti pi profondi , per lelevato assorbimento del fascio ultrasonoro , risultano difficilmente distinguibili dai tessuti circostanti : la precaria finestra acustica non consente di valutare gli spazi mediastinici posteriori , che risultano cos scarsamente esplorabili . le caratteristiche individuali dei pazienti inoltre influiscono sullesito dellesame : lhabitus costituzionale , la presenza di patologie concomitanti o particolari conformazioni anatomiche possono non consentire unesplorazione ottimale . per la valutazione dei linfomi a localizzazione mediastinica lesame di riferimento resta sicuramente la tc , cui si aggiunge in alcuni casi la pet - tc , nonostante limportante dose di radiazioni ionizzanti cui tali indagini espongono i pazienti ( circa 20 msv la tc spirale , 8 msv la sola tc del torace e 10 - 12 msv la pet - tc )  . 
6a , b ecostruttura linfonodale : linfonodo reattivo ( a ) e linfonodo neoplastico ( b )  . patients characteristics , including body habit , presence of concurrent disease or particular anatomical conformation . 
 ct , integrated by pet - ct in some cases , remains the reference standard for evaluation of mediastinal lymphomas despite the high dose of ionising radiation ( approximately 20 msv for spiral ct , 8 msv for chest ct and 1012 msv for pet - ct )  . 
in order to reduce the frequency of ct follow - up scans during treatment , and consequently total radiation exposure , while closely monitoring the disease , the use of mediastinal us could be extend to include evaluation of mediastinal involvement , with chest x - ray being limited to the study of the hilar regions . 
although conventional x - ray is the first - line examination for diagnosis of mediastinal disease , the gold standard for evaluation of this region is ct despite its suboptimal specificity due to poor characterisation of residual tissue after therapy . 
in this context , us is ideal for evaluating mediastinal adenopathies . advantages include good compliance , absence of patient risks , low cost , easy reproducibility and dynamic imaging with multiplanar capabilities and good qualitative and quantitative information . 
disadvantages include operator dependence , limited accessibility of some mediastinal regions and dependence on the individual patients characteristics . nonetheless , poor accessibility of some mediastinal regions should not be regarded as a limiting factor since treatment has proved successful in all disease locations . 
 our study indicates the essential superiority of us over x - ray , particularly in some anatomical districts such as the supraaortic , prevascular and paratracheal regions and the aortopulmonary window . 
in the future , greater experience with mediastinal us may help reduce the frequency of follow - up ct scans . conclusioni per individuare il trend della patologia , in senso positivo o negativo , ed operare quindi le opportune correzioni terapeutiche , di grande utilit per il clinico rilevare in tempi precoci variazioni a carico delle masse neoplastiche a livello mediastinico . 
nonostante la radiologia tradizionale costituisca un primo livello nella diagnosi delle malattie mediastiniche , il gold standard per la valutazione di questa regione rappresentato tuttora , nella pratica clinica , dalla tc , pur con i limiti di specificit dovuti allinsufficiente caratterizzazione tissutale di formazioni residue dopo terapia . 
sono infatti numerosi i vantaggi della metodica : la buona compliance e lassenza di rischi per il paziente , i bassi costi , lagevole ripetibilit , lo studio diagnostico di tipo dinamico che permette una valutazione multiplanare , gli apprezzabili risultati qualitativi e quantitativi , pur a fronte di limiti quali la dipendenza delle informazioni ottenibili dallesperienza delloperatore , il difficile accesso in alcuni distretti mediastinici e lesplorazione difficoltosa in presenza di sfavorevoli caratteristiche individuali del paziente . 
la scarsa accessibilit di alcune regioni mediastiniche agli ultrasuoni non deve tuttavia essere considerata limitante per lutilizzo dellecotomografia , perch il successo del trattamento analogo a livello di tutte le localizzazioni di malattia . nella nostra casistica si pone laccento sulla sostanziale superiorit dellecotomografia rispetto alla radiografia standard , con particolare riferimento ad alcuni distretti anatomici , quali le regioni sovraaortica , prevascolare e paratracheale e la finestra aortopolmonare . 
tali considerazioni spingono a proporre lindagine ultrasonografica come esame importante nel follow - up in corso di terapia dei pazienti affetti da linfomi con localizzazione mediastinica , da affiancare ai controlli tc programmati , la cui frequenza non incrementabile per le note problematiche legate allesposizione a radiazioni ionizzanti . 
gerardo , via pergolesi 33 , i - 20052 monza , italy 3scuola di specializzazione in fisica sanitaria , universit degli studi di milano , via celoria 16 , i - 20133 milan , italy correspondence to : a . 
the aim of this study was to calibrate monitors used in soft - copy review of diagnostic images in a pictures archiving and communication system ( pacs ) and to assess critical quality assurance ( qa ) parameters through appropriate checks . 
barco [ cathode ray tube ( crt ) and liquid crystal display ( lcd ) ] and eizo ( lcd ) monitors were evaluated . calibration and qa controls were carried out during acceptance tests on the systems and every 6 months according to the task group 18 ( tg18 ) report by the american association of physicists in medicine ( aapm )  . 
la calibrazione e i controlli sono stati effettuati in fase di accettazione e successivamente con cadenza semestrale seguendo le indicazione del report tg18 dellamerican association of physicists in medicine ( aapm )  . alcuni dei parametri considerati sono : luminanza massima , rapporto di contrasto , risposta di luminanza , risoluzione spaziale e risposta angolare . 
comunque assolutamente necessario procedere con i controlli di qualit periodici per garantire il perdurare delle caratteristiche di qualit . parole chiave interpretazione diagnostica soft - copy calibrazione monitor aapm tg18 a . 
international papers strongly recommend periodic checks and suggest reference procedures and values ; among them , the american association of physics in medicine ( aapm ) with its task group 18 ( tg18 ) has exhaustively dealt with the issue , thus becoming de facto the main source of reference [ 3 , 4 ]  . materials and methods simultaneously with the installation of the pacs system ( agfa impax ) , 15 reporting stations were set up , each provided with a low - resolution monitor allowing access to the radiological information system ( ris ) database not considered in this study and two high - resolution monitors displaying medical images . 
crt monitors are provided with conventional crts found in tv sets : a brush of accelerated electrons is run along the entire monitor surface and its intensity modulated to generate the required grey level . 
the lcd technology is based , instead , on the ability of liquid crystals to rotate when voltage is applied : the image is then generated by a matrix of liquid crystals that , based on the angle they form , mitigate the light emitted by the backlighting lamp to a varying degree . 
the reporting stations are currently installed in various hospital departments ( radiodiagnostic , accident and emergency ) and at a hospital a few miles away belonging to the same hospital trust . the aapm tg18 document suggests , in addition to the essential calibration , that a number of parameters should be checked , each with different techniques of varying complexity and accuracy levels . 
the decision about which kind of measurement to perform for every characteristic to evaluate was based on a compromise between quality of result and ease of performance , considering the contingent conditions of reporting rooms , which are always very busy and unable to lay out complex experimental setups [ 5 ]  . allinterno di un sistema pacs , come quello recentemente installato presso lospedale s . 
gerardo di monza , il percorso degli esami radiologici pu ora prescindere dalle pellicole radiografiche e seguire un iter completamente digitale.la visualizzazione delle immagini digitali al momento della refertazione viene affidata ad una coppia di monitor ad elevate prestazioni . 
i monitor di visualizzazione entrano anchessi a far parte della catena delle immagini digitali e bisogna evitare che siano questi ultimi il collo di bottiglia che porti ad un degrado della qualit finale dellesame . 
i monitor crt ( cathode ray tube ) sono dotati dei classici tubi catodici di tipo televisivo : un pennello di elettroni accelerati viene fatto scorrere lungo lintera superficie del monitor e modulato in intensit per generare il livello di grigio richiesto . 
la tecnologia lcd ( liquid crystal display ) si basa invece sulla capacit dei cristalli liquidi di ruotare se percorsi da corrente : limmagine viene quindi generata da una matrice di cristalli liquidi che a seconda dellangolo che formano attenuano in modo maggiore o minore la luce emessa dalla lampada retroilluminante . 
le stazioni di refertazione sono attualmente dislocate sia in diversi reparti dellospedale ( radiodiagnostica , pronto soccorso ) , che in un ospedale situato a qualche chilometro di distanza appartenente alla medesima azienda ospedaliera . il documento aapm tg18 suggerisce , oltre alla indispensabile calibrazione , molteplici parametri da controllare , ognuno con diverse tecniche di varia complessit e livello di accuratezza . 
all monitors included in this study are dedicated to reporting ; therefore , they must meet the tightest tolerances of primary monitors . in order to calibrate monitors and subsequent luminance measurements , the x - rite dtp - 92 colorimeter / photometer was used in combination with the medical pro software package ( barco )  . 
we chose to use mostly the phantoms suggested by aapm tg18 and available in digital imaging and communications in medicine ( dicom ) format on the internet [ 3 ]  . images were displayed on stations with the same software normally used by radiologists for reporting ( agfa impax ) , taking care to use the window and level standard values , without any look - up table ( lut ) that the software might apply . 
the just noticeable difference ( jnd ) is introduced for this purpose [ 7 ]  . si basata su di un compromesso tra la qualit del risultato e la semplicit di esecuzione , tenendo conto della situazione contingente delle sale di refertazione , sempre molto impegnate e senza la possibilit di allestire setup sperimentali complessi [ 5 ]  . i sistemi di visualizzazione vengono classificati in sistemi primari e sistemi secondari [ 3 ] ; si definiscono primari i sistemi di visualizzazione utilizzati per la refertazione , sono secondari invece i sistemi che visualizzano immagini mediche per scopi diversi dalla refertazione , tra cui la visualizzazione su console di unit diagnostiche o la consultazione da parte di medici non radiologi . 
tutti i monitor considerati in questo studio sono dedicati alla refertazione , pertanto devono rispondere alle pi strette tolleranze indicate per i monitor primari . per la calibrazione dei monitor e le successive misure di luminanza stato utilizzato il colorimetro / fotometro x - rite dtp - 92 in combinazione con il pacchetto software medical pro ( barco )  . per alcune misure di luminanza stato utilizzato inoltre il fotometro telescopico hagner s3 , caratterizzato dal fatto di poter misurare la luminanza a distanza . 
si scelto nella maggioranza dei casi di utilizzare i fantocci suggeriti da aapm tg18 e disponibili in formato dicom su internet [ 3 ]  . la visualizzazione delle immagini sulle stazioni stata affidata al medesimo software normalmente in uso ai radiologi per la refertazione ( agfa impax ) , avendo per laccortezza di utilizzare i valori standard di finestra e livello , senza utilizzare nessuna lut ( look up table ) che il software table 1 tolerances suggested by american association of physicists in medicine task group 18 ( aapm tg18 ) for primary and secondary displays primary displays secondary displays geometrical distortion , % maximum luminance , cd / m2 contrast ratio luminance response , % luminance uniformity , % resolution noise veiling glare distorsioni geometriche , % luminanza massima , cd / m2 contrasto risposta di luminanza , % uniformit di luminanza , % risoluzione rumore veiling glare > 170 > 250 0 ( cid : 2 ) cx ( cid : 2 ) 4 3 / 4 inserts ( cid : 3 ) 3 inserts > 170 > 250 0 ( cid : 2 ) cx ( cid : 2 ) 4 3 / 4 inserti ( cid : 3 ) 3 inserti tabella 1 tolleranze indicate da aapm tg18 per sistemi di visualizzazione primari e secondari monitor primari monitor secondari > 100 > 100 0 ( cid : 2 ) cx ( cid : 2 ) 6 2 / 4 inserts ( cid : 3 ) 1 inserts > 100 > 100 0 ( cid : 2 ) cx ( cid : 2 ) 6 2 / 4 inserti ( cid : 3 ) 1 inserto a . 
before calibrating , the x - rite contact photometer must be placed in the middle of the screen . with barco crt monitors , the procedure is repeated for every monitor of the pair . 
eizo monitors are instead controlled by the workstation as if they were a single monitor ( because of software and hardware limitations ) ; hence , the calibration curve applied will be the same for both monitors . 
in this case , the calibration will be performed first on the left then on the right monitor , and the quality of the greyscale curve will be checked to ensure it is within limits . 
the only possible correction is reduction of the brightness setting of the brightest monitor in order to equalize maximum luminance ( lmax ) as much as possible before the actual calibration . 
unfortunately , the user is given no information on the corrections made . geometric distortions deflections of the electron beam along its path often cause geometric distortions to appear on crt monitors that do not maintain the height / width ratios in the different screen areas . this is a known defect , and high - quality monitors usually incorporate appropriate correction circuits . 
partendo dal modello di barten della visione umana definita la curva dicom che associa un valore di luminanza tra 0 , 05 e 4000 cd / m2 a ognuno dei 1023 jnd che si suppongono discernibili dallocchio umano [ 8 ]  . 
la calibrazione fa in modo che , dato un intervallo di luminanza minima - massima , i valori di luminanza dei livelli di grigio intermedi si dispongano su jnd equispaziati , ottenendo quindi la linearizzazione percezionale [ 9 ]  . la procedura di calibrazione gestita in modo automatico dal software medical pro attualmente installato su tutte le stazioni destinate alla refertazione . 
i monitor eizo sono invece pilotati dalla workstation come se fossero un monitor unico ( a causa di limitazioni software e hardware ) , pertanto la curva di calibrazione applicata sar la stessa per entrambi i monitor . 
lunica correzione possibile ridurre limpostazione di luminosit ( brightness ) del monitor pi luminoso in modo da pareggiare lmax il pi possibile prima di procedere alla calibrazione vera e propria . 
i monitor barco lcd dispongono di un sensore incorporato che controlla in tempo reale la luminanza massima in modo da garantire una maggiore stabilit nel tempo di tale parametro . questo sensore anche utilizzato per la calibrazione dal software medical pro al posto del fotometro x - rite . 
purtroppo nessuna informazione viene fornita allutente sulle correzioni effettuate . distorsioni geometriche le deflessioni del pennello elettronico lungo il suo percorso portano spesso i monitor crt a presentare delle distorsioni di tipo geometrico in cui non sono mantenuti i rapporti altezza / larghezza nelle diverse aree dello schermo . 
according to the tg18 , under optimal conditions , the smallest insert observable in total darkness must be the same as can be observed under standard reporting conditions ; i.e. 
light conditions of the room should be adjusted to achieve this condition . however , the measurements were taken in a less - than - optimal reporting situation , with a good number of ambient lights [ 10 ]  . luminance and contrast the lmax and minimum luminance ( lmin ) are measured during the calibration procedure while a preset value can be set in the software . 
secondo il tg18 in condizioni ottimali il minimo inserto visibile in condizioni di buio totale deve essere lo stesso visibile nelle condizioni standard di refertazione ; i riflessi luminosi non devono cio interferire con la visione dei dettagli a basso contrasto . 
nelle misure effettuate stata comunque considerata una situazione di refertazione non ottimale , accendendo un buon numero di luci ambientali [ 10 ]  . luminanza e contrasto la luminanza massima ( lmax ) e quella minima ( lmin ) vengono misurate durante la procedura di calibrazione ed possibile impostare nel software un valore prefissato . 
nei monitor barco come lmax viene impostato un valore corrispondente a quello dichiarato dalla casa costruttrice come valore calibrato ; tale luminanza inferiore alla luminanza massima che il monitor in grado di emettere , in modo che sia possibile nel tempo mantenere la luminanza calibrata nonostante linevitabile decadimento dovuto alluso [ 11 ]  . 
i monitor eizo invece , per poter rispettare i limiti minimi di luminanza richiesti vanno impostati alla massima luminanza possibile ( brightness = 100% nella maggioranza dei casi ) , rendendo cos impossibile compensare il deterioramento che viene inoltre accelerato dagli alti valori di brightness impostati . 
il rapporto di contrasto ( cr ) dipende fortemente dal valore di lmin ; questultimo non pu essere abbassato a piacere poich renderebbe indistinguibili i dettagli a bassa luminanza , fig . 
eizo monitors , on the other hand , must be set to the highest lmax possible ( brightness = 100% in most cases ) to meet lmin requirements , thus making it impossible to compensate for deterioration , which is also accelerated by the high brightness values set . 
the contrast ratio ( cr ) depends largely on the lmin value ; it cannot be decreased at will because it would make the low luminance details vague , blurred by the ambient light reflections . 
these images are made up of 20% luminance background and of a central square of luminance spanning from black to white in 16 intervals with equally spaced grey levels , one per image . 
these measurements are taken by placing the contact photometer in the middle of the image . the luminance interval ( lmin - lmax ) is divided into 16 jnd equally spaced levels , resulting in a luminance vs . 
the same medical pro calibration software can automatically check the calibration following a similar procedure : the result is a percentage value indicating the degree of matching between the response curve and the dicom curve . 
this test is used for the periodic qa checks and , sometimes , to decide whether to recalibrate a monitor if it is out of tolerance . luminance uniformity luminance uniformity is often poor in crt monitors because of the different paths electrons have to travel before reaching the various spots of the screen . 
in barco monitors ( both crt and lcd ) , the evaluation was performed separately for each monitor of the pair while with eizo monitors , the five squares are distributed between both monitors , thus providing an overall uniformity for that pair . 
the tolerance suggested by aapm tg18 is 30% , as the maximum difference . spatial resolution the resolution capability of a monitor is not merely its speciconfusi dai riflessi della luce ambiente . 
le tolleranze indicate da aapm tg18 sono : lmax > 170 cd / m2 e cr > 250 . risposta di luminanza le misure di verifica della calibrazione sono state effettuate utilizzando i 16 fantocci geometrici aapm tg18 - ln ( fig . 3 ) , e il fotometro a contatto x - rite . 
queste immagini sono composte da uno sfondo di luminanza 20% e da un quadrato centrale di luminanza variabile dal nero al bianco in 16 intervalli a livelli di grigio equispaziati , uno per ogni immagine . 
maggiormente significativi sono i valori di risposta di contrasto , cio dl / l vs . jnd , la cui tolleranza consigliata del 10% rispetto al relativo valore nella curva dicom . 
lo stesso software di calibrazione medical pro in grado di effettuare un controllo automatico sulla calibrazione con un sistema analogo : il risultato fornito un valore percentuale indicante la corrispondenza della curva di risposta con la curva dicom . 
tale test viene utilizzato per i controlli di qualit periodici ed eventualmente per decidere di ricalibrare un monitor qualora fosse fuori dalle tolleranze . uniformit di luminanza luniformit di luminanza spesso carente nei monitor crt , a causa del differente percorso che gli elettroni devono percorrere per giungere nelle diverse regioni dello schermo . 
luniformit di luminanza stata misurata utilizzando un fantoccio geometrico incluso nel software medical pro ; composto da uno sfondo con luminanza 20% e da 5 aree quadrate ( centro e 4 angoli ) con luminanza 100% . 
nei monitor barco ( sia crt che lcd ) la valutazione stata effettuata separatamente per i due monitor della coppia , mentre per i monitor eizo i 5 quadrati sono distribuiti sui due monitor , fornendo quindi un valore di uniformit complessivo della coppia . 
la tolleranza proposta da aapm tg18 del 30% come massima differenza . risoluzione spaziale la capacit risolutiva di un monitor non semplicemente la dimensione del pixel dichiarata ma deve includere la capacit di controllare lemissione del singolo pixel indipendentemente da quelli vicini . 
resolution was evaluated through a visual test using the tg18 - cx phantom ( fig . 4 ) composed of a number of graphic inserts , with different levels of grey covering the entire surface of the monitor and showing in the middle a caption reporting the scores to be assigned to the resolution of those inserts . 
the average luminance in the middle of two test objects composed of horizontal and vertical bands , alternatively black and white , at the monitors nominal resolution ( nyquist frequency ) was measured with a hagner telescopic photometer . 
the two objects are expected to have a different average luminance because the electron beam is required to vary extremely quickly from minimum to maximum intensity and vice versa to draw the vertical bands , generating a lower average luminance because the maximum intensity condition has not been reached yet . 
these inserts are located in the middle and in the four corners of the tg18 - qc phantoaccording to aapm tg18 , the percentage difference between the average luminance cannot exceed 30% in the middle of the monitor . 
monitor response at half the frequency ( two black bands and two white bands ) was probed using the inserts contained in the same phantom . noise background noise on screen is one of the possible factors limiting resolution of low - contrast details . 
i punteggi variano da 0 ( perfetto ) a 9 ( gravi problemi di risoluzione ) ; sono presenti anche valori negativi che indicano un problema di funzionamento del monitor . 
con il fotometro telescopico hagner si misurata la luminanza media al centro di due oggetti test , composti da righe orizzontali e verticali alternativamente bianche e nere , alla risoluzione nominale del monitor ( frequenza di nyquist )  . 
ci si aspetta che i due oggetti abbiano luminanza media differente , in quanto al pennello elettronico sono richiesti rapidissimi passaggi dallintensit minima a quella massima e viceversa , per rappresentare le linee verticali , generando una luminanza media inferiore a causa del mancato raggiungimento della condizione di intensit massima . 
stata indagata anche la risposta del monitor a una frequenza dimezzata ( 2 linee nere e 2 linee bianche ) tramite gli inserti presenti nel medesimo fantoccio . rumore il rumore di fondo nella visualizzazione a schermo uno dei fattori che pu limitare la risolvibilit di dettagli a basso contrasto . 
la tolleranza suggerita che siano visibili 3 dettagli su 4 . i riflessi che avvengono allinterno della superficie del monitor producono un aumento della luminanza nelle aree scure adiacenti o circondate da aree ad alta luminanza . 
la luminanza percepita quindi fortemente variabile in funzione dellangolo di visione , provocando una degradazione del contrasto , arrivando perfino a valori negativi ( inversione del contrasto )  . per ovviare a questo problema necessario conoscere le fig . 
therefore , the perceived luminance is a heavily variable as a function of the viewing angle , causing some contrast degradation , even reaching negative values ( contrast inversion )  . 
lmax and lmin were measured with the hagner telescopic photometer aimed at the centre of the tg18 - ln phantoms , considering only the two extreme luminance values ( 0% and 100% )  . 
according to aapm tg18 , the cr for angles other than the perpendicular should not drop by more than 30% below the limit suggested for the contrast ( 250 ) ; hence , the maximum cr value allowed is 175 . anatomical images ten radiologists of different ages and experiences were recruited for this evaluation . 
the tg18 - qc pattern results from the wellknown society of motion picture and television engineers ( smpte ) phantom , which is currently the industrial standard to test television chains ; the use of this pattern is recommended by the international literature for day - to - day qa [ 12 , 13 ]  . results and discussion during acceptance tests , save for a few exceptions , monitors fig . 
6 tg18 - gv : nellangolo rappresentato un dettaglio degli inserti a basso contrasto presenti nel centro del fantoccio . caratteristiche del monitor in modo da definire un intervallo angolare in cui luminanza e contrasto rimangono entro i limiti di accettabilit . 
lmax e lmin sono state misurate con il fotometro telescopico hagner puntato sul centro dei fantocci tg18 - ln limitatamente ai due valori di luminanza estremi ( 0% e 100% )  . 
secondo aapm tg18 , il rapporto di contrasto per angoli diversi dalla normale non deve scendere di oltre il 30% rispetto al limite suggerito per il contrasto ( 250 ) ; quindi il massimo valore di cr consentito 175 . immagini anatomiche per questa valutazione sono stati arruolati dieci medici radiologi con diversa et ed esperienza . 
stato chiesto loro di valutare due immagini di riferimento : una radiografia toracica e una articolazione del ginocchio , in quattro modalit : su pellicola ( gold standard ) , su monitor barco crt , su monitor barco lcd e su monitor eizo lcd . 
nonetheless , the evolution of quality over time was followed by half - yearly checks . geometric distortions by their nature , lcd monitors are not affected by distortions , and this has been confirmed by the measurements always showing a 0% distortion . 
the measurements taken on crt monitors are always well within the 2% tolerance ( fig . 7 ) ; the slightly increasing trend that may be noticed may , in the future , require manually correcting the image geometry with the supplied software . ambient reflections no evident specular reflections were found because of the correct monitor position ; the presence of diffuse reflections caused by the light emitted by the adjacent ris monitor , by any view boxes and by other ambient lights . 
eizo monitors are significantly less sensitive to this effect , owing to the absence of glass protections , which is typical of lcd monitors . as a result , taking into account the presence of ambient light sources is crucial ; when possible , they must be switched off before using monitors for medical purposes . 
the different quality of monitors stands out immediately : medical models feature a high lmax , as well as the ability to keep it constant over a long period . eizo monitors are , instead , pushed to the limit from the very first day to meet the minimum requirements , determining their quick deterioration . 
il pattern tg18 - qc deriva dal ben noto fantoccio smpte che attualmente lo standard industriale per la verifica delle catene televisive ; lutilizzo di tale pattern raccomandato dalla letteratura internazionale come controllo di qualit giornaliero [ 12 , 13 ]  . risultati e discussione nel corso delle prove di accettazione , salvo piccole eccezioni , i monitor sono risultati entro le tolleranze consigliate . 
si comunque proceduto a seguire landamento nel tempo della qualit grazie allesecuzione di controlli con cadenza semestrale . distorsioni geometriche i monitor lcd per loro natura non risentono di distorsioni e ci stato confermato dalle misure che indicano una distorsione sempre pari allo 0% . 
this shows that calibration is crucial and requires a number of conditions to be met : the photometer must be in a correct position , the screen must be sufficiently clean and sufficient monitor warm up is necessary . 
the quality factor of the curve did not deteriorate much over time , and only occasionally did the monitor need to be recalibrated after the tolerances were no longer met . 
nel giro di un anno la luminanza massima di molti di questi calata al di sotto del valore di tolleranza di 170 cd / m2 , fatto che ne ha resa necessaria la sostituzione . 
i rapporti di contrasto dei monitor barco , sia crt che lcd si mantengono invece stabili su valori decisamente alti . risposta di luminanza in fase di accettazione stata acquisita la curva di luminanza e di contrasto per ogni monitor . 
8a , b andamento temporale della luminanza massima , aa monitor barco , bb monitor eizo . left monitor dicom right monitor left monitor dicom right monitor gray level / livello di grigio , jnd gray level / livello di grigio , jnd fig . 
luminance decay against time is consequently independent of the single monitor . spatial resolution visual test of spatial resolution confirmed the excellent resolution capability of the monitors in question , also over time . values measured on lcd monitors are constantly 0 , and on crt monitors , they stay within the tolerance range without decreasing over time . 
the luminance method was applied to crt monitors only during the acceptance to probe even more deeply the actual spatial resolution . the monitor proved scarcely able to represent pairs of lines at the nominal resolution along the horizontal axis . 
this realisation and the success of the tg18 - cx visual test let us assume that a structure such as the pairs of vertical lines can push the monitor to its limits but under perfectly theoretical conditions , which cannot be repeated in clinical usage . 
those frequencies cannot be distinguished by the average observer , who can always rely on software instruments to magnify the area of interest . noise on barco monitors , three inserts out of four are visible while on eizo monitors , only two are visible . 
no variations of this parameter were observed over time . the test is strongly affected by contrast resolution , which is lower on eizo monitors because of a lower maximum luminance . 
the noise generated by monitors , especially lcds , is predictably negligible ; hence , the inability to view the third insert , needed to meet tolerances , is really due to poor contrast . dal documento aapm tg18 , riportata sul grafico quale riferimento . 
ci indice della criticit della calibrazione che richiede molte accortezze , quali un corretto posizionamento del fotometro , una buona pulizia dello schermo e un sufficiente tempo di preaccensione del monitor . il fattore di qualit della curva non ha subito decadimenti particolari nel tempo e solo occasionalmente stato necessario ricalibrare il monitor a seguito di un mancato rispetto delle tolleranze . 
ovviamente il monitor finir per lavorare su di un range di jnd molto inferiore . uniformit di luminanza le misure di uniformit sono risultate sempre contenute nel 30% di tolleranza indicata . 
il degrado della luminanza in funzione del tempo risulta quindi indipendente dal singolo monitor . risoluzione spaziale il test visuale di risoluzione spaziale ha confermato lottima capacit risolutiva dei monitor in oggetto , anche nel corso del tempo . 
questa constatazione , unita al buon risultato ottenuto dal test visuale tg18 - cx , inducono a pensare che una struttura come le coppie di linee verticali siano in grado di portare il monitor ai suoi limiti , ma in condizioni del tutto teoriche e non presenti nellutilizzo clinico . 
tali frequenze infatti non sono neppure discernibili da un osservatore medio che potr sempre contare sullutilizzo di strumenti software per magnificare larea di interesse . veiling glare angular response all monitors were found to be within tolerance values , allowing three inserts to be distinguished . rumore table 2 shows the maximum angles from the perpendicular that maintain the luminance values acceptable . 
si pu quindi sconsigliare lutilizzo diagnostico dei monitor per angoli superiori a 30 per gli eizo e 50 per i barco . with eizo and greater than 50 with barco monitors is not to be recommended . immagini anatomiche anatomical images radiologists evaluations were analysed using students t test for paired samples , comparing the scores obtained with the different types of monitors with those obtained on film ( table 3 )  . 
eizo monitors were judged better than film , but in nearly all cases , the score was comparable although the test was carried out with highresolution images , which are not usually reported on these workstations . conclusions measurements taken confirm the good quality of the system and its suitability to that specific application [ 14 , 15 ]  . 
the quality of the greyscale curve declines over time , making it essential to repeat the le valutazioni dei radiologi sono state analizzate utilizzando il test t di student per campioni appaiati , confrontando i punteggi ottenuti dai diversi tipi di monitor con quelli del film ( tabella 3 )  . 
solo in pochi casi la qualit del monitor stata giudicata inferiore a quella della pellicola e neppure sono mancati casi in cui i monitor barco venissero valutati meglio del gold standard . 
i monitor eizo non sono mai risultati migliori del film , ma nella quasi totalit dei casi il giudizio risultato equivalente , anche se il test effettuato riguarda immagini ad alta risoluzione che solitamente non vengono refertate su queste stazioni di lavoro . conclusioni le misure effettuate confermano la buona qualit del sistema e ladeguatezza allutilizzo specifico [ 14 , 15 ]  . 
the measurements taken also enabled us to use the monitors in tasks suitable for their potential : eizo monitors , for instance , are not used in the reporting of high - resolution examinations while barco monitors showed their high quality even in the most critical applications . 
the periodic 6 - monthly checks are currently assigned to the medical physics expert , but it is not to be ruled out , that in the future , part of them could be performed by the radiographer . misurazioni effettuate hanno inoltre permesso di assegnare i monitor a compiti adeguati alle loro potenzialit : i monitor eizo ad esempio non vengono utilizzati per la refertazione di esami ad alta risoluzione . 
de cicco2 1unit operativa di radiologia domenico noto , azienda ospedali civili riuniti giovanni paolo ii , sciacca , italy 2unit operativa di neuroriabilitazione intensiva , fondazione salvatore maugeri , azienda ospedali civili riuniti giovanni paolo ii , sciacca , italy correspondence to : f . 
barbiera , via lanza 2 , i - 92019 sciacca , italy , tel . : + 39 - 0925 - 962203 , fax : + 39 - 0925 - 962316 , e - mail : radiologia@ospedaledisciacca.it * authors contributed equally to this study / * gli autori hanno contribuito in parti uguali alla realizzazione di questo studio received : 8 february 2006 / accepted : 22 march 2006 / published online : 11 august 2006 abstract purpose . 
in 1 year ( march 2004march 2005 ) 47 patients with oropharyngeal dysphagia due to different types of neurological deficit and who required rehabilitation were studied . all patients underwent : ( 1 ) clinical history assessment , ( 2 ) speech therapy assessment and ( 3 ) vfss using digital fluoroscopy ( 25 frames per second )  . 
patients were divided according to the waxman classification into seven levels of dysphagia , and the most suitable type of feeding was selected ( normal diet , restricted diet , artificial nutrition )  . 
in 13 / 47 ( 28% ) patients , vfss identified changes at the oral ( three patients ) or pharyngeal stage ( three patients ) or both ( seven patients ) but with no signs of silent aspiration . 
our experience shows that vfss precisely classifies the degree of dysphagia that conditions the dietary management of each neurologically compromised patient . key words dysphagia videofluorography swallow study rehabilitation riassunto obiettivo . 
tutti i pazienti sono stati sottoposti a : ( 1 ) valutazione clinico - anamnestica ; ( 2 ) valutazione logopedica ; ( 3 ) studio videofluorografico della deglutizione attraverso un sistema di fluoroscopia digitale ( 25 frame per secondo )  . 
tutti i pazienti sono stati classificati secondo la classificazione di waxmann in 7 livelli di disfagia e su questa base stata scelta la tipologia di nutrizione pi appropriata ( dieta libera , dieta con limitazione , nutrizione non orale )  . 
la videofluorografia ha confermato la presenza di aspirazione in 21 / 47 pazienti ( 44% ) di cui 4 ( 8% ) non sospettabili sulla base della valutazione clinico - logopedica in 13 / 47 ( 28% ) pazienti la videofluorografia ha dimostrato la presenza di alterazioni della fase orale ( 3 pz ) o della fase faringea ( 3 pz ) o di entrambe ( 7 pz ) ma senza segni di aspirazione silente . 
la nostra esperienza dimostra che attraverso lesame videofluorografico della deglutizione si pu ottenere una precisa classificazione della entit della disfagia e , su questa base , scegliere la dieta pi opportuna per ogni singolo paziente . parole chiave disfagia videofluorografia riabilitazione introduction introduzione f . 
the incidence of dysphagia following cerebral infarct varies from 25% to 45% [ 13 ] while it has been estimated that around 50% of patients with parkinsons disease are dysphagic [ 4 ]  . 
in these patients , the videofluorography swallow study ( vfss ) is crucial , given its ability to study disruptions in the swallowing process and , above all , to identify the presence of aspiration of food , which may cause aspiration pneumonia or choking [ 5 , 6 ]  . 
the aim of this study was to demonstrate the important role played by vfss in the management of dysphagic patients and especially in the selection of the type of diet the patient can maintain ( oral , artificial or mixed ) when they are admitted to the neurorehabilitation ward and after rehabilitative and speech therapy . materials and methods the study was based on a series of 47 patients observed during the period march 2004march 2005 suffering neurological deficit varying in nature ( table 1 ) and varying degrees of oropharyngeal dysphagia . 
lincidenza di disfagia dopo infarto cerebrale stata stimata in percentuali variabili da 25% al 45% [ 13 ] , mentre si calcola che circa il 50% dei pazienti con malattia di parkinson presentino disfagia [ 4 ]  . in questi pazienti lesame videofluorografico della deglutizione considerato di fondamentale importanza in quanto consente di studiare lalterazione della dinamica deglutitoria e soprattutto di identificare la presenza di aspirazione del materiale alimentare che pu causare polmonite ab - ingestis o soffocamento [ 5 , 6 ]  . scopo del presente lavoro quello di dimostrare il ruolo centrale dellesame videofluorografico della deglutizione nel management del paziente disfagico ed in particolare nella selezione del tipo di alimentazione che il paziente pu mantenere ( orale o artificale o mista ) al momento dellammissione nel reparto di neuroriabilitazione e dopo terapia riabilitativa e logopedica . materiali e metodi il presente lavoro si basa su una casistica di 47 pazienti osservati nel periodo compreso tra marzo 2004 e marzo 2005 e con deficit neurologici di vario tipo ( tabella 1 ) e differente grado di disfagia orofaringea . i pazienti , 27 maschi e 20 femmine , presentavano et compresa tra 16 e 80 anni e venivano inviati per un programma di riabilitazione neuromotoria . 
in realt il numero di pazienti disfagici , osservati nellarco di tempo considerato nel presente lavoro , stato di 62 su un totale di 270 pazienti ricoverati : di questi 62 pazienti , 3 sono stati esclusi in quanto non valutabili dal punto di vista logopedico e radiologico per il grave deficit cognitivo ; in altri 12 pazienti stata fatta la valutazione logopedica , ma non stato possibile eseguire lo studio videofluorografico con sufficiente attendibilit diagnostica ( pazienti irrequieti , con postura del capo non adeguata o impossibilitati a trattenere anche minime quantit di mdc nel cavo orale )  . tutti i 47 soggetti sono giunti alla nostra osservazione entro i primi 6 - 12 mesi dallesordio della disfagia e sono stati sottoposti a : 1 . 
valutazione logopedica con lanalisi di : livello di vigilanza , prassie bucco - linguo - facciale , sensibilit e motricit dellapparato linguo - buccale , attuazione di prove di deglutizione , ricerca di eventuali segni di inalazione e riflessi di protezione , studio delle funzioni correlate con la deglutizione ( fonazione e respirazione )  . sulla base della valutazione clinica e logopedica si sono suddivisi i pazienti in : f . 
speech assessment with analysis of the level of awareness , oral - buccal - lingual apraxia , sensitivity and motility of the lingual - buccal apparatus , swallowing trials , search for possible signs of aspiration and protection reflexes and study of functions correlated with swallowing ( phonation and breathing )  . 
to correlate the clinical data with the speech assessment and vfss findings , we used the dysphagia severity rating scale proposed by waxman et al [ 7 ] ( table 2 ) in which each patient has a different degree of dysphagia ( 7 points ) based on clinical findings ( more - or - less severe dysphagia , lengthening of meal times , nutritional state ) , speech assessment ( presence / absence of cough reflex , presence and degree of voluntary cough , ability to learn swallowing techniques and compensatory strategies ) and the findings of vfss ( presence / absence of aspiration , aspiration of one or more consistencies , amount of material aspirated , presence of cough reflex )  . 
on this basis , all patients were classified using the 7 - point scale , and as a result , the most appropriate type of diet was selected , as follows : free diet restricted diet ( semisolid homogenised with liquid restrictions ; semiliquid with a higher water content than the first ) nothing - by - mouth diet [ percutaneous endoscopic gastrostomy ( peg ) or nasogastric tube ] during their hospital stay , patients underwent speech therapy and a rehabilitation programme aimed , among other things , at regaining correct posture and in some cases ( patients with aspiration ) , a second vfss . 
on the basis of clinical course and degree of recovery obtained , modification to the hospital diet was assessed at the time of discharge . results results of the speech assessment showed suspected dysphagia with aspiration ( cough , gurgly voice , desaturation , low - grade fever , feeling of choking ) in 29 / 47 patients ( 61.7% ) while in the remaining cases , alterations to the oral and pharyngeal phases of swallowing were present but signs indicating aspiration were absent . 
in 13 / 47 patients ( 28% ) , vfss demonstrated the presence of alterations to the oral phase ( three cases ) or the pharyngeal phase ( three cases ) or both ( seven cases ) but without silent aspiration . 
in the remaining 13 patients ( 28% ) , vfss showed no swallowing alterations . on the basis of clinical and radiological findings , the patients were classified according to the waxman dysphagia dellesame videofluorografico il radiologo non era a conoscenza del risultato della valutazione logopedica ( studio in cieco )  . al fine di correlare il dato clinico con quello logopedico e videfluorografico abbiamo utilizzato la classificazione della disfagia di waxmann [ 7 ] ( tabella 2 ) nella quale ogni paziente ha un grado differente di disfagia ( 7 livelli ) a seconda di dati clinici ( disfagia pi o meno grave , allungamento del tempo del pasto , stato nutrizionale ) , valutazioni logopediche ( presenza / assenza del riflesso della tosse , presenza e entit della tosse volontaria , capacit di apprendimento delle tecniche di deglutizione e possibilit di compenso ) e risultati dellesame videofluorografico ( presenza / assenza di aspirazione , aspirazione di una o pi consistenze , entit del materiale aspirato , presenza del riflesso tussigeno )  . 
su questa base tutti i pazienti sono stati classificati in 7 livelli di disfagia e , di conseguenza , si selezionata la tipologia di nutrizione pi opportuna suddivisa come segue : dieta libera ; dieta con limitazioni ( dieta semisolida omogenea con restrizione di liquidi ; dieta semiliquida a maggiore contenuto idrico rispetto alla prima ) ; nutrizione non orale ( peg o sondino naso - gastrico )  . durante il ricovero i pazienti sono stati sottoposti a training logopedico oltre che a programma riabilitativo mirato anche al recupero della postura e in alcuni casi ( pazienti con aspirazione ) a rivalutazione videofluorografia . 
sulla base dellevoluzione clinica e del grado di recupero ottenuto si valutata , allatto della dimissione , la possibilit di modificare lapproccio nutrizionale avuto durante il ricovero . risultati i risultati della valutazione logopedica hanno fatto rilevare il sospetto di disfagia con aspirazione ( tosse , voce gorgogliante , desaturazione , rialzo termico , senso di soffocamento ) in 29 / 47 pazienti ( 61 , 7% ) mentre nei restanti casi erano presenti alterazioni della fase orale e faringea della deglutizione in assenza di segni che facessero sospettare la presenza di aspirazione . la videofluorografia ha confermato la presenza di aspirazione in 21 / 47 pazienti ( 44% ) : il confronto fra i dati della videofluorografia e quelli della valutazione clinico - logopedica ha fatto rilevare che solo in 17 casi il sospetto clinico di aspirazione stato confermato , mentre in 4 casi ( 8% ) lesame radiologico ha dimostrato la presenza di aspirazione non sospettata clinicamente . 
in 13 / 47 ( 28% ) pazienti la videofluorografia ha dimostrato la presenza di alterazioni della fase orale ( 3 pz ) o della fase faringea ( 3 pz ) o di entrambe ( 7 pz ) , ma senza segni di aspirazione silente . 
nei restanti 13 / 47 ( 28% ) pazienti la videofluorografia non presentava alterazioni della dinamica deglutitoria . sulla base della valutazione clinica e radiologica alla ammissione stata effettuata la classificazione waxmann del grado di disfagia dei pazienti e si sono adottate le seguenti strategie nutrizionali : 1 . 
in 1 / 13 patients , despite the lack of vfss evidence of significant swallowing alterations , the presence of cognitive alterations and the minimal autonomy related to psychophysical deficit suggested placing the patient on a semisolid diet with restrictions on solids ; at the end of the rehabilitation period , with the return of a certain degree of autonomy , the patient was placed on a free diet . 
two other patients , one of whom admitted with an in situ tracheostomy cannula and the other with a peg , followed a diet with a restriction of some consistencies ( in the patient with tracheostomy cannula ) and mixed ( via the peg and oral with restrictions ) during their hospital stay as a precautionary measure and to enable their return to an adequate nutritional state . 
the 13 patients with alterations of the oral and / or pharyngeal phase of swallowing but without silent aspiration were classified as grades 2 ( nine patients ) and 3 ( four patients ) and placed on a diet with restrictions of some consistencies . 
22 penetrazione laringea sub - epiglottica con presenza di mezzo di contrasto anche nel ventricolo di morgagni ( rischio di aspirazione ) ; ristagno di mdc in faringe . classificazione di waxmann sono stati trattati come segue : 10 / 13 pazienti sono stati lasciati in dieta libera . 
in 1 / 13 pazienti - pur non essendovi evidenza videofluorografica di alterazioni deglutitorie significativela presenza di alterazioni dello stato cognitivo e la scarsa autonomia legata al deficit psico - fisico ha suggerito di porre il paziente a dieta semisolida omogenea con limitazione dei solidi ; al termine del periodo riabilitativo , con il recupero di un certo grado di autonomia il paziente stato posto a dieta libera . 
altri 2 pazienti erano portatori di cannula endotracheale e peg al momento della ammissione ; durante la degenza si effettuata una alimentazione con limitazione di alcune consistenze ( nella paziente con cannula endotacheale ) e mista ( attraverso la peg e orale con limitazione ) a scopo precauzionale e per consentire il recupero dello stato nutrizionale . 
i 13 pazienti che presentavano alterazione della fase orale e / o faringea della deglutizione ma senza fenomeni di aspirazione silenziosa sono stati classificati nei livelli 2 ( 9 pz ) e 3 ( 4 pz ) della classificazione di waxmann e posti in dieta con limitazione di alcune consistenze . 
i 21 pazienti con aspirazione silenziosa venivano classificati come segue : 1 pazienti venivano classificati nei livelli 5 ( 4 pz ) ( fig . 2 ) e 4 ( 7 pz ) della classificazione di waxmann ; 10 di questi pazienti venivano posti allatto del ricovero a dieta con limitazione e alla dimissione 2 venivano posti a dieta libera . 
44 aspirazione di mezzo di contrasto con opacizzazione della trachea . stay while one patient had the peg removed after rehabilitation and at discharge was placed on a diet with restrictions . 
interrupting artificial feeding in the remaining six patients , even after rehabilitation , was not possible . discussion the role of videofluorography in identifying swallowing alterations has been codified in a number of studies [ 46 , 8 ] , which also demonstrated how the examination is crucial for revealing aspiration , particularly aspiration not followed by the cough reflex ( silent aspiration )  . 
a relatively large case series also recently demonstrated how the risk of developing pneumonia is closely correlated with the degree of swallowing dysfunction appreciable at vfss ; in fact , the risk of contracting pneumonia in such patients was calculated to be four times greater than in patients with normal swallowing in the event of laryngeal penetration , ten times greater in the event of aspiration and 13 times greater in the event of silent aspiration [ 9 ]  . 
this is why the type of dysphagia needs to be classified on the basis of degree and type of penetration or aspiration of contrast material so as to identify the type of diet the patient should maintain to avoid severe respiratory complications . over the years , a number of different scales have been developed to assess the degree of dysphagia , which take into account the findings of videofluorography : the waxman scale [ 7 ] , the rosenbek penetration - aspiration scale [ 10 ] ; the ott scale , which distinguishes four levels of dysphagia ( 03 ) [ 11 ] ; and the daniels scale , with five increasing levels of dysphagia ( 05 ) [ 12 ]  . 
of all of these , the waxman scale [ 7 ] appeared to be the most useful in that it correlates vfss findings to the degree of dysphagia and then to the type of diet the patient can maintaindeed , careful classification of the patients enabled us to place the clinical and vfss findings in one of the seven waxman degrees of dysphagia and zienti come il rischio di sviluppare polmoniti sia strettamente correlato al grado di disfunzione della deglutizione apprezzabile alla valutazione videofluorografica ; in particolare il rischio di polmonite stato quantizzato , rispetto ai pazienti con deglutizione normale , in 4 volte superiore in caso di penetrazione laringea , 10 volte superiore in caso di aspirazione e 13 volte superiore in caso di aspirazione silenziosa [ 9 ]  . da quanto sopra deriva la necessit di classificare il tipo di disfagia sulla base della entit ed il tipo di penetrazione o aspirazione del mdc cos da individuare correttamente il tipo di alimentazione che il paziente deve mantenere per evitare gravi complicanze respiratorie . 
 da questo punto di vista nel corso degli anni sono nate diverse scale di valutazione del grado di disfagia che tengono conto dei reperti videofluorografici : la scala penetrazione - aspirazione di rosenbek [ 10 ] , la scala di ott che distingue quattro livelli di disfagia ( 03 ) [ 11 ] e la scala di daniels in cinque livelli crescenti di disfagia ( 05 ) [ 12 ]  . tra tutte , la scala elaborata da waxmann [ 7 ] ci sembrata pi utile in quanto correla i reperti videofluorografici al grado di disfagia e , successivamente , al tipo di alimentazione possibile . 
lattenta classificazione dei pazienti ci ha consentito , infatti , di inquadrare i reperti videofluorografici e clinici in una delle sette categorie di waxmann e di impiegare le indicazioni dietetiche previste , in quasi tutti i casi . dalla nostra esperienza risulta chiaro che il ruolo dellesame videofluorografico fondamentale nel management del paziente disfagico in quanto consente un preciso grading dellalterazione il che influenza strettamente la gestione nutrizionale e la scelta tra alimentazione orale e non orale . si possono , inoltre , trarre alcune considerazioni : 1 . 
 our findings clearly demonstrate the crucial role of vfss in the management of the dysphagic patient in that the study enables precise staging of the alteration , which directly influences dietary management and the choice between an oral or artificial diet . 
vfss enabled patients with aspiration to be identified with extreme certainty and greater accuracy than the clinical assessment ( 17 aspirations out of the 29 suspected ) ; our data confirm the findings in the literature [ 46 ] with regard to the ability to identify aspirations in the absence of clinical suspicion , as occurred in four patients in our study . 
the possibility of radiologically reassessing the patient before discharge and thus monitoring the degree of recovery from dysphagia offers promise in identifying the type of diet the patient will be able to maintain at home at the end of rehabilitation . conclusions we feel that our findings demonstrate the central role of vfss in classifying the degree of dysphagia , the knowledge of which influences dietary management of the patient affected by neurological deficit . 
nonetheless , since complex patients are involved , at times with severe cognitive deficit and a limited degree of autonomy , the dietary and rehabilitative approach needs to be carried out ad hoc through the assessment of a multidisciplinary team in which the radiologist is a vital member . 
tuttavia in alcuni casi lapproccio suggerito stato modificato sulla base del grado di deficit cognitivo generale del paziente , del grado di autonomia nella gestione complessiva dellalimentazione e dello stato nutrizionale complessivo al momento della ammissione in reparto . 3 la possibilit di rivalutare radiologicamente il paziente prima della dimissione e monitorare cos il grado di recupero della disfagia appare uninteressante potenzialit ai fini della individuazione della tipologia di nutrizione che il paziente potr mantenere al domicilio al termine del trattamento riabilitativo . conclusioni riteniamo che la nostra esperienza sia dimostrativa del ruolo centrale dellesame videofluorografico della deglutizione nella classificazione della entit della disfagia il che condiziona la gestione nutrizionale del paziente affetto da deficit neurologico . tuttavia , poich si tratta di pazienti complessi , con a volte grave deficit cognitivo e limitazione del grado di autonomia , lapproccio nutrizionale - riabilitativo dovr a volte essere fatto ad hoc tramite valutazione di una equipe multidisciplinare di cui il radiologo deve assolutamente fare parte . 
berletti minerva medica , torino ( 2006 ) isbn 88 - 7711 - 525 - 4 published online : 8 september 2006 scrivere il referto di un esame radiologico una vera arte , un compito certamente pi difficile di quanto possa sembrare . 
in effetti possiamo dire che oggi pi che mai il referto il vero atto medico del radiologo . gli autori , e in particolare schiavon , si sono resi conto di questa importanza e da anni hanno dedicato allargomento interventi , pubblicazioni varie e hanno anche organizzato un congresso . 
dalla palma , presidente della sirm , e con una breve nota di ringraziamento , nella quale impressiona il gran numero di personalit e strutture intervistate e coinvolte nella redazione del testo : esponenti del mondo della filosofia , della psicologia , della scienza delle formazioni , un italianista di fama nazionale e televisiva , esperti di fisica , di medicina legale e un buon numero di esponenti del mondo manageriale . 
naturalmente sono quelli che maggiormente attirano lattenzione , linteresse e la curiosit del lettore , il quale improvvisamente si accorge di essere completamente coinvolto e intento a cercar di capire dove , quale , di che tipo lerrore , e... 
dalla modalit di impostazione del referto , dallimpiego di alcune espressioni si pu , ad esempio , riconoscere un radiologo veneto discendente dal mitico lenarduzzi e differenziarlo da un radiologo lombardo discendente dal maestro ratti o da un ligure discendente dallancor pi mitico vallebona vi sono espressioni , create e care ai nostro maestri , che , con tutta la buona volont non si riescono a sradicare . 
e cos riaffiorano ( anche nel testo che leggiamo ) le ombre ( lombra cardiaca , lombra mediastinica , lombra renale , lombra dellilo ) o gli emidiametri destro e sinistro del cuore . 
le ombre andavano bene ai tempi di busi e di balli , quando il radiologo , immerso nel buio pi completo della sala , scrutava sullo schermo radioscopico e vedeva soltanto e veramente delle ombre . 
quelle che abbiamo oggi sono splendide immagini chiarissime , spesso multiplanari o addirittura tridimensionali , che ci portano in un mondo completamente diverso da quello del bellissimo libro dei cacciatori di ombre di donizetti . 
e i diametri ! i diametri ci riconducono ai tempi fumosi , complicatissimi e spesso inconcludenti di alcuni dei grandi maestri come perona , palmieri o cignolini , in cui il radiologo era costretto a basarsi su queste misurazioni complesse e difficili , che non portavano mai a qualche cosa di clinicamente sicuro . 
oggi , che sul cuore sappiamo praticamente tutto , un buon radiologo deve avere il coraggio di parlare di ventricolo , di atrio , di aorta , di arteria polmonare . questopera inoltre ricca di dati ed elementi utili , di osservazioni critiche , di spunti che costringono a riflettere e a pensare , esposti sempre in maniera chiara e originale . 
non v dubbio che la conoscenza dei dati clinici e degli essenziali elementi anamnestici con un chiaro quesito da parte del medico possono influenzare e orientare in maniera determinante la stesura del referto e il giudizio diagnostico finale . 
gli autori riportano da schopenhauer che : non vi nulla di pi facile che scrivere in modo che nessuno possa capire , come nulla pi difficile che esprimere pensieri significativi in modo che ognuno possa comprenderli e da calvino che parlare oscuramente lo sa fare ognuno , ma chiaro pochissimi . 
opportuno avere sempre presente il referto deve essere chiaro , corretto , conciso , completo , coerente e competente , alle quali aggiungono una settima c , condiviso . nel testo che segue ognuno di questi punti viene analizzato , criticato , commentato ed esemplificato in maniera esauriente e originale . 
facile comprendere lestrema importanza di questo capitolo e la grande utilit che pu avere la sua analisi e la conoscenza di ogni risvolto che pu avere nellesercizio della nostra professione . 
attraverso una serie di utili consigli vengono esposti e analizzati problemi medicolegali che possono riguardare lattivit del radiologo , espressa proprio nella sua manifestazione pi importante : la refertazione dellesame . si deve sottolineare che tutto quanto fin qui esposto riguarda non soltanto gli esami radiologici tradizionali , ma anche le nuove metodiche , come lecografia , la tc e la rm . alla fine del libro viene proposto un capitolo di suggerimenti , nel quale i diversi principi esposti vengono book review riassunti e considerati in senso generico . 
auflage ( volume della serie rrr referenz - reihe radiologie ) gabriele benz - bohm ( ed ) georg thieme verlag , stuttgart , new york ( 2005 ) isbn 3 - 13 - 107492 - 2 published online : 8 september 2006 questo volume della serie rrr referenz - reihe radiologie ( collana di riferimenti radiologici ) giunto alla sua seconda edizione e , da quanto precisato nell ' introduzione da parte della curatrice , risulta molto ampliato rispetto alla prima . 
diviso in 10 capitoli ( caratteristiche della tecnica e delle radiazioni ; anatomia speciale del prematuro ; scheletro ; torace e mediastino ; apparato digerente ; apparato urinario ; ecografia speciale ; rmn ; tac ; angiografia ed interventistica ) , scritti dalla " crema " dei radiologi pediatrici tedeschi , il volume affronta i vari temi di questo delicato settore della radiologia , offrendo al lettore una descrizione precisa e sintetica della materia e divenendo cos opera di riferimento giornaliero , che evita spesso il ricorso a trattati pi ampi dedicati alla materia . il testo , nella sua sinteticit in grado di fornire con grande precisione i cardini dell ' argomento , correlato da tabelle di sintesi e da incisi a lato della pagina , ben indicati da frecce e caratteri evidenziati in blu , in cui vengono sintetizzati i punti fondamentali relativi all ' argomento trattato . di grande utilit pratica risultano le tre pagine in cui vengono raccolte , prima dellindice del volume , tutte le abbreviazioni utilizzate successivamente nel testo , evitando cos il rompicapo che le stesse possono provocare nel loro riscontro . le immagini sono riprodotte con la precisione e bellezza spettacolare , di cui si fa vanto la casa editrice , siano esse in bianco e nero o a colori : molto belli e dettagliati anche i disegni , esplicativi o di sintesi di particolari situazioni , nonch le tavole anatomiche . ogni capitolo correlato da una bibliografia sintetica , che consente di ulteriormente ampliare quanto descritto nello stesso . l ' indice analitico , infine , permette di individuare con immediatezza gli argomenti ricercati . questo volume pu essere considerato un testo di riferimento e pertanto denominarlo bigino risulterebbe termine assai riduttivo . 
ad esempio , delle 100 pagine in cui viene trattato lo scheletro , ben 17 si occupano del grave e sempre pi ricorrente problema del bambino maltrattato , in tutte le sue sfaccettature . notevole parte , inoltre , dedicata , non solo all ' ecografia , ma soprattutto alla rm , in particolare nello studio dell ' encefalo . 
sono sicuro che , qualora tradotto in italiano o inglese , la diffusione dello stesso sarebbe veramente significativa , proprio per le sue caratteristiche di sintesi , precisione e corretta spiegazione della materia . 
passariello1 1dipartimento di scienze radiologiche , 2dipartimento di chirurgia toracica , universit degli studi la sapienza , viale regina elena 324 , i - 00161 roma , italy correspondence to : f . 
nine patients with emphysema were studied by low - dose chest mdct ( 64 - slice somatom sensation cardiac , siemens ) with a collimation of 64x0.6 mm and a slice thickness of 1 mafter treatment , mdct scans were repeated at 7 and 30 days . 
volume assessment at 30 days showed a 29% reduction in rul volume in patient a , a 15% reduction in patient b , a < 1% reduction in patient c and a 30% reduction in patient d . 
in patients undergoing endobronchial valve placement , mdct with dedicated software allows for a better evaluation of volume reduction of a single lobe and of the whole lung . key words lung volumetric assessment endobronchial valve placement mdct emphysema riassunto obiettivo . 
nove pazienti con enfisema sono stati sottoposti a un esame tcms a bassa dose , ( somatom sensation cardiac , siemens , 64 strati ) : collimazione 64x0 , 6mm ; spessore di strato 1 mdopo il trattamento , lindagine tc stata ripetuta a distanza di 7 e 30 gg . 
quattro pazienti , ( a - d ) , in cui la tc ha evidenziato una maggior condizione enfisematica a carico del lobo superiore di destra ( lsd ) , sono stati sottoposti a sve . 
la valutazione volumetrica con tcms , in pazienti sottoposti a sve , effettuata con software dedicato , consente di valutare leffettiva rvp sia globale che per singolo lobo . parole chiave volumetria polomonare by - pass valvolari endobronchiali tcms enfisema f . 
its pathophysiology is characterised by dilatation of air spaces distal to the terminal bronchioles , and it is determined by irreversible , destructive changes in the alveolar walls [ 13 ]  . 
lung transplant provides better long - term results , but the number of organ donors is inadequate , and the selection of suitable candidates is problematic ( advanced age or concomitant disease ) [ 79 ]  . 
lung - volume reduction surgery ( lvrs ) aims to reduce total lung volume by 20%30% , thus improving chestwall elastic recoil ; however , this method presents the obvious limitation of subjecting patients , who are often elderly and with evident restrictive ventilatory defects , to a major surgical operation [ 4 ]  . 
more recently , several authors have suggested a less invasive procedure known as bronchoscopic lung - volume reduction ( blvr ) , which involves the placement of valves inside the bronchi leading to the most affected lobe . this procedure is both highly reliable , with low intraand postoperative morbidity and mortality , and reversible in that the valves may be removed at any time [ 10 ]  . 
it is thought that the implantation of one - way endobronchial valves allows the air to flow out but not to flow in , resulting in collapse of the nonventilated parenchyma [ 11 , 12 ]  . 
the aim of our study was to assess with a 64 - slice ct scanner lung - volume changes in patients undergoing blvr and to correlate these data with spirometric tests in order to confirm the true clinical benefits of the procedure . 
 materials and methods patient selection between june and september 2005 , nine men aged 6070 years referred to us from the department of thoracic surgery and respiratory medicine were studied by low - dose multislice chest computed tomography ( ct )  . 
the reason for referral was to evaluate the extent of emphysema to assess eligibility for endobronchial - valve placement ( emphasys endobronchial valve , redwood city , ca , usa )  . 
selection criteria for valve placement were severe limitation in daily activities despite adequate medical therapy , severe dyspnoea , radiological evidence of heterogeneous emphysema , forced expiratory volume in 1 s ( fev1 ) < 35% after administration of bronchodilators , residual volume ( rv ) > 180% and age lenfisema polmonare rappresenta una delle principali cause dinvalidit e di mortalit del mondo occidentale ; la fisiopatologia caratterizzata dalla dilatazione degli spazi aerei posti distalmente ai bronchioli terminali e determinata da alterazioni distruttive irreversibili delle pareti alveolari [ 13 ]  . 
ne consegue una riduzione della compliance polmonare e del ritorno elastico della parete toracica , che si manifesta con uniperinflazione aerea e un allungamento della fase espiratoria . il trattamento per via chirurgica principalmente rappresentato dal trapianto polmonare e dalla riduzione volumetrica del lobo maggiormente affetto [ 46 ]  . 
entrambe le metodiche presentano dei limiti ; il trapianto garantisce migliori risultati a lungo termine , ma subordinato allinadeguato numero di donatori di organi e alla difficolt di selezionare dei possibili candidati allintervento ( et avanzata o patologie concomitanti ) [ 79 ]  . 
la riduzione volumetrica polmonare per via chirurgica , ha come intento quello di poter ridurre il volume polmonare totale di circa il 20%30% , migliorando in tal modo il ritorno elastico della parete toracica ; tuttavia essa presenta lovvio limite di dover sottoporre un paziente , spesso anziano e con una evidente patologia restrittiva polmonare , ad un severo intervento chirurgico [ 4 ]  . recentemente diversi autori hanno proposto un intervento meno invasivo , rappresentato dallo stenting valvolare per via endobronchiale , eseguito con posizionamento di valvole allinterno dei bronchi del lobo maggiormente affetto . 
tale intervento si presenta molto affidabile , con bassa morbilit e mortalit intra e post operatoria e in pi reversibile , dal momento che le valvole possono essere rimosse in qualsiasi momento [ 10 ]  . 
ipoteticamente lapplicazione per via endobronchiale di valvole unidirezionali ( blvr , bronchoscopic lung volume reduction ) , consente allaria di uscire , ma non di entrare , determinando un collabimento del parenchima disventilato [ 11 , 12 ]  . 
lo scopo del nostro studio stato quindi di valutare , utilizzando una apparecchiatura tc a 64 strati , le modificazioni volumetriche nei pazienti trattati e di correlare tali dati con le prove spirometriche , per poter verificare il reale beneficio clinico . materiali e metodi selezione dei pazienti nel periodo compreso tra giugno e settembre 2005 , 9 pazienti , tutti uomini di et compresa tra i 60 e 70 anni , provescanning protocol procedura chirurgica between 35 and 75 years . 
exclusion criteria included radiological evidence of homogeneous emphysema and isolated bullae , being a smoker , paco2 > 50 mmhg , dlco < 20% , productive cough , and small airway disease . 
efficacy of the intervention was assessed by multidetector - row ct ( mdct ) scans repeated 7 and 30 days after the procedure to evaluate lung - volume reduction , and by spirometry and the 6 - min walking test , to evaluate clinical improvement . 
correct positioning was then checked by fibreoptic bronchoscopy . examinations were performed with a 64 - slice ct scanner ( somatom sensation cardiac 64 , siemens ag , forchheim , germany ) with 32 detectors and a gantry rotation speed of 33 ms . 
images were then reconstructed with the combi ( combined reconstruction ) protocol : one with a 1 - mm slice thickness , 1 - mm reconstruction interval and the b70 reconstruction algorithm to assess lung parenchyma , and the other with a 5 - mm slice thickness , 5 - mm reconstruction interval , and the b30 algorithm to study the mediastinuthe raw data sets were all saved on our pacs ( lifeweb ferrania imaging technologies ) for further processing , if needed . image analysis and volume assessment axial images were analysed on a dedicated workstation by a radiologist experienced in chest pathology . 
the preoperative evaluation took into account the presence and distribution of emphysema , establishing : site ( topographic location within the lobes and identification of the most affected lobe ) , features ( centrilobular , panlobular , or paraseptal ) , presence of isolated bullae and possible presence of focal alterations suspicious for malignancy . 
the volume of the most affected lobe and of the entire right and left lung was then calculated . postprocessing was carried out on a dedicated workstation ( vitrea 2.8 , vital images )  . 
il motivo della richiesta era la valutazione della patologia enfisematosa di cui erano affetti , al fine di valutare lidoneit allintervento di posizionamento di valvole endobronchiali ( emphasys endobronchial valve , redwood city , ca )  . 
i criteri di inclusione per lo stenting valvolare prevedevano una severa riduzione dello svolgimento delle attivit quotidiane , nonostante una adeguata terapia medica , dispnea severa , valutazione radiologica di enfisema eterogeneo , fev1 < 35% dopo somministrazione di broncodilatatori , vr > 180% , et compresa tra 35 e 75 anni . 
i criteri di esclusione prevedevano evidenza radiologica di enfisema omogeneo e di bolle aeree isolate , lessere fumatore , paco2 > 50 mmhg , dlco < 20% , tosse produttiva , malattia delle piccole vie aeree . lo stesso esame era poi eseguito a distanza di 7 e 30 giorni dopo lintervento , contemporaneamente a test clinici , quali la spirometria e il test del cammino in 6 min , per verificare lefficacia dellintervento , valutabili a livello radiologico con una riduzione del volumi polmonari e a livello clinico con un miglioramento della sintomatologia . il protocollo per lintervento di stenting valvolare per via endobronchiale stato precedentemente approvato dal comitato etico . 
i pazienti sono stati informati adeguatamente e in anticipo sulla possibilit , che se la procedura per via endobronchiale fosse fallita , la riduzione volumetrica per via chirurgica sarebbe rimasta come ultima scelta . 
tutti i pazienti sono stati sedati con lidocaina per prevenire linsorgenza della tosse . attraverso il broncoscopio sono stati individuati i bronchi segmentali target , su cui attraverso un catetere guida , sono state posizionate le valvole . 
il paziente , al momento dellesame , stato invitato a sdraiarsi in posizione supina sul lettino con le braccia dietro la testa ed stato informato sulla necessit di dover mantenere unapnea respiratoria per pochi secondi . 
i parametri tecnici di acquisizione dello studio utilizzati sono stati su tutti i pazienti : collimazione 64x0 , 6 mm , spessore di strato 1 mm ; al fine di ridurre le radiazioni ionizzanti stata eseguita una scansione care dose , per la modulazione automatica della dose in base alla diversa attenuazione corporea . 
1a axial computed tomography ( ct ) image : postprocessing on the dedicated workstation ( vitrea 2.8 , vital images ) to evaluate right upper lobe volume by manual tracing of the lobe profile ( arrow )  . 
1a immagine assiale tc , fase di postprocessing eseguita su workstation dedicata ( vitrea 2.8 , vital images ) ; elaborazione del volume polmonare del lobo superiore del polmone destro , con traccia manuale del profilo del lobo ( freccia )  . 
in ogni caso i dati grezzi sono stati salvati sul nostro sistema pacs ( lifeweb ferrania imaging technologies ) , per successive eventuali elaborazioni . analisi delle immagini e valutazione volumetrica le immagini assiali sono state analizzate su una workstation dedicata , da un radiologo esperto di patologia toracica . 
la valutazione pre - operatoria ha preso in considerazione la presenza e la distribuzione dellenfisema in maniera qualitativa , distinguendo : sede ( localizzazione topografica nei lobi e lobo maggiormente colpito ) , caratteristiche ( centrolobulare , panlobulare e parasettale ) ; presenza di bolle isolate ; eventuale presenza di alterazioni focali sospette per essere di natura eteroplasica . 
successivamente stato effettuato il calcolo della volumetria per il lobo maggiormente affetto dallenfisema e per lintero polmone destro e sinistro . la fase di postprocessing stata eseguita su una workstation dedicata ( vitrea 2.8 , vital images )  . 
la valutazione post operatoria stata eseguita a distanza di 7 e 30 giorni ed ha preso in considerazione la valutazione della riduzione volumetrica ed eventuali patologie infiammatorie , secondarie allintervento ( processi broncopneumonici ) , oltre che la presenza di pneumotorace . risultati nella valutazione pre - operatoria lindagine tc ha confermato la presenza di enfisema in tutti i pazienti . 
in 6 la distribuzione stata prevalente a carico del lobo superiore del polmone di destra ; in 2 a livello del lobo superiore di sinistra , in 1 a livello del lobo inferiore destro . 
in 7 pazienti le caratteristiche morfologiche hanno evidenziato un tipo di enfisema di natura panlobulare ; in 1 paziente il riscontro prevalente stato di tipo parasettale ( anche se con alcune aree a carattere panlobulare ) ; in 1 paziente lenfisema stato definito come centrolobulare . 
in 3 pazienti sono state identificate bolle isolate ; in due pazienti localizzate a livello controlaterale rispetto il polmone maggiormente affetto ed in 1 paziente a livello del lobo inferiore , mentre la maggiore fig . 
isolated bullae were identified in three patients : in two , they were contralateral to the most affected lung ; in one , they were in the lower lobe whereas the emphysema was prevalently distributed in the ipsilateral upper lobe . in one patient , a spiculated nodular formation was detected , which was considered to be suspicious for malignancy . 
 in agreement with the thoracic surgeons , only five patients were considered eligible for treatment ; in four of them , preoperative evaluation indicated panlobular emphysema , with prevalent distribution in the right upper lobe ; in one patient , image analysis identified paraseptal emphysema , most prominent in the left upper lobe . 
in un paziente si riscontrata una formazione nodulare a margini spiculati , considerata sospetta per essere di natura eteroplasica . in accordo con i chirurghi toracici , in base ai criteri di inclusione precedentemente esposti , sono stati cos sottoposti a trattamento unicamente 5 pazienti ; in quattro la valutazione pre - operatoria ha dimostrato un enfisema con caratteristiche di tipo panlobulare e con maggior estensione a carico del lobo superiore di destra , cos sottoposto a trattamento ; in un paziente lanalisi delle immagini ha messo in evidenza un enfisema di tipo parasettale , pi evidente a livello del lobo superiore di sinistra . 
non si sono riscontrate invece modificazioni volumetriche per lintero polmone e per il polmone controlaterale . negli stessi pazienti stato rilevato un miglioramento delle condizioni cliniche , del fev1 , del volume residuo ( vr ) e della capacit polmonare totale ( cpt )  . 
i risultati dei dati volumetrici ed il confronto con i risultati spirometrici del pre e post trattamento sono riportati in tabella 2 e nella figura 4 . discussione lintroduzione nella pratica clinica della tc multistrato consente di ottenere in ununica apnea respiratoria informazioni su tutto il parenchima polmonare , con una alta qualit di immagine e con possibilit di un imaging isotropico e volumetrico . 
lelevata risoluzione spaziale con strati contigui consente , infatti , di delineare lanatomia polmonare e di poter ottenere informazioni volumetriche con eccellente dettaglio anatomico fornendo al radiologo ed al clinico importanti informazioni aggiuntive rispetto allutilizzo delle immagini 2d . 
la possibilit inoltre con le apparecchiature di ultima generazione di modulare la dose , ottenendo valori di esposizione poco pi alti di un esame radiografico standard , pone lindicazione per un utilizzo routinario della tc in molte applicazioni e nella valutazione dellenfisema [ 15 ]  . diversi autori hanno , infatti , valutato lutilizzo della tc nella identificazione , caratterizzazione e valutazione preoperatoria dei pazienti con enfisema [ 5 , 16 ]  . 
3a , b axial computed tomography ( ct ) images a before and b after endobronchial valve placement in a patient with severe emphysema predominant in the right upper lobe . 
3a , b immagini assiali tc : prima ( a ) e dopo 7 gg ( b ) dallintervento di inserimento di valvole endobronchiali in paziente con enfisema pi evidente in corrispondenza del lobo superiore destro . 
possibile evidenziare il progressivo collasso del lobo superiore ( punta di freccia ) , con relativo spostamento anteriore della grande scissura ( freccia )  . 2500 2000 1500 1000 fev 1 ( l / s ) cv ( l ) vt6m vol ( cc ) paz a paz b paz c paz d paz a paz b paz c paz d fig . 
4a , b rappresentazione grafica dellandamento del fev1 , della cv ( a ) , del walking test e del volume polmonare ( b ) nei pazienti sottoposti a trattamento valvolare endobronchiale . tire lung or contralateral lung was observed . the patients showed improved clinical condition , fev1 , rv and total lung capacity ( tlc )  . 
results of lung volume assessment and correlation with spirometric tests before and after treatment are shown in table 2 and in figure 4 . discussion the introduction of mdct into clinical practice enables us to study the entire lung parenchyma during a single breathhold with good image quality and the possibility of obtaining isotropic and 3d images . 
the high spatial resolution with lavori in letteratura abbiano dimostrato limportanza di una misurazione volumetrica e funzionale dellenfisema prima e dopo un trattamento di resezione polmonare , poca attenzione stata posta nelle modificazioni volumetriche successive ad un intervento di riduzione volumetrica del lobo affetto eseguita per via endobronchiale [ 4 , 17 , 18 ]  . linnovazione tecnologica nel campo della chirurgia toracica ha consentito , infatti , la creazione di valvole unidirezionali , introdotte per via endobronchiale , che consentono allaria di uscire durante lespirazione , ma non di entrare . lo scopo del nostro studio stato quindi quello di validare la tesi empirica di un collasso parziale del lobo , in cui sono posizionate le valvole con una conseguente riespansione anche del lobo contiguo , non pi sottoposto alleffetto massa di iperinflazione del parenchima trattato . 
nei pazienti sottoposti allintervento stato individuato a distanza di 7 gg una riduzione volumetrica del lobo superiore pari a 26% per a , 24% per b , < 3% per c e 22% per d ; a distanza di 30 gg a ha ottenuto una riduzione totale del lobo superiore di destra table 1 functional assessment before and after bronchoscopic volume lung reduction patient / evaluation f . 
furthermore , the capability of latest - generation devices to modulate the dose so that exposure is only slightly higher than that delivered by standard radiographic equipment supports the routine use of ct in many applications , including the assessment of emphysema [ 15 ]  . several authors have evaluated the use of ct in the identification , characterisation and preoperative assessment of emphysema [ 5 , 16 ]  . 
although recent reports demonstrated the importance of volumetric and functional measurements of emphysema before and after lung - reduction surgery , little pari al 29% , b del 15% , c < 1% e d pari al 30% . 
la valutazione volumetrica del polmone di destra e di sinistra , non ha invece mostrato apprezzabili modificazioni ; ci potrebbe confermare lipotesi di una migliore compliance elastica della parete toracica , conseguenza di una riduzione della compressione locale da parte di parenchima disventilato sul tessuto circostante [ 19 ]  . 
la correlazione con i dati spirometrici ha confermato una evidente riduzione del fev1 , della cv e dei valori del walking test , sia dopo sette , ma soprattutto nel controllo a distanza di 30 gg . non necessariamente come dimostrato dai dati del nostro studio si assiste ad un miglioramento contemporaneo dei parametri di funzionalit respiratoria . 
 technological advances in thoracic surgery have led to the development of one - way endobronchial valves , which allow air to flow out during expiration , but not to flow the aim of our study was to validate the empirical hypothesis that valve placement leads to partial collapse of the affected lobe and consequently to an increase in volume of the contiguous lobe , which is no longer compressed by the hyperinflated parenchyma . 
seven days after the procedure , upper - lobe volumes were found to be reduced by 26% in patient a , 24% in patient b , < 3% in patient c and 22% in patient d ; at 30 days , overall volume reduction of the right upper lobe was 29% for patient a , 15% for patient b , < 1% for patient c and 30% for patient d . volume assessments of the right and left lung did not reveal significant changes ; this could validate the hypothesis that chest - wall compliance improves as a result of the reduction in local compression by the nonventilated parenchyma [ 19 ]  . 
correlation with the spirometric data confirmed a marked reduction in fev1 and vc , and an improvement in the 6 - min walking test results after 7 days and especially after 30 days . 
there is still no certainty of a real and coincident correlation between radiological and clinical data ; for example , a minimal volume change of the right upper lobe in patient c corresponded to a effective clinical response , quantified as a 9% improvement in fev1 , a 70% improvement in vc and an over 90% increase in the distance covered in 6 m however , the actual volume changes and the fact that symptoms , in particular subjective breathlessness , improved confirms the clinical benefit of the technique and supports the use of ct for both the preoperative and postoperative evaluation of patients . 
in our experience , ct allowed for accurate patient selection in terms of not only assessment of emphysema but also detection of concurrent disease that , although decisive for patient selection , may be missed on standard chest radiograph . 
furthermore , the preoperative ct study lazione tra i dati radiologici ed i dati clinici , dal momento che , come dimostrato nel paziente ( c ) alla minima variazione radiologica volumetrica del lobo superiore del polmone di destra , a livello clinico corrisponde una efficace risposta , valutabile con un miglioramento del fev1 pari al 9% , della cv del 70% ed un incremento superiore al 90% nella distanza percorsa nel test del cammino . 
tuttavia il miglioramento sintomatologico , in particolare con una riduzione soggettiva della dispnea , conferma il reale beneficio clinico di questa tecnica , associato alla reale modificazione volumetrica conseguente al trattamento e propongono lutilizzo della tc non solo nella valutazione preoperatoria dei pazienti , ma anche nel post operatorio . nella nostra esperienza personale la tc ha consentito una accurata selezione dei pazienti , sia in termini di enfisema , che di eventuali patologie correlate , spesso non diagnosticate dal semplice esame standard del torace e che invece rappresentano criteri decisionali nellinclusione dei pazienti al trattamento . lo studio preoperatorio con tc ha inoltre permesso di definire con un maggiore grado di sicurezza rispetto al semplice esame radiografico standard le caratteristiche dellenfisema [ 20 ] , il lobo maggiormente colpito e leventuale presenza di bolle aeree isolate o diffuse , anchesse criteri di selezione del paziente al trattamento . in tutti i pazienti il protocollo di studio ha previsto lesecuzione di una tc del torace a bassa dose , sia nella valutazione pre - operatoria , che in quella postoperatoria . 
lutilizzo di un basso dosaggio , cos come riportato da altri autori , limita la qualit di immagine , con conseguente perdita di informazioni in particolare quando si vuol studiare linterstizio polmonare [ 21 ]  . 
sebbene nel nostro studio non sia stata eseguita una valutazione quantitativa sulla qualit di immagine , lutilizzo di un sistema di modulazione automatica della dose , possibile sulla nostra apparecchiatura , ha permesso di acquisire immagini ad alta risoluzione , considerate dal radiologo di buona qualit e comunque adeguata per was more accurate than the chest radiograph in defining the extent of emphysema [ 20 ] , the most affected lobe and the possible presence of isolated or diffuse bullae , which are also selection criteria for treatment . for all patients , the study protocol involved low - dose chest ct both in the preoperative and in postoperative assessment . 
use of low doses , as reported by other authors , reduces image quality , causing loss of data , especially in the study of the pulmonary interstitium [ 21 ]  . 
although our study did not include a quantitative evaluation of image quality , the use of our scanners automatic dose - modulation option made it possible to acquire high - resolution images , which our radiologist judged to be of good diagnostic quality . 
these high - performing scanners are able to acquire images with high spatial resolution during a single 4 - s breath - hold , an especially important consideration in patients with severe emphysema . 
several commercially available systems can now provide immediate volumetric information on lung parenchyma and offer the possibility of automatic segmentation without requiring manual tracing . the application of these systems may prove essential for the assessment of postoperative respiratory function in candidates for surgical or endobronchial volume reduction and in patients undergoing more - extensive lung resections , such as cancer patients . 
few clinical and radiological studies have dealt with this topic , and all of them were conducted on small series ; nonetheless , the results were encouraging not only in terms of surgical and clinical response but also as regards the correlation between mdct and respiratory function tests . 
a second limitation is that , unlike previous authors [ 2224 ] , we made no quantitative evaluation of the extent of emphysema ; however , our patients lung function tests had already revealed a restrictive disorder for which the patients were receiving medication ; in addition , the main aim of our study was to demonstrate the reduction in lung volume following treatment and the advantages of mdct in preand postoperative evaluation of these patients . 
possibile che il progresso tecnologico in termini di apparecchiature consentir , tramite lintroduzione di filtri di convoluzione sempre pi sofisticati , di ridurre ulteriormente la dose , fino a quella di un semplice esame radiografico . 
ultimo punto riguarda il ridotto tempo di scansione ottenibile con una apparecchiatura a 64 detettori e con tempo di rotazione di 0 , 33 secondi . proprio in pazienti con una importante dispnea , lutilizzo di una apparecchiatura cos performante , consente di ottenere immagini in singola apnea di circa 4 secondi , e con elevata risoluzione spaziale . 
allo stato attuale diversi sistemi commerciali consentono di ottenere informazioni immediate volumetriche sul parenchima polmonare , con possibilit di una segmentazione automatica , senza il bisogno di una segmentazione manuale come eseguita nel nostro caso ; lapplicazione di tali sistemi appare essenziale nel calcolo della funzionalit respiratoria post operatoria sia in pazienti che andranno incontro a riduzione volumetrica per via chirurgica o endobronchiale , sia , come gi dimostrato , nei pazienti da sottoporre a resezioni polmonari pi ampie , come nei pazienti con neoplasia . al nostro studio bisogna riconoscere dei limiti ; primo fra tutti la ridotta numerosit della popolazione in studio , che non ha permesso di affrontare una analisi statistica . 
pochi studi clinici e radiologici hanno affrontato largomento ed ognuno con casistiche limitate ; nonostante comunque il piccolo campione studiato , i risultati ottenuti sono stati incoraggianti , sia per la risposta chirurgica e quindi sintomatologica del paziente , sia per la correlazione tra tc multistrato e test respiratori . probabile che un follow - up pi lungo ed un campione pi ampio di soggetti consentir una miglior comprensione fisiopatologica delle modificazioni che si riscontrano nel polmone in corso di enfisema e dopo il trattamento . un secondo limite riguarda la mancanza di valutazione quantitativa del grado di enfisema , cos come riportato da precedenti lavori [ 2224 ] ; tuttavia i pazienti gi presentavano test funzionali che evidenziavano una patologia di tipo ostruttivo , per la quale erano in trattamento medico ; inoltre lo scopo dello studio prevedeva principalmente la dimostrazione della tesi di riduzione volumetrica conseguente al trattamento ed i vantaggi della tecnica di tc multistrato nellimaging pree post - operatorio di questi pazienti . 
anche in questo caso dato il limitato numero di pazienti incluso e quindi la limitata correlazione statistica , un secondo giudizio , peraltro su dati in cui lelaborazione viene effettuata da un software dedicato , non avrebbe probabilmente aggiunto sostanziali modifiche . conclusions technological advances , in particular , 64 - slice scanners , in le innovazioni tecnologiche delle nuove apparecchiature tc in particolare a 64 strati , consentono una accurata definiconclusioni f . 
lung - volume assessment by means of dedicated software , used in addition to spirometry , enables the radiologist to calculate the volume of a single lobe or lung region and to determine the real improvement both in general terms as indicated by spirometry and in the treated parenchyma . 
larger patient populations and longterm follow - up are needed to determine the real benefit of bronchoscopic lung - volume reduction and the real correlation between ct findings and clinical tests . zione pree post - operatoria nei pazienti con enfisema trattati con un nuovo approccio di valvole endobronchiali . 
la valutazione volumetrica effettuata su software dedicati consente inoltre , in aggiunta alla spirometria , di poter effettuare un calcolo per singolo lobo , o regione polmonare , con possibilit di valutare leffettivo miglioramento sia in termini globali , cos come emerge dalla spirometria , ma anche sul parenchima trattato . 
un maggior numero di pazienti ed un follow - up a lungo termine consentir di valutare leffettivo beneficio del trattamento endobronchiale , cos come una pi reale correlazione tra tc e test clinici . radiol med ( 2006 ) 111 : 878 doi 10.1007 / s11547 - 006 - 0084 - 6 ecografia clinica p . 
nei due capitoli dedicati ai mezzi di contrasto in ecografia , vengono trattati i principi fisici , le tecnologie e le metodiche di visualizzazione dei mezzi di contrasto . larticolazione dei capitoli che segue quindi divisa per distretti anatomici . il primo volume dedicato alla testa , al collo e al torace ; in questo volume compreso anche lapparato locomotore . 
nella parte testa , collo e torace sono trattati i capitoli dedicati alle ghiandole salivari , alle malattie diffuse della tiroide , alle neoplasie della tiroide , alle paratiroidi ed ai linfonodi . 
la trattazione del rene , separata dalladdome superiore , nella parte dedicata allapparato urinario , consente una valutazione dinsieme delle patologie delle vie urinarie . nel terzo volume sono trattati lapparato genitale femminile , la pediatria , lapparato vascolare . 
muzzio1 1dipartimento di scienze medico diagnostiche e terapie speciali , 2dipartimento di scienze mediche e chirurgiche , clinica chirurgica 3 , universit degli studi di padova , istituto oncologico veneto , via giustiniani 2 , i - 35128 padova , italy 3dipartimento di radioterapia e medicina nucleare , azienda ospedaliera di padova , istituto oncologico veneto , padova , italy correspondence to : d . 
prognosis and treatment of esophagus and cardia cancer ( ecc ) depend on the precision with which the disease is staged according to the american joint committee of cancer ( ajcc ) criteria . 
imaging modalities normally used in clinical staging are esophagography , esophagoscopy , endoscopic ultrasound ( eus ) , computed tomography ( ct ) and positron emission tomographyct fusion ( ct - pet )  . 
fifty - six patients with ecc diagnosed by x - ray of the upper digestive tract , endoscopy and biopsy were staged using eus , chest and abdomen ct scan , and ct - pet . thirty - four patients in stage ii and 18 patients in stage iii underwent surgery after neoadjuvant chemotherapy ; four patients in stage iv were treated with the positioning of an endoprosthesis after chemoradiotherapy . 
in the 52 patients who had surgery , follow - up included digestive tract x - ray , endoscopy and ct of the chest and abdomen every 68 months for the first 3 years . 
in all 56 patients , endoscopy , eus , ct and ct - pet in combination were crucial in determining the site of disease , locoregional extent and depth of esophageal wall penetration ( t ) , and any involvement of the mediastinal lymph nodes ( n1 ) , extrathoracic lymph nodes ( m1 ) or hepatic metastases . 
in the locoregional staging of ecc before chemotherapy , we were able to differentiate t2t3 from t4 in 40 patients ; t4 disease was found in 12 potentially resectable cases . 
the specificity of ct in detecting small lymph nodes in the mediastinum was less than 50% while for ctpet , it was more than 80% ; eus revealed sensitivity higher than 90% but a low specificity in seven cases . 
le tecniche di imaging in uso nella pratica clinica sono : lesofagografia , lendoscopia , lecoendoscopia ( eus ) , la tomografia computerizzata ( tc ) e la tomografia ad emissione di positroni fusa con la tc ( tc - pet )  . queste metodiche combinate tra loro sono determinanti non soltanto per la diagnosi , ma soprattutto per la stadiazione ed il follow - up dopo trattamenti multimodali . 
cinquantasei pazienti portatori di cec , diagnosticato con esame radiologico del tubo digerente prime vie e con endoscopia con biopsia , sono stati stadiati mediante eus , tc toraco - addominale e tc - pet . 
trentaquattro in stadio ii e 18 in stadio iii hanno eseguito chemioterapia neoadiuvante e poi sono stati operati ; 4 in stadio iv sono stati sottoposti a posizionamento di endoprotesi dopo chemio - radioterapia . 
la endoscopia , la eus , la tc e la tc - pet combinate ci hanno permesso di determinare in tutti i 56 casi la sede , lestensione locoregionale e il grado di infiltrazione della parete dellesofago ( t ) , la presenza di metastasi linfonodali mediastiniche ( n1 ) , extratoraciche ( m1 ) e / o epatiche . 
nella stadiazione loco - regionale del cec prima della chemioterapia si differenziato il t2 - t3 dal t4 in 40 casi ; si evidenziato un t4 in 12 casi solo potenzialmente resecabili . 
per quanto riguarda i piccoli linfonodi metastatici nel mediastino la tc ha dimostrato specificit inferiore al 50% , la tc - pet specificit maggiore dell80% , la eus sensibilit di oltre il 90% ma bassa specificit in 7 casi . 
x - ray of the upper digestive tract and chest and abdomen ct scan are useful in preliminary evaluation of ecc . endoscopy is particularly indicated for evaluating tumour morphology , taking biopsies for a histological diagnosis and the early diagnosis of anastomotic recurrences . 
ct - pet is extremely useful in identifying small mediastinal metastatic lymph nodes ( n1 ) or extrathoracic lymph nodes ( m1 ) and hepatic metastases ( 1 cm ) , which may escape multislice ct . 
pet alone is useful for identifying residual or recurrent tumour in the esophageal wall when an endoprosthesis is in place . key words esophagus and cardia cancer x - ray upper digestive tract endoscopy endoscopic ultrasound computed tomography positron emission tomography ct fusion effettuata mediante endoscopia , eus , tc toraco - addominale e tc - pet nei casi dubbi ha posto la indicazione allintervento chirurgico . 
la tc - pet consente di identificare le piccole metastasi linfonodali intramediastiniche ( n1 ) ed extratoraciche ( m1 ) e le lesioni epatiche di dimensioni 1 cm , non sempre identificabili con la tc anche con la tecnica multistrato . 
la pet da sola trova indicazione nellidentificare persistenza o ripresa di malattia nella parete dellesofago sede di endoprotesi . parole chiave tumore dellesofago e del cardias tubo digerente , prime vie endoscopia eco - endoscopia tomografia computerizzata tomografia ad emissione di positroni - tc introduction introduzione esophagus and cardia cancer ( ecc ) is relatively rare and has a predilection for the male gender . 
in the last 20 years , there have been signs of a considerable increase in the incidence of ecc [ 7 , 8 ] associated with a decline in the epidermoid type ( which tends to affect the median and lower third of the esophagus and is related to alcohol abuse , smoking and vitamin and mineral dietary deficiencies ) and an increase in the proportion of adenocarcinomas , localised mainly in the distal portion and cardia , which develop mainly in barretts epithelium [ 912 ]  . there is no consensus concerning treatment protocols and survival curves for patients suffering from this disease . while patients operated for early squamous cell carcinoma can reach perfectly acceptable 5 - year survival rates of between 57% and 78% , patients with locally advanced disease have a far lower survival rate , ranging from 6% to 14% at 5 years [ 2 , 1315 ]  . 
this is partly due to the fact that more than a third of patients have extensive disease already at diagnosis ( involving the tracheal - bronchial tree , laryngeal nerves , vascular structures and / or regional or remote lymph nodes ) , which usually rules out any radical or even palliative surgery [ 16 ]  . 
after postoperative chemoradiotherapy or neoadjuvant chemotherapy , these patients survive a median of 9 months or so , with 5 - year survival rates of between 20% and 30% in those who have undergone surgery [ 13 , 14 , 1721 ]  . 
negli ultimi 20 anni si notato un notevole aumento dellincidenza del cec [ 7 , 8 ] associato ad una diminuzione del tipo epidermoide ( che interessa preferenzialmente il terzo medio e inferiore dellesofago , legato allabuso di alcol , tabacco e a deficit nutrizionali di vitamine e minerali ) e ad un aumento delladenocarcinoma localizzato principalmente nel tratto distale e nel cardias , che insorge sopratutto su lesione di barrett [ 912 ]  . in letteratura non vi uniformit sui protocolli terapeutici e sulle curve di sopravvivenza dei pazienti affetti da tale patologia . 
se da un lato i carcinomi squamosi early stage operati , possono raggiungere tassi di sopravvivenza accettabili tra il 57% e il 78% a 5 anni , dallaltro pazienti con malattia localmente avanzata hanno bassa sopravvivenza , con un range a 5 anni , che varia dal 6% all14% [ 2 , 1315 ]  . ci in parte dovuto al fatto che oltre un terzo dei pazienti si presenta alla diagnosi oramai con malattia estesa ( interessamento dellalbero tracheo - bronchiale , dei nervi laringei , delle strutture vascolari o dei linfonodi regionali o a distanza ) e non pu essere sottoposto ad un trattamento chirurgico radicale o palliativo [ 16 ]  . 
in questi pazienti , dopo chemio - radioterapia postoperatoria o dopo solo chemioterapia neoadiuvante viene riportata una sopravvivenza mediana intorno ai 9 mesi , con tassi di sopravvivenza a 5 anni d . 
thus , despite significant improvements in diagnostic imaging techniques [ 2232 ] , the advent of new surgical strategies [ 21 ] and new chemotherapeutic preparations [ 3339 ] , the survival rate remains low . 
 this paper discusses the indications for endoscopic ultrasonography ( eus ) , computed tomography ( ct ) , positron emission tomography ( pet ) and ct - pet fusion in pretreatment staging of ecc and in the follow - up after multimodality treatment . 
 materials and methods from january 2002 to september 2005 , 56 patients with ecc came under our observation for a joint surgical and oncological assessment ; 47 were men and nine were women , with an age ranging between 39 and 74 ( mean 63 ) years . 
the ecc was first diagnosed by barium - swallow upper - digestive - tract x - ray ; in the more severe cases with major dysphagia for solid and liquid foods , upper digestive tract x - ray with gastrografin was preferred . 
in all cases , ecc was staged using eus , chest - abdomen ct and ctpet before deciding the type of treatment . eus was performed using 7.5 mhz or 20 mhz probes capable of producing an excellent representation of the five layers of the esophageal wall . 
for ct , we used the spiral technique and 5 - mm slab thicknesses , 5 - mm increments , 3to 2 - mm reconstructions and a pitch of 1.5 after intravenous administration with an automatic injector of 100 ml of iopamiro 370 at a rate of 3 ml / s . 
the last 12 cases were evaluated using a multislice ( 16 - slice ) ct apparatus with the following acquisition parameters : thickness 3 mm , reconstruction index 1 mm , pitch 1.5. 
ct - pet images were acquired after injecting a dose of 710 mci ( 240370 mbq ) of 18 - fluorodeoxyglucose ( fdg ) at intervals of 6090 min with an acquisition time of 4 min / bed , using a ct voltage of 80110 kev , depending on the thickness of the adipose layer . 
image processing and fusion were done automatically using a specific clinical programme . patients were staged as follows : 34 in stage ii , 28 of them in stage iia ( t3 n0 ) and six in stage iib ( t2 n1 ) ; 18 in stage iii ( six t3 n1 and 12 t4 n0 ) ; four in stage iv ( m1 )  . 
the 34 patients ( 61% ) who underwent esophagectomy and the 18 ( 32% ) patients with advanced but potentially resectable disease were given neoadjuvant chemotherapy before surgery . postoperative radiotherapy was provided in 12 cases ( 18 stage iii and four stage ii ) in which there was some doubt as to surgical radicality . 
four patients ( 7% ) were treated with chemoradiotherapy and subsequently fitted with an endoprosthesis because they had hepatic metastases ( three cases ) and extracompartmental lymph node metastases ( three cases ) ; the follow - up tra il 20% e 30% in quelli operati [ 13 , 14 , 1721 ]  . 
la reale efficacia di tali strategie terapeutiche combinate o no non ancora supportata da validi e certi risultati e le linee guida terapeutiche non sono standardizzate [ 14 ]  . 
nonostante , quindi , i significativi miglioramenti delle tecniche diagnostiche di immagine [ 2232 ] , di nuove strategie di approccio chirurgico [ 21 ] e lutilizzo di nuovi preparati chemioterapici [ 3339 ] , la sopravvivenza rimane comunque mediamente bassa . scopo di questo lavoro quello di puntualizzare le indicazioni alla eco - endoscopia ( eus ) , alla tomografia computerizzata ( tc ) , alla tc associata alla tomografia ad emissione di positroni ( tc - pet ) sia nella stadiazione pre - trattamento del cec sia nel follow - up dopo trattamento multimodale . materiali e metodi da gennaio 2002 a settembre 2005 56 pazienti affetti da cancro dellesofago e del cardias , sono occorsi alla nostra osservazione nellambito di una valutazione collegiale chirurgico - oncologica . 
di questi 47 sono maschi e 9 femmine , di et compresa tra 39 e 74 anni con et mediana di 63 anni . il cec stato diagnosticato preliminarmente mediante esame radiologico del tubo digerente dopo assunzione di sospensione di solfato di bario ; nei casi di grave ed importante disfagia per i cibi solidi e liquidi si ritenuto di eseguire lesame radiologico delle prime vie del tubo digerente mediante gastrograf successivamente i pazienti sono stati sottoposti ad endoscopia per la valutazione morfologica del tumore e la diagnosi istologica mediante ago - biopsia . 
in tutti i casi il cec stato stadiato mediante eus , tc toraco - addominale e tc - pet prima di decidere il tipo di terapia . lindagine eus si avvalsa dellutilizzo di sonde da 7 , 5 mhz o 20 mhz in grado di produrre una ottimale rappresentazione dei 5 strati della parete dellesofago . 
la tc si avvalsa della tecnica a spirale con spessore dello strato di 5 mm , con avanzamento di 5 mm e ricostruzioni a 23 mm e con pitch di 1 , 5 , dopo somministrazione endovenosa mediante iniettore automatico di 100 ml di iopamiro 370 alla velocit di 3 ml / s . 
gli ultimi 12 casi sono stati valutati mediante apparecchio tc multistrato ( 16 strati ) con i seguenti parametri di acquisizione : spessore di 3 mm , indice di ricostruzione 1 mm , pitch 1 , 5 . 
le immagini tc - pet sono state acquisite dopo avere iniettato una dose di 710 mci ( 240370 mbq ) di 18 - fdg a distanza di 6090 minuti con un tempo di acquisizione di 4 min / bed , servendosi di un voltaggio della tc di 80110 kv in base allo spessore del pannicolo adiposo . lelaborazione e la fusione delle immagini sono state ottenute in automatico mediante lutilizzo di programma clinico specifico . 
chemotherapy was administered according to the following protocol : 5 - fluorouracil 1 , 000 mg / m2 in continuous infusion on days 1 and 5 ; 100 mg / m2 cisplatin on day 1 , for each cycle of treatment . 
radiotherapy was given in a dose of 45 gy / 25 fr . follow - up involved 52 operated patients and involved endoscopy and chest - abdomen ct every 68 months for the first 3 years . 
ct - pet was performed in 12 cases with a clinical picture and / or morphological signs on ct suspicious of recurrence ( small nodular formations in the intramediastinal lymph nodes , structural irregularities in the liver )  . results in all 56 cases , combining eus , ct and ct - pet enabled us to accurately establish site and locoregional extent of the ecc , degree of infiltration of the esophageal wall and involvement of the mediastinal and / or extracompartmental lymph nodes , as well as identifying any small hepatic metastases all fundamental factors in determining the most suitable treatment and an accurate prognosis . as for cancer site , it was located at the esophagogastric junction in 23 patients ( 41% of the total ) , in the middle thoracic portion of the esophagus in 14 ( 25% of the total ) , the upper thoracic portion in 16 ( 29% of the total ) and in the cervical esophagus in three ( 5% of the total )  . 
in the nodal staging , we differentiated n0 from n1 ( 12 cases ) ; although n1 cases are operable , their prognosis is not good . using ct - pet , we found four cases of small hepatic metastases 0.51.5 cm in size ( three cases ) and extrathoracic lymph node metastases in the subdiaphragmatic region ( three cases )  . 
at the time of reassessment 4 weeks after completing the treatment , three of these patients had persistent esophageal - wall disease demonstrated by pet ( two cases ) and ct - pet ( one case )  . 
 as for the presence of small metastatic lymph nodes in the mediastinum , in 12 n1 cases , ct demonstrated specificity of less than 50% ( five cases ) while ct - pet had a specificity higher than 80% ( ten cases ) ; eus sensitivity was higher than 90% ( 11 cases ) , even for periesophageal lymph nodes less than 1 cm , but its specificity was limited in seven cases ( less than 60% )  . 
quattro pazienti ( 7% ) sono stati trattati con chemio - radioterapia e successivamente sottoposti a posizionamento di endoprotesi in quanto presentavano metastasi epatiche ( 3 casi ) e metastasi linfonodali extracompartimentali ( 3 casi ) ; il follow - up stato eseguito in 2 casi solo mediante pet . 
la chemioterapia stata somministrata secondo il seguente schema : 5 - fluorouracile : 1000 mg / m2 in infusione continua dai giorni 1 e 5 e cisplatino : 100 mg / m2 nel giorno 1 , per ogni ciclo di terapia . 
la radioterapia con il seguente dosaggio : 45 gy / 25 fr . il follow - up stato effettuato nei 52 pazienti operati mediante endoscopia e tc toraco - addominale ogni 68 mesi per i primi 3 anni . 
la tc - pet stata eseguita in 12 casi in rapporto al quadro clinico sospetto e / o allaspetto morfologico alla tc dubbio per recidiva ( piccole formazioni nodulari linfonodali intramediastiniche , disomogeneit della struttura del fegato )  . risultati la eus , la tc e la tc - pet combinate hanno permesso di determinare in tutti i 56 casi con precisione la sede e lestensione loco - regionale del cec , il grado di infiltrazione della parete dellesofago , il coinvolgimento dei linfonodi mediastinici e / o extracompartimentali e di evidenziare anche piccole metastasi epatiche , tutti fattori determinanti per la scelta del trattamento pi idoneo e per una prognosi accurata . per quanto riguarda la sede del cancro in 23 pazienti ( 41% del totale ) la localizzazione era a livello della giunzione esofago - gastrica , in 14 ( 25% del totale ) della porzione toracica media , in 16 pazienti ( 29% del totale ) dellesofago toracico superiore ed in 3 ( 5% del totale ) dellesofago cervicale . 
nella stadiazione delln , abbiamo differenziato ln0 dalln1 ( 12 casi ) ; pur essendo ln1 operabile , la prognosi non buona . abbiamo riscontrato mediante tc - pet la presenza in 4 casi sia di piccole metastasi epatiche 0 , 51 , 5 cm ( 3 casi ) sia di metastasi ai linfonodi extratoracici della regione sottodiaframmatica ( 3 casi )  . 
 preoperative restaging of the 18 patients in stage iii given neoadjuvant chemotherapy ( performed a mean 40 days after completing the treatment ) using endoscopy , eus and ct of the chest and abdomen associated with ct - pet in dubious cases demonstrated good response to chemotherapy in all cases , so this group of patients also underwent surgery . 
 discussion prognosis and treatment of ecc relies on accurate staging according to the criteria of the american joint committee on cancer ( ajcc ) system based on the use of endoscopy , eus , ct and ct - pet [ 1 , 40 ]  . 
the first layer corresponds to the boundary echo and superficial mucosa ; the second to the mucosa , including the muscularis mucosae ; the third to the submucosa ; the fourth to the muscularis propria and the fifth to the adventitia [ 29 , 41 , 42 ]  . 
 ct scan of the chest and abdomen , especially using multislice techniques , was the basic imaging method we used not only for global staging of the tumour ( remote metastases ) but also for an assessment of its spread into the mediastinum . obliteration of the layers of mediastinal fat prior to any neoadjuvant treatment is an important predictor of spread of ecc beyond the esophageal wall . 
mediastinal fat is particularly abundant at the level of the lower third of the esophagus and cardia , where it acts as a sort of barrier to tumour aggression on adjacent anatomical structures , i.e. 
in addition to tumour spread into mediastinal fat , another ( albeit less reliable ) criterion to consider is the craniocaudal extension of the esophageal mass : the greater the extension of the tumour in the sagittal direction , the more likely it is to be a case of t4 ( 12 cases observed )  . even with modern multislice ct techniques , it is not always possible to evaluate infiltration of the vascular structures , and the aorta in particular , with any certainty since endolumento della rivalutazione eseguita a distanza di quattro settimane dalla fine dei trattamenti , persistenza di malattia intraparietale dimostrata con la pet ( 2 casi ) e con la tc - pet ( 1 caso )  . per quanto riguarda la presenza di piccoli linfonodi metastatici nel mediastino , la tc ha dimostrato , su 12 casi n1 , specificit inferiore al 50% ( 5 casi ) , la tc - pet una specificit maggiore dell80% ( 10 casi ) , la eus elevata sensibilit oltre il 90% ( 11 casi ) per i linfonodi periesofagei anche di dimensioni inferiori ad 1 cm , ma bassa specificit in 7 casi ( < 60% )  . 
in 2 casi nel tempo prechirurgico si ottenuta la diagnosi di metastasi linfonodale peritumorale mediante ago - biopsia eus mirata . per quanto riguarda i 3 casi di cec in cui erano presenti linfonodi extracompartimentali metastatici sottodiaframmatici , la tc ha evidenziato piccoli linfonodi ( diametro inferiore a 15 mm ) ma non stata in grado di dimostrare la presenza di eventuali metastasi ben evidenti alla tc - pet . la ristadiazione pre - operatoria dei 18 pazienti in stadio iii sottoposti a chemioterapia neoadiuvante effettuata dopo 40 giorni mediamente dalla fine del trattamento , con endoscopia , con eus e con tc toraco - addominale , associata a tc - pet nei casi dubbi , ha dimostrato in tutti una buona risposta alla chemioterapia per cui anche questo gruppo di pazienti stato sottoposto a intervento chirurgico . la recidiva a livello della anastomosi esofago - gastrica stata identificata in 16 dei 52 pazienti mediante endoscopia associata alla biopsia in un arco di tempo compreso tra i 3 e i 12 mesi . 
il gran vantaggio delleus la sua capacit di evidenziare i singoli strati che compongono la parete esofagea : si ottenuta infatti , nei nostri casi , una precisa rappresentazione dei 5 strati della parete dellesofago . 
va ricordato che il primo corrisponde al limite tra lo strumento e la superficie mucosa , il secondo strato alla mucosa inclusa la muscolaris mucosae , il terzo strato alla sottomucosa , il quarto alla muscolaris propria e il quinto strato allavventizia [ 29 , 41 , 42 ]  . 
so we have to rely on nonspecific signs , such as the degree of deformity in the circumference of the descending aorta or more than 90 of contact between the neoplastic solid tissue and the vessel wall . any involvement of the trachea and / or left main bronchus is ascertained by endoscopy in the absence of endoluminal non solo per una stadiazione complessiva del tumore ( metastasi a distanza ) , ma anche per una valutazione della sua estensione nel mediastino . 
in fact , ct very clearly identified the esophageal mass but failed to distinguish any involvement of peritumoural lymph nodes because , in the absence of planes of cleavage , it was incapable of discriminating neoplastic solid tissue from lymph nodes , which had the same densitometric values . 
any lymph node and hepatic micrometastases less than 1 cm in size of a dubious nature on the ct scan , even using the multislice technique , were easily detected by ct - pet , ruling out surgery for four patients who were treated with chemoradiotherapy alone . 
oltre allo spread tumorale nel grasso mediastinico , un criterio anche se meno valido , potrebbe essere quello della estensione cranio - caudale della massa esofagea : tanto pi esteso in senso sagittale il tumore tanto pi probabile che si tratti di un t4 ( 12 casi osservati )  . 
anche con le moderne tecniche tc multistrato non sempre possibile valutare con certezza la infiltrazione delle strutture vascolari , soprattutto laorta , in quanto non si mai in presenza , come avviene per i vasi venosi , di proliferazioni trombotiche endoluminali . 
bisogna quindi basarsi su segni aspecifici come per esempio il grado di deformazione della circonferenza dellaorta discendente o un contatto superiore ai 90 tra il tessuto solido neoplastico e la parete del vaso . 
il coinvolgimento della trachea e / o del bronco principale di sinistra di pertinenza della endoscopia in assenza di proliferazioni endoluminali , che sono bene evidenziate dalla tc , anche con il concorso della endoscopia virtuale . valutazioni diagnostiche delln leus lunica tecnica in grado di distinguere il tessuto tumorale esofageo dalla linfoadenopatia mediastinica contigua alla massa ( n1 )  . 
diagnostic certainty can come only from direct morphological examination and / or biopsy using endoscopy [ 43 ]  . evaluation of locoregional recurrence identification of locoregional recurrence in the operated cases posed problems of interpretation . 
la tc , infatti , ha evidenziato molto bene la massa esofagea , ma non ha distinto il coinvolgimento dei linfonodi contigui al tumore , in quanto non stata in grado di discriminare , in assenza di piani di clivaggio , il tessuto solido tumorale dai linfonodi che presentavano eguali valori densitometrici . 
piccole metastasi linfonodali e lesioni epatiche inferiori al centimetro , di natura dubbia alla tc anche con la tecnica multistrato , sono state individuate facilmente con la tc - pet , escludendo dalla terapia chirurgica 4 pazienti , sottoposti esclusivamente alla chemio - rafig . 
la certezza diagnostica deriva solo dallaccertamento morfologico diretto e / o bioptico mediante endoscopia [ 43 ]  . valutazione della recidiva locoregionale la identificazione della recidiva locoregionale nei casi operati ha offerto delle problematiche di interpretazione . 
nella nostra casistica quando la recidiva si localizzata a livello della anastomosi ( 16 casi ) , la sua identificazione e valutazione avvenuta prevalentemente per via endoscopica con ago - biopsia anche di piccole irregolarit della superficie mucosa . 
b fluorodeoxyglucose - positron emission tomography ( fdg - pet ) shows gross recurrence of the ecc , with a nodular appearance in the sagittal direction , metastatic lymph nodes in the left clavicular region and small ( 1 - cm ) metastases in the liver ( arrows )  . 
bb la fdg - pet mostra grossolana recidiva del cec con aspetti nodulari in senso sagittale , linfonodi metastatici in regione clavicolare sinistra e piccole metastasi epatiche ( 1 cm ) ( frecce )  . 
bb la fdg - pet rileva piccolo iperaccumulo del tracciante metabolico in corrispondenza della parete laterale destra del tratto esofageo sede dellendoprotesi ( frecce )  . first imaging methods to use for preliminary evaluation of ecc . 
endoscopy is indispensable for a direct view of the tumour , for taking biopsies and thus establishing a histological diagnosis and ensuring early detection of small recurrences at sione diretta del tumore , per il campionamento bioptico e quindi per la diagnosi istologica e per lidentificazione precoce di piccole recidive in sede di anastomosi dopo la chirurgia ; leus per differenziare il t2 - t3 dal t4 , per evidend . 
ct - pet provides morphological and functional information that enables even very small recurrences to be identified and located both in iatrogenic fibrotic periesophageal areas and in lymph nodes and intraand extrathoracic organs . even using the multislice technique , even when ct revealed liver lesions of 1 cm or more , it was unable to determine their benign or malignant nature with certainty . 
each imaging modality has its advantages and limitations , but in combination , they are crucial in determining the most appropriate treatment for patients with ecc . ziare piccoli linfonodi peritumorali e per biopsie mirate . lendoscopia , leus , la tc e la tc - pet combinate trovano indicazione per una precisa stadiazione pre - operatoria , essendo lintervento chirurgico altamente demolitivo . 
la tc - pet infatti , offre informazioni morfologiche e funzionali che consentono di evidenziare e localizzare anche piccole recidive sia nelle aree fibrotiche iatrogene periesofagee sia nei linfonodi e negli organi intra ed extratoracici . la tc , anche nei pazienti valutati con la tecnica multistrato , quando ha dimostrato la presenza di lesioni epatiche del diametro 1 cm non stata in grado di determinare con certezza la natura benigna - maligna di queste . 
the test , consisting of 17 screendetected cancer cases and 133 negative controls , was validated by a panel of expert readers defining a minimum standard as to sensitivity for cancer and recall rate of negative controls . 
radiologist training needs to be considered a priority , at least in those screening programmes that are still to be implemented in one third of the country . key words mammography screening diagnosis proficiency test riassunto obiettivo . 
il test , costituito da 17 casi di carcinomi diagnosticati allo screening e da 133 controlli negativi , stato validato da lettori esperti e prevede di superare uno standard relativo alla sensibilit e al tasso di richiamo a approfondimento tra i controlli negativi . 
laccuratezza misurata al test funzione della esperienza , rilievo non inaspettato , che suggerisce che limpiego corrente in screening di radiologi generalisti , con scarsa esperienza in mammografia e senza un congruo addestramento , dovrebbe essere riconsiderato . laccuratezza al test migliora sensibilmente con laddestramento ( stage formativi e consultazione di casistiche didattiche ) : molti programmi di screening in italia sono stati varati senza che si provvedesse a una adeguata formazione dei radiologi : almeno per i programmi che ancora devono iniziare in un terzo del territorio nazionale auspicabile che la formazione sia un punto di forza . parole chiave mammografia screening diagnosi test di accuratezza s . 
several such tests have been developed over time at the centro per lo studio e la prevenzione oncologica ( cspo ) of florence , and a large number of italian radiologists have been tested , either as a measure of quality control of ongoing national screening programmes , within courses held at cspo or on individual request for self - evaluation purposes [ 13 ]  . the aim of the present study was to review the series of tests taken to assess the success rate and its association to a variety of indicators of reader experience defined at the time of the test . 
however , because requests for the test have been increasing in recent years , probably as a result of the large number of organised screening programmes implemented on a national basis [ 4 ] , the series available for review has grown much larger , allowing for more stable estimates , and deserved a new evaluation . nella istruzione di un nuovo programma e tra i controlli di qualit di un servizio di screening mammografico , uno dei problemi quello di valutare laccuratezza diagnostica dei radiologi . 
diversi test del genere sono stati realizzati nel tempo presso il centro per lo studio e la prevenzione oncologica ( cspo ) di firenze , e ad essi si sono sottoposti nel tempo molti radiologi italiani , sia nellambito di programmi di controllo di qualit di programmi di screening nazionali , sia nel corso di tirocini pratici presso il cspo , sia per autoverifica personale [ 13 ]  . 
scopo dello studio qui riportato stato quello di rivedere la casistica dei test sostenuti , per verificare il tasso di superamento degli stessi , e la correlazione tra superamento e una serie di indicatori di esperienza dei lettori , raccolti in occasione del test . 
dato il notevole incremento della richiesta di sottoporsi al test negli anni pi recenti , verosimilmente in concomitanza della diffusione nazionale di programmi organizzati di screening di popolazione [ 4 ] , la casistica oggi disponibile molto pi ampia ed per questo stata rivalutata , consentendo stime assai pi stabili . materials and methods materiali e metodi the proficiency test was developed at cspo in 1996 . 
it consists of a set of 150 cases of bilateral , two - view screening mammography : a consecutive series of 133 negative cases ( diagnosed as negative within the florence province programme that screens women aged 5069 years and confirmed negative for at least one further screening round ) was seeded with 17 consecutive cancer cases detected by screening during the same year and confirmed by histology . 
the test was validated by a panel of four expert radiologists ( at least 20 , 000 mammograms read ) who were invited , as in current screening practice , to provide a report of : ( a ) negativity or ( b ) referral for further diagnostic assessment . 
average panel results ( sensitivity = 95% , range 88%100% ; referral rate = 8% , range 3%14% ) were lowered to make the test easier and were taken as reference standards . referral rate was determined only on negative controls . 
a test was passed when sensitivity was 80% and the referral rate was 15% . information on previous experience in mammography reading was available for most radiologists taking the test . experience was expressed as : ( a ) years of experience in mammography reading , ( b ) overall number of mammograms read and ( c ) number of mammograms read over the last year . after the test , radiologists were given their overall result and sensitivity and referral rate results , but not the results for the il test oggetto di questo studio stato sviluppato al cspo nel 1996 . 
esso consta di 150 casi di mammografia bilaterale in due proiezioni : una serie consecutiva di 133 casi negativi ( diagnosticati come tali nellambito del programma di screening della provincia di firenze , che invita le donne residenti dai 50 ai 69 anni , e rimasti tali almeno a un controllo biennale ) stata arricchita con 17 casi consecutivi di carcinoma , diagnosticati in occasione dello screening durante lo stesso anno e istologicamente confermati . 
dalla casistica consecutiva sono stati esclusi casi con radiogrammi di cattiva qualit ( cattiva esposizione , sfumature da movimento , artefatti , posizionamento inadeguato ) e carcinomi sintomatici o in categoria t2 o superiore . 
il test stato validato da 4 radiologi esperti ( almeno 20.000 mammografie lette ) ai quali stato richiesto , come nella pratica di screening , di formulare un giudizio alternativo tra un referto negativo o di indicazione a un ulteriore accertamento diagnostico . 
i risultati medi ( sensibilit media = 95% , range 88%100% ; tasso di richiamo medio = 8% , range 3%14% , questultimo calcolato escludendo i casi di carcinoma ) ottenuti da questo panel di esperti sono stati scontati per facilitare il superamento del test e hanno costituito lo standard di riferimento del test , che si ritiene superato solo quando siano raggiunti sia lo standard di sensibilit ( 80% ) che il tasso di richiamo ( 15% )  . 
the digital version was validated by a panel of four radiologists who confirmed the previously adopted reference standards . the study analysed the results obtained , their association with indicators of experience and the results of repeat tests taken by radiologists who had failed the first attempt . 
statistical analysis of the differences observed was based on the chi - square test ( 2 ) , statistical significance being set at p < 0.05. results during 19972005 , 537 first tests were taken by volunteering radiologists . 
only one third reported having > 10 years experience , approximately one third had read < 5 , 000 mammograms overall and one third had read < 1 , 000 mammograms during the previous year . test results are reported in table 2 . 
tests were usually repeated after a period of training , mostly after consulting a teaching dvd atlas provided by cspo , with several thousand mammography cases visible as an atlas or as an interactive test . 
 discussion use of a standard set to test diagnostic accuracy is definitely simpler , faster , and more feasible compared with conventional methods for measuring diagnostic accuracy , such as determining cancer detection rate or interval cancer frequency . 
on the contrary , a proficiency test is almost immediate , may be easily repeated and , if in sia come anni di esperienza in mammografia , che ( b ) come numero di mammografie totali lette , che ( c ) numero di mammografie lette nellultimo anno . 
i radiologi che hanno sostenuto il test sono stati informati solo sui valori si sensibilit e tasso di richiamo raggiunti e sulleventuale superamento del test , ma non sono stati rivelati i risultati nei singoli casi , il che ha consentito la ripetizione del test , a richiesta . 
data la grande richiesta di sostenere il test da parte dei radiologi italiani , piuttosto voluminoso e difficilmente decentrabile , esso stato trasposto in versione digitale ( fotografia digitale dei casi analogici ) su dvd , il che ha consentito il sostenimento del test al computer , e anche linvio a distanza . 
lo studio analizza i test sostenuti , in particolare confrontando il tasso di superamento con i diversi indicatori di esperienza raccolti , e valutando landamento del test nel tempo per i radiologi che lo hanno sostenuto pi volte , non avendolo superato . 
lanalisi statistica delle differenze osservate si basata sul test chi - quadrato ( 2 ) : la significativit statistica stata posta ad un valore di p < 0 , 05 . risultati dal settembre 1997 al dicembre 2005 sono stati sostenuti 537 primi test di screening , da parte di radiologi volontari , per la gran parte gi con esperienza di refertazione in mammografia , generalmente clinica . 
solo un terzo dei soggetti riferisce una esperienza da oltre 10 anni , circa il 30% riferisce a pi di 5000 mammografie totali lette , circa un terzo ha letto pi di 1000 mammografie nellultimo anno . 
il test stato superato al primo tentativo da 176 radiologi ( 32 , 7% ) , e non stato superato da 361 radiologi , dei quali da 220 per insufficiente sensibilit ( 60 , 9% ) , da 57 per eccessivo tasso di richiamo ( 15 , 7% ) e da 84 per entrambe le ragioni ( 23 , 2% )  . 
il superamento del test risultato essere correlato con lesperienza , quando questa misurata in base agli anni di esperienza di mammografia ( cut - off 5 anni : 2 per trend = 4 , 17 , p = 0 , 04 ) , o in termini di mammografie totali lette ( 2 per trend = 11 , 8 , p = 0 , 002 ) , o in termini di mammografie lette per anno ( 2 per trend = 6 , 27 , p = 0 , 04 )  . 
la ripetizione del test avvenuta per lo pi dopo un periodo di addestramento , in particolare dopo la consultazione di un dvd didattico , fornito dal cspo , contenente diverse migliaia di casi consultabili in forma di atlante o come test interattivo . 
la tabella 3 mostra i risultati di questi test ripetuti : un chiaro e significativo miglioramento della prestazione evidente sia al secondo ( 2 = 15 , 1 , p < 0 , 0001 ) che al terzo ( 2 = 10 , 2 , p < 0 , 01 ) tentativo . discussione luso di set standard in forma di test per la valutazione s . 
such an attitude is common in a clinical setting where excess investigations are not a major problem ( the presence of the woman allows immediate further investigation ) , and missing a cancer , often a symptomatic one , is unacceptable . 
on the contrary , in a screening setting , specificity is extremely imdellaccuratezza diagnostica certamente pi semplice e agile di misurazioni reali della sensibilit , basate sul tasso diagnostico di carcinoma , o sul tasso di carcinomi di intervallo . queste ultime misure , infatti , richiedono casistiche assai voluminose , difficili da ottenere per singoli operatori , e che comunque richiedono un lungo periodo di osservazione , mentre un test pressoch istantaneo , ripetibile , fruibile in qualsiasi momento e , specie in formato digitale , in qualsiasi luogo . la condizione del test , peraltro , introduce spesso una deformazione psicologica in chi lo sostiene , che spesso adotta una soglia di sospetto pi bassa , nel tentativo di aumentare al massimo la sensibilit . 
an excess referral rate increases economic and psychological costs ( reading is not immediate , and women have to be recalled , which is a major cause of anxiety ) , which may be unacceptable . 
in a scenario with a cancer prevalence < 1% , raising sensitivity by 10% detects one additional cancer every 2 , 000 screened cases whereas a 10% increase in referral rate implies 200 additional referrals per each additional cancer detected [ a referral positive predictive value ( ppv ) of 0.5% compared with 10%20% , as currently observed in screening ]  . 
the psychological effect associated with taking the test is almost unavoidable , and the observed sensitivity and referral rates are likely to be different from those obtained in current practice . 
thus , observed test results have no absolute value but are useful for comparison between tested subjects or to evaluate the same operator over time . the series examined is fairly large , and if we consider the total number of italian radiologists and the small minority involved in reporting mammography , a sample of over 500 cases is sufficiently representative of the national situation . although access to the test is on a voluntary basis , which might suggest a selection of less experienced but more strongly motivated radiologists , a large proportion of tests was taken by all radiologists working in the same radiology department or local health trust or even region . 
the option of employing properly trained general radiologists might turn out to be an acceptable and more feasible policy compared with the implementation of independent screening units on a national basis . rore falso negativo , che a un falso positivo : questo un atteggiamento tipico dello scenario clinico , ove un approfondimento diagnostico in pi non riveste un problema ( anche perch la donna presente e lapprofondimento immediato ) , mentre un errore diagnostico in un carcinoma , spesso sintomatico , decisamente da evitare . 
in uno scenario di screening , invece , la specificit ha una importanza molto maggiore , e un tasso di richiamo eccessivo , che comporta un aumento dei costi economici e psicologici ( la lettura differita e il richiamo causa un notevole stress alla donna ) pu risultare inaccettabile . 
in un contesto dove la prevalenza di carcinoma inferiore all1% , un aumento della sensibilit del 10% comporta lidentificazione aggiuntiva di un carcinoma ogni 2000 esami circa , mentre un aumento del 10% del tasso di richiamo comporta 200 richiami in pi per ogni carcinoma aggiuntivo diagnosticato ( un valore predittivo positivo dello 0 , 5% , rispetto al 10%20% comunemente osservato nello screening )  . 
leffetto psicologico del test su chi lo sostiene quasi inevitabile , e i valori di sensibilit e di tasso di richiamo ottenuti non saranno uguali a quelli che loperatore ha nella pratica corrente . 
considerato il numero generale di radiologi e il fatto che una piccola minoranza di essi referta la mammografia nella propria pratica professionale , un campione di oltre 500 casi risulta abbastanza rappresentativo della realt nazionale . 
anche se laccesso al test formalmente volontario , il che potrebbe suggerire una selezione tra i meno esperti ma certo i pi volonterosi , buona parte dei test sono stati sostenuti dallinsieme dei radiologi di una unit operativa di radiologia diagnostica , o di una asl , o addirittura di una regione . 
assumendo che il campione abbia una sua significativit , colpisce subito il dato dellesperienza in mammografia . soprattutto il dato relativo alle mammografie lette nellultimo anno , con oltre un terzo dei radiologi che leggono meno di 1000 , e poco meno di un terzo meno di 2000 mammografie lanno , rende ragione della realt italiana , peraltro ben nota , che vede la mammografia non affidata a centri senologici dedicati , ma a radiologi generalisti , nellambito delle varie metodologie diagnostiche afferenti a una unit operativa ospedaliera o territoriale standard , senza che questi radiologi siano stati previamente sottoposti ad un adeguato addestramento . 
questa una realt , pi che una scelta , che peraltro , come si vede dallanalisi dei risultati , potrebbe dover essere riconsiderata , per lo meno nel senso di garantire a tutti gli operatori addetti un congruo periodo di addestramento specifico . 
limpiego di radiologi generalisti propriamente addestrati potrebbe essere una soluzione accettabile e pi facilmente realizzabile su scala nazionale rispetto alla creazione di unit di screening indipendenti . nonostante il test sia stato creato con lintento di non renderlo troppo difficile ( i carcinomi erano stati effettivamente diagnosticati nella pratica corrente , i livelli di sensibilit e tasso di richiamo ottenuti dagli esperti sono stati ragionevolmente scontati ) , il tasso di superamento stato effettivamente modesto ( 32 , 7% )  . 
the number of mammograms read in the previous year is probably a better indicator of experience compared with number of years of experience or total mammograms read , as it accounts for recent intensive experience . 
consulting an interactive teaching atlas containing a large number of cases ( which would require decades to be seen in current practice ) allowed most radiologists to pass the test : the cumulative rate of success was 32.7% after one test , 66.7% after two and 89.3 % after three attempts . overall , with the limits inherent to the artificial nature of the test , the study suggests that : 1 . 
training is still possible , however , particularly if we consider that one third of italy has still to be covered by organised screening [ 4 ]  . rare il test dipende assai di pi dallesperienza che non dalla sua difficolt intrinseca . 
il numero di mammografie lette nellultimo anno un indicatore migliore degli anni di esperienza o delle mammografie totali lette , in quanto rende ragione di una esperienza recente e concentrata nel tempo : i radiologi di minore esperienza ( < 1000 mammografie / anno ) hanno risultati ( superamento del test nel 22 , 7% ) decisamente inferiori a radiologi di non tanto maggior esperienza ( > 2000 mammografie / anno ) che ottengono risultati pi che doppi ( superamento del test nel 54 , 8% )  . il ruolo fondamentale dellesperienza ribadito dallevidenza relativa a chi ripete il test , non avendolo superato , dopo un periodo ( in genere breve ) di addestramento su un supporto didattico congruo . 
la visione in un atlante interattivo di un numero elevato di casi , cosa che nella realt professionale avrebbe richiesto decenni o non si sarebbe mai avverata , ha consentito alla maggioranza dei radiologi di superare il test . 
se si generalizzano i risultati di chi ha ripetuto il test alla totalit della casistica , il tasso di superamento complessivo del test del 36 , 7% con un tentativo , del 66 , 7% con due tentativi , dell89 , 3% con tre . 
arrigo , via venezia 16 , i - 15100 alessandria , italy 2dipartimento di medicina sperimentale , sezione di diagnostica per immagini e radioterapia , universit di genova , largo rosanna benzi 10 , i - 16132 genova , italy correspondence to : f . 
zandrino , corso volpini 111 , i - 14057 isola dasti ( at ) , italy , tel . : + 39 - 0131 - 206481 , fax : + 39 - 0131 - 206682 , e - mail : zandrino@libero.it received : 17 october 2005 / accepted : 31 march 2006 / published online : 11 august 2006 abstract purpose . 
at mammography , five were not detected , three presented as spiculated masses , two as masses with irregular margins , two as spiculated masses with microcalcifications , two as distortions , one as a cluster of microcalcifications and one as asymmetric density . 
in three , core biopsy was made : in the first , a complex sclerosing lesion with atypical cells was suggested , in the second differential diagnosis between tubular carcinoma and sclerosing adenosis was proposed and in the third a tubular carcinoma . 
alla mammografia 5 non erano visibili , 3 si presentavano come masse spiculate , 2 come masse con contorni irregolari , 2 come masse spiculate con microcalcificazioni , 2 come distorsioni , 1 come gruppo di microcalcificazioni ed 1 come densit asimmetrica . 
in 3 stato fatto un prelievo bioptico mediante core - biopsy con le seguenti diagnosi : lesione sclerosante complessa con cellule atipiche ; lesione con diagnosi differenziale da porsi tra carcinoma tubulare ed adenosi sclerosante ; carcinoma tubulare . 
this particular subtype of invasive ductal carcinoma occurs with a prevalence of 1%10% of all mammary adenocarcinomas [ 26 ] , with a reported mortality of 2% [ 7 ]  . since prognosis is better than for other histological types [ 4 , 5 , 8 ] , it seems to be useful to improve the detection and presurgical characterisation of this type of lesion [ 9 ]  . 
the purpose of this paper is to present the histopathological , clinical , mammographic and ultrasonographic ( us ) features of a series of 16 cases of tubular carcinoma and to review the current literature . il carcinoma tubulare della mammella un adenocarcinoma invasivo , relativamente raro , ben differenziato . 
la prevalenza dell1%10% di tutti gli adenocarcinomi della mammella [ 26 ] , e la mortalit del 2% [ 7 ]  . essendo la prognosi pi favorevole rispetto agli altri tipi istologici [ 4 , 5 , 8 ] , utile , in fase pre - chirurgica , identificare e caratterizzare questo tipo di lesione [ 9 ]  . 
lo scopo del nostro lavoro presentare gli aspetti istopatologici , clinici , mammografici ed ecografici di una serie di 16 casi di carcinoma tubulare e analizzare la letteratura disponibile su tale lesione . materials and methods a retrospective review of 560 consecutive histologically proven breast carcinomas was made . 
in this series of patients , whenever a suspicious lesion was detected on mammography , us or supplementary projections were done . mammographic examinations were performed with dmr ( ge medical systems , milwaukee , wi , usa ) and mammomat 2000 ( siemens , erlangen , germany ) units . 
us was also performed in patients with a dense mammographic pattern . us - guided fine - needle aspiration cytology ( fnac ) was made for us - detectable lesions while stereotactic - guided biopsy ( 14 - gauge core biopsy ) was performed for lesions not detected with us . 
 materiali e metodi stata effettuata una revisione di 560 casi consecutivi di carcinoma della mammella , tutti con diagnosi istologica , osservati nel periodo compreso tra gennaio 2000 e gennaio 2005 . 
le mammografie sono state eseguite con apparecchiature dmr ( ge medical systems , milwaukee , wi , u.s.a. ) e mammomat 2000 ( siemens , erlangen , germania )  . lecografia veniva inoltre eseguita in pazienti con quadro mammografico di tipo denso . 
lagoaspirato ecoguidato stato eseguito sulle lesioni visibili allecografia , mentre nei casi di lesioni non identificabili allecografia veniva eseguita una core - biopsy sotto guida stereotassica con ago tranciante da 14 gauge . 
in tutti i casi di carcinoma tubulare sono stati registrati ed analizzati i dati clinici delle pazienti , cos come i reperti mammografici , ecografici ed istopatologici . results sixteen pure tubular carcinomas were found ( 2.86% ) in 14 women ( age 5510 years , meanstandard deviation )  . 
a nonspecific diagnosis positive for carcinoma was made in 11 cases without identification of the histological type while atypical cells were found in two cases ( table 1 )  . 
in three patients , core biopsy was made : in the first , a complex sclerosing lesion with atypical cells was suggested ; in the second , differential diagnosis between tubular carcinoma and sclerosing adenosis was proposed ; in the third , a tubular carcinoma was diagnosed . lesions associated with tubular carcinoma found at histology and the results of fnac and core biopsy in our series are listed in table 2 . 
1a - c craniocaudal ( a ) and mediolateral oblique ( b ) views demonstrate an irregularly shaped density in the upper - external quadrant of the left breast ( arrows )  . 
1a - c le proiezioni cranio - caudale ( a ) e medio - laterale oblique ( b ) dimostrano un addensamento con contorni irregolari nel quadrante supero - esterno della mammella sinistra ( frecce )  . 
in 11 lesioni stata fatta una diagnosi generica di carcinoma , senza identificazione del tipo istologico , mentre in 2 la diagnosi stata cellule atipiche ( tabella 1 )  . 
in 3 pazienti stata fatta una core - biopsy con le seguenti diagnosi micro - istologiche : lesione sclerosante complessa con cellule atipiche ; lesione con diagnosi differenziale da porsi tra carcinoma tubulare ed adenosi sclerosante ; carcinoma tubulare . 
adenosi sclerosanteb dcis , atypical hyperpalsia papillary hyperplasia , sclerosing adenosis , lcis sclerosing adenosis atypical lobular hyperplasia , lcis cribriform intraductal carcinoma dcis , atypical ductal hyperplasia atypical ductal hyperplasia cribriform intraductal carcinoma papillary intraductal carcinoma radial scar cribriform invasive carcinoma sclerosing adenosis cdis , iperplasia atipica iperplasia papillare , adenosi sclerosante , clis adenosi sclerosante iperplasia lobulare atipica , clis carcinoma cribriforme intraduttale cdis , iperplasia duttale atipica iperplasia duttale atipica carcinoma cribriforme intraduttale carcinoma papillare intraduttale radial scar carcinoma cribriforme invasivo adenosi sclerosante dcis , ductal carcinoma in situ ; lcis , lobular carcinoma in situ afine - needle aspiration cytology ; bcore biopsy tabella 1 risultati clinici ed istopatologici nella nostra casistica di 16 carcinomi tubulari puri in 14 pazienti paziente chirurgia linfonodi reperti istopatologici associati cdis , carcinoma duttale in situ ; clis , carcinoma lobulare in situ acitologia ; bcore biopsy discussion histopathology diagnosis of tubular carcinoma with fnac is often difficult . 
although not pathognomonic , some distinctive cytological features of tubular carcinoma can be identified : increased cellularity , angular cellular clusters with sharp borciate ai carcinomi tubulari identificate allesame istopatologico ed i risultati dellesame citologico e micro - istologico pre - operatori sono elencate in tabella 2 . le dimensioni dei carcinomi tubulari allesame istopatologico sono state di 9 , 74 , 9 mm ( da 4 , 9 a 20 , 0 mm )  . 
l ' ingrandimento ( b ) evidenzia un nodulo con contorni irregolari . all ' esame ecografico ( c ) questo piccolo carcinoma tubulare si presenta come una lesione ipoecogena , con contorni irregolari , senza attenuazione del fascio ultrasonoro . 
quadro citologico ( d ) ed istologico ( e )  . ders , oval cells perpendicularly arranged along the edges of the clusters , dispersed single epithelial cells with minimal atypia and few bare oval nuclei in the background [ 10 ]  . 
in the report by mitnick and coworkers , fnac was performed in 36 tubular carcinomas , with positive results for tubular carcinoma in 15 ( 42% ) , suspicion in three , atypia in ten and false negative discussione istopatologia la diagnosi citologica di carcinoma tubulare in genere difficile . 
found , in fnac of 15 tubular carcinomas , ten carcinomas and five cases with atypia [ 5 ]  . histological characteristics of tubular carcinoma are an orderly distribution of branched angular tubules in a loose , fibrous stroma , with low nuclear grade and no evidence of vascular or lymphatic invasion [ 10 ]  . 
core biopsy is recommended when tubular carcinoma is considered to be a possible diagnosis since fewer lesions are diagnosed as atypia and fewer false negatives can occur [ 5 , 9 ]  . 
nevertheless , in our series , a correct diagnosis of tubular carcinoma was made in only one case . other histological types of carcinoma can be found in association with tubular carcinoma , with a prevalence of ductal carcinoma in situ [ 13 , 7 , 9 ]  . 
the high frequency of ductal carcinoma in situ ( 12.5% in our series ) and cribriform carcinoma ( 19% ) associated with tubular carcinoma raised the question about a possible relationship among these lesions [ 4 ]  . 
calcifications , which are usually seen in intraductal carcinoma , are found on microscopy in 50% of tubular carcinomas [ 3 ] : this association suggests a correlation of tubular carcinoma and intraductal carcinoma [ 9 ]  . associated benign lesions are fibrocystic changes , atypical ductal hyperplasia , sclerosing adenosis and radial scar [ 9 ]  . 
radial sclerosing lesions ( radial scar , a central area of sclerosis composed of collagen and elastic tissue surrounded by stellate duct proliferation ) , presenting in many cases as spiculated masses , often mimic tubular carcinoma on histopathology . 
some authors have discussed the potential malignancy of radial scar and suggest progression from radial scar to tubular carcinoma [ 11 , 12 ]  . the age of our population is close to the mean age reported in other similar studies [ 4 , 5 , 8 ]  . 
the mean size of tubular clinical features aumentata cellularit , clusters cellulari con morfologia angolata e contorni netti , cellule ovalari disposte perpendicolarmente lungo i bordi dei clusters , cellule epiteliali sparse con minime atipie , rari nuclei ovali sullo sfondo [ 10 ]  . 
 [ 9 ] , la citologia stata effettuata in 36 carcinomi tubulari , con risultato positivo per carcinoma tubulare in 15 , sospetto in 3 , diagnosi di atipia in 10 e diagnosi falsamente negativa in 8 ( 22% )  . 
 [ 5 ] sono stati sottoposti ad esame citologico 15 carcinomi tubulari , con diagnosi di carcinoma in 10 e di atipia in 5 . le caratteristiche istologiche sono quelle di una serie di strutture tubulari angolate e ramificate disposte in modo ordinato allinterno di uno stroma connettivale lasso , con basso grado nucleare ; non presente invasione di vasi sanguigni o linfatici [ 10 ]  . 
alcuni autori raccomandano la core - biopsy nel caso si sospetti un carcinoma tubulare , in quanto con esame micro - istologico possibile evitare diagnosi generiche di atipia e sono rari i falsi negativi [ 5 , 9 ]  . 
ciononostante nella nostra casistica , solo in 1 caso stata fatta una diagnosi corretta di carcinoma tubulare . accanto al carcinoma tubulare vengono descritti altri tipi istologici , prevalentemente il carcinoma duttale in situ [ 13 , 7 , 9 ]  . 
lelevata incidenza di associazione con il carcinoma in situ ( 12 , 5% nella nostra casistica ) ed il carcinoma cribriforme ( 19% ) hanno fatto supporre un possibile rapporto tra queste lesioni [ 4 ]  . 
le calcificazioni , generalmente presenti nel carcinoma intraduttale , sono identificate allesame microscopico nel 50% dei carcinomi tubulari [ 3 ] : questa associazione fa supporre una relazione tra carcinoma tubulare e carcinoma intraduttale [ 9 ]  . si associano al carcinoma tubulare anche lesioni benigne quali alterazioni di tipo fibrocistico , iperplasia duttale atipica , adenosi sclerosante e radial scar [ 9 ]  . 
le lesioni radiali sclerosanti ( radial scar , unarea di sclerosi centrale costituita da tessuto collagene ed elastico e circondata da proliferazione cellulare con forma stellata ) , che si presentano in genere alla mammografia come masse spiculate , simulano spesso allesame istopatologico il carcinoma tubulare . alcuni autori hanno discusso sulla potenziale malignit della radial scar , ipotizzando un passaggio da radial scar a carcinoma tubulare [ 11 , 12 ]  . aspetti clinici let media delle nostre pazienti con carcinoma tubulare poco si discosta da quella riportata in studi simili [ 4 , 5 , 8 ]  . la dimensione media dei carcinomi tubulari allesame istopatologico descritta in letteratura di circa 1 cm [ 2 , 4 , 5 , 9 , 1315 ]  . 
nella nostra casistica stata di 9 , 7 mla maggior parte dei carcinomi tubulari non sono palpabili per le piccole dimensioni [ 4 , 9 ]  . lincidenza delle metastasi linfonodali va dallo 0% al 29% [ 2 , 48 , 14 ]  . 
moreover , the very low incidence of lymph node involvement is likely related to the low invasiveness of the primary lesion rather than the small size of most tubular carcinomas at the time of diagnosis [ 4 ]  . 
l ' ecografia dimostra un piccolo nodulo ipoecogeno con attenuazione posteriore ( d )  . all ' esame istologico carcinoma tubulare associato a radial scar . bassa incidenza sarebbe correlabile pi alla scarsa invasivit del tumore primitivo che alle piccole dimensioni della maggior parte dei carcinomi tubulari alla diagnosi [ 4 ]  . 
suggested no axillary nodal dissection in patients with a tubular component > 90% [ 16 ]  . no difference in presentation or clinical outcome of pure tubular carcinoma is observed in comparison with mixed forms [ 7 ]  . 
due to the low invasiveness of tubular carcinoma , breast preservation therapy , with or without radiotherapy , is probably the best approach [ 4 , 17 ] , in particular when no nodal metastasis is found [ 18 ]  . 
in a series of tubular carcinomas sized 3 cm or less , a low rate of recurrence was observed ( 4% ; 5.4 years mean follow - up ) after only lesion excision without radiotherapy [ 19 ]  . mammography the sensitivity of mammography in detecting tubular carcinoma ranges from 65% to 100% [ 2 , 5 , 9 , 15 ]  . 
according to most authors , the small size of the lesions on mammography is not specific enough to differentiate tubular carcinoma from other small carcinomas [ 2 , 4 ]  . 
only one case in our series presented as a cluster of microcalcifications without a mass . according to mitnick et al . , the presence of microcalcifications alone is the rarest mammographic pattern of tubular noma tubulare [ 4 , 17 ] , in particolare quando non sono presenti localizzazioni linfonodali [ 18 ]  . 
in carcinomi tubulari di dimensioni uguali o inferiori a 3 cm stata osservata una percentuale di recidiva bassa ( 4% , follow - up medio di 5 , 4 anni ) anche dopo semplice escissione della lesione senza radioterapia [ 19 ]  . mammografia la sensibilit della mammografia nellidentificazione del carcinoma tubulare varia dal 65% al 100% [ 2 , 5 , 9 , 15 ]  . 
come osservato nella nostra casistica , la maggior parte dei lavori descrivono il carcinoma tubulare come una piccola lesione spiculata senza microcalcificazioni ( tabella 2 ) [ 2 , 4 , 10 ]  . 
 [ 5 ] hanno identificato microcalcificazioni con aspetto radiologico maligno nel 24% delle lesioni . questo risultato potrebbe dipendere dallutilizzo dellingrandimento radiologico , effettuato nella maggior parte delle pazienti [ 5 ]  . 
not available afour lesions with microcalcifications ; bfour cases with calcifications suggestive of malignancy ; ctwo lesions with microcalcifications tabella 2 aspetti clinici e mammografici del carcinoma tubulare della mammella : dati estratti dalla letteratura carcinomi tubulari , n [ voce bibliografica ] palpabili mx negativa masse con contorni densit mal definiti / spiculati noduli microcalcificazioni / asimmetriche , rotondeggianti / masse con distorsioni ovali microcalcificazioni mx , mammografia ; n.a. , dato non disponibile aquattro lesioni con microcalcificazioni ; bquattro casi con microcalcificazioni ; cdue lesioni con mirocalcificazioni carcinoma [ 9 ]  . 
the only report suggesting typical radiological and clinical features of tubular carcinoma argued that a small , nonpalpable , spiculated mass is distinctive enough to differentiate tubular carcinoma from other malignancies [ 14 ]  . 
as in our series , all authors describe most tubular carcinomas as hypoechoic masses with ill - defined margins and posterior acoustic shadowing [ 5 , 9 , 15 ]  . 
the two missed lesions were 6 mm and 8 mm on histopathology [ 5 ]  . differential diagnosis a precise differential diagnosis between tubular carcinoma and other histological subtypes cannot be reached with mammography . 
small size and spiculated margins , the most frequent pattern of tubular carcinoma on mammography , are nonspecific characteristics that do not allow differentiation from other small - sized malignant lesions . 
the appearance of tubular carcinoma at colour - doppler us was not systematically evaluated in our series , and data were not recorded ; moreover , no large series of tubular carcinomas studied with colour - doppler us are available in the literature . 
 on magnetic resonance imaging ( mri ) , some authors observed enhancement patterns that could help to differentiate tubular carcinoma from other histological subtypes [ 2123 ]  . the most frequently observed is a thin rim of delayed peripheral enhancement [ 22 , 23 ]  . cytological diagnosis is difficult due to the possible occurrence of false negatives or nonspecific results [ 4 , 24 , 25 ] , also observed in our series , and for the sometimes difficult differential diagnosis with radial scar [ 24 , 26 ]  . 
notwithstanding the larger amount of material that allows a microhistological assessment , core biopsy , did not give , in our series , a definite diagnosis in two out of three patients . 
 [ 5 ] , in un tentativo di caratterizzare il carcinoma tubulare , hanno osservato come spicule lunghe , frequentemente associate alla radial scar , sono identificabili in circa la met dei carcinomi tubulari . ecografia sono rare le pubblicazioni in cui stato descritto laspetto ecografico dei carcinomi tubulari [ 5 , 9 , 20 ]  . 
le due lesioni non visualizzate misuravano , allesame istopatologico , 6 e 8 mm [ 5 ]  . diagnosi differenziale la mammografia non permette una sicura diagnosi differenziale tra carcinoma tubulare ed altri tipi istologici . 
le piccole dimensioni ed i contorni spiculati , che sono laspetto mammografico di buona parte dei carcinomi tubulari , sono caratteristiche aspecifiche , che non ne consentono la differenziazione da altre lesioni maligne di piccole dimensioni . 
anche la possibile associazione con la radial scar rappresenta una caratteristica aspecifica , non essendo state riscontrati aspetti radiologici particolari nelle radial scars associate a carcinoma tubulare [ 11 ]  . 
le caratteristiche dei carcinomi tubulari al color - doppler non sono state valutate in modo sistematico nella nostra casistica , e non sono al momento disponibili in letteratura descrizioni su ampie casistiche . 
dal punto di vista citologico la diagnosi di carcinoma tubulare complessa , per la possibile presenza di false negativit o di diagnosi aspecifiche [ 4 , 24 , 25 ] , osservate anche nella nostra casistica , e per la diagnosi differenziale talora difficile con la radial scar [ 24 , 26 ]  . 
la core - biopsy , nonostante la maggior quantit di materiale prelevato che consente una diagnosi micro - istologica , non ha fornito , nella nostra casistica , risultati sicuri in 2 casi su 3 . 
pertanto probabile che nella diagnosi pre - operatoria solo la vacuum - assisted biopsy possa consentire un prelievo sufficientemente accurato . conclusioni in conclusione , si pu affermare come la prognosi favorevole con possibilit di un approccio terapeutico minimamente invasivo al carcinoma tubulare rendono necessaria una vaf . 
this can probably only be obtained with histological sampling ( if possible , vacuum - assisted biopsy ) , allowing precise differential diagnosis between tubular carcinoma and other histological subtypes and benign associated lesions . lutazione pre - operatoria il pi possibile accurata . 
bruschi , institute of radiology pugd , via colugna 50 , i - 33100 udine , italy , tel . : + 39 - 043 - 2559266 , fax : + 39 - 043 - 2559867 , e - mail : radiologia.segr@med.uniud.it received : 7 september 2005 / accepted : 1 april 2006 / published online : 11 august 2006 abstract purpose . 
in a blind in vitro analysis , 28 great saphenous veins , obtained after stripping surgery from 28 patients with chronic venous insufficiency , were examined with a digital us scanner atl - hdi5000 , linear 5 to 12 - mhz broadband probe , compound imaging technique and analogic - digital zooming . 
the samples , fixed in formalin , were sent to the pathology laboratory : sections were obtained at the same level of the sonographic planes , and images were obtained by digital camera mounted on an optical microscope . measurements obtained at histology were considered as the gold standard . 
if these preliminary data are confirmed in vivo , sonography could be used to discriminate patients eligible for conservative treatment instead of surgery . key words ultrasound parietal thickness varicose veins endovenous laser treatment riassunto obiettivo . 
in una analisi in cieco sono stati esaminati in vitro 28 pezzi operatori di grande safena , provenienti da altrettanti pazienti affetti da insufficienza venosa cronica e sottoposti a stripping chirurgico . 
stato utilizzato un ecografo digitale atl - hdi 5000 , con sonda ecografica lineare broadband con frequenza variabile da 5 a 12 mhz , tecnica compound imaging e tecnica zoom ad , ottenendo sia scansioni assiali che longitudinali . 
nella nostra esperienza preliminare , lo studio in vitro delle vene varicose permette un riconoscimento almeno morfologico delle pareti ipotrofiche , ponendo le basi per lo studio in vivo nella selezione dei pazienti candidati a trattamento conservativo , piuttosto che chirurgico . parole chiave ecografia spessore parietale vene varicose trattamento laser endovascolare introduction introduzione e . 
it has been widely demonstrated that vascular morphology depends on general factors , such as age , and on particular factors , such as variations in resistance , pressure and flow strictly depending on local haemodynamics [ 2 ]  . 
 [ 3 ] noted that hypertrophy may be caused by derangement of smooth muscle cells ( due to mutation from a contractile phenotype to a less elastic phenotype ) and extracellular matrix accumulation , as well as by increased vasa vasorum due to hypoxia and some growth factors ( tgf )  . 
 [ 6 ] suggest that changes in the cellular matrix of the vein wall can be considered the first cause of varicosity while valve failure would be merely a consequence of these modifications . 
 histological remodelling phenomena account for cellular proliferation , cellular migration , and extracellular matrix biosynthesis due to biochemical factors produced by mural cells ( endothelial cells , smooth muscle cells , fibroblasts ) and by circulation elements ( platelets , leucocytes ) [ 2 ]  . 
therefore , the hyperdynamic flow , related more frequently in vivo to incontinence of the saphenofemoral junction of the great saphenous vein ( gsv ) , may induce parietal changes , which result in a succession of atrophic and clearly hypertrophic segments in the same vein wall [ 3 ]  . 
 the wall of normal veins displays , on histological section with masson 's trichrome , regular arrangement in three layers : intima ( thin and regular ) , muscularis ( which supports the vein wall and is made up of smooth muscle cells clustered in circular bundles or surrounded by extracellular matrix ) and adventitia ( outermost layer , at times interrupted by vasa vasorum )  . the intima and muscularis are usually involved in veinwall remodelling , and these two tunicae present an overall normal thickness ranging from 460 m to 690 m [ 3 ]  . 
this ultrastructural normal aspect , according to some authors , correlates with a three - layer pattern at sonographic imaging [ 7 , 8 ] : the adventitia appears hyperechoic ; the media hypoechoic ; the intima hyperechoic [ 7 ]  . 
initially , the wall reacts with an accumulation of smooth muscle cells and fibrous tissue , which causes disappearance of the typical three - layer pattern due to loss of separation between intima and media [ 2 ] and causes a thickening of these two layers , ranging from 800 to 1 , 380 m [ 3 ]  . 
 subsequently , ischaemia due to insufficient flow from vasa vasorum leads to a progressive loss of smooth muscle cells and matrix , which causes a reduction of wall thickness ( 115350 m ) [ 3 ]  . 
a hypotrophic vein wall is two times thinner than a regular le vene varicose colpiscono circa un quarto della popolazione adulta dei paesi occidentali , rappresentando una causa considerevole di morbidit e di accesso ai servizi di assistenza medica . 
stato largamente dimostrato che la morfologia vascolare strettamente legata a fattori generali come let e a fattori particolari che possono mostrare modificazioni legate a variazioni emodinamiche locali che comprendono la resistenza , la pressione e il flusso [ 2 ]  . 
 [ 3 ] hanno posto lattenzione sullipertrofia dovuta alla disorganizzazione delle cellule muscolari lisce ( con mutazione da un fenotipo contrattile verso un fenotipo meno elastico ) e allaccumulo di matrice extracellulare , nonch sullincremento dei vasa vasorum sotto lo stimolo ipossico e sotto lo stimolo di alcuni fattori di crescita ( tgf )  . 
altri autori [ 4 , 5 ] hanno enfatizzato come possa essere importante laumento della produzione di collagene di tipo i a scapito di quella di tipo iii e v e la diminuzione dellelastina . 
 [ 6 ] sostengono che i cambiamenti della matrice cellulare della parete della vena possono essere considerati come la prima causa delle varicosit e linsufficienza valvolare come lesito di tali cambiamenti . 
sicuramente i principali fenomeni di rimodellamento da un punto di vista istologico possono essere riassunti in : proliferazione cellulare , migrazione cellulare , biosintesi di matrice extracellulare , dovuti a fattori biochimici prodotti da cellule parietali ( cellule endoteliali , cellule muscolari lisce , fibroblasti ) e da elementi circolanti ( piastrine , leucociti ) [ 2 ]  . 
possibile quindi affermare che la parete di una vena che viene sottoposta ad un regime iperdinamico ( condizione pi frequente in vivo il caso della vena grande safena a monte della quale si instauri una incontinenza della crosse safeno - femorale ) va incontro ad una serie di trasformazioni parietali compensatorie costituite da una alternanza di segmenti atrofici ad altri pi francamente ipertrofici [ 3 ]  . 
la parete della vena sana presenta , alla sezione istologica con colorazione tricromica di masson , una regolare tristratificazione : lintima ( sottile e regolare ) , la muscolare ( che fa da struttura portante della parete ed costituita da cellule muscolari lisce disposte a bande circolari o impacchettate con matrice extracellulare ) e lavventizia ( la pi esterna )  . 
pi esternamente , lavventizia disposta a nastri viene interrotta talora dai vasa vasoruintima e muscolare sono di solito coinvolte nel rimodellamento della parete della vena e nel complesso queste due tuniche raggiungono uno spessore complessivo massimo di 460690 m [ 3 ]  . 
almeno inizialmente la parete risponde con un accumulo di cellule muscolari lisce e tessuto fibroso , che porta alla scomparsa della caratteristica tristratificazione , per la perdita della netta separazione fra intima e media [ 2 ] e un complessivo aumento dello spessore parietale a carico di intima e e . 
the succession of alternating hypertrophic and atrophic segments probably explains the typical tortuosity of varicose veins . analysis of wall features and thickness has never been considered really relevant until now , mainly for two reasons : first , because the only available treatment up to now was radical saphenectomy ; and second , because a presurgical histological study after percutaneous biopsy is probably too invasive a procedure for a low - risk disease . the advent of digital sonography with a high - frequency , high - resolution broadband probe has introduced many important advantages in clinical practice , such as decreased noise , absence of signal distortion and optimal focusing of the ultrasound ( us ) beathe quality of the resulting sonographic image is usually better than conventional us with regard to axial and lateral resolution , contrast , absence of noise and artefacts [ 10 ] , permitting the evaluation of submillimetric structures , such as vein walls , until now never really considered by sonography . the thickness of pathological vein walls is potentially very relevant for the surgeon , who may consider the option of conservative treatment , e.g. 
in fact , surgical excision of varicose portions characterised by parietal hypoplasia may be very difficult due to their fragility . on the contrary , a thick muscle wall makes evlt ineffective , increasing the risk of recurrences [ 11 ]  . 
 successivamente fenomeni ischemici dovuti allinsufficiente apporto dei vasa vasorum , portano alla perdita di cellule muscolari lisce e matrice con assottigliamento complessivo della parete ( 115350 m ) [ 3 ] , mentre le cellule muscolari lisce e la componente extracellulare sono quasi completamente scomparse . 
allo studio delle caratteristiche e dello spessore parietale , non mai stata data la giusta importanza , sia perch lunico trattamento proposto era quello della safenectomia radicale , con leliminazione del tronco venoso superficiale principale , sia perch presupponeva uno studio istologico preoperatorio , improponibile nellinquadramento di una patologia che rarissimamente mette a rischio la vita del paziente . 
la recente introduzione degli ecografi digitali con sonde ad alta frequenza , larga banda ed alta risoluzione , ha permesso nella pratica clinica una serie di vantaggi non indifferenti , tra cui la riduzione del rumore di fondo , la mancata distorsione del segnale , lottimale focalizzazione del fascio di ultrasuoni . 
la qualit dellimmagine definitiva di solito nettamente superiore allimmagine ecografica convenzionale in termini di risoluzione spaziale assiale e laterale , di contrasto , di annullamento del rumore di fondo e di eliminazione degli artefatti [ 10 ] , cos da poter valutare strutture submillimetriche , come la parete delle vene , finora mai considerata in ecografia . 
la conoscenza dello spessore della parete delle vene un dato potenzialmente interessante sia per lecografista , che si trova alle prese con misurazione submillimetriche , che per il chirurgo che abbia optato per il trattamento conservativo e nella fattispecie per il trattamento laser endovascolare ( evlt ) di recente introduzione . 
infatti i tratti varicosi caratterizzati da spiccata ipoplasia parietale risultano difficilmente asportabili con le tecniche invasive , proprio a causa della loro fragilit . per contro , lassenza di una spessa parete muscolare rende efficace il trattamento endovascolare , riducendo al minimo il rischio di recidive [ 11 ]  . 
during postprocessing with appropriately calibrated software , we were able to perform further sonographic measurements , extending our assessment to other segments not considered at the beginning . the formalin - fixed samples were sectioned by the pathologist axially and longitudinally , at the same level of the sonographic planes . 
the samples were embedded in paraffin and cut at a thickness of 5 m , then stained with hematoxylin - eosin and masson 's trichrome to better visualise wall layers . 
histological measures were calculated using dedicated software ( olympus dp soft version 3.2 ) with magnification of a digital camera olympus mounted on an optical microscope ( axioscop , zeiss , ag , germany )  . 
su queste ultime sono state effettuate da 1 a 3 misurazioni progressive ( con un massimo di 3 misurazioni nelle vene pi lunghe e con un valore medio di 2 , 39 misurazioni su ciascuna vena , per un totale di 67 spessori parietali )  . 
in accordo con quanto riportato in letteratura [ 3 ] , abbiamo considerato come normale una vena con spessore parietale compreso tra 0 , 4 e 0 , 7 mm , ipertrofica una con valori superiori a 0 , 8 mm e ipotrofica una con spessore inferiore a 0 , 35 mle misurazioni lungo il tratto safenico sono state riferite al collega anatomopatologo come distanza , espressa in centimetri , dal filo chirurgico legato a monte , in corrispondenza della crosse safeno - femorale ( punto 0 )  . 
in post - processing con lutilizzo di un software dedicato , opportunamente calibrato , abbiamo potuto compiere ulteriori misurazioni ecografiche , estendendo le nostre valutazioni ad altri tratti non considerati in un primo momento . 
i reperti , fissati in formalina , sono poi stati inviati presso il laboratorio di anatomia patologica , dove sono stati sottoposti a sezione in corrispondenza delle riferite scansioni ecografiche , secondo una direzione longitudinale e assiale . 
i prelievi sono stati inclusi in paraffina e sezionati ad uno spessore di 5 m e quindi sottoposti a colorazione con ematossilina - eosina e tricromica di masson , in modo da evidenziare con maggiore contrasto la parete e lavventizia . 
complete agreement was demonstrated in hypotrophy when the central hypoechoic layer only was considered us with the two hyperechoic lines us with central hypoechoic line only histology tabella 1 ecografia versus istologia : risultati . 
stata verificata una totale concordanza nei casi di ipotrofia quando stata considerata la sola linea ipoecogena centrale spessore ecografico con le due linee iperecogene spessore ecografico della sola linea centrale ipoecogena istologia diagnosis hypotrophy , n normotrophy , n hypertrophy , n total , n diagnosi ipotrofia , n normotrofia , n ipertrofia , n totale , n essa compresa e quindi non realmente parte dello spessore parietale , abbiamo diagnosticato 32 tratti safenici come ipotrofici ( valore medio di 0 , 28 mm ) , contro i 29 diagnosticati nelle corrispondenti sezioni istologiche ( valore medio di 0 , 279 mm )  . 
lecografia ha fatto diagnosi di normotrofia in 23 casi ( valore medio di 0 , 52 mm ) , mentre listologia ne ha diagnosticati 22 ( valore medio di 0 , 56 mm ) , confermando 19 dei casi individuati allecografia ( 86% ) , mentre 4 / 23 furono dimostrati poi essere ipertrofici allistologia , rappresentando una sottostima ecografica . 
lecografia ha permesso la diagnosi di 12 casi di ipertrofia ( valore medio di 0 , 86 mm ) , mentre lesame istologico ne ha riconosciuti 16 ( valore medio di 0 , 99 mm )  . 
the graphic shows , on the basis of measurements considering the hypoechoic layer only , higher dispersion of measurements in normotrophy and hypertrophy relative to hypotrophy even though for some authors , the values in hypotrophy are too small to be evaluated . 
sonography classified 23 segments as normotrophic ( mean value 0.52 mm ) as against 22 identified at histology ( mean value 0.56 mm ) ; 19 / 23 cases ( 86% ) considered normotrophic by sonography were confirmed by histology whereas 4 / 23 were hypertrophic at histology ( sonographic underestimation )  . 
the k - test statistic demonstrated excellent agreement ( 0.91 ) for the diagnosis of hypotrophy , good agreement ( 0.7 ) for the diagnosis of hypertrophy and fair - to - good agreement ( 0.47 ) for the diagnosis of normotrophy . 
furthermore , the more - or - less irregular morphology of vein walls and their inhomogeneous echogenicity , not distinguishable from surrounding tissue , do not always allow in vivo measurements of parietal thickness . 
3a , b histologically normal structure of a venous wall stained with masson 's trichrome ( a ) showing the different tunicae : intima ( i ) , media ( m ) and adventitia ( a )  . 
3a , b immagine istologica di normale struttura di parete venosa colorata con tricromica di masson ( a ) che mostra le differenti tuniche : lintima ( i ) , la media costituita da cellule muscolari lisce disposte a bande circolari o impacchettate con matrice extracellulare ( m ) e lavventizia ( a )  . 
secondo la nostra interpretazione , limmagine ecografica di un normale spessore parietale mostra una linea centrale ipoecogena ( i + m ) compresa da due linee iperecogene ( cfr doppia freccia ) : la pi interna che corrisponde allinterfaccia sangue - parete ( punte di freccia ) , la pi esterna che corrisponde allinterfaccia gel - avventizia ( a )  . pi o meno irregolare , e lecogenicit disomogenea , non sempre distinguibile dai tessuti circostanti , rendono agevole , in vivo , la sua misurazione . 
lautore , in particolare , ha dimostrato che il thi migliora la risoluzione spaziale laterale e lo spessore di fetta in funzione della profondit , mentre il compound quella di contrasto ed il riconoscimento dei bersagli anecogeni , infine che lhsonoct migliora la risoluzione spaziale laterale . 
ci nonostante la risoluzione spaziale massima per un trasduttore da 512 mhz , , sia pure con limpiego di tali algoritmi , sempre e comunque superiore a 0 , 4 mm , valore limite nel nostro studio tra una parete normotrofica ed una ipotrofica . 
daltra parte nel nostro studio basato sul confronto istologico , abbiamo constato che , anche se spessori inferiori a tale limite sono di difficile misurazione , possibile riconoscere perlomeno morfologicamente interfacce e . 
quindi , pur non essendo possibile effettuare attendibilmente misure quantitative al di sotto del limite di risoluzione del sistema , abbiamo potuto dimostrare in modo abbastanza chiaro che interfacce ad intervalli minori possono essere evidenziate . 
un ulteriore limite nella valutazione sperimentale rappresentato dalle difficolt nel far coincidere le misurazioni ecografiche con quelle effettuate nel preparato istologico , a causa delleterogeneit parietale , in particolare nei segmenti ipertrofici e normotrofici . 
lo studio ha messo inoltre in evidenza che la misura nellimmagine ecografica della parete venosa , comprendendo le due interfacce ecogene , non correla con la misura istologica , dato che produce una netta sovrastima globale . 
4a , b longitudinal histological sections ( a ) of a hypertrophic vein wall . derangement of the layers , mostly due to accumulation of smooth muscle cells and fibrous tissue green on masson 's trichrome corresponds to a thickened hypoechoic central line ( double arrow ) at ultrasound ( b )  . 
la disorganizzazione delle tuniche , in gran parte dovuta allaccumulo di cellule muscolari lisce e tessuto fibroso riconoscibile come verde alla colorazione tricromica , corrisponde nellimmagine ecografica ( b ) ad una linea centrale ispessita ( doppia freccia )  . 
this process , produced by haematic vaporisation , is presumably caused by the chromophore role of haemoglobin , which absorbs all of the laser 's beam energy [ 15 ] and consequently generates steam bubbles . 
furthermore , this procedure has fewer serious side effects than closure with radiofrequency ( molecular agitation produced by radiofrequency waves ) , which is associated in 16% of cases with major skin paraesthesias due to injury to surrounding nervous structures [ 16 ]  . 
in addition , evlt can be used in association with other treatments . nevertheless the first experiences with these procedures have demonstrated some limits . some authors have noted one the one hand the possibility of incomplete endothelial coagulations and early reopening of a treated venous portion and , on the other , the possibility of undesirable perforations causing haematoma and pain [ 16 , 17 ]  . 
it is even possible to assume that the knowledge of parietal thickness could be useful to sura che debba essere effettivamente considerata . oggi possiamo quindi ipotizzare una capacit dei nuovi ecografi digitali di riconoscere non solo la dilatazione del lume della vena grande safena , ma anche di potere correttamente riconoscerne lo spessore della parete . 
laffacciarsi di nuove tecniche terapeutiche che mirano a un trattamento conservativo , cio a sigillare la vena patologica , pi che a eliminarla , presupporrebbe , infatti , una corretta conoscenza della sua parete . 
gi noto infatti , fin dagli anni 80 , presso i sostenitori della terapia sclerosante [ 12 ] che le iniezioni endovasali di sostanze sclerosanti sono pi efficaci nei cosiddetti vecchi varicosi , proprio in relazione alla loro fibrosi cronica parietale , che esita in una ipotrofia parietale , piuttosto che nei giovani varicosi che , specie se donne in et fertile , vanno preferenzialmente indirizzati alla chirurgia tradizionale . 
 [ 13 ] hanno proposto un nuovo sistema conservativo per il trattamento delle varici dei tronchi safenici , utilizzando lenergia fornita dal laser attraverso una fibra ottica introdotta da una guida allinterno del vaso ( evlt )  . 
tale sottile fibra laser introdotta fino al punto di patologia , con linvio di flussi laser in tutte le direzioni , provoca un surriscaldamento del contenuto e quindi del contenente . 
 [ 14 ] hanno sottolineato come sia proprio la presenza del sangue allinterno del vaso che contribuisce alla dispersione omogenea del calore allinterno del vaso e sulle sue pareti , ottenendo cos , attraverso una vaporizzazione ematica , presumibilmente per la presenza dellemoglobina che si comporta come un cromoforo in grado di assorbire tutta lenergia del fascio laser [ 15 ] e di conseguenza di produrre bolle di vapore , una fotosclerosi dei due endoteli contrapposti e quindi una fibrosi da contatto connettivale tra le due pareti . 
carbonizzazioni e perforazioni dello strato connettivale sono stati dimostrati in vitro da proebstle in un recente lavoro [ 15 ] che distingueva , utilizzando un modello sperimentale , la trombosi del sangue vaporizzato dal danno parietale esercitato dal fascio laser diretto , in grado di penetrare fino a 0 , 3 mm di spessore . 
tale procedura , ha recentemente destato un notevole interesse , poich , unisce i vantaggi della scleroterapia ( poco invasiva e meno traumatica della chirurgia , brevi tempi di ospedalizzazione e anestesia locale ) a un minor numero di recidive . 
inoltre , essa gravata da meno effetti collaterali gravi rispetto al trattamento che utilizza radiofrequenze ( agitazione molecolare da onde a radiofrequenza ) , che si associa nel 16% dei casi a importanti parestesie cutanee da danno delle strutture nervose circostanti [ 16 ] e pu essere utilizzata in combinazione ad altri trattamenti . 
diversi autori hanno evidenziato le possibilit di incomplete coagulazioni endoteliali e ricanalizzazione precoce del tratto venoso trattato e per contro le possibilit di complicanze , soprattutto le indesiderate perforazioni , causa di ematomi e dolore [ 16 , 17 ]  . 
the digital us scanner used as a microscope in vivo would allow one to measure the thickness of pathological veins before treatment in order to distinguish patients with marked hypertrophy more frequent in young people suitable for surgery or longer exposition times with laser therapy from those with parietal atrophy caused by chronic disease . 
the second group of patients can be treated conservatively with evlt , reducing laser exposition time proportionally to the thickness of the hypotrophic wall . da a radiofrequenza , che pur utilizzando un principio differente dallevlt ( agitazione molecolare prodotta da onde a radiofrequenza ) , produce una embolizzazione del vaso e la pi antica scleroterapia che potrebbe utilizzare una quantit di sostanza sclerosante adeguata al vaso da trattare . 
bellelli , via ombrone 4 , i - 00198 rome , italy , tel . : + 39 - 06 - 6837243 , fax : + 39 - 06 - 6837346 , e - mail : abellelli@libero.it received : 8 april 2006 / accepted : 9 may 2006 / published online : 11 august 2006 abstract purpose . 
the mri exam was performed on a 0.2 - tesla ( t ) magnet and a 1.5 - t magnet using t1 - weighted spin echo ( se ) , t2 - weighted se and fat - suppression scans [ short - tau inversion recovery ( stir ) ] in axial , sagittal and coronal planes . 
out of 77 patients , 35% of the whole sample , 37 had a tear of the posteromedial fascicle ( type ii lesion ) , and the remaining 40 had anterolateral fascicle tears ( type iii )  . 
l ' esame rm stato eseguito con magnete permanente 0 , 2 tesla e superconduttivo 1 , 5 tesla , con sequenze se t1 e t2 pesate , e sequenze ottenute con la soppressione del segnale del tessuto adiposo ( stir ) , su piani assiali , sagittali e coronali . 
in 77 pazienti , che rappresentavano il 35% dei pazienti riesaminati , 37 mostravano una lesione del fascio postero - mediale ( ii grado di lesione )  . i restanti 40 pazienti mostravano una lesione del fascio anterolaterale , lesione di iii grado . 
l ' esame rm permetterebbe cos di qualificare il tipo di danno a livello del lcp e quindi come incida sulla stabilit articolare . key words posterior cruciate ligament injury mri knee stability parole chiave lesione del legamento crociato posteriore rm stabilit di ginocchio introduction introduzione many studies have reported that the posterior cruciate ligament ( pcl ) has biomechanical and neurosensory features that make it the main stabiliser in internal rotation and flexion of the knee . 
as with the anterior cruciate ligament ( acl ) , the pcl is composed of two molti studi in letteratura hanno sottolineato che il legamento crociato posteriore ( lcp ) ha delle caratteristiche biomeccaniche e neurosensoriali tali da renderlo il principale stabilizzatore nei movimenti di rotazione interna e flessione di ginocchio . 
in particular , studies on ligament strength show that the al is stronger than the pm portion ( 70% versus 30% ) and has the main role until knee flexion reaches 90 [ 79 ]  . in 1992 , for the first time , gross et al . 
the main limitation of t1 - weighted mri scans lies in their low contrast resolution , which does not allow one to distinguish between intraligament oedema and haematoma , the sign of a ruptured ligament . 
moreover , the gross classification does not consider the anatomical site of the injury , an important factor , especially in view of the different roles that the al and pm fascicles play in knee biomechanics . 
 this study proposes a new anatomical classification of acute traumatic pcl injuries using morphological t1weighted mri scans and high - contrast signal sequences , such as se t2 - weighted and fat - suppression images [ shorttau inversion recovery ( stir ) ]  . 
by using mri , we aimed to distinguish the more significant ligament injuries from those which are biomechanically less important . materials and methods we conducted a retrospective study of 4 , 000 mri scans carried out between june 2000 and june 2003 and selected 220 patients with acute pcl traumatic injury after car or sport accidents . 
ages ranged from 14 to 64 , with a mean age of 32.7. out of 220 patients , 125 underwent a new mri exam 1 - 3 months after sustaining the injury . 
la principale limitazione delle sequenze se t1 pesate dovuta alla bassa risoluzione di contrasto , che non permette di distinguere l ' edema intra - legamentoso della lesione , dall ' ematoma , segno di rottura a livello del legamento . 
inoltre , la classificazione di gross non considera la sede anatomica di lesione , dato di fondamentale importanza , in modo particolare in considerazione delle differenti caratteristiche biomeccaniche dei fasci al e pm . l ' obiettivo del nostro studio di proporre una nuova classificazione delle lesioni acute traumatiche del lcp , utilizzando sia sequenze morfologiche se t1 pesate , sia sequenze ad alto contrasto come le sequenze se t2 pesate e le sequenze ottenute con la soppressione del segnale del tessuto adiposo . 
attraverso l ' esame rm il nostro intento quello di poter distinguere le lesioni legamentose meno significative , da quelle che lo sono di pi da un punto di vista biomeccanico . materiali e metodi abbiamo condotto uno studio retrospettivo su 4000 risonanze magnetiche di ginocchio , da giugno 2000 a giugno 2003 . abbiamo preso in considerazione nel nostro studio 220 pazienti , 157 maschi e 63 femmine , con lesione traumatica acuta del lcp dopo incidente stradale o sportivo , con et compresa tra 14 e 64 anni , con una media di 32 , 7 anni . 
i 220 pazienti in cui stata evidenziata la lesione a carico del lcp erano stati precedentemente valutati clinicamente e classificati come pazienti con instabilit posteriore . abbiamo incluso nello studio sia pazienti con lesione isolata di lcp , che pazienti con lesione complessa , associata a patologia meniscale , osteocondrale o capsulare . l ' esame rm stato eseguito a pazienti supino , con ginocchio flesso tra 0 e 15 , con magnete permanente 0 , 2 tesla ( artoscan , esaote , genova , italia ) e superconduttivo 1 , 5 tesla ( simphony siemens , erlangen , germania ) , usando sequenze se t1 e se t2 pesate e sequenze ottenute con la soppresione del grasso ( stir ) , su piani assiali , sagittali e coronali . 
lo spessore di strato stato compreso tra 3 e 5 mm , con gap di 0 mm e fov variabile tra 170200 mm ; la matrice stata di 256x256 sul magnete 0 , 2 tesla , mentre era di 512x512 su magnete 1 , 5 tesla . 
the thin area with intermediate signal intensity appearing within the pcl on t1 - weighted images but not on t2 - weighted scans was considered to be due to the magic angle effect . 
we did not take into account the high - intensity signal within the ligament due to eosinophil degeneration in arthritic knees . results fifty - five of the 220 patients ( 25% of cases ) had an area of increased signal intensity within the pcl on both t1and t2 - weighted images ; the edges appeared homogeneously continuous and hypointense ( gross type i )  . 
in the last 22 patients ( 10% ) , we detected complete tear of the pcl , which presented no continuity of the remaining ligament fibres on high - contrast images . on the basis of our results , we propose a new classification of pcl traumatic injuries , which takes in consideration the extent of remaining fibres and the exact anatomical site of the lesion . 
this way , we can distinguish between posteromedial pcl ligament injuries on the dorsal aspect from those of the anterolateral fascicle ( ventral aspect )  . discussion the mri classification of posterior ligament injuries was first published by gross et al [ 10 ]  . 
il piano assiale stato posizionato parallelo al piano tibiale o meniscale , mentre il piano coronale stato allineato alla tangente bicondiloidea posteriore . in rm stata considerata come lesione del lcp il caso in cui la banda a bassa intensit del segnale , che rappresenta il legamento , appariva discontinua , con un ' area di alta intensit del segnale che coinvolgeva uno dei due versanti del legamento . 
abbiamo escluso dallo studio l ' iperintensit del segnale intra - legamentosa , nelle ginocchia artrosiche , dovuta alla degenerazione eosinofila . risultati il nostro studio ha condotto ai segmenti risultati . 
cinquantacinque dei 220 pazienti , ( il 25% dei casi ) ha mostrato nelle immagini rm un ' area di iperintensit del segnale all ' interno del legamento , sia nelle immagini t1 che in quelle t2 pesate , con margini omogeneamente continui e ipointensi ( di i grado secondo gross )  . 
sebbene in questi casi lo spessore del legamento risultato lievemente aumentato , il decorso intraarticolare del legamento apparso normale , cos come la rappresentazione del tessuto adiposo all ' interno della gola inter - condiloidea . 
il legamento appariva continuo nelle sequenze se t2 pesate ed in quelle ottenute con la soppressione del segnale del tessuto adiposo . lo spessore del legamento era lievemente aumentato , tuttavia i margini risultavano continui , anche se modicamente disomogenei . 
in accordo con la classificazione di gross , le lesioni del lcp appartenenti al iii grado , ossia lesioni complete , sono state individuate in 77 pazienti ( 35% )  . 
nella nostra esperienza abbiamo confrontato le sequenze se t1 pesate con le sequenze ad alto contrasto in rm e abbiamo notato che le lesioni del legamento sul versante ventrale ( sede del fascio al ) sono state evidenziate in 40 casi , poco pi della met , mentre quelle sul versante dorsale ( sede del fascio pm ) , sono state osservate in 37 casi . 
nei rimanenti 22 pazienti ( il 10% ) abbiamo identificato una lesione completa del lcp , con soluzione di continuo completa del legamento , senza evidenza di fibre residue nelle sequenze ad alto contrasto . quindi , in relazione a quanto stato evidenziato nel nostro studio , abbiamo deciso di proporre una nuova classifitable 1 magnetic resonance imaging ( mri ) grading of posterior cruciate ligament ( pcl ) injuries on spin - echo ( se ) t1 - weighted images [ 10 ] a . 
both edges of the ligament show homogeneously low signal intensity the high signal intensity is within the ligament and in one of two edges ; partial lesion the high signal intensity involves the entire ligament ; complete lesion tabella 1 gradi di lesione in rm delle lesioni del lcp , con sequenze se t1 - pesate [ 10 ] grado immagine rm il legamento crociato posteriore mostra omogenea bassa intensit del segnale ed continuo iperintensit del segnale all ' interno del legamento . 
type ii includes partial lesions , in which high signal intensity is seen in the central portion within the ligament as well as one of the two ventral or dorsal margins of the ligament . 
type iii injury is described as high signal intensity extending to the entire ligament and including both the dorsal and the ventral aspects ; this situation has been classified by gross as a complete injury . 
the gross classification presents some important limitations , in particular , the lack of comparison between t1weighted se images and high - contrast sequences , such as t2 - weighted se or stir scans . 
mri techniques with fat suppression and t2 se can make the diagnosis easier because the intact fascicle which shows high signal intensity on the t1 - weighted scan displays low signal intensity on t2 - weighted images , as the signal of the intraligament oedema is abated by the contrast of the sequence . 
in questo modo possiamo distinguere tra lesioni del fascio pm del lcp , sul versante dorsale , da quelle del versante ventrale , sede del fascio al . discussione la classificazione delle lesioni traumatiche del lcp , basata sui dati rm , stata pubblicata per la prima volta da gross et al . 
in questo modo sono stati distinti tre differenti gradi di lesione ( tabella 1 )  . nel i grado , gli autori hanno incluso le lesioni intra - legamentose , dove il lcp era continuo , normale e a bassa intensit del segnale sia sul versante dorsale che su quello ventrale . 
nel ii grado sono state catalogate le lesioni parziali , in cui l ' iper - intensit del segnale interessava la porzione interna del legamento ed uno dei due versanti , ventrale o dorsale . 
secondo gross questa rappresentava una lesione completa . la classificazione di gross ha , per , alcune importanti limitazioni , prima tra tutte l ' assenza di comparazione tra le sequenze se t1 pesate e le sequenze ad alto contrasto , come le se t2 o le sequenze stir . 
in accordo con quanto ottenuto dai nostri studi ed in altre recenti esperienze descritte in letteratura [ 1115 ] , l ' integrazione ed il confronto tra due differenti sequenze a diverso contrasto di fondamentale importanza . 
nelle sequenze se t1 pesate l ' edema , l ' ematoma e la soffusione siero - ematica hanno la stessa intensit del segnale sia nelle lesioni acute che nelle lesioni subacute . 
b rappresentazione del lcp da cadavere durante l ' estensione . ion , a correct classification of pcl injuries needs to take into account the anatomical site of injury on mri . 
for this reason , our new classification of traumatic pcl injuries considers four different types of acute lesion ( table 2 ) : type i : the high signal intensity is within the ligament in both sequences . 
le tecniche di rm ad alto contrasto , ( se t2 pesate e stir ) , possono essere d ' aiuto per una diagnosi migliore , per il fatto che il fascio legamentoso non interessato dalla lesione , che comunque table 2 anatomical classification of acute posterior cruciate ligament ( pcl ) injuries [ 11 ] type magnetic resonance imaging normal pcl : homogeneously low signal intensity on all sequences intraligament lesion : high signal intensity within the ligament . 
the ventral and dorsal ligament edges have homogeneously low signal intensity partial lesion : high intensity signal on the dorsal edge of the ligament , which is the anatomical site of the posteromedial ( pm ) fascicle . 
the anterolateral ( al ) fascicle shows low signal intensity partial lesion : high signal intensity on the ventral edge of the ligament , which is the anatomical site of the anterolateral ( al ) fascicle ( the main one in knee biomechanics )  . 
il fascio al mostra bassa intensit del segnale normale lesione parziale : iperintensit del segnale sul versante ventrale del legamento , sede anatomica del fascio al ( prevalente nella biomeccanica di ginocchio )  . 
4a - c type ii : high signal intensity involves the internal portion of the ligament and its dorsal border [ posteromedial ( pm ) fascicle ] on the t1 - weighted scan ( a )  . 
4a - c ii grado : iperintensit del segnale in sede intralegamentosa e con coinvolgimento del margine dorsale ( fascio pm ) nelle sequenze se t1 pesate ( a )  . 
in questi casi infatti le sequenze se t2 pesate o fat sat sono in grado di abbattere il segnale dell ' edema intra - legamentoso . al momento attuale non stata ancora ben sottolineata l ' importanza della biomeccanica e della sede anatomica di lesione . 
il legamento , da un punto di vista anatomico , composto da due differenti gruppi di fasci fibrosi , il fascio al ed il fascio pm , distinti per la loro inserzione su tibia e femore . 
il fascio al situato sul versante ventrale del lcp , mentre il fascio pm si trova sul versante dorsale . secondo noi , la corretta classificazione delle lesioni del lcp deve prendere in considerazione la reale sede anatomica del danno in rm . 
per questo motivo la nuova classificazione anatomica delle lesioni del lcp descrive 4 tipi differenti di lesione traumatica acuta ( tabella 2 )  . i grado : l ' alta intensit del segnale si trova all ' interno del legamento , in entrambe le sequenze . 
5a , b type iii : high signal intensity involves the internal portion of the ligament and its ventral border [ anterolateral ( al ) fascicle ] on t1and t2 - weighted images ( a )  . 
5a , b iii grado : nelle sequenze se t1 pesate l ' iperintensit del segnale evidente sull ' interno del legamento e nel versante ventrale ( fascio al ) ( a )  . 
mri detection of a traumatic injury affecting the ventral edge of the pcl , the anatomical site of the al fascicle , denotes more severe damage in relation to clinical - arthrometric and stress radiography data [ 21 ]  . 
we are strongly convinced it is clinically and prognostically important to be able to recognise on the first mri examination injuries that are very likely to develop into serious knee instability . 
questo causa un incremento nell ' instabilit di ginocchio che , nell ' arco di poco tempo , portano ad un precoce danneggiamento delle altre strutture articolare . in questi casi il chirurgo ortopedico ha maggiori difficolt nel ripristinare l ' assetto articolare corretto con la sola ricostruzione isolata del lcp . 
per questo motivo molto importante essere in grado definire quale sia il reale danno a carico del lcp nella fase acuta e sub - acuta . , ed eventuali lesioni capsulari associate . 
nel caso in cui in rm si osserva una lesione del fascio ventrale del legamento , sede anatomica del lcp , questa ha una gravit maggiore , in relazione ai dati clinico - artrometrici e alle radiografie in stress [ 21 ]  . siamo convinti che essere in grado di riconoscere al primo esame rm il tipo di lesione che svilupper nel tempo una grave instabilit di ginocchio da quelle meno significative da un punto di vista biomeccanico rappresenta una fattore clinico e prognostico importante . 
la rm ha evidenziato 26 traumi minori e 55 maggiori . la rm e l ' ecografia hanno mostrato una concordanza completa in 71 pazienti ( sede , tipo , entit della lesione )  . 
sono stati riportati 10 falsi negativi all ' ecografia , di cui 6 lesioni minori e 4 maggiori . la sensibilit dell ' ecografia nell ' identificare correttamente i traumi muscolari risultata dell ' 87 , 65% . 
la rm rileva le lesioni misconosciute all ' ecografia e valuta con maggiore precisione l ' entit e la sede del danno muscolare . parole chiave muscoli lesioni muscolari ecografia risonanza magnetica introduction introduzione acute muscle injuries are frequently observed in both amateur and professional athletes . 
the risk of injury is increased by poor preparation , inadequate warmup , excessive fatigue , slippery or hard playing surfaces , exposure to cold , muscle tension due to emotional factors ( performance anxiety ) and previous muscle injuries le lesioni muscolari acute sono di frequente osservazione nelle discipline sportive praticate sia a livello amatoriale che agonistico . 
le discipline pi a rischio sono il calcio , l ' atletica leggera e tutti gli sport in cui la muscolatura viene sollecitata in maniera massimale anche se per brevi periodi . i fattori di rischio sono l ' inadeguata preparazione , lo scarso riscaldamento , l ' eccessivo affaticamento , i terreni di gioco scivolosi o pesanti , l ' eccessiva esposizione al freddo , la tentable 1 classification of muscle injuries minor traumas major traumas indirect delayed - onset muscle soreness , traumas lengthenings , contractures strains direct traumas mild contusions severe contusions ( haematomas ) ttaabbeellllaa 11 classificazione dei traumi muscolari traumi minori traumi maggiori traumi indiretti traumi diretti doms elongazioni contratture contusioni lievi distrazioni contusioni gravi ( ematomi ) [ 1 , 2 ]  . 
in sports such as football or athletics , the posterior thigh muscles [ 3 , 4 ] , adductors [ 1 ] , femoral quadriceps [ 1 , 5 ] and sural triceps [ 1 , 3 , 5 ] are most commonly involved . muscle injuries can be distinguished into minor or major injuries [ 6 ]  . 
they may also be classified according to mechanism of injury into muscle strains , the origins of which are internal and triggered by exaggerated or untimely contraction ; and contusions , the causes of which are external . 
major injuries comprise first - , secondor third - degree strains produced by an indirect trauma and severe contusions resulting in the formation of a haematoma [ 7 , 8 ] ( table 1 )  . diagnostic imaging is indispensable for classifying the type of muscle injury , estimating the extent of the lesion , predicting possible complications and establishing recovery times . 
magnetic resonance imaging ( mri ) allows for correct diagnosis of even small muscle injuries and enables detection of even minimal muscle alterations that are not macroscopically detectable [ 815 ]  . the aim of our study was to analyse and compare the sensitivity of ultrasound ( us ) in relation to mri and define a diagnostic algorithm for the study of muscle injuries . materials and methods between june 2003 and june 2004 , we studied 81 professional and amateur football players with a mean age of 26.2 ( range 1832 years ) who presented with lower - limb muscle injuries ( 27 lower - leg and 54 thigh injuries )  . 
thirteen patients reported intense muscle pain developing within hours or 1 day of exercise , but they could not remember the kind of trauma sustained and the time of occurrence . 
negli sport come il calcio o l ' atletica leggera i muscoli maggiormente colpiti sono quelli posteriori della coscia [ 3 , 4 ] , gli adduttori [ 1 ] , il quadricipite femorale [ 1 , 5 ] e il tricipite surale [ 1 , 3 , 5 ]  . le lesioni traumatiche muscolari si possono distinguere , in relazione alla loro gravit , in traumi minori e traumi maggiori [ 6 ]  . 
un altro criterio classificativo considera il meccanismo lesivo e consente di differenziare i traumi di tipo distrattivo , la cui causa intrinseca al muscolo stesso sottoposto ad una contrazione esagerata o mal collocata nel tempo , dai traumi di tipo contusivo , la cui causa esterna . 
i traumi minori comprendono la contrattura , l ' elongazione , la doms ( delayed onset muscle soreness ) , indotti da un meccanismo indiretto , e la contusione lieve , indotta da un agente lesivo che agisce dall ' esterno . 
i traumi maggiori consistono , in relazione alla gravit , in distrazioni di primo , secondo e terzo grado , conseguenza di un trauma indiretto , e in contusioni gravi che comportano la formazione di un ematoma [ 7 , 8 ] ( tabella 1 )  . la diagnostica per immagini delle lesioni muscolari indispensabile per classificare il tipo di trauma , valutare l ' entit del danno , l ' eventuale insorgenza di complicanze e stabilire i tempi di recupero . 
la risonanza magnetica ( rm ) permette una diagnosi corretta nelle lesioni muscolari traumatiche , anche di piccole dimensioni , e permette di rilevare le minime alterazioni muscolari non macroscopicamente obiettivabili [ 815 ]  . lo scopo del nostro lavoro di analizzare e confrontare il valore diagnostico , in termini di sensibilit , dell ' ecografia ( us ) rispetto alla risonanza magnetica e di definire un algoritmo diagnostico nello studio dei traumi muscolari . materiali e metodi nel periodo tra giugno 2003 e giugno 2004 sono stati studiati 81 calciatori professionisti e dilettanti con et media di 26 , 2 anni ( range 1832 anni ) che presentavano un quadro clinico suggestivo di lesione muscolare traumatica , localizzata all ' arto inferiore ( 27 traumi della gamba e 54 della coscia )  . 
tredici pazienti riferivano la comparsa di un dolore muscolare intenso poche ore dopo o il giorno successivo rispetto allo svolgimento dell ' attivit sportiva , non ricordavano il momento e il tipo di trauma . 
ventisei pazienti riferivano la comparsa di un dolore muscolare acuto , violento , comparso durante lo svolgimento dell ' attivit sportiva in assenza di un trauma diretto ; 10 atleti non avevano sospeso l ' attivit , mentre negli altri 16 il dolore era stato cos intenso da costringerli a sospendere la gara o l ' allenamento . 
trentadue pazienti riferivano con precisione un trauma diretto , come lo scontro con un avversario o l ' impatto con il terreno , in seguito al quale era insorto il dolore muscolare . 
thirty - two patients reported having sustained direct traumas , such as collisions or falls , which had brought on the paof these , 29 had suspended their activity immediately after the trauma . 
physical examination was carried out to assess the site of pain , muscle tenderness , muscular hypertonia , functional impotence , reduced muscle strength , increased muscle volume , subcutaneous swelling and ecchymosis . all patients underwent us shortly after the injury ( 672 hours afterwards ) and mri within 5 days . 
to correctly evaluate lesion size , additional scans were performed in all planes and with various sequences , depending on the extent and anatomical characteristics of the lesions . diagnostic sensitivity of us was compared with that of mri , which was considered the gold standard . 
the study group was subdivided on the basis of patient history and clinical findings , physical examination and subsequent diagnostic investigations ( table 1 )  . results mri detected 26 minor and 55 major traumas in the 81 patients examined . 
 the seven patients with doms ( three in the medial head of the gastrocnemius , four in the rectus femoris ) , all of whom had a poor level of fitness , had diffuse pain in the affected muscle , which had typically appeared the day after athletic activity . 
con l ' esame obiettivo sono stati valutati la sede del dolore spontaneo , la dolorabilit alla palpazione del muscolo interessato , l ' eventuale presenza di ipertono muscolare , l ' impotenza funzionale , la riduzione della forza muscolare , l ' aumento del volume del muscolo leso , la tumefazione del tessuto sottocutaneo e l ' eventuale presenza di un ' ecchimosi sottocutanea . tutti i pazienti sono stati sottoposti ad ecografia nelle ore successive al trauma ( da 6 ad un massimo di 72 ore ) e a risonanza magnetica entro cinque giorni dal trauma . 
l ' ecografia stata condotta con apparecchio technos ( esaote biomedica , genova , italia ) , utilizzando transduttori lineari con frequenze comprese tra 7 , 5 e 13 mhz . 
sono state eseguite scansioni assiali e longitudinali del muscolo leso e dei muscoli adiacenti ; sono stati inoltre valutati la fascia muscolare , i tendini , le aponeurosi ed il tessuto sottocutaneo nella regione anatomica in esame . 
sono state effettuate sequenze assiali se t1pesate ( tr 400 ms , te 20 ms ; spessore di strato 8 mm , intervallo tra gli strati 0 , 8 mm ) , assiali se t2 - pesate ( tr 3000 ms , te 100 ms ; spessore di strato 8 mm , intervallo tra gli strati 0 , 8 mm ) e coronali stir ( short time inversion recovery ) ( tr 3600 ms , te 20 ms , ti 90 ms ; spessore di strato 7 mm , intervallo tra gli strati 0 , 7 mm )  . 
abbiamo considerato come falsi negativi sia i casi in cui l ' ecografia non ha evidenziato alcuna lesione dimostrata dalla risonanza magnetica sia i casi nei quali l ' ecografia ha sottostimato il grado della lesione . 
sulla base dei dati clinico - anamnestici , dell ' esame obiettivo e delle successive indagini diagnostiche , abbiamo suddiviso il gruppo di pazienti secondo la classificazione riportata in tabella 1 . risultati la risonanza magnetica , negli 81 pazienti , ha evidenziato 26 traumi minori e 55 maggiori . 
fra i traumi minori sono state evidenziate 7 doms , 5 elongazioni , 6 contratture e 8 contusioni lievi ; 26 traumi maggiori sono risultati distrazioni mentre 29 erano ematomi ( tabella 2 )  . 
 the five patients with lengthening ( rectus femoris in four patients , medial head of the gastrocnemius in one ) had acute localised muscle pain during exercise that was exacerbated by contraction or stretching . 
mri revealed a similar pattern to that seen in patients with doms ; us agreed with mri in terms of both lesion site and extent in four cases whereas it failed to detect any alteration in a fifth patient . the six patients with contractures ( medial head of gastrocnemius in four patients , rectus femoris in two patients ) had diffuse muscle pain that developed within hours or days of physical exercise and was brought on by muscle contraction against resistance . 
in all these patients , mri showed slight signal hyperintensity in axial se t2 - weighted and coronal stir sequences whereas us revealed vague echostructural inhomogeneity in five patients and was negative in one case ( one false negative )  . among the eight patients in whom mri revealed mild contusions ( signal hyperintensity in se t1 - weighted sequences , se t2 - weighted and stir sequences ) , five suffered intense pain in the affected muscle but had no history of a direct trauma whereas three suffered pain in the area of the contusion . 
la risonanza magnetica mostrava iperintensit di segnale del muscolo dolorante nelle sequenze se t2 - pesate assiali e nelle stir coronali , dovuta unicamente all ' edema e nessuna alterazione dell ' intensit del segnale nelle sequenze se t1 - pesate assiali . 
l ' ecografia era risultata negativa in 3 atleti ( falsi negativi ) , mentre negli altri 4 permetteva di rilevare una ipoecogenicit disomogenea , dovuta all ' edema , della struttura muscolare in esame . nei 5 pazienti con elongazione ( in 4 localizzata nel muscolo retto femorale ed in 1 nel muscolo gemello mediale ) , il quadro clinico era caratterizzato da un dolore muscolare acuto insorto durante l ' attivit sportiva , ben localizzato , esacerbato dalla contrazione o dall ' allungamento muscolare ; tutti i pazienti ricordavano con precisione il momento dell ' insorgenza del dolore . 
la risonanza magnetica mostrava un quadro del tutto simile a quello dei pazienti con doms ; l ' ecografia concordava con la risonanza magnetica sia per quanto riguardava la sede che per l ' entit della lesione in 4 pazienti , mentre in un altro paziente non dimostrava alcuna alterazione del muscolo . i 6 pazienti con contrattura ( 4 pazienti con localizzazione a livello del gemello mediale e 2 a livello del retto femorale ) riferivano un dolore muscolare mal localizzato , insorto a distanza di qualche ora o qualche giorno dall ' attivit sportiva , che compariva durante la contrazione muscolare contro resistenza . 
in tutti questi pazienti , la risonanza magnetica mostrava una lieve iperintensit di segnale nelle sequenze se t2 - pesate assiali e nelle stir coronali ; l ' ecografia mostrava una sfumata disomogeneit ecostrutturale muscolare in 5 pazienti , mentre risultava negativa in uno ( 1 falso negativo )  . degli 8 pazienti con quadro rm di piccola contusione ( iperintensit del segnale nelle sequenze se t1 - pesate , se t2 - pesate e stir ) , 5 lamentavano dolore intenso al muscolo interessato , ma senza il ricordo anamnestico di trauma diretto e 3 riferivano dolore nella sede del trauma contusivo ; l ' ecografia , eseguita dopo poche ore dal trauma , aveva confermato il quadro contusivo in 7 pazienti evidenziando una zona di ipoecogenicit associata a piccole aree iperecogene a livello del muscolo dolente ; in 1 paziente , invece , l ' ecografia era risultata negativa . 
la sede delle piccole contusioni era il muscolo retto femorale in 3 pazienti , il muscolo vasto intermedio in 2 pazienti , il muscolo gemello mediale in 2 pazienti ed il muscolo bicipite femorale in 1 paziente . ventisei pazienti mostravano un quadro clinico caratterizzato da dolore acuto insorto durante l ' attivit sportiva , impotenza funzionale e riduzione della forza . 
l ' esame obiettivo evidenziava impotenza funzionale e riduzione di forza in alcuni casi di grado lieve ( i grado ) , in altri moderato ( ii grado ) o grave ( iii grado )  . 
us and mri provided the following results : in 22 subjects , the two techniques were perfectly concordant in identifying and estimating the extent of the lesions and detecting discontinuity of tertiary bundles , reactive muscle oedema , interstitial haemorrhage and / or haematoma . 
in another four patients , mri produced the same results as those described above whereas us was negative in two patients and detected only discontinuity of muscle fibres in the other two , thus underestimating the degree of the lesion . 
 twenty - nine subjects with a history of severe direct trauma had functional impotence , strength reduction , muscle hypertonia and increased muscle volume proportional to pain intensity ; five patients had evidence of extensive subcutaneous ecchymosis distal to the lesion . 
in all these patients , both us and mri correctly detected a haematoma , with varying characteristics depending on stage of development . the sites of muscle injury were as follows : 11 in the rectus femoris , nine in the medial head of the gastrocnemius , four in the vastus lateralis , three in the vastus intermedius and two in the biceps femoris . 
us sensitivity by type of trauma was 57% for doms , 80% for lengthening , 83% for contracture , 84% for muscle strain , 87.5% for mild contusion and 100% for severe contusion ( table 3 )  . no fornito i seguenti risultati : in 22 pazienti le due tecniche sono state perfettamente concordi nell ' identificare e stimare l ' entit della lesione evidenziando discontinuit dei fasci terziari , edema muscolare reattivo , emorragia interstiziale e / o un ematoma . 
in altri 4 pazienti la risonanza magnetica aveva fornito gli stessi risultati sopra descritti , mentre l ' ecografia era risultata negativa in 2 pazienti , mentre negli altri 2 aveva mostrato soltanto una discontinuit delle fibre muscolari , sottostimando il grado della lesione . 
la localizzazione delle lesioni muscolari era la seguente : 11 nel muscolo retto femorale , 9 nel muscolo gemello mediale , 4 nel muscolo vasto laterale , 3 nel muscolo vasto intermedio e 2 nel muscolo bicipite femorale . in sintesi , la risonanza magnetica e l ' ecografia hanno mostrato una concordanza completa in 71 pazienti circa la sede , il tipo e l ' entit della lesione . 
non stato possibile calcolare i valori di specificit e di accuratezza diagnostica poich i pazienti non sintomatici ed ecograficamente negativi non sono stati sottoposti alla risonanza magnetica , per ovvii motivi di costi e tempi di attesa . 
sono stati calcolati i valori di sensibilit dell ' ecografia nei 2 sottogruppi di pazienti con lesioni maggiori ( 55 casi ) e con lesioni minori ( 26 casi ) , ottenendo un valore di sensibilit del gruppo con lesioni maggiori del 92 , 72% e di quello con lesioni minori del 76 , 92% . 
stata , inoltre , calcolata la sensibilit dell ' ecografia in considerazione del tipo di trauma , ottenendo valori del 57% per le doms , dell ' 80% per le elongazioni , del l ' 83% per le contratture , dell ' 84% per le distrazioni , dell ' 87 , 5% per le contusioni lievi , del 100% per le contusioni gravi ( tabella 3 )  . discussione traumatic muscle lesions are particularly common in football players and mainly affect the lower limbs , particularly the thigh and calf . 
the most frequently affected muscles are the posterior thigh muscles [ 3 , 4 ] , the adductors [ 1 ] , femoral quadriceps [ 1 , 5 ] and sural triceps [ 1 , 3 , 5 ]  . 
the decision whether to treat conservatively or surgically is crucial given that appropriate treatment can reduce recovery times and increase the chances le lesioni traumatiche muscolari sono particolarmente ricorrenti nei calciatori e interessano prevalentemente l ' arto inferiore , la coscia e il polpaccio . 
i muscoli pi frequentemente interessati sono quelli posteriori della coscia [ 3 , 4 ] , gli adduttori [ 1 ] , il quadricipite femorale [ 1 , 5 ] e il tricipite surale [ 1 , 3 , 5 ]  . 
in our experience , only a combination of imaging and accurate clinical examination makes it is possible to achieve correct diagnosis . in our study , we observed only 26 minor traumas out of a total of 81 traumas ( 32% )  . 
in letteratura non esiste una classificazione univoca dei traumi muscolari e risulta ancora pi complessa quella dei traumi minori , i quali presentano quadri di imaging sovrapponibili . nella nostra esperienza , soltanto attraverso l ' integrazione dell ' esame strumentale con un accurato esame clinicoanamnestico possibile formulare una diagnosi esatta . nel nostro studio abbiamo osservato solo 26 traumi minori su 81 traumi complessivi ( 32% )  . 
 [ 22 ] hanno osservato , in un gruppo di 40 pazienti con doms , che l ' ecografia ha una bassa sensibilit nell ' individuare le alterazioni muscolari della doms . nei sette pazienti con doms da noi studiati , la risonanza magnetica ha correttamente individuato l ' edema muscolare , ma il pattern aspecifico e per un suo corretto inquadramento necessaria l ' integrazione con dati di laboratorio , quali il dosaggio della creatin - chinasi [ 19 ]  . 
de smet [ 4 ] sostiene che la risonanza magnetica attendibile solo se preceduta da un valido inquadramento clinico che permetta di scegliere le sequenze pi adatte e orientare correttamente le scansioni ed ha osservato valori di specificit e sensibilit rispettivamente del 80% e del 83% nella diagnosi delle contratture e delle elongazioni . 
questo dato da ricondurre all ' edema muscolare di grado elevato che facilita l ' identificazione della lesione . in queste situazioni l ' ecografia dimostra una diffusa ipoecogenicit con dislocazione dei fasci terziari . 
in seven of our patients with doms , although mri correctly detected the muscle oedema , the pattern was nonspecific and the correct diagnosis required integration with laboratory findings such as creatine - kinase assay [ 19 ]  . de smet [ 4 ] states that mri is reliable only if it is preceded lit della ecografia sale ancora , seppur di pochi punti percentuali , nelle contusioni di lieve entit . 
nella nostra esperienza , l ' imaging ecografico delle piccole contusioni non si differenzia considerevolmente da quello degli altri traumi minori ; in tutti i casi , infatti , il quadro caratterizzato da imbibizione by a thorough clinical assessment that enables selection of the most appropriate sequences and scan planes and has observed specificity and sensitivity values of 80% and 83% , respectively , for diagnosis of contractures and lengthening . this may also be correlated to the presence of considerable muscle oedema , which facilitates lesion identification . 
in our experience , the us imaging pattern of small contusions does not differ much from that of other minor traumas ; in all cases , it shows muscle oedema without interruption of the continuity of muscle fibres . 
differentiation was only possible on the basis of the patient 's history . as regards major traumas , us had an 84% sensitivity in identifying muscle strathe extent of tissue alterations affects the us pattern . 
the reason for such underestimation was that both lesions were located deep in the femoral quadriceps and therefore less amenable to us . us examination enables identification of typical lesions of muscle strains : discontinuity of tertiary bundles , reactive oedema and haematoma . 
in first - degree strains , the involvement of a small number of myofibrils can make it difficult to recognise the lesion with us so that the use of mri is necessary . 
more - severe lesions ( second and third degree ) with involvement of a larger number of myofibrils , will exhibit a hypoechoic or anechoic haematoma , which can remain localised or extend along the bundles . 
immediately after a trauma , the true extent of a lesion may be masked by a hyperechoic haemorrhage whereas us done 4872 h afterwards will reveal the evolution of the haematoma and the extent of the area affected [ 7 , 10 , 23 , 24 ]  . 
in cases of complete tears of the muscle belly , the retracted muscle bundles have the typical us appearance of a bell clapper surrounded by a hypoechoic haematoma [ 7 , 25 ]  . 
a dynamic examination is fundamental in all cases and particularly in first - degree strains , as it enables detection of the separation and dislocation of tertiary bundles and evaluation of the effective extent of the lesion [ 7 , 24 , 26 ]  . muscle strains are recognised at mri by changes in muscle volume and composition , variations in signal intensity and pathological alterations of surrounding tissues [ 1 , 27 , 28 ]  . 
coronal or sagittal images along the muscle belly axis make it possible to define the extent of the lesion [ 3 , 4 , 15 ]  . an important finding of our study was that in all patients with severe contusion , the haematoma was detected by both us and mri , leading to a sensitivity of 100% for us . 
la differenziazione stata possibile solo sulla base dei dati anamnestici . per quanto riguarda i traumi maggiori , l ' ecografia ha ottenuto un valore di sensibilit dell ' 84% nell ' individuazione delle distrazioni . 
l ' entit delle alterazioni tissutali condiziona l ' aspetto del quadro ecografico ; in effetti , abbiamo notato differenze significative tra le distrazioni di iii grado , sempre individuabili , e quelle di i grado . 
in 2 atleti , considerati come dei falsi negativi , l ' ecografia , pur sottostimandone l ' entit , aveva effettivamente localizzato la lesione , valutata poi alla risonanza magnetica come di ii grado . 
la sottostima da imputarsi al fatto che tutte e due le lesioni si trovavano in regioni profonde del quadricipite femorale e quindi pi difficilmente esplorabili con la tecnica ecografia . lo studio ecografico consente di evidenziare le lesioni caratteristiche delle distrazioni muscolari : la discontinuit dei fasci terziari , l ' edema reattivo e l ' ematoma . 
nelle lesioni di maggiore gravit ( ii e iii grado ) , in cui coinvolto un numero maggiore di miofibrille , si evidenzia un ematoma ipoo anecogeno che pu rimanere localizzato o estendersi lungo le fasce . 
nel periodo immediatamente successivo al trauma , lo spandimento emorragico iperecogeno pu mascherare l ' entit reale della lesione ; un ' ecografia eseguita dopo 4872 ore rivela l ' evoluzione dell ' ematoma e l ' estensione dell ' area lesa [ 7 , 10 , 23 , 24 ]  . 
in caso di rottura completa del ventre muscolare , i fasci muscolari retratti appaiono all ' ecografia con la tipica immagine a batacchio di campana , circondati dall ' ematoma ipoecogeno [ 7 , 25 ]  . 
l ' esame dinamico fondamentale in tutti i casi e particolarmente nelle distrazioni di primo grado , dal momento che consente di rilevare l ' allontanamento e la dislocazione dei fasci terziari e di valutare l ' effettiva estensione della lesione [ 7 , 24 , 26 ]  . 
le distrazioni sono riconoscibili alla risonanza magnetica per i cambiamenti del volume e dell ' architettura muscolare , per le variazioni dell ' intensit del segnale , per le alterazioni patologiche a carico dei tessuti circostanti [ 1 , 27 , 28 ]  . 
le immagini coronali o sagittali , lungo l ' asse del ventre muscolare , permettono di definire l ' estensione della lesione [ 3 , 4 , 15 ]  . 
un dato importante , rilevato dal nostro studio , che in tutti i pazienti con contusione grave , l ' ematoma stato evidenziato sia dall ' ecografia che dalla risonanza magnetica : abbiamo ottenuto quindi un valore di sensibilit dell ' ecografia pari al 100% . 
secondo peetrons [ 12 ] possibile una classificazione ecografica delle lesioni muscolari maggiori in 4 tipi , in base alla percentuale di muscolo coinvolto , ma essenziale operare una distinzione tra lesioni che hanno provocato un ematoma e lesioni che hanno provocato solo una lacerazione delle fibre muscolari . 
questo un criterio fondamentale per stabilire il periodo di inattivit dell ' atleta che sar di 12 settimane per le lesioni senza ematoma e di almeno 4 settimane per i traumi con ematoma . nel nostro studio abbiamo osservato 29 atleti con contua . 
this is a fundamental criterion when establishing the length of the recovery period , which is usually 12 weeks for lesions without haematoma and at least 4 weeks for those with haematoma . in our study , there were 29 patients with severe contusion , and us was able not only to locate the haematoma but also to evaluate its extent in total agreement with mri . 
in contrast , in the study of deeper muscles , kolouris and connell [ 31 ] identified a discrepancy between us and mri in the evaluation of hamstring injuries and observed that mri is more accurate in assessing the extent of injury . these studies confirm that us is limited in the study of muscle injury in that its resolution is limited to the tertiary bundle , it is unable to identify pathological alterations to the secondary and primary bundles and myofibrils and cannot evaluate deep muscle planes [ 17 , 23 ]  . conclusions us is the first - line technique in the study of muscle traumas , as it is readily available , has a good cost - benefit profile , enables assessment of muscle dynamics and provides reliable assessment of the extent of damage . 
disadvantages of us are that its resolution is limited to the tertiary bundle , it is unable to identify alterations to secondary and primary bundles and myofibrils and cannot visualise deep muscle planes . 
 mri has many advantages , including multiplanar capabilities , panoramic views , ability to evaluate deep muscle planes and to detect lesions missed by us . sione grave , l ' ecografia stata in grado non solo di localizzare l ' ematoma , ma anche di valutarne l ' estensione in perfetto accordo con la risonanza magnetica . 
l ' ecografia mostra una sensibilit per i traumi maggiori molto pi elevata rispetto a quella per i traumi minori , ci deriva dal fatto che la capacit dell ' ecografia nella valutazione di questi ultimi limitata ai casi in cui l ' edema muscolare importante . 
in queste situazioni , si pu valutare con maggiore accuratezza l ' estensione della lesione , la percentuale di muscolo coinvolto , la grandezza della cicatrice e le eventuali complicanze [ 7 , 12 , 29 ]  . 
 [ 30 ] hanno esaminato 17 pazienti con lesione acuta del retto femorale , valutandola sia con la risonanza magnetica che con ecografia : in tutti i casi le due tecniche si sono rivelate concordi nel localizzare la lesione e nel valutarne l ' entit . 
nella valutazione di muscoli pi profondi , invece , kolouris e connell [ 31 ] hanno evidenziato una certa discrepanza fra la valutazione delle lesioni dei muscoli hamstring con l ' ecografia e con la risonanza magnetica , osservando che quest ' ultima fornisce una stima pi corretta dell ' entit del danno . 
questi studi confermano che l ' ecografia ha dei limiti nello studio della patologia muscolare : il potere di risoluzione limitato al fascio terziario , l ' incapacit di identificare le alterazioni patologiche del fascio secondario , del fascio primario e delle miofibrille , la difficolt nel valutare i piani muscolari profondi [ 17 , 23 ]  . conclusioni l ' ecografia rappresenta la tecnica di prima istanza nello studio dei traumi muscolari per la facile accessibilit , per il favorevole rapporto costo - beneficio , per la possibilit di valutare la dinamica del muscolo e per l ' affidabilit nella stima dell ' entit del danno . 
questa tecnica presenta alcuni svantaggi : il potere di risoluzione limitato al fascio terziario , l ' incapacit di identificare le alterazioni del fascio secondario , primario e delle miofibrille , la difficolt nel valutare i piani muscolari profondi . 
mri provided additional information , identifying the possible absence of cochlear nerve and excluding other central nervous system ( cns ) diseases . key words cochlear implants auditory brainstem implants cochlear nerve magnetic resonance imaging computed tomography riassunto obiettivo . 
inoltre la rm ha permesso di ottenere informazioni aggiuntive valutando leventuale assenza della componente cocleare dellviii nervo cranico ed escludendo altre patologie del snc . parole chiave impianti cocleari impianti acustici al tronco nervo cocleare risonanza magnetica tomografia computerizzata introduction introduzione r . 
sensorineural hearing loss has constituted both a diagnostic and surgical challenge , only in part overcome by computed tomography ( ct ) , which has allowed identification of congenital cochleovestibular abnormalities and indirectly of cochlear nerve agenesis , but it is unable to detect abnormalities affecting the central nervous system ( cns ) [ 1 ]  . 
following the development of abis for direct stimulation of cochlear nuclei in patients with sensorineural hearing loss , magnetic resonance imaging ( mri ) became crucial to confirm inner ear alterations demonstrated by ct and verify the integrity of the auditory pathways up to the temporal cortex [ 2 ]  . 
when the cochlear nerve is aplastic or hypoplastic , there is no possibility of restoring hearing by direct stimulation of the organ of corti or of the auditory nerve fibres using traditional cochlear implants . 
 materials and methods we examined ct and mri studies of 17 patients ( 12 females and five males ) aged between 2 and 34 years ( mean age 14 years ) with congenital inner ear disease and abnormalities of the cochleovestibular systethe presence of disorders constituting exclusion criteria for surgery , in particular , dysmetabolic disease and cortical malformations , had already been ruled out ( even by means of preliminary mri ) in all these candidates for abi . 
all patients had undergone preliminary audiometric testing ( tympanometry and impedance audiometry ) that excluded middle ear disorders ; they were then subjected to electrocochleography to assess function of the cells of the organ of corti and promontory electrostimulation to check the presence of stimulable neuronal fibres . 
all patients selected for abi were then studied by ct ( siemens , somatom plus 4 , germany ) and mri ( siemens , symphony maestro , germany )  . 
ct was performed to obtain a detailed study of the petrous bone using a bone algorithm in the axial and coronal planes ( sequential technique , slice thickness 1 mm , 140 kv , 77 ma )  . 
mri was performed with proton - density ( pd ) and t2 - weighted axial turbo spin echo ( tse ) scans ( tr 2800 te 24 / 121 , thickness 5 mm ) ; coronal t2 - weighted , ( tr 4850 , te 108 , thickness 4 mm ) and sagittal t1weighted scans ( tr 440 , te 12 , thickness 4.5 mm ) to confirm the absence of other cns diseases that could contraindicate the use of abi . 
inner ear structures were examined in the lanacusia congenita o la grave ipoacusia legata ad alterazioni dellorecchio rappresentano unimportante causa di disabilit che , fino a non molti anni fa , aveva come unico trattamento la logoterapia , che consentiva ai pazienti solo una limitata capacit di inserimento sociale . 
lipoacusia neurosensoriale ha infatti rappresentato un problema diagnostico oltre che chirurgico , solo in parte risolto dallavvento della tomografia computerizzata ( tc ) ; questa metodica ha consentito di evidenziare le alterazioni congenite vestibolo - cocleari , ed in maniera indiretta lagenesia del nervo cocleare , ma non le alterazioni a livello del sistema nervoso centrale ( snc ) [ 1 ]  . 
per le protesi elettriche impiantabili a livello cocleare , la metodica tc ha rappresentato lindagine essenziale per escludere la presenza di malformazioni a livello delle strutture cocleari ed in particolare per verificarne la regolare perviet . 
in seguito allapprontamento di protesi impiantabili a livello del tronco encefalico , per stimolare direttamente i nuclei cocleari nei pazienti affetti da ipoacusia neurosensoriale , la risonanza magnetica ( rm ) divenuta metodica radiologica fondamentale sia per confermare eventuali alterazioni gi documentate da indagine tc , a livello delle strutture dellorecchio interno , che per verificare lintegrit delle vie acustiche fino alla corteccia temporale [ 2 ]  . 
quando il nervo cocleare aplasico o ipoplasico , infatti , non vi alcuna possibilit di ripristinare la percezione uditiva con la stimolazione diretta dellorgano del corti delle fibre del nervo acustico mediante i tradizionali impianti cocleari . 
in questo lavoro sono state esaminate le indagini tc ed rm di pazienti affetti da sordit congenita sottoposti ad impianto di protesi al tronco encefalico ( auditory brainstem implants : abi ) , al fine di verificarne laffidabilit nellindividuare le cause del deficit acustico . materiali e metodi nel nostro studio sono state esaminate le indagini tc ed rm di 17 pazienti ( 12 femmine e 5 maschi ) di et compresa tra i 2 ed i 34 anni ( et media 14 anni ) , affetti da patologia congenita dellorecchio interno , aventi anomalie riscontrate a carico del sistema cocleo - vestibolare . 
per tutti questi pazienti candidati ad abi per anacusia erano gi state escluse ( anche tramite indagine rm preliminare ) patologie che rappresentavano criterio di esclusione per il trattamento chirurgico , in particolare la patologia dismetabolica e la malformativa corticale . 
tutti i pazienti hanno eseguito test audiometrici preliminari ( timpanometria e limpedenzometria ) che hanno escluso patologie a carico dellorecchio medio ; sono stati quindi sottoposti ad elettrococleografia per valutare la funzionalit delle cellule dellorgano del corti e ad elettrostimolazione del promontorio per verificare la eventuale presenza di fibre neuronali stimolabili . 
in agreement with the literature , among the inner ear malformations eligible for abi , we considered the following cochlear deformities : michel deformity , cochlear aplasia , common cavity , cochlear hypoplasia , type i incomplete partition ( ip - i ) and type ii incomplete partition ( ip - ii , mondini deformity ) [ 3 , 4 ]  . 
lindagine tc stata espletata per lo studio di particolare delle rocche con algoritmo per osso secondo i piani assiale e coronale ( tecnica sequenziale , spessore di strato 1 mm , kv 140 , ma 77 )  . 
lindagine rm stata eseguita mediante scansioni assiali tse , dp e t2 pesate ( tr 2800 te 24 / 121 , spessore 5 mm ) ; coronale t2 pesata , ( tr 4850 , te 108 , spessore 4 mm ) e sagittale t1 pesata ( tr 440 , te 12 , spessore 4 , 5 mm ) allo scopo di confermare lassenza di altre patologie del snc che potrebbero rappresentare controindicazione allutilizzo degli abi . 
le strutture dellorecchio interno sono state esaminate secondo il piano assiale , con sequenza 3dft - ciss ( constructive interference in steady state ) : 28 slices , tr 17 , 3 , te 6 , 9 , spessore 0 , 65 , flip angle 70 , matrice 358512 * ( interp ) , nex 2 , fov 100100 ; queste ultime sequenze sono state esaminate anche mediante algoritmo di ricostruzione volumetrico mip ( maximum intensity projection ) , per ottenere la migliore visualizzazione globale delle strutture labirintiche . 
the straight line corresponds to the parasagittal mr scan ( c ) to identify the components of the cochleo - vestibulo - facial nerve bundle . three dimensional reconstruction of ( 3d ) fig . 
the internal implant converts the code into electrical signals such as those produced by the organ of corti , which are sent to the abi electrodes adjacent to the cochlear nuclei in order to stimulate the nerve fibres ; at this point the brain recognises the signals as sounds , producing an auditory sensation [ 11 , 1518 ]  . 
axial magnetic resonance ( mr ) scan ( a ) : the straight line corresponds to the left parasagittal mr scan ( b ) that demonstrates the absence of the cochlear nerve ( arrow )  . 
in accordo con i dati presenti in letteratura , nellinsieme delle malformazioni dellorecchio interno passibili di intervento per impianto acustico al tronco , abbiamo considerato le seguenti malformazioni cocleari : deformit di michel , aplasia cocleare , cavit comune , ipoplasia cocleare , incompleta partizione di tipo i ( ip - i ) ed incompleta partizione di tipo ii ( ip - ii , deformit di mondini ) [ 3 , 4 ]  . 
i pazienti sono stati quindi sottoposti ad intervento chirurgico di impianto al tronco ( auditory brainsteam implant : abi ; cochlear ltd , australia ) [ 57 ] , nel periodo compreso tra il febbraio 2000 ed il marzo 2005 [ 5 , 710 ]  . 
tale impianto fornito di una componente esterna , costituita da un microfono posizionato in sede retroauricolare , in grado di ricevere le onde sonore dellambiente e di inviarle , dopo averle trasformate in segnale digitale , ad un elaboratore del linguaggio con un trasmettitore collocato nel tessuto sottocutaneo in sede temporale superficiale ; il trasmettitore invia il codice attraverso la pelle allimpianto interno collocato in una nicchia ossea immediatamente al di sotto del r . 
limpianto interno converte il codice in segnali elettrici , simili a quelli prodotti dallorgano del corti , che sono inviati agli elettrodi posizionati a livello dei nuclei cocleari al fine di stimolare le fibre nervose ; a questo punto i segnali vengono riconosciuti come suoni dal cervello producendo una sensazione uditiva [ 11 , 1518 ]  . 
3a - d mr findings ( a ) : unique cavity , in parasagittal left reconstruction in which is impossible to distinguish the cochlear and vestibular components ( b ) and right reconstruction with regular findings of vii ( arrow ) and viii nerve ( black arrow ) ( c )  . 
3a - d paziente studiato mediante rm ( a ) : cavit unica , nelle ricostruzioni parasagittali sinistra senza differenziazione delle componenti cocleare e vestibolare ( b ) , e destra con riconoscimento del vii ( freccia ) e viii n.c. 
abi is contraindicated in cases of enlarged lateral recess of the fourth ventricle , a possible cause of electrode instability ; in the case of cochlear nuclei ischaemia ; and radiation exposure ( gamma - knife or stereotactic irradiation ) , which may have damaged the floor of the risultati tutti i 17 pazienti inviati dalla clinica otorinolaringoiatrica con sospetto clinico di malformazione a carico dellorecchio interno hanno trovato conferma delle loro anomalie congenite mediante tc e / o rm . 
in particolare la rm ha permesso di ottenere informazioni aggiuntive per la corretta valutazione dei nervi cocleari in 10 pazienti su 17 . discussione il sistema abi fornisce a pazienti con grave compromissione della funzionalit uditiva lopportunit di ricevere informazioni uditive mediante una stimolazione diretta dei nuclei cocleari [ 57 , 9 ] ; da ci ne consegue la necessit di reperire per mezzo della rm eventuali controindicazioni allintervento . 
limpianto abi risulta infatti controindicato nel caso in cui ci si trovi di fronte ad un recesso laterale del quarto ventricolo allargato , possibile causa di instabilit degli elettrodi , in caso di sequele ischemiche dei nuclei cocleari , ed in caso di esposizione a radiazioni ( gamma - knife o irradiazione stereotassica ) che potrebbero aver lesionato il pafourth ventricle . 
cochlear aplasia , a relatively rare malformation ( bilateral in 50% of cases ) , may be recognised as a bony vesicle on the ventral surface of the internal auditory canal ( iac ) a malformation due to arrested development before the fourth week of gestation which has been equally well documented on ct and mri [ 1 , 3 , 4 ]  . 
similarly , patients with common cochleovestibular cavity a malformation due to developmental arrest at the fourth week had normal recognition of the components of the vii and viii cranial nerves but without separation of the cochlear and vestibular components , the latter well documented on mri only . 
the common cavity , generally containing fluid , is instead readily depicted by ct and mri . in ip - i or cystic cochleovestibular deformity arising from developmental arrest at the fifth week the cochlea lacked the entire modiolus and cribriform area , resulting in a cystic formation that was well visualised with both techniques ; in this case the cochleovestibular dimensions are normal , but the internal architecture is completely deranged . 
nella sezione parasagittale si evidenzia lassenza della componente cocleare dellviii nervo cranico ( b )  . lated and cystic and might , according to some authors , represent a subsequent and therefore more differentiated form of the common cavity deformity [ 19 ]  . 
in the only patient examined by us , the iacs were within normal limits . ip - ii or mondini deformity is due to a developmental arrest arising later than in ip - i , generally at the seventh week , since the dimensions of the cochlea and vestibule are normal and the internal architecture is less deranged . 
the cochlea presents only one and a half turns , and the defect in the interscalar septum is located between the middle turn and the apical turn , which are fused and appear as an apical cystic formation . 
the basal portion of the modiolus and the basal turn are present with a normal iac in 60% of cases [ 1 , 68 ] ; this implies that the spiral ganglion is more likely to be present and more abundant than in ip - i . 
in all cases of ip - ii ( mondini deformity ) , the vestibular aqueduct appears dilated and symmetric given that embryonic development of this passage occurs at a later stage than the previous deformity . ip - ii was well demonstrated on both ct and mri although mri was also able to recognise agenesis of the right cochlear nerve in one patient . 
laplasia cocleare , malformazione piuttosto rara ( bilaterale nel 50% dei casi ) , pu essere riconosciuta come una vescicola di osso compatto sulla superficie ventrale del condotto uditivo interno ( cui ) malformazione che pu essere ricondotta ad un arresto dello sviluppo embrionario prima della quarta settimana di vita intrauterina stata altrettanto bene documentata sia mediante indagine tc che rm [ 1 , 3 , 4 ]  . 
analogamente , i pazienti che presentavano una cavit comune cocleo - vestibolare che rappresenta entrambe le strutture malformazione riconducibile ad un arresto della differenziazione embrionale generalmente nella quarta settimana di gestazione presentavano regolare riconoscimento delle componenti del vii e viii nervo cranico di cui per non risultavano separate le componenti cocleare e vestibolare , queste ultime ben valutabili solo con rm ; la cavit comune , generalmente a contenuto liquido , invece facilmente riconoscibile sia alla tc che alla rm . 
 nella ip - i o malformazione cocleo - vestibolare cistica che si verifica per arresto della differenziazione alla quinta settimana la coclea era sprovvista dellintero modiolo e dellarea cribriforme , esitando quindi in una formazione cistica , ben visualizzata da entrambe le metodiche ; in questo caso le dimensioni cocleovestibolari sono regolari , ma larchitettura interna completamente sovvertita . nellunico paziente che presentava ip - i , la rm ha documentato la presenza di un nervo vestibolare senza la componente cocleare . 
analogamente in entrambe le metodiche il vestibolo risultava dilatato , anchesso di forma cistica , e potrebbe , secondo alcuni autori , rappresentare una fase successiva e quindi pi differenziata della cavit comune [ 19 ] ; nellunico paziente da noi esaminato i cui risultavano nei limiti . 
 lip - ii o deformit di mondini rappresenta un arresto dello sviluppo pi tardivo rispetto alla ip - i , generalmente alla settima settimana , in quanto le dimensioni della coclea e del vestibolo sono normali e larchitettura interna pi organizzata . 
la coclea presenta solo un giro e mezzo , il difetto del setto interscalare tra il giro medio ed il giro apicale che risultano fusi tra loro e si presentano come una formazione cistica apicale . 
la porzione basale del modiolo ed il giro basale sono presenti con cui regolare nel 60% dei casi [ 1 , 68 ] ; da ci deriva la maggior probabilit che il ganglio spirale sia presente ed in maggior quantit rispetto alla ip - i . 
in tutti i casi di ip - ii ( deformit di mondini ) lacquedotto vestibolare si presenta dilatato e simmetrico , poich lo sviluppo embriologico di tale condotto avviene in tempi successivi alla malformazione precedente . 
in the two patients with cochlear hypoplasia in our study , well depicted on both ct and mri , mri documented the absence of the cochlear component of the eighth cranial nerve . in our experience ct was able to provide immediate visualisation of malformations of the bony labyrinth ; mri , performed with 3d acquisitions with a slice thickness less than 1 mm was able to provide similar information on bony structures , as described in the literature [ 20 , 21 ]  . 
mri is therefore fundamental for assessing the cochlear nerve ( with function demonstrated by clinical and laboratory tests ) in patients with malformed cochlea complete in its basal and middle turn only as the patients can undergo surgical treatment with a less invasive but equally effective procedure , such as the cochlear implant . 
 conclusions our study confirms mri as the primary imaging method in patients with hearing loss , as it is more complete than ct in recognising inner ear malformations eligible for treatment with auditory brainstem implant and in assessing deaf patients for central causes such as neoplastic diseases ( typically neurofibromatosis ) or metabolic - demyelinating diseases . ct is nonetheless useful and should be used to assess abnormalities of the middle and external ear , which can be effectively visualised by this modality only . vano ipoplasia cocleare , ben evidente in tc e rm , si documentata mediante rm lassenza della componente cocleare dellviii nervo cranico . nella nostra esperienza la tc si dimostrata la metodica in grado di fornire immediata visualizzazione delle malformazioni del labirinto osseo ; la rm se eseguita con studio 3d acquisita con spessore della sezione inferiore ad 1 mm , stata in grado di fornire analoghe informazioni riguardo le strutture ossee , cos come in letteratura [ 20 , 21 ]  . 
la rm risulta quindi di fondamentale importanza per i pazienti con coclea malformata completa solo del giro basale ed intermedio per il riconoscimento del nervo cocleare ( con funzionalit documentata dai test clinico - laboratoristici ) , perch possono essere trattati chirurgicamente con un intervento meno invasivo , ma altrettanto efficace , come limpianto cocleare . conclusioni lo studio della nostra casistica ha permesso di confermare la rm come metodica principale di imaging nei pazienti affetti da anacusia , in quanto pi completa della tc nel riconoscere le malformazioni dellorecchio interno che indirizzino al trattamento con impianto protesico al tronco , e nel valutare pazienti anacusici per cause di tipo centrale , come patologie neoplastiche ( tipicamente la neurofibromatosi ) o metabolico - dismielizzanti . 
post - traumatic adrenal contusion / haematoma may arise not only because of a direct trauma but also as a consequence of a sudden increase in the pressure in the inferior vena cava system - adrenal veins . 
this is why adrenal haemorrhage is not directly proportional to the trauma : compression of the inferior vena cava leads to increased pressure in the adrenal venous circulation , which supports the parenchymal lesion . 
the right adrenal gland is more frequently injured than the left gland : it can be easily compressed between the liver , spine and kidney , and its venous drainage flows directly into the inferior vena cava . key words adrenal injuries blunt abdominal trauma pediatrics riassunto le lesioni pediatriche della ghiandola surrenale , in seguito a traumi chiusi toraco - addominali , sono rare e frequentemente associate a lesioni epato - renali . 
di fatto , questa patologia pu non essere direttamente proporzionale allentit del trauma stesso : la vena cava inferiore , se compressa , pu causare un incremento della pressione venosa retrograda intrasurrenalica con conseguente danno ghiandolare . parole chiave lesioni surrenali traumi chiusi toracoaddominali pediatria introduction introduzione thoracoabdominal trauma in children rarely causes isolated adrenal injuries : the adrenal glands are anatomically protected and their involvement is usually linked to multiorgan trauma [ 15 ]  . 
the aim of this paper is to present imaging findings and possible direct and indirect adrenal [ 6 ] involvement in children with blunt abdominal trauma [ 2 , 3 ]  . nei bambini i traumi toraco - addominali chiusi causano raramente lesioni surrenaliche isolate : il surrene un organo anatomicamente protetto e , nella maggior parte dei casi , il suo coinvolgimento legato ad un traumatismo toraco - addominale multiorgano [ 15 ]  . 
scopo del lavoro di sottolineare come nel politrauma la lesione surrenalica associata debba sempre essere presa in considerazione [ 2 , 3 ] anche perch il danno surrenalico pu avere una genesi indiretta legata allimprovviso aumento della pressione intra - addominale [ 6 ]  . three patients , ages ranging from 3 to 11 years , came to our hospital after sustaining a blunt abdominal trauma in a car crash in two cases and a household accident in one case . all patients had regular haemodynamic status . 
on admismateriali e metodi sono stati esaminati tre pazienti , 2 politraumatizzati della materials and methods sion , the first patient had multiple skin lesions and pain in the right hypochondriu neurological examination was negative . 
the third patient presented sternal and epigastric trauma with anterior thoracic pain . all ultrasound ( us ) examinations were performed with a us scanner ( atl 5000 , philips , eindhoven , netherlands ) equipped with multifrequency probes ( 57.5 mhz ) and doppler parameters set to the highest sensitivity . 
computed tomography ( ct ) examination with contrast medium injection was performed in one case only with a single - slice scanner ( aquilon , toshiba , tokyo , japan ) and using specific paediatric settings . 
the contrast medium ( iomeron 300 ) was administered as a 2 ml / kg bolus infused at a rate of 2 ml / s . two patients underwent magnetic resonance imaging ( mri ) using a 1.5 - t magnet ( edge , philips , eindhoven , netherlands ) , with 16mt gradient and a phased - array coil . 
us follow - up showed a cystic evolution ( typical of a focal haemorrhagic lesion ) of a nodule that disappeared in 5 months . the third patient underwent us and mri examri was performed because of vanilmandelic acid in the urine . 
la seconda paziente , politraumatizzata della strada , con trauma cranico e contusione toraco - addominale destra , presentava esami clinico - laboratoristici di base nei limiti della norma ; lo stick urinario era negativo per ematuria . 
il terzo paziente presentava contusione sternale ed epigastrica . gli esami ecografici sono stati eseguiti con apparecchiatura ( atl 5000 , philips , eindhoven , olanda ) dotata di trasduttori multi - frequenza da 5 a 7 , 5 mhz , e utilizzando parametri doppler impostati al massimo della sensibilit . 
lesame stato effettuato senza e con mezzo di contrasto ( iomeron 300 ) somministrato alla dose di 2 ml / kg con una velocit di iniezione di 2 ml / s . 
gli esami di rm sono stati effettuati con apparecchiatura da 1 , 5 t ( edge , philips , eindhoven , olanda ) , gradienti da 16 mt e bobina phased - array . 
i quadri us e di rm erano orientativi per emorragia surrenalica . discussione le lesioni della ghiandola surrenale , nei traumi chiusi toraco - addominali , sono rare e frequentemente associate a lesioni epato - renali . 
gli esiti , che si consolidano entro un termine massimo di 6 mesi dallevento traumatico , variano dalla restitutio ad integrum , come nei nostri tre casi , fino allesito calcifico [ 6 ]  . in letteratura non sono descritte lesioni traumatiche bilaterali e di fatto non sono riportate sequele di insufficienza funzionale . nel i caso la diagnosi non presentava difficolt per laumento volumetrico omogeneo della ghiandola . 
il nodulo permane ipointenso e senza ce ; il segnale intracistico verosimilmente ascrivibile ad un contenuto spurio . unilateral , no endocrine consequences have been reported in the literature . in the first case described here , the diagnosis of contusion was straightforward due to the homogeneous increase in gland volume . 
this is why adrenal haemorrhage is not directly proportional to the trauma : compression of the inferior vena cava leads to an increase in the pressure of the adrenal venous circulation , which supports the parenchymal lesion [ 6 ]  . 
the right adrenal gland is more frequently injured than the left gland , as it can be easily pressed between the liver , spine and kidney , and its venous drainage flows directly into the inferior vena cava . differential diagnosis between adrenal hemorrhage and other paediatric adrenal masses , especially neuroblastoma , is based upon both temporal criteria ( volume and structural changes of the lesion over time ) and doppler findings : intraparenchymal flow is not present in the adrenal haemorrhage whereas it is usually appreciable in neuroblastoma . 
iodine - 131 - metaiodobenzylguanidine ( mibg ) scintigraphy is required in cases of doubt . tica non legata alla sola contusione diretta , ma verosimilmente anche ad un improvviso aumento pressorio del sistema vena cava inferiore - vene surrenaliche . 
di fatto , questa patologia pu non essere direttamente proporzionale allentit del trauma stesso : la vena cava inferiore , se compressa , pu causare un incremento della pressione venosa retrograda intrasurrenalica con conseguente danno ghiandolare [ 6 ]  . 
inoltre la lesione traumatica del surrene quasi esclusivamente a destra : sia perch pi facilmente compresso tra fegato , rachide e rene , sia perch il drenaggio venoso del surrene destro stesso sbocca direttamente nella vena cava inferiore . la diagnostica differenziale con il neuroblastoma e si basa sia sul criterio temporale ( evoluzione lacunare e dimensionale riduttiva ) sia sui rilievi doppler : nella emorragia surrenalica non presente flusso al cd , mentre nel neuroblastoma la velocit di flusso ben evidente . 
garlaschi springer verlag italia ( 2006 ) isbn 88 - 470 - 0518 - 3 published online : 11 october 2006 il volume ecografia dellapparato osteoarticolare focalizzato sullimpatto dellecografia in campo muscoloscheletrico con particolare riferimento alla patologia in ambito reumatologico . come noto , infatti , significativi sono limpatto sociale delle malattie reumatiche , in costante aumento e causa spesso di alterazioni altamente invalidanti , e limportanza clinica di una diagnosi precoce . 
il ricorso a tecniche ecografiche sempre pi sofisticate ed avanzate ha reso possibile la definizione di importanti aspetti iniziali di malattia , quali il coinvolgimento sinoviale , conformazione del panno e il danno cartilagineo articolare . 
luso poi , sempre pi frequente , di metodiche contrastografiche ha consentito , inoltre , lacquisizione di parametri di significativa importanza , sia in campo clinico che terapeutico , di valutazione oggettiva ( grado di iperemia e qualit dei flussi ) delle fasi di attivit e di quiescenza delle patologie infiammatorie croniche articolari . lopera si articola in tre sezioni ben distinte : nella prima ( capitoli 1 - 3 ) vengono considerati i principali aspetti tecnologici e le molteplici procedure di esame con i relativi rilievi anatomici sia in condizioni basali che con le tecniche doppler . 
la seconda sezione ( capitoli 4 - 5 ) rappresenta senza dubbio , per la rigorosa e puntuale analisi della semeiotica ecografica dellapparato locomotore e per i molteplici quadri patologici presentati , resi ancora pi preziosi da uneccellente iconografia quanto mai dimostrativa e scelta con rigorosa cura , il punto pi innovativo e didattico di tutta la monografia , a cui contribuisce in maniera decisamente significativa il ruolo dellecografia nel monitoraggio della terapia in corso di malattie infiammatorie croniche articolari ( capitolo 6 )  . 
nellultima sezione , infine , ( capitolo 7 ) sono trattate , soprattutto in chiave prospettica , alcune interessanti applicazioni dellecografia in campo terapeutico che , se opportunamente affinate , potranno rappresentare in un prossimo futuro un prezioso ed insostituibile complemento per una sempre pi precisa ed essenziale programmazione terapeutica . in conclusione , si tratta di un testo chiaro , caratterizzato da una bibliografia precisa e aggiornata e da una iconografia curata nei minimi particolari , da cui traspaiono lesperienza e lelevata capacit didattica degli autori . 
lesions were found along the posteromedial wall of the proximal gastric pouch and ranged in size from 10 to 25 monly two patients were symptomatic at the time of diagnosis ; in most cases , diverticula were discovered during studies performed as part of the standard follow - up protocol . 
we do not believe these lesions to be clinically important ; at present , our patients are no longer followed up for this problem and undergo diagnostic examinations only if and when they develop symptoms . key words morbid obesity vertical - banded gastroplasty diverticula riassunto obiettivo . 
i diverticoli erano localizzati lungo la parete postero - mediale della tasca prossimale dello stomaco operato e avevano diametro variabile tra 10 e 25 msolo due pazienti erano sintomatici al momento della diagnosi ; nella maggior parte dei casi i diverticoli vennero riscontrati durante un follow - up routinario . 
riteniamo che queste lesioni siano da considerare non clinicamente importanti ; attualmente , i nostri pazienti non sono pi sottoposti a follow - up per questo problema e vengono controllati solo se e quando insorge sintomatologia . parole chiave obesit patologica gastroplastica verticale diverticoli introduction introduzione vertical - banded gastroplasty ( vbg ) is a gastric restriction procedure that is commonly used to control excessive weight in morbidly obese patients , the recently established laparoscopic feasibility of which has greatly contributed to increase diffusion , especially in europe [ 1 , 2 ]  . 
although generally regarded as a safe operation , vbg is not free from postoperative complications , and a variety of problems , such as leaks , staple - line disruptions , band erosion , reflux oesophagitis and pouch dilatations , with or without stenosis , have been described in the literature [ 3 , 4 ]  . 
diverticula arising from the la gastroplastica verticale ( vertical banded gastroplasty , vbg ) una tecnica chirurgica utilizzata nei pazienti con obesit patologica al fine di ridurre leccessivo introito di cibo che ha avuto , soprattutto in relazione alla possibilit di esecuzione mediante accesso laparoscopico , una notevole diffusione , specialmente in europa [ 1 , 2 ]  . 
non esiste consenso in letteratura sul meccanismo con cui queste alterazioni vengono a crearsi , sul loro significato clinico e sulla necessit o meno di un trattamento delle stesse [ 47 ]  . scopo di questo lavoro descrivere i reperti radiografici osservati in un gruppo di pazienti operati di vbg che hanno sviluppato diverticoli della tasca gastrica prossimale e riportare le variazioni degli stessi durante il follow - up clinicoradiologico . tecnica chirurgica nella gastroplastica verticale secondo mclean [ 2 ] , lo stomaco viene diviso in due parti , creando una piccola tasca prossimale lungo la piccola curva , in comunicazione con lo stomaco distale attraverso uno stretto stoma . 
il peso medio al momento dellintervento era di 103 kg ( range 90130 kg ) ; lindice di massa corporea ( body mass index , bmi ) era di 40 ( range 3550 ) kg / m2 . 
due pazienti erano state sottoposte alla vbg dopo complicanze insorte a seguito di un intervento di bendaggio gastrico per via laparoscopica . gli esami radiografici sono stati eseguiti come parte di un programma standard di follow - up postoperatorio : un primo controllo era effettuato in terza giornata , utilizzando mezzo di contrasto iodato idrosolubile ; studi baritati erano quindi ottenuti a 1 , 4 e 12 mesi dallintervento , o ogniqualvolta si sospettavano complicanze . 
allinizio dellindagine venivano somministrati al paziente 3060 ml di una sospensione liquida di bario 125% p / v per identificare eventuale dilatazione esofagea , reflusso gastroesofageo , e analizzare volume e forma della tasca gastrica prossimale , valutando anche il diametro del passaggio allo stomaco distale . stato possibile sottoporre a follow - up radiografico 7 / 12 pazienti per un periodo variabile tra 14 e 53 mesi ; quattro pazienti sono state esaminate due volte . 
viene creata una piccola tasca verticale lungo la piccola curva dello stomaco usando una suturatrice meccanica . proximal gastric pouch are an additional possible finding that has been reported in a few papers only [ 46 ]  . 
no consensus exists regarding the possible mechanism causing these lesions , their clinical significance and the need for treatment [ 47 ]  . the purpose of this paper is to describe the radiographic findings we observed in a series of patients with vbg who developed diverticula of the proximal gastric pouch and to describe changes observed during clinical and radiological follow - up . surgical technique in vbg , according to mclean [ 2 ] , the stomach is divided into two parts , creating a proximal pouch by two applications of an endovascular gastrointestinal anastomosis ( endo - gia ) 60 stapler from an end - to - end anastomosis window 7 cm below the angle of his along the lesser curvature against a 12mm ewald tube . 
the examination involved swallowing 3060 ml of a liquid barium suspension at 125% w / v to detect possible oesophageal dilatation and gastrooesophageal reflux and to evaluate pouch volume , size and shape , as well as the outlet diameter . radiological follow - up of the diverticula was possible in 7 / 12 cases ( range 1152 months ) ; four patients were examined twice during this period . 
images were reviewed by two radiologists who evaluated size and location of the diverticula and , although it was not possible to evaluate precisely dimensional changes due to different radiological projections and patient sizes , formulated a consensus opinion about enlargement of these lesions over time . correlation of the presence of diverticula with symptoms , postoperative weight loss and clinical history was obtained in all cases . 
le immagini radiografiche sono state quindi esaminate retrospettivamente da due radiologi che hanno valutato grandezza e posizione dei diverticoli e , bench non fosse possibile effettuare una misurazione precisa di eventuali modificazioni volumetriche dei diverticoli nel tempo , hanno cercato di esprimere , in consenso tra loro , una opinione su questo argomento . abbiamo correlato la presenza dei diverticoli con i sintomi del paziente , la perdita di peso postoperatoria , e la storia clinica . 
i diverticoli sono stati identificati in terza giornata postoperatoria in 6 pazienti ; sono stati dimostrati al controllo a 1 mese in 3 casi , e allesame effettuato a 4 mesi dallintervento nei 3 soggetti rimanenti . 
2a , b patient 2 : a anteroposterior ( ap ) radiograph obtained 1 month after vertical - banded gastroplasty ( vbg ) showing a diverticulum about 16 mm in largest diameter arising from the proximal gastric pouch . 
2a , b paziente 2 : a radiogramma ap ottenuto 1 mese dopo lintervento dimostra la presenza di un diverticolo di circa 16 mm di diametro massimo a partenza dalla tasca gastrica prossimale . 
one patient ( patient 1 ) presented with dysphagia at the first follow - up study at 34 months , when the diverticulum appeared unchanged ; symptoms had subsided at the second follow - up study , when the diverticulum was slightly enlarged . 
la maggior parte dei pazienti era del tutto asintomatica ; solo due soggetti ( le pazienti n 6 e 12 della tabella 1 ) avevano episodi ricorrenti di vomito al momento della diagnosi . 
stenosi del passaggio tra la parte prossimale dello stomaco e la parte distale dello stesso venne dimostrata in uno dei due casi ( paziente 12 ) al primo esame di follow - up , a 7 mesi di distanza . 
una paziente ( paziente 1 ) si present al primo esame di follow - up a 34 mesi accusando disfagia , e il diverticolo appariva immodificato rispetto allesame precedente ; i sintomi erano scomparsi al secondo follow - up , mentre il diverticolo appariva lievemente aumentato di volume . 
due pazienti ( caso 4 e caso 7 ) svilupparono reflusso gastroesofageo a 29 e 19 mesi dopo lintervento : in una ( paziente 4 ) il diverticolo non aveva subito modificazioni rispetto al primo esame ; nellaltra ( paziente 7 ) si osserv lieve aumento di volume . 
abbiamo osservato sviluppo di diverticoli della tasca prossimale lungo tutta la nostra esperienza di 79 interventi di vbg , e non solo allinizio della nostra casistica ( tabella 1 )  . quattro pazienti sono state sottoposte a nuovo intervento chirurgico . 
in two of them ( patients 2 and 4 ) , the diverticulum was unchanged from its first demonstration while it was slightly enlarged in the remaining one ( patient 7 )  . 
development of pouch diverticula was observed in all 79 patients who underwent vga and not only in the early cases ( see table 1 )  . four patients underwent a second surgical procedure . two were converted to biliopancreatic diversion : one because of new weight gain ( patient 5 ) ; the other for outlet stenosis ( patient 12 )  . 
two ( patients 8 and 11 ) requested restoration because of intolerance to the operation ; both underwent gastrogastrostomy ( one at our institution , the other at another hospital )  . 
the diverticula were not immediately visible , nor were they specifically searched for during surgery . creatica : una per aumento di peso ( paziente 5 ) ; laltra per la stenosi dello stoma ( paziente 12 )  . 
due ( pazienti 8 e 11 ) hanno richiesto una nuova operazione per intolleranza allintervento ; entrambe sono state sottoposte a gastrostomia ( una nel nostro reparto ; una in un altro ospedale )  . 
agli interventi , i diverticoli non erano immediatamente visibili n sono stati specificatamente ricercati . non abbiamo dimostrato correlazione tra presenza dei diverticoli , sintomi , perdita di peso postoperatoria e storia clinica delle pazienti . 
nella nostra casistica abbiamo avuto 2 / 12 pazienti con vomito ( 16 , 7% ) , 2 / 12 con reflusso gastroesofageo ( 16 , 7% ) , e 1 / 12 con stenosi dello stoma ( 8 , 3% )  . 
lanalisi statistica non ha dimostrato table 1 summary of clinical and radiographic findings following vertical - banded gastroplasty ( vbg ) diagnosis symptoms approximate first diameter , follow - up , months diameter , second follow - up , months symptoms approximate symptoms approximate diameter , no . 
no. in the series 1 month 1 month 4 months no 4 months no 3 days vomiting 3 days 4 months no 1 month 3 days 3 days 3 days vomiting 3 days go , gastrooesophageal reflux ; = , no changes tabella 1 riassunto dei reperti clinici e radiologici diagnosi nella serie operatoria 1 mese 1 mese 4 mesi 4 mesi 3 giorni 3 giorni 4 mesi 1 mese 3 giorni 3 giorni 3 giorni 3 giorni vomito vomito g - e , gastroesofageo ; = , referti invariati dysphagia go reflux go reflux > ( 28 ) go reflux vomiting > ( 16 ) > ( 15 ) disfagia riflusso g - e = vomito riflusso g - e > 28 riflusso g - e = no . 
no. sintomi diametro sintomi approssimativo , primo follow - up , mesi diametro approssimativo , secondo follow - up , mesi sintomi diametro approssimativo , there was no statistical correlation between the presence of diverticula and symptoms , postoperative weight loss and clinical history . 
they are considered to be a consequence of excessive pressure within the proximal gastric pouch that develops after surgery or , according to mason , due to a too narrow stoma between the proximal pouch and the distal stomach [ 7 ]  . 
although suturing was always performed against a 12 - mm ewald tube to ensure correct placement , this was probably not the case in all patients , thus leading to the development of diverticula at the crossing of the staple lines . 
moreover , at least in two patients , the fact that the vbg was done after a previous gastric banding could explain difficulties in obtaining correct alignment of the staple lines . 
however , it must be said that we encountered this kind of complication throughout our experience with this kind of operation , not only at the beginning , and this stands against this hypothesis . 
another possible explanation could be the follow - up protocol we adopted : in fact , all patients in our series underwent four radiographic studies in the first postoperative year even if they were symptom free . this closer follow - up strategy than that followed in most published series could possibly lead to demonstration of nonsymptomatic changes , as diverticula were demonstrated in most of our patients . the second point to discuss is the clinical significance of these lesions . 
vengono considerati conseguenza dellaumento della pressione allinterno della tasca gastrica prossimale che si sviluppa dopo lintervento o , secondo linterpretazione di mason , il risultato di uno stoma troppo ristretto tra la tasca prossimale e lo stomaco distale [ 7 ]  . 
bench le stesse siano state sempre effettuate lungo un tubo di ewald da 12 mm per garantire un corretto posizionamento , questo non stato , verosimilmente , possibile in tutti i casi , consentendo quindi il formarsi dei diverticoli allincrocio delle linee di sutura . 
inoltre , almeno in due pazienti , il fatto che lintervento di vbg sia stato effettuato dopo un precedente intervento di bendaggio gastrico pu spiegare ulteriori difficolt al corretto posizionamento delle linee di sutura . 
possibile inoltre ipotizzare che i diverticoli siano il risultato della doppia curva di apprendimento affrontata dai chirurghi ( quella durante lapprendimento della tecnica laparotomica di intervento e , in un secondo momento , quella per lapprendimento della tecnica laparoscopica )  . 
tuttavia , il fatto che i diverticoli si siano presentati in pazienti operati durante tutto larco della nostra esperienza , e non solo allinizio , sembra contrastare con questa possibilit . 
unaltra possibile spiegazione sta nel tipo di protocollo di follow - up radiologico utilizzato nei nostri casi : infatti , tutti i pazienti sono stati sottoposti a quattro indagini radiologiche nel primo anno dopo lintervento , anche se asintomatici . 
questa una strategia di controllo molto pi serrata di quella seguita nella maggioranza degli altri centri che pu aver portato alla dimostrazione di reperti non sintomatici , come si sono dimostrati essere i diverticoli nella maggior parte dei nostri casi . il secondo punto da discutere il significato clinico di queste lesioni . 
three other patients developed symptoms during follow - up ( one dysphagia and two gastrooesophageal reflux ) , but diverticula were unchanged in two of them and only slightly increased in the remaining one . 
more in detail , patient 1 , who had dysphagia , had an unchanged diverticulum at the first follow - up examination whereas the lesion was slightly enlarged at follow - up after about 1 year , when her symptom had disappeared . 
in addition , no differences were observed between frequency and type of long - term complications in vbg patients with diverticula and those without them . to conclude , our results show that , in our series , diverticula of the proximal pouch in patients who undergo vbg are a relatively common finding . 
the cause of diverticula were , most probably , problems in obtaining good alignment of the suture lines along the lesser curvature of the stomach at surgery while demonstration of diverticula was related to the strict follow - up protocol adopted . 
from a clinical point of view , we do not believe these lesions to be important ; at present , our patients are no longer followed up for this problem and undergo diagnostic examinations only if and when they develop symptoms . tra la sintomatologia osservata nei soggetti con diverticoli rispetto a quella dei pazienti senza questo tipo di lesioni . inoltre , non abbiamo osservato alcuna correlazione tra storia clinica e variazioni volumetriche dei diverticoli nel tempo . 
in uno dei due pazienti sintomatici il vomito scomparve dopo pochi mesi , mentre laltro ebbe persistenza dei sintomi a fronte di mancato incremento volumetrico della lesione . tre ulteriori soggetti hanno sviluppato sintomi durante il follow - up ( uno disfagia e due reflusso gastroesofageo ) , ma i diverticoli rimasero immodificati in due e solo lievemente aumentato nel rimanente . 
specificatamente , la paziente 1 , che aveva disfagia aveva il diverticolo immodificato al primo controllo , mentre lo stesso era lievemente ingrandito allindagine successiva , a circa un anno , ma con scomparsa della sintomatologia . per concludere , i nostri risultati mostrano che , nella nostra casistica , i diverticoli della tasca prossimale nei pazienti operati per vbg sono un reperto relativamente comune . lo sviluppo di queste lesioni stato in relazione , verosimilmente , a difficolt ad ottenere un corretto allineamento delle linee di sutura lungo la piccola curva durante lintervento chirurgico , e la loro dimostrazione stata il risultato dello stretto programma di follow - up da noi utilizzato . 
lagalla sezione di diagnostica per immagini , dipartimento di biotecnologie mediche e medicina legale , policlinico universitario , via del vespro 127 , i - 90127 palermo , italy , correspondence to : t.v. 
 + 39 - 091 - 6552335 / 6552336 , fax + 39 - 091 - 6552337 , e - mail : tv.bartolotta@unipa.it received : 14 february 2006 / accepted 11 april 2006 / published online : 11 october 2006 abstract purpose . 
however , the growing use of us in the study of the neck and thyroid has markedly increased the detection rate of nonpalpable thyroid nodules , with values reaching almost 70% of the study sample in some series [ 25 ]  . 
most thyroid incidentalomas are of no clinical significance since thyroid cancer is rare , accounting for 1.5% of all malignancies and 0.23% of all cancer deaths in the usa in 2001 [ 6 ]  . moreover , surgical and autoptic studies have demonstrated that occult thyroid cancers are more frequent than clinically manifest forms reaching 36% of the population studied in some countries which confirms the limited biological significance of this cancer [ 7 , 8 ]  . a new sonographic technique real - time spatial compound sonography ( cs ) has recently been introduced that uses electronic beam steering of a transducer array to acquire several ( three to nine ) partially overlapping scans of an object from different view angles [ 9 ]  . 
the application of this technique to the study of the thyroid has been shown to improve image quality compared with conventional sonography , mainly thanks to the reduction in background noise , spurious echoes and other acoustic artefacts [ 10 ]  . the primary aim of this study was to assess the incidence and sonographic patterns with both greyscale and colour doppler us of thyroid nodules detected by neck sonography in an adult population with no suspicion of thyroid disease . 
the secondary aim was to evaluate the possible benefit of cs technology in terms of improved image quality . materials and methods patients and lesions between january and october 2003 , after obtaining approval from the ethics committee , we carried out hrus and cs of the thyroid on 704 consecutive patients ( 400 women and 304 men ; age range 1793 years ; mean : 60.615 years ) who had been referred to us for colour doppler us of the epiaortic trunks and had no history , symptoms or signs of thyroid disease . 
all patients were inhabitants of the province of palermo ( northern sicily ) , an area with a low iodine intake ( mean daily urinary iodine excretion less than 50 - 100 g / g of creatinine )  . 
in order of frequency , the clinical indications for colour doppler us of the carotids were : vertigo ( 211 ) , previous cerebral stroke ( 181 ) or tia ( 157 ) , generalised atherosclerosis ( 130 ) , type i or ii diabetes mellitus ( 126 ) , headache ( 58 ) and previous trauma ( 15 )  . 
informed consent was obtained from all patients before the us study . lecografia ad alta risoluzione in scala di grigi ( us ) e limpiego dei moduli color e power - doppler consentono unaccurata valutazione delle strutture anatomiche della tiroide , fornendo , al contempo , informazioni di carattere funzionale sulla ghiandola normale e su quella patologica , con particolare riguardo alla sua vascolarizzazione [ 1 ]  . tuttavia , in diretta conseguenza del sempre crescente utilizzo dellus nello studio del collo e della tiroide , si registrato un netto incremento del riscontro di noduli tiroidei non palpabili , fino a valori che , in alcune casistiche , raggiungono quasi il 70% della popolazione in studio [ 25 ]  . 
la maggioranza degli incidentalomi tiroidei , infatti , non riveste significato clinico di rilievo , in quanto il cancro della tiroide poco frequente , rappresentando l1 , 5% di tutte le neoplasie maligne e lo 0 , 23% di tutte le morti per cancro nel 2001 negli stati uniti damerica [ 6 ]  . 
inoltre , studi chirurgici e autoptici hanno messo in evidenza come il cancro occulto della tiroide sia pi frequente di quello clinicamente manifesto raggiungendo in alcuni paesi anche il 36% della popolazione presa in esame a conferma della sua limitata rilevanza biologica [ 7 , 8 ]  . recentemente , stata introdotta una nuova tecnica ecografica real time spatial compound sonography ( cs ) la quale sfrutta la possibilit di angolare elettronicamente il fascio ultrasonoro generato dalla linea di elementi trasduttori al fine di effettuare diverse ( da tre a nove ) scansioni in parte sovrapposte di un oggetto da diversi angoli di vista [ 9 ]  . 
lapplicazione di tale tecnica allo studio ecografico della tiroide ha mostrato una migliore qualit dellimmagine rispetto allecografia convenzionale , principalmente grazie alla riduzione del rumore di fondo , degli echi spuri e di altri artefatti acustici [ 10 ]  . lobiettivo primario di questo lavoro stato quello di valutare lincidenza e gli aspetti ecografici sia in scala dei grigi che color - doppler dei noduli tiroidei riscontrati in una popolazione adulta , senza sospetto clinico di malattia tiroidea , sottoposta a studio ecografico del collo . 
quale obiettivo secondario , stato valutato leventuale apporto fornito dalla tecnologia cs in termini di qualit dellimmagine . materiali e metodi pazienti e lesioni tra gennaio e ottobre 2003 , ottenuta lapprovazione da parte del comitato etico , 704 pazienti consecutivi ( 400 donne e 304 uomini ; 1793 anni ; media : 60 , 615 anni ) inviati presso il nostro istituto per essere sottoposti ad uno studio ecocolor - doppler dei tronchi epiaortici e i quali non presentavano anamnesi , sintomi o segni clinici di patologia tiroidea sono stati sottoposti ad hrus e cs della tiroide . 
tutti i pazienti erano cittadini della provincia di palermo , nella sicilia settentrionale , che costituisce unarea a bassa increzione iodica ( escrezione media giornaliera di iodio con le urine inferiore a 50100 g / g di creatinina )  . 
once adjusted , the technical parameters remained unchanged throughout the us study . for each nodule detected , the study was completed with the acquisition of two images , one with conventional us and the other with the cs technique . 
both images were saved on the units hard disk and then transferred as raw data to a personal computer ( pc ) connected to the us unit via an ethernet connection . on - site analysis at the end of each examination , the radiologists , who were unaware of the final diagnosis , determined the number , size ( measured in three diameters ) , site , echogenicity ( hyper - , isoor hypoechoic ) , echostructure ( solid , cystic , mixed ) , margins ( well - defined , irregular , ill - defined ) and the presence of hyperechoic spots ( coarse calcification or microcalcifications ) in all nodules detected [ 11 ]  . 
in detail , the following were considered malignant sonographic characteristics on greyscale us : ( a ) punctate microcalcifications defined as hyperechoic spots less than 2 mm with or without acoustic shadows , ( b ) markedly hypoechoic echostructure ( increased hypoechogenicity relative to adjacent muscle ) , ( c ) irregular or microlobulated margins and ( d ) anteroposterior diameter greater than longitudinal diameter [ 12 ]  . 
the presence and pattern of flow at colour and power doppler imaging were assessed and classified as follows : type i , no flow ; type ii , perinodular flow only ; type iii , intranodular flow only ( iiia ) or intraand perinodular flow ( iiib ) [ 13 ]  . 
clinical , sonographic and cytological results were filed separately and used for statistical evaluation using the chi - square and fisher exact tests , with statistical significance set at p < 0.05. off - site analysis digital images stored on the pc were reviewed by two expert radiologists who had not taken part in the us examinations and were blinded to the final diagnosis and examination technique . 
la regolazione relativa al guadagno colore stata effettuata innalzando lamplificazione fino ad ottenere , in ogni singolo paziente , la comparsa di segnali casuali di natura artefattuale e poi abbassando lievemente lamplificazione fino alla loro scomparsa . 
una volta regolati , i parametri tecnici sono stati mantenuti invariati per tutta la rimanente durata dellindagine ecografica . ogni studio stato completato , per ogni nodulo individuato , con lacquisizione di due immagini , la prima ottenuta con metodica ecografica convenzionale e la seconda con tecnica compound . 
entrambe le immagini sono state archiviate sul disco rigido in dotazione allunit ecografica e poi inviati , sotto forma di dati grezzi , ad un personal computer ( pc ) collegato allecografo attraverso una connessione in standard ethernet . analisi on - site al termine di ogni indagine ecografica gli esaminatori , non a conocenza della diagnosi finale , hanno valutato consensualmente numero , dimensioni ( misurate in 3 diametri ) , sede , ecogenicit ( iper - , isoo ipo - ecogena ) , ecostruttura ( solida , cistica , mista ) , margini ( ben definiti , irregolari o sfumati ) e la presenza di spots iperecogeni ( calcificazioni grossolane o microcalcificazioni ) in tutti i noduli individuati [ 11 ]  . 
the two radiologists expressed their evaluation in consensus and graded image quality on a 5 - point scale ; statistical analysis was performed using the t test for paired samples . reference standard nodules displaying at least one criterion of malignancy at us or colour doppler us underwent fna . 
aspirates were fixed in alcohol and stained with papanicolaou staall cytological samples were examined and graded according to papanicolaou into classes from 0 to 5 : class 0 indicates inadequate sample ; class 1 absence of atypical or abnormal cells ; class 2 atypical cytological findings but no evidence of malignancy ; class 3 cytological findings suggestive of , but not conclusive for , malignancy ; class 4 strongly suggestive of malignancy ; and class 5 conclusive for malignancy . results we detected 711 thyroid nodules in 233 patients ( 33.1% ) , with an age range of 1793 years and a mean age of 59.415.3 years ( table 1 )  . 
no statistically significant difference was found between the number of nodules located in the right so ; tipo ii , flusso esclusivamente perinodulare ; tipo iii , flusso esclusivamente intranodulare ( iiia ) o intra e perinodulare ( iiib ) [ 13 ]  . 
gli accertamenti clinici , ecografici e citologici sono stati archiviati separatamente e utilizzati per valutazioni statistiche tramite i test chi - quadro ed esatto di fisher con livello di significativit statistica fissato a p inferiore a 0 , 05 . analisi off - site le immagini digitali archiviate allinterno della stazione di lavoro basata su pc sono state retrospettivamente riesaminate da due esperti radiologi , non coinvolti negli esami ecografici e tenuti alloscuro riguardo la diagnosi finale e la tecnica utilizzata per ogni esame . 
agli esaminatori stato chiesto di valutare , per ciascuna immagine , la qualit complessiva in relazione alla presenza o meno di artefatti , quali : ( a ) la granulosit dellimmagine artefatto anche noto con il termine di coherent wave interface o speckle e riconducibile ad un eccessivo rumore di fondo ; ( b ) la presenza di echi spuri o clutter ; ( c ) le ombre acustiche laterali e altri artefatti acustici ; ( d ) il grado di evidenza delle lesioni ( visibilit e chiarezza dei noduli rispetto al parenchima ghiandolare adiacente ) [ 15 ]  . 
i due radiologi hanno espresso , in consenso , la loro valutazione graduandola su una scala di cinque livelli e lanalisi statistica stata effettuata utilizzando il t test per campione doppio . standard di riferimento nei noduli con almeno un criterio di malignit positivo allecografia e / o al color - doppler stato effettuato laspirato con ago sottile ( fna )  . 
in accordo con questa classificazione , la classe 0 indica women : no nodules women : detected nodules men : no nodules men : detected nodules donne : noduli assenti donne : noduli presenti uomini : noduli assenti uomini : noduli presenti age range / fasce det fig . 
2a high - resolution ultrasonography ( hrus ) axial image in a 31 - year - old woman shows a slightly hypoechoic , ill - defined , 6 - mm nodule ( large arrow ) on the anterior aspect of the right lobe ; artefactual shadowing is clearly appreciable on the common carotid ( small arrows ) , as is speckle ( grainy appearance ) ( large arrow )  . 
2a immagine assiale in scala di grigi convenzionale in una donna di 31 anni che evidenzia , nel lobo destro in sede anteriore , un nodulo lievemente ipoecogeno a margini mal definiti del diametro di 6 mm ( freccia grande ) ; la presenza di ombre acustiche laterali chiaramente apprezzabile in corrispondenza del tronco carotideo comune ( frecce piccole ) cos come laspetto complessivamente granuloso dellimmagine . 
type ii colour doppler pattern was statistically more common in nodules less than 10 mm in diameter ( p < 0.001 ) ( table 5 )  . none of the nodules had microcalcifications , irregular or microlobulated margins , marked hypoechogenicity or longitudinal diameter greater than anteroposterior diameter . 
416 / 711 noduli sono stati rilevati in 143 donne ( 58 , 5% ) e 295 / 711 noduli in 90 uomini ( 41 , 5% ) ( p < 0 , 001 )  . 67 / 233 ( 28 , 7% ) pazienti presentavano un singolo nodulo mentre i rimanenti 166 / 233 ( 72 , 3% ) avevano multiple formazioni nodulari . 
non sono state , di contro , osservate differenze statisticamente significative tra il numero dei noduli localizzati nel lobo destro e quelli localizzati nel lobo sinistro : 372 / 711 ( 52 , 3% ) nel lobo destro , 336 / 711 ( 47 , 4% ) nel sinistro e i restanti 3 ( 0.4% ) noduli in sede istmica . lintervallo dimensionale dei noduli individuati stato di 0 , 184 , 1 cm ( media : 1 , 1 cm )  . 
in clinical practice , us is increasingly regarded as an extension of the physical examination of the thyroid and , as a result , the number of endocrinologist referrals for incidentally discovered thyroid nodules is likely to reach epidemic proportions , raising several concerns as to the management of these patients [ 6 ]  . 
the low malignancy and mortality rates associated with thyroid neoplasms and the fact that benign nodules tend to remain so for a long time call for a thorough assessment of the costbenefit ratio of the use of different diagnostic tools in nodular thyroid pathology [ 16 ]  . in agreement with the literature , the incidence of thyroid nodules observed in our series is high approximately one third of the population examined with a higher incidence in women and in old age . 
however , the incidence in the general population is most probably even higher , given that our study excluded patients with a positive history or clinical signs and symptoms of thyroid pathology . 
a study on us screening for thyroid nodules in 101 middle - aged women without previous thyroid disease living in southern finland detected thyroid nodules or echostructural abnormalities in 35.6% of subjects [ 17 ] , and the same authors reported the presence of sonographic thyroid abnormalities in 69 / 253 risultato statisticamente pi frequente nei noduli di diametro inferiore a 10 mm ( p < 0 , 001 ) ( tabella 5 )  . 
in nessuno dei noduli presi in esame stata riscontrata la presenza di microcalcificazioni , margini irregolari o microlobulati , ipoecogenicit marcata o diametro longitudinale maggiore di quello antero - posteriore . 
nella pratica clinica , lecografia assume sempre pi la valenza di vera e propria estensione dellesame obiettivo della tiroide e , per questo , verosimile che il numero di soggetti che si rivolgono agli endocrinologi per noduli tiroidei scoperti in maniera incidentale tenda progressivamente ad aumentare , assumendo proporzioni di vera e propria epidemia e sollevando non poche preoccupazioni in relazione alla gestione di tali pazienti [ 6 ]  . 
infatti , alla luce dei bassi tassi sia di malignit che di morte per neoplasie tiroidee nonch della constatazione che noduli tiroidei benigni tendono a rimanere tali a lungo , appare necessaria una corretta valutazione del rapporto costo / beneficio nellimpiego dei diversi presidi diagnostici nella patologia nodulare tiroidea [ 16 ]  . in accordo con quanto riportato in letteratura , lincidenza di noduli tiroidei osservata nella nostra serie elevata , pari a circa un terzo del campione in esame , con una maggiore incidenza nel sesso femminile ed in et avanzata . 
tuttavia , lincidenza nella popolazione generale da ritenersi , con tutta probabilit , ancora maggiore , dal momento che dal nostro studio sono stati esclusi i pazienti con anamnesi positiva o sintomi e segni clinici di patologia tiroidea . 
unindagine ecografica eseguita nella finlandia meridionale in 101 donne det media senza precedenti patologie tiroidee , ha dimostrato noduli tiroidei o anomalie ecostrutturali nel 35 , 6% dei soggetti [ 17 ] mentre gli stessi autori hanno rivelato la presenza di anomalie ecografiche della tiroide in 69 / 253 ( 27 , 3% ) soggetti selezionati casualmente [ 2 ]  . 
infine , una valutazione ecografica di eventuali anomalie tiroidee , condotta su 547 soggetti sovrappeso in unarea cittadina con introito giornaliero di iodio relativamente basso , ha evidenziato la presenza di alterazioni tiroidee nel 56% dei soggetti [ 18 ]  . in particolare , nella nostra casistica circa i due terzi dei noduli riscontrati misurava meno di 10 mm e la grande maggioranza ( 93% ) misurava meno di 20 mm , a conferma del fatto che i noduli tiroidei non palpabili sono di frequente riscontro allo studio ecografico della tiroide , anche in una popolazione non a rischio per patologia tiroidea . 
tuttavia , nonostante uno studio abbia dimostrato come lecografia convenzionale modifichi la gestione clinica nel 63% dei pazienti , lutilit della stessa nel predire la malignit dei noduli tiroidei risulta tuttora controversa , soprattutto se raffrontata con la biopsia ad ago sottile [ 19 ]  . 
finally , a sonographic assessment of possible thyroid abnormalities in 547 overweight individuals from an urban area with relatively low daily iodine intake found thyroid alterations in 56% of subjects [ 18 ]  . 
 in our series , about two thirds of the nodules detected were smaller than 10 mm , and the large majority ( 93% ) were smaller than 20 mm , confirming the fact that nonpalpable thyroid nodules are a common finding on thyroid sonography , even in a population not at risk of thyroid pathology . nonetheless , although it has been demonstrated that conventional us affects clinical management in 63% of patients , its usefulness in predicting the malignancy of thyroid nodules is still controversial , above all when compared with fna [ 19 ]  . indeed , as regards the relative risk of malignancy attributable to the various us findings in nonpalpable thyroid nodules both greyscale and colour doppler it has been shown that there is considerable overlap between benign and malignant lesions [ 14 , 2022 ]  . 
in particular , whereas some authors found that greyscale sonography findings had no value in predicting malignancy [ 23 ] , other more recent studies have suggested a positive predictive role for the presence of irregular or ill - defined margins , microcalcifications , marked hypoechogenicity and anteroposterior diameter greater than longitudinal diameter [ 14 ]  . 
no malignant lesions were found in our series , which indirectly confirms the high specificity of the findings of greyscale sonography and the very low rates of malignant thyroid nodules in the general adult population [ 12 , 16 , 2428 ]  . as regards colour doppler analysis , our series exhibited all the different vascular patterns described in the literature , with a statistically significantly higher prevalence of perinodular flow , the most common flow pattern especially in nodules smaller than 10 mbecause intranodular vascularity was reported as an independent risk factor for malignancy in one study , we decided to regard as suspicious all lesions with intralesional vascularity ( patterns iiia and iiib ) [ 13 , 14 ]  . 
this finding is essentially in agreement with previous reports and confirms that the colour doppler patterns , similar to the greyscale us findings mentioned above , are not highly predictive of malignancy when considered individually [ 22 ]  . 
it should also be noted that the availability of increasingly efficient colour doppler techniques has improved sensitivity in detecting intranodular flow but has also decreased , in terms of specificity , the possibility of characterising thyroid nodules using the colour mode only . 
 a technique described by whittingham in 1997 , spatial cs , has led to improvements in overall image definition , which some studies have reported to be useful for diagnostic purposes , for example , in the field of rotator - cuff imaging [ 2931 ]  . 
although some authors expressed concern about the possible elimination of potentially il rischio relativo di malignit attribuibile ai diversi reperti ecografici sia in scala di grigi che color - doppler nei noduli tiroidei non palpabili , stata dimostrata una non trascurabile sovrapposizione tra le lesioni benigne e quelle maligne [ 14 , 2022 ]  . 
in particolare , nonostante alcuni lavori non abbiano dimostrato la validit dei reperti ecografici in scala di grigi in quanto elementi predittivi di malignit [ 23 ] , altri e pi recenti studi suggeriscono un ruolo predittivo positivo per la presenza rispettivamente di margini irregolari o sfumati , microcalcificazioni , ipoecogenicit marcata e , infine , per la presenza di un diametro antero - posteriore del nodulo maggiore di quello longitudinale [ 14 ]  . 
nella nostra casistica non sono state riscontrate lesioni maligne , il che indirettamente conferma lalta specificit attribuita ai reperti ecografici in scala di grigi , cos come il tasso molto basso , riportato da svariati autori , di neoplasie maligne fra i noduli tiroidei nella popolazione generale adulta [ 12 , 16 , 2428 ]  . per quanto attiene lanalisi color - doppler , nella nostra serie sono stati osservati tutti i differenti pattern di vascolarizzazione descritti in letteratura , con una prevalenza pi alta e statisticamente significativa per il flusso perinodulare , che si rivelato molto pi frequente soprattutto nei noduli di diametro inferiore a 10 mpoich in uno studio la presenza di vascolarizzazione intranodulare era stato riportato come un fattore di rischio indipendente di malignit , ai fini di questo studio si deciso di considerare sospetta ogni lesione che presentasse vascolarizzazione intralesionale ( pattern iiia e iiib ) [ 13 , 14 ]  . 
tale osservazione risulta sostanzialmente in accordo con alcuni dati precedentemente pubblicati , confermando come i pattern color - doppler , allo stesso modo dei gi considerati reperti ecografici in scala di grigi , quando considerati isolatamente , non risultino altamente predittivi di malignit [ 22 ]  . 
in particolare , andrebbe osservato come la diffusione di tecniche color - doppler sempre pi efficaci abbia consentito un incremento della sensibilit nel rilevamento del flusso intranodulare ma , al contempo , abbia condotto ad un decremento , in termini di specificit , della possibilit di caratterizzazione dei noduli tiroidei con il semplice modulo colore . la cs una tecnica ultrasonografica le cui basi sono state descritte da whittingham nel 1997 e la cui applicazione ha condotto ad un miglioramento della definizione dellimmagine che alcuni studi hanno riportato essere utile anche ai fini diagnostici , ad esempio in ambito muscolo - scheletrico ed in particolare nel distretto anatomico della cuffia dei rotatori [ 2931 ]  . 
anche uno studio della ghiandola tiroidea condotto con tale tecnica , ha riportato una minore presenza di artefatti ed una pi alta capacit di evidenziare noduli rispetto allecografia convenzionale [ 10 ]  . 
tuttavia , alcuni autori hanno sollevato alcune perplessit riguardo la possibile eliminazione di artefatti di potenziale rilevanza diagnostica , come , ad esempio , lombra acustica posteriore , segno suggestivo di malignit nelle lesioni mammarie [ 32 ]  . 
anche nella nostra esperienza la cs ha fornito una qualit delle imdiagnostic artefacts such as the posterior shadow , a sign suggestive of malignancy in breast lesions [ 32 ] it has recently been shown that the use of spatial cs does not significantly affect the radiologists assessment of solid breast lesions [ 33 ]  . 
even in our experience , spatial cs of thyroid nodules provided an image quality that was subjectively judged to be better than that of conventional us , and the technique is routinely used at our institution . 
on the other hand , better image quality does not necessarily and immediately translate into a higher detection rate or better diagnostic efficacy , and therefore , further studies are required to assess these aspects . 
although reflecting clinical management , this approach may have led to a slight underestimation of the true number of thyroid cancers even if the number is expected to be extremely small . 
finally , a double - blind review of cases would have enabled us to calculate intraand interobserver variability even if this was only a secondary aim of our study . in conclusion , our series of incidentally discovered thyroid nodules exhibited a high incidence of nodules 1 cm or less in size that were hypoechoic , with exclusively peripheral vascularity and a benign nature . 
daltra parte , anche una migliore qualit dellimmagine pu non tradursi necessariamente ed immediatamente , nella pratica clinica , in un pi elevato tasso di identificazione di noduli tiroidei o in una maggiore efficacia diagnostica e , pertanto , ulteriori studi saranno necessari al fine di valutare tali aspetti . il nostro studio presenta alcune limitazioni . 
in primo luogo , nella nostra casistica non stata disponibile la verifica istologica , anche in relazione al fatto che nei pazienti presi in esame non era presente alcuna indicazione allintervento chirurgico . 
tale approccio , per quanto conforme alla pratica della gestione clinica , pu tuttavia lievemente sottostimare il numero reale di tumori della tiroide , sebbene ci si aspetti che tale cifra sia estremamente bassa . 
una revisione dei casi in doppio cieco , infine , avrebbe consentito il calcolo della variabilit intraed inter - osservatore , anche se questa era una finalit solo secondaria del nostro lavoro . in conclusione , nella nostra casistica di noduli della tiroide riscontrati incidentalmente , risultata elevata lincidenza di noduli di dimensioni pari o inferiori al centimetro , ipoecogeni , con vascolarizzazione esclusivamente periferica e di natura benigna . 
tali reperti suggeriscono , operativamente , un approccio conservativo per questo tipo di noduli , a meno che non siano presenti evidenti segni di malignit allo studio ecografico in scala dei grigi . 
pilon2 1dipartimento di scienze medico - diagnostiche e terapie speciali , sezione di radiologia , 2istituto di clinica chirurgica i , 3istituto di clinica chirurgica ii , universit di padova , italy correspondence to : g . 
barbiero , via fogarine 59 , i - 30030 foss ( ve ) , italy , tel . : + 39 - 0424 - 888795 , fax : + 39 - 0424 - 888791 , e - mail : giuliobarbiero@katamail.com received : 14 january 2006 / accepted : 10 may 2006 / published online : 11 october 2006 abstract purpose . 
the aim of the study was to evaluate quantitatively the main morphological changes of the abdominal aortic aneurysm ( aaa ) - endograft ( eg ) complex following endovascular repair of infrarenal aaa and to evaluate the functional consequences of these changes in terms of rate of complications ( endoleaks and thrombosis )  . 
eighty - five patients ( m / f = 82 / 3 ; mean age at time of operation 70.53.5 years , range 49.989.6 years ) who underwent endovascular treatment of infrarenal aaa between april 1997 and october 2004 with a follow - up of at least 1 month were considered . 
morphological and dimensional changes involved the diameter ( six cases ) and length ( 14 cases ) of aaa proximal neck , diameter ( 36 cases ) and length ( 51 cases ) of the aneurysm sac and shape of the stent - graft ( 47 cases )  . 
aaa growth was correlated with the diameter of the aneurysm sac while shrinkage was correlated with the eg used . during follow - up after endovascular repair , patients require careful evaluation of the morphological and dimensional features of the aaa and eg to promptly identify any changes that can anticipate major complications and even conversion to conventional surgery . 
valutare quantitativamente le principali modificazioni morfologiche del complesso aneurisma ( aaa ) - endoprotesi ( ep ) dopo trattamento endoluminale degli aaa infrarenali , e le loro conseguenze funzionali in termini di incidenza di complicanze ( endoleak e trombosi )  . 
sono stati considerati 85 pazienti consecutivi ( m / f = 82 / 3 ; et media al momento dellimpianto 70 , 53 , 5 anni , range 49 , 989 , 6 anni ) portatori di aaa infrarenale sottoposti fra aprile 1997 e ottobre 2004 a posizionamento di ep per via transfemorale con follow - up post - procedurale di almeno 1 mese . sono stati riesaminati criticamente 408 esami angio - tc eseguiti sia in fase preoperatoria che postoperatoria . 
modificazioni morfodimensionali significative hanno interessato il diametro ( 6 casi ) e la lunghezza ( 14 casi ) del colletto prossimale dellaaa , il diametro ( 36 casi ) e la lunghezza ( 51 casi ) della sacca aneurismatica , e la morfologia dello stentgraft ( 47 casi )  . 
nel follow - up dei pazienti trattati necessaria unaccurata valutazione dei parametri morfodimensionali sia dellaaa che dellep allo scopo di coglierne precocemente eventuali modificazioni che possano precedere importanti conseguenze funzionali , fino alla necessit di conversione laparotomica . parole chiave aneurisma aortico addominale trattamento endovascolare endograft endoleak c . 
after endoluminal treatment , the proximal neck , the major site of fixation of the eg to the aortic wall [ 4 , 5 ] , may become progressive dilated [ 6 , 7 ] , facilitating the development of a secondary type - 1 endoleak ( el ) , that is , persistence of perigraft blood flow ( table 1 ) [ 8 , 9 ] , with resulting expansion of the aaa and increased risk of rupture [ 1012 ]  . 
after complete endoluminal exclusion , the aneurysm sac shrinks progressively , especially in larger aaa and with certain types of eg , sometimes to the point of complete reabsorption of perigraft thrombotic material [ 1321 ]  . 
in some cases , however , the aaa enlarges after eg implantation , an indication of persistent pressurisation of the aneurysm sac , even in the absence of endotension dopo esclusione di un aneurisma dellaorta addominale ( aaa ) dal torrente circolatorio mediante endoprotesi ( ep ) [ 13 ] , si verifica un rimodellamento del complesso aaa - ep nel tempo , che interessa il colletto sottorenale , la sacca aneurismatica e lep stessa . 
il colletto prossimale , sito principale di fissazione dellep alla parete aortica [ 4 , 5 ] , dopo trattamento endoluminale pu subire una progressiva dilatazione [ 6 , 7 ] , che favorisce linsorgenza di endoleak ( el ) , ossia persistenza di flusso sanguigno periprotesico ( tabella 1 ) [ 8 , 9 ] , di tipo i secondario , con conseguente espansione dellaaa e , quindi , aumentato rischio di rottura [ 1012 ]  . 
la sacca aneurismatica , dopo esclusione endoluminale completa , generalmente subisce una progressiva contrazione ( shrinkage ) nel tempo , specie negli aaa pi grandi e con certi tipi di ep , talvolta fino al completo riassorbimento del materiale trombotico periprotesico [ 1321 ]  . 
talvolta , per , si verifica unespansione dellaaa post - ep , indice di pressurizzazione persistente della sacca aneurismatica , anche in assenza di el ( endotension ) [ 2224 ]  . 
lep , infine , pu presentare distorsioni ed table 1 classification based on aetiology of the endoleak ( adapted from [ 36 ] ) endoleak cause of the perigraft blood flow insufficient seal at the graft ends at the proximal end at the distal end insufficient seal of an iliac occlusion device retrograde blood flow from aortic branches ( lumbar arteries , inferior mesenteric artery , hypogastric arteries , accessories renal arteries ) graft disconnection or graft disintegration graft disconnection fabric tears minor ( < 2 mm ) major ( 2 mm ) type 4 type 5 endoleak of undefined origin unknown cause graft wall porosity ( within 30 days after endovascular repair ) persistent aneurysm sac pressurisation with no evidence of the source of leakage ( endotension ) tabella 1 classificazione eziologica degli endoleak ( da [ 36 ] , modificata ) endoleak causa del flusso periprotesico incompleto contatto della protesi con la parete vascolare a livello delle sue estremit dellestremit prossimale dellestremit distale da inefficienza di un dispositivo di occlusione iliaca da flusso retrogrado attraverso vasi viscerali ( arterie lombari , mesenterica inferiore , ipogastriche , renali accessorie ) per disconnessione dei moduli protesici o per rottura della protesi per disconnessione dei moduli protesici per rottura del tessuto protesico : minore ( < 2 mm ) maggiore ( 2 mm ) tipo iv tipo v endoleak di origine indeterminata visualizzazione dellendoleak senza determinazione della sua origine da porosit del tessuto protesico ( nei primi 30 giorni dopo il posizionamento dellendoprotesi ) pressurizzazione persistente della sacca aneurismatica senza rifornimento dimostrabile ( endotension ) type 1 type 2 type 3 tipo i tipo ii tipo iii c . 
finally , the eg may develop distortions and kinking [ 2528 ] and in some cases structural disintegration , with a risk of type - 3 el and a need for repeat endovascular procedures or surgical conversion . 
alternatively , it may migrate distally , resulting in type - 1 el secondary to loss of proximal seal [ 2934 ]  . the aim of this study was to quantitatively evaluate the main morphological changes affecting the aaa - eg complex after endovascular treatment and the functional consequences of these changes in terms of rate of complications ( in particular , el and thrombosis )  . 
in addition , we assessed whether the morphological and structural changes could be related to the preprocedural size of the aaa and to the type of eg used . angolazioni nel tempo [ 2528 ] , fino ad arrivare in certi casi ad un cedimento strutturale , che pu determinare la comparsa di un el di tipo iii e , quindi , la necessit di procedure endovascolari secondarie o conversione chirurgica , oppure pu presentare una migrazione distale con conseguente el di tipo i secondario da perdita del sealing prossimale [ 2934 ]  . scopo di questo lavoro valutare quantitativamente le principali modificazioni morfologiche del complesso aaa - ep dopo trattamento endoluminale , e le loro conseguenze funzionali in termini di incidenza di complicanze ( in particolar modo el e trombosi )  . 
inoltre si intende valutare se tali modificazioni morfofunzionali possano essere messe in relazione alle dimensioni preprocedurali dellaaa e al tipo di ep impiegato . materials and methods we studied 85 consecutive patients with infrarenal aaa ( m / f = 82 / 3 ; mean age at time of operation 70.53.5 years , range 49.989.6 years ) who underwent transfemoral eg placement between april 1997 and october 2004 and were followed up for at least 1 month . 
seven eg models from different manufacturers were used ( table 2 ) , of which one ( 1.2% ) was a straight aortoaortic tubular eg and 84 ( 98.8% ) were bifurcated aortobiiliac eg . 
the follow - up included a colour - doppler ultrasound ( us ) scan at discharge or a ct angiography scan on day 7 , followed by periodic ct angiography studies at 1 , 3 , 6 and 12 months and yearly thereafter . 
in 10 pazienti ( 11 , 8% ) il trattamento endovascolare stato eseguito pur non rispettando completamente i criteri suggeriti dalla letteratura [ 5 ] in quanto vi era controindicazione assoluta allintervento chirurgico tradizionale e laaa era ad alto rischio ( 4 pazienti con colletto sottorenale breve , 1 paziente con colletto prossimale ampio , 1 paziente con diametro dellaaa > 7 cm e infine 4 pazienti con esili arterie renali accessorie originanti dal colletto prossimale o dalla sacca aneurismatica )  . 
sono stati utilizzati 7 modelli di ep di case costruttrici diverse ( tabella 2 ) , di cui solo una ( 1 , 2% ) di tipo tubulare retto aorto - aortico , e 84 ( 98 , 8% ) di tipo biforcato aorto - bisiliaco . 
il follow - up dei pazienti trattati prevedeva un controllo mediante eco - color - doppler alla dimissione o lesecuzione di un angio - tc in settima giornata , seguita da controlli periodici mediante angio - tc a 1 , 3 , 6 , 12 mesi e quindi annualmente . 
di ogni esame tc sono stati valutati : diametro e lunghezza del colletto prossimale aortico , diametri anteroposteriore ( ap ) , laterolaterale ( ll ) e trasverso massimo ( mtr ) dellaaa ( che corrisponde al diametro trasverso minimo in una sezione ellittica del vaso ) , lunghezza dellaaa , morfologia e lunghezza ed eventuale dislocazione longitudinale dellep , presenza di el e / o trombosi endoprotesica . 
eg migration was evaluated by comparison to the first postprocedural ct scan by measuring the length of the infrarenal aorta not covered by the stent - grael and endoluminal thrombosis were assessed by comparing the ct scans before and after contrast administration and by performing densitometric measurements in equivocal cases [ 35 ]  . 
in acquiring the data , we followed the standards proposed by the ad hoc committee for standardized reporting practices in vascular surgery of the society for vascular surgery / 1nternational society for cardiovascular surgery ( svs / iscs ) [ 36 ]  . 
changes in diameter or length of at least 5 mm relative to preprocedural values were considered significant , in part to minimise intraand interobserver differences in measurement , as suggested by svs / iscs [ 36 ]  . statistical analysis a log - rank test was used to measure the relationship between changes in aaa size relative to preprocedural size , considering all 85 patients divided into two groups on the basis of a 50 - mm cut - off transverse diameter of the aaa . 
la dislocazione dellep stata valutata rispetto alla prima tc post - procedurale , misurando la lunghezza della porzione aortica sottorenale non ricoperta dallo stent - gralel e la trombosi endoluminale sono stati valutati confrontando le scansioni tc pree post - mdc e mediante misurazioni densitometriche nei casi dubbi [ 35 ]  . nellacquisizione dei dati si sono seguiti gli standard proposti dal ad hoc committee for standardized reporting practices in vascular surgery of the society for vascular surgery / international society for cardiovascular surgery ( svs / iscs ) [ 36 ]  . si considerata significativa una variazione di diametro e / o lunghezza di almeno 5 mm rispetto ai valori preposizionamento , anche allo scopo di minimizzare le differenze di misurazione intraed inter - osservatore , come suggerito dalla svs / iscs [ 36 ]  . analisi statistica stata valutata , mediante log - rank test , la relazione fra le modificazioni dimensionali dellaaa rispetto alle dimensioni pre - procedurali , considerando tutti gli 85 pazienti suddivisi in 2 gruppi in base a un diametro trasverso soglia dellaaa di 50 m stata valutata , sempre mediante log - rank test , anche la relazione fra le modificazioni dimensionali dellaaa rispetto al tipo di ep impiegato , considerando un sottogruppo di 75 pazienti cui era stato impiantato uno dei 3 modelli di ep maggiormente utilizzati ( aneurx , talent ed excluder )  . 
statistical significance was set at p < 0.05. risultati table 4 available follow - up data follow - up patients repair 1 month 3 months 6 months 1 year 2 years 3 years 4 years 5 years 6 years 7 years follow - up procedura 1 mese 3 mesi 6 mesi 1 anno 2 anni 3 anni 4 anni 5 anni 6 anni 7 anni le caratteristiche morfodimensionali preprocedurali degli aaa trattati sono presentate in tabella 3 . 
la durata media della procedura in sala operatoria stata di circa 120 minuti , con un tempo medio di scopia di 15 , 41 , 4 min ( range 558 min )  . 
la durata media del follow - up risultata di 34 , 459 , 3 mesi ( range 189 mesi )  . modificazioni morfodimensionali del colletto prossimale si verificato un aumento significativo ( > 5 mm ) del diametro del colletto prossimale in 2 pazienti ( 2 , 4% ) e un aumento ai limiti della significativit ( 5 mm ) in altri 4 pazienti ( 4 , 7% ) , di cui 2 con discesa e angolazione dellep e un caso con rottura dello stent prossimale ( vanguard )  . 
non vi sono state variazioni significative ( > 5 mm ) della lunghezza del colletto prossimale nei controlli tc effettuati . un aumento significativo ( > 5 mm ) del diametro trasverso massimo ( mtr ) dellaaa si verificato in 7 pazienti ( 8 , 2% ) nel corso del follow - up ( range 718 mm ) , ed era sempre associato ad aumento significativo ( > 5 mm ) anche del diametro trasverso ap e / o ll dellaaa . 
the mean duration of the repair procedure was 120 min , with a mean fluoroscopy time of 15.41.4 ( range 558 ) mavailability of follow - up data at the time of the analysis ( 31 october 2004 ) is shown in table 4 . 
mean duration of follow - up was 34.459.3 ( range 189 ) months . changes in size and shape of the proximal neck a significant increase ( > 5 mm ) in proximal neck diameter was seen in two patients ( 2.4% ) and an almost significant increase ( 5 mm ) in another four ( 4.7% ) , two of whom had caudal migration and kinking of the eg and one with disintegration of the proximal stent ( vanguard )  . 
 changes in shape and size of the aneurysm sac a significant increase ( > 5 mm ) in the maximum transverse ( mtr ) diameter of the aaa was observed during follow - up in seven patients ( 8.2% ) ( range 718 mm ) , and it was constantly associated with a significant increase ( > 5 mm ) in the ap and / or ll transverse diameter of the aaa . 
the significant reduction in one of the three transverse diameters of the aaa was associated with absence of el in 25 cases and presence of type - 2 el in four cases . 
la velocit media di riduzione della lunghezza dellaaa fu di 22 , 5 mm / anno ( range 2 , 160 , 0 mm / anno )  . modificazioni morfologiche e dislocazioni dellendoprotesi modificazioni morfologiche dellep , da minime a rilevanti , sono state osservate in 47 pazienti ( 55 , 3% ) , di cui 24 in cui la modificazione morfologica era gi presente entro il 1 mese dallimpianto dellep ( comprese 5 virgin position )  . le modificazioni morfologiche potevano interessare il corpo principale ( 15 casi ) , una o entrambe le branche iliache ( 23 casi ) o tutte le componenti dellep ( 9 casi )  . 
alterazioni morfologiche dellasse longitudinale dellep ( concavit , convessit , angolazioni , rettilineizzazioni ) erano presenti in 37 casi ( 43 , 5% ) , mentre alterazioni morfologiche dellasse trasversale ( restringimenti , allargamenti ) erano presenti , invece , in 10 casi ( 11 , 8% )  . 
le alterazioni morfologiche risultarono associate in 12 casi ( 14 , 1% ) a trombosi endoprotesica di grado variabile ( da minima apposizione trombotica endoluminale fino allocclusione di branca ) e in 15 casi ( 17 , 6% ) ad el ( 2 el di tipo i , 10 el di tipo ii e 3 el di tipo iii )  . 
in 4 casi ( 4 , 7% ) di alterazione morfologica dellep si rese necessaria la conversione laparotomica : 2 casi per el di tipo ia , 1 caso per el di tipo iii da rottura dello stent pi craniale ( vanguard ) e 1 caso per eccessiva angolazione delle componenti protesiche ( anaconda )  . 
le valutazioni della lunghezza dellep sono state escluse dallo studio , essendo risultate le misure assai variabili nei controlli tc e , pertanto , giudicate non sufficientemente attendibili . si sono verificati 13 casi ( 15 , 3% ) di dislocazione caudale significativa ( > 5 mm ) dellep rispetto al 1 controllo tc post - procedurale , dei quali 4 con associato allargamento del colletto prossimale aortico , e 6 con discesa dellep particolarmente marcata ( > 15 mm ) e conseguente angolazione pi o meno accentuata delle sue componenti ( 2 aneurx , 2 talent , 1 vanguard e 1 anaconda )  . 
evaluations of eg length were excluded from the study because ct measurements were extremely variable and therefore not considered sufficiently reliable . there were 13 cases ( 15.3% ) of significant downward migration ( > 5 mm ) of the eg relative to the first postprocedural ct study ; four of these were associated with enlarged proximal aortic neck and six with particularly marked downward slippage of the eg ( > 15 mm ) and resulting angulation of its components ( two aneurx , two talent , one vanguard and one anaconda )  . 
in addition , there was one case ( 1.2% ) of significant upward migration ( > 5 mm ) of the eg ( talent ) at 3 months , with resulting exclusion of the ostium of a main renal artery and reduction in renal perfusion . 
 endoleaks a total of 36 el occurred in 32 patients ( 37.6% ) , with four ( 4.7% ) having two el of a different type ( types 1 and 2 )  . 
in detail , there were six type - 1 el ( four primary and two secondary ; two proximal and four distal ) and 26 type - 2 el ( 16 primary and ten secondary ; 11 el resolved spontaneously , seven remained essentially unchanged and eight worsened ) due to retrograde flow from the inferior mesenteric artery ( ten cases ) , lumbar arteries ( seven cases ) , hypogastric arteries ( six cases ) or mixed origin ( three cases )  . 
inoltre vi furono 3 el di tipo iii ( 2 primari e 1 secondario ) e un caso di verosimile el di tipo iv con scomparsa spontanea in 12 giornata post - procedurale . trombosi endoprotesica trombosi endoprotesica di qualsiasi entit ( da minima a occludente ) stata riscontrata in 23 pazienti ( 27 , 1% ) : a livello del corpo dellep in 7 pazienti , a livello di una singola branca iliaca protesica in 11 pazienti , a livello di entrambe le branche iliache dellep in 2 pazienti , e a livello sia di corpo che di branca iliaca dellep in 3 pazienti . 
kaplan - meier curves for freedom from aaa growth > 5 mm in relation to the diameter of the aaa sac [ 85 patients with follow - up 1 month , grouped according to aaa diameter < 50 mm ( n = 49 ) or 50 mm ( n = 46 ) ]  . 
confronto delle curve di kaplan - meier per la libert da riduzione dellaaa > 5 mm in relazione al diametro massimo trasverso iniziale dellaaa ( 85 soggetti con follow - up di almeno 1 mese , ripartiti in 2 gruppi : gruppo 1 , n = 49 , aaa < 50 mm ; gruppo 2 , n = 46 , aaa ( cid : 2 ) 50 mm )  . tion in sample size , which prevents accurate assessment of morphological changes in the aaa - eg complex and the incidence of the long - term complications of endovascular repair . 
moreover , the suggested anatomical and morphological criteria for selecting candidates to eg implantation were not always applied because the risk - benefit analysis indicated a clear advantage of endovascular repair compared with the risk of surgical conversion . proximal neck despite six cases ( 7.1% ) of increased proximal neck diameter , none of which were associated with type - 1a el , in most cases , proximal - neck diameter ( 79 cases , 92.9% ) and length ( 85 cases , 100.0% ) remained essentially stable , in agreement with the literature [ 7 ] , the proximal neck is the transition between the extraaneurysmal aorta ( healthy ) and the aneurysmal aorta ( diseased ) and , despite there not being any significant reduction in radial forces after repair , it is subjected to the additional centrifugal force exerted by the eg itself . 
 aneurysm sac changes in shape and size of the aneurysm sac involved the three transverse diameters ( maximum , ap and ll ) and the length of the aaa even though the most frequently used parameter for evaluating morphological changes of the sac is the mtr , which corresponds to the minimal transverse diameter in an elliptical section of the vessel . 
changes in ap and / or ll diameter were always associated with changes in mtr , but not vice versa , a sign that mtr is a more sensitive index of variations in the size of the aneurysm sac . there were 36 cases ( 42.4% ) of significant mtr changes , metro trasverso minimo in una sezione ellittica del vaso . 
infatti unalterazione del diametro ap e / o ll dellaaa era sempre associata ad alterazione del mtr , ma non viceversa , segno , quindi , che il mtr rappresenta un indice pi sensibile di variazione dimensionale della sacca aneurismatica . si ebbero 36 casi ( 42 , 4% ) di modificazioni significative del mtr , 30 dei quali ( 83 , 3% ) entro i primi 30 mesi di follow - up , e 51 casi ( 60 , 0% ) di modificazioni significative della lunghezza dellaaa , 38 dei quali ( 74 , 5% ) entro i primi 30 mesi di follow - up , dati che sembrerebbero confermare , come affermato dalla letteratura , che le variazioni dimensionali dellaaa , per lo meno quelle in senso riduttivo , si osservano prevalentemente nei primi mesi di follow - up , per poi assestarsi in una sorta di plateau , perch i fenomeni di rimodellamento di un aaa completamente escluso avvengono in tempi relativamente brevi . il riscontro relativamente precoce di crescita significativa del mtr e / o della lunghezza dellaaa , invece , verosimilmente dovuto allassociazione con el ( 100 , 0% dei casi di aumento del mtr e 80 , 0% dei casi di aumentata lunghezza dellaaa ) , il quale el si evidenzi piuttosto precocemente ( 100 , 0% degli el di tipo ii e iv , 71 , 4% degli el di tipo i e 66 , 7% degli el di tipo iii comparvero entro i primi 30 mesi )  . da questi dati si deduce che la maggior parte dei pazienti trattati ha presentato una riduzione del volume totale dellaaa ( anche se tale parametro non stato direttamente calcolato ) , in virt della diminuzione dei diametri trasversi e / o della lunghezza dellaaa stesso e , quindi , una diminuzione del rischio di rottura . 
tale progressiva riduzione volumetrica dellaaa spiegabile con il rimodellamento dello stesso alle nuove condizioni createsi dopo la sua esclusione endoluminale , che hanno portato ad una riduzione della pressione endoluminale , e quindi della forza radiale esercitata sulle pareti della sacca stessa . la stragrande maggioranza degli aaa in crescita significativa ( 11 / 13 , 84 , 6% ) era associata a persistenza di flusso c . 
 the relatively early finding of a significant increase in mtr and / or aaa length is , instead , probably due to the associated el ( 100.0% of cases of increased mtr and 80.0% of cases of increased aaa length ) , which developed relatively early ( 100.0% of types 2 and 4 , 71.4% of type 1 and 66.7% of type 3 el developed within 30 months )  . it can be inferred that the majority of patients experienced a reduction in the total volume of the aaa ( even though this parameter was not calculated directly ) given that the transverse diameters and / or length of the aaa were reduced , and therefore a lower risk of rupture . 
this progressive decrease in aaa volume can be explained by the aneurysm remodelling to adapt to the new conditions resulting from its exclusion , which led to reduced endoluminal pressure and radial pressure on the sac walls . the vast majority of aaa that enlarged significantly ( 11 / 13 , 84.6% ) were associated with persistent perigraft flow after eg implantation . 
the only two cases ( 2 / 13 , 15.4% ) without el were significant increases in length ( 1 and 4 months postprocedure , respectively ) , both of which were followed by equally significant shortening . 
even these cases could be accounted for by transient persistent pressurisation of the aaa during the early phases of follow - up , which was related to the transmission of pressure from the eg to the mural thrombus and aortic walls . 
these data seem to confirm the presence of el as the main risk factor for significant aaa growth even though only type - 1 and type - 3 el have been shown to be associated with aaa growth [ 3742 ]  . 
only types 1 , 2 and 3 were associated with significant aaa growth ; the only case of type - 4 el had disappeared at ct follow - up on day 12 , so its relatively short life did not allow any significant increase in aaa size . 
the controversial role of type - 2 el is discussed below . endograft over a period of 7 years , seven different types of eg were implanted , with heterogeneous distribution : some of them are no longer available ( vanguard , endologix , anaconda , passager ) , and others have undergone major redesign over the years ( talent )  . 
for this reason and because our experience with other models was too limited , our evaluation of the morphological and functional changes of the aaa in relation to type of eg was based on the three most commonly used models ( aneurx , talent , excluder )  . sanguigno periprotesico dopo limpianto di ep e gli unici 2 casi ( 2 / 13 , 15 , 4% ) osservati in assenza di el erano precoci aumenti significativi di lunghezza ( rispettivamente ad 1 e a 4 mesi dallimpianto dellep ) , seguiti , poi , entrambi da accorciamento altrettanto significativo . 
anche tali casi potrebbero essere spiegati con una transitoria persistente pressurizzazione dellaaa nelle prime fasi di follow - up , legata a trasmissione di pressione dallep al trombo murale e , quindi , sulle pareti aortiche . 
questi dati confermerebbero , pertanto , la presenza di endoleak quale fattore di rischio principale per la crescita significativa dellaaa , anche se dalla letteratura risulta che soltanto gli el di tipo i e iii sono statisticamente associati a crescita dellaaa [ 3742 ]  . 
soltanto i tipi i , ii e iii di el risultarono associati a crescita significativa dellaaa , poich lunico caso osservato di el di tipo iv scomparve al controllo tc in 12a giornata e quindi la sua durata relativamente breve non permise un significativo aumento delle dimensioni dellaaa . 
il ruolo controverso degli el di tipo ii , invece , discusso pi avanti . endoprotesi nellarco di 7 anni sono stati impiantati ben 7 tipi differenti di endoprotesi , con una distribuzione non omogenea : alcuni di essi sono stati ormai ritirati dal commercio ( vanguard , endologix , anaconda , passager ) , altri hanno subito delle modificazioni importanti nel corso degli anni ( talent )  . 
tutto ci riflette la continua evoluzione tecnica dei device disponibili e rappresenta un grosso problema nella valutazione dei risultati poich , ovviamente , rende assai difficili i confronti tra pazienti portatori di ep diverse . 
per questo nella valutazione delle modificazioni morfofunzionali degli aaa in rapporto al tipo di ep abbiamo considerato soltanto i 3 modelli da noi maggiormente utilizzati ( aneurx , talent , excluder ) in quanto le nostre esperienze con gli altri modelli erano numericamente troppo esigue . quanto alle modificazioni morfologiche dellep , in 5 pazienti ( 5 , 9% ) stato adottato volontariamente un posizionamento dellep con incrocio delle branche iliache ( virgin position ) allo scopo di preservare la perviet di una o entrambe le arterie ipogastriche . 
infatti , nonostante la grande disponibilit di misure diverse per ciascun tipo di ep , vi sono certe situazioni ( origine precoce di una o entrambe le arterie ipogastriche , marcata tortuosit di uno o entrambi gli assi iliaci ) in cui la scelta del tipo di ep risulta difficile e non priva di rischi . 
pertanto , in mancanza di misure adeguate dellep , allo scopo di ridurne la lunghezza sullasse longitudinale , pu venire eseguita tale procedura , che sfrutta la rotazione di 180 sul piano trasversale delle branche iliache allinterno della sacca aneurismatica per far atterrare lestremo distale delle branche iliache stesse a monte dellemergenza delle arterie ipogastriche . oltre a queste modificazioni morfologiche indotte , ci sono tutta una serie di altre alterazioni della morfologia dellep che possono influenzare negativamente la funzione del device , quali concavit , convessit , angolazioni , restringimenti , etc . 
despite the wide availability of different sizes for each eg model , there are some situations ( early origin of one or both hypogastric arteries , marked tortuosity of one or both iliac axes ) where the choice of eg is difficult and not free of risks . 
as a result , when adequate eg sizes are lacking , eg length can be reduced by applying this procedure , which exploits the 180 transverse rotation of the iliac branches inside the aneurysm sac to place the distal end of the iliac branches above the emergence of the hypogastric arteries . in addition to these induced morphological changes , there are changes in eg morphology that may negatively affect device function , such as concavity , convexity , kinking , narrowing , etc . 
according to virchows triad , one factors responsible for thrombosis is slow flow , which arises when the flow changes from laminar to turbulent , as occurs at bifurcations or just below vessel strictures . 
in one of our cases , there was complete thrombosis of an iliac branch of the eg , ascribable to inadequate expansion 1 month after implantation , which was successfully treated by percutaneous transluminal angioplasty ( pta )  . 
most morphological changes observed were , however , minimal and not associated with endoluminal thrombosis . even the el , and especially graft - related el ( types 1 and 3 ) , might have been promoted by a preexisting morphological change in the eg extremities or at the junction between the main body and the contralateral iliac branch in modular models : the first situation facilitates detachment of the eg from the vessel walls ; the second may lead to separation of the graft components . 
in particular , there was one case of proximal stent disintegration ( vanguard ) 51 months after implantation in a patient who had never shown changes in graft morphology or significant variations in the aaa or the proximal neck ; loss of adherence between the eg and the aorta wall resulted in a massive type - 3 el and the patient had to undergo late surgical conversion . it is interesting to note that some morphological changes of the eg could also have affected the adjacent vessel segments not covered by the graft , leading to sharp angulations or kinking . 
although not directly addressed in our study in part because of lack of adequate 3d ct reconstructions [ maximum intensity projection ( mip ) and volume rendering technique ( vrt ) ] for some patients this phenomenon occurred in at least three cases ( 3.5% ) and could have been due to remodelling not only of the aaa but also of the uncovla trombosi endoprotesica poteva essere favorita dallalterazione morfologica quando questa comportava una significativa alterazione del flusso endoprotesico , tale da favorire il deposito di materiale trombotico sulle pareti interne dellep , come in caso di angolazioni e / o restringimenti del calibro luminale . 
secondo la triade di virchow , infatti , uno dei fattori responsabili della trombosi rappresentato dal rallentamento del flusso , che si verifica laddove il regime di flusso , normalmente di tipo laminare , diventa di tipo vorticoso , come in corrispondenza delle biforcazioni o subito a valle dei restringimenti del calibro vasale . 
in un caso , poi , si verific la trombosi completa di una branca iliaca protesica , attribuibile alla scarsa espansione della stessa ad 1 mese dallimpianto , trattata poi con successo mediante pta . 
la maggior parte delle alterazioni morfologiche osservate , tuttavia , era di minima entit e non si associava a trombosi endoluminale . anche gli endoleak , specie i cosiddetti el graft - correlati ( tipi i e iii ) , potevano essere favoriti da una preesistente alterazione della morfologia dellep , quando questa interessava uno degli estremi dellep , oppure il sito di embricazione fra corpo principale e branca iliaca controlaterale nei modelli modulari , perch favorisce il distacco dellep dalle pareti vasali nel primo caso , o dei singoli elementi protesici nel secondo . 
in particolare si verific un caso di rottura dello stent prossimale dellep ( vanguard ) a 51 mesi dallimpianto in una paziente che non aveva mai mostrato prima alterazioni della morfologia del graft , n variazioni significative dellaaa o del colletto prossimale ; la perdita di ancoraggio fra lep e la parete aortica determin , di conseguenza , un el di tipo iii massivo e la paziente fu pertanto sottoposta a conversione chirurgica tardiva . interessante notare che talune alterazioni morfologiche dellep potevano talvolta ripercuotersi anche nei segmenti vasali adiacenti non ricoperti dal graft determinando brusche angolazioni o kinking vasali a stretto raggio di curvatura . 
tale fenomeno , pur non essendo stato affrontato direttamente nello studio , anche per la mancata disponibilit di adeguate ricostruzioni tc tridimensionali ( soprattutto mip e vrt ) per alcuni pazienti , si verific in almeno 3 casi ( 3 , 5% ) e potrebbe essere spiegato con il rimodellamento non solo dellaaa , ma anche dei segmenti aortici e iliaci non ricoperti adiacenti allep e sottoposti a brusche forze longitudinali e / o torsionali da parte del graft . la presenza di importanti alterazioni morfologiche dellep stata considerata fattore di indicazione sufficiente per la conversione laparotomica , come in un caso ( 1 , 2% ) di espianto dellep ( anaconda ) a 17 mesi di follow - up . 
un altro caso simile , con severa angolazione ( < 90 ) a livello del corpo principale dellep , tuttora sotto stretto follow - up . riteniamo che i casi di marcata alterazione morfologica dellep debbano essere tenuti sotto stretta osservazione , in quanto a rischio di rottura , anche se lunico caso riportato di rottura dellep non aveva mai presentato prima alterazioni della morfologia del graft . la valutazione delle alterazioni di lunghezza dellep non stata presa in considerazione a causa dellalta variabilit delle misure , legata sia alla variabilit intraed inter - osservatore , che alla non disponibilit di adeguate ricostruzioni tc tridimensionali per alcuni pazienti . interessanti , infine , sono le dislocazioni longitudinali c . 
we believe that cases of marked eg morphological change should be closely monitored due to the risk of rupture even if the only case of eg failure never showed alterations in graft morphology . changes in eg length were not evaluated due to the wide variation in measurements related both to intraand interobserver variability and to the lack of adequate 3d ct reconstructions for some patients . finally , longitudinal migrations of the eg were interesting . 
on release , the eg is usually positioned with the covered proximal portion just beneath the ostium of the more caudad renal artery so as to cover almost completely the infrarenal neck of the aaa and exploit the entire length available for eg fixation . 
however , even though eg deployment was technically correct , 13 cases ( 15.3% ) showed significant caudal migration ( > 5 mm ) , with marked downward slippage ( > 15 mm ) of the stent - graft in six ( 46.2% ) during follow - up . 
increased length of the infrarenal aorta not covered by the eg could be related to the physiological lengthening of the aortic walls as a result of progression of atherosclerotic disease . 
by increasingly weakening vessel walls , atherosclerosis induces both a lengthening and widening of the vessel whereas in most patients , the increase in length of this uncovered segment of the aorta was not matched by an increase in diameter , meaning that the lengthening was only apparent and most probably due to distal migration of the eg . 
therefore , an isolated lengthening of the uncovered infrarenal portion of the aorta can be considered an indirect sign of caudal migration of the eg and should be carefully assessed in relation to the length measured at early postprocedural ct follow - up , which becomes crucial . the six cases ( 7.1% ) of marked downward slippage ( > 15 mm ) of the eg were associated with more or less marked angulations of the device due to loss of proximal fixation and resulting distal migration ; in the presence of stable distal fixation , this caudal migration led to shortening and kinking inside the aneurysm sac with possible haemodynamic consequences , especially at the level of the iliac branches , and eg thrombosis ( three cases )  . the only case of significant upward migration ( > 5 mm ) of the eg at 3 months led to occlusion of the origin of one of the renal arteries . 
generalmente , al momento del rilascio , lep viene collocata posizionandone la parte prossimale ricoperta subito al di sotto dellostio dellarteria renale pi caudale , in modo da ricoprire pressoch completamente il colletto sottorenale dellaaa , allo scopo di sfruttare tutta la lunghezza disponibile per il fissaggio dellep . 
tuttavia , anche se il posizionamento endoprotesico stato tecnicamente corretto , in 13 casi ( 15 , 3% ) si verificata una migrazione caudale significativa ( > 5 mm ) dellep con marcata discesa ( > 15 mm ) dello stent - graft in 6 di essi ( 46 , 2% ) nel corso del follow - up . laumentata lunghezza della porzione aortica sottorenale non ricoperta dallep potrebbe essere legata al fisiologico allungamento delle pareti vasali aortiche in rapporto alla naturale progressione della malattia aterosclerotica . 
generalmente il processo aterosclerotico , determinando un progressivo sfiancamento delle pareti vasali , provoca sia un allungamento che un allargamento del calibro del vaso , mentre nella maggior parte dei pazienti allaumento di lunghezza di tale segmento aortico non ricoperto non corrispondeva un altrettanto aumento del suo diametro , segno che lallungamento , solo apparente , era dovuto , pi probabilmente , a migrazione distale dellep . 
pertanto , un allungamento isolato della porzione aortica sottorenale non ricoperta pu essere considerato un segno indiretto di dislocazione caudale dellep e , quindi , deve essere valutato attentamente comparandolo sempre con il valore al precoce controllo tc postimpianto , che assume cos unimportanza cruciale . nei 6 casi ( 7 , 1% ) di marcata discesa ( > 15 mm ) dellep si associarono , poi , angolazioni pi o meno accentuate del device , dovute a perdita dellancoraggio prossimale del graft , con conseguente migrazione distale dellep ; in presenza di un ancoraggio distale stabile , tale dislocazione caudale dellep determinava un accorciamento della sua lunghezza sullasse longitudinale e , pertanto , la formazione di kinking allinterno della sacca aneurismatica con possibili conseguenze emodinamiche per alterazione dei regimi di flusso , specialmente a livello delle branche iliache , con possibile trombosi endoprotesica ( 3 casi )  . lunico caso di risalita significativa ( > 5 mm ) dellep a 3 mesi dallimpianto ha portato a occlusione dellorigine di unarteria renale . 
tale fenomeno , assai inconsueto per la pi facile possibilit di migrazione distale del device a causa della direzione centrifuga del flusso ematico , potrebbe , tuttavia , essere spiegato con un ancoraggio distale stabile dellep subito a monte delle biforcazioni iliache , per cui il riequilibrio delle forze in gioco avrebbe potuto determinare una spinta risultante verso lalto e , quindi , la risalita dellep stessa . trombosi endoprotesica la trombosi endoprotesica ( 27 , 1% dei pazienti trattati ) complicanza abbastanza frequente nel trattamento endovascolare degli aaa . 
a livello delle branche iliache , essendo minore il calibro e per fattori emodinamici , pu portare a ostruzione completa e , quindi , a necessit di ulteriori procedure di ricanalizzazione , che comportano un rischio aggiuntivo per il paziente e non sempre risultano efficaci ( 4 casi di occlusione trombotica , 2 dei quali esitati in conversione chirurgica )  . 
interessante notare che la trombosi endoluminale si rese evidente alla tc entro i primi 2 anni dallimpianto in 18 / 23 pazienti ( 78 , 3% ) ed entro i primi 3 anni dallimc . 
at the level of the iliac branches , due to the smaller calibre and to haemodynamic factors , it may result in complete occlusion and in the need for further recanalisation procedures that , as well as being an additional risk , are not always successful ( four cases of thrombotic occlusion , two of which requiring surgical conversion )  . 
it is interesting to note that the thromboses became manifest at ct within the first 2 years of implantation in 18 / 23 patients ( 78.3% ) and within the first 3 years in 22 / 23 patients ( 95.7% ) and that the four cases of complete iliac branch occlusion all occurred within the first year of implantation . 
these data underline the importance of a close follow - up of patients treated with eg , above all during the first year of follow - up , even if a certain degree of risk of eg thrombosis remains . 
whereas it is accepted that types 1 and 3 require prompt treatment since they are associated with significant aaa growth and increased risk of rupture , the management of type - 2 el is more challenging , partly because they tend to resolve spontaneously . 
this would seem to indicate that early appearance is predictive of spontaneous el thrombosis and that the spontaneous closure is rare after more than 12 months , as has been reported in the literature [ 38 ]  . 
as regards the decision whether or not to treat type - 2 el , treatment is only indicated in the presence of aaa growth whereas a watchful , waiting approach is indicated in all other cases [ 3942 ]  . 
the presence of type - 2 el may be associated with stability or even reduction in aaa volume : we had 20 cases ( 76.9% ) of mtr stability and two ( 7.7% ) of significant mtr reduction ( > 5 mm ) despite the presence of type - 2 el , which confirms that the association pianto in 22 / 23 pazienti ( 95 , 7% ) , e che i 4 casi di trombosi completa di branca iliaca si verificarono tutti entro i primi 12 mesi dallimpianto . 
questi dati sottolineano , pertanto , limportanza di una stretta sorveglianza dei pazienti trattati con ep , specie nel 1 anno di follow - up , anche se , tuttavia , un certo rischio di trombosi endoluminale persiste sempre , essendosi verificata la comparsa di sottile apposizione trombotica endoprotesica in un paziente a 45 mesi di follow - up . endoleak la complicanza pi frequente ( 37 , 6% ) osservata dopo trattamento endovascolare di aaa infrarenale costituita dallendoleak ( incidenza media variabile in letteratura compresa fra 2 , 4% e 45 , 5% a seconda delle varie casistiche ) [ 9 , 37 ] , che stato definito come la comparsa di iperdensit nel materiale trombotico periprotesico alla tc in fase arteriosa e / o tardiva , espressione di persistente flusso sanguigno allinterno dellaaa , ma allesterno dellep . gli el primari ( entro 30 giorni dallimpianto ) di tipo i furono 4 / 6 ( 66 , 7% ) , il 75% dei quali scomparve dopo pta ( 2 casi ) o posizionamento di unestensione iliaca ( 1 caso )  . 
si ebbero , invece , soltanto 2 / 6 casi ( 33 , 3% ) di el secondari ( oltre 30 giorni dallimpianto ) di tipo i , confermando la necessit di uno stretto follow - up dei pazienti trattati , specie nel 1 mese dopo il posizionamento dellep , poich i difetti di adesione delle estremit dello stent - graft alle pareti vasali sono precoci a comparire . gli el di tipo ii furono i pi frequenti ( 26 / 36 el , 72 , 2% ) e il loro significato clinico rimane tuttora controverso [ 37 ]  . 
infatti , mentre un dato consolidato che gli el di tipo i e iii richiedono un trattamento obbligatorio e tempestivo in quanto associati ad un significativo aumento delle dimensioni dellaaa e , quindi , ad aumentato rischio di rottura , quelli di tipo ii costituiscono un problema di pi difficile gestione , anche perch spesso evolvono nella risoluzione spontanea . 
gli 11 casi ( 42 , 3% ) di risoluzione spontanea osservati si verificarono in un intervallo di tempo compreso fra 3 giorni e 7 mesi dalla comparsa dellel ed erano tutti el primari , ossia a comparsa precoce dallimpianto dellep . 
questo dato , perci , sembrerebbe indicare che un fattore predittivo per la trombosi spontanea dellel potrebbe essere la sua comparsa precoce nel post - impianto , e che la chiusura spontanea di un el rara dopo pi di 12 mesi dalla sua persistenza , in accordo con quanto gi descritto in letteratura [ 38 ]  . 
quanto al problema dellopportunit o meno del trattamento degli el di tipo ii , esso viene invocato solamente in caso di crescita dimensionale dellaaa , riservando un atteggiamento di sorveglianza negli altri casi [ 3942 ]  . 
noto che la presenza di el di tipo ii possa essere associata a stabilit o addirittura a riduzione volumetrica dellaaa : si verificarono , infatti , 20 casi ( 76 , 9% ) di stabilit e 2 casi ( 7 , 7% ) di riduzione significativa ( > 5 mm ) del mtr pur in presenza di el di tipo ii , a conferma che lassociazione fra crescita dellaaa e presenza di el non sempre valida per gli el di tipo ii . 
pertanto , di fronte ad un el di tipo ii , sembra essere giustificato un atteggiamento attendistico , a meno che non si associ un incremento significativo delle dimensioni dellaaa . vi furono 3 / 36 casi ( 8 , 3% ) di el di tipo iii , di cui 2 primari alla giunzione corpo - branca iliaca protesica , e 1 secondario dovuto alla rottura dello stent pi craniale a 51 c . 
consequently , in the presence of a type - 2 el , a watchful , waiting approach appears to be justified unless there is significant growth of the aaa . there were 3 / 36 cases ( 8.3% ) of type - 3 el . 
of these , two were primary , at the junction between body and iliac branch , and one was secondary due to failure of the cranial stent after 51 months ; the latter required surgical conversion and occurred with an eg model ( vanguard ) that is no longer on the market , a sign of the constant evolution of materials and devices . the only case of likely type - 4 el seen at postprocedure ct and no longer present on day 12 was probably related to graft - wall porosity ( talent ) and did not require treatment . there were no demonstrated cases of type - 5 el or endotension . 
mtr is therefore an important predictor of aaa growth , and aaa with a diameter < 50 mm are those that respond better to endovascular repair , in agreement with previous reports [ 4345 ]  . 
in fact , the three types of eg considered in the statistical analysis ( aneurx , talent and excluder ) all have a nitinol skeleton , and the talent and aneurx models , which showed a strongly significant difference in aaa shrinkage ( p = 0.0004 ) , are both covered with a dacron membrane . 
therefore , to understand such differences in aaa behaviour during follow - up , the structure of the stent - graft might be more important than the covering material . mesi di follow - up , esitato in conversione chirurgica , e associato a un modello di ep ( vanguard ) gi ritirato dal commercio , a conferma della continua evoluzione tecnica dei materiali e dei modelli di device disponibili . lunico caso di verosimile el di tipo iv osservato al controllo post - procedurale e scomparso in 12a giornata postimpianto era probabilmente legato a porosit del tessuto protesico dellep ( talent ) e non richiese alcun tipo di trattamento . 
non ci fu nessun caso dimostrabile di el di tipo v o endotension , ma si segnala un paziente portatore di un piccolo el di tipo ii da rifornimento retrogrado attraverso unarteria ipogastrica , trattato con successo mediante posizionamento di unestensione iliaca omolaterale a 12 mesi dallimpianto , che tuttavia present un ulteriore lieve incremento del mtr dellaaa ai limiti della significativit ( 5 mm ) nei controlli tc successivi , e che potrebbe essere spiegabile o con una persistenza dellel precedente ma con flusso assai ridotto ( inferiore alla soglia di sensibilit della tc ) , oppure con una pressurizzazione persistente della sacca aneurismatica ( endotension )  . analisi statistica dallanalisi statistica effettuata mediante log - rank test risultato , innanzitutto , che la crescita dimensionale dellaaa significativamente correlata con le sue dimensioni pre - procedurali ( p = 0 , 002 ) , ma non con il tipo di ep impiegato ( p = 0 , 97 )  . 
pertanto il mtr rappresenta un importante fattore predittivo per la crescita dellaaa , e gli aaa che meglio rispondono al trattamento endovascolare sono risultati quelli con diametro inferiore a 50 mm , in accordo con altri studi effettuati in letteratura [ 4345 ]  . 
resta aperta , tuttavia , la questione se trattare o meno aaa di diametro inferiore a 50 mm , poich attualmente il diametro limite per il trattamento chirurgico tradizionale degli aaa infrarenali varia proprio tra 5 , 0 e 5 , 5 cm [ 10 , 4648 ]  . lo shrinkage , invece , non sembra correlato con il diametro iniziale dellaaa ( p = 0 , 85 ) , mentre risulta significativamente correlato al tipo di stent - graft impiantato ( p = 0 , 0005 ) , in accordo con quanto descritto in letteratura , secondo cui , appunto , la riduzione volumetrica dellaaa maggiore con certi modelli di ep , in rapporto al tipo di materiale di rivestimento ( dacron o ptfe ) [ 1921 ]  . 
in realt , i 3 tipi di ep su cui stata effettuata lanalisi statistica ( aneurx , talent ed excluder ) , possiedono tutti uno scheletro in nitinol , e i modelli talent ed aneurx , fra i quali esiste una differenza marcatamente significativa di contrazione volumetrica dellaaa ( p = 0 , 0004 ) , sono entrambi rivestiti da una membrana in dacron . 
pertanto , per giustificare una tale differenza di comportamento dellaaa nel follow - up , potrebbe essere importante non tanto il materiale della membrana di rivestimento , quanto piuttosto la sua struttura , anche se questa soltanto unipotesi . conclusions despite its limitations , this study allows us to draw at least two conclusions : conclusioni da questo studio , pur con tutte le sue limitazioni , possiamo tuttavia tentare di trarre almeno 2 conclusioni : laaa e lep non sono 2 componenti separati , ma sono 2 elementi strettamente associati dal punto di vista spaziale e c . 
ct angiography is the ideal modality for the follow - up of patients after endovascular positioning of an aortobiiliac eg , as it allows accurate assessment of all morphological and functional parameters of the aaa - ep complex : the first postprocedure , ct examination , is fundamental , as it provides important quantitative data ( in particular , mtr and length of the proximal neck and aaa ) , which will serve as a reference standard for all subsequent follow - up examinations . interdipendenti da quello funzionale , e sono soggetti ad un processo di rimodellamento nel tempo anche molto significativo ; tale rimodellamento , statisticamente correlato al diametro dellaaa e al tipo di ep , interessa tanto laaa ( alterazioni dimensionali del colletto e della sacca ) quanto lep ( modificazioni di forma e dimensioni , migrazioni , rotture ) , e si pu accompagnare a conseguenze funzionali importanti ( el , trombosi endoprotesica ) che possono comportare la necessit di ulteriori procedure endovascolari e / o chirurgiche fino alla conversione laparotomica [ 49 ]  . a causa di questo processo di rimodellamento del complesso aaa - ep , e delle sue conseguenze funzionali , si rende necessario il rispetto di un rigoroso follow - up nei pazienti trattati , nel breve ma anche nel lungo termine . 
infatti alcune complicanze ( el , trombosi endoluminale ) sono pi frequenti nei primi tempi dallimpianto dellep , specie nel 1 anno , per cui questo richiede un follow - up particolarmente stretto in tale fase delicata ; tuttavia altre complicanze possono manifestarsi anche a distanza di diversi anni dallimpianto dellep , tanto da poter richiedere una conversione chirurgica tardiva . 
piga1 1medicina nucleare , policlinico universitario , universit degli studi , cagliari , italy 2unit operativa territoriale di nefrologia e dialisi , azienda usl 8 , cagliari , italy 3chirurgia generale , policlinico universitario , universit degli studi , cagliari , italy correspondence to : m . 
sixteen patients ( 11 women and five men ; age range 3580 years ) with severe hyperparathyroidism ( hp ) ( ten php , six shp ) were studied by ultrasound ( us ) , and , after i.v. 
double phase parathyroid scintigraphy identified 14 probable parathyroid glands , spect 23 ( 14 ectopic and nine eutopic ) and spect / ct confirmed all 23 probable parathyroid lesions , offering more precise localisation and an evident improvement in diagnostic accuracy . 
we believe 99mtc - sestambi spect / ct to be a more reliable presurgical method to study a patient subgroup affected by php or shp in whom conventional us and other scintigraphic methods have failed for intrinsic reasons due to the concomitant presence of multinodular goitre or ectopic parathyroid gland . 
in fact , anatomical information provided by ct enables precise localisation of the functional abnormalities highlighted by spect , and both are essential to a correct surgical approach . key words parathyroid scintigraphy spect / ct hyperparathyroidism riassunto obiettivo . 
sono stati sottoposti ad indagine spect / tc per lidentificazione e localizzazione prechirurgica delle paratiroidi 16 pazienti , 11 femmine e 5 maschi det compresa tra i 35 e 80 anni , affetti da iperparatiroidismo . 
nel raffronto tra accuratezza diagnostica delle spect e spect / tc la prima metodologia stata in grado di localizzare correttamente il 61% ( 14 / 23 ) delle formazioni patologiche mentre la spect / tc ha localizzato con precisione tutte le 23 formazioni nodulari captanti il radiofarmaco . 
si ritiene che la spect / tc rappresenti un approccio prechirurgico essenziale in tutti i pazienti affetti da iperparatiroidismo , primitivo o secondario , quando le altre metodologie tradizionali , ecografia e scintigrafia planare non possano essere dirimenti per la contemporanea presenza di gozzo multinodulare o di paratiroidi ectopiche . 
la singola metodologia spect , infatti , pur essendo in grado di identificare la ghiandola responsabile del quadro clinico risulta essere meno accurata dellindagine spect / tc nella sua precisa localizzazione e quindi meno affidabile per il chirurgo che deve pianificare lintervento . parole chiave scintigrafia parotidea spect / ct iperparatiroidismo introduction introduzione the recent introduction of hybrid scanners [ 1 ] in the field of diagnostic imaging procedures has provided a marked improvement in diagnostic methodologies compared with those offered by the use of single , uncoupled devices [ 2 ]  . 
over the last few years , clinical use of positron emission tomography computed tomography ( pet - ct ) scanners [ 4 ] has increased significantly because of its important advantages compared with pet alone in the diagnostic / prognostic evaluation of a variety of cancers in determining their real extension , response to chemotherapy and / or radiotherapy and precise delineation of target volumes to be irradiated . 
their use could be even more frequent than pet - ct for the widespread diffusion of scanners utilising gamma - emitting radiopharmaceuticals , especially if the added value of the fusion procedure in assuring clinical improvement in the interpretation of functional imaging was known [ 6 ]  . 
the aim of our study was to define the role and diagnostic accuracy of spect / ct as a methodology of choice in presurgical imaging of patients with primary ( php ) and secondary hyperparathyroidism ( shp ) with undetectable , dubious or ectopic parathyroid glands according to the recent report by gayed et al . 
in patients affected by php alone [ 7 , 21 ]  . la recente introduzione in diagnostica per immagini delle apparecchiature ibride [ 1 ] ha permesso un notevole affinamento delle metodiche diagnostiche rispetto a quelle ottenibili dalle singole macchine disaccopiate [ 2 ]  . 
in particolare ha avuto grande incremento limpiego dei tomografi pet - tc [ 4 ] di grande impatto clinico sia nella valutazione diagnostico / prognostica delle neoplasie che nel loro follow - up oltre che per la precisa delineazione dei volumi bersaglio nellapprontamento dei piani di trattamento radioterapico . 
il loro utilizzo potrebbe essere ancora pi capillare rispetto alla pet - tc , in funzione della pi estesa presenza della diagnostica medico - nucleare utilizzante radionuclidi gamma emittenti , ove fosse chiaro il valore aggiunto che tale procedura pu garantire alla diagnostica funzionale [ 6 ]  . 
lo scopo del presente studio stato quello di definire il ruolo e laccuratezza diagnostica della spect / tc come metodologia di scelta nella diagnostica pre - intervento chirurgico di pazienti affetti da iperparatiroidismo ( ip ) primitivo ( ipp ) e secondario ( ips ) con sede della lesione paratiroidea non precisata , dubbia od ectopica secondo le tecniche convenzionali in considerazione di quanto recentemente dimostrato da altri aa nellipp [ 7 , 21 ]  . materiali e metodi materials and methods between october 2004 and october 2005 , 94 patients with hyperparathyroidism ( hp ) were studied in our department . sixteen ( 11 women and five men , age range 3580 years , mean age 51 ) were selected for the study . 
 i criteri dinclusione nello studio comprendevano i seguenti requisiti : indicazione clinica alla paratiroidectomia in ip con paratiroidi ad incerta identificazione ecografica per concomitante patologia tiroidea multinodulare o in ip con ecografia negativa per ricerca di paratiroide / i in presenza di tiroide indenne o in ip con paratiroide ectopica dimostrata alla scintigrafia paratiroidea planare . diagnostic protocol esami tradizionali e spect / tc preliminarily , all patients underwent thyroid / parathyroid cdus exploration and subsequent thyroid scintigraphy after i.v. 
the ct scanner is a third - generation system with a lowpower x - ray tube and linear detector array located on the opposite side of the gamma camera slip gantry . spect acquisition parameters matrix size was 128128 over a 360 arc with a 3 step , 25 - s duration per frame and a zoom factor of 1 . 
total acquisition time was approximately 25 min . ct acquisition parameters tutti i pazienti sono stati preliminarmente sottoposti ad eco - color doppler della regione del collo ed a scintigrafia tiroidea dopo somministrazione e.v. 
lacquisizione tomografica emissiva stata eseguita dopo la prima ora dalla somministrazione con successiva rilevazione tomografica trasmissiva . acquisizione spect lindagine stata effettuata mediante impiego del sistema ibrido infinia - hawkeye ( general electric medical system , milwaukee , wi ) comprendente una gamma camera a doppia testa ed uno scanner tc . 
nellacquisizione spect sono stati adottati i seguenti i parametri : matrice dacquisizione 128128 pixel , zoom 1 , con proiezioni ottenute su campionamento angolare di 3 su 360 per un totale di 120 immagini . 
la ricostruzione delle immagini stata fatta mediante luso dalgoritmi matematici ( retroproiezione filtrata ) con filtro hann ottenendo delle sezioni secondo i tre assi dello spazio assiale , sagittale e coronale . acquisizione tc slice step was 5 mm , slice time 14 s , voltage 140 kv and current 2.5 ma , which gives a smaller patient radiation dose than conventional ct ( < 3 mgy )  . 
forty translo scanner tc un sistema di terza generazione dotato di un tubo a raggi x di bassa energia e con detettore lineare situato in situazione opposta rispetto al gantry della gamma camera . 
nella tomografia trasmissiva sono stati utilizzati i seguenti parametri : dimensione della slice di 5 mm e slice / time di 14 secondi , valori di tensione di 140 kv e valori di cor1001 a . 
total acquisition time was approximately 10 min ( table 2 )  . methodological problems the coregistration of spect / ct , particularly of the neck - thorax regions , can produce artefacts caused by possible patient movement . 
this issue must be clear to both nuclear medicine physicians and surgeons to avoid or reduce the possibility of topographic errors in parathyroid localisation . the radiation exposure caused by the coregistered spect / ct was respectively 3.3 and 3 msv ( icrp 80 , 2000 ) [ 10 ]  . 
in three patients with mng , a large calcific nodule ( 40 mm ) , as well as other nodularities , was located in the upper right thyroid lobe ( patient 1 ) and in the other two ( patients 8 and 13 ) were probable hyperplastic parathyroids . double - phase planar parathyroid scintigraphy showed 14 probable hyperfunctioning parathyroid glands , 12 in eutopic and two in ectopic sites , but these anatomic localisations were not certatwenty - three probable hyperfunctioning parathyroids were shown by spect , 14 in ectopic and nine in normotopic sites . 
la considerazione che la spect / tc della regione cervico - toracica deve essere eseguita per localizzare con precisione le paratiroidi al fine esclusivo di un intervento chirurgico pone un ulteriore problema . 
il paziente , infatti , posizionato sul tavolo chirurgico in estensione del collo tale da poter falsare i rapporti anatomici della ghiandola rispetto ai piani circostanti , ed ossei in particolare , ove anche lindagine scintigrafica non venisse condotta , nei limiti del possibile , nella medesima postura . 
tale evidenza deve essere ben chiara sia al medico nucleare sia alloperatore per minimizzare possibili misinterpretazioni topografiche . la dose di esposizione del paziente dovuta alla coregistrazione dellindagine spect / tc rispettivamente di 3 , 3 e 3 msv ( icrp 80 , 2000 ) [ 10 ]  . 
stato attuato in ogni paziente il controllo intraoperatorio dei livelli ematici di paratormone intatto ( ipth ) ed stata eseguita lindagine istologica di tutte le paratiroidi ablate . risultati lecografia rilevava la presenza di gozzo multinodulare ( gmn ) in 12 / 16 pazienti e risultava negativa negli altri 4 . 
case 1 , who underwent thyroidectomy for parathyroid carcinoma inside a calcific cyst located in the right thyroid lobe and for a concomitant follicular carcinoma in the eutopiche e 2 ectopiche , ma con insufficienti informazioni circa la loro reale posizione anatomica . la spect evidenziava la presenza di 23 sospette paratiroidi iperfunzionanti di cui 14 in sede ectopica e 9 normotopica . 
di queste va segnalata la presenza ( caso n 1 ) di una vasta area diperfissazione right parathyroid carcinoma + left thyroid follicular carcinoma hyperplastic parathyroid hyperplastic parathyroids parathyroid adenoma parathyroid adenoma parathyroid adenoma parathyroid adenoma hyperplastic parathyroids parathyroid adenoma hyperplastic parathyroids hyperplastic parathyroid hyperplastic parathyroids hyperplastic parathyroid hyperplastic parathyroid hyperplastic parathyroids hyperplastic parathyroids 1003 a . 
worthy of note is the fact that in shp patients , another five parathyroid glands negative at all scintigraphic and cdus procedures were resected . these negative parathyroid glands were virtually identical in size to those positive to spect / ct and were all located in normotopic sites . 
after parathyroidectomy , the intraoperative ipth measurements confirmed normal values in all patients . the histological report of resected lesions identified 16 hyperplastic glands , five adenomatous glands , one parathyroid carcinoma and one thyroid carcinoma ( table 3 )  . 
da segnalare lesecuzione di tiroidectomia totale ( caso n 1 ) per gmn affetto da k paratiroideo interno a cisti calcifica del lobo destro e concomitante carcinoma tiroideo 1004 a . 
2a , b paziente femmina di 60 anni affetta da iperpartiroidsmo primitivo . la spect / tc evidenzia la presenza di unarea di iperaccumulo del sestamibi situata sul mediastino antero - superiore retrostante il manubrio sternale destro . 
in patients affected by shp , sensitivity was 43% for planar scintigraphy and 64% for both spect and spect / ct . nevertheless , in comparison between diagnostic accuracy of spect and spect / ct , the first method was able to localise correctly 61% ( 14 / 23 ) of pathological lesions while spect / ct localised all 23 sestamibi - positive lesions with extreme precision . 
however , in the other spect - positive lesions , subsequent spect / ct provided surgeons with imporfollicolare del lobo sva tuttavia precisato che nei casi di pazienti affetti da ipt secondario sono state identificate ed asportate al tavolo operatorio altre 5 paratiroidi non evidenziate dalla scintigrafia perch non captanti il radioindicatore . tali ghiandole avevano di norma una dimensione sovrapponibile a quelle circostanti evidenziate sia dalla spect che dalla spect / tc ed erano tutte situate in sedi eutopiche . 
il reperto istologico delle ghiandole ablate ha permesso di identificare 16 iperplasie paratiroidee , 5 adenomi paratiroidei oltre ai citati carcinoma paratiroideo interno alla cisti calcifica e carcinoma follicolare della tiroide nello stesso paziente ( tabella 3 )  . 
tutte le altre ghiandole asportate , scintigraficamente fredde , erano paratiroidi iperplasiche . nella forme di ipt primitivo la sensibilit della scintigrafia planare nel rilevare le formazioni patologiche stata del 57% mentre la sensibilit della spect e della spect / tc stata in entrambe del 100% . 
nel raffronto tra laccuratezza diagnostica delle spect e spect / tc la prima metodologia stata in grado di localizzare correttamente il 61% ( 14 / 23 ) delle formazioni patologiche fissanti il sestamibi mentre la spect / tc ha localizzato con precisione tutte le 23 formazioni nodulari captanti . 
comunque anche per le formazioni correttamente localizzate dalla sola spect lindagine con macchina ibrida stata in grado di fornire al chirurgo pi precisi e preziosi dettagli topografici facilitanti il compito ablativo . 
ha inoltre sicuramente mutato lapproccio chirurgico nei tre pazienti con localizzazione retrotracheale . discussione nel campo della diagnostica preoperatoria del paziente affetto da iperparatiroidismo del tutto evidente che la certez1005 a . 
3a , b a 63 - year - old woman with primary hyperparathyroidism ( php ) and multinodular goitre with a large calcified cyst on the right upper lobe of the thyroid gland . 
single - photon computed tomography / computed tomography ( spect / ct ) shows an extensive area of sestamibi uptake within the cyst , which was diagnosed at histology as a parathyroid carcinoma . 
3a , b paziente femmina di 63 anni , affetta da iperparatiroidismo primario e gozzo multinodulare con voluminosa cisti a pareti calcifiche sul polo superiore del lobo destro della tiroide . 
la spect / tc dimostra la presenza di una vasta area di fissazione del sestamibi allinterno della cisti che lesame istologico ha rilevato essere un carcinoma paratiroideo . tant additional information concerning the precise localisation of lesions , thereby simplifying resection . 
this method certainly changed the surgical approach in three patients with parathyroid glands located in the retrotracheal space . discussion in the preoperative diagnosis of hp , it is evident that precise localisation of the glands responsible for the clinical picture is fundamental both for surgeon and patient . 
prior to clinical introduction of hybrid devices , the most accurate method for identifying and localising parathyroid glands was represented by pinhole spect [ 11 , 12 ]  . the recent development of new devices with the ability to acquire functional and anatomical information in a single scheduled examination has provided diagnostic imaging with a new procedure . 
in a selected group of patients in whom for intrinsic methodological causes us cannot be the main point of reference , this value has been taken on by scintigraphic methodologies . cervical - thorax planar scintigraphy has been confirmed as a suboptimal technique , with positive results in only 61% of cases and an inability to identify posterior or small - size lesions in thyroids affected by mng . spect performed in the same regions improved sensitivity of planar scintigraphy , as it was able to identify the pres1006 za di localizzazione delle paratiroidi responsabili del quadro clinico rappresenta un elemento di assoluto interesse ed importanza sia per il chirurgo sia , in ultima analisi , per il paziente . 
infatti , i precisi correlati topografici della ghiandola da ablare , facilitando il compito del chirurgo , implicano tempi pi brevi dintervento con ricadute positive sia sul paziente sia sulla gestione complessiva delle risorse . 
fino allavvento delle nuove macchine ibride , la metodologia pi accurata nellidentificare e localizzare le paratiroidi stata quella legata allimpiego del pinhole spect [ 11 , 12 ]  . la attuale procedura di fusione di immagini sta ormai identificando un nuovo settore di imaging offrendo la possibilit di potenziare , sommando , le varie tecnologie . 
nella selezionata casistica presentata e caratterizzata da pazienti nei quali lecografia non poteva rappresentare per motivi intrinseci alla metodica il punto di riferimento diagnostico principale le indagini scintigrafiche hanno assunto tale valore . 
la scintigrafia planare della regione cervico - toracica si rilevata positiva nel 61% dei casi non riuscendo ad identificare formazioni patologiche posteriori o di modeste dimensioni in un contesto tiroideo di gmn . 
solo le immagini di fusione hanno correttamente permesso di localizzare , in maniera inattesa , al suo interno la presenza della paratiroide responsabile del quadro clinico di iperparatiroidismo , peraltro , dovuto ad un a . 
spect / ct provided additional information concerning anatomical characterisation of lesions , important for surgical resection , in 39% of patients examined . the case of a patient affected by a large calcific thyroid nodule located in the upper pole of the right lobe was particularly interesting . 
in fact , abundant mitochondria - rich oxyphil cells present in thyroid oxyphil adenoma [ 13 , 14 ] or thyroid carcinoma [ 15 ] seem to assume a relevant mechanism of sestamibi uptake . 
 in our previous report , we hypothesised that in shp - positive parathyroid , scintigraphy exclusively identifies hyperfunctioning autonomous parathyroid glands refractory to vitamin d therapy and therefore the glands responsible for the so - called tertiary hp [ 16 ]  . 
subsequently , other authors confirmed this observation by demonstrating that supplementation with calcium and vitamin d in patients affected by shp inhibits parathyroid cells and , at same time , reduces sestamibi uptake by influencing the parathyroid membrane potential [ 17 , 18 ]  . 
furthermore , cell membrane expression of p - glycoprotein encoded by the human multiple drug resistant ( mdr ) gene certainly plays a relevant role in parathyroid uptake of lipophilic cationic complex [ 19 , 20 ]  . 
in conclusion , a large series of biological / molecular variable factors can influence sestamibi uptake , in particular in shp , contributing to the discussed false negative results that are , however , independent of size and site of the parathyroid glands . 
 conclusions spec / ct might currently represent the state of the art in the diagnostic flowchart to localise parathyroid glands when these are not in normotopic sites or , at any rate , undetectable at carcinoma paratiroideo . 
lo stesso caso presentava unulteriore piccolo focolaio di iperfissazione stabile del sestamibi sul polo inferiore controlaterale che allindagine istologica risultato essere un carcinoma tiroideo follicolare di 7 mm di diametro . 
tale ultima osservazione appare utile per precisare che lindagine scintigrafica con il sestamibi certamente molto sensibile nellindividuare le paratiroidi responsabili di un quadro clinico di iperparatiroidismo primitivo , ma non offre al pari unelevata specificit . 
cellularit in cui vi sia alto contenuto mitocondriale quali ladenoma a cellule ossifile [ 13 , 14 ] od i carcinomi tiroidei [ 15 ] risultano parimenti iperfissare il radiocatione . 
nei tre casi con paratiroidi ectopiche in tale sede , infatti , solo la spect / tc si dimostrata in grado di offrire un valido e risolutivo contributo preliminare allintervento chirurgico . 
certamente non rappresenta un difetto intrinseco della metodica quanto piuttosto la documentazione in immagini di un differente comportamento biologico / molecolare delle cellule paratiroidee stesse . in un precedente lavoro del nostro gruppo si prospettata lipotesi che la scintigrafia paratiroidea identifichi nellipt secondario esclusivamente quelle paratiroidi a pi elevata attivit funzionale ed autonome dallazione della terapia supplementare con vitamina d , quindi le paratiroidi responsabili dellipt terziario [ 16 ]  . 
altri autori hanno successivamente confermato tale ipotesi dimostrando che la terapia supplementare con calcio e vitamina d in grado di inibire la funzione delle cellule paratiroidee ed in concomitanza luptake del sestamibi [ 17 , 18 ]  . 
in aggiunta , lespressione a livello di membrana della p - glicoproteina codificata dal gene mdr sicuramente gioca un ruolo rilevante nella fissazione del complesso lipofilico allinterno delle cellule paratiroidee [ 19 , 20 ]  . 
in conclusione , una larga serie di variabili biologico / molecolari concorre probabilmente alla creazione di falsi negativi . certamente , comunque , non rappresentano fattori discriminanti n la dimensione delle ghiandole n la loro posizione . conclusioni la spec / tc rappresenta attualmente lo stato dellarte nella diagnostica per immagini delle paratiroidi quando esse non siano eutopiche od indocumentabili con ecografia . 
in our experience , lesion localisation improved in 39% of cases , with consequent and implicit additive value for the surgical approach . the practical result of this new method confirms its importance in surgery , as it identifies and locates parathyroid lesions not only in php , as recently reported by gayed et al . 
in this subtype of hp , despite the wellknown low sensitivity of spect / ct , it is fundamental to resect all sestamibi - positive hyperfunctioning parathyroid glands during surgical exploration and not only the larger and more conspicuous ones , which may also be negative to sestamibi . in conclusion , a wider diffusion of the spect / ct hybrid system should be encouraged given its usefulness not only in presurgical hp but also in all clinical events when the precise correlation between radionuclide signals and anatomical position is important for the correct diagnostic and therapeutic approach . za riportata la localizzazione delle lesioni migliora nel 39% dei casi con la conseguente implicita valenza positiva nei confronti dellapproccio chirurgico . 
in tali forme , infatti , nonostante il riconosciuto modesto livello si sensibilit della metodica , appare fondamentale che allatto chirurgico vengano sicuramente ablate le paratiroidi iperfissanti il radiocatione , quindi sicuramente iperfunzionanti , e non solo quelle iperplastiche , che per le loro stesse dimensioni sono facilmente reperite dal chirurgo ma che possono essere anche fredde . 
olivetti masson editore ( 2005 ) isbn 88 - 214 - 2729 - 3 published online : 11 october 2006 il nuovo libro si propone come un punto di riferimento nella diagnostica per immagini del fegato e delle vie biliari . larticolazione dei capitoli prevede infatti un progressivo passaggio delle nozioni di base per arrivare alla parte fondamentale del libro dedicata agli aspetti semeiologici delle varie patologie del fegato e delle vie biliari . a questo proposito il libro si divide in 8 parti e 25 capitoli . la prima parte dedicata allanatomia , dapprima normale , macro e microscopica , e successivamente radiologica , con particolare riferimento allecografia , alla tc e alla rm . 
una attenzione particolare viene riservata allanatomia vascolare e allanatomia segmentaria del fegato . la seconda parte dedicata alla clinica delle malattie epatiche e delle vie biliari , con una ulteriore distinzione tra le malattie epatiche focali nel paziente epatopatico e non epatopatico . la terza parte entra nel merito delle tecniche di immagine e la relativa semeiotica ed dedicata allecografia , alla tc , alla rm , alla medicina nucleare e alla colangio - pancreatografia retrograda endoscopia e rm . 
in questa parte vengono inoltre trattati i mezzi di contrasto in tc e rm . nella quarta , quinta e sesta parte viene estesamente trattata la patologia epatica e biliare , con grande ricchezza sia di informazioni sia di iconografia . nella settima parte viene trattata la radiologia interventistica del fegato con particolare riguardo alla chemioembolizzazione e alle tecniche ablative percutanee dellepatocarcinoma e delle metastasi epatiche , nonch alla tips e ai drenaggi biliari . lultima parte , infine , a testimoniare la completezza dellopera , dedicata al fegato trapiantato . il libro rappresenta un testo molto aggiornato e completo sulla diagnostica per immagini del fegato e delle vie biliari . riccardo manfredi istituto di radiologia policlinico g.b. 
passariello1 1dipartimento di scienze radiologiche , 2dipartimento di scienze cliniche , divisione di gastroenterologia , 3polo didattico pontino di latina , dipartimento di scienze radiologiche , universit di roma la sapienza , policlinico umberto i , viale regina elena 324 , i - 00166 roma , italy correspondence to : v . 
functional mri sequences acquired during the administration of oral contrast material can evaluate oesophageal transit , providing information on motility and morphology ; furthermore , this modality can properly visualise the typical functional and morphological alterations of motility disorders . key words oesophagus functional mri oesophageal motility riassunto obiettivo . 
la valutazione del transito esofageo con rm funzionale utilizzando sequenze turbo flash acquisite durante la somministrazione di mezzo di contrasto orale fattibile ed in grado di fornire informazioni valide sui dati morfologici e funzionali dell ' esofago ; inoltre , risultata essere metodica promettente nello studio delle alterazioni della funzionalit , gi oggetto di nostri altri studi . parole chiave esofago rm funzionale motilit esofagea v . 
currently , the main field of application is cardiac imaging [ 4 ] , a sector in which high - performing , state - of - the - art equipment is particularly needed . 
gastrointestinal imaging is , on the other hand , a relatively unexplored field : the study of gastrointestinal function requires levels of spatial and temporal resolution that have long remained a prerogative of videofluoroscopic techniques . 
current experience with fmri of the gastrointestinal tract is limited to some preliminary [ 57 ] and clinical studies [ 8 , 9 ] on deglutition and other more recent reports on the evaluation of gastric motility [ 10 ] ; only manabe et al . 
 [ 11 ] have investigated the use of fmri for the evaluation of oesophageal motility , introducing an optimised protocol based on t1 - weighted fast - field - echo ( ffe ) sequences . 
the aim of our study was to use fmri with t1 - weighted turbo fast low - angle shot ( flash ) sequences in the evaluation of oesophageal motility to define mri morphofunctional patterns of normality in healthy subjects and to gain preliminary experience in the study of patients with oesophageal motility disorders . materials and methods population sample thirty healthy volunteers ( 18 men and 12 women ; mean age 307 years ) with no history of oesophageal motility disorders or previous functional radiological or manometric investigation and seven patients ( three men and four women ; mean age 587 years ) with a radiological and manometric diagnosis of oesophageal motility disorder were enrolled in the study . 
subjects with general contraindications to mri were excluded ( one patient with a pacemaker and one with a metallic crystalline implant , and one healthy volunteer with claustrophobia )  . contrast material to obtain a suitable contrast agent for the examination , we tried to reproduce the physical properties of barium while ensuring that the mri signal would be good . 
we therefore tested different combinations of foods ( water , semolina and yoghurt ) mixed with gadolinium - diethylene triamine pentaacetic acid ( gd - dtpa ) 0.5 m ( 1 : 100 ) [ 8 , 9 ]  . 
although the mixture of semolina and gd - dtpa and that of yoghurt and gd - dtpa both had adequate physical properties and optimal mr signal intensity , we opted for the yoghurt - based medium because it was more palatable and easier to handle ( preparation and preservation )  . la risonanza magnetica ( rm ) funzionale ha costituito una delle principali sfide nel campo della diagnostica per immagini sin dalla seconda met degli anni ottanta [ 13 ] grazie al progressivo miglioramento in termini di risoluzione temporale e di risoluzione spaziale via via offerti dalle nuove apparecchiature . 
di contro , il campo gastroenterologico permane sostanzialmente inesplorato ; lo studio della funzionalit gastrointestinale richiede , infatti , livelli di risoluzione spazio - temporale che sono rimasti a lungo esclusiva prerogativa delle tecniche videofluoroscopiche . 
lattuale esperienza nel campo della rm funzionale del tratto gastrointestinale limitata ad alcuni studi preliminari [ 57 ] e clinici [ 8 , 9 ] sulla funzionalit della deglutizione e ad altri pi recenti articoli sulla valutazione della motilit gastrica [ 10 ] ; solo manabe et al . 
 [ 11 ] , nel 2004 , hanno proposto uno studio sullimpiego della rm funzionale nella valutazione della motilit esofagea , introducendo lottimizzazione di un protocollo basato su sequenze fast field echo t1 - pesate . 
lo scopo del nostro studio stato quello di utilizzare la rm funzionale con sequenze turbo flash t1 - pesate nella valutazione della motilit esofagea definendo dei pattern morfofunzionali rm di normalit in soggetti sani e di acquisire unesperienza nello studio di pazienti affetti da patologie della motilit esofagea . materiali e metodi popolazione campione trenta volontari sani ( 18 maschi e 12 femmine ; et media = 307 anni ) , con anamnesi negativa per la presenza di disturbi della motilit esofagea e non precedentemente sottoposti ad alcunindagine per lo studio funzionale radiologico e manometrico , e 7 pazienti ( 3 maschi e 4 femmine ; et media = 587 anni ) con diagnosi radiologica e manometrica positiva per patologia della motilit esofagea sono stati arruolati nella popolazione campione . 
sono stati esclusi dallo studio tutti i soggetti che presentavano controindicazioni generali allesecuzione di un esame rm ( 1 portatore di pace - maker ed 1 di protesi metallica del cristallino tra i soggetti patologici , un volontario sano con claustrofobia )  . mezzo di contrasto allo scopo di ottenere un mezzo di contrasto adeguato a questo tipo di esame , si cercato di emulare le propriet fisiche del bario e di ottenere , allo stesso tempo , un adeguato segnale rm . 
sono state quindi testate differenti miscele di sostanze alimentari ( acqua , semolino e yoghurt ) addizionate con gd - dtpa 0 , 5 m ( 1 : 100 ) [ 8 , 9 ]  . 
il mezzo di contrasto a base di acqua , pur presentando eccellenti caratteimaging protocol all examinations were performed on a 1.5 - t magnet ( magnetom vision , siemens , erlangen , germany ; gradients 25 mt / m2 , slew rate 800 t / m per second , rise time 400 s )  . the subjects had fasted for at least 4 h before the examination to obtain a baseline assessment of oesophageal motility . during the scan , the subjects lay prone inside the magnet with a four - channel phased - array coil positioned on their back . 
la miscela di semolino e gd - dtpa e quella di yoghurt e gd - dtpa presentavano entrambe propriet fisiche adeguate ed un ottimale intensit di segnale rm , tuttavia si optato per lutilizzo del contrasto a base di yoghurt per la miglior gradevolezza e facilit di gestione riscontrate ( modalit di preparazione e conservazione della miscela )  . protocollo di imaging tutti gli esami sono stati eseguiti utilizzando un magnete da 1 , 5 t ( magnetom vision , siemens , erlangen , germania ; gradienti 25 mt / m2 , slew rate 800 t / m / s , rise time 400 micro / s )  . 
i parametri tecnici delle sequenze dynamic tfl sono stati adeguatamente modificati in modo da ottenere una corretta visualizzazione dinamica della motilit esofagea : il numero di acquisizioni stato aumentato da 1 a 45 per ottenere un campionamento ripetuto nel tempo del lume esofageo , lo spessore della singola fetta stato aumentato a 20 mm per coprire le curvature dellesofago lungo il suo passaggio attraverso il torace e laddome . 
la quantit di contrasto somministrata per ogni sequenza dinamica non stata superiore a 1015 ml ed i soggetti sono stati istruiti ad effettuarne la deglutizione in un unico atto , sincronizzandosi con gli impulsi sonori che segnano linizio dellacquisizione di ogni sequenza . 
images show bolus progression in the oesophageal lumen in the sagittal plane 3 s ( a ) , 6 s ( b ) and 9 s ( c ) after the start of acquisition . 
le immagini dimostrano la progressione del bolo nel lume esofageo sul piano sagittale dopo 3 ( a ) , 6 ( b ) e 9 ( c ) secondi dallinizio dellacquisizione . 
si noti la continua e regolare progressione del mezzo di contrasto lungo lesofago e la completa apertura della giunzione gastroesofagea ( freccia ) al suo passaggio ( d )  . be synchronised with the sound signalling the beginning of the acquisition of each sequence . 
to formulate the normality patterns , the observers assessed healthy volunteer images for a predetermined set of morphofunctional parameters based on those commonly used in the main oesophageal motility studies , manometry and barium fluoroscopy [ 11 ]  . 
per elaborare i pattern di normalit , gli osservatori hanno valutato in tutti i volontari un insieme prestabilito di parametri morfofunzionali , con riferimento a quelli che sono i dati comunemente acquisiti con i principali esami utilizzati nello studio della motilit esofagea , ovvero la manometria e la fluoroscopia con bario [ 11 ]  . 
3a - d dynamic t1 - weighted turbo fast low - angle shot ( flash ) sequence acquired in the oblique axial plane during contrast medium transit through the gastrooesophageal junction . 
4a - d morphofunctional measurements on images obtained with dynamic t1 - weighted turbo fast low - angle shot ( flash ) sequences : oesophageal length is measured from the cricopharyngeal sphincter to the gastrooesophageal junction ( goj ) using images showing a completely opacified lumen ( a , line ) ; calibre is measured on the anteroposterior diameter of the oesophagus ( b , arrows ) ; transit time is calculated from the first appearance of the bolus in the lumen to when it reaches and crosses the goj . 
4a - d misurazioni morfofunzionali su immagini ottenute con sequenze dynamic turbo - flash t1 pesate : la lunghezza dellesofago misurata dallo sfintere cricofaringeo alla giunzione gastroesofagea impiegando le immagini in cui il lume risulta completamente opacizzato ( a , linea ) ; il calibro viene valutato misurando il diametro antero - posteriore dellesofago ( b , frecce ) ; il tempo di transito calcolato dal momento iniziale in cui il mezzo di contrasto appare nel lume al momento in cui raggiunge ed oltrepassa la giunzione gastroesofagea . 
i pattern di normalit ottenuti dagli esami effettuati nei volontari sani sono stati successivamente utilizzati come parametro di confronto nei pazienti con alterazioni della motilit esofagea ; in questultimo gruppo di pazienti la diagnosi rm stata raggiunta in consenso con quella radiologica e manometrica , permettendo una preliminare valutazione della validit diagnostica della metodica che sar oggetto di studi futuri . results the fmri study of oesophageal morphology and function with dynamic tfl sequences was possible in all subjects , providing satisfactory image quality . 
la durata media dellesame stata di 155 mil tratto di esofago compreso tra lo sfintere cricofaringeo e la gge stato correttamente visualizzato in tutti i soggetti . pattern di normalit i parametri morfologici sono stati correttamente valutati grazie alladeguata risoluzione spaziale della sequenza utilizzata ; per ogni parametro stato calcolato il valore medio ( m ) e la deviazione standard ( ds ) : lunghezza dellesofago : m = 22 , 8 cm , ds = 2 , 7 cm ; calibro dellesofago : m = 21 mm , ds = 1 , 8 mm . la risoluzione temporale ottenibile attraverso i valori di tr , te e flip angle impiegati stata di circa 2 , 5 frames per secondo . 
none of the patients with a diagnosis of oesophageal motility disorder had mri findings referable to hiatal hernia or gastrooesophageal reflux . discussion the study of oesophageal morphology and motility currently relies on a combined radiological and manometric saggio del bolo . alterazioni della motilit nei pazienti con diagnosi di alterazione della motilit esofagea stato possibile evidenziare reperti patologici corrispondenti a quelli riscontrati allesame videofluoroscopico convenzionale : la rm ha potuto correttamente diagnosticare 3 casi di acalasia ( di cui uno in fase precoce e due in fase avanzata ) e 4 casi di spasmo esofageo diffuso . 
limmagine a 4 secondi ( a ) evidenzia un rapido riempimento del lume esofageo ; il normale transito del mezzo di contrasto tuttavia interrotto da contrazioni terziarie intermittenti ( b ) che ne ostacolano la progressione , creando un tipico aspetto a cavaturaccioli ( freccia nellingrandimento )  . esofagea si sono evidenziate immagini riferibili ad ernia iatale n a reflusso gastroesofageo . discussione attualmente lo studio della morfologia e della motilit esofagea si basa su un approccio combinato radiologico e manometrico : se infatti lo studio della morfologia e delle sue variazioni durante gli eventi fisiologici stretta pertinenza della fluoroscopia con bario [ 12 , 13 ] , lanalisi dettagliata della motilit viene effettuata attraverso limpiego della manometria , metodica strumentale in grado di misurare con estrema precisione parametri difficilmente valutabili con tecniche differenti , quali lintensit della singola onda peristaltica o la pressione di apertura dello sfintere esofageo inferiore [ 14 , 15 ]  . 
tecniche alternative di studio funzionale , quale la valutazione con radionuclidi del transito esofageo , sono state proposte con discreto successo [ 16 ] ; tuttavia non esiste ad oggi alcuna metodica di imaging alternativa che risulti sufficientemente affidabile rispetto allapproccio radiologico tradizionale . 
il protocollo da noi proposto introduce una tecnica rm in grado di offrire unadeguata risoluzione spazio - temporale nella visualizzazione degli eventi fisiologici che si producono durante la peristalsi esofagea . 
la sequenza dynamic tfl impiegata nel nostro protocollo soddisfa in maniera soddisfacente i requisiti tecnici necessari ad un esame dinamico : i bassi valori di flip angle ( 8 ) e di tr ( 416 ms ) utilizzati in combinazione con il mezzo di contrasto orale a base di gd - dtpa , permettono la visualizzazione in tempo reale della motilit esofagea con unadeguata risoluzione di contrasto a livello del lume . 
i risultati ottenuti con la metodica hanno permesso la formulazione di pattern morfofunzionali rm di normalit in accordo con i reperti manometrici e radiologici riscontrati in letteratura [ 17 , 18 ] in soggetti sani ; lutilizzo dei pattern di normalit come parametro comparativo costituisce , inoltre , presupposto fondafig . 
whereas oesophageal morphology and its changes during physiological events is studied with the barium swallow [ 12 , 13 ] , motility is investigated by manometry , a technique able to provide precise measurements of parameters not assessable with other methods , such as intensity of the single peristaltic wave or the opening pressure of the lower oesophageal sphincter [ 14 , 15 ]  . alternative techniques proposed for functional studies , such as radionuclide assessment of oesophageal transit , have met with moderate success [ 16 ] ; nonetheless , no alternative imaging method has proved sufficiently reliable compared with conventional radiology . 
the protocol proposed by us introduces an mri technique able to offer adequate spatial and temporal resolution in visualising physiological events occurring during oesophageal peristalsis . the dynamic tfl sequence adequately fulfils the technical requirements of a dynamic study : the low flip angle ( 8 ) and tr ( 416 ms ) values used in combination with oral gddtpa - based contrast material allows real - time visualisation of oesophageal motility with adequate contrast resolution in the lumen . 
in these subjects , mri proved able to identify morphofunctional alterations typical of the conditions being studied , in agreement with findings of conventional videofluoroscopy . compared with conventional radiology , fmri of the oesophagus has several major advantages : multiplanar imaging capabilities with visualisation of the oesophagus and intrathoracic soft tissues in the different spatial planes , the possibility of making acquisitions with dedicated sequences and possible use of contrast material targeted to specific diagnostic queries and higher contrast resolution . 
with fmri , it would be possible to repeat examinations over time ( for example , in the follow - up before and after dilatation and / or myotomy in patients with achalasia ) and to study patients , such as pregnant women and younger subjects , in whom radiation exposure is contraindicated . the technique presented does , however , suffer some limitations : first , the spatial resolution of the sequences used , although adequate for assessing oesophageal function , is still too low to study alterations of the wall profile , as can be done with videofluoroscopy ; we should , however , add that the typical filling defect pattern ( as seen in one case of leiomyoma of the distal third ) and / or marked wall thickening at the level of the goj ( as in the case of cardia cancer ) could raise the suspicion of pseudoachalasia . 
second , the maximum temporal resolution [ 2.5 frames per second ( fps ) ] is still far below the standards currently obtainable with conventional radiology ( 2530 fps )  . 
a further technical limitation might be the possibility of imaging patients in the clinostatic position only , especially if we consider the range of positions and projections used in videofluoroscopy and manometry ; however , in the light of the results of our preliminary experience we can state that even the information obtained with the patients in the clinostatic position may be valuable for diagnosis . 
our next study , which is being conducted on patients with motility disorders , already involves double acquisitions with patients in the prone and supine position . mentale per la valutazione diagnostica dei pazienti con alterazioni della motilit . 
 rispetto allapproccio radiologico tradizionale , lesame rm funzionale dellesofago presenta alcuni importanti vantaggi : capacit di imaging multiplanare con visualizzazione dellesofago e dei tessuti molli endotoracici sui diversi piani dello spazio , possibilit di effettuare acquisizioni con sequenze dedicate ed eventuale impiego del mezzo di contrasto sulla base di precise richieste diagnostiche , superiore risoluzione di contrasto . 
il vantaggio maggiore tuttavia rappresentato dalla possibilit di esplorare la funzionalit dellesofago senza limpiego di radiazioni ionizzanti : lesposizione alle radiazioni [ 19 ] rimane ad oggi , nonostante lavvento delle apparecchiature digitali e la sensibile riduzione della dose di esposizione , la principale controindicazione allesame fluoroscopico ; al contrario , la rm funzionale potrebbe consentire esami ripetibili nel tempo ( richiesti ad esempio nei programmi di follow - up pre e post dilatazione e / o miotomia nei pazienti acalasici ) , permettendo inoltre lo studio di quei pazienti nei quali lesposizione alle radiazioni sconsigliata , quali le donne in gravidanza ed i soggetti pi giovani . 
 la tecnica da noi presentata non tuttavia priva di limiti : in primo luogo la risoluzione spaziale delle sequenze impiegate , seppur adeguata per la valutazione della funzionalit esofagea , risulta ancora troppo bassa per lo studio delle alterazioni del profilo della parete esofagea cosi come ottenibile con la videofluoroscopia ; bisogna , tuttavia , aggiungere che il tipico aspetto da difetto di riempimento ( come ad esempio riscontrabile in un caso di leiomioma del terzo distale ) e / o un evidente ispessimento parietale a livello della giunzione gastro - esofagea ( come nel caso di un tumore del cardias ) potrebbero porre il sospetto per pseudoacalasia . 
potrebbe risultare tecnicamente opinabile la possibilit di eseguire gli esami nel solo clinostatismo soprattutto considerando le differenti posizioni e proiezioni utilizzate negli studi videofluoroscopico e manometrico ; alla luce dei risultati acquisiti nella nostra esperienza preliminare possiamo ritenere , tuttavia , che anche le informazioni ottenute in posizione clinostatica possano comunque risultare valide ai fini diagnostici . 
lesperienza successiva gi in corso su una popolazione di pazienti patologici , gi prevede la duplice acquisizione in posizione prona e supina . conclusioni conclusions our study demonstrated that the evaluation of oesophageal transit with fmri using tfl sequences during the administration of oral contrast material is feasible and able to provide valuable morphological and functional information on the oesophagus ; in addition , this method is able to correctly identify alterations of oesophageal function and morphology in subjects with motility disorders . in conclusione il nostro studio dimostra che la valutazione del transito esofageo con rm funzionale utilizzando sequenze turbo flash acquisite durante la somministrazione di mezzo di contrasto orale fattibile ed in grado di fornire informazioni valide sui dati morfologici e funzionali dellesofago ; inoltre , questa metodica in grado di evidenziare correttamente le alterazioni della funzionalit e della morfologia esofagea presenti in soggetti affetti da disordini della motilit . 
even though the technique , above all in terms of performance of the equipment , is still far from being state - of - the - art and suffers some significant technical limitations , we believe this diagnostic approach to be feasible and to hold significant future prospects for the study of subjects with oesophageal disorders , whom we are currently studying with state - of - the - art equipment . rm funzionale risultato rapido , facilmente riproducibile , del tutto non invasivo e privo di disagio per il paziente . 
magnavita2 1istituto nazionale di riposo e cura dellanziano ( inrca ) , sede di roma , via cassia 1167 , i - 00189 roma , italy 2istituto di medicina del lavoro delluniversit cattolica del sacro cuore , roma , italy correspondence to : a . 
lastly , radiologists were frequently named as one of multiple defendants , together with medical ( or surgical ) doctors , in cases of patient death in roughly 6% of all cases . 
strict adherence to radiological standards may be a means of reducing the risk of legal action and obviating litigation . key words malpractice radiology liability error clinical risk management riassunto obiettivo . 
in altri termini , il 44% della coorte , rappresentativa dei radiologi italiani , ha ricevuto o ricever una citazione in giudizio riferita alla propria attivit professionale degli ultimi 10 anni . 
a questo riguardo la prima causa di errore in ordine di frequenza ancora la mancata diagnosi di lesioni dello scheletro immediatamente seguita , in ordine di frequenza , dalla mancata diagnosi di neoplasia , soprattutto della mammella . 
magnavita : a 12 - year follow - up study of malpractice claims against radiologists in italy introduction introduzione legal action taken against physicians for presumed malpractice is an increasing problem in all countries and in all specialties . 
radiological malpractice in italy has recently been the subject of a number of papers , including qualitative investigations in mammography [ 1 ] , methodological proposals [ 2 ] , and the presentation of case series [ 3 ]  . there is therefore the need to analyse the epidemiology of the phenomenon , which would also help to identify the most effective measures in terms of clinical risk management . this is the aim of the present study , which is based on the examination of a cohort of more than 4 , 000 radiologists accounting for over half of the members of the italian society of medical radiology ( sirm ) and who were studied over a period of 12 years . le denunce contro i medici per presunta malapratica ( malpractice nella letteratura anglosassone ) sono un problema di crescente gravit in tutti i paesi e in tutte le specialit . 
al tema della malapratica radiologica nel nostro paese sono state recentemente dedicate verifiche qualitative nel settore mammografico [ 1 ] e proposte metodologiche [ 2 ] , oltre naturalmente a segnalazioni casistiche [ 3 ]  . 
la popolazione di riferimento , costituita da tutti i radiologi che hanno stipulato lassicurazione , sensibilmente aumentata nel periodo di osservazione ( da 1400 iscritti nel 1993 a 4007 al 1 / 1 / 04 con incremento del 286% )  . 
essa si riferisce attualmente al 53% dei radiologi iscritti alla sirm e pu essere quindi ritenuta sufficientemente rappresentativa dellinsieme dei radiologi italiani ( tabella 1 )  . le denunce sono state classificate in ragione della causa che le aveva prodotte , come : ( 1 ) da presunto errore diagnostico ; ( 2 ) da presunto errore nella conduzione dellesame ; ( 3 ) da omissione di indicazioni diagnostiche e / o terapeutiche ; ( 4 ) da terapie con radiazioni ionizzanti e / o non ionizzanti ; ( 5 ) da responsabilit civile per infortuni subiti dal paziente durante lesecuzione dellesame ( per urti , cadute ecc . ) ; ( 6 ) da applicazione dellart . 
in questultima categoria sono stati compresi anche tutti i casi nei quali il radiologo non ha specificato la causa della denuncia , riservandosi di farlo in un secondo tempo ; nei casi in cui entro il 31 / 12 / 2005 stato possibile conoscere le specificazioni di tipologia e sede del sinistro si provveduto a correggere la relativa classificazione . i dati sono stati analizzati mediante statistiche descrittive , utilizzando lo statistical package for social sciences ( spss / pc 11 , 5 )  . per ottenere la stima delle denunce riferibili al periodo in esame , ma che perverranno allente assicuratore entro i termini di prescrizione delle denunce di responsabilit civile ( dieci anni dallevento lesivo ) , si proceduto nel modo seguente : ( 1 ) stata formulata lipotesi che la propensione a a . 
magnavita : a 12 - year follow - up study of malpractice claims against radiologists in italy materials and methods all malpractice claims regarding the professional activity of radiologists received by the insurance company from 1 january 1993 ( the beginning of the convention with sirm ) to 31 december 2004 were examined . 
the study population , made up of all sirm radiologists insured with the company , noticeably increased over the study period ( from 1 , 400 members in 1993 to 4 , 007 on 1 january 2004 , an increase of 286% )  . 
the population currently accounts for 53% of sirm members and can therefore be considered a sufficiently representative sample of all italian radiologists ( table 1 )  . claims were classified according to the cause that produced them , such as : ( 1 ) presumed diagnostic error ; ( 2 ) presumed error in performing the examination ; ( 3 ) failure to provide diagnostic and / or therapeutic indications ; ( 4 ) treatment with ionising and / or nonionising radiation ; ( 5 ) civil liability due to accidents suffered by the patient during the examination ( slip - and - fall accidents , etc . ) ; and ( 6 ) application of articles 589 of the italian penal code ( manslaughter ) and 590 ( culpable lesions ) and other articles of the penal or civil codes . 
the last of these categories also includes all of those cases in which the radiologist fails to specify the cause of the claim , deciding to do so at a later time ; cases in which the cause of the claim could be verified by 31 december 2005 were reclassified accordingly . 
in other words , since only 22% of claims relating to the 19931995 period were filed within 1 year of the event , we estimated that the claims filed in 2004 made up only 22% of all claims referring to 2004 , which will be filed within the prescribed ( 10 - year ) time period . 
in a similar way , we performed calculations for 2003 , 2002 , and so on , until 1995 . using similar reasoning , we estimated the cause of claims yet to be filed by examining frequency according to cause of claims filed at least 1 year after the alleged harmful event and applying those percentages to the number of claims expected to be filed . results in the study period , radiologists covered by insurance policies received 990 claims for alleged malpractice . 
the claims prevalently concerned radiologists working in the public hospital system ( 714 cases ; 72.1% ) whereas radiologists working in private facilities were subject to about a quarter of all denunciare i medici radiologi sia costante nel tempo ; ( 2 ) di conseguenza , stato assunto che landamento delle denunce nei primi anni di osservazione , tra il 1993 ed il 1995 , una volta esaurito leffetto delle denunce tardive , esprima landamento costante delle denunce nella coorte ; ( 3 ) stata calcolata la percentuale cumulativa delle denunce pervenute dopo uno , due , tre o pi anni nel periodo di osservazione anteriore al 1995 ; ( 4 ) tali percentuali cumulative sono state applicate come coefficienti alle osservazioni attuali . 
in altri termini , poich nel periodo 19931995 solo il 22% delle denunce pervenuta entro lanno di accadimento , si stimato che le denunce pervenute nel 2004 siano solo il 22% di quello che sar il numero totale di denunce riferibili al 2004 che perverranno entro i termini di prescrizione . 
in modo analogo si proceduto per gli anni 2003 , 2002 e precedenti , fino al 1995 . con lo stesso procedimento logico si proceduto a stimare la causa delle denunce non ancora pervenute , esaminando la frequenza per causa delle denunce pervenute dopo almeno un anno dallevento lesivo ed applicando tali percentuali agli eventi attesi . risultati nel periodo esaminato i medici radiologi coperti da polizze assicurative hanno ricevuto 990 denunce per presunta responsabilit civile . 
le denunce hanno riguardato prevalentemente medici operanti nelle strutture pubbliche ( 714 casi , 72 , 1% ) , mentre i radiologi che lavorano in strutture private hanno subito circa un quarto delle denunce ( 244 casi , 24 , 6% )  . 
i danni allegati consistevano prevalentemente in lesioni personali ( 661 casi , 66 , 8% ) , ma significativamente elevata la quota di decessi ( 329 casi , 33 , 2% )  . 
il numero pi elevato di denunce perviene dopo un anno dallevento lesivo ( 311 denunce , 31 , 4% ) , mentre quasi met perviene negli anni ancora successivi , in alcuni casi anche dopo oltre otto anni ( tabella 4 )  . lanalisi delle cause delle denunce ( tabella 5 ) consente di osservare che nella maggior parte dei casi ( 660 casi , 66 , 7% ) limputazione di errata diagnosi , in quanto si ipotizza incapacit o negligenza del radiologo nellinterpretazione dellesame radiologico . 
il presunto errore diagnostico del radiologo non solo la principale causa di contenzioso , ma anche quella che registra il maggior incremento percentuale rispetto allanalisi dei primi dati italiani da noi analiz1011 a . 
alleged diagnostic error is not only the main cause of legal action but also registers the greatest percentage increase with respect to the zati , quando tale categoria rappresentava un terzo delle denunce [ 4 ]  . 
va considerato inoltre che ad un presunto errore diagnostico del radiologo si riferiscono molte delle denunce provvisoriamente classificate nella categoria miscellanea , nelle quali spesso il radiologo viene citato per omicidio colposo o lesioni assieme a tutti gli altri medici che hanno avuto a che fare con il paziente , sulla base di presunta imperizia o negligenza nellesecuzione degli accertamenti radiologici ( tabella 5 )  . la sede pi frequente degli errori diagnostici lapparato scheletrico ( 44 , 5% ) ; seguono in ordine di frequenza la mammella ( 25 , 9% ) , il torace ( 11 , 4% ) , laddome ( 8 , 3% ) ( tabella 6 )  . 
tra le cause di errore , si conferma al primo posto la mancata o errata diagnosi di lesioni dellapparato osteoarticolare ( 279 casi ) , ma le denunce per mancata o ritardata diagnosi delle neoplasie ( 265 casi ) risultano ormai quasi altrettanto numerose , e laumento rispetto alle precedenti rilevazioni fa ritenere che in un prossimo futuro lordine abbia ad invertirsi . 
nellambito delle neoplasie , gli errori riguardano in primo luogo la mammella ( 171 casi , pari al 64% di tutte le neoplasie ) e , in minor misura , tumori polmonari o di altre sedi ( tabella 7 )  . 
le denunce per neoplasie mammaria registrano anche il maggiore incremento nume1012 percentuale cumulativa 23 , 3 54 , 7 71 , 9 85 , 6 93 , 2 98 , 0 99 , 3 99 , 8 100 , 0 percentuale totale a . 
it should also be borne in mind that alleged misdiagnosis accounts for many of claims provisionally classified in the miscellaneous category , in which legal action is often taken against the radiologist , along with all other physicians who came into contact with the patient , for manslaughter or culpable lesions on the basis of alleged inability or negligence in interpreting radiological findings ( table 5 )  . 
 the most common site of diagnostic errors was the skeletal system ( 44.5% ) , followed by the breast ( 25.8% ) , the chest ( 11.4% ) and the abdomen ( 8.3% ) ( table 6 )  . 
once again , the most common cause of error was misdiagnosis or failure to diagnose lesions to the osteoarticular system ( 279 cases )  . however , the number of claims made for misdiagnosis or failure to diagnose cancer ( 265 cases ) has almost reached the same level . 
with regard to cancer , the most common site of claims was the breast ( 171 cases , or 64% of all cancers ) followed to rico dalle prime osservazioni da noi condotte [ 4 ] , quando erano stati denunciati solo due casi . 
gli errori nelle tecniche e procedure radiologiche rappresentano percentualmente solo il 10 , 3% delle denunce ( 112 casi ) , circa la met dei quali ( 59 casi ) da attribuirsi a tecniche di radiologia interventistica . la latenza tra evento lesivo e denuncia varia nei diversi gruppi di eventi causali ( tabella 8 )  . 
mentre le lesioni conseguenti a presunti errori di tecnica o infortuni occorsi nella sala radiologica danno luogo a denuncia spesso entro lo stesso anno di accadimento e comunque entro il secondo anno solare dallevento , i procedimenti per malapratica si attivano a distanza maggiore dallevento . 
errors in radiological techniques and procedures accounted for only 10.3% of claims ( 112 cases ) , almost half of which ( 59 cases ) involved interventional radiological techniques . latency between the alleged error event and the filed claim varies according to cause ( table 8 )  . 
whereas lesions due to alleged technical errors or accidents in the radiology room are often associated with claims filed in the same year or at least within the second calendar year from the alleged event , malpractice claims tend to be filed at a greater distance from the alleged event . 
in particular , claims for failure per ottenere una stima del numero di denunce che si riferiranno a tutti gli eventi radiologici occorsi nel periodo di osservazione , possiamo tenere conto dellandamento dei tempi di latenza relativi agli anni pi lontani , anteriori al 1995 , per i quali si pu ormai ritenere estinto il diritto al risarcimento civile ( dieci anni )  . 
lesame di questi dati rafforza quanto sopra affermato circa la maggiore latenza delle denunce per neoplasia , che negli eventi pi remoti , sono di circa quattro anni ( tabella 9 )  . 
tenendo conto della latenza delle denunce , e facendo lipotesi che la propensione a denunciare i radiologi sia costante negli anni a venire , si pu stimare che verranno sporte almeno 1370 denunce per eventi riferiti ai dodici anni di osservazione , con un incremento del 38% ( 380 casi ) rispetto alle 990 denunce sinora perve1014 a . 
the significant latency of claims complicates assessment of the phenomenon overall and makes assessment of the incidence of risk particularly difficult since the number of claims in the study period is still incomplete . to obtain an estimate of the number of future claims relating to all radiological events that occurred during the study period , we examined the trend of latency times with regard to the earliest years of the study period ( prior to 1995 ) for which the right to civil compensation has expired ( 10 years )  . 
analysis of these data reinforces the observation that claims regarding cancer have a greater latency , which in some cases extends to around 4 years ( table 9 )  . 
il metodo di stima da noi applicato ci induce a ritenere che le denunce per errata diagnosi di neoplasia rappresenteranno la principale causa di contenzioso , una volta esaurita la latenza dei tempi di denuncia ( tabella 11 )  . sempre sulla base dellipotesi che la propensione a denunciare i radiologi si mantenga costante , il rischio di denuncia risulta superiore al 44 per mille ( tabella 10 )  . 
ci corrisponde al rischio di una denuncia ogni 22 , 7 anni di lavoro , ovvero , in altri termini , alla certezza statistica , per il radiologo medio , di essere denunciato almeno una volta nel corso della propria vita lavorativa . 
magnavita : a 12 - year follow - up study of malpractice claims against radiologists in italy verse analisi della coorte da noi effettuate in anni precedenti induce a ritenere che la propensione a denunciare i radiologi nel nostro paese sia in aumento . stant in the future , we estimated that at least 1 , 370 claims will be lodged for events occurring in the 12 - year study period , with an increase of 38% ( 380 cases ) compared with the 990 claims lodged to date ( table 10 )  . 
the method of estimation we employed suggests that claims for cancer misdiagnosis is the main cause of legal action once the latency cut - off period for claims has been reached ( table 11 )  . while maintaining the hypothesis that the tendency for legal action against radiologist remains constant , the risk of being sued is greater than 44 per 1 , 000 subjects ( table 10 )  . this figure corresponds to a risk of being sued every 22.7 working years or , in other words , statistical certainty that the average radiologist will be sued at least once in his or her working life . 
this estimate is once again higher than the values we calculated in this cohort in previous studies [ 47 ]  . progressive increase in the rate of claims lodged in the various analyses of the cohort we carried out in previous years suggests that the tendency to take legal action against radiologists in italy is increasing . discussion the most significant finding of our study was the high risk of being sued for malpractice . 
our estimate , which is based on the conservative hypothesis that there is no increase in the tendency of patients to sue physicians and therefore probably underestimates the phenomenon , suggests that half of the radiologists in our cohort have received or will receive a request for compensation or be taken to court in relation to the previous 10 years of their professional activity . 
in one third of cases , claims pertain to events in which the patient died , thus involving requests for very high amounts of compensation and possible severe criminal proceedings . further analysis suggests that the risk of legal complications for italian radiologists is progressively approaching that of the united states where it is estimated that 40% of radiologists are taken to court on average once every 5 years [ 8 ]  . 
in the united states , malpractice suits filed against radiologists account for 10%15% of the total charges against physicians for inability or negligence even though in the united states radiologists overall are less exposed to the risk of legal action than are surgeons but much more exposed than are internists and specialists [ 9 ]  . even though not all legal action ends with the physician being sentenced since in most cases there is insufficient evidence to convict the radiologist and an out - of - court settlement is reached the burden of worry , legal expense and time that each case inevitably causes the radiologist should be borne in mind . 
in a significant number of cases , the stress connected to legal action can cause alterations of a physical , psychological and behavioural nature [ 10 ] , prompting the radiologist to adopt a defensive attitude ( so - called defensive medicine ) [ 11 ] or an aggressive defensive approach [ 12 ]  . these are situations that were practically unknown to the preceding generation of radiologists and that have suddenly become inevitable and innate to our professional activity . an epidemiological analysis of malpractice suits filed discussione il dato di maggiore rilievo nella nostra indagine lelevato rischio di essere denunciati per malapratica . 
la nostra stima , che basata sullipotesi prudenziale che non ci sia un aumento della tendenza a denunciare i medici da parte dei pazienti e che pertanto verosimilmente sottostima il fenomeno , induce a ritenere che la met dei radiologi della nostra coorte abbia ricevuto o stia per ricevere una richiesta di risarcimento o una citazione penale relativa allattivit professionale svolta nellultimo decennio . 
in un terzo dei casi le denunce si riferiscono ad eventi nei quali si verificato il decesso del paziente ; ci determina richieste di risarcimento di importo molto elevato e possibili pesanti ripercussioni penale nostre successive analisi della coorte di radiologi italiani assicurati ci inducono a ritenere che il rischio di complicazioni giudiziarie per i radiologi italiani tenda progressivamente ad avvicinarsi a quello degli stati uniti dove si stima che il 40% dei radiologi sia citato in giudizio mediamente una volta ogni 5 anni [ 8 ]  . 
sempre negli usa le denunce contro i radiologi rappresentano il 10%15% del totale delle denunce contro i medici per imperizia o negligenza anche se , in quel paese , i radiologi risultano nel complesso meno esposti al rischio di citazioni rispetto ai chirurghi , ma molto pi esposti dei medici internisti e specialisti [ 9 ]  . anche se non tutte le denunce si concludono con la condanna del medico , in quanto nella maggior parte dei casi possibile raggiungere un accordo extragiudiziale , ovvero non risultano sussistere elementi di colpa da parte del sanitario , occorre considerare il carico di preoccupazioni , spese legali e tempo che ciascuna di queste denunce inevitabilmente comporta per il medico radiologo . 
in non pochi casi lo stress connesso con la denuncia pu determinare alterazioni sul piano fisico , psicologico e comportamentale [ 10 ] , inducendo nel radiologo atteggiamenti di difesa ( la cosiddetta medicina difensiva ) [ 11 ] o attivi ( di difesa aggressiva ) [ 12 ]  . 
si tratta , in ogni caso , di esperienze quasi sconosciute ai colleghi della generazione che ci ha preceduto , ed improvvisamente diventate inevitabili e connaturate allattivit professionale . lanalisi epidemiologica delle denunce sporte contro i radiologi italiani consente di individuare , accanto ad aspetti analoghi a quelli gi segnalati nei paesi anglosassoni , alcune peculiarit strettamente legate al nostro sistema giuridico e sociale . 
tra gli aspetti di analogia con le esperienze dei colleghi statunitensi possiamo annoverare la prevalenza delle denunce per errori diagnostici e , allinterno di questi , il sempre maggiore peso delle denunce per mancata identificazione di neoplasie . 
magnavita : a 12 - year follow - up study of malpractice claims against radiologists in italy against italian radiologists enables identification of a number of features unique to the italian legal and social system , as well as others similar to those already reported in englishspeaking countries . 
in contrast , the high latency period of claims is atypical and can only be partly explained by the time required to gather the medical documentation required for formulating a malpractice suit . in more than two thirds of cases , legal action is taken against the radiologist due to incorrect diagnosis . 
this trend is in line with the tendency already noted in series in the united states [ 8 , 1315 ]  . errors are inevitably linked to radiological and medical practice in general , just as they are to all other human activities . 
there is , however , a need to question the possible causes of errors in order to formulate the most appropriate preventive measures ( clinical risk management ) [ 16 , 17 ]  . 
since the relationship between errors and claims is not biunique in that there can be errors not followed by legal action and legal action not substantiated by errors , there is also the need to question the factors that promote legal action . one of the main causes of diagnostic errors in radiology is undoubtedly the increase in the number of examinations . 
in the united states , it has been estimated that the number of examinations and images produced has increased by 40% in the last 5 years , and a further increase of 28% is expected by 2008 [ 18 ]  . 
review studies show that the performance of radiologists ( reduction of false negatives and false positives ) is correlated neither with the increase in the number of examinations interpreted nor the level of professional experience [ 19 ]  . 
it is therefore likely that an increase in the number of examinations interpreted , without a similar increase in the time available for their interpretation , produces a reduction in performance . 
in contrast , it has been shown that when double reading is possible , for example for mammograms , the number of errors can be reduced [ 2022 ]  . the risk of legal action due to delayed diagnosis is also associated with the spread of screening programmes , such as those for breast cancer [ 23 , 24 ] , in which the retrospective diagnosis of disease on previous radiographs is often possible or suspected . 
this tends to falsely undervalue the difficulty of reaching the correct diagnosis at the time of the first examination . the group of alleged misdiagnosis includes most of the claims originating from manslaughter and serious bodily injury charges . 
in these cases , the radiologist is invariably involved due to objective responsibility with all the other physicians who came into contact with the patient in very 1018 in oltre due terzi dei casi il radiologo citato per errore di diagnosi . 
tra questi errori , lomessa diagnosi di neoplasia si appresta a divenire anche nel nostro paese la prima causa di denuncia , seguita dallomessa diagnosi di fratture , secondo la tendenza gi osservata nelle serie storiche negli stati uniti [ 8 , 1315 ]  . lerrore inevitabilmente legato alla pratica radiologica e medica in generale , cos come ad ogni altra attivit umana . 
poich tra errori e denunce la relazione non biunivoca , in quanto possono esservi sia errori non seguiti da denunce che denunce non sostanziate da errori , occorre anche interrogarsi sui motivi che favoriscono le denunce contro i medici . tra le cause degli errori diagnostici in radiologia , una delle principali senza dubbio laumento del numero degli esami . 
negli stati uniti si stima che il numero delle indagini e delle immagini prodotte sia aumentato del 40% negli ultimi 5 anni , ed un ulteriore aumento del 28% atteso per il 2008 [ 18 ]  . 
in italia non vi sono dati di questo genere , ma riteniamo che la tendenza sia similare . lincremento di produttivit richiesto in questi ultimi anni ai servizi di radiologia ha senzaltro ridotto il tempo che il radiologo pu dedicare sia al colloquio con il paziente sia alla refertazione e questo fatto , associato al ridotto tempo da dedicare allaggiornamento professionale , pu giustificare laumento del numero degli errori . 
studi di revisione dimostrano che la performance del radiologo ( riduzione dei falsi positivi e dei falsi negativi ) non correlata n con lincremento del numero di esami refertati , n con lesperienza professionale [ 19 ]  . 
al contrario , stato dimostrato che , laddove sia possibile una doppia lettura , come ad esempio per gli esami mammografici , possibile ridurre il numero di errori [ 2022 ]  . il rischio di denunce per ritardata diagnosi inoltre associato al diffondersi dei controlli sanitari periodici a fini preventivi quali quelli per il tumore della mammella [ 23 , 24 ] nei quali la diagnosi retrospettiva di malattia su radiogrammi precedenti spesso possibile o sospettabile , e questo porta falsamente a sottostimare la difficolt di porre la diagnosi corretta allepoca del primo esame . tra gli errori diagnostici presunti rientrano la maggior parte delle denunce originate da avvisi di garanzia per omicidio o lesioni colpose . 
in questi casi il radiologo viene invariabilmente coinvolto per responsabilit oggettiva assieme a tutti gli altri sanitari che hanno avuto contatti con il paziente , nei casi , invero molto frequenti , in cui il paziente , pervenuto in condizioni critiche in un dipartimento di urgenza o emergenza , sia stato sottoposto ad accertamenti radiologici . 
questo meccanismo ha causato anni addietro unepidemia di denunce contro i radiologi in francia [ 25 ] e negli stati uniti , tanto da rendere necessaria una riforma legislativa [ 9 ]  . un altro aspetto finora episodico , ma comunque allarmante rappresentato da denunce per mancato riconoscia . 
magnavita : a 12 - year follow - up study of malpractice claims against radiologists in italy common cases in which the patient arrives at the emergency department in critical conditions and undergoes radiological examinations . 
some years ago , this mechanism led to such an epidemic of malpractice suits against radiologists in france [ 25 ] and the united states that legislative reform was required [ 9 ]  . another feature to date episodic but nonetheless alarming is the malpractice claim for failure to diagnose a lesion in panoramic examinations , such as computed tomography ( ct ) , in the case of lesions outside the clinical setting that motivated and justified the examination or after review of examinations that were reassessed with modified reconstruction parameters in search of a lesion that only became apparent later . malpractice claims for failure to order further diagnostic examinations are still rather rare in italy and are generally associated with missed cancer diagnosis . 
this has led to a change in the main reason for radiological malpractice suits in the united states in recent years : whereas in the past , the radiologist was sued above all for having done something wrong , today they are sued for not having done something right [ 26 ]  . the number of malpractice claims filed against physicians for not having ordered radiological examinations has in fact increased , and in parallel , so , too , have the claims against radiologists for not having ordered further imaging examinations . 
nor can it be ruled out that in italy in the near radiologists will be sued for not having made use of new and promising diagnostic techniques , such as computer - assisted detection ( cad ) and teleradiology , or for not having called for a second opinion from an expert colleague [ 26 ]  . 
the fears shown by american physicians appear to be finding ground even in the italian situation . in our cohort , the frequency of claims for lesions caused by radiological procedures themselves ( such as contrastmedium reactions , organ rupture during barium enema , etc . ) account for only one tenth of the total . 
the cause of this type of problem may be attributed to the diffusion of interventional radiology and more in general to the need to administer drugs or contrast media during radiodiagnostic procedures . interventional radiology is in all countries a field with a high risk of litigation [ 27 ]  . 
a recent american study , the medmarx data report [ 28 ] based on the voluntary reporting of errors occurring between 2000 and 2004 in 314 hospitals , reported that medication errors in radiology departments cause harm to the patient with a frequency seven times higher than in other hospital departments . 
questa caratteristica differenzia sostanzialmente il nostro paese dagli stati uniti , dove questo tipo di denuncia ha avuto negli ultimi anni il maggiore aumento , rispetto alle altre cause di denuncia , anche a causa dellintroduzione di provvedimenti che , allo scopo di frenare la spesa sanitaria , obbligano i medici a limitare le prescrizioni [ 9 ]  . 
in questo modo si assistito , negli ultimi anni , ad un cambiamento della motivazione principale per le denunce di malapratica negli usa : se in passato il radiologo veniva denunciato soprattutto per aver fatto qualcosa di sbagliato , oggi lo si denuncia soprattutto per non avere fatto qualcosa di appropriato [ 26 ]  . 
sono infatti fortemente aumentate le denunce contro i medici per non avere ordinato esami radiografici , e parallelamente anche quelle contro i radiologi , per non avere prescritto ulteriori accertamenti radiografici o strumentali . 
non si pu escludere che , in un prossimo futuro anche in italia , i radiologi possano essere denunciati anche per non avere fatto ricorso a nuove , promettenti tecniche di ausilio alla diagnosi , come la computer assisted detection ( cad ) o la teleradiologia , o per non avere richiesto un secondo parere da parte di un collega esperto [ 26 ]  . 
i timori dei medici statunitensi non sembrano privi di fondamento neppure nella situazione italiana . nella nostra coorte , la frequenza di denunce per lesioni causate dalle stesse procedure radiologiche ( quali ad es . reazioni al mdc , rottura del viscere nel clisma opaco , etc . ) rappresenta solo un decimo del totale . 
la causa di questo tipo di problemi va attribuita alla diffusione delle tecniche di radiologia interventistica e pi in generale alla necessit di somministrare farmaci o mezzi di contrasto nel corso delle procedure radiodiagnostiche . le tecniche di radiologia interventistica rappresentano in tutti i paesi una branca ad elevato rischio di liti legali [ 27 ]  . una recente indagine statunitense , il medmarx data report [ 28 ] , basato sulla segnalazione volontaria degli errori verificatisi tra il 2000 e il 2004 in 314 ospedali , ha segnalato come la frequenza di errori nella somministrazione di farmaci nel reparto radiologico determini conseguenze dannose per il paziente con una frequenza sette volte maggiore che negli altri reparti dellospedale . 
anche se lamerican college of radiology ha formalmente protestato contro le conclusioni dellinchiesta [ 28 ] , ritenuta eccessivamente allarmante per il paziente ( i duemilatrenta errori censiti , a fronte di due milioni e mezzo di procedure radiologiche condotte nel periodo in esame , rappresentano un rischio pari allo 0 , 00008% ) , losservazione che gran parte degli errori di somministrazione o di prescrizione di farmaci avrebbe potuto essere eliminata con un migliore collegamento tra il dipartimento di radiologia e quello di provenienza del paziente merita una 1019 a . 
improved communications between radiologists and clinicians is at the same time desirable and imperative for overcoming the ambiguity that is often the origin of radiological errors [ 29 ]  . 
policies available on the italian market can be the loss occurrence type , with coverage for claims lodged for events that occurred in the insured year and up to the 10 - year cut - off period , or the claims made type , with coverage only for claims lodged in the insured year and with reference to it . 
claims made policies do allow for coverage of a prior period as part of an expressly requested additional clause but is limited to 2 or , at most , 4 years . 
it is worth bearing in mind when signing this type of contract that in radiology , a certain amount of malpractice claims are nonetheless beyond its contractual guarantees . the high frequency of claims in relation to deceased patients or patients suffering severe harm and the correlated onset of criminal liability should induce the radiologist to the strictest adherence of professional best practice at all times . 
it is therefore not superfluous to say that the radiologist must pay close attention to the information provided by the patient prior to performing the examination and always ask for a document of informed consent to be signed , especially in the case of contrast - enhanced or invasive examinations . 
the radiologist should inform the clinician of any problems arising in relation to the examination . maximum care should be dedicated to technique , devices and environmental characteristics of the radiology room in relation to which the law prescribes the precise safety features , for which the radiologist is responsible in his or her role as employer ( in a facility of his or her own property ) or as director ( if working in a public facility or a private facility of anothers ownership )  . 
il miglioramento della comunicazione tra il radiologo ed i clinici al tempo stesso desiderabile e imperativa per risolvere le ambiguit che sono spesso allorigine di errori radiologici [ 29 ]  . 
 responsabilit del clinico trasmettere al radiologo le informazioni rilevanti in modo chiaro ed adeguato ; compito del radiologo scegliere la procedura corretta , in base alla conoscenza della storia clinica del paziente . infine le nostre osservazioni confermano che altre cause di denunce , come gli infortuni subiti dai pazienti mentre si trovano allinterno della sala radiologica , limperizia nella esecuzione di terapie radianti , o altri problemi episodici ( raccolti nella categoria miscellanea ) hanno una prevalenza ridotta ( 7 , 8% )  . un aspetto molto rilevante per le sue conseguenze pratiche la latenza tra evento lesivo e denuncia . 
le polizze disponibili sul mercato italiano possono essere del tipo loss occurrence , con copertura delle denunce per sinistri avvenuti nellanno assicurato e fino alla prescrizione decennale , ovvero claims made , con copertura delle sole denunce pervenute nellanno assicurato e ad esso riferite . 
le polizze claims made prevedono , come clausola aggiuntiva da richiedere espressamente , la copertura di un periodo pregresso , per limitato a due o al massimo a quattro anni . 
bene sapere , sottoscrivendo questo tipo di contratti , che in radiologia una quota di sinistri rimane comunque al di fuori delle garanzie contrattuali . lelevata frequenza di denunce riferite a pazienti deceduti o con lesioni gravi , e la correlata insorgenza di responsabilit penali , devono indurre il radiologo alla pi scrupolosa adesione alle buone pratiche professionali in ogni momento della propria attivit . 
non sembri quindi pleonastico ricordare che il medico radiologo dovr quindi porre sempre la massima attenzione alle notizie fornite dal paziente prima dellesecuzione dellesame , e richiedere sempre la sottoscrizione di un valido consenso informato soprattutto nel caso di esecuzione di esami contrastografici o invasivi . 
la massima cura dovr essere dedicata alla tecnica , alle apparecchiature e alle caratteristiche ambientali della sala radiologica ; si ricordi , al proposito , che la legge prevede precise caratteristiche di sicurezza degli ambienti radiologici , delle quali il radiologo responsabile nella sua veste di datore di lavoro ( in una struttura di sua propriet ) o di dirigente ( se opera in una struttura pubblica o di altrui propriet )  . 
magnavita : a 12 - year follow - up study of malpractice claims against radiologists in italy should be performed with the necessary care and possibly integrated with reviews and comparisons of interpretations of other colleagues . 
in formulating the findings , the radiologist should indicate any unexpected or relevant data , as well as all limitations to interpretation and any further examinations that would be required to clarify the diagnostic query . the radiologist - patient relationship should also be given significant attention , especially since many malpractice claims are prompted by inadequate information flows regarding diagnostic possibilities and results . 
unfortunately , precisely this aspect , which is highly qualifying for the radiologist and reassuring for patients in relation to the level of attention and professionalism with which they will be examined , is increasingly impeded and sacrificed by the need for productivity imposed by radiology departments . 
yet paradoxically in the healthcare setting , and especially in radiology characterised by rapid technological development , the best defence for facing the phenomenon of legal action seems to be the improvement of those human characteristics that lie at the heart of the medical vocation . zione e possibilmente integrata da revisioni e confronti con linterpretazione di altri colleghi . 
nel formulare il referto il radiologo avr cura di segnalare eventuali dati inaspettati o rilevanti , cos come tutti i limiti interpretativi e gli eventuali ulteriori accertamenti che dovessero essere necessari per chiarire il quesito diagnostico . si raccomanda in particolare di curare al massimo il rapporto tra il medico radiologo ed il paziente , giacch molte denunce scaturiscono proprio da una inadeguatezza dei flussi informativi circa le possibilit diagnostiche ed i risultati raggiunti . 
purtroppo proprio questo aspetto , che quanto mai qualificante per il radiologo e rassicurante per il paziente circa lattenzione e la professionalit con cui verr esaminato , sempre pi ostacolato e sacrificato dalle esigenze di produttivit poste ai servizi di radiologia . 
gortenuti1 1dipartimento di radiologia , 2divisione di chirurgia clinicizzata , 3servizio di anatomia patologica , azienda ospedaliera di verona , p.le stefani 1 , i - 37126 verona , italy correspondence to : g . 
nineteen patients ( 13 men and six women ) scheduled for cea between march and august 2004 were imaged on a 1.5 - t scanner ( magnetom symphony , siemens , erlangen , germany )  . 
the protocol included four types of sequences [ t1 , t2 , proton density ( pd ) and three - dimensional time of flight ( 3d - tof ) ]  . 
therefore , it may be useful in evaluating and guiding the treatment of haemodynamically nonsignificant stenoses with a potential embolic risk and , in the future , to assess coronary plaque . key words magnetic resonance atherosclerosis carotid arteries atherosclerotic plaque riassunto obiettivo . 
19 pazienti ( 13 maschi e 6 femmine ) , programmati per intervento di tea , sono stati sottoposti ad esame rm ( 1 , 5 t magnetom symphony , siemens , erlangen , germania ) nel periodo compreso tra marzo e agosto 2004 . 
la rm ha identificato la presenza del core lipidiconecrotico con sensibilit e specificit del 91 , 6% e del 95 , 0% , rispettivamente , mentre la sola emorragia intraplacca con sensibilit e specificit rispettivamente del 91 , 6% e del 100% . 
la rm in grado di identificare i segni di instabilit delle placche carotidee con elevata sensibilit e specificit . potrebbe quindi essere utile nella valutazione delle stenosi emodinamicamente non significative con un potenziale rischio emboligeno , in modo di scegliere il miglior trattamento e nel futuro per la valutazione delle placche a livello coronarico . parole chiave risonanza magnetica aterosclerosi arterie carotidi placche aterosclerotiche g . 
randomised trials have demonstrated the efficacy of thromboendarterectomy ( tea ) compared with medical therapy in symptomatic patients with stenosis > 70% [ north american symptomatic carotid endarterectomy trial ( nascet ) 1991 ] [ 2 , 3 ]  . 
asymptomatic patients with stenoses greater than 60% treated with surgery have been shown to be less likely to develop ipsilateral transient ischaemic attacks ( tia ) at 5 years compared with medically treated patients [ 4 , 5 ]  . 
a number of studies [ 614 ] have demonstrated that magnetic resonance imaging ( mri ) has a high sensitivity and specificity for characterising atherosclerotic plaque composition in vivo and ex vivo . 
 [ 13 ] were the first to use three - dimensional time of flight ( 3d - tof ) bright - blood sequences for evaluating the fibrous cap whereas yuan et al . 
 [ 9 ] were the first to assess the in vivo accuracy of mri in identifying the presence of a soft core composed of a lipid - rich acellular region and / or the presence of haemorrhage . 
 this paper presents an mri - histology correlation study of the morphological features of carotid plaque , with special reference to the presence of lipid - rich core and intraplaque haemorrhage , in symptomatic patients with stenosis greater than 70% undergoing tea . materials and methods the study is based on the evaluation of tissue contrast features at mri . 
 between march and august 2004 , we prospectively studied 19 patients with symptomatic carotid disease ( 13 men and six women , mean age 72.5 years , age range 5981 ) using mri and mr angiography . 
patients had carotid stenosis greater than 70% diagnosed with doppler sonography and were symptomatic for the presence of at least one tia or minor stroke during the 3 months before surgery . 
we first assessed plaque morphology and then performed mr angiography of the neck veslaterosclerosi caratterizzata dal progressivo accumulo , a livello dellintima della parete arteriosa , di lipidi , proteine ed esteri del colesterolo con conseguente formazione di placche che possono ostruire il lume vasale o fissurarsi determinando occlusione acuta del vaso o provocare disseminazione emboligena [ 1 ]  . 
attualmente vivo linteresse per la valutazione delle lesioni a livello carotideo in considerazione dellalto impatto socio - economico che rappresenta lo stroke ( circa 4 , 5 milioni di morti allanno e 5 disabili su 1000 persone , nel mondo )  . 
trials randomizzati hanno dimostrato lefficacia della trombo - endo - arterectomia ( tea ) rispetto alla terapia medica nei pazienti sintomatici con stenosi > 70% ( nascet 1991 ) [ 2 , 3 ]  . 
in questi ultimi stato riportato che le stenosi superiori al 60% trattate chirurgicamente presentano una minor possibilit di sviluppare tia omolaterale a distanza di 5 anni , rispetto ai pazienti trattati farmacologicamente [ 4 , 5 ]  . studi autoptici hanno mostrato come il rischio clinico dellaterosclerosi non correli solo con il grado di stenosi , ma anche con le caratteristiche morfologiche delle placche . recentemente numerose pubblicazioni [ 614 ] hanno dimostrato che la rm in grado di caratterizzare in vivo ed ex vivo la composizione delle placche aterosclerotiche con alta sensibilit e specificit . 
in particolare hatsukami [ 13 ] stato il primo ad utilizzare le sequenze 3d tof bright - blood per la valutazione del cappuccio fibroso , mentre il gruppo di yuan [ 9 ] ha valutato per primo in vivo laccuratezza della rm nellidentificare la presenza di un core soffice composto da una regione acellulare ricca di lipidi e / o con presenza di emorragia . lo scopo di questo lavoro descrivere la nostra esperienza nella valutazione comparata rm / istologia , delle caratteristiche morfologiche delle placche carotidee , in particolare riguardo la presenza di core lipidico ed emorragia intraplacca , in pazienti sintomatici , con stenosi superiore al 70% , sottoposti ad intervento di tea . materiali e metodi lo studio si basa sulla valutazione delle caratteristiche di contrasto tissutale delle immagini rm . 
listologia stata considerata come gold standard . nel periodo compreso tra marzo e agosto 2004 abbiamo studiato prospetticamente 19 pazienti ( 13 maschi , et media 72 , 5 anni , range 5981 anni ) con sintomatologia suggestiva di patologia carotidea , mediante rm e angio - rm , che successivamente sono stati sottoposti a intervento di tea . tutti i pazienti presentavano una stenosi carotidea superiore al 70% diagnosticata mediante eco - doppler , sintomatica per la presenza di almeno un tia o un minor stroke nei 3 mesi precedenti lintervento chirurgico . lindagine rm stata condotta con un magnete da 1 , 5 t g . 
si utilizzato un protocollo desame standard come segnalato in letteratura , ottenendo immagini in 4 differenti pesature ( time of flight - tof , t1 , densit protonica e t2 pesate )  . 
dalla parte centrale di una fetta macroscopica dello spessore di 3 mm corrispondente alla scansione rm , sono state ricavate sezioni istologiche da 5 micron colorate con ematossilina - eosina . 
tutti i pazienti sono stati preventivamente informati del tipo di procedura e di studio al quale venivano sottoposti . per ogni tipo di sequenza abbiamo effettuato 4 scansioni ( immagini ) della placca , dello spessore e con intervallo di 3 mm , che poi sono state correlate con le corrispondenti sezioni istologiche derivanti dal pezzo operatorio . 
le immagini rm sono state analizzate da due radiologi , i quali congiuntamente hanno valutato tutte le sequenze ed espresso un parere comune in merito alla presenza di core lipidico / necrotico ed emorragia intraplacca.il muscolo sternocleidomastoideo stato preso come riferimento relativamente allintensit del segnale , per linterpretazione delle immagini . 
il core lipidico necrotico si individua come unarea di iperintensit di segnale nelle sequenze t1 , di segnale intermedio ( isointenso ) nelle sequenze 3d tof e di intensit variabile nelle sequenze t2 e dp . 
surgical specimens were fixed in formaldehyde , decalcified and embedded in parafffrom the central portion of a 3 - mm section corresponding to the mri slab , 5 - micron histological sections were obtained and stained with haematoxylin and eosin ( h&e )  . 
all patients were informed beforehand of the type of procedure and study they would undergo . for each sequence , we obtained four scans ( images ) of the plaque , with a thickness and interval of 3 mm , which were then correlated with the corresponding histological sections . 
the lipid - rich necrotic core appears as an area of high signal intensity on t1 , intermediate signal intensity ( isointense ) on 3d - tof and variable intensity on t2 and pd . 
in four patients , we evaluated three histological sections . mri identified the presence of a lipid - rich necrotic core and / or intraplaque haemorrhage in 45 / 56 regions as against 48 / 56 detected at histology , resulting in three false negatives and no false positives . 
sensitivity and specificity of mri were thus high , 91.6% and 100% , respectively , with a k value of 0.945 ( one false negative and no false positives )  . histology identified calcifications in 40 / 56 sections whereas mri detected them in 32 / 40 , showing a sensitivity and specificity of 80% and 93.7% , respectively , with a k value of 0.652 ( eight false negatives and one false positive )  . comparative mri and histological evaluation of the fibrous cap was possible in 23 / 41 cases only ; three sections showed cap ulceration , all of which were identified by mri . there were no false negatives and four false positives . 
un valore di k > 0 , 75 indica una buona concordanza ; k compreso fra 0 , 40 e 0 , 75 indica concordanza media [ 16 ]  . risultati due pazienti sono stati esclusi dallo studio a causa di artefatti da movimento durante lesecuzione della rm . 
in 4 pazienti sono state valutate 3 sezioni istologiche . la presenza di core lipidico - necrotico e / o emorragia intraplacca stata rilevata alla rm in 45 / 56 regioni , 48 / 56 invece il riscontro allistologia ; 3 quindi i falsi negativi mentre table 3 correlation between magnetic resonance imaging ( mri ) and histology lipid - rich core intraplaque haemorrhage calcifications disrupted fibrous cap g . 
previous reports indicate , however , that vulnerable plaque is characterised by a lipid - rich necrotic core and / or haemorrhagic focus and separated from the vessel lumen by an unstable fibrous cap [ 18 , 21 , 22 ]  . 
studies on the coronary arteries [ 2325 ] have demonstrated that progression of atherosclerosis is sporadic and related to the fissuring or rupture of plaque composed of an acellular core separated from the lumen by a thin fibrous cap . 
given the histological similarity of carotid and coronary plaque , we can assume that similar phenomena of plaque instability underlie tias that arise in patients with haemodynamically nonsignificant stenoses but vulnerable plaque . conventional angiography , such as ct and mr angiography , are unable to assess the morphological characteristics of plaque other than the presence of ulcerations ; in addition , the degree of stenosis does not correlate with plaque composition . as a consequence , an imaging method is needed that is able to provide this type of information . 
moreover , it can also provide information regarding the stage of the plaque : in early stages the lipid - rich necrotic core is surrounded by a fibrous cap with or without calcification whereas in later stages , the plaque may grow and become complex , with loss of continuity of its surface ( fibrous cap disruption ) and presence of haemorrhage or thrombus ; finally , it may calcify and become chronic . 
il core lipidico - necrotico stato riscontrato da solo in 34 / 56 regioni alla rm , mentre istologicamente in 36 / 56 sezioni con sensibilit e specificit rispettivamente del 91 , 6% e del 95 , 0% , con valore di k = 0 , 848 ( 3 falsi negativi e 1 falso positivo )  . 
la rm ha identificato la sola emorragia intraplacca in 11 / 56 sezioni , listologia in 12 / 56 sezioni , con elevate sensibilit e specificit , rispettivamente del 91 , 6% e del 100% ( valore di k 0 , 945 ) ( 1 falso negativo e nessun falso positivo )  . istologicamente le calcificazioni sono state riscontrate in 40 / 56 sezioni ; la rm le ha individuate in 32 / 40 , con sensibilit e specificit rispettivamente dell80% e del 93 , 7% con valore di k = 0 , 652 ( 8 falsi negativi e 1 falso positivo )  . la valutazione comparata rm / istologia del cappuccio fibroso stata possibile solamente in 23 / 41 casi ; 3 sezioni presentavano ulcerazione del cappuccio , tutte identificate dalla rm . 
quindi anche le stenosi lievi e medie possono determinare un infarto del miocardio : non solo il grado di stenosi a determinare levento , ma anche le caratteristiche della placca . 
a lr - nc is identified by hiper - isotense signal on t1 - weighted image ( arrow ) , and b hipo - isotense signal on time - of - flight ( tof ) image ( arrow )  . 
la rm inoltre pu fornire informazioni riguardo lo stadio evolutivo della placca : negli stadi precoci si osserva la presenza del core lipidico e / o necrotico circondato da un cappuccio fibroso con o meno delle calcificazioni , successivamente la placca pu crescere e diventare complessa con possibile difetto della sua superficie ( interruzione del cappuccio fibroso ) e presenza di emorragia o trombo ; infine pu calcificare e cronicizzarsi . noi abbiamo valutato pazienti con stenosi superiore al 70% sintomatici , quindi concettualmente 2 volte a rischio , con alta possibilit di riscontrare placche complesse . 
infatti nell85 , 7% delle sezioni istologiche abbiamo riscontrato segni di vulnerabilit ( core lipidico - necrotico ed emorragia ) individuati con alta sensibilit ( 93 , 7% ) e specificit ( 100% ) dalla rm , senza nessun falso positivo . 
il core , considerato separatamente , il componente pi rappresentato ( 64 , 3% delle sezioni ) mentre lemorragia da sola si rileva in circa 1 / 5 delle sezioni . 
la rm ha dimostrato alta specificit e sensibilit nellidentificazione di entrambi i componenti . valutazione a parte merita il frequente riscontro di calcificazioni ( 40 / 56 sezioni istologiche )  . 
esse non sono certo marker di vulnerabilit , ma indicano piuttosto lo stadio evolutivo avanzato ( cronico ) delle placche in esame : il dato spiegabile con il criterio di selezione scelto per la popolazione in studio e cio la presenza di una stenosi superiore al 70% . 
da sottolineare lalto numero di falsi negativi [ 8 ] nel riconoscimento delle calcificazioni , giustificabile con i limiti intrinseci della metodica . analogamente a quanto riportato in letteratura [ 9 , 26 , 27 ] , confermiamo che la distinzione tra core lipidico ed emorragia intraplacca si effettua fondamentalmente tramite la valutazione combinata delle sequenze t1 e tof . 
come illustrato nelle figure 1 e 2 sia il core lipidico che lemorragia appaiono iperintense in t1 , ma lemorragia pu essere distinta dal core lipidico in quanto appare iperintensa anche nelle sequenze tof , le quali rappresentano quindi le sequenze discriminanti . 
mri proved to have high specificity and sensitivity in the identification of both components . the high frequency of calcifications observed ( 40 / 56 histological sections ) requires separate analysis . 
worthy of note is the high number of mri false negatives [ 8 ] in recognising calcifications , which is explained by the methods intrinsic limits . in agreement with the literature [ 9 , 26 , 27 ] , our study confirms that the distinction between lipid core and intraplaque haemorrhage essentially relies on the combined assessment of t1 and tof sequences . 
adjacent sternocleidomastoid ( scm ) muscle both on t1 - weighted ( a ) ( arrow ) and time - of - flight ( tof ) ( b ) ( arrow ) sequences but isointense on pdw ( c ) ( arrow )  . 
this is fundamental in that fresh and recent haemorrhage could represent the real sign of vulnerability . placca fresca , recente e vecchia , con alta sensibilit utilizzando le quattro diverse sequenze , enfatizzando per , in particolare , il ruolo delle t2 e dp [ 28 ]  . 
questo dato fondamentale perch lemorragia fresca e recente , a differenza di quella vecchia , potrebbe rappresentare il vero segno di vulnerabilit . la scelta del trattamento di questi pazienti , dovrebbe quindi tener conto non solo del grado di stenosi , ma anche della composizione della placca : di fronte ad una placca vulnerabile , anche in assenza di una stenosi significativa , si potrebbe prospettare lipotesi di un atteggiamento interthe choice of treatment in these patients should therefore take into account not only the degree of stenosis but also plaque composition : vulnerable plaque , even without significant stenosis , could prompt an interventional approach rather than medical therapy alone . 
in addition , the information on plaque morphology could be used to choose between open or endovascular surgery on the basis of embolism risk , which can also influence the choice of the cerebral protection device to be used . 
possiamo inoltre sfruttare le informazioni derivate dallo studio della placca per scegliere fra la chirurgica classica piuttosto che lapproccio endovascolare sulla base del rischio emboligeno , che pu influenzare anche la scelta del dispositivo di protezione cerebrale da utilizzare . 
un limite dello studio rappresentato dal numero limitato di pazienti , tutti sintomatici , quindi con placche prevalentemente complesse . lutilizzo di bobine di superficie dedicate , aumentando il potere risolutivo , dovrebbe consentire una miglior accuratezza nella discriminazione delle diverse componenti tissutali . 
pensiamo inoltre che quando gli sviluppi tecnologici lo permetteranno , interessante sar lapplicazione di questa metodica allo studio delle placche coronariche . conclusioni conclusions the results of our study are in agreement with the literature data and confirm that mri has good sensitivity and specificity in identifying the various components of atherosclerotic plaque and , in particular , markers of plaque vulnerability . further studies are , however , needed to validate the method and allow mri to be increasingly used in clinical practice . i risultati del nostro studio sono in linea con i dati riportati in letteratura e hanno confermato che la rm presenta buona sensibilit e specificit nellidentificazione delle varie componenti della placca aterosclerotica , in particolare dei markers di vulnerabilit . 
biondetti1 1department of radiology , 2department of surgery zonda , 3department of hematology and bone transplantation , 4department of surgery beretta est , fondazione irccs , ospedale maggiore policlinico , mangiagalli e regina elena , via f . 
lamarmora 18 , i - 20122 milan , italy , e - mail : alemos@sirm.org received : 27 march 2006 / accepted : 15 june 2006 / published online : 11 october 2006 abstract purpose . 
sedation was required in 31 / 88 ( 35% ) ct examinations in group 1 and in 6 / 46 ( 13% ) in group 2 . conventional angiography was performed in 20 / 88 ( 22% ) cases in group 1 and in 6 / 46 ( 13% ) in group 2 . 
mdct can reduce the need for sedation and conventional angiography in children after liver transplantation . there is no effect on patient motion artefacts . key words multidetector ct hepatic transplantation motion artefacts riassunto obiettivo . 
nel periodo antecedente a settembre 2000 , abbiamo utilizzato la tc a singolo detettore ( sdtc ) , dopo questo periodo , la tc a detettori multipli ( tcmd )  . 
centoventisei bambini di et inferiore ai 6 anni , sono stati sottoposti a 134 tc addominali , 88 eseguite con sdtc ( 65% ; gruppo 1 ) e 46 con tcmd ( 35% ; gruppo 2 )  . 
nel gruppo 1 , la sedazione era necessaria in 31 delle 88 ( 35% ) tc effettuate , mentre nel gruppo 2 in 6 delle 46 ( 13% )  . 
gli artefatti da movimento sono stati individuati in 8 su 88 tc effettuate nel gruppo 1 ( 9% ) e in 4 su 46 nel gruppo 2 ( 8% )  . 
the introduction of multidetector ct ( mdct ) has again created a wealth of new imaging opportunities [ 35 ]  . the ability to acquire multiple projections of raw data with each gantry rotation permits thinner slices with higher resolution and shorter scanning times . 
further advantages of mdct versus single - detector row ct ( sdct ) include more consistent contrast enhancement and higher quality of angiographic reconstructions . the increased speed of mdct , the resulting shorter scan times and the increased ability for vascular reconstructions should decrease the need for sedation and conventional angiography studies as well as the frequency of motion artefacts in infants and younger children . 
the aim of this retrospective study was to address the question of whether or not mdct compared with sdct can decrease sedation needs , the frequency of conventional angiograms and patient motion artefacts in paediatric imaging . 
 materials and methods patients between january 1993 and june 2005 , 126 children 6 years of age or younger underwent 134 ct studies for evaluation of suspected postoperative complications after liver transplantation on the basis of abnormal clinical and sonographic findings . 
 con lintroduzione della tc a detettore singolo ( tcsd ) e successivamente con lintroduzione della tc spirale , alla fine degli anni 80 , luso di questa metodica nei pazienti in et pediatrica notevolmente aumentato [ 1 , 2 ]  . 
inoltre , lavvento della tcmd ha determinato un pi consistente enhancement vascolare ed una pi elevata qualit delle ricostruzioni tridimensionali . lincremento della velocit di scansione ed il risultante breve tempo di acquisizione , nonch la capacit di ottenere ricostruzioni vascolari tri - dimensionali , dovrebbe portare ad una riduzione del bisogno di sedazione , del numero di angiografie convenzionali e degli artefatti da movimento nei pazienti pediatrici . 
precedenti lavori hanno evidenziato che la frequenza di sedazione nei pazienti pediatrici sottoposti a tcmd era inferiore al 5% , rispetto al 30% ottenuto con la tcsd [ 68 ]  . 
quindi , noi abbiamo realizzato questo studio retrospettivo per rispondere alla domanda se limpiego della tcmd , confrontata con la tcsd , pu determinare una riduzione del bisogno di sedazione , della frequenza della angiografie convenzionali e degli artefatti da movimento nei pazienti pediatrici . materiali e metodi pazienti da gennaio 1993 a giugno 2005 , 126 pazienti pediatrici di et inferiore ai 6 anni , sono stati sottoposti a 134 tc delladdome per la valutazione di eventuali complicanze post - trapianto epatico , sulla base dei risultati clinici e di laboratorio e sulla base dei quadri ecografici . 
la tc viene utilizzata quando lecografia e / o la risonanza magnetica non sono diagnostiche , oppure quando richiesta una ulteriore valutazione . abbiamo retrospettivamente rivisto gli esami tc ed i dati clinici del gruppo di pazienti inclusi nel nostro studio . 
inoltre , abbiamo scelto i pazienti con trapianto epatico , in quanto la popolazione studiata nei due periodi era molto simile . questi pazienti avevano simili indicazioni per lesame tc e simili indicazioni per langiografia . 
inoltre , abbiamo utilizzato let di 6 anni come et massima , in quanto eravamo interessati nel valutare il bisogno di sedazione in questo gruppo di pazienti , in quanto solitamente i pazienti pi grandi non hanno bisogno di essere sedati . 
eighty - eight of the 134 studies ( 65% ) were performed with a sdct scanner ( group 1 ) whereas the remaining 46 studies ( 35% ) were performed with a mdct scanner ( group 2 )  . 
in order to reduce patient agitation resulting from venipuncture performed just prior to the administration of contrast material , an intravenous catheter was in place when the child arrived in the ct suite . 
nonionic contrast material ( iopamidolo bioindustria 300 mg / ml , l.i.m , bologna , italy ) was administered at a dose of 2.0 ml / kg by using a power injector ( envision ct , medrad , indianola , pa , usa )  . 
patients examined with an sdct or mdct scanner received intravenous contrast material by means of power injection , using a flow rate of 1.52 ml / s for a 22 - gauge intravenous catheter and 23 ml / s for a 20 - gauge intravenous catheter . 
for the arterial phase , the empiric delay ranged from 12 to 30 s whereas for the portal venous phase , the empiric delay ranged from 60 to 80 s after the start of the intravenous contrast injection . tube current and kilovoltage were adjusted according to body weight [ 9 ]  . 
with the sdct scanner , images were acquired with 5 - mm collimation , 5 - mm reconstruction interval , 10 - mm / s table speed and gantry rotation of 1s . 
mdct examinations were acquired with 0.8 - s gantry rotation , 2.5 - mm collimation , 2.5 - mm reconstruction interval and a table speed of 15 mm / s . 
ct scans were obtained during breath holding at suspended inspiration in cooperative patients and during quiet respiration in children who were unable to comply with breath - holding instructions and in patients who were sedated . for the study and analysis of complex vascular structures and anastomoses , we used multiplanar reformation ( mpr ) and three - dimensional ( 3d ) reconstructions images . 
considerata la natura retrospettiva dello studio , il consenso informato non stato necessario . esami tc durante un periodo complessivo di 12 anni , centoventisei pazienti di et inferiore a 6 anni , sono stati sottoposti a 134 esami tc delladdome . 
in quanto 8 pazienti sono stati ritrapiantati , ciascuno di essi stato sottoposto a 2 esami tc . ottantotto delle 134 tc ( 65% ) sono state effettuate con tcsd ( gruppo 1 ) , mentre le rimanenti 46 sono state effettuate con una tcmd ( gruppo 2 )  . 
lintervallo di tempo medio tra trapianto epatico ed esecuzione della tc stato di 47 giorni ( range , 7 giorni - 3 mesi )  . nel gruppo 1 , tutti gli esami tc sono stati effettuati con limpiego di una tc spirale ad una singola filiera di detettori ( highspeed ct / i , ge medical systems , milwaukee , wi , usa ) , mentre nel gruppo 2 , mediante lutilizzo di una tc a 4 file di detettori ( lightspeed q / xi , ge medical system , milwaukee , wi , usa )  . 
allo scopo di ridurre lagitazione dei piccoli pazienti , che altrimenti si avrebbe se lago cannula per linfusione del mezzo di contrasto fosse posizionato nella sala tc , lago cannula viene posizionato prima che il paziente arrivi alla sala tc . 
stato utilizzato mezzo di contrasto non - ionico ( iopamidolo bioindustria , 300 mg / ml , lim , bologna , italia ) , alla dose di 2 , 0 ml / kg , somministrato mediante una pompa automatica di iniezione ( envision ct , medrad , indianola , pa , usa )  . 
indipendentemente dal tipo di tc utilizzata ( singolo o multi - detettore ) , abbiamo utilizzato un flusso di iniezione pari a 1 , 52 ml / s con un ago cannula di 22 - gauge e di 23 ml / s con un ago cannula di 20 - gauge . 
tutti gli esami tc sono stati eseguiti allo scopo di evidenziare eventuali complicanze vascolari , per questo motivo abbiamo utilizzato una fase tc arteriosa ed una venosa . abbiamo utilizzato un ritardo empirico per iniziare la scansione nei pazienti che pesavano meno di 10 kg ( n = 23 ) , mentre per i pazienti pi grandi abbiamo utilizzato un sistema di sincronizzazione automatico ( smart prep , ge medical systems , milwaukee , wi , usa )  . 
per la fase arteriosa , il ritardo empirico stato dai 12 ai 30 secondi dallinizio dellinfusione del mezzo di contrasto , mentre per la fase portale dai 60 agli 80 secondi . la corrente del tubo ed il voltaggio sono stati aggiustati in base al peso del paziente [ 9 ]  . 
con la tcsd , le immagini sono state acquisite a 5 mm di collimazione , 5 mm di intervallo di ricostruzione e 10 mm / s di velocit di scorrimento del tavolo , con una rotazione del gantry di 1 secondo . 
con la tcmd , le immagini sono state acquisite a 2 , 5 mm collimazione , 2 , 5 mm di intervallo di ricostruzione , 15 mm / s di velocit di scorrimento del tavolo , con una rotazione del gantry di 0 , 8 secondi . 
 oral chloral hydrate ( major pharmaceuticals , rosemont , il , usa ) at a dose of 50100 mg / kg was administered for infants 18 months of age or younger , and intravenous pentobarbital sodium ( farmotal , farmacia spa , milan , italy ) at a dose of 6 mg / kg was administered for children older than 18 months [ 10 , 11 ]  . 
explanation of the procedure and reassurance by both staff and parents was used in nonsedated patients . angiography to assess the frequency of conventional angiography after the introduction of mdct , two paediatric radiologists retrospectively and independently reviewed records from our computerised database . 
 before the introduction of the mdct scanner at our institution , conventional angiography was performed in all cases in which vascular complications were suspected on sdct . since mdct became available , conventional angiography has been performed only to confirm positive mdct angiography findings . 
negative findings are confirmed by sonography . patient motion artefacts motion - related artefacts were randomly assessed in sedated and nonsedated patients by two paediatric radiologists who were blinded to each others reading with respect to sedation and sdct or mdct technology . 
an artefact was considered present if it degraded visualisation of major vascular structures , including hepatic veins , hepatic artery , portal vein , celiac axis and splenic vein on one or more images . 
a consensus was also obtained to determine whether diagnostic quality was good enough to avoid ct repetition . records of every patient were reviewed for any repeated exzienti collaboranti , e durante respirazione libera nei pazienti che non erano in grado di collaborare o che non erano in grado di apprendere le istruzioni per mantenere lapnea , e nei pazienti sedati . per lo studio e lanalisi di strutture vascolari complesse e delle anastomosi , abbiamo utilizzato le ricostruzioni multiplanari ( mpr ) e le ricostruzioni 3d . 
dopo lacquisizione dei dati , i dati grezzi sono stati retro - ricostruiti a 1 , 25 mm di collimazione e 1 , 25 mm di intervallo di ricostruzione ed inviati alla workstation ( advantage windows 3.1 , ge medical systems , milwaukee , wi , usa ) per le riformattazioni in immagini 3d . 
abbiamo usato 2 algoritmi di ricostruzione 3d : volume rendering ( vr ) e maximum intensity projection ( mip )  . sedazione per la valutazione della frequenza di sedazione , due radiologi pediatrici , retrospettivamente ed indipendentemente hanno valutato i dati clinici relativi ai pazienti inclusi nello studio . 
i pazienti sedati ed intubati , provenienti dalla terapia intensiva , non sono stati inclusi nello studio . nei bambini di 18 mesi di et e nei pazienti pi piccoli , abbiamo utilizzato idrato di cloro per os ( major pharmaceuticals , rosemont , ill , usa ) , per la sedazione , alla dose di 50100 mg / kg , mentre per i bambini pi grandi abbiamo utilizzato pentobarbitale sodico ( farmotal , farmacia spa , milano , italia ) , alla dose di 6 mg / kg [ 10 , 11 ]  . 
nei pazienti non sedati , si proceduto alla spiegazione della procedura e a riassicurarli , con laiuto del personale e dei genitori . angiografie convenzionali per la valutazione della frequenza delle angiografie convenzionali , dopo lintroduzione della tcmd , due radiologi pediatrici , retrospettivamente e indipendentemente hanno rivisto la frequenza delle angiografie eseguite prima e dopo lintroduzione della tcmd , estraendo i dati dal nostro database . 
i pazienti sottoposti ad angiografia allo scopo di eseguire procedure interventistiche , non sono stati inclusi nella stima della frequenza delle angiografie . prima dellintroduzione della tcmd nel nostro istituto , langiografia stata eseguita in tutti i casi sospetti per complicanze vascolari . 
the total time required for interpretation for all images was approximately 3 h for reader 1 and 3 h 20 min for reader 2 . statistical analysis students t test was used to compare mean patient ages in groups 1 and 2 . 
all statistical calculations were performed by using software package of spss version 11.00 ( spss , chicago , il , usa )  . results clinical the patient population consisted of 74 boys and 52 girls . clinical findings that prompted ct scanning after us and / or mri examination included abnormal liver function tests , abdominal pain , fever and gastrointestinal signs , including distension , vomiting and diarrhoea . 
un artefatto da considerarsi presente se esso degrada la visualizzazione delle maggiori strutture vascolari , incluse le vene sovra - epatiche , larteria epatica , la vena porta , il tronco celiaco o la vena splenica , su una o pi scansioni . 
gli artefatti non relativi al movimento ( ad esempio , volume parziali , artefatti da scalini ) sono stati ignorati nella valutazione delle immagini . le immagini tc sono state valutate alla work station ( advantage windows 3.1 , ge medical systems , milwaukee , wi , usa )  . 
il tempo totale necessario per linterpretazione di tutte le immagini stato di 3 ore per losservatore 1 e di 3 ore e 20 minuti per losservatore 2 . analisi statistica abbiamo utilizzato il test t di student per confrontare le medie delle et dei pazienti nel gruppo 1 e 2 . 
il test di pearson ( 2 test ) stato utilizzato per confrontare la frequenza di sedazione , il numero di angiografie e gli artefatti da movimento , tra i gruppi 1 e 2 . 
una terza misurazione stata effettuata per lanalisi degli artefatti da movimento . per esprimere la variabilit inter - osservatore nella valutazione degli artefatti da movimento , abbiamo determinato la concordanza complessiva e quella chance - correlata ( cohen statistico )  . 
tutti i calcoli statistici sono stati effettuati mediante limpiego di un software package spss versione 11.0 ( spps , chicago , il , usa )  . risultati clinica la popolazione dei pazienti consisteva in 74 maschi e 52 femmine . 
the overall agreement between the two readers in the assessment of patient motion artefacts was 100% whereas the chance - corrected agreement was expressed as almost perfect for both groups : = 0.83 ( 95% ci = 0.7550.912 ) for group 1 , and = 0.84 ( 95% ci = 0.7340.946 ) for group 2 . 
nei pazienti di et inferiore ad 1 anno , la sedazione stata necessaria in 21 su 28 ( 75% ) delle tcsd eseguite ( due pazienti sono stati sottopoti a 2 tc ciascuno ) ed in 4 su 8 delle tcmd ( 50% ) eseguite ( due pazienti sono stati sottoposti a due tc ciascuno ) ( p < 0 , 001 )  . 
nei pazienti fra 1 e 6 anni di et la sedazione stata necessaria in 10 su 62 tcsd eseguite ( 16% ) e in 2 su 38 ( 5% ) tcmd ( quattro pazienti sono stati sottoposti a 2 tc ciascuno )  . 
1 motion artefacts detected in a 5 - year - old boy with situs inversus following split - liver transplantation by using single - detector computed tomography ( sdct )  . 
non abbiamo ottenuto una differenza statisticamente significativa riguardo la frequenza dellangiografia in relazione allet e al bisogno di sedazione ( p > 1 )  . artefatti da movimento 8 delle 88 tc effettuate nel gruppo 1 ( 9% ) e 4 delle 46 tc effettuate nel gruppo 2 ( 8% ) , presentavano artefatti da movimento . 
la concordanza complessiva tra i due osservatori nellindividuare gli artefatti da movimento era del 100% , mentre la concordanza chance - correlata stata espressa come quasi perfetta per entrambi i gruppi : = 0 , 83 ( 95% ic = 0 , 755 a 0 , 912 ) per il gruppo 1 , e = 0 , 84 ( 95% ic = 0 , 734 a 0 , 946 ) , per il gruppo 2 . 
il ruolo delle ricostruzioni tri - dimensionali ( 3d ) nella valutazione delle strutture vascolari continua ad evolvere diventando sempre pi importante sia per quanto riguarda la valutazione delle complicanze vascolari che per quanto riguarda la cura del paziente . 
le principali applicazioni della tcmd nella valutazione dei pazienti pediatrici dopo trapianto epatico , includono le stenosi vascolari , le trombosi ed i difetti di perfusione del fegato trapiantato . nel nostro studio , nei pazienti da 0 a 6 anni di et , il bisogno di sedazione passato dal 35% con la tcsd , al 13% con la tcmd . 
b le ricostruzioni arteriografiche 3d ottenute dopo dilatazione ed il posizionamento di uno stent , dimostrano perviet del by - pass aorto - epatico ( frecce ) e perviet dellarteria epatica ( teste di freccia )  . discussion mdct plays an important role in the evaluation of vascular complications after liver transplantation . 
the role of 3d volume imaging in the evaluation of vascular structures continues to evolve , with this technique becoming increasingly important in the detection of vascular complications as well as in decisions about patient care . 
the principal clinical applications of mdct in the evaluation of children following liver transplantations include vascular stenoses , thromboses and perfusion defects of the transplanted liver . in our study , for patients in the 06 age range , the sedation rate changed from 35% with single - detector ct to 13% with the mdct scanners . 
pappas et al also reported that they did not need to sedate any patients aged 26 years when mdct was performed whereas in the 16 age group , we had a sedation rate of 5% . 
following surgery , liver transplant recipients are often anxious and agitated , and they are more likely to need sedation than is a random population consisting of some patients who are acutely ill and some who are asymptomatic or relatively asymptomatic . 
decreasing sedation rates would reduce the incidence of potential complications in addition to decreasing parental anxiety , increasing patient throughput and decreasing costs . conventional angiography has remained the standard of reference for the evaluation of vascular complications in liver transplant recipients , but given its invasiveness and cost , a noninvasive alternative technique for evaluating postoperative complications would be of clinical value . 
the advantages of reducing or obviating the need for conventional angiography include decreased frequency of complications such as haematoma formation and vascular injuries [ 13 , 14 ]  . other advantages of ct over conventional angiography include shorter acquisition times , superior 3d rendering and greater range of coverage , which increase detection of vascular and nonvascular abnormalities [ 15 , 16 ]  . 
 in addition to better 3d technology for vascular reconstructions and increased operator ability , we believe that the reduction of angiography rates did not depend on a different fixed protocol used after the advent of mdct at our institution . rather , this reduction simply reflects a different diagnostic aperano estremamente agitati . 
nei risultati ottenuti da pappas et al . , non ci sono stati pazienti che hanno avuto bisogno di sedazione nella fascia di et compresa fra i 2 ed i 6 anni , quando sono stati sottoposti a tcmd , mentre nella fascia di et fra 1 e 6 anni , noi abbiamo avuto una percentuale di sedazione pari al 5% . 
dopo un importante intervento chirurgico , quale il trapianto epatico , i piccoli pazienti sono spesso ansiosi ed agitati e perci necessitano maggior sedazione rispetto ad una popolazione random di pazienti , caratterizzata da alcuni pazienti nella fase acuta della malattia ed altri asintomatici o relativamente asintomatici . 
diminuendo il bisogno di sedazione , si reduce lincidenza di potenziali complicanze , oltre a diminuire lansiet dei genitori , il tempo di ricovero ed i costi . langiografia convenzionale rappresenta il gold standard nella valutazione delle complicanze vascolari dopo trapianto epatico , ma considerata la sua invasivit , ed i costi , una modalit alternativa non - invasiva sarebbe necessaria per la valutazione delle complicanze vascolari nei pazienti pediatrici dopo trapianto epatico . 
tali vantaggi consentono di valutare sia le complicanze vascolari che quelle non - vascolari del fegato trapiantato [ 15 , 16 ]  . oltre ad una miglior tecnologia 3d e ad una maggiore abilit delloperatore nelleffettuare le ricostruzioni vascolari , noi crediamo che la riduzione del numero di angiografie dopo lintroduzione della tcmd nel nostro istituto , non sia dipesa da un differente protocollo fisso utilizzato , ma da un differente approccio diagnostico . 
infatti , prima dellavvento della tcmd tutti i pazienti con sospette complicanze vascolari sono stati sottoposti ad ecografia e successivamente ad angiografia , mentre dopo lavvento della tcmd tutti i pazienti con sospette complicanze vascolari sono stati sottoposti ad ecografia e successivamente a tcmd . 
langiografia veniva effettuata solo per confermare i quadri tc positivi . sebbene la tc , come langiografia , richiedano luso di radiazioni ionizzanti , la dose al paziente della tc pi bassa rispetto a quella di un esame angiografico convenzionale ( 6 msv per una tc bifasica delladdome contro 12 msv per un esame angiografico convenzionale della vascolarizzazione epatica )  . 
per minimizzare la dose di radiazioni abbiamo seguito i criteri alara ( as low as reasonable achievable ) per i pazienti pediatrici sottoposti ad esame tc [ 17 , 18 ]  . 
la tcmd dovrebbe essere effettuata con parametri di acquisizione che provvedano proach : before the advent of mdct , all patients with suspected vascular complications underwent sonography and subsequently conventional angiography whereas after the advent of mdct , all patients with suspected vascular complications underwent sonography and mdct . 
conventional angiography was performed only to confirm positive mdct findings . although ct , like conventional angiography , requires the use of ionising radiation , the dose from mdct is still several times lower than that from conventional angiography ( 6 msv for a double mdct acquisition of the upper abdomen vs 12 msv for a conventional angiogram of the liver vasculature )  . 
these acquisition parameters should include the use of low milliamperage and kilovoltage settings ( a higher kilovoltage will be needed in patients with larger body habitus : > 50 kg ) , appropriate section thickness and a faster table speed [ 4 , 5 , 19 , 20 ]  . 
mdct has the advantage over mri of shorter acquisition times , which means a reduced need for sedation and the ability to scan extremely ill patients who cannot tolerate the long imaging times for mri examinations . 
 in our study , the difference between sedation and angiography rates was statistically significant ( p0.001 ) whereas the difference with regard to patient motion artefacts was not ( p ( cid : 2 ) 1 )  . 
one explanation is the small sample size . the relatively small number of patients is due to the fact that we limited our study to patients with liver transplantations rather than studying all patients who had ct scans . 
before and after the introduction of mdct , we aggressively used restraining straps and blankets in infants and young children and verbal reassurance and explanation of the procedure in older children . 
finally , another possible explanation for the lack of difference in motion artefacts between sdct and mdct may be the correlation between patient movements and scanning speed : in a faster scanner , patient motion may potentially create artefacts on more slices than on a slower scanner . 
questi parametri di acquisizione dovrebbero includere luso di un basso amperaggio e voltaggio ( un voltaggio pi alto necessario nei pazienti con maggiore abito corporeo : > 50 kg ) , appropriati spessori di collimazione e unalta velocit di scorrimento del tavolo [ 4 , 5 , 19 , 20 ]  . 
anche le acquisizioni multifasiche non sono utilizzate nella routine nei pazienti pediatrici e non dovrebbero essere eseguite se non c una specifica indicazione clinica ( es , studio vascolare )  . la tcmd ha anche guadagnato una larga approvazione come alternative alla risonanza magnetica ( rm ) nella diagnosi delle anomalie vascolari nei pazienti pediatrici . 
la tcmd ha il vantaggio rispetto alla rm di minori tempi di acquisizione , che significa un ridotto bisogno di sedazione e labilit di esaminare pazienti estremamente malati che non possono tollerare i lunghi tempi di acquisizione della rm . esiste il rischio di esposizione alle radiazioni con la tcmd , ma il rischio di una prolungata sedazione maggiore rispetto a quello delle radiazioni [ 5 ]  . nel nostro studio , la differenza fra la frequenza di sedazione ed il numero di angiografie convenzionali stata statisticamente significativa ( p0 , 001 ) , mentre quella degli artefatti da movimento non lo stata ( p ( cid : 2 ) 1 )  . 
una pu essere dovuta al limitato campione di pazienti , in quanto abbiamo limitato il nostro studio a pazienti pediatrici dopo trapianto epatico , piuttosto che a tutti i pazienti pediatrici che hanno effettuato un esame tc . 
prima e dopo lintroduzione della tcmd , abbiamo utilizzato in maniera piuttosto aggressiva fasce di contenimento e coperte , nonch la riassicurazione verbale nei pazienti pi piccoli e la spiegazione della procedura nei pazienti pi grandi . 
infine , unaltra possibile spiegazione per la mancanza di una differenza statisticamente significativa negli artefatti da movimento , fra tcsd e tcmd pu essere la correlazione tra i movimenti del paziente e la velocit di scansione : in una tc veloce , i movimenti del paziente possono potenzialmente creare artefatti su pi immagini rispetto a quelli creati in una tc pi lenta . il maggior limite di questo lavoro rappresentato dal fata.a. 
 to che il criterio per sedare o no il paziente stato definito dal radiologo insieme allanestesista e alla nurse della tc , e quindi soggetto ad una certa variabilit . conclusions the higher performance of mdct compared with sdct , the capability of 3d arteriographic reconstructions , better contrast enhancement as well as the speed of the examinations have allowed a reduction in the need for sedation and the number of conventional angiograms . 
a larger population is necessary to determine a significant effect on patient motion artefacts . conclusioni la capacit di effettuare ricostruzioni tri - dimensionali , il migliore enhancement vascolare , cos come la maggior velocit di acquisizione delle immagini , della tcmd rispetto alla tcsd , ha determinato come conseguenza una riduzione della frequenza di sedazione e del numero di angiografie . non c un apparente effetto sugli artefatti da movimento . con il continuo sviluppo della tecnologia tc , realistico sperare in unulteriore riduzione della frequenza di sedazione e delle angiografie convenzionali . 
volpe3 1diagnostica per immagini di euganea medica , via colombo 13 , i - 35020 albignasego ( pd ) , italy 2servizio di radiologia , ospedale civile , monselice ( pd ) , italy 3unit operativa di chirurgia del piede e della caviglia , casa di cura abano terme ( pd ) , italy correspondence to : l . 
the aim of this paper is to demonstrate the efficacy of the dynamic study of the forefoot during lateral compression of the metatarsal heads ( mulders manoeuvre ) in the visualisation of mortons neuroma . 
clinical evaluation , which is fundamental for accurate diagnosis , can make use of dynamic us in the first instance in order to confirm clinical signs and identify the correct site and number of masses . 
in our opinion , mr maintains a primary role in differential diagnosis with other diseases ( mainly stress fractures , bursitis , ganglion cysts or tendon tumour sheaths )  . key words foot ultrasound magnetic resonance , neuroma mortons neuroma riassunto obiettivo . 
nei 40 avampiedi esaminati lecografia dinamica ha permesso di riconoscere 37 formazioni espansive intermetatarsali ( 2 doppie localizzazioni ) , dimostrandosi maggiormente efficace rispetto alla sola valutazione statica che ne ha dimostrate solo 25 . negli avampiedi studiati con rm si avuta la conferma del sospetto ecografico di formazione espansiva in 16 su 22 rilievi ecografici con valutazione dinamica ; uno dei 6 casi non confermati alla rm stato successivamente operato ottenendo il riscontro chirurgico del neuroma . 
la valutazione clinica , fondamentale ai fini della diagnosi , pu giovarsi dellecografia dinamica come metodica strumentale di primo impiego per la verifica del sospetto clinico , lidentificazione della sede e del numero di localizzazioni ; la rm a nostro parere mantiene un ruolo determinante nella diagnosi differenziale con altre patologie ( principalmente fratture da stress , borsiti , ganglio sinoviale o tumore gigantocellulare delle guaine tendinee )  . parole chiave piede , ecografia piede , risonanza magnetica neuroma di morton l . 
the aim of our study was to compare clinical , surgical and magnetic resonance ( mr ) findings with results obtained from examination of the intermetatarsal spaces with both static and dynamic ultrasound ( us )  . 
 la sindrome di morton una metatarsalgia neuralgica causata dallintrappolamento del nervo digitale comune nel canale metatarsale ; il traumatismo continuo determina un processo flogistico perineurale che a sua volta induce proliferazione connettivale endo - perineurale e degenerazione ialina dellendonevrio [ 1 ]  . 
la diagnostica per immagini viene impiegata per confermare il sospetto clinico , la sede ed il numero di localizzazioni e nella diagnosi differenziale con altre patologie [ 24 ]  . 
scopo del nostro lavoro stato quello di comparare i dati clinici , i rilievi chirurgici ed i riscontri rm con i risultati ottenuti dallesame degli spazi intermetatarsali con ecografia sia statica che dinamica , questultima ottenuta valutando ecograficamente gli effetti della compressione laterale delle teste metatarsali , in pazienti con sospetto clinico di neuroma di morton . materials and methods a total of 40 forefeet were studied in 38 patients ( 29 women and nine men , age range 2574 years ) , all with a clinical picture of interdigital neuritis ( table 1 )  . 
 following us assessment , mr was performed on 26 / 40 forefeet using dedicated low - field equipment ( esaote artoscan ) to obtain spin echo ( se ) t1 sequences ( tr / te 400 / 20 ms ) , se t2 sequences ( tr / te 2 , 600 / 90 ms ) and short - tau inversion recovery ( stir ) sequences ( tr / te / ti 1 , 580 / 22 / 65 ms ) , with a slice thickness of 4 mm in the sagittal , coronal and axial planes . a total of 20 intermetatarsal spaces were surgically explored in 18 forefeet ( two spaces were examined simultaneously in two cases )  . 
surgical treatment consisted of neurecmateriali e metodi sono stati studiati con ecografia in tutto 40 avampiedi in 38 pazienti ( 29 femmine e 9 maschi , di et compresa tra i 25 ed i 74 anni ) , con quadro clinico riferibile a neurite intermetatarsale ( tabella 1 )  . 
alla valutazione statica si associata lanalisi dinamica ottenuta con la compressione laterale delle teste dei metatarsi eseguita manualmente come nella manovra di mulder ; simultaneamente si registrato il quadro ecografico in sequenze di video - clip . alla valutazione ecografica seguita una rm di 26 / 40 avampiedi , utilizzando un apparecchio dedicato a basso campo ( esaote artoscan ) , ottenendo acquisizioni se t1 ( tr / te 400 / 20 ms ) , se t2 ( tr / te 2600 / 90 ms ) e stir ( tr / te / ti 1580 / 22 / 65 ms ) secondo i piani sagittale , coronale e assiale , con spessore di strato di 4 mm . table 1 summary of all cases forefeet , n tabella 1 sintesi della casistica in esame avampiedi , n clinical evaluation static and dynamic sonography surgery chirurgia valutazione clinica ecografia statica e dinamica l . 
lesplorazione chirurgica dei 20 spazi intermetatarsali ha rilevato in 19 la presenza di un neuroma di morton ; in un caso si riscontrato un ganglio sinoviale in continuit con la guaina del tendine flessore del 4 dito . complessivamente sono state identificate con ecografia nei 40 avampiedi esaminati ( tabella 2 ) 37 formazioni espansive di cui 36 formazioni nodulari solide ed 1 a contenuto fluido ( in 4 pazienti erano presenti 2 localizzazioni : in 2 pazienti nello stesso avampiede mentre negli altri due erano localizzate una per ciascun avampiede )  . 
a paziente supino si esplora il versante plantare con lieve inclinazione caudo - craniale della sonda ; la mano libera dellesaminatore esegue la manovra di compressione dei metatarsi . tomy through the intermetatarsal dorsal approach , with excision of the nerve at least 1 cm above the proximal margin of the deep transverse metatarsal ligament and repositioning of the nerve stump in the oblique adductor muscle . 
 results correlazione ecografia statica , ecografia dinamica , rm all patients had symptoms of intermetatarsal neuritis characterised by episodic pain in the distal metatarsal region , nei 40 avampiedi esplorati erano riconoscibili con ecografia statica ( es ) 25 / 37 ( 67 , 6% ) delle formazioni espansive ritable 2 correlation of data between static sonography , dynamic sonography and magnetic resonance ( mr ) in all cases investigated lesions / forefeet , n 25a / 40 37b / 40 static sonography dynamic sonography aone forefoot with double localisation btwo forefeet with double localisation tabella 2 confronto tra i dati relativi alla ecografia statica , ecografia dinamica e rm nel riscontro di formazioni espansive intermetatarsali in tutti i pazienti considerati nello studio lesioni / avampiedi , n 25a / 40 37 b / 40 ecografia statica ecografia dinamica aun avampiede con duplice localizzazione bdue avampiedi con duplice localizzazione 16a / 26 16 a / 26 l . 
in one case , a synovial ganglion was found in continuity with the flexor tendon sheath of the fourth toe . us identified 37 masses in the 40 forefeet examined ( table 2 )  . 
thirty - six were solid nodular formations , and one had a fluid content ( two localisations were present in four patients : two on the same forefoot in two patients , and one on each forefoot in the other two )  . 
in all cases , there was correlation between the site of the us finding and clinical examination ( pain on compression of the site )  . correlation between static us , dynamic us and mr in the 40 forefeet examined , static us ( sus ) detected 25 / 37 ( 67.6% ) of masses identified by us : 24 of these were small hypoechoic masses , with a diameter ranging from 4 mm to 9 mm , prominent on the plantar side at the level of the intermetatarsal space . 
sequenza di immagini ottenute prima ( a ) , e nel corso ( b , c ) della compressione laterale dei metatarsi che evidenzia il neuroma ( frecce ) riconoscibile solo nella fase dinamica , protrudere dallo spazio intermetatarsale per effetto della spremitura . scontrate complessivamente con ecografia : di esse 24 erano masserelle solide ipoecogene rispetto ai tessuti circostanti , con diametro variabile da 4 a 9 mm , prominenti sul versante plantare in corrispondenza dello spazio intermetatarsale . altre 12 formazioni ( 32 , 4% ) con analoghe caratteristiche ecografiche sono risultate riconoscibili solo con ecografia dinamica ( ed )  . 
3a sonography shows a fluid - filled mass , with mild inner echoes , protruding onto the plantar aspect at the third intermetatarsal space ( arrows ) and fixed during the dynamic manoeuvre related to the synovial cyst . 
b axial magnetic resonance ( mr ) t1 - weighted and coronal short - tau inversion recovery ( stir ) images confirm the presence of a fluid - filled mass in the intermetatarsal space ( arrows )  . 
3a scansione ecografica che dimostra una formazione a contenuto liquido con echi interni prominenti sul versante plantare tra le teste del 3 e 4 metatarso , immobile alla valutazione dinamica , compatibile con ganglio sinoviale . 
b acquisizioni rm assiale t1 e coronale stir confermano la presenza di formazione a contenuto fluido nel terzo spazio intermetatarsale . sonographic features were detected by dynamic us ( dus ) only . 
quindici di esse corrispondevano a formazioni solide nello spazio intermetatarsale , ipointense nelle acquisizioni t1 e t2 pesate con i caratteri del neuroma ( in un paziente era presente duplice localizzazione nello stesso avampiede ) ; in 8 di esse si associava il rilievo di una fig . 
4a , b longitudinal ultrasound ( us ) scan ( a ) in correspondence with the third metatarsal head showing thickening of the capsular structures on the plantar aspect related to capsulitis . 
fifteen of these were solid masses in the intermetatarsal space and hyperintense in the t1and t2 - weighted images with the appearance of neuroma ( one patient had two localisations on the same forefoot ) ; in eight of these , the finding was associated with fluid distension of the intermetatarsal bursa . 
 one of the six lesions seen on us and not confirmed on mr was later identified by surgical exploration , which identified a 5 - mm diameter neuromatous mass ; the other five received conservative treatment . 
1 , legamento trasverso metatarsale profondo e superficiale ; 2 , fascio neurovascolare ; 3 , muscolo e tendine lombricale ; 4 , tendini flessori delle dita ; 5 , borse intermetatarsali ; 6 , muscoli interossei . sistently confirmed the presence of a mass , with identification of 20 masses in 18 forefeet ( in two cases , there were two localisations on the same forefoot ) : 13 / 20 ( 65% ) were recognised using sus whereas 7 / 20 ( 35% ) were only seen on dus . among patients who underwent surgery , mr assessments were available for ten forefeet : mr correctly identified 8 / 11 lesions ( 72.7 % ) ( one patient had double localisation on the same forefoot ) ; in 2 / 11 ( 18.2% ) , results were uncertain , even on retrospective analysis ; in 1 / 11 ( 9.1% ) , results were negative . 
the neurovascular bundle and lumbrical muscle pass through the lower compartment immersed in abundant fat tissue whereas in the higher level , also immersed in fat , we can find the tendons of the interosseous muscles and the intermetatarsal bursa . 
in its relationship with the deep transverse metatarsal ligament , the latter is most prominent distal to the level of the second or third space beyond which it comes into contact with the neurovascular bundle [ 5 ]  . ( 9 , 1% )  . 
nella loggia inferiore decorre il fascio neurovascolare ed il muscolo lombricale immersi in unatmosfera adiposa , mentre nella loggia superiore , immersi anchessi nel tessuto adiposo , si trovano i tendini dei muscoli interossei e la borsa intermetatarsale ; questultima nel suo rapporto con il ligamento trasverso metatarsale profondo si dimostra maggiormente prominente distalmente a livello del 2 e 3 spazio ove , superandolo , giunge a contatto con il fascio neurovascolare [ 5 ]  . la metatarsalgia neuralgica nella sindrome di morton dunque da considerarsi una sindrome da impingement del nervo digitale comune nello spazio intermetatarsale legata a dislocamento dorsale del nervo tra le teste metatarsali a seguito della pressione plantare e successivamente pinzato tra le teste stesse , in associazione o meno a concomitante borsite . 
il continuo traumatismo determina un processo flogistico perineurale che induce proliferazione connettivale endo - perineurale e degenerazione ialina dellendonevrio [ 1 ] con ingrossamento del nervo che alimenta il meccanismo di impinl . 
 mulders manoeuvre is important in the diagnosis of the neuroma and consists of lateral compression of the metatarsal heads in alternation with plantar pressure , which tends to induce plantar dislocation of the intermetatarsal mass provoking a clicking sensation ( mulders click )  . 
 in our series , the efficacy of us in identifying intermetatarsal masses improved with the use of dynamic assessment , the sensitivity in the group with surgical correlation being 65% for sus and 100% for dus . 
these results seem to be inferior to those reported in the literature in terms of static assessment but are aligned with the best results obtained by dynamic assessment in the most recent series [ 69 ]  . 
one justification for the different sensitivity values of static us in our series may be its limited use , with only the plantar approach and without using the usual manual interdigital compression . 
dynamic assessment with lateral compression of the metatarsal heads , in our opinion , increases us sensitivity , allowing recognition of the neuroma as it is expelled from the intermetatarsal canal , which it entered due to plantar pressure or attraction by neighbouring structures , such as the intermetatarsal bursa ( which at surgery is often indistinguishable from the nerve )  . 
 in our series , mr had a sensitivity of 72.7% in the examination of intermetatarsal masses , which is lower than that found in studies using high - field equipment [ 10 ]  . 
in our experience , in particular , the most effective sequences for recognising the neuroma proved to be se t1 and t2 in the axial and coronal planes whereas identification with stir sequences was affected by oedema of plantar soft tissue that can mask the neuroma [ 10 ]  . 
in the five cases that did not undergo surgery , mr was nonetheless able to account for the clinical picture , with findings compatible with mechanical support capsulitis in three cases and a stress fracture of the third metatarsal in two . 
tale entit anatomo - patologica comunemente conosciuta come neuroma di morton . la manovra di mulder , importante manovra semeiologica nella diagnosi del neuroma , consiste nella compressione laterale delle teste metatarsali alternata a pressione plantare , che tende a provocare la lussazione plantare della massa intermetatarsale evocando una sensazione di scatto ( click di mulder ) ; tale meccanismo rende maggiormente evidente allecografia la massa stessa durante la spremitura dallo spazio intermetatarsale apparendo questa maggiormente prominente sul versante plantare . nella nostra casistica lecografia si rilevata maggiormente efficace nel riscontro di formazioni espansive intermetatarsali , quando si utilizzata la valutazione dinamica con una sensibilit nel campione con verifica chirurgica del 65% per la es e del 100% per la ed ; tali risultati appaiono inferiori a quanto riportato in letteratura per quello che riguarda la valutazione statica mentre si allineano ai migliori risultati ottenuti nelle casistiche della letteratura anche pi recente considerando la valutazione ecografica dinamica [ 69 ]  . 
una giustificazione alla discrepanza nei valori di sensibilit della ecografia statica pu essere ricercata nelluso limitato di tale metodo considerando nella nostra casistica il solo approccio plantare senza lutilizzo delle usuali tecniche di compressione manuale interdigitale . 
la valutazione dinamica con compressione laterale delle teste metatarsali aumenta a nostro giudizio comunque la sensibilit della metodica , consentendo il riconoscimento della masserella neuromatosa nella sua espulsione dal canale intermetatarsale , ove la stessa risalita a causa della pressione plantare o per attrazione da parte di strutture limitrofe come la borsa intermetatarsale ( che al tavolo operatorio risulta spesso indissociabile dal nervo )  . lindagine rm ha dimostrato nella nostra serie una sensibilit del 72 , 7% nel riscontro di formazioni espansive intermetatarsali ; tale dato risulta inferiore a quanto riportato in letteratura con indagini eseguite peraltro utilizzando apparecchiature ad alto campo [ 10 ]  . 
in particolare nella nostra esperienza le sequenze pi efficaci nel dimostrare il neuroma sono risultate le se t1 e t2 nei piani assiale e coronale , mentre le sequenze stir hanno presentato maggiori problemi nellidentificazione della massa neuromatosa in relazione alla presenza concomitante di edema dei tessuti molli plantari che pu mascherarla [ 10 ]  . 
in 5 casi non operati infine la rm ha potuto comunque giustificare il quadro clinico riscontrando in 3 di essi un quadro compatibile con una capsulite meccanica da appoggio ed in 2 una frattura da durata del 3 metatarso . conclusioni diagnosis of neuroma remains prevalently based on clinical findings , partly in consideration of the high incidence of la diagnosi di neuroma resta legata prevalentemente ai rilievi clinici , anche in considerazione dellelevata incidenza di riscontro di formazioni compatibili con neuromi tanto alconclusions l . 
diagnostic imaging ( us and mr ) is used to confirm the clinical diagnosis , site and number of localisations and in differential diagnosis with other conditions [ 6 , 10 , 12 ]  . in our experience , us remains the initial modality for assessing the forefoot of the patient with neuralgic metatarsalgia ; dynamic assessment allows easy identification of intermetatarsal masses . 
mr should be left as a second - level investigation to be carried out after us has failed to justify the clinical findings ; when other conditions need to be ruled out , such as stress fractures , bursitis or synovitis ; or to better characterise capsulitis , a synovial ganglion or a giant cell tumour of the tendon sheath . la ecografia che alla rm in pazienti asintomatici [ 11 , 12 ]  . la diagnostica per immagini ( ecografia e rm ) , viene impiegata per confermare il sospetto clinico , la sede ed il numero di localizzazioni e nella diagnosi differenziale con altre patologie [ 6 , 10 , 12 ]  . lecografia nella nostra esperienza resta metodica di primo impiego nella valutazione dellavampiede del paziente con metatarsalgia neuralgica consentendo con la valutazione dinamica il facile riscontro di processi espansivi intermetatarsali . 
da pozzo , istituto di radiologia , via giustiniani 5 , i - 35128 padova , italy , e - mail : spoxius@libero.it received : 24 february 2006 / accepted : 28 june 2006 / published online : 11 october 2006 abstract purpose . 
progressive multifocal leukoencephalopathy ( pml ) is a serious disorder that primarily affects individuals with a suppressed immune systefew semiological elements help clearly distinguish pml from other diseases included in the differential diagnosis . 
the purpose of this study was to confirm the diagnostic value of conventional mri sequences combined with diffusion - weighted imaging ( dwi ) in pml to identify those patients with worst prognosis . 
dwi permits more accurate differentiation of the disease progression front , which exhibits low signal intensity in apparent diffusion coefficient ( adc ) maps , from the central gliotic area of demyelinisation , characterised by high adc values . 
the addition of dwi sequences to conventional mri seems to be a valid method for accurately diagnosing pml and establishing the degree of disease progression . key words progressive multifocal leukoencephalopathy diffusion - weighted magnetic resonance imaging riassunto obiettivo . 
le sequenze pesate in diffusione permettono pi accuratamente di differenziare il fronte di progressione della malattia che appare ipointenso nelle mappe adc , dalla porzione centrale di demielinizzazione con riparazione gliotica ( elevati valori adc )  . 
le sequenze convenzionali associate alle nuove sequenze pesate in diffusione sembrano in grado di porre diagnosi di pml e di fornire indicazioni sul grado di progressione della malattia . parole chiave leucoencefalopatia multifocale progressiva risonanza magnetica diffusion - weighted s . 
among these disorders , progressive multifocal leukoencephalopathy ( pml ) is caused by the reactivation of human polyomavirus jc ( jcv ) , which remains latent in the organism and becomes pathogenic and aggressive in conditions of serious iatrogenic immunodeficiency as in leukaemia and lymphoma or of immunodepression mediated by other pathogens influencing the immune system directly as in patients with acquired immunodeficiency syndrome ( aids )  . 
jcv selectively affects oligodendrocytes , causing their cytolysis and consequent demyelination [ 2 ]  . the clinical presentation of pml is usually characterised by a progressive decline in mental status ( cognitive function ) often associated with neurological focal , motor and visual deficits ( homonymous hemianopsia )  . cerebral magnetic resonance imaging ( mri ) permits a noninvasive study of the brain and accurately reveals multifocal lesions of the white matter , which are hypointense on t1 - weighted sequences and hyperintense on t2 , often located in the grey / white interface with involvement of u - fibres [ 3 ]  . the most affected area is the semioval centre although the posterior cranial fossa is involved in about one third of all cases [ 4 ]  . 
in particular , reaction to contrast agents at the lesion margins seems to be related to improvement in the patients immunocompetence and the presence of mass effect to particularly aggressive forms [ 5 ]  . neuropathological studies have identified areas with different stages of white matter demyelination in the same lesion . 
older lesions and the central portion of more recent ones are characterised by almost total breakdown of the myelin sheath and axonal damage , with an increase in extracellular space . 
other studies [ 6 ] have recently discovered some interesting peculiarities of pml lesions in diffusionweighted sequences : although homogeneous on t1 and t2 , these lesions are characterised by different signal intensity on diffusion - weighted sequences and different apparent diffusion coefficient ( adc ) values . the aim of this paper is to illustrate the differences between gliotic and necrotic components and active disease elements in order to help establish the diagnosis and assess disease progression in response to treatment . pazienti immunocompromessi presentano spesso disturbi neurologici [ 1 ] dovuti ad malattie opportunistiche o neoplastiche la cui diagnosi differenziale affidata , oltre che alla neuroimmagine , anche ad esami laboratoristici del liquor e del sangue , alla risposta alle terapie e alla evoluzione nel tempo delle lesioni.fra queste malattie la pml legata alla riattivazione di un virus jc che , ubiquitario nellorganismo , diventa patogeno ed aggressivo in condizioni di grave immunodepressione iatrogena , come nelle leucemie e nei linfomi , o mediata da altri agenti patogeni che agiscono direttamente sul sistema immunitario , come nei pazienti aids . 
la presentazione clinica caratterizzata da un declino progressivo delle funzioni cognitive spesso associato a deficit neurologici focali motori o visivi ( emianopsia omonima )  . la rm cerebrale permette lo studio non invasivo dellencefalo riconoscendo con elevata sensibilit lesioni multifocali della sostanza bianca che appaiono ipointense nelle sequenze t1 pesate , iperintense in t2 , spesso localizzate a livello dellinterfaccia fra sostanza grigia e bianca con coinvolgimento delle fibre ad u [ 3 ]  . 
in particolare , la risposta al mezzo di contrasto ai margini delle lesioni sembra in relazione al miglioramento della immunocompetenza del soggetto mentre la presenza di effetto massa sembra essere correlata a forme particolarmente aggressive [ 5 ]  . studi neuropatologici hanno evidenziato allinterno della stessa lesione aree con differenti stadi di demielinizzazione della sostanza bianca . 
le lesioni pi datate cos come la porzione centrale delle lesioni recenti sembrano caratterizzate da una quasi totale perdita di mielina e da un danno assonale con allargamento degli spazi extracellulari . 
recentemente alcuni autori [ 6 ] hanno rilevato alcune specificit delle sequenze pesate in diffusione nellanalisi delle lesioni della pml evidenziando come esse , pur apparendo omogenee in t1 e t2 , siano invece caratterizzate da diverse intensit di segnale nella sequenza pesata in diffusione e da diversi valori di adc . lo scopo di questo studio di sottolineare le differenze fra componenti gliotiche o necrotiche e componenti in fase attiva di malattia al fine di aiutare a porre diagnosi e per valutare la progressione della malattia in risposta alle terapie effettuate . materials and methods we examined four men aged between 40 and 50 years affected by pml with identification by polymerase chain reaction ( pcr ) of jcv sequences in csf . 
in two patients , ( dl and tp ) , the disease rapidly led to death while the other two patients ( cm and ad ) presented a chronic course and are sono stati esaminati 4 pazienti , tutti di sesso maschile , con et compresa tra 40 e 50 anni , affetti da pml e con identificazione nel liquor cerebrospinale delle sequenze del jcv . 
the study protocol included axial single - shot echoplanar diffusion images [ 5 , 871 / 99 / 1 ( tr / te / excitation ) ] , fov = 24 cm , matrix 128128 , b - value of 0 and 1 , 000 s / ( mm ) ^2 in each cardinal plane . case 1 dl , a 53 - year - old man treated in 1989 for chronic lymphatic leukaemia ( cll ) with chemotherapy ( fludarabine + cyclophosphamide ) and splenic radiotherapy , presented in february 2004 with slowly progressive motor deficits in the right upper limb and language disorder with onset 1 week previously . 
tutti i pazienti sono stati esaminati mediante uno scanner marconi picker medical system 1 , 0 t polaris con sequenze sagittali ed assiali t1 spin echo ( 434 / 12 / 2 [ tr / te / excitation ] ) , axial dual echo fast spin echo ( fse , etl = 4 ) ( 3473 / 20 and 120 / 2 [ tr / te / excitation ] ) , axial fluid attenuated inversion recovery ( flair ) fast spin echo ( fse , etl = 8 ) ( 2100 / 6000 / 80 / 1 [ ti / tr / te / excitation ] )  . 
il paziente progressivamente peggiorato e successivi controlli rm condotti dopo 7 e 24 gg rispetto al primo esame confermano una progressione delle lesioni senza comparsa di contrast enhancement ; un successivo controllo liquorale tramite pcr evidenzia positivit per jc virus . 
lobiettivit neurologica allingresso evidenziava : parziale disorientamento s / t , anosognosia , deficit di attenzione , comprensione , denominazione , ripetizione , memoria , calcolo , ragionamento astratto e tendenza alla confabulazione ed alla perseverazione . 
the patient refused further examination and died 2 months later . case 2 tp , a 55 - year - old man , was accompanied to the emergency room in a state of agitation after knocking down a pedestrian proceeding from the left while aboard his moped . 
approximately 2 weeks later , an outpatient brain mri was performed , disclosing lesions in the right occipitaltemporal and left occipital white matter that did not react to contrast mediuthe patient was admitted to the neurology department . 
neurological examination performed on admission showed partial s / t disorientation ; anosognosia , cognitive impairment with deficits in attention , comprehension , denomination , repetition , memorisation , calculation , abstract reasoning and a tendency to confabulate and perseverate . brain ct revealed an extensive hypodense lesion in the right temporoparietooccipital subcortical region and , less markedly , in the left occipital region . 
biohumoral tests showed moderate pancytopenia [ white blood cell count ( wcc ) 2.70109 / l ( nv 4.411 ) , neutrophils 33% , red blood cell count ( rcc ) 3.191 , 012 / l ( nv 4.55.9 ) , haemoglobin ( hb ) 103g / l ( nv 140175 ) , haematocrit ( ht ) 32% ( nv 4150 ) , platelets ( plt ) 139109 / l ( nv 150450 ) ] , with an increase in erythrocyte sedimentation rate ( esr ) [ 80 mm / h ( nv 228 ) ] and normal fibrinogen , c - reactive protein ( crp ) and a - 2 globulin values . 
a follow - up brain mri performed roughly 1 month after the previous one showed an increase in lesion dimensions , which had extended to the right parietal and insularfrontal and left temporal regions . 
the patient underwent lumbar puncture , which proved positive for jcv . the patient rapidly deteriorated and died 1 month later . case 3 cm , a 42 - year - old man admitted to hospital due to dimmed eyesight lasting about 15 min with inability to distinguish surrounding objects , reported having experienced progressive decline in eyesight over the previous 2 months . 
in view of a selective fall in cd4 lymphocytes ( approximately 90 ) and a diminished cd4 / cd8 ratio ( 01 ) , human immunodeficiency virus ( hiv ) - acquired immunodeficiency syndrome associated with chronic hepatitis c virus ( hcv ) - related hepatopathy was suspected and diagnosed . 
la biopsia ossea risult positiva per linfoma non - hodgkuna rmn di controllo eseguita ad un mese dimostr un ingrandimento delle lesioni che si estendevano ora alle regioni parietale ed insulo - frontale destre e alla temporale dell altro lato . 
alla luce di un calo selettivo dei linfociti cd4 ( circa 90 ) con ridotto rapporto cd4 / cd8 ( 01 ) venne fatta sospettata e quindi confermata la diagnosi di sindrome da immunodeficienza acquisita da hiv con epatopatia cronica hcv correlata . 
fluid - attenuated inversion recovery ( flair ) , t1 - weighted and diffusion - weighted ( dw ) images and apparent diffusion coefficient ( adc ) maps show an area ( arrows ) of reduced adc values in the anterior portion of the right occipitotemporal lesion , consistent with active progression front not seen in the previous examination ( not shown )  . 
la rmn celebrale eseguita 12 giorni dopo mostr plurime aree di alterato segnale in sede iuxta - corticale , ipointense in t1 ed iperintense in t2 , con aspetto a stampo per coinvolgimento delle fibre ad u in sede fronto - occipitale , prive di effetto massa ed areagenti al mdc . 
anche in questo caso le immagini pesate in diffusione risultarono di scarso significato . discussione lo stato di immunodepressione predispone allinsorgenza di infezioni opportunistiche del sistema nervoso centrale ( snc ) , quali lencefalite da cytomegalovirus , le infezioni da toxoplasma e da jc virus . 
tra queste il quadro rm della pml ben conosciuto , caratterizzato da lesioni della sostanza bianca multifocali , povere di elementi specifici [ 79 ] tanto che la diagnosi rimane basata sul riconoscimento mediante pcr delle sequenze virali nel liquor . gli studi neuropatologici identificano allinterno delle lesioni aree con differenti stadi di demielinizzazione della sostanza bianca : le lesioni pi datate cos come la porzione fig . 
3 paziente 3 : immagini flair , t1w , dwi e mappe adc del primo esame e dei successivi controlli a 12 e 16 mesi ; le molteplici lesioni della sostanza bianca sottocorticale non mostrano significativi margini di accrescimento e le immagini evidenziano sfumati segni di atrofia cerebrale . region and occipital region bilaterally . 
brain mri , performed 12 days later , showed multiple areas of altered signal in the juxtacortical region , hypointense on t1 and hyperintense on t2 images , with a punched - out appearance due to involvement of u - shaped fibres in the frontooccipital region , with no mass effect and no reaction to contrast mediu since the findings were highly suggestive of pml , a lumbar puncture was performed and tested positive for jcv . 
diffusion - weighted images ( dwis ) were of little significance . case 4 ad , a 55 - year - old hiv - positive man with left pml - related hemiplegia , was hospitalised due to onset of recurrent seizures that failed to respond to medication . 
brain mri showed extensive signal alteration in the white matter of the semioval centre , with thinning of the corpus callosuin this case , too , dwis were of little significance . baseline 7th day 22th day discussion fig . 
accordingly , the diagnosis of pml is based on identification by pcr of viral sequences in the csf . neuropathological studies have detected some areas within the lesions with different stages of white matter demyelination : older lesions and the central portion of more recent ones are characterised by almost total breakdown of the myelin sheath and by axonal damage , with an increase in extracellular space ; conversely , the most medial portion of lesions consists of oligodendrocytes with nuclear inclusions and partially destroyed myelin , with relative sparing of axons . despite histopathological heterogeneity , mri signal alterations that characterise pml in conventional sequences seem to show homogeneous low signal intensity on t1weighted images and hyperintensity on t2 - weighted images , irrespective of the lesions progression and as a consequence of the altered ratio between myelin content and water . 
some authors have recently reported that analysis of the evolution of a lesion with diffusion - weighted mri can supply important new semiological elements [ 3 , 6 ]  . 
comparison of dwi sequences and adc maps shows different signal behaviour between the lesion centre and the extending margin particular , dwi presents some signal features that are similar to flair , i.e. 
margin hyperintensity and central hypointensity . on adc maps , signal intensity is elevated in the central area ( values : 1 , 710125 ) whereas at the lesion margins , it seems to further distinguish the more recent portion ( growth margin ) with reduced water diffusibility compatible with cytotoxic oedema ( values : 561135 ) from less recent portions that have intermediate values ( 940135 )  . 
in particular , adc maps show an elevated signal in the centre of the lesion and low signal intensity at the edge of the lesions expansion towards the white matter . 
 [ 10 ] correlated lesion stage with dwi / adc signal and interpreted these findings as both increased water diffusibility in the central areas of glial repair and reduced diffusibility because of cytotoxic oedema within active viral infectious areas . 
 [ 3 ] also described this peculiar pattern of development of pml using diffusion tensor imaging ( dti ) without , however , considering the relationship between findings obtained with dwi sequences and adc maps . our study confirms signal heterogeneity of lesions on adc maps : although lacking in neuropathological correlation , close comparison of lesions serially and temporally suggests that the alterations detected on adc maps strictly centrale delle lesioni recenti sono caratterizzate da una quasi totale perdita di mielina e da un danno assonale con allargamento degli spazi extracellulari ; la parte pi mediale delle lesioni invece sembra costituita da oligodendrociti con inclusioni intranucleari e mielina parzialmente distrutta , con relativo risparmio degli assoni . 
 a dispetto della eterogeneit istopatologica , le alterazioni di segnale rm che caratterizzano la pml nelle sequenze standard appaiono omogeneamente ipointense in t1 ed iperintense in t2 indipendentemente dalla fase evolutiva della lesione , come conseguenza dellalterato rapporto tra contenuto in mielina e acqua . alcuni autori hanno recentemente segnalato che nella pml le sequenze pesate in diffusione possono fornire nuovi ed utili elementi semeiologici [ 3 , 6 ]  . 
il confronto delle immagini rm nei pazienti con un decorso aggressivo della malattia evidenzia un progressivo aumento delle dimensioni delle lesioni che risultano sostanzialmente omogeneamente iperintense in t2 ed ipointense in t1 . 
nelle mappe adc il segnale elevato nella porzione centrale ( valori : 1710125 ) mentre , in corrispondenza dei margini delle lesioni , sembra ulteriormente differenziare la porzione pi recente ( margine di accrescimento ) , caratterizzata da ridotti valori di diffusibilit dellacqua compatibili con edema citotossico ( valori : 561135 ) , da porzioni meno recenti che presentano valori intermedi ( 940135 )  . 
 [ 10 ] hanno correlato lo stadio della lesione con il segnale in dwi / adc e hanno interpretato tali reperti come unaumentata diffusibilit dellacqua nelle aree centrali di riparazione gliotica e come una ridotta diffusibilit per presenza di edema citotossico nelle aree di infezione attiva del virus . 
 [ 3 ] hanno sottolineato questo caratteristico sviluppo della pml , grazie allutilizzo del tensore ( dti ) , senza porre per attenzione al confronto tra i risultati ottenuti con le sequenze in diffusione e le mappe adc . il nostro studio conferma leterogeneit di segnale delle lesioni nelle mappe adc : il confronto seriato e temporalmente ravvicinato delle lesioni , sebbene privo di correlato neuropatologico , sembra dimostrare che le alterazioni rilevate nelle mappe adc corrispondono strettamente alla maturit delle lesioni . 
in particular , the areas of lesion extension , initially characterised by a low adc signal ( cytotoxic oedema ) will evolve at subsequent examinations into high adc signal areas typical of vasogenic oedema and glial repair , and new cytolysis regions will emerge peripherally to the previous ones . 
these dwi features , which characterise the acute stages of the disease , were not detectable in patients 3 and 4 , whose lesions progressed very little over time , with a prevalence of atrophic degeneration . 
in these cases , the progression front was probably too tenuous , if not absent , and could not , therefore , be detected on mri . our results highlight the importance of all information provided by dwi and related adc maps and , together with previous findings , introduce new , early semiological and neuroradiological elements that are specific for this disease . mente caratterizzate da basso segnale adc ( edema citotossico ) evolvono , nei successivi controlli , ad aree ad alto segnale adc tipico delledema vasogenico e della riparazione gliotica e che nuove aree di citolisi si aggiungono perifericamente alle precedenti . 
questi aspetti nelle sequenze in diffusione , che caratterizzano le fasi acute della malattia , non sembrano essere evidenti nei pazienti 3 e 4 , in cui le lesioni presentano una lenta evolutivit , con prevalenza di aspetti degenerativo - atrofici . 
in questi casi il fronte di accrescimento troppo sfumato , se non assente , quindi non apprezzabile nelle immagini rmn . i nostri risultati confermano limportanza delle informazioni ottenibili con la sequenza pesata in diffusione e le relative mappe adc e , assieme alle segnalazioni gi presenti in letteratura , introducono un elemento semeiologico neuroradiologico nuovo , precoce e specifico di questa patologia . conclusions conclusioni diffusion - weighted sequences add new semiological elements in the neuroradiological diagnosis of pml and seem to correctly identify disease progression , which is not otherwise detectable on conventional sequences . 
further studies are needed to assess the clinical impact and reliability of these new elements in evaluating response to treatment in future clinical and pharmacological trials . le sequenze pesate in diffusione aggiungono nuovi elementi semeiologici nella diagnosi neuroradiologica di pml e sembrano in grado di riconoscere il fronte di progressione della malattia altrimenti non apprezzabile nelle sequenze convenzionali . 
fugazzola1 1vascular and interventional radiology , department of radiology , 2vascular surgery , 3anesthesiology , university of insubria , viale borri 57 , i - 21100 varese , italy correspondence to : d . 
we selected 19 patients ( mean age 66.3 years , range 4580 ) : five with complete occlusion of the infrarenal aorta and both common iliac arteries ( cias ) , four of which were associated with occlusion of the external iliac arteries ( eias ) ; three with complete occlusion and three with severe stenosis of the distal aorta with occlusion or stenosis of the cias and eias ; and eight with focal severe stenosis of the infrarenal aorta . 
immediate technical success was 94.7% ( 18 / 19 cases )  . we observed two cases of distal embolism treated with thrombolysis , one case of mild renal failure and one case of transient angina abdominis . 
during the follow - up ( mean 19.6 months ; range 648 ) , 2 / 18 ( 11.1% ) occlusions of an iliac stent occurred 1 and 3 months after the procedure ( treated with local intra - arterial thrombolysis )  . 
direct stenting is a feasible and safe option for the treatment of infrarenal abdominal aortic steno - occlusions , especially in patients at high surgical risk , with good early and late clinical results . key words aorta stent endovascular riassunto obiettivo . 
abbiamo selezionato 19 pazienti ( et media 66 , 3 anni , range 4580 ) : 5 con occlusione completa dellaorta addominale sottorenale e delle arterie iliache comuni ( aics ) , associata in 4 casi ad occlusione delle arterie iliache esterne ( aies ) ; 3 occlusioni e 3 stenosi severe dellaorta distale con occlusione o stenosi delle aics e aies ; 8 stenosi focali severe dellaorta sottorenale . 
si sono verificate 2 embolie distali , 1 insufficienza renale moderata e 1 angina abdominis transitoria . durante il follow - up ( medio 19 , 6 mese ; range 648 ) , abbiamo osservato 2 / 18 ( 11 , 1% ) occlusioni di uno stent iliaco a 1 e 3 mesi dalla procedure ( trattate con fibrinolisi loco - regionale )  . 
soprattutto in pazienti ad elevato rischio chirurgico , con buoni risultati a breve e medio termine . parole chiave aorta stent endovascolare introduction introduzione infrarenal aortic occlusive disease is an uncommon consequence of atherosclerosis and mainly affects middleaged women with dyslipidaemia and a history of smoking [ 1 ]  . 
usual management involves endarterectomy for focal lesions or surgical bypass for more extensive aortoiliac disease and is characterised by established and favourable long - term results ( 90% 5 - year and 75% 10year patency rates ) [ 2 , 3 ] even if burdened by a nonnegla malattia occlusiva dellaorta sottorenale una infrequente sequela dellaterosclerosi e colpisce soprattutto soggetti di sesso femminile di et media , con dislipidemia e anamnesi di tabagismo [ 1 ]  . 
il trattamento convenzionale prevede lendoarterectomia , per le lesioni focali , o il by - pass chirurgico per la patologia aorto - iliaca estesa , ed caratterizzato da buoni risultati a lungo termine ( perviet del 90% a 5 anni e del 75% a 10 anni ) [ 2 , 3 ] , anche se gravato da d . 
its limited invasiveness , lower morbidity and costs in comparison to surgery and favourable early and long - term results [ 712 ] have subsequently established the role of pta as the first - choice strategy in the management of focal infrarenal aortic stenosis [ 11 ]  . in most literature reports , indications for stenting have been unsuccessful or complicated pta , such as elastic recoil of the wall and stenosing or occluding flap ( secondary stenting ) [ 4 , 5 , 1322 ]  . 
subsequent data on primary or direct stenting ( stenting with or without balloon predilation , respectively ) in the elective management of focal lesions [ 4 , 13 , 1727 ] , infrarenal aortic occlusions [ 13 , 2528 ] or aortoiliac lesions [ 20 , 23 , 26 , 27 ] were more limited and conflicting . 
the aim of this paper is to present our experience in the elective management of steno - occlusive abdominal aortic disease with direct stenting . materials and methods over the past 4 years , we treated 19 patients ( 12 men and seven women ; mean age 66.3 years , range 4580 ) with leriche syndrome ( claudication and femoral pulselessness associated in some cases with impotence ) who were unfit for open conventional repair owing to poor cardiorespiratory function . 
five patients had complete occlusion of the infrarenal aorta and both common iliac arteries ( cia ) [ in four cases associated with bilateral occlusion of the external iliac arteries ( eia ) ; in three cases the aorta was occluded immediately below the origin of renal artery and in two cases at level of the middle aorta ; in four cases , a rich collateral circulation recanalised the common femoral arteries and in one case the eias ]  . 
eight cases had severe focal stenosis ( > 70% ) of the infrarenal aorta due to a calcified eccentric plaque , which was ulcerated in two . none of the patients had a history of previous surgical or endovascular intervention in the aortic or iliac vessels . risk factors included smoking ( n = 11 ) , hypertension ( n = 9 ) , cardiac disease ( n = 12 ) , hyperlipidaemia ( n = 6 ) , diabetes ( n = 4 ) and pulmonary disease ( n = 7 )  . 
symptoms were classified according to the classification of the society of vascular surgery and the international society of cardiovascular surgery ( svs / iscs ) : four patients were class 2 ( moderate claudication ) , eight were class 3 ( severe claudication ) and seven were class 4 ( ischaemic rest pain )  . 
 preprocedural assessment was performed in all patients with multislice computed tomography ( ct ) angiography ( mscta ) ( lightspeedplus , ge , milwaukee , wi , usa e aquilion 64 , toshiba , tokio , japan ) , with scans obtained at baseline and in un tasso non trascurabile di complicanze maggiori ( 5%10% dei casi ) [ 2 , 3 ]  . dalle prime esperienze riportate in letteratura a partire dal 1980 , riguardanti il trattamento percutaneo di stenosi focali dellaorta sottorenale mediante angioplastica ( pta ) in pazienti ad elevato rischio chirurgico [ 46 ] , lapproccio endovascolare stato proposto come alternativa alla chirurgia . 
la mini - invasivit , la riduzione della morbidit e dei costi rispetto alla chirurgia e i buoni risultati immediati e a distanza [ 712 ] hanno consolidato il ruolo della pta come approccio di prima scelta nel trattamento delle stenosi focali dellaorta sottorenale [ 11 ]  . in letteratura , nella maggior parte delle casistiche , le indicazione allo stent sono state linsuccesso o le complicanze della pta , quali il ritorno elastico di parete e il flap stenosante o occludente ( secondary stenting ) [ 4 , 5 , 1322 ]  . successivamente sono stati pubblicati dati pi limitati e contraddittori relativi al primary stenting o al direct stenting ( stenting rispettivamente con o senza pre - dilatazione ) nel trattamento endovascolare delle lesioni focali [ 4 , 13 , 1727 ] , e al trattamento delle occlusioni complete dellaorta sottorenale [ 2226 ] e delle lesioni aorto - iliache [ 20 , 23 , 26 , 27 ]  . 
scopo di questo lavoro presentare la nostra esperienza nel trattamento endovascolare della malattia stenoocclusiva dellaorta sottorenale mediante direct stenting . materiali e metodi negli ultimi 4 anni abbiamo selezionato 19 pazienti ( 12 maschi e 7 femmine ; et media 66 , 3 anni , range 4580 ) , sintomatici per sindrome di leriche ( claudicatio e assenza dei polsi femorali , associati in alcuni casi a impotenza ) , ad elevato rischio chirurgico per patologie cardio - respiratorie . 
i pazienti erano portatori delle seguenti lesioni : 5 occlusioni complete dellaorta sottorenale e di entrambe le aics in 4 casi associate allocclusione bilaterale delle aies , in 3 casi laorta era occlusa subito a valle dellorigine delle arterie renali e in 2 casi a livello del tratto intermedio , in 4 casi ricchi circoli collaterali ricanalizzavano le arterie femorali comuni ( afcs ) e in 1 caso le aies ; 3 occlusioni complete e 3 stenosi severe ( > 70% ) dellaorta distale con occlusione bilaterale delle aics e delle aies ; 8 stenosi focali severe ( > 70% ) dellaorta sottorenale da placca eccentrica calcifica , in 2 casi ulcerata . 
nessun paziente era stato sottoposto in precedenza a trattamenti chirurgici o endovascolari a livello aorto - ilaco . i pazienti presentavano i seguenti fattori di rischio : tabagismo ( n = 11 ) , ipertensione ( n = 9 ) , patologie cardiache ( n = 12 ) , iperlipidemia ( n = 6 ) , diabete ( n = 4 ) e patologie polmonari ( n = 7 )  . 
i pazienti sono stati classificati in base alla severit dei sintomi in accordo con la classificazione svs / iscvs ( society of vascular surgery and international society of cardiovascular surgery ) : 4 pazienti categoria 2 ( claudicatio moderata ) , 8 categoria 3 ( claudicatio severa ) e 7 categoria 4 ( dolore a riposo )  . la valutazione pre - trattamento stata effettuata in tutti i d . 
all patients underwent cardiac and pulmonary monitoring during the procedure and received antithrombotic prophylaxis consisting of heparin ( 5 , 000 iu ) followed by light - molecular - weight heparin in prophylactic doses for 30 days after the procedure and life - long antiplatelet or anticoagulant therapy . 
procedures were performed in 14 cases in the vascular suite under local anaesthesia ( 10 ml 2% carbocaine at the site of arterial access ) and in five cases ( all total occlusions of the infrarenal aorta ) in the operating theatre with surgical stand - by . all patients underwent percutaneous access of the common femoral artery using a 5 - f introducer sheath ( cordis , miami , fl , usa ) and a 0.035 ( 300 - cm ) hydrophilic curved guidewire ( glidewire , terumo , tokyo , japan ) unilaterally in eight cases with focal stenosis and bilaterally in the remaining cases . 
before stent implantation , the hydrophilic guidewire was exchanged with a 0.035 ( 300cm ) stiff wire ( amplatz , boston scientific , ratingen , germany ) , and the lesion was crossed with a 12 - f , 30 - cm - long sheath ( cook , bloomington , in , usa ) in order to enable the protected advancement of the hand - crimped stent and thus avoid dislodgement . 
the stents were first dilated to the minimum diameter enabling sealing to the vessel wall ( 12 - mm powerflex , cordis , miami ) and progression along the 12 - f sheath . postdilation by in - stent pta [ using balloons with a diameter ranging between 16 and 18 mm ( powerflex , cordis , miami ) ] was then performed in order to achieve an adequate vessel diameter and sufficient stent shortening , stopping the postdilation when the patient reported pa for lesions involving the bifurcation and the iliac vessels , direct kissing stenting of the cias was performed with self - expandable stents ( 1012 mm in diameter , 60 mm in length ) ( smart , cordis , miami )  . 
in tutti i casi durante la procedura stato effettuato il monitoraggio cardio - respiratorio ; stata inoltre effettuata la profilassi antitrombotica con somministrazione di eparina ( 5000 iu ) ; successivamente eparina a basso peso molecolare in dosi profilattiche per 30 giorni seguita da terapia antiaggregante o anticoagulante . 
le procedure sono state effettuate in 14 casi in sala angiografica in anestesia locale ( 10 ml di carbocaina 2% nel sito di accesso arterioso ) e in 5 casi ( tutti i pazienti con occlusione completa dellaorta sottorenale ) in sala operatoria con stand - by chirurgico . in tutti i pazienti stato effettuato un cateterismo percutaneo dellafc , utilizzando un introduttore vascolare 5 fr ( cordis , miami , usa ) e una guida idrofilica curva 0 , 035 ( glidewire terumo , tokyo , giappone ) , monolaterale in 8 casi con stenosi focale e bilaterale nei restanti casi . 
prima dello stenting la guida idrofilica stata sostituita con guida rigida 0 , 035 ( amplatz , boston scientific , ratingen , germania ) e le lesioni sono state superate con introduttore 12 fr lungo 30 cm ( cook , bloomington , in , usa ) , al fine di garantire lavanzamento protetto dello stent crimpato su pallone ed evitarne dislocazioni . 
gli stents sono stati dapprima dilatati al minimo diametro necessario a garantirne lancoraggio alla parete aortica ( 12 mm powerflex , cordis , miami , usa ) e la progressione lungo lintroduttore 12 fr . 
successivamente stata effettuata una pta intra - stent ( mediante palloni con diametro compreso tra 16 e 18 mm powerflex , cordis , miami , usa ) finalizzata a raggiungere un calibro sufficiente del vaso e a ottenere un adeguato accorciamento dello stent , arrestando la post - did . 
1a - f a 59 - year - old patient with a complete occlusion of the infrarenal aorta and both common iliac arteries ( cias ) and external iliac arteries ( eias )  . 
a preprocedural multislice computed tomography angiography ( mscta ) , volume rendering ( vr ) reconstruction ; mscta clearly shows the collateral circulation that , through deep iliac circumflex arteries ( black arrows ) and inferior epigastric arteries ( white arrows ) allows recanalisation of the common femoral arteries . 
e , f mscta , maximum intensity projection ( mip ) ( e ) and vr ( f ) reconstruction 1 year after the procedure confirms the patency of the aorta and iliac arteries . fig 1a - f paziente di 59 anni con occlusione completa dellaorta sottorenale e di entrambe le aics e aies . 
a atcms pre - procedura , ricostruzioni vr : occlusione completa dellaorta sottorenale con circoli collaterali che , attraverso le arterie circonflesse iliache profonde ( frecce nere ) e epigastriche superficiali ( frecce bianche ) ricanalizzano le arterie femorali comuni ; b angiografia pre - procedura espletata dopo superamento dellocclusione ; c direct stenting con posizionamento di 2 stents espandibili su pallone in aorta ( frecce bianche ) e di 3 stents autoespandibili in entrambi gli assi iliaci ( frecce nere ) ; d langiografia espletata dopo il trattamento , mostra la completa ricanalizzazione dellaorta sottorenale , delle aics e aies . 
2a - c a 70 - year - old patient with a complete occlusion of the infrarenal aorta and both common iliac arteries ( cias ) and external iliac arteries ( eias )  . 
a preprocedural multislice computed tomograph angiography ( mscta ) , curved multiplanar reconstruction ( mpr ) : occlusion of the abdominal aorta just below the origin of renal arteries ( bilaterally 2 renal arteries are present )  . 
c angiography performed after the direct stenting shows complete recanalisation of the infrarenal aorta , cias and eias and patency of renal arteries . fig 2a - c paziente di 70 anni con occlusione completa dellaorta sottorenale e di entrambe le aics e aies ; a angio - tcms , ricostruzioni mpr curve dellorigine delle arterie renali ( bilateralmente sono presenti 2 arterie renali ) ; b angiografia pre - procedura espletata dopo superamento dellocclusione , con guida angiografica che guadagna il lume vero dellaorta ; c angiografia espletata dopo il direct stenting che mostra la completa ricanalizzazione dellaorta sottorenale , delle aics e delle aies e la perviet delle arterie renali . lesions involving the eias , two additional stents ( 9 - mm diameter , 60 - mm long ) ( smart , cordis , miami ) were deployed to completely reconstruct the iliac vessels . 
in all nine cases in whom the lesion involved the origin of the inferior mesenteric artery ( which was patent before the procedure in 4 / 9 cases ) , the stent covered its ostium . immediate postprocedure results were assessed with angiography ( anteroposterior and lateral views ) in order to assess stent patency and deployment as well as the lumen of the target vessel , and angiograms of the distal leg vessels were also obtained to exclude complications such as distal embolis follow - up was performed with colour - doppler ultrasound ( cdus ) and clinical assessment 1 , 3 , 6 and 12 months after the procedure and yearly thereafter . 
1d , 2c latazione quando il paziente riferiva la comparsa di dolore . per le lesioni che coinvolgevano la biforcazione aortica e i vasi iliaci stato effettuato un direct kissing stenting delle aics , mediante stents autoespandibili ( diametro 1012 mm , lunghezza 60 mm ) ( smart , cordis , miami , usa )  . 
in 5 pazienti con lesioni che coinvolgevano le aies , sono stati embricati altri 2 stents ( diametro 9 mm , lunghezza 60 mm ) ( smart , cordis , miami , usa ) per completare la ricostruzione degli assi iliaci . 
nei 9 casi in cui la lesione coinvolgeva lemergenza dellarteria mesenterica inferiore ( che risultava pervia prima della procedura in 4 / 9 casi ) lostio stato coperto dallo stent . il risultato immediato stato controllato mediante angiografia ( in proiezione antero - posteriore e latero - laterale ) al fine di valutare lapertura e la perviet degli stents e il calibro vasale raggiunto ; stata inoltre effettuata unangiografia degli arti inferiori per escludere complicanze emboliche distali . 
in nine cases , the ostium of the inferior mesenteric artery was covered by the stent , with ensuing total occlusion of the artery in all cases ( in five patients , the artery was already occluded ) ; no patient experienced major complications due to inferior mesenteric artery occlusion with the exception of one case of transient angina abdominis . patients were discharged after a mean of 3.2 ( range 37 ) days . 
during the follow - up performed in the 18 patients achieving immediate procedural success ( mean 19.6 months ; range 648 ) , 2 / 18 ( 11.1% ) occlusions of the iliac stent occurred 1 and 3 months after the procedure . 
 discussion endovascular treatment of infrarenal aortic stenosis has been proposed [ 28 , 29 ] as a viable alternative to surgical revascularisation since the 1980s , and data available have established the safety and effectiveness of pta , with an 85% primary and a 90% secondary patency rate [ 712 ] together with a low ( 3% ) risk of major complications , such as vessel - wall rupture and distal and visceral embolism [ 21 ]  . 
on the other hand , data on aortic stenting mainly focus on its use for the management of unsuccessful or complicated pta procedures [ 4 , 5 , 1318 , 23 , 30 , 31 ]  . primary or direct stenting has been proposed as a result of the favourable results obtained in the iliac arteries , including its higher patency rates in comparison with pta [ 6 , 32 ] and has been employed selectively in complex stenotic lesions ( ulcerated , eccentric , irregular and / or calcific plaques ) [ 4 , 13 , 1727 ] and , more rarely , in total occlusions [ 20 , 2326 ]  . the rationale for direct stenting is based on the frequent early post - pta elastic recoil due to the extensive presence of elastic fibres in the aortic wall [ 13 ] and on the risk of rupture due to the large diameter of the aorta , according to the laplace law [ 19 ] ; the more uniform distribution of radial forces during stenting should decrease the risk of rupture in comparison to balloon pta [ 20 , 22 , 24 ]  . 
in un caso con occlusione completa dellaorta sottorenale e di entrambe le aics e aies non stato possibile valicare locclusione , nonostante plurimi tentativi retrogradi e anterogradi e il paziente stato trattato , durante la stessa seduta con by - pass axillo - bifemorale . non si sono verificate rotture di arteria ; abbiamo osservato 2 / 18 ( 11 , 1% ) embolie distali , trattate con successo mediante fibrinolisi loco - regionale e 1 lieve insufficienza renale dovuta a microembolia a livello dellarteria renale destra ( in questo caso lecd ha dimostrato una ridotta perfusione del parenchima renale )  . 
in 9 casi lostio dellarteria mesenterica inferiore stato coperto dallo stent con conseguente occlusione dellarteria in tutti i casi ( in 5 / 9 pazienti larteria era gi precedentemente occlusa ) ; non abbiamo osservato complicanze severe legate allocclusione dellarteria mesenterica inferiore con eccezione di una angina abdominis transitoria . 
peraltro i dati riguardanti lo stenting sono per la maggior parte focalizzati sul suo utilizzo nelle complicanze e negli insuccessi della pta [ 4 , 5 , 1318 , 23 , 30 , 31 ]  . lo stenting , primario o diretto , stato proposto alla luce dei favorevoli risultati ottenuti a livello iliaco con maggiori tassi di perviet rispetto alla sola pta [ 6 , 32 ] ed stato utilizzato di prima intenzione nelle lesioni stenosanti complesse ( placche ulcerate , eccentriche , irregolari e / o calcifiche ) [ 4 , 13 , 1727 ] e pi raramente nelle occlusioni complete [ 20 , 2326 ]  . il razionale del direct stenting basato sullalta frequenza del ritorno elastico di parete post - pta , dovuto allelevata presenza di fibre elastiche a livello della parete aortica [ 13 ] , e sul rischio di rottura dovuto al grosso calibro dellaorta , in accordo con la legge di laplace , [ 19 ] ; la pi uniforme distribuzione delle forze di dilatazione radiale durante lo stenting potrebbe diminuire il rischio di rottura rispetto alla sola dilatazione con pallone [ 20 , 22 , 24 ]  . 
inoltre , il posizionamento dello stent senza pre - dilatazione ( direct stenting ) , metodica applicata in questo studio e in alcuni altri riportati in letteratura , riduce il rischio di embolizzad . 
in our study , at an overall average follow - up of 17.4 ( range 148 ) months , we observed two occlusions ( 11.1% ) successfully treated with thrombolysis and pta . our choice of a balloon - expandable stent for aortic lesions is based on its superior radial force and the fact that it can be further dilated after the initial expansion in order to achieve a haemodynamically satisfying diameter [ 33 ]  . dilatation must be carefully guided by the patients symptoms , as the occurrence of pain during dilation is mostly due to the initial rupture of the intima and media . 
moreover , it is well known that balloon - expandable stents enable a quicker , more precise and accurate deployment than self - expandable stents given their reproducible shortening [ 24 ]  . 
to date , the availability of large - size stents ( with diameters up to 25 mm ) enables the management of a larger number of patients whereas in the past , only subjects with a small aorta could be treated [ 13 ]  . among the complications , the most feared aortic wall rupture is quite rare , and only sporadic cases have been reported after pta [ 34 ] and stenting [ 35 ]  . 
other reported complications , excluding those at the access site due to large diameter of the devices , may include distal or visceral embolisation [ 13 , 20 , 21 , 27 ]  . 
in any case , a careful preprocedural evaluation is of crucial importance in order to assess the patency of all relevant splanchnic vessels ( coeliac trunk , superior mesenteric artery , hypogastric arteries )  . among the causes of late clinical failure , with recurzione distale [ 1927 ]  . 
inoltre , la forza radiale dello stent in acciaio medicale consente una dilatazione uniforme e simmetrica della parete arteriosa , anche in vasi calcifici , evitando , soprattutto in caso di lesioni eccentriche , che la dilatazione interessi solo la porzione sana della parete . stato riportato il trattamento mediante primary o direct stenting dellaorta addominale in circa 100 casi ( nel 75% si trattava di stenosi focali ) con un successo clinico del 96% , definito come risoluzione o miglioramento dei sintomi , e come risultato emodinamico , e con tassi di perviet primaria del 90% e secondaria del 100% [ 4 , 13 , 1927 ]  . nella nostra casistica abbiamo ottenuto il successo tecnico in 18 / 19 casi ( 94 , 7% )  . 
nel nostro studio ad un follow - up medio di 17 , 4 mesi ( range 148 ) abbiamo osservato 2 occlusioni ( 11 , 1% ) di stents trattate con successo mediante fibrinolisi loco - regionale e pta . la nostra scelta di stents espandibili su pallone per le lesioni aortiche basato sulla loro superiore forza radiale e sul fatto che possono essere ulteriormente dilatati dopo lespansione iniziale , al fine di raggiungere un calibro del vaso emodinamicamente significativo [ 33 ]  . 
attualmente la disponibilit di stents di grosso calibro ( con diametro fino a 25 mm ) permette il trattamento di un maggior numero di pazienti , mentre in passato potevano essere sottoposti a stenting solo pazienti con small aorta [ 13 ]  . tra le complicanze , la pi temuta la rottura della parete aortica che in realt piuttosto rara e sono riportati in letteratura solo casi sporadici dopo pta [ 34 ] o stenting [ 35 ]  . inoltre , attualmente tale complicanza gestibile per via endovascolare con stents ricoperti . 
inoltre nel bypass chirurgico larteria mesenterica inferiore quasi sempre sacrificata , con un 2% di incidenza di ischemia intestinale [ 36 ]  . nella nostra esperienza abbiamo osservato solo 1 caso di angina abdominis transitoria . 
however , in - stent neointimal hyperplasia is less frequent in the aorta than in other vascular districts , as this is a high - flow vessel [ 1 ]  . 
accordingly , in most cases , restenotic lesions can be treated with a percutaneous approach , with secondary patency rates approaching 100% [ 13 , 23 , 27 ]  . 
such favourable results of endovascular treatment of aortic atherosclerotic occlusive disease are complemented by its advantages over open surgical revascularisation , basically due to its limited invasiveness , avoidance of general anaesthesia and use of laparotomy , with the consequent reductions in mortality and morbidity [ 19 ]  . 
similarly , the endovascular approach does not interfere with sexual function [ 26 ] , which , especially in men , may be compromised in up to 30% of cases after surgery [ 37 ]  . finally , the endovascular strategy does not preclude open surgical management in case of procedural failure or disease progression and , for total occlusions of the aortoiliac vessels , endovascular recanalisation of the aorta and even only one of the two iliac vessels may reduce the invasiveness of the subsequent surgical procedure ( femorofemoral crossover instead of aortobifemoral bypass )  . in conclusion , on the basis of the present study and the current literature , endovascular treatment , and in particular , direct stenting , may be considered the treatment of choice for steno - occlusive lesions of the abdominal aorta , as it provides excellent early and late clinical results with very low complication rates . 
the treatment of total occlusions of the abdominal aorta and lesions involving the bifurcation and the iliac vessels is feasible and safe but warrants additional studies , with larger series and longer follow - up . tra le cause di insuccesso clinico a distanza , con recidiva o peggioramento dei sintomi , oltre alla progressione prossimale o distale della patologia aterosclerotica [ 19 , 20 ] o alla insufficiente espansione dello stent [ 18 ] , va ricordata liperplasia intimale , la quale pu causare , quando determina una stenosi critica , anche locclusione trombotica dello stent [ 13 , 20 , 23 , 27 ]  . 
la maggior parte dei casi di restenosi pu comunque essere trattata con approccio percutaneo con tassi di perviet secondaria che raggiungono il 100% [ 13 , 23 , 27 ]  . tali favorevoli risultati del trattamento endovascolare nella patologia aterosclerotica occlusiva dellaorta addominale sono supportati anche dai vantaggi nei confronti dellapproccio chirurgico , legati alla minore invasivit e alla possibilit di evitare lanestesia generale con conseguente riduzione della morbidit e mortalit [ 19 ]  . 
peraltro lapproccio endovascolare non interferisce con la funzione sessuale [ 26 ] la quale , sopratutto nei maschi pu essere compromessa in pi del 30% dei casi dopo chirurgia [ 37 ]  . infine , la strategia endovascolare non preclude il trattamento chirurgico in caso di insuccesso o di progressione della patologia e , per le occlusioni complete dellaorta e delle arterie iliache , la ricanalizzazione dellaorta e anche di un solo asse iliaco , pu ridurre linvasivit della procedura chirurgica ( cross - over femoro - femorale anzich by - pass aorto - bifemorale )  . in conclusione , sulla base dei risultati di questo studio e dei dati della letteratura , il trattamento endovascolare , e in particolare il direct stenting , pu essere considerato la terapia di prima scelta per la patologia steno - occlusiva dellaorta addominale , con buoni risultati clinici immediati e a medio termine , con bassi tassi di complicanze . 
fugazzola1 1vascular and interventional radiology , department of radiology , 2vascular surgery , 3surgery , 4anesthesiology , university of insubria , viale borri 57 , i - 21100 varese , italy correspondence to : d . 
in 23 haemodynamically stable patients , computed tomography ( ct ) angiography was performed before treatment , and aaa exclusion was carried out as an urgent procedure ; 15 patients with haemorrhagic shock were examined by ultrasound ( us ) only in the emergency room whereas the procedure was carried out in emergency and planned using angiography . 
endovascular treatment of aaas is a good therapeutic alternative even in urgency and emergency conditions where correct planning ensures technical results comparable with those obtained under elective conditions . key words abdominal aortic aneurysm rupture endovascular treatment stent - grafts riassunto obiettivo . 
in 23 pazienti , emodinamicamente stabili , stata eseguita unangio - tc pre - trattamento e la procedura stata effettuata in urgenza ; 15 pazienti , in shock emorragico , sono stati valutati con la sola ecografia e la procedura stata effettuata in emergenza e pianificata mediante angiografia . 
il trattamento endovascolare una valida alternativa terapeutica per gli aaa anche in urgenza e emergenza , dove un corretto planning consente di ottenere risultati tecnici comparabili a quelli in elezione . parole chiave aneurisma aorta addominale rottura trattamento endovascolare stent - grafts d . 
although significant progress has been made in the fields of diagnosis , surgery and anaesthesiology , mortality among patients with aaa rupture who arrive at the hospital alive is 45%70% [ 2 ]  . 
since 1990 , the year of the first aortic stentgraft implanted by parodi , an alternative treatment , which is less invasive especially for patients at high surgical risk , has emerged [ 3 ]  . 
over the last decade , elective endovascular treatment of aaas has continuously spread , with shortand middle - term results comparable , according to some authors , to open surgery [ 4 ]  . 
improvements in devices and growing experience have led to suggestions for the use of endovascular treatment instead of surgery even in emergency settings , with the aim of reducing mortality [ 5 ]  . 
we describe our centres experience in urgency and emergency endovascular treatment of aaas . le emergenze dellaorta addominale costituiscono la terza causa di morte nei paesi industrializzati e il 95% di queste legato alle rotture di aaa . 
nonostante importanti progressi in campo diagnostico , chirurgico e anestesiologico , la percentuale di mortalit tra i pazienti con rottura di aaa che arrivano vivi in ospedale del 45%70% [ 2 ]  . 
dal 1990 , data della prima endoprotesi aortica impiantata da parodi si aperta una nuova alternativa al trattamento degli aaa , meno invasiva specialmente nei pazienti ad alto rischio chirurgico [ 3 ]  . 
nel corso dellultimo decennio si assistito ad una costante diffusione del trattamento endovascolare degli aaa in elezione con buoni risultati a breve e medio termine , secondo alcuni , comparabili con lesperienza di chirurgia open [ 4 ]  . 
il perfezionamento dei devices e la crescente esperienza degli operatori ha consentito di proporre il trattamento endovascolare come alternativa terapeutica alla chirurgia , anche in emergenza , con lo scopo di ridurre la mortalit [ 5 ]  . 
questo lavoro si propone di presentare lesperienza del nostro centro nella gestione e nel trattamento endovascolare in urgenza e emergenza degli aaa . materials and methods materiali e metodi between july 2001 and october 2005 , 193 patients underwent aaa exclusion with stent - grafts at our centre . 
the patients presented with the following comorbidities : hypertension ( 19 cases ) , ischaemic cardiac disease ( 17 cases ) , chronic obstructive pulmonary disease ( 16 cases ) , chronic kidney failure ( five cases ) and diabetes ( five cases )  . 
on the basis of the severe comorbidities and urgency of the disease , 23 patients were classified american society of anesthesiology ( asa ) 4e ( urgent cases with severe systemic disease that is a constant threat to life ) whereas 15 patients with symptomatic aaa and thrombus fissure were classed asa 3e ( urgent cases with moderate or severe systemic disease with functional limitations )  . 
more specifically , the studies were conducted with multislice ct ( lightspeedplus , ge , milwaukee , wi , usa ; aquilion 64 , toshiba , tokyo , japan ) with unenhanced , arterialand venous - phase durante il periodo compreso tra luglio 2001 e ottobre 2005 , presso il nostro centro sono stati trattati 193 pazienti con endoprotesi per esclusione di aaa . 
i pazienti presentavano le seguenti comorbidit : ipertensione ( 19 casi ) ; cardiomiopatia ischemica ( 17 casi ) ; broncopneumopatia cronica ostruttiva ( 16 casi ) ; insuffucienza renale cronica ( 5 casi ) e diabete ( 5 casi )  . 
in relazione alle severe comorbidit e allurgenza della patologia , 23 pazienti sono stati classificati grado e4 di rischio operatorio ( pazienti trattati in urgenza con patologia sistemica severa in costante pericolo di vita ) in accordo con la classificazione della american society of anesthesiologist ( asa ) e 15 pazienti , con aaa sintomatico e fissurazione del trombo , grado e3 ( pazienti trattati in urgenza , con moderata o severa patologia sistemica limitante lattivit )  . 
pi specificatamente gli studi sono stati eseguiti con tc multistrato ( lightspeedplus , ge , milwaukee , usa ; aquilion 64 , toshiba , tokyo , giappone ) attraverso scansioni ottenute in condizioni basali , fase arteriosa , venosa e rid . 
b , c enhanced ct , arterial phase : haemorrhage in the mural thrombus with compression of the true lumen ( b , arrow ) and penetration of contrast medium into the thrombus ( c , arrow )  . 
b , c tc con mdc , fase arteriosa : lematoma del trombo causa compressione del vero lume ( b , freccia ) ; mdc penetra il trombo ( c , freccia )  . 
the venous - phase study is done to identify any increase in density of the retroperitoneal haematoma to determine whether it is fed ; in addition , leakage of contrast material is better visualised in this phase . 
in patients with haemorrhagic shock ( 15 / 38 patients ) , the diagnosis was established by ultrasonography ( us ) ( atl 5000 , philips medical systems , best , the netherlands ) performed in the emergency room , and the costruzioni mip ( maximum intensity projection ) e vr ( volume rendering )  . 
la fase venosa stata effettuata in relazione alla possibilit di individuare lincremento di densit dellematoma retroperitoneale per stabilire se rifornito ; inoltre lo stravaso attivo di mdc viene meglio evidenziato in fase venosa . 
nei casi di shock emorragico ( 15 / 38 pazienti ) , la diagnosi stata ottenuta con ecotomografia ( atl 5000 , philips medical systems , best , olanda ) eseguita in pronto soccorso e la procedura stata realizzata in emergenza efd . 
a , b computed tomography ( ct ) : a unenhanced phase ; b arterial phase : abdominal aortic aneurysm ( aaa ) rupture due to posterior sac tear resulting from conflict with the lumbar spine ( black arrow ) , with penetration of the contrast agent into the thrombus ( white arrow )  . 
a , b tc ( senza mdc : a ; fase arteriosa : b ) aaa rotto tamponato per lacerazione posteriore della sacca da conflitto con il rachide ( freccia nera ) ; mdc penetra il trombo ( freccia bianca )  . 
in these cases , evaluation of neck size and stent - graft selection was carried out by angiography with software allowing measurements starting from a precise calibre , such as that of the catheter , and using a ruled catheter to measure length . 
mean aneurysm diameter was 85 mm ( range 45120 mm ) , mean length of the proximal neck was 20 mm ( range 1030 mm ) and mean neck diameter was 25 mm ( range 1830 mm )  . 
in 21 patients , the aneurysm involved the abdominal aorta only ; in ten , both the common iliac arteries ; and in seven , the abdominal aorta and one common iliac artery . 
il diametro medio degli aneurismi era di 85 mm ( range 45120 mm ) , la lunghezza media del colletto prossimale di 20 mm ( range 1030 mm ) e il diametro medio dello stesso di 25 mm ( range 1830 mm )  . 
in 5 / 15 cases treated as emergencies , we used a catheter with a latex occlusive balloon ( equalizer , boston scientific , ratingen , germany ) ( diameter : 2733 mm ) placed in juxtarenal position with transfemoral percutaneous access because of the patient rapidly deteriorating haemodynamic status . 
in 2 / 5 cases , this allowed us to complete the procedure with positioning of a bifurcated stent - grathirty - two bifurcated stent - grafts [ 27 excluder ( gore , flagstaff , az , usa ) and five zenith ( cook , bloomington , in , usa ) ] and six aortouniiliac devices ( zenith ) were implanted . 
in selecting stent - grafts , we chose devices 23 mm larger than the diameter of the aneurysm neck and the calibre of the common iliac arteries for proximal and distal fixation , respectively . 
 our operating room is equipped with the following devices for the emergency / urgency treatment of aneurysms : two aortouniiliac kits with occluders ( diameters : 30 and 32 mm ) and four bifurcated endografts , including two for infrarenal fixation ( diameters : 28 and 30 mm with two leg measuring 12 cm and 14 cm in length ) and two endografts for suprarenal fixation ( diameters : 30 and 32 mm )  . 
endograft extensions are also available and are also interchangeable , if necessary . the main body of the device was introduced through the common femoral artery that had been isolated surgically . transfemoral access for placement of the contralateral leg of bifurcated devices was percutaneous in 29 patients and surgical in three owing to the small calibre of the iliofemoral axis . 
le procedure sono state espletate da un team di radiologi interventisti , chirurghi vascolari e anestesisti in sala operatoria con angiografo portatile . tutte le procedure sono state realizzate in anestesia generale . in 5 / 15 casi trattati in emergenza stato utilizzato un catetere fornito di pallone occlusore in lattice ( equalizer , boston scientific , ratingen , germania ) , con diametro di 2733 mm , posizionato in sede iuxta - renale mediante accesso percutaneo transfemorale poich le condizioni emodinamiche del paziente erano in rapido peggioramento allarrivo in sala operatoria . 
sono state impiantate 32 endoprotesi biforcate ( 27 excluder - gore , flagstaff , az , usa e 5 zenith - cook , bloomington , in , usa ) e 6 devices aorto - uni - iliaci ( zenith - cook , bloomington , in , usa )  . 
sono state adottate per la scelta dellendoprotesi dimensioni superiori di 23 mm rispetto al diametro del colletto dellaneurisma e al calibro delle arterie iliache comuni per lancoraggio rispettivamente prossimale e distale . presso il nostro centro , per il trattamento degli aneurismi in emergenza / urgenza sono disponibili in sala operatoria i seguenti device : 2 kit aorto - monoiliaci con rispettivo occlusore ( diametri 30 e 32 mm ) e 4 protesi biforcate , di cui 2 ad ancoraggio sottorenale ( diametri 28 e 30 cm con 2 bracci protesici lunghi 12 e 14 cm ) e 2 protesi ad ancoraggio sovrarenale ( diametri 30 e 32 mm con i rispettivi bracci protesici )  . 
laccesso transfemorale per il posizionamento del braccio protesico controlaterale del device biforcato stato percutaneo in 29 pazienti e chirurgico in 3 casi , in relazione al ridotto calibro dellasse iliaco - femorale . 
successivamente stata avanzata una guida superstiff 0 , 035 300 cm ( back - up meier , boston scientific , ratingen , germania ) e il corpo principale dellendoprotesi stato posizionato attraverso un introduttore da 18 f ( cook , bloomington , in , usa )  . 
in the 32 / 38 cases treated with bifurcated stent - grafts , the contralateral leg was positioned through a 12 - f introducer ( cook ) and a 0.035 - ( 185 - cm ) super - stiff guide ( back - up meier , boston scientific ) in the common femoral artery . 
use of a catheter with a low - pressure latex balloon ( diameter : 2733 mm ) ( equalizer , boston scientific ) allowed sealing of the endograft at the proximal and distal fixation sites and at the points of junction of the graft segments . 
in 6 / 38 patients , additional endovascular procedures on the iliac axis were necessary to advance the device [ percutaneous transluminal angioplasty ( pta ) in five cases , pta and stenting in one case ]  . 
nonionic isosmolar contrast material ( iodixanol , visipaque , amersham , saluggia , italy ) was administered to all patients ( range : 80160 ml , mean 100 ml )  . 
antithrombotic prophylaxis consisted of intraprocedural heparin ( 5 , 000 iu i.v. ) and low - molecularweight heparin in prophylactic doses for 30 days followed by antiaggregants or anticoagulants ( depending on the clinical data )  . 
an ultra - short term antibiotic prophylaxis was administered during induction of anaesthesia with the aim of achieving a reasonable concentration during endograft positioning ( vancomycin , abbott , latina , italy ; 1 g i.v. ) ; in patients with kidney failure or a history of intolerance , ciprofloxacin and cefazolin were used . 
in all patients , follow - up was performed with ct angiography according to the european collaborators on stent - graft techniques for abdominal aortic aneurysm repair ( eurostar ) protocol at 1 , 3 , 6 and 12 months and yearly thereafter [ 6 ]  . 
 trattati con device aorto - uni - iliaco ( zenith - cook , bloomington , in , usa ) , in 5 / 6 casi la procedura stata completata con il posizionamento di un dispositivo occlusore ( cook , bloomington , in , usa ) nellarteria iliaca comune controlaterale ; in 1 / 6 casi in urgenza la procedura stata completata con lutilizzo di spirali metalliche embolizzanti ( vortex - 35 , boston scientific , ratingen , germania , 78 mm ) nella arteria iliaca comune controlaterale steno - occlusa , al fine di preservare la perviet dellarteria iliaca interna . 
in 32 / 38 casi trattati con endoprotesi biforcata , la gamba controlaterale stata posizionata mediante introduttore 12 f ( cook , bloomington / in / usa ) e guida superstiff 0 , 035 185cm ( back - up meier , boston scientific , ratingen , germania ) attraverso larteria femorale comune ; quando stata utilizzata lendoprotesi zenith , lintroduttore era parte integrante del device . 
lutilizzo di un catetere fornito di pallone in lattice ( 2733 mm di diametro ) a bassa pressione ( equalizer , boston scientific , ratingen , germania ) ha consentito di sigillare lendoprotesi a livello degli ancoraggi prossimale e distale e le connessioni dei segmenti protesici . 
in 6 / 38 pazienti sono state necessarie procedure endovascolari aggiuntive a livello dellasse iliaco al fine di consentire lavanzamento del device ( pta in 5 casi , pta e stenting in 1 caso )  . il successo tecnico immediato stato dimostrato dallo studio angiografico eseguito al termine della procedura . 
 stato utilizzato in tutti i pazienti un mezzo di contrasto nonionico , isoosmolare ( iodixanolo , visipaque , amersham , saluggia , italia ) ( range 80160 ml , media 100 )  . 
la profilassi antitrombotica durante la procedura stata praticata con somministrazione di eparina ( 5000 ui endovena ) , seguita da eparina a basso peso molecolare in dosi profilattiche nei 30 giorni successivi , e successivamente con antiaggregante o anticoagulante ( in base ai dati clinico - anamnestici del paziente )  . 
la profilassi antibiotica stata realizzata secondo un protocollo ultra breve al momento dellinduzione dellanestesia per ottenere unadeguata concentrazione del farmaco al momento del posizionamento dellendoprotesi ( vancomicina , abbott , lt , italia ; 1 g ev ) ; in pazienti con insufficienza renale o pregressa intolleranza stata utilizzata ciprofloxacina e cefazoline . 
in tutti i pazienti il follow - up stato realizzato mediante angio - tc in accordo al protocollo eurostar a 1 , 3 , 6 , 12 mesi e in seguito annualmente [ 6 ]  . una angio - tc pre - dimissione stata eseguita nei 15 pazienti che , emodinamicamente instabili allingresso , erano giunti a diagnosi mediante la sola ecotomografia . results mean procedure time was 105 min ( range : 65235 )  . 
abbiamo osservato 7 lievi rialzi ( > 2 mg / dl ) dei livelli di creatinina sierica ( 2 / 7 pazienti con preesistente insufficienza renale cronica sono stati trattati con dialisi temporanea ) e 2 infezioni di d . 
there were 4 / 38 deaths ( 10.5% ) , one on the first postoperative day due to arrhythmia , two on the second and one on the fourth postoperative day due to multiorgan failure . 
mean postoperative hospital stay was 7.7 ( range : 230 ) days ; 11 / 38 patients required admission to the intensive care unit ( mean hospital stay 13 days , range : 230 days )  . 
five endoleaks were identified during follow - up ( 12% ) : four type - 2 endoleaks were identified at 3 months ( one arising from the inferior mesenteric artery , which thrombosed after 8 months ; three from the iliolumbar arteries , of which one thrombosed after 6 months and two were under observation at the time of writing ) ; one type - 3 endoleak ( detected at 12 months ) due to disjunction of the contralateral leg , which was corrected by positioning an extension . 
the total percentage of types - 1 and 3 endoleaks related to technical failure was 2.9% , which is comparable with the percentage measured under elective conditions ( 4% )  . 
in one patient , the endograft leg occluded after 6 months and was treated with femorofemoral bypass surgery . during follow - up , shrinkage of the aneurysm sac was observed in 10 / 34 cases whereas the remaining 24 / 34 cases showed no change in size ; no cases of aneurysm sac growth ( endotension ) were observed . discussion despite major progress in the fields of surgery , radiology , anaesthesia and intensive care , morbidity and mortality of ruptured aaas have remained high over the past 10 years as a result of the advanced age and frequent cardiorespiratory comorbidity of patients affected by this disease [ 7 ]  . 
the development of increasingly versatile devices and growing operator experience have extended the clinical and morphological indications for endovascular treatment , which is now being proposed as an alternative to open surgery , even in emergency settings [ 2 , 7 , 928 ]  . endovascular treatment presents some analogies with the modern approach to patients with severe haemorrhage . 
the primary goal is to use the least invasive procedure to rapidly control the source of bleeding and reduce the risk of prolonged hypoperfusion , hypothermia , coagulopathy and acidosis [ 29 ]  . 
abbiamo osservato 4 / 38 decessi ( 10 , 5% ) : uno nella i giornata post - operatoria a causa di un aritmia cardiaca , due in ii e uno in iv giornata post - operatoria a causa di una insufficienza multiorgano . 
la degenza post - operatoria media stata 7 , 7 giorni ( range 230 giorni )  . il ricovero in terapia intensiva stato necessario in 11 / 38 pazienti ( degenza media 13 giorni , range 230 giorni )  . 
durante il follow - up si sono osservati 5 endoleaks ( 12% ) : 4 di tipo ii documentati 3 mesi dopo la procedura ( 1 dallarteria mesenterica inferiore trombizzato in 8 mesi ; 3 dalle arterie ileo - lombari di cui uno trombizzato in 6 mesi e 2 sotto osservazione ) ; 1 di tipo iii ( dimostrato 12 mesi dopo la procedura ) dovuto alla sconnessione della gamba protesica controlaterale e corretto con il posizionamento di una prolunga protesica . non abbiamo osservato endoleak di tipo i . 
la percentuale totale di endoleaks di tipo i e iii direttamente correlate allinsuccesso tecnico della procedura stata del 2 , 9% , comparabile con quella riscontrata nella nostra esperienza in elezione ( 4% )  . 
durante il follow - up abbiamo osservato una riduzione della sacca aneurismatica in 10 / 34 casi e dimensioni invariate in 24 / 34 casi ; non abbiamo osservato casi di incremento volumetrico della sacca ( endotension )  . discussione nonostante gli importanti progressi nel campo di chirurgia , imaging , anestesia e metodiche di terapia intensiva , negli ultimi dieci anni la percentuale di morbilit e mortalit tra i pazienti con rottura di aaa rimane ancora elevata , soprattutto in considerazione dellet avanzata e della frequente comorbidit cardio - respiratoria dei pazienti con questo tipo di patologia [ 7 ]  . 
il trattamento chirurgico in elezione degli aaa in centri specializzati ha attualmente una mortalit di poco inferiore al 5% , che raggiunge il 50%65% in emergenza [ 8 ]  . 
lo sviluppo di devices sempre pi versatili e la crescente esperienza degli operatori ha esteso le indicazioni clinico - morfologiche del trattamento endovascolare permettendo allo stesso di proporsi come possibile alternativa terapeutica alla chirurgia open anche in emergenza [ 2 , 7 , 928 ]  . lalternativa endovascolare presenta alcune analogie con il moderno approccio al paziente con emorragia severa . lobiettivo primario infatti il rapido controllo della fonte di sanguinamento , attraverso la procedura meno invasiva , per ridurre il conseguente rischio di prolungata ipoperfusione e quindi di ipotermia , coagulopatia e acidosi [ 29 ]  . 
in haemodynamically stable patients ( arterial pressure > 80100 mmhg ) , ct angiography can be performed to identify the signs of aaa rupture and assess feasibility of the endovascular approach , and planning of the procedure is done in urgent conditions [ 30 ]  . 
in patients with haemorrhagic shock ( arterial pressure < 80 mmhg ) who have a us diagnosis of aaa rupture and are ineligible for open surgery , planning of the endovascular procedure is done in emergency conditions with angiography only [ 11 , 14 , 23 ]  . 
 selection criteria for urgent endovascular treatment are clinical and radiological : lumbar pain and / or hypotension ( systolic pressure < 100 mmhg ) or haemorrhagic shock ( systolic pressure < 80 mmhg ) , and pulsatile abdominal masses were considered clinical signs of symptomatic or ruptured aaa . 
radiological criteria were evidence of retroperitoneal haematoma or signs of impending rupture on ct angiography ( precontrast hyperdense thrombus , crescent sign and / or contrast penetration in the thrombus ) or on us ( inhomogeneous and stratified structure of the thrombus )  . 
in our experience , we used aortouniiliac stent - grafts in urgent conditions in patients with anatomy precluding placement of a bifurcated device only : occlusion of an iliofemoral axis ; small aortic bifurcation preventing placement of two endograft leg . 
vascular reconstruction afforded by the stentgraft is less anatomical ; furthermore , use of a uniiliac device in emergency conditions guarantees that the bleeding stops after deployment of the iliac occluder , which is definitely faster than that of an endograft leg , to be used in cases of severe haemodynamic instability [ 29 ]  . 
 some authors [ 31 , 32 ] have analysed outcomes to identify the variables most frequently associated with high mortality . variables identified were age over 80 years , preoperative cardiac arrest , loss of consciousness and shock ( systolic pressure < 91 mmhg , haemoglobin < 8 g / dl , haematocrit < 25% )  . 
this may be an ethically and professionally correct approach that spares the patient unnecessary suffering , avoid raising unjustified hopes in the relatives and reduce unnecessary healthcare costs [ 31 , 32 , 33 ]  . 
as also reported by other authors , we found the time required for placement of the contralateral leg of a bifurcated endograft to be shorter than that needed to perform a surgical femorofemoral crossover , with reduction in operative times [ 10 , 28 ]  . 
however , in haemodynamically unstable patients , placement of an aortouniiliac stent - graft is faster and safer . stabilisation of the patients haemodynamic conditions after tamponade and resuscitation procedures may provide the tre nei pazienti con sospetto di rottura di aaa essenziale seguire liter diagnostico meno time consuming . 
nel paziente emodinamicamente stabile ( pressione arteriosa > 80100 mmhg ) possibile eseguire unangio - tc per individuare i segni di rottura dellaaa e valutare la fattibilit dellapproccio endovascolare e il planning della procedura viene effettuato in urgenza [ 30 ]  . 
nel paziente con shock emorragico ( pressione arteriosa < 80 mmhg ) allorquando viene effettuata diagnosi di aaa rotto mediante ecografia e stabilito che il paziente non possiede gli estremi per lintervento di chirurgia open , il planning della procedura endovascolare viene effettuato in emergenza con la sola angiografia [ 11 , 14 , 23 ]  . 
 i criteri utilizzati per selezionare i pazienti per il trattamento endovascolare in urgenza sono clinici e radiologici . dolore lombare e / o ipotensione ( pressione sistolica < 100 mmhg ) o shock emorragico ( pressione sistolica < 80 mmhg ) e massa addominale pulsante sono stati considerati segni clinici di aaa sintomatico o rotto . 
i criteri radiologici utilizzati sono stati la presenza di ematoma retroperitoneale o segni di imminente rottura alla valutazione mediante angio - tc ( iperdensit del trombo in fase pre - contrastografica crescent sign e / o penetrazione del mezzo di contrasto nel contesto del trombo ) o mediante ecografia ( disomogeneit e stratificazione dellecostruttura del trombo )  . 
nella nostra esperienza le endoprotesi aorto - uni - iliache sono stati utilizzate , in urgenza , solamente in casi dove lanatomia non ha permesso il posizionamento di un device biforcato : occlusione di un asse iliaco - femorale , biforcazione aortica di diametro ridotto tale da non poter allogare due bracci protesici . 
questa endoprotesi consente una ricostruzione vascolare meno anatomica ; inoltre luso di un device monoiliaco in emergenza garantisce larresto del sanguinamento dopo il posizionamento dellocclusore iliaco , sicuramente pi veloce del posizionamento di un braccio protesico , da utilizzare in casi di severa instabilit emodinamica [ 29 ]  . 
tali variabili sono state identificate in : et superiore a 80 anni , arresto cardiaco preoperatorio , perdita di coscienza , shock ( pressione sistolica < 91 mmhg , emoglobina < 8 g / dl , ematocrito < 25% )  . 
questo potrebbe essere un atteggiamento corretto dal punto di vista etico e professionale al fine di evitare inutili sofferenze al paziente ed infondere inutili speranze nei suoi familiari , oltre che evitare costi assistenziali assolutamente inutili [ 3133 ]  . come riportato da alcuni autori in letteratura , nella nostra esperienza il tempo necessario al posizionamento della gamba controlaterale dellendoprotesi biforcata minore rispetto a quello richiesto per realizzare il cross - over chirurgico femoro - femorale , con una riduzione dei tempi operatori [ 10 , 28 ]  . 
the catheter was placed percutaneously and transfemorally by fixing it to a juxtarenal site through a 30 - cmlong 12 - f introducer to prevent any caudal displacement of the occlusive balloon caused by the arterial pressure . 
transfemoral access was preferred to brachial access owing to catheter calibre ( 7 f ) and the fact that attempting a third arterial access would have been too time consuming [ 24 ]  . 
although a number of studies have proposed local anaesthesia because of the small rate of cardiorespiratory complications [ 10 , 21 ] , our experience and that of other authors [ 15 , 16 ] make us inclined to use general anaesthesia to prevent unwanted movements and achieve better control of the patients haemodynamic conditions . use of muscle relaxants is , however , contraindicated since relaxation of the retroperitoneal musculature prevents control of the retroperitoneal haematoma [ 7 ]  . 
it should be noted that with uncontrolled haemorrhage , excessive fluid infusion may be dangerous and increase the bleeding ; controlled hypotension , on the other hand , slows the haemorrhage and can therefore facilitate the formation of a local clot . 
in emergency procedures , it reaches 28% , which can be explained by the need to cover the renal arteries in the absence of a neck in nonsurgical patients undergoing emergency endovascular treatment [ 10 ]  . 
exclusion of the inferior mesenteric artery ( and hence of the hypogastric artery ) in a hypoperfused patient can cause colonic ischaemia , in part as a result of a reduced compensation capacity of the other splanchnic vessels . 
la decisione anche in relazione allesperienza degli operatori . nella nostra casistica in accordo con lesperienza riportata in letteratura , il catetere fornito di pallone occlusore in lattice stato utilizzato solo in 5 / 15 casi di instabilit emodinamica del paziente appena arrivato in sala operatoria [ 16 ]  . 
il catetere stato posizionato per via percutanea transfemorale ancorandolo in sede iuxtarenale mediante un introduttore 12 f lungo 30 cm per prevenire dislocazioni caudali del pallone occlusore dovute alla pressione arteriosa . 
laccesso transfemorale stato preferito al brachiale in relazione al calibro del catetere di 7 f e perch il tentare il terzo accesso arterioso avrebbe comportato un eccessivo dispendio di tempo [ 24 ]  . 
malgrado molti studi suggeriscano luso dellanestesia locale in relazione alla bassa percentuale di complicanze cardio - respiratorie [ 10 , 21 ] , la nostra esperienza e quella di altri autori [ 15 , 16 ] propende per la scelta dellanestesia generale . 
bene sapere che , in presenza di un emorragia incontrollata , uneccessiva infusione di liquidi pu essere pericolosa causando un incremento del sanguinamento ; una ipotensione controllata invece rallentando lemorragia pu agevolare la formazione di un coagulo locale . le possibili complicanze del trattamento endovascolare sono sovrapponibili a quelle incontrate nel trattamento in elezione . particolare attenzione andrebbe inoltre riservata alla funzionalit renale e alleventuale ischemia splancnica nel caso del trattamento in emergenza . 
lo shock emorragico con ipoperfusione renale , la prolungata ipotensione e la sindrome compartimentale da ematoma retroperitoneale sono tutti fattori che contribuiscono al deterioramento della funzione renale connesso anche alluso del mezzo di contrasto . 
lincidenza dellinsufficienza renale post - operatoria varia tra il 2 , 5% e l8 , 3% per il trattamento in urgenza , in accordo con i dati eurostar e non differisce significativamente dal trattamento chirurgico . 
nelle emergenze raggiunge il 28% e questo pu essere anche giustificato dalla necessit di coprire le arterie renali in assenza di colletto in paziente non chirurgico che effettua lalternativa endovascolare in emergenza [ 10 ]  . 
lesclusione dellarteria mesenterica inferiore ( e in definitiva dellarteria ipogastrica ) in un paziente ipoperfuso pu causare unischemia colica anche in relazione alla minore capacit di compenso degli altri vasi splancnici . 
the frequency of type - 1 and type - 3 endoleaks , directly related to technical failure , is 3.7% , similar to that observed in elective procedures in our experience ( 4% ) and to that reported in the literature [ 26 ]  . 
review of the literature and consideration of the different techniques used by the various centres for planning the procedure ( from device selection to choice of anaesthesia ) shows that endovascular treatment is feasible and carries a high rate of shortand medium - term technical success , with only a few cases requiring surgical conversion [ 2 , 7 , 928 ]  . 
furthermore , reports comparing endovascular and surgical treatment have demonstrated that endovascular treatment significantly reduces mortality in addition to providing the benefits of a minimally invasive procedure ( less blood loss , shorter stays in intensive care units ) [ 2 , 12 , 17 , 18 ]  . 
 on the basis of our personal experience and the literature , we can conclude that the endovascular approach is a promising alternative for the treatment of aaas , under both urgency and emergency conditions . 
the use of bifurcated grafts , when possible , allows for better reconstruction of the vascular anatomy with shorter overall operative times compared with the placement of an aortouniiliac device and subsequent femorofemoral bypass . 
further studies with longer follow - up periods and larger series are required to evaluate the long - term results of endovascular treatment and compare it with surgical treatment in terms of morbidity , mortality and long - term success . sere trattata con una decompressione extraperitoneale translombare nei pazienti con segni di insufficienza dorgano correlata allaumento della pressione intra - addominale [ 7 , 11 ]  . la frequenza di endoleak tipo iiii direttamente correlata al fallimento tecnico della procedura del 3 , 7% , comparabile con quella riscontrata nella nostra esperienza nel trattamento in elezione ( 4% ) e con quella riportata in letteratura [ 26 ]  . 
dalla revisione della letteratura e considerando le differenti tecniche usate nei differenti centri per pianificare la procedura ( dalla scelta del device al tipo di anestesia ) , lapproccio endovascolare fattibile con unalta percentuale di successo tecnico immediato e a medio termine e in pochi casi richiede la conversione chirurgica [ 2 , 7 , 928 ]  . 
 inoltre le casistiche che comparano il trattamento endovascolare con la chirurgia rivelano una significativa riduzione in mortalit nel primo gruppo in aggiunta ai vantaggi correlati alla mini - invasivit della procedura ( minori perdite ematiche , riduzione dei tempi di degenza in unit di cura intensiva ) [ 2 , 12 , 17 , 18 ]  . 
in conclusione , da quanto si evince dallesperienza personale e dalla revisione della letteratura , possibile affermare che lapproccio endovascolare una promettente alternativa terapeutica per il trattamento sia in urgenza che in emergenza degli aaa . 
la scelta del tipo di protesi , biforcata o aorto - uni - iliaca , in urgenza e in emergenza dipende dalle condizioni emodinamiche del paziente , dallesperienza degli operatori e dalladeguato approvvigionamento di materiali . 
luso dellendoprotesi biforcata , dove possibile , permette una migliore ricostruzione dellanatomia vascolare con minori tempi operatori complessivi , rispetto al posizionamento di un device aorto - uni - iliaco con successivo bypass femoro - femorale . 
gandini1 1ospedale san giovanni battista , via genova 3 , i - 10126 torino , italy 2struttura complessa fisica sanitaria i , 3scdu di endocrinologia e malattie del metabolismo , istituto di radiologia , universit di torino , torino , italy correspondence to : c . 
at baseline and at 6 months follow - up , 290 male patients aged between 18 and 37 ( average age 27.3 years ) with left ( 266 cases ) or bilateral ( 24 cases ) varicocele underwent clinical assessment , doppler ultrasonography ( us ) , laboratory testing of free and total serum testosterone , leutenising hormone ( lh ) and follicle stimulating hormone ( fsh ) gonadotropins , inhibin b and spermiograin 223 cases , selective catheterisation of the spermatic vein was performed with a right transfemoral approach . 
technical success was achieved in 206 / 223 cases ; two phlebographic examinations ( immediately following administration of the sclerosing agent and after 1520 min ) showed prethrombotic endoluminal alterations of the internal spermatic veat 6 months follow - up , 172 / 206 patients ( 83.49% ) showed complete resolution of the varicocele whereas 34 / 206 ( 16.5% ) had only partial disengorgement of the pampiniform plexus . 
the following minor procedural complications were recorded : two cases of acute abdominal pain , three of vagal crisis during administration of sclerosing agent that resolved spontaneously and two of spermatic cord inflammation that resolved within days after medical therapy . we recorded no statistically significant differences with regard to riassunto obiettivo . 
in condizioni basali e ad un follow - up di 6 mesi 290 pazienti maschi di et compresa tra 18 e 37 anni ( vm 27 , 3 ) con varicocele sinistro ( 266 casi ) o bilaterale ( 24 casi ) sono stati valutati con esame clinico , esame doppler , dosaggio su siero di testosterone libero e totale , delle gonadotropine lh ed fsh e dellinibina b e con lo spermiogramma . 
duecentosei su 223 pazienti sono stati sottoposti a trattamento radiologico del varicocele ; in 194 casi stato utilizzato esclusivamente lhydroxy - poliethoxy - docanol ( atossisclerol ) , mentre in 12 casi ( 5 , 8% ) in aggiunta ad esso sono stati impiegati 5 ml di alcol assoluto ed una spirale di gianturco ( cook 10 mmx50 mm da 0 , 038 pollici )  . 
nei 206 pazienti trattati stato ottenuto il successo tecnico immediato ; due successivi controlli flebografici ( immediatamente dopo ed a 1520 minuti dalla somministrazione degli agenti scleroembolizzanti ) hanno dimostrato le alterazioni pretrombotiche endoluminali della vena spermatica interna . 
il follow - up a 6 mesi dal trattamento radiologico percutaneo ha rilevato la scomparsa del varicocele in 172 / 206 pazienti ( 83 , 49% ) , mentre in 34 / 206 pazienti ( 16 , 5% ) la detensione del plesso pampiniforme stata soltanto parziale . 
sono stati registrati i seguenti piccoli effetti collaterali del trattamento : 2 casi di algia addominale acuta e 3 casi di crisi vagale durante liniezione dello sclerosante , regrediti spontaneamente , e 2 casi di flogosi irritativa c . 
the principal technical limitation to percutaneous treatment is related to difficult selective catheterisation of the spermatic vein due to anatomic alterations , spasms and intimal dissection of the vemoreover , when the cremasteric vein is incontinent , inguinal surgical ligation provides better results . 
in the majority of cases , administration of at least 3 ml sclerosing agent at 3% ensures occlusion of the gonadic vein above the abdominal collaterals , which are responsible for long - term recurrence if not treated . 
 key words varicocele sclerotherapy seminal parameters dosimetry del funicolo spermatico risolta in pochi giorni con terapia medica . non sono state registrate differenze statisticamente significative per quanto concerne la volumetria testicolare ed i dosaggi ormonali sierici tra il gruppo di pazienti trattati e non trattati . 
la somministrazione di almeno 3 ml di sclerosante concentrato al 3% assicura nella gran parte dei pazienti locclusione della vena gonadica a monte dei circoli collaterali addominali , responsabili , se non trattati , di recidiva a distanza ; negli altri casi , possono essere utilizzati a completamento terapeutico lalcol assoluto e le spirali metalliche . 
riteniamo , infine , che le manovre radiologiche siano controindicate ed ingiustificate nei casi in cui si rendano necessari prolungati cateterismi con elevata irradiazione delle gonadi . parole chiave varicocele scleroterapia parametri seminali dosimetria introduction introduzione varicocele , a varicose dilatation of the pampiniform plexus veins , is a very common disorder that is clinically relevant in 5%20% of males and often associated with dyspermia ( 30%45% ) and infertility ; hence , the indication for treatment of paediatric patients and / or adolescents [ 1 ]  . 
the disorder is caused by inversion of blood flow within the internal spermatic vein , which drains into the renal vein on the left side and , in 90% of cases , directly into the inferior vena cava on the right side . 
primary varicocele is due to valve incompetence and favoured by the retroperitoneal location of the internal spermatic vein ( absence of contiguous muscles facilitating centripetal blood flow ) and by renal - vein abnormalities ; it is associated with factors related to the vessel wall . symptomatic varicocele is rare and secondary to expansile processes of the retroperitoneum , kidney and pancreas . due to the above - mentioned anatomical factors , varicocele is more common on the left side . 
venous reflux occurs more frequently through the internal spermatic vein or via aberrant collateral branches originating from the renal vein , intraor perirenal branches , inferior vena cava , lumbar veins or iliac vein and anastomosed caudally with the internal spermatic vediagnostic imaging is instrumental in the diagnosis and staging of varicocele . 
ultrasonography is highly sensitive in detecting subclinical forms , and il varicocele , dilatazione varicosa del plesso pampiniforme , unaffezione molto frequente che ha rilevanza clinica nel 5%20% della popolazione maschile ed spesso associata a dispermia ( 30%45% ) ed infertilit ; di qui lindicazione al trattamento di pazienti pediatrici e / o adolescenti [ 1 ]  . 
questa patologia dovuta allinversione di flusso nella vena spermatica interna , che a sinistra sbocca nella vena renale e , a destra , nel 90% dei casi direttamente nella vena cava inferiore . 
la forma primitiva dovuta ad incontinenza valvolare , favorita dalla sede retroperitoneale della vena spermatica interna ( assenza di muscoli contigui favorenti la progressione centripeta del sangue ) e da alterazioni anatomiche della vena renale , ed associata a fattori costituzionali della parete vasale . 
il reflusso venoso avviene pi spesso attraverso la vena spermatica interna o in alternativa lungo collaterali venosi aberranti con essa anastomizzati caudalmente originanti dalla vena renale , da rami intrao peri - renali , dalla vena cava inferiore , dalle vene lombari o dalle vene iliache . la diagnostica per immagini ha un ruolo fondamentale nella diagnosi e nella stadiazione del varicocele e si avvale delle seguenti tecniche : flussimetria doppler , eco - tomografia e flebografia . 
selective phlebography of the renal vein or internal spermatic vein enables identification of incompetent valves at the ostial level , determination of degree of reflux of contrast medium as well as mapping of the venous drainage system , which serves to distinguish among the different types of varicocele ( table 1 )  . 
 percutaneous treatment , which can be performed as an outpatient procedure and involves a moderate radiation dose [ 4 ] , is always indicated in types i and iii but only indicated in types ii and iv if the catheter can be advanced above the collaterals close to the inguinal ligament . 
in advanced cases , with associated ectasia of the posterior pampiniform plexus draining into the iliac system , surgery is the only feasible treatment due to impossibility of selectively catheterising the relevant veins . 
 the through a right transfemoral or brachial rarely jugular is selectively internal spermatic vein approach , catheterised using simmons or cobra catheters , depending on whether the right or left side is being treated . 
the use of detachable balloons and metallic coils reduces embolisation time but is not recommended in the presence of a rich network of collaterals due to possible risk of early recurrence ; in such cases , sclerosing agents should be used . 
 complications secondary to percutaneous therapy are relatively rare and unimportant ; the most serious include development of phlebothrombosis of the pampiniform plexus and of the venous districts connected to the internal spermatic vein and the possible migration of embolisation material , or parts thereof , into the systemic circulation . current legislation on radiation protection [ 5 ] requires compliance with the principle of justification for medical extable 1 types of left - sided varicocele based on anatomical variants c . 
schematicamente , i metodi attualmente utilizzati nella terapia percutanea del varicocele sono locclusione meccanica con spirali di gianturco o palloncini staccabili e la sclerosi con un agente chimico . con approccio femorale destro o brachiale , raramente giugulare , si cateterizza selettivamente la vena spermatica interna , utilizzando per lo pi cateteri di tipo cobra se il trattamento da effettuare a sinistra e di tipo simmons se a destra . 
sulla base della preventiva flebografia , eseguita facendo compiere al paziente la manovra di valsalva , si decide il trattamento pi idoneo in base al tipo di variante anatomica dimostrata . 
luso dei palloncini staccabili e delle spirali metalliche riduce il tempo di trombizzazione , ma non indicato nei casi in cui sia presente una ricca rete di collaterali per la possibile insorgenza di recidiva precoce ; in questi casi opportuno limpiego di farmaci sclerosanti . le complicanze conseguenti alla terapia percutanea sono relativamente poco frequenti e di scarsa importanza ; tra le pi gravi , vi sono lassai rara comparsa di flebotrombosi del plesso pampiniforme e dei distretti venosi anastomizzati con la vena spermatica interna e la possibile migrazione nel cirsingle testicular vein without valves afferent collateral retroperitoneal vessels to the ascendant lumbar vein and / or retroaortic vessels adjacent to the lumbar vessels tributary of the inferior vena cava duplication of internal spermatic vein into parallel main branches with communicating cross - connecting branches afferent collaterals to renal and / or perirenal veins bifurcation of renal vein showing preand retroaortic course tabella 1 tipologie di varicocele sinistro secondo le varianti anatomiche vena testicolare singola senza valvole circoli collaterali retroperitoneali afferenti alla vena lombare ascendente e / o vasi retroaortici contigui alle vene lombari tributari della vena cava inferiore suddivisione della vena spermatica interna in due rami principali paralleli ampiamente anastomizzati tra loro circoli collaterali afferenti alle vene renali e / o perirenali vena renale biforcata a decorso antero e retro - aortico c . 
 materials and methods clinical and seminal evaluation two hundred and ninety patients aged 1837 years ( mean age 27 years ) referred to our division of andrology for low fertility or testicular pain were diagnosed with varicocele . all patients were assessed on entry and 6 months later after associated urogenital , endocrine and internal disorders had been excluded . 
serum concentrations of total ( radioimmunoassay , sorin biomedica , saluggia , italy ) and free ( radioimmunoassay , diagnostic products corporation , los angeles , ca , usa ) testosterone , follicle stimulating hormone ( fsh ) and leutenising hormone ( lh ) gonadotropin ( immunoradiometric assay , radim s.p.a. , rome , italy ) and inhibin b ( enzyme - linked immunosorbent assay , oxford bio - innovation ltd , upper heyford , uk ) were measured . 
seminal examinations were carried out in accordance with the world health organisation ( who ) criteria [ 9 ]  . sperm concentration was obtained using a makler chamtable 2 doppler - based grading system according to dubin [ 8 ] modest and transitory reflux during a valsalva manoeuvre ( normal report ) persistent reflux finishing before the end of a valsalva manoeuvre reflux persisting during a valsalva manoeuvre baseline reflux not changing during a valsalva manoeuvre tabella 2 grading doppler del varicocele secondo dubin [ 8 ] modesto e transitorio reflusso venoso durante la manovra di valsalva ( quadro fisiologico ) reflusso venoso persistente che si esaurisce prima del termine della manovra di valsalva reflusso venoso persistente durante lintera manovra di valsalva reflusso venoso presente in condizioni basali che non muta durante la manovra di valsalva grade 0 grade 1 grade 2 grade 3 grado 0 grado 1 grado 2 grado 3 colo sistemico dei mezzi embolizzanti o delle loro parti . lattuale normativa di radioprotezione [ 5 ] impone losservanza del principio di giustificazione per le esposizioni mediche attraverso la valutazione dei potenziali vantaggi diagnostici o terapeutici complessivi prodotti rispetto al danno alla persona che lesposizione potrebbe causare . lefficacia tecnico - clinica del trattamento radiologico del varicocele deve essere quindi valutata alla luce di misure dosimetriche . 
alcuni studi presenti in letteratura [ 6 , 7 ] riportano valori di dose alle gonadi che permettono di escludere linduzione di danni di tipo deterministico e valori di dose efficace che , pur rientrando nellordine di grandezza proprio di altre indagini diagnostiche , meritano una particolare attenzione nei confronti del rischio probabilistico . materiali e metodi valutazione clinico - seminale in 290 pazienti di et compresa tra 18 e 37 anni ( vm 27 anni ) , giunti al servizio di andrologia del nostro ospedale per ipofertilit o dolenza testicolare , stata posta diagnosi di varicocele . 
sono state quindi dosate le concentrazioni sieriche di testosterone totale ( radioimmunoassay sorin biomedica , saluggia , italia ) e libero ( radioimmunoassay diagnostic products corporation , los angeles , usa ) , delle gonadotropine fsh ed lh ( immunoradiometricassay radim s.p.a. , roma , italia ) e di inibina b ( enzyme - linked immunosorbent assay oxford bio - innovation ltd . , upper heyford , uk )  . 
gli esami seminali sono stati effettuati secondo i criteri del who [ 9 ]  . la concentrazione degli spermatozoi stata determinata utilizzando la camera di makler , mentre la loro morfologia stata valutata con colorazione di papanicolaou ; il grado di motilit stato stimato soggettivamente . duecentosei pazienti sono stati sottoposti a scleroembolizzazione radiologica della vena spermatica interna , mentre in 17 pazienti stata effettuata la sola flebografia spermatica . 
treatment effectiveness was assessed by two consecutive postprocedure phlebographic examinations and subsequently by doppler examination of the spermatic cord ; in particular , partial regression of the varicocele by at least one grade was defined as a reduction in the degree of incompetence . 
 radiological procedure this procedure was carried out using an angio diagnost 3 dvi ( philips medical systems , best , the netherlands ) radiological device . following local anaesthesia ( 5 ml of 2% lidocaine ) , the right common femoral vein was accessed and a 5f vascular introducer positioned . 
selective catheterisation of the renal vein with 5f cobra catheter and 0.035hydrophilic guidewire ( terumo corporation , tokyo , japan ) was performed using the seldinger technique . contrast medium ( ultravist 370 , schering ag , berlin , germany ) was administered with the patient performing a valsalva manoeuvre to demonstrate valve incompetence of the internal spermatic vein . subsequently , selective analysis of the internal spermatic vein was performed with the catheter tip positioned just above the inguinal ligament ; morphology of the collateral circulation and incontinence of the cremasteric vein were accurately evaluated [ 10 ]  . a simmons 5f hydrophilic catheter was used for rightsided selective spermatic phlebography ; in these patients , the ipsilateral transfemoral approach made it difficult to achieve deep catheterisation , which was only possible with the more expensive coaxial technique . following digital massage of the testis to drain the opacified veins of the pampiniform plexus , occlusion of the internal spermatic vein was obtained by administering a 50% mixture of hydroxy - poliethoxy - docanol ( atoxysclerol ) at 2% or 3% and contrast material via the catheter placed with the tip caudal to the origin of the collaterals just above the inguinal ligament . during treatment , the patient was instructed to compress the internal spermatic vein to avoid thrombosis of the testicular circulation and to perform a valsalva manoeuvre to prevent reflux of the sclerosing agent into the renal vein . the same technique was performed on right - sided varicoceles . 
at the end of the procedure , two successive phlebographic examinations were performed at 1520 min 12 cases of persistent patency of the spermatic vein , 5 ml of alcohol and a 0.038 - ( 10 mmx50 mm ) metallic coil were also used . 
following removal of the angiographic catheter and vascular introducer , the puncture site was briefly compressed ; the patient was then sent to the day hospital for 34 h of clinical observation . 
in anestesia locale ( 5 ml di lidocaina al 2% ) stata eseguita la puntura della vena femorale comune destra con successivo posizionamento di introduttore vascolare di 5f . con tecnica di seldinger stato eseguito il cateterismo selettivo della vena renale con catetere cobra di 5f e guida idrofila di 0 , 035 pollici ( terumo corporation , tokyo , japan )  . 
per dimostrare lincontinenza valvolare della vena spermatica interna stata eseguita uniniezione di mdc ( ultravist 370 , shering ag , berlin , germany ) in corso di manovra di valsalva . 
stato quindi effettuato lo studio selettivo della vena spermatica interna , sospingendo in tutti i casi lapice del catetere appena al di sopra del legamento inguinale ; stato possibile valutare precisamente lanatomia dei circoli collaterali e lincontinenza della vena cremasterica [ 10 ]  . 
a destra , per la flebografia spermatica selettiva , stato impiegato un catetere idrofilo tipo simmons di 5f ; in questi pazienti lapproccio vascolare transfemorale omolaterale ha reso difficoltoso in molti casi il cateterismo profondo , che stato reso possibile solo grazie alla pi costosa tecnica coassiale . 
previo massaggio digitale del proprio testicolo da parte del paziente , volto a drenare le vene del plesso pampiniforme opacizzate , stata ottenuta locclusione della vena spermatica interna somministrando una miscela al 50% di hydroxy - poliethoxy - docanol ( atossisclerol ) al 2% o 3% e contrasto attraverso il catetere posizionato con apice caudalmente allorigine dei circoli collaterali , poco al di sopra del legamento inguinale . 
durante il trattamento il paziente effettuava contemporaneamente una compressione occlusiva della vena spermatica interna allinguine , per evitare la trombosi del circolo testicolare , e la manovra di valsalva per evitare il reflusso dellagente sclerosante in vena renale . la stessa tecnica stata applicata ai varicoceli di destra . 
al termine della procedura sono stati eseguiti 2 successivi controlli flebografici a distanza di 1520 min 12 casi di persistente perviet della vena spermatica sono stati utilizzati anche 5 ml di alcol ed una spirale metallica da 0 , 038 pollici di 10 mmx50 mprevia rimozione del catetere angiografico e dellintroduttore vascolare stata effettuata una breve compressione del sito di puntura ; il paziente stato quindi rinviato in day hospital per unosservazione clinica di 34 ore . 
alla dimissione sono stati consigliati riposo a letto per 48 ore ed astensione da attivit fisica gravosa per almeno 30 giorni . analisi statistica sono stati utilizzati il t - test e c2 test . valutazione dosimetrica sono state effettuate misure in vivo nel corso di 20 procedure . 
per le misure stata utilizzata una camera trasmissiva diamentor m4kdk a doppio canale ( ptw , freiburg ) con misura simultanea del kerma in aria ( kair ) sullasse centrale del fascio ( area di misura di 13x13 mm ) e del dap ( area di misura di 140x140 mm )  . 
a double diamentor m4kdk transmission chamber ( ptw - freiburg ) was used to measure simultaneously the air kerma ( kair ) on the central beam axis ( field size 13x13 mm2 ) and the dose area product ( dap ) ( field size 140x140 mm2 )  . 
the chamber was positioned at the x - ray tube window during the procedures after checking that it did not interfere with beam characteristics and actions required for the interventional procedure . 
appropriately calibrated singlepack kodak x - omat v films ( 25.4x30.5 cm2 ) positioned between the bed and the patient were used to measure entrance surface dose ( esd ) and effective dose ( ed )  . 
film optical density , correlated to the dose according to the calibration curve used , was read using a densitometer for point measurements ( pehamed densoquick 2 ) and an agfa arcus ii scanner . 
complete distribution of the esd obtained in this way can be used to derive a reliable estimate of effective dose by defining an equivalent configuration of static exposures and using the ods - 60 calculation software ( distributed by rti electronic ) , which measures organ dose in a phantom for conventional radiological examinations ( thus with rectangular fields and based on dap ) on the basis of data obtained with monte carlo simulations . 
1 rappresentazione schematica della disposizione della strumentazione di misura utilizzata per le valutazioni di dose in corso di procedura angiografica . rificato la totale assenza di interferenza con le caratteristiche del fascio e con le azioni necessarie per lo svolgimento dellintervento . 
per le valutazioni della dose in ingresso ( di ) e per la stima della dose efficace ( de ) sono state utilizzate pellicole kodak tipo x - omat v impacchettate in busta singola di dimensioni 25 , 4x30 , 5 cm , opportunamente calibrate e posizionate tra il lettino e il paziente . 
la lettura dei valori di densit ottica delle pellicole , correlati alla dose secondo la curva di taratura realizzata , stata effettuata con un densitometro a punto ( pehamed densoquick 2 ) e con uno scanner agfa arcus ii . la distribuzione completa della dose in ingresso superficiale cos ottenuta pu essere utilizzata per effettuare una stima attendibile della dose efficace , attraverso la definizione di una configurazione equivalente di esposizioni statiche e lutilizzo del programma di calcolo ods - 60 distribuito dalla rti electronic , che determina la dose assorbita dagli organi di un fantoccio virtuale per esami radiologici tradizionali ( quindi con campi rettangolari e in base al dap ) , sulla base di dati ottenuti da simulazioni monte carlo . 
le calibrazioni e le letture dei dosimetri sono state effettuate dalla ditta x - gammaguard di saronno , con valori espressi in termini di equivalente di dose personale hp ( 0 , 07 )  . 
tali valori sono stati convertiti in kerma in aria utilizzando i coefficienti di conversione tabulati [ 12 ] e riferendosi al fascio di taratura utilizzato di energia media 58 kev . 
i valori di kerma in aria sono stati a loro volta convertiti in equivalente di dose alle gonadi tramite lopportuno coefficiente di conversione [ 12 ] pari a 1 , 6 , considerando che la tensione media di lavoro di 80 kv per lapparecchio utilizzato corrisponde ad unenergia media di 44 kev . risultati non sono state registrate differenze statisticamente significative per quanto concerne la volumetria testicolare ( 1825 cc ) ed i dosaggi ormonali sierici nei 2 gruppi studiati . 
in 206 / 223 pazienti ( 92 , 37% ) stato possibile effettuare con successo il trattamento radiologico percutaneo del varicocele ; i controlli flebografici hanno dimostrato le incipienti alterazioni pretrombotiche endoluminali della vena spermatica interna . 
in 17 / 223 pazienti ( 7 , 6% ) ci si limitati ad eseguire la flebografia selettiva diagnostica della vena spermatica a causa di dissezioni intimali invalicabili accidentalmente indotte o per la presenza nel letto da occludere di grossi collettori venosi comunicanti con il circolo sistemico . la durata media della procedura percutanea stata di 30 min ; il secondo controllo flebografico ha comportato un ulteriore impegno di 20 min della sala angiografica . 
il tempo medio di radioscopia continua stato 6 , 3 minuti ( range : 510 min )  . locclusione stata ottenuta mediamente con 3 fiale di atossisclerol ; in 12 pazienti , data la persistente opacizzazioc . 
air kerma values were in turn converted into gonad dose equivalent using the appropriate conversion coefficient of 1.6 [ 12 ] , considering that the mean working current 80 kv for the device used corresponds to a mean energy of 44kev . 
 results there were no significant differences regards testicular volume ( 1825 cc ) and serum hormone levels in the two groups . in 206 / 223 patients ( 92.37% ) , percutaneous treatment of the varicocele was successful ; postprocedural phlebography showed incipient endoluminal prethrombotic alterations of the internal spermatic vein 17 / 223 patients ( 7.6% ) , only diagnostic selective phlebography of the spermatic vein was performed due to either accidental intimal dissections or the presence of large venous vessels communicating with systemic circulation . 
mean duration of the percutaneous procedure was 30 min whereas the second phlebographic examination entailed a further 20 min in the angiography suite . mean duration of continuous fluoroscopy was 6.3 min ( range 510 min )  . 
 occlusion was obtained with an average of three vials of aetoxysclerol ; in 12 patients , treatment was completed with a metallic coil ( 0.038cook coil , 10 mmx50 mm ) and 5 ml of absolute alcohol due to persistent opacification of the varicocele during phlebography . 
in all 206 patients , follow - up at 6 months showed a significant increase in sperm concentration and motility , with negligible morphological changes ( table 3 )  . 
2 distribuzione e collocazione sul fantoccio virtuale della dose superficiale dingresso . ne del varicocele al controllo flebografico , sono stati impiegati a completamento terapeutico una spirale metallica ( cook di 10x50 mm da 0 , 038 pollici ) e 5 ml di alcol assoluto . 
because the procedures did not involve radiography , fluoroscopy time was the most significant exposure parameter ; variability in fluoroscopy time was relatively low , with a standard deviation of 24% . 
figure 2 shows an example of the entrance dose distribution obtained from radiographic films and an indication of its position on the phantom , which we used to calculate the effective dose by defining an equivalent configuration of four static fields . in particular , the gonad doses calculated by the ods - 60 programme corresponded to the direct readings of the thermoluminescent dosimeters , with a 20% tolerance . 
 discussion and conclusions varicocele is a disorder characterised by dilatation of the pampiniform plexus veins secondary to hypertension of the left renal vein ; the presence of circulation between the two gonadal veins may cause increased contralateral pressure with development of milder varicocele on the right side as well . 
27 / 206 , 13 , 1% ; p < 0 , 001 ]  . il trattamento percutaneo ha indotto complicanze minori in 5 pazienti : 2 algie addominali acute di pochi minuti in corso di iniezione di alcol , 2 flogosi del funicolo spermatico trattate con successo mediante terapia medica ed 3 brevi crisi vagali risoltesi spontaneamente . 
nei pazienti che costituivano il gruppo di controllo dello studio e nei 17 pazienti non trattati in corso di follow - up non si sono verificate variazioni significative n dal punto di vista clinico , n per quanto concerne i parametri seminali . 
poich le procedure effettuate non prevedevano lutilizzo della modalit grafia , il tempo di fluoroscopia stato il parametro di esposizione pi significativo per caratterizzare la complessit dellintervento ; la variabilit di tale grandezza risultata essere piuttosto bassa , con un valore di deviazione standard percentuale del 24% . la figura 2 mostra un esempio di distribuzione di dose in ingresso ottenuta dalle pellicole radiografiche e unindicazione della sua collocazione sul fantoccio virtuale utilizzato per il calcolo della dose efficace , attraverso lutilizzo di una configurazione equivalente di quattro campi statici . 
in particolare si verificata la corrispondenza dei valori di dose alle gonadi ricavate dal programma di calcolo ods - 60 con quelli ottenuti dalle misure dirette tramite dosimetri a termoluminescenza , entro una tolleranza del 20% . discussione e conclusioni il varicocele unaffezione caratterizzata dallectasia del plesso pampiniforme secondaria ad uno stato ipertensivo della vena renale sinistra ; lesistenza di circoli tra le 2 vene gonadiche spiega il possibile incremento pressorio controlaterale con insorgenza di varicocele anche a destra , seppur di grado inferiore . 
la possibile associazione tra varicocele ed infertilit ha suggerito di intraprendere sulla popolazione adolescenziale programmi di diagnosi preventiva e di terapia precoce . il trattamento percutaneo del varicocele una procedura paragonabile per efficacia alla correzione chirurgica [ 13 ] ; nellanalisi della giustificazione alla procedura e del rapporto costi / benefici vanno tuttavia considerati il rischio di danno probabilistico radio - indotto , la giovane et dei pazienti ed il basso grado di gravit della patologia . 
nella popolazione in studio , assume quindi una particolare rilevanza radioprotezionistica valutare la dose di esposizione , la dose efficace e la dose equivalente ; per calcolare queste grandezze in corso di procedure interventistiche ( ad elevata dinamicit , non standardizzabili e condotte per lo pi in fluoroscopia ) , si preferito utilizzare un metodo sperimentale in quanto pi preciso del metodo monte carlo . nel campione considerato , significativo seppur limitato , le dosi rilevate rientrano nellordine di grandezza di alcune cation of the procedure and cost - benefit ratio , one has to consider probabilistic risks , patients young age and limited severity of varicocele . 
in the population examined , it was therefore important to evaluate exposure dose , effective dose and equivalent dose ; in order to calculate these doses during interventional procedures ( highly dynamic , nonstandardised and performed using fluoroscopy ) , we opted for an experimental method as it is more accurate than the monte carlo method . 
 in our study population , which was small but significant , doses obtained were within the range of other diagnostic procedures , such as computed tomography , nuclear medicine , etc . 
although definitely below the lower threshold corresponding to the deterministic effect of temporary sterility ( 150 msv ) , gonad dose values recorded ( always lower than 1 msv ) were slightly higher than those previously reported , probably because of the short time of direct exposure of the inguinal region during some phases of the procedure . 
to optimise exposure during the angiographic procedure , we adopted adequate field - limiting measures , never used radiography and limited fluoroscopy time to a maximum of 10 m the principal technical limitation to percutaneous treatment of varicocele is related to the difficulty in achieving selective catheterisation of the internal spermatic vein due to anatomical factors , vasospasm or intimal dissections . 
in cases of incontinence of the cremasteric vein demonstrated by phlebography , in which selective radiological treatment is rarely achievable and which presents a high incidence of recurrences [ 10 , 14 ] , the patient was referred for surgical ligation . 
 in agreement with other authors [ 15 ] , we believe administration of at least 3 ml of sclerosing agent at 3% to be the most effective therapy , allowing occlusion of the pelvic segment of the gonadal vein and simultaneous treatment of abdominal collaterals , at times responsible for long - term recurrence . unlike other authors [ 16 , 17 ] , we used large - calibre metallic coils to rapidly complete the treatment of very large gonadal veins only . 
direct fluoroscopic control during injection of sclerosing agent allowed us to minimise complications associated with possible migration of the embolising agent in the pampiniform plexus and / or systemic circulation . despite the encouraging results obtained in the seminal parameters ( significantly increased sperm concentration and motility ) , fertility of the patients treated cannot be guaranteed [ 1823 ] ; as demonstrated by numerous studies , this depends on several female and male variables related to spermatogenesis and ovulation . 
i valori di dose equivalente alle gonadi registrati ( sempre inferiori al msv ) , pur essendo nettamente al di sotto della prima soglia corrispondente alleffetto deterministico di sterilit temporanea ( 150 msv ) , risultano lievemente superiori a quelli riportati in altri lavori , probabilmente a causa della breve esposizione diretta della regione inguinale durante alcune fasi della procedura . durante le manovre angiografiche , per ottimizzare le esposizioni sono sempre state effettuate opportune diaframmature del campo , non mai stata utilizzata la grafia e si deciso di limitare i tempi di fluoroscopia ad un massimo di 10 min . le difficolt al cateterismo selettivo della vena spermatica interna , correlate a problematiche anatomiche , fenomeni di vasospasmo o a dissezioni intimali , rappresentano la principale limitazione tecnica alla terapia radiologica percutanea del varicocele . nei casi di incontinenza della vena cremasterica dimostrata alla flebografia , in cui come suggerito dalla letteratura [ 10 , 14 ] il trattamento radiologico pu essere assai di rado condotto selettivamente e presenta una considerevole incidenza di recidiva , si preferito inviare il paziente al chirurgo per la legatura inguinale . nella nostra esperienza e secondo altri autori [ 15 ] la condotta terapeutica che garantisce i migliori risultati la somministrazione di almeno 3 ml di sclerosante concentrato al 3% , che consente di occludere la vena gonadica nella porzione pelvica e di trattare contemporaneamente i circoli collaterali addominali , responsabili talvolta di recidiva a distanza . a differenza di quanto descritto in altri lavori [ 16 , 17 ] le spirali metalliche di grande calibro sono state impiegate esclusivamente per completare rapidamente il trattamento di vene gonadiche assai voluminose . 
il controllo fluoroscopico diretto durante liniezione dello sclerosante ha consentito di ridurre al minimo le complicanze correlabili ad indesiderate migrazioni del farmaco nel plesso pampiniforme e / o nel circolo sistemico . gli incoraggianti risultati seminali ottenuti ( significativo incremento di concentrazione e motilit degli spermatozoi ) non ci consentono tuttavia di fare sicure valutazioni circa la fertilit dei pazienti trattati [ 1823 ] ; essa infatti , cos come dimostrato da numerosi studi , dipende da molte variabili maschili e femminili che influenzano la spermatogenesi e lovulazione . ipotizzabile infine che significative variazioni morfologiche delle cellule germinali si rendano apprezzabili solo in corso di follow - up pi estesi nel tempo . il trattamento percutaneo del varicocele si conferma tecnica affidabile , efficace , mininvasiva ed economica attuabile in regime ambulatoriale . 
muzzio1 1dipartimento di scienze medico diagnostiche e terapie speciali , universit degli studi di padova , via giustiniani 2 , i - 35128 padova , italy 2dipartimento di radioterapia e medicina nucleare , azienda ospedaliera di padova , padova , italy correspondence to : d . 
computed tomography ( ct ) , magnetic resonance ( mr ) and positron emission tomography ( pet ) have a very important role in the diagnosis of malignant pleural mesothelioma ( mpm ) in the choice of chemoradiotherapy alone or in combination with surgery and in evaluating possible recurrence . 
we considered 28 patients suffering from mpm whose mean survival after diagnosis was 1518 months . sixteen of these patients had radiotherapy or chemoradiotherapy alone , according to standard protocols , while 12 also underwent surgery . 
nine of the 12 patients who underwent pleuropneumonectomy had no significant survival advantage over the mean survival in the 16 who were not operated whereas the other three lived 13 years longer . 
ct , pet and ct - pet are indicated for diagnosis and , above all , for staging of mpm , in the selection of patients who might benefit from surgery after neoadjuvant therapy and also in identifying small recurrences and / or remote metastases . being highly specific , pet is essential in the follow - up of patients undergoing chemoradiotherapy alone and / or surgery . 
la tomografia computerizzata ( tc ) , la risonanza magnetica ( rm ) e la tomografia per emissione di positroni ( pet ) hanno un ruolo molto importante nella diagnosi del mesotelioma pleurico maligno ( mpm ) , nella scelta del trattamento chemioradioterapico semplice o combinato , eventualmente associato alla chirurgia , e nella identificazione di eventuali recidive . 
in 3 pazienti lincremento della sopravvivenza stato compreso tra 1 e 3 anni rispetto alla media . a questo proposito vengono riportati 2 casi sottoposti a terapia chirurgica dopo una risposta ottimale ai trattamenti chemioradio . 
la tc , la pet e la tc - pet hanno indicazioni nella diagnosi e soprattutto nella stadiazione del mpm , nella accurata selezione dei pazienti da sottoporre alla chirurgia dopo terapia neoadiuvante e nellidentificare la presenza di eventuali piccole recidive e / o di metastasi a distanza . 
mpm has a multifocal origin in a single district and advances by contiguity to serous membranes and adjacent tissues , metastasising only at a late stage . this particular clinical - biological behaviour makes careful staging essential . 
the international mesothelioma interest group ( imig ) [ 4 , 5 ] has recently developed a new staging system based on the traditional tnm staging system , with a view to obtaining a reliable prediction of survival according to tumour extension ( local or distant ) and lymph node involvement , so as to identify patients with a similar prognosis and thus select candidates for surgery . current imaging techniques play a fundamental role in evaluating mpm not only for diagnostic purposes , but also for ensuring a precise tumour staging [ 6 ]  . 
opinions differ in the literature as regards mean survival of patients after multimodal treatment , in the absence of nodal involvement or involvement of the contralateral hemithorax , of pulmonary metastases or subdiaphragmatic spread [ 7 , 8 ]  . materials and methods we reviewed a series of 28 patients with mpm who had been treated over the past 3 years and who survived 1518 months after treatment : 16 were treated with radiotherapy alone ( generally for palliative purposes ) or with chemotherapy and radiotherapy according to specific , partly standardised protocols . in addition to chemoradiotherapy , the other 12 patients also underwent demolitive surgery ( pleural decortication or pleuropneumonectomy with diaphragmatic and at pericardial reconstruction )  . 
all patients underwent ct , which revealed the following findings : relapsing unilateral pleural effusion nodes or plaques of pleural thickening nodosity of the interlobar pleura ring coarctation of the hemithorax calcific plaques obliteration of extrapleural fat planes foci of costal osteolysis hemidiaphragmatic encasement subdiaphragmatic nodosity although suggestive of the disease , these tomographic signs are not specific to the diagnosis of mpm . 
diagnosis was a lungo si discusso sulla eziologia e sulla epidemiologia del mesotelioma pleurico maligno ( mpm ) [ 1 , 2 ] e delle sue correlazioni radiologiche e anatomopatologiche [ 3 ]  . 
recentemente infatti linternational mesothelioma interest group ( imig ) [ 4 , 5 ] ha formulato un nuovo metodo di stadiazione basato sul tradizionale tnm . lobiettivo quello di ottenere informazioni certe sulla sopravvivenza a seconda della estensione del tumore ( locale o a distanza ) e del coinvolgimento linfonodale ; si possono cos inserire i pazienti nellambito di prestabilite categorie che hanno prognosi simile , al fine di identificare con maggiore accuratezza i soggetti potenzialmente resecabili . le tecniche di imaging attualmente disponibili nella valutazione del mpm sono fondamentali non solo per la corretta diagnosi , ma anche per una precisa stadiazione del tumore [ 6 ]  . 
lo scopo di questo lavoro quello di evidenziare : ( 1 ) il ruolo che le metodiche di imaging radiologico , la tomografia computerizzata ( tc ) , la risonanza magnetica ( rm ) , la tomografia per emissione di positroni ( pet ) e la tc - pet hanno nella diagnosi e nella valutazione complessiva del mpm , alla luce della scelta del trattamento terapeutico pi idoneo ( di tipo conservativo o di tipo altamente demolitivo , come la pleuropneumonectomia ) ; ( 2 ) leventuale beneficio che tale intervento pu apportare alla sopravvivenza del paziente . per quanto riguarda la sopravvivenza media dei pazienti sottoposti a trattamento multimodale , in assenza di coinvolgimento linfonodale , dellemitorace controlaterale , di metastasi polmonari e di estensione sottodiaframmatica della malattia , i pareri in letteratura sono discordanti [ 7 , 8 ]  . materiali e metodi abbiamo rivisitato 28 casi di mpm occorsi alla nostra osservazione negli ultimi 3 anni , la cui sopravvivenza media stata di 1518 mesi . 
sedici di questi pazienti sono stati sottoposti a semplici modalit di trattamento : soltanto terapia radiante generalmente usata per una terapia palliativa , chemioterapia e radioterapia secondo precisi schemi in parte standardizzati . 
dodici di questi pazienti sono stati sottoposti a terapia multimodale che oltre ai trattamenti di chemio - radioterapia prevede un approccio chirurgico aggressivo ( decorticazione pleurica o pleuropneumonectomia associata a ricostruzione del diaframma e a volte del pericardio )  . 
tutti i nostri pazienti sono stati valutati mediante tc che complessivamente ha evidenziato i seguenti elementi semeiologici : versamento recidivante monolaterale ; ispessimento nodulare o a placca della pleura ; nodulazioni della pleura interlobare ; coartazione ad anello di un emitorace ; placche calcifiche ; obliterazione dei piani di grasso extrapleurici ; d . 
d micronodulazioni pleuriche diffuse di diametro inferiore ai 5 mm . table 1 characteristics of pleural thickening pleural thickening morphology plaque nodular focal mass simple uni - bilateral calcific micro < 5 mm macro 520 mm > 3 cm tabella 1 caratteristiche dellispessimento pleurico ispessimento pleurico morfologia a placca nodulare massa focale semplice mono - bilaterale calcificata micro < 5 mm macro 520 mm > 3 cm focolai di osteolisi costale ; encasement dellemidiaframma ; nodulazioni sottodiaframmatiche . questi segni tomografici messi in evidenza indirizzano verso un sospetto diagnostico , ma non sono tuttavia specifici per la diagnosi di mpm . 
our study has attempted to highlight the numerous and in some cases unusual morphological patterns of mpm detectable by ct and underline the need for accurate staging of the disease to ensure a correct prognostic and therapeutic approach . 
 despite careful clinical and radiological assessment of our 12 mpm patients subjected to pleuropneumonectomy or pleural decortication , nine had no significant benefit in terms of survival [ 5 , 6 , 1618 ] compared with the mean survival of the 16 patients who did not undergo surgery . 
in un caso infine , abbiamo osservato una formazione nodulare in sede intrascissurale a sinistra senza , tuttavia , segni di versamento pleurico loco - regionale , la cui diagnostica differenziale col fibroma pleurico non stata possibile ( la diagnosi stata posta esclusivamente allintervento chirurgico )  . discussione numerose sono le casistiche di mpm riportate in letteratura [ 5 , 6 , 918 ] con risultati spesso sconfortanti ai fini dellincremento della media della sopravvivenza sia nei pazienti d . 
on this issue , it is worth noting two particularly unfortunate cases of very short survival after surgery despite the fact that surgery was absolutely indicated given the patients positive response to chemotherapy and radiotherapy . 
nel nostro lavoro abbiamo cercato di sottolineare i numerosi e a volte inusuali aspetti morfologici del mpm con la tc e la necessit di una precisa stadiazione ai fini di un corretto approccio prognostico e terapeutico . 
nonostante la valutazione clinico - radiologica estremamente accurata dei 12 pazienti con mpm , sottoposti ad intervento chirurgico di pleuropneumonectomia o di decorticazione pleurica , in 9 di questi d . 
8 la tc dimostra la presenza di placca pleurica nodulare in sede intrascissurale sinistra di mpm ( freccia ) , con minimo versamento . non si sono ottenute significative variazioni dellincremento della sopravvivenza in rapporto alla media dei 16 pazienti non operati [ 5 , 6 , 1618 ]  . 
paziente di 57 anni deceduta dopo 5 mesi . treatment for mpm make us sceptical as to the genuine efficacy of pleuropneumonectomy : although three of our operated patients lived a little longer , the survival gain ( 13 years ) was too limited to justify such aggressive treatment . pet and ct - pet are important in staging disease and identifying recurrences , providing important information on : active pleural nodulation for well - aimed biopsy mediastinal lymph node involvement metastases ( intrathoracic and / or distant ) clearly , the poor spatial resolution of pet is overcome by fusion with ct anatomical images . va , inoltre , ricordata limportanza della pet e della tcpet sia nella stadiazione sia nella identificazione delle recidive . 
tale metodica fornisce , inoltre , importanti informazioni su : nodulazioni pleuriche attive ai fini di una biopsia mirata ; coinvolgimento dei linfonodi mediastinici ; metastasi ( toraciche e / o a distanza )  . naturalmente la insufficiente risoluzione spaziale della pet compensata dalle tecniche di fusione con le immagini anatomiche della tc . conclusions ct , pet and ct - pet have an important , combined role in conclusioni la tc , la pet e la tc - pet trovano nel mpm , prima e dopo terapia semplice o multimodale , un uso combinato che ha come importantissimo scopo sia la selezione di pazienti nel d . 
10a , b pleuropneumectomia sinistra con scomparsa in b delle estese placche pleuriche , evidenti alla tc nel versante mediastinico in a ( frecce )  . paziente di anni 51 deceduto dopo 8 mesi dallintervento . evaluating mpm before and after treatment , in selecting patients potentially eligible for surgery and in detecting recurrences after surgery . 
being highly specific , pet proves helpful for accurate tumour staging and is also essential in the follow - up of patients after chemoradiotherapy both to identify residual neoplastic foci or thoracic recurrences and to detect remote metastasis . it is important to emphasise that thoracic recurrences are often hidden at ct by anatomical structures ( hila , diaphragm , etc . ) or by major anatomical disruption resulting from surgery . each imaging modality has its advantages and limitations , and we recommend their combined use in order to select the most appropriate treatment options . periodo pre - chirurgico , sia la evidenziazione di eventuali recidive nel periodo post - chirurgico . 
per la sua elevata specificit la pet indicata ai fini di una accurata stadiazione del tumore ed indispensabile nel monitoraggio dei pazienti sottoposti a chemio - radioterapia per identificare foci neoplastici residui , recidive intratoraciche ed eventuali metastasi extra - compartimentali . 
 importante sottolineare che le recidive intratoraciche spesso sono nascoste alla stessa tc o dalle strutture anatomiche ( ili , diaframma , etc . ) o dagli importanti sovvertimenti anatomici dovuti alla chirurgia . 
patients with primary mediastinal masses and cysts will usually undergo surgical resection ; radiological and clinical features should prompt limited biopsy specimens followed by oncologic consultation , and chemotherapy or radiotherapy when appropriate . 
the objective of this review was to examine the role of diagnostic imaging in the management of masses of the anterior mediastinum . key words anterior mediastinum thymus neoplasm germcell tumours mesenchymal tumours cysts riassunto i tumori del mediastino sono frequentemente asintomatici e riscontrati incidentalmente ad una radiografia del torace di routine . 
in relazione con il gruppo neoplastico , i tumori primitivi del mediastino anteriore pi comuni sono il timoma , il teratoma ed il linfoma ; tutte le altre lesioni sono rare . 
i pazienti con masse primitive mediastiniche e cisti andranno in genere incontro a resezione chirurgica ; le caratteristiche radiologiche e cliniche dovrebbero suggerire una biopsia limitata della lesione seguita da consulenza oncologica e chemioterapia o radioterapia quando opportune . 
scopo di questa revisione esaminare il ruolo dellimaging diagnostico nella gestione di una massa del mediastino anteriore . parole chiave mediastino anteriore timo , tumore tumori a cellule germinali tumori mesenchimali cisti introduction introduzione a wide spectrum of conditions can present as a space - occupying lesion in the anterior mediastinuthese may be divided into two groups : inflammatory processes and expansile lesions . 
the latter include heterogeneous groups : neoplasms ( primary or secondary ) and nonneoplastic congenital - dysembryoplastic or acquired lesions . unampia variet di condizioni pu presentarsi come lesione occupante spazio nel mediastino anteriore . 
si distinguono processi flogistici e lesioni espansive ; tra queste ultime si riconoscono gruppi eterogenei : condizioni neoplastiche ( primitive o secondarie ) e lesioni , congenite - disembriogenetiche o acquisite , non tumorali . inflammation flogosi mediastinitis and mediastinal abscesses mediastiniti ed ascessi mediastinici a.m. 
more often , the infection reaches the anterior compartment via locoregional spread ( from other mediastinal compartments , from the cervical region or from the lung and pleural cavity )  . 
infections may be classified into diffuse mediastinitis and mediastinal abscesses on the basis of their extension [ 2 ] ; they may be acute or chronic . acute infections are caused by bacteria and in some cases become organised into abscesses ; they are accompanied by symptoms and signs ( retrosternal pain , fever ) and are often fulminant and fatal . 
chronic infection results from mycosis or tubercular processes ; although chronic mediastinitis typically has insidious onset and is not associated with clinical manifestations , some patients show symptoms and signs of obstruction or compression of one or more mediastinal structures . 
in addition to chronic inflammation of infectious origin , there is also a group of mediastinal diseases of unknown origin characterised by accumulation of dense fibrous tissue ( at times systemic ) consisting of granulomatous inflammation . the major radiographic manifestation of acute mediastinitis is mediastinal widening , generally more evident in the upper portions ; the affected region is smoothly marginated . air may be seen in the mediastinum and in the soft tissues of the neck ; there may be pneumoor hydrothorax and multiple abscesses [ 4 ]  . 
the main signs are oesophageal thickening , obliteration and increased density of fat planes , areas of perioesophageal hypodensity , localised fluid collections , single or multiple abscesses , presence of extraluminal gas or contrast material [ 2 , 5 , 6 ] , pleural or pericardial effusion and lymphadenopathy . 
stenosis or occlusion of the superior vena cava may be demonstrated with ct . in rapporto agli altri distretti mediastinici , le flogosi con origine nel mediastino anteriore sono relativamente rare . 
pi spesso il compartimento anteriore raggiunto per diffusione loco - regionale dallevento infettivo ( da altre sezioni del mediastino , dalla regione cervicale oppure dal polmone e dal cavo pleurico )  . 
nella loggia anteriore possono avere origine mediastiniti da causa chirurgica , sequele di interventi di cardiochirurgia o chirurgia esofagea [ 2 ] : in particolare , le mediastiniti postoperatorie si verificano approssimativamente nello 0 , 5%1% dei pazienti sottoposti a sternotomia mediana per interventi di cardiochirurgia [ 3 ]  . 
le infezioni acute sono causate da batteri e talvolta si organizzano in ascessi : si accompagnano a segni e sintomi ( dolore retrosternale , febbre ) e , spesso , sono fulminanti e letali . 
linfezione cronica deriva da micosi o processi tubercolari ; sebbene le mediastiniti croniche siano caratteristicamente insidiose nella comparsa e non associate a manifestazioni cliniche , alcuni pazienti presentano sintomi e segni correlati allostruzione o compressione di una o pi strutture mediastiniche . 
in aggiunta ai casi di infiammazione cronica di origine infettiva , esiste inoltre un gruppo di mediastinopatie ad eziologia sconosciuta caratterizzate dallaccumulo di tessuto denso fibroso ( talvolta a carattere sistemico ) , che consiste in una infiammazione granulomatosa . la maggiore manifestazione radiografica delle mediastiniti acute costituita da un allargamento del mediastino , in genere pi pronunciato negli ambiti superiori ; la regione alterata si presenta con margini lisci e netti . 
la stenosi / occlusione della vena cava superiore pu essere dimostrata con la tc . space - occupying lesions table 1 illustrates the nosography of space - occupying lesions of the anterior mediastinuprimary tumours account la tabella 1 descrive la nosografia delle lesioni occupanti lesioni occupanti spazio a.m. 
in relazione alla clinica , il 75% delle masse mediastiniche in soggetti asintomatici risulta benigno , mentre nei pazienti sintomatici quasi i 2 / 3 sono maligne ; inoltre , l85% dei pazienti affetti da un tumore maligno sintomatico , mentre solo il 46% dei casi di natura benigna si associa a sintomi [ 8 ]  . 
globalmente , il 60% delle lesioni espansive del mediastino presenta sintomi correlati alla compressione o allinvasione diretta delle strutture circostanti , o a sindromi paraneoplastiche ( tabella 2 )  . i pi comuni sintomi da compressione sono dolore toracico , tosse e dispnea ; sindrome della vena cava superiore , sindrome di horner , raucedine e deficit neurologici sono pi frequentemente associati a patologie maligne [ 8 ]  . 
i tumori metastatici sono pi comuni rispetto alle neoplasie primitive e , nel caso del mediastino anteriore , rappresentano pi frequentemente il coinvolgimento linfatico da parte di tumori della mammella e del polmone . tumori le neoplasie del mediastino sono rare , si possono presentare ad ogni et , ma prevalentemente nel iii e iv decennio di vita [ 8 , 9 ]  . 
lincidenza dei tumori primitivi del mediastino , valutata su 1900 pazienti , risultata del 25 , 3% per i tumori neurogenici , 23 , 3% per il timoma , 15 , 3% per i linfomi , 12 , 2% per i tumori a cellule germinali [ 8 ]  . 
oesophagogram reveals an oesophageal tear and a massive leak of barium into the mediastinuc computed tomography ( ct ) scan shows a perioesophageal mediastinal abscess with bilateral pleural effusion and air . 
della componente epiteliale sono stati distinti , nel timo a maturazione , sei sottotipi cellulari ; uno di questi ( tipo vi ) forma i corpuscoli di hassall , caratteristici del timo . 
questi ultimi , localizzati nella zona midollare dei lobuli timici , sono fortable 1 space - occupying lesions benign hyperplasia follicular or lymphoid hyperplasia thymolipoma cysts : congenital , acquired ( inflammation ) lymphoepithelial thymoma ( benign ) mature teratoma immature teratoma ( age < 15 years ) mature teratoma with immature component less than 50% of volume a.m. 
with regard to clinical presentation , 75% of mediastinal masses in asymptomatic subjects are benign whereas almost 2 / 3 of those in symptomatic patients are malignant ; in addition , 85% of patients with a malignancy are symptomatic whereas only 46% of cases of benign disease are associated with symptoms [ 8 ]  . 
overall , 60% of expansile mediastinal lesions are associated with symptoms related to compression or direct invasion of neighbouring structures or paraneoplastic syndromes ( table 2 )  . the most common symptoms resulting from compression are chest pain , cough and dyspnoea . 
metastatic tumours are more common than primary tumours , and those involving the anterior mediastinum are more frequently related to lymphatic involvement by breast or lung cancers . tumours mediastinal tumours are rare , and they may present at any age although predominantly in the third and fourth decades [ 8 , 9 ]  . 
sino allet di 20 anni , pi dell80% della ghiandola costituita da tessuto linfoide : successivamente il suo volume si riduce e lorgano va incontro ad involuzione adiposa , che dopo i 60 anni diviene completa . 
inoltre , tessuto timico ectopico pu essere riscontrato in ogni compartimento mediastinico e nel collo , e spesso risulta indistinguibile dal grasso mediastinico : questa distribuzione giustifica la presentazione dei timomi al di fuori del mediastino anteriore e la possibile persistenza di sintomatologia miastenica dopo timectomia [ 10 ]  . la patologia timica la causa pi comune di allargamento del mediastino anteriore ; laumento di volume del timo riconosce cause molteplici : iperplasia , timoma , carcinoma , carcinoide , timolipoma , cisti , linfomi , seminoma , lesioni secondarie . 
in children , percentages are 43% , 18% and 39% , respectively [ 9 ]  . these differences are justified by a higher incidence of thymic tumours and lymphomas in adults and neurogenic tumours in children . 
therefore , both the subjects age and the site of presentation will help establish a tentative diagnosis . masses of thymic origin thymus the thymus arises from the ventral portion of the third branchial pouch , as do the parathyroid glands . 
six cellular subtypes of the epithelial component have been identified in the mature thymus ; one of these ( type vi ) forms hassall bodies , which are characteristic of the thymus . these are located in the thymic medulla and are formed by concentrically arranged , involuted epithelial cells ; the core cells are keratinized , calcific or necrotic and may undergo lysis with the consequent formation of cysts . 
its weight at birth is on average 22 g , increasing to 35 g at puberty . until the age of 20 years , more than 80% of the gland is made up of lymphoid tissue ; subsequently , it shrinks and undergoes fatty involution , which is complete after the age of 60 years . various events can cause its enlargement ( stress ) or shrinkage . 
in addition , ectopic thymic tissue may be found in every mediastinal compartment and in the neck and is often indistinguishable from mediastinal fat . this distribution accounts for the fact that thymomas may present outside the anterior mediastinum and the possible persistence of myasthenia , even after thymectomy [ 10 ]  . thymic disease is the most common cause of widening of the anterior mediastinuthymic enlargement may have a variety of causes : hyperplasia , thymoma , carcinoma , carcinoid tumour , thymolipoma , cysts , lymphoma , seminoma or secondary lesions . although lymphoma , carcinoid tumours and germ - cell tumours may develop within the thymus , only thymomas , thymic carcinomas and thymolipomas arise from thymic precursors . cellule germinali possano svilupparsi nel timo , soltanto i timomi , i carcinomi del timo ed i timolipomi derivano da precursori timici . iperplasia timica vera liperplasia timica vera , contrapposta alliperplasia linfoide associata alla miastenia gravis , definita come incremento nella dimensione della ghiandola in presenza di una normale architettura istologica [ 11 ]  . 
possibile il riscontro anamnestico di eventi causa di stress , quali la sepsi o le ustioni massive . la maggior parte dei soggetti , alla diagnosi , sono bambini o giovani adulti [ 11 ] : in genere asintomatica . 
anche la rm ha un valore limitato [ 17 ]  . timolipoma un tumore infrequente a lenta crescita , benigno , costituito da una commistione di tessuto adiposo maturo e di tessuto timico linfoepiteliale ; rappresenta l1%10% di tutti i tumori del timo [ 18 ]  . 
sono state formulate diverse ipotesi patogenetiche : potrebbe rappresentare un lipoma che insorge nel timo , un tumore misto ( lipoma e timoma ) o uniperplasia timica vera che successivamente regredisce per involuzione aditrue thymic hyperplasia true thymic hyperplasia , as opposed to the lymphoid hyperplasia associated with myasthenia gravis , is defined as enlargement of the gland in the presence of normal histological architecture [ 11 ]  . 
it may result from atrophy due to antiblastic chemotherapy ( generally for lymphoma or germ - cell tumour ) [ 13 ] or from hypercortisolism [ 14 ] ; more frequently , however , there is no correlation with other conditions and the finding is incidental . 
there may be a history of stressful events , such as sepsis or massive burns . at diagnosis , most subjects are children or young adults [ 11 ] , and the condition is usually asymptomatic . 
in children , the radiological finding , which is often incidental , is sufficient for diagnosis , and the condition requires no treatment . in adults , a tentative diagnosis may be made in the presence of a history of anticancer treatment ; without this , biopsy is mandatory . 
microscopicamente , i centri germinativi sono assenti ( anche in presenza di miastenia gravis ) , le calcificazioni e la degenerazione cistica dei corpuscoli di hassall sono comuni [ 20 ]  . 
laspetto radiografico consiste di una massa del mediastino anteriore con forma a campana [ 7 , 18 , 19 ] , con estensione in uno o entrambi gli emitoraci ; pu simulare cardiomegalia o elevazione del diaframma , mentre , quando di piccole dimensioni , ha forma rotonda o ovale . 
per la consistenza molle pu modificare forma ( che si adegua alle strutture circostanti ) e posizione in relazione al diverso decubito del paziente [ 19 ]  . alla tc mostra caratteristicamente lattenuazione del grasso , o densit equivalente a grasso e tessuti molli . 
spesso la massa connessa al timo da un piccolo peduncolo [ 19 ]  . la rm dimostra lelevata intensit di segnale , tipica del grasso , nella sequenza se t1 pesata ed aree di segnale intermedio correlate ai tessuti molli [ 21 ]  . 
le caratteristiche peculiari del timolipoma evidenti alla radiografia ed alla tc consentono la diagnosi differenziale nella maggior parte dei casi ; quando la componente adiposa predominante pu essere confuso con un lipoma mediastinico . cisti timiche le cisti del timo sono infrequenti e rappresentano solo il 3% di tutte le masse del compartimento anteriore [ 8 ]  . 
le masse si proiettano sul lato destro del mediastino . con alcune condizioni neoplastiche ( linfoma di hodgkin , seminoma , carcinoma timico ) , per una parte dopo trattamento ( radioterapia , toracotomia ) [ 22 , 23 ]  . 
la maggior parte deriva probabilmente dai residui del dotto timofaringeo fetale e possono , pertanto , essere riscontrate in qualsiasi punto , dal collo sino al mediastino anteriore , lungo il percorso di migrazione embriologica del timo [ 24 ]  . circa la met delle cisti congenite sono di riscontro occasionale nei primi due decenni di vita [ 24 ] , mentre le cisti reattive si presentano in soggetti adulti asintomatici . 
nella parete cistica pu essere identificato focalmente tessuto timico normale e questo riscontro necessario per la diagnosi e per la diagnosi differenziale con alcune condizioni neoplastiche ( timoma , linfoma di hodgkin , tumori a cellule germinali ) che possono presentare un aspetto cistico dominante [ 25 ]  . il quadro radiologico , al radiogramma del torace , consiste in un espanso del mediastino antero - superiore a margini lisci , ben circoscritto [ 26 ] ; quando ha dimensioni cospicue pu simulare una cardiomegalia . 
presenta una bassa intensit di segnale nelle sequenze rm t1 pesate , che diviene alta sulle immagini t2 [ 21 ] , se non complicata ( emorragia intracistica , contenuto proteinaceo )  . timoma linfoepiteliale le neoplasie del timo costituiscono il 30% di tutte le masse del mediastino anteriore in et adulta [ 27 ] , mentre sono meno comuni nel bambino , dove rappresentano solo il 15% degli espansi di questa sede [ 28 ]  . 
tuttavia , dal momento che il timoma evolutivo privo delle caratteristiche citologiche ed istologiche di malignit e dal punto di vista microscopico identico al timoma capsulato , la dizione di timoma invasivo da preferirsi a timoma maligno [ 29 ]  . 
inoltre , in presenza di atipia citologica ed elevato indice mitotico sono pi propriamente definiti carcinomi timici . il timoma il tumore primitivo pi comune del mediastino anteriore : rappresenta circa il 20% di tutti i tumori del mediastino . 
ha una lieve predilezione per il sesso femminile , con et media di incidenza nel v - vi decennio di vita [ 30 ] , mentre di raro riscontro nellinfanzia e nelladolescenza . la variabilit istologica ha suggerito diverse classificatomy [ 15 ]  . 
even magnetic resonance imaging ( mri ) is of limited value [ 17 ]  . thymolipoma this is an uncommon , slow - growing , benign tumour made up of a mixture of mature adipose tissue and lymphoepithelial thymic tissue ; it accounts for 1%10% of all thymic tumours [ 18 ]  . 
it may be a lipoma arising in the thymus , a mixed tumour ( lipoma and thymoma ) or a true thymic hyperplasia that subsequently regresses due to fatty involution . 
there is no gender predilection , and although it may manifest at any age , young adults seem to be most commonly affected by the disease [ 19 ]  . 
microscopically , it has no germinal centres ( even in the presence of myasthenia gravis ) , and calcification and cystic degeneration of the hassall bodies are common [ 20 ]  . the radiographic pattern consists of a bell - shaped mass within the anterior mediastinum [ 7 , 18 , 19 ] extending to one or both hemithoraces ; it may mimic cardiomegaly or diaphragmatic elevation . 
given the soft texture of these tumours , they may vary in shape ( adapting to the surrounding structures ) and location , depending on the patients position [ 19 ]  . at ct , they typically have fat attenuation or density of fat and soft tissue . 
mri reveals high signal intensity , typical of fat , in the t1 - weighted spin - echo ( se ) sequence and areas of intermediate signal related to soft tissues [ 21 ]  . the peculiar radiographic and ct features of thymolipoma allow differential diagnosis in most cases ; when the fatty component prevails , the condition may be mistaken for a mediastinal lipoma . thymic cysts thymic cysts are uncommon , accounting for only 3% of all anterior mediastinal masses [ 8 ]  . 
they may be congenital or acquired . the latter are due to inflammation or are associated with certain neoplasms ( hodgkins lymphoma , seminoma , thymic carcinoma ) , in some cases after treatment ( radiotherapy , thoracotomy ) [ 22 , 23 ]  . acquired cysts are thought to derive from dilatation of the epithelial component due to inflammation of the thymic parenchyma . 
most cysts probably derive from remnants of the foetal thymopharyngeal duct and may therefore be found anywhere between the neck and the anterior mediastinum along the embryological migration route of the thymus [ 24 ]  . 
in ambito clinico la classificazione anatomo - chirurgica di masaoka , che distingue iv stadi ( i , macroscopicamente capsulato senza invasione microscopica della capsula ; ii , invasione macroscopica nel tessuto adiposo circostante o a livello della pleura mediastinica o invasione microscopica della capsula ; iii , invasione macroscopica negli organi adiacenti pericardio , grossi vasi , polmone ; iva , disseminazione pleurica o pericardica , ivb , metastasi linfatiche o ematogene ) , condiziona leventuale integrazione terapeutica ( radioterapia e chemioterapia adiuvanti ) ed ha dimostrato una valida correlazione prognostica [ 31 ]  . circa la met di questi tumori sono asintomatici e di riscontro casuale alla radiografia del torace . 
nei pazienti sintomatici , il 40% presenta miastenia gravis , mentre in altri casi lamentano dolore toracico e sintomi da emorragia acuta intralesionale o compressione delle strutture mediastiniche [ 30 ]  . la maggior parte ( 90% dei casi ) origina nella porzione superiore del mediastino anteriore , che corrisponde alla sede usuale del timo , situata in prossimit della giunzione tra il cuore ed i grossi vasi ( pu tuttavia presentarsi in ogni altra sede , dal collo allangolo cardiofrenico ) ; ben capsulata ed ha forma rotonda o lievemente lobulata , di dimensioni in genere variabili tra 5 e 15 cm di diametro [ 32 ]  . 
generalmente si presenta come lesione solida , talvolta ha aspetto cistico [ 25 ] : fino ad 1 / 3 dei casi mostra necrosi , emorragia , calcificazioni ed aree di colliquazione cistica . 
nonostante questa difficolt chirurgica , laspetto citologico rimane benigno [ 32 ]  . linvasivit documentata dal riscontro istologico , sul pezzo operatorio , di cellule tumorali oltre il confine della capsula : indipendentemente dalla dimensione , almeno 1 / 3 dei timomi presenta infiltrazione della capsula con conseguente estensione nel grasso mediastinico [ 32 ]  . 
a seconda delle casistiche [ 29 , 32 , 33 ] , nel 30%50% dei casi viene documentata linfiltrazione della pleura , del polmone , della parete toracica , del pericardio , dei grossi vasi mediastinici , dellatrio destro o del diaframma : la prognosi correla con linvasione extracapsulare . 
inoltre , si pu verificare lestensione attraverso il diaframma in addome o nel retroperitoneo e linsemenzamento metastatico loco - regionale della pleura omolaterale e del pericardio [ 29 ]  . 
la diffusione linfonodale e sistemica sono evenienze rare [ 32 ] e possono verificarsi sia nella forma invasiva sia , eccezionalmente , nella variante capsulata . sebbene la stadiazione chirurgico - patologica proposta da masaoka sia universalmente accettata , alcuni punti di controversia rimangono : in particolare , molti timomi capsulati presentano una tendenza a recidivare localmente nonostante unexeresi radicale . 
by contrast , cysts associated with a neoplasm are frequently symptomatic . most congenital cysts are smaller than 6 cm , spherical , unior multilobulated and have thin walls without an inflammatory component . 
cyst walls may show foci of normal thymic tissue , a finding necessary for diagnosis and differentiating these cysts from neoplasms ( thymoma , hodgkins lymphoma , germ - cell tumours ) presenting with a predominantly cystic appearance [ 25 ]  . chest radiography shows a smoothly marginated , wellcircumscribed expansile mass of the anterosuperior mediastinum [ 26 ] ; when large , it may mimic cardiomegaly . 
thymomas are the most frequent neoplasms of the thymic epitheliuthey may have benign or malignant behaviour . however , because malignant thymomas do not display the cytological and histological features of malignancy and fig . 
in alcuni casi , la bassa attenuazione non assoluta , ma i valori di attenuazione sono analoghi a quelli dei tessuti molli a causa del contenuto mucoso o calcio . includendo anche il concetto di adesivit oltre quello di invasivit locale . 
alcuni timomi , infatti , presentano una reazione fibrosa pericapsulare priva di cellule neoplastiche , che si manifesta microscopicamente sotto forma di aderenze fibrose con la pleura parietale o con il pericardio : queste aumentano il rischio di recidiva locale . radiologicamente il timoma ha forma rotonda od ovale ed i margini sono lisci o lobulati , ben demarcati . 
si presenta nel mediastino antero - superiore , origina da uno dei lobi timici e , nella presentazione pi usuale , cresce verso un lato della linea mediana ; pu , peraltro , protrudere da entrambi i lati del mediastino ( lestensione bilaterale comunque meno comune ) e dislocare il cuore ed i grossi vasi posteriormente . le calcificazioni , non molto frequenti ( 20% nella nostra esperienza ) , sono pi spesso a grano di sale ( a differenza del teratoma dove in genere sono laminari ) [ 35 ]  . 
la densit omogenea [ 35 ] , tranne nelle lesioni di maggior dimensione nelle quali la presenza di emorragia , necrosi o formazioni cistiche produce aree focali di ipodensit [ 26 ]  . 
anche la tc non consente una distinzione preoperatoria attendibile del carattere di invasivit , a meno di grossolani reperti , suggestivi di infiltrazione , a carico dei grossi vasi , della pleura o di irregolarit delle interfacce con il polmone [ 35 ]  . 
lintensit di segnale rm intermedia ( equivalente o superiore a quella del muscolo scheletrico ) nella sequenza t1 pesata ed incrementata ( prossima a quella del tessuto adiposo ) nella t2 . 
la rm particolarmente utile nel dirimere un dubbio di invasione di strutture vascolari . alla diagnosi la met dei timomi risulta sintomatica : si rivelano per sintomi locali ( compressione ed invasione di strutture toraciche in 1 / 3 dei pazienti : dolore toracico , tosse , dispnea , disfagia ; sindrome della vena cava superiore ) o sistemici ( sindromi paraneoplastiche )  . 
la miastenia gravis la condizione paraneoplastica pi frequente : circa il 10%15% dei soggetti miastenici ha il timoma e circa il 35%40% dei pazienti affetti da timoma presenta la miastenia [ 32 ]  . lasportazione del timo migliora la clinica del paziente in molte delle condizioni morbose paraneoplastiche ; in particolare , circa il 70% dei pazienti miastenici con timoma mostra un graduale miglioramento dei sintomi in seguito alla timectomia [ 33 ] ; la remissione completa varia dal 7% al are microscopically identical to the encapsulated forms , the term invasive thymoma is preferred to malignant thymoma [ 29 ]  . 
moreover , in the presence of cytological atypia with high mitotic index , these thymomas are more appropriately referred to as thymic carcinomas . thymoma is the most common primary tumour of the anterior mediastinum and accounts for about 20% of all mediastinal tumours . 
in clinical practice , masaokas surgical - pathological classification into four stages ( i encapsulated tumour without gross or microscopic invasion of the capsule ; ii macroscopic invasion into the mediastinal fat or pleura or microscopic invasion into the capsule ; iii gross invasion of the adjacent organs : pericardium , great vessels , lung ; iva pleural or pericardial spread , ivb lymphatic or haematogenous metastases ) affects adjuvant treatment ( adjuvant radiotherapy and chemotherapy ) and has proved to correlate well with prognosis [ 31 ]  . about half of these tumours are asymptomatic and incidentally discovered at chest radiography . 
the others complain of chest pain and symptoms of acute intralesional haemorrhage or compression of mediastinal structures [ 30 ]  . most tumours ( 90% ) originate in the upper portion of the anterior mediastinum , which corresponds to the usual location of the thymus , near the junction between the heart and the great vessels ( they may , however , arise anywhere between the neck and the cardiophrenic angle )  . 
tumours are well encapsulated , have rounded or slightly lobular shape and are generally between 5 cm and 15 cm in diameter [ 32 ]  . they usually manifest as a solid lesion , and in some cases they have cystic appearance [ 25 ]  . 
up to 1 / 3 show necrosis , haemorrhage , calcification and areas of cystic colliquation . they may be encapsulated , more or less adherent to the surrounding structures , or frankly invasive [ 32 ]  . 
irrespective of size , at least 1 / 3 of thymomas show capsular infiltration with extension into the mediastinal fat [ 32 ]  . depending on the case series [ 29 , 32 , 33 ] , 30%50% of cases show evidence of infiltration of the pleura , lung , chest wall , pericardium , great mediastinal vessels , right atrium or diaphragprognosis is correlated with extracapsular invasion . 
in addition , the tumour may extend through the diaphragm into the abdomen or retroperitoneum , or there may be locoregional metastatic seeding of the ipsilateral pleura and pericardium [ 29 ]  . 
quasi il 15% dei pazienti con timoma sviluppa un secondo tumore : sarcoma di kaposi , chemodectoma , mieloma multiplo , leucemia acuta e carcinomi ( polmone , colon )  . una resezione chirurgica completa considerata il trattamento di scelta . 
in generale , una terapia complementare di associazione ( radioterapia , chemioterapia ) prescritta in tutti i casi dopo resezione chirurgica , ad eccezione dei timomi ben capsulati , che allesame istologico non presentano invasione microscopica e senza aderenze mediastiniche al tempo operatorio [ 38 ]  . 
per il rischio di persistenza / recidiva , indispensabile un programma di controlli radiologici seriati a lungo termine . carcinoma timico questa dizione riservata a quei tumori epiteliali maligni del timo che possiedono caratteristiche citologiche ed istologiche patognomoniche di malignit : atipia nucleare / citologica , elevata attivit mitotica , necrosi [ 39 ]  . 
in genere si presentano come massa bulky ( 515 cm ) del mediastino anteriore , dai margini lobulati o poco definiti [ 40 ] ed alla diagnosi invadono i piani adiposi circostanti e le strutture adiacenti . 
la distinzione tra carcinoma primitivo e metastatico pu essere difficile sulla base del solo esame istologico : lassenza di un tumore polmonare lelemento discriminativo di maggiore affidabilit ( essendo infrequente la presenza di secondariet di origine extrapolmonare ) [ 41 ]  . alla tc possono presentare una attenuazione omogenea da tessuti molli o eterogenea per la presenza di necrosi ; come il timoma , pu avere aspetto cistico [ 25 ] ; sono presenti calcificazioni nel 10%40% dei casi . 
linfoadenopatie mediastiniche sono presenti nel 40% dei casi [ 42 ]  . dal punto di vista clinico , la maggior parte dei pazienti sono asintomatici alla diagnosi ; al confronto con il timoma , non sono comuni le manifestazioni paraneoplastiche . 
la prognosi infausta , condizionata da una crescita intratoracica progressiva ( metastasi pleuriche , polmonari ed ai linfonodi mediastinici , cervicali ed ascellari ) e dalla metastatizzazione a distanza ( osso , fegato ) nella maggior parte dei pazienti . 
quando si riscontri alla tc una massa timica bulky con invasione dei grossi vasi , coinvolgimento linfonodale , paralisi del nervo frenico o metastasi ematogene , dovrebbe essere considerata lipotesi diagnostica del carcinoma timico piuttosto che il timoma invasivo [ 43 ]  . neoplasie neuroendocrine del timo comprendono diverse variet , con caratteristiche comuni sotto laspetto istologico e immunoistochimico : verosimile che rappresentino uno spettro di differenziazione , piuttosto che entit nosografiche specifiche . 
listotipo pi frequente il carcinoide ( tipico e atipico ) , identico nellistologia e nella clinica ( sindromi paraneoplastiche ) ad ogni altra presentazione extratimica . interessano ogni fascia di et , con media alla diagnosi di a.m. 
allo studio coronale rm fast spin - echo t1 - pesata , il timoma capsulato si manifesta come una massa del mediastino anteriore omogenea , lobulata . although masaokas surgical - pathological staging system is universally accepted , there are still some controversial points : in particular , many encapsulated thymomas tend to recur locally despite radical resection . 
to shed light on this controversial aspect , in 1996 , a japanese team [ 34 ] revised masaokas classification to include the notion of adhesiveness as well as local invasiveness . 
some thymomas , in fact , have a fibrous pericapsular reaction free of tumour cells , which manifests microscopically in the form of fibrous adhesions to the parietal pleura or pericardiu these adhesions increase the risk of local recurrence . radiologically , thymomas have a rounded or oval shape and smooth or lobulated , well - defined margins . 
possono , altre volte , essere presenti segni e sintomi di compressione e invasione locale , o sindromi paraneoplastiche , queste ultime presenti in circa 1 / 3 dei pazienti [ 44 ]  . 
contrast - enhanced computed tomography ( ct ) scan demonstrates an inhomogeneous anterior mediastinal mass with focal areas of calcification , nodular left pleural thickening and a left pleural effusion ; the mass is associated with focal obliteration of fat planes in close relationship to the aortic arch and main pulmonary artery . 
una scansione tc dopo mezzo di contrasto dimostra una massa disomogenea del mediastino anteriore con aree focali di calcificazione , ispessimento nodulare pleurico sinistro e versamento pleurico sinistro ; la massa associata con obliterazione focale dei piani adiposi , in stretta prossimit con larco aortico e larteria polmonare . 
computed tomography ( ct ) scan : large , well - defined mass adjacent to ascending aorta and superior vena cava contains solid component . it presents inhomogeneous attenuation with foci of calcification . 
at surgery , a thymoma was found , with solid areas surrounded by a thick , fibrous capsule . right posterior mediastinal thymic tissue is present due to lobulated pleural implants , without pleural effusion . 
tc : una grossa massa a margini ben definiti adiacente al tratto ascendente dellaorta ed alla vena cava superiore ha contenuto solido : mostra attenuazione disomogenea per presenza di focali calcificazioni . 
the common denominator of disorders associated with thymoma is a condition of autoimmunity neuromuscular syndromes haematological disorders autoimmune diseases endocrine disorders miscellaneous myasthenia gravis eaton - lambert syndrome paraneoplastic polyneuropathy limbic encephalopathy stiff - person syndrome pure erythrocyte aplasia aplastic anaemia low platelet count hypogammaglobulinaemia t lymphocytosis t - cell deficiency syndrome agranulocytosis hyperthyroidism hyperparathyroidism addisons disease panhypopituitarism cushings syndrome thyroiditis hypertrophic osteoarthropathy nephrotic syndrome primary glomerulonephritis with minimal lesions polymyositis systemic lupus erythematosus hashimoto thyroiditis rheumatoid arthritis pernicious anaemia scleroderma graves disease sjgrens syndrome sarcoidosis tabella 3 sindromi paraneoplastiche e condizioni associate a timoma . 
la funzione del timo la produzione di linfociti t immunocompetenti , per proliferazione e maturazione di linfociti immaturi del midollo osseo : nellambito della patologia il comune denominatore delle condizioni associate a timoma rappresentato da un carattere di autoimmunit sindromi neuromuscolari disordini ematologici malattie autoimmuni disordini endocrini miscellanea miastenia gravis sindrome di eaton - lambert polineuropatia paraneoplastica encefalopatia limbica sindrome di stiff - person aplasia eritrocitaria pura anemia aplastica piastrinopenia ipogammaglobulinemia linfocitosi t sindrome da deficienza di cellule t agranulocitosi polimiosite lupus erythematosus sistemico tiroidite hashimoto artrite reumatoide anemia perniciosa sclerodermia malattia di graves sindrome di sjgren sarcoidosi ipertiroidismo iperparatiroidismo morbo di addison panipopituitarismo sindrome di cushing tiroidite osteoartropatia ipertrofica sindrome nefrosica glomerulonefrite primitiva a lesioni minime sent in the anterosuperior mediastinum , originate from one of the thymic lobes and typically project onto one side of the midline . 
more rarely , however , they may extend on either side of the mediastinum and displace the heart and great vessels posteriorly . calcifications , which are relatively infrequent ( 20% in our experience ) , are often salt - grain calcifications ( unlike teratoma , where they are usually laminar ) [ 35 ]  . 
density is homogeneous [ 35 ] except in larger lesions in which the presence of haemorrhage , necrosis or cysts produces focal areas of low density [ 26 ]  . 
nonetheless , ct is not capable of reliably identifying invasiveness prior to surgery unless there are evident signs of infiltration of the great vessels or pleura or irregularities of the interfaces with the lung [ 35 ]  . 
signal intensity at mri is intermediate ( equal to or higher than that of skeletal muscle ) in t1 - weighted sequences and increased ( approaching that of fat ) in t2 . 
cystic areas with fluid la radiografia del torace pu risultare anche normale , evenienza plausibile in caso di sindrome di cushing ; invece , tumori non secernenti o men si presentano pi frequentemente come grosse masse del mediastino anteriore [ 45 ]  . alla tc la densit omogenea o , pi frequentemente , eterogenea per necrosi , emorragia e calcificazioni puntiformi e distrofiche [ 7 ] ; possono assumere il mezzo di contrasto . 
le metastasi ossee sono tipicamente osteoblastiche . tumori con origine da cellule staminali : neoplasie a cellule germinali il termine si riferisce ad un gruppo di tumori identici per aspetto istologico ad alcune neoplasie del testicolo e dellovaio , che nellinsieme si ritiene derivino da elementi germinali primitivi comuni . 
costituiscono un insieme eterogeneo di lesioni , benigne e maligne , a localizzazione mediastinica come conseguenza di un errore disembriogenetico per interruzione della migrazione , per la colonizzazione delle gonadi , di elementi staminali lungo la cresta urogenitale . si riscontrano pertanto lungo la linea mediana del corpo , dalla ghiandola pineale allarea presacrale , poich questa linea direttiva corrisponde alla cresta urogenitale embrionale . 
il mediastino rappresenta la sede pi comune delle localizzazioni extragonadiche e , in particolare , il compartimento anteriore il pi frequentemente interessato : infatti , content show hypointense signal in t1 and hyperintense in t2 . 
symptoms may be local ( compression and invasion of thoracic structures in 1 / 3 of patients : chest pain , cough , dyspnoea , dysphagia ; superior vena cava syndrome ) or systemic ( paraneoplastic syndromes )  . 
about 10%15% of myasthenic subjects have thymoma , and about 35%40% of patients with thymoma have myasthenia [ 32 ]  . thymic resection improves the clinical picture in many paraneoplastic syndromes . 
almost 15% of patients with thymoma develop a second tumour : kaposis sarcoma , chemodectoma , multiple myeloma , acute leukaemia and carcinomas ( lung , colon )  . complete surgical resection is the treatment of choice . 
in general , adjuvant therapy ( radiotherapy , chemotherapy ) is prescribed in all cases after resection , except for well - encapsulated thymomas not showing microscopic invasion at histology and mediastinal adhesions at surgery [ 38 ]  . 
due to the risk of persistence and recurrence , long - term radiological follow - up is mandatory . thymic carcinoma features the term thymic carcinoma is reserved for those malignant epithelial tumours of the thymus that have cytological and histological of malignancy : nuclear / cytological atypia , high mitotic index , necrosis [ 39 ]  . they form a diverse group of rare and aggressive epithelial tumours with a strong tendency towards early local invasion and metastatic spread . pathognomic there are no distinctive radiological features . 
thymic carcinomas generally present as a bulky mass ( 515 cm ) of the anterior mediastinum , with lobulated or poorly defined margins [ 40 ] , which at diagnosis are found to invade surrounding fat planes and adjacent structures . 
as in the case of thymoma , it may have a cystic appearance [ 25 ] ; calcification is present in 10%40% of cases . mediastinal lymphadenopathy is seen in 40% of cases [ 42 ]  . most patients are clinically asymptomatic at presentation . compared with thymoma , paraneoplastic manifestations are rare . 
prognosis is poor as a result of progressive intrathoracic growth ( metastases to the pleura and lungs and mediastinal , cervical and axillary lymph nodes ) and distant metastasis ( bone , liver ) in most patients . 
nella maggior parte dei casi , la neoplasia diviene manifesta nel giovane adulto , con et media alla diagnosi tra iii e v decennio [ 47 ]  . sono classificati in forme benigne ( 80% dei casi ) e maligne . 
i tumori maligni comprendono i seminomi , i tumori non seminomatosi ed una parte dei teratomi ( teratomi immaturi in et superiore a 15 anni ; teratomi con trasformazione maligna )  . 
in et pediatrica , invece , si presentano con uguale distribuzione per sesso . i tumori benigni sono in genere asintomatici ; al contrario , quasi la totalit dei pazienti affetti da neoplasie maligne manifesta sintomi : dolore toracico , dispnea , tosse , febbre o manifestazioni da compressione ed invasione delle strutture mediastiniche circostanti . tra le forme benigne listotipo pi comune il teratoma maturo , che rappresenta il 45%75% dei casi [ 48 ]  . 
inoltre , i tumori a cellule germinali del mediastino hanno propensione a sviluppare una componente di malignit non germinale ( rabdomiosarcoma , adenocarcinoma , tumori neuroectodermici ) che pu diventare listologia prevalente . 
stata descritta la relazione tra queste neoplasie e la sindrome di klinefelter ( cariotipo 47 , xxy ) , con unassociazione pari a quasi l8% dei casi [ 49 ]  . 
pi del 95% dei radiogrammi sono alterati , per la presenza di una massa localizzata il pi delle volte in mediastino anteriore ( solo 3%8% in mediastino posteriore ) [ 50 ]  . 
sebbene debba essere sempre effettuato lesame obiettivo del testicolo , incluso lapprofondimento ecotomografico , la presenza di una massa isolata in mediastino anteriore senza coinvolgimento retroperitoneale non suggestiva per lorigine primitiva testicolare . 
the most frequent histological type is carcinoid tumour ( typical and atypical ) , which is histologically and clinically ( neoplastic syndromes ) identical to all other extrathymic presentations . these tumours can affect all age groups , with a mean age at diagnosis of 45 years . 
half are well circumscribed or encapsulated ; the others invade the pleura , pericardium , diaphragm and mediastinal structures . chest radiography may even be normal , which is understandable in the case of cushings syndrome ; in contrast , nonsecreting tumours or men more frequently appear as large masses of the anterior mediastinum [ 45 ]  . ct attenuation is homogeneous or , more often , heterogeneous due to necrosis , haemorrhage and punctuate and dystrophic calcification [ 7 ]  . 
bone metastases are typically osteoblastic . tumours originating from stem cells : germ - cells neoplasms the term refers to a group of tumours with identical histology to some neoplasms of the testicle and ovary , all of which are believed to derive from common primary germ cells . they form a diverse group of benign and malignant lesions that develop in the mediastinum as a consequence of an embryogenic error interrupting the migration of stem cells along the urogenital ridge to the gonads . they therefore arise along the midline between the pineal gland and the presacral area , as this line corresponds to the embryonic urogenital ridge . 
la tc pu , inoltre , dimostrare la presenza di metastasi polmonari o lirregolarit dellinterfaccia con il polmone segno di infiltrazione per contiguit , oltre allestensione alla parete toracica , il coinvolgimento linfonodale mediastinico e le metastasi a distanza [ 40 , 51 ]  . la valutazione sierologica di - fetoproteina ( - fp ) e gonadotropina corionica umana ( - hcg ) utile nella gestione del paziente con sospetto clinico - radiologico di tumore maligno a cellule germinali , sia nella diagnosi sia nel follow - up . 
lelevazione di questi marcatori , singola o congiunta , si riscontra nell80%85% dei tumori non seminomatosi ; la sola - fp alterata nel 60%80% dei casi , mentre dal 30% al 50% di questi tumori presenta un incremento di hcg [ 52 ]  . 
i seminomi puri possono associarsi ad una alterazione della - hcg ( a bassi livelli ) con - fp mai mossa . teratoma un tumore rappresentato da tessuto derivante da uno o tutti e tre i foglietti embrionali germinali : queste linee non costituiscono una struttura organizzata . 
riconosce tre tipi istologici : ( 1 ) maturo , ( 2 ) immaturo e ( 3 ) con trasformazione maligna . i teratomi maturi costituiscono la variante pi comune : rappresentano il 70% dei tumori a cellule germinali del mediastino in et pediatrica ed il 60% nelladulto [ 46 , 50 ]  . sono ben delimitati rispetto alle strutture mediastiniche circostanti e sono uni o multicistici ; le forme solide sono di riscontro raro [ 53 ]  . 
alla diagnosi il paziente in genere asintomatico , e la diagnosi dunque incidentale al radiogramma del torace , ma , poich presentano dimensioni cospicue ( 810 cm ) , possono causare sintomi locali [ 53 ]  . 
dal punto di vista istologico , sono costituiti da tessuti adulti ben differenziati , arrangiati in modo irregolare , di derivazione ectodermica ( cute e suoi annessi quali denti , peli , ghiandole sudoripare e sebacee ) , mesodermica ( cartilagine , osso , grasso , tessuto muscolare liscio ) ed endodermica ( epitelio bronchiale , intestinale e tessuto pancreatico )  . 
altri tessuti relativamente comuni includono : cartilagine , grasso , tessuto muscolare liscio e mucosa respiratoria ; osso , denti , tessuto gliale e mucosa enterica sono di meno frequente riscontro . 
moeller [ 54 ] ha riportato la frequenza delle componenti a maggiore rappresentazione : liquido 88% , grasso 76% , calcificazioni 53% , i tre elementi of thymic tissue at the periphery of some of these tumours suggests that they originate in the thymus . 
in most cases , the tumour becomes manifest in the young adult , with an age range at diagnosis between the third and fifth decade [ 47 ]  . these tumours are classified into benign ( 80% of cases ) and malignant forms . 
benign tumours include mature teratomas and those mature teratomas with an immature component less than 50% of the volume . malignant tumours include seminomas , nonseminomatous tumours and some teratomas ( immature teratomas in subjects older than 15 years ; teratomas with malignant transformation )  . 
in children , instead , they are equally distributed between genders . benign tumours are generally asymptomatic whereas nearly all patients with malignant disease report symptoms : chest pain , dyspnoea , cough , fever or signs of compression and invasion of the surrounding mediastinal structures . among benign forms , the most frequent histological type is mature teratoma , which accounts for 45%75% of cases [ 48 ]  . 
moreover , germ - cell tumours of the mediastinum tend to develop a non - germ - cell malignant component ( rhabdomyosarcoma , adenocarcinoma , neuroectodermal tumours ) , which may become the predominant histological component . 
at diagnosis , a primary tumour of the gonads must be excluded even though a single metastatic localisation in the anterior mediastinum is rare . a correlation has been reported between these tumours and klinefelters syndrome ( karyotype 47 , xxy ) in 8% of cases [ 49 ]  . 
haematological malignancies ( commonly nonlymphoid leukaemia and occasionally malignant histiocytosis ) have also been found to be associated , in particular , with teratomas . germ - cell tumours of the mediastinum are more frequently identified at standard chest radiography . 
although physical and sonographic evaluation of the testicle should always be carried out , presence of an isolated mass in the anterior mediastinum without retroperitoneal involvement is not suggestive of a primary testicular origradiological presentation of malignant nonseminomatous tumours is generally of a large mass of the anterior mediastinum with smooth , lobulated or irregular margins . 
at ct , they have inhomogeneous attenuation with extensive areas of low density ; there may be focal calcification . contiguous fat planes are usually infiltrated ; pleural and pericardial effusion are common . 
ct can also reveal the presence of lung metastases or irregularity of the interface with the lung , a sign of invasion via contiguous spread , as well as extension to the chest wall , involvement of mediastinal lymph nodes and distant metastases [ 40 , 51 ]  . testing of serum - fetoprotein ( - fp ) and - human choria.m. 
il tessuto pancreatico presenta unincidenza relativamente alta [ 55 ] e pu indurre complicanze da rottura spontanea ( per autodigestione da fluidi pancreatici ) con spandimento negli organi cavi vicini : bronchi , pleura , pericardio o polmone [ 46 , 53 ]  . i teratomi immaturi sono costituiti dagli stessi tessuti differenziati della variet matura in associazione a tessuto poco organizzato di tipo fetale : il tessuto neuroepiteliale primitivo il pi comune . 
hanno comportamento variabile , in relazione allet del paziente : nellinfanzia la prognosi favorevole ( in et inferiore a 15 anni sono assimilabili alla controparte matura ) ; nelle altre et della vita hanno frequentemente comportamento aggressivo ( recidiva locoregionale o ripresa a distanza ) , che conduce allexitus il paziente . i teratomi con trasformazione maligna o teratomi maligni o teratocarcinomi contengono una componente francamente maligna , pi frequentemente sarcoma ( angiosarcoma , rabdomiosarcoma ) , in associazione a tessuto fetale o a tessuti adulti ben differenziati ; altre variet sono ladenocarcinoma , il carcinoma a cellule squamose , il carcinoma indifferenziato o una variante mista in associazione ad un tumore maligno a cellule germinali . 
sono aggressivi e causano la morte entro pochi mesi dalla diagnosi per diffusione locale , metastasi a distanza o come conseguenza di entrambe [ 47 ]  . la maggior parte dei teratomi del mediastino dimostrata sul radiogramma standard del torace come massa localizzata a livello del compartimento anteriore , in stretta prossimit allorigine dei grossi vasi ed al cuore [ 7 , 54 ] ; in genere protrudono da un lato della linea mediana e possono raggiungere dimensioni ragguardevoli . 
alla radiologia tradizionale , in una percentuale variabile sino al 26% dei casi , si possono evidenziare calcificazioni prevalentemente laminari e , raramente , osso strutturato o denti [ 53 ]  . 
in particolare , quando siano dimostrate strutture ossee o denti nellambito di una massa riscontrata alla radiologia tradizionale o la combinazione di grasso , fluido , tessuti molli e calcificazioni alla tc consentita la diagnosi nella maggior parte dei teratomi cistici maturi , con un elevato grado di confidenza . 
le complicanze , che possono risultare evidenti sia al radiogramma standard sia alla tc , includono latelectasia , come conseguenza dellostruzione bronchiale , la polmonite ostruttiva o da rottura [ 51 , 54 ] ed il versamento secondario a rottura spontanea nello spazio pleurico o pericardico [ 57 ]  . 
pure seminomas may be associated with altered - hcg ( at low levels ) with unchanged - fp . teratoma this tumour is made up of tissue arising from one or all three of the germ - cell layers of the embryo . 
at diagnosis , patients are generally asymptomatic , so these tumours tend to be incidentally discovered at chest radiography ; however , because they can reach a remarkable size ( 810 cm ) , they may give rise to local symptoms [ 53 ]  . in addition , an abrupt increase in size leading to sudden retrosternal pain is not necessarily an indication of malignancy , as it may be caused by intracystic haemorrhage . histologically , these tumours consist of irregularly arranged , well - differentiated adult tissues of ectodermal ( skin and annexes such as teeth , hairs , sweat and sebaceous glands ) , mesodermal ( cartilage , bone , fat , smooth muscle tissue ) and endodermal origin ( bronchial and intestinal epithelium and pancreatic tissue )  . 
of these , the ectodermal elements prevail , in particular , skin and annexes . other relatively common tissues include cartilage , fat , smooth muscle tissue and respiratory mucosa ; bone , teeth , glial tissue and intestinal mucosa are less frequent . moeller [ 54 ] reported the frequency of the most common components : fluid 88% , fat 76% , calcification 53% and a combination of the three elements 39% . 
ginecomastia ed incremento dei valori ematici di estradiolo e - hcg sono stati descritti raramente [ 61 ] : circa il 10% dei pazienti affetti da seminoma puro pu presentare un incremento sierologico dei livelli di - hcg , ma non mai presente lelevazione della - fp . radiograficamente si manifestano , per lo pi , come masse bulky , lobulate , che protrudono da uno o ambo i lati del mediastino anteriore . 
le metastasi pi frequenti hanno sede polmonare e intratoracica , ai linfonodi regionali ; quelle extratoraciche interessano in genere losso [ 7 , 59 ]  . raccomandato un vigile protocollo di osservazione , senza chirurgia , per le masse residue dopo chemioterapia , a meno che se ne dimostri la crescita . disembriomi non seminomatosi comprendono il tumore del seno endodermico o del sacco vitellino , il coriocarcinoma ed il carcinoma embrionario o , infine , la variante mista [ 62 ]  . 
circa l85% dei casi interessa gli uomini , con et media di 29 anni ; si associa la sindrome di klinefelter sino nel 20% dei pazienti [ 50 ]  . 
frequente il riscontro di valori elevati della lattato deidrogenasi ( ldh ) e di marcatori sierici : - fp nel 60%80% dei pazienti e - hcg nel 30%50% dei casi [ 63 ]  . 
teratomas with malignant transformation , malignant teratomas or teratocarcinomas contain a frankly malignant component , most commonly sarcoma ( angiosarcoma , rhabdomyosarcoma ) , associated with foetal tissue or welldifferentiated adult tissue . 
they are aggressive and lead to death within months of diagnosis as a result of local spread , distant metastasis or both [ 47 ]  . most mediastinal teratomas are depicted by standard chest radiography as a localised mass in the anterior compartment close to the heart and the origin of the great vessels [ 7 , 54 ]  . in general , they extend to one side of the midline and may reach a large size . 
at ct , they have well - defined , smooth or lobulated margins . mature teratomas are encapsulated and display heterogeneous attenuation due to the combination of soft tissue , fluid , fat and calcific components . 
in particular , a finding of bone structures or teeth within a mass at conventional radiology or of a combination of fat , fluid , soft tissue and calcification at ct will allow a confident diagnosis in most mature cystic teratomas . complications visible at both conventional radiology and ct include atelectasia resulting from bronchial obstruction , pneumonia due to obstruction or rupture [ 51 , 54 ] and effusion due to spontaneous rupture of the pleural or pericardial space [ 57 ]  . 
some teratomas may be complicated by synchronous or metachronous leukaemia , which may condition prognosis . seminoma this is the second most frequent germ - cell tumour of the the most common teratoma and mediastinum after ( 35%40% ) among the pure forms [ 59 ]  . 
sono frequentemente invasivi alla diagnosi , con quasi il 90% dei pazienti che presenta sintomi . radiologicamente assumono laspetto di grosse masse irregolari del mediastino anteriore , spesso con densit disomogenea alla tc per la presenza di estese aree centrali ipodense dovute a necrosi , emorragia e degenerazione cistica . pu essere presente invasione delle strutture adiacenti , compresa la parete toracica , diffusione ai linfonodi loco - regionali ed a distanza . 
alla diagnosi l85%95% dei pazienti presenta metastasi evidenti , a distanza ; le sedi pi comuni includono polmone , pleura , linfonodi , fegato e , meno frequentemente , losso [ 59 ]  . espansi linfonodali i linfonodi del mediastino anteriore possono essere interessati da eventi loco - regionali e sistemici . 
la patologia sistemica ( linfomi ) vede coinvolti spesso i linfonodi del mediastino anteriore e , contestualmente , il timo che organo linfatico . i linfonodi normalmente non sono visibili alla radiografia del torace per le piccole dimensioni e perch indovati nel tessuto adiposo . 
il loro ingrandimento pu dislocare la pleura producendone una bozzatura visibile : questo reperto pu risultare evidente , qualora coinvolga le catene mammarie interne , determinando una alterazione della linea extrapleurica anteriore ( lea )  . 
lecotomografia , pur se non consente di esaminare tutte le sezioni del mediastino , condivide la sensibilit della tc nella valutazione del mediastino anteriore , con elevata specificit nello studio delle linfoadenopatie . quando le linfoadenopatie sono associate ad una massa polmonare centrale , ad una lesione cavitaria od infiltrativa , essenziale la diagnosi di natura di tale lesione , ottenibile con broncoscopia a fibre ottiche , con biopsia endobronchiale , transbronchiale o fna . 
infine , quando non siano accessibili n per via broncoscopica , n percutanea , o quando i campioni siano inadeguati o non diagnostici , pu essere indicata la mediastinoscopia con biopsia escissionale . il trattamento delle linfoadenopatie mediastiniche legato alla malattia di base . linfoma primitivo mediastinico il linfoma un tumore mediastinico piuttosto comune e pu a.m. 
contrast - enhanced computed tomography ( ct ) of an 11 - year - old girl , large , complex mass containing multiple cysts , fat and dystrophic calcification displaces the mediastinum to leit contains fatty fluid , debris and focal fatty density ( mean attenuation 96 hounsfield units )  . 
invasion of adjacent structures is very rare [ 46 ] , but distant metastases are possible . the most frequent sites for metastasis are lung , chest and regional lymph nodes ; extrathoracic metastases usually involve bone [ 59 , 7 ]  . 
residual masses after chemotherapy should undergo close monitoring without surgery unless their growth has been documented . nonseminomatous dysembryomas these include tumours of the endodermal sinus or yolk sac , choriocarcinomas , embryonal carcinomas or the mixed variant [ 62 ]  . 
these are malignant tumours , which are symptomatic and affect young adults [ 46 ]  . about 85% of cases occur in men with a mean age of 29 years ; klinefelters syndrome is associated in up to 20% of patients [ 50 ]  . 
patients usually have elevated lactate dehydrogenase ( ldh ) and serum markers : - fp in 60%80% of patients and - hcg in 30%50% [ 63 ]  . essere riscontrato nel compartimento anteriore , medio e posteriore [ 64 ]  . 
rappresenta circa il 20% di tutti i tumori del mediastino nelladulto ed il 50% nel bambino ; nel 5% dei casi il mediastino lunica sede di malattia ed in questo caso la diagnosi differenziale pu essere difficile sulla base del solo reperto radiologico . 
in genere , la diagnosi conseguente allescissione di una linfoadenopatia periferica palpabile , qualora sia presente , ed il coinvolgimento toracico accertato dagli esami di diagnostica per immagini . molti dei linfomi primitivi del mediastino originano nel timo , alternativamente da linfonodi : si tratta per lo pi di linfomi di hodgkin , o , meno comunemente , di linfomi nonhodgkinfrequentemente sono confinati nel mediastino al momento della diagnosi : il 50%70% dei pazienti affetti da malattia linfomatosa di altra sede con coinvolgimento mediastinico presenta la malattia di hodgkin e solo nel 15%25% un linfoma non - hodgkin [ 65 ]  . 
tra i linfomi non - hodgkin , le varianti pi frequenti , che possono interessare primariamente il mediastino anteriore , sono il linfoma a grandi cellule b ed il linfoma linfoblastico . 
 la scintigrafia con gallio e soprattutto la fdg - pet sono utili nella stadiazione e nel follow - up dopo trattamento . linfoma di hodgkin si presenta con distribuzione bimodale , con picco di incidenza nelladolescenza e nella giovane et adulta , e dopo il vi decennio . 
i sintomi sistemici ( che identificano la categoria b : febbre , calo ponderale superiore al 10% del peso corporeo negli ultimi 6 mesi , sudorazioni notturne , prurito ) sono evidenti nel 20%30% dei casi , mentre la maggior parte dei soggetti non mostra una sintomatologia n sistemica n locale ed il riscontro pertanto incidentale alla radiografia del torace . talvolta , tuttavia , pu essere presente dolore toracico , tosse , dispnea , disfagia da invasione o da effetto massa sulle strutture mediastiniche . 
dal punto di vista macroscopico si manifesta come conglomerato linfonodale o come massa polilobulata a nonseminomatous germ - cell tumours of the mediastinum more commonly occur in the anterior compartment and appear as lobulated masses enclosed by a thin capsule . 
they are frequently invasive at diagnosis , with almost 90% of patients reporting symptoms . the radiological appearance is of a bulky irregular mass in the anterior mediastinum , often with inhomogeneous density at ct due to the presence of large hypodense central areas related to necrosis , haemorrhage and cystic degeneration . there may be invasion of adjacent structures , including the chest wall , as well as spread to the locoregional lymph nodes and distant metastasis . 
at diagnosis , 85%95% of patients have distant metastasis ; the most common sites include lung , pleura , lymph nodes , liver and , less commonly , bone [ 59 ]  . lymph node masses lymph nodes of the anterior mediastinum may be affected by locoregional and systemic events . 
systemic diseases ( lymphomas ) often involve nodes of the anterior mediastinum and thymus , a lymphatic organ . lymph nodes are not normally visible on chest radiographs due to their small size and the fact that they are embedded in fat . 
ultrasonography , though not allowing inspection of all sections of the mediastinum , has the same accuracy as ct in evaluation of the anterior mediastinum and high specificity in the study of lymphadenopathy . when node enlargement is associated with a central pulmonary mass , cavitary or infiltrative disease , it is mandatory to determine the nature of the lesion by fibreoptic bronchoscopy , endobronchial or transbronchial biopsy or fineneedle aspiration ( fna )  . 
finally , if the nodes are not amenable to bronchoscopic or percutaneous techniques , or in the event of inadequate or nondiagnostic sampling , mediastinoscopy with excisional biopsy may be indicated . 
 treatment of mediastinal lymphadenopathy depends on the underlying disease . primary mediastinal lymphoma lymphoma is a relatively common mediastinal tumour , which may be seen in the anterior , middle or posterior compartment [ 64 ]  . 
quando ha sede timica possono essere presenti , prima o dopo trattamento , aree cistiche [ 25 ]  . in circa il 70% dei casi la radiografia del torace patologica : di questi , il 90% presenta un interessamento linfonodale bilaterale . 
la diffusione per stazioni linfonodali contigue , linteressamento di un singolo gruppo segnalato solo nel 15% dei casi : il mediastino anteriore ed i linfonodi paratracheali rappresentano le sedi pi usuali . 
la variante pi comune ( scleronodulare ) si presenta tipicamente come massa lobulata ben delimitata nel mediastino antero - superiore [ 65 ]  . nel 12% dei pazienti si osserva invasione polmonare , che quasi sempre associata con linteressamento adenopatico ilare . 
alla tc , dal 71% all85% delle nuove diagnosi di linfoma di hodking presenta localizzazione toracica [ 67 ] : la massa mediastinica pu essere costituita da formazioni multiple arrotondate con densit dei tessuti molli ( linfoadenopatie ) , da una massa bulky ( conglomerati adenopatici ) o da una lesione solida ( e pi raramente cistica ) timica [ 25 ]  . 
alla rm rappresentato da una massa relativamente omogenea , con bassa intensit di segnale nella sequenza t1 , analoga al tessuto muscolare , e segnale intermedio o alto nelle immagini in t2 , come da tessuto adiposo . 
nelle sequenze in t2 liperintensit di segnale pu essere in relazione con edema , infiammazione , fibrosi precoce o tessuto granulomatoso , mentre il tessuto fibroso denso ( dopo trattamento ) ha bassa intensit . 
alle lesioni bulky dopo terapia spesso residuano masse fibrose che non possono essere distinte dalle persistenze tumorali alla tc : in questi casi le sequenze t2 di rm possono segnalare un incremento diffuso dellintensit o aree focali iperintense , che correlano con la malattia recidiva [ 68 ]  . infine , deve essere ricordato che una massa mediastinica in progressione o di nuova presentazione dopo trattamento pu rappresentare una recidiva tumorale , o in alternativa uniperplasia timica o una cisti del timo . linfoma non - hodgkin alla presentazione generalmente gi malattia diffusa ; la classificazione istopatologica ha un valore prognostico superiore allestensione anatomica di malattia . 
l85% dei pazienti presenta , alla diagnosi , uno stato di malattia avanzato che generalmente si accompagna a sintomi sistemici ( categoria b ) , linfoadenopatie generalizzate o malattia extranodale . il linfoma a grandi cellule b interessa primitivamente il mediastino , nella fascia di et del giovane adulto ed occasionalmente nel bambino , con prevalenza del sesso femminile in entrambi i casi . 
in general , diagnosis is achieved through excision of a palpable peripheral node , when present , and chest involvement is established by diagnostic imaging . many primary mediastinal lymphomas arise in the thymus rather than in lymph nodes . 
they may cause bulky enlargement of mediastinal nodes , at times associated with ipsilateral interstitial oedema due to lymphatic or venous obstruction . gallium scintigraphy and especially fluorodeoxyglucose positron emission tomography ( fdg - pet ) are useful in disease staging and follow - up after treatment . hodgkins lymphoma hodgkins lymphoma has a bimodal age distribution , with an incidence peak in adolescence and early adulthood and after the sixth decade of life . 
systemic symptoms ( which identify category b : fever , weight loss more than 10% of body weight during the previous 6 months , night sweats , itching ) are present in 20%30% of cases whereas most subjects have no systemic or local symptoms , and the disease is incidentally discovered at chest radiography . 
the most common variant ( nodular sclerosing ) typically presents as a well - circumscribed lobulated mass in massa tumorale , con possibile infiltrazione della vena cava superiore , delle vie aeree , della parete toracica o di altre strutture adiacenti . 
la diffusione extratoracica , alla presentazione , un reperto inusuale . anche i linfomi linfoblastici sono altrettanto aggressivi nella presentazione : si tratta di forme ad alto grado che si presentano nei primi due decenni di vita , pi spesso nelladolescente di sesso maschile . 
il linfoma linfoblastico condivide molte caratteristiche cliniche con la leucemia linfoblastica associata a massa mediastinica : questultima presentazione ne pu rappresentare la fase leucemica . entrambi gli istotipi si manifestano come massa bulky , non capsulata , con franco atteggiamento invasivo che interessa il timo e pu infiltrare le strutture adiacenti . le caratteristiche radiologiche sono varie . 
quasi il 50% ha un coinvolgimento linfonodale tipicamente a salto e non interessa le stazioni mediastiniche anteriori e paratracheali : infatti , a differenza del linfoma di hodgkin , interessa meno elettivamente il compartimento mediastinico anteriore ed ha una maggiore predilezione per la diffusione linfonodale a stazioni non contigue ed ematogena . pu verificarsi infiltrazione polmonare ( in meno del 5% dei pazienti in fase tardiva , indifferentemente dallassociazione con unadenopatia ilare ) , pleurica o pericardica [ 68 ]  . 
the mediastinal mass may consist of multiple rounded formations with soft tissue density ( lymphadenopathy ) , a bulky mass ( nodal conglomerates ) or a solid thymic lesion ( less commonly cystic ) [ 25 ]  . 
calcification is typically present after treatment [ 65 ]  . at mri , the mass is relatively homogeneous , with low signal intensity similar to muscle in t1 and intermediate or high signal intensity similar to fat in t2 . 
the high t2 signal intensity may be related to oedema , inflammation , early fibrosis or granulomatous tissue whereas the dense fibrous tissue ( after treatment ) has low signal intensity . 
in these cases , mri t2 sequences may reveal a diffuse increase in signal intensity or focal areas of hyperintensity , which are correlated with disease recurrence [ 68 ]  . finally , it should be remembered that a progressing or new mediastinal mass after treatment may represent tumour recurrence , thymic hyperplasia or a thymic cyst . non - hodgkins lymphoma at presentation , the disease is already diffuse ; histopathological classification has greater prognostic significance than anatomical extension of disease . 
at presentation , 85% of patients have advanced disease , usually with systemic symptoms ( category b ) , generalised lymphadenopathy or extranodal disease . large b - cell lymphoma is a primary mediastinal lymphoma seen in young adults and occasionally children , with prevalence in females in both cases . 
i soggetti affetti da carcinoma mammario , in particolar modo coloro che sviluppano una recidiva a livello della parete toracica in sede parasternale , dovrebbero essere indagati ponendo attenzione allarea retrosternale poich queste recidive possono rappresentare una diretta estensione dai linfonodi della catena mammaria interna . la diagnosi differenziale si pone con la malattia linfoproliferativa ( linfoma o leucemia ) , che pu coinvolgere questa catena linfonodale ; in genere , per , per tali patologie ematologiche non si verifica il loro interessamento in assenza di coinvolgimento delle stazioni in sede toracica . lindagine radiologica ricopre un importante ruolo nella detezione di linfoadenopatie della catena mammaria interna , dal momento che questo gruppo linfonodale non pu essere indagato attraverso la mediastinoscopia . un ingrandimento dei linfonodi di questa stazione rappresenta una delle cause di alterazione della lea , che in questo caso presenta margine dentellato o lobulato . 
enlargement of sternal or internal mammary chain nodes these nodes , especially those located in the first three intercostal spaces , tend to be involved by metastatic spread from breast cancer . 
subjects with breast cancer , in particular , those who develop a recurrence in the parasternal chest wall , should undergo careful investigation of the retrosternal region , as these recurrences may represent a direct extension from nodes of the internal mammary chain . differential diagnosis should consider lymphoproliferative diseases ( lymphoma or leukaemia ) , which may affect the internal mammary chain general , however , involvement by these diseases does not occur without involvement of the thoracic node groups . imaging is instrumental in the detection of internal mammary chain lymphadenopathy , which cannot be investigated with mediastinoscopy . 
enlargement of this node group is one of the causes of an altered ael , which in this case displays a serrated or lobulated margless frequently , the interface may appear linear , with a band of increased density separating the lung from the anterior chest wall . 
enlargement of diaphragmatic nodes ( anterior and middle ) or cardiophrenic angle nodes these lymph nodes cannot be visualised at radiography unless they are massively enlarged since they are surrounded by areolar tissue and fat , which mask minimal enlargement . when they become visible at the level of the cardiophrenic angle , they produce a mass with lateral convexity on frontal views and posterior convexity on laterolateral ( ll ) views . 
in the case of suspected lymphadenopathy , a ct ( or us ) examination may be useful in differentiating involvement of the diaphragmatic nodes from a pericardial fat pad . mesenchymal abnormalities mesenchymal abnormalities include cystic and solid lesions . mediastinal mesenchymal masses originate from the mediastinal connective tissue , account for about 5% of all mediastinal masses and include benign and malignant lesions originating from fat , fibrous tissue , smooth and striated muscle , blood and lymphatic vessels , bone and nerve tissue [ 69 ]  . around 55% are malignant ; there is no gender predilection [ 70 ]  . 
in caso di sospetta linfoadenopatia , lindagine tc ( ovvero con us ) risulta utile al fine di differenziare un interessamento dei linfonodi diaframmatici da un cuscinetto adiposo pericardico . anomalie mesenchimali le anomalie mesenchimali comprendono lesioni cistiche e lesioni solide . 
gli espansi mesenchimali mediastinici originano dagli elementi di tessuto connettivo del mediastino . rappresentano circa il 5% di tutte le masse mediastiniche ed includono lesioni benigne e maligne ad origine dal tessuto adiposo , fibroso , dal muscolo liscio e striato , dai vasi sanguigni e linfatici , da osso e da tessuto nervoso [ 69 ]  . 
circa il 55% maligno ; non si riscontra predilezione di sesso [ 70 ]  . possono presentarsi in qualunque compartimento , con leccezione delle formazioni nervose ( a sede elettiva paravertebrale ) , ma il mediastino anteriore rappresenta la sede pi frequente . 
tuttavia , la localizzazione mediastinica meno comune rispetto alle sedi extratoraciche . espansi ad origine dal tessuto adiposo il riscontro di tessuto adiposo nel mediastino frequente e ben documentabile soprattutto dallo studio tc , che , per intrinseche caratteristiche , ne permette una precisa dimostrazione . 
nonetheless , mediastinal location is less common than extrathoracic sites . masses originating from adipose tissue adipose tissue is frequently found in the mediastinum and is particularly well documented by ct thanks to the intrinsic properties of this technique . 
adipose tissue may be seen in a wide spectrum of conditions some nonpathological ( lipomatosis , fat pads ) , others expressing local disease ( lipomas , well - differentiated liposarcoma ) , and others expressing frankly progressive disease ( undifferentiated liposarcoma )  . lipomatosis is a diffuse and excessive accumulation fat in typical sites of the mediastinum ; it is seen in obesity and associated with hypercortisolism , as in cushings syndrome , ectopic adrenocorticotropic hormone ( acth ) production and as a side effect of treatment ( long - term steroids )  . 
the posteroanterior ( pa ) view shows symmetrical widening , with homogenous appearance , of the supra - aortic and supra - azygotic mediastinal portions , with normal and smoothly marginated appearance ( at times lobulated ) of the anterior junction line ( ajl )  . 
the finding may be confirmed by mri , which will reveal marked t1 hyperintensity with classical suppression in the fat - suppressed short tau inversion recovery ( stir ) sequences . pericardial fat pads are the most common mediastinal fat depositions . 
on pa and ll chest radiographs , they appear as faint triangular opacities of the cardiophrenic angles , more frequently on the lethe interface they form with the lung may have convex or concave shape . on the lateral radiogram , they appear as an extrapleural triangular opacity at the level of the anterior cardiophrenic angle . 
often , radiography is unable to discern the adipose nature of the pericardial pad , but this can be readily demonstrated by ct [ 40 , 71 ] , which provides differential diagnosis with pericardial cyst , thymolipoma and morgagnis hernia . extrathymic lipoma is the most common nonneurogenic mesenchymal tumour of the mediastinum and accounts for 2% of all mediastinal masses [ 28 ]  . 
at standard chest radiography , they appear as poorly radiopaque masses ( due to the typical low density of fat ) ; at times bulky , they are multilobulated and change their shape according to the patients position . the pliable nature of these masses accounts for the lack of symptoms , even in the presence of very large masses . 
nella proiezione pa visibile uno slargamento , simmetrico , di aspetto omogeneo , delle porzioni sovra - aortica e sovra - azygotica del mediastino , con aspetto regolare ed a margini lisci ( talvolta lobulati ) della linea di giunzione anteriore ( lga )  . 
nei casi dubbi , la tc diagnostica : si evidenzia un diffuso incremento del tessuto cellulo - adiposo mediastinico ; il dato pu essere confermato alla rm dal riscontro di marcata iperintensit nella sequenza t1 con classica soppressione nelle scansioni inversion recovery con soppressione ( stir )  . i cuscinetti adiposi paracardiaci sono i pi frequenti accumuli adiposi del mediastino . 
spesso la natura adiposa del cuscinetto pericardico non viene definita con sicurezza dallindagine radiografica , ma facilmente dimostrata dalla tc [ 40 , 71 ] , che discrimina la diagnosi differenziale con : cisti pericardica , timolipoma , ernia di morgagni . il lipoma extratimico il pi comune tumore mesenchimale non neurogenico a localizzazione mediastinica e rappresenta il 2% di tutte le masse del mediastino [ 28 ]  . 
al radiogramma standard del torace appaiono come masse poco radioopache ( per la tipica ipodensit del grasso ) , anche voluminose , polilobulate che modificano la forma con il decubito del paziente . 
la presenza di aree eterogenee , con valori di densit tipici dei tessuti molli , dovrebbe suggerire lipotesi diagnostica del liposarcoma . i liposarcomi sono molto rari ; in alcuni casi lorigine nel timo , ma la sede pi frequente il mediastino posteriore . la diagnosi avviene in et adulta ( v decennio )  . 
in many cases , the disease is locally advanced at presentation and manifests with the signs and symptoms of compression ( dyspnoea , chest pain , cough ) ; vena cava syndrome is uncommon . 
the well - differentiated forms show areas with fat density associated with areas of soft tissue density whereas the poorly differentiated forms have the typical homogenous density of soft tissue . 
less frequent , the other form is seen in adults and mostly affects the lower anterior mediastinum away from the neck [ 73 ]  . mediastinal cystic lymphangioma or cystic hygroma is a histologically benign proliferation of lymphatic vessels , which may have an infiltrative growth pattern . 
pathogenic hypotheses include a developmental abnormality or a hamartomatous or neoplastic origit is a typical childhood tumour : in 50% of cases it is present at birth , and 90% are diagnosed by the time the individual is 2 years old . 
they tend to enlarge as the child develops , in particular during puberty . a total of 95% of lymphangiomas affect the neck or axilla , and 10% extend in the anterosuperior mediastinum or , less commonly , in other mediastinal compartments . 
it is located in the anterior mediastinum in 37% of cases but may occur as gensegni e sintomi da compressione ( dispnea , dolore toracico , tosse ) ; la sindrome della vena cava rara . 
alla tc le forme ben differenziate presentano zone a densit adiposa associate ad aree dalla attenuazione dei tessuti molli , mentre le varianti scarsamente differenziate hanno la caratteristica densit omogenea dei tessuti molli . raramente si osservano metastasi extratoraciche . espansi ad origine dai vasi sanguigni i tumori vascolari del mediastino includono emangiomi , emangioendoteliomi , emangiopericitomi benigni e maligni ed angiosarcoma [ 70 ]  . 
la diagnosi conseguita con tc o rm : nel 30% dei casi si riconoscono fleboliti . langiografia utile per lembolizzazione prima della resezione chirurgica completa [ 72 ]  . espansi ad origine dai vasi linfatici sono nel complesso rari . 
si distinguono del linfangioma a sede mediastinica due variet clinicopatologiche : la prima , igroma cistico , nel mediastino alto , a prevalente presentazione nellinfanzia ( la diagnosi occorre in genere entro i 2 anni di et ) ; laltra forma , meno frequente , di riscontro in et adulta , per lo pi nel mediastino anteriore basso a distanza dal collo [ 73 ]  . il linfangioma cistico mediastinico o igroma cistico una proliferazione istologicamente benigna di vasi linfatici che pu assumere una crescita infiltrativa . 
raramente ( 17% dei casi ) , il linfangioma si presenta come tumore primitivo del mediastino in et adulta , ed in questa evenienza nel 37% dei casi ha sede nel mediastino anteriore , o come linfangiomatosi generalizzata con interessamento multifocale estensivo di diversi apparati [ 74 ]  . 
it may infiltrate tissue cleavage planes , but more frequently , it surrounds and displaces mediastinal structures and adjacent vessels [ 74 ]  . the most common ct finding , seen in around 60% of cases , is a smoothly marginated cystic mass with homogeneous fluid attenuation ; multilobulated cystic dilatations , which represent wide lymphatic vascular spaces and vary in diameter from a few millimetres ( 12 mm ) to several centimetres , are present in around 1 / 3 of cases [ 75 ] and have thin or thick septa that may show mild contrast enhancement . less frequent findings include calcification , homogeneous soft tissue density and spiculated margins . 
other lymphatic tissue tumours include lymphangiosarcoma and lymphangiopericytoma . pericardial mesothelial cysts these are congenital developmental abnormalities that derive from the persistence and fusion of anterior pericardial recesses ( ventral and parietal ) of the coelomic cavity . 
they have fluid content and a thin wall of connective tissue lined by a thin layer of mesothelial - like cells , are spherical or ovoid in shape and mostly unilobulated . these cysts are rare [ 8 ] , typically asymptomatic and therefore tend to be discovered incidentally at chest radiography . they occur in adulthood in the fourth and fifth decade . at chest radiography , they appear as rounded , unilobular , smoothly marginated and well - defined homogeneous masses at the level of the cardiophrenic angles , above all on the right . 
if the patients position is modified , they tend to take up a gravity - dependent position , and if there is communication with the pericardium , their fluid content can be seen to move . ct can demonstrate their benign nature : they appear as well - defined unilobulated masses with fluid density and a thin peripheral rim ; they do not enhance [ 65 ]  . 
alla tc , laspetto pi comune , che si riscontra circa nel 60% dei casi , di una massa cistica a margini lisci che ha densit omogenea , prossima a quella dellacqua ; le dilatazioni cistiche multiloculate , che rappresentano ampi spazi vascolari linfatici , variano di diametro da pochi millimetri ( 12 mm ) a diversi centimetri , sono presenti circa in 1 / 3 dei casi [ 75 ] e mostrano setti sottili o spessi che possono assumere debolmente il mezzo di contrasto . 
la resezione parziale pu essere causa di un chilotorace . altri tumori del tessuto linfatico includono il linfangiosarcoma ed il linfangiopericitoma . cisti mesoteliali pericardiche sono anomalie congenite di sviluppo che derivano dalla persistenza e fusione dei recessi pericardici anteriori ( ventrale e parietale ) della cavit celomatica . 
si presentano in et adulta , nel iv e v decennio di vita . al radiogramma del torace appaiono come masse uniloculate a margini lisci , ben definite , rotondeggianti , omogenee , a livello degli angoli cardiofrenici , soprattutto a destra . possibile la presentazione atipica in sede di mediastino superiore . 
modificando la postura del paziente , tendono ad assumere la posizione declive e , peraltro , qualora esista una comunicazione con il pericardio , pu aversi lo spostamento del contenuto liquido . 
alla tc ne viene documentata la natura benigna : risultano masse ben definite , uniloculate che presentano densit di tipo fluido ed un fine cercine periferico ; non assumono il mezzo di contrasto [ 65 ]  . 
talora la densit disomogenea o tipica dei tessuti molli per la presenza di un contenuto viscoso : in tal caso difficile la diagnosi differenziale con un processo neoplastico [ 40 ]  . 
diagnosis can also be confirmed by us [ 78 ] , which will demonstrate the hypoechoic nature and low - grade absorption of ultrasound waves . pericardial cysts can undergo clinical and radiological monitoring . 
in fact , detection of a space - occupying lesion in the anterior mediastinum is often coincidental to chest radiography performed for other purposes whereas in other cases , examination is carried out due to local ( and sometimes systemic ) clinical symptoms . ct is the modality that yields diagnosis in most cases ( more easily in differentiated forms ) while providing reliable assessment of disease extent . 
the remaining imaging techniques currently have a complementary role . integration of imaging with clinical findings ( signs and symptoms of myasthenia gravis , hyperparathyroidism , paraneoplastic syndromes ) and laboratory data ( autoantibodies against postsynaptic acetylcholine receptors , - fp , - hcg , ldh , thyroid hormones , pth , acth ) is nonetheless fundamental in the diagnostic work - up . 
infatti , la diagnosi di processi occupanti spazio nel mediastino anteriore spesso incidentale ad un radiogramma eseguito per altre ragioni , mentre in altri casi lindagine conseguente ad una sintomatologia clinica locale ( e talvolta sistemica )  . la tc la metodica che consente , nella larga maggioranza dei casi , di conseguire la diagnosi ( pi semplice nel caso delle forme differenziate ) , al contempo di un valido bilancio di estensione della malattia . 
le altre tecniche di imaging rivestono , attualmente , ruoli complementari . nelliter diagnostico comunque fondamentale lintegrazione con i dati clinici ( segni e sintomi di miastenia gravis , iperparatiroidismo , sindromi paraneoplastiche ) e di laboratorio ( autoanticorpi contro i recettori post - sinaptici dellacetilcolina , - fp , - hcg , ldh , ormoni tiroidei , pth , acth )  . 
in ogni caso , ove indicato , la tc offre unutile guida allaccertamento bioptico . ringraziamenti gli autori ringraziano , per gli importanti e competenti suggerimenti , i colleghi : f . 
for qualitative evaluation , one experienced reader graded the opacification of renal arteries as excellent , good or poor ; for quantitative evaluation , mdct and dsa were independently evaluated for the number of renal arteries and the presence , location and degree of stenosis in random order by three readers . 
on the basis of consensus readings , calculations of sensitivity , specificity , accuracy , positive predictive value ( ppv ) and negative predictive value ( npv ) for detection of degree of stenosis were made by using dsa findings as the standard of reference . 
although 72 of these were also classified as normal with ct angiography , one was overestimated by one grade ; at dsa , 16 arteries were classified as moderately stenotic ; in two arteries , there was an overestimation of one grade . 
in two patients , all three readers detected multiple severe stenoses on both modalities , with a string - of - beads appearance typical of fibromuscular dysplasia . accessory arteries were correctly identified as such by all three readers on either dsa or mdct . 
la valutazione quantitativa stata effettuata in modo indipendente da tre lettori che hanno determinato , sia con langio - tc che con la dsa , il numero delle arterie renali , la presenza , la localizzazione e il grado di eventuali stenosi . sensibilit , specificit , accuratezza , ppv e npv , per la valutazione del grado di stenosi , sono stati calcolati sulla base di una lettura consensuale usando i risultati della dsa come gold standard . 
per quanto riguarda la valutazione quantitativa 73 arterie sono state classificate come normali con la dsa ; 72 di queste sono state classificate come normali anche con langio - tc , in 1 caso la tc ha sovrastimato la stenosi di un grado . 
i valori di sensibilit , specificit , e accuratezza per quanto riguarda il grado di stenosi sono stati rispettivamente del : 100% , 98 , 6% , e 96 , 9% , con un ppv e npv del 97 , 6% e 100% . 
mdct angiography is very accurate and robust , even for the assessment of renal artery stenosis , and has the potential to become a viable substitute , in most cases , for diagnostic catheter - based dsa . key words computed tomography angiography comparative studies renal arteries stenosis hypertension fibromuscular dysplasia rispettivamente del : 100% , 97 , 3% , e 97 , 8% , con un ppv e un npv del 98 , 2% e 97 , 8% . 
langio tcms una metodica accurata e valida nella valutazione delle stenosi delle arterie renali e possiede le potenzialit per diventare un valido sostituto della dsa diagnostica . parole chiave tomografia computerizzata angiografia , studi comparativi arterie renali stenosi ipertensione displasia fibromuscolare introduction introduzione renal artery stenosis is the most common cause of secondary hypertension [ 1 ] , and its detection is an important goal for the physician since the condition is potentially treatable [ 2 ]  . although still considered the standard modality in the evaluation of stenosis of renal arteries , digital subtraction angiography ( dsa ) is not free of risks , and less invasive techniques such as magnetic resonance ( mr ) angiography and computed tomography ( ct ) angiography have been suggested as possible alternatives for assessment of renal artery disease , with very promising results . 
mr angiography , in view of the absence of ionising radiation and the possibility of using nonnephrotoxic contrast media , is widely accepted for detection of diseases of renal arteries and aorta [ 3 ]  . 
single - slice spiral ct , in particular with thin collimations , has proved to provide accurate assessment of renal artery stenoses [ 47 ]  . the limitation of single - slice ct lies in the obvious compromise : thin collimation / long scan time ( with resulting opacification of renal veins and venous structures and possible breathing artefacts affecting the quality of the examination ) , thicker collimation / short scan time ( therefore excellent arterial enhancement but limited spatial resolution and resulting poor depiction of accessory arteries )  . 
the advent of multislice ct ( msct ) has made it possible to acquire large body volumes with high spatial resolution and very short scan times and , therefore , to have excellent depiction of small vessels , including accessory arteries and segmental branches [ 810 ] without opacification of the renal veins . the aim of our study was to determine the utility of msct angiography in identifying and quantifying stenoses of renal arteries . 
 materials and methods during a period of 8 months , 50 patients , who had been studied by dsa following color doppler ultrasonography indication , underwent ct angiography of the abdominal aorta . the study was approved by the ethics committee , and informed consent was given by all patients . 
indications for angiography included vascular disorders , such as stenosis of renal arteries , abdominal aorta aneurysms and steno - occlusive disease of the aortoiliac axis . in all 50 patients , renal arteries were evaluated with both techniques , with dsa and ct angiography being performed within 7 days of each other ( 27 days : mean interval 4.3 days ) and dsa being performed first in all cases . 
for each injection , 20 ml nonionic contrast medium was administered ( iomeprol 350 mgi / ml : iomeron 350 , bracco , milan , italy ) at a rate of 20 ml / s . 
the ct examination was performed after administration of 80 ml contrast material injected at a rate of 4 ml / s with an automatic injector ( envision ct , medrad , indianola , pa , usa )  . 
a high - concentration nonionic iodinated contrast agent was used ( iomeron 400 , bracco , milan , italy ) followed by 20 ml saline injected at a flow rate of 4 ml / s via a second injector connected to the intravenous cannula with a y - shaped adaptor . 
scan parameters were as follows : collimation 4x1 mm , table speed 10 mm / s , gantry rotation time 0.5 s , 120 kvp , 80 mas , ctdiw ( weighted ct dose index ) 7.9 mgy ; reconstruction interval 1 mm , matrix 512x512 with a slice thickness of 1.25 mvoxel size was therefore 0.60.7x0.60.7x1.25 min all patients , scan delay was calculated using the bolus test : 20 ml of contrast material was injected at a flow rate of 4 ml / s followed by f . 
i pazienti ( 39 uomini , 11 donne ) di et compresa tra 43 e 79 anni ( et media 63 anni ) sono stati selezionati per lo studio in base ad una storia di ipertensione secondaria ( pressione arteriosa superiore a 160 / 90 mmhg , in trattamento con farmaci anti - ipertensivi )  . 
tutti i pazienti presentavano una funzionalit renale normale ( creatinemia media di 0 , 97 mg / dl )  . lindicazione ad eseguire lesame angiografico comprendeva disordini vascolari quali : stenosi delle arterie renali , aneurismi dellaorta addominale e patologia steno - occlusiva dellasse aorto iliaco . in tutti i cinquanta pazienti le arterie renali sono state valutate con entrambe le tecniche ; dsa e angio - tc sono state eseguite nellarco di 7 giorni ( 27 giorni : intervallo medio 4 , 3 giorni ) e la dsa stata eseguita come primo esame in tutti i casi . 
stato utilizzato un approccio transfemorale ( 36 punture dellarteria femorale destra e 14 sinistra ) per posizionare un catetere di 5f pigtail a livello delle arterie renali . per ogni iniezione sono stati utilizzati 20 ml di contrasto non ionico ( iomeprol 350 mgi / ml : iomeron 350 , bracco , milano , italia ) con un flusso di 20 ml / s . 
le immagini sono state ottenute inizialmente nella proiezione antero - posteriore ; successivamente sono state eseguite proiezioni oblique ( 15 sinistra e destra anteriore )  . se ritenuta necessaria dal radiologo , stata effettuata anche una cateterizzazione selettiva dellarteria renale . tomografia computerizzata multistrato ( tcms ) langio - tc stata eseguita in tutti i pazienti usando una tc spirale a 4 detettori ( siemens volume zoom , forcheim , germania ) con un tempo di rotazione del gantry di 0 , 5 s . 
this was followed by a series of lowdose scans ( 120 kvp , 10 ma ) at the same level ( coeliac trunk ) every 2 s from 12 s and up to 35 s after bolus administration . 
 image analysis all images were analysed on a dedicated console ( kayak xu 800 , hewlett - packard ) using three - dimensional ( 3d ) reconstruction software ( vitrea 2.6 , vital images inc , plymouth , mn , usa ) , which the radiologists had been using for at least 6 months . 
the 3d image analysis was done directly by the readers without using predefined reconstructions : analysis of each case took approximately 510 malthough not considered necessary for diagnostic purposes , bone segmentation was carried out to provide the vascular surgeons with easy - to - read images . 
the readers also noted the level of the stenosis : 1 , ostium ; 2 , proximal renal artery ; 3 , segmental renal arteries ; 4 , accessory renal artery . 
sensitivity , specificity , accuracy , 80 ml di mezzo di contrasto ad una velocit di flusso di 4 ml / s , con un iniettore automatico ( envision ct , medrad , indianola , pa , usa )  . 
stato utilizzato un mezzo di contrasto iodato non ionico ad alta concentrazione ( iomeron 400 , bracco , milano , italia ) seguito da 20 ml di soluzione salina iniettata con una velocit di flusso di 4 ml / s con un secondo iniettore connesso con una valvola ad y alla cannula endovenosa . del voxel la collimazione usata stata di 4x1 mm , la velocit del tavolo di 10 mm / s , il tempo di rotazione del gantry di 0 , 5 secondi ; i kvp impiegati sono stati 120 , i mas 80 , il ctdiw ( weighted ct dose index ) di 7 , 9 mgy ; lintervallo di ricostruzione utilizzato stato di 1 mm ; le dimensioni della matrice di 512x512 con uno spessore di strato di 1 , 25 mm : le dimensioni 0 , 60 , 7x0 , 60 , 7x1 , 25 min tutti i pazienti il tempo di ritardo per una corretta somministrazione del mezzo di contrasto , stato calcolato con il bolus test che ha previsto una preliminare iniezione di 20 ml di mezzo di contrasto con velocit di flusso di 4 ml / s seguita da 20 ml di soluzione salina . sono state conseguentemente effettuate una serie di scansioni a bassa dose ( 120 kvp , 10 ma ) allo stesso livello ( tronco celiaco ) ogni 2 secondi a partire da 12 e fino a 35 secondi dopo la somministrazione del bolo . risultate quindi sono analisi delle immagini tutte le immagini sono state analizzate su una console dedicata ( kayak xu 800 , hewlett - packard ) utilizzando un software di ricostruzione 3 - d ( vitrea 2.6 , vital images inc , plymouth , mn , usa ) , con il quale i tre radiologi avevano unesperienza di utilizzo di almeno 6 mesi . 
the confidence interval for each sample was also assessed . results on the basis of the qualitative analysis , arterial opacification was considered excellent in 39 patients and good in the remaining 11 . 
the renal veins were opacified in six cases but did not , however , affect correct evaluation of the renal arteries from their origin to their intraparenchymal branches : choice of a correct window and use of different 3d reconstructions permitted correct interpretation and differentiation of arterial and venous vessels , in particular at the level of the distal intraparenchymal branches where superimposition often occurs . in five patients with cardiac insufficiency , image quality was considered good , with limited arterial enhancement . in the 50 patients considered , all readers correctly identified the 99 main renal arteries and all 15 accessory arteries ( six for the right kidney and nine for the left kidney )  . 
perfect interobserver agreement was achieved with regard to location of the stenosis in all cases , with excellent correlation with dsa : 19 stenoses located at the ostium of the main artery , two in the proximal main renal artery and two in both the proximal main renal artery and the segmental branches ( fibromuscular dysplasia ) ; there were no cases of stenosis of accessory renal arteries . 
in two patients , all table 1 digital subtraction angiography ( dsa ) and multidetector - row computed tomography ( mdct ) findings : interobserver agreement ( k values ) a , 1st observer ; b , 2nd observer ; c , third observer tabella 1 concordanza tra gli osservatori tra dsa e tcms . 
gli osservatori hanno descritto anche il punto di stenosi coinvolto : 1 , lostio ; 2 , larteria renale prossimale ; 3 , le arterie renali segmentarie ; 4 , larteria renale accessoria . 
il grado di stenosi stato classificato come : 1 , normale ; 2 , malattia moderata ( 0%50% di stenosi ) ; 3 , stenosi di grado severo ( > 50% ) ; 4 , occlusione . analisi statistica i risultati dellanalisi quantitativa sono stati raccolti in un database ( excel per office 2000 ; microsoft corporation ; redmond , washington ) e analizzati per calcolare la concordanza tra gli osservatori mediante un test statistico di cohen ( spss 8.0 per windows ; spss ; chicago , iii ) : scarsa concordanza ( k < 0 , 01 ) , bassa concordanza ( k = 0 , 010 , 20 ) , discreta concordanza ( k = 0 , 210 , 40 ) , buona concordanza ( k = 0 , 410 , 60 ) , sostanziale concordanza ( k = 0 , 610 , 80 ) , concordanza quasi perfetta ( k = 0 , 811 , 00 ) [ 11 ]  . nove arterie sono state valutate di grado diverso da tutti e tre i radiologi che hanno successivamente raggiunto un consensus . 
sono stati calcolati i valori di sensibilit , specificit , accuratezza , valore predittivo positivo ( ppv ) e valore predittivo negativo ( npv ) nellidentificazione del grado di tutte le stenosi e delle stenosi significative ( > 50% ) , rispetto alla dsa che invece stata considerata come gold standard . 
stata inoltre effettuata la valutazione dellintervallo di confidenza per ogni campione . risultati in base allanalisi qualitativa , lopacizzazione arteriosa stata considerata eccellente in 39 pazienti , buona nei restanti 11 : in nessun caso stata considerata scarsa . 
le vene renali sono risultate opacizzate in 6 casi , senza tuttavia determinare limitazioni per la corretta valutazione delle arterie renali nella loro interezza , dallorigine sino alle diramazioni intraparenchimali : la scelta di una corretta finestra e luso di differenti ricostruzioni 3d hanno , infatti , permesso una corretta interpretazione e differenziazione dei vasi arteriosi da quelli venosi , in particolare a livello delle diramazioni distali intraparenchimali dove la sovrapposizione spesso presente . 
in 5 pazienti con insufficienza cardiaca , la qualit delle immagini stata considerata buona con un limitato enhancement arterioso . nei 50 pazienti studiati , tutti i lettori hanno correttamente identificato le 99 arterie renali principali e tutte le 15 arterie accessorie ( 6 per il rene di destra e 9 per il rene di sinistra )  . 
per tutti gli osservatori , laccordo per le due tecniche stato quasi perfetto ( k = 0 , 811 ) , con un valore di k compreso tra 0 , 82 e 0 , 95 . 
mean time for analysis of dsa images was approximately 5 ( range 46 ) min with readings performed on hard copy . discussion stenosis of renal arteries is one of the most common causes fig . 
angio - tcms con ricostruzioni mip ( a ) e dsa ( b ) : presenza di una stenosi severa bilaterale ( frecce ) delle arterie renali dovuta ad una placca dellostio parzialmente calcifica . lettori nella localizzazione della stenosi stato ottenuto in tutti i casi , con unottima correlazione con la dsa : 19 stenosi localizzate allostio dellarteria principale , due nella porzione prossimale dellarteria renale principale e due sia nella porzione prossimale sia nei rami segmentari ( displasia fibro - muscolare ) ; non stato trovato nessun caso di stenosi a livello delle arterie renali accessorie . 
i valori di sensibilit , specificit , e accuratezza rispetto al grado della stenosi sulla base della lettura consensuale sono stati del 100% ( ic 95% : 91 , 4100 ) , of secondary hypertension . 
detection and , above all , correct quantification of the degree of renal artery stenosis is fundamental for planning appropriate treatment : antihypertensive drugs , angioplasty or , more rarely , surgical treatment [ 12 ]  . noninvasive diagnostic techniques such as ultrasonography , fig . 
multidetector - row computed tomography ( mdct ) axial curved multiplanar reconstruction and coronal thin maximum intensity projection ( mip ) ( a , b ) show a < 50% stenosis of the proximal left renal artery due to a calcified plaque ( arrow )  . 
le ricostruzioni delle curve assiali multiplanari e coronali mip a spessore sottile evidenziano una stenosi inferiore al 50% della porzione prossimale dellarteria renale sinistra determinata da una placca calcifica ( freccia ) ( c )  . 
nellidentificazione e nella valutazione del grado delle stenosi severe ( restringimento del lume del 50100% ) la sensibilit , la specificit e laccuratezza sono state del 100% ( ic 95% : 86 , 3100 ) , 97 , 3% ( ic 95% : 92 , 199 , 7 ) e 97 , 8% ( ic 95% : 95 , 8100 ) , rispettivamente , con un valore predittivo positivo e negativo del 98 , 2% ( ic 95% : 8199 , 9 ) e 97 , 8% ( ic 95% : 95 , 9100 )  . le arterie renali accessorie sono state identificate correttamente dai tre lettori sia con la dsa che con langio - tc . il tempo medio per la valutazione delle immagini di tcms su stazioni di lavoro dedicate stato di 12 minuti circa ( compreso tra 10 e 15 minuti ) per i tre lettori . 
il tempo medio di lettura per lanalisi delle immagini di dsa stato di circa 5 minuti ( compreso tra 4 e 6 minuti ) con lanalisi eseguita su pellicola . discussione la stenosi delle arterie renali rappresenta la pi comune causa di ipertensione secondaria . 
la sua identificazione , ed in particolare una corretta quantificazione del grado di stenosi delle arterie renali risulta di fondamentale importanza per una corretta pianificazione del trattamento : terapia farmacologia antiipertensiva , angioplastica o , raramente , trattamento chirurgico [ 12 ]  . 
tecniche diagnostiche non invasive , come lecografia , langio - rm e langio - tc , si sono dimostrate accurate nella valutazione dellipertensione nefrovascolare con lo scopo di ridurre il numero delle angiografie diagnostiche [ 3 , 5 , 7 , 1317 ]  . lecografia di solito utilizzata come tecnica di prima istanza per la valutazione della stenosi delle arterie renali [ 13 ]  . 
il limite principale della ecografia rappresentato dalla difficile finestra acustica in un ampio numero di pazienti e da una relativa bassa sensibilit e specificit nella visualizzazione delle arterie renali accessorie [ 15 ]  . langio - rm una tecnica valida che offre risultati eccellenti nella valutazione delle arterie renali [ 3 , 14 , 15 ] ; il suo limite principale tuttavia limpossibilit di visualizzare i rami segmentari delle arterie renali principali e in alcuni casi le arterie renali accessorie che possono essere responsabili , anche se in un limitato numero di pazienti , di ipertensione nefrovascolare [ 16 ]  . la tc spirale singolo strato stata proposta da molti autori per la valutazione delle arterie renali con risultati promettenti , ma con molte limitazioni ; infatti il rapporto tra il volume da analizzare e la risoluzione spaziale lungo lasse z non permette , in molti casi , la visualizzazione dellorigine delle arterie renali principali e delle arterie accessorie . 
multidetector - row computed tomography ( mdct ) angiography ( a ) demonstrates irregular appearance of the ventral branch of the right renal artery with a string - of - beads morphology typical of fibromuscular dysplasia . 
il quadro tc confermato dalla dsa ( b )  . mr angiography and ct angiography have proved to be accurate in assessment of secondary hypertension and provide valuable alternatives to diagnostic angiography [ 3 , 5 , 7 , 1317 ]  . ultrasonography is normally used as a first - line approach in evaluation of renal artery stenoses [ 13 ]  . 
its main limitations are a difficult acoustic window in a large number of patients and relatively low sensitivity and specificity in depicting accessory renal arteries [ 15 ]  . mr angiography is a valuable technique that offers excellent results in assessment of renal arteries [ 3 , 14 , 15 ] ; its main limit is an inability to depict segmental branches of main renal arteries and in some cases accessory renal arteries , which may be responsible of secondary hypertension in a small number of patients [ 16 ]  . 
 single - slice spiral ct has often been proposed for evaluation of renal arteries with promising results , but it also suffers many limitations since the ratio between the volume to be analysed and spatial resolution in the z axis often does not allow visualisation of the origin of main renal arteries and accessory arteries . 
in addition , long scan times result in enhancement of the renal veins and possible breathing artefacts in patients with limited breath - hold capabilities , with resulting poor quality of 3d reconstructions , which have become integral to diagnosis for both clinicians and radiologists . the recent introduction of mdct has considerably shortened acquisition times of increasingly large volumes , with excellent longitudinal resolution and near - isotropic voxels , which allow for high - quality multiplanar and volume rendered reconstructions , reduced contrast material , excellent arterial enhancement without venous - system interference and fewer motion artefacts . 
in view of these advantages , this study toria , con una conseguente relativa limitata qualit delle ricostruzioni tridimensionali , che sono diventate parte integrante della diagnostica sia per i radiologi che per i clinici . la recente introduzione della tcms ha incrementato notevolmente la velocit di acquisizione di volumi sempre pi ampi , con una eccellente risoluzione longitudinale e con voxels pressoch isotropici , che permettono ricostruzioni multiplanari e volume rendering di alta qualit , una riduzione nella somministrazione di mezzo di contrasto , una ottima opacizzazione arteriosa , scevra da sovrapposizione del sistema venoso ed una riduzione degli artefatti da movimento . in virt dei suddetti vantaggi della tcms lo scopo di questo studio stato di verificare la capacit diagnostica della angio - tcms nella valutazione delle arterie renali . nella maggior parte dei pazienti del nostro studio , la qualit delle immagini stata considerata eccellente e con un ottimo enhancement anche nei pazienti con associate altre patologie cardio - vascolari , senza sovrapposizioni da parte del sistema venoso . 
nei pochi casi di sovrapposizione stata comunque possibile una ottima visualizzazione del sistema arterioso grazie alle ricostruzioni 3d . nellanalisi quantitativa , laccordo tra gli osservatori stato quasi perfetto sia per langio - tc che per la dsa ; nella valutazione del grado di stenosi , langio - tc ha mostrato unelevata specificit ( 97 , 8% ) , anche quando ristretta a stenosi severe ( > 50% )  . un caso considerato come stenosi di primo grado con dsa stato considerato di secondo grado con la tcms : questo errore diagnostico non ha modificato il programma terapeutico n i valori di sensibilit o specificit per le stenosi di grado severo ( > 50% )  . 
al contrario , in altri due pazienti , langio - tcms ha sovrastimato la stenosi di un grado aimed at evaluating the diagnostic capabilities of mdct angiography in assessment of renal arteries . in the majority of the patients examined , image quality was considered excellent , with optimal enhancement , even in patients with other associated cardiovascular disorders , without overlapping venous enhancement . 
in quantitative analysis , interobserver agreement was almost perfect for both mdct and dsa ; in evaluating degree of stenosis , mdct angiography had high specificity ( 97.8% ) , even when restricted to severe stenoses ( > 50% )  . 
one case assessed as a grade 1 stenosis with dsa was considered a grade 2 stenosis with mdct : this diagnostic error did not change treatment or sensitivity or specificity for severe stenoses ( > 50% )  . 
moreover , the use of protocols with thin collimations , which are routinely adopted with the latest generations of mdct ( 16 , 64 slices ) , may further improve spatial resolution and allow more accurate distinction between wall calcifications and residual lumen in severe stenosis . 
in addition , in order to limit diagnostic errors in the case of wall calcification , use of a soft window will allow differentiation between residual lumen and calcific atherosclerotic plaque . 
on the other hand , visualisation of vessel walls is a major advantage of ct not only over dsa but also over other noninvasive modalities , as it allows evaluation both of plaque components and , in particular , wall calcification and also of the residual lumen . there are two important limitations of mdct angiography : the use of ionising radiation , and iodinate contrast material . 
as regards nonionic iodinated contrast material , it should be noted that it is less nephrotoxic compared with ionic contrast agents and that it is well tolerated even by high - risk patients [ 18 ]  . 
it has recently been demonstrated that nonionic iodinated contrast agents can be used , even in patients with impaired renal function , without significant risk of nephrotoxic effects [ 19 ]  . 
the advent of new and faster ct scanners ( 16 , 64 slices ) means that even less contrast material is required to obtain good assessment of renal arteries , and use of saline solution injected immediately after the contrast bolus allows further reduction in volume to be injected and greater bolus uniformity . 
as regards the use of ionising radiation , we need to consider that the dose used in ct is significantly lower than that used for dsa and that the protocol used in this study was optimised with respect to previous studies , maintaining , however , a high diagnostic quality [ 20 ]  . 
recent studies have demonstrated the possibility of using low - dose protocols for other body districts , with excellent results in terms of image quality and diagnostic capabilities [ 21 , 22 ]  . one limitation of our study is the small number of patients f . 
inoltre , luso di protocolli con collimazioni sottili , che sono impiegati routinariamente con le ultime generazioni di tcms ( 16 , 64 strati ) possono ulteriormente migliorare la risoluzione spaziale e permettere una pi accurata distinzione tra calcificazioni parietali e lume residuo nel caso di stenosi severe ; inoltre , per limitare errori diagnostici nel caso di calcificazioni parietali , lutilizzo di una finestra morbida consente la differenziazione tra lume residuo e placca aterosclerotica calcifica . 
daltra parte , la visualizzazione delle pareti dei vasi un importante vantaggio della tc , nei confronti non solo della dsa , ma anche delle altre metodiche non invasive , in virt della capacit di valutare sia i costituenti della placca ed in particolare le apposizioni calcifiche parietali , sia il lume residuo . ci sono due importanti limitazioni dellangio - tc : luso di radiazioni ionizzanti e di mezzo di contrasto iodato . 
per quanto riguarda il mezzo di contrasto iodato non ionico bisogna ricordare che la sua nefrotossicit limitata rispetto ai mezzi di contrasto ionici , e che ben tollerato anche in pazienti ad alto rischio [ 18 ]  . nella nostra popolazione di studio non stato rilevato nessun incremento dei livelli sierici di creatinina dopo lesecuzione dellesame . 
 stato recentemente dimostrato come anche nei pazienti con una funzionalit renale compromessa i mezzi di contrasto iodati non ionici possono essere somministrati senza significativi rischi di nefrotossicit [ 19 ]  . 
inoltre la quantit di mezzo di contrasto utilizzata con la tcms inferiore rispetto a quella utilizzata con la tc spirale a singolo strato , grazie allincremento di velocit nellacquisizione delle immagini . 
lintroduzione di nuove e pi veloci tc ( 16 , 64 strati ) permette inoltre di ridurre ancor di pi la quantit di mezzo di contrasto necessario per ottenere una buona valutazione delle arterie renali . inoltre , luso di una soluzione salina iniettata subito dopo il bolo di mezzo di contrasto permette una ulteriore riduzione del volume da iniettare ed una maggior uniformit del bolo . 
per quanto riguarda luso di radiazioni ionizzanti , bisogna tener presente che la dose utilizzata in tc significativamente inferiore rispetto a quella utilizzata per la dsa e che il protocollo utilizzato in questo studio stato ottimizzato rispetto agli studi precedentemente pubblicati mantenendo tuttavia una elevata qualit diagnostica [ 20 ]  . 
sono stati ultimamente pubblicati degli studi che hanno dimostrato la possibilit di utilizzare protocolli a bassa dose di radiazioni ionizzanti per altre parti del corpo con risultati eccellenti in termini di qualit di immagini e di capacit diagnostiche [ 21 , 22 ]  . una limitazione del nostro studio rappresentata dal basso numero di pazienti con stenosi significative ( 50%100% ) e leterogeneit della nostra popolazione che includeva pazienti con patologie diverse : ipertensione nefrovascolare , malattia occlusiva aorto - iliaca , patologia dilatativa . 
a further major advantage of mdct is the possibility of visualising the entire aortoiliac axis , including the renal arteries , in a single examination without loss of enhancement or superimposition of the venous system using thin collimations and high spatial resolution . 
in conclusion , this study demonstrated that mdct angiography is very accurate and reproducible in evaluation of renal arteries and stenosis and has the potential to replace digital angiography . tato dalla possibilit di visualizzare in un unico esame lintero asse aorto - iliaco , comprese le arterie renali senza perdita di enhancement o senza sovrapposizione del sistema venoso , usando collimazioni sottili ed elevata risoluzione spaziale . difficile determinare quale sar il ruolo dellangiotcms rispetto allangio - rm . 
villari1 1sezione di radiodiagnostica , dipartimento di fisiopatologia clinica , universit degli studi , azienda ospedaliero - universitaria di careggi , viale morgagni 85 , i - 50134 firenze , italy 2sezione di radiodiagnostica , universit degli studi , azienda ospedaliero - universitaria senese , policlinico le scotte , siena , italy 3unit clinica operativa di radiologia , universit degli studi , ospedale di cattinara , trieste , italy correspondence to : s . 
 + 39 - 055 - 4377673 , fax : + 39 - 055 - 431970 , e - mail : s.colagrande@dfc.unifi.it received : 23 june 2005 / accepted : 23 july 2005 / published online : 11 april 2006 abstract diffusion - weighted ( dw ) imaging has for a number of years been a diagnostic tool in the field of neuroradiology , yet only since the end of the 1990s , with the introduction of echoplanar imaging ( epi ) and the use of sequences capable of performing diffusion studies during a single breath hold , has it found diagnostic applications at the level of the abdomen . 
the inherent sensitivity to motion and the magnetic susceptibility of dw sequences nonetheless still create problems in the study of the abdomen due to artefacts caused by the heartbeat and intestinal peristalsis , as well as the presence of various parenchymal - gas interfaces . 
with regard to focal liver lesions , a review of the literature reveals that dw imaging is able to differentiate lesions with high water content ( cysts and angiomas ) from solid lesions . 
with regard to the latter , although there are differences between benign forms [ focal nodular hyperplasia ( fnh ) , adenoma ] and malignant forms [ metastasis , hepatocellular carcinoma ( hcc ) ] in their apparent diffusion coefficient ( adc ) in the average values for histological type , there is a significant overlap in values when lesions are assessed individually , with the consequent problem of their correct identification . 
one promising aspect is the possibility of quantifying the degree of fibrosis in patients with chronic liver disease and cirrhosis given that the deposit of collagen fibres restricts the motion of water molecules and therefore reduces adc values . 
here it is possible to differentiate mucin - producing tumours of the pancreas from pseudocystic forms on the basis of adc values even though the limited spatial resolution of dw imaging does not enable the identification of small lesions . 
in addition , given that renal parenchyma has significantly variable adc values on the basis of the anatomic section and physiological conditions , the possibility of assessing functional alterations is currently being studied . 
with regard to musculoskeletal applications , the absence of riassunto in ambito neuroradiologico , limaging pesato in diffusione pu essere considerato una realt diagnostica da vari anni , mentre a livello addominale , soltanto verso la fine degli anni 90 , quindi con lintroduzione delle tecniche eco - planari ( epi ) , si sono rese disponibili sequenze tali da consentire studi di diffusione a respiro sospeso . 
lintrinseca sensibilit al movimento e alla suscettivit magnetica delle sequenze diffusione - pesate ( diffusion - weighted , dw ) rende tuttavia problematico , ancora oggi , lo studio delladdome superiore , per gli artefatti secondari allattivit cardiaca e alla peristalsi intestinale , nonch per la presenza di varie interfacce parenchimi - gas . 
per quanto riguarda la patologia epatica focale , una revisione della letteratura indica che limaging dw permette di differenziare le lesioni ad elevato contenuto acquoso ( cisti ed angiomi ) da quelle solide . 
fra queste ultime , pur esistendo differenze di coefficiente apparente di diffusione ( adc ) , nei valori medi per tipo istologico , fra forme benigne ( fnh , adenoma ) e maligne ( metastasi , hcc ) , si rileva anche unampia sovrapposizione di valori fra le lesioni valutate singolarmente , con conseguenti problemi in termini di caratterizzazione . 
interessanti le possibilit di quantificare il grado di fibrosi nei pazienti con epatopatia cronica e cirrosi , dal momento che la deposizione di fibre collagene , restringendo i movimenti molecolari dellacqua , comporta una riduzione dei valori di adc ; anche in questo settore , gli studi sono tuttavia da considerare preliminari , lontani da una reale applicazione in ambito clinico . 
nel retroperitoneo , distretto meno interessato da artefatti da movimento , altrettanto degna dattenzione risulta la possibilit di differenziare fra neoplasie pancreatiche mucinose e forme pseudocistiche , in funzione dei valori di adc , anche se la limitata risoluzione spaziale propria delle immagini dw , non permette il rilievo di lesioni di piccole dimensioni . 
inoltre , dal momento che il parenchima renale presenta valori di adc molto variabili in funzione della porzione anatomica e delle condizioni fisiologiche , allo studio la possibilit di valutare le alterazioni della funzione ; ad oggi stata riscontrata una buona correlazione tra i valori di adc e lentit del filtrato glomerulare . 
greater agreement has been found regarding sensitivity of the technique in assessing response of these tumours to chemotherapy : tumour necrosis is thought to increase adc whereas the persistence of vital neoplastic tissue tends to lower it . one of the most promising applications of dw imaging is without doubt the assessment of vertebral collapse where a high adc has been shown to be associated with an osteoporotic cause and a low adc with a neoplastic cause . 
in particular , no significant difference in adc is noted between normal hypercellular bone marrow and hypercellular bone marrow secondary to lymphomatous infiltration whereas this difference is significant between hypocellular , normocellular and haematopoietic hypercellular bone marrow . 
with regard to the study of joints , the limited structure dimensions , particularly cartilage , creates technical difficulties related to spatial resolution and an adequate signal - to - noise ratio , problems that can only be solved by further technological developments . 
si cos tentato di utilizzare limaging dw nella caratterizzazione delle neoplasie dei tessuti molli , anche se con risultati per il momento controversi . maggiore accordo vi invece sulla sensibilit di tale tecnica nella valutazione della risposta di questi tumori alla chemioterapia : la necrosi tissutale determinerebbe , infatti , un aumento delladc , diminuito viceversa al permanere di tessuto neoplastico vitale . una delle applicazioni pi promettenti dellimaging dw senza dubbio la valutazione dei crolli vertebrali , nei quali si dimostrato un adc elevato ove la causa sia porotica e ridotto ove neoplastica ; anche in questo caso si riscontra tuttavia un discreto overlap fra gli adc delle due popolazioni . 
in particolare non si rileva significativa differenza di adc tra il midollo ipercellulare normale e quello secondario ad infiltrazione linfomatosa , mentre tale differenza significativa tra il midollo ipocellulare , quello normocellulare e lipercellulare emopoietico . 
in ambito articolare , le ridotte dimensioni delle strutture di interesse e in particolare delle cartilagini , determinano difficolt tecniche inerenti la risoluzione spaziale ed un adeguato rapporto s / r , problemi che impongono ulteriori sviluppi della tecnologia . 
the main field of application of the earliest studies in molecular diffusion was neuroradiological [ 1 ] , where diffusion - weighted ( dw ) sequences play an important part in the diagnosis of cerebral ischaemia in the hyperacute phase ( 06 h )  . 
at the same time , other applications were developed regarding both the characterization of brain tumours ( with the possibility of differentiating the cystic / oedematous lesions from solid forms ) and more recently the assessment of demyelinating diseases . from applications limited to neuroradiology , steps have progressively been taken in other body regions , such as the abdomen [ 24 ] and musculoskeletal system [ 57 ] both in adults and paediatric patients . 
this interest is justified by the potential of dw imaging to qualitatively and quantitatively assess molecular motion on the basis of microstructural organization of the region being studied . i recenti avanzamenti tecnologici nel settore delle apparecchiature a risonanza magnetica ( rm ) hanno portato allo sviluppo di macchine commerciali con prestazioni sempre pi elevate , sia per quanto riguarda lintensit / omogeneit di campo magnetico , che le caratteristiche dei gradienti , rendendo possibili studi con tecniche avanzate , come la valutazione del parametro diffusione ( d )  . 
i primi studi sulla diffusione molecolare hanno avuto come principale campo dapplicazione lambito neuroradiologico [ 1 ] , ove le sequenze diffusione - pesate ( diffusion - weighted , dw ) rappresentano un momento importante nella diagnostica dellischemia cerebrale in fase iperacuta ( 06 ore )  . 
parallelamente , sono state sviluppate altre applicazioni riguardanti sia la caratterizzazione delle lesioni neoplastiche encefaliche ( con la possibilit di differenziare le forme cistiche / edematose dalle solide ) sia , pi di recente , la valutazione delle malattie demielinizzanti . da tali applicazioni circoscritte allambito neuroradiologico si progressivamente passati allo studio di altri distretti corporei , come laddome [ 24 ] e lapparato muscolo scheletrico [ 57 ] sia nelladulto che nel piccolo paziente . tale interesse risulta giustificato dalle potenzialit dellimas . 
the following summary is limited to the main technical problems related to the region being studied . in the abdomen the study of the diffusion parameter became possible with the development of ultrafast sequences . this enabled the entire acquisition to be made in a single breath hold , thus eliminating respiratory artefacts and reducing those resulting from vascular pulsation and intestinal peristalsis , which lower the signal - to - noise ratio ( snr ) and create difficulties for quantitative analysis . those ultrafast sequences were echoplanar imaging ( epi ) sequences , which , thanks to acquisition of around 3060 ms per image , reduce artefacts due to macroscopic physiological motion . the high acquisition speed , however , does have its limits , especially with regard to spatial resolution . 
the abdominal organs and their relative focal lesions do not possess a characteristic structural organization like the fibres of white matter . the sequences adopted for the study of the abdomen , therefore , are very similar to those used for the study of ischaemic disease in the brain . technically speaking , based on our experience and in line with other studies on the subject [ 24 ] , the mr examination should be performed with a high - field superconductive magnet ( 1.5 t ) equipped with 2330 mt / m intensity gradients , a slew rate of 150 mt / m per second and phased - array surface coils . 
all sequences are acquired in breath hold to reduce respiratory artefacts . after a preliminary conventional study with t1and t2weighted sequences , dw images are acquired using a variation of the spin - echo sequence ( stejskal and tanner sequence ) performed with the single - shot echoplanar technique ( se - epi - ssh )  . this entails a classic spin - echo sequence characterized by two successive radiofrequency impulses at 90 and 180 , to which two additional gradients known as motion - probing gradients ( mpgs ) equal in breadth and duration but opposite in direction are added immediately before and after the 180 impulse . the amount of weighting in diffusion of the sequence is ging dw nella valutazione quali - quantitativa del movimento molecolare in funzione dellorganizzazione microstrutturale del distretto esaminato . scopo di questa rassegna quello di presentare le principali esperienze maturate con lacquisizione in diffusione in ambito addominale e muscolo - scheletrico , compresi alcuni cenni sulla patologia pediatrica . note di tecnica per quanto riguarda i fondamenti dellimaging dw dal punto di vista fisico , tecnico e semeiotico si rimanda ad una precedente nota [ 8 ]  . 
di seguito ci limitiamo a ricordare i principali problemi di natura tecnica , in funzione del distretto da esaminare . a livello addominale lo studio del parametro diffusione stato permesso dallo sviluppo di sequenze ultraveloci che consentono lintera acquisizione in una singola apnea , in modo da eliminare gli artefatti respiratori e ridurre quelli secondari a pulsatilit vascolare e peristalsi intestinale , che diminuiscono il rapporto segnale / rumore ( s / r ) e rendono problematica lanalisi quantitativa . 
tali sequenze ultraveloci sono rappresentate dalle sequenze eco - planari ( echo - planar imaging , epi ) che , grazie a tempi di acquisizione dellordine di 3060 ms per immagine , riducono gli artefatti dovuti ai movimenti fisiologici macroscopici . 
pertanto le sequenze adottate in ambito addominale possono essere assimilate a quelle che si utilizzano per lo studio delle malattie ischemiche encefaliche . dal punto di vista tecnico , sulla base della nostra esperienza ed in accordo con quanto riportato dagli autori che si sono occupati dellargomento [ 24 ] , necessario eseguire lesame rm con un magnete superconduttivo ad alto campo ( 1 , 5 t ) , dotato di gradienti di intensit pari a 2330 mt / m e slew rate di 150 mt / m / s , e con bobine di superficie del tipo phased - array . 
tutte le sequenze sono acquisite a respiro sospeso ( breath - hold , bh ) per ridurre gli artefatti secondari ai movimenti respiratori . dopo un preliminare studio tradizionale tramite sequenze t1 e t2 - pesate , vengono acquisite le immagini dw , avvalendosi di una variante della sequenza spin eco ( sequenza di stejskal e tanner ) assunta con tecnica eco - planare del tipo single - shot ( se - epi - ssh ) : sullo schema di una classica spin eco , caratterizzata da due impulsi di radiofrequenza in sucs . 
a field lines in the air ( 1 ) and in the context of the object immersed in the magnetic field ( 2 ) with the consequent deviation of the magnetic field itself . 
b single - shot echoplanar ( se - epi ) image with b = 0 of a cylindrical phantom containing water ; three smaller cylinders containing ultrasound gel are placed around it . 
c note deformation of the cylinder in the se - epi image weighted in diffusion ( b = 800 s / mm2 ) without the presence of the cylinders , which already shows the sequence sensitivity to magnetic susceptibility . 
c notare la deformazione del cilindro nella immagine se - epi pesata in diffusione ( b = 800 s / mm2 ) senza la presenza dei cilindri , che gi evidenzia la sensibilit della sequenza alla suscettibilit magnetica . 
d dopo riposizionamento dei cilindri tale reperto pi accentuato a livello dellinterfaccia tra cilindro di acqua e cilindro di gel ( freccia nera ) come deformazione focale del profilo del fantoccio nella direzione della codifica di fase . defined by the factor b [ expressed in seconds per millimetre squared ( s / mm2 ) ] , which summarizes gradient characteristics according to the formula b = 2g22 ( - / 3 ) where is the gyromagnetic constant , g and are the breadth and the duration of the diffusion gradients , respectively , and is the time interval between their application [ 8 ]  . 
diffusion produces a reduction in cessione a 90 e 180 , vengono applicati , immediatamente prima e dopo limpulso a 180 , due gradienti aggiuntivi uguali per ampiezza e durata , ma contrari per verso , detti gradienti di diffusione ( motion probing gradients , mpgs )  . lentit della pesatura in diffusione della sequenza definita dal fattore b ( espresso in s / mm2 ) , che riassume le caratteristiche dei gradienti , secondo la formula : b = 2g22 ( - / 3 ) dove la costante giromagnetica , g e sono rispettivas . 
adc is usually measured by linear regression analysis with the following formula : adc = ln ( s0 / s ) / b the other parameters of the sequence generally used are : tr 1 , 8005 , 000 ms , 60 < te < 120 , matrix 128x128256 , number of measurements 1 , indicative band width 2 , 080 hz / pixel , number of sections acquired 1220 , slice thickness 58 mm , and field of view ( fov ) variable based on the build of the patient . chemical - shift artefacts are reduced by systematically using fat presaturation . 
in addition , where possible the use of parallel imaging can be an advantage , a technique capable of reducing acquisition time or increasing the matrix and therefore the spatial resolution without altering the acquisition time and without significant loss in image quality ( sensitivity encoding , sense ) [ 9 ]  . in the postprocessing phase , the various dw images are used to obtain the respective adc maps on which quantitative analysis of the signal is performed by positioning a region of interest ( roi ) on the structure being studied . as we shall see , the choice of the b value and therefore the degree of weighting in diffusion is a problem that cannot be overlooked and for which no solution has yet been found . various studies using different b values have yielded different results of these , we would like to draw particular attention to the significant differences reported in the literature between adc values of the normal and cirrhotic liver as well as focal hepatic lesions . 
the current tendency is to use a superconductive magnet with a field intensity of 1.5 t , gradients of 30 mt / m , slew rate of 150 mt / m per second and a 4channel phased - array coil [ 10 ]  . 
various sequences have been proposed to reduce sensitivity to magnetic susceptibility typical of epi and improve spatial resolution in acquisitions that require an fov smaller than those used for the abdomen . the first attempts were made with conventional spin - echo sequences , which have a high snr and very low magnetic susceptibility , but acquisition times were calculated in minutes , with a consequent increase in sensitivity to motion artefacts [ 1 ]  . mente lampiezza e la durata dei gradienti di diffusione e lintervallo di tempo intercorrente fra la loro applicazione [ 5 ]  . 
la diffusione produce una riduzione dellintensit di segnale in ogni pixel data da : s = s0e - bxadc dove s rappresenta lintensit di segnale nel pixel dopo lapplicazione dei gradienti di diffusione per un dato valore di b , s0 lintensit di segnale con b pari a 0 , mentre adc il coefficiente di diffusione apparente ( espresso in mm2 / s )  . ladc viene di solito misurato per mezzo dellanalisi della regressione lineare calcolata sulla base della seguente formula : adc = ln ( s0 / s ) / b gli altri parametri della sequenza generalmente utilizzati sono i seguenti : tr 18005000 ms , 60 < te < 120 ms , matrice 128x128256 , numero di misure 1 , ampiezza di banda indicativa 2080 hz / pixel , numero di sezioni acquisite 1220 , spessore di strato 58 mm , fov variabile in funzione della corporatura del paziente . 
inoltre , ove disponibile , pu essere vantaggioso utilizzare una tecnica di acquisizione parallela delle immagini ( parallel imaging ) che consente di ridurre il tempo di acquisizione o , alternativamente , di aumentare la matrice e quindi la risoluzione spaziale a parit di tempo di acquisizione , senza significativo degrado della qualit delle immagini ( sensitivity encoding , sense ) [ 9 ]  . 
in fase di postprocessing , dalle varie acquisizioni diversamente pesate in diffusione vengono ottenute le rispettive mappe adc sulle quali possibile eseguire lanalisi quantitativa del segnale posizionando una regione di interesse ( region of interest , roi ) sulla struttura in esame . come vedremo , la scelta del valore di b e quindi il grado di pesatura in diffusione un problema non trascurabile e non ancora risolto ; se osserviamo la letteratura riguardante largomento , infatti , vari autori utilizzando valori diversi di b , sono giunti a risultati non sovrapponibili . 
fra tutti vogliamo ricordare le notevoli differenze riportate in letteratura fra i valori di adc del fegato normale e cirrotico , nonch delle lesioni focali epatiche . in ambito retroperitoneale i problemi legati ai movimenti respiratori , cardiaci e peristaltici si rendono meno evidenti . attualmente la tendenza quella di utilizzare un magnete superconduttivo con intensit di campo di 1 , 5 t , gradienti pari a 30 mt / m , velocit di raggiungimento del picco di 150 mt / m / s , con limpiego di una bobina phased - array a 4 canali [ 10 ]  . 
le immagini di diffusione vengono acquisite utilizzando una sequenza se - epi - ssh ( tr = 2883 , te = 61 , flip angle = 90 , fov variabile , matrice = 128x256 ) , con valori di b pari a 0 e a 500 s / mm2 ( comunque possibile acquisire con pi alti valori di b ) ; viene impiegata inoltre una tecnica di acquisizione parallela delle immagini . table 1 summary of results of most cited studies . 
numero di pazienti studiati , valori di fattore b utilizzati ( in ordine crescente ) e valori di adc delle principali lesioni focali epatiche riportati dagli autori citati nel testo . 
other sequences commonly reported in the literature , especially for the study of cartilage , are based on steady - state free precession ( ssfp )  . these sequences have relatively short acquisition times although they are not directly comparable with epi - dw sequences , as well as a high snr . 
however , performing quantitative assessments of diffusion is not a simple matter : the b value in this type of sequence , required to calculate adc , is not clearly definable without knowing the t1 and t2 relaxation times [ 12 ]  . most studies in the literature , however , reveal that currently , the most commonly used sequences are se - epi - ssh [ 13 ] , with a configuration not unlike that presented above with regard to the study of the abdomen and retroperitoneu the use of dedicated coils , a slice thickness of 46 mm , fov relative to the segment being studied and a matrix of 128x128256 are obviously required . the most important parameter is still the b value , which , as noted earlier , determines the real weighting in diffusion of the sequence . 
in letteratura sono state proposte sequenze di vario tipo al fine di ridurre la sensibilit alla suscettibilit magnetica , propria della tecnica epi , e di migliorare la risoluzione spaziale in acquisizioni che necessitano di fov inferiori a quelli usati per laddome . i primi tentativi sono stati effettuati con sequenze spin eco convenzionali , che presentano elevato rapporto s / r e scarsa suscettibilit magnetica , ma tempi di acquisizione calcolabili in minuti , con incremento conseguente della sensibilit agli artefatti da movimento [ 1 ]  . 
per ovviare a tale inconveniente sono state pertanto usate da alcuni autori sequenze single - shot multi - eco ( rapid acquisition with relaxation enhancement , rare , o half - fourier acquired single - shot turbo spin - eco , haste ) , anche se i dati in proposito sono ancora scarsi [ 11 ]  . 
altre sequenze frequentemente riportate in letteratura , soprattutto nella valutazione delle cartilagini , sono quelle basate sulla steady - state free precession ( ssfp ) : esse presentano tempi di acquisizione relativamente brevi , anche se non sovrapponibili a quelli delle epi - dw , ed elevato rapporto s / r . 
non tuttavia semplice eseguire valutazioni quantitative della diffusione ; in questo tipo di sequenze , infatti , il valore del fattore b , necessario per il computo delladc , non chiaramente definibile senza conoscere i tempi di rilassamento t1 e t2 [ 12 ]  . attualmente , come si evince dalla maggior parte degli s . 
one of the promising applications in the study of the liver involves characterization of focal hepatic lesions following the demonstration of differences in mean adc values measured in hepatocellular carcinoma ( hcc ) , metastases and hepatic angiomas studi presenti in letteratura , vengono utilizzate sequenze seepi - ssh [ 13 ] , configurate in modo non dissimile da quanto gi riportato in precedenza a proposito del distretto addominale e retroperitoneale . ovviamente necessario lutilizzo di bobine dedicate , spessore di strato compreso tra 4 e 6 mm , fov relativo al segmento in esame e matrice 128x128256 . 
b , c in diffusion - weighted ( dw ) images with b = 0 and 1 , 000 s / mm2 , the signal remains high even with elevated weighting in diffusion . 
 [ 15 ] studied 74 hepatic lesions ( 48 hcc , 15 metastases and 11 angiomas ) ( table 1 ) using the sequences and the protocol mentioned above [ 15 ] , with some minor variations . since high b values ( > 400 s / mm2 ) reduce the signal intensity of the liver with resulting poor image quality , the authors chose to use b values equal to 1.6 , 16 and 55 s / mm2 , thus obtaining three sets of differently weighted dw images , even though with such low b values the image proves to be more influenced by phenomena of perfusion and t2 relaxation time ( shine through ) than by diffusion . 
all types of lesions ( hcc , metastases and angiomas ) showed mean adc values higher than those of normal and cirrhotic liver except for some cases of metastases . the mean adc value of angiomas was the highest , followed by that of hcc and metastases ( table 1 )  . 
ad esempio , la milza ha basso adc , che sta ad indicare ridotta mobilit delle molecole di acqua , mentre la corticale renale presenta valori di adc estremamente alti . lutilit clinica delle immagini dw a livello addominale non ancora chiara , sebbene esistano potenziali applicazioni nello studio del fegato , del pancreas e dei reni . 
hanno studiato 74 lesioni epatiche ( 48 hcc , 15 metastasi ed 11 angiomi ) ( tabella 1 ) avvalendosi , a parte piccole variazioni , delle sequenze e del protocollo ricordato nel paragrafo precedente [ 15 ]  . 
a turbo spin echo ( tse ) t2 - weighted image ( te = 80 ms ) showing degenerative nodule of about 3 cm in diameter in the iii hepatic segment , which has induced thrombosis in the portal branch of the left lobe . 
b , c single - shot echoplanar diffusion - weighted ( se - epi - dw ) images obtained with b = 0 and 1 , 000 s / mm2 in which the solid malignant lesion is clearly visible with a necrotic component at its centre ; the surrounding parenchyma has a relatively hyperintense signal owing to reduced diffusion of water molecules induced by the thrombosis . 
a immagine tse t2 pesata ( te = 80 ms ) nella quale si apprezza nodulo degenerativo del diametro di circa 3 cm nel iii segmento epatico , inducente trombosi nella diramazione portale per il lobo sinistro . 
b , c immagini se - epi - dw ottenute con b = 0 e 1000 s / mm2 nelle quali si rende ben evidente la lesione solida maligna che presenta al centro una componente necrotica ; il circostante parenchima presenta segnale relativamente iperintenso , in relazione alla diminuita diffusivit protonica indotta dalla trombosi . 
d mappa adc : il valore di adc dellepatocarcinoma risulta pari a 1 , 130 , 18x10 - 3 mm2 / s . the problem of the correct choice of the b value therefore appears vital , and in those years , it was tackled most convincingly by yamada et al . 
this microscopic motion in biological tissue includes not only molecular diffusion of water but also microcirculation of blood ( perfusion )  . due to the random organization of the capillary network at the level of the voxel , even the microcirculation of blood can be considered incoherent motion . 
this corresponds to the true diffusion coefficient ( d ) only when diffusion is the only type of motion present ( as occurs in a test tube containing distilled water )  . 
 [ 17 ] performed a thorough analysis of the phenomenon , showing that adc values calculated withmagine , tali autori hanno ritenuto di utilizzare valori di b pari a 1 , 6 , 16 e 55 s / mm2 ottenendo tre set di immagini diversamente pesate in diffusione , anche se , con valori di b cos bassi , limmagine risulta maggiormente influenzata da fenomeni di perfusione e dal tempo di rilassamento t2 ( shine through ) piuttosto che dalla diffusione . 
tutti i tipi di lesioni ( hcc , metastasi ed angiomi ) hanno evidenziato valori medi di adc superiori a quelli del fegato normale e cirrotico a parte alcuni casi di metastasi ; in particolare ladc medio degli angiomi risultato il pi alto , seguito da quelli dellhcc e delle metastasi ( tabella 1 )  . 
examinations with low b values suffer significantly from t2 effects ( shine through ) , and calculation of adc on the maps obtained contains a significant amount of nondiffusional ivim , often derived from perfusion . it would therefore be fair to say that such examinations are little weighted in d and heavily weighted in t2 and perfusion , and the same authors suggest it is worthwhile assessing the effective d and the perfusion fraction f with multiple measurements and an increasing b value ( up to 1 , 100 s / mm2 ) ( table 1 )  . it should be borne in mind , however , that measurements performed with b > 1 , 000 s / mm2 provide low - quality images , with an unfavourable snr and low spatial resolution . 
in addition , repeated measurements with increasing b values lengthens acquisition times , creating problems of repeatability , particularly with less - than - cooperative patients . other relevant considerations can be found in a thoroughly conducted study [ 18 ]  . 
the authors made acquisitions with varying b values on a phantom containing two liquids with known adc values ( of course , adc = d in these cases ) , namely , water ( 2.29x10 - 3 mm2 / s ) and acetone ( 3.98x10 - 3 mm2 / s )  . the aim was to obtain a definite reference parameter for the measurements performed . 
the acquisitions demonstrate significant differences between normal and cirrhotic liver ( table 1 ) and between benign and malignant hepatic lesions . with a threshold of 1.6x10 - 3 mm2 / s and a maximum b value < 846 s / mm2 , a sensitivity of 98% and a specificity of 80% are achieved in differentiating malignant from benign lesions . a revision of the subject was recently performed in a particularly interesting manner , both from the technical and conceptual point of view [ 4 ]  . 
the study involved 66 patients affected by 52 cystic , angiomatous , solid benign and malignant focal hepatic lesions with diameters between 1 and 15 cm ( table 1 )  . assessment was also extended to the normal and cirrhotic parenchyma . 
the study showed there is no statistically significant difference between adc values ( of normal parenchyma , cirrhotic parenchyma and focal hepatic lesions ) measured in the three directions , unlike in the brain and kidneys . 
a causa dellorganizzazione casuale della rete capillare a livello del voxel , infatti , anche la microcircolazione del sangue pu essere considerata un movimento incoerente . pertanto , ladc include tanto gli effetti della diffusione propriamente detta o vera , quanto quelli della perfusione ( pseudodiffusione ) ; corrisponde al coefficiente di diffusione vero ( d ) soltanto quando la diffusione lunico tipo di movimento presente ( come avviene nella provetta contenente acqua distillata )  . 
yamada ha effettuato unanalisi approfondita del fenomeno , dimostrando che i valori di adc , calcolati senza tener conto degli effetti della perfusione , risultano poco utili per differenziare le lesioni epatiche [ 17 ]  . 
la valutazione con bassi valori di fattore b risente infatti fortemente degli effetti del t2 ( shine through ) ed il calcolo delladc sulle mappe cos ottenute contiene una quota importante di ivim non diffusionali , specie derivanti da perfusione : si potrebbe dunque affermare che queste valutazioni sono poco pesate in d e molto in t2 e in perfusione , mentre secondo gli stessi autori viceversa utile valutare la d effettiva e la frazione di perfusione f tramite multiple misure con b crescenti ( fino a 1100 s / mm2 ) ( tabella 1 )  . 
 tuttavia da ricordare che le misure con b > 1000 s / mm2 comportano immagini di bassa qualit con rapporto s / r sfavorevole e bassa risoluzione spaziale ; inoltre , le misure ripetute a b crescenti allungano i tempi di acquisizione con problemi di ripetibilit , in particolare in pazienti non completamente collaboranti . altre considerazioni interessanti si possono trovare in un lavoro condotto in modo molto rigoroso [ 18 ] : questi autori hanno provato acquisizioni a vari b , sempre con presenza di un fantoccio contenente due liquidi con valori noti di adc ( naturalmente in questi casi adc = d ) , ovvero acqua ( 2 , 29x10 - 3 mm2 / s ) e acetone ( 3 , 98x10 - 3 mm2 / s ) ; questo al fine di ottenere un parametro di riferimento certo per le misure effettuate . 
essi hanno dimostrato differenze significative fra il fegato normale e quello cirrotico ( tabella 1 ) e fra lesioni epatiche benigne e maligne ; ponendo un valore soglia a 1 , 6x10 - 3 mm2 / s con b massimo < 846 s / mm2 , essi hanno ottenuto una sensibilit del 98% ed una specificit del 80% nel differenziare le lesioni maligne dalle benigne . recentemente stata operata una revisione della materia in modo particolarmente interessante , sia dal punto di vista tecnico che concettuale [ 4 ]  . 
nella maggior parte dei lavori precedenti , i valori di adc venivano misurati in una sola direzione , assumendo implicitamente che , a differenza dellencefalo e del rene , la diffusione a livello epatico fosse isotropica . 
questo studio ha coinvolto 66 pazienti portatori di 52 lesioni focali epatiche cistiche , angiomatose , solide benigne e maligne con diametri compresi fra 1 e 15 cm ( tabella 1 ) ; la valutazione stata estesa anche ai parenchimi normale e s . 
b , c in the single - shot echoplanar diffusion - weighted ( se - epi - dw ) images obtained with b = 0 and 1 , 000 s / mm2 , lesions are identifiable . 
b , c nelle scansioni se - epi - dw ottenute con b = 0 e 1000 s / mm2 le lesioni risultano percepibili . d mappa adc : il valore di adc della lesione di maggiori dimensioni risulta pari a 1 , 50 , 07x10 - 3 mm2 / s . lastly the study showed that malignant lesions ( hcc and metastases ) have the lowest adc values , benign hepatocellular lesions have intermediate adc values whereas angiomas and cysts have the highest adc values . 
lesions with adc between 1 and 2x10 - 3 mm2 / s may be either benign or malignant , with a considerable amount of overlap . this fact tends to invalidate , at least in part , the usefulness of dw images for characterizing focal hepatic lesions if the possibility of differentiating cysts and angiomas is excluded . in fact , hepatic cysts have the highest adc values owing to their fluid content , with unrestricted movement of water molecules , whereas angiomas have high adc values owing to their rich vascular component . to summarize , a number of significant limitations to dw images need to be recognized with reference to the sequences used . the se - epi - ssh has limited spatial resolution and low snr and therefore cannot be used to assess lesions with a diameter less than 1 cseveral studies suggest the possibility that new fast sequences will improve image quality and reduce artefacts associated with echoplanar acquisitions [ 19 ]  . in addition , the use of magnets with a 3t field intensity could improve the signal in diffusion [ 20 ]  . 
moreover , if on the one hand very high b values reduce image quality , then the mid - range values usually used ( 300500 s / mm2 ) lead to cirrotico . 
questi autori dimostrano che non esistono differenze statisticamente significative fra i valori di adc ( del parenchima epatico normale , cirrotico e delle lesioni focali epatiche ) misurati nelle tre direzioni , al contrario di quanto avviene nellencefalo e nel rene . 
pertanto , il fegato ha un pattern di diffusione isotropico , probabilmente per la sua organizzazione strutturale casuale , e non risulta quindi necessaria ladozione di gradienti di diffusione multi - direzionali , ovvero lungo le tre direzioni dello spazio . 
si dimostra inoltre che le misure dei valori di adc sono tanto pi accurate , quanti pi valori di b vengono utilizzati lungo la direzione della selezione dello strato ( quattro valori di b crescenti : 0 , 134 , 267 e 400 s / mm2 )  . 
infine risultato che le lesioni maligne ( hcc e metastasi ) presentano i pi bassi valori di adc , le epatocellulari benigne valori di adc intermedi , mentre angiomi e cisti i valori di adc pi alti . pi precisamente le lesioni con adc superiore a 2x10 - 3 mm2 / s risultano benigne , mentre quelle con valori inferiori a 1x10 - 3 mm2 / s , maligne . 
questo fatto inficia dunque , almeno in parte , lutilit delle immagini dw nella caratterizzazione delle lesioni focali epatiche , se si esclude la possibilit di differenziare cisti ed angiomi . 
le cisti epatiche presentano infatti i pi alti valori di adc per il loro contenuto fluido , the risk of overestimating the values due to inclusion of the perfusion fraction f . 
the interstitial deposit of collagen fibres within the lobule modifies the content and motility of water molecules ; this may be considered the expression of mr diffusion , probably causing a restriction of the movement of water molecules , which is evident by the reduction of adc values with respect to healthy parenchyma . 
the same study also showed a correlation between serum levels of hyaluronic acid ( a marker of hepatic fibrosis ) , the child - pugh classification and adc values . in the retroperitoneum and with regard to the pancreas , epi - dw sequences can be useful in differentiating mucinproducing tumours from other histological types [ 23 . 
alcuni studi suggeriscono la possibilit che le nuove sequenze rapide possano migliorare la qualit dellimmagine e ridurre gli artefatti correlati alle acquisizioni eco - planari [ 19 ] ; inoltre , ladozione di magneti con intensit di campo pari a 3t , potrebbe potenzialmente migliorare il segnale in diffusione [ 20 ]  . 
inoltre , se da un lato valori molto alti di fattore b comportano riduzione della qualit di immagine , i valori medi solitamente utilizzati ( pari a 300500 s / mm2 ) comportano il rischio della sovrastima dei valori per inclusione della frazione di perfusione f . 
tale reperto da ascrivere allaccumulo progressivo di matrice fibrillare extracellulare ( prevalentemente collagene di tipo i e iii ) che caratterizza le epatopatie croniche di qualunque etiologia e che raggiunge il suo massimo grado di espressione nella cirrosi ( epatopatia cronica sclerogena ) , contraddistinta dalla formazione di noduli rigenerativi , circondati da setti fibrotici , che sovvertono larchitettura del parenchima epatico . 
la deposizione interstiziale di fibre collagene nel contesto del lobulo epatico modifica il contenuto e la mobilit delle molecole di acqua , delle quali pu essere considerata espressione la diffusione rm , determinando verosimilmente una restrizione del movimento molecolare dellacqua che si rende evidente con la riduzione dei valori di adc rispetto al parenchima sano . 
la possibilit di quantificare in maniera non invasiva , senza cio il ricorso alla biopsia epatica , il grado di fibrosi , ha spinto recentemente alcuni autori a valutare lutilit dellimaging rm pesato in diffusione nella diagnosi di cirrosi e nella quantificazione della fibrosi epatica nei soggetti affetti da epatopatia cronica e da cirrosi . 
in particolare , si cercato di definire se sia possibile distinguere i vari gradi di fibrosi ed infiammazione nei pazienti con epatite cronica c , acquisendo sequenze dw bh con cinque valori di fattore b crescenti , compresi tra 50 e 250 s / mm2 , applicati lungo i tre assi [ 21 ]  . 
questi autori riferiscono di non aver trovato nessuna correlazione tra i valori di adc e i gradi di fibrosi ed infiammazione / attivit di malattia , determinati istologicamente tramite biopsia epatica , indicando che le modificazioni tissutali indotte dallepatite c non sembrano poter essere quantificate con dwi . 
unfortunately the limited spatial resolution obtained with epi sequences precludes assessment of small masses . therefore , small peripheral mucin - producing tumours ( situated near the peripheral ductal branches ) cannot be visualized . the kidney is a good candidate for dw imaging given the relative hypomotility of water molecules within the organ . recent studies [ 2529 ] report adc values of normal renal parenchyma to be higher than those of other abdominal parenchyma in relation to high blood flow and function of fluid transportation performed by the kidney . the first mean adc value of normal renal parenchyma reported in the literature was 3.54x10 - 3 mm2 / s [ 26 ]  . 
an experimental study on animals assessed the effect of intravenous administration of high - viscosity nonionic contrast material on flow in the renal cortex and medulla , showing that following administration , there is a transitory reduction in adc values both in the cortex and medulla . this is explained by a reduction in perfusion induced by the contrast material itself . 
the study showed an adc value at the upper pole significantly higher than at the central portion using low b values whereas no significant differences were obtained with high b values . this is explained by anisotropic diffusion in that structures in the polar regions are oriented parallel to the gradients whereas in the central portion , they are perpendicular . 
furthermore , there is a greater amount of cortex than medulla in the renal poles , and it should be borne in mind that blood flow in the cortex is about ten times greater than in the medulla . 
high adc values at the poles are therefore justified by the greater perfusion , an effect noted when low b values are used whereas with high b values , that effect is almost entirely eliminated [ 25 ]  . 
in fact , it has already been mentioned that as b increases , the weighting in d increases , and the effects secondary to nondiffusional ivim , such as perfusion , are almost entirely eliminated . it should be borne in mind that the findings presented so far refer to studies published in the last 10 years obtained with different devices ( some commercially available , others dedicated to experimental studies ) and using different examination techniques ( different b values , hydrated and dehydi fibrosi epatica ) , la classificazione di child - pugh ed i valori di adc . in ambito retroperitoneale , per quel che riguarda il pancreas , le epi - dw possono essere utili per differenziare i tumori mucinosi dalle altre variet istologiche [ 23 , 24 ]  . 
usando un fattore di diffusione b massimo pari a 300 s / mm2 , ladc medio del fluido viscoso ( contenuto sia nelle cavit cistiche che nei dotti pancreatici principali ) dei tumori mucinosi risultato sostanzialmente pi basso di quello dei tumori sierosi e del liquido cerebrospinale , ma con valori prossimi a quelli delle pseudocisti pancreatiche . 
tuttavia , dal momento che queste ultime non sono associate alla presenza di materiale viscoso allinterno del wirsung , il calcolo delladc effettuato allinterno della lesione cistica e del dotto principale potrebbe essere utile per differenziare le pseudocisti ( wirsung con segnale relativamente elevato , maggiore della lesione cistica ) dai tumori mucinosi ( wirsung con segnale relativamente ridotto , comunque uguale a quello della lesione cistica )  . 
pertanto piccoli tumori mucinosi periferici ( localizzati in prossimit dei rami duttali periferici ) non possono essere apprezzati . il rene si presta ad essere studiato con limaging in d per lipomobilit relativa alla sede . 
nella letteratura recente sono riportati valori di adc del parenchima renale normale [ 2529 ] superiori a quelli degli altri parenchimi addominali , in relazione allelevato flusso sanguigno e alla funzione di trasporto di fluidi del rene . 
questo dato confermato da altri autori [ 25 ] che hanno misurato i valori di adc al polo superiore , inferiore e in sede mesorenale , utilizzando diversi valori di b , compresi tra 317 e 932 s / mm2 . 
risultato che , a tutti i livelli indagati , utilizzando b elevati si ottengono valori di adc significativamente pi bassi ( 1 , 361 , 51x10 - 3 mm2 / s )  . 
c apparent diffusion coefficient ( adc ) map : adc value measured at the middle portion of the right kidney is 2.290.13x10 - 3 mm2 / s . ( se - epi - dw ) fig . 
questo pu essere spiegato dalla diffusione anisotropica , in quanto nelle regioni polari le strutture renali sono orientate in modo parallelo alla direzione dei gradienti , mentre in regione mesorenale risultano perpendicolari . 
inoltre , a livello dei poli renali esiste prevalenza della corticale rispetto alla midollare e deve essere ricordato che il flusso ematico nella corticale circa 10 volte superiore rispetto a quello nella midollare . 
gli alti valori di adc ai poli sono pertanto giustificati dalla maggior perfusione , effetto che si risente quando si utilizzano valori bassi di fattore b , mentre quando si utilizzano valori di b elevati tale contributo pressoch totalmente eliminato [ 25 ]  . 
infatti gi stato ricordato che allincremento del fattore b , aumenta la pesatura in d e si riducono gli effetti secondari ai cosiddetti ivim non diffusionali , quali la perfusione . importante sottolineare che i dati fin qui riportati fanno riferimento a lavori pubblicati negli ultimi 10 anni ottenuti con differenti apparecchiature ( alcune disponibili in commercio , altre dedicate solo a studi sperimentali ) ed utilizzando tecniche desame diverse ( diversi valori di b , pazienti idratati o disidratati , differente sede di posizionamento della roi )  . 
da sottolineare che , in tale studio , la roi stata posizionata in sede mesorenale a livello della giunzione cortico - midollare e non selettivamente a sede midollare e corticale in quanto , come gi evidenziato da altri autori [ 25 ] , risulta difficile posizionare la roi separatamente nelle due sedi , a causa della scarsa risoluzione spaziale delle epi e della presenza , seppur modesta , di artefatti da movimento . le potenzialit dellimaging in diffusione sono state valutate anche in varie situazioni patologiche , quali le alterazioni della funzione , le infezioni renali , la idro / pionefrosi e le masse tumorali [ 10 , 2532 ]  . nello studio della funzione renale [ 27 , 29 ] si dimostrata una buona correlazione tra i valori di adc e lentit del filtrato glomerulare ( gfr ) [ 29 ]  . 
limaging in diffusione potrebbe pertanto rappresentare un metodo nuovo e non invasivo per valutare linsufficienza renale acuta ( ira ) e cronica ( irc ) , e il parenchima in pazienti con stenosi dellarteria renale ( ras )  . 
nella irc i valori di adc risultano ridotti , rispetto al rene sano , sia nella corticale che nella midollare , a seguito della perdita di nefroni , con conseguente riduzione della mobilit dellacqua . 
si ipotizzato che la riduzione dei valori di adc sia secondaria allischemia renale e alledema intracellulare a cui consegue , a sua volta , la riduzione del movimento delle molecole dacqua . 
d apparent diffusion coefficient ( adc ) map : adc value measured at the level of the hydronephrotic pelvis is 3.140.24x10 - 3 mm2 / s . ( se - epi - dw ) fig . 
d mappa adc : il valore di adc misurato a livello della pelvi idronefrotica pari a 3 , 140 , 24x10 - 3 mm2 / s . the potential of dw imaging has also been assessed in a variety of pathological situations , such as functional alterations , kidney infections , hydro / pyonephrosis and tumours [ 10 , 2532 ]  . 
dw imaging might therefore be a useful noninvasive technique for assessing acute and chronic kidney failure and the parenchyma in patients with renal artery stenosis . adc values in chronic kidney failure are lower than those in the normal kidney , both in the cortex and medulla , owing to loss of nephrons and the consequent reduction in water motility . adc values in acute kidney failure are lower than those in the normal kidney , both in the cortex and medulla , but higher than those measured in chronic kidney failure . 
adc values in renal artery stenosis are lower than those in the normal kidney due to reduction in perfusion , which is particularly evident in perfusione particolarmente evidente a livello corticale [ 25 ]  . altri autori confermano valori di adc significativamente minori sia nel parenchima dei pazienti con insufficienza renale acuta e cronica , che a livello della corticale dei reni con stenosi dellarteria renale [ 28 ]  . 
le flogosi sembrano determinare riduzione dei valori di adc : questo riscontrato sia nei casi di pielonefrite , che di ascesso o cisti renale ascessualizzata [ 10 , 31 ]  . 
another study has confirmed significantly lower adc values both in the parenchyma of patients with acute and chronic kidney failure and in the renal cortex in patients with renal artery stenosis [ 28 ]  . 
in fact , in the pyonephrotic kidney , the collecting system is full of high viscosity and high cellularity purulent material , which produces reduction in diffusion , high dw signal and low adc values . dw imaging may also play a part in the assessment of kidney masses . 
further studies , however , are required to determine the possible differences in adc values between cystic and / or necrotic renal tumours and complex renal cysts . the use of dw sequences for the study of thoracoabdominal districts in paediatric patients is often hindered by motion artefacts secondary to free - breathing acquisitions in the case of uncooperative or sedated patients . 
a recent study examined the abdomen of eight paediatric patients with no clinical / humoural evidence of diffuse disease of the abdominal parenchyma ( se - epi - ssh with b set to 300 , 600 and 800 s / mm2 ) [ 33 ]  . 
values obtained with maximum b value were in line with those reported in the literature in adult patients with sequences acquired in breath hold or with respiratory triggering [ 16 , 18 , 34 ]  . 
if these findings are confirmed by further studies , the possibility of performing dw imaging in paediatric and sedated patients would have significant developments in the multimodal approach to abdominal paediatric disease . 
using a similar sequence to the one described above , a low value of adc was found in neuroblastomas ( 1.1x10 - 3 mm2 / s ) , suggestive of restricted diffusion [ 35 ]  . 
the study justifies this figure by suggesting that cellular barriers in a tissue characterized by significantly high cellular density with a high nucleus / cytoplasm ratio has a limiting effect on water molecule diffusion . 
the study of kidney diseases caused by reflux or stenosis of the pyeloureteral junction might also benefit from dw imaging if the existence of a correlation between parenchymal adc and biohumoural pas . 
sono tuttavia necessari ulteriori studi , per determinare le possibili differenze nei valori di adc tra i tumori renali cistici e / o necrotici e le cisti renali complesse . luso delle sequenze dw per lo studio dei distretti toracoaddominali in pediatria frequentemente gravato da artefatti da movimento secondari ad acquisizioni a respiro libero in caso di pazienti poco collaboranti o sedati . 
i valori ottenuti con b massimo sono risultati sovrapponibili a quelli riportati in letteratura in pazienti adulti con sequenze acquisite a respiro sospeso o con trigger respiratorio [ 16 , 18 , 34 ]  . 
utilizzando una sequenza analoga a quella descritta precedentemente , stato riscontrato un basso valore di adc nei neuroblastomi ( 1 , 1x10 - 3 mm2 / s ) , secondo un quadro di diffusione ristretta [ 35 ]  . 
per giustificare tale dato , gli autori , hanno ipotizzato leffetto limitante , sulla diffusivit protonica , delle barriere cellulari , in un tessuto caratterizzato da una notevole densit cellulare , ad alto rapporto nucleo / citoplasma . 
markedly hyperintense cyst with b = 0 appears isointense to renal parenchyma with increased weighting in diffusion ( b = 500 s / mm2 ) ; had the image been obtained with b = 1 , 000 s / mm2 , the cyst would have had a low signal . 
la cisti , marcatamente iperintensa a b = 0 , risulta isointensa al parenchima renale allaumentare del peso in diffusione ( b = 500 s / mm2 ) ; ove fosse stato acquisito b = 1000 la cisti sarebbe risultata a basso segnale . 
d mappa adc : il valore di adc misurato a livello della cisti 3 , 010 , 16x10 - 3 mm2 / s . fisico , dimostrando un aumento di tale parametro in condizioni di stress fisico , probabilmente in relazione ad un aumento della microcircolazione ( pseudodiffusione ) , oltre che della componente extra - cellulare [ 5 ]  . anche in questo distretto ha suscitato interesse la possibilit di discriminare le lesioni benigne dalle maligne . 
alcuni autori riportano un valore medio di adc significativamente maggiore nelle lesioni benigne ( 1 , 71x10 - 3 mm2 / s ) , rispetto alle maligne ( 1 , 08x10 - 3 mm2 / s ) [ 38 ]  . 
questi risultati sono tuttavia contraddetti da una recente pubblicazione in cui , basandosi su una limitata casistica ( 29 lesioni , di cui 16 benigne e 13 maligne ) , non si riscontrata una significativa differenza tra questi due gruppi ; infatti le lesioni benigne hanno presentato un adc compreso tra 1 e 2 , 9x10 - 3 mm2 / s , mentre le maligne fra 0 , 9 e 2 , 3x10 - 3 mm2 / s [ 41 ]  . 
altri ancora giungono a conclusioni intermedie : in una casistica pi ampia ( 60 lesioni ) , utilizzando b = 1000 s / mm2 , stato rilevato come solo nel caso di lesioni mixomatose il dwi non perrameters of renal function is confirmed [ 28 ]  . musculoskeletal applications in recent years , a number of advanced mr acquisition techniques have been proposed [ 37 ] , including dw imaging sequences , particularly for the study of soft tissue [ 38 ] , bone marrow [ 7 ] and joints ( effusion and cartilage ) [ 6 , 39 ]  . 
b single - shot echoplanar diffusion - weighted ( se - epi - dw ) image obtained with b = 0 s / mm2 : the lesion is moderately hypointense . 
one study [ 38 ] reports a mean adc value significantly higher in benign lesions ( 1.71x10 - 3 mm2 / s ) than in malignant lesions ( 1.08x10 - 3 mm2 / s )  . 
yet another study [ 42 ] came to an intermediate conclusion : based on a larger case series ( 60 lesions ) and using a b value of 1 , 000 s / mm2 , dw imaging was unable to successfully differentiate myxomatous lesions only whereas it was particularly effective in differentiating cartilaginous lesions . 
infine da ricordare , a proposito dellelevato valore di adc delle lesioni benigne , che stato sottolineato come gli alti valori riscontrati negli angiomi dei tessuti molli siano dovuti ad una significativa componente di pseudodiffusione o sloshing effect ( perfusione ovvero ivim non diffusionali ) , prodotta dal flusso ematico allinterno della lesione [ 43 ]  . se la caratterizzazione tumorale non ha offerto finora dati univoci , vi maggiore accordo sullefficacia del dwi nel follow - up post - chemio e radioterapia . 
secondo alcuni studi il dwi sarebbe infatti in grado di caratterizzare i tessuti neoplastici e di differenziare le aree di necrosi ( ad elevato adc ) dai residui tumorali ( a basso adc ) [ 44 , 45 ]  . 
sono stati inoltre riscontrati valori di adc significativamente superiori ( p < 0 , 0019 ) nelledema dei tessuti muscolari perilesionali e negli igromi , rispetto alle recidive tumorali [ 46 ]  . la rm presenta unelevata sensibilit nella valutazione delle alterazioni ossee , che nella maggior parte dei casi determinano una riduzione del segnale nelle sequenze t1 ed un aumento in quelle t2 - pesate ; la specificit della metodica s . 
b - d immagini a b crescenti pari a 300 , 600 , 800 s / mm2 . tipico artefatto da suscettibilit magnetica a livello della bolla gastrica ( punte di freccia bianche )  . 
progressivo calo del segnale degli organi parenchimali allaumentare di b , pi evidente a livello del parenchima renale ( frecce bianche ) , in relazione alla maggior diffusivit protonica . while findings regarding tumour differentiation have not been unequivocal to date , there is greater consensus regarding the effectiveness of dw imaging in postchemotherapy and radiotherapy follow - up . 
studies have shown that dw imaging is capable of characterizing neoplastic tissue and differentiating areas of necrosis ( high adc ) from tumour residue ( low adc ) [ 44 , 45 ]  . 
in addition , significantly higher adc values have been found ( p < 0.0019 ) in the oedema of perilesional muscular tissue and in hygromas than in relapsed tumour [ 46 ]  . mr has high sensitivity in assessment of bone alterations , which in most cases cause a signal decrease in t1 and an increase in t2 although its specificity in distinguishing oedema from inflammation or tumours is low . 
assessment of normal adc values in bone obviously refers to bone marrow , given the absence of diffusion in the compact component of bone . values reported in the literature vary according to intrinsic factors ( yellow marrow has a lower diffusible water component than red marrow ) and technical factors , such as sequences and , above all , b values used . 
i valori riportati in letteratura variano in relazione a fattori intrinseci ( il midollo giallo ha una componente di acqua diffusibile inferiore al midollo rosso ) e a fattori tecnici , come le sequenze utilizzate e soprattutto i valori di b adottati . 
il midollo osseo normale presenta un adc inferiore a 0 , 5x10 - 3 mm2 / s , con valori compresi tra 0 , 15 e 0 , 35x10 - 3 mm2 / s [ 4749 ] ; in particolare il midollo rosso avrebbe un adc superiore rispetto al midollo giallo [ 47 ]  . valori di adc pi elevati sono stati rilevati da nonomura et al . 
che hanno valutato il midollo a livello della cresta iliaca , distinguendo tre gradi di normalit midollare : ipocellulare ( 0 , 31 mm2 / s ) , normocellulare ( 0 , 82 mm2 / s ) e ipercellulare ( 1 , 29 mm2 / s ) , questultimo presente nei bambini [ 7 ]  . 
a turbo spin echo ( tse ) t2 - weighted axial image showing moderately hyperintense homogenous mass ( white arrows ) originating from the posterior mediastinum ; some axillary lymphonodal packets are also present ( black arrowheads )  . 
b single - shot echoplanar diffusion - weighted ( se - epi - dw ) sequence with fat signal suppresion and b = 0 ( tr 6 , 890 , te 95 , epi factor 88 ) confirms the presence of the formation described and the right axillary lymphonodal packet ; both appear with elevated signal intensity . 
c with the application of a diffusion gradient of 800 s / mm2 , there is a significant loss of axillary lymph node signal but not of the mediastinal mass , which maintains high signal intensity . 
d apparent diffusion coefficient ( adc ) map : high lymph node signal as a consequence of high diffusion ( ) at that level , most likely due to colliquative phenomena . 
a immagine tse t2 sul piano assiale che mostra una massa omogenea e modicamente iperintensa ( frecce bianche ) che origina dal mediastino posteriore ; sono presenti anche dei pacchetti linfonodali ascellari ( punte di freccia nere )  . 
b la sequenza seepi - dw con soppressione del grasso a b = 0 ( tr 6890 , te 95 , epi factor 88 ) conferma la presenza della formazione descritta e del pacchetto linfonodale ascellare destro ; entrambe appaiono ad elevata intensit di segnale . 
c con lapplicazione di un gradiente di diffusione di 800 s / mm2 si ottiene una notevole perdita di segnale dei linfonodi ascellari , ma non della massa mediastinica che mantiene elevata intensit di segnale . 
 [ 7 ] , who assessed marrow at the level of the iliac crest , distinguishing three degrees of marrow normality : hypocellular ( 0.31x10 - 3 mm2 / s ) , normocellular ( 0.82x10 - 3 mm2 / s ) and hypercellular ( 1.29x10 - 3 mm2 / s ) , the last of which was measured in paediatric patients . 
su questo presupposto si basata la valutazione con dwi dei crolli vertebrali , nel tentativo di distinguere quelli su base osteoporotica da quelli secondari , soprattutto qualora sia presente edema dellintero soma e / o dei peduncoli vertebrali [ 51 ]  . 
acquisition of single - shot echoplanar diffusion - weighted ( se - epi - dw ) images using orthogonal diffusion gradients oriented along the anteroposterior axis ( ap , corresponding to the phase code ) , laterolateral ( ll , corresponding to the read code ) and craniocaudal ( crc , corresponding to the slice selection ) direction , respectively . 
axial dw slice image , obtained along the longitudinal axis of the soleus muscle and the lateral belly of the triceps sura , shows greater hypointensity ( white arrowhead ) than shown by dw images acquired along the other two directions in space . 
acquisizione di immagini se - epi - dw utilizzando gradienti di diffusione orientati rispettivamente lungo lasse antero - posteriore ( ap , corrispondente alla codifica di fase : phase ) , latero - laterale ( ll , corrispondente alla codifica di lettura : read ) e lungo la direzione cranio - caudale ( crc , corrispondente alla selezione di strato : slice ) , tra loro ortogonali . 
this is the basis for using dw imaging to assess vertebral collapse in the attempt to distinguish those due to osteoporosis and those secondary in nature , especially when oedema is present in the entire vertebral body and / or peduncles [ 51 ]  . 
when assessed with the same sequence , vertebral collapse due to osteoporosis tends to have a hypointense signal in dw sequences ( i.e. high diffusion high adc ) while vertebral collapse due to metastasis has a hyperintense signal in dw ( low diffusion low adc ) [ 53 ]  . 
il crollo vertebrale su base osteoporotica tende inoltre , se valutato con la stessa sequenza , a presentare ipointensit di segnale nelle sequenze dw ( ossia elevata diffusione alto adc ) , mentre il cedimento vertebrale secondario a metastasi presenta iperintensit di segnale nelle dw ( bassa diffusione basso adc ) [ 53 ]  . 
ricordando che le sequenze se - epi - ssh permettono di quantificare ladc , nelle casistiche con b sovrapponibili e tendenzialmente elevati ( 8801000 s / mm2 ) , tale valore varia tra 1 , 61 e 1 , 94 nei crolli osteoporotici e tra 0 , 69 e 0 , 82x10 - 3 mm2 / s in quelli su base sostitutiva [ 49 , 54 ]  . infine , castillo et al . 
riportano dati contraddittori rispetto a quanto fin qui descritto [ 55 ] , anche se in tale studio erano presenti ben 9 / 15 mestastasi sclerotiche note , nelle quali la scarsa presenza di protoni diffusibili determina un basso segnale sia nellimmagine di riferimento ( b = 0 ) che nellimmagine dw ; il rischio di falsi negativi nelle lesioni ripetitive osteosclerotiche stato riportato anche da altri autori [ 49 ]  . 
a single - shot echoplanar diffusion - weighted ( se - epi - dw ) image with fat signal suppression in the sagittal plane with b = 0 s / mm2 . 
 [ 55 ] , however , report findings contradictory to the above even though in the study there were as many as 9 / 15 known sclerotic metastases , in which the limited presence of diffusible water molecules produces a low signal in both reference images ( b = 0 ) and dw images . 
it should also be borne in mind that this limitation is , in part , linked to the use of sequences in the studies mentioned that do not enable calculation of adc . we have already noted that adc of bone marrow may vary in healthy subjects on the basis of the degree of adipose involution . 
this would justify the higher adc value in haematopoietic marrow in the paediatric population , particularly those under 5 years of age , when both axial and appendicular skeletons ( with the exception of phalanxes ) are hypercellular [ 56 ]  . 
dw imaging is able to distinguish those different characteristics of the marrow [ 7 ] since areas of high cellularity have a higher adc value than hypocellular areas . abbiamo gi accennato in precedenza a come ladc midollare possa variare nei soggetti sani in funzione del grado di involuzione adiposa ; tale dato giustificherebbe il valore pi alto di questo indice nel midollo ematopioetico e nella popolazione infantile , in special modo al di sotto dei 5 anni , quando sia lo scheletro assile che quello appendicolare ( ad eccezione delle falangi ) ipercellulare [ 56 ]  . 
in particolare stato dimostrato come non vi sia una significativa differenza di adc tra il normale midollo ipercellulare e quello con ipercellularit secondaria ad infiltrazione linfomatosa , mentre tale differenza significativa tra il midollo ipocellulare , quello normocellulare e lipercellulare emopoietico [ 7 ]  . 
d mappa adc : larea edematosa presenta adc superiore a quello del circostante midollo osseo . it has been shown that there is no significant difference in adc between normal hypercellular marrow and marrow with hypercellularity resulting from lymphomatous infiltration while the difference is significant between hypocellular marrow , hypercellular haematopoietic marrow [ 7 ]  . 
tali valori sono in linea con quanto riportato da baur [ 11 ] : utilizzando b di 750 s / mm2 , vengono riferiti valori di adc pari a circa 1 , 37 e 0 , 95x10 - 3 mm2 / s in caso di edema midollare e metastasi , rispettivamente . 
a in the single - shot echoplanar ( se ) t1 - weighted image at the time of diagnosis , an extensive area of bone marrow hypointensity at the level of the posterior portion of the ankle is evident ( black arrowhead )  . 
b single - shot echoplanar diffusion - weighted ( se - epi - dw ) image obtained with b = 300 s / mm2 : diffuse medullary hyperintensity ( white arrowheads )  . 
a nella se t1 pesata al momento della diagnosi evidente una estesa area di ipointensit della midollare ossea a livello della porzione posteriore del calcagno ( punte di freccia nere )  . 
there have been no clinical studies regarding the use of dw imaging in bone tumours , with the exception of the above - mentioned work on vertebral metastasis and the relatively few studies regarding lymphomatous infiltration . 
however , thanks to a number of experimental studies , including the above - mentioned study by lang et al . [ 44 ] , we know that adc can be a sensitive indicator of the response of those tumours to treatment ( given the progressive increase in adc in the necrotic component )  . 
in a study performed on patients affected by vertebral metastasis and in a time interval ranging from 1 to 6 months following radioallutilizzo del dwi nelle neoplasie ossee , ad eccezione di quanto gi riportato a proposito delle metastasi vertebrali e dei pochi lavori sulla valutazione dellinfiltrazione linfomatosa . 
 [ 44 ] , sappiamo tuttavia che ladc pu essere un sensibile indicatore della risposta di tali neoplasie ai trattamenti ( dato il progressivo aumento delladc della componente necrotica )  . 
in uno studio condotto su pazienti affetti da metastasi vertebrali , in un intervallo di tempo compreso tra 1 e 6 mesi dopo terapia radiante , gli autori [ 58 ] hanno riscontrato , sulle immagini dw , una iperintensit di segnale delle metastasi nella valutazione pre - terapeutica , in relazione ai bassi valori di adc delle lesioni ripetitive ( 0 , 78x10 - 3 mm2 / s )  . 
nelle successive indagini in corso di terapia si evidenziata progressiva perdita di segnale , per laumento di diffusivit causato dallincremento dellacqua extra - cellulare e dalla diminuzione della componente cellus . 
 [ 58 ] found in dw images a hyperintense signal of the metastasis in the pretreatment assessment in relation to the low adc values of repetitive lesions ( 0.78x10 - 3 mm2 / s )  . 
examinations performed during therapy revealed progressive loss of signal owing to the increase in diffusivity caused by the increase in extracellular water and the decrease in the cellular component ( 1.22x10 - 3 mm2 / s )  . these changes first appeared 1 month after the beginning of therapy , which , if confirmed , would place dw imaging at a distinct advantage over bone scintigraphy , which cannot be performed prior to 6 months due to the false hyperfixation produced in that period by bone repair . 
in the same way , using a b value of 1 , 000 s / mm2 , an adc value was shown at the peak effectiveness of treatment to be about three times higher than the value when treatment began [ 59 ]  . cartilaginous structures have the potential to be well assessed by dw imaging thanks to their high water content [ 39 ]  . 
however , only in recent years and thanks to the development of new acquisition techniques have targeted studies been performed in this respect . the technical limitation regards the short t2 of cartilage , which necessitates dw sequences with a relatively low te and appropriate spatial resolution . 
in this sense , the se - epissh sequences , despite having a very short te , are unable to provide high resolution images while ssfp sequences , as stated above , have the disadvantage of not enabling the calculation of adc . 
the patellar cartilage showed a progressive reduction in signal intensity with an increasing b value while the signal for synovial fluid was totally absent due to high diffusion [ 61 ]  . 
this finding appears to vary in relation to the nature of the effusion : a significant difference between adc of effusion of a degenerative and inflammatory nature was found ( 2.4x10 - 3 mm2 / s and 3.1x10 - 3 mm2 / s , respectively ) [ 6 ]  . the authors of the study explain this finding as the result of hyaluronidase activity present in inflammatory forms , which causes reduction in hyaluronic acid concentration , with a consequent decrease in viscosity . the dependence of dw images on diffusivity of water molecules and the possibility of quantifying their movement therefore hold great promise for increasing the global ability of mr to distinguish benign from malignant lesions . 
in some cases , as in assessment of vertebral fractures and follow - up of soft tissue tumours or lymphomas undergoing adjuvant treatment , a large number of studies have been published , and the preliminary results obtained to date appear encouraging . 
work done to date regarding use of dw imaging to asses cartilage , although limited to in vitro studies , seems to offer developments in the near future in line with technological progress in hardware ( coils with a higher snr ) and software ( new sequences ) in response to clinical and therapeutic needs of increasingly molecular imaging techniques . nonetheless , an increase in spatial resolution will be relare ( 1 , 22x10 - 3 mm2 / s )  . 
tali modificazioni sono comparse un mese dopo linizio della terapia e questo rappresenterebbe , se confermato da altri studi , un vantaggio rispetto alla scintigrafia ossea , che non pu essere eseguita prima di 6 mesi a causa della falsa ipercaptazione prodotta in tale periodo dallattivit osteoriparativa . 
allo stesso modo , si dimostrato , utilizzando b = 1000 s / mm2 , un aumento delladc di circa 3 volte rispetto al valore di partenza nella fase di massima efficacia terapeutica [ 59 ]  . le strutture cartilaginee sono potenzialmente ben valutabili con dwi , a causa del loro contenuto idrico [ 39 ] : alcuni autori hanno stabilito una correlazione tra la perdita di proteoglicani che avviene nei processi degenerativi e laumento delladc cartilagineo [ 60 ]  . 
in questo senso le sequenze se - epi - ssh , pur con te brevissimo , non consentono immagini ad elevata risoluzione , mentre quelle ssfp presentano , come accennato in precedenza , lo svantaggio di non permettere il calcolo delladc . 
stata recentemente utilizzata una sequenza steady - state dw a cui stato applicato un eco - navigatore per ridurre gli artefatti da movimento ed aumentare la risoluzione spaziale : la cartilagine rotulea ha presentato progressiva riduzione dellintensit di segnale allaumentare del valore di b , mentre il liquido sinoviale risultato completamente privo di segnale per lelevata diffusione [ 61 ]  . 
tale reperto sembra variare in relazione alla natura del versamento : stata riscontrata una significativa differenza tra adc dei versamenti di natura degenerativa ed infiammatoria ( rispettivamente 2 , 4x10 - 3 mm2 / s e 3 , 1x10 - 3 mm2 / s ) [ 6 ]  . 
questo dato stato spiegato dagli autori come il prodotto dellattivit della ialuronidasi presente nelle forme infiammatorie , che determina una riduzione della concentrazione dellacido ialuronico con conseguente decremento della viscosit . la dipendenza dellimmagine dw dalla diffusivit dei protoni dellacqua e la possibilit di quantificarne il movimento risulta quindi molto promettente nellaumentare la capacit globale della rm nel distinguere le lesioni benigne da quelle maligne . in alcuni casi , come nella valutazione delle fratture vertebrali e nel follow - up delle neoplasie dei tessuti molli o dei linfomi in corso di terapia adiuvante , la letteratura offre unampia casistica ed i risultati preliminari ottenuti ad oggi sembrano incoraggianti . 
quanto riportato nella valutazione con dwi delle cartilagini , sebbene limitato a studi in vitro , sembra poter avere sviluppo nel prossimo futuro parallelamente ai progressi tecnologici sia nel campo del hardware ( bobine di quadratura a pi elevato s / r ) che del software ( nuove sequenze ) , rispondendo alle esigenze cliniche e terapeutiche di un imaging sempre pi molecolare . 
sar comunque necessario aumentare la risoluzione spaziale ed eliminare linfluenza del flusso capillare sulla genesi del segnale di diffusione , in modo da poter stabilire valori di adc quired along with elimination of the effects of capillary flow on the genesis of the diffusion signal to establish reliable adc values that are reproducible in the various districts and individual pathologies . 
dw sequences also need to be validated on a broad number of cases in an attempt to define sequences and b values that guarantee optimal results in the musculoskeletal district . recently , a number of studies were performed on animals using a diffusion sequence with steady - state sequential acquisitions known as line scan , both before and after induction of femoral ischaemia [ 62 ]  . 
the study showed a variation of adc values in the epiphysis characterized by an initial reduction ( 26% in the first 3 h ) followed by progressive increase ( + 27% after 5 h ; + 75% after 96 h )  . 
these findings are suggestive of early cytotoxic oedema , which would justify the initial restricted diffusion and consequent cellular lysis , which , in contrast , would tend to increase water molecule diffusion . these findings may have a significant impact in paediatrics regarding treatment of legg - calv - perthes disease , in which knowledge of the extension of ischaemia and its duration is vital for establishing the most appropriate treatment . the results seem to confirm anecdotal evidence : medullary oedema with a mean adc of 1.22x10 - 3 mm2 / s compared with a normal value of 0.56x10 - 3 mm2 / s [ 50 ]  . 
however , dw imaging does not appear to be able to differentiate between an increase in adc present in inflammatory foci and an increase due to medullary oedema and osteochondritis [ 63 ]  . 
appare necessaria la validazione delle sequenze dw su pi ampie casistiche , nel tentativo di definire le sequenze ed i valori di b che garantiscano i migliori risultati nel distretto muscolo - scheletrico . recentemente sono stati condotti alcuni studi su animali , utilizzando una sequenza di diffusione ad acquisizioni sequenziali in steady - state , nota come line - scan , prima e dopo induzione di ischemia femorale [ 62 ]  . 
gli autori hanno riscontrato nellepifisi una variazione dei valori di adc caratterizzata da una iniziale diminuzione ( 26% nelle prime 3 ore ) , seguita da un progressivo aumento ( + 27% dopo 6 ore , + 75% dopo 96 ore ) , tali risultati sono stati posti in relazione ad un precoce edema citotossico , che giustificherebbe la iniziale diffusione ristretta , e ad una successiva lisi cellulare , che , al contrario , determinerebbe laumento della diffusivit protonica . questi risultati potrebbero avere un significativo impatto in pediatria , sul trattamento della malattia di legg - calvperthes , nella quale importante conoscere lestensione dellischemia e la sua durata , al fine di stabilire la terapia pi appropriata . 
questi risultati sembrano confermati , ad ora , da riscontri aneddotici : edema midollare con adc medio di 1 , 22x10 - 3 mm2 / s , rispetto a valori di normalit di 0 , 56x10 - 3 mm2 / s [ 50 ]  . 
un aumento della diffusione rilevabile anche nelle osteomieliti , a causa verosimilmente delliperemia , dellaumento dello spazio extracellulare e della presenza di foci necrotici ; non sembra tuttavia che il dwi permetta di differenziare laumento delladc presente nei focolai flogistici da quello delledema midollare e delle osteocondriti [ 63 ]  . 
orsola malpighi , azienda ospedaliero - universitaria di bologna , via massarenti 9 , i - 40138 bologna , italy 2servizio sanit pubblica , direzione generale sanit e politiche sociali , regione emilia - romagna , via a . 
the italian decree of law 187 / 2000 provides for many fulfilments relevant to justification and optimisation of medical exposures that can complicate the daily work of radiology departments if considered as mere legal requirements . 
to this end , the emiliaromagna region carried out an initial assessment of medical exposures to its population in 2001 followed by a second survey taking into account new dosimetric evaluations . 
study of distribution of the entrance skin dose for different examinations in single hospitals showed no systematic differences in kilovoltage settings versus dose whereas the number of examinations tended to be inversely proportional to dose . 
these trends could be explained by the fact that in hospitals where many examinations of the same type are performed , operators , equipment and procedures are well integrated , leading to a level of specialisation that allows efficient interaction in order to deliver an optimal dose . 
the evaluations performed in this study show that this type of analysis heavily relies not only on the cooperation of all professionals responsible for patient radiation protection but riassunto obiettivo . 
il decreto legislativo 187 / 2000 prevede numerosi adempimenti relativi alla giustificazione ed alla ottimizzazione delle esposizioni mediche che , se vissuti come meri obblighi di legge , possono appesantire il lavoro quotidiano delle unit operative di radiologia . 
al contrario , la normativa dovrebbe essere unutile occasione per analizzare ed ottimizzare le modalit operative adottate : in questa ottica la regione emilia - romagna ha effettuato nel 2001 una prima valutazione di dose alla popolazione regionale dovuta alle esposizioni a scopo medico e , successivamente , una seconda raccolta dati che tenesse conto delle nuove valutazioni dosimetriche eseguite . 
si sono innanzitutto definite le tipologie di esame che si volevano prendere in considerazione : in radiodiagnostica convenzionale e tomografia computerizzata si sono identificati dodici differenti esami distinti in macroaggregati e facilmente identificabili . 
nello studio delle distribuzioni delle dosi di ingresso per i vari esami nelle singole aziende sanitarie in generale non si evidenziano differenze sistematiche dei kv impostati in funzione della dose , mentre si pu notare che il numero di prestazioni eseguite tende ad essere inversamente proporzionale alla dose erogata . 
questi andamenti potrebbero essere spiegati con il fatto che , laddove si eseguono molti esami appartenenti ad una medesima tipologia , gli operatori , le apparecchiature e le procedure sono ben integrati tra di loro e raggiungono una specializzazione che permette di interagire in maniera ideale per erogare una dose ottimale . 
le valutazioni effettuate dimostrano che in questa tipologia di analisi sono fondamentali sia la collaborazione tra tutte le figure professionali che si occupano della radioprotezione del paziente sia lesperienza maturata nel corso delle varie campagne di raccolta dati , in quanto la fase di censimento dei dati molto delicata e pu inficiare , se non condotta accuratamente , tutte le elaborazioni posteriori . parole chiave dosimetria misura esposizione radioprotezione paziente introduction introduzione by supplying regulatory tools to verify whether the principles of justification and optimisation are effectively implemented in clinical practice , decree of law no . 
in this context , studies investigating the doses delivered to patients , clearly in conjunction with analysis of image quality , are becoming increasingly important at both the national [ 25 ] and international level [ 610 ]  . it is therefore hoped that the many requirements provided for by this decree will not merely be seen as legal duties but as an opportunity to analyse , or reanalyse , our working practices : in particular , article 12 of the decree requires that regions carry out assessments of medical exposures as regards the regions population and specific reference groups in the region , that such assessments be carried out periodically and that the results be sent to the ministry of health . 
187 / 2000 richiede che le regioni provvedano a valutare le esposizioni a scopo medico con riguardo alla popolazione regionale e a gruppi di riferimento della stessa e che tale valutazione sia effettuata periodicamente ed inviata al ministero della salute . 
per ottemperare al citato obbligo di legge la regione emilia - romagna ha effettuato una prima valutazione di dose nel 2001 , relativa allanno 2000 [ 11 ] e , anche se non espressamente richiesto dalla legislazione , ha effettuato una nuova raccolta dei dati relativa allanno 2002 , sfruttando il fatto che entro il 31 / 12 / 2002 i responsabili degli impianti radiologici avvalendosi degli esperti in fisica medica dovevano verificare il rispetto dei livelli diagnostici di riferimento ( ldr ) e quindi potessero rendere disponibili dati dosimetrici pi attendibili . 
in questo lavoro sono illustrati i risultati di questa seconda raccolta dati , per evidenziare la variabilit che si pu riscontrare tra le dosi correlate ad esami di radiologia diagnostica anche in una regione come lemilia - romagna , in cui il livello quali - quantitativo delle prestazioni sanitarie erogate si presenta omogeneo a livello territoriale ; sono stati inoltre effettuati un confronto ed una analisi dei dati raccolti rispetto a quanto riscontrato in analoghe indagini riportate in letteratura . materials and methods the first pitfall in analysing the radiation dose imparted by the various imaging procedures is of a lexical nature and related to the possible ambiguity of terms such as radiological examination of the head , chest , spine , etc : even european un primo rischio che si presenta nellaffrontare la problematica delle dosi da radiazioni ionizzanti impartite nelle varie prestazioni diagnostiche di tipo lessicale e consiste nel riscontramateriali e metodi g . 
the first problem when planning our dose survey among the 19 emilia - romagna region hospitals was therefore to specify the types of examinations we intended to consider . as regards conventional radiology and computed tomography ( ct ) , we selected 12 easily identifiable examinations divided into macroaggregates , which are shown in the first column of table 1 . 
the survey on the number of examinations was carried out by asking each hospital for their list of examinations divided by type and grouped by the ministry of health nomenclature code : examination types and codes are shown in the second column of table 1 . if incomplete , data provided by a given hospital were extrapolated and completed on the basis of means or proportions derived from the other hospitals . 
with regard to private healthcare facilities under contract with the regional health system , we extrapolated frequencies of ambulatory services from data of the regional specialised ambulatory healthcare . as concerns dosimetry , considering that all radiological equipment undergoes regular quality control tests as part of quality assurance programmes implemented by equipment holders and medical physics experts , each hospital was asked to provide , for each of the examinations in table 1 , some technical parameters related to both the examination protocol and the equipment . the hospitals were also invited to supply the numerical value of dosimetric measurements made by the medical physics experts : entrance skin dose ( esd ) for conventional and paediatric imaging , ct dose index ( ctdi ) and doselength product ( dlp ) for ct . in particular , they were asked to supply the mean of these values as measured on all x - ray tubes performing a given examination , weighted for the number of exams performed on each equipment . 
il primo problema che si dovuto affrontare quando si pianificata la rilevazione delle dosi nelle 19 aziende sanitarie dellemilia - romagna ( as ) stato quindi specificare quali erano le tipologie di esame che si volevano prendere in considerazione . 
per quanto riguarda la radiodiagnostica convenzionale e la tomografia computerizzata ( tc ) si sono identificati dodici differenti esami distinti in macroaggregati e facilmente identificabili , che sono riportati nella prima colonna della tabella 1 . 
la raccolta dei dati relativi al numero di prestazioni stata effettuata richiedendo direttamente alle as il dettaglio del numero di esami suddivisi per tipologia e raggruppati per codice del nomenclatore ministeriale : tali tipologie e codici sono riportati nella seconda colonna della tabella 1 . 
i dati forniti da una determinata as , qualora siano risultati incompleti , sono stati estrapolati e completati in base a medie o a proporzioni effettuate sui dati delle altre as . 
relativamente alle strutture sanitarie che operano in regime di convenzione con il servizio sanitario regionale , si sono ottenute le frequenze delle prestazioni ambulatoriali tramite unestrapolazione della banca dati dellassistenza specialistica ambulatoriale regionale . 
per quanto attiene al discorso pi propriamente dosimetrico , premesso che regolari controlli di qualit sulle apparecchiature radiologiche vengono eseguiti allinterno dei programmi di garanzia della qualit predisposti dai responsabili degli impianti radiologici congiuntamente con gli esperti in fisica medica , ad ognuna delle as sono stati richiesti , per gli stessi esami descritti nella tabella 1 , alcuni parametri tecnici relativi sia al protocollo di esecuzione dellesame sia allapparecchiatura . 
le as sono state altres invitate a fornire il valore numerico delle grandezze dosimetriche misurate dagli esperti in fisica medica : dose di ingresso ( entrance skin dose , esd ) per la radiodiagnostica convenzionale e pediatrica , indice di dose tc ( computed tomography dose index , ctdi ) e prodotto dose - lunghezza ( dose - length product , dlp ) per la tc . 
in particolare , stata richiesta la media di queste grandezze effettuata su tutti i tubi radiogeni che eseguivano un dato esame , pesata in base al numero di esami effettuati su ogni apparecchiatura . 
ad esempio , la grandezza esdaddome relativa allesame addome in radiodiagnostica convenzionale stata calcolata per ogni as in base alla formula : esdaddome = n i = 1 ( ni / p ) ( esdaddome ) i dove : n = numero di tutte le apparecchiature radiologiche di quella as su cui viene eseguito lesame addome ; ni = numero di prestazioni ( cio di esami addome ) eseguite con lapparecchiatura radiologica i - esima ; ( esdaddome ) i = esd valutata ai sensi dellart . 
figures 1 and 2 show , for the various examinations , esd distributions ( in adult conventional radiology ) among single hospitals , versus a significant technical parameter ( kvp ) and the number of procedures performed , respectively . 
it should be noted that in both figures data are shown in increasing order of esd so that the order of the hospitals changes in each figure and is not the same as that shown in the tables . 
lordine da 1 a 19 il medesimo per entrambe le tabelle ed stato attribuito casualmente alle singole as . nelle figure 1 e 2 sono mostrate , per i vari esami , le distribuzioni delle esd ( nella radiologia convenzionale adulti ) tra le singole as in relazione , rispettivamente , a un parametro tecnico significativo ( kv impostati ) ed al numero di prestazioni eseguite . 
si fa notare che in entrambe le figure i dati sono sempre stati ordinati per esd crescente e quindi lordine delle singole as varia in ogni grafico e non lo stesso di quello riportato nelle tabelle . nella tabella 4 , infine , sono mostrati i rapporti esdmassima / esdminima per le varie proiezioni e tra le varie as , confrontandoli con valori di letteratura [ 13 , 14 ]  . discussion discussione data collected lend themselves to different possible analyses and interpretations , but two aspects will be discussed here : the first is related to the characteristics of each hospital and the second concerns the variability of examinations among all hospitals . to analyse the first aspect , figure 1 shows , for the various examinations , esd distributions in single hospitals in relation to a significant technical parameter ( kvp )  . 
to see if there is a more significant variable influencing esds , figure 2 shows , for the various examinations , esd distributions in single hospitals in relation to the number of examinations performed . 
it can be noted that the number of examinations tends to be inversely related to esd ( in other words , the dose imparted in a given examination tends to be lower in hospitals performing many examinations of the same type )  . 
this could be accounted for by the fact that where many examinations of the same type are performed , there is excellent integration among radiologists , equipment and procedures , enabling a level of specialisation that allows delivery of an optimal dose , that is , a dose which , associated with a correctly optimised and justified radiological procedure [ 15 ] , is the lowest possible dose compatible with a good quality image . 
this statement is consistent with the concept of clinical competence , whereby quantity may help attain a greater competence : the statistical significance of this trend will be studied more in depth when we have all data from our next survey , which are expected to be collected more accurately by single hospitals . as regards variability of examinations among all hospitals , table 4 illustrates the esdmax / esdmin ratio . 
because all hospitals perform the same examinations , we coni dati che sono stati raccolti si prestano a diverse possibili analisi ed interpretazioni : in questa sede verranno discussi essenzialmente due aspetti , il primo pi legato alle caratteristiche della singola as , il secondo riguardante la variabilit degli esami tra tutte le as . per analizzare il primo aspetto , nella figura 1 sono mostrate per i vari esami le distribuzioni delle esd nelle singole as in relazione a un parametro tecnico significativo ( kv impostati ) : come si pu notare dai vari grafici , in generale non si evidenziano differenze sistematiche di questo parametro tra le diverse realt della regione in funzione della dose . 
per vedere se esiste una variabile pi significativa nellinfluenzare le dosi in ingresso , nella figura 2 sono state riportate , per i vari esami , le distribuzioni delle esd nelle singole as in relazione al numero di prestazioni eseguite : in questo caso si pu notare che il numero di prestazioni tende ad essere inversamente proporzionale alla esd erogata ( cio la dose per un dato esame tende ad essere pi bassa nelle as in cui vengono effettuati molti esami di quel tipo )  . 
questi andamenti potrebbero essere spiegati con il fatto che , laddove si eseguono molti esami appartenenti ad una medesima tipologia , gli operatori , le apparecchiature e le procedure sono ben integrati tra di loro e raggiungono una specializzazione che permette di interagire in maniera ideale per erogare una dose ottimale , intesa come quella dose che , associata ad una procedura radiologica correttamente ottimizzata e giustificata [ 15 ] , risulta la pi bassa possibile e fornisce unimmagine di buona qualit . 
questa affermazione risulta peraltro coerente con il concetto di competenza clinica , secondo il quale la quantit pu aiutare a raggiungere una maggiore competenza : la significativit statistica di questo andamento verr studiata in maniera pi approfondita quando si avranno a disposizione i dati relativi alla successiva indagine , che dovrebbero essere raccolti in maniera pi accurata dalle singole as . per quanto riguarda la variabilit degli esami considerati tra tutte le as , nella tabella 4 si esamina il parametro rapporto esdmassima / esdminima : questo un indicatore gi utilizzato in letteratura [ 13 , 14 ] per valutare lampiezza delle differenze tra diverse strutture di radiologia . 
2 distribuzioni delle dosi in ingresso ( esd ) tra le singole aziende sanitarie regionali ( as ) in relazione al numero di prestazioni eseguite per i vari esami . sidered correct to analyse those with the lowest and highest esd values . the esdmax / esdmin ratios obtained for the various projections and hospitals were compared with those reported in the literature : in urography and abdomen examinations these ratios were found to be similar to those previously reported for a large italian hospital [ 13 ] , which has more than one radiology department and where one would expect ratios to be lower as a result of greater uniformity among departments . 
in the chest lat ( lateral ) , lumbar spine ap ( anteroposterior ) , pelvis ap , skull ap / pa ( posteroanterior ) and skull lat projections , ratios observed in the present study were almost double those reported in the italian study [ 13 ] ; in the chest pa , lumbar spine lat and lumbosacral spine views , ratios obtained in this study were definitely higher . 
the cause of this greater dispersion could be a physiological scale factor in that , as stated above , it is natural there are greater differences in clinical protocols and radiological equipment among hospitals of a region than there are within a single hospital , and it is just as natural that these differences will be reflected in broader esd ranges . 
 [ 14 ] for lumbar spine ap , pelvis ap , skull ap / pa , skull lat and abdomen views but highi rapporti esdmassima / esdminima ottenuti in questo studio per le varie proiezioni e tra le varie as sono confrontati con quelli riportati in letteratura : si nota che , negli esami urografia e addome , questi rapporti sono dello stesso ordine di grandezza di quanto riscontrato in uno studio italiano in un grande ospedale [ 13 ] dove sono presenti pi unit operative di radiologia e dove ci si aspetta effettivamente che tali rapporti siano minori , in quanto dovrebbe esserci pi uniformit tra le varie unit operative ; nelle proiezioni torace lat , rachide lombare ap , pelvi ap , cranio ap / pa e cranio lat i rapporti riscontrati nel presente lavoro sono circa il doppio di quanto riportato nello studio italiano [ 13 ] ; nelle proiezioni torace pa , rachide lombare lat e rachide lombosacrale , infine , i rapporti riscontrati in questo lavoro sono decisamente pi elevati rispetto allo studio italiano citato . la causa di questa maggiore dispersione potrebbe risiedere in un fisiologico fattore di scala , in quanto , come sopra accennato , inevitabile che allinterno di una regione siano presenti , rispetto a quanto avviene in un ospedale seppure grande , maggiori differenze nei protocolli clinici e nelle apparecchiature radiologiche tra le diverse as ed altrettanto inevitabile che queste differenze si riflettano in pi ampi intervalli di esd . 
per verificare se questa ipotesi di un fattore di scala potesse essere considerata valida , si esteso il confronto ad un altro studio effettuato su una dimensione a scala nazionale [ 14 ] : in effetti si riscontrato , come riportato nella tabella 4 , che nelle proiezioni rachide lombare ap , pelvi ap , cranio ap / pa , cranio lat e addome i rapporti esdmassima / esdminima sono inferiori a quanto riportato nello studio della realt nazionale , mentre nelle proiezioni torace lat , torace pa e rachide lombare lat i rapporti riscontrati nel nostro studio sono superiog . 
this finding might have been expected , as it is known [ 16 ] that the chest is a physically complex radiographic object , partly in view of the many different diseases that may affect it , and that therefore there may be many differences in clinical protocols even in a relatively uniform territory such as our region . 
although the esds imparted by single chest examinations are low , widespread use of this examination ( in terms of number of examinations , it accounts for approximately 50% of all conventional radiography studies ) urgently calls for its homogenisation and optimisation . 
questi confronti , pur con tutti i limiti connessi ad una analisi semi - quantitativa come quella qui presentata , sembrano dimostrare che il fattore di scala abbia comunque un ruolo fondamentale nella variabilit dei valori di esd tra le varie as e che soprattutto lesame torace quello pi critico , nel senso che presenta le maggiori differenze nelle esd . 
questo risultato del resto era abbastanza prevedibile in quanto ben noto [ 16 ] che il torace un oggetto radiografico fisicamente complesso anche per le molte e diverse patologie che possono riscontrarsi e quindi possono essere presenti molte differenze a livello di protocollo clinico pur in un ambito territorialmente abbastanza omogeneo come quello della nostra regione : sicuramente vero che le esd del singolo esame toracico sono basse , per la sua diffusione alquanto ampia ( infatti come numero di prestazioni rappresenta circa il 50% di tutte le indagini radiografiche tradizionali ) rende importante una sua omogeneizzazione ed ottimizzazione . it is hoped that the numerous duties provided for by decree auspicabile che i numerosi adempimenti previsti dal d . 
lgs. 187 / 2000 non vengano interpretati come un mero assolvimento conclusioni conclusions table 3 average dose - length product ( dlp ) values ( mgy cm ) for computed tomography ( ct ) examinations provided by emilia - romagna region hospitals tabella 3 valori di dlp medi ( in mgy cm ) per gli esami tc forniti dalle varie aziende sanitarie della regione emilia - romagna g . 
evaluations performed in our study demonstrate that both cooperation of all professionals involved in patient radiation protection and experience gained in the various surveys are fundamental in this type of study in that data collection is a very delicate phase which , if not accurately conducted , can invalidate all subsequent data processing . 
a new survey of the same hospitals for the years after 2002 will allow us to make a more accurate quantitative evaluation of the issues addressed in this study . di obblighi di legge , ma contribuiscano a migliorare realmente le prestazioni di radiologia diagnostica . 
in particolare , il monitoraggio della dose alla popolazione pu essere un utile strumento di ottimizzazione in quanto permette di effettuare valutazioni pragmatiche e non teoriche , usando dati raccolti sul campo , senza appesantire le normali attivit lavorative : le regioni infatti possono raccogliere i dati in maniera efficiente , nel senso di usare valutazioni che devono essere comunque effettuate dal responsabile dellimpianto radiologico e dallesperto in fisica medica . 
most mediastinal abnormalities are initially suspected following chest radiography ; the need for further investigation and the most appropriate imaging modality are largely dictated by the tentative diagnosis made on this examination . 
although routine chest radiography initiates the evaluation of mediastinal disorders , it is rarely diagnostic : notable exceptions are teeth or bones within a mass , which are diagnostic of a teratoma ; air / fluid levels suggest an oesophageal origin , hernia , cyst , or abscess . 
however , even sct with contrast material is occasionally diagnostic ( a confident diagnosis can be made of some lesions such as mature teratoma and mediastinal goiter ) but is usually sufficient for preoperative evaluation before mediastinotomy or mediastinoscopy : it is instrumental in planning further diagnostic workup . 
thyroid scanning with radioactive iodine is useful in identifying and evaluating masses of suspected thyroid origthe role of fluorodeoxyglucose positron emission tomography ( fdg - pet ) in mediastinal diseases continues to be evaluated : it has potential for differentiating between benign and malignant disease and is expected to play a more extensive role in the imaging of mediastinal neoplasms in the future . 
in this paper , the radiological features of masses located in the anterior mediastinum are discussed , with particular reference to radiographic and ct patterns useful to the clinicians everyday practice . key words anterior mediastinum standard radiography computed tomography magnetic resonance imaging positron emission tomography sonography riassunto il mediastino suddiviso in compartimenti ( anteriore , medio , posteriore ) sulla base della radiografia del torace in proiezione laterale . 
la maggior parte delle alterazioni mediastiniche sono sospettate alla radiografia del torace ; lopportunit di ulteriori indagini e la metodica ottimale di imaging sono dettate dalla diagnosi presuntiva formulata con tale esame . 
sebbene un radiogramma standard del torace inizi la valutazione delle malattie del mediastino , raramente diagnostico : fanno eccezione denti o osso entro una massa , che sono patognomonici per teratoma ; livelli aria - fluido suggeriscono lorigine esofagea , unernia , cisti o ascessi . 
tuttavia , anche la stc con mezzo di contrasto occasionalmente diagnostica ( possono essere formulate diagnosi presuntive di alcune lesioni , come il teratoma maturo e lo struma ) , ma in genere sufficiente per una valutazione preoperatoria prima di una mediastinotomia o di una mediastinoscopia : utile nella programmazione di ulteriori indagini diagnostiche . 
il ruolo della fdg - pet nelle malattie del mediastino in corso di valutazione : ha un valore potenziale nella diagnosi differenziale tra malattia benigna e maligna ed in futuro rivestir un ruolo pi estensivo nellimaging delle neoplasie mediastiniche . in questo lavoro , vengono enfatizzate le caratteristiche radiologiche delle masse localizzate nel mediastino anteriore , con particolare riferimento alla semeiotica radiologica , alla radiografia del torace ed alla tomografia computerizzata , utili nella pratica clinica quotidiana . parole chiave mediastino anteriore radiologia tradizionale tomografia computerizzata risonanza magnetica tomografia ad emissione di positroni ecotomografia s.m. 
to fully understand the nosography of this region , it is therefore indispensable to make some considerations on its contents and semantics , which are functional to the study techniques and their signs . il mediastino anteriore uno spazio del torace pressoch virtuale che , tuttavia , pu essere sede di numerose condizioni patologiche . 
sono dunque indispensabili , ai fini della piena comprensione della nosografia , alcune premesse sul contenuto e sulla semantica topografica , funzionali alla analisi delle tecniche di studio ed allapprendimento della semeiotica ad esse relative . contents of the anterior mediastinum different structures are present in the anterior mediastinum : containing structures ( muscles , ligaments , fat tissue ) , vessels , lymphatic tissue , parenchymatous organs : the transversus thoracis or triangularis sterni , which is located on the inner side of the anterior chest wall , delimits the mediastinum anteriorly , but its thickness is not sufficient to produce a corresponding radiological pattern . 
the inner aspect of the muscle is lined by the endothoracic fascia . the sternopericardial ligaments , upper and lower , extend between the pericardium and the sternal manubrium and xiphoid process , respectively . the internal thoracic artery or internal mammary artery originates from the first portion of the subclavian artery , descends behind the first six costal cartilages and ends at the sixth intercostal space giving rise to the musculophrenic artery and the superior epigastric artery ; it is accompanied by a chain of lymph nodes and by two satellite veins . the anterior mediastinal arteries ( which distribute to the areolar tissue and anterior mediastinum lymph nodes to the remains of the thymus and to the pericardium ) and the sternal branches ( for the transversus thoracis and the posterior aspect of the sternum ) are collateral branches of the internal mammary artery . the lymph node stations of the anterior mediastinum are the parietal lymph nodes of the internal mammary chains and the diaphragmatic chains , and the visceral lymph nodes of the anterior mediastinum chains . the lymph nodes of the internal mammary chain or sternal lymph nodes may vary from six to ten in number . 
they are located deep in the thoracic cavity on the endothoracic fascia in the intercostal spaces ivi between the costal cartilages , adjacent to the medial or lateral margin of the internal mammary vessels . 
the efferent collectors flow into a single trunk , which reaches the internal jugular vein directly at its confluence with the subclavian vein or the thoracic duct on the left and the subclavian trunk on the right . the anterior mediastinum lymph nodes form different clusters : right and left anterior mediastinum lymph nodes , lymph nodes of the transverse chain , and diaphragmatic nodes ; they receive lymphatic collectors from the diaphragm , pleura , heart , pericardium , lung , thymus , thyroid and trachea . 
they are connected to the sternal lymph contenuto del mediastino anteriore nel mediastino anteriore si descrivono diverse entit : strutture di contenimento ( muscoli , legamenti , tessuto adiposo ) , vasi , tessuto linfatico , organi parenchimatosi . il muscolo trasverso del torace o triangolare dello sterno , che situato sulla faccia interna della parete toracica anteriore , limita anteriormente il mediastino , ma il suo spessore non sufficiente a dare un corrispettivo reperto radiografico . 
la faccia interna del muscolo rivestita dalla fascia endotoracica . i legamenti sternopericardici , distinti in superiore ed inferiore , sono tesi tra pericardio e , rispettivamente , manubrio sternale e processo xifoideo . larteria toracica interna o mammaria interna origina dalla prima porzione dellarteria succlavia , discende in basso , dietro le prime sei cartilagini costali , e termina al vi spazio intercostale dando larteria muscolofrenica e larteria epigastrica superiore ; accompagnata da una catena di linfonodi e da due vene satelliti . le arterie mediastiniche anteriori ( che si distribuiscono al tessuto areolare ed ai linfonodi del mediastino anteriore , ai residui del timo ed al pericardio ) ed i rami sternali ( per il muscolo trasverso del torace e la faccia posteriore dello sterno ) sono rami collaterali dellarteria mammaria interle stazioni linfonodali del mediastino anteriore sono rappresentate da linfonodi parietali , delle catene mammarie interne e delle catene diaframmatiche , e da linfonodi viscerali , delle catene del mediastino anteriore . i linfonodi della catena mammaria interna o linfonodi sternali variano da sei a dieci . 
sono situati in profondit alla parete toracica , sulla fascia endotoracica negli spazi intercostali i - vi , tra le cartilagini costali , adiacenti al margine mediale o laterale dei vasi mammari interni . 
i collettori efferenti confluiscono in un tronco unico che raggiunge direttamente la vena giugulare interna alla sua confluenza con la vena succlavia oppure a sinistra il dotto toracico e a destra il tronco succlavio . i linfonodi mediastinici anteriori costituiscono diversi raggruppamenti : linfonodi mediastinici anteriori di destra , di sinistra , della catena trasversa , diaframmatici ; ricevono collettori linfatici che provengono dal diaframma , dalla pleura , dal cuore , dal pericardio , dal polmone , dal timo , nodes , and they send efferent collectors to the bronchomediastinal trunks . the anterior mediastinum also contains , at its lower boundary , the anterior and middle diaphragmatic lymph nodes or cardiophrenic angle nodes . 
the anterior diaphragmatic lymph nodes are situated on the diaphragm dome in front of the anterior leaflet of the central tendon behind the xiphoid process and the cartilage of the seventh rib . 
the middle diaphragmatic lymph nodes are found close to the point of innervation of the diaphragm by the phrenic nerve ; they are often missing on the left side . the thymus is a lymphoepithelial organ located for the most part in the anterior mediastinum with a small portion in the neck . 
it is a transient organ that is considerably developed in the foetus and during the early years of life ; in the young adult it undergoes fatty involution , with wide individual variability . occasionally , the anterior mediastinum may contain the lower pole of the thyroid or an ectopic parathyroid that has migrated with the thymus . topography of the anterior mediastinum to map its contents , the anterior mediastinum may schematically be divided into two regions : upper and lower . 
at this level , behind the anterior chest wall and lateral to the sternum , lie the parietal nodes of the internal mammary chain ( or sternal nodes ) , arranged along the course of the mammary vessels and , at a deeper level , three prevascular chains of the anterior mediastinum ( anterior mediastinal lymph nodes )  . 
finally , the anterior mediastinum communicates widely with the neck , middle mediastinum and , through the sternal fasciae of the diaphragm , with the preperitoneal lax connective tissue of the anterior abdominal wall . radiological anatomy of the anterior mediastinum . compartment definitions : anatomical and radiological classification the mediastinum extends from the sternum to the spine in an anteroposterior direction and from the thoracic inlet to the diaphragm in a craniocaudal direction . 
sono connessi con i linfonodi sternali ed inviano collettori efferenti ai tronchi broncomediastinici . il mediastino anteriore contiene anche , al suo limite inferiore , i linfonodi diaframmatici anteriori e medi o dellangolo cardiofrenico . i linfonodi diaframmatici anteriori sono posti sulla convessit del muscolo al davanti della fogliola anteriore del centro tendineo , dietro il processo xifoideo e la cartilagine costale della vii costa . 
i collettori efferenti si connettono con i linfonodi mediastinici e con i linfonodi sternali . i linfonodi diaframmatici medi sono prossimi al punto di innervazione del diaframma da parte del nervo frenico ; sono spesso assenti dal lato sinistro . il timo un organo linfoepiteliale localizzato , per la maggior parte , nel mediastino anteriore e , per una piccola porzione , nel collo . 
un organo transitorio , notevolmente sviluppato nel feto e nei primi anni della vita postnatale : va incontro ad involuzione adiposa nel giovane adulto , peraltro con un notevole grado di variabilit individuale . occasionalmente , nel mediastino anteriore pu localizzarsi il polo inferiore della tiroide o una paratiroide ectopica migrata con il timo . topografia del mediastino anteriore per collocare topograficamente il contenuto , per ragioni di semplicit , il mediastino anteriore pu , del tutto schematicamente , essere distinto in due distretti : superiore ed inferiore . superiormente rappresentato dalla loggia timica . 
a questo livello si localizzano , a ridosso della parete toracica anteriore e di lato allo sterno , i linfonodi parietali della catena mammaria interna ( o linfonodi sternali ) , disposti lungo il decorso dei vasi mammari e , pi profondamente , tre catene linfonodali prevascolari del mediastino anteriore ( linfonodi mediastinici anteriori )  . 
inferiormente rappresentato dal tessuto adiposo extra - pericardico con lestensione laterale , pi o meno pronunciata , dei cuscinetti adiposi paracardiaci . agli angoli cardiofrenici , si localizzano alcuni gruppi dei linfonodi parietali delle catene diaframmatiche ( linfonodi diaframmatici anteriori e medi o dellangolo cardiofrenico )  . infine , il mediastino anteriore comunica ampiamente con collo , mediastino medio e , attraverso i fasci sternali del diaframma , con il tessuto lasso preperitoneale della parete addominale anteriore . anatomia radiologica del mediastino anteriore . 
definizioni di compartimento : classificazione anatomica e radiologica il mediastino si estende , in senso antero - posteriore , dallo sterno alla colonna vertebrale e , in senso cranio - caudale , s.m. 
the mediastinum may be divided into three compartments on the basis of the lateral chest radiograph : anterior , central or middle and posterior [ 3 , 4 ]  . 
the anterior mediastinum lies behind the sternum and in front of the great vessels and pericardiuit is laterally bounded by the mediastinal pleurae in contact with the anteromedial portion of each lung . 
inferiorly it extends to the diaphragm whereas its upper boundary is arbitrarily considered the plane extending from the inferior margin of the manubrium to the lower edge of the fourth dorsal vertebra . 
however , because the anterior edge of the ascending aorta and brachiocephalic veins are difficult to visualise radiologically and the anterior mediastinal masses often project onto the heart , felsons classification [ 2 ] included among the disorders affecting this compartment lesions predominantly located in the region bounded posteriorly by a fig . 
anatomicamente suddiviso in superiore ( al di sopra del piano che passa per larticolazione manubrio - corpo sternale e la superficie inferiore della iv vertebra dorsale ) ed inferiore ( ulteriormente distinto nei compartimenti anteriore o prevascolare , medio o cardiovascolare e posteriore o postvascolare )  . 
tuttavia , sussistono molti punti criticabili in questa classificazione , come sottolineato da heitzman [ 1 ] e felson [ 2 ] : , in realt , basata solo parzialmente sullanatomia del mediastino , dal momento che lunica struttura di riferimento per la sepimentazione il pericardio , anche perch non esistono piani anatomici o fasciali che suddividano il mediastino in compartimenti definiti ; pertanto , una suddivisione anatomica ha un corrispettivo radiologico modesto . radiologicamente , nonostante lesistenza di numerose classificazioni , nessuna tra queste ha trovato unanime consenso . 
il mediastino pu essere distinto , sulla base del radiogramma del torace in proiezione laterale , in tre compartimenti : anteriore , centrale o medio e posteriore [ 3 , 4 ]  . 
infine , pi di recente , fraser ha definito compartimento mediastinico anteriore la regione situata al davanti di una linea tracciata , sempre sul radiogramma laterale del torace , lungo il margine anteriore della silhouette cardiaca ed i vasi brachiocefalici [ 4 ]  . line passing through the anterior edge of the trachea and the posterior margin of the heart . 
more recently , fraser defined the anterior mediastinal compartment as the region situated in front of a line drawn on the lateral chest radiograph along the anterior margin of the cardiac silhouette and the brachiocephalic vessels [ 4 ]  . in conclusion , although the best classification is still a matter of debate , alternative classifications to that proposed by heitzman appear to have limited clinical utility . study techniques and semeiotics chest imaging offers two main possibilities to study this region : conventional radiology ( cr ) and computed tomography ( ct )  . 
cr has an important role given that the first step in identifying a mediastinal disorder is standard chest radiography , the semeiotics of which relies on the assessment of mediastinal lines . 
in addition , sonography and nuclear medicine functional imaging are useful tools for further investigation in selected cases and in some instances may even become the examinations of choice ( endocrine disorders , diseases of the thyroid and parathyroid , ectopic glands )  . standard radiography pathological processes affecting the anterior mediastinum are frequently silent , and consequently , their radiological detection is incidental : around 40% of mediastinal masses are asymptomatic [ 5 ]  . 
radiographs with posteroanterior ( pa ) and laterolateral ( ll ) views represent the basic imaging modality in the study of the chest and mediastinuchest radiography allows localisation of the pathological process : orthogonal projections can demonstrate site , size and density and the presence of calcifications , thereby narrowing the range of differential diagnoses . 
moreover , fluoroscopic examination and opacification of the oesophagus will provide valuable complementary information . a mediastinal mass may : be directly visible and protrude unior bilaterally from the mediastinum and have varying degrees of lobulation ; cause an increase in the density of part of the mediastinum ; change the appearance of the mediastinal lines . 
in particular , judging normality on a radiograph presupposes adequate knowledge of the pattern formed by the mediastinal lines the interfaces between lung and mediastinum that can be observed in specific conditions . 
it should be recalled that radiologically , a line is formed when certain conditions are met : there is a difference in density between two adjacent tissues ; the two tissues are separated by a curved surface ; the beam hits this surface tangentially . these conditions occur often in chest radiography where the s.m. 
il ruolo della rt importante , poich il primum movens alla identificazione di una patologia mediastinica la radiografia standard del torace , la cui semeiologia affidata alla valutazione delle linee mediastiniche . la comprensione anatomica stata , peraltro , enormemente facilitata dallavvento delle tecniche di imaging tc e di risonanza magnetica ( rm ) che consentono la precisa localizzazione delle varie strutture mediastiniche . 
inoltre , lecotomografia e limaging funzionale di medicina nucleare costituiscono , in casi selezionati , utili strumenti informativi di approfondimento diagnostico , talvolta rappresentando persino esami di elezione ( patologia endocrina , tiroidea e paratiroidea , in ghiandole ectopiche )  . radiogramma standard i processi patologici del mediastino anteriore sono frequentemente silenti nella sintomatologia ed il riscontro radiologico pertanto incidentale : infatti circa il 40% delle masse mediastiniche asintomatico [ 5 ]  . 
le radiografie in proiezione pa e ll rappresentano lindagine radiologica base nello studio del torace , in generale , e del mediastino , nello specifico . la rt consente , in prima istanza , di localizzare il processo patologico : le proiezioni ortogonali definiscono la sede , la dimensione , la densit , la presenza di calcificazioni restringendo il ventaglio delle diagnosi differenziali . 
inoltre , lesame radioscopico e la opacizzazione dellesofago offrono valide informazioni complementari . la massa mediastinica pu essere direttamente visibile e protrudere monoo bilateralmente dal mediastino con contorni pi o meno polilobulati , determinare un aumento di densit di un distretto del mediastino o modificare la morfologia delle linee mediastiniche . in particolare , il giudizio di normalit dellimmagine radiografica presuppone una adeguata conoscenza della semeiotica basata sulle linee mediastiniche , rappresentate dalle interfacce polmone - mediastino osservate in particolari condizioni . 
it is therefore possible , based on the knowledge of the plane to which the mediastinal lines belong , to have a provisional three - dimensional ( 3d ) analysis of the mediastinum in a single view . 
normal morphology of these lines is a reliable indication of normality of the single mediastinal compartments : the presence of space - occupying lesions will , instead , cause their displacement or cancellation . 
 pa radiogram : anterior junction line or anterior commissure as the two lungs approximate anteromedially , they are separated by four layers of pleura and a variable amount of fat , giving rise to a septum of varying thickness : the anterior junction line ( ajl ) or anterior mediastinal line or anterior commissure . 
this line is present when there is apposition of the pleurae behind the sternusince the anterior portions of the lungs join only from the sternal manubrium downwards , the ajl ends superiorly at the sternal angle of louis ( notch for second rib )  . 
2 una radiografia del torace in proiezione postero - anteriore mostra sia la linea di giunzione anteriore sia la linea di giunzione posteriore . tessuti siano separati da una superficie curva , il fascio incidente colga tangenzialmente tale superficie . 
queste condizioni si verificano spesso nellosservazione radiografica delle riflessioni dei polmoni ( aerati e quindi radiotrasparenti ) sul mediastino ( radiopaco ) : da qui la formazione di numerose linee , definite linee mediastiniche . 
nel mediastino anteriore riconosciamo linee in proiezione frontale ( linea di giunzione anteriore ) e laterale ( linea retrosternale ) [ 1 , 3 ]  . risulta cos possibile , avvalendosi della conoscenza del piano di appartenenza delle linee mediastiniche , una analisi presuntiva tridimensionale del mediastino in una sola vista . una regolare morfologia di tali linee affidabilmente indicativa della normalit dei singoli compartimenti mediastinici : la presenza di lesioni occupanti spazio determina invece un loro spostamento o la loro cancellazione . radiogramma in proiezione pa : linea di giunzione anteriore o commissura anteriore nellaccostarsi antero - medialmente , i due polmoni restano separati da quattro strati di pleura e da una certa quantit di tessuto adiposo interposto , venendosi a formare un setto di spessore variabile : la linea di giunzione anteriore ( lga ) o linea mediastinica anteriore o commissura anteriore . 
questa presente nel caso si realizzi il mutuo contatto delle pleure in sede retrosternale . dal momento che le porzioni anteriori dei polmoni si congiungono solo dal manubrio sternale in gi , la lga ha , come suo limite superiore , langolo sternale del luys ( ii incisura costale )  . 
in basso , si estende , per una lunghezza variabile ( 58 cm ) , sino al punto di contatto del cuore con i tessuti molli retrosternali . si apre verso lalto per delimitare la loggia timica . 
al contrario , lincapacit di visualizzare una lga precedentemente presente ( con ampliamento dellombra mediastinica anteriore o superiore ) , su indagini radiografiche seriate , suggerisce linterposizione di un espanso tra i margini anteriori dei due polmoni e induce il sospetto di malattia mediastinica . 
by contrast , the inability to see an ajl that was previously present ( with widening of the anterior or superior mediastinal shadow ) on serial radiographs suggests the interposition of a mass between the anterior margins of the two lungs and raises the suspicion of mediastinal disease . 
at the caudate end of the ajl , the presence of fat pads produces simple marginal lines at the paracardiac level , which are due to creation of the interface conditions described above . ll radiograph : retrosternal line or anterior extrapleural line in front of the ajl , behind the sternum , the pleural interfaces diverge as they create each pleural cavity . 
in normal individuals , the anterior portion of the lungs reaches the anterior chest wall , and therefore , the retrosternal area in front of the heart is completely radiolucent in the true lateral view . 
the opaque line with soft - tissue density ( the retrosternal line or anterior extrapleural line , ael ) , which evenly follows the profile of the sternum , is made up of retrosternal fat , internal s.m. 
inoltre , da sottolineare che , poich lo spessore della lga variabile , la sua valutazione non affidabile a meno di un suo grossolano ispessimento . allestremo caudale della lga , per effetto della presenza di cuscinetti adiposi , si possono riscontrare , a livello paracardiaco , linee semplici marginali , dovute allinstaurarsi delle condizioni di interfaccia precedentemente descritte . radiogramma in proiezione ll : linea retrosternale o linea extrapleurica anteriore al davanti della lga , dietro lo sterno , le interfacce pleuriche divergono nel costituire ciascuna cavit pleurica . 
negli individui normali la porzione anteriore dei polmoni raggiunge la parete toracica anteriore e , pertanto , larea retrosternale , al davanti del cuore , interamente radiotrasparente in proiezione laterale vera . la banda opaca , della densit delle parti molli ( che rappresenta la linea retrosternale o linea extrapleurica anteriore , lea ) uniformemente addossata al profilo dello sterno , costituita da tessuto adiposo retrosternale , vasi e linfatici mammari interni , nervi intercostali . 
inferiormente , in sede paracardiaca , la presenza di un cuscinetto adiposo pu determinare un analogo scostamento della linea . inoltre , anche nella proiezione laterale , come gi nella frontale , la presenza di cuscinetti pu determinare la formazione di linee semplici . frequente nei soggetti brachitipi obesi la presenza di opacit triangolari con apice al processo xifoideo e base verso il parenchima , sostenute dai triangoli opachi parasternali , che corrispondono a cuscinetti adiposi particolarmente sviluppati . il cuscinetto adiposo prepericardico pu raggiungere notevoli dimensioni ( 35 cm in altezza , 10 cm in lunghezza ) e pu protrudere su ambo i lati del mediastino , pi frequentemente a sinistra . 
3 radiografia laterale del torace con evidenza della linea retrosternale normale . in sintesi , le formazioni del mediastino anteriore determinano lo slargamento della lga , mentre nel radiogramma in proiezione ll si possono apprezzare come opacit che occupano lo spazio chiaro retrosternale , con ondulazioni della s.m. 
inferiorly , the presence of a fat pad at the paracardiac level may produce similar displacement of the line . in addition , in the lateral view as well as in the frontal view , the presence of fat pads may cause the formation of simple lines . 
a frequent finding in endomorph obese subjects is the presence of triangular opacities with the apex at the xiphoid process and the base towards the parenchyma ; these are related to opaque parasternal triangles corresponding to particularly developed fat pads . 
i dati rilevabili dalla radiografia del torace permettono pertanto , quasi sempre , di accertare la presenza e la sede di una patologia mediastinica , ma , tranne poche eccezioni , non consentono di definirne natura , organo di origine e presenza di eventuali caratteri infiltrativi . 
la rt per queste ragioni raramente diagnostica , con poche eccezioni : calcificazioni linfonodali a guscio duovo , se pur rare , sono fortemente suggestive di silicosi , linfoma trattato e sarcoidosi ; il riscontro di denti o osso o di un livello grasso / fluido nel contesto di una massa sono patognomonici di teratoma ; la presenza di livelli idro - aerei suggerisce lorigine esofagea , unernia , lesioni cistiche o ascessi [ 7 ]  . 
le immagini assiali eliminano il problema relativo alla sovrapposizione dei tessuti a densit simile : la dimostrazione degli spazi mediastinici e delle stazioni linfonodali in essi contenute ed il rapporto con le strutture circostanti ed i vasi sanguigni costituiscono lapporto qualitativamente pi importante della tc . le indicazioni comprendono la caratterizzazione , sulla base dei valori di attenuazione , di lesioni ( cistiche / solide , densit adiposa / calcificazioni ) individuate sul radiogramma standard e la valutazione del mediastino in pazienti con sospetto clinico e radiografia standard negativa [ 8 , 9 ]  . i vantaggi di questa procedura possono essere raggruppati in due categorie generali : ( 1 ) la descrizione fine e analitica del dettaglio anatomico , garantita dalla rappresentazione per strati , associata alla acquisizione volumetrica spirale ed alla fruibilit di nuovi punti di vista ( mpr ) e di tecniche innovative di post processing ; ( 2 ) lanalisi densitomefig . 
4 una radiografia laterale del torace evidenzia una linea retrosternale anomala ( banda retrosternale ) dovuta a lipomatosi . from a few exceptions , it cannot define its nature , organ of origin or the presence of possible infiltrative features . 
for these reasons , chest radiography is seldom diagnostic , with few exceptions : although uncommon , egg - shell lymph node calcifications are strongly suggestive of silicosis , treated lymphoma and sarcoidosis ; the finding of teeth , bone or a fat / fluid level within a mass are diagnostic of teratoma ; the presence of air / fluid levels suggests an oesophageal origin , a hernia , cystic lesions or abscesses [ 7 ]  . 
axial imaging avoids the problem of overlapping tissues with similar density : demonstration of mediastinal spaces with their lymph nodes , relations with surrounding structures and blood vessels represent the most important contributions of ct . 
indications include characterisation of lesions ( cystic / solid , adipose density / calcification ) previously identified on standard x - ray based on attenuation values and assessment of the mediastinum in patients with a clinical suspicion of disease but negative standard x - ray [ 8 , 9 ]  . advantages of ct fall into two major categories : ( 1 ) a fine analytical depiction of anatomical detail guaranteed by cross - sectional imaging associated with volumetric spiral acquisition and availability of new points of view [ multiplanar reformatting ( mpr ) ] and innovative postprocessing techniques ; ( 2 ) densitometric analysis ( with good preservation of spatial resolution ) and enhancement patterns . 
with its higher contrast resolution compared with chest radiography , ct allows distinction between the different mediastinal organs and visualises the lesion directly ; this leads to better localisation of a mediastinal mass , allows assessment of its extension and relations with neighbouring organs and determination of attenuation values of mass content , which , if integrated with data on the site of origin , often allows the nature of the mass to be defined . 
 most masses involving the anterior mediastinum and most cystic lesion , even when benign , require surgical resection . the choice among the various options [ video - assisted thoracic surgery ( vats ) , standard cervical mediastinoscopy , anterior mediastinotomy , thoracoscopy , thoracotomy ] is dictated by the nature of the lesion as demonstrated by ct , by whether a biopsy or resection is required and by the surgeons ability and experience . 
in sintesi , la tc per la sua superiore risoluzione di contrasto rispetto alla rt permette di distinguere i differenti organi mediastinici visualizzando direttamente la lesione ; ci porta ad una migliore definizione topografica ( localizzazione ) delle alterazioni mediastiniche , a valutare lestensione , i rapporti con gli organi vicini e a rilevare dati di attenuazione del contenuto della massa stessa che , integrati con la sede di origine , consentono non di rado di definirne la natura . 
la tc inoltre importante per il conseguimento dellaccertamento bioptico . la maggior parte delle masse a localizzazione mediastinica anteriore e delle lesioni cistiche , anche quando benigne , richiede una resezione chirurgica . 
la scelta tra le diverse opzioni ( video assisted thoracic surgery - vats , mediastinoscopia cervicale standard , mediastinotomia anteriore , toracoscopia , toracotomia ) indirizzata dai riscontri alla tc , evocativi della natura della lesione , dallintento perseguito di biopsia o resezione e dalla abilit ed esperienza del chirurgo . 
la diagnosi definitiva delle lesioni a sede nel mediastino anteriore , comunque , pi spesso ottenuta con lago - aspirato ( fine needle aspiration biopsy - fnab ) percutaneo sotto guida tc . 
tuttavia , lindagine tc in genere sufficiente per la valutazione preoperatoria ( esame di diagnostica per immagini di ultimo livello ) , prima di indirizzarsi verso una procedura invasiva per laccertamento della diagnosi , poich utile nella visualizzazione delle eventuali linfoadenopatie mediastiniche o ilari , nella diagnosi differenziale con anomalie vascolari e nella discriminazione di varianti anatomiche [ 7 ]  . semeiotica tc la diagnosi differenziale delle patologie mediastiniche alla tc avviene sulla base di numerose osservazioni , che includono la localizzazione , lidentificazione della struttura di origine , la definizione della unicit , multifocalit o del carattere di processo diffuso , la dimensione e la forma , lattenuazione ( grasso , fluido , tessuto molle o una combinazione di questi ) , la presenza di calcificazioni ed il contrast enhancement . 
gli espansi mediastinici possono presentare attenuazione bassa , alta o pari a quella del grasso ; le lesioni a bassa attenuazione hanno densit superiore a quella del grasso , ma inferiore a quella del muscolo , mentre lalta attenuazione contraddistingue una densit superiore a quella del muscolo . 
le lesioni con la densit del grasso includono : lipomatosi e cuscinetti adiposi , timolipoma , teratoma , lipoma e liposarcoma , ernie di tessuto adiposo [ 10 ]  . 
low - attenuation lesions are generally cystic and include congenital or acquired cysts , cystic thymoma , germ - cell tumours , lymphangioma and haemangioma , mediastinal abscesses , mediastinitis , haematoma , cystic struma , and primary or secondary necrotic tumours [ 11 ]  . high - density masses contain calcium or are rich in iodine or blood . 
they include : calcific lymphadenopathy ( granulomatous infections , sarcoidosis , silicosis ; adenocarcinoma and sarcoma metastasis ; treated lymphoma ) , partially calcific primary tumours ( germ - cell tumours , thymoma ) , struma ( calcific or rich in iodine ) , vascular atheroma and mediastinal haematoma [ 12 ]  . finally , after administration of contrast material , enhancing masses are strongly vascular [ 13 ] and include thyroid and parathyroid , carcinoid tumour , lymphangiomas and haemangiomas , partially necrotic inflammatory and neoplastic lesions . in conclusion , diagnostic accuracy of ct is higher than that of conventional chest radiography : in a study of 128 patients , the correct diagnosis was achieved in 48% of cases with ct and in only 36% of cases with chest radiography [ 14 ]  . magnetic resonance imaging mri has not been as extensively used as ct in the study chest pathology . 
the reason is that it has suffered from certain technical limitations mainly artefacts caused by respiratory and cardiac movements but technological progress has made gating systems available and allowed the acquisition of fast sequences that are virtually free of artefacts and are used in cardiovascular imaging . 
considering its multiplanar capabilities and excellent depiction of musculoskeletal structures , mri is generally suitable for studying processes located near the lung apex and spine and in the chest wall , as well as disorders of the mediastinal vessels and peridiaphragmatic regions . 
 however , its high capacity for tissue differentiation allows discrimination , in a single examination , between pleural involvement and parenchymal consolidation and mediastinal and chest - wall involvement , which may be associated . 
in addition , it can distinguish atelectasia within an area of increased parenchymal density [ 15 ]  . le masse ad alta densit contengono calcio , ovvero hanno un alto contenuto di iodio oppure sono in relazione con un elevato tenore ematico ; includono : linfoadenopatie calcifiche ( infezioni granulomatose , sarcoidosi , silicosi ; metastasi da adenocarcinoma e sarcoma ; linfoma dopo trattamento ) , tumori primitivi parzialmente calcifici ( tumori a cellule germinali , timoma ) , struma ( calcifico o ad alto contenuto di iodio ) , ateromasie in lesioni vascolari , ematoma mediastinico [ 12 ]  . infine , dopo somministrazione di mezzo di contrasto , le masse con enhancement hanno alto tenore vascolare [ 13 ] ed includono tiroide e paratiroidi , carcinoidi , linfangiomi ed emangiomi , lesioni infiammatorie e neoplastiche parzialmente necrotiche . in conclusione , laccuratezza diagnostica di questa metodica risulta superiore alla radiografia tradizionale del torace : in uno studio su 128 pazienti , la diagnosi corretta stata conseguita nel 48% dei casi sulla base della tc e solo nel 36% dei casi al radiogramma toracico [ 14 ]  . risonanza magnetica a causa di alcuni limiti tecnici , la rm non stata utilizzata in modo estensivo nello studio della patologia toracica al contrario della tc . 
sussistevano infatti alcuni svantaggi nelle sue applicazioni al distretto toracico , principalmente gli artefatti generati dai movimenti respiratorio e cardiaco , sebbene oggi i progressi tecnologici abbiano reso disponibili sistemi gating e permesso lacquisizione di sequenze veloci , quasi del tutto prive di artefatti , utilizzate nella dimostrazione della patologia cardiovascolare . in considerazione della possibilit di rappresentazione multiplanare e della eccellente dimostrazione delle strutture muscoloscheletriche , la rm , in termini generali , idonea alla valutazione di processi localizzati in prossimit dellapice del polmone , della colonna vertebrale , nella parete toracica ed , infine , adeguata allo studio di patologie che coinvolgano i vasi mediastinici e le regioni peridiaframmatiche . 
ha , invece , applicazioni pi limitate nella valutazione della patologia del parenchima polmonare , a meno che sia associato un interessamento pleurico . tuttavia , a suo favore , lelevata capacit di differenziazione tessutale consente , in un unico esame , la discriminazione dellinteressamento pleurico dal consolidamento parenchimale , dal coinvolgimento mediastinico e della parete toracica , che possono essere associati . 
consente , inoltre , di distinguere nel contesto di un addensamento parenchimale la componente atelectasica [ 15 ]  . le indicazioni a questa modalit di imaging multiplanare sono , dunque , nellambito del distretto toracico limitate : pu essere utilizzata come indagine di terzo livello , complementare alla tc , ma senza che rivesta un carattere routinario [ 16 , 17 ]  . solo in casi selezionati si dimostra superiore alla tc e potrebbe , pertanto , rappresentare lindagine di seconda istanza , nello specifico di : valutazione di lesioni vascolari ; definizione del coinvolgimento vascolare da parte di processi patologici estrinseci ; studio della parete toracica e del mediastino posteriore ; imaging delle regioni paravertebrali , indications for mri are therefore limited : it may be used as a third - level investigation to complement ct but not as a routine study [ 16 , 17 ]  . 
these cases are assessment of vascular lesions ; definition of vascular involvement by extrinsic pathological processes ; study of the chest wall and posterior mediastinum ; imaging of the paravertebral regions , nerve plexuses and neurogenic tumours ; recognition , within the mediastinal structures and heart , cleavage planes and their invasion by tumours , particularly when the ct findings are doubtful ; and differential diagnosis between fibrous tissue and persistence or recurrence of neoplasm after radiation therapy . 
it is therefore useful when there is a contraindication to iodinated contrast agents , in imaging of the posterior mediastinum and to assess invasion of tissues , vessels and the heart . 
its limitations on the other hand are : claustrophobia , compliance of debilitated patients , examination times , high cost and limited availability . in conclusion , where mri provides the same diagnostic information as ct , ct should be preferred . 
the gas was directly insufflated into the anterior mediastinum through a curved needle inserted just above the throat and pushed along the posterior surface of the sternum for a few centimetres [ 19 ]  . 
on the other hand , it gave clear depiction of contours of a normal or pathological thymus thereby providing useful topographic information for planning surgical thymectomy . sonography transthoracic ultrasound imaging of the mediastinum is not very commonly used although its convenience and low cost make it a useful and rapid complement to radiography in selected cases [ 20 ]  . 
it has sensitivity close to that of ct , in particular in the study of certain regions ( supra - aortic , pericardiac , prevascular , paratracheal ) [ 20 ] ; in the study of mediastinal lymphadenopathy , its specificity is higher . 
questultimo aspetto di particolare rilievo per il monitoraggio dei pazienti affetti da linfoma , dopo trattamento [ 18 ]  . inoltre , nel confrontare questa metodica con la tc , lutilizzo del mezzo di contrasto in rm consentito anche nei pazienti con ridotta funzionalit renale . 
pertanto utile quando vi sia controindicazione alla somministrazione di mezzo di contrasto iodato , nellimaging del mediastino posteriore e per la valutazione dellinvasione dei tessuti , dei vasi e del cuore . 
ha inoltre il vantaggio di non utilizzare radiazioni ionizzanti . tuttavia presenta alcuni limiti : claustrofobia , compliance del paziente debilitato , tempi di esecuzione , costi e disponibilit sul territorio . in conclusione , qualora la rm offra informazioni diagnostiche equivalenti a quelle della tc , questultima rappresenta lo studio pi appropriato . 
lossigeno insufflato negli spazi del mediastino in quantit di 200300 cm3 , avvolgeva gli organi mediastinici , rendendone possibile lesatta delimitazione radiologica . il gas era direttamente insufflato nel mediastino anteriore attraverso un ago ricurvo , infisso subito al di sopra del giugulo e la cui punta veniva sospinta per qualche centimetro lungo la faccia posteriore dello sterno [ 19 ]  . 
possedeva peraltro il merito di dimostrare con chiarezza i contorni del timo , normale o patologico , fornendo utili informazioni di topografia preliminari ad uneventuale procedura chirurgica di timectomia . ecotomografia lo studio ecotomografico del mediastino per via transtoracica non molto diffuso , ma per la praticit ed il basso costo , costituisce , in situazioni selezionate , un utile e rapido esame complementare alla radiografia [ 20 ]  . consente una agile chiarificazione di reperti radiologici dubbi , senza richiedere unulteriore esposizione a radiazioni ionizzanti . presenta una sensibilit prossima a quella della tc nello studio , in particolare , di alcuni distretti ( sovraaortico , pericardico , prevascolare , paratracheale ) [ 20 ] ; per lo studio delle linfoadenopatie mediastiniche la sua specificit sembra superiore . tuttavia non consente di esaminare tutte le sezioni del mediastino . 
la finestra acustica per laccesso al mediastino ottenuta con approccio sovrasternale ( al giugulo ) [ 21 ] o parasternale [ 20 ] ; sporasonographic anatomy of the mediastinum has already been described in the literature [ 21 ]  . 
the acoustic window for access to the mediastinum is obtained with a suprasternal ( jugulum ) [ 21 ] or parasternal [ 20 ] approach ; in rare cases , an infrasternal approach may be useful [ 21 ]  . 
6a - d sonographic access to the mediastinum is obtained via the suprasternal ( a ) and parasternal ( b , c ) approach , though occasionally also the infrasternal ( d ) path is chosen . 
the great vessels and their position relative to the heart on the various levels serve as landmarks in the mediastinu suprasternal examination is carried out with the patient lying supine . 
6a - d laccesso ecografico al mediastino ottenuto attraverso lapproccio soprasternale ( a ) e parasternale ( b , c ) , sebbene venga scelta occasionalmente anche la via infrasternale ( d )  . 
b lo stesso giovane paziente , con malattia di hodgkin scleronodulare , dopo trattamento ( chemioterapia e radioterapia ) : lecotomografia dimostra unevidente riduzione della massa bulky . in a nonpathological context , lymph nodes , fat and connective tissue are depicted as homogeneous hypoechoic structures . 
the main conditions are : emphysema ( it reduces suprasternal , parasternal and aortopulmonary windows ) ; presence of fibrous tissue ( due to inflammation , surgery , radiotherapy ) ; skeletal deformations of the spine ( which may affect the patients decubitus or insonation ) , movement artefacts . 
as a result , in the event of systemic disease , sonography is penalised by its exploration limits . quality of the examination is strictly operator dependent ( adequate knowledge of mediastinal anatomy and considerable experience in image interpretation are required ) and depends on patient compliance . 
also , because the ultrasound wave is progressively attenuated as it penetrates tissue , deep lesions might not be differentiated from surrounding structures , with resulting false negative results . despite these limitations , sonography of the anterosuperior mediastinum may yield good results . 
according to the literature , sonography in expert hands is almost as reliable as ct and may therefore take on an intermediate role between chest radiography and ct [ 22 ]  . 
lindagine inoltre resa pi informativa con lintegrazione del segnale color doppler , che migliora la qualit e laccuratezza dellimaging b - mode . in un contesto non patologico , i linfonodi ed il tessuto adiposo e connettivo sono rappresentati come strutture ipoecogene a segnale omogeneo . 
le principali condizioni sono rappresentate da : enfisema ( riduce le finestre sovrasternale , parasternale ed aortopolmonare ) ; presenza di tessuto fibroso ( conseguente ad infiammazione , chirurgia , radioterapia ) ; deformazioni scheletriche del rachide ( che possono condizionare il decubito del paziente o linsonazione ) ; artefatti da movimento . 
pertanto , nellevenienza di una patologia sistemica , il limite di esplorabilit penalizza la metodica ultrasonografica . la qualit dellesame strettamente operatore dipendente ( indispensabile una adeguata conoscenza dellanatomia mediastinica ed richiesta una considerevole esperienza nella interpretazione delle immagini ) , oltre ad essere condizionata dalla compliance del paziente . 
finally , anterior mediastinal masses may be biopsied under sonographic guidance , especially if located at the edges of the sternum ; sonography minimises the risk of bleeding , as vessels are readily recognised without any need for contrast material ; complications are rare [ 25 ]  . nuclear medicine thyroid and parathyroid scintigraphy having excluded disorders affecting endocrine glands ( thyroid and parathyroid ) from the discussion , this section does not describe in detail the relevant nuclear medicine function investigations that are requested in conditions of ectopia . positron emission tomography : pet the role of fluorodeoxyglucose positron emission tomography ( fdg - pet ) in assessment of mediastinal pathology is controversial . 
8 la sezione assiale di un esame pet - ct di un uomo con tumore del polmone dimostra accumulo di fdg nei linfonodi del mediastino anteriore , che successivamente stato dimostrato essere metastatico . lapproccio soprasternale consente unaccessibilit soddisfacente nel 90%95% dei casi [ 20 ]  . lecotomografia , da quanto riportato in letteratura , a discrezionalit di un operatore esperto , quasi altrettanto affidabile della tc e , pertanto , potrebbe assumere una posizione intermedia tra radiografia del torace e tc [ 22 ]  . 
stato anche descritto luptake fisiologico , in assenza di patologia intrinseca , da parte del timo [ 2931 ] , che si verifica frequentemente in et pediatrica . non noto , in quanto sono descritti in letteratura dati contraddittori , leffetto della terapia ( chemioterapia , radioterapia a fasci esterni , radioterapia metabolica ) sulluptake da parte del timo : questultimo pu essere riscontrato prima o dopo trattamento e si verifica nei soggetti pi giovani con ghiandole pi voluminose . 
in one series , the mean differential uptake ratio for fdg by the normal thymus was 1.47 , which is considerably lower than the 2.5 associated with malignancy [ 29 ] ; this was confirmed by another study that concluded that physiological uptake of fdg by the thymus is substantially lower than that seen in the presence of disease [ 31 ]  . fdg uptake by thymic malignancies has , in fact , been reported [ 2628 ]  . 
quantitative analysis could potentially be very useful in those cases in which location and gross morphological features of the lesion at radiological imaging do not allow qualitative differentiation between physiological and pathological uptake [ 31 ]  . fdg - pet is indicated both for diagnosis and monitoring response to treatment [ 31 ]  . 
it has also been used to characterise thymoma and thymic hyperplasia , but its usefulness in this clinical context is still to be validated [ 27 ]  . in conclusion , fdg - pet has potential in the differential diagnosis between benign and malignant lesions and is expected to have a role in imaging mediastinal diseases in the future . invasive procedures : diagnostic confirmation histological diagnosis of mediastinal expansile processes is nearly always necessary to plan the most appropriate treatment . 
traditional exploratory surgery is no longer required , thanks to the development of radiological and nonradiological minimally invasive techniques and improved immunohistochemistry and electron microscopy techniques [ 3336 ]  . ct - guided percutaneous needle biopsy , whether fnab for cytological diagnosis or core - needle biopsy for histological definition , is currently standard procedure in initial assessment of most mediastinal masses . 
although fnab is generally appropriate for recognising carcinomatous lesions , coreneedle biopsy in indicated in the majority of cases , in particular when lymphoma or thymoma are suspected [ 34 , 37 , 38 ]  . guidance with ct or colour doppler us prevents puncturing vascular structures , so complications are rare . surgical procedures are still required in select cases [ 33 , 39 ]  . 
in una casistica , il differential uptake ratio medio per fdg uptake da parte del timo normale era di 1 , 47 , un valore considerevolmente inferiore al minimo di 2 , 5 associato con il riscontro di malignit [ 29 ] ; il dato stato confermato da un ulteriore studio , che ha concluso che luptake timico fisiologico di fdg consistentemente inferiore rispetto allassunzione in presenza di malattia [ 31 ]  . 
lanalisi quantitativa potrebbe potenzialmente essere molto utile nei casi in cui non fosse possibile , sulla sola base della localizzazione e dei caratteri macroscopici morfologici della lesione allimaging radiologico , differenziare qualitativamente luptake fisiologico dal patologico [ 31 ]  . fdg - pet indicata sia nella diagnosi sia nel monitoraggio della risposta alla terapia [ 31 ]  . 
in particolare , senzaltro utile nella stadiazione whole - body dei linfomi e nella valutazione , dopo trattamento , della malattia residua nei tumori a cellule germinali [ 26 ]  . 
stata inoltre utilizzata nella caratterizzazione del timoma e delliperplasia timica , ma la sua utilit in questo contesto clinico rimane da validare [ 27 ]  . in conclusione , fdg - pet ha potenzialit nella diagnosi differenziale tra lesioni benigne e maligne e , verosimilmente , guadagner un ruolo nellimaging delle patologie mediastiniche . procedure invasive per laccertamento della diagnosi laccertamento della diagnosi istologica dei processi espansivi del mediastino quasi sempre indispensabile per la pianificazione del trattamento adeguato . 
oggigiorno non sono pi necessarie procedure chirurgiche tradizionali esplorative per limplementazione di tecniche radiologiche e non - radiologiche mini - invasive e di metodiche di immunoistochimica e di microscopia elettronica pi efficaci [ 3336 ]  . lagobiopsia percutanea sotto guida tc , sia con aspirato con ago sottile ( fine needle aspiration biopsy - fnab ) per laccertamento citologico , sia con tecnica agobioptica ( core needle biopsy ) per la definizione istologica , rappresenta attualmente lo standard nella valutazione iniziale della maggior parte delle masse mediastiniche . 
sebbene la fnab sia in genere idonea nel riconoscimento delle lesioni carcinomatose , lagobiopsia indicata nella maggioranza dei casi , in particolare nel sospetto di linfoma e di timoma [ 34 , 37 , 38 ]  . 
la toracoscopia , in anestesia generale , una procedura miniinvasiva che garantisce unaccuratezza diagnostica pari a quasi il 100% nella maggior parte dei compartimenti mediastinici [ 40 ] : tuttavia dovrebbe essere riservata agli accertamenti bioptici che non possono essere conseguiti con la mes.m. 
finally , thoracotomy is almost never required for diagnosis and is indicated in select cases for exploratory purposes . diagnostic integration : laboratory tests some mediastinal lesions release substances into the bloodstream that may be measured using laboratory techniques . these markers can confirm a diagnostic suspicion , evaluate response to treatment , or indicate disease persistence or recurrence . 
some germ - cell tumours produce - fetoprotein ( fp ) , - human chorionic gonadotropin ( - hcg ) and lactate dehydrogenase ( ldh ) ; assessment of their serum levels is indispensable in evaluation of anterior mediastinal masses in males [ 4 ]  . 
in addition , in the appropriate clinical setting , evaluation of thyroid hormones , of adrenocorticotropic hormone ( acth ) and parathyroid hormone ( pth ) may help establish the nature of the lesion ( table 1 )  . conclusions diseases of the mediastinal comprise a wide spectrum of benign and malignant entities that share the same anatomical location within the chest . 
this division , which is based on the lateral chest x - ray , allows a rational , topographic approach to the differential diagnosis and helps plan further imaging studies and clinical investigations to select the definitive cytohistological procedure and the most appropriate treatment strategy . knowledge of pathology , demographics , clinical presentations , imaging patterns and treatment options is indispensable for correct management of anterior mediastinal masses , which often require a multidisciplinary approach . 
infine , la toracotomia non quasi mai necessaria per laccertamento diagnostico e , a titolo esplorativo , indicata in casi selezionati . integrazioni diagnostiche : esami di laboratorio alcune lesioni del mediastino rilasciano sostanze in circolo che possono essere misurate mediante tecniche di laboratorio . 
alcuni tumori a cellule germinali producono - fetoproteina ( - fp ) , - gonadotropina corionica umana ( - hcg ) e lattato deidrogenasi ( ldh ) : il loro dosaggio sierico indispensabile nella valutazione di masse del mediastino anteriore in soggetti di sesso maschile [ 4 ]  . inoltre , la valutazione degli ormoni tiroide , di acth e del pth pu , in casi selezionati sulla base dei dati clinici , indirizzare la diagnosi di natura ( tabella 1 )  . conclusioni le malattie del mediastino comprendono diverse entit nosografiche , di natura benigna e maligna , che condividono la stessa localizzazione anatomica nel torace . 
questa divisione , basata sul radiogramma laterale del torace , consente di stabilire razionalmente lambito delle diagnosi differenziali , di pianificare le ulteriori indagini di diagnostica per immagini e gli approfondimenti clinici per delineare laccertamento cito / istologico definitivo e la strategia terapeutica pi appropriata . la conoscenza della patologia , della demografia , delle presentazioni cliniche , dei dati di imaging e delle opzioni di trattamento indispensabile per una corretta gestione delle masse del mediastino anteriore , che spesso richiede un approccio multidisciplinare . 
in particolare , con il miglioramento delle tecniche di imaging nellindirizzare la diagnosi e nel guidare le procedure bioptiche , la maggior parte dei pazienti non richiede pi una toracotomia esplorativa prima della pianificazione del trattamento . ringraziamenti gli autori ringraziano , per gli importanti e competenti suggerimenti , i colleghi : f . 
of radiology , azienda ospedaliera universitaria , parma , italy 3dibimel , sezione di scienze radiologiche , universit di palermo , palermo , italy 4irccs santa lucia , roma , italy 5u.o. 
cademartiri , viale rustici 2 , i - 43100 parma , italy , tel . : + 39 - 052 - 1961833 , fax : + 39 - 052 - 1703381 , e - mail : filippocademartiri@hotmail.com received : 28 august 2004 / accepted : 5 march 2005 / published online : 11 april 2006 abstract conventional coronary angiography is the gold standard for the diagnosis of coronary artery anomalies . 
recently , the increasing performance of diagnostic techniques , such as electron beam tomography ( ebt ) , magnetic resonance ( mr ) and , more recently , multislice computed tomography ( msct ) , has enabled their application to cardiac imaging . 
we report the incidence and pathophysiology of coronary artery anomalies based on the capabilities of recent diagnostic tools with the aim of improving an accurate and noninvasive diagnostic approach . key words multislice computed tomography coronary artery anomalies magnetic resonance angiography electron beam tomography riassunto la coronarografia convenzionale rappresenta la tecnica di scelta per la diagnosi delle anomalie delle arterie coronarie . 
il loro riscontro , anche se in una percentuale molto bassa di individui , , nella maggior parte dei casi , occasionale durante lesecuzione di una coronarografia diagnostica nei pazienti con sospetta malattia coronarica ostruttiva . 
questultima , ricopre un ruolo importante nellimaging diagnostico coronarico grazie allelevata risoluzione temporale e spaziale raggiunte ed alla capacit di visualizzare in modo tridimensionale la complessa anatomia dei vasi epicardici . 
riportiamo lincidenza e la fisiopatologia specifica delle anomalie coronariche alla luce delle moderne tecniche diagnostiche , al fine di privilegiare , ove possibile , un approccio diagnostico accurato e non invasivo . parole chiave tomografia computerizzata multistrato anomalie delle arterie coronarie angiografia con risonanza magnetica tomografia ad emissione di elettroni introduction introduzione from the beginning of the 1990s , a variety of techniques other than conventional coronography ( cc ) have been used in diagnostic imaging of the coronaries [ 16 ]  . 
recently , multislice computed tomography ( msct ) has rapidly gained credibility thanks to the quality of the results obtained and its reproducibility in dallinizio degli anni novanta , diverse tecnologie , alternative alla coronarografia convenzionale ( cc ) , si sono affiancate nellimaging diagnostico delle coronarie [ 16 ]  . 
di recente , la tomografia computerizzata multistrato ( tcms ) ha rapidamente guadagnato credibilit per la qualit dei risultati ottenuti e per la loro rithe morphological assessment of coronary arteries [ 712 ]  . one of the strengths of msct of the coronaries is the threedimensional ( 3d ) visualisation of the epicardial vessels [ 13 ]  . for this reason , there has been much interest in the application of msct to assess coronary artery anomalies ( caas )  . the anatomical details and the physiopathological characteristics of most caas are not well known . 
although the medical community and the general public are increasingly aware that in special cases , caas can be fatal ( typically among young athletes under physical stress ) [ 14 ] , there are very few studies in the literature regarding their clinical significance , possible diagnostic protocol and the possibility of treatment and follow - up . 
the anatomical landmark shows the region of interest in an axial section at the level of the coronary artery origins from the left sinus of valsalva . the right coronary artery ( acd ) arises from the right sinus of valsalva and passes anteriorly in the right atrioventricular groove . 
the left coronary artery arises from the left sinus of valsalva and divides into the left anterior descending artery ( ada ) in the interventricular groove and the circumflex artery ( acx ) in the posterior atrioventricular groove . 
larteria coronaria sinistra ( acs ) , che origina dal seno di valsalva sinistro , dopo un breve tratto , si divide in arteria discendente anteriore ( ada ) , che si porta anteriormente , seguendo il decorso del solco interventricolare e arteria circonflessa ( acx ) che , invece , segue il decorso del solco atrio - ventricolare posteriore . 
per questa ragione , la tcms ha suscitato interesse nellapplicazione alla valutazione delle anomalie coronariche ( ac )  . i dettagli anatomici e le caratteristiche fisiopatologiche di gran parte delle ac non sono ben conosciute . 
sebbene la comunit medica e la popolazione siano sempre pi consapevoli che le anomalie coronariche possono , in casi particolari , essere fatali ( tipicamente nei giovani atleti durante sforzo fisico ) [ 14 ] , esistono solo pochi dati in letteratura sulla loro rilevanza clinica , sulleventuale protocollo diagnostico e sulle possibilit di trattamento e follow - up . 
 [ 15 ] , secondo la quale lac rappresenta un pattern coronarico con una caratteristica ( origine , numero , numero di ostii , decorso , etc . ) , che viene raramente riscontrata nella popolazione generale . epidemiologia in accordo con i dati riportati in letteratura , che si basano su reperti di angiografie eseguite per sospetta malattia ostruttiva coronarica , le ac coinvolgono circa l1 , 3% della popolazione generale [ 1618 ]  . 
i reperti autoptici , invece , forniscono unincidenza pi bassa : in 18.950 autopsie , alexander e griffith [ 19 ] hanno riscontrato solo 54 anomalie coronariche ( 0 , 3% )  . bisogna per considerare che le autopsie negli stati uniti non sono eseguite routinariamente , ma quasi esclusivamente per motivi medico - legali : dopo una morte violenta o avvenuta in ambiente non ospedaliero . 
inoltre , dal 1960 , il numero di autopsie per le morti in ospedale diminuito dal 50% al 10% [ 20 ] , motivo per cui , lincidenza delle ac fornita dagli studi autoptici risulterebbe poco affidabile [ 15 ]  . secondo uno studio effettuato da barriales villa et al . , che hanno rivalutato 13.500 coronarografie , riscontrando 75 pazienti con 75 anomalie coronariche , il sesso maggiormente interessato dalle ac sarebbe quello maschile ( 54 maschi vs 21 femmine ) [ 3 , 21 ]  . 
 [ 15 ] , which states that a caa is a coronary pattern with a characteristic ( origin , number , number of ostia , course , etc . ) which is rarely encountered in the general population . epidemiology according to data present in the literature , which are based on angiographic findings performed for suspected coronary obstructive disease , caas affect about 1.3% of the general population [ 1618 ]  . 
it should be borne in mind , however , that autopsies are not performed routinely in the united states but almost exclusively for medicallegal purposes after a violent death or death not in the hospital environment . 
in addition , since 1960 , the number of autopsies following death in hospital has fallen from 50% to 10% [ 20 ] , a reason for which the incidence of coronary anomalies provided by postmortem studies is somewhat unreliable [ 15 ]  . 
although they are rare , caas may be frequently present in association with other congenital heart diseases ( tetralogy of fallot [ 23 ] , transposition of the great arteries [ 24 ] , pulmonary atresia with intact ventricular septum [ 25 ] , rubella syndrome , hurlers syndrome and friedrichs ataxia [ 2630 ] )  . 
haemodynamically significant anomalies may induce angina pectoris , syncope , arrhythmia , myocardial infarct and sudden death and promote the onset and progression of coronary atherosclerosis [ 31 ]  . 
the study examining the largest number of cases , performed in north america at the cleveland clinic on 126 , 595 patients who underwent coronary angiography , reported a 1.3% incidence of caas ( 1 , 686 ) [ 18 ]  . 
while the cleveland study reported an 87% incidence for anomalies in the origin and course of the coronary arteries and 13% for coronary fistulas , the hunsottoposti ad angiografia coronarica per sospetta malattia ischemica , le ac sono pi comuni nelle donne ( 7 , 6% ) rispetto agli uomini ( 4 , 8% ) [ 15 , 22 ]  . sebbene rare , le ac possono frequentemente presentarsi in associazione con altre malattie congenite del cuore ( tetralogia di fallot [ 23 ] , trasposizione completa dei grossi vasi [ 24 ] , atresia della polmonare con setto interventricolare intatto [ 25 ] , sindrome della rubeola , sindrome di hurler e sindrome di friedrich [ 2630 ] )  . 
le anomalie che risultano emodinamicamente significative possono indurre : angina pectoris , sincope , aritmia , infarto miocardico , morte improvvisa e favorire linsorgenza e la progressione della malattia aterosclerotica coronarica [ 31 ]  . lo studio pi consistente dal punto di vista numerico , effettuato nel 1990 in nord america presso la cleveland clinic su 126.595 pazienti sottoposti ad angiografia coronarica , riporta una incidenza di ac dell1 , 3% ( 1.686 ) [ 18 ]  . 
mentre , nello studio della cleveland clinic lincidenza per le anomalie di origine e decorso delle arterie coronarie dell87% e per le fistole coronariche del 13% , in quello ungherese vengono riportati valori differenti , rispettivamente , del 95% e 5% [ 18 , 32 ]  . questa differenza nella frequenza e distribuzione delle anomalie congenite delle arterie coronarie pu essere attribuita ad un fattore genetico , come topaz et al . 
suggeriscono [ 33 ] : unarteria del cono indipendente pu essere identificata nel 27% dei pakistani , nel 38% dei britannici e nel 50% degli americani . nella popolazione giapponese , la frequenza di una origine separata dellarteria coronaria destra dal seno aortico di valsalva ( ad esempio , una dallarteria del cono e laltra dal tronco comune dellarteria coronaria destra ) solo del 10% [ 34 ]  . 
il 90% della popolazione cinese e di quella americana hanno nel circolo coronarico una dominanza destra [ 35 , 36 ]  . questi sono i risultati di due studi anatomo - patologici riportati da cheng ed effettuati da un gruppo di cardiologi cinesi a pechino e shanghai [ 36 ]  . 
 [ 32 ] , che hanno identificato , nel 52% dei casi valutati , una origine separata dellarteria coronaria discendente sinistra e dellarteria circonflessa nel seno coronarico sinistro ( ad esempio , assenza del tronco comune sinistro ) , mentre velican et al . 
 [ 42 , 43 ]  . classe i : ac benigne quelle pi frequenti di questo gruppo sono in genere silenti dal punto di vista clinico [ 18 ] e non sono state correlate allischemia miocardica o alla morte improvvisa , sebbene sia stata spesso riscontrata una loro associazione con diversi gradi di malattia aterosclerotica [ 31 , 44 ]  . 
il chirurgo , infatti , potrebbe erroneamente reciderla non conoscendone il decorso anomalo [ 38 ]  . la comunicazione intercoronarica una rara anomalia che si pu confondere con un vaso collaterale pervio , indicativo di una probabile ostruzione dellarteria coronaria . unorigine indipendente della discendente anteriore sinistra e della circonflessa pu essere relativamente frequente [ 15 ]  . 
la sua incidenza aumenta in caso di coesistenza di malattia valvolare aortica o dominanza sinistra . la pi frequente tra le ac considerata lorigine ectopica dellarteria circonflessa dallarteria coronaria destra o dal seno di valsalva destro . la sua rilevanza clinica correlabile alla possibilit di compressione durante un intervento chirurgico di sostituzione valvolare . 
 [ 45 ] in differenti sottotipi ( tipo iiv ) , in base alla distribuzione e al decorso nel solco interventricolare dei rami settali e diagonali , non associabile ad eventi sfavorevoli se non coesiste una malattia aterosclerotica [ 46 ]  . i ponti miocardici ( i.e. , myocardial bridges ) sono suddivisi in 5 gruppi ( da 1 a 5 ) a seconda della loro lunghezza ( < o > 1 cm ) e della riduzione di calibro durante la contrazione cardiaca ( < 25% , 50% o > 75% )  . 
varianti : acd che origina dal seno aortico anteriore sinistro , con decorso anomalo ( passando tra ap ed ao o posteriormente ) : acs che origina dal seno aortico anteriore destro , con decorso anomalo ( passa anteriormente o posteriormente allaorta o tra aorta ed arteria polmonare o presenta un decorso intramiocardico ) : arteria coronaria unica : la potenziale compressione di un singolo vaso coronarico se passa tra aorta ed arteria polmonare , pu provocare episodi ischemici , infarto miocardico e morte improvvisa acx che origina dal seno aortico anteriore destro o dal tratto prossimale della acd , con decorso anomalo ( posteriormente al decorso della ao ) : non emodinamicamente significativa . 
pu essere incidentalmente compressa durante una sostituzione valvolare acd , arteria coronaria destra ; acs , arteria coronaria sinistra ; acx , arteria coronaria circonflessa ; ada , arteria discendente anteriore ; ao , aorta ; ap , arteria polmonare f . 
right ventricle , right atrium , coronary sinus , superior vena cava , pulmonary artery , left atrium , left ventricle , multiple ( right and / or left ventricle ) rca , right coronary artery ; lca , left coronary artery ; lad , left anterior descending coronary artery ; lcx , left circumflex artery tion of congenital anomalies of the coronary arteries may be due to genetic factors , as topaz et al . 
multiple ostia in the right aortic sinus can be identified in another 10% of the same population ( 6% with three ostia and 4% with four )  . ninety percent of the chinese and american population have a right dominant coronary circulation [ 35 , 36 ]  . 
nelladulto questanomalia si pu associare ad ischemia miocardica , ad un pi elevato rischio di malattia coronarica e ad un sovraccarico cardiaco [ 50 ]  . la presenza di ununica arteria coronaria , distinta in diversi sottotipi r o l ( i - ii - iii ) , si associa ad ischemia miocardica per la inadeguata capacit di sostenere adeguatamente la normale circolazione coronarica [ 29 ]  . unulteriore ac da considerarsi benigna , sebbene in grado di provocare ischemia miocardica , latresia coronarica che , di solito , si associa ad altre malformazioni congenite tabella 3 anomalie dellanatomia intrinseca e della terminazione tipo di anomalia coronarica reperto anatomico rilevanza clinica anomalie dellanatomia intrinseca episodi ischemici miocardici a riposo f . 
if only the physiopathological and clinical consequences of caas are taken into account , this classification proves to be incomplete [ 41 ]  . there are a number of classifications in the literature that propose an anatomical and physiopathological approach to caas . 
this classification divides caas into four classes based on their clinical relevance ( table 1 ) , thus facilitating management and clinical follow - up . below is a summary of the main features of this classification . 
 [ 42 , 43 ]  . nei bambini con sindrome da rubeola , di hurler e di friederich [ 26 , 27 ]  . classe iii : associazione tra malattie coronariche e morte improvvisa le ac in quanto ad origine , decorso e distribuzione sono state , recentemente , ritenute la principale causa del 5%35% di morti improvvise nei giovani [ 50 ] , mentre una brusca ed acuta angolazione nel tratto prossimale di unarteria coronaria e la presenza di setti simili a delle valvole in prossimit del suo ostio , in assenza di malattia coronarica , possono essere la causa di una morte improvvisa [ 5153 ]  . 
il 19% delle morti improvvise nei giovani atleti sono da considerare imputabili alla presenza di una ac [ 43 ] , infatti , in presenza di una ac , un intenso sforzo fisico pu essere causa di morte improvvisa . i sottotipi di arterie coronarie uniche rappresentano una rara condizione nella quale la perfusione cardiaca totalmente affidata ad unarteria coronaria destra che origina f . 
its incidence increases with concomitant aortic valvular disease or left coronary artery dominance . the most common caa is probably the ectopic origin of the lcx from the rca or the right sinus of valsalva . 
quando presente questa ac il rischio di un evento fatale per il soggetto portatore elevato [ 54 , 55 ]  . il significato clinico dellorigine ectopica dellarteria coronaria sinistra dal seno di valsalva destro , dipende dalla sua posizione e dal decorso anatomico rispetto allaorta e allarteria polmonare . 
multiplanar reconstruction in an oblique coronal plane parallel to the long axis of the right coronary artery ( acd ) and the ectopic origin of the circumflex artery ( acx ) ( a )  . 
the acx first passes between the aortic root ( ao ) and the right atrium ( ad ) , and then passes between the ao and the left atrium ( as ) to enter the left atrioventricular groove ( b , arrowhead ) and to follow the lateral wall of the left ventricle . 
nelle immagini si pu anche apprezzare il tratto distale dellarteria circonflessa anomala che seguendo il decorso del solco atrio - ventricolare sinistro ( b , punta di freccia ) , subito dopo , si porta sulla parete laterale del ventricolo sinistro ( vs )  . 
panoramic three - dimensional ( 3d ) volume - rendering reconstruction of the heart shows the left anterior descending coronary artery ( ada ) that follows the anterior interventricular groove . 
the 3d volume - rendering and magnified multiplanar reconstructions in an orthogonal plane at the ada long axis ( 13 ) show the intramyocardial course of the artery ( 2 )  . 
vd , ventricolo destro ; vs , ventricolo sinistro ; ada , arteria coronaria discedente anteriore . basis of the distribution and course within the interventricular groove of the septal and diagonal branches , is not associated with unfavourable events in the absence of atherosclerosis [ 46 ]  . 
myocardial bridges are divided into five groups ( from 1 to 5 ) on the basis of their length ( < 1 or > 1 cm ) and their reduction in diameter during cardiac contraction ( < 25% , 50% or > 75% )  . 
the presence of a single coronary artery , which may be classified into subtypes r or l ( i - iiiii ) , is associated with myocardial ischaemia owing to the inability to adequately sustain normal coronary circulation [ 29 ]  . 
multiplanar reconstruction in a para - axial plane at the level of the origin of the right ( acd ) and left ( acs ) coronary arteries ( a )  . 
ricostruzione multiplanare sul piano assiale obliquo allemergenza ed al tratto prossimale delle arterie coronarie destra ( acd ) e sinistra ( acs ) dai rispettivi seni coronarici di valsalva ( a ) e ricostruzione 3d volume rendering panoramica del cuore ( b )  . 
hanno per primi suggerito che unarteria circonflessa anomala presenta un maggiore grado di stenosi rispetto alle arterie normali , ma la localizzazione ed il grado di stenosi nellarteria anomala non influenzano la sopravvivenza del soggetto [ 17 ]  . allo stesso modo , garg et al . 
 [ 33 ] , hanno rilevato unincidenza del 28%33% di malattia coronarica nelle arterie anomale e , soprattutto , unincidenza di malattia coronarica in pazienti con ac pari al 66%68% , suggerendo che queste due condizioni sono indipendenti . imaging non invasivo e metodi di screening sin ad oggi , la coronarografia convenzionale ha rappresentato la tecnica di scelta per la diagnosi delle anomalie coronariche [ 28 , 64 , 65 ]  . la diagnosi di ac stabilita , molto spesso , sulla base dellimpossibilit di trovare la coronaria nella normale sede anatomica e , quindi , di cateterizzarne lostio . negli ultimi anni si sono sviluppate altre tecniche nellimaging diagnostico cardiologico come , ad esempio , lecocardiografia transtoracica ( ett ) e transesofagea ( ete ) , langio - rm coronarica , la electron beam tomography ( ebt ) e la tcms [ 1 , 2 , 64 , 6669 ]  . ecocardiografia trans - esofagea e trans - toracica lett , che trova indicazione soprattutto in campo pediatrico , non sempre fornisce risultati diagnostici affidabili . 
caas are believed to be responsible for 19% of sudden death in young athletes [ 43 ] since intense physical activity in the presence of a caa may lead to sudden death . 
subtypes of single coronary arteries are a rare condition in which cardiac perfusion is performed entirely by a single rca , which arises from a normal left coronary artery , usually passing between the pulmonary artery and aorta . 
the clinical significance of the ectopic origin of the lad from the right sinus of valsalva depends on its position and anatomical course with respect to the aorta and the pulmonary artery . the septal subtype of these caas is the most commonly encountered whereas the between variety is rare but more dangerous [ 56 ]  . 
 [ 42 ] , patients with this anomaly have a high risk of malignant ventricular arrhythmias and require prompt treatment . the anomalous origin of the rca from the pulmonary artery is rare , and its clinical course is generally benign although it may be associated with sudden death , particularly after physical exertion [ 59 , 60 ]  . class iv : association between coronary artery anomalies and superimposed coronary artery disease there have been no studies to date that show that caas are more susceptible to coronary artery disease . 
 [ 61 ] found that an anomalous circumflex artery has an earlier and greater degree of atherosclerosis than a nonanomalous vessel . this predilection has been observed only in vessels arising from the right side with a retroaortic course [ 61 ]  . 
 [ 17 ] were the first to suggest that an anomalous circumflex artery has a greater degree of stenosis than normal arteries although the location and degree of stenosis in the anomalous artery do not influence survival of the subject . 
5 three - dimensional ( 3d ) volume - rendering reconstruction of the heart shows the anomalous origin and course of the right coronary artery ( acd )  . the anomalous artery arises as a separate vessel from the left sinus of valsalva . 
it then has an interarterial course between the aortic root ( ao ) and right ventricular outflow tract ( tevd ) to enter the right atrioventricular groove ( arrowhead )  . the anatomic landmark shows the coronary artery anomaly . 
5 anomalia coronaria destra ad origine dal seno coronario sinistro . ricostruzione 3d volume rendering del cuore nella quale si osserva lanomala emergenza dellarteria coronaria destra ( acd ) ed il suo anomalo decorso . 
lanomala arteria coronaria destra ( acd ) origina dal seno coronario sinistro e , passando tra aorta ascendente ( ao ) e tratto di efflusso del ventricolo destro ( tevd ) , segue il decorso del solco atrio - ventricolare destro ( punta di freccia )  . in basso a sinistra schematizzata lanomalia descritta . 
tevd , tratto di efflusso del ventricolo destro ; ao , aorta ascendente ; ad , atrio destro ; as , atrio sinistro ; vs , ventricolo sinistro . riportiamo lesperienza di davis et al . 
the diagnosis of a caa is often established on the impossibility of finding the coronary arteries in their normal anatomical position and therefore to catheterise the ostiu in recent years , other techniques in cardiologic diagnostic imaging have been developed , such as transthoracic echocardiography ( tte ) , transoesophageal echocardiography ( tee ) , magnetic resonance angiography ( mra ) , electron beam tomography ( ebt ) and msct [ 1 , 2 , 64 , 6669 ]  . transthoracic and transoesophageal echocardiography tte , which is used mainly in paediatric radiology , does not always provide reliable diagnostic results . 
although this technique is able to identify the course of and territory supplied by caas , and after colour doppler integration supply information regarding the direction of blood flow [ 70 , 71 ] , it has some important shortcomings . 
when performed on adult patients , it proves difficult to obtain diagnostic images owing to the interposition of the bones of the ribcage ( ribs and sternum ) , pulmonary parenchyma and subcutaneous adipose tissue [ 5 , 72 ]  . 
nella ricerca e valutazione dellorigine delle arterie coronarie , la rm pu fornire informazioni pi complete rispetto allangiografia convenzionale , specialmente nei pazienti con altre anomalie cardiache congenite concomitanti [ 1 , 7577 ]  . 
la maggiore limitazione dellangio - rm consiste nellincompleta visualizzazione dei vasi coronarici , in particolar modo dei loro tratti distali . questo ne limita lutilizzo a scopo diagnostico per la valutazione delle fistole , dellorigine delle arterie coronarie diversa dai seni aortici ( i.e. , da un ventricolo o dallarteria polmonare ) e dei vasi collaterali . electron beam tomography la electron beam tomography ( ebt ) , utilizzata estensivamente per la valutazione del calcio coronarico [ 7881 ] , sfrutta una elevata risoluzione temporale ( 50100 ms ) e la sincronizzazione prospettica della scansione in apnea con lecg . 
questo permette di ridurre gli artefatti da movimento cardio - respiratorio e la dose assorbita dal paziente [ 2 ]  . sulla base di queste caratteristiche stato riconosciuto il razionale per limpiego clinico nellimaging vascolare cardiaco [ 82 , 83 ]  . uno dei primi gruppi a studiare la possibilit di valutare le anomalie coronariche ed il loro decorso mediante ebt stato quello di ropers et al . 
le maggiori limitazioni allutilizzo dellebt nella pratica clinica sono lelevato costo dellapparecchiatura e la limitata diffusione sul territorio nazionale ( si contano ad oggi circa 150 scanner ebt nel mondo , due terzi dei quali negli stati uniti )  . coronary magnetic resonance angiography tomografia computerizzata multistrato mra [ 1 , 7577 ] is a highly promising technique since no ionising radiation is used . 
in the study of the origin of the coronary arteries , mra can provide more complete information than conventional angiography , particularly in patients with other concomitant congenital cardiac anomalies [ 1 , 7577 ]  . the main limitation of mra is incomplete visualisation of the coronary vessels , particularly their distal tracts . 
laccuratezza diagnostica di questa metodica ed il suo verosimile utilizzo estensivo consentiranno di apprezzare la prevalenza delle ac nelle differenti popolazioni . i vantaggi della tcms risiedono essenzialmente nellelevata accuratezza diagnostica ed anatomica . 
these characteristics reduce cardiac - respiratory motion artefacts and the dose absorbed by the patient [ 2 ]  . this has been the basis for the techniques clinical use in cardiac vascular imaging [ 82 , 83 ]  . one of the first groups to study the possibility of assessing caas and their course with ebt was ropers et al . 
ebt therefore has a relatively high diagnostic accuracy in the study of caas with a limited radiation exposure estimated at 1.1 msv [ 84 ]  . the main limitations to the use of ebt in clinical practice are the high costs of equipment and the limited number of scanners in italy ( there are approximate 150 in the world , two thirds of which are in the united states )  . multislice computed tomography in a few short years , msct has revolutionised coronary diagnostics by highlighting the feasibility of a noninvasive approach . 
the diagnostic accuracy of the technique and its likely widespread use excellently place it to appreciate the prevalence of caas in different populations . the advantages of msct lie primarily in its high level of diagnostic and anatomical accuracy . 
the complex and tortuous coronary anatomy can be readily visualised with msct [ 13 ]  . despite the excellent results , even this technique suffers from a series of limitations . 
multiplanar reconstructions with maximum intensity projections ( mip ) of the anomalous left coronary artery ( acs ) in a para - axial ( a ) and coronal oblique ( b ) plane . 
the anomalous acs then runs anteriorly and on the left , passing below the right ventricular outflow tract ( tevd ) within the myocardium ( a , b , arrowheads )  . 
ricostruzioni multiplanari con proiezione di massima intensit ( mip ) dellarteria coronaria sinistra ( acs ) anomala su un piano para - assiale ( a ) e coronale obliquo ( b )  . 
la acs nasce dal seno coronario di destra e si porta anteriormente e verso sinistra , passando al di sotto del tratto di efflusso del ventricolo destro ( tevd ) , nel contesto del muscolo cardiaco ( punte di freccia in a e b )  . 
knowledge of the characteristics and consequences of different types of anomalies enables the radiologist to appropriately assess the finding . the indication of performing a noninvasive coronographic examination for suspected caa or to exclude the presence of a caa must be carefully considered in cooperation with the clinic in order to avoid exposing young patients to excessive ionising radiation . conclusioni le anomalie coronariche sono un reperto relativamente frequente nel contesto dellimaging diagnostico non invasivo coronarico . la conoscenza delle caratteristiche e delle conseguenze dei differenti tipi di anomalie permette al medico radiologo di giudicare in modo adeguato il reperto . 
passariello1 1dipartimento di scienze radiologiche , universit degli studi di roma la sapienza , viale regina elena 324 , i - 00161 roma , italy 2sezione di statistica , 3oncologia medica , dipartimento di medicina sperimentale e patologia , universit degli studi di roma la sapienza , roma , italy correspondence to : f . 
the aim of this study was to assess the efficacy of a computer - aided detection ( cad ) system in the identification of lung metastases and to compare the volumetric cad measurements with unidimensional observer measurements in the evaluation of treatment response in oncology patients . 
multislice ct scans were performed before and after the injection of contrast material with a high - resolution protocol ( collimation 4x1 mm , 100 mas , 120 kv )  . 
tutti gli esami sono stati eseguiti prima della somministrazione di mdc , con un protocollo di tc multistrato ( tcms ) ad alta risoluzione del torace ( collimazione 4x1 mm , 100 mas , 120 kv )  . 
successivamente , i noduli , identificati come lesioni target ( noduli pi significativi presenti sia prima che dopo chemioterapia ) , sono stati rivalutati mediante un sistema computerizzato in grado di effettuare il calcolo del volume della lesione . 
ventiquattro noduli ( dt : 518 mm nel primo studio e 420 mm nel follow - up ) sono stati inclusi in questa valutazione . gli osservatori hanno espresso un giudizio di risposta alla terapia sovrapponibile in 21 noduli : 8 considerati aumentati e 13 stabili . le misurazioni degli osservatori e le misurazioni automatiche fornite dal sistema cad differivano in 3 noduli : due considerati stabili dai radiologi e aumentati dal cad e uno considerato aumentato dai radiologi e stabile dal cad . 
computed tomography ( ct ) and , in particular , multislice technology , has long been considered a highly sensitive method for identification of lung metastases and evaluation of response to therapy during oncologic follow - up examinations ; in fact , its high spatial resolution and high contrast resolution at the level of the lung parenchyma make it an essential diagnostic tool in oncologic patients undergoing chemotherapy . the international criteria that define oncologic response were initially established in the 1980s by the world health organisation ( who ) [ 3 ] and recently modified as response evaluation criteria in solid tumours ( recist ) criteria [ 4 ]  . recist criteria only take into consideration the largest diameter of the significantly more important lesions ( five for each organ ; up to ten per patient in the case of multiorgan secondary lesions ) and subdivide response to therapy into four categories : stable disease , when there is a < 30% reduction or < 20% increase in maximum diameter or in the sum of diameters of more than one lesion ; partial response , when there is a > 30% reduction ; progressive disease , when there is a > 20% increase ; complete response , in the case of complete tumour disappearance [ 4 ]  . although they are simple and easy to use , these criteria present some limitations in clinical practice , namely : difficulty in measuring the lesion ( extremes of the lesion in the axial plane ) ; different slice thickness used in successive exams ( often performed in different facilities or with different devices ) ; ill - defined lesion margins ; type of measurement performed ( manual or electronic ) ; selection of the target lesions . 
a consequence of these limitations is often high intraand interobserver variability , with a highly variable judgement of response to therapy both between the different observers who evaluate follow - up studies and for the same observer reevaluating previous exams [ 58 ]  . 
in science , this variability is often avoided by consensus : readings by two or more observers , and calculation of the mean among all readings ( or , in this case , among measurements ) ; in practice , however , this is often not possible . one alternative proposed in identification of lung nodules and their measurement is the utilisation of computerised reading devices , which map the lesions and measure their volume . 
at present , these computer - aided detection ( cad ) il ruolo della diagnostica per immagini in oncologia andato ampliandosi negli ultimi anni , sia per lidentificazione e stadiazione della neoplasia primitiva , sia nella rivalutazione dei pazienti in corso di trattamenti chemioo radioterapici [ 1 , 2 ]  . 
la tc , ed in particolare la nuova tecnologia multistrato , gi da tempo considerata tecnica di elevata sensibilit nella identificazione delle metastasi polmonari e nella valutazione della risposta alla terapia nel corso dei follow - up oncologici ; infatti , lelevata risoluzione spaziale , associata alla elevata risoluzione di contrasto a livello del parenchima polmonare , ne consentono un essenziale strumento diagnostico in pazienti oncologici in corso di chemioterapia . i criteri internazionali che definiscono la risposta oncologica sono stati inizialmente definiti negli anni 80 dalla who [ 3 ] e recentemente modificati come criteri recist [ 4 ]  . 
i criteri recist prendono in considerazione unicamente il diametro maggiore delle lesioni significativamente pi importanti ( 5 per organo ; fino ad un massimo di 10 per paziente , nel caso in cui le lesioni secondarie siano multiorgano ) , suddividendo la risposta alla terapia in 4 differenti categorie : stabilit di malattia , nel caso in cui vi sia una riduzione inferiore al 30% o un incremento inferiore al 20% del diametro massimo o della somma dei diametri di pi lesioni ; risposta parziale , nel caso in cui la riduzione sia superiore al 30% ; progressione di malattia , nel caso di un incremento superiore al 20% ; risposta totale , nel caso di scomparsa totale della lesione [ 4 ]  . sebbene semplici e di facile utilizzo , i limiti di applicabilit nella pratica clinica sono rappresentati dalla difficolt di misurazione ( estremi massimi di lesione sul piano assiale ) , dal diverso spessore di strato utilizzato tra esami successivi ( spesso eseguiti in strutture o con apparecchiature diverse ) , dai margini di lesione non ben definibili , dal tipo di misurazione effettuata ( manuale o elettronica ) , dalla scelta delle lesioni target . 
conseguenza di tali limiti spesso rappresentata da unalta variabilit intraed interosservatore , con un giudizio di risposta alla terapia ampiamente variabile sia tra diversi osservatori che valutano studi successivi , sia nellosservatore stesso quando si trovi a rivalutare esami precedenti [ 58 ]  . 
during a 14 - month period ( january 2004 to march 2005 ) , 67 oncologic patients from the department of clinical oncology ( age range : 3562 years ) undergoing chemotherapy were evaluated using a four - detector - row multislice ct ( msct ) scanner ( volume zoom , forcheim , germany )  . to create a database , we included only patients undergoing chemotherapy with a diagnosis of lung metastasis on the first examination and a ct scan performed during the two chemotherapy cycles . 
exclusion criteria were : patients with studies performed with different acquisition protocols ; patients who did not have lung metastases at the first study ; those who did not have a complete response at the second study ( absence of nodular formations within the lung parenchyma )  . 
thus , nine patients with breast cancer were selected for our database . the acquisition protocol we used was the so - called combi - mode , with the following technical features : 4x1 - mm collimation , 100 mas , 120 kv . 
a una delle alternative proposte nella identificazione dei noduli polmonari e nella loro misurazione lutilizzo di sistemi di lettura computerizzati ; essi forniscono , infatti , una mappa delle lesioni ed una loro misurazione volumetrica . allo stato attuale , tali sistemi , denominati cad , vengono applicati principalmente alla valutazione dei noduli polmonari , sia nella loro identificazione , sia per la quantificazione del tempo di crescita in studi successivi [ 911 ]  . lo scopo del nostro studio stato provare a integrare tali sistemi nella valutazione dei pazienti oncologici , identificando eventuali differenze tra una lettura effettuata da diversi osservatori ed una valutazione volumetrica effettuata con il nostro sistema computerizzato . materiali e metodi popolazione di pazienti e protocollo di acquisizione lo studio da noi proposto uno studio retrospettivo ; per la raccolta dati e per lelaborazione degli stessi stata comunque richiesta lautorizzazione da parte del comitato etico . durante un periodo di 14 mesi ( gennaio 2004 , marzo 2005 ) , 67 pazienti oncologici ( et compresa tra 35 e 62 anni ) , provenienti dal dipartimento di oncologia clinica ed in corso di trattamento chemioterapico , sono stati valutati utilizzando un apparecchiatura di tc multistrato ( tcms ) a 4 banchi di detettori ( volume zoom , forcheim , germany )  . nella creazione del database sono stati inclusi nello studio unicamente i pazienti in corso di chemioterapia , con una diagnosi di metastasi polmonari al primo controllo e con una tc eseguita durante due cicli di chemioterapia . 
criteri di esclusione sono stati : pazienti con studi effettuati con protocolli di acquisizione diversi ; pazienti che non presentavano metastasi polmonari nel primo studio o con una risposta completa nel secondo studio ( assenza di formazioni nodulari nel parenchima polmonare )  . 
lobiettivo per linclusione stato di valutare sia lutilizzo di un sistema computerizzato , sia la risposta ad un determinato chemio farmaco per i pazienti oncologici . sono stati cos selezionati , nel nostro database , 9 pazienti con tumore della mammella . il protocollo di acquisizione stato condotto utilizzando una tecnica definita combi con le seguenti caratteristiche tecniche : collimazione 4x1 mm , 100 mas , 120 kv . 
lo spessore di strato utilizzato stato di 1 , 25 mm con un intervallo di ricostruzione di 1 mm ed algoritmo di ricostruzione b60 per lo studio del parenchima ; la valutazione del mediastino stata effettuata utilizzando ricostruzioni con spessore di strato di 5 mm e con algoritmo b30 . la scansione iniziale sul parenchima polmonare stata effettuata nelle sole condizioni basali ; successivamente le pazienti sono state valutate per un follow - up completo con un esame tc completo di torace - addome - pelvi e , dove richiesto , del cranio , dopo iniezione di mezzo di contrasto organo iodato non ionico . 
in order to reduce any intraand interobserver variability , nodule images were enlarged by 150%200% . the third radiologist then measured ( based on the numerical measurement provided by each observer ) the percentage difference between the single lesions , using the recist criteria , and providing a general opinion on disease improvement , worsening or stability , considering both response of the single nodules and response for each patient , summing the size of every single nodule . 
thus , 24 metastases were considered in the first reading and 24 in the second . statistical analysis in the evaluation of response to treatment , the recist criteria were taken into consideration ( both for single nodules and for each patient )  . 
in agreement with volumetric measurements , partial response was defined as > 65% reduction ; progressive disease was defined as one having > 73% volume increase ; stable disease was defined as one having < 73% increase and < 65% reduction ( table 1 ) [ 12 ]  . 
reading table 1 treatment response categories state trasferite sul nostro sistema di archiviazione pacs ( lifeweb , ferrania imaging technologies )  . selezione delle metastasi tutti i casi selezionati sono stati trasferiti su una console dedicata fornita di un sistema computerizzato di identificazione dei noduli polmonari ( lungcheck , r2 technology , inc )  . 
in un primo momento la lettura dei casi avvenuta in consensus tra due radiologi , ponendo a confronto su un doppio monitor il primo studio ed il follow - up , per identificare le lesioni target su cui successivamente effettuare le misurazioni.un terzo radiologo , non incluso nella valutazione dei casi , ha creato il database , registrando su una tabella la sede delle lesioni target scelte dai radiologi . successivamente , a distanza di una settimana , i due radiologi , singolarmente , hanno valutato le dimensioni delle lesioni prescelte secondo lasse massimo trasversale di ogni lesione ; per ridurre leventuale variabilit di misurazione intra ed interosservatore , le immagini dei noduli sono state ingrandite del 150%200% . il terzo radiologo ha quindi calcolato ( sulla risposta numerica di misurazione fornita da ciascun osservatore ) la percentuale di differenza tra le singole lesioni , utilizzando i criteri recist e fornendo quindi un giudizio complessivo di miglioramento , peggioramento e stabilit di malattia , considerando sia la risposta per singoli noduli , sia la risposta per paziente , effettuando la somma delle dimensioni di ogni singolo nodulo . 
nei casi di disaccordo nella valutazione della risposta stata effettuata una seconda lettura in consensus . category unidimensional criteriaa volumetric criteriab complete response tumour disappearance tumour disappearance partial response stable disease > 30% reduction in diameter > 65% reduction in volume size between that for partial and progressive disease size between that for partial and progressive disease progressive disease > 20% increase in in diameter > 73% increase in volume abased on response evaluation criteria in solid tumours ( recist ) guidelines . 
in patients with multiple lesions , individual maximum diameters were summed bbased on recist guidelines tabella 1 categorie di risposta al trattamento categorie criteri unidimensionalia criteri volumetricib risposta completa scomparsa delle lesioni scomparsa delle lesioni risposta parziale riduzione > 30% del diametro massimo riduzione > 65% del volume stabilit di malattia dimensioni comprese tra risposta parziale e progressione di malattia dimensioni comprese tra risposta parziale e progressione di malattia progressione di malattia aumento > 20% del diametro massimo aumento > 73% del volume asecondo i criteri recist . 
thus , kappa - type statistics were used to determine very poor ( k < 0.01 ) , poor ( k 0.010.20 ) , moderate ( k 0.210.40 ) , good ( k 0.410.60 ) , very good ( k 0.610.80 ) and excellent ( k 0.811.00 ) agreement [ 13 ]  . 
considering results for each patient , there was one case of disagreement between the radiologists , who stated the patient was stable , and cad , which revealed disease progression . 
consensus results between the observers and between cad and the observers are shown in table 4 . discussion an objective evaluation of response to therapy in oncologic patients is an important goal in routine radiological activity both concerning clinical decisions on patient treatment and trials on new chemotherapeutic agents [ 2 ]  . 
in the past few years , assessment and follow - up of oncologic patients has successivamente , i tre radiologi in consensus hanno valutato le dimensioni volumetriche effettuate dal sistema computerizzato ed hanno valutato la risposta alla terapia , anche in questo caso sia per nodulo che per paziente . 
sono state cos prese in considerazione 24 metastasi nella prima lettura e 24 nella seconda lettura . analisi statistica nella valutazione della risposta al trattamento sono stati presi in considerazione i criteri recist ( sia per i noduli singoli , sia per paziente )  . 
in accordo con le misurazioni volumetriche , la risposta parziale stata definita come una riduzione superiore al 65% ; la progressione di malattia come aumento del volume superiore al 73% ; la stabilit di malattia come aumento inferiore al 73% e diminuzione inferiore al 65% ( tabella 1 ) [ 12 ]  . i risultati delle letture sono stati inseriti in un foglio di lavoro excel e valutati , utilizzando un programma statistico ( spss 8.0 per windows ; spss ; chicago , iii ) , in termini di accordo inter - osservatore e di concordanza tra le misurazioni del singolo osservatori con i criteri recist e le misurazioni volumetriche del cad . bassa ( k < 0 , 01 ) , ( k = 0 , 010 , 20 ) , stata cos utilizzata una statistica k per determinare scarsa discreta ( k = 0 , 210 , 40 ) , buona ( k = 0 , 410 , 60 ) , sostanziale concordanza ( k = 0 , 610 , 80 ) e concordanza quasi perfetta ( k = 0 , 811 , 00 ) [ 13 ]  . 
per ogni test statistico effettuato stato definito come parametro di significativit un limite di p < 0 , 005 . risultati ventiquattro noduli sono stati inclusi in questa valutazione . il diametro dei noduli valutati risultato compreso tra 5 e 18 mm nel primo studio e 4 e 20 mm nel follow - up . 
considerando i risultati per paziente , in un caso si avuta una discordanza di valutazione tra i radiologi , per i quali il paziente era stabile , e il cad , per il quale cera stata una progressione di malattia . 
1a , b metastatic lung nodule in right upper lobe , evaluated with two multislice computed tomography ( msct ) studies before and after chemotherapy . agreement between the first ( a ) and second observer ( b ) in evaluation of therapy response . 
based on response evaluation criteria in solid tumours ( recist ) criteria , both radiologists considered the disease as stable . fig 1a , b nodulo polmonare di natura secondaria localizzato a livello del lobo superiore del polmone di destra , valutato con due esami tcms prima e dopo chemioterapia . 
a case of disagreement between the two observers : the first ( a ) considered the nodule decreased in size while the second ( b ) considered the nodule as stable after therapy . 
il consensus , effettuato in un secondo momento , ha classificato il nodulo come stabile dopo terapia . marked improvement in survival and quality of life ; second , greater availability of increasingly sophisticated ct scanners , which results in a rise in requests for radiological examinations . 
these fundamentals result in a need to evaluate an increasing number of ct exams and in particular to study , even at short time intervals , exams of oncologic patients undergoing chemotherapy or actinic therapy . in spite of the growing diagnostic reliability of latest - generation scanners in diagnosis and staging , the large number of exams to be read and compared with previous exams often performed with different imaging techniques raises the issue of objective variability of opinions [ 7 ]  . 
nevertheless , as revealed in clinical practice and reported in our study , interobserver variability , even on a limited number of patients and with readers asked to define a response to therapy , proved to be wide and , in our study , involved two out of nine patients . 
in fact , according to recist criteria , measurement of the greatest transverse diameter of a lesion involves the obvious limitation of selecting the slice on which to perform the measurement [ 5 ] ; furthermore , although metastatic lung diseases generally consist of often spherical nodular masses , eccentric growth in the tra le molteplici ragioni di questo incremento nel numero di esami vi sono certamente due fondamenti . 
primo , terapie pi aggressive , con farmaci antiblastici di maggior successo nelle forme secondarie , con una prognosi per il paziente maggiormente favorevole ed un netto miglioramento della durata e della qualit di vita . 
il risultato che ne deriva rappresentato dalla necessit di valutare un numero crescente di esami tc ed in particolare di valutare , spesso a distanze ravvicinate , esami di pazienti oncologici in corso di terapie chemioterapiche o attiniche . 
nonostante la sempre maggior affidabilit diagnostica delle apparecchiature di ultima generazione nella diagnosi e stadiazione , lelevato numero di esami da dover giornalmente valutare , ponendo a confronto esami precedenti , peraltro spesso eseguiti con tecniche di studio diverse , pone il radiologo di fronte ad una oggettiva variabilit di giudizio [ 7 ]  . uno dei sistemi adottati nel nostro dipartimento per la valutazione di tutti i pazienti oncologici e per ridurre la variabilit intra ed interosservatore di utilizzare protocolli standard , e quindi riproducibili e confrontabili negli studi successivi . nonostante ci , cos come evidenziato nella pratica clinif . 
questi sistemi hanno dimostrato una ampia affidabilit diagnostica , con valori di sensibilit superiori al 90% e superiori alla valutazione dello stesso radiologo [ 911 ]  . una della caratteristiche tecniche di questi sistemi la valutazione automatica delle dimensioni del nodulo in termini di volume , consentendo quindi di comparare studi diversi ; lutilizzo di tali sistemi computerizzati allo stato attuale stato limitato alla identificazione dei noduli polmonari e alla valutazione del tempo di crescita . 
both observers agreed in classifying the nodule as progressive disease according to response evaluation criteria in solid tumours ( recist ) criteria ( a , b ) ; computer - aided detection ( cad ) volumetric measurements classified the disease as stable ( c )  . fig 3a - c metastasi polmonare localizzata a livello del lobo medio del polmone di destra . 
i due radiologi ( a , b ) concordavano , in accordo con i criteri recist , sulla progressione di malattia , mentre le misurazioni volumetriche effettuate dal cad consideravano stabile la patologia secondaria dopo terapia ( c )  . longitudinal plane is not uncommon [ 2 ]  . 
this leads to interobserver variability among measurements and , therefore , an overor underestimation of response to therapy according to recist criteria . several authors have evaluated the role of three - dimensional ( 3d ) measurements of lung nodules and liver metastases ; results provided by volumetric measurements present , in some cases , considerable differences from uniand bidimensional measurements , in particular when the lesion margins are ill defined [ 12 , 14 ]  . 
nonetheless , one limitation of using a method for volumetric measurement is the long time needed to calculate the volume , mainly due to the manual drawing of the lesion edges ; furthermore , small or ill - defined lesions are often associated with manual errors in volume calculation [ 15 ]  . the aim of our study was to apply a computerised system capable of automatically performing a volumetric measurement of a lesion to the evaluation of oncologic patients with f . 
one technical features of these systems is automatic assessment of nodule size in terms of volume , which allows for comparison between different studies ; at present , use of these techniques is limited to identification of lung nodules and assessment of their growth pattern . 
this role has proved to be extremely significant , providing better evaluation of volumetrically increased nodules that remain stable in the transverse plane [ 16 ]  . in our study , we took into consideration interobserver consensus , thus obtaining fair agreement between the response to therapy assessed by the observers and that assessed through the measurement ; nevertheless , the observers recorded three cases in which images had to be reevaluated to reach a consensus . 
this means that interobserver variability plays an important role in establishing therapy success . on the other hand , other authors have shown that volume measurement by a computerised system is reliable and reproducible among different studies [ 17 ]  . 
thus , the possibility of evaluating response to therapy using a computerised technique able to provide volumetric results represents valuable support for the radiologist , as it reduces reading times and provides more objective evaluation , even for lung metastases . conclusions cad volumetric measurements easily allow for objective , reproducible and automated volume measurement , thus limiting interobserver variability in evaluation of response to treatment of oncologic patients . 
furthermore , a different selection of more spherical or irregular metastases could modify results of our volumetric measurement , leading to greater agreement or disagreement between observers and cad . thus , our study only presents preliminary results and aimed at assessing the possibility of using computerised techniques even in the evaluation of response to treatment of oncologic patients . 
a second limitation found in the daily application of these systems is the need to use thin - collimation protocols , indispensable for cad image processing , which lead to difficult comparisons with exams performed in other centres and with different protocols . 
for this reason , our study had to be retrospective , performed only on patients who had undergone high - resolution exams that could be analysed by cad . two considerations need to be made about this limitation : first is that the need to use thin - slice protocols for correct evaluation of lung nodules has recently been demonstrated gliorare la valutazione di noduli cresciuti volumetricamente , ma stabili su un piano di misurazione trasversale [ 16 ]  . nel nostro studio , prendendo in considerazione il consensus tra gli osservatori , si ottenuta una buona concordanza tra la risposta alla terapia dei lettori e quella fornita da una misurazione volumetrica ; nonostante ci , tra gli osservatori vi sono stati 3 casi in cui le immagini sono state rivalutate per raggiungere un accordo . ne consegue che la variabilit interosservatore gioca un ruolo importante nello stabilire il corretto andamento di una terapia . di contro , la valutazione del volume di un sistema computerizzato stata dimostrata da altri autori essere invece una misura affidabile e quindi riproducibile nella comparazione tra i diversi studi [ 17 ]  . la possibilit di valutare la risposta alla terapia utilizzando un sistema computerizzato , in grado di fornire risultati volumetrici , rappresenta , quindi , un valido ausilio per il radiologo , riducendo il tempo di lettura e fornendo una valutazione maggiormente oggettiva anche per le metastasi polmonari . conclusioni le misurazioni volumetriche effettuate dal cad consentono con facilit una misurazione oggettiva , riproducibile ed automatica in termini volumetrici , riducendo , quindi , la variabilit interosservatore nella valutazione della risposta al trattamento dei pazienti oncologici . il nostro studio presenta alcune limitazioni , prima fra tutte il limitato numero di pazienti e di metastasi valutate . 
 [ 5 ] ; the second consideration concerns the increasing use of multislice ct scanners , with collimations that are either fixed or variable to less than 1 mm , that allow for storage on increasingly sophisticated pacs systems of submillimetre raw data that can thus be used by cad systems . the final limitation is that cad could be unable to identify all nodules chosen as target lesions . 
in our study , this did not happen , as cad identified all nodules selected as target lesions ; however , it is still possible to manually add a lesion not identified by cad on which the system can perform the same volumetric measurement . integration of new systems capable of automatically comparing two studies on different screens and providing the percentage of variation of a lesion paves the way for routine use of cad systems , even in evaluation of lung metastases , as well as representing a valuable second reading in screening programmes and identification of lung nodules . mm , con possibilit di archiviare su sistemi pacs sempre pi performanti i dati grezzi submillimetrici , utilizzabili quindi anche dai sistemi cad . infine , unultima limitazione che il cad potrebbe non identificare tutti i noduli scelti come lesioni target . 
nel nostro studio ci non si verificato , avendo il cad identificato tutte le formazioni nodulari prescelte come lesioni target ; nonostante ci , possibile aggiungere manualmente una lesione non identificata dal cad , su cui viene eseguita dal sistema la stessa misurazione volumetrica . 
lupattelli3 1dipartimento di patologia umana ed oncologia , sezione di radiologia oncologica , medicina nucleare e radioterapia , universit degli studi di siena , policlinico santa maria alle scotte , viale bracci 2 , i - 53100 siena ( si ) , italy 2radiologia universitaria , azienda ospedaliera universitaria senese , italy 3dipartimento di scienze chirurgiche , radiologiche , odontoiatriche e medico - legali , universit degli studi di perugia , italy 4ge healthcare , italy correspondence to : l . 
the purpose of this study was to test the reproducibility of the three - dimensional ( 3d ) advanced lung analysis software ( 3d - ala , ge healthcare ) in the estimation of pulmonary nodule volume . 
we retrospectively reviewed the unenhanced multislice ct scans ( lightspeed pro 16 ge ) of 77 patients with a solitary pulmonary nodule ( n = 71 ) or metastatic pulmonary disease ( n = 6 )  . 
the following acquisition parameters were used : slice thickness 0.625 mm , reconstruction interval 0.4 mm , pitch 0.562 : 1 , 140 kv , 300 mas , field of view 13 cm , bone kernel . for each of the 103 nodules three , 3d volume measurements were obtained by the 3d - ala software . 
the reproducibility of nodule segmentation was evaluated according to a visual score ( 1 = optimal , 95% ; 2 = fair , 9095% ; 3 = poor , 90% ) by three observers working in consensus . 
ala - 1 software allowed segmentation in all nodules ( type 1 segmentation n = 43 , type 2 n = 35 , type 3 segmentation n = 25 )  . 
sono stati analizzati retrospettivamente gli esami tc multistrato ( lightspeed pro 16 ge ) , senza mdc per vena di 77 pazienti ( 71 con noduli polmonari solitari e 6 con noduli secondari )  . 
i parametri di acquisizione utilizzati per il calcolo volumetrico sono stati i seguenti : spessore di strato 0 , 625 mm , intervallo di ricostruzione 0 , 4 mm , pitch 0 , 562 : 1 , 140 kv , 300 mas , fov 13 cm , algoritmo di ricostruzione ad elevata risoluzione spaziale . 
per ciascun dei 103 noduli , impiegando il software ala1 , sono state effettuate tre misurazioni volumetriche . tre osservatori in consensus , in ogni misurazione , hanno assegnato alla segmentazione dei contorni di ciascun nodulo , un punteggio visivo ( 1 = ottimo , 95% ; 2 = discreto , 9095% ; 3 = scarso , 90% )  . 
tale software ha consentito una segmentazione di tipo 1 in 43 noduli , una segmentazione di tipo 2 in 35 noduli ed una segmentazione di tipo 3 in 25 noduli . 
il calcolo volumetrico risultato identico , nelle tre misurazioni consecutive , in 62 dei 103 noduli : 16 dei 19 noduli circoscritti ( 84 , 2% ) , 31 dei 45 noduli iuxta - vascolari ( 68 , 8% ) e 15 dei 39 noduli iuxta - pleurici ( 38 , 4% )  . ala1 non ha effettuato una ricostruzione riproducibile in termini di volume in 41 dei 103 noduli , di cui 3 dei 19 ( 15 , 7% ) circoscritti , 14 dei 45 ( 31 , 1% ) iuxta - vascolari , 24 dei 39 ( 61 , 5% ) iuxta - pleurici e dei quali 2 su 41 ( 4 , 8% ) sono stati segmentati dal software con qualit 1 , 15 su 41 ( 36 , 5% ) con qualit 2 ed infine 24 su 41 ( 58 , 5% ) con qualit di segmentazione pari a 3 . 
il calcolo volumetrico con il software ala1 riproducibile per tutti i noduli in riferimento alle dimensioni e al tipo . il software ala1 ha consentito un calcolo volumetrico affidabile e riproducibile nei noduli circoscritti e nella maggior parte dei noduli iuxta - vascolari . 
relativamente ai noduli iuxta - pleurici , in particolare quelli adiacenti alla pleura diaframmatica , la riproducibilit del calcolo volumetrico non ancora sufficiente e necessita di miglioramenti tecnici . parole chiave tomografia computerizzata multistrato nodulo polmonare solitario introduction introduzione characterisation of pulmonary nodules , above all those measuring 510 mm , is difficult if not impossible with positron emission tomography ( pet ) , multiphasic dynamic computed tomography ( ct ) or percutaneous needle biopsy [ 13 ]  . 
with the exception of completely calcified nodules and / or fat - containing nodules , which are considered benign , all other nodules are to be considered potentially malignant , and it is crucial to monitor their possible growth which , if demonstrated , calls for a histological diagnosis with percutaneous biopsy , video - assisted thorascopy or surgical intervention . 
currently , ct is instrumental in evaluating the growth parameter of a pulmonary nodule , as it allows two - dimensional ( 2d ) comparison of diameters in the axial plane . 
however , the asymmetrical or minimal growth of some malignant nodules may be missed if a manual , and therefore nonreproducible , ct measurement is used [ 4 ]  . 
the aim of this study was to demonstrate the reproducibility of volume measurement of pulmonary nodules using the advanced lung analysis software ( ala - 1 , ge healthcare )  . materials and methods patient selection our series comprised 77 nonconsecutive patients ( 42 men , 35 women ) aged 4582 years ( mean age 65 years ) studied by ct between january and december 2004 . 
inclusion criteria were solid , noncalcific pulmonary nodules according to the definition reported in the literature [ 5 ] , solitary pulmonary nodules ( spn ) with peripheral ground - glass attenuation and central cavitation . 
in six patients , oncological follow - up revealed multiple repetitive lesions . overall , we retrospectively evaluated 103 nodules ranging in size between 3.8 mm and 30 mm , 19 of which were well cirla caratterizzazione di un nodulo polmonare , specie di quelli con dimensioni comprese tra 5 mm e 10 mm , difficile se non impossibile con metodiche quali la tomografia computerizzata ad emissione di positroni ( pet ) , la tc dinamica multifasica o lagobiopsia percutanea [ 13 ]  . ad eccezione di alcuni noduli polmonari completamente calcifici e / o contenenti tessuto adiposo e pertanto considerati benigni , tutti gli altri sono da considerarsi potenzialmente maligni e perci diventa cruciale la valutazione delleventuale aumento delle loro dimensioni che , se dimostrato , impone una diagnosi istologica con biopsia percutanea , videotoracoscopia oppure lintervento chirurgico . allo stato attuale , la tc riveste un ruolo fondamentale nella valutazione del parametro crescita di un nodulo polmonare , poich consente una comparazione bidimensionale dei diametri su piani di scansione assiali . 
tuttavia , la crescita asimmetrica o di lieve entit di alcuni noduli maligni pu essere misconosciuta , impiegando un tipo di misurazione manuale con tc e quindi non ripetibile [ 4 ]  . obiettivo del nostro studio quello di dimostrare la ripetibilit del calcolo volumetrico dei noduli polmonari utilizzando un software automatico advanced lung analysis ( ala1 , ge healthcare )  . materiali e metodi selezione dei pazienti la serie comprende 77 pazienti ( 42 maschi e 35 femmine ) non consecutivi di et compresa tra 45 e 82 anni ( media 65 anni ) , sottoposti a tc nel periodo compreso tra gennaio 2004 e dicembre 2004 . criteri di inclusione : noduli polmonari solidi non calcifici secondo la definizione riportata in letteratura [ 5 ] , noduli polmonari solitari con ground - glass periferico e con cavitazione centrale . 
criteri di esclusione : noduli con diametro > 30 mm , noduli con atelectasia adiacente , noduli calcifici ed a componente mista ( solido - liquida )  . settantasette pazienti presentavano un nodulo polmonare solitario ( nps ) riscontrato casualmente in corso di un esame radiologico del torace o tc del torace effettuati per altri cumscribed , 45 juxtapleural and 39 juxtavascular ( table 1 )  . acquisition parameters examinations were conducted with a 16 - detector - row multislice ct ( msct ) scanner ( lightspeed pro 16 ge healthcare , milwaukee , wi , usa )  . 
the study to identify the pulmonary nodules was carried out with an unenhanced lowdose ct scan of the chest with volumetric caudocranial acquisition from the costophrenic sulci to the thoracic inlet and with a single inspiratory breath - hold . 
the study to measure nodule volume was performed by including the single nodule in the acquisition and using the following acquisition parameters : slice thickness 0.625 mm , reconstruction interval 0.4 mm , pitch 0.562 : 1 , 140 kv , 300 mas , field of view 13 cm and high spatial resolution reconstruction algorith ala software the ala software segments the pulmonary nodule by combining watershed segmentation and shape analysis techniques . 
different shape - analysis algorithms are used depending on the type of nodule ( which is recognised automatically ) : ( a ) well - circumscribed ( no or minimal connections with vessels or pleura ) ; ( b ) juxtavascular ( adhering to vascular structures ) ; ( c ) juxtapleural ( adhering to pleural structures )  . 
in 6 pazienti , in follow - up oncologico , sono state rilevate lesioni ripetitive multiple . complessivamente sono stati valutati retrospettivamente 103 noduli con dimensioni variabili da 3 , 8 mm a 30 mm e di cui 19 circoscritti , 45 iuxta - pleurici e 39 iuxta - vascolari ( tabella 1 )  . parametri di acquisizione lesame stato condotto con apparecchiatura tc multistrato a 16 banchi di detettori ( lightspeed pro 16 ge healthcare , milwaukee , usa )  . 
per la ricerca dei noduli polmonari stato effettuato un esame tc del torace a bassa dose con acquisizione volumetrica senza mdc per via endovenosa , dagli sfondati costo - frenici allo stretto toracico superiore , in senso caudo - craniale , in singola apnea inspiratoria . 
lo studio per il calcolo volumetrico del nodulo stato effettuato includendo nel volume di acquisizione il singolo nodulo ed utilizzando i seguenti parametri di acquisizione : spessore di strato 0 , 625 mm , intervallo di ricostruzione 0 , 4 mm , pitch 0 , 562 : 1 , 140 kv , 300 mas , campo di vista ( field of view ) 13 cm ed algoritmo di ricostruzione ad elevata risoluzione spaziale . software ala il software da noi utilizzato segmenta il nodulo polmonare mediante un sistema combinato di segmentazione spartiacque e di tecnica di analisi della forma . 
il concetto di segmentazione la chiave del funzionamento del software advanced lung analysis ( ala )  . il risultato di una segmentazione automatica , ottenuta dopo che loperatore ha posto un repere di identificazione sul nodulo , fornisce una calcolo , anchesso automatico , del volume del nodulo stesso . 
after the radiologist has placed a marker on the nodule , the segmentation process starts with automatic display of a region of interest around the nodule that includes surrounding anatomic structures such as vessels , pleural surface and mediastinuthis box is oversampled to obtain isotropic voxels for more accurate volume estimation . 
lultimo step lanalisi della forma , che applica la giusta segmentazione per isolare il nodulo dai vasi e / o dalla pleura circostanti . and retrospectively analysed on a workstation ( advantage windows 4.2 , ge healthcare ) and were classified based on type and algorithm used by the software into well circumscribed ( n = 19 ) , juxtavascular ( n = 45 ) and juxtapleural ( n = 39 ) if in contact with the mediastinal , diaphragmatic pleura , fissures or chest wall . 
of the 103 nodules studied , ten ( 9.7% ) had a diameter < 5 mm , 25 ( 24.27% ) had a diameter of 5 mm and < 10 mm , 41 ( 39.8% ) had a diameter of 10 mm to < 15 mm , 14 ( 13.6% ) had a diameter of 15 to < 18 mm and 13 ( 12.62% ) had a diameter 18 and < 30 mm . the case series comprised 98 solid nodules , three excavated nodules ( two juxtavascular and one juxtapleural ) , one well - circumscribed excavated nodule with peripheral ground - glass attenuation , and one well - circumscribed solid nodule with peripheral ground - glass attenuation . 
accuracy and reproducibility of nodule contour segmentation over three consecutive measurements was evaluated in consensus by three radiologists ( lv , ms , mam ) with different levels of experience in chest ct ( 25 , 16 , 5 years )  . 
on the basis of the softwares ability to outline the nodule contours as accurately as possible , a visual score was assigned ( 1 = optimal , 95% ; 2 = fair , 9095% ; 3 = poor , 90% )  . 
reproducibility of volume measurement was assessed by comparing the values in mm3 estimated in the three measurements obtained for each nodule . statistical analysis correlations were made between the three measurements and between each measurement and diameters . 
repeatability of the calculation of mean volume mm3 in the three measurements obtained for each nodule ( well circumscribed , juxtavascular and juxtapleural ) was determined by simple variance analysis ( anova test )  . 
standard deviation in parentheses measurement nodules anova test 1 , 422.69 ( 1 , 678.68 ) 1 , 451.23 ( 1 , 702.76 ) 1 , 411.00 ( 1 , 650.18 ) p > 0.05 tabella 2 volume medio ( in mm3 ) dei noduli i nelle tre misurazioni . 
i numeri in parentesi indicano la deviazione standard misurazione noduli anova test 1422 , 69 ( 1678 , 68 ) 1451 , 23 ( 1702 , 76 ) 1411 , 00 ( 1650 , 18 ) p > 0 , 05 l . 
dei 103 noduli studiati , 10 ( 9 , 7% ) presentavano un diametro < 5 mm , 25 noduli ( 24 , 27% ) 510 mm , 41 ( 39 , 8% ) 1015 mm , 14 ( 13 , 6% ) 1518 mm ed infine 13 ( 12 , 62% ) 1830 mm . nella casistica sopradescritta sono compresi 98 noduli solidi , 3 noduli escavati ( 2 iuxta - vascolari ed 1 nodulo iuxtapleurico ) , 1 nodulo escavato con ground - glass periferico , circoscritto , ed 1 nodulo solido con ground - glass periferico , anchesso circoscritto . impiegando il software ala1 sono state effettuate tre misurazioni volumetriche automatiche consecutive per ciascuno dei 103 noduli studiati . 
laccuratezza e la riproducibilit nella segmentazione dei contorni di ogni nodulo , in tre misurazioni consecutive , stata valutata , in consensus da tre radiologi ( lv , ms , mam ) con diversa esperienza in tc del torace ( 25 , 16 , 5 anni )  . in base alla capacit del software di delineare i contorni del nodulo nel modo pi fedele possibile , stato assegnato un punteggio visivo ( 1 = ottimo , 95% ; 2 = discreto , 90%95% ; 3 = scarso , 90% )  . 
la riproducibilit nel calcolo del volume stata valutata confrontando i valori in mm3 stimati nelle tre misurazioni ottenute per ciascun nodulo . analisi statistica stata effettuata la correlazione tra le tre misurazioni e tra ciascuna misurazione e i rispettivi diametri . 
la riproducibilit nel calcolo del volume medio in mm3 nelle tre misurazioni ottenute per ciascun nodulo ( circoscritto , iuxta - vascolare e iuxtapleurico ) stata determinata con lanalisi della varianza semplice ( anova test )  . 
un valore p inferiore a 0 , 05 stato considerato statisticamente significativo . risultati la segmentazione stata possibile in tutti i noduli analizzacon il software ala1 stata ottenuta una segmentazione di tipo 1 in 43 noduli , una segmentazione di tipo 2 e 3 rispettivamente in 35 e 25 noduli . 
il software ala1 ha consentito una segmentazione ottima ( tipo 1 ) separando correttamente il nodulo dalla parete toracica . table 3 mean volume ( in mm3 ) of nodules after dimension classification in three measurements . 
standard deviation in parentheses measurement circumscribed ( n = 19 ) juxtapleural ( n = 39 ) juxtavascular ( n = 45 ) tabella 4 volume medio ( in mm3 ) dei noduli , dopo stratificazione per tipo , nelle tre misurazioni . 
the size distribution of the 41 nodules in which ala - 1 failed to provide a repeatable volume reconstruction was : 0 nodules < 5 mm , 11 nodules 5 to < 10 mm , 19 nodules 10 to < 15 mm , two nodules 15 to < 18 mm and nine nodules 18 to < 30 mm . of the 41 nodules with nonreproducible volume reconstruction , 22 showed a difference , expressed in mm3 , over the three measurements < 5% , nine 5% to < 10% and ten 10% . 
these 41 nodules included one excavated nodule ( juxtapleural ) and the solid nodule with peripheral groundglass attenuation ( well circumscribed ) , for which the difference , expressed in mm3 , over the consecutive measurements was < 5% . in the nodules for which ala - 1 yielded identical volume calculation , repeatability of the three measurements correlated with repeatability of the segmentation ; in contrast , in the nodules without repeatable volume calculation , none of the three measurements correlated with segmentation repeatability . 
the mean volume ( in mm3 ) of the well - circumscribed , juxtavascular and juxtapleural in the three measurements is shown in table 2 . figures 6 and 7 show the correlation between nodule volume and diameter , respectively . 
 discussion the introduction of singleand multislice ct scanners in clinical practice has substantially increased detection rates of pulmonary nodules [ 6 ] , and it has been reported that in 23%66% of subjects participating in screening programmes , at least one solitary pulmonary nodule is discovered , most with a diameter of 510 mm [ 7 , 8 ]  . previous reports have highlighted the potential utility of computer - aided detection ( cad ) in increasing sensitivity of misu misu misu misu misu misu itlineformisu misu itlineformisu misu itlineformisu misu qinear qinear qinear fig . 
nei 41 noduli sopradetti rientrano 1 nodulo escavato ( iuxtapleurico ) ed il nodulo solido con ground - glass periferico ( circoscritto ) che presentavano comunque una differenza , espressa in mm3 , tra le tre misurazioni consecutive < 5% . nei noduli in cui ala1 consentiva un identico calcolo del volume , la riproducibilit delle tre misurazioni si correlava con una riproducibilit di segmentazione ; viceversa nei noduli senza riproducibilit nel calcolo del volume , ciascuna delle tre misurazioni non si correlava , in nessun caso , con una riproducibilit di segmentazione . il volume medio ( in mm3 ) dei noduli circoscritti , iuxtavascolari e iuxta - pleurici nelle tre misurazioni riportato nella tabella 2 . nelle figure 6 e 7 riportata rispettivamente la correlazione tra volume e diametro del nodulo . 
lanalisi statistica non ha dimostrato una differenza statistica significativa tra i volumi dei noduli riscontrando quindi una forte correlazione ( p < 0 , 01 )  . measure 1 / misurazione 1 diameter / diametro measure 2 / misurazione 2 diameter / diametro measure 3 / misurazione 3 volume volume volume diameter / diametro fig . 
gli atti della letteratura , infatti , riportano che dal 23% al 66% di soggetti inclusi in un programma di screening presentano almeno un nodulo polmonare solitario nella maggior parte con diametro compreso tra i 5 ed i 10 mm [ 7 , 8 ]  . alcuni autori hanno sottolineato la potenziale utilit del cad ( computer - aided detection ) nel consentire un incremento della sensibilit nella identificazione dei noduli polmonari con tcms e nel ridurre il tasso di falsi positivi , con possibilit di rimpiazzare nella doppia lettura il secondo radiologo [ 9 , 10 ]  . 
 because lung cancer often presents as an spn and treatment at an early stage ( ia ) is associated with relatively good outcomes ( 5 - year survival around 60%70% ) , the current tendency is to overdiagnose and treat even very small nodules [ 11 ]  . 
moreover , hospitalisation for surgical removal of an spn costs around us$ 25 , 000 [ 15 ] , so that the aim of treatment is to find a balance between early and unnecessary resections . the main objective in the management of an spn is therefore twofold : ( a ) achieve early resection of malignant forms , and ( b ) minimise the number of benign nodules that are surgically removed . 
characterisation of an spn often relies on morphological criteria ( dimensions , shape , contours , internal characteristics ) of high - resolution ct , which is far more sensitive than the standard ct technique in evaluating features typically associated with benignity or malignancy [ 16 ]  . 
to date , however , only three such criteria have been widely accepted as indicators of benignity : ( a ) intralesional calcification with benign pattern ; ( b ) presence of intralesional fat ; ( c ) absence of volume increase for over two years . 
have , however , reported that the likelihood of a small noncalcified nodule ( 4 mm ) displaying perceptible growth at ct follow - up after 1 year is 1% ; they therefore recommend that nodules 4 mm in patients with no previous history of malignancy should undergo a first ct follow - up scan after 12 months at least rather than 36 months and be followed up for at least 2 years thereafter to ascertain the nodule is stable [ 17 ]  . 
 [ 4 ] reported a case of a nodule with an asymmetrical growth that was considered to have a doubling time compatible with benignity on the basis of 2d measurements but compatible with malignancy on the basis of 3d measurements , thus underlining that some malignant nodules may have an asymmetrical growth that may be missed with conventional 2d measurements . moreover , manual measurements may prove extremely imprecise and nonreproducible , especially when carried out on small nodules with only minimal growth [ 18 ] even if done with standard window and level values . 
the possibility of assessing the growth of an spn by means of automatic a tuttoggi , tuttavia , solo tre criteri radiologici tc sono largamente accettati come indice di benignit : ( a ) calcificazioni intralesionali con pattern benigno ; ( b ) presenza di tessuto adiposo intralesionale ; ( c ) assenza di crescita volumetrica per un periodo superiore a due anni . la valutazione delle caratteristiche morfologiche di un nps con tc ad alta risoluzione pu aiutare il radiologo nel differenziare forme benigne da forme maligne ed evitare ulteriori , costosi approfondimenti diagnostici . 
hanno comunque riportato che esiste una probabilit 1% che un piccolo nodulo non calcifico ( 4 mm ) possa presentare una crescita percettibile in un follow - up con tc entro 12 mesi e raccomandano quindi nei noduli con dimensioni 4 mm , in pazienti con anamnesi negativa per patologia neoplastica , un primo controllo tc ad almeno 12 mesi di distanza piuttosto che 36 mesi ed un follow - up con durata di almeno due anni per accertarne la stabilit [ 17 ]  . il follow - up mediante tc , basato sul rilievo della crescita del nodulo , si realizza con la misurazione comparativa bidimensionale del diametro su piani di scansione assiali . 
tuttavia poich la crescita di un nodulo un fenomeno tridimensionale , il calcolo diretto del volume , pu essere estremamente pi accurato della misurazione bidimensionale dei suoi diametri . a tal proposito , yankelevitz et al . 
 [ 4 ] hanno riportato un nodulo con crescita asimmetrica il cui tempo di raddoppiamento era stato considerato compatibile con un pattern benigno sulla base delle misurazioni bidimensionali e che invece presentava un tempo di raddoppiamento compatibile con un pattern maligno sulla base delle misurazioni volumetriche , sottolineando cos che alcuni noduli maligni possono presentare una crescita asimmetrica che pu essere misconosciuta dalle misurazioni bidimensionali convenzionali . inoltre leffettuazione di misurazioni manuali pu essere estremamente imprecisa e non ripetibile sopratutto su noduli di piccole dimensioni e con modesto incremento dimensionale [ 18 ] anche se effettuata con valori standard di finestra e di livello . la possibilit di valutare la crescita dimensionale di un nps mediante il calcolo volumetrico automatico , piuttosto che tramite il confronto manuale di due successive misurazioni della lesione sul piano assiale , una tecnica di recente introduzione e non ancora validata su ampie casistiche [ 1820 ]  . attualmente , sono in commercio alcuni software che consentono il calcolo volumetrico delle lesioni nodulari parenchimali polmonari con tcms . 
le problematiche concernenti , il calcolo automatico 3d di un nodulo , con tali software , sono essenzialmente legate a : ( a ) modalit destrazione del volume ; ( b ) posizionamento del marker o repere che individua il nodulo . lelevata riproducibilit o ripetibilit di un software 3d dipende dal fatto che lestrazione del volume viene eseguita automaticamente mediante lutilizzo di differenti algoritmi volume estimation rather than by manually comparing two successive measurements of cross - sectional diameter is a recent technique that has not yet been validated on a wide scale [ 1820 ]  . 
in particular , ala - 1 had higher reproducibility in volume calculation of well - circumscribed nodules compared with juxtavascular and juxtapleural nodules and higher reproducibility in volume calculation in nodules with type 1 segmentation compared with those with type 2 and 3 segmentation . the close correlation between volumes demonstrates the extreme reproducibility of ala - 1 unlike diameter measurements , which do not correlate with volume . 
 as regards nodule size in relation to nodule type in the various subgroups , variance analysis demonstrated that no statistically significant difference exists ( p > 0.05 ) between mean volumes in the three measurements relative to nodule type or there is no correlation between reproducibility of volume calculation and nodule type . 
mentre ala1 non ha effettuato una ricostruzione riproducibile in termini di volume in 41 su 103 noduli , di cui il 15 , 7% rappresentati da noduli circoscritti , il 31 , 1% da noduli iuxta - vascolari , il 61 , 5% da noduli iuxtapleurici . 
dei 41 noduli con segmentazione non riproducibile il 4 , 8% sono stati segmentati dal software con qualit 1 , il 36 , 5% con qualit 2 ed infine il 58 , 5% con qualit di segmentazione pari a 3 . 
i risultati ottenuti evidenziano lesistenza di una correlazione tra riproducibilit volumetrica e qualit di segmentazione e tra riproducibilit volumetrica e tipo di nodulo . in particolare ala1 presenta una percentuale di riproducibilit nel calcolo volumetrico pi elevata nei noduli circoscritti rispetto a quelli iuxta - vascolari e iuxta - pleurici ed una percentuale di riproducibilit nel calcolo del volume pi elevata nei noduli con qualit di segmentazione pari ad 1 rispetto a quelli con segmentazione di tipo 2 e 3 . 
la stretta correlazione tra i volumi dimostra che ala1 estremamente riproducibile differentemente dalla misurazione dei diametri che non correlano con il volume . relativamente alle dimensioni in relazione al tipo di nodulo nei vari sottogruppi , lanalisi della varianza ha dimostrato che non esiste alcuna differenza statistica significativa ( p > 0 , 05 ) tra le medie dei volumi nelle tre misurazioni in relazione alle dimensioni e al tipo di nodulo , ovvero non esiste una correlazione tra riproducibilit del calcolo volumetrico e dimensioni in relazione al tipo di nodulo . i dati della nostra serie , confermano la affidabilit e la riproducibilit del software . 
la riproducibilit del software stata valutata nel calcolo volumetrico di una singola acquisizione tc e non prende in considerazione due differenti acquisizioni tc ( ad esempio , eventuale controllo a distanza ) che potrebbero inficiare la comparazione del volume ; 2 . 
volume calculation with ala - 1 has therefore the potential to enter decision - making algorithms for management of spn and replace the less accurate 2d measurement in the ct follow - up of indeterminate or benign - appearing nodules . 
ciatto2 1ambulatori oncologici raphael , via vittorio emanuele ii , i - 25011 calcinato ( bs ) , italy 2centro per lo studio e la prevenzione oncologica , firenze , italy correspondence to : v . 
after evaluation by internal and external reviewers of cancers detected by ultrasonography performed to confirm negative mammography , we determined the additional cancer detection rate of ultrasonography and the cost of the protocol . 
the evidence is insufficient to recommend this policy in routine screening practice but suggests that , at least in current clinical practice , adding ultrasonography in dense breasts may be useful despite the substantial costs . key words breast cancer diagnosis screening ultrasonography dense breast riassunto obiettivo . 
levidenza ancora insufficiente per poter raccomandare lintroduzione di questa procedura nella corrente pratica di screening , in attesa dei risultati degli studi controllati in corso , ma suggerisce che , almeno nella pratica clinica corrente , lintegrazione ecografica nei seni densi pu essere utile , sia pure associata a costi non trascurabili . parole chiave carcinoma mammario diagnosi screening ecografia seno denso introduction introduzione v . 
this limit is evident in both clinical [ 1 ] and screening practice : dense breast tissue is considered the major determinant of reduced screening sensitivity in women younger than 50 years and is significantly associated with the risk of interval cancer at any age [ 25 ]  . 
the accuracy of ultrasonography is not dependent on age [ 6 ] , and ultrasonographic diagnosis of cancer occult to mammography due to the masking effect of dense breasts is not a rare event . 
routine association of ultrasonography and mammography in women with dense breasts has been advocated although clinical studies on this policy [ 4 , 711 ] do not allow for a reliable evaluation of its cost effectiveness , and specifically designed prospective controlled studied on this subject are ongoing [ 12 , 13 ]  . 
at the raphael clinic , a centre for cancer prevention and early diagnosis , multimodal breast diagnosis has been employed on women self referring for symptoms or screening since 1986 . 
in the present study , we reviewed a consecutive series of 17 , 883 subjects undergoing the protocol and evaluated the results of routine ultrasonography of dense breasts . materials and methods the study analyses a consecutive series of 17 , 883 women aged between 25 and 96 years undergoing breast palpation and mammography from january 2000 to september 2004 . four reporting radiologists classified breast density according to the volume occupied by fibroglandular opacity [ breast imaging reporting and data system ( bi - rads ) d1 = 025 , d2 = 2650 , d3 = 5175 , d4 = 76100 ]  . 
subjects with negative mammography and in the d3 - d4 classes underwent bilateral ultrasonography using an aloka pro sound ssd - 5500 echograph with a linear multifrequency 5to 10 - mhz probe . 
in order to confirm the negativity of mammography , cancer cases diagnosed by ultrasonography alone were first reviewed by four internal reviewers and thereafter by an expert external reviewer with more than 20 years mammography experience and more 200 , 000 mammograms read ( sc )  . 
cancer cases reviewed as even minimally suspicious at mammography ( bi - rads 35 ) were excluded from evaluation of the benefits of association of ultrasonography , as their la densit radiologica della mammella costituisce un limite notevole per la sensibilit della mammografia . 
questo limite ben evidente sia in ambito clinico [ 1 ] che di screening : il seno denso considerato il principale movente della ridotta sensibilit ed efficacia dello screening mammografico al di sotto dei 50 anni e si associa significativamente al rischio di carcinoma di intervallo a qualsiasi et [ 25 ]  . 
laccuratezza della ecografia mammaria non risente dellet [ 6 ] e la diagnosi ecografica di carcinomi non evidenziati alla mammografia per leffetto mascherante del seno denso un evento non raro . 
lassociazione sistematica dellecografia mammaria alla mammografia in donne con seno denso proposta da molti , ma gli studi clinici che riportano simili esperienze [ 4 , 711 ] non consentono una valutazione precisa dei costi e dei benefici di una simile procedura , per cui sono in corso studi controllati appositamente disegnati [ 12 , 13 ]  . presso gli ambulatori raphael , un centro di prevenzione e diagnosi precoce delle patologie oncologiche , in atto dal 1986 una attivit di diagnostica senologica integrata su donne che accedono spontaneamente per sintomi o con finalit preventive . 
nel presente studio abbiamo rivisto una serie consecutiva di 17.883 casi e valutato i risultati dellassociazione sistematica dellecografia nei casi con seno denmateriali e metodi lo studio considera una serie consecutiva di 17.883 donne di et compresa fra i 25 e i 96 anni sottoposte a esame senologico e mammografia dal gennaio 2000 al settembre 2004 . i 4 radiologi che hanno interpretato i radiogrammi hanno definito la densit del seno in base alla percentuale di volume mammario occupato da densit fibroghiandolare ( birads d1 = 025 , d2 = 2650 , d3 = 5175 , d4 = 76100 )  . i soggetti con mammografia negativa classificati nelle categorie d3 e d4 sono stati esaminati con ecografia mammaria bilaterale : liter diagnostico stato completato con altri accertamenti ( esami radiologici mirati , citologia , core biopsy , biopsie chirurgiche ) in base alle eventuali alterazioni evidenziate su base clinico - ecografica . 
la revisione avvenuta in cieco , attraverso la valutazione dei casi di carcinoma mescolati a controlli negativi , selezionati in modalit casuale tra casi con seno denso approfonditi e risultati effettivamente negativi , in un rapporto cancri / controlli di 1 : 1 per la revisione interna e di v . 
as far as surgical biopsy is concerned , two alternatives were considered , namely : ( a ) outpatient procedure under local anaesthesia , or ( b ) hospitalisation . results out of 17 , 883 mammographies performed between january 2000 and september 2004 , 167 cancers were detected , with a detection rate of 0.93% ( 167 / 17 , 883 ) in the overall series . suspicious mammographies were 257 and diagnosed cancers 138 : negative mammography was reported in 11 , 177 cases ; 6 , 449 were classified as d3 - 4 ( 36.5% of the total mammographies performed and classified as not suspicious : 6 , 449 / 17 , 626 ) and ultrasonography was performed . 
il revisore , oltre a indicare le alterazioni sospette per le quali avrebbe richiesto un approfondimento diagnostico , ha espresso secondo la classificazione bi - rads [ 14 ] sia il livello di sospetto che la densit radiologica del seno . 
i casi di carcinoma identificati dal revisore come portatori di alterazioni con livello anche minimo di sospetto ( bi - rads 35 ) sono stati esclusi dalla valutazione dei vantaggi dellecografia e la loro diagnosi stata attribuita alla mammografia che , per queste categorie diagnostiche , condiziona correntemente un completamento ecografico . i risultati dellassociazione dellecografia alla mammografia negativa in seno denso sono stati espressi in termini di tasso diagnostico di carcinoma : stata anche verificata la percentuale di approfondimenti diagnostici ( citologia , core biopsy , biopsia chirurgica ) richiesti a seguito di sospetto ecografico , ma con esito negativo . 
il costo della biopsia chirurgica stato computato in base a due alternative : ( a ) biopsia escissionale ambulatoriale in anestesia locale e ( b ) biopsia in regime di ricovero ospedaliero con breve anestesia generale . risultati su un totale di 17.883 mammografie eseguite dal gennaio 2000 al settembre 2004 sono stati diagnosticati 167 carcinomi , con un tasso diagnostico di carcinoma di 0 , 93% ( 167 / 17.883 ) nella serie complessiva . 
the remaining 18 cancers were mixed with 72 negative controls and were examined by the external reviewer , who identified three cancers as radiologically suspicious ( birads r3 = 2 , r4b = 1 ) and also reported abnormalities in 15 negative controls ( bi - rads r3 = 10 , r4a = 4 , r4b = 1 )  . 
overall , after excluding symptomatic cases and cases reviewed as suspicious at mammography , the cancers for which mammography was confirmed to be negative at review and diagnosis could be attributed to ultrasonography only were 15 , accounting for a detection rate of 0.23% ( 15 of 6 , 449 cases ) and for 8.9% of total cancers detected . 
table 2 shows the distribution of cancers detected by ultrasonography alone by age , stage , morphology ( mass , calcifications ) and radiological density according to the external reviewer . 
a seguito di tale approfondimento sono stati diagnosticati altri 29 carcinomi , con una prevalenza di 0 , 44% ( 29 / 6.449 ) , pari al 17 , 3% dei carcinomi totali diagnosticati . 
i dati dettagliati per classi di et sono riportati nella tabella 1 . dei 29 carcinomi diagnosticati dallecografia in casi con mammografia negativa , le mammografie originali erano disponibili in 25 casi . 
i restanti 18 casi , mescolati a 72 controlli negativi , sono stati valutati dal revisore esterno , che ha identificato altri 3 carcinomi effettivamente sospetti alla mammografia ( bi - rads r3 = 2 , r4b = 1 ) , formulando un sospetto anche in 15 controlli ( bi - rads r3 = 10 , r4a = 4 , r4b = 1 )  . 
a setable 2 distribution of cancers detected by ultrasonography alone in mammographically negative dense breasts , according to age , density at external review , morphology , staging ( ptnm ) and histological type case no . density morphology histological type mass mass mass mass mass mass mass mass mass mass mass mass mass mass mass nodulo nodulo nodulo nodulo nodulo nodulo nodulo nodulo nodulo nodulo nodulo nodulo nodulo nodulo nodulo 1 ( i + ) 1 ( i + ) nos , not otherwise specified tabella 2 distribuzione dei carcinomi identificati dallecografia in mammografie negative con seno denso , in funzione dellet , densit radiologica alla revisione , morfologia , stadio ( ptnm ) e istotipo caso densit morfologia istotipo ductal nos ductal nos ductal nos ductal nos ductal nos lobular tubular papillary mucinous lobular ductal nos medullary lobular ductal nos ductal nos duttale duttale duttale duttale duttale lobulare tubulare papillare mucinoso lobulare duttale midollare lobulare duttale duttale v . 
cost analysis details are reported in table 3 . discussion ultrasonography is often employed in both clinical and screening settings for further investigation of cases with negguito di questa revisione , i carcinomi la cui diagnosi stata effettivamente attribuita allecografia , escludendo quelli sintomatici o identificati come sospetti dai revisori , sono rimasti 15 , pari ad un tasso diagnostico di 0 , 23% ( 15 su 6.449 casi sottoposti ad ecografia di principio per seno denso ) , corrispondenti all 8 , 9% dei carcinomi totali diagnosticati . la tabella 2 riporta la distribuzione di questi carcinomi per et , stadio , morfologia ( massa , calcificazioni ) e densit radiologica alla revisione . 
la categoria pt risultata pt1a in 3 casi , pt1b in 8 casi , pt1c in 3 casi e pt2 in 1 caso . lapprofondimento ecografico ha generato in totale 405 esami citologici , 15 core biopsy , 66 biopsie chirurgiche . 
most published studies ( summarised in table 4 ) although poorly comparable due to differences in age distribution , criteria to define a dense breast , ultrasonographic equipment and accuracy [ 15 , 16 ] report a strikingly similar detection rate of mammographically occult cancers ( 0.3%0.4% ) and a false positive rate ranging between 1% and 3% . 
in our experience , the detection rate at initial analysis of crude data was 0.44% and the false positive rate was 1.25%. unfortunately , these series are not drawn from current screening practice but are based on subjects self referring to mammography , not necessarily in the screening age range ( 5069 )  . 
thus , it is difficult to evaluate from these clinical series ( for which the underlying risk and expected incidence are uncertain ) what might be the benefit of adding ultrasonography for dense breasts in screening practice in terms of additional cancer detection rate . 
to answer this question we will have to wait for the results of ongoing prospective controlled trials [ 12 , 13 ]  . the present consecutive series , although based on self referring subjects in a wide age range , is rather large and may provide some information , at least in a clinical scenario , of the likelihood of ultrasonography detecting mammographically occult breast cancer in dense breasts . 
on the other hand , it is a retrospective study , which is unable to provide definitive evidence to recommend the use of ultrasonography in current practice in cases of negative mammography and dense breasts . 
i dati dettagliati della analisi dei costi sono riportati nella tabella 3 . discussione lecografia frequentemente impiegata , sia in ambito clinico che di screening , per il completamento diagnostico in casi sostanzialmente negativi alla mammografia , ma ad elevato rischio di errore mammografico per la presenza di seno denso . 
la gran parte delle esperienze pubblicate ( le principali , assieme al presente studio , sono indicate nella tabella 4 ) , nonostante la discutibile comparabilit delle casistiche per la diversa composizione in et , la variabilit nella definizione di seno denso e nella accuratezza diagnostica ecografica [ 15 , 16 ] , riportano un tasso diagnostico di carcinoma occulto alla mammografia sorprendentemente simile , variabile tra lo 0 , 3% e lo 0 , 4% , e un tasso di falsi positivi variabile tra l1% e il 3% . 
nella presente esperienza il tasso diagnostico in prima analisi stato di 0 , 44% , con un tasso di falsi positivi di 1 , 25% . purtroppo queste esperienze non si riferiscono a casistica di screening , ma a casistica ad accesso spontaneo e spesso in et diversa da quella in cui lo screening correntemente raccomandato ; nel presente studio in particolare il 45 , 1% della popolazione esaminata aveva et inferiore ai 50 anni . quindi difficile valutare in base ai risultati di queste casistiche ( a rischio di malattia e prevalenza incerti ) quale possa essere il vantaggio relativo , in termini di tasso diagnostico di carcinoma , dellintroduzione del completamento ecografico nei seni densi nella routine dello screening . 
per tale motivo auspicabile che siano completati gli studi controllati in corso [ 12 , 13 ] , disegnati in un contesto di screening , che sono gli unici a poter rispondere al quesito . la casistica in esame , una serie clinica con afferenza spontanea di ogni et , comunque piuttosto ampia e pu fornire interessanti elementi quanto alla probabilit , sia pure in un contesto clinico , di diagnosi ecografica di carcinoma occulto alla mammografia . 
si tratta comunque di uno studio retrospettivo e , come tale , non pu costituire elemento definitivo per sostenere che lecografia in caso di mammografia negativa con seno denso radiologico sia una procedura raccomandabile di routine . 
quindi doveroso sottolineare i limiti di questa analisi , al fine di evitare che il lettore giunga a conclusioni affrettate . a tal fine , oltre a fornire il dato osservazionale grezzo , si operata una accurata revisione radiologica , che non risulta essere stata condotta nella maggioranza degli altri studi citati . 
inadequate interpretation or inaccurate reporting ( negative instead of suspicious ) of mammograms in cases selected for ultrasonography might bias the results when analysis is based on the original report . 
in the present study , review identified eight of 25 cancers that turned out to be suspicious at mammography . this peculiar aspect of our study , together with a different age distribution , may at least partially account for the lower cancer detection rate at ultrasonography observed with respect to other literature reports . 
it must be noted that the review process , though blind and with cancers mixed with negative controls , is likely to influence the reader , who is aware of reviewing a series with a high cancer prevalence , and might therefore lower the threshold for suspicion to maximise sensitivity at the expense of specificity : in fact , in our study , the external reviewer had a recall rate of 20.8% ( 15 / 72 ) among negative controls , definitely higher compared with the usual recall rate in screening , although suspicion was mild in most cases ( 14 / 15 < r4b , no r4c - r5 )  . 
external review does not discount that mammography was actually reported as negative or the benefit of ultrasonography but suggests that this benefit might also be obtained , to some extent , by improving the accuracy of mammography , particularly if this is suboptimal . 
the aim of external review is not to minimise the advantages of ultrasonography but to suggest caution in drawing definitive conclusions , and we believe that review may be useful for such a purpose . 
in agreement with previous literature reports , ultrasonography detected at least 15 additional cancers , a detection rate of 0.23% among subjects undergoing ultrasonography , or 8.98% of all cancers detected in the study cohort . 
detection rate was age dependent , being highest in the 50to 59 - year age group ( 0.32% ) ; this finding is interesting , as screening is currently recommended in this age group . 
the benefits of ultrasonography are further underlined by the limited stage of cancers detected , with 11 of 15 cases measuring less than 1 cm in size , a sign of a favourable prognosis . 
because ultrasonography is not highly specific , its extensive use implies unnecessary diagnostic procedures leading to a negative final diagnosis [ 9 , 10 , 15 ]  . grafie precedenti un carcinoma di intervallo . 
una insufficiente qualit della lettura mammografica , infatti , o una non accurata codifica ( negativo anzich sospetto ) dei casi avviati allecografia , potrebbe falsare i dati ove la valutazione prenda come riferimento la refertazione originale della mammografia . 
questa caratteristica dello studio , oltre alla diversa composizione in et , pu almeno in parte spiegare il minor tasso diagnostico di carcinoma allecografia osservato in questa , rispetto alle altre serie della letteratura ( tabella 4 )  . 
opportuno considerare che la conduzione di una revisione , sia pure cieca e con aggiunta di controlli negativi , modifica comunemente latteggiamento del revisore che , consapevole dello scopo della revisione , pu abbassare il livello di sospetto nellintento di aumentare la sensibilit a spese della specificit : nel presente studio il revisore esterno , infatti , ha avuto un tasso di falsi positivi tra i controlli del 20 , 8% ( 15 / 72 ) , sia pure con livelli di sospetto prevalentemente bassi ( 14 / 15 < r4b , nessun r4c - r5 )  . 
la revisione nulla toglie al fatto che quella mammografia stata di fatto considerata negativa e al beneficio conseguente allecografia , ma avverte che i benefici dellecografia potrebbero essere ottenuti , almeno in parte , anche da un miglioramento della accuratezza diagnostica mammografica . 
lintento della revisione non di sminuire i meriti dellecografia , ma di indurre cautela nelle conclusioni , e ci sembrato utile adottare questo controllo , preferendo presentare , oltre al dato osservazionale grezzo , una stima pi prudenziale dei benefici dellecografia . il contributo diagnostico dellecografia in questo studio non stato trascurabile . 
confermando i risultati della letteratura , lecografia ha diagnosticato 15 carcinomi aggiuntivi , pari ad un tasso del 0 , 23% nei soggetti sottoposti ad ecografia e al 8 , 98% dei carcinomi diagnosticati complessivamente nella coorte in esame . 
il tasso diagnostico dipende dallet , con il valore pi elevato nella fascia det 5059 anni ( 0 , 32% ) ; il dato appare significativo , se si considera che tale popolazione rientra fra quella a cui si rivolge lo screening . 
il beneficio dellecografia ulteriormente valorizzato dal fatto che le neoplasie diagnosticate hanno una distribuzione per stadio molto favorevole , con ben 11 casi su 15 di diametro inferiore ai 10 mm , foriera di una buona prognosi . la non assoluta specificit dellecografia implica ovviamente che un suo uso esteso comporti un certo numero di accertamenti inutili , non esitanti in diagnosi di carcinoma [ 9 , 10 , 15 ]  . 
lanalisi dei costi relativa a questo carico diagnostico addizionale stata necessariamente condotta in base a un preciso tariffario , pur consapevoli che una simile stima solo indicativa , in quanto non tiene conto di una notevole variabilit dei tempi e dellorganizzazione di lavoro nei diversi contesti . 
this is definitely higher compared with the average cost ( around euro 5 , 000 ) reported per cancer detected at screening [ 17 ] , but a higher cost was indeed expected , as ultrasonography was looking for cancers missed at mammography , which are likely to be difficult to detect and to have a low prevalence . 
it is impossible to compare costs to prognostic benefit obtained by additional cancer detection at ultrasonography , as we ignore whether and to what extent mortality was affected by the earlier diagnosis by ultrasonography . 
it is difficult to draw any conclusion concerning a screening scenario from the present experience , as the study population is selected ( self referring ) and is likely not representative of a general screening population in terms of breast cancer risk and dense - breast frequency . 
the proportion of subjects with dense breast over age 50 and thus sent for ultrasonography were 25.7% : assuming the cost subsequent to ultrasonography to be independent of age , the cost per woman examined would be around euro 15 , 00 . 
this is a substantial increase ( + 30% ) of screening costs , the most recent reliable published report being around euro 50 , 00 per woman in 1995 [ 17 ] , which may be presently estimated to be around euro 60 , 00 ( adjusted for devaluation )  . 
on the other hand , additional cancers detected by ultrasonography in women over age 50 were seven , compared with 112 detected at mammography , thus accounting for a relative sensitivity increase of 6.2%. 
overall , the present experience confirms the possibility of ultrasonography detecting mammographically occult cancer in dense breasts [ 1 , 18 ] although we agree with kopans [ 1922 ] that this does not constitute sufficient evidence to support the introduction of this policy in current screening practice . 
tale costo decisamente pi elevato del costo per carcinoma diagnosticato ( intorno ad euro 5000 ) comunemente riportato in esperienze di screening [ 17 ] , ma un costo pi elevato ovviamente atteso quando si ricerchino carcinomi aggiuntivi , sfuggiti alla mammografia , verosimilmente pi difficili da identificare e a bassa prevalenza . 
 impossibile quantificare il beneficio della diagnosi ecografica in termini prognostici , in quanto non possibile stimare se , ed eventualmente quanto , lanticipazione diagnostica ottenuta abbia influenzato di fatto la mortalit . assai difficile trarre da questa esperienza indicazioni valide per uno scenario di screening , dato che la popolazione in esame autoselezionata e ha verosimilmente una prevalenza di seno denso e un rischio di carcinoma diverso da una popolazione di screening . 
pu essere comunque utile , a solo fine speculativo , tentare un confronto approssimativo . se si considerano le donne di 50 anni ed oltre , i soggetti avviati allecografia per seno denso sono stati il 25 , 7% : assumendo che il costo dellintegrazione con ecografia sia omogeneo per et , il costo per donna esaminata risulterebbe di circa euro 15 , 00 e configurerebbe un aumento consistente ( + 30% ) dei costi dello screening , la cui ultima stima attendibile in letteratura , risalente al 1995 , intorno a euro 50 , 00 per donna esaminata [ 17 ] , stimabile attualmente intorno a euro 60 , 00 ( correzione in base al tasso di svalutazione )  . 
di contro , i carcinomi aggiuntivi diagnosticati dalla sola ecografia in et di 50 o pi anni sono stati 7 rispetto a 112 sospettati dalla mammografia , pari ad un aumento relativo di sensibilit del 6 , 2% . complessivamente la presente esperienza conferma la possibilit che lecografia identifichi neoplasie maligne occulte alla mammografia in seni densi [ 1 , 18 ] , anche se , come ripetutamente stigmatizzato da kopans a distanza di anni [ 1922 ] , non costituisce una evidenza sufficiente a raccomandare lintroduzione di questa procedura nella corrente pratica di screening . 
il disegno di studio , con lattenta revisione critica dei radiogrammi originalmente refertati come negativi , ha ridimensionato in parte il contributo dellecografia , ma non si deve dimenticare che nella realt clinica ( in assenza di una revisione ) i carcinomi occulti alla mammografia , asintomatici e sospettati grazie alla sola integrazione ecografica sono stati ben 27 ( 16 , 1% )  . 
the aim of this study was to determine the spin - lattice relaxation or t1 time constant of phosphocreatine in the human gastrocnemius muscle , both at rest and during aerobic exercise , when a clinical nuclear magnetic resonance ( nmr ) scanner is available . 
protocols and models were tested on a phantom containing 1 kg of distilled water in which were dissolved 5.88 g sodium chloride , 1 cm3 phosphoric acid and 7.58 g sodium phosphate dodecahydrate . 
tutti i protocolli ed entrambi i modelli sono stati testati su un fantoccio contenente 1 kg dacqua distillata in cui erano disciolti 5 , 88 g di cloruro di sodio , 1 cm3 di acido fosforico e 7 , 58 g di fosfato di sodio dodecaidrato . 
sul fantoccio , i valori medi di t1 sono risultati molto diversi per i protocolli ed i modelli matematici utilizzati , variando da 0 , 61 s a 7 , 20 s . 
correggendo il valore di t1 ottenuto sui volontari , mediante i risultati del fantoccio , si ricava un valore di t1 pari a 5 , 73 s , confrontabile con quello riportato in letteratura per la sola condizione di riposo . key words t1 time constant progressive saturation muscle , exercise phosphocreatine parole chiave costante di tempo t1 saturazione progressiva muscolo , esercizio fosfocreatina introduction introduzione clinical nuclear magnetic resonance ( nmr ) devices are often also used for research purposes after appropriate software and hardware has been added . 
1h - mrs is used to diagnose brain masses [ 1 ] or in oncology [ 2 , 3 ] whereas 31p or 13c spectroscopy is used in the diagnosis and follow - up of epilepsy and other neuropsychile apparecchiature di risonanza magnetica nucleare per uso clinico sono spesso utilizzate anche a scopo di ricerca , aggiungendo opportuni software ed hardware . 
ci avviene , in particolare , per quelle unit rmn ampliate per rendere possibili anche indagini di spettroscopia in vivo ( mrs ) , tecnica che negli ultimi anni sta ricevendo sempre maggiore attenzione . 
finally , 31p spectroscopy is used to study muscle function and / or metabolism both in healthy volunteers [ 7 ] and in patients [ 810 ]  . in 31p spectra obtained from muscle tissue , the prevailing lines are those of inorganic phosphate ( pi ) , phosphocreatine ( pcr ) and three phosphoric groups of adenosine triphosphate ( - atp , - atp , and - atp )  . 
furthermore , the absolute position of the pi peak depends on intracellular ph , which can thus be determined by measuring the chemical shift of pi relative to a fixed line ( typically the pcr line )  . 
in the absence of disease , at the start of moderate exercise , the pcr is hydrolysed to provide part of the energy needed for contraction ; consequently , the intensity of its peak drops , only to stabilise as soon as aerobic metabolic processes are able to produce all the necessary energy . 
 these phenomena can be observed clearly only with a good signal - to - noise ratio , obtained by adding as many spectra as possible although within an inevitably limited time . 
as a consequence , short repetition times ( trs ) are used , which , however , make the spectra partially saturated and therefore require the peak intensity to be corrected to obtain information on the absolute concentrations of metabolites . 
to this end , with the muscle at rest , the ratio between the area subtended by a spectrum acquired with a long tr and that of a spectrum acquired with a short tr is calculated . 
this ratio is then used to correct the areas of all the spectra acquired with a short tr during the experimental stage [ 15 , 17 , 18 ]  . 
application of this method must , however , take into account the intrinsic characteristics of the material being studied , quantised through the spin - lattice relaxation or t1 time constant . 
the t1 values reported in the literature have been obtained both on isolated muscles [ 13 , 19 ] and in vivo [ 20 , 21 ] but only during isometric exercises without doing any mechanical work [ 22 ] and , sometimes , with varying intracellular ph [ 21 ]  . the purpose of our work was to estimate , with the saturation recovery technique , the apparent t1 [ 23 ] on humans by using a clinical scanner adapted for 31p spectroscopy studies . 
in particular , the possible variations of the apparent t1 of phosphocreatine ( t1 ) between rest and moderate aerobic exercise were investigated . materials and methods the experiment was conducted with a 1.5 tesla magnetic resonance ( mr ) scanner ( magnetom sp 4000 , siemens , erlangen , germany ) equipped with a 5 - cm surface coil that can be tuned to detect both hydrogen ( 1h ) and phosphorus nuclei ( 31p )  . 
the spectroscopy sequence available on the nmr unit , provided by the manufacturing company , merely excited the spettroscopie al 31p o al 13c sono utilizzate nella diagnosi e nel follow - up di epilessia ed altre patologie neuro - psichiatriche [ 46 ]  . 
infine , la spettroscopia al 31p utilizzata per studiare la funzione e / o il metabolismo muscolare , sia in volontari sani [ 7 ] che in pazienti [ 810 ]  . negli spettri al 31p ottenuti dal tessuto muscolare , le righe che predominano sono quelle del fosforo inorganico ( pi ) , della fosfocreatina ( pcr ) e dei tre gruppi fosforici delladenosintrifosfato ( - atp , - atp e - atp )  . 
inoltre , la posizione assoluta del picco del pi dipende dal ph intracellulare , che quindi pu essere determinato misurando il chemical shift del pi rispetto ad una riga fissa ( tipicamente quella della pcr )  . 
in assenza di patologie , allinizio di un esercizio moderato , la pcr viene idrolizzata per fornire una parte dellenergia necessaria per la contrazione e quindi lintensit del suo picco si riduce , per stabilizzarsi appena i processi metabolici di tipo aerobico sono in grado di produrre tutta lenergia necessaria . 
il picco del fosforo inorganico presenta lo stesso andamento , ma speculare , mentre le dimensioni dei rimanenti picchi rimangono invariate , risultando quindi indipendenti dal lavoro muscolare . questi fenomeni sono chiaramente osservabili solo con un buon rapporto segnale - rumore , ottenuto sommando il maggior numero possibile di spettri , anche se in un tempo necessariamente limitato . 
di conseguenza , sono utilizzati tempi di ripetizione ( tr ) brevi che , per , rendono gli spettri parzialmente saturati e quindi , volendo ottenere informazioni sulle concentrazioni assolute dei metaboliti , lintensit dei picchi deve essere corretta . 
a tal fine , sul muscolo a riposo , viene calcolato il rapporto tra larea sottesa da uno spettro acquisito con un tempo di ripetizione lungo e quella di uno spettro acquisito con il tr breve , che poi viene utilizzato per correggere le aree di tutti gli spettri acquisiti con il tr breve in fase sperimentale [ 15 , 17 , 18 ]  . 
lapplicazione di questo metodo , per , non pu prescindere dalle caratteristiche intrinseche del materiale in studio , quantizzate dalla costante di tempo di rilassamento spin - reticolo o t1 . 
i valori di t1 finora riportati dalla letteratura sono stati ottenuti sia su muscoli isolati [ 13 , 19 ] che in vivo [ 20 , 21 ] , ma effettuando solamente esercizi di tipo isometrico , ovvero senza compimento di lavoro meccanico [ 22 ] e , a volte , con variazione di ph intracellulare [ 21 ]  . scopo del presente lavoro stimare , applicando la tecnica della saturazione progressiva , il t1 apparente [ 23 ] in vivo sulluomo utilizzando un tomografo per uso clinico adattato agli studi di spettroscopia al 31p . 
in particolare , sono state indagate le possibili variazioni del t1 apparente della fosfocreatina ( t1 ) tra le condizioni di riposo e di esercizio aerobico moderato . materiali e metodi la sperimentazione stata condotta con un tomografo rmn ( magnetom sp 4000 , siemens , erlangen , germania ) da 1 , 5 v . 
the time required for each measurement ( tmis ) included : a first waiting interval lasting one tr the time required by the prescans ( nps ) , namely , the number of excitations of the tissue caused by radiofrequency ( rf ) pulses with homogeneous tr intervals without collecting the mr signal the time required to excite the tissue and record the mr signal for a certain number of acquisitions ( nacq ) , each one tr apart . 
having to automatically acquire nmis spectra , the nmr unit operated for a ta time equal to ta = nmis tmis to which the time required for software reconstruction and saving of the spectra on the scanners computer ( about 1 s for each spectrum ) had to be added . the entire experiment involved a first set of in vitro measurements followed by the in vivo experiments . 
the parameters of the different acquisition protocols were selected in order to acquire of all spectra under saturation conditions , assuming that t1 = 6 s [ 22 ]  . in vitro experimental protocols a first set of measurements was made by placing the surface coil over a phantom consisting of a plexiglas cylinder containing 1 kg of distilled water in which 5.88 g nacl , 1 cm3 h3po4 and 7.58 g na3po412h2o were dissolved . 
the first measurement protocol ( long protocol ) was aimed at determining the t1 time constant of the solution contained in the phantom and was repeated six times on different days . 
the tr , initially set at 601 ms , was increased by 500 ms for every set whereas the number of the spectra acquired for each set was reduced from 24 in the first to 19 in the 13th set . 
the parameters of the three short protocols were fixed so as to be very different from one another although meeting the following conditions : total time of each protocol had to be less than 8 min information on the greatest number of trs had to be acquired within the available time tesla , dotato di una bobina di superficie del diametro di 5 cm , sintonizzabile sia per i nuclei di idrogeno ( 1h ) che per quelli di fosforo ( 31p )  . 
non era possibile controllare esattamente alcuni parametri cruciali , come ad esempio il flip angle . il software di gestione permetteva lacquisizione automatica di una serie di spettri ( misure ) , tutti acquisiti con lo stesso tr . 
il tempo necessario per ogni misura ( tmis ) comprendeva : un primo intervallo di attesa , della durata di un tempo di ripetizione ( tr ) ; il tempo impiegato dai pre - scans , ovvero dal numero nps di eccitazioni del tessuto con impulsi rf ad intervalli temporali omogenei tr , senza la raccolta del segnale rm ; il tempo impiegato per leccitazione del tessuto e la registrazione del segnale rm per un certo numero di acquisizioni nacq , tutte distanziate di un tempo tr . 
 conseguentemente tmis era pari a : tmis = tr + npstr + nacqtr + = tr ( 1 + spn + nacq ) + dove lintervallo di tempo aveva una durata di circa 1 , 26 s . 
dovendo acquisire automaticamente nmis spettri , lunit rmn veniva impegnata per un tempo ta pari a : ta = nmis tmis a cui si aggiungeva il tempo necessario per la ricostruzione software ed il salvataggio degli spettri sul computer del tomografo ( circa 1 secondo per ogni spettro )  . lintera sperimentazione ha previsto una prima serie di misure effettuate in vitro , alle quali hanno fatto seguito gli esperimenti in vivo . 
i parametri dei diversi protocolli di acquisizioni sono stati selezionati imponendo che tutti gli spettri fossero acquisiti in condizioni di saturazione , ipotizzando un t1 = 6 s [ 22 ]  . protocolli sperimentali in vitro una prima serie di misure stata effettuata posizionando la bobina di superficie su un fantoccio formato da un cilindro di plexiglass contenente 1 kg dacqua distillata in cui erano disciolti 5 , 88 g di nacl , 1 cm3 di h3po4 e 7 , 58 g di na3po412h2o . 
il primo protocollo di misura ( protocollo lungo ) ha avuto lo scopo di determinare il t1 della soluzione contenuta nel fantoccio ed stato ripetuto 6 volte in giorni diversi . 
il tr , inizialmente fissato a 601 ms , stato incrementato di 500 ms ad ogni serie , mentre il numero di spettri acquisiti per ciascuna serie stato ridotto da 24 nella prima a 19 nella tredicesima . per ciascun tr sono stati considerati solamente gli ultimi 16 spettri acquisiti . successivamente , sono stati effettuati 3 protocolli corti , v . 
the number of prescans fell from 25 in the first to three in the sixth measurement whereas the tr increased in each measurement from 601 ms to 5 , 601 ms with 1 - s increments . 
in the third protocol ( protocol c ) , 65 spectra were acquired with seven separate sets , all with three acquisitions and no prescan ; the first two sets supplied ten spectra , the remaining sets nine . 
for this protocol , only the last six spectra were taken into consideration for every tr . in vivo experimental protocol the protocol on volunteers was aimed at estimating the t1 time constant of muscle tissue both at rest and during moderate aerobic exercise . 
each subject was adequately informed on the experimental protocol and gave written informed consent to the spectroscopic examination . the subjects lay in a supine position on an ergometer made of nonferromagnetic materials and consisting of a support table upon which two independent pedals are hinged [ 25 ]  . 
power output was measured with appropriate force and position transducers applied to the ergometer . in the spectroscopy measurements , the surface coil was applied to the subjects right leg at the level of the medial gastrocnemius muscle . 
the protocol involved repeating the rest / exercise sequence three times , with an interval of at least 5 min between the repetitions , each phase lasting about 7 min measured using a chronometer . 
in the exercise phases , acquisition of the spectra was started at least 10 s after the beginning of the exercise . data processing after acquisition , the spectra were transferred to a workstation ( o2 , silicon graphics , usa )  . 
i parametri dei 3 protocolli corti sono stati fissati in modo che essi fossero molto diversi fra loro , pur soddisfacendo le seguenti condizioni : il tempo totale di ciascun protocollo doveva essere inferionel tempo a disposizione dovevano essere acquisite le informazioni per il maggior numero possibile di tr ; per ogni diverso valore di tr dovevano essere acquisiti alre a 8 minuti ; meno 16 fid . con il primo protocollo ( protocollo a ) sono stati acquisiti 6 spettri raccolti con misure distinte , tutte aventi nacq = 16 . il numero di pre - scans diminuiva da 25 per la 1a a 3 per la 6a misura , mentre il tr aumentava , per ciascuna misura , da 601 ms a 5601 ms con incrementi di 1 s . il secondo protocollo ( protocollo b ) ha consentito di raccogliere 107 spettri con 5 serie diverse , tutte effettuate con 1 acquisizione e nessun pre - scan . 
solamente gli ultimi 16 spettri di ciascun tr sono stati utilizzati . con il terzo protocollo ( protocollo c ) sono stati acquisiti 65 spettri con 7 serie distinte , tutte aventi 3 acquisizioni e nessun pre - scan ; le prime due serie hanno fornito 10 spettri , le altre 9 . 
per questo protocollo sono stati considerati solo gli ultimi 6 spettri di ogni tr . protocollo sperimentale in vivo il protocollo sui volontari aveva lo scopo di stimare la costante di tempo t1 del tessuto muscolare sia in condizioni di riposo che durante esercizio aerobico moderato . 
 stato scelto a priori di utilizzare il protocollo di acquisizione c in quanto presentava caratteristiche simili ai parametri di acquisizione utilizzati per gli studi sulla funzione muscolare nelluomo . alla sperimentazione hanno partecipato 4 soggetti sani ( 3 maschi e 1 femmina , di et , massa corporea ed altezza medi rispettivamente di : 25 , 33 , 1 anni , 73 , 511 , 6 kg e 1775 cm ( ds ) ) nessuno dei quali svolgeva attivit fisica abituale e / o con intenti sportivi . 
ogni soggetto stato adeguatamente informato sul protocollo sperimentale ed ha sottoscritto il consenso informato allindagine spettroscopica . i soggetti erano distesi supini su un apposito ergometro costruito con materiali non ferro - magnetici , formato da una tavola , che funge da supporto , sulla quale sono imperniati due pedali indipendenti [ 25 ]  . 
con i piedi vincolati ai pedali , i volontari effettuano un lavoro compiendo delle flessioni plantari che estendono gli elastici applicati sullergometro . per la presente sperimentazione le flessioni erano sincrone , alla frequenza di 50 battute al minuto e per unestensione di circa 30 gradi . 
la potenza istantanea sviluppata stata misurata attraverso appositi trasduttori di forza e di posizione applicati sullergometro . per le misure di spettroscopia , la bobina di superficie stata applicata sulla gamba destra dei soggetti , a livello del muscolo gastrocnemio mediale . 
the area under a precise spectrum peak , and its position were determined with the time - domain varpro fitting routine of the mrui software [ 26 ] using the appropriate starting values and without any prior conditions . the single peak for both 1h and 31p was considered for the spectra obtained on the phantom whereas the water ( for 1h ) and phosphocreatine and inorganic phosphate peaks ( for 31p ) were evaluated for the spectra obtained in vivo . 
for protocol c , considering the greater temporal complexity of the sequence used , the s values were interpolated with two modified models : s ( tr ) = s0 ( cid : 2 ) 1 - b e 2tr + ( cid : 3 ) + 2s0 ( cid : 2 ) 1 - b e tr ( cid : 3 ) data acquired for every volunteer were processed separipetere tre volte , con un intervallo di attesa di almeno 5 minuti tra luna e laltra , la successione riposo - esercizio della durata di circa 7 minuti ciascuno , controllato mediante un cronometro . 
nei periodi di esercizio la registrazione degli spettri iniziata almeno 10 s dopo linizio dellesercizio . trattamento dei dati dopo lacquisizione , gli spettri sono stati trasferiti su una stazione di lavoro ( o2 , silicon graphics , usa )  . 
successivamente , lintensit di segnale s , ovvero larea sottesa da un preciso picco spettrale e la sua posizione sono state determinate nel dominio temporale tramite il sottoprogramma di fitting varpro del software mrui [ 26 ] , utilizzando gli appropriati valori iniziali e senza limposizione di condizioni a priori . 
per gli spettri ottenuti sul fantoccio stato considerato lunico picco presente sia per l1h che per il 31p , mentre per gli spettri ottenuti in vivo sono stati valutati il picco dellacqua ( per l1h ) e quelli della fosfocreatina e del fosforo inorganico ( per il 31p )  . 
la distanza ( in p.p.m. ) fra il picco della pcr ed il picco del pi ha consentito di calcolare il valore del ph utilizzando la seguente formula [ 14 , 27 ] : ph = 6 , 75 + log 3 , 27 5 , 69 i diversi valori di s calcolati per il 31p allo scopo di determinare il t1 apparente sono stati approssimati , per tutti i protocolli , attraverso due modelli matematici , in funzione del parametro temporale t specifico del protocollo in esame : s ( t ) = s0 ( cid : 2 ) 1 - bet t1 ( cid : 3 ) il primo modello ha interpolato i valori secondo una curva esponenziale semplice , imponendo b = 1 , e quindi restavano da determinare i parametri s0 e t1 . 
il secondo modello , al fine di compensare il fatto che limpulso rf di eccitazione non investe uniformemente il campione indagato , ha utilizzato una funzione esponenziale saturata , in cui era da stimare anche il coefficiente di correzione di saturazione b , che dovrebbe risultare compreso tra 0 e 1 [ 22 ]  . per il protocollo lungo eseguito sul fantoccio , le 13 intensit di segnale s sono state interpolate con i due modelli matematici , assumendo come tempo t il tempo di misura di ciascuno spettro ( t = tmis = 2tr + ) , variabile da 2 , 46 s a 14 , 46 s con incrementi di 1 s . 
per confronto , la stessa metodologia di analisi stata applicata utilizzando solamente le intensit di segnale s acquisite con i 5 tr pi piccoli . i dati raccolti con i tre protocolli corti sono stati elaborati mediante analisi specifiche per ciascuno di essi . 
il segnale di ogni spettro acquisito con il protocollo a per ciascuno dei 6 diversi valori di tr stato diviso per il numero di acquisizioni ( n = 16 )  . 
 all statistical evaluations and interpolations were carried out with the spss software ( spss inc . , chicago , il , usa )  . the probability value p = 5% was assumed to indicate statistical significance . 
per il protocollo b , i valori s sono stati interpolati con i due modelli matematici assumendo come tempo t il tempo di misura di ciascuno spettro ( t = tmis = 2tr + ) , variabile da 2 , 46 s a 3 , 66 s con incrementi di 0 , 2 s . 
per il protocollo c , considerando la maggiore complessit temporale della sequenza utilizzata in questo test per lacquisizione degli spettri , i valori di s sono stati interpolati con due modelli modificati : s ( tr ) = s0 ( cid : 2 ) 1 - b e 2tr + ( cid : 3 ) + 2s0 ( cid : 2 ) 1 - b e tr ( cid : 3 ) results in vitro estimation of t1 the values of signal s obtained with the 13 different trs during a repetition are illustrated in figure 1 , which also depicts the curves described by the two different mathematical models , using as parameters t1 , s0 , and b those obtained by the nonlinear regression estimation on the same data . 
t1 and s0 values obtained with the two mathematical models were significantly different ( wilcoxon test , n = 6 , p < 0.05 ) , using both the complete set of values and only five values . 
the estimation performed by applying the simple exponential model to just five data gave significantly lower results ( wilcoxon test , n = 6 , p < 0.05 ) than by applying the same model to 13 values . i dati acquisiti su ogni volontario sono stati elaborati separatamente per ciascuna ripetizione del protocollo , mantenendo distinte le informazioni ottenute durante i periodi di riposo e di esercizio , ottenendo quindi per ciascun soggetto 6 set di spettri . 
 tutte le valutazioni statistiche e le interpolazioni sono state effettuate tramite il software spss ( spss inc , il , usa )  . il valore di probabilit p = 5% stato assunto come soglia di significativit statistica . 
sono riassunti i risultati ottenuti applicando entrambi i modelli matematici , utilizzando , per le stime , tutti i 13 valori a disposizione oppure solamente i 5 dati acquisiti con minore tr stima su 13 dati stima su 5 dati v . 
see text for details tabella 2 valori medi ( ds ) del tempo impiegato per una successione completa di misure e parametri t1 , s0 , r2 e b ( quando opportuno ) , stimati applicando i due modelli matematici ai dati ottenuti sul fantoccio con i tre diversi protocolli corti di acquisizione ( a , b e c ) protocollo a protocollo b protocollo c a valori medi di 3 sole ripetizioni . 
vedi testo per i dettagli una ripetizione sono illustrati nella figura 1 , in cui sono rappresentate anche le curve descritte dai due diversi modelli matematici , utilizzando come parametri t1 , s0 e b quelli ottenuti dalle stime per regressione non lineare sugli stessi dati . 
la tabella 1 riporta i valori medi dei parametri stimati con entrambi i modelli matematici , utilizzando tutti i 13 valori a disposizione oppure solamente quelli corrispondenti ai cinque tr pi piccoli . i valori di t1 e quelli di s0 ottenuti con i due modelli matematici sono risultati significativamente diversi ( test di wilcoxon , n = 6 , p < 0 , 05 ) , sia utilizzando lintera serie di valori che solamente 5 dati . 
le stime effettuate applicando il modello esponenziale semplice su 5 soli dati hanno fornito risultati significativamente inferiori ( test di wilcoxon , n = 6 , p < 0 , 05 ) rispetto allo stesso modello applicato su 13 valori . la tabella 2 riassume i tempi medi di esecuzione dei tre diversi protocolli corti ( a , b e c ) ed i parametri t1 , s0 e b ottenuti applicando i due modelli matematici , oltre al valore medio dei coefficienti di determinazione r2 . 
la curva continua e quella spezzata sono rispettivamente quelle predette attraverso il modello esponenziale semplice ed esponenziale saturato applicati agli stessi dati . table 2 summarises the mean time required for the three different short protocols ( a , b , and c ) and the parameters t1 , v . 
the t1 constant and the s0 values estimated with the simple exponential model are significantly smaller for protocol c ( kruskal - wallis test , n = 18 ; p < 0.005 for both parameters )  . 
 for protocol a , both the t1 constant and the s0 values estimated through the simple exponential are significantly lower than those predicted with the saturated exponential model ( wilcoxon test , n = 6 , p < 0.05 for both parameters )  . 
indeed , the nonlinear regression procedure succeeded in concluding the estimation of the saturated exponential model parameters in a finite number of iterations ( < 300 ) in only one case for protocol b ( for which , however , the value of coefficient b is meaningless : b = 7.96 ) and in only three out of six repetitions of protocol c . in vivo experimentation figure 2 illustrates , for a typical subject , the mean spectra obtained with different tr values during one of the three repetitions , both at rest ( a ) and during exercise ( b )  . 
it can be seen that compared to rest , the spectra obtained during exersui dati dei protocolli b e c non stato effettuato alcun test statistico di confronto , data la bassa numerosit dei risultati ottenuti con lesponenziale saturata . 
si pu notare che rispetto al riposo , gli spettri ottenuti durante esercizio presentano il picco della fosfocreatina pi piccolo e quello del fosforo inorganico pi grande , mentre le righe spettrali dellatp sono rimaste pressoch invariate . 
la stima dei parametri con il modello esponenziale saturata si conclusa con valori accettabili ( b1 ) solamente in 2 prove su 12 sia per i dati acquisiti a riposo che per quelli registrati durante esercizio . 
la stessa tabella riassume anche i valori di potenza e ph intratable 3 average values ( sd ) of parameters t1 , s0 and r2 obtained with the simple exponential model on volunteers . 
summary of average power developed by the right leg during exercises and intracellular ph calculated for the two conditions tabella 3 valori medi ( ds ) di t1 , s0 e r2 ottenuti con il modello esponenziale semplice sui volontari . 
2a , b spettri medi ricavati dagli ultimi 6 spettri acquisiti da un soggetto in funzione dei 7 tempi di ripetizione utilizzati : a a riposo , b durante esercizio . cise have a lower phosphocreatine peak and a higher inorganic phosphate peak whereas the atp spectrum lines are virtually unchanged . 
table 3 shows the mean t1 and s0 values obtained for phosphocreatine with the simple exponential model in resting and exercise conditions . the estimation of parameters with the saturated exponential model ended with acceptable values ( b1 ) in only two tests out of 12 for data acquired during both rest and exercise . 
table 3 also summarises power and intracellular ph values in both conditions . the kruskall - wallis test revealed that there were no significant differences in t1 and ph values between rest and exercise ( n = 24 , p = ns for both parameters )  . 
furthermore , for each tr , the first spectra collected were discarded for an overall time more than three times the hypothetical t1 = 6 s [ 22 ] , this to ensure that all data came from a sample under saturated conditions . the two mathematical models used to estimate the t1 are based on different physical interpretations of the systethe saturated exponential model describes better the phenomecellulare nelle due condizioni . 
al contrario , il valore di s0 durante esercizio risultato significativamente inferiore rispetto a quello a riposo ( test di kruskall - wallis a 1 coda , n = 24 , p < 0 , 05 ) , con una differenza , in media , dell11 , 3% . discussione stima del t1 in vitro una stima attendibile del t1 possibile solamente se le informazioni sono state ottenute su un ampio range di valori di tr e se , per ciascuno di essi , stato raccolto un numero sufficiente di spettri , tale da garantire le condizioni di saturazione ed un buon rapporto segnale - rumore . 
inoltre , sono stati scartati , per ciascun tr , i primi spettri raccolti , per un tempo complessivo superiore a 3 volte il t1 presunto di 6 s [ 22 ] , a garanzia che tutti i dati provenissero da un campione in condizioni saturate . 
i valori di t1 stimati con questo modello sono risultati circa doppi rispetto allesponenziale semplice , mentre i valori di s0 sono risultati simili , anche se quelli ottenuti con lesponenziale semplice sono sistematicamente minori ( tabella 1 )  . 
t1 values estimated with this model were almost double compared with the simple exponential whereas the s0 values were similar , even though those obtained with the simple exponential are systematically lower ( table 1 )  . 
furthermore , we tried to obtain the same signal - to - noise ratio as with the long protocol by having at least 16 readings of the relaxation signal for every tr value . the t1 and s0 values calculated by applying the simple exponential model to the data obtained with all three protocols ( a , b , and c ) are smaller than the corresponding parameters obtained with the long protocol . 
this result may be partially explained by the lower extension and lower maximum values of the trs under study with these protocols . furthermore , protocol c , despite the use of a modified version of the mathematical models allowing for the greater complexity of the acquisition , provided decidedly lower parameters than those obtained with protocols a and b . 
estimation of parameters , through saturated regression , on the data of protocol a gave higher values than those obtained with the simple exponential curve ( table 2 ) , similarly to what has been observed for the long protocol , in which the tr extension was larger . 
moreover , the power developed by the subjects with their right legs alone was lower than that reported by other authors as being the threshold for lactate production , namely 4.84.6 w [ 12 , 28 ]  . it should be noted that the time constant t1 during exercise can be estimated only if pcr - splitting processes which occur at the beginning of the exercise are no longer underway , i.e. 
anche per i protocolli corti stata garantita la condizione di saturazione , inserendo un adeguato numero di pre - scan o scartando degli spettri , nonostante i limiti di tempo imposti . 
inoltre , si cercato di ottenere lo stesso rapporto segnale - rumore del protocollo lungo , mantenendo ad almeno 16 il numero di letture del segnale di diseccitazione per ogni valore di tr . i valori di t1 e di s0 , calcolati applicando il modello esponenziale semplice ai dati ottenuti con tutti e tre i protocolli ( a , b e c ) , sono pi piccoli dei corrispondenti parametri ottenuti dal protocollo lungo . 
questo risultato pu essere parzialmente spiegato dalla minore estensione e dai valori massimi pi piccoli dei tr indagati con questi protocolli . inoltre il protocollo c , nonostante lutilizzazione di una versione modificata dei modelli matematici che tenga conto della maggiore complessit dellacquisizione , ha fornito parametri decisamente inferiori rispetto ai protocolli a e b . 
ci pu essere un indice del fatto che , in sequenze rm di acquisizione temporalmente pi complesse , altri fattori , oltre al t1 , possono essere importanti nel determinare lintensit del segnale raccolto . la procedura di stima dei parametri dellesponenziale saturata non riuscita a terminare correttamente per tutte le ripetizioni del protocollo b e per la met delle ripetizioni del protocollo c . 
la stima dei parametri , mediante regressione saturata , sui dati del protocollo a ha fornito valori superiori a quelli ottenuti tramite lesponenziale semplice ( tabella 2 ) , analogamente a quanto gi osservato per il protocollo lungo , in cui lestensione di tr indagata era maggiore . sperimentazione in vivo gli esperimenti condotti sui volontari sono stati eseguiti in modo da ottenere le informazioni relative alla costante di tempo t1 del muscolo gastrocnemio sia in condizioni di riposo che durante esercizio aerobico . 
questultima condizione stata raggiunta , in quanto non c stata variazione nei valori di ph fra la condizione di riposo e quella di esercizio . inoltre , la potenza sviluppata dai soggetti con la sola gamba destra risultata minore della potenza riportata da altri autori come soglia di produzione del lattato , pari a 4 , 84 , 6 w [ 12 , 28 ]  . va sottolineato che la stima della costante di tempo t1 in corso di esercizio possibile esclusivamente se non sono pi in atto i processi di scissione della pcr che avvengono allinizio dellesercizio , cio la concentrazione della pcr deve rimanere costante . 
precedenti studi [ 11 , 2931 ] hanno descritto il processo di scissione della pcr con un andamento esponenziale , la cui costante di tempo risulta di circa 20 s . assumendo tale costante di tempo e che il segnale della pcr durante esercizio aerobico diminuisca al massimo del 20% v . 
assuming such a time constant and that the pcr signal during aerobic exercise drops by a maximum of 20% compared with the rest phase [ 12 , 28 ] , after 30 s from the start of the process , the remaining signal will be around 85% of the rest signal instead of 80% . 
the 10 - s wait before beginning acquisitions and the time required for acquisition of the spectra to be discarded ensured an interval from the beginning of the exercise of at least 30 s . the relative decline of the s0 value of pcr between rest and exercise , estimated with the simple exponential model , is around 11.3%. 
moreover , during exercise , the signal comes from spectra collected during movement of the limb , a source of noise due to the noncomplete homogeneity of the main magnetic field b0 or to morphological changes of the muscle . 
this value is comparable with the value reported at rest by newcomer ( 5.6 s ) under the same acquisition conditions ( b0 = 1.5 t with surface coil [ 22 ] )  . rispetto al riposo [ 12 , 28 ] , dopo 30 s dallinizio del processo il segnale residuo sar circa l85% di quello a riposo , invece dell80% . 
i 10 s di attesa prima di iniziare le acquisizioni , assieme al tempo impiegato per lacquisizione degli spettri da scartare , hanno garantito un intervallo dallinizio dellesercizio di almeno 30 s . la diminuzione relativa del valore di s0 della pcr fra riposo ed esercizio , stimato con il modello esponenziale semplice , di circa 11 , 3% . 
inoltre , durante esercizio , il segnale proviene da spettri raccolti durante il movimento dellarto , che fonte di rumore dovuto alla non perfetta omogeneit del campo magnetico principale b0 od a modifiche morfologiche del muscolo . 
il valore di probabilit del test statistico ( p = 0 , 722 ) e la relativamente piccola deviazione standard , rassicurano sul fatto che la costante di tempo effettivamente non cambia tra le due condizioni . 
di conseguenza , negli studi di quantizzazione della pcr , appropriato utilizzare lo stesso coefficiente di correzione sia per il riposo che per lesercizio . la costante spin - reticolo misurata sul muscolo in vivo con la presente sperimentazione stata ottenuta con una sequenza rm complessa , risultando essere di 0 , 91 s . 
supponendo che le procedure di analisi abbiano introdotto gli stessi errori sistematici sia nei dati acquisiti in vivo che in quelli ottenuti in vitro , si pu calcolare il valore teorico ottenibile in condizioni ideali , a parit di modello esponenziale semplice . 
utilizzando la costante di tempo t1 ottenuta in vitro con il protocollo lungo ( 3 , 84 s ) ed il valore di t1 ottenuto in vitro a parit di protocollo di acquisizione ( protocollo c ; 0 , 61 s ) , il valore vero del t1 apparente della pcr in vivo pu essere calcolato come : 0 , 91 ( 3 , 84 / 0 , 61 ) = 5 , 73 s . 
tale valore sovrapponibile a quello riportato da newcomer a riposo ( 5 , 6 s ) nelle stesse condizioni di acquisizione ( b0 = 1 , 5 t con bobina di superficie [ 22 ] )  . conclusions conclusioni the main purpose of our work was to estimate the time constant t1 during aerobic exercise using a clinical mr scanner . lo scopo principale del presente lavoro era di stimare la costante di tempo t1 durante esercizio aerobico , utilizzando un v . 
franchi maggi 5 , i - 27100 pavia , italy , tel . : + 39 - 038 - 5022838 , fax : + 39 - 038 - 5028597 , e - mail : g.beluffi@smatteo.pv.it received : 11 july 2005 / accepted : 18 october 2005 / published online : 11 april 2006 abstract purpose . 
among the 16 , 326 patients who underwent x - ray study of the hands , nine ( six males and three females , 0.055% of the total ) presented the typical radiological features of kirners deformity ; no patient had a family history of the disorder except for one , in whom the suspicion of familiarity could not be verified radiologically . 
although rare , as also shown by our study , kirners deformity must be recognised and properly diagnosed in order to spare the subject unnecessary surgical procedures . key words kirners deformity dystelephalangy nail phalanx riassunto obiettivo . 
il portatore non lamenta generalmente n dolore n impotenza funzionale delle dita : al massimo denunciata una difficolt a compiere particolari movimenti . allesame radiologico la falange ungueale del v dito ( quasi sempre bilateralmente ) composta da una epifisi la cui porzione distale , di solito dal lato palmare , pu presentare un piccolo sprone osseo . 
la diafisi pi piccola che di norma , incurvata in senso palmare , deviata di 1050 dallasse normale in senso radiale , e non saldata con lepifisi , con possibile e frequente aumento dello spazio epifisometafiso - diafisario . la struttura ossea in corrispondenza della zona epifiso - metafisaria presenta talvolta segni di sclerosi , in particolare in corrispondenza della porzione metafisaria ungueale . 
distal phalanx bilateral changes are more pronounced on the left side where the shaft is more bent towards the palm ( a , b ) than on the right side ( c , d ) in relation to the dysmorphic epiphysis . 
alterazione bilaterale della falange ungueale con aspetto pi angolato della diafisi dal lato sinistro ( a , b ) , meno evidente a destra ( c , d ) con aspetto tozzo dellepifisi basale . 
fiori : clinical and radiological findings in kirners deformity patients and methods pazienti e metodi at our paediatric radiology section , an average of 1 , 040 wrist and hand x - ray films are taken each year , not only to study bone age but also to assess morphologic and pathologic changes in the presence of trauma , haematological disorders , bone syndromes and dysplasias , dysmorphic features , rheumatic disorders , etc . 
over a period of 23.5 years a total of 23 , 996 such procedures were performed with an average of 36% follow - up examinations without significant differences between males and females . 
ba : a boy aged 13 years and 5 months , karyotype 46 xy , with hypophyseal dwarfism , referred for radiological assessment of bone age , which corresponded to 10.5 years according to greulich and pyle ( g&p ) [ 10 ] standards . 
hm : a boy aged 9 years and 11 months , karyotype 46 xy , referred for bone - age evaluation ( equal to 9.5 years according to g&p ) for suspected fetal alcohol syndrome . 
sf : a girl with turners syndrome , karyotype 46 xo , seen for the first time at the age of 2 years and 3 months for evaluation of bone age ( 1 year and 10 months )  . 
a very small epiphysis appeared at the age of 5 years and 1 month , and the presumptive diagnosis of kirners deformity was confirmed when the girl was 8 years and 2 months . 
in 23 anni e mezzo sono stati eseguiti 23.966 radiogrammi di questo tipo con una percentuale di controlli nel tempo del 36% , senza significative differenze tra maschi e femmine . 
s.f. , femmina affetta da sindrome di turner , con cariotipo 46 xo , che allet di 2 anni e 3 mesi esegue il primo radiogramma del carpo per la determinazione dellet scheletrica ( pari ad 1 anno e 10 mesi )  . 
sin dal primo controllo si osserva dismorfia della falange ungueale del v dito che spiccatamente ipoplasica rispetto a quella delle altre dita , lievemente incurvata in senso radiale , con comparsa della epifisi ( puntiforme ) al controllo eseguito allet di 5 anni e 1 mese e definizione certa della anomalia di kirner al controllo intorno agli 8 anni e 2 mesi di vita ed in quelli successivi . 
3a - h changes over time of kirners deformity between age 2 years and 3 months and 13 years and 4months in a patient affected by turners syndrome . the first x - ray film shows lack of the epiphysis and a dysmorphic , hypoplastic phalanx shaft slightly bent towards the radial side ( a )  . 
follow - up x - ray films show the appearance of a small epiphysis at the age of 5 years and 1 month ( b ) , which slowly enlarges ( c ) , becoming stubby and dysmorphic with the typical appearance of kirners deformity at the age of 8 years and 2 months ( d )  . 
3a - h evoluzione nel tempo della anomalia di kirner dallet di 2 anni e 3 mesi a quella di 13 anni e 4 mesi in portatrice di sindrome di turner . 
comparsa di una piccola epifisi a 5 anni e 1 ese ( b ) con successivo ingrandimento della stessa ( c ) , tozza e dismorfica , che assume laspetto tipico dellanomalia di kirner allet di 8 anni e 2 mesi ( d ) con aspetto sempre pi evidente nei successivi controlli ( e , f ) , fino allultimo allet di 13 anni e 4 mesi ( g , h ) in cui lepifisi tozza , con sprone al suo margine infero - superiore e la diafisi angolata . 
affected by acute lymphatic leukaemia ( all ) , he first underwent an x - ray study of his left hand at the age of 14 years in order to establish bone age ( equal to 12 years ) as a follow - up in the treatment protocol of his disease . 
r.a. , maschio , cariotipo 46 xy , dellet attuale di 17 anni , portatore di lla stato sottoposto per la prima volta allet di 14 anni allo studio rx per la determinazione dellet scheletrica ( pari a 12 anni ) , quale controllo nel contesto del protocollo terapeutico della emopatia da cui era affetto . 
la falange distale del v dito appariva corta e tozza con deviazione volare e presentava una unghia dismorfica a vetrino dorologio . il controllo radiologico dimostr una epifisi falangea regolare g . 
the same findings were detected in the right hand . in all nine patients , the distal phalanges of the little fingers had a stubby , dysmorphic appearance with palmar and radial bending and showed dysmorphic , eagle - claw - like nails . 
in contrast , the x - ray findings in the other eight patients were typical for kirners deformity : bending of nail - phalanx shaft , irregular diaphyseal thickening with loss of normal bone texture and straight epiphysis with a spur on the volar surface projecting towards the shaft where it fits into a groove . ed una diafisi piccola e deviata medialmente con aspetto quasi ad uncino , tipico della anomalia di kirner . 
in tutti e 9 i pazienti le falangi distali del v dito della mano presentano un aspetto tozzo , deviazione in senso palmare e radiale ed unghia corta e dismorfica . 
nei rimanenti 8 pazienti , il quadro radiologico risultava essere quello tipico della anomalia di kirner documentato da : incurvamento della falange ungueale , irregolare addensamento struttura ossea diafisaria , epifisi mai incurvata , ma dotata di sprone ( becco ) , al quale corrisponde una incisura metafisaria , scavata quasi a colpo dunghia e con aspetto chiaramente dismorfico dellunghia . discussion and conclusions in all nine cases ( one of which was already reported [ 9 ] ) , there were no doubts about the diagnosis . 
all nine displayed the typical clinical and radiological features of kirners deformity ( dysmorphic , painless deformity of nail phalanges with bent shafts ) associated with delayed bone age , except in case 2 . 
both the clinical and especially radiological diagnosis in these cases , as well as in the other cases reported in the literature , are straightforward and can usually be made between 8 and 14 years of age ( 6 and 14 in the present series ) [ 18 , 10 , 1228 ]  . discussione e conclusioni in tutti e nove i casi da noi osservati ( di cui uno gi descritto ) [ 9 ] non possono sorgere dubbi sulla diagnosi . 
sono infatti riscontrabili clinicamente e radiologicamente le alterazioni gi descritte e tipiche della sindrome di kirner ( dismorfia della falange ungueale del v dito non accompagnata da sintomatologia dolorosa , incurvamento , malformazione radiologica tipica ) , associate ad un ritardo , pi o meno evidente , della maturazione scheletrica , ad eccezione del caso n . 
4 paziente affetto da lla : evoluzione nel corso di 3 anni ( dallet di 14 a quella di 17 anni ) della anomalia di kirner con progressiva riduzione della cartilagine di coniugazione e persistente deviazione mediale della diafisi , dallaspetto quasi ad uncino . even though kirners deformity involves swelling and a stubby appearance of the little fingers , it is usually painless . 
in only four cases reported in the literature were symptoms present : two , reported by staheli [ 18 ] had dull , aching pain and progressive myositis ossificans ; and two , reported by dykes [ 23 ] had pain for a few months and nail phalanx redness . the deformity is usually sporadic but may be inherited as an autosomal dominant trait [ 17 , 29 ] with scarce or incomplete penetrance [ 22 ]  . it affects females more frequently than males with a 2 : 1 ratio and is often associated with delayed bone age . 
dystelephalangy changes were reported in some members of the mothers family but not in the fathers , so the girl has to be considered heterozygous for the deformity [ 30 ]  . there is no agreement about the aetiopathogenesis of kirners deformity , which is still unknown and controversial [ 17 , 2931 ] , with hypotheses being : juvenile osteomalacia [ 5 ] ; aseptic necrosis on the basis of biopsy findings , which showed lysis between the diaphysis and epiphysis of the terminal phalanx with diminished trabecular bone and decreased thickness of the zone of provisional calcification [ 15 ] ; osteochondrosis of possible vascular origin [ 7 ]  . 
recently , it has been suggested that kirners deformity is a congenital defect of growth - cartilage to be classified among the genotypic osteochondrodysplasias ( such as a epiphyso - metaphyseal acrodysplasia ) [ 4 , 26 , 31 ]  . in the only case in the literature studied by magnetic resonance imaging ( mri ) , it has been suggested that a chronic inflammatory disorder or vascular dysfunction of the soft tissues of the distal phalanges could be the cause of the deformity [ 30 ]  . this hypothesis has been criticised on the grounds of technical artefacts during mri examination [ 32 , 33 ]  . 
our cases provide no information to confirm or refute the above hypotheses , nor do they suggest an alternative hypothesis . the apparently positive family history in one ( case 3 ) suggests the hereditary nature of the deformity . significant reports from the point of view of heredity are g . 
fiori : clinical and radiological findings in kirners deformity di osservazione variabile tra gli 8 ed i 14 anni ( nei nostri pazienti tra i 6 ed i 14 anni ) semplice e non dovrebbe far sorgere dubbi [ 18 , 10 , 1228 ]  . 
 [ 18 ] erano presenti disturbi clinici e soggettivi : in uno , in forma di marcata dolorabilit , nellaltro associata ad una miosite ossificante progressiva , ed in due di quelli descritti da dykes arrossamento e dolore per brevi periodi di tempo [ 23 ]  . 
lanomalia presenta in genere caratteri di sporadicit o di familiarit secondo una modalit di trasmissione autosomica dominante [ 17 , 29 ] a bassa od incompleta penetranza [ 22 ] e colpisce pi frequentemente le femmine dei maschi , con un rapporto di 2 : 1 , associandosi in genere ad un ritardo della maturazione scheletrica . 
di raro riscontro in america ed in europa ( 0 , 055% nella nostra casistica ) , con la sola eccezione dellinghilterra , ove raggiunge una incidenza dello 0 , 25% [ 29 ]  . 
lo stesso autore ha descritto un caso di distelefalangia esteso a quasi tutte le dita ( esclusi i pollici e lindice della mano destra ) come possibile espressione di uno stato omozigote del gene responsabile , poich verificatosi in una paziente in cui genitori ed il nonno materno ( nonch un fratello ) risultavano portatori di deformit di kirner bilaterale [ 8 ]  . un altro caso di distelefalangia politopica di tutte le dita ( una bambina con iniziale interessamento delle falangi distali allet di 2 mesi ed espressione completa dellalterazione a 5 anni ) stato descritto recentemente da brune [ 30 ]  . 
circa leziopatogenesi della anomalia , ancora oscura e fonte di discussione [ 17 , 29 , 30 , 31 ] , sono state fatte alcune ipotesi : da una osteomalacia giovanile [ 5 ] , ad una necrosi asettica [ 15 ] , giustificata da un reperto istopatologico post - bioptico , che ha dimostrato solo unirregolarit della zona di passaggio tra cartilagine e zona diafisaria con riduzione dellosso trabecolare ed in particolare con ridotto spessore della zona di calcificazione provvisoria ; ad una osteocondrosi di natura vascolare [ 7 ]  . 
attualmente lorientamento della letteratura che si tratti di una alterazione congenita della cartilagine di accrescimento nel contesto generale delle ostecondrodisplasie genotipiche ( acrodisplasia epifiso - metafisaria ) [ 4 , 26 , 31 ]  . 
stata avanzata anche lipotesi , nellunico caso studiato con rm , che un processo infiammatorio cronico od una alterazione della vascolarizzazione siano la causa della anomalia [ 30 ]  . 
non vi sono dati utili , nei casi osservati , per confermare o negare una di tali ipotesi , n siamo in grado di poter avanzare una nostra ipotesi eziopatogenetica . 
fiori : clinical and radiological findings in kirners deformity those by blank and girdany [ 17 ] , who described seven cases ( four males and three females ) , all belonging to the same family , and sugiura [ 8 ] , which suggest homozygosity of the deformity ; on the contrary , the case of brune et al . 
per quanto riguarda invece la percentuale pi elevata di manifestazione nelle femmine rispetto ai maschi , con un rapporto 2 : 1 ( addirittura di 5 : 1 nella casistica di dykes ) [ 23 ] , con interessamento quasi sempre bilaterale , mentre nei maschi spesso monolaterale ( lato destro pi del sinistro ) [ 29 ] , non possibile esprimere un giudizio , data lesiguit nel numero dei nostri pazienti , in cui anzi si potrebbe parlare di rapporto invertito maschi / femmine , pari infatti a 6 : 3 ( sei maschi e tre femmine ) e con riscontro bilaterale in tutti i casi . 
lanomalia stata invece segnalata in associazione con una grande variet di malformazioni sia dellapparato scheletrico , quali cifosi e ginocchio valgo [ 1 ] , deformit del dito indice e della metafisi radiale [ 12 ] , aspetto mongoloide [ 2 ] , piede cavo [ 3 ] , osteomielite [ 7 ] e miosite ossificante progressiva [ 18 ] , nonch di quello cardiovascolare ( difetto settale interventricolare , dotto arterioso pervio , difetto settale atriale tipo ostium secundum , stenosi valvolare polmonare [ 29 ] )  . 
nellet adulta la forma evolve con fusione delle due porzioni della falange , lasciando come reliquato solo un danno estetico dovuto allincurvamento od una lieve difficolt ad eseguire quei movimenti che richiedono luso del v dito , come suonare strumenti tipo pianoforte o violino e scrivere a macchina . 
the encouraging results , if validated by larger series , support the use of pet / ct in patients with carcinoma of unknown primary origin and negative conventional imaging results . key words metastatic cancer occult primary cancer 18f - fdgpet / ct riassunto obiettivo . 
la popolazione in studio costituita da 38 pazienti consecutivi con malattia tumorale metastatica provata istologicamente in cui le tecniche di diagnostica per immagini convenzionale erano risultate negative o non conclusive nella identificazione della lesione primitiva . 
the site of the occult primary tumour often remains unidentified after common imaging investigations ( chest x - ray , abdominal and pelvic ct , mammography in women ) ( 20%27% of cases ) [ 1 , 2 ] or autopsy ( 30%82% ) [ 24 ]  . 
reasons for the low rate of detection of the primary cancer include a lesion size smaller than the spatial and contrast resolution of the technique used or involution of the primary mass due to limited angiogenic competence [ 5 ]  . 
conventional contrast studies of the upper and lower digestive tract , for example , have a low diagnostic yield , and mammography , although routinely ordered , is often unhelpful because very few patients with cup have a primary mass in the breast [ 6 ]  . recent papers have reported that the median survival for patients with cup is approximately 12 months and that detection of the primary tumour and initiation of therapy can prolong survival to 23 months [ 7 ]  . 
the aim of this study was to assess the role of 18f - fdg - pet / ct in the identification of occult primary tumours . materials and methods the patient population consisted of 38 consecutive patients ( 22 men , 16 women ; mean age 5911 years , range 4177 years )  . 
the uptake period was 6090 meach patient then underwent a pet / ct scan with a dedicated hybrid scanner ( discovery ls , general electric ; biograph sensation 16 , siemens ) and whole - body scans ; attenuation correction was la sindrome del tumore primitivo occulto ( cup ) consiste nella presenza di una malattia tumorale metastatica per la quale sia impossibile identificare la sede primitiva della neoplasia dopo una attenta indagine anamnestica , esame obbiettivo negativo , negativi gli esami clinico - diagnostici ( emocromo completo , funzionalit renale , epatica , pancreatica , test delle urine , radiogramma del torace , tc addominale e pelvica e mammografia nelle donne , psa negli uomini ) [ 1 ]  . 
nella maggior parte dei casi , impossibile identificare la sede del tumore primitivo occulto con altri esami ( 20%27% ) di diagnostica per immagini pi diffusi ( radiografia del torace , tc addominale e pelvica , mammografia nelle donne ) [ 1 , 2 ] o allautopsia ( 30%82% ) [ 24 ]  . le motivazioni alla base dello scarso potere di identificazione della sede del tumore primitivo comprendono le dimensioni al di sotto del potere di risoluzione , sia spaziale che di contrasto , delle tecniche utilizzate o linvoluzione della massa primitiva a causa di uno scarso potere angiogenetico [ 5 ]  . 
la mammografia , nonostante sia utilizzata nella routine diagnostica , spesso non utile in quanto un limitato numero di pazienti con cup hanno una massa primitiva a livello mammario [ 6 ]  . recenti lavori riportano come la sopravvivenza mediana dei soggetti con cup sia attorno ai 12 mesi e che la identificazione della sede primitiva di malattia con la possibilit di intraprendere un iter terapeutico sposti la sopravvivenza mediana a 23 mesi [ 7 ]  . 
successivamente ogni soggetto stato sottoposto a scansione pet - tc con tomotabella 1 dati clinici , istopatologici e di diagnostica per immagini dei casi con tumore primitivo a livello toracico caso n sesso et , anni biopsia pet / tc tumore occulto dopo pet / tc dimensioni lesione , mm brain metastasis adenocarcinoma supraclavicular node metastasis mediastinal and neck node metastasis adenocarcinoma adenocarcinoma supraclavicular node metastasis sacral vertebra metastasis melanoma adenocarcinoma left humerus metastasis squamous cell carcinoma area of increased uptake in left lung area of increased positive mediastinal and neck nodes , area in right lung positive neck nodes , area of uptake on chest skin multiple areas of uptake in bone and left lung multiple metastases to bone , right adrenal gland , right lung lymph nodes , lung left lung left lung right lung melanoma left lung right lung lung lung laterocervical node spinous cell metastasis brain metastasis carcinoma epithelial carcinoma bone , lung diangostica convenzionale metastasi cerebrale metastasi linfonodo sovraclaveare metastasi linfonodali ( mediastinici e cervicali ) metastasi linfonodo sovraclaveare metastasi ossea vertebra sacrale adenocarcinoma adenocarcinoma adenocarcinoma area di aumentato uptake polmonare sx area di aumentato uptake polmonare sx linfonodi mesiatinici e cervicali , area polmone dx polmone sx polmone sx polmone dx melanoma linfonodi cervicali , area cute torso melanoma adenocarcinoma metastasi omero carcinoma squamocellulare metastasi linfonodali laterocervicali metastasi cerebrale carcinoma spinocellulare carcinoma epiteliale multiple aree captanti a livello osseo e polmonare sx multiple meta . 
the low rate of detection of the primary tumour site has two main causes . first , the size of the primary lesion may be smaller than the resolution power of conventional imaging procedures ( such as ct or gastrointestinal contrast studies ) , or the lesions may be located in sites such as the abdomen , pelvis , head and neck . 
epatiche , area positiva gastrica negativa carcinoma a cellule transizionali adenocarcinoma carcinoma a cellule fusate carcinoma squamocellulare carcinoma squamocellulare negativa linfonodi adiacenti mammella sx area mediastinica ignoto linfonodi cervicali dx ignoto ignoto ignoto ignoto ignoto ignoto carcinoma epiteliale linfonodo ascellare sx ignoto carcinoma epiteliale linfonodo pelvico ignoto adenocarcinoma surrene dx , linfonodi ignoto adenocarcinoma adenocarcinoma mucoide adenocarcinoma mucoide carcinoma epiteliale mano sx area mediastinica ed ossea cute ignoto ignoto cute peritoneo fegato ignoto carcinoma epiteliale cute carcinoma indifferenziato linfonodo mediastinico ignoto ignoto ignoto na , non applicabile suggested that the primary tumour may disappear after having given rise to metastasis because of its angiogenic incompetence , which leads to marked apoptosis and cell turnover [ 5 ]  . whereas no diagnostic procedures can identify a primary lesion that has disappeared due to marked apoptosis , it has been demonstrated that fdg - pet can improve primary - site detection in patients with cup and a negative diagnostic workup . 
although the number of patients in our study was small , our preliminary results show that pet / ct is more sensitive than pet alone , with a 53% detection rate for occult cancers missed by other techniques discussione la cup una sindrome caratterizzata da una prognosi infausta . 
la identificazione della sede primitiva di neoplasia , comunque , pu essere importante in quanto permette di impostare una terapia specifica , migliorando significativamente la prognosi ( 1 anno vs 2 anni ) [ 7 ]  . 
le dimensioni delle lesioni primitive possono essere inferiori al potere di risoluzione delle procedure diagnostiche ( tc o studi contrastografici del tratto gastrointestinale ) o trovarsi in sedi quali laddome , la pelvi , la testa - collo . 
la pet - tc ( sezioni transassiali della pet , tc e immagine di fusione pet - tc ) ha evidenziato unarea di patologico accumulo a livello dellipofaringe ( indicata da freccia nella immagine mip )  . nari di piccole dimensioni [ 1 ]  . 
laltro motivo dipende dalle caratteristiche biologiche del tumore primitivo : stato ipotizzato che la massa tumorale primitiva possa scomparire dopo aver dato luogo a metastatizzazione a causa di uno scarso potere angiogenetico che porta a marcata apoptosi e turn over cellulare [ 5 ]  . mentre nessuna procedura diagnostica pu identificare una lesione primitiva che scomparsa per marcata apoptosi , stato dimostrato come la pet con fdg possa migliorare la identificazione della sede primitiva della lesione in pazienti con cup e una flow - chart diagnostica peraltro negativa . 
al momento presente , sono pochi gli studi che hanno valutato il valore aggiunto della pet - tc rispetto alla fdgpet convenzionale per lidentificazione della sede del tumore occulto [ 810 ]  . 
i nostri risultati preliminari , nonostante il campione sia limitato , mostrano che la pet - tc pi sensibile della pet convenzionale , rilevando il 53% dei tumori occulti che non erano stati evidenziati con le altre tecniche [ 1113 ]  . 
le limitate informazioni morfologiche fornite dalla pet da sola , rendono necessaria lintegrazione con i dati anatomici forniti dalla tc per migliorare la definizione anatomica delle aree di aumentata captazione del tracciante . 
the limited morphological information provided by pet alone requires integration with the anatomical data supplied by ct in order to improve the anatomical definition of the areas of increased tracer uptake . 
pet / ct scanners capture anatomical and functional data in a single registration [ 14 ] , overcoming the limitations of patient repositioning and time interval between scans when the images are fused a posteriori [ 15 ]  . 
the primary tumour was identified in 7 / 29 cases ( two carcinomas of the nasopharynx , one plasmocytoma of the tongue base , one lung carcinoma , two breast cancers ) , showing 24% sensitivity . 
this result is not , however , comparable with our findings because alberini included 22 patients with another positive conventional test : this means that pet provided additional information in 4 / 44 cases only . 
although the population was not selected ( in contrast to our study population ) , the sensitivity of pet was 37.8% , which is significantly lower than that reported in our study . although the heterogeneity of patients could be seen as a potential limitation to our study , with patients referred from different centres and affected by different types of tumours , this fact does not undermine the value of our study , as the diagnostic - therapeutic approach to unknown primary tumours has not yet been codified by international guidelines . 
the head and neck , abdomen and pelvis are highly complex anatomical districts and are characterised by areas of physiological tracer uptake ( for example , by the tonsils , lymphatic tissue , stomach , bowel and ureters )  . small areas of increased uptake due to a small primary tumour may be mistaken for areas of physiological uptake . the careful comparison of metabolic and anatomical information , made possible by pet / ct , considerably improves the sensitivity of the procedure , allowing visualisation of even small areas of asymmetrical tracer distribution or areas of increased focal uptake that would otherwise be considered physiological . 1154 dallintervallo di tempo tra gli scans quando le immagini sono fuse a posteriori [ 15 ]  . 
la sede del tumore primitivo stata identificata in 7 / 29 ( 2 carcinomi del nasofaringe , 1 plasmocitoma della base della lingua , 1 carcinoma polmonare , 2 carcinomi della mammella ) con una sensibilit del 24% . 
questo risultato , comunque , non confrontabile con il nostro studio in quanto alberini ha incluso nel campione 22 soggetti che avevano un altro test convenzionale positivo : questo significa che la pet ha fornito indicazioni aggiuntive solamente in 4 / 44 casi . 
nonostante la popolazione non fosse stata selezionata , al contrario di quella descritta nel nostro studio , la sensibilit della pet risultata del 37 , 8% , che sensibilmente inferiore a quella da noi riportata . sebbene un potenziale limite al presente studio sia rappresentato dal fatto che il campione preso in esame eterogeneo in quanto costituito da pazienti provenienti da centri diversi e portatori di neoplasie di diverso tipo , tuttavia questo non toglie valore allo studio in quanto lapproccio diagnostico - terapeutico in casi di tumore primitivo ignoto non ancora codificato da norme internazionali . 
alcune sedi quali il capo - collo , laddome e la pelvi , sono anatomicamente molto complesse e caratterizzate da varie sedi di fisiologico uptake del tracciante ( per esempio le tonsille , il tessuto linfatico , lo stomaco , lintestino , gli ureteri )  . 
the main cause of acute chest pain , which accounts for 6.5% of urgent medical examinations in emergency rooms in italy , is acute coronary syndrome ( acs )  . 
we performed this prospective study to evaluate the diagnostic accuracy of a 16 - channel computed tomography ( ct ) scanner with dedicated software in a group of patients with chest pain and medium to low risk of acs materials and methods . 
this study involved a selected group of 31 patients reporting chest pain with a medium to low probability of acs , defined on the basis of preliminary tests [ electrocardiogram ( ecg ) and serum cardiac markers ]  . 
msct identified the presence of occlusions and significant ( > 50% ) or nonsignificant stenoses in the main coronary segments , with a sensitivity of 65% , a specificity of 98.8% , a positive predictive value ( ppv ) of 81.2% , a negative predictive value ( npv ) of 97.3% and an accuracy of 96.4%. 
in patients with a medium to low coronary risk , msct is a more accurate indicator of the need for coronary angiography than is exercise stress testing , which is less expensive but has lower predictive values . key words coronary arteries coronarography ct angiography multislice ct riassunto obiettivo . 
causa predominante del dolore toracico acuto , che motiva il 6 , 5% delle visite mediche urgenti effettuate nei pronto soccorso italiani , la sindrome coronarica acuta ( sca )  . 
lo studio analizza laccuratezza diagnostica della tc multistrato ( tc - ms ) a 16 canali in un gruppo selezionato di 31 pazienti con dolore toracico a medio - bassa probabilit di sca , cos definiti in base agli accertamenti preliminari ( elettrocardiogramma e markers biochimici )  . 
utilizzando come gold standard la coronarografia , eseguita nelle 24 ore successive allesame tomodensitometrico , la tc - ms ha identificato nei segmenti coronarici principali la presenza di occlusioni , stenosi significative ( > 50% ) e non ( < 50% ) con sensibilit : 65% ; specificit : 98 , 8% , vpp : 81 , 2% ; vpn : 97 , 3% , accuratezza : 96 , 4% . 
le stenosi significative e le occlusioni sono state invece accertate con sensibilit : 71 , 4% ; specificit : 99 , 6% ; vpp : 93 , 7% ; vpn : 97 , 7% ; accuratezza : 97 , 5% . 
the predominant cause is acute coronary syndrome ( acs ) , the management of which has le visite per dolore toracico acuto rappresentano circa il 6 , 5% di tutte le prestazioni effettuate nei pronto soccorso ( ps ) italiani cos come nelle emergency room statunitensi ; tale percentuale in costante aumento : nel 2000 si attestava al 5 , 1% . 
it has been calculated that in the united states 1 , 200 , 000 coronary angiography examinations are performed each year , 35% of which are followed by a revascularisation procedure . 
even though the morbidity and mortality rates of coronary catheterisation are very low ( 1% and 0.1% , respectively ) , there is a keen interest in alternative , less invasive and less expensive procedures possessing the same diagnostic accuracy in detecting or ruling out acute myocardial infarction ( ami ) and unstable angina . electrocardiogram ( ecg ) , serum cardiac markers and echocardiography can identify subjects with ami , for whom immediate reperfusion is indicated , and those with unstable angina . 
a proportion of patients presenting with chest pain are , however , considered at medium to low risk due to the absence of ischaemic st - segment abnormalities and negative serum markers , and these patients require additional investigations , such as exercise testing [ 1 ]  . 
because the advent of multidetector technology has considerably expanded the diagnostic potential of computed tomography ( ct ) , we performed this prospective study to evaluate the diagnostic accuracy of a 16 - channel ct scanner with dedicated software in a group of patients with chest pain and medium to low risk of acs . materials and methods between november 2004 and october 2005 , we recruited 31 subjects ( 19 men , 12 women ; mean age 59 years , age range 2670 years ) who presented to the emergency department with chest pain that was defined as medium to low probability of acs on the basis of first - line investigations ( ecg , and ischaemia markers : troponin , creatine phosphokinase and myoglobin ) but for whom a clinical suspicion of myocardial ischaemia persisted . 
subjects who had undergone previous myocardial revascularisation procedures or coronary angioplasty , with or without stent placement , were excluded . within 24 h of presentation , all patients ( who had given their informed consent for inclusion ) were studied by multislice ct ( msct ) and coronary angiography . 
further exclusion criteria for the ct study were heart rate > 70 bpm ( even after heart - rate - lowering drugs ) ; arrhythmia ; severe cardiac insufficiency ; pacemaker , defibrillators or valve implants ; history of adverse reactions to contrast material ; renal or respiratory insufficiency ; and pregnancy . the risk factors of the 31 patients selected for the study were hypertension ( 17 ) , cigarette smoking ( 6 ) , dyslipidaemia ( 6 ) , family history of ami ( 4 ) and diabetes ( 2 )  . 
all patients with heart rate higher than 65 bpm were treated with a heartrate - lowering agent ( metoprolol 100 mg / day ) whereas those with borderline heart rates around or just above 65 bpm received a - blocker ( metoprolol tartrate 5 mg ) intravenously 15 min before the examination . the ct study was performed with a 16 - channel multidetector scanner ( sensation cardiac , siemens ) using the following parameters : collimation 0.75 mm , voltage 120 kvp , rotation time 375 ms , slice thickness 0.75 mcardiac acacuta ( sca ) , nei cui confronti da una gestione attendista si passati ad una strategia aggressiva che prevede limmediata esecuzione di unindagine coronarografica e di uneventuale angioplastica . 
ancorch il cateterismo coronarico comporti una morbilit ed una mortalit assai ridotte ( rispettivamente nellordine dell1% e dello 0 , 1% ) permane alto linteresse per procedure alternative che assicurino non - invasivit e riduzione dei costi economici , identificando o escludendo con equivalente accuratezza diagnostica linfarto miocardico acuto ( ima ) e langina instabile . elettrocardiogramma , markers biochimici , ecocardiogramma consentono di individuare sia i soggetti con ima , nei quali generalmente indicata una terapia immediata di riperfusione , sia quelli con angina instabile . 
esiste , tuttavia , una quota di pazienti con dolore toracico definiti a mediobasso rischio , per lassenza di alterazioni ischemiche del tratto st dellelettrocardiogramma e la negativit dei markers biochimici , nei quali si rende necessaria lesecuzione di altre indagini , che attualmente consistono in test da stress [ 1 ]  . 
poich lintroduzione della tecnologia multidetettore ha comportato un incremento notevole delle potenzialit diagnostiche della tc , ci siamo proposti , in questo studio prospettico , di valutare laccuratezza diagnostica di una apparecchiatura a 16 canali e software dedicato in un gruppo di pazienti con dolore toracico a medio - basso rischio di sca . materiali e metodi nel periodo compreso tra novembre 2004 e ottobre 2005 , sono stati reclutati 31 soggetti ( 19 maschi , 12 femmine ; et media 59 anni con range : 2670 anni ) pervenuti allosservazione in ps per dolore toracico definito a medio - bassa probabilit di sca in base alle indagini di prima istanza ( tracciato elettrocardiografico e markers di ischemia : troponina , creatinfosfochinasi e mioglobina ) ma nei quali persisteva il sospetto clinico di ischemia miocardia . 
nellarco di 24 ore dalla presentazione in ps , tutti i pazienti ( dai quali era stato ottenuto il preventivo consenso informato per linserimento nel presente studio ) sono stati sottoposti a esame tc multidetettore ( tc - md ) e coronarografia . 
ai fini dellindagine tomodensitometrica altri criteri di esclusione sono stati : frequenza cardiaca ( fc ) > 70 bpm ( anche dopo bradicardizzazione farmacologia ) ; aritmia ; insufficienza cardiaca grave ; presenza di pacemaker , defibrillatori o protesi valvolari ; pregresse reazioni avverse al mezzo di contrasto ; insufficienza renale o respiratoria ; stato di gravidanza . nel gruppo selezionato di 31 soggetti i fattori di rischio 1055 l . 
coronary angiography images were obtained with volume rendering ( vr ) and curvilinear multiplanar reconstruction ( mpr ) three - dimensional ( 3d ) techniques . in the comparison between msct and coronary angiography , the latter being considered the gold standard , we evaluated 16 coronary segments according to the american heart association classification [ 3 ]  . 
two operators , one radiologist and one cardiologist , independently evaluated each segment for assessability and image quality ( excellent , fair and poor ) and the presence of occlusions and stenoses , which they ranked as nonsignificant ( 50% ) or significant ( 51%99% ) based on a subjective visual judgement for msct and a quantitative judgement for coronary angiography [ 4 ]  . results at msct , 383 ( 81.7% ) of the 469 segments visualised by coronary angiography were considered assessable ; the intermediate branch was present in four subjects only ( table 1 )  . if only the main segments were considered right coronary erano ipertensione ( 17 ) , fumo di sigaretta ( 6 ) , dislipidemia ( 6 ) , familiarit per ima ( 4 ) e diabete ( 2 )  . 
per coloro invece che presentavano una frequenza borderline e cio intorno o di poco superiore ai 65 bpm si optato per la somministrazione endovena di un - bloccante ( metoprololo tartrato 5 mg ) 15 minuti prima dellesecuzione dellesame . lindagine tc stata effettuata con una apparecchiatura multidetettore a 16 canali ( sensation cardiac , siemens ) , utilizzando i seguenti parametri : collimazione 0 , 75 mm , voltaggio 120 kvp , tempo di rotazione 375 ms , spessore di strato 0 , 75 mlattivit cardiaca stata costantemente monitorata durante lindagine mediante tracciato elettrocardiografico in singola derivazione . per liniezione di mezzo di contrasto ( mdc ) organo - iodato non ionico ( concentrazione 350400 mgi / ml ) si utilizzato un iniettore automatico collegato ad unagocannula da 1820 gauge inserita in una vena antecubitale del braccio . utilizzando la tecnica del bolus tracking con roi nellaorta ascendente sono stati iniettati 100 ml di mdc , seguiti da 40 ml di soluzione fisiologica , ad una velocit di infusione di 4 ml / s . la ricostruzione delle immagini stata effettuata mediante gating cardiaco retrospettivo , individuando la finestra temporale ottimale per ogni ramo coronarico ( solitamente 400 , 350 o 300 ms prima del successivo complesso qrs elettrocardiografico ) [ 2 ]  . 
sono state ricostruite sezioni assiali dello spessore effettivo di 0 , 75 mm con overlapping di 0 , 5 mle immagini coronarografiche sono state ottenute con tecnica 3d volume rendering ( vrt ) e multiplanare ( mpr ) curvilinea . ai fini del confronto tra tcmd e coronarografia , assunta quale gold standard , sono stati considerati 16 segmenti coronarici secondo la classificazione dellamerican table 1 number and percentage of coronary artery segments considered assessable on computed tomography ( ct ) in 31 patients tabella 1 numero e percentuale dei segmenti coronarici giudicati valutabili alla tc in 31 pazienti segments assessable , n ( % ) segmenti variabili , n ( % ) 1 . 
di ogni segmento due operatori , un radiologo e un cardiologo , hanno considerato in maniera indipendente la valutabilit e la qualit iconografica ( eccellente , discreta e scadente ) nonch la presenza di occlusioni o di stenosi classificate in non significative ( 50% ) e significative ( 51%99% ) in base ad un giudizio visuale soggettivo per la tc - md e quantitativo per la coronarografia [ 4 ]  . risultati alla tc - md sono stati giudicati valutabili 383 ( 81 , 7% ) dei 469 segmenti visualizzati alla coronarografia , essendo il ramo intermedio presente solo in 4 soggetti ( tabella 1 )  . 
considerando esclusivamente i segmenti principali e cio arteria coronaria destra ( segmenti 1 , 2 , 3 ) , tronco comune ( segmento 5 ) , arteria discendente anteriore ( segmenti 6 , 7 , 8 ) , arteria circonflessa ( segmenti 11 , 13 ) e , laddove presente , ramo intermedio ( segmento 16 ) , i segmenti considerati valutabili sono stati 277 di 283 ( 97 , 9% ) : in 4 / 31 ( 12 , 9% ) pazienti si avuto un segmento non valutabile , pi frequentemente il 13 ; in 1 paziente ( 3% ) i segmenti non esaminabili sono stati due . 
la tabella 3 mostra i risultati del confronto tc - coronarografia limitatatotal total tc , n positiva negativa totale , n tc , n positive negative totale , n fig . 
1a - c a 40 - year - old man , a heavy smoker with a family history of coronary disease , admitted to the emergency room with precordial chest pain ; sinusal rhythm with 60 bpm at electrocardiogram ( ecg ) , myocardial ischaemia markers negative , no regional wall motion abnormalities at echocardiography . 
computed tomography ( ct ) ( a , b ) shows about 50% tubular stenosis with focal , severe narrowing of the lumen of the anterior descending artery ( ada ) , segment 1 ( arrowheads )  . 
alla coronaro - tc ( a , b ) : stenosi tubulare di circa il 50% , cui segue focale serrato restringimento del lume dellarteria discendente anteriore ( ada ) prossimale , segmento 1 ( punta di freccia )  . 
2a - c a 70 - year - old woman with hypertension admitted to the emergency room with precordial chest pain radiating to the neck ; sinusal rhythm with 63 bpm and minimal st depression at electrocardiogram ( ecg ) , absence of serum cardiac markers , normal ventricular wall motion at echocardiography . 
paziente maschio , et 56 anni , diabete mellito nid , iperteso , con scintigrafia miocardica da sforzo ( eseguita 3 mesi prima ) dubbia per coronaropatia ; pervenuto in ps con dolore toracico retrosternale . 
4a , b a 70 - year - old man with hypertension and hypercholesterolaemia admitted to the emergency room with precordial chest pain ; sinusal rhythm with 58 bpm , absence of ischaemic signs at electrocardiogram ( ecg ) ; absence of serum cardiac markers . 
computed tomography ( ct ) ( a ) shows eccentric calcific plaque of the middle circumflex artery ( segment 13 ) ( arrow )  . the vessel lumen is not well assessable , but a > 50% stenosis ( arrow ) is suggested . 
alla tc ( a ) : placca eccentrica calcifica dellarteria circonflessa , tratto medio , segmento 13 ( freccia ) ; il lume non ben valutabile ma si ipotizza una stenosi > 50% . 
alla coronarografia ( b ) : stenosi della lunghezza di circa 15 mm e del 90% circa nel punto critico ( freccia )  . particular , it reports the number of subjects with at least one occlusion or stenosis > 50% identified by ct with respect to the number of those identified by coronary angiography . 
inoltre , la coronarografia documenta come in 3 di 6 segmenti ( 50% ) vi sia una riopacizzazione da circolo collaterale del tratto distale alla stenosi . la tabella 5 presenta , infine , unanalisi della casistica che considera il singolo paziente piuttosto che i segmenti coronarici ; in particolare , riporta il numero dei soggetti con almeno una occlusione o una stenosi > 50% identificati alla tc rispetto a quanti individuati dalla coronarografia . 
nei pazienti ( 16 / 31 ) , nei quali la qualit dimmagine risultata eccellente per tutti i segmenti , i valori di sensibilit , specificit , valore predittivo positivo e negativo , accuratezza sono pari al 100% . discussione lesame dei pazienti che si presentano in ps per dolore toracico acuto richiede tempo e risorse economiche finalizzati allaccertamento di una sca che risulta , al termine di tutte le indagini , inesistente in una percentuale tuttaltro che trascurabile di casi . 
la valutazione clinica ( intensit , irradiazione , durata ) del dolore toracico , daltra parte , facilmente soggetta ad errori interpretativi e insufficiente per la diagnosi ; ne consegue che lapproccio tradizionale a tali pazienti preveda lelettrocardiogramma e il dosaggio dei markers biochimici . 
lalterazione di questi ultimi unitamente allelevazione elettrocardiografica del tratto st corrobora il sospetto clinico di infarto miocardico , che comunque diagnosticabile anche in assenza di precoci alterazioni elettrocardiografiche qualora vi sia innalzamento di troponina , creatinfosfochinasi e mioglobina . 
quando anche tali markers siano negativi , i pazienti definiti a medio - basso rischio di sca debbono essere avviati a test da stress elettrocardiografici , ecocardiografici o scintigrafici per evidenziare la presenza o lassenza di malattia coronarica significativa [ 5 , 6 ]  . 
5a - c a 58 - year - old woman with hypertension and a previous history of smoking admitted to the emergency room with precordial chest pain and dyspnoea ; absence of ischemic alterations at electrocardiogram ( ecg ) , absence of serum cardiac markers . 
although poor quality of computed tomography ( ct ) image ( a ) should have induced the reader to avoid any judgement , a severe stenosis ( arrow ) of the right coronary artery ( segment 2 ) is suggested . 
si ipotizza comunque lesistenza di una stenosi critica da placca calcifica del segmento 2 , sottovalutando erroneamente lipotesi di una occlusione con riopacizzazione a valle da parte dellemisistema coronarico sinistro , come invece dimostrato dalla coronarografia ( punte di freccia ) ( b , c )  . partment with acute chest pain in order to identify an acs that very often turns out to be nonexistent . 
the clinical assessment ( intensity , radiation , duration ) of chest pain is , on the other hand , often prone to misinterpretation and is inadequate for the diagnosis , with the result that the conventional approach to these patients includes an ecg and serum cardiac markers . abnormal serum cardiac markers associated with st elevation supports the clinical suspicion of myocardial infarction , which can , however , be diagnosed even without early electrocardiographic alterations if the troponin , creatine phosphokinase and myoglobin levels are high . 
where these markers are negative , patients considered at medium to low risk of acs must proceed to electrocardiographic , echocardiographic or nuclear stress testing to determine the presence or absence of significant coronary disease [ 5 , 6 ]  . 
the diagnostic reference standard for the assessment of vascular anatomy and the detection of stenosing lesions is coronary angiography , which , in addition to a diagnostic phase , allows immediate myocardial reperfusion by means of angioplasty . 
the invasiveness and high cost of this examination have , however , promoted research into alternative , noninvasive diagnostic techniques . the technological evolution of spiral ct has made available multislice scanners with 4 , 8 , 16 and , recently , 64 detector rows that offer a temporal and spatial resolution that enable analysis of the coronary arteries with a high level of anatomical detail . 
linvasivit e lelevato costo di tale indagine hanno indirizzato tuttavia gli sforzi della ricerca verso tecniche diagnostiche alternative non invasive . levoluzione tecnologica della tc spirale ha reso disponibili apparecchiature multistrato a 4 , 8 , 16 e , recentemente , 64 file di detettori caratterizzate da risoluzione spaziale e temporale tali da consentire lanalisi delle coronarie con elevato dettaglio anatomico . 
in letteratura , dallanno 2000 ad oggi , sono stati pubblicati diversi studi sullidentificazione di placche coronariche mediante tc - md a 4 o 16 canali [ 711 ]  . 
nella quasi totalit dei lavori sono state prese in considerazione le stenosi che comportano riduzione > 50% o occlusione del lume coronarico ; attualmente , con lutilizzo di scanner a 64 slice , si iniziano a valutare anche stenosi < 50% [ 1214 ]  . 
peraltro , in questa classe di pazienti che presentano angina instabile , due o pi fattori di rischio , incremento della troponina sierica , slivellamento st > 1 mm , aritmie o instabilit emodinamica , le pi recenti linee - guida cardiologiche suggeriscono lesecuzione in prima istanza dellangiografia coronarica e della rivascolarizzazione , se indicata [ 16 , 17 ]  . la presente casistica considera esclusivamente soggetti giunti allosservazione in ps con un dolore toracico acuto 1061 l . 
in this class of patients with unstable angina , two or more risk factors , increased serum troponin , st deviation > 1 mm , arrhythmias or haemodynamic instability , the most recent cardiological guidelines recommend coronary angiography and revascularisation , if indicated , as the first - line approach [ 16 , 17 ]  . our study included only subjects presenting to the emergency department for acute chest pain and at medium to low risk for acs associated with one , two or more risk factors for coronary disease ( age , smoking , dyslipidaemia , arterial hypertension , diabetes mellitus ) but with ischaemia markers and ecg not indicative of q and non - q ami . 
in the 31 patients enrolled , all of the 15 ( or 16 if the intermediate branch was present ) segments were analysed ; subsequently , the main coronary branches only were considered , that is , the right coronary artery ( segments 1 , 2 , 3 ) , left main branch ( segment 5 ) , anterior descending or interventricular artery ( segments 6 , 7 , 8 ) , circumflex artery ( segments 11 and 13 ) and , where present , the intermediate branch ( segment 16 )  . 
 compared with the interesting statistical results of previous reports , the sensitivity of msct ( 71.4% for occlusions and stenoses > 50% ) identified in our study is among the lowest reported in the literature [ 18 , 19 , 2125 ] and higher only than the value reported by hoffmann et al . 
the false negatives are due to underestimation of the degree of stenosis in poor - quality images or to misinterpretation in cases of revascularisation of the branch below the stenosis due to collateral circulation ; in particular , the latter situation led to three stenoses being interpreted as significant rather than as occlusions . 
msct therefore takes on an important role in the diagnostic workup of this class of cardiological patients , as it guides towards coronary angiography more accurately than does exercise stress testing which , though definitely less expensive , has decidedly lower predictive values [ 2628 ]  . 
si sono esclusi i pazienti portatori di bypass aorto - coronarico o pace - maker e i soggetti sottoposti in passato ad interventi di angioplastica , con o senza posizionamento di stents coronarici . 
nei 31 pazienti arruolati , sono stati analizzati tutti i 15 ( o 16 in presenza di ramo intermedio ) segmenti ; successivamente sono stati presi in considerazione solo i rami coronarici principali , ovverosia arteria coronaria destra ( segmenti 1 , 2 , 3 ) , tronco comune ( segmento 5 ) , arteria discendente anteriore o interventricolare ( segmenti 6 , 7 , 8 ) , arteria circonflessa ( segmenti 11 e 13 ) e , qualora presente , il tronco intermedio ( segmento 16 )  . 
il dato in linea con quanto emerge da altri studi effettuati con tc - md a 12 e 16 canali [ 1820 ]  . rispetto a precedenti interessanti risultati statistici , nel presente studio emerge una sensibilit della tc - md ( 71 , 4% per le occlusioni e le stenosi > 50% ) che si posiziona ai limiti inferiori del range dei valori riportati in letteratura [ 18 , 19 , 2125 ] , risultando superiore soltanto a quella ( 63% ) di hoffmann et al . 
essi sono dovuti a sottostima dellentit della stenosi in immagini di qualit scadente o ancora ad errore interpretativo in casi di riabitazione del ramo a valle della stenosi per fenomeni di collateralit : in particolare , questultima evenienza responsabile di tre stenosi interpretate come significative e non , in realt , come occlusioni . 
per quanto concerne invece la specificit per le occlusioni e le stenosi > 50% , il nostro dato ( 99 , 6% ) appare incoraggiante essendo il range delle percentuali fino ad oggi riportate oscillante fra l86% ed il 98% . i valori predittivi negativo ( 97 , 7% ) e positivo ( 93 , 7% ) , superiori a quanto mediamente riportato in letteratura [ 18 , 19 , 2125 ] , implicano che un paziente , pervenuto in ps per dolore toracico con media - bassa probabilit di sca e tc - md negativa per stenosi significativa , abbia solo il 2 , 3% di probabilit di avere in realt una coronaropatia significativa ; allopposto , un soggetto con stenosi giudicata emodinamicamente significativa alla tc ha una probabilit superiore al 93% di conferma coronarografica . 
in questa classe cardiologica di pazienti la tc - md assume quindi un ruolo rilevante nellalgoritmo diagnostico indirizzando verso un approfondimento coronarografico pi correttamente di quanto non possa un test da sforzo , sicuramente meno costoso , ma caratterizzato da valori predittivi decisamente inferiori [ 2628 ]  . 
inoltre se , meno dettagliatamente ma in maniera clinicamente pi congrua , non si considerino i rami coronarici principali bens i singoli pazienti si ottiene una predittivit positiva del 100% . 
of the 467 patients examined by cdus , 159 ( 34% ) showed no signs of renal artery stenosis ( ras ) or nephropathy and were therefore started on medical therapy . 
nel periodo 20002004 sono giunti alla nostra osservazione 467 pazienti ( 205 donne e 262 uomini , range 2096 anni ) , per essere sottoposti ad indagine ecd , in quanto presentavano ipertensione arteriosa resistente alla terapia e / o segni di insufficienza renale . 
dei 467 pazienti esaminati mediante ecd , 159 ( 34% ) non presentavano indici di stenosi delle arterie renali n segni ecografici di nefropatia , ed hanno proseguito un iter terapeutico medico . 
dai risultati ottenuti e alla luce dei dati della letteratura , si riconferma il ruolo fondamentale svolto dallecd nei pazienti con sospetta o accertata patologia nefro - vascolare , fornendo dati importanti sullemodinamica renale di insostituibile utilit per un corretto approccio clinico - terapeutico . parole chiave eco color doppler ipertensione nefrovascolare arterie renali introduction introduzione atherosclerotic renal artery stenosis ( ras ) is associated with two common clinical syndromes , renovascular hypertension and ischaemic nephropathy , which imply a significant worsening of renal function in the presence of stenosis [ 13 ]  . the exact prevalence of ras is unknown , but many data indicate that it is high among elderly , chiefly caucasian , subjects and those with diabetes , diffuse atherosclerosis and rela stenosi aterosclerotica dellarteria renale ( sar ) si associa a due comuni sindromi cliniche : lipertensione nefro - vascolare e la nefropatia ischemica , intendendo per questultima una significativa riduzione della funzione renale in presenza della stenosi [ 13 ]  . lesatta prevalenza della stenosi aterosclerotica dellarteria renale non nota , ma molti dati fanno ritene1115 g.c. 
renal angiography and renal colour - doppler ultrasound ( cdus ) studies performed during coronary angiography or peripheral arteriography have demonstrated that ras is present in 20%30% of more than 10 , 000 investigations [ 1 ]  . 
a large number of patients with serum creatinine higher than 1.7 mg / dl have been found to have ras in prospective and retrospective studies , and ras has been demonstrated in 5%10% of patients with acute renal failure [ 1 ]  . 
this prevalence becomes higher when other clinical signs of atherosclerosis coexist , with rates of 12%24% in patients undergoing coronary angiography , 11% in stroke patients and 40% in patients with peripheral vascular disease [ 3 ]  . 
few studies have assessed atherosclerotic ras in patients with abdominal aorta aneurysm ( aaa ) , another manifestation of atherosclerotic disease . the high prevalence of atherosclerotic ras has raised the need to identify a noninvasive , inexpensive modality with high diagnostic accuracy in order to reduce the use of invasive investigations , such as arteriography , which nonetheless remains the gold standard in the diagnosis of ras [ 2 , 4 , 5 ]  . diagnostic imaging offers several noninvasive methods for selecting patients for angiography : cdus , captopril renal scintigraphy , spiral computed tomography ( ct ) angiography , and magnetic resonance ( mr ) angiography [ 2 , 3 , 511 ]  . cdus of the renal arteries is currently the main screening tool for ras , and many international studies have demonstrated a sensitivity of 83%96% , a specificity of 76%98% , a positive predictive value ( ppv ) of 81%92% and a negative predictive value ( npv ) of 94%98% [ 58 , 1115 ]  . 
the advantages of cdus are that it is inexpensive , reproducible , noninvasive and has good npv when performed in selected populations ; its known disadvantages , on the other hand , are operator dependence , patient preparation and cooperation , scars , obesity , bowel gas and vascular abnormalities , in particular , the presence of anatomical variants of the renal artery [ 2 , 4 , 6 , 1517 ]  . the primary aim of our study was to evaluate the role and usefulness of cdus in analysing overall renal morphology and haemodynamics in patients with hypertension and nephropathy ; the secondary aim was to assess the association between atherosclerotic ras and aaa . materials and methods between 2000 and 2004 , 467 patients ( 205 women and 262 men , age range 2096 years ) with refractory hypertension and / or signs of renal failure ( serum creatinine > 2.5 mg / dl ) were referred to our department for renal cdus . the patients were examined by a single radiologist on a philips ssd 800 and hitachi h21 scanner using a 3.5 - mhz convex - array transducer and colourand power - doppler mode . 
sonograms were assessed for renal morphology , overall renal blood flow and abdominal aorta . 1116 re che essa sia assai frequente nei soggetti anziani , specie di razza caucasica , diabetici , con patologia aterosclerotica polidistrettuale e con insufficienza renale . 
studi angiografici renali e di eco color doppler ( ecd ) renale , eseguiti in corso di coronarografia o di arteriografia periferica , hanno documentato una frequenza di sar nel 20%30% di oltre 10000 indagini [ 1 ]  . 
analisi retrospettive o prospettiche in pazienti con creatininemia superiore a 1 , 7 mg / dl dimostrano presenza di sar in molti casi , cos come in casi di insufficienza renale acuta si ha la sua dimostrazione nel 5%10% dei casi [ 1 ]  . 
poich la stenosi aterosclerotica dellarteria renale pu indurre un danno renale progressivo , in alcune casistiche essa causa di ingresso in dialisi nel 15%30% dei pazienti uremici [ 1 ]  . 
in pazienti anziani non selezionati stata dimostrata mediante ecd una prevalenza del 6 , 6% ; la prevalenza maggiore quando coesistono altre manifestazioni cliniche dellaterosclerosi : nei pazienti sottoposti a coronarografia la prevalenza varia dal 12% al 24% , nei pazienti con ictus dell11% , nei pazienti con malattia vascolare periferica intorno al 40% [ 3 ]  . 
esistono pochi dati che valutino levidenza della sar nei pazienti portatori di aneurisma dellaorta addominale , che rappresenta altra manifestazione di patologia aterosclerotica . lelevata prevalenza di tale patologia ha sollevato la necessit di individuare una metodica non invasiva , a basso costo e con elevata accuratezza diagnostica al fine di limitare il ricorso ad unindagine strumentale invasiva , quale larteriografia che rimane , comunque , la metodica gold standard nella diagnosi della stenosi delle arterie renali [ 2 , 4 , 5 ]  . 
limaging attualmente ha a disposizione alcune metodiche non invasive , come indagini di screening e selezione per la successiva angiografia : lecd , la scintigrafia renale con captopril , langio - tc spirale , langiorm [ 2 , 3 , 511 ]  . 
attualmente lecd delle arterie renali risulta la principale metodica di screening nella diagnosi di stenosi delle arterie renali con numerosi lavori in ambito internazionale che attribuiscono a tale metodica valori di sensibilit pari all83%96% , di specificit 76%98% , valore predittivo positivo 81%92% e valore predittivo negativo 94%98% [ 58 , 1115 ]  . 
in tutti i pazienti , per una pi ampia e corretta valutazione vascolare , stata esaminata laorta addominale , valutandone calibro , morfologia e flusso ( diametro normale < 2 , 5 cm , ectasia 2 , 53 cm , aneurisma > 3 cm )  . risultati dei 467 pazienti esaminati mediante ecd , 159 ( 34% ) non presentavano indici di stenosi delle arterie renali n segni ecografici di nefropatia , ed hanno proseguito un iter terapeutico medico ; in particolare uno di questi pazienti era portatore di protesi aorto - renale pervia . 
i restanti 308 pazienti ( 66% ) mostravano invece segni ecografici di stenosi emodinamicamente significativa o di nefropatia . in particolare , 174 pazienti ( 37 , 2% ) mostravano solo segni di nefropatia , con riduzione del flusso ematico in sede ilare ed ostiale ed un aumento dell ir ; in 123 pazienti ( 26 , 3% ) era presente nefropatia severa con ir 0 , 80 e tra questi un paziente presentava aneurisma dellarteria renale ed un paziente necrosi corticale acuta , confermati successivamente con esame tc ; 51 ( 10 , 9% ) invece presentavano nefropatia moderata con ir di 0 , 75 . in 134 pazienti ( 28 , 7% ) veniva evidenziata una sar emodinamicamente significativa ; 58 dei 134 pazienti ( 12 , 4% ) con sar presentavano segni di nefropatia associata ; in 2 di questi era gia stato posizionato stent , 2 hanno eseguito an1117 fig . 
to obtain a more exhaustive and correct vascular assessment , the calibre , morphology and flow pattern of the abdominal aorta were also evaluated in all patients ( normal diameter < 2.5 cm , ectasia 2.53 cm , aneurysm > 3 cm )  . results of the 467 patients examined with cdus , 159 ( 34% ) had no indices of ras or sonographic signs of nephropathy and were started on medical therapy ; in particular , one of them had a patent aortorenal stent . 
the remaining 308 patients ( 66% ) had sonographic evidence of haemodynamically significant stenosis or nephropathy . in detail , 174 patients ( 37.2% ) showed signs of nephropathy only , with reduced hilar and ostial flow and increased ri . nephropathy was severe in 123 patients ( 26.3% ) , with ri 0.80 ; among them , one had renal artery aneurysm and one had acute cortical necrosis , later confirmed by ct . 
altri 22 pazienti non hanno eseguito angiografia ; in particolare , 1 aveva gi posizionato stent renale , 1 presentava iperplasia mio - intimale , 10 pazienti avevano gi una completa occlusione dellarteria renale , mentre 10 pazienti sono al momento in attesa di eseguire angiografia ed eventuale intervento di rivascolarizzazione . 
in totale , quindi , 33 pazienti , con sar e senza segni di nefropatia ( 7% del totale e 44% dei pazienti con segni ecd di stenosi ) non hanno eseguito esame angiografico . quarantre pazienti ( 9 , 2% del totale e 56% dei positivi per stenosi allecd ) hanno invece eseguito angiografia che ha confermato la diagnosi in 39 dei 43 pazienti . 
di questi , 24 ( 5 , 1% del totale e 55 , 8% dei positivi per stenosi ) hanno posizionato stent con ripristino ottimale della vascolarizzazione renale nel 92% dei casi ( solo in 2 casi erano presenti dopo la procedura interventistica segni clinico - laboratoristici e bioptici di ateroembolismo colesterinico , con quadro di severa compromissione dellemodinamica renale allecd in entrambi i casi )  . diciannove pazienti ( 4% del totale e 44 , 2% dei positivi per stenosi ) invece non hanno posizionato stent : di questo ultimo gruppo 7 avevano gi posizionato stent ( 2 completamente chiusi e 5 restenosi ) , in 3 sussistevano problemi anatomici con biforcazione precoce dellarteria renale , 1 paziente presentava middle aortic syndrome , in 1 caso si era verificata complicanza ( dissezione in corso di procedura ) , in 1 un caso langiografista non riusciva a superare la stenosi , 1 paziente presentava dissezione aortica , in 1 caso si trattava di stenosi lieve - moderata e 2 erano falsi positivi . in particolare , lecd ha permesso di svelare in una giovane paziente una grave situazione anatomica , verosimilmente su base congenita , definita come middle aortic syndrome o coartazione aortica sottodiaframmatica . 
dopo una gestosi , nella paziente era insorta una severa ipertensione arteriosa e , dopo unindagine ecografia risultata negativa per alterazioni morfologiche renali , era stata sottoposta ad indagine ecd che rilevava marcato decremento degli ir arteriosi intraparenchimali ( ir = 0 , 45 ) con polso parvus et tardus in sede ilare e segni di stenosi emodinamicamente significative di entrambe le arterie renali . 
this was associated with signs of nephropathy in 58 patients ( 12.4% ) ; two had already had a stent implanted , two underwent angiography and one had polycystic kidneys , whereas the remaining 56 were excluded from angiography because of signs of severe nephropathy ( longitudinal diameter < 8 cm , ri > 0.80 and poor parenchymal perfusion on cd and pd imaging )  . 
a further 22 patients did not undergo angiography , as one had a renal stent , one had myointimal hyperplasia , ten had complete occlusion of the renal artery , whereas ten were on the waiting list for angiography and possible revascularisation at the time of this writing . 
therefore , a total of 33 patients with ras and without evidence of nephropathy ( 7% of the total and 44% of patients with cd signs of stenosis ) did not undergo angiography . forty - three patients ( 9.2% of the total and 56% of those with ras on cdus ) underwent angiography , which confirmed the diagnosis in 39 . 
of these , 24 ( 5.1% of the total and 55.8% of those with stenosis ) underwent renal stenting , with optimally restored renal perfusion in 92% of cases ( only two had clinical , laboratory and biopsy evidence of cholesterol atheroembolism , with severely impaired renal haemodynamics on cdus , after the interventional procedure )  . nineteen patients ( 4% of the total and 44.2% of those with stenosis ) did not undergo stent implantation : seven of these already had stents ( two of which completely occluded and five with restenosis ) , three patients presented early bi1118 g.c. 
b indagine tc con mdc : assenza di opacizzazione in sede corticale renale bilaterale ( c )  . furcation of the renal artery , one patient had middle aortic syndrome , one developed a complication ( dissection during the procedure ) , in one , the operator could not negotiate the stenosis , one had aortic dissection , one had mild to moderate stenosis and two were false positives . 
following a gestosis , the patient developed severe arterial hypertension and , after a negative sonography for morphological renal alterations , was studied by cdus , which revealed marked reduction of intraparenchymal arterial ri ( ri = 0.45 ) with parvus - tardus pulse in the hilum and signs of haemodynamically significant stenoses of both renal arteries . 
il 56% di questi presentava una marcata riduzione del flusso a livello delle arterie renali e nel 30% erano presenti stenosi emodinamicamente significative delle arterie renali ed in un caso ( 6% ) unarteria renale era occlusa . per quanto riguarda la presenza di aneurisma dellaorta addominale ( aaa ) operato e non , i dati raccolti sono i seguenti : laaa era presente in 19 pazienti dei 333 senza segni di sar ( 5 , 7% ) con netta prevalenza nei pazienti con nefropatia ( 16 casi ) rispetto ai pazienti senza nefropatia ( 3 casi ) mentre laaa era presente in 15 dei 134 pazienti con segni 1119 g.c. 
this translates into a marked clinical benefit for the patient as well as considerable economic savings , given that out of the 467 patients studied by cdus , 88% did not undergo angiography as against 22% who did undergo angiography . 
if we exclude patients who underwent angiography before cdus ( cdus follow - up ) and those with a previously implanted stent , the remaining 65% underwent stent revascularisation , with excellent outcome in all patients except two , one of whom develop cholesterol atheroembolism after the procedure . in some cases , cdus revealed severe impairment of renal circulation in patients with poor renal compensation who showed only minor laboratory alterations ( serum creatinine 1.62.5 mg / dl )  . 
in addition to being valuable for analysing renal morphology and volume and assessing blood flow on cdus and pdus , evaluation of the ri proved to be reliable for determining renal function 1120 di sar ( 11 , 2% ) ; la presenza di aaa operato stata di 5 pazienti tra i 333 senza segni di sar ( 1 , 5% ) ed in particolare pi frequente tra i pazienti nefropatici ( 4 casi ) che nei non nefropatici ( 1 caso ) mentre la presenza di aaa operato nei pazienti con segni di sar stata di 10 pazienti sui 134 ( 7 , 46% )  . discussione dai risultati ottenuti si riconferma lutilit dellecd nellesaminare i pazienti con ipertensione e nefropatia ; lo studio dellemodinamica renale con ecd si rilevata in alcuni casi indagine insostituibile per un corretto approccio clinico - terapeutico [ 2 , 5 , 7 , 9 , 15 ]  . lecd ha evitato langiografia nei pazienti negativi e in quelli con stenosi occlusiva dellarteria renale e con segni di grave nefropatia , nei quali lintervento avrebbe potuto aggravare il deficit renale ( tossicit del mdc , ateroembolismo colesterinico , ecc . ) con indubbio vantaggio clinico per il paziente e risparmio economico . 
infatti del totale dei 467 pazienti esaminati allecd l88% non stato sottoposto ad esame angiografico e solo il 22% ha eseguito angiografia . escludendo i pazienti che hanno eseguito lindagine angiografica prima dellecd ( monitoraggio ecd ) e i pazienti con pregresso posizionamento di stent , i restanti 65% sono stati sottoposti ad intervento di rivascolarizzazione con stent con ottimi risultati in tutti i pazienti ( ad eccezione di 2 casi , uno dei quali presentava ateroembolismo colesterinico dopo la procedura )  . lindagine ecd ha rilevato in diversi casi una compromissione del circolo renale di diversa entit in pazienti con labile compenso renale che presentavano modeste alterazioni laboratoristiche ( creatininemia 1 , 62 , 5 mg / dl ) ; in questi casi il corretto approccio terapeutico ha sicuramente ritardato lesaurimento della funzionalit renale ed il ricorso al trattamento dialitico con il miglioramento della qualit della vita  . 
si tratta di una sindrome non comune in letteratura , descritta in giovane et ( 628 anni ) come variet rara di coartazione aortica ( 0 , 2%2% delle coartazioni aortiche ) , associata ad aneurisma dellaorta addominale e a stenosi della arteria renale . 
rarely reported in the literature , the syndrome has been described in young subjects ( 628 years old ) as a rare variant of aortic coarctation ( 0.2%2% of aortic coarctations ) and to be associated with aaa and ras . 
multiple causes have been recognised , including congenital and secondary to takayasus syndrome , von recklinghausens neurofibromatosis , coeliac disease and autoimmune disorders ( dermatitis herpetiformis , insulindependent diabetes ) , and clinical onset is usually renovascular hypertension and claudication [ 2224 ]  . in 16 patients with a clinical and laboratory diagnosis of cholesterol atheroembolism , the results of cdus provided a good indication of the severity of their clinical condition : in 12 patients , the condition arose after interventional procedures ( five after angiography , four after coronary angiography , one after coronary artery stenting , one after subclavian artery stenting and one after left renal artery stenting ) and in four patients following administration of coumadall patients had signs of severely impaired parenchymal flow due to cholesterol emboli clogging the small arteries and causing inflammatory reactions , intimal proliferation , vessel fibrosis and luminal obliteration . 
despite being nonspecific findings on cdus , these alterations translate into increased resistive index in the intrarenal arteries and reduced parenchymal and hilar flow in the renal arteries . in nine cases , cdus diagnosed renal artery occlusion in patients with modest chronic renal failure whose renal function had rapidly declined ; thus , it avoided the need for further investigations and enabled selection of patients for medical therapy . 
the study of the renal artery should not be limited to the ostial region ( where the majority of stenoses are present ) but should include an overall assessment of renal arterial circulation to detect possible alterations and evaluate the feasibility of an appropriate revascularisation procedure . 
patients with severe impairment of renal haemodynamics who were unlikely to benefit from revascularisation were therefore excluded from angiography [ 17 , 25 ]  . our experience highlights the association between aaa and ras , an important factor for vascular surgeons and angiographers considering endovascular treatment for aortic aneurysms and for selecting the most appropriate strategy when the two diseases coexist [ 2630 ]  . this study reconfirms the crucial role of cdus in monitoring patients who have undergone stent revascularisation procedures [ 25 ]  . 
in addition , it raises the problem of contrast medium toxicity in patients with poor renal function . cdus consistently revealed haemodynamically significant stenosis of the stent site with parvus - tardus pulse below the stenosis , in most cases due to the presence of fibrointimal celiaco e malattie autoimmuni ( dermatite erpetiforme , diabete insulino dipendente ) ; spesso ad esordio clinico con ipertensione nefrovascolare e claudicatio [ 2224 ]  . in 16 pazienti con diagnosi clinico - laboratoristica di ateroembolismo colesterinico , lecd ha fornito risultati che ben rispecchiano la gravit del quadro clinico : in 12 pazienti il quadro insorto dopo manovre interventistiche ( 5 dopo angiografia , 4 dopo coronarografia , 1 dopo posizionamento di stent in arteria carotide , 1 in arteria succlavia e 1 in arteria renale sinistra ) e in 4 pazienti dopo somministrazione di coumadin tutti i pazienti sono stati rilevati segni di grave compromissione del circolo parenchimale dovuto agli emboli di colesterolo il cui impianto nelle piccole arterie seguito da una reazione infiammatoria , proliferazione dellintima , fibrosi dei vasi colpiti e obliterazione del lume vasale . 
tali alterazioni , pur essendo aspecifiche allecd , si traducono con lincremento dellindice di resistenza arterioso intrarenale e con riduzione del flusso parenchimale e ilare nelle arterie renali . in 9 casi lecd ha diagnosticato locclusione di unarteria renale , in pazienti con modesta insufficienza renale cronica che avevano presentato rapido aggravamento della funzionalit renale evitando in questi ulteriori indagini e sottoponendoli alla sola terapia medica . 
lo studio dellarteria renale non deve esaurirsi alla sede ostiale ( ove presente la maggior parte delle stenosi ) ma deve valutare globalmente il circolo arterioso renale per evidenziare eventuali alterazioni e per valutare la possibilit di proporre al paziente un corretto intervento di rivascolarizzazione . 
sono pertanto stati esclusi dallangiografia i pazienti che presentavano severa compromissione dellemodinamica renale , che probabilmente non avrebbero tratto beneficio dal trattamento di rivascolarizzazione [ 17 , 25 ]  . la nostra esperienza mette in risalto lassociazione fra la patologia aneurismatica dellaorta addominale e la stenosi dellarteria renale , dato sicuramente molto importante per il chirurgo vascolare e per langiografista , alla luce del trattamento endovascolare degli aneurismi aortici e sulla scelta strategica pi opportuna in presenza contemporanea delle due patologie [ 2630 ]  . in questo studio viene riaffermato linsostituibile ruolo svolto dallecd nel monitoraggio dei pazienti che hanno subito interventi di rivascolarizzazione con stent [ 25 ]  . 
in questi pazienti lindagine angio - tc ha fornito , nella nostra esperienza , informazioni sul corretto posizionamento dello stent ma risultato difficile lo studio della perviet dello stent stesso , senza tener conto della tossicit del mdc in questi pazienti dalla gi labile funzionalit renale . lesame ecd ha rilevato stenosi emodinamicamente significative in corrispondenza dello stent con presenza di polso parvus et tardus nel tratto a valle la stenosi in tutti i pazienti , nella maggior parte dovuto alla presenza di iperplasia fibrointimale o a placche aterosclerotiche site prossimalmente o distalmente rispetto allo stent . 
cdus identified patients with restenosis ( seven cases ) , all of which were confirmed by angiography and subsequently treated by repeat revascularisation ( placement with another co - axial stent or with the use of a cutting balloon )  . conclusioni di rivascolarizzazione ( posizionando un altro stent coassiale o con lutilizzo del cutting ballon )  . conclusions on the basis of our results and data from previous reports , we confirm that cdus plays a fundamental role in patients with suspected or known renovascular disease , as it provides important information on renal haemodynamics and guides the clinician towards the most appropriate clinical and therapeutic approach [ 2 , 57 , 1416 ]  . our results indicate that cdus , with appropriate selection criteria , is able to discriminate between patients who should or should not undergo angiography , leading to considerable economic savings and benefits for the patient , who can thus be directed to the most appropriate treatment plan [ 16 ]  . 
in addition , our findings point to a close association between the presence of aaa and ras and confirm the need for close collaboration between the vascular surgeon , the interventional radiologist and the nephrologist in planning the most appropriate treatment approach in these patients with associated diseases [ 2630 ]  . cdus proved its value in confirming the diagnosis of cholesterol embolism suspected on the basis of clinical and laboratory findings . 
these patients carry a poor prognosis , with 1 - year mortality rates between 64% and 87% , and monitoring by imaging may certainly help the clinician deal with this complex disease given that angiography is strongly contraindicated both because it is an interventional manoeuvre and because it requires heparin administration [ 3133 , 34 ]  . in the patient with middle aortic syndrome , cdus revealed the alteration in renal haemodynamics , thus justifying the use of a more panoramic modality , such as ct angiography , which shed light on the patients complex anatomical situation [ 2224 ]  . another important contribution of cdus was to substantiate the clinical and laboratory suspicion of acute renal failure in a septic patient with cortical necrosis . 
use of contrast medium , justified by the need to shed light on what was clearly a very critical clinical situation , enabled prompt treatment ( haemodialysis and plasmapheresis ) , with partial recovery of renal function [ 35 ]  . cdus confirmed its value in monitoring nephropathic patients and those who have undergone renal artery revascularisation procedures [ 17 , 25 ]  . 
our experience also highlights the importance of cdus in monitoring patients in whom renal artery occlusion or stenosis has been diagnosed at angiography : in these patients , clinical and laboratory monitoring should be combined with follow - up by cdus , which is able to effectively reveal disease progression and provide the clinician with the data required for a more appropriate treatment strategy . 1122 dai risultati ottenuti e alla luce dei dati della letteratura , si riconferma il ruolo fondamentale svolto dallecd nei pazienti con sospetta o accertata patologia reno - vascolare , fornendo dati importanti sullemodinamica renale di insostituibile utilit per un corretto approccio clinico - terapeutico [ 2 , 57 , 1416 ]  . dai nostri risultati si evince come ecd , alla luce dei criteri adottati , possa essere metodica in grado di discriminare i pazienti che devono o non devono essere sottoposti ad esame agiografico , con risparmio dei costi e beneficio per il paziente che potr quindi essere indirizzato al pi opportuno iter terapeutico [ 16 ]  . 
i nostri dati mettono inoltre in risalto unelevata associazione fra la presenza dellaaa e la sar : questo vuole riconfermare lutilit di una stretta collaborazione fra il chirurgo vascolare , il radiologo interventista e il nefrologo nel programmare la strategia terapeutica pi opportuna in questi pazienti con patologia associata [ 2630 ]  . lecd si dimostrata metodica utile nel confermare la diagnosi di ateroembolismo colesterinico , gi prospettata in base ai dati clinico - laboratoristici . 
questi sono pazienti a prognosi infausta con mortalit ad 1 anno variabile dal 64% all87% in cui il monitoraggio strumentale pu essere certamente di ausilio al clinico nellaffrontare una cos complessa patologia , dal momento che in tali pazienti fortemente controindicata langiografia , sia perch manovra interventistica sia per la somministrazione di eparina [ 3133 , 34 ]  . nella paziente con la middle aortic syndrome lecd ha svelato lalterazione emodinamica a livello renale , giustificando il ricorso a metodiche pi panoramiche come langiotc che ha rilevato la complessa situazione anatomica da cui era affetta [ 2224 ]  . occorre senzaltro rimarcare limportanza dellecd nel confermare lipotesi clinico - laboratoristica di insufficienza renale acuta in corso di sepsi nella paziente affetta da necrosi corticale ; lutilizzo del mdc , giustificato dalla necessit di chiarire una situazione che allesordio era molto critica , ha consentito la tempestiva terapia ( emodialisi e plasmaferesi ) con parziale recupero della funzione renale [ 35 ]  . la metodica ecd si dunque riconfermata di indubbio valore nel monitoraggio dei pazienti nefropatici e di quelli sottoposti ad intervento di rivascolarizzazione allarteria renale [ 17 , 25 ]  . 
souza de oliveira ir , widman a , molinar lj et al ( 2000 ) color doppler ultrasound : a new index improves the diagnosis of renal artery stenosis . ultrasound med biol 26 : 4147 12 . 
frauchiger b , zierler r , bergelin ro et al ( 1996 ) prognostic significance of intrarenal resistance indices in patients with renal artery interventions : a preliminary duplex sonographic study . cardiovasc surg 4 : 324330 18 . 
mehta m , darling rc 3rd , roddy sp et al ( 2004 ) outcome of concomitant renal artery reconstructions in patients with aortic aneurysm and occlusive disease . vascular 12 : 381386 29 . 
tali caratteri radiografici non sono correlati ad alcun tipo istologico particolare . parole chiave lesioni non palpabili linfonodi intra - mammari carcinoma della mammella introduction introduzione intramammary lymph nodes are the most common cause of nonpathological masses seen on conventional mammography , being detected in about 30% of breasts , mostly in the upper - outer quadrants [ 1 ]  . 
the typical mammographic features of intramammary lymph nodes ( size smaller than 15 mm , round or oval shape , regular margins , low density , association with a vascular branch and , frequently , a radiolucent centre ) have been clearly established [ 17 ] , and their detection does not warrant further diagnostic investigation i linfonodi intra - mammari costituiscono la causa pi frequente di formazione espansiva con significato non patologico riscontrata allesame mammografico convenzionale : sono infatti presenti in circa il 30% delle mammelle , prevalentemente nei quadranti supero - esterni [ 1 ]  . 
i caratteri mammografici tipici dei linfonodi intra - mammari ( dimensioni inferiori a 15 mm , morfologia rotondeggiante od ovalare , contorni regolari , bassa densit , associazione con una diramazione vascolare e frequente area centrale di maggiore radio - trasparenza ) sono chiaramente codificati da lungo tempo in letteratura [ 17 ] ed il 1078 e . 
however , in rare cases , breast cancers may exhibit mammographic features compatible with intramammary lymph nodes [ 1 , 2 , 9 , 11 ]  . the aim of this study was to verify the existence of mammographic criteria justifying a suspicion of atypical intramammary lymph node , assess whether certain histological types of breast cancer are more likely to display the radiographic features of intramammary lymph nodes , and consider the most appropriate diagnostic strategy in women with node - like mammographic findings . materials and methods we retrospectively reviewed the previous mammograms of women who received a diagnosis of breast cancer between january 1997 and december 2004 . 
the mammograms had been performed 6 months to 3 years before the diagnosis and depicted findings morphologically similar to intramammary lymph nodes at the site where the cancer was subsequently detected . 
the imaging equipment consisted of a dedicated conventional radiology unit ( mammomat 3000 , siemens , erlangen , germany ) combined with a high - spatialresolution detector , gadolinium screen ( kodak min r 2000 ) and single - coated film ( kodak m35 series x - omat processor )  . 
examinations performed with different equipment and techniques were excluded from the study . during the examination that aroused the suspicion of a neoplastic lesion , all lesions were studied by fine - needle cytology ( 2023 gauge ) under sonographic guidance ( seven cases ) or stereotactic guidance ( in one case in which the lesion was poorly visualised on ultrasound )  . 
all lesions subsequently underwent surgical excision and histological examination . loro riscontro non richiede approfondimenti diagnostici , che si rendono necessari solo in caso di incremento volumetrico in un controllo mammografico successivo [ 813 ]  . 
sebbene raramente , tumori della mammella in fase iniziale possono presentare caratteri mammografici sovrapponibili a quelli tipici dei linfonodi intra - mammari [ 1 , 2 , 9 , 11 ]  . scopo di questo lavoro ricercare se esistano criteri di semeiotica mammografica che consentano di porre il sospetto di linfonodo intra - mammario atipico , valutare se alcuni istotipi tumorali si manifestino preferenzialmente con caratteri radiografici simili a quelli dei linfonodi intra - mammari ed esaminare quale sia la strategia diagnostica pi opportuna quando si riscontrino reperti di aspetto compatibile con quello dei linfonodi intra - mammari . materiali e metodi sono stati retrospettivamente valutati i radiogrammi espletati in un periodo compreso tra i sei mesi e tre anni prima della diagnosi di tumore della mammella , posta tra gennaio 1997 e dicembre 2004 nei quali , nella sede in cui sarebbe stata in seguito identificata la neoplasia , era presente un reperto con i caratteri morfologici di un linfonodo intra - mammario . 
tutte le pazienti considerate hanno eseguito un esame mammografico standard , con almeno due proiezioni ( cranio - caudale e medio - laterale obliqua ) per mammella ; lindagine stata espletata con apparecchiatura radiologica convenzionale dedicata ( mammomat 3000 , siemens , erlangen , germania ) associata a detettore ad alta risoluzione spaziale , con schermo di rinforzo in gadolinio ( kodak min r 2000 ) e pellicola monoemulsionata ( kodak m35 series x - omat processor )  . 
le indagini espletate con modalit differenti da quella citata sono state escluse dallo studio . tutte le lesioni , nel corso dellindagine in cui ne stata sospettata la natura neoplastica , sono state sottoposte ad esame citologico con ago sottile ( calibro 2023 g ) , espletato sotto guida ecografica ( 7 casi ) o stereotassica ( nel singolo caso in cui la lesione non risultava chiaramente riconoscibile allesame ecografico )  . 
it is difficult to identify any lesion at all at the site of interest ( arrow and arrowhead ) on the mediolateral oblique mammogram ( a )  . only the craniocaudal view ( b ) shows an 8 - mm , faint , round opacity in the upper - outer quadrant that can be evaluated as an intramammary lymph node ( arrows )  . 
sul radiogramma in proiezione medio - laterale obliqua ( a ) risulta difficilmente identificabile , nel punto di interesse ( freccia e testa di freccia ) , una qualsiasi lesione . 
solo la proiezione cranio - caudale ( b ) dimostra nel versante supero - esterno tenue opacit rotondeggiante di 8 mm valutabile come linfonodo intra - mammario ( freccia )  . 
in the craniocaudal view ( b ) , an oval opacity with regular margins can be seen ( arrow ) projecting along a vascular branch , with a radiolucent central area ( 8 mm )  . 
in fase pre - diagnostica la proiezione medio - laterale obliqua ( a ) non consente di identificare lesioni sospette nellarea di interesse ( freccia e testa di freccia )  . 
in proiezione cranio - caudale ( b ) si identifica tenue opacit ovalare a contorni regolari ( freccia ) proiettatesi su diramazione vascolare , con nucleo radio - trasparente ( 8 mm )  . 
mediolateral oblique mammogram shows an 8 - mm , faint , round opacity in the upper quadrant ( arrow ) , with multilobular margins and radiolucent centre ( a ) interpreted as an intramammary lymph node . 
radiogramma in proiezione medio - laterale obliqua che dimostra nel versante superiore tenue opacit rotondeggiante di 8 mm ( freccia ) a contorni pluriciclici e nucleo radiotrasparente ( a ) interpretata come linfonodo intra - mammario . 
a minute ( 4 mm ) , round opacity with a radiolucent central area projecting along a small vascular branch can be seen in the upper - outer quadrant on both views ( arrows in a , b )  . 
minuta ( 4 mm ) opacit rotondeggiante con nucleo radiotrasparente , proiettantesi su piccola diramazione vascolare , riconoscibile nel versante supero - esterno in entrambe le proiezioni ( frecce in a , b )  . 
il controllo effettuato a distanza di 24 mesi ( c , d ) dimostra lieve incremento volumetrico ( 6 mm ) con densit lievemente aumentata . at baseline mammography , all masses showed round or oval shape and low density , with well - defined margins in seven cases ( two of which lobulated ) , and one case showed slightly indistinct margins . 
le dimensioni delle formazioni erano comprese fra 4 e 8 mm ( tabella 1 )  . lindagine in cui stata riconosciuta la reale natura neoplastica delle neoformazioni stata espletata da 6 a 36 mesi dopo la prima . 
a small ( 5 mm ) , faint , round opacity with radiolucent central area projecting along a vascular branch is seen in the upper - outer quadrant on the mediolateral oblique view ( arrow in a ) , which is even more evident on the craniocaudal view ( arrow in b )  . 
piccola ( 5 mm ) e tenue opacit rotondeggiante con nucleo radiotrasparente , proiettantesi sul decorso di diramazione vascolare nel versante supero - esterno , apprezzabile in proiezione medio - laterale obliqua ( freccia in a ) e meglio in proiezione cranio - caudale ( freccia in b )  . 
in the prediagnostic phase , the minute ( 6 mm ) , faint opacity visible in the upper - outer quadrants on both views ( arrows in a , b ) could easily be interpreted as an intramammary lymph node because of its site , density , radiolucent centre and proximity to vascular structures . 
in fase pre - diagnostica , la minuta ( 6 mm ) e tenue opacit apprezzabile in entrambe le proiezioni ( frecce in a , b ) nel versante supero - esterno facilmente interpretabile come linfonodo intramammario , in virt della sede , delle densit , del nucleo radiotrasparente e delle prossimit con le strutture vascolari . 
dopo 24 mesi ( c , d ) la lesione presenta contorni sfumati ; sensibilmente aumentata di volume ( 20 mm ) e di densit , questultima disomogenea . discussion discussione the radiographic features traditionally ascribed to intramammary lymph nodes [ 17 ] still hold good , as they are based on pathology findings and experience : the finding of small round or oval masses with regular margins and low density ( especially if located along a vascular branch and relatively radiolucent in their central portion ) does not justify a suspicion of neoplastic disease . 
a number of benign conditions can cause lymph nodal eni caratteri radiografici tradizionalmente attribuiti ai linfonodi intra - mammari [ 17 ] , in quanto fondati su dati anatomopatologici e condizionati dalla esperienza , rimangono validi a tuttoggi : il riscontro di formazioni espansive rotondeggianti od ovalari di piccole dimensioni , a margini regolari e dotate di bassa densit ( specie se situate lungo una diramazione vascolare e relativamente radio - trasparenti nella porzione centrale ) non autorizza a porre il sospetto di patologia neoplastica . 
the prediagnostic mediolateral oblique view shows , in the area in question ( arrow in a ) , an almost imperceptible , very small ( 4 mm ) and faint opacity , marked with an arrow on the craniocaudal view ( b )  . 
in fase pre - diagnostica , nella proiezione medio - laterale obliqua pressoch impercettibile nella zona di interesse ( freccia in a ) la minuta ( 4 mm ) e tenue opacit contrassegnata con freccia nella proiezione cranio - caudale ( b )  . 
a distanza di 36 mesi ( c , d ) la lesione risulta sensibilmente aumentata di volume ( 10 mm ) e densit , presentando contorni sfumati . largement and increased radiographic density , for example , inflammation of the breast skin ( at times related to implants ) or systemic inflammation ( rheumatoid arthritis )  . 
lymph nodal enlargement and increased density may also result from lymphoproliferative disease or from metastasis from cancers located in other parts of the breast , or rarely , elsewhere in the body ( melanomas , pulmonary , gastric and ovarian cancers ) [ 9 ]  . 
 inoltre causare nel tempo incremento nelle dimensioni e nella densit radiografica dei veri linfonodi intra - mammari , come avviene in caso di flogosi cutanee proprie della ghiandola mammaria ( anche sostenute dalla presenza di protesi ) o su base sistemica ( quali lartrite reumatoide ) ; analogo reperto pu essere causato da malattie infettive fra cui la tubercolosi . 
infine , possono ingrandirsi ed aumentare in densit i linfonodi intra - mammari sede di localizzazione di malattia linfoproliferativa o di metastasi , a partire da tumori primitivi posti in altre porzioni della mammella o , raramente , altrove ( melanomi ; tumori polmonari , gastrici ed ovarici ) [ 9 ]  . to our knowledge , the possibility that early primary secondo i dati a nostra disposizione , la possibilit che 1083 e . 
the retrospective review of our small case series did not reveal any correlation between histological type of cancer and node - like radiographic patterns , nor did it indicate the existence of new semiological criteria . 
it did , however , highlight a need for careful assessment of the known semiological criteria . owing to their small size , initial cancers with misleadingly similar features to intramammary lymph nodes may exhibit suspicious features in one radiographic view only . 
in addition , it should be noted that slight blurring of tumori primitivi della mammella esordiscano con quadri mammografici simili a quelli dei linfonodi intra - mammari non stata trattata in letteratura , sebbene sia noto che i carcinomi mucinosi possono presentare margini netti [ 7 ]  . la valutazione retrospettiva di questa casistica per quanto numericamente limitata non ha evidenziato correlazioni fra specifici istotipi tumorali e caratteri radiografici simil - linfonodali . 
non sono inoltre emersi criteri semeiologici nuovi ma la necessit di dedicare estrema attenzione a quelli gi noti . in conseguenza delle loro esigue dimensioni , lesioni tumorali in fase precoce e con caratteri ingannevolmente simili a quelli dei linfonodi intra - mammari possono mostrare segni sospetti in una sola proiezione radiografica . 
gists were located in the stomach in 18 / 27 patients , in the small bowel in seven , in the oesophagus in one and in the rectum in one . 
in 15 / 27 cases , the lesions exhibited extramural growth , and in 17 / 27 cases , they were homogeneous after contrast medium injection . the borders were regular in 17 cases . 
in 1983 , in - depth studies recognised gists as discrete entities , which allowed them to be differentiated from smooth con il termine di tumori stromali gastrointestinali ( gist , dallinglese gastro - intestinal stromal tumor ) si comprende attualmente un eterogeneo gruppo di neoplasie mesenchimali del tratto gastroenterico , che sono state a lungo oggetto di controversie riguardanti la loro nomenclatura , istogenesi , diagnosi e prognosi . 
fino al 1983 , infatti , erano inclusi in questa categoria di tumori anche i leiomiomi , leiomioblastomi , leiomiosarcomi , schwannomi e neurofibromi . nel 1983 studi pi approfonditi hanno conferito a questi tu1103 c . 
histological and immunohistochemical characteristics of gists are well defined and include the presence of spindle cells ( less commonly epithelioid cells and rarely both ) and positivity for the cd117 membrane protein [ 113 ]  . despite being the most common type of gastrointestinal mesenchymal tumour [ 1 , 2 , 5 , 9 , 10 ] , gists are rare and account for only 0.1%3% of all gastrointestinal neoplasms [ 8 , 1416 ]  . 
gists generally affect individuals over the age of 50 years and are rare in subjects younger than 40 years [ 2 , 5 , 10 , 12 ] , with a slight , though debated , predilection for males [ 2 , 4 , 5 , 17 , 18 ]  . 
gists arise in the stomach in 60%70% of cases , in the small bowel in 20%25% , in the rectum in 5% and very rarely in the oesophagus , colon and appendix [ 25 , 8 , 9 , 12 , 16 , 1925 ]  . 
regardless of the location and in the absence of haemorrhagic complications related to mucosal ulcerations , the clinical symptoms of gist are nonspecific and tend to develop late , when the tumour has reached a large size . diagnostic imaging and especially computed tomography ( ct ) are instrumental in the detection and staging of gist , as has been reported in the literature [ 1 , 5 , 8 , 9 , 12 , 13 , 19 , 23 , 24 , 26 , 27 ]  . 
the aim of this study was to evaluate the potential of multislice ct ( msct ) in the identification and characterisation of gist . materials and methods we retrospectively evaluated the ct images of 27 patients ( 15 men and 12 women , aged 3774 years ) studied between april 2001 and may 2004 and found to be affected by gist . the definitive diagnosis was provided by histological examination of the surgical specimen . 
given the retrospective design of the study , approval by the ethics committee was not required . in 15 cases , ct was carried out to investigate submucosal disease suspected at endoscopy and involving the oesophagus in one case , the stomach in 13 cases , and the rectum in one case . 
in genere interessano soggetti che hanno oltre 50 anni di et e sono di raro riscontro in pazienti di et inferiore ai 40 anni [ 2 , 5 , 10 , 12 ] , con una discussa lieve predominanza per il sesso maschile [ 2 , 4 , 5 , 17 , 18 ]  . 
indipendentemente dalla loro localizzazione ed in assenza di complicanze emorragiche legate ad ulcerazioni della mucosa , la sintomatologia clinica dei gist aspecifica e tardiva e compare soltanto quando i tumori raggiungono considerevoli dimensioni . la diagnostica per immagini e soprattutto la tomografia computerizzata ( tc ) hanno un ruolo fondamentale nel riconoscimento e nella stadiazione dei gist come riportato in letteratura [ 1 , 5 , 8 , 9 , 12 , 13 , 19 , 23 , 24 , 26 , 27 ]  . 
scopo del presente lavoro valutare le possibilit della tc multistrato ( tc - ms ) nella identificazione e caratterizzazione di tali tumori . materiali e metodi sono state valutate retrospettivamente le immagini tc di 27 pazienti ( 15 uomini e 12 donne ) , di et compresa tra 37 e 74 anni , studiati tra aprile 2001 e maggio 2004 e risultati affetti de gist . 
trattandosi di uno studio retrospettivo non stato necessario richiedere il consenso alla commissione etica per il suo svolgimento . lesame tc era stato eseguito in 15 casi per lo studio di una patologia sottomucosa sospettata con endoscopia in 1 caso a livello esofageo , in 13 casi a livello dello stomaco , in 1 caso a livello del retto . 
all patients received contrast material for the study of the bowel loops : two sachets of negative contrast material ( duogas , bracco , milan , italy ) for the study of the oesophagus ( one case ) 400600 cc of water or duogas for the study of the stomach ( 13 cases ) 400600 ml diluted methylglucamine diatrizoate ( gastrografin ) in the remaining 13 cases the data acquired were retrospectively transferred to a workstation ( hp kayak xu800 , hewlett packard , palo alto , ca , usa ) running dedicated reconstruction software ( vitrea 2.6 , vital images , minneapolis , mn , usa )  . 
multiplanar ( coronal , sagittal and oblique ) and three - dimensional ( 3d ) reconstructions were obtained with multiplanar reformatting ( mpr ) and volume rendering ( vr ) techniques . 
two cases of disagreement about the possible origin of large masses were resolved by consensus . the images were assessed for : morphological changes to the intestinal wall caused by the presence of a recognisable , expansile mass in the wall portion of the gastrointestinal tract involved by the tumour ( oesophagus , stomach , duodenum , jejunum , ileum , colon and rectum ) tumour size origin and growth pattern relative to the affected organ ( defined as intraor extraluminal , depending on whether more or less than half of the tumour lay inside or outside the lumen ) attenuation , defined as homogeneous or heterogeneous according to enhancement characteristics margin morphology of both mucosal and extramural components ( regular if smooth or lobular ; irregular if finely jagged ) presence of mucosal ulcerations at the lesion site relationship with adjacent structures or organs ; infiltration was hypothesised in the absence of a clear cleavage plane presence of distant metastases evidence of lymph nodes ; any node detected on the scans was considered pathological , regardless of diameter presence of ancillary findings , such as intestinal occlusion or ascites additional information provided by the mpr and vr reconstructions aiding identification and characterisation of the tumour and definition of its growth pattern and relationship with adjacent structures c . 
il mdc uroangiografico non - ionico ( iopromide , ultravist 370 , schering , berlino , germania ) stato iniettato nella vena cubitale attraverso un ago da 1618 gauge , utilizzando un iniettore automatico ( mk - iv , medrad , pittsburgh , pennsylvania , usa ) con i seguenti parametri : volume 1 , 5 ml / kg di peso corporeo ( fino ad un massimo di 130 ml ) , velocit di iniezione 3 , 5 ml / s . 
in tutti i casi stato somministrato mezzo di contrasto per lo studio delle anse intestinali : due bustine di mezzo di contrasto negativo ( duogas , bracco , milano , italia ) per lo studio dellesofago ( 1 caso ) ; 400600 ml di acqua o duogas per lo studio dello stomaco ( 13 casi ) ; 400600 ml di soluzione diluita di diatrizoato e metilglucamina ( gastrografin ) nei restanti 13 casi . i dati acquisiti sono stati trasferiti retrospettivamente ad una workstation ( hp kayak xu800 , hewlett packard , palo alto , california , usa ) dotata di un software di ricostruzione dedicato ( vitrea 2 , 6 , vital images , minneapolis , minnesota , usa )  . 
i programmi applicativi utilizzati sono stati il multiplanar reformatting ( mpr ) e il volume rendering ( vr ) che hanno consentito di ottenere ricostruzioni multiplanari ( coronali , sagittali e oblique ) e tridimensionali . 
la durata complessiva del postprocessing stata di 15 minuti . tutte le immagini assiali e le ricostruzioni sono state valutate in cieco da due radiologi esperti , non coinvolti direttamente nellesecuzione dellesame tc . 
in due casi divergenze collegate alla possibile origine di voluminose neoformazioni sono state risolte in consenso . nella valutazione delle immagini sono stati considerati : alterazioni morfologiche della parete intestinale determinate dalla presenza di un riconoscibile processo espansivo nel suo contesto ; tratto dellapparato gastroenterico ( esofago , stomaco , duodeno , digiuno , ileo , colon e retto ) interessato dalla patologia ; dimensioni del tumore ; origine e sviluppo del tumore rispetto alla parete del viscere interessato ( considerato intrao extraluminale a seconda che pi o meno di met lesione si sviluppasse allinterno o allesterno del lume del viscere ) ; densitometria del tumore , differenziata in omogenea o disomogenea a seconda delle caratteristiche di impregnazione ; morfologia dei margini sia sul versante mucoso che extraparietale ( regolari , se lisci o lobulati ; irregolari , se finemente frastagliati ) ; presenza di ulcerazioni mucose nella sede della lesione ; 1105 c . 
in tutti i casi esaminati la lesione neoplastica era unica . le dimensioni del tumore variavano da un diametro minimo di 1 , 5 cm ad un massimo di 21 cm ( dimensione media di 7 , 4 cm )  . 
in particolare in 10 dei 18 gist gastrici ( 55% ) lindagine tc ha mostrato una crescita di tipo endofitico mentre nei restanti 8 / 18 casi ( 45% ) lo sviluppo era prevalentemente ex1107 fig . 
presenza di profonda ulcerazione mucosa ( freccia vuota )  . results ct findings in the 27 patients with gist are reported in table 1 . the most frequently affected site was the stomach ( 18 / 27 , 67% ) , and in particular , the cardias in one case , the fundus in one case , the body in 13 cases and the antrum in three cases . in seven cases ( 26% ) , the disease involved the small bowel , and specifically , the jejunum in four cases , the duodenum in one case and the ileum in two cases . 
in detail , gastric lesions ranged from 1.5 cm to 15 cm ( mean 5.3 cm ) , intestinal lesions from 4 cm to 21 cm ( mean 12 cm ) , the oesophageal lesion was 12 cm and the rectal lesion was about 10 cm . in 23 out of 27 cases , an extramucosal origin was suggested by the presence of raised mucosa at the lesion site c . 
tutte le lesioni dopo iniezione del mezzo di contrasto hanno mostrato un persistente incremento di densit , che si manifestato in maniera omogenea in 17 / 27 casi e disomogenea in 10 casi . 
in nessun caso sono state evidenziate calcificazioni intralesionali . i margini sono risultati regolari in 18 / 27 casi ( 67% ) ed irregolari nei restanti 9 casi ( 33% )  . 
i margini delle lesioni gastriche si sono dimostrati regolari sia sul versante mucoso che extraparietale in 14 / 18 casi , in particolare in 6 / 8 gist a sviluppo extraparietale e in 8 / 10 a sviluppo endoluminale . 
in nessun caso stata evidenziata uninfiltrazione delle strutture vascolari o degli organi contigui alla neoplasia . lesioni metastatiche , in tutti i casi localizzate a livello epatico , erano gi evidenti al momento della diagnosi in 5 / 27 pazienti ( 18% )  . 
in particular , 10 / 18 gastric gists ( 55% ) showed an endophytic growth pattern whereas the remaining 8 / 18 cases ( 45% ) had a prevalently extragastric growth pattern . 
the intestinal gists exhibited extramural growth pattern in 5 / 7 cases ( 71% ) , a constant feature in lesions larger than 11 cthe remaining two lesions had intraluminal growth . 
a in correspondence with the posterior wall of the gastric body , there is a small , high - attenuation mass showing regular borders and intraluminal growth ( arrowheads )  . 
b , c nel lobo sinistro del fegato presente una metastasi che appare tenuemente iperdensa nelle scansioni precoci ( b ) ed ipodensa nelle scansioni tardive ( c ) ( frecce vuote )  . the lesions could always be detected on the precontrast scans , and they showed homogeneous appearance in 23 cases , and there was a heterogeneous appearance in four cases of large tumours . 
in no case was infiltration of vascular structures or adjacent organs observed . metastatic lesions , involving the liver in all cases , were detected at presentation in 5 / 27 patients ( 18% )  . 
the primary tumours ranged in size from 1.5 cm to 11 cm . lymph node enlargement was detected in only one gist of the gastric fundus , and the lesions were located in the perigastric node stations . 
il tumore primitivo aveva diametro compreso tra 1 , 5 e 11 cm . la presenza di linfonodi aumentati di volume stata riscontrata soltanto in 1 gist del fondo gastrico e le lesioni erano localizzate nelle stazioni perigastriche . 
per differenziare i gist da altri tumori mesenchimali sono comunemente utilizzati altri markers immunochimici come ls100 tipico degli schwannomi , e lactina e desmina caratteristiche dei veri tumori a cellule muscolari lisce , i leiomiomi ed i leiomiosarcomi [ 1 ]  . lesatta prevalenza dei gist sconosciuta e secondo miettinem e lasota [ 2 ] di 1020 casi per milione di persone , senza alcuna associazione geografica , etnica , razziale o occupazionale [ 1 , 2 , 14 ]  . tali tumori possono originare dalla parete di qualunque segmento del tratto gastroenterico , dallesofago the mpr and vr reconstructions were of no help in diagnosing the tumour , which was always appreciable on the axial scans , or in characterising it . 
other markers that are commonly used to differentiate gists from other mesenchymal tumours include s100 for schwannomas and actin and desmin for true smooth muscle tumours , leiomyomas and leiomyosarcomas [ 1 ]  . the exact prevalence of gist is unknown , but according to miettinem and lasota [ 2 ] , it is 1020 cases per million people , without any geographic , ethnic , racial or occupational association [ 1 , 2 , 14 ]  . gists can arise from the wall of any portion of the gastrointestinal tract , from the oesophagus to the anus [ 5 , 28 ] , and represent the most common type of mesenchymal tumour , if we exclude the oesophagus , where 25% are gists and 75% are leiomyomas [ 5 , 28 ]  . 
the gist locations found in our study ( stomach 67% ; small intestine 26% ; oesophagus 4% ; rectum 4% ) reflect the data reported in the literature [ 15 , 9 , 12 , 13 ]  . the size of these tumours varies widely , from several millimetres , in which case they are generally an incidental finding , to 30 cm or more [ 1 , 5 ]  . 
the gastric tumours in our series ranged in size from 1.5 cm to 15 cm ( mean 5 cm ) , the oesophageal gist was 12 cm , the intestinal gists were 421 cm ( mean 12 cm ) and the rectal gist was 10 cm . gists often originate in muscularis propria ( outer wall layers ) ; those arising in the muscularis mucosae ( deeper wall layers ) are rare and more frequently involve the colon [ 5 ]  . according to some authors , tumour origin from the muscularis propria or muscularis mucosae will influence its exophytic growth pattern , which was seen in 15 / 27 of our cases , or intraluminal growth pattern , which was seen in the remaining 12 cases . clinical presentation depends on lesion site and size . small tumours are generally asymptomatic and discovered incidentally during surgery , endoscopy or imaging studies . symptomatic gists tend to be large and , depending on location , can give rise to dysphagia , nausea , vomiting , abdominal pain and a palpable mass [ 1 , 5 , 28 ]  . 
whatever the site , erosion of the mucosa overlying the lesion may lead to gastrointestinal bleeding . biologically , gist are considered potential malignancies with different degrees of risk ( very low , low , intermediate , high ) on the basis of size and mitotic rate [ 3 , 2931 ] ; thus , all gists require surgical treatment . diagnostic imaging is instrumental in the detection of gists and relies on conventional radiology , ultrasound ( us ) , ct , magnetic resonance ( mr ) and positron emission c . 
in rapporto allorigine dagli strati pi esterni della parete ( muscolaris propria ) o dagli strati pi interni ( muscolaris mucosae ) , secondo alcuni autori si determinerebbe uno sviluppo esofitico riscontrato nella nostra casistica in 15 / 27 casi o endoluminale riscontrato nei 12 restanti casi . la presentazione clinica di questi tumori variabile a seconda della sede e dimensione della patologia . 
indipendentemente dalla loro localizzazione lerosione della mucosa sovrastante il tumore pu generare emorragie gastrointestinada un punto di vista biologico i gist sono considerati tumori potenzialmente maligni con differente grado di rischio ( molto basso , basso , intermedio , alto ) valutato in base alle dimensioni e tasso mitotico [ 3 , 2931 ] , pertanto tutti necessitano di una terapia chirurgica . nel riconoscimento dei gist la diagnostica per immagini ha un ruolo fondamentale e si basa su : radiologia tradizionale , ultrasonografia ( us ) , tomografia computerizzata ( tc ) , risonanza magnetica ( rm ) , tomografia ad emissione di positroni ( pet )  . 
il ruolo della tc nello studio dei gist stato ampiamente sottolineato in letteratura [ 1 , 5 , 8 , 9 , 12 , 13 , 19 , 23 , 24 , 26 , 27 ] e la metodica interviene nel riconoscimento del tumore e nel valutare la sua estensione o eventuale disseminazione metastatica . 
la morfologia tc dei gist nella nostra esperienza risultata priva di specificit ed i tumori apparivano come processi espansivi a partenza extramucosa , con modesto enhancement dopo iniezione di mezzo di contrasto . 
le lesioni di piccole dimensioni avevano aspetto omogeneo , quelle voluminose pi spesso disomogeneo in rapporto ad emorragie e necrosi intratumorali . nellambito delle diagnosi differenziali , in presenza di un sospetto gist riconosciuto alla tc devono essere consi1111 c . 
the role of ct in the study of gist has been widely emphasised in the literature [ 1 , 5 , 8 , 9 , 12 , 13 , 19 , 23 , 24 , 26 , 27 ] and consists of recognising the tumour and assessing its extension and possible metastatic spread . 
in our experience , the ct appearance of gist was found to lack specificity , with the tumours manifesting as expansile masses with extramucosal origin and moderate enhancement after contrast medium injection . 
small lesions appeared homogeneous whereas larger lesions were more often heterogeneous due to intralesional haemorrhage and necrosis . the differential diagnosis of a suspected gist detected on ct should include both benign lesions , such as lipomas , cystic duplications , leiomyomas , schwannomas or neurofibromas , and malignant lesions , such as leiomyosarcomas , lymphomas or adenocarcinomas [ 32 ]  . 
lymphomas cause varying degrees of wall thickening , which may be diffuse , often multiple , with a hypodense appearance of the submucosal infiltrates , and often associated with lymphadenopathy . 
carcinomas may exhibit broad - based intraluminal vegetations or varying degrees of concentric or eccentric wall thickening , always associated with mucosal alterations and frequently with lymphadenopathy [ 32 , 33 ]  . in a study by dematteo et al . 
only 18% of gists in our series ( three gastric and two ileal ) showed secondary liver lesions at presentation , and no correlation was found between tumour size and metastasis , which was also seen in a gist measuring 1.5 cthis finding supports the view that metastatic spread is influenced by mitotic rate rather than tumour size . in agreement with previous reports [ 1 , 5 , 12 ] finding limited evidence of nodal spread associated with sarcomas , only one out of 27 patients in our series had metastatic lymphadenopathy . use of multislice ct with mpr and vr reconstructions proved particularly useful in the study of large gists , as it enabled better definition of tumour origin and relationships with adjacent organs . 
conversely , the technique appears to offer no additional contribution to disease characterisation compared with conventional modalities . derate lesioni benigne come lipomi , duplicazioni cistiche , leiomiomi , schwannomi o neurofibromi e lesioni maligne come leiomiosarcomi , linfomi o adenocarcinomi [ 32 ]  . 
una diagnosi differenziale tra gist ed altri tumori sottomucosi come leiomiomi , schwannomi , neurofibromi e leiomiosarcomi impossibile in base alla morfologia tc ed a tale fine indispensabile lesame istologico . 
la diagnosi differenziale con altre forme maligne come linfomi ed adenocarcinomi in genere possibile . i linfomi determinano ispessimenti parietali di vario grado , diffusi , spesso multipli , con aspetto in genere ipodenso degli infiltrati sottomucosi , associati spesso a linfoadenopatie . 
nei carcinomi possono riscontrarsi vegetazioni endoluminali a larga base dimpianto o ispessimenti parietali concentrici o eccentrici di vario grado , sempre con alterazione della mucosa e frequenti linfoadenopatie [ 32 , 33 ]  . in uno studio di dematteo et al . 
le lesioni epatiche apparivano ipo - isodense rispetto al parenchima nelle scansioni di base e mostravano dopo iniezione di mdc un enhancement disomogeneo per la presenza di aree centrali necroticocolliquative . 
solo il 18% dei gist nella nostra casisitica ( tre tumori a sede gastrica e due a sede ileale ) presentavano al momento della diagnosi lesioni secondarie epatiche e non stata trovata alcuna correlazione tra dimensioni del tumore e presenza di metastasi , che sono state infatti riconosciute anche in un gist di 1 , 5 c tale dato depone per un rischio di disseminazione metastatica in rapporto al tasso mitotico piuttosto che alle dimensioni della lesione . in accordo con la letteratura [ 1 , 5 , 12 ] che sottolinea la scarsa evidenza di diffusione linfonodale associata a sarcomi , nei nostri risultati in un solo caso su 27 pazienti erano presenti linfoadenopatie metastatiche . limpiego della tc multistrato , per la possibilit di fornire ricostruzioni mpr ed in vr , risultato particolarmente utile nello studio dei gist voluminosi per meglio definire lorigine del tumore ed i rapporti con gli organi contigui . 
gallo dipartimento integrato dei servizi diagnostici e per immagini , struttura complessa di radiologia ii , azienda policlinico , via del pozzo 71 , i - 41100 modena , italy correspondence to : m.g. 
with regard to lesion site , virtual endoscopy proved to be as informative as real endoscopy . the virtual endoscope was able to cross severe stenoses with a residual lumen of 3 mfollow - up studies were performed in 15 patients treated with laser and cryotherapy . 
the technique , with the integration of mpr images , can be proposed as a preliminary study to obtain accurate characterisation of stenoses , to shorten the time required for the subsequent endoscopic procedure and to plan the most appropriate treatment . key words tracheobronchial stenosis virtual bronchoscopy fiberoptic bronchoscopy riassunto obiettivo . 
lendoscopio virtuale stato in grado di superare stenosi serrate con lume residuo fino a 3 mm . il follow - up stato eseguito in 15 pazienti sottoposti a laser e crio - terapia . 
essa , integrata dalle immagini mpr , pu essere proposta come indagine preliminare per una accurata caratterizzazione della patologia stenotica , per abbreviare i tempi di esecuzione della successiva procedura endoscopica e per la pianificazione del trattamento terapeutico pi appropriato . parole chiave stenosi tracheobronchiale broncoscopia virtuale fibrobroncoscopia 1064 m.g. 
in these patients , a reproducible and objective noninvasive technique could prove useful , not only for a correct diagnosis , but also for guiding towards the most appropriate treatment and for evaluating response to treatment during follow - up [ 2 , 3 ]  . the standard diagnostic procedures used in stenotic lesions of the central airway include computed tomography ( ct ) and flexible bronchoscopy [ 24 ]  . 
although these techniques are highly precise and reliable , they both suffer some technical limitations that could lead to inaccurate characterisation of the airway disease [ 3 ]  . multidetector spiral ct allows the acquisition of thin - slice axial sections of entire body volumes during a single breathhold [ 57 ] , thus eliminating respiratory artefacts [ 8 ] even in poorly cooperating patients with severe airway disease [ 9 ]  . the possibility of obtaining multiplanar and three - dimensional ( 3d ) images has enabled the demonstration of focal airway stenoses and the localisation of lesions in endobronchial , peribronchial or submucosal sites [ 10 , 11 ]  . furthermore , 3d techniques have led to virtual bronchoscopy , a reconstruction technique that exploits the natural contrast between endoluminal air and surrounding tissue [ 9 , 12 ] , allowing navigation through the tracheobronchial tree [ 10 ] with the same endoluminal perspective as real endoscopy [ 13 , 14 ]  . 
virtual bronchoscopy is not operator dependent [ 14 ] , and reports have demonstrated a 63%100% sensitivity and a 61%99% specificity for the identification of central stenoses [ 1 , 3 , 9 , 12 ]  . 
these include the inability to perform biopsies for histological assessment [ 1 , 3 , 9 , 15 ] and to provide a chromatic distinction between normal mucosa and pathological tissue [ 1 , 8 ] and to determine its texture [ 10 , 16 ]  . conventional endoscopy therefore remains the gold standard for the identification and characterisation of airway lesions of any size [ 10 ]  . endoscopy , on the other hand , may fail to provide sufficient information owing to the lesion completely obstructing a bronchus and preventing the study of branches beyond , because of the presence of severe bleeding following a biopsy attempt , or because some concomitant condition ( advanced age , tracheomalacia , etc . ) advises against its use where not absolutely needed [ 10 ]  . 
in addition , it is an uncomfortable , poorly tolerated procedure that requires local sedation [ 8 , 9 ] and is associated with a 0.8% morbidity [ 3 , 10 , 17 ] and occasionally with well - known complications ( oxygen desaturation and tachycardia ) caused by the procedure itself or by the anaesthetic [ 8 , 9 ]  . 
nonetheless , the indisputable diagnostic and therapeutic value of the technique justifies its relative invasiveness and the risk of complications [ 9 , 18 , 19 ]  . le patologie ostruenti le vie respiratorie centrali , che siano di natura benigna o maligna , sono unimportante causa di mortalit e morbilit [ 1 , 2 ]  . le principali cause di compromissione locale delle vie aeree includono : neoplasie intraluminali , compressioni estrinseche , tracheomalacia , tessuto granulomatoso aberrante e fibrosi [ 1 ]  . spesso , soggetti con lesioni di questo tipo , coinvolgenti il tratto respiratorio prossimale , esordiscono con un quadro di ostruzione bronchiale parziale o completa [ 2 ]  . 
in tali pazienti un metodo non invasivo , riproducibile ed oggettivo potrebbe rivelarsi utile non solo per una corretta diagnosi , ma anche per indirizzare verso unappropriata terapia e ad una successiva valutazione della risposta al trattamento [ 2 , 3 ]  . le procedure diagnostiche standard a cui vengono sottoposti i pazienti con patologie del torace , determinanti stenosi delle vie aeree centrali , includono la tc e lendoscopia con broncoscopio flessibile [ 24 ]  . 
sebbene queste metodiche siano altamente precise ed attendibili , entrambe presentano dei limiti tecnici che potrebbero essere causa di una inaccurata caratterizzazione della patologia aerea [ 3 ]  . la tc spirale multidetettore ha permesso lacquisizione di sezioni assiali a spessore sottile di interi volumi corporei , nel tempo di una singola breve apnea [ 57 ] , eliminando gli artefatti da respiro [ 8 ] anche in pazienti con gravi patologie aeree e , conseguentemente , con ridotta capacit collaborativa [ 9 ]  . la possibilit di ottenere immagini multiplanari e tridimensionali ha consentito di dimostrare stenosi focali delle vie aeree e di localizzare la lesione in sede endobronchiale , peribronchiale o sottomucosa [ 10 , 11 ]  . le tecniche 3d , inoltre , hanno permesso un tipo di ricostruzione , la broncoscopia virtuale , che , sfruttando il naturale contrasto tra il contenuto aereo endoluminale e il tessuto circostante [ 9 , 12 ] , consente di navigare allinterno dellalbero tracheo - bronchiale [ 10 ] , riproducendo la stessa prospettiva endoluminale del reale esame endoscopico [ 13 , 14 ]  . 
la broncoscopia virtuale non un esame operatore - dipendente [ 14 ] e numerosi lavori in letteratura ne hanno dimostrato una sensibilit nella individuazione di stenosi centrali del 63%100% e una specificit del 61%99% [ 1 , 3 , 9 , 12 ] ; ci nonostante , lindagine virtuale attualmente non pu sostituire quella convenzionale , in quanto non scevra da limiti . 
limite intrinseco alla metodica lincapacit di eseguire prelievi bioptici per lanalisi isto - citologica del tessuto [ 1 , 3 , 9 , 15 ] e di operare una distinzione cromatica tra la mucosa normale e il tessuto patologico [ 1 , 8 ] n tanto meno di valutare la sua consistenza [ 10 , 16 ]  . attualmente lendoscopia reale rimane lesame goldstandard nellindividuazione e tipizzazione delle lesioni delle vie aeree anche di piccole dimensioni [ 10 ]  . a volte la broncoscopia non d informazioni sufficienti , in quanto la lesione ostruisce completamente un bronco e di conseguenza non possono essere valutati i rami a valle 1065 m.g. 
furthermore , we believe that it may be suggested as a valuable tool both for planning treatment and for evaluating response to treatment during the follow - up of patients with cancer and those with severe stenosis , whose clinical conditions are severely compromised . materials and methods between december 2004 and october 2005 , 25 patients ( 16 men and nine women , age range 6083 years ) underwent virtual endoscopy at our radiology department . 
si aggiunga che lendoscopia convenzionale una procedura scomoda e poco tollerata dai pazienti , motivo per cui richiesta la sedazione locale [ 8 , 9 ] ; inoltre associata ad una morbilit dello 0 , 8% [ 3 , 10 , 17 ] e , in rari casi , a ben note complicanze ( desaturazione di ossigeno e tachicardia ) , intrinseche alla procedura stessa , nonch allanestesia [ 8 , 9 ]  . 
lindiscutibile valore diagnostico e terapeutico della procedura , tuttavia , ne giustifica la relativa invasivit ed il rischio di complicanze [ 9 , 18 , 19 ]  . in considerazione a ci , quindi , possiamo ritenere le due tecniche non antagoniste tra loro , bens indagini complementari [ 9 , 20 ] , che , integrando le loro informazioni , sono in grado di fornire unaccurata caratterizzazione pre - operatoria delle patologie tracheo - bronchiali , che si potr poi tradurre in un aumento dellefficacia terapeutica delle stesse [ 11 ]  . lo scopo del nostro studio non quello di proporre la broncoscopia virtuale come alternativa a quella reale , ma come indagine preliminare nellabbreviare i tempi di esecuzione della successiva procedura endoscopica . 
crediamo , inoltre , possa essere proposta sia come valido strumento per pianificare il trattamento terapeutico pi appropriato nel post - operatorio come follow - up per la valutazione della risposta al trattamento in pazienti oncologici e nei pazienti con gravi patologie stenosanti , le cui condizioni cliniche siano molto compromesse . materiali e metodi nel periodo compreso tra dicembre 2004 e ottobre 2005 sono stati sottoposti a broncoscopia virtuale , presso la struttura complessa di radiologia ii dellazienda ospedalierouniversitaria di modena , 25 pazienti ( 16 uomini e 9 donne , di et compresa tra i 60 e gli 83 anni ) , con sospetta patologia steno - ostruttiva tracheo - bronchiale , dopo preliminare valutazione radiologica tradizionale e clinica . 
diffusione extrabronchiale della lesione stenosante 1 ( stenosi < 25% ) ; 2 ( 25% < stenosi > 50% ) ; 3 ( 50% < stenosi > 75% ) ; 4 ( stenosi > 75% )  . about 500 hu for optimal representation of the airmucosa interface , a bronchoscopic study of the tracheobronchial tree was performed . the endoscopic images obtained were mounted as a sequence to simulate endoscopic navigation . 
this confirmed the presence of a lesion , the degree of obstruction and the patency and accessibility of the airway beyond the obstruction , as shown in table 1 . image processing and interpretation took 15 min on average , providing the endoscopist with the data needed to guide conventional bronchoscopy . 
this reduced bronchoscopy times by almost 10 min , with increased patient comfort . images obtained with virtual bronchoscopy before and after treatment were then compared with the findings of fiberoptic bronchoscopy . 
 conventional bronchoscopy conventional bronchoscopy was performed by an experienced pneumologist with a videobronchoscope ( epk - 1000 ; pentax , tokyo , japan ) and with the patient under local anaesthesia . before conventional endoscopy , the virtual bronchoscopy images were made available to the bronchoscopist to provide guidance on the examination path and the precise position of the region of interest . the conventional bronchoscopy images of the stenoses were then compared with those obtained with virtual bronchoscopy . 1068 diametro delle vie aeree normali adiacenti , in : grado 1 : restringimento del lume fino al 25% ; grado 2 : dal 25% al 50% ; grado 3 : fino al 75% ; grado 4 : superiore al 75% . in seguito , utilizzando il programma di endoscopia virtuale , con algoritmo di ricostruzione volume rendering e valore soglia di rappresentazione dellinterfaccia aria - mucosa intorno a 500 uh , stato condotto uno studio broncoscopico attraverso lalbero tracheobronchiale . le immagini endoscopiche cos realizzate sono state montate in sequenza , realizzando in tal modo la simulazione della visione endoscopica in movimento . 
 results concordance of the virtual bronchoscopy and fiberoptic bronchoscopy was evaluated with regard to identification , localisation and characterisation of the tracheobronchial stenoses before and after treatment . results before treatment the quality of the virtual endoscopy images was excellent for all patients , who were able to maintain the breath - hold for the entire duration of scan ( 68 s ) , even in cases of severe respiratory failure . among the 25 patients with proximal airway obstruction studied by chest ct with image processing followed by conventional bronchoscopy , four had benign lesions and 21 had malignant lesions ( table 2 )  . 
 a patient with a finding of aortic arch aneurysm had been referred for virtual bronchoscopy to confirm a suspicion of mediastinal mass , possibly causing extrinsic compression of table 2 tracheobronchial obstructive lesions lesion patients , n malignant lesion : squamocellular carcinoma small cell cancer adenocarcinoma mesothelioma benign lesion : sarcoidosis tracheomalacia endobronchial lipoma right aortic arch aneurysm patologia patologie maligne : squamocellulare microcitoma adenocarcinoma mesotelioma patologie benigne : sarcoidosi tracheomalacia lipoma endobronchiale aneurisma dellarco aortico destro - posto tabella 2 patologie ostruttive tracheo - bronchiali pazienti , n risultati pre - trattamento la qualit delle immagini endoscopiche virtuali stata eccellente in tutti i pazienti , che sono stati in grado di mantenere lapnea per lintera durata della scansione ( 68 s ) , anche nei casi di grave insufficienza respiratoria . dei 25 pazienti con patologia ostruente le vie aeree prossimali , sottoposti a tc del torace con elaborazione delle immagini e successiva broncoscopia reale , 4 hanno presentato lesioni di natura benigna e 21 di natura maligna ( tabella 2 )  . 
 nel caso del riscontro dellaneurisma dellarco aortico , il paziente ci stato inviato per lesecuzione dellindagine virtuale nel sospetto di massa mediastinica quale causa di compressione estrinseca delle vie aeree prossimali , in un quadro di insufficienza respiratoria e radiografia del torace apparentemente negativa . 
in questo caso , il paziente aveva gi eseguito fibrobroncoscopia che aveva rivelato un bomb in corrispondenza della parete posteriore della trachea , con riduzione del lume di circa il 50% . 
si trattava , infatti , di un aneurisma dellaorta toracica con arco destro - posto che , anzich localizzarsi anteriormente rispetto alla trachea e passare a ponte al di sopra del bronco principale sinistro , si dirigeva posteriormente alla trachea e allesofago , con normale decorso nel restante tratto discendente . per quanto riguarda la sede delle stenosi , la broncoscopia virtuale ha fornito informazioni sovrapponibili a quelle della broncoscopia reale . 
in particolare sono state riscontrate 4 stenosi della trachea , 5 del bronco principale destro , 4 del bronco intermedio , 12 nel bronco principale sinistro , 6 del bronco lobare superiore sinistro , 4 del bronco lobare inferiore sinistro . 
sono state riscontrate : 1 stenosi di grado i ; 5 di grado ii ; 2 di grado iii ; 18 di grado iv . tali dati sono risultati sovrapponibili alla valutazione semiquantitativa descritta in broncoscopia reale . 
in un unico caso lesame fibrobroncoscopico non ha confermato lesistenza di una stenosi di grado i , documentata allindagine virtuale , al iii medio della trachea , attribuibile alla presenza di secrezioni viscose , con una concordanza pari al 96% . lendoscopio virtuale stato in grado di passare oltre stenosi di grado iv , con lume residuo fino a 3 mm , riuscendo ad esplorare zone inaccessibili al broncoscopio flessibile . con lausilio delle immagini assiali e delle mpr , abbiamo documentato lestensione intraed extra - bronchiale , la perviet o meno delle vie aeree a valle ed il coinvolgimento delle strutture mediastiniche da parte della lesione stenosante . 
ct with mpr and virtual bronchoscopy demonstrated the presence of the stenosis and its vascular origthe stenosis was in fact caused by a thoracic aorta aneurysm with a right - sided aortic arch that , instead of lying anterior to the trachea and passing over the left main bronchus , ran posterior to the trachea and oesophagus , with an otherwise normal course in the remaining descending branch . as concerns the site of stenosis , virtual bronchoscopy provided identical data to those yielded by conventional bronchoscopy . 
in detail , there were four stenoses of the trachea , five of the right main bronchus , four of the intermediate bronchus , 12 of the left main bronchus , six of the left upper lobar bronchus and four of the left lower lobar bronchus . stenoses involving the left upper lobar bronchus and the left lower lobar bronchus were seen in cases with obstruction of the main bronchi . 
degree of stenosis was as follows : one grade - 1 stenosis five grade - 2 stenoses two grade - 3 stenoses eighteen grade - 4 stenoses these data were very similar to those obtained from the semiquantitative evaluation at conventional bronchoscopy . only in one case did fiberoptic bronchoscopy fail to confirm a grade - 1 stenosis of the middle third of the trachea that the virtual study had detected , possibly as a result of the presence of viscous secretions ; agreement was 96% . the virtual endoscope was able to pass through grade - 4 stenoses , with a residual lumen of 3 mm , thus succeeding in table 3 follow - up of benign lesions sarcoidosis tracheomalacia endobronchial lipoma therapy drug conservative surgical resection stenosis degree pretherapy posttherapy * the patient underwent follow - up , without treatment tabella 3 follow - up lesioni benigne terapia grado di stenosi pretrattamento posttrattamento farmacologica sarcoidosi tracheomalacia conservativa lipoma endobrochiale resenzione chirurgica * la paziente non stata sottoposta a terapia , ma solo a follow - up strumentale 1070 risultati post - trattamento il follow - up con broncoscopia virtuale dopo trattamento stato eseguito su 15 dei 25 pazienti ; 9 sono deceduti alcune settimane dallinizio della terapia . tra le lesioni stenosanti di natura benigna ( tabella 3 ) il caso dellaneurisma aortico non stato compreso nel programma di controllo a distanza . 
esso consente : visualizzazione diretta del lume e della mucosa dellalbero tracheo - bronchiale con la possibilit di individuare eventuali alterazioni quali tumori o lesioni granulomatose ; biopsia di lesioni endobronchiali evidenti o sospette ; spazzolamento , lavaggio o biopsia di parti del polmone per la coltura e lesame citologico [ 19 ]  . la broncoscopia in grado di visionare e valutare cambiamenti della mucosa relativi a qualsiasi patologia : infiammazioni acute e croniche , lesioni endoluminali , compressioni ab - estrinseco , etc . il broncoscopio flessibile attualmente in grado di identificare e tipizzare le lesioni delle vie aeree centrali con una percentuale di falsi positivi molto bassa ( 0 , 8% ) e unaccurata diagnosi istologica nel 94% dei casi [ 10 ]  . ci sono casi per in cui la broncoscopia non in grado di fornire informazioni sufficienti ; ci si verifica quando la massa neoplastica ostruisce completamente un bronco . 
sn 4 intermedio indagine tc 0 main bronchus 4 upper lobar bronchus 1 left main bronchus 4 upper lobar bronchus 4 left main bronchus 2 intermediate bronchus bronchus 4 lower lobar bronchus post - trattamento 0 br . 
the data obtained were provided to the endoscopist before conventional bronchoscopy , resulting in reduced examination times ( approximately 10 min )  . results after treatment follow - up virtual bronchoscopy was performed on 15 of the 25 patients ; nine had died a few weeks after beginning treatment . 
the patient affected by tracheomalacia , with stenoa partire dagli anni 90 levoluzione tecnologica ha consentito di produrre apparecchiature tc che permettono lo studio di un intero volume anatomico con una singola acquisizione in apnea respiratoria [ 22 ] , grazie alla possibilit di associare la rotazione continua del sistema tubo - detettori alla consensuale traslazione longitudinale del tavolo porta paziente [ 23 , 24 ]  . la tc multistrato rappresenta unevoluzione della tc spirale , con importanti innovazioni per quel che riguarda la composizione dei detettori ; soprattutto lutilizzo di multiple file di detettori , invece che di ununica fila , ha aumentato di molto non solo laccuratezza diagnostica , ma anche la qualit delle immagini e delle ricostruzioni tridimensionali , gra1071 m.g. 
3a - g pretreatment evaluation of left hilar lesion ( a ) causing a grade - 4 stenosis of the ipsilateral main bronchus , at a distance of 40 mm from the carina , and upper - lobe subatelectasis ( be )  . 
3a - g valutazione pre - trattamento di lesione ilare sinistra ( a ) che provoca stenosi di grado 4 del bronco principale omolaterale ad una distanza di 40 mm dalla carena e subatelettasia del lobo superiore ( b - e )  . 
the technique in fact , allows : direct visualisation of the lumen and mucosa of the tracheobronchial tree , with the possibility of detecting alterations such as tumours or granulomatous lesions biopsy of evident or suspicious endobronchial lesions brushing , lavage or biopsy of parts of the lung for cytologic analysis [ 19 ] bronchoscopy is able to detect and assess mucosal changes resulting from any disease : acute and chronic inflammations , endoluminal lesions , extrinsic compression , etc . the flexible bronchoscope can currently identify and characterise central airway lesions with very few false positive results ( 0.8% ) and provide an accurate histological diagnosis in 94% of cases [ 10 ]  . however , in some cases , bronchoscopy is unable to provide sufficient information , such as when a bronchus is completely obstructed by a neoplastic mass . 
there are very few absolute contraindications to bronchoscopy : unstable cardiovascular status , severe cardiac arrhythmia , very severe refractory hypoxaemia , congestive heart failure and bronchial spasm [ 18 , 21 ]  . la tc spirale multistrato ha inoltre aperto interessanti prospettive nellinterpretazione di peculiari aspetti della patologia bronco - polmonare , con lausilio dei software di ricostruzione e lo sviluppo della tecnica endoscopica virtuale [ 24 ]  . 
sono inoltre in grado di fornire preziose informazioni sui rapporti che la lesione contrae con le strutture mediastiniche adiacenti [ 11 ]  . ct imaging broncoscopia virtuale beginning in the 1990s , technological progress has led to the production of ct scanners permitting the study of an entire anatomical volume during a single breath - hold [ 22 ] , thanks to the association of the constant rotation of the tube - detector system with the simultaneous longitudinal translation of the patient table [ 23 , 24 ]  . la broncoscopia virtuale con tomografia computerizzata spirale rappresenta una tecnica complementare sul piano diagnostico a quella tradizionale , con interessanti risultati nella individuazione delle lesioni endobronchiali [ 24 , 26 ]  . 
4a - e after chemotherapy , multiplanar reconstructions ( mpr ) demonstrate patency of the left main bronchus , which appears to have reduced calibre ( grade - 3 stenosis ) and to be stretched anteriorly ( ac )  . 
in particular , the use of multiple rows of detectors instead of a single row has significantly improved not only diagnostic accuracy but also the quality of images and 3d reconstructions . 
these scanners can , in fact , acquire up to 64 slices per rotation , with a tube rotation time equal to or less than 0.5 s , which results in faster scans compared with single - slice spiral ct and the possibility of studying large volumes with thin slices [ 24 ]  . the advantages are many and include improved temporal resolution , resulting from reduced acquisition time for each rotation and fewer voluntary and involuntary motion artefacts ( peristalsis , breathing , etc . ) , and reduced volumetric acquisition time [ 24 ]  . 
furthermore , they provide valuable information on the relationships between the lesion and the surrounding mediastinal structures [ 11 ]  . virtual bronchoscopy spiral ct virtual bronchoscopy is a complementary diagnostic technique to conventional bronchoscopy , which has shown interesting results in the detection of endobronchial lesions [ 24 , 26 ]  . 
by exploiting the natural contrast between luminal content and surrounding tissue [ 2 , 3 ] , the 3d reconstruction software allows the user to navigate through the tracheobronchial tree [ 10 ] with same endoluminal perspective as conventional endoscopy [ 13 , 14 ]  . the bronchoscopic images are displayed on the monitor alongside the multiplanar ct axial , sagittal and coronal oblique reference sections , on which the position and direction of the virtual bronchoscope is marked with a cursor . 
this makes it possible to explore the tracheobronchial tree from the inside by moving the cursor on the reference ct sections or by navigating the airway with a kind of virtual airplane , whose direction and speed are controlled through a mouse [ 27 ]  . virtual endoscopy images have a strong visual impact and provide a more complete and panoramic view not only of the 1074 naturale contrasto tra il contenuto aereo endoluminale e il tessuto circostante [ 2 , 3 ] , consente di navigare allinterno dellalbero tracheo - bronchiale [ 10 ] , riproducendo la stessa prospettiva endoluminale del reale esame endoscopico [ 13 , 14 ]  . tali visioni broncoscopiche vengono rappresentate su monitor consensualmente alle sezioni tc multiplanari sul piano assiale , sagittale e coronale obliquo di riferimento , sulle quali vengono evidenziate , mediante un cursore , la posizione e lorientamento del broncoscopio virtuale . 
possibile in tal modo esplorare linterno dellalbero tracheobronchiale spostando il cursore sulle sezioni tc di riferimento o navigando al suo interno utilizzando una sorta di aeroplano virtuale , la cui direzione e velocit vengono regolate utilizzando il mouse [ 27 ]  . lendoscopia virtuale fornisce immagini di grande impatto visivo e fornisce una visione pi panoramica e completa non solo dellalbero tracheo - bronchiale ma anche di tutti gli organi e strutture circostanti [ 28 ]  . 
la adeguata selezione di questo parametro assume una notevole rilevanza in considerazione del fatto che le dimensioni apparenti di una lesione e la sua stessa morfologia , variano nella rappresentazione endoscopica virtuale in funzione del valore soglia prescelto . 
in particolare , quanto pi il valore negativo , tanto pi le dimensioni della lesione risulteranno sovrastimate . la possibilit di percezione delle lesioni molto piccole dipende strettamente dal pitch e dalla collimazione , in un rapporto ormai noto , e cio quanto pi bassi sono tanto pi alta sar la capacit di individuare piccole lesioni [ 24 ]  . 
nel nostro studio stato scelto un protocollo che prevedeva collimazione di 1 , 25 mm con sovrapposizione di 0 , 6 mm , tempo di rotazione di 0 , 6 secondi e noise index di 11 . 
tuttavia , nonostante lottimizzazione dei parametri di scansione , ricostruzione e visualizzazione finalizzata alla riduzione degli artefatti ed al miglioramento della definizione dellimmagine , si avr sempre una certa distorsione dellimmagine correlata alleffetto della rappresentazione prospettica [ 24 ]  . 
da ci deriva come la misura delle dimensioni di una lesione sia maggiormente attendibile sulle ricostruzioni assiali trasverse o multiplanari ( mpr ) bidimensionali che non sulle ricostruzioni broncoscopiche virtuali [ 24 ]  . vantaggi la broncoscopia virtuale ha senzaltro una migliore tolleranza da parte del paziente rispetto a quella convenzionale , risultando un metodo non invasivo , privo delle controindicazioni e delle complicanze proprie della fibrobroncoscopia e affidabile nella valutazione delle patologie delle vie aeree [ 12 , 26 ]  . 
in our study , we used a value of 500 hu as the threshold for optimal representation of the air - mucosa interface , based on our experience and the data reported in the literature . correct selection of this parameter is very important in view of the fact that in the virtual endoscopic representation , the apparent size and morphology of a lesion will change depending on the threshold value selected . 
in particular , the more negative the value , the greater the overestimation of lesion size . the conspicuity of very small lesions closely depends on pitch and collimation , with lower values improving the ability to identify small lesions [ 24 ]  . 
however , despite optimisation of the scan , reconstruction and visualisation parameters to reduce artefacts and improve image resolution , some image distortion will always occur as a result of the perspective rendering effect [ 24 ]  . 
for this reason , measurement of lesion size is more reliable on 2d transverse or axial mpr than on virtual bronchoscopy reconstructions [ 24 ]  . advantages virtual bronchoscopy is no doubt better tolerated by patients than is conventional endoscopy , as it is noninvasive , does not have the contraindications or complications of fiberoptic bronchoscopy , and is very reliable in the assessment of airway disease [ 12 , 26 ]  . 
as a consequence , this technique has become the modality of choice to evaluate response to medical or surgical treatment in cancer patients or to follow up patients who have undergone self - expanding stent placement as a palliative procedure [ 5 , 13 ]  . 
in addition , unlike conventional bronchoscopy [ 14 ] , virtual bronchoscopy is not operator dependent and can be performed in patients in poor clinical condition or in subjects with severe airway stenosis that precludes endoscopic investigation [ 1 ]  . 
in these cases , during the endoscopic simulation , segments can be explored that are not accessible to the bronchoscope , such as those distal to nonnegotiable stenoses or to segmental occlusions , allowing one to establish an indication for procedures that would never have been proposed ( endoscopic laser therapy , stent placement ) [ 6 , 27 ]  . a further major advantage of virtual bronchoscopy is the simultaneous visualisation of both the airway lumen and the mediastinal and hilar structures . 
this is made possible by the display , alongside each virtual image , of the axial and multiplanar ( sagittal and coronal oblique ) reference images that not only enable the correct localisation of endobronchial disease but also provide anatomical and pathological information on structures outside the airway , thereby allowing easier distinction between stenosis and extrinsic compression [ 12 , 26 ]  . 
the images displayed on the monitor can be rotated in different directions to visualise the relationships between mediastinal structures and the airway [ 5 , 12 ]  . the main indication for virtual bronchoscopy is the study of tracheobronchial stenoses , as the technique enables the precise evaluation of the site , size and degree of obstruction , with the possibility of studying the inner surface of the tracheobronchial tree even beyond a subtotal stenosis [ 12 , 29 ]  . risposta ad eventuali terapie o procedure chirurgiche o nel follow - up di pazienti a cui sono stati posizionati stent autoespandibili a scopo palliativo [ 5 , 13 ]  . 
in aggiunta , la broncoscopia virtuale ha come vantaggio , rispetto allendoscopia reale , di non essere una procedura operatore - dipendente [ 14 ] e di poter essere eseguita in pazienti in cattive condizioni cliniche o in soggetti con stenosi severe delle vie aeree , tali per cui non sarebbe possibile lesecuzione dellindagine endoscopica [ 1 ]  . 
in tali casi , durante la simulazione endoscopica , quindi possibile esplorare segmenti non raggiungibili dal broncoscopio , come quelli a valle delle stenosi non superabili o delle occlusioni segmentarie , consentendo di porre lindicazione a procedure altrimenti non proponibili ( laserterapia endoscopica , stent ) [ 6 , 27 ]  . un ulteriore grande vantaggio diagnostico della tecnica virtuale dato dalla possibilit di visualizzare simultaneamente sia il lume delle vie respiratorie che le strutture extra luminali mediastiniche ed ilari . 
questo grazie alla disponibilit , accanto ad ogni immagine virtuale , di immagini assiali e multiplanari ( sagittali e coronali oblique ) di riferimento che consentono non solo una esatta localizzazione di eventuali patologie endobronchiali , ma anche di avere informazioni anatomiche e patologiche sulle strutture esterne alle vie aeree , distinguendo , in tal modo , pi facilmente una stenosi endoluminale da una compressione estrinseca [ 12 , 26 ]  . 
le immagini sul monitor possono essere ruotate secondo diverse angolazioni , aiutando nella visualizzazione dei rapporti tra le strutture mediastiniche e le vie aeree [ 5 , 12 ]  . la principale indicazione della broncoscopia virtuale rappresentata dallo studio delle stenosi dellalbero tracheo - bronchiale , in quanto tale tecnica consente di definire esattamente la sede di ostruzione , lentit ed il grado con possibilit di visualizzare e studiare dallinterno lalbero tracheo - bronchiale oltre una stenosi anche subtotale [ 12 , 29 ]  . 
tali informazioni risultano piuttosto preziose in caso di stenosi neoplastica , nella pianificazione preoperatoria o in previsione del posizionamento di stent endobronchiali per prevenire la completa chiusura del lume bronchiale [ 12 ]  . 
virtual bronchoscopy may precede conventional studies and provide a map of the patients airway anatomy , thus reducing the time needed for bronchoscopic investigation of obstructing lesions [ 1 , 16 ]  . limitations partial volume artefacts , difficulties in visualising bronchial branches with complex orientation , and the differential diagnosis between vegetative lesions and mucosal irregularities on the one hand , and endoluminal pseudolesions related to the presence of mucus , secretions and mucosal sloughing ( responsible for partial volume artefacts ) on the other hand , are the main limitations of the technique [ 24 ]  . another important clinical and diagnostic pitfall is the inability to detect early mucosal changes ( small surface lesions , such as ulcers or plaques ) or the submucosal lymphatic permeation seen in some neoplasms [ 1 , 2 ]  . in addition , data provided by virtual bronchoscopy images alone do not allow distinction between stenoses due to endobronchial masses or to extrinsic compression ; the analysis of surrounding tissues and the study of the length of the stenosis requires simultaneous visualisation of the mpr [ 24 ]  . further limitations include the inability to biopsy the tissue for histological examination [ 1 , 3 , 9 , 15 ] and the fact that , although virtual bronchoscopy can assess the shape of a lesion [ 3 ] , it is unable to distinguish the colour of normal mucosa and pathological tissue [ 1 , 8 ] or to show their texture [ 10 , 16 ]  . conclusions ct and fiberoptic endoscopy are highly sensitive techniques for the evaluation of stenoses of the tracheobronchial tree , although they both present technical limitations that may lead to inaccurate characterisation [ 3 ]  . the introduction of volumetric spiral ct , including mpr and virtual bronchoscopy , has dramatically improved accuracy in establishing the site , extension and severity of endobronchial abnormalities [ 1 ] , thus providing important information for treatment planning . 
virtual bronchoscopy cannot , however , be regarded as an alternative to conventional bronchoscopy but , rather , as its integration , given that the two techniques are complementary [ 27 ]  . our study , performed on obstructive disease of the proximal airway , has demonstrated the high diagnostic accuracy of virtual bronchoscopy , which , when integrated with mpr images , provides data for the detailed classification of tracheobronchial diseases before and after treatment . 
anche la diffusione linfatica sottomucosa presente in alcune forme neoplastiche difficilmente identificabile [ 1 , inoltre , con il solo apporto di informazioni fornite dalle immagini di broncoscopia virtuale non si in grado di distinguere tra stenosi determinate da masse a sviluppo endobronchiale o sottomucoso da compressione ab - estriseco ; la valutazione dei tessuti vicini e la stima dellestensione in lunghezza di una stenosi richiede la simultanea visualizzazione di immagini di ricostruzione multiplanare [ 24 ]  . limite intrinseco alla metodica inoltre lincapacit di eseguire prelievi bioptici per lanalisi isto - citologica del tessuto [ 1 , 3 , 9 , 15 ] e , sebbene riesca a valutare la forma lesionale [ 3 ] , non in grado di distinguere il colore tra la mucosa normale e il tessuto patologico [ 1 , 8 ] n la sua consistenza [ 10 , 16 ]  . conclusioni la tc e lendoscopia a fibre ottiche sono tecniche altamente sensibili per la valutazione delle stenosi dellalbero tracheo - bronchiale , ma entrambe non scevre da limiti tecnici che possono essere causa di una inaccurata caratterizzazione della patologia aerea [ 3 ]  . con lintroduzione della tc spirale volumetrica , inclusa lmpr e la broncoscopia virtuale , sono stati fatti passi avanti nellaccuratezza con cui viene determinata la sede , lestensione e la severit delle anomalie endobronchiali [ 1 ] , fornendo , in tal modo , preziose informazioni per la successiva decisione sul trattamento pi appropriato da intraprendere . 
tuttavia lapproccio virtuale non pu essere considerato come alternativo alla valutazione broncoscopica convenzionale , bens integrativo , ritenendo le due tecniche complementari tra loro [ 27 ]  . il nostro studio , condotto su patologie ostruenti le vie aeree prossimali , ha dimostrato una elevata capacit diagnostica della broncoscopia virtuale che , integrata con immagini mpr , ha fornito utili informazioni per una classificazione dettagliata della patologia tracheo - bronchiale prima e dopo trattamento . 
in considerazione di ci , le ricostruzioni multiplanari e di broncoscopia virtuale possono essere proposte come indagini preliminari per una accurata caratterizzazione della lesione stenotica , per abbreviare i tempi di esecuzione della successiva procedura endoscopica e per la pianificazione del trattamento terapeutico pi appropriato . la broncoscopia virtuale , oltre che utile nel pre - operatorio , pu essere considerata un eccellente metodo riproducibile ed oggettivo per la successiva valutazione della risposta al trattamento in soggetti con stenosi severe delle vie aeree ed in condizioni cliniche tali per cui non sia possibile lesem.g. 
sofia 78 , 95123 catania , italy correspondence to : luca macarini , ab : via conte giusso 3 , 70125 bari , italy , tel : + 39 - 88 - 1736089 , fax : + 39 - 88 - 1733866 e - mail : l.macarini@unifg.it received : 27 december 2005 / accepted : 20 april 2006 / published online : 20 december 2006 abstract purpose . 
the study was performed with a 1.5 - tesla device and characterised by three phases : the first phase without contrast material ( unenhanced mri ) , the second after the administration of ferumoxides ( spio - mri ) , and the third , a double - contrast study following the injection of a bolus of paramagnetic contrast material ( dc - mri )  . 
the sensitivity of unenhanced mri , spiomri and dc - mri in identifying and characterising hepatic focal lesions was assessed , together with the diagnostic increment of one technique with respect to the others . 
lo studio rm stato effettuato con apparecchiatura da 1 , 5 tesla ed consistito di tre fasi : la prima senza mdc ( rm - base ) , la seconda dopo infusione di mdc spio , a contrasto singolo ( rm - spio ) e la terza dopo iniezione a bolo di mdc paramagnetico , a doppio contrasto ( rm - dc )  . 
sono stati valutati la sensibilit della rmbase , della rm - spio e della rm - dc nellidentificazione e nella caratterizzazione delle lesioni focali epatiche e lincremento diagnostico di una metodica rispetto alle altre . 
su 67 pazienti in 14 casi ( 20 , 8% ) erano presenti alterazioni della captazione e della distribuzione del mdc spio di tipo acde che limitavano lesame rm - spio . 
in 5 casi si trattava di fibrosi confluente , in 2 casi di cirrosi scompensata , in 1 caso di trombosi vascolare e in 1 caso di esiti cicatriziali di un paziente operato di 1087 l . 
twenty - three patients had a total of 68 lesions , which consisted of 37 dysplastic nodules ( dn ) , 19 hcc nodules , two relapses of hcc following chemoembolisation , two hcc associated with portal thrombosis , one cancer - cirrhosis , two angiomas and five small cysts . 
spio - luda were the result of vascular thrombosis in one case and fibrosis in four cases . in all of these cases , dc - mri proved useful for diagnosis . 
in our study , the combined use of two contrast agents , negative and positive , provided greater diagnostic confidence and caused no side effects in the patients . key words mr liver cirrhosis contrast medium resezione epatica per hcc . 
ventitre pazienti erano portatori di 68 lesioni rappresentate da : 37 noduli displasici , 19 noduli hcc , 2 recidive di hcc dopo chemioembolizzazione , 2 hcc associati a trombosi portale , 1 cancrocirrosi , 2 angiomi e 5 piccole cisti . 
la sensibilit nellidentificazione delle lesioni stata per la rm - base , la rm - spio e la rm - dc rispettivamente del 57 , 3% , 67 , 6% e 75% . 
lutilizzo combinato dei due mezzi di contrasto , negativi e positivi , consente una migliore confidenza diagnostica e nella nostra esperienza non ha determinato reazioni secondarie nei pazienti . parole chiave rm fegato cirrosi mezzi di contrasto introduction introduzione liver - specific superparamagnetic iron oxide ( spio ) contrast agents are used in the identification and characterisation of focal hepatic lesions and have been proposed for use in the staging of patients undergoing surgical procedures for focal hepatic malignancies [ 18 ]  . 
the use of ferumoxides has been proposed in the follow - up of patients with liver cirrhosis for the detection of hepatocellular carcinoma ( hcc ) , with excellent results [ 911 ]  . 
a number of studies claim that the dynamic study with the injection of a bolus of paramagnetic gadolinium - diethylenetriaminepentaacetic acid ( gd - dtpa ) contrast material is more effective , particularly in detecting small ( < 1.5 cm ) hcc [ 1215 ]  . 
other studies , in contrast , report better results with spio - enhanced imaging , [ 16 , 17 ] while still others have found increased diagnostic accuracy with the combined use of the two contrast agents [ 1822 ]  . reservations regarding the use of spio agents in liver cirrhosis are based on the possibility that the significant subversion of liver structure and function , intrahepatic vascular alterations , presence of inflammatory processes , and steatosis can alter the action of ferumoxides and reduce their effectiveness and that well - differentiated hcc can take up spio in the same way as regenerative nodules ( rn ) and dysplastic nodules ( dn ) , thus making their differentiation difficult [ 2329 ]  . 
the alterations in liver uptake and distribution of spio that can oc1088 i mezzi di contrasto ( mdc ) superparamagnetici epatospecifici a base di particelle di ossido di ferro ( spio ) sono utilizzati per lidentificazione e la caratterizzazione delle lesioni focali epatiche e il loro uso stato proposto per lo staging di pazienti da sottoporre a trattamenti chirurgici o loco - regionali per lesioni focali epatiche maligne [ 18 ]  . luso di mdc spio stato proposto anche nel follow - up dei pazienti portatori di cirrosi epatica per lidentificazione dellepatocarcinoma ( hcc ) con ottimi risultati [ 911 ]  . 
alcuni autori sostengono la superiorit dello studio dinamico dopo iniezione a bolo di mdc paramagnetico gd - dtpa soprattutto nellidentificazione dei piccoli ( 1 , 5 cm ) hcc [ 1215 ] , altri invece hanno rilevato migliori risultati con i mdc spio [ 16 , 17 ] ed altri infine hanno verificato un miglioramento diagnostico nellutilizzo combinato dei due mezzi di contrasto [ 1822 ]  . le riserve sulluso dei mdc spio nella cirrosi epatica si basano sulla possibilit che in tale patologia il notevole sovvertimento della struttura e delle funzioni epatiche , le alterazioni vascolari intraepatiche , la presenza di processi flogistici e la steatosi possano alterare il meccanismo dazione dei mdc spio , riducendone lefficacia e che gli hcc ben differenziati possano captare i mdc spio come i noduli di rigenerazione ( nr ) e i noduli displasici ( nd ) rendendone difficile lidentificazione e la caratterizzazione [ 2329 ]  . 
spio - luda appear on the mr images either as areas of hyperintense signal in which the contrast medium is absent or as areas of hypointense signal in which the contrast medium is more concentrated . 
nonetheless , spio - luda are an expression of changes to the liver parenchyma that occur in cirrhosis , and their recognition helps in understanding the causes of disease and provides useful information regarding its stage [ 30 ]  . as the survival of patients with cirrhosis is related to timely and appropriate treatment of hcc and its recurrences [ 31 , 32 ] , in cirrhotic patients with spio - luda or problems of focal lesion characterisation on spio - mri , completion of the mr examination with an injectable paramagnetic contrast medium having extracellular distribution could prove useful . 
to this end , several studies have proposed the sequential use during the same examination of spio contrast media and gadolinium chelates to exploit the increased contrast obtained with the simultaneous use of negative superparamagnetic and positive paramagnetic contrast media [ 2022 , 33 ]  . the aim of our study was to assess the role of doublecontrast mri ( dc - mri ) in the study of the cirrhotic liver with the sequential use of negative superparamagnetic spio and gadolinium - based positive paramagnetic contrast media and to compare the findings with unenhanced mri and spio - mri , paying special attention to those cases in which spio - luda were present on spio - mri . materials and methods sixty - seven patients affected by liver cirrhosis and scheduled for liver transplant underwent dc - mri . 
the screening protocol for all patients involved liver ultrasound ( us ) and alpha - fetoprotein testing every 6 months , and mri every 12 months or , in the presence of a hepatic nodule at us or elevated alpha - fetoprotein levels , every 6 months . 
the mri study was performed with a 1.5 - tesla device and consisted of three phases : the first without the use of contrast medium ( unenhanced mri ) ; the second , the single - contrast phase , after spio administration ( spio - mri ) ; and the third , the double - contrast study , after the injection of paramagnetic contrast medium ( dc - mri )  . 
sulle immagini rm le acde sono rappresentate da aree di segnale iperintenso in cui assente mdc o aree di segnale ipointenso in cui il mdc pi concentrato , possono simulare la presenza di lesioni focali o impedirne il riconoscimento , rendere difficoltosa la loro caratterizzazione o la valutazione delle loro dimensioni . 
in ogni caso le acde sono espressione di alterazioni del parenchima epatico che si verificano nella cirrosi e il loro riconoscimento permette la comprensione delle cause che le hanno determinate fornendo utili informazioni sullo stadio della malattia [ 30 ]  . siccome la sopravvivenza dei malati di cirrosi legata al trattamento tempestivo e adeguato dellepatocarcinoma e delle sue recidive [ 31 , 32 ] nei casi di cirrosi epatica in cui lesame rm eseguito con mdc spio presenti alterazioni del tipo delle acde o problemi di caratterizzazione di lesioni focali , potrebbe risultare utile completare lesame rm con limpiego di un mdc paramagnetico a distribuzione extracellulare , iniettabile a bolo . 
a tal fine alcuni autori hanno proposto lutilizzo sequenziale , nella stessa seduta di rm , dei mdc spio e dei chelati del gadolinio , per sfruttare lincremento di contrasto che si ottiene dallazione contemporanea di mezzi di contrasto superparamagnetici negativi e paramagnetici positivi [ 2022 , 33 ]  . scopo del nostro lavoro stato quello di valutare il contributo nello studio del fegato cirrotico della tecnica rm a doppio contrasto ( rm - dc ) , con impiego sequenziale di mdc superparamagnetici negativi spio e paramagnetici positivi a base di gadolinio , confrontandolo con gli esami rm di base e con mdc spio , con particolare attenzione a quei casi in cui allesame rm - spio si verificano alterazioni del comportamento contrastografico del tipo delle acde . materiali e metodi abbiamo studiato con rm - dc 67 pazienti affetti da cirrosi epatica in lista dattesa per trapianto epatico . 
in tutti i pazienti il protocollo di screening prevedeva lesecuzione semestrale di un esame ecografico del fegato , il dosaggio dellalfa - feto - proteina e lesecuzione annuale , o in seguito allidentificazione con lecografia di un nodulo epatico da caratterizzare o a un rialzo dellalfa - feto - proteina , dellesame rm . 
lo studio rm stato effettuato con apparecchiatura da 1 , 5 tesla ed consistito di tre fasi : la prima senza mdc ( rm - base ) , la seconda dopo infusione di mdc spio , a contrasto singolo ( rm - spio ) e la terza dopo iniezione a bolo di mdc paramagnetico , a doppio contrasto ( rm - dc )  . 
the slice thickness in all sequences was 8 mm with a 1 mm gap , the matrix was 128256 , and the field of view ( fov ) was 380450 mm with reconstruction algorithm of the matrix ( rfov ) of 3 / 4 . 
 we assessed the sensitivity of unenhanced mri , spiomri and dc - mri in detecting and characterising focal hepatic lesions , as well as the diagnostic increment of one technique with respect to the others . 
to assess the detection rate , we considered the number of lesions identified by unenhanced mri , spio - mri and dc - mri and calculated the sensitivity of the various techniques . 
to assess lesion characterisation , we evaluated three groups of images : the first group consisted of the unenhanced mr images , the second group consisted of the unenhanced mr images plus the spio - mr images , and the third group consisted of the images from the second group plus the dc - mr images . 
the following lesion parameters were considered : signal intensity ( si ) on the unenhanced mr images , rate of signal loss ( rsl ) on the spio - mr images compared with the unenhanced - mr images , and type of contrast enhancement ( ce ) on the dc - mr images . 
the structural characteristics of lesions , as revealed by the double - contrast technique , and in particular , vascularity and the presence of areas of necrosis or a pseudocapsule , were also assessed . the final diagnosis for lesion identification was made by radiologists working in consensus on the basis of the overall assessment of radiological examinations before and after mri and clinical - radiological follow - up in all cases . 
with regard to lesion characterisation , the definitive diagnosis was made on the basis of histological findings in 38 cases , lipiodol - ct in 13 cases , multidetector ct in 17 cases and clinical - radiological follow - up in all cases . 
liver and kidney function tests , blood tests and urinalysis were performed in all patients in the 2448 h before and after the mr study . results 1090 focal lesions were absent in 44 / 67 patients and present in 23 / 67 patients . 
in tutte le sequenze lo spessore di strato stato di 8 mm con gap di 1 mm , la matrice di 128256 , il campo di vista ( fov ) 380450 mm , con algoritmo di ricostruzione della matrice ( rfov ) di 3 / 4 . 
per lidentificazione stato considerato il numero delle lesioni identificate dalla rm - base , dalla rm - spio e dalla rm - dc calcolando rispettivamente la sensibilit delle varie metodiche . 
per la caratterizzazione sono stati valutati separatamente tre gruppi di immagini : il primo gruppo stato quello delle immagini ottenute dalla rm - base , il secondo da quello delle immagini della rm - base insieme a quelle della rm - spio ed il terzo da quello delle immagini della rm - base insieme a quelle della rm - spio e della rm - dc . 
sono stati considerati i seguenti parametri delle lesioni : sulle immagini rm di base le caratteristiche dintensit di segnale ( si ) ; in quelle dopo mdc spio la percentuale di perdita di segnale ( pps ) delle lesioni rispetto allesame di base ; in quelle a doppio contrasto il tipo di enhancement contrastografico ( ce ) dopo iniezione a bolo . 
sono state rilevate , inoltre , le caratteristiche strutturali delle lesioni che la tecnica a doppio contrasto ha permesso di evidenziare ed in particolare la vascolarizzazione , la presenza di aree di necrosi o di una pseudocapsula . la diagnosi finale per lidentificazione di lesione stata posta da un consenso di radiologi sulla base della valutazione complessiva delle indagini radiologiche pree post - rm e del follow - up clinico - radiologico in tutti i casi . 
per quanto riguarda la caratterizzazione delle lesioni la diagnosi definitiva stata fatta sulla base di accertamenti istologici in 38 casi , della lipiodol - tc in 13 casi , della tc multi - detettore in 17 casi e del follow - up clinico - radiologico in tutti i casi . in tutti i pazienti sono stati controllati prima e dopo 2448 h l . 
unenhanced magnetic resonance imaging ( mri ) ( a ) with t1 - weighted fast low - angle shot ( flash ) sequence reveals an irregular , multinodular appearance suspicious for recurrence at the site of resection ( arrows )  . 
la rm di base ( a ) , con sequenza flash t1w , dimostra un aspetto irregolarmente pluri - nodulare a livello della sede del pregresso intervento sospetto per recidiva locale ( frecce )  . 
these cases exhibited focal or diffuse areas of lack of spio uptake , which either simulated the presence of lesions or reduced the ability of spio to identify the in particular , in the case of decompensated cirrhosis , there was failure to absorb the contrast medium by the entire liver . 
in the patient with vascular thrombosis , dc - mri identified two hcc within an area of lack of spio uptake due to reduced blood flow , from which the two hcc could not be dissociated . 
in 14 casi ( 20 , 8% ) erano presenti alterazioni della captazione e della distribuzione del mdc spio tipo acde , di cui 9 in pazienti privi di lesioni e 5 in pazienti con lesioni focali . acde in pazienti privi di lesioni focali in 44 pazienti la rm non ha evidenziato le lesioni focali epatiche e tale dato stato confermato dal follow - up clinico - radiologico a 1 anno . 
in questi casi erano presenti aree focali o diffuse di mancata captazione del mdc spio che potevano simulare la presenza di lesioni o ne riducevano la capacit di identificazione del mdc spio . 
in tutti questi casi il completamento dellesame rm con la tecnica a doppio contrasto ha chiarito il quadro rm , confermando lassenza di lesioni focali . acde in pazienti con lesioni focali in 23 pazienti con lesioni focali epatiche erano presenti acde in 5 casi ( 21 , 7% )  . 
nel paziente con trombosi vascolare la rm - dc ha identificato due hcc allinterno di unarea di assenza di captazione del mdc spio per ridotto apporto vascolare e dalla quale i due hcc non erano dissociabili . 
contrast - enhanced computed tomography ( ct ) ( a ) demonstrates slight enhancement at the level of the right - lobe nodule ( arrows ) , suspicious for recurrence . 
unenhanced magnetic resonance imaging ( mri ) ( b ) with a spinecho ( se ) t2 - weighted sequence shows slight hyperintensity at the level of the nodule in the right lobe ( arrows )  . 
2a - d ripresa di malattia in paziente sottoposto a chemioembolizzazione di un hcc del lobo destro e piccolo hcc consensuale del lobo sinistro . la tc ( a ) con mdc dimostra un tenue ce a livello del nodulo del lobo destro trattato con chemioembolizzazione ( frecce ) sospetta per recidiva di malattia . 
la rm - spio ( c ) dimostra unassenza di captazione di mdc spio a livello della sede del nodulo sottoposto a chemioembolizzazione ( frecce ) e unaltra lesione nodulare del lobo sinistro ( punta di frecce )  . 
non sono stati identificati con tutte le tre metodiche rm 17 nd , riscontrati allesame istologico effettuato sui pezzi operatori dei pazienti sottoposti ad intervento chirurgico di resezione epatica , in quanto i nd presentavano caratteristiche di segnale e contrastografiche simili a quelle del parenchima epatico cirrotico limitrofo . identificazione delle lesioni in 23 / 67 pazienti sono state identificate 68 lesioni del diametro medio di 2 , 8 cm ( range 0 , 710 cm )  . 
le lesioni erano rappresentate da : 37 nd , 19 hcc , 2 recidive di hcc dopo chemio - embolizzazione , 2 hcc associati a trombosi portale , 1 cancro - cirrosi , 2 angiomi e 5 piccole cisti . 
la sensibilit nellidentificazione di lesione focale di diametro minimo di 1 cm circa stata rispettivamente per la rm - base , la rm - spio e la rmdc del 57 , 3% , 66 , 1% e 75% ( tabella 1 )  . 
la rm di base non ha identificato 7 hcc , 17 nd , le 2 recidive hcc dopo chemio - embolizzazione , i 2 hcc associati a trombosi portale e la cancro - cirrosi . 
unenhanced magnetic resonance imaging ( mri ) with fast low - angle shot ( flash ) t1 - weighted ( a ) and spin - echo ( se ) t2weighted ( b ) sequences reveals a slightly hyperintense nodule on the t2weighted images ( arrows )  . 
superparamagnetic iron oxide ( spio ) - mri ( c ) reveals an uptake of ferumoxide , which is barely distinguishable from the surrounding liver parenchyma ( arrows )  . 
lesame rm di base , con sequenze flash t1w ( a ) e se t2w ( b ) , dimostra una lesione nodulare discretamente iperintensa in nelle immagini t2w ( frecce )  . allesame rm - spio ( c ) la lesione presenta una captazione di mdc spio ed mal differenziabile dal parenchima epatico limitrofo ( frecce )  . 
4a - d massive cancer - cirrhosis of the left lobe with portal thrombosis . unenhanced magnetic resonance imaging ( mri ) ( a ) performed with t1weighted sequence shows a diffuse signal alteration in the left lobe , which appears inhomogeneously hypointense ( arrows )  . 
superparamagnetic iron oxide ( spio ) - mri ( b ) performed with t2 * - weighted sequence reveals lack of spio uptake by the left lobe ( arrows )  . 
4a - d cancro - cirrosi massiva del lobo sinistro con trombosi portale . lesame rm di base ( a ) , con sequenza t1w , dimostra una diffusa alterazione di segnale del lobo sinistro del fegato che appare disomogeneamente ipointenso ( frecce )  . 
lesame rm - spio ( b ) , con sequenza t2 * w , dimostra lassenza di captazione di mdc spio da parte del lobo sinistro epatico ( frecce )  . 
all three mri techniques missed 17 dn identified at histology of the resected liver specimens in that dn present signal and enhancement characteristics similar to those of the surrounding cirrhotic liver parenchyma . lesion identification in 23 / 67 patients , a total of 68 lesions with a mean diameter of 2.8 cm ( range 0.710.0 cm ) were identified . 
la rm - dc non ha identificato 17 nd . caratterizzazione delle lesioni i risultati ottenuti sul piano della caratterizzazione delle lesioni sono stati rispettivamente per la rm di base del 20 , 5% , per la rm - spio del 63 , 2% e per la rm - dc del 73 , 5% ( tabella 2 )  . 
la rm - base non ha caratterizzato nessun nd , nessuna recidiva hcc dopo chemio - embolizzazione , i due hcc associati a trombosi portale e la cancro - cirrosi , mentre ha caratterizzato solo 8 noduli hcc , e 1 angioma ; la rm - spio non ha caratterizzato 2 noduli hcc , 2 riprese di malattia in hcc trattati con chemio - embolizzazione , 1 angioma , i 2 hcc associati a trombosi portale e la cancro - cirrosi , mentre la rm - dc ha caratterizzato correttamente tutte le lesioni ad eccezione dei 17 nd , che non erano stati identificati , e di un angioma che stato erroneamente interpretato come un hcc . 
le formazioni cistiche sono state valutate correttamente con tutte tre le metodiche ma erano di difficile interpretazione con la rm - spio , se questa non era confrontata con le immagini di base senza mdc . incremento diagnostico lincremento diagnostico della rm - spio sulla rm base e della rm - dc sulla rm - spio stato rispettivamente nellidentificazione del 9% e del 8 , 9% ( tabella 1 ) e nella caratterizzazione del 42 , 7% e del 10 , 3% ( tabella 2 )  . reazioni avverse in nessun caso abbiamo registrato effetti collaterali indesiderati a seguito della somministrazione sequenziale dei due mdc , n si sono verificate alterazioni dei parametri bioumorali nei controlli a 2448 h . discussione i mezzi di contrasto superparamagnetici hanno migliorato notevolmente laccuratezza diagnostica della rm nello studio delle lesioni focali epatiche consentendo di identificare lesioni di dimensioni al di sotto del cm con elevata accuratezza diagnostica [ 35 , 3436 ]  . 
anche per quanto riguarda la caratterizzazione delle lesioni la rm - spio risultata valida e competitiva con la rm eseguita con mezzi di contrasto paramagnetici iniettabili a bolo [ 17 ]  . 
tuttavia ancora oggi non esistono risultati definitivi su quale sia il mdc e la tecnica di studio rm migliore per lo studio della cirrosi epatica e per lidentificazione e caratterizzazione degli hcc [ 10 , 1214 , 18 , 19 , 37 ]  . 
le due tecniche di studio rm pi utilizzate sono rappresentate da quella dinamica dopo iniezione a bolo di mdc paramagnetico gd - dtpa e da quella con mdc spio epatospecifico . 
i dati della letteratura che riguardano queste due metodiche sono discordanti . lo studio dinamico dopo iniezione a bolo di mdc gd - dtpa secondo alcuni presenta una maggiore sensibilit 1093 l . 
la rm - base ( a ) dimostra una piccola lesione nodulare tenuemente iperintensa ( frecce )  . la rm - spio ( b ) dimostra una mancata captazione della lesione ( frecce )  . 
la rm - dc ( c ) dimostra un marcato ce del piccolo hcc in fase arteriosa . consisted of 37 dn , 19 hcc , two hcc recurrences after chemoembolisation , two hcc associated with portal thrombosis , one cancer - cirrhosis , two angiomas and five small cysts . 
unenhanced mri failed to identify seven hcc , 17 dn , the two hcc recurrences following chemoembolisation , the two hcc associated with portal thrombosis and the cancer - cirrhosis . 
spiomri failed to identify two hcc , 17 dn , the two hcc recurrences following chemoembolisation and the two hcc nellidentificazione di piccoli hcc di diametro inferiore al cm [ 12 ]  . 
innanzitutto in tutti i lavori pubblicati su tale argomento la maggior parte dei piccoli hcc identificati come aree di enhancement vascolare in fase arteriosa non hanno avuto una conferma istologica [ 16 ]  . 
inoltre alcuni autori hanno dimostrato che oltre il 52% di queste aree di enhancement vascolare in fase arteriosa nel fegato cirrotico sono da riferire a shunt artero - venosi o a nd e non a hcc e che quindi lo studio con gd - dtpa altafig . 
unenhanced mri failed to characterise the dn , the hcc recurrences following chemoembolisation , the two hcc associated with portal thrombosis and the cancer - cirrhosis , whereas it correctly characterised only eight hcc and one angioma . 
spio - mri failed to characterise two hcc , the two hcc recurrences following chemoembolisation , one angioma , the two hcc associated with portal thrombosis and the cancer - cirrhosis . 
la rm - dc ( b ) dimostra un piccolo hcc ( freccia ) che presenta ce dopo iniezione di mdc paramagnetico . mente sensibile ma molto poco specifico [ 16 , 37 ]  . 
hanno rilevato che le piccole aree di enhancement vascolare in fase arteriosa con i mdc gd - dtpa che non presentano captazione di mdc spio sono sempre hcc e quindi nel loro lavoro hanno concluso che lutilizzo di entrambi i mdc migliora laccuratezza diagnostica della metodica [ 19 ]  . anche lo studio rm del fegato cirrotico con mdc spio pu presentare delle limitazioni in relazione alla presenza di acde causate con meccanismi patogenetici diversi daltable 1 identification of focal lesions : sensitivity of unenhanced magnetic resonance imaging ( mri ) , superparamagnetic iron oxide ( spio ) - mri and double - contrast ( dc ) - mri in 23 patients histological type no . 
with regard to lesion characterisation , spio - mri has also proved a valid technique , which is competitive with mri performed with injectable paramagnetic contrast media [ 17 ]  . 
nonetheless there are still no definitive findings as to which contrast medium and mri technique is best suited for studying liver cirrhosis and identifying and characterising hcc [ 10 , 13 , 14 , 18 , 19 , 37 ]  . 
the two most widely used techniques are dynamic mri following a bolus injection of paramagnetic gd - dtpa , and mri with liver - specific spio . the literature data on these two techniques are in disagreement . 
according to some studies , dynamic mri following a bolus injection of gd - dtpa has greater sensitivity in the identification of small hcc with a diameter less than 1 cm [ 12 ]  . 
firstly , in all the studies on the subject , the majority of small hcc identified as areas of arterial - phase enhancement were not confirmed histologically [ 16 ]  . 
in addition , some studies have shown that more than 52% of these areas of arterial - phase enhancement in the cirrhotic liver are caused by arteriovenous shunts or dn and not hcc , such that the study with gd - dtpa is highly sensitive but poorly specific [ 16 , 37 ]  . 
found that the small areas of arterial - phase enhancement with gd - dtpa that fail to take up spio are always hcc , leading the authors to conclude that the use of both contrast media increases diagnostic accuracy [ 19 ]  . spio - mri of the cirrhotic liver also suffers some limitations due to the presence of spio - luda caused by a variety of pathogenetic mechanisms , including fibrosis , vascular thrombosis , hepatitis and steatosis [ 20 ]  . 
such alterations may mimic the presence of focal lesions , prevent their detection or affect the assessment of lesion size or the results of lo1096 la fibrosi , dalle trombosi vascolari , delle epatiti e dalla steatosi [ 20 ]  . 
tali alterazioni possono simulare la presenza di lesioni focali , impedirne lidentificazione , alterare la valutazione delle loro reali dimensioni o dei risultati dei trattamenti loco - regionali con importanti risvolti clinici [ 2426 , 28 , 38 ]  . 
il meccanismo dazione dei mezzi di contrasto spio si basa sulla captazione selettiva delle nanoparticelle di ossido di ferro da parte delle cellule del sistema reticolo - endoteliale , in particolare dalle cellule di kupffer epatiche , presenti nel fegato normale e in alcune lesioni focali benigne e assenti nelle lesioni maligne [ 34 , 36 ]  . 
perch il mezzo di contrasto spio agisca sono fondamentali la presenza di un numero adeguato di cellule di kupffer con normale attivit macrofagica e la presenza di un normale flusso vascolare epatico per garantire un sufficiente apporto di mdc . 
le varie patologie alla base delle acde nel fegato cirrotico determinano , con meccanismi patogenetici differenti , alterazioni che agiscono direttamente sul funzionamento del mdc spio e quindi sulla sua captazione intracellulare e sulla sua distribuzione nel parenchima epatico . la fibrosi epatica non presenta al suo interno cellule di kupffer e allesame rm - spio rappresentata da aree iperintense di mancata captazione di mdc , di aspetto reticolare o focale , localizzate pi frequentemente in sede sottodiaframmatica , associate spesso a retrazione della superficie parenchimale . 
la fibrosi epatica assume un aspetto confluente nel 14% dei pazienti con cirrosi avanzata , con problemi di diagnosi differenziale con la cancro - cirrosi [ 28 , 39 ]  . le alterazioni vascolari , e in particolare la riduzione di afflusso vascolare conseguenti alle trombosi , possono determinare zone di ridotta o assente captazione di mdc spio , che assumono una morfologia legata al territorio tributario della struttura vascolare interessata e a volte possono simulare la presenza o impedire il riconoscimento di lesioni focali . 
alcuni autori , in particolare , hanno sottolineato la comparsa di pseudo - lesioni in pazienti studiati con mezzi di contrasto spio in sede peritumorale [ 29 , 40 ]  . tali pseudo - lesioni sarebbero determinate da riduzione del flusso vascolare conseguente a shunt vascolari peritumorali o da riduzione dellazione macrofagica per sofferenza l . 
the mechanism by which spio contrast agents act is based on the selective uptake of iron oxide nanoparticles by the cells of the reticuloendothelial system , especially the kupffer cells , which are present in the normal liver and in some benign focal lesions but absent in malign lesions [ 34 , 36 ]  . 
for the spio contrast agents to act , a sufficient number of kupffer cells with normal macrophagic activity must be present together with normal liver blood flow to guarantee a sufficient supply of contrast medium . the various conditions responsible for spio - luda in the cirrhotic liver cause changes that act directly on the functioning of the spio agent and therefore on its intracellular uptake and its distribution in the liver parenchyma . kupffer cells are not present within hepatic fibrosis so that at spio - mri , fibrosis appears as hyperintense areas of lack of spio uptake , with reticular or focal appearance , most frequently located directly below the diaphragm and often associated with retraction of the parenchymal surface . 
hepatic fibrosis has a confluent appearance in 14% of patients with advanced cirrhosis , thus complicating the differential diagnosis with cancer - cirrhosis [ 28 , 39 ]  . vascular changesand in particular , reduced blood supply due to thrombosiscan lead to areas of reduced or abdelle cellule di kupffer e si localizzano caratteristicamente in sede periferica , a valle della lesione neoplastica . 
gli esami bioptici di tali pseudo - lesioni hanno dimostrato parenchima epatico normale , ma anche aree di linfangite carcinomatosa e di flogosi peri - tumorale [ 40 ]  . 
sulle immagini rm dopo iniezione di mezzo di contrasto spio tali aree creano un alone peri - tumorale di assenza di mdc che non distinguibile dalla lesione neoplastica e ne impedisce la definizione dei margini . 
tale sovradimensionamento rende lesame non utilizzabile per una corretta valutazione topografica e volumetrica della lesione , soprattutto quando si voglia eseguire un trattamento locale di termoablazione o alcolizzazione in cui necessario stabilire con certezza i margini della lesione da trattare . 
such pseudolesions are thought to be caused by a reduction in blood flow resulting from peritumoural vascular shunts or a reduction in macrophage activity due to kupffer - cell damage , and they are characteristically found peripherally and distal to the neoplastic lesion . biopsy of pseudolesions has revealed normal liver parenchyma but also areas of carcinomatous lymphangitis and peritumoural inflammation [ 40 ]  . 
on spio - mr images , the areas of reduced or absent uptake produce a peritumoural halo of nonenhancement , which is indistinguishable from the neoplastic lesion and prevents definition of its margins . 
this size overestimation makes the examination useless for the correct assessment of lesion position and size , especially when planning local treatments such as thermoablation or alcohol ablation , which require precise definition of lesion margins . 
indeed , a reduction in spio uptake has been reported in areas of liver parenchyma that have undergone radiotherapy of a tumour and where there is a resulting postactinic hepatitis [ 41 ]  . decompensated cirrhosis in the acute phase may also exhibit reduced spio uptake due to inflammatory processes hampering the macrophagic activity of kupffer cells . 
in acute hepatitis , reduced spio uptake translates into an 11% signal loss compared with a 75% signal loss in normal liver parenchyma and a 52% loss in cirrhotic tissue [ 25 ]  . 
in hepatitis , there is a reduction in the function of the kupffer cells and a reduction in their number per gram of tissue [ 25 , 34 ]  . 
in addition , reduced hepatic clearance of spio is thought to be the result of reduced opsonisation , which is found in hepatitis and cirrhosis , because plasma fibronectin , an opsonin believed to enhance the phagocytic activity of kupffer cells , is reduced [ 25 ]  . in all of these cases where enhancement patterns may give rise diagnostic uncertainty , it might be worthwhile to assess not only intracellular spio uptake but also the dynamic vascular enhancement patterns after administration of gadolinium - based media . 
one study has proposed the combined use of two - positive and negative - contrast media , reporting better contrast - to - noise ratio between the liver and the lesion using the double - contrast technique [ 42 ]  . 
this approach enables the two contrast media to be administered in the same session without significant lengthening of examination times and with the summing of the different enhancement effects of the two media [ 20 ]  . 
moreover , the double - contrast technique can be seen as the completion of the spio - mr examination , and therefore the administration of gd - dtpa is optional and should be used only when diagnostic uncertainties are present . 1098 sentare una mancata o ridotta captazione del mdc spio per linibizione su base flogistica dellattivit macrofagica delle cellule di kupffer . 
nelle epatiti acute si ha una ridotta captazione di spio che si traduce in una perdita di segnale dell11% , rispetto alla perdita di segnale del 75% riscontrata nel parenchima epatico normale e del 52% in quello cirrotico [ 25 ]  . 
nellepatite , infatti , si ha una riduzione della funzionalit delle cellule di kupffer ed una loro riduzione numerica per grammo di tessuto [ 25 , 34 ] ; inoltre la ridotta clearance epatica di spio sarebbe imputabile anche ad una ridotta opsonizzazione , riscontrabile nelle epatiti come anche nella cirrosi , poich la fibronectina plasmatica , unopsonina che potenzierebbe lattivit fagocitaria delle cellule di kupffer , ridotta [ 25 ]  . in tutti questi casi in cui il comportamento contrastografico pu determinare delle incertezze diagnostiche pu essere utile valutare , oltre che la captazione del mdc spio intra - cellulare , anche il comportamento vascolare dinamico delle lesioni dopo somministrazione a bolo di mdc paramagnetici a base di gadolinio . 
a tale proposito alcuni autori hanno proposto luso combinato di due mezzi di contrasto , positivi e negativi , dimostrando un incremento del rapporto c / n tra fegato e lesione con la tecnica a doppio contrasto [ 42 ]  . 
tale metodologia consente la somministrazione di due mdc nella stessa seduta , senza un significativo allungamento del tempo desame e con sommazione degli effetti contrastografici differenti dei due mdc [ 20 ]  . 
 [ 21 ] hanno evidenziato con la tecnica a doppio contrasto una migliore definizione della caratteristiche strutturali interne delle lesioni , che ha consentito una diagnosi di natura pi agevole . 
 [ 22 ] hanno rilevato che la rm - dc pi accurata nellidentificazione delle lesioni rispetto alla rm - spio , mentre non hanno riscontrato differenze statisticamente significative nella caratterizzazione , pur segnalando una pi agevole confidenza diagnostica . tale autore , tuttavia , non ha utilizzato nel suo studio sequenze ge t2 * w in apnea , che si sono dimostrate successivamente le pi sensibili nellidentificazione delle lesioni quando utilizzato mdc spio [ 7 , 11 ]  . 
 [ 33 ] hanno rilevato , invece , una maggiore sensibilit della rmspio rispetto alluso combinato dei due mezzi di contrasto nellidentificazione delle lesioni ed hanno concluso che luso del doppio mezzo di contrasto non necessario nello studio del fegato cirrotico . nella nostra esperienza su pazienti con cirrosi epatica i maggiori vantaggi della doppia somministrazione di mdc si sono ottenuti quando erano presenti alterazioni della captazione e della distribuzione del mdc spio del tipo delle acde conseguenti a fibrosi , trombosi vascolare , cirrosi scompensata , trattamenti di chemio - embolizzazione o quando erano necessarie ulteriori informazioni per caratterizzare una lesione focale . 
in their study , however , the authors did not use ge t2 * - weighted breath - hold sequences , which have since proved to be more sensitive for lesion detection with spio - mri [ 7 , 11 ]  . 
in cases exhibiting spio - luda , the action of spio was reduced , creating difficulties in lesion detection and characterisation . completion of the mr study with a bolus injection of paramagnetic contrast material proved crucial for obtaining information useful for the diagnosis . 
this was done during the same mr session , immediately after the assessment of the spio - mr images , without having the patient leave the mri room and therefore without further time delays and logistic problems of rescheduling an mr examination . in lesion identification , the greater enhancement obtained with the sequential administration of two contrast media improved the diagnostic confidence of dc - mri with respect to spio - mri by 8.9%. 
in most cases , spio - luda were caused by fibrosis or vascular thrombosis , and the administration of gd - dtpa facilitated the recognition and delimitation of the focal lesions in the areas of nonuptake of spio . the sensitivity values in identifying focal lesions for the three techniques , and in particular the contrast - enhanced examinations , were not particularly high due to the failure to identify 17 low - grade dn , which were identified only at histology of the surgical specimen . 
these dn could not even be identified on review of the unenhanced and contrast - enhanced mr images in that they were not distinguishable from cirrhotic liver parenchyma or regenerative nodules . even in cases without focal lesions , dc - mri proved useful in the event of spio - luda preventing definitive exclusion of focal lesions . in lesion characterisation , administration of the two contrast media enabled the study of the lesions vascular dynamics , thus adding information that produced a diagnostic increment of 10.3% with respect to spio - mri . 
the additional information regarded the lesions structural characteristics , such as the presence of areas of necrosis or a pseudocapsule or the type of vascular enhancement , which permitted better assessment of the hcc and the two cases of hcc recurrence after chemoembolisation and differentiation from dn . 
with regard to characterisation of focal lesions , there may be problems differentiating between hcc and dn because both can display a hyperintense signal in the nonenhanced t1 - weighted sequences whereas they diserano presenti acde lazione del mdc spio era ridotta con difficolt di identificazione e caratterizzazione delle lesioni . 
stato possibile effettuare ci nella stessa seduta rm , immediatamente dopo la valutazione delle immagini ottenute con mdc spio , senza far uscire il paziente dalla sala di rm e , quindi , senza ulteriore perdita di tempo e senza problemi organizzativi relativi alla riprogrammazione dellesame nellidentificazione delle lesioni il maggior contrasto ottenuto dalla doppia somministrazione ha migliorato la confidenza diagnostica della rm - dc rispetto alla rmspio dell8 , 9% . 
nella maggior parte di questi casi erano presenti acde determinate da fibrosi o da trombosi vascolari in cui lapporto del mdc gd - dtpa iniettato a bolo ha consentito di riconoscere e delimitare meglio le lesioni focali nellambito di aree di mancata captazione di mdc spio . 
i valori di sensibilit nella identificazione delle lesioni focali delle tre metodiche di studio rm , in particolare di quelle contrastografiche , non sono risultati particolarmente elevati per la mancata identificazione di 17 nd a basso grado , che sono stati rilevati solo dallesame istologico su pezzo operatorio . 
questi nd non sono stati identificati neanche in una rivalutazione a posteriori delle immagini rm di base e contrastografiche , in quanto non erano differenziabili dal parenchima epatico cirrotico o dai noduli di rigenerazione . 
anche nei casi in cui non erano presenti lesioni focali la rm - dc stata utile quando alla rm - spio erano presenti alterazioni del tipo delle acde che non consentivano di escludere con certezza la presenza di lesioni focali . nella caratterizzazione delle lesioni , la doppia somministrazione di mdc permette di studiare la dinamica vascolare delle lesioni aggiungendo informazioni diagnostiche che hanno determinato un incremento diagnostico rispetto alla rm - spio del 10 , 3% . 
si sono aggiunte informazioni sulle caratteristiche strutturali delle lesioni , come la presenza di aree di necrosi o di una pseudocapsula o il tipo di enhancement vascolare , che hanno consentito una migliore valutazione degli hcc , della ripresa di malattia nei due casi di hcc sottoposti ad intervento di chemio - embolizzazione e della diagnosi differenziale con i nd . 
nellambito della caratterizzazione delle lesioni focali esistono in alcuni casi problemi di diagnosi differenziale tra i nd e gli hcc , poich possono apparire entrambi di segnale iperintenso nelle sequenze pesate in t1 senza mdc , mentre mostrano un comportamento di segnale variabile nelle sequenze pesate in t2 [ 43 ]  . 
tale comportamento contrastografico stato tipico in tutti i nd da noi evidenziati , successivamente sottoposti a controllo istologico , e differisce in maniera sostanziale da quello dei noduli di hcc , che non presentano captazione di mdc . 
these enhancement patterns were seen in all dn we encountered , which were later confirmed histologically , and differed substantially from those of hcc nodules , which presented no uptake of the contrast mediuthe greater uptake of spio by dn could be due to increased activity of the kupffer cells [ 44 ] and the different vascularisation , which is portal in dn but prevalently arterial in hcc [ 45 ]  . 
in the case of angiomas , however , the dc - mri examination failed to demonstrate the typical centripetal enhancement pattern after the bolus injection of gd - dtpa due to the hyperintensity of the signal typical of angiomas in t1 - weighted sequences after spio administration , which tends to mask the subsequent action of the paramagnetic contrast mediuin angiomas , spio remains in the extracellular space within the vascular sinusoids in a nonbonded forin this form , spio presents paramagnetic characteristics , with a hyperintense signal in t1 - weighted sequences known as t1 effect , characteristics that are typical of angiomas . 
this was , however , one of the first cases studied , and a more careful assessment of the images before and after contrast enhancement would have avoided the error . in our study there were no side effects or changes in biohumoural parameters after the administration of the contrast media . 
this time interval does not compromise the contrast features of spio in that its maximum liver concentration occurs within the fourth hour whereas its plasma half - life of about 10 min enables the delayed administration of gd - dtpa without pharmacological interaction of the two contrast media [ 42 , 46 ]  . 
in addition , the distribution and elimination of the two contrast media have completely different pathways , and pharmacological studies have demonstrated no interactions between the two contrast media in vitro [ 21 , 42 ]  . conclusions the combined sequential use of two - negative and positivecontrast media improves diagnostic confidence and in our study caused no side effects in patients . 
nonetheless , the technique can be useful to complete the spio - mr examination in cases of diagnostic uncertainty and in the presence of spioluda , increasing the diagnostic accuracy of the method . 1100 il differente tipo di vascolarizzazione che nei nd di tipo portale mentre negli hcc di tipo prevalentemente arterioso [ 45 ]  . 
nella nostra esperienza tutti gli hcc hanno presentato un enhancement vascolare arterioso tipico ed un wash - out in fase portale , mentre i nd non hanno presentato enhancement vascolare . 
negli angiomi invece , con la tecnica a doppio contrasto , non stato possibile dimostrare il comportamento vascolare tipico di enhancement centripeto dopo iniezione a bolo di gd - dtpa a causa delliperintensit di segnale tipico degli angiomi nelle sequenze pesate in t1 dopo iniezione di mdc spio che mascherava la successiva azione del mdc paramagnetico . 
in questa forma il mdc spio ha un comportamento paramagnetico , presenta un segnale iperintenso nelle sequenze pesate in t1 , denominato t1 effetto , e tale comportamento contrastografico tipico appunto degli angiomi . 
si trattava peraltro di uno dei primi casi studiati ed una pi attenta valutazione delle immagini prima e dopo contrasto avrebbe consentito di non incorrere in tale errore . nel nostro studio non si sono verificate reazioni secondarie o alterazioni dei parametri bio - umorali dopo somministrazione dei due mezzi di contrasto . 
tale distanza di tempo non pregiudica leffetto contrastografico del mdc spio , in quanto questultimo presenta la massima concentrazione epatica entro la 4a h , mentre la sua emivita plasmatica di circa 10 min consente la somministrazione ritardata di gd - dtpa senza interazione farmacologica dei due mdc [ 42 , 46 ]  . 
la distribuzione e leliminazione dei due mdc segue peraltro strade completamente diverse e studi farmacologici non hanno rilevato interazioni tra i due mdc in vitro [ 21 , 42 ]  . conclusioni lutilizzo sequenziale - combinato dei due mezzi di contrasto , negativi e positivi , consente una migliore confidenza diagnostica e nella nostra esperienza non ha determinato reazioni secondarie nei pazienti . 
carriero published online : 20 december 2006 ours is a life marked by fortune ; we have had the luck to be born in a country kissed by the sun and embraced by the sea , protected by the mountains and , above all , enriched with well - being . 
 its in this land whose very name italia suggests elegance , historical respect and reverence for ardent intelligence , an unusual coexistence in the imagination that we have our big family of more than 8 , 000 people united under the banner of sirm . 
 in todays world where alliances always have an archaic flavour or unbridled interests , in which the driving element is for ones own selfish means with the philosophy that theres no such thing as a free lunch , being united by a ray is a story of other times . 
if it were a ray of sunshine , there might be some justification , but that an invisible ray that doesnt heat or illuminate , and which is even hidden behind the unidentified x , can be the link for 8 , 000 people could be nothing short of a miracle . it was therefore spontaneous to ask to whose merit do we owe these 50 years of familiar militancy in the rm ? it brings to mind the words of my teacher , when trying desperately to make me grow up , who told me that when battles are lost , its always the fault of the generals and not the soldiers . from here we can use the opposite logic that the success of sirm is the result of the general presidents , members of the directive council and secretaries who conceptualised it , realized it and made the organisation grow in tune with the times . but wars arent won without soldiers , and in addition to the infinite recognition to our generals , its possible that the merit of this large organisation is due to the daily work of the 8 , 000 people of sirm . 
its a group of big - hearted professionals who , with ever - increasing difficulty , and in silence , support the radiology departments , which are , like it or not , the heart of the italian public and private health services . 
in this way , when one day my students at the school of specialisation in novara were in a discussional mood , something occurred to me like a bolt out of the blue our radiologists in sirm are , among other equally great things , a group of very generous people . 
in so doing , i was at the same time expanding my own personal efforts for humanitarian causes . it always happens that dreams are the first hope of man , also because dreaming doesnt cost anything and opens new horizons , allowing us to sail on a sea of optimism and to help those burnt by the aridness of daily life . 1170 la nostra una vita nel marchio della fortuna ; abbiamo avuto la fortuna di nascere in un paese baciato dal sole , accarezzato dal mare , protetto dalle montagne ma soprattutto ricco del suo benessere . ed in questo paese , che anche nel suo nome italia impone eleganza , rispetto storico , riverenza per le fervide intelligenze , nella coesistenza inusuale della fantasia , la nostra una grande famiglia di pi di 8000 persone unite nel segno della sirm . nel mondo doggi in cui le coalizioni hanno sempre il sapore dellarcaico o dellinteresse sfrenato , in cui lelemento conduttore il proprio torna conto nella filosofia che nulla debba essere fatto senza un immediato ritorno , sentirsi uniti da un raggio una storia daltri tempi . 
fosse un raggio di sole pur avrebbe una giustificazione ma che un raggio invisibile che non riscalda e non illumina e che addirittura nascosto dietro allignota x , fosse il filo dunione di 8000 persone non pu che essere un miracolo . stato spontaneo quindi domandarsi in questi cinquantanni di militanza familiare nella rm , di chi fosse il merito . 
allora vengono alla mente le frasi del mio maestro quando un giorno , nel tentativo disperato di farmi crescere , mi disse che quando si perdono le battaglie la colpa sempre dei generali e non dei soldati ; da qui la deduzione opposta che la realt sirm merito dei vari generali - presidenti membri del consiglio direttivo , segretari che lhanno pensata , voluta , realizzata , fatta crescere e proiettata al passo con i tempi . ma senza i soldati le guerre non si vincono e cos , oltre alla infinita riconoscenza ai nostri generali , forse il merito di questa grande societ sta anche nel lavoro quotidiano di 8000 radiologi : il popolo della sirm . 
un popolo di professionisti , dal cuore grande , che nel silenzio e in difficolt sempre maggiori regge i reparti di radiologia che sono , piaccia o no , il cuore della sanit pubblica e privata italiana . 
johns catholic school di watamu , kenya . between telephone calls , work dinners , congresses and days spent among scans and x - rays , this dream of coming together in the name of that unknown ray called x , to help whoever came knocking on our door , was born . so in that way , partly as a joke , and partly as a bet , the idea radiology for life was born . 
im not so sure yet what it will become , but i know that this group of people who are willing to support humanitarian efforts have been diffused all over italy , so much so that my e - mail inbox is full of mail every day from the people who have made sirm great , wanting to tell me of their availability . 
in 2006 , radiology for life adopted st . johns catholic school in the watamu area of kenya ; all of this resulted from a humanitarian trip to watamu that i had previously made . 
johns school was seriously incomplete and lacking in materials . two classrooms were missing ( 80 children were forced to have their lessons under a tree , even during the rainy season )  . 
it needed all the necessary furniture for the classrooms ( desks , blackboards , chairs etc . ) , all manner of sanitation was missing and it also needed an office , which could be used as a refectory when needed . 
i exchanged tra una telefonata e una cena di lavoro , tra un congresso ed una giornata tra risonanze tac e radiografie nato il sogno di mettersi insieme nel nome di quel raggio , incognita x , per aiutare chi bussa alla nostra porta . cos , un po per scherzo e un po per scommessa , nata a novara unidea , la radiologia per la vita : non so bene ancora cosa sia , so solo che la voce di questo gruppo che vuole essere di supporto ad iniziative umanitarie , si sparsa a macchia dolio nelle radiologie di tutta italia , tanto che la mia e - mail piena ogni giorno di disponibilit da parte di quel popolo radiologico che ha fatto grande la sirm . 
john era gravemente carente ed incompiuta : mancavano due classi ( 80 bambini erano costretti a far lezione sotto un albero anche durante il periodo delle piogge ) , mancavano quindi tutti gli arredi relativi alle classi ( banchi , lavagne , sedie , ecc . ) , mancavano tutti i servizi sanitari , mancava un office che potesse essere usato alloccorrenza come refettorio . 
the 400 little children probably wont ever ask themselves what these letters stand for and wont ever know what radiology for life is , but a little piece of the hearts of italian radiologists now beats in watamu . the man who doesnt dream is finished , and so id like to continue dreaming of the idea to build an entire school totally sustained by radiology for life , and who knows , maybe the unknown x can bring luck again . mio amico frate agapius , responsabile della scuola , bastato uno sguardo e cos dopo appena un anno la scuola cattolica di st . 
john ha tutto ci di cui necessitava ( classi , servizi igienici , refettorio ) e tutto grazie al grande cuore degli amici radiologi italiani . quello che mi preme per sottolineare che per realizzare tutto questo , su un budget complessivo necessario di 13000 euro , il presidente della sirm e tutto il consiglio direttivo ha deliberato di contribuire come societ alla costruzione della scuola donando una cifra sino a 5000 euro , ma ne sono bastati solo 3000 , al resto ci ha pensato il popolo della sirm . cos a fine dicembre sar a watamu ad inaugurare le nuove strutture della scuola di st . 
fortuna amirsys inc , salt lake city , utah ( 2005 ) isbn 1 - 4160 - 2333 - x ; isbn 0 - 8089 - 2324 - 2 ( international english edition ) published online : 20 december 2006 this volume is part of a series dedicated to diagnostic imaging in the various fields of radiology . 
it is made up of 1043 pages and 3000 illustrations some of which dealing with macroscopic and micro pathology specimens , often supplemented with beautiful explicative and very precise drawings . 
tali caratteri radiografici non sono correlati ad alcun tipo istologico particolare . parole chiave lesioni non palpabili linfonodi intra - mammari carcinoma della mammella introduction introduzione intramammary lymph nodes are the most common cause of nonpathological masses seen on conventional mammography , being detected in about 30% of breasts , mostly in the upper - outer quadrants [ 1 ]  . 
the typical mammographic features of intramammary lymph nodes ( size smaller than 15 mm , round or oval shape , regular margins , low density , association with a vascular branch and , frequently , a radiolucent centre ) have been clearly established [ 17 ] , and their detection does not warrant further diagnostic investigation i linfonodi intra - mammari costituiscono la causa pi frequente di formazione espansiva con significato non patologico riscontrata allesame mammografico convenzionale : sono infatti presenti in circa il 30% delle mammelle , prevalentemente nei quadranti supero - esterni [ 1 ]  . 
i caratteri mammografici tipici dei linfonodi intra - mammari ( dimensioni inferiori a 15 mm , morfologia rotondeggiante od ovalare , contorni regolari , bassa densit , associazione con una diramazione vascolare e frequente area centrale di maggiore radio - trasparenza ) sono chiaramente codificati da lungo tempo in letteratura [ 17 ] ed il 1078 e . 
however , in rare cases , breast cancers may exhibit mammographic features compatible with intramammary lymph nodes [ 1 , 2 , 9 , 11 ]  . the aim of this study was to verify the existence of mammographic criteria justifying a suspicion of atypical intramammary lymph node , assess whether certain histological types of breast cancer are more likely to display the radiographic features of intramammary lymph nodes , and consider the most appropriate diagnostic strategy in women with node - like mammographic findings . materials and methods we retrospectively reviewed the previous mammograms of women who received a diagnosis of breast cancer between january 1997 and december 2004 . 
the mammograms had been performed 6 months to 3 years before the diagnosis and depicted findings morphologically similar to intramammary lymph nodes at the site where the cancer was subsequently detected . 
the imaging equipment consisted of a dedicated conventional radiology unit ( mammomat 3000 , siemens , erlangen , germany ) combined with a high - spatialresolution detector , gadolinium screen ( kodak min r 2000 ) and single - coated film ( kodak m35 series x - omat processor )  . 
examinations performed with different equipment and techniques were excluded from the study . during the examination that aroused the suspicion of a neoplastic lesion , all lesions were studied by fine - needle cytology ( 2023 gauge ) under sonographic guidance ( seven cases ) or stereotactic guidance ( in one case in which the lesion was poorly visualised on ultrasound )  . 
all lesions subsequently underwent surgical excision and histological examination . loro riscontro non richiede approfondimenti diagnostici , che si rendono necessari solo in caso di incremento volumetrico in un controllo mammografico successivo [ 813 ]  . 
sebbene raramente , tumori della mammella in fase iniziale possono presentare caratteri mammografici sovrapponibili a quelli tipici dei linfonodi intra - mammari [ 1 , 2 , 9 , 11 ]  . scopo di questo lavoro ricercare se esistano criteri di semeiotica mammografica che consentano di porre il sospetto di linfonodo intra - mammario atipico , valutare se alcuni istotipi tumorali si manifestino preferenzialmente con caratteri radiografici simili a quelli dei linfonodi intra - mammari ed esaminare quale sia la strategia diagnostica pi opportuna quando si riscontrino reperti di aspetto compatibile con quello dei linfonodi intra - mammari . materiali e metodi sono stati retrospettivamente valutati i radiogrammi espletati in un periodo compreso tra i sei mesi e tre anni prima della diagnosi di tumore della mammella , posta tra gennaio 1997 e dicembre 2004 nei quali , nella sede in cui sarebbe stata in seguito identificata la neoplasia , era presente un reperto con i caratteri morfologici di un linfonodo intra - mammario . 
tutte le pazienti considerate hanno eseguito un esame mammografico standard , con almeno due proiezioni ( cranio - caudale e medio - laterale obliqua ) per mammella ; lindagine stata espletata con apparecchiatura radiologica convenzionale dedicata ( mammomat 3000 , siemens , erlangen , germania ) associata a detettore ad alta risoluzione spaziale , con schermo di rinforzo in gadolinio ( kodak min r 2000 ) e pellicola monoemulsionata ( kodak m35 series x - omat processor )  . 
le indagini espletate con modalit differenti da quella citata sono state escluse dallo studio . tutte le lesioni , nel corso dellindagine in cui ne stata sospettata la natura neoplastica , sono state sottoposte ad esame citologico con ago sottile ( calibro 2023 g ) , espletato sotto guida ecografica ( 7 casi ) o stereotassica ( nel singolo caso in cui la lesione non risultava chiaramente riconoscibile allesame ecografico )  . 
it is difficult to identify any lesion at all at the site of interest ( arrow and arrowhead ) on the mediolateral oblique mammogram ( a )  . only the craniocaudal view ( b ) shows an 8 - mm , faint , round opacity in the upper - outer quadrant that can be evaluated as an intramammary lymph node ( arrows )  . 
sul radiogramma in proiezione medio - laterale obliqua ( a ) risulta difficilmente identificabile , nel punto di interesse ( freccia e testa di freccia ) , una qualsiasi lesione . 
solo la proiezione cranio - caudale ( b ) dimostra nel versante supero - esterno tenue opacit rotondeggiante di 8 mm valutabile come linfonodo intra - mammario ( freccia )  . 
in the craniocaudal view ( b ) , an oval opacity with regular margins can be seen ( arrow ) projecting along a vascular branch , with a radiolucent central area ( 8 mm )  . 
in fase pre - diagnostica la proiezione medio - laterale obliqua ( a ) non consente di identificare lesioni sospette nellarea di interesse ( freccia e testa di freccia )  . 
in proiezione cranio - caudale ( b ) si identifica tenue opacit ovalare a contorni regolari ( freccia ) proiettatesi su diramazione vascolare , con nucleo radio - trasparente ( 8 mm )  . 
mediolateral oblique mammogram shows an 8 - mm , faint , round opacity in the upper quadrant ( arrow ) , with multilobular margins and radiolucent centre ( a ) interpreted as an intramammary lymph node . 
radiogramma in proiezione medio - laterale obliqua che dimostra nel versante superiore tenue opacit rotondeggiante di 8 mm ( freccia ) a contorni pluriciclici e nucleo radiotrasparente ( a ) interpretata come linfonodo intra - mammario . 
a minute ( 4 mm ) , round opacity with a radiolucent central area projecting along a small vascular branch can be seen in the upper - outer quadrant on both views ( arrows in a , b )  . 
minuta ( 4 mm ) opacit rotondeggiante con nucleo radiotrasparente , proiettantesi su piccola diramazione vascolare , riconoscibile nel versante supero - esterno in entrambe le proiezioni ( frecce in a , b )  . 
il controllo effettuato a distanza di 24 mesi ( c , d ) dimostra lieve incremento volumetrico ( 6 mm ) con densit lievemente aumentata . at baseline mammography , all masses showed round or oval shape and low density , with well - defined margins in seven cases ( two of which lobulated ) , and one case showed slightly indistinct margins . 
le dimensioni delle formazioni erano comprese fra 4 e 8 mm ( tabella 1 )  . lindagine in cui stata riconosciuta la reale natura neoplastica delle neoformazioni stata espletata da 6 a 36 mesi dopo la prima . 
a small ( 5 mm ) , faint , round opacity with radiolucent central area projecting along a vascular branch is seen in the upper - outer quadrant on the mediolateral oblique view ( arrow in a ) , which is even more evident on the craniocaudal view ( arrow in b )  . 
piccola ( 5 mm ) e tenue opacit rotondeggiante con nucleo radiotrasparente , proiettantesi sul decorso di diramazione vascolare nel versante supero - esterno , apprezzabile in proiezione medio - laterale obliqua ( freccia in a ) e meglio in proiezione cranio - caudale ( freccia in b )  . 
in the prediagnostic phase , the minute ( 6 mm ) , faint opacity visible in the upper - outer quadrants on both views ( arrows in a , b ) could easily be interpreted as an intramammary lymph node because of its site , density , radiolucent centre and proximity to vascular structures . 
in fase pre - diagnostica , la minuta ( 6 mm ) e tenue opacit apprezzabile in entrambe le proiezioni ( frecce in a , b ) nel versante supero - esterno facilmente interpretabile come linfonodo intramammario , in virt della sede , delle densit , del nucleo radiotrasparente e delle prossimit con le strutture vascolari . 
dopo 24 mesi ( c , d ) la lesione presenta contorni sfumati ; sensibilmente aumentata di volume ( 20 mm ) e di densit , questultima disomogenea . discussion discussione the radiographic features traditionally ascribed to intramammary lymph nodes [ 17 ] still hold good , as they are based on pathology findings and experience : the finding of small round or oval masses with regular margins and low density ( especially if located along a vascular branch and relatively radiolucent in their central portion ) does not justify a suspicion of neoplastic disease . 
a number of benign conditions can cause lymph nodal eni caratteri radiografici tradizionalmente attribuiti ai linfonodi intra - mammari [ 17 ] , in quanto fondati su dati anatomopatologici e condizionati dalla esperienza , rimangono validi a tuttoggi : il riscontro di formazioni espansive rotondeggianti od ovalari di piccole dimensioni , a margini regolari e dotate di bassa densit ( specie se situate lungo una diramazione vascolare e relativamente radio - trasparenti nella porzione centrale ) non autorizza a porre il sospetto di patologia neoplastica . 
the prediagnostic mediolateral oblique view shows , in the area in question ( arrow in a ) , an almost imperceptible , very small ( 4 mm ) and faint opacity , marked with an arrow on the craniocaudal view ( b )  . 
in fase pre - diagnostica , nella proiezione medio - laterale obliqua pressoch impercettibile nella zona di interesse ( freccia in a ) la minuta ( 4 mm ) e tenue opacit contrassegnata con freccia nella proiezione cranio - caudale ( b )  . 
a distanza di 36 mesi ( c , d ) la lesione risulta sensibilmente aumentata di volume ( 10 mm ) e densit , presentando contorni sfumati . largement and increased radiographic density , for example , inflammation of the breast skin ( at times related to implants ) or systemic inflammation ( rheumatoid arthritis )  . 
lymph nodal enlargement and increased density may also result from lymphoproliferative disease or from metastasis from cancers located in other parts of the breast , or rarely , elsewhere in the body ( melanomas , pulmonary , gastric and ovarian cancers ) [ 9 ]  . 
 inoltre causare nel tempo incremento nelle dimensioni e nella densit radiografica dei veri linfonodi intra - mammari , come avviene in caso di flogosi cutanee proprie della ghiandola mammaria ( anche sostenute dalla presenza di protesi ) o su base sistemica ( quali lartrite reumatoide ) ; analogo reperto pu essere causato da malattie infettive fra cui la tubercolosi . 
infine , possono ingrandirsi ed aumentare in densit i linfonodi intra - mammari sede di localizzazione di malattia linfoproliferativa o di metastasi , a partire da tumori primitivi posti in altre porzioni della mammella o , raramente , altrove ( melanomi ; tumori polmonari , gastrici ed ovarici ) [ 9 ]  . to our knowledge , the possibility that early primary secondo i dati a nostra disposizione , la possibilit che 1083 e . 
the retrospective review of our small case series did not reveal any correlation between histological type of cancer and node - like radiographic patterns , nor did it indicate the existence of new semiological criteria . 
it did , however , highlight a need for careful assessment of the known semiological criteria . owing to their small size , initial cancers with misleadingly similar features to intramammary lymph nodes may exhibit suspicious features in one radiographic view only . 
in addition , it should be noted that slight blurring of tumori primitivi della mammella esordiscano con quadri mammografici simili a quelli dei linfonodi intra - mammari non stata trattata in letteratura , sebbene sia noto che i carcinomi mucinosi possono presentare margini netti [ 7 ]  . la valutazione retrospettiva di questa casistica per quanto numericamente limitata non ha evidenziato correlazioni fra specifici istotipi tumorali e caratteri radiografici simil - linfonodali . 
non sono inoltre emersi criteri semeiologici nuovi ma la necessit di dedicare estrema attenzione a quelli gi noti . in conseguenza delle loro esigue dimensioni , lesioni tumorali in fase precoce e con caratteri ingannevolmente simili a quelli dei linfonodi intra - mammari possono mostrare segni sospetti in una sola proiezione radiografica . 
the main cause of acute chest pain , which accounts for 6.5% of urgent medical examinations in emergency rooms in italy , is acute coronary syndrome ( acs )  . 
we performed this prospective study to evaluate the diagnostic accuracy of a 16 - channel computed tomography ( ct ) scanner with dedicated software in a group of patients with chest pain and medium to low risk of acs materials and methods . 
this study involved a selected group of 31 patients reporting chest pain with a medium to low probability of acs , defined on the basis of preliminary tests [ electrocardiogram ( ecg ) and serum cardiac markers ]  . 
msct identified the presence of occlusions and significant ( > 50% ) or nonsignificant stenoses in the main coronary segments , with a sensitivity of 65% , a specificity of 98.8% , a positive predictive value ( ppv ) of 81.2% , a negative predictive value ( npv ) of 97.3% and an accuracy of 96.4%. 
in patients with a medium to low coronary risk , msct is a more accurate indicator of the need for coronary angiography than is exercise stress testing , which is less expensive but has lower predictive values . key words coronary arteries coronarography ct angiography multislice ct riassunto obiettivo . 
causa predominante del dolore toracico acuto , che motiva il 6 , 5% delle visite mediche urgenti effettuate nei pronto soccorso italiani , la sindrome coronarica acuta ( sca )  . 
lo studio analizza laccuratezza diagnostica della tc multistrato ( tc - ms ) a 16 canali in un gruppo selezionato di 31 pazienti con dolore toracico a medio - bassa probabilit di sca , cos definiti in base agli accertamenti preliminari ( elettrocardiogramma e markers biochimici )  . 
utilizzando come gold standard la coronarografia , eseguita nelle 24 ore successive allesame tomodensitometrico , la tc - ms ha identificato nei segmenti coronarici principali la presenza di occlusioni , stenosi significative ( > 50% ) e non ( < 50% ) con sensibilit : 65% ; specificit : 98 , 8% , vpp : 81 , 2% ; vpn : 97 , 3% , accuratezza : 96 , 4% . 
le stenosi significative e le occlusioni sono state invece accertate con sensibilit : 71 , 4% ; specificit : 99 , 6% ; vpp : 93 , 7% ; vpn : 97 , 7% ; accuratezza : 97 , 5% . 
the predominant cause is acute coronary syndrome ( acs ) , the management of which has le visite per dolore toracico acuto rappresentano circa il 6 , 5% di tutte le prestazioni effettuate nei pronto soccorso ( ps ) italiani cos come nelle emergency room statunitensi ; tale percentuale in costante aumento : nel 2000 si attestava al 5 , 1% . 
it has been calculated that in the united states 1 , 200 , 000 coronary angiography examinations are performed each year , 35% of which are followed by a revascularisation procedure . 
even though the morbidity and mortality rates of coronary catheterisation are very low ( 1% and 0.1% , respectively ) , there is a keen interest in alternative , less invasive and less expensive procedures possessing the same diagnostic accuracy in detecting or ruling out acute myocardial infarction ( ami ) and unstable angina . electrocardiogram ( ecg ) , serum cardiac markers and echocardiography can identify subjects with ami , for whom immediate reperfusion is indicated , and those with unstable angina . 
a proportion of patients presenting with chest pain are , however , considered at medium to low risk due to the absence of ischaemic st - segment abnormalities and negative serum markers , and these patients require additional investigations , such as exercise testing [ 1 ]  . 
because the advent of multidetector technology has considerably expanded the diagnostic potential of computed tomography ( ct ) , we performed this prospective study to evaluate the diagnostic accuracy of a 16 - channel ct scanner with dedicated software in a group of patients with chest pain and medium to low risk of acs . materials and methods between november 2004 and october 2005 , we recruited 31 subjects ( 19 men , 12 women ; mean age 59 years , age range 2670 years ) who presented to the emergency department with chest pain that was defined as medium to low probability of acs on the basis of first - line investigations ( ecg , and ischaemia markers : troponin , creatine phosphokinase and myoglobin ) but for whom a clinical suspicion of myocardial ischaemia persisted . 
subjects who had undergone previous myocardial revascularisation procedures or coronary angioplasty , with or without stent placement , were excluded . within 24 h of presentation , all patients ( who had given their informed consent for inclusion ) were studied by multislice ct ( msct ) and coronary angiography . 
further exclusion criteria for the ct study were heart rate > 70 bpm ( even after heart - rate - lowering drugs ) ; arrhythmia ; severe cardiac insufficiency ; pacemaker , defibrillators or valve implants ; history of adverse reactions to contrast material ; renal or respiratory insufficiency ; and pregnancy . the risk factors of the 31 patients selected for the study were hypertension ( 17 ) , cigarette smoking ( 6 ) , dyslipidaemia ( 6 ) , family history of ami ( 4 ) and diabetes ( 2 )  . 
all patients with heart rate higher than 65 bpm were treated with a heartrate - lowering agent ( metoprolol 100 mg / day ) whereas those with borderline heart rates around or just above 65 bpm received a - blocker ( metoprolol tartrate 5 mg ) intravenously 15 min before the examination . the ct study was performed with a 16 - channel multidetector scanner ( sensation cardiac , siemens ) using the following parameters : collimation 0.75 mm , voltage 120 kvp , rotation time 375 ms , slice thickness 0.75 mcardiac acacuta ( sca ) , nei cui confronti da una gestione attendista si passati ad una strategia aggressiva che prevede limmediata esecuzione di unindagine coronarografica e di uneventuale angioplastica . 
ancorch il cateterismo coronarico comporti una morbilit ed una mortalit assai ridotte ( rispettivamente nellordine dell1% e dello 0 , 1% ) permane alto linteresse per procedure alternative che assicurino non - invasivit e riduzione dei costi economici , identificando o escludendo con equivalente accuratezza diagnostica linfarto miocardico acuto ( ima ) e langina instabile . elettrocardiogramma , markers biochimici , ecocardiogramma consentono di individuare sia i soggetti con ima , nei quali generalmente indicata una terapia immediata di riperfusione , sia quelli con angina instabile . 
esiste , tuttavia , una quota di pazienti con dolore toracico definiti a mediobasso rischio , per lassenza di alterazioni ischemiche del tratto st dellelettrocardiogramma e la negativit dei markers biochimici , nei quali si rende necessaria lesecuzione di altre indagini , che attualmente consistono in test da stress [ 1 ]  . 
poich lintroduzione della tecnologia multidetettore ha comportato un incremento notevole delle potenzialit diagnostiche della tc , ci siamo proposti , in questo studio prospettico , di valutare laccuratezza diagnostica di una apparecchiatura a 16 canali e software dedicato in un gruppo di pazienti con dolore toracico a medio - basso rischio di sca . materiali e metodi nel periodo compreso tra novembre 2004 e ottobre 2005 , sono stati reclutati 31 soggetti ( 19 maschi , 12 femmine ; et media 59 anni con range : 2670 anni ) pervenuti allosservazione in ps per dolore toracico definito a medio - bassa probabilit di sca in base alle indagini di prima istanza ( tracciato elettrocardiografico e markers di ischemia : troponina , creatinfosfochinasi e mioglobina ) ma nei quali persisteva il sospetto clinico di ischemia miocardia . 
nellarco di 24 ore dalla presentazione in ps , tutti i pazienti ( dai quali era stato ottenuto il preventivo consenso informato per linserimento nel presente studio ) sono stati sottoposti a esame tc multidetettore ( tc - md ) e coronarografia . 
ai fini dellindagine tomodensitometrica altri criteri di esclusione sono stati : frequenza cardiaca ( fc ) > 70 bpm ( anche dopo bradicardizzazione farmacologia ) ; aritmia ; insufficienza cardiaca grave ; presenza di pacemaker , defibrillatori o protesi valvolari ; pregresse reazioni avverse al mezzo di contrasto ; insufficienza renale o respiratoria ; stato di gravidanza . nel gruppo selezionato di 31 soggetti i fattori di rischio 1055 l . 
coronary angiography images were obtained with volume rendering ( vr ) and curvilinear multiplanar reconstruction ( mpr ) three - dimensional ( 3d ) techniques . in the comparison between msct and coronary angiography , the latter being considered the gold standard , we evaluated 16 coronary segments according to the american heart association classification [ 3 ]  . 
two operators , one radiologist and one cardiologist , independently evaluated each segment for assessability and image quality ( excellent , fair and poor ) and the presence of occlusions and stenoses , which they ranked as nonsignificant ( 50% ) or significant ( 51%99% ) based on a subjective visual judgement for msct and a quantitative judgement for coronary angiography [ 4 ]  . results at msct , 383 ( 81.7% ) of the 469 segments visualised by coronary angiography were considered assessable ; the intermediate branch was present in four subjects only ( table 1 )  . if only the main segments were considered right coronary erano ipertensione ( 17 ) , fumo di sigaretta ( 6 ) , dislipidemia ( 6 ) , familiarit per ima ( 4 ) e diabete ( 2 )  . 
per coloro invece che presentavano una frequenza borderline e cio intorno o di poco superiore ai 65 bpm si optato per la somministrazione endovena di un - bloccante ( metoprololo tartrato 5 mg ) 15 minuti prima dellesecuzione dellesame . lindagine tc stata effettuata con una apparecchiatura multidetettore a 16 canali ( sensation cardiac , siemens ) , utilizzando i seguenti parametri : collimazione 0 , 75 mm , voltaggio 120 kvp , tempo di rotazione 375 ms , spessore di strato 0 , 75 mlattivit cardiaca stata costantemente monitorata durante lindagine mediante tracciato elettrocardiografico in singola derivazione . per liniezione di mezzo di contrasto ( mdc ) organo - iodato non ionico ( concentrazione 350400 mgi / ml ) si utilizzato un iniettore automatico collegato ad unagocannula da 1820 gauge inserita in una vena antecubitale del braccio . utilizzando la tecnica del bolus tracking con roi nellaorta ascendente sono stati iniettati 100 ml di mdc , seguiti da 40 ml di soluzione fisiologica , ad una velocit di infusione di 4 ml / s . la ricostruzione delle immagini stata effettuata mediante gating cardiaco retrospettivo , individuando la finestra temporale ottimale per ogni ramo coronarico ( solitamente 400 , 350 o 300 ms prima del successivo complesso qrs elettrocardiografico ) [ 2 ]  . 
sono state ricostruite sezioni assiali dello spessore effettivo di 0 , 75 mm con overlapping di 0 , 5 mle immagini coronarografiche sono state ottenute con tecnica 3d volume rendering ( vrt ) e multiplanare ( mpr ) curvilinea . ai fini del confronto tra tcmd e coronarografia , assunta quale gold standard , sono stati considerati 16 segmenti coronarici secondo la classificazione dellamerican table 1 number and percentage of coronary artery segments considered assessable on computed tomography ( ct ) in 31 patients tabella 1 numero e percentuale dei segmenti coronarici giudicati valutabili alla tc in 31 pazienti segments assessable , n ( % ) segmenti variabili , n ( % ) 1 . 
di ogni segmento due operatori , un radiologo e un cardiologo , hanno considerato in maniera indipendente la valutabilit e la qualit iconografica ( eccellente , discreta e scadente ) nonch la presenza di occlusioni o di stenosi classificate in non significative ( 50% ) e significative ( 51%99% ) in base ad un giudizio visuale soggettivo per la tc - md e quantitativo per la coronarografia [ 4 ]  . risultati alla tc - md sono stati giudicati valutabili 383 ( 81 , 7% ) dei 469 segmenti visualizzati alla coronarografia , essendo il ramo intermedio presente solo in 4 soggetti ( tabella 1 )  . 
considerando esclusivamente i segmenti principali e cio arteria coronaria destra ( segmenti 1 , 2 , 3 ) , tronco comune ( segmento 5 ) , arteria discendente anteriore ( segmenti 6 , 7 , 8 ) , arteria circonflessa ( segmenti 11 , 13 ) e , laddove presente , ramo intermedio ( segmento 16 ) , i segmenti considerati valutabili sono stati 277 di 283 ( 97 , 9% ) : in 4 / 31 ( 12 , 9% ) pazienti si avuto un segmento non valutabile , pi frequentemente il 13 ; in 1 paziente ( 3% ) i segmenti non esaminabili sono stati due . 
la tabella 3 mostra i risultati del confronto tc - coronarografia limitatatotal total tc , n positiva negativa totale , n tc , n positive negative totale , n fig . 
1a - c a 40 - year - old man , a heavy smoker with a family history of coronary disease , admitted to the emergency room with precordial chest pain ; sinusal rhythm with 60 bpm at electrocardiogram ( ecg ) , myocardial ischaemia markers negative , no regional wall motion abnormalities at echocardiography . 
computed tomography ( ct ) ( a , b ) shows about 50% tubular stenosis with focal , severe narrowing of the lumen of the anterior descending artery ( ada ) , segment 1 ( arrowheads )  . 
alla coronaro - tc ( a , b ) : stenosi tubulare di circa il 50% , cui segue focale serrato restringimento del lume dellarteria discendente anteriore ( ada ) prossimale , segmento 1 ( punta di freccia )  . 
2a - c a 70 - year - old woman with hypertension admitted to the emergency room with precordial chest pain radiating to the neck ; sinusal rhythm with 63 bpm and minimal st depression at electrocardiogram ( ecg ) , absence of serum cardiac markers , normal ventricular wall motion at echocardiography . 
paziente maschio , et 56 anni , diabete mellito nid , iperteso , con scintigrafia miocardica da sforzo ( eseguita 3 mesi prima ) dubbia per coronaropatia ; pervenuto in ps con dolore toracico retrosternale . 
4a , b a 70 - year - old man with hypertension and hypercholesterolaemia admitted to the emergency room with precordial chest pain ; sinusal rhythm with 58 bpm , absence of ischaemic signs at electrocardiogram ( ecg ) ; absence of serum cardiac markers . 
computed tomography ( ct ) ( a ) shows eccentric calcific plaque of the middle circumflex artery ( segment 13 ) ( arrow )  . the vessel lumen is not well assessable , but a > 50% stenosis ( arrow ) is suggested . 
alla tc ( a ) : placca eccentrica calcifica dellarteria circonflessa , tratto medio , segmento 13 ( freccia ) ; il lume non ben valutabile ma si ipotizza una stenosi > 50% . 
alla coronarografia ( b ) : stenosi della lunghezza di circa 15 mm e del 90% circa nel punto critico ( freccia )  . particular , it reports the number of subjects with at least one occlusion or stenosis > 50% identified by ct with respect to the number of those identified by coronary angiography . 
inoltre , la coronarografia documenta come in 3 di 6 segmenti ( 50% ) vi sia una riopacizzazione da circolo collaterale del tratto distale alla stenosi . la tabella 5 presenta , infine , unanalisi della casistica che considera il singolo paziente piuttosto che i segmenti coronarici ; in particolare , riporta il numero dei soggetti con almeno una occlusione o una stenosi > 50% identificati alla tc rispetto a quanti individuati dalla coronarografia . 
nei pazienti ( 16 / 31 ) , nei quali la qualit dimmagine risultata eccellente per tutti i segmenti , i valori di sensibilit , specificit , valore predittivo positivo e negativo , accuratezza sono pari al 100% . discussione lesame dei pazienti che si presentano in ps per dolore toracico acuto richiede tempo e risorse economiche finalizzati allaccertamento di una sca che risulta , al termine di tutte le indagini , inesistente in una percentuale tuttaltro che trascurabile di casi . 
la valutazione clinica ( intensit , irradiazione , durata ) del dolore toracico , daltra parte , facilmente soggetta ad errori interpretativi e insufficiente per la diagnosi ; ne consegue che lapproccio tradizionale a tali pazienti preveda lelettrocardiogramma e il dosaggio dei markers biochimici . 
lalterazione di questi ultimi unitamente allelevazione elettrocardiografica del tratto st corrobora il sospetto clinico di infarto miocardico , che comunque diagnosticabile anche in assenza di precoci alterazioni elettrocardiografiche qualora vi sia innalzamento di troponina , creatinfosfochinasi e mioglobina . 
quando anche tali markers siano negativi , i pazienti definiti a medio - basso rischio di sca debbono essere avviati a test da stress elettrocardiografici , ecocardiografici o scintigrafici per evidenziare la presenza o lassenza di malattia coronarica significativa [ 5 , 6 ]  . 
5a - c a 58 - year - old woman with hypertension and a previous history of smoking admitted to the emergency room with precordial chest pain and dyspnoea ; absence of ischemic alterations at electrocardiogram ( ecg ) , absence of serum cardiac markers . 
although poor quality of computed tomography ( ct ) image ( a ) should have induced the reader to avoid any judgement , a severe stenosis ( arrow ) of the right coronary artery ( segment 2 ) is suggested . 
si ipotizza comunque lesistenza di una stenosi critica da placca calcifica del segmento 2 , sottovalutando erroneamente lipotesi di una occlusione con riopacizzazione a valle da parte dellemisistema coronarico sinistro , come invece dimostrato dalla coronarografia ( punte di freccia ) ( b , c )  . partment with acute chest pain in order to identify an acs that very often turns out to be nonexistent . 
the clinical assessment ( intensity , radiation , duration ) of chest pain is , on the other hand , often prone to misinterpretation and is inadequate for the diagnosis , with the result that the conventional approach to these patients includes an ecg and serum cardiac markers . abnormal serum cardiac markers associated with st elevation supports the clinical suspicion of myocardial infarction , which can , however , be diagnosed even without early electrocardiographic alterations if the troponin , creatine phosphokinase and myoglobin levels are high . 
where these markers are negative , patients considered at medium to low risk of acs must proceed to electrocardiographic , echocardiographic or nuclear stress testing to determine the presence or absence of significant coronary disease [ 5 , 6 ]  . 
the diagnostic reference standard for the assessment of vascular anatomy and the detection of stenosing lesions is coronary angiography , which , in addition to a diagnostic phase , allows immediate myocardial reperfusion by means of angioplasty . 
the invasiveness and high cost of this examination have , however , promoted research into alternative , noninvasive diagnostic techniques . the technological evolution of spiral ct has made available multislice scanners with 4 , 8 , 16 and , recently , 64 detector rows that offer a temporal and spatial resolution that enable analysis of the coronary arteries with a high level of anatomical detail . 
linvasivit e lelevato costo di tale indagine hanno indirizzato tuttavia gli sforzi della ricerca verso tecniche diagnostiche alternative non invasive . levoluzione tecnologica della tc spirale ha reso disponibili apparecchiature multistrato a 4 , 8 , 16 e , recentemente , 64 file di detettori caratterizzate da risoluzione spaziale e temporale tali da consentire lanalisi delle coronarie con elevato dettaglio anatomico . 
in letteratura , dallanno 2000 ad oggi , sono stati pubblicati diversi studi sullidentificazione di placche coronariche mediante tc - md a 4 o 16 canali [ 711 ]  . 
nella quasi totalit dei lavori sono state prese in considerazione le stenosi che comportano riduzione > 50% o occlusione del lume coronarico ; attualmente , con lutilizzo di scanner a 64 slice , si iniziano a valutare anche stenosi < 50% [ 1214 ]  . 
peraltro , in questa classe di pazienti che presentano angina instabile , due o pi fattori di rischio , incremento della troponina sierica , slivellamento st > 1 mm , aritmie o instabilit emodinamica , le pi recenti linee - guida cardiologiche suggeriscono lesecuzione in prima istanza dellangiografia coronarica e della rivascolarizzazione , se indicata [ 16 , 17 ]  . la presente casistica considera esclusivamente soggetti giunti allosservazione in ps con un dolore toracico acuto 1061 l . 
in this class of patients with unstable angina , two or more risk factors , increased serum troponin , st deviation > 1 mm , arrhythmias or haemodynamic instability , the most recent cardiological guidelines recommend coronary angiography and revascularisation , if indicated , as the first - line approach [ 16 , 17 ]  . our study included only subjects presenting to the emergency department for acute chest pain and at medium to low risk for acs associated with one , two or more risk factors for coronary disease ( age , smoking , dyslipidaemia , arterial hypertension , diabetes mellitus ) but with ischaemia markers and ecg not indicative of q and non - q ami . 
in the 31 patients enrolled , all of the 15 ( or 16 if the intermediate branch was present ) segments were analysed ; subsequently , the main coronary branches only were considered , that is , the right coronary artery ( segments 1 , 2 , 3 ) , left main branch ( segment 5 ) , anterior descending or interventricular artery ( segments 6 , 7 , 8 ) , circumflex artery ( segments 11 and 13 ) and , where present , the intermediate branch ( segment 16 )  . 
 compared with the interesting statistical results of previous reports , the sensitivity of msct ( 71.4% for occlusions and stenoses > 50% ) identified in our study is among the lowest reported in the literature [ 18 , 19 , 2125 ] and higher only than the value reported by hoffmann et al . 
the false negatives are due to underestimation of the degree of stenosis in poor - quality images or to misinterpretation in cases of revascularisation of the branch below the stenosis due to collateral circulation ; in particular , the latter situation led to three stenoses being interpreted as significant rather than as occlusions . 
msct therefore takes on an important role in the diagnostic workup of this class of cardiological patients , as it guides towards coronary angiography more accurately than does exercise stress testing which , though definitely less expensive , has decidedly lower predictive values [ 2628 ]  . 
si sono esclusi i pazienti portatori di bypass aorto - coronarico o pace - maker e i soggetti sottoposti in passato ad interventi di angioplastica , con o senza posizionamento di stents coronarici . 
nei 31 pazienti arruolati , sono stati analizzati tutti i 15 ( o 16 in presenza di ramo intermedio ) segmenti ; successivamente sono stati presi in considerazione solo i rami coronarici principali , ovverosia arteria coronaria destra ( segmenti 1 , 2 , 3 ) , tronco comune ( segmento 5 ) , arteria discendente anteriore o interventricolare ( segmenti 6 , 7 , 8 ) , arteria circonflessa ( segmenti 11 e 13 ) e , qualora presente , il tronco intermedio ( segmento 16 )  . 
il dato in linea con quanto emerge da altri studi effettuati con tc - md a 12 e 16 canali [ 1820 ]  . rispetto a precedenti interessanti risultati statistici , nel presente studio emerge una sensibilit della tc - md ( 71 , 4% per le occlusioni e le stenosi > 50% ) che si posiziona ai limiti inferiori del range dei valori riportati in letteratura [ 18 , 19 , 2125 ] , risultando superiore soltanto a quella ( 63% ) di hoffmann et al . 
essi sono dovuti a sottostima dellentit della stenosi in immagini di qualit scadente o ancora ad errore interpretativo in casi di riabitazione del ramo a valle della stenosi per fenomeni di collateralit : in particolare , questultima evenienza responsabile di tre stenosi interpretate come significative e non , in realt , come occlusioni . 
per quanto concerne invece la specificit per le occlusioni e le stenosi > 50% , il nostro dato ( 99 , 6% ) appare incoraggiante essendo il range delle percentuali fino ad oggi riportate oscillante fra l86% ed il 98% . i valori predittivi negativo ( 97 , 7% ) e positivo ( 93 , 7% ) , superiori a quanto mediamente riportato in letteratura [ 18 , 19 , 2125 ] , implicano che un paziente , pervenuto in ps per dolore toracico con media - bassa probabilit di sca e tc - md negativa per stenosi significativa , abbia solo il 2 , 3% di probabilit di avere in realt una coronaropatia significativa ; allopposto , un soggetto con stenosi giudicata emodinamicamente significativa alla tc ha una probabilit superiore al 93% di conferma coronarografica . 
in questa classe cardiologica di pazienti la tc - md assume quindi un ruolo rilevante nellalgoritmo diagnostico indirizzando verso un approfondimento coronarografico pi correttamente di quanto non possa un test da sforzo , sicuramente meno costoso , ma caratterizzato da valori predittivi decisamente inferiori [ 2628 ]  . 
inoltre se , meno dettagliatamente ma in maniera clinicamente pi congrua , non si considerino i rami coronarici principali bens i singoli pazienti si ottiene una predittivit positiva del 100% . 
the encouraging results , if validated by larger series , support the use of pet / ct in patients with carcinoma of unknown primary origin and negative conventional imaging results . key words metastatic cancer occult primary cancer 18f - fdgpet / ct riassunto obiettivo . 
la popolazione in studio costituita da 38 pazienti consecutivi con malattia tumorale metastatica provata istologicamente in cui le tecniche di diagnostica per immagini convenzionale erano risultate negative o non conclusive nella identificazione della lesione primitiva . 
the site of the occult primary tumour often remains unidentified after common imaging investigations ( chest x - ray , abdominal and pelvic ct , mammography in women ) ( 20%27% of cases ) [ 1 , 2 ] or autopsy ( 30%82% ) [ 24 ]  . 
reasons for the low rate of detection of the primary cancer include a lesion size smaller than the spatial and contrast resolution of the technique used or involution of the primary mass due to limited angiogenic competence [ 5 ]  . 
conventional contrast studies of the upper and lower digestive tract , for example , have a low diagnostic yield , and mammography , although routinely ordered , is often unhelpful because very few patients with cup have a primary mass in the breast [ 6 ]  . recent papers have reported that the median survival for patients with cup is approximately 12 months and that detection of the primary tumour and initiation of therapy can prolong survival to 23 months [ 7 ]  . 
the aim of this study was to assess the role of 18f - fdg - pet / ct in the identification of occult primary tumours . materials and methods the patient population consisted of 38 consecutive patients ( 22 men , 16 women ; mean age 5911 years , range 4177 years )  . 
the uptake period was 6090 meach patient then underwent a pet / ct scan with a dedicated hybrid scanner ( discovery ls , general electric ; biograph sensation 16 , siemens ) and whole - body scans ; attenuation correction was la sindrome del tumore primitivo occulto ( cup ) consiste nella presenza di una malattia tumorale metastatica per la quale sia impossibile identificare la sede primitiva della neoplasia dopo una attenta indagine anamnestica , esame obbiettivo negativo , negativi gli esami clinico - diagnostici ( emocromo completo , funzionalit renale , epatica , pancreatica , test delle urine , radiogramma del torace , tc addominale e pelvica e mammografia nelle donne , psa negli uomini ) [ 1 ]  . 
nella maggior parte dei casi , impossibile identificare la sede del tumore primitivo occulto con altri esami ( 20%27% ) di diagnostica per immagini pi diffusi ( radiografia del torace , tc addominale e pelvica , mammografia nelle donne ) [ 1 , 2 ] o allautopsia ( 30%82% ) [ 24 ]  . le motivazioni alla base dello scarso potere di identificazione della sede del tumore primitivo comprendono le dimensioni al di sotto del potere di risoluzione , sia spaziale che di contrasto , delle tecniche utilizzate o linvoluzione della massa primitiva a causa di uno scarso potere angiogenetico [ 5 ]  . 
la mammografia , nonostante sia utilizzata nella routine diagnostica , spesso non utile in quanto un limitato numero di pazienti con cup hanno una massa primitiva a livello mammario [ 6 ]  . recenti lavori riportano come la sopravvivenza mediana dei soggetti con cup sia attorno ai 12 mesi e che la identificazione della sede primitiva di malattia con la possibilit di intraprendere un iter terapeutico sposti la sopravvivenza mediana a 23 mesi [ 7 ]  . 
successivamente ogni soggetto stato sottoposto a scansione pet - tc con tomotabella 1 dati clinici , istopatologici e di diagnostica per immagini dei casi con tumore primitivo a livello toracico caso n sesso et , anni biopsia pet / tc tumore occulto dopo pet / tc dimensioni lesione , mm brain metastasis adenocarcinoma supraclavicular node metastasis mediastinal and neck node metastasis adenocarcinoma adenocarcinoma supraclavicular node metastasis sacral vertebra metastasis melanoma adenocarcinoma left humerus metastasis squamous cell carcinoma area of increased uptake in left lung area of increased positive mediastinal and neck nodes , area in right lung positive neck nodes , area of uptake on chest skin multiple areas of uptake in bone and left lung multiple metastases to bone , right adrenal gland , right lung lymph nodes , lung left lung left lung right lung melanoma left lung right lung lung lung laterocervical node spinous cell metastasis brain metastasis carcinoma epithelial carcinoma bone , lung diangostica convenzionale metastasi cerebrale metastasi linfonodo sovraclaveare metastasi linfonodali ( mediastinici e cervicali ) metastasi linfonodo sovraclaveare metastasi ossea vertebra sacrale adenocarcinoma adenocarcinoma adenocarcinoma area di aumentato uptake polmonare sx area di aumentato uptake polmonare sx linfonodi mesiatinici e cervicali , area polmone dx polmone sx polmone sx polmone dx melanoma linfonodi cervicali , area cute torso melanoma adenocarcinoma metastasi omero carcinoma squamocellulare metastasi linfonodali laterocervicali metastasi cerebrale carcinoma spinocellulare carcinoma epiteliale multiple aree captanti a livello osseo e polmonare sx multiple meta . 
the low rate of detection of the primary tumour site has two main causes . first , the size of the primary lesion may be smaller than the resolution power of conventional imaging procedures ( such as ct or gastrointestinal contrast studies ) , or the lesions may be located in sites such as the abdomen , pelvis , head and neck . 
epatiche , area positiva gastrica negativa carcinoma a cellule transizionali adenocarcinoma carcinoma a cellule fusate carcinoma squamocellulare carcinoma squamocellulare negativa linfonodi adiacenti mammella sx area mediastinica ignoto linfonodi cervicali dx ignoto ignoto ignoto ignoto ignoto ignoto carcinoma epiteliale linfonodo ascellare sx ignoto carcinoma epiteliale linfonodo pelvico ignoto adenocarcinoma surrene dx , linfonodi ignoto adenocarcinoma adenocarcinoma mucoide adenocarcinoma mucoide carcinoma epiteliale mano sx area mediastinica ed ossea cute ignoto ignoto cute peritoneo fegato ignoto carcinoma epiteliale cute carcinoma indifferenziato linfonodo mediastinico ignoto ignoto ignoto na , non applicabile suggested that the primary tumour may disappear after having given rise to metastasis because of its angiogenic incompetence , which leads to marked apoptosis and cell turnover [ 5 ]  . whereas no diagnostic procedures can identify a primary lesion that has disappeared due to marked apoptosis , it has been demonstrated that fdg - pet can improve primary - site detection in patients with cup and a negative diagnostic workup . 
although the number of patients in our study was small , our preliminary results show that pet / ct is more sensitive than pet alone , with a 53% detection rate for occult cancers missed by other techniques discussione la cup una sindrome caratterizzata da una prognosi infausta . 
la identificazione della sede primitiva di neoplasia , comunque , pu essere importante in quanto permette di impostare una terapia specifica , migliorando significativamente la prognosi ( 1 anno vs 2 anni ) [ 7 ]  . 
le dimensioni delle lesioni primitive possono essere inferiori al potere di risoluzione delle procedure diagnostiche ( tc o studi contrastografici del tratto gastrointestinale ) o trovarsi in sedi quali laddome , la pelvi , la testa - collo . 
la pet - tc ( sezioni transassiali della pet , tc e immagine di fusione pet - tc ) ha evidenziato unarea di patologico accumulo a livello dellipofaringe ( indicata da freccia nella immagine mip )  . nari di piccole dimensioni [ 1 ]  . 
laltro motivo dipende dalle caratteristiche biologiche del tumore primitivo : stato ipotizzato che la massa tumorale primitiva possa scomparire dopo aver dato luogo a metastatizzazione a causa di uno scarso potere angiogenetico che porta a marcata apoptosi e turn over cellulare [ 5 ]  . mentre nessuna procedura diagnostica pu identificare una lesione primitiva che scomparsa per marcata apoptosi , stato dimostrato come la pet con fdg possa migliorare la identificazione della sede primitiva della lesione in pazienti con cup e una flow - chart diagnostica peraltro negativa . 
al momento presente , sono pochi gli studi che hanno valutato il valore aggiunto della pet - tc rispetto alla fdgpet convenzionale per lidentificazione della sede del tumore occulto [ 810 ]  . 
i nostri risultati preliminari , nonostante il campione sia limitato , mostrano che la pet - tc pi sensibile della pet convenzionale , rilevando il 53% dei tumori occulti che non erano stati evidenziati con le altre tecniche [ 1113 ]  . 
le limitate informazioni morfologiche fornite dalla pet da sola , rendono necessaria lintegrazione con i dati anatomici forniti dalla tc per migliorare la definizione anatomica delle aree di aumentata captazione del tracciante . 
the limited morphological information provided by pet alone requires integration with the anatomical data supplied by ct in order to improve the anatomical definition of the areas of increased tracer uptake . 
pet / ct scanners capture anatomical and functional data in a single registration [ 14 ] , overcoming the limitations of patient repositioning and time interval between scans when the images are fused a posteriori [ 15 ]  . 
the primary tumour was identified in 7 / 29 cases ( two carcinomas of the nasopharynx , one plasmocytoma of the tongue base , one lung carcinoma , two breast cancers ) , showing 24% sensitivity . 
this result is not , however , comparable with our findings because alberini included 22 patients with another positive conventional test : this means that pet provided additional information in 4 / 44 cases only . 
although the population was not selected ( in contrast to our study population ) , the sensitivity of pet was 37.8% , which is significantly lower than that reported in our study . although the heterogeneity of patients could be seen as a potential limitation to our study , with patients referred from different centres and affected by different types of tumours , this fact does not undermine the value of our study , as the diagnostic - therapeutic approach to unknown primary tumours has not yet been codified by international guidelines . 
the head and neck , abdomen and pelvis are highly complex anatomical districts and are characterised by areas of physiological tracer uptake ( for example , by the tonsils , lymphatic tissue , stomach , bowel and ureters )  . small areas of increased uptake due to a small primary tumour may be mistaken for areas of physiological uptake . the careful comparison of metabolic and anatomical information , made possible by pet / ct , considerably improves the sensitivity of the procedure , allowing visualisation of even small areas of asymmetrical tracer distribution or areas of increased focal uptake that would otherwise be considered physiological . 1154 dallintervallo di tempo tra gli scans quando le immagini sono fuse a posteriori [ 15 ]  . 
la sede del tumore primitivo stata identificata in 7 / 29 ( 2 carcinomi del nasofaringe , 1 plasmocitoma della base della lingua , 1 carcinoma polmonare , 2 carcinomi della mammella ) con una sensibilit del 24% . 
questo risultato , comunque , non confrontabile con il nostro studio in quanto alberini ha incluso nel campione 22 soggetti che avevano un altro test convenzionale positivo : questo significa che la pet ha fornito indicazioni aggiuntive solamente in 4 / 44 casi . 
nonostante la popolazione non fosse stata selezionata , al contrario di quella descritta nel nostro studio , la sensibilit della pet risultata del 37 , 8% , che sensibilmente inferiore a quella da noi riportata . sebbene un potenziale limite al presente studio sia rappresentato dal fatto che il campione preso in esame eterogeneo in quanto costituito da pazienti provenienti da centri diversi e portatori di neoplasie di diverso tipo , tuttavia questo non toglie valore allo studio in quanto lapproccio diagnostico - terapeutico in casi di tumore primitivo ignoto non ancora codificato da norme internazionali . 
alcune sedi quali il capo - collo , laddome e la pelvi , sono anatomicamente molto complesse e caratterizzate da varie sedi di fisiologico uptake del tracciante ( per esempio le tonsille , il tessuto linfatico , lo stomaco , lintestino , gli ureteri )  . 
of the 467 patients examined by cdus , 159 ( 34% ) showed no signs of renal artery stenosis ( ras ) or nephropathy and were therefore started on medical therapy . 
nel periodo 20002004 sono giunti alla nostra osservazione 467 pazienti ( 205 donne e 262 uomini , range 2096 anni ) , per essere sottoposti ad indagine ecd , in quanto presentavano ipertensione arteriosa resistente alla terapia e / o segni di insufficienza renale . 
dei 467 pazienti esaminati mediante ecd , 159 ( 34% ) non presentavano indici di stenosi delle arterie renali n segni ecografici di nefropatia , ed hanno proseguito un iter terapeutico medico . 
dai risultati ottenuti e alla luce dei dati della letteratura , si riconferma il ruolo fondamentale svolto dallecd nei pazienti con sospetta o accertata patologia nefro - vascolare , fornendo dati importanti sullemodinamica renale di insostituibile utilit per un corretto approccio clinico - terapeutico . parole chiave eco color doppler ipertensione nefrovascolare arterie renali introduction introduzione atherosclerotic renal artery stenosis ( ras ) is associated with two common clinical syndromes , renovascular hypertension and ischaemic nephropathy , which imply a significant worsening of renal function in the presence of stenosis [ 13 ]  . the exact prevalence of ras is unknown , but many data indicate that it is high among elderly , chiefly caucasian , subjects and those with diabetes , diffuse atherosclerosis and rela stenosi aterosclerotica dellarteria renale ( sar ) si associa a due comuni sindromi cliniche : lipertensione nefro - vascolare e la nefropatia ischemica , intendendo per questultima una significativa riduzione della funzione renale in presenza della stenosi [ 13 ]  . lesatta prevalenza della stenosi aterosclerotica dellarteria renale non nota , ma molti dati fanno ritene1115 g.c. 
renal angiography and renal colour - doppler ultrasound ( cdus ) studies performed during coronary angiography or peripheral arteriography have demonstrated that ras is present in 20%30% of more than 10 , 000 investigations [ 1 ]  . 
a large number of patients with serum creatinine higher than 1.7 mg / dl have been found to have ras in prospective and retrospective studies , and ras has been demonstrated in 5%10% of patients with acute renal failure [ 1 ]  . 
this prevalence becomes higher when other clinical signs of atherosclerosis coexist , with rates of 12%24% in patients undergoing coronary angiography , 11% in stroke patients and 40% in patients with peripheral vascular disease [ 3 ]  . 
few studies have assessed atherosclerotic ras in patients with abdominal aorta aneurysm ( aaa ) , another manifestation of atherosclerotic disease . the high prevalence of atherosclerotic ras has raised the need to identify a noninvasive , inexpensive modality with high diagnostic accuracy in order to reduce the use of invasive investigations , such as arteriography , which nonetheless remains the gold standard in the diagnosis of ras [ 2 , 4 , 5 ]  . diagnostic imaging offers several noninvasive methods for selecting patients for angiography : cdus , captopril renal scintigraphy , spiral computed tomography ( ct ) angiography , and magnetic resonance ( mr ) angiography [ 2 , 3 , 511 ]  . cdus of the renal arteries is currently the main screening tool for ras , and many international studies have demonstrated a sensitivity of 83%96% , a specificity of 76%98% , a positive predictive value ( ppv ) of 81%92% and a negative predictive value ( npv ) of 94%98% [ 58 , 1115 ]  . 
the advantages of cdus are that it is inexpensive , reproducible , noninvasive and has good npv when performed in selected populations ; its known disadvantages , on the other hand , are operator dependence , patient preparation and cooperation , scars , obesity , bowel gas and vascular abnormalities , in particular , the presence of anatomical variants of the renal artery [ 2 , 4 , 6 , 1517 ]  . the primary aim of our study was to evaluate the role and usefulness of cdus in analysing overall renal morphology and haemodynamics in patients with hypertension and nephropathy ; the secondary aim was to assess the association between atherosclerotic ras and aaa . materials and methods between 2000 and 2004 , 467 patients ( 205 women and 262 men , age range 2096 years ) with refractory hypertension and / or signs of renal failure ( serum creatinine > 2.5 mg / dl ) were referred to our department for renal cdus . the patients were examined by a single radiologist on a philips ssd 800 and hitachi h21 scanner using a 3.5 - mhz convex - array transducer and colourand power - doppler mode . 
sonograms were assessed for renal morphology , overall renal blood flow and abdominal aorta . 1116 re che essa sia assai frequente nei soggetti anziani , specie di razza caucasica , diabetici , con patologia aterosclerotica polidistrettuale e con insufficienza renale . 
studi angiografici renali e di eco color doppler ( ecd ) renale , eseguiti in corso di coronarografia o di arteriografia periferica , hanno documentato una frequenza di sar nel 20%30% di oltre 10000 indagini [ 1 ]  . 
analisi retrospettive o prospettiche in pazienti con creatininemia superiore a 1 , 7 mg / dl dimostrano presenza di sar in molti casi , cos come in casi di insufficienza renale acuta si ha la sua dimostrazione nel 5%10% dei casi [ 1 ]  . 
poich la stenosi aterosclerotica dellarteria renale pu indurre un danno renale progressivo , in alcune casistiche essa causa di ingresso in dialisi nel 15%30% dei pazienti uremici [ 1 ]  . 
in pazienti anziani non selezionati stata dimostrata mediante ecd una prevalenza del 6 , 6% ; la prevalenza maggiore quando coesistono altre manifestazioni cliniche dellaterosclerosi : nei pazienti sottoposti a coronarografia la prevalenza varia dal 12% al 24% , nei pazienti con ictus dell11% , nei pazienti con malattia vascolare periferica intorno al 40% [ 3 ]  . 
esistono pochi dati che valutino levidenza della sar nei pazienti portatori di aneurisma dellaorta addominale , che rappresenta altra manifestazione di patologia aterosclerotica . lelevata prevalenza di tale patologia ha sollevato la necessit di individuare una metodica non invasiva , a basso costo e con elevata accuratezza diagnostica al fine di limitare il ricorso ad unindagine strumentale invasiva , quale larteriografia che rimane , comunque , la metodica gold standard nella diagnosi della stenosi delle arterie renali [ 2 , 4 , 5 ]  . 
limaging attualmente ha a disposizione alcune metodiche non invasive , come indagini di screening e selezione per la successiva angiografia : lecd , la scintigrafia renale con captopril , langio - tc spirale , langiorm [ 2 , 3 , 511 ]  . 
attualmente lecd delle arterie renali risulta la principale metodica di screening nella diagnosi di stenosi delle arterie renali con numerosi lavori in ambito internazionale che attribuiscono a tale metodica valori di sensibilit pari all83%96% , di specificit 76%98% , valore predittivo positivo 81%92% e valore predittivo negativo 94%98% [ 58 , 1115 ]  . 
in tutti i pazienti , per una pi ampia e corretta valutazione vascolare , stata esaminata laorta addominale , valutandone calibro , morfologia e flusso ( diametro normale < 2 , 5 cm , ectasia 2 , 53 cm , aneurisma > 3 cm )  . risultati dei 467 pazienti esaminati mediante ecd , 159 ( 34% ) non presentavano indici di stenosi delle arterie renali n segni ecografici di nefropatia , ed hanno proseguito un iter terapeutico medico ; in particolare uno di questi pazienti era portatore di protesi aorto - renale pervia . 
i restanti 308 pazienti ( 66% ) mostravano invece segni ecografici di stenosi emodinamicamente significativa o di nefropatia . in particolare , 174 pazienti ( 37 , 2% ) mostravano solo segni di nefropatia , con riduzione del flusso ematico in sede ilare ed ostiale ed un aumento dell ir ; in 123 pazienti ( 26 , 3% ) era presente nefropatia severa con ir 0 , 80 e tra questi un paziente presentava aneurisma dellarteria renale ed un paziente necrosi corticale acuta , confermati successivamente con esame tc ; 51 ( 10 , 9% ) invece presentavano nefropatia moderata con ir di 0 , 75 . in 134 pazienti ( 28 , 7% ) veniva evidenziata una sar emodinamicamente significativa ; 58 dei 134 pazienti ( 12 , 4% ) con sar presentavano segni di nefropatia associata ; in 2 di questi era gia stato posizionato stent , 2 hanno eseguito an1117 fig . 
to obtain a more exhaustive and correct vascular assessment , the calibre , morphology and flow pattern of the abdominal aorta were also evaluated in all patients ( normal diameter < 2.5 cm , ectasia 2.53 cm , aneurysm > 3 cm )  . results of the 467 patients examined with cdus , 159 ( 34% ) had no indices of ras or sonographic signs of nephropathy and were started on medical therapy ; in particular , one of them had a patent aortorenal stent . 
the remaining 308 patients ( 66% ) had sonographic evidence of haemodynamically significant stenosis or nephropathy . in detail , 174 patients ( 37.2% ) showed signs of nephropathy only , with reduced hilar and ostial flow and increased ri . nephropathy was severe in 123 patients ( 26.3% ) , with ri 0.80 ; among them , one had renal artery aneurysm and one had acute cortical necrosis , later confirmed by ct . 
altri 22 pazienti non hanno eseguito angiografia ; in particolare , 1 aveva gi posizionato stent renale , 1 presentava iperplasia mio - intimale , 10 pazienti avevano gi una completa occlusione dellarteria renale , mentre 10 pazienti sono al momento in attesa di eseguire angiografia ed eventuale intervento di rivascolarizzazione . 
in totale , quindi , 33 pazienti , con sar e senza segni di nefropatia ( 7% del totale e 44% dei pazienti con segni ecd di stenosi ) non hanno eseguito esame angiografico . quarantre pazienti ( 9 , 2% del totale e 56% dei positivi per stenosi allecd ) hanno invece eseguito angiografia che ha confermato la diagnosi in 39 dei 43 pazienti . 
di questi , 24 ( 5 , 1% del totale e 55 , 8% dei positivi per stenosi ) hanno posizionato stent con ripristino ottimale della vascolarizzazione renale nel 92% dei casi ( solo in 2 casi erano presenti dopo la procedura interventistica segni clinico - laboratoristici e bioptici di ateroembolismo colesterinico , con quadro di severa compromissione dellemodinamica renale allecd in entrambi i casi )  . diciannove pazienti ( 4% del totale e 44 , 2% dei positivi per stenosi ) invece non hanno posizionato stent : di questo ultimo gruppo 7 avevano gi posizionato stent ( 2 completamente chiusi e 5 restenosi ) , in 3 sussistevano problemi anatomici con biforcazione precoce dellarteria renale , 1 paziente presentava middle aortic syndrome , in 1 caso si era verificata complicanza ( dissezione in corso di procedura ) , in 1 un caso langiografista non riusciva a superare la stenosi , 1 paziente presentava dissezione aortica , in 1 caso si trattava di stenosi lieve - moderata e 2 erano falsi positivi . in particolare , lecd ha permesso di svelare in una giovane paziente una grave situazione anatomica , verosimilmente su base congenita , definita come middle aortic syndrome o coartazione aortica sottodiaframmatica . 
dopo una gestosi , nella paziente era insorta una severa ipertensione arteriosa e , dopo unindagine ecografia risultata negativa per alterazioni morfologiche renali , era stata sottoposta ad indagine ecd che rilevava marcato decremento degli ir arteriosi intraparenchimali ( ir = 0 , 45 ) con polso parvus et tardus in sede ilare e segni di stenosi emodinamicamente significative di entrambe le arterie renali . 
this was associated with signs of nephropathy in 58 patients ( 12.4% ) ; two had already had a stent implanted , two underwent angiography and one had polycystic kidneys , whereas the remaining 56 were excluded from angiography because of signs of severe nephropathy ( longitudinal diameter < 8 cm , ri > 0.80 and poor parenchymal perfusion on cd and pd imaging )  . 
a further 22 patients did not undergo angiography , as one had a renal stent , one had myointimal hyperplasia , ten had complete occlusion of the renal artery , whereas ten were on the waiting list for angiography and possible revascularisation at the time of this writing . 
therefore , a total of 33 patients with ras and without evidence of nephropathy ( 7% of the total and 44% of patients with cd signs of stenosis ) did not undergo angiography . forty - three patients ( 9.2% of the total and 56% of those with ras on cdus ) underwent angiography , which confirmed the diagnosis in 39 . 
of these , 24 ( 5.1% of the total and 55.8% of those with stenosis ) underwent renal stenting , with optimally restored renal perfusion in 92% of cases ( only two had clinical , laboratory and biopsy evidence of cholesterol atheroembolism , with severely impaired renal haemodynamics on cdus , after the interventional procedure )  . nineteen patients ( 4% of the total and 44.2% of those with stenosis ) did not undergo stent implantation : seven of these already had stents ( two of which completely occluded and five with restenosis ) , three patients presented early bi1118 g.c. 
b indagine tc con mdc : assenza di opacizzazione in sede corticale renale bilaterale ( c )  . furcation of the renal artery , one patient had middle aortic syndrome , one developed a complication ( dissection during the procedure ) , in one , the operator could not negotiate the stenosis , one had aortic dissection , one had mild to moderate stenosis and two were false positives . 
following a gestosis , the patient developed severe arterial hypertension and , after a negative sonography for morphological renal alterations , was studied by cdus , which revealed marked reduction of intraparenchymal arterial ri ( ri = 0.45 ) with parvus - tardus pulse in the hilum and signs of haemodynamically significant stenoses of both renal arteries . 
il 56% di questi presentava una marcata riduzione del flusso a livello delle arterie renali e nel 30% erano presenti stenosi emodinamicamente significative delle arterie renali ed in un caso ( 6% ) unarteria renale era occlusa . per quanto riguarda la presenza di aneurisma dellaorta addominale ( aaa ) operato e non , i dati raccolti sono i seguenti : laaa era presente in 19 pazienti dei 333 senza segni di sar ( 5 , 7% ) con netta prevalenza nei pazienti con nefropatia ( 16 casi ) rispetto ai pazienti senza nefropatia ( 3 casi ) mentre laaa era presente in 15 dei 134 pazienti con segni 1119 g.c. 
this translates into a marked clinical benefit for the patient as well as considerable economic savings , given that out of the 467 patients studied by cdus , 88% did not undergo angiography as against 22% who did undergo angiography . 
if we exclude patients who underwent angiography before cdus ( cdus follow - up ) and those with a previously implanted stent , the remaining 65% underwent stent revascularisation , with excellent outcome in all patients except two , one of whom develop cholesterol atheroembolism after the procedure . in some cases , cdus revealed severe impairment of renal circulation in patients with poor renal compensation who showed only minor laboratory alterations ( serum creatinine 1.62.5 mg / dl )  . 
in addition to being valuable for analysing renal morphology and volume and assessing blood flow on cdus and pdus , evaluation of the ri proved to be reliable for determining renal function 1120 di sar ( 11 , 2% ) ; la presenza di aaa operato stata di 5 pazienti tra i 333 senza segni di sar ( 1 , 5% ) ed in particolare pi frequente tra i pazienti nefropatici ( 4 casi ) che nei non nefropatici ( 1 caso ) mentre la presenza di aaa operato nei pazienti con segni di sar stata di 10 pazienti sui 134 ( 7 , 46% )  . discussione dai risultati ottenuti si riconferma lutilit dellecd nellesaminare i pazienti con ipertensione e nefropatia ; lo studio dellemodinamica renale con ecd si rilevata in alcuni casi indagine insostituibile per un corretto approccio clinico - terapeutico [ 2 , 5 , 7 , 9 , 15 ]  . lecd ha evitato langiografia nei pazienti negativi e in quelli con stenosi occlusiva dellarteria renale e con segni di grave nefropatia , nei quali lintervento avrebbe potuto aggravare il deficit renale ( tossicit del mdc , ateroembolismo colesterinico , ecc . ) con indubbio vantaggio clinico per il paziente e risparmio economico . 
infatti del totale dei 467 pazienti esaminati allecd l88% non stato sottoposto ad esame angiografico e solo il 22% ha eseguito angiografia . escludendo i pazienti che hanno eseguito lindagine angiografica prima dellecd ( monitoraggio ecd ) e i pazienti con pregresso posizionamento di stent , i restanti 65% sono stati sottoposti ad intervento di rivascolarizzazione con stent con ottimi risultati in tutti i pazienti ( ad eccezione di 2 casi , uno dei quali presentava ateroembolismo colesterinico dopo la procedura )  . lindagine ecd ha rilevato in diversi casi una compromissione del circolo renale di diversa entit in pazienti con labile compenso renale che presentavano modeste alterazioni laboratoristiche ( creatininemia 1 , 62 , 5 mg / dl ) ; in questi casi il corretto approccio terapeutico ha sicuramente ritardato lesaurimento della funzionalit renale ed il ricorso al trattamento dialitico con il miglioramento della qualit della vita  . 
si tratta di una sindrome non comune in letteratura , descritta in giovane et ( 628 anni ) come variet rara di coartazione aortica ( 0 , 2%2% delle coartazioni aortiche ) , associata ad aneurisma dellaorta addominale e a stenosi della arteria renale . 
rarely reported in the literature , the syndrome has been described in young subjects ( 628 years old ) as a rare variant of aortic coarctation ( 0.2%2% of aortic coarctations ) and to be associated with aaa and ras . 
multiple causes have been recognised , including congenital and secondary to takayasus syndrome , von recklinghausens neurofibromatosis , coeliac disease and autoimmune disorders ( dermatitis herpetiformis , insulindependent diabetes ) , and clinical onset is usually renovascular hypertension and claudication [ 2224 ]  . in 16 patients with a clinical and laboratory diagnosis of cholesterol atheroembolism , the results of cdus provided a good indication of the severity of their clinical condition : in 12 patients , the condition arose after interventional procedures ( five after angiography , four after coronary angiography , one after coronary artery stenting , one after subclavian artery stenting and one after left renal artery stenting ) and in four patients following administration of coumadall patients had signs of severely impaired parenchymal flow due to cholesterol emboli clogging the small arteries and causing inflammatory reactions , intimal proliferation , vessel fibrosis and luminal obliteration . 
despite being nonspecific findings on cdus , these alterations translate into increased resistive index in the intrarenal arteries and reduced parenchymal and hilar flow in the renal arteries . in nine cases , cdus diagnosed renal artery occlusion in patients with modest chronic renal failure whose renal function had rapidly declined ; thus , it avoided the need for further investigations and enabled selection of patients for medical therapy . 
the study of the renal artery should not be limited to the ostial region ( where the majority of stenoses are present ) but should include an overall assessment of renal arterial circulation to detect possible alterations and evaluate the feasibility of an appropriate revascularisation procedure . 
patients with severe impairment of renal haemodynamics who were unlikely to benefit from revascularisation were therefore excluded from angiography [ 17 , 25 ]  . our experience highlights the association between aaa and ras , an important factor for vascular surgeons and angiographers considering endovascular treatment for aortic aneurysms and for selecting the most appropriate strategy when the two diseases coexist [ 2630 ]  . this study reconfirms the crucial role of cdus in monitoring patients who have undergone stent revascularisation procedures [ 25 ]  . 
in addition , it raises the problem of contrast medium toxicity in patients with poor renal function . cdus consistently revealed haemodynamically significant stenosis of the stent site with parvus - tardus pulse below the stenosis , in most cases due to the presence of fibrointimal celiaco e malattie autoimmuni ( dermatite erpetiforme , diabete insulino dipendente ) ; spesso ad esordio clinico con ipertensione nefrovascolare e claudicatio [ 2224 ]  . in 16 pazienti con diagnosi clinico - laboratoristica di ateroembolismo colesterinico , lecd ha fornito risultati che ben rispecchiano la gravit del quadro clinico : in 12 pazienti il quadro insorto dopo manovre interventistiche ( 5 dopo angiografia , 4 dopo coronarografia , 1 dopo posizionamento di stent in arteria carotide , 1 in arteria succlavia e 1 in arteria renale sinistra ) e in 4 pazienti dopo somministrazione di coumadin tutti i pazienti sono stati rilevati segni di grave compromissione del circolo parenchimale dovuto agli emboli di colesterolo il cui impianto nelle piccole arterie seguito da una reazione infiammatoria , proliferazione dellintima , fibrosi dei vasi colpiti e obliterazione del lume vasale . 
tali alterazioni , pur essendo aspecifiche allecd , si traducono con lincremento dellindice di resistenza arterioso intrarenale e con riduzione del flusso parenchimale e ilare nelle arterie renali . in 9 casi lecd ha diagnosticato locclusione di unarteria renale , in pazienti con modesta insufficienza renale cronica che avevano presentato rapido aggravamento della funzionalit renale evitando in questi ulteriori indagini e sottoponendoli alla sola terapia medica . 
lo studio dellarteria renale non deve esaurirsi alla sede ostiale ( ove presente la maggior parte delle stenosi ) ma deve valutare globalmente il circolo arterioso renale per evidenziare eventuali alterazioni e per valutare la possibilit di proporre al paziente un corretto intervento di rivascolarizzazione . 
sono pertanto stati esclusi dallangiografia i pazienti che presentavano severa compromissione dellemodinamica renale , che probabilmente non avrebbero tratto beneficio dal trattamento di rivascolarizzazione [ 17 , 25 ]  . la nostra esperienza mette in risalto lassociazione fra la patologia aneurismatica dellaorta addominale e la stenosi dellarteria renale , dato sicuramente molto importante per il chirurgo vascolare e per langiografista , alla luce del trattamento endovascolare degli aneurismi aortici e sulla scelta strategica pi opportuna in presenza contemporanea delle due patologie [ 2630 ]  . in questo studio viene riaffermato linsostituibile ruolo svolto dallecd nel monitoraggio dei pazienti che hanno subito interventi di rivascolarizzazione con stent [ 25 ]  . 
in questi pazienti lindagine angio - tc ha fornito , nella nostra esperienza , informazioni sul corretto posizionamento dello stent ma risultato difficile lo studio della perviet dello stent stesso , senza tener conto della tossicit del mdc in questi pazienti dalla gi labile funzionalit renale . lesame ecd ha rilevato stenosi emodinamicamente significative in corrispondenza dello stent con presenza di polso parvus et tardus nel tratto a valle la stenosi in tutti i pazienti , nella maggior parte dovuto alla presenza di iperplasia fibrointimale o a placche aterosclerotiche site prossimalmente o distalmente rispetto allo stent . 
cdus identified patients with restenosis ( seven cases ) , all of which were confirmed by angiography and subsequently treated by repeat revascularisation ( placement with another co - axial stent or with the use of a cutting balloon )  . conclusioni di rivascolarizzazione ( posizionando un altro stent coassiale o con lutilizzo del cutting ballon )  . conclusions on the basis of our results and data from previous reports , we confirm that cdus plays a fundamental role in patients with suspected or known renovascular disease , as it provides important information on renal haemodynamics and guides the clinician towards the most appropriate clinical and therapeutic approach [ 2 , 57 , 1416 ]  . our results indicate that cdus , with appropriate selection criteria , is able to discriminate between patients who should or should not undergo angiography , leading to considerable economic savings and benefits for the patient , who can thus be directed to the most appropriate treatment plan [ 16 ]  . 
in addition , our findings point to a close association between the presence of aaa and ras and confirm the need for close collaboration between the vascular surgeon , the interventional radiologist and the nephrologist in planning the most appropriate treatment approach in these patients with associated diseases [ 2630 ]  . cdus proved its value in confirming the diagnosis of cholesterol embolism suspected on the basis of clinical and laboratory findings . 
these patients carry a poor prognosis , with 1 - year mortality rates between 64% and 87% , and monitoring by imaging may certainly help the clinician deal with this complex disease given that angiography is strongly contraindicated both because it is an interventional manoeuvre and because it requires heparin administration [ 3133 , 34 ]  . in the patient with middle aortic syndrome , cdus revealed the alteration in renal haemodynamics , thus justifying the use of a more panoramic modality , such as ct angiography , which shed light on the patients complex anatomical situation [ 2224 ]  . another important contribution of cdus was to substantiate the clinical and laboratory suspicion of acute renal failure in a septic patient with cortical necrosis . 
use of contrast medium , justified by the need to shed light on what was clearly a very critical clinical situation , enabled prompt treatment ( haemodialysis and plasmapheresis ) , with partial recovery of renal function [ 35 ]  . cdus confirmed its value in monitoring nephropathic patients and those who have undergone renal artery revascularisation procedures [ 17 , 25 ]  . 
our experience also highlights the importance of cdus in monitoring patients in whom renal artery occlusion or stenosis has been diagnosed at angiography : in these patients , clinical and laboratory monitoring should be combined with follow - up by cdus , which is able to effectively reveal disease progression and provide the clinician with the data required for a more appropriate treatment strategy . 1122 dai risultati ottenuti e alla luce dei dati della letteratura , si riconferma il ruolo fondamentale svolto dallecd nei pazienti con sospetta o accertata patologia reno - vascolare , fornendo dati importanti sullemodinamica renale di insostituibile utilit per un corretto approccio clinico - terapeutico [ 2 , 57 , 1416 ]  . dai nostri risultati si evince come ecd , alla luce dei criteri adottati , possa essere metodica in grado di discriminare i pazienti che devono o non devono essere sottoposti ad esame agiografico , con risparmio dei costi e beneficio per il paziente che potr quindi essere indirizzato al pi opportuno iter terapeutico [ 16 ]  . 
i nostri dati mettono inoltre in risalto unelevata associazione fra la presenza dellaaa e la sar : questo vuole riconfermare lutilit di una stretta collaborazione fra il chirurgo vascolare , il radiologo interventista e il nefrologo nel programmare la strategia terapeutica pi opportuna in questi pazienti con patologia associata [ 2630 ]  . lecd si dimostrata metodica utile nel confermare la diagnosi di ateroembolismo colesterinico , gi prospettata in base ai dati clinico - laboratoristici . 
questi sono pazienti a prognosi infausta con mortalit ad 1 anno variabile dal 64% all87% in cui il monitoraggio strumentale pu essere certamente di ausilio al clinico nellaffrontare una cos complessa patologia , dal momento che in tali pazienti fortemente controindicata langiografia , sia perch manovra interventistica sia per la somministrazione di eparina [ 3133 , 34 ]  . nella paziente con la middle aortic syndrome lecd ha svelato lalterazione emodinamica a livello renale , giustificando il ricorso a metodiche pi panoramiche come langiotc che ha rilevato la complessa situazione anatomica da cui era affetta [ 2224 ]  . occorre senzaltro rimarcare limportanza dellecd nel confermare lipotesi clinico - laboratoristica di insufficienza renale acuta in corso di sepsi nella paziente affetta da necrosi corticale ; lutilizzo del mdc , giustificato dalla necessit di chiarire una situazione che allesordio era molto critica , ha consentito la tempestiva terapia ( emodialisi e plasmaferesi ) con parziale recupero della funzione renale [ 35 ]  . la metodica ecd si dunque riconfermata di indubbio valore nel monitoraggio dei pazienti nefropatici e di quelli sottoposti ad intervento di rivascolarizzazione allarteria renale [ 17 , 25 ]  . 
souza de oliveira ir , widman a , molinar lj et al ( 2000 ) color doppler ultrasound : a new index improves the diagnosis of renal artery stenosis . ultrasound med biol 26 : 4147 12 . 
frauchiger b , zierler r , bergelin ro et al ( 1996 ) prognostic significance of intrarenal resistance indices in patients with renal artery interventions : a preliminary duplex sonographic study . cardiovasc surg 4 : 324330 18 . 
mehta m , darling rc 3rd , roddy sp et al ( 2004 ) outcome of concomitant renal artery reconstructions in patients with aortic aneurysm and occlusive disease . vascular 12 : 381386 29 . 
manenti , tel : + 39 - 06 - 20902401 , fax : + 39 - 06 - 20902404 , e - mail : guggi@tiscali.it received : 9 november 2005 / accepted : 30 march 2006 / published online : 20 december 2006 abstract purpose . 
morphological imaging was obtained with t2 - weighted turbo spin - echo ( tse ) sequences with and without fat suppression [ spectral presaturation with inversion recovery ( spir ) ] and an axial dynamic t1 - weighted spir fast - field echo ( ffe ) sequence during intravenous administration of contrast material . 
our preliminary results indicate that dwi is useful for characterising tissue in the different regions of the prostate gland and in distinguishing normal from cancerous tissues , given its ability to detect early changes in the structural organisation of prostate tissue . key words prostate mr diffusion adc value of prostate tumours 1124 riassunto obiettivo . 
la diffusione un processo fisico basato sul movimento casuale delle molecole dacqua noto come moto browniano . limaging pesato in diffusione ( dwi ) quindi una tecnica di risonanza magnetica ( rm ) che fornisce indirettamente informazioni sulle propriet biofisiche dei tessuti come la densit , lorganizzazione cellulare e la microstruttura , che determinano la diffusibilit delle molecole dacqua . 
limaging morfologico stato condotto mediante sequenze tse t2 - pesata con e senza tecnica di soppressione del segnale del grasso ( spir ) ed una sequenza dinamica ffe assiale t1 - pesata spir durante somministrazione ev di mdc . 
il valore medio di adc della porzione centrale della ghiandola ( 1512 , 07124 , 8510 - 3 mm2 / s ) risultato significativamente pi basso del valore di adc della porzione periferica ( 1984 , 11226 , 2310 - 3 mm2 / s ) ( p < 0 , 01 )  . 
currently , mri is the only technique able to assess molecular diffusion in vivo and provide information about such biophysical properties of tissue as cell organisation , density and microstructure [ 1 ]  . rapid changes in diffusion properties can be identified by calculating the diffusion coefficient ( dc )  . 
however , in biological systems , this coefficient is known as apparent diffusion coefficient ( adc ) because its value is influenced not only by the brownian motion of water molecules , but also by other polluting factors , including perfusion and t2 tissue signals [ 2 ]  . diffusion mri techniques have been mainly used in the field of neuroradiology to evaluate cerebral ischaemia , stroke and multiple sclerosis , and to differentiate normal from cancerous tissue . 
all patients received ten punctures according to the division of the prostate into octants ( one puncture each for apex , intermediate zone and base , and two punctures on each side for the lateral zones )  . 
at least two samples were obtained from zones presenting abnormal features on mri and trus . mri study the mri examination was performed using a 1.5 - tesla magnet ( philips gyroscan intera ; best , the netherlands ) equipped with a master dynamic gradient system ( 30 mt / m maximum power and 150 mt / m / ms slew rate ) using a multichannel surface coil . le immagini di risonanza magnetica ( rm ) pesate in diffusione ( dwi ) sono sensibili alla diffusione molecolare dovuta ai movimenti casuali delle molecole dacqua in grado di produrre in un gradiente di campo magnetico unalterazione incoerente della fase che determina unattenuazione dellintensit di segnale . 
attualmente la rm la sola tecnica in grado di valutare il processo di diffusione molecolare in vivo e consente di ottenere quindi informazioni sulle propriet biofisiche dei tessuti come lorganizzazione , la densit cellulare e la microstruttura [ 1 ]  . rapidi cambiamenti nelle caratteristiche di diffusione possono essere identificati calcolando il coefficiente di diffusione ( dc )  . 
tuttavia , nei sistemi biologici si deve parlare di coefficiente apparente di diffusione ( adc ) poich il valore del coefficiente misurato determinato non solo dal moto browniano delle molecole dacqua , ma anche da altri fattori inquinanti , fra cui la perfusione del microcircolo e la componente t2 tissutale [ 2 ]  . finora le tecniche di diffusione rm sono state abitualmente utilizzate nel campo della neuroradiologia per la valutazione dellischemia cerebrale , dello stroke , della sclerosi multipla e per differenziare tessuto sano da quello tumorale . 
per lo studio degli organi addominali le sequenze pesate in diffusione non sono ancora entrate nella pratica clinica soprattutto per motivi di natura tecnica : movimenti respiratori , suscettibilit magnetica dellaria nei polmoni e nellintestino , e artefatti da pulsatilit e da movimento rappresentano le cause pi comuni della scarsa qualit delle immagini pesate in diffusione anche a livello addominale . lavvento delle sequenze eco - planari a singolo impulso ultraveloci ( epi ) in associazione con le tecniche di acquisizione in una singola apnea , hanno consentito la fattibilit delle sequenze pesate in diffusione anche a livello addominale [ 3 , 4 ]  . lo scopo del nostro studio stato quello di valutare laffidabilit del dwi nella valutazione delladc delle differenti regioni anatomiche della ghiandola prostatica e di testare la possibilit di eseguire una differenziazione fra tessuto sano e maligno sulla base del valore di adc . materiali e metodi pazienti sono stati studiati 10 volontari ( et media 56 anni , range : 4072 anni ; psa totale medio 4 , 65 , range 2 , 86 , 5 ng / ml ) e 19 pazienti ( et media 65 anni , range : 4585 anni ; psa totale medio 25 , 15 , range 4 , 346 ng / ml ) con tumore prostatico , precedentemente diagnosticato con esame ecotomografico trans - rettale ( trus ) e successivamente confermato con biopsia trus - guidata eseguita entro un mese dopo lesecuzione dellesame rm . 
in tutti i pazienti sono stati effettuati 10 prelievi secondo suddivisione in ottanti della prostata ( 1 prelievo per apice , zona intermedia , basale e 2 per la zona laterale per ciascun lato )  . 
le immagini pesate in diffusione sono state ottenute prima delliniezione di mezzo di contrasto con una sequenza single - shot spin - echo epi inversion recovery ( te 64 , tr 2500 , fa 90 , fov 140 mm , matrice 6464 , spessore di sezione 3 mm , intervallo 1 mm , 15 slice , valore b = 0 , 125 , 250 , 375 and 500 s / mm2 ) , nsa 6 e tempo di acquisizione 4 minuti e 22 secondi , orientata sul piano assiale e con lutilizzo della tecnica di codifica del segnale sense . misurazione adc la mappa di adc stata ottenuta per ogni slice calcolando il valore di adc dei singoli pixel utilizzando un algoritmo illustrato dalla seguente equazione : adc ( mm2 / s ) = 1 / 1000ln [ is ( b0 ) / is ( bx ) ] dove is ( b0 ) rappresenta lintensit del segnale nelle acquisizioni con fattore b uguale a 0 e is ( bx ) rappresenta lintensit del segnale nelle acquisizioni con fattore b uguale ad ognuno dei valori utilizzati . 
sulle immagini assiali della sequenza pesata in diffusione con valore b = 0 , che non contengono dati sulla diffusione molecolare , ma sono caratterizzate da una pesatura t2 , sono state posizionate roi ( regioni di interesse ) bilaterali e simmetriche , di forma ellittica e con area equivalente ad 8 pixel , a livello della zona centrale , della periferia e dellarea tumorale ( area focale di ipointensit relativa rispetto al parenchima circostante ) , quando presente , avendo cura di evitare calcificazioni e cisti . 
in particolare stato adottato il paired students t test per valutare le differenze statistiche fra i valori di adc delle differenti aree prostatiche sane e malate allinterno dei due gruppi separatamente ; mentre stato adottato lunpaired students t test per valutare le differenze statistiche dei valori di adc delle singole aree prostatiche fra i due gruppi . 
on the axial dwis with b = 0 , which do not contain data on molecular diffusion but are characterised by t2 weighting , we positioned symmetrical elliptical regions of interest ( rois ) measuring 8 pixels bilaterally in the central and peripheral zone and in the tumour area ( a relatively hypointense focal area compared with the surrounding parenchyma ) , when present , taking care to avoid calcifications and cysts . 
data were processed using the statistical package for social sciences , version 12 ( spss , chicago , il , usa )  . results 1128 all examinations were diagnostic , and no case was excluded from the study . nei 19 pazienti il valore medio del coefficiente apparente di diffusione ( adc ) della zona centrale della ghiandola prostatica ( 1512 , 07124 , 8510 - 3 mm2 / s ) risultato statisticamente pi basso del valore medio di adc della zona periferica sana ( 1984 , 11226 , 2310 - 3 mm2 / s ) e il valore medio di adc dei tumori ( 958 , 97168 , 9810 - 3 mm2 / s ) risultato statisticamente pi basso del valore medio di adc della zona periferica sana ( p < 0 , 01 )  . 
la variabilit dei risultati ottenuti legata soprattutto alla differente pesatura in diffusione delle immagini , rappresentata dal valore del fattore b che racchiude le principali caratteristiche dei gradienti di diffusione ( ampiezza , durata e intervallo di tempo ) [ 1 , 58 ]  . la pesatura in diffusione ideale di una sequenza epi rappresenta ancora un problema irrisolto . 
se vengono utilizzati valori bassi di fattore b ( 30100 s / mm2 ) pu risultare una sovrastima del valore di adc per laggiunta della componente t2 tissutale e della perfusione del microcircolo al valore puro di diffusione delle molecole dacqua . 
 [ 9 ] hanno proposto una sequenza pesata in diffusione con fattore b di 0 e 1000 ed elevata risoluzione spaziale , acquisita mediante bobina di superficie : contrariamente agli altri lavori apparsi in letteratura sullutilizzo delle sequenze pesate di diffusione nella diagnosi del tumore prostatico , il tessuto neoplastico stato in questo caso individuato grazie al suo segnale iperintenso nelle immagini con fattore b 1000 , senza la costruzione di mappe di adc a causa della riduzione del rapporto segnale / rumore e quindi della qualit delle immagini , determinati dalla forte pesatura in diffusione [ 9 ]  . nel nostro lavoro , al fine di ottenere una valutazione quantitativa della restrizione del coefficiente di diffusione apparente , abbiamo deciso di utilizzare un valore massimo di fattore b pari a 500 e di costruire mappe di adc grazie al buon rapporto segnale / rumore . 
4 valore medio adc ( 10 - 3 mm2 / s ) con relativo valore di deviazione standard nella zona periferica ( zp ) , nella zona centrale ( zc ) e nelle aree tumorali dei 19 pazienti esaminati . discussion mri is currently the only technique capable of measuring molecular diffusion in vivo . 
la mappa di adc stata ottenuta dai dati acquisiti con i singoli valori di fattore b . la difficolt aggiuntiva nella valutazione dei valori di adc nella ghiandola prostatica risiede nella disomogeneit del suo parenchima , soprattutto per quanto riguarda la porzione centrale sia essa normale che adenomatosa , spesso sede di alterazioni di natura sia cistica che fibro - calcifica le2350 2150 1950 1750 1550 1350 1150 2350 2150 1950 1750 1550 1350 1150 1130 g . 
if low b - values ( 30100 s / mm2 ) are used , the adc value can be overestimated owing to the t2 tissue component and perfusion adding to the pure diffusion value of water molecules . 
conversely , the use of high b - values ( > 1 , 000 s / mm2 ) produces extremely noisy images due to the short t2 value of abdominal tissues . a recent paper by shimofusa et al . 
unlike other reports investigating the use of dwi for prostate cancer detection , the study identified cancerous tissue based on its hyperintense signal on images with b - value = 1 , 000 , without the production of adc maps because of the lower signal - tonoise ratio ( snr ) and poorer image quality generated by the high diffusion weighting [ 9 ]  . in our study , in order to quantitatively evaluate the reduction in apparent diffusion coefficient , we decided to use a maximum b - value = 500 and to produce adc maps based on the good snr . 
a useful expedient to obtain good - quality images with high diffusion weighting was to acquire the sequence with different b - values ( 0 , 125 , 250 , 375 and 500 )  . the adc map was obtained from the data acquired with the individual b - values . an additional problem in assessing prostate adc values lies in the heterogeneity of its parenchyma ; this is especially true of its central portion , which , whether normal and adenomatous , is often the site of cystic and fibrotic - calcific changes related to inflammation . 
the peripheral zone may also undergo structural fibrotic changes related to chronic inflammation ; the differential diagnosis between these changes and cancer is not easy with noninvasive imaging techniques , and often biopsy is required although a radiated rather than nodular appearance and slow wash - in and wash - out following contrast administration are strongly indicative of fibrotic disease . 
to measure the adc values of different portions of the gland , we carefully selected the areas to be sampled and avoided positioning the roi on tissues with calcifications or cysts , whose signals in t2 - weighted sequences are necessarily altered . our series revealed a statistically significant difference between the adc values of the central zone and those of the healthy peripheral zone and , more importantly , a dramatic reduction in the adc values in focal malignant lesions compared with the healthy peripheral zone . 
zonal differentiation within the prostate based on t2 signal intensity and adc values was confirmed by the histological sections exhibiting larger luminal spaces in the peripheral zones , which accounts for the greater concentration and freedom of movement of water molecules in the periphery compared with the centre [ 10 ]  . 
conversely , the marked reduction of adc values in cancerous areas is explained with the replacement of normal tissue , composed of water - rich acinar structures , by cancerous tissue , usually consisting of small components packed into a small amount of stroma : derangement of normal tissue structure is associated with the presence of cellular elements with increased nucleus - to - cytoplasm ratio , resulting in extremely reduced motility of water molecules [ 11 ]  . gate essenzialmente a fenomeni flogistici . 
anche la porzione periferica pu essere sede di alterazioni strutturali di natura prevalentemente fibrotica , legate anchesse a fenomeni flogistici cronici , la cui diagnosi differenziale con lesioni di natura neoplastica non cos agevole con le tecniche di imaging non invasive , risolvendosi spesso solo con il prelievo bioptico , anche se un aspetto radiato piuttosto che nodulare e un comportamento contrastografico con wash - in e washout lenti sono fortemente indicativi di patologia fibrotica . per la misurazione dei valori di adc nelle differenti porzioni della ghiandola , stata quindi posta particolare attenzione nella scelta delle aree campionabili evitando di posizionare la roi su tessuto interessato da calcificazioni o cisti , il cui segnale nelle sequenze t2 - pesate risulta inevitabilmente alterato . dalla nostra casistica si riscontrata una differenza statisticamente significativa fra i valori di adc dellarea centrale e della zona periferica sana , ma ancora pi importante la netta riduzione dei valori di adc nelle lesioni focali maligne rispetto alla zona periferica sana . 
la differenziazione regionale allinterno della ghiandola prostatica in base allintensit di segnale nelle sequenze t2 - pesate e ai valori di adc stata confermata nello studio delle sezioni istologiche dalla dimostrazione di spazi luminali di maggiori dimensioni a livello della porzione periferica , che rende ragione della maggiore concentrazione e libert di movimento delle molecole dacqua in questa zona rispetto alla zona centrale [ 10 ]  . 
viceversa , la netta riduzione del valore di adc allinterno di unarea tumorale trova giustificazione nella sostituzione delle strutture acinari ricche di acqua del tessuto normale da parte di tessuto neoplastico , costituito comunemente da un pattern cellulare a piccoli elementi fittamente stipati allinterno di una esigua quantit di stroma : alla perdita della normale architettura tissutale , si associa anche la presenza di elementi cellulari con rapporto nucleo / citoplasma aumentato , quindi con motilit delle molecole dacqua estremamente limitata [ 11 ]  . gi alcuni lavori pubblicati negli ultimi hanno dimostrato la capacit di distinguere tessuto neoplastico sano e canceroso nella regione periferica e di transizione della ghiandola prostatica da parte del dwi con sequenze epi sulla base del valore di adc [ 12 , 13 ]  . 
per aumentare la capacit delle sequenze pesate in diffusione di differenziare regioni distinte , sane e patologiche , della ghiandola prostatica stato fondamentale lutilizzo di sequenze epi con risoluzione spaziale elevata , ottenuta con ladozione di spessori di strato sottili ( 3 mm ) e di fov di piccole dimensioni ( 140 mm2 ) seppur con una matrice di acquisizione ridotta ( 6464 )  . 
per lacquisizione di tale sequenza , stato preferito lutilizzo della bobina di superficie multicanale in sostituzione della bobina endorettale , che abbiamo deciso di non utilizzare a causa degli inevitabili artefatti gi codificati in letteratura oltre che per ridurre il disconfort del soggetto sottoposto allesame [ 14 ]  . la maggiore differenza statistica fra i valori di adc delle lesioni cancerose e del tessuto sano della zona periferica della ghiandola prostatica risultante dalla nostra casistica rispetto a lavori apparsi recentemente in letteratura 1131 g . 
however , to enhance the ability of dwi to differentiate between healthy and cancerous regions of the prostate gland , the use of epi sequences with high spatial resolution , obtained with thin sections ( 3 mm ) and small fov ( 140 mm2 ) , albeit with a reduced acquisition matrix ( 6464 ) , proved essential . 
for this sequence , we decided to use a multichannel surface coil instead of an endorectal coil to avoid the inevitable artefacts already described in the literature and to minimise the subjects discomfort [ 14 ]  . the greater statistical difference between the adc values of cancerous lesions and those of healthy tissue of the peripheral zone observed in our study is likely to be related to the higher spatial resolution of the sequence ( 13.23 - mm3 voxel ) and resulting reduction in partial volume effects when measuring the adc value : sato et al . 
the higher spatial resolution did , however , require a increased number of acquisition averages in the diffusion - weighted sequence , with a resulting time of 4 min 22 s , that is , 1 min 16 s and 1 min longer than the sequences used by sato et al . 
and by hosseinzadeh et al . , respectively , but causing in no case the appearance of motion artefacts in the prostate gland . conclusions our preliminary results suggest that thin - slice diffusionweighted mri can be used to acquire data on tissue characterisation of the different anatomical zones of the prostate gland . 
diffusion - weighted mri also proved to be reliable and reproducible in differentiating normal and cancerous tissue , allowing detection of early changes in the structural organisation of prostatic tissue . 
comparative studies on adc values of cancer tissue with fibrotic areas or with localised , especially granulomatous , prostatitis will be of great assistance in understanding the real value and possible role of diffusion - weighted sequences in the noninvasive characterisation of structural alterations in the peripheral zone . 
although larger series are needed to confirm the difference between the adc values of benign and malignant lesions , our study suggests that diffusion - weighted mri could be used to integrate the standard prostate imaging protocol , adding only a few minutes to the total mri examination time . 1132 verosimilmente legata alla maggiore risoluzione spaziale della sequenza utilizzata ( voxel di 13 , 23 mm3 ) , e alla conseguente riduzione degli effetti di volume parziale durante la misurazione del valore di adc : sato et al . 
 [ 12 ] per differenziare il tessuto sano da quello canceroso mediante il valore di adc hanno proposto una sequenza con fattore b di 0 - 300 - 600 , matrice di 119192 e fov di 250 mm2 , ma uno spessore di strato di 7 mm con voxel risultante di 2 , 11 , 37 mm ( 19 , 11 mm3 ) , utilizzando una bobina di superficie [ 12 ] ; mentre hosseinzadeh et al . 
 [ 13 ] per lo stesso fine hanno proposto una sequenza con fattore b di 0 - 1000 , matrice di 8080 , fov di 200 mm2 e spessore di strato di 3 mm con voxel risultante di 2 , 52 , 53 mm ( 18 , 75 mm3 ) , utilizzando una bobina trans - rettale insieme ad una bobina di superficie per la pelvi per aumentare il rapporto segnalerumore e consentire la costruzione di mappe di adc con lutilizzo di sequenze maggiormente pesate in diffusione [ 13 ]  . 
la maggiore risoluzione spaziale ha comportato tuttavia la necessit di aumentare il numero delle medie di acquisizione della sequenza pesata in diffusione con un tempo risultante di 4 minuti e 22 secondi , nettamente superiore a quello delle sequenze utilizzate da sato et al . 
 [ 12 , 13 ] , rispettivamente di 1 minuto e 16 secondi e di 1 minuto , senza tuttavia determinare in nessun caso la comparsa di artefatti da movimento a livello della ghiandola prostatica . conclusioni i nostri risultati preliminari indicano la possibilit di poter utilizzare le sequenze rm di diffusione a strato sottile per acquisire dati sulla caratterizzazione tissutale delle differenti regioni anatomiche della ghiandola prostatica . 
la diffusione in rm inoltre risultata essere una metodica affidabile e riproducibile per differenziare il tessuto prostatico normale da quello neoplastico , riuscendo a distinguere cambiamenti precoci nellorganizzazione strutturale del tessuto prostatico . 
studi comparativi sui valori di adc del tessuto neoplastico con aree fibrotiche o con localizzazioni di prostatite , soprattutto nella sua variante granulomatosa , saranno di grande ausilio per capire leffettiva utilit e il possibile ruolo delle sequenze pesate in diffusione nella diagnostica non invasiva di natura delle alterazioni strutturali della porzione periferica . 
inches2 1dipartimento di scienze oncologiche e chirurgiche , sezione di senologia , universit degli studi di padova , via gattamelata 64 , i - 35128 padova , italy 2dipartimento di scienze medicodiagnostiche e terapie speciali , universit degli studi di padova , via giustiniani 2 , i - 35128 padova , italy correspondence to : l . 
practical aspects with psychological and clinical involvement are discussed with tree diagrams . key words human error perception interpretation system riassunto viene proposto un approccio sistematico allerrore umano nella diagnostica per immagini ( di ) , riconoscendo la sua collocazione nelle attivit personali della percezione e della interpretazione e nelloperativit globale del sistema . 
nellinquadramento che si sviluppa secondo ramificazioni ad albero sono discussi gli aspetti pratici alla luce di contributi provenienti dagli studi psicologici e dalla evoluzione della realt clinica e sanitaria . parole chiave errore umano percezione interpretazione sistema introduction introduzione the approach to the study of human error in diagnostic imaging ( di ) requires a preliminary introduction regarding the concept of error in modern medicine . human error is always placed inevitably in relation with truth [ 1 , 2 ] , which in a strictly scientific field should be considered , as the epistemologist blandino states , as only probable and approximate , connected to theories which change and at times crumble , to be replaced by other , more valid ones . 
it can safely be said that there is currently such a huge quantity of knowledge in medicine and biology that the medical practitioner is increasingly exposed to a great number and variety of errors . traditionally two relevant aspects have been indicated : errors in medicine , considered as the collection of theories regarding health and the state of disease in humans ; for the scientific representation of errors in medicine the statistics and theory of bayes [ 3 ] are used medical practitioners errors , in that as humans . 
they are subject to making mistakes according to a distinction made by epistemologists , the term mistake is more accurate for errors related to the activity in which theoretical notions are applied whereas errors in the lapproccio allo studio degli errori commessi dalluomo nella diagnostica per immagini ( di ) impone una preliminare introduzione sulle connotazioni che il concetto di errore assume nella moderna attivit medica . lerrore da sempre messo inevitabilmente in rapporto con la verit [ 1 , 2 ] che in ambito strettamente scientifico da intendersi , come indica lepistemologo blandino , come soltanto probabile e approssimata , connessa a teorie che si modificano e talora crollano , sostituite da altre pi valide : anche le verit diagnostiche sono quindi relative e commisurate al sapere di un determinato tempo . 
non vi dubbio che allo stato attuale via sia una tale mole di conoscenze in medicina e biologia che si pu tranquillamente affermare che loperato del medico sempre pi esposto ad errori , molteplici e di varia natura . tradizionalmente se ne indicano due aspetti rilevanti : gli errori della medicina , considerata come insieme di teorie che riguardano la salute e lo stato di malattia delluomo , per la rappresentazione scientifica dei quali ci si avvale soprattutto della statistica e del teorema di bayes [ 3 ] ; gli errori dei medici , in quanto essere umani e come tali soggetti a sbagliare . secondo una distinzione degli epistemologi , proprio il termistrict sense regard science as such . 
in this paper , the term error will be used for simplicity , even if in reference to mistakes . medical tradition also commonly makes a distinction between two reference errors : cognitive : concerning diagnosis and prognosis operative : concerning decision making and therapy di is slightly removed from this picture , as the use of perception is particularly relevant but also because given the progressive expansion of the discipline , the search for errors needs to be placed in an organized and technologically advanced context , with a much broader range of devices than in the early days of the first applications of the coolidge tube when medicine was almost totally reliant on the personal abilities of the practitioners . like other fields , such as nuclear engineering or aviation [ 4 , 5 ] , a radiology department stands out for its organizational and operative peculiarities as a system , with features similar to any company such as logistics , finance and the hiring and training of personnel . 
some errors may be present in the planning and organizational phase such that reason and rasmussen [ 6 ] classifies them as latent errors with delayed effects and differentiates them from active errors in which there is a momentary and welldefined participation by the operator . 
seeking out these remote errors is an important feature of the modern management of the radiology department because when they appear , the operator is in fact programmed to make a mistake . 
the operator is the victim of a mediocre project , insufficient maintenance , inappropriate materials and managerial decisions which create situations of risk , such as unrealistic workloads , inappropriate training or exasperating turnaround times . 
since the error appears with the last operator , whether he or she is the direct or indirect cause , all errors should be considered human error and in di the radiologists error . in this context the aim of this study was to unify the problems and the causes of errors , taking into consideration the crucial moment of image assessment in radiology when the radiologist utilizes the characteristics of his or her personality , culture and training [ 7 ]  . characteristics of the radiologists activities the radiological act as it currently stands can be defined as a technical - methodological procedure performed on a patient in an organizational and decision - making system , the outcome of which is the image obtained to achieve diagnosis . 
it is here , in fact , that to reach a decision , the radiologist makes use of a process of perception and cognition , features which occur simultaneously and are interdependent , and yet , as wood [ 8 ] notes , are separate mental activities . 
inches : systematic approach to human error in radiology ne sbaglio pi corretto ad indicare gli errori inerenti alle attivit in cui le nozioni teoriche sono applicate , mentre gli errori in senso stretto riguardano la scienza come tale . nellelaborato il termine errore sar usato per comodit e consuetudine , anche se riferito agli sbagli . ancora nella tradizione medica si soliti configurare due errori di riferimento : cognitivo : concerne la diagnosi e la prognosi ; operativo : concerne gli aspetti decisionali e la terapia . la di se ne discosta parzialmente essendo per essa rilevante lapporto della percezione , ma anche perch , nella progressiva espansione della disciplina , la ricerca dellerrore va inquadrata in un contesto organizzato e tecnologicamente avanzato , che prospetta tipologie pi ampie rispetto allepoca artigianale delle prime applicazioni del tubo di cooledge , coeve ad una medicina legata alle pressoch assolute capacit personali dei medici . la radiologia , intesa quale servizio o unit operativa che dir si voglia , analogamente ad altre strutture di lavoro , come lingegneria nucleare o laviazione [ 4 , 5 ] , si identifica allo stato attuale per peculiarit gestionali e operative come sistema , al quale sono attribuiti analogamente a qualsiasi azienda almeno gli ambiti organizzativi della logistica , della finanza , dellassunzione e della preparazione del personale . il sistema come tale pu essere sottoposto ad analisi e verifica ai fini della ricerca degli errori e della loro correzione mediante procedure adatte . alcuni errori possono essere gi insiti in fase di progettazione e organizzazione , tanto che reason [ 6 ] li classifica come errori latenti , con effetti ritardati , differenziandoli dagli errori attivi in cui vi una partecipazione momentanea e bene circostanziata delloperatore . 
la ricerca di questi errori remoti rappresenta allora uno dei punti notevoli della moderna conduzione del reparto , perch , quando compaiono , loperatore in realt stato programmato per sbagliare : egli vittima ultima di un progetto mediocre , di una manutenzione insufficiente , di materiali inidonei , di decisioni della dirigenza che si esplicano in situazioni di rischio , quali ad esempio gli irrealistici carichi di lavoro , laddestramento non adeguato o i tempi di produzione esasperati . 
poich di fatto lerrore si manifesta nelle circostanze con lultimo operatore , ne sia egli la causa diretta o indiretta , tutti gli errori in definitiva debbono essere considerati riferiti alluomo : per la di al radiologo . sotto questo profilo lo scopo del lavoro di unificare problematiche e cause di errore prendendo in considerazione nella di il momento cruciale della valutazione del documento , allorquando il radiologo partecipa mettendo in campo le proprie caratteristiche di personalit , cultura e preparazione [ 7 ]  . caratteristiche dellattivit del radiologo latto radiologico allo stato attuale pu essere definito come procedura tecnico - metodologica condotta sul paziente , avvenuta in un sistema organizzativo - decisionale , che si esprime nel documento dimmagine ottenuto per conseguire il fil . 
inches : systematic approach to human error in radiology mental phenomena at play is required : the phenomenon of perception the perceptual process transforms retinal stimulation at the higher centres into conscious perception , allowing images to be extracted and translated in conscious assessment by simplifying and organizing the information from the peripheral regions . 
according to attneave , quoted by wood [ 8 ] , this process is limited , not being sufficiently capable in the sense of capacity to transmit all information which reaches the retina . 
hake , also cited by wood [ 8 ] , instead believes that perception is governed by the centre in that it is linked to the search for a meaning connecting the fragmentary retinal images . 
in fact , the meaning and even the writing of a sentence itself may be considered absolutely precise even though obvious errors may be found when the text is carefully revised [ 9 ]  . 
lastly , it is worth noting that perception is substantially different from simple vision , in which only the peripheral activities of the observer take part in relation to the nervous system . the phenomenon of cognition the cognitive part of diagnostic decision making is less well known yet fascinating , given the models proposed for explaining the mental stages which lead to a conclusion . 
qui , infatti , che il radiologo si avvale di un procedimento percettivo e conoscitivo , simultanei e interdipendenti , da considerare tuttavia con wood e tuddenham [ 8 ] come attivit psichiche separate , per giungere ad una decisione . 
per rendere comprensibile e al tempo stesso unitaria la classificazione a seconda della tipologia e delle cause di errore indispensabile un richiamo sintetico alle conoscenze dei fenomeni psichici in gioco . il fenomeno della percezione il processo percettivo trasforma in percezione cosciente i modelli della stimolazione retinica ai centri superiori ; consente di estrarre immagini e di tradurle in valutazione conscia , mediante una semplificazione e organizzazione delle informazioni provenienti dalla periferia . 
secondo atteneave , citato da wood [ 8 ] il processo limitato , non essendo sufficientemente capace , nel senso proprio di capiente , per trasmettere tutte le informazioni che raggiungono la retina . hake , pure citato da wood [ 8 ] ritiene invece che la percezione sia diretta dal centro in quanto legata alla ricerca di un significato che colleghi le frammentarie immagini retiniche . 
per la lettura ; i rettangoli indicano le conoscenze che dallalto in basso divengono via via pi profonde ; in un caso clinico linterprete potrebbe trascurare leziologia di un broncogramma , ad esempio metastasi polmonari , se lattivazione particolarmante frequente per le cause infettive . pointed out that classic cognitivism recognizes the diagnostic process as inductive reasoning aimed at identifying rules and categories , beginning with concrete examples : the dialogue between the areas of research of concrete examples ( variable features ) and rules ( fixed features ) leads to the selection of facts , to remembering the theories based on the facts and to test the hypotheses . 
the classic model , however , has the defect , particularly for medical applications , of overlooking the concrete and pragmatic aspects , in essence , situations such as emergencies in which the examinations are necessarily limited owing to time constraints . 
 the most complete and appropriate model for our purposes which explains the relationship between real facts and the examination of the mind is the model proposed by raufaste and eyrolle [ 10 , 11 ] in 1998 . 
long - term memory has an enormous capacity for storage and contains declarative knowledge ( theory ) and procedural knowledge ( know - how ) interconnected by various types of logical systems in an active for for example , the processes of classification of part / whole relations become increasingly rapid in acquisition times as the knowledge is implemented and interconnected . 
generally speaking , by bringing ones attention to a certain level of knowledge , that level is activated , and once more overcoming a certain threshold , awareness is achieved . working memory involves a variety of aspects , including phonological , visual and spatial . 
infine da osservare come la percezione si differenzi in modo sostanziale dalla semplice visione , al cui fenomeno partecipano solo le attivit periferiche dellosservatore in rapporto alle caratteristiche del sistema nervoso . il fenomeno cognitivo la parte cognitiva della decisione diagnostica meno conosciuta , affascinante per i modelli proposti a spiegare le tappe mentali che portano alla conclusione . 
premesso che lambito mentale molto ampio e a margini poco definiti , doveroso accennare come il cognitivismo classico riconosca il procedimento diagnostico come ragionamento induttivo teso ad identificare regole e categorie partendo da esempi concreti : il dialogo fra gli spazi di ricerca degli esempi concreti ( tratti variabili ) e delle regole ( tratti fissi ) conduce a selezionare i fatti , a ricordare le teorie a partire dai fatti e a verificare le ipotesi . 
il modello classico ha per il difetto , soprattutto per le applicazioni mediche , di trascurare gli aspetti concreti e pragmatici , in sostanza le situazioni , ad esempio le emergenze , in cui le indagini sono necessariamente limitate per motivi di tempo . 
per spiegare meglio il rapporto tra fatti reali e lindagine della mente risulta pi completo e adatto alle nostre esigenze , perch dedicato alla radiologia , il modello del francese roufaste del 1998 [ 10 , 11 ]  . 
la memoria a lungo termine ha vasta possibilit di stoccaggio ed duratura nel tempo : contiene le conoscenze dichiarative ( del sapere teorico ) e procedurali ( del saper fare ) interconnesse da diversi tipi di sistemi logici in forma attiva . 
cos ad esempio i processi di classificazione o di relazione parte / tutto divengono sempre pi rapidi nei tempi di acquisizione mano a mano che tali conoscenze sono implementate e interconnesse . 
unlike long - term memory , which is activated autonomously and without the intervention of the conscious , working memory highly depends on the attention system capable of authorizing the implementation of higher functions , such as symbolic calculations or the decision to implement or inhibit automatisms [ 10 , 11 ]  . 
 analysis of radiographs involves the fundamental function of working memory , that is , the maintenance of a representation constructed from empirical data , in our case from the characteristics of the radiographs , and from information recalled from long - term memory , which we could call experience . 
la memoria di lavoro ha apporti differenti , anche fonologici , visivi e spaziali ; permette di stoccare un numero piccolo di informazioni ( da 3 a 7 indipendenti una dallaltra )  . 
la risoluzione di problemi complessi , come quello della valutazione dei radiogrammi , abbisogna di parametri molto pi numerosi : il problema risolto con lassociazione nella memoria di lavoro di pi sottoinsiemi , che sono trattati come singole unit . 
contrariamente alla memoria a lungo termine , che attivata autonomamente e senza lintervento della coscienza , la memoria di lavoro dipende fortemente dal sistema attenzionale in grado di autorizzare la messa in opera di funzioni superiori come il calcolo simbolico , la decisione di una gestione o linibizione degli automatismi [ 10 , 11 ]  . 
ai fini dellosservazione dei radiogrammi interviene la funzione fondamentale della memoria di lavoro ovvero il mantenimento di una rappresentazione , costruita a partire dai dati empirici , nel caso nostro dalle caratteristiche dei radiogrammi , e da informazioni recuperate dalla memoria a lungo termine , che in modo arbitrario , ma di immediata acquisizione potremmo chiamare esperienza . 
knowledge - based activity : the diagnostic process is guided by analytical reasoning , in the awareness of already stored knowledge , to tackle new situations , which require a considerable application effort . human error in diagnostic imaging : types and causes having introduced the areas of human activity where errors may occur , the causes may now be sought in the three major groups of error : ( 1 ) perceptual errors , ( 2 ) cognitive errors and ( 3 ) system errors . 
these are summarised in figure 3 . the scientific principle of assessment is also valid for human error , that is to say , that similar to the bayes formula , errors are either false positives or false negatives . 
la diagnostica orientata da un ragionamento analitico , nella consapevolezza di un bagaglio di conoscenze gi immagazzinate , per far fronte a situazioni nuove , che richiedono quindi uno sforzo applicativo non comune . according to berlin [ 12 ] , 70% of lawsuits brought against radiologists are related to the non - identification of radiological signs , 60% of which are due to perceptual errors . with regard to the causes , a distinction is made between errori umani nella di : tipologie e cause introdotti gli ambiti delle attivit umane in cui avviene lerfig . 
inches : systematic approach to human error in radiology rore , possibile ricercarne le cause nei tre grandi insiemi : ( 1 ) linsieme degli errori percettivi , ( 2 ) linsieme degli errori cognitivi , ( 3 ) linsieme degli errori del sistema . 
per gli errori di sistema o latenti qui ribadito che lerrore umano di ripercussione , essendo la causa antecedente al momento dellavvenimento , gi radicata nel sistema organizzativooperativo . errori percettivi secondo berlin il 70% [ 12 ] delle pratiche legali contro i radiologi connesso ad una non identificazione di segni radiologici , con un contributo del 60% degli errori percettivi . quanto alle cause , si distinguono errori di identificazione con errata attribuzione ed errori di non identificazione . errori di identificazione con errata attribuzione sono errori poco frequenti : si verificano allorquando il radiologo segnala una lesione che non sussiste , scambiando ad esempio un reperto normale con uno patologico . 
barbaum [ 13 ] riporta , ad esempio , che nella ricerca di un corpo estraneo per la presunta ingestione di un bottone da parte di un bimbo di 1 anno in un radiogramma standard del torace , un radiologo ha malamente interpretato un nucleo di ossificazione sternale attribuendolo al bottone . 
le verifiche endoscopiche e il pasto opaco hanno dimostrato linconsistenza dellaffermazione . come gi osservato sono gli errori pi frequenti : il riconoscimento postumo di un nodo polmonare sul radiogramma del torace la situazione pi nota . 
questa possibilit di errore risulta chiara se ci si cimenta con i disegni paradossali di esher o con il libro per bambini di handford the great waldo [ 14 ]  . 
nellultimo libro , i bambini sono sfidati a trovare il mostriciattolo waldo nascosto tra mille disegni : difficile da riconoscere , ma una volta scoperto o rifugiatisi nelle soluzioni , alla fine la sua inconfondibile sagoma e la relativa posizione risultano ovvie . 
nellesempio la percezione fortemente influenzata dalle aspettative ; perci trovare waldo pi semplice che vedere un piccolo nodo polmonare sul radiogramma toracico , perch waldo sicuramente c , mentre per il nodo polmonare non abbiamo indizi . 
 [ 13 ] report an example in the search for a foreign body in a standard chest x - ray due to the presumed ingestion of a button by a 1 - year - old child . 
endoscopic examinations and an opaque bolus proved the affirmation incorrect . non - identification errors these are the most frequent kinds of errors , with the late recognition of a pulmonary node on a chest x - ray being the most common situation . 
the possibility of making this kind of error is clear if one tests oneself with the paradoxical drawings of escher or with the childrens books by handford wheres wally ? [ 14 ]  . 
in one of the most recent books , children are challenged to find wally hidden among hundreds of figures ; he is extremely difficult to recognize , but once identified his , unmistakable outline and his position are in the end obvious . 
in this example , perception is strongly influenced by expectations , so that finding wally is much easier than identifying a small pulmonary nodule on a chest x - ray because wally is definitely there while a pulmonary nodule may or may not be present . 
with regard to errors in this activity , kopans and daniel [ 15 , 16 ] note cases reported by psychologists of the difficulty and frustration of contingent situations often present in daily life , such as the fruitless search for a bunch of keys which we are unable to see but need to get into our house . 
the example can easily be related to a mammographic sign indicated by a colleague or recognized at a later time . specific causes as well as non - describable factors , the erroneous perception that leads to the non - identification on radiographs is connected to specific , numerous and heterogeneous causes . 
indeed , the final error is perceptual , but the complexity of mental behaviour is such that causal factors which are a part of technical and cognitive features play a part . 
with reference to berlin [ 18 ] , in one case of non - identified pulmonary nodule , the judicial assessment commission admitted that the lesion was difficult to diagnose since the x - rays were overexposed , thus suggesting the responsible error . 
the difficulty of diagnosing lesions at the level of the last cervical and first thoracic vertebrae is also well known , particularly in trauma patients , unless an optimal patient position is found . 
berlin [ 18 ] reports the case of a patient who fell from a ladder and underwent radiography of the cervical vertebrae on three separate occasions owing to the persistence of symptoms . 
unfortunately , the lesion identified on a later occasion in another hospital was a fracture / luxation of c6 - c7 , with the patient ending up a permanent quadriplegic . 
inches : systematic approach to human error in radiology screening : in quello mammografico infatti notevole la variabilit anatomica , spesso la struttura di difficile analisi , e il numero di casi da interpretare pu essere enorme . 
per gli errori in questa attivit kopans [ 15 , 16 ] ricorda , su segnalazione degli psicologi , le difficolt e le frustrazioni delle situazioni contingenti spesso presenti nella vita quotidiana , come la ricerca infruttuosa di un mazzo di chiavi , che non siamo in grado di vedere , ma che sono necessarie per entrare in casa . 
lesempio si ricollega con facilit ad un segno mammografico indicato da un altro operatore o riconosciuto in tempi successivi . cause specifiche oltre a fattori non descrivibili , lerrata percezione che porta alla non identificazione sui radiogrammi connessa a cause specifiche , numerose ed eterogenee . 
le tipologie sono indicate in figura 4 . gli errori tecnici [ 17 ] sono bene esemplificati dalla lesione non vista per sovra o sottoesposizione o per errato posizionamento del paziente . 
con riferimento a berlin [ 18 ] , in un caso di nodulo polmonare non visto , la commissione di valutazione giudiziaria ammise che era difficile diagnosticare la lesione perch le radiografie erano sottoesposte , sottintendendo cos lerrore responsabile . 
sempre berlin riporta il caso di un paziente , caduto dalle scale di casa e sottoposto a radiografie della colonna cervicale in tre fasi diverse , a causa di una persistente sintomatologia : il primo esame identific le prime 4 vertebre cervicali , il secondo dimostr anche c5 con sole note artrosiche . 
the anomalies outside the privileged area of the examination are not infrequent and include thoracic lesions identified during an abdominal examination ; or pleural , pulmonary or mediastinal lesions incidentally encountered during a radiological study of the spine , thoracic bones or shoulder girdle . 
in a litigation caused by lack of identification of an arteriovenous malformation of the bone marrow , berlin [ 19 ] underlines the difficulty of establishing whether the error was due to non - perception or lack of knowledge by the radiologist , and such difficulties can be extended to other cases . the attempt to explain non - identification with other causes dates back to the 1960s and the work of several authors , such as smith and tuddenham , which was later commented by wood , and raufaste and eyrolle [ 8 , 10 , 11 ] : several lesions were not identified owing to another lesion present in the image which had caught the attention of the radiologist . 
following the same line of research , berbaum [ 20 ] investigated the phenomenon on his own colleagues , introducing an artificial nodular lesion in chest x - rays already marked by original lesions . 
he defined this perceptual error satisfaction of search , thus recognizing the argument of cognitive psychologists who note that radiologists tend to be satisfied after finding a lesion , and if they continue the assessment of the image , it is only to restrict the field of study to what they have identified . the quantity of perceptual errors varies according to the examination in question . 
 [ 21 ] , in an interval of 16to 40 - mm nodules , recognized a minimum error of 20% ! in a specific study on the efficiency of health services , watcher et al . 
 [ 22 ] reports similar types of errors in other organs and systems recognized by other researchers : the errors in emergency situations have a low percentage of 3%6% ( robin ) , the sensitivity of identifying ureteral stones , many of which are recognizable a posteriori , is 46% ( levine ) and the error rate in contrast enema is 15% ( wesses )  . a first critical examination of perceptual errors , in the light of case series and studies which go beyond the individual case with legal implications , demonstrates that the real difficulty is the error without specific cause . 
it should be toracica individuata in corso di addome diretto , o ancora le lesioni pleuriche , polmonari o mediastiniche incidentalmente comparse in corso di studio radiografico della colonna vertebrale , delle ossa del torace o del cingolo scapolare . 
berlin in un suo articolo , in un caso di contenzioso per la mancata individuazione alla mielografia di una malformazione arterovenosa del midollo spinale [ 19 ] , sottolinea che vi sono concrete difficolt a stabilire se nel caso lerrore fosse dovuto alla non percezione o alla scarse conoscenze del radiologo e che tali perplessit sono da estendere ad altre evenienze . il tentativo di spiegare la non identificazione con ulteriori cause risale agli anni 60 con le osservazioni di alcuni autori , quali smith e tuddenham , raccolte e commentate successivamente da wood e raufaste [ 8 , 10 , 11 ] : alcune lesioni potevano non essere individuate a causa di unaltra lesione presente nel documento che aveva catturato lattenzione del radiologo . 
berbaum [ 20 ] nello stesso filone di ricerca ha indagato il fenomeno con i propri collaboratori , introducendo una lesione nodulare artificiale nei radiogrammi del torace gi interessati da altre lesioni originali : lidentificazione di queste ultime , per il disturbo delle altre , diminuiva in modo significativo . 
egli ha chiamato un tale errore percettivo satisfaction of search , riconoscendo la tesi degli psicologi cognitivisti che notano che i radiologi tendono ad essere soddisfatti dopo la ricerca di una lesione e proseguono semmai oltre solo restringendo il campo dindagine su ci che hanno visto . in termini applicativi la quantit di errori percettivi varia a seconda delle indagini in questione . 
interessante notare come in una revisione della casistica personale di radiogrammi del torace e nel confronto con altri autori , quekel [ 21 ] riconosca in un intervallo da 16 a 40 mm delle dimensioni dei noduli un errore minimo del 20% ! in uno studio specifico sullefficienza delle prestazioni sanitarie watcher [ 22 ] riporta analoghe tipologie di errore relative ad altri organi ed apparati riconosciute da altri ricercatori : gli errori in emergenza hanno percentuali minime del 3%6% ( robin ) ; la sensibilit di rilevamento dei calcoli ureterali , molti dei quali riconoscibili a posteriori , del 46% ( levine ) ; la quota derrore nel clisma opaco del 15% ( wesses )  . una prima considerazione critica sugli errori percettivi , alla luce di casistiche e studi che esulano dal caso singolo con risvolti giudiziari , mette in evidenza come il vero rebus sia lerrore senza specifica causa . 
 [ 23 ] hanno studiato gli effetti della doppia e tripla lettura su un campione di 60 casi di clisma opaco a doppio contrasto , in cui erano presenti 46 lesioni . 
the radiologist is aware of the routine and surrounding working conditions ; a temporary loss of attention causes the slip , which may be considered a small monitoring error . a ( perceptual ) error caused by capture occurs when the radiologists attention is attracted by a sign and is satisfied by this first finding , using a more common working framework rather than a less common one . alongside this type of slip is one in which the error is so obvious that it is known as a description error . 
it is an error characteristic of reporting when a semantic field related to one technique is used to describe another , for example , a ct study described with echogenicity - based terminology , or an ultrasound described with density - based terminology . other slips , such as associative activation ( answering the telephone when the doorbell rings ) or loss of activation , described as the temporary loss of memory while performing a precise task ( the search for an object ) , appear to be circumstantial , more common with the use of high - tech equipment or procedures , and can immediately be made up for . 
therefore , performing tasks without disturbances or distractions is the best prevention for this type of error . in contrast , a series of factors of various natures may help create situations which foster the occurrence of slips . 
the most common psychological elements include fatigue , loss of sleep , alterations caused by alcohol or drugs , or emotional states such as boredom , frustration , fear , anxiety or anger all conditions which cause concern and distract ones atl . 
inches : systematic approach to human error in radiology errori cognitivi gli errori della fase cognitiva sono stati classificati da reason e rasmussen [ 6 ] con il riferimento ad ogni livello attenzionale come basati sulla capacit intellettiva ( skill based ) , sul metodo cognitivo ( rule based ) e sulla conoscenza ( knowledge based )  . 
errore caratteristico nella refertazione quando si utilizzi una semantica relativa ad una metodica per descriverne unaltra : ad esempio , un referto con terminologia basata sullecogenicit per unindagine tc o invece sulla densit per unecografia . altre sviste quali lattivazione di associazioni ( associative activation ) come rispondere al telefono quando suona il campanello o la mancanza dattivazione ( loss of activation ) ovvero la temporanea perdita di memoria in corso di unazione ben precisa , come la ricerca di un oggetto , appaiono circostanziali , pi frequenti nel corso dimpiego di tecnologie o procedure , e di solito dimmediato recupero . tuttavia va sottolineato come le sviste sono spesso causate da interruzioni di precedenti attivit che erano in atto . pertanto lo svolgimento dellattivit senza disturbi e distrazioni la migliore prevenzione per tali tipologie derrore . per contro , una serie di fattori di diversa natura pu innescare situazioni favorevoli alle sviste . 
gli elementi psicologici pi frequenti comprendono la fatica , la perdita di sonno , le alterazioni da alcool o droghe oppure stati emozionali , quali la noia , la frustrazione , la paura , lansia , la collera , tutte condizioni che conducono alla preoccupazione che distrae lattenzione . a queste condizioni possono contribuire altri fattori esterni quali leccesso di lavoro e le relazioni interpersonali difficili o ambientali come il rumore , la calura , stimoli visivi eccessivi . 
in addition to these conditions , there may also be external factors , such as an excessive workload , difficult interpersonal relations or an unfavourable working environment characterised by excessive noise , heat or visual stimuli . in di , therefore , a large number of slips can be the result of working environments inappropriate for the study of radiographic images , excessive workloads , difficult conditions in the department and so on . rule - based and knowledge - based errors errors related to the two cognitive activities , commonly known as psychological errors , have been studied less than others and are more difficult to assess . 
these errors are due to an erroneous assessment of a contingent situation for which an incorrect procedure is applied . figure 5 shows how those errors , more correctly defined mistakes , are both recognition and decision making in nature . 
mistakes are therefore specifically cognitive errors , distinct from the more general term errors , which includes all types and therefore perceptual and system errors as well . figure 5 also shows how recognition errors are usually based on the most commonly used reasoning , adopted due to mental laziness on the part of the radiologist who has trouble breaking from known and repeated implementation models . included with this mental laziness or preconceived positions are mistaken choices , which piattelli - palmarini [ 24 ] defines deadly sins and which are summarized in figure 5 and outlined here below : biased memory leads to overgeneralizations of the ordinary or undergeneralizations of facts due to emotive reactions connected with personal success or contradictory experiences . heuristic availability prompts us to use only the primary information which comes to mind . confirmation bias supports our working hypotheses , causing us to ignore all contradictory information . overconfidence leads us to overestimate ourselves and our own abilities . anchoring highlights the influence that our first impression , or first judgement , has and how it becomes impossible to completely suppress it in later revisions . probability blindness refers to the mental procedure incapable of taking into consideration probabilistic estimates . it is so dangerous that piattelli - palmarini warns that any probabilistic intuition of any person not specifically versed in probability calculations has more than 50% probability of being incorrect [ 24 ]  . reconsideration under suitable scripts , lastly , is the seventh of the mental procedures which lead to cognitive errors . 
it is thought that a type of event or situation is judged all the more frequently the easier it is to mentally imagine and the greater the emotional impact it has as well as the more our judgement of probability is biased by the scripts . together with these seven deadly sins of the mind are two inevitable corollaries , situations contingent to the work phases : errori nellambito dellattivit basata sulle regole ( ruled based activity ) e dellattivit basata sulla conoscenza ( knowlodge based activity ) gli errori inerenti le due attivit cognitive , denominati comunemente psicologici , sono i meno studiati e i pi difficili da inquadrare : spesso sono identificabili pi cause che insieme contribuiscono allerrore . 
la figura 5 evidenzia ancora come lerrore di riconoscimento si basi di solito sul ragionamento usato pi frequentemente , cui si ricorre a causa della pigrizia mentale del radiologo , che stenta a staccarsi da schemi applicativi noti e ripetuti . 
clinical information may play a determining role in this assessment . before taking into consideration this aspect , which is fundamental in daily practice , it is worth noting the three main types of error in di associated with mental tunnels , as proposed by fitzgerald [ 26 ] : availability bias : the ease of remembering a diagnosis specific to the case compromises the possibility of making a correct diagnosis . regret bias : a distorted probability of diagnosis influences the correctness in the unconscious desire that something regrettable occurs . framing bias : the frame of the examination ( the request , case history , etc . ) unduly influences the report . a study in 1992 by norman et al . 
the first phase of the experiment involved 50 radiologists called on to assess 50 radiographs 25 from patients affected by bronchiolitis and 25 from normal patients with the radiologists being asked to search for signs of disease such as hyperinsufflation , bronchiolar thickening and perihilar opacities . the signs were found in both diseased and healthy patients in compliance with the suggested provisional diagnosis . 
in the second phase of the experiment , the radiographs were accompanied with a brief clinical history of normality or pathology , and three radiologists were specifically asked what the real probability of bronchiolitis was . the experiment showed the consistent effect of the clinical history on both attribution of a diagnostic category and the radiological signs on uncertain radiographs . 
at any rate , there is no doubt that knowledge of clinical history and signs regarding physical examination increase the possibility of radiological interpretation . further useful information can be gleaned from a comparison of previous radiological studies performed on a patient , as studied by berbaum et al . 
 [ 28 ] and berlthe american college of radiology has included the procedure among the published quality standards , stating that comparison with relevant previous examinations and reports should be part of the radiologic consultation and report when appropriate and available . 
inches : systematic approach to human error in radiology centrarsi su una sola fonte dinformazione ; il capovolgimento sotto affaticamento ( reversion under stress ) il fenomeno nel quale i comportamenti appresi di recente sono sostituiti con altri pi vecchi , ma pi familiari anche se inadatti al problema in esame . 
gli errori di riconoscimento avvengono quando una anomalia vista dal radiologo , ma non apprezzata come tale ; in realt riconoscere unanomalia richiede pi una memoria visiva che unacuit visiva . 
le informazioni cliniche possono giocare un ruolo determinante in questo giudizio . prima di prendere in considerazione tale aspetto , fondamentale nella pratica quotidiana , vale la pena di riportare da fitzgerald [ 26 ] i tre principali tipi di errore nella di connessi ai tunnel mentali : influenza della memoria ( avaibility bias ) : la facilit di ricordare una diagnosi specifica al caso pregiudica la possibilit della diagnosi corretta ; influenza emotiva ( regret bias ) : una distorta probabilit di diagnosi influenza la correttezza , nellinconscio desiderio che avvenga qualcosa che dispiace ; influenza del contorno ( framing bias ) : la cornice dellesame ( richiesta , anamnesi etc ) influenza eccessivamente il referto . a tale riguardo uno studio di norman del 1992 [ 27 ] dimostra empiricamente limportanza delle informazioni cliniche . 
lesperimento riguarda 50 radiologi esperti chiamati a valutare in una prima fase 50 radiogrammi , 25 di pazienti affetti da bronchiolite e 25 normali , chiedendo loro di ricercare i segni della malattia quali iperinsufflazione , ispessimento bronchiolare , opacit perilari . 
in una seconda fase stato chiesto esplicitamente ai tre radiologi quale fosse in ogni radiogramma , cui era stata aggiunta una breve storia clinica di normalit o di patologia , la probabilit reale di bronchiolite . 
a livello operativo nessuno osa in ogni caso mettere in dubbio che la conoscenza della storia clinica e dei segni relativi allesame obiettivo aumentino le possibilit dinterpretazione radiologica . altre informazioni utili possono ancora essere desunte dal confronto con i precedenti studi radiografici eseguiti sul paziente , come sperimentato da berbaum e berlin [ 28 ]  . lamerican college of radiology ha posto la procedura tra gli standard di qualit pubblicati affermando che il confronto con gli esami precedentemente eseguiti dal paziente , quando possibile , parte integrante dellindagine radiologica l . 
inches : systematic approach to human error in radiology space and time can be resolved with the picture archiving communication system ( pacs ) [ 29 ] which with the storage of digital data ( and therefore not analogical images ) could nonetheless give rise to revisions and evaluations after the fact , both regarding the use of the same in the broadest possible sense and the perception and interpretation of the derived images . if knowledge of the history and a comparison of previous data are valid methods for eliminating the radiologists decision - making errors , then reading previous radiological reports could also prove useful . 
this proposal , however , opens up an important debate regarding alliterative errors [ 30 ] , defined as an erroneous and reiterated diagnosis by several radiologists on the basis of the report of the first radiologist . the first such case reported by berlin [ 30 ] of a case of lung cancer , which on several occasions was labelled as bronchopneumonia , was followed by an important debate . 
the theory of berlin [ 31 ] which states that if a radiologist fails to recognize an anomaly or incorrectly interprets its meaning , then the possibility of the next radiologist repeating the same error is noticeably increased is supported by smith , cited by berlin [ 31 ] , who states that radiologists are induced to read the report of a colleague before assessing the radiograph . 
laspetto relativo allarchiviazione , di difficile applicazione nello spazio e nel tempo , potr essere risolto dai sistemi digitali pacs [ 29 ] , che tuttavia con la memorizzazione dei dati numerici ( non immagini analogiche quindi ) potranno dare luogo a revisioni e giudizi postumi , sia sullutilizzazione degli stessi in tutta la loro ampiezza che sulla percezione e interpretazione delle immagini ricavate . se la conoscenza della storia e il confronto con i dati precedenti sono metodi validi per eliminare gli errori decisionali del radiologo , anche la lettura dei referti precedenti allegati potrebbero rientrare in questa ottica . laffermazione apre tuttavia limportante dibattito relativo allerrore alliterativo [ 30 ] , individuando in questo unerronea e reiterata diagnosi da parte di pi radiologi sulla base del referto del primo radiologo . 
alla prima segnalazione di berlin su un caso di tumore polmonare , in pi circostanze etichettato come broncopolmonite [ 30 ] segu un importante dibattito . la teoria di berlin [ 31 ] che , se un radiologo non vede unanomalia o le attribuisce un significato sbagliato , la possibilit che il successivo radiologo ripeta il medesimo errore notevolmente aumentata sostenuta anche da smith , citato da berlin [ 31 ] , il quale afferma che i radiologi sono portati a leggere il riscontro del collega prima dellapproccio al radiogramma . tuttavia lamerican college of radiology nello standard for communication mette in evidenza che il paragone con precedenti esami e referti , dove possibile , parte integrante dellanalisi radiologica . 
in una successiva revisione berlin [ 32 ] riporta da autori diversi analisi sperimentali in contraddizione , riconoscendo che hunter e boyle dimostrano lutilit nel leggere i precedenti referti nel 60% dei casi e un considerevole aiuto nel 22%24% dei casi , mentre white segnala solo 1 , 5% di utilit e un 48% di inutilit . 
scelta della soglia del sospetto . ferred to for the following discussion . in the strategy outlined in model 1 , the threshold of suspicion would lead to a high specificity for the test and a good level of sensitivity but with 19% more false negatives than model 2 . 
on the other hand , the threshold of suspicion is lower in model 2 , thus introducing more recognizable minimal signs , even in healthy breasts , and therefore reducing the specificity of the test to the point of increasing false positives by 4 , 110 cases ( out of a total of 17 , 000 )  . 
so a diagnostic gain of 19% ( no small thing ! ) is counterbalanced by the anxiety of being called for follow - up multiplied by ten times and the implementation of invasive procedures multiplied by five . 
on the basis of current knowledge and experience , since in both cases the radiologist knows in advance the nature of the error , either false positive or false negative , and plans his or her work activity on the basis of this assessment , the error may be defined as strategic , especially if the choice is shared in the context of coordinated decision making and operations . 
as a result , in the implementation phase of breast screening , the error becomes operatively present , with a meaning similar to that codified by federspil and vettor [ 1 , 2 ] in clinical activity , when there is a choice of two alternative therapies . prevedere la diversa distribuzione degli errori : la figura 7 , di facile comprensione nei passaggi dai valori assoluti a quelli percentuali [ 39 ] , funge da riferimento per le successive argomentazioni . 
nellipotesi di una scelta strategica che si uniformi al modello 1 la soglia del sospetto comporterebbe unelevata specificit del test ed una buona sensibilit , ma con una quota di falsi negativi del 19% in pi rispetto al modello operativo 2 . 
daltro canto nel modello operativo 2 , ove la soglia del sospetto si abbassa , introducendo quindi molti segni minimi riconoscibili anche in mammelle sane , la specificit del test si riduce al punto di aumentare di ben 4410 casi ( su 17000 donne ! ) i falsi positivi . 
poich , sulla base delle attuali conoscenze ed esperienze , in entrambi i casi il radiologo conosce preventivamente il versante dellerrore , falso positivo o falso negativo , e anche in base a tale valutazione progetta la sua attivit , lerrore pu essere definito strategico , a maggior ragione se la scelta condivisa nellambito di un coordinamento operativo - decisionale . 
inches : systematic approach to human error in radiology system errors it has already been mentioned how system errors have repercussions in the end on the final medical assessment , with the characteristic connotations of human error [ 7 ]  . 
it has been recognized that the system as such has peculiarities in which the cause of errors is to be found . generally speaking , we would intuitively expect a wellconstructed system to be equipped with defences , barriers and mechanisms which reduce the risk of errors to a minimu nonetheless , those mechanisms , which reason and rasmussen [ 6 ] liken to barriers filled with holes , are extremely vulnerable when the holes line up on the same trajectory . the inevitable error is accompanied by the personal application , which is not able to correct the error owing to a series of preexisting causes , such as those summarized in figure 6 . system errors are therefore prevented above all by adopting a safety system which renders their occurrence difficult for individual work and by performing constant research which is accompanied by daily efficiency [ 40 ]  . there are numerous suggestions for achieving this in di . 
those which draw on applicative criteria include protocols for reducing reliance on memory , improvement of access to information through the patients case history , use of systems impermeable to errors to avoid homonymy , and training . measures more specifically related to the department include maintenance of quality standards , systematic control of equipment , revision of reports and appropriate instructions to users . federspil e vettor [ 1 , 2 ] nellattivit clinica , allorch si mettano in alternativa due scelte terapeutiche . errori di sistema gi stato dato risalto a come gli errori di sistema si ripercuotano alla fine sulla valutazione medica conclusiva , con le connotazioni caratteristiche dellerrore umano [ 7 ]  . 
allerrore inevitabile si aggiunge lapplicazione personale , che non in grado di correggerlo per una serie di concause tra le quali si riconoscono in modo immediato quelle raccolte in figura 6 . 
la prevenzione degli errori di sistema si attua quindi sopratutto mediante un apparato di sicurezza che li renda di difficile evenienza per il lavoro individuale e mediante una costante ricerca che proceda di pari passo con lefficienza giornaliera [ 40 ]  . nella di molti sono i suggerimenti in questo senso . 
tra quelli che si rifanno ai criteri applicativi generali si citano i protocolli per ridurre la fiducia nella memoria , il miglioramento allaccesso dellinformazione mediante la cartella clinica del paziente , luso di sistemi impermeabili agli errori ad evitare omonimie , laddestramento . 
per quanto riguarda pi specificamente il reparto si ricordano il mantenimento degli standard di qualit , il controllo sistematico delle attrezzature , la revisione dei referti , le idonee segnalazioni agli utenti . conclusions considerazioni conclusive error is a characteristic feature of human activity . 
the latin saying errare humanum est may be applied to all activity , and di is no exception . perhaps less well known is the medieval addition to the saying attributed to saint bernard sed perseverare diabolicum indicating that errors upset our balance and therefore need to be corrected or , better still , prevented . to achieve these aims in a complex system such as di , simple recognition of the error does not appear to be sufficient . preliminary knowledge of the types and causes of errors relating to human activity and the system is required , as well as their application in a daily analysis to improve both standards of service and personal satisfaction . lerrore rappresenta una connotazione caratteristica delle azioni umane . 
de vargas macciucca , via alberico albricci 28 , i - 00194 roma , italy , tel . : + 39 - 338 - 5924034 , fax : + 39 - 06 - 49970212 , e - mail : marinadevargasm@hotmail.com received : 2 february 2005 / accepted : 7 november 2005 / published online : 3 march 2006 abstract purpose . 
we retrospectively reviewed 35 patients ( 23 men and 12 women ; mean age 66.6 years ) presenting with acute cholecystitis who were assessed by emergency ultrasonography ( us ) ( 30 / 35 cases ) and spiral ct ( 12 / 35 cases ) ; all patients underwent emergency surgery . 
the us signs were analysed and classified as major criteria ( wall thickening and stratification , distension , murphys sign ) , minor criteria ( bile stones , sludge , and biliary tract dilatation ) , and complication signs ( gas collections , aerobilia , fluid collection , difficult or missed identification of the gallbladder )  . 
imaging results were compared with histological findings ( gold standard ) , and accuracy , sensitivity , specificity , and positive and negative predictive values ( ppvs and npvs ) were assessed for each modality . 
if more than two major signs associated with one minor sign or at least one sign of complication are present at us , ct is mandatory to recognise and thoroughly evaluate the type of complication and indicate appropriate treatment . key words acute cholecystitis complicated cholecystitis us spiral ct riassunto scopo . 
sono stati esaminati retrospettivamente 35 pazienti ( 23 uomini e 12 donne ; et media 66 , 6 anni ) affetti da colecistite acuta , valutati in urgenza con esame ecografico ( 30 / 35 casi ) e tc ( 12 casi ) e sottoposti a intervento chirurgico urgente . 
i parametri ecografici sono stati analizzati e classificati come criteri maggiori ( ispessimento parietale , stratificazione parietale , sovradistensione , segno di murphy ecografico ) , minori ( calcoli o sedimenti nella colecisti o nelle vie biliari , dilatazione delle vie biliari ) e segni di complicazione ( raccolte gassose , aerobilia , raccolte fluide , difficile o mancato riconoscimento della colecisti )  . i risultati dellimaging sono stati confrontati con quelli dellesame istologico ( gold standard ) e sono state valutate accuratezza , sensibilit , specificit , valore predittivo positivo ( vpp ) e negativo ( vpn ) delle rispettive metodiche . 
lecografia ha dimostrato una accuratezza del 66 , 6% , una sensibilit del 37 , 5% , una specificit del 70% , un vpp del 100% e un vpn del 58 , 3% . 
in presenza di pi di due segni maggiori e uno minore o di almeno un segno di complicazione allecografia , lesame tc raccomandabile per il riconoscimento e la completa valutazione del tipo di complicazione , fornendo la giusta indicazione allintervento chirurgico urgente . parole chiave colecistite acuta colecistite complicata ecografia tc - spirale m . 
de vargas macciucca et al . : ultrasonographic and spiral ct evaluation of simple and complicated acute cholecystitis introduction introduzione the clinical suspicion of acute cholecystitis is one of the most frequent conditions facing the radiologist in emergency settings , as well as being the fourth cause of hospitalisation in patients presenting with acute abdomen [ 1 ] and the chief indication for emergency cholecystectomy [ 2 , 3 ]  . 
women under the age of 50 years are up to three times more frequently affected by cholecystitis than men whereas after this age , the incidence is the same in both genders . 
the most common cause of cholecystitis is lithiasis with cystic duct obstruction ( 90%95% )  . mucosal hypersecretion due to irritation resulting from increased bile concentration leads to oedema associated with wall thickening , followed by increased intraluminal pressure , wall distension , and stretching of the thin wall vessels with subsequent ischaemia and necrosis of the parietal mucosa . bacterial superinfection , which develops in 50%70% of cases , can lead to complications of acute cholecystitis such as empyema , gangrene , necrosis , and perforation [ 46 ]  . 
a patient without a history of biliary symptoms will rarely develop acute cholecystitis , whereas fewer than 15% of patients with gallstones present symptoms and fewer than 5% develop acute cholecystitis [ 7 ]  . in 5%10% of cases , cholecystitis is non - calculous . 
this situation mainly occurs in patients who have suffered serious injuries or burns , debilitated patients , patients with long - term hospitalisation in icu , or those undergoing prolonged parenteral feeding ; in these patients , systemic shock or the continued use of vasoconstrictors apparently causes ischaemia of the gallbladder wall mucosa , which is characterised by terminal blood supply and insufficient collateral circuits . 
according to another theory , however , non - calculous cholecystitis is due to mechanical obstruction of the cystic duct ( already narrowed by oedematogenic events ) by bile that has become dense and viscous following prolonged biliary stasis [ 8 , 9 ]  . 
other risk factors include overeating , diabetes , sepsis , cardiac arrest , atherosclerosis , aids , and chemotherapy via the hepatic artery [ 10 ]  . because symptoms are non - specific , it is difficult to clinically differentiate biliary colic from acute inflammation , but differential diagnosis is fundamental for establishing the correct therapeutic approach . 
treatment of acute cholecystitis is a matter of debate due to the different therapeutic options : emergency surgery ( laparotomy or laparoscopy ) , or delayed surgery after antibiotic treatment [ 11 , 12 ]  . 
further diagnostic investigation using imaging studies is therefore necessary for early diagnosis and timely treatment , above all when there is a suspicion of complications of acute cholecystitis . ultrasonography ( us ) is usually the first - choice modality [ 13 ] : the association of hepatobiliary us alone with clinical assessment allows accurate diagnosis in the majority of cases of calculous cholecystitis thanks to its very high sensitivity in the detection of cholelithiasis ( 95% ) [ 14 ] ; in addition , this examination has a low cost , is reproducible , and allows recognition and exclusion of numerous other causes of abdominal pain . both computed tomography ( ct ) and magnetic resonance il sospetto clinico di colecistite acuta rappresenta uno dei quesiti pi frequentemente sottoposti al radiologo in urgenza nonch la quarta causa di ricovero ospedaliero per pazienti che si presentano con addome acuto [ 1 ] ed la principale indicazione alla colecistectomia eseguita in urgenza [ 2 , 3 ]  . al di sotto dei 50 anni le donne sono fino a 3 volte pi colpite degli uomini , mentre oltre tale et lincidenza uguale nei due sessi . 
lipersecrezione mucosa determinata da fenomeni irritativi per iperconcentrazione della bile alla base del conseguente edema con ispessimento delle pareti , cui segue laumento della pressione intraluminale e la distensione delle pareti , con stiramento dei sottili vasi parietali , fenomeni alla base della successiva ischemia e poi della necrosi della mucosa parietale ; la sovrainfezione batterica , che si verifica solo nel 50%70% dei casi , invece causa delle possibili complicanze della colecistite acuta quali lempiema , la gangrena , la necrosi e la perforazione [ 46 ]  . 
raro che un paziente senza storia di sintomatologia biliare possa sviluppare una colecistite acuta , mentre al contrario meno del 15% dei pazienti con colelitiasi presentano sintomi clinici e meno del 5% sviluppano una colecistite acuta [ 7 ]  . nel 5%10% dei casi la colecistite invece alitiasica . questa evenienza si verifica essenzialmente in pazienti che hanno subito gravi traumi , defedati , ustionati , ricoverati a lungo in reparti di rianimazione o con alimentazione per via parenterale protratta nel tempo , nei quali lo shock sistemico o luso prolungato di vaso - costrittori indurrebbero unischemia della mucosa parietale della colecisti , che possiede una irrorazione di tipo terminale , con scarsi circoli collaterali ; tuttavia , secondo unaltra teoria le colecistiti alitiasiche sarebbero causate dallostruzione meccanica del dotto cistico ( gi ridotto di calibro per fenomeni edemigeni ) da parte di bile divenuta densa e vischiosa in seguito a prolungata stasi biliare [ 8 , 9 ]  . 
altri fattori di rischio includono liperalimentazione , il diabete , la sepsi , larresto cardiaco , laterosclerosi , laids e la chemioterapia attraverso larteria epatica [ 10 ]  . a causa dellaspecificit dei sintomi difficile differenziare clinicamente una colica biliare da una infiammazione acuta , ma la diagnosi differenziale fondamentale per una corretta pianificazione terapeutica . 
il trattamento delle colecistiti acute oggetto di discussione in quanto esistono differenti opzioni terapeutiche : lintervento chirurgico ( per via laparotomica o laparoscopica ) eseguito in urgenza o differito dopo terapia antibiotica [ 11 , 12 ]  . 
pertanto , specie quando si sospettino le complicanze della colecistite acuta , al fine di una precoce diagnosi e di un tempestivo trattamento terapeutico , si impone la necessit di approfondimenti diagnostici con esami strumentali . generalmente lecografia lindagine strumentale di prima istanza [ 13 ] : associando la sola ecografia epato - biliare alla valutazione clinica possibile una diagnosi accurata nella maggior parte dei casi , specie se la colecistite su base litiasica , grazie alla elevatissima sensibilit di questa metodica nel rilievo di colelitiasi ( 95% ) [ 14 ] ; inoltre tale esame presenta un basso costo , riproducibile e permette il riconoscimento e lesclusione di numerose altre cause di dolore m . 
de vargas macciucca et al . : ultrasonographic and spiral ct evaluation of simple and complicated acute cholecystitis ( mr ) normally add a small amount of information to the evaluation of acute cholecystitis but can be very useful in the case of subacute manifestations , in the presence of palpable pseudo - masses in the right hypochondrium , of clinical us discrepancy , and in the case of complications such as empyemic cholecystitis , abscesses , emphysematous cholecystitis , necrotic - gangrenous cholecystitis , perforation , biliary - enteric fistula or biliary ileus [ 2 , 6 , 7 , 15 , 16 ]  . the aim of this study was to review cases seen over 1 year of complicated acute cholecystitis evaluated by emergency us and ct and treated with emergency surgery in order to recognise specific and non - specific signs of complication and compare them with those reported in the literature . another aim was to define a correct diagnostic protocol involving the use of ct in selected cases . materials and methods in this study , we retrospectively examined the cases of 35 patients ( 12 women and 23 men ) aged between 27 and 99 years ( mean age 66.6 years ) suffering from acute cholecystitis who underwent imaging examinations between april 2003 and april 2004 . all patients underwent surgery and histological examination was considered the gold standard . 
patients whose diagnostic examinations performed at other sites were not available , patients who did not undergo emergency surgery due to medical contraindications , and patients whose operation was delayed for more than 48 h after presentation were excluded . clinical signs ( pain , murphys sign , fever , palpable swelling , jaundice ) and laboratory signs ( neutrophilic leukocytosis , bilirubinaemia , transaminasemia , glycaemia ) at the time of diagnosis were considered . 
ct spiral examinations ( siemens somatom plus 4 , erlangen , germany ) were performed before and after iv injection of contrast agent , with scans during the venous phase ( 60to 70 - s delay ) without oral contrast agent . 
in one case , ct was performed without iv injection of contrast agent due to reported allergy to iodinated contrast agents . the parameters analysed in ultrasonographic examinations were the following : focal or diffuse wall thickening ( more than 3 mm ) ; stratification or double parietal profile ; distension ( maximum transverse diameter greater than 5 cm ) ; stones in the gallbladder or biliary tract ; sludge or endoluminal aggregates ; parietal , endoluminal or extra - parietal gas collections ; aerobilia ; free or sacculated pericholecystic or abdominal fluid collections ; intraand extra - hepatic biliary tract dilatation ; presence of ultrasonographic murphys sign ; and possible associated findings . 
difficulty or inability to recognise the gallbladder was also evaluated . ultrasonographic signs were divided into three groups : major criteria of simple acute cholecystitis ( msac ) , minor criaddominale . sia la tc che la rm generalmente aggiungono un numero esiguo di informazioni alla valutazione delle colecistiti acute , ma possono essere molto utili in caso di manifestazioni subacute , in presenza di pseudomasse palpabili in ipocondrio destro , in caso di discrepanza clinico - ecografica e nei casi di complicazioni quali la colecistite empiematosa , lascessualizzazione , la colecistite enfisematosa , la colecistite necrotico - gangrenosa , la perforazione , la fistola bilioenterica o lileo biliare [ 2 , 6 , 7 , 15 , 16 ]  . questo studio si propone di esaminare i casi di colecistite acuta complicata , valutati in urgenza con ecografia e tc e sottoposti ad intervento chirurgico in urgenza nel corso di un anno , al fine di riconoscere i segni specifici ed aspecifici di complicanza da noi riscontrati e confrontarli con una revisione della letteratura . 
ci proponiamo inoltre di definire un corretto iter diagnostico che preveda limpiego della tc in casi selezionati . materiali e metodi in questo studio sono stati esaminati retrospettivamente 35 pazienti ( 12 donne e 23 uomini ) di et compresa tra 27 e 99 anni ( et media 66 , 6 anni ) affetti da patologia acuta della colecisti e sottoposti ad esami strumentali nel periodo compreso tra laprile 2003 e laprile 2004 . 
sono stati pertanto esclusi i casi in cui non sono risultati disponibili gli esami diagnostici eseguiti in altre sedi , i pazienti non sottoposti ad intervento chirurgico in urgenza per controindicazioni mediche e quelli il cui intervento chirurgico stato differito oltre le 48 ore dalla diagnosi . sono stati considerati i segni clinici ( dolore , manovra di murphy , febbre , tumefazione palpabile e ittero ) e laboratoristici ( leucocitosi neutrofila , bilirubinemia , transaminasemia , glicemia ) al momento della diagnosi . 
per gli esami ecografici sono state utilizzate due differenti apparecchiature ( au5 esaote , italia e logiq 400 , ge medical system , milwaukee , wi , usa ) con sonda convex da 3 , 55 mhz . 
per riferita allergia al mdc iodato . i parametri analizzati negli esami ecografici sono stati i seguenti : ispessimento parietale diffuso o focale ( considerando ispessita la parete quando superiore a 3 mm ) , stratificazione o doppio contorno parietale , sovradistensione ( diametro traverso massimo maggiore di 5 cm ) , calcoli nella colecisti o nelle vie biliari , presenza di sedimenti o aggregati endoluminali , raccolte gassose parietali , endoluminali o extraparietali , aerobilia , raccolte fluide pericolecistiche o endo - addominali libere o saccate , dilatazione delle vie biliari intraed extra - epatiche , presenza di segno di murphy ecografico ed eventuali reperti associati . 
msac , major criteria of simple acute cholecystitis ; msac , minor criteria of simple acute cholecystitis ; cc , complication criteria msac msac wall thickening > 3 mm wall stratification overdistension > 5 cm ultrasonographic murphys sign biliary sludge or stones biliary tract dilatation calculous cholecystitis biliary lithiasis pericholecystic or abdominal fluid collections parietal , endoluminal or extraparietal gas collections aerobilia difficult or missed recognition of the gallbladder tabella 1 segni ecografici di colecistite . 
mcas , criteri maggiori di colecistite acuta semplice ; mcas , criteri minori di colecistite acuta semplice ; cc , criteri di complicanza mcas mcas ispessimento parietale > 3 mm stratificazione parietale sovradistensione > 5 cm murphy ecografico sedimenti o aggregati biliari dilatazione delle vie biliari litiasi della colecisti litiasi delle vie biliari raccolte fluide pericolecistiche o endoaddominali raccolte gassose parietali , endoluminali o extraparietali aerobilia difficile o mancato riconoscimento della colecisti teria of simple acute cholecystitis ( msac ) , and complication criteria ( cc ) ( table 1 )  . 
for ct in addition to the us parameters , with the exception of murphys sign , we evaluated the presence of free air and alterations to vascularisation of the liver parenchyma adjacent to the gallbladder , both being signs of complication . 
the final histological examinations , considered as the gold standard , were compared with us and ct results , and accuracy , sensitivity , specificity , ppv , and npv for each modality were measured . 
the association with neutrophilia was observed in 28 / 35 ( 80% ) of cases ; in 11 / 35 cases , patients had elevated bilirubinaemia ( 31.4% ) and in 9 / 35 cases increased transaminases ( 25.7% ) ; 21 / 35 patients ( 60% ) had hyperglycaemia on admission , and four of them had a history of diabetes mellitus . 
in the selected group , hepatobiliary us was performed as the first - choice diagnostic examination in 30 / 35 patients , followed by ct in seven cases ; in five cases presenting with acute abdomen , ct was performed as the first - choice examination . 
on the whole , 30 patients were studied with us and 12 by ct ; in 7 / 35 cases , both us and ct were performed . us findings us revealed diffuse thickening of the gallbladder wall in gni ecografici sono stati inoltre suddivisi in tre gruppi : criteri maggiori di colecistite acuta semplice ( mcas ) , criteri minori di colecistite acuta semplice ( mcas ) e criteri di complicanza ( cc ) ( tabella 1 )  . 
per la tc oltre agli stessi parametri utilizzati per lecografia , fatta eccezione per il segno di murphy ecografico , stata valutata la presenza di aria libera e le alterazioni della vascolarizzazione del parenchima epatico adiacente alla colecisti , entrambi segni di complicazione . in base ai reperti riscontrati negli esami diagnostici sono stati distinti due gruppi di patologie : colecistiti acute semplici e colecistiti acute complicate . 
gli esami istologici definitivi , considerati il gold standard , sono stati confrontati con i risultati degli esami ecografici e tc e sono state misurate accuratezza , sensibilit , specificit , valore predittivo positivo ( vpp ) e negativo ( vpn ) delle rispettive metodiche . 
nei casi sottoposti sia ad ecografia che a tc stata inoltre valutata la concordanza tra le due metodiche e lentit dei dati aggiuntivi forniti da una metodica rispetto allaltra . risultati i pazienti presentavano clinicamente dolore in ipocondrio destro o epigastrio in 32 / 35 casi ( 91 , 4% ) , manovra di murphy positiva in 22 / 35 casi ( 62 , 8% ) , febbre in 18 / 35 casi ( 51 , 4% ) , tumefazione palpabile in 6 / 35 casi ( 17 , 1% ) , ittero in 2 / 35 casi ( 5 , 7% )  . 
si associava leucocitosi neutrofila in 28 / 35 casi ( 80% ) ; in 11 / 35 casi i pazienti presentavano un innalzamento dei valori di bilirubinemia ( 31 , 4% ) e in 9 / 35 casi si rilevava un incremento dei valori delle transaminasi ( 25 , 7% ) ; 21 / 35 pazienti ( 60% ) avevano iperglicemia al momento del ricovero e 4 di questi pazienti presentavano storia di diabete mellito . 
nel gruppo selezionato , lecografia epatobiliare stata eseguita come primo esame diagnostico in 30 / 35 pazienti , seguita dalla tc in 7 casi ; in 5 casi la tc stata eseguita come esame di prima istanza , trattandosi di gravi quadri di addome acuto . 
ct also revealed three cases of acute pancreatitis ( 25% ) characterised by pancreatic enlargement , with peripancreatic fat tissue hyperdensity in two of these cases . in one patient with suspected thoracic aortic dissection , the detection of cholecystitis was an occasional finding revealed by ct . 
ct allowed the detection of three cases of simple acute cholecystitis and nine cases of complicated acute cholecystitis ( table 2 )  . surgical treatment and histology all patients underwent laparotomic or laparoscopic cholecystectomy within 2448 h of diagnosis . 
in addition to cholecystectomy , the following procedures were performed : three viscerolyses , two hepatic resections , one intestinal resection , one abdominoplasty for laparocele , one umbilical hernial repair , one peritoneal lavage , three abdominal collection drainages , one main biliary tract drainage , one duodenal fistula repair , and one choledochoplasty with kehr tube positioning . 
la tc ha inoltre individuato 3 casi di pancreatite acuta ( 25% ) caratterizzata da aumento volumetrico del pancreas con iperdensit del tessuto adiposo peripancreatico in 2 di questi casi . in un paziente con sospetta dissecazione dellaorta toracica il riscontro di colecistite stato un reperto occasionale rilevato allesame tc . 
la tc ha permesso di distinguere 3 casi di colecistite acuta semplice e 9 casi di colecistite acuta complicata ( tabella 2 )  . interventi chirurgici ed istologia tutti i pazienti sono stati sottoposti a colecistectomia per via laparotomica o laparoscopica nelle 2448 ore successive alla diagnosi . 
de vargas macciucca et al . : ultrasonographic and spiral ct evaluation of simple and complicated acute cholecystitis table 2 comparison between ultrasonography ( us ) , computed tomography ( ct ) , and histological findings * acute and recurrent cholecystitis are included in the group of simple cholecystitis tabella 2 confronto tra i risultati dellecografia , della tc e dellesame istologico simple cholecystitis * complicated cholecystitis neoplasms colecistiti semplici * colecistiti complicate neoplasie 24 / 30 ( 80% ) 6 / 30 ( 20% ) ecografia 24 / 30 ( 80% ) 6 / 30 ( 20% ) * il gruppo di colecistiti semplici comprende le acute e le croniche riacutizzate table 3 ultrasonographic signs in simple cholecystitis 3 / 12 ( 25% ) 9 / 12 ( 75% ) 3 / 12 ( 25% ) 9 / 12 ( 75% ) histology 14 / 35 ( 40% ) 20 / 35 ( 57.1% ) 1 / 35 ( 2.8% ) istologia 14 / 35 ( 40% ) 20 / 35 ( 57 , 1% ) 1 / 35 ( 2 , 8% ) patient thickening stratification distension murphys sign sludge biliary tract dilatation lithiasis tabella 3 segni ecografici riscontrati nelle colecistiti non complicate paziente ispessimento stratificazione sovradistensione murphy sedimenti dilatazione vvbb litiasi three cases of acute calculous cholecystitis ; 11 cases of recurrent chronic cholecystitis ( with inflammatory infiltrates associated with fibrosis ) , ten of which were calculous and one non - calculous ; and 20 complicated cases of cholecystitis ( 14 calculous and six non - calculous )  . 
the final histological examination revealed one case of undifferentiated carcinoma epatiche , 1 resezione intestinale , 1 addominoplastica per laparocele , 1 riduzione di ernia ombelicale , 1 lavaggio peritoneale , 3 drenaggi di raccolte addominali , 1 drenaggio della via biliare principale , 1 sutura di fistola duodenale e 1 coledoco - plastica con posizionamento di tubo di kehr . 
of the 20 complicated forms , histology diagnosed 13 cases of phlegmonous cholecystitis , two cases of empyema , two cases of necrotic and gangrenous cholecystitis , two cases of abscess , and one case of duodeno - cholecystic fistula . 
us had a sensitivity of 37.5% , a specificitable 4 ultrasonographic signs in complicated acute cholecystitis colecistite acuta litiasica , 11 casi di colecistite cronica riacutizzata ( con infiltrati flogistici associati a fibrosi ) , 10 delle quali su base litiasica e 1 alitiasica , 20 colecistiti complicate ( 14 litiasiche e 6 alitiasiche )  . in 1 caso lesame istologico definitivo ha dimostrato la presenza di un carcinoma indifferenziato della colecisti associato a litiasi . 
delle 20 forme complicate 13 sono risultate allesame istologico come colecistiti flemmonose , 2 casi di empiema , 2 casi di colecistite acuta necrotica e gangrenosa , 2 casi di ascesso e 1 caso di fistola colecisto - duodenale . 
de vargas macciucca et al . : ultrasonographic and spiral ct evaluation of simple and complicated acute cholecystitis ty of 70% , an accuracy of 66.6% , a ppv of 100% , and an npv of 58.3%. 
in the seven cases studied by both modalities , us and ct yielded similar results as regards the presence of complications ; however , ct provided additional information compared with us , as it detected the presence of fluid in four cases , identified the gallbladder in two cases in which it was not recognizable by us , and demonstrated the presence of intraparietal air in one case , endoluminal air in three cases and free air in one case . 
it also demonstrated biliary tract dilatation in two cases , a biliary - enteric fistula in one case , and intrahepatic fluid and gas due to cholecystitis in one case . 
unlike us , ct also permitted identification of three cases of oedematous pancreatitis . discussion considering that more than 20% of cases clinically classified as cholecystitis correspond to other diseases , the role of emergency diagnostic imaging is to support the clinical diagnosis of acute cholecystitis and to identify the complications of cholecystitis , thus indicating the correct therapeutic approach [ 7 ]  . 
in cases of simple cholecystitis , surgical treatment is normally delayed , and the patient is treated with antibiotics during the acute phase ; emergency surgery within 2448 h , however , prevents the formation of adhesions , which may complicate laparoscopic surgery and increase the rate of conversion into laparotomy [ 12 , 17 , 18 ]  . 
emergency surgery , especially when laparoscopic , therefore implies shorted hospitalisation and reduced post - operative complications and overall hospitalisation costs [ 11 , 12 , 17 , 1824 ]  . clinically , three quarters of patients suffering from acute cholecystitis have a history of biliary colic and pain in the right hypochondrium , which may irradiate to the right shoulder and to the epigastric region , accompanied by nausea , vomiting , and dyspepsia . 
con la tc sono stati identificati 3 vn , 9 vp , nessun fp e nessun fn . lecografia ha dimostrato una sensibilit del 37 , 5% , una specificit del 70% , una accuratezza del 66 , 6% , un vpp del 100% e un vpn del 58 , 3% . 
la tc ha invece presentato una sensibilit del 100% , una specificit del 100% , una accuratezza del 100% ( tabella 5 )  . nei 7 casi sottoposti sia ad ecografia che a tc le due metodiche sono risultate concordi per la presenza di complicanze , ma la tc ha fornito in ogni caso maggiori informazioni rispetto allecografia rilevando la presenza di versamento fluido in 4 casi , identificando la colecisti in 2 casi in cui allecografia non era riconoscibile , diagnosticando la presenza di aria intraparietale in 1 caso , endoluminale in 3 casi , di aria libera in 1 caso , ha mostrato la presenza di dilatazione delle vie biliari in 2 casi , la fistola bilio - enterica in 1 caso , la presenza di raccolta fluido - gassosa intraepatica ad origine dalla colecisti in 1 caso . 
la tc ha inoltre consentito rispetto allecografia lidentificazione di 3 casi di pancreatite edematosa . discussione il ruolo della diagnostica per immagini in emergenza nella patologia acuta della colecisti quello di supportare la diagnosi clinica , considerando che oltre il 20% dei casi classificati clinicamente come colecistite corrispondono a unaltra patologia , e di identificare le complicanze indicando il corretto approccio terapeutico [ 7 ]  . 
generalmente se la colecistite non risulta complicata , il trattamento chirurgico viene differito , sottoponendo il paziente in fase acuta a terapia antibiotica ; tuttavia , lintervento chirurgico in emergenza entro 2448 ore previene la formazione di aderenze che possono complicare lintervento chirurgico laparoscopico aumentandone la percentuale di conversione in laparotomia [ 12 , 17 , 18 ]  . 
in our study , fewer patients underwent ct than us , and the majority of them had complicated cholecystitis suspected at us or on the basis of clinical findings ; therefore , it was not possible to accurately assess the major and minor criteria using ct in simple cholecystitis . forty percent of patients with acute cholecystitis develop complications such as empyema , pericholecystic abscesses , necrotic - gangrenous cholecystitis , emphysematous cholecystitis , haemorrhagic cholecystitis , perforation , cholecysticenteric fistula , and biliary ileus [ 3638 ]  . 
 we also detected one case of duodeno - cholecystic fistula . in this case , the gallbladder was not recognisable at us whereas both aerobilia and a coarse calcification ( 3 cm ) between the liver and the pancreatic head were well visible in the second duodenal portion ; also , ct had some difficulties in that the stone could not be precisely localised in the intestinal lumen , and neither free air or endoperitoneal fluid collection could be seen . 
however , the presence of aerobilia guito in laparoscopia , comporta riduzione dei tempi di ospedalizzazione , delle complicanze post - operatorie e dei costi complessivi del ricovero [ 11 , 12 , 17 , 1824 ]  . clinicamente tre quarti dei pazienti affetti da colecistite acuta presentano una storia di coliche biliari , dolore in ipocondrio destro , che si pu irradiare alla spalla destra ed in sede epigastrica , accompagnato da nausea , vomito e dispepsia . 
il segno di murphy generalmente positivo e vi possono essere reperti di laboratorio aspecifici ( leucocitosi , iperbilirubinemia , iperamilasemia e aumento dei livelli di transaminasi ) [ 25 , 26 ]  . 
nel nostro studio i pazienti sottoposti a tc sono stati in numero ridotto rispetto ai casi sottoposti ad ecografia ed erano quasi tutti casi di colecistiti complicate sospettate allesame ecografico o in base al quadro clinico , pertanto non stato possibile valutare con accuratezza i criteri maggiori e minori con tale metodica nelle colecistiti semplici . 
nel 40% dei pazienti con colecistite acuta si sviluppano complicanze quali lempiema , gli ascessi pericolecistici , la colecistite necrotico - gangrenosa , la colecistite enfisematosa , la colecistite emorragica , la perforazione , la fistola colecisto - enterica e lileo bilare [ 3638 ]  . 
de vargas macciucca et al . : ultrasonographic and spiral ct evaluation of simple and complicated acute cholecystitis and inability to identify the gallbladder , perforated and collapsed , correctly prompted towards emergency surgery during which and a duodeno - cholecystic fistula with a stone impacted in the second duodenal portion was detected . 
in our study , no cases of haemorrhagic cholecystitis or biliary ileus were observed . the fundamental aim of diagnostic imaging , especially in emergency settings , is to differentiate simple and complicated cholecystitis in order to plan laparoscopic or laparotomic surgery within 48 h . 
to detect the complicated forms , we suggest a classification that takes into account the main us signs divided into major criteria ( wall thickening > 3 mm , wall stratification , positive sonographic murphys sign , and distension ) , minor criteria ( biliary sludge , calculous cholecystitis and biliary tract dilatation ) and criteria for complicated forms ( aerobilia , fluid and gas collections , or missed recognition of the gallbladder ) ( table 1 )  . 
this classification differs from mirviss classification [ 9 ] ( table 6 ) first of all because it takes into consideration sonographic criteria but also because the presence of stones and biliary sludge is considered a minor criteria ; this depends on the fact that , despite being a frequent sonographic finding , biliary sludge is not specific for cholecystitis . 
the analysis of associations between major and minor signs , on the other hand , did not show a significant difference between the two groups of simple and complicated cholecystitis . 
indeed , in 5 / 16 cases ( 31.2% ) of complicated cholecystitis and in 10 / 14 cases ( 71.4% ) of simple cholecystitis , an association of at least two major criteria and one minor was found . 
in 4 / 16 cases ( 25% ) of complicated cholecystitis and in 4 / 14 cases ( 28.5% ) of simtable 6 computed tomography ( ct ) signs of acute cholecystitis ( mirviss classification ) ct signs of acute cholecystitis [ mirvis et al . 
in questo caso allesame ecografico non risultava riconoscibile la colecisti , mentre erano ben evidenti sia laerobilia che la presenza di una struttura calcifica grossolana ( 3 cm ) situata tra il fegato e la porzione cefalica pancreatica , nella sede della seconda porzione duodenale ; anche la tc ha presentato alcune difficolt diagnostiche in quanto la formazione litiasica non risultava localizzabile con certezza nel lume intestinale e non erano visibili aria libera n versamento fluido endoperitoneale ; tuttavia la presenza di aerobilia e il mancato riconoscimento della colecisti , in quanto perforata e collassata , hanno indirizzato correttamente la paziente al trattamento chirurgico urgente ed stata riscontrata una fistola colecisto - duodenale con calcolo indovato a livello della seconda porzione duodenale . 
nella nostra casistica non sono stati riscontrati casi di colecistite emorragica n di ileo biliare . lobiettivo fondamentale che si propone la diagnostica per immagini soprattutto in emergenza quello di distinguere le colecistopatie semplici dalle complicate al fine di poter programmare lintervento chirurgico per via laporoscopica o laparotomica , entro le 48 ore . 
a tal fine per poter individuare le forme complicate proponiamo una classificazione che tiene conto dei principali segni ecografici suddivisi in criteri maggiori ( ispessimento parietale > 3 mm , aspetto stratificato della parete , segno ecografico di murphy positivo e sovradistensione ) , criteri miniori ( sedimenti biliari , litiasi della colecisti o delle vie biliari e dilatazione delle vie biliari ) e criteri riconducibili a forme complicate ( areobilia , raccolte fluide , raccolte gassose o mancato riconoscimento della colecisti ) ( tabella 1 )  . 
una classificazione basata su tali criteri differisce da quella di mirvis riportata in letteratura [ 9 ] ( tabella 6 ) , innanzitutto perch prende in considerazione dei criteri ecografici , ma anche perch la presenza di calcoli e sedimenti biliari considerata un criterio minore poich , sebbene con tale metodica sia molto frequentemente riscontrata , non specifica di colecistite : nel nostro studio calcoli e aggregati biliari erano presenti quasi sempre ( 98% della popolazione esaminata ) sia nelle forme complicate che in quelle semplici . nella nostra esperienza lecografia ha riscontrato segni sicuri di complicanza in 6 / 16 casi ( 37 , 5% )  . 
tra le colecistiti semplici i segni di complicazione non sono mai stati rilevati . lanalisi delle associazioni tra segni maggiori e minori invece non ha dimostrato una significativa differenza tra i due gruppi di colecistiti , semplici e complicate . 
infatti in 5 / 16 casi ( 31 , 2% ) di colecistite complicata e in 10 / 14 casi ( 71 , 4% ) di colecistite semplice stata riscontrata lassociazione di almeno 2 criteri maggiori e uno minore . 
in 4 / 16 casi ( 25% ) di colecistite complicata e in 4 / 14 casi ( 28 , 5% ) di colecistite semplice erano presenti allecografia meno di due criteri maggiori . 
 these data indicate that us can distinguish complicated and simple cholecystitis only in the presence of definite signs of complication ; in the majority of cases , however , these signs are not present . 
we believe that ct would have provided the correct diagnosis of complication in many of these cases given its higher accuracy and sensitivity . indeed , ct yielded no fp or fn results . 
because the majority of complications revealed by us showed at least two major criteria associated with at least one minor criterion or at least one criterion for complication ( overall 68.7% ) , further ct investigation after us is justified under these circumstances . numerous cases of phlegmonous cholecystitis were present ( 9 / 16 cases ) among the cases assessed as simple cholecystitis by us and as complicated cholecystitis by histological examination . although this form is not regarded as a complicated form in the literature , we decided to include these cases in the group of complications because involvement of the entire thickness of the organ causes serositis with peritoneal effusion in the majority of cases . 
if the emergency approach is based on medical treatment with delayed surgery , the peritoneal reaction may lead to the formation of synechia and fibrosis , with development of adhesions . 
phlegmonous cholecystitis therefore requires immediate surgical treatment because adhesions hinder possible surgical treatment during quiescence and increase the incidence of complications ; there is also a higher risk of laparoscopy being converted into laparotomy [ 12 ]  . conclusions assessment of acute cholecystitis in an emergency department requires careful analysis of the clinical findings and relies on us as first - choice examination . us allows the recognition of signs which , taken singularly , are poorly specific ; this directs the patient to further investigation by ct , which is indispensable for the complete recognition and evaluation of complicated forms . 
trenta , radiologists at the emergency department of policlinico umberto i , rome , for their help in collecting the case series . solo in presenza di segni certi di complicanza , ma nella maggior parte dei casi questi non sono presenti ; infatti , nella nostra casistica , allecografia sono risultati falsi negativi ( fn ) un terzo dei casi e la maggior parte di tali pazienti non stata sottoposta ad ulteriori indagini . 
poich allecografia la maggior parte delle complicanze presentavano almeno due criteri maggiori associati ad almeno uno minore oppure almeno un criterio di complicanza ( globalmente il 68 , 7% ) , si pu considerare giustificato in tali circostanze approfondire lesame ecografico con un esame tc . 
sebbene la letteratura non tratti questa forma come complicata , abbiamo ritenuto di includere tali casi nel gruppo delle complicazioni in quanto linteressamento a tutto spessore del viscere determina comunque una sierosite con presenza nella maggior parte dei casi di versamento peritoneale . 
in tali pazienti , se la scelta terapeutica in urgenza il trattamento medico e lintervento chirurgico viene differito , la reazione peritoneale nel tempo pu determinare la formazione di sinechie e fibrosi con lo sviluppo di briglie aderenziali . 
le colecistiti flemmonose necessitano pertanto di un trattamento chirurgico immediato , in quanto le aderenze rendono leventuale intervento in fase di quiescenza pi difficoltoso e aumentano lincidenza di complicanze ; in questi casi inoltre pi elevato il rischio di conversione dellintervento da laparoscopico a laparotomico [ 12 ]  . conclusioni la valutazione in un dipartimento di emergenza della patologia acuta della colecisti necessita di una attenta analisi dei dati clinici e si avvale dellutilizzo dellecografia come metodica di prima istanza . 
lecografia permette il riconoscimento di alcuni segni che sono poco specifici singolarmente , indirizzando il paziente allapprofondimento con tc che risulta indispensabile per riconoscere e valutare in maniera completa le forme complicate . 
in particolare , quando allecografia sono identificati almeno due segni maggiori e uno minore e quando sono presenti i segni specifici di complicanza , lapprofondimento con tc imprescindibile in quanto permette di selezionare i pazienti candidati ad intervento in urgenza rispetto a quelli da trattare con terapia antibiotica ed intervento differito ; inoltre la distinzione tra le diverse forme di colecistite aiuta il chirurgo nella scelta della tecnica operatoria pi indicata ( laparoscopica o laparotomica )  . ringraziamenti si ringraziano per la collaborazione nel reperimento della casistica il dott . 
gargiulo2 1dipartimento integrato dei servizi diagnostici e per immagini , azienda policlinico , via del pozzo 71 , i - 41100 modena , italy 2cattedra di chirurgia vascolare , azienda policlinico , modena , italy correspondence to : m.g. 
 + 39 - 059 - 4222238 , fax : + 39 - 059 - 4224349 , e - mail : amorico.mariagrazia@policlinico.mo.it received : 27 june 2005 / accepted : 18 october 2005 / published online : 3 march 2006 abstract purpose . 
magnetic resonance angiography ( mra ) has recently become instrumental in the diagnosis of arterial disease in various body districts and is gaining an increasingly important role in the study of peripheral vascularisation . 
mra was performed with a philips intera 1.5 t , with acquisitions from the coeliac trunk to the feet . for acquisitions of the feet and ankles we used unenhanced timeof - flight ( tof ) sequences with a head coil . 
digital subtraction angiography ( dsa ) remains the reference standard for planning treatment and for the follow - up of these patients , but it is invasive and potentially harmful as the iodinated contrast larteriopatia occlusiva periferica degli arti inferiori una patologia cronica e progressiva con unincidenza negli uomini con pi di 55 anni che va dal 4 , 5% al 8 , 8% [ 1 , 2 ]  . 
la diagnosi solitamente ottenuta mediante un accurato esame clinico , ma la scelta di uneventuale terapia non pu prescindere da precise informazioni morfologiche dellalbero arterioso ottenibili esclusivamente attraverso la diagnostica strumentale . 
because peripheral arterial occlusive disease is an increasingly common chronic condition , a large number of mostly elderly patients would have to undergo numerous angiographic examinations and endovascular procedures , both of which carry a high risk of complications , such as puncture of the arteries , iodinated contrast agent toxicity , and protracted exposure to radiation [ 3 ]  . 
for these reasons , it has become necessary to identify an alternative modality that is less invasive than dsa and able to evaluate the number and severity of lesions in order to plan treatment . 
recent technological innovations , such as continuous table movement and very fast three - dimensional ( 3d ) sequences [ contrast - enhanced mr angiography ( ce - mra ) ] [ 2 , 5 ] , have enabled the study of the arteries from the abdominal aorta to the foot with a single injection of contrast material . 
these improvements have overcome the limitations of the previous time - of - flight ( tof ) and phasecontrast sequences , such as long acquisition times ( ats ) and flow and movement artefacts . 
the aim of this study was to assess the reliability of mra in the evaluation of lesions caused by peripheral arterial occlusive disease , using intraarterial dsa as the reference standard . materials and methods patients from november 2003 to august 2004 , a total of 30 patients ( 11 women and 19 men ; age range , 4984 years ; mean age , 72 years ) underwent digital subtraction angiography and mra of the lower limbs . 
two patients had concurrent renal artery stenosis ( one on the right and one on the left ) , and another two had aneurysm of the infrarenal aorta , one of whom with concurrent aneurysm of the left popliteal artery . 
two patients were excluded after dsa because they had a pacemaker , and another was excluded due to a history of surgery with insertion of artificial crystalline made of non - mr - compatible material . 
poich larteriopatia periferica una patologia cronica in aumento , un numero elevato di pazienti , la maggior parte anziani , dovrebbe essere sottoposto a numerose angiografie e procedure endovascolari che comportano un elevato rischio di complicanze sia legate alla procedura , come la puntura dei vasi arteriosi , che alla tossicit del mezzo di contrasto ed allesposizione prolungata alle radiazioni ionizzanti [ 3 ]  . 
per tutti questi motivi si sentita lesigenza di una metodica di studio delle arterie degli arti inferiori meno invasiva e che possa essere utilizzata come valutazione del numero e della severit delle lesioni in previsione di un trattamento . la risonanza magnetica una tecnica di indagine non invasiva che non impiega radiazioni ionizzanti n mezzo di contrasto potenzialmente nefrotossico [ 4 ]  . 
recentemente sono state introdotte innovazioni tecnologiche come , per esempio , il movimento automatico del lettino porta - paziente e sequenze tridimensionali molto veloci ( ce - mra ) [ 2 , 5 ] che rendono possibile lo studio dellalbero arterioso dal tripode celiaco ai vasi del piede con ununica iniezione di mezzo di contrasto . 
ci ha permesso di superare le limitazioni delle precedenti sequenze time - of - flight e phase - contrast come i lunghi tempi di acquisizione , gli artefatti da flusso e da movimento . 
scopo di questo lavoro analizzare quale sia la reale affidabilit della angio - rm nella valutazione delle lesioni causate dallarteriopatia occlusiva periferica , utilizzando come standard di riferimento langiografia digitale a sottrazione ( dsa )  . materiali e metodi pazienti dal novembre 2003 allagosto 2004 , 30 pazienti di et compresa tra i 49 ed gli 84 anni ( 11 donne e 19 uomini ; et media 72 anni ) sono stati sottoposti ad angiografia digitale e ad angio - rm degli arti inferiori . 
due casi , inoltre , mostravano la presenza concomitante di stenosi dellarteria renale ( 1 a destra ed 1 a sinistra ) , in 2 casi un aneurisma dellaorta sottorenale in uno dei quali era concomitante un aneurisma dellarteria poplitea sinistra . 
the disease staging classification adopted by us was the following : mild stenosis ( not haemodynamically significant ) with less than 50% reduction in vessel lumen ; moderate stenosis ( haemodynamically significant ) with 50%90% reduction in lumen ; severe stenosis with a reduction in vessel lumen greater than 90% . the percentage of stenosis was automatically calculated by software present in the image - processing work stations connected to the angiographic and mr instruments ( viewforum , philips )  . patients included in the study underwent dsa performed with digital equipment ( philips integris allura , philips medical system , the netherlands ) , following a pre - established procedure . 
the examination procedure involved positioning of a 4 - french universal flash ( uf ) catheter into the abdominal aorta through a retrograde , generally right - sided , transfemoral approach , or a brachial approach according to the seldinger technique , after the local administration of 10 cc of lidocaine hydrochloride 200 mg ( bioindustria , italy )  . 
the catheter was positioned in the external or common iliac artery to perform selective arteriography of the limb , with anteroposterior ( ap ) projections for the thigh and leg vessels , and both ap and laterolateral ( ll ) projections for the feet vessels . 
images of the lower limb vessels , from the femoral artery to those of the feet , were obtained sequentially with separate injections of contrast material delayed relative to acquisition , on the basis of the level of study and the speed of flow . 
the mean dose of contrast agent injected was approximately 200 cc ( iomeprol 300 mg / i / ml , iomeron bracco , or iopamidol 300 mg / i / ml , iopamiro bracco ; iodixanol 270 mg / i / ml , visipaque nycomed amersham in selected subjects ) , with a range from 160 to 300 cc according to the diagnostic requirements of the single case . 
the entire examination had a mean duration of 60 min . mra was performed by using a 1.5 - t whole - body mri unit ( intera philips gyroscan 1.5 tesla , philips medical system )  . for each patient , a complete history was taken to exclude the presence of contraindications . 
the patient was positioned supine with both feet inside the coil usually used for the study of the head ( head coil ) for a preliminary evaluation of the foot arteries with an unenhanced 3d - tof sequence . 
due pazienti sono stati esclusi dallo studio dopo lesecuzione della dsa , perch portatori di pace - maker ed uno per presenza di anamnesi positiva per intervento con inserimento di cristallino artificiale di materiale non rm - compatibile . 
tre pazienti erano gi stati precedentemente sottoposti a procedure di rivascolarizzazione ( 1 by - pass femorofemorale destro - sinistro con bypass femoro - popliteo bilaterale , 1 by - pass femoro - popliteo destro , 1 endoprotesi hemoban in femorale superficiale destra ) e tre pazienti avevano subito lamputazione di uno o pi dita di un piede . la classificazione da noi seguita distingue : stenosi lievi ( non emodinamicamente significative ) con riduzione del lume vasale inferiore al 50% ; stenosi moderate ( emodinamicamente significative ) con riduzione del lume vasale superiore al 50% , ma inferiore al 90% ; stenosi gravi con riduzione del lume vasale superiore al 90% . la percentuale di stenosi stata calcolata automaticamente da un programma di calcolo presente nelle stazioni di elaborazione delle immagini correlate allangiografo philips allura ed allapparecchio di rm ( viewforum , philips medical systems , olanda )  . angiografia digitale i pazienti inclusi nello studio sono stati sottoposti a dsa con tecnica di sottrazione dimmagine eseguita con angiografo digitale ( integris allura philips , philips medical systems , olanda ) , seguendo una procedura prestabilita . 
lesecuzione dellesame prevede il posizionamento di un catetere uf ( universal flash ) di 4 f nellaorta addominale per mezzo di un approccio transfemorale retrogrado , generalmente destro , o brachiale , con ago munito di mandrino secondo la tecnica seldinger , ovviamente dopo aver eseguito nella sede lanestesia locale con 10 cc di lidocaina cloridrato 200 mg . 
il catetere viene posizionato in arteria iliaca esterna o comune per lesecuzione di unarteriografia darto selettiva ; si effettuano riprese dei vasi di coscia e di gamba in a - p e del piede sia in a - p che in ll . 
le immagini del riempimento degli arti inferiori dallarteria femorale a quelle del piede sono state ottenute sequenzialmente con iniezioni separate di mezzo di contrasto ritardate rispetto allacquisizione in base al livello di studio ed alla velocit del flusso . 
la dose media di mezzo di contrasto iniettato per via endoarteriosa di 200 cc circa ( iomeprolo 300 mg i / ml , iomeronr bracco oppure iopamidolo 300 mg i / ml , iopamiror bracco ; iodixanolo 270 mg i / ml , visipaquer , amersham , in soggetti selezionati ) , con un range variabile da 160 a 300 cc a seconda delle esigenze diagnostiche del singolo caso . 
the parameters used were : tr 23 ms , te 9.2 ms , flip angle 20 , thickness 0.7 mm , matrix 304x258 , field of view ( fov ) 30 c the sequence yielded around 150 images , which were processed in 3d at the workstation view forum 3.1 ( philips medical systems )  . 
subsequently , the head coil was replaced with a body coil , with mobytrack , a specific attachment for this type of study which supports and immobilises the legs at the level of the knees and holds the feet perpendicular to the legs . 
positioning of the entire lower limb in the same plane is important , as the coronal acquisition might otherwise miss arterial segments not perfectly included in the field of view . 
the arterial study was performed with three 40to 45 - cm acquisitions , with 3d coronal sequences from the coeliac trunk to the feet , each lasting around 3035 s , with an overlap of 13 c automatic table movement means the examination can be performed in a time compatible with the persistence of the paramagnetic contrast agent in the arterial vessels . first , a 2d fast - field - echo centring sequence was performed in the three planes in all three positions , on which the following sequences were planned . 
the parameters of this sequence were : tr 6.1 ms , te 1.5 ms , flip angle 35 , thickness 1.5 mm , matrix 512x384 , fov 43 cthen , circulation time ( ct ) , or bolus timing , was evaluated by injecting 2 cc of contrast agent followed by 15 cc of saline solution with a special mr - compatible injector ( spectris mr injector , medrad )  . 
this was followed by a fast - field - echo sequence ( tr 7 , te 0.87 , flip angle 40 , thickness 80 mm , matrix 256x256 , fov 53 cm ) in which a single coronal image at the level of the abdominal aorta and its bifurcation was acquired at intervals of 1 s for about 50 times , repeating the same image at regular intervals until the contrast agent was visualised . 
scan delay ( sd ) was obtained by subtracting half of the at from the ct plus half of the injection time ( it ) : sd = ct + ( it / 2 ) - ( at / 2 )  . an initial unenhanced 3d sequence serving as a mask was performed from the feet to the abdomen . 
around 40 - 50 cc of paramagnetic contrast agent ( gadodiamide ; omniscan nycomed amersham ) [ 15 ] was then injected as a bolus divided in two fractions : 50% was injected at a speed of 1 ml / s and 50% at a speed of 0.5 ml / s , followed by 15 ml of saline solution injected at a speed of 1 ml / s . 
this part of the examination lasted 2025 mparameters used in this sequence were : tr 6.1 ms , te 1.5 ms , flip angle 35 , thickness 1.5 mm , matrix 512x384 , fov 43 cthe 3d sequence used by us was designed to acquire a centric k space , the centre of the k space being acquired during the enhancement peak , which provides the best contrast between blood and static tissue . 
approximately 200 images were obtained , which were reconstructed with 3d maximum image projecte superconduttivo ( philips gyroscan intera 1 , 5 tesla , olanda ) dotato di gradienti con intensit massima di 30 mtesla / m ed una rapidit di salita ( slew rate ) di 150 mtesla / m / ms . 
il paziente viene posizionato supino , con entrambi i piedi allinterno della bobina solitamente usata per lo studio dellencefalo ( head ) , per eseguire una preliminare valutazione delle arterie del piede attraverso un sequenza 3d - tof senza mezzo di contrasto . 
la sequenza viene eseguita su un piano coronale obliquo perpendicolare allasse maggiore del piede e la banda di presaturazione necessaria per impedire il contemporaneo rilevamento del flusso venoso posta caudalmente alla acquisizione . 
successivamente si effettua un cambio di bobina : si sostituisce la bobina head e si utilizza la bobina body per la trasmissione e la ricezione del segnale ed un supporto dedicato per lo studio degli arti inferiori , moby - track , che sostiene ed immobilizza le gambe a livello delle ginocchia , che sono leggermente sollevate , e mantiene i piedi su uno stesso piano perpendicolare a quello delle gambe . 
il posizionamento di tutto larto inferiore su uno stesso piano un accorgimento importante poich altrimenti lacquisizione nel piano coronale potrebbe non includere segmenti arteriosi non perfettamente compresi nel campo di vista . 
lo studio arterioso viene eseguito in 3 acquisizioni da 4045 cm con sequenze 3d coronali dal tripode ai piedi , ognuna della durata di circa 3035 secondi , con sovrapposizione di 13 cil movimento automatizzato del letto porta - paziente permette di eseguire questo tipo di esami in un tempo compatibile con la permanenza del mezzo di contrasto paramagnetico nei vasi arteriosi . 
si esegue inizialmente una sequenza di centratura 2d turbo field echo sui 3 piani in tutte le 3 posizioni sulla quale verranno pianificate tutte le sequenze successive con i seguenti parametri : tr 6 , 1 ms , te 1 , 5 ms , flip angle 35 , spessore 1 , 5 mm , matrice 512x384 , campo di vista 43 csi procede poi ad una valutazione del tempo di circolo o bolus timing iniettando 2 cc di contrasto seguiti da 15 cc di soluzione fisiologica con un apposito iniettore rm - compatibile ( spectris mr injector , medrad , indianola , pa )  . 
si esegue una sequenza fast field echo ( tr 7 , te 0 , 87 , flip angle 40 , spessore 80 mm , matrice 256x256 , campo di vista 53 cm ) , in cui una singola immagine coronale , a livello dellaorta addominale e della sua biforcazione acquisita ad intervalli di 1 secondo per circa 50 volte ripetendo la stessa immagine ad intervalli costanti fino alla visualizzazione del mezzo di contrasto a quel livello . 
il tempo di ritardo scansione ( trs ) si ottiene sottraendo al tempo di circolo ( tc ) la met del tempo di acquisizione ( ta ) , dopo avervi sommato la met del tempo di iniezione table 1 evaluation form right left m . 
the total time spent by the patient inside the mr room , which included examination time , technical time for sequence formulation , and coil changing , was about 4045 min . data collection and analysis mra and dsa examinations were independently evaluated by three radiologists who used grading sheets to assess vessel state : no stenosis , or obstruction . 
the vessel being studied was drawn with parallel lines along a segment that includes the vessel above and below the stenosis and stenotic segment ; the computer analyses the segment and calculates the percentage of stenosis . 
conventional angiography examinations were evaluated by another radiologist blinded to the patients clinical history and results of mra . table 2 patent arteries with regular lumen artery common iliac external iliac internal iliac common femoral superficial femoral deep femoral popliteal anterior tibial posterior tibial peroneal dorsalis pedis plantaris pedis total mra , magnetic resonance ; dsa , digital subtraction angiography tabella 2 arterie pervie di calibro regolare vaso angio - rm angiografia a . 
si iniettano poi circa 4050 cc di contrasto paramagnetico ( gadodiamide omniscanner amersham , uppsala ) in un unico bolo suddiviso in due frazioni : il 50% viene iniettato ad una velocit di 1 ml / s , il restante ad una velocit di 0 , 5 ml / s ; seguito da 15 ml di fisiologica iniettata ad una velocit di 1 ml / s . 
la seconda sequenza con contrasto ha uno start - delay calcolato in base al bolus timing e viene eseguita dal tripode celiaco ai piedi . lapnea richiesta solo durante lacquisizione a livello addominale ed ha una durata di circa 25 s . 
il software della macchina sottrae automaticamente la sequenza con contrasto alla maschera per eliminare il segnale del tessuto adiposo e si ottiene unimmagine angio - rm delle arterie degli arti inferiori . 
la durata di questa parte dellesame di circa 2025 m i parametri utilizzati per eseguire questa sequenza sono : tr 6 , 1 ms , te 1 , 5 ms , flip angle 35 , spessore 1 , 5 mm , matrice 512x384 , campo di vista 43 cla sequenza 3d da noi utilizzata impostata per acquisire lo spazio k in senso centrico , grazie infatti allopzione centra , per cui viene acquisito il centro dello spazio k durante il passaggio del picco di contrasto massimo , che fornisce il contrasto migliore tra sangue e tessuto stazionario . 
il tempo totale che il paziente trascorre allinterno della stanza del magnete , che comprende il tempo desame , il tempo tecnico di impostazione delle sequenze e di cambio delle bobine di circa 4045 minuti . raccolta ed analisi dei dati gli esami di angio - rm e di angiografia sono stati valutati da 3 medici radiologi in modo indipendente attraverso la compilazione di schede , in cui si richiede di valutare lo stato del vaso : pervio , stenosi , ostruzione . 
la percentuale di stenosi stata ottenuta tramite un programma di calcolo inserito in speciali stazioni di elaborazione delle immagini , in cui viene disegnato con linee parallele il vaso in esame per un tratto che comprende il vaso a valle e a monte della stenosi ed il segmento stenotico ; il computer analizza il segmento in esame e calcola la percentuale di stenosi . 
langio - rm stata valutata da 2 medici radiologi in doppio cieco , senza conoscere la storia clinica del paziente ed i risultati dellangiografia tradizionale , utilizzando le rielaborazioni 3d delle immagini e le immagini originarie . 
si sono ottenute due schede ( tabella 1 ) per ogni paziente , una per la rm ed una per la angiografia , che sono state poi confrontate per valutare il livello di corrispondenza . 
i vasi arteriosi esaminati sono : aorta sottorenale , iliaca comune , iliaca esterna , iliaca interna , femorale comune , femorale superficiale , femorale profonda , poplitea , tibiale anteriore , tibiale posteriore , interossea , dorsale e plantare del piede . 
nei 2 pazienti con bypass se ne valutata anche la perviet , lo stato degli stessi e dei territori a valle . thus , two tables were obtained ( table 1 ) for each patient : one for mra and one for dsa , which were compared in order to evaluate the level of concordance . 
arterial vessels examined were : infrarenal aorta ; common iliac artery ; external and internal iliac arteries ; common , superficial , and deep femoral arteries ; popliteal artery ; anterior and posterior tibial arteries ; peroneal artery ; and dorsalis and plantaris pedis arteries . 
in the two patients with bypass , we also evaluated the patency and state of the bypass grafts and territories downstream . results mra was easy to perform and well tolerated although patients with trophic lesions , especially of the heels , had some difficulty staying motionless in the supine position because of pa this problem concerned above all the tof sequences , in which both feet have to be kept motionless inside the head coil for about 7 mas a result , because this sequence requires a very high level of patient cooperation , it was only performed in 15 . 
however , it did not yield important additional information compared with the contrast - enhanced study , except in one case . table 3 occlusions artery common iliac external iliac internal iliac common femoral superficial femoral deep femoral popliteal anterior tibial posterior tibial peroneal dorsalis pedis plantaris pedis total mra , magnetic resonance ; dsa , digital subtraction angiography tabella 3 occlusioni a . 
questo problema ha riguardato soprattutto lesecuzione delle sequenze tof , in cui entrambi i piedi devono rimanere immobili allinterno della bobina per circa 7 min . questa sequenza infatti non stata eseguita in tutti i pazienti , ma solo in 15 , in quanto necessita di estrema collaborazione e non ha dato informazioni rilevanti o aggiuntive rispetto allo studio con mezzo di contrasto , tranne in un singolo caso . 
sono stati visualizzati 552 vasi di calibro regolare con langio - rm e 547 con la dsa ( tabella 2 )  . si ottenuta una corretta visualizzazione di tutti i vasi fino alla caviglia , mentre per quanto riguarda le arterie del piede , in 3 casi , il precoce ritorno venoso ha reso difficile linterpretazione dellesame , ma grazie alla rielaborazione delle immagini alla workstation siamo sempre riusciti ad espritable 4 stenoses artery < 50% > 50% > 90% < 50% > 50% > 90% common iliac external iliac internal iliac common femoral superficial femoral deep femoral popliteal anterior tibial posterior tibial peroneal dorsalis pedis plantaris pedis total mra , magnetic resonance angiography ; dsa , digital subtraction angiography tabella 4 stenosi a . 
correct visualisation of all vessels was obtained up to the ankle whereas in the foot arteries , early venous return in three cases made interpretation of the examination difficult ; nonetheless , image processing enabled a judgement on the vessel to be expressed in all cases . 
discordances regarded the infrapopliteal arteries , with mra visualising the obstruction of 28 anterior tibial arteries , 35 posterior tibial , 14 peroneal , and 23 dorsalis pedis as against the 30 anterior tibial , 33 posterior tibial , 18 peroneal , and 22 dorsalis pedis visualised by dsa . 
in the cases of obstruction , the visibility of the collateral circulations was better with dsa , which was able to depict even tiny branches , but was also judged good with mra , which depicts the larger vessels only . 
evaluation of the foot arteries was more difficult : the tof sequences , if correctly performed , give information on the larger vessels perpendicular to the acquisition plane ; the contrast - enhanced sequences are also informative but they have a smaller field of view . 
as regards the nine ( 1.25% ) discordant segments , mra overestimated two ( 0.3% ) ( two occlusions of the posterior tibial arteries ) and underestimated six ( 1% ) ( two anterior tibial and four peroneal arteries )  . 
mra documented one occlusion of dorsalis pedis artery not visualised by dsa . overall , in the cases studied , mra had a specificity of 99.6% and a sensitivity of 96% ( table 5 )  . 
there was absolute concordance between the two imaging procedures in il numero di occlusioni valutato in angio - rm stato 140 e con la dsa 143 ( tabella 3 )  . 
le discordanze sono nelle arterie infrapoplitee , in particolare langio - rm ha riconosciuto lostruzione di 28 arterie tibiali anteriori , 35 tibiali posteriori , 14 interossee , 23 dorsali del piede contro le 30 tibiali anteriori , 33 tibiali posteriori , 18 inter - ossee , 22 dorsali del piede della dsa . 
nelle stenosi inferiori al 50% langio - rm ne ha riconosciute 13 , la dsa 14 : con questultima stata riconosciuta una stenosi dallarteria femorale profonda che risultava pervia alla angio - rm . 
nei casi di ostruzione la visibilit dei circoli collaterali stata migliore con la dsa , che stata in grado di evidenziare tutti i rami anche di piccolissimo calibro , ma stata giudicata buona con langio - rm , che mette in evidenza solo vasi di calibro maggiore . con entrambe le metodiche comunque possibile evidenziare il punto di ricanalizzazione del vaso . 
la valutazione pi difficoltosa stata quella dei vasi del piede : le sequenze tof danno informazioni , se correttamente eseguite , sui vasi di maggior calibro e perpendicolari al piano dacquisizione ; le sequenze con contrasto danno buone informazioni , ma hanno un campo di vista inferiore . 
dei 9 ( 1 , 25% ) segmenti discordanti , 2 ( 0 , 3% ) sono stati sovrastimati con langio - rm ( 2 occlusioni delle tibiali posteriori ) e 6 ( 1% ) sono stati sottostimati ( 2 tibiali anteriori e 4 inter - ossee )  . 
la permanenza totale del paziente nella sala del magnete stata in media di 45 mil tempo necessario alla rielaborazione ed alla stampa delle immagini alla workstation philips stato di 2030 mcome gi detto in caso di importante contaminazione venosa stato necessario un tempo maggiore . 
non si sono avute reazioni ai mezzi di contrasto in nessuno dei pazienti . discussione la tecnica di angio - rm da noi utilizzata , basata sullacquisizione di 3 volumi lievemente sovrapposti , iniettando il contrasto con uniniezione bifasica , ha permesso unaccurata valutazione dellalbero arterioso dal tripode celiaco alle arterie del piede . 
rispetto allangiografia , la rm offre importanti vantaggi nello studio di pazienti anziani con patologia arteriosa periferica , in quanto si possono evitare i rischi associati alla puntura arteriosa , al mezzo di contrasto iodato ed alle radiazioni ionizzanti . 
none of the patients experienced reactions to the contrast agent . discussion mra , based on the acquisition of three slightly overlapping volumes with two - phase injection of contrast agent , allowed table 6 data from the literature m . 
nellesecuzione dellangiorm si pu incorrere in alcuni pitfalls soprattutto perch per ottenere un esame angio - rm di elevata qualit con contrasto fondamentale la perfetta coordinazione tra linizio dellacquisizione e larrivo del mezzo di contrasto nella regione da studiare . 
non si sono avute comunque sostanziali discordanze tra angio - rm e dsa nella rilevazione della perviet dei vasi e nella valutazione della presenza di malattia aterosclerotica in tutti i territori esaminati . 
nello 0 , 3% dei casi si rilevata una sovrastima della gravit delle lesioni in angio - rm rispetto alla dsa [ 6 ]  . la sovrastima causata dal defasamento degli spin prodotto dal flusso turbolento che si crea nelle stenosi e da effetti di volume parziale . 
questo effetto risulta accentuato nelle ricostruzioni mip in cui non sempre si riesce a distinguere il ridotto calibro della porzione stenotica del vaso dal segnale di fondo [ 7 ]  . 
1 occlusione dellarteria iliaca esterna sinistra ( freccia ) e stenosi in serie di entrambe le arterie femorali superficiali . an accurate evaluation of the arterial tree from the coeliac trunk to the foot arteries . 
in comparison with conventional angiography , mr offers important advantages in the study of elderly patients with peripheral arterial disease , enabling one to avoid the risks associated with arterial puncture , iodinated contrast material , and radiation . 
this effect increases in the mip reconstructions , in which it is not always possible to distinguish the reduced calibre of the stenotic portion of the vessel from the background signal [ 7 ]  . 
la causa principale della sottostima nei casi da noi esaminati stata data da effetti di volume parziale , anche in questo caso dovuti alla difficolt di rilevare attraverso strati sottili i ridotti calibri dei vasi stenotici [ 6 , 8 ]  . 
langio - rm si rivelata una metodica dotata di alta specificit , ma di inferiore sensibilit , quindi praticamente sovrapponibile alla dsa nella valutazione dei vasi di calibro regolare , mentre in caso di lesione steno - ostruttiva la dsa pi accurata , anche se nel nostro studio abbiamo rilevato una buona concordanza . nessuno dei pazienti ha avuto reazioni alla somministrazione della gadodiamide [ 9 ]  . 
non ci sono stati artefatti da materiale metallico in quanto un solo paziente aveva uno stent hemoban ( nitinolo ) in femorale superficiale destra che per non ha causato alterazioni del segnale . 
alcuni pazienti di questo studio , tutti arteriopatici allo stadio iii e iv di la fontaine ( tranne due ) , hanno avuto difficolt a tollerare limmobilit a causa del dolore , ma questo problema si presentato sia durante lesecuzione dellangio - rm sia durante la dsa . 
per quanto riguarda la sequenza tof , ha fornito una discreta valutazione dei vasi del piede , ma come tipo di sequenza mostra alcune limitazioni : i vasi non perfettamente perpendicolari al piano di acquisizione possono avere una bassa intensit di segnale e simulare una patologia [ 10 ] ; la pulsatilit del flusso pu produrre artefatti se non si utilizza un gating cardiaco ; il flusso retrogrado di alcuni vasi collaterali o ricostruiti pu essere anchesso saturato e mascherare di conseguenza il vero livello dellostruzione o sovrastimare la lunghezza del segmento stenotico [ 11 , 12 ]  . solo 15 dei pazienti esaminati ha potuto eseguire questo tipo di sequenza a causa dei gi citati problemi legati allimmobilit in posizione supina . 
mra proved to have higher specificity but lower sensitivity and is therefore practically identical to dsa in the evaluation of normal vessels ; however , in the case of steno - obstructive lesions , dsa is more accurate , even if we recorded a good agreement in our study . 
no patient had a reaction to gadodiamide [ 9 ]  . there were no artefacts due to metallic material , as only one patient had a hemoban stent ( nitinol ) in the right superficial femoral artery , which did not cause signal alterations . a non - negligible limitation of this examination is the immobility requested of the patient . 
some patients in our study , all but two with fontaine stage iii and iv disease , had difficulty keeping immobile because of pain , but this problem arose during both mra and dsa . as regards the tof sequence , despite providing a discrete evaluation of the foot vessels , it does suffer some limitations : vessels not perfectly perpendicular to the acquisition plane may have low signal intensity and simulate disease [ 10 ]  . flow pulsatility can produce artefacts if cardiac gating is not used . 
 the retrograde flow of some collateral or reconstructed vessels can also be saturated and disguise the true level of obstruction or overestimate the length of the stenotic segment [ 11 , 12 ]  . only 15 of our patients could be studied with this sequence because of difficulty lying motionless in the supine position . 
it does , however , have some limitations , some of which will be eliminated by technological advances with fast gradients and specific contrast agents for vascular studies whereas others will remamra may be proposed for selected indications : screening asymptomatic patients with a family history of diabetes or risk factors for atherosclerosis , patients with renal insufficiency , elderly patients , and for surveillance of bypass grafts . conclusions in our experience based on a limited number of patients , mra can be considered an alternative modality to dsa in the management of patients with steno - occlusive disease of the peripheral arteries , as it allows screening of candidates for interventional [ percutaneous angioplasty ( pta ) or stent ] or surgical procedures ( bypass graft )  . the pre - requisite for this is to optimise the injection time on the basis of each patients cardio - circulatory parameters , as also shown by westenberg et al . 
da quanto detto si evince che , nonostante la metodica si sia rivelata sicura , affidabile e sopportabile per la maggior parte dei pazienti , presenta pur sempre dei limiti , alcuni dei quali saranno eliminati dallavanzamento della tecnologia con gradienti ancora pi veloci e mezzi di contrasto dedicati allo studio vascolare , mentre altri invece resteranno invalicabili . 
tutto questo rende langio - rm un esame proponibile solo ad alcuni pazienti : screening nei pazienti asintomatici con familiarit diabetica o con fattori di rischio per aterosclerosi , insufficienza renale , pazienti anziani , controllo di bypass . conclusioni nella nostra esperienza , limitata al numero di casi da noi studiati , langio - rm pu essere considerata una metodica alternativa alla dsa nel management dei pazienti con patologia steno - ostruttiva del circolo arterioso periferico , selezionando i candidati a procedura interventistica ( pta o stent ) o chirurgica ( bypass )  . la condizione imprescindibile su cui si fonda questa affermazione lottimizzazione del tempo di iniezione ad ogni singolo paziente , in base ai suoi parametri cardio - circolatori come dimostrato anche da westenberg et al . 
bacci1 1sezione di chemioterapia , 2servizio di radiologia , dipartimento di oncologia muscoloscheletrica , istituti ortopedici rizzoli , via pupilli 1 , i - 40136 bologna , italy correspondence to : a . 
between march 1997 and december 2001 at our department of musculoskeletal oncology , 231 patients with peripheral osteosarcoma received a central venous catheter to allow infusion therapy and blood sampling . 
in ten cases ( 4.3% ) , radiology showed damage to the alveolar interstitium typical of inflammatory forms , whereas in the remaining three ( 1.3% ) it depicted nodular opacities , which required differentiation between lung metastases and septic emboli . 
the most common complications of the use of central venous catheters include infection and venous thrombosis whereas pulmonary septic emboli are rare . key words osteosarcoma pulmonary metastases pulmonary nodules differential diagnosis riassunto obiettivo . 
da marzo 1997 a dicembre 2001 , presso il nostro dipartimento di oncologia muscoloscheletrica stato inserito un catetere venoso centrale per permettere terapia infusionale e prelievi ematici a 231 pazienti affetti da osteosarcoma periferico . 
in 10 casi ( 4 , 3% ) il quadro radiologico era di impegno interstizio alveolare tipico delle forme flogistiche , mentre nei restanti 3 ( 1 , 3% ) si osservarono delle opacit nodulari che posero problemi di diagnosi differenziale fra metastasi polmonari ed emboli settici . 
le complicanze pi frequenti dellutilizzo del catetere venoso centrale sono le infezioni e le trombosi venose , mentre gli emboli settici polmonari sono rari . parole chiave osteosarcoma metastasi polmonari noduli polmonari diagnosi differenziale introduction introduzione the use of hickman , broviac and groshong indwelling central venous catheters ( cvcs ) , which remain in place for months or years , is highly important for cancer patients who require prolonged chemotherapy and support infusion therapies . 
venous catheters connected with their port to a central venous line , such as infuse - a - port or port - a - cath , have also been used with success . 
vein perforation , generally caused by the insertion of the catheter or its incorrect positioning , is fortunately an uncommon immediate or early complication . the most frequent late complications are venous thrombosis of the vessel in which the catheter is positioned and infections . 
the incidence of thrombosis varies between 12% and 74% and is higher in cancer patients [ 1 ]  . the thrombosis may be located in the vessel or in the catheter lumen ; only one - third of thromboses are symptomatic . 
a number of studies [ 24 ] have demonstrated that thrombosis is one of the main risk factors for catheter infection whereas infections occur in 15%20% of cases [ 5 , 6 ]  . septic emboli resulting from catheter infection are a wellknown but uncommon complication , even in children and adolescents [ 6 , 7 ]  . the central venous line is a very important tool for delivering chemotherapy , especially in children . 
many studies have demonstrated the usefulness and feasibility of prolonged use , up to years , of a cvc even in children , with an acceptable incidence of complications : displacement , occlusion and infection [ 710 ]  . 
the aim of this study was to highlight the problems of differential diagnosis and the possible solutions using conventional radiography and computed tomography ( ct )  . materials and methods between march 1997 and december 2001 , the division of chemotherapy of our institution treated 253 patients with adjuvant chemotherapy for primitive bone sarcomas . 
anche i cateteri venosi collegati con la propria porta ad un catetere venoso centrale tipo infuse - a - porth o port - a - cath sono impiegati con successo . entrambi i tipi di cateteri sono associati a possibili complicazioni sia precoci che tardive . 
alcuni studi [ 24 ] hanno dimostrato che la trombosi uno dei principali fattori di rischio per linfezione del catetere venoso centrale , mentre le infezioni sono una complicanza che si verifica in circa il 15%20% dei casi [ 5 , 6 ]  . 
gli emboli settici conseguenti a infezioni del catetere sono una complicanza ben conosciuta , pur se non frequente , anche nei pazienti pediatrici od adolescenti [ 6 , 7 ]  . il catetere venoso centrale uno strumento molto importante per la somministrazione della chemioterapia in particolare nei bambini . 
molti studi hanno dimostrato lutilit e la fattibilit di un uso prolungato , talora per anni , del catetere venoso centrale anche in pazienti pediatrici con una percentuale accettabile di complicanze : dislocazioni , occlusioni ed infezioni [ 710 ]  . 
lo scopo del lavoro di evidenziare le problematiche di diagnosi differenziale e le possibilit di soluzione mediante radiologia tradizionale e tomografia computerizzata ( tc )  . materiali e metodi dal marzo 1997 al dicembre 2001 , presso lunit operativa di chemioterapia del nostro istituto sono stati trattati con chemioterapia adiuvante per osteosarcoma primitivo 253 pazienti ; 34 di questi pazienti presentavano metastasi alla diagnosi : 31 al polmone e 3 in altri segmenti ossei ; in 219 pazienti la lesione era localizzata . 
a 231 pazienti fu inserito un catetere venoso centrale : 228 cvc tipo broviac e 3 port - a - cath ; 22 pazienti tutti adulti rifiutarono il posizionamento del catetere . 
a second radiological assessment of the primary cancer with ct and mri , and chest ct was performed at the end of preoperative chemotherapy ( lasting four to six cycles , depending on the protocol ) , and before surgery . 
given the prolonged anticancer treatment typical of osteosarcoma ( 3643 weeks , depending on the protocol ) , conventional chest radiography was performed every 2 months to exclude the development of metastasis . in order to prevent catheter occlusions , 5 cc of heparin solution ( 20 cc of saline plus 3 cc of sodium heparin equal to 5 , 000 iu diluted in 100 cc of saline ) was injected into the central venous line every other day . 
patients with neutropoenia ( neutrophils < 500 / mm3 ) received granulocyte colony stimulating factors ( gcsf ) , together with a broad - spectrum antibiotic such as trimethoprim , sulfamethoxazole or ceftriaxone , at a dose of 5 g / kg per day until the white blood cell count ( wbc ) reached a concentration of at least 10 , 000 / mm3 . 
he received neoadjuvant chemotherapy from july 1998 to june 1999 after a broviac central venous catheter had been positioned in the left subclavian veone week after the last chemotherapy cycle ( cisplatin ) , the patient developed chills and fever ( 39c ) while at home . 
central and peripheral blood cultures were performed , both of which were positive for staphylococcus aureus and , on the basis of the antibiogram , antibiotic therapy with 200 mg / day teicoplanin was started , leading to remission of the fever . 
three weeks , later the patient was admitted for removal of the cvc and final staging with lung ct . the material sampled from the catheter after removal showed colonies of s . 
ct , performed on the same day ( 20 july 1999 ) , revealed the presence of a nodular opacity in the posterior segment of the lower right lobe that was suggestive of pulmonary metastasis from osteosarcoma teteri , usati sia per la somministrazione della chemioterapia che per prelievi ematici , furono posizionati prima dellinizio dei cicli di chemioterapia , previo consenso informato . tutti i pazienti ricevettero chemioterapia secondo i protocolli in uso al momento , ior / os - n4 o ior / os - n5 , impiegati dal 1997 al 2001 con i seguenti farmaci : adriamicina , cisplatino , ifosfamide , pi metotrexate nei pazienti al di sotto dei 40 anni ( precedentemente riportati in dettaglio [ 11 , 12 ] )  . la stadiazione radiologica di base comprendeva tc e rm del tumore primitivo osseo , scintigrafia ossea e tc del torace per escludere lesioni sostitutive secondarie polmonari . una seconda valutazione radiologica con tc ed rm del tumore primitivo e tc del torace stata eseguita al termine della chemioterapia preoperatoria ( durata 46 cicli a seconda del protocollo ) , prima dellintervento chirurgico . 
dato il prolungato trattamento antiblastico tipico dellosteosarcoma ( da 36 a 43 settimane a seconda del protocollo ) uno studio radiologico tradizionale del torace veniva eseguito ogni 2 mesi per escludere la comparsa di metastasi . a scopo profilattico , per evitare occlusioni del catetere , a giorni alterni si iniettavano nel catetere venoso centrale 5 cc di soluzione eparinata ( 20 cc di soluzione fisiologica pi 3 cc di eparina sodica pari a 5000 ui diluiti in 100 cc di soluzione fisiologica )  . 
in caso di neutropenia ( neutrofili inferiori o uguali a 500 / mm3 ) fattori stimolanti la crescita di colonie ( gcsf ) , congiuntamente ad un antibiotico ad ampio spettro tipo trimethoprim , sulfametoxazolo o cefriaxone , venivano somministrati ai pazienti alla dose di 5g / kg / die fino a che la concentrazione dei globuli bianchi ( wbc ) non raggiungeva il valore di 10000 / mm3 . tredici pazienti su 231 ( 5 , 6% ) svilupparono uninfezione del catetere venoso centrale . 
in 10 pazienti ( 4 , 3% ) lindagine radiologica tradizionale e / o la tc del torace evidenziarono un impegno interstizio - alveolare tipico delle forme flogistiche e nei restanti 3 ( 1 , 3% ) opacit nodulari , alcune delle quali a margini regolari che posero problemi di diagnosi differenziale con lesioni metastatiche . caso 1 maschio , 7 anni , con osteosarcoma osteoblastico localizzato allomero destro . 
una settimana dopo lultimo ciclo di chemioterapia ( cisplatino ) , il paziente ebbe , a domicilio , un episodio di febbre ( 39c ) con brividi ; esegu due emocolture : una da prelievo centrale e una da prelievo periferico . 
although the patient was asymptomatic , the recent cvc infection was thought to have caused the spread of a septic embolus to the lung , so antibiotic therapy with teicoplanin ( 200 mg / day ) was administered for a further 15 days . 
shortly after the end of the last cycle of postoperative chemotherapy , he developed a fever ( 40c ) and was treated at home with a broad - spectrum antibiotic that failed to resolve the fever . two - view chest radiography performed at the patients home was negative . 
on june 15 , the patient was admitted , and the cultures of material sampled from the central venous line and a peripheral vein both revealed the presence of pseudomonas fluorescens sensitive to piperacillthe central venous line was removed . 
poco dopo il termine dellultimo ciclo di chemioterapia postoperatoria , ebbe febbre ( 40c ) e inizi a domicilio terapia antibiotica a largo spettro che non ne determin la remissione . il radiogramma tradizionale del torace eseguito in due proiezioni al domicilio del paziente era negativo . 
il 15 giugno il paziente fu ricoverato e le emocolture del materiale prelevato dal catetere centrale a da vena periferica evidenziarono entrambe la presenza di psudomonas fluorescens sensibile alla piperacillina . 
nello stesso giorno il paziente esegu , come da protocollo , tc del torace di fine cura , che evidenzi la presenza di tre opacit nodulari nel polmone destro : la prima nel segfig . 
1a , b the first computed tomography ( ct ) study shows a nodular opacity measuring 15 mm in diameter with slight irregular margins in the posterobasal segment of the right lower lobe ( a )  . 
1a , b il primo esame evidenzia una opacit nodulare di 15 mm di diametro a margini leggermente irregolari nel segmento posterobasale del lobo inferiore di destra ( a )  . 
2a - d the first computed tomography ( ct ) study shows three subpleural nodular opacities ( arrows ) , two measuring a few millimetres : the first has paracardiac location in the middle lobe and no feeding vessel sign ; the second is in the laterobasal segment of the right lower lobe ( a ) ; the third is larger and located in the posterobasal segment of the right lower lobe ( b )  . 
2a - d il primo esame tc evidenzia tre opacit nodulari subpleuriche ( frecce ) di cui due millimetriche : la prima localizzata in sede paracardiaca a livello del lobo medio senza il segno del vaso che nutre , la seconda nel segmento laterobasale del lobo inferiore destro ( a ) , la terza opacit , di dimensioni superiori , nel segmento posterobasale del lobo inferiore di destra ( b )  . 
dopo somministrazione di terapia specifica , il secondo esame tc eseguito tre mesi dopo , non evidenzia pi alcuna lesione ( c , d )  . case 3 a 17 - year - old girl with fibroblastic osteosarcoma of the right femur . 
chest radiography demonstrated two nodular opacities in the middle third of the right lung field , both of which where strongly suspicious for metastases . chest ct performed immediately confirmed the presence of the lesions , which were subpleural and with slightly irregular margins , one in the anterior segment and the other in the posterior segment of the lower lobe . 
chemioterapia neoadiuvante dal dicembre 2000 attraverso un catetere centrale tipo broviac . terminato il nono ciclo di chemioterapia , la paziente ebbe un episodio di neutropenia febbrile risoltosi con somministrazione di terapia antibiotica a largo spettro e di fattori stimolanti la crescita . 
la tc del torace eseguita immediatamente conferm la presenza delle due lesioni entrambe subpleuriche e con margini lievemente irregolari , una nel segmento anteriore e laltra nel segmento posteriore del lobo inferiore . 
la paziente a tuttoggi viva , senza lesioni sostitutive . discussione losteosarcoma un tumore osseo estremamente maligno tipico dei bambini e degli adolescenti con un picco tra i 15 ai 25 anni [ 13 ]  . 
il polmone rappresenta , infatti , la pi frequente sede ( 90% di tutte le sedi di metastasi ) di metastasizzazione di questo tumore alla diagnosi e durante il follow - up [ 17 ]  . 
3a - c the first computed tomography ( ct ) study shows two subpleural nodular opacities ( arrows ) : the first in the apical segment of the right lower lobe ; the second , clearly inflammatory , in the lateral segment of the middle lobe ( a )  . 
the second ct study performed after 20 days shows disappearance of the two opacities , with moderate residual subpleural fibrosis in the middle lobe and the presence of a new opacity with irregular and fluffy margins contiguous with a pulmonary vessel in the posterolateral segment of the right lower lobe ( b )  . 
3a - c il primo esame tc evidenzia due opacit nodulari subpleuriche ( frecce ) : la prima nel segmento apicale del lobo inferiore destro , la seconda chiaramente flogistica nel segmento laterale del lobo medio ( a )  . 
il secondo esame tc , eseguito a 20 giorni di distanza , mostra la scomparsa delle due opacit con modesto esito fibrotico subpleurico nel lobo medio e la presenza di unaltra opacit , a contorni irregolari e sfrangiati in continuit con un vaso polmonare nel segmento posterolaterale del lobo inferiore destro ( b )  . 
the lung is often the first site of metastatic spread of many solid tumours [ 16 ] , and this is particularly true for osteosarcoma . the lung is the most frequent site of metastasis ( 90% of all metastasis sites ) of this tumour at presentation and during follow - up [ 17 ]  . osteosarcoma of the extremities tends to metastasize within the first 5 years of diagnosis in 30%40% of cases [ 13 ]  . 
early diagnosis of such lesions may in some cases dramatically increase the survival of patients after appropriate treatment : thoracic surgery and / or chemotherapy . all osteosarcoma protocols require ct scans at baseline and after treatment and conventional chest radiography every 2 months during chemotherapy . 
despite not having the same reliability as ct in detecting metastasis , conventional chest radiography in two views anteroposterior ( ap ) and laterolateral ( ll ) is just as effective in identifying metastatic lesions with a diameter of 1 cm or more , it is easy to perform and it is inexpensive . 
in addition , entrance dose and absorbed dose are much lower than in ct : 1.9 mgy / msi versus 30 mgy / msi , ctdiw / 650 mgy / msi - cm dlp . development of lung metastases during treatment is a rare but possible occurrence . 
mediastinal lymph nodes are rarely involved [ 13 ]  . oncological patients who are free of disease may , however , develop pulmonary nodules of varying size , shape and location , as has been reported by many studies [ 1824 ]  . 
almost all of cases reported were treated either with drugs with minimal bone marrow toxicity and high pulmonary toxicity or drugs and radiotherapy [ 19 ]  . many underwent segmental or lobar resection . 
histology revealed not only metastatic nodules but also the presence of nodular lesions of other natures : nodular nonspecific intersitial pneumonia or fibrosis in patients with peripheral infusion of drugs with high pulmonary toxicity , such as bleomycin [ 2024 ] or nodular lesions caused by infection by opportunistic organisms ( cryptococcus neoformans , aspergillus fumigatus , or others ) in patients treated with radiotherapy who became immunocompromised . infectious processes may also include septic emboli developing inside the cvc or migrating there from other sites . 
the finding of areas of pulmonary consolidation of varying shape on ct scans of cancer patients represents a challenge , and the differential diagnosis is one of the radiologists routine tasks . 
 lung nodules identified in our three patients were both subpleural and parenchymal ; they had similar density but ri metastatici osservati su un radiogramma del torace e / o una tc del torace in pazienti con tumore primitivo extrapolmonare da considerarsi segno prognostico infausto . 
una diagnosi precoce di lesioni sostitutive pu talora dare lopportunit di una lunga sopravvivenza al paziente dopo adeguata terapia : chirurgia toracica e / o chemioterapia . tutti i protocolli per gli osteosarcomi richiedono una tc di base allinizio del trattamento , unaltra alla fine e radiogrammi tradizionali del torace ogni due mesi durante il lungo periodo della chemioterapia . 
il radiogramma tradizionale del torace in due proiezioni ( p - a e l - l ) pur non avendo la stessa affidabilit della tc nel rilevare metastasi egualmente efficace nellevidenziare lesioni sostitutive di diametro pari o superiore al centimetro , di facile esecuzione , poco costoso , inoltre la dose allingresso / dose assorbita sono di molto inferiori a quelle di una tc : 1 , 9 mgy / msi versus 30 mgy / msi , ctdiw / 650 mgy / msi - cm dlp . 
i linfonodi mediastinici sono raramente coinvolti [ 13 ]  . tuttavia in pazienti neoplastici liberi da malattia possibile osservare la comparsa di noduli polmonari di dimensioni , forma e sede variabile ; tale comparsa stata descritta in molti lavori [ 1824 ]  . 
quasi tutti questi pazienti sono stati trattati o solo con farmaci a minima tossicit midollare ed ad alta tossicit polmonare o con farmaci e radioterapia [ 19 ]  . molti di questi pazienti furono sottoposti a resezione chirurgica segmentaria o lobare . 
lesame istologico rivel non solo la presenza di noduli metastatici , ma anche la presenza di lesioni nodulari di differente natura : polmoniti interstiziali nodulari aspecifiche e fibrosi polmonare interstiziale nodulare in pazienti con infusione periferica di farmaci ad alta tossicit polmonare come la bleomicina [ 2024 ] o lesioni nodulari di natura infettiva provocate da organismi opportunisti ( criptococcus neoformans , aspergillus fumigatus o altri ) in pazienti precedentemente trattati con radioterapia e divenuti ospiti immunocompromessi . 
i sintomi clidifferent shape , being rounded or lobulated but never reticular ; their margins were smooth and regular or fluffy and irregular , but never hazy ; size varied between 3 mm and 25 mthe two patients in whom the catheter was removed immediately and s . 
lung metastases from any primary cancer are well - marginated nodular opacities that are at times very calcific ( especially in osteosarcoma ) ; they tend to have peripheral location but have no predilection for the upper , middle or lower lobes [ 25 ]  . in our three cases , all pulmonary nodules simulating metastases appeared during chemotherapy in patients with broviac cvcs positioned in the subclavian vein for the delivery of chemotherapy , transfusions or blood sampling . 
none of the three patients had symptoms of pulmonary embolism . three cases of pulmonary septic embolism out of 231 patients with cvc is indeed a small percentage ( 1.3% ) , in agreement with other studies [ 6 ]  . 
pulmonary infection could have been the cause rather than the consequence of cvc infection but , given that culture of material retrieved from the distal lumen of the catheter was positive in two out of three cases , the cause of the haematopulmonary infection was far more likely to have been the cvc . the three cases described demonstrate that cvc infection can cause pulmonary septic embolism with variable imaging features . 
le metastasi polmonari , qualunque sia lorigine del tumore primitivo , sono opacit nodulari a margini netti , ben definiti , talora molto calcifiche ( specialmente nellosteosarcoma ) ; sono generalmente localizzate perifericamente ma senza predilezione di sede tra lobo superiore , medio od inferiore [ 25 ]  . nei nostri tre pazienti tutti i noduli polmonari che simulavano metastasi apparvero in corso di chemioterapia in pazienti con catetere venoso centrale tipo broviac inserito in una vena succlavia ed utilizzato per chemioterapia , trasfusioni o prelievo ematico . 
tre casi di emboli settici polmonari su 231 pazienti portatori di cvc una piccola percentuale ( 1 , 3% ) , in accordo coi risultati di altri studi [ 6 ]  . 
la flogosi polmonare potrebbe essere stata la causa e non la conseguenza dellinfezione del catetere venoso centrale , ma dal momento che lesame colturale effettuato sul materiale prelevato dal lume distale del catetere fu positivo in due dei tre casi , sembr estremamente pi probabile che la causa dellinfezione emato - polmonare fosse il catetere venoso centrale . 
nondimeno , aree polmonari di aumentata densit , in soggetti neutropenici e portatori di catetere venoso centrale deve suggerire , tra le altre ipotesi diagnostiche , la possibilit di unembolizzazione settica nodulare secondaria alla presenza ed allinfezione del catetere venoso centrale . 
in addition , the specificity of ct in evaluating the nature of a pulmonary nodule , especially when small , is still limited , and only biopsy or nodule excision are able to reveal the benign or malignant nature of the nodule through histological examination whereas evolution of the radiological finding can only guide the diagnosis between benign or malignant nodular opacities [ 26 ]  . conclusions correct diagnosis of a pulmonary opacity is fundamental for the prognosis and choice of treatment ( surgical ) in a patient with a doubtful lung lesion . 
 in the presence of a cvc , any change in shape ( rounded , lobulated , irregular , fluffy ) in relationship with a vessel ( contiguous , distal ) and in location ( apical , medial , basal ) of an area of lung consolidation seen on serial ct scans , both during and after chemotherapy , is an important clue for pulmonary septic embolism secondary to cvc infection regardless of antibiotic treatment . catheter replacement , central and peripheral blood cultures , administration of specific antibiotic treatment based on blood cultures and ct follow - up of nodular opacities must always be given priority with respect to surgical treatment . mento che possono essere normali o non specifici [ 22 ] come pure laspetto radiologico di noduli polmonari a differente eziologia . 
inoltre la specificit della tc , nella valutazione di natura , benigna o maligna , di un nodulo polmonare , specialmente se questo di piccole dimensioni , ancora limitata e solo la biopsia o lasportazione del nodulo sono in grado di rivelare la vera natura della lesione permettendone lesame istologico , mentre levoluzione del reperto radiologico pu unicamente orientare la diagnosi tra opacit nodulare benigna o maligna [ 26 ]  . conclusioni la corretta diagnosi di una opacit polmonare di vitale importanza per la prognosi ed il differente trattamento terapeutico ( chirurgico ) nel caso di paziente con lesione polmonare dubbia per metastasi . 
simonetti1 1dipartimento di diagnostica per immagini e radiologia interventistica , universit degli studi di roma tor vergata , roma , italy 2sezione di medicina nucleare , universit degli studi di roma tor vergata , roma , italy correspondence to : g . 
sergiacomi , policlinico tor vergata , viale oxford 81 , i - 00133 roma , italy , tel . : + 39 - 06 - 20902372 , fax + 39 - 06 - 20902404 , e - mail : sergiacomigianluigi@tin.it received : 22 march 2005 / accepted : 23 august 2005 / published online : 3 march 2006 abstract purpose . 
the purpose of this study was to evaluate efficacy of multislice computed tomography ( msct ) and single photon emission computed tomography ( spect ) - ct with tc - 99m sestamibi in the assessment of solitary pulmonary nodules of uncertain significance . 
between september 2003 and august 2004 , 23 patients with a solitary pulmonary nodule detected on ct underwent spect - ct using tc - 99m sestamibi as a radiotracer . nodules with positive scintigraphy were immediately subjected to biopsy or surgical resection . 
scopo del nostro studio valutare lefficacia diagnostica dellutilizzo della tomografia computerizzata multistrato ( tcms ) e della spect - tc con tc - 99m sestamibi nella gestione di lesioni nodulari polmonari di incerto significato . 
ventitre pazienti , nel periodo tra settembre 2003 e agosto 2004 , con un nodulo polmonare solitario sospetto , hanno eseguito una tcms del torace e successiva spect - tc utilizzando come radiotracciante il tc - 99m sestamibi . 
ci potrebbe permettere di anticipare la tipizzazione istologica e leventuale trattamento chirurgico in pazienti con nps riscontrato allesame tc del torace . parole chiave tc multistrato nodulo polmonare sestamibi spect g . 
this technique can now perform thinslice imaging of the lung parenchyma , and it employs software providing information on size , volume , density and contrast enhancement of pulmonary nodules as well as calculating automatically and accurately any change in size by comparing studies conducted at different times . a few nodules can be defined as probably benign ( presence of fat or benign calcification ) and not requiring further investigation ; others are highly suspicious for malignancy ( spiculated margins , malignant calcification , diameter > 2 cm ) and require immediate surgical excision or biopsy [ 1 ]  . 
the majority of pulmonary nodules ( 70% ) , however , cannot be characterised with certainty , even after a ct scan . encouraging results in lung cancer diagnosis have been obtained with single photon emission computed tomography ( spect ) using 99mtc - hexakis - 2 - methoxy - isobutyl - isonitrile ( sestamibi ) , a lipophilic cation used in myocardial perfusion scintigraphy [ 24 ]  . this technique currently makes use of new devices equipped with an x - ray tube - detector system for ct scans . scintigraphy is carried out using a hybrid g - camera allowing acquisition of spect and ct images during the same session . 
preliminary results of the clinical application of this new diagnostic tool in the study of different neoplasms , including thoracic applications , have been very interesting [ 5 , 6 ]  . 
this allows for a more effective and accurate preclinical diagnosis of disease , given that the early diagnosis of lung cancer is still the only means to improve patient survival [ 7 , 8 ]  . 
our aim was to correlate the results of msct and sestamibi spect - ct in the early diagnosis of lung cancer and to assess their concordance with histological examination . materials and methods between september 2003 and august 2004 , we selected 23 patients ( 17 men and six women ) aged between 40 and 82 years ( mean age 63.2 ) with an spn ranging from 0.8 cm to 2 cm in size ( mean size 1.3 cm ) discovered at ct performed to investigate a chest radiography finding or as a diagnostic examination for another condition . our study excluded nodules with tomodensitometric characteristics highly suggestive of either benign or malignant disease . 
the examinations were conducted with a lightspeed 16 msct scanner ( ge medical systems , milwaukee , wi , usa ) capable of obtaining slice thicknesses between 10 mm and 0.625 mthe examination was performed durla diagnostica per immagini ha raggiunto , grazie agli imponenti progressi tecnologici , importanti risultati nella valutazione del nodulo polmonare solitario ( nps ) con lausilio della tomografia computerizzata multistrato ( tcms )  . 
attualmente tale tecnica in grado di poter eseguire esami sul parenchima polmonare a strato sottile e si avvale di software in grado di fornire informazioni su dimensioni , volume , densit e contrast enhancement delle nodularit polmonari nonch di calcolarne automaticamente in modo accurato le modificazioni dimensionali , comparando esami eseguiti in tempi diversi . 
alcuni noduli possono essere definiti come benigni con alta probabilit ( presenza di grasso o calcificazioni benigne ) e non richiedere ulteriori approfondimenti ; altri risultano altamente sospetti per malignit ( margini spiculati , calcificazioni maligne , diametro superiore a 2 cm ) e richiedono immediata escissione chirurgica o biopsia [ 1 ]  . 
ciononostante la maggior parte dei noduli polmonari ( 70% ) non sono caratterizzabili con certezza anche dopo un esame tc . incoraggianti risultati nella diagnostica della neoplasia polmonare sono stati ottenuti con la tomografia computerizzata ad emissione di singolo fotone ( spect ) con lutilizzo di tc - 99m - hexakis - 2 - metossi - isobutil - isonitrile ( sestamibi ) , un catione lipofilico in uso nella diagnostica scintigrafica miocardica perfusionale [ 24 ]  . 
i risultati preliminari dellimpiego clinico di questa nuova apparecchiatura diagnostica nello studio di diverse neoplasie , comprendendo alcune applicazioni anche a livello toracico , si sono dimostrati molto interessanti [ 5 , 6 ]  . 
tutto ci ci consente di fare una diagnosi pre - clinica di malattia molto pi efficace e accurata , dato che la diagnosi precoce delle neoplasie del polmone rimane lunico mezzo per aumentare la sopravvivenza dei pazienti affetti [ 7 , 8 ]  . il nostro studio incentrato sulla caratterizzazione di lesioni nodulari solitarie di incerto significato rinvenute allesame radiografico del torace , o incidentalmente durante lesecuzione di esami tc , integrando lesame scintigrafico con le informazioni fornite dalla tcms . 
lo scopo correlare i risultati forniti dalla tcms e dalla spect - tc con sestamibi nella diagnosi precoce del cancro polmonare e valutarne la concordanza con lesame istologico . materiali e metodi nel periodo compreso tra settembre 2003 e agosto 2004 sono stati selezionati 23 pazienti di cui 17 uomini e 6 donne di et compresa tra i 40 e 82 anni ( et media 63 , 2 anni ) , con un nps di dimensioni comprese fra 0 , 8 e 2 cm ( dimensioni medie 1 , 3 cm ) , individuato mediante esame tc , eseguito come approfondimento diagnostico di una precedente radiografia del torace o come accertamento diagnostico per altra patologia . 
images were processed on an advantage w 4.1 workstation supplied by the same company using multiplanar reconstructions ( mpr ) , volume rendering ( vr ) and automatic segmentation software ( ala ) , which provides an accurate assessment of size , shape and density once the operator has selected the region of interest and performed three - dimensional ( 3d ) segmentation with removal of adjacent structures . volume and morphology data obtained at the first measurement were compared with those of the follow - up examinations . 
risk assessment was not performed on 2d criteria , such as changes in lesion diameter , but on 3d criteria , so that when the volume doubles , the lesion diameter actually increases by only 26% . all patients then underwent ( within 5 days ) lung scintigraphy with positive indicator using a vg millennium ( ge medical systems ) dual - head gamma camera equipped with an x - ray tube - detector system similar to a third - generation ct [ 9 ]  . 
this hybrid system has shown to improve the diagnostic accuracy of a large number of spect examinations [ 5 ]  . twenty minutes after the iv administration of about 740 mbq of tc - 99m sestamibi , spect - ct of the chest was performed . 
after prefiltering with a metz filter , the spect images were reconstructed in a ramp filter in order to produce transaxial sections from which coronal and sagittal images were derived . patients with positive nodules at scintigraphy underwent invasive procedures for histological characterisation ( ctguided or bronchoscopic biopsy , video - assisted thoracoscopic excision )  . patients with negative nodules at scintigraphy underwent repeat ct after 34 months with reassessment of volume by means of ala software and subsequent histological characterization ( ct - guided or bronchoscopic biopsy , video - assisted thoracoscopic excision )  . in these cases , follow - up ct was always performed with the same acquisition parameters as the first examinacon caratteristiche tomodensitometriche altamente probanti per benignit o malignit . 
gli esami sono stati condotti con una tc multistrato light - speed 16 ( ge medical systems , milwakee , wi , usa ) in grado di ottenere spessori di strato variabili da 10 mm a 0 , 625 mlesame stato effettuato in ununica apnea prima e dopo somministrazione di mezzo di contrasto organo - iodato non ionico ( 80100 ml , flusso di 34 ml / s , ritardo di 2530 s dallinizio della somministrazione ) previo consenso informato del paziente , con i seguenti parametri tecnici di acquisizione : spessore di strato pari a 5 mm ( retroricostruibile fino a 0 , 625 mm ) , velocit di avanzamento 11 , 25 mm / rotazione , tempo di rotazione di 0 , 5 s , tempo complessivo di acquisizione pari a 1215 s , 120140 kv , 200240 ma . 
le immagini sono state successivamente rielaborate su una workstation advantage w 4.1 fornita dalla stessa ditta mediante ricostruzioni multiplanari ( mpr ) , volumetriche ( vr ) e software di segmentazione automatica ( ala ) che permette , selezionando la regione di interesse ed effettuando una segmentazione tridimensionale con eliminazione delle strutture adiacenti , di ottenere una corretta valutazione di dimensioni , forma e densitometria . 
i valori ottenuti con le prime rilevazioni sono stati confrontati con quelli dei follow - up successivi , utilizzando come elemento di comparazione il criterio volumetrico accanto a quello morfologico . 
laccertamento del rischio non viene effettuato sulla base di elementi bidimensionali , come le modificazioni del diametro della lesione , ma si fonda su criteri tridimensionali , cos quando il volume raddoppia , il suo diametro aumenta in realt solo del 26% . tutti i pazienti sono stati successivamente ( entro 5 giorni ) sottoposti a tomoscintigrafia polmonare con indicatore positivo , impiegando una gamma camera a doppia testata vg millenium ( ge medical systems , milwakee , wi , usa ) equipaggiata con un complesso tubo radiogeno - detettori analogo ad una tc di iii generazione [ 9 ]  . 
per la spect sono state acquisite 120 immagini planari ( una ogni 3 ) della durata di 25 s ognuna su un arco di rotazione di 360 , utilizzando una matrice 128x128 . 
subito dopo , senza muovere il paziente dal lettino della gamma camera , stata effettuata una tc a bassa risoluzione del torace , su un campo di vista di 40 cm , esattamente uguale a quello acquisito dalla spect . 
i parametri utilizzati da questa tc sono fissi e non modificabili dalloperatore : spessore di strato di 1 cm , tempo di acquisizione pari a circa 14 s per sezione , 140 kv e 2 , 5 ma . 
le immagini spect e tc ottenute sono state elaborate in una workstation dedicata ( integra , ge medical system ) che permette in maniera rapida di ottenere una fusione precisa delle corrispondenti sezioni dei due studi , funzionale ( spect ) ed anatomico ( tc )  . 
dopo una pre - filtrazione con un filtro di metz , le immagini spect sono state ricostruite in un filtro a rampa per produrre sezioni transassiali , dalle quali sono state poi ottenute le immagini coronali e sagittali . 
ct computed tomography , spect single photon emission computed tomography patient size ( spn ) , cm site ( spn ) spect histology age , years a + / - , indeterminate ; - , doubt about its benignity ; + , doubt about its malignancy r , right ; l , left lower r lobe lingula lower r lobe lower r lobe upper l lobe upper l lobe upper l lobe lower r lobe l hilar lower r lobe lingula lower r lobe lower r lobe lower r lobe upper l lobe upper r lobe upper r lobe middle lobe upper l lobe upper r lobe upper r lobe middle lobe lower l lobe negative negative negative negative positive negative positive positive positive positive negative positive positive negative positive positive negative negative negative positive negative positive negative fibrous lesion adenocarcinoma fibrous lesion fibrous lesion adenocarcinoma fibrous lesion adenocarcinoma metastasis adenocarcinoma undifferentiated carcinoma fibrous lesion gi granuloma squamous cell carcinoma hamartoma adenocarcinoma adenocarcinoma hamartoma hamartoma granuloma adenocarcinoma granuloma adenocarcinoma granuloma tabella 1 caratteristiche riassuntive dei 23 pazienti con nodulo polmonare solitario : dato radiologico , scintigrafico ed istopatologico paziente dimensioni ( nps ) , cm sede ( nps ) aspetto tca spect istologia et , anni 1 , 5 0 , 8 1 , 1 1 , 0 1 , 5 1 , 5 1 , 0 0 , 9 1 , 2 2 , 0 1 , 0 1 , 2 1 , 4 1 , 3 2 , 0 1 , 5 1 , 4 2 , 0 1 , 0 1 , 1 1 , 1 0 , 9 1 , 3 a + / - , indeterminato ; - , dubbio sulla benignit ; + , dubbio sulla malignit dx , destro ; sn , sinistro lobo inf . 
the smallest lesion ( 0.8 cm ) , this adenocarcinoma , measured 1 cm at ct followup after 3 months , with a significant increase in volume and therefore suspicious for malignancy . table 1 summarises the characteristics of the 23 patients in our study . 
ct indicators of suspected benignity are well - defined contour , nonspiculated margins , fat tissue or calcification in more than 10% of the nodule and contrast enhancement < 20 hu . 
ct indicators of suspected malignancy are blurred contour , spiculated margins , eccentric microcalcifications and contrast enhancement > 20 hu . nodules were considered doubtful as to benignity or malignancy when they showed a prevalence of no more than two criteria whereas they were considered indeterminate when they displayed no prevalence of any of the classification criteria . on the basis of the correlation between the scintigraphic and histological results , the sestamibi spect - ct scan had 90.9 % sensitivity ( se ) , 91.6 % specificity ( sp ) , diagnostic accuracy of 91.3 % with positive predictive value ( ppv ) of 90.9 % and negative predictive value ( npv ) of 91.6 % ( table 2 )  . in evaluating the scintigraphic scan , the simultaneous ct acquisition was always decisive in identifying nodular lesions with no uptake and contributed to the differential diagnosis in nodules with uptake located in perihepatic sites . spect acquisition alone without the aid of the anatomical ct images was decisive in all enhancing lung lesions other than those with perihepatic distribution . 
the ct scan with volumetric evaluation after 34 months showed absolute concordance with the histological findings ( table 3 )  . i pazienti portatori di noduli non captanti alla tomoscintigrafia sono stati sottoposti ad un nuovo controllo tc a distanza di 34 mesi con rivalutazione volumetrica mediante software ala e successiva tipizzazione istologica ( biopsia tc guidata o broncoscopica , escissione videotoracoscopica )  . 
in questi casi lesame tc di controllo stato sempre eseguito rispettando gli stessi parametri di acquisizione dellesame precedente ( kv , ma , spessore di strato , intervallo di ricostruzione , ecc ) al fine di ottenere una comparazione attendibile . 
tutti i pazienti , anche quelli portatori di lesioni non captanti rivalutati a 34 mesi , hanno optato , previo consenso informato , per la definitiva caratterizzazione istologica piuttosto che sottoporsi ad ulteriori controlli per un lungo periodo di tempo . risultati dei 23 noduli polmonari solitari rinvenuti allesame tc , 11 sono risultati positivi allesame scintigrafico , 12 sono risultati negativi . 
questultimo , risultata la lesione pi piccola ( 0 , 8 cm ) della nostra casistica , al successivo controllo tc a 3 mesi , presentava dimensioni pari a 1 cm , con un incremento volumetrico significativo e pertanto sospetto per malignit . 
dopo lasportazione chirurgica il referto istologico ha confermato la natura maligna della lesione ( carcinoma anaplastico )  . localizzata nel lobo diagnostic investigation based on standard chest radiography and sputum cytology failed to deliver the expected results in reducing mortality [ 1113 ]  . over the last few years , the concept of early diagnosis of lung cancer has been reconsidered as a following the encouraging results obtained with multidetector computed tomography ( mdct ) [ 8 , 14 , 15 ] and the use complementary nuclear medicine methods . 
indici tc di sospetta benignit sono la presenza di limiti ben definiti , margini non spiculati , presenza di tessuto adiposo o metaplasia calcifica in pi del 10% del nodulo , un contrast enhancement < 20 uh . 
abbiamo classificato le formazioni nodulari come dubbie per benignit o malignit quando presentavano una prevalenza di non oltre 2 criteri , mentre quelle definibili come indeterminate quando non era possibile evidenziare una prevalenza dei criteri classificativi utilizzati . 
extremely rapidly growing masses are probably inflammatory while slowgrowing masses fall under the category of so - called pseudodiseases , a term indicating diseases that never progress over a lifetime either because they do not increase in size and may in fact regress naturally ( type i pseudodiseases ) or bepari a 91 , 6% ( tabella 2 )  . 
la consensuale acquisizione tc nella valutazione dellesame scintigrafico risultata sempre determinante nellindividuazione delle lesioni nodulari non captanti ed ha orientato verso una diagnosi differenziale nei casi di nodularit captanti localizzate in sede peri - epatica . la sola registrazione spect non adiuvata dallimmagine anatomica tc risultata determinante in tutte le lesioni polmonari captanti a distribuzione diversa da quella peri - epatica . 
lo studio tc , mediante valutazione volumetrica a 34 mesi , ha presentato una concordanza assoluta con il dato istologico ( tabella 3 )  . discussione il convenzionale esame radiografico del torace , primo ed essenziale momento diagnostico per tutta la patologia polmonare , non si dimostrato sufficientemente sensibile e specifico per assicurare una diagnosi precoce [ 10 ]  . 
in passato , esami diagnostici basati sulla radiografia standard del torace e sullesame citologico dellescreato non hanno dato i risultati sperati nella riduzione della mortalit [ 1113 ]  . negli ultimi anni si sta assistendo ad una profonda revisione del concetto di diagnosi precoce del cancro del polmone grazie agli incoraggianti risultati ottenuti con la tomografia computerizzata multistrato [ 8 , 14 , 15 ] , ed a tecniche complementari di medicina nucleare . 
nella differenziazione tra lesioni benigne e maligne in tc sono stati individuati criteri qualitativi , basati sulla morfologia del nodulo e quantitativi , rappresentati dalla densit , caratteristiche di enhancement dopo somministrazione di mdc organoiodato e dalla velocit di crescita della lesione nei casi sottoposti a controllo . 
in particolare lassenza di crescita in un periodo di almeno 2 anni un indicatore attendibile di benignit [ 16 ]  . luso del tempo di raddoppio , che per le lesioni sferiche definito come un incremento del 26% del diametro , si basa sullosservazione che le lesioni benigne raddoppiano il proprio volume in un tempo inferiore a 30 gg e superiore a 450 gg . 
infatti , le formazioni a rapidissimo accrescimento sono di probabile significato infiammatorio , mentre quelle a lenta crescita rientrano nel novero della cos detta pseudomalattia ove con tale termine si intenda una malattia che non progredir mai in senso negativo nel corso della vita , o perch non aumenta mai di dimensioni , potendo regredire naturalmente ( pseudomalattia di tipo i ) o perch progredisce cos lentamente che non si svilupperanno mai i sintomi nel corso della vita ( pseudomalattia di tipo ii ) [ 17 ]  . 
un nps che cresca ad una velocit tale che il suo tempo di raddoppio volumetrico previsto si collochi tra i 30 ed i 450 gg deve essere sottoposto ad ulteriori accertamenti in quanto sospetto per malignit . 
the mechanism through which the uptake occurs in cancer cells is still uncertain , and some studies [ 19 ] have suggested a role for the negative potential of cell and mitochondrial membranes , passive diffusion , and the lipophilic nature of methoxyisobutyl isonitrile ( mibi )  . 
in agreement with the literature [ 21 , 22 ] , in all cases examined and subsequently subjected to histopathology , the use of spect with tc - 99m sestamibi often provided support for radiological suspicion , but in selected highly suspicious cases , it prompted surgical excision rather than follow - up . 
the results suggest , consistent with the literature data [ 21 , 22 ] , that sestamibi has a high specificity in the identification of malignant lung lesions . although our preliminary data are based on a small case series and require confirmation by larger studies , they suggest that spect - ct can detect true positive cases at an early stage . 
therefore , the possible inclusion of scintigraphy in the characterisation of spns might lead to ct being reserved for the follow - up of scintigraphically negative lesions only . with regard to sensitivity , spect yielded one false negative result , in an 8 - mm lesion ( the smallest in our series )  . however , even biological factors such as cancer aggressiveness and the possible presence of multidrug resistance may affect results . 
the mechanism for the uptake of tc - 99m sestamibi a lipophilic cation that is taken up by cancer cells is related to regional blood flow and to the activity of mitochondria , where it is almost exclusively concentrated [ 23 , 20 ]  . 
it has also been observed that cancer cells , characterised by chemoresistance , show a rapid washout of tc - 99m sestamibi , which is linked to increased expression of certain membrane p - glycoproteins [ 24 , 25 ]  . the main benefit of spect - ct fusion imaging was , in our experience , the correct characterisation of perihepatic lesions affected by the normal uptake of the radiopharmaceutical by the liver . 
our work , although based on a limited number of patients , confirms preliminary results on the potential of this new technique in the differentiation of lung lesions with lipophilic cationic radiopharmaceuticals based on technetium [ 5 ]  . 
the use of this radiotracer would also assist in cost - benefit decision making for pamibi un radiofarmaco impiegato solitamente nello studio della perfusione miocardica , ma dimostratosi efficace nella diagnostica oncologica . 
il meccanismo attraverso il quale avviene lup - take nella cellula neoplastica ancora incerto ed alcuni studi [ 19 ] suggeriscono il ruolo del potenziale negativo delle membrane cellulari e mitocondriali , della diffusione passiva , e della natura lipofilica del mibi . 
nellambito del nostro studio lapplicazione della spect - tc con tc - 99m sestamibi , sulla base di precedenti in letteratura [ 21 , 22 ] , in tutti i casi da noi esaminati e successivamente sottoposti a diagnosi anatomo - patologica , ha fornito da un lato spesso un conforto al sospetto radiologico , dallaltro , in casi selezionati fortemente sospetti , ha fatto optare per lescissione chirurgica piuttosto che per il follow - up . 
i nostri dati preliminari , ottenuti in una casistica limitata , se confermati in studi successivi con linclusione di un numero maggiore di pazienti , suggeriscono che la spect - tc in grado di identificare precocemente i casi veri positivi . 
per quanto riguarda la sensibilit della spect - tc , va sottolineato che lunico caso risultato falso negativo stato osservato in una lesione di 8 mm ( la pi piccola dimensionalmente nella nostra casistica )  . 
infatti il meccanismo di accumulo del tc - 99m sestamibi , che un catione lipofilico che viene captato dalle cellule neoplastiche , in relazione al flusso ematico distrettuale ed alla attivit mitocondriale , dove si concentra quasi esclusivamente [ 23 , 20 ]  . stato poi osservato che nelle cellule neoplastiche , caratterizzate da chemioresistenza , si ha un rapido wash - out del tc - 99m sestamibi , legato ad unaumentata espressione di alcune p - glicoproteine di membrana [ 24 , 25 ]  . 
il vantaggio pi importante della fusione delle immagini spect - tc , nella nostra esperienza , si avuto nella corretta caratterizzazione delle lesioni peri - epatiche , condizionate dal normale up - take del radiofarmaco da parte del fegato . 
il nostro lavoro , anche se basato su un numero non elevato di pazienti , conferma le acquisizioni preliminari circa le potenzialit di questa nuova tecnica nella diagnostica differenziale delle lesioni polmonari con radiofarmaci cationici lipofilici tecneziati [ 5 ]  . 
lutilizzo di questo radiotracciante sarebbe inoltre di ausilio nellottica decisionale costo - beneficio , nei pazienti che , per la compromessa funzionalit cardio - polmonare , risulterebbero ad alto rischio per una toracotomia . 
another important aspect is that spect is more widely available and less expensive than positron emission tomography - fluorodeoxyglucose ( pet - fdg ) , an examination that is sensitive and specific in diagnosis and staging of lung cancer [ 27 , 28 ] but currently limited by its cost and poor availability . 
pet - fdg , however , has an accuracy of 90% and sensitivity and specificity of 91% and 68% , respectively , in the differential diagnosis of spns [ 29 ]  . 
 ritorio delle apparecchiature ed il basso costo dellesame , se confrontato con la pet - fdg , esame dimostratosi sensibile e specifico nella diagnosi e nella stadiazione delle neoplasie polmonari [ 27 , 28 ] , ma attualmente limitato dai costi e dalla scarsa reperibilit su territorio . 
bazzocchi istituto di radiologia , policlinico universitario a gestione diretta , universit degli studi di udine , via colugna 50 , i - 33100 udine , italy correspondence to : r.m. 
fifty patients with 111 hcc foci ( 61 confirmed histologically , 46 confirmed by percutaneous interventional procedures , four confirmed by ct follow - up of at least 6 months ) underwent mdct with a double arterial phase and a portal venous phase after administration of contrast material with a high iodine concentration ( 400 mgi / ml , 2 ml / kg , 5 ml / s )  . two radiologists independently evaluated the images in three distinct reading sessions ( early arterial phase ( eap ) , late arterial phase ( lap ) and double arterial phase ) to determine presence , number and degree of suspicion of hcc . 
sensitivity and ppv increase progressively when passing from eap to lap to double - arterial - phase images obtained with contrast material with a high iodine concentration . however , the difference in sensitivity between lap and the double arterial phase was not statistically significant . 
cinquanta pazienti con 111 foci di hcc ( 61 confermati istologicamente , 46 tramite procedure intervenzionali percutanee , 4 confermati mediante un follow - up in tc di almeno 6 mesi ) hanno eseguito una tc con una doppia fase arteriosa ed una fase venosa portale con un apparecchio multidetettore dopo somministrazione di mdc ad elevata concentrazione di iodio ( 400 mgi / ml , 2 ml / kg , 5 ml / s )  . 
due radiologi hanno valutato indipendentemente le immagini in tre sessioni di lettura separate ( fase arteriosa precoce ( fap ) , tardiva ( fat ) ed entrambe le fasi arteriose combinate ) per determinare la presenza , il numero ed il livello di sospetto di hcc . 
la sensibilit media ed il vpp per lidentificazione dellhcc sono risultati rispettivamente 83 , 8% e 93 , 5% per la fap , 90 , 5% e 94 , 8% per la fat , 94 , 1% e 95 , 1% per le due fasi arteriose combinate . 
la sensibilit ed il vpp incrementano progressivamente passando dallanalisi delle immagini della fap , a quelle della tardiva e delle due fasi arteriose combinate ottenute con mdc ad elevata concentrazione di iodio . 
as a result , it has only been possible to study the characteristics of the phenomenon since the advent of spiral computed tomography ( ct ) scanners , which permit analysis of the passage of cm in the liver vasculature within seconds of its arrival in the hepatic artery ( during the so - called arterial phase ) [ 39 ]  . 
with the introduction of multidetector ct ( mdct ) scanners , the passage of cm can even be studied by acquiring two arterial phases , one very early [ early arterial phase ( eap ) ] and one immediately after [ late arterial phase ( lap ) ] [ 1013 ]  . 
over the last few years , several studies have attempted to determine whether the contribution of the two arterial phases to the identification of hcc was the same as that of a single arterial phase or whether it improved ct sensitivity in diagnosing hcc [ 11 , 13 ] , or , again , whether either of the two phases was better than the other , making the other redundant . 
some studies found that lap yields a higher sensitivity than eap in identifying hcc [ 13 ] although the use of the two phases in combination improves the final result . 
while there is some agreement on the importance of administering the cm with a sufficiently high flow rate [ 1417 ] , there is no agreement on the optimal concentration and amount of iodine allowing the best visualisation of hcc in the two different phases . 
recently , various studies have compared aortic and hepatic enhancement obtained with cm at different iodine concentrations [ 1822 ] , emphasising the benefits linked to the use of higher iodine concentrations not only to determine a more intense and earlier aortic enhancement but also improved contrast difference between hypervascular lesions and the liver parenchyma [ 19 , 20 , 22 ]  . the purpose of our study was to evaluate whether mdct with double arterial phase and high - iodine - concentration cm increases the techniques sensitivity in the identification of hepatocellular carcinoma . materials and methods the study included 50 consecutive patients ( 32 men , 18 women , aged between 38 and 81 years ; mean age : 68 years ) referred for ct for the suspicion of at least one hcc based on ultrasound findings . 
forty - three patients had histologically confirmed chronic liver disease ( 23 with hepatitis c , 12 with hepatitis b , one with hepatitis b and c , one with primary biliary cirrhosis , six with a toxic - dysmetabolic liver disease ) ; seven patients had a sonographically suspicious lesion without known chronic liver disease . 
all patients gave their informed consent , and the study was approved by our ethics lepatocarcinoma ( hcc ) presenta generalmente una ricca vascolarizzazione arteriosa in seguito a fenomeni di neoangiogenesi intratumorale , direttamente proporzionale al grado di malignit della lesione [ 1 ]  . 
in queste lesioni vi pertanto uninversione del bilancio artero - portale a favore di un incremento dellinput arterioso [ 2 ] , che fa s che il mezzo di contrasto ( mdc ) , giunto attraverso larteria epatica , arrivi molto precocemente allinterno dellhcc ed altrettanto precocemente ne venga dismesso . 
la precocit del fenomeno tale che solo lavvento delle tc spirali , che hanno permesso lanalisi del passaggio del mdc nel fegato e nei suoi vasi nei primi secondi dallarrivo in arteria epatica ( durante cio la cosiddetta fase arteriosa ) , ha consentito di analizzarne le caratteristiche [ 39 ]  . 
lintroduzione delle tc multidetettore ha consentito di poter studiare il passaggio del mdc addirittura mediante lacquisizione di due fasi arteriose , una molto precoce ( fase arteriosa precoce ) ed una immediatamente successiva ( fase arteriosa tardiva ) [ 1013 ]  . 
negli ultimi anni diversi lavori hanno cercato di stabilire se lapporto delle due diverse fasi arteriose nellidentificazione dellhcc fosse sovrapponibile a quello di una singola fase arteriosa , oppure se migliorasse la sensibilit della tc nella diagnosi dellhcc [ 11 , 13 ] ; oppure , ancora , se una sola delle due fasi fosse migliore e rendesse superfluo lapporto dellaltra . 
alcuni lavori hanno sottolineato che la fase arteriosa tardiva fornisce una sensibilit maggiore rispetto a quella precoce nellidentificazione dellhcc [ 13 ] , anche se lassociazione delle due migliora il risultato finale . 
mentre vi un certo accordo nel sottolineare limportanza dellintroduzione del mdc ad un flusso sufficientemente elevato [ 1417 ] , non vi sufficiente accordo nel ritenere quali siano le concentrazioni e la quantit di iodio ottimali per la migliore visualizzazione dellhcc nelle due diverse fasi . 
recentemente , in letteratura sono comparsi diversi studi che hanno confrontato lenhancement aortico ed epatico ottenuto con mdc a differenti concentrazioni di iodio [ 1822 ] , sottolineando i vantaggi nellimpiego di maggiori concentrazioni di iodio non solo nel determinare un enhancement aortico pi intenso e precoce , ma conseguentemente anche una migliore differenza di contrasto tra lesioni ipervascolari e parenchima epatico [ 19 , 20 , 22 ]  . 
lo scopo del nostro lavoro stato valutare se lo studio mediante tc multidetettore con doppia fase arteriosa e mdc ad elevata concentrazione di iodio determini un miglioramento in termini di sensibilit nellidentificazione dellepatocarcinoma . materiali e metodi in questo studio sono stati inclusi 50 pazienti consecutivi ( 32 maschi , 18 femmine ; et compresa tra 38 ed 81 anni ; et media , 68 anni ) inviati allesame tc per il sospetto di almeno un hcc sulla base di reperti ecografici . 
in patients with biopsy - proven hcc , only one nodule was biopsied ; consequently , those patients presenting with other lesions with the same imaging characteristics as the biopsied lesion were considered to be affected by multifocal hcc . 
forty - six nodules in 15 patients were confirmed by response to interventional percutaneous procedures such as chemoembolization , radiofrequency , and alcoholization ( meant as a reduction in size and / or total or partial absence of arterial vascularity at ct follow - up 1 month after the procedure and every 6 months for at least 1 year )  . 
four lesions in four patients were confirmed by ct follow - up alone ( size increase of the lesion and / or increase of the number of nodules at 3 , 6 , 9 and 12 months ) , as the patients poor clinical condition precluded surgery or percutaneous procedures . 
in ten patients without focal hepatic lesions on ct ( suspected at previous ultrasonography ) , absence of the tumour was confirmed by ct follow - up at 6 months from the first examination and negative - fetoprotein test . 
acquisition parameters were as follows : collimation 3 mm , slice thickness 5 mm , reconstruction interval 5 mm , pitch 5.5 ( table feed 33 mm / s ) ; 135 kv ; 175 mas . iodine concentration administered before examination , all patients received 500 ml of water orally and a non - ionic water - soluble contrast agent with a high intravenously ( 400 mgi / ml iomeprol ) ( iomeron 400 , bracco , milan , italy ) at a 5 - ml / s flow rate and a 2 - ml / kg volume ( volume range 100160 ml ; mean 138.5 ml ) using an automatic injector ( envision ct injector , medrad , indianola , pa , usa )  . 
to determine the best time to start the ct scan after administering the cm , we used contrast tracking software ( sure start , toshiba , tokyo , japan ) : a region of interest ( roi ) , which calculates blood density before the cm , is positioned within the abdominal aorta at the level of a plane passing through the diaphragm ; the operator establishes a density threshold in hounsfield units ( hu )  . 
the contrast bolus is then administered and , as soon as the established density threshold is reached , the ct scan starts automatically . three scans of the entire liver were performed with an acquisition time of 710 s ( 9 s on average ) , depending on the craniocaudal diameter of the liver . 
the first scan ( eap ) was obtained in the craniocaudal direction , the second ( lap or portal inflow phase ) in the caudocranial direction with a 5s delay between the two acquisitions . 
tutti i pazienti hanno firmato il consenso informato ; lo studio stato approvato dal nostro comitato etico ed ha seguito i principi della dichiarazione di helsinki [ 23 ]  . in 40 pazienti su 50 stato riscontrato almeno un hcc , per un totale di 111 lesioni ( con un numero medio di lesioni per paziente pari a 2 , 77 )  . 
la conferma istologica di hcc stata ottenuta per 61 noduli in 21 pazienti ( mediante trapianto di fegato per 16 lesioni in 8 pazienti , mediante resezione parziale per 8 lesioni in 4 pazienti , mediante biopsia percutanea con ago da 18 gauge per 37 noduli in 9 pazienti )  . nei pazienti con conferma bioptica di hcc , un unico nodulo stato sottoposto a biopsia ; pertanto sono stati considerati portatori di hcc multifocale quei pazienti che presentavano altre lesioni con le medesime caratteristiche allimaging della lesione sottoposta a biopsia . 
la conferma per i rimanenti 50 noduli in 19 pazienti stata ottenuta con un follow - up , che teneva conto di dati di laboratorio ( ad esempio , test per la funzionalit epatica e dosaggio dell - fetoproteina ) e radiologici . quarantasei noduli in 15 pazienti sono stati confermati grazie alla risposta a terapie percutanee intervenzionali come chemioembolizzazione , radiofrequenza ed alcolizzazione ( intesa come riduzione delle dimensioni e / o assenza completa / parziale di vascolarizzazione arteriosa al controllo tc ad 1 mese dalla procedura ed ogni 6 mesi per un periodo medio di almeno 1 anno )  . quattro lesioni in 4 pazienti sono state confermate solamente mediante un follow - up con tc ( aumento dimensionale della lesione e / o del numero di noduli a 3 , 6 , 9 e 12 mesi ) , in quanto non suscettibili di intervento chirurgico n di terapie percutanee a causa delle scadenti condizioni cliniche del paziente . 
nei 10 pazienti privi di lesioni focali epatiche allesame tc ( sospettate ad un precedente esame ecografico ) , la conferma di assenza di patologia neoplastica stata ottenuta mediante tc di controllo a 6 mesi di distanza dal primo esame e valori negativi di - fetoproteina . 
tutti gli esami tc sono stati eseguiti mediante un apparecchio multidetettore a 4 canali di acquisizione ( data acquisition system , das , aquilion , toshiba , tokio , giappone ) , a matrice asimmetrica di detettori e tempo di rotazione del gantry di 0 , 5 s . 
i parametri di acquisizione sono stati i seguenti : collimazione , 3 mm ; spessore di visualizzazione , 5 mm ; intervallo di ricostruzione , 5 mm ; pitch , 5 , 5 ( avanzamento del lettino , 33 mm / s ) ; 135 kv ; 175 mas . prima dellesame a tutti i pazienti sono stati somministrati per os 500 ml di acqua e per via endovenosa un mdc non ionico idrosolubile ad elevata concentrazione di iodio ( iomeprolo 400 mgi / ml ) ( iomeron 400 , bracco , milano , italia ) ad un flusso di 5 ml / s e con un volume pari a 2 ml / kg ( volume compreso tra 100160 ml ; media , 138 , 5 ml ) , mediante iniettore automatico ( envision ct injector , medrad , indianola , pennsylvania , usa )  . 
per determinare il momento ideale in cui iniziare lacquisizione tc dopo la somministrazione di mdc stato impiegato un software di contrast tracking ( sure start , toshiba , tokio , giappone ) : una region of inter.m. 
la fase arteriosa precoce evidenzia un voluminoso hcc caratterizzato da un disomogeneo enhancement ( a ) ; in fase arteriosa tardiva sono ben evidenti sia un incremento della sua cospicuit che la comparsa di una piccola lesione ipervascolare ( freccia ) ( b )  . 23 s on average ( range , 1925 s )  . 
fifteen seconds after the end of lap , the third scan was performed ( portal venous phase ) in the craniocaudal direction , with a mean delay of 62 s ( range , 5075 s )  . 
in the 12 patients undergoing surgery ( transplantation or partial resection ) , data obtained in eap were used for rest , roi , che calcola la densit ematica prima del mdc , viene posizionata allinterno dellaorta addominale , a livello di un piano passante per il diaframma ; loperatore fissa un valore di densit soglia in unit hounsfield , hu ; quindi si somministra il bolo di mdc e , dopo il raggiungimento del valore soglia di densit preimpostato , la scansione tc inizia automaticamente . sono state effettuate 3 scansioni sullintero fegato con un tempo di acquisizione di 710 s ( media , 9 s ) , a seconda del diametro cranio - caudale del fegato . 
la prima scansione ( fase arteriosa precoce ) stata ottenuta con direzione craniocaudale , la seconda ( fase arteriosa tardiva o portal inflow phase ) con direzione caudo - craniale ed un ritardo tra le due acquisizioni di 5 s . 
il ritardo medio dallinizio della somministrazione di mdc stato di 21 s ( range , 1635 s ) per la fase arteriosa precoce e di 34 s ( range , 2850 s ) per la fase arteriosa tardiva . 
dopo 15 s dal termine della fase arteriosa tardiva , stata effettuata la terza scansione ( fase venosa portale ) con direzione cranio - caudale ed un ritardo medio di 62 s ( range , 5075 s )  . nei 12 pazienti sottoposti ad intervento chirurgico ( trapianto o resezione parziale ) i dati ottenuti durante la fase arteriosa precoce sono stati utilizzati per ricostruzioni vascolari tridimensionali ( maximum intensity projection e / o volume rendering )  . le immagini della fase arteriosa precoce , tardiva e delle due fasi arteriose combinate ( double arterial phase ) sono state valutate separatamente ed indipendentemente da due radiologi esperti . 
per ogni paziente sono state impiegate da entrambi i lettori tre sessioni di lettura separate : per evitare bias da apprendimento nelle prime due venivano proposte in modo randomizzato le immagini della fase arteriosa precoce e tardiva ; nellultima sessione venivano proposte le immagini delle due fasi arteriose combinate . 
tra ogni sessione di lettura trascorso un periodo di tempo pari a due settimane . la valutazione delle immagini stata eseguita su una workstation dedicata ( anet , toshiba , tokio , giappone ) impiegando un livello della finestra pari a 100 hu ed unampiezza pari a 250 hu . 
i due lettori erano a conoscenza che la maggior parte dei pazienti avevano unepatopatia medica cronica ed erano soggetti a rischio di hcc , ma non conoscevano ulteriori informazioni cliniche ( ad esempio , livelli di - fetoproteina ) n riscontri ecografici . 
per ogni fase ciascun lettore ha indicato dimensioni , posizione e numero delle lesioni ed ha fornito in modo soggettivo un grado di sospetto per la presenza di hcc mediante una scala a 5 punti : punteggio 0 , assenza di hcc ; punteggio 1 , hcc probabilmente assente ; punteggio 2 , hcc possibilmente presente ; punteggio 3 , hcc probabilmente presente ; punteggio 4 , hcc sicuramente presente . 
una lesione veniva definita hcc quando nelle immagini delle fasi arteriose appariva come un nodulo di enhancement focale , omogeneo o disomogeneo , privo dei criteri tipici per una lesione benigna . 
the readers used three separate reading sessions for each patient : in order to avoid learning bias in the first two , eap and lap images were submitted to their judgement at random ; in the last session , images of the two arterial phases combined were examined . 
two weeks elapsed between each reading session . image assessment was carried out on a dedicated workstation ( anet , toshiba , tokyo , japan ) with a window level of 100 hu and width of 250 hu . 
if the two readers disagreed , images were re - assessed with a third reader to reach a consensus . all lesions , subsequently confirmed to be hcc , with a degree of suspicion > 2 were considered true positives . 
 table 1 k values for degree of agreement between the two readers as to presence ( score 24 ) or absence ( score 01 ) of lesions reading session reader 1 vs . 
lettore 2 tutte le lesioni , con successiva conferma di hcc , con un grado di sospetto superiore o uguale a 2 sono state considerate veri positivi ; tutte quelle con punteggio da 0 a 1 , con conferma di hcc , sono state considerate falsi negativi . 
un valore di p inferiore a 0 , 05 con un test a due code stato considerato significativo . risultati in 40 pazienti su 50 arruolati stato riscontrato almeno un hcc , per un totale di 111 lesioni . 
trentanove tumori su 111 presentavano un diametro maggiore o uguale a 20 mm , 72 / 111 presentavano un diametro inferiore ai 20 mm ( range , 5220 mm ; media , 22 mm )  . 
i valori k dei due lettori hanno mostrato una concordanza moderata nellanalisi della fase arteriosa precoce ( k = 0 , 49 ) , ed elevata nella valutazione sia della fase arteriosa tardiva ( k = 0 , 62 ) che delle due fasi arteriose combinate ( k = 0 , 78 ) ( tabella 1 )  . 
la sensibilit ed il valore predittivo positivo medi nellidentificazione di hcc sono risultati rispettivamente pari a 83 , 8% e 93 , 5% per le immagini della fase arteriosa precoce , 90 , 5% e 94 , 8% per la fase arteriosa tardiva , 94 , 1% e 95 , 1% per la double arterial phase . 
lanalisi delle immagini della sola fase arteriosa tardiva e di quelle delle due fasi arteriose combinate ha consentito lidentificazione di un numero significativamente superiore di hcc , rispetto alla valutazione della sola fase arteriosa precoce ( p < 0 , 05 )  . 
la valutazione delle immagini della double arterial phase ha evidenziato valori di sensibilit e valore predittivo positivo superiori rispetto a quelli della sola fase arteriosa tardiva , anche se entrambi con differenza non statisticamente significativa ( p > 0 , 05 ) ( entrambi i lettori hanno identificato 4 lesioni in 4 pazienti perse nella fase arteriosa tardiva ) ( tabella 2 e 3 )  . 
in lap , the two readers identified 12 tumours in six patients , which had a seguito di fenomeni di neoangiogenesi intratumorale nellhcc si riscontrano vasi tumorali , spesso irregolari e tortuosi , con un apporto vascolare prevalentemente arterioso rispetto al fegato sano circostante ( arterializzazione ) [ 2 ]  . 
per la corretta identificazione di tale neoplasia necessaria pertanto lacquisizione di una fase arteriosa , durante la quale vi unelevata quantit di mdc allinterno dei vasi arteriosi , oltre che lacquisizione di una fase venosa portale pi tardiva , in cui la neoplasia ha gi dismesso il mdc apparendo quindi ipodensa rispetto al parenchima circostante [ 2426 ]  . negli ultimi anni molti autori hanno riconosciuto come la fase arteriosa migliori la sensibilit nellidentificazione table 2 sensitivity and positive predictive value ( ppv ) of the two readers in the three reading sessions . 
evaluation of double arterial phase images had higher sensitivity and positive predictive value than achieved with lap alone although the difference was not statistically significant ( p > 0.05 ) ( both readers identified four missed lesions in four patients in lap ) ( tables 2 and 3 )  . 
the two readers mean degree of suspicion was higher with images of lap and the double arterial phase as compared with eap alone ( 3.48 in eap ; 3.67 in lap ; 3.63 in double arterial phase )  . 
 discussion following intratumoral neoangiogenesis phenomena , often irregular and tortuous tumour vessels are present within hccs , leading to predominantly arterial supply relative to the surrounding healthy liver ( arterialization ) [ 2 ]  . 
as a consequence , correct ct identification of this tumour requires acquisition of an arterial phase during which a large amount of cm is contained in the arteries , as well as acquisition of a later portal venous phase during which the neoplasm has already eliminated the cm and appears hypodense against the surrounding parenchyma [ 2426 ]  . 
over the last few years , several authors have recognised that the arterial phase improves sensitivity of ct in the identification of hcc , both with single - slice [ 3 , 59 ] and multislice scanners [ 1113 ] ; others have reported that higher iodine concentrations increase aortic endellhcc , sia con tc a singolo strato [ 3 , 59 ] , sia con apparecchi multidetettore [ 1113 ] ; altri hanno descritto come maggiori concentrazioni di iodio determinino un incremento in particolar modo dellenhancement aortico , risultando in una migliore differenza di contrasto tra strutture iperdense , come vasi arteriosi e tumori ipervascolari ( ad esempio , hcc ) , ed il parenchima epatico [ 1822 , 27 , 28 ]  . in questo studio abbiamo eseguito un esame tc in modalit trifasica con un apparecchio multidetettore , come gi descritto da altri autori [ 10 , 12 , 13 ] , per aumentare la capacit di identificare le lesioni ipervascolari . abbiamo acquisito due fasi arteriose ( precoce e tardiva ) in ununica apnea inspiratoria ed una fase venosa portale . a differenza di laghi et al . 
 [ 12 , 13 ] , in questo studio abbiamo deciso di valutare solamente il contributo delle immagini ottenute nelle due fasi arteriose , anche se la fase venosa portale stata acquisita in tutti i pazienti . 
sulla base di osservazioni della letteratura , che evidenziavano lefficacia delle alte concentrazioni dei mdc [ 22 , 29 ] , si scelto di utilizzare un mdc ad alta concentrazione per valutare se questo migliori lidentificazione dellhcc , che notoriamente , nella maggior parte dei casi , una neoplasia ipervascolarizzata . 
per ovviare ai problemi legati alla velocit di circolo dei pazienti abbiamo impiegato un software di contrast - tracking . i nostri risultati hanno evidenziato unelevata sensibilit per lidentificazione dellhcc in fase arteriosa precoce ( 83 , 8% ) , tardiva ( 90 , 5% ) e nella double arterial phase ( 94 , 1% ) , sebbene non vi fosse una differenza statisticamente significativa tra la fase arteriosa tardiva e la double arterial phase , come peraltro gi riscontrato da laghi et al . 
our study used a three - phase mdct examination , as already reported by other authors [ 10 , 12 , 13 ] , to increase our ability to detect hypervascular lesions . 
 [ 12 , 13 ] , we decided to evalli e valutabili durante la fase arteriosa tardiva , probabilmente a causa di un intervallo di tempo necessario affinch il sangue arterioso contrastato , proveniente dallarteria epatica , si distribuisca allinterno dellinterstizio della lesione [ 10 ]  . 
va inoltre sottolineato che dai nostri dati sono emersi valori di sensibilit maggiori , se paragonati a quelli ottenuti in altri studi in cui siano state valutate le due fasi arteriose [ 11 , 13 ] , e questa differenza risulta pi marcafig . 
2a - d computed tomography ( ct ) images of a 52 - year - old patient with hepatitis - c - virus - related liver disease , with a transjugular intrahepatic portosystemic shunt ( tips ) , candidate for liver transplantation . 
in early arterial phase , a 25 - mm hepatocellular carcinoma ( hcc ) nodule can be clearly seen near the hepatic hilum , characterised by early enhancement ( a ) and increased conspicuity in the late arterial phase ( b )  . 
in fase arteriosa precoce evidente un nodulo di hcc di 25 mm in prossimit dellilo epatico caratterizzato da un precoce enhancement ( a ) , la cui cospicuit risulta maggiore in fase arteriosa tardiva ( b )  . 
on the basis of literature reports , which showed the effectiveness of high concentrations of cm [ 22 , 29 ] , we decided to use a high - concentration cm to determine whether it can improve identification of hcc , which is known to be a hypervascular tumour . 
our results indicated a high sensitivity in the identification of hccs in eap ( 83.8% ) , in lap ( 90.5% ) and in the double arterial phase ( 94.1% ) although there was no statistically significant difference between lap and the double arterial phase , as already noted by laghi et al . 
it should be noted that our study found higher sensitivity values compared with those obtained by other studies assessing both arterial phases [ 11 , 13 ] , and that this difference is more dramatic in eap . 
in our view , this greater sensitivity may be due to the higher concentration of contrast agent employed since high - concentration cm administered at a high flow rate leads to an earlier and more intense arterial enhancement peak [ 27 , 30 ]  . 
since the hepatic artery is one of the main branches of the aorta , concentration of cm in the hepatic artery , and consequently in the hcc , is thought to be equivalent to concentration in the aorta . 
therefore , it can be assumed that during the arterial phase , the contrast difference between a hypervascular lesion and the liver is comparable to that between the aorta and the liver [ 18 ]  . 
thus , lesions such as hcc , which is supplied by the hepatic artery , have a more intense and earlier enhancement during the arterial phase when a high - concentration contrast agent is used . 
the above may also explain the greater increase in sensitivity in hcc identification during eap as compared with the late phase when our results are compared with those reported in the literature . 
 [ 18 ] , who found greater aortic enhancement and a more dramatic contrast difference between lesion and liver during eap using higher concentrations of cm ( 370 mgi / ml )  . 
 [ 20 ] , who studied patients with chronic liver disease , even though only one arterial phase was used in these studies . finally , our results agree with those of a recent pilot study by marchiano et al . 
nevertheless , as suggested by other reports [ 11 , 13 , 22 ] , we used other methods to obtain confirmation , such as clinical history , ct follow - up and response ta nella fase arteriosa precoce . 
secondo noi questa maggiore sensibilit potrebbe essere dovuta alla maggiore concentrazione del mdc impiegato , in quanto mdc ad elevata concentrazione , somministrati con elevata velocit , determinano un picco di enhancement arterioso pi precoce e pi intenso [ 27 , 30 ]  . 
dato che larteria epatica rappresenta uno dei principali rami dellaorta , si suppone che la concentrazione di mdc in arteria epatica , e quindi nellhcc , sia equivalente a quella aortica . 
si pu allora presumere che in fase arteriosa la differenza di contrasto tra una lesione ipervascolare e fegato sia paragonabile a quella tra aorta e fegato [ 18 ]  . quindi lesioni come lhcc , vascolarizzato dallarteria epatica , presentano verosimilmente un enhancement pi intenso e precoce in fase arteriosa quando si impieghi un mdc ad elevata concentrazione . quanto sopra pu spiegare anche il pi marcato incremento di sensibilit nellidentificazione dellhcc in fase arteriosa precoce rispetto a quella tardiva , quando si confrontano i nostri risultati con quelli medi riscontrati in letteratura . peraltro altri studi confermerebbero la nostra ipotesi , come quello di awai et al . 
 [ 18 ] , che hanno evidenziato un maggiore enhancement aortico ed una pi marcata differenza di contrasto tra lesione e fegato in fase arteriosa precoce utilizzando concentrazioni di mdc pi elevate ( 370 mgi / ml )  . conclusioni simili sono emerse anche in lavori di sultana et al . 
execution of a single arterial phase followed by a portal venous phase would imply considerable dose savings for each examination ( 6.03 msv ) , an important aspect if we consider that these patients undergo periodic ct scans during follow - up . 
we believe that further studies are needed to compare different concentrations of cm with the same amounts of iodine administered to the same subject , especially in hcc candidates for liver transplantation , with the possibility of establishing the precise number of lesions on the explanted liver . conclusions the use of high - iodine - concentration cm ( 400 mgi / ml ) produces an increase in the number of hccs identified during the arterial phase , both early and late . 
in ct studies of patients with more or less known chronic liver disease and suspected hcc , we suggest that high - iodine - concentration cm should be used ( 400 mgi / ml )  . 
considering that these patients undergo periodic follow - up examinations , and considering the dose delivered by a double arterial phase acquisition , we believe acquisition of lap alone , followed by the portal venous phase , to be indicated . 
 riteniamo siano necessari ulteriori studi per paragonare differenti concentrazioni di mdc con medesime quantit di iodio somministrate allo stesso soggetto , soprattutto in pazienti con hcc candidati a trapianto di fegato , con la possibilit quindi di calcolare il numero esatto di lesioni sul fegato espiantato . conclusioni limpiego di mdc ad elevata concentrazione di iodio ( 400 mgi / ml ) consente di incrementare il numero di hcc identificati in fase arteriosa sia precoce che tardiva . 
negli studi tc di pazienti con epatopatia medica cronica nota o meno e sospetto di hcc , suggeriamo lutilizzo di un mdc ad elevata concentrazione di iodio ( 400 mgi / ml )  . tenuto conto del fatto che questi pazienti sono sottoposti a frequenti controlli e della dose a cui sarebbero esposti in caso di acquisizione di una doppia fase arteriosa , riteniamo che sia indicata lacquisizione della sola fase arteriosa tardiva seguita dalla fase venosa portale . 
bassiano , via dei lotti 14 , i - 36061 bassano del grappa ( vi ) , italy 2istituto di radiologia , ospedale cattinara , universit di trieste , strada di fiume , i - 34100 trieste , italy 3u.o. 
typical computed tomography ( ct ) pattern has been widely described and consists of peripheral parenchymal consolidations with air bronchogram with or without surrounding ground - glass - like opacities . 
the purpose of this article is to describe the less frequent imaging pattern of this disease represented by single or multiple focal lesions ( nodules or masses that place diagnostic problems with malignancy ) , bronchocentric pattern ( parenchymal consolidations with peribronchovascular distribution ) , atoll sign ( central area of ground - glass - like density and peripheral area of consolidation ) , nodular lesions ( poorly defined micronodular pattern ) , linear and band - like opacities ( subpleural linear opacities that can have disposition parallel or perpendicular in relation to the pleura ) , perilobular pattern ( thickening of the interlobular septa with reticular pattern ) and progressive fibrotic pattern ( irregular thickening of the interlobular septa with associated ground - glasslike appearance and consolidations )  . key words organizing pneumonia hrct cryptogenic organizing pneumonia idiopathic interstitial pneumonias boop riassunto la polmonite organizzata ( op ) unentit clinico - patologica che , se idiopatica , fa parte delle polmoniti interstiziali secondo la classificazione ats / ers del 2002 ( 50% dei casi , chiamata polmonite organizzata criptogenetica o cop )  . 
op is a relatively common condition accounting for approximately half la polmonite organizzata ( op ) unentit clinico - patologica descritta per la prima volta da davison e collaboratori nel 1983 [ 1 ]  . 
il termine op , recentemente entrato nelluso comune , da preferire perch meglio descrive gli aspetti anatomopatologici ed evita confusione diagnostica con altre patologie delle vie aeree ( per esempio , la bronchiolite obliterante ) [ 3 ]  . 
according to the new classification introduced by he american thoracic society / european respiratory society ( ats / ers ) in 2002 [ 5 ] , the idiopathic form of op falls within the idiopathic interstitial pneumonias and is called cryptogenic organizing pneumonia ( cop )  . 
op may , however , also be secondary to several conditions since it represents the lungs reaction to different pathogenic noxae ( op reaction pattern ) ( table 1 )  . over the years , many papers have appeared in the radiological literature that describe new high - resolution computed tomography ( hrct ) patterns of op [ 6 , 7 ]  . 
the aim of this paper is to review the recent literature data describing typical and atypical clinical , pathological and especially ct and hrct aspects of op . za frequente , rappresentando circa la met dei casi diagnosticati di malattia delle piccole vie aeree , ed idiopatica nel 50% dei casi [ 4 ]  . 
secondo la nuova classificazione dellats / ers del 2002 [ 5 ] , la forma idiopatica della op fa parte delle polmoniti interstiziali idiopatiche e denominata cop ( polmonite organizzata criptogenetica )  . la polmonite organizzata pu essere tuttavia anche secondaria , associata cio a molte situazioni rappresentando una peculiare modalit di risposta del polmone a diverse noxae patogene ( op reaction - pattern ) ( tabella 1 )  . 
scopo di questo lavoro quindi di proporre i dati recenti della letteratura , descrivendone gli aspetti clinici , anatomopatologici , ma soprattutto tc ad alta risoluzione ( hrct ) , sia tipici sia quelli meno comuni . clinical presentation the frequency of the disease is identical in men and women and , even though no sure relation exists with cigarette smoking , it is prevalent in non - smokers , with a 2 : 1 ratio [ 8 ]  . 
there may also be weight loss , mild fever and myalgia . they will usually have been treated with one or more courses of antibiotics without symptom resolution or improvement [ 9 ]  . 
although a definitive diagnosis of op cannot be made on the sole basis of clinical findings , the combination of a compatible clinical history and adequate hrct signs may enable diagnosis in 80% of cases . 
transbronchial biopsy and bronchoalveolar lavage can confirm the diagnosis , but imaging alone may be sufficient for a diagnostic hypothesis of op [ 6 ]  . histology histologically , op presents with polyps of lax connective tissue within the terminal or respiratory bronchioles , alveolar ducts and the alveoli , with patchy distribution , usually without changes in pulmonary architecture and with temporal homogeneity . 
the bronchiolar component may be lacking , and there may be signs of chronic interstitial inflammation , but istologia clinica la frequenza della malattia sovrapponibile tra maschi e femmine e , anche se non esiste una sicura relazione con il fumo di sigaretta , prevalente nei non fumatori , con un rapporto 2 : 1 [ 8 ]  . 
i pazienti presentano caratteristicamente un quadro clinico di infezione delle basse vie aeree con malessere di recente insorgenza ( di solito da meno di 3 mesi ) , con grado variabile di tosse e dispnea . 
in alcuni casi si pu osservare una rapida evoluzione verso la fibrosi , ma esiste anche la possibilit di una polmonite organizzata sovrapposta alla fibrosi polmonare idiopatica ( fpi )  . 
non possibile porre una diagnosi di certezza di op solo in base al quadro clinico , ma lassociazione di una storia clinica compatibile e i segni hrct adeguati possono porre diagnosi nell80% dei casi . 
la biopsia transbronchiale e il lavaggio bronchiolo - alveolare possono confermare la diagnosi , anche se in alcuni casi limaging radiologico pu essere da solo sufficiente nellipotesi diagnostica di questa malattia [ 6 ]  . il quadro istologico della op rappresentato da polipi di r . 
miscellaneous inflammatory bowel disease primary biliary cirrhosis polyarteritis nodosa haematological malignancies myelodysplastic syndrome t - cell leukaemia lymphoma radiotherapy exposure to toxic substances aspiration syndrome extrinsic allergic alveolitis acute and chronic eosinophilic pneumonia wegeners disease a . 
varie malattie infiammatorie intestinali cirrosi biliare primitiva poliarterite nodosa tumori ematologici sindrome mielodisplasica leucemia a cellule t linfoma radioterapia esposizione a sostanze tossiche sindrome da aspirazione alveolite allergica estrinseca polmonite eosinofila acuta e cronica malattia di wegener d . 
finally , it should be noted that foci of intraluminal op typical of op are also seen in association with inflammation and fibrosis in about half of the cases of non - specific interstitial pneumonia ( nsip ) [ 12 ]  . typical hrct pattern the typical pattern is present in 60%90% of cases [ 13 , 14 ] and characterised by bilateral patchy areas of consolidation , often triangular or polygonal in shape , in the peripheral subpleural regions , but also peribronchial , and often located in the lower lobes . 
in 60% of cases scattered ground - glass opacities are associated and at times ( 10%50% ) small ill - defined centrinodular lobules that are often peribronchial [ 13 , 15 ]  . 
the prevalently subpleural location of op calls for a differential diagnosis with chronic eosinophilic pneumonia [ 9 , 16 ]  . other diseases may display similar radiological patterns and , in particular , bronchioloalveolar carcinoma , lymphoma , vasculitis , and infections ( above all tuberculous and atypical mycobacteriosis )  . 
even the symptoms may be suggestive of cancer due to the presence of haemoptysis if the lesions are cavitated , a relatively common occurrence in this type of op . some hrct signs may , however , help in differentiating the two diseases : peripheral location , predominant oval or trapezoidal shape ( unlike cancer , which is usually rounded ) and the frequent presence of satellite lesions are suggestive of op [ 15 , 1822 ]  . 
nel 60% dei casi coesistono aree sparse di ground - glass e talvolta ( 10%50% ) piccoli noduli centrolobulari a margini mal definiti , spesso peribronchiali [ 13 , 15 ]  . le lesioni tendono a progredire e a cambiare sede nel tempo e sono stati descritti casi di remissione spontanea . 
la localizzazione prevalente subpleurica pone la op in diagnosi differenziale con la polmonite eosinofila cronica [ 9 , 16 ]  . altre malattie possono manifestarsi con aspetti radiologici simili ed in particolare , il carcinoma bronchiolo - alveolare , il linfoma , le vasculiti , e le infezioni ( soprattutto tubercolari e le micobatteriosi atipiche )  . in tali casi la diagnosi certa si ottiene solo con manovre invasive ( biopsia transbronchiale ) [ 5 , 16 ]  . 
 da ricordare che gli addensamenti presenti in caso di op secondaria ad amiodarone , hanno la particolarit di essere iperdensi per la presenza di iodio nel farmaco [ 17 ] ; tale aspetto rende la diagnosi certa . pattern hrct atipico lesione focale ( unica o multipla ) la op pu presentarsi come unica lesione parenchimale , frequentemente localizzata nei lobi superiori , a contatto con la pleura e le scissure ( pleural tags , nel 38% dei casi ) ( fig . 2 ) o lungo il fascio bronco - vascolare . queste caratteristiche radiologiche sono pressoch sovrapponibili a quelle del cancro del polmone e la diagnosi definitiva richiede il ricorso alla biopsia o allasportazione chirurgica . anche la sintomatologia pu a volte far pensare alla neoplasia per la presenza di emoftoe nel caso in cui le lesioni siano cavitate , evento non raro in questo tipo di op . 
1a , b bilateral peripheral air - space consolidation , more evident on the right , with air bronchogram in a patient with typical organizing pneumonia ( a )  . 
1a , b addensamento parenchimale bilaterale , pi esteso a destra , in sede mantellare posteriore , con broncogramma aereo ( a ) in paziente con forma tipica di op . 
in the presence of multiple lesions , the differential diagnosis is complex and includes metastasis , lymphoma , infections ( including septic emboli ) , wegeners disease and kaposis sarcoma . 
although uncommon as a single sign ( about one - third of cases ) , it is seen in association with the classical form in 33% of patients [ 6 , 14 ]  . 
this presentation raises problems in the differential diagnosis with other lung diseases with a similar hrct pattern , in particular , vasculitis ( churg - strauss syndrome and wegeners disease ) , diffuse bronchiolo - alveolar carcinoma and chronic eosinophilic pneumonia . 
 [ 16 ] have , however , noted that patients with op are more likely to have bronchocentric lesions associated with ground - glass - like opacity than are those with chronic eosinophilic pneumonia . 
the association of bronchocentric op with polymyositis and dermatomyositis has been frequently reported , such that the finding of areas of bronchocentric consolidation in patients with these diseases may be sufficient for a diagnosis of op [ 18 , 24 , 25 ]  . atoll sign in 1996 , voloudaki et al . 
this presentation is exactly the opposite to that of nodules with a halo sign and has therefore been called reversed halo sign [ 14 ] , or atoll sign [ 27 ]  . 
described it in about 19% of patients in their series [ 14 ]  . micronodular lesions the predominant feature of this rare presentation of op is the presence of micronodular lesions ( smaller than 1 cm ) , with ill - defined margins and often peribronchial location . the micronodules represent areas of op around the mucusfilled bronchioles , surrounded by relatively healthy lung . they are seen in around one third of patients with op in association with other signs but constitute the only sign in 9% of cases only [ 15 ]  . the presence of such nodules may be the only clue for the differential diagnosis with chronic eosinophilic pneumonia , in which nodules are rare [ 28 ]  . linear and band - like opacities this is a rare presentation which is possible both in isolation or combined with other more common patterns . 
two types have been described , usually in association with parenchymal consolidation . the first type is characterised by linear opacities extending in a radial manner along the bronchi towards the pleura , measuring 24 cm in length and 12 mm in width , located in the lower lobes and bilaterally in 50% of cases . 
6 segno dellatollo : lesione nodulare nel lobo inferiore sinistro in cui il quadro a vetro smerigliato disposto centralmente e la parte consolidativa periferica , patognomonico per op . ma classica [ 6 , 14 ]  . 
questo tipo di presentazione pone dei problemi di diagnosi differenziale con altre malattie del polmone che hanno un quadro hrct simile , in particolare le vasculiti ( sindrome di churg - strauss e malattia di wegener ) , il carcinoma bronchiolo - alveolare diffuso e la polmonite eosinofila cronica . arakawa e collaboratori [ 16 ] hanno comunque constatato una frequenza maggiore di lesioni broncocentriche associate a vetro smerigliato nei pazienti con op rispetto a quelli con polmonite eosinofila cronica . stata descritta la frequente associazione di questa forma nelle polimiositi e dermatomiositi , al punto che la presenza di addensamenti broncocentrici in pazienti con queste malattie pu essere sufficiente per la diagnosi di op [ 18 , 24 , 25 ]  . segno dellatollo voloudaki e collaboratori [ 26 ] hanno descritto nel 1996 due casi di op con aree di vetro smerigliato centrale circondato da un alone addensativo conformato ad anello . 
tale presentazione esattamente lopposto di quella dei noduli con halo sign ed stata quindi denominata reversed halo sign [ 14 ] o , per limmagine che ricorda , segno dellatollo [ 27 ]  . 
questo segno tipico della op poich , fino ad ora lunica malattia in cui stato segnalato ; kim e collaboratori lo descrivono in circa il 19% dei pazienti della loro casistica [ 14 ]  . lesioni micronodulari in questa rara forma di presentazione dellop , laspetto prevalente rappresentato dalle lesioni microdulari ( inferiori al centimetro ) e a margini mal definiti , spesso peribronchiali . i micronoduli rappresentano aree di polmonite organizzata attorno ai bronchioli ripieni di muco , circondati da polmone relativamente sano . 
la presenza di noduli di questo tipo pu essere lunico elemento di diagnosi differenziale con la polmonite eosinofila cronica dove i noduli sono poco frequenti [ 28 ]  . opacit lineari e a banda presentazione rara , possibile sia come quadro isolato che in combinazione con gli altri aspetti pi comuni . 
ne sono stati descritti 2 tipi , di solito in associazione con gli addensamenti parenchimali . il primo tipo caratterizzato da opacit lineari che si estendono in modo radiale lungo i bronchi e verso la pleura , lunghe 24 cm , con spessore di 12 mm , localizzate nei lobi inferiori , bilaterali nel 50% dei casi . 
they are related to inflammation along the segmental bronchial branches , and there is no fibrosis [ 6 ]  . perilobular lesions this pattern was first described by murata et al . 
in 1989 [ 30 ] as disease localisation in the peripheral portions of the secondary pulmonary lobule ; it is related to the accumulation of exudate of op along the walls of the peripheral alveoli adjacent to the interlobular septa . at hrct , there may be apparent thickening of the septa themselves due to their proximity with the site of the inflammation , contributing to a coarse reticular pattern with polygil secondo tipo costituito da opacit lineari subpleuriche , senza relazioni con i bronchi , parallele alla superficie pleurica , con lunghezza di 34 cm e spessore di 12 mm , a volte conformate ad arco o con broncogramma aereo allinterno , bilaterali e prevalenti nei lobi inferiori , anche se possono essere presenti nei lobi superiore e medio [ 29 ]  . 
sono determinate da flogosi localizzata lungo i rami bronchiali segmentari e non presente fibrosi [ 6 ]  . lesioni perilobulari questo quadro stato descritto per la prima volta da murata e collaboratori nel 1989 [ 30 ] come localizzazione della malattia a livello delle porzioni periferiche del lobulo polmonare secondario ; esso cio determinato dallaccumulo di essudato della polmonite organizzata lungo la parete degli alveoli periferici , adiacenti ai setti interlobulari . 
in hrct vi pu essere un apparente ispessimento dei setti stessi per la loro vicinanza alla sede della flogosi con realizzazione di un quadro di tipo reticolare sfumato a morfologia poligonale . 
the distribution of these findings corresponds to the typical subpleural and peripheral distribution of op , and this presentation is usually associated with the use of nitrofurantoin and tends to progress to fibrosis [ 3133 ]  . fibrosi progressiva pica della op , con sede mantellare e periferica e di solito questa presentazione associata alluso di nitrofurantoina e presenta unevoluzione verso la fibrosi [ 3133 ]  . progressive fibrosis this radiological pattern was first described in 1973 by gosink et al . 
it is important to bear in mind these different appearances to be able to consider , among the various possible differential diagnoses and in an appropriate clinical setting , even the possibility of a primary or secondary op not presenting with a conventional pattern . pur essendo ormai conosciute da lungo tempo le alterazioni tipiche della op , sempre pi numerose sono le descrizione dei differenti patterns di presentazione di questa malattia . 
ospedali civili riuniti , sciacca ( agrigento ) , italy 4servizio di radiologia , ospedale san massimo , penne ( pescara ) , italy 5facolt di medicina e chirurgia , dipartimento di internistica clinica e sperimentale f . 
stanzione 18 , i - 80129 napoli , italy , tel . : + 39 - 348 - 3738149 , fax : + 39 - 081 - 5584521 , e - mail : roberto.grassi@libero.it received : 27 october 2004 / accepted : 11 july 2005 / published online : 3 march 2006 abstract this paper examines the diagnostic potential of multislice computed tomography enteroclysis ( msct - e ) to detect and assess different diseases affecting the small bowel , emphasising the increasingly important role assumed by the technique in the study of this anatomical region . 
after a short summary of the technical aspects , we discuss the different findings that can be observed during an msct - e study and that enable detection of small - bowel disease and , if necessary , assessment of the extent and stage of disease . key words small bowel multidetector - row ct enteroclysis malabsorption crohns disease bowel neoplasms riassunto gli autori in questo lavoro esaminano le potenzialit diagnostiche della enteroclisi tc multistrato ( e - msct ) nella identificazione e nella valutazione di estensione delle differenti patologie dellintestino tenue , sottolineando il ruolo sempre pi importante che tale tecnica assume nello studio di differenti condizioni morbose che interessano tale distretto anatomico . 
dopo un breve richiamo delle nozioni di tecnica vengono esaminati i differenti rilievi osservabili e da ricercare nel corso di un esame e - msct al fine di ottenere lidentificazione della patologia e , nel caso sia necessario , lestensione e la stadiazione delle differenti patologie del piccolo intestino . parole chiave intestino tenue enteroclisi - tc multistrato malassorbimento morbo di crohn neoplasie intestinali introduction introduzione despite the widespread diffusion and technological evolution of endoscopy , the small bowel is still the only portion of the gastrointestinal tract that cannot be completely explored with endoscopic techniques . 
as a consequence , the diagnosis of small - bowel disease is still mostly established by radiology , which enables the study of both the bowel lumen and wall as well as of extraparietal structures . 
singleor double - contrast enteroclysis has high levels of sensitivity and specificity in the diagnosis of small - bowel disease , as has been widely documented over the past decades . 
forced bowel loop distension through naso - intestinal intubation allows a more accurate study compared with that obtained nonostante la diffusione e levoluzione tecnologica della endoscopia , lintestino tenue rimane , attualmente , lunica parte del tratto gastro - enterico non esplorabile completamente con le metodiche endoscopiche . 
di conseguenza , la diagnosi delle malattie del piccolo intestino ancora affidata in gran parte alla radiologia che offre uno studio sia del versante endoluminale , sia della parete , nonch del versante extraparietale . le metodiche pi utilizzate per lo studio di gran parte delle malattie gastro - intestinali sono la radiologia tradizionale , lecografia ( us ) e la tomografia computerizzata ( tc )  . 
volumetric acquisition can rapidly explore , in a single inspiratory breath - hold , the entire abdomino - pelvic region , almost completely eliminating artefacts due to respiration and intestinal peristalsis . 
multislice ct enteroclysis ( msct - e ) is a recent technique in small - bowel imaging , which combines the advantages of bowel distension with naso - intestinal intubation with those of spiral ct [ 68 ]  . 
the aim of the present review was to define the indications for msct - e in the most common clinical settings and to illustrate the diagnostic gains provided by msct - e compared with other imaging modalities . accurato rispetto a quello ottenibile con lopacizzazione per os del piccolo intestino [ 4 , 5 ]  . 
lacquisizione volumetrica in grado di esplorare rapidamente , in unica apnea inspiratoria , tutto il distretto addomino - pelvico , eliminando quasi completamente gli artefatti respiratori e da peristalsi intestinale . 
lenteroclisi - tc multistrato ( e - tcms ) una recente metodica di studio dellintestino tenue , che somma i vantaggi della distensione intestinale , ottenuta con lintubazione naso - enterica , a quelli propri della tc spirale [ 68 ]  . 
scopo del presente articolo definire le indicazioni della e - tcms nelle pi frequenti situazioni cliniche e di precisare il guadagno diagnostico di tale metodica rispetto alle altre metodiche di imaging . malabsorption malassorbimento the diagnosis of malabsorption is essentially based on clinical and laboratory findings . 
in some cases , the radiologist is called upon to demonstrate the intestinal disorder causing the malabsorption [ 9 , 10 ]  . the intestinal conditions most commonly responsible for malabsorption include : parasite infection ; small - bowel diverticular disease ; short bowel syndromes ( both post - surgical and due to reduced mucosal surface , such as diffuse chronic enteritis , chronic intestinal ischaemia , radiation enteritis ) ; jejuno - colic fistulae ; and adult coeliac disease ( acd )  . 
tra le malattie intestinali di interesse radiologico pi frequentemente causa di malassorbimento ricordiamo : le parassitosi ; la diverticolosi del tenue ; le sindromi da intestino corto ( sia post - chirurgico che da riduzione della superficie mucosa utile allassorbimento , come le enteriti croniche diffuse , la ischemia intestinale cronica , lenterite da radiazioni ) ; le fistole digiuno - coliche ; la malattia celiaca delladulto ( mca )  . 
frequentemente si tratta di malattie croniche e note da tempo ; pertanto non esiste indicazione elettiva allo studio radiologico dellintestino tenue e le alterazioni dimostrabili radiologicamente hanno in genere unimportanza marginale [ 912 ]  . 
infatti , i linfonodi mesenterici e la irregolarit delle pliche digiunali residue sono reperti frequentemente riscontrabili anche in pazienti con mca scompensata , ma non complicata ; talvolta , un breve follow - up clinico contribuisce a risolvere il problema diagnostico [ 21 ]  . small - bowel occlusion mechanical occlusion of the small bowel ( sbo ) , a very common clinical condition , may be complete or partial . 
conventional ct has high levels of sensitivity in both the diagnosis of locclusione meccanica dellintestino tenue ( sbo ) , condizione clinica molto frequente , pu essere completa oppure parziale . 
ben evidente la riduzione del numero delle valvole conniventi nelle anse prossimali dellintestino tenue ( a ) , ed il relativo incremento delle pliche nelle anse pelviche ( b ) , che presentano anche aspetto ipotonico e diffusamente digiunizzato . 
inoltre , non si rende necessaria la somministrazione di contrasto endoluminale perch la distensione intestinale , provocata dallo stato occlusivo , consente gi uno studio ottimale delle anse , grazie al contrasto naturale tra il lume ipodenso ( per il contenuto liquido della stasi ) e la parete resa iperdensa dal mdc iodato e.v. 
spiral computed tomography with iv iodinated contrast agent without further opacification of the bowel loops , which appear distended ( a ) ; normal parietal contrast enhancement ; small collection due to perihepatic ascites . 
esame tc spirale con mdc iodato e.v , senza ulteriore opacizzazione delle anse intestinali , che appaiono distese ( a ) ; regolare ce parietale ; piccola falda di ascite periepatica . 
reperti suggestivi per sbo da aderenze ; conferma chirurgica . of sbo , above all in the case of complex adherence syndromes [ 8 , 22 ]  . in partial sbo , the diagnosis may prove challenging . 
there may also be an indication for mr enteroclysis or convenmultiplanari possono , talvolta , rendere pi agevole la diagnosi di sbo , soprattutto nel caso di sindromi aderenziali complesse [ 8 , 22 ]  . in caso di sbo parziale , la diagnosi pu essere impegnativa . 
consente di escludere problemi di perfusione parietale e permette anche un giudizio accurato sul mesentere ( a )  . ben apprezzabile la transizione tra le anse distese a monte della ostruzione e lintestino vuoto a valle . 
il contrasto endoluminale positivo consente una dimostrazione ottimale della natura estrinseca della patologia ostruente . tional enteroclysis , which allow progression of the endoluminal contrast material to be followed through the bowel loops [ 2 , 22 , 27 , 28 ]  . possono trovare indicazione anche la rm - enteroclisi o il clisma del tenue tradizionale , che permettono di seguire la progressione del mdc endoluminale attraverso le anse intestinali [ 2 , 22 , 27 , 28 ]  . crohns disease crohns disease was and still is the most common field of application of all small - bowel imaging techniques . 
total parenteral nutrition and new effective drugs , even non - conventional drugs such as infliximab ( anti - alpha tnf ) , have changed the role of surgery ; surgery now tends to be used less frequently and as an elective procedure . 
 all imaging modalities provide a fundamental contribution to the correct diagnosis and staging of crohns disease . patients with the disease do not form a clinically homogeneous group , and they may be very different from one another , with different clinical situations influenced by individual expression of the disease and possible previous surgical procedures ; this does not always allow a uniform and reproducible clinical and radiological standardisation of the disease [ 37 ]  . 
msct - e is usually used immediately after us or even as a first - line examination and is therefore gradually replacing conventional enteroclysis [ 38 , 39 ]  . 
luminal distension with neutral contrast material and iv administration of iodinated contrast macampo di studio e di applicazione pi frequente di tutte le metodiche di imaging dellintestino tenue . una malattia infiammatoria cronica , ancora oggi ad ezio - patogenesi sconosciuta , che pu interessare qualsiasi parte del tratto gastro - enterico , prediligendo lileo e il colon . 
la storia naturale della malattia caratterizzata da fasi di riacutizzazione alternate a periodi di remissione clinica [ 2936 ]  . la nutrizione parenterale totale e nuovi farmaci efficaci , anche non convenzionali come linfliximab ( anti - alfa tnf ) , hanno modificato il ruolo della chirurgia ; si tende ad intervenire chirurgicamente sempre meno , in elezione , e sono sempre pi praticate stricturoplastiche , dilatazioni pneumatiche delle stenosi , drenaggi percutanei di raccolte ascessuali . per una corretta diagnosi e stadiazione della malattia risulta fondamentale lapporto di tutte le metodiche di imaging . 
i pazienti con malattia di crohn non costituiscono , infatti , una entit clinica omogenea e possono essere molto differenti tra loro , con situazioni cliniche polimorfe , influenzate dalla espressivit individuale della malattia e da eventuali precedenti terapie chirurgiche ; ci non sempre permette una standardizzazione clinico - strumentale uniforme e riproducibile [ 37 ]  . 
follow - up of patients with known crohns disease , even those with good response to medical therapy , is mostly carried out by ultrasound so as to exclude the appearance of complications with insidious course [ 4042 ]  . 
recurrence of crohns disease requires a similar diagnostic workup to that used for the initial diagnosis since lesion course in recurring disease is known to mimic the course at initial onset . however , it should be recalled that crohns disease is not the only cause of bowel - wall thickening . 
attualmente , la e - tcms viene di solito utilizzata subito dopo lesame ecografico od addirittura in prima istanza , sostituendo cos gradualmente il clisma del tenue tradizionale [ 38 , 39 ]  . la distensione del lume con mdc neutro e la somministrazione di mdc iodato e.v. 
il follow - up del paziente con malattia di crohn gi nota affidato oggi prevalentemente allesame ecografico , anche in caso di buona risposta al trattamento medico , allo scopo di escludere la comparsa di complicanze a sviluppo insidioso [ 4042 ]  . 
noto , infatti , che la naturale evoluzione delle lesioni in caso di recidiva mimi il decorso della malattia allesordio . comunque opportuno ricordare che la malattia di crohn non lunica causa di ispessimento delle pareti intestinali . 
nei casi in cui clinica e rilievi tc non siano concordi , pu risultare opportuno ricorrere allintegrazione diagnostica ( eventualmente in follow - up ) con le metodiche di contrastografia baritata . esse permettono uno studio morfologico accurato del rilievo mucoso , la cui semeiotica , consolidata da pluriennale esperienza , risulta senzaltro pi ricca di elementi diagnostici [ 49 ]  . lenteroclisi - rm una metodica emergente , ancora in via di sviluppo . 
essa sta fornendo ottimi risultati nello studio delle malattie del piccolo intestino , grazie alla sua eccellente risoluzione di contrasto ed alla possibilit di ottenere immagini sia statiche che dinamiche . 
transverse ultrasound scan ( a ) and spiral computed tomography ( ct ) ( b , c ) with intestinal opacification with positive contrast material in a patient with crohns disease in the descending colon , which shows thickened walls and narrowed lumen ( a , c )  . 
a hypoechoic fistula ( a ) originating in the colon and directed posteriorly feeds a large abscess collection with mixed content ( even gaseous ) in adjacent soft tissues , with involvement of the ileo - psoas muscle and reaching as far as the gluteal region . 
esame ecografico in scansione trasversale ( a ) e tc spirale ( b , c ) con opacizzazione intestinale con mdc positivo in paziente con localizzazione di malattia al colon discendente , che presenta pareti ispessite e lume ristretto ( a , c )  . 
tramite fistoloso ipoecogeno ( a ) a partenza colica e diretto posteriormente ; alimenta ampia raccolta ascessuale a contenuto misto ( anche gassoso ) nei tessuti molli contigui , con coinvolgimento del muscolo ileo - psoas e sconfinamento fino alla regione glutea . 
spiral computed tomography enteroclysis with negative endoluminal contrast material ( methylcellulose ) ; the intubation catheter is clearly visible in the jejunum ( a , b ) ; administration of iv iodinated contrast material . 
e - tc spirale con mdc endoluminale negativo ( metilcellulosa ) ; visibile il catetere da intubazione in sede digiunale ( a , b ) ; somministrazione di mdc iodato e.v. 
demonstration of extraluminal fistulas by spiral computed tomography after injection of iodinated contrast material through the outer opening of the fistulas ( a ) and after intestinal opacification with positive oral contrast material ( b )  . 
dimostrazione dei tramiti fistolosi extraluminali con esami tc spirale eseguiti dopo iniezione di mdc iodato attraverso lorifizio esterno delle fistole ( a ) , e dopo opacizzazione intestinale con mdc positivo per os ( b )  . 
there are , however , no definite findings allowing differential diagnosis with other forms of enteritis or crohns disease even if the wall enhancement is more intense at the mucosal level ( a )  . 
non ci sono comunque elementi sicuri che permettano una diagnosi differenziale con altre enteriti e con la malattia di crohn , anche se il ce parietale pi intenso lungo il versante della mucosa ( a )  . 
la paziente stata controllata successivamente ( dopo guarigione clinica ) con un altro esame radiografico , che ha dimostrato la regressione delle alterazioni in precedenza visibili . cy between clinical and ct findings , it may be useful to resort to diagnostic integration ( even during the follow - up ) with barium studies . 
these allow an accurate morphological study of the mucosa which , as years of experience have shown , is undoubtedly richer in diagnostic clues [ 49 ]  . mr - enteroclysis is an emerging technique that is still developing . 
the absence of ionising radiation and encouraging initial results herald a growing clinical role for mr enteroclysis [ 2 , 5057 ]  . intestinal bleeding in acute bleeds , endoscopy , nuclear medicine , and angiography preserve their fundamental role in both diagnostic and therapeutic terms [ 5860 ]  . 
however , in this field , we should recall the emerging role of capsule endoscopy [ 62 ]  . intestinal ischaemia mesenteric ischaemia is a complex disease [ 6365 ]  . 
in the acute phase , there is no indication for msct - e , and the method should be regarded as contraindicated until the intestinal distress and consequent risk of infarction and / or perforation is overcome . 
in addition , multiplanar reconstructions can provide impressive angiography - like images of the vascular arches [ 58 ]  . intestinal neoplasms small - bowel neoplasms are rare and often manifest with non - specific symptoms and signs , such as nausea , abdominal pain , occult blood , and weight loss . 
inoltre , le ricostruzioni multiplanari possono offrire suggestive immagini simil - angiografiche delle arcate vascolari [ 58 ]  . neoplasie intestinali i tumori dellintestino tenue sono rari e spesso si presentano con segni e sintomi aspecifici , come nausea , dolore addominale , sanguinamento occulto , dimagrimento . 
la scarsa incidenza di queste malattie e lintroduzione recente della metodica e - tcms non hanno ancora consentito lacquisizione di una casistica ampia e valida , dalla quale attingere aspetti di semeiotica utili per linquadramento diagnostico . 
i rilievi della e - tcms sono quasi sempre aspecifici , cos come lo sono , daltronde , anche quelli dellesame baritato . la diagnosi viene posta solo con lintegrazione dei dati clinico - polistrumentali . 
in reality , our experience has shown that patients with systemic mesenchymal diseases have a greater incidence of intestinal involvement , such as crohns enteritis , in that there is probably a common autoimmune basis . 
after years of experience with the technique , we feel we can propose msct - e as the examination of choice studying the small bowel , in particular , in partial occlusions , crohns disease , chronic occult bleeds , chronic ischaemia , and tumours . 
la e - tcms ( a ) dimostra la presenza di lesioni infiltranti a livello di alcune anse digiunali , in un paziente studiato per follow - up di neoplasia gi nota , nel quale erano presenti inoltre lesioni secondarie di pertinenza linfonodale a livello dei linfonodi inguinali ( b )  . gangrenoso , in letteratura descritta una incidenza minima di enteriti sincrone alla patologia mesenchimale principale . in realt , nella nostra esperienza abbiamo verificato che i pazienti portatori di mesenchimopatie sistemiche hanno una frequenza maggiore di coinvolgimento intestinale , come lenterite di crohn , in quanto hanno verosimilmente una comune base autoimmune . 
le nuove apparecchiature tc spirale multidetettore consentono scansioni veloci e collimazioni sempre pi sottili con conseguente miglioramento delle ricostruzioni multiplanari e 3d . attualmente , la nostra esperienza , ormai pluriennale , ci consente di proporre le - tcms come esame di scelta nello studio del piccolo intestino , in particolare nellocclusione parziale , nella malattia di crohn , nei sanguinamenti occulti cronici , nellischemia cronica e nelle neoplasie . la metodica non oggi proponibile nei pazienti in cui necessaria unanalisi accurata del rilievo mucoso intestinale e questo senza ombra di dubbio costituisce una limitazione importante della metodica , specie nelle patologie nelle quali sia richiesta unaccurata valutazione della integrit dello strato mucoso del piccolo intestino . se questa rappresenta lunica limitazione dal punto di vista diagnostico , non va dimenticato che esistono anche degli svantaggi , che si estrinsecano prevalentemente in alcune limitazioni tecniche , che non sono solo costituite dalla intubazione , indispensabile , naso - intestinale , ma anche dal fatto che molto spesso , quando non adeguatamente standardizzata , la metodica pu risultare limitata nella definizione diagnostica in relazione alla distensione sub - ottimale dellileo , legata alla impossibilit di seguire fluoroscopicamente la progressione del mezzo di contrasto neutro fino al colon destro . accanto allalto costo globale dellesame esistono , infine , secondo alcuni autori , problematiche relative alla probabile maggiore invasivit biologica della metodica . tuttavia unanalisi accurata della dose totale assorbita durante un esame baritato del piccolo intestino e durante un esame msct per lo studio del tenue fornisce risultati sorprendenti . 
baseline spiral computed tomography , with intestinal opacification with iodinated contrast mediu clear demonstration of the segment of pelvic ileum with diffusely and evenly thickened walls and stenotic lumen , responsible for the patients occlusive symptoms . 
although this is the techniques only diagnostic limitation , it should be recalled that there are also some disadvantages , prevalently due to technical shortcomings : as well as requiring naso - intestinal intubation , the technique , when not adequately standardised , may very often suffer diagnostic limitations as a fig . 
however , a careful analysis of the total dose absorbed during a barium study and an msct study of the small bowel provides surprising results . considering that the total absorbed dose during a radiographic study of the small bowel consists not only of the dose delivered while acquiring the radiograms but also that delivered during fluoroscopy , total absorbed dose is represented by the sum of the dose delivered during naso - intestinal intubation , the dose absorbed during fluoroscopic assessment of the passage of the contrast agent , and the dose absorbed while acquiring the radiograms , with a mean absorbed dose in depth of around 24.48 mgy for each procedure . 
as regards ct enteroclysis , total absorbed dose is represented by the sum of the dose delivered during fluoroscopy for naso - jejunal intubation and the dose delivered during a single volumetric scan from the diaphragm to the pelvis , with a mean absorbed dose in depth of around 11.2 mgy for each procedure if a single - slice spiral scanner is used . 
even if this figure is multiplied by the number of detector rows used in msct , the total absorbed dose during msct - e will be similar to , if not lower than , that delivered by conventional small - bowel enteroclysis . experience has taught us that modalities performed with naso - intestinal intubation allow a more accurate assessment of the small bowel compared with examinations requiring oral administration of contrast material . 
in this sense , even msct - e confirms that the luminal distension obtained with direct administration of suitable contrast material allows better assessment not only of the bowel wall but of all structures surrounding hollow abdominal organs and is undoubtedly an important diagnostic tool . 
rollandi ga , curone pf , biscaldi e et al ( 1999 ) spiral ct of the abdomen after distention of small bowel loops with transparent enema in patients with crohns disease . 
dallapiccola 962 pp , 835 illustrazioni springer - verlag berlin , heidelberg ( 2005 ) isbn 3 - 540 - 67997 - 9 published online : 10 march 2006 ricordo che dellintenzione e desiderio di scrivere un volume sullargomento , ne sentii parlare dal castriotascanderbeg nel lontano 1994 , durante una delle pause del congresso annuale della european society of paediatric radiology in corso di svolgimento a bruxelles . 
da allora ho seguito la progressiva evoluzione del progetto e la sua gestazione nel corso di 12 anni , come specificato nella prefazione , le difficolt incontrate nella sua preparazione e nel reperimento del materiale da utilizzare , fino al compimento di questo , che considero il tipico miracolo italiano , frutto di una perseveranza e di una dedizione totale allopera da parte del castriota - scanderbeg , autore di buona parte del testo . lopera veramente monumentale divisa in due parti : la prima , in 9 capitoli ( di cui uno solo a due mani ) , dedicata alla trattazione delle anomalie di cranio , torace , rachide , pelvi , ossa lunghe , mani , piedi , articolazioni ed anomalie generalizzate dello scheletro ; la seconda , alla trattazione e descrizione di 111 sindromi . 
la base concettuale della prima parte costituita dalla descrizione dei singoli segni o alterazioni morfologiche dei componenti ossei della porzione di scheletro interessata ed in studio , segni che possono rientrare in un pi vasto quadro di alterazioni e lesioni e che , proprio per il loro aspetto , talvolta anche sfumato , devono o dovrebbero far sospettare , allosservatore attento , la presenza di una peculiare patologia . 
ad esempio , il primo capitolo , dedicato al cranio , suddiviso in 6 sezioni ( anomalie di forma e / o dimensioni del cranio ; anomalie dello sviluppo e dellossificazione ; anomalie della base cranica ; anomalie delle ossa facciali e dei seni paranasali ; anomalie della mandibola ; calcificazioni endocraniche ) , a loro volta comprendenti diverse sottosezioni , quali : microcefalia , macrocefalia , craniosinostosi , per la sezione dedicata alle anomalie di forma e / o dimensioni del cranio ; oppure difetti dellossificazione , ritardata chiusura e / o incompleta ossificazione delle suture e delle fontanelle , ossa wormiane , ispessimento del cranio , per la sezione dedicata alle anomalie dello sviluppo e dellossificazione . 
tale impostazione si ripete nei capitoli successivi , risultando pi o meno ampia , ma sempre esauriente , a seconda della porzione di scheletro in studio . ciascuna parte comprende la descrizione del quadro clinico e radiologico di base ; quello delle sindromi , displasie o malattie in cui lalterazione pu esser rintracciata [ ciascuna riportata con la relativa classificazione omim ( on line mendelian inheritance in man ) e mappatura genica , ove conosciuta ] ; una sinossi radiologica delle proiezioni necessarie per lo studio della parte e dei segni in base ai quali porre la diagnosi ; un elenco delle associazioni con malattie , sindromi , displasie , anomalie cromosomiche , per finire con una bibliografia aggiornata , nonostante la tumultuosa evoluzione delle conoscenze e delle scoperte , soprattutto in campo genetico . la seconda parte , scritta in collaborazione con il prof . dallapiccola , uno dei massimi genetisti italiani , che con il suo contributo rende lopera completa da un punto di vista clinico , dedicata alla descrizione di 111 sindromi , displasie ed anomalie cromosomiche : di ciascuna vengono elencate sinteticamente eponimi od altre denominazioni , frequenza , genetica , sintesi analitica delle caratteristiche cliniche e radiologiche , diagnosi differenziale e bibliografia : il tutto corredato da immagini cliniche e radiografiche significative e dimostrative . 
questa parte non comprende le possibili sindromi o displasie , ritrovabili in altri testi e a cui si fa preciso riferimento nellintroduzione . le immagini , sia cliniche che radiologiche , sono riprodotte con notevole fedelt , ad eccezione di alcune non originali , riprese da altri testi . 
esse forniscono una rappresentazione completa per giungere , in modo relativamente semplice , ad una interpretazione di quadri talvolta se non spesso tenui od oscuri e rari e perci non facilmente apprezzabili ed interpretabili sia dal radiologo generale che dal clinico . forniscono inoltre la possibilit di un apprendimento esaustivo a coloro che desiderano approfondire o illustrare correttamente tali aspetti . il testo infine completato da un minuzioso ed esaustivo indice analitico . 
simonetti1 1dipartimento di diagnostica per immagini e radiologia interventistica , universit degli studi di roma tor vergata , viale oxford 81 , i - 00133 roma , italy 2dipartimento di neuroscienze , universit degli studi di roma tor vergata , roma , italy correspondence to : f . 
the regions of interest were manually positioned on rcbf maps over the caudate nucleus , the putamen , the external and internal globus pallidus , and over the ventrolateral nucleus of the thalamus . 
our study suggests that pw - mri is a valuable tool for assessing haemodynamic changes in pd patients . haemodynamic change pattern may be useful in the early diagnosis of pd . key words parkinsons disease perfusion - weighted magnetic resonance imaging basal ganglia riassunto obiettivo . 
sono state posizionate manualmente delle regioni di interesse ( roi ) sulle mappe di rcbf a livello del caudato , putamen , pallido interno / esterno e a livello del nucleo ventrolaterale del talamo . 
 diagnosis of pd is currently a clinical diagnosis only based on history , disease course , and finding of at least two of the three basic symptoms of the disease , i.e. 
on the other hand , in the early stages of disease , it is impossible to arrive at a firm differential diagnosis between idiopathic pd and atypical vascular or degenerative parkinsonisms on the sole basis of clinical data and initial response to treatment . 
indeed , early pd , when symptoms are extremely mild and blurred , may show only a subtle , if any , response to dopaminergic therapy , just as subjects with atypical parkinsonism may exhibit a limited response to therapy , which can persist for 12 years . 
diagnosis based only of such criteria was found to be incorrect in 25% of cases subjected to post - mortem histological examination at the united kingdom brain bank in london [ 2 ]  . 
interest in such a differential diagnosis lies mainly in the different course of these diseases and in the significant difference in how they respond to treatment . the neuroimaging techniques that have been used in the differential diagnosis mainly rely on nuclear medicine methods , such as single proton emission computed tomography ( spect ) and positron emission tomography ( pet ) [ 38 ]  . although these methods are extremely sensitive in the early detection of depleted nigrostriatal nerve endings and useful in the follow - up [ 911 ] , their limited availability ( in particular in the case of pet ) , high costs , limited supply of tracers , and poor spatial resolution make them unsuitable as a routine aid in the early diagnosis of pd . for instance , pet is unable to distinguish , within the basal nuclei , the putamen from the globus pallidus and even less the lateral from the medial pallidus . 
current theories on the pathogenesis of pd assign often diametrically opposed functions to these structures . an examination comprising them without differentiation [ 12 , 13 ] carries a risk of underestimating those alterations . the reduced metabolism of the external pallidus should correspond to increased activity of the internal pallidus without affecting total activity [ 14 ]  . the purpose of this study was to use perfusion - weighted magnetic resonance imaging ( pw - mri ) as a diagnostic method to identify a specific perfusion pattern in the basal ganglia of pd subjects . il morbo di parkinson ( mdp ) una patologia neurodegenerativa caratterizzata da una preferenziale degenerazione dei neuroni dopaminergici a livello della pars compacta della sostanza nigra ( sn ) [ 1 ] e di altri pigmenti tronco - encefalici , nonch dalla comparsa , nei neuroni coinvolti nel processo degenerativo , di inclusioni citoplasmatiche note come corpi di levy . la diagnosi di morbo di parkinson attualmente una diagnosi clinica basata solo sulla anamnesi , sul decorso della malattia e sul riscontro obiettivo di almeno due dei tre sintomi cardine della patologia , vale a dire della rigidit , di una ipo - bradicinesia e del tremore a riposo con caratteristica asimmetria tra i lati corporei in termini di epoca di comparsa ed intensit dei sintomi ; inoltre di estrema rilevanza , sempre ai fini diagnostici , la risposta ai farmaci dopaminergici nel tempo . 
daltro canto , in fase iniziale del tutto impossibile arrivare a porre una diagnosi differenziale certa tra il morbo di parkinson idiopatico ed i parkinsonismi atipici su base vascolare o degenerativa , sulla sola base dei dati clinici e della risposta farmacologica iniziale ; infatti , nei pazienti con parkinson iniziale ( early pd ) il cui quadro clinico sia quindi estremamente lieve e sfumato , la risposta alla terapia dopaminergica pu non essere eclatante anche quando presente , cos come in soggetti affetti da parkinsonismi atipici si pu osservare una risposta alla terapia farmacologica che , ancorch ridotta , perdura per uno - due anni . una diagnosi effettuata esclusivamente sulla base di tali criteri , ha portato ad errore nel 25% dei casi sottoposti postmortem ad esame autoptico - istologico presso la brain bank di londra [ 2 ]  . 
linteresse per una tale diagnosi differenziale risiede principalmente nella differente evoluzione delle patologie sopra citate e nella notevole differenza della rispettiva risposta ai farmaci . fino ad oggi le tecniche di neuroimaging utilizzate per la valutazione di tale diagnosi differenziale si sono basate principalmente sulla utilizzazione di metodiche di medicina nucleare quali la spect e la pet [ 38 ]  . 
pur essendo tali metodiche altamente sensibili nel rilevare precocemente una deplezione delle terminazioni nervose nigro - striatali e utili nel follow - up della malattia [ 911 ] , la loro ridotta disponibilit ( in particolare della pet ) , gli alti costi , la ridotta disponibilit di traccianti e la scarsa risoluzione spaziale le rendono inadatte come supporto routinario alla clinica nella diagnosi precoce di mdp . 
infatti , ad esempio , la pet non permette di distinguere allinterno dei nuclei della base il putamen dal globo pallido e tanto meno il pallido laterale da quello mediale . molti degli studi effettuati con pet nel mdp hanno in effetti studiato le variazioni di densit recettoriale , fibre dopaminergiche o le variazioni di metabolismo nel nucleo lenticolare . 
in order to avoid any interferences caused by the effect of dopaminergic drugs on the basal ganglia flow [ 16 ] , the subjects were studied after at least 20 days withdrawal of dopaminergic treatment . 
a sagittal t2 - tse sequence ( slice thickness 3 mm , number of averages 3 ) was used to identify the two commissures on which we performed an axial t1 - se sequence , a t2 - weighted sequence using cerebral spinal fluid ( csf ) suppression [ axial fluid attenuated inversion recovery ( flair ) ] with a fov 230 mm , te 150 ms , tr 6 , 000 ms , ti 2 , 000 ms , slice thickness 5 mm , gap 1 mm , matrix 256x256 , number of averages 1 , acquisition time approximately 3 m the pw - mri study was carried out using a gradient echo ( ge ) , t2 * - weighted sequence with tr 620 ms , te 30 ms , al diminuito metabolismo del pallido esterno dovrebbe corrispondere un aumento di attivit in quello interno con una somma dei due probabilmente invariata [ 14 ]  . lo scopo di questo studio stato quello di utilizzare la rm perfusionale come metodica diagnostica per identificare uno specifico pattern di perfusione a livello dei gangli della base nei soggetti affetti da mdp . materiali e metodi sono stati studiati 20 soggetti affetti da parkinson idiopatico ( 11 maschi , 9 femmine ; et media 683 , 2 anni ) secondo i criteri della brain bank [ 15 ] , con una durata di malattia variabile tra i 52 , 4 anni . 
sono stati esclusi da questo gruppo di pazienti in studio , soggetti che presentavano altre patologie neurologiche e che non rispondevano alla terapia acuta . per evitare interferenze possibili dovute allazione dei farmaci dopaminergici sul flusso dei gangli della base [ 16 ] , i soggetti sono stati studiati dopo un periodo di interruzione del trattamento dopaminergico di almeno venti giorni . 
finally , the axial t1 sequence was repeated after administration of gadoliniu gadolinium diethylene triamine pentaacetate ( g - dtpa ) paramagnetic contrast material ( magnevist , schering , berlin , germany ) was injected as a bolus , 0.4 mmol / kg , while the subjects held their eyes closed , via an 18 - gauge needle cannula inserted into an antecubital vein of the arm using an automatic injector with a 4 ml / s flow rate . perfusion data were processed by dedicated software on an independent console to obtain perfusion maps to calculate cerebral blood flow ( rcbf ) according to the technique previously reported by apruzzese et al . 
then , ten - pixel regions of interest ( rois ) were manually placed on the rcbf maps in the following regions : head of the caudate nucleus ( cn ) , putamen ( pu ) , external and internal globus pallidus ( egp / igp ) separately , and ventrolateral nucleus of the thalamus ( th )  . 
perfusion data were calculated as the average of every roi positioned on the basal nuclei and powm on every side and values obtained were transformed into logarithms and normalised as a percentage of the value obtained with the ipsilateral powm . in order to determine asymmetries ( contrast effect ) , the normalised data of each nucleus were expressed as the ratio between the nucleus being studied and the corresponding contralateral nucleus according to the formula : ( r nucleus l nucleus ) / ( r nucleus + l nucleus ) * 100 . 
in pd patients , the formula was expressed as : [ ( less affected side more affected side ) / ( less affected side + more affected side ) * 100 ] based on their clinical asymmetry . 
we used the third section of the unified parkinsons disease rating scale ( updrs ) as a score of pd motor disability , ranging from 0 ( normal ) to 180 ( maximum disability ) [ 18 ]  . 
in order to compare contrast data in the two groups , the factors group and nuclei were used . results all patients with pd showed a significant ( f ( 1 / 37 ) = 7.98 ; p < 0.01 ) difference in the average rcbf on the better side ( bs ) 1 , 5 tesla ( philips medical - system , best , olanda ) con intensit di gradiente di 30 mt con profilo sinusoidale e con una bobina in quadratura standard dedicata per lo studio dellencefalo . abbiamo eseguito una sequenza t2 tse sul piano sagittale ( spessore di strato 3 mm , numero di medie 3 ) per la individuazione delle due commessure sulle quali abbiamo impostato una sequenza t1 se assiale , una sequenza t2 pesata con la tecnica della soppressione liquorale ( flair assiale ) con fov 230 mm , te di 150 ms , tr = 6000 ms , ti = 2000 ms , spessore di strato 5 mm gap di 1 mm , matrice 256x256 , numero di medie 1 con un tempo di acquisizione di 3 minuti circa . lo studio rm perfusionale stato effettuato utilizzando una sequenza gradient echo ( ge ) t2 * pesata con un tr = 620 ms , te = 30 ms , spessore di strato = 7 , gap = 0 mm , numero di medie 1 , flip angle = 40 , matrice = 128x128 , tempo di acquisizione = 1 , 17 min , al fine di ottenere una serie di immagini dinamiche a suscettibilit di contrasto t2 pesate acquisite durante il passaggio di mdc . 
il mdc paramagnetico gadolinio - dtpa ( magnevist , shering , berlino , germania ) stato iniettato a bolo , 0 , 4 mmol / kg mentre i soggetti tenevano gli occhi chiusi , mediante iniettore automatico con una velocit di flusso pari a 4 ml / s ed attraverso un ago - cannula del calibro di 18 gauge posizionato in una vena antecubitale del braccio . 
i dati di perfusione sono stati elaborati tramite software dedicato su consolle indipendente , ottenendo quindi mappe di perfusione per il calcolo del flusso ematico cerebrale ( rcbf ) , in accordo a quanto gi descritto da apruzzese et al . 
in un precedente lavoro [ 17 ]  . sono state quindi posizionate manualmente sulle mappe dellrcbf delle roi di 10 pixel a livello della testa del nucleo caudato ( cn ) , a livello del putamen ( pu ) , a livello del globo pallido esterno ed interno ( gpe / gpi ) separatamente , ed a livello del nucleo ventrolaterale del talamo ( th )  . 
i dati della perfusione sono stati calcolati come la media di ogni roi posizionata a livello dei nuclei della base e di quelle posizionate a livello della wpom di ogni lato , i valori ottenuti sono stati trasformati in logaritmo , e quindi normalizzati come percentuale del valore ottenuto con la wpom omolaterale . 
per la determinazione delle asimmetrie ( effetto contrasto ) le normalizzazioni dei dati relativi a ciascun nucleo sono stati espresse come rapporto tra il nucleo in esame ed il corrispondente controlaterale , secondo la formula : ( nucleo dx - nucleo sn ) / ( nucleo dx + nucleo sn ) * 100 . 
nei pazienti con morbo di parkinson la formula stata espressa come : [ ( lato meno affetto - lato pi affetto ) / ( lato meno affetto + lato pi affetto ) * 100 ] in accordo con la loro asimmetria clinica . 
abbiamo usato la sezione iii dellupdrs ( unified parkinsons disease rating scale ) come score di disabilit motoria nel parkinson che va da 0 ( normale ) a 180 ( massimo livello di disabilit ) [ 18 ]  . 
seven patients with pd had abnormal contrast values in all nuclei under study whereas these were normal in all control subjects . traendo lo score del lato meno affetto da quello del lato pi affetto . per lanalisi dei dati stato utilizzato un software statistica per windows . 
la correzione di greenghouse geisser stata utilizzata quando necessario . in caso di significativit di un fattore con pi di due livelli od in caso di significativit di interazioni tra fattori stato utilizzato il tukey test come post hoc . per comparare i dati contrasto , nei due gruppi sono stati usati i seguenti fattori : gruppo , nuclei . discussion the diagnosis of pd remains largely clinical and is based on the presence of the three characteristic pathognomonic symptoms . 
pet and spect are still , however , very costly methods , use of which is restricted to a small number of highly specialised centres ; hence , they cannot be proposed as screening tests . 
over the last few years , various researchers have used pw - mri to measure cbf in several diseases of neuroradiological concern . the literature does not provide information on possible altered flow values detected in degenerative diseases such as pd with pd patients controls fig . 
2 il grafico evidenzia una asimmetria interemisferica dei valori di rcbf nei gangli della base nei pazienti con mdp non presente nei soggetti controllo . risultati nella totalit dei pazienti con mdp si dimostrato una significativa ( f ( 1 / 37 ) = 7 , 98 ; p < 0 , 01 ) differenza dellrcbf medio nel lato migliore ( bs ) vs . 
la differenza risulta essere simile in tutti i nuclei studiati cos che nessuna interazione significativa gruppox nuclei stata riscontrata . sedici dei 20 pazienti con mdp hanno mostrato un anormale indice di contrasto nel putamen e 13 hanno mostrato la stessa anormalit nel talamo . solamente 5 soggetti controllo hanno mostrato valori anormali nel putamen e nessuno nel talamo . 
sette pazienti con mdp hanno mostrato valori anormali di contrasto in tutti i nuclei studiati , mentre in nessuno dei soggetti controllo si evidenziato tale riscontro . discussione la diagnosi di morbo di parkinson rimane prevalentemente clinica e si basa sulla presenza della caratterizzazione della triade sintomatologia patognomonica . 
a possible explanation of this result might be the loss of dopamine in the affected side , which could result in a maximum increase in the baseline rcbf on this side , an asymmetry that was never observed in control subjects . 
wolfson conducted a cerebral perfusion study using pet on patients affected by mild - to - moderate forms of unilateral and bilateral pd , finding blood flow asymmetry in the basal ganglia on both sides and with respect to controls . 
other pet studies , on the other hand , did not find any asymmetry between homologous basal ganglia regions but only when the rcbf in basal ganglia was compared with the flow in critical cortical areas [ 19 ]  . 
we were also able to detect a perfusion asymmetry in most pd patients ( 65%80% ) by analysing homologous sub - regions of basal nuclei , thanks to the higher resolution of mri relative to pet . we believe that future studies analysing the ratio between cortical areas and basal ganglia might be useful to better demonstrate the abnormalities found in our investigation . pochi centri ad elevata specializzazione , non quindi proponibile il loro impiego come esame di screening . la rm , al contrario , una metodica molto pi diffusa , meno dispendiosa e disponibile in numerosi centri di diagnostica per immagini . 
tuttavia , a tuttoggi , non sono riportati dati in letteratura circa la possibilit di evidenziare alterati valori di flusso nellambito di patologie degenerative come la mdp mediante la rm perfusionale . il nostro lavoro , unico nel suo genere , si propone di valutare alterazioni perfusionali a livello dei nuclei della base , nei soggetti con mdp . i nostri dati mostrano una notevole asimmetria dellrcbf tra il lato pi affetto ed il lato meno affetto nei nuclei della base dei pazienti con mdp . 
una possibile spiegazione di tale risultato , a nostro giudizio , potrebbe essere dovuta alla maggior perdita di dopamina nel lato affetto , che potrebbe indurre un incremento massimale del rcbf in questo lato in condizioni basali . 
tale asimmetria non stata , infatti , mai osservata nei soggetti controllo . una relativa asimmetria dellrcbf nei gangli della base , stata riportata anche in altri studi con pet nei pazienti con mdp unilaterale o bilaterale . 
wolfson ha condotto uno studio di perfusione cerebrale , valutata con la pet , su pazienti affetti da forme lievi / moderate di mdp mono e bilaterale ed ha evidenziato una asimmetria del flusso a livello dei gangli della base di entrambi i lati e rispetto ai controlli . 
altri studi con pet , invece , non hanno mostrato asimmetria tra regioni dei gangli della base omologhe , ma solamente se lrcbf dei nuclei della base erano comparati con quelli di aree corticali critiche [ 19 ]  . il nostro studio conferma in parte questi dati , come precedentemente descritto , attraverso una metodica molto meno dispendiosa quale la risonanza magnetica perfusionale . 
stato inoltre possibile , grazie alla pi alta risoluzione della rm rispetto alla pet , rivelare unasimmetria perfusionale nella maggioranza dei pazienti con mdp ( 65%80% ) esaminando subregioni omologhe dei nuclei della base . 
riteniamo che studi futuri , che analizzeranno la ratio tra le aree corticali ed i gangli della base , potrebbero essere utili per dimostrare ancor meglio le anormalit evidenziate nel nostro lavoro . conclusioni conclusions pw - mri appears to be a valid method in the diagnosis of pd since it can identify blood flow asymmetry in the basal nuclei in both hemispheres . 
the low cost of this method , its wide availability , and its higher resolution compared with other functional imaging modalities ( pet , spect ) make pw - mri extremely useful and expedient as a support tool for the early diagnosis of pd . la rm perfusionale sembra essere una metodica valida per la diagnosi di mdp , essendo in grado di individuare unasimmetria di flusso a livello dei nuclei della base dei due emisferi . 
bacci1 1sezione di chemioterapia , 2servizio di radiologia , dipartimento di oncologia muscoloscheletrica , istituti ortopedici rizzoli , via pupilli 1 , i - 40136 bologna , italy correspondence to : a . 
between march 1997 and december 2001 at our department of musculoskeletal oncology , 231 patients with peripheral osteosarcoma received a central venous catheter to allow infusion therapy and blood sampling . 
in ten cases ( 4.3% ) , radiology showed damage to the alveolar interstitium typical of inflammatory forms , whereas in the remaining three ( 1.3% ) it depicted nodular opacities , which required differentiation between lung metastases and septic emboli . 
the most common complications of the use of central venous catheters include infection and venous thrombosis whereas pulmonary septic emboli are rare . key words osteosarcoma pulmonary metastases pulmonary nodules differential diagnosis riassunto obiettivo . 
da marzo 1997 a dicembre 2001 , presso il nostro dipartimento di oncologia muscoloscheletrica stato inserito un catetere venoso centrale per permettere terapia infusionale e prelievi ematici a 231 pazienti affetti da osteosarcoma periferico . 
in 10 casi ( 4 , 3% ) il quadro radiologico era di impegno interstizio alveolare tipico delle forme flogistiche , mentre nei restanti 3 ( 1 , 3% ) si osservarono delle opacit nodulari che posero problemi di diagnosi differenziale fra metastasi polmonari ed emboli settici . 
le complicanze pi frequenti dellutilizzo del catetere venoso centrale sono le infezioni e le trombosi venose , mentre gli emboli settici polmonari sono rari . parole chiave osteosarcoma metastasi polmonari noduli polmonari diagnosi differenziale introduction introduzione the use of hickman , broviac and groshong indwelling central venous catheters ( cvcs ) , which remain in place for months or years , is highly important for cancer patients who require prolonged chemotherapy and support infusion therapies . 
venous catheters connected with their port to a central venous line , such as infuse - a - port or port - a - cath , have also been used with success . 
vein perforation , generally caused by the insertion of the catheter or its incorrect positioning , is fortunately an uncommon immediate or early complication . the most frequent late complications are venous thrombosis of the vessel in which the catheter is positioned and infections . 
the incidence of thrombosis varies between 12% and 74% and is higher in cancer patients [ 1 ]  . the thrombosis may be located in the vessel or in the catheter lumen ; only one - third of thromboses are symptomatic . 
a number of studies [ 24 ] have demonstrated that thrombosis is one of the main risk factors for catheter infection whereas infections occur in 15%20% of cases [ 5 , 6 ]  . septic emboli resulting from catheter infection are a wellknown but uncommon complication , even in children and adolescents [ 6 , 7 ]  . the central venous line is a very important tool for delivering chemotherapy , especially in children . 
many studies have demonstrated the usefulness and feasibility of prolonged use , up to years , of a cvc even in children , with an acceptable incidence of complications : displacement , occlusion and infection [ 710 ]  . 
the aim of this study was to highlight the problems of differential diagnosis and the possible solutions using conventional radiography and computed tomography ( ct )  . materials and methods between march 1997 and december 2001 , the division of chemotherapy of our institution treated 253 patients with adjuvant chemotherapy for primitive bone sarcomas . 
anche i cateteri venosi collegati con la propria porta ad un catetere venoso centrale tipo infuse - a - porth o port - a - cath sono impiegati con successo . entrambi i tipi di cateteri sono associati a possibili complicazioni sia precoci che tardive . 
alcuni studi [ 24 ] hanno dimostrato che la trombosi uno dei principali fattori di rischio per linfezione del catetere venoso centrale , mentre le infezioni sono una complicanza che si verifica in circa il 15%20% dei casi [ 5 , 6 ]  . 
gli emboli settici conseguenti a infezioni del catetere sono una complicanza ben conosciuta , pur se non frequente , anche nei pazienti pediatrici od adolescenti [ 6 , 7 ]  . il catetere venoso centrale uno strumento molto importante per la somministrazione della chemioterapia in particolare nei bambini . 
molti studi hanno dimostrato lutilit e la fattibilit di un uso prolungato , talora per anni , del catetere venoso centrale anche in pazienti pediatrici con una percentuale accettabile di complicanze : dislocazioni , occlusioni ed infezioni [ 710 ]  . 
lo scopo del lavoro di evidenziare le problematiche di diagnosi differenziale e le possibilit di soluzione mediante radiologia tradizionale e tomografia computerizzata ( tc )  . materiali e metodi dal marzo 1997 al dicembre 2001 , presso lunit operativa di chemioterapia del nostro istituto sono stati trattati con chemioterapia adiuvante per osteosarcoma primitivo 253 pazienti ; 34 di questi pazienti presentavano metastasi alla diagnosi : 31 al polmone e 3 in altri segmenti ossei ; in 219 pazienti la lesione era localizzata . 
a 231 pazienti fu inserito un catetere venoso centrale : 228 cvc tipo broviac e 3 port - a - cath ; 22 pazienti tutti adulti rifiutarono il posizionamento del catetere . 
a second radiological assessment of the primary cancer with ct and mri , and chest ct was performed at the end of preoperative chemotherapy ( lasting four to six cycles , depending on the protocol ) , and before surgery . 
given the prolonged anticancer treatment typical of osteosarcoma ( 3643 weeks , depending on the protocol ) , conventional chest radiography was performed every 2 months to exclude the development of metastasis . in order to prevent catheter occlusions , 5 cc of heparin solution ( 20 cc of saline plus 3 cc of sodium heparin equal to 5 , 000 iu diluted in 100 cc of saline ) was injected into the central venous line every other day . 
patients with neutropoenia ( neutrophils < 500 / mm3 ) received granulocyte colony stimulating factors ( gcsf ) , together with a broad - spectrum antibiotic such as trimethoprim , sulfamethoxazole or ceftriaxone , at a dose of 5 g / kg per day until the white blood cell count ( wbc ) reached a concentration of at least 10 , 000 / mm3 . 
he received neoadjuvant chemotherapy from july 1998 to june 1999 after a broviac central venous catheter had been positioned in the left subclavian veone week after the last chemotherapy cycle ( cisplatin ) , the patient developed chills and fever ( 39c ) while at home . 
central and peripheral blood cultures were performed , both of which were positive for staphylococcus aureus and , on the basis of the antibiogram , antibiotic therapy with 200 mg / day teicoplanin was started , leading to remission of the fever . 
three weeks , later the patient was admitted for removal of the cvc and final staging with lung ct . the material sampled from the catheter after removal showed colonies of s . 
ct , performed on the same day ( 20 july 1999 ) , revealed the presence of a nodular opacity in the posterior segment of the lower right lobe that was suggestive of pulmonary metastasis from osteosarcoma teteri , usati sia per la somministrazione della chemioterapia che per prelievi ematici , furono posizionati prima dellinizio dei cicli di chemioterapia , previo consenso informato . tutti i pazienti ricevettero chemioterapia secondo i protocolli in uso al momento , ior / os - n4 o ior / os - n5 , impiegati dal 1997 al 2001 con i seguenti farmaci : adriamicina , cisplatino , ifosfamide , pi metotrexate nei pazienti al di sotto dei 40 anni ( precedentemente riportati in dettaglio [ 11 , 12 ] )  . la stadiazione radiologica di base comprendeva tc e rm del tumore primitivo osseo , scintigrafia ossea e tc del torace per escludere lesioni sostitutive secondarie polmonari . una seconda valutazione radiologica con tc ed rm del tumore primitivo e tc del torace stata eseguita al termine della chemioterapia preoperatoria ( durata 46 cicli a seconda del protocollo ) , prima dellintervento chirurgico . 
dato il prolungato trattamento antiblastico tipico dellosteosarcoma ( da 36 a 43 settimane a seconda del protocollo ) uno studio radiologico tradizionale del torace veniva eseguito ogni 2 mesi per escludere la comparsa di metastasi . a scopo profilattico , per evitare occlusioni del catetere , a giorni alterni si iniettavano nel catetere venoso centrale 5 cc di soluzione eparinata ( 20 cc di soluzione fisiologica pi 3 cc di eparina sodica pari a 5000 ui diluiti in 100 cc di soluzione fisiologica )  . 
in caso di neutropenia ( neutrofili inferiori o uguali a 500 / mm3 ) fattori stimolanti la crescita di colonie ( gcsf ) , congiuntamente ad un antibiotico ad ampio spettro tipo trimethoprim , sulfametoxazolo o cefriaxone , venivano somministrati ai pazienti alla dose di 5g / kg / die fino a che la concentrazione dei globuli bianchi ( wbc ) non raggiungeva il valore di 10000 / mm3 . tredici pazienti su 231 ( 5 , 6% ) svilupparono uninfezione del catetere venoso centrale . 
in 10 pazienti ( 4 , 3% ) lindagine radiologica tradizionale e / o la tc del torace evidenziarono un impegno interstizio - alveolare tipico delle forme flogistiche e nei restanti 3 ( 1 , 3% ) opacit nodulari , alcune delle quali a margini regolari che posero problemi di diagnosi differenziale con lesioni metastatiche . caso 1 maschio , 7 anni , con osteosarcoma osteoblastico localizzato allomero destro . 
una settimana dopo lultimo ciclo di chemioterapia ( cisplatino ) , il paziente ebbe , a domicilio , un episodio di febbre ( 39c ) con brividi ; esegu due emocolture : una da prelievo centrale e una da prelievo periferico . 
although the patient was asymptomatic , the recent cvc infection was thought to have caused the spread of a septic embolus to the lung , so antibiotic therapy with teicoplanin ( 200 mg / day ) was administered for a further 15 days . 
shortly after the end of the last cycle of postoperative chemotherapy , he developed a fever ( 40c ) and was treated at home with a broad - spectrum antibiotic that failed to resolve the fever . two - view chest radiography performed at the patients home was negative . 
on june 15 , the patient was admitted , and the cultures of material sampled from the central venous line and a peripheral vein both revealed the presence of pseudomonas fluorescens sensitive to piperacillthe central venous line was removed . 
poco dopo il termine dellultimo ciclo di chemioterapia postoperatoria , ebbe febbre ( 40c ) e inizi a domicilio terapia antibiotica a largo spettro che non ne determin la remissione . il radiogramma tradizionale del torace eseguito in due proiezioni al domicilio del paziente era negativo . 
il 15 giugno il paziente fu ricoverato e le emocolture del materiale prelevato dal catetere centrale a da vena periferica evidenziarono entrambe la presenza di psudomonas fluorescens sensibile alla piperacillina . 
nello stesso giorno il paziente esegu , come da protocollo , tc del torace di fine cura , che evidenzi la presenza di tre opacit nodulari nel polmone destro : la prima nel segfig . 
1a , b the first computed tomography ( ct ) study shows a nodular opacity measuring 15 mm in diameter with slight irregular margins in the posterobasal segment of the right lower lobe ( a )  . 
1a , b il primo esame evidenzia una opacit nodulare di 15 mm di diametro a margini leggermente irregolari nel segmento posterobasale del lobo inferiore di destra ( a )  . 
2a - d the first computed tomography ( ct ) study shows three subpleural nodular opacities ( arrows ) , two measuring a few millimetres : the first has paracardiac location in the middle lobe and no feeding vessel sign ; the second is in the laterobasal segment of the right lower lobe ( a ) ; the third is larger and located in the posterobasal segment of the right lower lobe ( b )  . 
2a - d il primo esame tc evidenzia tre opacit nodulari subpleuriche ( frecce ) di cui due millimetriche : la prima localizzata in sede paracardiaca a livello del lobo medio senza il segno del vaso che nutre , la seconda nel segmento laterobasale del lobo inferiore destro ( a ) , la terza opacit , di dimensioni superiori , nel segmento posterobasale del lobo inferiore di destra ( b )  . 
dopo somministrazione di terapia specifica , il secondo esame tc eseguito tre mesi dopo , non evidenzia pi alcuna lesione ( c , d )  . case 3 a 17 - year - old girl with fibroblastic osteosarcoma of the right femur . 
chest radiography demonstrated two nodular opacities in the middle third of the right lung field , both of which where strongly suspicious for metastases . chest ct performed immediately confirmed the presence of the lesions , which were subpleural and with slightly irregular margins , one in the anterior segment and the other in the posterior segment of the lower lobe . 
chemioterapia neoadiuvante dal dicembre 2000 attraverso un catetere centrale tipo broviac . terminato il nono ciclo di chemioterapia , la paziente ebbe un episodio di neutropenia febbrile risoltosi con somministrazione di terapia antibiotica a largo spettro e di fattori stimolanti la crescita . 
la tc del torace eseguita immediatamente conferm la presenza delle due lesioni entrambe subpleuriche e con margini lievemente irregolari , una nel segmento anteriore e laltra nel segmento posteriore del lobo inferiore . 
la paziente a tuttoggi viva , senza lesioni sostitutive . discussione losteosarcoma un tumore osseo estremamente maligno tipico dei bambini e degli adolescenti con un picco tra i 15 ai 25 anni [ 13 ]  . 
il polmone rappresenta , infatti , la pi frequente sede ( 90% di tutte le sedi di metastasi ) di metastasizzazione di questo tumore alla diagnosi e durante il follow - up [ 17 ]  . 
3a - c the first computed tomography ( ct ) study shows two subpleural nodular opacities ( arrows ) : the first in the apical segment of the right lower lobe ; the second , clearly inflammatory , in the lateral segment of the middle lobe ( a )  . 
the second ct study performed after 20 days shows disappearance of the two opacities , with moderate residual subpleural fibrosis in the middle lobe and the presence of a new opacity with irregular and fluffy margins contiguous with a pulmonary vessel in the posterolateral segment of the right lower lobe ( b )  . 
3a - c il primo esame tc evidenzia due opacit nodulari subpleuriche ( frecce ) : la prima nel segmento apicale del lobo inferiore destro , la seconda chiaramente flogistica nel segmento laterale del lobo medio ( a )  . 
il secondo esame tc , eseguito a 20 giorni di distanza , mostra la scomparsa delle due opacit con modesto esito fibrotico subpleurico nel lobo medio e la presenza di unaltra opacit , a contorni irregolari e sfrangiati in continuit con un vaso polmonare nel segmento posterolaterale del lobo inferiore destro ( b )  . 
the lung is often the first site of metastatic spread of many solid tumours [ 16 ] , and this is particularly true for osteosarcoma . the lung is the most frequent site of metastasis ( 90% of all metastasis sites ) of this tumour at presentation and during follow - up [ 17 ]  . osteosarcoma of the extremities tends to metastasize within the first 5 years of diagnosis in 30%40% of cases [ 13 ]  . 
early diagnosis of such lesions may in some cases dramatically increase the survival of patients after appropriate treatment : thoracic surgery and / or chemotherapy . all osteosarcoma protocols require ct scans at baseline and after treatment and conventional chest radiography every 2 months during chemotherapy . 
despite not having the same reliability as ct in detecting metastasis , conventional chest radiography in two views anteroposterior ( ap ) and laterolateral ( ll ) is just as effective in identifying metastatic lesions with a diameter of 1 cm or more , it is easy to perform and it is inexpensive . 
in addition , entrance dose and absorbed dose are much lower than in ct : 1.9 mgy / msi versus 30 mgy / msi , ctdiw / 650 mgy / msi - cm dlp . development of lung metastases during treatment is a rare but possible occurrence . 
mediastinal lymph nodes are rarely involved [ 13 ]  . oncological patients who are free of disease may , however , develop pulmonary nodules of varying size , shape and location , as has been reported by many studies [ 1824 ]  . 
almost all of cases reported were treated either with drugs with minimal bone marrow toxicity and high pulmonary toxicity or drugs and radiotherapy [ 19 ]  . many underwent segmental or lobar resection . 
histology revealed not only metastatic nodules but also the presence of nodular lesions of other natures : nodular nonspecific intersitial pneumonia or fibrosis in patients with peripheral infusion of drugs with high pulmonary toxicity , such as bleomycin [ 2024 ] or nodular lesions caused by infection by opportunistic organisms ( cryptococcus neoformans , aspergillus fumigatus , or others ) in patients treated with radiotherapy who became immunocompromised . infectious processes may also include septic emboli developing inside the cvc or migrating there from other sites . 
the finding of areas of pulmonary consolidation of varying shape on ct scans of cancer patients represents a challenge , and the differential diagnosis is one of the radiologists routine tasks . 
 lung nodules identified in our three patients were both subpleural and parenchymal ; they had similar density but ri metastatici osservati su un radiogramma del torace e / o una tc del torace in pazienti con tumore primitivo extrapolmonare da considerarsi segno prognostico infausto . 
una diagnosi precoce di lesioni sostitutive pu talora dare lopportunit di una lunga sopravvivenza al paziente dopo adeguata terapia : chirurgia toracica e / o chemioterapia . tutti i protocolli per gli osteosarcomi richiedono una tc di base allinizio del trattamento , unaltra alla fine e radiogrammi tradizionali del torace ogni due mesi durante il lungo periodo della chemioterapia . 
il radiogramma tradizionale del torace in due proiezioni ( p - a e l - l ) pur non avendo la stessa affidabilit della tc nel rilevare metastasi egualmente efficace nellevidenziare lesioni sostitutive di diametro pari o superiore al centimetro , di facile esecuzione , poco costoso , inoltre la dose allingresso / dose assorbita sono di molto inferiori a quelle di una tc : 1 , 9 mgy / msi versus 30 mgy / msi , ctdiw / 650 mgy / msi - cm dlp . 
i linfonodi mediastinici sono raramente coinvolti [ 13 ]  . tuttavia in pazienti neoplastici liberi da malattia possibile osservare la comparsa di noduli polmonari di dimensioni , forma e sede variabile ; tale comparsa stata descritta in molti lavori [ 1824 ]  . 
quasi tutti questi pazienti sono stati trattati o solo con farmaci a minima tossicit midollare ed ad alta tossicit polmonare o con farmaci e radioterapia [ 19 ]  . molti di questi pazienti furono sottoposti a resezione chirurgica segmentaria o lobare . 
lesame istologico rivel non solo la presenza di noduli metastatici , ma anche la presenza di lesioni nodulari di differente natura : polmoniti interstiziali nodulari aspecifiche e fibrosi polmonare interstiziale nodulare in pazienti con infusione periferica di farmaci ad alta tossicit polmonare come la bleomicina [ 2024 ] o lesioni nodulari di natura infettiva provocate da organismi opportunisti ( criptococcus neoformans , aspergillus fumigatus o altri ) in pazienti precedentemente trattati con radioterapia e divenuti ospiti immunocompromessi . 
i sintomi clidifferent shape , being rounded or lobulated but never reticular ; their margins were smooth and regular or fluffy and irregular , but never hazy ; size varied between 3 mm and 25 mthe two patients in whom the catheter was removed immediately and s . 
lung metastases from any primary cancer are well - marginated nodular opacities that are at times very calcific ( especially in osteosarcoma ) ; they tend to have peripheral location but have no predilection for the upper , middle or lower lobes [ 25 ]  . in our three cases , all pulmonary nodules simulating metastases appeared during chemotherapy in patients with broviac cvcs positioned in the subclavian vein for the delivery of chemotherapy , transfusions or blood sampling . 
none of the three patients had symptoms of pulmonary embolism . three cases of pulmonary septic embolism out of 231 patients with cvc is indeed a small percentage ( 1.3% ) , in agreement with other studies [ 6 ]  . 
pulmonary infection could have been the cause rather than the consequence of cvc infection but , given that culture of material retrieved from the distal lumen of the catheter was positive in two out of three cases , the cause of the haematopulmonary infection was far more likely to have been the cvc . the three cases described demonstrate that cvc infection can cause pulmonary septic embolism with variable imaging features . 
le metastasi polmonari , qualunque sia lorigine del tumore primitivo , sono opacit nodulari a margini netti , ben definiti , talora molto calcifiche ( specialmente nellosteosarcoma ) ; sono generalmente localizzate perifericamente ma senza predilezione di sede tra lobo superiore , medio od inferiore [ 25 ]  . nei nostri tre pazienti tutti i noduli polmonari che simulavano metastasi apparvero in corso di chemioterapia in pazienti con catetere venoso centrale tipo broviac inserito in una vena succlavia ed utilizzato per chemioterapia , trasfusioni o prelievo ematico . 
tre casi di emboli settici polmonari su 231 pazienti portatori di cvc una piccola percentuale ( 1 , 3% ) , in accordo coi risultati di altri studi [ 6 ]  . 
la flogosi polmonare potrebbe essere stata la causa e non la conseguenza dellinfezione del catetere venoso centrale , ma dal momento che lesame colturale effettuato sul materiale prelevato dal lume distale del catetere fu positivo in due dei tre casi , sembr estremamente pi probabile che la causa dellinfezione emato - polmonare fosse il catetere venoso centrale . 
nondimeno , aree polmonari di aumentata densit , in soggetti neutropenici e portatori di catetere venoso centrale deve suggerire , tra le altre ipotesi diagnostiche , la possibilit di unembolizzazione settica nodulare secondaria alla presenza ed allinfezione del catetere venoso centrale . 
in addition , the specificity of ct in evaluating the nature of a pulmonary nodule , especially when small , is still limited , and only biopsy or nodule excision are able to reveal the benign or malignant nature of the nodule through histological examination whereas evolution of the radiological finding can only guide the diagnosis between benign or malignant nodular opacities [ 26 ]  . conclusions correct diagnosis of a pulmonary opacity is fundamental for the prognosis and choice of treatment ( surgical ) in a patient with a doubtful lung lesion . 
 in the presence of a cvc , any change in shape ( rounded , lobulated , irregular , fluffy ) in relationship with a vessel ( contiguous , distal ) and in location ( apical , medial , basal ) of an area of lung consolidation seen on serial ct scans , both during and after chemotherapy , is an important clue for pulmonary septic embolism secondary to cvc infection regardless of antibiotic treatment . catheter replacement , central and peripheral blood cultures , administration of specific antibiotic treatment based on blood cultures and ct follow - up of nodular opacities must always be given priority with respect to surgical treatment . mento che possono essere normali o non specifici [ 22 ] come pure laspetto radiologico di noduli polmonari a differente eziologia . 
inoltre la specificit della tc , nella valutazione di natura , benigna o maligna , di un nodulo polmonare , specialmente se questo di piccole dimensioni , ancora limitata e solo la biopsia o lasportazione del nodulo sono in grado di rivelare la vera natura della lesione permettendone lesame istologico , mentre levoluzione del reperto radiologico pu unicamente orientare la diagnosi tra opacit nodulare benigna o maligna [ 26 ]  . conclusioni la corretta diagnosi di una opacit polmonare di vitale importanza per la prognosi ed il differente trattamento terapeutico ( chirurgico ) nel caso di paziente con lesione polmonare dubbia per metastasi . 
de vargas macciucca , via alberico albricci 28 , i - 00194 roma , italy , tel . : + 39 - 338 - 5924034 , fax : + 39 - 06 - 49970212 , e - mail : marinadevargasm@hotmail.com received : 2 february 2005 / accepted : 7 november 2005 / published online : 3 march 2006 abstract purpose . 
we retrospectively reviewed 35 patients ( 23 men and 12 women ; mean age 66.6 years ) presenting with acute cholecystitis who were assessed by emergency ultrasonography ( us ) ( 30 / 35 cases ) and spiral ct ( 12 / 35 cases ) ; all patients underwent emergency surgery . 
the us signs were analysed and classified as major criteria ( wall thickening and stratification , distension , murphys sign ) , minor criteria ( bile stones , sludge , and biliary tract dilatation ) , and complication signs ( gas collections , aerobilia , fluid collection , difficult or missed identification of the gallbladder )  . 
imaging results were compared with histological findings ( gold standard ) , and accuracy , sensitivity , specificity , and positive and negative predictive values ( ppvs and npvs ) were assessed for each modality . 
if more than two major signs associated with one minor sign or at least one sign of complication are present at us , ct is mandatory to recognise and thoroughly evaluate the type of complication and indicate appropriate treatment . key words acute cholecystitis complicated cholecystitis us spiral ct riassunto scopo . 
sono stati esaminati retrospettivamente 35 pazienti ( 23 uomini e 12 donne ; et media 66 , 6 anni ) affetti da colecistite acuta , valutati in urgenza con esame ecografico ( 30 / 35 casi ) e tc ( 12 casi ) e sottoposti a intervento chirurgico urgente . 
i parametri ecografici sono stati analizzati e classificati come criteri maggiori ( ispessimento parietale , stratificazione parietale , sovradistensione , segno di murphy ecografico ) , minori ( calcoli o sedimenti nella colecisti o nelle vie biliari , dilatazione delle vie biliari ) e segni di complicazione ( raccolte gassose , aerobilia , raccolte fluide , difficile o mancato riconoscimento della colecisti )  . i risultati dellimaging sono stati confrontati con quelli dellesame istologico ( gold standard ) e sono state valutate accuratezza , sensibilit , specificit , valore predittivo positivo ( vpp ) e negativo ( vpn ) delle rispettive metodiche . 
lecografia ha dimostrato una accuratezza del 66 , 6% , una sensibilit del 37 , 5% , una specificit del 70% , un vpp del 100% e un vpn del 58 , 3% . 
in presenza di pi di due segni maggiori e uno minore o di almeno un segno di complicazione allecografia , lesame tc raccomandabile per il riconoscimento e la completa valutazione del tipo di complicazione , fornendo la giusta indicazione allintervento chirurgico urgente . parole chiave colecistite acuta colecistite complicata ecografia tc - spirale m . 
de vargas macciucca et al . : ultrasonographic and spiral ct evaluation of simple and complicated acute cholecystitis introduction introduzione the clinical suspicion of acute cholecystitis is one of the most frequent conditions facing the radiologist in emergency settings , as well as being the fourth cause of hospitalisation in patients presenting with acute abdomen [ 1 ] and the chief indication for emergency cholecystectomy [ 2 , 3 ]  . 
women under the age of 50 years are up to three times more frequently affected by cholecystitis than men whereas after this age , the incidence is the same in both genders . 
the most common cause of cholecystitis is lithiasis with cystic duct obstruction ( 90%95% )  . mucosal hypersecretion due to irritation resulting from increased bile concentration leads to oedema associated with wall thickening , followed by increased intraluminal pressure , wall distension , and stretching of the thin wall vessels with subsequent ischaemia and necrosis of the parietal mucosa . bacterial superinfection , which develops in 50%70% of cases , can lead to complications of acute cholecystitis such as empyema , gangrene , necrosis , and perforation [ 46 ]  . 
a patient without a history of biliary symptoms will rarely develop acute cholecystitis , whereas fewer than 15% of patients with gallstones present symptoms and fewer than 5% develop acute cholecystitis [ 7 ]  . in 5%10% of cases , cholecystitis is non - calculous . 
this situation mainly occurs in patients who have suffered serious injuries or burns , debilitated patients , patients with long - term hospitalisation in icu , or those undergoing prolonged parenteral feeding ; in these patients , systemic shock or the continued use of vasoconstrictors apparently causes ischaemia of the gallbladder wall mucosa , which is characterised by terminal blood supply and insufficient collateral circuits . 
according to another theory , however , non - calculous cholecystitis is due to mechanical obstruction of the cystic duct ( already narrowed by oedematogenic events ) by bile that has become dense and viscous following prolonged biliary stasis [ 8 , 9 ]  . 
other risk factors include overeating , diabetes , sepsis , cardiac arrest , atherosclerosis , aids , and chemotherapy via the hepatic artery [ 10 ]  . because symptoms are non - specific , it is difficult to clinically differentiate biliary colic from acute inflammation , but differential diagnosis is fundamental for establishing the correct therapeutic approach . 
treatment of acute cholecystitis is a matter of debate due to the different therapeutic options : emergency surgery ( laparotomy or laparoscopy ) , or delayed surgery after antibiotic treatment [ 11 , 12 ]  . 
further diagnostic investigation using imaging studies is therefore necessary for early diagnosis and timely treatment , above all when there is a suspicion of complications of acute cholecystitis . ultrasonography ( us ) is usually the first - choice modality [ 13 ] : the association of hepatobiliary us alone with clinical assessment allows accurate diagnosis in the majority of cases of calculous cholecystitis thanks to its very high sensitivity in the detection of cholelithiasis ( 95% ) [ 14 ] ; in addition , this examination has a low cost , is reproducible , and allows recognition and exclusion of numerous other causes of abdominal pain . both computed tomography ( ct ) and magnetic resonance il sospetto clinico di colecistite acuta rappresenta uno dei quesiti pi frequentemente sottoposti al radiologo in urgenza nonch la quarta causa di ricovero ospedaliero per pazienti che si presentano con addome acuto [ 1 ] ed la principale indicazione alla colecistectomia eseguita in urgenza [ 2 , 3 ]  . al di sotto dei 50 anni le donne sono fino a 3 volte pi colpite degli uomini , mentre oltre tale et lincidenza uguale nei due sessi . 
lipersecrezione mucosa determinata da fenomeni irritativi per iperconcentrazione della bile alla base del conseguente edema con ispessimento delle pareti , cui segue laumento della pressione intraluminale e la distensione delle pareti , con stiramento dei sottili vasi parietali , fenomeni alla base della successiva ischemia e poi della necrosi della mucosa parietale ; la sovrainfezione batterica , che si verifica solo nel 50%70% dei casi , invece causa delle possibili complicanze della colecistite acuta quali lempiema , la gangrena , la necrosi e la perforazione [ 46 ]  . 
raro che un paziente senza storia di sintomatologia biliare possa sviluppare una colecistite acuta , mentre al contrario meno del 15% dei pazienti con colelitiasi presentano sintomi clinici e meno del 5% sviluppano una colecistite acuta [ 7 ]  . nel 5%10% dei casi la colecistite invece alitiasica . questa evenienza si verifica essenzialmente in pazienti che hanno subito gravi traumi , defedati , ustionati , ricoverati a lungo in reparti di rianimazione o con alimentazione per via parenterale protratta nel tempo , nei quali lo shock sistemico o luso prolungato di vaso - costrittori indurrebbero unischemia della mucosa parietale della colecisti , che possiede una irrorazione di tipo terminale , con scarsi circoli collaterali ; tuttavia , secondo unaltra teoria le colecistiti alitiasiche sarebbero causate dallostruzione meccanica del dotto cistico ( gi ridotto di calibro per fenomeni edemigeni ) da parte di bile divenuta densa e vischiosa in seguito a prolungata stasi biliare [ 8 , 9 ]  . 
altri fattori di rischio includono liperalimentazione , il diabete , la sepsi , larresto cardiaco , laterosclerosi , laids e la chemioterapia attraverso larteria epatica [ 10 ]  . a causa dellaspecificit dei sintomi difficile differenziare clinicamente una colica biliare da una infiammazione acuta , ma la diagnosi differenziale fondamentale per una corretta pianificazione terapeutica . 
il trattamento delle colecistiti acute oggetto di discussione in quanto esistono differenti opzioni terapeutiche : lintervento chirurgico ( per via laparotomica o laparoscopica ) eseguito in urgenza o differito dopo terapia antibiotica [ 11 , 12 ]  . 
pertanto , specie quando si sospettino le complicanze della colecistite acuta , al fine di una precoce diagnosi e di un tempestivo trattamento terapeutico , si impone la necessit di approfondimenti diagnostici con esami strumentali . generalmente lecografia lindagine strumentale di prima istanza [ 13 ] : associando la sola ecografia epato - biliare alla valutazione clinica possibile una diagnosi accurata nella maggior parte dei casi , specie se la colecistite su base litiasica , grazie alla elevatissima sensibilit di questa metodica nel rilievo di colelitiasi ( 95% ) [ 14 ] ; inoltre tale esame presenta un basso costo , riproducibile e permette il riconoscimento e lesclusione di numerose altre cause di dolore m . 
de vargas macciucca et al . : ultrasonographic and spiral ct evaluation of simple and complicated acute cholecystitis ( mr ) normally add a small amount of information to the evaluation of acute cholecystitis but can be very useful in the case of subacute manifestations , in the presence of palpable pseudo - masses in the right hypochondrium , of clinical us discrepancy , and in the case of complications such as empyemic cholecystitis , abscesses , emphysematous cholecystitis , necrotic - gangrenous cholecystitis , perforation , biliary - enteric fistula or biliary ileus [ 2 , 6 , 7 , 15 , 16 ]  . the aim of this study was to review cases seen over 1 year of complicated acute cholecystitis evaluated by emergency us and ct and treated with emergency surgery in order to recognise specific and non - specific signs of complication and compare them with those reported in the literature . another aim was to define a correct diagnostic protocol involving the use of ct in selected cases . materials and methods in this study , we retrospectively examined the cases of 35 patients ( 12 women and 23 men ) aged between 27 and 99 years ( mean age 66.6 years ) suffering from acute cholecystitis who underwent imaging examinations between april 2003 and april 2004 . all patients underwent surgery and histological examination was considered the gold standard . 
patients whose diagnostic examinations performed at other sites were not available , patients who did not undergo emergency surgery due to medical contraindications , and patients whose operation was delayed for more than 48 h after presentation were excluded . clinical signs ( pain , murphys sign , fever , palpable swelling , jaundice ) and laboratory signs ( neutrophilic leukocytosis , bilirubinaemia , transaminasemia , glycaemia ) at the time of diagnosis were considered . 
ct spiral examinations ( siemens somatom plus 4 , erlangen , germany ) were performed before and after iv injection of contrast agent , with scans during the venous phase ( 60to 70 - s delay ) without oral contrast agent . 
in one case , ct was performed without iv injection of contrast agent due to reported allergy to iodinated contrast agents . the parameters analysed in ultrasonographic examinations were the following : focal or diffuse wall thickening ( more than 3 mm ) ; stratification or double parietal profile ; distension ( maximum transverse diameter greater than 5 cm ) ; stones in the gallbladder or biliary tract ; sludge or endoluminal aggregates ; parietal , endoluminal or extra - parietal gas collections ; aerobilia ; free or sacculated pericholecystic or abdominal fluid collections ; intraand extra - hepatic biliary tract dilatation ; presence of ultrasonographic murphys sign ; and possible associated findings . 
difficulty or inability to recognise the gallbladder was also evaluated . ultrasonographic signs were divided into three groups : major criteria of simple acute cholecystitis ( msac ) , minor criaddominale . sia la tc che la rm generalmente aggiungono un numero esiguo di informazioni alla valutazione delle colecistiti acute , ma possono essere molto utili in caso di manifestazioni subacute , in presenza di pseudomasse palpabili in ipocondrio destro , in caso di discrepanza clinico - ecografica e nei casi di complicazioni quali la colecistite empiematosa , lascessualizzazione , la colecistite enfisematosa , la colecistite necrotico - gangrenosa , la perforazione , la fistola bilioenterica o lileo biliare [ 2 , 6 , 7 , 15 , 16 ]  . questo studio si propone di esaminare i casi di colecistite acuta complicata , valutati in urgenza con ecografia e tc e sottoposti ad intervento chirurgico in urgenza nel corso di un anno , al fine di riconoscere i segni specifici ed aspecifici di complicanza da noi riscontrati e confrontarli con una revisione della letteratura . 
ci proponiamo inoltre di definire un corretto iter diagnostico che preveda limpiego della tc in casi selezionati . materiali e metodi in questo studio sono stati esaminati retrospettivamente 35 pazienti ( 12 donne e 23 uomini ) di et compresa tra 27 e 99 anni ( et media 66 , 6 anni ) affetti da patologia acuta della colecisti e sottoposti ad esami strumentali nel periodo compreso tra laprile 2003 e laprile 2004 . 
sono stati pertanto esclusi i casi in cui non sono risultati disponibili gli esami diagnostici eseguiti in altre sedi , i pazienti non sottoposti ad intervento chirurgico in urgenza per controindicazioni mediche e quelli il cui intervento chirurgico stato differito oltre le 48 ore dalla diagnosi . sono stati considerati i segni clinici ( dolore , manovra di murphy , febbre , tumefazione palpabile e ittero ) e laboratoristici ( leucocitosi neutrofila , bilirubinemia , transaminasemia , glicemia ) al momento della diagnosi . 
per gli esami ecografici sono state utilizzate due differenti apparecchiature ( au5 esaote , italia e logiq 400 , ge medical system , milwaukee , wi , usa ) con sonda convex da 3 , 55 mhz . 
per riferita allergia al mdc iodato . i parametri analizzati negli esami ecografici sono stati i seguenti : ispessimento parietale diffuso o focale ( considerando ispessita la parete quando superiore a 3 mm ) , stratificazione o doppio contorno parietale , sovradistensione ( diametro traverso massimo maggiore di 5 cm ) , calcoli nella colecisti o nelle vie biliari , presenza di sedimenti o aggregati endoluminali , raccolte gassose parietali , endoluminali o extraparietali , aerobilia , raccolte fluide pericolecistiche o endo - addominali libere o saccate , dilatazione delle vie biliari intraed extra - epatiche , presenza di segno di murphy ecografico ed eventuali reperti associati . 
msac , major criteria of simple acute cholecystitis ; msac , minor criteria of simple acute cholecystitis ; cc , complication criteria msac msac wall thickening > 3 mm wall stratification overdistension > 5 cm ultrasonographic murphys sign biliary sludge or stones biliary tract dilatation calculous cholecystitis biliary lithiasis pericholecystic or abdominal fluid collections parietal , endoluminal or extraparietal gas collections aerobilia difficult or missed recognition of the gallbladder tabella 1 segni ecografici di colecistite . 
mcas , criteri maggiori di colecistite acuta semplice ; mcas , criteri minori di colecistite acuta semplice ; cc , criteri di complicanza mcas mcas ispessimento parietale > 3 mm stratificazione parietale sovradistensione > 5 cm murphy ecografico sedimenti o aggregati biliari dilatazione delle vie biliari litiasi della colecisti litiasi delle vie biliari raccolte fluide pericolecistiche o endoaddominali raccolte gassose parietali , endoluminali o extraparietali aerobilia difficile o mancato riconoscimento della colecisti teria of simple acute cholecystitis ( msac ) , and complication criteria ( cc ) ( table 1 )  . 
for ct in addition to the us parameters , with the exception of murphys sign , we evaluated the presence of free air and alterations to vascularisation of the liver parenchyma adjacent to the gallbladder , both being signs of complication . 
the final histological examinations , considered as the gold standard , were compared with us and ct results , and accuracy , sensitivity , specificity , ppv , and npv for each modality were measured . 
the association with neutrophilia was observed in 28 / 35 ( 80% ) of cases ; in 11 / 35 cases , patients had elevated bilirubinaemia ( 31.4% ) and in 9 / 35 cases increased transaminases ( 25.7% ) ; 21 / 35 patients ( 60% ) had hyperglycaemia on admission , and four of them had a history of diabetes mellitus . 
in the selected group , hepatobiliary us was performed as the first - choice diagnostic examination in 30 / 35 patients , followed by ct in seven cases ; in five cases presenting with acute abdomen , ct was performed as the first - choice examination . 
on the whole , 30 patients were studied with us and 12 by ct ; in 7 / 35 cases , both us and ct were performed . us findings us revealed diffuse thickening of the gallbladder wall in gni ecografici sono stati inoltre suddivisi in tre gruppi : criteri maggiori di colecistite acuta semplice ( mcas ) , criteri minori di colecistite acuta semplice ( mcas ) e criteri di complicanza ( cc ) ( tabella 1 )  . 
per la tc oltre agli stessi parametri utilizzati per lecografia , fatta eccezione per il segno di murphy ecografico , stata valutata la presenza di aria libera e le alterazioni della vascolarizzazione del parenchima epatico adiacente alla colecisti , entrambi segni di complicazione . in base ai reperti riscontrati negli esami diagnostici sono stati distinti due gruppi di patologie : colecistiti acute semplici e colecistiti acute complicate . 
gli esami istologici definitivi , considerati il gold standard , sono stati confrontati con i risultati degli esami ecografici e tc e sono state misurate accuratezza , sensibilit , specificit , valore predittivo positivo ( vpp ) e negativo ( vpn ) delle rispettive metodiche . 
nei casi sottoposti sia ad ecografia che a tc stata inoltre valutata la concordanza tra le due metodiche e lentit dei dati aggiuntivi forniti da una metodica rispetto allaltra . risultati i pazienti presentavano clinicamente dolore in ipocondrio destro o epigastrio in 32 / 35 casi ( 91 , 4% ) , manovra di murphy positiva in 22 / 35 casi ( 62 , 8% ) , febbre in 18 / 35 casi ( 51 , 4% ) , tumefazione palpabile in 6 / 35 casi ( 17 , 1% ) , ittero in 2 / 35 casi ( 5 , 7% )  . 
si associava leucocitosi neutrofila in 28 / 35 casi ( 80% ) ; in 11 / 35 casi i pazienti presentavano un innalzamento dei valori di bilirubinemia ( 31 , 4% ) e in 9 / 35 casi si rilevava un incremento dei valori delle transaminasi ( 25 , 7% ) ; 21 / 35 pazienti ( 60% ) avevano iperglicemia al momento del ricovero e 4 di questi pazienti presentavano storia di diabete mellito . 
nel gruppo selezionato , lecografia epatobiliare stata eseguita come primo esame diagnostico in 30 / 35 pazienti , seguita dalla tc in 7 casi ; in 5 casi la tc stata eseguita come esame di prima istanza , trattandosi di gravi quadri di addome acuto . 
ct also revealed three cases of acute pancreatitis ( 25% ) characterised by pancreatic enlargement , with peripancreatic fat tissue hyperdensity in two of these cases . in one patient with suspected thoracic aortic dissection , the detection of cholecystitis was an occasional finding revealed by ct . 
ct allowed the detection of three cases of simple acute cholecystitis and nine cases of complicated acute cholecystitis ( table 2 )  . surgical treatment and histology all patients underwent laparotomic or laparoscopic cholecystectomy within 2448 h of diagnosis . 
in addition to cholecystectomy , the following procedures were performed : three viscerolyses , two hepatic resections , one intestinal resection , one abdominoplasty for laparocele , one umbilical hernial repair , one peritoneal lavage , three abdominal collection drainages , one main biliary tract drainage , one duodenal fistula repair , and one choledochoplasty with kehr tube positioning . 
la tc ha inoltre individuato 3 casi di pancreatite acuta ( 25% ) caratterizzata da aumento volumetrico del pancreas con iperdensit del tessuto adiposo peripancreatico in 2 di questi casi . in un paziente con sospetta dissecazione dellaorta toracica il riscontro di colecistite stato un reperto occasionale rilevato allesame tc . 
la tc ha permesso di distinguere 3 casi di colecistite acuta semplice e 9 casi di colecistite acuta complicata ( tabella 2 )  . interventi chirurgici ed istologia tutti i pazienti sono stati sottoposti a colecistectomia per via laparotomica o laparoscopica nelle 2448 ore successive alla diagnosi . 
de vargas macciucca et al . : ultrasonographic and spiral ct evaluation of simple and complicated acute cholecystitis table 2 comparison between ultrasonography ( us ) , computed tomography ( ct ) , and histological findings * acute and recurrent cholecystitis are included in the group of simple cholecystitis tabella 2 confronto tra i risultati dellecografia , della tc e dellesame istologico simple cholecystitis * complicated cholecystitis neoplasms colecistiti semplici * colecistiti complicate neoplasie 24 / 30 ( 80% ) 6 / 30 ( 20% ) ecografia 24 / 30 ( 80% ) 6 / 30 ( 20% ) * il gruppo di colecistiti semplici comprende le acute e le croniche riacutizzate table 3 ultrasonographic signs in simple cholecystitis 3 / 12 ( 25% ) 9 / 12 ( 75% ) 3 / 12 ( 25% ) 9 / 12 ( 75% ) histology 14 / 35 ( 40% ) 20 / 35 ( 57.1% ) 1 / 35 ( 2.8% ) istologia 14 / 35 ( 40% ) 20 / 35 ( 57 , 1% ) 1 / 35 ( 2 , 8% ) patient thickening stratification distension murphys sign sludge biliary tract dilatation lithiasis tabella 3 segni ecografici riscontrati nelle colecistiti non complicate paziente ispessimento stratificazione sovradistensione murphy sedimenti dilatazione vvbb litiasi three cases of acute calculous cholecystitis ; 11 cases of recurrent chronic cholecystitis ( with inflammatory infiltrates associated with fibrosis ) , ten of which were calculous and one non - calculous ; and 20 complicated cases of cholecystitis ( 14 calculous and six non - calculous )  . 
the final histological examination revealed one case of undifferentiated carcinoma epatiche , 1 resezione intestinale , 1 addominoplastica per laparocele , 1 riduzione di ernia ombelicale , 1 lavaggio peritoneale , 3 drenaggi di raccolte addominali , 1 drenaggio della via biliare principale , 1 sutura di fistola duodenale e 1 coledoco - plastica con posizionamento di tubo di kehr . 
of the 20 complicated forms , histology diagnosed 13 cases of phlegmonous cholecystitis , two cases of empyema , two cases of necrotic and gangrenous cholecystitis , two cases of abscess , and one case of duodeno - cholecystic fistula . 
us had a sensitivity of 37.5% , a specificitable 4 ultrasonographic signs in complicated acute cholecystitis colecistite acuta litiasica , 11 casi di colecistite cronica riacutizzata ( con infiltrati flogistici associati a fibrosi ) , 10 delle quali su base litiasica e 1 alitiasica , 20 colecistiti complicate ( 14 litiasiche e 6 alitiasiche )  . in 1 caso lesame istologico definitivo ha dimostrato la presenza di un carcinoma indifferenziato della colecisti associato a litiasi . 
delle 20 forme complicate 13 sono risultate allesame istologico come colecistiti flemmonose , 2 casi di empiema , 2 casi di colecistite acuta necrotica e gangrenosa , 2 casi di ascesso e 1 caso di fistola colecisto - duodenale . 
de vargas macciucca et al . : ultrasonographic and spiral ct evaluation of simple and complicated acute cholecystitis ty of 70% , an accuracy of 66.6% , a ppv of 100% , and an npv of 58.3%. 
in the seven cases studied by both modalities , us and ct yielded similar results as regards the presence of complications ; however , ct provided additional information compared with us , as it detected the presence of fluid in four cases , identified the gallbladder in two cases in which it was not recognizable by us , and demonstrated the presence of intraparietal air in one case , endoluminal air in three cases and free air in one case . 
it also demonstrated biliary tract dilatation in two cases , a biliary - enteric fistula in one case , and intrahepatic fluid and gas due to cholecystitis in one case . 
unlike us , ct also permitted identification of three cases of oedematous pancreatitis . discussion considering that more than 20% of cases clinically classified as cholecystitis correspond to other diseases , the role of emergency diagnostic imaging is to support the clinical diagnosis of acute cholecystitis and to identify the complications of cholecystitis , thus indicating the correct therapeutic approach [ 7 ]  . 
in cases of simple cholecystitis , surgical treatment is normally delayed , and the patient is treated with antibiotics during the acute phase ; emergency surgery within 2448 h , however , prevents the formation of adhesions , which may complicate laparoscopic surgery and increase the rate of conversion into laparotomy [ 12 , 17 , 18 ]  . 
emergency surgery , especially when laparoscopic , therefore implies shorted hospitalisation and reduced post - operative complications and overall hospitalisation costs [ 11 , 12 , 17 , 1824 ]  . clinically , three quarters of patients suffering from acute cholecystitis have a history of biliary colic and pain in the right hypochondrium , which may irradiate to the right shoulder and to the epigastric region , accompanied by nausea , vomiting , and dyspepsia . 
con la tc sono stati identificati 3 vn , 9 vp , nessun fp e nessun fn . lecografia ha dimostrato una sensibilit del 37 , 5% , una specificit del 70% , una accuratezza del 66 , 6% , un vpp del 100% e un vpn del 58 , 3% . 
la tc ha invece presentato una sensibilit del 100% , una specificit del 100% , una accuratezza del 100% ( tabella 5 )  . nei 7 casi sottoposti sia ad ecografia che a tc le due metodiche sono risultate concordi per la presenza di complicanze , ma la tc ha fornito in ogni caso maggiori informazioni rispetto allecografia rilevando la presenza di versamento fluido in 4 casi , identificando la colecisti in 2 casi in cui allecografia non era riconoscibile , diagnosticando la presenza di aria intraparietale in 1 caso , endoluminale in 3 casi , di aria libera in 1 caso , ha mostrato la presenza di dilatazione delle vie biliari in 2 casi , la fistola bilio - enterica in 1 caso , la presenza di raccolta fluido - gassosa intraepatica ad origine dalla colecisti in 1 caso . 
la tc ha inoltre consentito rispetto allecografia lidentificazione di 3 casi di pancreatite edematosa . discussione il ruolo della diagnostica per immagini in emergenza nella patologia acuta della colecisti quello di supportare la diagnosi clinica , considerando che oltre il 20% dei casi classificati clinicamente come colecistite corrispondono a unaltra patologia , e di identificare le complicanze indicando il corretto approccio terapeutico [ 7 ]  . 
generalmente se la colecistite non risulta complicata , il trattamento chirurgico viene differito , sottoponendo il paziente in fase acuta a terapia antibiotica ; tuttavia , lintervento chirurgico in emergenza entro 2448 ore previene la formazione di aderenze che possono complicare lintervento chirurgico laparoscopico aumentandone la percentuale di conversione in laparotomia [ 12 , 17 , 18 ]  . 
in our study , fewer patients underwent ct than us , and the majority of them had complicated cholecystitis suspected at us or on the basis of clinical findings ; therefore , it was not possible to accurately assess the major and minor criteria using ct in simple cholecystitis . forty percent of patients with acute cholecystitis develop complications such as empyema , pericholecystic abscesses , necrotic - gangrenous cholecystitis , emphysematous cholecystitis , haemorrhagic cholecystitis , perforation , cholecysticenteric fistula , and biliary ileus [ 3638 ]  . 
 we also detected one case of duodeno - cholecystic fistula . in this case , the gallbladder was not recognisable at us whereas both aerobilia and a coarse calcification ( 3 cm ) between the liver and the pancreatic head were well visible in the second duodenal portion ; also , ct had some difficulties in that the stone could not be precisely localised in the intestinal lumen , and neither free air or endoperitoneal fluid collection could be seen . 
however , the presence of aerobilia guito in laparoscopia , comporta riduzione dei tempi di ospedalizzazione , delle complicanze post - operatorie e dei costi complessivi del ricovero [ 11 , 12 , 17 , 1824 ]  . clinicamente tre quarti dei pazienti affetti da colecistite acuta presentano una storia di coliche biliari , dolore in ipocondrio destro , che si pu irradiare alla spalla destra ed in sede epigastrica , accompagnato da nausea , vomito e dispepsia . 
il segno di murphy generalmente positivo e vi possono essere reperti di laboratorio aspecifici ( leucocitosi , iperbilirubinemia , iperamilasemia e aumento dei livelli di transaminasi ) [ 25 , 26 ]  . 
nel nostro studio i pazienti sottoposti a tc sono stati in numero ridotto rispetto ai casi sottoposti ad ecografia ed erano quasi tutti casi di colecistiti complicate sospettate allesame ecografico o in base al quadro clinico , pertanto non stato possibile valutare con accuratezza i criteri maggiori e minori con tale metodica nelle colecistiti semplici . 
nel 40% dei pazienti con colecistite acuta si sviluppano complicanze quali lempiema , gli ascessi pericolecistici , la colecistite necrotico - gangrenosa , la colecistite enfisematosa , la colecistite emorragica , la perforazione , la fistola colecisto - enterica e lileo bilare [ 3638 ]  . 
de vargas macciucca et al . : ultrasonographic and spiral ct evaluation of simple and complicated acute cholecystitis and inability to identify the gallbladder , perforated and collapsed , correctly prompted towards emergency surgery during which and a duodeno - cholecystic fistula with a stone impacted in the second duodenal portion was detected . 
in our study , no cases of haemorrhagic cholecystitis or biliary ileus were observed . the fundamental aim of diagnostic imaging , especially in emergency settings , is to differentiate simple and complicated cholecystitis in order to plan laparoscopic or laparotomic surgery within 48 h . 
to detect the complicated forms , we suggest a classification that takes into account the main us signs divided into major criteria ( wall thickening > 3 mm , wall stratification , positive sonographic murphys sign , and distension ) , minor criteria ( biliary sludge , calculous cholecystitis and biliary tract dilatation ) and criteria for complicated forms ( aerobilia , fluid and gas collections , or missed recognition of the gallbladder ) ( table 1 )  . 
this classification differs from mirviss classification [ 9 ] ( table 6 ) first of all because it takes into consideration sonographic criteria but also because the presence of stones and biliary sludge is considered a minor criteria ; this depends on the fact that , despite being a frequent sonographic finding , biliary sludge is not specific for cholecystitis . 
the analysis of associations between major and minor signs , on the other hand , did not show a significant difference between the two groups of simple and complicated cholecystitis . 
indeed , in 5 / 16 cases ( 31.2% ) of complicated cholecystitis and in 10 / 14 cases ( 71.4% ) of simple cholecystitis , an association of at least two major criteria and one minor was found . 
in 4 / 16 cases ( 25% ) of complicated cholecystitis and in 4 / 14 cases ( 28.5% ) of simtable 6 computed tomography ( ct ) signs of acute cholecystitis ( mirviss classification ) ct signs of acute cholecystitis [ mirvis et al . 
in questo caso allesame ecografico non risultava riconoscibile la colecisti , mentre erano ben evidenti sia laerobilia che la presenza di una struttura calcifica grossolana ( 3 cm ) situata tra il fegato e la porzione cefalica pancreatica , nella sede della seconda porzione duodenale ; anche la tc ha presentato alcune difficolt diagnostiche in quanto la formazione litiasica non risultava localizzabile con certezza nel lume intestinale e non erano visibili aria libera n versamento fluido endoperitoneale ; tuttavia la presenza di aerobilia e il mancato riconoscimento della colecisti , in quanto perforata e collassata , hanno indirizzato correttamente la paziente al trattamento chirurgico urgente ed stata riscontrata una fistola colecisto - duodenale con calcolo indovato a livello della seconda porzione duodenale . 
nella nostra casistica non sono stati riscontrati casi di colecistite emorragica n di ileo biliare . lobiettivo fondamentale che si propone la diagnostica per immagini soprattutto in emergenza quello di distinguere le colecistopatie semplici dalle complicate al fine di poter programmare lintervento chirurgico per via laporoscopica o laparotomica , entro le 48 ore . 
a tal fine per poter individuare le forme complicate proponiamo una classificazione che tiene conto dei principali segni ecografici suddivisi in criteri maggiori ( ispessimento parietale > 3 mm , aspetto stratificato della parete , segno ecografico di murphy positivo e sovradistensione ) , criteri miniori ( sedimenti biliari , litiasi della colecisti o delle vie biliari e dilatazione delle vie biliari ) e criteri riconducibili a forme complicate ( areobilia , raccolte fluide , raccolte gassose o mancato riconoscimento della colecisti ) ( tabella 1 )  . 
una classificazione basata su tali criteri differisce da quella di mirvis riportata in letteratura [ 9 ] ( tabella 6 ) , innanzitutto perch prende in considerazione dei criteri ecografici , ma anche perch la presenza di calcoli e sedimenti biliari considerata un criterio minore poich , sebbene con tale metodica sia molto frequentemente riscontrata , non specifica di colecistite : nel nostro studio calcoli e aggregati biliari erano presenti quasi sempre ( 98% della popolazione esaminata ) sia nelle forme complicate che in quelle semplici . nella nostra esperienza lecografia ha riscontrato segni sicuri di complicanza in 6 / 16 casi ( 37 , 5% )  . 
tra le colecistiti semplici i segni di complicazione non sono mai stati rilevati . lanalisi delle associazioni tra segni maggiori e minori invece non ha dimostrato una significativa differenza tra i due gruppi di colecistiti , semplici e complicate . 
infatti in 5 / 16 casi ( 31 , 2% ) di colecistite complicata e in 10 / 14 casi ( 71 , 4% ) di colecistite semplice stata riscontrata lassociazione di almeno 2 criteri maggiori e uno minore . 
in 4 / 16 casi ( 25% ) di colecistite complicata e in 4 / 14 casi ( 28 , 5% ) di colecistite semplice erano presenti allecografia meno di due criteri maggiori . 
 these data indicate that us can distinguish complicated and simple cholecystitis only in the presence of definite signs of complication ; in the majority of cases , however , these signs are not present . 
we believe that ct would have provided the correct diagnosis of complication in many of these cases given its higher accuracy and sensitivity . indeed , ct yielded no fp or fn results . 
because the majority of complications revealed by us showed at least two major criteria associated with at least one minor criterion or at least one criterion for complication ( overall 68.7% ) , further ct investigation after us is justified under these circumstances . numerous cases of phlegmonous cholecystitis were present ( 9 / 16 cases ) among the cases assessed as simple cholecystitis by us and as complicated cholecystitis by histological examination . although this form is not regarded as a complicated form in the literature , we decided to include these cases in the group of complications because involvement of the entire thickness of the organ causes serositis with peritoneal effusion in the majority of cases . 
if the emergency approach is based on medical treatment with delayed surgery , the peritoneal reaction may lead to the formation of synechia and fibrosis , with development of adhesions . 
phlegmonous cholecystitis therefore requires immediate surgical treatment because adhesions hinder possible surgical treatment during quiescence and increase the incidence of complications ; there is also a higher risk of laparoscopy being converted into laparotomy [ 12 ]  . conclusions assessment of acute cholecystitis in an emergency department requires careful analysis of the clinical findings and relies on us as first - choice examination . us allows the recognition of signs which , taken singularly , are poorly specific ; this directs the patient to further investigation by ct , which is indispensable for the complete recognition and evaluation of complicated forms . 
trenta , radiologists at the emergency department of policlinico umberto i , rome , for their help in collecting the case series . solo in presenza di segni certi di complicanza , ma nella maggior parte dei casi questi non sono presenti ; infatti , nella nostra casistica , allecografia sono risultati falsi negativi ( fn ) un terzo dei casi e la maggior parte di tali pazienti non stata sottoposta ad ulteriori indagini . 
poich allecografia la maggior parte delle complicanze presentavano almeno due criteri maggiori associati ad almeno uno minore oppure almeno un criterio di complicanza ( globalmente il 68 , 7% ) , si pu considerare giustificato in tali circostanze approfondire lesame ecografico con un esame tc . 
sebbene la letteratura non tratti questa forma come complicata , abbiamo ritenuto di includere tali casi nel gruppo delle complicazioni in quanto linteressamento a tutto spessore del viscere determina comunque una sierosite con presenza nella maggior parte dei casi di versamento peritoneale . 
in tali pazienti , se la scelta terapeutica in urgenza il trattamento medico e lintervento chirurgico viene differito , la reazione peritoneale nel tempo pu determinare la formazione di sinechie e fibrosi con lo sviluppo di briglie aderenziali . 
le colecistiti flemmonose necessitano pertanto di un trattamento chirurgico immediato , in quanto le aderenze rendono leventuale intervento in fase di quiescenza pi difficoltoso e aumentano lincidenza di complicanze ; in questi casi inoltre pi elevato il rischio di conversione dellintervento da laparoscopico a laparotomico [ 12 ]  . conclusioni la valutazione in un dipartimento di emergenza della patologia acuta della colecisti necessita di una attenta analisi dei dati clinici e si avvale dellutilizzo dellecografia come metodica di prima istanza . 
lecografia permette il riconoscimento di alcuni segni che sono poco specifici singolarmente , indirizzando il paziente allapprofondimento con tc che risulta indispensabile per riconoscere e valutare in maniera completa le forme complicate . 
in particolare , quando allecografia sono identificati almeno due segni maggiori e uno minore e quando sono presenti i segni specifici di complicanza , lapprofondimento con tc imprescindibile in quanto permette di selezionare i pazienti candidati ad intervento in urgenza rispetto a quelli da trattare con terapia antibiotica ed intervento differito ; inoltre la distinzione tra le diverse forme di colecistite aiuta il chirurgo nella scelta della tecnica operatoria pi indicata ( laparoscopica o laparotomica )  . ringraziamenti si ringraziano per la collaborazione nel reperimento della casistica il dott . 
inches2 1dipartimento di scienze oncologiche e chirurgiche , sezione di senologia , universit degli studi di padova , via gattamelata 64 , i - 35128 padova , italy 2dipartimento di scienze medicodiagnostiche e terapie speciali , universit degli studi di padova , via giustiniani 2 , i - 35128 padova , italy correspondence to : l . 
practical aspects with psychological and clinical involvement are discussed with tree diagrams . key words human error perception interpretation system riassunto viene proposto un approccio sistematico allerrore umano nella diagnostica per immagini ( di ) , riconoscendo la sua collocazione nelle attivit personali della percezione e della interpretazione e nelloperativit globale del sistema . 
nellinquadramento che si sviluppa secondo ramificazioni ad albero sono discussi gli aspetti pratici alla luce di contributi provenienti dagli studi psicologici e dalla evoluzione della realt clinica e sanitaria . parole chiave errore umano percezione interpretazione sistema introduction introduzione the approach to the study of human error in diagnostic imaging ( di ) requires a preliminary introduction regarding the concept of error in modern medicine . human error is always placed inevitably in relation with truth [ 1 , 2 ] , which in a strictly scientific field should be considered , as the epistemologist blandino states , as only probable and approximate , connected to theories which change and at times crumble , to be replaced by other , more valid ones . 
it can safely be said that there is currently such a huge quantity of knowledge in medicine and biology that the medical practitioner is increasingly exposed to a great number and variety of errors . traditionally two relevant aspects have been indicated : errors in medicine , considered as the collection of theories regarding health and the state of disease in humans ; for the scientific representation of errors in medicine the statistics and theory of bayes [ 3 ] are used medical practitioners errors , in that as humans . 
they are subject to making mistakes according to a distinction made by epistemologists , the term mistake is more accurate for errors related to the activity in which theoretical notions are applied whereas errors in the lapproccio allo studio degli errori commessi dalluomo nella diagnostica per immagini ( di ) impone una preliminare introduzione sulle connotazioni che il concetto di errore assume nella moderna attivit medica . lerrore da sempre messo inevitabilmente in rapporto con la verit [ 1 , 2 ] che in ambito strettamente scientifico da intendersi , come indica lepistemologo blandino , come soltanto probabile e approssimata , connessa a teorie che si modificano e talora crollano , sostituite da altre pi valide : anche le verit diagnostiche sono quindi relative e commisurate al sapere di un determinato tempo . 
non vi dubbio che allo stato attuale via sia una tale mole di conoscenze in medicina e biologia che si pu tranquillamente affermare che loperato del medico sempre pi esposto ad errori , molteplici e di varia natura . tradizionalmente se ne indicano due aspetti rilevanti : gli errori della medicina , considerata come insieme di teorie che riguardano la salute e lo stato di malattia delluomo , per la rappresentazione scientifica dei quali ci si avvale soprattutto della statistica e del teorema di bayes [ 3 ] ; gli errori dei medici , in quanto essere umani e come tali soggetti a sbagliare . secondo una distinzione degli epistemologi , proprio il termistrict sense regard science as such . 
in this paper , the term error will be used for simplicity , even if in reference to mistakes . medical tradition also commonly makes a distinction between two reference errors : cognitive : concerning diagnosis and prognosis operative : concerning decision making and therapy di is slightly removed from this picture , as the use of perception is particularly relevant but also because given the progressive expansion of the discipline , the search for errors needs to be placed in an organized and technologically advanced context , with a much broader range of devices than in the early days of the first applications of the coolidge tube when medicine was almost totally reliant on the personal abilities of the practitioners . like other fields , such as nuclear engineering or aviation [ 4 , 5 ] , a radiology department stands out for its organizational and operative peculiarities as a system , with features similar to any company such as logistics , finance and the hiring and training of personnel . 
some errors may be present in the planning and organizational phase such that reason and rasmussen [ 6 ] classifies them as latent errors with delayed effects and differentiates them from active errors in which there is a momentary and welldefined participation by the operator . 
seeking out these remote errors is an important feature of the modern management of the radiology department because when they appear , the operator is in fact programmed to make a mistake . 
the operator is the victim of a mediocre project , insufficient maintenance , inappropriate materials and managerial decisions which create situations of risk , such as unrealistic workloads , inappropriate training or exasperating turnaround times . 
since the error appears with the last operator , whether he or she is the direct or indirect cause , all errors should be considered human error and in di the radiologists error . in this context the aim of this study was to unify the problems and the causes of errors , taking into consideration the crucial moment of image assessment in radiology when the radiologist utilizes the characteristics of his or her personality , culture and training [ 7 ]  . characteristics of the radiologists activities the radiological act as it currently stands can be defined as a technical - methodological procedure performed on a patient in an organizational and decision - making system , the outcome of which is the image obtained to achieve diagnosis . 
it is here , in fact , that to reach a decision , the radiologist makes use of a process of perception and cognition , features which occur simultaneously and are interdependent , and yet , as wood [ 8 ] notes , are separate mental activities . 
inches : systematic approach to human error in radiology ne sbaglio pi corretto ad indicare gli errori inerenti alle attivit in cui le nozioni teoriche sono applicate , mentre gli errori in senso stretto riguardano la scienza come tale . nellelaborato il termine errore sar usato per comodit e consuetudine , anche se riferito agli sbagli . ancora nella tradizione medica si soliti configurare due errori di riferimento : cognitivo : concerne la diagnosi e la prognosi ; operativo : concerne gli aspetti decisionali e la terapia . la di se ne discosta parzialmente essendo per essa rilevante lapporto della percezione , ma anche perch , nella progressiva espansione della disciplina , la ricerca dellerrore va inquadrata in un contesto organizzato e tecnologicamente avanzato , che prospetta tipologie pi ampie rispetto allepoca artigianale delle prime applicazioni del tubo di cooledge , coeve ad una medicina legata alle pressoch assolute capacit personali dei medici . la radiologia , intesa quale servizio o unit operativa che dir si voglia , analogamente ad altre strutture di lavoro , come lingegneria nucleare o laviazione [ 4 , 5 ] , si identifica allo stato attuale per peculiarit gestionali e operative come sistema , al quale sono attribuiti analogamente a qualsiasi azienda almeno gli ambiti organizzativi della logistica , della finanza , dellassunzione e della preparazione del personale . il sistema come tale pu essere sottoposto ad analisi e verifica ai fini della ricerca degli errori e della loro correzione mediante procedure adatte . alcuni errori possono essere gi insiti in fase di progettazione e organizzazione , tanto che reason [ 6 ] li classifica come errori latenti , con effetti ritardati , differenziandoli dagli errori attivi in cui vi una partecipazione momentanea e bene circostanziata delloperatore . 
la ricerca di questi errori remoti rappresenta allora uno dei punti notevoli della moderna conduzione del reparto , perch , quando compaiono , loperatore in realt stato programmato per sbagliare : egli vittima ultima di un progetto mediocre , di una manutenzione insufficiente , di materiali inidonei , di decisioni della dirigenza che si esplicano in situazioni di rischio , quali ad esempio gli irrealistici carichi di lavoro , laddestramento non adeguato o i tempi di produzione esasperati . 
poich di fatto lerrore si manifesta nelle circostanze con lultimo operatore , ne sia egli la causa diretta o indiretta , tutti gli errori in definitiva debbono essere considerati riferiti alluomo : per la di al radiologo . sotto questo profilo lo scopo del lavoro di unificare problematiche e cause di errore prendendo in considerazione nella di il momento cruciale della valutazione del documento , allorquando il radiologo partecipa mettendo in campo le proprie caratteristiche di personalit , cultura e preparazione [ 7 ]  . caratteristiche dellattivit del radiologo latto radiologico allo stato attuale pu essere definito come procedura tecnico - metodologica condotta sul paziente , avvenuta in un sistema organizzativo - decisionale , che si esprime nel documento dimmagine ottenuto per conseguire il fil . 
inches : systematic approach to human error in radiology mental phenomena at play is required : the phenomenon of perception the perceptual process transforms retinal stimulation at the higher centres into conscious perception , allowing images to be extracted and translated in conscious assessment by simplifying and organizing the information from the peripheral regions . 
according to attneave , quoted by wood [ 8 ] , this process is limited , not being sufficiently capable in the sense of capacity to transmit all information which reaches the retina . 
hake , also cited by wood [ 8 ] , instead believes that perception is governed by the centre in that it is linked to the search for a meaning connecting the fragmentary retinal images . 
in fact , the meaning and even the writing of a sentence itself may be considered absolutely precise even though obvious errors may be found when the text is carefully revised [ 9 ]  . 
lastly , it is worth noting that perception is substantially different from simple vision , in which only the peripheral activities of the observer take part in relation to the nervous system . the phenomenon of cognition the cognitive part of diagnostic decision making is less well known yet fascinating , given the models proposed for explaining the mental stages which lead to a conclusion . 
qui , infatti , che il radiologo si avvale di un procedimento percettivo e conoscitivo , simultanei e interdipendenti , da considerare tuttavia con wood e tuddenham [ 8 ] come attivit psichiche separate , per giungere ad una decisione . 
per rendere comprensibile e al tempo stesso unitaria la classificazione a seconda della tipologia e delle cause di errore indispensabile un richiamo sintetico alle conoscenze dei fenomeni psichici in gioco . il fenomeno della percezione il processo percettivo trasforma in percezione cosciente i modelli della stimolazione retinica ai centri superiori ; consente di estrarre immagini e di tradurle in valutazione conscia , mediante una semplificazione e organizzazione delle informazioni provenienti dalla periferia . 
secondo atteneave , citato da wood [ 8 ] il processo limitato , non essendo sufficientemente capace , nel senso proprio di capiente , per trasmettere tutte le informazioni che raggiungono la retina . hake , pure citato da wood [ 8 ] ritiene invece che la percezione sia diretta dal centro in quanto legata alla ricerca di un significato che colleghi le frammentarie immagini retiniche . 
per la lettura ; i rettangoli indicano le conoscenze che dallalto in basso divengono via via pi profonde ; in un caso clinico linterprete potrebbe trascurare leziologia di un broncogramma , ad esempio metastasi polmonari , se lattivazione particolarmante frequente per le cause infettive . pointed out that classic cognitivism recognizes the diagnostic process as inductive reasoning aimed at identifying rules and categories , beginning with concrete examples : the dialogue between the areas of research of concrete examples ( variable features ) and rules ( fixed features ) leads to the selection of facts , to remembering the theories based on the facts and to test the hypotheses . 
the classic model , however , has the defect , particularly for medical applications , of overlooking the concrete and pragmatic aspects , in essence , situations such as emergencies in which the examinations are necessarily limited owing to time constraints . 
 the most complete and appropriate model for our purposes which explains the relationship between real facts and the examination of the mind is the model proposed by raufaste and eyrolle [ 10 , 11 ] in 1998 . 
long - term memory has an enormous capacity for storage and contains declarative knowledge ( theory ) and procedural knowledge ( know - how ) interconnected by various types of logical systems in an active for for example , the processes of classification of part / whole relations become increasingly rapid in acquisition times as the knowledge is implemented and interconnected . 
generally speaking , by bringing ones attention to a certain level of knowledge , that level is activated , and once more overcoming a certain threshold , awareness is achieved . working memory involves a variety of aspects , including phonological , visual and spatial . 
infine da osservare come la percezione si differenzi in modo sostanziale dalla semplice visione , al cui fenomeno partecipano solo le attivit periferiche dellosservatore in rapporto alle caratteristiche del sistema nervoso . il fenomeno cognitivo la parte cognitiva della decisione diagnostica meno conosciuta , affascinante per i modelli proposti a spiegare le tappe mentali che portano alla conclusione . 
premesso che lambito mentale molto ampio e a margini poco definiti , doveroso accennare come il cognitivismo classico riconosca il procedimento diagnostico come ragionamento induttivo teso ad identificare regole e categorie partendo da esempi concreti : il dialogo fra gli spazi di ricerca degli esempi concreti ( tratti variabili ) e delle regole ( tratti fissi ) conduce a selezionare i fatti , a ricordare le teorie a partire dai fatti e a verificare le ipotesi . 
il modello classico ha per il difetto , soprattutto per le applicazioni mediche , di trascurare gli aspetti concreti e pragmatici , in sostanza le situazioni , ad esempio le emergenze , in cui le indagini sono necessariamente limitate per motivi di tempo . 
per spiegare meglio il rapporto tra fatti reali e lindagine della mente risulta pi completo e adatto alle nostre esigenze , perch dedicato alla radiologia , il modello del francese roufaste del 1998 [ 10 , 11 ]  . 
la memoria a lungo termine ha vasta possibilit di stoccaggio ed duratura nel tempo : contiene le conoscenze dichiarative ( del sapere teorico ) e procedurali ( del saper fare ) interconnesse da diversi tipi di sistemi logici in forma attiva . 
cos ad esempio i processi di classificazione o di relazione parte / tutto divengono sempre pi rapidi nei tempi di acquisizione mano a mano che tali conoscenze sono implementate e interconnesse . 
unlike long - term memory , which is activated autonomously and without the intervention of the conscious , working memory highly depends on the attention system capable of authorizing the implementation of higher functions , such as symbolic calculations or the decision to implement or inhibit automatisms [ 10 , 11 ]  . 
 analysis of radiographs involves the fundamental function of working memory , that is , the maintenance of a representation constructed from empirical data , in our case from the characteristics of the radiographs , and from information recalled from long - term memory , which we could call experience . 
la memoria di lavoro ha apporti differenti , anche fonologici , visivi e spaziali ; permette di stoccare un numero piccolo di informazioni ( da 3 a 7 indipendenti una dallaltra )  . 
la risoluzione di problemi complessi , come quello della valutazione dei radiogrammi , abbisogna di parametri molto pi numerosi : il problema risolto con lassociazione nella memoria di lavoro di pi sottoinsiemi , che sono trattati come singole unit . 
contrariamente alla memoria a lungo termine , che attivata autonomamente e senza lintervento della coscienza , la memoria di lavoro dipende fortemente dal sistema attenzionale in grado di autorizzare la messa in opera di funzioni superiori come il calcolo simbolico , la decisione di una gestione o linibizione degli automatismi [ 10 , 11 ]  . 
ai fini dellosservazione dei radiogrammi interviene la funzione fondamentale della memoria di lavoro ovvero il mantenimento di una rappresentazione , costruita a partire dai dati empirici , nel caso nostro dalle caratteristiche dei radiogrammi , e da informazioni recuperate dalla memoria a lungo termine , che in modo arbitrario , ma di immediata acquisizione potremmo chiamare esperienza . 
knowledge - based activity : the diagnostic process is guided by analytical reasoning , in the awareness of already stored knowledge , to tackle new situations , which require a considerable application effort . human error in diagnostic imaging : types and causes having introduced the areas of human activity where errors may occur , the causes may now be sought in the three major groups of error : ( 1 ) perceptual errors , ( 2 ) cognitive errors and ( 3 ) system errors . 
these are summarised in figure 3 . the scientific principle of assessment is also valid for human error , that is to say , that similar to the bayes formula , errors are either false positives or false negatives . 
la diagnostica orientata da un ragionamento analitico , nella consapevolezza di un bagaglio di conoscenze gi immagazzinate , per far fronte a situazioni nuove , che richiedono quindi uno sforzo applicativo non comune . according to berlin [ 12 ] , 70% of lawsuits brought against radiologists are related to the non - identification of radiological signs , 60% of which are due to perceptual errors . with regard to the causes , a distinction is made between errori umani nella di : tipologie e cause introdotti gli ambiti delle attivit umane in cui avviene lerfig . 
inches : systematic approach to human error in radiology rore , possibile ricercarne le cause nei tre grandi insiemi : ( 1 ) linsieme degli errori percettivi , ( 2 ) linsieme degli errori cognitivi , ( 3 ) linsieme degli errori del sistema . 
per gli errori di sistema o latenti qui ribadito che lerrore umano di ripercussione , essendo la causa antecedente al momento dellavvenimento , gi radicata nel sistema organizzativooperativo . errori percettivi secondo berlin il 70% [ 12 ] delle pratiche legali contro i radiologi connesso ad una non identificazione di segni radiologici , con un contributo del 60% degli errori percettivi . quanto alle cause , si distinguono errori di identificazione con errata attribuzione ed errori di non identificazione . errori di identificazione con errata attribuzione sono errori poco frequenti : si verificano allorquando il radiologo segnala una lesione che non sussiste , scambiando ad esempio un reperto normale con uno patologico . 
barbaum [ 13 ] riporta , ad esempio , che nella ricerca di un corpo estraneo per la presunta ingestione di un bottone da parte di un bimbo di 1 anno in un radiogramma standard del torace , un radiologo ha malamente interpretato un nucleo di ossificazione sternale attribuendolo al bottone . 
le verifiche endoscopiche e il pasto opaco hanno dimostrato linconsistenza dellaffermazione . come gi osservato sono gli errori pi frequenti : il riconoscimento postumo di un nodo polmonare sul radiogramma del torace la situazione pi nota . 
questa possibilit di errore risulta chiara se ci si cimenta con i disegni paradossali di esher o con il libro per bambini di handford the great waldo [ 14 ]  . 
nellultimo libro , i bambini sono sfidati a trovare il mostriciattolo waldo nascosto tra mille disegni : difficile da riconoscere , ma una volta scoperto o rifugiatisi nelle soluzioni , alla fine la sua inconfondibile sagoma e la relativa posizione risultano ovvie . 
nellesempio la percezione fortemente influenzata dalle aspettative ; perci trovare waldo pi semplice che vedere un piccolo nodo polmonare sul radiogramma toracico , perch waldo sicuramente c , mentre per il nodo polmonare non abbiamo indizi . 
 [ 13 ] report an example in the search for a foreign body in a standard chest x - ray due to the presumed ingestion of a button by a 1 - year - old child . 
endoscopic examinations and an opaque bolus proved the affirmation incorrect . non - identification errors these are the most frequent kinds of errors , with the late recognition of a pulmonary node on a chest x - ray being the most common situation . 
the possibility of making this kind of error is clear if one tests oneself with the paradoxical drawings of escher or with the childrens books by handford wheres wally ? [ 14 ]  . 
in one of the most recent books , children are challenged to find wally hidden among hundreds of figures ; he is extremely difficult to recognize , but once identified his , unmistakable outline and his position are in the end obvious . 
in this example , perception is strongly influenced by expectations , so that finding wally is much easier than identifying a small pulmonary nodule on a chest x - ray because wally is definitely there while a pulmonary nodule may or may not be present . 
with regard to errors in this activity , kopans and daniel [ 15 , 16 ] note cases reported by psychologists of the difficulty and frustration of contingent situations often present in daily life , such as the fruitless search for a bunch of keys which we are unable to see but need to get into our house . 
the example can easily be related to a mammographic sign indicated by a colleague or recognized at a later time . specific causes as well as non - describable factors , the erroneous perception that leads to the non - identification on radiographs is connected to specific , numerous and heterogeneous causes . 
indeed , the final error is perceptual , but the complexity of mental behaviour is such that causal factors which are a part of technical and cognitive features play a part . 
with reference to berlin [ 18 ] , in one case of non - identified pulmonary nodule , the judicial assessment commission admitted that the lesion was difficult to diagnose since the x - rays were overexposed , thus suggesting the responsible error . 
the difficulty of diagnosing lesions at the level of the last cervical and first thoracic vertebrae is also well known , particularly in trauma patients , unless an optimal patient position is found . 
berlin [ 18 ] reports the case of a patient who fell from a ladder and underwent radiography of the cervical vertebrae on three separate occasions owing to the persistence of symptoms . 
unfortunately , the lesion identified on a later occasion in another hospital was a fracture / luxation of c6 - c7 , with the patient ending up a permanent quadriplegic . 
inches : systematic approach to human error in radiology screening : in quello mammografico infatti notevole la variabilit anatomica , spesso la struttura di difficile analisi , e il numero di casi da interpretare pu essere enorme . 
per gli errori in questa attivit kopans [ 15 , 16 ] ricorda , su segnalazione degli psicologi , le difficolt e le frustrazioni delle situazioni contingenti spesso presenti nella vita quotidiana , come la ricerca infruttuosa di un mazzo di chiavi , che non siamo in grado di vedere , ma che sono necessarie per entrare in casa . 
lesempio si ricollega con facilit ad un segno mammografico indicato da un altro operatore o riconosciuto in tempi successivi . cause specifiche oltre a fattori non descrivibili , lerrata percezione che porta alla non identificazione sui radiogrammi connessa a cause specifiche , numerose ed eterogenee . 
le tipologie sono indicate in figura 4 . gli errori tecnici [ 17 ] sono bene esemplificati dalla lesione non vista per sovra o sottoesposizione o per errato posizionamento del paziente . 
con riferimento a berlin [ 18 ] , in un caso di nodulo polmonare non visto , la commissione di valutazione giudiziaria ammise che era difficile diagnosticare la lesione perch le radiografie erano sottoesposte , sottintendendo cos lerrore responsabile . 
sempre berlin riporta il caso di un paziente , caduto dalle scale di casa e sottoposto a radiografie della colonna cervicale in tre fasi diverse , a causa di una persistente sintomatologia : il primo esame identific le prime 4 vertebre cervicali , il secondo dimostr anche c5 con sole note artrosiche . 
the anomalies outside the privileged area of the examination are not infrequent and include thoracic lesions identified during an abdominal examination ; or pleural , pulmonary or mediastinal lesions incidentally encountered during a radiological study of the spine , thoracic bones or shoulder girdle . 
in a litigation caused by lack of identification of an arteriovenous malformation of the bone marrow , berlin [ 19 ] underlines the difficulty of establishing whether the error was due to non - perception or lack of knowledge by the radiologist , and such difficulties can be extended to other cases . the attempt to explain non - identification with other causes dates back to the 1960s and the work of several authors , such as smith and tuddenham , which was later commented by wood , and raufaste and eyrolle [ 8 , 10 , 11 ] : several lesions were not identified owing to another lesion present in the image which had caught the attention of the radiologist . 
following the same line of research , berbaum [ 20 ] investigated the phenomenon on his own colleagues , introducing an artificial nodular lesion in chest x - rays already marked by original lesions . 
he defined this perceptual error satisfaction of search , thus recognizing the argument of cognitive psychologists who note that radiologists tend to be satisfied after finding a lesion , and if they continue the assessment of the image , it is only to restrict the field of study to what they have identified . the quantity of perceptual errors varies according to the examination in question . 
 [ 21 ] , in an interval of 16to 40 - mm nodules , recognized a minimum error of 20% ! in a specific study on the efficiency of health services , watcher et al . 
 [ 22 ] reports similar types of errors in other organs and systems recognized by other researchers : the errors in emergency situations have a low percentage of 3%6% ( robin ) , the sensitivity of identifying ureteral stones , many of which are recognizable a posteriori , is 46% ( levine ) and the error rate in contrast enema is 15% ( wesses )  . a first critical examination of perceptual errors , in the light of case series and studies which go beyond the individual case with legal implications , demonstrates that the real difficulty is the error without specific cause . 
it should be toracica individuata in corso di addome diretto , o ancora le lesioni pleuriche , polmonari o mediastiniche incidentalmente comparse in corso di studio radiografico della colonna vertebrale , delle ossa del torace o del cingolo scapolare . 
berlin in un suo articolo , in un caso di contenzioso per la mancata individuazione alla mielografia di una malformazione arterovenosa del midollo spinale [ 19 ] , sottolinea che vi sono concrete difficolt a stabilire se nel caso lerrore fosse dovuto alla non percezione o alla scarse conoscenze del radiologo e che tali perplessit sono da estendere ad altre evenienze . il tentativo di spiegare la non identificazione con ulteriori cause risale agli anni 60 con le osservazioni di alcuni autori , quali smith e tuddenham , raccolte e commentate successivamente da wood e raufaste [ 8 , 10 , 11 ] : alcune lesioni potevano non essere individuate a causa di unaltra lesione presente nel documento che aveva catturato lattenzione del radiologo . 
berbaum [ 20 ] nello stesso filone di ricerca ha indagato il fenomeno con i propri collaboratori , introducendo una lesione nodulare artificiale nei radiogrammi del torace gi interessati da altre lesioni originali : lidentificazione di queste ultime , per il disturbo delle altre , diminuiva in modo significativo . 
egli ha chiamato un tale errore percettivo satisfaction of search , riconoscendo la tesi degli psicologi cognitivisti che notano che i radiologi tendono ad essere soddisfatti dopo la ricerca di una lesione e proseguono semmai oltre solo restringendo il campo dindagine su ci che hanno visto . in termini applicativi la quantit di errori percettivi varia a seconda delle indagini in questione . 
interessante notare come in una revisione della casistica personale di radiogrammi del torace e nel confronto con altri autori , quekel [ 21 ] riconosca in un intervallo da 16 a 40 mm delle dimensioni dei noduli un errore minimo del 20% ! in uno studio specifico sullefficienza delle prestazioni sanitarie watcher [ 22 ] riporta analoghe tipologie di errore relative ad altri organi ed apparati riconosciute da altri ricercatori : gli errori in emergenza hanno percentuali minime del 3%6% ( robin ) ; la sensibilit di rilevamento dei calcoli ureterali , molti dei quali riconoscibili a posteriori , del 46% ( levine ) ; la quota derrore nel clisma opaco del 15% ( wesses )  . una prima considerazione critica sugli errori percettivi , alla luce di casistiche e studi che esulano dal caso singolo con risvolti giudiziari , mette in evidenza come il vero rebus sia lerrore senza specifica causa . 
 [ 23 ] hanno studiato gli effetti della doppia e tripla lettura su un campione di 60 casi di clisma opaco a doppio contrasto , in cui erano presenti 46 lesioni . 
the radiologist is aware of the routine and surrounding working conditions ; a temporary loss of attention causes the slip , which may be considered a small monitoring error . a ( perceptual ) error caused by capture occurs when the radiologists attention is attracted by a sign and is satisfied by this first finding , using a more common working framework rather than a less common one . alongside this type of slip is one in which the error is so obvious that it is known as a description error . 
it is an error characteristic of reporting when a semantic field related to one technique is used to describe another , for example , a ct study described with echogenicity - based terminology , or an ultrasound described with density - based terminology . other slips , such as associative activation ( answering the telephone when the doorbell rings ) or loss of activation , described as the temporary loss of memory while performing a precise task ( the search for an object ) , appear to be circumstantial , more common with the use of high - tech equipment or procedures , and can immediately be made up for . 
therefore , performing tasks without disturbances or distractions is the best prevention for this type of error . in contrast , a series of factors of various natures may help create situations which foster the occurrence of slips . 
the most common psychological elements include fatigue , loss of sleep , alterations caused by alcohol or drugs , or emotional states such as boredom , frustration , fear , anxiety or anger all conditions which cause concern and distract ones atl . 
inches : systematic approach to human error in radiology errori cognitivi gli errori della fase cognitiva sono stati classificati da reason e rasmussen [ 6 ] con il riferimento ad ogni livello attenzionale come basati sulla capacit intellettiva ( skill based ) , sul metodo cognitivo ( rule based ) e sulla conoscenza ( knowledge based )  . 
errore caratteristico nella refertazione quando si utilizzi una semantica relativa ad una metodica per descriverne unaltra : ad esempio , un referto con terminologia basata sullecogenicit per unindagine tc o invece sulla densit per unecografia . altre sviste quali lattivazione di associazioni ( associative activation ) come rispondere al telefono quando suona il campanello o la mancanza dattivazione ( loss of activation ) ovvero la temporanea perdita di memoria in corso di unazione ben precisa , come la ricerca di un oggetto , appaiono circostanziali , pi frequenti nel corso dimpiego di tecnologie o procedure , e di solito dimmediato recupero . tuttavia va sottolineato come le sviste sono spesso causate da interruzioni di precedenti attivit che erano in atto . pertanto lo svolgimento dellattivit senza disturbi e distrazioni la migliore prevenzione per tali tipologie derrore . per contro , una serie di fattori di diversa natura pu innescare situazioni favorevoli alle sviste . 
gli elementi psicologici pi frequenti comprendono la fatica , la perdita di sonno , le alterazioni da alcool o droghe oppure stati emozionali , quali la noia , la frustrazione , la paura , lansia , la collera , tutte condizioni che conducono alla preoccupazione che distrae lattenzione . a queste condizioni possono contribuire altri fattori esterni quali leccesso di lavoro e le relazioni interpersonali difficili o ambientali come il rumore , la calura , stimoli visivi eccessivi . 
in addition to these conditions , there may also be external factors , such as an excessive workload , difficult interpersonal relations or an unfavourable working environment characterised by excessive noise , heat or visual stimuli . in di , therefore , a large number of slips can be the result of working environments inappropriate for the study of radiographic images , excessive workloads , difficult conditions in the department and so on . rule - based and knowledge - based errors errors related to the two cognitive activities , commonly known as psychological errors , have been studied less than others and are more difficult to assess . 
these errors are due to an erroneous assessment of a contingent situation for which an incorrect procedure is applied . figure 5 shows how those errors , more correctly defined mistakes , are both recognition and decision making in nature . 
mistakes are therefore specifically cognitive errors , distinct from the more general term errors , which includes all types and therefore perceptual and system errors as well . figure 5 also shows how recognition errors are usually based on the most commonly used reasoning , adopted due to mental laziness on the part of the radiologist who has trouble breaking from known and repeated implementation models . included with this mental laziness or preconceived positions are mistaken choices , which piattelli - palmarini [ 24 ] defines deadly sins and which are summarized in figure 5 and outlined here below : biased memory leads to overgeneralizations of the ordinary or undergeneralizations of facts due to emotive reactions connected with personal success or contradictory experiences . heuristic availability prompts us to use only the primary information which comes to mind . confirmation bias supports our working hypotheses , causing us to ignore all contradictory information . overconfidence leads us to overestimate ourselves and our own abilities . anchoring highlights the influence that our first impression , or first judgement , has and how it becomes impossible to completely suppress it in later revisions . probability blindness refers to the mental procedure incapable of taking into consideration probabilistic estimates . it is so dangerous that piattelli - palmarini warns that any probabilistic intuition of any person not specifically versed in probability calculations has more than 50% probability of being incorrect [ 24 ]  . reconsideration under suitable scripts , lastly , is the seventh of the mental procedures which lead to cognitive errors . 
it is thought that a type of event or situation is judged all the more frequently the easier it is to mentally imagine and the greater the emotional impact it has as well as the more our judgement of probability is biased by the scripts . together with these seven deadly sins of the mind are two inevitable corollaries , situations contingent to the work phases : errori nellambito dellattivit basata sulle regole ( ruled based activity ) e dellattivit basata sulla conoscenza ( knowlodge based activity ) gli errori inerenti le due attivit cognitive , denominati comunemente psicologici , sono i meno studiati e i pi difficili da inquadrare : spesso sono identificabili pi cause che insieme contribuiscono allerrore . 
la figura 5 evidenzia ancora come lerrore di riconoscimento si basi di solito sul ragionamento usato pi frequentemente , cui si ricorre a causa della pigrizia mentale del radiologo , che stenta a staccarsi da schemi applicativi noti e ripetuti . 
clinical information may play a determining role in this assessment . before taking into consideration this aspect , which is fundamental in daily practice , it is worth noting the three main types of error in di associated with mental tunnels , as proposed by fitzgerald [ 26 ] : availability bias : the ease of remembering a diagnosis specific to the case compromises the possibility of making a correct diagnosis . regret bias : a distorted probability of diagnosis influences the correctness in the unconscious desire that something regrettable occurs . framing bias : the frame of the examination ( the request , case history , etc . ) unduly influences the report . a study in 1992 by norman et al . 
the first phase of the experiment involved 50 radiologists called on to assess 50 radiographs 25 from patients affected by bronchiolitis and 25 from normal patients with the radiologists being asked to search for signs of disease such as hyperinsufflation , bronchiolar thickening and perihilar opacities . the signs were found in both diseased and healthy patients in compliance with the suggested provisional diagnosis . 
in the second phase of the experiment , the radiographs were accompanied with a brief clinical history of normality or pathology , and three radiologists were specifically asked what the real probability of bronchiolitis was . the experiment showed the consistent effect of the clinical history on both attribution of a diagnostic category and the radiological signs on uncertain radiographs . 
at any rate , there is no doubt that knowledge of clinical history and signs regarding physical examination increase the possibility of radiological interpretation . further useful information can be gleaned from a comparison of previous radiological studies performed on a patient , as studied by berbaum et al . 
 [ 28 ] and berlthe american college of radiology has included the procedure among the published quality standards , stating that comparison with relevant previous examinations and reports should be part of the radiologic consultation and report when appropriate and available . 
inches : systematic approach to human error in radiology centrarsi su una sola fonte dinformazione ; il capovolgimento sotto affaticamento ( reversion under stress ) il fenomeno nel quale i comportamenti appresi di recente sono sostituiti con altri pi vecchi , ma pi familiari anche se inadatti al problema in esame . 
gli errori di riconoscimento avvengono quando una anomalia vista dal radiologo , ma non apprezzata come tale ; in realt riconoscere unanomalia richiede pi una memoria visiva che unacuit visiva . 
le informazioni cliniche possono giocare un ruolo determinante in questo giudizio . prima di prendere in considerazione tale aspetto , fondamentale nella pratica quotidiana , vale la pena di riportare da fitzgerald [ 26 ] i tre principali tipi di errore nella di connessi ai tunnel mentali : influenza della memoria ( avaibility bias ) : la facilit di ricordare una diagnosi specifica al caso pregiudica la possibilit della diagnosi corretta ; influenza emotiva ( regret bias ) : una distorta probabilit di diagnosi influenza la correttezza , nellinconscio desiderio che avvenga qualcosa che dispiace ; influenza del contorno ( framing bias ) : la cornice dellesame ( richiesta , anamnesi etc ) influenza eccessivamente il referto . a tale riguardo uno studio di norman del 1992 [ 27 ] dimostra empiricamente limportanza delle informazioni cliniche . 
lesperimento riguarda 50 radiologi esperti chiamati a valutare in una prima fase 50 radiogrammi , 25 di pazienti affetti da bronchiolite e 25 normali , chiedendo loro di ricercare i segni della malattia quali iperinsufflazione , ispessimento bronchiolare , opacit perilari . 
in una seconda fase stato chiesto esplicitamente ai tre radiologi quale fosse in ogni radiogramma , cui era stata aggiunta una breve storia clinica di normalit o di patologia , la probabilit reale di bronchiolite . 
a livello operativo nessuno osa in ogni caso mettere in dubbio che la conoscenza della storia clinica e dei segni relativi allesame obiettivo aumentino le possibilit dinterpretazione radiologica . altre informazioni utili possono ancora essere desunte dal confronto con i precedenti studi radiografici eseguiti sul paziente , come sperimentato da berbaum e berlin [ 28 ]  . lamerican college of radiology ha posto la procedura tra gli standard di qualit pubblicati affermando che il confronto con gli esami precedentemente eseguiti dal paziente , quando possibile , parte integrante dellindagine radiologica l . 
inches : systematic approach to human error in radiology space and time can be resolved with the picture archiving communication system ( pacs ) [ 29 ] which with the storage of digital data ( and therefore not analogical images ) could nonetheless give rise to revisions and evaluations after the fact , both regarding the use of the same in the broadest possible sense and the perception and interpretation of the derived images . if knowledge of the history and a comparison of previous data are valid methods for eliminating the radiologists decision - making errors , then reading previous radiological reports could also prove useful . 
this proposal , however , opens up an important debate regarding alliterative errors [ 30 ] , defined as an erroneous and reiterated diagnosis by several radiologists on the basis of the report of the first radiologist . the first such case reported by berlin [ 30 ] of a case of lung cancer , which on several occasions was labelled as bronchopneumonia , was followed by an important debate . 
the theory of berlin [ 31 ] which states that if a radiologist fails to recognize an anomaly or incorrectly interprets its meaning , then the possibility of the next radiologist repeating the same error is noticeably increased is supported by smith , cited by berlin [ 31 ] , who states that radiologists are induced to read the report of a colleague before assessing the radiograph . 
laspetto relativo allarchiviazione , di difficile applicazione nello spazio e nel tempo , potr essere risolto dai sistemi digitali pacs [ 29 ] , che tuttavia con la memorizzazione dei dati numerici ( non immagini analogiche quindi ) potranno dare luogo a revisioni e giudizi postumi , sia sullutilizzazione degli stessi in tutta la loro ampiezza che sulla percezione e interpretazione delle immagini ricavate . se la conoscenza della storia e il confronto con i dati precedenti sono metodi validi per eliminare gli errori decisionali del radiologo , anche la lettura dei referti precedenti allegati potrebbero rientrare in questa ottica . laffermazione apre tuttavia limportante dibattito relativo allerrore alliterativo [ 30 ] , individuando in questo unerronea e reiterata diagnosi da parte di pi radiologi sulla base del referto del primo radiologo . 
alla prima segnalazione di berlin su un caso di tumore polmonare , in pi circostanze etichettato come broncopolmonite [ 30 ] segu un importante dibattito . la teoria di berlin [ 31 ] che , se un radiologo non vede unanomalia o le attribuisce un significato sbagliato , la possibilit che il successivo radiologo ripeta il medesimo errore notevolmente aumentata sostenuta anche da smith , citato da berlin [ 31 ] , il quale afferma che i radiologi sono portati a leggere il riscontro del collega prima dellapproccio al radiogramma . tuttavia lamerican college of radiology nello standard for communication mette in evidenza che il paragone con precedenti esami e referti , dove possibile , parte integrante dellanalisi radiologica . 
in una successiva revisione berlin [ 32 ] riporta da autori diversi analisi sperimentali in contraddizione , riconoscendo che hunter e boyle dimostrano lutilit nel leggere i precedenti referti nel 60% dei casi e un considerevole aiuto nel 22%24% dei casi , mentre white segnala solo 1 , 5% di utilit e un 48% di inutilit . 
scelta della soglia del sospetto . ferred to for the following discussion . in the strategy outlined in model 1 , the threshold of suspicion would lead to a high specificity for the test and a good level of sensitivity but with 19% more false negatives than model 2 . 
on the other hand , the threshold of suspicion is lower in model 2 , thus introducing more recognizable minimal signs , even in healthy breasts , and therefore reducing the specificity of the test to the point of increasing false positives by 4 , 110 cases ( out of a total of 17 , 000 )  . 
so a diagnostic gain of 19% ( no small thing ! ) is counterbalanced by the anxiety of being called for follow - up multiplied by ten times and the implementation of invasive procedures multiplied by five . 
on the basis of current knowledge and experience , since in both cases the radiologist knows in advance the nature of the error , either false positive or false negative , and plans his or her work activity on the basis of this assessment , the error may be defined as strategic , especially if the choice is shared in the context of coordinated decision making and operations . 
as a result , in the implementation phase of breast screening , the error becomes operatively present , with a meaning similar to that codified by federspil and vettor [ 1 , 2 ] in clinical activity , when there is a choice of two alternative therapies . prevedere la diversa distribuzione degli errori : la figura 7 , di facile comprensione nei passaggi dai valori assoluti a quelli percentuali [ 39 ] , funge da riferimento per le successive argomentazioni . 
nellipotesi di una scelta strategica che si uniformi al modello 1 la soglia del sospetto comporterebbe unelevata specificit del test ed una buona sensibilit , ma con una quota di falsi negativi del 19% in pi rispetto al modello operativo 2 . 
daltro canto nel modello operativo 2 , ove la soglia del sospetto si abbassa , introducendo quindi molti segni minimi riconoscibili anche in mammelle sane , la specificit del test si riduce al punto di aumentare di ben 4410 casi ( su 17000 donne ! ) i falsi positivi . 
poich , sulla base delle attuali conoscenze ed esperienze , in entrambi i casi il radiologo conosce preventivamente il versante dellerrore , falso positivo o falso negativo , e anche in base a tale valutazione progetta la sua attivit , lerrore pu essere definito strategico , a maggior ragione se la scelta condivisa nellambito di un coordinamento operativo - decisionale . 
inches : systematic approach to human error in radiology system errors it has already been mentioned how system errors have repercussions in the end on the final medical assessment , with the characteristic connotations of human error [ 7 ]  . 
it has been recognized that the system as such has peculiarities in which the cause of errors is to be found . generally speaking , we would intuitively expect a wellconstructed system to be equipped with defences , barriers and mechanisms which reduce the risk of errors to a minimu nonetheless , those mechanisms , which reason and rasmussen [ 6 ] liken to barriers filled with holes , are extremely vulnerable when the holes line up on the same trajectory . the inevitable error is accompanied by the personal application , which is not able to correct the error owing to a series of preexisting causes , such as those summarized in figure 6 . system errors are therefore prevented above all by adopting a safety system which renders their occurrence difficult for individual work and by performing constant research which is accompanied by daily efficiency [ 40 ]  . there are numerous suggestions for achieving this in di . 
those which draw on applicative criteria include protocols for reducing reliance on memory , improvement of access to information through the patients case history , use of systems impermeable to errors to avoid homonymy , and training . measures more specifically related to the department include maintenance of quality standards , systematic control of equipment , revision of reports and appropriate instructions to users . federspil e vettor [ 1 , 2 ] nellattivit clinica , allorch si mettano in alternativa due scelte terapeutiche . errori di sistema gi stato dato risalto a come gli errori di sistema si ripercuotano alla fine sulla valutazione medica conclusiva , con le connotazioni caratteristiche dellerrore umano [ 7 ]  . 
allerrore inevitabile si aggiunge lapplicazione personale , che non in grado di correggerlo per una serie di concause tra le quali si riconoscono in modo immediato quelle raccolte in figura 6 . 
la prevenzione degli errori di sistema si attua quindi sopratutto mediante un apparato di sicurezza che li renda di difficile evenienza per il lavoro individuale e mediante una costante ricerca che proceda di pari passo con lefficienza giornaliera [ 40 ]  . nella di molti sono i suggerimenti in questo senso . 
tra quelli che si rifanno ai criteri applicativi generali si citano i protocolli per ridurre la fiducia nella memoria , il miglioramento allaccesso dellinformazione mediante la cartella clinica del paziente , luso di sistemi impermeabili agli errori ad evitare omonimie , laddestramento . 
per quanto riguarda pi specificamente il reparto si ricordano il mantenimento degli standard di qualit , il controllo sistematico delle attrezzature , la revisione dei referti , le idonee segnalazioni agli utenti . conclusions considerazioni conclusive error is a characteristic feature of human activity . 
the latin saying errare humanum est may be applied to all activity , and di is no exception . perhaps less well known is the medieval addition to the saying attributed to saint bernard sed perseverare diabolicum indicating that errors upset our balance and therefore need to be corrected or , better still , prevented . to achieve these aims in a complex system such as di , simple recognition of the error does not appear to be sufficient . preliminary knowledge of the types and causes of errors relating to human activity and the system is required , as well as their application in a daily analysis to improve both standards of service and personal satisfaction . lerrore rappresenta una connotazione caratteristica delle azioni umane . 
gargiulo2 1dipartimento integrato dei servizi diagnostici e per immagini , azienda policlinico , via del pozzo 71 , i - 41100 modena , italy 2cattedra di chirurgia vascolare , azienda policlinico , modena , italy correspondence to : m.g. 
 + 39 - 059 - 4222238 , fax : + 39 - 059 - 4224349 , e - mail : amorico.mariagrazia@policlinico.mo.it received : 27 june 2005 / accepted : 18 october 2005 / published online : 3 march 2006 abstract purpose . 
magnetic resonance angiography ( mra ) has recently become instrumental in the diagnosis of arterial disease in various body districts and is gaining an increasingly important role in the study of peripheral vascularisation . 
mra was performed with a philips intera 1.5 t , with acquisitions from the coeliac trunk to the feet . for acquisitions of the feet and ankles we used unenhanced timeof - flight ( tof ) sequences with a head coil . 
digital subtraction angiography ( dsa ) remains the reference standard for planning treatment and for the follow - up of these patients , but it is invasive and potentially harmful as the iodinated contrast larteriopatia occlusiva periferica degli arti inferiori una patologia cronica e progressiva con unincidenza negli uomini con pi di 55 anni che va dal 4 , 5% al 8 , 8% [ 1 , 2 ]  . 
la diagnosi solitamente ottenuta mediante un accurato esame clinico , ma la scelta di uneventuale terapia non pu prescindere da precise informazioni morfologiche dellalbero arterioso ottenibili esclusivamente attraverso la diagnostica strumentale . 
because peripheral arterial occlusive disease is an increasingly common chronic condition , a large number of mostly elderly patients would have to undergo numerous angiographic examinations and endovascular procedures , both of which carry a high risk of complications , such as puncture of the arteries , iodinated contrast agent toxicity , and protracted exposure to radiation [ 3 ]  . 
for these reasons , it has become necessary to identify an alternative modality that is less invasive than dsa and able to evaluate the number and severity of lesions in order to plan treatment . 
recent technological innovations , such as continuous table movement and very fast three - dimensional ( 3d ) sequences [ contrast - enhanced mr angiography ( ce - mra ) ] [ 2 , 5 ] , have enabled the study of the arteries from the abdominal aorta to the foot with a single injection of contrast material . 
these improvements have overcome the limitations of the previous time - of - flight ( tof ) and phasecontrast sequences , such as long acquisition times ( ats ) and flow and movement artefacts . 
the aim of this study was to assess the reliability of mra in the evaluation of lesions caused by peripheral arterial occlusive disease , using intraarterial dsa as the reference standard . materials and methods patients from november 2003 to august 2004 , a total of 30 patients ( 11 women and 19 men ; age range , 4984 years ; mean age , 72 years ) underwent digital subtraction angiography and mra of the lower limbs . 
two patients had concurrent renal artery stenosis ( one on the right and one on the left ) , and another two had aneurysm of the infrarenal aorta , one of whom with concurrent aneurysm of the left popliteal artery . 
two patients were excluded after dsa because they had a pacemaker , and another was excluded due to a history of surgery with insertion of artificial crystalline made of non - mr - compatible material . 
poich larteriopatia periferica una patologia cronica in aumento , un numero elevato di pazienti , la maggior parte anziani , dovrebbe essere sottoposto a numerose angiografie e procedure endovascolari che comportano un elevato rischio di complicanze sia legate alla procedura , come la puntura dei vasi arteriosi , che alla tossicit del mezzo di contrasto ed allesposizione prolungata alle radiazioni ionizzanti [ 3 ]  . 
per tutti questi motivi si sentita lesigenza di una metodica di studio delle arterie degli arti inferiori meno invasiva e che possa essere utilizzata come valutazione del numero e della severit delle lesioni in previsione di un trattamento . la risonanza magnetica una tecnica di indagine non invasiva che non impiega radiazioni ionizzanti n mezzo di contrasto potenzialmente nefrotossico [ 4 ]  . 
recentemente sono state introdotte innovazioni tecnologiche come , per esempio , il movimento automatico del lettino porta - paziente e sequenze tridimensionali molto veloci ( ce - mra ) [ 2 , 5 ] che rendono possibile lo studio dellalbero arterioso dal tripode celiaco ai vasi del piede con ununica iniezione di mezzo di contrasto . 
ci ha permesso di superare le limitazioni delle precedenti sequenze time - of - flight e phase - contrast come i lunghi tempi di acquisizione , gli artefatti da flusso e da movimento . 
scopo di questo lavoro analizzare quale sia la reale affidabilit della angio - rm nella valutazione delle lesioni causate dallarteriopatia occlusiva periferica , utilizzando come standard di riferimento langiografia digitale a sottrazione ( dsa )  . materiali e metodi pazienti dal novembre 2003 allagosto 2004 , 30 pazienti di et compresa tra i 49 ed gli 84 anni ( 11 donne e 19 uomini ; et media 72 anni ) sono stati sottoposti ad angiografia digitale e ad angio - rm degli arti inferiori . 
due casi , inoltre , mostravano la presenza concomitante di stenosi dellarteria renale ( 1 a destra ed 1 a sinistra ) , in 2 casi un aneurisma dellaorta sottorenale in uno dei quali era concomitante un aneurisma dellarteria poplitea sinistra . 
the disease staging classification adopted by us was the following : mild stenosis ( not haemodynamically significant ) with less than 50% reduction in vessel lumen ; moderate stenosis ( haemodynamically significant ) with 50%90% reduction in lumen ; severe stenosis with a reduction in vessel lumen greater than 90% . the percentage of stenosis was automatically calculated by software present in the image - processing work stations connected to the angiographic and mr instruments ( viewforum , philips )  . patients included in the study underwent dsa performed with digital equipment ( philips integris allura , philips medical system , the netherlands ) , following a pre - established procedure . 
the examination procedure involved positioning of a 4 - french universal flash ( uf ) catheter into the abdominal aorta through a retrograde , generally right - sided , transfemoral approach , or a brachial approach according to the seldinger technique , after the local administration of 10 cc of lidocaine hydrochloride 200 mg ( bioindustria , italy )  . 
the catheter was positioned in the external or common iliac artery to perform selective arteriography of the limb , with anteroposterior ( ap ) projections for the thigh and leg vessels , and both ap and laterolateral ( ll ) projections for the feet vessels . 
images of the lower limb vessels , from the femoral artery to those of the feet , were obtained sequentially with separate injections of contrast material delayed relative to acquisition , on the basis of the level of study and the speed of flow . 
the mean dose of contrast agent injected was approximately 200 cc ( iomeprol 300 mg / i / ml , iomeron bracco , or iopamidol 300 mg / i / ml , iopamiro bracco ; iodixanol 270 mg / i / ml , visipaque nycomed amersham in selected subjects ) , with a range from 160 to 300 cc according to the diagnostic requirements of the single case . 
the entire examination had a mean duration of 60 min . mra was performed by using a 1.5 - t whole - body mri unit ( intera philips gyroscan 1.5 tesla , philips medical system )  . for each patient , a complete history was taken to exclude the presence of contraindications . 
the patient was positioned supine with both feet inside the coil usually used for the study of the head ( head coil ) for a preliminary evaluation of the foot arteries with an unenhanced 3d - tof sequence . 
due pazienti sono stati esclusi dallo studio dopo lesecuzione della dsa , perch portatori di pace - maker ed uno per presenza di anamnesi positiva per intervento con inserimento di cristallino artificiale di materiale non rm - compatibile . 
tre pazienti erano gi stati precedentemente sottoposti a procedure di rivascolarizzazione ( 1 by - pass femorofemorale destro - sinistro con bypass femoro - popliteo bilaterale , 1 by - pass femoro - popliteo destro , 1 endoprotesi hemoban in femorale superficiale destra ) e tre pazienti avevano subito lamputazione di uno o pi dita di un piede . la classificazione da noi seguita distingue : stenosi lievi ( non emodinamicamente significative ) con riduzione del lume vasale inferiore al 50% ; stenosi moderate ( emodinamicamente significative ) con riduzione del lume vasale superiore al 50% , ma inferiore al 90% ; stenosi gravi con riduzione del lume vasale superiore al 90% . la percentuale di stenosi stata calcolata automaticamente da un programma di calcolo presente nelle stazioni di elaborazione delle immagini correlate allangiografo philips allura ed allapparecchio di rm ( viewforum , philips medical systems , olanda )  . angiografia digitale i pazienti inclusi nello studio sono stati sottoposti a dsa con tecnica di sottrazione dimmagine eseguita con angiografo digitale ( integris allura philips , philips medical systems , olanda ) , seguendo una procedura prestabilita . 
lesecuzione dellesame prevede il posizionamento di un catetere uf ( universal flash ) di 4 f nellaorta addominale per mezzo di un approccio transfemorale retrogrado , generalmente destro , o brachiale , con ago munito di mandrino secondo la tecnica seldinger , ovviamente dopo aver eseguito nella sede lanestesia locale con 10 cc di lidocaina cloridrato 200 mg . 
il catetere viene posizionato in arteria iliaca esterna o comune per lesecuzione di unarteriografia darto selettiva ; si effettuano riprese dei vasi di coscia e di gamba in a - p e del piede sia in a - p che in ll . 
le immagini del riempimento degli arti inferiori dallarteria femorale a quelle del piede sono state ottenute sequenzialmente con iniezioni separate di mezzo di contrasto ritardate rispetto allacquisizione in base al livello di studio ed alla velocit del flusso . 
la dose media di mezzo di contrasto iniettato per via endoarteriosa di 200 cc circa ( iomeprolo 300 mg i / ml , iomeronr bracco oppure iopamidolo 300 mg i / ml , iopamiror bracco ; iodixanolo 270 mg i / ml , visipaquer , amersham , in soggetti selezionati ) , con un range variabile da 160 a 300 cc a seconda delle esigenze diagnostiche del singolo caso . 
the parameters used were : tr 23 ms , te 9.2 ms , flip angle 20 , thickness 0.7 mm , matrix 304x258 , field of view ( fov ) 30 c the sequence yielded around 150 images , which were processed in 3d at the workstation view forum 3.1 ( philips medical systems )  . 
subsequently , the head coil was replaced with a body coil , with mobytrack , a specific attachment for this type of study which supports and immobilises the legs at the level of the knees and holds the feet perpendicular to the legs . 
positioning of the entire lower limb in the same plane is important , as the coronal acquisition might otherwise miss arterial segments not perfectly included in the field of view . 
the arterial study was performed with three 40to 45 - cm acquisitions , with 3d coronal sequences from the coeliac trunk to the feet , each lasting around 3035 s , with an overlap of 13 c automatic table movement means the examination can be performed in a time compatible with the persistence of the paramagnetic contrast agent in the arterial vessels . first , a 2d fast - field - echo centring sequence was performed in the three planes in all three positions , on which the following sequences were planned . 
the parameters of this sequence were : tr 6.1 ms , te 1.5 ms , flip angle 35 , thickness 1.5 mm , matrix 512x384 , fov 43 cthen , circulation time ( ct ) , or bolus timing , was evaluated by injecting 2 cc of contrast agent followed by 15 cc of saline solution with a special mr - compatible injector ( spectris mr injector , medrad )  . 
this was followed by a fast - field - echo sequence ( tr 7 , te 0.87 , flip angle 40 , thickness 80 mm , matrix 256x256 , fov 53 cm ) in which a single coronal image at the level of the abdominal aorta and its bifurcation was acquired at intervals of 1 s for about 50 times , repeating the same image at regular intervals until the contrast agent was visualised . 
scan delay ( sd ) was obtained by subtracting half of the at from the ct plus half of the injection time ( it ) : sd = ct + ( it / 2 ) - ( at / 2 )  . an initial unenhanced 3d sequence serving as a mask was performed from the feet to the abdomen . 
around 40 - 50 cc of paramagnetic contrast agent ( gadodiamide ; omniscan nycomed amersham ) [ 15 ] was then injected as a bolus divided in two fractions : 50% was injected at a speed of 1 ml / s and 50% at a speed of 0.5 ml / s , followed by 15 ml of saline solution injected at a speed of 1 ml / s . 
this part of the examination lasted 2025 mparameters used in this sequence were : tr 6.1 ms , te 1.5 ms , flip angle 35 , thickness 1.5 mm , matrix 512x384 , fov 43 cthe 3d sequence used by us was designed to acquire a centric k space , the centre of the k space being acquired during the enhancement peak , which provides the best contrast between blood and static tissue . 
approximately 200 images were obtained , which were reconstructed with 3d maximum image projecte superconduttivo ( philips gyroscan intera 1 , 5 tesla , olanda ) dotato di gradienti con intensit massima di 30 mtesla / m ed una rapidit di salita ( slew rate ) di 150 mtesla / m / ms . 
il paziente viene posizionato supino , con entrambi i piedi allinterno della bobina solitamente usata per lo studio dellencefalo ( head ) , per eseguire una preliminare valutazione delle arterie del piede attraverso un sequenza 3d - tof senza mezzo di contrasto . 
la sequenza viene eseguita su un piano coronale obliquo perpendicolare allasse maggiore del piede e la banda di presaturazione necessaria per impedire il contemporaneo rilevamento del flusso venoso posta caudalmente alla acquisizione . 
successivamente si effettua un cambio di bobina : si sostituisce la bobina head e si utilizza la bobina body per la trasmissione e la ricezione del segnale ed un supporto dedicato per lo studio degli arti inferiori , moby - track , che sostiene ed immobilizza le gambe a livello delle ginocchia , che sono leggermente sollevate , e mantiene i piedi su uno stesso piano perpendicolare a quello delle gambe . 
il posizionamento di tutto larto inferiore su uno stesso piano un accorgimento importante poich altrimenti lacquisizione nel piano coronale potrebbe non includere segmenti arteriosi non perfettamente compresi nel campo di vista . 
lo studio arterioso viene eseguito in 3 acquisizioni da 4045 cm con sequenze 3d coronali dal tripode ai piedi , ognuna della durata di circa 3035 secondi , con sovrapposizione di 13 cil movimento automatizzato del letto porta - paziente permette di eseguire questo tipo di esami in un tempo compatibile con la permanenza del mezzo di contrasto paramagnetico nei vasi arteriosi . 
si esegue inizialmente una sequenza di centratura 2d turbo field echo sui 3 piani in tutte le 3 posizioni sulla quale verranno pianificate tutte le sequenze successive con i seguenti parametri : tr 6 , 1 ms , te 1 , 5 ms , flip angle 35 , spessore 1 , 5 mm , matrice 512x384 , campo di vista 43 csi procede poi ad una valutazione del tempo di circolo o bolus timing iniettando 2 cc di contrasto seguiti da 15 cc di soluzione fisiologica con un apposito iniettore rm - compatibile ( spectris mr injector , medrad , indianola , pa )  . 
si esegue una sequenza fast field echo ( tr 7 , te 0 , 87 , flip angle 40 , spessore 80 mm , matrice 256x256 , campo di vista 53 cm ) , in cui una singola immagine coronale , a livello dellaorta addominale e della sua biforcazione acquisita ad intervalli di 1 secondo per circa 50 volte ripetendo la stessa immagine ad intervalli costanti fino alla visualizzazione del mezzo di contrasto a quel livello . 
il tempo di ritardo scansione ( trs ) si ottiene sottraendo al tempo di circolo ( tc ) la met del tempo di acquisizione ( ta ) , dopo avervi sommato la met del tempo di iniezione table 1 evaluation form right left m . 
the total time spent by the patient inside the mr room , which included examination time , technical time for sequence formulation , and coil changing , was about 4045 min . data collection and analysis mra and dsa examinations were independently evaluated by three radiologists who used grading sheets to assess vessel state : no stenosis , or obstruction . 
the vessel being studied was drawn with parallel lines along a segment that includes the vessel above and below the stenosis and stenotic segment ; the computer analyses the segment and calculates the percentage of stenosis . 
conventional angiography examinations were evaluated by another radiologist blinded to the patients clinical history and results of mra . table 2 patent arteries with regular lumen artery common iliac external iliac internal iliac common femoral superficial femoral deep femoral popliteal anterior tibial posterior tibial peroneal dorsalis pedis plantaris pedis total mra , magnetic resonance ; dsa , digital subtraction angiography tabella 2 arterie pervie di calibro regolare vaso angio - rm angiografia a . 
si iniettano poi circa 4050 cc di contrasto paramagnetico ( gadodiamide omniscanner amersham , uppsala ) in un unico bolo suddiviso in due frazioni : il 50% viene iniettato ad una velocit di 1 ml / s , il restante ad una velocit di 0 , 5 ml / s ; seguito da 15 ml di fisiologica iniettata ad una velocit di 1 ml / s . 
la seconda sequenza con contrasto ha uno start - delay calcolato in base al bolus timing e viene eseguita dal tripode celiaco ai piedi . lapnea richiesta solo durante lacquisizione a livello addominale ed ha una durata di circa 25 s . 
il software della macchina sottrae automaticamente la sequenza con contrasto alla maschera per eliminare il segnale del tessuto adiposo e si ottiene unimmagine angio - rm delle arterie degli arti inferiori . 
la durata di questa parte dellesame di circa 2025 m i parametri utilizzati per eseguire questa sequenza sono : tr 6 , 1 ms , te 1 , 5 ms , flip angle 35 , spessore 1 , 5 mm , matrice 512x384 , campo di vista 43 cla sequenza 3d da noi utilizzata impostata per acquisire lo spazio k in senso centrico , grazie infatti allopzione centra , per cui viene acquisito il centro dello spazio k durante il passaggio del picco di contrasto massimo , che fornisce il contrasto migliore tra sangue e tessuto stazionario . 
il tempo totale che il paziente trascorre allinterno della stanza del magnete , che comprende il tempo desame , il tempo tecnico di impostazione delle sequenze e di cambio delle bobine di circa 4045 minuti . raccolta ed analisi dei dati gli esami di angio - rm e di angiografia sono stati valutati da 3 medici radiologi in modo indipendente attraverso la compilazione di schede , in cui si richiede di valutare lo stato del vaso : pervio , stenosi , ostruzione . 
la percentuale di stenosi stata ottenuta tramite un programma di calcolo inserito in speciali stazioni di elaborazione delle immagini , in cui viene disegnato con linee parallele il vaso in esame per un tratto che comprende il vaso a valle e a monte della stenosi ed il segmento stenotico ; il computer analizza il segmento in esame e calcola la percentuale di stenosi . 
langio - rm stata valutata da 2 medici radiologi in doppio cieco , senza conoscere la storia clinica del paziente ed i risultati dellangiografia tradizionale , utilizzando le rielaborazioni 3d delle immagini e le immagini originarie . 
si sono ottenute due schede ( tabella 1 ) per ogni paziente , una per la rm ed una per la angiografia , che sono state poi confrontate per valutare il livello di corrispondenza . 
i vasi arteriosi esaminati sono : aorta sottorenale , iliaca comune , iliaca esterna , iliaca interna , femorale comune , femorale superficiale , femorale profonda , poplitea , tibiale anteriore , tibiale posteriore , interossea , dorsale e plantare del piede . 
nei 2 pazienti con bypass se ne valutata anche la perviet , lo stato degli stessi e dei territori a valle . thus , two tables were obtained ( table 1 ) for each patient : one for mra and one for dsa , which were compared in order to evaluate the level of concordance . 
arterial vessels examined were : infrarenal aorta ; common iliac artery ; external and internal iliac arteries ; common , superficial , and deep femoral arteries ; popliteal artery ; anterior and posterior tibial arteries ; peroneal artery ; and dorsalis and plantaris pedis arteries . 
in the two patients with bypass , we also evaluated the patency and state of the bypass grafts and territories downstream . results mra was easy to perform and well tolerated although patients with trophic lesions , especially of the heels , had some difficulty staying motionless in the supine position because of pa this problem concerned above all the tof sequences , in which both feet have to be kept motionless inside the head coil for about 7 mas a result , because this sequence requires a very high level of patient cooperation , it was only performed in 15 . 
however , it did not yield important additional information compared with the contrast - enhanced study , except in one case . table 3 occlusions artery common iliac external iliac internal iliac common femoral superficial femoral deep femoral popliteal anterior tibial posterior tibial peroneal dorsalis pedis plantaris pedis total mra , magnetic resonance ; dsa , digital subtraction angiography tabella 3 occlusioni a . 
questo problema ha riguardato soprattutto lesecuzione delle sequenze tof , in cui entrambi i piedi devono rimanere immobili allinterno della bobina per circa 7 min . questa sequenza infatti non stata eseguita in tutti i pazienti , ma solo in 15 , in quanto necessita di estrema collaborazione e non ha dato informazioni rilevanti o aggiuntive rispetto allo studio con mezzo di contrasto , tranne in un singolo caso . 
sono stati visualizzati 552 vasi di calibro regolare con langio - rm e 547 con la dsa ( tabella 2 )  . si ottenuta una corretta visualizzazione di tutti i vasi fino alla caviglia , mentre per quanto riguarda le arterie del piede , in 3 casi , il precoce ritorno venoso ha reso difficile linterpretazione dellesame , ma grazie alla rielaborazione delle immagini alla workstation siamo sempre riusciti ad espritable 4 stenoses artery < 50% > 50% > 90% < 50% > 50% > 90% common iliac external iliac internal iliac common femoral superficial femoral deep femoral popliteal anterior tibial posterior tibial peroneal dorsalis pedis plantaris pedis total mra , magnetic resonance angiography ; dsa , digital subtraction angiography tabella 4 stenosi a . 
correct visualisation of all vessels was obtained up to the ankle whereas in the foot arteries , early venous return in three cases made interpretation of the examination difficult ; nonetheless , image processing enabled a judgement on the vessel to be expressed in all cases . 
discordances regarded the infrapopliteal arteries , with mra visualising the obstruction of 28 anterior tibial arteries , 35 posterior tibial , 14 peroneal , and 23 dorsalis pedis as against the 30 anterior tibial , 33 posterior tibial , 18 peroneal , and 22 dorsalis pedis visualised by dsa . 
in the cases of obstruction , the visibility of the collateral circulations was better with dsa , which was able to depict even tiny branches , but was also judged good with mra , which depicts the larger vessels only . 
evaluation of the foot arteries was more difficult : the tof sequences , if correctly performed , give information on the larger vessels perpendicular to the acquisition plane ; the contrast - enhanced sequences are also informative but they have a smaller field of view . 
as regards the nine ( 1.25% ) discordant segments , mra overestimated two ( 0.3% ) ( two occlusions of the posterior tibial arteries ) and underestimated six ( 1% ) ( two anterior tibial and four peroneal arteries )  . 
mra documented one occlusion of dorsalis pedis artery not visualised by dsa . overall , in the cases studied , mra had a specificity of 99.6% and a sensitivity of 96% ( table 5 )  . 
there was absolute concordance between the two imaging procedures in il numero di occlusioni valutato in angio - rm stato 140 e con la dsa 143 ( tabella 3 )  . 
le discordanze sono nelle arterie infrapoplitee , in particolare langio - rm ha riconosciuto lostruzione di 28 arterie tibiali anteriori , 35 tibiali posteriori , 14 interossee , 23 dorsali del piede contro le 30 tibiali anteriori , 33 tibiali posteriori , 18 inter - ossee , 22 dorsali del piede della dsa . 
nelle stenosi inferiori al 50% langio - rm ne ha riconosciute 13 , la dsa 14 : con questultima stata riconosciuta una stenosi dallarteria femorale profonda che risultava pervia alla angio - rm . 
nei casi di ostruzione la visibilit dei circoli collaterali stata migliore con la dsa , che stata in grado di evidenziare tutti i rami anche di piccolissimo calibro , ma stata giudicata buona con langio - rm , che mette in evidenza solo vasi di calibro maggiore . con entrambe le metodiche comunque possibile evidenziare il punto di ricanalizzazione del vaso . 
la valutazione pi difficoltosa stata quella dei vasi del piede : le sequenze tof danno informazioni , se correttamente eseguite , sui vasi di maggior calibro e perpendicolari al piano dacquisizione ; le sequenze con contrasto danno buone informazioni , ma hanno un campo di vista inferiore . 
dei 9 ( 1 , 25% ) segmenti discordanti , 2 ( 0 , 3% ) sono stati sovrastimati con langio - rm ( 2 occlusioni delle tibiali posteriori ) e 6 ( 1% ) sono stati sottostimati ( 2 tibiali anteriori e 4 inter - ossee )  . 
la permanenza totale del paziente nella sala del magnete stata in media di 45 mil tempo necessario alla rielaborazione ed alla stampa delle immagini alla workstation philips stato di 2030 mcome gi detto in caso di importante contaminazione venosa stato necessario un tempo maggiore . 
non si sono avute reazioni ai mezzi di contrasto in nessuno dei pazienti . discussione la tecnica di angio - rm da noi utilizzata , basata sullacquisizione di 3 volumi lievemente sovrapposti , iniettando il contrasto con uniniezione bifasica , ha permesso unaccurata valutazione dellalbero arterioso dal tripode celiaco alle arterie del piede . 
rispetto allangiografia , la rm offre importanti vantaggi nello studio di pazienti anziani con patologia arteriosa periferica , in quanto si possono evitare i rischi associati alla puntura arteriosa , al mezzo di contrasto iodato ed alle radiazioni ionizzanti . 
none of the patients experienced reactions to the contrast agent . discussion mra , based on the acquisition of three slightly overlapping volumes with two - phase injection of contrast agent , allowed table 6 data from the literature m . 
nellesecuzione dellangiorm si pu incorrere in alcuni pitfalls soprattutto perch per ottenere un esame angio - rm di elevata qualit con contrasto fondamentale la perfetta coordinazione tra linizio dellacquisizione e larrivo del mezzo di contrasto nella regione da studiare . 
non si sono avute comunque sostanziali discordanze tra angio - rm e dsa nella rilevazione della perviet dei vasi e nella valutazione della presenza di malattia aterosclerotica in tutti i territori esaminati . 
nello 0 , 3% dei casi si rilevata una sovrastima della gravit delle lesioni in angio - rm rispetto alla dsa [ 6 ]  . la sovrastima causata dal defasamento degli spin prodotto dal flusso turbolento che si crea nelle stenosi e da effetti di volume parziale . 
questo effetto risulta accentuato nelle ricostruzioni mip in cui non sempre si riesce a distinguere il ridotto calibro della porzione stenotica del vaso dal segnale di fondo [ 7 ]  . 
1 occlusione dellarteria iliaca esterna sinistra ( freccia ) e stenosi in serie di entrambe le arterie femorali superficiali . an accurate evaluation of the arterial tree from the coeliac trunk to the foot arteries . 
in comparison with conventional angiography , mr offers important advantages in the study of elderly patients with peripheral arterial disease , enabling one to avoid the risks associated with arterial puncture , iodinated contrast material , and radiation . 
this effect increases in the mip reconstructions , in which it is not always possible to distinguish the reduced calibre of the stenotic portion of the vessel from the background signal [ 7 ]  . 
la causa principale della sottostima nei casi da noi esaminati stata data da effetti di volume parziale , anche in questo caso dovuti alla difficolt di rilevare attraverso strati sottili i ridotti calibri dei vasi stenotici [ 6 , 8 ]  . 
langio - rm si rivelata una metodica dotata di alta specificit , ma di inferiore sensibilit , quindi praticamente sovrapponibile alla dsa nella valutazione dei vasi di calibro regolare , mentre in caso di lesione steno - ostruttiva la dsa pi accurata , anche se nel nostro studio abbiamo rilevato una buona concordanza . nessuno dei pazienti ha avuto reazioni alla somministrazione della gadodiamide [ 9 ]  . 
non ci sono stati artefatti da materiale metallico in quanto un solo paziente aveva uno stent hemoban ( nitinolo ) in femorale superficiale destra che per non ha causato alterazioni del segnale . 
alcuni pazienti di questo studio , tutti arteriopatici allo stadio iii e iv di la fontaine ( tranne due ) , hanno avuto difficolt a tollerare limmobilit a causa del dolore , ma questo problema si presentato sia durante lesecuzione dellangio - rm sia durante la dsa . 
per quanto riguarda la sequenza tof , ha fornito una discreta valutazione dei vasi del piede , ma come tipo di sequenza mostra alcune limitazioni : i vasi non perfettamente perpendicolari al piano di acquisizione possono avere una bassa intensit di segnale e simulare una patologia [ 10 ] ; la pulsatilit del flusso pu produrre artefatti se non si utilizza un gating cardiaco ; il flusso retrogrado di alcuni vasi collaterali o ricostruiti pu essere anchesso saturato e mascherare di conseguenza il vero livello dellostruzione o sovrastimare la lunghezza del segmento stenotico [ 11 , 12 ]  . solo 15 dei pazienti esaminati ha potuto eseguire questo tipo di sequenza a causa dei gi citati problemi legati allimmobilit in posizione supina . 
mra proved to have higher specificity but lower sensitivity and is therefore practically identical to dsa in the evaluation of normal vessels ; however , in the case of steno - obstructive lesions , dsa is more accurate , even if we recorded a good agreement in our study . 
no patient had a reaction to gadodiamide [ 9 ]  . there were no artefacts due to metallic material , as only one patient had a hemoban stent ( nitinol ) in the right superficial femoral artery , which did not cause signal alterations . a non - negligible limitation of this examination is the immobility requested of the patient . 
some patients in our study , all but two with fontaine stage iii and iv disease , had difficulty keeping immobile because of pain , but this problem arose during both mra and dsa . as regards the tof sequence , despite providing a discrete evaluation of the foot vessels , it does suffer some limitations : vessels not perfectly perpendicular to the acquisition plane may have low signal intensity and simulate disease [ 10 ]  . flow pulsatility can produce artefacts if cardiac gating is not used . 
 the retrograde flow of some collateral or reconstructed vessels can also be saturated and disguise the true level of obstruction or overestimate the length of the stenotic segment [ 11 , 12 ]  . only 15 of our patients could be studied with this sequence because of difficulty lying motionless in the supine position . 
it does , however , have some limitations , some of which will be eliminated by technological advances with fast gradients and specific contrast agents for vascular studies whereas others will remamra may be proposed for selected indications : screening asymptomatic patients with a family history of diabetes or risk factors for atherosclerosis , patients with renal insufficiency , elderly patients , and for surveillance of bypass grafts . conclusions in our experience based on a limited number of patients , mra can be considered an alternative modality to dsa in the management of patients with steno - occlusive disease of the peripheral arteries , as it allows screening of candidates for interventional [ percutaneous angioplasty ( pta ) or stent ] or surgical procedures ( bypass graft )  . the pre - requisite for this is to optimise the injection time on the basis of each patients cardio - circulatory parameters , as also shown by westenberg et al . 
da quanto detto si evince che , nonostante la metodica si sia rivelata sicura , affidabile e sopportabile per la maggior parte dei pazienti , presenta pur sempre dei limiti , alcuni dei quali saranno eliminati dallavanzamento della tecnologia con gradienti ancora pi veloci e mezzi di contrasto dedicati allo studio vascolare , mentre altri invece resteranno invalicabili . 
tutto questo rende langio - rm un esame proponibile solo ad alcuni pazienti : screening nei pazienti asintomatici con familiarit diabetica o con fattori di rischio per aterosclerosi , insufficienza renale , pazienti anziani , controllo di bypass . conclusioni nella nostra esperienza , limitata al numero di casi da noi studiati , langio - rm pu essere considerata una metodica alternativa alla dsa nel management dei pazienti con patologia steno - ostruttiva del circolo arterioso periferico , selezionando i candidati a procedura interventistica ( pta o stent ) o chirurgica ( bypass )  . la condizione imprescindibile su cui si fonda questa affermazione lottimizzazione del tempo di iniezione ad ogni singolo paziente , in base ai suoi parametri cardio - circolatori come dimostrato anche da westenberg et al . 
conte3 1fisica sanitaria , 2radiologia , ospedale di circolo e fondazione macchi , viale borri 57 , i - 21100 varese , italy 3dipartimento di scienze cliniche e biologiche , universit dellinsubria , i - 21100 varese , italy correspondence to : s . 
la dose efficace media in un esame angio - tc varia da 18 , 8 msv a 28 , 8 msv , secondo il protocollo adottato . lottimizzazione ha portato alla stesura di una tabella che riporta i valori di corrente al tubo da utilizzare in funzione della corporatura del paziente in fase arteriosa . 
le procedure per esami angio - tc impartiscono dosi rilevanti al paziente ; tuttavia possibile ottenere una riduzione delle stesse senza influenzare la qualit dellimmagine . parole chiave tomografia computerizzata multistrato dose efficace introduction introduzione during the last few years , multislice computed tomography ( msct ) has spread rapidly and is now widely used as a diagnostic technique . 
 negli ultimi anni la tomografia computerizzata multistrato ( tcms ) si rapidamente diffusa nei differenti centri e ormai largamente impiegata come tecnica diagnostica , quindi necessario approfondire sia lottimizzazione dei protocolli di acquisizione , sia laspetto radioprotezionistico inevitabilmente collegato . 
assessment of the computed tomography dose index ( ctdiw ) was performed on polymethylmethacrylate ( pmma ) cylindrical phantoms with diameters of 1632 cstatistical data analysis was performed using the programme statistica 6.1 produced by statsoft , italia s.r.l. four different protocols used in angio - ct were considered : the thoracic aorta protocol , the thoracic - abdominal aorta protocol , the abdominal aorta protocol and the endoprosthesis protocol . 
each ct examination consisted of a digital projection radiograph ( topogram ) and two or three series ( baseline , arterial , venous ) , depending on the clinical query . 
the algorithm uses the monte carlo dosimetric data of the national radiological protection board ( nrpb ) , uk , described in the report sr250 [ 1 , 2 ]  . 
calculation of organ dose is based on the nctdiair value , measured on the scanners rotation axis and multiplied by an international commission on radiation units ( icru ) factor that provides soft - tissue dose di acquisizione , in modo tale da ridurre la dose al paziente , garantendo allo stesso tempo una buona qualit dellimmagine . 
lottimizzazione stata eseguita dapprima su un paziente di corporatura standard e in un secondo momento estesa a pazienti con differenti corporature . materiali e metodi il presente studio stato effettuato su un apparecchio lightspeed plus 4 - slice ct scanner ( general electric , milwaukee , wi , usa ) ; per le misure dosimetriche stato impiegato un elettrometro rti solidose 400 con camera a ionizzazione capintec pc4p da 10 cle valutazioni dellindice di dose in tomografia computerizzata ( ctdiw ) sono state eseguite con fantocci cilindrici in polimetilmetacrilato ( pmma ) da 16 cm e 32 cm di diametro . 
lanalisi statistica dei dati stata effettuata col programma statistica 6.1 prodotto da statsoft italia srl . sono stati considerati quattro differenti protocolli impiegati in esami angio - tc : protocollo aorta toracica , protocollo aorta toraco - addominale , protocollo aorta addominale , protocollo endoprotesi . 
ogni esame tc costituito da una proiezione digitale radiografica ( topogramma ) e da due o tre serie ( basale , arteriosa , venosa ) , secondo il quesito clinico . 
x - ray tube voltage is 120 kv , pitch is 1.5 and rotation time is 0.8 s for all protocols thoracic aorta abdominal - thoracic aorta abdominal aorta endoprosthesis 160 ; 184a ; 232 ; 240 5 ; 10 ; 20a 184a ; 240 5 ; 10 ; 15 ; 20a 160 ; 184a ; 224 10 ; 20a 184a ; 200 ; 224 ; 240 10 ; 15 ; 20a tube load , mas nominal beam collimation , mm aparameters relating to baseline series only tabella 1 parametri tecnici dei differenti protocolli di acquisizione . 
la tensione del tubo radiogeno di 120 kv , il pitch di 1 , 5 e il tempo di rotazione del tubo di 0 , 8 s per tutti i protocolli aorta toracica aorta toraco - addominale aorta addominale endoprotesi 160 ; 184a ; 232 ; 240 5 ; 10 ; 20a 184a ; 240 5 ; 10 ; 15 ; 20a 160 ; 184a ; 224 10 ; 20a 184a ; 200 ; 224 ; 240 10 ; 15 ; 20a carico del tubo , mas larghezza nominale del fascio , mm aparametri relativi solo alla serie basale s . 
dosimetric calculations were performed using the monte carlo data set number 19 ; for each patient , scan length and position were evaluated on the basis of the topogram . the arterial series of each protocol was optimised for a standard patient ( weighing 70 kg ) , following the procedure shown in figure 1 . 
during the arterial phase , a series was obtained with the minimum number of images on the liver ; parameters of additional series initially coincide with those of the clinical protocol . 
the process is repeated and allows identification of the lowest tube current value needed to obtain good image quality for each protocol . this value will correspond to the maximum acceptable image noise . 
in boones study , tube current values are adapted to the patients abdominal diameter ; fantoccio utilizzato per generare il data set permette la selezione interattiva del punto di inizio e di fine della scansione . il foglio di calcolo contiene una lista di nctdiair e nctdiw per specifici modelli di scanner in differenti condizioni ( ad esempio , tensione del tubo , collimazioni del fascio )  . 
il calcolo della dose al singolo organo basato sul valore di nctdiair , misurato sullasse di rotazione dello scanner , moltiplicato per un fattore icru che permette di ottenere la dose nei tessuti molli [ 3 ]  . 
la dose efficace in una serie ( dserie ) si ottiene sommando tutte le dosi agli organi , ciascuna moltiplicata per il relativo fattore di peso wt [ 4 ]  . 
i calcoli dosimetrici sono stati effettuati con il data set monte carlo numero 19 ; per ogni paziente la lunghezza e la posizione della scansione sono state valutate dal topogramma . la serie arteriosa di ogni singolo protocollo stata ottimizzata , per paziente standard ( peso 70 kg ) , seguendo la procedura descritta in figura 1 . 
se la serie di immagini giudicata di buona qualit quella sul paziente successioriginal protocol protocollo di acquisizione originale images on the liver immagini sul fegato noise evaluation image quality evaluation va lutazione rumore va lutazione qualit dellimmagine other patient lower ma altro paziente riduzione ma previous step noise and ma values are considered optimal considero rumore e ma del passo precedente come valori ottimali fig . 
 the equation that best interpolates the data is the following : deq = - 0.003 x weight2 + 0.58 x weight + 1.8 ( r2 = 0.809 ) where deq is the patients equivalent diameter , i.e. 
in boone [ 6 ] i valori di corrente al tubo vengono adattati al diametro addominale del paziente ; tale lavoro si riferisce a pazienti pediatrici , ma possibile estrapolare i dati per diametri maggiori . 
as before , the equivalent diameter is a function of the patients weight and is obtained experimentally . equation 2 shows that optimal tube load ( mas ) is a function of optimal noise value , of patient weight and of tube table 3 tube current values related to patient weight weight , kg tube current , ma tabella 3 valori di corrente al tubo da impostare in base al peso del paziente peso , kg corrente al tubo , ma dove : k una costante di proporzionalit ; = ( kv ) il coefficiente di attenuazione lineare a un dato valore di tensione del tubo , questo valore stato ricavato da [ 8 ] ; deq il diametro equivalente . 
come in precedenza il diametro equivalente funzione del peso del paziente ed ricavato sperimentalmente . dalla ( 2 ) si evidenzia che il valore ottimale di carico al tubo ( mas ) funzione del valore ottimale di rumore , del peso del paziente e della tensione al tubo attraverso il coefficiente di attenuazione  . in breve : rumore peso il valore ottimale di carico al tubo pu essere calcolato quindi dalla seguente formula : rumore peso peso dove k1 una costante di proporzionalit scelta in base ai valori relativi al paziente standard . 
il valore di carico al tubo determina automaticamente la corrente in ma poich il tempo di rotazione del tubo stato giudicato adeguato . risultati risultati delle valutazioni dosimetriche in tabella 2 sono riportati i valori di dose efficace per i differenti protocolli . 
la dose efficace media per una serie ( dserie ) effettuata con protocollo toracico pari a 14 , 9 msv , con protocollo toraco - addominale 9 , 4 msv , con protocollo addominale 10 , 9 msv e con protocollo endoprotesi 12 , 4 msv . la dose efficace media per un esame effettuato con protocollo toracico pari a 28 , 5 msv , con protocollo toraco - addominale 18 , 8 msv , con protocollo addominale 24 , 4 msv e con protocollo endoprotesi 28 , 8 msv . risultati dellottimizzazione i parametri di acquisizione della serie arteriosa dei protocolli angio - tc ottimizzati per un paziente standard sono i seguenti : tensione al tubo 120 kv ; tempo di rotazione 0 , 8 sec ; modalit spirale ; larghezza nominale del fascio 10 mm ; spessore di fetta 2 , 5 mm ; 15 mm / rot ; pitch 1 , 5 ; corrente al tubo 235 ma . 
tube load value automatically determines the current in ma because tube rotation time has been considered appropriate . 24 , 0 26 , 0 30 , 0 32 , 0 34 , 0 28 , 0 de q [ cm ] fig . 
 optimisation arterial series acquisition parameters of optimised angio - ct protocols for a standard patient are as follows : tube voltage 120 kv , rotation time 0.8 s , ; spiral acquisition , nominal beam width 10 mm , slice thickness 2.5 mm ; 15 mm / rotation , 1.5 pitch and tube current 235 ma . 
3 relazione tra rumore e diametro equivalente ( deq ) dopo la procedura di ottimizzazione . esame non ottimizzato aveva portato al valore di dose efficace media di 14 , 9 msv . 
la riduzione di dose ottenuta in fase arteriosa quindi dellordine del 45% . la figura 3 e la figura 4 riportano i dati di rumore e di diametro equivalente nel campione dei pazienti esaminati con protocollo ottimizzato e nel campione dei pazienti esas . 
dose reduction obtained during the arterial phase is therefore about 45% . figures 3 and 4 show noise and equivalent diameter values in the study patients examined using the optimised protocol and the original protocol , respectively . 
il test per la correlazione dei ranghi di spearman ha , infatti , dato un valore di r uguale a 0 , 43 nel caso del protocollo ottimizzato e a 0 , 86 nel caso del protocollo non ottimizzato . 
questo significa che il rumore ottenuto sulle immagini dipende dal diametro del paziente se i parametri di acquisizione non vengono adattati alle dimensioni del paziente , mentre questa correlazione viene persa nel caso del protocollo ottimizzato . 
sui grafici di figura 3 e figura 4 sono inoltre riportate le rette interpolanti i dati , che confermano come il rumore ( e quindi la qualit dellimmagine ) nel protocollo ottimizzato indipendente dal diametro del paziente ( r2 = 0 , 24 )  . 
la metodologia proposta di facile applicabilit e pu essere estesa anche ad altre tipologie di esame . secondo la tipologia di esame considerato , e del quesito clinico cui deve rispondere , il livello ottimale di rumore dellimmagine sar differente . 
cova1 1unit clinica operativa di radiologia , universit degli studi , ospedale di cattinara , strada di fiume , 447 , i - 34149 trieste , italy 2unit clinica operativa di neurologia , universit degli studi , ospedale di cattinara , trieste , italy 3brain center for neuroscience , trieste , italy 4dipartimento di fisica , universit degli studi di trieste , italy correspondence to : m . 
sono stati studiati 7 pazienti ( 6 di sesso maschile e 1 di sesso femminile ) affetti da una forma di morbo di parkinson lateralizzata acinetico - rigida , sottoposti ad un esame di risonanza magnetica funzionale . 
inoltre , stata riscontrata in alcuni casi lassenza di attivazione a livello delle aree premotoria e supplementare motoria ; quando invece presente , lattivazione era caratterizzata da intensit superiore durante il movimento della mano sana . 
le alterazioni osservate riflettono verosimilmente una riorganizzazione corticale secondaria al deficit sottocorticale allorigine della malattia . parole chiave risonanza magnetica risonanza magnetica funzionale encefalo morbo di parkinson introduction introduzione parkinsons disease is a neurological disorder characterised by progressive loss of dopaminergic neurons from the subcortical circuit of the basal nuclei , which presents clinically with four cardinal signs : tremor at rest , bradykinesia , rigidity and postural instability . 
the prevalence of the disease is approximately 120180 cases in 100 , 000 individuals per il morbo di parkinson un disordine neurologico caratterizzato da una progressiva perdita di neuroni dopaminergici del circuito sottocorticale dei nuclei della base e si manifesta clinicamente con quattro segni cardinali : tremore a riposo , bradicinesia , rigidit ed instabilit posturale . 
the diagnosis is mainly clinical although morphologicalfunctional brain imaging , with both radiological and nuclear medicine techniques , may be of particular assistance despite the lack of specificity and the variability of imaging signs . although the disease has always been considered to affect subcortical ganglia , the effects of dopamine depletion are widespread and also involve non - dopaminergic neurons in areas of the brain crucial for motor control . only a few published reports have investigated functional magnetic resonance imaging ( mri ) in the assessment of cortical activation in patients with parkinsons disease . 
in particular , the studies focused on cortical activation during simple motor tasks , prevalently evaluating the primary sensory - motor area , the supplementary motor area and the pre - motor area , with only limited information on parietal lobes . 
this study aimed to assess cortical activation patterns with functional mri in patients with lateralised parkinsons disease during a relatively complex motor task in order to better understand pathophysiological mechanisms of the disease and detect possible clinical effects of functional mri on these subjects . materials and methods this study assessed two groups of subjects : the first included 11 healthy volunteers ( six men and five women ) aged between 23 and 37 with no neurological disease ; the second consisted of seven patients ( six men and one woman ) aged between 56 and 82 years with parkinsons disease . 
 patients were receiving a mean l - dopa dose of 42064.54 mg / day , and none of them was taking dopamine agonists : all were continuing their normal work activities . both the healthy volunteers and patients received adequate information about the functional study , which they underwent only after giving their consent . 
volunteers were univocally identified by a number : numbers from 1 to 7 identified lanno nella popolazione caucasica [ 1 ] , ma considerando la prevalenza nei soggetti di oltre 65 anni di et si arriva all1% e al 2% oltre gli 80 anni . leziologia multifattoriale , in particolare correlata sia a fattori genetici che ambientali . 
la diagnosi principalmente clinica , anche se le metodiche di imaging cerebrale morfo - funzionale , sia radiologiche che di medicina nucleare , possono fornire un notevole ausilio nonostante lestrema aspecificit ed incostanza dei segni riscontrabili . 
nonostante tale patologia sia sempre stata considerata di pertinenza ganglionare , nettamente sottocorticale , gli effetti della perdita di dopamina sono diffusi e coinvolgono anche i neuroni non - dopaminergici in regioni encefaliche che costituiscono elementi cruciali per il controllo motorio . 
in letteratura sono stati pubblicati pochi lavori riguardanti lattivazione corticale valutata con risonanza ( rm ) funzionale in pazienti affetti da morbo di parkinson . in particolare , in questi studi stata posta lattenzione sullattivazione corticale durante lesecuzione di compiti motori semplici , valutando prevalentemente larea sensitivo - motoria primaria , larea supplementare motoria e larea premotoria , con solo pochi dati riguardanti i lobi parietali . con questo studio abbiamo voluto valutare i pattern di attivazione corticale in rm funzionale in soggetti affetti da morbo di parkinson lateralizzato durante lo svolgimento di un compito motorio relativamente complesso con lobiettivo di comprendere meglio i meccanismi fisiopatologici di tale malattia ed in secondo luogo di ricavare una possibile ricaduta clinica della rm funzionale in questi soggetti . materiali e metodi in questo studio sono stati valutati due gruppi di soggetti : il primo comprendeva 11 volontari sani ( 6 maschi e 5 femmine ) di et compresa tra i 23 ed i 37 anni , non affetti da patologie neurologiche , mentre il secondo gruppo comprendeva sette pazienti ( 6 maschi e 1 femmina ) di et compresa tra i 56 e gli 82 anni , affetti da morbo di parkinson . 
tutti i pazienti presentavano una sindrome ad esordio asimmetrico , acinetico - rigida , ipertonica , atremorigena cui si associava una bradicinesia importante ed ipomimia : il tempo medio di diagnosi di malattia di parkinson era di 12 , 82 , 37 mesi . nella tabella 1 sono riportate le caratteristiche di lateralizzazione riscontrate clinicamente per ciascun paziente . 
i pazienti assumevano una quantit di l - dopa media di 42064 , 54 mg / die e nessuno era in terapia con farmaci dotable 1 lateralisation detected clinically in each patient with parkinsons disease tabella 1 lateralizzazione riscontrata clinicamente in ciascun paziente affetto da morbo di parkinson patient lateralisation paziente lateralizzazione right left right right left right left destra sinistra destra destra sinistra destra sinistra m . 
lattivazione viene espressa in variazione percentuale del contrasto bold tra fase di attivazione e di riposo soggetto no . mano destra mano sinistra m1 omolaterale m1 controlaterale m1 omolaterale m1 controlaterale the healthy volunteers who performed the motor task with both hands , volunteers 810 performed it with their right hand only whereas volunteer 11 performed it with the left hand only . 
likewise , parkinsons disease patients were identified by a progressive number from 1 to 7 , and all performed the motor task with both hands . the examinations were conducted on a standard clinical mri unit with a 1.5 - t intera master super - conductive magnet ( philips , gyroscan intera 1.5 t master , best , the netherlands )  . 
an echo planar fast field echo ( ffe ) sequence ( tr / te : 3 , 200 / 45 ms , slice thickness 4 mm , gap 0 , fov 200 mm , matrix 64x64 ) was used for functional acquisition , with evaluation of cortical activation exploiting the blood oxygen level dependent ( bold ) contrast . finger tapping was selected as the activation test : in this test , the subject is asked to tap the thumb on the pulp of each of the other fingers in turn , and then start over again particular , during the whole sequence , made up of 150 trs lasting a total of 8 min , the subject was asked to alternate a phase of rest and a phase of activity for three consecutive times with the same hand . 
in particular , we chose an inversion recovery sequence ( tr / te / ti : 1 , 786 / 15 / 350 ) characterised by fov , slice thickness and gaps similar to those used for the functional sepaminoagonisti : tutti continuavano la loro normale attivit lavorativa . 
i volontari sono stati individuati univocamente da un numero : i numeri da 1 a 7 individuano i volontari sani che hanno eseguito il compito motorio con entrambe le mani , i volontari 8 , 9 , 10 con la sola mano destra mentre il volontario 11 con la sola mano sinistra . 
 gli esami sono stati condotti su un tomografo clinico standard dotato di un magnete superconduttivo da 1 , 5 t ( philips , gyroscan intera 1 , 5 t master , best , olanda )  . 
per lacquisizione funzionale stata utilizzata una sequenza echo planar ffe ( tr / te : 3200 / 45 ms , spessore di strato = 4 mm , gap = 0 , fov = 200 mm , matrice 64x64 ) , con valutazione dellattivazione corticale sfruttando il contrasto bold ( blood oxygen level dependent )  . 
per quanto riguarda la prova di attivazione stato scelto il finger tapping : cio stato chiesto al soggetto di toccare con il polpastrello del primo dito , in successione , il polpastrello del secondo , terzo , quarto e quinto dito e , quindi , ricominciare da capo . 
activation is expressed as a percentage change of the blood oxygen level dependent ( bold ) contrast between activity and rest phases tabella 3 risultati relativi allattivazione dellarea motoria supplementare e dellarea premotoria nei volontari sani : confronto tra movimento della mano destra e sinistra . 
activation is expressed as a percentage change of the blood oxygen level dependent ( bold ) contrast between activity and rest phases subject no . affected hand healthy hand m1 ipsilateral m1 contralateral m1 ipsilateral m1 contralateral tabella 5 risultati relativi allattivazione dellarea motoria primaria ( m1 ) nei pazienti parkinsoniani : confronto tra movimento della mano sana e della mano malata . 
a p value of 0.01 was considered to indicate statistical significance . in all subjects , the presence or absence of activation was evaluated at the level of the primary motor area , the pre - motor area and supplementary motor area of the parietal and occipital lobes . 
each area was studied as a cluster if voxels were contiguous and made the structure identifiable or as a spherical volume if the voxels were separate but could be related to the same brodmann area . 
graphically , pixels corresponding to cortical activation were overlaid onto the anatomic sequence to enable more accurate localisation . results tables 2 , 3 and 4 show numerical results for each healthy volunteer and each area . 
in all patients considered , we observed activation of the primary motor area contralateral la durata complessiva di 8 minuti , stato chiesto al soggetto di alternare una fase di riposo ad una fase di attivit e questo per tre volte consecutive con la stessa mano . 
successivamente stata acquisita una sequenza anatomica , in particolare abbiamo scelto una sequenza inversion recovery ( tr / te / ti = 1786 / 15 / 350 ) caratterizzata da fov , spessore di strato e gap analoghi a quelli utilizzati per la sequenza funzionale . 
in tutti i soggetti stata valutata la presenza o assenza di attivazione in corrispondenza dellarea motoria primaria , dellarea premotoria e supplementare motoria , dei lobi parietali e dei lobi occipitali . 
ciascuna area stata studiata come cluster , se i voxels erano contigui e dunque rendevano individuabile la struttura , oppure come volume sferico , se i voxels erano separati , ma riconducibili alla stessa area di brodmann . 
da un punto di vista grafico i pixels relativi allattivazione corticale sono stati sovrapposti alla sequenza anatomica , di modo da consentire una loro pi corretta localizzazione . risultati nelle tabelle 2 , 3 e 4 sono riportati i risultati numerici relam . 
intensity of the activation signal was higher , on average , in the primary motor area contralateral to the moving hand , as compared with the ipsilateral primary motor area . 
signal intensity of the primary ipsilateral and contralateral motor areas was higher during movement of the affected hand as compared with the healthy tivi a ciascun volontario sano e ciascuna area . 
accanto a queste attivazioni stata rilevata unattivazione a livello dellarea motoria omolaterale e a livello dei lobi parietali . nelle tabelle 5 , 6 e 7 sono riportati i risultati relativi ai pazienti parkinsoniani . 
in tutti i pazienti studiati stata osservata la presenza di attivazione in corrispondenza dellarea motoria primaria controlateralmente al lato in esame e in 6 pazienti su 7 lattivazione delle aree motoria supplementare e premotoria . table 6 results concerning activation of the supplementary motor area and pre - motor area in parkinsons patients : comparison between movement of the healthy hand and affected hand . 
activation is expressed as a percentage change of the blood oxygen level dependent ( bold ) contrast between activity and rest phases tabella 6 risultati relativi allattivazione dellarea motoria supplementare e dellarea premotoria nei pazienti parkinsoniani : confronto tra movimento della mano sana e della mano malata . 
activation of the supplementary motor and pre - motor areas was observed in six patients out of seven during movement of the healthy hand and in five patients out of seven during movement of the affected hand . 
in the remaining two cases , activation of the parietal cortex ipsilateral to the moving hand and activation of the parietal cortex were observed , respectively , bilaterally when the task was performed with the healthy hand , and contralateral when performed with the affected hand . 
the disease has always been regarded as gangliar and subcortical , but , despite the neurochemical lesion being mainly located in the nigrostriatal dopaminergic system , the effects of dopamine depletion are widespread and also affect neuronal pathways that are not strictly dopaminergic but more like bridges to areas of the brain crucial for motor control [ 3 ]  . 
with this study , we aimed at evaluating cortical acaccanto a queste attivazioni stata rilevata unattivazione a livello dellarea motoria omolaterale al movimento , della corteccia parietale e della corteccia occipitale . il segnale proveniente dallarea motoria primaria omolaterale stato osservato in 5 pazienti su 7 durante il movimento della mano sana e in 6 pazienti su 7 durante il movimento della mano malata : lintensit del segnale di attivazione mediamente maggiore in corrispondenza dellarea motoria primaria controlaterale alla mano in movimento rispetto allarea motoria primaria omolaterale . 
nei restanti due casi stata osservata rispettivamente unattivazione della corteccia parietale omolaterale alla mano in movimento e lattivazione della corteccia parietale bilateralmente quando il compito stato svolto con la mano sana e controlateralmente se stato svolto con la mano malata . 
tale affezione sempre stata considerata di pertinenza ganglionare , nettamente sottocorticale ma , anche se la lesione neurochimica localizzata prevalentemente a livello del sistema dopaminergico nigrostriatale , gli effetti della perdita di dopamina sono diffusi e coinvolgono vie neuronali anche non strettamente dopaminergiche , trait - dunion con regioni encefaliche cruciali per il controllo motorio [ 3 ]  . 
con questo studio , pertanto , abbiamo voluto valutare le caratteristiche dellattivazione corticale in soggetti affetti da morbo di parkinson durante lo svolgimento di un compito motorio relativamente complesso , confrontandole con quelle osservate in un gruppo di controllo costituito da volontari sani e , avendo i pazienti un morbo di parkinson lateralizzato , stato possibile effettuare un confronto tra le attivazioni ottenute con la mano sana e quella malata . in condizioni normali , con il semplice finger tapping , si osserva lattivazione della corteccia pree post - rolandica controlaterale , legate , la prima al movimento della mano , la seconda principalmente al contatto intrinseco interfalangeo . spesso si riconosce inoltre lattivazione in sede frontale delle aree supplementare motoria e premotoria : generalmente lattivazione dellarea motoria supplementare si distingue in una parte anteriore bilaterale simmetrica ed una posteriore , controlateralmente alla mano che esegue il compito motorio fig . 
2 marcata attivazione bilaterale della corteccia parietale durante il movimento della mano malata . tivation characteristics in patients with parkinsons disease during a relatively complex motor task , comparing them with those observed in a control group consisting of healthy volunteers . 
under normal conditions , simple finger tapping activates the preand post - rolandic contralateral cortex , the former being linked to hand movement , the latter mainly to intrinsic contact between phalanges . 
furthermore , frontal activation of the supplementary motor and pre - motor areas is often detected : activation of the supplementary motor area is generally subdivided into a symmetrical , bilateral , anterior portion and a posterior portion , contralateral to the hand performing the motor task [ 4 ]  . 
in healthy volunteers , the expected activation pattern was confirmed , but other areas were also activated , in particular , the ipsilateral primary motor cortex and the parietal cortex . activation corresponding to the primary motor cortex ipsilateral to the moving hand is neurologically justified by the partial hemilateral non - specificity of motor operations . 
when the task becomes slightly more complex and sequential , as with finger tapping , the ipsilateral primary motor cortex and parietal lobes , which are involved in the perception of movement in space , are also activated [ 6 ]  . before evaluating the results obtained in parkinsons patients , it should be noted that any differences between patients and healthy volunteers might be related not only to the disease but also to age . 
in this respect , it is important to note that the healthy volunteers age range was 2337 years whereas patients age range was 5682 years and that there were no functional mri investigations of brain activation induced by finger tapping performed under the same conditions by healthy subjects of different ages . 
nonetheless , the only published study contrasting the extent of activation of primary motor areas during a motor task , as assessed by spectroscopy in subjects of different ages , showed no statistically significant difference in activation among the different age groups [ 7 ]  . 
this hyperactivity of the contralateral primary motor area has already been described in the literature [ 8 , 9 ] and could represent the neural substrate for the difficulty experienced by the patient with parkinsons disease to perform the movement . 
it could therefore be assumed that this hyperactivity reflects the cortical re - organisation , which compensates for the subcortical deficit causing the disease . activation of the primary motor cortex ipsilateral to the hand examined has been also observed in parkinsons patients , but [ 4 ]  . 
nei volontari sani stato confermato il pattern di attivazione atteso , ma stata rilevata unattivazione a livello di altre aree , in particolare a livello della corteccia motoria primaria ipsilaterale e della corteccia parietale . la presenza di attivazione in corrispondenza della corteccia motoria primaria omolaterale alla mano in movimento giustificata , neurologicamente , dalla parziale aspecificit emilaterale delle operazioni motorie : infatti , a ciascuna mano sono affidate differenti abilit e le istruzioni per eseguire un determinato movimento possono in parte risiedere nelluno o nellaltro emisfero ed essere richiamate con la stessa modalit che implica lattivazione dellarea motoria supplementare . 
ovviamente , essendo ridotto il feed - back tattile , il segnale omolaterale riguarda la sola area rolandica deputata al controllo del movimento [ 5 ]  . altra ipotesi per spiegare tale attivazione legata al tipo di compito motorio : nel caso di un movimento molto semplice ( ad es . , apertura e chiusura della mano ) si osserva generalmente lattivazione della sola area motoria primaria controlaterale . 
quando il compito diviene modestamente pi complesso e sequenziale , come accade con il finger tapping , si pu osservare lattivazione anche della corteccia motoria primaria omolaterale e dei lobi parietali , che sono coinvolti nella percezione del movimento nello spazio [ 6 ]  . prima di valutare i risultati ottenuti nei pazienti parkinsoniani , va sottolineato come eventuali differenze rispetto ai volontari sani potrebbero essere legate non solo alla malattia , ma anche al fattore et . 
riguardo a questultimo aspetto , va ricordato come la fascia det dei volontari sani sia 2337 anni , mentre quella dei pazienti 5682 anni e come non esistano in letteratura studi di rm funzionale sullattivazione cerebrale indotta da finger tapping , eseguito nelle medesime condizioni da soggetti sani , ma di et diverse . 
va comunque sottolineato come lunico lavoro pubblicato , nel quale stata messa a confronto lentit di attivazione delle aree motorie primarie durante un compito motorio , valutata con spettroscopia , in soggetti di diverse fasce di et , non ha dimostrato una differenza di attivazione statisticamente significativa tra i diversi gruppi [ 7 ]  . 
in tutti i parkinsoniani stata osservata la presenza dellattivazione della corteccia motoria primaria controlateralmente alla mano in esame . ma , mediamente , a tale livello lintensit di segnale superiore rispetto a quanto osservato nei volontari sani : questa iperattivit dellarea motoria primaria controlaterale gi stata descritta in letteratura [ 8 , 9 ] e potrebbe rappresentare il substrato neurale della difficolt del paziente con morbo di parkinson nellesecuzione del movimento . 
moreover , taking into account the hemispheric dominance and equal distribution of parkinsons disease between the dominant and non - dominant hand , the correlation between observations and the disease seems plausible . 
 an explanation of our results might be that there is a recovery of the activity of the ipsilateral motor cortex , compensating for the contralateral deficit , as demonstrated by two positron emission tomography ( pet ) studies on patients with central , lateralised and ischaemic deficits [ 10 , 11 ]  . 
in addition to the contralateral motor cortex , the ipsilateral cortex appears to undergo a process of compensatory functional re - organisation . another phenomenon has been described that could account for this asymmetry in activation , which is also seen in subjects with motor deficits : the presence of associated movement of the hand contralateral to the one being studied [ 12 ]  . 
although this was not done in our study , we did carefully monitor the subjects visually during image acquisition , and no movement of the contralateral hand in either volunteers or patients was detected . as regards activation of the supplementary motor and premotor cortices , these were activated in six parkinsonian patients , with asymmetric activation ( more intensely during the movement of the healthy hand )  . 
this could be explained by the speed of movement . a recent study observed that the intensity of activation of the pre - motor and supplementary motor areas in healthy volunteers is directly proportional to the speed of the hand movement [ 2 ]  . 
this aspect has already been observed in the literature [ 2 , 79 ] and is explained in the following manner : the parietal cortex integrates different sensory , motivational and attentional inputs and can therefore provide the basis for the generation of sensory - guided movements in parkinsons patients without requiring circuitry between the basal ganglia and frontal cortex , which are altered in these patients . as a consequence , hyperactivity of the parietal lobes , along with activation of the pre - motor and supplementary motor areas , would be a compensatory mechanism for the hypoactivity of striatofrontal projections , thus creating an intact alternative pre - motorparietal circuit . 
in this respect , we found no difference in the activation of the parietal lobes between movement of the affected and healthy hands in parkinsons patients , and this suggests that these alterations entrambe le mani . 
inoltre , tenendo conto della dominanza emisferica , e dellequa distribuzione del morbo di parkinson , alla mano dominante e non , la correlazione possibile tra quanto osservato e la patologia sembra plausibile . 
una spiegazione dei nostri risultati pu essere data dal fatto che vi sia un recupero dellattivit della corteccia motoria ipsilaterale , compensatoria al deficit controlaterale , come dimostrato in due studi eseguiti con tecnica pet su pazienti con deficit centrale , lateralizzato , ischemico [ 10 , 11 ]  . 
in letteratura stato descritto un ulteriore fenomeno che potrebbe essere alla base di questa asimmetria di attivazione che presente anche nei soggetti con deficit motorio : la presenza di movimento associato della mano opposta a quella in esame [ 12 ]  . 
esso non ha messo in evidenza movimenti della mano controlaterale , n nei volontari , n nei parkinsoniani . riguardo allattivazione delle cortecce motoria supplementare e premotoria , nei parkinsoniani si dimostrata la loro attivazione in sei casi , con attivazione asimmetrica ( maggiore intensit di attivazione durante il movimento della mano sana )  . 
in un recente studio si osservato come in volontari sani lintensit di attivazione delle aree premotoria e supplementare motoria sia direttamente proporzionale alla velocit del movimento della mano [ 2 ]  . 
questo aspetto gi stato osservato in letteratura [ 2 , 79 ] ed giustificato dal fatto che la corteccia parietale viene ritenuta un integratore di diversi input sensitivi , motivazionali ed attenzionali che possa pertanto fornire le basi per la genesi di movimenti guidati da stimoli sensoriali nei pazienti affetti da morbo di parkinson senza la necessit dei circuiti tra gangli della base e corteccia frontale , che sono alterati in questi pazienti . 
pertanto liperattivazione dei lobi parietali , insieme allattivazione delle aree premotoria e supplementare motoria , rappresenterebbe un meccanismo di compenso per lipofunzione delle proiezioni striato - frontali , creando un circuito alternativo integro premotorio - parietale . 
a tale riguardo , non abbiamo rilevato una differenza di attivazione dei lobi parietali tra movimento della mano malata e movimento della mano sana nei parkinsoniani , e questo suggerisce come tali alterazioni possano essere rilevabili anche in una fase preclinica . 
another hypothesis is that the impairment of the connections between basal nuclei and cortex , responsible for parietal cortical hyperactivity , changes the perception of movement in space in patients during the movement of both the healthy and affected hands . another observation concerns the activation of the occipital lobes . 
this was absent in healthy volunteers . these results are certainly interesting from a neuropathological point of view but might also have clinical relevance , in particular , in the differential diagnosis between the different types of parkinsonism , and suggest an anatomical - physiological basis for the rehabilitation of these patients . 
more precisely , functional mri could be used in the differential diagnosis of parkinsons disease and cortico - basal degeneration , which has been shown to have a different activation pattern [ 13 ]  . 
consequently , the technique could be useful in the initial stages of the disease , when the cortico - basal degeneration may be impossible to distinguish clinically from parkinsons disease although the therapeutic approach and prognosis are completely different . the main limitation of our study , shared by all studies of this kind , concerns the small number of patients studied , which precludes a statistical analysis of the results . 
however , the problems in finding patients with a lateralised akineticrigid form of parkinsons who can endure a functional mri examination should be considered . rattivit corticale parietale , determini nei pazienti una alterata percezione del movimento nello spazio , presente sia durante il movimento della mano sana che della mano malata . altra osservazione quella relativa allattivazione dei lobi occipitali : un primo commento riguarda il fatto che non stato chiesto n ai volontari sani n ai pazienti di tenere gli occhi chiusi durante lacquisizione delle immagini . 
questa spiegazione trova ulteriore conferma dallosservazione sperimentale della presenza di ampi movimenti del capo nei parkinsoniani allinizio del compito motorio , movimenti eseguiti involontariamente per permettere il controllo visivo del movimento . conclusioni con questo lavoro abbiamo descritto i patterns di attivazione corticale durante un compito motorio in pazienti affetti da morbo di parkinson lateralizzato confrontandoli con quelli nei volontari sani e inoltre , nellambito dello stesso paziente , abbiamo confrontato lattivazione ottenuta con la mano sana con quella della mano malata . 
inoltre stata riscontrata , in alcuni casi , lassenza di attivazione a livello delle aree premotoria e supplementare motoria , verosimilmente secondarie allimpaccio motorio . dove , invece , tali zone si attivavano lo facevano con intensit superiore durante il movimento della mano sana . 
infine , si osservata una spiccata tendenza di questi pazienti al controllo visivo del movimento con conseguente attivazione dei lobi occipitali , assente nei volontari sani . questi risultati sono sicuramente interessanti da un punto di vista neurofisiopatologico , ma potrebbero anche rivestire un ruolo nellambito clinico , in particolare nella diagnosi differenziale tra i diversi parkinsonismi e suggerire una base anatomo - fisiologica per il recupero fisiatrico di questi pazienti . 
pi precisamente la rm funzionale potrebbe essere impiegata nella diagnosi differenziale tra morbo di parkinson e degenerazione cortico - basale , nella quale si dimostrato un diverso pattern di attivazione [ 13 ]  . 
pertanto tale tecnica potrebbe risultare utile nelle fasi iniziali di malattia , quando la degenerazione cortico - basale pu essere clinicamente indistinguibile dal morbo di parkinson , ma il suo approccio terapeutico e la prognosi sono completamente diverse . il principale limite del nostro lavoro , comune a tutti i lavori simili pubblicati , riguarda la non elevata numerosit dei pazienti che non consente una valutazione statistica dei risultati . 
simonetti dipartimento di diagnostica per immagini e radiologia interventistica , universit di roma tor vergata , policlinico , viale oxford 81 , i - 00133 rome , italy correspondence to : e . 
magnetic resonance imaging ( mri ) and mrsi were performed on 39 patients with prostate - specific antigen ( psa ) levels greater than 4 ng / ml and suspicious findings at trans - rectal ultrasound ( trus )  . 
at mrsi , cancer was defined as possible if the ratio of choline plus creatine to citrate exceeded mean normal peripheral zone values by two standard deviations ( sd ) or as definite if that ratio exceeded the normal value by three sd . 
mri and mrsi alone had sensitivity , specificity , positive and negative predictive values and diagnostic accuracy in the detection of prostate cancer equal to 85% , 75% ; 53% , 89% ; 65% , 88% ; 77% , 74% ; and 69% , 79% , respectively . 
trentanove pazienti con valori di antigene specifico prostatico ( psa ) superiori a 4 ng / ml e reperti sospetti allecografia transrettale ( trus ) sono stati sottoposti ad imaging morfologico con risonanza magnetica ( rm ) e a rmsp . 
alla rmsp la presenza di tumore stata definita come possibile se il rapporto tra colina pi creatina su citrato era maggiore di 2 ds rispetto al valore medio della zona periferica normale o certa se tale rapporto superava di 3 ds quello normale . 
la rm e la rmsp singolarmente hanno evidenziato sensibilit , specificit , valore predittivo positivo e negativo e accuratezza diagnostica per lindividuazione del cancro prostatico del 85% , 75% e 53% , 89% e 65% , 88% e 77% , 74% e 69% , 79% rispettivamente . 
laggiunta della rmsp alla rm permette una significativa pi alta specificit nellindividuazione del tumore rispetto alla sola rm e pu essere indicata come modalit risolutiva prima della biopsia nei pazienti con elevati valori di psa ed ecografia transrettale ( trus ) sospetta . key words prostate prostatic neoplasm biopsy magnetic resonance imaging magnetic resonance spectroscopy imaging parole chiave prostata neoplasia prostatica biopsia risonanza magnetica spettroscopia con risonanza magnetica introduction introduzione the growing incidence of prostate cancer , together with an increasing ageing population in the western world , has made prostate tumour an acute medical and socioeconomic problein 2004 in the united states , the american cancer society estimated 230 , 000 new cases of prostate carcinoma as well as 29 , 900 cases of deaths related to this disease [ 1 ]  . screening with digital rectal examination associated with evaluation of serum prostate - specific antigen ( psa ) and trans - rectal ultrasound ( trus ) , possibly complemented by biopsy [ 2 ] , has enabled physicians to identify cancers at an earlier stage than in the past , drawing attention to the possibility of using treatments ( brachytherapy , radiotherapy , thermoablation and cryosurgery ) that have less aggressiveness and morbidity than radical prostatectomy . hence , locating and staging the cancer accurately as well as assessing its biological aggressiveness have become crucial . magnetic resonance imaging ( mri ) improves the evaluation of zonal morphology and prostate anatomy although there is still no agreement over diagnostic accuracy of mri in identifying prostate cancer lesions . studies conducted to date have shown extreme variations in accuracy levels , with values ranging from 75% to 90% , with peaks of 97% [ 3 ] in locating known lesions , but showing a remarkable drop with lesions < 5 mm [ 4 ]  . 
however , morphologic evaluation alone had poor specificity ( 55% ) [ 5 ] because of a high number of false positives . these can be attributed to several conditions ( post - biopsy haemorrhage , the effects of conservative treatments ) or diseases ( prostatitis ) affecting the prostate tissue , which can mimic cancer and its typical t2 appearance as an area of low signal intensity against a background of relatively hyperintense normal peripheral zone . 
the introduction of proton mr spectroscopic imaging ( mrsi ) has enabled direct correlation of anatomical and morphological findings with functional information on tissue metabolism . initial studies show the potential of combined mri and mrsi in determining the presence of prostate cancer with higher accuracy , view its extension and estimate its biological aggressiveness [ 6 ]  . 
therefore , the purpose of our study was to compare the diagnostic performance of mrsi and morphological imaging alone in the identification and localisation of prostate tumours by comparing it prospectively with histopathologic findings . materials and methods patients between april and october 2004 , 39 patients underwent , after giving informed consent , mri of the prostate with associated spectroscopic evaluation at our department . 
lamerican cancer society ha stimato negli stati uniti , nel 2004 , 230.000 nuovi casi di carcinoma prostatico e 29.900 casi di decesso ad esso correlati [ 1 ]  . 
lo screening con esplorazione digito - rettale associato alla valutazione dellantigene prostatico specifico ( psa ) sierico e allo studio con ecografia transrettale ( trus ) , eventualmente completato dalla biopsia [ 2 ] ha consentito lindividuazione delle neoplasie ad uno stadio pi precoce rispetto al passato , richiamando inoltre lattenzione sulla possibilit di utilizzare degli strumenti terapeutici ( brachiterapia , radioterapia , termoablazione e criochirurgia ) meno aggressivi e gravati da minore morbilit rispetto alla prostatectomia radicale . 
diviene in tal modo di fondamentale importanza la precisa localizzazione e stadiazione della neoplasia e la valutazione della sua aggressivit biologica . limaging con risonanza magnetica ( rm ) consente una migliore valutazione della morfologia e dellanatomia zonale della prostata sebbene riguardo al valore di accuratezza diagnostica della rm nellidentificazione delle lesioni prostatiche neoplastiche non vi sia ancora accordo tra i vari autori . 
gli studi finora effettuati hanno mostrato unampia variabilit dei risultati di accuratezza con valori che vanno dal 75% al 90% , raggiungendo il 97% [ 3 ] nella localizzazione di lesioni gi note , ma mostrando una sensibile flessione per lesioni di diametro inferiore ai 5 mm [ 4 ]  . 
la sola valutazione morfologica , per , si rilevata scarsamente specifica ( 55% ) [ 5 ] , a causa di un elevato numero di falsi positivi , i quali possono essere attribuiti alla presenza di alcune condizioni ( emorragia post - bioptica , effetti delle terapie conservative ) o patologie ( prostatiti ) che interessano il tessuto prostatico e che sono in grado di mimare la lesione neoplastica con il tipico aspetto di area a bassa intensit di segnale nelle sequenze t2 pesate nel contesto della relativa iperintensit della zona periferica normale . lintroduzione della spettroscopia dellidrogeno con rm ( rmsp ) ha permesso di correlare il dato anatomico e morfologico direttamente con informazioni di tipo funzionale relative al metabolismo tissutale . 
studi iniziali mostrano la potenzialit della combinazione del rm e rmsp nel determinare in maniera pi accurata la presenza del carcinoma prostatico , visualizzarne lestensione e stimarne laggressivit biologica [ 6 ]  . 
lo scopo del nostro studio stato , pertanto , verificare la performance diagnostica della spettroscopia dellidrogeno rispetto al solo imaging morfologico nella individuazione e localizzazione della patologia tumorale prostatica , confrontandola prospetticamente con il dato istopatologico . materiali e metodi pazienti da aprile ad ottobre 2004 , 39 pazienti si sono recati presso il nostro dipartimento per essere sottoposti , previo consenso g . 
for the morphological study , t2 - weighted turbo spin echo ( tse ) images with high spatial resolution were acquired in the transverse plane ( using the spir fat - suppression technique as well ) and in the coronal plane , including the entire prostate and seminal vesicles in the acquisition volume . 
acquisition parameters were the following : tr 4 , 000 ms , te 130 ms ( tr 4 , 750 ms , te 90 ms in the spir sequence ) , 2.5 - mm slice thickness without gap between the slices , four excitations , 190 mm field of view ( fov ) and acquisition and reconstruction matrices of 240 and 256 , respectively . the morphological study was completed with the acquisition of ten t1 - weighted fast - field echo ( ffe ) spir post - contrast dynamic sequences ( tr 20 ms , te 5 m , 20 flip angle , 3 - mm slice thickness )  . the volume to be studied by spectroscopy was selected on the basis of the axial t2 morphological images , trying to include the prostate parenchyma and exclude as much periglandular fat as possible . 
to this end , a double spin - echo point - resolved spatially localized spectroscopic ( press ) sequence was used , along with a te ( 136 ms ) optimised for quantitative evaluation of citrate and choline with multivoxel analysis . 
let media dei pazienti esaminati di 68 , 5 aa ( range 5582 anni ) con un valore medio di psa sierico di 24 , 15 ng / ml ( range 4 , 344 ng / ml )  . tecnica rm le immagini sono state acquisite con unapparecchiatura ad elevata intensit di campo ( 1 , 5 t ) ( philips gyroscan intera , best , netherlands ) equipaggiata con gradienti da 30 mt / m , utilizzando la bobina del corpo per la trasmissione degli impulsi e una bobina di superficie ( c1 ) , posizionata a livello della pelvi , per la ricezione del segnale . 
allo scopo di ridurre la motilit intestinale e migliorare cos la qualit delle immagini sono stati somministrati per via i.glucagone o nbutilbromuro di ioscina ( buscopan fl 20 mg / ml )  . 
per lo studio morfologico sono state acquisite immagini tse t2 pesate ad alta risoluzione spaziale sul piano trasversale ( utilizzando anche la tecnica della soppressione del grasso spir ) e su quello coronale includendo nel volume dacquisizione lintera prostata e le vescicole seminali . 
i parametri di acquisizione sono stati i seguenti : tr 4000 ms , te 130 ms ( tr 4750 ms , te 90 ms nella sequenza spir ) , spessore di sezione 2 , 5 mm senza intervallo tra le sezioni , 4 eccitazioni , fov di 190 mm e una matrice di acquisizione e di ricostruzione rispettivamente di 240 e 256 . 
lo studio morfologico stato completato con lacquisizione di 10 sequenze dinamiche post - contrastografiche ffe t1w spir ( tr 20 msec , te 5 msec , flip angle 20 , spessore di sezione 3 mm )  . il volume sottoposto ad analisi spettroscopica stato selezionato , sulla base delle immagini assiali t2 morfologiche , cercando di includere il parenchima prostatico e di escludere il pi possibile il grasso perighiandolare . 
a tal fine stata utilizzata una sequenza press ( double spin - echo point - resolved spatially localized spectroscopic sequence ) con un te ( 136 ms ) ottimizzato per la valutazione quantitativa del citrato e della colina con analisi multivoxel . 
sono stati utilizzati i seguenti parametri di acquisizione : tr 1.600 ms , te 136 ms , ampiezza spettrale 1.000 hz , con risoluzione spettrale nominale del voxel di 0 , 24 cm3 . 
il tempo di acquisizione della sequenza spettroscopica stato di 18 minuti . le sequenze post - contrastografiche sono state utilizzate nella valutazione di rapidi potenziamenti di segnale , in particolare della porzione periferica della ghiandola , sincroni con la normale omogeneizzazione della porzione adenomiomatosa , suggestivi di lesione produttiva con neoangiogenesi anarchica nel contesto . image analysis was carried out by an experienced radiologist ( es ) in mri of abdominal - pelvic structures . 
1a - d dynamic t1 - weighted fast - field ( ffe ) sequence : the acquisitions at time 0 ( a ) and 20 s after contrast medium infusion ( b ) show a focal early wash - in ( arrow ) in the middle - left peripheral portion . 
acquisizione a 20 s dalla somministrazione del mezzo di contrasto che evidenzia precoce wash - in focale ( freccia ) a carico della porzione periferica paramediana sinistra ( b )  . 
presence of a rounded homogeneously hypointense area esperto ( es ) nellimaging rm delle strutture addomino - pelviche il quale era a conoscenza del fatto che i pazienti avevano lesioni sospette per neoplasia alla trus . 
i confini della zona periferica sono stati definiti grazie alla visualizzazione delle bande di bassa intensit di segnale nelle immagini t2 dipendenti riferibili alla capsula e alla pseudocapsula prostatica [ 7 ]  . 
by using dedicated software for spectroscopic analysis , values of the integral of the areas underlying citrate , choline and creatine peaks were calculated . to detect the presence of cancer , the ratio between the integral of choline plus creatine and the integral of citrate was calculated for each voxel . 
la presenza di unarea omogeneamente ipointensa a margini rotondeggianti e limiti sfumati stata considerata come sospetta per neoplasia . i singoli voxel del volume selezionato nella sequenza spettroscopica sono stati rappresentati bidimensionalmente in forma grafica di griglia sovrapposta alle corrispondenti immagini traverse t2 pesate . 
in tutti i pazienti sono stati effettuati 10 prelievi secondo suddivisione in ottanti della prostata ( 1 prelievo per apice , zona intermedia , basale e 2 per la zona laterale per ciascun lato )  . almeno 2 campionamenti sono stati ottenuti dalle zone che presentavano caratteristiche patologiche alla rm ed alla rmsp . 
in all patients , ten samples were taken according to the subdivision of the prostate into octants ( one sample from the apical , intermediate and basal areas and two samples for the lateral area on each side )  . at least two samples were taken from the areas that presented pathological features at mri and mrsi . 
samples were classified based on the sampling site , and histological analysis was carried out for each site . every biopsy sample was analysed by a single pathologist specialized in prostatic diseases . statistical analysis findings were processed with the statistical package for social sciences , version 12 ( spss , chicago , il , usa )  . 
results obtained with mri and mrsi used separately and in combination and with biopsy were analysed in terms of sensitivity , specificity , positive ( ppv ) and negative predictive value ( npv ) and accuracy values . statistically significant differences in the comparison between mri and mrsi and mri and mrsi combined versus biopsy were processed through an c2 test according to pearson , assuming a value of p0.05 , to indicate a significant correlation . 
although images had been classified as normal , equivocal or suspicious for cancer , in the statistical analysis , results were divided into two groups only : positive ( suspicious and equivocal , > 3 sd and < 23 sd > ) and negative ( normal and < 2 sd )  . results findings of the dynamic post - contrast acquisitions were not used in statistical evaluation of the selected sample . 
twentysix out of 39 patients ( 66% ) showed focal and / or unilateral or bilateral signal hypointensity in the t2 - weighted images whereas the remaining 13 ( 33% ) showed no pathological signal alterations . 
in particular , as regards location within the gland , mri detected five suspicious areas at the apical level ( three on the right , two on the left ) , eight at the intermediate level ( three on the right , five on the left ) , nine at the lateral level ( five on the right , four on the left ) and four at a basal level ( two on the right , two on the left )  . of the 26 patients in whom mri was suggestive of a neoplastic lesion , histology confirmed adenocarcinoma in 17 [ 65% true positive ( tp ) ] but diagnosed benign lesions in nine [ 35% false positive ( fp ) ]  . 
ciascun campione bioptico stato analizzato da un unico anatomo - patologo specialista nella patologia prostatica . analisi statistica i dati sono stati elaborati utilizzando lo statistical package for social sciences , versione 12 ( spss , chicago , illinois )  . 
i risultati ottenuti rispettivamente dalla rm , dalla rmsp , dalla combinazione rm - rmsp e dalla biopsia sono stati tabulati calcolando i valori di sensibilit , specificit , valore predittivo positivo ( vpp ) e negativo ( vpn ) ed accuratezza . i valori di significativit statistica nel confronto tra i risultati della rm e della rmsp e della combinazione rm - rmsp rispetto alla biopsia sono stati elaborati mediante test c2 secondo pearson , assumendo un valore di p0 , 05 indicante una correlazione significativa . sebbene le immagini fossero state classificate come normali , equivoche o sospette per neoplasia , ai fini dellanalisi statistica i risultati sono stati suddivisi in due soli gruppi ovvero positivi ( sospetti ed equivoci , > 3 ds e < 23 ds > ) e negativi ( normali , < 2 ds )  . risultati i dati relativi alle acquisizioni dinamico - contrastografiche non sono stati utilizzati nella valutazione statistica del campione selezionato . ventisei pazienti dei 39 totali ( 66% ) presentavano ipointensit di segnale di tipo focale e / o diffusa mono o bilateralmente nelle immagini t2 dipendenti , mentre i rimanenti 13 ( 33% ) non mostravano alterazioni di segnale di carattere patologico . in particolare , in riferimento alla localizzazione a livello della ghiandola , con la rm sono state attribuite 5 aree sospette a livello dellapice ( 3 a destra , 2 a sinistra ) , 8 in regione intermedia ( 3 a destra , 5 a sinistra ) , 9 in sede laterale ( 5 a destra , 4 a sinistra ) e 4 in regione basale ( 2 a destra , 2 a sinistra )  . dei 26 pazienti con rm suggestiva per lesione discariocinetica , 17 ( 65% vp ) hanno trovato conferma istologica di adenocarcinoma , mentre 9 ( 35% fp ) hanno avuto diagnosi di lesione benigna , in 5 casi di natura flogistica ( prostatiti acute o croniche ) nei rimanenti 4 di iperplasia nodulare benigna . 
of the 13 patients without suspicious findings for cancer , histology confirmed the absence of neoplastic lesions in ten [ 77% true negative ( tn ) : seven cases of benign nodular hyperplasia and three of granulomatous prostatitis ] but detected adenocarcinoma in 3three [ 23% false negative ( fn ) ] ( table 1 )  . based on these results , mri had a 85% sensitivity , 53% specificity , with 65% ppv and 77% npv and 69% accuracy with a p = 0.013 ( table 2 )  . 
mrsi identified pathological spectra ( cho + cr / cit ratio > 3 sd or < 23 sd > ) in 17 patients ( 43% ) whereas spectrum analysis was normal ( cho + cr / cit ratio < 2 sd ) in the remaining 22 ( 57% )  . 
la rmsp ha individuato spettri di tipo patologico ( rapporto cho + cr / cit > 3 ds o < 23 ds > ) in 17 pazienti ( 43% ) , mentre nei restanti 22 ( 57% ) lanalisi spettrale risultata nei limiti della normalit ( rapporto cho + cr / cit < 2 ds )  . 
in base alla suddivisione in ottanti 2 erano le aree sospette a livello dellapice prostatico , 7 in regione intermedia ( 3 a destra , 4 a sinistra ) , 7 in sede laterale ( 4 a destra , 3 a sinistra ) 1 in regione basale destra . 
la rmsp ha dimostrato una sensibilit del 75% con specificit del 89% , un vpp del 88% , un vpn del 74% con unaccuratezza del 87% con un valore di p = 0 , 000 ( tabella 2 )  . combinando i risultati della rm con quelli della rmsp abbiamo avuto una sensibilit del 70% con una specificit del 89% , vpp 88% , vpn 74% , unaccuratezza del 79% e un valore di p = 0 , 000 ( tabella 2 )  . riguardo alla localizzazione delle lesioni non si evidenziata una significativa correlazione tra i risultati ottenuti ai prelievi bioptici e quelli evidenziati alla rm e alla rmsp , se non per quanto riguarda i risultati dellistologia e della rmsp nella zona intermedia e laterale ( p = 0 , 003 e p = 0 , 002 rispettivamente )  . table 1 correlation of magnetic resonance imaging ( mri ) , mr spectroscopic imaging ( mrsi ) and biopsy findings tabella 1 correlazione tra rm , rmsp e reperti bioptici biopsy findings mrsi risultati biopsia rmsp negative positive negative positive negativi positivi negativi positivi negative positive total negativi positivi totale table 2 sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) , and accuracy of magnetic resonance imaging ( mri ) , mr spectroscopic imaging ( mrsi ) and combined mri / mrsi tabella 2 sensibilit , specificit , vpp , vpn , accuratezza della rm , rmsp e rm / rmsp combinate mri , % mrsi , % mri / mrsi , % rm , % rmsp , % rm / rmsp , % sensibilit specificit accuratezza sensitivity specificity accuracy g . 
3a - d ipointensit focale nelle immagini assiali t2pesate nella zona periferica in sede mediana sinistra . lo spettro ottenuto dallarea di alterata intensit di segnale ha evidenziato unelevazione della colina e una riduzione del citrato compatibile con la presenza di cancro ( d )  . 
4a - d diffusa alterata intensit di segnale nella zona periferica a destra nelle immagini t2 - pesate ( piano assiale , a e c e piano coronale , b )  . 
in contrast , our study aimed at assessing the ability of mri and mrsi to identify and localise prostate cancer by prospectively correlating findings of single techniques with biopsy results . the decision to use a surface coil was dictated by the need to obviate the poor tolerability of the examination carried out with an intracavitary coil and to improve the quality of images corrupted by specific artefacts of endorectal coils ( coil flare , straight line , glandular distortion ) [ 18 ]  . as regards the morphological evaluation alone , mri had 85% sensitivity and 53% specificity in the detection of lesions values in line with those reported in the literature . il carcinoma prostatico rappresenta a tuttoggi unimportante causa di morte per cancro nella popolazione maschile [ 1 ]  . 
di contro a questa rilevante mortalit , ci sono molti casi di tumore a decorso subclinico rilevati casualmente al solo esame autoptico [ 9 ]  . le tecniche diagnostiche non invasive correntemente disponibili presentano delle forti limitazioni nel discriminare forme latenti da forme aggressive e progressive [ 10 , 11 ]  . la trus ha dimostrato di non essere in grado di individuare fino al 30% delle lesioni palpabili allesplorazione rettale e di avere unelevata percentuale di falsi positivi in quanto solo il 20% delle lesioni ipoecogene ( principale caratteristica ecografia di malignit ) sono realmente maligne [ 12 ] ; la biopsia ecoguidata inoltre presenta unelevata percentuale di errori nel campionamento per il fatto che solo una piccola parte della ghiandola prostatica viene effettivamente analizzata cosicch molti pazienti che presentano valori di psa alterati ( > 4 ng / ml ) e che sono sottoposti ad un campionamento sistematico lungo i sestanti prostatici hanno esito negativo allesame bioptico e sono candidati ad un ulteriore prelievo [ 13 , 14 ]  . con lemergere di nuove strategie terapeutiche meno invasive rispetto alla prostatectomia radicale , si sente , inoltre , maggiormente lesigenza di una precisa localizzazione e valutazione dellestensione della malattia , per eseguire una terapia mirata che incrementi lefficacia del trattamento , riducendone la morbilit . 
le esperienze sinora pubblicate , aggiungendo al dato morfologico quello metabolico mediante limaging spettroscopico 3d , evidenziano un miglioramento nellindividuazione della neoplasia e nella definizione della stadiazione , volume e grado di aggressivit [ 8 , 16 , 17 ]  . 
 la maggior parte dei dati in letteratura deriva per da studi di tipo retrospettivo , in cui laccuratezza diagnostica della rm e della rmsp stata valutata facendo riferimento al dato istopatologico ottenuto dopo prostatectomia radicale . 
nel nostro studio abbiamo voluto invece valutare la capacit della rm e della rmsp nellindividuazione e localizzazione del tumore prostatico , correlando prospetticamente i risultati ottenuti dalle singole tecniche con quelli bioptici . la scelta di utilizzare una bobina di superficie si imposta per ovviare alla scarsa tollerabilit dellesame eseguito con bobina endocavitaria e per migliorare la qualit dellimmagine corrotta da artefatti specifici delle bobine endorettali ( coil flar , straight line , distorsione ghiandolare ) [ 18 ]  . per quanto riguarda la sola valutazione morfologica la rm ha mostrato , nellidentificazione delle lesioni , una sensibilit del 85% e una specificit del 53% , valori questi sostanzialmente sovrapponibili a quelli riportati in letteratura . la rmsp , con laggiunta del dato metabolico ha mostrato mrsi , with the addition of metabolic data , proved to be significantly more specific in identifying tumours as compared with mri alone ( 89% versus 53% )  . 
a positive result at mri and mrsi indicates the likely presence of cancer ( ppv 88% ) whereas a negative result excludes it with a slightly lower likelihood ( npv 77% )  . 
presumably , this is affected by the number of false negatives , which are likely due to limitations of the current multivoxel technology , with a nominal voxel value that is still high ( 0.24 cm3 ) , and is typical of 1.5t magnetic fields , with a possible summation of the tumour 's metabolic findings with those of surrounding healthy tissue in the sampled volume . 
another possible explanation of the failed identification of tumour lesions by mrsi may be the low biological aggressiveness of some cancers . some authors [ 19 ] have already reported that small , lowgrade tumours ( gleason scores 4 and 5 ) may be missed owing to slight alterations of citrate and choline . 
il valore nominale del voxel infatti ancora elevato ( 0 , 24 cm3 ) , caratteristico per campi magnetici di 1 , 5t , e ci potrebbe determinare un effetto di sommazione del dato metabolico tumorale con quello del tessuto sano circostante nel volume campionato . unaltra possibile spiegazione della non avvenuta identificazione delle lesioni tumorali da parte la rmsp pu essere correlata alla scarsa aggressivit biologica di alcune neoplasie . infatti , gi stato riportato da alcuni autori [ 19 ] come piccoli tumori a basso grado ( gleason 4 e 5 ) possono non essere rilevati per le lievi alterazioni del citrato e della colina . 
iniziali studi clinici , che vedono lapplicazione di campi magnetici ad alta intensit ( 3t ) , dimostrano come sia possibile aumentare sensibilmente la risoluzione spettrale , con un sostanziale incremento di accuratezza nellidentificazione spaziale del dato metabolico [ 20 ]  . la scarsa correlazione complessiva tra i risultati della rm e della rmsp e quelli istopatologici , riguardo la localizzazione spaziale delle lesioni , fa s che il nostro studio non sia in grado di rispondere alla domanda se la localizzazione del tumore prostatico influenzi la capacit delle due tecniche di rilevare il tumore stesso . 
in realt , per lintrinseca difficolt della biopsia ecoguidata di garantire una precisa corrispondenza tra il sito del prelievo e le aree sospette alla rm e alla rmsp , solo lanalisi istologica dopo prostatectomia radicale in grado di rispondere realmente a tale quesito . conclusions conclusioni although ours was a preliminary study carried out on a small sample of patients and larger series are needed to confirm our findings , we can reasonably conclude that the combination of mri and mrsi also considering the greater tolerability of an examination that avoids the use of endorectal coils may be indicated as a problem - solving modality for two categories of patients : those whose high serum psa and suspicious trus finding , which make them candidates for biopsy ; and those who have already undergone multiple biopsies , all with a negative outcome but persistent serum psa alterations , and are therefore candidates for further biopsy . 
identification of suspicious areas on mri and mrsi also provides the possibility of performing targeted biopsies of those areas , under direct mri guidance , and to remove the error inherent in correlating mri with trus findings [ 21 ]  . in conclusione , sebbene il nostro sia certamente uno studio iniziale , realizzato su di una piccola popolazione campione e che siano necessarie pi ampie serie di pazienti per confermare quanto da noi evidenziato , ragionevole ritenere che lassociazione rm / rmsp , anche in relazione alla maggiore tollerabilit di un esame che evita lutilizzo della bobina endorettale , possa essere indicata come modalit problem - solving in quei pazienti che , con elevati valori di psa sierico e trus sospetta per lesione neoplastica , sono candidati al prelievo bioptico oppure in coloro che , gi sottoposti a biopsie multiple con esito negativo , per il persistere delle alterazioni sieriche del psa , rientrano in una zona di incertezza e sono candidati ad unulteriore biopsia . 
cova1 1unit clinica operativa di radiologia , universit degli studi di trieste , ospedale di cattinara , strada di fiume 447 , i - 34149 trieste , italy 2unit operativa di radiologia , ospedale umberto i , mestre , venezia , italy 3dipartimento di economia e tecnica aziendale , universit degli studi di trieste , trieste , italy correspondence to : f . 
the sum of equipment costs ( amortisation and service contract ) , variable costs ( supplies and related services ) and personnel costs ( radiologist , radiographer and nurse ) was determined . 
intra - arterial dsa costs more than mra , mainly because of the higher costs of supplies used during the procedure and higher personnel costs ( as a result of the longer duration of intra - arterial dsa )  . 
mra provides good diagnostic accuracy in the evaluation of arteries of the lower extremities , and its biological cost is far lower than that of intra - arterial dsa ( mra is noninvasive , it does not use ionising radiation , and the contrast medium is safe )  . 
its lower cost is another argument in favour of the use of mra instead of intra - arterial dsa in the evaluation of lower - extremity arterial disease . key words magnetic resonance angiography intra - arterial digital subtraction angiography cost analysis peripheral vascular disease riassunto obiettivo . 
stato calcolato il costo differenziale delle due indagini , ovvero la somma dei costi delle apparecchiature ( ammortamenti e manutenzione ) , dei costi variabili ( materiali e servizi connessi ) e dei costi del personale ( medico , tecnico e infermieristico )  . 
il costo differenziale dellangio - rm risultato di 186 , 14 euro ( costi delle apparecchiature 50 , 80 euro , costi variabili 75 , 04 euro , costi del personale 60 , 30 euro ) , mentre quello dellangiografia digitale risultato di 238 , 18 euro ( costi delle apparecchiature 57 , 60 euro , costi variabili 90 , 13 euro , costi del personale 90 , 45 euro )  . 
langiografia digitale ha un costo superiore allangio - rm , determinato essenzialmente dal maggior costo del materiale utilizzato durante la procedura e dal maggior costo del personale ( legato , a parit di figure professionali coinvolte , alla maggior durata dellesame angiografico )  . 
langio - rm fornisce una buona accuratezza diagnostica nella valutazione delle arterie degli arti inferiori ed ha un costo biologico nettamente inferiore allangiografia digitale ( assenza di invasivit , assenza di radiazioni ionizzanti , mezzo di contrasto con pi elevato margine di sicurezza )  . 
il minor costo dellangio - rm costituisce un ulteriore elemento che porta a preferire questa indagine allangiografia digitale nella diagnostica delle arteriopatie degli arti inferiori . parole chiave angio - rm angiografia digitale intra - arteriosa analisi dei costi arteriopatia periferica f . 
technological advancements have allowed this technique to provide high levels of sensitivity and specificity as compared with digital subtraction angiography ( dsa ) as the gold standard even though the reported ranges are extremely wide ( sensitivity : 71%100% , specificity : 63%100% ) [ 111 ]  . 
most papers have , therefore , considered its diagnostic contribution and emphasised its advantages of having a negligible biological cost . by contrast , this study analyses and compares the costs of mra and intra - arterial dsa in the evaluation of lower - limb arteries . 
it should be noted that activities and costs related to these two examinations pertain largely to the radiology department even if some aspects , particularly related to dsa , concern other departments as well . 
the costs were also analysed from the point of view of the producer ( the hospital ) , whose aim it is to provide the patient with the best and most cost - effective clinical approach . 
our study presents a cost - identification analysis , namely , a partial assessment of the two examinations in question , concentrating on their effect on costs ( input ) [ 12 ]  . 
clinical consequences of the two different approaches have , instead , been excluded from our study ( output )  . langiografia mediante risonanza magnetica ( angio - rm ) ha trovato negli ultimi anni applicazione sempre pi ampia nella valutazione delle arteriopatie degli arti inferiori . 
i progressi tecnologici hanno fatto s che tale indagine possa garantire una sensibilit ed una specificit elevate , utilizzando langiografia digitale come gold standard , pur se i lavori pubblicati riportano un range di percentuali assai ampio ( sensibilit tra il 71% e il 100% , specificit tra il 63% e il 100% ) [ 111 ]  . 
molti lavori hanno preso quindi in considerazione il suo apporto diagnostico ed hanno sottolineato i suoi benefici , per il trascurabile costo biologico . in questo lavoro abbiamo ritenuto opportuno analizzare invece i costi dellangio - rm nella valutazione delle arterie degli arti inferiori , raffrontati a quelli dellangiografia digitale per via arteriosa . 
va rilevato come attivit e costi relativi a queste due indagini sono in larga misura di pertinenza dei servizi di radiologia , ma per talune , langiografia digitale in particolare , riguardano anche altri reparti . 
i costi sono inoltre stati considerati dal punto di vista del produttore ( lospedale ) che ha lobiettivo di fornire al paziente lapproccio clinico migliore e con il miglior rapporto costo - efficacia . 
lanalisi dei costi effettuata del tipo cost - identification , ovvero una valutazione parziale con lobiettivo di valutare , analizzando le due indagini considerate , gli effetti sui costi ( input ) [ 12 ]  . 
non sono invece state considerate le conseguenze cliniche dei due diversi approcci ( output )  . materials and methods examination technique magnetic resonance angiography mra examinations were performed using a 1.5 - t intera master superconductive magnet ( philips medical systems , eindhoven , the netherlands ) with 30 mt / m power gradients and 150 mt / m per millisecond rise time . 
the use of automated table movement ( mobitrack , philips ) allowed us to study the aorto - iliac , femoral - popliteal and popliteal - tibial vascular regions in a sequence . 
for evaluation of each vascular region , we used three - dimensional ( 3 - d ) fast spin echo ( fse ) t1 - weighted sequences with the following parameters : tr = 6 ms , te 1.5 ms , flip angle 35 , slice thickness 1.5 mm ( 3 mm with 1.5 - mm overlap ) , field of view 430 mm , matrix 512512 , number of slices 70 and acquisition time 28 s . 
overall acquisition time of successive sequences , including the time needed for two table movemateriali e metodi tecnica desame angio - rm gli esami di angio - rm sono stati eseguiti utilizzando un magnete superconduttivo da 1 , 5 t intera master ( philips medical systems , eindhoven , olanda ) con gradienti di potenza pari a 30 mt / m e velocit di raggiungimento del picco di 150 mt / m / ms . 
limpiego dello spostamento automatico del tavolo ( mobitrack ; philips ) ha consentito di studiare in successione il distretto vascolare aorto - iliaco , quello femoropopliteo e quello popliteo - tibiale . 
sono state impiegate per la valutazione di ciascun distretto vascolare sequenze 3d fse t1 pesate utilizzando i seguenti parametri : tr = 6 ms , te 1 , 5 ms , flip angle = 35 , spessore di strato pari ad 1 , 5 mm ( 3 mm con sovrapposizione di 1 , 5 mm ) , campo di vista = 430 mm , matrice = 512x512 , numero di strati = 70 , tempo di acquisizione paf . 
this was followed by injection of 0.2 mmol / kg of 0.5 m paramagnetic contrast agent ( gadobenate dimeglumine , multihance , bracco , milan , italy ) with a 0.5 - ml / s flow rate . 
post - processing included subtraction of images acquired before and after the contrast agent injection and 3 - d reconstructions based on the maximum intensity projection ( mip ) algorithm . dsa ( intra - arterial ) dsa examinations were carried out using the integris allura system for diagnostic angiography and interventional radiology ( philips medical systems , eindhoven , the netherlands )  . 
examinations were performed with intra - arterial catheterisation , usually after puncture of the femoral artery and insertion of the distal end of the catheter into the infrarenal abdominal aorta . 
straight - end or pigtail catheters , which can endure high flows of contrast material , were employed . intra - arterial examinations were performed using two different methods : the bolus - chasing technique ( in which synchronised table movement during injection of the contrast agent allows passage of the contrast through the aorto - iliacfemoral - popliteal region to be monitored ) , and the successive fields technique . 
choice of technique was based on the result of the preliminary test to determine the knee arrival time ( kat ) or the number of seconds it takes for a small amount of injected contrast agent to reach the popliteal artery at the knee joint . we noted that when the kat is very short ( 46 in . ) , image quality , especially at the abdominal level , is affected by blurring of the vessel caused by the table moving too fast . hence , the bolus - chasing technique is only used when the kat exceeds 7 s whereas for shorter kats , the classical multiple - fields technique is adopted . 
in the bolus - chasing technique , the examination is carried out with a single injection of 80 ml of non - ionic monomeric contrast medium ( 350370 mgi / ml ) with a 79 ml / s flow rate through an automatic injector , and the table moves automatically at a speed determined by result of the preliminary test . 
stato preliminarmente valutato con liniezione di 2 ml di mezzo di contrasto ( mdc ) ( 0 , 5 ml / s ) in una vena del braccio il tempo necessario per iniziare ad ottenere lopacizzazione dellaorta addominale . 
sono state quindi effettuate con spostamento del tavolo le sequenze maschera ( senza somministrazione del mdc ) sui tre tratti a partire da quello popliteo - tibiale . sono stati poi iniettati 0 , 2 mmol / kg di un mdc paramagnetico 0 , 5 m ( gadobenato dimeglumina , multihance , bracco , milano , italia ) con flusso di 0 , 5 ml / s . 
sono state quindi acquisite le sequenze dopo iniezione di mdc a partire dal distretto aorto - iliaco , secondo il tempo di ritardo calcolato mediante il bolo preliminare di 2 ml . 
il post processing prevedeva la sottrazione di immagine tra quelle acquisite prima e dopo iniezione di mdc e ricostruzioni 3d secondo lalgoritmo mip ( maximun intensity projection )  . angiografia digitale ( accesso arterioso ) gli esami sono stati effettuati utilizzando il sistema per angiografia diagnostica e radiologia interventistica integris allura ( philips medical systems , eindhoven , olanda )  . 
sono stati utilizzati cateteri ad estremit diritta o pig tail in grado di sostenere flussi elevati di mdc . gli esami con accesso arterioso sono stati eseguiti con due diverse modalit : tecnica bolus chasing ( che grazie al movimento sincronizzato del tavolo durante liniezione del mdc consente di seguire lavanzamento del bolo di mdc nel distretto aorto - iliaco - femoro - popliteo ) o tecnica per campi successivi . 
la diversa tecnica di esecuzione si basata sul risultato del preliminare test per determinare il kat ( knee arrival time ) che stabilisce , una volta iniettato un piccolo quantitativo di mdc , quanti secondi esso impiega a giungere nellarteria poplitea a livello della rima articolare del ginocchio . 
si verificato che quando il kat molto breve ( 46 s ) la qualit delle immagini soprattutto a livello addominale risulta essere subottimale per la presenza di sfumatura del vaso causata dalleccessiva velocit del movimento di avanzamento del tavolo . 
nella tecnica bolus chasing lesame viene effettuato con ununica iniezione di 80 ml di un mdc monomero non ionico ( 350370 mgi / ml ) ad un flusso di 79 ml / s eseguita mediante apposito iniettore automatico e il tavolo scorre automaticamente ad una velocit che si basa sul risultato del test preliminare . 
if motion artefacts are present , images can be processed with pixel shift software so as to minimise them . if motion artefacts are more substantial , images can be visualised at any rate without subtracting the skeletal structures . 
an automatic injector is again used to administer 30 ml of contrast agent at a 15 - ml / s flow rate for each field , for a total of 150 ml . 
the full cost of the two studies was thus obtained ( the sum of differential and common costs )  . equipment cost was calculated based on the time it was used ; when analysing the cost of technology , the purchase price of the various machines ( which can be strongly affected by substantial discounts ) and the adequacy of the system for calculating amortisation are clearly important . 
purchase prices were derived from hospital administration records . amortisation was calculated on a time basis based on a constant annual value . the lifetime of the radiological units was estimated as 8 years , i.e. 
procedure duration was estimated based on times envisaged by the radiology information system ( ris ) in use at our unit . in evaluating the differential cost , we merely considered the technical act , disregarding any other costs related to patient management , as inclusion of the latter would have required an explicit description of the relations between the radiology department and other cost centres of the hospital and the part played by radiological examinations in inpatient treatment . therefore , our analysis was restricted to costs incurred by the radiology unit . 
this made it possible to draw comparisons over time ( with respect to budget targets ) and space ( with respect to other hospitals : independent , with separate accounting , or private ) [ 13 ]  . 
 the cost of fixed - asset utilisation ( technology ) was related to their time of use , and the cost of radiologists , technologists and nurses was related to their specific activity meadelliniezione del mdc e la seconda durante liniezione del mdc , ottenendo quindi due serie di 15 immagini ciascuna . 
ad ogni campo desame vengono iniettati , sempre mediante iniettore automatico , 30 ml di mdc con un flusso di 15 ml / s , per un totale di 150 ml . analisi economica stato calcolato il " costo differenziale " ( definito come la somma dei costi delle apparecchiature , dei costi variabili e dei costi del personale ) delle due indagini . 
 stato inoltre calcolato il " costo comune " , il quale raggruppa quelle componenti del costo che risultano sovrapponibili per ogni tipo di esame svolto allinterno dellunit clinico operativa di radiologia . 
stato cos possibile ottenere il " costo pieno " delle due indagini ( somma del costo differenziale e di quello comune )  . il costo delle apparecchiature stato calcolato in base al tempo di utilizzo delle stesse ; nel valutare il costo della tecnologia emerge limportanza del costo dacquisto delle diverse attrezzature ( che pu essere influenzato notevolmente da sconti significativi ) e della congruit del sistema di calcolo dellammortamento . 
la durata della vita lavorativa delle apparecchiature radiologiche stata considerata di otto anni , il loro tempo tecnologico massimo . sono poi stati valutati i costi variabili , relativi ai materiali impiegati e ai servizi legati a tale impiego . 
la durata delle procedure stata stimata facendo riferimento ai tempi previsti nel sistema informativo radiologico ( ris ) in uso presso la nostra struttura . nella valutazione del costo differenziale ci siamo limitati a considerare latto tecnico , ignorando tutti gli altri costi legati alla gestione del paziente . 
ci infatti avrebbe comportato lesplicitazione di tutti i rapporti tra la radiologia e gli altri centri di costo dellazienda ospedaliero - universitaria e , per i pazienti ricoverati , anche del ruolo che le indagini radiologiche hanno sul processo di cura . 
they are therefore not decisive for comparing costs but play a role in determining the full cost of the two methods . the sections below provide a detailed analysis of the differential costs of mra and dsa , with the methods in use at our radiology unit . 
as stated above , three components of the differential cost were considered , namely : ( 1 ) equipment costs ( amortisation and service contract ) ; ( 2 ) variable costs ( supplies and related services ) ; ( 3 ) personnel costs . it should be noted that every patient undergoing dsa must have a blood test to evaluate serum glucose and creatinine levels and coagulation profile [ quick time , international normalised ratio ( inr ) and platelets ]  . in addition , patients with a history of allergy ( 1% of cases ) require pre - medication with steroids associated with antih1 and anti - h2 . the cost of these procedures does not pertain to the radiology unit however , because the procedures are mandatory before contrast - enhanced angiography and because their cost is not negligible , they will be briefly discussed . finally , it should be recalled that costs of supplies and equipment are inclusive of 20% vat . results differential costs magnetic resonance angiography 1 . 
we took into account the cost of a 1.5 - t intera master mri device ( philips medical systems , eindhoven , the netherlands ) with 30 - mt / m power gradients and a rise time of 150 mt / m per millisecond . 
there follows an annual cost of 3 , 021.25 euro considering 8 years of amortisation . then the cost of the dry printer ( kodak dryview 8700 laser imager ) was included . 
come gi evidenziato , sono state prese in considerazione 3 componenti del costo differenziale , e precisamente : ( 1 ) costi delle apparecchiature ( ammortamenti e manutenzione ) ; ( 2 ) costi variabili ( materiali e servizi connessi ) ; ( 3 ) costo del personale . va rilevato che ogni paziente sottoposto ad angiografia digitale deve eseguire preliminarmente un prelievo ematico per valutare i livelli sierici di glucosio e creatinina e il suo assetto emocoagulativo ( tempo di quick , inr e piastrine )  . nell1% dei casi inoltre , essendoci unanamnesi allergica positiva , si esegue anche una preparazione farmacologica ( basata sullimpiego di cortisonici associati ad anti - h1 ed anti - h2 )  . 
i costi legati a tali procedure non sono di pertinenza dellunit clinico operativa di radiologia ; tuttavia queste sono fondamentali ai fini dellesecuzione dellesame angiografico con mezzo di contrasto ed avendo comunque un certo rilievo economico verranno in seguito brevemente prese in considerazione . si ricorda infine che i costi di materiali ed apparecchiature sono comprensivi di iva al 20% . risultati costi differenziali angio - rm 1 . 
abbiamo preso in considerazione il costo di un apparecchio di rm da 1 , 5 t intera master con gradienti di potenza pari a 30 mt / m e velocit di raggiungimento del picco di 150 mt / m / ms . 
by estimating the average annual cost of a radiologist , technologist and nurse ( totalling 158 , 744 euro ) and subtracting the time off work for ordinary , special and sick leave ( 30% on average ) from the annual hourly debt , the actual work time amounted to 35 weeks / year , which results in a daily cost of 905 euro and an hourly cost of 120.73 euro for these three professionals taken together . 
valutando il costo medio annuo per un medico , per un tecnico sanitario di radiologia medica ( tsrm ) e per un membro del pool infermieristico ( complessivamente 158.744 euro ) e sottraendo al debito orario annuo le assenze per congedo ordinario , straordinario e malattie ( 30% in media ) , risulta un tempo lavorativo effettivo pari a 35 settimane / anno , da cui si deriva il costo giornaliero ( 905 euro ) e orario ( 120 , 73 euro ) di queste tre figure professionali considerate assieme . 
limpegno temporale del medico legato alle diverse attivit che egli deve svolgere in prima persona , ovvero : presenza fisica in fase di acquisizione dellesame , analisi delle immagini alla consolle , refertazione , correzione e sigla del referto . 
va inoltre preso in considerazione il tempo preliminare allesame ; in particolare , il paziente deve sottoscrivere un apposito questionario mediante il quale se ne verifica lidoneit allaccesso al magnete . 
perci , in base alla nostra esperienza , il tempo medico previsto per un angio - rm degli arti inferiori di 30 minuti . ogni indagine di angio - rm per lo studio degli arti inferiori comporta un impegno temporale del tsrm e dellinfermiere anchesso stimabile in 30 minuti . 
ne risulta quindi un costo personale per esame di 60 , 30 euro . da quanto esposto risulta come , sommando tutti i parametri considerati , il costo differenziale di unangio - rm degli f . 
the cost of the contrast agent injector ( mark 4 , medrad , indianola , usa ) , 8 , 374.29 euro , corresponds to 1 , 046.78 euro per year for 8 years of amortisation . 
the following factors were taken into account : supplies used prior to the procedure ( sterile gloves , disinfection material , sterile cloth , sterile cloths without slit , a sheet with slit for femoral access ) : total cost of preparatory supplies was 1.10 euro supplies used during the procedure ( surgical gauzes , 10 - cc luerlock syringe , 20 - cc syringe , needle for arterial puncture , lidocaine , guidewire , introducer , catheter )  . 
essa viene utilizzata dallunit clinico operativa di radiologia con contratto daffitto triennale comprensivo di installazione , interfacciamento ai sistemi di archiviazione delle immagini e manutenzione , per un costo pari a 13.785 , 00 euro annui . 
alla stampante a secco confluiscono peraltro le due apparecchiature tc in uso nellunit clinico operativa di radiologia ; nel nostro caso langiografo digitale utilizza la stampante per circa il 30% della sua attivit ; ne deriva quindi un costo annuo di 4135 , 50 euro . 
va inoltre considerato il costo delliniettore del mezzo di contrasto ( mark 4 , medrad , indianola , usa ) pari a 8.374 , 29 euro , il che corrisponde a 1.046 , 78 euro annui per 8 anni di ammortamento . 
vanno presi in considerazione i seguenti fattori : materiale preparatorio allesecuzione dellesame ( guanti sterili , materiale per disinfezione , telino sterile , teli sterili senza spacco , lenzuolo con taglio accesso femorale )  . 
il costo totale del materiale preparatorio di 1 , 10 euro . materiale utilizzato durante la procedura ( garze sterili , siringa luerlock da 10 cc , siringa da 20 cc , ago da puntura arteriosa , lidocaina , guida , introduttore , catetere )  . 
estimating the overall average annual cost for a radiologist , a technologist and a nurse at 158 , 744 euro and subtracting ordinary , special and sick leaves ( 30% on average ) from the annual hourly debt , the actual work time is 35 weeks / year , with a daily cost of 905 euro and an hourly cost of 120.73 euro for these three professionals taken together . the radiologists time commitment depends on the different activities he / she has to perfor in the case of an angiographic examination , the radiologists time includes performing the procedure from the end of patient preparation to positioning the catheter into the aorta , as well as being physically present during the acquisition phase , assessing images on screen , removing the catheter , performing the compression , reporting , correcting and signing the written report . hence , based on our experience , intra - arterial lower limb angiography involves 45 min of the radiologists time . 
these costs are unaffected by variations in the total number of yearly examinations . common costs are those associated with support staff and support materials . we were unable to conduct a detailed analysis of support activities to quantify the contribution of these production factors to the different procedures ( random principle ) , but we could , as an alternative , use the means method , assuming that each radiological procedure uses these services to the same extent , i.e. 
that the specific features of each procedure do not affect the common costs . support staff costs were calculated considering all professionals not described so far . they consist of one consultant , one chief technologist , three technologists , three auxiliary staff , four file clerks and seven assistants and administrative assistants . 
per ogni esame se ne impiegano in media 120 ml ( diaco , sodio cloruro 0 , 9% ) usando flaconi da 250 ml ( al costo di 0 , 25 euro per flacone ) , con un costo per esame di 0 , 10 euro . pellicole : nella documentazione delle angiografie degli arti inferiori previsto luso di una pellicola 3543 cm ( dvb kodak ) , per un costo di 2 , 97 euro ( le confez ioni da 500 hanno un costo di 1489 , 40 euro )  . 
valutando il costo medio annuo complessivo di un medico , un tsrm e un infermiere in 158.744 euro e sottraendo al debito orario annuo le assenze per congedo ordinario , straordinario e malattie ( in media 30% ) , risulta un tempo lavorativo effettivo pari a 35 settimane / anno , da cui si deriva un costo giornaliero di 905 euro e orario di 120 , 73 euro di queste tre figure professionali considerate assieme . 
nel caso dellesame angiografico il tempo medico prevede lesecuzione della procedura dal termine della preparazione del paziente fino al posizionamento del catetere in aorta , la presenza fisica in fase di acquisizione dellesame , la verifica a monitor delle immagini acquisite , la rimozione del catetere , la compressione , la refertazione dellesame , la correzione e la sigla del referto . 
ne risulta quindi un costo personale per esame di 90 , 45 euro . da quanto esposto risulta come , sommando tutti i parametri considerati , il costo differenziale di una indagine angiografica degli arti inferiori risulti presso il nostro istituto di 238 , 18 euro . costi comuni abbiamo indicato con lespressione costi comuni i costi dei fattori produttivi , interni allunit clinico operativa di radiologia , che garantiscono unattivit di supporto a tutti gli atti diagnostici svolti nellunit operativa . 
non siamo in grado di effettuare unanalisi dettagliata delle attivit di supporto per quantizzare il contributo di questi fattori produttivi alle diverse procedure ( criterio causale ) , per cui rimane lalternativa di ricorrere al metodo della media , ipotizzando che ciascuna indagine radiologica usufruisca in pari misura di tali servizi , vale a dire che le specificit di ciascuna indagine non siano una determinante dei costi di cui stiamo trattando . 
non riteniamo che il loro lavoro sia influenzato dalla tipologia degli esami svolti e quindi il loro costo complessivo stato ripartito proporzionalmente su tutte le indagini eseguite presso lunit clinico operativa di radiologia . i materiali di supporto comprendono computer , stampanti , registratori , materiale elettrico e di rete , materiale di cancelleria e materiale di pulizia . 
ne risulta un costo comune aggiuntivo per esame di 5 , 62 euro . costo pieno sommando i costi differenziali con i costi comuni , stato calcolato il costo pieno di ciascuna indagine ( tabella 1 ) , ovvero : 1 . 
angio - rm : 186 , 14 euro + 5 , 62 euro = 191 , 76 euro ; 2 angiografia digitale : 238 , 18 euro + 5 , 62 euro = 243 , 8 euro . in tutti i pazienti che vengono sottoposti ad indagini contrastografiche che prevedono limpiego di un mezzo di contrasto organo iodato ( in questo caso si tratta dellesame angiografico ) viene effettuato un prelievo ematico per valutarne i livelli sierici di glucosio e creatinina a cui vengono aggiunti i parametri emocoagulativi ( inr , assetto piastrinico ) per un costo complessivo di 7 , 50 euro per esame . 
questo costo non rientra tra i costi differenziali dellesame , per riteniamo non scorretto sottolineare come langiografia venga cos a pesare maggiormente sui costi allesterno della struttura radiologica rispetto allangio - rm . 
opportuno infine ricordare , come gi precedentemente accennato , che circa nell1% dei pazienti che vanno sottoposti ad esami angiografici , essendoci unanamnesi allergica positiva , si esegue anche una preparazione farmacologica ( basata sullimpiego di cortisonici associati ad anti - h1 ed anti - h2 ) , per un costo di 4 euro , in media 0 , 04 euro in pi per ogni esame . 
 complessivamente , questi costi esterni alla radiologia ammontano a 7 , 54 euro . discussione e conclusioni lanalisi dei costi ha dimostrato un costo superiore del 27 , 1% dellangiografia digitale rispetto allangio - rm nella valutazione delle arterie degli arti inferiori ( 243 , 8 versus 191 , 76 euro )  . 
factors with the highest impact are supplies used in the procedure and personnel costs ( owing to the longer examination time )  . decisions concerning the treatment to be pursued after ascertaining that treatment is indeed required will also affect overall cost calculation [ 1417 ]  . indeed , mra may or may not evidence lesions requiring surgical treatment with percutaneous transluminal angioplasty ( pta ) and / or stent placement . 
in this event , the cost of the mra examination should be subsequently added to the costs of intra - arterial angiography and the costs for 1 day in hospital after the interventional procedure ( however , costs of hospitalisation are not included in our analysis , which assesses only costs incurred by the radiology unit )  . it would also be possible to perform angiography first , combining the diagnostic and therapeutic parts in one session , thereby avoiding mra examination . this approach would have to take into account an additional hypothetical , non - predictable workload for the section and delays or cancellations of scheduled examinations because of possible complications arising during the procedure . in our personal experience , however , diagnostic and interventional procedures are usually performed separately . it should also be noted that after diagnostic angiography with arterial catheterisation , the patient is usually hospitalised to monitor possible complications such as bleeding . therefore , external costs of hospitalisation need to be considered . on the other hand , an alternative to manual compression and hospitalisation is to apply an intra - vasal haemostatic device ( angio - seal ) [ 18 ]  . if the device is applied at the end of the procedure , the patient can be mobilised immediately without serious complications or longer examination times [ 19 ]  . 
however , the use of this device entails a cost of 139.44 euro , which , although cheaper than 1 day in hospital , directly weighs on our units budget . 
if it were used , it would then have considerable impact on the full costs of dsa . hospitalisation may also be avoided by monitoring the patient for 5 h after a 15 - min compression and then conducting a telephone follow - up up to the seventh day [ 20 , 21 ]  . 
a further alternative to avoid hospitalisation may be to perform the diagnostic examination with a brachial as opposed to femoral access . the cost analysis we conducted employed a technique often applied in industry , which ensures a rigour that reduces fattori che incidono maggiormente sono rappresentati dal costo del materiale utilizzato durante la procedura e dal costo del personale ( questultimo legato , a parit di figure professionali coinvolte , alla maggior durata dellesame angiografico )  . 
vi infatti la possibilit che langio - rm non evidenzi lesioni passibili di trattamento interventistico mediante pta e / o posizionamento di stent , oppure al contrario che dimostri lesioni suscettibili di tali trattamenti . 
in questo secondo scenario , al costo dellesame angio - rm andrebbero successivamente aggiunti i costi relativi allesame angiografico eseguito mediante accesso arterioso e i costi relativi ad un giorno di degenza post - procedura interventistica ( i costi di degenza non rientrano peraltro nella nostra analisi che valuta i costi allinterno dellunit clinico operativa di radiologia )  . vi sarebbe peraltro la possibilit di eseguire in primis langiografia , effettuando cos in ununica seduta sia la parte diagnostica che la parte terapeutica , evitando in tal modo lesame di angio - rm . 
tale atteggiamento dovrebbe tenere in considerazione il carico di lavoro ipotetico aggiuntivo non prevedibile della sezione e eventuali ritardi o annullamento di esami programmati a causa di eventuali complicanze insorte in corso di procedura . 
esso tuttavia presenta un costo di 139 , 44 euro che , pur essendo inferiore al costo di un giorno di degenza , grava direttamente sul budget dellunit clinico operativa di radiologia . qualora utilizzato , esso graverebbe quindi considerevolmente sui costi pieni dellangiografia digitale . la degenza ospedaliera pu anche essere evitata eseguendo al termine di una compressione di 15 minuti un monitoraggio temporaneo del paziente per 5 ore e successivamente un follow - up telefonico fino alla 7a giornata [ 20 , 21 ]  . 
 unulteriore alternativa per evitare la degenza pu essere costituita da un esame diagnostico eseguito con accesso brachiale anzich femorale . lanalisi dei costi da noi condotta fa riferimento a una tecnica ampiamente utilizzata in campo industriale che garantisce un rigore che riduce le approssimazioni dei metodi tradizionali di calcolo dei costi [ 22 ]  . 
they reflect the situation in our department at a certain moment in time , but costs in other radiology departments may be extremely different to assess , either owing to different costs of equipment used or simply because of different organisation , which can affect variable costs ( different materials used ) or personnel costs ( possible different roles )  . 
in our view , this does not detract from the validity of our analysis , but it emphasises its value in suggesting a model that can be easily applied to other settings rather than for reported cost data . this can therefore be a stimulus for similar cost evaluations of these two techniques , possibly considered within more complex diagnostic protocols or in comparison with other techniques , such as ct angiography . clearly , a cost analysis is only the first step towards a cost - effectiveness analysis , which considers both the diagnostic benefits , including impact on therapy , and biological costs . the high diagnostic accuracy of mra in the evaluation of lower - limb arteries is an established fact [ 111 ]  . 
in addition , mra no doubt offers clear advantages over angiography if biological costs are considered since it is non - invasive , involves no ionising radiation and makes use of a paramagnetic contrast agent , which is safer than organic iodinated products . 
these elements led to a quick transition from dsa to mra in the diagnosis of lower - limb arterial disease in our department . in 2004 , mra was carried out on all patients with this clinical condition , reserving dsa to a mere 4% of cases , i.e. those with contraindications to mri . tuata in questo campo applicativo risulta comparabile ad un solo lavoro in letteratura [ 23 ] , mentre negli altri [ 14 , 24 ] si fa riferimento alle tariffe di angio - rm ed angiografia digitale e non ad una valutazione dei costi reali . 
vi sono soprattutto differenze operative che giustificano questa diversit , tra le altre il computo del costo del mantenimento del paziente sorvegliato per 34 ore in un letto predisposto nel servizio di radiologia dopo leffettuazione dellangiografia e il minimo coinvolgimento del medico radiologo ( stimato in soli 7 minuti ) per leffettuazione e la refertazione dellangio - rm . 
essi rappresentano una fotografia della situazione nella nostra struttura in un certo momento storico , ma evidente che in altre strutture i costi possono essere estremamente diversi , vuoi per il costo diverso delle apparecchiature utilizzate o semplicemente per diverse scelte organizzative che possono incidere ad esempio sui costi variabili ( per diversi materiali utilizzati ) o sui costi del personale , qualora ne sia previsto un diverso impiego . 
ci non toglie a nostro giudizio validit a tale analisi , ma sottolinea il suo valore non tanto per i dati grezzi di costo riportati quanto per il suggerimento di un modello facilmente applicabile in altre realt operative . ci pu quindi essere stimolo per analoghe valutazioni dei costi di queste due tecniche , eventualmente inserite anche in protocolli diagnostici pi articolati o in raffronto ad altre indagini , quale langio - tc . 
chiaro che unanalisi dei costi rappresenta solo un primo tassello per giungere ad unanalisi costo - efficacia , che consideri quindi da un lato i benefici diagnostici , includendo le ricadute terapeutiche , e dallaltro i costi biologici . 
lelevata accuratezza diagnostica dellangio - rm nella valutazione delle arterie degli arti inferiori un dato acquisito [ 111 ]  . non vi dubbio inoltre che , per quanto riguarda i costi biologici , langio - rm offre chiari vantaggi rispetto allangiografia , per lassenza di invasivit , lassenza di radiazioni ionizzanti e lutilizzo di un mezzo di contrasto paramagnetico , pi sicuro di un prodotto organo - iodato . 
the purpose of this study was to investigate supraspinatus tendon sonographic morphology in a population of young overhead athletes in correlation with main pathologic models of secondary shoulder impingement syndrome . 
between april and may 2004 , 20 subjects ( ten professional basketball players and ten non - athlete controls of the same age , weight and height ranges ) underwent bilateral , standardised , sonographic sholulder examination to evaluate supraspinatus echotexture , supraspinatus and subacromial bursa thickness , subacromial space width ( cutoff of 7 mm ) and dynamic anterior impingement beneath the acromial marg results . 
despite the study limitations , ultrasonography ( us ) is able to detect subacromial space narrowing in young overhead athletes as early shoulder impingement sign , according to the continuum impingement - instability pathologic model . key words sport lesions sonographic examination of shoulder sonographic examination of tendons subacromial impingement syndrome shoulder instability riassunto obiettivo . 
quattro giocatori mostravano segni e sintomi di instabilit di spalla atraumatica destra ( 2 casi ) o di impingement del sovraspinato con dolore anteriore di spalla ( 1 spalla destra ed 1 spalla sinistra )  . 
nonostante i limiti dello studio , lecografia in grado di individuare il restringimento dello spazio subacromiale in atleti overhead giovani come segno precoce di impingement del sovraspinato , in accordo con il modello patogenetico del continuum impingement - instabilit . parole chiave lesioni da sport spalla , ecografia tendini , ecografia sindrome di impingement subcromiale instabilit introduction introduzione r . 
among shoulder pathologies , secondary supraspinatus impingement syndrome frequently affects so - called overhead sports players , sometimes with the risk of compromising athletic careers [ 1 ]  . during the last few years , increasing understanding and subtler classification of pathologic mechanisms responsible for this condition have enabled more appropriate orthopaedic treatments than in the past , leading to more acceptable clinical and functional results [ 2 ]  . 
all subjects underwent preliminary clinical examination as previously described [ 7 ] , including neer and hawkins tests for anterior and posterior impingement and apprehension and relocation tests for instability . standardised morphological study of the supraspinatus tendon was performed by a senior sonographer with 10 - years experience in musculoskeletal radiology targeted to tendon il movimento overhead , caratterizzato dalla elevazione del braccio al di sopra dei 90 associato ad abduzione ed extrarotazione , caratteristico di diverse discipline sportive e la sua biomeccanica ed i riflessi patologici sullarticolazione di spalla sono stati ampiamente studiati negli ultimi anni [ 1 ]  . fra le patologie di spalla la sindrome da impingement secondario del tendine sovraspinato affligge di frequente i giocatori di sport cosiddetti overhead , comportando il rischio di compromettere la carriera atletica [ 1 ]  . 
negli ultimi anni una comprensione progressiva ed una classificazione pi approfondite dei meccanismi patogenetici responsabili di questa condizione hanno permesso trattamenti ortopedici pi appropriati rispetto al passato con risultati pi accettabili in termini clinici e di recupero funzionale [ 2 ]  . 
il tendine del sovraspinato coinvolto elettivamente nei disordini secondari della cuffia dei rotatori , indipendentemente dalla loro etiologia [ 3 ]  . lecografia , specialmente in mani di operatori esperti , considerata attualmente indagine di primo livello nella diagnosi di impingement o di lesioni del sovraspinato , in virt della elevata risoluzione spaziale , del buon contrasto tissutale e della multiplanariet , con risultati sovrapponibili a quelli della rm specie nellindividuazione e nella quantificazione delle lesioni tendinee [ 4 ]  . 
i criteri di esclusione erano rappresentati da alterazioni muscolari dovute a precedenti traumi di spalla , malattie sistemiche muscoloscheletriche e / o assunzione corrente di fans o di steroidi . tutti i soggetti sono stati sottoposti in via preliminare ad una visita clinica come precedentemente descritto [ 7 ] , comprendente i test di neer e di hawkins per limpingement anteriore e posteriore ed i test di apprensione e di relocation per linstabilit . 
lo studio morfologico standardizzato del tendine sovraspinato stato eseguito da un ecografista esperto con 10 anni di esperienza nel settore muscoloscheletrico , mirato ai siti tendinei ed extra - tendinei tipicamente coinvolti nella sindrome da impingement [ 8 ]  . 
lo spessore del tendine stato misurato in prossimit dellinserzione sulla testa omerale , fra la superficie inferiore della borsa ed il margine inferiore visibile della struttura fibrillare . and extra - tendon sites of typical involvement in impingement syndrome [ 8 ]  . 
examination starts with the probe placed parallel to the longitudinal tendon axis ( paracoronal plane ) with the shoulder internally rotated and extended by positioning the arm behind the back in order to increase the extent of supraspinatus lateral to the acromial shadow [ 9 ]  . 
lo spessore del tendine stato misurato dalla sutable 1 tendon echotexture was interpreted based on a division into grades , following neers classification of tendon tears [ 10 ] tendon echotexture fibrillar hypoechoic hyperechoic iiia partial tear iiib full - thickness tear tabella 1 lecostruttura del tendine stata interpretata in base ad una suddivisione in gradi , operata sulla falsariga della classificazione di neer delle lesioni tendinee [ 10 ] ecostruttura del tendine fibrillare ipoecogena iperecogena iiia iiib lesione parziale lesione a tutto spessore table 2 criteria for interpretation of the other sonographic parameters considered [ 9 , 10 ] increased reduced tendon thickness , mm bursal thickness , mm subacromial space , mm anterior impingement spazio subacromiale , mm impingement anteriore normal present normale presente absent assente tabella 2 criteri di interpretazione degli altri parametri ecografici considerati [ 9 , 10 ] spessore del tendine , mm spessore della borsa subacromiale , mm aumentato ridotto r . 
dynamic passive abduction of the arm from this position assesses tendon integrity and the presence or absence of tendon impingement beneath the acromial margrepositioning the arm in the original posiperficie inferiore della borsa perpendicolarmente fino al margine inferiore dove lecostruttura fibrillare risulta chiaramente visibile . 
la sonda successivamente posizionata perpendicolarmente al maggior asse tendineo ( piano parasagittale ) , con lavambraccio flesso a 90 sul braccio , a sua volta addotto al tronco con la mano poggiata sulla spalla controlaterale . la successiva abduzione passiva del braccio volta a stabilire lintegrit del tendine e la presenza od assenza di impingement del tendine al di sotto del margine dellacromion . 
none of the sonographic measurements is pathological in relation to the criteria selected mean bursal thickness , mm tendon thickness , mm subacromial space , mm impingement echotexture spessore borsa , mm spessore tendine , mm spazio subacromiale , mm impingement ecostruttura dx , spalla destra ; sx , spalla sinistra ; ds , deviazione standard r , right shoulder ; l , left shoulder ; sd , standard deviation tabella 4 reperti ecografici nei soggetti di controllo non - atleti . 
4a , b player with right shoulder paon the basis of the selected cut - off , the width of the right subacromial space is moderately reduced ( a ) whereas the contralateral space ( b ) is normal . 
measurement of the subacromial space in the parasagittal scan , lateral to the acromion shadow cone , between the point of entry of the tendon at dynamic abduction and the humeral - head cortical fig . 
misurazione dello spazio subacromiale in scansione parasagittale , effettuato lateralmente al cono dombra dellacromion fra il punto di ingresso del tendine alla manovra dinamica di abduzione e la corticale della testa omerale r . 
imaging was interpreted following criteria reported in tables 1 and 2 , according to expected clinical and sonographic patterns previously described [ 9 , 10 ] and choosing a cut - off of 7 mm for subacromial space width . 
statistical analysis on measurements lacking a definite cut - off for pathology ( tendon and bursal thicknesses ) was based on a two - tailed u mann - whitney test whereas the one - tailed fisher exact test was used for the other parameters . 
four players showed symptoms and positive clinical signs of secondary supraspinatus impingement syndrome at an early stage : two right shoulder atraumatic instability ( previous spontaneous subluxation episode in one case ) , and anterior shoulder pain in one right assenza di restringimento per lampiezza dello spazio subacromiale . 
lanalisi statistica sulle misure che difettavano di un cut - off patologico definito ( spessori del tendine e della borsa ) si avvalsa di un test u di mann - whitney a due code , mentre per gli altri parametri stato utilizzato il test esatto di fisher ad una coda ; in entrambi i casi il livello di significativit era fissato al 5% ( p < 0 , 05 )  . risultati tutti i soggetti erano destrimani . 
quattro giocatori mostravano sintomi e test clinici positivi suggestivi di impingement secondario del sovraspinato ad uno stadio iniziale : 2 instabilit atraumatiche di spalla destra ( episodio di sublussazione spontanea in 1 caso ) e dolore anteriore di spalla a carico di 1 spalla destra e di 1 spalla sinistra ( con scrosci articolari nel primo caso )  . 
i rilievi ecografici sono riportati neltabella 6 classificazione secondo belling srensen e jrgensen dellimpingement della cuffia dei rotatori secondo il modello del continuum impingement - instabilit ( modificata da [ 2 ] ) r . 
no significant sonographic differences were found between players and controls in tendon echotexture ( all cases classified as grade 0 ) and in dynamic anterior impingement testing ( absent in all cases )  . 
gli spessori del tendine e della borsa subacromiale non mostrano significativa differenza fra le spalle di destra e di sinistra allinterno dello stesso gruppo e fra i 2 gruppi ( p > 0 , 05 ) ( tabella 5 )  . 
lo spazio subacromiale risulta ridotto in ampiezza sia a destra ( a ) che a sinistra ( b ) , pur in assenza di alterazioni ecostrutturali a carico del tendine sovraspinato e della borsa subacromiale . discussion basketball is considered an overhead sport due to repeated shooting and passing , throwing - like movements , studied in the throwing model , characterised by typical elevation - abduction - extrarotation of the arm , which lead to functional overload and mechanical stress of the supraspinatus [ 11 ]  . 
neers pathogenic model , which explains primary supraspinatus impingement as a consequence of a primitive narrowing of the subacromial space , cannot alone explain the association of supraspinatus lesions with shoulder instability in overhead players [ 12 ] , as demonstrated by the fact that surgical intervention on instability does provide symptom relief and acceptable functional recovery whereas acromionplasty and / or surgical - cuff decompression fails this goal in most cases [ 13 ]  . 
a supraspinatus tendon can be damaged , primarily as a consequence of an eccentric tensile overload responsible of tears [ 15 ] or when chronically impinged during throwing movement between the humeral head and the postero - superior capsular labrum in the so - called postero - superior impingement ( or internal impingement ) [ 13 , 16 ]  . 
according to the continuum impingement - instability pathologic and clinical model , supraspinatus impingement in overhead athletes would be a consequence of a primary narrowing of the subacromial space that occurs in a spectrum of clinical conditions , which vary from pure impingement as described by neer , usually more frequent in older athletes ( > 35 years ) , to pure instability , prevailing in young athletes ( 1835 years )  . 
in our series , in comparison to a control group of ten non - athlete subjects with no clinical or sonographic evidence of supraspinatus impingement , 6 / 10 modello del lancio , caratterizzati dalla tipica elevazione - abduzione - extrarotazione del braccio e responsabili di sovraccarico funzionale e di stress meccanico del tendine sovraspinato [ 11 ]  . 
il modello patogenetico di neer , che riconduce limpingement primario del sovraspinato ad un restringimento primitivo dello spazio subacromiale , non in grado di spiegare da solo lassociazione delle lesioni tendinee con linstabilit di spalla [ 12 ] , come dimostrato dal fatto che la correzione chirurgica dellinstabilit determina un beneficio sintomatico ed un recupero funzionale accettabili , mentre lacromionplastica e / o la decompressione chirurgica della cuffia perlopi falliscono questo obiettivo [ 13 ]  . in pi , stata riportata una caratteristica prevalenza di lesioni tendinee del versante articolare piuttosto che di quello bursale negli atleti overhead giovani con spalla dolorosa [ 14 ]  . 
il danno tendineo pu essere primitivo , come conseguenza di un sovraccarico tensivo ( eccentric tensile overload ) responsabile di lacerazioni [ 15 ] o di un impingement cronicamente ripetuto , durante il movimento di lancio , fra la testa omerale ed il labbro capsulare postero - superiore , nel cosiddetto impingement postero - superiore ( o impingement interno ) [ 13 , 16 ]  . secondo il modello patogenetico e clinico del continuum impingement - instabilit limpingement del sovraspinato negli atleti overhead sarebbe conseguenza di una primitiva riduzione in ampiezza dello spazio subacromiale nel contesto di uno spettro continuo di condizioni cliniche variabili dal puro impingement come descritto da neer , di solito pi frequente negli atleti vecchi ( > 35 anni ) , fino allinstabilit pura , prevalente negli atleti giovani ( 1835 anni ) , passando attraverso una serie di condizioni intermedie con caratteristiche miste , ciascuna delle quali richiede un trattamento adeguato ( tabella 6 ) [ 2 , 14 ]  . nella nostra esperienza in confronto ad un gruppo di controllo di 10 soggetti non atleti senza evidenza clinica od ecografica di impingement del sovraspinato , 6 giocatori di basket su 10 hanno mostrato un significativo restringimento dello spazio subacromiale allecografia : di quello destro ( spalla dominante ) in 2 casi asintomatici ed in 1 caso di spalla dolorosa , di quello sinistro ( spalla non dominante ) in 1 caso di r . 
6 the subacromial space of the right shoulders ( dominant ) in the players group is reduced in a significant number of cases compared with the control group ( no reduction )  . 
6 lo spazio subacromiale delle spalle di destra ( dominanti ) nel gruppo dei giocatori ridotto in un numero significativo di casi rispetto al gruppo di controllo ( non riduzione )  . 
us + , spazio subacromiale < 7 mm ; us - , spazio subacromiale 7 msullasse delle ordinate riportato il numero di spalle osservate . basketball players showed sonographic subacromial space narrowing : right dominant in two asymptomatic cases and in one case of painful shoulder , left non - dominant in one case of painful shoulder and bilaterally in two cases of right atraumatic instability . 
because of the insufficient accuracy of this sonographic measurement , despite the fact that reliability comparable to standard radiography has been reported [ 19 ] , we preferred a lower cut - off to be sure of effective space narrowing . 
in particular , no tendon thickness measurements were considered pathologic because of its high interobserver variability reported to be as high as 56% in one study on asymptomatic subjects [ 20 ]  . the same considerations were extended to subacromial bursa thickness . 
the data obtained are in agreement with the concept of a primitive space narrowing in athletes as a cause of supraspinatus impingement , independently from clinical onset and before the sonographic evidence of tendon damage becomes visible . meanwhile 